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Plants have been used as medicine for millennia. Out of the estimated 2,50,000 to 3,50,000 plant species identified so far, about 35,000 are used worldwide for medical purposes. It has been confirmed by WHO that herbal medicines serve the health needs of about 80 percent of the world's population; especially for millions of people in the vast rural areas of developing countries. Meanwhile, consumers in developed countries are becoming disillusioned with modern health care and are seeking alternatives. The recent resurgence of plant remedies results from several factors: 1) The effectiveness of plant medicines; 2) The side effect of most modern drugs; 3) The development of science and technology.
Qualitative phytochemical screening of the successive leaf extracts of Rhodomyrtus tomentosa revealed the presence of certain phytoconstituents such as glycoside, tannins, high amount of saponins, phytosterols, carbohydrates, steroids, flavonoids and phenolic compounds .
Qualitative phytochemical screening and ethnobotanical survey on the successive leaf extracts 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.
Free radicals play an important role in various pathological conditions such as tissue injury, inflammation process, neurodegenerative diseases, cancer and aging. The compounds that can scavenge free radicals have great potential in ameliorating these disease processes. Antioxidants thus play an important role to protect the human body against damage by reactive oxygen species (Halliwell and Gutterigde, 1989).
Recently natural products and drugs as antioxidant agents have received much attention. Many plant extracts and plant products have been shown to have significant antioxidant activity (Couladis et al. , 2003, Lee et al. , 2001). A large number of plants possessing strong antioxidant activity are known in the literature. Natural antioxidants, which are distributed widely in vegetables, fruits and other plant foods, have attracted special interest all over the world.
The two plants selected for the present study possess several ethnomedical uses related to its antioxidant activity. Qualitative tests indicated the presence of phenolic compounds, tannins, flavonoids and terpenoids in these extracts. Plants containing these constituents are known to possess antioxidant properties. Hence the different plant extracts of Mallotus philippensis and Rhodomyrtus tomentosa leaves were evaluated for in vitro antioxidant studies using 5 different methods.
Among the five leaf extracts (petroleum ether, chloroform, ethyl acetate, aqueous alcoholic (70%) and water) of Rhodomyrtus tomentosa tested for invitro antioxidant activity, the aqueous alcoholic extract and the ethyl acetate extract exhibited potent antioxidant activity with low IC50 values in the scavenging of ABTS, DPPH and Nitric oxide and Hydrogen peroxide radicals. The potent antioxidant activity of the aqueous alcoholic extract may be due to the high phenolic content such as tannins an flavonoids.
Among the four leaf extracts (petroleum ether, chloroform, ethyl acetate, methanolic) of Mallotus philippensis tested for invitro antioxidant activity, the methanolic extract exhibited potent antioxidant activity with low IC50 values in the scavenging of ABTS, DPPH and Nitric oxide and Hydrogen peroxide radicals. However these IC50 values were found to be higher or comparable with those obtained for the standards used. The potent antioxidant activity of the methanolic extract may be due to the high phenolic content such as tannins.
To check the safety profile of the extract it was subjected to acute toxicity study which confirmed the absence of any toxicity or mortality at a higher dose of 2000mg/kg. Thus the extract can be classified in to the safe drug category according to the "Global harmonised Classification System" quoted in the OECD guidelines 1996.
Based on the acute toxicity studies of all extracts two dose levels (100mg/kg and 200mg/kg) were selected for acute models and three dose levels (100mg/kg, 200mg/kg and 400mg/kg) were selected for chronic models to evaluate the gastric antiulcer and invivo antioxidant activity in ulcer models. The same doses were used to study the hepatoprotective and invivo activity of the methanolic extract of Mallotus philippensis.
The gastric antiulcer activity of the five different extracts of Rhodomyrtus tomentosa and four different extracts of Mallotus philippensis were evaluated by absolute induced gastric mucosal damage.
Administration of absolute alcohol induced haemorrhagic gastric lesions in the gastric mucosa of the control group. The ethyl acetate, aqueous alcoholic and the water extracts of Rhodomyrtus tomentosa reduced these lesions as evidenced by a significant (P< 0.01) reduction in the ulcer index when compared with the control group. At 100 mg/kg b.w. the ethyl acetate, aqueous alcoholic and water extracts of Rhodomyrtus tomentosa showed a protection index of 45.33%, 58.66 and 40.66% respectively and at 200 mg/kg b.w. the extracts showed protection index of 48%, 62% and 55.33% respectively. The results were comparable to Omeprazole which reduced the ulcer index significantly. Histopathological study of the aqueous alcoholic extract confirmed the antiulcer activity. The rats treated with absolute alcohol showed loss of gland architecture with erosion of the epithelial layer and evident edema and infiltration by inflammatory cell. The rats treated with the aqueous alcoholic extract at 100 mg/kg, b.w showed no ulceration but intactness of gastric epithelium was not restored. However at 200 mg/kg the rats showed significant regenerative changes. Minimal oedema and infiltration were seen in one area. Omeprazole treated groups showed no ulceration in gastric mucosa, glands were regular and minimal inflammation was observed. However the petroleum ether and the chloroform extracts did not exhibit significant gastric antiulcer activity.
