STUDIES ON GASTRIC ULCER INDUCED OXIDATIVE STRESS AND ITS AMELIORATION BY CLERODENDRUM PHLOMIDIS LINN IN EXPERIMENTA RATS

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STUDIES ON GASTRIC ULCER INDUCED OXIDATIVE STRESS AND

ITS AMELIORATION BY

CLERODENDRUM PHLOMIDIS

LINN. IN

EXPERIMENTA RATS

S. Gopinathan* and N. Rameela

Pharmaceutical Biotechnology Lab, Department of Biotechnology, Srimad Andavan Arts and Science College (Autonomous), Tiruchirappalli -625 005, Tamil Nadu, India.

ABSTRACT

Gastric ulcer is a major health problem both in terms of morbidity and mortality, affecting about 10% of the global population. Plant drugs are proved their efficacy as preventive as well as curative agents for gastric ulcer disease conditions. Objectives: The present study was aimed to evaluate the amelioration potential of Clerodendrum phlomidis Linn. leaf extract on oxidative stress induced by alcohol induced gastric ulcer in rats. Experimental design: Animal model in vivo study by using albino Wistar rats was carried out. The rats were divided into four groups comprising of six rats in each group and treated as follow. Group I: Healthy control, Group II: Disease control (ulcer was induced by 50% alcohol), Group III: Ulcer induced rats treated with C. phlomidis leaf extract, Group IV: Ulcer induced rats treated with ranitidine. Results obtained: Alcohol induced ulcerated rats showed increased level of ulcer index and total acidity and showed significant reduction in the levels of total protein and total carbohydrate. The activities of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase and the level of non enzymatic antioxidant reduced glutathione were decreased where as the lipid peroxidation and alkaline phasphatase were increased in diseased rats and these results had given enough evidence of increased „oxidative stress‟ during the pathogenesis of gastric ulcer

induced by alcohol. After treatment with C. phlomidis leaf extract activities of antioxidant enzymes and the level of non enzymatic antioxidant were increased and levels of lipid peroxidation and alkaline phosphatase were decreased in the rats. Conclusion: Treatment with C. phlomidis leaf extract significantly inhibited the alcohol induced ulcer congestion,

Volume 4, Issue 8, 2001-2020. Research Article ISSN 2277– 7105

*Correspondence for Author S. Gopinathan Pharmaceutical Biotechnology Lab, Department of Biotechnology, Srimad Andavan Arts and Science College (Autonomous), Tiruchirappalli -625 005, Tamil Nadu, India. Article Received on 06 June 2015,

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hemorrhage and necrosis in diseased stomach which was evidenced from the resumption of various physiological and enzymological parameters. The results of the present study substantiated that the use of C. phlomidis leaf extract is ameliorated the damages caused by oxidative stress and beneficial in curing gastric ulcer. Among the phytochemicals present in the extract, ursolic acid and lupeol were identified as active pharmacological molecules responsible for the amelioration of oxidative stress, gastroprotective and ulcer curative properties.

KEYWORDS: Gastric ulcer, Clerodendrum phlomidis leaf extract, oxidative stress, antiulcer activity, preclinical study.

INTRODUCTION

Peptic ulcer is considered as a major health problem, both in terms of morbidity and mortality and it is very common in the present day life of industrialized and civilized countries. Available statistical reports indicate that10% or more of adult population are affected within their life time and peptic ulcer affects individuals invariably from 20 to 60 years of age with males being predominantly affected.[1,2] Clinical observation refers that the painful sores or ulcers in the lining of the stomach is called as gastric ulcer or in the first part of the small intestine is called as duodenal ulcer and both are collectively called as peptic ulcer.

