Evaluation of Antidiabetic Activity of Helicteres

“EVALUATION OF ANTICLASTOGENIC AND ANTIGENOTOXICITY EFFECT OF ISORHAMNETIN "

Synopsis for registration of M.pharm Dissertation

Submitted to RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE KARNATAKA In partial fulfillment of the requirement for the Degree of

Master of pharmacy In Pharmacology

Under the Guidance of Dr. RAJU KONERI M.PHARM.,Ph.D Dept.of Pharmacology.

BY G.RAMYA DURGA I M. PHARM.

Department of Pharmacology Karnataka College of Pharmacy, Bangalore-64 2010-12

1 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name of the candidate and address G.RAMYA DURGA, D/O G.SRINIVAS RAO, D.NO-10-13-14,CHENCHUPET, TENALI,GUNTUR(Dist) A.P

Karnataka College of Pharmacy 2. Name of the Institution Bengaluru-560064 Karnataka, India

3. Course of study and subject M.Pharm-Pharmacology

4. Date of the admission 31-05-2010

5. Title of the topic “Evaluation of Anticlastogenic and Antigenotoxicity Effect Of Isorhamnetin ”

2 6.0 Brief resume of the intended work

6.1 - Need for the study:

A clastogen is an agent that can cause one of two types of structural changes. A clastogen can cause breaks in chromosomes that result in the gain, loss, or rearrangements of chromosomal segments. A clastogen can also cause sister chromatid exchanges, which are "homologous chromatid strand interchanges and reunions [that occur] during DNA replication"[1]. Cytotoxic relating to substances that are toxic to cells, Cell-killing. Genotoxic describes a poisonous substance which harms an organism by damaging its DNA. Mutagenic is capable of inducing mutation. Sister chromatid exchange (SCE) is a sophisticated cytomolecular technique that is commonly applied in a search for clastogenicity or genotoxicity. An SCE analysis will tell us whether the chromosomes and thus DNA of a particular interest group has undergone some genetic damage compared to a control group. Many studies have shown, however, that any increase in frequency of SCEs beyond the threshold indicates that something in that persons existence is or has caused genetic damage, which can lead to ill health. Many environmental agents, at home or at work, can increase the number of SCEs, for example, UV light, X-rays, nicotine and alcohol etc [2]. It is widely recognized that clastogenicity or genotoxicity mostly may be due to various environmental and dietary mutagens that has resulted in greater emphasis on toxicological studies that mainly include chronic toxicity, carcinogenicity, teratogenicity and mutagenicity [ 3]. Many a times these defects occur not only due to the presence of genotoxic agents but also due to lack of antimutagenic/antcarcinogenic agents in our diet [3, 4]. The best way to minimize these effects is identifying the antimutagens and anticarcinogens in our diet and increasing their use [ 4]. Micronucleus antimutagenic assay is a well established method to study the mutagens and antimutagens [5, 6]. Insufficient intakes of many micronutrients found in fruits and vegetables, such

3 as folic acid, vitamins C and B6 etc. may lead to DNA damage, cancer, and degenerative disease [7]. Natural antioxidant compounds in the human diet may prevent the genetic effects of mutagens and carcinogens and many studies have shown that an increased intake of fruits or vegetables rich in antioxidants decreases the level of oxidative DNA damage [8, 9]. 6.2 - Review of Literature:

Clastogenic factors (CFs) are chromosome-damaging agents with low molecular weight(<30,000 daltons) that cause chromosome aberrations, sister chromatid exchanges, DNA strand breakage, and gene mutation. For more than 30 years they have been known to be an indirect effect of ionizing radiation [16][17]. Because of their persistence in the blood of irradiated persons many years after exposure [18], CF have been considered risk factors for late effects of ionizing irradiation such as cancer and leukemia [19].The presence of CF in plasma has also been described in patients with congenital breakage syndromes such as ataxia teleangiectasia, Fanconis anemia, and Bloom's syndrome, which are all associated with a high incidence of malignancies [20]. The superoxide anion radical is implicated in clastogenic factor formation and action [21]. This has been demonstrated particularly for CF derived from the plasma of patients with human immunodeficiency virus (HIV) [22]. Recently, the presence of CF was reported in patients infected with the HIV-1 virus [23]. Antioxidants are phytochemicals, vitamins and other nutrients that protect our cells from damage caused by free radicals. Free radicals are formed as part of our natural metabolism but also by environmental factors, including smoking, pesticides, pollution and radiation. Antioxidants have the property to neutralize free radicals and protects from cell damage. Antioxidants can be found in most fruits and vegetables but also culinary herbs and medicinal herbs can contain high levels of antioxidants. Therefore we need to continuously supply our body with antioxidants. The action of free radicals could increase the risk of diseases such as cancer and health problems and could accelerate ageing. Numerous studies with plant phytochemicals show that phytochemicals with antioxidant activity may reduce risk of cancer and improve heart health.

