DRUG INDUCED ULCER ROLE OF NIRMALI {StrychnospotatorumlAnn.)
THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PitlLOSOPHV (MEDICINE) OF THE UNIVERSITY OF NORTH BENGAL
h a j e n v h a k u m a k a q k a w a l QA<^cbS, qas DEPARTMENT OF BIOCHEMISTRY NORTH BENGAL MEDICAL COLLEGE & HOSPITAL MAY, 2002 J 55235 23 SEP 2003 Hiis w4M*k is llc«flcatc "ill 0 T H e ir P O K E W 0 K n ^de woric emfoclieif in tfiis tdesis su^itte Q¥fiife presenting tfie worfi, QHedicaC CofTege a n tC ^ospitaC a ru C 0 r . < T . ‘K. Cfiatterjee, <^egistrar, (University o f o f 7rtfi <^BengaC for tdeir fiefpfuf suggestions ana ^uU&nce in course o f tde investigation, am afso tfianlcfuf to a ff tfie teacfiers aruf researcfi scfiofars o f tfie department o f <3iocfiemistry, O f jrtfi (JU U Tm yiLA XV.MJk.'R JAgTLAy/JAL) COKTENTK Page itrmoDUcnoN 01 PLAN OF RESEARCH WORK 14 CHAP1ER - I DeYetopment of dn% induced experimental iiteer 16 Role of Nlrmali IStydm os potatmvm Linn.) CHAPTER - II Studies on Acid - Pepsin durii^ uteeratkm 24 Role of Nirmali IStryt^nos potatorum Linn.) CHAPTER - III Studies on dissolved gastrin mucin durii^ 30 ulceration Role of Nirmali ISbychnos potatonm Unn.l CHAPTER - IV Studies on gastric mucosal mucus durii^ 40 ulceration Role of Nirmali {Stydm as potattMvm Linn.) CHAPTER • V Studies on Upid peroxidation and on Nucleic 46 acid in g ^ tric mucosa durii% uteeration Role of Nirmali ISbydums potatonm Linn.) DISCUSSION 53 SUMMARY 65 REFERENCES 69 INTRODUCTION Peptic vlcei represents a major health problem, both in terms of morbidity and mortality. Because of its frequency and worldwide distribution, peptic ulcer continues to be a subject of numerous investigation, both experimental and clinico pathological. In this respect peptic ulcer occupies a place secondary to carcinoma in the field of gastroenterology. Quineke (i) was probably the first to use the term “Peptic ulcer” which is an excoriated area of gastroduodenal mucosa caused by digestive action of gastric juice. DRUG INDUCED ULCER It is now well accepted that drugs like analgesic, antipyretic, anti inflammatory, steroid, histamine etc. can induce gastroduodenal ulcers in man and animals. (1) # INDOMETHACIN In 1962 indomethacin was introduced as a synthetic, non steroidal and inflammatory, analgesic and antipyretic agent for the treatment of rheumatoid arthritis, degenerative joint disorder and other inflammatory conditions. Wanka et al. (2) and Kaig et al. (3) for the first time reported that indomethacin caused peptic ulcer in human subject. There are also reports (4, 5, 6) suggesting that the patients taking indomethacin sometimes develop gastro intestinal symptoms. Ballabio (7), however, found no evidence of ulcer but only bleeding in patients taking indomethacin as drugs. In animal experiment it was noted (8, 9) that indomethacin caused gastric, duodenal, antral and jejunal ulceration, haemorrhage and perforation in rats and guineapigs. # ASPIRIN It was demonstrated (10, 11) that parenteral administration of aspirin does not produce gastric haemorrhage in animals or man, while other workers have shown that direct contact of aspirin with gastric mucosa is not necessaiy for production of gastric bleeding (12,13,14). Brodie, Chase (15) and Djahanguiri (16) reported that subcutaneous or intraperitonial injection of aspirin produced 90 - 100% ulcerogenic response in rats. Furthermore, dose dependent response of aspirin by oral or intraperitonial route was also demonstrated (15). There was observation (15) that aspirin induced gastric ulcers were significantly reduced by antacids, anticholinergic, ganglion blocking agent and vagatomy, but not by central nervous system depressant. Catecholamines, however, were involved in aspirin induced gastric ulceration (17). There were suggestions (18, 19) that hypothalamic pituitaiy, adrenal system is involved in salicylates induced gastric ulcer. Salicylates mimic in many respect the pharmacological properties of cortisone (20) but the evidence is conflicting regarding the release of cortisone by the action of salicylates on the pituitary adrenal axis. In a comprehensive review of the subject. Smith (21) has concluded that the majority of effects of salicylates are due to their intrinsic properties rather than to secondary endocrine influences. # ANALGIN AND PARACETAMOL Analgin and paracetamol are generally thought to be safe antipyretic and analgesic having anti inflammatory activity too. Paracetamol was originally used in late 19* century and in the last few years it is widely used as an antipyretic and (2) analgesic agent. Adverse effect following therapeutic dosages have rarely been recorded. There is, however, a study on 41 cases of acute paracetamol poisoning in which one died of gastrointestinal haemorrhage and acute massive necrosis of the liver (22). Analgin, on the other hand, has been shown to cause a granuloc3^osis as a severe toxic effect (23,24). # PHENYLBUTAZONE A non-steroid, synthetic compound “phenylbutazone” was introduced in 1949 for the treatment of rheumatoid arthritis, muscoloskeletal diseases and allied disorders. Meuer (25) in a survey of 3934 patients treated with phenylbutazone reported 40 proved cases of peptic ulcers, 9 cases of haemorrhage and an additional 424 individuals also developed gastrointestinal symptoms including epi-gastric pain. Others who reported development of peptic ulcer after constant use of phenylbutazone were Leonard (26), Deseze and Levemievx (27) and Kem et al. (28) Kirener (29) told that Phenylbutazone is a good example in which dosages level employed are of utmost importance. Low dosages appear to be well tolerated. A high dosage of the same compoimd have been observed to produce gastroduodenal ulcer both in experimental animals and in human. Epigastric pain, nausia, vomiting, acute ulcer, activation of pre existing peptic ulcer or development of peptic or duodenal ulcer are the common features after prolonged use of phenylbutazone (30 - 34). Experimental studies also showed that mice, rats, guineapigs and dogs were vulnerable to peptic ulcer after oral or parenteral administration of phenylbutazone (35 - 37). # GLUCOCORTICOIDS Numerous reports are there showing ^ucocorticoids viz. hydrocortisone, cortisone, prednisolone and ACTH produce gastric ulcer in normal rats (38,39), the Shay rats (40), dogs (41, 42) and in man (43, 44). Khan et al.(45) have reported extension and perforation of the ulcers as well as reulceration of a healed ulcer area in rats receiving cortisone. Several mechanisms have been put forward to explain the pathogenesis of glucocorticoid induced gastric ulcer (46 - 49). Somewhere it was considered that anticholinergic drug, methapopolamine bromide prevent the steroid induced ulcer, while somebody suggested that the anti polgistic properties of steroid may be (3) concerned for gastric ulceration. There were also suggestions that diminution of tissue resistance related to the anti inflammatoiy action of corticosteroid is responsible in the genesis of ulceration. Histamine is also considered involved in corticoid induced gastric ulcer. Levis (50) observed vascular dilatation resulting in localized haemorrhagic thrombosis in corticoid induced gastric ulceration. Sanyal and co-workers (51) reported that anti 5 HT agent (cyproheptadine) fail to prevent prednisolone induced gastric ulcer. # HISTAMINE Of the many substances that have been used to the experimental production of ulcer in laboratory, histamine is perhaps the only physiological agent. Popielski in 1920 (52) demonstrated that histamine is a powerful stimulator of gastric secretion. Injected intravenously histamine stimulates acid secretion not only from the intact stomach but also from totally denervated gastric pouch. Evidence ha^ been accimiulating that it might even be physiological stimulant of the acid secreting parietal cell. More recently in the investigation of the so called histamine releasor, experimentalist have noted the occurrence of gastric ulceration in the cat (53) and also in some small animals such as rats, mice etc. (54, 55). Ulcerogenesis has been attributed to the endogenous histamine released from the tissue. Similarly, following injection of gastrotoxin and its anaphylaxis due to horse serum, endogenous histamine is held responsible for production of ulcer in the stomach (55). # DIMAPRTT On the basis of universally recognized hypothesis about the involvement of H2 receptors in the pathogenesis of peptic ulcer, an appropriate rat model has been designed with dimaprit as specil5c H2 receptor agonist. Dimaprit was administered intraperitonially or through intra venous route to 24 hours fasted rats and the animals were sacrificed four hours after the injection. The drugs for studying their gastroprotective effect were given 30 minutes before dimaprit. The procedure is extremely simple and rapid.Its feasibility and specificity are added advantage. It is veiy useful for evaluating not only the absolute potency of a drug given by any route but also of other pharmacodynamic parameter, particularly the duration of action which seems to be an important criterion in selecting new potentially H2 antagonistic drugs (56). (4) Recently, Del Soldato et al. (57) have shown that dimaprit also induces duodenal ulcer in guineapigs. # RESERPINE Reserpine produces severe haemorrhagic ulcer at the glandular part of the stomach which has been attributed to significant degranulation of gastric mast cell and consequent liberation of histamine. These events are thought to be cholinei^cally mediated (58). The morphological changes in gastric mucosa are very similar to the destructive changes found in the mucosa of human gastric ulcer (59). # SEROTONIN Serotonin ulcer, one of the chemically induced experimental gastric ulcer, has been described by WTilhelmi (60), Hedinger and Veraguth (61). Since then it has been widely used for the investigation of the etiology of peptic ulcer disease and as a tool in the search for new anti-ulcer drug. # DULCEROZINE Kurebayashi et a l (62) reported that acute perforating duodenal ulcer can be produced in the rat following single oral administration of dulcerozine, a compound structurally related to non-steroidal anti-inflammatory drug such as phenylbutazone and other known to cause gastro intestinal damages in man and in animals. It has been proposed that prolonged gastric hyper secretion might be an important factor contributing to the pathogenesis of duodenal ulcer in this species. The dulcerozine induced duodenal ulcer in rats is a useful model for studying the pathogenesis of duodenal ulcer and testing the anti- ulcer drug from the practical and pathogenic standpoint because 1. The lesions develop are analogous to the clinical disease with respect to location and histology. 2. The factor producing the pathologic changes is similar in man and animals used. 3. The drug treatment and a surgical operation effective in animals could be clinically useful. 4. It is extremely simple to perform the massive production of ulcer and the results are obtained within 18 hours. (5) # CYSTEAMINE Experimental duodenal ulcer in rats induced by cysteamine hydrochloride was first described by Selya and Szabo (63). The pathogenic mechanism leading to ulceration has not yet been fully explained, but both protective and aggressive factor influencing the resistance of the duodenal mucosa seen to be involved. Gastric emptying is delayed and serum gastrin concentration is increased. Cysteamine induced ulcer resemble duodenal ulcer in man to its location, histopathology and some aspects of pathophysiology. The development of duodenal ulcer in response to cysteamine is inhibited by the anticholinergic agents, antacid, prostaglandin and H2 receptor antagonist. Since multiple dosing is necessary to prevent cysteamine induced ulcers, the usefulness of the model in a screening programme is limited by the large quantity of drug required. Hence, cysteamine has also been used in mice to produce duodenal ulcer which can be used for the evaluation of anti-ulcer drug overcoming the above mentioned drawback seen in rats. # ENDOTOXIN (LIPOPOLYSACCHARIDE) Administration of endotoxin produce a moderate degree of gastric mucosal damage in rats ((64). The lesions remained confined to the glandular mucosa and consisted of smaU punctiform lesions, erosions and petechial haemorrhages. The characteristic feature of these lesions was a typical submucosal ecchymosis in the glandular stomach observed in about 30% of the animals. Pretreatment with ranitidine, pirenzepine, proglumide, sucralfate and naloxone provide significant protection(6s). # MPTP Szabo et al. (66) have shown that the parkinsonism inducing agents i-methyl- 4-phenyl-i,2,3,6 tetra hydro pyridine (MPTP) given in multiple daily doses induces solitaiy or doubled (“kissing”) duodenal ulcer in a rat in a dose dependent manner. MPTP produces duodenal ulcer by impairing defense in the duodenal bulb. Dopamine agonist like bromocriptine, lergotrile and monoamine oxidase inhibitors like pargyline and deprenyl have been shown to prevent MPTP induced duodenal ulcers in rats. # ACETIC ACID Acetic acid produces gastric ulcer in rats. This model of ulcer, resembling (6) human peptic ulcer, may be quite useful for the study of human ulcer and the evaluation of pharmacological agents used for this disease (67). # HYDROCHLORIC ACID Robert et a l (68) observed that 1 ml of 0.6 N hydrochloric acid can produce gastric erosions and ulcers in albino rats which can be inhibited by pretreatment with prostaglandins. # ETHYL ALCOHOL 1 ml of 80% ethyl alcohol when administered orally to 12 hour fasted rats can induce gastric ulcers in rats. The ulcer formation is through the generation of free radicals (69). # SODIUM HYDROXIDE 1 ml of 0.6 N sodium hydroxide can produce gastric lesions in rats (70). # OTHERS Chemicals like silver nitrate, formalin, nicotine, epinephrine, hypertonic saline etc. can induce gastric ulcers in experimental animals like rats, cats and dogs. The ulcers, though resembled human gastric ulcer grossly and histologically, healed rapidly and completely within 2 -3 weeks (68,71-74). MECHANISM OF DRUG INDUCED ULCERATION Various factors have been recognized to explain the mechanism of drug induced ulceration. ACID - PEPSIN The stomach has a pivotal role in the digestive process, functioning both as a reservoir and a mill by virtue of gastric glands secreting hydrochloric acid and pepsin. Acidification of ingested food initiated the process of digestion by creating optimal conditions for peptic digestions of proteins. Parietal cells of stomach secrete hydrochloric acid while chief cells formed pepsinogen which is activated into pepsin in the acid medium of gastric juice. Both acid and pepsin have proteolytic actions on living tissue and are capable of autodigestion of gastroduodenal mucosa (75). For several decades, the dictim “no acid - no ulcer” has dominated the pharmacological basis of ulcer therapy, and the drugs used, reduced acid secretion. (7) However, it was observed that in 45-75% patients of duodenal ulcer acid secretion was within normal limits, whereas in gastric ulcer patients add secretion was either normal or subnormal (76). Patients of ZoUinger-Ellison syndrome, characterized by abnormally high acid secretion, show minimal incidence of peptic ulceration (77). It was, therefore, apparent that peptic ulcer was not solely induced by the offensive factors of the acid and pepsin. In experimental studies also same picture was found out.Nicoloff(78) showed that ulcerogenic effect of indomethacin in albino rats did not seem to be related to acid hypersecretion. Lynch et a l (79) reported an increase, no change or a decrease in gastric secretion and acidity by salicylates depending upon the preparation and species studied. There are reports that aspirin caused decrease in volume and acidity of gastric secretion in rat (80,81). There is also a report that aspirin given by mouth in customary dosage has no effect on hydrochloric acid secretion (82). Similar findings were also noted by other workers in experimental gastro-duodenal ulcerations (83 - 88). MUCUS SECRETION Mucus is one of nature’s perfections, protecting the gastrointestinal tract from infective, chemical and physical insults. It has been commented that it encloses the gastric juice in the stomach as if it was an impermeable porcelain vase. Mucus is secreted into the gastroduodenal lumen by surface epithelial cells and mucus neck cells (goblet cells) and submucosal Brunner’s glands. The secretion has two components, a water insoluble gel adherent to the mucosal surface and soluble mucus in the lumen. The latter can either be secreted directly into the lumen or may be derived from the mucus gel by proteolytic degradation or mechanical shearing during digestion (89). Mucus consists of about 1% by weight of salts and other dialyzable components, 0.5 - 1% of free proteins and a similar quantum of carbohydrate rich glycoproteins and 95% or more of water. The glycoprotein component of mucus is responsible for the characteristic viscous gel forming property, believed to be important for the functional role of mucus. Native human mucus glycoprotein has a high molecular weight of about two million daltons and is formed by polymerization of four glycoprotein subunits, joined by disulphide bridges. Each subunit consists of a protein core with about 150 side chains, studded along the length of the protein core, projecting out like bristles of a test tube brush. About 25% of the protein core, which is bereft of these carbohydrate side chains, is non-glycosylated, and the disulphide (8) LUMENpR 2^ * ^Pepain^r - — Mucus . r Mucus-bicarbonate barrier t BCO, secretion.) J13S03333333“™ ®'”““ Cell ■igration/regeneration { MUcosal prostagiandint j Mucosal blood flow Gastric muconi bamm bridges, 78 per moleciile, are located in this region. Disulphide bond splitting reagents, like mercaptoethanol and proteolytic enzymes, break the native glycoprotein molecule into its four component subunits which are water soluble and do not have the viscous gel forming characteristics of the parent glycoprotein molecule. The linkage between carbohydrate and protein in the mucus glycoprotein is o-glycosidic and the monosaccharide involved is N-acetyl galactosamine, which is attached to either serine or threonine. Apart from these two, the other predominant amino acid in the protein core is proline which does not permit alpha-helix conformation within the peptide chain, providing the requisite close packing of the carbohydrate chains found in these glycoproteins. The carbohydrate side chains comprise over 80% by weight of the glycoprotein molecule and their presence is compatible with the high degree of hydration essential for the special rheological properties of the molecule. There are approximately 600 carbohydrate side chains per molecule of native glcoprotein and each branched side chain consists of about 15 oligosaccharide units composed of galactose, fucose (methyl pentose), N-acetyl glucosamine, N-acetyl galactosamine and sialic acid, with traces, if any, of mannose and no uronic acid , which distinguish them from serum glycoproteins and proteo glycans, respectively. The carbohydrate chains are often negatively charged due to the presence of ester sulphate and sialic acid, the latter being located in the terminal position. The most important sialic acid is N-acetyl neuraminic acid. Rejection of the neighbourly negatively charged group of glycoproteins results in molecular expansion and increase in viscosity. An interesting feature of mucus glycoprotein is that the structure of the terminal parts of the sugar chain is similar to that determining the specificity of ABO blood groups on the erythrocyte surface (90 - 92). Mucus has several functions which contribute significantly to cytoprotection. a) Lubrication and mechanical protection: The mucus gel covers the entire surface of the gastroduodenal mucosa with a variable thickness of less that 500 pm, lubricating the mucosa and forming the first line of defense against noxious gastric contents. It exists in a dynamic balance between production on one hand and degradation by pepsin and shear forces on the other (93). b) Mixing barrier: Mucus is readily permeable to hydrogen ions and provides a mixing barrier at the mucosal surface, preventing the relatively small amounts of bicarbonate ions (9) LUMEN INCREASED HCO./HOCOS OOTPUT Jl !