protective effect of origanum majorana l. 'marjoram' on...

May 19, 2009 11:20 WSPC WS-AJCM SPI-J000 00703

The American Journal of Chinese Medicine, Vol. 37, No. 3, 531–545© 2009 World Scientific Publishing Company

Institute for Advanced Research in Asian Science and Medicine

Protective Effect of Origanum majoranaL. ‘Marjoram’ on Various Modelsof Gastric Mucosal Injury in Rats

Tawfeq Al-Howiriny,∗ Abdulmalik Alsheikh,‡ Saleh Alqasoumi,∗,†

Mohammed Al-Yahya,∗ Kamal ElTahir† and Syed Rafatullah†

∗Department of Pharmacognosy

†Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC)College of Pharmacy, King Saud University

P.O. Box 2457, Riyadh 11451, Saudi Arabia

‡Department of Pathology (32), P.O. Box 2925King Khalid University Hospital

King Saud University, Riyadh 11461, Saudi Arabia

Abstract: ‘Marjoram,’ Origanum majorana L., a culinary aromatic medicinal herb is known topossess various therapeutic properties. We evaluated the antiulcerogenic activity of the ethanolextract in hypothermic restraint stress-, indomethacin-, necrotizing agents- (80% ethanol,25% NaCl and 0.2 M NaOH) induced ulcers and basal gastric acid secretion using pylorusligated Shay rat-model. Marjoram at doses of 250 and 500 mg/kg of body weight, signif-icantly decreased the incidence of ulcers, basal gastric secretion and acid output. Further-more, the extract replenished the ethanol-induced depleted gastric wall mucus and nonproteinsulfhydryls (NP-SH) contents and significantly lowered the increase in the concentration ofmalondialdehyde (MDA). Ulcer preventing potential was further confirmed by histopatholog-ical assessment. An acute toxicity test showed a large margin of safety of the extract in mice.The phytochemical screening of aerial parts of marjoram revealed the presence of volatile oil,flavonoids, tannins, sterols and/or triterpenes.

Keywords: Marjoram; Origanum majorana; Antiulcer; Antisecretory; Antioxidant.

Introduction

Peptic ulcer is one of the major gastro-intestinal diseases caused by multiple factors, includ-ing stress, smoking, nutritional deficiencies, noxious agents such as alcohol, nonsteroidal

Correspondence to: Dr. Syed Rafatullah, Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC),College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia. Tel: (+966) 1-467-7207,Fax: (+966) 1-467-7245, E-mail: [email protected]; [email protected]

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532 T. AL-HOWIRINY et al.

anti-inflammatory drugs (NSAIDs), Helicobacter pylori infection, etc. (Belaiche et al.,2002). The creation of ulcer in the stomach is due to acid and breakdown of mucosaldefense. Local mechanisms implicated in mucosal defense are; mucus-like alkaline secre-tions, mucosal hydrophilicity, rapid epithelial cell renewal, rich mucosal blood flow, mucosalsulfhydryls and increased resistance of gland cells in deep mucosa to acid and peptic activ-ity (Konturek, 1985). Long before the advent of modern medical treatment to cure diseasesincluding gastric disorders, herbs, spices, vegetables, medicinal plants were utilized as com-bative agents (Kamal, 1975). Since the last two decades, considerable number of scientificreports has appeared in the literature on the potential gastric antiulcer properties of medicinaland aromatic plants (Al-Yahya et al., 1989; Rafatullah et al., 1990, 1995; Al-Howiriny et al.,2003; Al-Howiriny, 2008a, 2008b; Al-Mofleh et al., 2005, 2007; Alqasoumi et al., 2008).

Origanum majorana L., family Lamiaceae, known by the names sweet marjoram or mar-joram and locally as Marzanjosh, is an ancient aromatic medicinal plant and a culinary herb.Usually it is grown as an annual or biennial, 30–60 cm tall, with square, branched, tomen-tose stems; sometimes occurring as a subshrub. Leaves appear elliptic, entire or toothed,petiolate, opposite, grayish-pubescent, 0.75–3 cm long. The shrub appears late summer tomid-autumn. Marjoram is native to North Africa, Middle East and parts of India (Stuart,1979). In Arabian traditional, Unani, Ayurvedic and Malay-Chinese medicine, the plant isconsidered to be a tonic and is used to treat liver disorders, sterility and epilepsy (Perry,1980). In the Middle East and Eurasian Turkey, marjoram is considered to be useful for thetreatment of gastro-intestinal disturbances, like hyperacidity, gastritis, ulcer and preventingspasms in the digestive tract (Dogan et al., 2004). In combination products, the aerial partsof marjoram or the oil are used for stimulating appetite, as a digestive aid, antispasmodic,antiflatulent, astringent, for “strengthening of the stomach,” and to cure acute and chronicgastritis. Marjoram has gained generally-regarded-as-a-safe (GRAS) status in USA (Jellinet al., 2000). Apart from its widespread culinary use, it is also suggested that an infusion ofthe fresh herb is beneficial in treating upset stomach and indigestion, headache, colic, andnervous complaints, as well as cough and other respiratory ailments. Marjoram is reportedto contain various chemical constituents including phenolic glycosides, flavonoids, pheno-lic compounds, tannins and an essential oil (Lust, 1980; Assaf et al., 1987; Dorman andDeans, 2000). Vagi et al. (2005) have obtained phenolic and triterpenoid antioxidants fromOriganum majorana herb.

