CHAPTER 6 IN-VITRO PHARMACOLOGICAL STUDIES

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1 95 CHAPTER 6 IN-VITRO PHARMACOLOGICAL STUDIES The biological experiments like in-vitro and in-vivo screening models for various diseases may be applied to discover new molecules. These contemporary techniques include bio evaluation, clinical trials, toxicological studies etc. The use of disease induced animal models or the genetically transformed disease models are in use to validate the claims of these medicines, but ethical issues and cost effectiveness of these models have discouraged their common use. Besides, these disease induced models very often get reversed to the normal condition in due course of time as a part of natural healing, which also makes it difficult in interpreting the result of a test drug. Therefore, in-vitro mechanism based screening of herbal medicine is mandatory in the initial phases of plant drug research before taking them to in-vivo study to evaluate their efficacy. Special statistical tools must be used to evaluate their efficacy of the collected data, otherwise it may be difficult to reach any conclusion or to frame a hypothesis [50]. Symplocos cochinchinensis (Lour.) is traditionally used to treat various diseases. Since, no literature is available on the biological effects of this plant, the present study was carried out to screen the plant extracts for various in-vitro pharmacological studies like in-vitro cytotoxic activity, in-vitro anti-inflammatory activity and in-vitro anti-snake venom activity for n-hexane, chloroform, ethyl acetate and methanol extracts.

2 IN-VITRO CYTOTOXIC ACTIVITY Introduction Cancer continues to represent the largest cause of mortality in the world and claims over 6 million lives every year [51]. Drug development programmes involve preclinical screening of a vast number of chemicals for their specific and non specific cytotoxicity against many types of cells. Use of in-vitro assay system for the screening of potential anticancer drug has been a common practice almost since the beginning of cancer therapy in The National Cancer Institute now routinely measure the growth inhibitory properties of every compound under test against a panel of human tumor cell lines which are representative of major human tumor types. There are number of advantages in in-vitro test using cell cultures which includes analysis of species specificity, feasibility of using only small amount of test substance and facility to do mechanistic studies. A novel anticancer drug should possess cytotoxicity at low concentration against cancerous cell lines and should be safe against normal cell lines even at higher concentration [52]. The direct antitumor activity of the plant extracts can be tested under in-vitro conditions using cultured or fresh preparation of various cancer cell types. Once the activity has been detected, the study has to be followed up vigorously to establish therapeutic efficacy and safety [53] Materials and Methods Method : MTT Assay methanol extracts Plant materials used: n-hexane, chloroform, ethyl acetate and

3 97 Cell culture Human breast cancer- MDA-MB-231, Colon cancer-sw 620, Liver cancer Hep G 2 were obtained from National centre for cell science (Pune, India). Chemicals Dubelcco s Modified Eagle s Minimum Essential Medium (DMEM), Foetal Calf Serum (FCS), trypsin (0.25%) were from D. Dutscher (Brumath, France). 3- (4, 5 dimethyl thiazo l -2-yl)- 2,5 diphenyl tetrazolium bromide (MTT) and dimethylsulfoxide (DMSO). Principle This colorimetric assay is based on the capacity of mitochondria succinate dehydrogenase enzymes in living cells to reduce the yellow water soluble substrate 3- (4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) into an insoluble, blue colored formazan product which is measured spectrophotometrically. Only viable cells with active mitochondria reduce significant amounts of MTT [54] since reduction of MTT can only occur in metabolically active cells, the level of activity is a measure of the viability of the cells[55]. Cell lines Human breast cancer- MDA-MB-231, Colon cancer-sw 620, Liver cancer Hep G 2 were obtained from National centre for cell science (Pune, India). Stock culture of these cell lines were cultured in RPMI or DMEM supplemented with 10% inactivated newborn calf serum, Penicillin (100 IU/ml), Streptomycin (100ìg/ml) and Amphotericin (5 ìg/ml) under humidified atmosphere of 5% CO 2 at 37 0 C until confluent. The cells were