■ MUCUS INCREASED MUCOSAL BLCNOD FLOW Prostaglandins and cytoprotection firam miving with the bulk of hydrogen ions in the lumen, thus confining the neutralization at the mucosal surface, ensuring a pH gradient across the mucus layer . The glycoprotein molecules appear to retard the diffusion of hydrogen ions, the rate of diffusion being four times slower than that in unstirred water. However, this retardation of hydrogen ion diffusion across the mucus layer is unlikely to be the sole factor protecting the mucosa from acid since this would require the renewal of a mucus layer of at lease 04 mm thickness, ten times each second, in order to maintain epithelial surface neutrality (94). c) Unidirectional flux of hydrogen ions: The mucus facilitates unidirectional flux of hydrogen ions from the gastric glands into the lumen. The passage of acid secreted by the parietal cells appears to occur through mucus channels which are highly sulphated. These channels, with high negative charge, contributed by sulphate and sialic acid radicals, produced predominantly by the neck mucus cells, behave as cation exchangers. Periodically, instead of an increasing pH gradient across mucus, there is a sudden drop in pH. A concentration gradient of sodiimi ions appear to be generated across the mucus layer by the continuous activity of Na/K-ATPase at the baso-lateral membrane of mucus cells. Sodium diffusing across this gradient win generate a diffusion potential positive at the cell facing surface of the mucus gel. It is postulated that this potential moves hydrogen ions into the lumen and retards back diffusion (95). d) Prevention of back diffusion of pepsin and pepsinogen activation: Since mucus exploits the phenomenon of phase separation, it attenuates back diffusion of macromolecules like pepsin from gastric juice. Mucus also has a fimction in transporting pepsinogen and preventing its activation into pepsin (96). e) Repair of superficial mucosal damage: Following damage of surface cells, there is rapid release of large amounts of mucus and plasma proteins which, together with the cellular debris, form a coating over the destroyed area, providing a favourable micro-environment for repair and restitution (97). f) Antibacterial activity: Identification of bacteria in human stomach in gastritis indicates that normal gastric mucus may have antibacterial activity (98). The ability of mucus to afford mucosal protection appears to depend upon its (10) thickness commonly known as mucus gel thickness. It has been reported that the gel is resistant to hypertonic saline, bile, ethanol and nonsteroidal anti-inflammatoiy drugs and its secretion is augmented by chemical irritants, carbachol, carbenoxolone, prostaglandins and secretin (99). It has also been suggested that bicarbonate secretion (100), intrinsic barrier properties of epithelial cells (101), mucosal blood flow (102), acid-base balance and acid secretion (103), endogenous prostaglandins (104), Helicobacter pylori (105), dietary factors (106), nonsteroidal anti-inflammatory drugs (107) etc. are somehow related with the mucus gel thickness. Role of gastric mucus both in clinical and experiment ulcers has been studied by several workers. Patients of gastric ulcer have an abnormal mucus gel structure containing less polymeric glycoproteins (108) and certain pepsins, more prevalent in duodenal ulcer patients induce marked digestion of the glycoprotein matrix (i09).In experimental system, aspirin has been shown to reduce the rate of synthesis and secretion of mucus (110,111). It seems possible, as Menguy suggested (110), that it is the loss of mucus iDarrier’ that permits the back diffusion of hydrochloric acid into the aspirin-damaged gastric mucosa. Davenport (112) showed that aspirin damaged gastric mucosal barrier which allows back diffusion of hydrogen ion. He suggested that when the mucosa is damaged by salicylates histamine is released and capillary permeability increases which is followed by bleeding. While studying the mucus secretion in vagally denervated gastric antral pouch of dog during administration of indomethacin for twenty one days, Menguy showed (110) that indomethacin lowered the rate of secretion of gastric antral mucus and diminished the amoimt of carbohydrate incorporation into the mucosubstance. Working on phenylbutazone induced gastric ulcer in albino rats, number of workers have given importance to the mucus barrier whose absolute or relative deficiency acts as a factor in the production of ulceration (113 - 116). Ziaidi et a l observed a steady decrease of mucin in the gastric juice after thirty days of phenylbutazone treatment in guinea pigs (117). UPID PEROXIDATION Role of lipid peroxidation in gastric mucosa has been extensively studied in the pathogenesis of peptic ulcer. It has been found out that lipid peroxidation mediated by active oxj^en species plays an important role in the pathogenesis of peptic ulcer (118 -123). Antioxidants, however, act as anti-ulcer agents by inhibiting lipid peroxidation through decrease of reactive oxj^en metabolites (124 -126). (11) Mechanism of oxidative damage to DNA Chemical agents like quercetin, rebamipide, zinc acexamate etc. also exert their anti-ulcer activity by inhibiting lipid peroxidation (127 -132). Guth (133 - 134) explained that oxygen derived free radicals, specially the superoxide radical, play an important role in ischaemic gastric mucosal lesions, particularly in presence of hydrochloric acid. Ischaemia is followed by utilization of high energy compounds like adenosine tri phosphate (ATP), resulting in accumulation of adenosine mono phosphate (AMP) in the presence of reduced oxidative phosphorylation. AMP is further catalyzed to hypoxanthine, which tends to accumulate. Ischaemia also converts the enzyme xanthine dehydrogenase to xanthine oxidase form. The latter requires the presence of oxygen for its activity, and when oxygen is available after rapid reperfusion, the enzyme acts on hypoxanthine to produce superoxide radicals, hydrogen peroxide and hydroxyl radicals. These free radicals, each containing an unpaired electron in their outer shell, are highly reactive and are potent oxidizing and/or reducing agents, including ceU wall damage and the release of intracellular lysosomal enzymes. DRUG INDUCED ULCER - ROLE OF NATURAL PRODUCTS The use of some plant and mineral drugs, including vegetable banana, narikelkhand (coconut) and tamrabhasma (copper preparation), has been advocated in Ayurveda for the therapy of a symptom complex akin to the modem version of peptic ulcer syndrome. Some of these have been subjected to experimental evaluation. Vegetable banana (Musa paradisiaca) has been extensively investigated by Sanyal and his coworkers for nearly three decades. The antiulcerogenic activity of unripe green banana was first reported in 1961 (135) and later confirmed on a variety of experimental models (136 - 137). Though it was initially postulated that the anti ulcer effect was due to the high serotonin content of green banana (135), it was later realized that the effective dose of banana did not contain sufficient serotonin to justify this postulate (138). Further studies not only confirmed the anti-ulcer activity of banana but also showed that this effect was not due inhibition of acid-pepsin output but was associated with augmentation of gastroduodenal mucosal protective factors (139 - 142). Some of the active principles likely to be responsible for the anti-ulcer action of banana have been identified (143 -146). Tamrabhasma inhibits acid-pepsin secretion and also exerts significant mucosal protection in experimental ulcer (147 -149). Narikelkhand also exerts (12) significant anti-ulcer effect due to mucosal protection (150). Anti-ulcer activity of amlaki {Emblica officinalis linn.) (151 -154), black tea extract (155), Piper longum Linn. (156), Ul-409, Cauvery-100 and PK-2001 (multiconstituent herbal preparations) (157 - 159), Symplocos racemosa (160), Synclisia scabrida (161), Shankha Bhasma (162), Rhamnvs triquerta (163) etc. were also studied and reported in the literature. From pilot experiments we came to know that Nirmali (Strychnos potatorum linn.) exerts anti-ulcer effect on experimental ulcers as induced by 1. Aspirin and 2. Indomethacin. Since two experimental models are not sufficient to evaluate the anti-ulcer property of a plant, it was thought worthwhile to study the same in more experimental ulcer models. The present work, thus, was an attempt to evaluate the anti-ulcer activity of Nirmali (strychnos potatorum linn.) and its effect on various biochemical parameters in drug induced experimental ulcer models. ## (13) PLAN OF KE8EAKGH WOKK PLAN OF RESEARCH WORK The work embodied in the thesis covers the studies on the role of Nirmali iStrychnos potatorum linn.) in experimental ulcers. Gastric ulcers were induced in experimental animals (rats, guinea pigs and mice) by various ulcerogenic drugs and effect of Nirmali {Strychnos potatorum linn.) on the ulcers and on different biochemical parameters was studied. The following scheme of work was followed in course of the investigations. CHAPTER-I : DEVELOPMENT OF EXPERIMENTAL ULCER : ROLE OF NIRMALI (Strychnos potatorum Linn.) a) Development of gastric ulcers in rats by the drugs like, 1. Salicylic acid 2. Aspirin 3. Paracetamol 4. Indomethacin (14) 5. Hydrocortisone 6. Prednisolone in guinea pigs by the drug 7. Phenylbutazone and in mice by the drug 8. Histamine b) Evaluation of antiulcerogenic property of Nirmali (Strychnos potatorum Linn.) in these ulcer models. CHAPTER-II : STUDIES ONACID-PEPSINDURING ULCERATION; ROLE OF NIRMALI iStrychnos potatorum Linn.) Collection of gastric juice and determination of its rate of secretion, acidity and peptic activity during ulceration as well as after treatment with Nirmali (Strychnos potatorum Linn.) CHAPTER-III : STUDIES ON DISSOLVED GASTRIC MUCIN DURING ULCERATION : ROLE OF NIRMALI (Strychnos potatorum linn.) Analysis of dissolved gastric mucin by estimating its constituent carbohydrate components like, total hexoses, hexosamine, methyl pentose and sialic acid during ulceration as well as after treatment with Nirmali (strychnos potatorum Linn.) CHAPTER-IV : STUDIES ON GASTRIC MUCOSAL MUCUS DURING ULCERATION : ROLE OF NIRMALI iStrychnos potatorum linn.) Isolation of gastric mucosal mucus and its analysis during ulceration as well as after treatment with Nirmali (Strychnos potatorum Linn.) CHAPTER-V ; STUDIES ON LIPID PEROXIDATION AND ON NUCLEIC ACID IN GASTRIC MUCOSA DURING ULCERATION : ROLE OF NIRMALI iStrychnos potatorum linn.) Studies on gastric lipid peroxidation (Lipid peroxides) and nucleic acid (DNA) during ulceration as well as after treatment with Nirmali (Strychnos potatorum Linn.) 1 5 5 2 3 5 *' CHAPTER - I DEVELOPMENT OF EXPEfflMENTAL ULCER ROLE OF NIRMALI (Strychnos potatorum Linn.) CHAPTER - 1 To evaluate the anti-ulcer property of Nirmali iStrychnos potatorum Linn.), gastric ulcers were produced in experimental animals by knovm ulcerogenic drugs and the effect of Nirmali (Strychnos potatorum Linn.) on such ulcer was observed. EXPERIMENTAL ANIMALg Experiments were conducted on healthy albino rats (90 -150 g), guinea pigs (450 - 700 g) and albino mice (30 - 35 g) of either sex. DRUGS AND SOLUTION$ 1. Salicylic acid (Alta Laboratory): 30 mg/ml suspension was made in olive oil fresh at the time of use. 2. Aspirin (Indosal Chemical Corporation): 30 mg/ml suspension was made in olive oil fresh at the time of use. (16) 3. Paracetamol (East India Pharmaceutical): 30 mg/ml suspension was made in distilled water fresh at the time of use. 4. Indomethacin (Indocid, Merck Sharp and Dohme): 5 mg/ml suspension in distilled water was prepared fresh before injection. 5. Hydrocortisone (Roussel Pharmaceutical): Stock solution supplied in vials of 25 mg/ml was used. 6. Prednisolone acetate (Deltacortil, Pfizer): Stock solution supplied in the vials of 20 mg/ml was used. 7. Phenylbutazone sodiimi (Butazolidins, Geigy): 40 mg/ml solution in distilled water was prepared fresh at the time of use. 8. Histamine acid phosphate (Evan Medical) 9. Pentobarbital Sodium (Hembutal, Abbot): 1% solution in distilled water was prepared at the time of use. 10. Mepyramine maleate (May and Baker). 11. Anesthetic ether (Industrial solvents and chemicals) INDUCTION OF ULCER Drugs Animals Dose of Route of Reference drugs injection Salicylic acid Rat 100 mg/kg Intraperitoneal Djahanguri (8) Aspirin Rat 100 mg/kg Intraperitoneal Djahanguri (8) Paracetamol Rat 100 mg/kg Intraperitoneal Ram (164) Indomethacin Rat 25 mg/kg Intraperitoneal Lee et al. (9) Hydrocortisone Rat 30 mg/kg Intraperitoneal Robert 8c Nezamis (165) Prednisolone kat 30 mg/kg Intraperitoneal Robert & Nezamis (165) Phenylbutazone Guinea 50 mg/kg Oral Sanyal et al. -pig (137) Histamine Mice 33 micro Intraperitoneal Elliott 8c g/mouse Reward (166) (17) NIRMAU jStnjchnos potatorum Powdered seed of Nirmali (Strychnos potatorum linn.) was iised as test drug. DRUG INDUCED ULCERS; EFFECT OF NIRMALI (Struchnos potatorum linn.)| Rats / guinea pigs / mice under study were divided into three groups: Drug : When the animals were acclimatized with laboratory conditions, drugs (in specific dose for specific animal) was given to them through specific route as mentioned in the table (page - 17) at about 9 A.M. once daily for consecutive three days. These days the animals took normal diet and water. Animals, received the drug through oral route e.g. phenylbutazone group for guinea pigs, were provided with the drug through a fine catheter. Only in case of histamine group mice received an additional intraperitoneal injection of mepyramine maleate in the dose of 2.5 mg/mouse 20 minutes before the histamine injection. This was to protect the animals from the potentially lethal effects of large doses of histamine necessary to produce gastric ulcer (167). After given the third dose of ulcerogenic drug, animals were kept on fasting for a period of 24 hours when no food but water was supplied ad libitum. When the fasting period was over, animals were anesthetized with pentobarbitone sodiimi (40 mg/kg), abdomen opened and the pyloric end of the stomach was ligated avoiding nearby vessels and nerves. Abdomen was then closed and the animals left in cages for a period of 4 hours when water was withheld. Duration of anesthesia and operative technique were same for all animals as suggested by Sanyal et al. (168). After 4 hours the animals were sacrificed with ether, abdomen opened and the stomach was exposed. The cardiac end was ligated and the stomach was dissected out, fi:eeing it firom all of its attachments. The content of the stomach was evacuated into a centrifuge tube and centrifuged for 10 minutes at 3000 x g. The supernatant was collected and utilized for the subsequent studies (For Chapters II and III). Evacuated stomachs were then opened along the greater curvature, carefully washed with water, examined macroscopically and microscopically for the presence of ulcers and kept for subsequent studies (For Chapters IV and V). Drug and Nirmali iStrychnos potatorum Linn.) : Powdered seeds of Nirmali (18) STANDARIZATION OF ANTI-ULCER DOSE OF NIRMALI (Strychnos potatorum Linn.) AGAINST ASPIRIN INDUCED EXPERIMENTAL ULCERS IN ALBINO RATS X ■o c o o Dose of Nirmali {Strychnos potatorum Linn.) in g/kg/day orally for three consecutive days along with Aspirin (100 mg/kg/day, intraperitoneaily) {Strydmos potatorum Linn.) in the dose of i g/kg of body weight was given to the animals orally with the help of a fine catheter once daily for consecutive three days alongwith the ulcerogenic drugs , normal diet and water. This dose (i g/kg of body weight of the animals) was finalized after a pilot experiment which showed that in this oral dose Nirmali (strychnos potatorum linn.) exerted maximum anti-ulcer effect. When the third dose of Nirmali iStrychnos potatorum Linn.) was over, animals were kept on fasting for a period of 24 hours when no food but water was supplied ad libitum. Rest experimental procedure was same as that of “Drug” group. Control: Random controls, received three successive injections of vehicles of drugs, were kept on fasting for a period of 24 hours when no food but water was supplied ad libitum. Rest experimental procedure was same as that of “Drug” group. Animals died in any group during the course of experiment were immediately separated, abdomen opened, stomach dissected out and examined for the presence of ulcers, if any. ULCER INDEX Ulcer index was calculated following the method described by Robert et al. (169). Each and every stomach was examined with the help of a magnifying glass and ulcer index was scored for the group as the sum of the followings; a) Percentage incidence (divided by number of animals) of animals with ulcers. b) Average severity per group scored in pluses (From a scale of 0 to 4+) and c) Average number of ulcers per stomach (The presence of at least one area of erosion of the gastric mucosa was taken as the criterion for positive appearances of ulcer). Evaluation of pathology was always by the “BLIND” method. RESULTSI Results are given in and from Table - 1 to Table - 8. Salicylic add induced ulcer : Effect of Nirmali CStrychnos potatorum Linn.) Table - 1 shows the effect of Nirmali {Strychnos potatorum Linn.) on (19) o o o 5 I % VO .3 O vn | - S .3 •S s d _o ^ '5 ' 02m w o "S 'e I §. Os >n d 00 os (S fW3 i c o < :! m o (S o r<^ • n c n V) 1-H «n m < n cn m T3 op S l l c 5 •fMo & Io •3 + t l S I U oo V5 ^ i / salicylic acid induced peptic ulcer in albino rats. Salicylic acid in the dose of lO O mg/kg/day when given intraperitoneally to the rats for three consecutive days produced massive ulcers in the glandular part bf the stomach. All animals, received salicylic acid, developed ulcers (100%) which were mainly superficial in nature. Few penetrating ulcere were, however, noted. Frank intra gastric hemorrhage was noticed m 90.9% of the animals. In this group all together 33 rats were investigated, out of which adhesion was foimd in 4 animals, acute dilatation in 6 animals and 5 animals showed perforation. Oedema of the mucosal and sub mucosal layers was also observed in some animals. 