International Journal of Antimicrobial Agents 18 (2001) 85 88 www.ischemo.

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Antibacterial activity of black myrobalan (Terminalia chebula Retz) against Helicobacter pylori
F. Malekzadeh a, H. Ehsanifar a, M. Shahamat b, M. Levin b, R.R. Colwell b,c,*
b a Department of Microbiology and Biological Sciences, Uni6ersity of Tehran, Tehran, Iran Center of Marine Biotechnology, Biotechnology Institute, Uni6ersity of Maryland 701 East Pratt Street, Baltimore, MD 21202, USA c Department of Cell and Molecular Genetics, Uni6ersity of Maryland, College Park, MD 20742, USA

Received 24 October 2000; accepted 6 February 2001

Abstract The effect of ether, alcoholic and water extracts of black myrobalan (Teminalia chebula Retz) on Helicobactor pylori were examined using an agar diffusion method on Columbia Agar. Water extracts of black myrobalan showed signicant antibacterial activity and had a minimum inhibitory concentration (MIC) and minimum bacteriocidal concentration (MBC) of 125 and 150 mg/l, respectively. The extract was active after autoclaving for 30 min at 121C. Plant powder (incorporated in agar) gave higher MIC and MBC values (150 and 175 mg/l, respectively). Water extracts of the black myrobalan at a concentration of 1 2.5 mg/ml inhibited urease activity of H. pylori. The results show that black myrobalan extracts contain a heat stable agent(s) with possible therapeutic potential. Other bacterial species were also inhibited by black myrobalan water extracts. 2001 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved.
Keywords: Helicobacter pylori ; Antibacterial activity; Black myrobalan

1. Introduction Helicobctor pylori is an important cause of chronic gastritis, peptic ulceration and gastric cancer in humans [1 5]. The signicance of these infections and the need for effective therapeutic agents have led to the development of several drug treatment regimens including colloidal bismuth subcitrate (CBS), together with antibiotics, such as amoxycillin and metronidazole [6,7]. A complete cure does not always follow such therapy and resistant strains may develop leading to relapse [8 16]. The antibacterial activity of several plant extracts have been tested against H. pylori. In 1996, Tabak et al. [17] showed that aqueous extracts of thyme and alcoholic extracts of cinnamon were effective against H. pylori. Earlier, in 1994, Diker and coworker [18]
* Corresponding author. Tel.: + 1-703-292-8000; fax: + 1-703-2929232. E -mail address: colwell@umbi.umd.edu (R.R. Colwell).

showed that extracts of both black and green tea had bactericidal activity against H. pylori within 5 min of exposure. Since black myrobalan powder is used in traditional medicine in southern and central parts of Iran as a remedy for human gastritis and peptic ulcers, we have looked at the antibacterial activity of black myrobalan powder and an extract against H. pylori and other pathogenic bacteria.

2. Materials and methods

2.1. Preparation and e6aluation of extracts

The dried powdered fruit of black myrobalan (10 g), was extracted by mechanical maceration with water, petroleum ether or ethanol at 35 C for 24 h. The extracted liquids were ltered and the ltrates concentrated under vacuum, followed by drying (40 C). Paper discs, 6 mm diameter (BBL, Becton Dickinson, Cockeysville, MD) were impregnated with selected con-

0924-8579/01/$20 2001 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S 0 9 2 4 - 8 5 7 9 ( 0 1 ) 0 0 3 5 2 - 1

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F. Malekzadeh et al. / International Journal of Antimicrobial Agents 18 (2001) 85 88

centrations of the extracts. The discs were dried and the amount of each extract in each disc was calculated. The agar diffusion method used Columbia Agar with 5% added debrinated sheep blood and incubation of the inoculated plates under microaerophilic conditions. Discs were placed on plates on which 0.1 ml of a suspension of H. pylori in saline phosphate buffer (108 109 cfu/ml) had been spread.

2.2. Bacterial isolates

Ten clinical isolates of H. pylori were obtained from the Firoozabadi Hospital in Tehran, Iran. Identication was by colony morphology, Gram staining, microaerophilic growth (at 37 C), oxidase, catalase, urease, nitrate, H2S and hippurate hydrolysis tests and nalidixic sensitivity [19,20].

Vibrio cholerae 01, clinical isolates from Bangladesh; Salmonella typhimurium from the US Department of Agriculture, Beltsville, Maryland; Vibrio 6ulnicus, an isolate from seafood; Ralstonia pickettii ATCC 27512; and Bre6undimonas diminuta ATCC 19146). H. pylori NCTC RSB6 and 33098 were grown on blood agar plates for 3 days under microaerophilic conditions. Suspensions of the cultures were prepared in saline phosphate buffer (ltered and autoclaved). Fresh blood agar plates were seeded with 0.1 ml of the suspensions (108 109 cells per ml). All other bacterial species were grown on TSA and/ or blood agar incubated at 37C for 24 h, after which suspensions were prepared and adjusted to 0.1 OD Plates were lawned with 0.1 ml of the suspensions. Black myrobalan antibacterial activity against the set of 11 strains was evaluated, using the methods described above.