The aim of the present study was to evaluate the antiulcerogenic activity profile of theethanolic extract of marjoram O. majorana, by using various in vivo experimental gastriculcer models in rodents, as well as histopathological assessment, biochemical parametersand acute toxicity determination.

Materials and Methods

Plant Material and Extraction

The fresh aerial parts of marjoram used in this study were purchased from a local vegetablemarket of Riyadh, and identified by an expert taxonomist. A specimen was deposited in

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ANTIULCEROGENIC ACTIVITY OF MARJORAM 533

the Medicinal, Aromatic and Poisonous Plants Research Center of this College for futurereference.

The shade dried aerial parts of the plant (500 g) were coarse powdered and macerated in3 liters of 96% ethanol for 72 hours using percolation method. The solvent was then removedat 40◦C under reduced pressure in a rotavapor. The extract was suspended in distilled waterbefore administration.

Animals

Wistar albino rats of either sex, approximately in the same age, having weight range 150–200 g and fed on standard chow diet were used. They were divided into groups of 6 animalseach, the distribution of animals in groups, the sequence of trials and the treatment to eachgroup were randomized. For acute toxicity and LD50 studies, Swiss albino mice were used.The ulcerogenic drugs and necrotizing agents were freshly prepared before administration.The stomach was removed, opened along the greater curvature, washed with saline and theinner surface was examined with a 6.4× binocular magnifier. Lesions were also assessed by2 observers, unaware of experimental protocol. The conduct of experiments and the proce-dure of sacrifice (using ether) were approved by the Ethics Committee of the ExperimentalAnimal Care Society, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.

Gastric lesions induced by the drugs used in this study were multiple in each stomach.They were evaluated singly according to their dimensions and severity and scored between0, no visible ulcers, and 10, deep lesion with diameter greater than 8 mm, in each stomach.The scores for each single stomach were then totaled (Valcavi et al., 1982).

Phytochemical Screening

A preliminary phytochemical screening of aerial parts of marjoram was conducted to deter-mine the presence or absence of alkaloids, cardiac glycosides, flavonoids, tannins, coumarins,anthraquinones, saponins, volatile oil, volatile bases, cyanogenic glycosides, glucosinolates,sugars, sterols and/or triterpenes according to the methods described by Farnsworth (1966).

Hypothermic Restraint Stress-Induced Ulcers

The method of Senay and Levine (1967) was followed with slight modification. The animalswere fasted for 36 hours with access to water ad libitum. One hour after receiving oralmarjoram extract at the doses 250 or 500 mg/kg of body weight, the rats were immobilizedin restraint cages and placed inside a ventilated refrigerator maintained at a temperature of2–4◦C. After 3 hours, they were taken out and sacrificed. The stomachs were excised andexamined for the severity of intraluminal bleeding according to the following arbitrary scale:0, no blood detectable; 1, thin blood follows the rugae; 2, thick blood follows the rugae, 3,thick blood follows the rugae with blood clots in certain areas; and 4, thick blood (Chiuet al., 1984). After wiping off the blood, the total area of lesions in each stomach was scoredas described above.

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Indomethacin-Induced Gastric Ulcers

Indomethacin was suspended in 1% carboxymethylcellulose sodium (CMC) in water(6 mg/ml) and administered p.o. at a dose of 30 mg/kg (0.5 ml/100 g of body weight) torats fasted for 36 hours (Bhargawa et al., 1973), Marjoram extract was administered orally250 and 500 mg/kg body weight 30 min before indomethacin. The rats were killed 6 hoursafter indomethacin administration.

Gastric Lesions Induced by Necrotizing Agents (Cytoprotection Studies)

The experiments were done on Wistar albino rats fasted for 36 hours with access to drinkingwater ad libitum. The following necrotizing agents were administered orally in a volume of1 ml: 80% ethanol, 0.2 M NaOH and 25% NaCl (Robert et al., 1983). The marjoram extractin the doses of 250 or 500 mg/kg body weight was administered orally 30 min before thenecrotizing agent treatment.

Pylorus Ligated (Shay) Rats

The animals were fasted for 36 hours with access to water ad libitum before the pylorus wasligated under light ether anesthesia, care being taken not to cause bleeding or to occludeblood vessels (Shay et al., 1945). Marjoram extract was (250 or 500 mg/kg body weight)administered immediately after pylorus ligation by intraperitoneal injection. The animalswere sacrificed 6 hours after the pylorus ligation, stomachs were removed, and contentswere collected, measured, centrifuged, and subjected to analysis for titratable acidity against0.01 N NaOH to pH 7. Each stomach was examined for lesions as described above.

Gastric Wall Mucus Determination

Gastric wall mucus was determined according to a modified procedure of Corne et al. (1974).The glandular segments of the stomach which included corpus and antrum were separatedfrom the rumen of the stomachs and weighed. Each segment was transferred immediatelyto 10 ml of 0.1% w/v Alcian blue (0.16 M sucrose solution buffered with 0.05 ml of sodiumacetate at pH 5). Tissue was stained for 2 hours in Alcian blue and excess dye was removed bytwo successive rinses with 10 ml of 0.2 M sucrose, first for 15 min and then for 45 min. Dyecomplexed with the gastric wall mucus was extracted with 10 ml of 0.5 M MgCl2 which wasshaken intermittently for 1 min at 30 min intervals for 2 hours. Four ml of blue extract werethen shaken vigorously with an equal volume of diethyl ether. The resulting emulsion wascentrifuged at 3,600 r.p.m. for 10 min and the absorbance of the aqueous layer was recordedat 580 nm. The quantity of Alcian blue extracted per gram of wet glandular tissue was thencalculated.

