PHARMACOGNOSY MAGAZINE (PHCOG MAG.) · Sajjad Khan. M, Salma Khanam, Deepak M and Shivananda B.G....

PHARMACOGNOSY MAGAZINE (PHCOG MAG.)

An official publication of Phcog.Net

Volume 2, Issue 8, Oct-Dec, 2006

Pharmacognosy Network Worldwide (Phcog.Net)

Al-Ameen College of Pharmacy, Bangalore, Karnataka, India.

www.phcog.net

Abstracted and Indexed in Index Copernicus, CABI, Indian Science Abstracts, Chemical Abstracts (CAS), Cambridge Scientifc Abstracts (CSA), Medicinal and Aromatic Plant Abstracts (MAPA), Google Scholar

[PHCOG MAG. ISSN: 0973-1296]

PHCOG MAG.publishes high quality Medicinal Plant Research papers.

Easier & faster manuscript handling and &

One of the highly indexed publication in medicinal plants research

<<SUBMISSIONS

< QUERIES < REVISED MANUSCRIPT SUBMISSIONS

<PROOFS < ACCEPTANCE LETTERS

ALL IN JUST ONE E-MAIL !!!

All Communications with just a send click to

[email protected]

PHARMACOGNOSY MAGAZINE (PHCOG MAG.)

A Peer Reviewed Publication of Phcog.Net

Volume 2, Issue 8, Oct-Dec, 2006

EDITOR–IN-CHIEF:

SHIVANANDA B.G.

Dept. of Pharmacognosy, Al-Ameen College of Pharmacy,

Bangalore, India

ASSOCIATE EDITORS

MUEEN AHMED K. K.

Dept. of Pharmacognosy, Al-Ameen College of

Pharmacy, Bangalore, India

ARUN KUMAR H. S.

Dept. of Cardiovascular Sciences Bioscience Institute

University College Cork Cork, Ireland

MUHAMMAD NABEEL GHAYUR

Department of Medicine, McMaster University,

Ontario, Canada.

EDITORIAL BOARD MEMBERS

HUKKERI.V.I (India), JERRY COTT (USA), DIXIT V.K. (India), RASHEED ZAFAR (Jordan), SYED RAFATULLAH (Saudi Arabia), RAINA R. (India), LUZ MARIA SANCHEZ (Cuba),

RANA A.C. (India), RAFI KHAN (Papua New Guinea), RULA DARWISH (Jordan), SALMA KHANAM (India), ANNIE SHIRWAIKAR (India), CHRISTOPHER WIARTE (Malaysia),

GANAPTHY.S (India)

TEXT EDITORS

VANAJA. K. (India) & BHUVANESHWARI. J (India)

COPY EDITORS

GOVINDARAJAN. R. (India) & CAMILA GAMBINI PEREIRA (Brazil)

EDITORIAL OFFICE

The Editor, PHCOG MAG. Al-Ameen College of Pharmacy,

Hosur road, Bangalore 560 027, Karnataka, India e.mail : [email protected] ; website : www.phcog.net

[PHCOG MAG. ISSN: 0973-1296]

Abstracted and Indexed in Index Copernicus, CABI, Indian Science Abstracts,Chemical Abstracts (CAS), Cambridge Scientifc Abstracts CSA), Medicinal and Aromatic Plant Abstracts (MAPA), Google Scholar

SPONSORS

Al- Ameen College of Pharmacy, Opp. Lal bagh Main Gate, Hosur road, Bangalore 560 027 www.alameenpharmacy.edu

Association of Pharmaceutical Teachers of India H.Q : Al- Ameen College of Pharmacy, Hosur road, Bangalore 560 027 www.aptiindia.org

Contents Inside • Editorial – Plant science and its noble contribution – Dr. Arun kumar H.S

Peer review process – PHCOG MAG.

• PHCOG MAG.: Letters to the editor

• PHCOG MAG.: General Article

• Fuzzy logic and modern analytical tool coupling in comprehensive evaluation of traditional and

complementary medicine.

K. Jayaram Kumar

• PHCOG MAG.: Review Article

Herbal options for Contraception: A Review

Absar A. Qureshi, Dhirendra B. Sanghai and Padgilwar S.S.

• PHCOG MAG.: Research Article

Inhibition of nitric oxide and interferon-� production by iridoids and triterpenes from the roots of

Himatanthus sucuuba,

Marinete S. de Siqueiraa, Milade dos S. Cordeiroa, Elaine C. Rosasb, Maria das Graças O. M.

Henriquesb, Antonio C. Sianib


In vitro and In vivo inhibitory effects of Piper longum fruit extracts on mouse Ehrlich ascites

carcinoma

K.K. Hullatti, Uma.D.Murthy, B.R. Shrinath


Development of HPTLC method for estimation of charantin in herbal formulations

Patel P.M., Patel K.N., Patel N.M. and Goyal R.K.


Cost effective medium for callus initiation from Catharanthus roseus leaves

Namdeo A.G. Mahadik R.R and Kadam S.S.


Analgesic and Anti-Inflammatory activities of Lagenaria siceraria Stand. Fruit juice extract in rats

and mice

B.V. Ghule, M.H. Ghante, A.B. Upaganlawar and P.G. Yeole


Anti-inflammatory activity of Plumbago capensis

Charles Dorni A.I., Vidyalakshmi K.S., Hannah R.Vasanthi., Rajamanickam G.V and Dubey G.P


Hypoglycemic and antihyperglycemic effect of alcoholic extract of Euphorbia leucophylla and its

fractions in normal and in alloxan induced diabetic rats

Satyanarayana T, Katyayani B.M., Hema Latha E., Anjana A. Mathews, Chinna Eswaraiah M.


Antioxidant activity of a new diarylheptanoid from Zingiber officinale

Sajjad Khan. M, Salma Khanam, Deepak M and Shivananda B.G.


Evaluation of Anti-inflammatory activity of Nyctanthes arbor-tristis and Onosma echioides.

Amrite Omkar, Thurackal Jeeja and Gadgoli Chhaya

• INFORMATION FOR AUTHORS –PHARMACOGNOSY MAGAZINE [PHCOG MAG.]

• List of Reviewers – PHCOG MAG.

BRINGING GLOBAL MEDICINAL PLANT RESEARCHERS TOGETHER: PHCOG.NET

PHARMACOGNOSY MAGAZINE

ABOUT PHCOG MAG. [ISSN: 0973-1296] Published quarterly, Pharmacognosy Magazine (PHCOG MAG.) serves the need of different scientists and

others involved in medicinal plant research and development. Each issue covers different topics in natural

product drug discovery, and also publishes manuscripts that describe investigations, clinical reports,

methods, techniques and applications of all forms of medicinal plant research and that are of broad

readership interest to users in industry, academia, and government. Phcog Mag. is a must-read magazine for

medicinal Plant researchers —and it's an Open Access Publication.

PHCOG.NET – Contact details

Prof. B. G. Shivananda

Director, Phcog.net &

Editor, Phcog Magazine

Dept. of Pharmacognosy,

Al-Ameen College of Pharmacy,

Hosur road, Bangalore, Karnataka, India

Ph: 0091-80-22234619

Fax: 0091-80-22225834

Mueen Ahmed K. K.

Project Co-ordinator, Phcog.net &

Associate Editor, Phcog Magazine

Dept. of Pharmacognosy,


Hosur road, Bangalore, Karnataka, India

Ph: 0091-9845655732:

E.mail: [email protected]

Subscription details

Subscription cost : Rs. 1000/- per year (4 issues/year)

Send D. D/ Cheque in favour of “The Principal, Al-Ameen College of Pharmacy”

Payable at Bangalore, Karnataka, INDIA.

Send your subscription request along with D.D or Cheque to following address

Prof. B.G. Shivananda,

Editor, Phcog mag.

Al-Ameen College of Pharmacy, Opp. Lalbagh Main Gate,

EDITORIAL EDITORIAL EDITORIAL EDITORIAL

PLANT SCIENCE AND ITS NOBLE CONTRIBUTION

This month the scientific community is overwhelmed about the announcement of Nobel Prize in Medicine to the discovery of RNAi technique, which has revolutionized the way researchers have adopted to silence genes. Further refinement and understanding of this technique will pave way for novel category of therapeutics in future. I am delighted to share with our readers that much before RNA interference was well characterized, the phenomenon was well known in plant genetics to reduce gene expression and was referred as post transcriptional gene silencing, transgene silencing and quelling. Single-stranded antisense RNA was introduced into plant cells and hybridized to the homologous single-stranded "sense" messenger RNA and the

resulting dsRNA is responsible for reducing gene expression. RNA interference is distinct from other gene-silencing phenomena in that the RNAi based gene silencing is inheritable from cell to cell in plants, worms and as well as in eukaryotes. Hence, I would not be wrong in mentioning that plant base research work has continued to contribute directly or indirectly to the development of medical sciences and hope and trust such fundamental work in plants is also recognized by the Nobel foundation some day. One of the major hindrances in the development of most developing and underdeveloped countries is the population explosion. Although several allopathic contraceptive pills are available, many affordable and safe alternatives are intensively investigated and the Mother Nature is at the forefront with a solution. Compiling the herbal options for contraception, Qureshi et al., systematically review the plants with anti-fertility activity in the current issue. Refinement in Natural Product research needs time tested and robust purification/quantification procedures and HPTLC is one such technology gaining increasing utility in Pharmacognosy research. Adopting this technology Patel et al., report the utility of HPTLC for estimation of Charantin, which is phytoconstituent present in Momordica charantia, possessing hypoglycemic activity. The reported assay would be an important tool in the quality control of antidiabetic polyherbal formulations consisting of Charantin. Also reported in this issue is the “hypoglycemic and antihyperglycemic effect of alcoholic extract of Euphorbia leucophylla” by Satyanarayana et al. Incidence of diabetes is on constant rise globally and so are the associated complications. Hence search for phytoconstituents with antidiabetic activity and their evaluations techniques are timely and need of the hour. Pain and inflammatory disease are yet another therapeutic area wherein natural product research has continued to contribute effective drugs. In the current issue, Ghule et al., Dorni et al., and Omkar et al., report anti-inflammatory activity of Lagenaria siceraria fruit juice extract, Plumbago capensis, Nyctanthes arbortristis and Onosma echioides respectively. The Editorial team of Phcog Mag is indeed delighted to bring in these fascinating and interesting sciences to its readers and we hope you do enjoy reading this issue. We are open to your suggestions, comments and critic. Arun Kumar HS Associate Editor (Phcog Mag)

Dr. ARUN KUMAR H. S. is a post doctoral fellow at Dept. of Cardiovascular Sciences, Bioscience Institute,

University College Cork, Cork, Ireland

PHCOG MAG.: Peer review Process

Pharmacognosy Magazine - (ISSN 0973-1296: PHCOG MAG.) is a quarterly publication of Phcog.Net, the first issue was released on January 1st, 2005 (for more details: www.phcogmag.com). PHCOG MAG. (An Official Publication of Phcog.Net) is an open access publication series of Phcog.Net, which comprises of contributions to Medicinal Plant research with special reference to Phcog.Net and certain aspects of information with prior knowledge of editorial board. It publishes Special Article FOCUS (App. 5000 words), Short reviews (App. 3000 words), Clinical reports on a Plant (App. 2000 words), Recent advances in Assay methods and techniques (App. 5000 -6000 words), Plant review (App. 1000 words) and Research articles. All submitted articles are subjected to peer review. Phcog Mag being subjected to indexing in different databases, contributions and manuscripts with high quality of scientific content and language submitted exclusively to PHCOG MAG. would undergo possible reviewing. Manuscripts not meeting the scientific standards will not be considered for the reviewing process. Editors will be asking referees to advice on

the scientific merit as well as the likely appeal the paper will have for broad Natural Product researcher’s readership. Editors usually give reviewers 21 days to complete their review process. Although editors always hope for a quick turnaround, this is not always possible. However, editors will be in contact with the referees once paper is sent to them, with weekly reminders of their due date. Once all the reviews are in-house, the Editor handling the manuscript will most likely make a decision within a day or two. The editor will then contact the corresponding author with the decision. The entire review process of the articles submitted to PHCOG MAG is done online and digitally. Reviewers invest precious time in the belief that they are making important contributions to the scientific process. Author’s criticism or negative comments on reviewer’s comments will be subjected to the cancellation of publication. PHCOG MAG follows communication only through e.mail. Authors can also submit their articles/manuscripts either in Document or Plain ASCII text format through e. mail (see below)

Prof B. G. Shivananda Editor-in-Chief, PHCOG MAG. Dept. of Pharmacognosy, Al-Ameen College of Pharmacy, Hosur road, Bangalore, Karnataka, India

Mueen Ahmed K K [email protected]

Arun Kumar H .S [email protected]

Nabeel Ghayur [email protected]

Associate Editors, PHCOG MAG.

Submitted Manuscript Assignment of manuscript Number & Reviewers

Reviewer 1 Reviewer 2/3 Comments (if any)

Sent to Authors for possible revision On receipt of revised manuscript

Editorial reviewProof Copy to authors

Final publication

Pharmacognosy Magazine ISSN: 0973-1296

Vol 2, Issue 8, Oct - Dec, 2006 200

PHCOG MAG.An official Publication of Phcog.Net

PHCOG MAG.: Letters to the editor ARE MULTIPLE NAMES MANDATORY TO REFER A VEGETABLE CRUDE DRUG? Using too many names (trade names, common names and vernacular names) to refer to a single vegetable crude drug and single name to refer more than one crude drug, is a common practice in India, which leads to confusion, deception and also gives room for fraudulent activities especially in the crude drug industry. The crude drugs are the basis for the drug preparations of the major Indian systems of medicines such as Siddha, Ayurveda and Unani. The ambiguous names may cause adultaretion which in turn lead to harmful/side effects to the health of the public who depend on the Indian systems of medicine for various health disorders/diseases. To correct the existing problems, the existence of too many names to refer to a single crude drug and single name applied to more than one crude drug should be streamlined and some selected names for each and every vegetable crude drug must be introduced. This is essential since the common public, crude drug dealers, medicine men and others cannot understand the botanical names which are applied to every single plant. India is not only a home of many languages but also rich in medicinal plants. Naming is an art, which is as old as humanity. Without a name, one cannot communicate the things that are in use. ‘Too much of anything is good for nothing’ cautions a proverb. It is applicable in this case also. Say for example, under the trade name ‘Gaozaban’ (Bengali & Hindi names), a well known Unani drug is sold in the market, which is derived from 5 different plants/plant parts viz. Onosma bracteatum Wall, Anisomeles indica Kuntze, A. malabarica R. Br., Caccinea glauca Savi and Macrotomia benthami DC. It is not clear as to which crude drug, out of these five crude drugs is genuine or this name is applied to. Like this, there are many crude drug samples

that are sold in the crude drug market under the single name. Since, the crude drugs are linked with the healthcare of people, unauthenticated/adulterated crude drug may create health disorders or it may even be fatal. Recently Government of India has set up a Department viz. AYUSH (Ayurveda, Yoga and Naturopathy, Unani, Siddha and Homeopathy) under Ministry of Health and Family Welfare, which is a gateway for information of all these systems and also regulates them. As per the Indian Systems of Medicine (ISM), there are three major systems in practice. They are Siddha, Ayurveda and Unani. The basic literature of these systems is available in Tamil, Sanskrit and Arabic languages respectively. Therefore, it is suggested that single name representing a system/language to refer a single crude drug may be made mandatory. In addition to that, Hindi - the national language of India spoken by majority of the people in India and English - an international language spoken by majority of the people in the world may also be considered as mandatory. Altogether, each and every crude drug that is sold in the Indian crude drug market will have five different names. In the light of the above facts, it is suggested that department of AYUSH should come forward to constitute a committee comprising of eminent/experienced persons representing different Indian systems of medicine such as Siddha, Ayurveda and Unani to carefully examine the existing names/synonyms etc., that are in current usage. They can assign some selected names (mandatory names) to each crude drug. This will establish a specific penta-nomial (5 names) system for every single crude drug, which is derived from 5 different languages such as Tamil, Sanskrit, Arabic, Hindi and English. The proposed system is just like the binomial (two names) system that are available for every single plant species

based on ICBN (International Code of Botanical Nomenclature), where there is no confusion in the dealing of plants.

Exceptions may be given in some cases. Say for example, if a plant is endemic to a particular area or region in India, a local language or vernacular name may be considered as mandatory language along with the proposed five names. An example is given below for easy understanding; Cycas beddomei Dyer is endemic to Cuddapah and Tirumala hills in Andhra Pradesh. In such case, the local name or vernacular name of this plant, ‘Perita’ in ‘Telugu’ may be considered as mandatory to refer this particular plant species, apart from the penta-nominal system. While selecting a valid trade/vernacular names, preference should be given for names that are in use for a very long period (popular/familiar names) or names having specific meaning. Also the names that are short, simple, easy to remember and quite meaningful may be preferred over other ordinary names. The proposed system will streamline the vegetable crude drug industry to certain extent; thereby the existing confusion, fraudulent activities, repetitions etc. could be checked on one hand and on the other, this will ensure a healthy life to the people of India who depend on Indian systems of medicine for their healthcare.

A.B.D. SELVAM Pharmacognosy Unit, Botanical Survey of

India, Howrah – 711103.

[email protected] *******

CAN THE TERM ORGANOLEPTIC BE CONSIDERED EQUIVALENT TO ZOOLOGICAL? Pharmacognosy is an interdisciplinary subject concerned with organoleptic, botanical, physical, chemical and biological (pharmacological) characters of crude drugs. Of the above-mentioned five parameters, except organoleptic, the remaining four parameters represent the four different branches of




Science such as botany, physics, chemistry and pharmacology respectively. The term organoleptic literally means impression on sense organs viz. eyes, nose, tongue, ears and touch. The organoleptic study is a qualitative method where worker applies sight, smell, taste, touch and hearing to record the characteristic features of crude drugs. In this study, size, shape including external and internal colours and markings, fractures, texture, odour and taste are noted down. The methodology of this study is generally known as Organoleptic and the data so obtained from this study is referred to as Sensory characters.

Since Organoleptic study is totally based on the sense organs of human beings, who are an important member of animal kingdom, the term Zoological seems to be more appropriate for this study than the term Organoleptic in a broader sense. Further, the term Organoleptic is less familiar as compared to Zoological.

In the light of the above mentioned facts, it is suggested that the term ‘Organoleptic’ may be renamed as ‘Zoological’ followed by sensory characters within brackets for the better understanding, i.e. Zoological (Sensory characters). In addition, the proposed term is expected to bring uniformity among the other

pharmacognostic parameters by representing one of the branches of Science (Zoology) on one hand and familiarity on the other. It is further suggested that before renaming the term ‘Organoleptic’ as ‘Zoological’, it could be discussed among the pharmacognosists to arrive at a suitable conclusion in this regard. Once, the renaming is accepted, it may be written as Zoological (Sensory characters) as is adopted in the case of pharmacological studies, which is often written as Biological (pharmacological).

A.B.D. SELVAM Pharmacognosy Section, Botanical Survey

of India, Howrah – 711103. [email protected]

PLANTS OF THE QUR’AN - (Edition 2003 ) ISBN-81-900290-9-6

(Illustrated by Plant Photographs and Drawings)

MEDICINAL PLANTS OF PROPHETIC TRADITIONS (Edition 2004) ISBN-81-2900290-5-3 (Medicinal, Food and Aromatic Plants) (Illustrated by Plant Photographs and Drawings)

By Dr. Mohammad Iqtedar Husain Farooqi

(Retd Dy Director (Scientist) & Head, Plant Chemistry Division, National Botanical Research Institute, Lucknow, INDIA) Secretary, Urdu Scientific Society, C-3/2 Shahid Apartments, Golaganj, Lucknow-226018 Tel; 9839901066, 0522-2610683S; Email: [email protected]

(Some of the plants described in the above two books include Cedar, Date Palm, Olive, Black cumin, Manna (Plant Product), Camphor Plant, Euphorbia, Costus, Senna, Aloes etc)

The books referred to above contain scientific descriptions (Botany, Chemistry & Medicinal Properties) of Medicinal, Food and Aromatic Plants mentioned in Quran and Sunnah with relevant references of Verses and Ahadith. (Prophetic Sayings). Eminent Islamic scholars and scientists have extensively reviewed both the books worldwide. Magazines, Journals and Newspapers including Economic Botany, New York Times, have published detailed reviews. These books are considered as the best and most authentic works on the subject and important addition to scripture botany. Internationally known Islamic thinker Late Maulana Abul Hasan Ali Nadwi, Founder-Member, Rabita Al-Islami, Makkah Mukarramah, has written scholarly Preface for both the books and the renowned Botanist Dr. S.K. Jain (Former Director, Botanical Survey of India, Calcutta) has contributed the Introduction to the books.

Sidrah Publishers C-3/2 Shahid Apartments, Golaganj Lucknow-226018, India Tel; 0522-2610683, 9839901066 E-mail: [email protected] or [email protected]

Or write to Dr. M. I. H. Farooqi Shahid Apartments, Golaganj,Lucknow-226018 Tel; 9839901066, Email: [email protected]

Price (in India): Plants of Quran : Rs160/-(PB) & Rs 200/-(HB) Medicinal Plants of Prophet Muhammad: Rs 150/-(PB) & Rs 200/-(HB).




PHCOG MAG.: General Article Fuzzy logic and modern analytical tool coupling in

comprehensive evaluation of traditional and complementary medicine.

K. Jayaram Kumar Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra. Ranchi-835215. India.

Correspondence -

Traditional medicines (TM) and complementary and alternative medicines (CAM) are attracting more and more attention within the context of health care provision and health sector reforms. W.H.O is promoting its inclusion in health policies because of its positive features, which include diversity and flexibility; accessibility and affordability in many parts of the world; broad acceptance among many populations in developing countries; comparatively low cost and growing economic importance. With out critical assessment of what should be integrated and what should not, we risk developing a health care system that costs more, is less safe and fails to address the management of chronic disease in publicly responsible manner (1). The priority areas of research according to the committee on Science and Technology to the house of Lords, include • Effects of each individual therapy: efficacy, safety

and cost effectiveness. • Research into mechanisms of action of individual

therapies including patterns of response to treatment.

• Research into TM/CAM genre itself, including social research into motivation of patients seeking TM/CAM and usage patterns of TM/CAM

• Research into new strategies which are sensitive to the TM/CAM paradigm

• Research into efficacy of diagnostic methods used • Research into implementation and effects of

TM/CAM in specific health care settings (2). In the present scenario the quality control of herbal medicines mainly concentrate on use of modern analytical tools include droplet counter current chromatography (DCCC), preparative thin layer chromatography (PTLC), centrifugal thin layer chromatography (CTLC), overpressure layer chromatography (OPLC), gel filtration, vacuum liquid chromatography (VLC), preparative high performance liquid chromatography (PHPLC), etc. The parameters

studied usually like total ash value, Acid insoluble ash. Alcohol soluble ash, assay for total alkaloids, glycosides, total volatile oils etc. Can these parameters alone decide the fate of TM/CAM medicines practiced for many decades? Ayurveda (in India), Kampo (in Japan) etc and in almost all the countries have their own traditional system of medicine have been used successively for centuries. For example in Ayurveda the treatise Charaka samhita dates to 7th century B.C. The classification of medicinal plants found in Charaka samhita can be grouped under 3 main categories • Naama-Roopa jnaana (Identification,

Pharmacognosy) • Guna Karma Jnaana (Pharmacology) • Proyoga jnaana(Application, Pharmaceutics, and

Pharmacodynamics ) For example the pharmacological aspects are explained on the basis rasa (taste), guna (properties) and Karma (action) (7). Most of the standards in TM/CAM are expressed in linguistic terms and the knowledge is passed to students by teacher by experiential learning. Do these methods have no significance in the standardization of TM/CAM medicines? Traditional knowledge has to be studied and understood with reference to its foundation. Use of intelligent techniques like fuzzy logic and neural logic can be effectively used to scientifically present explanation given by experts in traditional system in scientific terms. The characteristics of fuzzy logic which is put to use it in the present context of standardization is as follows i) Fuzzy systems are suitable for uncertain or approximate reasoning, especially for the system with a mathematical model that is difficult to derive, ii) Fuzzy logic allows decision making with estimated values under incomplete or uncertain information. Fuzzy logic has been applied to many different types of problems since introduced by Zadeh in 1965. Unlike Boolean logic, fuzzy logic is suited




to evaluating subjective situations. For agriculture, the subjectivity of fuzzy logic is particularly appealing (3-5).

Field conditions – weather, the position and intensity of the sun and dust, just to name a few – and crop conditions – size, shape, weed and pest pressure – combine to create a difficult situation for conventional evaluation methods. For example, given a pile of fruit, we can separate apples and oranges. If we are looking at an apple X, our description might include red, a hint of green and a mostly round shape. If we look at an apple Y, the description might include green, smooth and oblong. An orange might have an orange color, a rough texture and round shape as descriptors. While we can verbally describe apples and oranges, it’s much more difficult to create a mathematical description of an apple or orange that will apply for all situations. The variability common in agriculture makes fuzzy logic appealing. In classification of TM medicine fuzzy logic modern analytical tool coupling can be successively used. The %w/w of phytoconstiutents and also linguistic variables can be given as input and different rules are made to connect these variables to get a known output. Mamdani and sugeno fuzzy rule based systems can be used in classification. For example in figure- I mamdani fuzzy rule based system is used where fifteen inputs are connected with a set of thirty five rules to classify the samples into five varieties.

Figure- I A model fuzzy inference system editor.

This will result in increased recording and preservation of indigenous knowledge of herbal pharmaceuticals including development of digital libraries. With development of sophisticated H.P.L.C equipment where fractionation is done automatically if there is arrangement where information regarding the status of the herbal drug is indicated it helps in authentication of the plant and also identification of it from adulterants and substitutes. Through fuzzy logic this can be achieved and we can get a comprehensive evaluation of TM/CAM medicine both with regards to active constituents and also clinical evaluation. References 1. Jones WB. Alternate medicine- learning from the

past, examining the present, advancing to the future. Journal of the American Medical Association, 280 (18):1616-1618(1998).

2. House of Lords. United Kingdom. Select Committee on Science and Technology. Session 1999-2000. 6th Report. Complementary medicine and Alternate Medicine .London, Stationary Office.2000 (HL Paper 123)

3. Ribeiro, R.A. Fuzzy evaluation of the thermal quality of buildings. Computer-Aided Civil and Infrastructure Engineering, 14: 155-162 (1999).

4. Wang, J. and V. Allada. Fuzzy logic approach for serviceability evaluation. Intelligent Engineering Systems through Artificial Neural Networks, 6: 1075-1080(1996).

5. Zhang, Q. and J.B. Litchfield. Knowledge representation in a grain drier fuzzy logic controller. J. Agric. Engg Res. 57: 269-278(1994).

6. Zhang, Q. and J.B. Litchfield. Applying fuzzy mathematics to product development and comparison. Food Technology: 108 – 115(1991).

7. Venugopal S N. Medicinal plants of Caraka samhita – A close up”. Amruth, 6 :5-14 (2001).




PHCOG MAG.: Review Article Herbal options for Contraception: A Review Absar A. Qureshi*, Dhirendra B. Sanghai and Padgilwar S.S.

S.G.S.P.S. Institute of Pharmacy, Hingna Road, Kaulkhed, Akola- 444 004 (M.S.), India. * For Correspondence - [email protected]

ABSTRACT - Due to existing and overwhelming growth rate of world population, oral contraceptive have become need of the time. But steroids have various side effects. This forced us to review the existing options of plants having anti-fertility activity. Plant plethora is rich source of plants having anti-fertility activity. In this review we have also covered the plants having anti-fertility activity by different mechanism in both male and female. KEY WORDS: Anti-fertility, abortifacient, anti-implantation, spermicidal, estrogen. INTRODUCTION The extraordinary growth of the world population stands as one of the significant events of the modern era to think over. The current world population is around 6.46 billion and that of India in particlualar is around 1.1 billion (1). One of the critical problems of the developing countries like India is its geometrical increase in human population. Today we understand that our sheer numbers have increased so much that they are straining Earth’s capacity to supply food, energy and raw materials. Advances in medicine and public health have led to a significant decrease in mortality and an increased life expectancy. This population explosion will have negative impact on our economic policies and would simultaneously misbalance our socio-economic infrastructure. Thus the control of human fertility in the sense of its limitation is the most important and urgent of all-biosocial and medical problem confronting mankind today. Contraception is literally the prevention of conception, but generally is taken to mean the prevention of pregnancy (2). The development of new fertility regulating drug from medicinal plants is an attractive proposition, because from times immemorial humans have relied on plants and their products as sources of drugs and therapeutic agents, although in recent times, synthetic drugs are used extensively in modern medicine. However many modern medicines are developed through the clues obtained from phytochemicals. More over the phytochemicals even today are important resources for medicine. The plant products are becoming more popular than the synthetic drugs. In recent times it is mainly attributed to their low toxicity and long standing experience of exposure of these drugs in ethnic medicine system like Ayurveda.

