pomegranate: cultivation, pomological properties

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FOOD SCIENCE AND TECHNOLOGY

POMEGRANATE CULTIVATION, COMPOSITION, ANTIOXIDANT PROPERTIES, AND HEALTH BENEFITS

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FOOD SCIENCE AND TECHNOLOGY

POMEGRANATE CULTIVATION, COMPOSITION, ANTIOXIDANT PROPERTIES, AND HEALTH BENEFITS RAFAT A. SIDDIQUI AND

MOHAMMED AKBAR EDITORS


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Library of Congress Cataloging-in-Publication Data ISBN: H%RRN

Published by Nova Science Publishers, Inc. † New York


CONTENTS Foreword

vii Mohammed Abdul Sattar Khan

Preface

xi

Acknowledgments Chapter 1

Chapter 2

Chapter 3

Pomegranate: Cultivation, Antioxidant Properties and Its Health Benefits Leonardo Sepúlveda, Juan Buenrostro-Figueroa, Juan A. Ascacio-Valdés, Antonio F. Aguilera-Carbó and Cristóbal N. Aguilar Anti-Oxidant and Therapeutic Properties of Pomegranate for Health Benefits Mohammed Mahboob, Pitta Venkata Prabhakar, Bugata Lakshmi Sai Pratyusha and Addi Utkarsh Reddy Pomegranate: A Health Promoting Food Antonia Kotsiou and Christine Tesseromatis


xiii 1

29

53

vi Chapter 4

Chapter 5

Contents The Effect of Aphis Punicae on Pomegranate Trees in Tunisia: Biology and Natural Enemies Lassaad Mdellel, Amani Tlili, Rihem Adouani and Monia Ben Halima Kamel Beneficial Effects of Pomegranate on the Cardiovascular System Nathalie Tristão Banhos Delgado, Wender do Nascimento Rouver and Roger Lyrio dos Santos

Chapter 6

Pomegranate: Health Benefits on Brain Functions Saida Haider, Zehra Batool and Saiqa Tabassum

Chapter 7

What Can We Learn About the Traits of Aril Juice by Studying Wide Collections of Diverse Pomegranate Fruits? Rachel Amir, Doron Holland and Li Tian

Chapter 8

Chapter 9

Pomegranate: Cultivation, Antioxidant Properties, Health Benefits and Effects of Industrial Processing Marina Cano-Lamadrid, Patricia Navarro-Martinez, Santiago Lopez-Miranda, Aneta Wojdyło, Angel. A. Carbonell-Barrachina and Antonio Jose Perez-Lopez Pomegranate: Cultivation, Pomological Properties, Processing, Global Market and Health Benefits Laila Hussein, Mostafa Gouda and Eid Labib

69

89

147

203

231

267

About the Editors

303

Index

307


FOREWORD Pomegranate is a wonderful and delicious fruit of paradise, which is mentioned in the holy Quran and in the Bible. This fruit has a beautiful, shiny red and spherical shape with crown-like structure on its apex and is categorized as a berry. Its botanical name is Punica granatum and it grows on deciduous shrub between 5 and 8 meters tall. Pomegranate belongs to the order Myrtales and family Lythraceae or Punicaceae. The family of Punicaceae consists of single genus Punica with two species, namely Punica granatum and protopunica. The name pomegranate originates from Latin words pomum means “apple” and granatum means “seeded”. In ancient days, pomegranate was cultivated mostly in Iran and India now it is widely cultivated in different parts of the world, including the Mediterranean region, Europe, and North and South America. The pomegranate tree is tolerant to almost all soil types, but thrives in a fairly dry climate and produces best quality fruits in arid regions. The pomegranate fruit is self-fertile and its production takes about 2-3 years. The fruit has medium to thick rind and chambers inside the pomegranate comprise of beautiful arranged soft, juicy, ruby-colored arils, which encapsulate medium to hard nutritiously edible seeds. The arils mainly contain carbohydrate, protein and fat. They are also the natural source of fiber, vitamins (C, E and K), potassium and folate.


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Mohammed Abdul Sattar Khan

Traditionally, the potion of pomegranate has proven to be beneficial for health. In virtue of its nutritional, therapeutic, and ornamental values, it has been known as a remedy for many ailments since ancient times. Scientifically, different parts of the pomegranate fruit (e.g., peel, arils and seeds) contain several active ingredients. The peel is an important source of bioactive compounds (e.g., phenolics, flavonoids, ellagitannins and quercetin), complex polysaccharides, and minerals; arils mainly contain water, carbohydrates (e.g., fructose and glucose), pectin, organic acid, and bioactive compounds such as phenolics and flavonoids (e.g., anthocyanins); seeds mainly comprise of the oily components, triacylglycerols (e.g., linolenic acid) and sterols. The pomegranate extract or juice, because of the presence of the biologically active polyphenols, is rich in anti-oxidant, anti-inflammatory, anti-viral, and anti-aging properties. The bioactive compounds, independently or collectively, also have been demonstrated to exert therapeutic effects against cancers and neurological diseases. The pomegranate is aphrodisiac food, and its extract or juice also fortifies the immune system, prevents anemia, reduces oxidative stress, controls hyperglycemia, lowers blood pressure and cholesterol, freshens skin and teeth, and protects prenatal brain. Economically, pomegranate fruit is considered as a health-promoting food, and it has found a significant place in the international commercial markets. Commercially, pomegranate juice is not only sold as a meat marinade or sourcing agent in salads but also marketed as a natural fruit juices and as pomegranate-flavored beverages. Owing to its popularity, establishment of commercial orchards of pomegranate have grown in huge numbers in order to meet the rising global demand. Now, this fruit is cultivated in both Southern and in Northern hemispheres. The advancement in research of pomegranate is updated time to time through the literary means in the form of review articles or books. In the past, several books were published to disseminate the health benefits of pomegranate. However, this book assimilates the latest knowledge on pomegranate fruit and its cultivation, pest control, storage. Most importantly, the health benefits of pomegranate in the form of chapters. The authors have done a good job by extracting core and up-to-date


Foreword

ix

information from the most recent research articles on pomegranate, and presented to the readers in a succinct manner. I leave you with this healthful quote “If you want health, you must fall in love with pomegranate.”

Mohammed Abdul Sattar Khan, PhD Massachusetts General Hospital Harvard Medical School Shriners Hospital for Children Boston, MA.



PREFACE Recently for the prevention and treatment of many neurologic, metabolic and other chronic diseases, the focus has shifted from allopathic Western medicine to alternative and complementary medicine in many countries including the United States of America. In fact, some dietary supplements including herbs, vegetables, nuts and fruits have demonstrated promising outcomes in improving human health. Among them, pomegranate is one of the exotic fruits that has been known for its valuable effects. Pomegranate (Punica granatum), one of the oldest known fruits belongs to the family Lythraceae. It is believed to have originated in Persia and have several varieties. At present, pomegranates are grown in many Middle Eastern, Asian, European and other Western countries, including the United States of America. Pomegranates have been used for thousands of years in treating a wide variety of diseases in ancient times and currently used to treat many complications, including; pregnancy, coronary heart disease, atherosclerosis, prostate cancer and male infertility. The beneficial effects of pomegranate have been revealed in the writings of many cultures and religions, since they were grown in abundance in all ancient civilizations. Pomegranates have been appreciated for their beautiful color and flavor, and health benefits since ancient times. The edible juicy ruby red arils are full of antioxidants and are considered the healthiest part of


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Rafat A. Siddiqui and Mohammed Akbar

the fruit. Fortunately, the century-old description of the pomegranate as a healthy fruit among all others still holds its integrity due to the presence of antioxidants, vitamins B6 and C, minerals, and fiber. Accumulated evidence suggests that naturally occurring phytocompounds, such as polyphenolic antioxidants found in pomegranate may potentially hinder neurodegeneration and improve memory, cognition and other brain functions. The effects of pomegranate as a medicine in neurological and other diseases are discussed in this book. This book will benefit students at various levels of academia, scientists in several disciplines (such as alternative medicine, nutrition, neuroscience, agriculture, food science, and medicine) and many others interested in this discipline. This book may become a part of the curriculum at various universities globally.


ACKNOWLEDGMENTS We are grateful to the authors of the book chapters for sharing their research expertise to provide evidence-based information on pomegranate and its beneficial effects on various health conditions. We are indebted to our families for their understanding and unconditional support, allowing us to spend extra time on completing this book. Many thanks are due to our teachers, colleagues, postdoctoral fellows and students, who inspired us to investigate and put together the potential health and medicinal values of pomegranate as a natural food product for human health. We are thankful to the Virginia State University, Petersburg, VA and the National Institutes of Health, Bethesda, MD, for permitting us to use our personal time to finish this book in a successful manner. Thanks are due to the staff of Nova Publishers for their patience and assistance at various stages of the publication of this book.



In: Pomegranate Editors: Rafat A. Siddiqui et al.

ISBN: 978-1-53614-119-1 © 2018 Nova Science Publishers, Inc.

Chapter 1

POMEGRANATE: CULTIVATION, ANTIOXIDANT PROPERTIES AND ITS HEALTH BENEFITS Leonardo Sepúlveda1, PhD, Juan Buenrostro-Figueroa2, PhD, Juan A. Ascacio-Valdés1, PhD, Antonio F. Aguilera-Carbó3, PhD and Cristóbal N. Aguilar1,, PhD 1

Food Research Department, Autonomous University of Coahuila, Saltillo, Coahuila, México 2 Research Center in Food and Development, A.C. Cd. Delicias, Chihuahua, México. 3 Department of Animal Nutrition, Agrarian Autonomous University “Antonio Narro,” Saltillo, Coahuila, México



Corresponding Author Email: [email protected].


2

L. Sepúlveda, J. J. Buenrostro-Figueroa, J. A. Ascacio-Valdés et al.

ABTSRACT Pomegranate (Punica granatum L.) in Mexico is a marginal fruit and ornamental tree. Despite its wide adaptation, pomegranate is consumed sporadically and seasonally, usually as fresh fruit and as decoration in Mexican food. In Mexico, pomegranate is also used in traditional herbolaria taking advantage of all parts of the fruit and the shrub. For example, leaves and stem are used as antidysenteric while root as astringent and anthelmintics. This chapter discusses the origin, botanical and biodiversity of pomegranate fruit in Mexico. General aspects of its cultivation, economic importance, nutritional composition, as well as some products derived from this fruit are highlighted. The antioxidant and antimicrobial characteristics from pomegranate were explained in detail. Finally, major research and development work on the use of pomegranate against diabetes and obesity problems are also discussed.

1. INTRODUCTION Pomegranate is a fruit with a remarkable high content of carbohydrates, vitamins and minerals. This fruit is cultivated in a great variety of regions in the world, from the northern hemisphere through the Mediterranean to the Middle East. This fruit since ancient times symbolizes prosperity and ambition. It also appeared in ancient Greece in the coins of Side in Pamphylia an ancient region of the Mediterranean coast south of Asia Minor (Sear, 1978). Currently it is used in wide culinary range forming part of typical food in every corner of the world such as salads, ice cream and consomme with meat. In Mexico, pomegranate forms part of the ingredient for a traditional dish called “chiles en nogada”. This fruit attracts attention for its various benefits for human health. For example, fractions obtained from sweet, sour and sour pomegranate peel were reported to have antifungal and antibacterial activities (Rosas-Burgos et al. 2016). In addition, pomegranate juice was evaluated as a potential chemopreventive agent for prostate cancer and the action of ellagic acid on proliferation by regulation of the cell cycle or induction of apoptosis (Naiki-Ito et al. 2015). The benefits of pomegranate to human health can be attributed mainly to a group of compounds called


Pomegranate

3

polyphenols. These polyphenols are currently classified into condensed tannins and hydrolysable tannins. Condensed tannins such as proanthocyanidins, prodelphinidins, profisetinidins, etc. are oligomers of flavan-3-ol units through carbon-carbon bonds such as epicatechin, catechin, fisetinidiol. Hydrolysable tannins, belong to the gallotannins (galoyl groups linked by ester bond generally bound to glucose) and the ellagitannins (derivatives of hexahydroxydiphenic acid linked to a polyol via an ester bond). Finally, florotannins that are oligomeric compounds of the phloroglucinol present in brown algae (Quideau et al. 2011). The novelty of these compounds is due to the different biological activities present. For example antioxidants (Bharathi and Jagadeesan, 2015), treatment and prevention of free radicals by neuronal damage (Farbood et al. 2015) and anthelmintic activity (Mondal et al. 2015). The objective of this chapter is to highlight the general aspects of cultivation, botanical and biodiversity, as well as economic importance, uses and chemical composition of the pomegranate fruit. The role of pomegranate fruit and its polyphenol compounds for the prevention and treatment of obesity and diabetes are also discussed.

2. POMEGRANATE CULTIVATION 2.1. Origin, Botany and Biodiversity Pomegranate (Punica granatum L.) belongs to the Punicacea family, native to central Asia, notably Iran (Figure 1). Its scientific name was derived from the latin Pomuni (apple) granatus (grainy), which means seeded apple (Erkan and Kader 2011). The origin and genetic diversity of pomegranate can be classified into three mega-centres (primary, secondary and tertiary) and five macro-centres (Middle Eastern, Mediterranean, Eastern Asian, American and South African) (Teixeira da Silva et al. 2013). Pomegranate cultivation was extended to the east (India, China) and west (Mediterranean countries such as Turkey, Egypt, Tunisia, Morocco


4


and Spain). It was introduced into Central America, Mexico and South America by the Spanish during the 1500 and 1600s (Pande and Akoh 2016).

Figure 1. Punica granatum var. Wonderful (ref?).

Pomegranate grows as a shrub or small tree of abundant foliage that naturally trends to develop multiple trunk with a bushy appearance, generally spiny and able to grow up to 3-8 m high (Holland et al. 2009). Pomegranate plant has green, small, narrow leaves with smooth and shiny surface slightly undulated, with short stems. The flower is composed of 5 - 7 bright orange petals arranged in a flared form, having three flower types (hermaphrodite, male and intermediate) (Holland et al. 2009). Pomegranate fruit is developed from the ovary and it is a fleshy berry (6.25 to 12.5 cm wide with a weight range from 200-650 g) with a broad range color from reddish yellow to green with reddish zones. It has a smooth leathery skin which is separated by a white, spongy, astringent and thin inedible membranes appeared as a number of cells, each packed full of angular seeds contained in a juicy pulp sac called Arils. The arils are prismatic, thick with a woody consistency, fleshy or pulpy, very juicy with bittersweet flavor and come in color ranging from intense ruby to white (García-Viguera and Perez-Vicente 2004; Erkan and Kader 2011). The


Pomegranate

5

arils vary in size and hardness while the skin varies among the cultivars growth in several parts of the world (Holland et al. 2009). According to Stover and Mercure (2007), there are around 500 pomegranate cultivars in the world. However, many of them have similar genotype with different names (according to the region). Differences are established according to potential market, preferred use and consumer preference, taking into account important parameters such as fruit size, husk color (yellow to purple, with pink and red as most common), aril color (from white to red), seed hardness, maturity, juice content, acidity, sweetness and astringency. Amongst the most common pomegranate variants and its ‘local name’ are as follows; “Wonderful” in EEUU, “Molar de Elche/Valenciana” in Spain, “Ahmar/Aswad/Halva” from Iraq, “Mangulati” from Saudi Arabia, “Red Loufani/Ras el Bahgl” from Israel & Palestine and “Apaseo/Apaseo tardía” in Mexico (Herrera-Hernández et al. 2013).

2.2. Cultivation Pomegranate planting is favorable in climates with altitude below 1000 m, where winters are mild (with a minimal temperature of -12°C) and summers are long and hot without rain (useful during last stages of fruit development, tolerating up to 45-48°C). The plant likes the light and reacts negatively to excessive shading. However, direct sunlight exposure for long time can produce sunburn damages (Pande and Akoh 2016). Likewise, high relative humidity or rain are not suitable for its cultivation as it will result in less tasty and mild sweetness fruit (Erkan and Kader 2011). Pomegranate can be cultivated from seeds, but the most common propagation method is by hardwood cuttings. The fruit is non-climacteric i.e., does not ripen after cutting off the tree, even with ethylene treatment. Hence, it is recommended to harvest the fruit at a fully ripe stage or ripe consumption, to avoid difficulties in its marketing. Commercial value depends on variety and quality of the fruit, presence of defects (sunburn, cracked), sugar content, arils size and seed hardness (Pande and Akoh 2016). Pomegranate fruit is also prone to insect attack including ants, pomegranate butterfly (Virachola isocrates), leaf-footed bug (Leptoglossus


6


zonatus) and fruit flies (Drosophila melanogaster). Moreover, microbial attack is also of concern due to the resulting effect i.e., grey mold by Botrytis cinerea (the most economically important), fruit rotting caused by Alternaria alternata, Aspergillus niger, Penicillium implicatum, Nematospora, Coniella granati, wilt caused by Fusarium oxysporum and bacterial blight by Xantomonas axanopodis (Pande and Akoh 2016). To control these pathogens, on-field application of fungicides during developing stages are necessary. The application of postharvest fungicides are carried out to control localized infections or to protect the fruit during postharvest processes and storage (Erkan and Kader 2011). Lately, various researches were carried out focusing on the development of postharvest technologies that can improve and extend the storage life of fruit quality. Technologies such as heat treatment, fungicides (to control decay), biocontrol (using natural antimicrobial agents), packaging combined with edible coatings and organic acids, gamma irradiation, modified packaging as well as controlled atmosphere storage were used to maintain postharvest quality and to alleviate chilling injury symptoms which occur during the storage of pomegranate below 5-7°C, depending on the variety and storage duration (Munhuweyi et al. 2016).

2.3. Economic Importance According to the Food and Agriculture Organization of the United Nations (FAO 2014), India is the largest producer and consumer of pomegranate, Spain is the country with highest productivity (18.5 t/ha), while Iran and India are leading exporters with values of 60,000 and 35,176 t/year respectively. Pomegranate production in Mexico was 4774 ton in 2015 with a yield of 6.88 t/ha with Oaxaca, Guanajuato e Hidalgo the main producers that contributed 70% of national production (SIAP 2015). Production was increased due to dissemination of the functional and medicinal properties, as well as the development of new products such as juices, nectars, teas, food supplements, wine, liquor, pills, creams, facial and body oils (Mercado Silva et al. 2011).


Pomegranate

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2.4. Composition The variety, geographical location, agronomic practices, seasonal and maturity differences, as well as processing and storage conditions, greatly affect the composition of both fruits and juice (Melgarejo-Sánchez et al. 2015). Some researchers reported on the studies about compositional analysis of several pomegranate varieties, even its parts such as peel, seed, juice, leaves and flowers (Robledo et al. 2008; Ferrara et al. 2011). The edible fraction (arils) corresponds to 55-60% of total fruit weight and contains 80% juice and the rest are seeds (Erkan and Kader 2011). One example for food value of pomegranate fruit (raw and juice) is shown in Table 1 (USDA, 2016). Table 1. Food value of pomegranate fruit “Wonderful” (raw and juice) Pomegranate (100 g) Proximates Water Energy Protein Total lipid (fat) Carbohydrate, by difference Fiber, total dietary Sugars, total Minerals Calcium, Ca Iron, Fe Magnesium, Mg Phosphorus, P Potassium, K Sodium, Na Zinc, Zn Vitamins Vitamin C, total ascorbic acid Thiamin Riboflavin Niacin

Unit g kcal g g g g g

Raw Value 77.9 83 1.67 1.17 18.7 4 13.7

Juice Value 86 54 0.15 0.29 13.1 0.1 12.7

mg mg mg mg mg mg mg

10 0.3 12 36 236 3 0.35

11 0.1 7 11 214 9 0.09

mg mg mg mg

10.2 0.07 0.05 0.29

0.1 0.02 0.02 0.23


8

L. Sepúlveda, J. J. Buenrostro-Figueroa, J. A. Ascacio-Valdés et al. Table 1. (Continued)

Pomegranate (100 g) Proximates Vitamin B-6 Folate, DFE Vitamin E (alpha-tocopherol) Vitamin K (phylloquinone) Lipids Fatty acids, total saturated Fatty acids, total monounsaturated Fatty acids, total polyunsaturated

Unit mg µg mg µg

Raw Value 0.08 38 0.6 16.4

Juice Value 0.04 24 0.38 10.4

g g g

0.12 0.09 0.08

0.08 0.06 0.05

DFE, dietary folate equivalents: USDA National Nutrient Database for Standard Reference Release.

Pomegranate juice is rich in sugars, organic acids, vitamins, polysaccharides and essential minerals. The phenolic content is higher than those reported for red wine, cranberry, strawberry, blueberry, orange juice and green tea. The antioxidant activity of pomegranate juice is up to 3-fold higher than red wine and green tea (Gil et al. 2000). Punicalagin, punicalin, anthocyanins and ellagic acid are the main compounds present in pomegranate. Changes in phenolic compounds profile in pomegranate juice are a result from juice extraction processes (Calani et al. 2013; Nuncio-Jáuregui et al. 2015a). Polyphenolic compounds have great pharmaceutical interest because they exhibit several biological properties, which were directly responsible for the increased consumption of pomegranate juice and products over the last few years (Ascacio-Valdés et al. 2011).

2.5. Product Derived from Pomegranate Pomegranate is consumed as whole fruit, juice, or processed into various products. Arils is used as ingredient in cuisine in baked goods, energy bars, yogurt, ice cream, salad dressings and its dehydrated form for easy conservation. Seed is used to obtain an oil rich fraction containing


Pomegranate

9

punicic acid (81%), oleic, palmitic, linoleic and stearic acids. Pomegranate extracts are used in skin care product and health supplements (Pande and Akoh 2016). Pomegranate juice has been reported to have several health benefits including controlling obesity, diabetes, hypertension, cardiovascular disease, hypercholesterolemia, antiatherogenic effect, anticancer and antimicrobial activity (Seeram et al. 2005a; Çam and Hışıl 2010; Al-Muammar and Khan 2012). Interestingly, the above nutraceutical properties are not limited to the edible part of pomegranate fruit. In fact, the non-edible parts of fruit and tree (i.e., leaf, flower, peel, seeds and stems), although considered as waste, contain even higher amounts of specific nutritionally valuable and biologically active components as compared to the edible fruit (Viuda-Martos et al. 2011; Akhtar et al. 2015). Table 2 shows some results on the health benefit of pomegranate fractions. Pomegranate peel is rich in ellagitannins as punicalin, punicalagin, gallic acid, ellagic acid and glycosides (Ascacio-Valdés et al. 2011). There are reports about valorization of pomegranate peels in biotechnological process to obtain enzymes such as tannase, pectinase and ellagitannase (Buenrostro-Figueroa et al. 2014a; de la Cruz et al. 2014) and bioactive compounds using fungal strains (Sepúlveda et al. 2012; BuenrostroFigueroa et al. 2014b; Sepúlveda et al. 2016). Since the yield of bioactive compounds recovered is affected by extraction type and conditions, pomegranate polyphenols extraction has been evaluated by different methods, such as maceration, pulsed ultrasound-assisted extraction, ultrasound-assisted extraction, microwave-assisted extraction, enzymeassisted extraction and fermentation-assisted extraction (Aguilera-Carbó et al. 2008; Ascacio-Valdés et al. 2014; Çam and İçyer 2015; Sood and Gupta 2015; Akhtar et al. 2015). According to Kazemi et al. (2016), one ton of fresh pomegranate generates 669 kg of by-products, including 78% peel and 22% seeds. It is estimated that more than 782,730 ton of peels are generated annually in the world, important source of molecules with biological activity that can be valorized into interesting compounds for food, cosmetic and pharmaceutic industries.


10 L. Sepúlveda, J. J. Buenrostro-Figueroa, J. A. Ascacio-Valdés et al. Table 2. Health benefits from pomegranate fractions Pomegranate fraction Juice Juice

Peel

Whole fruit extract

Peel

Polyphenols (capsules)

Husk extract

Juice

Main Results Significant decrease in interleukin-6 and C-reactive protein in plasma It increases the levels of insulin in the blood, consequently, it decreases the glucose levels. The combination of pomegranate peel extract and black bean peel extract have the ability to ameliorate hyperglycemia by inhibiting oxidative stress-induced damage to the pancreas. The punicalagin present in the extract is a promising therapeutic nutrient for the treatment of metabolic disorders, particularly non-alcoholic fatty liver disease. The poor bioavailability of pomegranate polyphenols, its bifidogenic effect observed after pomegranate peel extract consumption suggests the involvement of the gut microbiota in the management of host metabolism by polyphenolic compounds. The polyphenols reduces lipid peroxidation in patients with type 2 diabetes, but with no effects in healthy controls, and specifically modulates liver enzymes in diabetic and nondiabetic subjects. The pomegranate husk extract has an antiinflammatory action on adipocytes and macrophages but seems to be not able to reduce the inflammatory vicious cycle between both cells. The results showed a significant decrease in insulin resistance, body weight, hip circumstance, waist circumstance in intervention group whereas no significant changes were found for serum glucose, HbA1C, hs-CRP in this group.

Health benefit of pomegranate Antiinflammatory Antioxidant

Reference

Antioxidant

Wang et al. 2015

Antioxidant and Antiinflammatory

Zou et al. 2014

Antioxidant and Antiinflammatory

Neyrinck et al. 2013

Antiinflammatory

Basu et al. 2013

Antiinflammatory

Winand and Schneider, 2014

Antiinflammatory

Babaeian et al. 2013


Sohrab et al. 2014 Rezaei et al. 2013

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3. ANTIMICROBIAL ACTIVITY Antimicrobial activity is one of the most studied human health benefits of pomegranate fruit. Devatkal et al. (2013) reported the antibacterial activity of pomegranate peel against bacteria isolated from poultry meat where Pseudomonas stutzeri, an opportunistic bacteria isolated from poultry meat, showed sensitivity in vitro to aqueous extract of pomegranate peel. In another research by Malviya et al. (2014), pomegranate peels of Ganesh variety were subjected to extraction using different solvents viz. water, methanol and ethanol either alone or in combination with water and the antibacterial activity of peel extracts was tested against Staphylococcus aureus, Enterobacter aerogenes, Salmonella typhi and Klebsiella pneumoniae. The extract showed maximum antibacterial activity against S. aureus while minimum activity was recorded when tested against K. pneumoniae. On the other hand, Gullon et al. (2016) highlighted on the antibacterial activity of pomegranate peel flour against Listeria monocytogenes, Listeria innocua, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Salmonella sp. bacterial strains. From a total of eight phenolic compounds determined from the HPLC analysis, Punicalagin was the main component found in pomegranate peel flour (16.67 mg/g) followed by ellagic acid (0.15 mg/g). In other study, major polyphenols of pomegranate arils and peel by-products were extracted in 50% (v/v) aqueous ethanol, characterized and used in microbiological assays in order to test antimicrobial activity against clinically isolated human pathogenic microorganisms. Pomegranate phyto-complex extracts were shown to have an effective antimicrobial activity, as evidenced by the inhibitory effect on bacterial growth of two important human pathogens, often involved in foodborne illness (Pagliarulo et al. 2016). Moreover, the hydroalcoholic extract of pomegranate fruit peel showed activity against the dermatophyte fungi Trichophyton mentagrophytes, T. rubrum, Microsporum canis and M. gypseum. They concluded that the crude extract and punicalagin had a greater antifungal activity against T. rubrum, indicating that the pomegranate is a good target for study to obtain a new antidermatophyte medicine (Foss et al. 2014).


12 L. Sepúlveda, J. J. Buenrostro-Figueroa, J. A. Ascacio-Valdés et al.

4. ANTIOXIDANT PROPERTIES OF POMEGRANATE (PUNICA GRANATUM L.) Recently, pomegranate consumption has been reported to provide protection against cell oxidation, preventing the development of chronic diseases (Gil et al. 2000; Kalaycıoğlu and Erim, 2017). Cell oxidation is a process carried out by reactive oxygen species (ROS) and reactive nitrogen species (RNS), known as free radicals (Ascacio-Valdés et al. 2011), which can cause significant damage to biomolecules like DNA (Priyadarsini et al. 2002). It has been reported that the formation of free radicals that affect human cellular health was due to exposure to radiation, pollution, smoking, drugs etc. (Ascacio-Valdés et al. 2011). In recent years there has been increased interest in the development of research on pomegranate and its derivatives (juice, shells etc.) because of the compounds it contains (Ozgen et al. 2008). The antioxidant property of pomegranate is due to the fact that it has chemical compounds of natural origin, known as phytochemical compounds or bioactive compounds (Wilson and Hagerman, 1990). Within these bioactive compounds are the polyphenolic compounds or tannins, which are non-nitrogen compounds and form bonds with macromolecules such as proteins and carbohydrates (Ascacio-Valdés et al. 2013). Polyphenolic compounds are considered as metabolites of secondary metabolism of plants and function as protective agents. The polyphenolic compounds or tannins are classified into four groups: gallotannins, ellagitannins, complex tannins and condensed tannins (Aguilera-Carbó et al. 2008). Ellagitannins are the most abundant compounds in pomegranate, mainly in the juice and shells (Seeram et al. 2005b). It has been reported that ellagitannins possess important biological properties, and the pomegranate contains some of these with the highest biological activity reported. One of the biological properties of pomegranate ellagitannins is the antioxidant property (Masci et al. 2016), which is currently of great relevance because many chronic diseases are caused by the presence of free radicals, and the pomegranate ellagitannins are able to inhibit them (Amakura et al. 2000). This antioxidant capacity of


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pomegranate ellagitannins has been attributed to their molecular structure, because they have highly reactive groups such as hydroxyls and carbonyls, which can accept electrons donated by the oxidant and form a stable complex difficult to dissociate, representing the antioxidant activity of these compounds (Feldman et al. 2003; Feldman et al. 2005). Due to the reactivity of pomegranate ellagitannins, they have become highly interesting compounds in the field of research (extraction, separation, identification, characterization, purification and biological evaluation). In the literature, there are reports about the extraction and recovery of the pomegranate ellagitannins as well as their characterization and identification. The properties of these reported compounds have been specifically evaluated for biological properties. It has been demonstrated that ellagitannins in pomegranate juice possess high free radicals (ABTS, DPPH) inhibition activities (Mena et al. 2016). Other works about the antioxidant activity of pomegranate ellagitannins mention that juice, shells and arils of the pomegranate have ellagitannins with antioxidant properties, such as punicalagin, punicalin, ellagic acid hexoside, ellagic acid pentoside. These compounds were evaluated as antioxidants using FRAP (ferric reducing antioxidative power) and TEAC (trolox equivalents antioxidant capacity) techniques, showing an antioxidant activity of 9.1 mmol/L and 24.5 mmol/L, respectively (Fischer et al. 2011, Mphahlele et al. 2016). Other pomegranate shell ellagitannins such as galloyl-hhdphexoside, pedunculagin I, pedunculagin II have been evaluated as antioxidants using other free radical scavenging techniques, such as oxygen radical absorbance capacity (ORAC), with an antioxidant activity of 338 mmol trolox kg -1 of dry shell. This technique in combination with the ABTS and DPPH techniques provides very important information about the antioxidant potential of the bioactive compounds that the pomegranate contains (Nuncio-Jáuregui et al. 2015b). It is important to mention that not only the pomegranate juice, shells and arils have antioxidant potential. It has been shown that some products derived from pomegranate juice also have antioxidant activity. For example, pomegranate jelly was shown to have inhibition percentage of 41.8% for the ABTS radical and 44.8% for the DPPH radical. This indicates that the


14 L. Sepúlveda, J. J. Buenrostro-Figueroa, J. A. Ascacio-Valdés et al. bioactive compounds present in the juice (ellagitannins) remain viable and active after the processing of jelly (Ventura et al. 2013). This further highlights the potential of pomegranate as one of the fruits with the greatest antioxidant activity in the world.

5. DIABETES AND OBESITY PROBLEMS In Mexico, more than 4 million people were diagnosed with diabetes, of which the main causes are obesity, reduced physical activity, metabolic disorders and genetics. Pomegranate promises to be one of the natural sources to obtain compounds with potential anti-diabetic properties. For example, Banihani et al. (2014) demonstrated the direct effect of fresh pomegranate juice on fasting serum glucose and insulin levels in type 2 diabetes patients. Blood samples from 85 participants with type 2 diabetes were collected after a 12-hour fast, then 1 and 3 hours after administration of 1.5 mL of pomegranate juice per kg body weight. The results demonstrated decreased fasting serum glucose, increased β-cell function, and decreased insulin resistance among type 2 diabetes participants, 3 hours after pomegranate juice administration (p < 0.05). In another report by Wu et al. (2013), pomegranate husk extract, punicalagin and ellagic acid showed potent inhibition activity on fatty acid synthase with halfinhibitory concentration values (IC50) of 4.1 µg/ml (pomegranate husk extract), 4.2 µg/ml (4.50 µM, punicalagin) and 1.31 µg/ml (4.34 µM, ellagic acid), respectively. Evaluation on antihyperglycemic activity using relevant animal models and in vitro methods showed that the diabetic rats treated with ethyl acetate fraction of P. granatum leaves showed a dose dependent reduction of fasting blood glucose and normalized the lipid profile and liver antioxidant status in comparison to diabetic control group. This research concluded that the ethyl acetate fraction of P. granatum leaves appears to possesses antihyperglycemic activity along with antihyperlipidemic and antioxidant potential may prove beneficial for the management of diabetes and associated complications (Patel et al. 2014). On the other hand, type 2 diabetes has now been linked with the activation


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of the redox-sensitive transcription factor NF-κB. High cellular levels of ROS lead to the activation of this transcriptional factor. Therefore, pomegranate extracts with potent ROS scavenging activity, are able to inhibit the activation of NF-κB and diminish the development of type 2 diabetes complications (Banihani et al. 2013). Figure 2 shows the possible mechanism of action of ellagic acid, a potent antioxidant present in pomegranate fruit, to inhibit the activation of the NF-kB complex.

Figure 2. Possible inhibition of the polyphenolic compound on the IkB kinase (IKK).

Zhao et al. (2016) investigated the effect of pomegranate extract as intervention on immune function and the underlying mechanism involved in inflammation and oxidative stress in rats with high fat diet induced obesity. It was shown that the pomegranate extract was effective in improving immune function in high fat diet fed rats by inhibiting inflammation and decreasing oxidative stress. The effect of pomegranate vinegar on adiposity was also investigated in high-fat diet-induced obese rats (Ok et al. 2013). The results suggest pomegranate vinegar prevented hepatic lipid deposition by overflow of lipid to the liver resulting from


16 L. Sepúlveda, J. J. Buenrostro-Figueroa, J. A. Ascacio-Valdés et al. abnormalities of peripheral lipid storage in high fat status. It suggests that activated protein kinase activation of pomegranate vinegar might act as an energy sensor between liver and adipose tissue to improve metabolic health. The in vivo anti-hyperglycemic activity of Punica granatum (pomegranate) fruit peel extract was evaluated using Caenorhabditis elegans (Barathikannan et al. 2016). The results showed that ethyl acetate extract supplemented C. elegans worms showed inhibition of lipid accumulation similar to acarbose indicating good hypoglycemic activity. Normal worms (without supplemented with ethyl acetate extract) showed highest hypoglycaemic activity by increasing the lifespan of the worms. As a consequence, pomegranate peel extract could be used in preventing the incidence of long-term complication of diabetics. Another researcher reported that pomegranate juice consumption increases the binding of high-density lipoproteins to paraoxonase 1 (PON1), thus increasing the catalytic activity of this enzyme (Estrada-Luna et al. 2017). Decreased levels of PON1 are associated with higher levels of cholesterol. The effects of pomegranate juice on body weight, cholesterol and triacylglycerol were studied through 5 months of supplementation. The results suggest that pomegranate juice protects against the effects of a high-fat diet in body weight, and decrease cholesterol levels. Rodriguez et al. (2015) demonstrated the protective effect of pomegranate and green tea extracts against endoplasmic reticulum stress, oxidative stress and protein degradation induced by high-fat diet in skeletal muscle. The effects of pomegranate juice and seed powder on the levels of plasma glucose and insulin, inflammatory biomarkers, lipid profiles were studied by Rouhi et al. (2017) where it was suggested that the pharmacological, biochemical, and histopathological profiles of pomegranate juice treated rats obviously indicated its helpful effects in amelioration of diabetes associated complications. Choi et al. (2014) elucidated the Xanthigen mechanism of an anti-obesity activity in high-fat diet fed mice where it was reported that Xanthigen attenuates high fat diet-induced obesity through downregulation of peroxisome proliferator-activated receptor γ and activation of the AMP-activated protein kinase pathwa. Six pomegranate varieties (Mridula, Bhagwa, Ganesh, Jyoti, G-137, Kandhari) grown in semiarid


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regions of India were studied for biochemical parameters including total antioxidant content and antioxidant activities. Overall, antioxidant composition of pomegranate, especially total phenolic and total anthocyanin content, could provide an attractive strategy to manage postprandial hyperglycemia (Kaur et al. 2014). On the other hand, Kojadinovic et al. (2016) investigated the effect of 6-week long pomegranate juice consumption on modification of lipid peroxidation and phospholipid fatty acid composition of plasma and erythrocytes in subjects with metabolic syndrome. Twenty-three women, aged 40-60, were enrolled and randomly assigned into two groups: intervention group- consuming 300 mL of juice per day for 6 weeks- and the control one. It was concluded that the positive impact of pomegranate juice consumption on lipid peroxidation and fatty acid status in metabolic syndrome subjects suggest potential anti-inflammatory and cardio-protective effects.

CONCLUSION The pomegranate fruit has many traditional uses ranging from the relief of a stomach colic to accompanying typical culinary dishes. All types of pomegranate fruit variants can be used, since it contains high content of polyphenolic compounds. Polyphenols were identified to be responsible for the antibacterial and antifungal activity. Pomegranate fruit also offers a preventive alternative for the control and possible cure of diabetes.

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Rosas-Burgos, E, Burgos-Hernández, A, Noguera-Artiaga, L, Kačániová, M, Hernández-García, F, Cárdenas-López, JL, Carbonell-Barrachina, A. Antimicrobial activity of pomegranate peel extracts as affected by cultivar. Journal of the Science of Food and Agriculture, 2016, 97, 802-810. Rouhi, S, Sarker, M, Rahmat, A, Alkahtani, S, Othman, F. The effect of pomegranate fresh juice versus pomegranate seed powder on metabilic inides, lipid profile, inflammatory biomarkers, and the histopathology of pancreatic islets of Langerhans in sreptozotocin-nicotinamide induced type 2 diabetic Sprague-Dawley rats. BMC Complementary and Alternative Medicine, 2017, 17, 156, 1-13. Sear, DR. Greek coins and their values (Volume 1: Europe). Second edition. London: Englnad: Seaby Publication LTD; 1978. Seeram, NP, Adams, LS, Henning, SM, Niu, Y, Zhang, Y, Nair, MG, Heber, D. In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice. The Journal of Nutritional Biochemistry, 2005a, 16 6, 360-367. Seeram, N, Lee, R, Hardy, M, Heber, D. Rapid large scale purification of ellagitannins from pomegranate husk, a by-product of the commercial juice industry. Separation and Purification Technology, 2005b, 41, 1, 49–55. Sepúlveda, L, Aguilera-Carbó, A, Ascacio-Valdés, JA, Rodríguez-Herrera, R, Martínez-Hernández, JL, Aguilar, CN. Optimization of ellagic acid accumulation by Aspergillus niger GH1 in solid state culture using pomegranate shell powder as a support. Process Biochemistry, 2012, 47, 12, 2199-2203. Sepúlveda, L, de la Cruz, R, Buenrostro, JJ, Ascacio-Valdés, JA, AguileraCarbó, AF, Prado, A, Rodríguez-Herrera, R, Aguilar, CN. Effect of different polyphenol sources on the efficiency of ellagic acid release by Aspergillus niger. Revista Argentina de Microbiología, 2016, 48, 1, 71-77.


26 L. Sepúlveda, J. J. Buenrostro-Figueroa, J. A. Ascacio-Valdés et al. SIAP. Anuario Estadístico de la Producción Agrícola (Granada). [Statistical Yearbook of Agricultural Production (Pomegranate)] Servicio de Información Agroalimentaria y Pesquera. (2015) http://infosiap.siap.gob.mx/aagricola_siap_gb/ientidad/index.jsp. Accessed 01/03/2017. Sohrab, G, Nasrollahzadeh, J, Zand, H, Amiri, Z, Tohidi, M, Kimiagar, M. Effects of pomegranate juice consumption on inflammatory markers in patients with type 2 diabetes: A randomized, placebo-controlled trial. Journal of Research in Medical Sciences, 2014, 19, 215-220. Sood, A, Gupta, M. Extraction process optimization for bioactive compounds in pomegranate peel. Food Bioscience, 2015, 12, 100-106. Stover, E, Mercure, EW. The Pomegranate: A New Look at the fruit of paradise. Hortscience 2007, 42, 5, 1088-1092. Teixeira da Silva, JA, Rana, TS, Narzary, D, Verma, N, Meshram, DT, Ranade, SA. Pomegranate biology and biotechnology: A review. Scientia Horticulturae, 2013, 160, 85-107. USDA. National Nutrient Database of Standard. United States Department of Agriculture (2016) https://ndb.nal.usda.gov/ndb/foods/show/2359? manu=&fgcd=&ds= Accessed 01/03/2017. Ventura J, Alarcón-Aguilar, F, Roman-Ramos, R, Campos-Sepúlveda, E, Reyes-Vega, ML, Boone-Villa, D, Jasso-Villagómez EI, Aguilar, CN. Quality and antioxidant properties of a reduced-sugar pomegranate juice jelly with an aqueous extract of pomegranate peels. Food Chemistry, 2013, 136, 1, 109–115. Viuda-Martos, M, Ruiz-Navajas, Y, Fernández-López, J, Sendra, E, SayasBarberá, E, Pérez-Álvarez, JA. Antioxidant properties of pomegranate (Punica granatum L.) bagasses obtained as co-product in the juice extraction. Food Research International 2011, 44, 5, 1217-1223. Wilson TC, Hagerman EA, Quantitative determination of Ellagic Acid. Journal of Agricultural and Food Chemistry, 1990, 38, 1678–1683. Winand, J, Schneider, YJ. The anti-inflammatory effect of a pomegranate husk extract on inflamed adipocytes and macrophages cultivated independently, but not on the inflammatory vicious cycle between adipocytes and macrophages. Food & Function, 2014, 5, 310-318.


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Wu, D, Ma, X, Tian, W. Pomegranate husk extract, punicalagin and ellagic acid inhibit fatty acid synthase and adipogenesis of 3T3-L1 adipocyte. Journal of Functional Foods, 2013, 5, 633-641. Wuang, JY, Zhu, C, Qian, TW, Guo, H, Wang, DD, Zhang, F, Yin, X. Extracts of black bean peel and pomegranate peel ameliorate oxidative stress-induced hyperglycemia in mice. Experimental and Therapeutic Medicine, 2015, 9, 43-48. Zhao, F, Pang, W, Zhang, Z, Zhao, J, Wang, X, Liu, Y, Wang, X, Feng, Z, Zhang, Y, Sun, W, Liu, J. Pomegranate extract and exercise provide additive benefits on improvement of immune function by inhibiting inflammation and oxidative stress in high fat diet induced obesity rats. The Journal of Nutritional Biochemistry, 2016, 32, 20-28. Zou, X, Yan, C, Shi, Y, Cao, K, Xu, J, Wang, X, Chen, C, Luo, C, Li, Y, Gao, J, Pang, W, Zhao, J, Zhao, F, Li, H, Zheng, A, Sun, W, Long, J, Szeto, M-Y, Zhao, Y, Dong, Z, Zhang, P, Wang, J, Lu, W, Zhang, Y, Liu, J, Feng, Z. Mitochondrial dysfunction in obesity-associated nonalcoholic fatty liver disease: The protective effects of pomegranate with its active component punicalagin. Antioxidants & Redox Signaling, 2014, 21, 1557-1570.





Chapter 2

ANTI-OXIDANT AND THERAPEUTIC PROPERTIES OF POMEGRANATE FOR HEALTH BENEFITS Mohammed Mahboob*, PhD, Pitta Venkata. Prabhakar PhD, Bugata Lakshmi Sai Pratyusha and Addi Utkarsh Reddy, PhD Applied Biology Division, CSIR-Indian Institute of Chemical Technology, TS, Hyderabad, India

ABSTRACT Pomegranate is a nutrient rich fruit which represents a reservoir of bioactive phytochemicals with exceptional medicinal values. It consumes world-wide as a delicious and tasty fruit and thus has high commercial value. Its pharmacognistic study reveals presence of active ingredients in different parts of the plant. Taking the activities of these

*

Corresponding Author E-mail: [email protected].


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M. Mahboob, P. V. Prabhakar, B. L. S. Pratyusha et al. chemical constituents into concern, the entire pharmacological profile containing therapeutic uses along with adverse drug reactions and toxicity are reported. It’s conceivable to state that the nutritive value of the juice from the pomegranate is of good value and an important source of nutrients to be considered. It is utilized widely to cure various diseases and ailments specially treating the assortment of disease over various societies and cultures. The anthocyanin pigments, which are responsible for red colour, can be used as colouring agents. Urolithin-A is termed as the most dynamic compound responsible for the anti-inflammatory activity of pomegranate. The synergistic activity of the pomegranate constituents seems, by all accounts, to be better than that of single constituents. Clinical research demonstrates that when pomegranates used as a routine diet, it helps in preventing different diseases and ailments. Its nutritive value, cosmetic value, anti-inflammatory, anti-carcinogenic, anti-diabetic, anti-microbial activities, and other therapeutic values makes it a plant with incredible uses. The anti-oxidants activity of pomegranate is the major mechanism behind the curative properties for various altered physiological systems. During the last decade, pomegranate fruit has been gaining a widespread reputation as a dietary supplement as well as a functional food due to emerging scientific evidence on potential health benefits, including prevention and/or treatment of cardiovascular ailments, neurological disorders, oncologic diseases, dental problems, inflammation, ulcer, arthritis, microbial infection, anticancer, obesity, diabetes, acquired immune deficiency syndrome, neuroprotective effect, and erectile dysfunction.

