Role of Anthocyanins in Neurodegenerative diseases . .... Figure 1: Basic Structure of Anthocyanidin . ..... may retain their basic anthocyanin structure (26,27).
In Vitro Antioxidant Activity of Amaranthus gangeticus Ethanol Extract Submitted for completion of the B.Pharm Degree at the Department of Pharmaceutical Sciences, North South University, Bangladesh.
Priota Islam ID: 1110088046 Department of Pharmaceutical Sciences NORTH SOUTH UNIVERSITY December 2014
Table of Contents LIST OF FIGURES ....................................................................................................... 3 LIST OF TABLES ......................................................................................................... 4 ACKNOWLEDGEMENT ............................................................................................. 5 ABSTRACT ................................................................................................................... 6 1.
Introduction............................................................................................................. 8 References ................................................................................................................ 10
2.
3.
Literature Review ................................................................................................. 14 2.1.
Introduction ................................................................................................... 14
2.2
Occurrence .................................................................................................... 14
2.3
Chemistry ...................................................................................................... 15
2.4
Pharmacokinetics & Bioavailability.............................................................. 16
2.5
Health Benefits .............................................................................................. 18
2.5.1
Role of Anthocyanins in Cardiovascular diseases ................................. 18
2.5.2
Role of Anthocyanins in Neurodegenerative diseases ........................... 20
2.5.3
Role of Anthocyanins in Visual Acuity ................................................. 21
2.5.4
Role of Anthocyanins in Cancer ............................................................ 22
2.5.5
Role of Anthocyanins in Diabetes and Obesity ..................................... 25
2.5.6
Miscellaneous Effects of Anthocyanins ................................................. 27
2.6
Conclusion and future direction .................................................................... 28
2.7
References ..................................................................................................... 28
Study ..................................................................................................................... 45 3.1
Abstract ......................................................................................................... 45
3.2
Introduction ................................................................................................... 46
3.3
Materials & Methods ..................................................................................... 47
3.3.1
Collection and preparation of Plant material ......................................... 47
3.3.2
Extraction process .................................................................................. 47
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3.3.3
Chemicals Used ..................................................................................... 47
3.3.4
In vitro antioxidant assays of the ethanol, ethyl acetate and toluene
extracts of Amaranthus gangeticus ..................................................................... 47 3.3.5
High performance liquid chromatography (HPLC) system ................... 49
3.3.6
Assay for total phenolic content of the ethanol extract of Amaranthus
gangeticus ............................................................................................................. 51 3.3.7
Assay for total flavonoid content of the ethanol extract of Amaranthus
gangeticus ............................................................................................................. 51
4.
3.3.8
Phytochemical screening ....................................................................... 52
3.3.9
Statistical Analysis ................................................................................. 52
3.4
Results ........................................................................................................... 52
3.5
Discussions .................................................................................................... 55
3.5.1
DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging activity..... 56
3.5.2
NO· scavenging activity ........................................................................ 56
3.5.3
H2O2 scavenging activity ...................................................................... 57
3.5.4
Reducing activity ................................................................................... 57
3.5.5
Total flavonoid content and total Phenolic Content............................... 58
3.5.6
HPLC-DAD analysis of phenolic contents in leaves extracts................ 58
3.6
Conclusion ..................................................................................................... 58
3.7
References ..................................................................................................... 59
Conclusion ............................................................................................................ 63
Page 2 of 63
LIST OF FIGURES Figure 1: Basic Structure of Anthocyanidin ................................................................ 16 Figure 2: HPLC chromatogram of a standard mixture of polyphenolic compounds. Peaks: 1, gallic acid; 2, (+)-catechin; 3, vanillic acid; 4, caffeic acid; 5, (–)epicatechin; 6, p-coumaric acid; 7, rutin hydrate; 8, ellagic acid; 9, myricetin; 10, querceti ......................................................................................................................... 53 Figure 3: HPLC chromatogram of the ethanol extract of Amaranthus gangeticus. Peaks: 1, rutin hydrate. ................................................................................................. 53 Figure 4: Effect of Amaranthus gangeticus extract on various antioxidant assays. .... 55
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LIST OF TABLES Table 1: Presence of various bioactive molecules in the extract of Amaranthus gangeticus. ................................................................................................................... 52 Table 2: Contents of Polyphenolic compounds in the ethanol extract of Amaranthus gangeticus (n=3). ......................................................................................................... 54 Table 3: IC50 values of Amaranthus gangeticus extracts in different antioxidant assays such as DPPH method, NO- scavenging method and H2O2 scavenging. Values are expressed as average of duplicate experiments ...................................................... 54
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ACKNOWLEDGEMENT First and foremost, I would take the opportunity to thank my project supervisor, Dr. Hasan Mahmud Reza, Associate Professor and Chairman, Department of Pharmacy, North South University, for his patience, motivation, enthusiasm, and immense knowledge. His guidance helped me in all the time of research. I could not have imagined having a better advisor and mentor for my undergraduate thesis. I would like to thank you from the bottom of my heart for your support and understanding over these past four years of my undergraduate study.
I would also like to show my gratitude to Dr. Md Ashraful Alam, Assistant Professor, Department of Pharmacy, North South University, for his undying support and motivation that gave me the courage and strength to complete this thesis work. Without his assistance and dedicated involvement in every step throughout the process, this paper would have never been accomplished.
Getting through my thesis work required more than academic support, and I have many, many people to thank for listening to and, at times, having to tolerate me over the past four years. I cannot begin to express my gratitude and appreciation for their friendship. They have been a wonderful support for me and mentioning a few names would not be enough.
Most importantly, none of this could have happened without my family. Their consistent support has made me come this far. I cannot thank them enough for bearing me and being beside me through highs and lows even when I was unable to be beside them due to my workload with studies and thesis work. Every time I was ready to quit, they did not let me and I am forever grateful. This dissertation stands as a testament to your unconditional love and encouragement.
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ABSTRACT Purpose: This study evaluated the in vitro antioxidant potential of the ethanol, ethyl acetate and toluene extracts of Amaranthus gangeticus.
Methods: The different antioxidant assays, including DPPH free radical scavenging, nitric oxide scavenging, hydrogen peroxide scavenging and reducing power, were studied. Moreover, high-performance liquid chromatography (HPLC) coupled with diodearray detection was used to identify and quantify the phenolic compounds in the ethanol extract of this leafy vegetable.
Results: Amaranthus gangeticus extracts showed effective hydrogen peroxide radical scavenging and nitric oxide scavenging activity. However, reducing power of ferric ion and DPPH radical scavenging activity was not significant compared to the standard antioxidant activity. Rutin hydrate was abundantly detected in the extract of Amaranthus gangeticus.
Conclusion: Our investigation suggests that Amaranthus gangeticus show competent antioxidant activity though a further modified HPLC-DAD analysis is required to specifically identify and quantify the actual compounds responsible for such antioxidant activities of its extracts.
