RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES



EVALUATION OF HEPATOPROTECTIVE POTENTIAL OF HYDROALCOHOLIC EXTRACT OF SANTALUM ALBUM LINN LEAVES

MASTER OF PHARMACY DISSERTATION PROTOCOL

SUBMITTED TO THE

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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE

BY

DEEPAK.TK

Under The Guidance of

DR. KARUNAKAR HEGDE M.Pharm, Ph.D.

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DEPARTMENT OF PHARMACOLOGY,

SRINIVAS COLLEGE OF PHARMACY, MANGALORE– 574 143

2012 – 2014

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

| | | |

|1.0 |NAME AND ADDRESS OF THE CANDIDATE |DEEPAK.TK |

| | |THEKKINKATTIL (H) |

| | |CHULLIYODE (PO) |

| | |MALAPPURAM (DIST) |

| | |KERALA-679332 |

| | | |

|2.0 |NAME OF THE INSTITUTION |SRINIVAS COLLEGE OF PHARMACY |

| | |VALACHIL, |

| | |MANGALORE.-574 143 |

| | | |

|3.0 |COURSE OF STUDY AND SUBJECT |MASTER OF PHARMACY IN PHARMACOLOGY |

| | | |

|4.0 |DATE OF ADMISSION TO COURSE |14/05/2012 |

| | |EVALUATION OF HEPATOPROTECTIVE POTENTIAL OF HYDROALCOHOLIC EXTRACT OF |

|5.0 |TITLE OF THE TOPIC |SANTALUM ALBUM LINN LEAVES |

| | | |

| | |

|6.0 |BRIEF RESUME OF THE INTENDED WORK : |

| |NEED FOR THE STUDY: |

| |The liver is the largest organ in the body, often the target organ for chemically induced injuries. Several important factors|

| |are known to contribute to the liver’s susceptibility. First, most xenobiotics enter the body through the gastrointestinal |

| |(GI) tract and, after absorption, are transported by the hepatic portal vein to the liver. Thus the liver is the first organ |

| |perfused by chemicals that are absorbed in the gut. The second factor is the high concentration in the liver of xenobiotic |

| |metabolizing enzymes, primarily the cytochrome P450-dependent mono-oxygenase system. Although most bio-transformations are |

| |detoxication reactions, many oxidative reactions produce reactive metabolites that can induce oxidative stress and may cause |

| |lesions within the liver. Such agents are always associated with the disturbance of hepatocytes resulting in generation of |

| |Reactive Oxygen Species (ROS)1. |

| |Maintenance of a healthy liver is a crucial factor for overall health and well being. But it is abused by poor drug habits, |

| |alcoholism and prescribed or over-the-counter drugs which can eventually lead to various liver ailments like hepatitis, |

| |cirrhosis and alcoholic liver disease. Hepatoprotection is the ability to prevent damage to the liver2. |

| |Paracetamol is a widely used drug for fever and headaches but when used in high doses or chronic use in therapeutic doses may|

| |cause liver damage. It causes liver damage by decreasing the levels of glutathione (an endogenous antioxidant). It leads to |

| |imbalance between antioxidants levels and free radical generation in the body3. |

| |Liver diseases are one of the fatal diseases in the world today. They pose a serious challenge to international public |

| |health. Modern medicines have a little to offer for alleviation of hepatic diseases and it is chiefly the plant based |

| |preparations which are employed for the treatment of liver disorders4. |

| |Many folk remedies from plant origin are tested for its potential hepatoprotective effect in experimental animal model. |

| |Carbon tetrachloride (CCL4) and paracetamol induced hepatotoxicity models are widely used for the study of hepatoprotective |

| |effects of drugs and plant extracts5. |

| |All the currently available hepatoprotective molecules showed side effects, attempts should be made to find new drugs without|

| |side effect or fewer side effects. Medicinal plants used for the treatment of liver disorders by tribal practitioners and |

| |traditional medicine systems have been shown to possess promising hepatoprotective activities in the animal models. This |

| |reveals that evaluation of herbal drugs is still needed to explore its benefits in the treatment of liver disorders. |

| |Criteria for selection of the plant: |

| |Derived natural products such as flavonoids, terpenes, alkaloids, etc have received considerable attention in recent years |

