GENOTOXICITY STUDIES & Elucidation OF mechanism of LIPOVEDIC A POLYHERBAL FORMULATION FOR ITS

ANTIHYPERCHOLESTEROLEMIC ACTIVITY

SYNOPSIS FOR

M.PHARM DISSERTATION

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA

BY

SWETHA.S

I M.PHARM

DEPARTMENT OF PHARMACOLOGY

VISVESWARAPURA INSTITUTE OF PHARMACEUTICAL SCIENCES

BANGALORE-560070

(2009-2010)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1.0 / Name of the candidate and address(in block letters) / SWETHA.S
D/o SATYANARAYANA. T
# 4, OFFICERS QUARTERS,
SUBRAMANYAPURA,
BANGALORE-560061
2.0 /

Name of the institution

/

VISVESWARAPURA INSTITUTE OF PHARMACEUTICAL SCIENCES

3.0 /

Course of study and subject

/

MASTER OF PHARMACY IN

PHARMACOLOGY

4.0 /

Date of the admission

/ 27/06/2009
5.0 /

Title of the topic:

GENOTOXICITY STUDIES & Elucidation OF mechanism of LIPOVEDIC, A POLYHERBAL FORMULATION FOR ITS ANTIHYPERCHOLESTEROLEMIC ACTIVITY.
6.0
7.0
/ BRIEF RESUME OF THE INTENDED WORK
6.1 NEED FOR THE STUDY
Hypercholesterolemia is not a disease but a metabolic derangement that can be secondary to many diseases and can contribute to many forms of disease, most notably cardiovascular disease. It is closely related to the terms "hyperlipidemia" and "hyperlipoproteinemia".1
Earlier studies have confirmed that the polyherbal formulation, lipovedic, increases serum HDL level, serum glucose level and significant decrease in serum cholesterol, serum LDL, serum VLDL levels and hence has anti-obesity activity.2 It is hypothesized that, as a result of these findings drug can be used as antihypercholesterolemic. The mechanism of antihypercholesterolemic activity is yet to be understood. This study is an attempt to elucidate the mechanism of action of the poly herbal formulation Lipovedic.
Genotoxicity refers to alteration in-gross structure or content of chromosomes (Clastogenicity) OR base pair sequencing of DNA (Mutagenicity) by exposure to toxic agents. Micronucleus test (MNT) and chromosomal aberration test (CAT) using Mouse bone marrow test system is extensively used cytogenetic tests for detection of anticlastogenetic agents. All chemicals that produce DNA damage leading to mutations or cancer are described as genotoxic.3 The present study is taken up to evaluate genotoxicity of a polyherbal formulation, Lipovedic.
6.2  REVIEW OF LITERATURE:
Lipovedic is a polyherbal formulation, developed by Biovedic labs, Bangalore and is intended to use in obesity and hypercholesterolemia. Following table shows the plant constituents with their uses:
Sl. No. / Sanaskrit name / Botanical name / Actions
1 / Guggulu / Commiphora mukul 4 / Very effective in controlling obesity and cholesterol
2. / Triphala / Terminalia chebula
Terminalia belerica
Emblica officinalis 5 / Reduces high blood pressure, improves blood circulation, Increase digestion, assimilation and reduce fat
3. / Trikatu / Piper longum
Piper nigrum
Zingeber officinalis 6 / Increases digestive enzymes, improves metabolism
4. / Nagakesara / Mesua ferra 7 / Used extensively as an anti-inflammatory medicine
5. / Chitraka / Plumbago zeylanica 8 / Gradually reduces hypercholesterolemia.
6. / Musta / Cyperus rotandus 9 / harmonizes the liver, spleen, and pancreas
7. / Vidanga / Embelia ribes 10 / Supports digestive functions and it’s a mild laxative
8. / Chavya / Piper cubeba 11 / Rectifies if there is any problem in digestive system
9. / Hapushpa / Juniperus communis 12 / an anti-inflammatory agent
10. / Ativisha / Aconitum heterophyllum13 / an anti-inflammatory agent
11. / Pata / Cissampelos pareira14 / an anti-inflammatory agent
