Bactericidal activity of medicinal plants, employed for the treatment of gastrointestinal ailments, against Helicobacter pylori

Journal of Ethnopharmacology 121 (2009) 286–291

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Bactericidal activity of medicinal plants, employed for the treatment of gastrointestinal ailments, against Helicobacter pylori Syed Faisal Haider Zaidi a,b , Kazuki Yamada b , Makoto Kadowaki a , Khan Usmanghani c , Toshiro Sugiyama b,∗ a b c

Division of Gastrointestinal Pathophysiology, Institute of Natural Medicine, University of Toyama, Toyama 930-0194, Japan Department of Gastroenterology and Hematology, Faculty of Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan Department of Basic Clinical Sciences, Faculty of Eastern Medicine, Hamdard University, Karachi 74600, Pakistan

a r t i c l e

i n f o

Article history: Received 15 June 2008 Received in revised form 24 September 2008 Accepted 1 November 2008 Available online 8 November 2008 Keywords: Anti-Helicobacter pylori activity Pakistani herbs Mallotus phillipinensis Minimum bactericidal concentration

a b s t r a c t Aim of the study: Helicobacter pylori infection plays a crucial role in the pathogenesis of peptic ulcer, and gastric cancer. The current PPI-based triple regimens for the eradication of Helicobacter pylori faces uprising resistance problem demanding for the search of novel candidates. Medicinal plants have always been a source of lead compounds for drug discovery. In the present study, we evaluated the anti-Helicobacter pylori activity of 50 commonly used Unani (traditional) medicine plants from Pakistan that are extensively utilized for the cure of gastrointestinal disorders to explore the natural source for pilot compounds against Helicobacter pylori. Materials and methods: Total seven clinical isolates and one standard strain were employed to examine the bactericidal effects of medicinal plants. Helicobacter pylori was isolated from the antral biopsy specimens and confirmed through the standard microbiology procedures. Minimum bactericidal concentration (MBC) of the active plants was determined at the concentration range from 7.8 to 500 ␮g/ml. Results: Among the herbs evaluated, more than 50% inhibited the growth of eight strains at the concentration of 500 ␮g/ml. The 70% aqueous-ethanol extracts of Curcuma amada Roxb., Mallotus phillipinesis (Lam) Muell., Myrisctica fragrans Houtt., and Psoralea corylifolia L. demonstrated strong anti-Helicobacter pylori activity with MBC value ranged from 15.6 to 62.5 ␮g/ml. The most potent bactericidal activity was exhibited by Mallotus phillipinesis (Lam) Muell. which completely killed the bacteria at the concentration of 15.6–31.2 ␮g/ml. Conclusion: The results revealed significant anti-Helicobacter pylori activity of medicinal plants which could be the potential source of new bactericidal agents. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Helicobacter pylori, a Gram-negative bacterium, is one of the most widespread infections in human worldwide that persistently infects up to 50% of the world’s population (Suerbaum and Michetti, 2002). Infections have been reported to be higher in the developing than in the developed countries with up to almost 100% of the population being infected in some developing nations (Marshal and Gilman, 1999). The infection is associated with atrophic gastritis, duodenal ulcer or gastric ulcer, and gastric adenocarcinoma at a later stage (Covacci et al., 1999). Eradication of the organism has been shown to result in ulcer healing, prevention of peptic ulcer recurrence and may also reduce the prevalence of

∗ Corresponding author. Tel.: +81 76 434 7300; fax: +81 76 434 5027. E-mail address: [email protected] (T. Sugiyama). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.11.001

gastric cancer in high-risk populations (Sepulveda and Coelho, 2002). Several short course drug regimens have been prescribed for the eradication of Helicobacter pylori with different combinations of therapeutic agents such as antibiotics, bismuth subsalicylate, proton pump inhibitors and H2 -blockers (Hentschel et al., 1993). However, emerging resistance to antibiotics, especially clarithromycin and metronidazole limits their use in the treatment of infections in developed and more in developing countries (Sullivan et al., 1990; Lind et al., 1999; Sherif et al., 2004). Furthermore, undesirable side effects of the drugs and the significant cost of combination therapy, require the exclusive need to search alternative approaches of eradicating or preventing infections (O’Gara et al., 2000). Phytomedicine has proved to be an untapped treasure for the discovery of model compounds to cure different diseases including gastrointestinal (GI) disorders (Thompson and Ernst, 2002). Hence, several studies have been focused to evaluate the anti-Helicobacter