| |due to their diverse pharmacological properties including antioxidant effect. Free radical damage may lead to cell death. |

| |Antioxidants like flavonoids, alkaloids, terpenoids, etc may interact with these radicals and prevent the damage caused by |

| |them. Number of plants being used for the treatment of hepatic disorders. The wood, root, bark and leaves of the “Santalum |

| |album” plant used for the treatment of the liver disease like jaundice by the tribal healers6. Sandalwood is very beneficial |

| |for treating gastric irritability, jaundice, dysentery, tension and confusion. Previous reports have proved that sandal wood |

| |and root bark possessed abortifacient, hepatoprotective7, urinary antiseptic, stomachic, anti-viral and anti-herpetic |

| |activities. |

| |Flavonoids are the main active constituents present in leaves; eight flavonoids were isolated and identified as vicenin-2, |

| |vitexin, isovitexin8, orientin, isoorientin, chrysin-8-C-beta-D-glucopyranoside, chrysin-6-C-beta-D-glucopyranoside and |

| |isorhamnetin9. However, there is paucity of scientific data for the hepatoprotective activities of leaves of the plant. On |

| |the basis of use of leaves in Ayurveda for the treatment of jaundice and presence of bioactive flavonoids, it is hypothesized|

| |that the leaves of the Santalum album may possess hepatoprotective potential which needs to be investigated. |

| |Keeping the above information in view, in the present study attempt is made to evaluate the safety and efficacy of hydro |

| |alcoholic extract of the leaves of the Santalum album Linn for hepatoprotective activity. |

| | |

| | |

| |REVIEW OF LITERATURE: |

| |Name of the plant: Santalum album Linn |

| | |

| |Santalum album Linn. Belongs to family Santalaceae. |

| | |

| |Sanskrit synonym: Chandana |

| | |

| |Other name: Chandanam (Malayalam), Sandanam (Tamil), Chandan, Srikhanda |

| |(Bengali), Gandha (kanada). |

| | |

| |Parts used: Wood, root, leaves. |

| |Plant Description: |

| | |

| |Habitat: |

| |It is commonly found in the comparatively dry regions of peninsular India from Vindhya mountains southwards, especially in |

| |Mysore and Tamil Nadu, ascending to an altitude of c. 1,200 m. It has also been introduced into Rajasthan, parts of Uttar |

| |Pradesh, Madhya Pradesh and Orissa, where it has become naturalized at some places, but the sandalwood produced in these |

| |areas is usually of an inferior quality. |

| | |

| |Description: |

| |A small to medium-sized evergreen tree with drooping branches, sometimes attaining a height of 18 m and a girth of 2.4 m. |

| |Bark dark grey or nearly black or reddish, rough, with deep vertical bracks on old trees. Sapwood unscented and whitish |

| |yellow to white, but heart wood scented and light yellowish-brown (when freshly cut), dark brown or reddish-brown (upon |

| |exposure and ageing). Leaves elliptic-ovate or ovate-lanceolate, glabrous, thin, 1.5-8.0 cm x 1.6-3.2 cm or larger. Flowers |

| |brownish-purple, violet or straw-colored, unscented borne in axillary and terminal paniculate cymes. Drupes purple black, |

| |globose 1.3 cm diam., with hard ribbed endocarp. Seeds globose or ovoid. |

| | |

| | |

| | |

| |Chemical Constituents: |

| | |

| |Sandalwood oil |

| |The main constituent of sandalwood oil is santalol. Heartwood which contains 90% of santalol and is present as a mixture of |

| |two isomers, α- santalol and ß-santalol, the former predominating. The characteristic odor and medicinal properties of |

| |sandalwood oil are mainly due to the santalols. The other constituents reported in sandalwood oil include: the hydrocarbons |

| |santene, nor-tricycloekasantalene and a- and, ß-santalenes; the alcohols santenol and teresantalol; the aldehydes |

| |nor-tricycloekasantalal and isovaleraldehyde; the ketones l-santenone and santalone; and the acids teresantalic acid |

| |occurring partly free and partly in esterfied form, and a-and ß- santalic acids10. |

| |Leaves: |

| |Flavonoids are the main active constituent present in leaves; Eight flavonoids were isolated and identified as vicenin-2, |

| |vitexin8, isovitexin8, orientin, isoorientin, chrysin-8-C-beta-D-glucopyranoside, chrysin-6-C-beta-D-glucopyranoside and |

