CHAPTER 7 PHARMACOLOGICAL STUDIES

Transcription

1 108 CHAPTER 7 PHARMACOLOGICAL STUDIES Pharmacological screening is essential to evaluate the efficacy and potency of the plant drug. Methanolic extract was selected for the pharmacological screening based on the in-vitro pharmacological and preliminary phytochemical studies. 7.1 ACUTE TOXICITY STUDY Materials and Methods Plant extract Methanolic extract of leaves of Symplocos cochinchinensis (Lour.) Experimental animals Healthy Wistar Albino rats of either sex, weighing about g were procured from Animal House. The entire process was approved by the Institutional Animal Ethical Committee which is certified by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) IAEC/31/2007. The animals were kept in clean and dry polycarbonate cages and maintained in a well ventilated animal house with 12hrs light 12 hrs dark cycle. The animals were fed with standard pellet diet and water was given as

2 109 libitum. For experimental purpose the animals were kept fasting overnight but allowed for access to water. Acute toxic class method [73] Acute toxicity study was performed according to OECD guidelines 423 (Organization of Economic Co-Operation and Development). It is a stepwise procedure with three animals of single sex per step. Depending on the mortality and morbidity status of the animal, on average of 2-4 steps may be necessary to allow judgment on the test substance. The procedure is to fix a minimal number of animals, which allows acceptable database scientific conclusion. The method uses different defined doses (5, 50, 500, 2000mg/kg body weight) and the results allow a substance to be ranked and classified according to the Globally Harmonized System (GHS) for the classification of extracts which cause acute toxicity. Procedure Three healthy, Wistar Albino rats weighing gms were selected for the study. The rats were fasted over-night and provided with water ad libitum. Following the period of fasting, the animals were treated with the methanolic extract at the dose of 2000mg/kg body weight, orally. As most of the crude extracts possess LD 50 value more than 2000mg/kg body weight and this was used as starting dose. After oral administration, the rats were observed on hourly basis for 24hrs to access mortality and to detect any changes in the autonomic or behavioral responses viz. alertness, aggressiveness, spontaneous activity, irritability, tremor, corneal reflex, salvation, urination, respiration and convulsion etc.

3 110 The rats were observed regularly for 14 days to note the mortality or toxic symptoms. Since there was no death as per the guidelines, the study was repeated with the same dose to confirm the results. The flow chart depicts the procedure adopted for this method Results During the acute toxicity study, the methanolic extract was administered orally and animals were observed for mortality, changes in the autonomic nervous system, central nervous system and behavioral responses. There was no mortality observed even at 2000mg/Kg for the extract. All the animals were found to be normal and there were no gross behavioral changes till the end of the observation period. This observation revealed that the methanolic extract of the leaves was found to be very safe up to 2000mg/kg of body weight known as maximum tolerated dose (MTD) by acute toxicity model study as per OECD guidelines 423. Hence from this 1/10 th and 1/5 th of MTD was selected and the effective doses were fixed as 200 and 400 mg/kg for the further pharmacological studies.

4 Figure 7.1 OECD Guidelines 111

5 HEPATOPROTECTIVE ACTIVITY Introduction Liver is a vital, central largest organ in the body which is actively involved in many metabolic function, detoxification, excretory, storage, haematological function to regulate the body and also the frequent target for a number of toxicants. Human beings are exposed on a daily basis to certain toxic chemicals and pathogens, which cause serious health problems. As a result of the chemical reactions, the liver may generate several reactive species like free radicals. These reactive species forms covalent bond with the lipids of the tissue. Free radical initiated auto oxidation of cellular membrane lipids can lead to cellular necrosis and is now accepted to be important in connection with a variety of pathological conditions [74] such as heart disease, diabetes, gout and cancer [75]. However inbuilt protective mechanisms combat the hazardous reactions associated with the free radicals. Due to excessive exposure to hazardous chemicals, the free radicals generated will be so high such that they overpower the natural defensive system leading to hepatic damage and cause jaundice, cirrhosis and fatty liver, which remains as one of the serious health problems [76]. Reduction of these radicals by antioxidant molecules is crucial to the protection of cells against hepatic damage and various disorders. A range of conditions which can prevent the liver from performing its vital functions includes fat accumulation, alcohol misuse, viral infection, iron or copper accumulation, toxic damage and cancer. Among this the major toxic substance is carbon tetrachloride which cause acute or chronic liver disease. It is the widely accepted model by most of the research scientist for inducing liver damage. Toxicant has been found to induce extensive liver damage within a period of 24 hrs following the intra peritoneal administration [77].

6 113 Symplocos cochinchinensis (Lour.) is traditionally used to treat liver disorders. The present investigation is aimed to study about the hepatoprotective and in-vivo anti oxidant activity of the methanolic extract of the leaves against carbon tetrachloride induced hepatotoxicity in rats Materials and Methods Experimental animals 30 male Albino rats of Wistar strain weighing gms were used for the study. They were maintained under standard environmental condition (temperature C and 12hrs light/dark cycle) and they were allowed with standard laboratory feed and water ad libitum. The animals were given a week s time to get acclimatized with the laboratory condition. Initial body weights of all the animals were recorded. Procedure Experimental design [78] A total of 30 Albino rats were divided into 5 groups of 6 animals each. Group I : Vehicle control, received orally normal saline 5ml/kg body weight daily for 9 days. Group II : Carbon tetra chloride control (0.1ml/kg) Group III Group IV : Served as test and received methanolic extract of leaves (200mg/kg p.o) daily for 9 days : Served as test and received methanolic extract of leaves (400mg/kg p.o) daily for 9 days Group V : Animals served as standard received Silymarin(25mg/kg) oral [79] for 9 days.