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pylori effects of traditional herbal medicines from various parts of the world including Japan, Korea, Turkey, Taiwan, Cameron, etc. with promising bactericidal activities against Helicobacter pylori (Bae et al., 1998; Yesilada et al., 1999; Shin et al., 2004; Wang and Huang, 2005; Ndip et al., 2007). To the best of our knowledge, there is no report on the screening of herbal medicines, used for gastrointestinal disorders, against Helicobacter pylori from South-Asian countries like India, Pakistan, and Bangladesh where traditional medicine is extensively employed in daily life for curative, preventive and promotive health care design. Thus, in the present study we examined anti-Helicobacter pylori activities of 50 Unani medicinal plants from Pakistan which are widely used for the treatment of GI diseases in South-Asian countries. Aqueous-ethanol extract was obtained through plant extraction and minimum bactericidal concentrations were determined. 2. Materials and methods 2.1. Unani medicinal plants and extracts preparation Medicinal herbs used in this study are given in Table 1 with plant abbreviations, family, part used, therapeutic applications and % yield. Total 50 plants were purchased from local herbal market at Karachi, Pakistan. They were identified by Dr. Khan Usmanghani, Department of Basic Clinical Sciences, Faculty of Eastern Medicine, Hamdard University, Karachi, Pakistan. Medicinal plants were selected on the basis of their traditional use for the treatment of GI disorders (Usmanghani et al., 1997, 2005, 2006). Voucher authentic specimens have been deposited in the Museum of Materia Medica, Analytical Research Center for Ethnomedicines, Institute of Natural Medicine, University of Toyama, Toyama, Japan (see Table 1). Curcumin was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). The plants were air dried for at least one week and ground to fine powder. 70% ethanol was employed as a solvent for extraction of all herbs sample. Powdered plant material, 5–50 g, was sonicated twice with 50–100 ml of aqueous-ethanol for 48 h at room temperature. The slurry was then filtered using filter paper and the solvent was evaporated under reduced pressure. Plants extracts were stocked at 4 ◦ C prior to use. Each extract was dissolved at 100 mg/ml with dimethyl sulfoxide (DMSO) and diluted with brucella broth (BBLTM ; BD, Franklin Lakes, USA) liquid medium. Final concentration of DMSO was ≤1% in the mixture. Extraction yield (%, w/w) was calculated as the ratio of the weight of the extract to the weight of the crude herb powder. 2.2. Bacterial strains and culture conditions In the present study, seven clinical isolates of Helicobacter pylori (numbered PP-001, PP-002, PP-003, PP-005, PP-006, PP-007, and PP-010) were used from antral biopsies of Pakistani residents living in Toyama, Japan after informed consent was obtained and ethical approval from the hospital’s management board. One reference bacteria (ATCC 43504) was used as a standard strain of Helicobacter pylori. All clinical isolates were provided by the Division of Endoscopy, Toyama University Hospital (Toyama, Japan) and submitted to the Department of Gastroenterology and Hematology into seed tube (EIKEN, Tokyo, Japan) immediately. Three Japanese Helicobacter pylori strains (159A, 198C, and 696C) were also used from biopsies of Japanese patients visiting the hospital. Primary isolation was performed on commercial selective Pylori agar plates (Kyokuto, Tokyo, Japan) after incubation at 37 ◦ C, under humid microaerophilic condition (5% O2 , 10% CO2 , and 85% N2 at 37 ◦ C;

