Journal of Medicinal Plants Research

Journal of Medicinal Plants Research Volume 6 Number 18 ISSN 1996-0875

16 May, 2012

ABOUT JMPR The Journal of Medicinal Plant Research is published weekly (one volume per year) by Academic Journals. The Journal of Medicinal Plants Research (JMPR) is an open access journal that provides rapid publication (weekly) of articles in all areas of Medicinal Plants research, Ethnopharmacology, Fitoterapia, Phytomedicine etc. The Journal welcomes the submission of manuscripts that meet the general criteria of significance and scientific excellence. Papers will be published shortly after acceptance. All articles published in JMPR are peerreviewed. Electronic submission of manuscripts is strongly encouraged, provided that the text, tables, and figures are included in a single Microsoft Word file (preferably in Arial font).

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Editors Prof. Akah Peter Achunike Editor-in-chief Department of Pharmacology & Toxicology University of Nigeria, Nsukka Nigeria

Prof. Parveen Bansal Department of Biochemistry Postgraduate Institute of Medical Education and Research Chandigarh India.

Dr. Ugur Cakilcioglu Elazıg Directorate of National Education Turkey.

Dr. Ravichandran Veerasamy AIMST University Faculty of Pharmacy, AIMST University, Semeling – 08100, Kedah, Malaysia.

Dr. Jianxin Chen Information Center, Beijing University of Chinese Medicine, Beijing, China 100029, China.

Dr. Hassan Sher Department of Botany and Microbiology, College of Science, King Saud University, Riyadh Kingdom of Saudi Arabia.

Dr. Jin Tao Professor and Dong-Wu Scholar, Department of Neurobiology, Medical College of Soochow University, 199 Ren-Ai Road, Dushu Lake Campus, Suzhou Industrial Park, Suzhou 215123, P.R.China.

Dr. Pongsak Rattanachaikunsopon Department of Biological Science, Faculty of Science, Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand.

Dr. Sayeed Ahmad Herbal Medicine Laboratory, Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi, 110062, India.

Dr. Cheng Tan Department of Dermatology, first Affiliated Hospital of Nanjing Univeristy of Traditional Chinese Medicine. 155 Hanzhong Road, Nanjing, Jiangsu Province, China. 210029

Dr. Naseem Ahmad Young Scientist (DST, FAST TRACK Scheme) Plant Biotechnology Laboratory Department of Botany Aligarh Muslim University Aligarh- 202 002,(UP) India.

Dr. Isiaka A. Ogunwande Dept. Of Chemistry, Lagos State University, Ojo, Lagos, Nigeria.

Editorial Board Prof Hatil Hashim EL-Kamali Omdurman Islamic University, Botany Department, Sudan.

Dr. Arash Kheradmand Lorestan University, Iran.

Prof. Dr. Muradiye Nacak Department of Pharmacology, Faculty of Medicine, Gaziantep University, Turkey.

Prof Dr Cemşit Karakurt Pediatrics and Pediatric Cardiology Inonu University Faculty of Medicine, Turkey.

Dr. Sadiq Azam Department of Biotechnology, Abdul Wali Khan University Mardan, Pakistan.

Samuel Adelani Babarinde Department of Crop and Environmental Protection, Ladoke Akintola University of Technology, Ogbomoso Nigeria.

Kongyun Wu Department of Biology and Environment Engineering, Guiyang College, China.

Prof Swati Sen Mandi Division of plant Biology, Bose Institute India.

Dr.Wafaa Ibrahim Rasheed Professor of Medical Biochemistry National Research Center Cairo Egypt.

Dr. Ujjwal Kumar De Indian Vetreinary Research Institute, Izatnagar, Bareilly, UP-243122 Veterinary Medicine, India.

Instructions for Author Electronic submission of manuscripts is strongly encouraged, provided that the text, tables, and figures are included in a single Microsoft Word file (preferably in Arial font). The cover letter should include the corresponding author's full address and telephone/fax numbers and should be in an e-mail message sent to the Editor, with the file, whose name should begin with the first author's surname, as an attachment. Article Types Three types of manuscripts may be submitted: Regular articles: These should describe new and carefully confirmed findings, and experimental procedures should be given in sufficient detail for others to verify the work. The length of a full paper should be the minimum required to describe and interpret the work clearly. Short Communications: A Short Communication is suitable for recording the results of complete small investigations or giving details of new models or hypotheses, innovative methods, techniques or apparatus. The style of main sections need not conform to that of full-length papers. Short communications are 2 to 4 printed pages (about 6 to 12 manuscript pages) in length. Reviews: Submissions of reviews and perspectives covering topics of current interest are welcome and encouraged. Reviews should be concise and no longer than 4-6 printed pages (about 12 to 18 manuscript pages). Reviews are also peer-reviewed. Review Process All manuscripts are reviewed by an editor and members of the Editorial Board or qualified outside reviewers. Authors cannot nominate reviewers. Only reviewers randomly selected from our database with specialization in the subject area will be contacted to evaluate the manuscripts. The process will be blind review. Decisions will be made as rapidly as possible, and the journal strives to return reviewers’ comments to authors as fast as possible. The editorial board will re-review manuscripts that are accepted pending revision. It is the goal of the JMPR to publish manuscripts within weeks after submission.

Regular articles All portions of the manuscript must be typed doublespaced and all pages numbered starting from the title page. The Title should be a brief phrase describing the contents of the paper. The Title Page should include the authors' full names and affiliations, the name of the corresponding author along with phone, fax and E-mail information. Present addresses of authors should appear as a footnote. The Abstract should be informative and completely selfexplanatory, briefly present the topic, state the scope of the experiments, indicate significant data, and point out major findings and conclusions. The Abstract should be 100 to 200 words in length.. Complete sentences, active verbs, and the third person should be used, and the abstract should be written in the past tense. Standard nomenclature should be used and abbreviations should be avoided. No literature should be cited. Following the abstract, about 3 to 10 key words that will provide indexing references should be listed. A list of non-standard Abbreviations should be added. In general, non-standard abbreviations should be used only when the full term is very long and used often. Each abbreviation should be spelled out and introduced in parentheses the first time it is used in the text. Only recommended SI units should be used. Authors should use the solidus presentation (mg/ml). Standard abbreviations (such as ATP and DNA) need not be defined. The Introduction should provide a clear statement of the problem, the relevant literature on the subject, and the proposed approach or solution. It should be understandable to colleagues from a broad range of scientific disciplines.

Materials and methods should be complete enough to allow experiments to be reproduced. However, only truly new procedures should be described in detail; previously published procedures should be cited, and important modifications of published procedures should be mentioned briefly. Capitalize trade names and include the manufacturer's name and address. Subheadings should be used. Methods in general use need not be described in detail.

Results should be presented with clarity and precision. The results should be written in the past tense when describing findings in the authors' experiments. Previously published findings should be written in the present tense. Results should be explained, but largely without referring to the literature. Discussion, speculation and detailed interpretation of data should not be included in the Results but should be put into the Discussion section. The Discussion should interpret the findings in view of the results obtained in this and in past studies on this topic. State the conclusions in a few sentences at the end of the paper. The Results and Discussion sections can include subheadings, and when appropriate, both sections can be combined. The Acknowledgments of people, grants, funds, etc should be brief. Tables should be kept to a minimum and be designed to be as simple as possible. Tables are to be typed doublespaced throughout, including headings and footnotes. Each table should be on a separate page, numbered consecutively in Arabic numerals and supplied with a heading and a legend. Tables should be self-explanatory without reference to the text. The details of the methods used in the experiments should preferably be described in the legend instead of in the text. The same data should not be presented in both table and graph form or repeated in the text. Figure legends should be typed in numerical order on a separate sheet. Graphics should be prepared using applications capable of generating high resolution GIF, TIFF, JPEG or Powerpoint before pasting in the Microsoft Word manuscript file. Tables should be prepared in Microsoft Word. Use Arabic numerals to designate figures and upper case letters for their parts (Figure 1). Begin each legend with a title and include sufficient description so that the figure is understandable without reading the text of the manuscript. Information given in legends should not be repeated in the text. References: In the text, a reference identified by means of an author‘s name should be followed by the date of the reference in parentheses. When there are more than two authors, only the first author‘s name should be mentioned, followed by ’et al‘. In the event that an author cited has had two or more works published during the same year, the reference, both in the text and in the reference list, should be identified by a lower case letter like ’a‘ and ’b‘ after the date to distinguish the works. Examples: Abayomi (2000), Agindotan et al. (2003), (Kelebeni, 1983), (Usman and Smith, 1992), (Chege, 1998;

1987a,b; Tijani, 1993,1995), (Kumasi et al., 2001) References should be listed at the end of the paper in alphabetical order. Articles in preparation or articles submitted for publication, unpublished observations, personal communications, etc. should not be included in the reference list but should only be mentioned in the article text (e.g., A. Kingori, University of Nairobi, Kenya, personal communication). Journal names are abbreviated according to Chemical Abstracts. Authors are fully responsible for the accuracy of the references. Examples: Chikere CB, Omoni VT and Chikere BO (2008). Distribution of potential nosocomial pathogens in a hospital environment. Afr. J. Biotechnol. 7: 3535-3539. Moran GJ, Amii RN, Abrahamian FM, Talan DA (2005). Methicillinresistant Staphylococcus aureus in community-acquired skin infections. Emerg. Infect. Dis. 11: 928-930. Pitout JDD, Church DL, Gregson DB, Chow BL, McCracken M, Mulvey M, Laupland KB (2007). Molecular epidemiology of CTXM-producing Escherichia coli in the Calgary Health Region: emergence of CTX-M-15-producing isolates. Antimicrob. Agents Chemother. 51: 1281-1286. Pelczar JR, Harley JP, Klein DA (1993). Microbiology: Concepts and Applications. McGraw-Hill Inc., New York, pp. 591-603.

Short Communications Short Communications are limited to a maximum of two figures and one table. They should present a complete study that is more limited in scope than is found in full-length papers. The items of manuscript preparation listed above apply to Short Communications with the following differences: (1) Abstracts are limited to 100 words; (2) instead of a separate Materials and Methods section, experimental procedures may be incorporated into Figure Legends and Table footnotes; (3) Results and Discussion should be combined into a single section. Proofs and Reprints: Electronic proofs will be sent (email attachment) to the corresponding author as a PDF file. Page proofs are considered to be the final version of the manuscript. With the exception of typographical or minor clerical errors, no changes will be made in the manuscript at the proof stage.

Fees and Charges: Authors are required to pay a $600 handling fee. Publication of an article in the Journal of Medicinal Plant Research is not contingent upon the author's ability to pay the charges. Neither is acceptance to pay the handling fee a guarantee that the paper will be accepted for publication. Authors may still request (in advance) that the editorial office waive some of the handling fee under special circumstances. Copyright: © 2012, Academic Journals. All rights Reserved. In accessing this journal, you agree that you will access the contents for your own personal use but not for any commercial use. Any use and or copies of this Journal in whole or in part must include the customary bibliographic citation, including author attribution, date and article title. Submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, or thesis) that it is not under consideration for publication elsewhere; that if and when the manuscript is accepted for publication, the authors agree to automatic transfer of the copyright to the publisher. Disclaimer of Warranties In no event shall Academic Journals be liable for any special, incidental, indirect, or consequential damages of any kind arising out of or in connection with the use of the articles or other material derived from the JMPR, whether or not advised of the possibility of damage, and on any theory of liability. This publication is provided "as is" without warranty of any kind, either expressed or implied, including, but not limited to, the implied warranties of merchantability, fitness for a particular purpose, or non-infringement. Descriptions of, or references to, products or publications does not imply endorsement of that product or publication. While every effort is made by Academic Journals to see that no inaccurate or misleading data, opinion or statements appear in this publication, they wish to make it clear that the data and opinions appearing in the articles and advertisements herein are the responsibility of the contributor or advertiser concerned. Academic Journals makes no warranty of any kind, either express or implied, regarding the quality, accuracy, availability, or validity of the data or information in this publication or of any other publication to which it may be linked.

Journal of Medicinal Plants Research

International Journal of Medicine and Medical Sciences Table of Contents:

Volume 6

Number 18

16 May, 2012

ARTICLES Review Asian plants as a promising alternative to classic drugs in postmenopausal osteoporosis: A review of literature from clinical perspective Ioana Ilie, Razvan Ilie and Teodora Mocan.

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Research Articles Callus induction and plant regeneration from leaf explants of Falcaria vulgaris an important medicinal plant Jaberian Hamideh, Piri Khosro and Nazari Deljou Mohammad Javad.

Ethnobotanical survey of medicinal plants used in the treatment of gastrointestinal disorders in the Eastern Cape Province, South Africa O. O. Olajuyigbe and A. J. Afolayan

Bioactivity of natural compounds isolated from cyanobacteria and green algae against human pathogenic bacteria and yeast H. Al-Wathnani, Ismet Ara, R. R. Tahmaz, T. H. Al-Dayel and M. A. Bakir

Status and trade of crude drug in Uttarakhand Deepshikha Arya, G. C. Joshi and Lalit M. Tiwari.

Morin, a flavonoid, prevents lysosomal damage in experimental myocardial ischemic rats Khalid S. Al-Numair, Govindasamy Chandramohan, Chinnadurai Veeramani and Mohammed A. Alsaif

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Volume 6

Number 18 16 May, 2012

ARTICLES Effects of Psidium guajava on the metabolic profile of Wister rats Flávia M. V. Farinazzi-Machado, Élen Landgraf Guiguer, Sandra M. Barbalho, Maricelma da Silva Soares de Souza, Patrícia Cincotto dos Santos Bueno, Claudemir Gregório Mendes, Adriano Cressoni Araújo, Andressa Rezende Teixeira Rodrigues, Larissa Maria de Lara Lima, Nádia Sanches Marim, Anna Cláudia Saad Brunnati, Paula Martins Ribeiro da Silva and Isabella Taietti Sato.

Antihaemorrhagic Potential of Citrullus colocynthis Schrad (Cucurbitaceae) against Naja naja karachiensis (Black Pakistan Cobra) Venom MHHB Asad, MT Razi, G Murtaza, S Azhar, SA Khan, QNU Saqib and I. Hussain

High-performance liquid chromatography (HPLC) nano analysis of antioxidant compounds of Iranian medicinal plants M. Monajjemi, A. R. Ilkhani, A. L. Nurul Aminin A. Eghdami, F. Mollaamin and S. Rezaei

Essential oil composition and insecticidal activity of Evodia lepta (Spreng) Merr. root barks from China against two grain storage insects Cai Hong Jiang, Qi Zhi Liu, Shu Shan Du, Zhi Wei Deng and Zhi Long Liu.

An efficient protein preparation method compatible with 2-DE analysis of Panax quinquefolius root - a tissue riches in interfering compounds Sun Liwei Ma Rui, Lei Xiujuan, Jiang Rui, Wang Yingping and Zhao Daqing

Tocopherol and phytosterol profile of Sesbania grandiflora (Linn.) seed oil Huma Shareef, Ghazala H. Rizwani, Muhammad Zia-ul-Haq, Shakeel Ahmad and Hina Zahid

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Volume 6

Number 18 16 May, 2012

ARTICLES Composition and insecticidal activity of the essential oil of Cananga odorata leaves against Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) Jun Cheng, Kai Yang, Na Na Zhao, Xuan Gao Wang, Shu Ying Wang and Zhi Long Liu

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Composition and biological activities of the essential oil extracted from a novel plant of Cinnamomum camphora Chvar. Borneol Jianyu Su, Jianping Chen, Shengmei Liao, Lin Li, Liang Zhu and Lei Chen.

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Enhancement of compatible solute and secondary metabolites production in Plantago ovata Forsk. by salinity stress Zahra Haghighi, Naser karimi, Masoud Modarresi and Saeed Mollayi

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The hypolipidemic and antioxidant actions of aqueous extracts Of Ocimum basilicum and Ocimum suave in high fat fed Rats Umar, I. A., Mohammed, A., Dawud, F. A. Kabir, A. M., Sai J. V., Muhammad, F. S. and Okalor, M. E.

Feeding deterrents from the tubers of Boschniakia himalaica against the red flour beetle, Tribolium castaneum Jie Cao, Zhi Long Liu, Shu Shan Du and Zhi Wei Deng

Investigation of in vitro antifungal activity of honey Anyanwu, C.U

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Journal of Medicinal Plants Research Vol. 6(18), pp. 3399-3406, 16th May, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR011.804 ISSN 1996-0875 ©2012 Academic Journals

Review

Asian plants as a promising alternative to classic drugs in postmenopausal osteoporosis: A review of literature from clinical perspective Ioana Ilie1*, Razvan Ilie2 and Teodora Mocan3 1

Department of Endocrinology, University of Medicine and Pharmacy, ‘‘Iuliu Hatieganu”, 400349, Louis Pasteur, ClujNapoca, Romania. 2 Department of Obstetrics and Gynecology, University of Medicine and Pharmacy, ‘‘Iuliu Hatieganu”, 400124, ClujNapoca, Romania. 3 Department of Physiology, University of Medicine and Pharmacy, ‘‘Iuliu Hatieganu”, 400620, Clinicilor, Cluj-Napoca, Romania. Accepted 15 July, 2011

Osteoporosis is characterised by low bone mineral density (BMD) resulting in fragile bones with increased risk of fracture. According to statistics, 75 million people worldwide suffer from osteoporosis. However, the higher consumption of soy by the Asian population is thought to be one of the contributing factors for the lower incidence of osteoporosis-related fractures in the region. Various side effects were reported to be associated with the use of both estrogen or hormone replacement therapy, and other classical anti-fracture agents. Therefore, alternative approaches, and especially natural therapies to treat osteoporosis are currently under research. In traditional Chinese medicine, osteoporosis is considered to be a disorder caused by the insufficiency of kidney yang, and the herbs perceived to be able to tonify the kidney yang have been used for more than 1000 years as therapy. Within this context, we have systematically researched the specialty literature on the topic, in an attempt to check whether various Asian plants could constitute valid therapies in the prophylaxis and treatment of osteoporosis, especially postmenopausal one. The current evidence suggests that isoflavones as well as other compounds from Asian plants extracts may regulate bone turnover by complex mechanisms and increase BMD, thus, potentially, reducing the fracture rate. Key words: Osteoporosis, traditional Chinese medicine, Asian plants, bone turnover, osteoclast, osteoblast, estrogen, postmenopause.

