Completed Proforma for Registration of Subject for Dissertation

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA - BANGALORE

COMPLETED PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

“ANEXPERIMENTAL EVALUATION OF TRAPUSHA BEEJA (Cucumis sativus Linn.) AND ERVARU BEEJA (Cucumis melo var. utillissimus Duthie & Fuller)

W.S.R. TO ITS MOOTRALA KARMA, A COMPARATIVE STUDY”

BY

Dr. SOMIT KUMAR

B.A.M.S.

DEPT. OF P.G. STUDIES IN DRAVYAGUNA,

A.L.N.RAO MEMORIAL AYURVEDIC MEDICAL COLLEGE

AND P.G. CENTRE

KOPPA-577126

GUIDE:

DR. VINAYAKA S. BHAT

B.A.M.S., M.D. (Ay.),

ASST. PROFFESOR

DEPT OF P.G. STUDIES IN DRAVYAGUNA,

1. L.N.RAO MEMORIAL AYURVEDIC MEDICAL COLLEGE

AND P.G. CENTER

KOPPA – 577126

2009-2010

A.L.N.RAO MEMORIAL AYURVEDIC MEDICAL COLLEGEAND P.G. CENTER

KOPPA-577126

CHIKMAGALUR DISTRICT, KARNATAKA

RAJIV GANDHIUNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE

ANNEXURE I I

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name of the candidate:Dr. SOMIT KUMAR

and AddressS/o. dR. ABHOY KUMAR GHOSH

bELA BAGAN, MATHBARI,

dEOGHAR, JHARKHAND.

2. Name of the institution:A.L.N. Rao Memorial Ayurvedic Medical

College and P.GCenter, Koppa,

Chikmagalur DIST.

Karnataka - 577126.

3. Course of study and subject:Ayurveda Vachaspathi, M.D. (Ay.) (dRAVYA gUNA)

4. Date of admission of course:15th JUNE 2009

5. Title of the topic : AN EXPERIMENTAL EVALUATION OF

TRAPUSHABEEJA (Cucumis sativusLinn.) AND ERVARU BEEJA (Cucumis melo var.utillissimusDuthie & Fuller) W.S.R. TO ITS MOOTRALA KARMA, A COMPARATIVE STUDY”

6. Brief resume of the intended work:

6.1 Need for the study:

The earliest reference of the medicinal uses of plants is found in Rigveda and subsequently in Samhita kala. Charaka and Susruta Samhita oldest magnum opuses of Ayurveda provide a remarkable description of uses of plants based on their therapeutic uses as Balya, Vajeekarana, Mootrala etc..Mootrala or Mutravirechanahas been recognized as a pharmacological activity in the earliest written documents on Ayurveda viz.,the Bruhattrayes.1,2,3Acharya Yadavji Trikamji Acharya has clearly equated Mootrala and Mutravirechanam based on evidence in classics. Mutravirechanam or Mootrala karma can be equated with pharmacological activity of diuresis in contemporary medical science4. Drugs ascribed with the above said therapeutic activities are used singly or in combination as frontline therapeutic agents for pathologies, where urine flow is obstructed partially (Mutrakricchra) or completely (Mutraghata). These drugs are also used in Asmari, Udara, Sopha etc. as explained in Bruhattrayees.1,2,3

Diuresis is a therapeutic modality to treat edematous conditions like congestive heart failure, hypertension, pregnancy toxemia, ascites, nephriticsyndrome and some conditions of acute or chronic renal failure in conventional medical science5.

Diuretics bring about physiological alterations likeelectrolyte disequilibrium, metabolic alkalosis,acidosis and hyperuricemia, leading to acute medical emergencies like hypotension, stroke, arrhythmia, seizures, coma5 and chronic conditions likegout, tissue calcification, chronicfatigue syndrome, depression, pancreatitis5 and breast cancer6.Pondering over clinical need and significance, diuretics are indispensable therapeutic tool, but it is also evident that we do need safer and cost effective alternative.

Trapusha Beeja(Cucumis sativus Linn.)7 and Ervaru Beeja(Cucumis melo var. utilissimus Duthie & Fuller)7 has been identified in classics of Ayurveda and contemporary books possessing mootrala(diuretic) activity8-17. Both the drugs are included under Saka1,3,9 varga,Amradi varga9,Guduchyadi varga10,Ousadhi varga11,Pippaliyadi varga12in Bruhattrayes and various Nighantus.Considering these facts, it would pave a way for development of natural, safe, efficacious and cost effective diuretic, if not proved otherwise. The purpose of this study is to evaluate the comparative mootrala(diuretic) activity of the classically mentioned kalka of Trapusha Beeja (Cucumis sativus Linn.) andErvaru Beeja (Cucumis melo var. utilissimus Duthie & Fuller)and their hydro-alcoholic extractin animal models.

