Rajiv Gandhi University of Health Sciences s41

Rajiv Gandhi University of Health Sciences Karnataka, Bangalore.

SCREENING OF AREAL PARTS OF TERAMNUS LEBIALIS SPRENG, FOR HEPATOPROTECTIVE, ANTI ULCER AND ANTHELMINTIC ACTIVITIES.

A Protocol submitted to Rajiv Gandhi University of Health Sciences Karnataka, Bangalore. In partial fulfillment of the requirement for the award of

MASTER OF PHARMACY IN PHARMACOLOGY

Mr. MD RUSTAM HUSSAIN

Department of Pharmacology, National College of Pharmacy, Balraj-Urs Road, Shimoga-577 201. Karnataka-INDIA. RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BENGALURU

Annexure – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

Mr. MD RUSTAM HUSSAIN NATIONAL COLLEGE OF PHARMACY, Name and Address of the 01 BALRAJ-URS ROAD, Candidate SHIMOGA-577 201 KARNATAKA-INDIA

NATIONAL COLLEGE OF PHARMACY, 02 Name of the Institution BALRAJ-URS ROAD, SHIMOGA-577 201 KARNATAKA-INDIA Course of the Study 03 M. PHARM. (PHARMACOLOGY) Branch 04 Date of Admission to course 11-07-2012

SCREENING OF AREAL PARTS OF TERAMNUS LABIALIS SPRENG. FOR 05 Title of the Topic HEPATOPROTECTIVE, ANTI ULCER, AND ANTHELMINTIC ACTIVITIES.

Brief resume of the intended work Enclosure – I 6.1. Need for the Study 06 6.2. Review of the Literature Enclosure – II

6.3. Objective of the Study Enclosure – III

Materials and Methods Enclosure – IV 7.1. Source of data 7.2. Methods of collection of data Enclosure – V 07 7.3. Does the study require any Investigations on animals? Enclosure – VI If yes give details 7.4. Has ethical clearance been obtained form your institution Enclosure – VI – A incase of 7.3. 08 List of References (About 4 – 6) Enclosure – VII

09 Signature of the Candidate

The present research work is original and not published in any of the journals. This work can 10 Remarks of the Guide be carried out in our Pharmacology Department laboratory.

Name and Designation of Mr. T. SHEKSHAVALI (in Block Letters) M.Pharm . LECTURER, 11.1. Guide Department of Pharmacology, National College of Pharmacy, Balraj-Urs Road, Shimoga-577 201 Karnataka-INDIA

11.2. Signature 11

11.3. Head of the Department Dr. I. J. KUPPAST M.Pharm., Ph.D., F.I.C.

Head of the Department National College of Pharmacy, Balraj-Urs Road, Shimoga-577 201 Karnataka-INDIA

11.4. Signature

Remarks of the Principal The present study is permitted to perform in the Pharmacology Department laboratory of our Institution and the study protocol has been approved by IAEC. 12

12.1. Signature Principal

Enclosure – I

Brief resume of intended work:

6.1. Need for study:

World’s plant population has been used for medicinal purposes at one time or another. Most of the traditional health care systems of the world, like Egypt, Middle

East, India and China developed from 3000 BC onwards. Scientific evidence has shown that many of these herbs do have medicinal properties. Herbal products may act in a manner similar to pharmaceuticals yet without side effects1.

According to World Health Organization, medicinal plants would be the best source to obtain a variety of drugs. Therefore, such plants should be investigated to better understand their properties, safety and efficacy2.

The universal role of plants in the treatment of disease is exemplified by their employment in all the major systems of medicine. A complete understanding of medicinal plants involves a number of disciplines including Pharmacology3.

Medicinal plants being as an important natural resource and potentially safe drugs can play an important role in assuaging human health by contributing herbal medicines. The high cost of allopathic medicine and their potential side effects, encouraged the people to use the traditional medicine. The increasing demand of plant extracts to use in the cosmetic, food and pharmaceutical industries suggests that systematic studies of medicinal plants are very important in order to find active compounds and their use as a medicine for curing various diseases4. Botanical name: Teramnus labialis Spreng.

Synonyms: Teramnus parviflorus Spr., Glycine labialis Linn., G. parviflora Lam .

Family: Fabaceae.

Classical name: Mashaparni.

Vernacular names: English - Vogel-Tephrosis, Hindi - Mashparni, Vana Urada,

Kannada - Kadu uddu, Malayalam - Katt ulandu , Tamil - Katulandu. Telugu -

Karuminum, Adavi-vuddulu.

A widely spreading twining herb, stems slender, more or less appressedly hairy.