2.3. Experimental procedures

Three extract-impregnated discs were placed on each of the lawned plates and a disc saturated in distilled water served as control. Two lawned plates, without discs, were also included as additional controls. All plates were incubated under microaerophilic conditions for 72 h at 37 C, after which the diameter of the inhibition zone appearing around each disc was recorded. The same procedures were used to test strains of nine other bacterial species; these were also incubated at 37 C for 48 h. The MICs were determined by adding selected concentrations of autoclaved extracts and black myrobalan powder (0 400 mg/ml) to media heated to 50 C, before inoculation with H. pylori suspensions. MBCs were established by lack of growth upon reinoculation from extract-treated plates to Columbia Agar plates. 3. Results and Discussion As shown in Table 1, aqueous extracts of black myrobalan were signicantly more active than other extracts. There was no signicant difference between isolates in sensitivity to the extracts. The aqueous extract preserved its antibacterial activity after autoclaving for 30 min at 121C and was inhibitory at 125 150 mg/l. When the plant powder was tested directly against H. pylori, without grinding and/or extraction and using Colombia Agar plates, the MIC and MBC values were 150 and 175 mg/l, respectively, (Table 2). Aqueous extracts of black myrobalan at a concentration of 200 mg/l in saline phosphate buffer, inhibited growth of H. pylori after 3 h exposure and viable cells could not be detected after incubation for 10 days on Columbia Agar plates. In addition to inhibition of the
Table 1 Antibacterial effect of black myrobalan (T. chebula Retz) extracts tested against ten clinical isolates of H. pylori Isolate number Diameter of inhibition zone (mm)a Aqueous extract 1 2 3 4 5 7 8 9 10 20 21 21 21 23 21 21 26 22 Petroleum ether extract 10 11 10 10 12 12 10 10 10 Ethanol extract

2.4. Determination of urease acti6ity assay

Urease activity was measured by the method of Mobley et al. [20] and Hamilton-Miller and Gargan [21]. Briey, a bacterial suspension (50 100 ml) was added to a cuvette containing 0.25 1.0 ml of a 3 mmol phosphate buffer solution (PBS) (pH 6.8), 0.1 ml of phenol red (7 mg/ml), and 0.4 ml of urease (330 mg/ml). Color intensity was measured at different time intervals using a spectrophotometer (Shumatzu, Japan) at 560 nm. Additional tests were performed to evaluate the antibacterial activity of the water extract of black myrobalan against two strains of H. pylori (NCTC RSB6 and NCTC 33098) and nine strains of other pathogenic bacterial species (Escherichia coli strain 735, a clinical isolate from London, England; Pseudomonas aeruginosa ATCC 43495; E. coli K-12; Shigella dysenteriae and

16 19 18 17 15 20 21 21 15

a Diameter of the discs, each containing 400 mg of the extract, was 6 mm.

F. Malekzadeh et al. / International Journal of Antimicrobial Agents 18 (2001) 85 88 Table 2 Comparison of inhibition of growth of H. pylori by black myrobalan (T. chebula Retz) water extracts and powder (mg/l, measured in%) Strain number Control Concentration 25 1 5 9 10 Average 100 100 100 100 100 85/90 80/90 80/90 75/90 80/90 40 70/75 70/70 60/80 60/70 65/75 75 50/60 50/50 35/55 45/55 45 100 25/40 30/30 10/35 15/35 20/35 125 10/25 10/15 /20 /20 5/20 150 /10 /10 /10 / /5 175 / / / / /

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Values above the diagonal line indicate percent growth on aqueous extract and values below the line indicate percent growth on powder.

growth of H. pylori, the urease activity of the cultures was also reduced. With increasing concentrations of the extract (1.5 and 2.5 mg/ml), urease activity was decreased by 24 and 67%, respectively, within 60 min (data not shown). When aqueous extracts of black myrobalan were tested against other Gram-negative bacteria, six of 11 strains were weakly sensitive and four were very sensitive, while one was resistant (Table 3). The aqueous extract of Black myrobalan (Terminalia chebula Retz) has been shown to have uniform antibacterial activity against ten clinical strains of H. pylori, (Table 1) This activity was bactericidal after 3 h and was stable after autoclaving. Although Sato and coworkers [22] reported gallic acid and ethyl gallate in T. chebula Retz and have shown antibacterial activity of ethanol extracts of this plant against both methicillin resistant and sensitive Staphylococcus aureus and other bacteria, the components of T. chebula Retz aqueous extracts responsible for the observed bacteriocidal activity remain unknown. The antibacterial activity of aqueous extracts of black myrobalan against bacteria other than H. pylori was also demonstrated. Six of nine strains showed
Table 3 Results of agar diffusion tests to evaluate the antibacterial activity of water extracts of black myrobalan (T. chebula Retz) against H. pylori and other pathogenic bacteria Bacterial species E. coli K-12 V. cholerae V. 6ulnicus P. aeruginosa B. diminuta R. pickettii S. dysenteriae S. typhimurium E. coli 735 H. pylori RSB6 H. pylori 33098 Inhibitiona + + + +++ +++ + + + +++ +++ Growth + + + + + + + + + + +

moderate sensitivity, but the extract did not inhibit growth of P. aeruginosa. B. diminuta, R. pickettii, and H. pylori proved to be the most sensitive of the strains tested, with inhibition zones of 26 30 mm diameter. It is concluded that the traditional Iranian folk medicinal use of black myrobalan powder to treat gastric infections is substantiated by the antibacterial activity of its extracts against H. pylori.

Acknowledgements This work was supported, in part, by a grant from the University of Tehran, Tehran, Iran and by Ofce of Naval Research Grant No. 00014-95-1-1250. We thank Dr Cecil Felkner for helpful discussions.

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