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Orthosiphon stamineus Leaf Extract Protects AgainstEthanol-Induced Gastropathy in Rats

Mun Fei Yam,1,2 Lee Fung Ang,2 Ibrahim Muhammad Salman,2 Omar Ziad Ameer,2

Vuanghao Lim,2 Lai Man Ong,2 Mariam Ahmad,2 Mohd. Zaini Asmawi,2 and Rusliza Basir1

1Department of Human Anatomy, Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, Serdang, Selangor;and 2School of Pharmaceutical Sciences, Universiti Sains Malaysia, Pulau Pinang, Malaysia

ABSTRACT Orthosiphon stamineus Benth., which is used as a gastroprotective herbal remedy in Malaysia, was assessed

for its anti-ulcerogenic activity against ethanol-induced ulcers in rats. Fifty percent methanol was used to extract the oven-

dried O. stamineus leaves. The extract was then lyophilized with a rotary evaporator and freeze-dried. Oral administration of

O. stamineus methanolic extract (OSME) (125, 250, 500, and 1,000 mg=kg) was found to significantly decrease the ulcer

index (P< .01, P< .001, P< .001, and P< .001, respectively). Histological study of a section of the rat stomach also showed

a marked improvement in the gastric mucosal damage in groups receiving OSME. In order to further investigate the

gastroprotective mechanism of OSME, mucus secretion and lipid peroxidation level were estimated in vitro and ex vivo.

OSME exhibited dose-dependent stimulation of mucus secretion (r¼ 0.718, P< .001) and inhibition of lipid peroxidation in

rat gastric mucosal homogenates (both in vitro [r¼ 0.819, P< .05] and ex vivo [r¼ 0.981, P< .05]). It was concluded that the

gastroprotective mechanism of OSME was partly due to its ability to inhibit lipid peroxidation and stimulate gastric mucus

secretion.

KEY WORDS: � gastroprotection � high-performance liquid chromatography profile � lipid peroxidation � Orthosiphon

stamineus

INTRODUCTION

Gastroduodenal ulcers are common diseases. It isestimated about 10% of the world’s population will

have an ulcer at some point during their lives.1 The etiologyof gastroduodenal ulcers is influenced by various factors,such as acid-pepsin secretion, parietal cells, mucosal bar-rier, mucus secretion, blood flow, cellular regeneration, andendogenous protective agents (prostaglandins and epidermalgrowth factors).1 Many drugs are used for the treatment ofgastric ulcers, including antacids, proton pump inhibitors,and antihistamines, but most of these drugs produce severaladverse reactions.1 Owing to safety concerns about syntheticanti-ulcer drugs, many people prefer to take the effective andless toxic natural anti-ulcer agents from edible materialssuch as fruits, spices, herbs, and vegetables.2 Therefore, thedevelopment and utilization of more effective anti-ulcerdrugs of natural origins are of great interest.

Orthosiphon stamineus Benth (Lamiaceae) is a shrubindigenous to various parts of Malaysia and Indonesia. InMalaysia, it grows along forest edges and roadsides andon wasteland and is easily propagated through noddedstem cuttings. O. stamineus is used as a remedy for ca-tarrh of the bladder and as a medicine for various disor-ders such as nephritis, nephrolithiasis, hydronephrosis,vesical calculi, arteriosclerosis, rheumatism, inflammation,gout, and diabetes.3,4 Previous phytochemical investiga-tion of the plant revealed the presence of terpenoid andpolyphenolic compounds, which contributed to the thera-peutic effects of O. stamineus.5 Lipophilic flavonoidsisolated from O. stamineus showed radical scavengingactivity towards the diphenylpicrylhydrazyl radical andinhibition of 15-lipoxygenase from soybeans.6,7 Researchalso indicates that the flavones (sinensetin and 30-hydroxy-5,6,7,40-tetramethoxyflavone) isolated from members ofthe Orthosiphon genus exhibited diuretic activity in rats.8

The diterpenes isolated from members of the Orthosiphongenus have been shown to exhibit a suppressive effect oncontractile responses in rat thoracic aorta and nitric oxideinhibition and inhibitory activity against the inflammationinduced by a tumor promoter (12-O-tetradecanoylphorbol13-acetate) on mouse ears.9–13 However, a review of the

Manuscript received 2 January 2008. Revision accepted 30 June 2009.

