6 DISCUSSION. Plants and plant products have been used medicinally for various. ailments for years. WHO has estimated that about 36,000 plant

Transcription

1 168 6 DISCUSSION Plants and plant products have been used medicinally for various ailments for years. WHO has estimated that about 36,000 plant species are used worldwide for medical purposes out of the estimated 2,50,000 to 3,50,000 species identified so far, covering the health needs of more than 75 percent of the population. In developing countries the usage of herbal medicines is quite prevalent. Meanwhile in developed countries people are seeking alternate medical facilities as they are disillusioned with current health care. Harmful effects of most modern drugs and effectiveness of newly developed plant medicines due to improved science and technology has lead to resurgence of plant based remedies. In the present study, certain phytoconstituents such as glycosides, tannins, saponins, phytosterols, carbohydrates, steroids, flavonoids and phenolic compounds were detected during qualitative phytochemical screening of the successive leaf extractives of Rhodomyrtus tomentosa. Qualitative phytochemical screening and ethnobotanical survey on the successive leaf extractives of Mallotus philippensis revealed the presence of certain phytoconstituents such as glycoside, tannins, phenolic compounds, triterpenoids, flavonoids and carbohydrates. These phytoconstituents have diverse biological activities.

2 169 Free radicals are implicated in various pathological conditions like tissue injury, inflammatory conditions, neurodegenerative diseases, cancer and aging. The ability of Free radical scavenging compounds to ameliorate diseased conditions is appreciated. Thus the human body is protected by antioxidants against damage by reactive oxygen species [89]. Recently herbal products and synthetic drugs as antioxidant agents have received much attention. Many plant extractives and herbal products have been reported to possess significant antioxidant activity [90],[91]. The two plants selected for the present study possess several ethnomedical uses related to its antioxidant activity. Plants containing important phytoconstituents like phenolic compounds, tannins, flavonoids and terpenoids are known to possess antioxidant properties. Among the five leaf extractives (petroleum ether, chloroform, ethyl acetate, aqueous alcoholic (70%) and water) of Rhodomyrtus tomentosa tested for in vitro antioxidant activity, the aqueous alcoholic extractives and the ethyl acetate extractives exhibited potent antioxidant activity with low IC 50 values in the scavenging of hydrogen peroxide, nitric oxide, ABTS and DPPH radicals. The potent antioxidant activity of the aqueous alcoholic extractives may be due to the presence of phenolic compounds such as tannins and flavonoids.

3 170 Among the four leaf extractives (petroleum ether, chloroform, ethyl acetate, methanolic) of Mallotus philippensis tested for in vitro antioxidant activity, the methanolic extractives exhibited potent antioxidant activity with low IC 50 values in the scavenging of ABTS, DPPH, Nitric oxide and Hydrogen peroxide radicals. However, these IC 50 values were found to be higher or comparable with those obtained for the standards used. The potent antioxidant activity of the methanolic extractives may be due to the high phenolic content such as tannins. To check the safety profile of the extractives, it was subjected to acute toxicity study, which confirmed the absence of any toxicity or mortality at a higher dose of 2000 mg/kg. Thus the extractives can be classified in the safe drug category according to the Global harmonised Classification System quoted in the OECD guidelines 1996 [74]. The gastric anti ulcer activity of the five different extractives of Rhodomyrtus tomentosa and four different extractives of Mallotus philippensis were evaluated by ethanol induced gastric mucosal damage. The ethyl acetate, aqueous alcoholic and the water extractives of Rhodomyrtus tomentosa reduced the ethanol induced lesions in the gastric mucosa as was evident from the significant (p< 0.01) reduction in the ulcer index in comparison to the ulcerated control group. The results were comparable to Omeprazole, which

4 171 reduced the ulcer index significantly. Histopathological study of the aqueous alcoholic extractives confirmed the anti ulcer activity. The ethyl acetate and the methanolic extractives of Mallotus philippensis also reduced the lesions as was evident by a significant (p< 0.01) reduction in the ulcer index when compared with the control group. The results were comparable to Omeprazole (2 mg/kg b.w.) treated group of animals in which the ulcer index was reduced significantly. Histopathological study of the methanolic extractives confirmed the anti ulcer activity. Ethanol induced gastric mucosal damage is mediated by active oxygen and free radicals generated both exogenously and endogenously. Hydroxyl radicals interact with the cell membrane to produce conjugated dienes and lipid hydroperoxides, which are lipidderived free radicals. Thus, lipid peroxidation induced oxidative damage takes place. Ethanol treatment causes induction of oxidative stress intracellularly and leads to transition of mitochondrial permeability and depolarisation, which further leads to cell death in the gastric mucosa [92]. The active compounds of plants like flavonoids, terpenoids, tannins and phenolic compounds are regarded as possible active compounds against gastric lesions by acting as cytoprotectives or increasing antioxidant activity. Flavonoids could act by alteration of GSH metabolism or by quenching of reactive species. They could also