Gastric ulcer develops when the delicate balance between defensive (gastroprotective) factors and aggressive factors is lost. Secretion of acid and pepsin are considered as the aggressive factors and the mucin-bicarbonate secretion, mucus cell proliferation and healthy phospholipid layer, secretion of prostaglandins and gastro-duodenal epidermal healing factors are identified as the defensive factors. Psychological stress, alcohol consumption, H. pylori

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cellular functions, leading to various pathological conditions including cardiovascular dysfunction, neurodegenerative diseases, gastro-duodenal pathogenesis, metabolic dysfunction of almost all the vital organs, tumor and cancer and premature aging.[5] The ROS mediated oxidative stress results in oxidation of membrane lipoproteins, glycoproteins and oxidation of DNA and subsequently cell death results. ROS from damaged cells also attack the adjacent cells, resulting in injury and necrosis of tissues.

Peptic ulcer impairs the quality of life and cure of peptic ulcer is one of the most important medical challenges. Research advances during the last few years have offered new insight in the therapy and prevention of gastro-duodenal ulcer diseases by strengthening the mucosal defense system rather than attenuating the aggressive acid- pepsin factors. The drug therapy for gastric ulcers is aimed at preventing and opposing the effect of ulcer causing factors or provoking the effect of ulcer preventing factors like mucosal defenses and blood flow of the gastric mucosa.[6,7] A number of synthetic drugs are available for the treatment of gastric ulcer, but clinical evidences have shown the incidence of relapses, side effects and drug interactions. Phytochemicals available in the herbs are safe and proved their efficacy as preventive as well as curative agents. They have been acquired a greater appreciation because of their long time usage and rich heritage in traditional systems of medicine.

Clerodendrum phlomidis Linn. (synonyms: Clerodendrum multiflorum (Burm. f) O. Kuntz.,

and Volkameria multiflorum Burm. F.), belongs to the Botanical family of Lamiaceae. It is a

large bush or a small tree, growing in drier parts of India. Common names are: Tamil- Tazhuthalai; Sanskrit- Agnimantha; Hindi- Arni. Different parts of the plant are well regarded in Indian traditional systems of medicine and useful in the preparation of many famous Ayurvedic formulations.[8,9] Earlier we reported the antidiabetic activity [10,11] and antiobesity activity.[12] of C. phlomidis leaf extract. Further, the ameliorative effects of C.

phlomidis leaf extract against oxidative stress induced metabolic syndrome in obese rates was

also reported.[13] In this article, we report the gastroprotective, antiulcer and antioxidant potential of C. phlomidis leaf extract.

MATERIALS AND METHODES Preparation of plant extract

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professors of Botany department. The voucher specimen of the plant (No.SAAC-BT- 062) is deposited in the department for future reference.

Preparation of plant extract

1000 gm of C. phlomidis dried leaf was extracted with 80% aqueous ethanol. To one part of the plant material, six parts of aqueous ethanol was added in round bottom flask fitted with condenser and extracted at reflex temperature for 3 h. The aqueous ethanol extract was filtered and the filtrate was evaporated to dryness in a rotary- evaporator at low temperature. Paste from of the extract obtained was further processed and obtained as dry powder.

Quantification of phytochemicals

The leaf extract of C. phlomidis was subjected to analysis for important phytochemicals such as total alkaloids, total flavonoids, total phenolic compounds, total saponins, total tannins, ursolic acid and lupeol according to the standard methods.[13,14,15,16]

Experimental animals

Healthy Wistar strain of albino rats of both sexes, about three months old and weighing 150-200 g were obtained from Tamil Nadu Veterinary and Animal Sciences University, Chennai. The animals were allowed to acclimatize under laboratory conditions for a period of 5 days prior to the experiment. Animals were fed with standard rat chow pellets and water ad libitum. All the studies were conducted according to the Institutional Ethical Committee‟s Approval (No: 790/03/ac/CPCSEA).

Experimental design

Group I: Healthy control rats (without any treatment).

Group II: Disease control rats- gastric ulcer was induced by alcohol (10 ml /kg-bw of 50% ethanol- single dose) (Pre- treatment)

Group III: Alcohol induced ulcerated rats treated with C. phlomidis leaf extract (400 mg/kg-bw) for 21 days

Group IV: Drug control rats – Alcohol induced ulcerated rats treated with standard drug ranitidine (50 mg/kg-bw) for 21 days.

