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This thesis deals with the investigations carried out by the authors in their laboratory on the scientific validation of two Indian medicinal plants as potential antioxidant and Anti ulcer agents.
Traditional system of medicine
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.
1.1.1 Current Scenario of Herbal medicine world wide
In countries like Germany and France, many herbs and herbal extractives are used as prescription drugs and their sales in the European Union were around $6 billion in 1991 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 $1 billion and the export of plant-based crude drugs is about $80 billion (Kamboj, 2000).
The total number of plant species of all groups recorded from India is 45,000. Of these, seed-bearing plants account for nearly 15,000-18,000. 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 the 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 untapped wild medicinal plant generic resources together with vital ethnomedicinal leads offer greater scope for developing latest therapeutic agents (Dubey, 2004).
1.2 Peptic ulcers
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 (Crawford James, 2000).
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 10,00,000 patients are hospitalized yearly, and about 3000 people die each year due to 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 gastric ulcers. (Bose, 2003)
Gastric ulcers are caused mainly due to net imbalances in mucosal defensive and offensive 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, 2003). In gastric or duodenal peptic ulcers, the mucosa has been attacked by digestive juices to such an extent that the subjacent connective tissue layer (submucosa) gets exposed. 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.
Various hypotheses have been proposed to understand the biochemical changes taking place during ulcer generation. Increased gastric motility (Garrick et al., 1986), vagal over activity (Clive Ogle 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 levels during stress conditions are thought to be involved in ulcer generation. 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 known to be the most common cause of gastric ulcer in humans which exhibits active inflammation with epithelial damage accompanied by neutrophil migration.
The introduction of the term 'Cytoprotection' in 1979 by Andre Robert refers to protection by prostaglandins 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:
The exact mode of action of cytoprotective agents has not yet been established but various mechanisms have been suggested (Rosa and Vishwanath, 1991).
Increase in mucus secretion
Several studies have suggested that mucus may play an important role in protecting the mucosa from further damage after the initial damage 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. The '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'. 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
Formation 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 thereby reduce the volume of the irritant on specific sites of the mucosa (Takeuchi and Nobuharu, 1985).
Increased release of endogenous mediators of gastric cytoprotection Prostaglandin
Prostaglandins were the first endogenous compounds implicated in gastric cytoprotection. Prostaglandins 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. Prostaglandins also act by stimulating rapid resolution of disrupted surface epithelium (Hawkey and Rampton., 1985).
Sulfhydryl (SH) containing amino acids like L-cysteine and methionine and sulfhydryl 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 and their receptor actions is dependent on endogenous sulfhydryls (Szabo and Trier, 1981).
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
Oxygen derived free radicals, especially the superoxide radical is suggested to be involved in ischemic gastric mucosal damage which may cause lipid peroxidation (LPO) and damage to intracellular compounds. Antioxidants like vitamin E and selenium have shown 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 induces gastric vasoconstriction and increases 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 and Rampton, 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 30 minutes. 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)
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 stress, ethanol and NSAIDs (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).
Excess Reactive Oxygen Metabolite (ROM) production is directly related to the severity of gastritis in H.pylori patients (Davies and Rampton, 1994). Oxygen derived free radicals cause tissue injury through LPO (Lipid peroxidation). Oxygen handling cells have different systems like Superoxide dismutase 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 (Vanishree 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 and Sen, 1995). Antioxidants like Vitamin 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 et al., 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.
1.2.6. Role of stress in gastric ulcers
Stress is a common phenomenon that is experienced by most individuals. Stress is involved in the pathogenesis of a variety of diseases that includes depression and anxiety, immunosuppression, diabetes mellitus, male impotence, cognitive dysfunction, peptic ulcer, hypertension and ulcerative colitis (Hemnani and Parihar, 1998). Formation of excessive free radicals due to stressful conditions is a major internal threat to cellular homeostasis of aerobic organisms. These free radicals are extremely reactive and unstable and react with most of the intracellular molecules (Sevanian and Hochstein, 1985). They enhance the process of lipid peroxidation (Hulliwell and Gutteridge, 1984). During stress ulceration lesions rarely penetrate the muscularis mucosa and are therefore strictly erosions. Stress induced ulcers are produced by subjecting the animal to various form of stress, either in combination or singly, restraining the animals in small cage, 3rd degree burns, shocks, cold environment (Dua, 1989). Swimming continuously in cold water is also a method of inducing stress in rats. Irrespective of the technique used, ulcer incidence and pathology are remarkably similar indicating that with the exception of burning, which probably acts via histamine release all other techniques act through a common mechanism (Brodie, 1962). The cause of stress induced ulcer is clearly not understood. The most likely increased components are acid concentrations, reduced mucosal blood flow, reduced mucus secretion, reduced gastric epithelial cell turnover and activation of the hypothalamic pituitary - adrenal axis.
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 years back. Free radicals can be of two types depending upon its origin, namely, reactive oxygen species (ROS) and reactive nitrogen species (RNS).
Free radical generation
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. (Devasagayam, 2002)
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 radicals 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 component of the epithelial basement membrane, and thus promote mucosal damage and 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 superoxide dismutase, glutathione peroxidase, glucose oxidase and catalase. They help in maintaining the balance between generation of reactive oxygen species and its eradication.
Reactive oxygen species
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 (Ray 2002).
Hydrogen peroxide (H2O2)
Hydrogen peroxide is the most stable reactive oxygen metabolite. It is the least reactive and is 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 and 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
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. (Irshad, 2002)
Cells under aerobic conditions 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 (Irshad, 2002).
The first line of defense against O2.- and H2O2 mediated injury are antioxidant enzymes like SOD, GPx and CAT (Rana 2002).
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 that 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. 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.
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 is an 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.
Thus 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 (Somani, 1996).
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 like impotence, arrhythmia, gynacomastia, etc and cause drug interactions with other drugs on chronic administration besides being expensive.