INTRODUCTION Osteoporosis is the most common metabolic bone disorder and an important health care issue in both Caucasians and Asians, extremely likely to aggravate with aging populations around the world. Although osteoporosis commonly affects both older men and women, postmenopausal women represent the primary focus of the disease. Women's Health Initiative Study as well as The Million Women Study indicated that long-term estrogen or hormone replacement therapy (HRT) in postmenopausal women may enhance the risk of breast

*Corresponding author. E-mail: [email protected].

cancer, stroke, thrombosis and cardiovascular disease, even though significantly reducing vertebral and nonvertebral fracture risk (Rossouw et al., 2002; Beral et al., 2003). These findings pointed out against the use of HRT as first-line therapy for the prevention and therapy of postmenopausal osteoporosis. As various side effects were reported to be associated with the use of other antifracture agents such as bisphosphonates, selective estrogen receptor (ER) modulators, human parathyroid hormone-derived peptides and calcitonin (Papaioannou et al., 2007), alternative approaches and natural therapies in the management of osteoporosis have become worth exploring. Phytoestrogens are considered to be an effective

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option in preventing bone loss caused by the deficiency of either estrogen and/or androgen. The higher consumption of soy by the Asian population is thought to account for the lower incidence of osteoporosis-related fractures in the region (Glazier et al., 2001) and data from both human and animal studies using soy isoflavones sustain the hypothesis that a diet high in soy exerts beneficial effects on bone health (Arjmandi et al., 1996; Picherit et al., 2001; Chen et al., 2003). Mounting evidence shows positive effects of Chinese herbal drugs as primary or adjuvant treatment in human diseases. To exemplify, in chronic viral hepatitis C, antiviral treatment is associated to partial response and several adverse effects (Duncea and Pepene, 2008); nonetheless, randomized clinical trials reported improved virological response after interferon plus Chinese herbs compared to interferon alone (Zhao et al., 2011). In traditional Chinese medicine (TCM), osteoporosis is considered to be a disorder caused by the insufficiency of kidney yang; thus, herbs such as Fructus Ligustri Lucidi (Nv-Zhen-Zi), Hominis Placenta (Zi-Hec-Che), Herba Epimedii (Yin-Yang-Huo) and Rhizoma Drynariae (GuSui-Bu), thought to invigorate the kidney yang, have been used for more than 1000 years for the therapy of osteoporosis (Ma et al., 2011b; Zhang et al., 2010b). In addition, various Kampo medicines (i.e., Hachimijiogan, Juzentaihoto and Unkeito) were indicated to be useful for preventing postmenopausal osteoporosis (Hidaka et al., 1997; Hattori et al., 2010). In this context, the purpose of this review is to present and assess recent studies on the effects of several Asian plant extracts on bone turnover, bone mineral density (BMD) and bone health and discuss the potential mechanisms involved in their action. THE GENUS EPIMEDIUM Herba Epimedii is one of the most commonly used Chinese herbs in the prevention and treatment of osteoporosis in TCM. Epimedium (Berberidaceae), also known as Rowdy Lamb Herb, Barrenwort, Bishop's Hat, Fairy Wings, Horny Goat Weed, Xianlinpi, Yangheye and ) is a genus of about 52 Yin Yang Huo (Chinese: species of herbaceous plants of which more than 15 species are believed to help “nourishing the kidney and reinforcing the Yang”. The crude extracts and compounds from the aerial parts or roots have various biological functions . According to the literature, the high content of flavonoids and polysaccharides in Epimedium plants, Wang and Liu (2008) especially 8-prenylflavonoids, account for most of its pharmacological activities. In vivo and/or in vitro experiments demonstrated that Epimedium and its active compounds such as icariin, icaritin, desmethylicaritin, des-methylanhydroicaritin, icariside II, ikarisoside A, baohuoside-1, baohuoside II, epimedokoreanin B, breviflavone B, luteolin, hyperoside, epimedin

B or epimedin C benefit from wide-reaching pharmacological actions such as anti-oxidative, anti-tumor and anti-aging effects, and modulators of immunological functions (Ma et al., 2011a). Effects on proliferation and differentiation of boneforming cells The crude extract, total flavonoids and main flavonoid constituents from Herba epimedii have been shown to stimulate the proliferation of primary osteoblasts (Wang et al., 2002; Han et al., 2003; Meng et al., 2005; Zhang et al., 2008a) and osteoblast-like UMR106 cells (Meng et al., 2005; Xie et al., 2005). At concentrations of 1– 10 mg/l, the total flavonoids of Epimedium pubescens significantly augmented the number of osteoblasts and intensified mineralized tuberculation in vitro (Li et al., 2002b). H. epimedii extract has been shown to limit bone loss in an ovariectomized (OVX) rat model (Chen et al., 2011) by increasing cell proliferation and intensifying alkaline phosphatase (ALP) activity in primary rat calvarial osteoblasts (Li et al., 2002b; Han et al., 2003; Zhang et al., 2008a; Chen et al., 2009; Songlin et al., 2009). A recent study also proved that Epimedium wushanense, a common type of H. epimedii, is able to stimulate osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (Zhang et al., 2010a). The up-regulation on bone morphogenetic proteins (BMP) and Wnt-related regulators mRNA expression suggests involvement of both the BMPs- and Wnt-signaling pathways in H. epimedii-stimulated bone formation (Zhang et al., 2010a). Polysaccharides from Epimedium determined significant growth of cell proliferation rates and DNA synthesis in cultured mouse bone-marrow cells (Liu et al., 1991). The serum from old male rats continuously fed with the water extract of Epimedium (1,2 g/ml) for one month increased the proliferation and differentiation of newborn rat calvarial osteoblasts, an effect possibly mediated by the generation of a relatively large number of bone formation mediators such as BMP and leptin (Ma et al., 2002b). Moreover, Epimedium has been proved to enhance gonadal hormones secretion and promote an androgen-like effect on the proliferation and differentiation of osteoblasts. Further studies showed that various doses of icariin, the major flavonoid compound in H. epimedii, increase the proliferation and activity of cultured osteoblasts. Especially at a dose of 10 ng/ml, icariin had a depressive effect on ALP activity in the early stage while enhancing ALP activity in the later stage of osteoblast maturation (Wang et al., 2002). Notably, there seems to be a relationship between osteoporosis and trace-element deficiency and the efficacy of calcium (Ca), manganese (Mn) and zinc (Zn) supplementation on spinal BMD in postmenopausal women. Mineral elements such as

Ilie et al.

manganese , zinc and iron (Fe) appear surprisingly abundant in H. epimedii. In line with these data, the combination of Zn, Ca and Mn with icariin and total flavonoids significantly improves cell viability and intensifies ALP activity compared to each agent alone (Zhang et al., 2008a,b,c). Effects on osteoclastic cells metabolism When an Epimedium solution is parenterally administered, it promotes osteoclast apoptosis and inhibits bone resorption in a dose-dependent manner (Li et al., 2002a). −4 In vitro, total flavonoids (10 mol/l) of H. epimedii reduced the number of osteoclasts and directly inhibited osteoclast resorption activity (Zhang et al., 2004). Further studies have proved that the inhibitive effects of flavonoids on the proliferation of the RAW 264.7 cell line are bidirectional, and depend on their concentration and chemical structures. Ikarisoside A inhibits osteoclastogenesis in nuclear factor-κappaB (NF-κB)-stimulated RAW 264.7 cells as well as in bone marrow-derived macrophages. In fact, expression of osteoclast-specific genes, such as matrix metalloproteinase-9, tartrateresistant acid phosphatase, receptor activator of NF-κB (RANK) and cathepsin K is repressed by ikarisoside A (2.5–20 µM). These findings constitute an argument for possibly using ikarisoside A in the treatment of diseases involving abnormal bone lysis, such as osteoporosis, rheumatoid arthritis and periodontal bone erosion (Choi et al., 2010a). In rabbit bone-marrow cells, icariin at concentrations of 100, 50 and 10 µmol/l significantly inhibited the boneresorbing activity of osteoclasts and greatly reduced the number and surface area of resorption lacuna. Thus, icariin limits bone loss not only by suppressing the boneresorbing activity of mature osteoclasts but also by reducing the formation of osteoclast-like multinucleated cells (Zhang et al., 2007a). In UMR-106 cells, icariin mimicked 17β-estradiol in stimulating cell proliferation, ALP activity and osteoprotegerin (OPG)/RANKL mRNA expression via the ER suggesting that it could exert estrogen-like effects in promoting osteoblastic functions and inhibiting osteoclastogenesis (Xie et al., 2005; Zhang et al, 2007b). Mechanistic studies indicated that the oestrogenic effects of icariin on osteoblastic cells did not depend on estrogen responsive elements in the ER as icariin did not activate estrogen responsive elements-luciferase activity in UMR 106 cells, via the ERα- or the ERβ-mediated pathways and involved activation of the ER by rapid phosphorylation (Mok et al., 2010). Human and animal studies Oral administration of total flavonoid (TF) extracts of Epimedium to rats with osteoporosis significantly increasees

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increases the femur dry weight, femoral ash weight and the calcium and phosphorus contents of bone, augmenting the trabecular bone area and trabeculae thickness of the proximal tibia as well as the cortical bone area percentage in the middle section of the tibia (Ma et al., 2002a). The administration of a lyophilized aqueous extract of Epimedium to castrated male rats for 12 weeks significantly increased serum OPG concentration and BMD, and decreased the microcrack percentage per unit trabecular area. Epimedium prevented the loss of bone mass and improved bone structure in castrated male rats (Wang and Liu, 2008). Intragastric administration of 5 g/(kg day) Epimedium to male rats with Kidney-Yang insufficiency induced by prednisone intensified bone formation and helped rebuild the injured bone by increasing the serum BMP-7 content and up-regulating renal and femoral BMP-7 expression (Zhou et al., 2008). As proved by Zhang et al. (2007b), a preparation containing 60 mg icariin, 15 mg daidzein and 3 mg genistein was able to decrease bone loss in late postmenopausal women in a 24-month randomized, double-blind and placebo-controlled trial. The increase in vertebral and femoral neck BMD in the icariin-treated group was accompanied by suppression of urinary deoxypyridinoline levels, thus, suggesting that the boneprotective action of icariin is mediated by suppression of bone resorption. More recently, Chen et al. (2011) described the dosedependent effects and mechanisms of action of the TF fraction of H. epimedii extract on bone and mineral metabolism in OVX mice, showing that TF suppressed OVX-induced increase in urinary Ca excretion as well as loss of bone mass and strength at the distal femur in mice in a dose-dependent manner. It augmented total BMD and trabecular BMD of the distal femur in OVX mice, with the most effective dosages at 50-100 µg/g. In that study, there was an inverse correlation between the changes in urinary Ca excretion and the expression of renal Ca transport protein (CaBP-28K) and vitamin D receptor mRNA, which suggests for the first time that TF exerts additional effects on CaBP-28K m RNA expression in the kidney that may contribute to bone mass preservation in OVX mice. In addition, TF treatment increased type I collagen and osteocalcin (OC) mRNA expression and the OPG/receptor activator of NF-κB ligand (RANKL) mRNA ratio, and suppressed the growth in interleukin (IL)-6 mRNA induced by OVX in the femur of mice. The results indicated that the increase in the OPG/RANKL ratio by TF was different from that of estradiol and can be accounted for by its inductive effects on OPG mRNA expression, in vivo. In OVX rats, icariin augments the mRNA expression ratio of OPG/RANKL in tibia, following OVX (Mok et al., 2010). These results confirm the ones of a previous animal study (Xie et al., 2005) in which the extracts of H. epimedii increased trabecular BMD in OVX rats and also induced the expression of OPG mRNA and OPG/RANKL ratio, all

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these suggesting that it could modulate the process of osteoclastogenesis. It should be mentioned that icariin lacks uterotrophic effects.

ISOFLAVONES Soy isoflavone preparations, such as purified genistein and a soy extract (Novasoy®), have been proved to exert beneficial effects on bones. Genistein has been extensively studied as one of the main phytoestrogens. Due to its structure and the fact that it resembles 17βestradiol, genistein can compete with estradiol for the ER (Ma et al., 2011b). Various studies using cultured bone cells, OVX rat models and clinical trials supported the fact that genistein might provide an alternative to prevent postmenopausal bone loss (Ullmann et al., 2005; Atmaca et al., 2008). In a randomized, double-blind, placebocontrolled trial that enrolled 389 postmenopausal women with osteopenia, it was shown that genistein enhanced BMD by increasing urinary excretion of pyridinoline and deoxypyridinoline and increasing alkaline phosphatase and serum insulin-like growth factor (IGF)-1 levels (Marini et al., 2007). Apart from the systemic circulation, IGF-1 is abundantly found in both trabecular and cortical bone (Pepene et al., 2004a,b) to directly or indirectly mediate the effects of estrogens, parathyroid hormone, growth hormone and thyroid hormones (Pepene et al., 2001, 2003) and glucocorticoids (Pepene et al., 2010) on bone cells metabolism. To support isoflavones positive effects on bone via the IGF-1/IGF-1R pathway, it was reported that isoflavones extracted from Sophorae fructus, ® (Isocal ) are able to up-regulate the growth factors IGF-1 and transforming growth factor-ß in rat bone marrow cells (Joo et al., 2004). Novasoy® is a commercial isoflavone-enriched product that contains 40% isoflavones and 60% other naturally occurring soy proteins. The effects of genistein and ® Novasoy on tri-dimensional trabecular bone parameters and the expression of bone-specific genes in OVX mice were compared for the first time by Zhang et al. (2009), to conclude that the diet containing soy extract in the form ® of Novasoy was more effective in improving trabecular BMD and micro-architecture of the tibia in comparison to the diet containing purified genistein. Other bone-active soy isoflavones such as daidzein and glycitein are also ® found in Novasoy , and this might account for the different effects of the two isoflavones in the OVX rat (Picherit et al., 2000; Somjen et al., 2008). Additionally, purified genistein decreased RANKL-, carbonic anhydrase II- and cathepsin K-mRNA expression and increased the OPG/RANKL-mRNA ratio in the tibia head ® of OVX mice, whereas Novasoy stimulated OPG mRNA expression but had no effect on the OPG/RANKL-mRNA ratio (Zhang et al., 2009) thus suggesting that the two drugs exert distinct actions on osteoclastogenesis. Nevertheless, further studies are needed to confirm the bone protective effects of genistein and Novasoy in

humans. Comparative studies on the ability of the two wellknown phytoestrogen compounds, genistein and icariin to enhance differentiation and mineralization of cultured rat calvarial osteoblasts concluded on that, compared to genistein, icariin has a stronger effect, as demonstrated by ALP activity, OC secretion, calcium deposition and the number and area of mineralized bone nodules. The same applies for its ability to stimulate the gene expression of type 1 collagen α2, BMP-2, osterix (OSX), and Runx-2 (Ma et al., 2011b); the prenyl group on C-8 of icariin could play the active role in osteoblastic differentiation, and this may explain why icariin is more potent than genistein in stimulating differentiation of osteoblasts. However, they inhibited proliferation of osteoblasts to a similar degree (Ma et al., 2011b). FRUCTUS LIGUSTRI LUCIDI The source of the crude drug, fructus ligustri lucidi (FLL, Chinese name, Nvzhenzi) is the fruit of Ligustrum lucidum. In the OVX rat, FLL increases the calcium absorption rate and improves calcium balance as well as BMD in both sham- and OVX rats, thus, suggesting that the actions of FLL on bone might differ from other phytoestrogen-containing plants (Zhang et al., 2008d). Treatment of UMR-106 cells with FLL extracts increases the formation of calcified matrix and intensifies extracellular calcium and phosphorus depositions in timeand dose-dependent manner. The enhanced mineralization may, at least partially, explain the increase of cortical bone mass in the appendicular skeleton by promoting new bone formation. Recently, Li et al. (2010) showed that the ethanol extract of FLL significantly enhanced ALP activity, reduced the time needed for the mineralization of bone marrow stromal cells and increased the expression of several osteoblast differentiation regulators such as BMP-2, OPG and ßcatenin. Overall, these effects support the hypothesis that FLL improves calcium balance and stimulates osteoblastic differentiation to promote bone formation.

DIPSACUS ASPER WALL Dipsacus asper wall (DAW), belonging to Dipsacaceae, is a kind of perennial herb growing in moist fields and mountains. In a rat study, DAW increased bone density and improved bone histo-morphology (Liu et al., 2009). Several chemical constituents, in particular Dipsacus saponins, have been extracted from the root of DAW. Preliminary experiments showed that three of them, namely, Asperosaponin (ASA) acetyl, ASA IV and ASA VI, could stimulate cell proliferation in MC3T3-E1 osteoblasts, with the ASA VI displaying the most potent effect. In line with these data, Niu et al. (2011) recently demonstrated that the treatment of MC3T3-E1 and primary

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rat osteoblasts with ASA VI not only promoted proliferation and increased ALP activity but also stimulated bone nodules formation. The possible mechanism is that ASA VI induces osteoblast maturation and differentiation, and then increases bone formation via intensified BMP-2 synthesis, and activating p38 and extracellular signal-regulated kinase (ERK) 1/2.

Acanthopanax senticosus

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from the Fructus Cnidii (FC) plant was reported to exhibit estrogen-like effects, preventing osteoporosis in OVX rats (Li et al., 2002c). However, the bone-forming actions of FC on osteoporosis and the biological effects of osthole on bone cells are relatively unknown. Kuo et al. (2005) reported osthole-mediated cell differentiation through the BMP-2, p38 and ERK1/2 pathways in human osteoblast cells. Furthermore, enhanced cell proliferation and differentiation was demonstrated in osthole-treated osteoblasts isolated from neonatal Sprague-Dawley rat calvaria (Zhang et al., 2010b).