Address correspondence to: Mr. Mun Fei Yam, Unit Pharmacology, Department ofHuman Anatomy, Faculty of Medicine & Health Sciences, Universiti Putra Malaysia,43400 Serdang, Selangor, Malaysia, E-mail: yammunfei@yahoo.com

JOURNAL OF MEDICINAL FOODJ Med Food 12 (5) 2009, 1089–1097# Mary Ann Liebert, Inc. and Korean Society of Food Science and NutritionDOI: 10.1089=jmf.2008.0005

1089

current literature revealed that no documented reports areavailable so far on the evaluation of O. stamineus as a pos-sible anti-ulcer agent. Recent studies have shown that naturalantioxidants are able to reduce or prevent gastric ulcer.14

Because O. stamineus was proven to be a good antioxidantagent,15 experiments were therefore carried out to test theanti-ulcer property of O. stamineus leaf extract. The contentof the potential antioxidant and anti-ulcer components,namely, 30-hydroxy-5,6,7,40-tetramethoxyflavone, eupatorin,rosmarinic acid, and sinensetin (Fig. 1), were also determinedusing high-performance liquid chromatography (HPLC)analysis.

MATERIALS AND METHODS

Chemicals and reagents

Acetic acid, n-butanol, formaldehyde, and diethyl etherwere purchased from Merck (Darmstadt, Germany). Tris-HCl buffer, magnesium chloride, phosphate-buffered sa-line (PBS), Alcian blue, sucrose, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), 1,1,3-3-tetra-methoxypropane [malonaldehyde bis(dimethyl acetal)], andHarris hematoxylin were purchased from Sigma (St. Louis,MO, USA). Methanol and eosin were purchased from Riedel-de Haen (Seelze, Germany). Absolute ethanol and chloro-form were purchased from R&M Marketing (Essex, UK).Ferrous chloride was purchased from Fluka (St. Gallen,Switzerland). Xylene was purchased from Fisher Scientific(Loughborough, UK). Thiobarbituric acid (TBA) was pur-chased from Applichem (Darmstadt). Omeprazole was pur-chased from Hovid (Perak, Malaysia). The solvents used forHPLC, tetrahydrofuran and methanol (HPLC grade), wereobtained from Merck. The standard compounds of sinensetin,eupatorin, 30-hydroxy-5,6,7,40-tetramethoxyflavone, androsmarinic acid were purchased from Indofine Chemical Co.(Hillsborough, NJ, USA).

Animals

Male Sprague-Dawley rats (weighing 180–250 g) ob-tained from the animal house, School of PharmaceuticalSciences, Universiti Sains Malaysia, Palau Pinang, Malaysiawere used in this study. The animals were maintained at 28–308C, and food (normal laboratory chow, Gold Coin, Se-langor, Malaysia) and tap water were provided ad libitum.The animals were acclimatized to laboratory conditions for7 days before commencement of the experiments. Forty-eight hours before the experiments, the animals were de-prived of food. Free access to water was allowed until 1 hourbefore the beginning of experiments. All procedures in thisstudy were performed according to the animal ethicsguidelines of Universiti Sains Malaysia.

Plant material

O. stamineus plants were grown from cuttings using stan-dard agronomic practices at Kepala Batas, Penang, Malay-sia. The leaves of the plant were collected after they hadflowered. A voucher specimen (number 10106) was sub-mitted to the School of Biological Sciences Herbarium,Universiti Sains Malaysia.

