Global Journal of Pharmacology 5 (3): 176-180, 2011ISSN 1992-0075 IDOSI Publications, 2011

Corresponding Author: Mahboobeh Nakhaei Moghaddam, Department of Biology, Mashhad Branch, Islamic Azad University,Mashhad, Iran.

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In vitro Inhibition of Helicobacter pylori by Some Spices and Medicinal Plants Used in Iran

Mahboobeh Nakhaei Moghaddam

Department of Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran

Abstract: Helicobacter pylori is the most common cause of gastric infections worldwide. The antibacterialactivity of methanol extracts of Satureja hortensis L. (Lamiaceae), Artemisia dracunculus L. (Asteraceae),Carthamus tinctorius L. (Asteraceae), Apium petroselinum L. (Apiaceae), Citrus sinensis L. (Rutaceae) andPunica granatum L. (Punicaceae) were evaluated against H. pylori. Inhibition tests were carried out using thedisc diffusion method against 27 clinical isolates of H. pylori for initial screening and then agar dilution methodto determine minimum inhibitory concentration (MIC). Among the crude extracts, P. granatum showed thehighest inhibition (27.960.97mm) and the MICs of its butanol and aqueous fractions were 156 and 195.12g/mL, respectively. While its chloroform fraction had no activity against tested H. pylori isolates. All extractsexcept safflower preserved their anti-Helicobacter pylori activity at 121C. All the extracts were capable ofinhibiting the in vitro growth of H. pylori and the butanol fraction of P. granatum, showed excellent anti-H.pylori activity.

Key words: H. pylori Antibacterial activity Extract Medicinal plants

INTRODUCTION The present study was carried out to evaluate

After of Helicobacter pylori in 1982 by spices of native medicinal plants in Iran [SaturejaWarren and Marshall, it was shown that most hortensis L. (Lamiaceae), Artemisia dracunculus L.duodenal and peptic ulcers are caused by this organism. (Asteraceae), Carthamus tinctorius L. (Asteraceae),H. pylori is an important etiological agent of gastritis, Apium petroselinum L. (Apiaceae), Citrus sinensis L.peptic ulcer and gastric cancer [1, 2] Current therapy is (Rutaceae) and Punica granatum L. (Punicaceae)] thatbased on a combination of antibiotics, bismuth sub have been traditionally used in folk medicine.citrate and proton pump inhibitors [3]. Although thetriple therapy is considered highly efficient, only 80% MATERIALS AND METHODSof patients show absence of pathogen and theinfection sometimes returns after treatment Plant Materials: The names of plants, collection sites andcompletion [4, 5]. The main reason for eradication failure parts used in this study are illustrated in Table 1. Allcan be H. pyloris resistance to one of the antibiotics plants were identified by Herbarium of Department ofused for treatment [6]. Also chemical drugs have side Pharmacognosy, Mashhad Faculty of Pharmacyeffects such as diarrhea and colitis. Therefore it is (Mashhad University, Iran). A voucher specimen wasnecessary to introduce alternative remedial maintained for reference in the Herbarium.regimens. One of these resources is medicinal plantsthat some of their therapeutic properties have been Preparation of Extracts: Plants were dried at roomrecognized in folk medicine. Most people have temperature in the shade and ground separately beforepositive attitude toward natural products due to their extraction. Each material was extracted using macerationnatural origin and lesser toxicity [7]. Study of with methanol. The powder of materials was macerated inanti-Helicobacter pylori effects of these plants, methanol for 3 days and the mixture was subsequentlyespecially those that are traditionally used for gastric filtered and concentrated under reduced pressure at 40C.problems are important. Dried extracts were stored at 4C.

in vitro anti-Helicobacter pylori activity of some

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Table 1: Characteristics of the plants used in this studyPlant Scientific name Family Collection sites PartSafflower Carthamus tinctorius L. Asteraceae Khorasan FlowerSweet fennel Satureja hortensis L. Lamiaceae Mashhad- Khorasan LeavesTarragon Artemisia dracunculus L. Asteraceae Mashhad- Khorasan LeavesOrange Citrus sinensis L. Rutaceae North of Iran Peel of fruitPomegranate Punica granatum L. Punicaceae Saveh- Markazi Peel of fruitParsley Apium petroselinum L. Apiaceae Neishabur- Khorasan Seeds

Dried methanol extract of pomegranate rind was Determination of Temperature and pH Stability offurther fractionated by successive solvent extraction with Extracts and Constituents: Methanol extracts dissolvedn-butanol saturated with distilled water (three times) and in sterile distilled water and either were incubated atthen with chloroform (three times). The solvents were 80C for 30 min or autoclaved at 121C for 20 min. Inomitted under reduced pressure at 45C. Butanol (BuOH), order to determine pH stability, methanol extracts werechloroform (CHCl ) and aqueous (R-H O) fractions were mixed with 0.1M sterile phosphate buffer solution of3 2used for activity testing. various pH values (5, 6, 7 and 8) in separate tubes and