5 rats were died during the course of experiment. They showed the presence of perforated ulcers in their stomach. Ulcer index when calculated gave the value 25.1. In Nirmali (Strychnos potatorum linn.) fed group 35 rats were employed. Only 5 rats (14.2%) showed the presence of small ulcers in glandular part of the stomach. 10 rats (28.5%) had intra gastric hemorrhage . Dilatation of the blood vessels was found in 4 rats only. In other animals ulcers were attempting to heal as shown by more fibrosis and thickening. All these gave the ulcer index 14. Only 1 rat, died during the experiment, showed the presence of penetrating ulcers in the stomach. In other animals stomachs were almost normal microscopically. Aspirin induced nicer: Effect of Nirmali (Stri/c/mospotatonon linn.) Results of effect of Nirmali (Strychnos potatorum Linn.) on aspirin induced pepic ulcers in albino rats were given in Table - 2. Aspirin (100 mg/kg/day for consecutive three days intraperitoneally ) developed ulcers in glandular portion of stomach in all animals (100%). Ulcers were superficial in nature. Few were deep penetrating . In 82.3% cases ulcerated stomach was associated with frank intra gastric hemorrhage. 4 rats showed adhesion while acute dilatation was observed in 6 rats. 5 animals out of 34, died during the experiment, showed the presence of deep penetrating ulcers in their stomach. Ulcer index, after calculation, came 23. In the Numali (Strychnos potatorum linn.) fed rats (1 g/kg/day for three consecutive days alongwith aspirin) small ulcers were found present in only 14.2% of animals employed. Some of the rats (20%) had intra gastric hemorrhage. In other rats the stomach showed microscopically normal and the ulcers were attempting to heal. Only 1 rat out of 35 died during the experiment. Ulcer index came down to 7.3 (20) (U o m m I I VO a& 0 .B c 1 ’TS •i .3 3 I & < CO od (N o 00 fS § o 00 iz: irT •T) rt m CO cn *gI PP I . 1 2 f o o o o + I Q o I, fS A §■ e2 Io (§ U Paracetamol induced ulcer : Effect of NirmaU CStrychnos potatorum linn.) Results are summarized in Table - 3. Paracetamol in the dose of 100 mg/l^/day when given intraperitoneally to rats for three consecutive days produced ulcers in the glandular part of stomach. Incidence of ulceration was 100%. Hemorrhage was noticed in 100% of the ulcerated stomach. Ulcers were mainly superficial in nature though 5 animals had perforation and 6 had acute dilatation. Ulcer index came 23.5 rats out of 30 in this group died during the experiment. They showed the presence of deep penetrating ulcer with perforation. Nirmali {Strychnos potatorum linn.) treatment, on the other hand, decreased the ulcer index to 7.3. In this group out of 33 ,only 9 rats (27.2%) showed the presence of ulcers which were mainly superficial in nature. Incidence of hemorrhage was also reduced to 18.1% only. Ulcers, still present, were attempting to heal as shown by more fibrosis and thickening. MacroscopicaUy and microscopically stomachs appeared normal. Only 1 rat died during the experiment. Indomethacin induced ulcer : Role of Nirmali (Strychnos potatorum linn.) Indomethacin in the dose of 25 mg/kg/day for three consecutive days developed profuse superficial ulcers in glandular part of stomach of the animals (Table - 4). Incidence of ulceration was 100%. Out of 30 , 26 rats (86.6%) showed hemorrhage in the stomach. 7 had acute dilatation while perforation was found in 4 rats. Ulcer index was 22. 4 animals, died during experiment, showed perforation in stomach. When Nirmali (Strychnos potatorum Linn.) was given orally in the dose of 1 gm/kg/day for three consecutive days alongwith indomethacin, ulcer index came down to 8. 25.8% animals showed presence of superficial ulcers in their stomach. Ulcers were associated with frank intra gastric hemorrhage. Only 3 rats out of 33 used in this group showed adhesion. Stomachs of the rest animal.Q were microscopically normal. Death of 1 rat was due to presence of deep penetrating ulcers. Hydrocortisone induced ulcer : Effect of Nirmali (Strychnos potatorum linn.) Results are summarized in Table-5 . Rats received hydrocortisone in the (21) (S si 00 •S I ^ 3 OS 1 '^ 5Bi B % 13 ?2 'B ^ i o < , ( X I VO 00 vd 00 a «> VO I(U s I p E Io •8 m U 5 ra «n B i .3 I ^ cs l - i .a .3 I •a •■§ l | | S VO o 00 00 (N 00 & g) o o Tt •o I rrl «o cn (4-1o «n e6 o 0o 1 PQ i uI dose of 50 mg/kg/day intrapeiitoneally for three consecutive days developed ulcers in the glandular part of stomach. Incidence of ulceration was ioo 96. In 80% animals frank intra gastric hemorrhage was observed. 8 animals showed perforation while acute dilatation was observed in 7 rats. Ulcer index was 25. 5 rats out of 35, died during experiment, showed deep penetrating ulcers and perforation in the stomach. In Nirmali group 29 rats were used. Nirmali {Strychnos potatorum linn.) treatment along with hydrocortisone reduced the ulcer index to 14. 10 rats (344%) showed ulcer in this group. Ulcers were mainly superficial. Adhesion was found in 1 rat while 2 rats showed acute dilation. Other animals had normal stomach. 8 animals (27.6%) showed hemorrhage in the stomach. There was the death of only 1 rat during the experiment. The animal showed perforation in the stomach. Prednisolone induced ulcer : Effect of Nirmali iStrychnos potatorum Linn.) Results are given in Table - 6. It appears from the table that the rats when took prednisolone (30 mg/l^/day for 3 consecutive days intraperitoneaUy) developed massive gastric ulcers. Ulcers were localized mainly in the glandular part of stomach. Incidence of ulceration was 100%. All ulcers were accompanied by hemorrhage. Adhesion was found in 5 rats, acute dilation in 7 while 8 rats showed perforation. Ulcer index came 25.4 rats out of 33 died during experiment. When Nirmali (Strychnos potatorum linn.) was given (i g/kg/day orally for 3 consecutive days along with prednisolone) ulcer index came down to 10.2. 9 rats out of 32 used (28.1%) showed presence of ulcer in stomach.Ulcers formed were small, mostly superficial in nature and were attempting to heal as shown by more fibrosis and thickening of the mucosa. Adhesion was found in only 1 rat. 18.7% animals had hemorrhage along with ulcer. Stomach of the rest animals gave normal appearance under microscope.During the course of experiment there was the death of only 1 rat. Phenylbutazone induced ulcer: Effect of Nirmali iStrychnos potatorum Linn.) Effect of Nirmali (Strychnos potatorum linn.) on phenylbutazone induced peptic ulcers in guinea pigs was given in Table - 7. Table shows that phenylbutazone (100 mg/kg/day orally for 3 consecutive days) produced massive ulcers in the glandular part of stomach. Ulcers were developed in all animals (ioo%).Ulcers were superficial, deep penetrating associated with adhesion (8 animals), acute (22) .a (S t >o o I efl o o 00 B cn tn VO © S4, § O o 00 s; ■§ tN ■ra I r;3 m ;§ CO ON t*-l0 1-H § CO m (N fo cn 1 I0> 0^ 4> u 1 1) tn CO + jaI I £ c/3 eS Q c3 u (X § i l 1 s Xi "o.S(/I ►^7 -g e Is f I 1^3«u +1 *>5 m u £ 04Vh S.^ :d 00 IID .a n cn «n o (N o o Os 'f. CO «o o m Tt- T p& Io U dilatation (7 animals) and perforation (9 animals) . 74% animals showed hemorrhage in association with ulcer. 5 guinea pigs out of 27 , died during experiment, showed presence of deep penetrating ulcer and perforation in the stomach. Ulcer index was 25. Nirmali (.strychnos potatorum Linn.) in the oral dose of 1 g/kg/day for 3 consecutive days along with phenylbutazone reduced the occurrence and severity of ulcers. 31% animals showed presence of ulcers in their stomach. Ulcers were mainly superficial in nature though adhesion was noted in 3 guinea pigs. 4 animals out of 29 (13.7%) employed in this group showed intra gastric hemorrhage. Other guinea pigs had normal stomach when examined macroscopically and microscopically. Ulcer index reduced to 14. 2 guinea pigs died during experiment. They had perforation in the stomach. Histamine induced ulcer ; Effect of Nirmali (Strychnos potatorum Linn.) Results are summarized in Table - 8. Table shows that histamine in the dose of 33 micro gram / mouse/ day for 3 consecutive days when given intraperitoneally developed ulcers in the stomach of mice. Incidence of ulceration was 100%. 20 mice out of 39 (51.3%) showed intra gastric hemorrhage. Adhesion was found in 17 mice, acute dilatation in 9 and perforation was observed in 9 mice. Ulcer index came 28. Six mice died during the experiment. They had severe hemorrhage and perforation in their stomach. Nirmali (Strychnos potatorum lin n .), on the other hand, when given orally in the dose of 1 gm/kg/day for 3 consecutive days along with histamine reduced ulcer index to 10.1. Ten mice out of 40(25%) used in this group, showed presence of ulcer in their stomach. Adhesion was noted in 7 mice and acute dilatation was in 3 mice. Only 10% animals showed intra gastric hemorrhage. Ulcers in this group were mainly superficial and were attempting to heal as shown by more fibrosis and thickening of the stomach mucosa. Other animals (75%) showed normal stomach imder microscope. There was the death of 4 mice in this group. Those animals showed presence of deep penetrating ulcer in their stomach. ## (23) CHAPTeit - II STUDIES ON ACID - PEPSIN DURING ULCERATION ROLE OF NIRMALI (Stiychnos potatorum Linn.j CHAPTER - II To Study the effect of Niraiali {Strychnos potatorum Linn.) on the rate of gastric secretion, gastric acidity and peptic activity during drug induced ulceration, collected gastric juice samples (page - 18) were analysed by the following methodologies. Nature of ulcerogenic drugs, type of animals, doses of drugs and seeds of Nirmali iStrychnos potatorum linn.), route of administration, duration of anesthesia and operative techniques were essentially same as described in Chapter - 1. MATERIALSI Estimation of acid: a) 0.02 (N) aqueous solution of sodium hydroxide was prepared fresh at the time of estimation. b) Topfer’s reagent: 0.5% alcoholic solution of dimethyl amino azo benzene (24) (Riedal Dehae-nag-acelre-Haimover-Germany) was prepared and stored in glass stoppered bottle, c) 1% alcoholic solution of phenolphthalein (Merck, USA) was made and kept in a glass stoppered bottle. Estimation of peptic activity: a) 0.01 (N) and 0.06 (N) aqueous solution of hydrochloric acid were prepared from concentrated hydrochloric acid (Analar, BDH) with distilled water and kept in glass stoppered bottle separately. b) Haemoglobin solution (E. Merck) : 2% haemoglobin solution in o.o6(N) hydrochloric acid was prepared fresh before use. c) Trichloroacetic acid (Reanel, Budapest): 10% solution was made by dissolving 100 g trichloroacetic acid in 1 litre of distilled water and kept in glass stopperer bottle. d) Phenol reagent (Folin & Clocalteu’s reagent): Sodium tungstate (Analar, BDH) - - -loog Sodium molybdate (Analar, BDH) - - -25g Phosphoric acid ,85-90% (BDH) - - -50 ml Concentrated hydrochloric acid (Analar, BDH) - -100 ml Lithium sulphate (BDH) - - -150 g Bromine (E. Merck) - - -Few drop Sodium tungstate and sodium molybdate were dissolved in 700 ml of distilled water in a 2 litre round bottom flask and to this were added phosphoric and hydrochloric acid and then refluxed for 10 hours. Next lithium sulphate along with 50 ml of water and few drops of bromine water were added. It was then boiled for 15 minutes without condenser to remove excess of bromine. The mixture vs^s then cooled and distilled water was added to make up the volume to 100 ml and filtered. The stock solution was kept in refrigerator. e) Alkaline reagent : 2 g of sodium carbonate (Analar, BDH) was added to 100 ml of 0.1 (N) aqueous solution of sodium hydroxide (Analar, BDH). f) Alkaline mixture: To 100 ml of alkaline reagent 1 ml of 4% aqueous Potassium tartarate (E. Merck) and 1 ml of 2% aqueous copper sulphate (Analar, BDH) were added. This was prepared fresh before use. (25) METHODOLOGIES Determination of volume; Volumes of gastric juice samples were individually measured with the help of a 5 ml graduated pipette and expressed in terms of ml/ioog body weight of the animals/4h secretion. Determination of acidity: 0.2 ml of centrifuged blood free gastric juice sample was taken in a porcelain evaporating dish. It was diluted with 2 ml of distilled water. To it was added a drop of Topfer’s reagent when a pink colour developed. It was then titrated with 0.02 (N) sodium hydroxide until a salmon pink colour (a point in between pink and yellow) was observed. To it was next added a drop of 1% alcoholic solution of phenolpthalein and the titration was continued tiU it turned pink. The burette reading at this point was a measure of acidity. Acidity was expressed in terms of mEq/ml and the output was calculated by multiplying each value with the respective volume of gastric juice as expressed in terms of ml/ioog/4h. The aforesaid procedure to determine the gastric acidity was essentially same as described by Varley (170). Determination of peptic activity : Peptic activity of gastric juice was estimated employing a modified method of Anson (171). 1 ml of 1:250 (diluted with 0.01 N hydrochloric acid) gastric juice was added to 2.5 ml of 2% haemoglobin solution in 0.06 (N) hydrochloric acid. The mixture was incubated at 37 degree centigrade for 20 minutes. Immediately thereafter an equal volume of ice cold 0.6 N trichloro acetic acid was added. The mixture tubes were then kept in ice bath for another 15 minutes and after this period the samples were centrifuged to separate the precipitated proteins. 1 ml of clear supernatant was used to determine the concentration of liberated amino acids following the method of Lowry et cd. (172). The optical densities were measured with “Spectronic 20” absorptiometer set at 610 nm against a blank similarly prepared using 0.01 (N) hydrochloric acid instead of diluted gastric juice. The peptic activity was expressed in terms of Umol tyr / ml and output was calculated by multiplying each value with the respective volume of gastric juice as expressed in terms of ml/ioog/4h. Student’s “t” test was applied to evaluate the data. (26) RESULTSi Results are ^ven in and from Table - 9 to Table -16. Salicylic acid induced ulcer ; Effect of Nirmali iStrychnos potatorum linn.) on gastric secretion, gastric acidity and peptic activity. Table - 9 shows the effect of Nirmali (Strychnos potatorum Linn.) on rate of gastric secretion, gastric acidity and peptic activity in salicylic acid induced peptic ulcer in albino rats. It appears from the table that no significant change m gastric juice volimie, gastric acidity and peptic activity was observed in the animals received salicylic acid (100 mg/kg once daily for 3 consecutive days intraperitoneally) when compared to that of control values. In Nirmali fed group powdered seed of Nirmali (Strychnos potatorum linn.) was given to the rats in the dose of 1 g/kg/day for 3 consecutive days along with salicylic acid. A low trend in the rate of gastric secretion, output in gastric acidity and peptic activity was observed when compared to the values of saliqrlic acid treated group but the results were not statistically significant. Aspirin induced ulcer : Effect of Nirmali iStrychnos potatorum linn.) on gastric secretion, gastric acidity and peptic activity. Results are given in Table - 10. Aspirin, in the dose of 100 mg/kg once daily for 3 consecutive days when injected intraperitoneally to the albino rats, produced ulcer in the stomach (ulcer index, 23) but did not exert any significant effect in the rate of gastric secretion, gastric acidity and peptic activity when compared to the data of control animals. Nirmali (Strychnos potatorum Linn.) treatment in the oral dose of 1 gm/kg/day for three consecutive days along with aspirin reduced the ulcer index to 7.3 but did not exert any significant change in the values of rate of gastric secretion, gastric acidity and peptic activity as compared to that of aspirin treated group. Paracetamol induced ulcer : Effect of Nirmali (Strychnos potatorum linn.) on gastric secretion, gastric acidity and peptic activity. Table - 11 shows the effect of Nirmali (Strychnos potatorum liim.) on rate of gastric secretion, gastric acidity and peptic activity in paracetamol induced peptic ulcers in albino rats. Paracetamol, when given to the rats through intraperitoneal injection (dose, 100 mg/kg once daily for 3 consecutive days) did not exert any significant change in the values of gastric juice volume, gastric acidity and peptic (27) X <-N ■w f-. o 00 o V© 00 VO o 00 +1 ® +1 oo +1 00 I f QQ 1 p 00 r-H 0\ r - 2 .S m o« o On oo ON +1 00 +1 -a I 1 00 o L o >n r~ VO 0 « ON o o I , a u ’■« 13I. UI tcn oo I o CN 1 I tN S 5 t^ +1 00 +1 tS SS +t *B 1 ' 1 CIh so 00 «o & •S <=> ::^ d o^ ^ o ^ 1/^ VO 00 Os so 00 S Os o l - i s cn o cs fN +1 (S +1 22 d ^ o o OO cn § S S3 ®I—1 o o •« 1 00 o sS so O y . a CS +1 csi +1 CO* 1*s« jO c/5 . . 'co*0> % .3 § a CQK• r\ W*' C/3 +1 a ICO I P!h activity when compared to the values of control animals. Nirmali iStrydmos potatorum linn.) when given in the oral dose of i g/kg/day for 3 consecutive days along with paracetamol did not also exert any significant change in the values of gastric juice volume, gastric acidity and peptic activity when compared to that of paracetamol treated group. Indomethacin induced ulcer ; Effect of Nirmali iStrychnos potatorum linn.) on gastric secretion, gastric acidity and peptic activity. Table -12 shows the effect of Nirmali {Strychnos potatorum linn.) on rate of gastric secretion, gastric acidity and peptic activity in indomethacin induced peptic ulcer in albino rats. It appears fi:om the table that no significant change in gastric juice volume, gastric acidity and peptic activity v»^s observed in the animals received indomethacin (25 mg/kg once daily for 3 consecutive days intraperitoneally) when compared the data with that of control animals. In Nirmali fed group powdered seed of Nirmali (Strychnos potatorum linn.) was given to the rats in the dose of 1 g/kg/day for 3 consecutive days along with indomethacin. A low trend in the rate of gastric secretion, output in gastric acidity and peptic activity was observed when compared to that of indomethacin treated group but the results were not statistically significant. Hydrocortisone induced ulcer: Effect of Nirmali (Strychnos potatorum linn.) on gastric secretion, gastric acidity and peptic activity. Results are given in Table -13 . Hydrocortisone, in the dose of 50 mg/kg once daily for 3 consecutive days when injected intraperitoneally to the albino rats, produced ulcer in the stomach (ulcer index, 25) but did not exert any significant effect in the rate of gastric secretion, gastric acidity and peptic activity when compared the values to that of the control animals. Nirmali (Strydinos potatorum Linn.) treatment in the oral dose of 1 gm/kg/day for three consecutive days along with hydrocortisone reduced the ulcer index to 14 but did not exert any significant change in the values of rate of gastric secretion, gastric acidity and peptic activity as compared to the values of hydrocortisone treated group. Prednisolone induced ulcer : Effect of Nirmali iStrychnos potatorum linn.) on gastric secretion, gastric acidity and peptic activity. Table -14 shows the effect of Nirmali iStrychnos potatorum linn.) on rate of (28) .a is VO00 I O V©o ei) •?: S S (S 00 +1 0 0 + 1 00 +1 d .2 1 o £ •13 Dh D eu o r - o o 0 0 O • c (N cn cn f-H ^ c<^ +1 +1 m +1 OD I 'S § 1 ^ CN 2 «« rt O ^ O 4» d CN CN VO Os «n +1 t- ®o & 00 si 0 0 + 1 § a . V I s 2i o l l q o ^ >o CN § a 3 ^ 00 s: *3 VO ® IT) O «o o ii 51* S fl II CN S ^ D .3 CN 00 • l - - t/3 ® 'S (N O ti --S 'sl ro m cn % « II *0 ^ S. t(L>/5 a 3 bO s f + o m >n I 11 Q CN CN '‘B 24 o .. « ,£ s e2 a f o U 'B rt 2 .9 VO Ov VO ^ e ^ cs l | CS +1 (N +1 i ' 4 VO .s & ! © Ov a 00 VO o| >n II o u-> Ov cn m T~K/l § J 1 i f I I p I o ■I +• a Co Q U I 'f—w ^ *5 VO tN ss ®0. td> s CL, 'sO m II wi ;□ .3 (S ‘s:s iri fS II 00 se (S CN (N « re II T(U3 !