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Sanyo-Multigas Incubator, SANYO Electric Co., Ltd. Tokyo, Japan) for 3–7 days. Following primary selective isolation, Helicobacter pylori strains were identified by usual diagnostic procedures, i.e. according to colony morphology, Giemsa-staining, and positive oxidase, urease, and catalase reactions (Sugiyama et al., 1991). All Helicobacter pylori strains were subcultured in 10-ml brucella broth liquid culture with 10% FBS for 24–48 h under microaerophilic conditions on a gyratory shaker at 160 rpm with 100% humidity. The concentration of bacteria was estimated by using the formula as an absorbance of 0.1 = 108 bacteria/ml (Meyer et al., 2000). 2.3. Determination of minimum bactericidal concentration (MBC) For initial screening, plant extracts with the concentration of 500 ␮g/ml were used against seven clinical isolates and one reference Helicobacter pylori strain ATCC 43504. Extracts that showed 100% inhibition (no visual growth of bacterial colonies compared to control) at 500 ␮g/ml were further evaluated for MBC values with two fold dilutions. Final concentrations of each extract were 500, 250, 125, 62.5, 31.2, 15.6, 7.8 ␮g/ml in the mixture. Bactericidal activity of the plant extracts was determined by the method described earlier (O’Mahony et al., 2005) with minor modifications. Briefly, 100 ␮l of a suspension of 108 bacteria/ml was added to 900 ␮l of plant extract with different concentrations for 60 min. After incubation, 100 ␮l of this mixture was spread onto commercial selective Pylori agar plates under the conditions as presented above and the colonies formed were subsequently enumerated. For quality control and comparative analysis, the antibiotic amoxicillin (Sigma) was used as a positive control and DMSO as a negative control with each batch of plant extracts. All experiments were performed in duplicate. The MBC was defined as the minimum concentration of the test sample at which there was no visible growth of bacterial colonies at the latter cultivation. 3. Results 3.1. Extraction yield The % yield of extracts was calculated as explained in materials and methods. The herbal extracts from the 70% aqueous-ethanol exhibited a wide range of yield from 1.02% to 50.13% (w/w) (Table 1). A total of 13 herbs were found to have extraction yields greater than 10% including 5 herbs CML, CME, PDL, SAL, and TCR with more than 20% yield. On the other hand, a yield of less than 5% was observed for 14 herbs namely ASR, BADC, BNK, CCB, CSL, CRR, CVP, ECM, FRL, FVM, OML, PAL, PICL, and PNL. Of these herbs, Brassica nigra Koch. (BNK) exhibited least extraction yield of 1.02%. The maximum yield was obtained from the dried fruits of Prunus domestica L. (PDL) with 50.13%. The % yield differs dramatically in the two closely attached parts of the same plant Myristica fragrans Houtt., wherein seed yield (14.2%) is almost double of that of aril (7.6%). 3.2. Minimum bactericidal concentration values The MBC values for the ethanol extracts of 50 herbs against seven clinical isolates (PP-001, PP-002, PP-003, PP-005, PP-006, PP-007, and PP-010) and one reference Helicobacter pylori strain ATCC 43504 are given in Table 2. All these isolates were obtained from Pakistani residents living in Toyama and were diagnosed to have chronic gastritis. Among the evaluated herbs, 13 extracts (ASR, BADC, BNK, CTN, CML, CRR, MACL, PBL, PDL, RGL, SAL, TDR, and TCR) were solely inactive against all the strains tested after 60 min of incubation. Thirty plants including AGW, AGL, AAL, CME, CCL, CAR, CCR, CLL, CVP, ECM, FRL, FVM, GGL, MPM, MFHS, MFHA, NSL, ORW, OML, PPE, PAL, PICL,

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Table 1 List of Unani medicinal plants used in this study with % yield. Plant name

Abbreviation

Family

Voucher specimen number

Part used

Therapeutic application

% yield

Achillea millefolium L. Alpinia galangal Willd. Amomum subulatum Roxb. Anethum graveolens L. Artemisia absinthium L. Berberis aristata D.C. Brassica nigra (L.) Koch. Cinnamomum cassia Blume Cinnamomum tamala (Ham.) Nees Citrus medica L. Commiphora mukul Engler. Coriandrum sativum L. Cuminum cyminum L. Curcuma amada Roxb. Curcuma caesia Roxb. Curcuma longa L. Cuscuta reflexa Roxb. Cydonia vulgaris Pers. Eletteria cardamomum (L.) Maton Ficus religiosa L. Foeniculum vulgare Miller Glycyrrhiza glabra L. Mallotus philippinensis (Lam) Muell.

