ijapr - international journal of advances in pharmaceutical research

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M.S Latha et al. / International Journal of Advances in Pharmaceutical Research the process, which is followed by regeneration, growth and clonal proliferation, eventually leading to cancer (5). There is extensive evidence that the free radicals participate in NDEA induced hepatocarcinogenesis (6). Enzymes such as GST, GR and GPx reduce oxidative stress in the normal tissues. Extensive liver damage causes decrease in levels of these enzymes suggesting a high oxidative stress in the tissues, which may be one of the key factors in the etiology of cancer (7). They have been proved to cause numerous cellular anomalies, including but not limited to protein damage, deactivation of enzymatic activity, alteration of DNA and lipid peroxidation of membranes (8). Several herbal drugs like Abrus precatorius, Scutia myrtina, Funaria indica etc. have been evaluated for its potential as liver protectant against NDEA induced hepatocellular carcinoma in rats (9- 11). Chemoprevention, which is referred to as the use of nontoxic natural or synthetic chemicals to intervene in multistage carcinogenesis, has emerged as a promising and pragmatic medical approach to reduce the risk of cancer. Numerous components of plants, collectively termed “phytochemicals” have been reported to possess substantial chemopreventive properties. For many years cancer chemotherapy has been dominated by potent drugs that either interrupt the synthesis of DNA or destroy its structure once it has formed. Unfortunately, their toxicity is not limited to cancer cells and normal cells are also harmed (12). Use of plants and plant derivatives considered as alternative medicine for many ailments are on the increase on a global perspective. Woodfordia fruticosa is a traditional medicinal plant belongs to the family Lythraceae is used against a wide variety of diseases. All parts of the plant possess valuable medicinal properties such as anti inflammatory, antitumor, hepatoprotective and free radical scavenging activity but flowers are of maximum demand. Dried flowers are used as tonic in disorders of mucous membrane, hemorrhoids and in derangement of liver (13). Phenolics, particularly hydrolysable tannins and flavonoids were identified as major components in Woodfordia fruticosa flowers. In view of these the present study was undertaken to evaluate the chemopreventive effect of Woodfordia fruticosa flower extract on NDEA induced hepatocellular carcinoma in experimental rats. 2. MATERIALS AND METHODS 2.1 Chemicals N-nitrosodiethylamine (NDEA), silymarin, proliferating cell nuclear antigen (PCNA), cyclin D1, anti-mouse IgG horse radish peroxidase, streptavidin horse radish peroxidase conjugate and diaminobenzidine were purchased from Sigma Chemical Co., St. Louis, MO, USA. Assay kits for serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma glutamyl transferase (GGT) were purchased from Agappe Diagnostics, India. All other chemicals were of analytical grade. 2.2 Collection of plant material and preparation of plant extracts Woodfordia fruticosa flowers were collected from natural habitat (Kollam, Kerala, India) during November - January. Plant material was identified by Dr. V.T Antony, S.B College. A voucher specimen (Acc. No. 7566) is deposited at the herbarium of the Department of Botany, S.B College, Changanassery, Kottayam, Kerala. Flowers were shade-dried and powdered and 50 g of dried powder was soxhlet extracted with 400 mL of methanol for 48 h. The extract was concentrated under reduced pressure using a rotary evaporator and was kept under refrigeration. The yield of methanolic extract of Woodfordia fruticosa (MEWF) was 12.5 % (w/w). The concentrate was suspended in 5% Tween 80 for the present studies. 2.3 Animals and diets Male Wistar rats weighing 150-160 gm were used in this study. The animals were housed in polypropylene cages and had free access to standard pellet diet (Sai Durga Feeds, Bangalore, India) and drinking water. The animals were maintained at a controlled condition of temperature of 26–28 o C with a 12 h light: 12 h dark cycle. Animal studies were followed according to Institute Animal Ethics Committee regulations approved by Committee for the Purpose of Control and Supervision of Experiments on Animals (Reg. No. B 2442009/4) and conducted humanely. 2.4 Induction of hepatocellular carcinoma Hepatocellular carcinoma (HCC) was induced by oral administration of 0.02% NDEA (2ml, 5 days/week for 20 weeks (12). Silymarin at an oral dose of 100 mg/kg body weight was used as standard control in the experiment (14-15). Two different doses of MEWF (100mg/kg and 200 mg/kg) suspended in 5% Tween 80 were prepared for oral administration to the animals. It is reported that the extract of W. fruticosa flowers are safe up to the dose of 2000mg/kg, p.o (13). 2.5 Experimental design IJAPR / Dec. 2012/ Vol. 3 /Issue. 12 / 1322 – 1330 1323
