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Master Title

Pharmacological study of the possible protective effects of certain herbal plants against gastric and colon ulcer in experimental animals

Master Abstract

In the present investigation, the possible protective effects of three natural extracts, namely echinacea extract, green tea extract and boswellia extract were studied in comparison with ranitidine as a reference standard, against experimentally-induced gastric ulcer. Gastric ulceration was induced following pyloric ligation or ethanol model. Test drugs were intraperitoneally administered one hour before pyloric ligation to 48 hours fasted rats. The doses of drugs were ranitidine 20 mg/kg, echinacea extract 25 mg/kg, green tea extract 25 mg/kg and boswellia extract 200 mg/kg. In ethanol model, test drugs were administered intraperitoneally one hour before administration of ethanol (70 % p.o.) in same doses as before except for ranitidine (40 mg/kg). The possible subchronic antiulcerogenic effects of two weeks treatment with ranitidine (10 mg/kg) in combination with echinacea extract (25 mg/kg), green tea extract (25 mg/kg) or boswellia extract (200 mg/kg) on pyloric ligated-induced gastric ulcer were studied. In addition to the above protocol, the possible protective effects of echinacea extract (25 mg/kg), green tea extract (25mg/kg) and boswellia extract (200 mg/kg) were studied in comparison with prednisolone (2mg/kg) as a reference standard, against iodoacetamide-induced colon ulcer. Test drugs were intraperitoneally administered for one week. The possible subchronic antiulcerogenic effects of one week treatment with prednisolone (1mg/kg) in combination with echinacea extract (25 mg/kg), green tea extract (25 mg/kg) or boswellia extract (200 mg/kg) on iodoacetamide-induced colon ulcer were evaluated. The main findings of the present study can be summarized as follows:- I) Gastric Ulcer:- 1- Acute administration of test drugs namely ranitidine (20 mg/kg), echinacea extract (25 mg/kg), green tea extract (25 mg/kg) and boswellia extract (200mg/kg) against pyloric ligation-induced gastric ulcer significantly _____________________________________________Summary and Conclusions 190 reduced ulcer number, ulcer index and gastric acidity but did not exhibit any significant effect on mucin concentration. 2- Acute administration of echinacea extract and green tea extract on pyloric ligated-induced gastric ulcer significantly increased gastric mucosal glutathione content, while ranitidine and boswellia extract did not produce any significantly effect on gastric mucosal glutathione content. 3- Acute administration of these test drugs on pyloric ligated-induced gastric ulcer significantly reduced gastric mucosal malondialdehyde content and significantly increased superoxide dismutase activity in blood. 4- Subchronic administration of test drugs for two weeks on pyloric ligated-induced gastric ulcer significantly reduced ulcer number, ulcer index and gastric acidity. 5- Subchronic administration of ranitidine and green tea extract for two weeks on pyloric ligated-induced gastric ulcer significantly increased mucin concentration while echinacea extract and boswellia extract did not produce any significant effect on mucin concentration. 6- Subchronic administration of test drugs for two weeks on pyloric ligated-induced gastric ulcer significantly increased gastric mucosal glutathione content and superoxide dismutase activity in blood while gastric mucosal malondialdehyde content was significantly reduced. 7- Combination of ranitidine (10 mg/kg) and individual test drugs for two weeks on pyloric ligated-induced gastric ulcer significantly reduced ulcer number, ulcer index and. gastric acidity. All test drugs synergized the effect of ranitidine on ulcer number, ulcer index and. gastric acidity. _____________________________________________Summary and Conclusions 191 8- Combination of ranitidine (10 mg/kg) and individual test drugs for two weeks on pyloric ligated-induced gastric ulcer significantly increased mucin concentration. Green tea extract synergized the effect of ranitidine on mucin concentration while, there is no significant difference between the effect of ranitidine alone and combination of ranitidine with echinacea extract or boswellia extract on mucin concentration. 