Estimation of Non-Protein Sulfhydryl Groups (NP-SH)

Gastric mucosal NP-SH was measured according to the method of Sedlak and Lind-say (1968). The glandular part of the stomach was homogenized in ice-cold 0.02 M

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ethylenediaminetetraacetic acid (EDTA). Aliquots of 5 ml of the homogenates were mixedwith 4 ml of distilled water and 1 ml of 50% trichloroacetic acid (TCA) in 15 ml test tubes.The tubes were shaken intermittently for 10–15 min and centrifuged at 3,000 g. Two ml ofsupernatant were mixed with 4 ml of 0.4 M Tris buffer, pH 8.9, and 0.1 ml of 0.4% DTNB[5,5-dithio-bis-(2-nitrobenzoic acid)] and the sample was shaken. The absorbance was readwithin 5 min of addition of DTNB at 412 nm against a reagent blank with no homogenate.

Determination of Malondialdehyde (MDA)

The method reported by Utley et al. (1967) was followed. The extract was administered30 min before alcohol. The animals were killed 1 hour after ethanol administration. Thestomachs were removed and each tissue was homogenized in 0.15 M KCl (at 4◦C, Potter-Elvehjem type C homogenizer) to give a 10% w/v homogenate. Aliquots of homogenate1 ml in volume were incubated at 37◦C for 3 hours in a metabolic shaker. Then 1 ml of 10%aqueous trichloroacetic acid was added and mixed. The mixture was then centrifuged at800 g for 10 min. One ml of the supernatant was mixed with 1 ml of 0.67% 2-thiobarbituricacid in water and placed in a boiling water bath for 10 min. The mixture was cooled anddiluted with 1 ml distilled water. The absorbance of the solution was then read at 535 nm.The content of malondialdehyde (nmole/g wet tissue) (index of the magnitude of lipidperoxidation) was then calculated by reference to a standard curve of malondialdehydesolution.

Determination of LD50 in Mice

Swiss albino mice were divided into various groups and each group was treated orally with asingle dose of marjoram extract in a range of 2.5–15 g/kg body weight. Following treatments,the animals were observed for 6 hours and thereafter at intervals of 2–12 hours for up to 72hours. All behavioral changes and death during the observation period were recorded. Thepercentage of death in each group was then calculated. The LD50 was determined by usingthe methods outlined by Ghosh (1984).

Histopathological Studies

The gastric tissue was fixed in 10% ethanol buffered formalin and processed through gradedethanol, xylene and impregnated with paraffin wax; sections were made by microtome. Afterstaining with haemotoxylin and eosin stain (Culling, 1974), the sections were examinedunder a microscope by a person who was not aware of experimental protocols. The differenthistopathological indices were screened.

Statistical Analysis

The numbers shown are mean ± standard error of mean. The mean determination of treat-ment groups was compared statistically with the control by using ANOVA, followed byTukey-Kramer post-hoc test.

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Results

The preliminary qualitative phytochemical analyses revealed the presence of tannins, volatileoils, sterols and triterpenes in marjoram extract. The control animals subjected to hypother-mic restraint-stress showed considerable ulcerogenicity in the form of haemorrhagic mucosallesions in the glandular segment of the stomach. There was evidence of intraluminal bleed-ing in these animals. Treatment with marjoram extract produced significant decreases inintraluminal bleeding and gastric lesions at both doses (250 and 500 mg/kg, body weight)(Table 1).

Administration of indomethacin resulted in the production of gastric mucosal damagemainly in the glandular segment of the stomach. Pretreatment of the animals with marjoramextract was effective in significantly reducing the intensity of ulceration (Table 2).

The lesions induced by various necrotizing agents were grouped in varying sized patches,usually parallel to the major axis of the stomach. Oral administration of marjoram extractsignificantly reduced the severity of these lesions in a dose dependent manner (Table 3).

Pylorus ligation for 6 hours resulted in the accumulation of basal gastric secretory volume,an increase in the titratable acidity, and ulcers which were mainly confined to the forestomach.Marjoram extract produced a significant dose dependent decrease in the volume and acidityof gastric secretion and ulcer index (Table 4). Although the extract (250 mg/kg) reduced theseverity of the ulceration in animals, it was not statistically significant.

Lowered gastric wall mucus was observed in the animals treated with 80% ethanol andthis depletion of wall mucus was significantly reversed by pretreatment with both doses ofmarjoram extract (Table 5).

Table 1. Effect of Ethanol Extract of Marjoram on Hypothermic Restraint-Stress-Induced Intra-luminal Bleeding and Gastric Lesion in Rats

Treatment Dose Intraluminal Bleeding Gastric Lesions(mg/kg, Orally)

Score (Mean ± SE) Ulcer Index (Mean ± SE)

Control — 2.83 ± 0.40 18.33 ± 1.50Marjoram ext. 250 0.66 ± 0.51∗ 5.00 ± 5.47∗Marjoram ext. 500 0.33 ± 0.51∗ 3.33 ± 5.16∗

∗p < 0.05; ANOVA followed by Tukey-Kramer post-hoc test.

Table 2. Effect of Ethanol Extract of Marjoram on Gastric Lesions Inducedby Indomethacin

Treatment (n = 6) Dose (mg/kg) Orally Ulcer Index

Indomethacin — 41.50 ± 4.76Marjoram ext. + Indomethacin 250 13.50 ± 4.08∗∗∗Marjoram ext. + Indomethacin 500 11.66 ± 1.50∗∗∗

∗∗∗p < 0.001, ANOVA followed by Tukey-Kramer post-hoc test.

