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					HEPATO PROTECTIVE STUDY ON THE LUFFA CYLINDRICA
  LEAF EXTRACT IN PARACETAMOL AND ALCOHOL
          RENDERED RAT LIVER INJURY


        M. PHARM DISSERTATION PROTOCOL
               SUBMITTED TO THE




       RAJIV GANDHI UNIVERSITY OF HEALTH
        SCIENCES, KARNATAKA, BANGALORE

                        BY
          KUMBHAR PRAMOD BABURAO
                                        B.Pharm.

               UNDER THE GUIDANCE OF

            Dr. SHIVAKUMAR HUGAR
                                 M.Pharm., Ph.D.




      P. G. DEPARTMENT OF PHARMACOLOGY
        B.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

                                            KUMBHAR PRAMOD BABURAO
                                            S/o KUMBHAR BABURAO EKNATHRAO
      Name and Address of the
 01                                         ‘SHIR’ NIVAS SUSHILADEVI NAGAR
      Candidate                             OLD AUSA ROAD, LATUR,
                                            MAHARASHTRA.

                                            B.L.D.E.A’S COLLEGE OF PHARMACY,
02    Name of the Institution               BIJAPUR-
                                            KARNATAKA.

03    Course of the Study Branch            M.Pharm (Pharmacology)

04    Date of Admission to course                             30.06.2010
                                            HEPATO PROTECTIVE STUDY ON
                                            THE LUFFA CYLINDRICA LEAF
05    Title of the Topic                    EXTRACT IN PARACETAMOL AND
                                            ALCOHOL RENDERED RAT
                                            LIVER INJURY
      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
                                            Yes, Registration No: 1076/C/07/CPCSEA
         obtained form your institution     (Copy enclosed)
               in case of 7.3.
08    List of References                    Enclosure – VII


09    Signature of the Candidate
                                                      (KUMBHAR PRAMOD)
                                    The present research work is original and not
                                    published in any of the journals with best of
                                    my knowledge upon extensive literature
10   Remarks of the Guide
                                    review. This work will be carried out in the
                                    Pharmacology laboratory by the above said
                                    student under my supervision.
     Name and Designation of
     (in Block Letters)

     11.1. Guide
                                    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.

     11.2.Signature

11
     11.3.Co-Guide (if any)         -----------------------------

     11.4.Signature

     11.5. Head of the Department   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.

     11.6.Signature

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

                                    (Dr. N. V. Kalyane)
                                                          M. Pharm., Ph.D.
                               ENCLOSURE: I

06. Brief resume of the intended work
6.1. Need for the study
          Herbal drugs have been used since ancient times as medicines for the
treatment of various illnesses. Medicinal plants have played a key role in world
health. In spite of the great advances made in allopathic medicine in recent decades,
plants still make an important contribution to health care system. A large proportion
of the Indian population for their physical and psychological health problems depend
up on traditional systems of medicine. Medicinal plants have become the focus of
intense study in terms of conservation and as to whether their traditional uses are
supported by actual pharmacological effects or merely based on folklore1, 2.
                    In recent years many researchers have examined the effects of
plants used traditionally by native practitioners and herbalists to support liver function
and treat liver disorders. In most cases, research has confirmed traditional experience
and wisdom by discovering the mechanisms and mode of action of these plants as
well as reaffirming the therapeutic effectiveness of certain medicinal plants or their
extracts in clinical studies. The several hundred plants have been examined for use in
a wide variety of liver diseases. However, only few of them have been fairly well
researched3.
           The plant Luffa cylindrica (Linn.) M. Roem. Syn. Luffa aegyptica
belonging to family Cucurbitaceae is commonly called as Sponge gourd. Vegetable
Sponge is a large climber with slender, slightly hairy, furrowed stem, cultivated
throughout India. The literature reports indicated that the plant is known to possess
antiinflammatory4 and fungitoxic activities5, 6. Though the various parts of the plant
recommended in the traditional system of medicine for the treatment of various
diseases, much work on pharmacological activities on title plant has not been
investigated and reported in literature so far. The leaves of the plant are traditionally
used for the treatment of liver diseases.7 However, there is no report on hepato
protective efficacy of the leaves of the title plant available in the literature so far upon
extensive literature survey. Therefore, the present investigation is undertaken.
                             ENCLOSURE: II

