M. PHARM DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH
SCIENCES, KARNATAKA, BANGALORE
BY
SACHIN KAUSHIK
B.Pharm.
UNDER THE GUIDANCE OF
Dr. SHIVAKUMAR HUGARM.Pharm., Ph.D.
P. G. DEPARTMENT OF PHARMACOLOGYB.L.D.E.A’s COLLEGE OF PHARMACY,
BIJAPUR-586103
2010-11
Rajiv Gandhi University of Health Sciences,
Karnataka, Bangalore.
Annexure – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
01 / Name and Address of the Candidate / SACHIN KAUSHIKS/o JAGDISH KAUSHIK
VINOBA NAGAR, TALAPARA ROAD, BILASPUR, CHHATTISGARH
02 / Name of the Institution /
B.L.D.E.A’s COLLEGE OF PHARMACY,
BIJAPUR- 586103KARNATAKA
03 / Course of the Study Branch / M.Pharm (Pharmacology)
04 / Date of Admission to course / 19.06.2010
05 / Title of the Topic / EVALUATION OF ANTI-STRESS (ADAPTOGENIC) ACTIVITY OF LUFFA CYLINDRICA FRUIT EXTRACT
06 / Brief resume of the intended work
6.1. Need for the Study / Enclosure – I
6.2. Review of the Literature / Enclosure – II
6.3. Objective of the Study / Enclosure – III
07 / Materials and Methods
7.1. Source of data / Enclosure – IV
7.2. Methods of collection of data / Enclosure – V
7.3. Does the study require any
Investigations on animals?
If yes give details / Enclosure – VI
7.4. Has ethical clearance been
obtained form your institution
in case of 7.3. / Yes, Registration No: 1076/c/07/CPCSEA Dt27/7/2007
(Copy enclosed)
08 / List of References / Enclosure – VII
09 / Signature of the Candidate / (SACHIN KAUSHIK)
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 / Dr. SHIVAKUMAR HUGAR
M. Pharm., Ph. D.
Professor & H.O.D.
P.G. Department of Pharmacology,
B.L.D.E.A’S College Of Pharmacy,
Bijapur, Karnataka.
------
Dr. SHIVAKUMAR HUGAR
M. Pharm., Ph. D.
Professor & H.O.D.
P.G. Department of Pharmacology
B.L.D.E.A’s College Of Pharmacy,
Bijapur, 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. N. V. Kalyane
M.Pharm., Ph.D.
ENCLOSURE: I
06. Brief resume of the intended work
6.1. Need for the study
Adaptability is probably the most distinct characteristic of life. Dr. Hans Seyle1 defined stress as sum of all non-specific responses of the body to any demands made upon it; fundamentally it was a physiological response; primary object of which was to maintain life and reestablish the normal state. It is evident from the definition that stress cannot be avoided; no matter what one does or what happens to one; there arises a demand to provide the necessary energy to perform the task required to maintain life and to resist and adapt to changing external stimuli. The medicinal substance causing non-specifically increased resistance (SNIR) was variously named as adaptogen or athenkropic2. The plant adaptogen is defined as “Smooth pro-stressors which reduce reactivity of host defense systems and decrease damaging effects of various stressors due to increased basal level of mediators involved in the stress response”3. A number of plants possess adaptogenic activity due to diverse classes of chemical compounds.
Luffa cylindrica (Cucurbitaceae) is also known as torai. This is grown and harvested before maturity and eaten as a vegetable popular in Asia and Africa. The ripe dried fruit is also the source of the loofah or plant sponge. Luffa cylindrica is a sub-tropical plant, which requires warm summer temperatures and long frost-free growing season when grown in temperate regions. Luffa cylindrica has alternate and palmate leaves comprising petiole. The leaf is 13 and 30 cm in length and width respectively and has the acute-end lobe. It is hairless and has serrated edges. The flower of Luffa cylindrica is yellow and blooms on August-September. Luffa cylindrica is monoecious and the inflorescence of the male flower is a raceme and one female flower exists. Its fruit, a gourd, is green and has a large cylinder-like shape. The outside of the fruit has vertical lines and a reticulate develops inside of the flesh. Luffa cylindrica grows about 12 cm long. The stem is green and pentagonal and grows climbing. During a routine screening of this plant for medicinal activity, the
Cucurbita seeds were reported to contain about 35% of crude protein4 showing
anthelmintic, cytotoxic activities, besides as flavanoids5, 6. The ethanolic extract of the seeds of Luffa cylindrica showed more than 80% fungitoxic activity against Helminthosporium oryza7, 8, whereas the alcoholic extract of the leaves and roots of certain species of Cucurbitaceae showed anti-inflammatory activity similar to phenylbutazone9. Luffa cylindrica mainly contains alkaloids, flavonoids, saponins and steroids in moderate concentration. However, trace amount of tannins, glycosides and anthraquinone were noticed. The antistress property of the title plant has been also described in RN Chopra and SL Nayar’s Glosary of Indian Medicinal Plants10,11. As far as our literature survey could ascertain, there is no scientific investigation on antistress / adaptogenic activity of the Luffa cylindrica available till date. In view of this, the present study is undertaken.