Acanthopanax senticosus (AS), also called Siberian ginseng, is a widely used oriental herb that has been reported to exert immunomodulatory, hypoglycemic, antistress, anti-tumor, anti-allergic and anti-oxidant effects. It was reported that AS contains various flavonoids such as quercetin, quercitrin, rutin, and hyperin. Wattel et al. (2004) demonstrated that quercetin may inhibit osteoclastic differentiation, via a mechanism involving NF-κB and activator protein-1 (AP-1). Another group reported that quercetin has a stimulatory effect on osteoblastic activity through ERK- and ER pathway (Prouillet et al., 2004). Furthermore, in a 6-month, prospective randomized study, Hwang et al. (2009) investigated the effects of AS extract on biochemical markers of bone turnover and BMD in 81 Korean postmenopausal women with low bone mass aged less than 65. No significant changes in BMD were observed; nevertheless, the study showed that a 6-month treatment with AS extract may have a favorable effect on bone remodeling in women with reduced BMD, increasing serum OC by 23.3% and decreasing serum C-telopeptide by 8.2%.

Several studies have shown that statins, which are drugs with lipid-lowering effects, also have potentially beneficial effects on bone metabolism. Saiko-ka-ryukotsu-borei-to (SRB) is a traditional Japanese herbal medicine used to treat hyperlipidemia and inhibit aortic intimae thickening in hypertensive patients. Hattori’s group investigated the effect of SRB on bone metabolism using an OVX murine model and showed that the bone volume of the proximal tibia of SRB-treated mice is significantly greater than that of mice in the OVX group, as assessed by micro-CT, and that the protective effects of SRB on bone in OVX mice are explained by the suppression of bone resorption. The study also shows that levels of serum IL-6, a primary mediator of bone resorption, were significantly lower in the SRB group compared to the OVX group, thus, suggesting that SRB may suppress osteoclastogenesis by decreasing serum IL-6 level (Hattori et al., 2010).

Paeonia lactiflora Pallas

Achyranthes bidentata

Numerous studies have indicated that inflammatory cytokines play a major role in osteoclastogenesis, promoting bone resorption. Paeonol (2′-hydroxy-4′methoxyacetophenone) is the main active compound of the Paeonia lactiflora Pallas, a traditional Chinese herb. Paeonol has an anti-inflammatory effect, suppressing cyclooxygenase-2, nitric oxide synthase, cell surface adhesion molecules, IL-1β and tumor necrosis factor (TNF)-α genes expression and inhibiting the activity of ERK and p38 (Nizamutdinova et al, 2007). In the study by Tsai et al. (2008), paeonol was shown to inhibit not only osteoclastogenesis from bone marrow stromal cells and macrophages via attenuated RANKL-induced ERK, p38 and NF-κB activation but also the resorption activity of mature osteoclasts. Additionally, paeonol prevented bone loss induced by ovariectomy in vivo.

FRUCTUS CNIDII Osthole, one of the main components of the dried seeds

SAIKO-KA-RYUKOTSU-BOREI-TO

Using the bone organ culture system, He et al. (2010) demonstrated that Achyranthes bidentata (AB), another Chinese herbal drug shows potent inhibitory activity on PTH-induced bone resorption. Further research using the OVX rat model revealed that AB significantly prevented BMD loss without any estrogen-like side effects. The main active chemical constituents are oleanolic acid glycosides, ecdysone type compounds and allantoin. Oleanolic acid glycosides inhibit osteoclasts formation (Li et al., 2005) and ecdysone type compounds stimulate proliferation of osteoblast-like UMR106 cells (Li et al., 2001). Dioscorea spongiosa Several reports demonstrated that glycosides, diarylheptanoids and lignans contained by the 90% ethanol fraction of water extract of the rhizomes of Dioscorea spongiosa (DS) not only significantly stimulate osteoblastic proliferation but also powerfully inhibit osteoclastic formation and bone resorbtion (Yin et

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al., 2004b). Twenty-two glycosides were isolated from the 90% ethanol fraction of water extract of DS. While glycosides appear to stimulate osteoblastic proliferation and mineralization, diarylheptanoids and lignans are powerful inhibitors of osteoclastic formation and bone resorption, reporting a simultaneous weaker stimulatory activity of osteoblastic proliferation and mineralization (Yin et al., 2004a, b, 2010).

Carthamus tinctorius L The Honghwain (HHI), Carthamus tinctorius L. seed extract, specific of the Korean herbal medicine and Herbimycin A are a novel class of Src-tyrosine kinase inhibitors that decrease cyclooxygenase-mRNA levels as well as prostaglandin E2 production. Tyrosine kinase(s) seem to play a role in the signal transduction of cyclooxygenase -2 in mouse calvarial osteoblasts. HHI decreases dose-dependently the hypercalcemia induced in mice by IL-1β and partly acts against bone loss and micro-architectural changes in young OVX rats (Yuk et al., 2002). Another Korean herbal formulation, comprised of an herb of C. tinctorius L. seed and Hominis Placenta, which seems to inhibit IL-1ß-induced bone resorption is Honghwain-Jahage (Hong et al., 2002).

Cuscuta chinensis To-Sa-Za (TSZ) is the dry seed of Cuscuta chinensis Lam. It is suggested that TSZ has osteogenic effects. Sustaining these, it was demonstrated that the aqueous extract of TSZ mildly promoted the proliferation of human osteoblast-like MG-63 cells and increased ALP, collagen and BMP-2 mRNA expression and mineralization of MG63 cells (Yang et al., 2009). The clinical use of TSZ in the treatment of osteoporosis is further supported by a recent study, which demonstrates that kaempferol and hyperoside are the active compounds with an osteogenic effect in TSZ (Yang et al., 2011).

CONCLUSION To conclude, the use of alternative therapies sourced from some medicinal plants and natural products in preventing and treating postmenopausal osteoporosis while avoiding significant risks associated with hormone replacements and other anti-fracture therapies, might be achieved. Overall, experimental studies are promising, showing the systematic, beneficial activities of Epimedium’s metabolites and other Asian plants on bone metabolism. However, several questions need to be addressed; the optimal dosage and the mechanism of actions by which compounds from different plants have bone-protective effects are to be cleared, and the active constituents of these herbs as well as their pharmacological

and toxicity profiles should be further investigated. Moreover, the pharmacological studies so far have mostly been performed with animals. Therefore, there is an utmost urge for prospective, randomized, clinical studies in humans to confirm this traditional phytotherapy.

Abbreviations: AB, Achyranthes bidentata; ALP, alkaline phosphatase; AP-1, activator protein-1; AS, Acanthopanax senticosus; ASA, asperosaponin; BMD, bone mineral density; BMP, bone morphogenetic proteins; DAW, dipsacus asper Wall; DS, dioscorea spongiosa; ER, estrogen receptor; ERK, extracellular signal-regulated kinase; FC, fructus cnidii; FLL, fructus ligustri lucidi; HRT, hormone replacement therapy; IGF-1, insulin-like growth factor-1; IL, interleukin; NF-κB, nuclear factor-κappaB; OC, osteocalcin; OPG, osteoprotegerin; OSX, Osterix; OVX, ovariectomized; RANKL, receptor activator of NF-κB ligand; SRB, saikokaryukotsuboreito; TCM, traditional Chinese medicine; TF, total flavonoid; TNF, tumor necrosis factor; HHI, Honghwain; TSZ, To-Sa-Za.

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Journal of Medicinal Plants Research Vol. 6(18), pp. 3407-3414, 16 May, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1293 ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

Callus induction and plant regeneration from leaf explants of Falcaria vulgaris an important medicinal plant Jaberian Hamideh1*, Piri Khosro2 and Nazari Deljou Mohammad Javad3 1

Department of Biotechnology, Faculty of Agriculture, Payame Noor University, Tehran Branch, Tehran, Iran. 2 Department of Biotechnology, Bu-Ali Sina University, Hamadan, Iran. 3 Department of Horticulture, Mahabad Branch, Islamic Azad University, Mahabad, Iran. Accepted 18 January, 2012

Falcaria vulgaris is an important medicinal plant belonging to the family Apiaceae. The leaf of explants of this plant was cultured for callus induction and plant regeneration. The explants of this plant were cultured onto Murashig and Skoog (MS) medium supplemented with different concentrations of (alpha)naphtalene acetic acid (NAA), 2,4-dichlorophenoxy acetic acid (2,4-D), thidiazuron (TDZ), alone and in combination with 6- benzyladenine (BA) for callus induction. The highest callus was induced in medium -1 containing (0.5 and 1.0 mg L ) 2,4-D in combination with BA. These callus and leaf segments were transferred to MS medium supplemented with different combination of NAA and BA for indirect and -1 direct regeneration, respectively. The medium containing (1.0 mg L ) NAA in combination with (0.5 and -1 1.0 mg L ) BA showed the highest number of shoot and root formation in plant regeneration through -1 the callus. In direct regeneration, NAA with 1.0 mg L concentration was observed to be more potent -1 than with concentration of 0.5 mg L and showed highest root regeneration frequency (15.7%). In vitro raised plantlets were acclimatized onto natural condition with 90% survival. These results provide a basis for future studies on genetic improvement and could be applied to production of secondary metabolites through cell culture in Falcaria vulgaris. Key words: Falcaria vulgaris, plant growth regulators, callus induction, in vitro propagation, direct regeneration.

INTRODUCTION Plants are a valuable source of a wide range of secondary metabolites, which are used as pharmaceuticals, agrochemicals, flavors, fragrances, colours, biopesticides and food additives. Over 80% of the approximately 30,000 known natural product are of plant origin (Fowler and Scragy, 1988; Balandrin and Klocke, 1988; Phillipson, 1990). The advent of chemical analyses and the characterization of molecular structures have helped in precisely identifying these plants and correlating them with their activity under controlled experimentation (Ramachandra and Ravishankar, 2002). Biotechnological tools are important for multiplication and genetic enhancement of the medicinal plants by adopting

*Corresponding author. E-mail: yahoo.com. Tel: +989183146178.

hamideh_jaberian@

techniques such as in-vitro regeneration and genetic transformations (Tripathi and Tripathi, 2003). Plant cell culture has been successfully applied to produce large quantities of secondary metabolites from many plants (Taniguchi et al., 2002). Propagation of plants holds tremendous potential for the production of high-quality plant-based medicines (Murch et al., 2000). Induction of callus growth and subsequent differentiation and organogenesis is accomplished by the differential application of growth regulators and the control of conditions in the culture medium (Tripathi and Tripathi, 2003). Falcaria vulgaris (locally named ghazzyaghi/poghazeh), a member of Apiaceae family, is consumed as a vegetable in some regions of Iran including Hamedan Province, for healing of skin ulcer, stomach disorders including peptic ulcer, liver diseases and stones of kidney and bladder. Naturally, this plant exists for a short time, therefore, in

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Table 1. Effect of different growth regulators combinations on callus induction from leaves of Falcaria vulgaris. -1

BA 0 0 0 0 0 0 0 0 0 0 0 0 0 0.5 1 2 4 0.5 1 2 4 0.5 1 2 4

Growth regulators (mg L ) 2,4-D NAA 0 0 0.5 0 1 0 2 0 4 0 0 0.5 0 1 0 2 0 4 0 0 0 0 0 0 0 0 0.5 0 1 0 2 0 4 0 0 0.5 0 1 0 2 0 4 0 0 0 0 0 0 0 0

TDZ 0 0 0 0 0 0 0 0 0 0.5 1 2 4 0 0 0 0 0 0 0 0 0.5 1 2 4

Explant forming callus (%) 0 8.5 1 0 8.5 8.5 0 0 0 0 0 0 0 92.5 83.5 5.7 62.5 20 27 0 17 45 28 0 20

vitro tissue culture technique helps this plant to be more available in all seasons, and improve the leaf explants of wild F. vulgaris, produce cultivars with higher yield, tolerance and resistance to diseases. Callus production is also a necessary step for obtaining protoplasts used in protoplast fusion, a useful tool in genetic improvement of vegetatively propagated plants (Yamashita et al., 2002; Liu et al., 2005). Although F. vulgaris is a very valuable medicinal plant but to our knowledge, no method has been reported about in vitro study such as callus induction and in vitro propagation of this plant. Therefore, we conducted this study to assay the effects of plant growth regulators on callus induction, shoot regeneration from callus and direct regeneration from leaf explant in F. vulgaris.

Callus induction

MATERIALS AND METHODS

Tissue culture and plant propagation

Plant materials

Callus produced from leaf were used for indirect regeneration. After 4 weeks, for shoot regeneration of callus, the callus that have been best grown were dissected into small pieces and transferred to regeneration medium containing of half strength MS medium supplemented with different concentrations (0.5 and 1 mg L-1) of

The leaves of F. vulgaris were collected from Hamedan Province in the Western part of Iran and identified by the Botanic Laboratory of Bu-Ali Sina University in May, 2010.

The leaves of F. vulgaris were washed thoroughly with tap water, then the explants were surface sterilized in 70% (v/v) ethanol for 1 min, subsequently in 2% (w/v) sodium hypochlorite with tween-20 for 10 min. Finally, the explants were washed with sterile deionized water for three times. For callus induction, the sterilized leaves segments of 5 × 5 mm length were placed onto the surface of Murashig and Skoog (MS) medium (Murashig and Skoog, 1962) supplemented with different concentrations of (alpha)-naphtalene acetic acid (NAA), 2,4-dichlorophenoxy acetic acid (2,4-D), thidiazuron (TDZ), alone and in combination with 6-benzyladenine (BA) (Table 1). The pH was adjusted to 5.8 and autoclaved (121°C, 15 min). All media contained 0.8% agar, 3% sucrose and incubated at 25 ± 2°C, under two conditions of photoperiod (darkness and 16/8 h day/night). Then, the suitable callus was separated and transferred to fresh medium with the same composition. Callus induction rate was calculated after 4 weeks.

Hamideh et al.

NAA in combination with different concentrations (0.5, 1, 2, 5, 7 and 10 mg L-1) of BA (Table 2). Regenerated shoots were transferred to rooting medium, which consisted of half strength MS medium supplemented again with different concentration of NAA in combination with BA. Explants were maintained at 25 ± 2°C under a photoperiod of 16 h of cool-white florescent light and subcultured every two weeks.

Direct regeneration Leaves of F. vulgaris were used for direct regeneration without callus formation. Explants were cultured on half-strange MS medium supplemented with the various concentrations and combinations of NAA with BA for shoot and root direct regeneration (Table 2). Regeneration rate was calculated after 2 weeks and explants were maintained at 25 ± 2°C under a photoperiod of 16 h of cool-white florescent light. Shoot regeneration was estimated by percentage of callus forming shoots and number of shoots formed by leaf after 8 weeks of culture.

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induction. TDZ can be substituted with a demine type cytokines in various culture systems, including callus and micro propagation of many plants (Lu, 1993). In the present study, callus induction from leaf explant in medium containing of 2,4-D in combination with BA were better than medium containing TDZ and BA. These results showed that equal concentrations of auxin and -1 cytokinen (especially 0.5 and 1.0 mg L ), induced large and fragile callus which had better potential for regeneration. Similar result was reported by Pal and Dhar (1985) and Hitmi et al. (1998). Callus cultures are extremely important in plant biotechnology. Manipulation of the auxin to cytokinin ratio in the medium can lead to the development of shoots, roots, or somatic embryos from which whole plants can subsequently be produced. Callus cultures can also be used to initiate cell suspensions, which are used in a variety of ways in plant transformation studies.

Acclimatization After 8 weeks, plantlets with well-developed roots washed in distilled water to remove agar from their roots were transferred to plastic cups containing sand, fertile soil and vermiculite (1:1:1) and growth under greenhouse conditions with 90% humidity. After 2 month, the plants placed in shade under natural conditions.

Statistical analysis For all experiments there were three repeats and in each repeats five explants. Data were analyzed statistically using SAS 9.1 software. The mean values of different treatments were compared using Duncan’s multiple tests (At p< 0.05).

RESULTS AND DISCUSSION Callus induction The callus was produced from the explants after 4 weeks of culture. The callus induced in the dark condition produce less chlorophyll and these had most growth compared with light condition. The light did not aid callus growth. Thus, darkness was beneficial for callus induction in comparison of light condition. The highest callus induction was achieved when MS medium supplement -1 with 2,4-D in combination with BA (0.5 and 1.0 mg L ) was used (Figure 4A and B). Also the growth index increased when callus were exposed to MS medium -1 supplemented with 1.0 mg L TDZ in combination with -1 0.5 mg L BA (Figure 4C and D). Different hormonal treatments were studied for callus induction of F. vulgaris as shown in Table 1 and Figure 1, of which most of them led to callus induction. The results of the present study showed that, auxin alone and in combination with cytokinin can produce callus but 2,4-D was the most effective for callus induction. These results is in line with other research, same as many other plants, because 2,4D is the primary auxin which is used for the callus

Plant propagation through callus For plant regeneration from callus, callus was transferred to the development media; MS medium supplemented with different concentration of NAA and BA under light conditions (Table 2). After 2 weeks of culture, most callus started to turn to light green, then they became dark green (Figure 5A and B). The medium containing NAA -1 -1 (1.0 mg L ) and BA (0.5 and 1.0 mg L ) showed the highest number of shoot and root formation in this plant (Figure 7A to F). Among the various concentrations of BA -1 -1 (0.5 to 10 mg L ) tested, 0.5 and 1.0 mg L BA showed the highest shoot regeneration from callus (Figures 2, 3 and 7C and D). This rate decreased on media supplemented with higher or lower NAA concentrations. Root formation increased with higher NAA concentration. There are few reports about plant regeneration by callus in Apiaceae family (Tiwari et al., 2000; Makunga et al., 2005). Transition of callus to regeneration pathway was affected by interaction effect between BA and NAA. This interaction plays a key role in regeneration through indirect organogenesis pathway. Our finding is in agreement with Tiwari et al. (2000) reported micro propagation in Centella asiatica in the combination of NAA and BA for leaf and nodel explants. Data did not match those of Ayan et al. (2005) which reported that shoots of H. perforatum were rooted very intensively on -1 MS medium supplemented with 1 mg L of IAA. Direct regeneration Maximum rate of direct regeneration without callus formation was obtained with MS Medium supplemented with different concentrations of NAA and BA. In direct -1 regeneration, NAA with 1.0 mg L concentration was observed to be more potent than concentration of 0.5 mg

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Table 2. Effects of different growth regulator combinations on regeneration from callus and leaf of F. vulgaris. -1

Growth regulators (mg L )

Callus explants

BA

NAA

Explants forming shoot (%)

0 0.5 1 2 5 7 10 0.5 1 2 5 7 10

0 0.5 0.5 0.5 0.5 0.5 0.5 1 1 1 1 1 1

0 b 92.3 a 100 c 12.1 d 5.9 e 0 e 0 b 94.3 a 100 c 16.1 d 6.3 e 0 e 0

Leaf explants

Explant forming root (%)

e

Explants forming shoot (%)

Explant forming root (%)

0 0 0 0 0 0 0 b 31.8 c 0 a 56 c 0 c 0 c 0

0 0 0 0 0 0 0 b 7.33 c 0 a 15.9 c 0 c 0 c 0

c

0 b 5.7 a 13.1 c 0 c 0 c 0 c 0 b 48 a 88.3 c 5.7 d 2.88 e 0 e 0

Callus induction (%)

100

80 60

NAA+BA

40

2.4-D+BA

20

TDZ+BA

0 0+0

0.5+0.5

1+1

2+2

4+4

Hormones treatment (mg L-1)

Figure 1. Effect of different growth regulator combinations on callus induction from leaves of F. vulgaris.