Family planning has been promoted through several methods of contraception, but due to serious adverse effects produced by synthetic steroidal contraceptives, attention has now been focused on indigenous plants for possible contraceptive effect. Although contraceptives containing estrogen and progesterone are effective and popular, the risks associated to the drugs have triggered the need to develop newer molecules from medicinal plants. Hence, there is a need for searching suitable product from indigenous medicinal plants that could be effectively used in the place of pills. All combination oral contraceptives (COCs) contain both an estrogenic compound and a progestin. Over the years, the amounts and types of these components have changed in attempts to lower side effects and improve efficacy. Mechanism of steroid hormone action Estradiol, like other steroids, is thought to exert its action directly on the nucleus of the cell. As a consequence, an estrogen-response tissue must have estrogen receptor and nuclear acceptor sites to which activated receptor can bind. Upon entry into the cytosol of the cell (by diffusion), estradiol is bound to a specific receptor (ERc). In the cytoplasm, the estrogen receptor complex is activated (ERn) and translocated to the nucleus. This complex binds to acceptor sites in chromatin and enhances processes associated with differentiated functions of the responsive tissue, which include the production and utilization of messenger and other classes of RNA needed for the synthesis of constituent enzymic and secretory proteins, as well as the receptor itself. In some cells replication of DNA is also stimulated, followed by cell division. The concentration of estrogen receptors in most tissues is constitutive, but in some instances it is increased by estradiol. The




concentration of progesterone receptors in the uterus and other progesterone response tissues is markedly increased by estrogen. In fact, one of the recognized actions of estradiol is to stimulate synthesis of

progesterone receptors. The induction of progesterone receptors with estrogen can explain the synergistic action of these two hormones on the uterus.

Methods of contraception

Hormonal control of fertility The most effective method of contraception, the birth control pill, is based on oral administration of steroids. Estrogens and progestins are used either combined or, as with the “minipill”, progestins are used alone. In addition, various combinations of steroids can also be administered as long-acting injectable preparations

or via intrauterine systems. The pills to be effective via the oral route, estradiol and progesterone cannot be used since they are metabolized in the gastrointestional tract and liver. As a consequence, synthetic estrogens such as mestranol (50-100 µg/day) or ethinyl estradiol (20-50 µg/day) are

Desire for Contraception

Permanent No Yes

Sterilization Male/Female

Female Couple Male

Hormonal Contraceptive

Barrier Contraceptive

Intrauterine Device

Post-coital Contraception

Natural Family

Planning

Condom/ Spermicide

Combination Pills

Progestin Pill

Norplant Depo Provera Progesterone Copper T

Diaphragm Cervicalcap Condom Spermicide




used in combination with various synthetic progestins, such as norethindrone, norethindrone acetate, norgestrel, ethinodiol diacetate or norethynodrel (0.3 – 100 mg/day). The hormones are given in a cyclic fashion for 21 days, beginning on day 5 of the menstrual cycle, followed by 7 days of placebo treatment or no pills. The elevated estrogen and progestin levels inhibit the midcycle LH surge and ovulation by exerting negative feedback effects on the hypothalamus. Irregular LH peaks are sometimes observed, while FSH levels are usually suppressed. Ovarian progesterone production is diminished, but estrogens continue to be secreted. The effects on the endometrium are variable and depend on the type and dosage of the contraceptive. Rapid progression from proliferation to early secretory changes can be observed within a few days from the start of daily intake, followed by regressive changes. Secretory activity is either minimal or absent. The pregnancy rate for combined pills is approximately 2%. Risks The reason why some women may be reluctant to take combination oral contraceptives (COCs) consistently and correctly is a fear of possible adverse effects. Cardiovascular disease Historically, combination oral contraceptives have been associated with increased risks for myocardial infarction and stroke. Overall, oral contraceptives were found to multiply the effects of age and other risk factors for MI and stroke, rather than just add to them. Because cigarette smoking is far more prevalent among women of reproductive age than any of these other risk factors, it becomes by far the most important factor. Whereas early epidemiological studies of high-dose oral contraceptives found significantly increased risks of developing cardiovascular disease among users of combination oral contraceptives. Use of oral contraceptives by healthy women who do not smoke does not appear to be associated with an increased risk of either myocardial infarction or stroke. Hypertension As with the increased risks for MI and stroke, older formulations of combination oral contraceptives have been associated with significant elevations of blood pressure as well. The risk of hypertension appears to be much lower when estrogen and progestin doses are lowered. The mechanism for contraceptive-induced changes in blood pressure is still unclear, with alterations in plasma angiotensinogen and increases in sodium and water retention being noted. Although these are

primarily estrogenic effects, progestins may have a synergistic effect, as significant elevations in blood pressure have only been apparent in the combination products and not with either hormone alone. Thrombosis As doses of estrogenic were lowered to less than 50 µg, a marked drop in the incidence of fatal and nonfatal pulmonary embolism was noted, thus implying an estrogen dose-related effect. As with the other concerns for myocardial infarction, stroke, and hypertension, patient selection remains the most important method of reducing the incidence of these adverse effects. Women who are already at high risk for cardiovascular problems (hypertension, smoking and older than 35 years, or diabetes with vascular complications) or have already had a cardiovascular or thromboembolic event should not use combination oral contraceptives. Hepatomas It may occur in women taking oral contraceptive, the most common of which are focal nodular hyperplasias and liver cell adenomas. Hepatocellular cancer was also felt to be associated with combination oral contraceptives use (3). Here it is clear that estrogen and progesterone play a crucial role in anti-fertility activity but not without the serious side effects. So in this background we have reviewed some of the plants having anti-fertility activity in both male and female. Following is the list of plants available for anti-fertility activity with their parts used and somewhere mechanism of action to understand this activity. Alangium salviifolium The family Alangiaceae consists of twenty-two species out of which A. salviifolium (Linn.f) Wang is mainly used as medicine in India, China and Phillipines (4). Different parts of this plant are reported to possess acrid, astringent, emollient, anthelmintic, diuretic and purgative properties. It is also used externally in acute case of rheumatism, leprosy and inflammation. Applied externally and internally in case of rabid dog bite. Root bark is an antidote for several poisons. Fruits are sweet, cooling and purgative and used as a poultice for treating burning sensation and heamorrhage period (5). Daily administration of petroleum ether, ethyl acetate, chloroform, methanol or aqueous extracts of A. salvifolium at a dose of 100mg/kg body weight for eight days starting from the first day of pregnancy showed significant abortifacient activity in comparison to vehicle treated group. Interestingly, except petroleum ether and ethyl acetate extracts, all the extracts showed no anti-implantation activity. Eight




days of drug treatment lead to resorption of fertilized ovum as noticed by red spots in the horns of uterus. Among the extracts, chloroform extract was found to be least effective followed by petroleum ether extract. Methanol extract showed total resorption sites in two animals. Aqueous and ethyl acetate extracts have also shown good activity. These results indicate that A. salviifolium (Linn.f) Wang produced mainly abortifacient activity and less antiimplantation activity. It indicates that the herbal drugs may have anti-progesterone effect (6). Aloe vera Twenty samples of fresh ejaculate donated by healthy volunteers ranging in age from 20-30 years were obtained. Lyophilized A. barbadensis at concentrations of 7.5% and 10% proved to be spermicidal due to the multiple microelements (boron, barium, calcium, chromium, copper, iron, potassium, magnesium, manganese, phosphorus and zinc) which were toxic to the tail causing instant immobilization. These results suggest the possibility of using lyophilized A. barbadensis as a new, effective and safe vaginal contraceptive (7). Aristolochia tagala Aristolochia, a large genus of shrubs, rhizomatous perennial herbs often twining, is distributed in tropical and termerate regions of the world. A. tagala is a perennial herb highly prevalent in Himalayas, Bihar, Assam and southwards in forest cleanings. The root of the plant is reported to contain aristalochic acid, which possesses tumor-inhibiting activity and has been used in the treatment of cancer, snakebite and helmenthiasis (8). Preliminary phytochemical studies revealed that the ethanol extract showed the presence of alkaloids, saponins, flavonoid glycoside, steroids and phenolic compound. The ethanol extract of A. tagala showed significant reduction in the number of corpora lutea and increase in the number of resorptions in comparison to the control. The extract showed 72% antifertility activity on oral administration of 100mg/kg whereas a remarkable 100% antifertility activity resulted on the administration of 200mg/kg as compared to the untreated control group (9). Azadirachta indica Male albino rats were administered orally 100 mg/kg A. indica leaf powder with or without testoesterone. Suitable controls were maintained. Damaged semineferous tubules and abundance of vacuoles of varying size were observed in A. indica treated rats. The germ cells showed overall decrease in cytoplasmic ground substance. Leydig cells exhibited

characteristics of degeneration with condensed nuclei. Total count of spermatocytes, spermatids and Leydig cells were reduced. The cell and nuclear diameter of spermatogonia, spermatocytes, spermatids and Leydig cells were also reduced. From the result obtained, it can be observed that effects of Azadirachta indica on the testis are possibly due to gonadotrophic hormone deficiency, caused directly or indirectly (10). Neem oil proved spermicidal against rhesus monkey and human spermatozoa in vitro (11). In vivo studies showed that intravaginal application of neem oil prior to coitus can prevent pregnancy (11). Antifertility effect of neem oil has also been studied and suggested to be a novel method of contraception (12-14). Oral administration of aqueous extract of neem leaf also shows antifertility effect in mice (15). Purified neem seed extract has also been demonstrated to abrogate pregnancy in both baboons and bonnet monkeys, when administered orally (16). From the hexane extract of neem seed, an active fraction containing six components has been found to completely abrogate pregnancy in rodents when given orally up to a concentration of 10%, with no apparent side effect (17). Biophytum sanctivum Different extracts of B. sanctivum were tested for their antifertility activity. Ethyl acetate and n-butanol extracts significantly inhibited pregnancy in 4/6 rats with mean number of implants 3.0±1.92(P≤0.05). It is also understood that the chloroform and the ethanol extracts significantly inhibited pregnancy in 5/6 rats with a mean number of implants of 2.0±1.23(P≤0.05) and 6/6 rats with mean number of implants of 0.00±0.00 (P≤0.05), respectively (18). Calotropis procera 90% ethanolic and aqueous extracts of roots of C. procera produced on female Wister rats temporary and reversible modification on oestrous cycle characterized by absence of oestrous and metaoestrous phases and dioestrus stage prolonged. Therefore, extracts provoked inhibition of ovulation with consequent reduction of cyclicity (19). Ethanolic extract of roots shows 100% anti-implantation activity at the dose of 250mg/kg (20). Cardiospermum helicacabum Ethanolic extract of whole plant at a dose of 250mg and 500mg/kg body weight /day orally for day 1 to 7 of pregnancy showed significant decrease in the implantation sites. Animals treated with 500mg/kg body weight showed 60% inhibiton of implantation sites.




Table 1: Plants with anti-fertility activity.

Sl. No Common Name Botanical Name Family Parts used Action 1 Ankota Alangium salvifolium Alangiaceae Stem bark Abortifacient, anti-implantation 2 Kumari Aloe vera Liliaceae Latex Spermicidal 3 Nallayiswari Aristolochia tagala Aristolochiaceae Whole plant Anti-implantation 4 Neem Azadirachta indica Maliaceae Seed, oil Spermicidal, abortifacient 5 Lajalu Biophytum sanctivum Oxalidaceae Leaves Anti-implantation 6 Akada Calotropis procera Ascrophluariaceae Roots Anti-implantation 7 Kanphuti Cardiospermum

helicacabum Spindaceae Whole plant Anti-implantation, increase uterus weight, inhibit

sperm motility and decrease sperm count 8 Papai Carica papaya Cariaceae Seeds Inhibit sperm motility and decrease sperm count 9 Amaltas Cassia fistula Caesalpiniaceae Seeds Anti-fertility

10 Haldi Curcuma longa Zingiberaceae Rhizomes Anti-implantation, inhibit sperm motility 11 Akasbel Cuscuta reflexa Convolvulaceae Stem 12 Parvel Cyclea burmanni Menispermaceae Roots Resorption, estrogen effect 13 Jamun Eugenia jambolana Myrtaceae Flowers Decrease sperm count 14 Chobehyat (Pookwood) Guaiacum officinale Zygophyllaceae Aerial parts Abortifacient

15 Jangli-arandi Jatropha curcus Euphorbiaceae Fruits Abortifacient 16 Tulsi Ocimum sanctum Labiatae Leaves Decrease sperm count and sperm motility,

Abortifacient 17 Utranajutuka Pergularia daemia Asclepiadaceae Twig Anti-implantation, late abortifacient 18 ---- Stevia rebaudiana Compositae Whole Plant Decrease sperm count




After confirming the antiimplantation activity of ethanol extract, the extract was subjected for its estrogenic/antiestrogenic studies in immature overiectomised albino rats. The dose increase in uterine weight was observed (21). Carica papaya Manivannan et.al reported the structural changes in the testis and epididymis of rats followed by the treatment with the benzene chromatographic fraction of the chloroform extract of the seeds of C. papaya. Administration of benzene chromatographic fraction of the chloroform extract of the seeds of C. papaya at a dose of 10mg/rat/day for 150days showed a total inhibition of sperm motility, reduced sperm count and infertility. It is concluded that the inhibition of sperm motility by the drug could be due to other epididymal factors rather than the subcellular characteristics of the testis and epididymis (22). Udoh et.al has reported the activity of the alkaloid extract of C. papaya seeds on male reproductive physiology in rats. Each male rat was treated with C. papaya seed extract (10, 50 and 150mg/kg/day) daily for three days for fecundity study, semen analysis and testis histopathology respectively. Twenty male rats treated with C. papaya seed extract (10, 50 and 150mg/kg/day), abstained from sex for 7 days and divided into 4 groups were mated with fertile female rats. Another set of 30 male rats, divided into 4 groups, treated with the seed extract (10, 50 and 150mg/kg day-1), respectively, was used for semen analysis and testis histopathology. The results showed that the oral administration of C. papaya seed extract prevented ovum degeneration, and induced testicular cell lesion. These observations led to the conclusion that the C. papaya seed extract oral administration could induce reversible male infertility (23). Cassia fistula Oral administration of aqueous extract of seeds of C. fistula to mated female rats from day 1-5 of pregnancy, at the doses of 100 and 200 mg/kg body weight resulted in 57.14% and 71.43% prevention of pregnancy, respectively, whereas 100% pregnancy inhibition was noted at 500mg/kg body weight (24). Curcuma longa Petroleum ether and aqueous extracts of turmeric rhizomes show 100% antifertility effect in rats when fed orally (25). Implantation is completely inhibited by these extracts (26). Curcumin inhibits 5a-reductase, which converts testosterone to 5a-dihydrotestosterone, thereby inhibiting the growth of flank organs in hamster (27). Curcumin also inhibits human sperm

motility and has the potential for the development of a novel intravaginal contraceptive (28). Cuscuta reflexa Methanolic extract of C. reflexa stem has significantly increased the carbonic anhydrase activity in the uterus of mice. This is associated with elevated level of prosgesterone, which is supporting the antifertility effect of the plant (29). Cyclea burmanni The petroleum ether and chloroform extracts of the roots of C. burmanni have been found to possess significant antifertility effect in rats. Both these extracts exhibited partial and complete resorption of implants at 200 and 400 mg/kg body weight dose levels. In estrogenic activity study, both the extracts increased uterine weight and caused opening and cornification of vagina in immature rats. The present work justifies its effectiveness in preventing pregnancy in all rats when administered at 400 mg/kg p.o. (30). Eugenia jambolana Flowers of E. jambolana significantly decreased the fertilizing capacity of the male albino rats without any significant change in body or reproductive organ weight. It causes significant reduction in conversion of spermatogenesis at the early stages of meiosis leading to decrease in sperm count without any abnormality to spermatogenic cells, leydig interstitial cells and sertoli cells (31). Guaiacum officinale The hot aqueous extract of aerial parts caused abortion in the second and third trimesters only. At a dose of 480.75 mg/kg, the extract did not reduce the litter size in mice when given during the first trimester of pregnancy (32). Jatropha curcus Fruits when fed to the female rats at a dose of 3.3% of the diet exhibited 100% effect. Seeds, when fed to female rats at a dose of 3.3% of the diet exhibited 100% effect (33). Foetal resorption was observed with methanol, petroleum ether and dichloromethane extracts of fruits of J. curcus indicating the abortifacient property (34). Ocimum sanctum Treatment of albino rats with a benzene extract of O. sanctum leaves (250mg/kg body weight) for 48 days decreased total sperm count, sperm motility, and forward velocity. The percentage of abnormal sperm increased in caudal epididymal fluid and the fructose content decreased in the caudal plasma of the edpididymis and the seminal vesicles (35). Histological and biochemical studies on mice fed with leaves




showed evidence of mild impairment of spermatogenesis with significant reduction of seminal pH. There was also a decrease in the reducing substance, acid and alkaline phosphatases and mucoproteins. Treated male mice failed to fertilize females of proven fertility (36). The benzene extract of the leaves (100mg/kg) showed antifertility effect in 80% rats whereas the petroleum ether extract showed the effect in only 60% animals. The extract did not reveal an abortifacient activity (37). O. sanctum leaves have been reported to show abortifacient and antifertility activity. The aqueous extract at a dose of 100mg/kg showed antiimplantation action (38). Pergularia daemia Golam Sadik et.al. revealed that oral administration of the ethanol extract of P. daemia at a dose of 600, 400 and 200 mg/kg body weight daily was found to terminate pregnancy in the pre-implantation stage in mice. But when the dose was reduced to 100mg/kg-body weight, pregnancy occurred in 20% of the mice. and demonstrated that the extract was able to prevent implantation or cause resorption depending upon the dose. To identify the bioactive fractions, the steroidal fraction was separated from the ethanol extract and was tested for antifertility activity in mice. The fraction at a dose of 200mg/kg-body weight exhibited antifertility activity in mice at preimplantation stage. The possible cause of termination of pregnancy upon oral administration of the ethanol extract and its steroidal fraction from day 1 to day 9 of pregnancy might be due to antizygotic, antiblastocytic as well as antioestrogenic property. The ethanol extract also showed late abortifacient activity in the mice. In this case pregnant mice was treated with the extract at a dose of 600 and 800 mg/kg body weight daily for any two consecutive days from 12 to 13 of pregnancy, respectively. The former dose produced 50% abortifacient activity with 50% mortality as two of the mice died after oral administration of the extract. The extract at a dose of 600 mg/kg body weight exhibited 100% abortifacient activity without any mortality. The mice aborted all the fetuses within 48 hours of drug treatment. The present investigation clearly demonstrates that the ethanol extract and its steroidal fraction are able to prevent fertilization in the female mice (39). Stevia rebaudiana Aqueous extract of S. rebaudiana (Compositae) was fed orally to 3 groups of adult male Wistar albino rats for a period of 65 days at the doses of 100, 1000 and 5000 mg/kg body weight. A control group was also

maintained. The results revealed decrease in the epididymal sperm count, plasma testosterone concentration and decrease in organ body weight ratio of testis and cauda epididymis in the group fed with 5000 mg/kg body weight as compared to saline fed control group. This study revealed the anti-fertility effect of S. rebaudiana in adult male Wistar albino rats (40). DISCUSSION It is well known fact that estrogenic substances inhibit pregnancy by suppressing the level of both follicular stimulating hormone (FSH) and luteinizing hormone (LH) which in turn prevent the implantation. Estrogen and progesterone are the hormones responsible for histological and functional modifications of female genital tract. The exogenous administration of physiological doses of estrogen, in sexual immature rats, stimulated histoarchitecture of uterus (41). According to Laurence (42) any compound possessing estrogenic activity may exhibit antifertility activity, they act by suppressing gonadotrophin secretion, with consequent inhibition of ovulation. In immature female rats, hence the antiimplantation activity of these plants may be due to an imbalance in endogenous estrogen and progesterone levels. The loss of implantation caused by the extracts may be due to their anti-zygotic, blastocytotoxic or antiimplantation activity as described by Hafez (43). Isoflavones along with coumarins (also flavonoids) and lignans belong to a class of substances known as non-steroidal phytoestrogens, they produce infertility in animals (44). According to Miksicek (45) several commonly occurring flavonoids mimic the biological effects of 17 β-estradiol by virtue of their ability to bind to and activate the nuclear estrogen receptor. The primary way in which a woman can prevent microbial infection and pregnancy through intercourse is with effective vaginal contraceptives. However, formulations generally available are not as effective as some other birth control methods. So that spermicidal activity was also included in the present study to check that whether the plants can be used in the formulation of vaginal contraceptives. The results of the spermicidal activity tend to suggest that many plants showed spermicidal effect at lower concentration. It is interesting to note that use of herbal contraceptives generally did not lead to permanent sterility in rats, since discontinuation of the treatment allowed a prompt return to normal fertility. REFERENCES 1. http://www. esa. un. Org. Accessed- May 22, 05.




2. E.T. Herfindal, D.R. Gourley, Text Book of Therapeutics, Drug and Disease Management. 7th ed. Philadelphia, Lippincott Williams & Wilkins; 2019-25 (2000).

3. B.R. Mackenna, R. Callander, Illustrated physiology 6th ed. London, Churchill Livingstone; 225-45 (1997).

4. K.R. Kirtikar, B.P. Basu, Indian Medicinal plants, Vol II, Dehradun, International book distributor; 1236-39 (1987).

5. P.K. Warrier, Nambiar, Ramakutty, Indian Medicinal plants a compendium of 500 species, Vol I, Madras. Orient Long man Ltd.; 67 (1964).

6. V. Murugan, H. Shareef, G.V.S Rama Sarma, M. Ramanathan, B. Suresh. Anti-fertility activity of Alangium salvifolium. Indian Journal of Pharmacology. 32: 388-89 (2000).

7. Nandan Kumar Jha, I.K. Pandey, Anup Kumar Jha. Aloe vera: Ghritkumari. Phytopharma. 6(6): 03-22 (2005).

8. Anonymous. The wealth of India: Raw Materials, Vol 1. New Delhi, Publication and Information Directorate CSIR; 422 (1985).

9. S. Balaji, P. Pethiah Raj, J. Thomas and K. Asok Kumar. Antifertility activity of ethanol extract of Aristalochia tagala leaf. Indian J. Phrama. Sci. 66(6): 834-36(2004).

10. R. H. Aladakatti, R. N. Ahmed. Azadirachta indica A. juss induced changes in spermatogenic pattern in albino rats. J. Nat. Remed. 6(1): 62-7 (2006).

11. K.C. Sinha, S.S Riar, R.S Tiwari, A.K.Dhawan, J. Bardhan, P. Thomas, A.K. Jain and R.K. Jain. Neem oil as vaginal contraceptive. Indian J. Med. Res. 79: 131–36 (1984).

12. Kausik Biswas, Ishita Chattopadhyay, Ranajit K. Banerjee, Uday Bandyopadhyay. Biological activities and medicinal properties of neem (Azadirachta indica). Current sci. 82(11): 1336-45 (2002).

13. S. Upadhyay, S. Dhawan, M.G. Sharma and G.P. Talwar. Long term contraceptive effects of intrauterine neem treatment (IUNT) in bonnet monkeys: An alternative to intrauterine contraceptive device. Contraception. 49: 161–69 (1994).

14. S.N. Upadhyay, C. Kaushic, G.P. Talwar. Proc. R. Soc. London B, 242: 175–79 (1990).

15. V. Y. Despande, K. N. Mendulkar and N. L. Sadre. Male antifertility activity of Azdirachta indica in mice J. Postgrad. Med. 26: 167–70 (1980).

16. S. Mukherjee, N.K. Lohiya, R. Pal, M.G. Sharma and G.P. Talwar. Contraception. 53: 375–78 (1996).

17. S. Mukherjee, S. Garg and G. P. Talwar. Early post implantation contraceptive effects of a purified fraction of neem (Azadirachta indica) seeds given orally in rats. Possible mechanism involved. J. Ethnopharmacol. 67(3): 287–96 (1999).

18. D. B. Johnson, C. Dinesh Kumar, K. R. Arunkanth, D. Giles, M. Gopal and V. G. Hubert. Antifertility activity of Biophytum sanctivum. Indian Drugs. 40(9): 523- 25 (2003).

19. Clara Circosta, Rokia Sonago, Francesco Occhiuto. Effects of Calotropis precera on oestrous cycle and on oestrogenic functionality in rats. IL Framco.56: 373-78 (2001).

20. V.K. Jagdish, A.C. Rana. Preliminary study of antifertility activity of Calotropis procra roots in female rats. Fitoterapia. 73: 111-15 (2002).

21. R. Dhanwad, M. G. Patil, S. B. Patil and N. D. Satyanarayan. Antiimplantation activity of Cardiospermum helicacabum Linn. (Sapindaceae) in albino rats. Indian Drugs 42(11): 726-30 (2005).

22. B. Manivannan, P.K. Mishra, N. Pathak, S. Sriram, S.S. Bhande, S. Panneerdoss and N.K. Lohiya. Ultrastructural changes in the testis and epididymis of rats following treatment with the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya. Phytother. Res. 18(4): 285-89 (2004).

23. F.V. Udoh, P.B. Udoh and E.E. Umoh. Activity of alkaloid extract of Carica papaya seeds on reproductive in male wistar rats. Pharm. Biol. 43(3):563-67 (2005).

24. R. Yadav, G.C. Jain. Antifertility effects of aqueous extract of seeds of Cassia fistula in female rats. Adv. Contracept. 15(4): 293-301 (1999).

25. S.K.Garg. Effect of Curcuma longa (rhizomes) on fertility in experimental animals. Planta Med. 26: 225–27 (1974).

26. S. K. Garg, V. S. Mathur and R. R. Chaudhury. Screening of Indian plants for antifertility activity. Indian J. Exp. Biol. 16: 1077–79 (1978).

27. S. Liao, J. Lin, M.T. Dang, H. Zhang, Y.H. Kao, J. Fukuchi and R.A. Hiipakka. Growth suppression of hamster flank organs by topical application of




catechins, alizarin, curcumin, and myristoleic acid. Arch. Dermatol. Res. 293: 200–05 (2001).

28. T. Rithaporn, M. Monga and M. Rajasekharan. Curcumin: a potential vaginal contraceptive. Contraception. 68: 219–23 (2003).

29. U. K. Mazumdar, M. Gupta, D. K. Pal and S. Bhattachara. Induction of carbonic anhydrase by Cuscuta reflexa stem and Corchorus olitorius seed in mice. Indian J. Pharma. Sci. 65(4): 401-03 (2003).

30. S. K. Panda, S. K. Sahu, and G. K. Dash. Antifertility effect of Cyclea burmanni. Indian J. Pharm. Sci. 65(3): 305-07 (2003).

31. M. Rajasekaran, J. S. Bapana, S. Lakshmanan, A. G. Ramachandran Nair, A. J. Veliath and M. Panchanadam. Antifertility effect in male rats of oleanolic acid, a triterpine from Eugenia jambolana flowers. J. Ethnopharmacol. 24(1): 115-21 (1988).

32. N. V. Offiah and C. E. Ezenwaka. Antifertility properties of the hot extract of Guaiacum officinale. Pharm. Biol. 41(6): 454-57 (2003).

33. Nandan Kumar Jha, I.K. Pandey, Anup Kumar Jha. Jatropha curcus: Jangli Erand. Phytopharm 7(3): 3-9 (2006).

34. M. M. Goonasetera, V. K. unawwdana, K. Jayasena, S. G. Mohammed, S. Balasubramanium. Pregnancy terminating effect of Jatropha curcus in rats. J. Ethnopharmacol. 47(3): 117-23 (1995).

35. M. Ahmed, R.N. Ahmed, R.H. Aladakatti, M.G. Ghosesawar. Reversible anti-fertility effect of benzene extract of Ocimum sanctum leaves on sperm parameters and fructose content in rats. Basic Clin. Physiol. Pharmacol. 13(1): 51-9 (2002).

36. S. Kasinathan, S. Ramakrishnan, S.L. Basu. Antifertility effect of Ocimum sanctum L. Indian J. Exp. Biol. 10(1): 23-5 (1972).

37. S.K. Batta, G. Santhakumari. The anti-fertility effect of Ocimum sanctum and Hibiscus rosa sinensis. Indian J. Med. Res. 59(5): 777-81 (1971).

38. S.B. Vora, S.K. Garg, R.R. Chaudhaury. Antifertility screening of plants. Effect of six indigenous plants on early pregnancy in albino rats. Indian J. Med. Res. 57(5): 893-9 (1969).

39. Golam Sadik, M. A. Gafur, M. Shah Alam Bhuiyan, A. H. M. Khurshid Alam, M.Helal U. Biswas, Parvez Hassan, Abdul Mannan, M. Omar Faruk Khan and A. K. A.Chowdhury. Antifertility Activity of Pergularia daemia. The Sciences 1(1): 22-4 (2001).

40. S. Prasad, K. Jaykumar, Honnegowda, K. Narayana and S. Rao. Male reproductive toxicity of Stevia rebaudiana (bert.) in rats. Toxicol.Int. 12(1): 48 (2005).

41. L. Martin, C.A. Finn. The effects of intrauterine devices on preimplantation changes in the mouse uterus. J Endocrinol. 46(2): 19-20 (1970).

42. 42. D.R Laurence, A.L. Bacharach. Evaluation of Drug activities. London, Academic press; 808-09 (1964).