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CHAPTER: 1 INTRODUCTION
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1. Introduction Food is a very basic and vital necessity for survival, as it plays a crucial role in growth and maintenance of the human body. Several research findings support the hypothesis that in addition to supply of the basic nutrients, food can also contribute to various functions of the human body. There are certain fruits and vegetables that are capable of providing certain health benefits along with fulfilling the physiological needs and are thus referred to as “Functional Food” (1,2). The terminology “Functional Food” acts as a bridge between food and medicine due to the potential health beneficial role provided by the bioactive compounds and nutrients present in many of the plant origin foods available throughout the world. Such effects including, but are not limited to anti-carcinogenic, anti-inflammatory, anti-allergic, cardiovascular protective effects, with some recent findings reporting about their anti- allergic effects as well (3–7). Currently, researchers are focusing immensely on the protective effects against oxidative stress acquired from the natural dietary antioxidants (8). Natural antioxidants that are present in plants are capable of trapping free radicals from our body which are potentially harmful. Free radicals are species that exist independently having one or more unpaired electrons with which they react with other molecules by taking or supplying electrons and thus results in many pathological conditions (9). Researchers suggest that oxidative and free radical generated reactions are a prime contributor in degenerative processes like aging and various diseases like cancer, diabetes, atherosclerosis, etc
(10–12). Reactive oxygen species are constantly
generated in vivo through aerobic cellular respiration or are induced by external sources such as pollution, ionizing radiation and drugs (13). Living organisms defend themselves from such harm by either endogenous antioxidant defence systems or by taking dietary antioxidant widely available in natural foods (8,10). By enhancing the body’s natural antioxidant defences or by providing supplementary dietary antioxidants, the risk of occurrence of many chronic diseases and their progression can be prevented (14). Plants are rich sources of natural antioxidants like phenolic acids, flavonoids and anthocyanins which constitute an important class of antioxidants. Phenolic compounds present in foods are a prime focus for researchers due to their strong antioxidant effect as they in particular help to fight the formation of free radicals in the human body and thus slow down cellular aging or damaging process. These are a large and diverse group of secondary plant metabolites that are widely spread in the Page 8 of 63
plant kingdom, and include phenolic acids, flavonoids and tannins (15). Hydroxybenzoic acid and hydroxycinnamic acids constitute the majority of phenolic acids found in plant tissue (16). On the other hand, flavonoids are the most abundant in plant phenolics with a flavone nucleus as structural basis. The flavonoid groups are classified on the basis of hydroxylation of the nucleus and linked sugar that includesisoflavones, flavonols, flavonones, catechins, anthocyanins, dihydroflavonols, chalcones and quercetins. Tannins are capable of tanning leather, or precipitating gelatine from solutions. Many of these compounds are considered as potential protective factors in combating chronic degenerative diseases such as cataracts, diabetes mellitus, cancer, muscular degeneration, cardiovascular diseases and neurodegenerative diseases (17). Also, polyphenolic substructure containing antioxidant is called a polyphenol antioxidant. Fruits such as apples, blackberries, blueberries, grapes, cherries and vegetables such as cabbage, broccoli, celery, etc are main sources of polyphenol antioxidants. Numerous of these kinds of functional foods are available in Bangladesh since agriculture is the single largest producing sector of the economy of this country due to its fertile soil and ample water supply. Thus various types of crops are grown by our hardworking farmers and as a result many of the functional foods are widely available in our country at cheap prices. Among all the available fruits and vegetables Amaranthus gangeticus, locally known as Lal shak is thought to be a very potential source of antioxidants. This leafy vegetable is commonly grown across the Indian subcontinent and is eaten by the people as an ingredient of fresh salad or as a cooked item for lunch or dinner. It belongs to the family Amaranthaceae which is a rich plant family containing more than 700 species most of which showing health beneficial effects. For example, plants belonging to the Alternanthera genera of the Amaranthaceae family have been found to show significant health beneficial effects such as antioxidant, anti-diabetic, antimicrobial, wound healing, anti-inflammatory and numerous others (18–21). In Brazilian folk medicine, Alternanthera brasiliana is used against cough, inflammation and diarrhea, whereas its extract has been found to possess antioxidant activity and has found to contain a mixture of β-sitosterol, stigmasterol and spinasterol (18). The Reactive Oxygen species mediated apoptosis in β-cells due to glucotoxicity is prevented by Alternanthera paronychioides (22). The Achyranthes aspera from Amaranthaceae family showed hypoglycemic effect due to potent antioxidant activity Page 9 of 63
of the isolated extract in alloxan induced diabetic mice (23). Numerous other reports are available justifying the strong antioxidant properties of the plants belonging to the Amaranthaceae family for which Amaranthus Gangeticus is chosen to be the plant of this study as it shares the same family. Thus, the objective of the study is to evaluate the antioxidant activity of Amaranthus Gangeticus using
different antioxidant assays, including DPPH free radical
scavenging, nitric oxide scavenging, hydrogen peroxide and reducing power. In addition to that, high-performance liquid chromatography (HPLC) coupled with diode-array detection is used to identify and quantify the phenolic compounds in the ethanolic extract of this leafy vegetable.
References 1.
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antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet. 1993 Oct 23;342(8878):1007–11. 2.
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tocopherol, ascorbate, and beta carotene on low-density lipoprotein oxidation. Circulation. 1993 Dec;88(6):2780–6. 3.
Kaluza WZ, McGrath RM, Roberts TC, Schroeder HH. Separation of
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Watanabe J, Shinmoto H, Tsushida T. Coumarin and flavone derivatives from
estragon and thyme as inhibitors of chemical mediator release from RBL-2H3 Cells. Biosci Biotechnol Biochem. 2005 Jan;69(1):1–6. 6.
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Development and validation of oxygen radical absorbance capacity assay for lipophilic antioxidants using randomly methylated beta-cyclodextrin as the solubility enhancer. J Agric Food Chem. 2002 Mar 27;50(7):1815–21. 7.
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evaluate antioxidant activity. LWT-Food Science and Technology. 1995;28(1):25–30.
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health perspectives / edited by D. L. Madhavi, S. S. Deshpande, D. K. Salunkhe. SERBIULA (sistema Librum 20). 10.
Shafique S, Akhter N, Stallkamp G, de Pee S, Panagides D, Bloem MW.
Trends of under- and overweight among rural and urban poor women indicate the double burden of malnutrition in Bangladesh. Int J Epidemiol. 2007 Apr;36(2):449– 57. 11.
Bloem MW, Huq N, Gorstein J, Burger S, Kahn T, Islam N, et al. Production
of fruits and vegetables at the homestead is an important source of vitamin A among women in rural Bangladesh. Eur J Clin Nutr. 1996 Jul;50 Suppl 3:S62–7. 12.
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Huang D, Ou B, Hampsch-Woodill M, Flanagan JA, Prior RL. High-
throughput assay of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate fluorescence reader in 96-well format. Journal of Agricultural and Food Chemistry. 2002;50(16):4437–44. 14.
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evidence for the “antioxidant hypothesis.” Public health nutrition. 2004;7(03):407–22. 15.
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J A Manthey KG. Phenols in citrus peel byproducts. Concentrations of
hydroxycinnamates and polymethoxylated flavones in citrus peel molasses. Journal of agricultural and food chemistry. 2001;49(7):3268–73. 17.
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activities and triterpenoids isolated from Alternanthera brasiliana (L.) Kuntze leaves. Natural product research. 2013;27(18):1660–3.
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19.
Tan KK, Kim KH. Alternanthera sessilis red ethyl acetate fraction exhibits
antidiabetic potential on obese type 2 diabetic rats. Evidence-Based Complementary and Alternative Medicine. 2013;2013. 20.
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Pathak D, et al. Influence of Alternanthera brasiliana (L.) Kuntze on Altered Antioxidant
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Wu C-H, Hsieh H-T, Lin J-A, Yen G-C. < i> Alternanthera paronychioides
protects pancreatic β-cells from glucotoxicity by its antioxidant, antiapoptotic and insulin secretagogue actions. Food chemistry. 2013;139(1):362–70. 23.
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CHAPTER: 2 LITERATURE REVIEW
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2. Literature Review 2.1.Introduction Natural dietary antioxidants provide protective effects against oxidative stress which has become the prime focus of researchers nowadays (1). These are capable of trapping free radicals from our body which are potentially harmful. Free radicals are species that exist independently having one or more unpaired electrons with which they react with other molecules by taking or supplying electrons and thus results in many pathological conditions (2). Researchers suggest that oxidative stress and free radical generated reactions are a prime contributor in degenerative processes like aging and various diseases like cancer, diabetes, atherosclerosis, etc (3–5). Thus, discovery of natural compounds that combat against oxidative stress has become very important for the cure and management of many pathological conditions. Plants are rich sources of natural antioxidants like phenolic acids, flavonoids and anthocyanins which constitute an important class of antioxidants. Amaranthus Gangeticus is a red coloured leafy vegetable and its colour indicates the presence of one of the most common pigment, the ‘Anthocyanins’. These are an important group of water-soluble pigments belonging to the flavonoid family. They are the reason for the distinctive red, blue and purple pigments in most of the fruits and vegetables. They display numerous potential health benefits for which they have been investigated recently for their use as potential clinical treatments for several human disorders. They have both anti-inflammatory as well as antioxidant properties for which have proven effective in in vitro and in vivo models of various chronic conditions
such
as
cardiovascular
disease,
obesity,
ophthalmic
disorders,
atherosclerosis and type II diabetes (6–10). In this review, recent studies on the health beneficial effects of Anthocyanins are presented that includes its role in cardiovascular diseases, neurodegenerative diseases, visual acuity, cancer, diabetes and several other health-related issues. The chemical properties and pharmacokinetic nature of this valuable pigment is also revealed here.
2.2 Occurrence Anthocyanins are widely present in consumed foods, including berries, purple sweet potatoes, red rice and grapes, existing in relatively high concentrations compared to other dietary polyphenolic compounds (11). They contribute to the colours of many fruits and vegetables and are probably the most widespread food colours occurring as Page 14 of 63
red colours in fruit juices, wines and jams. These pigments have been found in edible plant materials including apple, berries (blackcurrant, boysenberry, blueberry, bilberry, strawberry, blackberry, raspberry, cranberry, elderberry, lingo berry, chokeberry etc.), black carrot, cabbage, cherry, radish, red onions and etc (12).