The ethyl acetate and the methanolic extracts of Mallotus philippensis reduced the lesions as evidenced by a significant (P< 0.01) reduction in the ulcer index when compared with the control group. At 100 mg/kg b.w. the ethyl acetate and the methanolic extracts showed a protection index of 22.18%, and 63.80% respectively and at 200 mg/kg b.w., the extracts showed protection index of 29.40% and 68.34% respectively. The results were comparable to Omeprazole (2 mg/kg b.w.) which reduced the ulcer index significantly. Histopathlogical study of the methanolic extract confirmed the antiulcer activity.
Ethanol induced gastric mucosal damage is produced by the generation of exogenous and endogenous active oxygen and free radicals. The process of lipid peroxidation is mediated by the interaction of hydroxyl radicals with the cell membrane producing lipid derived free radicals such as conjugated dienes and lipid hydroperoxides. These radicals are known to cause oxidative damage. Ethanol treatment induces intracellular oxidative stress and produces mitochondrial permeability transition and mitochondrial depolarisation which precede cell death in gastric mucosal cells. (Repetto, 2002).
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 cause inhibition of Ca2+ influx that signals the last step in cell death induced by glutamate. (Ishige K, 2001). The antiulcerogenic activity of all the plant extracts studied was confirmed by histopathological studies. The cytoprotective effect of the extracts could be partially due to the presence of flavonoids, tannins and other phenolic compounds and due to the reactive oxygen species scavenging property.
Among the five different extracts tested for invitro antioxidant activity and absolute alcohol induced gastric antiulcer activity the aqueous alcoholic (70%) extract of Rhodomyrtus tomentosa and the methanolic extract of Mallotus philippensis was found to be potent. Hence it was selected for antiulcer studies with Modified pyloric ligation and acetic acid induced chronic ulcer models including invivo antioxidant studies. Synthetic NSAIDs like aspirin cause mucosal damage by interfering with PG synthesis, increasing acid secretion and back diffusion of H+ ions and thus leading to breaking up of mucosal barrier. Ulcer index parameter was used for the evaluation of anti-ulcer activity since ulcer formation is directly related to factors such as reduction in gastric volume, decrease in free and total acidity. It is significant to note when the pH reached 3, the ulcer score appeared less. This is born out by the decrease in free acid, which might have contributed to the anti-ulcer property of the plant extract. (Malairajan et al., 2007)
It has been postulated that histamine may be involved in the formation of pylorus-ligated ulcers and play a mediating role in the gastric secretion, stimulated by gastrin, vagal excitation and cholinergic agents. (Jafri MA et al., 2001)
The defense mechanism of the gastrointestinal mucosa against aggressive factors, such as hydrochloric acid, bile acid and non-steroidal anti-inflammatory drugs, mainly consists of functional, humoral and neuronal factors. Mucus-alkaline secretion, mucosal microcirculation, and motility act as functional factors, while prostaglandins and nitric oxide act as humoral factors, and capsaicin sensitive sensory neurons act as neuronal factors. All the above factors are known to contribute to mucosal protection. (Yasuhiro Tsukumi and Susumu Okabe, 2001)
. Hydrochloric acid back diffuses through the compromised defense and destroys the cells, capillaries and vein causing hemorrhagic ulcers. It also causes release of histamine furthering acid output and thus further damaging the mucosa. Presence of acid causes decreases of gastric pH and leads to activation of pepsinogen to pepsin, which increases the size of lesion by its proteolytic action. The reduction of acid secretion is necessary for healing and prevention of esophageal reflux disease. (Dorababu M et al., 2006)
Aspirin causes mucosal damage by interfering with prostaglandin synthesis, increasing acid secretion and back diffusion of H+ ions. In pyloric ligation, the digestive effect of accumulated gastric juice and interference of gastric blood circulation are responsible for the induction of ulceration. Aspirin was administered to pylorus ligated rats, thus, aspirin further aggravated the acidity and resistance of gastric mucosa was decreased thereby causing extensive damage to the glandular region of the stomach. (Sanmugapriya E and Venkataraman S, 2007)
The mucosal defense mechanism may be due to the epithelial cells of the gastric mucosa, which are impermeable to hydrogen ions thereby forming a physical barrier. Carbohydrate/protein ratio also supports the above observation. The antiulcer effect is also supported by the decrease in the aggressive factors like pepsin and proteins and an increase in the resistance factors like pH, hexose, hexosamine, fucose and sialic acid.