7 114 On day 7 and 9, carbon tetra chloride 0.1ml/kg/day [80] of body weight i.p was given to all rats except the rats in group I. After 24 hours of the second dose of CCl 4, blood was collected from all the animals by retro-orbital plexus, since many of the biochemical and histological changes are known to manifest after 24 hours of CCl 4 administration [81]. The serum was separated by centrifugation at 2000 rpm for 10min and used for the assay of hepatic marker enzymes like Serum Glutamate Oxaloacetate Transaminase(SGOT), Serum Glutamate Pyruvate Transaminase(SGPT), Alkaline phosphatase(alp), Bilirubin and Total Protein. The SGOT, SGPT, serum alkaline phosphatase (ALP) and bilirubin were determined by using commercially available kits. Serum total protein was measured according to the method of Lowry [82]. After the collection of blood samples the rats in different groups were sacrificed and their livers were excised immediately and washed in icecold normal saline followed by 0.15M Tris-HCl (ph 7.4) blotted dry and weighed. A 10% w/v of homogenate was prepared in 0.15M Tris-HCl buffer and processed for the estimation of lipid peroxidation[83]. A part of homogenate after precipitating proteins with trichloro acetic acid (TCA) was used for the estimation of glutathione. The rest of the homogenate was centrifuged at rpm for 15min at 4 0 C. The supernatant thus obtained was used for the estimation of superoxide dismutase (SOD) and Catalase (CAT) activities. Estimation of lipid peroxidation Lipid peroxidation is commonly regarded as deleterious process [84] leading to structural modification of complex lipid protein assemblies, such as bio membranes and lipoproteins and is usually associated with cellular malfunction. During lipid per oxidation a polar oxygen moiety is introduced in the hydrophobic tails of unsaturated fatty acids. This process is

8 115 of dual consequence: the presence of hydroperoxy groups disturbs the hydrophobic lipid/lipid and lipid/protein interactions which leads to structural alteration of biomembranes and lipoproteins. Hydroperoxy lipids are the source for the formation of free radicals when free radicals are generated they can attack poly unsaturated fatty acid in cell membrane leading to a chain of chemical reaction called lipid peroxidation. As the fatty acid is broken down, the hydrocarbon gases and aldehyde are formed. The most common method used to asses malonodialdehyde (MDA) is the thiobarbituric acid assay(tbars) assay[85]. Reagents 20% Tri chloro acetic acid solution in water, 0.12 M thiobarbituric acid which is prepared freshly before use, Standard Solution: tetramethoxy propane (100 nmoles/ml) Procedure To 0.1 ml of the liver homogenate, 2.0 ml of 20% TCA was added. The contents were mixed well and centrifuged at 4000 rpm for 20 minutes. 2.0 ml of the supernatant was mixed with 2.0 ml of thiobarbituric acid reagent. Reagent blank and standards (5-20 nmoles) were also treated similarly. The contents were heated for 20 min in a boiling water bath. The tubes were cooled to room temperature and the absorbance was read at 532 nm in a Shimadzu UV Visible double beam spectrophotometer. The lipid peroxide content was expressed as moles MDA per 100 mg of protein. Catalase (CAT) Catalase is an enzyme present in the cells of plants, animals and aerobic bacteria[87]. Catalase is located in a cell organelle called peroxisome.

9 116 In animals catalase is present in all major body organs. The role of catalase is to scavenge hydrogen peroxide and prevent oxidative damage in the cell. Catalase is a haem containing protein that can convert hydrogen peroxide to water and oxygen in a two step reaction cycle. In the first step, one molecule of hydrogen peroxide is converted to water. The catalytic cycle begins with the oxidation of ferric haem by hydrogen peroxide to form the ferryl-oxo porphyrin/protein radical as intermediate. The catalase cycle is completed by the reduction of compound I to the ferric enzyme by hydrogen peroxide, resulting in the production of molecular oxygen [88]. Reagents Phosphate buffer (0.05 M, ph 7.0), Hydrogen Peroxide (5.0 mm) Procedure 3.0 ml of reaction mixture containing 1.9 ml buffer(ph 7.0), 1.0 ml of the substrate and 0.1 ml of diluted enzyme were used in this assay. The activity was measured as the change at 240 nm at 30 sec interval for 3min in a Shimadzu UV 160A UV Visible Recording Spectrophotometer. The activity was expressed as µmole of H 2 O 2 consumed/min/mg of protein [89]. Estimation of superoxide dismutase(sod) It is one of the most effective anti oxidant intracellular enzyme. In 1969 McCord and Fridivich proved the antioxidant activity of SOD [90]. It exist in several isoforms differing in the nature of active metal centre and aminoacid constituency, as well as their number of subunits, cofactors and other features. In human there are three forms of SOD namely cytosolic Copper-zinc SOD and mitochondrial Mn-SOD and extracellular SOD (EC- SOD). SOD destroys oxygen with remarkably high reaction rates by successive oxidation [87].