4 Antimutagenic effects of flavonoids (Isorhamnetin), chalcones and structurally related compounds on the activity of 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) and other heterocyclic amine mutagens from cooked food, Mutation . With respect to the mechanism(s) of antimutagenicity, the following results were obtained here. (1) 7-Methoxy- and 7-ethoxyresorufin-O-dealkylase activities in rat liver microsomes, linked to cytochrome P-450-dependent 1A1 and 1A2 monooxygenases catalyzing oxidation of IQ to N-hydroxy-IQ (N-OH- IQ), were effectively inhibited by 16 flavonoids(Isorhamnetin). Within the flavone and flavonol subgroups inhibitory effects increased in dependence on number and position of hydroxyl functions. Isoflavones and flavanones, however, as well as glycosides, were inactive. Hydroxyl groups at carbons 7, 3′, 4′, and 5′ generated antimutagenic compounds, a hydroxyl function at C5 was ineffective, but hydroxyls at C3 and 6 as well as methoxyl groups at C3′ (isorhamnetin) or 4′ (diosmetin) generated comutagenic compounds. 4. Cytosolic activation of IQ to mutagenic metabolites as determined by experiments with the hepatic S105 fraction comprises about 10% of the mutagenicity after activation by the combined microsomal and cytosolic fractions (S9). The pattern of inhibition as produced by 20 flavonoids(Isorhamnetin) was closely similar to that observed with the S9 fraction. 5. In various experiments designed for modulation of the mutagenic response, it could be shown that further mechanisms of flavonoid(Isorhamnetin) interaction with the overall mutagenic process may exist, such as interactions with biological membranes and effects on fixation and expression of DNA damage. 6.3 – Objective of Study: 1. Evaluation of anticlastogenic activity in mice Bone marrow cells, mice Peripheral blood cells and human lymphocyte culture 2. Evaluation of antigenotoxicity activity by sperm abnormality testing, tranplacental MN test in mice. 6.4-Source of Data:

Whole work is planned to generate data from laboratory studies i.e. experiments are performed as described in references. Experimental studies in journals and in

5 text books available with college, IISc and other libraries. Literature is searched from various web sites in the internet 7.0 MATERIALS AND METHODS

7.1-Experimental animals: Eight week old healthy laboratory bred, swiss albino mice (Mus musculus) of either sex, weighing 25±3 g will be included for the study and maintain under conventional laboratory conditions, at temperature 25± 20C with 12 hr natural light period. All animals will have a free access to food and water ad libitum. Model 1-Evaluation of anticlastogenic activity on bone marrow cells: Groups : Group 1: Control group animals treated with distilled water Group 2: Positive group animals treated with clastogen Group 3: Treated with Clastogen + flavonoids(Isorhamnetin) Group 4:Treated with drug without clastogen Six mice will be randomly selected for individual treatment groups and each group consisted of 3 males and 3 females. Different groups of animals will receive Extracted drug suspension for 7 consecutive days and on 7th day clastogens will administer after 1 hr of the last dose of extracted drug. The bone marrow sampling will be done after 24, 48 and 72 hr of clastogen treatment. The groups of animals receive only vehicle for 7 days will taken as negative control group and the group treated with acute dose of clastogen will consider as positive control group. To find out the effect of test drug on MN induction, Calculated dose of flavanoid and alkaloids will administered for 7 days and the MN frequency will evaluated .In the same way MN frequency is evaluated in citral treated clastogenic animals

Doses, Treatment and Sampling A suspension Of alkaloid/flavonoid/citral will made using tween 80 and distilled water and will be administered orally. The clastogens Cyclophosphamide (CP)[24], Mitomycin-C(MMC)[24] and