Induction of ulcer in rats

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sacrificed and checked for ulcer induction. From the same day, the rats in Group III and IV were treated with respective drugs. Both herbal drug and ranitidine were dissolved in distilled

water and given orally to the rats of respective groups. On 22ndday (24 h after the treatment) the rats were sacrificed. The rats were anaesthetized by using ether. The abdomen was opened by an incision of the sternum and the stomach was exposed. Passed a thread around the pyloric sphincter and applied a tight knot closed the abdomen wall and surgically cut and removed the stomach. The gastric fluid was collected in a graduated centrifuge tube and samples of stomach tissues were collected and stored for biochemical and enzymological analyses.

Determination of ulcer index in stomach[17,18]

The stomach was cut opened along the greater curvature and washed it slowly under running tap water, placed it on the glass slide and observed under microscope (10x) for ulcer mean score. The ulcer index was calculated by using the following formula.

Ulcer index = Total mucosal area / Total ulcerated area.

% of ulceration = T/C X 100 (Where, C = Ulcer index in the diseases Control Group; T = Ulcer index in the treated group.

Determination of total and free acidity in gastric fluid

The gastric fluid was centrifuged at 1000 rpm for 10 min. 1 ml of supernatant (gastric juice) was diluted to 10 ml with distilled water. The solution was titrated against 0.01N NaOH using Topfer‟s reagent as indicator to the end point when the solution turned to orange colour. The volume of NaOH needed was taken as corresponding to the free acidity.

Titration was further continued by adding two drops of 1% solution of phenolphthalein till the solution developed the pink colour. The volume of NaOH required was noted and was taken as corresponding to the total acidity.[19]

Acidity = Volume of NaOH x Normality x 100/0.1 (Acidity was expressed as mEq/L)

Estimation of total protein

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using a suitable blank. The standard curve was prepared with bovine albumin. The protein content was expressed as µg/ml of gastric juice.

Estimation of total carbohydrate

The dissolved total carbohydrate in the stomach tissue was determined by the method described by Venkataranganna et al..[21] Briefly, tissue homogenate was mixed with 3N sulphuric acid and sodium tungstate solution. The content was centrifuged and to the supernatant anthrone reagent was added. The OD of the reaction mixture was read at 540 nm. The total carbohydrate content was calculated from standard glucose solution and the results were expressed in terms of µg/ml of gastric fluid.

Determination of serum alkaline phosphatase (ALP)

ALP was assayed by the method of King.[22] In brief, the gastric fluid was mixed with 0.1M carbonate buffer (pH 10), 0.1M disodium phenyl phosphate and 0.1M magnesium chloride. The reaction mixture was incubated at 37°C for 15 min and the reaction was arrested by the addition of Folin‟s phenol reagent. To this 15% sodium carbonate was added and the colour

developed was read after 10 min at 640 nm. The enzyme activity was expressed as IU/ml.

Determination of lipid peroxidation (MDA content)

Lipid peroxidation was estimated by estimating the formation of malondialdehyde (MDA). Estimation of MDA was carried out by using the thiobarbutric acid reactive substance (TBARS) test. Assay of TBARS measures malondialdehyde (MDA) present in the sample, as well as malondialdehyde generated from lipid hydroperoxides by the hydrolytic conditions of the reaction was done by the method proposed by Ohkawa et al.[23] Briefly, to the tissue homogenate, 0.85N H2SO4 and 10% phosphotungstic acid were added and stirred well. The

content was centrifuged and to the supernatant, TBA (0.8%) was added and heated at 90ºC for 20 min. After cooling down the sediment developed in the TBA-MDA complex was extracted with butanol and absorbance was read at 532nm. The enzyme activity was expressed as nanomoles/min/mg tissue protein.