Leaves 3-foliate, leaflets membranous or subcoriaceous, 3.5– 6 x1.5 – 2.5 cm, the terminal slightly the largest, ovate-oblong or oblong – lanceolate, hairy beneath, base rounded or acute, stipels subulate, stipules ovate – lanceolate, deciduous. Flowers reddish, bisexual, in axillary few flowered lax racemes, 5-15 cm long, solitary or fascicled along a slender more or less hairy rachis. Pods 3-5 cm long, narrowly linear, straight or slight incurved, hairy when young, glabrous on maturity with a short stout beak bent upwards nearly at right angle with the pod. Seeds 8-12, oblong, truncate or slightly rounded at the ends, smooth and dark brown in colour.

Flowering and Fruiting: August – December.

Distribution: Found throughout the greater parts of the India. Punjab, West Bengal,

Gujarat, Maharashtra, Deccan, N. Circars, grows wild in the plains in southern parts of India. Also occurs in Sri Lanka,Bangladesh, Burma, Thailand, China, Vietnam,

Indonesia, Philippines,Madagascar and New Guinea.

Part(s) Used: Whole plant, root and fruit.

Chemical Constituents: Seed: Fraxidin , amino acids – lysine, leucine, isoleucine, arginine, valine, histidine; unsaturated fatty acids, minerals - potassium, magnesium, calcium, phosphorus, free phenols, tannins, L-DOPA, hydrogen cyanide, phytic acid; proteins.

Stem and aerial parts: Flavonol glycoside, Vitexin, bergenin, daidzin and 3-O- methyl-D-chiro –inositol5. Phytochemical screening of extract of Teramnus labialis showed presence of

Triterpenoids ,flavonoids, glycosides, tannins and alkaloids6 .

Actions and Uses: The fruit is bitter, cooling and sweet, dry; aphrodisiac, astringent to the bowels, antipyretic, tonic, galactagogue; causes Kapha, cures Vata, inflammation, biliousness, blood diseases, gout, Tridosha, fevers ,consumption, bronchitis, thirst, burning sensation; useful in paralysis, rheumatism and affections of the nervous system

(Ayurveda). Slightly astringent. In La Reunion considered very useful in haemoptysis and catarrh 7.

Other Uses of Teramnus labialis;

 In Scorpion bite (root) and body pain ( leaf ) traditionally in Utter Pradesh by

Tribes8.

 In Elephant diseases9.

 In the Formulation (one of the constituents) of Chyawanprash10.

 Teramnus labialis is one of the Life Promoting ( Jeevaniya ) drug in Charak

Samhita. It decreases Vata, Pitta and increases kapha. It is also one of the

important Rasayana drug in Charak Samhita11.

Vitexin compound 1 could inhibit choriocarcinoma via inducing cell apoptosis and suppressing the mTOR pathway12.

Bergenin exhibits Antihepatotoxic / Hepatoprotective, Antiulcer, Anti-HIV,

Immunomodulatory, Antiarrythemic, Neuroprotective and Anti-inflamatory activities13.

The medical utility of Teramnus labialis is very broad in the traditional system as mentioned above but it has not been scientifically studied for all its claims.

The claim of Hepatoprotective, Anti ulcer and Anthelmetic activity of areal parts of the plant Teramnus labialis has not been studied so far scientifically. Hence the present study is aimed to investigate Hepatoprotective, Anti ulcer and Anthelmetic activities of areal parts of the plant Teramnus labialis. Enclosure – II

6.2. Review of literature:

1. G. Alagumanivasagam, A. Kottai Muthu, R. Manavalan, studied In vivo

antioxidant and Lipid Peroxidation effect of Methanolic Extract of whole

plant of Teramnus labialis (Linn.) in Rat fed with high fat diet and

concluded that it has significant antioxidant and lipid peroxidation activity

and the plant extract is a significant source of natural antioxidant and may

helpful in preventing the progress of various oxidative stresses and further

investigation needed to isolate and identify the antioxidant compound of the

plant14.

2. G. Alagumanivasagam, A. Kottai Muthu, R. Manavalan, studied In-vitro

Free Radical Scavanging Activity of Methanolic Extract of Whole Plant of

Teramnus labialis (Linn.) and concluded that high antioxidant capacity

observed for methanolic extract of whole plant of Teramnus labialis (Linn.)

and suggest that it may play a role in preventing human diseases in which

free radicles are involved and can provide good protection against oxidative

damage15.

3. M.B. Viswanathan, D. Thangadurai, K. Tamil Vendan and N. Ramesh

carried out Chemical analysis and nutritional assessment of Teramnus

labialis (L.) Spreng and the report unveiled that Teramnnus Labialis seed

meal contained approximately equivalent levels of crude protein and lipids

as other leguminous seeds and high food energy value16.

