Summary HEPATOPROTECTIVE ACTIVITY OF AQUEOUS EXTRACT OF CAESALPINIA BONDUC AGAINST CCL4 INDUCED CHRONIC HEPATOTOXICITY.

INTRODUCTION The liver is a major target for the chemical induced toxicity, as it participates in the metabolism, detoxification, and secretory functions. Liver disease is a worldwide problem and in most of the cases, liver damage is due to oxidative stress. Liver damage if not managed at the right time, may progress from steatosis to chronic hepatitis, fibrosis, cirrhosis and even hepatocellular carcinoma [1]. The progressive and chronic liver assault triggers the progressive wound healing response which results in the irreversible excessive production of collagen fiber and alteration of the extracellular matrix complex [2-4]. Although the precise mechanisms of the pathogenesis of liver cirrhosis are incompletely understood, the role of free radicals and lipid peroxides in inducing this has garnered considerable attention [5]. The active management of liver disease is a major concern in medical science as there are no reliable drugs available in modern medicine. Thus, there is a phenomenal increase in the search for the effective natural products worldwide to treat or to prevent liver toxicity. Keeping up with the pace, we have undertaken the screening of the hepatoprotective activity of the leaves of locally available thorny plant-Kantakikaranja (Caesalpinia bonduc) (CB). CB belongs to the family Fabaceae, and this plant is found in the coastal areas of South Asian countries. Different parts of this plant are used for various purposes in ethnopharmacology. The leaves of CB are used extensively in folk medicine for live ailments. However, there are no scientific evidence to prove its hepatoprotective properties. Earlier we have shown that the CB can effectively prevent the acute hepatotoxicity [6]. The hepatoprotective effect of CB in chronic hepatotoxicity is not experimented upon. Therefore, the present study was designed to understand the role of CB in chronic hepatotoxicity conditions which leads to irreversible adverse changes in the liver structure and function. MATERIALS AND METHODS Procurement of plant materials and chemicals Leaves of CB were collected in the month of February from “Soans farms,” Moodabidiri, Mangalore District, Karnataka, India and was authenticated by a botanist from Mahatma Gandhi Memorial College, Udupi. A voucher specimen is deposited in the Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal (PP 585). CCl4 was procured from Merck Ltd. Mumbai, silymarin and other chemicals (ascorbic acid, 2,2’-diphenyl-1picrylhydrazyl(DPPH), Masson’s trichrome staining reagents) and assay kits for assessing the (AST, ALT, ALP, total protein, Bilirubin, TriniCLOT PTEXCEL and albumin) were procured from Durga laboratory Mangalore. Preparation of aqueous extract (Hot maceration method) Leaves of CB were shade dried and powered. Leaf powder (200 g) was then dissolved in 1500 ml of distilled water, and a decoction was prepared at 75-80 °C. The decoction was then cooled and filtered. Finally, the filtrate was evaporated to dryness using lyophilizer. Preparation of the test material Carboxy Methyl Cellulose (CMC) stock solution was prepared by dissolving 250 mg of CMC in 100 ml of PBS (Phosphate Buffer Solution). Dilution of aqueous extract of CB is prepared by dissolving 500 mg of aqueous extract in 10 ml CMC of 0.25 % w/v. α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging assay DPPH radical scavenging activity was measured by the spectrophotometric method [7]. To measure the DPPH scavenging activity of CB, 50 µl of DPPH and 50 µl CB in methanol was taken at the concentration of 200 µg/ml and serially diluted(25, 12.5, 6.25, 3.125, 1.625 µg/ml). An equal amount of DPPH and ascorbic acid in methanol was taken as the positive control. They were further incubated for 20 min, the decrease in absorbance of the test mixture (Due to quenching of DPPH free radicals) was read at 517 nm and the percentage inhibition was calculated using the formula given below [8]: control − test Inhibition % = × 100 control Animals Healthy female Albino rats (150-200 g) were maintained under standard environmental conditions at room temperature of 27 °C with 12 h day/night cycle in central animal house facility, Manipal University, Manipal. The animals were fed with a standard pellet diet and water ad libitum. Animal studies were approved by the institutional animal ethics committee (IAEC/KMC/34/). Toxicity study The toxicity test of aqueous extract of CB was done as per staircase methods [9]. The rats were divided into seven groups of six animals each. The control group received saline, and the other groups received single oral dose of 100, 500, 1000, 2000, 3000, and 4000 mg/kg body weight of CB extract respectively. Then, animals were observed for various toxic signs and symptoms continuously for the first four hours and further observed for seven days. Based on the observed results, the dose of 500 mg/kg body weight was considered as optimal and safe