6.2 Review of literature:

       The plant Luffa cylindrica (Linn.) M. Roem. Syn. L. aegyptica Mill. Ex Hook. f.
belonging to family Cucurbitaceae. It is commonly called as Torai in Hindi & Urdu,
Ghosali in Marathi, Bhol in Assamese, Jhinga in Bengali, Janhi in Oriya, Gisoda in
Gujarati Beerakaya in Telugu, Heeray kayi in Kannada, Peechinga in Malayalam,
Pirkanga in Tamil7.

       Syn: Luffa aegyptica, smooth Luffa, loofah, vegetable sponge, loofa, loufah
sponge, luffa8.

Description:

       The plant Luffa cylindrica (Linn.) M. Roem. Syn. L. aegyptica Mill. Ex Hook.
f. 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 5 or more meters. Stems are four-angled. Leaves are rounded-ovate to kidney-
shaped, 10-20 cm wide, shallowly 5- to 7-angled or lobed, with pointed tips and heart-
shaped bases. Calyx is green; lobes are ovate-lanceolate, about 1 cm long. Corolla is
yellow, 5-7 cm in diameter. Fruit is oblong, cylindric, smooth and green, upto 20 cm
long and 5 cm wide. Seeds are black, 1 cm long, smooth or slightly tubercled. Fruit is
sweet and larger than the common and bitter wild form9.



Medicinal uses of different parts of Luffa cylindrica.

Root                   : Hydragogue.

Leaves                 : Skin diseases, Liver Diseases and amenorrhea.

Flowers                : Abdominal pains, Cough10.

Seed                   : Anti-inflammatory11, 12, emetic and cathartic.

Tender Fruits         : Diuretic, Antidiabetic13.

Ripe Fruit            : Carminative & Anthelmic.

Fruit Juice           : Purgative.
Phytochemical constituents14, 15:

    Parts                           Phytochemical constituents

    Herb             Saponins Lucyoside A-H, Ginsenoside - Re & Rgl.

   Leaves            Machaerinic Acid Lactone its Acetate & Apigenin.

    Fruit                    Flavonoids, Lucyosides J, K, L & M.

    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 cylindrica 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
hepato protective efficacy of 70% hydro-alcoholic extract of Luffa cylindrica leaf
against different experimentally induced liver toxicity in Wistar rats with the
following objectives.

1. To prepare 70% ethanolic extract of Luffa cylindrica leaf.

2. To determine the presence of phytoconstituents in crude extract of Luffa cylindrica
leaf.

3. To study the acute toxicity of the test extract in mice.

4. To assess the hepato protective efficacy of the test extract against Paracetamol and
ethanol challenged liver injury in rats.
                                ENCLOSURE: IV
7. Materials and methods
7.1 Source of data
   Whole work is intended to explore the results from the pharmacology laboratory
of our institution. Albino rats and mice will be used for the screening purpose.
     The experiments, which involves the following steps


1. Collection of the leaves of Luffa cylindrica after the authentication from the
 botanist/ pharmacognocist.
2. Extraction of leaves of Luffa cylindrica using 70% hydro-alcohol.
3. Detection of presence of phytochemicals in the crude extract.
4. Evaluation of the crude extract of Luffa cylindrica for hepatoprotective activity
 against Paracetamol and ethanol intoxicated liver damage in rats.
                                ENCLOSURE: V

7.2 Method of collection of data

A) Chemical studies
             For this study, leaves of Luffa cylindrica will be collected from the
surrounding gardens of the Bijapur. Fresh leaves will be cleaned, shade dried at room
temperature and coarse powdered. Then the powdered material will be extracted with
70% hydro-alcohol by Soxhlet’s extraction method. Thereafter, the extract will be
concentrated using rotary flash evaporator and percentage yield of the same will be
recorded. The crude extract obtained will be subjected to preliminary phytochemical
screening following the standard procedures described in the literature16.