5 172 cause inhibition of Ca 2+ influx that signals the last step in cell death induced by glutamate [93]. Flavonoids, tannins and other phenolic compounds may have partially contributed to the cytoprotective effect of the extractives. Among the five different extractives tested for in vitro antioxidant activity by ethanol induced gastric Anti ulcer activity, the aqueous alcoholic (70%) extractives of Rhodomyrtus tomentosa and the methanolic extractives of Mallotus philippensis was found to be potent. Hence, it was selected for anti ulcer studies with Modified pyloric ligation, Forced swimming stress ulcer and acetic acid induced chronic ulcer models including in vivo antioxidant studies. Aspirin like synthetic NSAIDs are known to act as analgesics by inhibiting prostaglandin synthesis. This interference causes gastric mucosal damage and break up of the mucosal barrier by causing an increased secretion of acid and back diffusion of H + ions. An important parameter like ulcer inhibition is generally used for anti ulcer evaluation since this parameter has direct relation to gastrointestinal parameters like gastric volume reduction and acidity changes. With the increase in ph the ulcer score appeared less in the experimental animals. The reduction in free acid might have contributed to the ulcer protecting effect of the plant extractives [94]. Previous studies suggests that ulcers produced due to pyloric ligation may be due to the stimulating effect of gastrin, cholinergic agents and

6 173 vagal stimulation which causes histamine release and hence mediates gastric secretion [95]. The defense mechanism of the gastrointestinal mucosa against aggressive factors, such as hydrochloric acid, bile acid and nonsteroidal anti-inflammatory drugs, mainly consists of functional, humoral and neuronal factors. Functional factors include Mucousalkaline secretion and mucosal microcirculation, while prostaglandins and nitric oxide act as humoral factors, and capsaicin sensitive sensory neurons act as neuronal factors. These factors are known to contribute to mucosal protection [96]. Back diffusion of hydrochloric acid through the compromised defense causes destruction of the cells, capillaries and veins causing hemorrhagic ulcers. Histamine release furthering acid output also contributes to damage of the gastric mucosa. Presence of acid causes decrease of gastric ph which leads to activation of pepsinogen to pepsin, which causes a proteolytic action and increases the size of lesions. The reduction of acid secretion is necessary for healing and prevention of esophageal reflux disease [97]. Aspirin causes gastric mucosal damage by blocking prostaglandin synthesis, increasing acid secretion and back diffusion of H + ions. During pyloric ligation, the digestive effect of accumulated gastric juice and interference of gastric blood circulation are responsible for the induction of ulceration. Aspirin administered to pylorus-ligated

7 174 rats, further aggravated the acidity causing decreased resistance of gastric mucosa resulting in extensive damage to the glandular region of the stomach [98]. The gastric mucosal epithelial cells are impermeable to hydrogen ions and therefore form a physical barrier. This may have contributed to the mucosal defence mechanism as was evident from increased Carbohydrate/protein ratio and an increase in carbohydrates like hexose, hexosamine, fucose and sialic acid. From the results of anti secretory model it was found that both the extractives decreased the ulcer index in the doses used. The extractives decreased total carbohydrate, in the gastric juice compared with the ulcerated control group. On the other hand the extractives decreased the aggravating parameters such as gastric volume, total protein, total and free acidity in the gastric juice and increased the gastric ph. These findings suggested that the aqueous alcoholic (70%) extractives of Rhodomyrtus tomentosa and the methanolic extractives of Mallotus philippensis showed good anti ulcer activity in the acute aspirin plus pylorus ligated model. It is possible that the inhibitory effects of extractives are due, at least partly, to the presence of terpenoids. Terpenes were associated to be anti ulcerogenic activity in other plants. The mechanism by which triterpenes act as antiulcerogens may be, due to the activation of cellular protection, reduction of mucosal prostaglandins metabolism, cytoprotective action and reduction of gastric vascular permeability [99].

8 175 Acetic acid induced chronic ulcer model is similar to ulcers seen in human chronic ulcers and is most commonly used for healing study [100]. Free radical generation and induction of gastric obstruction and hyperacidity of the gastric juice by acetic acid produces chronic ulcers in rats and delays healing. Reactive oxygen species plays a role in the etiology and pathophysiology of inflammation and gastroduodenal ulcers [92]. From the data of chronic acetic acid induced ulcer model it was found that the aqueous alcoholic (70%) extractives of Rhodomyrtus tomentosa and the methanolic extractives of Mallotus philippensis effectively increased the enzymatic antioxidants such as SOD and CAT. Conversly, it decreased the LPO level which suggests an antioxidant effect of the extractives. Present studies showed an increase in the mucous production in the treated group of animals subjected to acetic acid induced gastric injury. The mucosa and the submucosa of the gastrointestinal tract is protected from inflammatory reactions by the mucous secreted by the gastric cells [78]. The severity of ulceration decreases with the increase in the production of mucous. The study revealed that the repeated dose administration is needed for healing chronic ulcers. Antiulcerogenic activity of MEMP and RTLE was also assessed by Forced swimming Stress induced gastric ulceration in rats at different dose levels. Gastric ulcers induced by stress are known to be caused

