Anti-Diarrhoeal Properties Of70% Alcoholic Extract Oftylophora Indica Leaves

INVESTIGATION ON THE GASTROPROTECTIVEAND

ANTI-DIARRHOEAL PROPERTIES OF70% ALCOHOLIC EXTRACT OFTYLOPHORA INDICA LEAVES

M. Pharm. Dissertation Protocol

Submitted to the

Rajiv GandhiUniversity of Health Sciences, Karnataka Bangalore

By

PARVATHI ABILASH GOUD

B. Pharm

Under the guidance of

Mr. RAJSHEKAR BHANDE

M. Pharm, (Ph. D)

PROFESSOR

POST GRADUATE DEPARTMENT OF PHARMACOLOGY

R.R.K.S.COLLEGE OF PHARMACY,

NAUBAD, BIDAR-585 402

2011-12

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE

Annexure-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1 / Name and address of the candidate / Mr. PARVATHI ABILASH GOUD
S/o P. MallaiahGoud
H. No. 4-1-13,
Balaji Road,
Village Korutla,
Dist. Karimnagar (Andhra Pradesh)
Ph. No. 9392660740
2 / Name of the institution / RRK’s College of Pharmacy,
Naubad, Bidar-585 402
(KarnatakaState)
3 / Course of the study branch / M. Pharm (Pharmacology)
4 / Date of admission to course / 22-11-2010
5 / Title of the topic / Investigation on the Gastroprotective and Anti-Diarrhoeal Properties of 70% alcoholic extract of TylophoraIndica Leaves
6 / Brief resume of the intended work:
6.1. Need for the study
6.2. Review of the Literature
6.3. Objective of the Study / Enclosure-I
Enclosure-II
Enclosure-III
7 / Materials and Methods:
7.1. Source of data
7.2. Methods of collection of data
7.3. Doest the study require any
investigations on animals?
If yes give details.
7.4. Has ethical clearance been
obtained form your institution
in case of 7.3. / Enclosure-IV
Enclosure-V
Enclosure-VI
Yes
REGISTRATION NO.361/01/C/CPCSEA.
(Copy enclosed)
8 / List of references (about 4-6) / Enclosure-VII
9 / Signature of the candidate / (PARVATHI ABILASH GOUD)
10 / Remarks of the Guide / The present research work is original and not published in any of the journals with best of my knowledge upon extensive literature review. This work will be carried out in the Pharmacology laboratory by the above said student under my supervision.
11 / Name and Designation of
(in block letters)
11.1. Guide
11.2. Signature
11.3. Co-Guide (if any)
11.4. Signature
11.5. Head of the Department
11.6. Signature / MR. RAJSHEKHAR BHANDE
M. Pharm. (Ph. D).
PROFESSOR
Department of Pharmacology,
RRK’s College of Pharmacy,
Naubad, Bidar-585 402
(Karnataka)
------
-No-
MR.RAJSHEKAR BHANDE
M.pharm.(ph.D).
Head of the Department of Pharmacology,
RRK’s College of Pharmacy,
Naubad, Bidar-585 402
(Karnataka).
12 / Remarks of the Principal
12.1 Signature / The present study is permitted to perform in the Pharmacology laboratory of our institution and the study protocol has been approved by IAEC.
(Dr. K.SRINIVAS RAO)
M.Pharm., Ph.D.
Principal,
R.R.K’S College Of Pharmacy,
Naubad,Bidar, Karnataka.

ENCLOSURE-I

06. Brief resume of Intended Work:

Need for the study:

Several plants and herbs are used in medicine to treat different types of gastrointestinal disorders including peptic ulcer and diarrrhoea. Ulcerative lesions of gastrointestinal tract are one of major side effects associated with the use of non-steroidal anti-inflammatory drugs, alcohol, and stress1. Therefore there is an urgent need to identify novel anti-ulcer agents derived from plant sources. The use of the medicinal plants for curing disease has been documented in history of all civilization.

Tylophoraindica (family: Asclepidaceae) commonly known as Antmul is a twining perennial plant distributed through southern and eastern plant of India2.

There are several plants very effective in treating ulcer / diarrhea, such plants include mermia emerginata3,vernonia cinerea4oroxylum indium vent5. One such plant Tylphoraindica, contain alkalods viz., Tylophorine, tylophorinine, tylophorinidine, sterols, flavanoids, wax, resins and tannins6. the active constituent mention above has reported many biological activities, such as anti inflammatory, anti-cancer, anti-oxidant, anti-asthmatic and anti-microbial activity7, 8, 9.However the literature reveals that no scientific data on anti-ulcer and anti diarrhoea.