ENCLOSURE: II
Review of literature12:
The plant Luffa cylindrica (Linn.) M. Roem. Syn. L. aegyptica Mill, belonging to family Cucurbitaceae is commonly called as Torai in Hindi & Urdu, Bhol in Assamese, Jhinga in Bengali, Janhi in Oriya, Gisoda in Gujarati Beerakaya in Telugu, Heeray kayi in Kannada, Peechinga in Malayalam, Pirkanga in Tamil.
Synonym13: Luffa aegyptica
Description14:
The plant Luffa cylindrica (Linn.) M. Roem. Syn. L. aegyptica Mill, belonging to family Cucurbitaceae is commonly called as Rajakoshaataki or Sponge gourd. Vegetable Sponge is a large climber with slender, slightly hairy, furrowed stem, cultivated throughout India. A climbing, hairy, smooth vine, reaching a length of 12 or more meters.
Stems : Four-angled
Leaves : Rounded ovate to kidney-shaped, 10-20 cm wide, shallowly 5 to 7 angled or lobed, with pointed tips and heart-shaped bases.
Calyx : Green
Lobes : Ovate, lanceolate, about 1 cm long.
Corolla : Yellow, 5-7 cm in diameter.
Fruit : Oblong, cylindric, smooth and green, 12 to 30 cm long, Sweet and larger than the common and bitter wild form.
Seeds : Black, 1 cm long, smooth or slightly tubercled.
Medicinal uses15, 16:
Some common medicinal uses of different parts of Luffa cylindrica
Root : Hydragogue.
Leave : Used for skin diseases, orchitis and amenorrhea.
Flowers : Abdominal pains, Cough.
Seed : Anti-inflammatory, emetic and cathartic.
Ripe Fruit : Carminative & Anthelmic.
Fruit Juice : Purgative.
Phytochemical constituents17-19:
Parts / Phytochemical constituentsHerb / Saponins Lucyoside A-H, Ginsenoside - Re & Rgl.
Leaves / Machaerinic Acid Lactone its Acetate & Apigenin.
Fruit / Lucyosides J, K, L & M.
Male flower / Apigenin Methyl Oleanolate, Oleanolic Acid, Glucose & Arabinose.
Seeds / Lucyoside N & P, Saponin Neutral Genin & Acetyl Gypsogenin & Acetyl Gypsogenin & its Lactone
Seed Kernels / Fatty Oil, Saponin, Bitter Crystalline Substance probably Cucurbitacin B.
Seed oil / α - Spinasterol & other Sterol.
There are few pharmacological properties of the Luffa cylindrical reported in the literature so far have been mentioned in the need for the study of the present work (ENCLOSURE: I).
ENCLOSURE: III
6.3 Objectives of the study
The current research programme is an attempt to establish the possible anti-stress (adaptogenic) efficacy of 70% hydro-alcoholic extract of Luffa cylindrica fruits in mice and rats with the following objectives.
1. To prepare 70% hydro- alcoholic extract of Luffa cylindrica fruits.
2. To detect the presence of phytoconstituents in crude extract of Luffa cylindrica fruits.
3. To study the acute toxicity of the test extract in mice.
4. To assess the anti-stress ( adaptogenic) efficacy of the test extract using various experimentally induced stress models in animals.
ENCLOSURE: IV
7. Materials and methods
7.1 Source of data
The present research work is intended to establish the pharmacological data from the pharmacology laboratory using Wistar rats and Swiss mice in B.L.D.E.A’s College of Pharmacy, Bijapur. The literature review upon the title plant has been thoroughly made using various medicinal plants text books, various national and international research journals from our college library and from the internet also. Standard experimental procedures have been reviewed extensively by referring several research articles as well as review articles.
The experiments, which involves the following steps
01. Collection of the fruits of Luffa cylindrica after the authentication from the botanist/ pharmacognocist.
02. Extraction of fruits of Luffa cylindrica using 70% hydro-alcohol.
03. Evaluation of the crude extract of Luffa cylindrica for anti-stress (adaptogenic) activity using following experimental stress models in animals.
01. Anoxia Tolerance Test
02. Swimming Endurance Test
03. Cold Restraint Stress in rats.
ENCLOSURE: V
Method of collection of data
A) Chemical studies
For this study, fruits of Luffa cylindrica will be collected from the surrounding gardens of the Bijapur. Fresh mature fruits will be cleaned and shade dried at room temperature. Then the powdered material will be extracted with 70% hydro-alcohol by suitable extraction procedure. Thereafter, the extracts will be concentrated using rotary flash evaporator and percentage yield of the same will be recorded. The crude extracts thus obtained will be subjected to preliminary phytochemical screening following the standard procedures described in the literature20.