Shoot and root regeneration (%)

120

100

a

b

80 60

indirect Indirectshoot shoot Indirectroot root indirect

40 20

a

c d

b 0

0

0.5+0.5

0.5+1

0.5+2

Hormone treatments (mg

0.5+5

0.5+7

0+10

L-1)

Figure 2. Effects of 0.5 mg L-1 NAA in combination with BA on regeneration from callus and leaf of Falcaria vulgaris.

Hamideh et al.

Figure 3. Effects of 1 mg L-1 NAA in combination with BA on regeneration from callus and leaf of F. vulgaris

A

B

C

D

Figure 4. (A and B) Large and white callus induced from leaf under the culture on MS basal medium containing TDZ and BA. (C and D) Yellowish callus induced from leaf under the culture on MS basal medium containing TDZ and BA.

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A

B

Figure 5. (A and B) Callus started to turn to light green after 2 week of callus culture on regeneration medium.

A

B

Figure 6. (A and B) Direct regeneration without callus formation from Falcaria vulgaris leaves and plantlets development of F. vulgaris.

-1

L NAA showed the highest root regeneration frequency (15.7%) and number of root (Figure 6A and B). Based on the results, application of NAA causing was producing of root and shoot in F. vulgaris. BA was the most important cytokinin used for regeneration of this plant. Because this hormone is effective in increasing of endogenous cytokinin plants. NAA was found to be the best for root induction. The findings are in agreement with those observed in other plant species such as Caphaelis ipecacuanha (Jha and Jha, 1989), Plantago ovata (Wakhlu and Barna, 1989). Effectiveness of BA + NAA for in vitro shoot regeneration and multiplication from shoot tip and leave cultures was reported in several other plants (Conver and Lits, 1987; Tokuhara and Mii, 1993). Our findings are compatible with those of Pretto and Santarem (2000), who reported that in Hypericum

perforatum, BA was found to be the most efficient in promoting shoot regeneration when leaves were used as the explants.

Acclimatization When the propagated plantlets were transferred to small plastic cups containing sand, fertile soil and vermiculite (1:1:1), and the humidity was maintained at approximately 90% by covering with plastic. After 2 month the plants were transferred to large pots and after acclimatization, the 100-day-old plants were transferred to field. Over 90% of the plantlets were successfully acclimatized and were similar to the mother plants.

Hamideh et al.

A

B

C

D

E

F

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Figure 7. (A and B) Shoot and root regeneration from callus after six weeks of culture. (C and D) Plantlets rooting and shoot regeneration of Falcaria vulgaris in half strength MS + NAA and (0.5 and 1) BA after two weeks of subculture. (E and F) Plantlets development of F. vulgaris.

Conclusion In this study, we reported for the first time a protocol for the successful callus induction, direct regeneration from leaf explants and regeneration through callus in F. vulgaris which would provide more homogenous source of medicine. The frequency of callus induction and regeneration from leaf explants described here was high enough to encourage us to carry out, protoplast culture genetic transformation and cell suspension culture for

improvement of oil and secondary metabolites quality and quantity.

REFERENCES Ayan AK, Çirak C, Kevseroglu K, Sokmen A (2005). Effects of explants types and different concentrations of sucrose and phytohormones on plant regeneration and hypericin content in Hypericum perforatum L. Turk. J. Agric., 29: 197–204. Balandrin MJ, Klocke JA (1988). Medicinal aromatic and industrial

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materials from plants. In: Bajajy PJ, editor. Biotechnol. Agriculture Forest. Med. Aromat. Plant, Berlin, 11: 1-36. Conver RA, Litz RW (1987). Progress in breeding papayas with tolerance to papaya ring spot virus. Proc. Fla. State Hort. Soc., 91: 182-184. Hitmi A, Barthomeuf C, Sallanon H (1998). Rapid mass propagation of Chrysanthemum cinerariaefolium Vis. by callus culture and ability to synthesise pyrethrins. Plant Cell Rep., 19: 156-160. Jha S, Jha TB (1989). Micropropagation of Caphaelis ipecacuanha. Plant Cell Rep., 8: 437-439. Liu J, Xu X, Deng X (2005). Intergeneric somatic hybridization and its application to crop genetic improvement. Plant Cell Tissue Organ Cult., 82: 19-44. Lu CY (1993). The use of thidiazuron in tissue culture. in vitro Cell Dev. Biol., 29: 92-96. Makunga NP, jager AK, Staden JV (2005). An improved system for the in vitro regeneration of Tapsia garaganica via direct organogenesisinfluence of auxin and cytokinins. Plant Cell Tissue, Organ Cult., 82: 271-280. Murashig T, Skoog F (1962). A revised medium for rapid growth and bioassay with tobacco tissue Cult. Physiol. Plants, 15: 473-497. Murch SJ, Krishna RS, Saxena PK (2000). Tryptophan is a precursor for melatonin and serotonin biosynthesis in in-vitro regenerated St. John's wort (Hypericum perforatum L. cv. Anthos) plants. Plant Cell Rep., 19: 698-704. Pal A, Dhar K (1985). Callus and organ development of pyrethrum, (Chrysanthemum cinerariaefolium Vis.) and analysis of their cytological status. Pyrethrum Post, 16: 3-11.

Phillipson JD (1990). Plants as source of valuable products, In: Charlwood BV, Rhodes MJC, editors. Secondary products from plant tissue culture. Oxford: Clarendon Press, pp. 1-21. Pretto FR, Santarem ER (2000). Callus formation and plant regeneration from Hypericum perforatum L. leaves. Plant Cell Tissue Organ Cult., 67: 107–113. Ramachandra RS, Ravishankar GA (2002). Plant cell cultures: chemical factories of secondary metabolites. Biotechnol. Adv., 20: 101-153. Taniguchi S, Imayoshi Y, Kobayashi E, Takamatsu Y, Ito H, Hatano T, Sakagaami H, Tokuda H, Nishino H, Sugita D, Yoshida T (2002). Production of bioactive triterpenes by Eriobotrya japonica calli. Phytochemistry, 59: 315-323. Tiwari KN, Sharma NC, Tiwari V, Singh BD (2000). Micropropagation of Centella asiatica (L), a valuable medicinal herb. Plant Cell Tissue Organ Cult., 63: 179-185. Tokuhara K, Mii M (1993). Micropropagation of Phalaenopsis sp. and Doritaenopsis sp. by cutting shoot tip of flower stalk bud. Plant Cell. Rep., 13: 7-11. Tripathi L, Tripathi JN (2003). Role of biotechnology in medicinal plants, Trop. J. Pharm. Res., 2(2): 243-253 Wakhlu AK, Barna KS (1989). Callus initiation, growth and plant regeneration in Plantago ovata Forsk. cv. Gl-2. Plant Cell, Tissue Cell Organ Cult., 17: 235-241. Yamashita k, Hisatsune Y, Sakamoto T, Ishizuka K, Tashiro Y (2002). Chromosome and cytoplasm analyses of somatic hybrids between onion (Allium cepa L.) and garlic (A.sativym L.). Euphytica, 125: 163167.

Journal of Medicinal Plants Research Vol. 6(18), pp. 3415-3424, 16 May, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1707 ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

Ethnobotanical survey of medicinal plants used in the treatment of gastrointestinal disorders in the Eastern Cape Province, South Africa O. O. Olajuyigbe and A. J. Afolayan* Phytomedicine Research Centre, Department of Botany, University of Fort Hare, Alice, 5700, South Africa. Accepted 11 April, 2012

An ethnobotanical survey of plants used for the treatment of gastrointestinal disorders was carried out in the Eastern Cape Province, South Africa. Information on the names of plants, used parts and methods of preparation was obtained from traditional medical practitioners, herbalist, hawkers in traditional medicines and rural dwellers, using semi-structured questionnaire. 36 plant species representing 24 families were found to be commonly used in the treatment of a variety of gastrointestinal disorders in this study. The family Fabaceae had the highest number of species being used for treating gastrointestinal disorders. 47.06% of the plants used in treating dysentery and other gastrointestinal disorders were used in the treatment of dysentery alone while 46.15% of the plants used to treat diarrhoea and other gastrointestinal disorders, were used in the treatment of diarrhoea alone. 30.3% of the different plants were implicated in the treatment of various stomach problems. Rationales for the choice of these plants were also identified. The leaves were the most commonly used parts, followed by roots and bark while decoctions and infusions are the most frequent methods of preparation. The traditional healers in this Province possess rich ethno-pharmacological knowledge and depend largely on naturally growing plant species. The documented medicinal plants can serve as a basis for further and future phytochemical and pharmacological studies. Key words: Medicinal plants, gastrointestinal disorders, dysentery, indigenous knowledge, over-exploitation. INTRODUCTION Traditionally, plants are reliable sources for the treatment of diseases in different parts of the world (Eisenberg et al., 1993; Cowan, 1999; Hostettmann et al., 2000). Their use contributes significantly to primary health care delivery (Holetz et al., 2002) as they are regarded as invaluable sources of pharmaceutical products (Olalde, 2005). Globally, medicinal plants have been unique sources of medicines and constituted the most common human use of biodiversity (Hamilton, 2004; Hiremath and Taranath, 2010). In African societies, the tradition of collecting, processing and applying plants and plantbased medications have been handed down from generation to generation. Traditional medicine, with medicinal plants as their most important component, are sold in market places or prescribed by traditional healers

*Corresponding author: Fax: +27866282295 ; Email: [email protected]

in their homes (Von Maydell, 1996). As a result of this strong dependence on plants as medicines, many ethnopharmacological studies have been conducted to determine their safety, efficiency and discovery of new active principles from them. Ethnopharmacology, the science of application of indigenous or local medicinal remedies, including plants for treatment of diseases (Gurib-Fakim, 2006; Pande et al., 2008), is the investigation of biologically active agents traditionally used by humans (Bruhn and Holmested, 1981). These active agents included plant mixtures, whole plants and a portion of a plant as well as special preparations from plant materials. Being a multidisciplinary science, successful research in ethnopharmacology requires the interaction of ethnobotanists, natural products chemists, pharmacologists, taxonomists, traditional healers and/or user communities. According to Vanden Berghe et al. (1986), Rojas et al. (1992) and Silva et al. (1996), the goal of ethnobotany or

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ethnopharmacology, therefore, is to utilize the impressive array of knowledge assembled by indigenous peoples about the plant and animal products they have used to maintain health. Although the use of medicinal plants for subsistence, home remedies, trade and alleviating human suffering (Kunwar et al., 2006) plays important roles in the lives of rural people (Ahmad, 2003), the non-sustainable collection methods have caused threat from harvesting and many valuable medicinal herbs are becoming rare due to their continuous utilization (Swe and Win, 2005) and over-exploitation for commercial purposes (Tabuti et al., 2003; Kamatenesi-Mugisha and Oryem-Origa, 2005). In addition to these endangering effects of human-plant relationships, traditional folk knowledge and folklore information from many different cultures, which are the sum of attitudes, opinions, beliefs and customs handed down from generation to generation in a given society, (Anonymous, 1993) which are important tools in revealing plants with useful medicinal properties (Balandrin et al., 1993), are neither often found in written form, nor organized and structured in ways accessible to science. As a result, this knowledge changes because of indigenous creativity, innovativeness and contact with other knowledge systems. Along with the traditional lifestyles, traditional usage and folk knowledge of plants are disappearing due to copying of westernized lifestyles and economic systems. Considering a sharp decrease in the biological species all across the globe and the increasing economic values placed on medicinal plants, documentation on ethnobotanical knowledge is a way to understand the use of different plant species to cure various ailments and means to conserve these natural resources. Globally, there is currently a renaissance of ethnobotanical surveys of medicinal plants and the need to screen specific parts of the plants (Paterson and Anderson, 2005; Igoli et al., 2005; Li and Vederas, 2009). Regardless of many ethnobotanical studies on medicinal plant resources in South Africa (Cunningham, 1988; Hutchings, 1989; Hutchings et al., 1996; Mander, 1998; Van Wyk et al., 1997; Van Wyk and Gericke, 2000; Appidi et al., 2008, Olorunnisola et al., 2011) and the world over, a large number of medicinal plants and associated indigenous uses still wait proper documentation (Tabuti et al., 2003). Although Appidi et al. (2008) had earlier reported some plants used in the treatment of diarrhoea, there is a lack of information on plants used in the treatment of gastrointestinal disorder such as dysentery while many plants relevant in treating diarrhoea and other gastrointestinal disorders are inexhaustible. This may be due to the thin line of distinction differentiating these infections especially diarrhea and dysentery. Hence, previous attentions may have been directed towards plants used in treating diarrhea while possibly and unknowingly addressing dysentery simultaneously. This ethnobotanical survey is, however, aimed at identifying

plants and part(s) that are used in the treatment of some gastrointestinal disorders as well as indicating their methods of preparation and rationale for their tradotherapeutic effects in the Eastern Cape Province, South Africa. MATERIALS AND METHODS The study area falls within the latitude 30o 00‟ to 34o 15‟S and longitudes 22o45‟ to 30o15‟E. It is bounded by the sea on the East and the drier Karroo (Semi-desert vegetation) in the West. The elevation ranges from sea-level to approximately 2200 m in the North and the vegetation is veld type, known as the Eastern Cape thorn veld (Acocks, 1975; Masika and Afolayan, 2003). This area consists of many villages which are generally classified as rural and poor with difficulty in distinguishing between gastrointestinal disorders such as diarrhoea and dysentery infections. Field visits for this study were carried out in May and July, 2011. Information was obtained from rural dwellers, traditional healers, hawkers of medicinal plant preparations and herbalists with the help of a semi-structured questionnaire and the guided field-walk method as described by Martin (1995) and Maundu (1995). The questionnaire was used to interview these individuals while the guided field-walk involved contacting and interviewing individuals recommended by other community members for their knowledge. The information collected included local names, the parts of the plant used and methods of preparation. The information was further validated by common response. The information from at least three or more respondents was considered as common response. Proper scientific identification of the plants and their uses in these communities were reaffirmed from the literature sources containing medicinal plants used in South Africa (Roberts, 1990; Hutchings et al., 1996; Van Wyk et al., 1997; Mander, 1998; Van Wyk and Gericke, 2000).

Intellectual property agreement statement Prior to the interviews, the informants were duly informed about the objectives of the research. With verbal agreement that this research shall not be used for commercial purposes but to enlighten and document medicinal plants used for the treatment of gastrointestinal disorder, the interview was granted.

RESULTS A total of 36 plant species distributed in 24 families were found to be used locally for treating various gastrointestinal disorders including diarrhoea, dysentery, abdominal cramps, gut disturbances, stomach disorders, upset and aches. The families are arranged in alphabetical order. Family names are followed by vernacular names or local names and plant part(s) used and their methods of preparations. The results are summarized in Table 1. The Fabaceae was represented by five plants, followed by Apiaceae, Asphodelaceae, Lamiaceae and Solanaceae (3 species each), Hyacinthaceae (2 species each) while other families (1 species each) were found to be used medicinally by the local communities. Of these plants, 19 (52.78%) different plants were indicated to be used for treating dysentery

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Table 1. Ethnomedicinal plants used in Eastern Cape, South Africa, for the treatment of gastrointestinal disorders.

Plant

Family

Local name

Used part

Uses

Acacia mearnsii

Fabaceae

Idywabasi

Bark and leaves

Dysentery

Acacia karroo Hayne

Fabaceae

Umngampunzi, umnga

Leaves, bark and gum

Dysentery, diarrhoea haemorrhage

Alepidea amatymbica Eckl. and Zeyh.

Apiaceae

Iqwili

Root/rhizome

Abdominal cramps

Decoction of the roots

Bulbine abyssinica A.Rich., B.

Asphodelaceae

Iyeza lipulayiti, utswelana, Uyakayakana

Leaves and roots

Dysentery

Decoctions of the leaves and roots

Brachylaena ilicifolia (Lam.) E. Phillips and Scheweick.,

Asteraceae

Umgqeba

Leaves

Diarrhoea

Infusion and decoction of the leaves

Bulbine latifolia (L.f.) Roem. et Schult.

Asphodelaceae

Ibhucu, ingcelwane

Root

Diarrhoea

Decoctions of the root

Bulbine asphodeloides Roem. et Schult.

Asphodelaceae

Umthi Uyakayakane

Tuber

Diarrhea, dysentery

Decoctions of the root/tuber

Cussonia spicata Thunb.,

Araliaceae

intsenge, umsenge,

umgezisa,

Leaves

Stomach complaints

Infusion

Curtisia dentata C.A.Sm., Olea

Cornaceae

umLahleni, Uzintlwa,

umGxina,

Bark

Stomach ailments

Decoctions of the bark

Apiaceae

Inyongwane, iphuzi

Roots and leaves

Stomach disorders, dysentery and diarrhoea

Infusion, decoction and concoction of the leaves and roots

Rutaceae

Iperepes

Root, bark, fresh leaves

Stomach complaints

A decoction of the aromatic leaves or roots

Menispermaceae

Umayisake

Roots, leaves

(Burm.f.)