The antiulcer activity of the extracts under study may be due to the presence of saponins, terpenoids, flavonoids and tannins. The antiulcer agent may protect the mucosa from acid effects by selectively increasing prostaglandin PGF2a, Protection against experimental ulcers may be due to the effect of prostaglandins in the parietal cells, as prostaglandins enhance the mucosal resistance, perhaps by increasing the secretion of mucous and bicarbonates, strengthening the mucosal barrier, decreasing the gastric motility, Increasing the release of endogenous mediators scavenging the free radicals, decreasing the release of endogenous amines and stimulation of cellular growth and repair.
The higher the mucin contents the lower is the free acidity. In case of refractory duodenal ulcers mucosal defense agents to be a new dimension in the treatment of gastro-duodenal diseases. Also hexosamine concentration is an index of mucin content of the gastric juice. The carbohydrate/protein ratio is a direct index of the dissolved mucosubstances in the gastric juice.
The increased carbohydrate ratio is a reliable index for an effective mucosal barrier. The increase in carbohydrate protein ratio is the direct reflection of mucin activity. This suggests the increase in glycoprotein content of the gastric mucosa. The decrease in the protein content of gastric juice suggests the decrease of leakage of plasma protein into gastric juice.
Removing oxygen derived free radicals by inhibiting their formation and by scavenging stimulates the healing of acute and chronic duodenal ulceration in the rat. Scavenging of oxygen derived free radical may be an added dimension in the treatment of stress induced acute gastric mucosal injury in humans. (Salim A, 1990)
It is possible that the inhibitory effects of extracts are due, at least partly, to the presence of terpenoids. Terpenes were associated to antiulcerogenic activity in other plants. Some triterpenes are known as antiulcer drugs and their action has been suggested to be due to the activation of cellular protection, reduction of mucosal prostaglandins metabolism-cytoprotective action and reduction of gastric vascular permeability. (Hosseinzadeh Hossein, 2002)
From the results of anti secretary model it was found that both the extracts decreased the ulcer area and ulcer index. The extract decreased total carbohydrate, in the gastric juice compared with the ulcerated control group. On the other hand the extracts decreased the aggravating parameters such as gastric volume, total protein, total and free acidity in the gastric juice and increased the gastric pH. The findings suggested that the aqueous alcoholic (70%) extract of Rhodomyrtus tomentosa and the methanolic extract of Mallotus philippensis extract showed good antiulcer activity in the acute aspirin plus pylorus ligated model.
Acetic acid induced chronic ulcer model, is similar to human chronic ulcers. In this model, acetic acid produced mucosal injury, which was confined to the glandular stomach. ( Umamaheshwari etal., 2007)
Acetic acid induced ulcer model is most commonly used for healing study. Acetic acid solution consistently induces penetrating ulcers and lacks adhesion of ulcer base to underlying liver. Ulcers are caused by hyper secretion of acid in this model. (Dharmani Poonam, 2003)
Oxygen-derived free radicals may play important role in delaying the healing of acetic acid induced chronic gastric ulcers in rats (Kanoko Hamaishi et al., 2006) Acetic acid is reported to produce ulcers by gastric obstruction leading to increase in acidic gastric juice. Perhaps increase in defensive mucosal factors may have beneficial role in protecting ulcers induced by acetic acid. (Sairam, 2003)
There are evidences for the participation of reactive oxygen species in the etiology and pathophysiology of human disease, such as neurodegenerative disorders, inflammation, viral infections, autoimmune gastrointestinal inflammation and gastric ulcer. (Rapetto MG, 2002)
The importance of mucous secretion as a response to gastric mucosal trauma has long been recognized and protective role of mucous on gastric mucosa. Has been investigated. More the production of mucous, the less is the degree of ulceration. Mucus also protects the mucosa and sub-mucosa from inflammatory reaction.(Annop A and Jagadeeshan M, 2003)
From the data of chronic acetic acid induced ulcer model it was found that the aqueous alcoholic (70%) extract of Rhodomyrtus tomentosa and the methanolic extract of Mallotus philippensis effectively increased the enzymatic antioxidants such as SOD and CAT on the other hand it decreased the LPO level.. Also it increased the different defensive factors of ulcer and mucus production, and decreased the ulcer area and ulcer index. The study revealed that the repeated dose administration is needed for healing chronic ulcers.
Based on the results of the invitro antioxidant studies ,the methanolic extract of Mallotus philippensis was subjected to hepatoprotective studies.