4 98 dissociated in 0.2% trypsin and 0.02% EDTA in phosphate buffer saline solution. The stock culture was grown in 25cm tissue culture flasks and cytotoxicity experiments were carried out in 96 well microtiter plates (Tarsons India, Kolkata, India). Procedure Cell lines in the exponential growth phase were washed, trypsinized and suspended in complete culture media. Cells were plated at 10,000 cells/well in 96 well microtiter plates and incubated for 24hrs during which a partial monolayer was formed. They were then exposed to various concentrations of the extract (1 100mcg/ml). Control wells received only maintenance medium. The plates were incubated at 37 C in a humidified incubator with 5% CO 2 for a period of 72 hrs and cells were periodically checked for granularity, shrinkage and swelling. After 72 hrs, the sample solution in wells was flicked off and 50 l of MTT dye was added to each well. The plates were gently shaken and incubated for 4 hrs at 37 0 C in 5% CO 2 incubator. The supernatant was removed and 50 l of propanol was added. The plates were gently shaken to solubilise the formed formazan. The absorbance was measured at 540nm [56]. The percentage growth inhibition was calculated using the following formula, Mean OD of individual test group Percentage growth inhibition = 100 (6.2) Mean OD of control group Values of absorbance were converted into percentage of residual viability. Usually, inhibition concentration 50% (GI 50 ) is chosen as the best biological marker of cytotoxicity. The GI 50 value represents the concentration of the tested extracts that caused 50% of cell inhibition.

5 Results The in-vitro cytotoxic activity of n-hexane, chloroform, ethyl acetate and methanol extracts was given in Table 6.1 Table 6.1 The in-vitro cytotoxic activity of various extracts of leaves of Symplocos cochinchinensis (Lour.) MDA-MB-231 ( g/ml) SW 620 ( g/ml) Hep G 2 ( g/ml) Extracts GI 50 TGI GI 50 TGI GI 50 TGI N-Hexane >100 >100 >100 >100 >100 >100 Chloroform >100 >100 >100 >100 >100 >100 Ethyl acetate 50 > >100 Methanol Average of 3 determinations, 3 replicates GI 50 - Drug concentration inhibiting 50% cellular growth TGI- Total cellular growth inhibition Discussion In in-vitro cytotoxic study of both ethyl acetate and methanol extracts showed significant activity against the three human cancer cell lines namely Human breast cancer- MDA-MB-231, Colon cancer-sw 620, Liver cancer Hep G 2. The n-hexane and chloroform extracts did not show any activity. Methanol extract showed greater cytotoxic effect against colon cancer cell lines SW 620 and HepG2 (liver cancer cell) with a GI 50 value below 20 g/ml when compared to ethyl acetate extract.

6 IN-VITRO ANTI-INFLAMMATORY ACTIVITY Introduction The inflammatory response involves a complex array of enzyme activation, mediator release, cell migration, tissue breakdown and repair[57] which are aimed at host defense and usually activated in most disease condition. Currently much interest have been paid in the searching of medicinal plants with anti-inflammatory activity which may lead to the discovery of new therapeutic agent that is not only used to suppress the inflammation but also used in diverse disease conditions where the inflammation response is amplifying the disease process. In this work the various extracts of Symplocos cochinchinensis (Lour.) were studied for its in-vitro anti-inflammatory activity Materials and Method Plant material n-hexane, chloroform, ethyl acetate and methanol extracts Method The human red blood cell membrane stabilization method Procedure [58] The blood was collected from healthy human volunteers who have not taken any NSAIDS for 2 weeks prior to the experiment and mixed with equal volume of Alsever s solution(2% dextrose, 0.8% sodium citrate, 0.5% citric acid and 0.42% NaCl) and centrifuged at 3,000 rpm. The packed cells were washed with isosaline and a 10% suspension was made. Various concentrations of extracts were prepared (, and 1000 mcg/ml) using distilled water and to each concentration 1 ml of phosphate buffer, 2 ml hyposaline and 0.5 ml of HRBC suspension were added. It was incubated at 37 0 C for 30 min and centrifuged at 3,000 rpm for 20 min. The haemoglobin