2.3 Chemistry Anthocyanins exist in fruits and vegetables as glycosides, with glucose, galactose, rhamnose, xylose or arabinose attached to an aglycon nucleus(13,14). Unlike other flavonoids, they carry a positive charge in acidic solution which makes it more stable in acidic environments such as the stomach than the aglycon alone(14). The anthocyanidins are the de-glycosylated or aglycon forms of anthocyanins, among which the six most common anthocyanidins skeleton includes- cyanidin, delphinidin, pelargonidin, malvidin, petunidin and peonidin (Fig 2). The sugar components of anthocyanins are usually attached to the anthocyanidin skeleton through the C3 hydroxyl group in ring C. Numerous anthocyanins are known that vary in the basic anthocyanidin skeleton, and the position and extent to which the glycosides are attached to the skeleton(13). Thus implicating their various pathways of absorption(15).The glycosides of the three nonmethylated anthocyanidins (cyanidin, delphinidin and pelargonidin) are the most widespread in nature, being80% abundant in pigmented leaves, 69% in fruits and50% in flowers. The most plentiful anthocyanins in the edible parts of plants are cyanidin, followed by pelargonidin, peonidin, delphinidin, petunidin, and malvidin(16,17). They are soluble in water and their intensity of colour depends upon pH and presence of chelating metal ions, mainly occurring as blue, purple or red. At pH 1-3 the flavylium cation is red n colour, at pH 5 the resultant carbinol pseudo base is colourless, and at pH 7-8 the blue purple quinoidal base is formed(18).
The polyphenolic and cationic nature of anthocyanins and their metabolites activate a variety of cellular responses among which the polyphenolic nature is principally responsible for their strong antioxidant and free radical scavenging activities, and their overall metabolic benefits.
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Figure 1: Basic Structure of Anthocyanidin
2.4 Pharmacokinetics & Bioavailability Recent bioavailability studies suggest that anthocyanins are absorbed rapidly from the stomach and small intestine, and are greatly metabolized resulting in very low concentrations of the parent compound being observed in plasma within only a few hours of consumption(19,20). In general, in both animals and humans, the anthocyanins are absorbed as intact glycosides, and their absorption and elimination is really quick. However, the efficiency of their absorption is relatively meagre (20,21). The absorption and metabolism of black raspberry anthocyanins in humans were investigated in a study by orally administering the black raspberry extracts at high doses (2.69 ± 0.085 g/day) Page 16 of 63
(22). Peak plasma levels of the four anthocyanins in black raspberries were observed within 2h of oral berry treatment and their elimination from plasma was found to follow first-order kinetics. They were excreted both as intact anthocyanins and as methylated derivatives in the urine within 4–8 h of intake. Overall, less than 1% of the administered dose of the berry anthocyanins was absorbed and excreted in urine. Similar results have been obtained in studies of the absorption and metabolism of anthocyanins in rodents (21). The biological activities of anthocyanins are closely correlated to their absorption and metabolism. Glycosylation and acylation patterns decrease the bioavailability of an anthocyanin; however, glycosidases present in the GI tract may hydrolyze anthocyanins into anthocyanidins, thereby increasing their biological potential but decreasing their stability. The presence of a glucose substituent compared to a galactose or arabinose on the cyanidin and peonidin anthocyanidins present in cranberry juice seems to make them more bioavailable as a percentage of the administered dose (23). Anthocyanins exist in the circulation and urine as intact, methylated, glucuronide derivatives
and/or
sulfoconjugated
forms
(24–26),
reaching
peak
plasma
concentrations between 1 and 3 h after consumption and depending on the individual compound and the food matrix. The metabolites remain in the urine for up to 24 h and may retain their basic anthocyanin structure (26,27). Pharmacokinetic evidence implies that parent glycosides and glucuronide derivatives are prominent in the bloodstream between 0 and 5 h but become increasingly methylated over time (6–24 h), which suggests that the bioactivity of anthocyanins are likely altered over time as a result of metabolic transformation (28). Several in vivo studies suggest that the food matrix has a significant effect on the absorption and metabolism of anthocyanins. The degree of individual variation in anthocyanin bioavailability may result from differences in xenobiotic metabolism in the GI tract, liver, and other tissues. Human polymorphisms have also been reported in the genes for catechol- Omethyltransferase, glutathione S-transferases, and UDP glucuronosyl transferase (29). The variation of human gut microflora may also play a important role in the bioavailability of anthocyanins. Microbiota present in the GI tract may metabolize anthocyanins, producing smaller, and more bioavailable end-products. Gut microflora have the ability to metabolize anthocyanins, however, the literature in this area is limited. Using a bacterial preparation imitating the normal human Page 17 of 63
microbiota population, it is possible to demonstrate the conversion of larger polyphenols to phenolic acids, which demonstrate similar antiinflammatory effects as the parent compounds (30). In addition, smaller phenolic acids and other anthocyanin metabolites have been found to contain greater chemical and microbial stability, suggesting that they may play an important role in the physiological effects and antioxidant activity observed in many studies (31).
2.5 Health Benefits The chemical nature and virtues of anthocyanins have made it available to play a number of beneficial roles in control of many disease conditions. Their roles have been significant in conditions such as heart disease, neurodegenerative diseases, in improving visual acuity, cancer, diabetes, obesity and many more. Some of such effects of anthocyanins are mentioned below: 2.5.1 Role of Anthocyanins in Cardiovascular diseases There is strong relation between oxidative stress protection and the role of anthocyanins in cardiovascular diseases. Study was conducted where four anthocyanins were isolated from elderberries and incorporated into the plasma lemma and cytosol of endothelial cells to directly examine the protective roles, since endothelial dysfunction is involved in initiation and development of vascular disease (32). The test result revealed that not only anthocyanin could be incorporated into endothelial cells but also significant oxidative stress protection was evident. Endothelium-dependent vaso-relaxation was provided by Delphinidin in the rat aorta, providing a pharmacological benefit that can be compared with red wine polyphenolics (33). Feeding of purified anthocyanins or anthocyanin rich extracts from elderberry or black currant showed little influence on the cholesterol levels or fatty acid patterns in the liver of a rat model, but the pigments were able to spare vitamin E (34). Capillary permeability have been found to be reduced by administration of crude anthocyanin extracts from bilberry both orally and via injection (35). Prevention of heart attacks through administration of grape juice or wine have been found to be strongly linked to roles of these anthocyanin rich compounds in reducing inflammation, enhancing capillary strength and permeability, inhibiting platelet formation and enhancing nitric oxide (NO) release (36). In addition, administration of black currant extract containing high concentration of anthocyanins Page 18 of 63
resulted in endothelial-dependent vasorelaxaton in rings of rat aorta in vitro (37). Also, when rats were pre-treated to be more susceptible to oxidative damage and then fed with anthocyanin-rich extracts, there was a significant reduction in lipid peroxidation indices and DNA damage was also observed (38). Postmenopausal women who participated in the Iowa Women’s Health study showed significant reduction in CVD mortality after being treated with strawberry extracts for a 16 year follow up period (39). Blueberries also revealed significant decrease in CHD mortality in an age and energy adjusted model. Red wine intake have been proven to reduce CVD mortality in several studies (40,41). A consistent doseresponsive cardiovascular preventive effect has been suggested in an analysis of wine consumption in relation to CVD risk (42). Red wine has proven to have more beneficial effects on lipid metabolism than the white one, probably due to its increased phytochemical content (43). There have been significant reductions in ischemia, blood pressure, lipid levels and inflammatory status in patients clinically diagnosed with vascular diseases when given relatively low-dose anthocyanin therapy (44–47).Commercial grape juice (10 mL/kg) has been shown to markedly inhibit platelet activity and experimental coronary thrombosis in vivo (48). Corn-derived anthocyanins made the myocardium less vulnerable to ischemia reperfusion injury in both ex vivo and in vivo studies as compared with the anthocyanin-free control (8). Anthocyanins may be effective in improving endothelial function by influencing NO levels. Chokeberry and bilberry anthocyanin-rich extracts have the ability to prevent loss of endothelium-dependent and NO mediated relaxation in porcine arteries in vitro (49). Protection from heart treatment was associated with a reduction in NF-kB with lyophilized grape powder for 4 wk (50).Human umbilical vein endothelial cells when treated with anthocyanins, regulated cholesterol distribution by interfering with the recruitment of TNF receptor-associated factors-2 in lipid rafts, thus inhibiting CD40induced proinflammatory signalling (51).Delphinidin has been shown to reduce the degree of apoptotic and necrotic cell death in cultured cardiomyocytes and also the infarct size after ischemia in rats. The process is mediated by the inhibition of signal transducers and activators of transcription1 activation (52). Anthocyanins may also protect against production of adhesion molecule induced by activated platelets. An investigation involving optimal platelet function revealed that anthocyanins and their colonic metabolites inhibited thrombin receptor-activating Page 19 of 63
peptide–induced platelet aggregation but did not influence the reactivity of platelet when strong agonists such as collagen and ADP were present (53). Increased levels of CRP due to low-grade chronic inflammation has been recognized as an independent risk factor for CVD (30). Among the adults in the United States, a significant inverse association between serum CRP and anthocyanin intakes has been found upon analysis of NHANES data (54). Data from the USDA flavonoid databases also indicated that anthocyanidin intakes were inversely linked with serum CRP concentration (54). By using anthocyanin-rich sweet cherries a recent clinical study showed a decrease in serum CRP after 4 wk of intervention (55).