> .r^ f s ’ + O o o o o «3It *5 Q ir> 1 § X) o e2 I o •5i l ^ ' i s U J3 Z ^ g _ Oh 'SJ r -4 >o at '^. CN fS oo' M «n r^ 0®t-' ^ 00 VO in o I« m m « & jd ^ * a, -ts I I 08 i op I § Si O 1 1 * ro «3 t/j *a Q m a V© A p I U gastric secretion, gastric acidity and peptic activity in prednisolone induced peptic ulcers in albino rats. Prednisolone, when given to the rats through intraperitoneal injection (dose, 30 mg/kg once daily for 3 consecutive days) did not exert any significant change in the values of gastric juice volume, gastric acidity and peptic activity when compared to that of control animals. Nirmali (Strychnos potatorum linn.) treatment in the oral dose of 1 gfkg/day for 3 consecutive days along with prednisolone did not also exert any significant change in the values of gastric juice volume, gastric acidity and peptic activity when compared to the values of prednisolone treated group though ulcer index came down to 10.2 from 25. Phenylbutazone induced ulcer: Effect of Nirmali (Strychnos potatorum linn.) on gastric secretion, gastric acidity and peptic activity. Table - 15, relating to the effect of Nirmali iStrychnos potatorum linn.) on rate of gastric secretion, gastric acidity and peptic activity in phenylbutazome induced peptic ulcer in guinea pigs, shows that phenylbutazone in the oral dose of 100 mg/kg once daily for 3 consecutive days produced gastric ulcers (ulcer mdex, 25) in the animals but it was not associated with any significant change in the levels of gastric juice volimie, gastric acidity and peptic activity in comparison to the control values. These parameters again showed no significant changes when Nirmali (Strychnos potatorum linn.) was given to the animals orally in the dose of 1 gm/kg/day for 3 consecutive days along with phenylbutazone (in comparison to the data obtained in phenylbutazone group). Ulcer index, however, came down to 14. Histamine induced ulcer : Effect of Nirmali iStrychnos potatorum Linn.) on gastric secretion, gastric acidity and peptic activity. Results are given in Table - 16. Histamine, in the dose of 33 micro gram/mouse once daily for 3 consecutive days when injected intraperitoneally to the mice, produced ulcer in the stomach (ulcer index, 28) but did not exert any significant effect in the rate of gastric secretion, gastric acidity and peptic activity when compared to that of control values. Nirmali {Strychnos potatorum linn.) treatment in the oral dose of 1 gm/kg/day for three consecutive days along with histamine reduced the ulcer index to 10.1 but did not exert any significant change in the values of rate of gastric secretion, gastric acidity and peptic activity as compared to the values of histamine treated group. ## (29) CHAPTER - III STUDIES ON DISSOLVED GASTRIC MUCIN DURING ULCERATION ROLE OF NiraiAALI (Stiychnos potatorum Linn.) CHAPTER - III To Study the effect of Nirmali iStrychnos potatorum linn.) on dissolved gastric mucin during drug induced ulceration, collected gastric juice samples (page - 18) were analysed by the following methodologies. Nature of ulcerogenic drugs, types of animals, doses of drugs and seeds of Nirmali (Strychnos potatorum Lirm.) route of administration, duration of anaesthesia and operative techniques were essentially same as described in Chapter - 1. MATERIALSi For dissolved gastric mucin: a) Ethyl alcohol: 95% ethyl alcohol (Bengal Chemical) was used. b) 0.1 (N) aqueous sodium hydroxide was prepared fresh at the time of analysis. c) 0.1 (N) sulphuric acid was prepared fresh at the time of analysis by diluting concentrated sulphuric acid stepwise with distilled water. (30) d) 6 (N) hydrochloric acid was prepared from concentrated hydrochloric acid (BDH, Analar) and kept in glass stoppered bottle. For total hexose estimation: a) Orcinol (E. Marck) : 1.6% in a solution of distilled water was prepared and stored in refrigerator. b) Sulphuric acid - water mixture : This was prepared by adding 3 volumes of concentrated sulphuric acid (Analar, BDH) to 2 volumes of distilled water slowly with constant stirring. It was kept in a glass stoppered bottle and stored in refrigerator. c) Orcinol - sulphuric acid mixture : This was made by adding one volume of 1.6% orcinol solution to 7.5 volumes of sulphuric acid - water mixture (3 : 2, v/v) only at the time of estimation. d) Galactose, Mannose (BDH) : These were used as standards for total hexose estimation. For hexosamine estimation : a) Hydrochloric acid : 0.2 (N) and 6 (N) solution were prepared from concentrated hydrochloric acid (Analar, BDH) by diluting with distilled water and stored in glass stoppered bottles separately. b) Sodium hydroxide (E. Merck) : 4 (N) solution was prepared by dissolving pallets in distilled water and stored in a glass bottle. c) Phenolpthalin (E. M erck): 1% alcoholic solution was prepared and stored in glass stoppered bottle. d) Acetyl acetone reagent: 0.3 ml acetyl acetone (E. Merck) and 9.7 ml of 1.5 (N) sodium carbonate (Analar, BDH) were mixed just before use. e) Ethanol (Bengal Chemical) was dehydrated with lime and distilled before use. f) Ehrlich’s reagent : This reagent was made by dissolving 1.6 g of p-dimethyl amino ben2aldehyde (E. Merck) in 30 ml of 95% ethanol and then was added 30 ml of concentrated hydrochloric acid. This reagent was kept in refrigerator. g) D(+) Glucosamine hydrochloride (BDH) was used as standard in hexosamine estimation. For methyl pentose estimation : a) Ethanol was dehydrated with lime and distilled before use. (31) b) Sulphuric acid - water mixture : 6 volumes of concentrated sulphuric acid (Analar, BDH) was added to i volume of distilled water slowly with constant stirring. It was kept in a glass stoppered bottle and stored in refrigerator. c) Cysteine reagent: 600 mg cysteine hydrochloride (E. Merck) was dissolved in 20 ml of distilled water. It was prepared weekly, stored in refrigerator. d) Methyl pentose standard : 5 mg of methyl pentose (Sigma) was dissolved in 50 ml of distilled water in a volumetric flask. 1 ml of this solution was diluted to 2.5 ml of distilled water to make it suitable for standard concentration. It was stored in refrigerator. For sialic acid estimation : a) Sodiimi periodate, meta (BDH) : 0.2 M in 9 M orthophosphoric acid (BDH) solution was prepared and stored in glass stoppered bottle. This was prepared every 20 days. b) Sodium arsenite (E. Merck) : 10% in a solution of 0.5 M sodium sulphate (Analar, BDH) and stored in glass stoppered botfle. This was prepared every 20 days. c) Thiobarbituric acid : 0.6% of thiobarbituric acid (E. Merck) in a solution of 0.5 M sodium sulphate (Analar, BDH) was prepared and filtered. It was kept in glass stoppered bottle. This was also prepared every 20 days. d) Cyclohaxanone (Reanel, Budapest) : Original soluion was used. METHODOLOGIES Analysis of dissolved gastric mucin Dissolved gastric mucin was analysed according to the method of Sanyal et al. (173). To 0.5 ml of clear gastric juice, 5 ml of 95% ethyl alcohol was added. After 10 minutes it was centrifuged at 5000 r.p.m. for a period of 10 minutes and then the supernatant was drained out. The precipitate was allowed to dry in the tube for next 10 minutes. The precipitate was dissolved in 0.5 ml of 0.1 (N) sodium hydroxide. 0.2 ml of this solution was then hydrolysed with 0.6 ml of 0.1 (N) sulphuric acid at 100 degree centigrade for 1 hour to liberate sialic acids ftt)m dissolved mucin. Remaining 0.3 ml of the solution was hydrolysed with 0.9 ml of 6 (N) hydrochloric acid at 100 degree centigrade for 2 hours to liberate neutral and amino sugars. The hydrolysates were then neutralized by 0.1 (N) sodium hydroxide and the volume was made upto 2 ml for first aliquot and 3 ml for the second aliquot. (32) Released sialic acid in the first aliquot was estimated by thiobarbituric acid method of Warren (I74). From the second aliquot hexosamine was measured by the method of Dische and Borenfreund (175) and total hexoses, methyl pentose were estimated by the method of Dische and Shettles (176). The sum of total hexoses, hexosamine, methyl pentose and siallic acid i.e. total carbohydrate was taken as the index of the level of dissolved gastric mucin as suggested by Menguy (177), and was expressed in terms of mg/mg of dissolved mucin. Estimation of total hexoses Total hexoses of dissolved gastric mucin was estimated by the method of Dische and Shettles (176) according to the following protocol: Reagent Blank Standard Unknown Water im l Nil Nil Galactose: Mannose Solution, i:i,(o.img/ml) Nil im l Nil Orcinol - Sulphuric Acid mixture (1: 7.5) 4-25 ml 4-25 ml 4.25 ml Sample (Neutrali2ed second Aliquot, page - 32) Nil Nil im l Contents of the tubes were then heated in a boiling water bath for exactly 15 minutes, cooled and the intensities of the developed colour were recorded at 540 nm using “Spectronic 20” againt the blank. The amount of total hexose in imknown sample was calculated on the basis of standard concentration and was expressed in terms of mg/mg of dissolved mucin. Estimation of hexosamine Hexosamine of dissolved gastric mucin was estimated by the method of Dische and Borenfreund (175) according to the following protocol. Reagent Blank IStandard Unknown Water im l Nil Nil Glucosamine Solution (0.05 mg/ml) Nil im l Nil Sample (Neutralized second Aliquot, page - 32) Nil Nil im l Acetyl acetone im l im l im l (33) Contents of the tubes were heated for 20 minutes in a boiling water bath and then cooled. Then Ethyl alcohol 3 ml ' 3 ml 3 ml Ehrlich’s reagent 1 ml 1 ml 1 ml Tubes were then kept at room temperature for 30 minutes and the intensities of the developed colour were recorded at 530 nm using “Spectronic 20” against the blank. The amount of hexosamine in unknown sample was calculated on the basis of standard concentration and was expressed in terms of mg/mg of dissolved mucin. Estimation of methyl pentose Methyl pentose of dissolved gastric mucin was estimated by the method of Dische and Shetteles (176) according to the following protocol: Reagent Blank IStandard Unknown Water im l Nil Nil Methyl pentose Solution (0.02 mg/ml) Nil im l Nil Sample (Neutralized second Aliquot, page - 32) Nil Nil im l Sulphuric acid: water Mixture 4-5 ml 4.5 ml 4-5 ml Contents of the tubes were heated in a boiling water bath for exactly 3 minutes and then colled. Then Cysteine reagent 0.1 ml 0.1 ml 0.1 ml Contents of the tubes were mixed thoroughly and kept at room temperature for 1 hour. After 1 hour intensities of the developed colour were recorded at 396 nm and at 430 nm using spectrophotometer setting the instrument with blank. One more vmknown tube was prepared exactly in the same way described above without adding cysteine reagent. This was a correction for any non-specific colour as suggested by Dische and Shetteles (176). The amount of methyl pentose in imknown sample was calculated from the difference in reading obtained at 396 nm and 430 nm and substracting the values without cysteine as under: Concentration of methyl pentose in unknown sample ( A - A ’) - ( B - B ’) X Strength of the standard solution x Dilution factor. (C - C’) (34) Where A = Optical density of unknown sample at 396 nm with cysteine A’ = Optical density of unknown sample at 430 nm with cysteine B = Optical density of iinknown sample at 396 nm without cysteine B’ = Optical density of unknown sample at 430 nm without cysteine C = Optical density of unknown solution at 396 nm C’= Optical density of unknown solution at 430 nm. The amount of methyl pentose in unknown sample was expressed in terms of mg/mg of dissolved mucin. Estimation of sialic acid Sialic acid of dissolved gastric mucin was estimated by the method of Warren (174) according to the following protocol: Reagent Blank [Standard Unknown Water im l Nil Nil Sialic acid solution (0.05 mg/ml) Nil im l Nil Sample (Neutralized first Aliquot, page - 32) Nil Nil im l Sodium periodate Solution 0.1ml 0.1ml 0.1ml Contents of the tubes were mixed up properly and kept for 20 minutes at room temperature, then Sodium arsenite solution iml iml im l Thiobarbituric acid solution 3ml 3inl 3m l Contents of the tubes were then heated in a boiling water bath for exactly 15 minutes, cooled, then, Cyclohexanone 4-3 ml 4-3 ml 4-3 ml Contents of the tubes were again mixed well and centrifuged. Cyclohexanone layer was carefully taken out and its colour intensity was recorded at 532 nm and at 550 nm using “Spectronic 20” against the blank. The amount of sialic acid in unknown sample was calculated from the difference in reading obtained at 532 nm and at 550 nm on the basis of standard concentration and expressed in terms of mg/mg of dissolved mucin. Student’s “t” test was applied to evaluate the data. (35) RESULTS Results are given in and from Table -17 to Table - 24. Salicylic acid induced ulcer : Effect of Nirmali iStrychnos potatorum linn.) on dissolved gastric mudn. Effect of N irm ali {Strychnos potatorum linn.) on the carbohydrate components of dissolved gastric mucin in salicylic acid induced peptic ulcers in albino rats was given in Table — 17. Salicylic acid in the dose of 100 mg/kg, when given to the rats intraperitoneally once daily for three consecutive days, caused a significant decrease in the levels of hexosamine (p< 0.002), methyl pentose (p< 0.001), sialic acid (p < 0.001) and thus in total carbohydrate (p < 0.001) of dissolved gastric mucin when compared to those values of control animals. Total hexose part of dissolved gastric mucin, however, did not show any change. When Nirmali {Strychnos potatorum linn.) was given to the rats orally in the dose of 1 g/kg/day for three consecutive days along with salicylic acid, significant increase in the levels of hexosamine (p < 0.001), methyl pentose (p < 0.01), sialic acid (p < 0.001) and thus in total carbohydrate (p < 0.001) of dissolved gastric mucin was observed when compared to those of salicylic acid treated group. No significant increase was, however, noted in total hexose concentration after Nirmali iStrychnos potatorum linn.) treatment. Aspirin induced ulcer ; Effect of Nirmali (Strychnos potatorum Linn.) on dissolved gastric mucin. Results are given in Table - 18. Aspirin in the dose of 100 mg/kg once daily for three consecutive days when given to rats intraperitoneally decreased the levels of total hexoses, hexosamine, methyl pentose, sialic acid and thus total carbohydrate of dissolved gastric mucin. Excepting total hexose all values were statistically significant (p < 0.001) when compared to those of control animals. In addition with aspirin when Nirmali (strychnos potatorum linn.) was given to rats in the oral dose of 1 gm/kg /day for three consecutive days, significant increase in the levels of hexosamine (p < 0.001), methyl pentose (p < 0.001), sialic acid (p < 0.001) and thus in total carbohydrate (p < 0.001) of dissolved gastric mucin was observed when compared the values to those obtained in aspirin treated group only. Total hexose part of dissolved gastric mucin in this case did not show any significant change in comparison to that of aspirin treated group. (36) * * «4H ■» * * * 00 q J tn (S •I P4 ^ £ I «n ® § »n cn cn cn cn %'BI' M « ^ M t 3 c/3(U M’S d •M ^ f I S o + ja .s o o I (ZJ ■« Q o ll .a I I p& f z e2 II I •^3 '■3 + &Q O u c/2 cn a .' -o « « # * *0 * » ^ 2 .a o o. 00 o 00 O I3 «g w§31. o . cn o © (S O e d +1 d +1 d -H I I % -fi g) g o * * * fo * V) o © ♦ © s 00 q (s q * S II o © o © So o® ^ c d -H d +1 d +1 2 ■o A ^ * « eS 2 .3 * CO * CN •r?_ CO« o * s O «o q ts q * Sq I "S "S O O o O o © € a d ** 'o d +1 d -H d +1 S . s * * I s * * -3 * '-H * 3 o »-H O o VO ® ^ ®. VO © i-^ o d o o o o' III ®- d +1 d +1 -H 0^ 1 « m CS ^ g O o q fS © o s o r-H o t-H © II H a o +1 d +1 d +1 ^ ' S . cn CO I (N r^’ Z .9 «n F—i4 "ts aI •o cn m m 1 - 1 ^ w I II I o o + ja o o I (/5 Q 00 o S o I I I < Paracetamol induced ulcer ; Effect of Nirmali (Strychnos potatorum. Linn.) on dissolved gastric mucin. Effect of Niraiali (Strychnos potatorum Linn.) on the carbohydrate components of dissolved gastric mucin in paracetamol induced peptic ulcers in albino rats was given in Table - 19. Paracetamol in the dose of 100 mg/kg, when given to the rats intraperitoneaUy once daily for three consecutive days, caused a significant decrease in the levels of hexosamine (p< 0.001), methyl pentose (p< 0.001), sialic acid (p < 0.001) and thus in total carbohydrate (p < 0.001) of dissolved gastric mucin when compared to those values of control animals. Total hexose part of dissolved gastric mucin, however, did not show any change. When Nirmali (Strychnos potatorum Linn.) was given to the rats orally in the dose of 1 g/kg/day for three consecutive days along with paracetamol, significant increase in the levels of hexosamine (p < 0.01), methyl pentose (p < 0.001), sialic acid (p < 0.001) and thus in total carbohydrate (p < 0.001) of dissolved gastric mucin was observed when compared to those of paracetamol treated group. No significant increase was, however, seen in total hexose concentration after Nirmali (Strychnos potatorum Linn.) treatment. Indomethadn induced ulcer : Effect of Nirmali (Strychnos potatorum linn.) on dissolved gastric mucin. Results are given in Table - 20. Indomethacin in the dose of 25 mg/kg once daily for three consecutive days when given to rats intraperitoneaUy decreased the levels of total hexoses, hexosamine, methyl pentose, sialic acid and thus total carbohydrate of dissolved gastric mucin. Excepting total hexose all values were statistically significant (p < 0.01 to p < 0.001) when compared to those of control animals. When Nirmali (strychnos potatorum Linn.) was given in addition with indomethacin to rats in the oral dose of 1 gm/kg /day for three consecutive days, significant increase in the levels of hexosamine (p < 0.001), methyl pentose (p < 0.01), sialic acid (p < 0.001) and thus in total carbohydrate (p < 0.001) of dissolved gastric mucin was observed when compared the values to those obtained in indomethacin treated group only. Total hexose part of dissolved gastric mucin in this case did not show any significant change in comparison to that of indomethacin treated group. (37) * * ♦ * ♦ « Tt fs J5 ®. oo p es o »—I o CN O e d +1 d -H d +1 s r ^&.