Oven-dried (458C) and powdered leaves of O. stamineuswere extracted with methanol:water (50:50 vol=vol) (for 8hours). The extract was lyophilized, and the resultant yieldwas found to be 6% (wt=wt). The dried O. stamineus me-thanolic extract (OSME) was kept in tightly stoppered bot-tles and stored in a dessicator until used.

HPLC analysis

HPLC analysis was performed using an Agilent Tech-nologies (Palo Alto, CA, USA) series 1100 system=seriesequipped with an automatic injector, a column oven, and adiode array ultraviolet detector. A LiChrosorb RP-18 col-umn (250 mm�4.6 i.d. mm; particle size, 5 mm) (Merck)was used. The temperature was maintained at 258C, with aninjection volume of 20mL and flow rate of 1 mL=minute. Allmarkers were separated using the reverse-phase LiChrosorbC-18 column with methanol:water:tetrahydrofuran (45:50:5by volume) as the mobile phase. The peaks were detected at340 nm and identified using standard substances, namely,rosmarinic acid, sinensetin, eupatorin, and 30-hydroxy-5,6,7,40-tetramethoxyflavone.16

Experimental groups

The animals were randomly divided into four majorgroups (n¼ 138). Group I was used to study the effect ofOSME on absolute ethanol-induced gastric membrane le-sions (ulcer index and histopathology studies), Group II wasallocated for in vivo determination of gastric wall mucuscontent, Group III was used in the ex vivo assessment oflipid peroxidation inhibition activity, and Group IV wasretained for the determination of the effect of OSME onFeCl2-induced lipid peroxidation in rat gastrointestinalhomogenate in vitro. Groups I, II, and III were further

FIG. 1. Chemical structure of the markers.

1090 YAM ET AL.

subdivided into seven (Groups II and III) or eight (Group I)subgroups of six animals each, namely, normal subgroup(Groups I, II and III), control subgroup (Groups I, II,and III), standard drug subgroup (received 30 mg=kg om-perazole p.o.) (Groups I and II), and OSME subgroups(received 125, 250, 500, or 1,000 mg=kg OSME p.o., re-spectively) (Groups I, II, and III). Group IV consisted of sixhealthy rats.

Absolute ethanol-induced gastric membrane lesions

Single doses of OSME (125, 250, 500, or 1,000 mg=kg, indistilled water), omeprazole (30 mg=kg, in distilled water),and distilled water (10 mL=kg) were administered orally tothe rats, 60 minutes prior to induction of gastric ulcers by1 mL of absolute ethanol (perorally).17 Each of the specifieddoses was calculated based on the body weight of the ani-mal, weighed individually using a triple beam balance(Navigator, Ohaus Scale Corp., Pine Brook, NJ, USA), andsubsequently dissolved in a specific premeasured volume ofdistilled water based on the animal’s body weight. The an-imals were sacrificed at the end of experiment using over-doses of the anesthetic, diethyl ether. The stomachs wererapidly removed, and each stomach was opened along thegreater curvature, gently washed with saline, and pinnedupon a cork plate with the mucosal surface turned upward;the lesions formed were observed and scored.17 The scoresof each single lesion was then summed up for determinationof the ulcer index according to the method described bySchiantarelli et al.18 using the following scale: 0¼ normalmucosa; 1¼ hyperemic mucosa up to three small patches;2¼ four to 10 small patches; 3¼more than 10 small or up tothree medium-sized patches; 4¼ four to six medium-sizedpatches; 5¼more than six medium-sized or up to threelarge patches; 6¼ four to six large patches; 7¼ seven to10 large patches; 8¼more than 10 large patches; and 10¼large patches of extensive necrotic zones. A ‘‘small patch’’is defined as an area of lesion up to 2 mm across (maximumdiameter), a ‘‘medium-sized patch’’ as between 2 and 4 mmacross, and a ‘‘large patch’’ as more than 4 mm across.