2.5.2 Role of Anthocyanins in Neurodegenerative diseases Anthocyanins have a strong antioxidant capacity, which appears to be highly effective in several models of neurodegenerative diseases (56–58). They have a high oxygen radical absorption capacity (ORAC) value, which is partially responsible for this neuroprotective effect (59,60). More specifically, anthocyanins act as antioxidants because of their ability to directly trap free radicals and to prevent formation of ROS in affected cells. For example, anthocyanins decrease the generation of ROS in in vitro models of alpha-beta peptide-induced toxicity, as well as in hydrogen peroxide injury (57,61). Moreover, by using the highly accurate method of electron spin resonance (ESR) spectroscopy it has been found that anthocyanins have a strong affinity to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH), alkyl, and hydroxyl free radicals in a dose dependent fashion (61). Initiation of inflammatory gene expression and following production of interleukins and pro-inflammatory cytokines is a common feature in neurodegeneration. Methods that target these inflammatory processes may be beneficial in limiting neuronal apoptosis associated with the disease. Anthocyanins display significant antiinflammatory properties, as they have been shown to inhibit various inflammatory biomarkers, including interleukin-8 (IL-8) (59). In addition to decreased IL-8 production, pomegranate anthocyanins hold back activation of nuclear transcription factor kappa B (NFκB), which is responsible for the expression of several proinflammatory genes. They were also shown to inhibit a number of other bio molecules which are responsible for the expression of a number of pro-inflammatory cytokines (62). Furthermore, cherry and blackberry anthocyanins have been proven to be Page 20 of 63
powerful cyclooxygenase-2 (COX-2) inhibitors which is an important proinflammatory enzyme employed in the synthesis of prostacyclins (63,64). At high concentrations (250 μg/mL),anthocyanins have been shown to inhibit up to 95% of COX activity (65). These cumulative data suggest that anthocyanins may have a significant role in preventing inflammatory processes that are associated with neurodegenerative disease. Anthocyanins have also been attributed with the capacity to modulate cognitive and motor function, to enhance memory, and to have a role in preventing age-related declines in neural function. Administration of isolated semi purified anthocyanins from purple sweet potato enhanced cognitive performance in mice, and also effectively inhibited lipid peroxidation in rat brain tissues (66). Administration of blueberry extracts with significant anthocyanin content (but not purified pigments), led to effective reversal of age-related deficits in various neural and behavioural parameters (memory and motor functions) (67). Further investigations demonstrated that anthocyanins were highly bioavailable in endothelial cells, which was linked to their roles in prevention of atherosclerosis and neurodegenerative disorders (32,68). 2.5.3 Role of Anthocyanins in Visual Acuity Visual acuity can be improved significantly through administration of anthocyanin pigments to animal and human subjects, and the role of these pigments has been particularly well documented in enhancing night vision or overall vision (69) Significantly improved night vision adaptation has been seen in human subjects following oral intake of anthocyanosides from black currants (70), and similar benefits were obtained after administration of anthocyanins from bilberries (71). Regeneration of rhodopsin (a G-protein-coupled receptor localized in the retina of the eye) was stimulated by three anthocyanins from black currant and formation of regeneration intermediate was accelerated by cyanidin 3-rutinoside (72). Thus, enhancement of rhodopsin regeneration has been proven to be at least one mechanism by which anthocyanins improve visual acuity. A positive effect of anthocyanins on vision improvement was suggested by early clinical trial studies (73–75) carried out in France and Italy. A controlled clinical trial of cyanoside chloride and Heleniene (Adaptinol, xanthophyll dipalmitate) on 31 patients suffering from functional disturbances of vision in the dark, reported that both agents significantly improved photopic visual acuity. In a similar clinical trial in Page 21 of 63
Germany, Difrarel®E (anthocyanosides and vitamin E) was given to thirty six patients with progressive myopia
for 14.5 months and in about half of them, an
increase in myopia was suppressed by approximately 50%, along with 29 patients showing stabilisation of fundus-alterations, and an overall improved and stable visual acuity was obtained. Anthocyanins from blackcurrant in the form of a concentrated extract powder were examined for their effects on asthenopia, an ophthalmological condition that has nonspecific symptoms such as fatigue, red eyes, eye strain, pain, in or around the eyes, blurred vision, headache and occasional double vision, and is a result of continuous exposure of the eye to video displays. Oral administration of blackcurrant anthocyanins to such victims at various doses was found to decrease the dark adaptation threshold in a dose dependent fashion (70). The photo oxidation of pyridinium disretinoid A2E, an auto fluorescence pigment accumulating in retinal pigment epithelial cells with age and in some retinal disorders was also found to be suppressed by scavenging singlet oxygen, upon administration of nine anthocyanins from bilberry extracts (76).In a very recent study, the ocular distribution of blackcurrant anthocyanins (BCAs) in rats and rabbits after oral, intravenous and intraperitoneal administration was demonstrated (77). BCAs were found to be absorbed and distributed in ocular tissues as intact forms and pass through the bloodaqueous barriers and blood-retinal barriers in both rats and rabbits. These studies revealed that oral intake of anthocyanins or anthocyanin rich extracts can be used as a drug for treating ophthalmological diseases such as myopia and glaucoma. Investigation of the effect of purified high-dose anthocyanin oligomers on nocturnal visual function and clinical symptoms were carried out on 60 patients with asthenopia and refractive errors in both eyes. Among the whole bunch 73.3% of patients were reported with improved symptoms (78).