TAXONOMY 

Kingdom: Plantae  Subkingdom: Tracheobionta



Super-Division: Spermatophyta  Division: Magnoliophyta o Class: Magnoliopsida o Subclass: Rosidae o Order: Myrtales


Anti-Oxidant and Therapeutic Properties of Pomegranate …

31

   

Family: Lythraceae (Punicaceae) Genus: Punica Species: Punica granatum Vernacular names: dadima or dalim (Sanskrit), Granaatappel (Dutch), granatapfel (German) (“Classification |USDA PLANTS,” n.d.) (“Pomegranate,” n.d.).

DESCRIPTION An erect deciduous spreading shrub or tree can be 8 to 10 meters high; stern, woody and thorny; the size of primary stem, 48 to 78 cm; wood is hard and light yellow. Fruit is globular, crowned by a tenacious calyx, having a hard external peel with a red tinge. Seeds are angular, with a fleshy aril which constitutes the edible part. Color differs from red to pinkish white (“Pharmacognosy: Punica granatum,” n.d.).


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M. Mahboob, P. V. Prabhakar, B. L. S. Pratyusha et al.

CULTIVATION

Agro-Climatic Requirements The pomegranate tree grows well under semi-arid-dry conditions, it is deciduous in zones of low winter temperature and an evergreen or in part deciduous in tropical and sub-tropical conditions. It can live through frost to a significant degree in a dormant stage, however, gets injured at a temperature underneath - 110 C. Sandy loam to deep loamy or alluvial soils which are well-drained, an altitude of 500m, above m.s.l are most suitable conditions for the cultivation of pomegranate (“Pomegranate,” n.d., “Pomegranate Farming Guide For Beginners | Agri Farming,” n.d.).

Methodology for Culturing Rooted cuttings or seedlings are set out in pre-fertilized pits 2 ft. (60 cm) deep and wide and are dispersed 12 to 18 ft. (3.5-5.5 m) separated apart, based upon the soil fertility. At first, the plants are cut into 24 to 30 in (60-75 cm) in length and after they branch out the lower branches



33

are pruned to give a reasonably clear main stem. In as much as fruits are borne just only at the tips of new growth, it is recommended that, for the initial 3 years, the branches are to be shortened every year to energize the most extreme number of new shoots on all sides, avoid straggly advancement, and accomplish a strong, well-framed healthy plant. After the third year, just only suckers and dead branches are removed (“Pomegranate,” n.d.).

Harvesting the Yield The fruits can’t be ripened off the tree even with ethylene treatment. The natural fruits ripen 6 to 7 months after blossoming of flowers. Fruit growers, mostly consider that the fruit is ready for harvest if it makes a metallic sound when tapped. The fruits must be picked before overripening when it tends to crack open if rained upon or under certain conditions of atmospheric humidity, or deficient water system. Obviously, one may accept that extreme part is the characteristic methods for seed discharge and dispersal. The fruits ought not to be pulled off, but rather clipped near the base in order to leave no stem to cause harm in handling and shipping. An excessive amount of sun exposure causes sunscald– brown, rusted blemishes and roughening of the peel and rind (“Pomegranate,” n.d.). The pomegranate (Punica granatum L.) is regarded as a fruit with high commercial value and one of the tasty and delicious fruit consumed throughout the world. This plant is native from the Himalayas in northern India to Iran and has been cultivated since ancient times, then distributed throughout the world and in the drier regions of the United States, including Arizona, arid regions of Southeast Asia, the East Indies and tropical Africa (Mandal, Bhatia, & Bishayee, 2017). The fruits of pomegranate are usually consumed as fresh, but with the advancement in science, it can be processed into products, like syrup and juices, sauces and jams (Viyar et al., 2017).


34


AYURVEDIC PROPERTIES In Ayurveda the pomegranate is considered “a pharmacy unto itself,” as the bark and roots believed to have anthelmintic and vermifuge properties, the peels a powerful astringent and cure for diarrhea and oral aphthae, and the juice a “refrigerant” and “blood tonic”(Lansky & Newman, 2007). Rasa: Madhura, Amla, Kashaya Guna: Lakhu, Snigda Virya: Ushna Vipaka: Madhura (“Pharmacognosy: Punica granatum,” n.d.)

ACTIVE INGREDIENTS Over the past decade, significant progress has been made in establishing the pharmacological mechanisms of pomegranate and the individual constituents responsible for them. The tree/fruit can be divided into several anatomical parts like leaf, flower, bark, roots, fruit, seed and



35

peel, studies show that consumption of extracts of all parts appears to have therapeutic properties each of which has interesting pharmacological activity (Gil, Tomás-Barberán, Hess-Pierce, Holcroft, & Kader, 2000; Jurenka, 2008).

Table 1. Principal bioactive constituents in different parts of pomegranate Peel Gallic acid Ellagic acid Punicalin Punicalagin Caffeic acid Ellagitannins Pelletierine alkaloids Luteolin Kaempferol Quercetin

Juice Aliphatic organic acids Gallic acid Ellagic acid Quinic acid Flavonols Amino acids Minerals EGCG Ascorbic acid

Seeds 3,3-Di-Omethylellagic acid 3,3,4-Tri-Omethylellagic acid Punicic acid Oleic acid Palmitic acid Stearic acid Linoleic acid Sterols Tocopherols

Flower Gallic acids Ursolic acid Triterpenoids Fatty acids

Leafs Sterols Saponins Flavanoids Tannins Piperidine alkaloids Flavone Glycoside Ellagitannin

Root & Bark Ellagitannins Piperidine alkaloids Pyrrolidine alkaloid Pelletierine alkaloids

(Sreekumar, Sithul, Muraleedharan, Azeez, and Sreeharshan, 2014)

Pomegranate is a rich source of anthocyanin pigments, Six anthocyanins delphinidin 3-glucoside and 3, 5-diglucoside, cyanidin 3-


36


glucoside and 3, 5-diglucoside and pelargonidin 3-glucoside and 3, 5diglucoside were found to be responsible for the red colour of pomegranate juice out of which Delphinidin-3, 5-diglucoside is known as a major anthocyanin present (DU, WANG, & FRANCIS, 1975; Hernández, Melgarejo, Tomás-Barberán, & Artés, 1999). A molecule, urolithine A (UA), restores the recycling procedure and delay ageing of the tissues altogether. Pomegranates don’t contain UA, yet a precursor which can be transformed over into UA by the intestinal microbiota (“The pomegranate fruit: a fountain of youth? | Recherche animale,” n.d.).

NUTRITIONAL VALUE Pomegranate is a nutrient rich fruit which represents a reservoir of bioactive phytochemicals with exceptional medicinal values. From the research carried till date, it’s conceivable to state that the nutritive value of the juice from the pomegranate is of good value and an important source of nutrients to be considered. The following nutrition information is for one medium sized raw pomegranate.



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The following information is about the nutrients in 100 gms of aril alone

(“Pomegranate Nutrition is Amazing,” n.d.).

COSMETIC VALUE Pomegranate has anatomical parts which are rich in therapeutically useful ingredients for skin repair. A study carried out by taking pomegranate seed oil, aqueous extracts of fermented juice, peel or seed cake had shown to stimulate keratinocyte proliferation in monolayer culture, pomegranate peel extract (and to a less extent, both the fermented juice and seed cake) stimulated type I procollagen synthesis and inhibited matrix metalloproteinase-1 production by dermal fibroblasts, but showed no proliferation effect on keratinocytes (Aslam, Lansky, & Varani, 2006). The pomegranate is the best anti-aging nourishment for softening the skin since it is rich in vitamin C, it shields skin from the wrinkling impacts of sun damage. Pomegranate juice contains ellagic acid, which battles free


38


radical damage. This juice additionally contains punicalagin, a super nutrient that builds the body’s capacity to save collagen, which influences skin to look plump and smooth (“October | 2013 | Beauty Vancouver,” n.d.).

THERAPEUTIC USES The extracts of all the parts of the plant appear to have a therapeutic effect. This plant relieves tridosha, tapeworm infection, increased digestion; increases fatigue, thirst, and aphrodisiacal. The juice of crisp leaves and young pomegranate is given in the treatment of dysentery. The powdered bark is given for removing roundworms. The unripe fruits and flowers are valuable in instigating vomiting, the peel of the fruit is given in the treatment of diarrhea and dysentery. They additionally strengthen the gums. Ripen fruit is tonic, purgative and enriches the blood. Pomegranate is likewise used in sore throat, sore eyes, urinary infections, coughs, arthritis, skin disorders, brain disorders and chest inconveniences. Seed decoction helps in treating syphilis (“Indian Medicinal Plants: By K. R. Kirtikar, B. D. Basu, and An I. C. S. In 4 ... - Kanhoba Ranchoddas Kirtikar - Google Books,” n.d.). The bloomed buds are astringent, and are given to diarrhea patients. Pomegranate is helpful in controlling the inflammation and fever. Sour pomegranate increases the appetite. Sweet pomegranate assists in building the intelligence. Pomegranate helps in controlling gum bleeding and cures indigestion (“Pharmacognosy: Punica granatum,” n.d.). During the last decade, pomegranate fruit has been gaining a widespread reputation as a dietary supplement as well as a functional food due to emerging scientific evidence on potential health benefits, including prevention and/or treatment of cardiovascular ailments, neurological disorders, oncologic diseases, dental problems, inflammation, ulcer, arthritis, microbial infection, obesity, diabetes, acquired immune deficiency syndrome and erectile dysfunction (Mandal et al., 2017). Pomegranate seed extract administered to Sprague Dawley rats elevated



39

the number and expanded diameter erythrocytes (Kafkas Üniversitesi. Veteriner Fakültesi., Karadenİz, & Bayraktaroğlu, 2009). Study of Pomegranate juice consumption on parameters related to metabolic health, inflammation and oxidative stress has shown promising results with increased hemopoiesis (Manthou et al., 2014). In a study conducted administering the methanolic extract of pomegranate peel showed potential to cure antiulcer activity via its higher antioxidant property (Moghaddam, Sharifzadeh, Hassanzadeh, Khanavi, & Hajimahmoodi, 2013).

Antioxidant Properties Epidemiological studies show that consumption of fruits and vegetables with high phenolic content correlate with reduced cardio, cerebrovascular diseases and cancer mortality. Phenolic compounds produce their beneficial effects by scavenging free radicals. In the past few years there has been an increasing interest in determining relevant dietary sources of antioxidant phenolics. Thus, red fruit juices such as grape, different berry juices and Pomegranate juice have become more popular because of their antioxidant property and attribution of important biological actions. Thus, the antioxidant and antitumoral activity of pomegranate bark tannins (punicacortein) and the antioxidant activity of the fermented pomegranate juice have been reported (Gil et al., 2000). The in vitro antioxidant activity of pomegranate has been attributed to its high polyphenolic content, specifically punicalagins, punicalins, gallagic acid, and ellagic acid (Johanningsmeier & Harris, 2011). A comparative investigation on pomegranate extract and its metabolite revealed that urolithin-A could be the most dynamic anti-inflammatory compound from pomegranate ingestion in healthy individuals, in colon inflammation, the impacts could be expected due to nonmetabolized ellagitannin-related fractions (Larrosa et al., 2010). Free radicals play an important role in some pathogenesis of serious diseases, such as neurodegenerative disorders, cancer, liver cirrhosis, cardiovascular diseases, atherosclerosis, cataracts, diabetes and


40


inflammation. Many naturally occurring antioxidative compounds from plant sources have been identified as free radical inhibitors, active oxygen scavengers or reducing agents. Antioxidant compounds that can scavenge free radicals have great potential in ameliorating these diseases (Pirinççioğlu, Kızıl, Kızıl, Kanay, & Ketani, 2014). During the past few decades, the use of natural antioxidants and plant extracts have received increased interest due to the concerns about possible ill health effects generated by the use of synthetic antioxidants (Nahm, Juliani, & Simon, 2012). The pomegranate is one of the oldest edible fruits and is widely grown in many tropical and subtropical countries. A vast number of in vivo, in vitro studies have produced a huge amount of scientific evidence regarding effective antioxidant property and consequent health benefits of pomegranate.

Effects on Altered Physiological Systems Evidence for the clinical benefits of pomegranate juice was reported in several studies. High blood pressure, or “hypertension,” is associated with high levels of oxidative stress in the paraventricular nucleus of the hypothalamus. Chronic treatment of hypertensive rats with pomegranate extract significantly reduced oxidative stress, increased the antioxidant defense system and decreased inflammation led to significant decrease in blood pressure and cardiac hypertrophy. It was also identified that Punicalagin rich Pomegranate extract reduced mitochondrial superoxide anion levels and increased the mitochondrial function by promoting mitochondrial biogenesis, improving mitochondrial dynamics and clearance in the paraventricular nucleus of hypertensive rats. Punicalagin rich Pomegranate extract administration at a dosage of 150 mg/kg/day for 8 weeks significantly inhibited hyperlipidemia and hepatic lipid deposition by suppressing mitochondrial protein oxidation, improved mitochondrial complex activity in the liver, restored oxidative stress and expression of pro-inflammatory cytokines such as tumour necrosis factor-alpha, interleukins in high fat diet induced non-alcoholic fatty liver disease



41

(NAFLD) Sprague–Dawley (SD) male rats (Zou et al., 2014). Similarly, Pomegranate juice administration prevented the development of NAFLD through improvement in activities of Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPx), Glutathione reductase (GR) enzymes, serum glycemic indices, triglycerides, and down regulation of hepatic pro-inflammatory and pro-fibrotic gene expressions. These beneficial effects were observed even in the presence of the main risk factors of non-alcoholic fatty liver disease (NAFLD) development, such as dietary high energy, fat, and sugar intakes (Noori, Jafari, & Hekmatdoost, 2017). In human athletes, a 21 days daily consumption of Pomegranate juice had decreased the LPO and protein carbonyls due to oxidative damage caused by the exercise (Fuster-Muñoz et al., 2016). The elevated oxidative stress present in many complicated pregnancies contributes to placental dysfunction and suboptimal pregnancy outcomes. Chen et al., (2012) reported that the pregnant women with singleton pregnancies were given 8 oz/day of Pomegranate juice until the time of delivery. Placental tissues from 12 patients (4 in the Pomegranate group and 8 in the control group) were collected for analysis of oxidative stress. The Pomegranate juice reduced labor induced oxidative stress in the term human placenta in vivo. The Hsp 90, marker of oxidative stress, was significantly lower in the placentas of women who received pomegranate juice compared with control pregnant women. In in vitro study the primary trophoblasts isolated from the same placental tissue, pomegranate juice reduced oxidative stress and apoptosis after exposing to hypoxia. High plasma LDL concentrations and LDL modifications like retention, oxidation and aggregation, blood platelet activation, macrophage foam cell formation and atherosclerotic lesions play a key role during early atherogenesis. Aviram et al., (2000) demonstrated that pomegranate juice alleviated major atherogenic risk factors in healthy humans and in atherosclerotic mice that may be attributable to its antioxidative properties. Increased systemic inflammation and oxidative stress are well established key players in the pathogenesis


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of atherosclerosis. Shema-Didi et al., (2012) investigated the effect of the 1 year intake of pomegranate juice, on oxidative stress, inflammation, and long-term clinical outcomes in 101 chronic haemodialysis (HD) patients. Pomegranate juice intake resulted a significant time response, decrease in polymorpho-nuclear leukocyte priming, protein oxidation, and lipid oxidation and levels of inflammatory biomarkers. The beneficial effects of the polyphenol rich fruits had been reported as cost effective strategies for health promotion. Randomized controlled trial was conducted in healthy adults aging 21 – 35 years who received pomegranate juice daily for 21 days and blood and urine was analyzed. Results demonstrated that pomegranate juice consumption enhanced the antioxidant status in humans by decreasing the oxidative damage in lipids by decreasing urinary TBARS and improving the antioxidant mechanisms when compared to a healthy control group (Gouda, Moustafa, Hussein, & Hamza, 2015). Braidy et al., (2013) demonstrated neuroprotective effect of Pomegranate extract in Parkinsonian-like syndrome neurotoxicity model; where Pomegranate extract exposure reverted the 1-methyl-4-phenyl1,2,3,6-tetrahydropyridin (MPTP) induced oxidative stress conditions like increased LPO, decreased GSH, increased activities of SOD, CAT, LDH enzymes and decreased GPx activity in human primary neuronal cell cultures. Pomegranate exhibits good antioxidant capacity against Carbon tetrachloride (CCl4) is one of the most commonly used hepatotoxins in the experimental study of liver diseases. CCl4 intoxication of rats demonstrated significant induction in lipid peroxidation and histomorphological alterations in liver, kidney and brain and increases in serum aspartate aminotransferase, alanine aminotransferase and total bilirubin, and decrease in albumin. Concomitant Pomegranate juice treatment reversed all the CCl4 induced damage to normal levels (Pirinççioğlu et al., 2014).



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Effect on Diabetes Table 2. Determined Beneficial Effects of Pomegranate in Diabetes Mellitus Activity Increased insulin sensitivity Inhibition of α-glucosidase Hypoglycaemic Restores cardiac GLUT-4 mRNA Up-regulation of mRNA expression of fatty acid transport protein, PPAR-, carnitine palmityl transferase-1, acyl-CoA oxidase, and 5-adenyl monophosphatase-activated protein kinase α-2; down-regulation of mRNA expression of acetyl-CoA carboxylase Reduces heart triglyceride and total cholesterol Inhibits lipopolysaccharide activation of NFκB, suppressing overexpressed cardiac fibronectin and collagen I and III mRNAs Reduces oxidative stress markers, lipid peroxides, and thiobarbituric acid- reactive substances Reduces total cholesterol, LDL cholesterol

Pomegranate plant parts Flower Flower Seeds

Test animals

Flower Flower

Zucker diabetic fatty rats Zucker diabetic fatty rats Streptozotocin-induced diabetic rats Zucker diabetic fatty rats Zucker diabetic fatty rats

Flower Flower

Zucker diabetic fatty rats Zucker diabetic fatty rats

Juice

Diabetic patients (Human) Diabetic patients (Human)

Juice

(Katz, Newman, & Lansky, 2007).

Pomegranate rind, flower and seeds have hypoglycemic activity. The hypoglycemic activity of seeds was reported in a study conducted by supplementing methanol extract of seeds to STZ induced diabetic rats (Das et al., 2001). Oral treatment with pomegranate seed juice for 21 days demonstrated significant protective effects on diabetes induced oxidative stress condition by decreasing lipid peroxidation (LPO), restored SOD, CAT enzyme activity and total antioxidant status (TAS) in streptozocinnicotinamide induced diabetic Sprague Dawley rats (Aboonabi, Rahmat, & Othman, 2014).


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Anticancer Activity Lansky & Newman (2007) discovered combining equal measures of Fermented pomegranate juice, Pomegranate peel extract, and Cold pressed seed oil brought about a 99-percent suppression of DU-145 prostate cancer cell proliferation over a Matrigel matrix. Various in vitro studies have explored the therapeutic effect of pomegranate extracts against a few other cancer cell lines. In HT-29 colon cancer cells, cyclooxygenase-2 (COX-2) expression is elevated through the activation of nuclear factor kappa-B (NF[kappa]B) by tumor necrosis factor-alpha (TNF-[alpha]), an inflammatory cell signaling cascades that might be a reason for cancer development and metastasis. Treatment of HT-29 colon cancer cells with Pomegranate juice, pomegranate tannins, or concentrated pomegranate punicalagin initiated a significant reduction in COX-2. Pomegranate juice treatment brought about the highest levels of COX-2 expression (79%) compared to that in treatment with single constituents. The impacts were credited to the synergistic activity of the bioactive constituents thought to be vital for pomegranate’s mitigating anti-inflammatory and anticarcinogenic action (Jurenka, 2008).

Antimicrobial Activity Extractions of ellagic acid, gallagic acid, punicallins, and punicalagins separated from Pomegranate showed antimicrobial action when tested against Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and other harmful microscopic organisms. Both aril and peel of the Pomegranate contained significant measures of vitamin C, yet the contents of the peel were altogether higher than the aril, Sun drying of the natural product peels increased retention of vitamin C and antimicrobial activities than the oven-dried parts (Braga et al., 2005; Opara, Al-Ani, & Al-Shuaibi, 2009). Research conducted by Dahham, Ali, Tabassum, & Khan, (2010) depicted the antibacterial and antifungal properties of Pomegranate peel extract (rind), seed extract, juice and whole



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fruit on the chosen bacteria and fungi demonstrating maximal antibacterial activity on Staphylococcus aureus and highest anti-fungal on Aspergillus niger. Pomegranate juice intake resulted in a significantly lower incidence rate of the second hospitalization due to infections and significant decrease in the progression of the atherosclerotic process (Shema-Didi et al., 2012).

TOXICITY AND DRUG-INTERACTION Past findings on the anti-flu action of P. granatum extracts has offered support to the ethnopharmacological application. In an examination, in the chick embryo life model, it was discovered that doses of the extract under 0.1 mg per embryo are not toxic. The doses of extract post repeated intranasal administration in Wistar rats, produced toxic impacts at higher doses (Vidal et al., 2003). Apart from its protection in health ailments, Pomegranate has also shown efficient protective antioxidant behavior towards various types of chemicals, pesticides and drugs. For example, the clinical efficacy of the widely used chemotherapeutic drug methotrexate (MTX) is limited due to its associated hepatotoxicity. Male Swiss albino mice exposed to MTX exhibited increased activities of alanine transaminase, aspartate transaminase, lactate dehydrogenase and alkaline phosphatase, enhanced ROS generation, lipid peroxidation, decrease reduced glutathione level, decreased superoxide dismutase, catalase and heme-oxygenase 1 enzymes activity, increased the expression of the apoptotic enhancer Rho/Cdc42, phosphorylation of SAPK/JNK, shift in the Bax/Bcl-2 ratio towards apoptosis and increase in the caspase 3 level. Administration of Pomegranate extract for 7 consecutive days before and after MTX challenge suppressed apoptosis, reduced ROS generation in hepatocytes by activating the Nrf2-ARE pathway and inhibited NF-κB as a consequence of which the antioxidant defence mechanism in the liver was up-regulated (Mukherjee et al., 2013).


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REFERENCES Aboonabi, A., Rahmat, A., & Othman, F. (2014). Antioxidant effect of pomegranate against streptozotocin-nicotinamide generated oxidative stress induced diabetic rats. Toxicology Reports, 1, 915–922. https://doi.org/10.1016/j.toxrep.2014.10.022. Aslam, M. N., Lansky, E. P., & Varani, J. (2006). Pomegranate as a cosmeceutical source: pomegranate fractions promote proliferation and procollagen synthesis and inhibit matrix metalloproteinase-1 production in human skin cells. Journal of Ethnopharmacology, 103(3), 311–8. https://doi.org/10.1016/j.jep.2005.07.027. Aviram, M., Dornfeld, L., Rosenblat, M., Volkova, N., Kaplan, M., Coleman, R., … Fuhrman, B. (2000). Pomegranate juice consumption reduces oxidative stress, atherogenic modifications to LDL, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein E-deficient mice. The American Journal of Clinical Nutrition, 71(5), 1062–76. Retrieved from http://www.ncbi.nlm. nih.gov/pubmed/10799367. Braga, L. C., Shupp, J. W., Cummings, C., Jett, M., Takahashi, J. A., Carmo, L. S., … Nascimento, A. M. A. (2005). Pomegranate extract inhibits Staphylococcus aureus growth and subsequent enterotoxin production. Journal of Ethnopharmacology, 96(1–2), 335–9. https:// doi.org/10.1016/j.jep.2004.08.034. Braidy, N., Selvaraju, S., Essa, M. M., Vaishnav, R., Al-Adawi, S., AlAsmi, A., … Guillemin, G. J. (2013). Neuroprotective Effects of a Variety of Pomegranate Juice Extracts against MPTP-Induced Cytotoxicity and Oxidative Stress in Human Primary Neurons. Oxidative Medicine and Cellular Longevity, 2013, 1–12. https://doi. org/10.1155/2013/685909. Chen, B., Tuuli, M. G., Longtine, M. S., Shin, J. S., Lawrence, R., Inder, T., & Michael Nelson, D. (2012). Pomegranate juice and punicalagin attenuate oxidative stress and apoptosis in human placenta and in human placental trophoblasts. American Journal of Physiology-



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Endocrinology and Metabolism, 302(9), E1142–E1152. https://doi. org/10.1152/ajpendo.00003.2012. Classification | USDA PLANTS. (n.d.). Retrieved March 15, 2018, from https://plants.usda.gov/java/ClassificationServlet?source=display&clas sid=PUGR2. Dahham, S. S., Ali, N., Tabassum, H., & Khan, M. (2010). Studies on Antibacterial and Antifungal Activity of Pomegranate (Punica granatum L.). & Environ. Sci, 9(3), 273–281. Retrieved from https://pdfs.semanticscholar.org/9aa0/7f932b470efa3beac9d801c3948a 52ddb721.pdf. Das, A. K., Mandal, S. C., Banerjee, S. K., Sinha, S., Saha, B. P., & Pal, M. (2001). Studies on the hypoglycaemic activity of Punica granatum seed in streptozotocin induced diabetic rats. Phytotherapy Research : PTR, 15(7), 628–9. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/11746848. Du, C. T., Wang, P. L., & Francis, F. J. (1975). Anthocyanins of Pomegranate, Punica granatum. Journal of Food Science, 40(2), 417– 418. https://doi.org/10.1111/j.1365-2621.1975.tb02217.x. Fuster-Muñoz, E., Roche, E., Funes, L., Martínez-Peinado, P., Sempere, J. M., & Vicente-Salar, N. (2016). Effects of pomegranate juice in circulating parameters, cytokines, and oxidative stress markers in endurance-based athletes: A randomized controlled trial. Nutrition, 32(5), 539–545. https://doi.org/10.1016/j.nut.2015.11.002. Gil, M. I., Tomás-Barberán, F. A., Hess-Pierce, B., Holcroft, D. M., & Kader, A. A. (2000). Antioxidant Activity of Pomegranate Juice and Its Relationship with Phenolic Composition and Processing. Journal of Agricultural and Food Chemistry, 48(10), 4581–4589. https://doi.org/ 10.1021/jf000404a. Gouda, M., Moustafa, A., Hussein, L., & Hamza, M. (2015). Three week dietary intervention using apricots, pomegranate juice or/and fermented sour sobya and impact on biomarkers of antioxidative activity, oxidative stress and erythrocytic glutathione transferase activity among adults. Nutrition Journal, 15(1), 52. https://doi.org/ 10.1186/s12937-016-0173-x.


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Hernández, F., Melgarejo, P., Tomás-Barberán, F. A., & Artés, F. (1999). Evolution of juice anthocyanins during ripening of new selected pomegranate (Punica granatum) clones. European Food Research and Technology, 210(1), 39–42. https://doi.org/10.1007/s00217 0050529. Johanningsmeier, S. D., & Harris, G. K. (2011). Pomegranate as a functional food and nutraceutical source. Annual Review of Food Science and Technology, 2(1), 181–201. https://doi.org/10.1146/ annurev-food-030810-153709. Jurenka, J. S. (2008). Therapeutic applications of pomegranate (Punica granatum L.): a review. Alternative Medicine Review : A Journal of Clinical Therapeutic, 13(2), 128–44. Retrieved from http://www.ncbi. nlm.nih.gov/pubmed/18590349. Kafkas Üniversitesi. Veteriner Fakültesi., N., Karadenİz, A., & Bayraktaroğlu, A. G. (2009). Veteriner fakultesi dergisi = The journal of the Faculty of Veterinary Medicine. Kafkas Üniversitesi Veteriner Fakültesi Dergisi (Vol. 15). Kafkas Üniversitesi, Veteriner Fakültesi Dergisi. Retrieved from https://www.cabdirect.org/cabdirect/ abstract/20093079827. Katz, S. R., Newman, R. A., & Lansky, E. P. (2007). Punica granatum: heuristic treatment for diabetes mellitus. Journal of Medicinal Food, 10(2), 213–7. https://doi.org/10.1089/jmf.2006.290. Kirtikar, K. R. , B. D. Basu, and An I. C. S. Indian Medicinal Plants In 4 ... - Kanhoba Ranchoddas Kirtikar - Google Books. (n.d.). Retrieved March 16, 2018, from https://books.google.co.in/books/about/Indian_ Medicinal_Plants.html?id=xArsSQAACAAJ&redir_esc=y. Lansky, E. P., & Newman, R. A. (2007). Punica granatum (pomegranate) and its potential for prevention and treatment of inflammation and cancer. Journal of Ethnopharmacology, 109(2), 177–206. https://doi. org/10.1016/j.jep.2006.09.006. Larrosa, M., González-Sarrías, A., Yáñez-Gascón, M. J., Selma, M. V., Azorín-Ortuño, M., Toti, S., … Espín, J. C. (2010). Anti-inflammatory properties of a pomegranate extract and its metabolite urolithin-A in a colitis rat model and the effect of colon inflammation on phenolic



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metabolism. The Journal of Nutritional Biochemistry, 21(8), 717–725. https://doi.org/10.1016/J.JNUTBIO.2009.04.012. Mandal, A., Bhatia, D., & Bishayee, A. (2017). Anti-Inflammatory Mechanism Involved in Pomegranate-Mediated Prevention of Breast Cancer: the Role of NF-κB and Nrf2 Signaling Pathways. Nutrients, 9(12), 436. https://doi.org/10.3390/nu9050436. Manthou, E., Georgakouli, K., Sotiropoulos, A., Deli, C., Kouretas, D., Koutedakis, Y., & Jamurtas, A. Z. (2014). Effects of Pomegranate Juice Supplementation on Complete Blood Count, Metabolic Health and Inflammation Markers. 4ο Συνέδριο Βιοχημείας Και Φυσιολογίας Της Άσκησης [4th Congress of Biochemistry and Physiology of Exercise. Retrieved from http://www.pe.uth.gr/ocs/index.php/ eevfa4-sefaa/vfa/paper/viewPaper/82. Moghaddam, G., Sharifzadeh, M., Hassanzadeh, G., Khanavi, M., & Hajimahmoodi, M. (2013). Anti-Ulcerogenic Activity of the Pomegranate Peel (Punica granatum) Methanol Extract. Food and Nutrition Sciences, 4(10), 43– 48. https://doi.org/10.4236/fns.2013.410A008. Mukherjee, S., Ghosh, S., Choudhury, S., Adhikary, A., Manna, K., Dey, S., … Chattopadhyay, S. (2013). Pomegranate reverses methotrexateinduced oxidative stress and apoptosis in hepatocytes by modulating Nrf2-NF-κB pathways. The Journal of Nutritional Biochemistry, 24(12), 2040–2050. https://doi.org/10.1016/j.jnutbio.2013.07.005. Nahm, H. S., Juliani, H. R., & Simon, J. E. (2012). Journal of Medicinally Active Plants Effects of Selected Synthetic and Natural Antioxidants on the Oxidative Stability of Shea Butter (Vitellaria paradoxa subsp. paradoxa). Journal of Medicinally Active Plants 1, (2), 69–75. https://doi.org/10.7275/R5BR8Q4R. Noori, M., Jafari, B., & Hekmatdoost, A. (2017). Pomegranate juice prevents development of non-alcoholic fatty liver disease in rats by attenuating oxidative stress and inflammation. Journal of the Science of Food and Agriculture, 97(8), 2327–2332. https://doi.org/10.1002/ jsfa.8042.


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October | 2013 | Beauty Vancouver. (n.d.). Retrieved March 15, 2018, from https://beautyvancouver.wordpress.com/2013/10/. Opara, L. U., Al-Ani, M. R., & Al-Shuaibi, Y. S. (2009). Physico-chemical Properties, Vitamin C Content, and Antimicrobial Properties of Pomegranate Fruit (Punica granatum L.). Food and Bioprocess Technology, 2(3), 315–321. https://doi.org/10.1007/s11947-0080095-5. Pharmacognosy: Punica granatum. (n.d.). Retrieved March 15, 2018, from http://p-cognosy.blogspot.in/2008/09/punica-granatum.html. Pirinççioğlu, M., Kızıl, G., Kızıl, M., Kanay, Z., & Ketani, A. (2014). The protective role of pomegranate juice against carbon tetrachloride– induced oxidative stress in rats. Toxicology and Industrial Health, 30(10), 910–918. https://doi.org/10.1177/0748233712464809. Pomegranate. (n.d.). Retrieved March 15, 2018, from https://www.hort. purdue.edu/newcrop/morton/pomegranate.html#Description. Pomegranate. (n.d.). Retrieved March 15, 2018, from http://nhb.gov. in/report_files/pomegranate/POMEGRANATE.htm. Pomegranate Farming Guide For Beginners | Agri Farming. (n.d.). Retrieved March 15, 2018, from http://www.agrifarming.in/ pomegranate-farming/. Pomegranate Nutrition is Amazing. (n.d.). Retrieved March 15, 2018, from http://www.amazing-pomegranate-health-benefits.com/pomegranatenutrition.html. Shema-Didi, L., Sela, S., Ore, L., Shapiro, G., Geron, R., Moshe, G., & Kristal, B. (2012). One year of pomegranate juice intake decreases oxidative stress, inflammation, and incidence of infections in hemodialysis patients: A randomized placebo-controlled trial. Free Radical Biology and Medicine, 53(2), 297–304. https://doi.org/ 10.1016/j.freeradbiomed.2012.05.013. Sreekumar, S., Sithul, H., Muraleedharan, P., Azeez, J. M., & Sreeharshan, S. (2014). Pomegranate fruit as a rich source of biologically active compounds. BioMed Research International, 2014, 686921. https://doi. org/10.1155/2014/686921.



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The pomegranate fruit: a fountain of youth ? | Recherche animale. (n.d.). Retrieved March 15, 2018, from https://www.recherche-animale.org/ en/pomegranate-fruit-fountain-youth. Vidal, A., Fallarero, A., Peña, B. R., Medina, M. E., Gra, B., Rivera, F., … Vuorela, P. M. (2003). Studies on the toxicity of Punica granatum L. (Punicaceae) whole fruit extracts. Journal of Ethnopharmacology, 89(2–3), 295–300. https://doi.org/10.1016/J.JEP.2003.09.001. Viyar, A. H., Qadri, R., Iqbal, A., Nisar, N., Khan, I., Bashir, M., & Shah, F. (2017). Evaluation of unexplored pomegranate cultivars for physicochemical characteristics and antioxidant activity. Journal of Food Science and Technology, 54(9), 2973–2979. https://doi.org/10. 1007/s13197-017-2736-z. Zou, X., Yan, C., Shi, Y., Cao, K., Xu, J., Wang, X., … Feng, Z. (2014). Mitochondrial Dysfunction in Obesity-Associated Nonalcoholic Fatty Liver Disease: The Protective Effects of Pomegranate with Its Active Component Punicalagin. Antioxidants & Redox Signaling, 21(11), 1557–1570. https://doi.org/10.1089/ars.2013.5538.





Chapter 3

POMEGRANATE: A HEALTH PROMOTING FOOD Antonia Kotsiou, PhD and Christine Tesseromatis, PhD Department of Medicine, National & Kapodistrian University of Athens, Athens Greece

ABSTRACT Pomegranate, Punica granatum Lin, or Punicaceae, a native fruit to Persia, has been grown since ancient times throughout the Mediterranean region. The biological properties of extracts obtained from several parts of the fruit have found various applications in ethnobotany. Flavonoids and tannins are the major pomegranate polyphenols and among them anthocyanins are in abundance. Anthocyanins are water soluble pigments and impart a red color to the fruit arils and also, along with tannins, give this fruit its mild, astringent flavor. The hydrolysable tannins are the most powerful antioxidants in various pomegranate parts. Other phytochemicals reported in pomegranate include organic acids, phenolic acids, fatty acids and alkaloids.

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A. Kotsiou and C. Tesseromatis These pomegranate bioactive compounds have proved to have preventive and curative potentials for many diseases like atherosclerosis, all types of cancer, diabetes, Alzheimer’s disease, and in addition are antibacterial, antifungal and anthelmintic agents.

INTRODUCTION The pomegranate, which is a native fruit to Persia, is one of the oldest fruits known to humans and has been cultivated since ancient times throughout the Mediterranean region and all over the world. Pomegranates are found in ancient writings and pictured in the ancient art of many cultures and religions.

The pomegranate anatomical compartments – including the seed, juice, peel, leaf, flower, bark, and roots – hold many healthy properties thanks to their high levels of mineral salts, vitamins and antioxidants. Pomegranate fruit presents strong health promoting properties, thus leading to an increased popularity as a functional food and nutraceutical source since ancient times.

1. Antioxidant Properties The edible parts of pomegranate fruit (about 50% of total fruit weight) comprise 80% juice and 20% seeds. Pomegranate fruit and mainly


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pomegranate juice is a rich source of hydrolysable tannins such as allotannins, ellagic acid tannins- ellagitannins- and gallotannins (these are termed as “punicalagins”). Punicalagins have the highest antioxidant activity. The acidic tannins provide the sour notes in taste of pomegranate juice [1]. It has been reported that the antioxidant level in pomegranate juice was higher than found in other fruit juices, e.g., blueberry, cranberry, orange and the antioxidant potential of pomegranate juice, induced through hydrosable tannins, is more than that of red wine and green tea [2]. The phenolic content of pomegranate juice is adversely affected by processing and pasteurization techniques [3]. The phytochemical content of the different pomegranate parts include flavonoids, tannins, anthocyanins, carotenoids polyphenols, cis-γ-Linolenic acid, punicic acid and fatty acids - mono-unsaturated (MUFA) and Diunsaturated (DiUFA) [4]. The degree of ripeness (low, low-medium, medium and medium-high) affects the physical and compositional changes, as well as antioxidant properties of pomegranate fruit. The lowest concentrations of flavonoids and hydrolysable tannins were recorded in skin and pellicles at mediumhigh maturity stage, which explains the decrease in the total phenols and reducing power during ripening of pomegranate. On the contrary, the highest concentration of flavonoids (165 mg of quercetin equivalents per 100 mL of juice) was determined in the juice at the most advanced ripening stage, concomitant with the highest total phenols (1695 mg of gallic acid equivalents per 100 mL of juice) [5]. Studies indicate the presence of compounds possessing antioxidant activity from peel and seeds of pomegranate [6]. Pomegranate peel is separated into outer leathery skin (PS), mesocarp (PM), and divider membrane (PD), and the total phenolic content follows the order PD > PM > PS. Gallic acid is the major phenolic compound in pomegranate peel, whereas kaempferol 3-O-glucoside is the major flavonoid [7].


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2. Cultivation Pomegranate tree is native to Persia (now Iran) since 3500 - 2000 B. C. It was cultivated in Egypt around the time of Moses. The generic term, Punica, was the Roman name for Carthage from whence the best pomegranates came to Italy. Spanish settlers brought the pomegranate to the U.S California in 1769. Pomegranate fruits were found in the prehistoric town of Akrotiri in Santorini Greece [8, 9]. The pomegranate plant is very adaptable and grows in regions ranging from temperate to tropical. The fruit is typically in season in the Northern Hemisphere from September to February and in the Southern Hemisphere from March to May. Pomegranate plants take 4 - 5 years to come into bearing. The best prospects for commercial fruit production exists in those countries where the summer is warm to hot and where rainfall is minimal during late summer/autumn. Rainfall can cause soft fruit and bring diseases. The main diseases reported are leaf spot and fruit rot. If water is available for irrigation the chances of rainfall splitting the fruit will be reduced. Very hot weather can lead to sunburn injury on fruit. Deep well drained soils are preferred [10]. Planting is usually done in spring (February-March) and July-August in sub-tropical and tropical regions respectively. High density planting with a spacing gives 2 - 2.5 times more yield than that obtained when the normal planting distance of 5 X 5 m. is adopted. Farmers have adopted a spacing of 2.5 X 4.5 m. Closer spacing increases disease and pest incidence. Weekly irrigation in summers and that during winters at fortnightly intervals is recommended. It can tolerate frost to a considerable extent in dormant stage, but is injured at temperature below - 11°C [10]. Fruit cracking is a serious disorder. This physiological disorder observed in young fruits is due to boron deficiency and that in fully grown fruits is mainly due to moisture imbalances. Spraying with calcium hydroxide soon after fruit set has been found to be beneficial. Pomegranate should be picked when fully ripe. Harvesting of immature or over mature fruits affects the quality of the fruits. The fruits become ready for picking 120 - 130 days after fruit set. The calyx at the distal end of the fruit gets



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closed on maturity. At maturity, the fruits turn yellowish-red and get suppressed on sides. Fruits can be stored in cold storage up to 2 months or 10 weeks at a temperature of 5°C. Longer storage should be at 10°C to avoid chilling injury and weight loss [11]. The most important work in commercial cultivation of pomegranate is plant protection against fruit insects like Virachova livia and Cryptoblabos gnidiella causing fruit rot. If fruits are not treated with organic phosphor spraying every 10 - 14 days every fruit will be infected. This is not environmentally friendly. The best way is biological control by treating with Bacillus Turgeniensis [12].

3. Health Benefits Historically, pomegranate has been extensively used for different ethnomedical uses, including the treatment of parasitic infections, ulcers, diarrhoea, dysentery, microbial infections and respiratory pathologies, as blood builder, stopping nose and gum haemorrhage and treating haemorrhoids [9, 13, 14, and 15]. Several studies suggested that pomegranate polyphenol-rich juice can exert antiatherogenic, antioxidant, antihypertensive, anti-inflammatory and anticarcinogenic effects and can help prevent or treat various disease risk factors. The positive health effect of pomegranate is due to the unique antioxidant activity of this fruit due to the total phenolic contents which are highly correlated to antioxidant activities, and the other important ingredients. Pomegranate wine, vinegar, and sour sauce obtained directly from pomegranate juice are also rising in interest [16]. All three pomegranate parts: seeds, peel, and juice, seem to have medicinal benefits. Several studies investigate its bioactive components as a means to associate them with a specific beneficial effect and develop future products and therapeutic applications. Many beneficial effects are related to the presence of ellagic acid, ellagitannins (including punicalagins), punicic acid, fatty acids, flavonoids, anthocyanidins,


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anthocyanins, estrogenic flavonols, and flavones, which seem to be its most therapeutically beneficial components. The antioxidant potential of pomegranate juice is more than that of red wine and green tea, which is induced through ellagitannins and hydrosable tannins [17]. Pomegranate juice can reduce macrophage oxidative stress, free radicals, and lipid peroxidation. Moreover, pomegranate fruit extract prevents cell growth and induces apoptosis, which can lead to its anticarcinogenic effects. In addition, promoter inhibition of some inflammatory markers and their production are blocked via ellagitannins [17]. Fermented pomegranate juice and cold-pressed pomegranate seeds possess antioxidant activity and can reduce prostaglandin and leukotriene formation by inhibition of cyclooxygenases and lipoxygenases [18].

3.1. Antimicrobial Activity Research indicates that pomegranates and their extracts are effective against a wide range of bacterial and viral pathogens. Nearly every part of the pomegranate plant has been tested for antimicrobial activities, including the fruit juice, peel, arils, flowers, and bark. Many studies have utilized pomegranate peel with success. There are various phytochemical compounds in pomegranate that have demonstrated antimicrobial activity, but most of the studies have found that ellagic acid and larger hydrolyzable tannins, such as punicalagins, have the highest activities. In some cases the combination of the pomegranate constituents offers the most benefit. The positive clinical results on pomegranate suppression of oral bacteria are intriguing and worthy of further study. Much of the evidence for pomegranates’ antibacterial and antiviral activities against food borne pathogens and other infectious disease organisms comes from in vitro cellbased assays, necessitating further confirmation of in vivo efficacy through human clinical trials [19]. 3.2. Cardiovascular Diseases Consumption of pomegranate juice for 6 weeks indicate a positive impact on lipid peroxidation and fatty acid status in subjects with



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metabolic syndrome and suggest potential anti-inflammatory and cardioprotective effects, partly attributed to the effects of polyphenols on lipid metabolism. A statistically significant decrease in the relative amount of arachidonic acid (P < 0.05) and an increase in the relative amount of saturated fatty acids (P < 0.05) were observed. In addition, pomegranate juice significantly increased the relative amount of total mono-unsaturated fatty acids (P < 0.05), and significantly decreased the levels of thiobarbituric acid reactive substances in erythrocytes (P < 0.05) [20]. Treatment with pomegranate hydroalcoholic extract improved coronary vascular reactivity and cardiovascular parameters (Systolic blood pressure reduced) in hypertensive Sham and ovariectomized female rats. The ovariectomy promoted an increasing in the superoxide anion levels and the treatment was able to prevent this elevation and reduce oxidative stress. Moreover, the treatment prevented the decreasing in plasmatic nitrite. A reduction in total cholesterol and LDL was observed in the treated Sham group. The treatment enhances the endothelium-dependent coronary relaxation in menopausal women where decline in estrogen levels promotes endothelial dysfunction and cardiovascular diseases and this suggests a therapeutic role of pomegranate [21]. Pomegranate juice is rich in bioactive phytochemicals with antioxidant, and anti-inflammatory and cardioprotective functions. The acute effects of juice consumption on blood pressure and markers of endothelial function were investigated and promising acute hypotensive properties were found. Consumption of pomegranate juice could be considered in the context of both dietary and pharmacological interventions for hypertension [22]. The potential cardioprotective benefits of pomegranate juice consumption were investigated and data showed significant reductions in both systolic [weighed mean difference (WMD): - 4.96mmHg, 95% CI: 7.67 to - 2.25, p < 0.001) and diastolic blood pressure (WMD: 2.01mmHg, 95% CI: - 3.71 to - 0.31, p = 0.021) after pomegranate juice consumption. Pomegranate juice reduced systolic blood pressure regardless of the duration (> 12 wks: WMD = - 4.36mmHg, 95% CI: - 7.89 to - 0.82, p = 0.016) and < 12 wks: WMD = - 5.83 mmHg, 95% CI: - 10.05 to - 1.61,


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p = 0.007) and dose consumed (> 240cc: WMD = - 3.62mmHg, 95% CI: 6.62 to - 0.63, p = 0.018) and < 240cc: WMD = - 11.01mmHg, 95% CI: 17.38 to - 4.65, p = 0.001, pomegranate juice per day) whereas doses > 240cc provided a borderline significant effect in reducing diastolic blood pressure [23]. Pomegranate peel extracts showed inhibitory effects against αglucosidase activity, lipase activity, and cupric ion-induced LDLcholesterol oxidation as well as peroxyl and hydroxyl radical-induced DNA scission [5]. Potent antioxidative effects of pomegranate juice against lipid peroxidation in whole plasma and in isolated lipoproteins (HDL and LDL) were assessed in humans and in apolipoprotein E–deficient mice after pomegranate juice consumption. In humans, pomegranate juice consumption decreased LDL susceptibility to aggregation and retention and increased the activity of serum paraoxonase (an HDL-associated esterase that can protect against lipid peroxidation) by 20%. In mice, oxidation of LDL by peritoneal macrophages was reduced by up to 90% and the size of their atherosclerotic lesions was reduced by 44% [24].