6 Nickel chloride(NiCl2)[25] dissolved in distilled water and administered by intra- peritoneal route.

Bone Marrow MN Test and Scoring The same experimental animals are used for both peripheral blood MN and bone marrow MN assays. The animals are killed by cervical dislocation. The femur and tibia will be excised. Bone marrow MN slides will prepared by using the modified method of Schmid [24]. Marrow suspension from femur and tibia bones prepared in 5% bovine serum albumin (BSA), will be centrifuged at 1000 rpm and the pellet has resuspended in BSA solution. A drop of this suspension will place on a clean glass slides and smears will prepared and the slides are air dried. The slides are fixed in methanol, stained with May-Grunwald-Giemsa and MN will identified in two forms of RBCs (i.e. polychromatic erythrocytes as PCEs and normochromatic erythrocytes as NCEs). About 2000 PCEs and corresponding NCEs per animals were scanned for the presence of MN. Statistical Analysis The statistical significance of the results will be tested using non paired t-test and one-way Anova [24]. Model 2- Evaluation of anticlastogenic activity on peripheral blood cells:

GROUPS :

GROUP 1: CONTROL GROUP ANIMALS TREATED WITH DISTILLED WATER

GROUP 2: POSITIVE GROUP ANIMALS TREATED WITH CLASTOGEN

GROUP 3: TREATED WITH CLASTOGEN + FLAVONOIDS(ISORHAMNETIN)

GROUP 4: TREATED WITH DRUG WITHOUT CLASTOGEN

Peripheral Blood MN Test and Scoring Peripheral blood smears are prepared from tail vein within 30 seconds after cervical dislocation of the animals. The tails of the animals will cut about 2 cm from the tip so as to allow free flow of blood. Then smears are made on clean glass slides and air dried. Blood will diluted using BSA suspending medium, if necessary. The slides will fixed in methanol and stained using Wright Giemsa stains [26]. About 2000 NCEs and the corresponding PCEs per animal will scored for the presence of MN.

7 Statistical Analysis The statistical significance of the results will be tested using non paired t-test and one-way Anova [24].

MODEL 3- EVALUATION OF ANTICLASTOGENIC ACTIVITY IN HUMAN LYMPHOCYTE CULTURE:

The chromosome aberrations test will be carried out using conventional techniques [27] .The human lymphocycyte cells will be collected from NCI (National Cancer Institute) Pune.The cultures are prepared by adding isolated lymphocytes at 1x106/ml at 5ml of complete medium, containing 78% RPMI 1640[28], 20% inactivated fetal bovine serum, antibiotics (penicillin and streptomycin) and 2% phytohemagglutinin (PHA) to stimulate cell proliferation. The cells are incubated for 50 hr at 37 °C, and colchicine will be added to the culture medium to a final concentration of 0.4 µg/ml 1 hr prior to harvesting. Cell harvesting will be carried out according to standard procedures; after hypotonic treatment with KCl for 10min, lymphocytes will be fixed in methanol: acetic acid (3:1). Slides will be stained with Giemsa .The mitotic index will be obtained by counting the number of metaphase cells in a total of 12,000 lymphocytes analyzed per treatment. The end points analyzed will be the mitotic index, the presence of structural chromosomal aberrations, which will recorded as chromatid type (including breaks or fragments, quadriradial and triradial) or chromosome type (including breaks or fragments, translocations, dicentrics and rings), and aberrant metaphases. Values will be obtained according to the following formula. % of Abnormal Metaphases = Number of cells containing at least one aberration x 100 Total number of metaphases

The concentrations of extracted drug (15, 30, 90, and 120 µg/ml culture medium) tested in combination with doxorubicin will be established on the basis of the results of the preliminary experiments with this vitamin (5–270 µg/ml culture medium). Doxorubicin will used to induce chromosomal aberrations. It will be diluted in distilled water, and added to the medium culture at the concentration of 0.2 µg/ml defined by preliminary experiments (0.05–40.0 µg/ml of

8 doxorubicin) and according to the literature data[28][29]. Human peripheral blood lymphocytes will be incubated for 24 hr before extracted drug and/or doxorubicin will be simultaneously added to the medium culture, and left until harvest. An untreated control culture will established as well. Separate pre or post treatment experiments with flavonoid and alkaloids will be performed. drug will be added to the cultures 2 hr before doxorubicin as pre treatment and 2 hr after doxorubicin as post treatment and incubated until harvested for 50 hr after PHA stimulation. Statistical analysis Differences in total number of chromosomal aberrations, percentage of abnormal metaphases, and mitotic index between treatments Will be analyzed statistically by analysis of variance. In cases in which P <0.05, the values of each treatment Will be compared by the Student–Newman– Keuls test with the software Sigma Stat 1.0. Model 4 -Evaluation of antigenotoxicity of isorhamnetin by using sperm abnormality test: Groups : Group 1: Control group animals treated with distilled water Group 2: Positive group animals treated with cyclophosphamide Group 3: Treated with cyclophosphamide + drug Eight to ten weeks old inbred swiss albino mice with an average body Weight of 22-24 g were utilized. In the first experiment (32) the animals were administered lead nitrate orally for five days. Six animals were used for the dose. Parallel controls and mitomycin-C as positive control were maintained. In the second experiment the three doses of drug were selected on therapeutic basis. Each set was administered orally with one dose for 35 days. All the animals were sacrificed on 35thday after treatment. Epididymal sperms were collected in physiological saline and stained with 1% eosin following the method The animals were orally fed with 3 doses of drug for 35 days, on every 7th