Centella asiatica (Linn.) Clausena anisata Hook.f. ex Benth.

(Willd.)

Cissampelos capensis L.f.,

intlaka,

kanoyayi,

the

Stomach diarrhoea

Preparation Infusions and concoctions of the bark; decoction of the bark

upset

and

and

Infusions and concoctions leaves, bark and gum

Infusion

of

the

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Table 1. Contd

Eucomis autumnalis (Mill.)

Hyacinthaceae

Isithithibala Esimathunzi,

Bulbs

Stomach ache and colic

Decoctions of the bulb

Ekebergia Sparrm.

Meliaceae

uManaye, wezinja

Bark

Dysentery

A decoction of the back

Foeniculum vulgare Mill., Hydnora Africana (Thunb.) Ipomoea crassipes Hook., Iconotis leonurus (L.) R. Br.

Apiaceae Hydronaceae Convolvulaceae Lamiaceae

Imbambosi Umavumbuka Ubhoqo Imvovo

Leaves and stem Whole plant Whole plant Whole plant

Stomach cramps Diarrhoea, dysentery Dysentery Dysentery

Infusion Infusions and decoctions Infusions and decoctions Infusion, decoction, rectum application

Mentha aquatica L., Mentha longifolia (L). L.

Lamiaceae

Inxina,

Leaves

Stomach aches

Infusion

Olea europaea L. subsp. africana (Mill.) P. S. Green

Oleaceae

Umnquma

Bark, leaves and roots

Diarrhoea

Infusions and decoctions of dried leaves, bark and roots.

Pelargonium Curtis

Geraniaceae

Umkumiso,

Root

Dysentery

Decoction

Persicaria lapathifolia (L.) Gray

Polygonaceae

Idolo lenkonyana

Roots and leaves or whole plant

Stomach diarrhoea

Plantago lanceolata (L.)

Plantaginaceae

Ubendlela

Leaves

Infusion

Achanthaceae

Impendulo

Roots, leaves

Diarrhoea, dysentery Stomach problems, haemorrhagic diarrhoea and amoebic dysentery

Balanophoraceae

umavumbuka

Whole plant

Diarrhea, dysentery

Infusion, Decoction

Schotia afra (L.) Thunb. Schotia latifolia Jacq. Schotia brachypetala Sond.

Fabaceae Fabaceae Fabaceae

Umgxam, Umgxam Ishimnumyane,

Bark and root, Bark and root Bark and roots

Diarrheoa Diarrheoa Dysentery, diarrhoea

Decoction Decoction Decoction

Strychnos henningsii Gilg,

Loganiaceae

umnonono, umnono,

Stem Bark

Stomach aches

Decoctions of the bark and infusions of the leaves

capensis

reniforme

Rubia petiolaris DC.

Sarcophyte Sparrm.,

sanguinea

umGwenya-

complaints

and

Infusion

Infusion, decoction and concoction

Olajuyigbe and Afolayan

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Table 1. Contd.

Syzygium cordatum Hochst.

Myrtaceae

Umsu

Bark, leaves and roots

Stomach complaints, diarrhoea

Decoction and concoction

Schizocarphus nervosus (Burch.) Van der Merwe

Hyacinthaceae

umagaqana, inkwitelu

Roots or rhizomes

Dysentery

Decoction of the roots

Solanum aculeastrum tomentosum L.

Solanaceae

Umthuma

Root, bark and berries

Dysentery

Infusion, decoction and concussion

Typhaceae

inqoboka, ingcongolo

Rhizomes

Diarrhoea, dysentery

Decoction of the rhizomes

Rhamnaceae

Umphafa

Bark, leaves and roots

Diarrhoea, Dysentery

Decoction of the roots; concoction of bark and leaves

Dun.,

Solanum

Typha capensis (Rohrb.) N.E.Br., Ziziphus mucronata mucronata Willd

Willd.

subsp.

and other gastrointestinal disorders. Of these 19 plants used to treat dysentery and other gastrointestinal disorders, 8 (42.11%) plants were implicated in the treatment of dysentery alone. These plants include Acacia mearnsii, Bulbine abyssinica, Ekebergia capensis, Ipomoea crassipes, Iconotis leonurus, Pelargonium reniforme, Schizocarphus nervosus and Solanum aculeastrum. In addition, 14 (38.89%) plants were used to treat diarrhoea along with other gastrointestinal disorders. While six (42.86%) of these plants: Brachylaena ilicifolia, Bulbine latifolia, Cissampelos capensis, Olea europaea, Persicaria lapathifolia and Schotia afra, were implicated in the treatment of diarrhoea alone; eight plants (22.22%): Acacia karroo, Hydnora Africana, Plantago lanceolata, Rubia petiolaris, Sarcophyte sanguine, Schotia brachypetala, Typha capensis and Ziziphus mucronata, were implicated in treating dysentery and diarrhoea as shown in Figure 1. Though Curtissia dentata alone was mentioned as being used in the

treatment of diarrhoea and stomach complaints, ten (27.78%) different plants were implicated in the treatment of various stomach problems. These plants include Alepidea amatymbica, Cussonia spicata, Centella asiatica, Clausena anisata, C. capensis, Eucomis autumnalis, P. lapathifolia, R. petiolaris, Syzygium cordatum and Strychnos henningsii. In different communities, many plants are given different names. Many of these plants, eventually, have more than one local name. These plants are prepared and mostly administered orally in different ways, except Iconotis leonurus having rectum application in addition to its oral administration, in different ways to treat gastrointestinal and its associated disorders. In their preparations for therapeutic purposes, whole plants as well as various parts of each plant species were either used singly or in combined forms. Parts used also depend on the plant under consideration and severity of ailments. Leaves constituted the majority of uses (30.77%), followed by roots

(25%) and bark (21.15%), whole plant (9.62%), rhizomes (5.77%), fruits (3.85%) as well as bulb and tuber (1.92%). The results are shown in Figure 2. Decoctions and infusions are the most frequently used methods of preparation as shown in Figure 3. DISCUSSION Over the last century, ethnobotany has evolved into a specific discipline that looks at the people– plant relationship in a multidisciplinary manner such as ecology, economic botany, pharmacology and public health (Balick, 1996). With extensive uses of medicinal plants, numerous drugs have been introduced into the international markets as a result of exploring ethnopharmacology and traditional medicines (Bussmann, 2002) which have expressed different pharmacological actions (Gregory, 2004). Hence, the traditional use of low profile and less known medicinal plants should

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Figure 1. Number of plants relevant in treating each gastrointestinal disorder.

Figure 2. Frequency of plant parts used for treating gastrointestinal disorders.

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Frequency of preparation methods (%)

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Figure 3. Frequency of different preparation methods for treating gastrointestinal disorders.

be documented to disseminate their therapeutic efficacy to pave the way for preparation of acceptable medicine and to reduce the pressure on overexploited species (Kala et al., 2006). In South Africa, up to 60% of the population consults traditional healers (van Wyk et al., 1997), especially in rural areas where traditional healers are more numerous and accessible than Western health-care providers. The traditional healer are found within a short distance and are familiar with the patient‟s culture while the environment and the costs associated with treatments are negligible (Rinne, 2001). While the loss of valuable medicinal plants due to population pressure, agricultural expansion and deforestation have been widely reported (Abebe, 2001; Berhan and Dessie, 2002), documenting indigenous knowledge becomes essential to preserve the traditional knowledge and valuable information passed verbally from generation to generation and can be lost whenever a traditional medical practitioner passes without conveying his knowledge about traditional medicinal plants. In this study, the number of indicated medicinal plants and their potential applications in the treatment of gastrointestinal disorders reflect the rich ethnomedicinal

knowledge in the Eastern Cape. Here, traditional medicine remains the main resource of phytotherapy for a large majority of the people. The wide spread use of the various plants could be attributed to cultural acceptability, efficacy, physical accessibility and economic affordability as well as playing a major role in the treatment of gastrointestinal disorders in comparison to modern medicine. Based on the difficulty in distinguishing between diarrhoea and dysentery by the local people, many of the plants have been used in treating either of these infections unknowingly, thereby, indirectly showing their multipurpose efficacies. For instance, 38.89% of the plants mentioned are used for diarrhoea and other gastrointestinal disorders while 52.78% were indicated as being used against dysentery and other gastrointestinal disorders. While 22.22% of these plants are used in treating dysentery and diarrhoea, 27.78% are used in the treatment of various stomach problems. There are overlaps in plants used in treating both infections. Plants used in treating different stomach problems are not an exemption. In addition to earlier report of Appidi et al. (2008), B. ilicifolia, C. capensis, P. lapathifolia and S. afra have been used in the treatment of diarrhoea locally. The prevalence of the use of leaves for the preparation of

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traditional herbal remedies as shown in this study corresponds with earlier reports in other studies (Brinkhaus et al., 2000; Yineger and Yewhalaw, 2007; Pradhan and Badola, 2008; Zainol et al., 2008). While the use of more than one plant or plants‟ parts in herbal preparations could be attributed to the additive or synergistic effect that extracts from the different plants are thought to have during treatment (Bussman and Sharon, 2006), Gessler et al. (1994) indicated that the use of concoctions suggests that the traditional medicines may only be active in combination due to the synergistic effects of several compounds that are acting singly. On the contrary, the use of bark, roots or uprooting the whole plant of a given species could be destructive means of obtaining the herbal remedies. These unfavorable extraction methods will eventually contribute to the loss of the forest trees. Though the methods of preparing these medicinal plants vary, decoction and infusion methods are highly reputed and valued by traditional healers in Southern African native population for its curative and palliative effects in the treatments of diseases generally (Watt and Breyer-Brandwyk, 1962; Hutchings, 1996) while active compounds in preparations taken anally are more effectively re-absorbed by the mucus membranes of the rectum (Van Wyk and Wink, 2004). Decoction of a part or combination of different parts could be more effective as more active phytochemicals are likely to be extracted by boiling. In agreement with Nanyingi et al. (2008) and Bekalo et al. (2009), there is a lack of standardization and quality control in orally administered traditional medicine. Against these parameters, oral dosages are estimated using lids, spoons, cups, pinches and handfuls while most preparations are often prescribed through estimation in term of a full, half or one-fourth of a cup, depending on the age, physical condition of the patient being treated, severity and type of infection. In addition, without scientific proofs from the traditional healers and local people, the rationales for the choice of some of these plants have been attributed to include some inherent properties of these plants. These attributes included being purgative, anti-dysenteric, anodyne, anti-inflammatory, carminative, demulcent, diaphoretic, emollient, styptic or astringent, refrigerant, stomachic, tonic and vasodilator. Usher (1984) and Koide et al. (1998) reported that the folk use of A. mearnsii (Fabaceae), Mentha aquatic (Lamiaceae), P. lanceolata (Plantaginaceae), P. lapathifolia (Polygonaceae), R. petiolaris (Achanthaceae), S. sanguine (Balanophoraceae), S. afra (Fabaceae) and S. latifolia (Fabaceae) as anti-dysenterics was due to their tannin content imparting astringent activity which helps to recuperate from diarrhoea and dysentery. Plants containing tannins are astringent, able to draw together or constrict body tissues and are effective in stopping the flow of blood or other secretions. Tannins strengthen veins by repairing the connective tissues surrounding veins and decrease capillary fragility. They are also known

as antimicrobial (Cowan, 1999) and triterpenoids are beneficial for inflammation (Cipak et al., 2006). The antiinflammatory activities may be due to the presence of alkaloids, flavonoids and saponins present in these plants like every other plants (Wong et al., 1992; Ono, 1994; Kerber, 1999; Fernanda et al., 2002; Fawole et al., 2009). The refrigerant, purgative and vasodilatory activities of these plants substantiate their ability to cause the blood to stop flowing and clog the arteries and veins as well as removing enough “heat” from the system (Littlewood, 1988; Lans, 2006). Conclusion Traditional knowledge of medicinal plants and their uses by indigenous cultures are not only useful for conservation of cultural traditions and biodiversity but also for community healthcare and drug development in the present and future. In this study, 36 plant species consisting of 24 families were used as ethnomedicines for gastrointestinal disorders in the Eastern Cape, South Africa. These plants treated diarrhoea, dysentery and various stomach problems. Reasons for the choice of these plants, plants‟ parts used and methods of preparations were indicated. Since traditional healers harvest roots and barks of some of these medicinal plants, there is need to educate them about the looming danger of wiping out some of the plant species if overexploited. Further investigation of ethnopharmacology is worthwhile to affirm their antimicrobial activities against bacteria in diarrhoea and dysentery, isolate the plants‟ active chemical compounds, and decipher their modes of action. ACKNOWLEDGEMENT The authors are grateful to the National Research Foundation (NRF) of South Africa for supporting this research. REFERENCES Abebe D (2001). Biodiversity conservation of medicinal plants: Problem and prospects. In Conservation and sustainable use of medicinal plants in Ethiopia Proceeding of The National Workshop on Biodiversity Conservation and Sustainable Use of Medicinal Plants in Ethiopia Edited by: Zewdu M, Demissie A. Addis Ababa: IBCR, 198203. Acocks JPH (1975). Veld Types of South Africa, Memoirs of Botanical Survey of South Africa Bot. Res. Inst., Dep. Agric. Water Suppl., Pretoria, p. 57. Ahmad M (2003). Ethnobotanical and taxonomic studies of economically important plants of Tehsil Attock. M. Phil. Thesis, Quaid-e-Azam Univ., Islamabad Pak., pp. 205-207. Anonymous (1993). Guidelines for the Conservation of Medicinal Plants. IUCN. WHO & WWF, Gland, Switzerland. Appidi JR, Greirson DS, Afolayan AJ (2008). Ethnobotanical study of plants used for the treatment of diarrhoea in the Eastern Cape, South Africa. Pak. J. Biol. Sci., 11(15): 1961-1963.

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Journal of Medicinal Plants Research Vol. 6(18), pp. 3425-3433, 16 May, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1746 ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

Bioactivity of natural compounds isolated from cyanobacteria and green algae against human pathogenic bacteria and yeast H. Al-Wathnani, Ismet Ara*, R. R. Tahmaz, T. H. Al-Dayel and M. A. Bakir Department of Botany and Microbiology, College of Science, King Saud University, P. O. Box 22452, Riyadh-11495, Kingdom of Saudi Arabia. Accepted 30 January, 2012

Analysis of bioactive natural compounds of algae such as cyanobacteria (Spirulina platensis; Nostoc linckia; Phormidium autumnale; Tolypothrix distorta and Microcystis aeruginosa) and green algae (Chlorella vulgaris and Dunaliella salina), and their activity against human pathogenic bacteria and yeast such as Salmonella suis ATCC 13076; Pseudomonas aeruginosa ATCC 27583; Escherichia coli ATCC 25922; Staphyllococcus aureus ATCC 25923; Bacillus subtilis ATCC 6633; Shigella sonnei ATCC 11060 and Candida albicans ATCC 10231 have been studied in Saudi Arabia. Extraction of algal metabolites was performed by using the mixture of three organic reagents, methanol:acetone:diethyl ether as 5:2:1 v/v, respectively. All metabolites of algae isolates had shown weak to strong antimicrobial activity toward one or more human pathogenic microorganisms. Almost all the algal extract showed strong activity against S. sonnei in agar well diffusion technique. Crude extract of cyanobacteria, T. distorta showed moderate to strong activity against S. aureus, B. subtilis and S. sonnei. Further, crude extract of all the algal metabolites have been analyzed using gas chromatography-mass spectrometry (GC-MS). Results indicated that the main component in the crude extracts of P. autumnale is 1-Hexyl-2-Nitrocyclohexane (91.7%); C. vulgaris is 2-Butanol, 3-methyl-, (S)(90.8%); S. platensis is Nitrocyclohexane-2-Hexyl-1 (92.1%); N. nostoc is Octadecanal (aldehyde) (86.8%); D. salina is 3-Methyl-2-(2-Oxopropyl) Furan (90.%); T. distorta is Boronic acid, Ethyl-, Dimethyl ester (83.9%) and lastly M. aeruginosa is (S)-(+)-1-Cyclohexylethylamine representing 91.9%, respectively. Further, in this study, the extracts of all the algal species especially T. distorta, P. automonate, C. vulgaris and D. salina have been found potential for the production of several compounds including biomedically important organic metabolites such as ethane,1,1-diethoxy-; butanal; heptanal and octanal. Further study for the purification of the potent compound will explain their usefulness in the pharmaceutical and biotechnological industry. Key words: Cyanobacteria, green algae, algal extract, gas chromatography-mass spectrometry (GC-MS), antimicrobial activity, human pathogenic bacteria, biomedical properties.