3.3. Chronic Obstructive Pulmonary Disease (COPD) Experimental and clinical evidences support polyphenol-mediated potential health benefits in smokers. Pomegranate juice is able to attenuate the damage provoked by cigarette smoke on cultured human alveolar macrophages. Cigarette smoke accounts for the outcome of several pathologies, even including lung cancer, cardiovascular disease and chronic obstructive pulmonary disease. Under healthy conditions, lung immune system becomes tolerant in response to various external stimuli. Cigarette smoke exposure alters the pulmonary immune equilibrium, thus leading to a condition of hyperactivation of the local innate and adaptive immunity. The potential targets of polyphenols in the course of COPD include the activation of T regulatory cells as well as the inhibition of the polymorphonuclear cell and monocyte respiratory burst and of the NF-kB pathway [25].



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3.4. Diabetes Increased free radicals production due to hyperglycemia produces oxidative stress in patients with diabetes. Pomegranate juice consumption of 200 ml daily for six weeks, decreased oxidized LDL and anti-oxidized LDL antibodies and moreover total serum antioxidant capacity and arylesterase activity of paraoxonase increased significantly [26]. It has been proved that the intake of pomegranate juice in patients with type 2 diabetes mellitus, produces a significant increase in both total and high-density lipoprotein cholesterol without statistically significant changes in serum triglyceride low-density lipoprotein cholesterol, fasting blood glucose, and blood pressure. There was also a significant reduction in serum interleukin-6 and unchanged tumor necrosis factor-α and highsensitivity C-reactive protein. The mean value of serum total antioxidant capacity (plasma concentrations of antioxidants) was substantially increased [27]. 3.5. Bone Health All pomegranate parts have beneficial effects on osteoporosis and are effective in preventing the development of bone loss induced by ovariectomy in mice. Such an effect could be partially explained by an improved inflammatory and oxidative status. The consumption of pomegranate peel extract limits the process of osteopenia in ovariectomized mice, significantly prevents the decrease in bone mineral and bone microarchitecture impairment. Moreover it appeared to substantially stimulate osteoblastic alkaline phosphatase activity, mineralization and the transcription level of osteogenic markers. Pomegranate may be effective in preventing the bone loss associated with ovariectomy in mice, and offers a promising alternative for the nutritional management of this disease [28]. Consumption of pomegranate induced bone-sparing effects in ovariectomized mice, both on femoral BMD and bone micro-architecture parameters and (whatever the part) up-regulated osteoblast activity and decreased the expression of osteoclast markers. It elicited a lower expression of pro-inflammatory markers and of enzymes involved in ROS


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generation, whereas the expression of anti-inflammatory markers and antioxidant actors was enhanced [29].

3.6. Cancer There is an immense interest for herbal formulations with cancer preventive effect because of the problems, generated with existing chemotherapeutic regimens. Research interest in pomegranate fruit, rich in polyphenols, and the underlying mechanism of its inhibition of cancer progression is increasing. Pomegranate has demonstrated anti-proliferative, anti-metastatic and anti-invasive effects on various cancer cell lines in vitro as well as in vivo animal models or human clinical trials [30]. The antioxidant activity, as well as suppression of inflammation, may contribute to the chemotherapeutic and chemopreventive effects, pomegranate has shown against cancer cell lines, both in vitro and in vivo [15]. The antioxidant pomegranate seed extract has a preventive effect on cisplatin-induced hepatotoxicity in rabbit and ameliorated cisplatininduced pathological changes [31]. In a multicenter study of the effects of pomegranate cold-pressed or supercritical CO(2)-extracted seed oil, fermented juice polyphenols and pericarp polyphenols on human prostate cancer, a significant antitumor activity of pomegranate-derived materials against human prostate cancer was demonstrated [32]. 3.7. Rheumatoid Arthritis Pomegranate juice significantly reduced and improved some blood biomarkers of inflammation and oxidative stress in Rheumatoid Arthritis patients and alleviated disease activity and clinical symptoms (joint dysfunction caused by inflammation and serious pain). Pomegranate reduced the Disease Activity Score (P < 0.001) which could be related to the decrease in swollen (P < 0.001) and tender joints (P = 0.001) count, the pain intensity (P = 0.003) and erythrocyte sedimentation rate levels (P = 0.03). It decreased Health Assessment Questionnaire score (P = 0.007) and



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morning stiffness (P = 0.04) and increased glutathione peroxidase concentrations (P < 0.001) [33].

3.8. Acute Pancreatitis Due to antioxidant and anti-inflammatory properties of pomegranate, it could be considered as a good candidate alternative medicine with beneficial effects on acute pancreatitis [34]. 3.9. Obesity In obesity the combination of pomegranate extract and exercise showed additive benefits on inhibition of high-fat-diet -induced body weight increase and improvement of the induced immune dysfunction, including (a) attenuating the abnormality of histomorphology of the spleen, (b) increasing the ratio of the CD4+:CD8+ T cell subpopulations in splenocytes and peripheral blood mononuclear cells (PBMC), (c) inhibition of apoptosis in splenocytes and PBMC, (d) normalizing peritoneal macrophage phenotypes and (e) restoring immunomodulating factors in serum. The immune dysfunction was associated with increased inflammatory cytokine secretion and oxidative stress biomarkers, and pomegranate and exercise combination effectively inhibited the inflammatory response and decreased oxidative damage [35].

POMEGRANATE AS FUNCTIONAL FOOD Pomegranate fruit presents strong anti-inflammatory, antioxidant, antiobesity, and antitumoral properties, thus leading to an increased popularity as a functional food and nutraceutical source since ancient times. According to the US International Food Information Council (IFIC), a food is considered as functional, when besides its nutritional value, it has been evidenced with one or more health beneficial properties ameliorating or preventing disease conditions [36].


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The seed oil of Pomegranate can be considered an interesting alimentary source of substances of nutraceutical value involved in the modulation of cholesterol metabolism [37]. It should be mentioned that although moderate consumption of pomegranate does not result in adverse effects, future studies are needed to assess safety and potential interactions with drugs that may alter the bioavailability of bioactive constituents of pomegranate as well as drugs [38].

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Singh, RP; Chindabara Murthy, KN; Javaprakasha, GK. Studies on the Antioxidant Activity of Pomegranate (Punica granatum) peel and seed extracts using in vitro Models. Journal of Agricultural and Food Chemistry, 2002, 50(1), 81-6. Ambigaipalan, P; de Camargo, AC; Shahidi, F. Phenolic Compounds of Pomegranate Byproducts (Outer Skin, Mesocarp, Divider Membrane) and Their Antioxidant Activities. J Agric Food Chem., 2016, 31, 64(34), 6584-604. Pomegranate: Department of Plant Sciences, University of California at Davis, College of Agricultural and Environmental Sciences, Davis CA., 2014, Retrieved January 2017. Morton, JF1; Morton, J. Pomegranate., p. 352–355. In: Fruits of warm climates. Julia F. Morton, Miami, FL., 1987. Agfact H3: Pomegranate growing: 1.42 First Edition 1983 J.F. Johnson. Division of Plant Industries. Pomegranate- National Horticulture Board - nhb.gov.in/report_ files/pomegranate/Pomegranate.htm. Blumenfeld, A; Shaya, F; Hillel, R. Cultivation of pomegranate. In: Melgarejo P, Martinez- Nicolas JJ, Martinez-Tome J (ed.) Production, processing and marketing of pomegranate in the Mediterranean region: Advances in research and technology. Zaragosa: CIHEAM, Options Mediterraneennes: Serie A, Seminairs Mediterraneennes, 2000, n. 42, 143-147. Sreeja, S; Hima, S; Parvathy, M; Juberiya, MA; Sreeja, S. Pomegranate Fruit as a Rich Source of Biologically Active Compounds. Biomed Res Int., 2014, 2014, 686921. Jahfar, M; Vijayan, KK; Azadi, P. Studies on a polysaccharidefrom the fruit rind of Punica granatum. Res. J. Chem. Environ, 7, 43, (2003). Lansky, EP(1); Newman, RA. Punica granatum (pomegranate) and its potential for prevention and treatment of inflammation and cancer. J Ethnopharmacol., 2007, Jan 19, 109(2), 177-206.


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[16] Kalaycıoğlu, Z; Erim, FB. Total phenolic contents, antioxidant activities, and bioactive ingredients of juices from pomegranate cultivars worldwide. Food Chem., 2017, Apr 15, 221, 496-507. [17] Zarfeshany, A; Asgary, S; Javanmard, SH. Potent health effects of pomegranate. Adv Biomed Res., 2014, Mar 25, 3, 100. [18] Shubert, YS; Lansky, EP; Neeman, I. Antioxidant and eicosanoid enzyme inhibition properties of pomegranate seed oil and fermented juice flavonoids. J Ethnopharmacol, 1999, 66, 11–7. [19] Howell, AB; D’Souza, DH. The pomegranate: effects on bacteria and viruses that influence human health. Evid Based Complement Alternat Med., 2013, 2013, 606212. [20] Kojadinovic, MI; Arsic, AC; Debeljak-Martacic, JD; Konic-Ristic, AI; Kardum, ND; Popovic, TB; Glibetic, MD. Consumption of pomegranate juice decreases blood lipid peroxidation and levels of arachidonic acid in women with metabolic syndrome. J Sci Food Agric., 2016. [21] Rouver, WD; Freitas-Lima, LC; de Paula, TD; Duarte, A; Silva, JF(4); Lemos, VS; et al. Pomegranate Extract Enhances Endothelium-Dependent Coronary Relaxation in Isolated Perfused Hearts from Spontaneously Hypertensive Ovariectomized Rats. Delgado NT, Front Pharmacol., 2017, Jan 4, 7, 522. [22] Asgary, S; Keshvari, M; Sahebkar, A; Hashemi, M; Rafieian-Kopaei, M. Clinical investigation of the acute effects of pomegranate juice on blood pressure and endothelial function in hypertensive individuals. ARYA Atheroscler., 2013 Nov, 9(6), 326-31. [23] Sahebkar, A; Ferri, C; Giorgini, P; Bo, S; Nachtigal, P; Grassi, D. Effects of pomegranate juice on blood pressure: A systematic review and meta-analysis of randomized controlled trials. Pharmacol Res., 2017 Jan, 115, 149-161. [24] Aviram, M; Dornfeld, L; Rosenblat, M; Volkova, N; Kaplan, M; Coleman, R; Hayek, T; Presser, D; Fuhrman, B. Pomegranate juice consumption reduces oxidative stress, atherogenic modifications to LDL, and platelet aggregation: studies in humans



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and in atherosclerotic apolipoprotein E–deficient mice Am J Clin Nutr, May 2000, 71 (5), 1062-1076. Magrone, T; Jirillo, E. Cigarette smoke-mediated perturbations of the immune response: A new therapeutic approach with natural compounds. Endocr Metab Immune Disord Drug Targets., 2016, Sep 27. [Epub ahead of print]. Sohrab, G; Ebrahimof, S; Sotoudeh, G; Neyestani, TR; Angoorani, P; Hedayati, M; et al. Effects of pomegranate juice consumption on oxidative stress in patients with type 2 diabetes: a single-blind, randomized clinical trial. Int J Food Sci Nutr., 2017 Mar, 68(2), 249255. Shishehbor, F; Mohammad Shahi, M; Zarei, M; Saki, A; Zakerkish, M; Shirani, F; Zare, M. Effects of Concentrated Pomegranate Juice on Subclinical Inflammation and Cardiometabolic Risk Factors for Type 2 Diabetes: A Quasi-Experimental Study. Int J Endocrinol Metab., 2016, Jan 30, 14(1), e33835. Spilmont, M; Léotoing, L; Davicco, MJ; Lebecque, P; Miot-Noirault, E; Pilet, P; et al. Pomegranate Peel Extract Prevents Bone Loss in a Preclinical Model of Osteoporosis and Stimulates Osteoblastic Differentiation in Vitro. Nutrients., 2015, Nov 11, 7(11), 9265-84. Spilmont, M; Léotoing, L; Davicco, MJ; Lebecque, P; Mercier, S; Miot-Noirault, E; et al. Pomegranate and its derivatives can improve bone health through decreased inflammation and oxidative stress in an animal model of postmenopausal osteoporosis. Eur J Nutr., 2014 Aug, 53(5), 1155-64. Panth, N; Manandhar, B; Paudel, KR. Anticancer Activity of Punica granatum (Pomegranate): A Review. Phytother Res, 2017, Feb 10 doi: 10.1002/ptr.5784 [Epub ahead of print]. Yildirim, NC; Kandemir, FM; Ceribasi, S; Ozkaraca, M; Benzer, F. Pomegranate seed extract attenuates chemotherapy-induced liver damage in an experimental model of rabbits. Cell Mol Biol (Noisy-legrand)., 2013, Feb 2, 59 Suppl, OL1842-7. Albrecht, M; Jiang, W; Kumi-Diaka, J; Lansky, EP; Gommersall, LM; Patel, A; Mansel, RE; Neeman, I; Geldof, AA; Campbell, MJ.


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A. Kotsiou and C. Tesseromatis Pomegranate extracts potently suppress proliferation, xenograft growth, and invasion of human prostate cancer cells. J. Med. Food., 2004, 7, 274–283. Ghavipour, M; Sotoudeh, G; Tavakoli, E; Mowla, K; Hasanzadeh, J; Mazloom, Z. Pomegranate extract alleviates disease activity and some blood biomarkers of inflammation and oxidative stress in Rheumatoid Arthritis patients. Eur J Clin Nutr., 2017 Jan, 71(1), 9296. Minaiyan, M; Zolfaghari, B; Taheri, D; Gomarian, M. Preventive Effect of Three Pomegranate (Punica granatum L.) Seeds Fractions on Cerulein-Induced Acute Pancreatitis in Mice. Int J Prev Med., 2014 Apr, 5(4), 394-404. Zhao, F; Pang, W; Zhang, Z; Zhao, J; Wang, X; Liu, Y; et al. Pomegranate extract and exercise provide additive benefits on improvement of immune function by inhibiting inflammation and oxidative stress in high-fat-diet-induced obesity in rats. J Nutr Biochem., 2016 Jun, 32, 20-8. Sharon, Ross. Functional foods: the Food and Drug Administration perspective1, 2, 3. Am J Clin Nutr, June 2000, vol. 71, no. 6 1735s1738s. Caligiani, A; Bonzanini, F; Palla, G; Cirlini, M; Bruni, R. Characterization of a potential nutraceutical ingredient: pomegranate (Punica granatum L.) seed oil unsaponifiable fraction. Plant Foods Hum Nutr., 2010 Sep, 65(3), 277-83. Viladomiu, M; Hontecillas, R; Lu, P; Bassaganya-Riera, J. Preventive and prophylactic mechanisms of action of pomegranate bioactive constituents. Evid Based Complement Alternat Med., 2013, 2013, 789764.




Chapter 4

THE EFFECT OF APHIS PUNICAE ON POMEGRANATE TREES IN TUNISIA: BIOLOGY AND NATURAL ENEMIES Lassaad Mdellel, PhD1,*, Amani Tlili2, Rihem Adouani, PhD1 and Monia Ben Halima Kamel, PhD1 1

High Institute of Agronomy of Chott-Mariem, Sousse University, Tunisia 2 Graduate School of Agriculture of Kef. Jendouba University. Tunisia

ABSTRACT Pomegranate is one of the important horticultural products throughout the Tunisian continent. The pomegranate trees are susceptible to several insect species that decrease the quality and quantity of its product, among which aphid is considered the most serious one. In this chapter, Aphis punicae Pass (Hemiptera: Aphididae) on pomegranate trees, its biology and its main natural enemies are presented. Results demonstrate that, after rearing of aphids on eight cultivars of *

Corresponding Author: Email: [email protected].


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L. Mdellel, A. Tlili, R. Adouani et al. pomegranate (Chefli, Kalii, Gabsi, Nebli, Rafrafi, Zehri, Garoussi and Tounsi), cultivar Tounsi is more sensitive to Aphis punicae attack. Therefore, cultivar Chefli is the most resistant. Winter pruning is one of technical practice which reduces the population of pomegranate aphid. The survey of the aphid population on pomegranate demonstrate the presence of lady beetles (Coleoptera, Coccinellidae), flower flies (Diptera, Syrphidae), aphid midge (Diptera: Cecodomyiidae), lacewing (Neuroptera, Chrysopidae) and parasites.

Keywords: pomegranate, Aphis puincae, biologie, natural enemies, technical practice, cultivars

1. INTRODUCTION Aphids are the major arthropod pests feeding on a wide range of horticultural crops (Blackman and Eastop 2006). They can damage plants by sucking the sap and injecting toxic saliva, which result in the blighting of buds, dimpling of fruits, curling of leaves and the appearance of discolored spots on the foliage (Hashmi 1994). Aphids further damage plants by excreting honeydew on their leaves which causes the development of sooty mold that ultimately hinders photosynthesis (Blackman and Eastop 2006). In Tunisia, aphids are considered one of the most serious and widespread pests in pomegranate orchards (BenHalima 2012). The pomegranate aphid, Aphis punicae Passerini (Hemiptera, Aphididae) is one of the key pests in pomegranate (Punica granatum L.) orchards. This aphid is distributed throughout the Mediterranean region, Middle East, Ethiopia, India and Pakistan. Both winged and wingless forms breed parthenogenetically and the entire life cycle takes 22 – 25 days; 12 – 14 generations per year were registered (Blackman and Eastop 2000). The morphology of oviparous and viviparous females of this aphid was studied in Japan in laboratory conditions (Sugimoto 2011). Both adult and nymphs feed on leaves, inflorescences and fruits from April to September (Moawad and Al-Barty 2011). This pest has a high reproductive capacity and it was observed from April to June (Ben Halima & Ben Hamouda 2004). It is


The Effect of Aphis Punicae on Pomegranate Trees in Tunisia

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well known for its ability to reduce plant vigor, facilitate the growth of mold on leaves and consequently reduce crop quality and yield (Rouhani et al. 2013). Because of its economic importance for pomegranate culture, the biology, ecology and control of A. punicae have been widely studied. Insecticides are a very important strategy for control of pomegranate aphid in Tunisia. However, extensive usage of insecticides to control this pest results in the development of resistance (Pavela et al. 2009; Zheng et al. 2009), and systemic insecticides can be absorbed by the tree’s root system and circulated throughout the rest of the plant (Rouhani et al. 2013). This has forced many researchers to find new and effective methods for safe control of this pest, such as the use of plant extracts (Endersby and Morgan 1991; Moawad and Al-Barty 2011). Cultural practices are also used to create unfavorable conditions for pest development, and constitute a major component of fruit pest management. Cultural practices involved in plant vigor are an important field of interest. The plant vigor hypothesis proposed by Price (1991) predicts that herbivorous insects will perform better on vigorously growing plants. Several studies have confirmed (Inbar et al. 2001; Teder and Tammaru 2002) or contradicted (Johnson et al. 2003) this hypothesis. Variations in insect responses, both among insect feeding guilds and within the sucking guild (Koricheva et al. 1998), highlight the complexity of plant–insect interactions. The susceptibility response of insect herbivores to the plant greatly depends on the plant and insect species. Indeed, aphids are very selective feeders, which choose their host plant based on visual, mechanical and chemical stimuli (Bernays 1994). The acceptance or rejection of a plant by aphids can involve the nature of the plant volatile chemicals, surface waxes, intracellular polysaccharides, mesophyll and phloem constituents (Niemeyer 1990; Caillaud and Via 2000). Therefore, the size of aphid populations varies from host to another and depending to plant vigor. Price (1991) predicts that herbivorous insects will perform better on vigorously growing plants. Several aphid species, including Rhopalosiphum padi L., Aphis citricola Van der Goot and Aphis pomi de Geer, have a preference for succulent terminal shoots of cherry apple trees (Sandström et al. 2000; Whitaker et al. 2006). On pomegranate tree, A. punicae was observed colonizing apices


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L. Mdellel, A. Tlili, R. Adouani et al.

of pomegranate shoots in spring. In order to control this pest, several methods were used like resistant cultivars and winter pruning. The development of the comprehensive pest management program to pomegranate in Tunisia is dependent on having good estimates of the demographic parameters of A. punicae on different cultivars of pomegranate. Indeed, there are several studies of the effect of host plants on the development rates and fecundities of various pest insects (Morgan et al. 2001). For this, to know the behavior of this aphid on different cultivated cultivars of pomegranate in Tunisia and the susceptibility or resistance of these cultivars to this aphid is a fundamental component of an integrated pest management program for any crop. For winter pruning, as training practice, used in all fruits orchards to shape fruit trees or to adjust crop load. It is generally seen as a means of controlling tree vegetative growth and vigor (Marini 1984), and therefore may also influence aphid performance. However, limited literature was found regarding the response of A. punicae to pomegranate tree vigor, and the effect of winter pruning as a possible alternative technique of aphid management. The aim of this chapter is to improve the available knowledge of regulating aphid population in relation to cultivars, vegetative growth, and to study the impact of winter pruning on natural enemies of A. punicae.

2. MATERIALS AND METHODS 2.1. Impact of Pomegranate Cultivars on Aphis Punicae Populations 2.1.1. Biological Material Aphis were collected from parthenoogenetic population on pomegranate in a field at Chott Mariem in Tunisia (36° 48’ N, 10°11’E) and reared for several generations on pomegranate in a green house at 21 ± 2°C, HR: 60 - 80% and under a long day photoperiod (L:D 14:10) (Troncoso et al. 2005). These aphids were needed to infest the pomegranate trees used in the experiments. Eight cultivars of pomegranate



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(Chefli, Kalii, Gabsi, Nebli, Rafrafi, Zehri, Garoussi and Tounsi) were used as host plants, which were established from pomegranate fragment of 20 cm from each cultivar in a 30 cm diameter plastic pots filled with suitable soil. At the 12th leaf stage, plants were infested with A. punicae.

2.1.2. Methods of Survey At the 12th leaf stage, five apterous A. punicae were placed on each of the plants. The plants were then placed in cages to prevent contamination by other aphids. For each treatment (cultivars) there were 20 replicates (plants). The plants were checked each five days in order to determine the mean numbers of wingless and winged adult, overall fecundity (total number of larvae produced after 20 days), the infestation rates (number of infested leaves/total number of leaves), the mean relative growth rate (MRGR) which was calculated according to Fisher (1920) and Radford (1967), in Leather and Dixon (1984) formula: MRGR = (Ln N tn – Ln N tn-1)/ΔT

Where N tn-1 = size of the population at the time tn-1, N tn = size of the population at the time tn, Δt = interval of time between the two evaluations of the size of population= 5 days. The specific age (T) is also calculated according to Ramade (2003) equation: T = log2/MRGR.

2.2. Response of A. Punicae Population to Pomegranate Tree Winter Pruning 2.2.1. Experimental Orchard We collected data in 2008 and 2009 in two experimental orchards with the pomegranate cultivar Kalii, with 4 × 5-m spacing between rows. The experimental orchards were located in Chott Erroman in Akouda (Coastal


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region of Tunisia: 35°52′16′′N, 10°34′16′′E) the plots were 1-km apart. Each orchard consisted of 100- trees. One of the plots was neatly pruned in February 2008; while the other one was not pruned.

2.2.2. Methods of Survey In order to determine the impact of winter pruning on A. punicae population, 20 infested trees were chosen at weekly intervals starting from mid-March until no more aphids were observed on trees (about mid-June), where eight shoots of 10 cm were collected from each tree. Fragments were collected from the upper and lower parts of infested trees and from different orientations (north, south, east and west). All fragments were examined under an ocular microscope in the laboratory in order to determine the mean number of wingless adults, the mean number of winged adults, overall fertility (total number of larvae produced), and the mean relative growth rate (MRGR), which was calculated according to Fisher (1921) and Radford (1967), in Leather and Dixon (1984) formula described in paragraph 2.1.2. The specific age (T) and the infestation rate (number of infested leaves/total number of leaves) were also determined. 2.2.3. Impact of Winter Pruning on A. Punicae Natural Enemies Mummified and living aphids from all samples were placed individually in plastic boxes with perforated top for aeration. They were transported to the laboratory and stored at room temperature until the emergence of parasitoids. Emerging parasitoids were counted and identified according to Tomanovic et al. (2012). Hoverflies, ladybirds and lacewing were counted and species were identified respectively, according to Stubbs and Falk (1983) and Rotheray (1991).

2.3. Statistical Analyses Mean separation of data corresponding to A. punicae population and natural enemies recorded on pruned and unpruned plots of the pomegranate



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was achieved using Duncan’s multiple range tests at the 5% level using SPSS (version 16.).

3. RESULTS 3.1. Impact of Cultivars on the Abundance of Aphis Punicae Results of the impact of eight pomegranate cultivars on the abundance of A. punicae are presented in Figure (1 and 2). The largest aphid population was recorded in decreasing order on the cultivars Tounsi, Garoussi, Zehri, Rafrafi, Guebsi, Nebli, Kalii and Chelfi (Figure 1). There was a significant difference between the levels of population after 20 days (F = 431.33, df = 7, ß = 0.001) of the cultivars. A significant difference was also recorded in a number of wingless individuals on the different pomegranate cultivars (Figure 2) (F = 687.35, df = 7, ß = 0.0013). The highest numbers of wingless individuals of A. punicae were recorded on Tounsi, Garoussi, Zehri and the lowest numbers on Nebli, Kalii, Guebsi, cheffi and Rafrafi. In terms of the numbers of the winged individuals, there a significant difference in the numbers recorded on the different cultivars (Figure 2) (F = 110.23, df = 7, ß = 0.002).

Figure 1. Number of Aphis punicae recorded on eight cultivars of pomegranate after twenty (20) days of initial infestation. Different letters with mean values in a chart indicate significant differences between the treatments by Duncan test at p = 0.05.


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Figure 2. Number of winged and wingless individuals of Aphis punicae recorded on eight cultivars of pomegranate after twenty (20) days of initial infestation. Different letters with mean values in a chart indicate significant differences between the treatments by Duncan test at p = 0.05.

3.2. Impact of Cultivars on the Mean Relative Growth Rate, Generation Time of A. Punicae and Infestation Rate The pomegranate plant cultivars differed in their impact on the mean relative growth rate (MRGR) of A. punicae. The highest MRGR was recorded on cultivars Tounsi (0.03 ± 0.01) and the lowest on cultivars Chelfi (0.024 ± 0.02) (Table 1). The shortest generation time was recorded on cultivar Tounsi (19.76 days) and the longest on cultivars Chelfi (28.75 days) (Table 1). Results demonstrated also that the infestation rate differed significantly (F = 3.57, df = 7, ß = 0.009) between cultivars. The highest infestation rate was recorded on Tounsi cultivar (83.75 ± 11.5) and the lowest on Kalii (49.11 ± 12.83).

3.3. Response of A. Punicae and Its Natural Enemies to Winter Pruning of Pomegranate Tree 3.3.1. Aphis Punicae Population Response Results of the impact of winter pruning on population of A. punicae on pomegranate trees were indicated from the total number of larvae produced each week during the infestation period, the mean number of wingless and



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winged aphids, means the relative growth rate, the specific age of aphids and leaf infestation rate. The results are shown in Table 2 and Figure 3. It was demonstrated that the weekly population densities of A. punicae were significantly higher on unpruned trees during April and early May, than on pruned trees (Figure 1; F = 39.11, df = 2, p = 0.000; F = 10.40, df = 2, p = 0.000; F = 8.31, df = 2, p = 0.002; F = 5.54, df = 2, p = 0.001; F = 23.09, df = 2, p = 0.010). The impact of winter pruning on the A. punicae population was also observed by the mean number of wingless and winged individuals. It was found that during the infestation period (April and May), the mean number of wingless individuals was significantly (p < 0.05) higher on unpruned than on pruned trees (Figure 1). Similarly, the mean number of winged individuals was much higher on unpruned trees (Figure 1) compared with pruned trees. There were significant differences in the mean number of winged aphids only in the middle of April until the beginning of May (F = 6.02, df = 2, p = 0.007; F = 7.7, df = 2, p = 0.002, F = 14.03, df = 2, p = 0.000). The winter pruning also affected the mean relative growth rate and the specific age of aphid (Table 1). We observed that A. punicae reproduced primordially on unpruned trees and the mean relative growth rate was 0.039 ± 0.028. The mean relative growth rate was 0.034 ± 0.018 on pruned trees. Also, the specific age of A. punicae is much shorter in the unpruned plot (17.22 days, compared to 20.55 days on pruned trees). We also observed a significant difference of infestation rates between pruned and unpruned trees (F = 5.36, df = 2, p = 0.007) (Table 1).

Figure 3. (Continued).



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Figure 3. Aphis punicae population on pomegranate trees in two treated plots. A, Weekly population densities; B, mean number of wingless aphids; C, mean number of winged aphids. Different letters with mean values in a chart indicate significant differences between the treatments by Duncan test at p = 0.05.

Table 1. MRGR, Generation Time (T) and infestation rate by Aphis punicae of eight pomegranate cultivars Cultivars Nabli Gabsi Garoussi Zehri Kallii Rafrafi Chelfi Tounsi

MRGR (M ± SEM) 0.266 ± 0.017 0.256 ± 0.02 0.0278 ± 0.02 0.0272 ± 0.01 0.245 ± 0.018 0.0262 0.024 ± 0.02 0.03 ± 0.01

Specific age 24 23.43 21.58 22.05 24.48 22.90 28.75 19.75

Infestation rate (%) (M ± SEM) 54.96 ± 11b 52.96 ± 12b 79.80 ± 9ab 73.39 ± 15ab 49.11 ± 12b 57.05 ± 9b 59.67 ± 20.85b 83.85 ± 11a



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3.4. Natural Enemies Response The following natural enemies of A. punicae on pruned and unpruned plots revealed the presence of predators belonging to four families: Coccinellidae: Coccinella Algeria Kovar (Figure 4A) and Hippodamia variegata Goeze; Syrphidae: Episyrphus balteatus De Geer (Figure 4B); Chrysopidae: Chrysoperla carnea Stephens (Figure 4C); and Cecidomyiidae: Aphidoletes aphidimyza Rondani. The investigation also revealed the presence of two parasitoids: Aphidius matricariae Haliday (Hymenoptera, Braconidae, Aphidiinae) and Lysiphlebus testaceipes Cresson (Hymenoptera, Braconidae, Aphidiinae). The study of the impact of winter pruning on the numerical importance of natural enemies demonstrated that the number of predators was higher in the unpruned plot than in the pruned plot (Table 3). With ANOVA no significant difference was detected between the mean number of C. algerica (p = 0.59), H. variegata (p = 0.072), A. aphidimyza (p = 0.34), C. carnea (p = 0.57) and E. balteatus (p = 0.38) in the two plots. Table 2. MRGR, specific age (T) and infestation rate of Aphis punicae on pomegranate trees in two different plots Pruned plot Unpruned plot MRGR 0.034 ± 0.018 0.039 ± 0.018 Specific age (T) (days) 20.55 ± 8.43 17.22 ± 5.45 Infestation rate 42.3 ± 8.6%b 66 ± 13.2%a Different letters with mean values in charts indicate significant differences between the treatments by Duncan test at p = 0.05.

Figure 4. Predators associated with Aphis punicae population on pomegranate trees. A, Coccinella algerica larva; B, Episyrphus balteatus larva; C, Chrysoperla carnea adult.


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Table 3. Mean number per week of Aphis punicae predators recorded on pomegranate trees in two different plots Predators Coccinella algerica Hippodamia variegata Episyrphus balteatus Chrysoperla carnea Aphidoletes aphidimyza

Prunod plot 2.78 ± 1.4 1.42 ± 0.68 1.76 ± 1.4 0.74 ± 0.52 0.92 ± 0.68

Unpruned plot 4.3 ± 2.6 2.3 ± 1.12 2.72 ± 2.3 1.14 ± 0.86 1.26 ± 0.91

4. DISCUSSION The aim of this chapter is to improve the available knowledge of regulating aphid population in relation to cultivars, vegetative growth, and to study the impact of winter pruning on natural enemies of A. punicae. Results indicate that the development and the reproduction of Aphis puniace differed significantly on the eight cultivars of pomegranate studied. A. punicae developed faster on Tounsi than on the other cultivars. Indeed, significant differences were recorded in population density, winged and wingless individuals mean relative growth rate, generation time and infestation rate of the different cultivars of pomegranate by A. punicae. Our results demonstrate that Tounsi cultivar was most susceptible host than Garoussi, Shari, Rafrafi, Guebsi, Nebli, Kali and Chelfi. This indicates that the cultivar Tounsi is the best of these plants as host of A. punicae in terms of the demographic parameters measured. These results indicate that cultivars of pomegranate can affect the reproductive performance of A. punicae. In this context, Saikia and Muniyappa (1989) indicate that the host plant variety can affect the reproductive performance of several species of aphids. Also, Nikolalkakis et al. (2003) reports a significant effect of the variety of the host plant on the intrinsic rate of increase, fertility and generation time of M. persicae feeding on pepper and tobacco, with the intrinsic rate of increase 0.24 – 0.29 and 0.23 – 0.29, and fecundity 32.2 and 37.7 on pepper and tobacco, respectively. Fecundity also ranged from 28.9 to 45.5 on the different varieties of tobacco.



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Similarly, Goundoudaki et al. (2003) studies in Greece also revealed an effect of host plant variety on the performance of M. persicae and that its performance on 11 varieties of oriental tobacco and 5 of Virginia tobacco at 20°C and under a 16 hour photophase differed significantly. In addition, Goundoudaki et al. (2003) report that the longevity of the green peach aphid ranges from 9.1 and 9.6 days, the intrinsic rate increase is 0.23 and percentage mortality between 27.9 and 52.5% on different varieties of Virginia tobacco, whereas, on oriental varieties longevity is between 7.3 and 9 days, intrinsic rate of increase ranges from 0.28 to 0.33 and percentage mortality between 10 and 47.9%. The impact of host plant varieties on the performance of aphids was demonstrated also in Sauge et al. (1998) works in France. Indeed, authors’ report that the intrinsic rate of increase of M. persicae on four varieties of peach (GF305; Summer grand, Molo konare and Wild) are respectively 0.2; 0.28; 0.26 and 0.36. The effect of different host plant varieties on the performance of other species of aphids is also well documented. Buriro et al. (1997) report a significant difference in terms of adult longevity, generation time and fertility of Schizaphis graminum Rondani, with of the four wheat cultivars tested Zagros the most resistant and Tajan the most susceptible. Similarly, the effect of host plant variety on biological parameters of aphids is reported by Razmjou et al. (2006) for five cultivars of cotton in which the highest offspring production per female of 29.6 was recorded on cultivar Sahel and lowest on Sea land. Goldasteh et al. (2012), in a similar study by S. graminum on four commercial cultivars of wheat grown under laboratory conditions, report that the development times of viviparous wingless aphids, total number of offspring produced per female and adult longevity differed significantly on the four wheat cultivars. For the response of the Aphis punicae population and their natural enemies to winter pruning, results showed that severe winter pruning applied to pomegranate trees decreased both at. punicae and its natural enemies. This effect is connected to the distribution of vegetative shoots and branches. Indeed, Simon et al. (2007) indicated that physiological and physical aspect of tree architecture affects the distribution and abundance of both phytophagous arthropods and natural enemies. Holb et al. (2001)


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found that strong pruning of apple trees resulted in less pest damage. Van Emden and Harrington (2007) found that aphids often feed preferentially on certain parts of a plant, and some plant parts may be more susceptible to damage. In some situations, pruning of plants may provide an appropriate means of reducing the effect of aphids. Deguine et al. (2004) prove that the removal of terminal shoots using a pruning knife in large scale field experiments on seven sites in Cameroon had no effect on sites with low aphid densities, but did reduce aphid densities and the proportion of leaves infested at the most heavily infested sites. The current decline of an aphid population on pomegranate can be explained also by reduction of aphid eggs after cutting branches. Based on the study of Gange and Liewelly (1998), normal winter pruning destroys 25% of Pterocallis alni aphids. However, when orchards are abandoned or damaged by frost, the aphid population is abundant. Several other factors can explain the decline of an aphid population on pomegranate trees, e.g., emigration and lower fecundity of winged forms, depressed performance and elevated mortality of aphids (Karley et al. 2004). Nevertheless, winter pruning is not always a factor in the aphid population reduction. Indeed, several works demonstrated that severe winter pruning applied to fruit trees increased the abundance of aphids. Grechi et al. (2006) indicated that pruning prolongs the period of active vegetative growth and the production of newly formed leaves available to aphid multiplication. Grechi et al. (2006) proved also that simultaneously intensive pruning peach trees increases shoots structure to a greater infestation degree. Thus, the proportion of suitable plant parts was available to aphids. Grechi et al. (2006) explained that host-plant pruning could exacerbate aphid infestation because it encouraged the growth of lush new shoots favored by herbivores. These authors showed that plants that were pruned and watered had greater aphid densities than plants that were either just watered or just pruned or untreated. Concerning the abundance of natural enemies, the present results demonstrated the presence of generalist predator species like those observed on Hyalopterus pruni Geoffrey on almond (Ben Halima et al. 2013) and Pterochloroides persicae Cholodkovsky (Mdellel & Ben Halima 2015). These predator species are commonly considered as potential biological agents for aphid



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control, especially as aphids develop resistance to many pesticides (Ben Halima et al. 2013). Our results demonstrated that predator number of the two plots responds in a similar manner to pest number. Indeed, the results revealed a higher number of identified predators on unpruned plots than on pruned plots. Dixon (2000) indicated that the slope of the relationship between the logarithm of the number of hibernating ladybirds and the logarithm of the number of cereal aphids recorded the previous summer was not significantly different, which supports the view that the abundance of aphid determines the abundance of ladybirds and not the reverse. This author also proved that in years when aphids are abundant, ladybirds and other natural enemies have suitable conditions for multiplication and the following year large number of enemies can reduce the aphid population. The relationship between aphids and their natural enemies can be affected by landscape complexity. Thies et al. (2005) and Östman et al. (2001) found that landscape complexity affected the relationship between predator and prey. In conclusion, resistant plants play a key role in Integrated Pest Management programs. Identification of resistant cultivars of host plants is therefore the first step in the development of an IPM program. The results obtained in this study revealed that of the eight cultivars of pomegranate studied, Chelfi is the most resistant and Tounsi the most susceptible. For the mass rearing of the natural enemies of A. punicae, in insectaries or under laboratory conditions, for release in augmentative biological control programs, Tounsi would be the best choice for mass producing aphid prey based on our results. However, more studies are needed on the chemical composition of the sap and the performance of A. puniace on different cultivars of pomegranate in the field, and the effect of the pomegranate on effectiveness of the natural enemies of A. punicae. The current study is also interested in the effect of winter pruning on A. punicae and its natural enemies. These results indicate that winter pruning is an effective cultural method for controlling A. punicae in pomegranate trees, but the data are insufficient to draw clear conclusions on the relative importance of winter pruning on the aphid population and the decline of its natural enemies. Further studies on the impact of winter pruning with several replications


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should be done in order to confirm our results. Also, the impact of climatic factors, cultivar, the growing shoots and nutrients in the leaf should be studied.

REFERENCES Ben Halima, K. M. (2012). Aphid fauna (Hemiptera, Aphididae) and their host association of Chott Mariem, Coastal area of Tunisia. Annals of Biological Research, 3, 1–11. Ben Halima, K. M. & Ben Hamouda, M. H. (2004). Aphids of fruit trees in Tunisia In: Simon JC, Dedryver CA, Rispe C, Hullé M, editors. Aphids in a New Millennium. Proceedings of the VIth International Symposium on Aphids, Rennes, 119–123. Ben Halima, K. M., Mdellel, L., Karboul, H. & Zouari, S. (2013). Natural enemies of Hyalopterus pruni species complex in Tunisia. Tunisian Journal of Plant Protection, 8, 119–126. Blackman, R. L. & Eastop, V. F. (2000). Aphid on the world’s crops. An identification and information guide. 2nd ed. England: John Wiley & Sons, 466 p. Blackman, R. L. & Eastop, V. F. (2006). Aphids on the world’s herbaceous plants and shrubs, volume 1, Hosts lists and key. Museum (UK): John Wiley and Sons, the Natural History, 1415 p. Buriro, A. S., Khuhro, R. D., Khuhro, I. U. & Nizamani, M. (1997). Demography green bug (Homoptera: Aphididae) on wheat. Pakistan Journal of Zoology, 29, 165–170. Deguine, J. P., Vaissayre, M. & Ferron, P. (2004). Aphid and white fly management in cotton growing: review and challenges for the future in Cotton production for the new millennium. Proc. World Cotton Res. Conf. - 3, Cape Town (South Africa), 9–13 March 2003. ARC, Institute for Industrial Crops, RSA. Dixon, A. F. G. (2000). Insect predator-prey dynamics ladybird beetles and biological control. Cambridge (UK): Cambridge University Press, 257 p.



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Endersby, N. M. & Morgan, W. C. (1991). Alternatives to synthetic chemical insecticides for use in crucifer crops. Biological Agriculture and Horticulture, 8, 33–52. Fisher, R. A. (1921). Some remarks on the methods formulated in a recent article on the quantitative analysis of plant growth. Annals of Applied Biology, 7, 367–372. Gange, A. C. & Liewelly, M. (1998). Egg distribution and mortality in the alder aphid, Pterocallis Alni. Entomologia Experimentalis et Applicata, 48, 9–14. Goldasteh, S., Talebi, A. S., Rakhshani, E. & Goldasteh, S. H. (2012). Effect of four wheat cultivars on life table parameters of Schizaphis graminum (Hemiptera, Aphididae). Journal of Crop Protection, 1, 121–129. Goundoudaki, S., Tsitsipis, J. A., Margaritopoulos, J. T., Zarpas, K. D. & Divanidis, S. (2003). Performance of the tobacco aphid Myzus persicae (Hemiptera: Aphididae) on oriental and Virginia tobacco varieties. Agricultural and Forest Entomology, 5, 285–291. Grechi, I., Sauphanor, B., Hilgert, N., Senoussi, R., Sauge, M. H., Chapelet, A., Lacroze, J. P. & Lescourret, F. (2006). Effect of winter pruning on the peach-Myzus persicae interactions. In OILB, Organisation Internationale de Lutte Biologique, Section Régionale Ouest Paléarctique, Groupe de travail. Protection intégrée en vergers, Workshop on Integrated plant protection in stone-fruit orchards. Hashmi, A. A. (1994). Insect pest management. Vol. 1–3. Islamabad: PARC, 2, 461–469. Holb, I. J., Gonda, I. & Bitskey, K. (2001). Pruning and incidences of diseases and pests in environmentally oriented apple growing systems: some aspects. International Journal of Horticultural Science, 7, 24–29. Inbar, M., Doostdar, H., Gerling, D. & Mayer, R. T. (2001). Induction of systemic acquired resistance in cotton by BTH has a negligible effect on phytophagous insects. Entomologia Experimentalis et Applicata, 99, 65–70. Jansson, R. K. & Smilowitz, Z. (1986). Influence of nitrogen on population parameters of potato insects; Abundance population growth, and


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within-plant distribution of the green peach aphid Myzus persicae (Homoptera: Aphididae). Environmental Entomology, 15, 49–55. Johnson, S. N., Elston, D. A. & Hartley, S. E. (2003). Influence of host plant heterogeneity in the distribution of a birch aphid. Ecological Entomology, 28, 533–541. Karley, A. J., Parker, W. E., Pitchford, J. W. & Douglas, A. E. (2004). The midseasoncrash in aphid populations: why and how does it occur? Ecological Entomology, 29, 383–388. Koricheva, J., Larsson, S., Haukioja, E. & Keininen, M. (1998). Regulation of woody plant secondary metabolism of resource availability: hypothesis testing by means of meta analysis. Oikos, 83, 212–226. Leather, S. R. & Dixon, A. F. G. (1984). Aphid growth and reproductive rate. Entomologia Experimentalis et Applicata, 35, 137–140. Marini, R. P. (1984). Vegetative growth of peach trees following three pruning treatments. Hort Science, 146, 287–292. Mdellel, L. & Ben Halima, K. M. (2015). Prospection and identification of natural enemies of Pterochloroides persicae Cholodovsky (Hemiptera, Aphididae) in Tunisia. Journal of Entomology and Zoology Studies, 3, 278–282. Moawad, S. S. & Al-Barty, A. M. F. (2011). Evaluation of some medicinal and ornamental plant extracts toward pomegranate aphid, Aphis punicae (Passerini) under laboratory conditions. African Journal of Agricultural Research, 6, 2425–2429. Nikolakakis, N. N., Margaritopoulos, J. T. & Tsitsipis, J. A. (2003). Performance of Myzus persicae (Hemiptera: Aphididae) clones on different host-plants and their host preference. Bulletin of Entomological Research, 93, 235–242. Östman, Ö., Ekbom, B., Bengtsson, J. & Weibull, A. C. (2001). Landscape complexity and farming practice influence the condition of polyphagous carabid beetles. Ecological Applications, 11, 480–488. Pavela, R., Nadezda, V. & Bozena, Š. (2009). Repellency and toxicity of three Impatiens species (Balsaminaceae) extract on Myzus persicae Sulzer (Homoptera: Aphididae). Journal of Biopesticides, 2, 48–51.