4. Yadava R.N., Jain S. studied and found a novel biologically active flavonol

glycoside (1) from the chloroform soluble fraction of the ethanol extract of the stems of Teramnus labialis which showed antibacterial and antifungal

activities17.

5. C. Sridhar, A V Krishnaraju, G V Subbaraju, studied and found that

methanolic extract of aerial part of Teramnus labialis showed potent

antiinflamatory activity and it was further fractionated which gave , vitexin,

bergenin, daidzin and 3-O-methyl-D- chiro- inositol the known

antiinflamatory compounds. Vitexin exhibited a dose-dependent inhibitory

activity on 5-lipoxygenase enzyme and moderate antioxidant activity18.

6. Fort DM, Rao K, Jolad SD, Luo J Carlson TJ, King SR. studied and found

that In vivo bioassay-guided fractionation, based on anti hyperglyceamic

activity of aqueous alcoholic extract of the aerial parts of Teramnus labialis

(Roxb.) Benth. (Fabaceae), yielded an active fraction containing a mixture

of coumarins. The major coumarin present in the active fraction was

identified as fraxidin19.

7. G. Alagumanivasagam, A. Kottai Muthu, R. Manavalan, studied

Hypolipidemic Effect of Methanolic Extract of whole plant of Teramnus

labialis (Linn) on High Fat Diet Fed Rats and concluded that the methanolic

extract of whole plant of Teramnus labialis sign deduces then plasma and

tissue lipid profile and lowers the risk of atherosclerosis in high fat diet fed

rats. Therefore, it is concluded that the methanolic extract of Teramnus

labialis effective against hyperlipidemic20.

8. P. K. Ingale in the Article L-Dopa bearing plants - stated the role of L-Dopa

in the Parkinson disease and Seed of Teramnus labialis posses L-Dopa

which is with or without peripheral deca decarboxylase inhibitor most

effective drug for the treatment of Parkinson disease21. 9. Michael A. Grusak studied Genetic Diversity for Seed Mineral

Composition in the Wild Legume Teramnus labialis and results suggested

that it would be beneficial to promote Teramnus labialis as a human food,

especially where it is currently being grown as a forage crop. Additionally,

this species could serve as a model for understanding the mechanisms

controlling Ca, Mg or K transport from vegetative tissues to developing

seeds. Interestingly, Teramnus labialis is closely aligned with soybean22.

10. V. Arinathan1, V.R. Mohan2, A. Maruthupandian2 and T.

Athiperumalsami, studied about Chemical Evaluation of Raw Seeds of

Certain Tribal Pulses in Tamil Nadu, India. Teramnus labialis is also

included in the study and concluded that investigated tribal pulses can be

used as protein sources to curtail with problem of protein deficiency in most

of the developing countries which may result in many child killer diseases23.

11. Alluri V. Krishnaraju, Tayi V. N. Rao, Dodda Sundararaju, Mulabagal

Vanisree, Hsin-Sheng Tsay, and Gottumukkala V. Subbaraju, studied for

Assessment of Bioactivity of Indian Medicinal Plants Using Brine Shrimp

(Artemia Salina) Lethal assay and found that extract of whole plant of

Teramnus labialis exhibits 182.5 Brine shrimp lethality(LC50, μg/mL,

24h)24. Enclosure – III

6.3. Objectives of study:

1. Collection and authentication of Teramnus labialis.

2. Drying of areal parts of Teramnus labialis.

3. Extraction of the plant using solvents – Water, Methanol and Chloroform.

4. Phytochemical investigations of extracts.

5. Screening of hepatoprotective, anti ulcer and anthelmitic actives.

Enclosure – IV

MATERIALS AND METHODS:

7.1. Source of data:

The required data will be obtained from:

1. Electronic data [internet].

2. Published Research Papers.

3. Review and Research Articles from Journal.

4. Library, National College of Pharmacy, Shimoga, Karnataka, India. Enclosure – V

7.2. Method of collection of data:

1. The plant of Teramnus labialis will be collected from local areas of Shimoga

district, Karnataka.

2. Evaluation of LD50.

3. Screening of Pharmacological activities – a) hepatoprotective, b) anti ulcer and c)

anthelminthic activies.

Evaluation of LD50 will be carried out as per the standard procedure as per OECD

guidelines25.

The different extracts dose will be fixed after obtaining LD50 to carry out the

above said activities.

a) Hepatoprotective activity (carbon tetrachloride model) will be carried out

against carbon tetrachloride induced hepatotoxicity in Wistar albino rats of both

sexes (weighing 150‐200gm). Thirty six rats divided randomly into six groups,

each comprising of six animals.

