“evaluation of antioxidant activity of viscum Articulatum Burm f.”




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“EVALUATION OF ANTIOXIDANT ACTIVITY OF Viscum Articulatum Burm.f.”


SYNOPSIS FOR REGISTRATION OF M.PHARM DISSERTATION


Submitted to

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,


KARNATAKA

In partial fulfillment

of the requirement for the Degree of

Master of pharmacy In Pharmacology
UNDER THE GUIDANCE OF

Mrs.GEETHA. K.M

Associate Professor and H.O.D

Dept of Pharmacology
BY

BINDU.V

I M.PHARM

Department of Pharmacology

DAYANAND SAGAR COLLEGE OF PHARMACY

BANGALORE-560078
2009-2010

ANNEXURE – I

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION


 
 


1.


Name of the Candidate

and Address


BINDU.V
BIMBA’, # 447,10th Cross, 4th Main,

Annapoorneswarinagar,

Bangalore-91




2.



Name of the Institution

DAYANANDA SAGAR COLLEGE OF PHARMACY, Shavige Malleswara Hills, Kumaraswamy Layout, Bangalore-560078.



3.


Course of Study and Subject


Master of Pharmacy in Pharmacology




4.


Date of Admission


25.05.2009




5.


Title of the Topic:


Evaluation   of   Antioxidant Activity of Viscum articulatum Burm.f.”





6.0

7.0

8.0

Brief resume of the intended work 

6.1 – Need for study: 

Free radicals are molecules or molecular fragments containing an unpaired electron in the valence shell and capable of existing independently. Due to unpaired electron status free radicals are extremely reactive species reacting rapidly with majority of organic compounds. The potentially reactive derivatives of oxygen, ascribed as ROS such as O2•−, H2O2 and •OH(free radicals) are being continuously produced inside the human body as a consequences of exposure to exogenous chemicals in our ambient environment, and/or a number of endogenous metabolic processes involving redox enzymes and bioenergetic electron transfer. Substances like drugs, radiation, tobacco smoking, inorganic particles etc., also contribute to enhanced free radical production.1,2

The ROS readily attack and induce oxidative damage to various biomolecules including proteins,lipids, lipoproteins and DNA This oxidative damage is a crucial etiological factor implicated in several chronic human diseases such as diabetes mellitus,cancer, atherosclerosis,arthritis, neurodegenerative diseases and also in the ageing process.1 However, in healthy conditions nature has endowed human body with enormous antioxidant potential. Subtle balance exists between free radical generation and antioxidant defense system to cope up with oxidative stress by various enzymes and vitamins at cellular level which prevent occurance of diseases. However factors tilting the balance in favour of excess free radical generation leads to widespread oxidative tissue damage and disease. Therefore trouble starts when there is an excess of free radicals and the defense mechanism lags behind. Overwhelming production of free radicals in response to exposure to toxic chemicals and ageing has necessitated for judicious antioxidant supplement to help alleviate free radical mediated damage2

Anitoxidants combat oxidation and are involved in prevention of cellular damage by directly reacting with free radicals, quenching them and/or chelating the catalytic metal ions.

Several synthetic antioxidants, e.g., BHA and butylated hydroxytoluene are commercially available but are quite unsafe and their toxicity is a problem of, are concern .Natural antioxidants, especially phenolics and flavonoids safe and also bioactive. Therefore, in recent years, considerable attention has been directed towards identification of plants with antioxidant ability that may be used for human consumption.1

Carbon tetrachloride is an exogenous toxin which produces cellular injury largely or entirely by free radical mechanism. It causes damage to all the organs but majorly affects the liver. The biological evidence for injury caused by oxidative stress induced by Carbon tetrachloride includes elevated levels of transaminases,alkaline phosphatates and certain antioxidant enzymes like Catalase, Superoxide dismutase, peroxidases etc.,3The leaves of Viscum articulatum has great medicinal value and is used in Kapha, Vata, diseases of blood, ulcer, epilepsy and biliousness which might be related to the antioxidant activity of the plant. Thus, the present study is done with an objective to evaluate the invitro and invivo antioxidant activity of natural herb Viscum Articulatum in Carbon tetrachloride induced oxidative stress in rats.