•§ U o I ■ X » ♦ 0 * * (N * t> 1 00 o fs) O o r ' P m p o O O o o O O I d -H d -H d +1 ti 0 * ♦ b *2 CA •S « CO ♦ 2 -5 \0 O fS o € cn X ,-H^ SO m o ^.s 'S I t i O O o d o o' ;s S1 *oI l l d +1 d +1 d +1 * s f « * * m * IT) 0I 8 00 © s I \o ^ cn o >n P 1 o d O o o o I 1 i d +1 d -H d +1 (U cn 00 I 13 o o O I o o o^ O O p § § U 2 « o O O r-^ O S H K o +1 •§ I d +1 d +1 SI■5 cn cn .s II (N ^ .s e S »n ■w S V-/ cn cn cn cn cn % • = II1 1 f o I ® o o + oo1-^ -T3 Q ON I I Io U v _ T3 !P » 4(- * * 's ii * I OJ £J O P 00 ® 00- pg 1 3 ^ ’32 1 ro o ^ o fS o *s I I 2^ o 6 +1 d +1 d +1 I u 0 •* * o VO * ts * lo ♦ 0 •» 1 •'3 •« 00 p o ^ ®. C/3 < 0 0 0 0 O O d +1 d +1 d +1 l i •xa e i . a cn * s ^ O * g P *CO Sq o I O o o O o 1 ^ 'S d +1 d +1 d -H «■ * * * •I a,« * M * rp (N O r—< O 1—1 o S -d in O CO O O O O o O O O d +1 d +1 d +1 0> m VO o00 © o S'-9 I s m o 00 o ^ O d O O o i i t 2 S O -H d +1 d +1 i | ^ ® a2 O OJX r^i Effect of Nirmali (Strydinos potatorum linn.) on the carbohydrate components of dissolved gastric mucin in hydrocortisone induced peptic ulcers in albino rats was given in Table - 21. Hydrocortisone in the dose of 50 mg/kg, when given to the rats mtraperitoneally once daily for three consecutive days, caused a significant decrease in the levels of hexosamine (p< 0.001), methyl pentose (p< 0.001), sialic acid (p < 0.001) and thus in total carbohydrate (p < 0.001) of dissolved gastric mucin when compared to those values of control animals. Total hexose part of dissolved gastric mucin, however, did not show any change. When Nirmali {Strychnos potatorum linn.) was given to the rats orally in the dose of 1 g/l^/day for three consecutive dajrs along with hydrocortisone, significant increase in the levels of hexosamine (p < 0.001), methyl pentose (p < 0.001), sialic acid (p < 0.01) and thus in total carbohydrate (p < 0.001) of dissolved gastric mucin was observed when compared to those of hydrocortisone treated group. No significant increase was, however, observed in total hexose concentration after Nirmali {Strychnos potatorum linn.) treatment. Prednisolone induced ulcer : Effect of Nirmali iStrychnos potatorum linn.) on dissolved gastric mucin. Results are given in Table - 22. Prednisolone in the dose of 30 mg/kg once daily for three consecutive days when given to rats intraperitoneally decreased the levels of total hexoses, hexosamine, methyl pentose, sialic acid and thus total carbohydrate of dissolved gastric mucin. Excepting total hexose all values were statistically significant (p < 0.001) when compared to those of control animals. In addition with prednisolone when Nirmali (strychnos potatorum Linn.) was given to rats in the oral dose of 1 gm/kg /day for three consecutive days, significant increase in the levels of hexosamine (p < 0.001), methyl pentose (p < 0.001), sialic acid (p < 0.001) and thus in total carbohydrate (p < 0.001) of dissolved gastric mucin was observed when compared the values to those obtained in prednisolone treated group only. Total hexose part of dissolved gastric mucin in this case did not show any significant change in comparison to that of prednisolone treated group. (38) * * VO * (N * os I cn r; 'O ®. 'O ®. 5 o cs o r-< O ts O Ie d +1 d -H d -H o TT d * D r- * rt * >n o s 0I 00 O * &l a «a K 1o rs) (X .S 1 tfl z .a cn V) s ts* ®I o IT) c^ fO cn ts “..I ^•| W CZ5 pM2 «&d czi 611 +1 .9 ^ f o + O «n I c/5 TS Q I fS 0^ .A o I0 p e2 go U 1 * * * * m ♦ ^ * cs tN P 00 P Os p CO o '—1 o C « * i> o r- * fS * r- o * 2 * o 1 1 ON P cn ® 00 p * * Cm IT) * 0> § 1 (N I p •n d 1 ^ 5 2 (N Oh q ■SB ^| . V l3 w t-H cn cn fS S O cn cn m “..I tti ■" T3 CZ5 • • :3 I +1* o o + I Q m I CI/3 (U a '^ § p-§ i< o fi I o I I C/5 o .a (2 u £ P-, y ■TI" 0 * * TT « 1 s * s * ::Z 0 tn P o Os ®. o> ^ o O O o o d +1 d +1 d +1 1W) I II * C/5 .3 * m 9 d _ W o * Q -o V 00 p o P (U .. 'o I ■s ■§ O O O o CO d +1 d +1 I o l l 1 ** § § o * * Ut ?■ o * o 0I ft Os p * I *ON ps d 1 o o s § o o c 4 d +1 d +1 d +1 o> 1 rS o 1 u o «o (S f1 t o •S :i «n <+H 2 w o o B oo 2 I0 ■3" * * g 00 * fo * V O 1 o o * o s I r- ®. m ®. * s .205 3 o o O O o © es I d +1 d +1 d +1 o T3 V VO ® *(N OS * s 0 I ^1 O O O O ’S d +1 d +1 d +1 w * od 5 1r> * ^ * ? Io * o ft 6 in o CN ®. *VO Sp d a O O O O o © a *’ s d +1 d +1 d +1 (L>* ■Su ■5 S ©ON cd0 o o o O ON P d §s g tU o O O o © V O +1 d +1 d +1 1«) ft ■ s l ‘m *’ s . .2 I a 3m § I o 3 2 (N00 ft I .a z .au j2 ® 3 C/3 VO 1/3 t3 S 'o > s | CO o 12 € PQ• #v « is II pq i/D 5_a ex«« •T3 C/D ■« cnD ll +1 1 3 pp 5 © Effect of Niraiali iStrychnos potatorum linn.) on the carbohydrate components of dissolved gastric mucin in phenylbutazone induced peptic ulcers in guinea pigs was given in Table - 23. Phenylbutazone in the dose of 100 mg/kg, when given to the guinea pigs orally once daily for three consecutive days, caused a significant decrease in the levels of hexosamine (p< 0.001), methyl pentose (p< 0.001), sialic acid (p < 0.001) and thus in total carbohydrate (p < 0.001) of dissolved gastric mucin when compared to those values of control animals. Total hexose part of dissolved gastric mucin, however, did not show any change. When Nirmali (Strychnos potatorum Linn.) was given to the guinea pigs orally in the dose of 1 g/kg/day for three consecutive days along with phenylbutazone, significant increase in the levels of hexosamine (p < 0.001), methyl pentose (p < 0.001), sialic acid (p < 0.01) and thus in total carbohydrate (p < 0.01) of dissolved gastric mucin was observed when compared to those of phenyl butazone treated group. No significant increase was, however, observed in total hexose concentration after Nirmali (Strychnos potatorum linn.) treatment. Histamine induced ulcer : Effect of Nirmali (Strychnos potatorum linn.) on dissolved gastric mucin. Results are given in Table - 24. Histamine in the dose of 33 microgram/mouse once daily for three consecutive days when given to mice intraperitoneally decreased the levels of total hexoses, hexosamine, methyl pentose, sialic acid and thus total carbohydrate of dissolved gastric mucin. Excepting total hexose all values were statistically significant (p < 0.001) when compared to those of control animals. In addition with histamine when Nirmali (strychnos potatorum Unn..)'w2LS given to mice in the oral dose of 1 gm/kg /day for three consecutive days, significant increase in the levels of hexosamine (p < 0.001), methyl pentose (p < 0.001), sialic acid (p < 0.001) and thus in total carbohydrate (p < 0.001) of dissolved gastric mucin was observed when compared the values to those obtained in histamine treated group only. Total hexose part of dissolved gastric mucin in this case did not show any significant change in comparison to that of histamine treated group. ## (39) C H A P TE R - IV STUDIES ON GASTRIC MUCOSAL MUCUS DURING ULCERATION ROLE OF NIRMALI (Stiychnos potatorum Linn.) CHAPTER - IV To Study the effect of Niraiali {Strychnos potatorum linn.) on gastric mucosal mucus during experimental ulcerations, collected stomachs (page - 18) were processed by the following methodologies. Nature of ulcerogenic drugs, types of animals, dose of drugs, seeds of Nirmali (Strychnos potatorum linn.) and their route of administration, duration of anaesthesia and operative techniques were essentially same as described in Chapter - 1. MATERIALSI Essentially same as described in Chapter - III (Page - 30) METHODOLOGIES Collection of gastric mucus from stomach was done by the technique of Menguy (177). The entire glandular mucosa of clean stomach was scraped off the underlying muscularis. Each mucosal sample was immediately frozen and then (40) lyophilized. After lyophilization, each sample of mucosa was carefully homogenized. One part of the homogenized sample was then hydrolysed with three parts of 6(N) hydrochloric acid at lOO degree centigrade for two hours to release neutral and aminated sugars (173). The hydrolysates were neutralized with 0.1 (N) sodium hydroxide, volume made up to 3 ml and 1 ml each was utilized for the estimation of total hexoses (176), hexosamine (175) and methyl pentose (176). Remaining part of the homogenized sample was hydrolysed with its three volumes of 0.1 (N) sulphuric acid at 100 degree centigrade for one hour to liberate the sialic acid (173)- The hydrolysate was neutralized with 0.1 (N) sodium hydroxide, volume made up to 1 ml and was used for the estimation of sialic acid by thiobarbituric acid method of Warren (174). Methodologies for the estimation of total hexoses, hexosamine, methyl pentose and sialic acid were essentially same as described in Chapter - III (page - 32 ) . The simi of total hexoses, hexosamine, methyl pentose and sialic acid i.e. total carbohydrates, taken as the index of the level of gastric mucus as suggested by Menguy (178), was expressed in terms of micro gram/ mg of dry tissue. Student’s “t” test was applied to evaluate the data. RESULTSI Results are given in and from Table - 25 to Table - 32. Salicylic acid induced ulcer : Effect of Nirmali iStrychnos potatorum linn.) on gastric mucosal mucus. Results are given in Table - 25. When salicylic acid (100 mg/kg) was given to albino rats intraperitoneally once daily for three consecutive days, significant decrease was observed in the levels of total hexoses (p < 0.01), hexosamine (p < 0.025), methyl pentose (p < 0.001) , sialic acid ( p < 0.001) and thus in total carbohydrates (p < 0.001) of gastric mucosal mucus when compared to the control data. Nirmali (Strychnos potatorum linn.) treatment (1 g/kg once daily for three consecutive days orally) along with the aforesaid doses of salicylic acid, on the other hand, significantly increased the levels of total hexoses (p < 0.01), hexosamine (p < 0.025), methyl pentose (p < 0.001), Sialic acid ( p < 0.001) and thus the total carbohydrate (p < 0.001) of gastric mucosal mucus when compared to those values obtained in salicylic acid treated rats. (41) * * * * ** CO 00 cn' -t Tf +1 (N +1 -H CO 3 ♦ * ♦ >-' * 00 I r*^ * •» (N (U O n o ' fS O m o' ' i 0 (S +1 -! +1 m' +1 1 I o g^ s 'B •g o * S) ^— D C/3 O * o * o ^ V r- * fn * ^ t3 &i 'o o o' o’ (U . . ^(U I « T) +1 <^‘ +1 'O +1 ^ t JS * «J . I o I u~i o o V 0 * cx VO 0\ o 1 t- <^' C 4' ^ +1 ^ +1 +1 (U * p. I * k5 o * * o t >—( 0\ * 00 00 '-' O U 00 fN (N 3 V +1 00 +1 -H +1 c« Dh cn 0) * o (U 8 I o O «T) I- fS O. S s ^ i ; i < n v_,vt-H 3Cfl • ^o 0 § Paracetamol induced ulcer : Effect of Nirmali iStrychnos potatorum Linn.) on gastric mucosal mucus. Results are given in Table - 27. When paracetamol (100 mg/kg) was given to albino rats intraperitoneally once daily for three consecutive days, significant decrease was observed in the levels of total hexoses (p < 0.025), hexosamine (p < 0.01), methyl pentose (p < 0.001) , sialic acid ( p < 0.001) and thus in total carbohydrates (p < 0.001) of gastric mucosal mucus when compared to the control data. Nirmali (Strychnos potatorum linn.) treatment (1 g/kg once daily for three consecutive days orally) along with the aforesaid doses of paracetamol, on the other hand, significantiy increased the levels of total hexoses (p < 0.01), hexosamine (p < 0.025), methyl pentose (p < 0.001), Sialic acid ( p < 0.001) and thus the total carbohydrate (p < 0.001) of gastric mucosal mucus when compared to those values obtained in paracetamol treated rats. Indomethacin induced ulcer : Effect of Nirmali (Strychnos potatorum linn.) on gastric mucosal mucus. Effect of Nirmali (Strychnos potatorum Linn.) on the carbohydrate components of gastric mucosal mucus in indomethacin induced peptic ulcers in (42) r * * * o * a a 4 ^ 1 ' 00 o ON »— ( o 1-H Tf rW H CO 10 1 +1 C S + 1 + 1 < D «■ * * o « A ^ S Io on Co C5 U < f! * * ♦ ^ *r - «+-( ? 1 * ( S o 3| 0 « cx ^ (50 d 1 a d +1 MD ^ * "S '—I +1 » ■Iin' t (U * § s il ' i OS •T) 0 d 3 g r-l Tj- * _ : u o s V H a r~: CN •1 fH +1 00 + ' CO a t S * ^ % -S • «> ^ 2 55 « CO CO 1 . 1 1^ fS » o •mI .s •S « ^ 3 ^ I) Cm U e s ,A o f2 I + i5 o albino rats was given in Table - 28. It appeared from the table that indomethacin when given to the rats intraperitoneally in the dose of 25 mg/kg/day for three consecutive days caused a significant decrease in the levels of total hexoses, hexosamine, methyl pentose, sialic acid and, thus, in total carbohydrate of gastric mucosal mucus. Values were statistically significant ( p < 0.025 to p < 0.001) when compared to those for control animals. In addition to this dose of indomethacin when Nirmali {Strychnos potatorum Linn.) in the dose of 1 g/kg/day orally was given to the rats, significant increase (p < 0.025 to p <0.001) was found in the levels of total hexoses, hexosamine, methyl pentose, sialic acid and in total carbohydrate of gastric mucosal mucus when compared to the values of indomethacin group. Hydrocortisone induced ulcer: Effect of Nirmali {Strychnos potatorum Linn.) on gastric mucosal mucus. Results are given in Table - 29. When hydrocortisone (50 mg/kg) was given to albino rats intraperitoneally once daily for three consecutive days, significant decrease was observed in the levels of total hexoses (p < 0.025), hexosamine (p < 0.01), methyl pentose (p < o.ooi) , sialic acid ( p < 0.001) and thus in total carbohydrates (p < 0.001) of gastric mucosal mucus when compared to the control data. Nirmali (Strychnos potatorum Linn.) treatment (1 g/kg once daily for three consecutive days oraUy) along with the aforesaid doses of hydrocortisone , on the other hand, significantly increased the levels of total hexoses (p < 0.01), hexosamine (p < 0.001), methyl pentose (p < 0.001), Sialic acid ( p < 0.001) and thus the total carbohydrate (p < 0.001) of gastric mucosal mucus when compared to those values obtained in hydrocortisone treated rats. Prednisolone induced ulcer : Effect of Nirmali iStrychnos potatorum linn.) on gastric mucosal mucus. Results are given in Table - 30. It appeared from the table that prednisolne when given to the rats intraperitoneally in the dose of 30 mg/kg/day for three consecutive days caused a significant decrease in the levels of total hexoses, methyl pentose, sialic acid and in total carbohydrate of gastric mucosal mucus. Values were statistically (43) * * * * * * O «n o -r +1 m’ ^ 0 't +1 (N o U H m m 1 « 2 S •S3 S 0> M a 3 •S -g (t> + ^ 5 OCA Q C»■§ I M •S 90 Q fNI § - I A •s; i -3 ^ ^ e2 p sC I o -§ U I ^ s « * r I * o t - 00 ^ CN vq I o ^ 2 'S V in +1 "=»■ p o o o a +1 (N Tt +1 - -P ?P ^ 5 S '-O u g o * n * * y I CO O o * 0) is -3 Os o' -aC/3 c/3 c +1 o 0 fS +1 (N +1 (U 1 -5 o a '^• * 1 10 B 3 o' * 1 * ^ V 0 * * . a fS 1-H CO g o * cn * * SCN I .2 o * '-' V3 < o o ON O V3 +1 (N +1 0 rn +1 I o ?■ o O h cn m o m c (U fs o «n o fS 1. li a '^-1 cn m (N 2 ® m CO “I pq C/3 • ■ -d(O c« + 1 * 3 + I I cu £i u cu significant ( p < 0.025 to p < 0.001) when compared to those for control animals. This dose of prednisolone, however, did not exert any effect on the level of hexosamine of gastric mucosal mucus. In addition to this dose of prednisolone when Nirmali {Strychnos potatorum linn.) in the dose of 1 g/kg/day orally was given to the rats, significant increase (p < 0.01 to p < 0.001) was found in the levels of total hexoses, methyl pentose, sialic acid and in total carbohydrate of gastric mucosal mucus when compared to the values of prednisolone group. Hexosamine part of gastric mucosal mucus did not show any significant change by Nirmali (Strychnospotatorum linn.). Phenylbutazone induced ulcer: Effect of Nirmali (Strychnospotatonan Linn.) on gastric mucosal mucus. Results are given in Table - 31. It appeared from the table that phenylbutazone when given to the guinea pigs orally in the dose of 100 mg/kg/day for three consecutive days caused a significant decrease in the levels of total hexoses, methyl pentose, sialic acid and in total carbohydrate of gastric mucosal mucus. Values were statistically significant (p < 0.001) when compared to those for control animals. This dose of phenylbutazone , however, did not exert any effect on the level of hexosamine of gastric mucosal mucus. In addition to this dose of phenylbutazone when Nirmali (Strychnos potatorum linn.) in the dose of 1 gAg/day was given orally to the guinea pigs , significant increase (p < 0.001) was found in the levels of total hexoses, methyl pentose, sialic acid and in total carbohydrate of gastric mucosal mucus when compared to the values of phenylbutazone group. Hexosamine part of gastric mucosal mucus did not show any significant change by Nirmali (Strychnos potatorum Linn.). Histamine induced ulcer : Effect of Nirmali (Strychnos potatorum linn.) on gastric mucosal mucus. Results are given in Table - 32. When histamine (33 microgram/mouse) was given to albino mice intraperitoneally once daily for three consecutive days, sigmficant decrease was observed in the levels of total hexoses (p < 0.025), hexosamine (p < 0.001), methyl pentose (p < 0.01), sialic acid ( p < 0.001) and thus in total carbohydrates (p < 0.001) of gastric mucosal mucus when compared to the control data. (44) * * * <4-(o I - (N 00o +1 os o « o + 1 « o + 1 a d o CA s o * c +1 o i «o +1 2 .Sf> 0 U i * 1 o I 0 ^‘1e« ®* * 100 * O n ^ . a VO •» i n 0 ON O m o cfl K +1 +1 § I a o* 1 J ®- 0 s "S 1 00 q ^ « X ^a ^f c« Oh « n 3 CN U 2 S 'O ^ I dJ M I + % o IIe <» o o Q 5. d M c I • ^ o I sa 13|M4 o s U I f IPi iz: 2 3 / ’^3 * P. I I - VO cn (S fT> XI o I u Nirmali (Strychnos potatorum linn.) treatment (i g/kg once daily for three consecutive days orally) along with the aforesaid doses of histamine, on the other hand, significantly increased the levels ofhexosamine(p < o.ooi), methyl pentose (p < 0.025), sialic acid ( p < 0.001) and the total carbohydrate (p < 0.001) of gastric mucosal mucus when compared to those values obtained in histamine treated mice. Total hexose part of gastric mucosal mucus did not show any significant change by Nirmali (Stryc/inos potatorum Linn.). ## (45) €HAI*TGK - V STUDIES ON LIPID PEROXIDATION AND ON NUCLEIC ACID IN GASTRIC MUCOSA DURING ULCERATION ROLE OF NIRMALI (Stiychnos poUAomm Una.1 CHAPTER - V In ulcer research much attention has recently been focused on oxj^en derived free radicals which play an important role in the pathogenesis of acute experimental gastric lesions induced by ischemia reperfusion (179), stress (180), ethanol (181) and non steroidal anti inflammatory drugs (182,118 - 123). Antioxidants, on the other hand, promote the healing of chronic gastric ulcer (124 -126). CbQTgen derived free radicals caused tissue injury through lipid peroxidation. Oxygen handling cells have different systems which are able to protect them against the toxic effects of ojQ^en derived free radicals. If the generation of free radical exceeds the ability of free radical scavenging enzymes to dismute the radicals the gastric mucosa maybe injured by free radicals (118,120-121). To study the effect of Nirmali (Strychnos potatorum linn.) on lipid peroxides during drug induced ulceration, collected stomachs of the a n im a ls were processed for the estimation of lipid peroxides. Stomachs were also processed for the (46) estimation of deoxy ribonucleic acid (DNA). Standard methodologies followed are given below. Nature of ulcerogenic drugs, type of animals, doses of drugs, seeds of Nirmali (Strychnos potatorum linn.) and their route of administration, duration of anaesthesia and operative techniques were essentially same as described in Chapter - 1. METHODOLOGIES Preparation of tissue homogenate Excised rat stomachs were washed with 0.9% sodium chloride. Tissue homogenates were prepared in a ratio of 1 g of mucosal tissue scrapped from the stomach to 9 ml of 1.15% potassium chloride by using Teflor Potter - Elvehjem homogenizer. Estimation of lipid peroxides This was estimated by the method of Okhawa et al. (183). To 0.2 ml of 10% (W/V) tissue homogenate were added 0.2 ml of 8.1 % sodium dodecyl sulfate, 1.5 ml of 20% acetic acid solution adjusted to pH 3,5 with sodium hydroxide and 1.5 ml of 0.8% aqueous solution of thio barbituric acid (TBA). The mixture was made up to 4.0 ml with distilled water and then heated in an oil bath at 95 degree centigrade for 60 minutes using a glass ball as a condenser. After cooling with tap water, 1.0 ml of distilled water and 5.0 ml of the mixture of n - butanol and pyridine (15:1, V/V) were added and shaken vigorously. After centrifugation at 4000 r.p.m. for 10 minutes, the organic layer was taken and its absorbance at 532 nm was measured. i,i,3,3, - tetramethoxy propane (TMP) was used as external standard and results were expressed in terms of n mol MDA (Malondialdehyde) per mg of tissue protein. TMP was collected from Tokyo Kasel Kogyo Ltd. , Tolq^o, Japan ; TBA from BDH, England and all other reagents were from E. Merck. Estimation of DNA (Deoxy ribonucleic acid) Before estimation, DNA was separated from the stomach tissue by the method of Schneider (184). Step - 1 : Removal of acid - soluble compounds. 