Methyl and isopropyl N-methylanthranilates attenuate diclofenac- and ethanol-induced gastric lesions in rats

Aims

Two natural alkaloids, methyl (M) and isopropyl (I) N-methylanthranilates, with recently demonstrated significant pharmacological activities, were assayed for their possible overall effect on intact gastric mucosa and their protective properties towards the onset of gastric lesions induced by diclofenac (a non-steroidal anti-inflammatory drug, NSAID) or ethanol.

Main methods

The influence of I and M on gastric mucosa integrity was assessed by oral administration in doses of 200 mg/kg. The gastroprotective action of I and M in doses of 50, 100 and 200 mg/kg was analyzed in the diclofenac and ethanol-induced gastric lesion models in rats. After the treatment, the stomachs of the animals were analyzed (captured by a digital camera). Ulcer scoring, morphometric and histopathological analyses of the stomachs were done.

Key findings

The oral application of these compounds on their own, even in quite high doses (200 mg/kg) did not induce gastric lesions. Both alkaloids exerted a very strong antiulcer activity, even in low doses (50 mg/kg), by decreasing the number of lesions caused by the application of either diclofenac or ethanol, eliminating them completely or reducing them to a form of mucosal hyperemia.

Significance

Their possible mechanism of action was discussed and due to their many positive properties including anxiolytic, antidepressant, antinociceptive, anti-inflammatory and gastroprotective activities, as well as a cheap and simple synthetic route for their preparation, methyl and isopropyl N-methylanthranilates, both alike, might represent a cost effective alternative sought for in the treatment of peptic ulcers and/or new safer NSAIDs for pain management.     

Tachykinins protect against ethanol-induced gastric lesions in rats

Subcutaneous pretreatment of rats with neurokinin (NK) A or the fragment NKA(4-10) reduced the degree of gastric lesions induced by oral administration of 96% ethanol. The protective effect of NKA(4-10) was dose-dependent. Arg-NKB, the water soluble derivative of NKB, was less effective than NKA or NKA(4-10) while [Me-Phe7]NKB, substance P (SP) and SP-methyl-ester were inactive. The NKA(4-10) antilesion effect was reversed by pretreatment with N-ethyl-maleimide, suggesting a possible involvement of sulphydryls in its action. Among the nonmammalian tachykinins, kassinin significantly reduced ethanol-induced lesions while eledoisin and physalaemin at equivalent molar doses were inactive. These results provide, for the first time, evidence that tachykinins and their derivatives exert gastroprotective activity toward ethanol-induced haemorrhagic lesions. Assuming a receptor-mediated mechanism, NK-2 sites could be involved.     

Cholecystokinin-8 protects gastric mucosa against ethanol-induced lesions in rats

Subcutaneous administration of cholecystokinin-8 (CCK-8, 10-100 micrograms/kg) reduces in a dose-dependent manner gastric lesions induced by 96% ethanol in rats, and CCK-4, CCK-7, and the CCK-8 nonsulfated form (all up to 100 micrograms/kg sc) were inactive. The presence of the entire molecule and sulfation of the tyrosine in position 2 are necessary for the mucosal protective properties of CCK-8 against 96% ethanol-induced gastric lesions. These effects are probably at least in part, due to a sulfhydryl-sensitive process.     

Protection by tetramethylpyrazine in acute absolute ethanol-induced gastric lesions

Acute oral administration of absolute ethanol (1.0 ml/kg) to fasting rats produced extensive necrosis of the gastric mucosa within 1 h. Pretreatment 30 min before administration of ethanol with oral tetramethylpyrazine (TMP) prevented this necrosis. Gross examination of the gastric mucosa of rats that received TMP showed fewer gastric lesions than that of rats who did not receive TMP. TMP pretreatment in rats exhibited superoxide scavenging activity in absolute ethanol-induced lipid peroxidation in gastric mucosal homogenates. TMP added in vitro to gastric homogenates made from control rats also showed scavenging activity. We conclude that the gastric protective mechanism of TMP could be attributed, at least in part, to its ability to inhibit lipid peroxidation and hence indirectly protect the gastric mucosa from oxidative stress.     