10 117 Under physiological conditions, a balance exists between the level of reactive oxygen species (ROS) produced during normal cellular metabolism and the level of endogenous antioxidants which serve to protect tissue from oxidative damage. Disturbance of this balance (either through increased production of RSO or decreased levels of antioxidants) produce a condition known as oxidative stress and leads to variety of pathological conditions. SOD removes damaging ROS from the cellular environment by catalyzing the dimutaion of two superoxide radicals to hydrogen peroxide and oxygen[91]. O O 2 SOD 2H + O 2 + H 2 O SOD estimation was therefore carried out for its ablity to inhibit spontaneous oxidation of epinephrine and adrenochrome. Procedure Sodium carbonate buffer (2.8ml 0.05mM) and 0.1 ml of tissue homogenate or sucrose (blank) was incubated at 30 O C for 45min. The absorbance was then adjusted to 0 to sample. The reaction was then initiated by adding 10 L of adrenaline solution (9mM). The change in the absorbance was measured at 480nm for 8-12min and the temperature was maintained at 30 O C throughout the assay. The results were expressed as unit (U) of SOD activity/mg of tissue [92]. Reduced Glutathione (GSH) 0.5ml of tissue homogenate, 20% TCA was added and precipitated. The contents were mixed well for complete precipitation of proteins and centrifuged. To an aliquot of clear supernatant 2ml of 5,5 -dithiobis [2- nitrobenzoic acid] (DTNB) reagent and 0.2ml buffer were added to make final volume 4ml. The absornace was measured at 412nm against blank contains TCA instead of sample. Amount of Glutathoine is expressed as nanomoles of GSH oxidized/mg of protein

11 118 Histopathological examination: The blood was collected by retro-orbital plexus and liver was removed, sliced and washed in saline. Liver pieces were preserved in 10% formasal (10% formaldehyde diluted with normal saline) for histopathological study. The piece of liver was processed and embedded in paraffin wax. Sections were made about 4-6 µm in thickness. They were stained with haematoxylin and eosin, mounted and observed under light microscope for histological changes Statistical Analysis The data were expressed as Mean ± SEM, statistical analysis was performed by one way ANOVA followed by Tukey-Kramer multiple comparison test, p values <0.05 were considered as significant. Highest significant difference test was performed with Graph pad instat software Results The effects of methanolic extract on liver weight, SGOT, SGPT, ALP, bilirubin and total protein levels in carbon tetra chloride induced liver damage in rats were summarized in Table 7.1 and Figures 7.2, 7.3. Administration of CCl 4 (0.1ml/kg b.wt), after 24 hrs of intoxication resulted in a significant elevation of serum marker enzymes SGPT, SGOT, ALP and Bilirubin and reduced protein content when compared with normal control. On administration of the methanolic extract of the leaves of Symplocos cochinchinensis (Lour.) and Silymarin(25mg/kg) the levels of the enzymes were found to retrieving towards normal level. The effect of extract on lipid peroxidation, SOD, glutathione content and catalase activity were shown in Table 7.2 and Figures 7.4, 7.5. Lipid peroxidation level was significantly (p<0.001) increased and SOD,

12 119 glutathione level as well as catalase activity were significantly (p<0.001) decreased in CCl 4 intoxicated rats when compared with those of the animals in normal control group. Rats treated with methanolic extract at the doses of 200 and 400 mg/kg significantly decreased the elevated lipid peroxide levels and restored the altered glutathione level and catalase activity towards the normal levels in a dose dependent manner. The results are well comparable with Silymarin (standard drug) treated group. Histopathological study of liver The vehicle control group showed normal cellular architecture with distinct hepatic cells, sinusoidal spaces and central vein [Figure.7.6(a)]. In the carbon tetra chloride treated groups disarrangement of normal hepatocytes with focal fatty changes vacuolization of cytoplasm, ballooning degeneration and some of the cells are seemed swollen and also centrilobular necrosis were seen [Figure 7.6(b)]. The methanolic extract (200, 400mg/kg) treated groups showed sign of protection as it was evident by the absence of necrosis and vacuoles, clearing of cytoplasm [Figure 7.6(c)]. Methanolic extract (400mg/kg) pretreatment showed the protection to maximum extent of hepatocytes from damage induced by CCl 4, with trace fatty changes in the hepatic parenchymal cells, lack of nectrosis and necrosis [Figure 7.6 (d)]. The standard treated group showed sign of protection, absence of degeneration of normal hepatocyte [Figure 7.6(e)].

13 Table 7.1 Hepatoprotective activity of the leaves of of Symplocos cochinchinensis ( Lour.) on CCl 4 induced liver S.No damage in rat Groups Liver weight/100gm b.w SGOT (IU/L) SGPT (IU/L) ALP (IU/L) Bilirubin (mg%) Total protein (gm%) 1. Control 3.8± ± ± ± ± ± CCl 4 Control 6.8 ± 0.28 a 182.5±1.33 a ±0.98 a ±1.74 a 2.17± 0.04 a 6.3±0.16 a Methanol extract (200mg/kg) Methanol extract (400mg/kg) 5.3 ±.0.2 a 80.5± 1.63 a ±1.48 a 224.0±1.82 a 0.69 ±0.03 a 7.5 ± 0.11 a 4.9 ± 0.32 a 55.0 ±1.08 a ±1.18 a 198.0±3.55 a 0.57 ±0.01 a 8.4 ± 0.11 a 5. Silymarin (25mg/kg) 4.2 ± 0.30 a c 46.0 ±1.14 ac ±2.45 a 193.5±1.52 ac 0.44 ±0.03 ac 8.8±0.13 ab Values are expressed as mean ± SEM n=6, a p < 0.001, when compared to control and CCl b 4 p < 0.01 when compared with CCl 4 induced control 0.05 compared to control Data were analyzed by using one way ANOVA followed by Tukey- Kramer multiple comparison test c p < 120