1 ml of 20% tissue homogenate was mbced with 2.5 ml of cold 10% trichloro acetic acid and centrifuged. The precipitate was resuspended in 2.5 ml of cold 10% trichloro acetic acid and centrifuged. The combined extracts constitute the acid soluble phosphorus fraction and was discarded. (47) Step - 2 :Removal of phospholipids The tissue residue was suspended in i.o ml of water, mixed with 4 ml of 95% ethanol and centrifuged. The residue was resuspended in 5 ml of ethanol and centrifuged. It was then extracted three times with three portions of 3:1 alcohol: ether at room temperature with brief stirring. The combined extracts constitute the phospholipid phosphorus fraction and was discarded. Step - 3 : Removal of ribonucleic acid (RNA) The residue was treated for 16-20 hours at 37 degree centigrade with 10 ml of 1 (N) potassium hydroxide. The solution was then neutralized with 6 (N) hydrochloric acid. Total volume was noted and the same amount of 5% trichloro acetic acid was added to get the precipitate of DNA and protein. The centrifuged precipitate was washed with 5 ml of 5% trichloro acetic acid and the extracts were combined to give the RNA fraction plus the inorganic phosphate derived from phosphoprotein. The extract was discarded. Step - 4: Removal of deoxy ribonucleic acid (DNA) The residue from the above step was suspended in 5 ml 5% trichloro acetic acid, heated for 15 minutes at 90 degree centigrade. The residue was resuspended in 2.5 ml of 5% trichloro acetic acid and centrifuged. The extracts were combined to form the DNA fraction. The residue was the ‘protein’ fraction and was discarded. Step - 5: Diphenylamine reaction for the estimation of DNA This was done by the method of Dische and Schwarz (185). The reagent was prepared by dissolving i.o g of diphenylamine in 98 ml of redistilled glacial acetic acid and then adding 2 ml of concentrated sulphuric acid. The reagent was kept cold and was used within 2 days after preparation. 5 ml of the above extract (from step - 4) were added to 2.5 ml of the reagent and the mixture was heated for 5 minutes in a boiling water bath. The sample was cooled and read at 540 nm in a Beckman spectrophotometer using white light and silica cells. A similarly treated blank was used for the zero setting. The DNA concentration was calculated by comparing the value with a standard DNA solution. Results were expressed in terms of mg per gram of mucosal tissue. DNA was collected from Sisco Research Laboratory Pvt. Ltd., Bombay and all other reagent were from E. Merck. Estimation of protein of mucosal tissue scrapped from the stomach This was done by biuret method (186). Reagents : (48) A. SULPHATE - SULPHITE SOLUTION: To a volumetric flask were added 208 gram of sodium sulphate, 70 gram of sodium sulphite, 900 ml of water and 2 ml of concentrated sulphuric acid. Volume was made up to one litre after proper mixing. pH of the solution was above 7. B. BIURET SOLUTION: To 45 gram of Rochele salt were added about 400 ml of 0.2 (N) sodium hydroxide and 15 gram of copper sulphate. Components were mixed well. 5 gram of potassium iodide were added and the volume of the solution was made up to one litre with 0.2 (N) sodium hydroxide. The prepared solution was diluted (1:4, V/V) with 0.2 (N) sodium hydroxide. Method: To 0.4 ml of tissue homogenate 6 ml of sulphate-sulphite solution were added. The solution was mixed well and 2 ml of the solution was immediately mixed with 5 ml of the biuret solution. After proper mixing the tube was places in a boiling water bath for 10 minutes. Tube was then cooled and the reading was taken in the absorptiometer at 555 nm. Standard tube was made by using standard protein collected from the Armour Laboratories, Sussek. All other reagents were from E. Merck. Student’s “t” test was applied to evaluate the data. RESULTS Results are given in and from Table-33 to Table-40. Salicylic add induced ulcer : Effect of Nirmali (Strychnos potatorum Linn.) on lipid peroxides and DNA of the gastric mucosa. Results are given in Table - 33. It appears from the table that salicylic acid in the dose of 100 mg/kg, when given to albino rats intraperitoneally once daily for 3 consecutive days, increased the lipid peroxide level of gastric mucosa. DNA content of the mucosa, on the other hand, found decreased under the treatment of salicylic acid. The results were significant (p (49) * * « * « * VO o F—( On ts o o 4H I +1 cd © O 1 o a . • ^ 12 C/) « ed * * * I O * g S ON * ro o ©■ 2 .S +1 +1 d & O +1 V ON d cs § I CO r t I tS & 0 O- g^ "O « e 9 i-i 1 u u u q I-S^ - 3 S 3 «n ;d e« § z|1 *>9 *0 c/5 CO I I C/5 U Aspirin induced ulcer : Effect of Nirmali iStrychnos potatorum linn.) on lipid peroxides and DNA of the gastric mucosa. Results are shown in Table - 34. Increase in the level of lipid peroxides of gastric mucosa was observed when aspirin in the dose of 100 mg/kg was given to rats intraperitoneaUy once daily for three consecutive days. DNA content of the gastric mucosa under this condition was found decreased. Results were statistically significant (p < 0.001) when compared to the control values. Nirmali (Strychnos potatorum linn.) treatment in the dose of 1 g/kg orally once daily for three consecutive days along with aspirin decreased the level of lipid peroxides and increased DNA content of the gastric nucosa. Results were statistically significant (p < 0.001) when compared to the values of aspirin group. Paracetamol induced ulcer : Effect of Nirmali (Strychnos potatorum linn.) on lipid peroxides and DNA of the gastric mucosa. Results of the effect of Nirmali (Strychnos potatorum linn.) on lipid peroxides and DNA content of gastric mucosa in paracetamol induced ulcer in albino rats are given in Table - 35. It appears from the table that paracetamol (100 mg/kg) when given to rats intraperitoneaUy once daily for three consecutive days caused an increase in the level of lipid peroxides of gastric mucosa. DNA content of the gastric mucosa, however, decreased imder the same condition. Results were statistically significant (p < 0.001) when compared to the control values. Nirmali (Strychnos potatorum linn.) treatment (1 orally for 3 consecutive days along with paracetamol) decreased the level of lipid peroxides and increased DNA content of the gastric mucosa. Results were statistically significant (p < 0,001) when compared to the values of paracetamol group. Indomethacin induced ulcer : Effect of Nirmali (Strychnos potatorum Linn.) on Upid peroxides and DNA of the gastric mucosa. Results are given in Table - 36. It appears from the table that indomethacin in the dose of 25 mg/kg, when given to albino rats intraperitoneaUy once daily for 3 consecutive days, increased the level of Upid peroxides in gastric mucosa. DNA content of the mucosa, on the other hand, found decreased under the treatment of indomethacin. The results were statisticaUy significant (p (50) * * * * Cf« *r> * e o • 5 § L • ^ ' O - S 'M Cfl a 2 * It •-a o 03 * * « II , s | £|e 01^ s . I » n s _ > fO O CO 2 O cCI ’® R CO CO CO V o i» “..I m ■ " 11 5/5 • ■ - o 0> u + 1 • = e + cc,£3 611 o I I Q I V3 jS 1 I U * * * * * * 00 o o +1 -H § t 'S? cti o A § c n *r> ■o p s m (S a O S' St O ;:t s I I . . I t ■§ & o o d 8 ^ I' -«s; -wa CO V ’S3 o< § s 4^ 1 ' ^ fS o ^ • 1 51 fN 00 S i 4 ) O' I .a I ^ 1) •s « -K 2 ? . a CM o 2 o m m ro I« g«< ® a t/ 5 5 I c/i +1 1*5o « u id «< cn + S M O Q VO •S o ,a f2 0 cS I < S J 6 1 U I Nirmali (Stnjchnos potatorum linn.) treatment (i g/kg/day orally for 3 consecutive days along with indomethacin) decreased the level of lipid peroxides and increased DNA content of the gastric mucosa. Results were statistically significant (p < o.ooi) when compared to the values of indomethacin group. Hydrocortisone induced ulcer : Effect of Nirmali (Strychnos potatorum Linn.) on lipid peroxides and DNA of the gastric mucosa. Results are shown in Table - 37. Increase in the level of lipid peroxides of gastric mucosa was observed when hydrocortisone in the dose of 50 mg/kg was given to rats intraperitoneally once daily for three consecutive days. DNA content of the gastric mucosa under this condition was found decreased. Results were statistically significant (p < 0.001) when compared to the control values. Nirmali {Strychnos potatorum linn.) treatment in the dose of 1 g/kg orally once daily for three consecutive days along with hydrocortisone decreased the level of lipid peroxides and increased DNA content of the gastric mucosa. Results were statistically significant (p < 0.001) when compared to the values of hydrocortisone group. Prednisolone induced ulcer : Effect of Nirmali iStrychnos potatorum Linn.) on Upid peroxides and DNA of the gastric mucosa. Results are shown in Table - 38. Increase in the level of lipid peroxides of gastric mucosa was observed when prednisolone in the dose of 30 mg/kg was given to rats intraperitoneally once daily for three consecutive days. DNA content of the gastric mucosa under this condition was foimd decreased. Results were statistically significant (p < 0.001) when compared to the control values. Nirmali (Strychnos potatorum Linn.) treatment in the dose of 1 g/kg orally (once daily for three consecutive days along with prednisolone) decreased the level of lipid peroxides and increased DNA content of the gastric mucosa. Results were statistically significant (p < 0.001) when compared to the values of prednisolone group. Phenylbutazone induced ulcer : Effect of Nirmali (Strychnos potatonmi Linn.) on lipid peroxides and DNA of the gastric mucosa. Results are given in Table - 39. It appears fi:om the table that phenylbutazone in the dose of 100 mg/kg, when given to guinea pigs orally once daily for 3 (51) * * * * * m * ‘o o t—H o \ (S o ©■ +1 +1 t «r> 0 c4 o o 03 T*H r - O s O cn cs 1 o d I 6 o 1 . 1 * al i I e« * © * a «« O On * *T—I ® .a ro c n 7 t O +1 d +1 0 0 I I fS O s +1 J ’S q s tS '=!• ■«t g ft I ft£ fft, - g § - i ;-i u |i U (U (N a t: I S *s w » n I - a o a ^ % I cn m cs ^ I a T3 U a a CA PO .9 *c =J ^ -W © « s & ^ a ^ CJ « 0 ’t^ g « X fS I s «n d w 9 3| fS Ia ' i ^ II g l *5 On 1 - 9 c<^ CO cnfS ®« Ss * 1 II of + I a m 00 X i I f2 I I U I * * * * 00 COCN (S O o' d +1 + 1 + 1 § o o o c«ca ,A ICO e2 ps! I uI consecutive days, increased the level of lipid peroxides in gastric mucosa. DNA content of the mucosa, on the other hand, found decreased under the treatment of phenylbutazone, The results were statistically significant (p Histamine induced ulcer : Effect of Nirmali CStrychnos potatorum linn.) on lipid peroxides and DNA of the gastric mucosa. Results are shown in Table - 40. Increase in the level of lipid peroxides of gastric mucosa was observed when histamine in the dose of 33 micro gram/mouse was given to mice intraperitoneally once daily for three consecutive days. DNA content of the gastric mucosa under this condition was found decreased. Results were statistically significant (p < 0.001) when compared to the control values. Nirmali (Strychnos potatorum Linn.) treatment in the dose of 1 g/kg orally (once daily for three consecutive days along with histamine), on the other hand, decreased the level of lipid peroxides and increased DNA content of the gastric mucosa. Results were statistically significant (p < 0.001) when compared to the values of histamine group. ## (52) IM 8 C II S 8 1 0 K DISCUSSION The profile of Nirmali, as revealed from Ayurvedic literature (187), is as under; BOTANICAL NAME Strychnos potatorum linn. INDIAN NAMES Hindi -Nelmal Bengali - Nirmali Sanskrit -Kataka Punjabi - Nirmali Tamil - Telankottai Telugu - Chettu FAMILY Loganiaceae ABOUT THE PLANT Middle sized plant with branches, height up to 40 feet, oval shaped leaves with small stalk, light yellow flowers having aroma, fruits contain one / two seed (s). (53) , V •; . ■ ‘ ' ‘^>Yf *' ‘fe i.--' *- •»,%•<'.«. • ^-.--'■A v:\»»; .. „ Nirmali {strychnos potatorum Linn.) leaves . ' m - , - ,'w ' i s ;* w . ', V * Vmj!- ,, » y«, . J» >- j? 4'’-'^ <" Nirmali (strychnos potatorum Linn.) seeds DISTRIBUTION ; Occurred in plenty in Bihar, Orissa and southern part of India. MEDICINAL USE : Seeds are used as a local application in eye diseases, in dysentery and hyperacidity, in diabetes and gonorrhea (188). In connection with screening experiments of various plants, fiioits, seeds, roots etc. for having their anti- ulcer effect, if any, we noticed that seeds of Nirmali iStrychnos potatorum Linn.) exerted anti- ulcer effect on experimental ulcers induced by the drugs like aspirin and indomethacin. The observations tempted us to undertake the project in detail with a view to study the anti -ulcer effect of Nirmali (Strychnos potatorum Linn.) in other drug induced experimental ulcer models and to note the possible mechanism of the anti-ulcer effect. The present work, thus, was an attempt to evaluate the anti-ulcer activity of Nirmali (Strychnos potatorum linn.) and its effect on various biochemical parameters in experimental ulcer models induced by the drugs like; 1. Salicylic acid 2. Aspirin 3. Paracetamol 4. Indomethacin 5. Hydrocortisone 6. Prednisolone 7. Phenylbutazone and 8. Histamine. Salicylic acid induced ulcers : Role of Nirmali (Strychnos potatorum Linn). Brodie and Chase(i5) while working on the vilcerogenic role of salicylic acid in rats, observed a dose dependent response of salicylic acid in the production of gastric ulcer. In the present study it was also noticed (Table - 1) that salicylic acid in the dose of 100 mg/kg when given to rats intraperitoneaUy once daily for three consecutive days produced profuse ulcers in the glandular part of stomach. Ulcers were associated with frank intragastric haemorrhage. Dodd et al. (19) also found the intragastric haemorrhage with ulcers induced by salicylic acid. Conflicting reports are available in the literature regarding the effect of (5 4 ) salicylic acid on the volume of gastric juice, gastric acidity and peptic activity. lish et al. (81) reported that salicylic acid caused a decrease in volume and acidity of gastric secretion while Winkelman and Summerskill (82) foimd that salicylic acid had no effect on the rate of gastric secretion and gastric acidity. In the present study it was observed that salicylic acid had no significant effect on rate of gastric secretion, gastric acidity and peptic activity (Table - 9). Salicylic acid in the aforesaid dose was found to decrease significantly (p < 0.025 to p Aspirin induced ulcers : Role of Nirmali (Strychnos potatorum Linn). Table - 2 showed that aspirin in the dose of 100 mg/kg/day when given to rats intraperitoneally for three consecutive days induced profuse ulcers in glandular part of stomach and the stomachs were invariably accompanied by fi:’ank intragastric haemorrhage - an earlier observation made by Brodie and Chase (15), Djahanguiri (16) using different doses of aspirin in experimental animals. (55) While studying the effect of aspirin on the rate of gastric secretion, gastric acidity and peptic activity Paul et a l (80) observed that aspirin causes a decrease in volume and acidity of gastric secretion while Lynch (79) observed an increase, no change or a decrease in gastric secretion and gastric acidity by aspirin depending on the dose and species studied. We, however, observed no significant change in volume, acidity and peptic activity of gastric secretion by the dose of aspirin (100 mg/kg/day intraperitoneally for three consecutive days) we used in albino rats (Table -10). That aspirin reduces the secretion of gastric mucus was an earlier observation made by different workers (110,111). We also found that aspirin reduced the amount of dissolved gastric mucin as well as gastric mucosal mucus. The constituent carbohydrate components of dissolved gastric mucin and mucosal mucus viz. total hexoses, hexosamine, methyl pentose, sialic acid etc. showed a significant decrease (p < 0.025 to p < 0.001) in levels when compared to that of control values. The amount of dissolved mucin and mucosal mucus as represented by total carbohydrate (110) also showed a significant decrease (p < 0.001) by aspirin. (Tables -18,26). We have also noted that aspirin increased the concentration of lipid peroxides and decreased the amount of DNA of gastric mucosa (Table - 34). Results were statistically significant (p < 0.001) when compared to that of control values. Best et a l., however, observed (189) that aspirin had no effect on DNA content of gastric mucosa. Effect of Nirmali (Strychnos potatorum Linn.) on aspirin induced gastric ulcers in albino rats was not reported in the available literature. We have noted that powdered seeds of Nirmali iStrychnos potatorum Linn.) in the oral dose of 1 g /kg/day for three consecutive days along with aspirin could prevent gastric ulcer induced by aspirin(Table - 2). Ulcer index came down from 23 to 7.3. This anti-ulcer effect of Nirmali (Strychnos potatorum Linn.) was not related with gastric secretion, gastric acidity and peptic activity (Table -10) but had relation with mucus secretion, lipid peroxidation and DNA content of the stomach. We had seen that Nirmali (Strychnos potatorum Linn.) could increase the amount of gastric mucus (Tables - i8, 26), decrease lipid peroxides and increase DNA content of gastric mucosa (Table - 34) and thus exerted "its anti — ulcer effect. All changes were found statistically significant (p < 0.025 to p < 0.001). (56) Paracetamol induced ulcers : Role of Nirmali (Stryehnos potatorum liim). Studies on paracetamol for its ulcerogenic potency are few in the available literature. Proudfoot and Wright (22) when studied the cases of acute paracetamol poisoning noticed gastrointestinal haemorrhage. We found the presence of massive ulcers at the glandular part of the stomach of rats when the animals were treated with paracetamol (100 mg/kg/day intraperitoneally for three consecutive days). Most of the ulcers were associated with haemorrhage(Table - 3). We also found that the ulcerogenic dose of paracetamol could not change significandy the rate of gastric secretion, gastric acidity and peptic activity (Table - 11) but could decrease the gastric mucus as well as DNA content of gastric mucosa (Tables - 19, 27, 35) and could increase the level of lipid peroxides (Table - 35) . All the changes were statistically significant (p < 0.025 to p< 0.001). Effect of Nirmali iStrychnos potatorum Linn.) on paracetamol induced gastric ulcers in albino rats was not reported in the available literature. We have noted that powdered seeds of Nirmali {Stryehnos potatorum Linn.) in the oral dose of 1 g /kg/day for three consecutive days along with paracetamol could prevent gastric ulcer induced by paracetamol (Table - 3). This anti-ulcer effect of Nirmali iStrychnos potatorum Linn.) was not related with gastric secretion, gastric acidity and peptic activity (Table - 11) but had relation with mucus secretion, lipid peroxidation and DNA content of the stomach. We had seen that Nirmali (Stryehnos potatorum Linn.) could increase the amount of gastric mucus (Tables - 19, 27), decrease lipid peroxides and increase DNA content of gastric mucosa (Table - 35) and thus exerted its anti - ulcer effect. AU changes were found statistically significant (p < 0.025 to p < 0.001) when compared to that of paracetamol group. Indomethacin induced ulcers : Role of Nirmali {Stryehnos potatorum linn). In animal experiments, Djahanguri (8) and Lee et al. (9) showed that indomethacin caused gastric, duodenal, antral and jejunal ulcerations, haemorrhage and perforation. On studying this ulcerogenic property, Nicoloff (78) showed that ulcerogenic effect of indomethacin did not seem to be related to acid hypersecretion. On the other hand, Menguy (110) and Goel et al.