M.S Latha et al. / International Journal of Advances in Pharmaceutical Research Thirty six rats were divided into six groups, Group I - Normal control Group II - NDEA control (0.02% NDEA, 2 ml, 5days/week, p.o) Group III - Silymarin (100mg/kg b.w) + NDEA Group IV - MEWF (100mg/kg, b.w ) + NDEA Group V - MEWF (200mg/kg, b.w) + NDEA Group VI - MEWF (200 mg/kg) alone Daily doses of silymarin and MEWF treatments were started in groups III to V animals 1 week before the onset of NDEA administration and continued up to 20 weeks. Group VI served as drug control received MEWF alone for the entire period. The rats were sacrificed 48 h after the last dose of NDEA administration. 2.6 Serum enzyme analysis Blood was collected from neck blood vessels and kept for 30 min at 4 0 C. Serum was separated by centrifugation at 2500rpm at 4 0 C for 15 min. Quantifying the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma glutamyl transferase (GGT) by using a standard diagnostic kit (Agappe Diagnostic Ltd., India). Activities of these serum enzymes were measured by using semi autoanalyzer (RMS, India). 2.7 Tissue analysis Liver tissue was excised, washed thoroughly in icecold saline to remove the blood. Then the dissected livers were cut into separate portions for biochemical assays and immunohistochemical examination. 2.7.1 Biochemical assays Ten percent of homogenate was prepared in 0.1M Tris HCl buffer (pH – 7.4). The homogenate was centrifuged at 3000 rpm for 20 min at 4 o C and the supernatant was used for the estimation of glutathione-S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx) and total protein. GST (EC 2.5.1.18) activity was determined from the rate of increase in conjugate formation between reduced glutathione and CDNB (16). GR (EC 1.6.4.2) activity was assayed at 37 o C and 340 nm by following the oxidation of NADPH by GSSG (17). GPx (EC 1.11.1.9) activity was determined by measuring the decrease in GSH content after incubating the sample in the presence of H2O2 and NaN3 (18). Protein content in the tissue was determined using bovine serum albumin (BSA) as the standard (19). 2.7.2 Immunohistochemical analysis Immunohistochemical analysis of the two cancer markers, namely proliferating cell nuclear antigen (PCNA) and Cyclin D1 were conducted. Tissue sections were deparaffinised in three changes of xylene at 60 o C for 10min each and hydrated through a graded series of alcohol. For antigen retrieval evaluation, slides were placed in citrate buffer (pH 6.0) for three cycles of 5 min each in a microwave oven. The sections were then allowed to cool to room temperature and then rinsed with 1x tris buffered saline (TBS), and treated with 0.3% H2O2 in water for 10 min to block endogenous peroxidase activity. Non specific binding was blocked with 3% BSA in room temperature for 1 h. Subsequently the primary antibody was applied at predetermined dilutions (PCNA antibody diluted 1:500 / Cyclin D1antibody diluted 1:80) with 1% BSA in PBS for overnight at 4 o C. After this incubation washed thrice in PBS and incubated with anti-mouse horseradish peroxidase for 45 min. After triplicate washing with PBS, sections were incubated for 30 min with streptavidin–HRP complex. Then washed with PBS and incubated for 5–10 min in a solution of diaminobenzidine (6 mg/10mL 50mM Tris–HCl, pH 7.6) containing 0.01% H2O2. Counterstaining was performed with hematoxylin. Images were taken at original magnification of 100× ( Motic AE 21, Germany and Moticam 1000 camera). 2.8 Statistical analysis Results were expressed as mean ± S.D and all statistical comparisons were made by means of oneway ANOVA test followed by Tukey’s post hoc analysis and p-values less than or equal to 0.05 were considered significant. 3. RESULTS 3.1 Serum enzyme analysis The serum levels of AST, ALT, and GGT in group II were significantly (p ≤ 0.05) elevated by the administration of NDEA, when compared to normal control. The treatment of MEWF at a dose of 100 and 200 mg/kg showed a significant decrease (p ≤ 0.05) in AST, ALT, and GGT (Fig.1). Standard control drug, Silymarin at a dose of 100 mg/kg also prevented the elevation of serum enzymes. Treatment with MEWF at 200 mg/kg and Silymarin exhibited a protection of 98% and 80.7% in AST levels, 97% and 88% in ALT levels and 98.2% and 84% in GGT levels, respectively. 3.2 Liver morphology Fig.2 shows the morphological variations of rat livers in different experimental groups. NDEA treated rat IJAPR / Dec. 2012/ Vol. 3 /Issue. 12 / 1322 – 1330 1324
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