6.2. Review of Literature:

The review of literature shall include exhaustive screening of classical Ayurvedic literature where direct and indirect references are given on the diuretic activity of Trapusha(Cucumis sativus Linn.) and Ervaru(Cucumis melo var. utilissimus Duthie & Fuller)1-18.Review of Literature includes exhaustive screening of Classical literature regarding Mootrala activity. So it will be dealt in 2 chapters.

6.2.1.Drug review:

The botanical source of both is identified as seeds of Trapusha(Cucumis sativus Linn.)and Ervaru(Cucumis melo var. utilissimus Duthie & Fuller.).ItsMootrala (diuretic) activity is also well documented in various all Ayurvedic Classics and Nighantus1,2,3,9-18.An exhaustive screening of all the available references related to the experimental drug, especially books recommended by the Drugs and Cosmetics Act, Govt. of India and the Ayurvedic formulary of India will be done. Contemporary researches done by scientists in the field of Ayurveda and allied disciplines will also be reviewed to generate sufficient background information to undertake the study.As they are well known classical diuretic drugs it is consideredworth to study them pharmacologically also for assessment of diuretic activity toprovide experimental basis to clinical findings and also to know probablemechanism of action.

6.2.2.Review of literature regarding Mootrala karma:

A thorough screening of classical Ayurvedic texts, where references regarding Mootrala or Mutravirechaniya karmaareavailable, will be reviewed and recorded. Classics like Bruhattrayees etc. will be referred. Contemporary texts, Materia Medica of Ayurveda(Nighantus) and previous worksetc. will be reviewed for relevant information.

6.3. Objectives of the study:

Review of literature of two trial drugs as mentioned in Ayurveda and contemporary sciences

Pharmacognostic analysis of Trapusha(Cucumis sativus Linn.) and Ervaru(Cucumis melo var. utilissimus Duthie & Fuller).

Preparation of Hydro-alcoholic extract of Trapusha Beeja(Cucumis sativus Linn.) and Ervaru Beeja(Cucumis melo var. utilissimus Duthie & Fuller.) according to reference.

Phytochemical screening of the drugs Trapusha Beeja (Cucumis sativus Linn.) and Ervaru beeja(Cucumis melo var. utilissimus Duthie & Fuller) and their hydro-alcoholic extract.

To evaluate the diureticactivity of drugs as per classical reference and also their hydro-alcoholic extract by experimental study as per the reference protocol.

7. Materials and Methods

7.1. Source of Data

The source of data for the present study can be grouped into the following three;

1)Literary Source

2)Pharmacognostic Source

3)Phytochemical Source

4)Experimental Source

7.1.1. Literary Source

A thorough review of the available literature on the drug and Mootrala karma in Classical texts of Ayurveda, other compendiums, Nighantus will be done.Data will also be collected from periodicals, journals, magazines and papers published in the e-journals etc.Diuretic property of the drugs Trapusha beeja(Cucumis sativus Linn.) and Ervaru beeja(Cucumis melo var. utilissimus Duthie & Fuller) will be particularly reviewed according to the various principles of Ayurveda such as Rasa, Guna, Veerya, Vipaka, Panchamahabhoutika siddhanta as per references available.

7.1.2. PharmacognosticSource

Detailed Pharmacognostic screening of individual drugs Trapusha (Cucumis sativus Linn.) and Ervaru (Cucumis melo var. utilissimus Duthie & Fuller)and their powder after taxonomical identification of plants with help of various floras and herbarium seeking opinion from the botanist. The microscopically study of structures of crystals of hydro-alcoholic extracts will also be done.