Group I- (Normal control), will receive vehicle for 7 days.

Group II (Toxicant control) will receive carbon tetrachloride 1mg/kg/day p.o.

for 7 days.

Group III receives standard drug ‘Silymarin’100mg/kg; p.o. for 7 days with

carbon tetrachloride as of group II. Group IV and Group V and Group VI will receive Teramnus Labialis extracts

of water, methanol and chloroform respectively with carbon tetrachloride as of

group II for 7 days. Carbon tetrachloride, Silymarin and Plant extract will be

given concomitantly to the respective groups. All the animals will be killed on

day 7 under light ether anesthesia. The blood samples will be collected

separately by retro orbital vein and serum will be separated by centrifugation at

2500 rpm for 10 minutes, and assay will be done for ALP, Total Bilirubin

Serum Protein, ALT, AST and TP. Then animals will be sacrificed and

histopathological study will be done of dissected liver.

b) Anti ulcer activity will be carried out with Wistar albino rats of both sexes

(150-200 gm.). Rat will be fed standard rat pallet diet.

I) Pyloric Ligation method – Rats will be divided into five groups of six

animals.

Group I- control – will receive vehicle.

Group II- III, IV- will receive plant aqueous, methanolic and chloroform

extract respectively.

Group V - standard drug (Omeprazole 20mg/kg b.w.).

In this method Albino rats will be fasted in individual cages for 24 hours, and

then standard drug, plant extracts and control vehicle will be administered 30

min. prior to pylorus ligation under light ether anaesthesia and abdomen will be

opened and pylorus will be ligated. The abdomen will then be sutured, after 4

hours the animals will be sacrificed, the abdomen will be opened and

oesophageal end of the stomach will be tied and separated from the body and

gastric juice will be collected in to graduated centrifugation tube and

centrifugated at 1000 rpm for 10 min. and gastric volume will be noted and pH

will be recorded by pH meter. The supernatant contents will be subjected to analysis for total and free acidity. The stomach will be opened to observe ulcer

and severity will be graded.

II) Indomethacine Induced Ulcer Method- Wister Albino Rats weighing 150-

200gm. will be used and randomly allotted in to five groups of six animals.

Group I – (control). Indomethacine 20mg/kg body weight will be given orally,

after 24 hours of fasting, and then after 4 hours animal will be killed and

histological changes will be observed.

Group II, III & IV – (Treated). Plant aqueous, methanolic & chloroform

extract will also be given respectively orally for 5 days once daily. After

completion of 5 days, rats will be kept for 24 hours fasting and then

Indomethacine (20 mg/kg) will be given orally and after 4 fours animals will be

sacrificed and study will be done.

Group V – (standard) Omeprazole 20mg/kg will be administered intra

peritoneally as a standard for 6 days. On 6th day, animals will be fasted and

Indomethacine will be administered 4 hours after Omeprazole administration.

Rats will be killed after 6 hours, stomach dissected out then ulcer index and

histological changes will be observed.

c) Anthelminthic Activity will be carried out using Adult earthworms (Pheretima

posthuma) to evaluate anthelmintic activity in vitro, according to the method

(Ghosh et al). Pheretima posthuma will be placed in petridish containing four

different concentrations. Each petridish will be placed with 6 worms and

observed for paralysis (or) death. The mean time for paralysis will be noted, death time of worm (min) will be recorded. The test result will be compared

with reference compound - Albendazole (15mg/ml) treated sample.

Group I - Control (distilled water).

Group II- Reference (Albendazole 15mg/ml).

Group III, IV & V - Test (Plant aqueous, methanolic & chloroform extract).

Group VI – reference (albendazole 15mg/ml).

Enclosure – VI

7.3. Does the study require any investigation or intervention to be conducted on patients or other humans or animals?

As per the standard procedure, the study of the hepatoprotective, anti ulcer and

anthelminthic activities of areal parts of Teramnus labialis will be carried out

on the Wistar albino rats and Earth worms. Enclosure-VI A

7.4. Has ethical clearance been obtained from your institution?

Ethical clearance is provided by the Institution – Yes.

Clearance number: - NCP|IAEC|CL|105|05 / 12 – 13. Enclosure –VII

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24. Alluri V. Krishnaraju, Tayi V. N. Rao, Dodda Sundararaju, Mulabagal Vanisree, Hsin-Sheng Tsay, et al. Assessment of Bioactivity of Indian Medicinal Plants Using Brine Shrimp (Artemia Salina) Lethal assay. International Journal of Applied Science and Engineering 2005;3( 2): 125-134.

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