| |isorhamnetin9. |

| | |

| |Uses: |

| |Sandalwood is very beneficial for treating, gastric irritability, jaundice, dysentery, tension and confusion. It also acts as|

| |abortifacient, hepatoprotective6,7, urinary antiseptic, stomachic, anti-viral12, anti-herpetic and expectorant. Sandalwood |

| |paste is also used for healing inflamed skin, softens and soothes the skin, acts as an astringent which has great toning |

| |effect on the skin. |

| |6.3 OBJECTIVES OF THE STUDY: |

| |The main objective of the present study are as follows: |

| |Collection and authentication of the plant Santalum album leaves. |

| |To prepare the hydro alcoholic extract of the plant Santalum album leaves. |

| |Preliminary phytochemical screening of the extract. |

| |Assessment of acute toxicity study of leaves extract. |

| |To study the hepatoprotective activity of leaves extract using following animal models |

| |Carbon Tetrachloride (CCL4) induced liver toxicity |

| |Paracetamol induced liver toxicity |

| |6) Biochemical Estimation: |

| |Blood will be collected from the animals and serum will be evaluated for |

| |a) Serum glutamic-oxaloacetate transminase (SGOT) |

| |b) Serum glutamic-pyruvate transminase (SGPT) |

| |c) Alkaline phosphatase (ALP) |

| |d) Total Billirubin |

| |7) Assessment of oxidative stress by estimating the liver reduced glutathione enzyme (GSH), catalase, super oxide dismutase |

| |and lipid peroxidation level. |

| |8) To assess histopathological examination of liver. |

|7.0 |MATERIALS AND METHODS: |

| |7.1 SOURCE OF DATA: |

| |Experiment will be performed as described in the standard bibliography, literatures and text books. The reputed journals and |

| |publications are obtained from college library and through web search. |

| | |

| |7.2 COLLECTION OF MATERIAL |

| | |

| |7.2.1. COLLECTION OF PLANT MATERIAL AND PREPARATION OF EXTRACT |

| |The Santalum album leaves will be collected from Mysore district, and authenticated by a Taxonomist. The shade dried powdered|

| |leaves will be extracted by hot percolation method (Soxhlet apparatus) using 50 % hydro alcohol. The extract will be filtered|

| |through a cotton plug followed by Whattman filter paper No.1 and then concentrated to syrupy consistency by using a rotary |

| |flash evaporator at low temperature. The dried extract will be preserved in airtight containers and kept at 4-5°C until |

| |further use. |

| |7.2.2 DRUGS AND CHEMICALS |

| |Chemicals like carbon tetrachloride, EDTA, liquid paraffin, ethanol, xylene, haematoxylin, eosin and 10% ethanol buffered |

| |formalin of pure analytical grade will be procured from E. Merck (India) Ltd, Mumbai and drugs like Silymarin, paracetamol |

| |will be obtained from local suppliers. |

| |7.2.3. ANIMALS |

| |The albino rats (150-200 g) of either sex will be will be used for the experiment. They will be maintained under standard |

| |conditions (temperature 22 ± 2oC, relative humidity 50±5% and 12 h light/dark cycle). The animals will be housed in sanitized|

| |polypropylene cages containing sterile paddy husk as bedding. They will have free access to standard pellet diet and water ad|

| |libitum. All experimental protocols will be reviewed and approved by the Institutional Animal Ethical Committee prior to the |

| |initiation of the experiment and the care of the laboratory animals will be taken as per the CPCSEA regulations. |

| |7.3 EXPERIMENTAL METHODS |

| |7.3.1. ASSESSMENT OF ACUTE TOXICITY: |

| |Acute toxicity study of the hydro alcoholic extract of leaves of the plant Santalum album will be performed as per the OECD |

| |guidelines 425 at a limit dose of 2000 mg/kg or 5000 mg/kg. The doses will be administered by oral route in albino rats |

| |(150-200g). Animals will be observed individually at least once during the first 30 minutes after dosing, periodically during|

| |the first 24 hours (with special attention given during the first 4 hours), and daily thereafter, for total 14 days for sign |

