Methanol Extract of Hypericum
hookerianum Stem in Ehrlich
Ascites Carcinoma Bearing Mice

Santoshkumar H. Dongre, Shrishailappa Badami^*,

Senthilkumar Natesan and Raghu Chandrashekhar H.

Department of Pharmaceutical Chemistry,

J.S.S. College of Pharmacy, Rock Lands, Ootacamund – 643 001, T.N., India

ABSTRACT

The methanol extract of H. hookerianum (MEHH) was studied for in vitro antioxidant activity using several standard methods. It exhibited potent activity in ABTS, DPPH and hydrogen peroxide methods. The extract was studied for antioxidant and hepatoprotective properties in Ehrlich ascites carcinoma (EAC) bearing mice. Tumor control animals inoculated with EAC showed a significant alteration in the antioxidant and hepatoprotective parameters. The extract treatment at 100, 200 and 400 mg/kg body weight doses caused a significant reversal of the biochemical changes towards the normal when compared to tumor control animals in serum, liver and kidney indicating the potent antioxidant and hepatoprotective nature of the extract.

Keywords:Antioxidants, Free radicals, H. hookerianum, Hepatoprotective.

Introduction

Reactive oxygen species (ROS) have been implicated in initiating, accompanying or causing pathogenesis of many diseases. Therefore, increased antioxidant concentration at the cellular level could provide considerable protection against ROS (Polidori, 2003). Most of the known antioxidants are derived from the plants, probable due to their increased capacity to defend themselves from various sources of stress (Badami et al., 2005; Natesan et al., 2007; Gupta et al., 2004; Silva et al., 2005; Conforti et al., 2002; Cakir et al., 2003). Strong evidence indicates that ROS plays an important role in the initiation as well as promotion phase of carcinogenesis. Many cancer chemo preventive agents possess antioxidant potentials (Natesan et al., 2007; Gupta et al., 2004). The presence of tumors in the human body or in experimental animals is known to affect many functions of the vital organs specially the liver, even when site of the tumor does not interfere directly with organ functions (De Wys, 1982). High levels of free radicals and malondialdehyde (MDA), the end product of lipid peroxidation were reported in cancer tissues than in non-diseased organs. Hence, currently the reduction of avoidable endogenous and exogenous source of oxidative stress is potentially the most important means of preventive oxygen free radical related cancer (Natesan et al., 2007; Gupta et al., 2004).

The plants of the genus Hypericum (Family: Hypericaceae) are widely used in folk medicine. Several species belonging to Hypericum viz., H. perforatum (Silva et al., 2005), H. triquetrifolium (Conforti et al., 2002), H. hyssopifolium (Cakir et al., 2003), etc., are reported to possess potent antioxidant and hepatoprotective activity. Earlier studies carried out in our laboratories have shown strong in vitro cytotoxic properties of MEHH (Vijayan et al., 2003). The plant is also reported to possess wound healing and antibacterial properties (Mukherjee et al., 2000; Mukherjee and Suresh, 2001). Strong in vivo anticancer properties were observed against EAC induced tumor mice (Dongre et al., 2007). Except these, so far no other biological activities have been carried out on MEHH and no phytoconstituents have been isolated. Hence, in the present study MEHH was evaluated for in vitro antioxidant studies using various standard methods and for in vivo antioxidant and hepatoprotective nature in EAC tumor bearing mice. High performance liquid chromatography (HPLC) standardized extract containing 0.36 per cent w/w hyperoside was used.

Materials and Methods

Chemicals

5-Flurouracil (5-FU) was obtained from Ranbaxy Ltd., Guregaon, India. DPPH (2,2-diphenyl-1-picryl hydrazyl) and ABTS [2,2’-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt] were obtained from Sigma Aldrich Co, St Louis, USA. HPLC grade solvents and Ecoline diagnostic Kits were obtained from E-Merck Ltd., Mumbai. All chemicals used were of analytical grade.