14 Vehicle Control Carbon tetra chloride treated Methanol Extract(200mg/kg) Methanol Extract(400mg/kg) Silymarin (25mg/kg) SGOT(IU/L) SGPT(IU/L) ALP(IU/L) Figure 7.2 Effect of methanolic extract on SGOT, SGPT and ALP in CCl 4 induced hepatotoxicity in rats Vehicle Control Carbon tetra chloride treated Methanol Extract(200mg/kg) Methanol Extract(400mg/kg) Silymarin (25mg/kg) Liver weight Total Protein (g/dl) Bilirubin (mg/dl) Figure 7.3 Effect of methanolic extract on Liver weight, total protein and Bilirubin in CCl 4 induced hepatotoxicity in rats

15 Table 7.2 Effect of methanolic extract of the leaves of of Symplocos cochinchinensis (Lour. ) on antioxidants levels in CCl 4 -induced hepatotoxicity in rats S.No Treatment LPO (n mole of MDA/mg protein) SOD(µg/mg protein) CAT (µg/mg protein) Glutothione (µg/mg protein) 1. Control 0.88± ± ± ± CCl 4 Control 9.02 ± 0.53 a 48.17±1.86 a ±1.54 a 0.72 ±0.03 a 3. Methanol extract (200mg/kg) 4. Methanol extract (400mg/kg) 5.97 ± 0.52 ab 63.67±1.61 ab ±1.43 ab 1.98± 0.24 ab 3.92 ± 0.05 ab 70.17±1.89 ab 299.0±2.28 ab 3.31±0.19 ab 5. Silymarin (25mg/kg) 2.27 ± 0.48 ab 86.6±1.15 nsb 352.8±3.54 ns b 5.45±0.12 ns b n = 6; Values are expressed as mean ± SEM a P< 0.001; Vs Control b P< 0.001; Vs CCl 4 ns- non significant Data were analyzed by using one way ANOVA followed by Tukey - Kramer multiple comparison test. 122

16 Vehicle Control Carbon tetra chloride treated Methanol Extract(200mg/kg) Methanol Extract(400mg/kg) Silymarin (25mg/kg) LPO Glutothione Figure 7.4 Effect of methanolic extract on LPO and Glutothione in CCl 4 induced hepatotoxicity in rats Vehicle Control Carbon tetra chloride treated Methanol Extract(200mg/kg) Methanol Extract(400mg/kg) Silymarin (25mg/kg) SOD CAT Figure 7.5 Effect of methanolic extract on SOD and CAT in CCl 4 induced hepatotoxicity in rats

17 124 (a) Control showing normal hepatocytes (b) CCl 4 treated Hepatotoxin showing ballooning degeneration, vacuolization of cytoplasm (c) Methanol extract (200mg/kg) showing sign of protection, feathery degeneration, normal hepatocytes (d) Methanol extract treated (400mg/kg) showing absence of degeneration, slight fatty change (e) Standard treated showing sign of protection with normal hepatocytes Figure 7.6 Histopathological observation from hepatoprotective model

18 Discussion The liver damage in CCl 4 induced hepatotoxicity is mainly assessed by determining the serum enzyme levels. Liver is considered to be highly sensitive to toxic agents. CCl 4 administration causes necrosis or membrane damage of liver thereby release of enzymes into the circulation which can be determined by using serum. In the present study, it was observed that the animals treated with CCl 4 resulted in significant hepatic damage as shown by the elevated levels of marker enzymes. These changes in the marker level will reflect in hepatic structural integrity. The rise in the SGPT plays a vital role in the conversion of alanine to pyruvate and glutamate (amino acids to ketoacid) [93]. The pre treatment with extract and standard significantly attenuated the elevated levels of the serum markers. SGPT is more specific to liver and is thus a better parameter for detecting the liver injury [94]. The normalization of the serum markers by the extract suggests that they are able to condition the hepatocytes so as to protect the membrane integrity against CCl 4 induced leakage of marker enzymes into the circulation[95]. Serum ALP and bilirubin levels on the other hand are related to the function of hepatic cells. Increase in serum level of ALP is due to increased synthesis in presence of increasing biliary pressure [96]. Our findings suggest that the treatment with extract caused significant inhibition of ALP and bilirubin levels. Liver cell injury induced by CCl 4 involves the biotransformation of the toxin carbon tetrachloride by cytochrome P-450 to produce the trichloromethyl free radical, which causes peroxidative degradation in the adipose tissue resulting in fatty infiltration of the hepatocytes. Trichloromethyl free

19 126 radicals elicit lipid peroxidation of membrane lipids in the presence of oxygen generated by metabolic leakage from mitochondria. All these events culminate in loss of integrity of the cell membranes and damage of hepatic tissue [97]. Treatment with extract of the leaves significantly reduced the elevated liver enzymes and bilirubin level, indicating the hepatoprotective action. The production of reactive oxygen species due to the attack of CCl 4 leads to membrane damage, alteration in the structure and function of cellular membrane. In CCl 4 induced group the level of LPO is increased and the levels of catalses, SOD were decreased due to the liver damage caused by the high oxidative stress produced by CCl 4. The increased level of lipid peroxidation is due to tissue damage and failure of antioxidant defense mechanism to prevent formation of excessive free radicals. SOD has been reported as one of the most important enzymes in the enzymatic antioxidant defense system, decrease in enzyme activity of superoxide dismutase is a sensitive index in hepatocellular damage and is the most sensitive enzymatic index in liver injury[98]. It scavenges the superoxide anion to form hydrogen peroxide and thus diminishing the toxic effect caused by this radical. Catalase (CAT) found in all animal tissues is an enzymatic antioxidant and the highest activity is found in the red cells and liver. CAT protects the tissues from highly reactive hydroxyl radicals by decomposing the hydrogen peroxide [99]. Therefore reduction in the activity of CAT may result in a number of deleterious effects due to the assimilation of superoxide radical and hydrogen peroxide.