7 101 content of the supernatant solution was estimated spectrophotometrically at 560 nm. Diclofenac (50 mcg/ml) was used as reference standard and a control was prepared omitting the extracts. The percentage inhibition of lysis was calculated Results The results of in-vitro anti-inflammatory activity of extracts of Symplocos cochinchinensis (Lour.) by HRBC membrane stabilization method was tabulated in Table 6.2 and Figure 6.1. Table 6.2 In-vitro anti-inflammatory activity of extracts of Symplocos cochinchinensis(lour.) by HRBC membrane stabilization method S.No Treatment Conc(mcg/ml) Absorbance(540nm) %Inhibition 1. Control ± n-hexane Chloroform Ethyl acetate Methanol ± ± 0.01 ** 0.38 ± ± *** 0.33 ± *** 0.35 ± *** 0.25 ± *** 0.28 ± *** 0.29 ± *** 0.15 ± *** 0.19 ± *** 0.20 ± Diclofenac ± *** 73.9 Values are expressed as mean ± SEM. n=5 ***P<0.001 **P< 0.01 compared to control group

8 102 (%) Inhibition n-hexane Chloroform Ethyl Acetate Methanol Diclofenac Figure 6.1 Effect of various extracts on HRBC membrane stabilization method Discussion Among all the extracts tested the methanolic extract showed significant anti-inflammatory activity in a concentration dependent manner. Methanol extract at a concentration of 1000 mcg/ml showed 69.9% protection of HRBC in hypotonic solution. All the results were compared with standard Diclofenac which showed 73.9% protection. It is well known that the vitality of the cells depends on the integrity of their membranes. Exposure of red blood cell to hypotonic medium results in lysis of its membrane accompanied by haemolysis and oxidation of haemoglobin [59,60]. The haemolytic effect of hypotonic solution is related to excessive accumulation of fluid within the cell resulting in the rupturing of its membrane. It is therefore expected that compounds with membrane-stabilizing properties, should offer significant protection of cell membrane against injurious substances. The lysosomal enzymes released during inflammation produces a variety of disorders. The extracellular activity

9 103 of these enzymes is said to be related to acute or chronic inflammation. There is increasing evidence that lysosomal enzymes play an important role in the development of acute and chronic inflammation [61]. Most of the anti-inflammatory drugs exert their beneficial effects by inhibiting either release of these enzymes or by stabilizing lysosomal membrane, which is one of the major event responsible for the inflammatory process [62]. The extracts exhibited membrane stabilization effect by inhibiting hypotonicity induced lysis of erythrocyte membrane. The erythrocyte membrane is analogous to the lysosomal membrane [63] and its stabilization implies that the extract may as well stabilize the lysosomal membranes. Stabilization of lysosomal membrane is important in limiting the inflammatory response by preventing the release of lysosomal constituents of activated neutrophil such as bactericidal enzymes and proteases, which cause further tissue inflammation and damage upon extra cellular release [64]. Some of the NSAIDs are known to possess membrane stabilization properties which may contribute to the potency of their anti-inflammatory effect. Though the exact mechanism of the membrane stabilization by the extract is not known yet, hypotonicity-induced haemolysis may arise from shrinkage of the cells due to osmotic loss of intracellular electrolyte and fluid components. From this result, it is suggested that anti-inflammatory activity observed in this study, may be due to the ability of the extracts to interfere with the early phase of inflammatory reactions, which may stimulate or enhance the efflux of these intracellular components [65] there by exhibits the anti-inflammatory activity.