2.5.4 Role of Anthocyanins in Cancer In both in vitro and in vivo research trials, anthocyanins have been found to have significant ability in reducing cancer cell proliferation and inhibiting tumour formation (17,79–81).Several investigations have compared the anti proliferative effects of anthocyanins on normal as well as cancer cells and surprisingly revealed that they selectively inhibit the growth of cancer cells with insignificant effect on the
Page 22 of 63
growth of normal cells (69,70). Moreover, anthocyanidins have been found to be more potent inhibitors of cell proliferation than the anthocyanins (82). The ability of anthocyanin pigments to interfere with the process of carcinogenesis seems to be related to multiple potential mechanisms of action including inhibition of cyclooxygenase enzymes and potent antioxidant potential. Anthocyanins have been found to inhibit tumour formation by blocking activation of a mitogen-activated protein kinase pathway (83). Thus providing the first indication of a molecular basis for why anthocyanins display anti carcinogenic properties. Fruit extracts with significant anthocyanin concentrations were found to be effective against various stages of carcinogenesis in other studies (17,84–86). The antioxidant activity of anthocyanins is due to their phenolic structure (87). These effects have been verified in vitro using several cell culture systems including colon (88,89), endothelial (90), liver (91,92), breast (93,94) and leukemic cells (95), and keratinocytes (96). Anthocyanins have shown multiple antitoxicant and anticarcinogenic effects in these culture systems such as: directly scavenging reactive oxygen species (ROS), increasing the oxygen-radical absorbing capacity of cells, stimulating the expression of Phase II detoxification enzymes, reducing the formation of oxidative adducts in DNA, decreasing lipid peroxidation, inhibiting mutagenesis by environmental toxins and carcinogens, and reducing cellular proliferation by modulating signal transduction pathways. Anthocyanins have also been found to function by chelating metals and by direct binding to proteins in their ant carcinogenic functions (35). In a study, anthocyanins have also been proven to induce phase II antioxidant and detoxifying enzymes in cultured cells that contribute to its anti carcinogenic properties (97). In addition, apoptosis or programmed cell death, plays a major role in the development and growth regulation of normal cells, but is often malfunctioned in cancer cells. Anthocyanin-rich extracts from berries and grapes, and several pure anthocyanins and anthocyanidins, have been found to exhibit pro-apoptotic effects in multiple cell types in in vitro studies (94,96,98–101), which is via both intrinsic (mitochondrial) and extrinsic (FAS)pathways (100,102). Inflammation on the other has also been shown to play a role in the promotion of some types of cancer in animals, and probably in humans (103). Abnormal upregulation of two inflammatory proteins, nuclear factor kappa B (NF-rB) and cyclooxygenase-2 (COX-2), is a common phenomena in many cancers, and inhibition Page 23 of 63
of these proteins usually exhibit significant anti carcinogenic effects (101,102). Anthocyanins, through their ability to inhibit the mRNA and/or protein expression levels of COX-2, NF-rB and various interleukins, have shown anti-inflammatory effects in multiple cell types in vitro (104–108). The process of forming new blood vessels from the existing vascular network is known as Angiogenesis, which is an important factor in tumour growth and metastasis (109). Some of the most potent angiogenesis-activating molecules are members of a family of vascular endothelial growth factors (VEGF), whose expression is rapidly enhanced in developing tumours (109). Anthocyanins’ anti-angiogenic effects have been demonstrated using cultured endothelial cells (90), oral cancer cells (104) and mouse epidermal JB6 cells (109). Anthocyanins in all the cases have been shown to suppress angiogenesis through several mechanisms that include: inhibition of H2O2and tumour necrosis factor alpha (TNF-a)-induced VEGF expression in epidermal keratinocytes (90), and by reducing VEGF and VEGF receptor expression in endothelial cells (90). In addition, mouse epidermalJB6 cells when treated with an anthocyanin-rich extract from black raspberries resulted in down-regulation of VEGF expression (109). An early and critical invasion event is the degradation of basement membrane collagen by proteolysis. In order to facilitate degradation of the extracellular matrix barriers for successful invasion tumour and stromal cells have to secrete proteolytic enzymes. Degradation of the basement membrane is not only dependent on the amount of proteolytic enzymes present but also on the balance between activated proteases and their naturally occurring inhibitors. Matrix metalloproteinases (MMP) and plasminogen activators are involved in the regulation of degradation of the basement membrane (110). Anthocyanin extracts from different berry types, black rice and eggplant have been investigated for their ability to inhibit the invasion of multiple cancer cell types and were found to inhibit invasion of cancer cells by reducing the expression of MMP and urokinase-plasminogen activator (u-PA) (110). Induction of cellular differentiation can be used as a means of prevention and treatment of cancer through a cell-specific approach that is likely to be less toxic than standard radio/chemotherapy (111). Treatment of leukemic cells in vitro with anthocyanins (25–200 lg/ml) led to induction of differentiation as proved by: (a) Increased reduction of nitro blue tetrazolium (NBT), a functional marker for granulocyte/monocyte differentiation; Page 24 of 63
(b) Increased adherence of cells to plastic, suggesting differentiation of leukemic cells into a monocyte/macrophage-like phenotype; (c) Induction of naphthol AS-D chloroacetate activity, a marker for granulocytic differentiation; and, (d) An increase in the number of a-naphthyl acetate esterase positive cells, further indicating differentiation toward the monocytic/macrophagic lineage (111). Anthocyanins have also been found to induce differentiation in melanoma cells characterized by a significant increase in dendritic outgrowth along with a remodelling of the micro tubular network (112). 2.5.5 Role of Anthocyanins in Diabetes and Obesity Many recent studies suggest that consumption of fruits and vegetables, especially those rich in polyphenols, reduce the incidence of type-2 diabetes, a condition which is greatly associated with insulin resistance (113–115). Insulin resistance is a disorder where insulin inadequately stimulates glucose transport in skeletal muscle and fat, and also suppresses hepatic glucose production. Anthocyanins and anthocyanidins, have been found to protect pancreatic β-cells from glucose induced oxidative stress in a number of studies (116,117).The dimethoxy ether and the glycoside of leuco pelargonidin isolated from the bark of the Indian banyan tree Ficus bengalensis have been found to show significant hypoglycaemic, hypolipidemic and serum insulin-raising effects in moderately diabetic rats, being comparable with the effects of glibenclamide (an oral hypoglycaemic sulfonylureabased drug) (118–120). In addition, Cornus fruits (cherry) which is a rich source of anthocyanins, have been reported to possess anti-diabetic activity (121,122). Pelargonidin-3-galactoside and its aglycone, pelargonidin, have been reported to cause a 1.4-fold increase in insulin secretion. Several compounds present in grape skin or whole grapes have also been reported of being capable of enhancing insulin secretion as well as selectively inhibiting COX-2 enzymes (123). The study suggested that cherries, grapes, and berries containing anthocyanins might be useful in the prevention of type-2diabetes. Anthocyanin extracts were found to have potent alphaglucosidase inhibitory activity, suppressing the increase in postprandial glucose level in few in vitro and animal studies (124,125). Toxic reactions take place in organ tissues due to continuous hyperglycemia. Loss of lens opacity control is related to high levels of reducing sugars in in vitro and in vivo Page 25 of 63
studies, i.e. the formation of experimental diabetic cataract, which is a complication of diabetes occurring in about 10% of diabetic patients. It is proven that caloric and food intake greatly influence the progression of diabetes and diabetic complications (126). Flavonoids have been well known for their possible role in the prevention of diabetic cataracts (127). Inhibitory activities for lens opacity were shown by five anthocyanin monomers isolated from the extract of grape skin (126). Flavonoids have also been found to prevent or delay the occurrence of cataracts in rat lenses perfused with a high-glucose solution or in diabetic rabbits (127,128).Many naturally extracted or synthetic anthocyanin combinations with novel anti-cataract or anti-glaucoma activity have been reported in patents (129,130). Diabetes results in various microcirculatory disorders. Many of them may occur before the onset of microangiopathic lesion (thickening of capillaries in many areas including the eye in diabetics) and are assumed critical in the pathogenesis of microcirculatory
complications
involved
with
diabetes.
The
microvascular
permeability and the number of leucocytes sticking to the venular endothelium are increased in the diabetic microangiopathic condition (131,132). Delphinidin chloride showed increased microvascular permeability and a reduction of leucocytes adhering to the venular vessels in diabetic hamsters (133). Anthocyanosides from berries are in use nowadays in ophthalmology for their ability to improve vision and prevent diabetic retinopathy (134,135). Several flavonoids including anthocyanosides have been effective against experimentally induced capillary hyper filtration (136,137). In one animal study, it was shown that anthocyanosides can improve and even normalise capillary filtration of albumin (138). Endothelium-dependent vasorelaxaton by different vasodilator agonists is reduced in several pathological conditions including diabetes (134). One of the mechanisms resulting the dysfunction of the endothelium is a decreased release of nitric oxide (NO)(140). It was found that extracts from red wines, other grape products, and various plants that contain polyphenols (mainly anthocyanins), induced endothelium-dependent vasorelaxaton, probably through NO release or due to enhanced biological activity of NO( 33,141–143). A combination of anthocyanins extracted from bilberry(V. myrtillus L.) was reported to have biological and pharmacological properties, including vasorelaxaton(144)and ophthalmic activity (145). Increased level of triglyceride (hyper triglyceridemia) is a major feature of the insulin resistance syndrome and obesity is strongly associated with insulin resistance. Thus, a Page 26 of 63
reduction in this resistance is important in preventing the development of type-2 diabetes. It was demonstrated in a study that cyanidin 3-glucoside-rich purple corn colour may improve high fat diet-induced insulin resistance in mice (146). Consumption of Pomegranate juice (PJ) by diabetic patients resulted in anti oxidative effects in their serum, and the oxidative state of in their monocytes/macrophages levels, effects being attributed specifically to anthocyanins (147,148). Extracts of anthocyanin and procyanidins have been shown to decrease triglycerides and increase HDL-cholesterol levels in rats (116). The primary site of energy storage is the adipocytes that are known to accumulate triacylglycerol during nutritional excess. In recent years, it has been established that adipocyte dysfunction plays an important role in the development of obesity and insulin resistance. Adipocytes synthesise and secrete biologically active molecules called adipocytokines among which Adiponectin is an important one (149). In the obese and insulin resistant state the plasma adiponectin concentration and mRNA expression level have been found to decrease (150,151). Anthocyanins have shown to regulate obesity and insulin sensitivity associated with adipocytokine secretion in adipocytes providing a biochemical basis for the use of anthocyanins, which also in turn will have significant implications for preventing obesity and diabetes (146). Thus, studies demonstrate that anthocyanins modulated the gene expression of the adipocytokines in human and may have a unique therapeutic advantage for the regulation of adipocyte function (152,153). 2.5.6 Miscellaneous Effects of Anthocyanins Anthocyanins extracted from Hibiscus sp have been used in the cure for liver dysfunction and hypertension whereas bilberry (Vaccinium) anthocyanins have been in use for vision disorders, microbial infections, diarrhea, and diverse other health disorders (84,154,155).Anthocyanin treatment was found to down regulate the expression of enzymes involved in inflammation in the lungs (156). The antimicrobial activity of anthocyanins has been well recognized, including marked inhibition of aflatoxin biosynthesis (157). There has been experimental evidence revealing the benefits of anthocyanins in diabetes and pancreatic disorders. Such efficacy is attributed to the multiple, simultaneous biological effects these pigments exhibit in the body, including prevention of free radicals generation, decreased lipid peroxidation,
Page 27 of 63
reduced pancreatic swelling, and decreased blood sugar concentrations in urine and blood serum (158,159).