Microbiological Studies: In this study 27 clinical discs containing extracts were prepared after exposure toisolates of H. pylori were obtained from the the different thermal and pH conditions and tested asLaboratory of 17-Shahrivar Hospital (Mashhad-Iran). above for anti-Helicobacter pylori assay.These isolates were identified by colony morphology,Gram stain, microscopic morphology, calalase , oxidase , Phytochemical Screening: The methanol extract of S.+ +rapid urease , nalidixic acid resistance and H S [8]. Growth hortensis, A. dracunculus and C. tinctorius were+ -2inhibition was performed on Mueller-Hinton agar screened for alkaloids, flavonoids, tannins [14] and(Pronadisa-Madrid) containing 7% egg yolk emulsion and saponins [15].4 mg triphenyl tetrazolium chloride (Merck) by the filterpaper disc diffusion method according to Clinical and Statistical Analysis: Data were the means of inhibitionLaboratory Standards Institute (CLSI) guidelines [9, 10]. zones of antibacterial activity of plant extracts experimentsStandard discs containing 2 mg of herbal extract were which have been repeated three times. The results wereplaced on egg yolk emulsion agar plate, previously analyzed initially using analysis of variance (ANOVA)inoculated with 0.1 mL bacterial suspension in sterile and then Duncan test. A value of p < 0.05 was considerednormal saline. The turbidity of the bacterial suspension statistically significant.was equivalent to Mc-Farlands tube No. 4 (10 CFU/ mL)8

[11]. Inhibition growth assay was performed using RESULTSstandard commercial discs of amoxicillin (25 g/disk) and,metronidazole (5 g/disk) as positive control and discs All herbal extracts showed anti-Helicobacter pyloricontaining evaporated methanol and distilled water as activity at concentration of 2 mg/disc (Table 2). Amongnegative control. All plates were incubated for 2-3 days the crude extracts, pomegranate rind showed the highestat 37C under microaerophilic condition (10% CO ) and inhibition (27.960.97 mm) and orange peel (13.560.51290-100% humidity. Minimum inhibitory concentration mm) and Sweet fennel (13.700.45 mm) showed the least(MIC) values were determined by the agar dilution method activity against H. pylori clinical isolates. The effect offor four isolates of H. pylori with the highest sensitivity. pomegranate rind and safflower extracts were significantlyMICs were reported as the lowest concentration of the more than parsley and orange peel extracts (p < 0.05).plant extract that inhibited visible bacterial growth [12]. The extract of pomegranate rind has a significantThe minimum bactericidal concentrations (MBC) were MIC (312.5 g/mL) (Table 3). According to the results, theestablished by the lack of growth upon re- inoculation extracts of pomegranate rind, orange peel and sweetfrom extract- treated plates to Mueller- Hinton agar fennel had bacteriostatic effect, whereas safflower andplates [12]. All experiments were carried out three times. parsley seed showed bactericidal effect against H. pyloriAfter primary screening of anti-Helicobacter pylori isolates.experiments, the extract of pomegranate rind showed As the extract of pomegranate rind was the mostbetter activity against H. pylori, therefore the activity of efficient, it was decided to test its fractions againstthe fractions of pomegranate rind were evaluated like H. pylori isolates. The mean of inhibition zone ofpreceding methods. butanol fraction (25.30.86 mm) was more than aqueous

were incubated at room temperature for 3 [13]. Then,

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Table 2: Inhibition zone (meanSE) of herbal methanol extracts atconcentration 2 mg/disc against 27 H. pylori clinical isolates

Methanol extract MeanSE (mm)Carthamus tinctorius L.* 18.770.56Satureja hortensis L. 13.700.45Artemisia dracunculus L. 15.070.48Citrus sinensis L. 13.560.51Punica granatum L.* 27.960.97Apium petroselinum L. 16.440.57Amoxicillin (25 g/disk) 37.142.22Metronidazol (5 g/disk) 24.413.78*significant at p < 0.05

Table 3: MICs and MBCs of the herbal extracts against H. pylori clinicalisolates

Herbal extract MIC (g/mL) MBC (g/mL)Carthamus tinctorous L. 691.25 691.25Artemisia dracunculus L. 691.25 827.5Citrus sinensis L. 729 1041.5Punica granatum L. 312.5 338.5Apium petroselinum L 729 729

fraction (22.690.82 mm), but there was no significantdifference by Duncan test (P > 0.05). Chloroform fractionhad no activity against tested H. pylori isolates. TheMICs of butanol and aqueous fractions were 156 and195.12 g/mL, respectively.

Preliminary phytochemical screening of the methanolextracts indicated the presence of saponins in sweetfennel, saponins and tannins in tarragon and flavonoidsin safflower.