43. E.S.E. Hafez. Reproduction and Breeding Techniques for Laboratory Animals. Pheladelphia, Lea and Febiger; 16 (1970).

44. W.C. Evans. Pharmacognosy. 15th ed. London, WB Saunders Company Ltd; 247 (2001).

45. R.J. Miksicek. Commonly occurring plant flavonoids have estrogenic activity. Mol. Pharmacol. 44: 37-43 (1993).




PHCOG MAG.: Research Article Inhibition of nitric oxide and interferon-γγγγ production by

iridoids and triterpenes from the roots of Himatanthus sucuuba

Marinete S. Souzaa, Milade dos S. Cordeiroa, Elaine C. Rosasb, Maria das Graças O. M. Henriquesb, Antonio C. Sianib*

1Departamento de Química Orgânica, Instituto de Química, Fundação Universidade do Amazonas, Manaus, AM, Brazil; 2Laboratório de Química de Produtos Naturais e Farmacologia Aplicada, Far-Manguinhos, Fundação

Oswaldo Cruz , Rua Sizenando Nabuco 100, 21041-250, Rio de Janeiro, RJ, Brazil Corresponding Author : [email protected]

ABSTRACT – The iridoids β-dihydroplumericin (1), plumericin and isoplumericin were isolated from the roots of Himatanthus sucuuba, along with lupeol acetate and cinnamate. The isolated compounds and three different crude extracts from the roots exhibited in vitro immunoregulatory activity in the nitric oxide and interferon-γ inhibition assays, when tested below the cytotoxic concentrations. KEYWORDS – Himatanthus sucuuba, iridoid, triterpene, β-dihydroplumericin, immunoregulatory activity. INTRODUCTION The latex, the powdered dried bark, or the aqueous extracts of Himatanthus sucuuba Spruce (M. Arg.) Woodson. (Apocynaceae) (“sucuúba”, “belasco-capi”) are popularly used in the Amazonian countries (Brazil, Peru and Colombia) as agents for many purposes, such as vermifuge (anthelmintic), laxative, anti-tumoral, anti-fungal, anti-inflammatory, anti-anemic, anti-ulcer, analgesic, antitussive, aphrodisiac; and to treat arthritis, boils, hernias, swellings, skin tumors, gastritis and hemorrhoids (1,2). Such a plethora of somewhat conflicting ethnobotanical information (3) may lead to the supposition of this species as possessing general immunoregulatory ability. The iridoids fulvoplumierin, plumericin, isoplumericin and β-dihydroplumericinic acid as well as the triterpenes lupeol acetate, lupeol cinnamate and α-amyrin cinnamate have been isolated from latex and bark of Himatanthus sucuuba (3-5). Other compounds, as vanillic acid, p-coumaric acid, p-hydroxybenzoic acid, confluentic acid and 2’-O-methylpertaloic acid also have been isolated from the methanolic extract of the bark; the two latter compounds from an associated lichen (6). The present study reports the isolation of β-dihydroplumericin (11S-13-dihydroplumericin) (1), plumericine, isoplumericine and triterpenes from the roots of H. sucuuba. The immunoregulatory activity of these compounds as well as crude extracts from the bark was inferred by the ability to inhibit the in vitro production of nitric oxide and interferon-γ.

Roots of Himatanthus sucuuba were collected in the campus of the University of Amazonas, Manaus, in January 1995 and identified by the botanist A. F. Barbosa. A voucher specimen is deposited in the Herbarium of the Instituto de Ciências Biológicas da Universidade do Amazonas, Manaus, under No. 5436. All extracts were prepared by maceration at room temperature: n-hexane extract of the inner part of the root (extract A, 0.76%), acetone fraction (0.68%) from methanolic extract of the same (extract B, 3.2%), and methanol extract of the root bark (extract C, 8.6%). Extract yields were taken on dried plant basis. The triterpenes lupeol (0.52%), lupeol acetate (0.47%), and lupeol cinnamate (0.42%) were isolated from the hexane fraction of extract C, and β-dihydroplumericin was isolated from extract B (1.26%). All the compounds were purified by silica column chromatography. The mixture of plumericin and isoplumericin (0.39% total yield from extracts A and C) was separated by

O

COOCH3

O

O

OH

H

H

11

13

114

10

6

75

9

3

1




preparative TLC (silica), eluted twice with n-hexane:ethyl acetate 8:2. A large amount of a mixture of α- and β-amyrin cinnamates (35%) was also isolated from the chloroform extract of the hexane-defatted root bark, but it was not pharmacologically assayed. The isolated compounds were identified by spectroscopic methods and by comparing with data from the literature (3,7,8). 1H-NMR and 13C-NMR (including 2D) data for β-dihydroplumericin were obtained. As the source of ex vivo material, male Balb/c mice (20-30 g), from the CECAL-FIOCRUZ colony, were lodged in a room with controlled temperature and lighting, with free access to lab chow and tap water. All experiments were conducted in accordance with the ethical guidelines of the International Association for the Study of Pain (9) and the institutional guidelines for animal use. Cytoxicity was measured by the MTT method (10), and NO and IFN-γ productions were evaluated from murine macrophages and splenocytes respectively (11). All the values were obtained as a result of triplicate experiments. Peritoneal macrophages were harvested from mice injected 3 days before with 1 ml of 3% thioglycolate. The cell viability was determined by trypan blue and the viable cells (2.5 x 105 cells/well) were incubated in 96-wells

plates (2.5 x 106 cells/mL) with the samples in different concentrations during 20h. Stock MTT solution [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; 5 mg/mL; 22.5 µL/well] was then added, and the plates incubated at 37 °C during 4 h. DMSO (150 µL/well) was added and mixed thoroughly to dissolve the dark blue crystals and the absorbance was read at 540 nm in a plate reader (Biorad - 450). For evaluating the nitric oxide production, the macrophages obtained as described above, were incubated in a 96-wells flat bottom plate (2.5 x 106 cells/mL) and stimulated with or without IFN-γ enriched media plus lipopolysaccharide (30 ng/mL), in the presence of the samples at the non-cytotoxic concentrations. After 24 h, the supernatant was recovered and the nitrite production was determined by the Greiss method (12). For evaluating the interferon-γ production, the spleens cells were recovered from Balb/c male mice (13). The spleen mononuclear cells were isolated through Ficoll-Hypaque gradient and incubated (3 x 105 cells/well) with or without Concanavalin-A (5 µg/ml) plus the extracts or the pure compounds in different concentrations. After 72 h, the supernatant was recovered and used to analyze the INF-γ production by sandwich enzyme-liked immunosorbent assay (ELISA).

Table 1: Inhibition of nitric oxide and interferon-γγγγ production by extracts and isolated compounds from H.

sucuuba roots Sample* Cytotoxicity (%) NO inhibition (%) IFN-γ inhibition (%)

Extract A 0 83 0

Extract B 0 83 100

Extract C 0 84 4.5

Lupeol 0 14 62

Lupeol acetate 0 23 100

Lupeol cinnamate 0 7.0 100

Plumericin 0 80 100

β-Dihydroplumericin 100 µg 74.6 cytotoxic cytotoxic

β-Dihydroplumericin 10 µg 27.9 96 98

β-Dihydroplumericin 1 µg 22.5 2.5 92

*Macrophages were incubated with β-dihydroplumericin as liposomes in phosphatidylcholine (1:1 weight) in phosphate buffered saline (sonication at 50 Hz for 5 min), at the concentration of 50 mg/mL. The other samples were administered (100 µg/well) in Tween 20 (10 mg :1µL) suspended in PBS at a concentration of 50 mg/mL. This represents the first chemical study of H. sucuuba roots. Lupeol and its esters as well as plumericine and

isoplumericine, which were also reported in the bark and latex of H. sucuuba (5,8), were identified by




comparison of data with those reported in literature (3). β-dihydroplumericin is reported for the first time in this species. All three of the crude extracts A, B and C as well as plumericin inhibited NO production by more than 80%. Extracts A and C alone were not able to inhibit IFN-γ production. Samples that showed more than 30% of cytotoxicity were not further assayed. The activity shown by extracts A and B is probably related to the presence of plumericin and its geometric isomer. Extract A did not affect IFN-γ at the tested dose, probably due to an insufficient concentration of those compounds. Higher degrees of IFN-γ inhibition were observed with extracts rich in triterpene esters and iridoids. β-dihydroplumericin showed an evident dose-dependent immunoregulatory effect, when tested at doses below those toxic to the macrophages. β-dihydroplumericin was cytotoxic at 100 µg/mL and inhibited NO and IFN-γ at 10 µg/mL or higher (Table 1). Its occurrence may be associated with the high toxicity reported for the roots of H. sucuuba (14); the hydrogenation of the exocyclic C11–C13 double bond possible being responsible for enhancement in the cytotoxicity when compared to plumericin. In spite of the well-known chemical liability of the iridoids, the last five years have watched an increasing research involving the pharmacological activities of such compounds, either as glycosides or aglycone. Nevertheless, not many examples of immunoassays using these compounds are available in literature (15-17). β-Dihydroplumericin (1). White powder from CHCl3-MeOH; m.p. 150-152 oC (lit. 150-1 oC) [18]; IR bands (KBr): 1772, 1696, 1645, 1612 cm-1; 1H-NMR (200 MHz, CDCl3): δ 1.10 (t, J 7.3 Hz, H-14, 3H : methyl), 1.76 (m, H-13), 2.74 (dd, J 6.7, 1.8 Hz, H-11), 3.43 (dd, J 9.5, 5.8 Hz, H-9), 3.76 (s, 3H, COOCH3), 3.97 (td, J 9.5, 2.1, H-5), 4.40 (s, H-10), 5.58 (d, J 5.8 Hz, H-1), 5.66 (dd, J 5.5, 2.1 Hz, H-7), 6.08 (dd, J 5.5, 2.1 Hz, H-6), 7.43 (s, H-3); 13C-NMR (75 MHz, CDCl3): δ 176.5 (C-12), 166.6 (C-15), 152.7 (C-3), 141.4 (C-6), 126.4 (C-5), 108.6 (C-4), 106.0 (C-8), 101.4 (C-1), 86.7 (C-10), 53.8 (C-9), 51.6 (OMe), 48.8 (C-11), 37.9 (C-5), 22.7 (C-13), 11.9 (C-14). EI-GCMS m/z: 292 (M+, 30%), 231 (84), 203 (35), 193 (74), 175 (32), 139 (100). 13C and 1H-NMR data: corresponding to those reported for 1 isolated from Plumeria rubra var. alba (18) and Plumeria acutifolia (19); the β-orientation of the ethyl group being indicated by the H-10 singlet signal in 1H-NMR.

REFERENCES 1. P. Corrêa, Dicionário das Plantas Úteis do Brasil e

das Exóticas Cultivadas, (Imprensa Nacional, Ministério da Agricultura, Rio de Janeiro, 1975), vol. VI, pp.154-155.

2. L.F. Villegas, I.D. Fernández, H. Maldonado, R. Torres, A. Zavaleta, A.J., Vaisberg and G.B. Hammond. Evaluation of the wound-healing activity of selected traditional medicinal plants from Perú, J. Ethnopharmacol. 55(3): 193-200 (1997) and references therein.

3. Wood C.A., Lee K., Vaisberg A.J., Kingston D.G.I., Neto C.C., Hammond G.B. A Bioactive Spirolactone Iridoid and Triterpene from Himatanthus sucuuba. Chem. Pharm. Bull., 49(11): 1477-1478 (2001).

4. G.P.Perdue and R.N. Blomster. South American Plants III: Isolation of Fulvoplumierin from Himatanthus sucuuba (M. Arg.) Woodson (Apocynaceae). J. Pharm. Sci. 67(9): 1322-1323 (1978).

5. J.R.A. Silva, C.M. Rezende, A.C. Pinto, M.L.B. Pinheiro, M.C. Cordeiro, E. Tamborini, C.M. Young and V.S.Bolzani. Triterpenic esters from Himatanthus sucuuba (Spruce) Woodson, Quim. Nova 21(6): 702-704 (1998).

6. Y. Endo, H. Hayashi, T. Sato, M. Maruno, T. Ohta and S. Nozoe. Confluentic Acid and 2’-O-Methylperlatoic Acid, Monoamine Oxidase B Inhibitors in a Brazilian Plant, Himatanthus sucuuba. Chem. Pharm. Bull. 42(6): 1198-1201 (1994).

7. M.S. Siqueira. Estudo fitoquímico e farmacológico das raízes de Himatanthus sucuuba (Apocynacea). MSc Thesis, (Universidade Federal do Amazonas, Manaus, Brazil, 1999), pp. 1-120.

8. J.R.A. Silva. Contribuição ao estudo do látex de Himatanthus sucuuba: aspectos químicos e farmacológicos. PhD Thesis, (Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 2000), pp. 1-261.

9. M. Zimmerman. Ethical Guidelines for investigation of experimental pain in conscious animals. Pain 16(2):109-110 (1983).

10. T. Mosmann. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Meth. 65(1-2): 55-63 (1983).

11. J.E. Lima, A.L.F. Sampaio, M.G.M.O. Henriques, T.C.B. Fidalgo. Lymphocyte activation and cytokine production by Pisum sativum agglutinin




(PSA) in vivo and in vitro. Immunopharmacol. 41: 147-155 (1999).

12. L.C. Green, D.A. Wagner, J. Glogowski, P.L. Skipper, J.S. Wishnok and S.R. Tannenbaum. Analysis of nitrate, nitrite, and [15N]Nitrogen in biological fluids. Anal. Biochem. 126(1):131-8, (1982).

13. C.D. Mills, K. Kincaid, J.M. Alt, M.J. Heilman and A.M. Hill. M-1/M-2 macrophages and the Th1/Th2 paradigm, J. Immunol. 164(12): 6166-6123 (2000).

14. R.E. Schultes. De plantis toxicariis e mundo novo tropicale commentationes. XIX. Biodynamic apocynaceous plants of the northwest Amazon. J. Ethnopharmacol. 1(2): 165-192 (1979).

15. V.T. Mathad, K. Raj, A.P. Bhaduri, R. Sahai, A. Puri, L.M. Tripathi and V.M.L. Srivastava. Bioorg. Studies on the Profile of Immunostimulant Activities of Modified Iridoid Glycosides. Med. Chem. 6(5): 605-611 (1998).

16. H-J. Jeong, H-Na Koo, H-J. Na, M-S. Kim, S-H. Hong, J-W. Eom, K-S Kim, T.Y. Shin and H-M. Kim. Inhibition of the TNF-α and IL-6 Production by Aucubin through Blockade of NF-κΒ Activation in RBL-2H3 Mast Cells. Cytokine 18(5): 252-259 (2002).

17. W-L. Chang, H-Y. Wang, L-S. Shi, J-H. Lai and H-C. Lin. Immunosupressive Iridoids from the Fruits of Gardenia jasminoides. J. Nat. Prod. 68(11): 1683-1685 (2005).

18. G. Albers-Schönberg, H. Schmid. Über die Struktur von Plumericin, Isoplumericin, β-Dihydroplumericin und der β-Dihydroplumericinsäure. Helv. Chim. Acta 44: 1447-1473 (1961).

19. F. Abe, R.F. Chen and T. Yamauchi. Chem. Pharm. Bull. 36(8): 2784-2789 (1988).

CRN SEEKS BOTANICALS EXPERT —Leading Association to Increase Emphasis on Science Behind Supplements—

The Council for Responsible Nutrition (CRN), a Washington, D.C.-based trade association representing dietary supplement industry ingredient suppliers and manufacturers, founded in 1973, is in search of a Botanicals Expert to increase the awareness of the evolving botanical research base that supports an appropriate role for plant-based dietary supplements and to use that science as the basis for influencing and responding to regulatory and legislative initiatives, industry activities and media coverage of botanical and herbal dietary supplements. The ideal candidate will: provide scientific expertise in botany, pharmacognosy and toxicology for evaluating issues related to current practices and viewpoints of opinion leaders; evaluate proposed government or industry actions and recommend appropriate CRN positions and actions; build rapport with the botanical and natural products chemists’ communities on issues and identify experts who can provide assistance to CRN on matters of concern. The ideal candidate will exercise ingenuity and initiative in developing CRN position statements on botanical and herbal product issues and submitting comments to government agencies on proposed regulatory actions that impact plant-based dietary supplements. The new scientist will also ensure that CRN’s legislative or other policy positions are based on a sound scientific rationale along with analyzing emerging research results and attempts to put new data into perspective for CRN members, for regulators and legislators, for the media, and for the public. The new scientist will support an appropriate role for plant-based dietary supplements with a variety of audiences including regulators, legislators and the media. The new scientist will be expected to: help increase the awareness of the evolving research base that supports botanical supplements; represent CRN before regulatory agencies and government institutions such as the Food and Drug Administration (FDA), the Office of Dietary Supplements, the National Center for Complementary & Alternative Medicine, and the Federal Trade Commission; interact with members of Congress in hearings and briefings; be a resource for media inquiries regarding botanical research or other issues; organize and provide staff support for CRN member working groups on botanical or regulatory issues; and serve on industry-related committees (e.g. AOAC, etc.). The new scientist will also organize botanical/regulatory components of The Conference: CRN’s Annual Symposium on Dietary Supplements, The Workshop: CRN’s Day of Science and other CRN-sponsored events. The ideal candidate will possess a Doctor of Philosophy in Plant Biology, Organic Chemistry, Botany, Pharmacognosy, Toxicology or Natural Medicine or related discipline and minimum of five years relevant experience in the field of Botanical Science or Natural Products Chemistry. He or she must be familiar with current federal regulation of dietary supplements and previous dietary supplement industry or FDA experience is preferred. In addition, excellent written and oral communication skills are required with some history of publication in peer-reviewed journals. Must have the ability to multi-task and work in collegial, fast-paced, fluid environment as a team player with other staff and with member company representatives. Interested candidates should visit http://www.crnusa.org for a full job description or contact Marie Hooper, CRN’s Director of Administration and Human Resources, at [email protected] for more information.




PHCOG MAG.: Research Article In vitro and In vivo inhibitory effects of Piper longum fruit

extracts on mouse Ehrlich ascites carcinoma Hullatti K.K.1*, Uma.D.Murthy2, Shrinath B.R.3

1Department of Pharmacognosy and Phytochemistry, National college of Pharmacy, Shimoga - 577 201, India. 2Department of pharmacognosy, Govt. College of Pharmacy Bangalore-560 027, India.

3Principal Research Scientist, Central Animal Facility, Indian Institute of Sciences, Bangalore. India. *Corresponding author - [email protected]

ABSTRACT - In order to screen the In vitro and In vivo inhibitory effects of Piper longum fruit extracts on mouse Ehrlich ascites carcinoma., Crude extracts of Piper longum fruits, (petroleum ether (60°–80°), chloroform, ethanol 95%, and water extract) were tested for short term in vitro cytotoxic activity using Ehrlich ascites carcinoma cells, and In vivo cytotoxicity using the Swiss albino mice intraperitoneally inoculated with Ehrlich ascites carcinoma cells. Pet ether and chloroform extracts have shown the significant cytotoxic action on the EAC cell lines. Petroleum ether extract increased the life span of tumor bearing animals by 44.44%. Chloroform extract at a dose of 200 mg/kg body weight increased the life span of tumor bearing animals by 33.33%. Combination therapy of both the extracts increased the life span by 87.5%. These results clearly indicate the further detailed work on these extracts can provide us with an effective non-toxic anti tumor agents. KEY WORDS - Piper longum, short-term in vitro cytotoxicity, Ehrlich ascites carcinoma. INTRODUCTION Piper longum Linn. is an important medicinal plant used traditionally in Indian medicine. Fruits of Piper longum are known as “Pippali” in Sanskrit. Fruits are used as Carminative, liver tonic, abortifacient and used in the treatment of joint pains. Decoction of the fruit is used extensively in acute and chronic bronchitis (1). Piper longum is one of the components of formulations used for the treatment of gonorrhea, menstrual pain, tuberculosis, respiratory tract infections and arthritic conditions (2). Alcoholic extract of Piper longum fruits has shown the promising immunomodulatory and antitumor activity (3), however no antitumor activity was reported on other extracts of piper longum fruits, hence it was decided to investigate the antitumor activities of petroleum ether and chloroform extracts of Piper longum fruits. MATERIALS AND METHODS Authenticated dried fruits of P. longum were obtained form M/s Indian Herb, herbal manufacturing unit, Bangalore. Ehrlich ascites carcinoma cells were initially procured form Department of Radiobiology, Kasturba Medical College, Manipal. It was propagated by serial transplantation into the intraperitonial cavity of female Swiss albino mice. Ascites fluid from the intraperitonial cavity of the donor animals was aseptically aspirated using hypodermic syringe7-8 days

after tumor inoculation. The aspirate was diluted in Dulbeco’s modified Eagle’s medium to get a concentration of 5×106 cells/ml and 0.2 ml of this tumor cell suspension (1×106 cells) was injected in the intraperitonial cavity of mice (3). Extraction Fruits were course powdered and extracted with Petroleum ether (60°–80°C) Chloroform, Ethanol 95%, successively in Soxhlet apparatus and water extract was prepared by macerating the powder for 7 days in chloroform water. Solvents were distilled off under vacuum. In Vitro Cytotoxic Studies. All the animal experiments were performed as per the OECD guidelines. The animals were maintained under standard conditions with food and water ad libitum. 100 mg of Petroleum ether and Chloroform extract were dissolved in 10 ml of dimethyl sulfoxide, where as alcohol extract was dissolved in distilled water. This stock solutions were diluted with phosphate buffered saline (pH 7.2), (small amount of DMSO does not have toxic effect on Ehrlich ascites carcinoma) (4). The short-term in vitro cytotoxic studies were carried out using Ehrlich ascites carcinoma cells (5). Cells were aspirated form the intraperitonial cavity of Swiss albino mice and washed three times with normal saline, and cell count was done using neubeur’s counter. Suspension containing approximately 1 x 106




cells was taken in each tube containing different concentration of extracts and the volume was made up to 1 ml with phosphate buffered saline. All the tubes were incubated at 37° for 3 h. The percentage of dead cells was determined by using trypan blue exclusion method (6). In Vivo Studies Petroleum ether and Chloroform extracts were prepared for i.p. injection according to NCI (National Cancer Institute) protocol (7). Petroleum ether and Chloroform extracts were prepared for i.p injection by resuspending them in 0.1% tween 80. Experimental animals were prepared by injecting 1x106 cells of EAC into the intraperitonial cavity of female Swiss albino mice. Treatment was started 24 h. after the tumor transplantation. Petroleum ether extract was administered at doses 50, 100 and 200 mg/kg body weight, i.p once daily for 10 days Chloroform extract was administered at the dose of 200 mg/kg body wt., i.p once daily for 10 days. Petroleum ether extract at a dose of 50 mg/kg body wt, was administered i.p. 1, 5 and 9th day and Chloroform extract on remaining days for 10 days at a dose of 200 mg/kg body wt. Doses were selected based on the toxicity studies. All the control groups received 0.1% tween 80 in normal saline (Since all the test materials were prepared using 0.1% tween 80 as stated above). All the mice were weighed on the day of tumor inoculation and every day before treatment. The tumor response was assessed on the basis of median survival time (MST) and % increase in life span (% ILS) . Activity was assessed on the basis of % T/C (‘T’ and ‘C’ represent the number of days that treated and control animals survived, respectively) (reproducible % T/C ≥ 125 confirms the activity against tumor.). Statistical analysis was done by student “t” test. Toxicity Studies Three groups of 6 Swiss albino mice each received 0.1% Tween 80 in normal saline, 30 mg/kg-body weight of Petroleum ether extract, and 200 mg/kg body weight of Chloroform extract everyday for 10 day. The hematological measurements were done on day 0, 2, 4, 6 8 and 10 by obtaining peripheral blood from retro-orbit puncture, using neubeur’s chamber. Hematological studies after treatment with pet ether extract and chloroform extracts to the normal animals were performed. Three groups of Swiss albino mice each comprising 6 animals were given with 0.1% Tween 80 (control group), 30 mg/kg body weight of pet ether

extract and 200 mg / kg body weight of Chloroform extract. Peripheral blood (0.5 ml) was collected from all the three groups on day 0, 2, 4, 6, 8 and 10th post treatment days. Total WBC count and differential count was determined at histopathology department at Institute of Animal Health and Veterinary Biologicals, Bangalore. Statistical analysis Results were expressed as the mean±standerd deviation. Statistical evaluation was done by Student’s t-test and the difference was considered statistically significant at P<0.001. Results Short-term in vitro cytotoxicity activities of the extracts are given in table 1. Both Petroleum ether and Chloroform extracts have shown the significant cytotoxic action on the EAC cell lines. Whereas alcoholic extract did not show marked cytotoxic action. The same result has been proved in the animal studies also. Petroleum ether extract at a dose of 50 mg/kg body weight the life span of tumor bearing animals of 44.44% whereas the higher doses has shown toxic effects. Whereas chloroform extract at a dose of 200 mg/kg body weight increased the life span of tumor bearing animals of 33.33%.Interestingly, combination therapy of both the extracts has shown the synergistic effect by increasing the life span by 87.5%, but where as the increase in body weight was not significantly controlled. n the important observation the effective doses of both the extracts did not show any significant changes in total WBC count, and differential count. Discussion Among currently available drugs, synthetic drugs do have potential adverse reactions and which can be minimized to greater extent through natural compounds. (As supported by Vinca alkaloids and taxol), Still there are many natural drugs which are yet to be explored scientifically. Fruits of P. longum were used successfully in the treatment of asthma and bronchitis. Still many uses have been indicated in Ayurvedic literature. Treatment of tumors is one of them. All the parameters of our studies showed that both pet ether extract and chloroform extract at a doses of 50 mg/kg body weight and 200 mg/kg body weight respectively has got significant effect on growth of tumor ascites both in vitro and in vivo. Both the extracts in the effective doses have significantly increased the life span of tumor bearing animals.




Table 1: Short term in vitro cytotoxic effect of Piper longum fruit extracts on Ehrlich ascites carcinoma cells.

% Death* Extracts 10 ppm 100 ppm 1000 ppm

Petroleum ether Chloroform Alcohol

22.17 19.20 9.02

70.16 31.91 18.47

100.00 100.00 23.75

*Approx .1 x 106 EAC cells were incubated with the extract in 1ml PBS at 37°C for 3 hrs.

Table 2: Effect of Piper longum fruit extracts on Ehrlich ascites carcinoma bearing animals. Treatment Dose

(mg/kg body wt) MST a (days)

% ILSb

Control PPE

- 50

100

9±0.8 13±2.3 11±1.9

- 44.44 22.22

Control PCE

- 100 200

12±1.2 12±2.8 16±3.1

- 0.0

33.33 Control

PPE + PCE -

50 + 200 8±1.4

15±2.5 -

87.5 *Approx. 1 x 106 cells of EAC inoculated into the intraperitonial cavity of female

Swiss albino mice. Average of 6 animals ± SEM, P<0.001. (Student “t” test), a Median survival time, b percentage increase in life span.

Table 3: Effect of Piper longum fruit extracts on peripheral WBC count.

Total WBC (cells / mm3)* Days Control PPE (30mg/kg) PCE (200mg/kg)

0 2 4 6 8

10

8400±0.24 8500±0.35 8500±0.23 8400±0.24 8400±0.30 8500±0.24

8400±0.32 8000±0.30 8500±0.34 8700±0.32 8500±0.28 8600±0.30

8500±0.28 8200±0.30 6500±0.32 6800±0.28 8400±0.32 8200±0.34

* Counts are average of 6 animals ± SEM, P<0.001. (Student “t” test)

Table 4: Effect of P.longum fruit extracts on differential count of Swiss albino mice. % Of cells *

Control PPE (30mg/kg) PCE (200mg/kg) Days

L N L N L N 0 2 4 6 8

10

69±0.35 70±0.56 70±0.23 68±0.47 69±0.89 69±0.56

31±0.45 30±0.56 30±0.26 31±0.45 30±0.69 31±0.58

69±0.42 65±0.75 70±0.46 75±0.25 73±0.69 72±0.45

30±0.38 34±0.55 30±0.26 24±0.35 27±0.24 28±0.48

70±0.58 69±0.45 70±0.64 71±0.68 70±0.75 69±0.63

30±0.23 30±0.45 29±0.75 29±0.56 29±0.45 31±0.35

* Counts are average of 6 animals ± SEM, P<0.001. (Student “t” test) L – Lymphocytes ; N – Neutrophils.




Combination of these two extracts has shown a synergistic effect by increasing the life span of animals by 87.5%. Moreover the hematological studies showed that the effective doses of these extracts have no effect on the normal blood cells.The antitumor activity observed against EAC cells may be due to its cytotoxic effect as observed in short term in vitro cytotoxicity studies. Previous studies have shown that many of the steroidal molecules have got the antitumor properties (9, 10), especially β sitostirol (11, 12). Some of the Triterpinoidal compounds from plant sources have shown the inhibitory action on cancer cell lines (13, 14). The phytochemical investigation of Piper longum fruit extracts shown the presence of steroids, β-sitostirol and triterpinoids in petroleum ether and chloroform extracts. Hence the observed antitumor property of Piper longum fruit extracts may be due to the presence of steroidal and triterpinoidal molecules in the extracts. These results clearly indicate the further detailed work on these extracts can provide us with an effective non-toxic anti tumor agents. ACKNOWLEDGMENTS We are very much grateful to Mr. R.K Agarwal, Chairman, M/s Natural Remedies Pvt Ltd., for providing us with the plant material. The authors are thankful to Prof. R. Naik, Chairman, Department of Microbiology IISC, Bangalore, and Dr. B.V. Kumaraswamy, Asst. Professor, Dept. of Indian Medicine, Kidwai Memorial institute of Oncology, Bangalore for their timely help. We thank Prof. P. Umadevi, Dept. of Radiobiology, Kasturba Medical College, Manipal for providing us with Ehrlich ascites carcinoma cells. References 1. K.R Kiritkar, B.D Basu., Indian Medicinal Plants,

2nd Edition, International book distributors, New Delhi, 3: 2128-2130 , (1991).

2. Y.N Singh, Kava an overview, Journal of Ethnopharmacology, 37: 18-45, (1992).

3. E.S Sunila, G. Kuttan, Immunomodulatory and antitumor activity of Piper longum Linn. And Piperine, Journal of Ethnopharmacology, 90: 339-346, (2004).

4. R.A Raja Naresh, N. Udupa, P. Uma Devi., Toxicity and antitumor activity of niosomal bleomycine in tumor bearing mice. Indian Journal of Experimental Biology, 34(Aug): 764-772 (1996).

5. Studies on Culture of ascites tumor cells in chemically defined media; Cancer cells in culture. University of Tokyo Press, Tokyo., 51. (1968).