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Table 3. Effect of Ethanol Extract of Marjoram on Gastric Lesions Induced by VariousNecrotizing Agents

Treatment Dose Ulcer Index (Mean ± SE)(n = 6) (mg/kg, Orally)

80% EtOH 0.2 M NaOH 25% NaCl

Control — 7.00 ± 0.63 7.16 ± 0.75 7.16 ± 1.32Marjoram ext. 250 4.16 ± 0.75∗ 7.83 ± 0.40∗ 2.00 ± 0.89†

Marjoram ext. 500 2.16 ± 0.75† 2.16 ± 0.75‡ 1.50 ± 0.83†

∗p < 0.05; †p < 0.01; ‡p < 0.001, ANOVA followed by Tukey-Kramer post-hoc test.

Table 4. Effect of Ethanol Extract of Marjoram on the Basal Gastric Secretion, Acidity and Ulcer-ation in 6 hours Pylorus-Ligated Shay Rats

Treatment Dose Gastric Secretion at 6 hours (Mean ± SE)(mg/kg, i.p.)

Volume of Gastric Titratable Acidity Ulcer IndexSecretion (ml) (mEq/l)

Control — 10.66 ± 1.03 130.55 ± 8.27 2.33 ± 0.51Marjoram ext. 250 5.33 ± 1.01† 92.77 ± 12.36∗ 1.33 ± 0.51Marjoram ext. 500 3.00 ± 0.89‡ 64.10 ± 7.20‡ 0.66 ± 0.51∗

∗p < 0.5; †p < 0.01; ‡p < 0.001, ANOVA followed by Tukey-Kramer post-hoc test.

Table 5. Effect of Ethanol Extract of Marjoram on 80% Ethanol-InducedGastric Wall Mucus (GWM) Changes in Rats

Treatment (n = 6) Oral Dose GWM (µµµg/g of Wet(mg/kg, Orally) Glandular Tissue)

Control — 409.95 ± 40.5880% Ethanol — 182.35 ± 12.46‡,a

Marjoram ext. + 80% Ethanol 250 355.25 ± 47.30†,b

Marjoram ext. + 80% Ethanol 500 336.65 ± 18.97‡,b

aAs compared to control group. bAs compared to 80% ethanol only group.†p < 0.01; ‡p < 0.001, ANOVA followed by Tukey-Kramer post-hoc test.

The gastric mucosal NP-SH concentrations were significantly decreased following theadministration of 80% ethanol. Treatment of animals with marjoram extract at the higherdose (500 mg/kg) significantly replenished the ethanol-induced decrease in NP-SH level(Table 6).

A significant increase in MDA level in stomach homogenate was observed after thealcohol treatment. Pretreatment of rats with a higher dose (500 mg/kg) of marjoram extractsignificantly reduced lipid peroxidation by decreasing MDA level. However, the lower dose(250 mg/kg) of extract lowered MDA level insignificantly in stomach tissue (Table 6).

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Table 6. Effect of Ethanol Extract of Marjoram on Levels of Non-Protein Sulfhydryls (NP-SH) andMalondialdehyde (MDA) in Glandular Stomach of Rats Treated with 80% Ethanol

Groups (n = 6) Oral Dose NP-SH MDA(mg/kg, Orally) (µµµmol/g of (µµµmol/g of Wet

Wet Glandular Tissue) Glandular Tissue)

Control — 5.06 ± 0.16 1.22 ± 0.2780% Ethanol — 2.40 ± 0.15‡,a 3.53 ± 0.31‡,a

Marjoram ext. + 80% Ethanol 250 2.91 ± 0.24b 2.98 ± 0.43b

Marjoram ext. + 80% Ethanol 500 3.55 ± 0.29†,b 1.96 ± 0.41‡,b

aAs compared to control group. bAs compared to 80% ethanol only group. †p < 0.01; ‡p < 0.001, ANOVAfollowed by Tukey-Kramer post-hoc test.

The oral LD50 dose in mice was 10.625 g/kg marjoram extract. The histological assess-ment of gastric tissue supported the pharmacological and biochemical findings on the abilityof marjoram extract to prevent induced gastric ulcers in rats (Figs. 1–4).

Discussion

The results of the present investigation provided substantial evidence for an antiulcer, antise-cretory and cytoprotectiveeffects of an ethanol extract of marjoram. Cold plus restraint stressprovoked gastric ulceration in all control stressed animals. It has previously been demon-strated (Bhargawa et al., 1980) that simultaneous exposure to cold (2–4◦C) and restraintfor 3 hours results in gastric ulceration in 90% of rats. Hypothermic-restraint stress ulcershave been widely used as experimental models because of data reproducibility (Murakami

Figure 1. Section through gastric of control (untreated) rat showing normal appearance. Hematoxylin and eosinstain × 100.

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Figure 2. Section through gastric mucosa of rat treated with ethanol (80%, 1 ml/rat) showing haemorrhagic necrosiswith underlying regenerative mucosa. Hematoxylin and eosin stain × 100.