20 127 The non- enzymatic antioxidant glutathione present in the liver is one of the most abundant tripeptide. It is substrate for glutathione peroxidase (GPx) [100]. It removes free radical species such as hydrogen peroxide, superoxide radicals and maintains membrane protein thiols. The decreased level of GSH is associated with an enhanced lipid peroxidation in CCl 4 treated rats. Treatment with methanolic extract of Symplocos cochinchinensis (Lour.) significantly reversed these changes in dose dependent manner and the results are comparable with that of the Silymarin treated group. Hence the mechanism of hepatoprotection of the extract may be due to its antioxidant effect. Histopathological examination of the liver section of the rats treated with toxicant showed intense centrilobular necrosis and vascuolisation. The rats treated with extracts along with toxicant showed sign of protection against these toxicants to considerable extent as evident from formation of normal hepatic cards and absence of necrosis. Hepatotoxic effect of carbon tetrachloride is due to oxidative damage by free radical generation and antioxidant property is claimed to be one of the mechanisms of hepatoprotective activity of Symplocos cochinchinensis (Lour.). The co-administration of extract may induce the hepatocytes to resist the toxic effects of carbon tetrachloride. The results indicate that the methanolic extract at a dose level of 400mg/kg possess significant hepatoprotective activity in dose dependent manner.

21 ANTI-INFLAMMATORY ACTIVITY Introduction Inflammation is a local response of living mammalian tissues to the injury. It is a defence reaction of body in order to eliminate or limit the spread of injurious agents. The inflammatory response involves a complex array of enzyme activation, mediator release, fluid extravasations, cell migration, tissue breakdown and repair [101] which are aimed at host defence and usually activated in most disease condition. Oedema formation, leukocyte infiltration and granuloma formation represent such components of inflammation [102]. Oedema formation in the paw is because of a synergism between various inflammatory mediators that increase vascular permeability and the mediators that increase blood flow [103]. Narcotics e.g. opioids or non-narcotics e.g. salicylates and corticosteroids e.g. hydrocortisone are the drugs which are in use presently for the management of pain and inflammatory conditions. These drugs are very expensive to develop and possess well known side and toxic effects. On the contrary many medicines of plant origin had been used since long time without any adverse effects [104]. 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 in amplifying the disease process. Rat paw oedema is the most commonly used model for acute inflammation and carrageenaninduced paw oedema is widely used for determining the acute phase of inflammation. In this work the methanolic extract of Symplocos cochinchinensis (Lour.) was studied for its in-vivo anti-inflammatory activity using caragennan induced rat paw oedema model.

22 Materials and Methods Animals Wistar male Albino rats weighing gms were used for the in-vivo anti-inflammatory studies. The animals were housed under standard conditions of temperature (23 0 C ± 1 0 C), 12hrs light/dark cycle and fed with water ad libitium. Before performing the experiment the ethical clearance was obtained from institutional animal ethics committee. Procedure [105] The anti-inflammatory activity was studied for the methanolic extract by carrageenan induced paw oedema model in rats. The Wistar Albino rats were divided into 4 groups of 6 animals each. Group I served as control, Group II, III were given with methanolic extract at the dose of 200 and 400 mg/kg and Group IV was served as standard (Diclofenac 50 mg/kg). Carrageenan was injected into the sub planter aponeurosis of the right hind paw of rats. An hour before carrageenan injection the animals were given with the extract and standard orally. The paw volumes were measured before and three hours after carrageenan administration by volume displacement method. The percentage inhibition of oedema was calculated for each group with respect to its vehicle treated control group Statistical Analysis The data s were expressed as mean ± SEM, statistical analysis was performed by one way ANOVA followed by Tukey-Kramer multiple comparison test, p values <0.05 were considered as significant. Highest significant difference test performed with Graph pad instat software.

23 Results The methanolic extract of the leaves was selected for the evaluation of in- vivo anti-inflammatory activity by carrageenan induced paw oedema model in rats. The extract showed significant anti-inflammatory activity (53%) at the dose of 400 mg/kg while the standard Diclofenac showed 75% inhibition of oedema. The results are tabulated in Table 7.3 and Figure 7.7. Table 7.3 Anti-inflammatory activity of the methanolic extract of the leaves of Symplocos cochinchinensis (Lour.) S.No. Treatment Dose(mg/kg) Oedema volume 1. Control ± Methanolic extract ± 0.029*** Methanolic extract ±0.012*** % inhibition 4. Diclofenac ±0.001*** 75 Values are expressed as mean ± SEM n=6, ***p < 0.001, when compared to control. Data were analyzed by using one way ANOVA followed by Tukey- Kramer multiple comparison test (%) Inhibition Vehicle Control Methanol Extract(200mg/kg) Methanol Extract(400mg/kg) Diclofenac(50mg/kg) Figure 7.7 Effect of methanolic extract on anti-inflammatory activity