| |of toxicity and/or mortality if any. |

| |7.3.2 CARBON TETRACHLORIDE(CCL4) INDUCED LIVER TOXICITY |

| | |

| |EXPERIMENTAL DESIGN : |

| |The albino rats (150-200 g) of either sex will be divided randomly into five groups of six animals each. The different groups|

| |will be assigned as below. |

| |Group I : Vehicle control (Tween-80, 1 ml/kg) |

| |Group II : Hepato toxic control (CCL4 0.7 ml/kg) |

| |Group III : Reference Standard Silymarin (100 mg/kg) + (CCl4 0.7 ml/kg) |

| |Group IV : Santalum album plant leaves extract (low dose) + (CCl4 0.7 ml/kg) |

| |Group V : Santalum album plant leaves extract (high dose) + (CCl4 0.7 ml/kg) |

| |All the animals except Group I will be intoxicated by the administration of CCl4 (1:1 of CCl4 in olive oil) on 2nd, 4th and |

| |6th day of treatment. All the treatment will be given orally once daily for seven days13. |

| |EVALUATION: |

| |On 7th day 24 hrs following CCL4 administration, blood will be collected through cardiac puncture and analyzed for various |

| |biochemical parameters. Animals will be sacrificed by euthanasia and liver will be dissected out and used for |

| |histopathological studies. |

| | |

| |BIOCHEMICAL ESTIMATION: |

| |The blood samples will be collected without any coagulant and allowed to clot for 10 minutes at room temperature. The blood |

| |will be allowed to centrifuge at 2500 rpm for 20 minutes at 30o C. The obtained serum is stored at 4oC for estimation of |

| |SGOT, SGPT, ALP and total billirubin content using commercially available diagnostic kits according to the manufactures |

| |instructions14. |

| |ASSEMENT OF OXIDATIVE STRESS: |

| |The liver will be isolated and rinsed s successively with the ice cold distilled water, sucrose solution (0.25M), followed by|

| |once again with distilled water. One gram of tissue will be homogenized with 10 ml of ice cold Tris-hydrochloride buffer. The|

| |homogenate will be centrifuged & used for the assay of determination of reduced glutathione enzyme (GSH)14. |

| |HISTOPATHOLOGICAL STUDIES : |

| |The liver will be allowed to fix in 10% formalin and processed through graded ethanol, xylene and impregnated with paraffin |

| |wax. Sections will be made using microtome. After staining with haematoxylene and eosin, the different histopathological |

| |indices will be determined. |

| | |

| |7.3.3 PARACETAMOL (PCM) INDUCED LIVER TOXICITY: |

| | |

| |EXPERIMENTAL DESIGN : |

| |The Wistar albino Rats (150-200 g) of either sex will be randomly divided into five groups of six each. The different groups |

| |will be assigned as follows. |

| |Group I : Vehicle control (distilled water 1 ml/kg) |

| |Group II : Hepatic toxic control (Paracetamol 2 g/kg) |

| |Group III : Reference Standard Silymarin (100 mg/kg) + Paracetamol (2 g /kg) |

| |Group IV : Santalum album leaves extract (low dose) + Paracetamol (2 g/kg) |

| |Group V : Santalum album leaves extract (high dose) + Paracetamol (2 g/kg) |

| | |

| |All the drug preparations will be done in distilled water and administered orally once daily for 7 days. On 5th day Group II,|

| |III, IV and V animals will be given with Paracetamol (2 g/kg body weight) in 40% sucrose solution. |

| |EVALUATION: |

| |On 7th day 24 hrs following paracetamol administration, blood will be collected through cardiac puncture and analyzed for |

| |various biochemical parameters. Animals will be sacrificed by euthanasia and liver will be dissected out and used for |

| |histopathological studies. All the biochemical, oxidative stress and histopathological parameters will be assessed as per |

| |CCl4 induced hepatotoxicity model. |

| |7.4 STATISTICAL ANALYSIS: |

| |The data will be expressed as mean value ± SEM and will be analyzed by the one-way ANOVA test. |

| |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. Study requires investigation on Wistar strain albino rats and mice. |

| |7.6 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION? |

| |Yes. Ethical clearance has been obtained. (Copy enclosed) |

| |Ref:SCP/CPCSEA/P15/F150/2012 |

| | |

|8.0 |LIST OF REFERENCES: |

| |Fernendez–checa, Kaplowitz N. Hepatic mitochondrial glutathione: transport and role in disease and toxicity. Toxicol Appl |