9 day after priming with drug 40 mg/kg lead nitrate was fed orally. All the animals were sacrificed on 35th day; slides were prepared The prepared slides were screened for the presence of various types of sperm head abnormalities such as amorphous,banana, hammer, hook like etc. A total of 2000 sperms per animal were scored for the incidence of sperm head abnormalities in controls and treated animals. Model 5- Anti genotoxicity effect of isorhamnetin by using tranplacental MN test: Groups : Group 1: Control group animals treated with distilled water Group 2: Positive group animals treated with cyclophosphamide Group 3: Treated with Cyclophosphamide + drug

Isorhamnetin suspended in Methyl cellulose was administered orally to pregnant mice daily from days 11 to 17 of gestation in two concentrations (23.6 and 70.07 mg/kg body wt) 2 h prior to cyclophosphamide injection. The 6- day treatment regime was used in connection with other aspects of the whole experiment. The Isorhamnetin concentrations were chosen according to the following sources: a chronic 26-week toxicity study and MN assay preclinical studies testing the doses inducing therapeutic effects (0.5-20.0 mg/kg body wt of bases, i.e. 1.7-70.07 mg/kg body wt of dipalmitate) The LD50 of

Dipalmitate is 355.6-406.2 mg/kg in mice and >1800 mg/kg in rats after p.o. administration. . The 2 h interval between isorhamnetin pretreatment and cyclophosphamide administration was used in accordance with our previous results . Cyclophosphamide was administered i.p. at a concentration of 10 mg/kg body wt The concentration of Cyclophosphamide was chosen according to teratological studies in mice . Control animals received Methyl cellulose and saline according to the same time schedule as used in the Cyclophosphamide-treated animals. On day 18 of pregnancy, the mice were killed. Maternal femur bone marrow smears were prepared according to Schmid . The peritoneal cavity and uterus were opened and four fetuses were used from each female, two from

10 the right and two from the left uteral hom . Livers from these fetuses were pooled and suspended in fetal calf serum by using a syringe. The cell suspensions were concentrated by centrifugation, spread on slides and stained with Giemsa and May—Gruenwald . Polychromatic erythrocytes (PCE), 1000 per female and 1000 per litter, were analysed for the presence of Micronucleus with 100X objective with oil immersion using a Jenaval microscope The number of PCE was scored in a visual field of 2000 normochromatic erythrocytes (NCE) and the index PCE/(PCE+NCE) was calculated. Statistical evaluation The Mann-Whitney-Wilcoxon test was used to compare experimental and control data in the MN test. 7.2-Does The Study Require Any Investigation To Be Conducted On Patients Or Animals? If So, Please Describe Briefly. Yes, the Two experimental models require usage of laboratory animals. 7.3-Has Ethical Clearance Been Obtained From Your Institution? Yes. Approved by IAEC.

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9 Signature of the candidate (G.RAMYA DURGA) 10 Remarks of the Guide: The topic selected for dissertation is satisfactory. Adequate equipment and chemicals are available to carry out the project work.

11 Name and Designation of Guide: Dr. RAJU KONERI Dept. of Pharmacology&DEAN,

15 Karnataka college of pharmacy, Bengaluru-64.

Signature of Guide:

(Dr. RAJU KONERI) Head of the Department: Dr.RAJU KONERI Professor & Dean. Dept of Pharmacology, Karnataka college of pharmacy, Bengaluru-64. Signature of HOD:

(Dr.RAJU KONERI)

12 Remarks of the Principal: All the required facilities will be provided to carry out dissertation work under the supervision of guide.

principal: Dr.K.RAMESH. principal Karnataka College of Pharmacy, Thirumenahalli, Bangalore –64.

Signature of the principal:

(Dr.K.RAMESH)