24 Discussion The present study was aimed to evaluate the anti-inflammatory activity of methanolic extract so as to exploit it as potent anti-inflammatory agent. The enzyme, phospholipase A2, is known to be responsible for the formation of mediators of inflammation such as prostaglandins and leukotrienes which by attracting polymerphonuclear leucocytes to the site of inflammation which would lead to tissue damage probably by the release of free radicals. Phospholipase A2 converts phospholipids in the cell membrane into arachidonic acid, which is highly reactive and is rapidly metabolized by cyclo oxygenase (prostaglandin synthesis) to prostaglandins, which are major components that induce pain and inflammation[106,107]. It is well known that carrageenan induced paw oedema is characterized by biphasic event with involvement of different inflammatory mediators. In the first phase (during the first 2 h after carrageenan injection), chemical mediators such as histamine and serotonin play a role, while in second phase (3 4 h after carrageenan injection) kinin and prostaglandins are involved[108]. There is increasing evidence that lysosomal enzymes play an important role in the development of acute and chronic inflammation. 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 events responsible for the inflammatory process [109].Our results revealed that administration of methanolic extract inhibited the oedema which is probably due to the inhibition of different aspects and chemical mediators of inflammation. So, the extract might be acting by either inhibiting the lysosomal enzymes or stabilizing the membrane. From the above studies it is quite apparent that the methanolic extract possesses significant anti-inflammatory activity. The study justifies its use in inflammation as suggested in the folklore medicines.

25 ANTI-SNAKE VENOM ACTIVITY Introduction Deaths due to snake venomation have been a major health problem since time immemorial. Snakebite is a major health hazard that leads to high mortality rate especially in India. The common poisonous snakes found in India are Cobra (Naja naja), Krait (Bangarus Caeruleus), Russell s viper (Daboia russelli) and Saw scaled viper (Echis Carinatus) [110]. About 35,000 to 50,000 people die of snakebite every year in India. Most of the venoms are neurotoxic in nature, considered to attack victim s central nervous system and usually results in heart failure. Antivenom immunotherapy is the only specific treatment against snake venomation. There are various side effects of antivenom such as anaphylactic shock, pyrogen reaction and serum sickness and its development from animal source is time consuming and expensive. Most of these symptoms may be due to the action of high concentrations of non-immunoglobulin proteins present in commercially available hyper immune antivenom [111]. This necessiates the search for antidotes from herbal sources without these side effects. Over the years many attempts have been made for the development of snake venom antagonists especially from plant sources. Although, use of plants against the effects of snake bites has been long recognized, more scientific attention has been given since last 20 years [112]. Several medicinal plants, which appear in old drug recipes or which have been passed on by oral tradition, are believed to be snakebite antidotes. These plants or their extracts or their decoctions or chewing leaves or topical application of its sap on to the bite area are some important procedures to counteract snake venom activity. In most cases the efficiency of this treatment regimen is unproven compared to the numerous folklore claims, the pharmacological and clinical investigations done on this same are meager[113].

26 Materials and Method Snake venom Lyophilized snake venom of Russell s viper (Daboia russelli) was obtained from CSIR Centre for Biochemical s, New Delhi and preserved at 4 0 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. Animals Swiss Albino mice weighing between 20-25gms were used for the study. They were maintained under standard environmental condition (temperature C and 12h light/dark cycle) and they were allowed with standard laboratory feed and water ad libitum. The animals were given a week s time to get acclimatized with the laboratory condition. Ethical clearance for the use of animals was obtained from the committee constituted for the purpose. In- vivo anti- snake venom activity Twenty four adult Swiss Albino mice of both sex were divided into four groups of six mice each. The control group was injected with only venom (lethal dose 61mcg/20g of the mice, i.p)[114], while the other groups were treated separately with venom, after 5 min of oral administration of anti snake venom serum (10mg/kg) and methanolic extract(200, 400mg/kg) respectively. The mice were observed for 24 hours for the number of mice which were survived.

27 Statistical Analysis The data s were expressed as mean ± SEM, statistical analysis was performed by one way ANOVA followed by Tukey-Kramer multiple comparison test, p values <0.05 were considered as significant. Highest significant difference test performed with Graph pad instat software Results In-vivo anti-snake venom activity of the methanolic extract was studied at two different dose levels of 200 and 400mg/kg body weight respectively. In control group, which was treated with only venom (61mcg/20g) no mice were survived. Mice treated with 200 and 400 mg/kg of extract recorded 66 and 83% survival and the standard showed 100% survival which was shown in the Table 7.4 and Figure 7.8. Table 7.4 Anti-snake venom activity of the methanolic extract of the leaves of Symplocos cochinchinensis (Lour.) S.No. Treatment Dose(mg/kg) % survival 1. Control Methanolic extract Methanolic extract Snake venom anti serum

28 Vehicle Control Methanol Extract(200mg/kg) Methanol Extract(400mg/kg) Snake venom antiserum (%) Survival Figure 7.8 Effect of methanolic extract on anti-snake venom activity Discussion The methanolic extract was screened for in-vivo anti-snake venom activity. The extract at 400mg/kg increased the percentage survival which was comparable to that of standard anti venom serum. It was observed that the survival of the mice increased progressively with increasing the dose of the extract in a dose dependant manner. The snake venom acts at peripheral neuromuscular junction either post synaptically by binding competitively with the acetylcholine receptor or presynaptically by preventing the release of acetylcholine transmitter from the nerve terminals. The probable mechanism of preventing the neurotoxic effect by Symplocos cochinchinensis (Lour.) may be by interfering with the acetylcholine receptor sites i.e. by the action of the neurotoxic substances in the venom at the acetylcholine receptor sites[115].