9- Combination of ranitidine (10 mg/kg) and individual test drugs for two weeks on pyloric ligated-induced gastric ulcer significantly increased gastric mucosal glutathione content and superoxide dismutase activity in blood while gastric mucosal malondialdehyde content was significantly reduced. All test drugs synergized the effect of ranitidine on gastric mucosal glutathione content, gastric mucosal malondialdehyde content and blood superoxide dismutase activity in blood. 10- Acute administration of test drugs namely ranitidine (40 mg/kg), echinacea extract (25 mg/kg), green tea extract (25 mg/kg) and boswellia extract (200 mg/kg) against ethanol-induced gastric ulcer significantly reduced ulcer number and ulcer index. 11- All above mentioned test drugs significantly increased gastric mucosal glutathione content and superoxide dismutase activity in blood on ethanol-induced gastric ulcer. While gastric mucosal malondialdehyde content were significantly reduced II) Colon Ulcer:- 1- Subchronic administration for one week of test drugs namely prednisolone (2 mg/kg), echinacea extract (25 mg/kg), green tea extract (25 mg/kg) and boswellia extract (200 mg/kg) against iodoacetamide-induced colon ulcer _____________________________________________Summary and Conclusions 192 significantly increased colonic mucosal glutathione content and significantly reduced colonic myeloperoxidase (MPO) activity. 2- Combination of prednisolone (1 mg/kg) and individual test drugs for one week against iodoacetamide-induced colon ulcer significantly increased colonic mucosal glutathione content and significantly reduced colonic myeloperoxidase (MPO) activity. All test drugs synergized the effect of prednisolone on colonic mucosal glutathione content and colonic myeloperoxidase (MPO) activity. Depending on results of the present study it can be concluded that:- 1- Echinacea extract, green tea extract and boswellia extract have protective effects against pyloric ligation and ethanol-induced gastric ulcer. 2- The acute protective effect of echinacea extract, green tea extract and boswellia extract could be mediated via reduced gastric acidity as well as their antioxidant activity. The gastroprotective effects of these drugs against ethanol-induced gastric ulcer could be attributed to their antioxidant activities 3- The gastroprotective effects of green tea extract after two weeks treatment could be attributed to reduce gastric acidity, increase mucin concentration as well as its antioxidant activity. However, the effect of echinacea extract and boswellia extract could be mediated through reduce gastric acidity in addition to their antioxidant activity. 4- The combination of ranitidine (10 mg/kg) with echinacea extract, green tea extract or boswellia extract is an effective tool to reduce the adverse effects of _____________________________________________Summary and Conclusions 193 ranitidine. It can also provide economic benefit where the combination produced the same effects of ranitidine (20 mg/kg) by using 10 mg/kg only. 5- Echinacea extract, green tea extract and boswellia extract have protective effects against iodoacetamide-induced colon ulcer after 7 days treatment. The protective effects of these test drugs could be mediated via their anti-inflammatory as well as antioxidant activity. 6- The combination of prednisolone (1 mg/kg) with echinacea extract, green tea extract or boswellia extract in colon ulcer is an effective tool to reduce the adverse effects of prednisolone and provide economic benefit where the combination produced the same effects of prednisolone (2 mg/kg) by using 1mg/kg only. 7- Although results of the present work revealed that echinacea extract, green tea extract and boswellia extract are effective antiulcer agents in gastric and colon ulcers in rats, yet studies of adverse and toxic effects then clinical investigations are required before they are tried for treatment in patients