AML AGW ASR AGL AAL BADC BNK CCB CTN CML CME CSL CCL CAR CCR CLL CRR CVP ECM FRL FVM GGL MPM

Asteraceae Zingiberaceae Zingiberaceae Apiaceae Asteraceae Berberidaceae Cruciferae Lauraceae Lauraceae Rutaceae Burseraceae Apiaceae Apiaceae Zingiberaceae Zingiberaceae Zingiberaceae Convolvulaceae Rosaceae Zingiberaceae Moraceae Apiaceae Papilionaceae Euphorbiaceae

TMPW 25801 TMPW 25680 TMPW 25682 TMPW 25684 TMPW 25687 TMPW 25690 TMPW 25695 TMPW 25703 TMPW 25704 TMPW 25705 TMPW 25708 TMPW 25710 TMPW 25711 TMPW 25712 TMPW 25713 TMPW 25714 TMPW 25715 TMPW 25716 TMPW 25721 TMPW 25724 TMPW 25725 TMPW 25727 TMPW 25733

Carminative, inflammations Expectorant, carminative Flatulence, diarrhea, anorexia Carminative, diuretic Inflammation of liver and spleen, dyspepsia Fevers, inflammation, digestive, Meningitis, paralysis, carminative Dyspepsia, flatulence, vomiting Indigestion, stomach ache, cardiac tonic Dyspepsia, flatulence, vomiting Bleeding Piles, Ulcer, Inflammation Dyspepsia, flatulence, brain tonic Dyspepsia, chronic diarrhea Inflammation, analgesic, skin diseases Dyspepsia, stomach and liver tonic Inflammation, jaundice Ulcers, liver and spleen inflammations Tonic, gastric ulcers, sore throat Dyspepsia, flatulence, cardiac tonic Ulcer, Skin Diseases Indigestion, gastritis, flatulence Asthma, gastric ulcer Intestinal worms, skin diseases, purgative

9.38 9.02 2.87 7.88 5.41 2.58 1.02 1.72 11.48 29.82 24.46 4.82 6.20 7.15 7.89 6.38 2.71 2.60 4.65 2.40 4.40 10.03 8.84

Matricaria chamomilla L. Melilotus officinalis (L.) Desr. Mentha arvensis L. Myristica fragrans Houtt. Myristica fragrans Houtt. Myrtus communis L. Nigella sativa L. Oligochaeta ramosa (Roxb.) Wagenitz Origanum majorana L. Parmelia perlata Esch. Pimpinella anisum L. Piper cubeba L. Piper longum L. Piper nigrum L. Polygonum bistorta L. Prunus domestica L. Psoralea corylifolia L. Rosa damascena Miller Ruta graveolens L. Solanum nigrum L. Syzygium aromaticum L. Tamarix dioica Roxb. Terminalia chebula Retz. Thymus serpyllum L. Trachyspermum ammi (L.) Sprague Trigonella foenum-graecum L. Zanthoxylum armatum D.C.

MACL MOD MAL MFHS MFHA MYCL NSL ORW OML PPE PAL PICL PLL PNL PBL PDL PSCL RDM RGL SNL SAL TDR TCR TSL TAS TFGL ZADC

Asteraceae Papilionaceae Lamiaceae Myristacaceae Myristacaceae Myrtaceae Ranunculaceae Asteraceae Lamiaceae Parmeliaceae Apiaceae Piperaceae Piperaceae Piperaceae Polygonaceae Rosaceae Papilionaceae Rosaceae Rutaceae Solanaceae Myrtaceae Tamaricaceae Combretaceae Lamiaceae Apiaceae Papilionaceae Rutaceae