(i4o) observed that ulcerogenic effect of indomethacin was related to mucus secretion as it lowered the rate of mucus secretion and diminished the amount of carbohydrate incorporation into the (57) mucosubstances which helped the drug to exert its toxic effect to damage the mucosa. In our experiment we observed that indomethacin in the dose of 25 mg/kg when given to rats intraperitoneally once daily for three consecutive days formed several ulcers at the glandular region of stomach (Table - 4). Formation of ulcer was not related with acid-peptic digestion as indomethacin with the said dose had no effect on rate of gastric secretion, gastric acidity and peptic activity (Table -12). On the other hand, we observed, indomethacin had a relation with gastric mucus secretion since it decreased the levels of total hexoses, hexosamine, methyl pentose and sialic acid (the constituent carbohydrate components of mucin) and thus the levels of dissolved gastric mucin and gastric mucosal mucus (Tables - 20, 28). In addition, indomethacin increased the levels of gastric lipid peroxides and decreased DNA content of gastric mucosa (Table - 36). AH changes were foimd statistically significant (p < 0.025 to p < 0.001) when compared to that of control values. Naito et al. (131), Joseph et a l (190) and Pandit et cd. (162) also showed that indomethacin induced gastric ulcer in albino rats was mediated by increasing gastric lipid peroxidation. Effect of Nirmali {Strychnos potatorum Linn.) on indomethacin induced gastric ulcers in albino rats was not reported elsewhere. We have noted that powdered seeds of Nirmali (Strychnos potatorum linn.) in the oral dose of 1 g /kg/day for three consecutive days along with the said dose of indomethacin could prevent gastric ulcer induced by indomethacin (Table - 4). Ulcer index came down from 22 to 8. This anti-ulcer effect of Nirmali (Strychnos potatorum linn.) was not related with gastric secretion, gastric acidity and peptic activity (Table -12 ) but had relation with mucus secretion, lipid peroxidation and DNA content of the stomach. We observed that Nirmali (Strychnos potatorum linn.) could increase the amount of gastric mucus (Tables - 20, 28), decrease lipid peroxides and increase DNA content of gastric mucosa (Table - 36) and thus exerted its anti - ulcer effect. All changes were found statistically significant (p < 0.025 to p < 0.001) when compared to that of indomethacin group. Hydrocortisone induced ulcers: Role of Nirmali iStrychnos potatorum linn). Ulcerogenic potency of hydrocortisone in man as well as in experimental animals has been studied elsewhere (38,39,43,44). We also observed that (58) hydrocortisone acetate in the dose of 50 mg/kg/day when given to rats intraperitoneally for three consecutive days produced massive ulcers of different sizes at the glandular region of stomach (Table - 5). Most of the ulcers were associated with haemorrhage. We found that this ulcerogenic dose of hydrocortisone had no effect on the rate of gastric secretion, gastric acidity and peptic activity (Table - 13) but decreased the levels of dissolved gastric mucin and gastric mucosal mucus (Tables - 21, 29). All carbohydrate components of mucus like total hexoses, hexosamine, methyl pentose and sialic acid were found decreased significantly(p < 0.025 to p < 0.001) in gastric juice and gastric mucosa after the administration of hydrocortisone in comparison to the control values. Seyle (48) observed a steady decrease in mucus secretion in gastric juice after hydrocortisone administration in albino rats. Ulcerogenic properly of hydrocortisone has not been explained earlier in terms of lipid peroxidation. We, however, noted that hydrocortisone in the said dose could increase the level of lipid peroxides and decrease the amount of DNA of gastric mucosa in albino rats (Table - 37). Results were statistically significant (p < 0.001) when compared to that of control values. Effect of Nirmali {Strychnos potatorum Linn.) on hydrocortisone induced gastric ulcers in albino rats was not reported in the available literature. We have noted that powdered seeds of Nirmali {Strychnos potatorum linn.) in the oral dose of 1 g /kg/day for three consecutive days along with the said dose of hydrocortisone could prevent gastric ulcer induced by hydrocortisone(Table - 5). Ulcer index was 25. After Nirmali (Strychnos potatorum linn.) treatment ulcer index came down to 14. This anti-ulcer effect of Nirmali (Strychnos potatorum Linn.) was not related with gastric secretion, gastric acidity and peptic activity (Table - 13) but had relation with mucus secretion, lipid peroxidation and DNA content of the stomach. We had seen that Nirmali (Strychnos potatorum Linn.) could increase the amount of gastric mucus (Tables - 21, 29), decrease lipid peroxides and increase DNA content of gastric mucosa (Table - 37) and thus exerted its anti - ulcer effect. All changes were found statistically significant (p < 0.025 to p < 0.001) when compared to that of hydrocortisone group. Prednisolone induced ulcers : Role of Nirmali iStrychnos potatorum Linn). Several reports (46-49) are available in the literature showing the (5 9 ) ulcerogenic potency of prednisolone in experimental animals. Researchers have noted that prednisolone induces gastric ulcer in rats. We also observed the presence of ulcers at glandular part of the stomach of albino rats when the animals received prednisolone (30 mg/kg/day intraperioneally) for three consecutive days. 4 rats out of 33 died during etperiment showed the extension and perforation of ulcers. This fin d in g s were in agreement with the earlier reported observation of Khan et al. (45). When studied the effect of prednisolone on acid-peptic as well as mucus factor, we found that this drug with the said dose had no effect on the rate of gastric secretion, gastric acidity and peptic activity (Table - 14). But the mucus factor represented by the levels of dissolved gastric mucin and gastric mucosal mucus was found significantly (p<0.025 to p < 0.001) decreased in comparison to that of control values (Table - 22, 30). Earlier, this was observed by Seyle (48) who stressed on the fact that diminution of tissue resistance is related to the anti inflammatory action of corticosteroids in the genesis of ulceration. Ulcerogenic property of prednisolone has not been explained earlier in terms of lipid peroxidation. We had noted that prednisolone in the said dose could increase the level of lipid peroxides and decrease the amount of DNA of gastric mucosa in albino rats (Table - 38). Results were statistically significant (p < 0.001) when compared to that of control values. Effect of Nirmali (Strychnos potatorum linn.) on prednisolone induced gastric ulcers in albino rats was not reported in the available literature. We found that powdered seeds of Nirmali (Strychnos potatorum linn.) in the oral dose of 1 g /kg/day for three consecutive days along with the said dose of prednisolone could prevent gastric ulcer induced by prednisolone (Table - 6). Ulcer index was 25. After Nirmali (Strychnos potatorum linn.) treatment ulcer index came down to 10.2. This anti-ulcer effect of Nirmali {Strychnos potatorum linn.) was not related with gastric secretion, gastric acidity and peptic activity (Table -14) but had relation with mucus secretion, lipid peroxidation and DNA content of the stomach. We had seen that Nmnali (Strychnos potatorum linn.) could increase the amoimt of gastric mucus (Tables - 22, 30), decrease lipid peroxides and increase DNA content of gastric mucosa (Table - 38) and thus exerted its anti - ulcer effect. All changes were found statistically significant (p < 0.025 to p < 0.001) when compared to that of prednisolone group. (60) Phenylbutazone induced ulcers : Role oflSirmaiiiStrychnospotatorum Linn). Ulcerogenic property of phenylbutazone in man as well as in experimental a n im a ls had been studied by various workers (25 - 37). We also observed that phenylbutazone in the dose of 100 mg/kg/day when given to guinea pigs orally for three consecutive days produced massive ulcers of different sizes at the glandular region of stomach (Table - 7)- Almost all ulcers were accompanied by haemorrhage. We found that this ulcerogenic dose of phenylbutazone had no effect on the rate of gastric secretion, gastric acidity and peptic activity (Table - 15) but decreased the levels of dissolved gastric mudn and gastric mucosal mucus (Tables - 23, 31). All carbohydrate components of mucus like total hexoses, hesosamine, methyl pentose and sialic acid were foimd decreased significantly(p < 0.025 to p < 0.001) in gastric juice and gastric mucosa after the administration of phenylbutazone in comparison to the control values. Zaidi et a l (117) observed a steady decrease in mucus secretion in gastric juice after phenylbutazone administration in guinea pigs. Ulcerogenic property of phenylbutazone has not been explained earlier in terms of lipid peroxidation. We, however, noted that phenylbutazone in the said dose could increase the level of lipid peroxides and decrease the amount of DNA of gastric mucosa in guinea pigs (Table - 39). Results were statistically significant (p < 0.001) when compared to that of control values. Effect of Nirmali iStrychnos potatorum Linn.) on phenylbutazone induced gastric ulcers in guinea pigs was not reported in the available literature. We found that powdered seeds of Nirmali {Strychnos potatorum Linn.) in the oral dose of 1 g /kg/day for three consecutive days along with the said dose of phenylbutazone could prevent gastric ulcer induced by phenylbutazone (Table - 7). Ulcer index was 25. After Nirmali (Strychnos potatorum Linn.) treatment ulcer index came down to 14. This anti-ulcer effect of Nirmali (Strychnos potatorum Unn.) was not related with gastric secretion, gastric acidity and peptic activity (Table - 15) but had relation with mucus secretion, lipid peroxidation and DNA content of the stomach. We had seen that Nirmali (Strychnos potatorum linn.) could increase the amount of gastric mucus (Tables - 23,31), decrease lipid peroxides and increase DNA content of gastric mucosa (Table - 39) and thus exerted its anti - ulcer effect. AH changes were found statistically signifi^cant (p < 0.025 to p < 0.001) when compared to that of phenylbutazone group. (61) Figure-1: Showing phenylbutazone induced ulcers in the stomach of guinea pig. Figure-2: Showing the effect of Nirmali (Strychnos potatorum Linn.) on phenylbutazone induced ulcers in the stomach of guinea pig. Histamine induced ulcers : Role of Nirmali iStrychnos potatorum linn). There are reports that histamine in specific dose when administered through specific route caused gastric and duodenal ulcers in man and animals (52 — 55)* Ulcerogenic potency of the drug was related to gastric hyper secretion. In our experiment when histamuie in the dose of 33 micro gram/mouse/day when given to mice intraperitoneally for three consecutive days produced massive ulcers at the glandular region of stomach (Table - 8). Ulcers were accompanied by haemorrhage and other pathologies like adhesion and acute dilatation of the blood vessels. We found that this ulcerogenic dose of histamine had no effect on the rate of gastric secretion, gastric acidity and peptic activity (Table - 16) but decreased the levels of dissolved gastric mucin and gastric mucosal mucus (Tables - 24, 32). All carbohydrate components of mucus like total hexoses, hexosamine, methyl pentose and sialic acid were found decreased significantly(p < 0.025 to p < 0.001) in gastric juice and gastric mucosa after the administration of histamine in comparison to the control values. Effect of ulcerogenic dose of histamine on lipid peroxidation was not reported elsewhere. We, had noted that histamine in the said dose could increase the level of lipid peroxides and decrease the amount of DNA of gastric mucosa in mice(Table - 40). Results were statistically significant (p < 0.001) when compared to that of control values. Effect of Nirmali {Strychnos potatorum Linn.) on histamine induced gastric ulcers in mice was not reported in the available literature. We foimd that powdered seeds of Nhmali iStrychnos potatorum linn.) in the oral dose of 1 g /kg/day for three consecutive days along with the said dose of histamine could prevent gastric ulcer induced by histamine (Table - 8). Ulcer index was 28. After Nirmali (Strychnos potatorum Linn.) treatment ulcer index came down to 10.1. This anti-ulcer effect of Nirmali iStrychnos potatorum Linn.) was not related with gastric secretion, gastric acidity and peptic activity (Table -16 ) but had relation with mucus secretion, lipid peroxidation and DNA content of the stomach. We had seen that Nirmali (Strychnos potatorum Unn.) could increase the amoimt of gastric mucus (Tables - 24, 32), decrease lipid peroxides and increase DNA content of gastric mucosa (Table - 40) and thus exerted its anti - ulcer effect. All changes were found statistically significant (p < 0.025 to p < 0.001) when compared to that of histamine group. ## (62) Concept of antiulcerogenic property of indigenous plants and fruits was not new. Several reports are now available in literature (70 - 86). In the present study we confirmed anti-ulcer property of Nirmali {Sttychnos potatorum linn.) and explored the possible mechanism behind this anti-ulcer property. Our observations were: 1. Nirmali {Strychnospotatorum linn.) could decrease the incidence and severity of gastric ulcers in ©cperimental ulcer models induced by the drugs like salicylic acid, aspirin, paracetamol, indomethacin, hydrocortisone, prednisolone, phenylbutazone and histamine. 2. Nirmali (Strychnos potatorum lirm.) had no efifect on rate of gastric secretion, gastric acidity and peptic activity during ulceration. 3. Nirmali (Strychnos potatorum liim.) increased the levels of gastric dissolved mucin and gastric mucosal mucus which were found decreased during ulceration. 4. Nirmali (Strychnos potatorum linn.) decreased the level of gastric lipid peroxides which was found increased during ulceration. 5. Nirmali (Strychnos potatorum linn.) increased the amount of DNA of gastric mucosa which was found decreased during ulceration. Thus, we conclude; * Nirmali (Strychnos potatorum linn.) has anti-ulcer effect against drug induced experimental ulcer^. {Nirmali (Strychnos potatorum linn.) could prevent the incidence and severity of ulcers as induced by different ulcerogenic drugs to the extent of 60 - 90%} (63) ** Anti-ulcer effect of Nirmali (Strychnos potatorum linn.) is through inhibition of gastric lipid peroxidation. { Ulcerogenic drugs could increase gastric lipid peroxidation thereby generate reactive oxygen metabolites. This could damage gastric cells as observed by various workers (118 - 123). This was reflected by decreased amount of DNA in gastric mucosa which, in turn, was responsible for decreased synthesis of gastric mucosubstances. In absence of proper protective layer of mucosubstances, ulcer developed in the stomach. Nirmali {Strychnos potatorum Linn.), on the other hand , could inhibit gastric lipid peroxidation thereby inhibit generation of reactive ojQ^en metabolites. This could protect the gastric cell from damage. DNA of gastric mucosa was, thus, found increased with a concomitant increase in the level of gastric mucosubstances. These mucosubstances gave a proper protection in the stomach for which ulcers could not develop. Anti-ulcer property of Nirmali (Strychnos potatorum Linn.) was thus explained by its anti-oxidative activity.} ## (64) K l I M i l l A K Y SUMMARY Role of Ninnali {Strychnos potatorum Linn.) in drug induced experimental ulcers was described. 1. Experimental gastric ulcers were induced in albino rats by the drugs like Salicylic acid -100 mg/kg, intraperitoneally (8) Aspirin -100 mg/kg, intraperitoneally (8) Paracetamol -100 mg/kg, intraperitoneally (164) Indomethacin - 25 mg/kg, intraperitoneally (9) Hydrocortisone - 50 mg/kg, intraperitoneally (165) Prednisolone - 30 mg/kg, intraperitoneally (165) in guinea pigs by Phenylbutazone -100 mg/kg, orally (137) and in mice by Histamine - 33 microgram/mouse. (166) intraperitoneally Drugs were given once daily for three consecutive days. (65) 2. Glandular part of the stomach of all animals (100%) developed massive iilcers by the drugs. In most of the cases (80 -100%) ulcers were accompanied by haemorrhage. Adhesion and acute dilatation were seen. 3. Powdered seed of Ninnali (Strychnos potatorum lin n .) in a dose of ig/kg/day, orally (exerted maximum activity as revealed from the pilot experiment) was given to the animals for three consecutive days along with the ulcerogenic drug. 4. Nirmali {Strychaws potatorum linn.) was found “antiulcerogenic” in aU the ulcer models studied since it reduced the rate of incidence and severity of ulcers (60 - 90%) induced by ulcerogenic drugs. 5. Gastric juice was collected from the animals and the rate of gastric secretion, gastric acidity and peptic activity were measured during ulceration as well as after treatment with Nirmali {Strychnos potatorum Linn.). 