Plant Material and Extraction

Hypericum hookerianum Weight and Arnot was collected in Ootacamund in July 2004 and authenticated by Dr. S. Rajan, Medicinal Plants Survey and Collection Unit, Ootacamund and retained for future reference. The stem was shade dried, powdered and extracted (190 g) with methanol (1.2 l) in a Soxhlet extractor for 18-20 h. The extract was concentrated to dryness under reduced pressure and controlled temperature (40-50 °C) to yield a dark brown solid weighing 40 g (21.04 per cent). The extract was preserved in a refrigerator at 4 °C till further use.

Animals

Healthy Swiss Albino mice weighing 25±2.0 g were obtained from J. S. S. College of Pharmacy animal house, Ootacamund. The mice were grouped and housed in polyacrylic cages and maintained under standard conditions (temp. 25±2 °C) with 12±1 h dark/light cycle. The animals were fed with rat pellet feed supplied by Hindustan Lever Ltd., Bangalore, India and water ad libitum. All procedures were reviewed and approved by CPCSEA, Chennai, India (No. JSSCP/IAEC/PH.D/Ph.Chem/03/2005-2006).

Tumor Cells

EAC cells were supplied by Amala Cancer Research Centre, Thrissur, KL, India. The cells were maintained in vivo in Swiss albino mice, by intraperitoneal transplantation. EAC cells aspirated from the peritoneal cavity of mice were washed with saline and given intraperitoneally to develop ascitic tumor.

Preparation of Suspensions and Solutions

MEHH and standard 5-FU were suspended in distilled water using sodium carboxy methyl cellulose (CMC, 0.3 per cent) and administered orally to the animals with the help of an intragastric catheter for in vivo activity. MEHH and the standards, ascorbic acid and rutin were dissolved in distilled DMSO separately and used for the in vitro antioxidant testing using six different methods. For the hydrogen peroxide method (where DMSO interferes), the extracts and the standards were dissolved in distilled methanol and used. The stock solutions were serially diluted with the respective solvents to obtain the lower dilutions.

In vitro Antioxidant Activity

The in vitro antioxidant activity of MEHH was assessed on the basis of scavenging of various radicals like DPPH, ABTS, nitric oxide, superoxide radical by alkaline DMSO and hydrogen peroxide methods (Badami et al., 2005; Natesan et al., 2007).

In vivo Antioxidant Activity

Swiss Albino mice were divided in to six groups (n = 6). All the animals were injected with EAC cells (2 x 10⁶ cells/mouse) intraperitoneally except the normal group. This was taken as day zero. Group I was served as normal and group II as tumor control. These two groups were received sodium CMC suspension (0.3 per cent). Group III as a positive control was treated with a suspension of 5-FU at 20 mg/kg body weight. Groups IV, V and VI were treated with MEHH at 100, 200 and 400 mg/kg body weight doses, respectively. All these treatments were given 24 h after the tumor inoculation, once daily for 14 days. After the last dose and 24 h fasting, six mice from each group were sacrificed for the study of biochemical parameters.

Blood was collected from the animals by retro-orbital puncture under mild anesthetic (diethyl ether) condition and the serum was separated by keeping it at 37 °C for half an hour and subjected for the estimation of superoxide dismutase (SOD, Misra and Fridovich, 1972), catalase (CAT, Beers and Sizer, 1952) and thiobarbituric acid reactive substances (TBARS, Ohkawa et al., 1979) and hepatoprotective parameters, aspartate amino transferase (ASAT), alanine amino transaminase (ALAT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), triglycerides (TGL), creatinine (CR), albumin, total protein (TP), total cholesterol (TC) and total bilirubin (TB) using Ecoline (E Merck Ltd., Mumbai, India) Kits.

After the collection of blood samples, the mice were sacrificed and their liver and kidney were excised, rinsed in ice-cold normal saline followed by cold 0.15 M Tris-HCl (pH 7.4) and blotted dry. A 10 per cent w/v homogenate was prepared in the buffer with Elvenjan homogenizer fitted with teflon plunger and centrifuged at 1500 rpm for 15 min at 4 ºC. The supernatants were used for the estimation of the antioxidant parameters.

Statistical Analysis

The significance of the in vivo

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