6.2 – About the plant
Viscum Articulatum were used in Bardia District of Nepal and was first documented in the book of ‘Medicinal Plants of Nepal’ marking the beginning of ethnobotonical studies on mistletoes.4
Viscum Articulatum belongs to the genus Visum and Family Loranthaceae. It is a group of semiparasitic shrubby type plant, generally growing over Diospyros & Brassica sp. distributed in the region of Himalaya, Assam, Khasia Hills, Burms, Central provinces, W.Peninsula, Celon, Malay peninsula, Java, Australia.5

The plant has a great medicinal value, it is used in Kapha, Vata,diseases of blood, Ulcer,epilepsy,biliousness. In Chotanagpur a preparation from the plant is given in fever attended with aching limbs.5 Plant bark mixed with Hen egg and pinus roxburghii leaf is used for ailment of bone dislocation. Paste prepared from all parts of plant is applied over the fractured portion of the body.4 It is also used in atropy, cachexia, leg ache.6 The plant is used for snake bites by Adivasis of Rayalaseema region of Andhra Pradesh.7 Other traditional uses of Viscum Articulatum are in Rheumatic arthritis, Urinary tract infection, leucorrhea, Epistaxis,Lumbar muscle strain,Low back pain,Bacillary dysentery,Uterine bleeding, Pyodermas, psoriasis,weakness,Lactation deficiency.8 The plant has also shown inhibition effect on superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanin.(FMLP).9 Some of the species of this genus have shown to pocess medicinal functions. The most well known is Viscum Album.L. which is frequently used as an alternative for Cancer treatment in Europe.10

A number of compounds including triterpenes-Betulin,oleanolic acid, lupeol stearate, lupeol palmitate, lupeol acetate, Alpha-amyrin, lupeol,betulinic acid,steroids-Beta-sitosterol.11,12 Flavanoids,12Phenolic glycosides13 ,tannic acid14,Olealonic acid,15Ceryl oleonolate, Mesoinositol16 have so far been isolated from Viscum Articulatum.

Based on the above said ethnomedical uses and chemical constituents present in the plant, The present work,would be done to evaluate the antioxidant activity of alcoholic extract of Viscum Articulatum using in vitro and in vivo model systems.



6.3 – Review of the literature


1.

Gurpreet Kaur has reported that free radicals are the main cause of oxidative damage of biomolecules,which is crucial eitiological factor of chronic human diseases and also reports regarding the use of natural antioxidants.


2.

K.N.Ansari reported above the balance between free radical generation and antioxidant defence system in healthy conditions and also the consequences of oxidative stress.


3.

Venukumar.M.R. and Latha.M.S. explains the induction of oxidative stress using CCl4


4.

Kunwar .R.M. et.al. reported that Viscum Articulatum was used for ailment of bone dislocation and bone fracture.


5.

Kirtikar.K.R. and Basu.B.D. reported the geographical distribution of Viscum Articulatum and the use of the same in Kapha,Vatha,diseases of blood,Ulcer,Epilepsy,biliousness and fever.


6.

Timoth.V.Johnson reported the use of plant in treatment of atropy ,cachexia and leg ache.


7.

Jonson.et.al. reported that the plant was used by Adhivasis for snake bite.


8.

Takeatsu reports the traditional uses of plant Viscum Articulatum .



9.

Yan-Lii Leu.et.al. reported that Viscum Articulatum shows inhibition of superoxide anion generation by neutrophils.


10.

Seifert.G. reported the use of Viscum Album in cancer therapy.


11.

Ray.S.et.al. reported the chemical constituents present in Viscum Articulatum.


12.

Wang.X.L.et.al. reported the chemical constituents present in Viscum Articulatum.


13.

Yang Li.et.al. reported the presence of two phenolic glycocides in the plant.


14.

William Dymock.et.al. reported the presence of tannic acid in the plant.


15.

Chandragouda.R.Patil isolated oleanolic acid from cuticular epithelium of the Viscum Articulatum.


16.

Mahendra Kumar Jain isolated ceryl oleonolate and mesoinositol from the plant.