Figure 3. Section through gastric mucosa of rat treated with marjoram extract (250 mg/kg) and ethanol (80%,1 ml/rat). No significant histopathological changes are noted. Hematoxylin and eosin stain × 100.

et al., 1985). Stress-induced ulcers are probably mediated by enhancement of acid secre-tion (Kitagawa et al., 1979), reduction in mucus production (Koo et al., 1986), disturbancesof gastric microcirculation (Levine, 1971; Guth, 1972) and alterations leading to abnormalmotility (Watanabe, 1966). Another important factor in the genesis of stress ulcer is the vagalover-activity, which increases gastric acid secretion and is often termed the aggressive factor(Goa and Monk, 1987).

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Figure 4. Section through gastric mucosa of rat treated with marjoram extract (500 mg/kg) and ethanol (80%,1 ml/rat). Unremarkable histopathological changes are noted. Haematoxylin and eosin stain × 100.

Our data showed that pre-treatment with marjoram extract (250 and 500 mg/kg bodyweight) significantly protected the gastric mucosa against hypothermic restraint-stressinduced ulcers, this protection could be due to inhibition of gastric mucus depletion and/ordecreased basal gastric acid secretion. It has also been suggested that the antiulcer effectof marjoram extract observed in this experimental model might result from a combinationof sedative, anxiolytic and antisecretory actions (File and Pearce, 1981). Indeed, marjoramcontains a volatile oil, which has sedative properties.

Non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin are one of thecommonly used ulcerogenic pharmacological agents (Rainsford, 1978; Garner, 1992). Theymay cause gastrointestinal ulceration, due to their ability to suppress prostaglandin synthesis(Wallace, 2001), acting as non-selective COX inhibitors. Cyclooxygenase-1 (COX-1) is con-stitutively expressed in the gastrointestinal tract in large amounts and has been reported tomaintain mucosal integrity through continuous generation of prostaglandins (Droy-Lefaix,1988; Halter et al., 2001). An antiulcerogenic effect of marjoram extract and protectionof gastric mucosa against indomethacin-induced gastric lesions was evident in this study.Besides, volatile oil phytochemical analyses of marjoram revealed the presence of tannins,flavonoids, sterols and triterpenes. The effect of marjoram may be related to its chemicalcomposition of flavonoids, tannins and sterols. There are many studies related to antiul-cerogenic properties of tannins (Al-Rehaily et al., 2002), flavonoids (Gracioso et al., 2002;Gonzales and Di Stasi, 2002), sterols and/or triterpenes (Kitazawa et al., 1980). Since tan-nins, flavonoids and sterols are found in marjoram, they may play roles in the gastroprotectiveaction through antioxidant properties.

Necrotizing agents including ethanol- and strong alkalies-induced gastric lesions arethought to occur as a result of direct insult of gastric mucosal cells, resulting in the gen-eration of free radicals and hyperperoxidation of lipids (Terano et al., 1989). Pretreatment

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with marjoram extract significantly and dose dependently protected gastric mucosa againstnecrotizing agents induced injury. These findings further indicate that marjoram extract mayenhance gastric mucosal defensive factors, such as mucus and/or prostaglandins (Whittle,1981).

Moreover, gastric acid is an important factor for the genesis of ulceration in pylorus-ligated rats (Shay et al., 1945). The activation of the vagus-vagal reflex by stimulation ofreceptors in the antral gastric mucosa in the hypersecretion model of pylorus ligature isbelieved to increase gastric acid secretion (Baggio et al., 2003). The current data clearlydemonstrated that marjoram extract significantly decreased the basal gastric acid secretionand ulceration.

The antiulcer, cytoprotective and antisecretory effects of marjoram extract in the presentinvestigation were accompanied by improvements in ethanol-induced depletion of gastricwall mucus and NP-SH and inhibition of MDA concentration in gastric tissue.

The synthesis of mucus that strengthens the mucosal barrier against noxious chemi-cals, also has an important role in protection of gastric damage. The continuous adherentmucus layer is also a barrier to luminal pepsin, thereby protecting the underlying mucosafrom proteolytic digestion (Allen and Flemstrom, 2005). The adherent mucus gel layeris a structural one to create a stable, undisturbed layer to support the surface by neu-tralization of aggressive acidity and acts as a protective physical barrier against luminalpepsin (Avila et al., 1996). Similarly, endogenous non-protein sulfhydryls compounds arethe main compounds in mucosal defence and protection against ethanol-induced gastricinjury (Szabo and Vattay, 1990). The gastric damage was accompanied by a decreasedconcentration of mucosal NP-SH compounds, due to the SH-group binding the free rad-icals formed by noxious agents (Szabo et al., 1981). Animals pretreated with marjoramextract showed a significant improvement in the depleted level of NP-SH concentration,thus indicating a strong participation of the endogenous SHs in gastroprotective effect of themarjoram extract. NP-SH compounds, on the other hand, are considered to be involved inscavenging oxygen-derived free radicals and controlling the production and nature of mucus(Allen et al., 1984; Salim, 1993). Additionally, mucus glycoproteins and lipids bound tomucin are also involved in antiradical processes (Gong et al., 1990; Hiraishi et al., 1993).Therefore, the presence of tannins, flavonoids and volatile oil compounds in marjoramare possibly the chemical constituents that prevent the loss of gastric mucus and NP-SH,since they are able to remove free radicals formed due to ethanol-induced mucosal ulcer(Sun et al., 1991; Andreo et al., 2006).

On the other hand, the treatment of animals with marjoram significantly decreased thelevel of MDA, a product of lipid peroxidation caused by free radical mediated injury ingastric tissue. All these evidences, therefore, indicate that marjoram ethanol extract exertscytoprotective and anti-gastric ulcer actions predominantly by its antioxidant property.