The methanolic extract of Mallotus philippensis exhibited good invivo antioxidant activity and has traditionally been used in liver diseases and hence was subjected to hepatoprotective activity using ethanol and CC4 induced hepatotoxoxicity. Liver is an important organ actively involved in metabolic functions and is a frequent target of a number of toxicants. (Meyer and Kulkarni, 2001). In the absence of reliable liver protective drugs in modern medicine, there are a number of medicinal preparations in Ayurveda, recommended for the treatment of liver disorders( Chatterjee, 2000). The principle causes of carbon tetra chloride and alcohol induced liver damage is lipid peroxidation and decreased activities of antioxidant enzymes and generation of free radicals (Castro etal., 1974) The antioxidant activity or the inhibition of the generation of free radicals is important in providing protection against hepatic damage. A number of plants heve been shown to possess hepatoprotective activity by improving antioxidant status( Shahjahan et al., 2004)
In both the models studied, pretreatment with the extract decreased the wet liver weight and wet liver volumes and caused a reduction in elevated biochemical parameter levels like serum SGPT, SGOT, SALP, Cholesterol, triglycerides, direct and total bilirubin, malondialdehyde (TBARS) and caused an increase in serum total protein levels, increase in ROS scavenging enzymes like Catalase and Super oxide dismutase. It also decreased the functional parameters like thiopentone induced sleeping time which confirmed the hepatoprotective effect of extract. In liver injury models in rats restoration of hepatic cells with minor fatty changes and absence of necrosis after treatment with extract was observed, indicating satisfactory hepatoprotection. The observed antioxidant and hepatoprotective activity of the extract may be due to the presence of flavonoids and other phenolic compounds present in the extract.
In the present studies the aqueous alcoholic extract of Rhodomyrtus tomentosa exhibited potent antioxidant and antiulcer properties when compared to the other extracts studied. Hence it was subjected to column chromatography on a silica gel column for isolation of its active constituents. One compound was isolated and its purity was confirmed as a single spot by TLC. The compound was subjected to spectral analysis for structural elucidation and was characterised as umbelliferone. This compound has not been reported earlier in this plant.
This thesis deals with the investigations carried out by the author in their laboratory on the scientific validation of two Indian medicinal plants as potential antioxidant, antiulcer and hepatoprotective agents.
1.1. Traditional system of medicine (Kamboj VP, 2000)
Herbal medicines have a long therapeutic history and are still serving many of the health needs of a large population of the world.
The WHO has recently defined traditional medicine (including herbal drugs) as comprising therapeutic practices that have been in existence, often for hundreds of years, before the development and spread of modern medicine and are still in use today. Traditional medicine is the synthesis of therapeutic experience of generations of practicing physicians of indigenous systems of medicine. The traditional preparations comprise medicinal plants, minerals, organic matter, etc. Herbal drugs constitute only those traditional medicines which primarily use medicinal plant preparations for therapy
1.1.1. Current Scenario of herbal medicine world wide (Kamboj VP, 2000)
In Germany and France, many herbs and herbal extracts are used as prescription drugs and their sales in the countries of European Union were around $6 billion in1991 and may be over $20 billion now. In USA, herbal drugs are sold in health food stores with a turnover of about $4 billion in 1996 which is anticipated to double by the turn of the century. In India, the herbal drug market is about $ one billion and the export of plant-base crude drugs is about $ 80 billion
1.1.2. Indian scenario (Dubey NK, 2004)
India is a land of immense biodiversity in which two out of eighteen hot spots of the world are located. India is also one of the twelve mega biodiversity countries in the world. The total number of plant species of all groups recorded from India is 45,000 (the total number may be even close to 60,000, as several parts of India are yet to be botanically explored). Of these, seed-bearing plants account for nearly 15,000-18,000. India enjoys the benefits of varied climate, from alpine in the Himalaya to tropical wet in the south and arid in Rajasthan. Such climatic conditions have given rise to rich and varied flora in the Indian subcontinent.
Herbal medicine is still the mainstay of about 75-80% of the world population, mainly in the developing countries, for primary health care because of better cultural acceptability, better compatibility with human body and lesser side effects. However the last few years have seen a major increase in their use in developed world. The plant kingdom is by far the most efficient "factory" of novel compounds to combat against various diseases, but many of them are still unexplored or confined in the various ethnic communities which remain to be scientifically validated. The vast and hitherto untapped wild medicinal plant generic resources together with vital ethnomedicinal leads offer greater scope for developing latest therapeutic agents.
1. 2. PEPTICULCER
1.2.1. Definition (Crawford James M, 2000)
Peptic ulcers are chronic, most often solitary, lesions that occur in any portion of the gastrointestinal tract exposed to the aggressive action of acid-peptic juices. It can be defined as a breach in the mucosa of the alimentary tract, which extends through the muscularis mucosa into the submucosa or deeper.