All the extracts with the exception of methanol extractof safflower preserved their anti-Helicobacter pyloriactivity at 80C or 121C against the four tested isolates.The activity of safflower extract was decreased 21-43%after autoclaving. Methanol extracts of pomegranaterind and parsley seed were stable at pH 5 and 6 with noreduction in anti-Helicobacter pylori activity. Theactivity of parsley extract was decreased 9.3-16% at pH 8.The effect of pH was not tested for other herbal extracts.

DISCUSSION

In the present study, among the plant crude extractstested, that of pomegranate peel which showed thehighest activity on H. pylori growth. The MIC ofpomegranate peel was 312.5 g/mL and it hadbacteriostatic effect. Several authors have previouslyreported that some extracts of plants exhibit an inhibitoryactivity against H. pylori [12, 16-19]. It was reported thatMICs for the aqueous garlic extract against nineteenstrains of H. pylori range from 2 to 5 mg/mL [19]. H. pylorigrowth in liquid medium was completely inhibited at 3-5

mg/mL by aqueous extract of thyme whereas in solidmedium complete inhibition was attained at 4-5 mg/mL[17]. The MIC of black myrabalan (Terminalia chebulaRetz) aqueous extract against H. pylori has been reportedto be 125 mg/L [12]. The methanol and chloroform extractsof Byrsonima crassa inhibited, in vitro, the growth ofH. pylori with MIC value of 1024 g/mL [20]. The MICs ofcrude urushiol extract from the sap of the Koreanlacquer tree (Rhus vernicifera Stokes) against 3 strainsof H. pylori ranged from 0.064 mg/mL to 0.256 mg/mL[21]. The MIC and MIC of oil extract of Chamomilla90 50recutita flowers against H. pylori were 125 and 62.5mg/mL, respectively [22].

Aqueous and butanol fractions of pomegranatepeel showed good activity on H. pylori clinical isolateswith MICs of 156 and 195.12 g/mL, respectively,but chloroform fraction had no effect on H. pylori.Methanol extract and hexane fractions of rhizome andleaves of Aristolochia paucinervis exhibited aninhibitory activity at a concentration of 128 g/mLagainst H. pylori. Hexane fraction of rhizomedemonstrated a higher inhibitory activity (MIC: 4 g/mL)than hexane fraction of leaves (MIC: 16 g/mL) [23]. Thedichloromethanic fraction of Calophyllum brasiliense,a medicinal tree that grows particularly in the hilly andforested regions of Brazil, appeared to have the mostactive and potent fraction in vitro against H. pylorigrowth with an MIC of 31 g/mL [24].

Many of chemical materials that are used todayfor preservation, coloring or flavoring of food productsare hazardous for human health.

Pomegranate peels are used as a medicinal plant aswell as for making dye. Anti-bacterial, anti-parasitic,antiviral effects [25-27], anti-oxidant and anti-mutagenicactivities [27, 28] have been reported for pomegranatepeel, up to now. The use of this extract is recommendedfor making food products instead of chemical coloringmaterial due to its useful effects. The presence of phenoliccompounds (proantocianin) and tannins (Ellagic tannins)in pomegranate peel have been reported and anti-microbial effects of these compounds have beenrecognized [29, 30]. It is possible that some of anti-Helicobacter pylori activity of pomegranate peel isrelated to tannin and phenolic compounds.

Although the activity of orange peel on H. pyloriwas less than other extracts, it can still be used forflavoring cakes and sweets instead of chemical agentssuch as vanilla that in high doses is poisonous [31]. Atraditional Iranian cake is made with a grated unpeeledorange. There is also a rice dish and a jam prepared withorange peels [32].

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Considered to be stable at 121C, all extracts except 9. Tabak, M., R. Armon and I. Neeman, 1999. Cinnamonsafflower are therefore suitable for process of food extract's inhibitory effect on Helicobacter pylori.preparation without any effect on anti-Helicobacter J. Ethnopharmacol., 67: 269-277.pylori of these herbal extracts. Whereas black and green 10. Clinical and Laboratory Standards Institute, 2010.tea lose their effectiveness at 85C for 5 min as was Performance Standards for Antimicrobialreported by Diker and Hascelik [33]. Susceptibility Testing; Twentieth Informational

The results of this study showed these herbs might Supplement. Wayne, PA: Clinical and Laboratoryhave a considerable potency at preventing or aiding the Standards Institute. CLSI document M100-S20.treatment of H. pylori infections. Available at: http://www.clsi.org.

ACKNOWLEDGMENT K. McColl, A. Price, G. Smith and L. Teare, 2002.

The author would like to appreciate the Research Antimicrob. Chemother., 49: 601-609.Deputy of Islamic Azad University- Mashhad Branch 12. Malekzadeh, F., H. Ehsanifar, M. Shahamat, M.for the financial support of this research Levin and R.R. Colwell, 2001. Antibacterial activity(Project No. 86182/304/39). of black myrobalan (Terminalia chebula Retz)

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