6. Joshi Kuncheria, S. Jayashree, K.K .Aravindakshan, and Girija Kuttan, Indian Journal of Pharmaceutical sciences., 56(2): 37-40. (1994).

7. H. Kuttan, D. M Vasudevan and R. Kuttan., Cancer letters, 41(3):307-314. (1988).

8. R.I Geran, N.H Greenberg, M.M McDonald, A.M Schumacher and B.J Abbott, Cancer Chemotherapy reports Part 3, 3(2): 21. (1972).

9. R.I Geran, N.H Greenberg, M.M McDonald, A.M Schumacher and B.J Abbott, Cancer Chemotherapy reports Part 3, 3(2): 48. (1972).

10. L.Golubovskaya, Z. Smirnova, V.Tolkachev, V .Rzheznikov, Cytotoxic steroids with antiestrogenic activity of the 11α-acyloxyestra-1,3,5(10)-triene series, Russian Journal of Bioorganic Chemistry, 32( 2): , 201-203 (2006).

11. K Kahlos, L Kangas, R.Hiltunen, Antitumor activity of some compounds and fractions from an n-hexane extract of Inonotus obliquus in vitro. Acta Pharm Fennica. 96:33–40. (1987).

12. H.C Chiang, T.H Tseng, C.J Wang, C.F Chen, W.S Kan., Experimental antitumor agents from Solanum indicum L. Anticancer Res. 11(5):1911-1917. (1991)

13. A.B Awad., beta-Sitosterol inhibits growth of HT-29 human colon cancer cells by activating the sphingomyelin cycle. Anticancer Res. 18(1A):471-473 (1998).

14. M.C Wani , J.P Schaumberg , H.L Taylor , J.B Thompson , M.E Wall ., Plant antitumor agents, 19. Novel triterpenes from Maprounea africana. Journal of Natural Products. 46(4):537-543. (1983).

15. Andrzej Witkowski and Jerzy Konopa, Binding of the cytotoxic and antitumor triterpenes, cucurbitacins, to glucocorticoid receptors of Hela cells, Biochim Biophys Acta. 674(2): 246-55. (1981).




PHCOG MAG.: Research Article

Development of HPTLC method for estimation of charantin in herbal formulations

Patel P.M.1, Patel K.N.2, Patel N.M.1 and Goyal R.K.*

1 Shri B.M. Shah College of Pharm. Educ. & Res., Modasa 383315, India. 2 Dept. of Pharmacognosy, L.M. College of Pharmacy, Ahmedabad India

* Dept. of Pharmacology, L.M. College of Pharmacy, Navrangpura,Ahmedabad 380009 India *For correspondence : [email protected]

ABSTRACT - Charantin is one of the phytoconstituent present in Momordica charantia Linn. M. charantia is known for its hypoglycemic activity from ancient times. In the present study an attempt has been made to develop a HPTLC method for quantitative estimation of charantin in small, big, dried fruits used in formulations and different marketed antidiabetic polyherbal formulations (PHF). This HPTLC method was found to be reproducible, accurate and precise and detect charantin concentration at nanogram level. The developed HPTLC method would be an important tool in the quality control method of polyherbal formulations. KEY WORDS - HPTLC, polyherbal formulations, charantin, antidiabetic INTRODUCTION Diabetes mellitus has recently been identified by Indian Council of Medical Research (ICMR) as one of the refractory diseases for which satisfactory treatment is not available in modern allopathic system of medicine and suitable herbal preparations are to be investigated(1). WHO has approved the use of traditional medicines as part of health programme. A herbal medicine is defined as a finished, labeled medicinal product that contains active ingredients as aerial or underground parts of plants or other plant material or combinations thereof. Approximately 3300 million people in the underdeveloped countries use medicinal plants on a regular basis while there has been great fascination for the herbal medicines and dietary food supplements in the developed countries(2). India has a rich heritage of usage of medicinal plants in the Ayurvedic, Siddha and Unani system of medicine with a mention of about 45,000 plants (3-4). A large number of plant preparations have been reported to possess antidiabetic activity over last several decades (5). A database of natural hypoglycemics collected by researchers in Mexico listed 800 plants. Researchers in India have documeted the use of over 150 plants in various families with hypoglycemic activity (6). A recent cross-cultural compendium cites over 1,200 medicinal plants used in diabetes (7). There are around 6000 herbal manufacturers in India. More than 4000 units are producing Ayurvedic medicines. Due to lack of Infrastructure, skilled manpower reliable methods and

stringent regulatory laws most of these manufacturers produce their products on very tentative basis. Herbal drug formulations have received a raw deal from the modern medicine due to many reasons. Momordica charantia Linn. Cucurbitaceae is a well known to possess antihyperglycemia, anticholesterol, immunosuppressive, antiulcerogenic, anti seperma -togenic and androgenic activities (8-9). One of the active principles reported to be responsible for various actions is charantin (10). All the three selected polyherbal formulations contain karela as one of the plants. The reference standard charantin had to be isolated, purified and the structure authenticated by various spectral analysis. There are no titrimetric, colorimetric, spectrophotometric or chromatographic methods available for quantitative estimation of charantin in different marketed antidiabetic PHFs. Therefore an attempt has been made to develop a HPTLC method because this method is fast, precise, sensitive and reproducible with good recoveries for standardization of polyherbal formulations. MATERIAL AND METHODS Equipment: A Cammag HPTLC system equipped with a sample applicator Linomat V, twin trough plate development chamber, TLC Scanner III and Wincats an integration software 4.02 (Switzerland). Chemicals: Analytical grade chloroform, benzene, methanol, formic acid ethyl acetate were obtained from S.D. Fine Chem Ltd (Mumbai, India). TLC aluminium plates pre-coated with silica gel 60 F 254 (10x 10 cms, 0.2 mm thick) used were obtained from E. Merck Ltd (Mumbai, India).




Sample preparation: Three polyherbal formulations were taken for the quantitative estimation of biomarkers like charantin, curcumin and swetiamarin. The selected PHFs were Mersina (J & J Dechane, Hyderabad), Diabecone (Himalaya Drug Co, Bangalore) and Madhuripu (LVG, Ahmedabad). 200 mg of these selected PHFs were taken for the quantitative estimation. HPTLC method for estimation of charantin Preparation of calibration curve of standard charantin One milligram of working standard charantin was dissolved in 100 ml of chloroform to yield stock solution of 100µg/ml concentration. Calibration curve from 20-600 ng /spot was prepared and checked for reproducibility, linearity and validating the proposed method. The correlation coefficient, coefficient of variance and the linearity of results were calculated. Sample preparation 200 mg of PHFs were taken and extracted in 10 ml of chloroform then the chloroform extract was filtered through Whatmann no. 42 filter paper. The final volume of the extract was made to 10ml with chloroform in volumetric flask. The charantin contents were analyzed after subjecting to HPTLC. The small and big karela were dried under shade and finely powdered. From that 50 mg of fine powder was taken and extracted by chloroform and filtered dried extract the volume make up to 2 ml with chloroform. Method Specifications Silica gel 60 F254 precoated plates (10 x 10 cm) were used with benzene: methanol (80:20) as solvent system. Sample was spotted on precoated TLC plates by using Linomat 5 spotter. Ascending mode was used for development of thin layer chromatography. TLC plates were developing up to 8 cms. The plates were sprayed with 10% sulphuric acid in alcohol and the reagent was prepared freshly, heated at 1300 C for 2-3 min and brought to room temperature. Violet spot with Rf 0.32 was visible and scanned under 536 nm. The contents of charantin in the selected PHFs were determined by comparing area of the chromatogram of PHFs with calibration curve of the working standard of charantin. RESULTS AND DISCUSSION Standard charantin showed single peak in HPTLC chromatogram. The calibration curve of charantin was obtained by spotting standard charantin on HPTLC plate. After development the plate was scanned at 536 nm (Fig.1). The calibration curve was prepared by plotting the concentration of charantin versus average

area of the peak (Fig.2). PHFs were analysed by the proposed method. The amount of charantin was computed from calibration curve and calibration curve was shown in Fig. 2.

Figure 1: HPTLC chromatogram of standard charantin

Figure 2: Calibration curve of peak area versus standard charantin concentration ranging from

20ng- 600ng/spot

0500

10001500

0 200 400 600 800

Concentration ng/spot

Are

a of

pea

k

Data from Table 3 revealed that Madhuripu contained highest quantity of the charantin. We found little less amount of charantin in Diabecone and Mersina. It may be due to varied factors like time of collections, age of the pant, processing conditions, incorrect identification of the plant, improper selection of the herb variety, addition of exhausted material and genetic variety of the plat material. Validation of HPTLC method 1. Linearity: A representative calibration curve of charantin was obtained by plotting peak area of charantin against the conc4ntration of charantin over the range of 20- 600 ng/spot. The correlation coefficient was found to be 0.9635. 2. Accuracy (% Recovery): The % recovery of charantin given in Table 3 was found to be 98.89 which is satisfactory. 3. Specificity: It was observed that the other herbs present in the formulations their constituents an excipients did not interfere with the peak of charantin. Therefore the method was specific. The spectrum of standard charantin spot and charantin in other sample was found to be similar or overlap.




Table 1: Linear range, co-relation coefficient and standard deviation for charantin Parameters Values

Linear range Standard Deviation (According to area) Correlation coefficient (According to area)

20-600 ng 3.752762

0.9635

Table 2: Percentage of charantin in different type of M.charantia fruits and its formulations by measuring area

in HPTLC method Sample Amount of charantin per ng/spot %w/w charantin

Small Karela 301 0.602 Big karela 235.88 0.4716 Fresh fruits 193.15 0.3863 Madhuripu 375.21 0.09356 Diabecone 257.68 0.06443 Mersina 252.18 0.06305

Table 3: Results of recovery study of the method

Amount of charantin added in ng

Amount of charantin found (ng)

% Recovery Average recovery

50 49.63±0.48 99.26 100 98.87±1.06 98.87 98.89 150 147.8±0.96 98.53

4. Limit of Detection: The minimum detectable limit was found to be 20 ng / spot. CONCLUSIONS The proposed HPTLC method was found to be rapid, simple and accurate for quantitative estimation of charantin in different marketed polyherbal formulations and small fruits, big fruits of M. charantia. The recovery values of charantin were found to be about 98.89%, which shows the reliability and suitability of the method. The lowest detectable limit of charantin in different formulations was found upto 20 ng/spot. REFERENCES 1. R.D. Leslie, R.B. Elliott, Early environmental

events as a cause of IDDM. Diabetes. 43: 843 (1994).

2. P.J. Campbell, M.G. Carlson, Impact of obesity on insulin action in NIDDM. Diabetes. 42: 405 (1993).

3. U. Smith, Insulin resistance in obesity, type II diabetes and stress. Acta Endocrin supp. 262: 67 (1984).

4. M. Krotkiewski, P. Bjrntorp, U. Smith, Impact of obesity on metabolism in men and women. J Clin Invest. 72: 1150 (1983).

5. J.A. Marshall, R.F. Hamman, J. Baxter, High-fat, low-carbohydrate diet and the etiology of non-

insulin-dependent diabetes mellitus. Am J Epidemiol. 134: 590 (1991).

6. J.A. Marshall, Dietary fat predicts conversion from impaired glucose tolerance to NIDDM. Diabetes Care. 17: 50-6 (1994).

7. R. Riales, M. Albrink, Effect of chromium chloride supplementation on the glucose tolerance and serum lipids, including HDL, of adult men. Am J Clin Nutr. 34: 2670-8 (1981).

8. P. Scartezzini, E. Speroni, Review on some plants of Indian traditional medicine with antioxidant activity. J Ethnopharmacol. 71(1-2): 23- 43 (2000).

9. A. S. Bourinbaiar, S. Lee-Huang, Potentiation of anti-HIV activity of anti- inflammatory drugs, dexamethasone and indomethacin, by MAP30, the antiviral agent from bitter melon. Biochem. Biophys. Res. Commun. 208(2): 779-85 (1995).

10. G.V. Senanayake, M. Maruyama, K.Shibuya, M. Sakono, N. Fukuda, T.Morishita, C.Yukizaki, M. Kawano, H. Ohta, The effects of bitter melon (Momordica charantia) on serum and liver triglyceride levels in rats, Journal of Ethnopharmacology. 91(2): 257-262 (2004).




PHCOG MAG.: Research Article Cost effective medium for callus initiation from

Catharanthus roseus leaves Namdeo A.G*., Mahadik R.R and Kadam S.S.

Department of Pharmacognosy and Phyto-Biotechnology, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune,India.

*Corresponding author: [email protected]

ABSTRACT - Catharanthus roseus (L.) G. Don. is well known for the anti-cancer agents vincristine and vinblastine, and anti-hypertensive agent ajmalicine. Low yields and high production cost of these alkaloids from intact plants motivated us to establish a cost effective medium for callus initiation. Healthy and friable callus is the prerequisite for any biotechnological investigation. Leaves of C. roseus were incubated in MS medium supplemented with different combinations of growth hormones. MSD medium containing 2,4 D (4.52 µM)+ Kn (4.65 µM) yielded good friable callus after five weeks of incubation. To economize the process, MS medium is prepared in normal water in stead of double distilled water, sucrose (3%) is replaced by market sugar (3%) and coconut water is added in place of growth hormones. Frequency of callus initiation is slightly improved by these modifications. Modified medium responded favorably for the production of good friable callus and high biomass in medium containing 10% coconut water after five weeks of incubation was observed. Higher concentration of coconut water however produced compact and hard callus. KEY WORDS: Catharanthus roseus, anti-cancer, tissue culture medium, callus, sucrose, market sugar, coconut water INTRODUCTION Catharanthus roseus (L) G. Don, the Madagascar periwinkle, produces several commercially valuable alkaloids including the anti-cancer compounds vincristine, vinblastine and the anti-hypertensive compound ajmalicine (1). The low yield and high market value of these compounds have made C. roseus as a model system for plant biotechnology. Attempts have been made to obtain high value bioactive compounds vincristine and vinblastine from cell and organ cultures of C. roseus, but the content was much lower than in the natural grown plants (2, 3). Several strategies have been studied for the production of monomeric and dimeric indole alkaloids by cell and organ cultures of C. roseus (4), optimization of nutrient media (5, 6), growth regulators (7), chemical treatment (8), elicitation (9, 10), precursor feeding (11-13) and large-scale cultivation in pilot scale bioreactor (14 -18). Due to low productivity and high production costs of these alkaloids by cultures of C. roseus, efforts were made to minimize the costs by addition of market sugar in stead of sucrose, normal water in place of double distilled water and coconut water in place of growth hormones.

MATERIALS AND METHODS Explants were collected form one-year-old plant of C. roseus. Leaves of plant have been used for initiation of cell cultures. Explants were kept under running tap water for 30 min and then washed with 10 % Dettol (Chloroxylenol 4.8 % w/v, Terpeniol 9 % v/v, and absolute alcohol 13.1 % v/v) for five min followed by thorough washing with distilled water to remove the traces of germicidal agent. The materials were then treated with 70 % ethyl alcohol for 2 min and subsequently with mercuric chloride (0.1 % w/v) solution for 2 min. Finally, the plant materials were thoroughly washed six times with sterilized distilled water. Disinfected explants were incised into small pieces (5-8 mm) and aseptically transferred to Murashige and Skoog’s (MS) (19) medium supplemented with combinations of growth hormones stated in Table 1. The medium pH was adjusted to 5.8 ± 0.02 using 0.1N HCl or 0.1N NaOH prior to addition of Agar-Agar (8 g-l) and autoclaved at 1210C for 20 min. Cultures were kept on culture rack at 26 ± 2°C and maintained 16 h photoperiod with white fluorescent light. Cultures were incubated for 5 weeks and the percent induction was calculated using the following equation: Frequency = No. of explants showing response X 100

Total No. of explants




RESULTS Figure 1. Illustrates the percent induction of callus from C. roseus leaves incubated for five weeks on MS medium supplemented with different combinations of growth hormones. Maximum percent (84 %) was observed on MSD medium supplemented with 2,4 D

(4.52 µM) + Kn (4.65 µM) followed by MSC medium containing 2,4 D (9.05 µM) + Kn (4.65 µM) after same period of incubation. The increase in dry weight of callus initiated from leaf explants of C. roseus on MS medium supplemented with different combinations of growth hormones is

Table 1. Combinations of growth hormones for initiation of C. roseus callus

Sl. No. Medium Growth hormones References 1. MSA 2,4 D (9.05 µM)+ Kn (0.93 µM) (20) 2. MSB 2,4 D (9.05 µM)+ Kn (2.32 µM) (14) 3. MSC 2,4 D (9.05 µM)+ Kn (4.65 µM) (20) 4. MSD 2,4 D (4.52 µM)+ Kn (4.65 µM) (10) 5. MSE NAA (0.54 µM) + BAP (6.46 µM) (21) 6. MSF NAA (10.74 µM) + BA (22.19 µM) (21)

Table 2. Influence of coconut water on % callus initiation

Coconut water (%)

MS medium in double distilled water + 3 % sucrose

MS medium in normal water + 3 % market sugar

5 % 55 58 10 % 72 80 15 % 48 56 20 % 42 50

Table 3. Comparison of medium cost Conventional Protocol Modified Protocol Medium component

& quantity Item Rate Cost (Rs.) Item Rate Cost (Rs.) Nutrients (As per MS medium)

Macro- Micro Nutrients

15/ lit. 15.00 Macro-Micro Nutrients

15/ lit. 15.00

Water (1.0 lit.)

Double Distilled Water

10/lit. 10.00 Normal Water

0.10/ lit. 0.10

Carbohydrate source (30 g)

Sucrose 300/Kg 9.00 Market Sugar

18/Kg 0.54

Hormones (0.5-2.0 ppm)

Auxin/ Kinetin

2/lit 2.00 Coconut Water

10/lit. 0.25

TOTAL COST 36.00/lit. 15.89/lit. exhibited in Figure 2. The callus initiation was observed on 10th day of incubation on MSA, MSB, MSC and MSD media, whereas it was on 15th day of incubation on MSE and MSF media. Maximum (124 mg) dry weight of callus was obtained on MSD medium after 30 days of incubation [Plate 1] followed by 98 mg on MSC medium after same period of incubation. Dry weight biomass of callus increased upto 30th day of incubation on MSC, MSD and MSF media and reduced thereafter on further incubation. To economize the medium cost, MS medium was prepared in normal water instead of double distilled

water, sucrose (3%) was replaced by market sugar (3%) and coconut water is used in place of growth hormones. Both medium responded favorably for callus induction in coconut water. Table (2) represents that maximum frequency of callus induction was observed at 10% coconut water in both cases. The medium prepared in normal water and containing 3% market sugar showed better callus initiation as compared to medium prepared in double distilled water with 3% sucrose. Maximum frequency of callus initiation (80%) was observed in modified medium in normal water, 3% market sugar containing 10% coconut water. However, higher concentrations of coconut water affected




Figure 1. The frequency of callus initiation from leaves of C. roseus in different medium after three

weeks of incubation.

0102030405060708090

100

MSA MSB MSC MSD MSE MSF

Different media for callus initiation

Perc

enta

ge o

f cal

lus i

nitia

tion

Figure 2. Increase in dry weight of callus initiated

from leaf explants of C. roseus on MS medium supplemented with different combinations of growth

hormones. adversely in both cases. Callus obtained from modified medium was greenish, friable, fast growing. Thin layer chromatography (TLC) of alcoholic extract of dried callus shows the presence of similar concentrations of active constituents as showed by alcoholic extract of dried leaves of C. roseus. Table (3) shows the comparison between the cost of conventional medium/ protocol and the cost of modified protocol. It clearly indicates that if the cost of macro and micronutrients remains constant (Rs. 15.00), the cost of modified medium will reduce from Rs. 36.00 to Rs. 15.89, which is approximately 25 times cheaper than the conventional protocol using double distilled water, sucrose and growth hormones. DISCUSSION Healthy and fast growing friable callus is the prerequisite of different biotechnological investigations. Callus consists of undifferentiated masses of cells developed on a semi-solid medium. The maintenance of such cultures depends on an adequate supply of nutrients, growth hormones and controlled sterile environment. The cells, although

undifferentiated, contain all the genetic information present in parent plant. By suitable manipulation of hormone and contents of the medium, it is possible to initiate the development of roots, shoots and complete plants from callus cultures (24). The nutritional requirements of plant cells and tissues vary from species to species and therefore a number of media have been devised for specific tissues by different workers (25, 26). Sucrose is main constituent of the medium and contributes major costs of the product. Growth studies of C. roseus established in different concentrations of market sugar and analytical grade sucrose revealed that the cells grew uniformly for a period of 30 days and packed cell volume, fresh weight, dry weight and ajmalicine content were found maximum in medium containing 4% market sugar (27). Present investigation revealed that the growth pattern and ajmalicine production cell cultures in medium containing market sugar, normal water and coconut water were almost similar than those grown in similar concentrations of sucrose and distilled water. Cell cultures of C. roseus were grown in 20 l bioreactor containing Zenk’s production medium with addition of market sugar and normal water produced ajmalicine that was also similar to the control medium supplemented with sucrose and distilled water (14). Similarly, large-scale cultivation of Panax ginseng was performed in a 2000 l bioreactor with normal water and market sugar (28). Reinhard et al., (29) reported the biotransformation and production of cardenoloids in 300 l bioreactor containing medium with market sugar by Digitalis lanata cells. Conclusion The best combination (MSD) of growth hormones for maximum frequency (84%) of callus initiation was found to be 2, 4 D (4.52 µM) + Kn (4.65 µM). The cells of C. roseus responded favorably when grown in medium containing market sugar (3%), prepared in normal water and exposed to coconut water in place of growth hormones. However, this protocol reduced the cost of medium by about 25 times. Present work will not only motivate to work on callus initiation and further biotechnological aspects of other medicinal plants but will also provide an opportunity to conserve the endangered species through micropropagation. References 1. P.R.H Moreno, R. Ven der Heijden, R.Verpoorte.

Cell and tissue cultures of Catharanthus roseus: A literature survey: II. Updating from 1988 to 1993. Plant Cell Tiss. Org. Cult. 2 (1), 1-25 (1995).

0

50

100

150

10 15 20 25 30 35Incubation period (Days)

Dry

wei

ght (

mg)

MSA MSB MSC MSD MSE MSF




2. Y. Miura, K. Hirata, N. Kurano, K. Miyamoto, and K. Uchida. Formation of vinblastine in multiple shoot culture of Catharanthus roseus. Planta Med. 54, 18-20, (1988).

3. W. Hofmann, K. H. Kubeczka, and F.C. Czygan. An improved method of isolation and quantitative determination of vincaleucoblastine from intact plants and tissue cultures of Catharanthus roseus. G. Don. Z. Naturforsch. 38c, 201-206, (1982).

4. K. Hirata, A. Yawanaka, N. Kurano, K. Miyamoto, and Y. Miura. Production of indole alkaloids in multiple shoot cultures of Catharanthus roseus (L.) G. Don. Agri. Biol. Chem. 51, 1311-1317, (1987).

5. R. Van der Heijden, R. Verpoorte, and H. J. G. Ten Hoopen. Cell and tissue cultures of Catharanthus roseus (L.) G. Don: a literature Survey. Plant Cell Tiss. Org.Cult. 18, 231-280, (1989).

6. M. Lounasmaa, and J. Galambos, Indole alkaloid production in Catharanthus roseus cell suspension cultures. In Progress in the Chemistry of Organic Natural Products. Vol. 55. W.Herz, , H. Grisebach, G.W. Kirby, and C. Tamm, (eds.), Springer-Verlag, Wien-New York. pp 89-115, (1989).

7. G. Ganapathi and F. Kargi. Recent advances in indole alkaloid production by Catharanthus roseus (Periwinkle). J. Exptl. Bot. 41, 259-267, (1990).

8. J. Zhao, W.H. Zhu, Q. Hu, X. W. He. Improved alkaloid production in Catharanthus roseus suspension cultures by various chemicals. Biotechnol Lett. 22, 1221-1226, (2000).

9. S. K. Rijhwani and J. V. Shanks. Effect of elicitor dosage and exposure time on biosynthesis of indole alkaloids by Catharanthus roseus hairy root cultures. Biotechnol. Prog. 14 (3), 442-449, (1998).

10. A. G. Namdeo, S. Patil, and D. P. Fulzele. Influence of fungal elicitors on production of ajmalicine by cell cultures of Catharanthus roseus. Biotechnol. Prog. 18, 159-162, (2002).

11. F. Naudascher, P. Doireau, A. V. Guillot and M. Thiersault. Time-course studies on the use of secologanin by Catharanthus roseus cell cultures in vitro. J. Plant Physiol. 134, 608-612, (1989).

12. P. R. H. Moreno, R. Van der Heijden, and R. Verpoorte. Effect of terpenoid precursor feeding and elicitation on formation of indole alkaloids in cell suspension cultures of Catharanthus roseus. Plant Cell Rep. 12, 702-705, (1993).

13. J. Zhao, Q. Hu, Y.Q. Guo and W. H. Zhu. Effects of stress factors, bioregulators and synthetic precursors on indole alkaloid production in

compact callus cluster cultures of Catharanthus roseus. Appl. Microbiol. Biotechnol. 55(6), 693-98, (2001).

14. D. P. Fulzele and M. R. Heble Large-scale cultivation of Catharanthus roseus cells: Production of ajmalicine in a 20-l airlift bioreactor. J. Biotechnol. 35(1), 1-7, (1994).

15. F. Kargi and M. Z. Rosenberg. Plant cell bioreactors: present status and future trends. Biotechnol. Prog. 3, 1-8, (1987)

16. W. Kries and E. Reinhard. The production of secondary metabolites by plant cells cultivated in bioreactor. Planta Med. 55, 409-416, (1989)

17. K. Panda, S. Mishra, V. S. Bisaria, and S. S. Bhojwani. Plant Cell Reactors- a perspective. Enzyme Microb. Technol. 11, 386-397, (1989).

18. J. E. Schlatmann, A. M. Nuutila, H. J. G. Ten Hoopen, R. Verpoorte and J. J. Heijnen. Scale-up of ajmalicine production by plant cell cultures of Catharanthus roseus. Biotechnol. Bioeng 41(2), 253-262, (1993).

19. F. Mascarenhas. Handbook of Plant Tissue Culture, ICAR, New Delhi, 27, (1993).

20. M. K. Razdan. An introduction to Plant Tissue Culture, Oxford and IBH Publishing Company, New Delhi, 31, (1995).

21. T. Murashige and F. Skoog. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497, (1962).

22. B. D. Benjamin, A.T. Sipahimalani, and M. R. Heble. Growth and alkaloid production in different cell lines of Catharanthus roseus (L.) G. Don. Indian J. Exptl. Bio. 28, 516-518, (1990).

23. Datta and P. S. Srivastava. Variation in vinblastine production by Catharanthus roseus during in vivo and in vitro differentiation. Phytochem. 46 (1), 35-137, (1997).

24. W. C. Evans. Plant cell and tissue culture; biochemical conversion; clonal propagation. In Pharmacognosy W. C. Evans, (ed.). 14th Edition. W.B. Saunders Company Ltd. U.K. pp 76-86, (2001).

25. O. L. Gamborg, R. A. Miller, and K. Ojman, Nutrient requirements of suspension cultures of soybean root cells. Exptl. Cell Res. 50, 151-8, (1968).

26. M. H. Zenk, H. El Shagi, H. Arens, J. Stockigst, E. W. Weiler, and D. Deus. Formation of indole alkaloids serpentine and ajmalicine in cell suspension cultures of Catharanthus roseus. In Plant Tissue culture and its Biotechnological Application. Barz, W., Reinhard, E. and Zenk,




M.H. (eds.), Springer-Verlag, Berlin. pp 27-44, (1977).

27. A.G. Namdeo, D. P. Fulzele, S. Patil, and S. S. Kadam. Influence of sucrose concentration on growth and ajmalicine production from Catharanthus roseus suspension cultures. Biosciences, Biotechnology Research Asia, 3(2), 341-345 (2005).

28. K. Ushiyama. Suspension cultures of Panax ginseng’s root tissue. Plant Biotechnology. Y.

Yamada and Y. Okada, (eds.). Kagaku Dozin, Tokyo. pp 97-104, (1986).

29. E. Reinhard, W. Kries, U. Barthlen, and U. Helmbold. Semicontinuous cultivation of Digitalis lanata cells: Production of β-methyldigoxin in a 300 l airlift bioreactor. Biotechnol. Bioeng. 34, 502-508, (1989).

SUBSCRIPTIONS – PHCOG MAG.

Pharmacognosy Magazine (PHCOG MAG. - An official publication of Phcog.Net www.phcog.net) [ISSN:

0973-1296] Published Quarterly, Pharmacognosy Magazine (Phcog Mag) serves the needs of scientists

and others involved in medicinal plant research and development. Each issue covers different topics in

natural product drug discovery, and also publishes manuscripts that describe investigations, clinical

reports, methods, techniques and applications of all forms of medicinal plant research and that are of

immediate interest to users in industry, academia, and government. Phcog Mag is a must-read magazine

for medicinal Plant researchers -and it's an open access publication. Phcog Mag. Currently indexed in

Index Copernicus, CABI, Indian Science Abstracts and being indexed in IPA, ISI, EBSCO, Asian Science

citation index, Embase (under evaluation) etc. Due to increased demand for a print copy world wide,

we are proud to inform about the print version of the PHCOG MAG. (Issue 6) is available now.

• http://www.phcog.net/phcogmag

As members are the key assets of Phcog.Net, All members of Phcog.net are requsted to download the

subscription form and recommend to your library.