Determination of reduced glutathione (GSH)

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immediately at 412nm against blank containing TCA instead of sample. A series of standard treated in a similar way were also run to determine glutathione content. The amount of reduced glutathione was expressed as moles of GSH oxidized/min/mg protein.[24]

Determination of superoxide dismutase (SOD)

SOD activity was assayed by inhibition of autocatalysed adrenochrome formation in the tissue homogenate. Briefly, ethanol was added to tissue homogenate and centrifuged. To the supernatant 0.6nM EDTA solution and 0.1 M phosphate buffer (pH 10.2) were added and mixed. The reaction was initiated by the addition of fresh epinephrine (1.8nM) and the absorbance was read at 480 nm. The reaction mixture without tissue homogenate was used as blank. The enzyme activity was expressed as U/ml.[25]

Determination of catalase (CAT)

CAT activity was assayed by the decomposition of H2O2 in the tissue homogenate. Briefly,

tissue homogenate in phosphate buffer was added with H2O2 to start the enzyme reaction.

Then potassium dichromate was added and incubated in boiling water bath for 10 min. After green colour development, the absorbance was read at 240nm at 60 sec intervals for 3 min. Activity of catalase was expressed as µ moles of H2O2 utilized/min/mg protein.[26]

Determination of glutathione peroxidase (GPx)

GPx activity was assayed by using the spectrophotometer method developed by Rotruck et al.[27] The reaction mixture (consisted of EDTA, reduced glutathione, sodium azide, H2O2,

tissue homogenate in phosphate buffer) was incubated at 37ºC. The reaction was arrested by adding TCA and centrifuged. To the supernatant, disodium hydrogen phosphate and DNTB were added and the colour developed was read at 420 nm immediately. The level of glutathione peroxidase was expressed as µmoles of glutathione oxidized/min/mg protein.

STATISTICAL ANALYSIS

The data obtained were subjected to statistical analysis and expressed as mean ± SD. The data were statically analyzed by one way analysis of various (ANOVA) and to compare the means of the studied groups with post hoc Duncan multiple range tests at 5% and 1% for those results where significant difference was indicated.

RESULTS AND DISCUSSION

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Table 1: Determination of phytochemicals in C. phlomidis leaf extract

Pytoconstituents* Quantity determined

Total poly phenolic compounds1 7.5±0.61

Total flavonoids2 6.2±1.16

Total alkaloids3 6.8±1.25

Total tannins (%) 2.5±0.35

Total saponins (%) 1.1±0.35

Ursolic acid (%) 2.21±0.05

Lupeol (%) 3.15±0.06

*Values are means of triplicate determination.

1. mg gallic acid equivalents/ 100 mg dry wt of extract 2. g rutin equivalents/ 100 g dry wt of extract

3. g colchicine equivalents/ 100 g dry wt of extract

The quantitative phytochemical analysis of C. phlomidis leaf extract revealed that it has considerable quantities of therapeutically important phytochemicals (Table-1). The use of phytoconstituents to treat major ailments has proved to be clinically effective and less relatively toxic than the existing synthetic drugs and serving as a tool in the prevention of peptic ulcer.[28]

Rats in group I were maintained as healthy control and the rats in the group II, III and IV were administered with 50% ethanol to induce the ulcer. After 24 hours of ulcer induction, an animal in the group II was sacrificed and checked its stomach for ulceration. Ulcer was noticed and the observation confirmed that ethanol treated rats were ulcerative. The leaf extract of C. phlomidis (plant drug) and ranitidine (reference drug) were administered in group III and group IV animals respectively for 21 days. On 22nd day (24 h after the treatment) the animals were sacrificed and the samples of gastric fluid and stomach tissues were collected for analyses.

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pathways, primarily in the liver. Studies have shown that ethanol consumption may result in increased generation of free radicals and formation of lipid peroxides. Ethanol is one of the pro-oxidants that induce intense damage in gastric mucosa. Gastric lesion formation may be due to stasis in gastric blood flow which contributes to the development of the hemorrhage and necrotic aspects of tissue injury.[29, 30]