Oleuropein prevents ethanol-induced gastric ulcers via elevation of antioxidant enzyme activities in rats

Purified oleuropein from olive leaf extract has been shown to have antioxidant effects in our recent studies. Thus, the aim of this study was to assess the antioxidant abilities of oleuropein in comparison with ranitidine in ethanol-induced gastric damages via evaluation of ulcer index inhibition, antioxidant enzyme activities, and lipid peroxidation level. Fifty-six adult male Sprague?CDawley rats were divided into seven equal groups as follows: control group, ethanol group (absolute ethanol 1?ml/rat), oleuropein group (12?mg/kg), and oleuropein (6, 12, and 18?mg/kg) plus ethanol groups, as well as ranitidine (50?mg/kg) plus ethanol group. Pretreatment with oleuropein (12 and 18?mg/kg) significantly increased the ulcer index inhibition (percent), in comparison with oleuropein (6?mg/kg). Glutathione peroxidase (GPx) activity was significantly lower in the ethanol group when compared with the other groups whereas, treatment of rats with oleuropein (12?mg/kg) significantly increased glutathione content in gastric tissue when compared with the other groups, and lipid peroxidation was significantly reduced in the oleuropein- (12 and 18?mg/kg) and ranitidine-treated animals. Superoxide dismutase (SOD) and catalase (CAT) activities were both much higher in oleuropein-treated rats than the ethanol group, and although there was a moderate increase in SOD and CAT activities in ranitidine-treated rats, the differences were not significant. These findings suggest that oleuropein has beneficial antioxidant properties against ethanol-induced gastric damages in the rat. Therefore, it seems that a combination regimen including both antioxidant and antisecretory drugs may be beneficial in prevention of ethanol-mediated gastric mucosal damages.     

苹果多酚对高尿酸血症大鼠抗氧化能力的影响

目的:高尿酸血症与许多现代慢性疾病关系密切,本文旨在研究苹果多酚对高尿酸血症大鼠体内抗氧化能力的影响.方法:采用对Wistar大鼠灌胃酵母膏及氧嗪酸钾的方法,制作高尿酸血症大鼠模型,再给其灌胃不同剂量苹果多酚.通过测定大鼠血清总抗氧化能力,脂质过氧化产物含量,超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶活力,探讨苹果多酚对高尿酸血症大鼠机体抗氧化能力的影响.结果:服用苹果多酚的高尿酸血症大鼠的血清总抗氧化能力显著升高,脂质过氧化产物水平相对降低;超氧化物歧化酶是受高尿酸血症和苹果多酚摄入影响最大的抗氧化酶,模型组酶活显著升高,而苹果多酚可以抑制该酶活力因大量尿酸生成引发的升高现象.结论:苹果多酚在适当剂量水平可以显著提高高尿酸血症大鼠的总抗氧化能力,抑制脂质过氧化,同时抑制因大量尿酸生成而引发的超氧化物歧化酶活升高,维护机体氧化还原平衡.     

Lipid peroxidation and antioxidant enzymes in vanadate-treated rats

Male Wistar rats received an aqueous solution of ammonium metavanadate (AMV) of 0.15 mg/V/ml concentration instead of water for 14 days. The erythrocyte count and haemoglobin level in blood were not changed; the haematocrit index was slightly increased. The spontaneous lipid peroxidation in kidney and liver homogenates was increased. The Fe(II)- or ascorbate-induced lipid peroxidation was more pronounced in the kidney than in the liver. No changes in lipid peroxidation were observed in erythrocytes after AMV treatment. The AMV treatment resulted in a decrease in the activity of the antioxidant enzymes, catalase and glutathione peroxidase in the kidney and liver; the cytosolic Cu,Zn-SOD and mitochondrial Mn-SOD were unchanged. The activity of the enzymes in blood was not changed. The results are discussed with a view to the participation of lipid peroxidation in vanadium toxicity.     

Protective effects of polygodial and related compounds on ethanol-induced gastric mucosal lesions in rats: structural requirements and mode of action

The methanolic extract from the leaves of Tasmannia lanceolata was found to potently inhibit ethanol-induced gastric lesions in rats. Through bioassay-guided separation, three known sesquiterpenes, polygodial, polygodial 12 alpha-acetal, and polygodial 12 beta-acetal, and a new sesquiterpene, methyl isodrimeninol, were isolated as the active constituents. Among them, polygodial showed very potent gastroprotective effects (ED(50)=0.028 mg/kg, po). From the gastroprotective effects of various reduction and oxidation derivatives of polygodial, the dialdehyde or diacetal structure was found to be essential for the strong activity. Since the gastroprotection of polygodial was attenuated by pretreatment with indomethacin, N-ethylmaleimide, N(G)-nitro-L-arginine methyl ester and ruthenium red, endogenous prostaglandins, sulfhydryl compounds, nitric oxide and vanilloid receptors may be involved in the protective activity.     