Peptic ulcer is one of the common diseases in human population. The incidence of peptic ulcers are increasing due to rapid development and civilizational constraints. The estimates of incidence of peptic ulcer vary ranging between 3-10%. An estimated 15,000 deaths occur each year due to PUD. Approximately four million people suffer from peptic ulcers in the United States. 350,000 new cases are diagnosed each year. Around 1000, 000 patients are hospitalized yearly, and about 3000 people die each year as a result of peptic ulcer disease. The male to female ratio for duodenal ulcer is 3:1 and gastric ulcer about 1.5 to 2:1. There has been a significant decrease in the prevalence of duodenal ulcers over the past decades but little change in the prevalence in the gastric ulcer. (Bose, 2003)
Gastric ulcers arise due to various factors. Etiology of gastric ulcers is still debeted, but, it is accepted that ulcers are caused due to net imbalances in mucosal offensive and defensive factors. Ulcer therapy is now mainly focused on limiting the deleterious effects of offensive acid secretion, but the search for new safer alternative drugs has rekindled the interest in cytoprotective drugs, which protect the gastric mucosa from damaging agents without influencing acid secretion or neutralizing intragastric acidity (Sairam K, 2003). In the area of a gastric or duodenal peptic ulcer, the mucosa has been attacked by digestive juices to such an extent as to expose the subjacent connective tissue layer (submucosa). This self-digestion occurs when the equilibrium between the corrosive hydrochloric acid and acid-neutralizing mucus, which forms a protective cover on the mucosal surface, is shifted in favor of hydrochloric acid.
The precise biochemical changes during ulcer generation are not clear yet, although various hypotheses have been proposed from time to time. Increased gastric motility (Garrick et al., 1986), vagal over activity (Cho et al., 1976), mast cell degranulation (Cho et al., 1979) decreased gastric mucosal blood flow (Hase and Moss, 1973, Kitagawa et al., 1979) and decreased prostaglandin level (Miller, 1987) during stress condition are thought to be involved in ulcer generation. Similarly role of oxygen derived free radicals has shown to play a role in experimental gastric damage induced by ischemia and reperfusion (Perry et al., 1986), hemorrhagic shock (Itoh and Guth, 1985) and ethanol administration (Salim 1990). Helicobarter pylori, a pathogen is now known to be the most common cause of gastric ulcer in humans which exhibits active inflammation with epithelial damage accompanied by neutrophil migration.
1.2.4. Gastric Cytoprotection
The introduction of the term 'Cytoprotection' in 1979 by Andre Robert refers to protection by prostaglandin against experimentally induced acute gastric lesions without affecting gastric secretion in the rat. Now the term 'Cytoprotection' is used in a broader sense to mean protection against gastric mucosal injury by a mechanism other than inhibition or neutralization of gastric acid.
Mechanism of Cytoprotection: (Rosa and Vishwanath, 1991)
The exact mode of action of cytoprotective agents has not yet been established but various mechanisms have been suggested.
Increase in mucus secretion
The relative importance of mucus as protective mechanism is still controversial. However, several studies have suggested that mucus may play an important role in protecting the mucosa from further damage after the initial insult by providing a thick 'cap' over the rapidly migrating epithelial cells favoring a rapid re-epithelalization of the mucosa (Morris et al., 1984; )
Increase in bicarbonate secretion
Vagal stimulation increases both acid and alkali secretion. This 'alkaline tide' during hydrogen ion secretion increases bicarbonate delivery to the surface epithelium. The rate of bicarbonate secretion is only 5 to 10 percent of the maximal acid output. Thus bicarbonate alone cannot lower sufficiently the hydrogen ion concentration but it can complement the action of mucus, forming what is known as the 'mucus-bicarbonate barrier' (Rees and Turnbery, 1982).
Strengthening of gastric mucosal barrier
The apical membrane or tight junctions between epithelial cells are relatively impermeable to hydrogen ions and therefore form a physical barrier to back diffusion of acid. It was called as 'gastric mucus barrier' (Davenport et al., 1964). More recent studies have shown the existence of surface-active phospholipids, which form a hydrophobic lining on the luminal surface of the gastric epithelium and retard the passage of water-soluble ions such as hydrogen ions (Hills et al., 1983).
Increase in mucosal blood flow
The mucosal microcirculation is extremely important in maintaining oxygenation and supplying nutrients. The anatomical design of the gastric vasculature is such that the 'alkaline tide' from secreting oxyntic cells is readily available to the basal aspect of surface epithelial cells. Thus if blood flow is adequate there can be an almost unlimited supply of bicarbonate neutralization of back-diffused hydrogen ions. In addition enhanced blood flow ensures that the absorbed injurious agent is diluted within the sub-epithelial capillaries.
Decrease in gastric motility
Formulation of mucosal folds relates closely to muscle action, especially circular muscle, an inhibiting effect of gastric motility may protect the gastric mucosa through flattening of folds. This will lead to an increase in the mucosal surface area exposed to ulcerogens and there by reduce the volume of the irritant on specific sites of the mucosa (rugal crests) (Takeuchi and Nobuharu, 1985).
Increased release of endogenous mediators of gastric cytoprotection
Prostaglandin was the first endogenous compounds implicated in gastric cytoprotection. Prostaglandin increase mucosal blood flow; this has been suggested to be responsible for their gastro protective effect (Gaskill et al., 1982). Various other mechanisms have also been postulated like dilution of noxious agents by prostaglandin stimulated mucus secretion, stimulation of basal bicarbonate secretion, increase in surface active phospholipids, decrease in gastric motility, stimulation of cyclic AMP and dissolution of gastric mucosal folds. Prostaglandin probably also have a repair function by stimulating rapid resolution of disrupted surface epithelium. This 'adaptive' cytoprotection is mediated by prostaglandin (Hawkey and Rampton., 1985).