Guggulu is reported to have lipid lowering activity, platelet aggregation inhibitory activity, anti-inflammatory and anti-bacterial activity. Cyperus rotundus is reported to have cytoprotective effect, anti-inflammatory,anti- pyretic and analgesic activity, anti-malarial and anti-obesity activity. Piper longum is reported to have anti-amoebic,isecticidal and acaricidal,immunomodulatory and anti tumor,fungicidal and thermogenic properties. Plumbago zeylanica is cytotoxic, antimicrobial and anti-plasmodial.2 Lipid lowering activity of Abutilon indicum(L.) leaf extracts in rats,15 antihyperlipidemic activity of the methanolic extracts of Garlic in triton X-100 induced hyperlipidaemic rats,16 antihyperlipidaemic activity of Sapindus emarginatus in triton WR-1339 induced albino rats17 have been studied recently.
The mechanism for antihypercholesterolemic activity of a drug may be due to inhibition of cholesterol absorption, inhibiton of cholesterol biosynthesis, bile acid sequestering property.
This study is an attempt to understand the effect of lipovedic on the genetic apparatus.
6.3  OBJECTIVE OF THE STUDY
1)  To evaluate genotoxicity of lipovedic by
a.  Chromosomal Aberration Test by using mouse bone marrow system.
b.  Micronucleus Test by using mouse bone marrow system.
2)  Elucidation of mechanism of anti hypercholesterolemic action of lipovedic by
a)  Cholesterol diet induced hypercholesterolemia in Rats.
b)  Triton induced hyperlipidemia.
c)  Effect of HMG CoA reductase inhibitors in vivo.
d)  In vivo test for ACAT inhibitory activity.
MATERIAL AND METHODS
7.1 Source Of Data :
Data will be generated by performing experiments on animals. The standard information is collected from various journals, standard Text books available in library of Visveswarapura Institute of Pharmaceutical Sciences, Indian Institute of Science, RGUHS digital library and from various standard websites.
Web sites: www.sciencedirect.com
www.pubmed.com
www.google.com
www.rguhs.ac.in/j-gate@helinet
7.2 METHOD OF COLLECTION OF DATA:
The data will be generated by performing the experiments using animal models like rats and rabbits.
7.3  METHODOLOGY :
7.3.1  Cholesterol diet induced hypercholesterolemia
Rationale: Rats are known to be susceptible to hypercholesterolemia and arteriosclerosis after excessive cholesterol feeding. Diet-induced hypercholesterolemia is useful only for detection of agents interfering with the adsorption, degradation and excretion of cholesterol. Agents interfering with cholesterol biosynthesis are less probable to be detected.18
Animals: 36 Adult male albino rats weighing 150-200g will be assigned into six groups of 6 animals each.
Group 01: Normal control (without any treatment).
Group 02: Vehicle treated (D.M water 10ml/kg p.o.).
Group 03: Vehicle treated (Groundnut oil 10ml/kg p.o.).
Group 04: High cholesterol diet p.o.
Group 05: Atorvastatin (7.2mg/kg p.o) +High cholesterol diet p.o.
Group 06: Lipovedic (180mg/kg p.o)+High cholesterol diet p.o
Blood and feces will be collected on days 7, 14, 21 and 28. Blood will be collected by retro orbital sinus under light ether anesthesia and analysed for total triglyceride, total cholesterol, HDL cholesterol, LDL cholesterol, VLDL cholesterol. Feces will be analysed for fecal cholesterol, fecal bile acid.20
7.3.2  Triton-induced hyperlipidemia
Rationale: The systemic administration of the surfactant Triton to mice or rats results in a biphasic elevation of plasma cholesterol and triglycerides. The mechanism of the Triton induced hypercholesterolemia in phase I is thought to be due to increased hepatic synthesis of cholesterol through the ability of Triton to interfere with the uptake of plasma lipids by the tissues. Drugs interfering with cholesterol biosynthesis were shown to be active in phase I, while drugs interfering with cholesterol excretion and metabolism were active in phase II.18
Animals: 30 adult male Sprague Dawley rats weighing 250-300 g will be randomly assigned to 6 groups of 6 animals each.
Group 01: Normal control (without any treatment).
Group 02: Animal treated with vehicle (D.M water 10ml/kg p.o).
Group 03: Animals treated with triton (200mg/kg i.p).
Group 04: Animals treated with Atorvastatin (7.2mg/kg p.o) + triton (200mg/kg i.p).
Group 05: Animals treated with Lipovedic (180mg/kg p.o) + triton (200mg/kg i.p).