2.6 Conclusion and future direction There are many other current studies being conducted to further evaluate the health beneficial effects of this extraordinary pigment, as researchers nowadays are focusing on extracting health benefits from functional foods. We can hope to see anthocyanins contributing significantly in the manufacture of therapeutics in the near future and facilitate people to benefit from the gifts of nature rather than artificial products.
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CHAPTER: 3 STUDY
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3. Study Abstract Purpose: This study evaluated the in vitro antioxidant potential of the ethanol, ethyl acetate and toluene extracts of Amaranthus gangeticus. Methods: The different antioxidant assays, including DPPH free radical scavenging, nitric oxide scavenging, hydrogen peroxide scavenging and reducing power, were studied. Moreover, high-performance liquid chromatography (HPLC) coupled with diode-array detection was used to identify and quantify the phenolic compounds in the ethanol extract of this leafy vegetable. Results: Amaranthus gangeticus extracts showed effective hydrogen peroxide radical scavenging and nitric oxide scavenging activity. However, reducing power of ferric ion and DPPH radical scavenging activity was not significant compared to the standard antioxidant activity. Rutin hydrate was abundantly detected in the extract of Amaranthus gangeticus. Conclusion: Our investigation suggests that Amaranthus gangeticus show competent antioxidant activity though a further modified HPLC-DAD analysis is required to specifically identify and quantify the actual compounds responsible for such antioxidant activities of its extracts.
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3.1 Introduction Food is a very basic and vital necessity for survival, as it plays a crucial role in growth and maintenance of the human body and there are certain fruits and vegetables that are capable of providing certain health benefits along with fulfilling the physiological needs and are thus referred to as “Functional Food” (1,2).Such effects including, but are not limited to anti-carcinogenic, anti-inflammatory, anti-allergic, cardiovascular protective effects, with some recent findings reporting about their anti- allergic effects as well (3–7). Currently, researchers are focusing immensely on the protective effects against oxidative stress acquired from the natural dietary antioxidants that are present in plants and are capable of trapping free radicals from our body which are potentially harmful (8). Many of the functional foods are from plant origin and they exhibit their function due to the presence of natural antioxidants like phenolic acids, flavonoids and anthocyanins which constitute an important class of antioxidants. Numerous of these kinds of functional foods are available in Bangladesh since agriculture is the single largest producing sector of the economy of this country due to its fertile soil and ample water supply. Among all the available fruits and vegetables Amaranthus gangeticus, locally known as Lal shak is thought to be a very potential source of antioxidants. This leafy vegetable is commonly grown across the Indian subcontinent belonging to the Amaranthaceae family which is a rich plant family containing more than 700 species most of which showing health beneficial effects. Thus, the objective of the study is to evaluate the antioxidant activity of Amaranthus gangeticus using
different antioxidant assays, including DPPH free radical
scavenging, nitric oxide scavenging, hydrogen peroxide scavenging and reducing power. In addition to that, high-performance liquid chromatography (HPLC) coupled with diode-array detection is used to identify and quantify the phenolic compounds in the ethanolic extract of this leafy vegetable.
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3.2 Materials & Methods 3.2.1 Collection and preparation of Plant material Amaranthus gangeticus was purchased from the local market in Dhaka city and the leaves were separated from the other plant parts prior to thorough washing with distilled water to remove any dirt. The leaves were shade dried and then ground into coarse powder with the help of a grinder. The powder was stored in an airtight container and kept in a cool, dark and dry place until further analysis was commenced. 3.2.2 Extraction process The dried powder of the leaves (200 g each) was extracted with 80% of ethanol, ethyl acetate and toluene separately in a Soxhlet apparatus at an elevated temperature (45±2 °C). The extracts were concentrated by evaporation under reduced pressure at 40°C using Buchi rotary evaporator to have gummy concentrate of deep green coloured extracts. 3.2.3 Chemicals Used Gallic acid (GA), (+)-catechin hydrate (CH), vanillic acid (VA), caffeic acid (CA), (-)-epicatechin (EC), p-coumaric acid (PCA), rutin hydrate (RH), ellagic acid (EA), myricetin (MC), kaempferol (KF), and quercetin (QU) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Acetonitrile (HPLC), methanol (HPLC), acetic acid (HPLC), and ethanol was obtained from Merck Inc. (Darmstadt, Germany). 2,2-diphenyl-1-picrylhydrazyl (DPPH), naphthyl ethylene diamine dihydrochloride, Folin-Ciocalteu reagent, gallic acid, quercetin were also purchased from Sigma-Aldrich, St. Louis, MO, USA. All other reagents are of standard laboratory grade. 3.2.4 In vitro antioxidant assays of the ethanol, ethyl acetate and toluene extracts of Amaranthus gangeticus 3.2.4.1 DPPH radical scavenging activity The free radical scavenging capacity of the extracts was determined using DPPH (9). DPPH solution (0.004% w/v) was prepared in 95% ethanol. All the three extracts of Amaranthus gangeticus were mixed separately with ethanol, ethyl acetate and toluene to prepare the stock solution (5 mg/mL). Freshly prepared DPPH solution (0.004% w/v) was taken in test tubes and the extracts
were added followed by serial dilutions (1 μg to 500 μg) to every test tube so that the final volume was 3 mL and after 10 min, the absorbance was read at 515 nm using a spectrophotometer (HACH 4000 DU UV –visible spectrophotometer). Ascorbic acid was used as a reference standard and was dissolved in distilled water to make the stock solution with the same concentration (5 mg/mL). Control sample was prepared containing the same volume without any extract and reference ascorbic acid. 95% ethanol, ethyl acetate and toluene were served as blanks for each extract type respectilvely. Percent scavenging of the DPPH free radical was measured by using the following equation: % Scavenging Activity=[(Absorbance of the control – Absorbance of the test sample)/ Absorbance of the control] X 100 The inhibition curve was plotted for duplicate experiments and represented as % of mean inhibition ± standard deviation. 3.2.4.2
Reducing power The reducing power of Amaranthus gangeticus was performed accordingly as previously described (10). Different concentrations of the three different extracts
(100 to 1000 μg) in 1 ml of distilled water were mixed with
phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 ml, 1%). The mixture was incubated at 50°C for 20 min. A portion (2.5 ml) of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml. 0.1%) and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. Ascorbic acid was used as the standard. Phosphate buffer (pH 6.6) was used as blank solution. The absorbance of the final reaction mixture of two parallel experiments was taken and is expressed as mean ± standard deviation. 3.2.4.3
Nitric oxide (NO) radical inhibition assay Nitric oxide radical inhibition can be estimated by the use of Griess Illosvoy reaction (9). In this investigation, Griess-Illosvoy reagent was modified by using naphthyl ethylene diamine dihydrochloride (0.1% w/v) instead of 1napthylamine (5%). The reaction mixture (3 ml) containing sodium 48
nitroprusside (10 mM, 2 ml), phosphate buffer saline (0.5 ml) and Amaranthus gangeticus extract (10 to 320 μg) or standard solution (ascorbic acid, 0.5 ml) was incubated at 25°C for 150 min. After incubation, 0.5 ml of the reaction mixture was mixed with 1 ml of sulfanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 min for completing diazotization. Then, 1 ml of naphthyl ethylene diamine dihydrochloride was added, mixed and allowed to stand for 30 min at 25°C. A pink colored chromophore was formed in diffused light. The absorbance of these solutions was measured at 540 nm against the corresponding blank solutions. 3.2.4.4