Naturally occurring sulfhydryl (SH) containing amino acids L-cysteine and methionine as well as sulfhydryls containing drugs protect rats from ethanol induced gastric lesions whereas sulfhydryl blocking drugs counteract the cytoprotective effect of PGE2. They proposed that endogenous sulfhydryls might be one of the mediators of cytoprotection. Synthesis of prostaglandin as well as prostaglandin's receptor actions is dependent on endogenous sulfhydryls (Szabo et al., 1984)
Epidermal Growth Factor
This polypeptide, a potent inhibitor of gastric acid secretion is found in salivary glands as well as other sources like duodenal mucosa and pancreas. It has been reported to have a cytoprotective action in non-antisecretory doses. Perhaps this effect is mediated through sulfhydryl group rather than prostaglandin or alkali secretion (Olsen et al., 1984).
Scavenging of Free Radicals
The involvement of oxygen derived free radicals, especially the superoxide radical in ischemic gastric mucosal damage has been suggested but the exact mechanisms are not yet defined. Probably free radicals result in lipid peroxidation (LPO) and damage to intracellular compounds. Antioxidants like vitamin E and selenium have been shown to have a protective effect on the gastric mucosa against stress and chemically induced lesions (Tariq, 1988)
Decreased release of endogenous mediators of gastric injury vasoactive amines and leukotrienes
In addition to mast cell and vasoactive amines, leukotrienes have been shown to induce gastric vasoconstriction and to increase vascular permeability. Mucosal levels of leukotrienes are increased after exposure to ethanol. In addition inhibition of synthesis of LTC4 and LTD4 in the gastric mucosa protects against damage by noxious agents (Hawkey et al., 1985)
Stimulation of cellular growth and repair
It is well known that rapid epithelial restitution of the damaged mucosal surface takes place by migration of cells from deep within the gastric pits, which recovers the denuded basal lamina. Following injury with agents like ethanol, aspirin and hyper tonic saline mucosal re-epithelialization occurs within as shorter 30 minutes. It should be noted that an intact basal lamina is vital for the cells to migrate during this repair process. The integrity of the basal lamina is maintained by a medium to high pH. On the other hand, if the luminal pH is low (acid) re-epithelialization is hampered (Morris and Wallace., 1981)
2.6 Free Radicals or Reactive Oxygen Metabolite in the Pathogenesis of Peptic Ulcer Disease
Oxygen derived free radicals play an important role in the pathogenesis of acute experimental gastric lesion induced by variety of stress, ethanol, NSAIDs; secretagogue induced duodenal ulceration (Bulger and Helton, 1998). Oxygen derived free radicals are detrimental to the integrity of gastric-duodenal mucosa in that they are responsible for maintaining ulceration (Salim, 1990)
Davies and Rampton (1994) studied that the excess Reactive Oxygen Metabolite (ROM) production was directly related to the severity of gastritis in H.pylori patients. Oxygen derived free radicals cause tissue injury through LPO (Lipid peroxidation). Oxygen handling cells have different systems (e.g.) SOD, peroxides and catalases that are able to protect them against the toxic effects of oxygen derived free radicals. If the generation of free radical exceeds the ability of free radical scavenging enzyme to dismute the radicals, the gastric mucosa may be injured by excessive free radicals. (Vanisree et al., 1996). The oxygen radicals in the gastro-intestinal tract may induce the suppression of a protective mechanism of the gastric mucosa inhibiting glucosamine synthetic activity, a possible cause of decreased mucosal protective capacity (Yoshihide et al., 1992)
Further, these oxygen-derived free radicals are detrimental to the integrity of gastric-duodenal mucus in that they are responsible for maintaining ulceration (Salim, 1990). Iron / Copper binding capacity with ferritin, transferrin, lactoferrin, plasmin and albumin disallows the existence of free metal ions that are known to intensify oxidative stress by promoting the generation of hydroxyl radical (OH-), a very deleterious reactive oxygen species (Chandan, 1995). Antioxidants like vit. E and selenium are shown to have a protective effect on the gastric mucosa against stress and chemically induced lesions
The antioxidant enzymes contain metal (Cu, Zn, Mn, Fe or Se) at the catalytic site. These cofactors are essential for enzymatic activity and have the potential to limit the expression of the enzyme activity (Somani and Rybak, 1996). The antioxidant enzymes also require trace metal cofactors for maximal efficiency, including selenium for glutathione peroxidase, copper, zinc or manganese for SOD and iron for catalase.
1.3. FREE RADICALS
No other discovery in the field of science had an impact on pathobiology with respect to the implication of free radicals in almost all the diseases within a discovery of 20 yrs back. Free radicals can be of two types depending upon its origin, namely, reactive oxygen species (ROS) and reactive nitrogen species (RNS).
