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International Journal of Pharmacognosy and Phytochemical Research 2013; 5(2); 113-119 Gastroprotective Efficacy of Folic Acid and Omeprazole in 1Samar Morjan , *1Shaza Al Laham , 2Rana Atieh 1Pharmacology & Toxicology Department - Faculty of pharmacy- Damascus University- Damascus-Syria 2Histopathological Department - Medicin faculty- Damascus University- Damascus-Syria ABSTRACT
Gastric and intestinal mucosal damage is the commonest adverse effect of NSAIDs. Their use is associated with a
significant risk of hemorrhage, erosions, and perforation of both gastric and intestinal ulcers. NSAID-induced ulcers can
be prevented largely through co-administration of a proton pump inhibitor to block acid secretion in the stomach. Also
there is a role for folic acid in the attenuation of indomethacin induced gastric ulceration. Study the effect of Folic acid and
its association with the antisecretory drug Omeprazole (Proton pump inhibitor) for their abilities to protect gastric mucosa
against the indomethacin-induced gastric ulcer. The experiments had been done on 10 white wistar rats for each group.
Gastric ulcer was induced by administration of indomethacin (20 mg/kg i.p.). Folic acid (2 mg/kg, orally) has been given
for the first group, while Omeprazole (20 mg/rat, orally) has been given for the second group and Folic acid (2 mg/kg,
orally) with Omeprazole (10 mg/rat, orally) has been given for the third group as a repeated administration (once daily for
7 days). The gastric ulcers in stomach's rat were examined histological and macroscopical .The ulcer Index and protective
Index were calculated. Then the glycoproteines of stomach's mucus gel was measured by spectrophotometer. The
combination-treated group (Folic acid and Omeprazole ) gave significant increase in the amount of gastric mucosa as
compared to Indomethacin group and in comparing to each drug alone the protective Index was increased while ulcer
Index retreat. It could be concluded that the combination-treated groups afford a good gastro-protective potential against
the gastric ulceration induced by indomethacin better than each drug alone. The antioxidant effects of folic acid and
omeprazole are involved and ameliorating the oxidative stress induced by indomethacin in the gastric mucosa.
Keywords: Ulcer, Omeprazole, Folic acid.
INTRODUCTION
degraded at low pH and must be given in enteric-coated Peptic ulcer occurs when there is an imbalance between the granules1. The coating is removed in the alkaline damaging effects of gastric acid and pepsin, and the duodenum, and the prodrug, a weak base, is absorbed and defense mechanisms, which protect the gastric and transported to the parietal cell canaliculus. There, it is duodenal mucosa from these substances1. The danger of converted to the active form. Clinical studies have shown epithelial arrosion and subsequent ulcer formation exists that PPIs reduce the risk of bleeding from an ulcer caused whenever the protective and reparative mechanisms are weakened and/or the chemical attack by the acid–pepsin Folic acid (or folate), is a water-soluble vitamin8. Folic mixture is too strong and persists for too long2. The acid is used to prevent or cure deficiency of folate which majority of gastric ulcers can be attributed to either H.
is due either to a decreased supply or to an increased pylori or NSAID-induced mucosal damage3, particularly requirement. Deficiency of folic acid leads to a in the elderly1. NSAIDs cause serious complications only megaloblastic anaemia because it is necessary for the in a small fraction of NSAID users. Hence, NSAID production of purines and pyrimidines, which are essential prophylaxis is advised for those older than 654, cases of precursors of deoxyribonucleic acid (DNA)24. Folate cardiac disease5, those with previous history of ulcers or modulates a number of disorders as a result of its anti- upper GI bleed4,5, and those using anticoagulants and gastroprotective activity of folic acid supplementation at Recent studies suggest that NSAID-induced ulcers in at- the basal requirement supplemental dose of 2 mg/kg diet risk patients can be prevented largely through co- against the lipid peroxidative activity of indomethacin was administration of a proton pump unhibitor6, such as Omeprazole that bind to the H+/K+-ATPase enzymesystem (proton pump) of the parietal cell and suppress the MATERIALS AND METHODS
secretion of hydrogen ions into the gastric lumen7. This Animals: Fifty male albino rats of the Wistar strain irreversibly inactivates the enzyme causing profound weighing between 180-250 g were used for this study. The inhibition of acid secretion: a single 20 mg dose reduces animals were separated randomly into ten cages of five rats gastric acid output by 90% over 24 h. Omeprazole is each where they were kept for four weeks before the start *Author for correspondence: Email: [email protected] Shaza Al Laham et.al./ Gastroprotective Efficacy of… Table 1: Effect of folic acid, omeprazole and combination between them on ulcer number, ulcer acuity, ulcer index,and preventive index in indomethacin- (INDO-; 20 mg/kg, i.p.) induced gastric injury.