TMPW 25735 TMPW 25737 TMPW 25738 TMPW 25740 TMPW 25741 TMPW 25742 TMPW 25744 TMPW 25748 TMPW 25750 TMPW 25752 TMPW 25755 TMPW 25757 TMPW 25758 TMPW 25759 TMPW 25762 TMPW 25764 TMPW 25765 TMPW 25768 TMPW 25769 TMPW 25777 TMPW 25781 TMPW 25782 TMPW 25784 TMPW 25786 TMPW 25787 TMPW 25789 TMPW 25794

Aerial part Rhizome Fruit Seed Aerial part Root Seed Bark Leaf Fruit Gum Seed Seed Rhizome Root Rhizome Seed Fruit Fruit Bark Seed Root Powder covering fruit Flower Fruit Leaf Seed Aril Fruit Seed Aerial part Aerial part Lichen Seed Fruit Fruit Fruit Root Fruit Seed Flower Leaf Fruit Flower bud Aerial part Fruit Aerial part Seed Seed Seed

Stomach tonic, colic, epilepsy Liver, stomach and spleen inflammation Stomachache, fevers, dysmennorhea Flatulence, dyspepsia, nausea Stomach tonic, headache, migraine Diarrhoea, indigestion, palpitation Stomach and liver tonic, skin diseases Fevers, cough, diarrhea Flatulence, colic, dyspepsia Diarrhoea, dysentery, amenorrhoea Dyspepsia, flatulence, colic Carminative, stomach tonic, antiseptic Diarrhoea, dyspepsia, cold Dyspepsia, flatulence, sorethroat Dysentry, diarrhoea, haematuria Dyspepsia, nausea, vomiting Vitiligo, constipation, anthelmintic Stomach and intestine tonic, inflammation Indigestion, flatulence, rheumatism Inflammation, liver cirrhosis, gastritis Carminative, toothache, antiseptic Diarrhoea, dysentery, liver inflammation Stomach and brain tonic, diarrhoea Flatulence, anthelmintic, epilepsy Flatulence, colic, dyspepsia Carminative, resolvent Indigestion, flatulence, depression

18.27 8.80 19.32 14.20 7.62 12.92 5.10 9.05 3.97 8.63 3.28 4.16 6.33 2.82 8.90 50.13 12.62 6.37 5.30 7.20 22.49 11.89 27.92 5.26 5.68 8.01 8.71

PLL, PNL, PSCL, SNL, TSL, TAS, TFGL, and ZADC completely inhibited the growth of Helicobacter pylori at 500 ␮g/ml in all strains. At the concentration of 62.5 ␮g/ml, seven herbs namely CAR, CLL, MPM, MFHA, PPE, PICL, and PSCL showed no growth of colonies. Of these herbs, four extracts of Curcuma amada Roxb. (CAR), Mallotus phillipinesis (Lam) Muell. (MPM), Myrisctica fragrans Houtt. (MFHA), and Psoralea corylifolia L. (PSCL) exhibited potent anti-Helicobacter pylori activity with MBC value at 15.6–62.5 ␮g/ml. The strongest bactericidal activity was demonstrated by Mallotus phillipinesis (Lam) Muell. (MPM) which completely arrest the growth of Helicobacter pylori at the concentration of 15.6–31.2 ␮g/ml. Furthermore, we evaluated the four most active extracts (CAR, MPM, MFHA, and PSCL) against three Japanese Helicobacter pylori strains namely 159A, 198C, and 696C obtained from Japanese patients visiting hospital, diagnosed with gastric and duodenal ulcer. The MBC values of these four extracts in Japanese strains were almost similar to that of Pakistani strains which are given in Table 3.