Determination of gastric wall mucus content

Gastric wall mucus was determined using the Alcian bluemethod.19 In brief, single doses of OSME (125, 250, 500, or1,000 mg=kg, in distilled water), omeprazole (30 mg=kg, indistilled water), and distilled water (10 mL=kg) were ad-ministered orally to the rats, 60 minutes before the inductionof gastric ulcers by orally administered absolute ethanol.17

Similar to the previous experiment, 1 hour following oraldosing the animals were sacrificed, and the stomach of eachanimal was excised, opened along the lesser curvature,weighed, and immersed in 0.1% (wt=vol) Alcian blue so-lution for 2 hours. The excess dye was then removed bytwo successive rinses in 0.25 M sucrose solution. The dye–gastric mucus complex was extracted with 10 mL of 0.5 Mmagnesium chloride for 2 hours. The blue extract was thenshaken vigorously with an equal volume of diethyl ether,

and the optical density of the aqueous phase was measuredat 605 nm using a spectrophotometer (model U-2000, Hi-tachi, Tokyo, Japan).20

Assessment of lipid peroxidation inhibitionactivity (ex vivo)

The effect of OSME on ethanol induced-lipid peroxida-tion was studied in rats using the method of Yuda et al.21 andLiu et al.22 with minor modification. In this method, singledoses of OSME (125, 250, 500, or 1,000 mg=kg, in distilledwater) and distilled water (control group) (10 mL=kg) wereadministered orally to the rats, 60 minutes prior to inductionof gastric ulcers. Animals that were administered distilledwater without induction of gastric ulcer served as the normalgroup. The rats were sacrificed using the anesthetic diethylether. The stomachs were dissected and washed with salinebefore being sliced and homogenized (3 minutes) with25 mM Tris-HCl buffer (pH 7.2) (10% wt=vol). Stomachhomogenate (100mL) was dispensed into test tubes and in-cubated in an intermittent shaking water bath (model-903,Protech, Selangor) for 1 hour at 378C. The homogenate wasadded to 1.5 mL of 25 mM Tris-HCl buffer, 1.5 mL of 1.0%TBA, and 1.5 mL of 20% acetic acid. The mixture was heatedfor 60 minutes at 958C, then cooled on ice, and centrifugedat 3,000 rpm (using Centrifuge 5403, Eppendorf GmbH,Engelsdorf, Germany). Supernatants were removed, and theabsorbance was measured on a spectrophotometer (modelU-2000, Hitachi) at 532 nm. The calibration curve wasplotted from 1,1,3-3-tetramethoxypropane. The percentageinhibition of lipid peroxidation in the samples was calcu-lated using the following formula:

Percentage inhibition of lipid peroxidation¼ [(A0�A1 / A0]

· 100

where A0 is the absorbance of the control treated group andA1 is the absorbance of the sample-treated groups.

FeCl2-induced lipid peroxidation in rat gastrointestinalhomogenate (in vitro)

The inhibitory effect of OSME on FeCl2-induced lipidperoxidation in normal rat gastrointestinal homogenate wasdetermined by TBA-malondialdehyde (MDA) adduct for-mation according to the method of Yuda et al.21 with minormodification. The healthy rats were terminally anesthetizedwith diethyl ether. The stomach was removed via abdominaldissection, and 10% (wt=vol) homogenate of the stomach in25 mM Tris-HCl buffer (pH 7.2) was prepared using a ho-mogenizer (MSE, Crawley, UK) under ice-cold (0–48C)conditions. A mixture containing 0.5 mL of normal ratstomach homogenate, 0.1 mL of 25 mM Tris-HCl buffer(pH 7.2), 0.05 mL of 4 mM FeCl2, and 0.05 mL of variousconcentrations (0.0625, 0.125, 0.25, 0.5, and 1 mg=mL) ofOSME, BHA, and BHT was incubated for 1 hour at 378C.After incubation, 9 mL of distilled water and 2 mL of 0.6%

GASTROPROTECTION BY O. STAMINEUS EXTRACT 1091

TBA were added to 0.5 mL of the incubated mixture andshaken vigorously. The mixture was then heated for 30 min-utes in a boiling water bath. After cooling, 5 mL of n-butanolwas added, and the mixture was again shaken vigorously. Then-butanol layer was separated by centrifugation at 1,000 g for10 minutes and collected for measurement at 532 nm (usingthe model U-2000 spectrophotometer [Hitachi]).