Parametric data were expressed as mean ± S.E.M. P < 0.05. As compared to control (CONT) (**). As compared toINDO (*). Figure 3: Pin-point petechial hemorrhages animals from contracting disease. They were fed with standard commercial rat pellets and allowed water ad of the experiment. The animals were housed understandard conditions of temperature (23 ± 2°C), humidity(55 ± 15%) and 12 hour light (7.00 am - 7.00 pm). The cages were constantly cleaned in order to prevent the IJPPR, Vol-5, Issue 2, June- August 2013,113-119 Shaza Al Laham et.al./ Gastroprotective Efficacy of… Table 2: Effect of folic acid, omeprazole as well as their combination on indomethacin-induced (INDO; 20 mg/kg, i.p.)gastric injury histoligically.
Values given as mean+SEM. P < 0.05. As compared to control (CONT) (**),As compared to INDO (*). Figure 4: Histological section of gastric mucosa in a rat from Group 1 (NORMAL). Microscopic grade: 0. Note: Thereis no disruption to the surface of epithelium with neither edema nor leucocytes infiltration of the submucosal layer(H&E stain, 20x magnification).
Grouping: The animals were divided into five groups of Operative procedure13:After seven days of treatment with the appropriate drug for each group, animals were Group One (NORMAL): Animals were treated with normal saline for seven days. They were called the The rat was anesthetized and a midline incision was made and the stomach is removed, then the stomach was opened Group Two (INDO): Animals were treated with normal along the greater curvature, stretched moderately by saline for seven days before indomethacin (CSPC Ouyi pinning on a cork board, and then the gastric mucosa was pharmaceutical.co) administration (20 mg/kg).
examined by naked eye and magnifying lens to: Group Three (FOLIC): Animals were treated with 2 mg/kg of folic acid (Ibn Sina laboratories) for seven *determine the ulcer acuity for each group. Gastric lesions days before indomethacin administration (20 mg/kg).
were scored according to the following system: Group Four (OMEPRAZOLE): Animals were treated 0: no lesion; 1 : <5 lesions, all <2 mm; 2 : <5 lesions, at with omeprazole (ASIA Pharmaceutical Industries) for least one lesion >2 mm; 3 : 5- 10 lesions, all <2 mm; 4 : 5- seven days (20 mg/rat)11 then indomethacin was 10 lesions, at least one lesion >2 mm; 5 : >10 lesions, all administrated (20 mg/kg). This group served as the <2 mm; 6 : >10 lesions, at least one lesion >2 mm14.
Each lesion was considered as an ulcer to calculate the Group Five (FOLIC + OMEPRAZOLE): Animals were ulcer index (UI) as described by Robert 15: treated with omeprazole (10 mg/rat) + folic acid (2 mg/kg) for seven days then indomethacin was a : percentage of animals with ulcers / 10 The route of administration for folic acid was oral. Folic c : average of number of ulcers per group animals acid was dissolved in normal saline to give a suspension.
Then the preventive index (P.I.) of a given drug was Omeprazole pellets were administrated by feeding tube calculated by the equation of Hano et al 16.
without dissolving to reach the duodenum in this form and U.I. of IND group - U.I. of pretreated group protect them from degradation at low pH in the stomach.
P.I. = ------------------------------------------------------X 100 Ulcer Induction12: Animals were singly housed and fasted Histopathological techniques: Each stomach was fixed for 36h in widemesh bottomcages, allowed free access to with 10% formalin solution for 24 hours. After fixation, water except for the last hour before the last dose of the five specimens were taken from each stomach. Then medication. Rats were injected intraperitoneally by sections were stained with conventional EH stain; at least indomethacin (20 mg/kg) 1 h after the last dose of the four sections were prepared from each animal. The stained medication and euthanized under deep ether anesthesia 4 h sections were examined under light microscope and the IJPPR, Vol-5, Issue 2, June- August 2013,113-119 Shaza Al Laham et.al./ Gastroprotective Efficacy of… Figure 5: Histological section of gastric mucosa in a rat Figure 6: Histological section of gastric mucosa in a rat from Group 2 (INDO). Microscopic grade: 3. Note: There from Group 3 (FOLIC). Microscopic grade: 3. Note: There is ulceration with inflammation and neutrophiles inflammation (H&E stain, 20 x magnifications).
infiltration (H&E stain, 40 x magnifications).