PHD Title

A Pharmacological study of the effects of ethanol consumption on normal heart and colon as well as on experimentally-induced inflammatory bowel disease in rodents

PHD Abstract

6. Summary and Conclusion The aim of the present study was running in two parallel aspects. First, to elucidate the possible mechanisms underlying the effects of ethanol consumption on normal colon and on colons with ulcerative colitis (UC). Second, to explore its effects on the heart. To achieve this goal, two sets of experiments were performed; an in vivo model on colon, and an in vitro model using HL-1 cardiomyocyte cell line. I. In vivo Experiments: This experiment was performed to demonstrate the effects of ethanol consumption on normal colon and on colons with experimentally-induced UC in rats. UC was induced at the 8th day of the experiment by intracolon injection of 0.1 ml of 2% N-ethylmaleimide (NEM; dissolved in 1% methylcellulose) at a depth of 6 cm from anus via a Nelaton's catheter. Rats were allocated into 4 groups. Animals were divided into a normal control group which received distilled water orally, an ethanol group which received ethanol (2.5 g/kg, orally) once daily for two weeks, an UC control group which received distilled water orally and single dose of 2% NEM via the intracolon route and an ethanol/UC group which received ethanol (2.5 g/kg, orally) once daily for two weeks and a single intracolon dose of 2% NEM. On the last day of the experiment, the rats were sacrificed by cervical dislocation. The colon was excised and the colitis was assessed macroscopically by measuring both colon length and colon mass index. The degree of colonic injury was assessed by measuring colonic reduced glutathione (GSH) content as an oxidative stress biomarker, as well as colonic myeloperoxidase (MPO), tumour necrosis factor-alpha (TNF-a) and total nitrate/nitrite (NOx) contents as inflammatory biomarkers, supported by colon macroscopic and microscopic examinations. The main findings of the in vivo experiments can be summarized as follows: 1. The intracolon injection of NEM led to a significant shortening of colon length as well as an increase in colon mass index. This was associated with a marked increase in the colonic contents of the pro-inflammatory cytokine TNF-a. Colonic MPO activity was also elevated indicating an increase in neutrophilic infiltration of the intestinal mucosa. Colonic tissue GSH was significantly reduced while NOx was significantly increased. Histopathological examination of the colon revealed severe ulcerative changes coupled with neutrophil infiltration. 2. Daily administration of oral ethanol (2.5 mg/kg/day) significantly decreased the colon length but did not significantly affect the colon mass index of normal animals, and markedly increased the colonic contents of the pro-inflammatory cytokine TNF-a. Colonic tissue GSH was significantly reduced while NOX was significantly increased compared with normal rats. The harmful effect of the drug was substantiated histologically as marked degenerative changes in the colon. 3. Daily administration of oral ethanol (2.5 mg/kg/day) worsened UC disease progression. Treatment of ethanol in UC rats led to a marked shortening of colon length as well as an increase in colon mass index compared to the UC control values. This was associated with a marked increase in the colonic contents of TNF-a. Colonic MPO activity was also elevated indicating an increase in neutrophilic infiltration of the intestinal mucosa. Colonic GSH was significantly reduced while NOX content was significantly increased. The inflammatory condition of the colon was also confirmed histologically. II. In vitro Experiments: The effect of ethanol was performed on isolated cardiac myocytes in vitro, through using the atrial cell cultures of HL-1 cells. In order to characterize the effects of ethanol on the calcium dependent steps of cardiac excitation-contraction coupling (EC-coupling), cells were exposed to 25 mM and 100 mM ethanol acutely, where Ca2+ spikes were recorded before and after cell exposure to 25 or 100 mM of ethanol for 5-10 minutes. In order to investigate the effect of chronic ethanol exposure on cardiac atrial cells, cells were plated and a time course of treatment was developed to characterize the changes associated with 25 mM and 100 mM ethanol exposure up to 72 hours by using western blotting technique to determine the expression of several proteins level as total L-type Ca2+ channel (LTCC), phosphorylation of the LTCC (phospho LTCC), T-type Ca2+ channel (TTCC) and Ryanodine receptors (RyRs). The main findings of the in vitro experiments can be summarized as follows: 1. HL-1 cells treated with 25 mM ethanol showed a reduction in Ca2+ transient amplitude and frequency. The reduction of Ca2+ transients was magnified upon increasing ethanol concentration to 100 mM. 2. HL-1 cells treated with 25 mM ethanol showed an increase in level of total LTCC after 24, 48 and 72 hours. Cyclic AMP stimulation also enhanced the protein level of LTCC in comparison to the cells without treatment. When Cyclic AMP stimulation treated to the cells with 25 mM ethanol for 72 hours also enhanced the level of LTCC as compared to treatment of 25 mM ethanol for 72 hours alone. 3. Concerning to phosphorylation of the LTCC, HL-1 cells treated with 25 mM ethanol revealed a significantly higher level of basal phosphorylation after 24, 48 and 72 hours. Cyclic AMP stimulation also enhanced the phosphorylation of the LTCC in comparison to the cells without treatment. 4. Treatment of HL-1 cells with 100 mM ethanol caused an increase in LTCC level after 24, 48 and 72 hours. The highest level of LTCC was after 48 hours of treatment, and then the level decreased after treatment of 100 mM ethanol for 72 hours as compared to 48 hour of treatment. Concerning to phosphorylation of the LTCC, the present study showed that HL-1 cells treated with 100 mM ethanol showed a significantly higher level of basal phosphorylation after 48 hours of treatment. 5. HL-1 cells treated with 25 mM ethanol showed a slight increase in TTCC expression after 24, 48 and 72 hours. Cyclic AMP stimulation also enhanced the expression of TTCC in comparison to the cells not subjected to ethanol treatment, and in comparison to the cells treated with 25 mM ethanol for 72 hours alone. 6. Treatment of the cells with 100 mM ethanol markedly increased the level of TTCC after 24, 48 and 72 hours. The level increased by increasing the duration of treatment to 72 hours. Cyclic AMP stimulation also enhanced the TTCC level in comparison to the cells without treatment, but not when compared to the cells treated with 100 mM ethanol for 72 hours alone. 7. HL-1 cells treated with 25 mM ethanol and 100 mM ethanol showed a significantly higher level of expression of RyR level after 24, 48 and 72 hours of treatment. Cyclic AMP stimulation also enhanced the expression of RyR level in comparison to the cells without treatment, but not when compared to the cells treated with 25 mM and 100 mM ethanol for 72 hours alone. In conclusion, the present study suggests that ethanol consumption has harmful effects on normal colon and may worsen UC disease progression through decrease in endogenous antioxidant capacity coupled with an increase in inflammatory progression. Additionally, ethanol consumption has harmful effects on the heart, where acute negative inotropic effects of ethanol were found on isolated atrial cells, while a positive inotropic phase and enhanced EC-coupling were found as increased the proteins expression of LTCC, phospho LTCC, TTCC and RyR after chronic treatment of ethanol, eventually leading to the development of alcoholic cardiomyopathy.