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Price, P. W. (1991). Evolutionary theory of host and parasitoid interactions. Biological Control, 1, 83–93. Radford, P. J. (1967). Description of some new or little known species of Aphis of Japan with a key to species. Transactions of the American Entomological Society, 92, 519–556. Ramade, F. (2003). Ecology elements, Fondamental Ecology [Eléments d’Écologie, Ecologie fondamentale]. 3rd ed. Paris (France): Dunod Editeur de savoirs, 704 p. Razmjou, J., Moharramipour, S., Fathipour, Y. & Mirhoseini, S. Z. (2006). Effect of cotton cultivar on performance of Aphis gossypii (Homoptera: Aphididae) in Iran. Journal of Economic Entomology, 99, 1820–1825. Rotheray, G. E. (1991). Larval stages of 17 rare and poorly known British hoverflies (Diptera: Syrphidae). Journal of Natural History, 25, 945– 969. Rouhani, M., Samih, M. A., Izadi, H. & Mohmmadi, E. (2013). Toxicity of new insecticides against pomegranate aphid, Aphis punicae. International Research Journal of Applied and Basic Sciences, 4, 496– 501. Saikia, A. K. & Muniyappa, V. (1989). Epidemiology and control of tomato leaf curl virus in southern India. Tropical Agriculture, 66, 350– 354. Sandström, J., Telang, A. & Moran, N. A. (2000). Nutritional enhancement of host plants by aphids-a comparison of three aphid species on grasses. Journal of Insect Physiology, 46, 33–40. Sauge, M. H., Kervella, J. & Pascal, T. (1998). Settling behaviour and reproductive potential of the green peach aphid Myzus persicae on peach varieties and a related wild Prunus. Entomologia experimentalis et applicata, 89, 233–242. Simon, S., Sauphanor, B. & Lauri, P. E. (2007). Control of fruit trees pests through manipulation of tree architecture. Pest Technology, 1, 33–37. Stubbs, A. E. & Falk, S. J. (1983). British hoverflies, an illustrated identification guide. British Entomological and Natural History Society. Cornwall: New Headland Printers.


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Sugimoto, S. I. (2011). The taxonomic identification of Aphis punicae Shunji 1922 (Hemiptera, Aphididae). Ecological Science, 14, 68–74. Teder, T. & Tammaru, T. (2002). Cascading effects of variation in plant vigor on the relative performance of insect herbivores and their parasitoids. Ecological Entomology, 27, 94–104. Thies, C., Roschewitz, I. & Tscharntke, T. (2005). The landscape context of cereal aphid-parasitoid interactions. Proceedings of the Royal Society of London B: Biological Sciences, 272, 203–210. Tomanović, Ž., Starý, P., Kavallieratos, N. G., Gagić, V., Plećaš, M., Janković, M., Rakhshani, E., Ćetković, A. & Petrović, A. (2012). Aphid parasitoid (Hymenoptera: Braconidae: Aphidiinae) in wetland habitats in western Palearctic: key and associated aphid parasitoid guilds. Annales de la Société Entomologique de France, 48, 189–198. Van Emden, H. F. & Harrington, R. (2007). Aphids as crop pests. Oxford: CAB International, 717 p. Whitaker, P. M., Mahr, D. L. & Clayton, M. (2006). Verification and extension of a sampling plan for apple aphid, Aphis pomi De Geer (Hemiptera: Aphididae). Environmental Entomology, 35, 488–496. Zheng, C., Weisser, W. W., Härri, S. A. & Ovaskainen, O. (2009). Hierarchical metapopulation dynamics of two aphid species in a shared host plant. The American Naturalist, 174, 331–341.




Chapter 5

BENEFICIAL EFFECTS OF POMEGRANATE ON THE CARDIOVASCULAR SYSTEM Nathalie Tristão Banhos Delgado, PhD Wender do Nascimento Rouver PhD and Roger Lyrio dos Santos*, PhD Department of Physiological Sciences, Health Sciences Center, Federal University of Espirito Santo, Vitoria, ES, Brazil

ABSTRACT Punica granatum L. is an ancient fruit that is still part of the diet in the Mediterranean area, the Middle East, and India. This fruit is a native shrub of occidental Asia and Mediterranean Europe, and it grows in round bushes or as small trees and has round, orange-sized fruits with a prominent calyx at the top. The pomegranate possesses a vast ethnomedical history, is widely used as a phytotherapeutic agent worldwide and has been used to ameliorate several diseases due to the phytochemistry and pharmacological actions of all its components. Ancient Egyptian culture regarded the pomegranate fruit as a symbol of

*



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N. T. B. Delgado, W. do N. Rouver and R. L. dos Santos ambition and prosperity, making it common practice to decorate sarcophagi with the fruit. According to ancient Ebers papyrus (1500 BC), the plant was used by Egyptians as a treatment for tapeworm and other parasitic infestations. In addition, the pomegranate was lauded in the Old Testament of the Bible, the Jewish Torah, and the Babylonian Talmud and is also known as a sacred fruit that conferred powers of fertility. The effects of the pomegranate may be related especially to its components; thus, the chemical composition is greatly impacted by not only the cultivar and habitat but also the climate, which affects the content of phenols, sugars, and organic acids. In the last decade, industry and agricultural production have been adapted to meet higher market demands for pomegranates. This fruit is rich in antioxidants of the polyphenol class, which includes ellagitannins and anthocyanins. In recent decades, it has been studied for many potential uses, including hypertension, oxidative stress, atherosclerosis, lipid peroxidation, hyperglycemia, immunomodulation, bacterial infection, fungal infection, parasitic infection, periodontal disease, cancer and food poisoning. Currently, the pomegranate has a potential application as a possible alternative to hormone replacement therapy due to its phytoestrogen content. There is increasing evidence regarding the link between the intake of fruits (including pomegranate) and lower incidence of certain types of diseases, and this effect is attributed to dietary polyphenols. The antioxidant capacity of the polyphenols from pomegranate was shown to be three times higher than that of red wine and green tea, based on the evaluation of free-radical scavenging. It was also shown to have significantly higher levels of antioxidants in comparison to commonly consumed fruit juices, such as grape, cranberry, grapefruit or orange juices. Although there are many studies on the benefits promoted by the pomegranate, mainly for its antioxidant characteristics, epidemiological and clinical studies are still insufficient. Therefore, the virtual explosion of interest in the pomegranate as a medicinal and phytotherapeutic product has caused knowledge about this fruit and its potential to become of great importance.

Keywords: Punica granatum L., cardiovascular benefit, polyphenols, antioxidant effect

1. INTRODUCTION Punica granatum L. is a small tree that belongs to the family Punicaceae. It is an infructescence originally from occidental Asia and


Beneficial Effects of Pomegranate on the Cardiovascular System

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Mediterranean Europe, popularly referred to as the pomegranate (Vidal et al., 2003). For centuries, the pomegranate has been used in ethnomedicine for several applications (Gracious Ross et al., 2001). Ancient Egyptian culture regarded the pomegranate as a symbol of ambition and prosperity. It was common practice to decorate sarcophagi with this plant. According to ancient Ebers papyrus (1500 BC), the plant was used by Egyptians as a treatment for tapeworm and other parasitic infestations. The pomegranate, which was lauded in ancient times in the Old Testament of the Bible, the Jewish Torah, and the Babylonian Talmud, was also known as a sacred fruit that was said to promote fertility (Jurenka, 2008). In recent decades, the pomegranate has been studied for many potential uses, including treatment of obesity, inflammation, diabetes, the regulation of blood lipid parameters, and hence, metabolic syndrome (Medjakovic and Jungbauer, 2013), as well as immunomodulation, atherosclerosis/arteriosclerosis, bacterial infection, fungal infection, parasitic infection, periodontal disease, and food poisoning (Gracious Ross et al., 2001; Prashanth et al., 2001; Schubert et al., 2002; Longtin, 2003). There has been great interest in the pomegranate as a medicinal and nutritional product mainly due to its high polyphenol content, which contributes to a strong antioxidant effect. The pomegranate has a concentration of 273 mg/100 g dry weight when compared to other fruits, whose concentrations vary from 30 to 230 mg/100 g dry weight (Jayakumar and Kanthimathi, 2011). The major polyphenols present in pomegranate include ellagitannins, which are hydrolyzed to ellagic acid in the organism (Gil et al., 2000; Seeram et al., 2004). There is an indication that the great therapeutic benefit of pomegranate is due to the high quality of its constituents, such as ellagic acid, flavonoids, and isoflavones (Jurenka, 2008). The antioxidant effect of the pomegranate is much more potent in relation to red wine, grape juice, orange juice and white wine due to the high amount (approximately 1.5%) and quality of polyphenols compared to other fruits (Ignarro et al., 2006). Substances such as polyphenols and phytoestrogens are secondary metabolites produced by plants whose function is to protect them from infection. Characterized by having one or more hydroxyls attached to one


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N. T. B. Delgado, W. do N. Rouver and R. L. dos Santos

or more phenolic rings, they may be associated with carbohydrate and organic acid molecules. Polyphenols are classified according to function and number of phenolic rings and structural elements connected to them; these families are divided into flavonoids (flavones, catechins, and anthocyanins), phenolic acids, lignans, stilbenes (resveratrol) and tannins (Singh et al., 2011). The concentration of polyphenols depends on the type of fruit, cultivation, processing and storage conditions (Manach et al., 2004). Intestinal absorption varies among individuals, and it is related to the consumed dietary content and the state of the intestinal microbiota (Adlercreutz et al., 1987). The metabolism occurs largely within the body and depends almost entirely on microorganisms in the intestinal microbiota. They are responsible for the production of enzymes that hydrolyze to glycosidic bonds (glycosidases) before being absorbed by the intestine (Bowey et al., 2003). Polyphenols have received much attention in the scientific community for their numerous biological effects, such as antioxidant, antiatherogenic, anti-inflammatory, antibiotic, antiallergenic, and anticarcinogenic, as well as their capability to modulate numerous enzyme systems (Jurenka, 2008). Another class of polyphenols that is present in the pomegranate is the phytoestrogens, described in 1940. There are several types of phytoestrogens; however, the largest category studied are the isoflavones, lignans, and coumestans (Lissin and Cooke, 2000). Estrogenic activity depends on the ability of these compounds to bind to the estrogen receptor, which in turn is determined by the presence of aromatic rings, as well as hydroxyl groups at specific sites (Martucci and Fishman, 1993). In addition, phytoestrogens may have antiestrogenic effects that may occur because of the competition for the same receptor or inhibition in estrogen synthesis. However, what will determine the estrogenic or antiestrogenic effects of these substances are the target tissues, as well as the time and level of exposure (Awoniyi et al., 1998; Mueller et al., 2004). One of the main characteristics of the pomegranate is its antioxidant effects. These effects are characterized by the ability to retard, prevent or remove oxidative damage to the affected molecule from an oxidizable substrate (Halliwell and Guttering, 2007). Such effects are mediated by the



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polyphenols, molecules that are able to eliminate or neutralize the free radicals present in the organism by acting as an exogenous antioxidant system. The antioxidant system can be classified as endogenous, or enzymatic, and exogenous, or nonenzymatic. The enzymatic antioxidant system is naturally present in the body. It is made up by the following enzymes: glutathione peroxidase, catalase, methionine reductase and superoxide dismutase (SOD). Cells possess two types of glutathioneperoxidase: one of them is selenium-dependent, and the other is not; the ratio between the two forms varies among different tissue types and species (Halliwell and Gutteridge, 1985). Catalase has iron and tocopherols (vitamin E) as important elements for its activity and is normally distributed in the cell membrane. SOD has the ability to disrupt the superoxide anion (O2•-) in hydrogen peroxide (H2O2) by offering the first line of defense against oxygen toxicity (Peskin and Winterbourn, 2000). It has copper, zinc, and manganese in its catalytic center and is divided into three isoforms: SOD-1 or copper-zinc dependent (CuZnSOD) is expressed in the cytoplasm, SOD-2 or manganese-dependent (MnSOD) is expressed in the mitochondrial space, and SOD-3 or EC-SOD was the most recently characterized and is expressed in the extracellular space and contains zinc in its catalytic center (Zelko, et al., 2002). The nonenzymatic or exogenous antioxidant system is formed mostly by essential nutrients. They are vitamin E, the family of tocopherols, ascorbic acid (vitamin C), beta-carotene and polyphenols (Halliwell, 1987). The antioxidant property occurs due to the resonance present in the aromatic rings of the polyphenol structure generating the sharing of electrons between the carbon atoms; thus, the free radicals become more stable substances. According to Halliwell and Gutteridge (2007), free radicals are defined as any species that presents unpaired electrons in its last orbital. Currently, the presence of free radicals in organic systems has attracted attention in research, as several chronic-degenerative diseases are related to high concentrations of these substances, which are produced by various intracellular systems; therefore, the enzymatic antioxidant system is unable to neutralize free radicals formed in excess, causing an imbalance between the enzymatic antioxidant system and the generation of free


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radicals. Thus, oxidative stress occurs, and it triggers the onset of various types of chronic-degenerative diseases (Baynes, 1991; Slater, 1994). It is noteworthy that within chronic-degenerative diseases, the cardiovascular diseases (CVDs) are the main cause of death around the world (Mozzafarian et al., 2016). The increase in oxidative stress in the cardiovascular system induces the activation of a series of inflammatory mediators such as adhesion molecules and pro-inflammatory cytokines in the arterial wall. In addition, oxidative stress promotes an increase in O2•- in endothelial cells, which induces endothelial dysfunction. Endothelial dysfunction is characterized by a reduction in nitric oxide (NO) bioavailability through NO reactions with O2•-, promoting the formation of more toxic species such as peroxynitrite (ONOO-), even at low concentrations (Zou and Ullrich, 1996) in the endothelial cells (Basu and Penugonda, 2008; Meyrelles et al., 2011). Due to the antioxidant properties of pomegranate polyphenols, their use promotes beneficial effects in the endothelium by reducing oxidative stress. The main antioxidant mechanism of polyphenols is the ability to improve the endothelial function by protecting NO against reactive oxygen species; increasing its bioavailability and consequently, vasodilation (Ignarro et al., 2006; de Nigris et al., 2007a); inhibiting the oxidation of low-density lipoprotein (LDL) (de Nigris et al., 2006); inhibiting elastase, which is a modulatory component of thrombin (Thring et al., 2009); and reducing the expression of vascular pro-inflammatory biomarkers such as thrombospondin and transforming growth factor–β1 (de Nigris et al., 2007b). In addition, they have antihypertensive activity promoting the reduction of angiotensin-converting enzyme activity (Mohan et al., 2010a) and reducing the thickness of the region between the intima-media layer (Davidson et al., 2009). Nevertheless, polyphenols also present antihyperglycemic activity by improving postprandial hyperglycemia in type II diabetes via inhibition of the activity of the α-glucosidase enzyme (Li et al., 2005). The pomegranate polyphenols have antiatherogenic, antihyperglycemic and anti-inflammatory properties (Jurenka, 2008), thus generating cardiovascular protection by improving endothelial function.



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Therefore, the frequent consumption of pomegranate plays an important role in health, as it is a great ally in the prevention of CVD.

2. BIOCHEMISTRY Fruits are rich sources of bioactive compounds. From the late 2000s to the present, significant progress has been made for a much more comprehensive understanding of some of the important pharmacological components of the pomegranate (Lansky and Newman, 2007; Fawole and Opara, 2013). During development and growth, the fruit goes through various stages of ripening, which result in a variety of physiological, biochemical and structural processes (Fawole and Opara, 2013).

2.1. Seed The pomegranate seeds are packed in arils that contain the red juice that constitutes 40% of the weight of the whole fruit (Viuda-Martos et al., 2010). The smallest components of pomegranate seed oil include sterols, steroids, and a key component of mammalian myelin sheaths, cerebroside (Tsuyuki et al., 1981). The concentration of phytosterols in pomegranate seed oil is approximately 3–4 times higher than the one observed in soybean oil, and the major phytosterols presented are b-sitosterol, campesterol, and stigmasterol (Kaufman and Wiesman, 2007). Between 12–20% of the total seed weight corresponds to seed oil, and approximately 80% of such oil is composed of octadecatrienoic fatty acids. This octadecatrienoic fatty acid has a high content of cis 9, trans 11, cis 13 acid (i.e., punicic acid), synthesized in situ from nonconjugated octadecadienoic fatty acid, linoleic acid (Hopkins and Chisholm, 1968; Hornung et al., 2002) itself about 7% of pomegranate seed oil. Fatty acid, which is present in the pomegranate seed oil comprises over 95% of the oil, of which 99% are present in triacylglycerols.


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Linolenic acid is the major fatty acid present in the seed of the pomegranate, followed by linoleic acid, oleic acid, palmitic acid, stearic acid, gadoleic acid, lignoceric acid, arachidic acid, and myristic acid (Elfalleh et al., 2011). The seed also includes lignins (Dalimov et al., 2003), products of cell wall components and hydroxycinnamic acids, and antioxidant lignin derivatives (Wang et al., 2004). Various organic acids, among them ascorbic acid, citric acid, and malic acid, are also reported to be present in the pomegranate seed coat (Viuda-Martos et al., 2010). The seed pod (aril) contains water (85%); sugars (10%), mainly fructose and glucose; and pectin (1.5%). Arils are also a rich source of bioactive compounds such as phenolics and flavonoids, mainly anthocyanins (Viladomiu et al., 2013).

2.2. Pericarp It is known that almost 67% of the pomegranate is the peel itself, which includes the mesocarp and divider membrane (Lansky and Newman, 2007). The pomegranate pericarp is a rich source of bioactive components, such as phenolics, flavonoids, ellagitannins, and proanthocyanidin compounds. Minerals, such as potassium (K), nitrogen (N), calcium (Ca), phosphorus (P), magnesium (Mg), and sodium (Na), as well as complex polysaccharides, are present in the pericarp (Viuda-Martos et al., 2010). Either flavonoids or tannins are more abundant in the peels of wildcrafted fruits in comparison with those of cultivated fruits (Ozcal and Dinc, 1993). Complex polysaccharides from the peels (Jahfar et al., 2003) and alkaloids, for example, pelletierine (Vidal et al., 2003), can also be present in the pericarp.

2.3. Leaves Tannins are present in pomegranate tree leaves, which also contain glycosides of apigenin, a flavone with progestin (Zand et al., 2000) and



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anxiolytic properties (Paladini et al., 1999). Leaves also represent a rich source of elements, such as N, K, Ca, and Fe (Lansky and Newman, 2007; Sharma et al., 2017). It is known that the content of minerals in pomegranate leaves can be different depending on the age of the plant (Fawole and Opara, 2013). For example, N is high in medium-aged plants; K, in young-aged plants; and Ca and Fe, in old leaves. There are other situations that may alter the content of minerals in leaves, for example, in July and August in the Northern Hemisphere, N and K are both low during flowering and fruit-setting; N further declines during fruit maturity, along with Mg, Fe and Zn (Munde et al., 1980; 1981).

2.4. Flower Studies are still in progress to elucidate the chemistry of these flowers that have also been ethnomedically employed. Nevertheless, it is known that the flowers of the pomegranate tree also contain compounds found in peels, i.e., gallic acid (Sadeghi et al., 2015), and seed, i.e., ursolic acid (Huang et al., 2005). Al-Muammar and Khan (2012) also found that pomegranate flower contains abundant polyphenols (gallic acid and ellagic acid) and triterpenes (oleanolic, ursolic, maslinic, and asiatic acids). In addition, the pomegranate flower contains one sterol (daucosterol) and one flavonoid (punicaflavone) that can be separated from the flower. According to Harzallah et al. (2016), the flower has a higher phenol content than the peel. On the other hand, Zhang et al. (2011) found that the phenolic content of the flower is lower than that of the peel.

2.5. Tree Bark and Roots The presence of alkaloids has made the bark of the pomegranate tree and its roots objects of study throughout history due to the potent physiological effect of its extracts (Lansky and Newman, 2007). However, it is worth mentioning that pomegranate fruits (excluding the peel) are not


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toxic, but its roots and bark are (Fuentes et al., 1985). The toxic activity is related to its alkaloid content (Ferrara et al., 1989).

2.6. Juice In addition to all parts of the pomegranate fruit, the juice also has several components that are important for health. In fresh pomegranate juice, several phenolic acids can be identified including gallic acid, chlorogenic acid, caffeic acid, ferulic acid, and coumaric acids, as well as flavonoids, such as catechin (also epicatechin and epigallocatechin (de Pascual-Teresa et al., 2000), phloridzin, and quercetin (Poyrazoğlu et al., 2002), tannins and anthocyanins (Ambigaipalan et al., 2017). The hydrolyzable tannins or more precisely, ellagitannins and gallotannins, which are present in all parts of the plant, constitute the most prevalent class in the pomegranate, and it is known that the bioactivity of the pomegranate is due to these compounds (Medjakovic and Jungbauer, 2013). Such characteristics make pomegranate juice, as well as whole fruit extract, components with high antioxidant activity (Kalaycioğlu and Erim, 2017). Punicalagin, another type of pomegranate polyphenol, is the link between ellagic acid and gallagic acid to a glucose molecule and such compounds (Wang et al., 2010; Medjakovic and Jungbauer, 2013). In industrialized pomegranate juice, the presence of punicalagin A (7.6-36.9 mg l-1) and punicalagin B (15.2–135.6 mg l-1) has been observed (Borges et al., 2010). Anthocyanins, which are potent antioxidant flavonoids, are present in pomegranate juice with its brilliant color, which increases in intensity during ripening (Hernandez et al., 1999), and declines after pressing (Perez-Vicente et al., 2002; Miguel et al., 2004). There is also the presence of some minerals in the pomegranate juice, such as Fe, Ca, Ce, Cl, Co, Cr, Cs, Cu, K, Mg, Mn, Mo, Na, Rb, Sc, Se, Sn, Sr, and Zn (Waheed et al., 2004).



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3. CULTIVATION There are more than 1000 pomegranate cultivars around the world (Levin, 1994). Important characteristics of pomegranate cultivars include fruit size, juice content, sweetness, acidity, and coloring (skin and arils). The pomegranate is native to the area stretching from Iran to the Himalayas in northern India and has been cultivated and naturalized over the whole Mediterranean region since ancient times. It has been reported to be grown commercially in several regions including India, Pakistan, Israel, Afghanistan, Iran, Egypt, China, Japan, the United States of American (USA), Russia, Australia, South Africa and Saudi Arabia and in the subtropical areas of South America (Holland et al., 2009). Indeed, the increasing knowledge about the positive effects of the pomegranate and the public awareness of the impact of food on health have greatly expanded the demand for the pomegranate fruit and its byproducts in the Western world (Di Nunzio et al., 2013). The pomegranate fruit growth pattern appears to be cultivar dependent. The ‘Mule’s Head’ cultivar follows a simple sigmoid curve, whereas the growth pattern of ‘Wonderful’ was linear (Shulman et al., 1984). However, for the Omani cultivars grown in the Al-Jabal Al-Akhdar area (Al-Yahyai et al., 2009) and the ‘Wonderful’ cultivar grown in Australia (Weerakkody et al., 2010), the researchers reported a linear fruit growth pattern. Nevertheless, in the ‘Malas-e-Torsh-e-Saveh’ cultivar grown in Iran, the average fruit weight and volume increased rapidly until 45 days after fruit set and then continued more slowly until the time of fruit harvest (Varasteh et al., 2008). The time taken for the fruit to reach harvest maturity varies with cultivar type, growing location and season (Shulman et al., 1984; Gil et al., 1995). Fruit ripens 5–8 months from fruit set, involving a sequence of changes in fruit characteristics from flowering to maturity and senescence. These changes include physical and structural, biochemical, physiological and elemental changes, reflecting differences in fruit appearance during maturation and ripening and maturation among cultivars (Ben-Arie et al., 1984; Shulman et al., 1984; Al-Maiman and Ahmad, 2002; Holland et al., 2009; Shwartz et al., 2009).


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A few studies have reported on the effects of cultivar difference, growing region and maturity status on fruit harvest maturity and eating quality (Ben-Arie et al., 1984; Al-Maiman and Ahmad, 2002). In addition, great differences in phenol contents and antioxidant activities related to different processing and/or cultivars have been demonstrated (Gil et al., 2000; Tezcan et al., 2009; Borochov-Neori et al., 2009). Climate conditions, ripening, and storage also affect the content of total phenols, sugars, and organic acids (Aarabi et al., 2008; Akbarpour et al., 2008; Shwartz et al., 2009). Polyphenol is the major antioxidant and functional health factor found in pomegranate arils and juice, and it mainly consists of ellagitannin (punicalagin), gallic acid, ellagic acid, anthocyanins, catechins, caffeic acid, and quercetin (Viuda-Martos et al., 2010). The abundance of these compounds depends on cultivar type, climate, and growing region (Melgarejo et al., 2000; Poyrazoğlu et al., 2002). The cultivar-related differences in phenolic content and antioxidant activity must be carefully taken into account when evaluating the health effects (Di Nunzio et al., 2013). Therefore, cultivar and habitat have a great impact on the chemical composition of the pomegranate. Important characteristics of pomegranate cultivars include fruit size, juice content, sweetness, acidity, and coloring (skin and arils). These parameters are essential for consumer preference, as well as for manufacturing processes. In any case, regular pomegranate consumption is certainly a good way to benefit from the bioactive effects of fruit-derived compounds. Pomegranate should be recommended by nutritionists as part of a healthy diet.

4. ANTIOXIDANT EFFECTS ON CARDIOVASCULAR DISEASES For many years, the scientific community has shown that changes in oxidative stress are associated with dietary factors and physical activity levels. Various modern lifestyle factors can promote an increase in reactive


Beneficial Effects of Pomegranate on the Cardiovascular System 101 oxygen species production, for example, i) high saturated fatty acid and sugar intake and ii) physical inactivity. In addition, iii) smoking, alcohol and lack of fresh fruits and vegetables (antioxidants) also intensify oxidative stress (Roberts et al., 2009). Furthermore, the pathophysiology of CVD, mainly hypertension and atherosclerosis, includes mechanisms of chronic inflammation and oxidative stress (Siti et al., 2015). The development of oxidative stress occurs through excessive synthesis of reactive oxygen species incapacitating the enzymatic antioxidant system and preventing it from neutralizing all these substances. The excess of reactive oxygen species promotes the deterioration of proteins, lipids and nucleic acids, thus promoting deleterious effects on organic structures, such as lipid peroxidation, cell membrane damage, impairment of cell function and DNA fragmentation (Halliwell and Gutteridge, 1985; Halliwell, 1987; Aruoma, 1994, Dröge, 2002). The antioxidant system has the function of inhibiting and/or reducing the formation of reactive oxygen species, as it neutralizes and repairs the damages they cause. It acts in a triad system, promoting prevention, scavenging, and repair (Madamanchi et al., 2005). Therefore, foods with higher concentrations of antioxidants such as Punica granatum L. have potentially better results against deleterious effects from reactive oxygen species. Among the different fruit species studied, the pomegranate has the highest concentration of polyphenols (Jayakumar and Kanthimathi, 2011). In addition, it has potent antioxidant activity in comparison to other fruits; these characteristics are related to the quality of the polyphenols present in the pomegranate (Ignarro et al., 2006). Ellagic acid is the polyphenol found in the highest concentration in the pomegranate and belongs to the class of hydrolyzable tannins (Cerdá et al., 2003a/b). The presence of high concentrations of polyphenols that have potent antioxidant activity make this fruit an important therapeutic agent in CVD. Studies have shown that the polyphenols present in the pomegranate are able to increase the activity and expression of endothelial nitric oxide synthase (eNOS) and reduce proliferation of vascular smooth muscle cells (Ignarro et al., 2006); they act as scavengers of the reactive oxygen species preventing the oxidation


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of the formed NO to ONOO-, which is a highly toxic substance (Van Den Ende et al., 2011; Rodrigues et al., 2012); they inhibit platelet aggregation via phospholipase Cγ2/protein kinase type C (PLCγ2-PKC) and protein kinase activated by mitogen/protein kinase B (MAPK/Akt) both in humans and in hypercholesterolemic mice (Chang et al., 2013); and finally they inhibit the oxidation of LDL (Aviram et al., 2000). Because their chemical structures favor the chelation of the zinc metal present in the structure of the enzyme (Aviram and Dornfeld, 2001; Mohan et al., 2010b), the polyphenols present in the pomegranate can also directly inhibit angiotensin-converting enzyme activity by attenuating the formation of angiotensin II and consequently reducing the formation of reactive oxygen species through the NADPH oxidase system. In addition, they reduce the progression of atherosclerosis and increase endotheliumdependent vasodilation as shown in the study by Machha and Mustafa (2005), which used male hypertensive animals treated with captopril and flavonoids (baicalein, flavone, and quercetin); there was a reduction in systolic blood pressure and improved acetylcholine-induced relaxation. Another study demonstrated that C57BL/6 mouse abdominal aortic rings incubated with quercetin and its metabolites were able to promote reduction in contraction induced by phenylephrine, an effect mediated at least in part via AMPK leading to subsequent activation of eNOS and increased NO production (Khoo et al., 2010; Shen et al., 2012). In another study carried out on aortic rings of male Wistar rats, the presence of ellagic acid was able to promote endothelium-mediated vasodilatation in a rapid and dose-dependent manner by inhibiting L-type calcium influx in vascular smooth muscle (Yilmaz and Usta, 2013). Another antioxidant action is the modulation that polyphenols exert on the sources that generate reactive oxygen species. Many studies have shown that these polyphenols may inhibit these systems by reducing the formation of reactive oxygen species as shown by Steffen et al. (2008); when incubating the flavonoid metabolite, epicatechin, in human umbilical vein endothelial cell culture, there was a reduction in NADPH oxidase system activity and formation of O2•-. Such results can be explained by


Beneficial Effects of Pomegranate on the Cardiovascular System 103 analyzing the chemical structures of these substances, as they are similar to that of apocynin, an inhibitor of the NADPH oxidase complex. Another study suggests that quercetin treatment is capable of enhancing eNOS activity by reducing the expression of the NADPH oxidase complex via reduced expression of the p47phox subunit (Sánchez et al., 2006). Xia et al. (2010) indicate that treatment with resveratrol (30 or 100 mg/kg) in male knockout mice for apolipoprotein E promoted downregulation in NADPH oxidase complex activity via a reduction in mRNA expression of the NOX2 and NOX4 subtypes. In addition, resveratrol was able to reduce the oxidation of tetrahydrobiopterin (BH4) and increase its biosynthesis by reversing eNOS uncoupling. These polyphenols may act on the formation of reactive oxygen species from the mitochondrial chain, such as epicatechin, which promotes the reduction of O2•- synthesis by the mitochondrial chain (Li et al., 2012). Lin et al. (2000) show that these same polyphenols were able to reduce the activity of the xanthine oxidase system by means of a competitive inhibition or “suicide substrate” in leukemic human myelocytic cells. Studies indicate that treatment with pomegranate seed oil reduces the formation of prostaglandins and leukotrienes by inhibiting the activity of the cyclooxygenase and lipoxygenase enzymes and, as a consequence, it reduces proinflammatory cytokines and reactive oxygen species from arachidonic acid metabolism (Schubert et al., 1999; Loke et al., 2010; Shingai et al., 2011). There are interactions between pomegranate and the enzymatic antioxidant system that have important effects on the body's defense against reactive oxygen species, showing that polyphenols promote influences on antioxidant enzymes by upregulation of SOD, catalase and glutathione in diabetic and hypertensive Wistar rats (Mohan et al., 2010b), in rats with diabetes alone (Bagri et al., 2009; Althunibat et al., 2010), or in animals with high levels of oxidative stress (Murthy et al., 2002; Celik et al., 2009). The polyphenols present in pomegranate are capable of influencing both the activity and expression of antioxidant enzymes. Antioxidant enzymes play an important role in the body, for example, they are


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important in oxygen metabolism by intercepting and reducing O2•- to H2O2 (Blake et al., 1987; Vincent et al., 2004). Catalase, in turn, is involved in the elimination of H2O2 and can be inactivated by O2•- and glycation of the enzyme. In addition, catalase acts in the detoxification of high concentrations of H2O2 (Zhang and Tan, 2000). In hypertensive animals, the turbulent flow on the endothelium itself may promote increased expression of responsive genes sensitive to oxidation, such as ELK-1 and p-CREB, which increase the production of reactive oxygen species. These effects promote a reduction in eNOS expression and consequently, an aggravation of endothelial dysfunction. However, a study by de Nigris et al. (2007) showed that pomegranate extract was effective in enhancing the expression of eNOS accompanied by a reduction in expression of oxidation-sensitive genes in hypercholesterolemic mice.

5. BENEFICIAL EFFECTS OF POMEGRANATE ON THE INFLAMMATORY SYSTEM The inflammatory process includes a series of physiological responses of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. During this process, various types of immune cells, blood vessels, and molecular mediators are involved, since the function of inflammatory processes is directed to eliminate the initial cause of cell injury, to clear out necrotic cells and tissues damaged by the original insult and the inflammatory process and to initiate tissue repair (Ferrero-Miliani et al., 2007). The inflammatory cells, which include neutrophils, macrophages and monocytes, may inflict damage to nearby tissues, an event thought to be of pathogenic significance in a large number of diseases such as emphysema, acute respiratory distress syndrome, atherosclerosis, reperfusion injury, malignancy and rheumatoid arthritis, known as chronic inflammatory diseases (Babior, 2000). In chronic inflammatory diseases, several


Beneficial Effects of Pomegranate on the Cardiovascular System 105 cytokines recruit activated immune and inflammatory cells to the site of lesions, thereby amplifying and perpetuating the inflammatory state. The mechanisms responsible appear to be multifactorial and are poorly understood, and the causes for the onset of inflammatory diseases are inconclusive. However, it is already known that there are both genetic and environmental factors involved (Barnes and Karen, 1997). Recent therapeutic advancements in understanding the molecular and cellular mechanisms of these disorders have highlighted strategies that aim to inhibit the harmful effects of upregulated inflammatory mediators and their associated signaling events in various types of inflammatory diseases such as osteoarthritis (Saklatvala, 2007; Alghasham and Rasheed, 2014). According to a review by Rasheed (2016), activated p38 mitogenactivated protein kinase (p38-MAPK), c-Jun N-terminal kinases (JNK) and nuclear factor (NF)-κB pathways regulate proinflammatory genes such as cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), matrix metalloproteinases (MMPs) and are major targets of the anti-inflammatory agents that may alleviate the clinical symptoms, such as natural agents. Among them, pomegranate juice is rich in polyphenolic compounds that have antioxidant and anti-inflammatory activities. The pomegranate is able to improve some blood biomarkers of inflammation (Ghavipour et al., 2017). The pomegranate is a safe and nontoxic therapy that delays inflammatory disease progression at an earlier stage. Indeed, this fruit has a number of identified bioactive molecules with anti-inflammatory effects. The anti-inflammatory components of pomegranate, i.e., punicalagin, punicalin, strictinin A and granatin B significantly reduce production of nitric oxide and prostaglandin E synthase (PGE2) by inhibiting the expression of pro-inflammatory proteins (Lee et al., 2008; Romier et al., 2008). Experimental studies show beneficial effects of pomegranate on the inflammatory process in various types of inflammatory diseases. To that end, Toklu et al. (2007) demonstrated that administration of pomegranate peel extract (50 mg kg-1) for 28 days in rats with liver fibrosis induced


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by bile duct ligation decreased malondialdehyde levels and myeloperoxidase activity as well as tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1β levels. Using a model of rheumatoid arthritis, pretreatment with pomegranate extract decreased the arthritis incidence and interleukin (IL)-6 and IL-1β levels in arthritic joints (Shukla et al., 2008). Larrosa et al. (2010) demonstrated that pomegranate intake and its main microbiota-derived metabolite urolithin-A decreased inflammation markers (iNOS, COX-2, and prostaglandin E synthase in colonic mucosa) and showed a downregulation in pathways of the inflammatory response. Based on these results, different hypotheses can arise: urolithin-A is the main active anti-inflammatory compound related to pomegranate consumption by healthy subjects, or urolithin-A is the only antiinflammatory compound that provided protection in induced colitis. In another study with a model of sodium sulfate-induced colon inflammation and ulceration, a pomegranate beverage was able to reduce the expression of proinflammatory cytokines (TNF-α and IL-1β), COX-2, and iNOS at the mRNA and protein levels (Kim et al., 2017). Asgary et al. (2013) also demonstrated the anti-inflammatory effects of pomegranate in the cardiovascular system. In his study, the consumption of pomegranate juice was associated with a significant reduction in a biomarker of endothelial function and vascular inflammation in hypertensive patients aged 30–67 years. Another study demonstrated that rats with high-fat diet-induced obesity suffer from immune dysfunction, and the combination of pomegranate extract (150 mg/kg) and exercise inhibited the inflammatory response by (i) increasing the ratio of the CD4+:CD8+ T-cell subpopulations, (ii) inhibiting apoptosis, (iii) normalizing peritoneal macrophage phenotypes and (iv) restoring the immunomodulating factors in serum (Zhao et al., 2016). In view of the studies cited above, it is possible to infer that pomegranate has anti-inflammatory effects in several types of systems, making this fruit a therapeutic agent in inflammatory diseases.


Beneficial Effects of Pomegranate on the Cardiovascular System 107

6. BENEFICIAL EFFECTS OF POMEGRANATE ON CARDIOVASCULAR DISEASES CVD are a group of diseases that affect the heart and blood vessels, such as coronary artery disease, myocardial infarction, angina pectoris, hypertension, congestive heart failure, and stroke, and are a leading cause of death and disability in the Western world (Thom, 1989). An estimated 85.6 million American adults (>1 in 3) have ≥1 type of CVD. Of these, 43.7 million are estimated to be ≥60 years of age. In addition, CVD kill more people than cancer (Mozzaffarian et al., 2016). Pharmacological treatment of CVD has improved greatly over the past decades, and several effective drugs, e.g., statins, beta-blockers, angiotensin-converting enzyme inhibitors/rennin-angiotensin-aldosterone system, and antiplatelets are commercially available. However, side effects and limited adherence to treatment can limit pharmacological effectiveness. Thus, functional foods are being increasingly used as an adjunct treatment or in the prevention of CVD (Tomé-Carneiro and Visioli, 2015). Dietary bioactive compounds, especially polyphenols, have been shown to mediate biological mechanisms that lead to the modulation of clinical biomarkers reflecting blood glucose, lipid profiles, blood pressure, and inflammation (e.g., Creactive protein (CRP)), which are routinely used in clinical practice. Therefore, functional foods such as pomegranate hold promise for CVD and can lower levels of biomarkers associated with disease progression (Basu et al., 2016).

6.1. On Atherosclerosis Atherosclerosis, a systemic disease process in which fatty deposits, inflammation, cells, and scar tissue build up within the walls of arteries, is the underlying cause of the majority of clinical cardiovascular events (Roger et al., 2011). Atherosclerotic coronary artery disease has become epidemic since the 20th century in industrialized countries and currently


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accounts for the highest percentage of deaths worldwide (American Heart Association, 2002), leading to a death rate of 1 in 7 people with coronary heart disease (Mozaffarian et al., 2016). Studies have shown that pomegranate extract treatment reduced multiple parameters associated with coronary heart disease, including both morphological and physiological effects of coronary artery atherosclerosis (Al-Jarallah et al., 2013). Pomegranate juice has antiatherogenic effects in four related components of atherosclerosis: it inhibits LDL aggregation, reduces the macrophage-mediated oxidation of LDL, and reduces oxidative stress in the cell-mediated oxidation of LDL as well as the uptake of oxidized LDL (Aviram et al., 2000). Nonetheless, pomegranate can create changes in the microbiota accompanied by an improvement in atherogenic markers, reduction in plasma LDL levels and reduction in LDL and very low-density lipoprotein (VLDL) levels in hypercholesterolemic animals (Hossin, 2009; Neyrinck et al., 2013). Bagri et al. (2009) demonstrated a reduction in levels of other atherogenic markers such as triglycerides and total cholesterol in diabetic rats treated for 21 days with aqueous pomegranate extract compared to the diabetes-only group. It is possible to observe the pomegranate effects on paraoxonase-1, also known as aromatic esterase 1, which is the major antiatherosclerotic component responsible for the protection of high-density lipoprotein (HDL) and LDL from lipid oxidation. Fuhrman et al. (2010) demonstrated that pomegranate juice treatment improves lipid levels by increasing the levels of the enzyme paraoxonase-1 coupled to HDL. In addition, the phytoestrogens present in the pomegranate can preserve and/or increase the activity of the enzyme paraoxonase-1 coupled to HDL (Aviram and Dornfeld, 2001). Pomegranate extract has effects on the development of spontaneous occlusive coronary artery atherosclerosis in the SR-BI/apoE double knockout mouse model of coronary heart disease, which was treated for two weeks with the extract (307.5 mL per L of drinking water). Treatment was able to reduce the size of atherosclerotic plaques in the aortic sinus and reduce the proportion of coronary arteries with occlusive


Beneficial Effects of Pomegranate on the Cardiovascular System 109 atherosclerotic plaques. In addition, the pomegranate treatment reduced levels of oxidative stress and monocyte chemotactic protein-1 in atherosclerotic plaques. Finally, the treatment with pomegranate extract was able to reduce lipid accumulation, macrophage infiltration and fibrosis in the myocardium (Al-Jarallah et al., 2013). Thus, it is possible to affirm that pomegranate has beneficial effects on atherosclerosis. The main effects of the pomegranate on atherosclerosis are summarized in Figure 1.

Figure 1. Pomegranate vascular actions. ROS, reactive oxygen species; VSM, vascular smooth muscle; LDL, low-density lipoprotein; NO, nitric oxide; PGI2, prostacyclin; EDHF, endothelium-derived hyperpolarizing factor.

6.2. On Hypertension Hypertension is a chronic condition which affects individuals’ health care needs. Hypertension, which is a more prevalent disease than the other pathological conditions, can accelerate both macrovascular and microvascular complications. Chronic activation of the rennin-angiotensinaldosterone system leads to hypertension and perpetuates a cascade of


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proinflammatory, prothrombotic, and atherogenic effects associated with organ damage. It has been indicated that angiotensin II, a substance formed during the rennin-angiotensin-aldosterone system cascade, may contribute to vascular hypertrophy and hypertension via stimulation of the NADPH oxidase system to increase reactive oxygen species in vascular cells. The increase in reactive oxygen species production may play a role in the elevation of blood pressure directly, by a vasoconstrictive effect, or indirectly, by reducing the activity of vasodilators such as NO and arachidonic acid metabolites. There is a relationship between hypertension and impaired endothelial function, leading to a decrease in NO bioavailability, which characterizes the endothelial dysfunction (Hamilton et al., 2001). With hypertension, there is an increase in O2•- generation leading to lower NO bioavailability. O2•- rapidly reacts with NO, forming ONOO- and decreasing NO bioavailability (Rey et al., 2001; Androwiki et al., 2015). It is known that oxidative stress is related to increased reactive oxygen species generation. Such an increase overloads the antioxidant system in an effort to neutralize these substances, which can induce the development and/or progression of chronic degenerative diseases such as hypertension (Beswick et al., 2001; Shah and Channon, 2004; Androwiki et al., 2015). Pomegranate can act as a therapeutic agent in hypertension due to its potent antioxidant activity, via polyphenols. These polyphenols are capable of protecting NO against O2•--mediated oxidation, thus increasing its biological actions such as endothelium-dependent relaxation (Ignarro et al., 2006). Pomegranate has a high antioxidant capacity due to its high polyphenol concentrations, more specifically hydrolyzable tannins, which are capable of scavenging free radicals (Aviram and Rosenblat, 2012). In addition to its scavenging effect, pomegranate is able to reduce angiotensin-converting enzyme activity (Santos et al., 2016), contributing to an environment with less oxidative stress (Aviram and Dornfeld, 2001; Mohan et al., 2010b). In addition to the angiotensin-converting enzyme inhibition, there is a decrease in bradykinin degradation (Shiuchi et al., 2002).


Beneficial Effects of Pomegranate on the Cardiovascular System 111 Such antioxidant characteristics of polyphenols confer protective effects on the blood vessels due to a reduction in oxidative stress in endothelial cells and improvements in vascular reactivity. Machha and Mustafa (2005) showed that chronic treatment with flavonoids improves endothelium-dependent relaxation, reduces systolic blood pressure and significantly improves endothelial function in hypertensive animals. The polyphenols of the pomegranate act as an exogenous antioxidant system by neutralizing reactive oxygen species and increasing NO bioavailability. It is known that an antioxidant is any substance that, when presented at a low concentration compared with those of any oxidizable substrate, significantly delays or prevents oxidation of that substrate (Halliwell and Gutteridge, 2007). The pomegranate has elevated concentrations of polyphenols with high antioxidant capacity and high concentrations of phytoestrogens that can confer cardioprotective effects similar to estrogen. Among the phytoestrogens that are known, urolithins A and B (enterophytoestrogens), which are microflora metabolites from ellagic acid that belong to the tannins class, display both estrogenic and antiestrogenic activities (Larrosa et al., 2006; Ryu et al., 2016). It knows that estrogen phosphorylates eNOS mainly via the serine (Ser)/threonine (Thr) kinase Akt (Forstermann and Sessa, 2011). In fact, Delgado et al., (2017) showed that the hormonal dysfunction induced by ovariectomy promotes a reduction in eNOS activity and that the treatment with pomegranate extract prevented this decrease. In addition, the treatment avoided the phosphorylation of Thr495, the residue in eNOS that was associated with a stimulus that elevated intracellular Ca+2 concentrations and increased eNOS activity. The eNOS protein can be phosphorylated on several Ser, Thr, and tyrosine (Tyr) residues. Phosphorylation of Ser1177 stimulates the flux of electrons within the reductase domain, increasing the Ca+2 sensitivity of the enzyme, and represents an additional and independent mechanism of eNOS activation (Fleming and Busse, 2003). Both extract and juice were effective in increasing vascular eNOS expression and plasma NOx levels and increased the relaxation response to acetylcholine in resistance arteries (de Nigris et al., 2007).