7.1.3. Phytochemical Source

Analytical study:Phytochemicalscreening of Trapusha Beeja (Cucumis sativus Linn.) and Ervaru Beeja (Cucumis melo var. utilissimus Duthie & Fuller)and their hydro-alcoholic extract. The analysis will be fragmented in various sub-headings carrying diversified parameters as (yes means for following of parameters while no stands for irrelevancy):

Organoleptic tests:

Parameters / Trapusha Beeja / Ervaru Beeja / Hydro-alcoholic extracts(Both Drugs)
Shape / Yes / Yes / No
Size / Yes / Yes / Nome
Colour / Yes / Yes / Yes
Taste / Yes / Yes / Yes
Fracture / Yes / Yes / No
Texture / Yes / Yes / No
Consistency / Yes / Yes / Yes

Physico-chemical tests:

Parameters / Trapusha Beeja / Ervaru Beeja / Hydro-alcoholic extracts
Total ash / Yes / Yes / Yes
Acid insoluble ash / Yes / Yes / Yes
Water soluble ash / Yes / Yes / Yes
Alcohol soluble extractives / Yes / Yes / No
Water soluble extractive / Yes / Yes / No
pH / No / No / Yes

Qualitative Tests:

Parameters / Trapusha Beeja / Ervaru Beeja / Hydro-alcoholic extracts(Both Drugs)
Glycosides / Yes / Yes / Yes
Alkaloids / Yes / Yes / Yes
Flavonoids / Yes / Yes / Yes
Sugar / Yes / Yes / Yes
Tannin / Yes / Yes / Yes
Phytosterol / Yes / Yes / Yes

Quantitative Tests:

Parameters / Trapusha Beeja / Ervaru Beeja / Hydro-alcoholic extracts(Both Drugs)
Flavonoid / Yes / Yes / Yes
Na+, K+ and Ca+ / Yes / Yes / Yes
Oil content / Yes / Yes / No

7.1.4) Experimental source:

Diuretic activity of Trapusha beeja(Cucumis sativus Linn.) and Ervaru beeja(Cucumis melo var.utilissimusDuthie & Fuller.) will be tested by In Vivo method. Kalka and standard hydro-alcoholic extract solution will be subjected to the experimental study on Wister Rat Models

7.2. Methods of Collection of data (Including sampling procedures if any)

7.2.1)Drug collection and preparation of hydro-alcoholic extract of Trapusha Beeja and Ervaru Beeja.

Samples will be collected and verified at the Dept. of Dravyaguna A.L.N.Rao memorial Ayurvedic medical college from Ayurvedic aspects and in Quality Control Laboratories from modern aspects. Powdered seeds (500 g) willbe packed in soxhlet’s apparatus. The drug will be defatted with petroleum ether (60-80°C) for about 30-35 complete cycles. Defatted material will be extracted with two litres of alcohol: water (7:3) mixture in soxhlet’s apparatus, concentrated under vacuum to get solid crude mass. This dried crude extract of ethanol: water (7:3) will be stored in desiccators and used for further experiment18.

7.2.2) Macroscopic and Microscopic study will be carried out at Quality Control Laboratory of A.L.N.Rao Memorial Ayurveda Medical College.

7.2.3) Phytochemical investigation and screening of individual drug study will be carried out at Quality Control Laboratory of A.L.N.Rao Memorial Ayurveda Medical College.

7.2.4) Experimental study will be carried out at Animal House of A.L.N.Rao Memorial Ayurveda Medical College