dose for further experiments. Treatment protocol Animals were divided into three groups namely; preventive, curative and prophylactic based on the treatment strategy of CB. The groups were further subdivided into four groups each: Group I control, Group II-CCl4 control, group III-CB+CCl4 and group IV-silymarin+CCl4. Each subgroup comprised of eight animals (n=8). The aqueous extract of CB/silymarin was administered orally by using the intragastric tube at the dose of 500 mg/kg and 30 mg/kg body weight once daily for eight weeks in the curative group and for four weeks in preventive and prophylactic groups respectively. The chronic hepatotoxicity/liver damage was induced by intraperitoneal (i. p) injection of CCl4 0.5 ml/kg body weight, 1:1 in olive oil, twice a week for four weeks in the preventive and prophylactic group and for eight weeks in the curative group [10]. Experimental design A. Preventive group Group I: control, Group II: CCl4 control, where CCl4 is administered twice a week, Group III: where CCl4 and CB are administered together, Group IV: CCl4 and silymarin are administered together. The drugs were administered for four weeks in all the treatment groups. B. Curative group Group I: control, Group II: CCl4 control, where CCl4 is administered for eight weeks, Group III: Animals were treated with CCl4 for eight weeks, along with CB extract for the last four weeks, Group IV: Animals were treated with CCl4 for eight weeks, along with silymarin for the last four weeks. The animals were treated for a total of eight weeks in all the treatment groups. C. Prophylactic group Group I: control, Group II: CCl4 control, where CCl4 is administered for four weeks, Group III: Rats were pretreated with CB for first four weeks followed by the CCl4 for the next four weeks, Group IV: Rats were pretreated with silymarin for the first four weeks followed by the CCl4 for the next four weeks. The animals were treated for a total of eight weeks in all the treatment groups. At the end of the treatment period, the animals were sacrificed by ether overdose. The liver tissues and blood was collected and processed for histological and biochemical analysis respectively. Histological analysis using Masson's trichrome stain Each formaldehyde-fixed sample was embedded in paraffin, cut into 5 μm-thick sections and stained with Masson’s trichrome according to the standard protocol [11]. Biochemical analysis The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP) levels, bilirubin, prothrombin time (PT), total serum proteins (TP) including albumin (ALB) and globulin (GLB) were estimated by using commercially available standard kits. Statistical analysis The results were presented as mean±standard error mean. One-way ANOVA was applied to compare between the groups, and Bonferroni’s post hoc test was applied for multiple comparisons using Graph pad prism software (Microsoft, San Diego, CA, USA). p0.05 was considered statistically significant. RESULTS DPPH radical scavenging activity Higher the dosage of CB, greater was the inhibition of DPPH free radical activity, i.e., 25 μg/ml of CB showed maximum inhibition (46.90 %) while the lower dose (1.625 μg/ml) failed to evoke any response. This indicated that the free radical was scavenged by CB in a concentration-dependent manner (table 1). Table 1: Effect of aqueous extract of CB on DPPH radical scavenging activity Concentrations of CB (μg/ml) DPPH ( % inhibition) 25 46.90±2.05 12.5 32.72±2.27 6.25 15.27±0.51 3.125 6.36±0.32 1.625 3.6±1.01 Results are expressed in mean±SEM, [n=3] Histological findings The Masson’s trichrome stain was used to assess the degree of fibrosis in the liver samples from different treated groups. Liver sections from the control group appeared normal without signs of excessive collagen deposition. In the preventive group (fig. 1), CCl4 treated sections (fig. 1-B) revealed increased deposition of collagen fibers around the congested central vein and a remarkable increase in thickness of the capsule indicating severe fibrosis. Liver sections also revealed the inflammatory changes with the micro and macrovesicular changes around the central vein suggesting centrilobular necrosis. The tissues from silymarin-treated groups (fig. 1-D) showed minimal sinusoidal congestion with near normal architecture without visible changes in the thickness of the liver capsule. Liver tissues exposed to the CB (fig. 1-C) greatly improved the morphology with mild inflammatory changes, with minimal collagen deposition around the central vein. The liver capsule greatly reduced in thickness indicating minimal fibrosis when compared to CCl4 treated groups. Near normal changes were observed in groups co-treated with silymarin and CCl4 (fig. 1-D). In the curative group (fig. 2), a liver tissue treated with CCl4 showed severe deposition of collagen fibers and congestion in the central vein (fig. 2-B). Excessive deposition of collagen fibers was also observed in the capsule contributing to its thickness when compared to the normal control (fig. 2-A).