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In a clinical study, it has been demonstrated that the consumption of 50 mL per day for 2 weeks (1.5 mmol of total polyphenols) of pomegranate juice can cause a 36% decrease in serum angiotensin-converting enzyme activity and a 5% decrease in systolic blood pressure in patients with hypertension. A similar dose also reduced serum angiotensin-converting enzyme activity (31%) in vitro (Aviram and Dornfeld, 2001). Given this context, pomegranate juice can offer a wide protection against CVD, which could be related to its inhibitory effect on oxidative stress and serum angiotensin-converting enzyme activity. Therefore, regardless of which part of the pomegranate fruit was used in the studies mentioned above, in general, its polyphenolic constituents are capable of promoting beneficial effects on hypertension. This contribution is related to the following mechanisms: (i) reduction in systolic blood pressure; (ii) increase in endothelium-dependent relaxation; (iii) reduction in oxidative stress and (iv) inhibition of eNOS phosphorylation at its inhibitory residue Thr495.

6.3. On Acute Myocardial Infarction Acute myocardial infarction is the most severe manifestation of coronary artery disease, which causes more than 2.4 million deaths in the USA, more than 4 million deaths in Europe and northern Asia (Nichols et al., 2014). In most cases, myocardial infarction is due to disruption of a vulnerable atherosclerotic plaque or erosion of the coronary artery endothelium (Thygesen et al., 2012; Libby et al., 2013). Acute myocardial infarction remains a leading cause of morbidity and mortality worldwide, despite substantial improvements in prognosis over the past decade (Reed et al., 2017). Notwithstanding, studies have demonstrated the beneficial effects of pomegranate on myocardial infarction. Mohan et al. (2010a) verified that the administration of pomegranate juice has a protective effect against isoproterenol-induced alterations in various cardiac, biochemical and histopathological parameters. In addition, the study showed that


Beneficial Effects of Pomegranate on the Cardiovascular System 113 pomegranate juice attenuates cardiac necrosis (induced by isoproterenol) in rats due to the protection of glutathione, vitamin C content, activity levels of cardiac SOD and catalase. Moreover, these cardiac cells became resistant to changes in Na+-K+, Mg+2 and Ca+2 ATPase. It is likely that the protective effect of pomegranate juice could be due to its ability to inhibit membrane lipid peroxidation and consequent alterations in the activity of various ATPases, and it is known that cardiac necrosis induced by isoproterenol is characterized by reductions in Na+-K+, and Mg+2 ATPase activity levels (Jadeja et al., 2010). The beneficial effects of pomegranate have also been demonstrated in clinical studies. Sumner et al. (2005) show that daily consumption of pomegranate juice for 3 months may decrease myocardial ischemia and improve myocardial perfusion in patients who have ischemic coronary heart disease. It is known that pomegranate juice consumption (240 mL/day) was able to slow down carotid intima–media thickness progression in subjects with coronary heart disease risk, increased oxidative stress, disturbances in the triglyceride-rich lipoprotein/HDL axis, low HDL cholesterol, increased blood pressure, or current cigarette smoking (Davidson et al., 2009). Pomegranate concentrate juice can also reduce LDL levels in diabetic patients presenting with hyperlipidemia (Esmaillzadeh et al., 2004).

6.4. On Diabetes The term diabetes mellitus describes a metabolic disorder of multiple etiology characterized by chronic hyperglycemia with disturbances in carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both (Alberti et al., 1998). The International Diabetes Federation (IDF) has estimated that the numbers of adults with diabetes should increase from 415 million in 2015 to 642 million by 2040. In addition, global spending to treat diabetes and its complications was estimated to be $673 billion in 2015 and is projected to increase to $802


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billion by 2040. Thus, diabetes is a growing clinical and public health problem. Hyperglycemia promotes vascular complications in most patients, mainly represented by atherosclerotic disease (macrovascular complications) and its sequelae (Paneni et al., 2013). In addition, diabetes can promote microvascular diseases such as retinopathy and nephropathy, which are a major cause of blindness and renal insufficiency, respectively (Paneni et al., 2013). The metabolic abnormalities caused by diabetes induce vascular dysfunction that predisposes this patient population to atherosclerosis (Beckman and Creager, 2002). Atherosclerosis is the most important complication of diabetes, and diabetes is an important risk factor for CVD. Notwithstanding, oxidative stress, which is defined as an imbalance between reactive oxygen species levels and antioxidant defenses (Wind et al., 2010; Ghio et al., 2012), may have a role in the onset and progression of diabetes and its complications (Martin-Gallan et al., 2003; Ceriello and Motz, 2004; Johansen et al., 2005; Whiteside, 2005). Endothelial dysfunction occurs mainly due to the increase in the concentration of reactive oxygen species. Among the reactive oxygen species, O2•- reacts with NO forming ONOO- and thus reducing the bioavailability of NO (Creager et al., 2003). Importantly, reduced NO bioavailability is a strong predictor of cardiovascular outcomes (Lerman et al., 2005) and markedly increases the risk of myocardial infarction, stroke, amputation, and death (Beckman and Creager, 2002). Overall, antioxidants were suggested as a possible treatment for diabetes (Johansen et al., 2005). However, glucose control remains a major focus in the management of patients with diabetes (Inzucchi et al., 2015). A review by Banihani et al. (2013), indicated that pomegranate extracts and their active components have great medical potential as they may provide an effective and safe treatment for type 2 diabetes and its pathological concerns. Pomegranate polyphenols can mainly improve the effects of hyperglycemia in two ways: (i) by improving fasting serum glucose and (ii) by reducing oxidative stress. In fact, Banihani et al. (2014), demonstrated that 3 hours after pomegranate juice consumption at 1.5 mL/kg by type 2 diabetes patients, fasting serum glucose levels were


Beneficial Effects of Pomegranate on the Cardiovascular System 115 decreased, β-cell function was increased, and insulin resistance was decreased. The hypoglycemic effects of the pomegranate polyphenols are related to the high α-glucosidase inhibitory activity (Li et al., 2005; Kaur et al., 2014) and exhibit interesting antiglucosidase and antiamylase activities in some pomegranate varieties (Bekir et al., 2016). According to Saxena and Vikram (2004), the flowering part of the pomegranate has been recommended as a remedy for diabetes. In fact, an experimental study demonstrated that the consumption of 60 g/kg body weight/day for 15 days of pomegranate seed did not exert a significant hypoglycemic effect in diabetic rats (Jelodar et al., 2007). Nekooeian et al. (2014) observed that treatment with pomegranate seed oil improved insulin secretion without changing fasting blood glucose in Sprague-Dawley rats with type 2 diabetes. In addition, Rosenblat et al. (2006) also observed in a clinical study that pomegranate juice consumption by diabetic patients does not worsen diabetic parameters but rather acts as an antiatherogenic agent. Diabetic rats had significantly higher levels of serum LDL, triglyceride, and total cholesterol (Nekooeian et al., 2014). Rosenblat et al. (2006) demonstrated that pomegranate has an antiatherogenic effect. This antiatherogenicity is manifested by antioxidant properties in serum and monocytes–macrophages. An experimental study showed that the consumption of 40 g/day of concentrated pomegranate juice for 8 weeks improved the lipid profiles of type II diabetic patients with hyperlipidemia (Esmaillzadeh et al., 2004). It is known that the main mechanism involved is by a decrease in oxidative stress through direct neutralization of the formed reactive oxygen species or an increase in the activity of the antioxidant enzymes (paraoxonase-1, SOD, catalase, and glutathione reductase). Alternatively, pomegranate may affect diabetic conditions by inhibiting or activating certain transcription factors (i.e., nuclear factor-κB and peroxisome proliferator–activated receptor γ) (Rosenblat et al., 2006). In addition, pomegranate flower ameliorates diabetes and obesityassociated fatty liver, at least in part, by activating hepatic expression of genes responsible for fatty acid oxidation. These effects were accompanied by enhanced hepatic gene expression of peroxisome proliferator-activated


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receptor (PPAR)-alpha, carnitine palmitoyltransferase-1 and acyl-CoA oxidase (ACO) and reduced stearoyl-CoA desaturase-1 (Xu et al., 2009). Bragri et al. (2009) also demonstrated the antidiabetic effect of pomegranate flowers and its effects on hyperlipidemia, pancreatic cells, lipid peroxidation and antioxidant enzymes in experimental diabetes, and in fact, he verified that the administration of an aqueous extract at doses of 250 mg/kg and 500 mg/kg for 21 days resulted in a significant reduction in fasting blood glucose, atherogenic index (total cholesterol, triglycerides, LDL, VLDL) and tissue lipid peroxidation levels coupled with elevation of HDL, glutathione-S-transferase content and antioxidant enzymes. It is important to note that the consumption of pomegranate can also improve subclinical inflammation in type 2 diabetes. The intervention of 50 g of concentrated pomegranate juice per day appeared to have favorable effects on IL-6 and increased the plasma concentrations of antioxidants in diabetic patients (Shishehbor et al., 2016). On the other hand, Faghihimani et al. (2016) observed that the consumption of 2000 mg pomegranate seed oil per day for 8 weeks had no effect on fasting blood sugar, insulin resistance and lipid profiles in diabetic patients. Although the pomegranate has paradoxical effects with regard to its antidiabetic activity, it is important to emphasize that the consumption of the varieties of pomegranate may provide an effective management strategy in diabetic patients by reducing the risk of diabetic complications.

6.5. On Stroke Stroke is classically characterized as a neurological deficit attributed to an acute focal injury of the central nervous system by a vascular cause, including cerebral infarction, intracerebral hemorrhage, and subarachnoid hemorrhage, and is a major cause of disability and death worldwide (Sacco et al., 2013). An ischemic stroke is a cerebrovascular event that reduces or blocks the flow of blood (and thus, oxygen and nutrients as well) to the brain, often resulting in temporary and/or permanent cellular damage. This damage occurs via many pathological processes: oxidative stress (El Kossi


Beneficial Effects of Pomegranate on the Cardiovascular System 117 and Zakhary, 2000), inflammation (Huang et al., 2006), excitotoxicity (Castillo, et al., 1997), and apoptosis (Du et al., 1996). Approximately 800,000 Americans experience a new or recurrent stroke each year (Go et al., 2014). The Trial of Org 10172 in Acute Stroke Treatment (TOAST) created a classification system that includes five categories: 1) large-artery atherosclerosis, 2) cardioembolism, 3) small-artery occlusion, 4) stroke of other determined etiology, and 5) stroke of undetermined etiology. (Adams Jr et al., 1993). Intravenous recombinant tissue plasminogen activator (tPA) is the standard treatment for acute ischemic stroke, but more than half the treated patients do not recover completely or die (Ciccone et al., 2013). Due to the high mortality rate in patients diagnosed with stroke, dietary supplementation with polyphenol-rich foods and beverages has received much attention. It is known that pomegranate has large amounts of polyphenols compared with other foods. Their high phenol content has made them a target for studies investigating the promotion of health. Recent studies have shown that pomegranate components can be used as an alternative treatment in stroke. It was observed that supplementation with pomegranate polyphenol-enriched extract in the drinking water of pregnant mice resulted in a significant decrease in hypoxia-ischemiainduced caspase-3 activation, which suggests that the polyphenols in the pomegranate juice are responsible for neuroprotection in the neonatal rodent brain against hypoxia-ischemia brain injury (Loren et al., 2005; West et al., 2007). Another study demonstrated that the preadministration of 250 mg/kg or 500 mg/kg pomegranate extract to rats can offer neuroprotective activity against cerebral ischemia/reperfusion brain injury via antioxidants due to the reduction in oxidative stress in the brain and enhancement in brain SOD, glutathione peroxidase and glutathione reductase activity. These effects are related to (i) anti-inflammatory activity, due to the reduction in brain TNF-α, NF-κB p65 and increase in brain interleukin (IL)-10 levels, and (ii) antiapoptotic activity, due to the reduction in brain caspase-3 and ATP-replenishing effects (Ahmed et al., 2014). Finally, the pomegranate seed extract (100, 200, 400 and 800 mg/2 mL/kg) exhibits therapeutic potential in ovariectomized rats with


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permanent cerebral hypoperfusion/ischemia, which is most likely related, at least in part, to its phytoestrogenic and antioxidative actions (Sarkaki et al., 2015). Therefore, studies demonstrate that the pomegranate components are a potential attraction in stroke treatment due to their neuroprotective actions. In addition, clinical studies are required for assessing the efficacy of pomegranate polyphenols in a clinical stroke population. Taken together, the beneficial pomegranate effects can protect against the development and/or progression of CVD and have important implications for their prevention. Thus, the characterization of pomegranate constituents may provide a new approach to the development of new forms of therapeutic intervention in the treatment of CVD.

7. NEW PERSPECTIVES ON THE TREATMENT OF CARDIOVASCULAR DISEASES WITH POMEGRANATE CVD have the highest mortality rate in populations around the world, and they mainly affect populations from developed countries. Because of this, CVD are considered so harmful, and knowledge about prevention and/or treatment forms are of great importance for health systems. It is important to notice that CVD kills more than the sum of all types of cancers. Currently, various medications are available for the treatment of CVD. However, its prevention remains the main objective for the reduction in statistics on cardiac diseases. For prevention, the goal is to stimulate changes in factors that are modifiable, such as lifestyle, physical activity, and nutrition. Within this perspective, the consumption of fruits and vegetables with potential bioactive effects (mainly antioxidant action) has become an ally in the prevention of CVD. The pomegranate is distinguished by its high amount of phenolic compounds with high antioxidant capacity. Therefore, its use is not restricted only to the prevention of CVD, but it can also be applied in


Beneficial Effects of Pomegranate on the Cardiovascular System 119 parallel with conventional treatments. Although pomegranate research has resulted in positive effects of the pomegranate, more specific clinical studies are needed to better understand the pivotal mechanism involved by which the pomegranate promotes its benefits. In addition, the characterization of pomegranate polyphenols may provide a new approach to the development of new forms of therapeutic intervention in the treatment of hypertension associated with hormonal dysfunction.

8. RECENT DISCOVERIES As already discussed, pomegranate is employed in folk medicine throughout humanity. However, due to the improvement of methodologies and techniques, the scientific community is increasingly able to further explore what was observed in popular culture. Currently, studies have shown the application of pomegranate in the most diverse types of diseases, such as diabetes, cancer, rheumatic diseases, inflammatory disorders and CVD. In addition to demonstrating the benefits of pomegranate, studies are even more capable of clarifying the molecular mechanisms involved in those benefits. Recently, studies have suggested that the intake of pomegranate can modulate gene expression in a tissue-specific manner (Nuñez-Sánchez et al., 2017). Indeed, advances in the field of oncology medicine have also raised interest in the benefits of using pomegranate. Cancer prevention via dietary agents is a promising field in oncology that has demanded great attention from scientists due to its ability to prevent or suppress cancers, the low cost, and easy availability. Thus, preclinical and clinical studies emphasize the role of the pomegranate in the prevention and treatment of skin, breast, prostate, lung, and colon cancers (Sharma et al., 2017). We emphasize that currently, diseases do not show up in an isolated form; rather, they are associated with other pathologies. Thus, the challenges are increasing in order to find the mechanisms by which the substances present in the pomegranate act in the prevention and treatment of the health-disease process.


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9. HIGHLIGHTS 

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Pomegranate in its varied presentations (juice, extract, whole fruit, seeds, leaf husks and roots) on the cardiovascular system, lipid profile, oxidative stress, atherosclerosis, and diabetes could represent prophylaxis and a new kind of parallel therapy for patients at risk and even for those currently affected by diseases. Identification of the mechanisms and intracellular pathways involved in the effects of pomegranate on CVD may contribute to the development of better forms of therapy. New information about the ingested concentration of pomegranate necessary to provide the fruit benefits will help physicians and patients in the treatment of CVD. Identification of new forms for the use of pomegranate may contribute to the protection of important organ systems, such as the cardiovascular, nervous, and renal systems. Clarifying the pomegranate responses in the cardiovascular system could provide new information that may contribute to the development of new drugs that can be used in CVD treatment aimed at enhancing the beneficial actions of its components. It is better to understand the effects of the main compounds that are present in pomegranate (e.g., tannins, flavonoids, phytoestrogens, anthocyanins, alkaloids) for a better treatment strategy associated with convective treatments of CVD. The characterization of pomegranate polyphenols may provide a new approach to the development of new forms of therapeutic intervention in the treatment of hypertension associated with hormonal dysfunction.

ACKNOWLEDGMENT We would like to thank Fabrício Bragança da Silva for the image kindly provided for this chapter.


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Steg PG, Wijns W, Bassand JP, Menasche P, Ravkilde J; Trials & Registries Subcommittee, Ohman EM, Antman EM, Wallentin LC, Armstrong PW, Simoons ML; Trials & Registries Subcommittee, Januzzi JL, Nieminen MS, Gheorghiade M, Filippatos G; Trials & Registries Subcommittee, Luepker RV, Fortmann SP, Rosamond WD, Levy D, Wood D; Trials & Registries Subcommittee, Smith SC, Hu D, Lopez-Sendon JL, Robertson RM, Weaver D, Tendera M, Bove AA, Parkhomenko AN, Vasilieva EJ, Mendis S; ESC Committee for Practice Guidelines (CPG), Bax JJ, Baumgartner H, Ceconi C, Dean V, Deaton C, Fagard R, Funck-Brentano C, Hasdai D, Hoes A, Kirchhof P, Knuuti J, Kolh P, McDonagh T, Moulin C, Popescu BA, Reiner Z, Sechtem U, Sirnes PA, Tendera M, Torbicki A, Vahanian A, Windecker S; Document Reviewers, Morais J, Aguiar C, Almahmeed W, Arnar DO, Barili F, Bloch KD, Bolger AF, Botker HE, Bozkurt B, Bugiardini R, Cannon C, de Lemos J, Eberli FR, Escobar E, Hlatky M, James S, Kern KB, Moliterno DJ, Mueller C, Neskovic AN, Pieske BM, Schulman SP, Storey RF, Taubert KA, Vranckx P, Wagner DR. Third universal definition of myocardial infarction. J Am Coll Cardiol. 2012 Oct 16;60(16):1581-98. doi: 10.1016/j.jacc.2012.08.001. Toklu HZ, Dumlu MU, Sehirli O, Ercan F, Gedik N, Gökmen V, Sener G. Pomegranate peel extract prevents liver fibrosis in biliary-obstructed rats. J Pharm Pharmacol. 2007 Sep;59(9):1287-95. doi: 0.1211/jpp. 59.9.0014. Tomé-Carneiro J, Visioli F. Polyphenol-based nutraceuticals for the prevention and treatment of cardiovascular disease: Review of human evidence. Phytomedicine. 2016 Oct 15;23(11):1145-74. doi: 10.1016/ j.phymed.2015.10.018. Tsuyuki H, Ito S, Nakatsukasa Y. Studies on the lipids in pomegranate seeds. Bull College Agric Vet Med Nipon Univ Japan. 1981;38:141-48. Van Den Ende W, Peshev D, De Gara L. Disease prevention by natural antioxidants and prebiotics acting as ROS scavengers in the gastrointestinal tract. Trends Food Sci & Technol 2011 Dec;22(12): 689-97. doi: 10.1016/j.tifs.2011.07.005.


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Chapter 6

POMEGRANATE: HEALTH BENEFITS ON BRAIN FUNCTIONS Saida Haider, PhD1, , Zehra Batool, PhD2 and Saiqa Tabassum, PhD1 1

Neurochemistry and Biochemical Neuropharmacology Research Unit, Department of Biochemistry, University of Karachi, Karachi, Pakistan 2 Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan

ABSTRACT The use of plant products and their extracts have shown tremendous potential to prevent or treat various pathophysiological conditions including neurodegenerative diseases. Plants are known to synthesize secondary metabolites which provide a defense mechanism in plants. These secondary metabolites including polyphenols have also shown protective effects against various diseases in animal and human studies through their anti-inflammatory and anti-oxidant effects. Pomegranate, a 

Corresponding Author Email:[email protected]


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S. Haider, Z. Batool and S. Tabassum nutritionally rich fruit, is known since centuries for its medicinal power and therapeutic qualities. In this chapter health benefits of pomegranate are presented, specifically in the context of its neurological potential to improve brain functioning and to ameliorate neurological disorders. The most potent pharmacological agents of pomegranate are punicalgins (ellagitannins) and punicic acid that are metabolized into ellagic acid and urolithins, respectively. These metabolites are responsible for their benevolent health effects. These bioactive constituents have anti-oxidant and anti-inflammatory properties and may act synergistically for numerous pharmaceutical properties. The major focus of this chapter is to elaborate the neurological effects of pomegranate as a neuroprotective agent and to further highlight its tremendous power in preventing, ameliorating, and delaying neurological disorders. Based on the facts outlined in this chapter, it is recommended to consume pomegranate fruit on a regular basis as a low cost, safer preventive strategy to maintain a healthy lifestyle.

Keywords: Alzheimer’s disease, Parkinson’s disease, anti-oxidant, ellagic acid, neuroprotection, punicalagins

INTRODUCTION The pomegranate, Punica granatum is an extraordinary and mystically unique fruit with a high nutritional value. This oldest known edible and highly distinctive fruit along with its juice, peel, and seeds has a unique medicinal power and therapeutic qualities (Jurenka, 2008; Dipak et al. 2012). In his chapter pomegranate is explained and defined comprehensively as a potent neuroprotective agent that has a tremendous therapeutic potential in improving brain function and treating various neurological disorders. Before presenting its neurological potential, readers are provided with additional information regarding its botanical and geographical profile, nutritional value, chemical composition, and metabolic fate so that its therapeutic potential can be clearly understood. Then evidence-based current information is presented regarding tremendous applications of pomegranate in various neurological conditions.


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Botanical Profile The pomegranate belongs to the Kingdom: Plantae (Angiosperms), Order: Myrtles, Family: Lythraceae (previously Puniacaceae), Genus: Punica, Species: Punica granatum and Punica protopunica (Fateh et al. 2013; Richard and Paul, 2016). The pomegranate, Punica granatum L. is the predominant member of two species and commonly named as ‘Anar’ (Zarfeshany et al. 2014). More than 500 cultivars of Punica granatum exist with specific characteristics such as fruit size, exocarp, and aril color (Hollebeeck et al. 2012).

Traditional Uses Punica granatum is the predominant specie of genus Punica family which is cultivated worldwide whereas Punica protopunica is only restricted to the Island of Socotra (Republic of Yemen) (Fateh et al. 2013). Pomegranates have been praised and appreciated since ancient times, owing to its beautiful, color and flavor, and extraordinary health benefits (Akbar et al. 2015). It has been known as a natural and holistic medicine for centuries and being used in the folk medicine of many cultures for treating a wide variety of diseases like sore throats, coughs, urinary infections, digestive disorders, skin disorders, arthritis, and to expel tapeworms (Kumari et al. 2012). Reports suggest that each part of pomegranate plant has tremendous therapeutic benefits in ethnomedicine such as the bark and roots are considered as anti-helminthic medicine, the fruit peel/rind has been used as a cure against oral aphthae, diarrhea, dysentery, and intestinal parasites, and believed to be a powerful astringent, the flowers are used as a traditional remedy for diabetes mellitus, while the juice and seeds are regarded as a tonic for blood, heart, throat, eyes, skin, and for various other purposes including to stop nose and gum bleeds, to firm-up sagging breasts, and to treat hemorrhoids (Lansky and Newman, 2007; Hollebeeck et al. 2012; Bhowmik et al. 2013).


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Historically, pomegranate has been used all over the world. In Greece, pomegranate juice was prescribed as a remedy for inflammation, intestinal worms, persistent coughs, diarrhea, and dysentery (Kumari et al. 2012). The Babylonians regarded pomegranate seeds as an agent of resurrection (Aviram, 2000). Persians believed that the seeds conferred strength and invincibility on the battlefield (Aviram, 2000). People of the Georgian Republic in Russia used pomegranate for arresting chronic mucous discharges, passive hemorrhages, night sweats, and diarrhea (Kumari et al. 2012). In Turkey (South Anatolia) the ashes of fruit peel are used to protect against skin infections (Dipak et al. 2012). In addition to its ancient historical uses, pomegranate is used in several systems of medicine for a variety of ailments. For examples: in the Unani system of medicine, pomegranate is served as a remedy for diabetes (Saxena and Vikram, 2004; Jurenka, 2008). In Iranian traditional medicine, pomegranate flowers are used as an astringent, hemostatic, antibacterial, antifungal, antiviral agent; as a remedy to wound healing, bronchitis, diarrhea, digestive problems, and diabetes; to enhance men sex power, and to treat dermal infected wounds (Dipak et al. 2012). In Chinese medicine, the seeds are revered for their powers to promote longevity and immortality, and the flowers are used for the treatment of injuries from falls and for reducing grey hair of young men (Lansky and Newman, 2007; Dipak et al. 2012). In Ayurvedic medicine, the pomegranate is considered “a pharmacy unto itself” and is used as an anti-parasitic agent, a “blood tonic,” and to heal aphthae, diarrhea, and ulcers (Jurenka, 2008; Pirbalouti et al. 2010; Dipak et al. 2012). More recently, the beneficial effect of pomegranate is realized in the modern medicine and new products are being developed using modern technology to improve its beneficial effects.

Medicinal Products of Pomegranate The pomegranate juice is one of its main products; however, regarding its greatest medicinal value all parts of the pomegranate fruit, arils, seeds and peel, including its leaves, husk, bark, and roots are now used



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(Medjakovic and Jungbauer, 2013; Jinu et al. 2016) and processed into various commercial products by food and beverage industries. These products include bottled juice (fresh or concentrated); powdered capsules and tablets which are derived from seeds, fermented juice, peel, leaf and flower; gelatin capsules of seed oil extracts; dry or beverage tea from leaves or seeds; powdered extracts and extracts based gels and ointments (Zarfeshany et al. 2014). A tremendous importance has been given worldwide to pomegranate peel and its extracts in food based preparations to be used as an anti-oxidant, protein and lipid stabilizer, antimicrobial, color and texture enhancer, and above all, as a nutraceutical and functional food component (Iqbal et al. 2008; Naveena et al. 2008; Altunkaya et al. 2013; Singh and Immanuel, 2014; Ismail et al. 2014; 2016). Different parts of pomegranate fruit are represented in Figure 1.

Figure 1. A semi-schematic drawing of a pomegranate fruit. (Adapted from Medjakovic and Jungbauer, 2013).

Nutritional Composition Pomegranates have an impressive nutrient profile containing almost all macro (carbohydrates, proteins, lipids) and micro (vitamins, minerals) nutrients. The edible seeds of pomegranate are an important source of dietary fiber (20%) and of powerful ingredients like punicic acid (65.3%), palmic acid (4.8%), stearic acid (2.3%), oleic acid (6.3%), and linoleic acid (6.6%). The nutritional benefits offered by the seed fiber and micronutrients are wasted by consumers who opt to discard the seeds (Schubert et al. 1999; Keservani et al. 2016).


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Table 1. Nutrition value of Punica granatum (per 100 g of Edible Portion) (Stowe, 2011; Zarfeshany et al. 2014) Nutrients Moisture (Water) Caloric Value (Energy) Macronutrients Carbohydrates Sugars Dietary Fiber Ash Protein Fat (Total lipids) Micronutrients Vitamins Vitamin C (Ascorbic acid) Vitamin E (Tocopherol) Vitamin K (Quinone) Vitamin B1 (Thiamine) Vitamin B2 (Riboflavin) Vitamin B3 (Niacin) Vitamin B5 (Pantothenic Acid) Vitamin B6 (Pyridoxine) Vitamin B9 (Folic Acid) Choline Minerals Phosphorus Iron Potassium Calcium Sodium Manganese Zinc Magnesium Copper Selenium Others Carotene Citric Acid Boric Acid

Units g kcal

Value per 100g 72.6-86.4 (78%) 65-85

g g g g g g

15.4-19.6 (14.5%) 13.67 (1.4%) 3.4-5.0 (5.1%) 0.36-0.73 0.5-1.67 (1.6%) 0.9-1.17 (0.1%)

mg mg mg mg mg mg mg mg µg mg

10-20 0.350 0.1 0.038 0.012-0.03 0.18-0.3 0.596 0.075 2.166 4.3-7.6

mg mg mg mg mg mg mg mg mg mg

8-37 0.3-1.2 236-259 3-12 3.0 3.0 0.12 12 0.07 0.6

mg mg mg

0.001 0.46-0.36 0.005



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A critical study of the nutritional values per serving of pomegranate showed that there is no specific tropical fruit or vegetable that could be compared to it (Richard and Paul, 2016). The nutritional profile of pomegranate per 100 g of edible portion is given in Table 1 (Stowe, 2011; Zarfeshany et al. 2014; Dipak et al. 2012; Richard and Paul, 2016).

Chemical Composition Pomegranate is rich in various types of valuable constituents in its different parts such as fruits, leaves, roots, seeds, peels, and arils. These ingredients obtained from various parts of the plant show pharmacological and therapeutic role in the disease management and cure via modulating certain biological processes (Rahmani et al. 2017). Pomegranate whole fruit is comprised of peel (50%), seeds (10%), and arils (40%) (Shastry et al. 2017). Research showed that pomegranate fruit, peel, and seeds are biochemically composed of more than 124 different phytochemicals including flavonoids, proanthocyanidins, ellagitannins and polyphenolic compounds (Shastry et al. 2017), anthocyanins, catechins, tannins, organic acids, alkaloids (Lansky and Newman, 2007; Wanget al. 2010; Medjakovic and Jungbauer, 2013), gallic acid, chlorogenic acid, caffeic acid, ferulic acid, vitamins B6, vitamin C, minerals, and fibers (Akbar et al. 2015). A brief overview of phyto-constituents obtained from different parts of pomegranate plant is summarized in Table 2.

Bioactive Phytochemicals Although pomegranate contains a variety of beneficial phytochemicals but the unique substances in pomegranates that are responsible for their pharmacological profile and therapeutic role are punicalgins (ellagitannins) and punicic acid (Richard and Paul, 2016) (see Figure 2).


Table 2. Phytochemical composition of pomegranate (Punica granatum) Compound Class Diverse Polyphenols

Compounds

Flavonoids

Flavones (Luteolin, Luteolin 7-O-Glucoside), Flavanones (Naringenin 7-O-Rutinoside, Epigenin), Flavanols (Catechin, Epicatechin And Epigallocatechin 3-Gallate) And Flavonols (Quercetin, Rutin And Kaempferol, Kaempferol-3-O-Glycoside, Kaempferol-3O-Rhamnoglycoside, Dihydrokaempferol-Hexoside), Flavan-3-Ols (Epicatechin And Epigallocatechin), Delphinidin, Cyanidin, Pelarogonidin, Phloridzin, Genistein, Diadzein, Genistin, And Diadzin, Anthocyanidins

Catechin, Ellagic Acid, Epicatechin, Gallocatechin, Epigallocatechin, Gallagic Acid, Ellagic Acid Glycosides, Catechin, Epicatechin, Epigallocatechin-3Gallate, Hydroxybenzoic Acids, Hydroxycinnamic Acids

Consumable Product Pomegranate pericarp, peel

Pharmacological Potential Anti-carcinogenic effect, anti-oxidant properties

Pomegranate pericarp, leaves, peel, arils

Anti-oxidant activity, anti-inflammatory activity, cancer-preventive effects

References Medjakovic and Jungbauer, 2013; Dipak et al. 2012; Zarfeshany et al. 2014; Jurenka, 2008; Fateh et al. 2013; Afaq et al. 2009; Hollebeeck et al. 2012; Amakura et al. 2000; Fischer et al. 2011; Lansky and Newman, 2007; Rahmani et al. 2017 Lansky and Newman, 2007; Kumari et al. 2012; Zarfeshany et al. 2014; Jurenka, 2008; Fateh et al. 2013; Afaq et al. 2009; Bhowmik et al. 2013; Hollebeeck et al. 2012; de Pascual-Teresa et al. 2000; Fischer et al. 2011; Medjakovic and Jungbauer, 2013; Poyrazoğlu et al. 2000; Yang and Wang, 2010


Compound Class Complete and Hydrolysable Tannins

Compounds

Anthocyanins

Pelargonidin 3 O-glucoside, Pelargonidin 3,5 O-diglucoside, Cyanidin 3 O-glucoside, Cyanidin 3,5 O-diglucoside, Delphinidin 3 O-glucoside, Delphinidin 3,5 Odiglucoside, cyanidin-pentoside-hexoside, cyanidin 3-rutinoside, cyanidin-3pentoside, cyanidin-3-hexoside

Ellagitannins (total ellagitannins, ellagic acid), Gallotannins (1,2,4,6-tetra-OgalloylD-glucose and 1,2,3,4,6-penta-Ogalloyl-D-glucose), Brevifolin, Brevifolin carboxylic acid, Corilagin, Castalagin, Casuarinin, Gallagyldilactone, Granatins, Granatin-A, Granatin-B, Lagerstannin-B, Pedunculagin, Pomegranatate, Punicafolin, Punicalin, Punicalagins, Punicalagin- A, Punicalagin-B, Tellimagranandin I

Consumable Product All parts of pomegranate plant (seeds, arils, fruit peels, leaves, flowers, tree bark, and roots)

Pharmacological Potential Anti-oxidant activity used in plastic surgeries, to prevent skin flap’s death, preservative activities

Pomegranate bark (stem), roots, peel and juice

Anti-oxidant potential, anti-inflammatory role, preservative activities

References Medjakovic and Jungbauer, 2013; Lansky and Newman, 2007; Gil et al. 2000; Li et al. 2006; Schubert et al. 1999; Seeram et al. 2005a; Tzulker et al. 2007; Hollebeeck et al. 2012; Jurenka, 2008; Fateh et al. 2013; Afaq et al. 2009; Zarfeshany et al. 2014; Kumari et al. 2012; Amakura et al. 2000; Fischer et al. 2011; Shastry et al. 2017; Borges et al. 2010; Martin et al. 2009; Rahmani et al. 2017; Mena et al. 2013; Singh et al. 2002; Keservani et al. 2016; Cerdá et al. 2006 Zarfeshany et al. 2014; Lansky and Newman, 2007; Hollebeeck et al. 2012; Fischer et al. 2011; Medjakovic and Jungbauer, 2013; Hernandez et al. 1999; Gil et al. 1995; Borochov-Neori et al. 2011; Alighourchi et al. 2008


Table 2. (Continued) Compound Class Volatile Compounds

Organic and Phenolic Acids

Compounds E-a-Bergamotene, E-b-Bergamotene, Bisabolene, Cadrene, Camphor, 3-Carene, b-Caryophyllene, p-Cumic aldehyde, pCymene, (Z,Z)-a-Farnesene, b-Farnesene, Fenchone, Furfural, Hexanal, Hexanol, cis-3-Hexenal, trans-2-Hexenal, cis-3Hexenol, Heptanal, Limonene, Menthol, Monoterpens (b-Myrcene, Nonanal, Octanal, a-Phellandrene, a-Pinene, bPinene, a-Terpinene, g-Terpinene, 4Terpineol, a-Terpineol, and limonene), aldehydes, esters, and alcohols Caffeic acid, Protocatechuic acid, Chlorogenic acid, Ctric acid, o-Coumaric acid, p-Coumaric acid, Ferulic acid, Gallic acid, Gallagic acid, Malic acid, Oxalic acid, (-)-Quinic acid, Sucinic acid, Tartaric acid, Ascorbic acid, Fumaric acid, Succinic acid, Malic acid, Ellagic acid

Consumable Product Pomegranate juice

Pomegranate bark (stem), roots, peel, juice

Pharmacological Potential

References Medjakovic and Jungbauer, 2013; Calin-Sanchez et al. 2011; VázquezAraújo et al. 2010; Melgarejo et al. 2011

Ismail et al. 2016; Akhtar et al. 2015; Bhowmik et al. 2013; Zarfeshany et al. 2014; Lansky and Newman, 2007; Kumari et al. 2012; Hollebeeck et al. 2012; Amakura et al. 2000; Fischer et al. 2011; Medjakovic and Jungbauer, 2013; Rahmani et al. 2017; Tezcan et al. 2009


Compound Class Fatty Acids

Lignans Sterols

Compounds Caproic acid (C6 : 0), Caprylic acid (C8 : 0), Capric acid (C10 : 0), Lauric acid (C12 : 0), Myristic acid (C14 : 0), Myristoleic acid (C14 : 1), Palmitic acid (C16 : 0), Palmitoleic acid (C16 : 1), Stearic acid (C18 : 0), Oleic acid (C18 : 1), Linoleic acid (C18 : 2), Punicic acid (C18 : 3, 9cis, 11-trans, 13-cis), a-Eleostearic acid (C18 : 3, 9-cis, 11trans, 13-trans), b-Eleostearic acid (C18 : 3, 9-trans, 11-trans, 13-trans), Catalpic acid (C18 : 3, 9-trans, 11-trans, 13-cis), Arachidic acid (C20 : 0), Gadoleic acid (C20 : 1), Lignoceric acid (C24 : 0), Nervonic acid (C24 : 1, 15-cis), Linolenic acid (18 : 3: 9-cis, 11-trans, 13-cis) Isolariciresinol, Matairesinol, Medioresinol, Pinoresinol, Secoisolariciresinol, Syringaresinol Estrogenic flavonols and flavones, β-sitosterol, campesterol, daucosterol, and stigmasterol

Estrogens

Cholesterol, Coumestrol Estrone, Estrone, Testosterone, Estriol

17-Alpha-Estradiol,

Triterpenoids

Ursolic acid, Maslinic acid, Asiatic acid, Oleanolic acid

Consumable Product Pomegranate seeds, seeds’oil

Pomegranate seeds Pomegranate seeds, seeds’ oil Pomegranate seeds Pomegranate Flowers and seeds


References Medjakovic and Jungbauer, 2013; Elfalleh et al. 2011; Pande and Akoh, 2009; Kaufman and Wiesman, 2007; Fadavi et al. 2006

Medjakovic and Jungbauer, 2013; Bonzanini et al. 2009 Dipak et al. 2012; Medjakovic and Jungbauer, 2013; Kaufman and Wiesman, 2007 Shastry et al. 2017; Zarfeshany et al. 2014; Lansky and Newman, 2007 Shastry et al. 2017


Table 2. (Continued) Compound Class Alkaloids

Sugars Amino acids Vitamins

Compounds Isopelletierine, Pseudopelletierine, NMethylisopelletierine, Piperidines, Pyrrolidines Glucose, Fructose, Sucrose, proline, methionine, and valine Vitamin C (Ascorbic acid), Vitamin E (αtocopherol) and Lipoic Acid

Consumable Product Pomegranate roots and bark pomegranate juice pomegranate juice Pomegranate seeds and juice

Minerals

Iron, calcium, magnesium, nitrogen, phosphorus, potassium and sodium iron, copper, sodium, magnesium and zinc

Pomegranate juice, peel, seeds

Others

β-Carotene

Pomegranate peel and juice


References Zarfeshany et al. 2014; Lansky and Newman, 2007; Kumari et al. 2012 Rahmani et al. 2017; Melgarejo et al. 2000 Rahmani et al. 2017 Medjakovic and Jungbauer, 2013; Bonzanini et al. 2009; Rahmani et al. 2017; Vroegrijk et al. 2011 Rahmani et al. 2017; Mirdehghan and Rahemi 2007; Shastry et al. 2017; Fateh et al. 2013, Medjakovic and Jungbauer, 2013; Bonzanini et al. 2009; Fateh et al. 2013 Fateh et al. 2013


Pomegranate and Brain Functions 



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Ellagitannins (punicalagin): Ellagitannins are the phenolic compounds which are basically the esters of ellagic acid formed by its conjugation with a glycoside moiety (Clifford and Scalbert, 2000; Hollebeeck et al. 2012). The most potent ellagitannins in the pomegranate are punicalagins which are found in two forms; alpha and beta punicalagins (see Figure 2). These are water-soluble and have high bioavailability. They are present in juice and peel of a pomegranate and have rich anti-oxidant potential which is three times greater than that of red wine and green tea. In vivo, punicalagins are hydrolyzed across the mitochondrial membrane into smaller phenolic compounds like ellagic acid which acts as a highly active carbonic anhydrase inhibitor (Medjakovic and Jungbauer, 2013; Richard and Paul, 2016). Punicic acid: Punicic acid (trichosanic acid) is the main polyunsaturated fatty acid (18:3; n-5) fatty acid found in pomegranate seed oil and arils. It is slowly absorbed in the intestine, but after that it is quickly converted to conjugated linoleic acid (having three conjugated double bonds). Reports showed that it has potent biological effects such as anticancer activity against prostate cancer cells, and helps in controlling obesity (Richard and Paul, 2016).

a) Punicalagins

b) Punicic Acid

Figure 2. Chemical structures of bioactive compounds of pomegranate (a) Punicalagins (Yuan et al. 2015) (b) Punicic acid (Zarfeshany et al. 2014).


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Saida Haider, Zehra Batool and Saiqa Tabassum

Bioavailability and Metabolic Fate of Pomegranate Ellagitannins Although pomegranate contains several compounds that may contribute to the observed benevolent health effects, the ellagitannins such as punicalagin appear to be the most promising candidates as a highly bioactive compound (Medjakovic and Jungbauer, 2013). Punicalagins are hydrolyzed to ellagic acid in the acidic stomach environment (GilIzquierdo et al. 2002; Hollebeeck et al. 2012). After pomegranate consumption, ellagic acid is released and cleared from the serum within 6 h independent of the form of administration (Seeram et al. 2008; Medjakovic and Jungbauer, 2013). Release of ellagic acid occurs via hydrolysis of ellagitannins which is not due to an enzymatic reaction but is pHdependent (Daniel et al. 1991; Larrosa et al. 2006a; Hollebeeck et al. 2012; Medjakovic and Jungbauer, 2013). Ellagic acid is quickly absorbed in the first portion of the gastrointestinal tract, then enters in the intestinal cells by passive diffusion, and binds irreversibly to deoxyribonucleic acid (DNA) and proteins (Whitley et al. 2003; Hollebeeck et al. 2012). Ellagic acid is subsequently (by successive losses of hydroxyl groups) biotransformed and metabolized in the intestine within 4–6 h (Seeram et al. 2006; Seeram et al. 2004; Medjakovic and Jungbauer, 2013) by gut microbiota that is already active in the jejunum and much more concentrated in the colon, to yield urolithins (6H-dibenzo[b,d]pyran-6-one derivatives) (Yuan et al. 2015), including urolithin D (3,4,8,9tetrahydroxy-6H-dibenzo[b,d]pyran-6-one), urolithin C (trihydroxy-6Hdibenzo[b,d]pyran-6-one), urolithin A and urolithin B (3-hydroxy-6Hdibenzo[b,d]pyran-6-one glucuronide) (Espín et al. 2007; Bialonska et al. 2010; Hollebeeck et al. 2012; Medjakovic and Jungbauer, 2013; Ismail et al. 2016) (see Figure 3). After absorption, the first glucuronidation takes place in the intestinal cells and then in the liver. Additional metabolism to diglucuronides and/or sulphates occurs by phase-2 enzyme conjugates [methylated (i.e., conversion of hydroxyl to methoxyl or methyl ether), sulfated, and glucuronidated forms] (Larrosa et al. 2006a; Espín et al. 2007; Hollebeeck et al. 2012; Yuan et al. 2015) and some of these metabolites also appear in



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bile (Hollebeeck et al. 2012). Urolithin A-glucuronide (main metabolite of ellagitannins) (González-Sarrías et al. 2010; Medjakovic and Jungbauer, 2013), urolithin B-glucuronide (Cerdá et al. 2004; Medjakovic and Jungbauer, 2013; Cerda et al. 2006) and dimethylellagic acid glucuronide (González-Sarrías et al. 2010; Medjakovic and Jungbauer, 2013) are the major metabolites seen in serum and urine while urolithin C (minor metabolite) (Cerdá et al. 2004; Medjakovic and Jungbauer, 2013) and urolithin D absorbed earlier in the intestine, then enters the enterohepatic circulation until their phenolic hydroxyl groups are decreased and the metabolites are able to enter serum and urine (Medjakovic and Jungbauer, 2013). The absorption of urolithins increases as their lipophilicity increases (Espín et al. 2007; Hollebeeck et al. 2012) as shown in Figure 4.

Figure 3. The catabolism of ellagitannin and punicalagin to form ellagic acid and then urolithins (Medjakovic and Jungbauer, 2013).

Figure 4. Intestinal absorption of urolithins increased by increasing lipophilicity (Hollebeeck et al. 2012).


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Non-Toxicity of Pomegranate Products Given that pomegranates have been consumed for several millennia, the normal consumption of the fruit, raw or as an ingredient, can be regarded as absolutely safe. Studies of pomegranate constituents in animals at concentrations and levels commonly used in folk and traditional medicine have shown no toxic effects (Cerdá et al. 2003; Medjakovic and Jungbauer, 2013). However, the question arises whether this safety extends to extracts or pure compounds that may be used in a more concentrated form as dietary supplements. It is suggested that pomegranate extracts appear to be safe in concentrations that have been used in traditional ethnomedicine and that are currently available as dietary supplements (Medjakovic and Jungbauer, 2013). Many studies have been carried out on the different components derived from pomegranate; however, no adverse effects have been reported (Vidal et al. 2003; Jurenka, 2008; Meerts et al. 2009; Medjakovic and Jungbauer, 2013; Zarfeshany et al. 2014) and no signs of toxicity, no mortality or treatment-related clinical signs, no histopathological effects (Azorín-Ortuño et al. 2008; Medjakovic and Jungbauer, 2013) and no toxic effect on blood chemistry analysis for kidney, liver, and heart function (Aviram et al. 2004; Jurenka, 2008) were observed. Pomegranate and the peel extracts, like other herbal extracts might have toxicological risks for its consumers that merit for systematic safety evaluation of the plant material (Ismail et al. 2016). Recently, only a single study has been referenced for the adverse genotoxic effects associated with pomegranate extract (Sanchez-Lamar et al. 2008). It has been realized that more studies that examine exposure over a long period of time may be helpful to understand any health risks from its long-term or high doses consumption (Medjakovic and Jungbauer, 2013).