The percentage inhibition of lipid peroxidation in thesamples was calculated using the following formula:

Percentage inhibition of lipid peroxidation¼ [(A0�A1 / A0]

· 100

where A0 is the absorbance of the control group and A1 is theabsorbance of the sample-treated groups.

Histopathological study

Immediately after the ulcer index was recorded, thestomachs were preserved in 10% formalin for histopatho-logical studies. The specimens were then processed for de-hydration and clearing steps using an automatic tissueprocessor (model Citadel 1000, Shandon, Cheshire, UK).After processing, the tissues were embedded in paraffin witha Histo-Center II-N (Barnstead=Thermolyne, Dubuque, IA,USA) and sectioned at 5 mm thickness using a Histocut 820(Reichert-Jung, Nussloch, Germany). The sections werestained with hematoxylin and eosin. The mucosal injuryevaluation was performed double-blind under light micros-copy for histopathological changes such as edema, erosion,ulceration, and necrosis. The severity of histopathologicalchanges was quantified according to an arbitrary scale(between – to þþþþ).

Statistical analysis

Statistical analysis involved the use of Statistical Packagefor Social Sciences (SPSS) software (SPSS, Chicago, IL,USA). Data were expressed as mean� SEM values andwere analyzed statistically by one-way analysis of variance.Significant differences between the means of groups weredetermined using the LSD multiple comparison test, andP< .05 was taken as significant. The direction and magni-tude of the correlation coefficient (r) between variables werealso quantified using Pearson’s correlation test, and P< .05was considered as statistically significant.

RESULTS

HPLC analysis

The percentages of rosmarinic acid, sinensetin, eupatorin,and 30-hydroxy-5,6,7,40-tetramethoxyflavone in OSME were7.58%, 0.2%, 0.34%, and 0.24%, respectively (Fig. 2).

Effects of OSME on absolute ethanol-inducedgastric lesions

Treatment with absolute ethanol produced extensive gas-tric lesions in the glandular mucosa of the stomachs of the

control group. Hemorrhagic red bands or patches were ob-served along the axis in corpus or glandular stomach, theportion that secretes HCl and pepsin. Pretreatment withOSME (125, 250, 500, or 1,000 mg=kg) and omeprazole(30 mg=kg) decreased the ethanol-induced formation of gas-tric lesions. No gross lesion was observed in the forest-omach of either control or treated groups (Fig. 3). The ulcerindex was found to be 9.0� 0.45 in the control group 1 hourafter ethanol administration. Pretreatment with omeprazole(30 mg=kg) (ulcer index¼ 1.8� 0.48) and OSME in in-creasing doses of 125 mg=kg (ulcer index¼ 6.7� 0.61),250 mg=kg (ulcer index¼ 4.5� 0.56), 500 mg=kg (ulcerindex¼ 2.8� 0.31), and 1,000 mg=kg (ulcer index¼ 1.17�0.31) significantly inhibited the formation of gastric lesions(Table 1). The inhibition of ethanol-induced mucosal dam-age by OSME was dose-dependent (r¼ 0.948). Histologicalexamination of gastric mucosa showed the appearance ofethanol-induced lesions in the form of detachment of thesurface of epithelium of gastric pits (Fig. 4b). Pretreatmentwith OSME (125, 250, 500, or 1,000 mg=kg) and omepra-zole (30 mg=kg) before administration of absolute etha-nol showed more superficial gastric erosions compared tothe control group that was pretreated with distilled water(Fig. 4). Table 2 summarizes the histological changes seenafter treatment with ethanol and OSME. Oral administrationof absolute ethanol induced severe necrosis, hemorrhage,congestion, and edema in stomach sections. These injurieswere markedly ameliorated by omeprazole and OSME.