Effect of stobadine on indomethacin- and ethanol-induced stomach lesions and gastric secretion.

Stobadine was found to inhibit the ulcerogenic activity of indomethacin in relation to the dose but was ineffective against the direct necrotizing action of ethanol. It also inhibited gastric acid secretion when administered intraduodenally. Although stobadine is considered to be a scavenger of free radicals, our results indicate that, under the given experimental conditions, it is rather the inhibition of gastric acid secretion that is responsible for its antiulcerogenic effect. The preliminary results do not allow the exclusion of other mechanisms for explaining its antiulcerogenic effect.     

Region specific increase in the antioxidant enzymes and lipid peroxidation products in the brain of rats exposed to lead

The objective of this study is to determine the effect of lead (pb) on antioxidant enzymes and lipid peroxidation products in different regions of rat brain. Wistar male rats were treated with lead acetate (500 ppm) through drinking water for a period of 8 weeks. Control animals were maintained on sodium acetate. Treated and control rats were sacrificed at intervals of 1st, 4th and 8th week and the whole brains were dissected on ice into four regions namely the cerebellum, the hippocampus, the frontal cortex and the brain stem. Antioxidant enzymes namely catalase and superoxide dismutase in all the four regions of brain were determined. In addition, lipid peroxidation products were also estimated. The results indicated a gradual increase in the activity of antioxidant enzymes in different regions of the brain and this response was time-dependent. However, the increase was more in the cerebellum and the hippocampus compared to other regions of the brain. The lipid peroxidation products also showed a similar trend suggesting increased effect of lead in these two regions of the brain. The data indicated a region-specific oxidative stress in the brain exposed to lead.     

Effects of traditional herbal medicine on gastric mucin against ethanol-induced gastric injury in rats

The effect of the traditional herbal medicine, Rikkunshi-to and its component crude drugs, Zingiberis Rhizoma and Glycyrrhizae Radix, on the gastric mucin was studied using a method developed to separate and quantify the mucin localized in the different layers of rat gastric mucosa. The oral administration of spray-dried extract to Rikkunshi-to (1000 mg/kg), Zingiberis Rhizoma (500 mg/kg) and Glycyrrhizae Radix (500 mg/kg) significantly prevented gastric mucosal damage induced by 70% ethanol in rats. In ethanol-treated rats the mucin content of the deep mucosa was reduced, and the reduction of the deep corpus mucin content was significantly inhibited by pretreatment of Rikkunshi-to and Zingiberis Rhizoma. Rikkunshi-to and Glycyrrhizae Radix pretreatment increased the surface mucin content by 140 and 146%, respectively. The effect on the gastric mucin by each drug differed in the different layers of the gastric mucosa.     

Isolation of a wild Morchella spp. strain and the effects of its extract on ethanol-induced gastric mucosal lesions in rats

A Morchella spp. strain was isolated from a wild morel mushroom, and the effects of its mycelia extract on the ethanol-induced gastric mucosal lesions of rats were investigated in vivo. Sequence analysis of internal transcribed spacer suggested that this Morchella spp. strain (strain No. M1) was clustered together with M. conica in the phylogenetic tree. The superoxide dismutase (SOD) activity increased significantly compared to the control. However, the malondialdehyde (MDA) level and myeloperoxidase (MPO) activity decreased significantly compared to the control. These results indicated that M1 is one member of M. conica and the protective effects of M1 extract against the ethanol-induced gastric lesions may be related to the increased SOD activity and decreased MDA level and MPO activity in rats.     