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EFFECTS OF POMEGRANATE ON GENERAL HEALTH AND BODY The overwhelming inflammatory reactions bring deleterious health injuries which may lead to pathological condition (de Visser et al. 2017). Activation of immune cells against nervous system results in multiple sclerosis (Dendrou et al. 2015). In these autoimmune diseases there is increased production of interleukin (IL)-17 and interferon (IFN)- from Thelper cells (Kaskow and Baecher-Allan, 2018). The peel of pomegranate and its extract is used as diet supplement as punicalagin and derivatives of ellagic acid are more abundantly present in peel extract of pomegranate. In a study, peel extract of pomegranate was tested in animal model of type-1 diabetes and multiple sclerosis. It was found that the intraperitoneal administration of peel extract showed significant amelioration of inflammation that induced multiple sclerosis in rats and C57BL/6 mice. These researchers noticed reduced production of IL-17 and IFN- in extract supplemented rats suggesting the anti-inflammatory effects of phenolic compounds present in pomegranate extract (Stojanović et al. 2017). The main strategy of anti-cancer drugs involves anti-proliferation, antiangiogenesis and apoptotic activities. The fruit extract of pomegranate shows inhibitory activity on cell cycle, cell division and cell growth of breast and prostate cancer. It also intervenes in the signal transduction of P13k/AKT, P13K, Bcl, Bax, Bad, P38, JNK and caspase pathways. These pathways play a crucial role in the development of cancerous cell (Malik et al. 2005). Disturbances in metabolic activities of liver may cause several other pathological manifestations. It is found that the plant derived formulations exhibit protective and preventive role against liver diseases (Guan and He, 2015). Similarly, use of pomegranate is found to improve liver functions. Pomegranate peel extract showed protection in liver fibrosis induced by biliary obstruction in rats (Ibrahim et al. 2016). Moreover, extract of peel and seed prevented carbon tetrachloride (CCl4)-induced toxicity in an in


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vitro study (Ibrahim et al. 2016). These studies emphasized the hepatoprotective effects of anti-oxidant constituents present in pomegranate. The anti-diabetic activity of different products of pomegranate has also been shown previously (Salwe et al. 2015). The pomegranate is useful for the management of both type-I and II diabetes. Extracts of seed and peel of pomegranate showed a significant decrease in blood glucose levels in animal model of diabetes (Das and Barman, 2009). Reduced blood glucose was found in alloxan-induced diabetic rats following the administration of powder of pomegranate husk. The flower extract of pomegranate was also found to reduce plasma glucose level in genetically modified rat model of diabetes. The flower extracts reduced plasma glucose levels more significantly in sucrose-loaded rats. The suggested mechanism for glucose lowering effect of pomegranate involves inhibition of α-glucosidase enzyme which converts sucrose into glucose (Li et al. 2005). The aqueous extract of pomegranate peel was also tested in alloxan-induced diabetes and it lowered blood glucose, increased insulin levels, and enhanced function of pancreatic β-cells in diabetic rats. Thus, the use of pomegranate is also suggested in type-I diabetes because of possible regeneration of βcells in pancreas (Radhika et al. 2011). The antidiabetic activity of peel extract was also monitored in animal model of type-I diabetes. It was found that intraperitoneal administration of peel extract resulted in attenuation of diabetic condition by reducing the release of IL-17 and IFN (Khalil, 2011). It can, therefore, be concluded that pomegranate may provide protection against other autoimmune diseases as well. Studies suggest that the beneficial effects of pomegranate in diabetic condition are due to the presence of anti-diabetic compounds including punicalagin, ellagic acid, gallic acid, uallic acid, ursolic acid and oleanolic acid. Oleanolic acid and its derivates have shown to reduce blood glucose levels by delaying glucose absorption and decreasing glucose uptake in small intestine (Castellano et al. 2013). Ursolic acid enhances insulin secretion and sensitivity which may lead to decreased blood glucose levels in typeII-diabetes (Wu et al. 2014). Peroxisomes proliferator-activated receptor (PPAR)- stimulates glucose oxidation and increases insulin sensitivity.



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Gallic acid and its dimer ellagic acid activate genetic expression of PPAR mRNA and thus induce anti-diabetic activity (Li et al. 2008). Furthermore, the diabetes-associated coronary disease involves upregulation of genetic expression of nuclear factor B (NF-B), which induces expression of fibronectin mRNA resulting in an increased synthesis of collagen I and III factors. The supplementation of pomegranate flower extract in Zucker diabetic fatty rats resulted in reduced over-expression of fibronectin mRNA and down-regulation of collagen I and III mRNA expression suggesting its protective effects against diabetesassociated complications (Huang et al. 2005). In addition to pomegranate effects of diabetes, the supplementation of pomegranate seed and leaves extracts showed a significant decrease in food intake and body weight in rats fed on high-fat diet. Previous studies described the anti-obesity properties of pomegranate and its products (AlMatar et al. 2017). Elevated serum low-density lipoprotein, triacylglycerol and total cholesterol are often associated with obesity (Grundy, 2004). Extract of pomegranate leaves, seeds and flower has been tested by various scientists to find out the prevention against hyperlipidemia, obesity and related disorders (Lei et al. 2007; Mirmiran et al. 2010; Neyrinck et al. 2013). There are convincing data demonstrating a significant decrease in total cholesterol, low density lipoprotein (LDL)-cholesterol, LDL/HDL (High density lipoprotein) ratio as well as reduced total cholesterol/HDL cholesterol ratio demonstrating the anti-obesity activity of pomegranate (Medjakovic and Jungbauer, 2013). The different products of pomegranate have been tested in animal model of obesity as well as in obese patients and researchers observed a dramatic change in obesity biomarkers in these subjects (Mirmiran et al. 2010). The mechanism by which pomegranate can reduce the onset of obesity is because of two important features including inhibition of pancreatic lipase activity and decrease caloric intake (Lei et al. 2007). There are clinical evidences which show the decreased intestinal absorption and increased fecal excretion of fats following the administration of pomegranate extract by inhibiting lipase activity. Reduced fat absorption, increased fat excretion, and inhibition of energy intake are suggested to lower obesity risk (Al-Muammar and Khan,


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2012). Furthermore, activation of PPAR- and decreased triglyceride levels following the supplementation of pomegranate seed extract have also been demonstrated as a possible mechanism against obesity (Li et al. 2008). Activation of PPAR- has shown to decrease fatty acids and lipid content in circulation leading to reduced incidence of obesity and cardiovascular diseases (Aasum et al. 2002). Treatment of pomegranate extract and seed oil in diabetic obese mice resulted in activation of PPAR- receptors in these animals suggesting pomegranate has PPAR- agonist activity which may help to attenuate the abnormalities of hyperlipidemia (Al-Muammar and Khan, 2012). Obesity is a progressive inflammatory condition leading to increased oxidative stress and other related clinical manifestations (Freitas et al. 2018). The free radical scavenging properties of punicalagin, ellagic acid and anthocyanins is also suggested to be responsible for lipid lowering and anti-atherosclerotic property of pomegranate (Seeram et al. 2005b). The constituents of pomegranate have shown to reduce IL-6 release thereby decreasing the release of triglycerides from adipocytes and enhancing insulin sensitivity resulting in reduced symptoms of obesity (BenSaad et al. 2017). It is clear from these evidences that supplementation of pomegranate products in daily diet of obese patients may play a crucial role to subside the obesity and its related disorders. Pomegranate has also shown to improve cardiovascular performance. Juice of pomegranate and extract of its flower, leave, and seed have been demonstrated to improve lipid profile by decreasing total cholesterol and LDL/HDL ratio therefore, the use of pomegranate has been employed for delaying the onset of cardiovascular diseases (Aviram and Rosenblat, 2013). Increased oxidation of LDL activates pro-atherogenic activities (Forte et al. 2002). Paraoxigenase 1 (PON 1) is the enzyme which is present in bound form with HDL. Activated PON 1 inhibits the oxidation of LDL and enhances efflux of cholesterol from macrophages (Ikhlef et al. 2016). PON 1 also exhibits anti-oxidative and anti-inflammatory activities (Litvinov et al. 2012). Decreased activity of PON 1 is linked with diabetesassociated atherosclerosis (Kota et al. 2013). Supplementation of pomegranate juice in diabetic patients resulted in enhanced PON 1 activity and reduced serum LDL oxidation (Estrada-Luna et al. 2018). The isolated



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components of pomegranate including punicalagin, gallic acid, and ellagic acid showed activation of PON 1 in in-vitro studies (Aviram et al. 2008). Similarly, in another study, administration of pomegranate juice in apolipoprotein E-deficient mice resulted in increased PON 1 activity in serum, reduced LDL oxidation and diminished cholesterol esterification (Aviram et al. 2000). These studies support the anti-atherogenic activity of pomegranate via mediating anti-oxidant defense mechanism. Pomegranate has also shown to induce anti-oxidant effects by increasing the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase enzyme activities, and enhancing the levels of reduced glutathione (GSH) (Murthy et al. 2002; Braidy et al. 2013). These observations suggest that pomegranate improves lipid profile and reduces the inflammatory responses mediated by oxidized LDL. These effects can be very beneficial to reduce cardiovascular diseases.

POMEGRANATE AND BRAIN FUNCTIONS Pomegranate has shown to improve learning and memory, to provide neuroprotection against Alzheimer’s disease and also to improve mental functioning (Youdim et al. 2004; Said et al. 2013). The emerging data on pomegranate and their inherent polyphenols suggest benefits of pomegranate consumption ranging from neuro-protective effects to staving off effects of senescent neurodegeneration.

Mechanism of Neuroprotection Neurodegeneration observed in Parkinson’s, Alzheimer’s, and other neurodegenerative diseases are multifactorial, i.e., a complex set of toxic reactions, including inflammation, glutamatergic neurotoxicity, increase in iron and nitric oxide, depletion of endogenous anti-oxidants, reduced expression of trophic factors, dysfunction of the ubiquitin–proteasome system, and expression of pro-apoptotic proteins lead to the degeneration


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of neurons. The anti-oxidant and anti-inflammatory effects of pomegranate provide the beneficial effects on brain as outlined below:

Anti-Oxidant Effects As stated before, pomegranates are a rich source of unique and powerful anti-oxidants such as ascorbic acid, vitamin E, polyphenols, tannins, pro-anthocyanidins and flavonoids (Li et al. 2006; Rahimi et al. 2012; Richard and Paul, 2016), flavones, anthocyanins, catechins, organic acids, and tannins. These compounds scavenge free radicals and decrease oxidative stress and lipid peroxidation (Murthy et al. 2002; Jurenka, 2008; Said et al. 2013). Reports have shown that pomegranate phytochemicals are effective in maintaining an oxidative balance state essential for a healthy brain (Zhang et al. 2008; Sadeghi et al. 2010; Ashoush et al. 2013; Jinu et al. 2016; Rahmani et al. 2017). It has been reported that polyphenolic compounds present in different parts of pomegranate act synergistically to prevent oxidative stress-induced brain damages (Shastry et al. 2017). The predominant neuroprotective anti-oxidant in pomegranate is the punicalagin, a natural phenol formed by the oxidative linkages between galloyl or gallic acid groups on the pentagalloyl glucose, and it contributes for more than half of the anti-oxidant activity of pomegranate juice (Richard and Paul, 2016). This compound can slow memory loss, confusion, disorientation and other symptoms associated with dementia (Olajide et al. 2014). Ellagic acid protects the blood-brain barrier (BBB) from oxidative damage while the flavonoid, anthocyanin, penetrates the BBB and thus protects brain cells (Shin et al. 2006; Said et al. 2013). Several studies support that the anti-oxidant potential of pomegranate is 23 times the anti-oxidant capacity of either red wine or green tea, which may be valuable in improving brain function and preventing neurological disorders (Gil et al. 2000; Jurenka, 2008). The anti-oxidant properties of pomegranate polyphenols may have beneficial effect by reducing oxidative stress through restoring the activities of anti-oxidant enzymes including SOD, CAT, glutathione peroxidase (GPx), GSH and glutathione Stransferase (Hartman et al. 2006; Leuner et al. 2010; Riaz et al. 2014; Jinu et al. 2016). Pomegranate reduces free radical-induced lipid peroxidation



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and improves mitochondrial dysfunction in brain which is the primary cause of all neurodegenerative disorders from dementia to Parkinsonism (Sreekumar et al. 2014; Jinu et al. 2016). Studies suggest that pomegranate juice has an anti-oxidant-driven neuroprotective effect conferred from mother to neonate thus protecting against hypoxia-induced ischemic brain injury (Jurenka, 2008). Evidence showed that polyphenols in pomegranate juice reduced the formation of cellular reactive oxygen species (ROS) and suppressed the detrimental effects of oxidative damage caused by aluminum and/or radiation via reducing Thio Barbituric Acid Reactive Substances (TBARS) level and Advanced Oxidation Protein Products (AOPPs) and enhancing GSH level and activities of SOD and CAT (Kaur et al. 2006; Said et al. 2013). Furthermore, it was suggested that the sugar fraction of pomegranate juice, which consists of conjugated sucrose, fructose and glucose may have significant anti-oxidant properties independent of phenolic compounds. Furthermore, pomegranate juice also inhibited the activation of oxidation-sensitive genes in response to cellular stress (Kwak et al. 2005; Jinu et al. 2016) and the modulation of endothelial nitric oxide synthase expression (Jinu et al. 2016). The other suggested possible mechanism responsible for neuroprotective effects of pomegranate might involve the potential of polyphenols and ellagic acid in protecting the tricarboxylic acid cycle enzymes from free radical attack, and to maintain the levels of mitochondrial energy production (Lee et al. 2010a; Choi et al. 2012; Ahmed et al. 2014), to stabilize mitochondrial membranes and to prevent mitochondrial damage by acting as an electron donor (Ou et al. 2010; Ahmed et al. 2014).

Anti-Inflammatory Effects Ellagitannins, ellagic acid and metabolic product of the hydrolysable tannins are the major stakeholders among pomegranate phytochemicals pool bearing anti-inflammatory properties (Ismail et al. 2016). Unlike antiinflammatory pharmacological drugs, pomegranate phytochemicals e.g., ellagic acid have prolonged onset of anti-inflammatory response and duration of inhibitory action. Granatin B among some tested hydrolysable tannins has also been suggested as standard marker in identifying anti-


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inflammatory properties of pomegranate. The compound was reported to inhibit carrageenan-induced mice paw edema by strongly inhibiting expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE-2) (Lee et al. 2010b; Ismail et al. 2016). Studies have also shown that pomegranates possess a high amount of copper that might also be involved in the production of brain neurotransmitters and support the immune system (Said et al. 2013). Pomegranate has been shown to decrease the inflammatory cytokine levels (Essa et al. 2015) production which might be responsible for its antiinflammatory activity (Jung et al. 2006; Lee et al. 2008; Ouachrif et al. 2012; Ahmed et al. 2014). Chronic dietary pomegranate supplementation has been observed to decrease the levels of IL-1β, TNF-α, and IL-6 in the brains of transgenic Alzheimer’s disease mice especially in the cerebral cortex and the hippocampus of mice (Essa et al. 2015) but the mechanism of reduction is uncertain, since its multiple active components such as anthocyanins, ascorbic acid, ellagic acid, gallic acid, fumaric acid, caffeic acid, catechin, epigallocatechin-3-gallate, quercetin, rutin, tannins, alkaloids, and flavonoids, have multifunctional action, thus making it pharmacologically complex (Essa et al. 2015). Some reports suggested that the mechanism of anti-inflammatory activity of pomegranate may include inhibition of cyclooxygenase and lipoxygenase enzymes, and matrix metalloproteinase (Schubert et al. 1999; Ahmed et al. 2005; Dipak et al. 2012; Ahmed et al. 2014). Other studies showed that pomegranate extract exerts an inhibitory effect on NF-κB activation and the downstream proinflammatory signaling pathway, possibly through its anti-oxidant effects, thus providing protection against neuro-inflammation (Ahmed et al. 2014). In addition, pomegranate extract has been found to suppress NF-κB translocation by inhibition of I-κB kinase, as well by induction of NF-κB inhibitory factor (Adams et al. 2006; Bishayee et al. 2013; Ahmed et al. 2014). Moreover, pretreatment with pomegranate was also found to increase the brain levels of anti-inflammatory cytokine IL-10 that protects the brain against ischemic injury and inhibits TNF-α production (Di Santo et al. 1995; Ooboshi et al. 2005; Ahmed et al. 2014). It is suggested that pomegranate decreases tissue inflammation via attenuating the potency of



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mononuclear phagocytes to mediate inflammatory responses, or by inhibiting secretion of many inflammatory cytokines and chemokines, or by opposing the effects of TNF-α (Schroeter and Jander, 2005; Ziebell and Morganti-Kossmann, 2010; Ahmed et al. 2014). Further studies showed that the anti-oxidant and anti-inflammatory activities of pomegranate may be attributed to caspase-3 inhibition (Ahmed et al. 2014) and downregulation of its expression that mediates the cleavage of proteins necessarily required for DNA repair and cell stability, causing cell death primarily by apoptosis (Liu et al. 1997; Loetscher et al. 2001; PachecoPalencia et al. 2008; Ahmed et al. 2014). Another study found the effectiveness of punicalagin in inhibiting the gene expression of microsomal prostaglandin E synthase-1 which is suggested as a possible remedy for neuro-degenerative diseases (Olajide et al. 2014; Sairam, 2015). On the basis of these outcomes it can be concluded that regular intake and consumption of pomegranate is helpful in preventing the neuroinflammation related to dementia.

POMEGRANATE AND BRAIN DISORDERS Cognitive Dysfunctions The beneficial effects of the pomegranate were studied extensively in preclinical models of Alzheimer’s disease. As summarized in Figure 5, research showed that pomegranate can improve memory, prevent postoperative memory dysfunction, and memory deficits in aged animals and humans (Bookheimer et al. 2013; Ismail et al. 2016). Markers of verbal and visual memory are improved following pomegranate consumption leading to increased brain activation (Richard and Paul, 2016). Studies have shown that phytochemicals derived from pomegranate triggers the inhibition of acetylcholinesterase (AChE) enzyme, increasing half-life of acetylcholine via reducing calcium (Ca2+) influx which is useful in enhancing cognitive skills, treating cognitive decline, improving memory, or related central nervous system (CNS) activities (Tripathi et al.


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2004; Akhlaghi et al. 2009; Dipak et al. 2012; Bekir et al. 2013; Dhingra and Jangra, 2014; Jinu et al. 2016). Reports revealed that daily allowance of 4% pomegranate juice reduces anxiety, improves memory, locomotor function and learning ability (Subash et al. 2015; Ismail et al. 2016), and restores the activities of membrane-bound enzymes in the brain (Subash et al. 2014; Rahmani et al. 2017). The neuroprotective features of pomegranate polyphenols showed that pomegranate can help fight and protect against Alzheimer’s disease (Richard and Paul, 2016). Studies showed that punicalagin is involved in inhibition of peroxynitrites formation and delaying the onset of Alzheimer’s disease via inhibiting the release and synthesis of neurotransmitters involved in short-term memory, sleep, mood, social recognition, and alertness (Sairam, 2015). It has been shown that pomegranate juice is useful for improvement of Alzheimer’s disease as its consumption in mice reduces accumulation of amyloid beta 42 (A42) protein deposition in the hippocampus by 50% (Hartman et al. 2006; Zarfeshanyet al. 2014) displaying improved learning of water maze accomplishments (Hartman et al. 2006; Jurenka, 2008; Keservani et al. 2016). The peel extracts of pomegranate have shown to be effective as gamma secretase modulator that favors diminution in Alzheimer’s disease pathogenesis (Ahmed, 2013; Ismail et al. 2016). Ellagic acid, an important component of pomegranate extract was reported to inhibit beta-secretase activity (Feng et al. 2009; Yuan et al. 2015), hence prevent the formation of amyloid beta proteins from precursor amyloid precursor protein (APP). Additionally, ellagic acid also suppresses the pro-inflammatory transcriptional factor, NF-kB activation pathway and positively modulates its cell signaling pathways (Larrosa et al. 2006b; Yuan et al. 2015). Several studies have reported neuroprotective effects of pomegranate against Alzheimer’s disease but the bioactive compounds responsible have not been characterized (Hartman et al. 2006; Choi et al. 2011; Rojanathammanee et al. 2013; Subash et al. 2014; Yuan et al. 2015). However, the physiologically relevant gut microbiota-derived metabolites of its constituent ellagitannins, urolithins (6H-dibenzo[b,d]pyran-6-one derivatives) are considered to be the responsible bioactive constituents as it



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is only compound of pomegranate that can penetrate BBB and absorbed by brain contributing to pomegranate’s anti-Alzheimer’s disease effects; however, further in vivo studies are required (Yuan et al. 2015). It might be possible that there are unidentified compounds, yet to be isolated from pomegranate, which are responsible for its neuroprotective effects. In vitro studies showed that urolithins have prevented β-amyloid fibrillation and methyl-urolithin B is protective against amyloid β1−42 induced neurotoxicity (Yuan et al. 2015). Moreover, in vitro studies have also reported anti-inflammatory (Epsin et al. 2013), anti-glycative and neuroprotective effects of urolithins (Verzelloni et al. 2011; Yuan et al. 2015). Chronic supplementation of 4% pomegranate ameliorated the Alzheimer’s disease pathogenesis by inhibiting oxidative damage, reducing lipid peroxides and protein carbonyl content via restoration of activities of anti-oxidant enzymes (Subash et al. 2014; Akbar et al. 2015; Essa et al. 2015; Keservani et al. 2016). Studies showed that in the brains of the pomegranate-fed mice levels of Aβ-stimulated TNFα is reduced with decreased transcriptional activity of NF of activated T-cells (NFAT) which is attenuated by punicalagin and ellagic acid thus inhibiting Alzheimer’s disease progression (Rojanathammanee et al. 2013; Akbar et al. 2015). The flavonoids in the pomegranate have the potential to improve mitochondrial dysfunction and to treat age-related cognitive impairment and Alzheimer’s disease in animals and humans (Aliev et al. 2010; Chakrabarti et al. 2011; Jinu et al. 2016) via increasing cyclic adenosine monophosphate (cAMP) levels in the brain that reduces the production of pro-inflammatory mediators and stimulates the transcriptional machinery necessary for mitochondrial biosynthesis (Schmitt-schillg et al. 2005; Scarmeas et al. 2007; Jinu et al. 2016). Punicalagin, the major polyphenol of pomegranate, reduces the inflammation of the microglial cells in the brain found in the Alzheimer’s disease. Apart from Alzheimer’s disease, punicalagin also curb the inflammation found in Parkinson’s disease and Rheumatoid arthritis as well as other types of dementias (Olajide et al. 2014; Sairam, 2015).


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Psychiatric Illnesses Depression being a chronic illness, affects people of all ages. Despite availability of many effective antidepressants, the existing therapy is often inadequate and, therefore, search for new and more effective antidepressants is required (Wichers et al. 2014). The drawbacks of current antidepressants have diverted the minds of researchers to pursue hunt for natural remedies to get more beneficial effects with lesser side effects and toxic consequences (McIntyre et al. 2014). The current popularity and research on pomegranate suggest its therapeutic benefit in various psychiatric diseases; however, not much research is performed on its effects on depression. Only, few authors have reported beneficial effects of pomegranate in depression. Shastry et al. (2017) used aqueous extract of whole fruit of pomegranate and reported antidepressant effects at 500 mg/kg after acute as well as chronic administration in adult rats. They used classical models for depression, the forced swim test, and tail suspension test. The dose of 250 mg/kg showed antidepressant activity only on chronic administration in tail suspension model. Naveen et al. (2013) and Riaz and Khan, (2017) also showed antidepressant like activity of pomegranate juice and peel in rodents. Jahromy et al. (2014) used pomegranate fruit extract in male mice followed by monitoring performance in forced swim test and they have reported antidepressant like activity following acute, short-term and repeated administration. Pomegranate is known to contain estrogenic compounds such as estrone and estradiol which have potential antidepressant activity (van Elswijk et al. 2004). Decreased monoamine oxidase activity, an enzyme responsible for degradation of serotonin, causes an increase in the concentration of serotonin available in synapse (Alia-Klein et al. 2008). Rahman et al. (2015) showed significant antidepressant effects of pomegranate pulp extract in both tail suspension test and forced swim test showing reduced immobility time. Others have also reported similar effects using pulp extract of pomegranate. MoriOkamoto et al. (2004) reported reduced immobility by pomegranate extract at 0.1 ml/kg of body weight whereas Kumar et al. (2008) showed a significant reduction in immobility time using pomegranate pulp extract at



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dosage of 250 mg/kg and 500 mg/kg. This rich polyphenolic and antioxidant content scavenge free radicals in depression-like condition and reduce oxidative stress causing attenuation of depression (Sarubbo et al. 2017). Secondly the estrogenic content in pomegranate seeds may inhibit the monoamine oxidase enzyme which may increase serotonin level in synapse to reduce depression like condition (Valdés-Sustaita et al. 2017). Pomegranate extract is also reported to have anxiolytic properties which are attributed to pomegranate polyphenols on CNS via their influence on neurotransmitters. The reported anxiolytic, antidepressant (Naveen et al. 2013; Keservani et al. 2016), and anti-nociceptive (González-Trujano et al. 2015) properties of pomegranate are thought to be mediated by pomegranate phytochemical-induced modulation of the activities of two important neurotransmitters of CNS i.e., gammaaminobutyric acid (GABA) and Glutamate. Anxiolytic activity is the result of binding of polyphenols to GABA benzodiazepines complex and the interactions between GABAergic and serotonergic systems (Hanrahan et al. 2011; Liaquat et al. 2018), while anti-depressive effects are the consequence of glutamate increment (Losi et al. 2004).

Motor Dysfunctions With advancing age, motor coordination is highly affected and impaired due to neuronal disturbance (Sato et al. 2018). The older subjects display limited functional mobility due to abnormalities in motor and psychomotor behaviors (Seidler et al. 2010). Use of polyphenols and related anti-oxidant compounds are suggested for healthy aging and to forestall deficits in memory and motor functions (Obrenovich et al. 2011). A study conducted by Dulcich and Hartman (2013) showed that supplementation of pomegranate juice improved locomotive behavior in mice. Effects of pomegranate and/or its product are not well studied on motor function; however, the individual anti-oxidant constituent present in this polyphenol-rich fruit have been investigated in animal model of impaired motor behavior (Magalingam et al. 2015). The pathogenesis of


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Parkinson’s disease is related to a number of factors including aging brain, exposure to toxin, genetic susceptibility leading to oxidative stress-induced dopaminergic degeneration and neuronal imbalance resulting in impaired motor coordination (Sveinbjornsdottir, 2016). It has been suggested that pomegranate extract can prevent neuronal damage in animal model of Parkinson’s disease induced by methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) (Braidy et al. 2013). Gallic acid and ellagic acid are thought to have anti-Parkinsonian effects which are mediated by their anti-oxidant activity (Farbood et al. 2015; Mansouri et al. 2017). In a study conducted by Sarkaki et al. in 2016, ellagic acid was given to 6hydroxydopamine (6-OHDA)-induced rat model of Parkinson’s disease at the dose of 50 mg/kg for ten consecutive days. Rats were infused with 6OHDA directly into medial forebrain bundle (MFB) to develop animal model of Parkinson’s disease. Treatment with ellagic acid exhibited significant restoration of motor deficits induced by 6-OHDA in Parkinson’s disease rat model. Impaired motor coordination and shortened stride-length that are main symptoms of Parkinson’s disease were successfully attenuated by the treatment of ellagic acid. Furthermore, infusion with 6-OHDA leads to increased activity of monoamine oxidase and increased generation of free radicals which may exacerbate dopaminergic neurodegeneration. Sarkaki et al. (2016) found increased malondialdehyde levels and decreased activities of GPx and SOD in rat brain with lesions in MFB which were considerably reverted by the administration of ellagic acid. The effects of ellagic acid on Parkinson’s disease models are also addressed in right MFB-lesioned rats that showed hyperalgesic responses to the stimulus in tail-flick and hot-plate tests, and memory and learning deficit in cognitive tests (Dolatshahi et al. 2015; Rahmani et al. 2017). Pomegranate juice extracts also reported to have neurotoxicity protective effect in primary human neurons in a dosedependent manner by attenuating MPTP-induced increase in extracellular lactate dehydrogenase (LDH) activity (Braidy et al. 2013; Rahmani et al. 2017). These scientists suggest that ellagic acid which is abundantly present in pomegranate may treat or prevent motor disturbance observed in Parkinson’s disease. Anthocyanin, which is also one of the polyphenolic



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constituent of pomegranate, has been tested in impaired motor function that is caused by spinal cord injury (Kim et al. 2011). It was found that anthocyanin rich extract exerts anti-inflammatory responses in rats and suppresses the proliferation of astrocytes. This anti-inflammatory response is produced by inhibiting the expression of IL-6, IL-1β, and TNF-α at the injured site of spinal cord resulting in re-myelination and recovery from locomotor dysfunction (Wang et al. 2012). These findings led to conclude that the anthocyanin rich fruit has critical impact on functions of CNS, suggesting that these fruits may help in improving the quality of life of patient suffering from spinal cord injuries.

Metal Neurotoxicity Exposure to heavy metal is considered as the major vulnerable factor to induce neurodegenerative diseases (Chin-Chan et al. 2015). These metals including lead, cadmium, and mercury induce neurotoxicity by interrupting the normal metabolism of biomolecules. At high concentration, heavy metals induce disruption of blood brain barrier and thus further increase the entrance of heavy metal in neurons (Kim et al. 2013). Treatment with heavy metal chelators exerts some adverse effects such as depletion of copper, zinc, and other minerals (Flora and Pachauri, 2010). Aslani et al. (2014) tested the chelating property of pomegranate pith extract for lead intoxication. These scientists reported a significant elimination of lead from kidney and bones with the highest removal from brain in female Wistar rats. Methanolic extract of pomegranate peel protected brain against aluminum toxicity via stimulating anti-oxidant activities and anti-apoptotic proteins such as Bcl-2 (Abdel Moneim, 2012). Therefore, it can be concluded that the extract of pomegranate may also have chelating properties for other divalent heavy metals as well and may provide an effective measure for preventing or reducing their neurotoxicity.


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Figure 5. Summarized health benefits of pomegranate on brain.

Other Brain Dysfunctions It has been shown that proton irradiation decreases neurogenesis in hippocampus (Raber et al. 2004). Moreover, cell proliferation biomarker BrdU (5-bromo-2'-deoxyuridine) expression was significantly reduced in hippocampus sub-granular of irradiated mice (Li et al. 2016). Proton radiation exposure is thought to be the most possible factor of neurodegeneration and its related behavioral deficits in astronauts. Researchers suggest the use of anti-oxidants and polyphenols to reduce these effects as oxidative stress is considered to induce radiation-mediated neurodegeneration (Huang et al. 2012; Mao et al. 2017). Supplementation of pomegranate juice for 3 months significantly increased neurogenesis and proliferation in non-irradiated mice but failed to attenuate radiationinduced injury in hippocampus. These scientists suggested a longer duration of supplementation in order to accomplish the protective effects against radiations. Behavioral deficits which were observed following the exposure of radiations include depression- and anxiety- like behaviors. The supplementation of pomegranate juice in these radiation-exposed mice



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significantly mitigated the neurobehavioral-deficits observed after 8-weeks of exposure (Dulcich and Hartman, 2013). Ellagic acid also showed to have significant antiepileptic activity that is the result of increase in GABAergic transmission in brain (Kaplan et al. 2001; Barger et al. 2007; Jinu et al. 2016). The possible underlying mechanism is not explored yet. It is suggested that the anti-oxidant and anti-inflammatory properties of pomegranate may be involved which provided protection against irradiation in these mice.

CONCLUSION Clinical and epidemiological studies related to the health beneficial effects of pomegranate on brain functions are sparse. However, this fruit is considered as “nature’s power fruit” that contains a significant anti-oxidant pool and is a plentiful source of high molecular weight active biomolecules. It is realized that several limitations exist in the proper utilization of fruit and its extracts owing to inadequate information on pomegranate biomolecules and drug interaction, organ and cell specific role of extracts, and fractionated compounds in disease management. Research from both basic and clinical domains clearly demonstrates the direct correlation between pomegranate compounds and their therapeutic potential on neurological disorders. Studies have shown that pomegranate is effective in preventing neurodegenerative changes through different mechanisms. The beneficial effects of pomegranate are ranged from improving primary neurological functions (memory, anxiety, depression, locomotion) to reducing aging and treating severe neurological disorders (like Alzheimer’s, Parkinson’s, Rheumatoid Arthritis and other severe dementias). Furthermore, only a few convincing studies are conducted to utilize the plant material as ready to use therapeutic and functional food ingredient. It is, therefore, important to develop safe and efficacious commercial preparations to improve quality of life. Based on the facts outlined in this chapter, it is recommended to consume pomegranate on a regular basis to maintain a healthy lifestyle.


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FUTURE PROSPECTS Extensive research on the effects of pomegranate and its parts has been done. Effects of whole fruit and its parts on animals and humans have been studied in various ailments and in different animal models as a treatment and/or prevention against a number of diseases. Despite the large amount of data available on medicinal properties of pomegranate, the studies emphasizing the effects of pomegranate on CNS are scantly addressed. This provides impetus to conduct and design studies on its effects on brain function to obtain insight into the underlying mechanism for the beneficial effects of pomegranate in psychological illnesses like depression and other diseases like Parkinson’s disease and Alzheimer’s disease. Oxidative stress is essentially thought to be implicated in almost all chronic diseases and the polyphenolic compounds are known to prevent oxidative stress and hence can act as therapeutics for these conditions. In order to study in detail, the effects of pomegranate in animal models of various neurological diseases may be developed and both pre-treatment and post-treatment effects of whole fruit and its separate parts should be investigated. The investigation must not be limited to its anti-oxidant and polyphenolic properties but following its administration, effects on different brain neurotransmitter should also be taken into account in all the neurological diseases. Furthermore, it is necessary to conduct both acuteand chronic-treatment studies to investigate the effects for short and longterm side effects. Studies of pomegranate in combination with other herbal extracts are also warranted to see whether enhanced synergistic beneficial effects can be obtained.

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Chapter 7

WHAT CAN WE LEARN ABOUT THE TRAITS OF ARIL JUICE BY STUDYING WIDE COLLECTIONS OF DIVERSE POMEGRANATE FRUITS? Rachel Amir, PhD1,4,*, Doron Holland, PhD2 and Li Tian, PhD3 1

Migal Galilee Technology Center, Kiryat Shmona, Israel Institute of Plant Sciences, Agricultural Research Organization, Newe Ya’ar Research Center, Ramat Yishay, Israel 3 Department of Plant Sciences, University of California, Davis, CA, US 4 Tel Hai College, Israel 2

ABSTRACT Pomegranate trees (Punica granatum L.) originated in Central Asia from where they were spread to other climatic regions. From ancient times, the adaption to different growth conditions, which has enabled the *



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R. Amir, D. Holland and L. Tian development of a wide range of local genotypes that have different phenotypes. In addition, farmers and breeders continue to select cultivars with desired traits. As a result, the fruits exhibit great divergence in their morphological and chemical traits. Studies have shown that pomegranate juice (PJ) has a high human health benefit traits, which include high antioxidant activity, reduction in stress-related chronic inflammatory diseases and age-related disorders such as cardiovascular diseases, cancer and neurodegeneration. This chapter is focused on the biodiversity of PJ and aril traits found in different pomegranate cultivars in different collections reported from different countries. We refer to antioxidant capacity, phenolic content, the components controlling the taste such sugar, organic acid, volatiles, as well as to seed hardness, aril color, and anthocyanins content. We also intend to show how studies conducted with a large collection of diverse cultivars could help elucidate different traits that contribute to the health benefits and marketing of the fruit.

INTRODUCTION Pomegranate trees (Punica granatum L.) originated in Central Asia (Iran, Turkmenistan and northern India) from where they were dispersed thousands of years ago to the Mediterranean basin, East Asia, North Africa, Europe, and later to North and South America (Fadavi et al., 2005). Today, pomegranate is cultivated throughout the world in many tropical and sub-tropical climatic regions (Holland et al., 2009; Verma et al., 2010). In particular, it has successfully adapted to the Mediterranean climate, different rain regimes and soils, and additional environmental conditions. From ancient times, this adaptation to different growth conditions has enabled the development of a wide range of local genotypes in different countries/regions that have different phenotypes. Farmers and plant breeders continue throughout the years to select pomegranate for its desired traits according to fruit qualities and a wide range of growing traits (Holland et al., 2009). Hence, these cultivars are characterized by a large variability in terms of domestic, wild and ornamental genotypes. As a result, pomegranate trees exhibit great divergence in growth characteristics, as well as many morphological and chemical traits, such as rind and aril color, fruit size, shape, flavor, seed


What Can We Learn About the Traits of Aril Juice by Studying … 205 hardness, juiciness, sugar/acid ratio, polyphenolic content, antioxidant activity, fatty acids and arils anthocyanins content. Aside from the genetic factors, several of these traits were also influenced by environmental conditions (Borochov-Neori et al., 2009; Schwartz et al., 2009a; BorochovNeori et al., 2011). Collectively, more than 5,000 wild and cultivated pomegranate varieties were reported in the literature that are maintained by germplasm repositories located in Asia, Africa, Europe and America (Still, 2006). It should be noted that a redundancy may exist among different pomegranate collections since, although most germplasms are landraces and local cultivars, some pomegranate accessions were collected from all over the world. In recent years, several studies involving pomegranate genotyping were conducted on the pomegranate germplasm. In one of these studies (Ophir et al., 2014), the pomegranate collection was shown to be divided into two major groups of accessions: one from India, China and Iran, and the other group composed of accessions mainly from the Mediterranean basin, Central Asia and California. It is estimated that overall probably only several hundreds of pomegranate varieties exist today worldwide (Ophir et al., 2014; Harel-Beja et al., 2015). Pomegranate has been employed extensively in folk medicine remedies of many cultures (Langley, 2000). The medicinal and therapeutic effects of the compounds extracted from pomegranate have been well recognized by many Mediterranean and Asian cultures, and used against diseases and symptoms such as stomach aches, diarrhea, dysentery, skin diseases and eye inflammations (Langley, 2000; Holland et al., 2009). The Western world is now recognizing the nutritional and medicinal potential embedded in the arsenal of metabolites produced in pomegranate. Studies have shown that pomegranate juice (PJ) has a high total phenolic content (Seeram et al., 2006a) associated with the scavenging of free radicals and reactive oxygen species (ROS) (Aviram et al., 2008). The antioxidant activity of total phenols in PJ has been associated with a reduction in stress-related chronic inflammatory diseases and age-related disorders


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such as cardiovascular diseases (Aviram and Rosenblat, 2012; Vlachojannis et al., 2015), carcinogenesis (Turrini et al., 2015) and neurodegeneration, as well as skin deterioration (Quideau et al., 2011). PJ is comprised mostly of the edible part of the fruit, the arils. The arils constitute 45-60% of the total fruit weight (Kulkarni and Aradhya, 2005) and are rich in sugars, organic acids, vitamins, minerals and polyphenols (Li et al., 2015). They can be consumed as fresh fruit and juice, but also as canned beverages, jellies, jams, flavorings and drink colorings. In some juicing industries, the fruit is extracted using a hydrostatic press, which also squeezes the pomegranate peels, a rich source of phenolic natural products such as hydrolysable tannins (HTs), in particular, punicalagin (Gil et al., 2000). Another important class of phenolic natural products in pomegranate peel and juice are anthocyanins, which contribute to the pink to red color of the arils and the skin peel (Seeram et al., 2006b). It was proposed that the synergistic or additive action of HTs and other phenolic compounds in pomegranate juice and fruit extracts (e.g., flavonoids, anthocyanins) accounts for the superior antioxidant and anticancer activities of PJ (Seeram et al., 2005). As with many other fruit species, PJ color impacts consumers’ first impressions and plays a primary role in their purchasing decisions, which are also determined by the flavor of PJ (Mena et al., 2011). Over the last few years, consumers have increasingly been attracted to PJ due to its high nutritional quality and healthy compounds. This review focuses on the biodiversity and variability of aril traits found in various pomegranate cultivars in different collections that were examined in different countries. We also intend to demonstrate how studies conducted with a large collection of diverse cultivars could help elucidate different traits that contribute to the health benefits and marketing of the fruit. Studying pomegranate collections is also important to meet the current market demand for quality fruits.


What Can We Learn About the Traits of Aril Juice by Studying … 207

ANTIOXIDANT CAPACITY AND PHENOLIC CONTENT IN DIFFERENT POMEGRANATE CULTIVARS EXAMINED IN DIFFERENT COLLECTIONS The beneficial health effects of PJ are attributed mainly to their high antioxidant activity. This activity is related to redox-controlling properties (Zhang et al., 2008), which play an important role in neutralizing free radicals (Caliskan and Bayazit, 2012). To better understand the range of antioxidant capacity in aril juice and the compounds that contribute to this activity, we selected and examined 29 pomegranate cultivars from a collection of about 150 varieties at the Newe Ya’ar Research Center, ARO (IBG, website: http://igb.agri.gov.il)] (Tzulker et al., 2007). The results showed that a high antioxidant activity correlated significantly to total polyphenol (R2 = 0.75) and total anthocyanin contents (R2 = 0.46), but not to the levels of individual HTs (punicalagin, punicalin, gallagic and ellagic acids) (Tzulker et al., 2007). Considering the significant (R2 = 0.46) but the relatively low correlation between total anthocyanin content and the antioxidant activity, it was proposed that additional polyphenols other than anthocyanins may contribute to the antioxidant activity of PJ. A wide range of antioxidant capacity (up to 3-fold difference) was detected from the cultivar that has the lowest content to the highest content, the range for the phenol content was up to 2.5-fold difference, and total anthocyanin content up to 33-fold were also observed for the 29 cultivars (Tzulker et al., 2007). High correlations between antioxidant activity, total phenols and total anthocyanins were also reported from eight cultivars from Chile (Sepúlveda et al., 2010), 13 cultivars from Tunisia (Zaouaya et al., 2012), six cultivars from the Mediterranean region of Turkey (Ozgen et al., 2008), 76 cultivars from the eastern Mediterranean region of Turkey (Caliskan and Bayazit, 2012) and 15 cultivars from Spain (Mena et al., 2011). Taken together, these results collectively suggest that when consuming the arils, the pomegranate cultivars that contain the highest antioxidant capacity and


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health benefits are those that have red or darker colored arils (i.e., anthocyanin pigments). Since the antioxidant mechanisms are quite complex in the food matrix, different antioxidant assays are usually used concurrently to determine the antioxidant capacity of food products. Several commonly used in vitro methods for determining antioxidant activity of food constituents are the Folin-Ciocalteu reducing capacity assay, the 1,1diphenyl-2- picrylhydrazyl radical scavenging assay (DPPH), the 2,20azinobis-(3-ethylbenzothiazoline- 6-sulphonate) radical scavenging (ABTS) assay, the oxygen radical absorbance capacity assay (ORAC), and the ferric reducing antioxidant power assay (FRAP) (Gil et al., 2000). In addition to the different analytical methods, differences in antioxidant capacity and total phenol content of PJ in the various studies may also be attributed by the genotype of the cultivars, the maturity stage, the environmental conditions, and the agricultural and environmental factors. Furthermore, the juicing process may also affect the antioxidant capacity of PJ (Akhavan et al., 2015b). Thus, for a large collection, it is always better to analyze fruits that grow under the same environmental conditions and using the same set of analytic method(s). Since a high correlation between total phenolic contents to antioxidant activity was reported from several studies measuring different cultivars [e.g., (Kaur et al., 2006; Mena et al., 2011; Caliskan and Bayazit, 2012; Fawole and Opara, 2013; Radunic´ et al., 2015); Table 1], it was proposed that the total phenolics assay is a suitable candidate for measuring the antioxidant capacity of PJ (Mena et al., 2011). Total phenolic contents were usually determined only by one method, the Folin-Ciocalteu reagent assay (Singleton and Rossi, 1965), and except for a few reports, total phenolic contents are reported as gallic acid equivalents (GAE). This compatibility allows comparison of the total phenolic contents of different cultivars from different countries. The levels of total phenols in PJ range, for example, by 1,245-2,076, 1,5004,500, 1,339-3,500 mg GAE/L (Ozgen et al., 2008; Mena et al., 2011; Zaouaya et al., 2012) (Table 1).