6. Rate of gastric secretion, gastric acidity and peptic activity were not significantly affected during ulceration and after treatment with Nirmali (Strychnos potatorum linn.). 7. Dissolved gastric mucins of the animals were analysed during ulceration and effect of Nirmali (Strychnos potatorum Linn.) on it was studied. 8. Level of dissolved gastric mucin in terms of its constituent carbohydrate components viz. total hexoses, hexosamine, methyl pentose and sialic acid was found decreased significantly (p < 0.025 to p < 0.001) during ulceration. Nirmali (Strychnos potatorum Linn.) treatment, on the other hand, increased significantly (p < 0.025 to p < 0.001) the level of dissolved gastric mucin. 9. Gastric mucosal mucus was collected from the ulcerated stomach and the amount was estimated. Effect of Ninnali (Strychnos potatorum Linn.) on the said parameter was studied. 10. Level of gastric mucosal mucus of the animals was found decreased (66) significantly (p < o.ooi) during ulceration . Nirmali {Strychnos potatorum linn.) treatment could significantly (p < o.ooi) increase the level of gastric mucosal mucus. 11. Anti-ulcer effect of Nirmali iStrychnos potatorum linn.) was, thus, not related with offensive factors like "acid-pepsin** but had relation with defensive parameters like ^^mucosubstances of gastric juice and gastric mucosa”. 12. Lipid peroxidation was studied in ulcerated stomach. Effect of Nirmali iStrychnos potatorum Linn.) on the said parameter was studied. 13. Level of lipid peroxides in stomach was found increased (p <0.001) during ulceration. Nirmali (Strychnos potatorum Linn.) treatment could significantly (p < 0.001) decrease the level of lipid peroxides. 14. DNA content of the gastric mucosa was estimated in ulcerated stomach as weU as in stomach after Nirmali iStrychnos potatorum Linn.) treatment. 15. DNA content of the gastric mucosa, decreased during ulceration, was found significantly increased (p < 0.001) by the treatment with Nirmali (Strychnos potatorum Linn.). 16.Ulcerogenic effect of drugs was thus explained in terms of lipid peroxidation which was protected by Nirmali (S try ch n os potatorum linn.). In conclusion it can be suggested that, 'UCcerogenic drugs couCcC increase gastric CipicCjjeroxicCation tfiereBy generate reactive oxygen metaBoCites. 'T'fiis couCcC cCamage gastric ceCCs as oBservecC By various 'workers (118 - 123), T'fiis was refCected By decreased amount of DMA in gastric mucosa wfiicfi, (67) tn turn, was resjjonstBCe fo r decreased synthesis of gastric mucosuBstances. In aBsence of proper protective Cdyer of mucosuBstances, ufcer devefoped in tfie stomacfi. !KirmaCi (Stryc^nosjfotatorumLinn.), on tfie other fiand, couCd infUBit gastric Cipidperoxidation tfiereBy indiBit generation of reactive oxygen metaBoCites. 7'fiis couCdprotect tfie gastric ceCCfrom damage, 'D^N'JA of gastric mucosa was, tfiuSy foun d increased wit fi a concomitant increase in tfie CeveC of gastric mucosuBstances, 7'fiese mucosuBstances gave a proper protection in tfie stomacfi for wfiicB uCcers couCdnot deveCop. J\nti-uCcerproperty ofJ^irmaCi (StrycAnosj?otatonim Linn.) was thus expCained By its anti-oxidative activity. ## ( 6 8 ) KEFEKEKCE8 REFERENCES 1. Quincke, H .: Quoted from Pathophysiology of Peptic ulcer. Ed. S. C. Skoiyna and H. L. Bockus, J. B. Lippin Cott Company, Philadelphia 1963. 2. Wanka, J., Jonls, L. T., Wood, P. H. and Dixon, A. T. Am. ’.Rheum. Dis., 23, 218,1964. 3. Kaig, J., Dryin, R. L., Paul, W. D. and Roth, J. I. : Am. J. Dig. Dis. , 16, 88, 1949. 4. Bilka, F. J., Wolhoim, I. And V\^Uams, G. B .: Minnasota Med. 47,777,1964. 5. Rothrmicn, N. O .: Arther Rheum., 6,295,1963. 6. Obrein, W. M .: Clin. Pharmacol. Ther., 9,94,1968. 7. Ballabio, C. B. : In International Symposium on Steroidal Anti inflammatory Drug, Ed. Garatinie and Dukes, M. N. G., Excerpts Medical Foundation., London, p - 342,1964. (69) 8. Djahanguiri, B .: J. Pharm. Pharmacol, 21,514,1969. 9. Lee, Y. H ., MoUison, K. W., and Cheng, W. D. : Arch. Int. Pharmacol, 191, 370,1971- 10. Anderson, K. W .: Arch. Int. Pharmacodyn., 152,372,1964. 11. Davidson, W. D. and Davis, R. E. : In The action of hormone on molecular processes. Ed. G. litwack and D. Kritchevsky, Wiley and Sons, New York, 1964. 12. Clark, B. B. and Adams, W. L .: Gastroenterology, 9,461,1947* 13. Levert, M. and Lambert, R .: Gastroenterology, 94,273, i960. 14. Cooper, C. N.(Jr.), Meade, R. C. and EUisiv, E. H .: Arch. Surg. (Chicago), 93, 171,1966. 15. Brodie, D. A. and Chase, B. J .: Gastroenterology, 53,604,1967. 16. Djahanguiri, B. D. J., Sodeghi, P., Hekmni, S. and Firouzababi, A .: Europ. J. Pharmacol, 2,345,1968. 17. Djahangoiri, B., Taubin, Hh. Z. and Landsberg, L : J. Pharm. Exp. Ther, 184, 163,1973- 18. Caravati, D. M. and Cosgrove, L. F .: Am. Intern. Med., 24,368,1964. 19. Dodd, K., Minot, A.S. and Arena, J. M .: Am. J. Dig. Dis., 53,1435,1937. 20. Hailman, H. F .: J. Clin. Endocrinol, 12,454,1952. 21. Smith, M. J. M .: J. Phar. Pharmacol, 5,81,1953. 22. Praddfoot, A. T. and Wright, N .: Brit. Med. J., 3,557,1970. 23. Knedilr, W. H .: Brit. Med. J ., 1,876,1946. 24. Huguiley, Jr. C. M .: J. Amer. Med. Assoc, 189,938,1964. 25. Mauer, E. F .; New. Eng. Med. J ., 253,404,1955. 26. Leonard, J. C .: Brit. Med. J. , 1,1311,1953. 27. Deseza, S. amd Levemievx, J .: Bull Etm’en. Soc. Med. (Paris), 69,389,1953. 28. Kern, F. (Sr.), Lukens, J. G. and Clark, G. M .: Clin. Res. Poc., 4,159,1956. 29. Kirsner, J. B .: Ann. Int. Med., 47,666,1957. 30 .Kuzeli, W. C., Schaffarzick, R. W., Brown, B. J. and Hankie, F. A. : JAMA, 149,729,1952. 31. Kuzeli, W. C., Schaffarzick, R. W., Wangler, W.E., Gandin, G. and Mankle, E. A .: Am. Arch. Intern. Med., 92,646,1953. 32. Brodie, B. B., Lowman, E. M., Bums, J. J., Lee, P. R., Chenkin, T., Goldman, A., Neiner, M. and Steela, J. M .: Am. J. Med., 16,181,1954. 33- Gramirer, L. W .: JAMA, 150,1332,1952. (70) 34- Maner, E. F .: New. Eng. J. Med. 253,404,1955. 35. Bonfil, S. and Hardonin, S .: C. R. S’eanc. Soc. Biol. J., 147,2016,1953. 36. Kirsner, J. B. and Ford, H .: Gastroenterology, 29,1,1955- 37. Menguy, R. and Desbaillets, L .: Am. J. Dig. Dis., 12,862,1967a. 38. Robert, A. and Nezamis, J. E .: Proc. Soc. Expt. Biol. Med., 100,596,1959. 39. Kowalewski, K .: Proc. Soc. Expt. Biol. Med., 98,9,1958. 40. Robert, A. and Nezamis, J. E .: Proc. Soc. Expt. Biol. Med., 98,9,1958. 41. Chaikof, K., Janke, W. H ., Pesards, P. C., Ponika, J. L and Brush, B. E .: Arch. Surg., 83,32,1961. 42. Nicoloff, D. M., Peter, E. T., Leonard, H. S. and Wangensteen, 0 . H. : J. Am. Med. Assoc., 191,383,1965. 43. Ivy, A. C., Grossman, M. I. And Brachrach, W. D .; In “Peptic Ulcer”, J & A Churchill Ltd., London, 1950. 44. Hall, B. D .: Gastroenterology, 25, 80,1953. 45. Kahn, D. S., Phillips, M. J. and Skoiyna, S. C .: Am. J. Park., 38,177,1961. 46. Robert, A. and Nezamis, J. E .: Proc. Soc. Expt. Biol. Med., 114,545,1963. 47. Robert, A. and Nezamis, J. E .: Abstract in “6* International Congress of Pharmacology”, Heleinki, 1975. 48. Seyle, H .: Gastroenterology, 26,221,1954. 49. Foley, W. A. and Glick, D .: Gastroenterology, 43,425,1962. 50. Levis, S. and Deersaerts, W .: Arch. Int. Pharmacol, 126,359, i960. 51. Sanyal, A. K. and Watson, N. G .: J. Physiol (London), 222,47,1971. 52. Popielski, L. and Arch, F. D .: Ges. Physiol, 178,214,1920. 53. Smith, A. N .: J. Physiol (London), 121,517,1953. 54. Franco-Browder, S., Masson, G. N. C. and Corcoran, A. C .; J. Allergy, 30, 1,1959. 55. Smith, A. N .: Brit J. Surg., 46,157,1958. 56. Delcoldato, P. A .: Pharmacol Res. Commun., 14,175,1982. 57. Del Seldato, P., Ghiorzi, A, Cereda, E. and Donetti, A. : Pharmacology, 30,45,1985- 58. Cho, C. H., Ogle, C. W. and Dai, S .: J. Pharmacol 37,823,1985. 59- Lambert, R., Skoryna, S. C. : Pathophysiology of peptic ulcer, Montreal, Me. Gill University Press, 253,1963. 60. Wilhelmi, G .: Pharmaca Helv. Physiol Pharmacol Acta. , 15,83,1957. 61. Hedinger, C. and Veraguth, F .: Schweizn. Med. Wschr., 37,1175,1957. (71) 62. Kurebayashi, Y., Asano, M., Hashizume, T and Akashi, A . : Jap. J. Pharmacol, 36,121,1984. 63. Selye, H. and Szabo, S .: Nature, 244,458,1973* 64. Kivilaakso, E., Fromm. M. D. and Silen, W .: Surgery, 84,70,1978. 65. Parmer, N. S .: Toxicol, 24,611,1986. 66. Szabo, S., Brown, A., Pihan, G., Dali, H. and Neumeyer, J. L : Proc. Soc. Exp. Biol Med., 180,557,1985. 67. Okabe, S., Roth, J. L. A. and Paeiffer, C. J .: Experientia., 27,146,1971. 68. Robert, A. , Nezamis, J. E. , Lancaster, C. and Hanchar, A. : Gastroenterol, 77,433,1979. 69. Paul, Arindam and Santani Devdas: Ind. J. Pharmacol, 34,44,2002. 70. Obi, E., Emeh, J. K., Orisakwa, 0 . E., Afonna, 0 . J., Ilondu, N. A. and Agbasi, P. U .: Ind. J. Pharmacol, 32,381, 2002. 71. Okabe, S. and Pfeiffer, C. J .: In “Peptic Ulcer”, Denmark, Copenhagen, 13, 1971. 72. Parmer, N. S., Tariq, M. and Ageel, A. M .: Int. J. Tissue Reactions, 8, 67, 1968. 73. Tariq, M., Parmer, N. S. and Ageel, A. M. : J. Pharmacol Expt. Therap., 241,602,1987. 74. Kimura, M. Saziki, R., Aravil, I., Tarumoto, Y. and Nakane, S. : J. Pharmacol, 36,390,1984. 75. Goel, R. K. and Bhattacharya, S. K .: Ind. J. Exp. Biol, 29,701,1991. 76. Blair, A. J., Feldman, M., Barnett, C., Walsh, J. H. and Richardson, C. T .: J. Clin. Invest, 79,582,1987. 77. Wolfe, M. M. and Soil, A. H .: New Eng. J. Med., 319,1707,1988. 78. Nicoloff, D. M .: Arch. Surg., 97,809,1968. 79- Lynch, A .: Gut, 5, 230,1964. 80. Paul, W. D., Dryer, R. L. and Routh, J. M. : J. Am. Phar. Assoc., 39, 21, 1950. 81. Lish, P. M., Dungan, K. W. and Robins, S. R .: Arch. Int. Pharmacodyn., 119,389,1959. 82. Winkelman, E. L And Summerskill, E. M. : Gastroenterology, 40, 56, 1961. 83. Schimid, E., Heinkel, T., Acer, O. and Zicha, L : In “International Symposia on Non-steroidal Anti-inflammatory drugs”, London, 440, i960. (72) 84* Bonfils, S., Hordonin, J. P., Rochia, C. and Delbarre, F. : Bull. Soc. Med. Hosp. Paris., 71,114,1955- 85. Shay, H .: Gastroenterology, 26,316,1954- 86. Brodie, D. A. and Hanson, H. M .: Gastroenterology, 2 8 ,353, i960. 87. Brodie, D. A., Marshall, R. W. and Moreno, O. M .: Am. J. Physiol, 202, 812,1962. 88. Menguy, R .: Am. J. Dig. D is., 5 » 9U, i960. 89. Allen, A. and Gamer, G .: Gut, 21, 249,1980. 90. Allen, A. : In “Physiology of the gastrointestinal tract” , Ed. By L. R. Johnson, (Raven Press, New York), 617,1981. 91. Allen, A .: Br. Med. Bull., 34,28,1978. 92. Scratchard, T .: Curr. Opin. Gastroenterol, 3,916,1987. 93. Clamp, R., Allen, H., Gibbons, R. A. and Robert, G. P .: Br. Med. Bull, 34, 25,1978. 94. Shorrock, C. J. and Rees, W. D. W .: Am. J. Med., 84 (Suppl 2A), 25,1988. 95. Smith, G. W. , Tasman-Jones, C., Wiggins, P. M. and Lee, S. P. : Gastroenterology, 87,1313,1985. 96. Edwards, P. A. W., Br. Med. B ull, 34,55,1978. 97. Wallace, J. L. and Whittle, B. J. R .: Gastroenterology, 91,603,1986. 98. Tasman-Jones, C. and Mistilis, S; P .: Med. J. Australia, 142,53,1985. 99. Kerss, A., Allen, A. and Gamer, A .: Clin. Sci., 63,187,1982. 100. Isenberg, J. I., Hogan, D. L., Koss, M. A. and Selling, J. A. , Gastroenterology, 91,370,1986. 101. Lichtenberger, L M., Richards, J. E. and Hills, B. A .: Gastroenterology, 88,308,1985. 102. Leung, F. W., Robert, A. and Guth, P. H. : Gastroenterology, 88, 1984, 1985. 103. Gannon, B., Browning, J. andO’Brien, P .: J. Anat., 135,667,1982. 104. Robert, A .: Gastroenterology, 77,761,1979. 105. McLean, A. L., Harconrt, D. M. and McNeil, J. J .: Drugs, 35,329,1988. 106. Jayarah, A. P., Tovey, F. and Clark, C. G .: Gut, 21,1068,1980. 107. Soli, A. H., Kurata, J. and McGuigan, J. E. : Gastroenterology, 96, 561, 1989. 108. Flemstrom, G .: In “Physiology of the gastrointestinal tract, Ed. By, M. J. Jackson, E. E. Jacobson and J. H. Walsh (Raven Press, New York), 1011, 1987. (73) 109* Pearson, J. P., Ward. R. and Allen, A .: Gut, 27,243,1986 110. Menguy, R .: Gastroenterology, 5 1,430» 1966. 111. Kent, P. W. and Allen, A .: Biochem. J., 106,645,1968. 112. Davenport, H. W .: Gut, 6,513,1965. 113. Hollander, F .: Gastroenterology, 1,401,1943- 114. Price, P. B. : Surg. Gynec. Obstet, 86,59, 1948. 115. Zaidi, S. H. and Mukheiji, B .: Ind. J. Med. Res., 46,27,1958. 116. Wolf, S. and Wolf, H. G .: In “ Human Gastric Function Oxford University Press, New York, 1943. 117. Zaidi, S. H., Singh, G. B. and Bajpai, R. P .: Ind. J. Med. Res., 49,16, 1961. 118. Esedov, E. M., Muradova, V. R. and Mamaev, S. N .: Terapevticheskii Arkhiv., 71,19,1999- 119. Bandyopadhyay, U., Das, D., Bandyopadhyay, D., Bhattacharya, M. and Baneijee, R. K .: Current Science, 76,55,1999. 120. linuma, S., Yoshikawa, T., Yoshida, N., Naito, Y. and Kondo, M ; Dig. Dis. Sci., 43,1657,1998. 121. Drake, I. M., Mapstone, N. P., Schorah, C. J., White, K. L M., Chalmers, D. M ., Dixon, M. F. and Axon, A. T. R .: Gut, 42,768,1998. 122. Figura, N .: J. Clin. GasfroentroZ., 25, 149,1997. 123. Figura, N .: Ita. J. Gastroentrol & Hepatology., 29,367,1997. 124. Alptekin, N., Seckin, S., Dogru Abbasoglu, S., Kocak Toker, N., Cevikbas, U. and Uysal, M .: Med. Sci. Res., 26, 595,1998. 125. Ito, M., Suzuki, Y., Ishihara, M. and Suzuki, Y. : Eu. J. Pharmacol, 3 5 4 ,189,1998. 126. Bing, S. R., Kinouchi, T., Kataoka, K. Kuwahara, T. and Ohnishi, Y .: Microbiology & Immunology, 4,745,1998. 127. Martin, M. J., LaCasa, C., AlarcondeLaLastra, C. Cabeza, J ., Villegas, I And Motivla, V .: Zeitschrift Fur Naturforschung., 53,82,1998. 128. Huh, K., Kwon, T. H., Kim, S. K. Kim, J. S. and Shin, U. S .: Arch. Pharmacol Res., zif 1, 1998. 129. Kokura, S., Yoshikawa, T., Naito, Y., Ichikawa, H., Takano, H., Takahashi, S., Tomii, T. Yoshida, N. and Kondo, M .: Dig. Dis. Sci., 42, 2566,1997. (74) 130. Linimia, S., Naito, Y., Yoshikawa, T., Takahashi, S., Takemura, T., Yoshida, N.and Kondo, M .: Dig. Dis. Sci., 43,35,1998. 131. Naito, Y., Yoshikawa, T., linuma, S., Yagi, N., Matsuyama, K., Boku, Y., Fujii, T., Yoshida, N., Kondo, M. and Sasaki, E .: Dig. Dis. Sci., 43, 83, 1998. 132. Tsutsui, Y., Nakamura, Y., Yamaguclii, S., Kavvanaka, N. and Sato, M : Pharmacol, 58,209,1999. 133. Guth, P. H .: In “Advances in nicer disease”, Ed. By., K. H. HolteraiuUer and J. R. Malagdada (Excerpta Medica, Amsterdam), 101,1980. 134. Guth, P. H .; In “Mechanisms of mucosal protection in upper gastro intestinal tracf, Ed. By., A. AUen, G. Flemstrom, A. Gamer, W. Silen, and L. A. Tumberg (Raven Press, New York), 253, 1984. 135. Sinha, S. N., San)^, R. K. and Sinha, Y. K .: Ind. J. Med. Res., 49,681, 1961. 136. Sanyal, A. K., Baneijee, C. R. and Das, P. K .: Arch. Int. Pharmacodyn., 155,244,1965. 137. Sanyal, A. BC., Gupta, K. K., and Chowdhuiy, N. K .: Arch. Int. Pharmacodyn., 149,393,1964. 138. Goel, R. K.,ChakrabartiAand Sanyal, A. K .: Planta Med. ,2,85,1985. 139. Best, R., Lewis, D. A. and Nasser, N. :BrJ. Pharmacol.^ 82,107,1984. 140. Goel, R- K., Govinda Das, D. and Sanjral, A. K .: Ind. J. Gastroenterol., 4, 249, 1985. 141. Goel, R. K., Gupta, S., Shankar, R. and Sanyal, A. K .: J. EthnopharmacoLy i8y 33, 1986. 142. Mukhopadhyay, K., Bhattacharya, D., Chakrabarti, A, Goel, R. K. and Sanyal, A. K .: J. Ethnopharmacol.f 21, 11, 1987. 143. Bhattacharya, S. K.and Ghosal, S .: Phytotherapy Res., 1,95,1987. 144. Goel, R. K., Tavares, I. A., and Bennett, A .: J. Pharm. Pharmacol, 4 1 ,747,1989. 145. Ghosal, S .: Phytochemistry., 24,1807,1985. 146. Ghosal, S. and Saini, K. S .: J. Chem. R es., 110 S, 1984. 147. Pandey, B. L., Goel, R. K. and Das, P. K .: Ind. J. Exp. Biol, 21,258, 1983. 148. Sanyal, A. K., Pandey, B. I., and Goel, R. K .: J. Ethnopharmacol, 5,79, 1982. (75) 149- Tavares, I. A., Goel, R. K. and Bennett, A .: Rec. Adv. Prostaglandins. Thromboxane Leukotrienes., 21,785,1990. 150. Goel, R. K., Biswas, D. C., Debnath, P. K. and Sanyal, A. K .: Ind. J. Pharmacol, 14 ,74> 1982. 151. Varma, M. D., Singh, R. H., Gupta, J. P. and Udupa, K. N .: In “International Seminar on Stress in Health and Diseases”, IMS, BHU, India, 1977. 152. Mitra, P. K. and Deb, S .: In “Prpc. Cf66* Session ofIndian Science Congress”, 1979. 153. Mitra, P. K .: In “Proa. O f 67^ Session o f Indian Science Congress” 1980. 154. Mitra, P. K .: In “Prof. Of 68^ Session of Indian Science Congress” 1981. 155. Maity, S., Vedasiromoni, J. R. and Ganguli, D. K .: Ind. J. Pharmacol. 2 9 ,55,1997. 156. Udupa, A. L., Lobo, O. and Srinivasan, K. K : Int. J. Pharmacol, 28, 50,1996. 157. Goel, R. K. and Kulkami, S. K .: Ind. J. Pharmacol, 28,38,1996. 158. Manonmani, S., Vishwanathan, V. P., Subramanian, S. and Govindasamy, S .: Ind. J. Pharmacol, 27, loi, 1995. 159. Mitra, P. K .: In “ Recent Advances in Animal Sciences”, Ed. By. S. K. Ghosal and Dipak Roy, (Orion Press International) 1,192,2001. 160. ArifuUah, H. M., Narada, KM. and Amruthraj, G .; Ind. J. Pharmacol, 20,1,1998. 161. Obi, E.,Emch, J. K., Orisakwe, O. E., Afonne, O. J ., liondu, N. A. and Agbasi, P. U .: Ind. J. Pharmacol, 32,381,2000. 162. Pandit, S., Sur, T. K., Jana, U., Bhattacharya, D. and Debnath, P. K .; Ind. J. Pharmaco., 32,378, 2000. 163. Goel, R. K. and Dasgupta, G .: Ind. J. Exp. Biol, tig, 230,1991. 164. Mitra, P. K. : Ph. D. thesis, University o f North Bengal, 1988. 165. Robert, A. and Nezamis, J. I .: Proc. Expt. Biol. Med., 98,9,1958. 166. Elliot, R. C. and Heward, G. J. F .: Pharmacol Res. Commun,^ 8,167, 1976. 167. Parmar, N. S. and Desai, Jagruti. K .: Ind. J. Pharmacol, 25,120, 1993- (76) 168. Sanyal, A. K., Debnath, P. K-, Bhattacharya, S. K. and Code, K. D .: In “Peptic Ulcer”, Ed. By, C. J. Pfeiffer (Munagaard, Copenhagan), 312, 1971. 169. Robert, A, Nezamis, J. E. and Phillips, J.P.: Gastroentererology, 5 5 > 481,1968. 170. Varley, H .: In “Practical Clinical Biochemistry”, Arnold Heinemann Publication, 32,1976. 171. Anson, M. L .: J. Gen. Physiol, 22,79,1938. 172. Lowry, O. M., Rosenbrough, W. L., Farr, A. L and Randall, R. S .: J. Biol Chem., 193,265,1951- 173. Sanyal, A. K., Mitra, P. K and Goel, R. K .: Ind. J. Exp. B iol, 21,78, 1983. 174. Warren, L .: J. B iol Chem., 234,1971,1959. 175. Dische, Z. and Borenfreund, E .: J. B iol Chem., 184,517,1950. 176. Dische, Z. and Schettles, L B .; J. B iol Chem., 175,595,1948- 177. Menguy, R .: Am. J. Surg., 117,806,1969. 178. Menguy, R .: Am. J. Dig. Dis., 5,911, i960. 179. Itoh, M. and Guth, P .: Gastroenterol, 88,1162,1984. 180. Cochron, R. A., Stefanko, J., Moore, C., Saik, R .: Surg. Forum., 28, 159,1982. 181. Mizui, T., Sato, H., Hirose, F and, Doteeuchi, M.: Life S ci., 41,755, 1987. 182. Pihan, G., Regillo, C. and Szabo, S .: Dig. Dis. Sci., 32,1395,1987. 183. Okhawa, H., Onoshi, N. and Yagi, K .: Anal. Biochem., 95,351,1979. 184. Schneider, W. C .: J. B iol Chem., 164,747,1946. 185. Dische, Z. and Schwarz, K .: Microchem. Acta., 2,13,1937. 186. Reinhold, J. G .: In “ Standard Methods in Clinical Chemistry”, Ed. By. M. Reiner, Academic Press, New York, 1,88. 187. Chopra, R. M. and Chopra, R. K .: In “Glossary of Indian Medicinal Plants”, CSIR, New Delhi, 7,1969. 188. Brucine, N .: Arch. Pharm. BerZ.,549,1609,1892. 189. Ralph, B., David, L. and Nasser, N .: Br. J. Pharmac., 82,107,1984. 190. Joseph, R. M., Varela, V., Kanji, V. K., Subramony, C. and Mihas, A. A. : Alimen. Pharmacol Therap., 13, 203,1999. ### (77) +1 e /-V e5J I II « o * o 8 00 P ®. * so O O O O o O d +1 d +1 d +1 ■o « T3 e ^ .a X £ .s * e« 1CO 0 ro * * 2 'Ti_ _ % A 1 o 0 ■s OS . O 1(5 0 * fn «n * d * '=*• 'o ^ B fs d 00 o | 1 I §