Protective effects of DIDS against ethanol-induced gastric mucosal injury in rats

The compound 4,4＇-diisothiocyanostilbene-2,2＇-disulfonic acid （DIDS） is an efficient anion exchanger inhibitor that can block the activities of anion exchanger 2 （AE2）, which plays an indispensable role in gastric acid secretion. DIDS also has potent anti-oxidative and antiapoptosis activities. This study aimed to investigate the effect of DIDS on ethanol-induced mucosal damage in rats and to evaluate the underlying mechanisms that mediate the action of the compound. The rats received 1 ml of absolute ethanol or saline orally. DIDS [50 mg/kg intravenous （i.v.）] was given 5 min before ethanol administration. Gastric lesions were evaluated macroscopically, microscopically, and electron microscopically at 60 min after ethanol challenge. Gastric myeloperoxidase （MPO） activity, malonyldialdehyde （MDA） level, prostaglandin E2 （PGE2） synthesis, and cyclooxygenase-2 （COX-2） expression were assessed. For the evaluation of the effect of DIDS on gastric acid secretion, histamine-stimulatory gastric acid secretion was examined with or without pretreatment of DIDS （50 mg/kg; i.v.）. Ethanol-induced gastric lesions were characterized by increasing gastric MDA level, MPO activity, and COX-2 expression, and decreasing PGE2 synthesis. It was found that DIDS significantly reduced the extent of gastric mucosal damage and reversed tissue MDA level and MPO activity. DIDS further enhanced the expression of COX-2 and reversed the decrease of PGE2. Our results suggested that DIDS is beneficial in rat model of gastric injury through mechanisms that involve inhibiting inflammatory cell infiltration and lipid peroxidation and up-regulating the COX-2/PGE2 pathway.     

Effect of malotilate on ethanol-induced gastric mucosal damage in capsaicin-pretreated rats

We studied the role of afferent sensory neurons in malotilate-mediated gastric mucosal protection. Intact and capsaicin sensory-denervated rats were used in the experiments. Gross gastric mucosal injury was assessed and evaluated as a main criterion of the gastroprotective effect of the tested substances. Besides malotilate, methyl-prostaglandin E2 was applied alone or in combination with malotilate to compare the effects and the mechanism of action of both substances. The results revealed that both malotilate as well as methyl-prostaglandin E2 exerted a significant protective action on 96% ethanol-induced gastric mucosal damage. However, there were no significant differences between intact and capsaicin-denervated rats. Only the use of 50% ethanol as a milder mucosal irritating agent resulted in significant differences in both groups of animals. We propose that malotilate (like methyl-prostaglandin E2) has a gastroprotective effect on ethanol-induced gastric mucosal injury. This effect is partly dependent on the sensory nervous system and the combination of both above substances has an additive effect.     

Effect of stress on the antioxidant enzymes and gastric ulceration

The effect of cold-restraint stress on the antioxidant enzymes of the rat gastric mucosa was studied with a view to finding out their role in stress induced gastric ulceration. Histological examination revealed stress induced extensive damage of the surface epithelial cell with lesions extending upto submucosa in some cases. Stress causes time-dependent increase in histamine and pepsin content but decrease in acid content of the gastric fluid with the progress of ulceration (ulcer index) for two hours. The tissue lipid peroxidation was significantly increased as evidenced by accumulation of malondialdehyde. Since lipid peroxidation results from the generation of reactive oxygen species, stress effect was studied on some antioxidant enzymes such as superoxide dismutase, peroxidases and prostaglandin synthetase as a function of time. The time dependent increase in stress ulcer correlates well with the concomitant increase in superoxide dismutase activity and decrease in peroxidase and prostaglandin synthetase activity. This creates a favourable condition for accumulation of endogenous H₂O₂ and more reactive hydroxyl radical (OH·). Administration of antioxidants such as reduced glutathione or sodium benzoate prior to stress causes significant decrease in ulcer index and lipid peroxidation and protection of gastric peroxidase activity suggesting the involvement of reactive oxygen species in stress induced gastric ulceration. This is supported by thein vitro observation that OH· can also inactivate peroxidase and induce lipid peroxidation. As prostaglandin is known to offer cytoprotection, stress-induced loss of prostaglandin synthetase activity appears to aggravate the oxidative damage caused by reactive oxygen species.Abbreviations ROS reactive oxygen species - GPO gastric peroxidase - SOD superoxide dismutase - MDA malondialdehyde - GSH reduced glutathione - TCA trichloroacetic acid     