Procedure :
Animal will be fasted for 18h before the experiment. Blood is collected at 0, 18, 24, 40, 48 h after triton administration by retro orbital retro orbital route under light ether anesthesia and analysed for total triglyceride, total cholesterol, HDL cholesterol, LDL cholesterol, VLDL cholesterol. Individual body weight was recorded on the day 0, 1, 2.20
7.3.3  Effect of HMG CoA reductase inhibitors in vivo
Rationale: β-hydroxy β-methyl glutaryl coA is the rate limiting step for cholesterol biosynthesis. It is seen that in man HMG coA reductase activity is higher at night than during day time, similar to the enzyme activity in rodents.
Animals: 18 Male heterozygous WHHL rabbits weighing 1.8 to 2.5 kg at an age between 8 and 20 weeks will be selected for the experiment.
Procedure :
Group 01: : 0.5% methylcellulose(vehicle control)
Group02: 0.5% methylcellulose+ standard drug( lovastatin7.2mg p.o)× 14 days
Group 03: 0.5% methylcellulose18 + LIPOVEDIC (140 mg/1.5kg p.o)2× 14 days
The test compounds will be suspended in 0.5% methylcellulose and are then administered each day orally by gavage in the afternoon to insure an increased plasma level at night. Blood samples will be taken in the morning without previous feeding. Two ml of blood are drawn from the outer ear vein 5 days prior to the beginning of treatment, on days 3 and 8 of treatment and 30 days after the end of treatment for the determination of biochemical parameters.
Six ml blood is drawn at the first and the last day of treatment and 10 days after the end of treatment for determination of biochemical parameters and lipoprotein profile. In order to obtain serum, blood is allowed to clot at room temperature and then centrifuged twice at 10, 000 rpm.
The following biochemical parameters are determined in non-frozen samples (kept at 4 °C):
Total cholesterol,
HDL-cholesterol,
Triacylglycerol,
Total bilirubin,
Creatinine
Amino transferase (ALAT),
Aspartate amino transferase(ASAT),
γ-glutamyl transferase (γ-GT) using commercially available kits.18
7.3.4  In vivo tests for ACAT inhibitory activity:
Rationale: Acyl coA cholesterol acyl transferase is an enzyme implicated as a key enzyme involved in the absorption of cholesterol via the gut. Therefore an ACAT inhibitor will have potential hypolipidemic and anti-atherosclerotic activities.19
Animals: 18 Adult Male Sprague-Dawley rats weighing 200–225 g will be selected and are fed with a diet containing 5.5% peanut oil, 0.5% cholic acid and 1.5% cholesterol with or without (controls) drugs for 1 week.
Procedure :
Group 01: Cholesterol diet
Group 02: Cholesterol diet + STANDARD drug (Ezetimibe 0.18 mg/200g p.o)
Group 03: Cholesterol diet + LIPOVEDIC( 180mg/kg p.o)
On the last day, food is removed at 8:00 AM and the isotopes are administered at 2:00 PM. [3H] cholesterol (13 μCi/rat) is given by oral gavage and [14C] cholesterol (1.5 μCi/rat) is given by tail vein injection.
The [3H] cholesterol is prepared as an emulsion by dissolving 125 mg cholesterol in 1625 mg olive oil. The oil phase is suspended by sonication in 25 ml of water containing 156 mg taurocholate (sodium salt). Each animal receives 1 ml. The intravenous dose is prepared by drying the labeled cholesterol (50 μCi), and then adding 300 μl warm ethanol followed by 12.5 ml of saline. Each animal receives 0.5 ml of this colloidal suspension. The rats are allowed to consume their respective diets at 3:00 PM and are sacrificed 48 h after the isotope administration.18
8.0 / 7.3.5: MICRONUCLEUS TEST
Animals: Swiss albino mice of either sex 8-10 weeks old, weighing 25-30g will be divided into 8 groups.
Group 01: (n = 6) Vehicle treated Animal
receive vehicle (10ml/kg) orally for 7 consecutive days
Group 02: (n =6) Clastogenic control
Challenge group will receive cyclophosphamide (i.p. 100 mg/kg). Bone marrow extraction from these animals will be done at 24 h after cyclophosphamide injection.
Group 3: (n =6) Clastogenic control