• Subscription details (Vol 2, 4 issues/year)

• Subscription cost : Rs. 1000/- per year (4 issues/year) and US $ 45 (International)

• Download subscription form at www.phcog.net/phcogmag/subscription_form.pdf

• Send D. D/ Cheque in favour of "The Principal, Al-Ameen College of Pharmacy" Payable at

Bangalore, Karnataka , INDIA.

For more details, please write to [email protected]

Send your subscription request along with D.D or Cheque to following address

Prof. B.G. Shivananda,

Editor, Phcog mag.


Opp. Lalbagh Main Gate, Hosur road, Bangalore 560 027,

Karnataka , India




PHCOG MAG.: Research Article Analgesic and Anti-Inflammatory activities of Lagenaria

siceraria Stand. fruit juice extract in rats and mice B.V. Ghule*, M.H. Ghante, A.B. Upaganlawar and P.G. Yeole

Institute of Pharmaceutical Education and Research, Borgaon (Meghe), Wardha 442 001, India *Corresponding author. Tel.: +91 7152 240284; Fax: +91 7152 241684

E mail : [email protected] (B.V. Ghule) ABSTRACT- Present work was undergone to investigate analgesic and anti-inflammatory effects of Lagenaria siceraria (Molina) Stand. fruit juice extract (LSFJE) in rats and mice. LSFJE was studied for its analgesic effect on acetic acid-induced writhing and formalin pain tests in mice. The anti-inflammatory effects were investigated employing the acute inflammatory models, i.e. ethyl phenylpropionate-induced ear edema, carrageenin- and arachidonic acid-induced hind paw edema, and also the albumin-induced paw edema in rats. LSFJE was also studied for its preliminary phytochemical screening and acute toxicity studies. LSFJE (150-300 mg/kg, p.o.) exhibited a dose-dependent inhibition of writhing and also showed a significant (P<0.001) inhibition of both phases of the formalin pain test, but with a less intense effect on the first than on the second phase. The effects of the extract were significantly (P<0.01) lower than those produced by morphine (10 mg/kg) and aspirin (300 mg/kg) in the same tests. LSFJE elicited significant (P<0.05) inhibitory effect on the ear edema formation at 30 min, 1 h and 2h after EPP-injection. In other acute inflammatory models, the extract significantly inhibited carrageenin- and arachidonic acid-induced hind paw edema. LSFJE also caused inhibition of albumin-induced paw edema over a period of 90 min. The extract did not produce mortality in dose up to 4.2 g/kg, p.o. and 0.29 g/kg, i.p. Preliminary phytochemical screening revealed the presence of flavonoids, cucurbitacin, saponins, proteins, and carbohydrates. The results obtained suggest marked analgesic and anti-inflammatory activity of the LSFJE. The results support the traditional use of this plant in some painful and inflammatory conditions. KEYWORDS- Lagenaria siceraria, Cucurbitaceae, Analgesic, Ant-inflammatory, Acute and chronic models INTRODUCTION Lagenaria siceraria (Molina) Standley syn. L. leucantha Rusby; L. Vulgaris Ser. (Family: Cucurbitaceae) is commonly known as Bottle gourd, an excellent fruit in the nature having composition of all the essential constituents that are required for normal and good health of humans (1). L. siceraria fruits are traditionally used for its cardioprotective, cardiotonic, general tonic, aphrodisiac and acts as alternate purgative, diuretic (2, 3). It also cures pain, ulcers, fever, and used for pectoral cough, asthma and other bronchial disorders (2). The fruits are edible and considered as good source of vitamin C, β-carotene, vitamin B-complex, pectin and also contain highest choline level- a lipotropic factor (1, 4, 5). Modern phytochemical screening methods showed the presence of triterpenoid cucurbitacins B, D, G, H (4, 6, 7) fucosterol, campesterol (8) and flavone C-glycosides (9). L. siceraria seeds are used in migraine type headache and pain and reported to contain saponins, essential fixed oils, vitamins (1, 5). Lagenin- a novel ribosome inactivating protein has been isolated from

the lyophilized water extract of seeds which is known to possess immunosuppressive, antitumour, antiviral, antiproliferative and anti-HIV activities (10). The purpose of the present study was, therefore, to evaluate the analgesic and anti-inflammatory effects of the L. siceraria fruit juice extract (LSFJE) using different acute and chronic models of pain and inflammation in mice and rats. The extract was also studied for its acute toxicity effects and preliminary phytochemical screening. MATERIALS AND METHODS Plant material The fresh fruits of Lagenaria siceraria Stand. (Mol.) were collected from Wardha district of Maharashtra state, India. Plant was authenticated by the authority of Botany Department, Nagpur University, Nagpur, India where a voucher specimen is deposited for future reference. The fresh, rind fruit (500g) of L. siceraria was squeezed to obtain juice extract. The extract was evaporated to dryness in vacuo (40ºC). The yield of LSFJE was obtained 22.5 % w/w.




Preliminary phytochemical screening LSFJE was studied for its preliminary phytochemical screening (6) for the detection of various plant constituents. Preparation of test drugs All test drugs were suspended in 10% v/v propylene glycol except in the ethyl phenyl propionate-induced ear edema model, where test drugs were dissolved in acetone. Experimental Animals Swiss albino mice (18–20 g) and Wistar rats (100-150 g) of either sex kept at the Laboratory Animal Center of the Institute of Pharmaceutical Education and Research, Wardha, India were used. The experimental protocol was initially approved from the Institutes animal ethics committee and then experimental studies were undergone according to their rules and regulations. The animals were housed under standard environmental conditions and had free access to standard pellet diet (Goldmohar brand, Lipton India Ltd.) and water ad libitum. Acute toxicity studies The 50% lethal dose of the LSFJE was estimated by the up- and -down method in mice (11). Doses were adjusted by a constant multiplicative factor viz. 4, for this experiment. The dose for each successive animal was adjusted up and down depending on the previous outcome. Mouse writhing assay The method of Koster et al. (12) was used. The LSFJE (150-300 mg/kg, p.o.) or acetylsalicylic acid (300 mg/kg, s.c.) was administered to mice 60 and 30 min, respectively, before intraperitoneal injection of acetic acid (0.6%, v/v in normal saline, 10 ml/kg). 10% v/v propylene glycol was used as the control. The number of writhes was counted for 15 min. Formalin test The method used was similar to that described previously by Shibata et al. (13). Twenty microlitre of 1% v/v formalin was injected subcutaneously into the right hind paw of mice. The time (in seconds) spent in licking and biting responses of the injected paw was taken as an indicator of pain response. Responses were measured for 5 min after formalin injection (first phase) and 15–30 min after formalin injection (second phase). The LSFJE (150-300 mg/kg, p.o.) and acetylsalicylic acid (300 mg/kg, s.c.) were administered 60 and 30 min, respectively, before formalin injection. Control animals received 10% v/v propylene glycol (10 ml/kg).

Ethyl phenylpropionate (EPP)-induced ear edema in rats Male rats weighing 100-150 g were used. Ear edema was induced by topical application of EPP at a dose of 1 mg/20 µl per ear to the inner and outer surfaces of both ears by means of an automatic micriliter pipette (14). Test drugs were applied topically in volumes of 20 µl just before the irritant. The control group received vehicle only. Before and at 30min, 1h and 2h after edema induction, the thickness of each ear was measured by vernier calipers. The percent inhibition of the edema formation of test substances was calculated. Carrageenin- and arachidonic acid–induced paw edema in rats Male rats weighing between 100-150 g were used. Paw edema was induced by an intradermal injection of carrageenin (1% in normal saline solution) (15) or arachidonic acid (0.5% in 0.2 M carbonate buffer, pH 8.4) into the plantar surface of the right hind paw of the rats, at a volume of 0.05 or 0.1 ml, respectively. The edema volume was determined using a plethysmometer (16) prior to and 1, 3 and 5 h after carrageenin injection, or 1 h after arachidonic acid injection. Test drugs were given 1 h prior to carrageenin or 2 h prior to arachidonic acid injection. The control group received vehicle only. Egg albumin-induced paw edema in rats This test was performed by a modification of Winter et al. (17) as described by Akah and Nwambie (18). Four groups of male and female wistar rats were pre-treated as follows, group 1, 10% propylene glycol; group 2 and 3, 150 and 300 mg/kg, p.o. of LSFJE; and group 4 with Diclofenac sodium (100 mg/kg, p.o.). After 30 min, each group was injected with 0.5 ml raw egg albumin sub-plantar to the left hind-paw. A digital plethysmometer was used to measure volume of paw oedema for a period 120 min, with readings taken at 30 min intervals, i.e. at 30, 60 and 90 min after albumin administration. Statistical analysis All data were expressed as mean±SEM and analyzed statistically by using Paired t-test and Dunnett’s t-test. A difference was considered significant at P value less than 0.05. RESULTS Acute toxicity studies In the acute toxicity test, signs of toxicity included lethargy, jerk, convulsions and death. The LD50 value of orally and intraperitoneally administered LSFJE in mice




was estimated to be 4.2 g/kg and 0.29 g/kg, respectively. Preliminary phytochemical screening Preliminary phytochemical screening of the LSFJE showed the presence of flavonoid glycosides, cucurbitacin saponins, proteins and carbohydrates. Analgesic studies The effect of LSFJE on acetic acid induced writhing is demonstrated in Table 1. The LSFJE (150 and 300 mg/kg, p.o.) showed the significant (P<0.01) reduction in the number of writhes induced by acetic acid in a dose-dependent manner. Aspirin (300 mg/kg, p.o.) exhibited inhibitory effect on writhing response. There was a significant, dose-dependent inhibition of both phases of the formalin induced pain response in mice, with a more potent effect on the second than the first phase (Table 2). Anti-inflammatory studies The activity of LSFJE on EPP-induced ear edema in rat is shown in Table 3. The LSFJE dose-dependently elicited significant (P<0.05) inhibitory effect on the edema formation at 30 min, 1 h, 2 h after EPP injection. Phenylbutazone, a COX –inhibitor, at a dose of 1 mg/kg also showed significant reduction of ear edema. The result of LSFJE against carrageenin-induced paw edema is shown in Table 4. The result shows that the LSFJE (150-300 mg/kg, p.o.) gave the significant (P<0.01) reduction of rat paw edema at all assessment times. Aspirin, a COX-inhibitor at the dose of 300 mg/kg, p.o. significantly reduced the paw edema. The activity of the LSFJE on arachidonic acid-induced hind paw edema in rats is shown in Table 5. LSFJE (150 and 300 mg/kg, p.o.) and also phenidone (60 mg/kg), a dual blocker of cyclooxygenase and lipooxygenase, exerted significant reduction of the paw edema whereas aspirin (300 mg/kg) did not show any effect. The effect of LSFJE on egg albumin-induced hind paw edema in rats is shown in Table 6. The result showed that the LSFJE caused dose-dependent inhibition of albumin-induced edema over a period of 90 min. DISCUSSION In this work, we have demonstrated the effect of LSFJE (150-300 mg/kg; p.o.) on acetic acid-induced writhing and formalin pain tests in mice. The methods for investigating analgesic effects of LSFJE were selected such that both centrally and peripherally mediated effects were investigated. The acetic acid-induced writhing response method elucidated peripheral activity, while the formalin test investigated both peripheral and central activity. Four different animal

models (i.e. ethyl phenylpropionate-induced ear edema, carrageenin- and arachidonic acid-induced hind paw edema, and also the albumin-induced paw edema in rats) were employed to investigate the potential anti-inflammatory activity of the LSFJE in this study. The mouse writhing assay is a useful test to evaluate mild analgesic non-steroidal anti-inflammatory agents. Acetic acid causes algesia by liberating endogenous substances including serotonin, histamine, PGs, bradykinin and substance P which stimulate pain nerve endings (19). Local peritoneal receptors are postulated to be partly involved in the abdominal constriction (writhing) response (20). The method has been associated with prostanoids in general, i.e. increased levels of PGE2 and PGF2α in peritoneal fluids (21) as well as lipoxygenase products by some researchers (22). Therefore, the LSFJE might inhibit the synthesis and/or release of these endogenous substances (Table 1). The formalin pain test is very useful for evaluating the mechanism of pain and analgesia. The formalin injection into rat paw produces localized inflammation and pain. The effect is biphasic in nature: an early neurogenic component followed by a late tissue-mediated response (23).Drugs which act mainly centrally, such as narcotic analgesics, inhibits both phases of pain in this model while peripherally acting drugs, such as aspirin or indomethacin, only inhibit the late phase (24). The formalin pain test assesses the way an animal responds to moderate, continuous pain generated by injured tissue (25). The LSFJE inhibited both phases of the formalin-induced pain with more potent effects on the second than the first phase (Table 2). EPP-induced ear edema test provides a skin inflammation model suitable for evaluation of topical and systemic anti-inflammatory agents. The majority of its activities appear to involve or depend on arachidonic acid release and metabolism and interaction with protein kinase C (26). It has a good predictive value to screen anti-inflammatory agents. Inflammatory mediators such as kinin, serotonin, and PGs are released by EPP (14). It was found that the LSFJE elicited significantly inhibitory effect on the edema formation at all assessment times. Phenylbutazone, a COX-inhibitor, showed a marked reduction of the ear edema (Table 3).




Table 1: Effect of LSFJE on Acetic Acid induced writhing

Group(s)

Dose (mg/kg)

No. of writhes (per 15 min)

Inhibition (%)

Control LSFJE

Aspirin

--- 150 300 300

37.0±0.70 31.6±0.48a

29.8±0.56 a

11.8±0.83 a

--- 14.50 19.50 68.10

Values are expressed as mean±SD. a P<0.01; significantly different from control; Dunnett’s t-test (n=6).

Table 2: Effect of LSFJE on Formalin Induced Pain

Group(s)

Dose

(mg/kg) 0-5 min

Inhibition

(%) 15-30 min

Inhibition

(%)

Control LSFJE

Morphine

--- 150 300 10

96.60±0.57 89.33±0.55 b 84.66±0.61 b

62.33±0.71 b

--- 7.52

12.70 33.47

91.16±0.70 85.33±0.40 a

70.50±1.28b

49.16±0.87b

--- 6.39

22.66

46.07

Values are expressed as mean±SEM. aP<0.0006, bP<0.0001; Significantly different from control; Paired t-test (n=6).

Table 3: Effect of LSFJE and Phenylbutazone on EPP-Induced Ear Edema in Rats

Time after topical application of EPP

30 min 1h 2h

Group(s)

Dose (mg/kg)

ED (um) EDI (%) ED (um) EDI (%) ED (um) EDI(%)

Control LSFJE PBZ

--- 0.5 1.0 1.0

290±12.0 261±10.02

14±6.3a 197±5.6a

--- 10.0

26.20 32.06

288±12.6 257±8.6b

205±6.3a

168±4.6a

--- 10.76 28.81 41.66

224±10.4 164±9.9a 153±6.7a 127±5.8a

--- 26.78 31.69 43.30

Values are mean±SEM. aP<0.01, bP<0.05; Significantly different from control; Dunnett’s t-test (n=6); PBZ: Phenylbutazone; ED: edema thickness; EDI: edema inhibition.

Table 4: Effect of LSFJE and Aspirin on Carrageenin-Induced Hind Paw Edema in Rats

Time after Carrageenin injection 1h 3h 5h

Group(s)

Dose

(mg/kg) EV (ml) EI (%) EV (ml) EI (%) EV (ml) EI (%) Control Aspirin LSFJE

--- 200 150 300

0.48±0.06 0.20±0.04a 0.39±0.03 0.28±0.02a

-- 58.33 18.75 41.66

0.72±0.08 0.28±0.03a

0.52±0.02 0.38±0.08a

--- 61.11 27.77 47.22

0.76±0.03 0.31±0.01a

0.55±0.03a

0.41±0.04a

--- 59.21 27.63 46.05

Values are expressed as mean±SEM. aP<0.01; Significantly different from control, Dunnett’s t-test (n=6); EV: edema volume; EI: edema inhibition.




Table 5: Effect of LSFJE, Phenidone and Aspirin on Arachidonic Acid Induced Hind Paw Edema in Rats

Time (1h) after arachidonic acid injection

Group(s)

Dose (mg/kg)

EV (ml) EI (%) Control PhenidoneAspirin LSFJE

--- 60

200 150 300

0.72±0.03 0.49±0.02b

0.56±0.01 0.64±0.04a

0.61±0.03b

--- 31.94 05.55 15.27 16.66

Values are expressed as mean±SEM. aP<0.01, bP<0.05, Significantly different from control; Dunnett’s t-test (n=6); EV: edema volume; EI: edema inhibition.

Table 6: Effects of LSFJE and Diclofenac Sodium on Egg Albumin-Induced Hind Paw Edema in Rats

Time after albumin injection

30 min 60 min 90 min

Group(s)

Dose

(mg/kg) EV (ml) EI (%) EV (ml) EI (%) EV (ml) EI(%)

Control LSFJE

Diclofenac

---

150 300 200

0.39±0.04

0.31±0.04 0.29±0.03 0.22±0.01a

---

20.51 25.64 43.58

0.48±0.02

0.37±0.02b 0.32±0.01a 0.28±0.01a

---

22.91 33.33 41.66

0.54±0.03

0.39±0.02a

0.35±0.04a

0.29±0.01a

---

27.77 35.18 46.29

Values are expressed as mean±SEM (n=6); aP<0.01, bP<0.05; Significantly different from control; Dunnett’s t-test; EV: edema volume; EI: edema inhibition

The paw edema induced by the subplantar injection of carrageenin in rats is biphasic, the first phase (1 h) involves the release of serotonin and histamine while the second phase (over 1 h) is mediated by prostaglandins, the cyclooxygenase products and the continuity between two phases is provided by kinins (27). The LSFJE might have inhibitory effects on the release and/or synthesis of inflammatory mediators especially the cyclooxygenase products (Table 4). Metabolism of arachidonic acid mediated through the lipooxygenase pathway is necessary for the edema formation (28). The LSFJE (150-300 mg/kg) as well as phenidone (60 mg/kg) a dual blocker of cyclooxygenase and lipooxygenase, and aspirin (200 mg/kg) exerted significant reduction of the paw edema. This suggests that the activity of LSFJE might be mediated via the cyclooxygenase and/ or lipooxygenase pathway (Table 5). The egg albumin-induced hind paw edema method is useful to detect activity in acute inflammation. The LSFJE also caused marked inhibition of egg albumin-induced hid-paw edema in rats (Table 6). The co-existence of analgesic and anti-inflammatory effects seen with this extract is well-defined for

various non-steroidal anti-inflammatory drugs (NSAIDs) particularly salicylates and their derivatives. It’s therefore interesting that the extract behaved like NSAIDs in this study which correlates well with the traditional application of the plant. The LSFJE does possess significant analgesic and anti-inflammatory effects in laboratory animals at the doses investigated. The results support the traditional use of this plant in some painful and inflammatory conditions and also suggest the presence of biologically active principals i.e. flavonoid glycosides, cucurbitacin saponins, proteins and carbohydrates. Several flavonoids and saponin glycosides isolated from medicinal plants have been discovered to possess significant analgesic and/ or anti-inflammatory effects (29). It is, therefore, possible that both the analgesic and anti-inflammatory effects observed with LSFJE may be attributed to its flavonoids and saponin glycoside components, shown to be present during phytochemical screening. Further studies are in progress to isolate and characterize the active principles of the extract. The oral and intraperitoneal LD50 obtained with this plant extract also suggest that it may have a




reasonable safety margin with regards to acute toxicity further justifying its wide application in various communities and lack of any reported side effects with the traditional use of this plant.

ACKNOWLEDGEMENTS We owe our thanks to the authority of Botany Department, Nagpur University, Nagpur for the authentication of plant specimen. The facilities provided by the Institute of Pharmaceutical Education and Research, Wardha during this course of study are gratefully acknowledged. REFERENCES 1. A.S.H.Rahman. Bottle Gourd (Lagenaria siceraria)-

a vegetable for good health. Natural Product Radiance 2(5): 249-250 (2003).

2. V.V. Shivarajan, I. Balachandra, Ayurvedic drugs and their Plant source, (Oxford and IBH Publishers, New Delhi, 1996) pp. 176-177.

3. K.R. Kirtikar, Indian Medicinal Plants, (Oriental Enterprises, Dehradun, India, 2001) pp. 722-723.

4. J.A. Duke, Handbook of Phytochemical and Constituents of GRASS herbs and other economic plants, (Boco, Raton, CRC Press, FL, 1999). pp. 98-119.

5. R. N. Chopra, I.C. Chopra, B.S. Verma, Supplement to Glossary of Indian Medicinal Plants, (Council of Scientific and Industrial Research, New Delhi, 1992) pp. 51.

6. W.C. Evans, Treese and Evans Pharmacognosy, (Balliere, Tindall, London 1996) pp. 388-433.

7. S. Sonja and S. Hermann. Analysis of Cucurbitacins in Medicinal Plants by HPLC-MS. Phytochem. Analysis 11: 121 (2000).

8. A. Shirwaikar and K.K. Sreenivasan. Chemical investigation and anti-hepatotoxic activity of the fruits of Lagenaria siceraria. Ind. J. Pharm. Sci. 58 (5): 197-202 (1996).

9. M.K. Baranowska and W. Cisowski. High Performance Liquid Chromatographic determination of Flavone C-glycosides in some species of the Cucurbitaceae family. J. Chromatography A 675: 240-243 (1994).

10. H.X. Wang and T.B. Ng. Lagenin, a novel ribosome-inactivating protein with ribonucleatic activity from bottle gourd (Lagenaria siceraia) seeds. Life Sciences 67(21): 2631-2638 (2000).

11. R.D. Bruce. An up- and -down procedure for acute toxicity testing. Fundam. Appl. Toxicol. 5: 151-157 (1985).

12. R. Koster. Acetic acid analgesic screening. Federation Proceedings 18: 418-421 (1959).

13. M. Shibata, T. Ohkubo, H. Takahashi and R. Inoki. Modified formalin test: characteristic biphasic response. Pain 38: 347-349 (1989).

14. R. Brattsand, A. Thalen, K. Roempke, L. Kallstrom and E. Gruvstad. Influence of 16α, 17α- acetal substitution and steroid nucleus flourination on the topical to systemic activity ratio of glucocorticoids. J. Steroid Biochem. 16: 779-786 (1982).

15. C.A. Winter, E.A. Risley and G.W. Nuss. Carrageenin-induced edema in hind paw of the rat as assay for anti-inflammatory drug. Proceed. Soc. Expetl. Biol. Med. 111: 544-547 (1962).

16. M.J. Dimartino, G.K. Campbell, C.E. Wolff and N. Hanna N. The pharmacology of arachidonic acid-induced rat paw edema. Agents and Actions 21: 303-305 (1987).

17. C.A. Winter, E.A. Risley and G.W. Nuss, Anti-inflammatory and antipyretic activities of indomethacin. J. Pharmacol. Exptl. Therap. 141: 369-376 (1963).

18. P.A. Akah and A.I. Nwambie. Evaluation of Nigerian traditional medicines: Plants used for rheumatic disorders. J. Ethnopharmacol. 42: 179-182 (1994).

19. H.O. Collier, L.C. Dinneen, C.A. Johnson and C. Schneider. The abdominal constriction response and its suppression by analgesic drugs in the mouse. Br. J. Pharmacol. 32: 295-310 (1968).

20. G.A. Bentley, S.H. Newton and J. Starr. Studies on the anti-nociceptive action of α-agonist drugs and the interaction with opioid mechanisms. Br. J. Pharmacol. 79: 125-134 (1983).

21. R. Derardt, S. Jougney, F. Delevalcee and M. Falhout. Release of prostaglandins E and F in an algogenic reaction and its inhibition. Eur. J. Pharmacol. 51: 17-54 (1980).

22. A.K. Dhara, V. Suba, T. Sen, S. Pal and A.K. Nag Chaudhary. Preliminary studies on the anti-inflammatory and analgesic activity of the methanolic fraction of the root extract of Tragia involucrate. J. Ethnopharmacol. 72: 265-268 (2000).

23. H. Wheeler-Aceto and A. Cowan. Neurogenic and tissue-mediated components of formalin-induced edema. Agents Action 34: 264-267 (1991).

24. A. R. Santos, V.C. Filho, R. Niero, A.M. Viana , F.N. Moreno, M.M. Campos and J.B. Calixto. Analgesic effects of callus culture extracts of selected species of Phyllanthus in mice. J. Pharm. Pharmacol. 46: 755-758 (1994).




25. A. Tjolsen, O.G. Berge, S. Hunskaar, J.H. Rosland and K. Hole. The formalin test: an evaluation of the method. Pain 51: 5-17.

26. Young JM, Young LM. Pharmacological methods in the Control of Inflammation. In: Spector J, Back N, eds. Alan R Liss Inc., New York, 215-217 (1989).

27. O.O. Adeymei, S.O. Okpo and O. Orpaka. The analgesic effect of the methanolic extract of Acanthus montanus. J. Ethnopharmacol. 90: 45 (2004).

28. J.M. Young, D.A. Spires, C.J. Bedord, B. Anger, S.D. Ballaron and L.M. De Young. The mouse ear inflammatory response to topical arachidonic acid. J. Invest. Dermatol. 82: 367-371 (1984).

29. J.A. Duke. Handbook of Biologically Active Phytochemicals and their Activities, (CRC Press, Boca, Raton, FL, 1992).

PHARMACOGNOSY MAGAZINE FEAUTURES

WEB BASED MANUSCRIPT HANDLING SYSTEM AT

www.phcogmag.com

For more information on manuscript submission, write to [email protected]

DOWNLOAD INSTRUCTIONS TO AUTHORS – www.phcogmag.com/instructions.pdf For latest issue : Visit www.phcog.net/phcogmag





Anti-inflammatory activity of Plumbago capensis Charles Dorni A.I*., Vidyalakshmi K.S., Hannah R.Vasanthi.,

Rajamanickam G.V and Dubey G.P Centre for Advanced Research in Indian System of Medicine (CARISM)

SASTRA Deemed University, Thanjavur, Tamil Nadu, India * For Correspondence: [email protected]

ABSTRACT - The HAEPC (Hydroalcoholic extract of Plumbago capensis) showed a maximum of 46.88% inhibitory action on Carrageenan induced paw edema at the dose of 250 mg/kg and inhibited the leukocyte migration in a dose dependent manner. The anti-inflammatory activity observed was comparable to the standard non-steroidal anti-inflammatory drug indomethacin (10mg/kg). Plumbago capensis, a lesser known Plumbago species has significant anti-inflammatory activity with potential constituents targeting different components of inflammatory process. KEY WORDS - Plumbago capensis, Plumbagin, Peritonitis, HPTLC, Prostaglandin, Carrageenan. INTRODUCTION Plants of the genus Plumbago, namely Plumbago capensis (PC), Plumbago rosea (PR) and Plumbago zeylanica (PZ), grown throughout India, have long been used traditionally in Indian folk medicine to treat inflammatory disorders such as rheumatoid arthritis, laryngitis and skin diseases such as leucoderma, ringworm, scabies, leprosy etc., Among the three species PZ has been widely assigned for various medicinal properties and is used in formulations of a number of ayurvedic compounds (1). The root of the three species are rich source of 1,4 naphthoquinone, Plumbagin, exhibiting a wide range of pharmacological activities such as anti-bacterial, anti-fungal, anti-coagulant, anti-fertility, anti-cancer activity, anti-helicobacter pylori activity (2,3). PZ and PR have been prescribed in the treatment of cancer in Siddha system of medicine (4). Plumbago capensis Thunb., a small scandent shrub, indigenous to S.Africa is a lesser known species in ethnopharmacognosy. Previous studies reported the presence of anti fungal protein acting against Trichosporium vesiculosum and Macrophomina phaseolina in the crude extract of P.capensis (5, 6). The present study was focused on the anti-inflammatory effects of the hydroalcoholic extract of Plumbago capensis root (HAEPC) in animal models of acute inflammation. The bioactive marker plumbagin has been quantified using HPTLC. MATERIALS AND METHODS Chemicals - Carrageenan and Indomethacin was obtained from Sigma-Aldrich, Germany. All the solvents used were of analytical grade procured from

E.Merck, Mumbai and the standard Plumbagin (RM 2947) was purchased from Hi-Media. Plant materials – Air dried roots of Plumbago capensis were collected from Nagercoil, Tamil Nadu, India and taxonomically identified by the Captain Srinivasa Murti Drug Research Institute (CSMDRI), Chennai and a voucher specimen was deposited in the CARISM herbarium. Preparation of the Hydroalcoholic Extract – 1000 gm of air dried roots was extracted by cold maceration process with 70% Hydroalcohol (Ethanol:Water; 70:30) for 72 hrs. The hydroalcoholic extract (HAEPC) was rotary evaporated at 50°C and concentrated in vaccuo to obtain a brown solid mass with a yield of 12.9% w/w. Phytochemical screening of the extract revealed the presence of quinones, flavonoids, terpenoids and amino acids (7). Acute Oral Toxicity – Acute oral toxicity of the HAEPC was studied in Swiss albino mice weighing 20-25 gm. HAEPC was administered orally at the dose of 100, 1000, 2000, 3000 or 4000 mg/kg to 6 groups of animals (8 mice in each). Control group received normal saline (5 ml/Kg orally or 0.1 to 0.125 ml for mice weighing 20-25 g). Signs of toxicity and mortality within 24-72 h were noted. CPCSEA Registration No.817/04/AC/CPCSEA. Estimation of Plumbagin in Hydroalcoholic Extract by HPTLC To determine the content of plumbagin in the HAEPC, 500 mg of HAEPC is made up to 25 ml with chloroform as naphthoquinones are reported to be easily soluble in chloroform (8, 9). The resulting solution is centrifuged at 3000 rpm for 15 minutes and the supernatant is




analyzed for plumbagin content. 40 µl of the filtered solution is applied on a 10 x 10 cm preactivated HPTLC Silica gel 60F254 plate. HAEPC and standard plumbagin were applied to the plate as 6 mm wide band with an automatic TLC applicator Linomat V with N2 flow (CAMAG, Switzerland), 8 mm from the bottom. Densitometric scanning was performed on CAMAG scanner III at 265 nm. The plates were pre-washed by methanol and activated at 60◦ C for 5 min prior to chromatography. The slit dimension was kept at 5 mm x 0.45 mm and 40 mm/s scanning speed was employed. The mobile phase consisted of toluene:ethyl acetate: methanol (8:1:1) and 10 ml of mobile phase was used per chromatography. Linear ascending development was carried out in 10 cm x 10 cm twin glass chamber saturated with the mobile phase. The analysis is repeated for six times and the possibility of interference from other components of HAEPC in the analysis is studied. Carrageenan induced Paw Edema. Male wistar rats weighing 120-150 gm were used. The animals were put on standard diet and water was provided ad libitum. The animals were fasted overnight before the experimentation. The rats were divided into five groups (n=6). The anti-inflammatory activity of HAEPC was assessed by the method described by Winter et al, (10). Rats in Group I were given normal saline and were treated as control. Rats in Group II were administered Indomethacin in normal saline at the dose of 10 mg/kg b.w. orally and were kept as standard (11). Rats in Group III to Group V were administered orally with the HAEPC in normal saline at the doses of 150, 250 & 350 mg/kg b.w. respectively. Since the LD 50 has not been determined during the acute toxicity study, the doses for this study were selected by trial and error method. The standard and HAEPC were given orally to the animals one hour prior to Carrageenan injection. Acute paw edema was induced by injecting 0.1ml of 1 % (w/v) Carrageenan solution, prepared in normal saline in sub-plantar region of the left hind paw of the rat. The perimeter of paw was measured by using vernier caliper. Measurements were taken at 0, 1, 2, 3 & 4 hours after the administration of the Carrageenan. % Inhibition of Edema = {[C-T}/C}*100 Where, C – Control Paw Edema ; T – Test Paw Edema Carrageenan Induced Peritonitis Inflammation was induced by the modified method of Griswold et al., 1987 (12). Male Swiss albino mice weighing 20-25 gm were divided into five groups (n=4).