Lipid peroxidation and activity of antioxidant enzymes in diabetic rats

We hypothesized that oxygen free radicals (OFRs) may be involved in pathogenesis of diabetic complications. We therefore investigated the levels of lipid peroxidation by measuring thiobarbituric acid reactive substances (TBARS) and activity of antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT)] in tissues and blood of streptozotocin (STZ)-induced diabetic rats. The animals were divided into two groups: control and diabetic. After 10 weeks (wks) of diabetes the animals were sacrificed and liver, heart, pancreas, kidney and blood were collected for measurement of various biochemical parameters. Diabetes was associated with a significant increase in TBARS in pancreas, heart and blood. The activity of CAT increased in liver, heart and blood but decreased in kidney. GSH-Px activity increased in pancreas and kidney while SOD activity increased in liver, heart and pancreas. Our findings suggest that oxidative stress occurs in diabetic state and that oxidative damage to tissues may be a contributory factor in complications associated with diabetes.     

Protective and therapeutic effects of Argyreia speciosa against ethanol-induced gastric ulcer in rats

The protective and therapeutic effects of Argyreia speciosa Sweet (Convolvulaceae) against ethanol-induced gastric ulcer in rats were evaluated. Ethanolic and water extracts of the aerial plant parts (200 mg/kg body weight) were orally administered daily for seven days prior to or after ulceration with one oral dose of 1 mL absolute ethanol on 24-h empty stomachs. Rats were divided into eleven groups. Group 1 served as control. To groups 2 and 3 each extract was administered. Groups 4 to 6 received each extract or ranitidine (100 mg/kg body weight) prior to ulcer induction. Groups 7 to 9 received each extract or ranitidine post ulcer induction. Groups 10 and 11 were gastric ulcerative rats after one hour and one week of ethanol induction. The evaluation was done through measuring ulcer indices: stomach acidity and volume, lesion counts, mucus, and prostaglandin E2 contents. Oxidative stress marker, i. e. malondialdehyde, glutathione, and superoxide dismutase, were estimated. Certain marker enzymes for different cell organelles, i. e. succinate and lactate dehydrogenases, glucose-6-phosphatase, acid phosphatase, and 5'-nucleotidase, were evaluated. The work was extended to determine the collagen content and the histopathological assessment of the stomach. Gastric ulcer exhibited a significant elevation of the ulcer index, antioxidant levels, collagen content, and the marker enzymes. The water extract attenuated these increments and was more potent as a protective agent, while the ethanol extract exhibited stronger therapeutic potency. In conclusion, A. speciosa acted as antiulcer agent. More detailed studies are required to identify the compounds responsible for the pharmacological effect.     

Lead-induced increase in antioxidant enzymes and lipid peroxidation products in developing rat brain

Pregnant rats were treated with 0.4% lead acetate through drinking water from 6th day of gestation and this treatment was continued till 21 post natal days (PND). Four regions of the brain namely hippocampus, cerebellum, frontal cortex and brain stem were dissected at 10, 20, 30 and 40 PND for estimation of lipid peroxidation products (LPP), catalase (CAT) and superoxide dismutase (SOD). The results indicate that there was a significant (P < 0.05) increase of LPP in exposed rats than their corresponding control at 10, 20 and 30 PND both in hippocampus and cerebellum. At PND 40, the LPP of control and exposed were found to be almost same in both the tissues indicating recovery from lead toxicity. CAT activity was significantly (P < 0.05) high in hippocampus of exposed rats up to PND 30 but up to PND 20 in cerebellum and frontal cortex. However, in brain stem, a significant (P < 0.05) increase in CAT activity was observed only at PND 10. A significant (P < 0.05) increase in SOD activity was observed up to PND 30 both in hippocampus and cerebellum on lead exposure. Frontal cortex exhibited a similar significant (P < 0.05) increase of SOD activity up to PND 20 and for brain stem up to PND 10. There was no significant change in the activity of antioxidant enzymes (CAT and SOD) and LPP in all the four brain tissues of control and exposed rats at PND 40 indicating recovery from lead-induced oxidative stress. This research work was presented as a poster in Annual Biomedical Research Conference for Minority Students (ABRCMS) at Dallas, Texas, USA, during November 10–13, 2004 and the abstract was printed on page 231 of the Conference Proceedings