B) Determination of acute toxicity
Fixed dose method21 (OECD guide line no. 420) of CPCSEA will be adapted to perform acute toxicity of the extracts. The Albino mice (Swiss Strain) weighing between 20-25 g will be utilized for this purpose.
C) Screening of anti-stress (adaptogenic) activity
Antistress activity of 70% hydro-alcoholic extract of fruits of Luffa cylindrica will be evaluated against various experimental stress models in animals as mentioned under22,23.
01. Anoxia Stress Tolerance Test in mice. {Acute stress model}
02. Swimming Endurance Test in mice. {Acute stress model}
03. Cold Restraint Stress in rats. {Chronic stress model}
The following biochemical parameters will be estimated using ErbaChem Semi auto analyzer.
01. Blood glucose
02. Cholesterol
03. Triglycerides
04. BUN
In addition to the above said biochemical parameters organs weight namely liver, testes, spleen and adrenal gland will be recorded.
Experimental design
Each group will be comprising of not less than six animals (mice/ rats).
Group 1- Control
Group 2- Standard
Group 3- Dose-1 1/25th of LD50
Group 4- Dose-2 1/10th of LD50
Group 5- Dose-3 1/5th of LD50
D) Statistical analysis
The data obtained from the above findings will be subjected to statistical analysis using one-way ANOVA followed by Turkey Kramer Multiple Comparison Test to assess the statistical significance of the results.
E) Work plan details
Total duration for the completion of proposed research work may be ten months
01. Authentication and collection of fruits of - One month.
Luffa cylindrica
02. Duration of experimentation on animals including - Five months.
preparation of crude extract
03. Literature review - Two months.
04. Dissertation writing and communication of research - Two months.
papers.
ENCLOSURE-VI
7.3 Does the study require any investigation or interventions to be conducted on patients or other humans and animals? if so please describe briefly.
The proposed study requires the investigation on male albino rats (Wistar Strain) weighing 150 – 200 g for the chronic stress model. Whereas albino mice of either sex (Swiss Strain) weighing 20-25 g will be utilized for the acute stress models and acute toxicity study.
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
The present study protocol is approved from Institutional Animal Ethics
Committee (IAEC certificate is enclosed).
ENCLOSURE: VII
8. List of references:
01. Selye H. The evaluation of the stress, American scientist, 1973; 61(69): 2695.
02. Breakhman II, Dardymov IV. New substances of plant origin which increase non specific resistance, Ann Rev Pharmacol, 1969; 419-21.
03. Panossian A, Wikman G, Wagner H. Plant adaptogens III, Earlier and more rescent aspects and concepts on their mode of action, Phytomedicine, 1999; 6(4): 287-300.
04. Dunnill PM, Fowden L, The amino acids of seeds of the cucurbitaceae, Phytochemistry,1965; 4: 933-44.
05. Ganju K, Puri B, Bioflavonoids from Indian vegetables and fruits, Indian J Med Res, 1959; 47: 563-570.
06. Seshadri T R, Vydeeswaran S. Chemical examination of Luffa echinata, Phytochemistry, 1971; 10: 667-669.
07. Dixit SN, Tripathi SC & Tripathi NN. Fungitoxicity of some seed extracts, Natl Acad Sci. Lett, 1978; 1: 287-288.
08. Dixit SN, Tripathi SC. Fungistatic properties of some seedling extracts, Curr Sci, 1975; 44: 279-280.
09. Peters R , Farias MR, Ribeiro-do-Vale RM. Anti-inflammatory and analgesic effects of cucurbitacins from W. ebracteata, Planta Med, 1997;63: 525–528
10. Chopra RN, Nayar SL, Chopra IC. Glossary of Indian Medicinal Plants, 1996. New Delhi, National Institute of Science Communication.
11. Agarwal SS, Singh VK. Immunomodulators : A review of studies on Indian Medicinal Plants and synthetic peptides; Part I: Medicinal Plants; 1999; PINSA,B-65, 179-204.
12. Yoganarasimhan SN. Medicinal plants of India, Vol. 1, Karnataka, 1996; 294p.
13. Mugisha MK et al. The oxytocic properties of Luffa cylindrica, African Journal of Ecology,45.
14. Keshavamurthy KR, Medicinal plants of Karnataka, 1994; 202.
15. Gitter S, Gallily R, Shohat B, Lavie D. Studies on the antitumer effect of cucurbitacins, Cancer Research, 1961; 21: 516-521.
16. Gitter S, Gallily R, Shohat B, Lavie D. Further studies on the antitumer effect of Cucurbitacins; Cancer Research, 1962; 22: 1038-1045.
17. Oboh IO, Aluyor EO. Review Luffa cylindrica an emerging cash crop, African Journal of Agricultural Research, 2009; 4(8): 684-688.
18. Pandey BP. Economic Botany, S Chand and Company Ltd, 2000; 75-76.
19. Ahmad MI, Goel HC, Rizvi MMA. Phytochemical Screening and High Performance TLC Anlysis of Some cucurbits, Science Alert, 2010; 4: 242-247.