Group I served as control. Group II served as standard and was dosed with Indomethacin (10 mg/kg, p.o) and Groups III – V were dosed with HAEPC at the doses of 150, 250, 350 mg/kg, p.o. Since the LD 50 has not been determined during the acute toxicity study, the doses for this study were selected by trial and error method. The standard drug and HAEPC were administered orally one hour prior to the induction of peritonitis. After one hour, Carrageenan (0.25 ml, 0.75 % w/v in saline) was injected intraperitoneally. Four hours later, the animals were sacrificed by cervical dislocation and 2 ml of Ca2+ and Mg2+ free phosphate-buffered saline (PBS) was injected into the peritoneal cavity. Following a gentle massage, peritoneal exudates were removed. The total leukocyte count was determined in a Neubauer chamber and the differential cell count was determined (13,14). The percentage of the leukocyte inhibition was calculated using the following formula: Leukocyte Inhibition (L I %) = (1- T/C) X 100 where ‘T ‘represents the treated groups’ leukocyte count and ‘C’ represents the control group leukocyte count. Inhibition of Neutrophil migration was calculated by the following equation: Inhibition of Neutrophils Migration = 100 – {[NT/NC] X 100}. Where, NT = Neutrophil counts of treated groups; NC = Neutrophil counts of Control group. Statistical analysis Results are expressed as mean ±S.D. and difference in means are determined by one-way ANOVA followed by post-hoc analysis with Dunnet’s t-test ; values P<0.05 were considered as statistically significant. RESULTS AND DISCUSSION Plumbago species is one of the most important medicinal plants grown throughout India. The whole plant and its root have been used as a folk medicine in India as well as in countries like Taiwan, Vietnam etc., for the treatment of rheumatic pain, menostasis, carbuncle, and injury by bumping.(15). Despite the traditional use of the species in rheumatic diseases, the anti-inflammatory activity of the Plumbago species has not been studied in detail in the past. Inhibition of carrageenan induced inflammation in rats is one of the most suitable test procedures to screen anti-inflammatory agents. The development of carrageenan induced inflammation is a biphasic event, the first phase occurs within an hour of injection is attributed to the release of histamine, 5-HT and kinins, while the second phase which can be measured around 3-4 hours time is related to the release of




prostaglandins (16). The presence of prostaglandin E2 in inflammatory exudates from the injected foot can be demonstrated at three hours time period and thereafter. (17) Indomethacin is used as standard reference drug as it is reported to inhibit inflammation by its effect upon plasma exudation associated with carrageenan mediated inflammation. (18). HAEPC showed a maximum of 46.88% edema inhibition at 3 hrs at the dose of 250 mg/kg and the effect lasted for 3 hrs for P.capensis. The inhibitory activity decreased after 3 hrs and above 250 mg/kg dose the anti-inflammatory effect recedes. Intraperitoneal injection of carrageenan leads to inflammation of the peritoneum resulting from carrageenan induced release of interleukin-1 from macrophages in the carrageenan insulated tissue. Interleukin-1, a pro-inflammatory cytokine, induce accumulation of polymorpho nuclear cells by a variety of processes including adhesion and cell mobility (19) Leukocyte aggregation is a fundamental event during inflammation. Cell migration occurs as a result of much different process including adhesion and cell mobility. In the present study the standard NSAID, Indomethacin (10mg/kg) has produced 63.36% of leukocyte inhibition and 42.16% inhibition of neutrophil migration. HAEPC exhibited a maximum of 48.85% leukocyte inhibition at 350 mg/kg dose and the effect increases dose dependently. The results suggest that

the HAEPC have potential constituents interacting with the different cellular processes of inflammation. As, plumbagin is reported to have cytotoxic activity (20) its quantity in the HAEPC is of greatest importance in terms of toxicity and biological activity. Hence, we quantified the amount of plumbagin in the hydroalcoholic extract and studied the acute toxicity in albino mice. The spot at Rf = 0.78 corresponding to plumbagin was observed in the chromatogram of the HAEPC along with other components. There is no interference from other components present in the chromatogram. The HAEPC was found to contain 0.43 % (w/w) of plumbagin in dried extract. The mice administered with the HAEPC showed no toxic signs or mortality up to a dose of 4000 mg/kg showing the safety of the extract. Plumbagin as a pure molecule also exhibited concentration dependent immunomodulatory activity on macrophages in BALB/c mice (21). Plumbagin which is also found as active ingredient in the common species of Plumbago, P.zeylanica, reported to have significant antioxidant activity. In an antioxidant and pulse radiolysis study (22), the PZ extracts (aqueous/alcoholic) and plumbagin showed significant antioxidant abilities in FRAP, ABTS and DPPH assays. The observed anti-inflammatory activity of HAEPC can also be attributed to the anti-oxidant potential of the naphthoquinone, plumbagin in the extract, as inflammation involves oxidative damage.

Table.1 - Effect of Plumbago capensis on carrageenan-induced paw edema in rats (n=6)

Groups Paw volume (cm, Mean ± SD) Edema value(cm, Mean ± SD)

1 hr 2 hr 3 hr 4 hr 1 hr 2 hr 3 hr 4 hr Control 0.62

±0.02 0.67

±0.03 0.74

±0.02 0.69

±0.02 0.21

±0.01 0.25

±0.02 0.32

±0.02 0.27

±0.01 Indomethacin 10 mg/kg

0.56* ±0.02

0.57* ±0.02

0.55* ±0.01

0.53* ±0.02

0.16* ±0.03

(23.81)

0.17* ±0.02 (32.0)

0.15* ±0.02

(53.13)

0.13* ±0.01

(51.85) PC 150 mg/kg 0.59

±0.01 0.62*

±0.002 0.66* ±0.02

0.63* ±0.02

0.18 ±0.02

(14.29)

0.21* ±0.03 (16.0)

0.25* ±0.03

(21.88)

0.22 ±0.03

(18.52) PC 250 mg/kg 0.56*

±0.04 0.58* ±0.02

0.56* ±0.02

0.58* ±0.03

0.17* ±0.03

(19.05)

0.19* ±0.02 (24.0)

0.17* ±0.02

(46.88)

0.19* ±0.04

(29.63) PC 350 mg/kg 0.56*

±0.02 0.58* ±0.02

0.61* ±0.01

0.60* ±0.02

0.16* ±0.02

(23.81)

0.18* ±0.01 (28.0)

0.21* ±0.01

(34.38)

0.20* ±0.01

(25.93) Values represent the mean ± S.D. of 6 animals for each group. Values in parenthesis indicate the percentage inhibition rate. *

Experimental groups compared with control (p<0.05)




Table.2 Effect of Plumbago capensis on Carrageenan induced Leukocyte Aggregation (n=4) Groups Total Leukocyte Count

( 106 Cells/cmm) % Leukocyte

Inhibition Neutrophils

(105mL-1) % Inhibition of

Neutrophil Migration

Control 16.13± 2.37 - 0.67±0.02 Indomethacin 10 mg/kg

5.91± 2.05* 63.36 0.39±0.01* 42.16

PC 150 mg/kg 12.83± 2.20 23.46 0.53±0.02* 22.64 PC 250 mg/kg 10.19± 0.66* 36.83 0.50±0.03* 25.37

PC 350 mg/kg 8.25± 2.30* 48.85 0.40±0.29* 40.30 Values are mean±SD; * Experimental groups were compared with control (p<0.05)

Fig.1 HPTLC chromatogram of Plumbago capensis

Thus the hydroacloholic extract obtained from the root of Plumbago capensis is found to have a significant anti-inflammatory activity due to the synergistic effect of multiple ingredients of the extract including the known bioactive marker plumbagin. ACKNOWLEDGEMENTS The authors wish to thank Prof.R.Sethuraman, Vice-Chancellor, SASTRA Deemed University for his constant support and encouragement for our research activities at the Centre for Advanced Research in Indian System of Medicine (CARISM). Research fellowship for the authors from the Drugs and Pharmaceuticals Division of the Department of Science and Technology, Govt. of India is gratefully acknowledged. REFERENCES 1. K.R. Kirtikar and B.D. Basu, Indian Medicinal

Plants, Vol.II. Bishen Mahendra Pal Singh, Dehradun, India.1466-1468 (1993).

2. M. Krishnaswamy and K.K.Purushottam. Plumbagin: a study of its anticancer, antibacterial and antifungal properties. Ind.J Exp Biol. 18: 876-877 (1980).

3. Yuan-Chuen Wang and Tung-Liang Huang. Anti-Helicobacter pylori activity of Plumbago

zeylanica. FEMS Imm. Med. Micro. 43(3):407-412 (2005).

4. Murugesa Mudaliar. Materia Medica, Govt. of Tamil Nadu, India. 311(1969).

5. D. Modhumita Ghosh, Thangamani, Manisha Thapliyal, R.Yashodha and K.Gurumurthi. Purification of a 20 KD antifungal protein from Plumbago capensis - a medicinal plant. Jl. Med. and Aro. Plant Sci. 24:16-18 (2002).

6. M.Ghosh, M.Thapliyal, D.Thangamani and R.Yasodha. In vitro antifungal activity of crude protein extracts of Plumbago capensis against Trichosporium vesiculosum. Indian Forester. 126:685-689 (2000).

7. C.K. Kokate, Practical Pharmacognosy, 108-109 (1994).

8. G.S.Siddhu and A.V.B.Sankaram. A new biplumbagin and 3-chloroplumbagin from Plumbago zeylanica. Tetrahedron letters. 12(26):2385:2388 (1971).

9. S.M. Zhong, P.G.Waterman and J.A.D.Jeffrevs. Naphthoquinones and triterpenes from African Diospyrus species. Phytochemistry. 123:1067-1072 (1984).

10. C.A.Winter, E.A. Risley and G.W. Nuss. Carrageenan-induced edema in the hind paw of the rats as an assay for anti-inflammatory drugs. Exptl. Biol. and Med. 111:544-547 (1962).

11. S. M. Hess and R.C Milonig, Assay for anti-inflammatory drugs. In: Lepawl H.Ward PA, editors. Inflammation, Mechanism and control. New York: Academy, 1-12 (1972).

12. D.E. Griswold, P.J. Marshall, E.F. Webb, R.Godfrey, J.Newton and M.J.Diamatrina. S K & F, 86002: A structurally novel anti-inflammatory agent that inhibits lipoxygenase and cyclooxygenase mediated metabolism of arachidonic acid. Biochem. Pharmacol. 36:3463-3470 (1987).

Plumbagin




13. M.M.Wintrobe, G.R.Lee, D.R.Boggs, T.C.Bothel, J.W.Athens, and J. Foerester, Clinical Hematology, fifth ed. Lea and Febiger, Philadelphia. 326 (1961).

14. F.E. D’Amour, F.R.Blood and D.A.Belden Jr, Manual for Laboratory work in Mammalian Physiology. Third ed. Chicago: The University of Chicago Press, pp 4 -6 (1965).

15. N.Y.Chiu and K.H.Chang, The illustrated Medicinal Plants of Taiwan. SMC, Taipei. (1986).

16. J.R.Vane and B.M.Botting, Overview, the mechanism of action of anti-inflammatory drugs. In: Vane Jr, Botting, eds. Clinical significance and potential of selective COX-2 inhibitors. London: William Harvey Press:1-18 (1998).

17. R.Vineagar, J.F.Traux, J.H.Selph, P.R.Johnston, A.H.Vinable and K.K.Mckenzie. Pathway to carrageenan-induced inflammation in hind limb of the rat. Fed.Proc. 46: 118 -126 (1987).

18. Maria Terezinha Zanin and Sergio H.Ferreira. Relationship between edema and plasma exudation in the rat paw carrageenan inflammation. Infla. Res. 8(6): 606-609 (1978).

19. C.J.Meade, G.A.Turner and P.E.Bateman. The role of polyphosphoinositides and their break down products in A23187 induced release of arachidonic acid from rabbit polymorphonuclear leucocytes. Biol. Che. Jl. 23: 425-436 (1986).

20. U.V. Singh, K.S.Bisht, S.Rao, P.Uma Devi and N. Udupa. Reduced toxicity and enhanced antitumour efficacy of Plumbagin using poly (lactic-co-glycolic) biodegradable injectable implant. Ind. J.Pharmacol. 29(3):168-172 (1997).

21. Kamal Mohammed Abdul and Rao Pinnti Rachender. Modulatory effect of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) on macrophage functions in BALB/c mice I. Potentiation of macrophage bactericidal activity. Imunopharmacol. 30(3):231-236(1995).

22. Jai C. Tilak, Soumyakanti Adhikari and P.A.Thomas Devasagayam. Antioxidant properties of Plumbago zeylanica, an Indian medicinal Plant and its active ingredient, plumbagin. Redox Report. 9(4):219-227 (2004).

MILESTONES AND ACHIEVEMENTS – PHCOG.NET - (2004 -2006) Pharmacognosy Network Worldwide is a non-profit network dedicated to Natural Products Research in order

to develop promising drugs. Phcog.net was started on July 6, 2004

• Development and launch of Website – www.phcog.net

• Initiation of Discussion forum – http://groups.yahoo.com/group/phcog/

• Started a forum – www.phcog.net/forum.php

• Started a New Online peer reviewed magazine – Pharmacognosy Magazine (PHCOG MAG).

Editorial team was finalized for the term of three years (2004-2007).

• Release of four issues in 2005.

• Project Phcog Refbase started in the month of May 2005.

• Release of 8th issue of Pharmacognosy Magazine in Oct 2006.

• Print version of Pharmacognosy Magazine

• Knowledge base section – http://www.phcog.net/knowledge

• Online web based manuscript handling system – http://www.phcogmag.com

• First issue of Phcog E –news – http://www.phcog.net/bulletin

• Phcog.Net Careers – http://www.phcog.net/careers

• Phcog.Net Global Activities – http://www.phcog.net/global





Hypoglycemic and antihyperglycemic effect of alcoholic extract of Euphorbia leucophylla and its fractions in normal

and in alloxan induced diabetic rats Satyanarayana T.*, Katyayani B.M., Hema Latha E., Anjana A. Mathews,

Chinna Eswaraiah M. Department of Pharmacognosy & Phytochemistry, Dept. of Pharmaceutical Sciences, Andhra University,

Visakhapatnam-530 003. Andhra Pradesh, INDIA. *Correspondence: [email protected] ; [email protected]

ABSTRACT - The hypoglycemic and antihyperglycemic effect of alcoholic extract of Euphorbia leucophylla was investigated in normal and alloxan induced diabetic rats. A single oral administration of alcoholic extract at doses 100, 250 and 500 mg kg-1 produced a significant blood glucose reduction in a dose dependent manner and elevated the serum insulin levels in normal and diabetic rats. A bioguided extraction and fractionation of alcoholic extract of the roots of E. leucophylla afforded a new flavone xylopyranoside, 4', 5-Dihyroxy-6, 7-dimethoxy flavone-3-O-β-D-xylopyranoside (ELR-05), identified as active principle together with the known compounds taraxerol, taraxerone, lupeol, 3-O-(z)-p-coumaroyl taraxerol. The isolated compound, 4', 5-Dihyroxy-6, 7-dimethoxy flavone-3-O-β-D-xylopyranoside obtained by ethylacetate fraction of alcoholic extract at doses 25, 50 and 75 mg/kg significantly reduced the blood glucose levels and increased the serum insulin levels in normal and diabetic rats. These data confirm the hypoglycemic and antihyperglycemic effect of alcoholic extract and its active isolated compound (ELR-05) in normal and diabetic rats.

KEYWORDS- Diabetes mellitus; Euphorbia leucophylla; Alloxan; Flavone xylopyranoside INTRODUCTION Diabetes is a serious metabolic disorder with micro and macrovascular complications that results in significant morbidity and mortality(1).Chronic hyperglycemia during diabetes causes glycation of body proteins that in turn leads to secondary complications affecting eyes, kidneys, nerves and arteries (2).These may be delayed, lessened or prevented by maintaining blood glucose values close to normal. The increasing number of ageing population, consumption of calories rich diet, obesity and sedentary life style have led to a tremendous increase in the number of diabetics world-wide. According to WHO projections, the prevalence of diabetes is likely to increase by 35%. Currently there are over 150 million diabetics worldwide and this is likely to increase to 300 million or more by the year 2025. Statistical projection about India suggests that the number of diabetics will rise from 15 million in 1995 to 57 million in the year 2025 making it the country with the highest number of diabetics in the world (3,4). Therefore, it is necessary to look for new solutions to manage this health problem. In modern

medicine, the beneficial effects on glycemic levels are well documented; the preventing activity of these drugs against progressive nature of diabetes and its micro and macrovascular complications was modest and not always effective. Insulin therapy affords effective glycemic control, yet its short comings such as ineffective-ness on oral administration, short shelf life, requirement of constant refrigeration, and in the event of excess dosage — fatal hypoglycemia —limits its usage. Treatment with sulfonylureas and biguanides is also associated with side effects (1). It is apparent that due to the side effects of the currently used drugs, there is a need for a safe agent. with minimal adverse effects, which can be taken for long durations. Recently, the search for appropriate hypoglycemic agents has been focused on plants used in traditional medicine partly because of leads provided by traditional medicine to natural products that may be better treatments than currently used drugs (5) In the indigenous system of medicine (Ayurveda), a mention was made on good number of plants for the cure of diabetes or ‘Madhumeha’ and some of them have been experimentally evaluated and




the active principles were isolated (6,7,8). However, search for newer antidiabetic drugs continues. The Euphorbiaceae family is one of the largest families in the plant kingdom. It comprises 263 genera and about 7300 species of almost cosmopolitan distribution. Euphorbia, the largest genus of Euphorbiaceae, with about 1600 species is characterized by the presence of milky latex. This genus has been the subject of numerous chemical studies (9, 10, 11). Plants belonging to Euphorbia species have been the subjects of many investigations for their biologically active components. Biological activities including skin irritant, tumor promotion and proinflammatory properties are attributed to the presence of specific classes of macro- and polycyclic diterpenes (12, 13, 14). Some species of Euphorbia have been used as medicinal plants for the treatment of skin diseases, gonorrhea, migraine, intestinal parasites, wart cures (15) and as anti-inflammatory in Indian folk medicinal system. However, no chemical investigation on Euphorbia leucophylla has been reported up to now. In the present study, we report the isolation and structure elucidation of a new flavone xyloside namely 4', 5-Dihyroxy-6, 7-dimethoxy flavone-3-o-β-D-xylopyranoside (compound ELR-05) from roots of E. leucophylla. In addition, taraxerol, taraxerone, lupeol, 3-(z)-p-coumaroyl taraxerol were isolated. MATERIALS AND METHODS Plant material The roots of the plant Euphorbia leucophylla was purchased from local nurseries and identified by Dr. M. Venkaiah, Associate Professor, Department of Botany, Andhra University. A voucher specimen (MN-108) is deposited in herbarium of our Department, Andhra University, Visakhapatnam. Animals Laboratory bred Sprague Dawley rats of either sex weighing 200-225 g were employed for the study. All animals were procured from National Institute of Nutrition, Hyderabad. The rats were maintained under standard laboratory conditions at 25 ± 2°C, relative humidity 50 ± 15% and normal photo period [12 h dark / 12 h light] were used for the experiment. Commercial pellet diet [Ratan Brothers, India] and water were provided ad libitum. The experimental protocol has been approved by the Institutional Animal Ethics committee and by the Regulatory body of the government [Regd no.516/01/A/CPCSEA].

Drugs Alloxan monohydrate was purchased from sigma chemicals (St Louis, USA). All other chemicals used for this study were of analytical grade Extraction and isolation procedure Freshly collected roots of E. leucophylla were cut into small pieces and shade dried. The dried roots were powdered in Wiley mill. The powdered root (1500 g) was extracted with ethyl alcohol (95% v/v) by process of continuous extraction (Soxhlation). The crude extract was evaporated to dryness in a rotary film evaporator. The yield of dried extract was 48g. The alcoholic extract was fractioned with hexane (10x250 ml), ether (4x150ml), chloroform (7x150ml), ethylacetate (4x150ml) and methanol (15x200ml). All fractions were concentrated and the percentage yields were: hexane 3%, ether 10%, chloroform 20%, methanol 1% and ethylacetate 1% (w/w) in terms of dry plant material. While concentrating the ethylacetate layer, a yellow compound was precipitated and it was filtered under vacuum and the filtrate was concentrated, purified and noted as a compound ELR-05 (yield 18.33%). Among all the fractions, the hexane fraction showed four distinct spots in thin layer chromatography over silica gel in different solvent systems and the hexane layer was chromatographed over silica gel (finer than 200#, ACME) column and eluted taking 1000 ml fractions, starting with hexane/ EtoAc, 90:10 (frs. 20-24) to afford compound ELR-01 (yield 0.011%), Hexane/EtoAc, 90:10(frs. 25-30) to afford compound ELR-02 (yield 0.016%), hexane/EtoAc , 85:15 (frs 31-34) to afford compound ELR-03 (yield 0.012%), hexane/EtoAc , 80:20 (frs. 37-40) to afford compound ELR-04 (yield 0.026%). Compound ELR-01, ELR-02, ELR-03 and ELR-04 were identified as taraxerol, taraxerone, lupeol and 3-(z)-p-coumaroyl taraxerol respectively and by comparing the data with known literature values of NMR and IR. Compound ELR-05 was identified as 4, 5-Dihyroxy-6,7-dimethoxy flavone-3-O-β-D-xylopyranoside. Toxicity evaluation in mice The alcohol extract was tested for its acute and short- term toxicity (if any) in mice. To determine acute toxicity, a single oral administration of the alcoholic extract at doses of 0.25, 0.5, 0.75 and 1.0 gm/kg were administrated to different groups of mice (5 mice were used for each group, control mice received Tween 80). Mortality and general behavior of the animals were observed periodically for 48 h. The animals were observed continuously for the initial 4 h




and intermittently for the next 6 h and then again at 24 h and 48 h following administration of the plant extract. The parameters observed were grooming, hyperactivity, sedation, loss of righting reflex, respiratory rate and convulsion. To study short-term toxicity, 3 groups of mice each containing 6 male mice (20-25 g, body weight) were used. Group 1 was kept as control and Group II and III received 200 and 400 mg/kg alcoholic extract respectively in 5% Tween 80. The extract was administrated daily for 14 days (p.o.). Control group received 5% Tween 80 in an identical manner. The behavior of the animals was observed daily for 1h in the forenoon (10 to 11 A.M.) for 14 days. Initial and final body weights, water and food intake, state of stool and body temperatures were observed. Induction of diabetes Animals were allowed to fast 18 h and were injected with alloxan monohydrate dissolved in sterile normal saline at a dose of 140 mg/kg body weight intraperitoneally. After 2 weeks, diabetic rats (250-350 mg/dl) were used for the experiment. Experimental design In the experiment a total number of 80 rats (40 normal and 40 diabetic surviving rats) were used. The rats were divided into 16 groups (8 normal and 8 diabetic), each group consisting of 5 animals. Group 1 normal rats treated with vehicle (1% Sodium CMC) and served as normal control, and Group 2, 3 and 4 normal rats were treated with alcoholic extract of E. leucophylla at a doses of 100, 250, and 500 mg/kg respectively, Group- 5, 6 and 7 normal rats treated with compound ELR-05 at doses 25, 50 and 75 mg/kg dose respectively. Group 8 normal rats treated with tolbutamide (40 mg/kg) served as standard reference. Group 9 diabetic rats treated with vehicle and served as diabetic control. Group- 10, 11and 12 diabetic rats were treated with alcoholic extract of E. leucophylla at doses of 100, 250, and 500 mg/kg respectively; Group- 13, 14 and 15 diabetic rats were treated with compound 5 at doses 25, 50 and 75mg/kg dose respectively. Group- 16 diabetic rats treated with 40 mg/kg dose of Tolbutamide. All the doses were administered orally. Oral glucose tolerance test After overnight fasting, 0 min blood sample (0.2 ml) was taken from the rats in normal control, diabetic control, normal + plant extract treated group, normal + compound ELR-05 treated, diabetic + plant extract treated, diabetic + compound ELR-05 treated groups by orbital sinus puncture(16). Glucose solution (2 g/kg) was administered orally immediately. Four more

samples were taken at 30, 60, 90 and 120 min after glucose administration (17). Estimation of blood glucose and serum insulin levels The rats were fasted for 18 h and blood samples were collected by puncture of retro-orbital plexus immediately with capillary tube under ether anesthesia into glass vials containing a small quantity of a mixture of potassium oxalate and sodium fluoride as an anticoagulant at 0 h (before treatment) and 2, 4, 6, 8, 12, 16, 24 h (after treatment). The plasma blood glucose levels were determined by using GOD--POD method (18). Serum insulin was determined by Radio Immunoassay (RIA) method. Statistical analysis All values were expressed as Mean ± SEM. The differences were compared using one-way analysis of variance (ANOVA) followed by Dunnet’s t test. P values <0.05 were considered as significant. RESULTS Compound ELR-01: colourless needles from CHCl3: MeOH, m.p. 280-2820c, [α]+0.780c (CHCl3, c1.14). The molecular formula was determined to be C30H50O by HR–MS. It gave a positive Liebermann- Burchard test and readily formed a monoacetate, m.p. 292-2940c. The 1H as well as 13C NMR- data is in agreement with those already reported earlier for taraxerol (19,20,21). 13C NMR spectrum of ELR-01 acetate was found to be identical with spectrum of taraxerol acetate. 1. Compound ELR-02: colourless needles from MeOH, m.p.238-2400c, [α]+ 11.60C (CHCl3, c 0.78). The molecular formula was determined to be C30H48O by HR–MS. The 1H as well as 13C NMR- data was found to be identical with spectrum of those already reported earlier for taraxerone (21, 22, 23). Compound ELR-03: colourless needles from CHCl3, m.p.210-2120c, [α] +270C (CHCl3, c 0.985). The molecular formula was determined to be C30H50O by HR–MS. The 1H as well as 13C NMR- data was found to be identical with spectrum of those already reported earlier for lupeol (23,24,25,26). Compound ELR-04: Amorphous powder from CHCl3, m.p. 2840C. The molecular formula was determined to be C39H56O3 by HR–MS. It gave a violet colour changing to purple in Liebermann- Burchard test. It gave monoacetate, m.p. 2480c. The 1H as well as 13C NMR spectrum of ELR-04 and ELR-04 acetate was identical with those already reported earlier for taraxerol. 13C NMR spectrum of ELR-01 acetate was found to be identical with spectrum of 3-o-(z)-p-coumaroyl taraxerol and its acetate respectively (27).