FIG. 2. (a) HPLC chromatogram of standard markers. Peaksare marked: RA, rosmarinic acid; TMF, 30-hydroxy-5,6,7,40-tetramethoxyflavone; SEN, sinensetin; and EUP, eupatorin. (b) HPLCchromatogram of OSME.

1092 YAM ET AL.

Effect of OSME on absolute ethanol-induced changesin gastric wall mucus

Treatment with absolute ethanol significantly (P< .05)decreased the Alcian blue binding to the gastric wall mucus(817.8� 28.35mg of Alcian blue=g of tissue) comparedto the normal group (1,002.7� 49.7mg of Alcian blue=gof tissue). Pretreatment with omeprazole (30 mg=kg)(1,207.0� 60.31 mg=g) and OSME at doses of 250 mg=kg(937.7� 46.7mg=g), 500 mg=kg (1,180.3� 68.2 mg=g), and1,000 mg=kg (1,260.3� 53.8 mg=g) significantly enhancedAlcian blue binding to the gastric wall mucus (Fig. 5).Pretreatment with OSME enhanced the Alcian blue bindingcapacity of gastric wall mucus in a dose-dependent manner(r¼ 0.718, P< .001).

Effect of OSME on FeCl2-induced lipid peroxidationin stomach tissue (in vitro)

In this study, Fe2þ was able to stimulate lipid peroxida-tion in stomach mucosa (Fig. 6). Lipid peroxidation isbelieved to primarily occur in cell organelles, especiallymicrosomes and mitochondria.21 As shown in Figure 6,various concentrations (0.0625, 0.125, 0.25, 0.5, and1 mg=mL) of OSME, BHT, and BHA inhibited FeCl2-stimulated lipid peroxidation in rat stomach homogenate (invitro) dose-dependently (r¼ 0.819, P< .05; r¼ 0.818,P< .05; r¼ 0.867, P< .05, respectively). The 50% effectiveconcentrations of OSME, BHT, and BHA are 0.157, 0.167,and 0.201 mg=mL, respectively.

Effect of OSME on tissue lipid peroxidationin ethanol-induced gastric lesions (ex vivo)

Table 3 shows the inhibitory effect of pretreatment withvarious doses of OSME (125, 250, 500, and 1,000 mg=kg) onethanol-induced stomach lipid peroxidation ex vivo. Treat-ment of rats with absolute ethanol significantly (P< .001)increased lipid peroxidation in stomach mucosa compared tothe control group as determined by MDA formation. Ratstreated with OSME at doses of 250, 500, and 1,000 mg=kgsignificantly (P< .01, P< .001, and P< .001, respectively)exhibited decreased formation of MDA with percent-age inhibitions of 16.3%, 32.6%, and 52.9%, respectively.Therefore, the inhibitory effect of OSME against theethanol-induced MDA level in stomach tissue homogenatewas in a dose-dependent manner (r¼ 0.981, P< .05).

DISCUSSION

Rat gastric mucosal damage induced by high concentra-tions of ethanol has widely been used to investigate gastro-protective effects of medicinal plants.23 There is substantialdocumentation of the physiological consequences of acuteethanol intoxication in gastric mucosa: (1) direct action ofextracellular and=or intracellular generated oxygen-derivedfree radicals such as superoxide anion, hydroxyl, peroxyl,and alcoholxyl radicals24–26; (2) increased conversion ofxanthine dehydrogenase to xanthine oxidase produces hy-poxanthine and leads to damage of the gastric mucosa by

Table 1. Effects of OSME on Absolute Ethanol

(AE)-Induced Gastric Lesions

Group, dose (mg=kg) Ulcer index Inhibition rate (%)

Normal (distilled water) — —Control (distilled waterþAE) 9.0� 0.45 —Omeprazole (30)þAE 1.83� 0.48c 79.13� 6.08AEþOSME

125 6.67� 0.61b 24.35� 6.07250 4.50� 0.56c 47.47� 7.24500 2.83� 0.31c 67.56� 3.481,000 1.17� 0.31c 86.93� 4.09

Data are mean�SEM values (n¼ 6).bP< .01, cP< .001, significant differences compared to control (using one-

way analysis of variance).