6.4 – Objective of the Study:

Based on the ethnomedical uses of the plant, antioxidant activity of the aqueous alcoholic of the plant will be scientifically validated. The study will prove whether the extract of Viscum Articulatum can ameliorate the tissue injuries inflicted by release of free radicals as a result of oxidative stress induced by CCl4.

The work consists of

A: Extraction of leaves of the plant in a soxlet apparatus with aqueous ethanol (70%)

B: Preliminary Phytochemical screening and toxicity studies of the extract.

C: Evaluation of antioxidant activity of the aqueous ethanolic extract

of Viscum Articulatum using invitro and invivo methods     

Materials and Methods:

7.1 Extraction :

The leaves of the plant would be cut in to small pieces, dried and extracted with aqueous alcohol by hot soxhlet extraction The extracts of the plant would be filtered, collected and concentrated by using a Rotary flash evaporator.



7.2 – Pharmacological studies:

 Maximum tolerable dose: The fraction obtained from the aqueous alcoholic extract would be subjected to Maximum Tolerable Dose determination using step-up/step-down method (OECD-125) at five doses. Swiss albino mice of either sex would be used for the purpose. The effects of extracts on the animals would be observed and those extract, which do not show significant changes in behaviour normal activity would be selected for further pharmacological investigations. The aqueous ethanolic extract of Viscum Articulatum is evaluated for its in vitro and invivo antioxidant activity.



1)Invitro

a]Assessment of Free radical scavenging Activity by

DPPH Assay

ABTS Assay

Nitric oxide scavenging assay

Hydrogen peroxide scavenging

Reducing ability

b]Determination Of Protein oxidation



2)Invivo

Albino rats would be used throughout this study. Rats would be divided into three groups containing six rats each. Group 1 would serve as control, group 2 would be administered with CCl4 (negative control), and group 3 would be administered with Viscum Articulatum extract orally for 7 days. The rats of the groups 1 and 2 would be simultaneously administered saline until the seventh day. On the seventh day, the rats of the groups 2 & 3 would be given a single oral dose of CCl4 (1:1) in olive oil at 2.0 g/kg of body weight 6 h after the last dose of extract/saline. After 24 h of CCl4 administration, rats would be sacrificed, the livers would be isolated and hepatic PMS (post mitochondrial supernatant) would be prepared and used to estimate the following parameters

1. a]Aspartate aminotransferase (AST)
b]Alanine aminotransferase (ALT)
c]Glutathione
d]Speroxide dismutase

e]Catalase



f]Glutathione peroxidase

2. Determination Of Lipid peroxidation


Histopathological studies: Histopathological changes produced in the liver by CCl4 such as necrosis, fatty changes, ballooning degeneration, etc will also be studied.

The results would be subjected to statistical analysis and the significance would be assessed using student t-test.



7.3 - Source of Data:

Journals such as :

Journal of Ethnopharmacology

Indian Journal of Forestry

Indian Journal of Pharmacology

Pharmacological and Toxicological Journals

Indian Journal of Physiology and Pharmacology

Journal of Clinical Pathology

Brazilian Journal of Medical and Biological Research

Internet browsing

Standard books such as:

Indian Medicinal plants by Kirtikar KR, Basu BD.

Pharmacographica Indica.

Medicinal plants Research in Asia.

Experimental Phamocology by M.N. Ghosh.

7.4- Method of Collection of data:

The plant material will be collected from and authenticated by a botanist from Tamilnadu Agricultural University. All chemicals and solvents used in the study would be procured from reputed manufacturers like Merck,BDK etc.,



7.5 - Does the study require any investigations or interventions to be conducted on  patients or other humans or animals? If so, Please describe briefly.

       Yes, the study requires investigation on albino rats.       



7.6Has ethical clearance been obtained from your Institution in case of 7.5?   

  Yes, the protocol is approved by CPCSEA.



LIST OF REFERENCES:-
1. K.N. Ansari., The free radicals--the hidden culprits--an update. Indian J Med Sci,1997;51(9):319-336 .