29 ANTI-CANCER ACTIVITY Introduction Cancer continues to represent the largest cause of mortality in the world and claims over 6 million lives every year [116]. An extremely promising strategy for cancer prevention today is chemoprevention, which is defined as the use of synthetic or natural agents (alone or combination) to block the development of cancer in humans. It is well recognized that allopathic drugs are not without danger as they produce serious side effects to the normal cells. The chemo therapeutic agents causes non-selective damage to the normal cells, therefore world wide research is now focussing on the investigation of best antitumor agents from various sources. Plants, vegetables and herbs used in the folk and traditional medicine have been accepted currently as one of the main source of cancer chemoprevention drug discovery and development. Around 60% of currently used anti-cancer agents are derived in one way or another from natural sources, including plants, marine organisms and micro-organisms. Molecules derived from natural sources (so-called natural products have played and continue to play a dominant role in the discovery of leads for the development of conventional drugs for the treatment of most human diseases. [117]. Experimental cancer models in animals employing transplantable tumors as well as chemical induced carcinogenesis which maybe used to evaluate the anticancer potential. Although many plants are used to treat tumors in Indian traditional systems of medicine, most of these plants are not scientifically evaluated. If a systematic and intensive ethno pharmacological study is carried out one or more plants used in various ethno medical practices are sure to provide effective anticancer drugs [118].

30 137 Since Symplocos cochinchinensis (Lour.) is traditionally used to treat tumors, the present study was aimed to investigate the anticancer property of the methanolic extract of the leaves of the plant using DAL(Dalton s ascitic lymphoma) cell lines Materials and Method Animals Swiss Albino mice weighing between 20-25gms were used for the study. They were maintained under standard environmental condition (temperature C and 12hrs light/dark cycle) and they were allowed with standard laboratory feed and water ad libitum. The animals were given a week s time to get acclimatized with the laboratory condition. Tumor cell lines Dalton s ascitic lymphoma (DAL) cells were obtained by the courtesy of Cancer Research Centre, Adyar, Chennai. They were maintained by weekly intra-peritoneal inoculation of 10 6 cells/ mouse. Anti-cancer activity After acclimatization, mature male Swiss Albino mice were divided into four groups (n=10). All the groups except group I were inoculated (i.p.) with DAL Cells of 1 x 10 6 cells/mouse weekly [119]. The day of DAL administration was considered as day 0.Group I served as control and was given with distilled water for 14 days, group II served as DAL control group and was inoculated (i.p.) with DAL Cells of 1 x 10 6 cells/mouse without drug treatment. Group III & IV Animals received methanolic extract (200 and 400 mg/kg) p.o for 14 consecutive days.

31 138 On day 15, five mice of each group were sacrificed 24hrs after the last dose and the rest were kept with food and water ad libitum to check the increase in the life span of the tumor hosts. The effect of methanolic extract on tumor growth and host s survival time were examined by studying the parameters like tumor volume, tumor cell count, viable tumor cell count, nonviable tumor cell count, mean survival time and increase in life span [120]. Determination of tumor volume The fluid was collected from the peritoneal cavity of the mice. The volume was measured by taking it in a graduated centrifuge tube and packed cell volume was determined by centrifuging at 1000rpm for 5 min. Determination of tumor cell count The fluid was taken in a RBC pipette and diluted to 1000 times. A drop of the diluted cell suspension was placed on the Neubauer s counting chamber and the number of cells in 64 small squares were counted. Estimation of viable tumor cell count The cells were then stained with Trypan blue (0.4% in normal saline) dye. The cells that did not take up the dye were considered viable and the stained as nonviable. They were counted and the viable and non viable cell count was estimated as follows [121] Cell count = (No. of cells x Dilution) / (Area x Thickness of liquid film) (7.1) Percentage increase in life span Percentage increase in life span was calculated by observing the mortality and the effect of extract on tumor growth. The mean survival time (MST) of each group consisting of 6 mice were noted [122].

32 139 Haematological studies Blood was drawn from each mouse under sterilised condition and the red blood cell (RBC) count, haemoglobin content (Hb) and white blood cell (WBC) count were determined using cell diluting fluids and a haemocytometer. Differential cell (DC) count was carried out using Leishman stained blood smears. Serum protein concentration was estimated by Lowry s method and packed cell volume (PCV) was determined by the method described by Docie et al [123] Statistical Analysis The data s were expressed as mean ± SEM, statistical analysis was performed by one way ANOVA followed by Tukey-Kramer multiple comparison test, p values <0.05 were considered as significant. Highest significant difference test performed with Graph pad instat software Results In the tumor control mice the haematological parameters on day 15 were found to be significantly altered when compared to normal group. The total WBC count, protein and PCV were found to be increased with a reduction of the haemoglobin and RBC. In the differential count of WBC, the percentage of neutrophils increased while the lymphocyte count decreased. The extract treated group showed significant effect on increasing life span and retrieving all the parameters to normal level. Effect of extract on mean survival time and tumor growth In the DAL control group the mean survival time was 12 ± 1.5days, while it increased to 28.2 ± 1.34 (200mg/kg) and 32.2±1.67 (400mg/kg) for