4. Discussion Gastric cancer evolves through a multistep mechanism in which Helicobacter pylori is supposed to play a vital role (Chung et al., 2001; Sugiyama and Asaka, 2004). WHO has classified Helicobacter pylori as a class I carcinogen and the eradication of this silent killer with antibiotic combinations has been reported to be beneficial in preventing gastric ailments especially cancer (IARC, 1994). However, increasing dilemma of antibiotic resistance, adverse effects and high costing has lead researchers to explore natural resources especially plant materials as an alternative source of antimicrobials. Hence, in this study we investigated the anti-Helicobacter pylori activity of 50 medicinal plants from Pakistan which are frequently prescribed for GI disorders in Unani system of medicine. Among the 13 plants that were totally inactive against any strain at the concentration of 500 ␮g/ml, three (CCB, TCR, and SAL) had been examined previously with strong to moderate anti-Helicobacter pylori activity

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Table 2 MBC (␮g/ml) of ethanol extracts of Unani medicinal plants on each tested strain. Plant name

ATCC

PP-001

PP-002

PP-003

PP-005

PP-006

PP-007

PP-010

Achillea millefolium L. Alpinia galangal Willd. Amomum subulatum Roxb. Anethum graveolens L. Artemisia absinthium L. Berberis aristata D.C. Brassica nigra Koch. Cinnamomum cassia Blume Cinnamomum tamala Nees Citrus medica L. Commiphora mukul Engler. Coriandrum sativum L. Cuminum cyminum L. Curcuma amada Roxb. Curcuma caesia Roxb. Curcuma longa L. Cuscuta reflexa Roxb. Cydonia vulgaris Pers. Eletteria cardamomum Maton Ficus religiosa L. Foeniculum vulgare Miller Glycyrrhiza glabra L. Mallotus philippinensis Muell. Matricaria chamomilla L. Melilotus officinalis Desr. Mentha arvensis L. Myristica fragrans Houtt. (Seed) Myristica fragrans Houtt. (Aril) Myrtus communis L. Nigella sativa L. Oligochaeta ramosa Wagenitz Origanum majorana L. Parmelia perlata Esch. Pimpinella anisum L. Piper cubeba L. Piper longum L. Piper nigrum L. Polygonum bistorta L. Prunus domestica L. Psoralea corylifolia L. Rosa damascena Miller Ruta graveolens L. Solanum nigrum L. Syzygium aromaticum L. Tamarix dioica Roxb. Terminalia chebula Retz. Thymus serpyllum L. Trachyspermum ammi Sprague Trigonella foenum-graecum L. Zanthoxylum armatum D.C. Amoxicillin

500 500 >500 250 250 >500 >500 500 >500 >500 125 500 250 31.2 250 62.5 >500 250 125 250 500 250 31.2 >500 500 500 62.5 31.2 500 62.5 500 250 62.5 125 62.5 125 62.5 >500 >500 31.2 500 >500 250 >500 >500 >500 250 250 500 250 25.0

500 500 >500 250 250 >500 >500 500 >500 >500 125 500 500 62.5 250 62.5 >500 250 125 250 500 125 15.6 >500 500 500 125 31.2 >500 62.5 500 250 62.5 250 62.5 62.5 62.5 >500 >500 31.2 500 >500 250 >500 >500 >500 250 125 500 250 25.0

500 500 >500 250 125 >500 >500 >500 >500 >500 250 500 250 31.2 250 62.5 >500 250 125 125 500 125 15.6 >500 500 500 125 31.2 >500 62.5 500 250 62.5 250 62.5 125 62.5 >500 >500 31.2 500 >500 250 >500 >500 >500 250 125 500 250 12.5

500 500 >500 250 125 >500 >500 500 >500 >500 125 500 250 31.2 250 62.5 >500 125 125 125 500 125 15.6 >500 250 500 125 62.5 >500 62.5 500 250 62.5 250 62.5 125 62.5 >500 >500 31.2 500 >500 250 >500 >500 >500 250 125 250 250 12.5

500 500 >500 250 125 >500 >500 >500 >500 >500 125 500 500 31.2 250 62.5 >500 250 125 250 500 125 15.6 >500 500 500 125 31.2 >500 62.5 500 250 62.5 250 62.5 125 125 >500 >500 31.2 500 >500 500 >500 >500 >500 250 125 500 500 12.5