INTRODUCTION Cyanobacteria or blue-green algae are a fascinating group of primitive phototrophic prokaryotic organisms whose long evolutionary history dates back to the Proterozoic era. These organisms, endowed with tremendous genome plasticity, are distributed in all

*Corresponding author. E-mail: [email protected] or iara@ ksu.edu.sa. Tel: +966 1478 9585 Ext. 1639. +966534509242.

possible biotypes of the world. These organisms have tremendous potential in environmental, management as soil conditioners. Due to their occurrence in diverse habitats, these organisms are the excellent material for investigation by the ecologists, physiologists, biochemists, pharmacists and molecular biologists. Accordingly, looking for cyanobacteria with antimicrobial activity has gained importance in recent years. Biologically active substances were proved to be extracted by cyanobacteria

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(Borowitzka, 1995; Kreitlow et al., 1999; Mundt et al., 2001; Volk and Furkert, 2006). Various strains of cyanobacteria are known to produce intracellular and extracellular metabolites with diverse biological activities such as antialgal (John et al., 2003), antibacterial and antifungal (Ghasemi et al., 2003, 2007; Isnansetyo et al., 2003; Jaki et al., 1999; Kundim et al., 2003; Soltani et al., 2005) and antiviral activity (Moore et al., 1989). Antibiotic resistance in bacteria is one of the emerging health related problem in the world nowadays. Plants and among them algae are valuable natural sources effective against infectious agents. Extensive efforts for the identification of bioactive compounds derived from natural resources have been made worldwide, in order to develop safe, nontoxic and efficient anti-microbial agents of valuable practice in pharmacology. Algae of marine and terrestrial origins have been the best choice among natural resources within aquaculture and agriculture fields. Screening bioactivity of algal crude extracts is mandatory in biomedical practice, where antibacterial (Tuney et al., 2006), antifungal (Moreau et al., 1998; Tang et al., 2002), antiviral (Serkedjieva, 2004) and even more antialgal (Hellio et al., 2002) activity have been assessed to these metabolites. Emergence concerns have been raised to establish structural and functional properties of the bioactive compounds described in algal crude extracts, up to date, over 2,400 bioactive metabolites have been isolated and identified from a diverse group of algal communities (Faulkner, 2001). Due to the pivotal role played by these organisms, it was considered worthwhile to examine growth parameters, physiological attributes and antimicrobial activity for possible biotechnological applications. Our goal in the current study is to analyze and identify the chemical components and structure of algal crude extracts which was previously determined to have a strong antibacterial activity. All the subjected extracts were originated from algae isolates obtained from different desert soil sources in Saudi Arabia.

preserved in the Phycology Laboratory, Botany Department, Faculty of Science, King Saud University, Saudi Arabia.

Extraction of algal biomass For the extraction of metabolites, dried algae biomass was mixed in a glass flask with methanol:acetone:diethyl ether as 5:2:1 volumes, respectively, and shaken for 3 days at about 20°C. The mixture was separated by filtration. Then, the combined solvents were evaporated to dryness and the residue re-dissolved in 2 ml distilled water to form stock solution as 50 mg/ml.

Bacterial bioassay Microbial indicators and growth conditions Seven microorganisms including Gram +ve, Gram -ve bacteria and yeast were used in this study. These are: S. suis ATCC 13076, P. aeruginosa ATCC 27583, E. coli ATCC 25922, S. aureus ATCC 25923, B. subtilis ATCC 6633, S. sonnei ATCC 11060 and C. albicans ATCC 10231. Bacterial strains and yeast were kindly provided from Microbiology Laboratory, Botany Department, Faculty of Science, King Saud University, Saudi Arabia. Bacterial bioassay was performed using agar well diffusion method with the extract of algae species. In each assay, fresh cultures were obtained by inoculating the strains on nutrient agar plates and incubated at 37°C for 18-24 h. About 2-3 colonies of bacteria were inoculated into nutrient broth and incubated overnight at 37°C. Bacterial turbidity of all the test organisms as 0.5 Mc Farland standard was obtained and then swabbed with sterile cotton swab onto Muller Hinton agar plates. The swabbed plates were allowed to dry, and then distant wells were made within agar to be loaded with the organic extracts.

Antimicrobial activity by the agar-well diffusion method For antagonistic activity of algal extracts, agar well diffusion technique was performed. For this reason, surface of Petri dishes was punched with 5 mm cut with sterile straw and bottom of each well was sealed with two drops of sterile water agar. About 100 µl of algal extract were transferred into each well. Wells loaded with the extracting solvents were used as controls. All the plates inoculated with bacteria were incubated at 37°C for 24 h. After incubation, the diameter of the inhibition zone was measured with scale and the results were recorded in mm (data not shown).

MATERIALS AND METHODS Sample preparation

Analysis of algal crude extracts using gas chromatographymass spectrometry (GC-MS)

Isolation and cultivation of algal species Seven algal strains were selected for screening of their antimicrobial activity belonging to cyanobacteria and green algae including C. vulgaris; S. platensis; N. linckia; P. autumnale; D. salina; T. distorta and M. aeruginosa. Algal species were isolated from different desert soils in Saudi Arabia according to standardized algae isolation procedure (Rippka et al., 1979; Vaara et al., 1979), and further identification was performed by scanning electron microscopy (SEM, JEOL, JSM, 6460), in addition to light microscopic morphology. Each isolate was sub cultured in suitable nutrition media (BG11; Rippka et al., 1979) for algae cultivation and allowed to flourish at 20-30°C under constant light for 2-4 weeks. Algal cells were collected in the exponential growth phase by filtration to be applied for extraction. All the algal strains were

The gas chromatography coupled with mass spectrometry detection technique allows good qualitative and quantitative analysis of the fractionated extracts with high sensitivity to smaller amounts of components. Accordingly, identification of the chemical constituents of fractionated extracts, for the selected microalgae which showed effective antibacterial activities against the test bacteria were analyzed. This was done by using 1 µl of each sample was injected into an RT x -5 column (30X0.32 nm) of GC-MS model (Perkin Elmer, Clarus 500, USA) and Helium (3 ml/min) was used as a carrier gas. The following temperature gradient program was used; 75°C for 2 min followed by an increase from 75 to 175°C at a rate of 50°C per min and finally 7 min at 175°C. The m/z peaks representing mass to charge ratio characteristics of the antimicrobial fractions were compared with those in the mass spectrum library

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Table 1. Antibacterial activities of crude extract from different soil algal species isolated in Saudi Arabia.

Crude extract (Acetone/Methanol/Di-Ethyl-ether, 5:2:1 v/v) Chlorella vulgaris Spirulina platensis Nostoc linckia Phormidium autumnale Dunaliella salina Tolypothrix distorta Microcystis aeruginosa

S. aureus _ _ + _ _ ++ +

B. subtilis + _ _ ++ _ +++ _

S. sonnei +++ +++ +++ +++ ++ +++ ++

(-) No activity, (+) low activity, (++) moderate activity, (+++) high activity.

of the corresponding organic compounds (Pandey et al., 2010). The chemical components of the extracts were analyzed in the central laboratory of King Saud University, Riyadh, Saudi Arabia. Identification of the chemical constituents of extracts were made using Perkin Elmer (Clarus 500, USA) gas chromatography coupled with (Clarus 500, USA) mass spectrometer (MS). Neither internal nor external chemical standards were used in this chromatographic analysis. Interpretation of the resultant mass spectra were made using a computerized library-searching program (NIST database) and by studying the fragmentation pattern of such compound resulted from mass spectrometry analysis. Concentration of such compound was calculated by the following formula: Compound concentration percentage= [P1/P2] x 100 Where, P1 is the peak area of the compound and P2 is whole peak areas in the fractionated extracts.

RESULTS Soil cyanobacteria isolated from the cultivated fields of different places in Saudi Arabia was evaluated for antimicrobial activity. Mixture of methanol, acetone and diethyl ether extracts from 7 algae were examined for antimicrobial properties against six bacteria and one fungus. Of total microalgae, 100% (5 cyanobacteria and 2 green algae) exhibited antimicrobial effects. Selected cyanobacteria with positive antimicrobial activities were C. vulgaris, S. platensis, N. linckia, P. autumnale, D. salina, T. distorta and M. aeruginosa. Antibacterial activities for all the crude extracts of algal species were examined (Table 1) and significant bioactivities against S. aureus, B. subtilis and S. sonnei were determined. All the algae species used in this study showed strong inhibition against S. sonnei. Considering fungi, no antimicrobial activity was observed in all the tested algae.

Chemical analysis of the potent algal extract To determine the active organic components within the described algal species, GC-MS analysis were performed for all of the potent crude extracts. Chemical composition and concentrations of the analyzed fractions are

presented in Table 2. In our data, the major peaks in the gas chromatogram were assigned with the highest percentage of the compound concentration in the total extract (REV values). For each algal species, the most 2 intensive fraction was recorded such as P. automonate: 1-Hexyl-2-Nitrocyclohexane; Cyclohexane; 1-(1,5Dimethylhexyl)-4-(4-Methylpentyl)-; S. platensis: 2Butanol; 3-Methyl-(S)-; 2-Hexanol (S)-; C. vulgaris: Bromoacetic acid; Pentadecyl ester; 1-Hexyl-2Nitrocyclohexane; N. linckia: Octadecanal; 1,37Octatriacontadiene; D. salina: 3-Methyl-2-(2-Oxopropyl) Furan; 1-Hexyl-2-Nitrocyclohexane; T. distorta: Boronic acid; Ethyl-Dimethyl ester; 7,9-Di-Ter-Butyl-1Oxaspiro(4,5) Deca-6,9-Diene-2,8-Dione; M. aeruginosa: (S)-(+)-1-Cyclohexylethylamine; Boronic acid, EthylDimethyl ester (Table 2). The resultant major peaks of the extract were surveyed by the use of the available data base PubChem, provided by the National Center for Biotechnology Information (NCBI) at http://pubchem.ncbi. nlm.nih.gov. Classification, biomedical features and biological assay activity was obtained for some of the resultant fractions, and data are represented in Table 3.

DISCUSSION Emergence of microbial diseases in pharmaceutical industries implies serious loss. Usage of commercial antibiotics for human disease treatment produces undesirable side effects. Cell extracts and active constituents of various algae may be potential bioactive compounds of interest in the pharmaceutical industry (Rodrigues et al., 2004). In the current research, combination of methanol, acetone and diethyl ether was the best solvents for extracting the bioactive compounds compared to acetone, methanol and diethyl ether alone (data not shown), meanwhile it gave the highest antimicrobial activities against the selected pathogens. This was in contrast with the study of Tuney et al. (2006). However, Das et al. (2005) examined acetone, ethanol and methanol extracts of other algae and showed from moderate to high activity against strains of virulent

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Table 2. The GC-MS analysis of different components in (methanol:acetone:diethyl ether) extracts of soil algal species.

S/N Compound Phormidium autumnale 1 Boronic acid, Ethyl-, Dimethyl ester 2 3,4-Hexanediol, 2,5-Dimethyl3 2-Butanol, 3-Methyl-, (S)4 2-Butanol, 3-Methyl5 Butanal 6 2-Thiophenecarboxylic acid, 5-(1,1-Dimethylethoxy)7 Cyclopropanepentanoic acid, 2-Undecyl-, Methyl Ester, Trans8 1,6-Anhydro-3,4-Dideoxy-.Beta.-D-Gluco-Hexopyranose 9 1-Hexyl-2-Nitrocyclohexane 10 Cyclohexane, 1-(1,5-Dimethylhexyl)-4-(4-Methylpentyl)11 1-Hexyl-1-Nitrocyclohexane

Rev* 843 772 882 862 874 854 804 773 917 903 870

Chlorella vulgaris 1 N-(3-MethylButyl) Acetamide 2 D-Mannoheptadecane-1,2,3,4,5-Pentaol (methyl ester-fatty acid) 3 2-Butanol, 3-Methyl-, (S)4 2-Hexanol, (S)5 5,9-Dodecadien-2-one,6,10-Dimethyl-, (E,E))6 Heptanal 7 3-Decyn-2-Ol 8 3-Nonyn-2-Ol 9 2(3H)-Furanone, 3-(15-Hexadecynylidene)Dihydro-4-Hydroxy-5-Methyl-, [ 10 3-Methyl-2-(3-Methylpentyl)-3-Buten-1-Ol 11 (3R,2E)-2-(Hexadec-15-Ynyliedene)-3-Hydroxy-4-Methylenebutanolide 12 4-Methyldocosane

776 769 908 905 773 772 763 755 887 884 893 879

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Spirulina platensis 1-Propanamine, N1-Methyl-2-Methoxy 1,2-Benzendicarboxylic acid, Bis(2-Ethoxyethyl) Ester 1-Dodecanol, 3,7,11-Trimethyl Hexadecen-1-Ol, Trans-93-Decyn-2-Ol 1,6;2,3-Dianhydro-4-Deoxy-.Beta.-D-Lyxo-Hexopyranose 3-Chloropropionic acid, Heptadecyl Ester Acetic acid, Chloro-, Hexadecyl Ester retsE lycedatneP ,dica citecaomorB 10-Heneicosene (C,T) 2(3H)-Furanone, 3-(15-Hexadecynylidene)Dihydro-4-Hydroxy-5-Methyl-, [ (3R,2E)-2-(Hexadec-15-Ynyliedene)-3-Hydroxy-4-Methylenebutanolide enaxeholcycortiN-2-lyxeH-1 1-Hexyl-1-Nitrocyclohexane 1-Propanamine, N1-Methyl-2-Methoxy 1,2-Benzendicarboxylic acid, Bis(2-Ethoxyethyl) Ester 1-Dodecanol, 3,7,11-Trimethyl

682 673 849 842 838 825 892 887 909 901 904 890 921 893 682 673 849

1 2

Nostoc linckia Boronic acid, Ethyl-, Dimethyl ester 3,4-Hexanediol, 2,5-Dimethyl-

848 772

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Table 2. Contd.

3 4 5 6

2-Butanone, 3-Hydroxy(S)-Isopropyl Lactate )EDYHEDLA( lanacedatcO eneidatnocairtatcO-1,37

828 797 868 861

1 2 3 4 5 6 7 8

Dunaliella salina 2-Aminononadecane 1,6;3,4-Dianhydro-2-Deoxy-.Beta.-D-Lyxo-Hexopyranose 2-Hexanol, Acetate Octanal 3-Methyl-2-(2-Oxopropyl)Furan 1-Hexyl-2-Nitrocyclohexane 2-Octadecyl-Propane-1,3-Diol Pentadecanal-

837 792 811 785 909 906 888 887

1 2 3 4 5 6

Tolypothrix distorta Boronic acid, Ethyl-, Dimethyl ester 3,4-Hexanediol, 2,5-Dimethyl7,9-Di-Ter-Butyl-1-Oxaspiro(4,5)Deca-6,9-Diene-2,8-Dione Silane,TrichlorodecylTetraethylene Glycol Diethyl Ether Ethane, 1,1-Diethoxy-

839 778 791 714 712 699

1 2 3 4 5 6 7 8 9 10 11 12

Microcystis aeruginosa (S)-(+)-1-Cyclohexylethylamine Octodrine Boronic acid, Ethyl-, Dimethyl ester 3,4-Hexanediol, 2,5-Dimethyl2-Butanone, 3-Hydroxy(S)-Isopropyl Lactate 2-Thiophenecarboxylic acid, 5-(1,1-Dimethylethoxy)Cyclobutanol 2-Dodecen-1-Yl(-)Succinic Anhydride Cyclohexanol, 4-Ethyl-4-Methyl-3-(1-Methylethyl)-, (1.Alpha.,3.Alpha.,4. Decane, 5,6-Bis(2,2-Dimethylpropylidene)-, (E,Z)Cyclohexanol, 4-Ethyl-4-Methyl-3-(1-Methylethyl)-, (1.Alpha.,3.Alpha.,4.A

919 842 849 772 831 801 786 790 793 785 782 780

*(Rev), % of the compound concentration in the total extract.

Table 3. Biomedical properties of different mass spectra components in crude extracts belonging to soil algal species.

Algal species T. distorta P. automonate C. vulgaris D. salina

Mass spectra compound Ethane, 1,1-DiethoxyButanal Heptanal Octanal

Classification Stearyl alcohol Aldehyde Aldehyde Caprylic Aldehyde

Compound ID in PubChem (CID)

Pharmacological and biomedical effects

7765 261 8130 454

(+) Ezra et al.,1998 (+) Gregory et al., 2006 (+) Cueto et al., 1992 (+) Singer, 2000

Reported properties (+), no reported activity (-).

pathogens Pseudomonas florescence, hydrophila, Vibrio anguillarum and E. coli.