Compound ELR-05: Yellow amorphous powder, m.p.193-1940C. The molecular formula was determined to be C39H56O3 by HR–MS, IR νmax (CHCl3) cm-1; 3350, 1640, 1620, 1595, 1295, 1215, 1085, 1060; MS m/z (rel. int.): 330 (M+, 100) (C22H22O11), 312 (30) (C22H22O10), 301 (10) (C21H21O10), 287 (95) (C20H19O10) 269 (22), 258 (6.6), 244 (15.5), 216 (5), 187 (7.2), 165 (12.4), 153 (10.0), 137 (5), 121 (33.3), 106 (7.6), 93 (11.7), 69 (16.5). 1H NMR (pyridine-ds): δ 3.43 (1H,dd, J=11.7, 8.5 Hz, H-5''b), 3.51 (1H, dd, J=7.7, 6.3 Hz, H-2''), 3.59 (1H, dd, J=7.7, 7.7 Hz, H-3''), 3.61 (1H, m, H-4''), 3.83 (3H, s, 7-OCH3), 3.95 (3H, s, 6-OCH3), 4.00 (1H, dd, J=11.7, 4.6 Hz, H-5''a), 4.95 (1H, d, J=6.3 Hz, H-1''), 6.64 (1H, s, H-8), 7.20 (2H, d, J=8.0 Hz, H-3', 5'), 7.50 (1H, s, 4-OH), 8.40 (2H, d, J=8.0 Hz, H-2', 6'), 13.13 (1H, s, 5-OH). 13C NMR (pyridine-d5, 100 MHz): δ 158.2 (C-2), 135.5 (C-3), 179.5 (C-4), 153.5 (C-5), 132.2 (C-6), 159.8 (C-7), 91.7 (C-8), 153.0 (C-9), 107.0 (C-10), 122.3 (C-1'), 132.4 (C- 2',6'), 116.5 (C-3', 5'), 162.2 (C-4'), 104.8 (C-1''), 73.7 (C-2''), 75.7 (C-3''), 70.2 (C-4''), 66.6 (C-5''), 60.9 (6-OCH3), 56.8 (7-OCH3). The structures of compounds were given in Fig.1. Effect on normal rats The effect of different doses of alcoholic extract of E. leucophylla on fasting blood sugar levels in normal rats were assessed at different time interval. The maximum percentage blood glucose reduction with 100, 250 and 500 mg/kg doses of E. leucophylla at 8 h were 32.69%, 40.08% and 47.49% respectively. Where as the treatment with the compound ELR-05 at a dose of 25, 50, 75 mg/kg produced maximum percentage reduction at 6h and were found to be 38.53%, 41.40% and 46.69% respectively. Tolbutamide 40 mg/kg dose produced 44.44% blood glucose reduction at 6 hr in normal rats and the results are shown in Table 1. In normal control animals, the serum insulin levels were found to be 16.12 ± 0.56 µU/ml at 8 h, where as the treatment of alcoholic extract of E. leucophylla and compound ELR-05 at dose of 500 mg and 50 mg /kg produced a significant increase in serum insulin levels and were found to be 40.13 ± 1.32 µU/ml and 41.13 ± 0.98 µU/ml respectively at 8 h when compared to control group. Similarly tolbutamide 40 mg/kg dose produced a significant rise in serum insulin level at 8 h in normal rats Effect on alloxan induced diabetic rats The antihyperglycemic effect of different doses of alcoholic extract of E. leucophylla on fasting blood glucose levels in diabetic rats were assessed at different time intervals. The percentage blood glucose reduction with 100, 250 and 500 mg/kg dose of E.

leucophylla at 6 h were 33.03%, 41.79% and 48.00% respectively. Where as the treatment with compound ELR-05 at a dose of 25, 50, 75 mg/kg produced maximum percentage reduction at 6h and were found to be 46.46%, 50.19% and 52.24% respectively. Tolbutamide (40 mg/kg) produced 48.65% blood glucose reduction in alloxan induced diabetic rats and the results are shown in table 2. In diabetic control animals, the serum insulin levels at 8 h were found to be 4.95 ± 0.43 µU/ml, the treatment of alcoholic extract of E. leucophylla and compound ELR-05 at a dose of 500 mg and 50 mg/kg produced a significant rise in serum insulin level at 8 h and were found to be 20.18 ± 1.35 µU/ml and 19.98 ± 0.93 µU/ml respectively, when compared to control group. Similarly tolbutamide 40 mg/kg dose produced a significant rise in serum insulin level at 8 h in and alloxan induced diabetic rats. Toxicity evaluation Although it is the normal practice to determine the LD50 value, now it is acceptable to limit the study with an acute toxicity test using several doses including reasonably high doses of the drugs. In the present study, acute toxicity was tested up to a high concentration of 1 gm/kg (two times more than the active dose). Even at this dose the herbal extract did not exhibit any sign of toxicity. Since the main purpose of the preliminary acute toxicity study is to get some idea on conspicuous behavioral changes and death, if any, and the alcoholic extract of E. leucophylla did not exhibit any toxic symptoms in the limited toxicity evaluation in male mice. Daily feeding for 14 days with the alcohol extract (200 and 400 mg/kg) did not result in any change in general behavior of the animals. Body temperature and state of the stool were also not influenced by the drug treatment. Body weight, weight of liver, kidneys and spleen and food and water intake were not significantly altered by the drug administration Effect on oral glucose tolerance test Administrations of the alcoholic extract of E. leucophylla (500 mg/kg) and compound ELR-05 (50 mg/kg) orally half an hour prior to glucose load showed improved glucose tolerance in normal rats. In control animals the percentage increase in blood glucose at 60 min are found to be 83.15%, where as the treatments with alcoholic extract (500 mg/kg) and compound ELR-05 (50 mg/kg) ameliorates the percentage increase in blood glucose significantly and the results were found to be 38.15% and 41.67% respectively at 60 min in normal rats. Glucose tolerance in alloxan-induced




HOH

24

123

45

678

9

10

1112

13

14

15

16

1718

1920

2122

23

25

27

2628

29 30

HO

H

diabetic rats exhibited a similar pattern to that of normal rats and the changes in the levels of blood glucose in diabetic control and experimental groups

after oral administration of glucose (2 g/kg) were shown in Table 4.

3029

2826

27

25

23

2221

2019

18 17

16

15

1413

1211

10

9

87

65

4321

H24

O

Taraxerol (ELR-01) Taraxerone (ELR-02) Lupeol (ELR-03)

2826

27

25

23

2221

2019

18 17

16

15

1413

1211

10

9

87

65

4321

OH

24

OHO

29 30

1,2,

3,

1,,2

,,

3,,

4,,

5,, 6

,,

O

OOHH3CO

H3CO

OH

OO

HO

OHOH

1 234

56

78 1

23

4

56

1,,

2,,

3,,

4,,

5,,

3-O-(Z) p-Coumaroyl taraxerol (ELR-

04) 4', 5-Dihydroxy-6,7-dimethoxy flavone-3-O-β-D-xylopyranoside

(ELR-05) Fig.1. Structures of compounds ELR-01 to ELR-05

DISCUSSION The present study was conducted to evaluate the hypoglycemic and antihyperglycemic activity of E. leucophylla a herbal drug, first identified by us, to get a berth in the group of antidiabetic herbal drugs. In this study, the alcoholic extract of E. leucophylla produced a dose dependent percentage blood glucose reduction in normal and diabetic groups. In normal treated groups a significant percentage blood glucose reduction was observed up to 18 h and maximum percentage blood glucose reduction was observed at 8 h, where as in diabetic groups a significant reduction in blood glucose was maintained up to 24 h and maximum at 6 h. The percentage blood glucose reduction produced by the extract in diabetic group is greater than the percentage reduction observed in normal treated groups and E. leucophylla (500 mg/kg) dose produced better percentage blood glucose reduction than the tolbutamide in normal and diabetic groups. The treatment with compound ELR-05 (50 mg/kg) produced a maximum percentage blood glucose reduction at 6 h in normal and diabetic rats. Different mechanisms of action to reduce blood glucose levels with the help of plant extracts already exist. Some plants exhibit properties similar to the well-known sulfonylurea drugs like tolbutamide; they

reduce blood glucose in normoglycemic animals (28,29). Some other plants act like biguanides such as metformin, which is an antihyperglycemic compound; they do not affect blood glucose in normal state (30, 31, 32, 33). We hypothesized that E. leucophylla could have a sulfonylurea-like mechanism since it decreased blood glucose in normoglycemic rats such as tolbutamide. It is also known that alloxan selectively destroys insulin-secreting β-cells in the islets of Langerhans and their effects are irreversible (34). In the present study, the dose of alloxan (140 mg/kg, i.p.) was selected in order to partially destroy the pancreatic β-cells. In these conditions, insulin was secreted but not sufficiently to regulate the blood glucose. Sulfonylurea compounds lower blood glucose in normal and type 2 diabetic animals by stimulating insulin release from β-pancreatic cells. In the present study, the treatments with alcoholic extract of E. leucophylla (500 mg/kg) and compound ELR-05 (50 mg/kg) significantly elevated the serum insulin levels in normal and alloxan induced diabetic rats. Study on underlying cellular mechanism by which the alcoholic extract of Euphorbia leucophylla stimulate the secretion of insulin from β-cells, cannot be defined.




Table 1 - Effects of oral administration of alcoholic extract of E. leucophylla roots on fasting blood glucose level in normal rats.

Blood glucose levels (mg/dl) at different hours Group

Dose (mg/kg) 0 h 2 h 4 h 6 h 8 h 12 h 18 h 24 h

Control

-----

87.90±2.05

86.12 ± 0.9 (2.02)

85.50±1.34 (2.73)

82.15±0.95 (6.54)

79.28±1.80 (9.80)

79.66±1.74 (9.37)

78.74±2.23 (10.42)

78.34±2.10 (10.87)

E. leucophylla 100 88.23±1.65 84.13±1.20 (4.64)

75.13±1.97 (14.84)*

63.15±1.35 (28.42)*

59.38±1.50 (32.69)*

61.32±0.95 (30.49)*

69.39±1.09 (21.35)*

78.45±0.97 (11.08)

250 90.30±1.97 81.15±1.79 (10.13)

66.57±1.77 (26.27)*

57.12±1.43 (36.74)*

54.1±1.12 (40.08)*

63.16±1.32 (30.05)*

74.65±1.41 (17.33)*

80.17±0.85 (11.21)

500 89.77±0.97 74.20±1.38 (17.34)

61.44±1.52 (31.55)*

51.41±1.06 (42.73)*

47.13±1.69 (47.49)*

58.70±0.29 (34.61)*

68.35±2.69 (23.86)*

74.17±3.12 (17.37)*

ELR-05 25 85.51±0.58 74.03±0.69 (3.42)

64.85±0.90 (24.16)*

55.12±2.13 (35.53)*

61.31±0.60 (28.30)*

69.24±1.58 (19.02)*

75.13±0.66 (12.13)

84.68±0.42 (0.97)

50 87.96±1.22 72.81±1.25 (17.22)

62.20±1.31 (29.28)*

51.54±1.48 (41.40)*

58.90±1.30 (33.03)*

61.31±1.01 (30.29)*

68.19±3.21 (22.47)*

71.54±1.30 (18.66)*

75 87.62±0.64 64.54±0.59 (26.34)*

51.79±0.74 (40.89)*

46.71±0.69 (46.69)*

50.72±1.83 (42.11)*

58.69±0.55 (33.01)*

65.43±1.43 (25.32)*

69.15±2.61 (21.07)*

Tolbutamide 40 88.44±1.95 69.35±1.67 (21.58)*

52.36±1.21 (40.79)*

49.13±1.65 (44.44)*

52.95±1.06 (40.12)*

63.87±1.45 (27.78)*

70.52±1.21 (20.26)*

79.16±0.69 (10.49)

All values are expressed as Mean ± SEM, n=5 ; Values given in the parenthesis are percent blood glucose reduction.; *Statistically significant P<0.05 compared to 0 h of their respective group.




Table 2 - Effects of oral administration of alcoholic extract of E. leucophylla roots on fasting blood glucose level in diabetic rats.

Blood glucose levels (mg/dl) at different hours Group

Dose (mg/kg) 0 h 2 h 4 h 6 h 8 h 12 h 18 h 24 h

Control ----- 367.09±2.0

359.75±1.4 (1.99)

341.31±9.7 (7.02)

339.9±8.6 (7.40)

333.87±2.5 (9.04)

328.68±1.1 (1046)

330.19±4.7 (10.05)

335.12±8.6 (8.70)

E. leucophylla 100 337.95±8.9 301.54±5.6 (10.77)

278.68±4.4 (17.53)*

226.3±4.6 (33.03)*

249.32±3.5 (26.25)*

265.2±1.95 (21.52)

269.98±3.7 (20.11)*

278.85±7.3 (17.48)*

250 371.38±4.2 328.62±5.2 (11.51)

298.52±2.9 (19.61)*

216.15±1.1 (41.79)*

221.32±8.6 (40.40)*

253.63±1.6 (31.70)

274.52±2.6 (26.08)*

285.74±5.1 (23.05)*

500 385.32±2.8 332.13±5.3 (13.80)*

261.61±4.6 (32.10)*

200.35±6.3 (48.00)*

213.56±5.4 (44.57)*

259.61±2.5 (32.62)

296.31±1.3 (23.10)*

302.31±2.5 (21.54)*

ELR-05 25 335.31±1.7 271.10±1.2 (19.14)*

230.54±5.7 (31.24)*

179.52±1.1 (46.46)*

201.17±0.9 (40.00)*

262.63±2.1 (21.67)

275.14±1.3 (17.94)*

285.12±1.8 (14.96)*

50 367.17±5.3 271.52±1.0 (26.05)*

219.51±2.1 (40.21)*

180.23±3.1 (50.91)*

235.45±2.1 (35.87)*

251.41±1.3 (31.52)

262.33±8.8 (28.55)*

274.31±1.5 (25.29)*

75 359.41±4.2 268.31±1.8 (25.34)*

223.51±2.3 (37.81)*

171.63±3.4 (52.24)*

198.56±8.5 (44.75)*

252.54±1.9 (29.730

265.18±1.1 (26.21)*

269.58±2.3 (24.99)*

Tolbutamide 40 368.54±3.4

289.51±1.6 (21.44)*

225.75±5.0 (38.74)*

189.23±1.6 (48.65)*

222.12±1.6 (39.72)*

256.67±9.8 (30.35)

286.45±2.1 (22.27)*

302.32±5.2 (17.96)*

All values are expressed as Mean ± SEM, n=5 ; Values given in the parenthesis are percent blood glucose reduction. ; *Statistically significant P<0.05 compared to 0 h of their respective group.




Table 3 - Effects of oral administration of alcoholic extract of E. leucophylla roots on fasting blood glucose level in normal and diabetic rats.

Serum insulin (µU/ml) in normal rats Serum insulin (µU/ml) in diabetic rats Treatment Dose

(mg/kg) 0 h 4 h 8 h 12 h 0 h 4 h 8 h 12 h Control

-------

18.10±0.17

16.23±0.35

16.12±0.56

15.42±2.1

5.32±0.51

4.91±0.32

4.95±0.43

5.01±0.52

E. leucophylla 500 19.36±0.58 32.14±1.2* 40.13±1.32* 28.56±0.9 4.89±0.56 9.31±1.23 20.18±1.35* 16.54±0.98 ELR-05 50 16.13±1.23 34.16±2.3* 41.13±0.98* 39.23±1.1* 4.46±0.23 10.31±0.97 19.98±0.93* 18.8±0.67* Tolbutamide 40 16.96±0.75 33.45±1.8* 43.15±1.12* 36.17±1.3* 4.57±0.67 9.67±0.71 21.32±0.54* 17.6±1.42*

All values are expressed as Mean ± SEM. *Statistically significant P<0.05 compared to 0 min of their respective group.

Table 4 - Oral glucose tolerance test in normal and experimental animals

Blood glucose levels (mg/dl) Groups 0 min 30 min 60 min 90 min 120 min

Normal control 89.12 ± 2.88 172.38 ± 6.02* 153.72 ± 5.82* 124.92 ± 3.14* 95.38 ± 3.28 Normal + E. leucophylla

(500mg/kg) 85.12 ± 1.23 132.61 ± 4.12* 113.67 ± 2.31* 99.58 ±3.17* 92.67 ±5.12

Normal + ELR-05

91.34 ± 3.54 128.69 ± 4.13* 116.52 ±3.71* 100.75± 1.57* 92.85 ±1.65

Normal + tolbutamide 95.63 ± 5.36 140.35 ± 1.21* 111.35 ± 5.85* 98.34 ±3.45* 92.17±4.35

Diabetic control 247.84 ± 4.46 322.24 ± 5.47* 377.01 ± 10.59* 349.28 ± 3.77* 316.66 ± 3.57*

Diabetic + E. leucophylla 120.64 ± 3.05 207.02 ± 5.49* 183.66 ± 7.66* 149.06 ± 5.11* 128.16 ± 3.64 Diabetic + ELR-05 135.67 ±5.64 199.67 ±6.89* 175.64 ±5.61* 141.28 ±3.33 116.61±5.52

Diabetic + Tolbutamide 122.12 ± 1.88

207.1 ± 4.62*

187.34 ± 5.93*

48.92 ± 4.95*

129.62 ± 1.92

All values are expressed as Mean ± SEM. *Statistically significant P<0.05 compared to 0 min of their respective group.




However, it may stimulate insulin secretion like the oral hypoglycemic agents i.e. by influencing depolarization of islet membrane and thereby the changes in the ion-flux, as most of the insulin releasing agents act. Further intensive study can project light on the molecular mechanism of action of insulin secretagogue activity of Euphorbia leucophylla. Flavonoids, sterols/triterpenoids, alkaloids and phenolics are known to be bioactive antidiabetic principles (7, 8, 35, 36). Flavonoids are known to regenerate the damaged beta cells in the alloxan diabetic rats (37). In the present study, a flavone xylopyranoside (compound ELR-05) exhibited dose dependent decrease in blood glucose levels and elevated the serum insulin levels in normal and alloxan induced diabetic rats. Further work is in progress to identify the possible mechanisms of action for its hypoglycemic and antihyperglycemic activities. REFERENCES 1. The Endocrine pancreas and the control of blood

glucose. In: Barbara Simmons, Susan Beasley. Eds. In H.P. Rang, M.M. Dale and J.M. Ritter’s, Pharmacology, 3rd edition, U.K., Longman Group Ltd., 403–410 (1991).

2. Sharma AK. Diabetes Mellitus and Its Complications: An Update. (1st ed. Macmillan, New Delhi, 1993)

3. H. King, R.E. Aubert and W.H. Herman. Global burden of diabetes 1995-/2025: prevalence, numerical estimates and projections. Diabetes Care 21: 1414-/1431(1998).

4. J.P. Boyle, A.A. Honeycutt, K.M. Narayan, T.J. Hoerger, L.S. Geiss, H. Chen and T.J. Thompson. Projection of diabetes burden through 2050: Impact of changing demography and disease prevalence in the US. Diabetes Care, 24: 1936-1940 (2001).

5. S.M. Rates. Plants as source of drugs. Toxicon 39 (5): 603–613 (2001).

6. J.K. Grover, S. Yadav and V. Vats , Journal of Ethnopharmacology. 81: 81-100(2002).

7. M.D. Ivorra, M. Paya, A. Villar. A review of Natural Products and Plants as Potent antidiabetic drugs. Journal of Ethnopharmacology, 27(3): 243–275 (1989).

8. B. Kameswara Rao, R. Giri, M.M. Kesavulu and Ch. Appa Rao. Herbal medicine: in the management of diabetes mellitus. Manphar Vaidhya Patrika, 1(4): 33–35 (1997).

9. A.K. Singla, K. Pathak. Phytoconstituents of Euphorbia species. Fitoterapia. 61: 483–516 (1990).

10. V.U. Ahmad, A.R. Jassbi. Three tricyclic diterpenoids from Euphorbia decipiens. Planta Medica, 64: 732–735 (1998).

11. G. Vogg, E. Mattes, J. Rothenburger, N. Hertkorn, S. Achatz and Jr. H. Sandermann. Tumor promoting diterpenes from Euphorbia leuconeura L. Phytochemistry, 51: 289–295 (1999).

12. J.A. Marco, J.F. Sanz-Cervera and A. Yuste. Ingenane and lathyrane diterpenes from latex of Euphorbia canariensis. Phytochemistry, 45: 563-570(1997).

13. Q.G. Ma, W.Z. Liu, X.Y. Wu, T.X. Zhou and G.W. Qin. Diterpenoids from Euphorbia fischeriana. Phytochemistry, 44 (4): 663-666 (1997).

14. F.J. Evans, S.E. Taylor, Progress in the Chemistry of Organic Natural Products, (Springer-Verlag,New York, 44, 1983) pp. 1–99.

15. T.Morgenstern, M. Bittner, M. Silva, P.Aqueveque and J. Jakupovic. Diterpenes and phloracetophenones from Euphorbia portulacoides. Phytochemistry, 41: 1149–1153 (1996).

16. B.H. Waynforth, Injection techniques, Experimental and surgical techniques in the rat, (Academic press, London, 1980) pp. 3-61.

17. K.B. Whittington, S. Soloman and N. Lu. 1991. Islet allografts in the cryptorchid testes of spontaneously diabetic BB/Word p rats: response to glucose, glipizide and arginine. Endocrinology, 128: 2671-2677(1991).

18. A.Kunst, B.Draeger and J.Ziegenhorn, Methods of Enzymatic Analysis, (Vol.6,VCH, Weinheim,W. Germany,1984) pp.178-185.

19. M. Zhi-da, M. Mizuno, T. Tanaka, M. Iinuma, X. Guang and Y.H. Qing. A diterpene from Euphorbia antiquorum. Phytochemistry, 28(2): 553-555 (1989).

20. A.S.R.Anjeneyulu, L.Ramachandrarow, C. Subrahmanyam and K.Suryanarayana Murty. Crystalline constituents from euphorbiaceae—XIII: The structure of a new triterpene from Euphorbia nerifolia L. Tetrahedron, 29(23): 3909 -3914 (1973).

21. M. Ahmed, H. Atallah, J. Nicholas, Triterpenoids and steroids of Euphorbia pilulifera. Phytochemistry, 11(5): 1860 (1972).




22. M.C.L. Elisabete, M.R.M. Jorge and B.D. Laurence. Pentacyclic triterpenes from Euphorbia stygiana. Phytochemistry, 63(4): 421-425 (2003).

23. D. R. Misra, D. B. Naskar, T. K. Ray and H. N. Khastgir Filices. Phytosterols in plants. Phytochemistry, 12(7): 1819-1820 (1973).

24. G.G. Antonio, M.F. Braulio, G. Pedro and G.R. Angel. Triterpenes from latex of Euphorbia balsamifera. Phytochemistry 15(3): 427 (1976).

25. A.N. Starratt. Cycloartenol and lupeol from Euphorbia esula. Phytochemistry, 12(1): 231 (1973).

26. D. Lavie, K. Mahendra, T. Jain and O. Orebamjo. Terpenoids—VII. Constituents of Euphorbia lateriflora Schum. and Thonn. Phytochemistry, 7(4): 657-660 (1968).

27. U. Kokpol, W. Chavasirui, V. Chittawong, M. Bruce, G.N. Cunningham and D.H. Miles. Long chain aliphatic alcohols and saturated carboxylic acids from heartwood of Rhizophora apiculata. Phytochemistry, 33(5): 1129-1131 (1993).

28. M.D. Ivorra, M.. Paya, A. Villar. Hypoglycemic and insulin release effects of tormentic acid, a new hypoglycemic natural product. Planta Medica, 54: 282–286 (1988).

29. S.N. Davis, D.K. Granner. Insulin, Oral Hypoglycaemic agents & the pharmacology of the Endocrine pancreas. In: J.G.Hardman, L.G.Limbird, Eds. L.S.Goodman, A.G. Gilman’s: The Pharmacological Basis of Therapeutics, 10th ed. New York, McMillan;1701-1704, (1996).

30. C.J. Bailey, C. Day, S.L Turner and B.A. Leaterdhale. Cerasee, a traditional treatment for diabetes, studies in normal and streptozotocin diabetic mice. Diabetes Research 2: 81–84 (1985).

31. L.S. Hermann, B. Schersten, P.O. Bitzen, T. Kjellstrom, F. Lindgarde and A. Melandev. Therapeutic comparison of metformin and sulfonylurea, alone and various mechanisms. Diabetes Care, 17: 1100–1109 (1994).

32. R.A. De Fronzo, A.M. Goodman. Efficacy of metformin in patients with non-insulin-dependent diabetes mellitus. New England Journal of Medicine, 333: 550–554 (1995).

33. M. Stumvoll, W. Nurjan, G. Periello, G. Dailey and J.E. Gerich. Metabolic effects of metformin in non-insulin-dependent diabetes mellitus. New England Journal of Medicine, 333: 541–549(1995).

34. J. Fisher.Drugs and chemicals that produce diabetes. Trends in Pharmacological Sciences, 6: 72–75 (1985).

35. Oliver-Bever, B., Medicinal Plants in Tropical West Africa, (Cambridge University Press, London, 1986) pp. 245–267.

36. Atta-Ur-Rhemann, Z. Khurshid. Medicinal plants with hypoglycemic activity. Journal of Ethnopharmacology, 26: 1–55 (1989).

37. B.K. Chakravarthy, S. Gupta, S.S. Gambir and K.D. Gode. Pancreatic beta cell regeneration. A novel antidiabetic mechanism of Pterocarpus marsupium Roxb. Indian Journal of Pharmacology, 12: 123–127 (1980).

JOIN AS REVIEWERS – PHCOG MAG.

PHCOG MAG. (An official magazine of Phcog.Net) recognizes the value and importance of the peer reviewer in the

overall publication process – not only in shaping the individual manuscript, but also in shaping the credibility and

reputation of a journal. Phcog Mag. is committed to the timely publication of all credible scientifically based

manuscripts submitted for publication. As such, the identification and selection of reviewers who have expertise and

interest in the topics appropriate to each manuscript are essential elements in ensuring a timely, productive peer

review process.

Phcog Mag. calls for reviewers who have keen interest in reviewing the manuscripts submitted. Medicinal plant

researchers (both from Academia and Industry are welcome), Send your resume along with the list of publications in

document format to [email protected] or [email protected] or [email protected]

Visit www.phcogmag.com, Register as author / reviewer and submit resumes for approval.

PHARMACOGNOSY MAGAZINE

(PHCOG MAG.) - An official publication of Phcog.Net


Hosur road, Bangalore 560 027, Karnataka, India





Antioxidant activity of a new diarylheptanoid from Zingiberofficinale

Sajjad Khan M1, Salma Khanam1*, Deepak M2 and Shivananda B.G.1 Department of Phamacognosy, Al – Ameen College of Pharmacy, Hosur road, Bangalore 560 027, India

Department of Phytochemistry, Natural Remedies Pvt. ltd., Hosur road, Bangalore, India For Correspondence: [email protected]

ABSTRACT - Rhizomes of Zingiber officinale commonly known as ginger are used popularly as spices. This drug is also used in traditional system of medicine as antiulcer and antioxidant. Many phytoconstituents had been isolated from oil and oleoresins of ginger. In the present study, a new diarylheptanoid was isolated from the spent ginger devoid of oleoresin. The spent ginger was extracted with 50% methanol and partitioned with butanol. Column chromatography and preparative HPLC of the butanolic extract resulted in isolation of a new compound which was characterized as 3, 5 diacetoxy-7-(3, 4 dihydroxy phenyl)-1-(3, 4 dihydroxy phenyl) heptane (ZA6). In vitro DPPH, superoxide scavenging bioassay indicated that, this diarylheptanoid is a potent antioxidant. KEY WORDS - Zingiber officinale, diarylheptanoids, DPPH assay, superoxide scavenging. INTRODUCTION Zingiber officinale Roscoe belonging to family Zingiberacea has been used as a spice and medicine for thousands of years (1). Utilization of plants and other crude preparations for therapeutic reason have several drawbacks including lack of marker compound to maintain uniformity of the herbal preparation. The isolation and characterization of marker compounds is one of the most important areas of research in medicinal plants. The isolation of markers permits structural determination of bioactive compounds that may enable production of synthetic material, incorporation of structural modification and rationalization of mechanism of action (2). Many ayurvedic formulations like “trikatu” are known to contain ginger extract as one of the ingredient. Gingerol and shogaol isolated from oleoresin are the only reported markers of ginger. Hence, in present study an attempt was made to isolate a new chemical constituent of Zingiber officinale from spent ginger extract using column chromatography and preparative HPLC and evaluate its anti-oxidant potential MATERIALS AND METHODS Raw material of fresh ginger was collected and authenticated at Natural Remedies Pvt. Ltd. The raw material was subjected to supercritical fluid extraction (SFE) to collect oleoresin devoid spent. The spent ginger obtained after SFE was extracted with 50% methanol in water for 4hrs. at 700C. The extract was partitioned with butanol and dried in vacuum tray drier at 70 0C. The butanolic extract was fractionated using silica gel and pet-ether, ethyl acetate, methanol

in varying concentration as stationary and mobile phase. The TLC of butanolic extracts and fractions of the column was optimized using precoated 60F254 silica plates with toluene: butanol: glacial acetic acid: water - 6:4:5:1 and pet-ether: chloroform: ethyl acetate: acetone - 2:5:1:2 with anisaldehyde as detecting agent. The fraction obtained from butanolic extract at 50-75% ethyl acetate in pet-ether was selected and refractionate using another silca column with pet-ether:chloroform:acetone:methanol in different concentration. The fraction obtained at 12.5-15% acetone in chloroform was collected and purified by preparative HPLC (shimadzu consisting of LC10ATVP pump, reodyne injector and class VP-6 software) as a new diarylheptanoid. The RP merk-C-18 (cromosil, 250х20mm, 5µ stationary phase) column with isocratic elution and 40% acetonitrile in water was used for purification of an isolated diarylheptanoid. The volume injected was 20ml of an isolated compound dissolved in methanol (HPLC grade). The detection was carried out at 205-330nm using photodiode array detector. An isolated compound was characterized by NMR (1H and 13C). An isolated compound was subjected to three different bioassay namely DPPH inhibition, superoxide scavenging and elastase inhibition for screening its antioxidant and antiulcer activity. RESULTS An isolated diarylheptanoid was purified by preparative HPLC and characterized using TLC and NMR.