1. 3. 1. Free radical generation (Devasagayam, 2002)
During normal biochemical reactions in our body there is a generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). When ROS and RNS exceed the total antioxidant activity it causes oxidative stress. It has been postulated that age-dependent diseases such as atherosclerosis, arthritis, cancer and neurodegenerative disorders involve oxygen free radicals (OFR) at least at some stage of their development.
The role of oxygen derived free radicals has been demonstrated in acute and chronic ulceration (Bast et al., 1991 ). Involvement of neutrophil in ulcer as been implicated in different models of gastrointestinal mucosal injury such as colitis, ischemia (Perry et al., 1986), reperfusion (Grisham et al., 1987) stress (Das et al., 1997) and ethanol (Salim 1990) induced ischemia/reperfusion. ethanol induced injury to the gastric and intestinal mucosa are substantially ameliorated in neutropenic animals. It is also widely accepted that oxygen derived free radical result in the lipid preoxidation and damage of cellular membrane with the release of intracellular component e.g. lysosomal enzymes leading to further tissue damage.
Another hypothesis is that free radicals cause degradation of hyaluronic acid, the principal compound of the epithelial basement membrane, and thus promote mucosal damage, the body has an effective mechanism to prevent and neutralize the free radical induced damage. It is done by a set of endogenous antioxidant enzymes such as superoxides dismutase, glutathione peroxidase, glucose oxidase and catalase. They help in maintaining the balance between the reactive oxygen species generation and its eradication.
1.3.2. Reactive oxygen species (Ray 2002)
Superoxide anions (O2-.)
Superoxide anion is the first reduction product of oxygen. O2.- is a relatively non-reactive species and dismutates to H2O2. This reaction either occurs spontaneously or is catalysed by intracellular enzyme SOD. The most important source of O2.- is oxidative enzymes, among which xanthine oxidase and NADPH/NADH oxidase are the most effective sources
Hydrogen peroxide (H2O2)
Hydrogen peroxide is the most stable reactive oxygen metabolite means it is the least reactive and the most readily detected. H2O2 may be generated directly by divalent reduction of O2 or indirectly by univalent reduction of O2.-. Hydrogen peroxide is the primary product of the reduction of O2 by numerous oxidases, such as xanthine oxidase, D-amino acid oxidase etc. In any system producing O2.-, substantial amount of H2O2 is formed. The H2O2 is decomposed to H2O and O2 the reaction is catalysed mainly by CAT and peroxidase. Experiments with antioxidant enzymes show that H2O2, rather than O2.- is the more essential species to induce cell injury
Hydroxyl radical (.OH)
Hydroxyl radical is highly reactive. This hydroxyl is produced following the reaction of O2.- and H2O2 in the presence of metallic ions such as Fe2+ / Cu+. Lipid is very susceptible to .OH attack and initiate lipid peroxidation (LPO). .OH is the most potent among ROM's, reacting with a wide range of macromolecules at a high rate constant. .OH is known to induce conformational changes in DNA including strand breaks, base modifications and enhanced expression of protooncogenes.
Reactive nitrogen species (Irshad 2002)
Nitric oxide rapidly undergoes addition, substitution, redox and chain terminating reactions. The target molecules of nitric oxide are intracellular thiol and metal containing proteins and low molecular weight thiols like glutathione and cysteine etc. Peroxynitrite (ONOO-) is another powerful oxidant that interacts with a wide range of targets to cause tyrosine nitration, thiol oxidation, lipid peroxidation, guanosine nitration, oxidation and ultimately cell death. The reaction of ONOO- with excess NO generates NO2, which can combine with more NO to form N2O3 to cause nitrosative stress
1. 4. Antioxidants (Irshad 2002)
Cells under aerobic condition are threatened to the insult of reactive oxygen metabolites (ROMs) that are efficiently taken care by the powerful antioxidant system in human body. The term antioxidant has been defined as any substance that delays or inhibits oxidative damage to a target molecule. Antioxidant enzymes, together with the substances that are capable of either reducing ROMs or preventing their formation, form a powerful reducing buffer which affects the ability of the cell to counteract the activation of oxygen metabolites. All reducing agents thereby form the protective mechanisms, which maintain the lowest possible levels of ROMs inside the cell.
Enzymic antioxidants ( Rana 2002)
The first line of defense against O2.- and H2O2 mediated injury are antioxidant enzymes like SOD, GPx and CAT.
Superoxide dismutase (SOD)
SOD is the most important enzyme because it is found virtually in all aerobic organisms. O2- is the only known substrate for SOD. SOD is considered to be a stress protein, which is synthesised in response to oxidative stress.
Glutathione dependent enzymes
The multiple physiological and metabolic functions of glutathione include thiol transfer reactions that protect cell membranes and proteins. Glutathione participates in reactions that destroy H2O2, organic peroxides, free radicals and certain foreign compounds.