In the present study, the oral administration of alcohol to the rats caused severe gastric mucosal damage by disruption its barrier and provokes rapid and strong stasis in the gastric mucosal blood flow in to mucosal capillaries, which contributes to the development of hemorrhage and necrosis resulting in tissue injury and hence the levels of aggressive factors such as ulcer index and total acidity were increased. The treatment with C. phlomidis leaf extract significantly decreased these factors, which was exhibited in the significant cytoprotective effect and healed the ulcer wounds in the stomach (Plate-1). Flavonoids are reported to possess the wound healing property by preventing the formation of lesions by various necrotic agents. Not only flavonoids, but also other phenolic compounds are known for their antioxidant activities, and the phenolic compounds are linked to the antiulcer property. In the present study, C. phlomidis leaf extract contains considerable quantity of phenolic compounds such as total flavonoids (6.2±1.16%), total polyphenolic compounds (7.5±0.61%), total tannins (2.5±0.35%), ursolic acid (2.21±0.05%) and lupeol (3.15±0.06%). Further, content of total saponins in the leaf extract was (1.1±0.35%) and saponins have been reported to promote ulcer healing by forming protective mucus barrier on the gastric mucosa. (Table-1).

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Group III Group IV

Plate 1: Photograph showing the ulceration in the stomach mucosa of experimental rats

Fig.1-a. Effect of Clerodendrum phlomidis on Ulcer Index in ethanol induced ulcerogenic rats.

Fig.1-b. Effect of Clerodendrum phlomidis on total acidity in ethanol induced ulcerogenic rats.

Fig.1-c. Effect of Clerodendrum phlomidis on total protein in ethanol induced ulcerogenic rats.

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Fig.2-a. Effect of Clerodendrum

phlomidis on superoxide dismutase in ethanol induced ulcerogenic rats.

Fig.2-b. Effect of Clerodendrum phlomidis on catalase in ethanol induced ulcerogenic rats.

Fig.2-c. Effect of Clerodendrum phlomidis on glutathione peroxidase in ethanol induced ulcerogenic rats.

Fig.2-d. Effect of Clerodendrum phlomidis on reduced glutathione in ethanol induced ulcerogenic rats.

Fig.2-e. Effect of Clerodendrum phlomidis on lipid peroxidation in ethanol induced ulcerogenic rats.

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In the present study, the ulcer index was significantly (p> 0.001) increased (3.23±2.1) in diseased rats (Group II) when compared to healthy rats (Group I). Administration of drugs decreased the ulcer index. Both plant drug and standard drug showed significant inhibition in ulcer index, and they were 0.31±0.22 and 0.66±0.13 in the rats treated with plant extract and ranitidine respectively [Fig.1-a].

Total acidity was increased in ethanol induced ulcerated rats. The increased level of total acidity was recorded higher in diseased rats (86.35±3.1 mEq/L) than healthy rats (38.25±4.2 mEq/L). After the treatment with C. phlomidis leaf extract and ranitidine the level of acidity was decreased in the diseased rats. The levels of total acidity in group III rats and group IV rats were 43.98±4.2 mEq/L and 38.98±4.6 mEq/L, respectively [Fig.1-b]. This may be due to the antacid effect and/or cytoprotective effect of C. phlomidis leaf extract in ethanol induced gastric ulcerogenesis.

The content of total protein in the stomach tissue was 9.65±1.6 µg/mg in healthy rats and the same was and was significantly (p>0.001) decreased in diseased rats (6.13±1.2 µg/mg). After the treatment with both plant drug and standard drug the total protein content was increased. The increment in the content of total protein was 17.56±0.44 µg/mg the in the rats administered with plant extract and 14.6±0.71 µg/mg in the rats treated with ranitidine. Similar trend was noticed in the content of total carbohydrates also. The total carbohydrate content in stomach tissue of the healthy rats was 165.00±7.1 µg/mg and the content was decreased to 135.66±6.9 µg/mg in diseased rats. The administration of drugs significantly (p>0.01) increased the content of total carbohydrate. The increment was more pronounced in the rats treated with plant drug (186.54±8.5 µg/mg) than the rats treated with ranitidine (177.43±6.1 µg/mg) [Fig. 1-c and 1-d].