10 IN-VITRO ANTI-SNAKE VENOM ACTIVITY Introduction Snake envenoming is a major public health issue in the rural tropics with large numbers of envenoming and deaths. In India there are about 216 different snake species of which 53 species are reported to be poisonous. Due to high cost and unavailability of anti venoms, the use of plant remedies for the treatment is the most common practice in India. Extracts from plants have been used among traditional healers, especially in tropical areas for snakebite for a long time [66]. Several medicinal plants, which appear in old drug recipes or which have been passed on by oral tradition are believed to be snakebite antidotes [67]. In almost any part of the world, where venomous snakes occur, numerous plant species are used as folk medicine to treat snakebite. However many of the reported studies lacks detailed scientific investigation, which is needed in the development of medicinal agents from plant sources. Symplocos cochinchinensis (Lour.) is traditionally used in the treatment of snake bites. Since, the anti-snake venom properties of this plant is not scientifically proved, the present study has been undertaken to investigate the in- vitro anti-snake venom activity of various extracts of the leaves Materials and Methods Plant extract n-hexane, chloroform, ethyl acetate and methanol extracts Snake venom Lyophilized snake venom of Russell s viper (Daboia russelli) was obtained from CSIR Centre for Biochemical s, New Delhi and preserved at

11 C. The snake venom was dissolved in 0.9%(w/v) saline, centrifuged and the supernatant was used whenever required. The venom concentration was expressed in terms of dry weight (mg/ml) of the stock venom. Method - Inhibition of in-vitro Human Red Blood Corpuscles lysis method Procedure n-hexane, chloroform, ethyl acetate and methanolic extracts at a concentration of 100,, and 1000mcg/ml of the leaves of Symplocos cochichinensis (Lour.) were used for the study. The hypo saline induced haemolysis was evaluated in-vitro by the method of Roelofsen et al and Balu et al [68,69]. This method was modified in the present study by venominduced haemolysis. Blood was collected from healthy human volunteers by vein puncture and heparin was used as an anti coagulant. The collected blood was washed three times with physiological saline solution to make a stock solution of 100mcg/ml. 1 ml of the venom, 1 ml phosphate buffer(ph 7.4) and 1 ml of 1% HRBC were taken in various tubes. Different concentrations of n-hexane, chloroform, ethyl acetate and methanolic extracts of the leaves (100,, and 1000mcg/ml) were added. The extracts were prepared by using saline and carboxy methyl cellulose (CMC) as suspending agent and the control was prepared omitting the extracts. The anti-snake venom serum(100mcg/ml) was used as standard. These mixtures were incubated at 37 0 C for 30 min and then centrifuged at 1000 rpm for 3 min. The absorbance of the supernatant was measured at 540 nm using spectrophotometer. The percentage inhibition of haemolysis was calculated by using the following formula A (Control) A (test) % inhibition = X 100 (6.2) A (Control)

12 Results The in-vitro anti-snake venom activity of various extracts of the leaves of Symplocos cochinchinensis (Lour.) were studied against Russell s viper venom. The results were tabulated in the Table 6.3 and Figure 6.2. Table 6.3 In-vitro anti-snake venom activity (Inhibition of human red blood cell lysis method) S.No Treatment Conc.(mcg/ml) % inhibition 1. Contorl Snake venom antiserum n-hexane extract Chloroform extract Ethyl acetate extract Methanol extract

13 (%) Inhibition n-hexane Chloroform Ethyl Acetate Methanol Snake venom antiserum Figure 6.2 In-vitro anti-snake venom activity Discussion In-vitro anti-snake venom activity was carried out by prevention of HRBC membrane lysis method. Haemolysis is one of the major causes of snake venomation. Most of the snake venom contains phospholipase and haemolysin[70], which acts on membrane associated phospholipids to liberate lysolecithin. Lysolecitihin acts on the membrane of HRBC causing haemolysis [71]. The extracts inhibit the haemolysis induced by Russell s viper venom in a concentration dependent manner. The percentage inhibition of haemolysis activity was found to be significant in methanol extract at a concentration of 1000mcg/ml. It showed 69.5% protection against venom induced haemolysis which may be due to the stabilization of the protein in the membrane of HRBC [72]. Hence, it may be suggested that the extract may interact with Russell s viper venom and stabilize the protein in the membrane.

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