Experimental Study Design and Protocol
Sample / Wister rats
Inclusion Criteria / 1) Healthy Male Wister albino rats weighing 150-200g .
2) Male Wister albino rats aged between 60-90 days.
Exclusion Criteria / 1) Male Wister albino rats used for other experiments.
2) Diseased Male Wister albino rats.
Method / In vivo method
Pre-experimental setup / The animals will have free access to water and food and will be acclimatized at least 1 week before starting the experiments. The experiment will be performed under standard conditions of temperature, light, humidity and noise. Six groups of six Male Wister Albino rats each weighing between 150-200g will be used. They will be deprived of any food and water for the preceding 12 hour and will be divided into six groups containing 6 animals in each group. All the animals will be weighed before the experiment.
Grouping / Groups / No.of Rats / Drug / Form / Dose
(orally) / Purpose
Group I / 6 / Normal saline aline / Liquid / 25ml/kg b.w. / Normal Control
Group II / 6 / Frusemide / Powdered tablet mixed in Saline. / 5mg/kg+25ml/kg b.w. / Standard Drug
Control
Group III / 6 / Trapusha beeja Kalka . / Kalka mixed in Saline. / 1g/kg+25ml/kg b.w. / Trapusha beeja Kalka diuretic activity
Group IV / 6 / Ervarubeeja
Kalka . / Kalka mixed in Saline. / 1g/kg+25ml/kg b.w / Ervaru beeja Kalka diuretic activity
Group V / 6 / Trapusha beeja Hydro-Alcocholic Extract. / Hydro-alcoholic extract in Saline. / 500mg/kg+25ml/kg b.w. / Trapusha beeja hydro-alcohol ext. diuretic activity
Group VI / 6 / Ervaru beeja Hydro-alcoholic Extract. / Hydro-alcoholic extract in Saline. / 500mg/kg +25ml/kg b.w. / Ervaru beeja hydro-alcohol ext. diuretic activity
Procedure / Immediately after administration of the drug, the rats will be placed, one in each metabolic cage, specially designed to separate urine and fecal matter and observed at room temperature. The animals will be denied food and water during the experiment. The urine volume will be collected after 1st, 2nd, 3rd, 4th, 5th and 24hour of the oral administration20,21,22.
Duration / 24 hours
Observations / The urine volume (ml/day) will be measured and then assayed for Na+, K+, Ca2+ and Cl- concentrations in mmol/l. The pH values will be analyzed using a pH meter23.
Calculations / 1)Urinary Volume Excretion
The volume of the urine excreted after 5hrs. and 24 hrs. of study by Group I ,II,III,IV,V and VI will be expressed as percent of the liquid administered giving rise to a measure of “Urinary excretion” (U.E)-independent of group weight.
Urinary excretion = Total urinary output/Total liquid administered x 100
The ratio of (U.E) in test group and control group will be denoted.
2) Diuretic Activity
Diuretic action, which will be used as the measure of degree of diuresis
Diuretic action= Urinary excretion in test group/Urinary excretion in control group x100
Diuretic activity= Diuretic action of drug/Diuretic action of frusemide x 100
3) Saliuretic, Natriuretic and Carbonic Anhydrase Inhibition
The sum ofNa+and Cl- excretion will be calculated as a parameter of saliuretic activity. The ratio Na+/ K+ will be calculated for natriuretic activity. The ratio Cl-/ Na++ K+(ion quotient) will be calculated to estimate carbonic anhydrase inhibition22.
Statistics / One way ANOVA followed by Post hoc Scheffe’s test.

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

Yes this being an experimental study conducted on healthy albino rats that are maintained on standard food and water, in our institution animal house (Reg. no. 191/99), test for electrolytes Na+, K+, Ca2+ and Cl- would be done with collected urine according to standard reference.

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

Yes, clearance has been obtained through the ethical committee formulated in the institution.

References:

1. AGNIVESHA elaborated by CHARAKA and DRIDHABALA – Charaka Samhitha with the Ayurveda–deepika-Commentary of Chakrapanidatta and edited by Vaidhya Jadavaji Trikamji Acharya. Published by Chaukhambha Subharathi Prakashan,Varanasi. Reprint edition 2005.page. 151
2. SUSRUTA – MAHARSI – Sursuta Samhita Edited with Ayurveda – Tattva – Sandipika by Kaviraja Ambikadutta Shastri Part – I Chaukhambha Sanskrit Sansthan Publication Varanasi.page 158,202,203
3. VAGBHATA – Astanga Hridaya with commentaries of Sarvangasundar by Arundatta and Ayurveda rasayana by Hemadri, Edited by Pt. Bhisagacharya Harishastri Paradkar vaidhya. 1st Edition. Published by KrishnadasAcademyVaranasi. – 2000.page. 104-105.
4. YADAVJI TRIKAMJI – Dravya guna vijnanam 6th Edition published by Sri Baidhyanath Ayurved Bhavan Limited,Nagpur – 1983,page.52.
5. BORON, WALTER F. (2004). Medical Physiology: A Cellular And Molecular Approach. Elsevier/Saunders. ISBN 1-4160-2328-3.page. 875.

6.J ALARGENT, A JMCELIGOT, AZIOGAS, CREID, JHESS, NLEIGHTON, DPEEL AND HANTON-CULVER. Hypertension, diuretics and breast cancer risk Journal of Human Hypertension (2006) 20, page.727–732.