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terpenoids, and saponins have been reported as gastroprotective agents in several anti-ulcer studies.[31] The cytoprotective and anti-ulcerogenic activities of flavonoids, tannins and triterpenes has been extensively studied.[32,33] It is suggested that these compounds may have the ability to stimulate the secretion of mucus, bicarbonate and prostaglandin, and also counteract with the deteriorating effects of ROS in gastrointestinal lumen.[34]

One factor that has been suggested as playing a central role in many bath ways of alcohol-induced damage is the excessive generation of free radicals, which result in a state called oxidative stress.[35] O2 is converted to reactive oxygen species (ROS) such as O2-, H2O2 and.

HO by univalent reduction of O2. ROS can damage or cause complete degradation (i.e., peroxidation) of essential molecules in the cells, including lipids, proteins and DNA. Both acute and chronic alcohol exposure can increase the production of ROS and enhanced the peroxidation.

Lipids that contain phosphate groups are known as phospholipids and they are essential components of the membranes that surround the cells as well as other cellular structures, such as the nucleus and mitochondria. Consequently, damage to the phospholipids will compromise the viability of the cells. The complete degradation (i.e. peroxidation) of lipids is a hall mark of oxidative stress induced by ROS. Alcohol ingestion caused damages to gastric mucosa and results in the enhanced activity of marker enzyme, alkaline phosphatase (ALP) in the blood.[36] In the present study, ALP activity was significantly (p≥0.01) increased in diseased rats (128.15±5.4 IU/L) when compared to healthy control rats (86.53±4.2 IU/L). Oral administration of plant extract and standard drug decreased the level of ALP activity and brought back to normalcy (87.96±9.04 IU/L, 81.55±8.03 IU/L in plant drug and ranitidine treated animals, respectively), which indicated that the gastric damage was cured in plant drug treated animals [Fig. 2-a]. This result proved that the plant drug acted as an antiulcer agent.

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Lipid peroxidation (LPO), a process induced by free radicals, lead to oxidative deterioration of polyunsaturated lipids. Under normal physiological conditions, only low level of LPO generate in the cells. The administration of ethanol induced the gastric ulcer in the rats by producing excessive generation of free radicals such as hydroxyl radical (.OH), superoxide radical (O2.-), peroxide radical (O2.=), and hydrogen peroxide (H2O2). All these radicals have a

great potential to react rapidly with lipids and in turn induce higher level of LPO.[37] Due to this reason, in the present study also increased level of LPO was observed in ulcerated rats (78.54±2.9 nmoles/mg) than the healthy rats (34.25±2.7 nmoles/mg). After the treatment with plant drug and ranitidine the level of LPO was decreased. The reduced levels of LPO were 33.83±2.2 nmoles/mg and 26.68±5.5 nmoles/mg in plant drug and ranitidine treated rats respectively [Fig.2-b]. LPO involves in the formation and propagation of lipid radicals and the uptake of oxygen and rearrangement of double bonds in unsaturated lipids that eventually results in destruction of membrane rich in unsaturated fatty acids. Therefore, it may not be surprising that membrane lipids are susceptible to peroxidative attack.[38] The results of present study revealed that the C. phlomidis leaf extract significantly decreased the lipid peroxidation in the stomach tissues of ulcerated rats, which suggests its efficacy as gastroprotective agent. Polyphenolic compounds present in the C. phlomidis leaf extract have been implicated in the stimulation of the PGE2 formation based on their action as

co-substrates for the peroxidase reaction.[39]

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To counter the excess ROS production, antioxidant enzymatic mechanisms have been evolved to protect the cells from ROS damage. Enzymes involved in the elimination of ROS include superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). SOD is present in cytosol and mitochondria of mammalian cells and provides the first line of defense against free radical damage. SOD catalyzes and helps in the rapid removal of superoxide radicals. Dismutation reaction catalyses the superoxide radicals and generates H2O2 molecules. Both CAT and GPx are helpful in further elimination of H2O2 . CAT is an

iron-containing enzyme which detoxifies H2O2 and other ROS molecules. One way that CAT

eliminates H2O2 is by catalyzing a reaction between two H2O2 molecules resulting in the

formation of H2Oand O2. In addition, CAT can promote the interaction of H2O2 with other

compounds that can serve as hydrogen donors so that the H2O2 can be converted to H2O

molecules.