33 140 extract treated groups (p<0.001). The increase in life span of tumor bearing mice treated with extract (200 and 400mg/kg) was found to be 44.5 and 64.8% respectively. Treatment with extract reduced the tumor volume, packed cell volume, and viable tumor cell count in a dose-dependent manner as compared to that of DAL control group. Further, nonviable tumor cell count was increased when compared with the DAL control [Table 7.5 and Figures 7.9 and 7.10]. Effect of extract on haematological parameters Haemoglobin content and RBC count in the DAL control group was decreased when compared to normal group. The haemoglobin content for DAL treated control was found to be 6.3 ± 0.5 and treatment with methanolic extract at the dose of 200 and 400 mg/kg increased the content to 9.2 ± 0.3 and 11.4 ±0.7(p<0.01). The RBC count was found to be increased to more or less normal level for extract treated group at 400mg/kg (p<0.001). The total WBC counts and protein were found to be increased in DAL control group 15.1±0.5 and 16.7±0.8 when compared with normal group. Administration of extract in DAL bearing mice reduced both WBC count and protein when compared with DAL control. In the differential count of WBC, increase of neutrophils and the lymphocyte count decreased in DAL control group. Treatment with extract at different doses changed these altered parameters more or less normal [Table 7.6 and Figures 7.11 and 7.12]..

34 Table 7.5 Effect of methanolic extract on survival time, life span, tumor volume, viable and non-viable cell count in DAL bearing mice S.No Treatment Survival time (Days) 1. Vehicle control (5ml/kg p.o) 2. DAL control (x10 6 cells/ml) 3. Methanol extract 200mg/kg 4. Methanol extract 400mg/kg Increase of life span Tumor volume (ml) Viable cell count x 10 6 cells/ml Non-viable cell count x 10 6 cells/ml ± 1.5 * ±0.25 * 9.7 ±0.32 * 1.7 ±0.74 * * 18.2 ± 1.34 * * ±0.61 * 3.1 ±0.45 * 2.6±0.25 * * 28.2 ± 1.34 * * ±0.851 * 2.4 ±0.32 * 2.8 ±0.43 * * Values are mean+ SEM expressed as (n=6) p*<0.05; **<0.01; ***<0.001; as compared with group II. P< 0.05 considered significant 141

35 Figure 7.9 Vehicle Control DAL control Methanol Effect of methanolic extract on survival time and increase of life span Vehicle Control DAL control Methanol Extract(200mg/kg) Methanol Extract(400mg/kg) Tumor volume (ml) Viable cell count Non viable cell count Figure 7.10 Effect of methanolic extract on Tumor volume, Viable cell count and Non viable count

36 Table 7.6 Effect of methanolic extract on haematological parameters in DAL bearing mice S.No Treatment Hb (g/dl) 1. Vehicle control (5ml/kg p.o) 2. DAL control (x10 6 cells/ml) 3. Methanol extract 200mg/kg RBC (106cells/mm3) WBC (103 cells/mm3 Total Protein (g/dl) PCV (mm) Differential cell count (%) Lymphocytes Neutrophils Basophils 14.3± ± ± ± ± ± ± ± ± 0.5* 2.3±0.2 * 15.1±0.5** 16.7±0.8** 27.4 ±0.2*** 33.4±0.7** 46.9±0.2** 3.7±0.7* 9.2 ± 0.3 * 3.8±0.7** 11.5±0.6** 12.1±0.4*** 21.6 ±0.9*** 49.5±0.3*** 39.2± ± Methanol extract 400mg/kg 11.4 ±0.7* 5.4±0.6*** 9.7 ±0.8 * 10.4±0.3*** 18.3 ±0.2*** 61.3±0.4*** 24.3±0.9** 2.7±0.4 Values are mean+ SEM expressed as (n=6) p*<0.05; **<0.01; ***<0.001; as compared with group II. P< 0.05 considered significant 143

37 Hb(g%) RBC WBC Total Protein PCV Vehicle Control Methanol Extract(200mg/kg) DAL control Methanol Extract(400mg/kg) Figure 7.11 Effect of methanolic extract on Hb, RBC, WBC, total Protein and PCV Vehicle Control DAL control Methanol Figure 7.12 Effect of methanolic extract on differential counts

38 Discussion The present study was carried out to evaluate the anti-cancer activity of methanolic extract (200 and 400mg/kg) of the leaves on DAL bearing mice. Reliable criteria for judging the value of any anti-cancer agent is the prolongation of life span of animals [124]. A decrease in tumor volume and viable tumor cell count as mentioned above finally reduced the tumor burden and enhanced the life span of DAL bearing mice. A significant increase in life span of animals was observed in the methanolic extract treated animals at the doses of 400 mg/kg, the tumor volume, packed cell volume, tumor cell count and also the haematological parameters were brought back to more or less normal levels. To evaluate the extract treatment indirectly inhibited the tumor cell growth, the effect of extract treatment was examined on the viable and nonviable cell counts against tumor bearing mice. Normally each mouse contains around intra peritoneal cells, 50% of which are macrophages. The extract treatment was found to enhance the non-viable cell count and decrease the viable cell count. It may be due to the absorption of extract by viable cells which leads the lysis of the cells through the activation of macrophages or some cytokinnin production in the peritoneal cavity. In cancer chemotherapy the major problem encountered are myelosuppression and anaemia [125,126]. The anaemia encountered in tumor bearing mice is mainly due to reduction in RBC or haemoglobin percentage and this may occur either due to iron deficiency or due to haemolytic or myelopathic conditions[127]. Treatment with extract brought back the haemoglobin content, RBC and WBC cell count near to normal values. This indicates that the methanolic extract possess a significant anti-cancer activity.