In the results of histopathological investigation, the gastric mucosa of rats revealed thatethanol treatment caused haemorrhagic necrosis to the gastric mucosa in rats. Pretreatmentwith marjoram showed no ulceration or haemorrhage in the gastric tissue. Our results arein corroboration with the anti-gastric ulcer activity of the extract observed in the studies onpharmacological and biochemical evaluation.

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In conclusion, the results of the present study showed that marjoram extract possessedgastroprotective activity as evidenced by its significant inhibitory effects on gastric ulcerformation induced by physical and chemical agents. Since the role of free radicals andantioxidants in the process of ulcer healing is well-defined, the gastroprotective potential ofOriganum majorana may be in part due to its strong antioxidant activity.

Acknowledgments

This work was supported by College of Pharmacy, Research Center (Grant # CPRC 201).The technical assistance of Mr. Malik Sawood Ahmed is thankfully acknowledged.

References

Al-Howiriny, T., M. Al-Sohaibani, K. ElTahir and S. Rafatullah. Prevention of experimentally-inducedgastric ulcers in rats by an ethanolic extract of “Parsley” Petroselinum crispum. Am. J. Chin.Med. 31: 699–711, 2003.

Al-Howiriny, T. Evaluation of hepatoprotective activity of broccoli “Brassica Oleracea” in rats. Hun-garian Med. J. 1(2): 147–158, 2008a.

Al-Howiriny, T. Protective effect of ‘Purslane’ on rat liver injury induced by carbon tetrachloride.Saudi Pharm. J. 16(3–4): 239–244, 2008b.

Allen, A., W.J. Cunliffe, J.P. Pearson, L.A. Sellers and R. Ward. Studies on gastrointestinal mucus.Scand. J. Gastroenterol. 93: S101–S113, 1984.

Allen, A. and G. Flemstrom. Gastroduodenal mucus bicarbonate barrier: protection against acid andpepsin. Am. J. Physiol. Cell 288: C1–C19, 2005.

Al-Mofleh, I.A., A.A. Alhaider, J.S. Mossa, M.O. Al-Sohaibani, S. Qureshi and S. Rafatullah. Phar-macological studies on ‘Clove’ Eugenia caryophyllata. Phcog. Mag. 1: 105–109, 2005.

Al-Mofleh, I.A., A.A. Alhaider, J.S. Mossa, M.O. Al-Sohaibani and S. Rafatullah. Aqueous suspensionof anise “Pimpinella anisum” protects rats against chemically induced gastric ulcers. World J.Gastroenterol. 13(7): 1112–1118, 2007.

Alqasoumi, S., T. Howiriny, M.A. Al-Yahya and S. Rafatullah. Gastroprotective effects of radish“Raphanus sativus” L. on experimental gastric ulcer models in rats. Farmacia LVI: 204–214,2008.

Al-Rehaily, A.J., T.A. Al-Howiriny, M.O. Al-Sohaibani and S. Rafatulah. Gastroprotective effects of“Amla” Emblica officinalis on in vivo test models in rats. Phytomedicine 9: 515–522, 2002.

Al-Yahya, M.A., S. Rafatullah, J.S. Mossa, A.M. Ageel, N.S. Parmar and M. Tariq. Gastroprotectiveactivity of ginger Zingiber officinale rocs., in albino rats. Am. J. Chin. Med. 17: 51–56, 1989.

Andreo, M.A., K.V.R. Ballesteros, C.A. da Hiruma-Lima, L.R.M. Rocha, A.R.M.S. Brito and W.Vilegas. Effect of Mouriri pusa extract experimentally induced gastric lesions in rodents: Roleof endogenous sulfhydryls compounds and nitric oxide in gastroprotection. J. Ethanopharmacol.107: 431–441, 2006.

Assaf, M.H., A.A. Ali, M.A. Makboul, I.P. Beck and R. Anton. Preliminary study of phenolic gly-cosides form Origanum majorana: quantitative estimation of arbsutin, cytotoxic activity ofhydroquinone. Planta Med. 53: 343–345, 1987.

Avila, J.R., C. Alarcón de la Lastra, M.J. Martin, V. Motilva, I. Luque, D. Delgado, J. Esteban andJ.R. Herrerias. Role of endogenous sulphydryls and neutrophil infiltration in the pathogenesisof gastric mucosal injury induced by piroxicam in rats. Inflamm. Res. 45: 83–88, 1996.

Baggio, C.H., C.S. Freitas, L. Rieck and M.C. Marques. Gastroprotective effects of a crude extract ofBaccharis illinita DC in rats. Pharmacol. Res. 47: 93–98, 2003.

Am

. J. C

hin.

Med

. 200

9.37

:531

-545

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

ST

ON

Y B

RO

OK

UN

IVE

RSI

TY

on

10/1

5/14

. For

per

sona

l use

onl

y.



Belaiche, J., A. Burette, M. De Vos, E. Louis, M. Huybrechts and M. Deltenre. Study group of NSAID-G1 complications. Observational survey of NSAID-related upper gastro-intestinal adverseevents in Belgium. Acta Gastroenterol. Belg. 65: 65–73, 2002.

Bhargawa, K.P., M.B. Gupta and K.K. Tangri. Mechanism of ulcerogenic activity of indomethacin andoxyphenbutazone. Eur. J. Pharmacol. 22: 191–195, 1973.

Bhargawa, K., M. Dass and M. Gupta. Study of central neurotransmitters in stress-induced gastriculceration in albino rats. Br. J. Pharmacol. 68: 765–772, 1980.