Scavenging of hydrogen peroxide The ability of the extracts to scavenge hydrogen peroxide was determined by the method based on the previously described method (11). Hydrogen peroxide (43 mM) was prepared in phosphate buffered saline (pH 7.4). Standards (ascorbic acid) and extract solutions were prepared at concentrations of 50 to 250 mM. Aliquots of standard or extract solutions (3.4 mL) were added to 0.6 mL of hydrogen peroxide solution. The reaction mixture was incubated at room temperature for 10 min, and the absorbance was determined at 230 nm. The percentage of scavenging was calculated as follows: % H2O2 Scavenging = 100 x (Absorbance of Control- Absorbance of Sample)/ Absorbance of Control
3.2.5 High performance liquid chromatography (HPLC) system Chromatographic analyses were carried out on a Thermo Scientific Dionex UltiMate 3000 Rapid Separation LC (RSLC) systems (Thermo Fisher Scientific Inc., MA, USA), coupled to a quaternary rapid separation pump (LPG-3400RS), Ultimate 3000RS autosamplier (WPS-3000) and rapid separation diode array detector (DAD-3000RS). Phenolic compounds were separated on an Acclaim® C18 (4.6 x 250 mm; 5µm) column (Dionix, USA) which was controlled at 30°C using a temperature controlled column compartment (TCC-3000). Data acquisition, peak integration, and calibrations were performed with Dionix Chromeleon software (Version 6.80 RS 10). 3.2.5.1
Chromatographic conditions The phenolic composition of the ethanol extract of Amaranthus gangeticus was determined by HPLC, as described previously with some modifications 49
(12). The mobile phase consisted of acetonitrile (solvent A), acetic acid solution pH 3.0 (solvent B), and methanol (solvent C). The system was run with the following gradient elution program: 0 min, 5%A/95%B; 10 min, 10%A/80%B/10%C; 20 min, 20%A/60%B/20%C and 30min, 100%A. There was a 5 min post run at initial conditions for equilibration of the column. The flow rate was kept constant throughout the analysis at 1 ml/min and the injection volume was 20 µl. For UV detection, the wavelength program was optimized to monitor phenolic compounds at their respective maximum absorbance wavelengths as follows: λ 280 nm held for 18.0 min, changed to λ 320 nm and held for 6 min, and finally changed to λ 380 nm and held for the rest of the analysis and the DAD was set at an acquisition range from 200 nm to 700 nm.The detection and quantification of GA, CH, VA, CA, and EC was done at 280 nm, of PCA, RH, and EA at 320 nm, and of MC, QU, and KF at 380 nm, respectively. 3.2.5.2
Standard and sample preparation A stock standard solution (100 µg/ml) of each phenolic compound was prepared in methanol by weighing out approximately 5mg of the analyte into 50 ml volumetric flask. The mixed standard solution was prepared by dilution the mixed stock standard solutions in methanol to give a concentration of 5 µg/ml for each polyphenols except (+)-catechin hydrate, caffeic acid, rutin hydrate (4 µg/ml) and quercetin (3 µg/ml). All standard solutions were stored in the dark at 5°C and were stable for at least three months. The calibration curves of the standards were made by a dilution of the stock standards (five set of standard dilutions) with methanol to yield 1.0 - 5.0 µg/ml for GA, CH, VA, EC, PCA, EA, MC, KF; 0.5 - 4.0 µg/ml for CH, CA, RH, and 0.25 - 3.0 µg/ml for QU.The calibration curves were constructed from chromatograms as peak area vs. concentration of standard. A solution of ethanol extract of Amaranthus gangeticus at a concentration of 5 mg/ml was prepared in ethanol by vortex mixing (Branson, USA) for 30 min. The samples were stored in the dark at low temperature (5°C). Spiking the sample solution with phenolic standards was done for additional identification of individual polyphenols. Prior to HPLC analysis, all solutions (mixed standards, sample, and spiked solutions were filtered through 0.20 µm nylon 50
syringe filter (Sartorius, Germany) and then degassed in an ultrasonic bath (Hwashin, Korea) for 15 min. 3.2.5.3
Peak characterization and quantification The compounds were identified by comparing with standards of each identified compound using the retention time, the absorbance spectrum profile and also by running the samples after the addition of pure standards. Quantification was performed by establishing calibration curves for each compound determined, using the standards. Linear calibration curves for standards (peak area vs concentration) were constructed with R2 exceeding 0.995. Data are reported as means ± standard deviations of triplicate independent analyses.
3.2.6 Assay for total phenolic content of the ethanol extract of Amaranthus gangeticus The concentrations of total phenols in extracts ware measured by a UV spectrophotometer based on a colorimetric oxidation/reduction reaction (13). The oxidizing reagent used was Folin-Ciocalteu reagent. Gallic acid was used as standard. 2.5 ml of Folin-Ciocalteu reagent (diluted 10 times with water) and 2 ml of Na2CO3 (75 g/1 L) were added to 0.5 ml of diluted extract (1 mg in 4 ml distilled water). The sample was incubated for 20 min at room temperature. For control sample, 0.5 ml distilled water was used. The absorbance was measured at 760 nm. These data were used to estimate the phenolic contents using a standard curve obtained from various concentration of gallic acid. The results were expressed as μg of gallic acid per mg of extract. 3.2.7 Assay for total flavonoid content of the ethanol extract of Amaranthus gangeticus Total flavonoid content was measured by the aluminium chloride colorimetric assay (14). An aliquot (1ml) of extracts or standard solution of quercetin (20, 40, 60, 80 and 100mg/l) was added to 10ml volumetric flask containing 4ml of distilled H2O. To the flask was added 0.3ml 5% NaNO2. After 5min, 0.3ml 10% AlCl3 was added. At 6th min, 2ml 1M NaOH was added and the total volume was made up to 10ml with distilled H2O. The solution was mixed well and the absorbance was measured against prepared reagent blank at 510 nm. 51
Total flavonoid content of Amaranthus gangeticus was expressed as mg of quercetin equivalents per 100g of fresh mass. Samples were analysed in duplicates. 3.2.8 Phytochemical screening Phytochemical screening of the extract was performed using the following reagents and chemicals: Presence of reducing sugars were determined using Molisch’s reagent, alkaloids were determined using Mayer's reagent, flavonoids were determined by using Mg and HCl, tannins were determined using ferric chloride and potassium dichromate solutions and saponins were determined by the ability to produce froth. Gum was tested using Molisch’s reagents and concentrated sulphuric acid. 3.2.9 Statistical Analysis All data are presented as mean ±Standard deviation (SD). IC50 values for scavenging of free radicals by the extracts were calculated from dose-response curve by using default analyzing tab of Graph Pad Prism Software (USA). 3.3 Results Table 1: Presence of various bioactive molecules in the extract of Amaranthus gangeticus.
Group
Ethanol extract of Amaranthus gangeticus
Reducing Sugar
++
Flavonoids
++
Tannins
++
Gums
+
Saponins
+
Alkaloids
--
Total Phenolic content
32.14 µg GAE/mg of extract
Total flavonoid content
18.6 µg GAE/mg of extract 52
Figure 2: HPLC chromatogram of a standard mixture of polyphenolic compounds. Peaks: 1, gallic acid; 2, (+)-catechin; 3, vanillic acid; 4, caffeic acid; 5, (–)-epicatechin; 6, p-coumaric acid; 7, rutin hydrate; 8, ellagic acid; 9, myricetin; 10, querceti
Figure 3: HPLC chromatogram of the ethanol extract of Amaranthus gangeticus. Peaks: 1, rutin hydrate.
53
Table 2: Contents of Polyphenolic compounds in the ethanol extract of Amaranthus gangeticus (n=3).
Polyphenolic
Amaranthus Gangeticus
compound
Content (mg/100 g of dry extract)
% RSD
RH
3.42
0.11
Table 3: IC50 values of Amaranthus gangeticus extracts in different antioxidant assays such as DPPH method, NO- scavenging method and H2O2 scavenging. Values are expressed as average of duplicate experiments
Sample
DPPH Scavenging NO
Scavenging H2O2
Scavenging
Method (µg/ml)
Method (µg/ml)
Method (µg/ml)
Ascorbic acid
14.76
~ 6.927
53.16
Ethanol Extract
169.3
0.008063
42.39
~ 0.002676
65.96
~ 0.0003934
286.0
Ethyl
Acetate ~ 479388
Extract Toluene Extract
~1.092e+017
54
Figure 4: Effect of Amaranthus gangeticus extract on various antioxidant assays.
a) Scavenging of DPPH radical by ascorbic acid and different extracts of Amaranthus gangeticus, b) Reducing power of ascorbic acid and different extracts of Amaranthus gangeticus, c) Scavenging of H2O2 radical by ascorbic acid and different extract of Amaranthus gangeticus, d) Scavenging of NO- radical by ascorbic acid and different extracts of Amaranthus gangeticus, Values are given as duplicate and expressed as Mean ± Standard error of mean.