Aeromonas

In this study, 4 cyanobacteria and 2 green microalgae were tested in compliance with the agar well diffusion

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method for their antibacterial agent production on various organisms that incite diseases of humans. The antimicrobial activity was maintained by using mixture of methanol, acetone and diethyl ether. It was found that, cyanobacteria T. distorta and P. autumnale had the highest antibacterial activity towards the tested bacteria. In a previous study by Rania and Taha (2008), 3 cyanobacteria (Anabaena oryzae, Tolypothrix ceytonica and Spirulina platensis) and 2 green microalgae (Chlorella pyrenoidosa and Scenedesmus quadricauda) were tested in compliance with the agar well diffusion method for their antibacterial and antifungal agent production on various organisms that incite diseases of humans and plants (Escherichia coli, Bacillus subtilis, Staphyllococcus aureus, Pseudomonas aeruginosa, Aspergillus niger, Aspergillus flavus, Penicillium herquei, Fusarium moniliforme, Helminthosporium sp., Alternaria brassicae, Saccharomyces cerevisiae, Candida albicans). In their study, the antimicrobial activity was maintained by using ethanol, acetone, diethyl ether and methanol. It was found that, Spirulina platensis and Anabaena oryzae had the highest antibacterial and antifungal activity towards the tested bacteria and fungi (Rania and Taha, 2008). Very recently, in another similar study, methanolic extracts of four marine algae of Algeria coast were investigated for antibacterial activity against six pathogenic bacteria (Bacillus subtilis, Listeria innocua, S. aureus, E. coli, Klebsiella pneumonia and Pseudomonas aeruginosa). Susceptibility assays using disc diffusion and broth micro dilution test for the determination of minimum inhibitory concentration (MIC) were employed to assess the antibacterial activity of methanolic extracts of algae. All algae extracts showed antibacterial activity against four of the six pathogenic bacteria tested with MIC values ranged between 0.25-3.0 mg/ml. In their study, extract of Rhodomela confervoïdes exhibited the highest activity against Bacillus subtilis (24.0 mm) and Cystoseira tamariscifolia exhibited the highest activity against Listeria innocua (19.67 mm) (Bedjou et al., 2011). On the other hand sufficient data regarding crude extracts of seaweed and terrestrial algae have been obtained in previous studies, revealing the antimicrobial properties of organic compounds derived from natural sources (Robles-Centeno et al., 1996; Manilal et al., 2009b; O'Sullivan et al., 2010; Wijesinghe and Jeon, 2011). Analysis of bioactive metabolites have been studied mostly with marine (Wijesinghe and Jeon, 2011) algal species (Caccamese et al., 1985; Lima-Filho et al., 2002). On the other hand, little is known for the terrestrial or soil originated algae, but some studies were subjected to cyanobacteria (Bloor and England, 1989; Burja et al., 2001). In the current study, all our isolates belonged to cyanophytae and chlorophytae groups of soil origins, assigned antibacterial effect of these species are presented in Table 1. The most effective activity was

recorded for T. distorta against S. aureus, B. subtilis and S. sonnei, followed by P. autumnale affecting B. subtilis and S. sonnei. In the present work, a simple chemical methodology to carry out the screening for natural functional compounds is presented. To do that, a strategy has been conducted including the use of unexplored natural sources (that is, algae and microalgae) together with environmentally clean extraction techniques and advanced analytical tools. The procedure also allowed estimating the functional activities of the different extracts obtained and even more important, to correlate these activities with their particular chemical composition. By applying this methodology, Plaza et al. (2010) reported that it is possible to carry out the screening for bioactive compounds in the algae Himanthalia elongata and the microalgae Synechocystis sp. Both algae produced active extracts in terms of both antioxidant and antimicrobial activity. In their study, the obtained pressurized liquid extracts were chemically characterized by GC-MS and HPLC-DAD. Different fatty acids and volatile compounds with antimicrobial activity were identified, such as phytol, fucosterol, neophytadiene or palmitic, palmitoleic and oleic acids. Based on the results obtained, ethanol was selected as the most appropriate solvent to extract this kind of compounds from the natural sources studied. Crude extract analysis of the described species using gas chromatography-mass spectrometry (GC-MS) had revealed several important organic volatile compounds as fatty acids in this study (Table 2). Similar to our findings, it was reported that the soil cyanobacteria isolated from the paddy fields of seven provinces in Iran was evaluated for antimicrobial activity. Aqueous, petroleum ether, and methanol extracts from 76 microalgae were examined for antimicrobial properties against four bacteria and two fungi. Of the total microalgae, 22.4% (17 cyanobacteria) exhibited antimicrobial effects (Soltani et al., 2005). Little privileges to the biomedical properties for these compounds have been assessed so far, however, the bioactivity of fatty acids has been approved in certain microorganisms and fouling organisms (Russel, 1991). Such compounds have showed antimicrobial properties (Katayama, 1960; Bloor and England, 1989; Khairy and El-Kassas, 2010). Similarly, in another study, the methanol, dichloromethane, petroleum ether, ethyl acetate extracts and volatile components of Spirulina platensis were tested in vitro for their antimicrobial activity (four Gram-positive, six Gram-negative bacteria and Candida albicans ATCC 10239). GC-MS analysis of the volatile components of S. platensis resulted in the identification of 15 compounds which constituted 96.45% of the total compounds. The volatile components of S. platensis consisted of heptadecane (39.70%) and tetradecane (34.61%) as major components. The methanol extract showed more potent antimicrobial activity than dichloromethane,

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Figure 1. Mass spectrum of ethane,1,1-diethoxy- (A); butanal (B); heptanal (C) and octanal (D) in the Figure 1. Mass spectrum of ethane,1,1-diethoxy- (A); butanal (B); heptanal (C) and methanol:acetone:diethyl ether extract of T. distorta; P. automonate; C. vulgaris and D. salina, respectively.

octanal (D) in the methanol:acetone:diethyl ether extract of T. distorta; P. automonate; C. vulgaris and D. salina, respectively.

petroleum ether, ethyl acetate extracts and volatile components (Karabay et al., 2007). These findings were correlated with our present observation, as shown in Table 2. In this study, similar chemical components with different percentage were detected for some species belonging to different groups probably due to the difference in polarity of the used solvents and to habitat environmental factors. The mass spectra of the compounds were investigated

with those similar in the PubChem database and some of our chemical components are reported to have a known biomedical value in the pharmacological fields (Table 3). Fractionated matrices of T. distorta crude extract, contained as stearyl alcohol and Ethane, 1,1-Diethoxywhich is known to demonstrate valuable therapeutic uses including anti-inflammatory, antipyretic, antithrombotic and analgesic effects (Figure 1). Interestingly, some of our resultant spectra compounds exhibited important

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biomedical features as described in Table 3. Fractionated constituents specified in each algal species were inspected for biomedical characteristics such as butanal was applied in controlling Bovine soles symptoms in cattle (Gregory et al., 2006); heptanal was investigated in vitro as biomarker for ozonation, suggesting an ozonation mechanism in lung cell lines of rats (Cueto et al., 1992). Finally, octanal was studied with olfactory receptor model, suggesting an interaction of octanal with such receptors in rat cell lines (Singer, 2000). In this preliminary study, assessing the biomedical characteristics of such compounds in the assayed species was still under prediction. Further, utilization of naturally occurring bioactive compounds from algae could obtain a wide alternatives of manufactured therapeutics. Interestingly, these findings would be the opening the new trends in biomedical and pharmaceutical industries in the region.

ACKNOWLEDGEMENT The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project No. RGPVPP-086. REFERENCES Bedjou F, Maiza-Benabdesselam F, Saidani K, Touati N (2011). Antibacterial activity of four species of Algerian algae. Nat. Prod. Ind. J., 7(2): 66-70. Bloor S, England RR (1989). Antibiotic production by the cyanobacterium Nostoc muscorum. J. Appl. Phycol., 1: 367-372. Borowitzka MA (1995). Microalgae as sources of pharmaceuticals and other biologically active compounds. J. Appl. Phycol., 7: 3-15. Burja AM, Banaigs B, Abou-Mansour E, Burgess JG, Wright PC (2001). Marine cyanobacteria-a prolific source of natural products. Tetrahedron, 57: 9347-9377. Caccamese S, Toscano RM, Furnari G, Cormaci M (1985). Antimicrobial activities of red and brown algae from Southern Italy Coast. Bot. Mar., 28: 505-507. Cueto R, Squadrito GL, Bermudez E, Pryor WA (1992). Identification of heptanal and nonanal in bronchoalveolar lavage from rats exposed to low levels of ozone. Biochem. Biophys. Res. Commun., 188:129-134. Das B, Pradhan J, Pattnaik P, Samantaray B, Samal S (2005). Production of antibacterials from the fresh water alga Euglena viridis (Ehren). World. J. Microbiol. Biotechnol., 21: 45-50. Ezra Y, Mordel N, Sadovsky E, Schenker JG, Eldor A (1989). Successful pregnancies of two patients with relapsing thrombotic thrombocytopenic purpura. Int. J. Gynaecol. Obstet., 29: 359-363. Faulkner DJ (2001). Marine natural products. Nat. Prod. Rep., 18: 1-4. Gregory N, Craggs L, Hobson N, Krogh C (2006). Softening of cattle hoof soles and swelling of heel horn by environmental agents. Food Chem. Toxicol., 44: 1223-1227. Ghasemi Y, Tabatabaei Yazdi M, Shokravi S, Soltani N, Zarrini G (2003). Antifungal and antibacterial activity of paddy-fields cyanobacteria from the north of Iran. J. Sci. Islamic Repub. Iran, 14: 203-209. Ghasemi Y, Moradian A, Mohagheghzadeh A, Shokravi S, Morowvat MH (2007). Antifungal and antibacterial activity of the microalgae collected from paddy fields of Iran: characterization of antimicrobial activity of Chroococcus disperses. J. Biol. Sci., 7: 904-910. Hellio C, Berge JP, Beaupoil C, Le Gal Y, Bourgougnon N (2002). Screening of marine algal extracts for anti-settlement activities

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Journal of Medicinal Plants Research Vol. 6(18), pp. 3434-3444, 16 May, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.11-1755 ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

Status and trade of crude drug in Uttarakhand Deepshikha Arya1, G. C. Joshi1 and Lalit M. Tiwari2* 1

Regional Research Institute (AY) CCRAS, Tarikhet, India. Department of Botany, DSB Campus, Kumaon University, Nainital, India.

2

Accepted 7 March, 2012

The present study deals on crude drug marketing in Tanakpur market, a virgin mandi of Uttarakhand. Survey of Tanakpur mandi was carried out in 2005 and 2008. A complete data of crude drugs in trade was gathered, 65 crude drug samples were collected from different traders of Tanakpur for authentication on taxonomical basis. After critical evaluation, it was found that some of the crude drugs were substituted/adulterated and some crude drugs were imported through Indo-Nepal Borders. Key words: Crude drugs; Uttarakhand; marketing.

INTRODUCTION Herbal medicines have been used for treating human ailments from time immemorial. It is well accepted that drugs derived from plants have negligible side effects as compared to their chemical counter parts; therefore, there is a growing global demand for medicines, pharmaceuticals, tonics, cosmetics and other products based on herbal raw material because of their efficacy, safety and minimal side effects. Nearly 75 to 80% of global populations still rely on herbal medicine for primary health care. The unprocessed material or crude drug, as it is commonly known is employed chiefly in the medicines on over-the-counter products (OTC), based on Ayurvedic, Unani, Siddha and other traditional system of medicine practiced in the Indian sub continent (Table 1). The raw material consist of either the whole plant or one of its vegetative parts, an exudate, fatty or volatile oil having specific therapeutic properties of yielding a physiologically active chemical compound. Trade in herbal raw material is flourishing day by day. The medicinal and aromatic plants from higher Himalayan Mountains and their products have a very long history of being utilized and traded in the lower Himalayan regions and plains of India. Uttarakhand State is a part of North Western Himalaya; it shares international boundary with china in the north and Nepal in the east. The nascent state has an area of 5.35 million ha (77°34'27” E to 81°02'22” E Longitudes and 28°53'24” N to 31°27'50” N Latitudes) and is very rich

in vegetation wealth (65% vegetation cover), which comprises of a vast range of important medicinal plants in natural condition. There is a flourishing market of crude drug material in this state. The setup consists of local, regional and central markets. The medicinal plant sector in the state is peacefully developing. Survey of available literature reveals that about 2500 medicinal plants from Indian sub continent are in local medical use and/or in commerce/trade, especially in the pharmaceutical industries; out of these, 1748 species are from Indian Himalayan region, most of which are found in Uttarakhand region (Anonymous, 1982, 1999; Bentley and Trimer, 1980; Bisht et al., 2008; Kala, 2008; Kirtikar and Basu, 1993; Nadkarni, 1954; Chopra et al., 1956, 1959; Nandarni, 1954; Sarin, 2003, 2008; Singh and Kumar, 2005). Study area The present study was carried out across the borderline between India and Nepal in Tanakpur Distt Champawat of Uttarakhand. It is one of the important entry points to India from Nepal. Some of medicinal plants brought to Tanakpur, Champawat district, are from Nepal. Tanakpur is a small town and a municipal board in Champawat distt in Uttarakhand State of India, located at 29.08° N 80.12° E on riverside of Sarda river banks (Figure 1). METHODOLOGY

*Corresponding author. E-mail: [email protected].

To gather information on quantity, rate, source etc, of the medicinal plants in trade, local traders and contractors of crude drugs were

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Table 1. The frequency of occurrence of plant in 1145 important herbal formulation.

S. no. 1

2 3 4 5 6 7 8 9 10 11

Botanical name Terminalia chebula Retz. Terminalia bellirica Roxb Emblica officinalis (Gaertn) Triphala

Common name Triphala (Haritaki) (Bibhitaka) (Amla)

Glycyrrhiza glabra Linn Piper longum Linn Adhatoda vasica Nees Withania somnifera Dunal Cyperus rotundus Linn Tinospora cordifolia (willd) Miers. Berberis aristata DC. Tribulus terrestris Linn Holarrhena antidysenterica Wall Boerhaavia diffusa Linn

Yashtimadhu Pipali Vasaka Ashwagandha Mustaka Guduchi Daruharidra Gokshura Kutaja Punarnava

Figure 1. Location of Tanakpur in Uttarakhand.

identified and interviewed. Crude drug samples were collected from different traders and dealers. The field studies were carried out in 2005 and December 2008. The information was gathered after extensive market study of Tanakpur. A complete check list of all crude drugs and plant parts used in trade was compiled from each traders shop. The information related to their, common trade name, part, rate and their source (that is, the region from where they have been collected) were gathered.

RESULTS AND DISCUSSION During the course of the present study, 65 crude drug samples were collected from different traders of Tanakpur region for identification on taxonomical basis (Table 2) Figure 2, 3 and 4. The Uttarakhand Forest Development Corporation (UAFDC) has established a

No. of herbal formulations 219

141 135 110 109 102 88 65 65 59 52

crude drug mandi at Aamwala Depot. Date of auction has been fixed permanently in these depots. Open auction system is being followed at this mandi. The Uttarakhand Forest Development Corporation was involved in the trade of medicinal and aromatic plant in 2003 to 2004. Collection agencies (UAFDC, Bhesaj Sangh and KMVN) collected the medicinal plant produce from their allotted region either from forest or farmers and bring it to existing mandis because marketing is only allowed at this mandi. The detail of the area, species permitted and royalty rate were to be conveyed to the Bhesaj Sangh, Kumoun Mandal Vikas Nigam, which were to get the medicinal plant parts collected through their registered members/village level cooperatives societies. These agencies receive the plant parts collected by the registered member against payment at specified rates. The forest department then verifies the material, charge royalty and issue transport permit. In Tanakpur forest depot mandi, the medicinal plant comes through KMVN, Pithoragarh, Champawat, Van Vikas Nigam, and Bhesj Sangh. This approach helps farmer for cultivation of medicinal plant. In Utttarakhand in particular, the National Plant Board has sanctioned 45 projects under commercial scheme during 2003-2004, out of which about 50% of the projects have been sanctioned directly to individual farmers for the cultivation of medicinal plants and remaining are sanctioned to government institutions and NGOs for production of planting material of medicinal plants and research in this sector. Herbal Research and Development Institute (HRDI), which is the nodal agency for medicinal plant sector in the state is working closely with several departments for the development of medicinal plants in the state in the basis of existing climatic conditions in the state and market potential of the species, 14 species have been identified by HRDI as focus species in Uttarakhand. The species are: Kuth (Saussurea lappa), kutki (Picrorhiza kurroa), chirayata

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Table 2. Description of collected crude drugs from Tanakpur mandi of Uttarakhand.

S. no 1 (2 samples from different traders)

2

Trade name Satawar

Charila

Sanskrit name

Botanical name

Diagnostic character

Satamuli

Asparagus Willd

Roots are cream to light brown in color and show a number of scars and protuberances of lateral rootlets beside numerous minute wrinkles on the external surface.

-

racemosus

Parmelia perforata.

Thallus consist of a flattened foliosa structure with a more or less deeply inside upper surface, yellowish white on top and black on the lower surface, leathery to touch; dedicated rhizoid arises from lower surface; odour and taste not distinct.

3 (2 samples from different traders)

Tallish patra

Talish

Taxus baccata Linn.

Leaves 15 to 25 mm long and 1 to 2 mm broad, with a prominent midrib and pointed apex occurring free or attached to twigs. Upper surface dark green, waxy, lower pale green in color and somewhat mealy. Taste astringent; odour turbenthinate.

4 (3 samples from different traders)

Meethi Vach

Satwa

Paris pollyphylla J. E. Smith.

Root stock annulate, sometime as large as potato and are odour less

Rubia cordifolia Linn.

The drug occurs as hard woody pieces of cylindrical root up to 1 cm in diameter. Outer surface of root is smooth, faintly striated longitudinally and rusty brown in colour. Surface of transversely cut root shows a closely adhering bark followed by reddish brown tissue full of minute pores and a hollow center.

5 (3 samples from different traders)

Manjistha

Manjistha

6 (2 samples from different traders)

Kapur kachri

Karchura

Hedychium spicatum Buch-Ham

Occurs as whole rhizome or its transversely cut pieces. The rhizome is tuberous, divided into nodes and internodes, reddish brown in colour, with round root scars or rootlets attached at the some places. In market it occurs with a mealy white and starchy cut surface.

7 (3 samples from different traders)

Sikakai

Soptala

Acacia concinna DC.

Pod thick, succulent, strap shaped straight, 3 to 4 by ¾ in., depressed between the seeds, the broad sutures narrowed to a short stalk.

Nardostachys jatamansi DC.

Stout, woody, frequently arched rhizome, 2.5 to 8 cm long and up to 1 cm thick, dark grey in colour; densely covered with reddish brown silky fibers which are matted together to form a network. Odour strong, with valerianaceous note; taste aromatic.

Cinnamomum tamala Nees

Ovate-oblong coriaceous leaves, strongly three nerved from the base, up to 18 cm long and 6 cm broad. Upper surface leathery, shining olive green in colour. Lower surface rough, bluishgreen. The leaf becomes hard and brittle on drying. Odour resembling that of cinnamoum; taste aromatic and astringent.

8 (3 samples from different traders)

9 (2 samples from different traders)

Jataman si

Dalchini

Jatamansi

Tamalaka

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Table 2. Contd.

10 (2 samples from different traders)

11 (2 samples from different traders)

12

13

14 (4 samples from different traders)

15 (3 samples different traders)

16 (3 samples from different traders)

17 (4 samples from different traders)

18 (4 samples from different traders)

Gur Vach

Bhadra

Acorus calamus Linn.

The whole rhizome is sub-cylindrical or laterally compressed, up to 20 cm long and 1 to 5 cm broad, having a light brown colour. The upper surface shows triangular leaf scars, while lower surface bears small raised circular remnants of roots.

Dry globular fruits, around 2 cm in diameter. Outer surface shriveled, soapy translucent, golden brown or light brown in colour. There is a heart shaped scars of grayish white or yellowish colour on the attached side. The fruit encloses a loose round or oval seed of black colour.

Retha

Phenila

Sapindus Gaertn.

mukorossi

Indrayan seed

MahendraVaruni

Citrullus Schrad.

colocythis

Tagar

Sameva, Tagar

Daruhaldi

Daruharidr a

Timur

Pappali

Kutki

Amla

Tumburu

Tikshnata ndula

Katuka

Adiphala, Amalki

Dry light and spongy piece of fruit. Outer surface smooth, yellowish brown in colour in which a number of seed are embedded. The dry pulp breaks into thin flakes. The seed ovoid, flattened, grayish brown in colour. Odour none.

Valeriana wallichi DC.