FIG. 3. Morphological appearance of various treatments againstethanol-induced gastric lesions: (a) normal, (b) distilled water(10 mL=kg), (c) omeprazole (30 mg=kg), (d) OSME (125 mg=kg),(e) OSME (250 mg=kg), (f) OSME (500 mg=kg), and (g) OSME(1,000 mg=kg).

GASTROPROTECTION BY O. STAMINEUS EXTRACT 1093

influencing the energy metabolism27; and (3) constrictiveeffects on blood vessels of the gastric mucosa producescongestion, tissue injury, and inflammation through lipox-ygenase and arachidonic acid mechanism.27–30 Furthermore,ethanol appears to stimulate gastric acid secretion by excit-ing sensory nerves in the gastric mucosa and promoting re-lease of gastrin and histamine. Gastrin and histamine, in turn,stimulate the proton pump via calcium-dependent and cyclicAMP-dependent pathways, respectively, with partial actionof gastrin to stimulate histamine secretion as an additionalpathway that eventually enhances the proton pump action.31

In this study, omeprazole, a proton pump inhibitor that

specifically binds to the Hþ=Kþ-ATPase enzyme system ofthe gastric parietal cell to suppress the secretion of hydrogenion into the gastric lumen,32 was chosen as a reference anti-ulcer drug because it has been shown to have good anti-ulcereffects on ethanol-induced gastric mucosal damage,33 whichwas clearly reiterated and demonstrated in our experiments.Because a previous study reported that absolute ethanolinduces lipid peroxidation and increases the tissue MDAlevels,22,34 in vitro and ex vivo stomach tissue lipid perox-idation models were also adapted for the study.

In the present study, pretreatment with OSME exhibited aremarkable concentration-dependent inhibition of ethanol-

FIG. 4. Light micrographs show the effect of OSME on ethanol-induced gastric lesions. (a) The stomach wall of the normal group has a normalappearance. (b) Administration of ethanol produced lesions in the form of gastric pits with detachment of the surface of the epithelium, andepithelial cells appeared to be vacuolated. (d and e) Pretreatment of rats with OSME at 125 or 250 mg=kg, respectively, partially protected againstethanol-induced lesions. (c, f, and g) Omeprazole (30 mg=kg) and OSME (500 or 1,000 mg=kg), respectively, almost completely prevented theformation of ethanol-induced lesions.

FIG. 5. Effect of omeprazole and different doses of OSME ongastric wall mucus content in rats. Data are mean� SEM values(n¼ 6). aP< .01, cP< .001, significant differences between treatedgroups compared to the control (distilled water plus absolute ethanol[AE]) group; #P< .05, ##P< .01, significant difference betweentreated groups compared to the normal group.

Table 2. Histopathological Evaluations

of the Effects of OSME on Absolute Ethanol

(AE)-Induced Gastric Lesions in Rats

Group, dose(mg=kg) Necrosis Hemorrhage Congestion Edema

Normal (distilledwater)

— — — —

Control (distilledwaterþAE)

þþþþ þþþþ þþþþ þþþþ

Omeprazole(30)þAE

— — þ þ

AEþOSME125 þþ þþ þþþ þþþ250 þ þ þþ þþ500 — þ þ þ1,000 — — þ þ

Rating scale was as follows: —, normal; þ, little effect; þþ, appreciable

effect; þþþ, severe effect; þþþþ intensively severe effect.