9 176 by both physical and psychological factors like degranulation of mast cells and decreased synthesis of prostaglandins [6]. Forced swimming stress induces generation of free radicals causing lipid peroxidation and mucousal damage accompanied by changes in antioxidant enzyme levels [101]. Stress induced increase in MDA levels is known to increase the free radical generation, mainly superoxide, hydrogen peroxide and hydroxyl radicals. This increases peroxidation of cell membrane lipids and induces cell degranulation. This causes the structural and functional integrity of cell membranes to be lost. If the hydrogen peroxide accumulated in the cytosol and mitochondria does not get scavenged by CAT increased generation of hydroxyl radicals takes place [102]. In the present study, forced swimming stress caused increased lipid peroxidation activity decreased SOD and catalase activities in the gastric tissues and blood. RTLE at 100 and 200 mg/kg induced a dose related reversal of the stress induced effects by causing a decrease in the thiobarbituric acid reactive species in the blood and gastric tissues in comparison to the stress control group. Antioxidant markers, superoxide dismutase and catalase were found to be significantly increased at 200 mg/kg. However MEMP at 100 mg/kg did not cause reversal of the stress induced changes. In gastrointestinal ulceration associated with tissue necrosis alkaline phosphatase activity is known to be increased [103]. The release of ALP plays an important role in tissue necrosis associated with

10 177 various models of gastrointestinal ulceration. In this study alkaline phosphatase activity was found to be increased in the stress control group of animals which was significantly reduced by RTLE at 200 mg/kg. Forced swimming stress induced an increase in the serum levels of cholesterol, AST, ALT and ALP and a decrease in triglycerides. This change in the enzyme levels may be possibly due to Hypothalamo- Pituitary-Axis and sympathetic stimulation causing liberation of catecholamines and glucocorticoids which causes inhibition of the immune system in the liver, kidney and other organs. This in turn causes mobilisation of lipids and increased cholesterol synthesis [104]. The levels of triglycerides decreased possibly because it acts as a source of energy during stressful conditions. The reversal of these stress induced changes is possibly due to an antioxidant effect of the extracts under study. Flavonoids, Terpenoids and phenolic compounds are reported to possess antioxidant and gastroprotective effects. Thus the gastroprotective effects of RTLE and MEMP may have been mediated through the free radical scavenging activity of these phytoconstituents present in the plant as was evidenced by the potential amelioration of oxidative stress markers. In the present studies the aqueous alcoholic extractives of Rhodomyrtus tomentosa exhibited potent antioxidant and anti ulcer

11 178 properties when compared to the other extractives studied. Hence, the constituents were separated by column chromatography on a silica gel column to isolate the active constituents from the extract. Three compounds were isolated and TLC confirmed its purity as a single spot. The compounds were subjected to spectral analysis for structural elucidation and was characterised as 7- hydroxy coumarin (umbelliferone), Psoralene and 8-hydroxy psoralene (xanthotoxin). These compounds are not been reported earlier from this plant. The strong antioxidant properties are known to be responsible for the therapeutic activities of coumarins. Hydroxy coumarins are phenolic compounds with free radical scavenging and metal chelating properties. Coumarins are involved in DNA repairing by acting as antimutagens. They are also involved in the restoration of damaged SOD, CAT and GPX activities including activation of enzymes like glutathione S-transferase and glutathione synthetase, which participate in immune defence. Coumarins are known to act as antioxidants by recombining radicals that initiate or propagate lipid peroxidation. They also undergo complexation with metal ions and forms chelate complexes as they form free radicals [105]. Recent reports suggest inhibitory activity of xanthotoxin against Helicobacter pylori. GC MS standardisation of the Aqueous alcoholic extractives of Rhodomyrtus tomentosa revealed the presence of Hexadecanoic acid methyl ester, 1,2-Benzenedicarboxylic acid, 3,7,11,15-Tetramethyl-2- hexadecen-1-ol, diisooctyl ester, 9,12-Octadecadienoic acid, methyl

12 179 ester, n-hexadecanoic acid, phytol, 9,12,15-Octadecatrienoic acid methyl ester and squalene. Some of these constituents are reported to possess antioxidant activity. The methanolic extractives of Mallotus philippensis exhibited good antioxidant activity but the anti ulcer activity was weaker than the aqueous alcoholic extractives of Rhodomyrtus tomentosa. The genus Mallotus contains betulin, a triterpene, which is known to possess antioxidant and wound healing properties. HPTLC separation of the methanolic extractives of Mallotus philippensis leaves and its comparison to the reference standard revealed betulin, an antioxidant in the extractives.