The drug occurs as sub-cylindrical, slightly arched and unbranched rhizomatic rootstock, yellowish brown to dark grey in colour with prominent ring like scars. The under surface is covered with thin tubular roots. Odour strong.

Berberis aristata DC.

Stem hard pale yellow hard, distinctly radiated with medullary rays. Bark pale yellow brown, closely and deeply furrowed and internally smooth.

Zanthoxylum Roxb.

Small spherical fruits of dark brown colour, upto 7 mm in diameter. The outer surface is covered with prominent oily tubercles, frequently dehiscing half way exposing an oily globular seed of shining black colour lined with white papery membrane. Odour acrid and aromatic.

alatum

Piper longum Linn.

Fruit cylindrical, blunt, straight twisted or slightly curved spikes with a rough beady surface of grayish brown or greenish black colour. Odour strong and spicy taste aromatic and pungent, leaving burning sensation in the mouth.

Picrorhiza kurroa Royle

Straight or slightly arched, cylindrical rhizome. Outer surface, grey or creamish brown in colour, bearing impressions of round root scars and numerous scales. Odour faint agreeable; taste very bitter and long lasting.

Emblica Gaertn.

Pieces of dry sub hexagonal fruit with hard fleshy and a wrinkled surface of yellowish brown or grayish green colour. The drug often contains triangular seeds of yellowish brown colour. Odour mid characteristic; taste acidic and astringent.

officinalis

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Table 2. Contd.

19 (2 samples from different traders)

20 (4 samples from different traders)

21 (3 samples from different traders)

Bidarikan d

Nepali chirayita

Kuth

Vidarikand

Kairata

Kustha

Pueraria tuberose DC.

Partially dry tuber or its dried transversely sliced pieces. The tuber is globular or pear shaped. The outer surface of the tuber is yellowish brown in colour, wrinkled and transversely fissured bearing beady spots. The pulp is a white spongy, highly fibrous tissue. Taste sweet mucilaginous. Sliced pieces are rough with creamish white fibrous pulp, which exfoliates in papery flakes.

Swertia chirayita (Roxb. Ex fleming) Karst.

Occurs as whole herb consisting of a short taproot, pieces of stem and leaves. The stems are topped with branched corymbose panicles bearing dry flowers or shining brown or dark green capsules. Stem cylindrical at lower and middle portion but bluntly quadrangular in upper parts having a smooth surface of rust brown or purplish green colour. Leaves broadly lanceolate , cordate at base. Tate bitter.

Sassurea lappa C. B. Cl

Dry root slightly arched or twisted with a rough surface of dull or light brown colour brown colour bearing longitudinal wrinkles and small tubercles. Surface of transversely sliced root is brownish white in colour with a thin ring representing periderm, followed by a woody portion with fine radial striations and central pith, which is hollow in some older roots.

22 (2 samples from different traders)

Sarpagan dha

sarpgandh a

Rauvolfia serpentina Benth ex Kurz.

Stout, cylindrical, tortuous, somewhat crooked roots, occurring whole or as transversely cut pieces. The external surface is rough, longitudinally fissured, yellowish brown in colour, bearing occasional root scars. In older roots, the bark becomes thick, corky and friable, and constitutes a substantial part of root biomass. Taste very bitter.

23 (2 samples from different traders)

Pashan bheda

Pashan bheda

Bergenia ciliata (haworth) Sternb.

Rhizome solid, barrel shaped, cylindrical, 1.5 to 3 cm long, and 1 to 2 cm in diameter with small root scars brown in colour.

Tinospora cordifolia (Willd.)Miers ex Hook.f & Thoms

Cylindrical soft stem, outer surface, grayish brown to almost black in colour, longitudinally wrinkled, warty and covered with raised lenticles. The bark peels off in thin papery flakes. Transversely cut surface of the stem shows a wedge shaped structure formed by radiating medullary rays. Very bitter taste.

Aegle marmelos Correa ex Roxb.

Peeled and dried unripe fruits cut into halves or quarters, the cut surface show a hardened sticky or glutinous pulp of light orange or honey colour, having a number of radially arranged cellsin each of which an oblong, compressed and hairy seed is embedded. Odour-fainty aromatic, taste mucilaginous, astringent and agreeable.

24 (3 samples from different trader)

25

Guduchi

Bail

Guduchi

Bilva

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Table 2. Contd.

26 (3 samples from different traders)

Rohini

kampillaka

Mallotus philippinensis (Lam.) Muell.-Arg.

The drug occurs as course resinous powder of crimson or brick red colour consisting of numerous spherical glands, thick walled stellate hair and cellular fragments of cell wall. Taste gritty and oily, the powder is inflammable and resists admixture with water.

List of medicinal plant auctioned in Tanakpur forest development corporation Madi (Year2007-2008) (Year 2007 – 2008) 120 107.23 100

Rate (Rs.)

80

KMVN Pithoragarh

65.133

KMVN Champawat

60

Pithoragarh Van Vikas Nigam

45

44.75

42.58

Tanakpur Van Vikas Nigam

40

20

22.3 19 14.6

27.5 22

27.6 11.32

14

10 4.1

8

Ka rip at ta

G ur j

la Am

Jh ul a

Te jp at ta Ka pu rk ac hr Pa i sh an Bh Ro ed hi ni Po w de r Se m al Ph oo l

M

os s

gr as s

0

Crude Drug Name

Figure 2. List of medicinal plant auctioned in Tanakpur forest development corporation Mandi.

(Swertia chirayata), lavender (Lavender angustifolia), sarpgandha (Rauvolfia serpentina), tagar (Valeriana wallichi), atish (Aconitum heterophyllum), kalihari (Gloriosa superba), jatamansi (Nardostachys jatamansi), bankakri (Podophyllum hexandrum), pangar (Aesculus hippocastanum), honeyplant (Ammi majus), holy thistle (Silybum marianum) and Herracleum candicans. Initially, HRDI had taken 7 Districts namely Uttarkashi, Chamoli, Dehradun, Nainital, Udham singh Nagar, Haridwar and Pithoragarh for cultivation of selected species under Agri Export Zone (AEZ). Availability of planting materials is the basic challenge in promotion of cultivation of medicinal plants in the state. To find out the solution, three herbal gardens at Muni-ki reti, Rishikesh, Selaqui (District Dehradun) and Mandal, Gopeshwar have been established by HRDI and establishment of many small nurseries and herbalgardens is being undertaken with the help of the Forest Department and others. However, during the course of study, it was observed that this was not being followed. Besides, these government agencies,

a number of traders, stockholders in the medicinal plant trade sector, also engage local people in collection of the medicinal plant. They get cash payment by selling medicinal plant. Due to this, the local collectors did not get this actual share due to the lack of right information about the price of the medicinal plant.After detail studies, it was observed that some of these crude drug like Nardostachys jatamansi, Picrorhiza kurroa, Arnebia benthami, Swertia chirata, Zanthoxylum armatum etc, are imported through Indo-Nepal borders.After collection of crude drug sample, to ensure correct identification (on Taxonomic bases) and authentication, detailed comparative studies of samples were carried out. And, it was observed that substituted and adulterated drug is also available in trade. Consequently, due to less availability of medicinal plants and to meet the growing demand of these plants, there is a gap in demand and supply that lead to adulteration and substitution for genuine material. Result obtained after critical study of the crude drug sample received for identification is shown in Table 3, 4,5

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Crude drug Traded in Tanakpur Mandi during 2008 400

350

300

Rates (Rs.)

250

200 375 350 150

300

300 275 200

100 170

65

46

80

60 20

27

35

40

40

26

48

35

25

40

20

15

15

Sa ta w ar C h a Ta r li s il a h P at ra Sa tw a M a Ka nj is p u tha rk ac hr i Si ka k Ja ta ai m an D si al ch G i ni ur va ch R et In ha dr ay a Sa n m ev D ar a uh al di Ti m u Pi r pp al i Ku tk i A Bi m la da N ep rik a al i C nd hi ra it M ed a Sa a la rp k Pa ga ri s h nd an ha B he da G ilo y B R oh ai l G in ir ip ow i de r

0

150

120

50

Crude drug nam e

Figure 3. Crude drug Traded in Tanakpur Mandi during 2008

and 6.

Daruharidra The root or root bark of Berberis aristata DC. is considered the true source of 'daruharidra' in Ayurvedic text. The root in this case is hard, cylindrical, and more or less krarled. Outer surface of this material is corky and greyishbrown. Berberis lycium Royle is a usual adulterant. It has smooth grayish white bark with deep yellow wood. The alkaloid content in this material is comparatively low.Part used: Root and root barkAction and uses: The drug is credited with diaphoretic, bitter tonic alterative and stomachic properties. It is used in the treatment of jaundice, hemorrhoids, urino-genital disorders and skin diseases. Chirayata S. chirata (Roxb ex Fleming) Karst. is an 'endangered'

plant in India. Stem of the plant is smooth, externally yellowish or purplish brown, it is quadrangular, it has bitter taste, and stem is porous. The market drug is mixed with other species Swertia angustifoloa Buch-Ham, having winged quadrangular stems, is less bitter and possesses no pith in their square stem Part uses: Whole plant. Action and uses: The drug is credited with bitter tonic febrifuge and stomachic properties. It is used in fever urinary disorders anorexia. Jatamansi Nardostachys jatamansi DC. is the true source of Jatamansi, it has dark grey rhizome crowned with reddish brown tufted fibrous basal portions of petioles in old radical leaves. Roots of various species of Selinum are used as jatamansi. The root in this case is covered with stiff leaf base of dusty green color which has camphoraceous odour. Part used: Root stockAction and uses: The drug is credited with sedative antispasmodic and anti-arrhythmic

Arya et al

Figure 4. A) Market sample of Chirayata (Swertia chirata (Roxb ex Fleming) Karst); B) Original sample of Chirayata (Swertia chirata (Roxb ex Fleming) Karst); C) Market sample of Jatamansi (Nardostachys jatamansi DC.); D) Original sample of Jatamansi (Nardostachys jatamansi DC.); E) Original sample of Hemvati vaca (Paris polyphylla J.E. Smith); F) Market sample of Hemvati vaca (Paris polyphylla J.E. Smith); G) Market sample of Indrayan seed (Citrullus colocynthis Schrad); H) Original sample of Manjistha (Rubia cordifolia Linn); I) Market sample of Manjistha (Rubia cordifolia Linn.); J) Market sample of Daruharidra (Berberis aristata DC); K) Original sample of Daruharidra (Berberis aristata DC.).

Table 3. Major trade companies in Tanakpur.

S. no 1 2 3 4 5 6 7

Trading company Agrawal Trading Company; Ward No- 1 Tanakpur Ratan Lal and Sons; G. B. Pant Marg, Tanakpur Distt. – Champawat. Jagdish Narayan Hari Mohan, Ward No- 2, Tanakpur Distt – Champawat. Himalayan Jari Booti Centre, Bus Station Road, Tanakpur A. S. Sarda Enterprises, Nehru Marg, Tanakpur Distt. – Champawat Sharda Brothers, Tanakpur Uttarakhand Forest Development Corporation (UAFDC), Aamwala Depot Punagiri Road, Tanakpur

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Table 4. List of herbal material auctioned from Tanakpur Forest Development Corporation Mandi year 2007-2008.

Medicinal plant name Moss Grass -do-doJhula -do-doAmla -doTejpatta -doKapurkachri Pashan Bhed Rohini Powder Semal Phool Gurj Karipatta

Rate (Rs.) 19/22.30/14.60/107.23/42.58/65.133/27.50/22/27.60/44.75/11.32/14/45/4.10/10/8/-

Collected by KMVN Pithoragarh KMVN Champawat Pithoragarh Van Vikas Nigam KMVN Pithoragarh KMVN Champawat Pithoragarh Van Vikas Nigam KMVN Pithoragarh KMVN Champawat KMVN Pithoragarh Pithoragarh Van Vikas Nigam Pithoragarh Van Vikas Nigam -doTanakpur Van Vikas Nigam -do-do-do-

Table 5. Crude drugs traded from the Tanakpur mandi.

S. no.

Trade name

Botanical name

Local name

Part used

Rate

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Satawar Charila Talish patra Satwa/Meethi vach Manjistha Kapur Kachri Sikakai Jatamansi Dalchini Gurvach Retha Indrayan Sameva Daruhaldi Timur Pippali Kutki Amla Bidarikand Nepali Chiraita Kustha Sarpgandha Pashan Bheda Giloy Bail giri Rohini power

Asparagus racemosus Willd Parmelia perforata Taxus baccata Linn Paris polyphylla J.E Smith Rubia cordifolia Linn Hedychium spicatum Buch-Ham Acacia concinna DC Nardostachys jatamansi DC Cinnamomum tamala Nees & Ebern Acorus calamus Linn Sapindus mukorossi Gaertn Citrullus colocynthis Schrad Valeriana wallichi DC Berberis aristata DC Zanthoxylum alatum Roxb Piper longum Linn Picrorhiza kurroa Royle ex Benth. Emblica officinalis Gaertn. Pueraria tuberosa DC. Swertia chirata Buch-Ham Saussurea lappa C.B. Cl Rauwolfia serpentine Benth ex Kurz Bergenia ciliata (Haworth) Sternb. Tinospora cordifolia (Willd) Miers ex Hook Aegle marmelos Correa ex Roxb Mallotus philippinensis Muell Arg.

Satawar Charila Talish patra Satwa Manjistha Kapur kachri sathi Sikakai Jatamansi Dalchini Vach Retha Indrayan Tagar, Sameva Kilmora, Daruhaldi Timur Pippali Kutki Amla Bidarikand Chiraita Kuth Sarpgandha Pashanbhed Giloy, Gurj Bail giri Rohini

Root Whole plant Leaves Rhizome Stem Rhizome Fruit Root Bark Rhizome Fruit Seed Root Stem Fruit Fruit Root Fruit Tuberous root Whole plant Root Root Rhizome Stem Fruit Fruit Power

300/65/46/350/60/20/27/275/35/40/40/120/170/26/48/150/375/35/25/300/300/200/20/15/15/80/-

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Table 6. Sample name with part used and substitute adulterant.

S. no. 1 2 3 4 5 6

Sample name Methi voch, satwa Manjistha Indrayan Seed Daruharidra Nepali Chiraita Jatamansi

Part Rhizome Stem Seed Stem Whole plant Root

Sample expected as Paris polyphylla J.E. Smith Rubia cordifolia Linn Citrullus colocynthis schrad Berberis aristata DC Swertia Chirata Buch-Ham Nardostachys jatamansi DC

properties. It is used in high blood pressure, palpitation of heart hysteria.

Substitute/adulterant Acorus calamus Linn Rubia cordifolia Linn stem Trichosanthes palmata Roxb Berberis lycium Royle Swertia angustifolia Buch-Ham Selinum sps.

Rubia cordifolia Linn is the truly traded manjistha; outer surface of root is smooth, rusty brown, and is faintly striated longitudinally. The drug is adulterated with pieces of stem of R. cordifolia Linn which are 4 angular, scabrid angles. Part used: Root Action and uses: The drug is credited with alterative expectorant and emmenagogue properties. It is used for curing cough, rheumatism and hepatic affections.

medicinal and aromatic plants in Uttarkhand in general, and Tanakpur in particular. It revealed the importance of market studies on crude drugs and to the primary processing industries where value addition is done, and the chain of flow of raw materials up to the mandis. The marketing opportunities and means of accessing the market have also been sorted out in the study from which both planning and operation of marketing can be developed. Detail market information makes the market more transparent, so that the traders can make more informed on choice of product and sale. It will also help in availability of genuine crude drug material in these markets, and it further strengthens the potency of herbal product as well as improves the efficacy of the indigenous health care practices.

Hemvati vaca

ACKNOWLEDGEMENTS

Paris polyphylla J. E. Smith rootstock is annulate, sometimes as large as a small potato and is substituted by Acorus calamus Linn, rhizome is woody branched, light, aromatic with distinct nodes and internodes, nodal region are broad with leaf scars. Part used: - Rhizome Action and uses: Rhizome is used as anthelmintic and tonic.

Authors are grateful to the Director General, CCRAS, New Delhi, and Head, Botany, D.S.B. Campus, Kumaon University, Nainital for providing necessary facilities during the tenure of the work.

Manjistha

Indrayan seed Citrullus colocynthis Schrad is truly traded Indrayan seed which is compressed ovoid, yellowish seeds, odour slight, intensely bitter. The drug is adulterated by Trichosanthes palmata Roxb whose seed are reddish brown and compressed. Part used: Fruit Action and uses: Colocynthis is in moderate dose, drastic hydrogogue cathartic, diuretic, in large doses emetic, and gastro intestinal irritant. It is used in puerperal disorders, abortifacient, and dropsy; oil from seed is useful in hair growth.

Conclusion The

present studies show the market strategies of

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Journal of Medicinal Plants Research Vol. 6(18), pp. 3445-3449, 16 May, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1760 ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

Morin, a flavonoid, prevents lysosomal damage in experimental myocardial ischemic rats Khalid S. Al-Numair, Govindasamy Chandramohan*, Chinnadurai Veeramani and Mohammed A. Alsaif Department of Community Health Sciences, College of Applied Medical Sciences, King Saud University, P.O. Box 10219, Riyadh 11433, Saudi Arabia. Accepted 7 March, 2012

The present study was designed to investigate the preventive effect of morin on lysosomal enzymes in isoproterenol (ISO) treated myocardial infarcted rats. Myocardial ischemia was induced by subcutaneous injection of ISO hydrochloride (85 mg/kg BW, twice at an interval of 24 h) for two consecutive days. The morin (40 mg/kg BW) was administered daily for 30 days and subsequently two th th doses of ISO administered on 29 and 30 days. The activities of lysosomal enzymes β-glucuronidase, β-N-acetylglucosaminidase, β-galactosidase, cathepsin-B and D were increased significantly (P 0.05) change in the level of serum total proteins, it significantly increased the serum albumin concentration, as compared to control rats. Administration of either of the extracts to HFD fed rats had no effect on these two parameters (Table 2). The serum and kidney superoxide dismutase (SOD) activity as well as catalase activity in liver and kidney of untreated HFD fed rats were significantly (p