500 500 >500 250 125 >500 >500 500 >500 >500 125 500 500 31.2 250 62.5 >500 250 125 125 500 125 31.2 >500 500 500 125 31.2 >500 125 500 500 62.5 125 62.5 125 125 >500 >500 31.2 500 >500 500 >500 >500 >500 500 125 500 500 25.0

>500 500 >500 250 250 >500 >500 >500 >500 >500 250 >500 500 31.2 250 62.5 >500 250 125 250 500 125 15.6 >500 500 500 125 31.2 >500 62.5 500 250 62.5 250 62.5 125 125 >500 >500 62.5 500 >500 500 >500 >500 >500 250 125 500 250 25.0

500 500 >500 500 250 >500 >500 >500 >500 >500 125 >500 500 62.5 250 62.5 >500 250 125 250 500 250 15.6 >500 >500 >500 125 31.2 >500 62.5 500 500 62.5 500 62.5 125 125 >500 >500 31.2 >500 >500 500 >500 >500 >500 500 250 500 500 25.0

(Bae et al., 1998; Malekzadeh et al., 2001; Bhamarapravati et al., 2003). This disparity may be due to the difference in solvent used for extraction, the method of bioassay employed, and the variation in plant species (Ogundare et al., 2006; Ndip et al., 2007). After initial screening at 500 ␮g/ml, further MBC determination of the active plants revealed four herbs (CAR, MPM, MFHA, and PSCL) that exhibited stronger antibacterial activities as com-

pared to others. Out of these four herbs, Mallotus phillipinesis (Lam) Muell. (MPM) and Psoralea corylifolia L. (PSCL) are only used medicinally while Curcuma amada Roxb. (CAR) and Myrisctica fragrans Houtt. (MFHS) are utilized both as medicinal plant and culinary spice. Interestingly, MPM and PSCL are being commonly used as an anthelmintic in Unani system of medicine. Particularly MPM is employed extensively for this purpose and liquid preparations

Table 3 Comparative MBC (␮g/ml) analysis of most active extracts between Pakistani and Japanese strains. Plant name