ENCLOSURE-II

6.2. Review of Literature:

The plant Tylophoraindica, (family Asclepidaceoe) commonly known as antmul is a twining perennial plant distributed through southern and eastern plant of India in plans, forests and hilly places10. The plant is found growing normally in Uttar Pradesh, Bengal, Assam, Orrisa, Himalayas and sub-himalayas in India. It is a branching climber or shrub that grows up to 1.5 meter leaves or obvate-oblong to ellipstic-oblong, 3-10 cm long and 1.5-7 cm wide11.

Chemical Constituents

The literature survey reveals that the plant contain major chemical constituents TylophorineKaempferol, -amyrin,-sitosetrol, and quercetin, tannins, flavanoids, other major alkaloide like tylophorindine, desmethylthlophorine, desmethyltylophornine and other alkaloids (+) septicine and (+) isotylocerebrine from fresh leaf12.

The literature reveals of Tylophoraindica has been posses diuretic activity13, hepato protective activity (paracetamol induced liver damage)14, immunomodulatory effect15, antiulcer activity (histamine and naproxen induced gastric ulcer)16. However the litrerature reveals that there is no scientific data on anti-ulcer and anti-diarrhoeal. Hence, purpose of safe compound with lesser side effects to treat gastrointestinal disorders by using herbal plants. Tylophoraindica use as herbal drug in Ayurveda traditional medicinal system, hence present study is purpose to investigate the TylophoraIndica leaves for anti-ulcer and anti diarrhoeal activity.

Review of literature till date regarding Tylophoraindica was carried out by referring chemical abstracts, biological abstracts, medicinal and aromatic plant abstracts and other scientific journals.

In view of this the present study is taken up to investigate the anti ulcer and anti diarrhea activity of tylophoraindica so this study is essential and justifiable.

ENCLOSURE-III

3). Objectives of the study:

1)The study is aimed to investigate and validate the anti-diarrhoeal and anti-ulcer activities of pharmacological methods of evaluation.

2)In view of this, the leaves of the Tylophoraindica have been selected for the present study.

ENCLOSURE-IV

7. Material & methods:

7.1 Source of data:

Whole work is aimed to generate data from the laboratory i.e. experiments on animals. Albino mice and rats will be used for this purpose.

The experiments, which involves:

1)Extraction of leaves of the Tylophoraindica with suitable solvent(70% alcohol).

2)Phytochemical investigation.

3)Evaluation of 70% alcoholic extract of Tylophoraindica leaves for anti-diarrhoeal and anti-ulcer potential by employing experimentally induced ulcer and diarrhoea models.

ENCLOSURE-V

7.2Methods of collection of data:

a)Chemical studies:

The leaves and Tylophoraindica were dried in shade and processed to a coarse powder. The powder is used for extraction purpose by using different solvents.

Pharmacological studies:

1)Determination of acute toxicity17:

Fixed dose method (OECD guide line No. 420) of CPCSEA will be followed to carry out LD50 of the extracts. For this purpose albino mice of either sex (20-25gm) will be used.

2)Evaluation of the extract for antidiarrhoel and antiulcer activities by different experimentally induced diarrhoea and ulcer models. The experimental models (screening models) will be used in the study are outlined as below.

Anti-diarrhoeal activity:

a)Castor-oil induced diarrhoea18:

1. Inclusion criteria: In the present study only healthy albino rats of either sex (wistar strain), 150-200g body weight are selected

Any animals not satisfying above criteria will be excluded.

1. Four groups of rats containing six animals each

b)Gastro-Intestinal motility test19.

1. Albino rats of either sex weighing 15-200gm should be selected
2. Four groups of rats containing six animals each.

Group-I: Control (0.2ml 2% w/v of gum acacia p.o)

Group-II:Atropine sulphate (5mg/kg. i.m)

Group-III:70% Ethanolic extract (p.o)

.

All the results were subjected for the statically analysis by ANOVA. Followed by student ‘t’ test

Anti-ulcer activity:

a)Aspirin induced ulcer20:

ii)Inclusion criteria: Only healthy albino rats of either sex weighing between 150-200g should be selected

iii)Four groups of rats containing six animals each.

Group I:Control (Aspirin 500mg/kg p.o)

Group II:Standard (Lansoprazole 8mg/kg p.o)

Group III:70% Ethanolic extract (p.o)

The test drugs should be administered orally in 2% gum acacia solutions 45min prior to aspirin treatment

After 4hrs animals were sacrificed and stomach is dissected out for scoring ulcer index.

b)Pylorous ligation induced gastric ulcer21:

i)Inclusion criteria: Only healthy albino rats of either sex weighing between 150-200g should be selected

ii)Four groups of rats containing six animals each.

Group I:Control

Group II:Standard (Ranitidine 50mg/kg)

Group III:70% Ethanolic extract

The drugs were given orally 2hrs prior to pylorus ligation as reported by shay (shay et.al. 1945). 5% w/v Acacia mucilage was used as vehicle at a dose of 5ml/Kg.