Analytical HPLC chromatogram of isolated compound ZA6. Peak no. Retention time Area of peak Concentration. 1 6.375 1334137 3.8249 2 13.420 71634 0.2054 3 14.068 745251 2.1366 4 15.182 596840 1.7111 5 15.770 1815950 5.2063 6 17.031 1116482 3.2009 7 17.641 1185774 3.3996 8 18.185 28013814 80.3151 Total 34879880 100.00

Preparative HPLC chromatogram of isolated compound ZA6

Peak no. Retention time Area of peak Concentration. 1 3.883 200694 0.1356 2 4.117 81728 0.0552 3 4.537 141506 0.0956 4 7.806 806391 0.5449 5 7.957 355987 0.2406 6 9.353 145772897 98.5112 7 12.107 5493 0.0037 8 13.270 91293 0.0617 9 13.512 519937 0.3514 Total 147975924 100.00

The physical characters of isolated diarylheptanoid were found to be sticky having melting point 53 0C. The structure was predicted as 3,5-diacetoxy-7-(3,5 dihydroxy phenyl)-1-(3,4 dihydroxy phenyl)heptane on the basis of NMR(1H and 13C).

Structure of an isolated new diarylheptanoid is predicted as:

O

OH

OHHO

HO

CH3 C

O

O C CH3

O

3,5 diacetoxy-7-(3,5 dihydroxy phenyl)-1-(3,4 dihydroxy phenyl)heptane.

Bioassay: Isolated diarylheptanoid was tested for its in vitro superoxide scavenging activity using PMS-NADH system(3), antioxidant activity DPPH system(4) and antiulcer activity using elastase inhibition activity(5). It showed to posses potent antioxidant and antielastase activities.

13C NMR

δ 47.88-49.16 Heptane δ115.12-115.24 CH3 of ester δ119.58 C-OH δ133.13 Aromatic carbons δ142.70 Aromatic carbons attached to heptane δ144.49 C=O

1H NMR

δ1.877-2.026 Singlet-CH3 of ester δ6.511-6.735 Aromatic region δ3.844-3.859 Doubletof heptane chain.




Percentage inhibition using different concentration of a new diarylheptanoid with standard Sample Concentration % inhibition Activity

10µg/ml 18.66 Diarylheptanoid

50µg/ml 35.43 10µg/ml 30.90 Gallic acid (std.)

50µg/ml 54.81

Superoxide scavenging

10µg/ml 15.55 Diarylheptanoid 50µg/ml 76.01

1.5µg/ml 14.56 Gallic acid (std.) 5µg/ml 66.69

antioxidant

Diarylheptanoid 5µg/ml - 50µg/ml 1.97 Ursolic acid (std.) 5µg/ml 26.34 20µg/ml 55.92

antielastase

DISCUSSION A 50% methanolic extract was prepared and standardized for pilot scale extraction. Methanolic extract of ginger has been reported for different pharmacological activities like antioxidant, antiviral and antielastase activity etc (6-7). An attempt was made to made to fractionate the aqueous methanolic extracts to isolate the phytoconstituents. TLC is one of the most commonly used procedures to select the phytoconstituents. Isolation and purification of chemical constituents Isolation of chemical constituents was done by fractionation using repeated column chromatography. The butanolic extract obtained from 50% methanolic extract was subjected to column chromatography using 17 different columns and 94 fractions ranging from 4-2 lts volume were collected. The fractions were rechromatographed on silica gel G and diaion HP-20 columns to get the isolated compound ZA6. Analytical HPLC method was developed at Natural Remedies (Bangalore) using HPLC (Shimadzu) with RP C-18 column and acetonitrile in d.w (30-65%) as mobile phase and PDA and ELSD detector. The detection was carried out at 205-330nm. The purification of the isolated compounds was done by using preparative HPLC which is a popular tool for isolation of phytoconstituents in industries. A gradient elution RP-HPLC separation of isolated compounds has been developed which gives better separation than isocratic separation (8). Semi preparative RP-HPLC was used as the first separation technique to isolate the pure compounds (9).

Characterization of isolated compounds Isolation and purification of compounds by column chromatography and preparative HPLC (9, 10). The 1H-NMR and 13C-NMR spectrums was used to characterize the isolated compounds (11). Isolation of different diarylheptanoids has been reported from different extracts of ginger (12-14). The structure of the isolated compound ZA6 in our study shows 3, 5 diacetoxy–7–[3, 4 dihydroxy phenyl]–1–[3, 4 dihyroxy phenyl] heptane. Evaluation of bioactivity of isolated compounds As ginger is widely used as stomachic (15), antiulcer (16) and antioxidant (10) and the diarylheptanoids are also reported as antiulcer (15), antifungal (17), prostaglandin inhibitor (18), antihepatotoxic (19), cytotoxic and apoptotic (20). The isolated compounds were tested for antiulcer and antioxidant activity by using invitro methods. The three bioassays namely DPPH inhibition (4), superoxide scavenging (3) and elastase inhibition (5) of isolated compound ZA6 was performed to determine antioxidant and antiulcer property. CONCLUSION Authenticated samples of rhizomes of Zingiber officinale Roscoe belonging to family Zingiberacea were selected for isolation of phytoconstituents. Spent ginger devoid of oleoresin was collected and extracted with water, methanol and aqueous methanol (50%) in different lots to select an extract with maximum number of phytoconstituents. A TLC system with toluene: butanol: glacial acetic acid: water (6:4:5:1 as mobile phase and anisaldehyde as detecting agent) was optimized to identify the




phytoconstituents. A 50% methanolic extract of spent ginger was found to contain maximum number of phytoconstituents and hence it was selected for isolation of markers. The methanolic extract was partitioned with butanol and the butanolic extract was fractionated by repeated column chromatography using silica gel and diaion HP-20 as stationary phase and the solvents with varying polarity as mobile phase. This method was found to be suitable for fractionation/isolation of phytoconstituents at commercial scale. A new method of HPLC analysis was developed and standardized for identification of the isolated compounds. On the basis of purity and yield one isolated compound was selected for further purification by preparative HPLC. An isolated compound was characterized as 3,5- diacetoxy–7–[3,4,dihydroxyphenyl]–1–[3,4, dihyroxy-phenyl] heptane (ZA6). In vitro antioxidant screening showed that, the isolated compound ZA6 was found to posses antioxidant activity with 15.55 and 76.01% inhibition and superoxide scavenging activity with 18.66 and 35.43% inhibition at 10 and 50µg/ml concentration whereas, in vitro antielastase screening showed that, the isolated compound ZA6 was found to posses antielastase activity with 1.97% inhibition at 50µg/ml concentration. ACKNOWLEDGEMENT We thank Dr. Amit Agarwal, Director, Natural Remedies Pvt. Ltd. Bangalore for providing the facility to isolate the active constituent from Zingiber officinale. REFERENCES 1. A.Akhila, R.Tiwari. Chemistry of ginger-A review.

CIMAP 6:143-56 (1984). 2. C.K.Kokate, A.P.Purohit. Pharmacognosy. Nirali

Prakashan, Pune 1996: 4th edn; 1-5. 3. G.C.Yen, H.Y.Chen. Antioxidant activity of various

tea extracts in relation to their antimutagenecity. J. Agri. Food Chem. 43: 27-32 (1995).

4. T.Vani, M.Rajani, S.Sarkar, C.J.Shishoo. Antioxidant properties of the ayurvedic formulations – Triphala and its constituents. Int. J. Pharmacog. 35: 313-7(1997).

5. J.Bieth, B.Spiess, C.G.Wermuth. The synthesis and analytical use of a highly sensitive and convenient substrate of elastase. Biochem Med.11:350-7 (1974).

6. Combest WC. Herbal Pharmacy - Ginger. U.S. Pharmacist 1998. (www.uspharmacist.com)

7. K.Bone. Ginger-Review. Phytotherapia 4:110-20(1997).

8. R.D.Hiserodt, S.G.Franzblav, R.T.Rosen. Isolation of 6, 8 and 10 gingerol from rhizome by HPLC and preliminary evaluation of inhibition of Mycobacterium avium and Mycobacterium tuberculosis. J. Agri. Food Chem. 46:2504- 8 (1998).

9. T.A.V.Beek, G.P.Lelyveld. Isolation and identification of five major sesquiterpene hydrocarbons of ginger. Phytochem Anal. 2: 26-34(1991).

10. Y. Sekiwa, K.Kuboto, A.Kobayashi. Isolation of novel glucosides related to gingerdiol from ginger and their anti-oxidant activities. J. Agri. Food Chem. 48:373-7(2000).

11. Y.Hori, T.Miura, Y.Hirai, M.N.Fukumura, Y.Nemoto, K.Toriizuka, Pharmacognostic studies on ginger and related drugs – part I: five sulphonated compounds from Zingiberis rhizome (Shokyo). Phytochemistry. 62:613-7 (2003).

12. N.Natakani, H.Kikuzaki, M.Kobayashi. Diarylheptanoids from rhizomes of Zingiber officinale. Phytochemistry. 30:3647-51(1991).

13. Z.L.Liu, J.Ma, X.Jin, L.Yang. Diarylheptanoids from rhizomes of zingiber officinale. Phytochemistry 65: 1137-43 (2004).

14. J.Yamahara, S.Hatakeyama, K.Taniguchi, M.Kawamura, M.Yoshikawa. Stomachic principles in ginger II. Pungent and anti-ulcer effects of low polar constituents isolated from ginger, the dried rhizome of zingiber officinale ROSCOE cultivated in Taiwan, the absolute stereostructure of a new diarylheptanoid. Yakugaku Zasshi 112: 645-55(1992).

15. J.Yamahara, M.Mochzuki, H.Q.Rong, H.Matsuda, H.Fujimura. Anti-ulcer effects in rats of ginger constituents. J. Ethnopharmacol. 23:299-304 (1998).

16. K.Endo, E.Kanno, Y.Oshima. Structures of anti-fungal diarylheptenones, gingerenones A, B, C and isogingerenone B, isolated from rhizomes ofZingiber officinale. Phytochemistry 29: 797-9 (1990).

17. K.Kiuchi, S.Iwakami, M.Shibbya, F.Hanaoka, U.Sankawa. Inhibition of prostaglandin and leukotriene biosynthesis by gingerols and diaryldeptanoids. Chem Pharm Bull. 40: 387-91 (1992).

18. H.Hikino, Y.Kiso, N.Kato, Y.Hamada, T.Shiori, R.Aiyama. Antihepatotoxic actions of gingerols and diarylhetanoids. J. Ethnopharmacol. 54: 31-9 (1985).





Evaluation of Anti-inflammatory activity of Nyctanthes arbor-tristis and Onosma echioides.

Amrite Omkar, Thurackal Jeeja and Gadgoli Chhaya* Department of Pharmacognosy,

Saraswati Vidya Bhavan’s College of Pharmacy, Dombivli – 421203, Maharashtra, India *Corresponding Author: [email protected]

ABSTRACT - Anti-inflammatory activity of the ethanolic extract of the orange tubular calyx of N.arbor-tristis and pet.ether extract of root bark of O.echioides was studied in albino rats of Wistar strain using the carrageenan induced paw edema model. The ethanolic extract of N.arbor-tristis (200 mg/kg, i.p.), isolated carotenoid from the extract N.arbor-tristis (200mg/Kg, i.p.) and pet.ether extract of O.echioides (5mg/Kg, i.p.) inhibited carrageenan induced rat paw edema. The results indicated that all the extract produced significant (p < 0.05) anti-inflammatory activity when compared with the standard drug (diclofenac sodium) and untreated control. KEYWORDS - Anti-inflammatory, Nycthanthes arbor-tristis, carotenoid, Onosma echioides INTRODUCTION Scientific interest in medicinal plants has burgeoned in recent times due to increased efficiency of new plant-derived drugs and rising concerns about the side-effects of conventional medicine. Inflammation is seen in conditions such as Alzheimer's disease, cancer, irritable bowel syndrome and hepatic diseases. It is believed that controlling inflammation may help to alleviate these conditions or even prevent them. Thus the present investigation was carried out to evaluate the anti-inflammatory potential of Nyctanthes arbor-tristis and Onosma echioides. Nyctanthes arbortristis Linn. z(Oleaceae) commonly known as Parijatak (Marathi) and Night Jasmine (English) is cultivated in gardens almost throughout India and in many other tropical countries (1). The leaves of Nyctanthes arbor-tristis Linn. are used extensively in Ayurvedic medicine for the treatment of various diseases such as sciatica, chronic fever, rheumatism and internal worm infections and as a laxative, diaphoretic and diuretic (2,3). Onosma echioides (Boraginaceae), known as Ratanjot, Laljari is a perennial herb growing to 0.3m. The leaves are made into a powder and given to children as a purgative. The flowers are used as a cordial and stimulant in the treatment of rheumatism and palpitations of the heart. The root is bruised and used as an external application to skin eruptions. A red dye obtained from the root, is used as an alkanna substitute (4). Lack of scientific data with respect to the pharmacological properties of the flowers of

Nyctanthes arbor-tristis and root bark of Onosma echioides encouraged for the evaluation of N.arbor-tristis and O.echioides for anti-inflammatory activity. The effect of the extract was also compared with the standard drug viz. diclofenac sodium. MATERIALS AND METHODS The fresh flowers of N. arbor-tristis were collected early in the morning from wildly grown trees from Dombivli, District Thane, Maharashtra, India and were authenticated at St.Xavier’s College, Mumbai. The dried O. echioides roots were procured from the local market (Princess Street) of Mumbai and were authenticated by Dr. A.M. Mujumdar, Head, Plant Sciences Division, Agharkar Research Institute, Pune. The orange coloured tubular calyx was separated from corolla and was dried in vacuum oven at temperature not exceeding 50oC. The dried root bark (outer covering of root) was separated from dried roots of O. echioides, dried at 100oC for 2 hours and coarsely powdered. Preparation of extract The dried tubular calyx of N.arbor-tristis was macerated with ethanol for 6-8 hours at room temperature, filtered and filtrate was concentrated under reduced pressure of 15mmHg at 40oC. Preliminary phytochemical screening of the extracts revealed presence of sugars and carotenoids. The extract was chromatographed on Silica gel (60–120 mesh) column eluted with mobile phase of ethyl acetate: isopropanol: water (65:25:10) to isolate a major carotenoid molecule corresponding a Rf of 0.4. The isolated carotenoid molecule was also evaluated for anti-inflammatory activity. The dried powdered material of O. echioides was extracted using Soxhlet




apparatus with petroleum ether (60 - 80oC). The extract was concentrated under vacuum to obtain semi-solid mass and qualitative phytochemical evaluation indicated presence of naphthoquinones. All the extracts were suspended in 5%w/v gum acacia (LOBA Chemie) for the present study. Animals Albino rats of Wistar strain of the either sex (150 – 200g), maintained under standard environmental conditions (270±20C, relative humidity 60±5% light-dark cycle of 12h) and fed with standard pellet diet and water ad libitum, were used for the present study. All the experimental protocols were approved by Institutional Animal Ethics Committee. Acute Toxicity study The animals were divided into control and test groups containing six animals each. The control group received the vehicle (5%w/v gum acacia) whereas the test group consisting of ethanolic extract of N.arbor-tristis and isolated carotenoid received increasing doses of 250, 500, 1000, 1500 (mg/Kg b.w.i.p.) whereas the test group consisting of pet.ether extract of O.echioides received increasing doses of 25, 50, 75(mg/Kg b.w.i.p.) and were observed for mortality till 48 hours and LD50 was calculated (5). Anti-inflammatory activity The rats were divided into 4 groups where six animals in each group were used for study. Acute inflammation was produced by the sub–plantar administration of 0.1ml of 1% w/v carrageenan (Sd fine – Chem Ltd.) in 5% w/v gum acacia in the right hind paw of rats (6). The Group I was treated with ethanol extract of N.arbortristis (200mg/Kg i.p.), Group II with isolated carotenoid (200mg/Kg i.p.), Group III with pet.ether extract of O.echioides (5mg/Kg i.p.), Group IV with diclofenac sodium (Sun Pharmaceuticals) (10mg/Kg i.p.) and Group V as Control (5%w/v gum acacia) was administered intraperitonially. The paw volume was measured before the injection and then at intervals of 30 min. for a period of 2 h after carrageenan injection using plethysmometer. The animals were pretreated with the extract 30min. before the administration of carrageenan. % inhibition of inflammation was calculated using the formula, where % inhibition = 100[Vc -Vt/Vc] ‘Vc’ represents oedema volume in control and ‘Vt’ oedema volume in treated with test extracts. Statistical Analysis Results are expressed as mean±s.d. The statistical analysis was performed by using unpaired Student’s t-

test for comparing test group with control group. P values less than 0.05 were considered statistically significant. RESULTS The orange tubular calyx of N.arbor-tristis macerated with ethanol. The average percentage yield of ethanolic extract of N.arbor-tristis was found to be 8.7 w/w. On preliminary phytochemical screening of N.arbor-tristis revealed the presence of glycosides, carotenoids. The LD50 was found to be 1500 mg/Kg i.p. for ethanol extract of N.arbor-tristis. The root bark of O.echioides oven-dried and extracted by continuous hot extraction process using soxhlet apparatus. The average percentage yield of pet.ether extract of O.echioides was found to be 5.7 w/w. On preliminary phytochemical screening of the root bark of O.echioides revealed the presence of napthoquinones. The LD50 was found to be 50 mg/Kg i.p.for pet.ether extract of O.echioides. The ethanolic extract of N.arbor-tristis, isolated carotenoid of N.arbor-tristis and the petroleum ether (60–80oC) extract of O.echioides exhibited significant (p<0.05) anti-inflammatory activity at doses of 200, 200 and 5 (mg/Kg b.w. i.p.) respectively. As shown in fig.1 ethanolic extract of N.arbor-tristis, isolated carotenoid and petroleum ether extract of O.echioides exhibited inhibition in rat paw oedema, 67.4, 70.0 and 85.76 % respectively whereas standard drug showed 96.7% of inhibition of inflammation. DISCUSSION Indigenous drug systems can be source of variety of new drugs which can provide relief in inflammation. The most widely used primary test to screen new anti-inflammatory agents measures the ability of a compound to reduce local edema induced in the rat paw by injection of an irritant agent(5). This edema depends on the participation of kinins and polymorphonuclear leukocytes with their pro-inflammatory factors including prostaglandins (7). The development of edema in the paw of the rat after the injection of carrageenan has been described as a biphasic event. The initial phase, observed around 1 h, is attributed to the release of histamine and serotonin; the second, accelerating phase of swelling is due to the release of prostaglandin-like substances. It has been reported that the second phase of edema is sensitive to both clinically useful steroidal and non-steroidal anti-inflammatory agents (8). Significant anti-inflammatory activity was observed for ethanolic extract of N.arbor-tristis, isolated carotenoid of




0

20

40

60

80

100

120

1

% re

duct

ion

in ra

t paw

oed

ema

standard

N.arbortristisextractisolatedcarotenoidO.echioides

*

* **

Fig. 1 Anti-inflammatory activity of ethanolic extract N.arbor-tristis, isolated carotenoid, pet.ether extract

O.echioides as compared against standard drug. N.arbor-tristis and petroleum ether extract of O.echioides in carrageenan induced oedema model. The red dye obtained from O.echioides is used as a substitute for Alkanna tinctoria. Alkanna tinctoria contains the napthoquinones, alkannin and shikonin which exhibit anti-inflammatory activity (9). Hence the anti-inflammatory activity in O.echioides can be attributed to the presence of napthoquinones as the pet. ether extract of O.echioides showed positive test for napthoquinones. The study indicates the potential of these herbal drugs as anti-inflammatory drugs. Such drugs can be explored in various inflammatory diseases. The activity may be attributed to the inhibition of the COX-2 enzyme or inhibition of the activation of transcription factors. It can be concluded that all the extracts have potential to be explored as anti-inflammatory agents. Further studies may reveal the exact mechanisms of action responsible for the anti-inflammatory activities of N.arbor-tristis and O.echioides. REFERENCES 1. The Wealth of India, Raw Materials; CSIR

Publication, Delhi, India. 7, (1966). 2. Saxena, R.S., Gupta, B., Saxena, K.K., Singh, R.

and Prasad, D.N., Study of Anti-Inflammatory Activity of the Leaves of Nyctanthes arbor-tristis Linn.-An Indian Medicinal Plant. J. Ethnopharmacol., 11: 319-30, (1987).

3. Saxena, R.S., Gupta, B., Saxena, K.K., Srivastava, V.K. and Prasad , D.N., Analgesic, Antipyretic and Ulcerogenic Activity of Nyctanthes arbor-tristis

Leaf Extract. J. Ethnopharmacol., 19: 193-200, (1987).

4. Information on Onosma echioides. Available on-line: http://www.ibiblio.org

5. Winter, C.A., Risley, E.A. and Nuss, G.W., Carrageenan-Induced Oedema in Hind Paw of Rat as an Assay for Anti-Inflammatory Drugs. Proc. Soc. Exp. Biol. Med., 111: 544-547, (1962).

6. Chakraborty., Devi, R.K.B. and Rita, S., Preliminary Studies on Anti-Inflammatory and Analgesic Activities of Spilanthes Acmella in Experimental Animal Models. Ind. J. Pharmacol., 36(3):148-150 (2004).

7. Damas, J., Remacle-Volon, G. and Deflandre, E., Further Studies of the Mechanism of Counter Irritation by Turpentine. Arch. Pharmacol., 332:196-200 (1986).

8. Beatriz, B., Gerardo, M., Antonio, J. L. and Jose, A. S. E. Anti-inflammatory Activity of Urera baccifera (Urticaceae) in Sprague Dawley Rats, Research paper available on-line: http://rbt.ots.ac.cr

9. Papageorgiou, V.P., Assimopoulou, A.N., Couladouros, E.A., Hepworth, D and Nicolaou, K.C., The Chemistry and Biology of Alkannin, Shikonin, and Related Naphthazarin Natural Products Angewandte Chemie International Edition. 38:270-300 (1999).




INFORMATION FOR AUTHORS –PHARMACOGNOSY MAGAZINE [PHCOG MAG.] (An official publication of Phcog.Net)

www.phcogmag.com

The Pharmacognosy Magazine PHCOG MAG. [ISSN: 0973-1296] (An Official Publication of Phcog.Net; Indexed in Index Copernicus and other databases) publication series of Phcog.Net comprises contribution on every aspect of medicinal plant research and is aimed at documenting the vast and rich resource the nature has provided us with. PHCOG MAG. consists of following general topics: News, Announcements, Special Articles, Invited Articles, Clinical reports, Plant reviews, Review Articles, Short Reviews, Research Articles, Short Communications, Meetings, Opportunities, Product review and Book review.

PHCOG MAG. welcomes contributions and manuscripts that describe investigations, clinical reports, methods, techniques and applications of all forms related to medicinal plant research and that are of immediate interest to users in industry, academia, and government. All contribution to Phcog Mag are reviewed editorially with the understanding that they have not been published previously and are not under consideration for publication elsewhere. Author/s is/are responsible for all statements made in their work and obtaining necessary permission to republish any previously published illustrations and/or other relevant materials. All contributions and manuscripts are subjected to peer review (Editorial, Phcog Mag Volume 2 Issue 7) and copyediting process.

Manuscript preparation

Manuscripts must conform to the “Uniform Requirements for Manuscripts Submitted to Biomedical Journals” http://www.icmje.org/. Contributions and Manuscripts must be written in English and submitted exclusively to PHCOG MAG. Manuscripts must be typewritten (double-spaced) with liberal margins and space at the top and bottom of the page. We highly encourage authors to submit manuscripts by e. mail (See submission); and contact the Assoc. editors for any necessary instructions/clarifications. Begin each of the following on a separate page: title page (including author’s name, affiliations and address for correspondence), abstract (including at least 4 key words), text (consisting of introduction, materials and methods, results, discussion, conclusion and acknowledgements), references, figure legends, tables and figures. Titles should be short, specific, and clear.

Beginning with the first page of text, number each page consecutively. If a manuscript is otherwise acceptable but the standard of English not satisfactory, the editorial office will, with the agreement of the authors, perform technical editing of the manuscript. All experimental procedure followed must be approved by appropriate animal ethics committee.

Tables

Tables should be presented on separate pages, Tables should have a short, one-line title in bold text. Symbols and abbreviations should be defined immediately below the table, followed by essential descriptive material as briefly as possible, in double-spaced text.

Figures/Photographs/Images

Figures/Images should be submitted as photographic quality scanned prints, and if possible attach an electronic version (EPS/TIFF/JPEG/PPT). Any specific orientation should be indicated and a brief cover legend should be included. Submitted photo (micro) graphs should be reproducible without reduction. If not feasible, indicate the desired percentage reduction on the back of the figure. Indicate the orientation, figure number, and first author's name on the reverse of each figure using a soft pencil. Original Photographs/scanned (high quality: 300 pixels) should be sent along with the manuscript.

References

Literature citations in the text must be indicated by Arabic numerals in parentheses. Number each reference separately in the order it appears in the text. List cited references at the end of the manuscript in the order of their appearance in the text. Designate submitted articles as “in press” only if its formally accepted for publication. Use “unpublished work” otherwise, listing all author’s involved. For personal communications, include name and date.

Format for references:

Reference style should be strictly adhered to the style of PHCOG MAG.

Use the following format for references Note: Reference style should be strictly adhered to the style of PHCOG MAG.




PHCOG MAG. FEATURESWEB BASED MANUSCRIPT HANDLING SYSTEM AT

www.phcogmag.com

Book reference 1. R.N. Chopra, S.L. Nayar, I.C. Chopra, Glossary

of Indian Medicinal Plants, (Council of Industrial and Scientific Research, New Delhi, 1956) pp. 1−329.

Chapter in books 2. E.C. Johannsen, L.C. Madoff, Infections of the

liver and biliary system. In: G.L. Mandell, J.C. Bennett, R. Dolin, eds. Mandell, Douglas, and Bennett’s: Principles and Practice of Infectious Disease. Vol 1. 6th ed. Philadelphia, PA: Elsevier; 951-952 (2001).

Journal reference 3. C.J. Rompelberg, J.T. Vogels, N. de-Vogel, G.C.

Bruijntjes-Rozier, W.H. Stenhuis, J.J. Bogaards and H. Verhagen. Effect of short-term dietary administration of eugenol in humans. Hum. Exp. Toxicol. 15(2): 129−35 (1996).

Note: Check journal abbreviations at: http://home.ncifcrf.gov/research/bja/ Download list of medline journals: ftp://ftp.ncbi.nih.gov/pubmed/J_Medline.zip Internet reference Author. "Title of Web Page." Title of the Site. Editor. Date and/or Version Number. Name of Sponsoring Institution. Date of Access <URL>. 4. FDA/CEDR resources page. Food and Drug

Administration Web site. Available at: http://www.fda.gov/cder/approval/index.htm. Accessed - April 7, 2003.

For online journals and periodicals Author. "Title of Article." Title of Publication Date: Page(s) or Section(s), if numbered. Date of Access <URL>.

5. Sven Wind and H.S. Arun Kumar. Apocynin: Mother Nature’s gift to combat oxidative stress. Phcog Mag. 1(4): 136-139 (2005). Accessed - February, 17, 2006 Available at: http://www.phcog.net/issue4.php

Submitting the Accepted Manuscript

After proofreading and making the final changes to accepted articles, submit a hard copy of the manuscript and an identical, electronic copy in MS Word on CD to the editor along with copyright form duly signed by all authors.

Submission

Authors can submit their articles/manuscripts either in Document or Plain ASCII text format through surface mail or online submission at www.phcogmag.com. Authors are advised to send their contributions or manuscripts online, for swifter communication.

By surface mail

SHIVANANDA B.G.

Editor, PHCOG MAG.


Hosur road, Bangalore 560 027,

Karnataka, INDIA.

By e- mail

Manuscripts can also be sent to the following e-mail

E mail addresses of Associate Editors [Phcog Mag.], for status of publication and any other information, please write to,

MUEEN AHMED K.K.

e.mail: [email protected]

ARUN KUMAR H. S.

e.mail: [email protected]

MUHAMMAD NABEEL GHAYUR

e. mail: [email protected]

*******

[email protected]




LAST COMPLIMENTARY COPY, TO RECEIVE REGULARLY, SUBSCRIBE NOW !!!

PHARMACOGNOSY MAGAZINE - SUBSCRIPTION FORM

2006 Subscription, (4 issues/year) Pharmacognosy Magazine (a quarterly publication of Phcog.net) and online access to Phcog Mag. comes with annual subscription. A subscription with delivery to an institution/individual must pay the subscription rate.

• Cost : (Institutional/Industry) � Rs. 1000/- for one year � Rs. 2000/- for two years � Rs. 3000/- for three years • Single Article—Rs.100 (US $ 5). Anything older than one year is Rs. 150.00, if

available. Please specify issue and article if applicable:

SUBSCRIPTION DETAILS Subscriber/Institution name:______________________________________________________________ Address: ______________________________________________________________________________________ ______________________________________________________________________________________ City/State/ZIP:__________________________________________________________________________ Province/Country/Postal code: ___________________________________________________________ Phone: ____________________________________ Fax: ( ) __________________________________ E-mail:________________________________________________________________________________ D.D/ Cheque No._______________________________ Bank Name : ______________________in favour of “The Principal, Al-Ameen College of Pharmacy” payable at Bangalore, India. For any other information, kindly write to [email protected] SEND COMPLETED FORM The Editor, Pharmacognosy Magazine, Al-Ameen College of Pharmacy, Opp. Lalbagh Main gate, Hosur road, Bangalore 560 027.India Fax : 0091-80-22225834 International Subscriptions may write to [email protected] for more details.

PHARMACOGNOSY MAGAZINE (PHCOG MAG.) · Sajjad Khan. M, Salma Khanam, Deepak M and Shivananda B.G....

Documents

Transcript of PHARMACOGNOSY MAGAZINE (PHCOG MAG.) · Sajjad Khan. M, Salma Khanam, Deepak M and Shivananda B.G....