Chiu, P.J.S., C. Gerhart, A.D. Brown and A. Barnett. Effects of a gastric antisecretory cytoprotectant2-methyl-8-(phenylmethoxy)imidazo (1,2-a)-pyridine-3-acetonitrile (Sch 28080) on cys-teamine, reserpine and stress ulcers in rats. Arzneim. Forsch. 34: 783, 1984.

Crone, S.J., S.M. Morrissey and R.J. Woods. A method for the quantitative estimation of gastric barriermucus. J. Physiol. 242: 116–117, 1974.

Culling, C.F.A. Handbook of Histopathogical and Histochemical Techniques, 3rd Ed. Butterworth andCo., London, 1974, pp. 73, 126, 159.

Dogan, Y., S. Baslar, G. Ay and H.H. Mert. The use of wild edible plants in Western and CentralAnatolia (Turkey). Econ. Bot. 58(4): 684–690, 2004.

Dorman, H.J. and S.G. Deans. Antimicrobial agents from plants: antibacterial activity of plant volatileoils. J. Appl. Microbiol. 88: 308–316, 2000.

Droy-Lefaix, M.T. Prostaglandins. In: P.B. Curtis-Prior (ed.) Biology and Chemistry of Prostaglandinsand Related Eicosanoids. Churchill Livingstone, New York, 1988, pp. 345–360.

Farnsworth, N.R. Biological and phytochemical screening of plants. J. Pharm. Sci. 55(3): 225, 1966.File, S. and J. Pearce. Benzodiazepines reduce gastric ulcers induced in rats by stress. Br. J. Pharmacol.

74: 593–599, 1981.Garner, A. Adaptation in the pharmaceutical industry, with particular reference to gastrointestinal

drugs and diseases. Review. Scand. J. Gastroenterol. 193: S83–S89, 1992.Ghosh, M.N. Fundamentals of Experimental Pharmacology. Scientific Book Agency, Calcutta, 1984,

pp. 153–158 and 187–189.Goa, K.L. and J.P. Monk. Emprostil: a preliminary review of its pharmacodynamic and pharmacokinetic

properties and therapeutic efficacy in the treatment of peptic ulcer disease. Drugs 3: 539–559,1987.

Gong, D.H., B. Turner, K.R. Bhaskar and J.T. Lamont. Lipid binding to gastric mucin: protective effectagainst oxygen radicals. Am. J. Physiol. 259: G681–G686, 1990.

Gonzalez, F.G. and L.C. Di Stasi. Anti-ulcerogenic and analgesic activities of the leaves of Wilbrandiaebracteata in mice. Phytomedicine 9: 125–134, 2002.

Gracioso, J.S., W. Vilegas, C.A. Hiruma-Lima and A.R. Souza Brito. Effects of tea from Turneraulmifolia L. on mouse gastric mucosa support the Turneraceae as a new source of antiulcerogenicdrugs. Biol. Pharm. Bull. 25: 487–491, 2002.

Guth, P.H. Gastric blood flow in restraint stress. Dig. Dis. 17: 807–813, 1972.Guth, P.H., G. Paulsen and H. Nagata. Histologic and microcirculatory changes in alcohol-induced

gastric lesions in the rats: effects of prostaglandin cytoprotection. Gastroenterology 87: 1083–1090, 1984.

Halter, F., A. Schmassmann and B.M. Peskar. Cyclooxygenase 2-implications on maintenance of gas-tric mucosal integrity and ulcer healing: controversial issues and perspectives. Gut 49: 443–453,2001.

Hiraishi, H., A. Terano, S. Ota, S. Mutoh, T. Sugimoto, T. Hirada, M. Razandi and K.J. Ivey. Role formucus glycoprotein in protecting cultured gastric mucosal cells against toxic oxygen metabo-lites. J. Lab. Clin. Med. 121: 570–578, 1993.

Jellin, J.M., P. Gregory, F. Batz and K. Hitchens. Pharmacist’s Letter/Prescriber’s Letter NaturalMedicines Comprehensive Database, 3rd ed. Therapeutic Research Faculty Stockton, CA, 2000,p. 709.

Kamal, H. Encyclopaedia of Islamic Medicine. General Egyptian Book Organization, Egypt, 1975.

Am

. J. C

hin.

Med

. 200

9.37

:531

-545

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

ST

ON

Y B

RO

OK

UN

IVE

RSI

TY

on

10/1

5/14

. For

per

sona

l use

onl

y.



Kitagawa, H., M. Fujiwara and Y. Osumi. Effects of water immersion stress on gastric secretion andmucosal blood flow in rats. Gastroenterology 77: 298–302, 1979.

Kitazawa, E., A. Sato, H. Takahashi, H. Kuwano and A. Ogiso. Novel diterpenelactones with anti-pepticulcer activity from Croton sublyratus. Chem. Pharm. Bull. 28: 227–234, 1980.

Konturek, S.J. Gastric cytoprotection. Scand. J. Gastroenterol. 20: 543–553, 1985.Koo, M.W.L., C.W. Ogle and C.H. Cho. Effect of verapamil, carbenoxolone and N-acetylcysteine on

gastric wall mucus and ulceration in stressed rats. Pharmacology 32: 326–234, 1986.Levine, R.J. A method for rapid production of stress ulcers in rats. In: C.J. Pfeiffer (ed.) Peptic Ulcer.