The animals were sacrificed 6hrs after pylorus ligation for observation of gastric lesion as described by Gupta (Gupta et.al. 1985).

iii)Now follow up as all the animals will be sacrificed

The data obtained from the above study will be subjected to statistical analysis using one-way ANOVA. Followed by student’s ‘t’ test.

Total duration for the completion of whole project may be eight months

1. Leaves collection and shade drying:One month
2. Duration of experimentation :Four months
3. Literature survey:One and half month
4. Thesis writing :One and half month

ENCLOSURE-VI

7.3.Does the study require any investigation or interventions to be conducted on patients or other humans or animals? If so, please describe briefly.

The above study requires investigation on animals i.e. albino mice for determination of LD50 and anti-diarrhoeal and anti-ulcer potential on albino rats and mice.

7.4.Has ethical clearance been obtained from your institution in case of 7.3?

The study is cleared from institutional animal ethics committee (IAEC certificate enclosed).

ENCLOSURE-VII

8.0 List of references:

1)Jaikumar s*, Ramaswamy S, Asokan B R,MohanT,GnanavelM,Anti-ulcer activity of Methonolic Extract of Jatrophacurcas (linn .) on Aspirin Induced Gastric lesions in Wister Strain rats.Research journals of pharmaceutical,Biological and Chemical Sciences.2010;1(4):p.g.no.886.

2)Mayankgupta,Hayat m. mukhtar,sayeedAhmed.phyto pharmacological and plant tissue culture.Overview of tylophoraIndica (burm f.)merill.journal of pharmaceutical sciences and research 2010;2:401-411.

3)Babu,A.V.;Sujatha.K.;valli,A.S.,Narayana,K.J.P.,Babu,B.H.;Sathyanarayana,P.V.V.,Journal of Biosciences,BiotechnologyReseach Asia 2009;Vol.6 no.2:pp. 835-838.

4)Mazumdaruk,Evalvation of anti-inflammatory activity of v.cinerea extracts in rats,phytomedicine,2003;10;185-188.

5)Lebranchu Y.New approaches to de novo Immunosuppression and Steroid elimination,Transplant proc.2008 dec.

6)M.ali,K.K.bhutani,Regional research Laboratory,C.S.I.R,Alkaloids from TylophoraIndica ,1989;vol(28):p.g. 3513-3517.

7)David CR, Mohammed S, Mahesh.N, anti-inflamatory activity of Tylophoraindica in albino rats.J. PharmaclToxicol 2006;1:490-492.

8)B.KrishnaReddy,M.Balaji,P.Umareddy,G.Sailaja,K.Vaidyanath and G.Narasimha .African Journal of Biochemistry Reseach.2009;vol.3(12):pp.393-397.

9)Gujrati ,Venkat NR, Kumar SM. Hepatoprotective Activity of alcoholic and aqueous extracts of leaves of TylophoraIndica (Linn.) in rat. Indian J.Pharmacol 2007;39:43-47.

10)Gupta,Mayank Journal of Pharmaceutical sciences and Research,2010:July 1st.

11)IHerb.com,EBSCO CAM Review Board;2011.

12)Guptha A.K., “Quality Standards of Indian Medicinal Plants”, ICMR, 2003; vol-1: 221-225.

13)MeeraR,DeviP,MuthumaniP,B.Eswarapriya Evaluation of Diuretic activity from Tylophoraindica leaves extracts.Journal of pharmaceutical sciences and Research.2009;vol.3(12):pp.393-397.

14)R.Malathi and M.Patrick Gomez, Journal of Pharmacology and Toxicology 2007; 2(8): 737-742.

15)T.Ganguly,L.PBadheka and K.B.Sainis.Immunomodulatory Effect of Tylophoraindica on Con A Induced lympho proliferation.Phytomedicine,2001;vol(8) issue 6:page 431.

16)Amagase k, Okabe S, on the mechanisms underlying histamine induction of gastric mucosal lesions in rat with partial gastric vascular occlusion. J.
Pharmacol. Sci 2003;92:124-136.

17) Mrs. PremaVeeraraghavan, Expert consultant, CPCSEA, OECD guideline No. 420.

18)Pulok K. Mukherjee, Das J, Balasubramanian R, KakaliSaha, Pal M, Saha BP, Antidiarrhoeal evaluation of Nelumbonucifera rhizome extract. Ind. J. Pharmacol 1995: 27: 262-64.

19)S.Meite,JDNguessan,CBhi,H.Fyapi,AJDjaman,and F GuedeGuina.Anti-diarrhoeal activity of the ethyl acetae extract of MorindaMorindoides in Rats,Tropical Journal of pharmaceutical Research,2009;8(3):201-207.

20)Kulkarni S.k.,Hand Book of Experimental pharmacology,Vallabhprakashan,New delhi,77;1987.

21)RobertA.turner.Screening methods in pharmacology,Academic press,Newyork,22.(1965).