In the present study, SOD activity was decreased in ulcerated rats (25.95±2.2 u/mg protein) when compared to healthy rats (56.43±4.3 u/mg protein). Rats treated with plant drug and ranitidine showed increased levels of SOD activity. The enhanced level of SOD activity was 62.35±5.5 u/mg protein in plant drug treated rats and the same was 55.25±7.9 u/mg protein in ranitidine treated rats [Fig.2- d]. The significant decreased activity of SOD in the ulcer induced animals may be due to an excessive formation of superoxide radicals. SOD owes its antioxidant properties to scavenge O2. radicals and plays an important role in protecting the

gastrointestinal mucosa.[41,42]

The main function of CAT includes catalyzing and/or decomposition of O2.- and H2O2. CAT

has highest turnover rates of all enzymes; one molecule of CAT can convert millions of molecules of H2O2 to O2 and H2O per second by acting at different sites in the metabolic

pathway of free radicals.[43] In the present study, the CAT activity was decreased in diseased rats (3.65±0.62 u/mg protein) when compared to healthy rats (6.86±0.92 u/mg protein). After the administration of plant extract and ranitidine the activity was increased. The increased CAT activity was found higher in the rats treated with plant drug (7.74±0.72 u/mg protein), than the rats treated with ranitidine (6.31±1.52 u/mg protein) [Fig.2-e]. The phytoconstituents especially flavonoids, tannins, polyphenolic compounds available in C. phlomidis leaf extract (Table 1) may be responsible for the enhanced CAT activity.

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peroxidation in mammalian cells by GPx is the removal of hydrogen peroxide generated by SOD in cytosol and mitochondria.[44] In the present study, GPx activity was decreased in the diseased rats (15.08±1.22 u/mg protein) when compared to healthy animals (32.06±1.92 u/mg protein). GPx inhibition results in H2O2 accumulation and subsequent increase in lipid

peroxidation and could be caused the damage to the gastric tissues. After the administration of both plant drug and ranitidine the level of enzyme activity was increased. The significantly increase in the activity of GPx in the rats treated with plant drug was 36.54±1.62 u/mg protein, whereas the same was 30.53±1.52 u/mg protein in the rats treated with ranitidine [Fig.2-f]. GPx activity is important for the elimination of hydrogen peroxide and lipid hydroperoxides in the gastric mucosal cells.[45] Thus, inhibition of this enzyme activity in the gastric mucosa by ethanol may result in the accumulation of hydrogen peroxide with subsequent oxidation of lipids. The reversal of GPx activity in ethanol treated animals after the treatment with C. phlomidis extract may therefore be due to the replenishment of glutathione content.

CONCLUSION

Oxidative stress plays an important role in the pathogenesis of various diseases including gastric ulcer and antioxidants are being reported to play a significant role in the protection of gastric mucosa against various necrotic agents including alcohol. ROS are involved in the pathogenesis of ethanol induced gastric mucosal injury in vivo. [46] Antioxidant enzymes such as SOD, CAT and GPx are known to be the first line cellular defense factors against oxidative damage by disposing O2. and H2O2, before their interaction to form more harmful

hydroxyl (OH·) radicals.[47]

In the present study, we observed the physiological damages due to oxidative stress during the gastric ulcer pathogenesis induced by alcohol in experimental rats, which was manifested by the decreased activities of antioxidant enzymes (SOD, CAT, GPx) with subsequent increase in the LPO and ALP activities. Treatment with C. phlomidis

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phlomidis leaf extract including ursolic acid and lupeol exert their effort by scavenging the free radicals and providing mucosal defense and gastroprotection.

ACKNOWLEDGEMENT

The authors are thankful to Dr. J. Radhika, Principal and Mr. N. Kasthurirengan, Secretary and Correspondence of the college for providing facilities to carry out this study.

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References

  1. s malondialdehyde