RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA

Annexure – II

PROFORMA FOR REGISTRATION OF SUBJECT FOR

DISSERTATION

1. / NAME OF THE CANDIDATE AND ADDRESS / Mr. YADVEER SINGH
DEPT. OF PHARMACOLOGY
SET’s COLLEGE OF PHARMACY
S.R.NAGAR,
DHARWAD-580002
2. /

NAME OF THE INSTITUTION

/ SET’s COLLEGE OF PHARMACY
S.R.NAGAR,
DHARWAD-580002
3. /

COURSE OF STUDY & SUBJECT

/ MASTER OF PHARMACY IN PHARMACOLOGY
4. /

DATE OF ADMISSION TO COURSE

/ JUNE-2008
5. /

TITLE OF THE TOPIC

HEPATOPROTECTIVE AND ANTIOXIDANT POTENTIAL OF CUSCUTA REFLEXA ROXB IN MICE.
6 /

BRIEF RESUME OF THE INTENDED WORK:

6.1 Need for the study:

The liver regulates several important metabolic functions and the hepatic injury is associated with distortion of these metabolic functions 1, along with that liver is the key organ of metabolism and excretion and it is continuously exposed to xenobiotics because of its strategic placement in the body. Many hepatotoxicants such as carbon tetrachloride (CCl4), nitrosamines, and polycyclic aromatic hydrocarbons require metabolic activation, particularly by liver cytochrome P450 (CYP) enzymes, to form reactive, toxic metabolites, which in turn cause liver injury in animals and humans 2. .
Thus, liver diseases remain one of the serious health problems. In spite of tremendous strides in the modern medicine, there are not much drugs available for the treatment of` Liver diseases3.
The general strategy for prevention and treatment of liver damage includes reducing the production of reactive metabolites by using antioxidants 4, 5. Antioxidants appear to act against diseases by raising the levels of endogenous defense [e.g., by up-regulating gene expressions of the antioxidant enzymes, such as superoxide dismutase (SOD), catalase, glutathione peroxidase, and lipid peroxidase] 6.
The liver is a vital organ of paramount importance involved in the maintenance of metabolic functions and detoxification from the exogenous and endogenous challenges, like xenobiotics, drugs, viral infections and chronic alcoholism. If during all such exposures to the above mentioned challenges the natural protective mechanisms of the liver are overpowered, the result is hepatic injury.
There are a number of medicinal preparations recommended in the Indian traditional system of medicine ‘‘Ayurveda” for the treatment of liver diseases. There are scientific claims to offer significant relief as hepatoprotective7.
The cuscuta reflexa Roxb. (Amarabela in Hindi and swarnalata in Bengali ) family convolvulaceae is a golden yellow dodder like parasite. The plant is common throughout India, found widely in the plains of West Bengal, growing on thorny or other shrubs as parasite annuals. Various parts of this plant were used in tribal medicine for the diseases like fits, melancholy, and insanity 8. It is also useful externally against itch and internally in protracted fevers, retension of wind and induraion of liver 9, 10. The plant is also used as anthelmintic, diuretic, cough, bronchitis and in the treatment of jaundice11.
The present study has been designed to investigate the “Hepatoprotective and antioxidant potential of Cuscuta Reflexa Roxb in Mice”
6.2 Review of literature:
Liver damage is always associated with cellular necrosis, increase in tissue lipid peroxidation and depletion in the tissue GSH levels. In addition serum levels of many biochemical markers like Serum gluatamate oxaloacetate transaminase (SGOT), Serum glulutamate pyruvate transminase (SGPT), triglycerides, cholesterol, bilirubin, alkaline phosphatase, are elevated12, 13.
Carbon tetrachloride exerts its damaging effect on the liver cells by conversion to free radicals i.e. conversion of CCl4 (carbon tetrachloride) to CCl.3 (trichloromethyl radicals) by the process of dehalogenation of carbon tetrachloride. This dehalogenation is mediated by cytochrome P-450, this process of production free radical setting the chain reaction of lipid peroxidation. This lipid peroxidation increases membrane permeability which ultimately causes cell ageing 14, 15.
Liver damage is taking place in three stages i.e. necrosis, fibrosis, and cirrhosis. Necrosis can be developed by the CCl4 and phenobarbiton administration. This is first step of liver damage. The liver sometimes displays lesions and acute or sub acute necrosis, which may be accompanied by regeneration. Depending on duration of the effect, cirrhosis becomes more prominent. The necrotic lesions can be caused by a variety of xenobiotics, such as tetrachloroethane, trinitrotoluene, and mixtures of chlorinated biphenyls and chlorinated naphthalenes16.
Plants are the basis of life on earth and are central to people’s livelihoods. Tribal people are the ecosystem people who live in harmony with the nature and maintain a close link between man and environment. Indian subcontinent is being inhabited by over 53.8 million tribal people in 5000 forest dominated villages of tribal community and comprising 15% of the total geographical area of Indian landmasses, representing one of the greatest emporia of ethno-botanical wealth 17.
Cuscuta reflexa Roxb. belongs to family convolvulaceae. A slender much brached leafless yellow interwinning parasite on shrubs and trees almost covering them, without contact with soil; fowers sometimes solitary or in clusters of 2-4; fruits capsules, 6-8mm diameter, depressed, globose with 2-4 black seeds11.