2. Gurpreet Kaur.et.al., Evaluation of antioxidant activity of Cassia siamea flowers. J Ethnopharmacol,2006;108:340-348.

3. Venukumar.M.R.,Latha.M.S.,Antioxidant effect of Coscininum fenestratum in CCl4 treated rats. Indian J Physiol Pharmacol,2002;46(2):223-228.

4. Kunwar.R.M.,Adhikari.N,Devakota.M.P.,Indigenous use of mistletoes in tropical & temperate regions of Nepal,Banka Janakari.2007;15(2):38-40.

5. Kirtikar.K.R. &Basu.B.D., Indian medicinal plants, International book distributors, Text Vol.III, 2184-2185.

6. Johnson.et.al., Taxonomic validation of crude drugs used for poisonous bites by Adivasis of Rayalaseema Region, Andhra Pradesh. Ethnobotonical leaflets,2008;12:934-937.

7. Takeatsu, North East Asia,UNESCO,Part III,.428.

8. Timoth.V.Johnson,CRC Ethnobotony Desk reference, 1999;885:28100

9. Yann-Lii Leu.et.al., The inhibition of superoxide anion generation by neutrophils from Viscum Articulatum. Chem Pharm Bull ,2004;52(7):858-860.

10. Seifert.G.Jasse.P.Laengler.A., molecular mechanisms of mistletoe plant extract induced apoptosis in acute lymphoblastic leukemia invivo and invitro. cancer lett.,2008;264,218-228.

11. Ray.S,Thakur.T.N,Ghosh.A,Barua.A.K., chemical investigation of Viscum Articulatum. J. Indian Chem. Soc.1984;61:727-728.

12. Wang.X.L.et.al., chemical investigation of Viscum Articulatum,.Hauxi Youxue Zazhi,1995;10:1-3.

13. Yang Li et.al., Two new phenolic glycosides from Viscum Articulatum. ‘Molecules 2008’,2008;13(10):2500-2508.

14. William Dymock,et.al., Pharmacographia Indica,Vol.III, 232.

15. Chandragouda.R.Patil.et.al., Protective effect of oleanolic aicd on gentamicin induced nephrotoxicity in rats, phytotherapy research,published online inWiley interscience, 2009

16. Mahendrakumar Jain,letter to editor, current science , 406.

17. Marja.P.Kahkonen.et.al., Antioxidant activity of plant extracts containing phenolic compounds. J Agricult Food Chem ,1999;47(10):3954-3962.

18. Chun-Ching lin.et.al., Evaluation of hepatoprotective and antioxidant activity of Boehmeria nivea var.nivea and B.nivea.Var.tenacissima. J Ethnopharmacol 1998;60:9-17.

19. Glauco Morales.et.al., Antioxidant activity of 50% aqueous ethanol extract from Acantholippia deserticola. Biol Res ,2008;41:151-155.

20. M.S. Miranda.et.al., Antioxidant activity of microalga spirulina maxima.Braz J Med Biol Res,1998;31:1075-1079.

21. Rajlakshmi.D,Banerjee.S.K,Sood.S,Maulik.S.K., Invitro and Invivo antioxidant activity of different extracts of the leaves of clerodendron colebrookianum walp in the rat.J Pharm Pharmacol,2003;55(12):1681-1986





9.



Signature of the candidate



(BINDU .V)


10.


Remarks of the Guide:

Requested for clearance and approval




11.


Name and Designation of:








11.1 Guide:  
 
 
 

Signature:



Mrs.GEETHA. K.M Associate Professor and H.O.D Dept of Pharmacology, Dayananda Sagar College of Pharmacy, Kumaraswamy layout, Bangalore – 560 078. 






11.2 Co-Guide: 
 


Signature


Not applicable





11.3 Head of the Department: 
 
 
 
 Signature



Mrs. GEETHA K.M. Associate Professor and H.O.D Dept of Pharmacology, Dayananda Sagar College of Pharmacy, Kumaraswamy layout, Bangalore – 560 078. 




12.


Remarks of the Chairman and Principal

Requested for clearance and approval






Signature                   


Dr. V. Murugan

Professor and principal,

Dept of Pharmaceutical Chemistry,

Dayananda Sagar College of Pharmacy,

Kumaraswamy layout,



Bangalore – 560 078.



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