Glutathione peroxidases (GPx) are selenoenzymes and catalyse the reduction of hydroperoxides at the expense of glutathione (GSH). In this process, H2O2 is reduced to H2O where as organic hydroperoxides are reduced to alcohols. Glutathione peroxidase resides in the cytosol and mitochondrial matrix. It acts as an enzyme protecting haemoglobin from oxidative destruction by H2O2. It catalyses reduction of H2O2 and organic hydroperoxides including those derived from unsaturated lipids.
Function of this enzyme is to regenerate glutathione (GSH), which has been converted to GSSG by oxidation and by thiol transfer reactions. Several haematological disorders are associated with decreased levels of glutathione reductase in red blood cells.
Catalase is an enzyme which catalyses the decomposition of H2O2 to form H2O and O2. CAT is a heme containing protein, which catalyses the following reaction. CAT is found to act 104 times faster than peroxidase. It is localized mainly in mitochondria and in subcellular response organelles. Glutathione peroxidase and catalase were found to be important in the inactivation of many environmental mutagens. This will increase the rate of consumption of oxygen, lowering the local oxidation product, making oxidative damage less likely. As a natural unavoidable consequence, it will increase cellular energy, which can be used for everything including the prevention or repair of damage.
Non-enzymatic antioxidants (Somani 1996)
Three non-enzymatic antioxidants of particular importance are:
Vitamin E is the major lipid-soluble antioxidant, and plays a vital role in protecting membranes from oxidative damage. Its primary activity is to trap peroxy radicals in cellular membranes.
Vitamin C or ascorbic acid is a water-soluble antioxidant that can reduce radicals from a variety of sources. It also appears to participate in recycling vitamine E radicals. Interestingly, vitamin C also functions as a pro-oxidant under certain circumstances.
Glutathione may well be the most important intracellular defense against damage by reactive oxygen species. It is a tripeptide (glutamyl-cysteinyl-glycine). The cysteine provides an exposed free sulphydryl group (SH) that is very reactive, providing an abundant target for radical attack. Reaction with radicals oxidizes glutathione, but the reduced form is regenerated in a redox cycle involving glutathione reductase and the electron acceptor NADPH.
In addition to these, the trace elements selenium, manganese, copper, and zinc also play important roles as nutritional antioxidant factors. Selenium is a factor for the enzyme glutathione peroxidase and manganese, copper and zinc are cofactors for S.O.D. zinc also act stabilize the cellular metallothione in pool which as direct free radical quenching ability the complex interactions of these different antioxidant system may imply that eventual therapeutic strategies will depend on combination therapy of various antioxidants rather than a single agent.
Thus in short antioxidants fall in to two classes (i) Preventive antioxidants (catalase and other peroxidases), which reduce the rate of chain initiation and (ii) chain breaking antioxidants (superoxide dismutase) which interfere with chain propagation. To conclude, if we focus on the dominant chemical process in our body based on simple quantity, the energy supply process of metabolism, the true beneficial effect of antioxidant is not the selective elimination of damaging free radical, but rather the enhancement of metabolism.
Allopathic drugs used in peptic ulcer are directed against a single luminal agent (Jagruti et al. 1997). Inspite of this, we have yet to discover effective anti-ulcer drugs, which not only heal the peptic ulcers but also effectively prevent their recurrence (Parmer and Jagruti, 1993) because with the new and potent anti-ulcer drugs, healing of peptic ulcer is usually achieved within six to eight weeks in most patients (Jorde et al. 1987) and 89% of gastric ulcer patients experience ulcer recurrence with one year of successful healing with conventional anti-ulcer therapy. (Sue et al. 1996). Allopathic drugs cause adverse effects, toxic effects and cause drug interactions with other drugs on chronic administration besides their availability at high cost.
1.5. Free Radicals and Hepatic damage
Free radicals are reactive molecules involved in many physiological processes and human diseases such as cancer, aging, arthritis, Parkinson syndrome, ischaemia, toxin induced reaction, alcoholism, liver injury etc. The damage to hepatic parenchymal cells, leading to hepatic injury, is due to oxidative stress within the cells caused by partially reduced free oxygen (PRFO) species such as O 2 (Superoxide anion),H 2 ,O2 , and OH (hydroxy free radical). The elevation of free radical levels seen during the liver damage is due to enhanced production of free radicals and decreased scavenging potential of the cells. A variety of intrinsic antioxidants (reduced glutathione, superoxide dismutase, glutathione-S-transferase etc.) are present in the organism, which protect them from oxidative stress.
Technically, the estimation of free radicals directly is not possible due to the transient nature of the free radicals. Thus estimations are usually done indirectly by measuring the "Antioxidant defense status" of the liver microsomes. Hepatoprotection by enzymatic free quenching is brought about by elevating the levels of antioxidant enzymes in tissues such as the Superoxide dismutase (SOD), Peroxidase and Catalase.