| |Pharm 2005;204:263-73. |

| |Ernest Hodgson. A Textbook of Modern Toxicology, A John Wiley and Sons, Inc. Publication, Third Edition, 2004;263-270. |

| |Mitchell JR, Jollow DJ, Potter WZ, Gillette JR, Brodie BB. Acetaminophen-induced hepatic necrosis. IV .Protective role of |

| |glutathione. J Pharmacol Exp Ther 1973;187:211-7. |

| |Karan M, Vasist K, Handa S. Antihepatotoxic activity of Swerita chirata on carbon tetrachloride induced hepatotoxicity in |

| |rats. Phytother Res 1999;13: 24-30. |

| |Suja SR, Latha PG, Pushpangadan P, Rajashekharan S. Aphrodisiac property of Helminthostachys zeylenica in mice. J Trop Med |

| |Plants 2002; 3:191-5. |

| |James A Duke, Mary Jo Bogenschutz-Godwin, Judi duCellier, Peggy-Ann K Duke. Handbook of Medicinal Herbs, Second Edition.2002;|

| |646-7. |

| |R.Chavda, K.R. Vadalia, R. Gokani, “Hepatoprotective Activity of Root Bark of Calatropis procera R.Br (Asclepediaceae)” |

| |International Journal of Pharmacology 2010; 6(6); 937-934. |

| |Yan C, Liu H, Lin L. Simultaneous determination of vitexin and isovitexin in rat plasma after oral administration |

| |of Santalum album L. leaves extract by liquid chromatography tandem mass spectrometry. Biomed Chromatogr. 2012. doi: |

| |10.1002/bmc.2780. |

| |Yan C, Lin L, Liu H, Lin Z, Chen P, Cai C, Zheng L. Study of flavonoids from leaves of Santalum Album. Zhongguo Zhong Yao Za |

| |Zhi.  |

| |2011 ;36(22):3130-3 |

| | |

| | |

| | |

| |Available from |

| | |

| |retrieved on12/11/2012. |

| |OECD, Guidelines for testing of chemicals, Acute oral toxicity, Environmental Health and Safety Monograph Series on Testing |

| |and Adjustment No. 425, 2001, 1. |

| |Benencia F, Courreges MC; Antiviral Activity of Sandalwood oil against Herpes Simplex Viruses 1 & 2, Phytomedicine, 1999, |

| |6(2),119-123 |

| |Chanchal K Roy, Jagdish V Kamath, Mohammed Asad. Hepatoprotective activity of Psidium guajava Linn. Leaf extract. Indian J |

| |Exp Biol 2006; 44: 305-11. |

| |Reitman S, Frankel SA. A colorimetric method for determination of SGOT and SGPT. Am J Clin Pathol 1957; 28:56-63. |

|9. |SIGNATURE OF THE CANDIDATE | |

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|10. |REMARKS OF THE GUIDE |Recommended and Forwarded |

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|11. |NAME AND DESIGNATION | |

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| | | |

| |11.1 GUIDE |DR. KARUNAKAR HEGDE |

| | |ASSISTANT PROFESSOR |

| | |DEPARTMENT OF PHARMACOLOGY |

| | |SRINIVAS COLLEGE OF PHARMACY |

| | |MANGALORE- 574 143 |

| | | |

| |11.2 SIGNATURE | |

| | | |

| |11.3 CO-GUIDE (IF ANY) |Not Applicable |

| | | |

| |11.4 SIGNATURE | |

| | | |

| | |MR. MOSES SAMUEL RAJAN |

| |HEAD OF THE |ASSOCIATE PROFESSOR |

| |DEPARTMENT |DEPARTMENT OF PHARMACOLOGY |

| | |SRINIVAS COLLEGE OF PHARMACY |

| | |MANGALORE- 574 143 |

| | | |

| |SIGNATURE | |

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|12 |12.1 REMARKS OF THE PRINCIPAL |Strongly Recommended and Forwarded for Favorable Action. |

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| | | |

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| |NAME AND SIGNATURE OF PRINCIPAL |DR. A. R. SHABARAYA |

| | |PRINCIPAL, |

| | |SRINIVAS COLLEGE OF PHARMACY |

| | |MANGALORE- 574 143. |

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