Munksgaard, Copenhagen, 1971, pp. 92–97.Lust, J.B. The Herb Book. Bantam Books, New York, 1980.Mizui, T. and M. Doteuchi. Effect of polyamines on acidified ethanol-induced gastric lesion in rats.

Japan J. Pharmacol. 33: 939–945, 1983.Murakami, M., S.K. Lam, M. Inada and T. Miyake. Pathophysiology and pathogenesis on acute gas-

tric mucosal lesions after hypothermic restraint stress in rats. Gastroenterology 88: 660–665,1985.

Parnaham, M.J. and K. Brune. Therapeutic control of inflammatory diseases. Agents Actions 21: 232–234, 1987.

Perry, L.M. Medicinal Plants of East and South East Asia: Attributed Properties and Uses. The MITPress, London, England, 1980, p. 190.

Rafatullah, S., M. Tariq, M.A. Al-Yahya, J.S. Mossa and A.M. Ageel. Evaluation of turmeric (Curcumalonga) for gastric and duodenal antiulcer activity in rats. J. Ethnopharmacol. 29: 25–34, 1990.

Rafatullah, S., A.M. Galal, M.A. Al-Yahya and M.S. Al-Said. Gastric and duodenal antiulcer andcytoprotective effects of Afromomum melegueta in rats. Int. J. Pharmacogn. 33: 311–316, 1995.

Rainsford, K.D. Gastric ulcerogenicity of non-steroidal anti-inflammatory drugs in mice with mucosasensitized by cholinomimetic treatment. J. Pharm. Pharmacol. 39: 669–672, 1978.

Robert, A., J.E. Nezamis, C. Lancaster, J.P. Davis, S.O. Field and A.J. Hanchar. Mild irritants preventgastric necrosis through adaptive cytoprotection mediated by prostaglandins. Am. J. Physiol.245: G113, 1983.

Robert, A., J.E. Nezamis, C. Lancaster and A.J. Hanchar. Cytoprotection by Pgs in rats. Preventionof gastric necroses produced by alcohol, HCl, NaOH, hypotonic and thermal injury. Gastroen-terology 77: 433–433, 1979.

Salim, A.S. Sulfhydryl-containing agents: new approach to the problem of refractory peptic ulceration.Pharmacology 46: 281–288, 1993.

Senay, E.C. and R.L. Levine. Synergism between cold and restraint for rapid production of stress ulcerin rats. Proc. Soc. Exp. Biol. Med. 124: 1221–1231, 1967.

Sedlak, J. and R.H. Lindsay. Estimation of total protein bound and nonprotein sulfhydryl group intissue with Ellman’s reagents. Anal. Biochem. 25: 192–205, 1968.

Shay, H. The simple goes the uniform production of gastric ulceration in rat. Gastroenterology 5:43–61, 1945.

Sun, S.B., T. Matsumoto and H. Yamada. Effects of a polysaccharide fraction from the roots of Bupleu-rum folcatum L. on experimental gastric ulcer models in rats and mice. J. Pharm. Pharmacol.43: 699–704, 1991.

Stuart, M. The Encyclopedia of Herbs and Herbalism. Orbis Publishing, London, 1979, p. 230.Szabo, S., J.S. Trier and P.W. Frankel. Sulfhydryl compounds may mediate gastric cytoprotection.

Science 214: 202–220, 1981.Szabo, S. and P. Vattay. Experimental gastric and duodenal ulcers. Advances in pathogenesis. Gas-

troenterol. Clin. North Am. 19: 67–85, 1990.Terano, A., H. Hiraishi, S. Ota, J. Shiga and T. Sugimoto. Role of superoxide and hydroxyl radicals in

rat gastric mucosal injury induced by ethanol. Gastroenterol. Jpn. 24: 488–493, 1989.Utley, H.G., F. Bernheim and P. Hochstein. Effect of sulfhydryl reagents on peroxidation in microsomes.

Arch. Biochem. Biophys. 118: 29–32, 1967.

Am

. J. C

hin.

Med

. 200

9.37

:531

-545

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

ST

ON

Y B

RO

OK

UN

IVE

RSI

TY

on

10/1

5/14

. For

per

sona

l use

onl

y.



Vagi, E., E. Rapavi, M. Hadolin, K. Vasarhelyine Peredi, A. Balazs, A. Blazovics and B. Simandi.Phenolic and triterpenoid antioxidants from Origanum majorana L. herb and extracts obtainedwith different solvents. J. Agric. Food Chem. 53: 17–21, 2005.

Valcavi, U., R. Caponi, A. Brambilla, F. Palmira and R. Fuwagali. Gastric anti-secretory, anti-ulcerand cytoprotective properties 9-hydroxy-19,20-bis-nor-prostanoic acid in experimental animals.Arzneim. Forsch. 32: 657–663, 1982.

Wallace, J. Mechanisms of protection and healing: current knowledge and future research. Am. J. Med.110: 19S–22S, 2001.

Watanabe, K. Some pharmacological factors involved in formation and prevention of stress ulcers inrats. Chem. Pharm. Bull. 14: 101–107, 1966.

Whittle, B.J. Temporal relationship between cyclooxygenase inhibition, as measured by prostacyclinbiosynthesis, and the gastrointestinal damage induced by indomethacin in the rat. Gastroen-terology 80: 94–98, 1981.

Am

. J. C

hin.

Med

. 200

9.37

:531

-545

. Dow

nloa

ded

from

ww

w.w

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omby

ST

ON

Y B

RO

OK

UN

IVE

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TY

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10/1

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l use

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protective effect of origanum majorana l. 'marjoram' on...

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