Challenge group will receive cyclophosphamide (i.p. 100 mg/kg). Bone marrow extraction from these animals will be done at 48 h after cyclophosphamide injection.
Group 4: (n =6) Clastogenic control
Challenge group will receive cyclophosphamide (i.p. 100 mg/kg). Bone marrow extraction from these animals will be done at 72 h after cyclophosphamide injection.
Group 5: (n = 6) Drug control
Received lipovedic (260 mg/kg) orally for 7 consecutive days
Group 6: (n =6) Treatment group
This will Received lipovedic (260 mg/kg) orally for 7 consecutive days. Followed by cyclophosphamide (i.p.100 mg/kg) as a challenge on 7th day. After 24 h of Cyclophosphamide injection bone marrow extraction will be performed.
Group 7: (n =6) Treatment group
Received lipovedic (260 mg/kg) orally for 7 consecutive days followed by cyclophosphamide (i.p.100 mg/kg) as a challenge on 7th day. After 48 h of Cyclophosphamide injection bone marrow extraction will be performed.
Group 8: (n =6) Treatment group Received lipovedic (260 mg/kg) orally for 7 consecutive days followed by cyclophosphamide (i.p.100 mg/kg) as a challenge on 7th day. After 72 h of Cyclophosphamide injection bone marrow extraction will be performed.
Extraction of bone marrow
The experimental animals will be sacrificed by cervical dislocation. Bone marrow will be collected from femur and tibia, smears will be prepared, slides will be stained using May-Grunwald’s and Giemsa stain and observed under compound light microscope.
7.3.6 CHROMOSOMAL ABERRATION TEST
Animals: Swiss albino mice of either sex 8-10 weeks old, weighing 25-30 g will be divided into 8 groups
Group 1: (n = 6) Vehicle treated Animal
This group will Receive vehicle (10 ml/kg) orally for 7 consecutive days
Group 2: (n =6) Clastogenic control Challenge group
This group will Receive cyclophosphamide (i.p. 100 mg/kg). Bone marrow extraction from these animals will be done at 24 h after cyclophosphamide injection.
Group 3: (n =6) Clastogenic control Challenge group
This group will Receive cyclophosphamide (i.p. 100 mg/kg). Bone marrow extraction from these animals will be done at 48 h after cyclophosphamide injection.
Group 4: (n =6) Clastogenic control Challenge group
This group will Receive cyclophosphamide (i.p. 100 mg/kg). Bone marrow extraction from these animals will be done at 72 h after cyclophosphamide injection.
Group 5: (n = 6) Drug control
This group will Receive lipovedic (260 mg/kg) orally for 7 consecutive days.
Group 6: (n =6) Treatment group
Received lipovedic (260 mg/kg) orally for 7 consecutive days. Followed by cyclophosphamide (i.p. 100 mg/kg) as a challenge on 7th day. After 24 h of Cyclophosphamide injection bone marrow extraction will be performed.
Group 7: (n =6) Treatment group
Received lipovedic (260 mg/kg) orally for 7 consecutive days followed by cyclophosphamide (i.p. 100 mg/kg) as a challenge on 7th day. After 48 h of Cyclophosphamide injection bone marrow extraction will be performed.
Group 8: (n =6) Treatment group
Received lipovedic (260 mg/kg) orally for 7 consecutive days followed by cyclophosphamide (i.p. 100 mg/kg) as a challenge on 7th day. After 72 h of Cyclophosphamide injection bone marrow extraction will be performed.
Animals will be sacrificed by cervical dislocation after 24h of administration of the clastogen. 90 min prior to death, each animal will be injected with 0.04% colchicines in a dose of 4 mg/kg i.p for mitotic arrest. Animals will be cut open and femur and tibia from both the legs will be quickly removed. For collection of bone marrow, the upper end of femur will be cut open. About 0.5 ml of 0.56% (or 0.075 M) hypotonic potassium chloride solution will be taken in a syringe and the needle will be inserted at the lower epiphysial end. The bone marrow will be flushed into a clean cavity block. Similarly tibial marrow will be also collected. Slides will be prepared and stained using Grunwald’s stain and observed under compound light microscope.3