1. BAPALAL G. VAIDYA – Some Controversial drugs in Indian Medicine 1st Edition –Chaukhambha Orientalia, Varanasi. ––1982.page.299.
2. THAKUR BALWANT SINGH AND CHUNEKAR K.C. - Glossary of vegetable drugs in Brhattrayi, 2nd Edition. Published by Chaukhambha Amarabharati Prakashan. Varanasi. – 1999.page. 57,79,81,191,469,
3. BHAVAMISHRA – Bhava Prakasha Nighantu Translated by Prof. K.R.Srikantha Murthy.Krishnadas Academy. Varanasi Publication. Page.315,389.
4. DHANVANTARI –NIGHANTU – Edited by Prof. P.V. Sharma Translated by Dr. Guruprasad Sharma. 2nd Edition Chaukhambha Orientalia – Varanasi– 1998.page.48
5. KAIYADEVA – Kaiyadeva nighantu – Edited and translated by Prof. P.V. Sharma and Dr. Guruprasada Sharma. 1st Edition Chaukhambha orientalia Varanasi. 1979.page 99-100.
6. NARAHARI PANDIT – Raja Nighantu, with Dravyaguna Prakashika Hindi Commentary. 1st Edition. Krishnadas Achademy Varanasi. – 1982.page. 227-228.
7. NRUPA MADANAPALA – Madanapala Nighantu. Published by Khemaraja. Sreekrishnadasa Prakashana Mumbai – 1990.page. 142-143
8. VARIERS VAIDYARATNAM P.S. – Indian Medicinal Plants a Compendium of 500 species. Vol. – published by Orient Longman Ltd. Madras,page. 227-234.
9. THE AYURVEDIC PHARMACOPOEIA OF INDIA PART – I VOL – II & IV 1st Edition Ministry of health and Family Welfare, Government of India.
10. KIRTIKAR K.R. and BASU B.D – Indian Medicinal Plants Vol. – II. 3rd Edition published by International book, distributors Dehradun – 1988.page.1143-1145.
11. NADKARNI K.M. – Indian Materia Medica. Vol. – I. 3rd Edition Popular Prakashan, Bombay. – 1995.page. 403-406.
12. KALPESH GAUR, M.L. KORI AND R.K. NEMA, Comparative Screening of Immunomodulatory Activity of Hydro-alcoholic Extract of Hibiscus rosa sinensis Linn. and Ethanolic Extract of Cleome gynandra Linn, Global Journal of Pharmacology, ISSN 1992-0075, 3 (2): 85-89, 2009.
13. GHOSH, M.N., 1984. Fundamentals of experimental pharmacology. Published by Scientific book agency. Calcutta.Pharmacologyonline 2: 72-87) Setty et al.86.
14. TWAIJ HAA, ELISHA EE AND AL-JEBOORY AA. Screening of Iraqi Medicine Plants for Diuretic Activity. Indian. J. Pharmac 1985; 73-76.
15. STANIC G, SAMARZIJA I, BLAZEVIC N. Time dependent diuretic response in rats treated with Juniper berry preparations, Phytotheraphy Research 1998; 12: 494-497.
16. MUKHERJEE PK, PAL M, SAHA K, SAHA BP. Diuretic activity of extract of the rhizomes of Nelumbo nucifera Gaertn. (Fam. Nymphaeaceae). Phytotheraphy Research 1996; 10: 424-425.
17. LIPSCHITZ WL, HADIDIAN Z, KERPESAR A. BIOASSAY OF DIURETICS. Pharmacol.Ex.Therap 1943; 79: 97-110.

Signature of the Candidate :

10.Remarks of the Guide :

11. Name and Designation :

(IN BLOCK LETTERS)

11.1 Guide :DR.VINAYAKA S. BHAT

B.A.M.S, M.D (Ay),

ASST. PROFFESOR,

DEPT OF P.G. STUDIES IN DRAVYAGUNA,

A.L.N.RAO MEMORIAL AYURVEDIC MEDICAL

COLLEGEAND P.G.CENTER, KOPPA – 577126

11.2 Signature :

11.3 Co – Guide (if any) :DR.PRASHANT KUMAR JHA

DIM, CIPR, PGDEE, M.Sc.(Medicinal plants), Ph.D.

LECTURER

DEPT OF P.G. STUDIES IN DRAVYAGUNA,

A.L.N.RAO MEMORIAL AYURVEDIC MEDICAL

COLLEGEAND P.G. CENTER, KOPPA – 577126

11.4 Signature :

11.5 Head of the Department : PROF. M.VIDYASAGAR

MD (Ay),PROFFESSOR AND HOD,

P.G DEPT. OF DRAVYAGUNA,

A.L.N.RAO MEMORIAL AYURVEDIC

MEDICALCOLLEGE AND P. G. CENTRE, KOPPA – 577126,

11.6 Signature :

12

12.1 Remarks of the Principal:

12.2 Signature:

1