Figure 7: Histological section of gastric mucosa in a rat Figure 8: Histological section of gastric mucosa in a rat from Group 4 (OMEPRAZOLE). Microscopic grade: 2.
from Group 5 (FOLIC + OMEPRAZOLE). Microscopic Note: There is surface mucosal abrasion with old grade: 2. Note: There is mucosal edema and Congestion haemorrhages and hemosiderin precipitation (H&E stain, (H&E stain, 20 x magnifications).
The severity and the degree of mucosal damage were All obtained values were expressed as mean + standard assessed according to modified Sedny scale, so that the error of mean (SEM). By using Mann-Whitney test the following grades were obtained: Grade (0): no mucosal proportion of histopathological changes and ulcer acuity in lesions; Grade (1): mucosal edema, congestion, and various groups of animals were compared. While the neutrophils infiltration; Grade (2): surface mucosal significance of differences between means of ulcers erosion. Grade (3): ≤ 2 Gastric ulcers. Grade (4): > 2 number and mucin content were calculated using the student’s t-test. P-values < 0.05 were considered Mucin Content Determination:The gastric mucin was significant. All analysis utilized Prizm4.
determined according to the method described byWinzler17. Briefly, to diluted samples orcinol (1.6%) and sulphuric acid (60%) were added, vortexed, and boiled for Macroscopic study: The macroscopic findings of the 10min. Mixtures were cooled in ice-cold water to stop the opened stomach are shown in Fig. 1. Numerous, tiny, pin- reaction and the absorbance was measured at 425 nm.
point petechial hemorrhages (figure 3) and large erosions(figure 2) were observed in the indomethacin administered STATISTICAL ANALYSIS
group (INDO, Group 2). Folic acid alone (FOLIC, Group IJPPR, Vol-5, Issue 2, June- August 2013,113-119 Shaza Al Laham et.al./ Gastroprotective Efficacy of… Table 3: Effect of folic acid, omeprazole as well as their combination on mucin secretion in indomethacin-induced(INDO; 20 mg/kg, i.p.) gastric injury. Values are means ± SEM; comparisons were carried out using Student t-test, P< 0.05. As compared to control (CONT)(**); INDO (*).
Values given as mean+SEM. P < 0.05. As compared to control (CONT) (**), INDO (*). 3) had almost no effects on the stomach, and the ulcer index, and decrease mucin content (75%) as coadministration of folic acid with omeprazole inhibited compared to control group. Research works on the effect indomethacin-induced ulcer formation (FOLIC + of indomethcin on the gastric mucosa are in agreement with our results. In a previous study, indomethacin Histological evaluation of gastric lesions decrease gastric mucin concentration by 55.7% and Group 1: Most rats in the control group revealed normal increase ulcer index12. Histological observation showed squamous gastric epithelium (figure 4). Only two of them comparatively extensive damage to the gastric mucosa, showed a mild degree of congestion of blood vessels.
and oedema and leucocytes infiltration of the submucosal Group 2: Histological observation of indomethacin layer in most of the animals in indomethacin group. There induced gastric lesions in ulcer control group pre-treated was a statistically significant difference on gastric erosion with normal saline only, showed comparatively extensive score between indomethacin group and control group (p damage to the gastric mucosa, and oedema and leucocytes infiltration of the submucosal layer (Figure 5). Most rats in The molecular basis for the gastrointestinal toxicity of indomethacin group experienced the gastric mucosal NSAIDs is widely believed to their inhibitory activity against cyclooxygenase, which causes them to block the Group 3: Rats that received pretreatment with only folic production of prostaglandins18. The physiological acid didn’t have enough protection of the gastric mucosa function of mucosal prostaglandins is cytoprotective, by as compared to indomethacin group. Six rats in the group inhibiting acid secretion, promoting the secretion of mucus of folic acid (2 mg/kg) had ulcers in the stomach and and strengthening resistance of the mucosal barrier to leucocytes infiltration of the submucosal layer.
back-diffusion of acid from the gastric lumen into the Group 4: Rats that received pretreatment with omeprazole submucosal tissues where it causes damage1. NSAIDs had comparatively better protection of the gastric mucosa significantly decreased the mucosal prosta-glandin E2 as seen by reduction in ulcer area, reduced or absent (PGE2) concentration. Charac¬teristically, the damage submucosal edema and leucocytes infiltration (Figure 2).