Table 1. Antioxidant activities, total phenolic contents, total anthocyanins and total HTs/punicalagin/total flavonoids in aril juices of different cultivars of different collections grown in different countries No. Cultivars

Country

Antioxidant capacity

Total Phenols content

Total anthocyanins

8

Iran

18.6–42.8 TEAC (mM)

2380–9300 mg GAE/L

815 and 654830 mg/L

10

Iran

13.6- 23.6 FRAP (mmol/100 mL)

220–1267 mg/L

1.8-175 mg/L

20

Iran

15.59–40.72 DPPH (% I)

5.56 -30.11 mg/100 g

10

Iran

2960–9850 mg GAE/kg PJ 220.0–1266.8 mg GAE/L

15

Spain

1500–4500 mg GAE/L

34 - 1075 mg/L

20

Spain

18

Spain

13

Tunisia

6

Tunisia

13.6–25.9 FRAP (mmol Fe2+/100 mL) 10–38 FRAP (mmol TE/L) 1.55 - 4.24 ABTS mmol Trolox kg−1FW; DPPH 6.39 - 7.74 mmol Troloxkg−1FW. 10.40–15.80 FRAP (mmol TE/L) 9.57–21.07 FRAP (mmol TE/L) 6.36- 8.65 FRAP (mmol TE/L)

1.8–175 mg/L

Total HTs Punicalagins/Total flavonoids Total tannines: 0.0150.032 (g/100g) 31-607 (mg/L) nd nd nd

Ref. (Mousavinejad et al., 2009) (Akhavan et al., 2015a) (Tehranifara et al., 2010) (Akhavan et al., 2015b) (Mena et al., 2011)

nd 1283- 1791 mg GAE 100 g−1dw. 1136.20–3581.10 mg GAE/L 1339.3–3500.6 mg GAE/L nd

(Alcaraz-Mármola et al., 2017)

nd

62.43– 276 mg/L 50.5 - 490 mg/L

28.15 - 48.27 mg/L

nd nd HTs: 1.97 - 3.38 mg tannic acid equivalent/mL


(Vegara et al., 2013) (Zaouaya et al., 2012)

(Elfalleh et al., 2011)

Table 1. (Continued) No. Cultivars

Country

6

Turkey

76

Turkey

6

India

7

Croatia

10

China

10 7 18

South Italy South Africa Morocco

Antioxidant capacity 4.63–10.9 FRAP (mmol TE/L) 7.4–17.5 FRAP (mmol Fe2+/kg) 4.86–7.67 FRAP (lmol TE/g) 404.9–450.1 TOSC (lmol TE/kg) 80-270 DPPH mg GAE/L 2.23- 6.35 (TEAC, mmoles/L) 0.33- 0.52 FRAP 100 μg/ml 35.21 to 76.45 ml PJ/g DPPH 14-16 μM TE/g of FW

Total Phenols content

Total anthocyanins

1245–2076 mg GAE/L

6 - 219 (CyE)/L

1080–9449 mg GAE/kg 876.2–1536.2 mg GAE/kg 1985.6–2948.7 mg GAE/L

1.1 and 63.0 mg CyE/100 g 6.45 and 458 mg of CyE/kg. 2.08 and 45.83 mg/100 g.

3150–7430 mg GAE/L

0.004–0.160 mg CyE/mL

627- 2829 (mg/L)

nd

187- 295 mg GAE/ g DW

58-322 μg C3gE /g DM

1385- 9476 mg GAE/L

64.1- 188.7 mg/L

84.9-91.1 (mg/100 g of nd FW) 5.21- 6.43 (GAE mg/g 5 Egypt 39-60 DPPH % nd FW) 29 Israel 2.6-9.2 mM 1.2-4.4 mM 1-371 (mg/L) Nd= not detected; FW, fresh weight; HTs, hydrolysable tannins; TF= total flavonoids. 6

Georgia

Total HTs Punicalagins/Total flavonoids nd nd TF: 8.23-23.99 (mg QE/100 g) nd HT: 0.89- 2.53 (TaE mg/mL) Punicalgin: 149-1042 (lg/mL) nd 326-783 μg GAE/ g DM HTs: 216.3 and 730.8 mg/L HTs: 22.8- 36.6 (mg/100 g of FW) TF: 9.64- 12.91 rutinequivalents mg/g FW 0.02-192 (mg/L)


Ref.

(Ozgen et al., 2008) (Caliskan and Bayazit, 2012) (Kaur et al., 2014) (Radunic´ et al., 2015)

(Li et al., 2015)

(Ferrara et al., 2014) (Fawole et al., 2012) (Hmid et al., 2016) (Pande and Akoh, 2009) (Ahmed et al., 2016) (Tzulker et al., 2007)

What Can We Learn About the Traits of Aril Juice by Studying … 211 Interestingly, six studies indicated that sour cultivars showed significantly higher antioxidant activity than sweet cultivars (Ozgen et al., 2008; Mousavinejad et al., 2009; Sepúlveda et al., 2010; Mena et al., 2011; Fawole et al., 2012; Zaouaya et al., 2012). However, Caliskan et al. (2012) showed that sweet cultivars had the highest levels of total anthocyanins and total antioxidant capacity. In the future, it is recommended to define the relationship between the acidities and sweetness of these cultivars to total anthocyanins and antioxidant activity in additional collections. Overall, the results obtained from studies performed on different cultivars suggest that phenols are the main contributors to antioxidant activity in PJ. Several studies also proposed that anthocyanins at least partially account for the antioxidant activity of PJ. Additional compounds, such as flavonoid precursors of anthocyanins may also contribute to the antioxidant capacity of PJ, their identity and synergistic interactions with anthocyanins remain to be determined.

VARIATIONS IN CHEMICAL COMPONENTS THAT AFFECT PJ TASTE AMONG DIFFERENT POMEGRANATE COLLECTIONS An analysis of large collections could also shed more light on the compounds that are responsible for and contribute to the taste of PJ. PJs differ in taste, ranging from sweet to sour (Melgarejo et al., 2000). This is related directly to the quality and quantity of the organic acids and sugars found in the arils. A great diversity in these components and their contents has been detected in different PJs collected from several regions around the world (examples are shown in Table 2). The desired taste of PJ varies, however, among different countries and regions. In North Africa and southern Spain, for example, almost all commercialized pomegranate belongs to the sweet varieties (Al-Kahtani, 1992), while in Russia and north Turkey, relatively sour cultivars are commercialized (Gabbasova and


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Abdurazakova, 1969; Mayuoni-Kirshinbaum and Porat, 2014; AlcarazMármola et al., 2017). PJ taste is determined mostly by the ratio of total soluble solids (TSS) and titratable acidity (TA) (Ben-Arie et al., 1984). Determination of TSS is important not only for establishing the organoleptic quality of the juice, but also because TSS content is the major parameter determining the cultivars that can be used for pomegranate-wine preparation (Matapathi et al., 2004). Acidity can play an important role in the perception of fruit quality. It affects the fruit’s sour taste but also its sweetness by masking the taste of the sugars. To gain more knowledge about the component that contributes to the taste of aril juices, we analyzed the TSS, TA, sugars and organic acids contents in the same 29 cultivars that were used previously for determination of antioxidant activity (Dafny-Yalin et al., 2010). The results showed that no significant difference exists in TSS levels among the 29 cultivars (TSS values ranged from 13.7 to 17.8 g/100g), suggesting that TSS is not the major factor determining the taste of PJ prepared from these cultivars. The TSS range is in accordance with other studies of pomegranate collections grown in different regions around the world (Table 2). In Spain, the TSS level ranged from 11.4 to 13.5 g/100 g (Melgarejo et al., 2000) and between 12.6 and 15.3 g/100 g (MartinezNicolas et al., 2016), while in cultivars collected in Turkey, the TSS ranged from 14.9 to 16.1 g/100 g (Poyrazolua et al., 2002) and in Russia, from 15.2 to 20.5 g/100 g (Gabbasova and Abdurazakova, 1969). The values obtained from the Russian collection are higher than those from other countries (Table 2). The reason for the higher TSS levels in the Russian cultivars is not known, but we previously reported that growth conditions could significantly affect the TSS level. Eleven cultivars that were grown in a Mediterranean climate in Israel having the same genetic background exhibited significantly higher TSS contents than those grown in the Israeli desert (Schwartz et al., 2009b). This suggests that colder climates in


What Can We Learn About the Traits of Aril Juice by Studying … 213 Russia might promote the accumulation of compounds contributing to the TSS level. Another explanation could be that the taste preference in Russia tends more to the sour scale, and therefore to balance it, popular Russian cultivars contain higher TSS. A strong positive correlation was found previously in PJs for TSS and sugar content (Schwartz et al., 2009b; Dafny-Yalin et al., 2010). Determination of the composition of sugars in the 29 cultivars revealed that glucose and fructose were the major sugars in pomegranate aril juice, which also contains a small amount of other sugars (Dafny-Yalin et al., 2010). The content of glucose and fructose varied from 4.8 to 6.6 g/100 g. Similar values were reported for six Spanish cultivars (Melgarejo et al., 2000), however, by analyzing a larger collection of 76 cultivars in Turkey, the range increased from 4.2 to 8.5 g/100g (Caliskan and Bayazit, 2012) (Table 2). While the levels of TSS and sugars varied up to about 1.4-fold in the 29 cultivars and up to 2-fold in the other collections, the levels of TA differed significantly among the 29 cultivars, from 0.22 to 1.97 g/100 g (by 9-fold) (Dafny-Yalin et al., 2010). These values are similar to the range found in the Spanish cultivars, which varied from 0.22 to 2.9 g/100 g (13fold) (Melgarejo et al., 2000). Thus, TA is apparently the main contributor to taste, which, as mentioned above, is determined by the ratio between TSS and TA. This ratio ranged significantly from 6.1 to 64.6 among these 29 cultivars (by about 10-fold) (Dafny-Yalin et al., 2010). The values for the Spanish cultivars showed that in sour-sweet cultivars, the TSS/TA ratio ranges from 17 to 28, while those considered to have a sour taste have values ranging from 32 to 96 (Melgarejo et al., 2000). However, the most abundant cultivars in Spain found on the market are the sweet cultivars having a relatively low ratio (Gil et al., 1995; AlcarazMármola et al., 2017).


Table 2. The levels of TSS, TA, glucose and fructose contents, TA, pH, citric and malic contents in aril juices of different cultivars of different collections grown in different countries No. Cultivars

Country

6

Turkey

7

Turkey

13

Turkey

76

Turkey

10

morocco

15

Spain

18

Spain

9

Spain

6

Spain

40

Spain

TSS 4.9-17 nd 139.6 160.6 g/L nd 15.2-17.6 nd nd nd 15 -18

nd

Glucose content

Fructose

5.8 - 7.62 g/100 mL 39.78 - 69.14 mg/mL

5.8 - 7.06 g/100 mL 45.49–93.63 mg/mL

nd

nd

4.2–8.5 g/100 g

6.5–11.2 g/100 g

6.9 - 8.6 g/100 g

7.8–10.4 g/100 g

67–89 g/L

76 - 96 g/L

61.4–65.0 g/L

65.3–68.0 g/L

45.3 - 60.1 g/L

52.6 - 73.4 g/L

4.83 - 6.03 g/ 100 g

8.43 - 10.07 g/ 100 g

5.53- 7.8 g/100 g

5.54- 8.24 g/100 g

TA

pH

0.5-3.8

2.98-3.68

nd

nd

4.58- 17.30 g/L nd

3.29- 3.93

2.4-4.18

nd

nd

nd

nd

nd

nd

nd

2.12 - 12.44

3.49 - 5.14

nd

nd

nd

Citric acid

Malic acid

Ref

0.2 -3.2 g/100 mL 3.93 - 13.06 mg/mL

0.9–1.5 mg/mL 0.285 - 4.11 mg/mL

0.33–8.96 g/L

0.56–6.86 g/L

nd

nd

0-3.22 g/100 g

0.31 - 1.56 g/100 g

0.6–18.54 g/L

0.89–1.63 g/L

2.3 - 2.8 g/L

1.3–1.4 g/L

1.1- 28.8 g/L

1.4 - 2.4 g/L

0.08–1.4 g/100 g

0.43–0.73 g/100 g

0.08- 2.46 g/100 g

0.08- 0.21 g/100 g

(Ozgen et al., 2008) (Tezcan et al., 2009) (Poyrazolua et al., 2002) (Caliskan and Bayazit, 2012) (Legua et al., 2012) (Mena et al., 2011) (Vegara et al., 2013) (Nuncio-Jáuregui et al., 2015) (MelgarejoSánchez et al., 2015) (Melgarejo et al., 2000)


No. Cultivars

Country

20

Spain

8

Croatia

20

Iran

13

Tunisia

12

Tunisia

6

Russia

3

Croatia

29

Israel

TSS 15.1- 17.7

12.5– 15.0% 11.37 15.07 14.33 16.30 13.1 19.9g/ 100 g 15.2 to 20.5 g/100 g 13.115.2% 13.7- 17.8 g/100 g

Glucose content

Fructose

4.21-5.83 (g 100 mL−1),

7.78-10.1 (g 100 mL−1),

nd

nd

nd

nd

nd

nd

5.7- 8.5 g/100 g

7.21 -10.61 g/100

nd

nd

6.4-7.4 g/100g

7.5-8.3 g/100g

4.8 -6.6 g/100g

4.9- 6.7 g/100g

TA

pH

1.44- 19.2 g citric acid L−1 0.37–0.59%

3.13- 6.78

Citric acid

Malic acid

Ref

0.07-1.90 (g 100 mL−1),

0.28-1.21 g 100 mL−1),

(AlcarazMármola et al., 2017) (Radunic´ et al., 2015)

2.81-3.80

nd nd

0.33 - 2.44 g 100 g−1 0.14 to 2.43%.

3.16 - 4.09 nd n.d

0.52%to 2.3%

nd

0.9-4.3%

2.6-3.4

0.22- 1.97

2.8-3.9

nd nd 0.1-3.2 g/ 100 g

nd nd 0.6-2.0 g/ 100 g nd

nd

nd 0.1-2.2 g/ 100 g

nd 0.02-0.59g/ 100 g

Nd- not detected.


(Tehranifara et al., 2010) (Zaouaya et al., 2012) (Hasnaouia et al., 2011) (Gabbasova and Abdurazakova, 1969) (Gadže et al., 2012) (Dafny-Yalin et al., 2010)

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It appeared that in general, sour cultivars were characterized more in cold regions, while sweet accessions having low TA values appeared more in regions having hot/dry conditions. For example, in northern regions such as Turkey and Russia, TA ranged from 1.73 to 4.6% (Poyrazolua et al., 2002) and 0.52 to 2.3% (Gabbasova and Abdurazakova, 1969), respectively, while in hot climates such as India, Egypt and Saudi Arabia, TA values dropped to 0.12 to 0.13%, 0.03 to 0.1%, and 0.02 to 0.14%, respectively (Al-Kahtani, 1992; Al-Maiman and Ahmad, 2002). This hypothesis is supported by a previous study showing that climatic conditions significantly affect the total TA level of several pomegranate cultivars. The TA levels decreased in a hot and dry climate compared to a Mediterranean climate for the same genotype (Schwartz et al., 2009b). On the other hand, the generally higher TA values found in the pomegranate cultivars grown in the northern countries could be attributed to the different demand by customers in these regions, which led breeders to breed for a sour taste in the aril juice than those found in India, Egypt and Saudi Arabia. The organic acid content, which contributes to the TA of PJ, was also examined in the 29 cultivars (Dafny-Yalin et al., 2010). Citric acid is the major organic acid found in many pomegranate cultivars and its level shows a strong and positive correlation to TA (R2 = 0.91, P < 0.01) (Dafny-Yalin et al., 2010). Citric acid was also the predominant organic acid found in all six accessions from Georgia (Pande and Akoh, 2009), 17 out of 25 cultivars from Iran (Aarabi et al., 2008), as well as most of the cultivars studied in other collections (Table 2). However, in some of the Spanish accessions, malic acid was found to be the predominant TA, followed by citric acid (Legua et al., 2000). In addition to TSS, sugars, TA and organic acids, PJ taste is also influenced by the feeling of astringency (Gil et al., 1995). Astringency is a dry mouthfeel sensation that usually comes from fruits that contain HTs. The major HT present in pomegranate fruits and responsible for the sensation of astringent mouthfeel is punicalagin, followed by the relatively small amount of other HTs, such as gallagic acid, ellagic acid and punicalin (Mayuoni-Kirshinbaum and Porat, 2014). However, the levels of


What Can We Learn About the Traits of Aril Juice by Studying … 217 punicalagin, gallagic acid and ellagic acid in PJ are very low in comparison to the levels in the fruit peels. For example, the levels of punicalagin, punicalin and gallagic acid in these juices decreased to about 6 x103, 2.5 x 104 and 103, respectively, when compared to their contents in the peel’s homogenate of the 29 cultivars. The level of ellagic acid was below the detection level under these conditions (i.e., below 0.5 mg/L) (Tzulker et al., 2007). However, despite these low levels, the effect of HTs influenced the astringent feeling in PJ taste. Pomegranate aroma also influences PJ taste. Volatiles directly affect the sensory quality of fresh and processed PJ products whose aroma is formed by a complex group of dozens of volatiles, including alcohols, aldehydes, ketones, monoterpenoids and linear hydrocarbons, which provide a mixture of various tastes (Mayuoni-Kirshinbaum and Porat, 2014). It was noted that by examining 18 Israeli cultivars, hexanal and its derivatives (hexanal, hexanol and (Z)-3-hexenol) and terpenes (β-pinene, limonene, α-terpineol and β-caryophyllene) were found to be key aroma volatiles in PJ (Mayuoni-Kirshinbaum and Porat, 2014). Eighteen of these compounds were also detected in nine Spanish cultivars, revealing that the most abundant compounds were trans-2-hexenal, 3-carene, α-terpinene and α-terpineol. The total concentration of these volatiles, identified and quantified using the hydrodistillation technique, ranged from 1.7 to 10.9 g kg−1. Overall consumer liking of PJs was associated with the presence of monoterpenes such as α-pinene, β-pinene, β-myrcene, limonene and γterpinene as revealed by a group of 30 consumers (Calin-Sanchez et al., 2011). However, no clear correlation was found between the total concentration of volatile compounds and PJ taste (Calin-Sanchez et al., 2011).

POMEGRANATE ARILS COLOR In addition to taste, PJ color also has a great impact on consumers’ purchasing decisions (Melgarejo et al., 2011; Mena et al., 2011). Color varies significantly between cultivars, from white-yellow through orange-


218


pink, to intense red and purple. The color examined by a colorimeter is divided into five parameters: L* defines lightness, a* green-red transition, b* blue-yellowness, C saturation, and H hue angle shown to be effective in predicting visual color appearance, where 0 or 360 = red purple, 90 = yellow, 180 = green, and 270 = blue. It was previously suggested that the a* values could serve as a valid estimate of anthocyanin concentration in aril juices (Borochov-Neori et al., 2009). By examining these color parameters in 29 different pomegranate cultivars, we found that TA, citric acid, TSS, glucose and fructose showed a significant negative correlation with the L* parameter with R2 values of 0.51, -0.53, -0.78, -0.58, -0.59, respectively. TA, citric acid and TSS significantly correlated to a* with R2 values of 0.60, 0.58, 0.55, respectively, while b* significantly correlated only to citric acid (R 2 = 0.57) (Dafny-Yalin et al., 2010). However, the relationship between the color parameters and the sour-sweet cultivars varies in different studies. For example, Caliskan and Bayazit (2012) examined 76 pomegranate cultivars in the Mediterranean region of Turkey and showed that the L* value was the highest in the sour and sour-sweet cultivars, while a* was the highest in the sweet cultivars, which had lower C and H values, as indicated by their red aril colors. However, an examination of 10 cultivars from southern Italy showed that L* was the lowest in the sour cultivars (Ferrara et al., 2014), and an examination of 13 cultivars from Tunisia showed that the sour and sour-sweet cultivars had more red-colored juices (Zaouaya et al., 2012). Cultivars from Spain, however, showed that in general, the juices having the highest values of green-red coordinate (a*) were those from the sweet cultivars, followed by the sour and sour-sweet cultivars. A similar pattern to that described for the a* values was found for the C* values, and the lightness of juices (the lower the L* value, the darker the juice) increased from sour-sweet to sour and sweet PJ (CalinSanchez et al., 2011). Zaouaya et al. (2012) also reported a correlation between the juice’s visual color and color indices of L*, a*, b*, emphasizing the effectiveness of the grading color scale in determining the color of PJ without using the digital system. The correlation was also observed between the visually


What Can We Learn About the Traits of Aril Juice by Studying … 219 observed juice color and individual anthocyanins emphasize the efficiency of the grading color scale in determining PJ color. The intensity of the color was significantly affected by climate in the growth habitat (Borochov-Neori et al., 2009; Schwartz et al., 2009b; Borochov-Neori et al., 2011). Aril juices from fruits grown in the hot and dry desert climate in Israel’s southern Arava Valley had a lighter color than those grown in a Mediterranean climate (Schwartz et al., 2009b). The color of aril juices from fruits grown in a Mediterranean climate was more intense and had more of the magenta-red color. In addition, by analyzing 11 cultivars grown in the southern Arava Valley during the fruits’ development, it was revealed that the fruits experienced different climatic conditions during their development and ripening (Borochov-Neori et al., 2009). The higher intensity of the red color of the arils obtained at the beginning (early July) and end (October) of the sampling period, compared to the extreme temperatures in late-July through September, could probably be attributed to anthocyanin degradation at high temperatures (Oren-Shamir and Nissim-Levi, 1999). Indeed, the intensity of the red color of the arils was found to be inversely related to the sum of heat units accumulated during fruit development and ripening (Borochov-Neori et al., 2009). Fruits from four cultivars were harvested from summer to winter from trees grown in Israel. All six anthocyanin pigments previously detected in pomegranates, including 3-mono- and 3,5-diglucosides of cyanidin, delphinidin and pelargonidin, were identified in the arils of these fruits (Borochov-Neori et al., 2011). The total anthocyanin content in the arils obtained in mid-July was notably lower than that measured in midDecember. Cyanidins were slightly more abundant than delphinidins in the arils of mid-July fruit, and were found at a significantly higher proportion than delphinidins and pelargonidins in the arils of mid-December fruit (Borochov-Neori et al., 2011). Nearly all of the anthocyanins were in the form of diglucosides in the arils of mid-July fruit, whereas most of the anthocyanins of mid-December arils (∼69%) were in the form of monoglucosides (Borochov-Neori et al., 2011). Thus, the anthocyanins in arils of mid-July and mid- December fruits differed markedly in content,


220


profile and level of glucosylation. This result is in agreement with the temperature stabilizing effect attributed to anthocyanin glucosylation (Welch et al., 2008). These studies indicate that the full genetic potential for color production in each cultivar is revealed only under particular growth conditions (Schwartz et al., 2009b). For example, higher temperatures in the desert climate, adversely affects anthocyanin content and consequently the color of the arils.

SEED HARDNESS Seed hardness should also be considered when the arils are eaten. An examination of 20 cultivars by experts who gave a score to seed hardness (from 1 = soft to 7 = hard) showed that nine of the cultivars had a score of 1 to 2, and only two a score of 7 (Alcaraz-Mármola et al., 2017). In general, sweet cultivars had the lowest and sour cultivars had the highest values in seed hardness (R2 = 0.68 for seed hardness and TA). It was also shown that the cultivars with high sensorial taste scores and low seed hardness values are preferred by consumers for consumption of fresh arils (Alcaraz-Mármola et al., 2017).

FUTURE PERSPECTIVES Investigations of different pomegranate cultivars from diverse collections over the past couple of decades have revealed correlations between the chemical components of PJ and their sensory and health beneficial traits. Despite the increasing knowledge of the compounds and their levels detectable in PJ, many compounds remain to be annotated. Recently, using several extraction methods and different analytical tools, the nature of several phenol compounds was reveled in 12 different cultivars (Brighenti et al., 2017). Yet, data are lacking from other cultivars


What Can We Learn About the Traits of Aril Juice by Studying … 221 in other collections, which could provide more knowledge about the diversity of these compounds. Chemical analysis should go hand-in-hand with genomic analysis in order to reveal the molecular basis underlying the production and accumulation of these compounds in PJ. In the past few years, progress has been made in revealing the genes in the biosynthetic pathway leading to HTs (Ono et al., 2016), and those regulating the levels of anthocyanin content (Ben-Simhon et al., 2011). Moreover, a detailed genetic map of pomegranate based on 1092 SNPs was published and 25 QTLs for fruit traits were determined. The map includes QTLs for total soluble solids (TSS), fruit weight and perimeter, seed hardness, aril color and weight, and plant height (Ophir et al., 2014; Harel-Beja et al., 2015). Despite the progress of metabolite research in pomegranate and the availability of new genomic approaches, high throughput studies that associate between metabolite content and gene expression have not yet been reported. Further studies are still required to gain a better understanding of the regulatory mechanisms controlling important pomegranate metabolites, which would facilitate breeding nutritious pomegranate cultivars that are also tasteful.

ACKNOWLEDGMENT The study of the authors is supported by the BARD, Binational Agricultural Research & Development Fund, project No. IS-4822-15 R.

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What Can We Learn About the Traits of Aril Juice by Studying … 225 Harel-Beja, R., Sherman, A., Rubinstein, M., Eshed R., Bar-Ya'akov I., Trainin T., Ophir R., Holland.D. (2015). A novel genetic map of pomegranate based on transcript markers enriched with QTLs for fruit quality traits. Tree Genetics and Genomics 11, 109. Hasnaouia, N., Marsa, M., Ghaffarib, S., Trific, M., Melgarejod, P., and Hernandezd, F. (2011). Seed and juice characterization of pomegranate fruits grown in Tunisia: Comparison between sour and sweet cultivars revealed interesting properties for prospective industrial applications. Industrial Crops and Products 33, 374–381. Hmid, I., Hanine, H., Elothmani, D., and Oukabli, A. (2016). The physicochemical characteristics of Morrocan pomegranate and evaluation of the antioxidant activity for their juices. Journal of the Saudi Society of Agricultural Sciences http://dx.doi.org/10.1016/j.jssas.2016.06.002. Holland, D., Hatib, K., and Bar-Ya'akov, I. (2009). Pomegranate: Botany, Horticulture, Breeding, in Horticultural Reviews. John Wiley & Sons, Inc.), 127-191. Kaur, G., Jabbar, Z., Athar, M., and Alam, M. S. (2006). Punica granatum (pomegranate) flower extract possesses potent antioxidant activity and abrogates Fe-NTA induced hepatotoxicity in mice. Food Chemistry and Toxicoogyl 44, 984-993. doi: S0278-6915(05)00375-3 [pii]10.1016/j.fct.2005.12.001. Kaur, C., Pal, R. K., Kar, A., Gad, C., Sen, C., Kumar, R., et al. (2014). Caracterization of antioxudant and hydpoglycemic potential of poemgranate grown in India: a preliminary investigation. Journal of Food Biochemistry 38(7), 397–406. doi: S0278-6915(05)00375-3 [pii]10.1016/j.fct.2005.12.001. Kulkarni, A. P., and Aradhya, S. M. (2005). Chemical changes and antioxidant activity in pomegranate arils during fruit development. Food Chemistry 93(2), 319-324. Langley, P. (2000). Why a pomegranate? British Medical Journal 321(7269), 1153-1154. Legua, P., Melgarejo, P., Abdelmajid, H., Martinez, J., Martinez, R., Ilham, H., et al. (2012). Total Phenols and Antioxidant Capacity in 10


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Moroccan Pomegranate Varieties. Journal of Food Science 71, C115120. Legua, P., Melgarejo, P., Martnez, M., and Hernandez, F. (2000). Evolution of sugars and organic acid content in three pomegranate cultivars (Punica granatum L). Options Mediterr. 49, 99–104. Li, X., Wasila, H., Liu, L., Yuan, T., Gao, Z., Zhao, B., Ahmad, I. (2015). Physicochemical characteristics, polyphenol compositions and antioxidant potential of pomegranate juices from 10 Chinese cultivars and the environmental factors analysis. Food Chemistry 175, 575-584. doi: 10.1016/j.foodchem.2014.12.003. Martinez-Nicolas, J. J., Melgarejo, P., Legua, P., Garcia-Sanchez, F., and Hernandez, F. (2016). Genetic diversity of pomegranate germplasm collection from Spain determined by fruit, seed, leaf and flower characteristics. Peer Journal 4, e2214. doi: 10.7717/peerj.2214. Matapathi, S. S., Patil, A. B., Jonees, P. N., and Savalgi, V. V. (2004). Studies on Screening of Pomegranate Cultivars for Wine Production. Karnataka Journal of Agriculture Science 17, 725-730. Mayuoni-Kirshinbaum, L., and Porat, R. (2014). The flavor of pomegranate fruit: a review. Journal Science and Food Agriculture 94, 21-27. doi: 10.1002/jsfa.6311. Melgarejo, P., Calin-Sanchez, A., Vazquez-Araujo, L., Hernandez, F., Martinez, J. J., Legua, P., Carbonell-Barrachina, A. A. (2011). Volatile composition of pomegranates from 9 Spanish cultivars using headspace solid phase microextraction. Journal of Food Science 76(1), S114-120. doi: 10.1111/j.1750-3841.2010.01945.x. Melgarejo, P., Salazar, D., Artes, F., and 2000 (2000). Organic acids and sugars composition of harvested pomegranate fruits. European Food Research and Technology 211, 185–190. Melgarejo-Sánchez, P., Martínez, J. J., Legua, P., Martínez, R., Hernández, F., and Melgarejo, P. (2015). Quality, antioxidant activity and total phenols of six Spanish pomegranates clones. Scientia Horticulturae 182, 65–72. Mena, P., Garcia-Viguera, C., Navarro-Rico, J., Moreno, D. A., Bartual, J., Saura, D., Marti, N. (2011). Phytochemical characterisation for


What Can We Learn About the Traits of Aril Juice by Studying … 227 industrial use of pomegranate (Punica granatum L.) cultivars grown in Spain. Journal Science and Food Agriculture 91, 1893-1906. doi: 10.1002/jsfa.4411. Mousavinejad, G., Emam-Djomeh, Z., Rezaei, K., and Khodaparast, M. H. H. (2009). Identification and quantification of phenolic compounds and their effects on antioxidant activity in pomegranate juices of eight Iranian cultivars. Food Chemistry 115, 1274–1278. Nuncio-Jáuregui, N., Nowicka, P., Munera-Picazo, S., Hernández, F., Carbonell-Barrachina, A., and Wojdyło, A. (2015). Identification and quantification of major derivatives of ellagic acid and antioxidant properties of thinning and ripe Spanish pomegranates. Journal of functional foods 12, 354–364. Ono, N. N., Qin, X., Wilson, A. E., Li, G., and Tian, L. (2016). Two UGT84 Family Glycosyltransferases Catalyze a Critical Reaction of Hydrolyzable Tannin Biosynthesis in Pomegranate (Punica granatum). PLoS One 11, e0156319. doi: 10.1371/journal. pone.0156319. Ophir, R., Sherman, A., Rubinstein, M., Eshed, R., Sharabi Schwager, M., Harel-Beja, R., Bar-Ya'akov, I., Holland, D. (2014). Single-nucleotide polymorphism markers from de-novo assembly of the pomegranate transcriptome reveal germplasm genetic diversity. PLoS One 9, e88998. doi: 10.1371/journal.pone.0088998. Oren-Shamir, M., and Nissim-Levi, A. (1999). Temperature and gibberellin effect on growth and anthocyanins pigmentation in Photinia leaves. Journal of Horticultural Science 74, 355-360. Ozgen, M., Durgac, C., Serce, S., and C., K. (2008). Chemical and antioxidant properties of pomegranate cultivars grown in the Mediterranean region of Turkey. Food Chemistry 111, 703–706. Pande, G., and Akoh, C.C. (2009). Antioxidant capacity and lipid characterization of six Georgia-grown pomegranate cultivars. Journal of Agriculture and Food Chimistry 57, 9427-9436. doi: 10.1021/jf901880p. Poyrazolua, E., Gokmen, V., and Artιk, N. (2002). Organic acids and phenolic compounds in pomegranates (Punica granatum L.) grown in Turkey. Journal of Food Composition and Analysis 15, 567–575.


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Quideau, S., Deffieux, D., Douat-Casassus, C., and Pouysegu, L. (2011). Plant polyphenols: chemical properties, biological activities, and synthesis. Angewandte Chemie International Edition in English 50, 586-621. doi: 10.1002/anie.201000044. Radunić R., Špika, R., Ban, S. G., Gadže, J., Díaz-Pérez, J. C., and D., M. (2015). Physical and chemical properties of pomegranate fruit accessions from Croatia. Food Chemistry 177, 53–60. Schwartz, E., Glazer, I., Bar-Ya'akov, I., Matityahu, I., Bar-Ilan, I., Holland, D., Amir, R. (2009a). Changes in chemical constituents during the maturation and ripening of two commercially important pomegranate cultivars. Food Chemistry 115, 965-973. Schwartz, E., Tzulker, R., Glazer, I., Bar-Ya'akov, I., Wiesman, Z., Tripler, E., Bar-Ilan, I. Fromm, H. Borochov-Neori, H. Holland, D. Amir, R. (2009b). Environmental conditions affect the color, taste, and antioxidant capacity of 11 pomegranate accessions' fruits. Journal of Agriculture and Food Chimistry 57, 9197-9209. doi: 10.1021/ jf901466c. Seeram, N. P., Adams, L.S., Henning, S. M., Niu, Y., Zhang, Y., Nair, M. G., Heber, D. (2005). In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice. The Journal of Nutritional Biochemistry 16, 360-367. doi: S0955-2863(05)00019-7 [pii]10.1016/ j.jnutbio.2005.01.006. Seeram, N. P., Schulman, R. N., and Heber, D. (2006a). Pomegranates: ancient roots to modern medicine. New York: CRC/Taylor & Francis. Seeram, N. P., Zhang, Y., Reed, J. D., Krueger, C. G., and Vaya, J. (2006b). Pomegranate Phytochemicals, in Pomegranates Ancient Roots to Modern Medicine, eds. N. P. Seeram, R. N. Schulman & D. Heber. CRC Press), 3–29. Sepúlveda, E., Sáenz, C., Peña, A., Robert, P., Bartolomé, B., and GómezCordovés, C. (2010). Influence of the genotye on the anthocyanin composition, antioxidant capacity and color of the Chilean


What Can We Learn About the Traits of Aril Juice by Studying … 229 pomegranate (Punica granatum L.) juices Chilean journal of agricultural resreach 70, 50-57. Singleton, V. L., and Rossi, J. A. (1965). Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. American Journal of Enology and Viticulture 16, 144-158. Still, D. W. (2006). Pomegranates: A Botanical Perspective, in Pomegranates: Ancient Roots to Modern Medicine, eds. N. P. Seeram, R. N. Schulman & D. Heber. CRC Press), 199-209. Tehranifara, A., Zareia, M., Nematia, Z., Esfandiyaria, B., and Vazifeshenas RB. (2010). Investigation of physico-chemical properties and antioxidant activity of twenty Iranian pomegranate (Punica granatum L.) cultivars. Scientia Horticulturae 126, 180–185. Tezcan, F., Gültekin-Özgüven, M., Diken, T., Özçelik, B., and Erim, F. (2009). Antioxidant activity and total phenolic, organic acid and sugar content in commercial pomegranate juices. Food Chemistry 115, 873– 877. Turrini, E., Ferruzzi, L., and Fimognari, C. (2015). Potential Effects of Pomegranate Polyphenols in Cancer Prevention and Therapy. Oxidative Medicine and Cellular Longevity, 938475. doi: 10.1155/2015/938475. Tzulker, R., Glazer, I., Bar-Ilan, I., Holland, D., Aviram, M., and Amir, R. (2007). Antioxidant activity, polyphenol content, and related compounds in different fruit juices and homogenates prepared from 29 different pomegranate accessions. Journal of Agriculture and Food Chemistry 55, 9559-9570. doi: 10.1021/jf071413n. Vegara, S., Mena, P., Marti, N., Saura, D., and Valero, M. (2013). Approaches to understanding the contribution of anthocyanins to the antioxidant capacity of pasteurized pomegranate juices. Food Chemistry 141, 1630-1636. doi: 10.1016/j.foodchem.2013.05.015. Verma, N., Mohanty, A., and Lal, A. (2010). pomegranate genetic resources and germplasms. Fruit, vegetable, and cereals science and biotechnology 4, 120-125. Vlachojannis, C., Erne, P., Schoenenberger, A. W., and ChrubasikHausmann, S. (2015). A critical evaluation of the clinical evidence for


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pomegranate preparations in the prevention and treatment of cardiovascular diseases. Phytotherapy Research 29, 501-508. doi: 10.1002/ptr.5280. Welch, C. R., Wu, Q., and Simon, J. E. (2008). Recent Advances in Anthocyanin Analysis and Characterization. Current Analytical Chemistry 4, 75-101. doi: 10.2174/157341108784587795. Zaouaya, F., Menab, P., Garcia-Viguerab, C., and Marsa, M. (2012). Antioxidant activity and physico-chemical properties of Tunisian grown pomegranate (Punica granatum L.) cultivars. Industrial Crops and Products 40, 81– 89. Zhang, Y., Wang, D., Lee, R., Henning, S. M., Seeram, N. P., and Heber, D. (2008). AGFD 150-Phenolic content of commercial pomegranate dietary supplements and their antioxidant capacity. Abstracts of Papers of the American Chemical Society 236.




Chapter 8

POMEGRANATE: CULTIVATION, ANTIOXIDANT PROPERTIES, HEALTH BENEFITS AND EFFECTS OF INDUSTRIAL PROCESSING Marina Cano-Lamadrid, PhD1, Patricia Navarro-Martinez, PhD2, Santiago Lopez-Miranda, PhD2, Aneta Wojdyło, PhD3, Angel A. Carbonell-Barrachina PhD1 and Antonio Jose Perez-Lopez, PhD2,* 1

*

Universidad Miguel Hernández de Elche (UMH), Escuela Politécnica Superior de Orihuela, Department of Agro-Food Technology, Research Group ‘Food Quality and Safety’, Ctra. Beniel, Orihuela, Alicante, Spain 2 Universidad Catolica de Murcia (UCAM), Department of Food Technology & Nutrition, Guadalupe, Murcia, Spain



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M. Cano-Lamadrid, P. Navarro-Martinez et al.

Wrocław University of Environmental and Life Sciences, Department of Fruit, Vegetable and Plant Nutraceutical Technology, Wrocław, Poland 3

ABSTRACT Considering the antioxidant capacity positively correlated with indicators such as polyphenols and flavonoids, pomegranate (Punica granatum L.) and pomegranate based products can be considered as functional food due to the huge amount of them such as hydrolysable tannins (punicalalgins, punicalins) and phenolic acids (gallic and ellagic acid). There are several groups of commercial interest such as cv. Mollar and cv. Wonderful. Among varieties, the functional capacity (polyphenols, punicic acid), sensory profile (color, sweetness, seed hardness) and consumer`s acceptance differ a lot. Moreover, the influence of different culture practices in the field such as thinning or regulated deficit irrigation (RDI), fruits cultivated under RDI are called “hydroSOStainable” products, allows us to increase as bioactive compounds as some sensory attributes. Nowadays, pomegranate is usually available on the market freshly or processed into beverages: concentrated, wine and mainly, juices. They are also available as dried arils, jams, jellies, and are used for candy production. During the industrial processing, especially pomegranate juice, the waste (mainly peel and carpelar membranes) is on average account for 50 % of the fresh weight. The huge quantity of beneficial compounds find into them could be included in new products as technologic ingredient (preservative or colorant) or functional one (pharmaceutics), taking advantage of increasing demand for natural products which could serve as alternative food ingredients. Recent studies concluded that pomegranate and pomegranate products have been specifically associated with inhibition of some kind of cancer (prostate, breast and lung), reduction of dyslipidaemia, cardiovascular issues, antioxidant stress effect and antidiabetic properties, among others. Otherwise, Pomegranate industrial processing and impact on bioactive components is a impact target on consumers.


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1. INTRODUCTION An increasing number of studies have suggested that consumption of fruits can play a protective role in reducing the risk of cancer, diabetes, and cardiovascular diseases, among other illnesses (Kurotani et al., 2013; Masala et al., 2012). Increasing evidence suggests that antioxidant properties are likely to be a contributing factor in the effects of those components in vivo (Kalt and Kushad, 2000). Recently, the huge interest in pomegranate has increased worldwide, especially in western countries. This may be due to its myriad of health benefits. However, current production and shipments are beginning to saturate existing and consolidated markets, such as Europe, the United States of America and the Middle East (Costa and Melgarejo, 2000). Therefore, pomegranate producers and traders are currently searching for new destinations for pomegranates. The Asian market represents the greatest opportunities, because as a result of different research projects, these markets are developing new products using this fruit. For this reason, they require an increasing volume of fresh pomegranate, as well as pomegranate juice (World Pomegranate Market Supply, Demand and Forecast, 2015). Pomegranate is an extraordinarily rich source of vitamin C and phenolic compounds, such as ellagic acid, punicalagin, and anthocyanins. Also, pomegranate juice shows an antioxidant activity three times higher than those of green tea and red wine (Gil et al., 2000; Mena et al., 2011).

2. POMEGRANATE PRODUCTION Pomegranate has been considered one of the most extended fruit trees since the earliest times. The original growing area was the Near East, from where it extended to the rest of Asia and the Mediterranean countries, arriving in South America and North America. Pomegranate farming is currently located in semi-arid zones, mild-temperature and subtropical


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climates (Galindo et al., 2013), and the major cultivation areas are India, Spain, Israel and the United States of America (Peña-Estévez et al., 2016). The expansion of pomegranate is due to its good adaptation to arid and semi-arid conditions, and to be able to maintain a proper production level in conditions of climate and soil in which other crops would not do so profitably. The improvement in growing techniques and plant material selection to increase the production and quality of fruit, and the discovery of a huge nutritional, pharmacological, functional and cosmetic properties of the pomegranate, has made this fruit highly demanded by the consumers. World pomegranate production has increased substantially in the last decade. Pomegranate is not only consumed as fresh fruit, but also as juice and other derivatives as blends with tea or nuts (McLean et al., 2014).

Botany Pomegranate (Punica granatum L.) belongs to the Punicaceae botanic family. It is a shrub, with dense vegetation and deciduous tree with a maximum height of between 3 and 4 m, although the shape and size of the trees could be very conditioned by the training and pruning systems (McLean et al., 2014). Branches tend to be thin and prickly, while the leaves are bright and dark green. Flowers are of a very intense orange-red color, and they appear between spring and summer, depending on the growing areas and climatic conditions. Fruits consist of hundreds of seeds surrounded by a very juicy red pigment. The fruit peel is generally smooth but coriaceous, and could be yellow, orange or red.

Climate Pomegranate is a very high temperature tolerant plant, and the optimal temperature for vegetative growth is 30 ºC or above for at least three months a year. Due to the Mediterranean origin of this species,


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pomegranate also tolerates drought, although controlled water supply is necessary to carry out a plantation with guarantees and to obtain stable and profitable production. During prolonged periods of drought, fruit production will be lost, and significant injury to young trees is likely to occur. In relation to low temperature limits, during winter dormancy, most pomegranate varieties can withstand temperatures up to -10 °C, and the most resistant ones can be safe without wood damage until -14 °C (McLean et al., 2014). However, like most fruit trees, pomegranates are very vulnerable to cold temperatures in the end-of-cycle or flowering stages in which early autumn or late spring frosts can damage vegetative and flowering organs.

Soil One of the main soil conditions for pomegranate growth is that they do not tolerate for long time soaked soils with little aeration. Long periods of high humidity can damage trees. Pomegranates are better adapted to deep and loamy soils than in sandy or clayey soils. They tolerate from moderately acidic to slightly alkaline soils and grow best in a soil pH range of 5.5 to 7.2 (McLean et al., 2014). Pomegranate plants are moderately salt tolerant and can withstand irrigation with water containing from 2000 to 2500 mg/L of salt.

Irrigation Pomegranate presents a considerable resistance to drought, as other xeromorphic plants, due to their relatively high content of apoplastic water in leaves, as well as other mechanisms of tolerance to drought conditions (Rodriguez et al., 2012). Although pomegranates are plants with high tolerance to drought, controlled water supply will ensure adequate vegetative development as well as good yield (Holland et al., 2009). In


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Israel, pomegranate is normally irrigated with 5000 to 6000 m3/ha per year (Schwartz et al., 2009). In Spain, one of the most important producers in Europe, pomegranate trees are irrigated with 4500-5500 m3/ha per year (Intrigliolo et al., 2011). As most tree crops, the most suitable irrigation system for pomegranates is drip irrigation. Alternatives, such as PRD (Partial Root Drying), have also been studied, but the additional cost of introducing this type of irrigation system is an important economic drawback (Parvizi and Sepaskhah, 2015). Sprinkler irrigation is not recommended as it would increase the spread of pests and diseases, as well as in increase of flower damage due to their high sensitive to humidity. The main problem caused by excessive irrigation, especially in autumn, is the risk of skin cracking due to excessive growth of the fruit, which negatively affects the fruit quality and its post-harvest management (McLean et al., 2014). Khattab et al. (2011) determined that an excess of irrigation increased the fruit size and the total soluble solids and the fruit firmness decreased. The new trend in irrigation strategies applied to pomegranate is to apply a controlled water deficit or deficit irrigation. Deficit irrigation induces a smaller final fruit size and a reduction in yield, as well as some physiological changes as an earlier ripening (Mellisho et al., 2012; Galindo et al., 2013). Water deficit during fruit growth may have a positive effect on some quality parameters, improving taste and increasing total soluble solids (Mpelasoka et al., 2001). The growth stage in which the water deficit is applied can affect the plant behavior. On one hand, when water restriction is applied at the end of the fruit development, during ripening, an increase in total soluble solids and fruit color intensity is observed (Laribi et al., 2013). On the other hand, water deficit during fruit linear growth stage may increase the concentration of some bioactive compounds such as anthocyanins (Laribi et al., 2013). Peña-Estevez et al. (2016) determined that deficit irrigation yielded fruits with better quality and healthy attributes.


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Fertilization Pomegranate is not very demanding as far as fertilization needs, as a common characteristic of a rustic plant. The appropriate time for annual P and K fertilization is leaf fall, while N rich fertilizers should be provided at the beginning of the vegetative period (McLean et al., 2014). It is desirable to supplement mineral supply with organic matter fertilizers every 2 or years. Both excess of irrigation or N fertilization in young plants can cause an excessive vegetative growth of trunk and branches with a thinner structure that can later condition the fruit production load due to the appearance of branches breaks. In adult plants, the high N fertilization in late summer or early fall will have a detrimental impact on fruit color, and may also increase tree susceptibility to low temperatures. A balanced supply of N, P, and K increases fruit size, yield and total soluble solids (Dhillon et al., 2011). One of the few deficiencies found in pomegranate is Zn, which appears as an unusual yellowing of the leaves. If necessary, foliar Zn fertilization in spring is recommended (MacLean et al., 2014).