1094 YAM ET AL.

induced gastric mucosal injury in vivo. Histopathologicalstudy showed that pretreatment with OSME prevents dam-age of gastric mucosa. Gross examination and ulcer indexstudy also showed that OSME significantly and dose-dependently prevents the surface epithelium hemorrhagiclesion produced by ethanol. This study further showed thatabsolute ethanol was able to induce lipid peroxidation andcaused a significant increase in MDA level in rat stomachtissue. OSME was able to inhibit lipid peroxidation in vitroand ex vivo in a dose-dependent manner. This finding agreedwith previous studies, indicating that decreased MDA for-mation is likely to play an important role in the prevention ofgastric lesions induced by absolute ethanol.22

Gastric mucus is an important protective factor for thegastric mucosa. It consists of a viscous, elastic, adherent,transparent gel formed by 95% water and 5% glycoprotein.Prostaglandin I2 and prostaglandin E2 appear to stimulatemucus secretion by a direct action on prostaglandin receptorson the gastric parietal cells.31 Gastric mucus is capable ofacting as an antioxidant and oxygen free radical scavenging

agent.35 A decrease in gastric mucus renders the mucosasusceptible to injuries induced by ulcerogenic agents.36,37

In the present study, gastric wall mucus content in theethanol-treated group was significantly lower than in thecontrol group. It was found that pretreatment with OSMEand omeprazole significantly increased the gastric mucuscompared to the control group. This finding indicates thatOSME enhanced gastric mucus synthesis and secretion inthe experimental rats.

Flavonoids, as secondary metabolites present in plants,have drawn the attention of researchers because of thewide range of their biological activities, which encom-passes the gastroprotective effect.38,39 The HPLC studyindicated that sinensetin, eupatorin, 30-hydroxy-5,6,7,40-tetramethoxyflavone, and rosmarinic acid are all present inOSME, accounting for a total of 8.36%. A previous studyindicated that there was a correlation among antioxidant,lipoxygenase, and xanthine oxidase inhibitory effects andgastroprotective effect of phenolic compounds.14 Thesefour compounds were proven to have antioxidant and freeradical scavenging effects, as well as xanthine oxidase andlipoxygenase inhibitory properties.15,40 Therefore, sinense-tin, eupatorin, 30-hydroxy-5,6,7,40-tetramethoxyflavone, androsmarinic acid can be considered to be the compounds inOSME that may act as gastroprotective agents becausethese compounds were reported to possess very potent an-tioxidant activities and to inhibit lipid peroxidation andxanthine oxidase. Moreover, sinensetin, eupatorin, and 30-hydroxy-5,6,7,40-tetramethoxyflavone from OSME alsoexhibit a structure with a C-2–C-3 double at the B ring andsubstitution of the C ring at C-2 (B ring) (Fig. 1), whichfulfills the minimum structural requirement for gastro-protective property mentioned by Ares et al.41

In conclusion, the present study demonstrates that oraladministration of O. stamineus extract exerts a dose-dependent gastroprotective effect on acute ethanol-inducedgastric lesions in the rat. This further supports the localtraditional belief that the extract of O. stamineus protectsagainst ulcers.

ACKNOWLEDGMENTS

This study was supported by an Intensifying ResearchPriority Areas grant from the Ministry of Science, Tech-nology and Innovation, Malaysia. The authors are indebtedto Prof. Zhari Ismail for various help.

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

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Table 3. Effect of OSME on Tissue Lipid

Peroxidation in Absolute Ethanol (AE)-Induced

Gastric Lesion (Ex Vivo)

Group, dose(mg=kg)

MDA(nmol=mg of protein)

Inhibitionrate (%)

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(distilled water plus AE) group (using one-way analysis of variance).

FIG. 6. Effect of various concentrations of BHT, BHA, and OSMEon lipid peroxidation (LPO).

GASTROPROTECTION BY O. STAMINEUS EXTRACT 1095

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GASTROPROTECTION BY O. STAMINEUS EXTRACT 1097