was observed along the long axis of the stomach and Group 5: The combination between folic acid and consisted mostly of hemorrhagic lesions, with a few non- omeprazole showed cytoprotective effects. Most rats in hemorrhagic lesions22. Suppression of prostaglandin this group experienced the gastric mucosal erosion with the synthesis is associated with reduction of gastric mucosal score 2. Mean gastric erosion scores were demonstrated in blood flow, disturbance of microcirculation, decrease in Table 2. Pretreatment with Omeprazole and folic acid 1 mucus secretion, lipid peroxidation, and neutrophil hour before administration of indomethacin resulted in a activation, which are involved in the pathogenesis of decrease in mean gastric erosion score. In addition, there gastrointestinal mucosal disorders18. All NSAIDs, except was a statistically significant difference between the aspirin, increased gastric motility at ulcerogenic doses, combination group and indomethacin group (p <0.05).
leading to the development of gastric lesions. Gastric Mucin Content Determination: Indomethacin, as shown in Table 3, leveled off mucin concentration in the gastric especially at specific sites on mucosal folding, leading to juice by 75% while folic acid, as well as omeprazole and their combination elevated it (24%, 79%, 95%), interaction. In addition, indomethacin caused oxyradical respectively, as compared to the indomethacin-treated rats.
production and lipid peroxidation in the gastric mucosa,probably resulting from the ischemic-reperfusion changes DISCUSSION
due to rhythmic hypercontraction of the stomach that is Experimental studies have demonstrated that nonsteroidal mediated by a vagal-cholinergic mechanism22.
anti-inflammatory drugs (NSAIDs) such as indomethacin Stress, nonsteroidal anti-inflammatory drugs, and H. pylori are capable of producing injury to gastrointestinal mucosa cause mucosal damage through a number of mechanisms, in experimental animals and humans18. In the present of which some reactive oxygen species (ROS) such as O2•- study, injecting indomethacin intraperitoneally (20 mg and OH• are now considered to be one of the major /kg) induced raise in ulcer acuity, number of ulcers and causative factors for mucosal lesions through oxidativedamage. Lipid peroxidation, an important parameter for IJPPR, Vol-5, Issue 2, June- August 2013,113-119 Shaza Al Laham et.al./ Gastroprotective Efficacy of… OH•-induced oxidative damage of membrane, is increased In addition to the gastroprotective effect of omeprazole in gastric lesions caused by ethanol, indomethacin, and through inhibiting gastric acid secretion, a recent study water immersion stress. Increased lipid peroxidation, showed antioxidant and antiapoptotic role of omeprazole increased protein oxidation, and decreased glutathione to block gastric ulcer through scavenging of endogenous level are also evident in restraint cold stress-induced hydroxyl radical associated lipid peroxidation and protein gastric lesions as a result of oxidative damage caused by oxidation, indicating that its antioxidant role plays a major the significant generation of OH• . Recent studies also part in preventing oxidative damage. omeprazole prevents indicate that programmed cell death or apoptosis plays a loss of membrane permeability and dysfunction of the significant role in gastric ulceration. Gastric mucosal cellular proteins, leading to survival of the functionally lesions caused by stress, indomethacin, ethanol, and H.
active cells. Moreover, it offers an antiapoptotic effect by pylori are also due to increased cell death by apoptosis 19.
blocking DNA fragmentation during ulceration19.
The gastroprotective activity of folic acid supplementation As a result, the effect of co-administration of folic acid and at the basal requirement supplemental dose of 2 mg/kg diet omeprazole in preventing indomethacin-induced gastric against the lipid peroxidative activity of indomethacin was ulcer can be explained through their antioxidant role. So mentioned10. Folate, an important factor in the de novo they are able to attenuate oxidative stress induced after synthesis of purines, and thymidine, Deoxyribonucleic indomethacin administration. Indomethacin produces an acid (DNA) stability, and apoptosis, is able to attenuate the increase in formation of ROS that catalyze lipid development of gastric ulcer. Increase the superoxide peroxidative and gastric epithelial damage, while folic acid dismutase and mucus concentration observed in the folic and omeprazole play antioxidant and antisecretory roles, acid pre-treated group suggests gastroprotective activity of scavenge ROS and reduce their harmful effects thus as a folates, because they are scavengers which mop up and result prevent gastric ulcer formation.
resist free radicals predisposing the stomach toinflammation. Moreover, this is underscored by a decrease CONCLUSIONS
in the MDA concentration observed in this group of The combination between folic acid and omeprazole have animals. This could mean that folate inhibits the lipid preventive effect against indomethacin-induced gastric peroxidative activity of indomethacin 9.
ulcer in rats. Using folic acid (2 mg/kg) for seven days is Folic acid (2 mg/kg) was used in previous studies for 21 ineffective in preventing indomethacin-induced gastric days. It showed a gastroprotective activity according to its the antioxidative and antisecretory properties 9, 10. Whilein our study it was used in that dose for 7 days and it didn’t REFERENCES
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