3.4 Discussions The secondary metabolites of plant origins are gaining immense consideration recently due to their wide range of biological activities. Thus intake of the functional foods for their benefits has markedly increased with the awareness of their safety and nil side-effects. In this study a wide range of established in vitro assays have been performed to evaluate the antioxidant and free radical scavenging properties of Amaranthus gangeticus extracts. Preliminary Phytochemical analysis of Amaranthus gangeticus extracts revealed the presence of reducing sugar, flavonoids, gums, tannins and saponins in the extracts (Table 1). Antioxidant properties are significantly associated with the presence of phenolic compounds and flavonoids (15,16). In 55
addition to that gums, tannins and saponins containing plants are considered to be rich sources of antioxidants (17–19). 3.4.1 DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging activity The DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging activity of the three different extracts of Amaranthus gangeticus are given in Table 3 and Figure 3. In the TLC-based qualitative antioxidant assay using DPPH spray, the extract of Amaranthus gangeticus showed low to moderate free radical scavenging properties as indicated by the presence of a mildly yellowish spot on a reddish purple background on the TLC plate. Though the ethanol extract showed more significant effect compared to the ethyl acetate and toluene extract. The IC50 values of the extracts were found to be 169.3 µg/ml, ~ 47938 µg/ml and ~1.092e+017 µg/ml respectively whereas IC50 for ascorbic acid was found to be 14.76 µg/ml, which is a well-known antioxidant. The IC50 values for the ethyl acetate and toluene extracts came out to be excessively high which is not possible to obtain in a laboratory experiment and should be withdrawn as a result of some technical errors. Though Amaranthaceae plant family possess strong antioxidant activities, Amaranthus gangeticus ethanol extract showed moderate DPPH free radical scavenging effect as compared to standard antioxidants. DPPH is pink in solution and is a stable free radical, capable of accepting one electron from antioxidant containing plant extracts and thus, neutralizing its free radical nature. The degree of decolourization indicates the scavenging activity of the plant extracts and can be measured using UV spectrophotometer (20,21). 3.4.2 NO· scavenging activity The direct scavenging of the NO· radical may be partially related to the suppression of its release, as all Amaranthus gangeticus extracts decreased the amount of nitrite generated from the decomposition of sodium nitroprusside in vitro. The scavenging of nitric oxide by the plant extract was increased prominently in a dose-dependent manner. The IC50 value of the extracts were 0.00806 µg/ml, ~ 0.002676 µg/ml and ~ 0.0003934 µg/ml respectively whereas the IC50 value of ascorbic acid was ~ 6.927 µg/ml (Table 3 and Figure 3). Nitric oxide is a water soluble gaseous molecule involved in inflammation, cancer and other pathological conditions (22,23). Various tissues can be harmed by the nitric oxide and super oxide anion. The NO· and .O-2 react to produce peroxynitrite (ONOO_) which exacerbate the toxicity and damage 56
profile as it leads to serious toxic reactions with biomolecules (24,25). By scavenging the reactive peroxynitrite, the chain of reactions initiated by excess NO generation can be prevented that will in turn help preventing the harmful effects of such radicals on the human body. 3.4.3 H2O2 scavenging activity The H2O2 scavenging activity of both ascorbic acid and Amaranthus gangeticus after 10mins incubation time increased with increased concentration of the sample. The ethanol and ethyl acetate extract showed almost similar H2O2 scavenging activity with that of ascorbic acid at similar concentrations. The IC50 values of the extracts were 42.39 µg/ml, 65.96 µg/ml and 286.0 µg/ml, whereas that for ascorbic acid was 53.16 µg/ml. (Table 3 and Figure 3) Upon the presence of iron ions hydrogen peroxide may give rise to hydroxyl ions which can turn toxic to the cells (26). Thus, H2O2 must be removed from the cells to provide antioxidant defense. Dietary polyphenols (especially compounds with the orthodihydroxy phenolic structure quercetin, catechin, gallic acid ester, caffeic acid ester) showed protection against hydrogen peroxide induced cytotoxicity in mammalian and bacterial cells (27). The presence of certain significant phenolic compounds such as rutin hydrate in the extracts of Amaranthus gangeticus may be involved in removing the H2O2. 3.4.4 Reducing activity Our data on the reducing power of the tested extracts suggests low properties. Like other antioxidant assays, the reducing power of Amaranthus gangeticus extracts increased with increasing the amount of samples. Figure 3 shows the reducing ability of Amaranthus gangeticus extracts in comparison with ascorbic acid. Presence of reductones is highly correlated with showing reducing properties by the extracts as they help in breaking the free radical chain by donating a hydrogen atom (28). This species may be absent or present in very small amounts in our extracts for which significant reducing activity was not observed. The antioxidant activity of plant extracts are accredited due to the presence of various antioxidants and their consequent mechanisms such as prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity and radical scavenging, thus absence of one such antioxidant doesn’t prevent an extract from showing strong antioxidant properties (28,29). 57
3.4.5 Total flavonoid content and total Phenolic Content The value of the total phenolic and flavonoid content were found to be 32.14 µg GAE/mg of extract and 18.6 µg GAE/mg of extract respectively. This value is coherent with other findings of the extract showing antioxidant properties. 3.4.6 HPLC-DAD analysis of phenolic contents in leaves extracts The phenolic compounds present in the extract of Amaranthus gangeticus were identified and quantified by HPLC-DAD system. Eleven different phenolic standards, C18 column with 250 mm length, and rapid separation LC (RSLC) systems were used in this study while other options like six standards,20 C18 column with 150mm length, and HP 1090, series II, liquid chromatography systems are also available for the detection of polyphenolic compounds (30,31). Wavelengths of 280, 320 and 380 nm were selected for the detection of all standards in this study. Figure 1 shows a good separation achieved within 30min using the above conditions described. Symmetrical, sharp and well-resolved peaks were observed for the eleven polyphenolic standards. The order of elution and the retention times for GA, CH, VA, CA, EC, PCA, RH, EA, MC, QU, and KF were 6.25, 13.69, 15.95, 16.24, 16.69, 19.89, 21.07, 21.79, 24.54, 26.12, and 27.13 minutes respectively. Figure 2 shows the chromatogram for the ethanol extract of Amaranthus gangeticus. A sharp peak of rutin hydrate is seen with an abundance of 3.42 mg per 100g of dry weight (Table 2) and no other significant peaks are observed. The described conditions may have been unsuitable for the separation of our extract for which the chromatogram lacked good separation of the peaks or may be some other compounds were present instead of the eleven standard used for which we could not obtain satisfactory HPLC profile. However, rutin hydrate is a potent antioxidant which may have also contributed to the antioxidant activity of the extracts of Amaranthus gangeticus.
3.5
Conclusion
All the extracts of Amaranthus gangeticus have showed competent antioxidant activities compared to the standard compounds in vitro. The extracts’ strong abilities as a scavenger of nitric oxide and hydrogen peroxide free radicals may be a result of various antioxidant mechanisms. Though a satisfactory HPLC profile was not obtained but hopefully a further modified HPLC-DAD analysis would be able to 58
identify and quantify the actual compounds responsible for such antioxidant activities of the Amaranthus gangeticus extract. Moreover, further work should be carried out using the Amaranthus gangeticus extracts to determine its in-vivo antioxidant activities
3.6
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CHAPTER: 4 CONCLUSION
62
4. Conclusion All the extracts of Amaranthus gangeticus have showed competent antioxidant activities compared to the standard compounds in vitro. The extracts’ strong abilities as a scavenger of nitric oxide and hydrogen peroxide free radicals may be a result of various antioxidant mechanisms. Though a satisfactory HPLC profile was not obtained but hopefully a further modified HPLC-DAD analysis would be able to identify and quantify the actual compounds responsible for such antioxidant activities of the Amaranthus gangeticus extract. Moreover, further work should be carried out using the Amaranthus gangeticus extracts to determine its in-vivo antioxidant activities.
63