B) Determination of acute toxicity
          Fixed dose method17 (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 hepatoprotective activity18, 19
           Hepatoprotective activity of 70% hydro-alcoholic extract of leaf of Luffa
cylindrica will be evaluated for the possible preventive and curative effects      on
paracetamol and ethanol induced liver damage in rats. Liver protective efficacy of the
test extract will be assessed by measuring the biochemical markers level such as
SGPT, SGOT, ALP, ACP and Bilirubin (Total and Direct). Further, histopathological
examination of the liver sections will be determined.


D) Experimental Design
       Each group will be comprising of not less than 6 rats each.
       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
E) 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.


F) Work plan details
      Total duration for the completion of proposed research work may be ten months


1. Authentication and collection of plant material            - One month.

2. Duration of experimentation on animals including            - Five months.
   preparation of crude extracts

3. Literature review                                          - Two months.
4. 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 albino rats of either sex
   (Wistar Strain) weighing 150 - 200 g for the hepatoprotective activity.
    Whereas albino mice of Swiss Strain weighing 20-25 g will be utilized for the
    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. Anonymous, Wealth of India, CSIR, Ed 2(5), New Delhi, 1985; 27p.

  02. Naik JB, Jadge DR. Antiinflammatory activity of ethanolic and aqueous
      extracts of Caralluma adscendens. Journal of Pharmacy Research, 2009; 2(7):
      1228-29.

  03. Scott Luper ND. A review of plants used in the treatment of liver disease.
      Part-I. Alternative Medicine Review, 1998; 3(6): 410.

  04. Susithra E, Rao MK, Ramseshu KV, Saraswathy A. Evaluation of anti-
      inflammatory activity of seeds extract of Luffa cylindrica. Journal of
      Pharmacy Research, 2010; 3(7): 1496-98.

  05. Dixit SN, Tripathi SC, Tripathi NN. Fungitoxicity of some seed extracts.
      Natl Acad Sci Lett, 1978; 1: 287-88.

  06. Dixit SN, Tripathi SC. Fungistatic properties of some seedling extracts. Curr
      Sci 1975; 44: 279-80.

  07. Kurian JC, Plants That Heal. 1995; 192p.

  08. Yoganarasimhan SN, Medicinal Plants of India. Vol 1, Karnataka, 1996;
      294-95.

  09. Mugisha MK . The oxytocic properties of Luffa cylindrica, African Journal of
      Ecology, Vol 45 Issue s3, 88-93

  10. Keshavamurthy KR. Medicinal Plants of Karnataka, 1994; 202p.

  11. Gitter S, Gallily R, Batia S, Lavie D. Studies on the antitumer effect of
      Cucurbitacins. Cancer Research, 1961; 21: 516-21.

  12. Gitter S, Gallily R, Batia S, Lavie D. Further studies on the antitumer effect
      of Cucurbitacins. Cancer Research, 1962; 22: 1038-45

  13. Oboh IO, Aluyor EO. review Luffa cylindrica an emerging cash crop.
      African Journal of Agricultural Research, 2009; 4(8): 684-88.

  14. Pandey BP, Economic Botany, S Chand and Company Ltd. 2000; 75p

  15. Ahmad MI, Goel HC, Rizvi MMA. Phytochemical screening and high
      performance TLC Anlysis of some cucurbits, Science Alert, 2010; 4: 242-47.

  16. Khandelwal KR. Practical Pharmacognosy, Nirali Prakashan, Pune, 12th
      Edition; 2004; 149p.
17. Mrs. Prema Veeraraghavan. Expert consultant, CPCSEA, OECD Guideline
    no. 2000; 420

18. Kanda SM, Yeliger VC, Maiti BC, Maity TK. Hepato protective and
    antioxidant role of Beberis tinctoria lesch leaves on paracetamol induced
    hepatic damage in rats. Iranian Journal of pharmacology and therapeutics,
    2005; 4: 64-9

19. Nadro MS, Arungbemi RM, Dahiru D. Evaluation of Moringa oleifera leaf
    extract on alcohol induced hepatotoxicity. Tropical Journal of Pharmaceutical
    Research, 2006; 5(1): 539-44.

				
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