The herb has an acrid, bitter; astringent to the bowels, aphrodiasiac, tonic and useful in the diseases of the eye and heart. The seeds have a bitter bad taste; sedative, diuretic, and liver and spleen diseases and stem are especially useful in bilious disorders.
The fresh plant yields scopaone, melanettin, quercetin and hyperoside. Some soluble phenolic compounds, hydroxycinnamic acid like 3,4-dicaffeolyquinic, 3,5-dicaffecoylquinic and 3-caffeoylquinic acids have been isolated from plant. The main active principles present in the plant are cuscutalin and cuscutin. The plant also contains reducing sugars. The seeds contain amrvelin, resins and reducing sugars18.
C. reflexa is parasitic on the above-ground parts of other plants. The leafless
stems coil around the host stems and petioles. C. reflexa contains only a small amount of chlorophyll and relies on obtaining photosynthetic assimilates from the phloem of its host. It forms specialized structures, called haustoria, which are used to acquire nutrients from its host plant19.
Cuscuta reflexa on preliminary analysis is found to contain 0.2% cuscutin (a colouring matter), 1% cuscutalin (lactone) and a large quantity of flavonoids20, 21.
Scientifically proven effect of cuscuta reflexa Roxb. Are; Methanolic fraction of c.reflexa stem exhibited a broad spectrum antibacterial acvivity against all the tested strains such as, S.auresus, S. boydii, P.aeruginosa, S.dysenteriae and E.coli with a zone of inhibition ranging from 16-24 mm22.
Methanolic extraction of C. reflexa roxb. depresses and reduce the ovarian steroidogenesis23. The petroleum ether extract of C.reflexa exhibited promising hair growth-promoting activity as reflected from follicular density, anagen/telogen ratio, and skin sections.24 Cuscuta reflexa also exhibits hematological benefits.25
6.3 Objectives of the study:
In vivo study:
To evaluate the effect of Cuscuta reflexa Roxb, on Liver Marker Enzymes, such as
·  Serum glutamate pyruvate transaminase (SGPT)
·  Serum glutamate oxaloacetate transaminase (SGOP)
·  Alkaline phosphatase ( ALP )
To evaluate the effect of Cuscuta reflexa Roxb, on Antioxidant Enzymes, such as
·  Catalase
·  Glutathione peroxidase
·  Superoxide dismutase (SOD)
In vitro study:
·  Lipid peroxidation
·  DPPH
7 / MATERIALS AND METHODS
7.1 Source of data:
The sources of data are from the laboratory experiments, which involve evaluation of pharmacological activities on experimental animals.
7.2 Method of collection of data: (Including sampling procedure if any)
1. Animals: Male Swiss mice weighing 23-25 g will be used. The animals will be maintained under control condition of temp. 23±2oC. The animals are randomized into expt. And control groups and housed each in sanitized polypropylene cages. They will be free access to standard pellets as diet and water.
2. Chemicals: Thiobarbituric acid (TBA), Dimethyl superoxide (DMSO), Carbon tetrachloride and all the other chemicals of analytical grade will be used according to the standard procedure.
3. Parameters to be studied:
i) Extraction of plant: Whole plant will be extracted with alcohol.
ii) Acute toxicity study: The guidelines described by OECD will be adopted for the determination of LD50 on mice. And 1/10th of LD50 will be taken as dose for the study.
iii) Liver toxicity: Hepatic toxicity will be induced by administration of CCl4 at a dose of 20µl/kg by i.p route.
iv) Liver Marker Enzymes: The effect of cuscuta reflexa Roxb. is evaluated on liver marker enzymes such as; Serum glutamate pyruvate transaminase (SGPT), Serum glutamate oxaloacetate transaminase (SGOT), and Alkaline phosphatase ( ALP ).
v) Antioxidant enzymes: The effect of cuscuta reflexa Roxb. is evaluated on liver Antioxidants enzymes such as; Catalase, Glutathione peroxidase and Superoxide dismutase (SOD).
vi) Lipid peroxidation and DPPH will also be measured as an in-vitro model.
7.3  DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR INVENTION TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS? IF SO, PLEASE, MENTION BRIEFLY.
Yes. The above study requires an investigation on adult male Swiss mice for evaluation of hepatoprotective and antioxidant effect of Cuscuta reflexa Roxb.
7.4  HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION INCASE OF 7.3?
The study is cleared from the ethical committee of the institution.The study is cleared from the ethical committee of the institution.

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19. Hibberd JM, Bungard RA, Press MC, Jeschke WD, Scholes JD, Quick WP Localization of photosynthetic metabolism in the parasitic angiosperm Cuscuta reflexa Planta 1998;205:506-513.
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23. Gupta M, Mazumder UK, Pal DK. Ooset of puberty and ovarian steroidogenesis following administration of methanolic extract of Cuscuta reflexs Roxb. Stem and Corhorus Olitorius Linn. Seed in mice. J Ethanopharmacol 2003;89:55-59.
24. Pandit, Shweta, Chauhan. Effect of Cuscuta reflexa Roxb on androgen-induced alopecia. J cosmetic dermatol 2008;7(3):199-204.
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