Pruning and Training Systems Actually, pruning and training are fundamental to regulate vegetative and reproductive development through a suitable canopy development and an effective use of light and other resources available to the plant. One of the most common variants in training of pomegranate in the choice of single or multiple trunk plant. The main advantage of using a single trunk lays in the ease of some cultivation techniques such as pruning. However, pruned plants with a multi-stem allow a better replenishment of vegetation in case of severe frost damage to the wood (McLean et al., 2014).


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Pomegranate trees require annual light pruning to stimulate the growth of productive shoots and avoid the accumulation of large quantities of old wood that require severe pruning that negatively affect production and viability of the plant in the medium and long term. Winter pruning is used to define the architecture of the tree, trying to keep its structure open for adequate aeration and illumination of the interior of the canopy. Pomegranates require at least 6 hours of direct sunlight a day to ensure good color and fruit productivity. Air circulation is important, especially in spring during flowering. Flowers may have pollination problems if they are in too humid surroundings. In summer, a lighter pruning can be done to eliminate unproductive vegetative shoots.

3. NUTRITIONAL COMPOSITION Pomegranate has many chemical compounds of high biological value, is a very important source of bioactive compounds such as polyphenols, flavonoids, elagitannins, proanthocyanidins and minerals, mainly K, N, Ca, P, Mg and Na (Table 1). The edible portion of the pomegranate represents about 50% of the total weight, in turn consists of 80% of fleshy part and 20% of seed. The composition of the grains is water (85%), sugars (10%), mainly fructose and glucose, organic acids (1.5%), mainly ascorbic acid, citric and malic, bioactive compounds such as polyphenols and flavonoids (mostly anthocyanins). Pomegranate grains are an important source of lipids because the seeds contain a number of fatty acids ranging from 12 to 20% of their total weight (dry weight). The fatty acid profile is characterized by a high content of unsaturated fatty acids, such as linoleic, linoleic, punicic, oleic, stearic and palmitic acid (Andreu Sevilla et al., 2008; Mena et al., 2011; Ullah et al., 2012).


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Table 1. Compositional and nutritional characteristics of pomegranate (Punica granatum L.) (USDA, 2016)

Nutrient Proximate composition Energy Water Protein Total lipid Carbohydrate Total dietary fiber Total sugars Minerals Calcium, Ca Iron, Fe Magnesium, Mg Phosphorus, P Potassium, K Sodium, Na Zinc, Zn Vitamins Vitamin C (ascorbic acid) Thiamin Riboflavin Niacin Vitamin B-6 Folate Vitamin E (α-tocopherol) Vitamin K (phylloquinone) Lipids Total saturated Total monounsaturated Total polyunsaturated Total trans Cholesterol

Unit

Content per 100 g

Content per 1 fruit (282 g)

kcal g g g g g g

83 77.93 1.67 1.17 18.70 4.0 13.67

234 219.76 4.71 3.30 52.73 11.3 38.55

mg mg mg mg mg mg mg

10 0.30 12 36 236 3 0.35

28 0.85 34 102 666 8 0.99

mg mg mg mg mg µg mg µg

10.2 0.067 0.053 0.293 0.075 38 0.60 16.4

28.8 0.189 0.149 0.826 0.211 107 1.69 46.2

g g g g mg

0.120 0.093 0.079 0 0

0.338 0.262 0.223 0 0


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4. BIOACTIVE COMPONENTS Anthocyanins Among phytochemicals, the anthocyanins (water-soluble vacuolar pigments) belong to the flavonoid group, and responsible for red, purple and blue colors in fruits and vegetables, including pomegranate. Structurally anthocyanins are glycosylated or acylated anthocyanidins, linked to sugars, which confer stability and water solubility to the molecule. Fruit exterior and juice colors are important characteristics to the consumer acceptance and commercial value. On the other hand, the antioxidant activity of them has been studied both in vitro and in vivo, concluding positive effects on human health (biological, pharmacological, anti-inflammatory, antioxidative, and chemoprotective properties) (Zhao et al., 2013). The anthocyanins profile and the quantity depend on the cultivar, place, ripening state and processing conditions of pomegranate and products, the values found in the bibliography are summarized in Table 2.

Ellagitannins Ellagitannins are the most characteristic group of high molecular weight polyphenols in pomegranate, especially in the peel, including hydrolyzable tannins, mainly punicalagin. The juice obtained by squeezing the whole fruit presents a higher concentration of ellagitannins than the commercial juices (mainly prepared from only arils), and contain the unique ellagitannin, punicalagin. Punicalagin is not absorbed intact into the blood stream, but is hydrolyzed to ellagic acid and rapidly metabolized into metabolites of short life. In this way, ellagitannins are metabolized into urolithins (bioactive compounds that might inhibit prostate cancer) by gut flora, which are conjugated in the liver and excreted in the urine (Li et al., 2015; Heber 2011).


Table 2. Summary of anthocyanin profiles in pomegranate and their products Variety

Country Matrix

Units

Lvbaoshi

China

Peel

Hongbaoshi

China

Peel

Moshiliu

China

Peel

Valenciana

Spain

Juice

Mollar

Spain

Juice

Piñon Tierno

Spain

Juice

Hizcaznar

Spain

Juice

Wonderful

Spain

Juice

Daqingpi

China

Flowers

mg/100 fw mg/100 fw mg/100 fw μM c3-OGlu μM c3-OGlu μM c3-OGlu μM c3-OGlu μM c3-OGlu undefined

Wonderful

Undef

Arils

Wonderful

Undef

Ovendried

Dp3, 5dG¥ 1.2

Cy3, 5dG 0.8

Pg3, 5dG 0.6

Dp3G

Cy3G

Pg3G 0.6

Cypent -

Tota lA 5.3

0.5

1.6

1.1

2.8

1.2

0.4

4.6

3.0

-

13.1

6.6

2.1

1.0

34.4

53.5

4.7

-

115

3.39.6 ndα 8.4 nd-4.6

19.2nd-13.3 63.7 8.2-56.8 2.7-14.9

nd10.8 nd

5.3-11.9

-

-

2.2-22.2

-

-

2.7-6.6

nd

2.9-4.8

-

-

8.8

16.735.7 100

nd

15.4

23.839.8 8.943.9 21.7112 119

12.6

-

-

37.3

57.4

nd

47.1

70.3

5.0

-

-

-

-

Detected -

-

Detected -

-

μg/g

-

-

-

-

-

-

-

251

μg/g

-

-

-

-

-

-

-

97.4


Reference Zhao et al. (2013) Zhao et al. (2013) Zhao et al. (2013) Legua et al. (2016) Legua et al. (2016) Legua et al. (2016) Legua et al. (2016) Legua et al. (2016) Zhang et al. (2011) Jaiswal et al. (2010) Jaiswal et al. (2010)

242


Variety

Country Matrix

Units

Anardana

Undef

μg/g

Sun-dried arils Juice

Dp3, 5dG¥ -

Cy3, 5dG -

Pg3, 5dG -

Dp3G

Cy3G

Pg3G

-

-

-

Cypent -

Tota lA 42.2

Reference

Jaiswal et al. (2010) Wonderful Brazil mg/100 50.8 65.8 3.0 3.3 33.5 2.8 159 Araujo dw Santiago et al. (2016) Wonderful Brazil Powder mg/100 14.6- 27.10.9-1.9 1.7-3.2 11.30.7-1.5 56.4- Araujo dw 27.0 57.4 20.4 111 Santiago et al. (2016) Mollar Spain Juice mg/L 78.4 190 21.6 62.9 221 64.7 3 641 Trigueros et al. (2014) Mollar Spain Yogurt mg/L 31.4- 103.10- 9.8533.10- 80.60- 16.351.25- 275- Trigueros 61.80 162.70 16.45 49.35 125.40 23.05 1.75 442 et al. (2014) Mollar/Wond Spain Juice mg/L 25.32 162.31 0.9 14.06 175.45 30.08 Berenguer erful (60:40) et al. (2016) ¥ Dp3,5dG: delphinidin-3,5-diglucoside; Cy3,5dG: cyanidin-3,5-diglucoside; Pg3,5dG: pelargonidin-3,5-glucoside; Dp3G: delphinidin-3glucoside; Cy3G: cyanidin-3-glucoside; Pg3G: pelargonidin-3-glucoside; Cy-pent: cyanidin-pentoside; αnd = no detected.


Pomegranate

243

There are certain nutraceutical products based on this compound (punicalagin), such as capsules, ampules and concentrates, among others. The quality of these products can be affected by many factors during their formulation and industrial processing. Zhang et al. (2009) compared 27 commercial products, and found that some of them presented no detectable contents of tannins, not guaranteeing the authenticity of the pomegranate supplements.

Punicic Acid Fatty acids are found in the woody part of the pomegranate seeds or arils. The lipid profile is very interesting and more than 90% of its content consists of polyunsaturated fatty acids (Alcaraz et al., 2015). In addition, there are essential fatty acids very important for the human diet, such as linoleic, linolenic, and arachidonic acids. The fatty acid characteristic of pomegranate is punicic acid (≈70%) which plays a very important role in biological properties, including antidiabetic, antiobesity, antiproliferative and anticarcinogenic activities against various forms of cancer (Aruna et al., 2016).

Alkaloids Alkaloids are carbon-based substances which can be used, among other treatments, to treat worms in the human gastrointestinal tract (anthelmintic activity). Several studies reported the presence of alkaloids in different parts of the pomegranate fruit, especially peel (Swarnakar et al., 2013). On the other hand, the barks and roots of pomegranate are rich sources of pseudopelletierine, pelletierine, isopelletierine, methyl-pelletierine 1-pelletierine and methyl isopelletierines (Al-Shahwany et al., 2013; Sreekumar et al., 2014).


244


5. HEALTH EFFECTS The intake of fruits and vegetables per day has been estimated to be lower than the recommended dietary intake (RDI) (Di Cagno et al., 2011). The international recommendation (WHO/FAO) reports a minimum of 400 g of fruit and vegetables per day to improve health, preventing chronic diseases, such as heart disease, some cancers, diabetes and obesity (FAO, 2016). Recent scientific publications (Orgil et al., 2014; Banihani et al., 2014; Roseblat et al., 2015) have demonstrated the health benefits of pomegranate juice, which has drastically increased consumer interest in this fruit and its based-products. Each of the beneficial properties for health is detailed below.

Anti-Mutagenic, Antitumor and Anticarcinogenic Activities

Figure 1. Main activities associate with the two key bioactive compounds present in pomegranates, punicalagin and ellagic acid.

There is recent scientific evidence on the antiproliferative, antimutagenic, anticancer, and antitumor activities associated with pomegranate and its derivatives both in vitro and in vivo studies (Adhami et al., 2012; Zahin et al., 2014; Masci et al., 2016; Cano-Lamadrid et al.,


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2016, Les et al., 2015, Orgil et al., 2014). These activities are mainly associated with two compounds present in pomegranates, punicalagin and ellagic acid, which are present in higher concentrations in non-edible parts, such as the peel (Figure 1).

Prevention of Oxidative Deterioration Numerous studies (Kalaycıoğlu et al., 2017) have shown that oxidative stress contributes significantly to the development and progression of a wide variety of diseases. Oxidative stress is defined as the imbalance between pro-oxidants and antioxidants, with the first ones predominating over the second ones. Recently, Kalaycıoğlu et al. (2017) reviewed the huge quantity of scientific studies about the antioxidant capacity of pomegranate, based products and compounds from the fruit. This activity is related to phenolic compounds, including punicalagin isomers (α and β), ellagic acid derivatives, and anthocyanins presents in different concentration in pomegranate parts. However, some authors (Haghighian et al., 2016) suggest that punicalagin from pomegranate peel is one of the main phytochemicals that contribute to the total antioxidant capacity of pomegranate juice. All of these compounds are known for their properties for the elimination of pro-oxidants (free radicals) and for the inhibition of lipid oxidation.

Diabetes Diabetes prevention and treatment are of high priority in medical research. Fruit extracts have been used extensively in this context because they are natural, safe, and readily available. Various studies in vitro and in vivo have linked pomegranate (Punica granatum L.), with type 2 diabetes prevention and treatment. A non-edible part of the pomegranate, the flowers, has been prescribed for the treatment of diabetes due to its α-


246


glucosidase inhibitory activity (Li et al., 2005), which help improving insulin resistance (Huang et al., 2005) and the inhibition of postprandial hyperglycemia (Li et al., 2008) in a rat genetic model of obesity and type 2 diabetes. Another point of view was recently raised by Banihani et al. (2013), who concluded that there is no enough scientific evidence to support the use of pomegranate for the clinical management of type 2 diabetes. On the other hand, some authors (Li et al., 2008; Banihani et al., 2014) concluded that pomegranate juice consumption can be an additional contribution to control glucose levels in type 2 diabetes patients (not affected by the sex of the patient and was less potent in elderly patients).

Protection of Obesity and Cardiovascular Disease Recent studies in vivo and in vitro have been carried out to study the effect of pomegranate fruits and its products (supplements, juices or extracts) on the reduction of risk for people with overweight and obesity. Several authors (Haghighian et al., 2016; Almuammar et al., 2012) concluded that anthocyanins, tannins, polyphenols, and flavonoids from different parts of the pomegranate (leaf, flower, peel, seed, and juices) present anti-obesity activity. Some effects demonstrated to be: i) an increase of weight loss and body fat oxidation, ii) abdominal fat reduction and iii) a decrease of serum cholesterol glucose and insulin (Almuammar et al., 2012). Dyslipidemia, one of the most relevant risk factors for cardiovascular disease, is characterized by high levels of triglycerides, total cholesterol, and low-density cholesterol (LDL), and/or low cholesterol levels of high density (HDL) (Fodor, 2011). Research about the effect of consumption of pomegranate and its products in animals and humans have increased in the last decade (Aviram et al., 2008; Lynn et al., 2012); Aviram et al., 2013; Roseblat et al., 2015), especially taking into account the fruit peel. Recently, a clinical study has investigated the potential effects of pomegranate peel in humans (Haghighian et al., 2016).


Pomegranate

247

The study consisted of two groups of obese women (with dyslipidemia, 30 < body mass index (BMI) > 35 kg/m2), one of them received 500 mg pomegranate skin extract daily (n = 19) and the other received placebo (n = 19) for 8 weeks. Different parameters were measured as total cholesterol, HDL, LDL and triglycerides to know the lipid profile of serum and blood pressure. The results obtained were encouraging: a decrease of total cholesterol, LDL and triglycerides, and an increase of HDL, compared to placebo.

Others Other effects of pomegranate and its components on human health have been demonstrated, such as: i) anthelmintic properties due to alkaloids compounds (Shah et al., 2012; Al-Shahwany et al., 2013; Sreekumar et al., 2014), ii) oral health maintenance due to the antibacterial activity of polyphenols (Batista et al., 2014; Oliveira et al., 2007; Michelin et al., 2005), iii) neuroprotective properties, against Alzheimer disease, due to the urolithins generated by the bacterial flora from the pomegranate ellagitannins (Yuan et al., 2016; Subash et al., 2015; Hartman et al., 2006), and iv) protection of human skin (inhibition of skin damage by ultraviolet rays) by pomegranate seed oil due to the functionality of catechin and ellagic acid (Melo et al., 2014; Badea et al., 2015).

6. NOVEL PRODUCTS AND FUTURE TRENDS The commercial value of pomegranate fruit (Punica granatum L.) is very high due to the fact that all parts of the fruit can be used and because of its chemical composition, nutritional value, and characteristic taste. In the USA, the sales of pomegranate products increased from $84,500 in 2001 up to $66,000,000 in 2005 (Vázquez-Araújo et al., 2015), because manufacturers were deeply involved in the development of new pomegranate based products. On the market right now, pomegranate is


248


commonly available as fresh fruits, processed into beverages (juices, concentrates or wine) or as an additive to jams, jellies, and candies (Jaiswal et al., 2010; Tehranifar et al., 2010; Tezcan et al., 2009). Research, development and innovation are carried out in all stages of food: production, processing and distribution. Nowadays, consumers are demanding novel and healthier ready-to-eat products, which characteristics should be as close as possible to those of the fresh product, and which are produced under environment friendly farming conditions. The tendency on pomegranate based products can be divided into the following trends: i) hydroSOStainable fruits, ii) healthy snacks, iii) dairy products, iv) spraydrying encapsulation, v) new beverages, vi) pomegranate wine, and, vii) packaging and film.

HydroSOStainable Products There are environmental friendly agronomic practices in olive-, almond-, pistachio- and pomegranate-orchards, which simultaneously reduces farmers’ expenses, and improve quality of fruits (bioactive compounds, sensory attributes and consumer acceptance). To enhance these fruit parameters, pomegranate trees are grown under regulated deficit irrigation (RDI). As a result, for example, pomegranates and its products with a special sensory profile and chemical composition, were obtained; these fruits are different from the rest of pomegranates in the market (a high content of bioactive compounds and/or high sensory quality), and are called “hydroSOStainable” products (Cano-Lamadrid et al., 2017a; Noguera-Artiaga et al., 2016; Mena et al., 2013a; Cano-Lamadrid et al., 2015; Cano-Lamadrid et al., 2016; Carbonell-Barrachina et al., 2015).

Healthy Snacks Snacks are one of the best ways to incorporate pomegranate arils into the diet. Dried pomegranate arils might be consumed as ready-to-eat


Pomegranate

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snacks (Alaei et al., 2015; Kingsly et al., 2006). To prolong the arils shelflife, different drying processes can be applied (Calín et al., 2013; 2014), such as convective drying, vacuum-microwave drying or combined drying (convective pre-drying followed by vacuum-microwave finish drying, CPD-VMFD). The appearance of the dried arils currently present in the market is mostly not acceptable for consumers due to the fact that drying had a significant negative impact on the final product quality, especially their appearance (Bialonska et al., 2007). The major problem is the internal browning of arils after drying. A previous study (Calín-Sánchez et al., 2013) showed that high quality dried arils could be prepared using appropriate drying methods, mainly the combined method (CPD-VMFD). This method consists of convective pre-drying and vacuum-microwave finish drying, which improve the functionality and sensory complexity of dried fruits, reducing the time and temperature during processing (Figiel, 2010). Recently, osmotic dehydration was used as a pre-treatment of combined method describe above, increasing the functionality and quality, even the consumer acceptance (Cano-Lamadrid et al. 2017b).

Dairy Products Fruit juices, especially from pomegranate, have a high potential to be used as an ingredient in the dairy sector, such as yogurts, fermented milks and cheese (Cano-Lamadrid et al., 2017c; Gumienna et al., 2016; Trigueros et al., 2014). Apart of the increase of functional properties when the pomegranate is added to dairy products, the special interest of this application is based on its anthocyanins, which are water-soluble pigments and can improve visual organoleptic attributes. Trigueros et al. (2012) concluded that yogurt, rich in pomegranate juice (40%) is a promising antioxidant food (rich in phenolic compounds) with an attractive color (a reddish color close to that expected by consumers). The direction of research of pomegranate based dairy products focuses on reducing anthocyanins degradation during the commercial shelf-life (CanoLamadrid et al., 2017; Trigueros et al., 2014).


250


Spray-Drying Encapsulation Spray-drying process has been used for decades to encapsulate food ingredients, evaporating the water quickly to preserve the quality of the main bioactive compounds (Gharsallaoui et al., 2007). Several studies (Miravet et al., 2016; Goula et al., 2012) support the application of this method in different fields of both food and pharmaceutical industries. Rober et al. (2010) encapsulated polyphenols and anthocyanins from pomegranate with maltodextrin (MD) or soybean protein isolates using spray drying. Experimental data showed that polyphenols encapsulating efficiency were significantly better when soybean protein was used, whilst that of anthocyanins was better with maltodextrin, improving their storage efficiency. Moreover, Miravet et al. (2016) studied the production and the functional parameters of pomegranate juice powder produced by spraydrying using prebiotic fibers (resistant dextrins as healthy drying-aid agents) as compared with the typical MD. These authors indicated that this novel way to produce pomegranate powder could be used in both the food (soft drinks and ice creams) and pharmaceutical industries as a nutraceutical supplement in the prevention of obesity. During the industrial processing of pomegranate, a huge amount of wastes is generated, such as seeds. The pomegranate seed oil presents 65– 80% conjugated fatty acids, the most important of which is 9-trans,11cis,13-trans-octadecatrienoic acid, called punicic acid, which has important biological properties (Eikani et al., 2012). A new protocol for the use of pomegranate seeds was developed by Goula et al. (2012) using skimmed milk powder as encapsulating agent of the seed oil. On the other hand, the major polyphenol in pomegranates, punicalagin, is hydrolyzed to ellagic acid and rapidly metabolized into short-life metabolites. The encapsulation of these compounds into nanoparticles can increase the real functionality of the fruit. In this case, the use of pomegranate bioactive compounds with this technique in polylactic-coglycolic acid nanoparticles would improve their anticancer activity through increased cellular uptake (Shirode et al., 2015).


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New Beverages The main problem of pomegranate beverages is the degradation of color during processing and storage. The selection of pomegranate cultivar, pasteurization treatment, storage conditions and use of additives are the key factors to obtain high quality beverages. Mollar de Elche pomegranate juice is fully integrated in the Spanish and international markets, but its color is unattractive due to its low color intensity, which worsens significantly after the pasteurization of juices. For this reason, farmers are now introducing non-native varieties, mainly Wonderful (with a complementary sensory profile and a very intense red garnet color), to incorporate a 10-20% percentage of these fruits in the production of pomegranate juice industry (Vázquez- Araújo et al., 2014). On the other hand, the conditions of heat treatment (temperature and time) have a strong influence on the color in pomegranate juices. Several studies (Mena et al., 2013b; Vegara et al., 2013a) were focused on determining the best processing conditions to keep the garnet color of pomegranate arils during their juicing, being the best option a low temperature pasteurization (65ºC and 30 s), enhancing anthocyanin content with positive effect on the attractive color (a* coordinate CIEL*a*b*, green-red coordinate). Moreover, a heat treatment plus refrigeration during storage might reduce anthocyanin degradation in pasteurized pomegranate juice, minimizing color loss (Vegara et al., 2013b). Another option to increase the quality of the juices is the addition of some ingredients or additives during the process. Vázquez-Araújo et al., (2015) studied the influence of macerating pomegranate albedo with pomegranate juice on the main chemical and sensory characteristics of the juice. The results showed both the improvement of complexity of sensory profile and antioxidant capacity, being useful in developing and promoting new “healthy” products based on pomegranate.


252


Pomegranate Wines Recently, many studies on the preparation of fruit wines, including pomegranates, have been performed and have used the same process as the one used in the preparation of grape wine. In the specific case of pomegranate, for the first time, Mena et al. (2012a) and Berenguer et al. (2016) characterized pomegranate varietal wines (Mollar de Elche, Wonderful and a coupage) and fermented with different Saccharomyces cerevisiae yeast strains, respectively. The variety used played an important effect on the phytochemical composition, the process and the final quality (sensory attributes) of the pomegranate wines. It is important to keep the desirable and characteristic color of the pomegranate arils because this will ensure consumers’ acceptance and their willingness to buy. Regarding the phytochemical profile of these novel quality products, pomegranate wine consumption could be a promising source of phenolic compounds with healthy properties (Mena et al., 2012a). Moreover, although no scientific evidence has been published detecting melatonin in pomegranate juice, Mena et al. (2012b) found this biogenic amine in pomegranate wine, enhancing the functional properties of this new product.

Packaging and Coating The time required for the separation of arils from the endocarp by consumers, has drastically limited the consumption of pomegranates. The consumer of today prefers to purchase fresh pomegranate ready-to-eat, and these types of products are becoming a new trend. On one side, active- and passive-modified atmosphere packaging combined with temperature storage slows down biochemical processes, increasing the shelf-life of arils (Banda et al., 2015). Moreover, the pre-treatment with distillery ethanol and brandy vapors to the arils, before modified atmosphere packaging, reduced the weight loss, maintained the anthocyanin content and the intensity of red color during the 14 days of shelf-life, inhibiting yeasts and molds as well as lactic acid bacteria (Kapetanakou et al., 2015).


Pomegranate

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On the other hand, the use of edible coatings used as natural antimicrobial agents are a novel way to maintain the quality of food. It might be one of the solutions to produce healthy and environmentally friendly foods as required by today's consumers. The use of pomegranate extracts as an antimicrobial agent, fixed in a Chitosan matrix, to obtain an active packaging improved the shelf-life of fresh strawberries (Duran et al., 2016).

CONCLUSION Consumption of pomegranate fruits and their based products has drastically increased in recent years due to the fact that many scientific publications showed their positive effects on health, preventing diseases and/or maintaining good health. In this sense, pomegranate and their based products are rich in bioactive compounds, such as polyphenols (ellagitanins, anthocyanins), fatty acids (punicic acid) and alkaloids (pelletrines), among others. Thus, an increasing demand of new and healthier ready-to-eat pomegranate based products is opening new markets for pomegranate growers and distributors. Therefore, pomegranate has still great potential for further development of novel products adapted to consumers’ demands and needs, especially in food industry; however, the pharmaceutical industry should not be forgotten because a huge amount of money is invested in new pharmaceutical products, and this could be an interesting option for farmers and companies processing pomegranates to improve their benefits. But, it is necessary to generate scientific knowledge to evidence healthy activity of pomegranate and based products, especially there is a need of clinical studies to prove the health benefits of pomegranate products in specific groups of consumers.


254


ACKNOWLEDGMENTS The authors are grateful to the projects AGL2013-45922-C2-1-R, AGL2013-45922-C2-2-R (Ministerio de Economía y Competitividad, Spain) and AGL2016-75794-C4-1-R (Ministerio de Economía, Industria y Competitividad, Spain). Author Marina Cano-Lamadrid was funded by an FPU grant from the Spanish Ministry of Education (FPU15/02158).

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Chapter 9

POMEGRANATE: CULTIVATION, POMOLOGICAL PROPERTIES, PROCESSING, GLOBAL MARKET AND HEALTH BENEFITS Laila Hussein*, PhD, Mostafa Gouda, MSc, and Eid Labib, MSc Department of Nutrition, National Research Center, Giza, Egypt

ABSTRACT Pomegranate has over 2000 years of cultivation history and has been described in divine scriptures, including the Holy Quran and the Old Testament. Pomegranate trees requires, an average annual water requirement of 20 m3, a dry climate, and a 5 – 8 years duration for the fruit development and ripening. Pomegranate fruits weight ranges 375 500 g and its edible juice arils makes up to 26.9 – 65.6% of the fruit weight. Pomegranate is the most difficult fruit to process for its juice due to the difficulty to separate the arils from the peels. Untreated pomegranate juices (PJ) contains an excessive turbidity and it must be clarified by addition of fining agents such as albumin, bentonite, gelatin kiesel gel, polyvenylpyrrolidone or ultrafiltration. In industrial *

Corresponding Author Email: [email protected]


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L. Hussein, M. Gouda and E. Labib processing, the juice is concentrated to reduce volume, proper storage to resist microbial and chemical deterioration, and packaging for year-round utilization. The industrial concentration is performed by removal and subsequent concentration of aroma compounds by distillation, which is then added back to the de-aromatized juice concentrate during reconstitution step to restore the original aroma profile. Processes such as different types of pasteurization, osmotic distillation, high hydrostatic pressure, microwave, and heating are applied either separate or in combination for the processing of pomegranate juice. Regardless of the processing technique, the PJ should pass specific criteria of quality control and storage stability, such as the microbiological safety, color, total soluble solids, anthocyanins content, total phenols contents, and antioxidant activities. Greater attentions have been directed to the bioactive properties of phytochemical compounds present in pomegranate. The active compounds in pomegranate possess antiatherosclerotic, anti-atherogenic, anti-carcinogenic, anti-diabetic, antidiarrheal and many others biological properties. It is anticipated that the use of PJ as non-pharmacological agent in the treatment of both infectious and non-communicable diseases will find wider application in the Millennium.

INTRODUCTION There is a growing interest at the international level from the nutraceutical, pharmaceutical, and cosmetic industries to include pomegranate on their lists of ingredients because of its nutritional value, bright color, sweet and sour flavor, and phytochemical composition for health properties. A number of consumable commercial products are now available that contained ingredients derived from pomegranate and popularity of these products are gaining on the global pomegranate market. Pomegranate (Punica granatum L., Punicaceae) is one of the most ancient world’s edible fruits which has two thousand years of cultivation history in China. The pomegranate, which is native in Iran was introduced in Egypt by the mid second millennium BC. In ancient Egypt (1600 BC), pomegranates (PG) were part of the fruit supplies required in a pharaoh’s residence. The fruits were painted on walls and tombs of king Tut Anch Amoun and Zoroastrian temples, to symbolize eternal life and fertility.


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Today it is one of the important fruits grown in Turkey, Iran, Middle East, Mediterranean, Europe, and USA.

Cultivation Pomegranate trees are drought tolerant and it requires hot and dry climate during fruit development and ripening. The trees need good, deep irrigation periodically and it can be grown up to an altitude of 500 meter above sea level in well drained, sandy loan to deep loamy soils. Pomegranate trees can be 12 to 20 feet in height (Figure 1), with ever green and pretty red flowers (Figure 2). The red fruit weighs between 375 – 500 g (Figure 3). The seeds in their casing or aril are the most desired part of the pomegranate juice and are consumed raw (Figure 4). The white flesh aril inside the thick skin is the casing of several hundred sweet juicy seeds. Pomegranate thrives well in regions with semi-arid to subtropical climatic conditions. It is native to Iran (Persia), but it is spread to Southwestern Asia, the Mediterranean region, including Egypt, South Europe and then to parts of the USA. For the last two decades, India is the largest producer and exporter in the world, followed by Turkey and Egypt (Borgues and Massini, 2007). Today pomegranate is one of the greatest commercially important fruit crop in term of growing area and production. Farmers cultivate the crop by carefully selecting varieties and adopting advanced technologies. Planting and propagation is done vegetatively by cuttings and layering in spring (February-March) and summer (July-August) in sub-tropical and tropical regions, respectively. Adopting a planting distance of 5 x 5 meters is usually followed in northern India which gives 2-2.5 times more yield than that obtained when closer spacing of 2.5 x 4.5 meters, which increases disease and pest incidence. About a month prior to planting, pits of 60 x 60 x 60 cm size are dug (at a spacing of 5 cm. in square system) and kept open under the sun for a fortnight. About 50 g of 5% carbaryl dust is placed on the bottom and sides of the pits as a precaution against termites. The pits are filled with top soil


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mixed with 20 kg farm yard manure and 1 kg Super-phosphate. After filling the pit, watering is done to allow the soil to settle down. Cuttings/air layers are then planted and staked. Irrigation is provided immediately after planting and first irrigation is provided in the middle of May followed by regular weekly irrigation in summers; however, during winters irrigation at fortnightly intervals is recommended. The average annual water requirement through drip irrigation is 20 cubic meters. Drip irrigation helps to save 44% on irrigation and also helps to increase the yield by 30-35%. Plants trained on a multi-stem system is more prevalent, since the crops trained on the single stem training system are more susceptible to pests viz. stem borer and shoot hole bore. Pruning is not much required except for removal of ground suckers, water shoots, cross branches, dead and diseased twigs, and also to give shape to the tree. A little thinning and pruning of old spurs is done to encourage growth of new ones. Pomegranate has a noticeable adaptation to drought stress. The levels of the green leaf volatile, trans-2-hexenal is increased in response to drought stress, suggesting a possible role of this compound in drought stress response in pomegranate inter-cropping (Catola et al., 2016).

Figure 1. A representative picture of Pomegranate tree with fruits.

Figure 2. Flower on Pomegranate tree that becomes fruits.


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Figure 3. Pomegranate Fruit.

Figure 4. Pomegranate Arils containing juice and seeds.

Since the orchard starts giving yield from 5th year onwards, it is proposed to take up inter-cropping with low growing vegetables, pulses or green manure crops, particularly off season vegetables, which would cost Rs.10000/per acre and would yield on average 6 tonnes/acre valued at Rs.30000.The main components are planting material, land preparation, input application (FYM, fertilizers, micro-nutrients liming material, plant protection chemicals etc.), power and labor on application of inputs, intercultural and other farm operations. The yield from the plantation is 4.0 tons per acre in the 5th year and up to 7 tons per acre in the 8th year onwards. In arid regions, inter-cropping is possible only during the rainy season, whereas winter vegetables are feasible in irrigated areas. To foster fruit


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setting the plant should be exposed to full sunlight. In addition, PG flowers are both male and female and accordingly planting two or more PG encourage pollinating insects and humming birds in spreading the pollen from flower to flower. Fertilized female flowers remain to become fruit and flowering starts in May and continue through early autumn. The fruits become ready for picking 120-130 days after fruit set. At maturity, the fruits turn yellowish-red. The majority of the growers sell their product either through trade agents at village level or commission agents in the market. High quality commercial cultivation of crop by using high quality planting material and drip irrigation leads to multiple benefits viz. synchronized growth, flowering and harvesting. Fruit cracking is a serious disorder and this physiological disorder which is observed in young fruits is due to boron deficiency whereas in fully grown fruits it is mainly due to moisture imbalances. However, spraying with calcium hydroxide soon after fruit set has been found to be beneficial. Table 1. Properties of the different parts of pomegranate fruits* Fruit part Whole fruit, g

Mean (Range) 374.9 (42.6 - 320.5) (175 - 250) 374.9 (137.1 - 738.2) 49.9 (26.9 - 65.6) 14.2 ± 2.6 2.6 ± 0.7 50.1 (34.4 -73.1) 34.7 (19.2 - 48.0)

Aril ratio, %, w/w Endocarp, mesocarp Epicarp Peel ratio, % w/w Juice yield (based on whole fruit) % w/w Juice yield 68.3 (50.9 - 78.8) (based on aril) % w/w Brix degree 15.6 (12.2 - 17.8) Total titratable acidity, g/L 11.5 (2.4- 30) The formol index, g/L 13.1 (4.0 -20.0) The sorbitol/xylitol, mg/l 115.5(16 - 423) The sorbitol/xylitol, mg/l 61 - 191 Viscosity, K 293.15 - 363.15 * Adopted from: Al Maiman, Ahmad, 2002; Ozen et al., 2014.

References Boz, 1988 Al Maiman, Ahmad, 2002 Al Maiman, Ahmad, 2002

Al Maiman, Ahmad, 2002 Al Maiman, Ahmad, 2002


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Pomological Properties The fruit weight and other parameters were studied in 45 pomegranate fruits grown in different regions of Persia (Al Maiman, Ahmad, 2002) and this study reported the mean value and the ranges of the different anatomic part of pomegranate (Table 1). The edible parts of pomegranate fruits comprise the arils (26.9–65.6%) whereas the non-edible part is peels (34.4–73.1%).The fruit juice yield ranges between 9.2–48.0%. The juice yield measured is about 332 L per ton of pomegranate fruit (Wonderful var.)

Brix Index One of the basic criteria used for the definition of fruit juices is certainly Brix degree, which indicates the % of water-soluble solids in pomegranate juice (PJ). Brix degree is measured with refractometer at 200C and the results ranged between 12.2 – 17.8 and are expressed in Brix (AlMaiman, Ahmad, 2002). The minimum brix degree of (PJ) should be 14.0. Brix degree with values lower than 14.0 was, however, found in 18% of 45 pomegranates collected from different regions. The Total Titratable Acidity It is determined potentiometrically by titrating aliquots of PJ (2 ml) with 0.1 NaOH until the pH reached 8.1 and the result is expressed as grams of citric acid per liter. The titrable acidity in PJ ranged between 2.430(Al Maiman, Ahmad, 2002). The Formol Index This is measured with a potentiometric titration of the acidity of the compounds formed by the reaction of formaldehyde and amino acids in the juice up to a pH of 8.1. The formol number of PJ varied between 4.0 and 20.0 (Table1).


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The Sugar Alcohols Sorbitol/Xylitol Contents An enzymatic kit is used to assay sugar alcohol contents which are reported in a range between 61 – 191 mg/L of PJ (Ozen et al., 2014). Heating processes, enzymatic action, and fermentation can affect the sorbitol/Xylitol content of PJ. The Viscosities of Pomegranate Juice Knowledge of the viscosity is of primary importance in the fruit juice industry. The viscosity is important for the operation of the major pieces of equipment used in fruit processing. It is important to estimate the viscosity of juices in order to make engineering calculations for proper design of the equipment and heat-transfer coefficients, evaporation rates and evaporator performance, pumping and pipe requirements. If the viscosity of the concentrate exceeds a threshold value, then the output products concentration must be reduced or the concentrate will “burn on” the inside of the evaporator, which would cause a loss of energy and product. Viscosity can become an important factor during the concentration of juices, especially in the production of high density concentrates. Viscosity of fruit juices changes with content of soluble and suspended solids. Pectin and sugar concentration are the main factors in changes of viscosity. Magerramov et al., (2007) reported the viscosity for full-ripe pomegranate fruit juice (11 and15.20 Brix) with different concentrations and at different temperatures. After elimination of suspended solids by filtering, viscosity of five pomegranates juices concentrations (20, 23, 30, 35, and 400 Brix) were obtained from the original concentrate using a rotary glass vacuum evaporator at a constant rotational speed in water bath at 400C. The viscosities of the pomegranate juice ranged 293.15 to 363.15 K at atmospheric pressure, when the measurement was done with a capillary flow technique. Increase in temperature from 293 up to 403 Kelvin decreases significantly the viscosity by a factor of 4–7. However, at low concentrations below230 Brix, the temperature has little effect on the viscosity (by a factor of 1–2). The viscosity is little affected (up to 1.5– 1.7%) by pressure (at pressure changing between 0.1 and 10 MPa) along the constant temperature.


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Pomegranate has been discussed not only for its arils but also for other parts of the tree and the fruit, such as its roots, barks, peels and flowers as well as its seeds (Lansky & Newman, 2007). Table 2. Phytochemicals in Pomegranate components Juice

Seed oil Pericarp (Peel, rind) Leaves Flowers Roots and bark

Anthocyanins, glucose, ascorbic acid, ellagic acid, gallic acid; caffeic acid; catechin, EGCG, quercetin, rutin, numerous minerals particularly iron, amino acids. 95% punicic acid, other fatty acids, sterols, ellagic acid Phenolic punicalagins, gallic acid and other fatty acids, catechin, EGCG, quercetin, rutin and other flavonols, flavones, flavonones, anthocyanidins Tannins (punicalin and punicafolin), flavones glycosides, including luteolin and apgenin Gallic acid, ursolic acid, triterpenoids, including maslinic and asiatic acid Ellagitannins, including punicalin and punicalagin; numerous piperidine alkaloids

Antioxidant Activity of Pomegranate Juice The PJ contained the greatest antioxidant activity among the commonly consumed polyphenol (PP)-rich beverages in the US market (Gil et al., 2000; Seeram et al., 2008). The antioxidant capacity of commercial pomegranate juice is three times higher than those of red wine and green tea. The soluble PP content ranges between 0.2 and 1.0% in PJ depending on the fruit variety.

Figure 5. Structure of typical flavonoids found in pomegranate.


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Table 3. Polyphenolic contents in fresh and frozen pomegranate juices Phenolic compound Group: anthocyanins Delphinidin 3,5-diglucoside Cyanidin 3,5-diglucoside Delphinidin 3-glucoside Cyanidin 3-glucoside Pelargonidin 3-glucoside Total anthocyanins Group: Gallagyl-type tannins Punicalagin B Punicalagin D Other Total gallagyl-type tannins Group: Ellagic acid derivatives Ellagic acid glucoside Ellagic acid Total ellagic derivatives Group: Other hydrolyzable tannins Galloyl glucose Compound C Other compounds Total hydrolyzable tannins Total Phenolics in PJ Total Phenolics in green tea

Pomegranate juices Fresh

Frozen

42.9 53.0 76 128.3 5.9 306.0

38.8 46.4 23.6 59.5 3.9 172.2

12.7 10.1 45.1 67.9

14.4 11.1 102.5 128.1

17.9 15.3 33.2

17.9 8.7 26.5

51.1 224.5 264.1 539.2 2117 1029

43.9 203.6 277.7 525.2 1808

Pomegranate juices consist of four major phenolic compounds: (1) anthocyanins, (2) gallagyl tannins, (3) ellagic acids and (4) hydrolyzable tannins; anthocyanin and flavan-3-ols are the two main groups (Figure 5). Pro-anthocyanidin is present in raw pomegranate in monomer and dimer forms in average concentrations of 0.81, 0.29 mg/100 g of edible part, respectively [USDA]. Phenolic compounds of pomegranate juice harvested at different maturity stages showed that total monomeric anthocyanin increased significantly from 14.7 – 15.8 up to 22- 32 mg per 100g juice pressed from unripe to fully ripe fruits, respectively. The same trend was found in the changes of total flavonoids associated with the ripening of the pomegranate. On the other hand, the concentration of total phenolic and total tannins in the juice decreased with the ripening of the fruit (Mphahlelea et al., 2014). Pomegranate anthocyanins are responsible for


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the bright red color of the juice but they are quite unstable, since freezing of the pure PJ resulted in a 41% loss in the total anthocyanidin content (López-Rubira, et al., 2005). The contents of phenolic compounds in fresh and frozen pomegranate juices are presented in Table (3) (Gil et al., 2000). Table 4. HPLC separation of 100% pomegranate juice and commercial PJ Peak

Poly phenol group/fractions

Anthocyanin 1 Delphinidin-3, 5-O-diglucoside 2 Cyanidin-3, 5-O-diglucoside 3 Delphinidin-3-O-glucoside 4 Cyanidin-3-O-glucoide 5 Petunidin-3-O-glucoside 6 Pelargonidin-3-O-glucoside 7 Cyanidin-O-(p- coumaroyl) triglucoside 8 Peonidin-3-O-glucoside 9 Malvidin-3-O-glucoside 10 Delphinidin-3-O-(600-O-acetyl)glucoside 11 Petunidin-3-O-(600-O-acetyl)glucoside 12 Unknown anthocyanin 13 Peonidin-3-O-(600-O-acetyl)glucoside 14 Malvidin-3-O-(600-O-acetyl)glucoside 15 Delphinidin-3-O-(600-O-p-coumaroyl)glucoside Anthocyanin 16 Vitisin A 17 Petunidin-3-O-(600-O-p-coumaroyl)glucoside 18 Peonidin-3-O-(600-O-p-coumaroyl)glucoside 19 Malvidin-3-O-(600-O-p-coumaroyl)glucoside Flavan-3-ols 1 Unknown 2 Procyanidin dimer B1 3 Procyanidin dimer 4 Catechin 5 Procyanidintrimer 6 Procyanidin dimer B2 7 Epicatechin

Pure PJ

commercial PJ beverages

15.8 ± 0.6 32.2 ± 0.5 17.7 ± 0.5 53.0 ± 1.1 ND 3.4 ± 0.1 ND ND ND ND ND ND ND ND ND

1.6 – 2.8 2.5 – 5.8 0.5 – 4.1 0.5 – 1.0 0.8 – 3.7 ND 3.3 – 4.0 0.8 – 1.8 0.7 – 7.2 0.2 – 0.4 ND– 0.4 0.1 - 0.1 ND 0.2 – 0.7 ND

ND ND ND ND

0.5 – 1.0 ND ND– 0.2 ND– 0.8

ND ND ND ND ND ND ND

ND 9.9 – 11.1 1.6 – 7.3 3.1 – 5.5 ND 2.2 – 6.8 1.7 – 4.7

ND – not detected Borges, Crozier (2012).


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Composition of Pure PJ Juice and PJ Beverages The commercial labelled pomegranate pure juice and its beverages often contain adulteration from other sources. HPLC separation of anthocyanin and ellagitannins in fresh and commercial PJ can provide a straight forward fingerprinting to assess the authenticity of pomegranate juice (Table 4). Data indicates that commercial PJ contained the expected ellagic tannins but they also contained flavan-3-ol monomers and procyanidin dimers and polymers; components not usually detected in 100% pomegranate beverages, but are constituents derived from red grapes (Borges, Crozier 2012). Pomegranate punicalagins are the poly phenolic group with the highest anti-oxidative activity (10.5 ascorbic acid equivalent antioxidant capacity (AEAC), compared with the respective activities of 1.4, 0.6 and 6.6 reported for anthocyanins, ellagic acids and hydrolyzable tannins, respectively (Gil et al., 2000). It is found that drying, processing of pomegranate arils and rind were associated with a reduction in punicalagins and ellagic acid, total poly phenols, total antioxidant activity and sensory quality (Calín-Sánchez et al., 2013). Vacuum–microwave drying at 240 or 360 W was the best drying treatment for arils compared with the other tested drying process, i.e., freeze drying, convective drying (50, 60, or 70°C), and a combined method of convective pre-drying and vacuum–microwave (Carbonell-Barrachina, 2012; Borges, Crozier 2012). Aromatic Compounds in Pomegranate Aroma is a large combination of substances that are directly responsible for its odor and taste and these are volatile organic compounds such as aldehydes, alcohols, ketones, esters, and lactones Due to their dissimilar molecular characteristics and concentrations, every single aroma compound has different contributions on the final aroma of a fruit. However, during industrial processing of juices such as in concentration by evaporation process, losses or chemical modification of these aroma compounds can occur, which leads to variation in the aroma quality of juices. The results of identified pomegranate juice aroma components by GC and GC-MS are listed in Table 5. Five esters, two aldehydes, one terpene and one alcohol were detected.


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Table 5. List of identified aroma components in the pomegranate juice Component 3-Methyl butanal Ethyl butyrate Isopentyl acetate n-Hexanol Amyl butyrate Diethyl allylmalonate Ethyl pelargonate α-Ionone Aldehyde C-20

Concentration (ppm) 9.76 2.65 15.24 4.53