CAR MPM MFHA PSCL Curcumin Amoxicillin

Pakistani strains

Japanese strains

PP-001

PP-002

PP-003

159A

198C

696C

62.5 15.6 31.2 31.2 50.0 25.0

31.2 15.6 31.2 31.2 50.0 12.5

31.2 15.6 62.5 31.2 25.0 12.5

31.2 31.2 31.2 31.2 25.0 25.0

15.6 15.6 31.2 15.6 25.0 12.5

15.6 15.6 31.2 31.2 25.0 25.0

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(Itrifal) are available in local markets to eradicate intestinal worms (Usmanghani et al., 1997). A previous report by Wang and Huang (2005) demonstrated the potent anti-Helicobacter pylori activity of Plumbago zeylanica, which is also employed for killing intestinal parasites in Taiwanese folk medicine. This correlation between anthelmintic medicinal plants and anti-Helicobacter pylori activity could be a trail for future screening of folk medicines in search for novel compounds against Helicobacter pylori. Previous study by Kumar et al. (2006) from India has demonstrated antibacterial activity of MPM on various organisms but there is no report on its anti-Helicobacter pylori activity till now. In our study, MPM (15.6–31.2 ␮g/ml) displayed the most potent bactericidal activity against all Helicobacter pylori strains which is comparable to amoxicillin MBC value (12.5–25.0 ␮g/ml). Evaluation of MPM chemical constituents against Helicobacter pylori growth is in progress which will be reported elsewhere. PSCL, in addition to its anthelmintic usage, is also helpful in relieving the complaints of constipation, piles, and anorexic conditions (Usmanghani et al., 1997). Yin et al. (2004) reported antibacterial activity of Psoralea corylifolia L. (PSCL) but so far no report has appeared on its anti-Helicobacter pylori activity. Our results clearly revealed the strong bactericidal effect of PSCL (31.2–62.5 ␮g/ml) which demand further analysis of its constituents for potent lead candidates against Helicobacter pylori. Curcuma species are widely known for their broad range of pharmacological activities. In Pakistan, three varieties of Curcuma species namely Curcuma amada Roxb. (CAR), Curcuma caesia Roxb. (CCR), and Curcuma longa L. (CLL) are available. CAR and CLL are commonly used as a spice in daily life while CCR is mainly employed for medicinal purposes especially as an alternate of turmeric or CLL (Usmanghani et al., 1997). Among these three species, CAR (31.2–62.5 ␮g/ml) and CLL (62.5 ␮g/ml) showed strong inhibition on Helicobacter pylori growth in all strains while CCR (250 ␮g/ml) exhibited weak bactericidal activity in contrast with the other two. Anti-Helicobacter pylori activity of CLL and its major polyphenolic chemical constituent, curcumin, has been documented earlier (Mahady et al., 2002) whereas, Siddaraju and Dharmesh (2007) recently reported the anti-Helicobacter pylori activity of CAR. However, no comparative study between the three species against Helicobacter pylori has been reported previously. Our results displayed the different bactericidal potential in the above mentioned species which might be due to the difference in antibacterial chemical constituents in them. The MBC value of curcumin revealed from our study is ranged from 25.0 to 50.0 ␮g/ml which is also comparable with amoxicillin. Similarly, two parts of Myristica fragrans Houtt. plant, seed (MFHS) and aril (MFHA) surrounding seed, are usually employed in both cuisine and traditional medicine. Both MFHS and MFHA are given to relieve flatulence, colic, gastrointestinal complaints like dyspepsia, diarrhoea, nausea and vomiting (Usmanghani et al., 1997; Azhar and Mazhar, 2003). Previously, two reports have revealed the anti-Helicobacter pylori activity of Myristica fragrans, one using seed (Mahady et al., 2005) and other aril (Bhamarapravati et al., 2003) with MIC value of 12.5 ␮g/ml. In our study, although both parts exhibited potent anti-Helicobacter pylori activity but aril of the seed (MBC 31.2–62.5 ␮g/ml) showed strong bactericidal activity than the seed itself (MBC 62.5–125 ␮g/ml). Likewise, the two types of cardamom spice namely Amomum subulatum Roxb (Black cardamom) and Eletteria cardamomum Maton (Green cardamom) are considered to be closely related species with almost similar name, usage (see Table 1) and belongs to the same family (Zingiberaceae) but anti-Helicobacter pylori activity differ quite prominently. The former showed no toxicity even at 500 ␮g/ml in all of the strains while later displayed complete inhibition of Helicobacter pylori growth at 125 ␮g/ml. Earlier, Mahady et al. (2005)

also reported antibacterial activity of Eletteria cardamomum against Helicobacter pylori with MIC value of 100 ␮g/ml. This is the first report on the comparative anti-Helicobacter pylori activities of the two types of cardamom against Helicobacter pylori and the differences in bactericidal activity between the two requires further research on the active constituents for possible new antimicrobials. Interestingly, green cardamom is employed traditionally more for dyspeptic symptoms than black cardamom. As Helicobacter pylori is considered to be one of the causative factors for dyspepsia, the comparatively strong bactericidal activity of green cardamom might explain its traditional use. Among the other active plant extracts, AAL, CME, FRL, GGL, NSL, PPE, PAL, PICL, PLL, PNL, TAS demonstrated appreciable bactericidal activities with MBC value range 62.5–250 ␮g/ml in any of the strains evaluated. Of them, six plants namely AAL, CME, FRL, PPE, PICL, and TAS are reported for the first time for their anti-Helicobacter pylori activity. Regarding GGL, NSL, PLL, and PNL, an earlier report by O’Mahony et al. (2005) reported the strong anti-Helicobacter pylori activity for the former three but no bactericidal activity of PNL was demonstrated in % inhibition. In our study, PNL was bactericidal at the MBC range of 62.5–125 ␮g/ml. This discrepancy might be due to the difference in the extraction procedure and the solvent used in the two studies. Additionally, Fukai et al. (2002) and Krausse et al. (2004) also reported the potent anti-Helicobacter pylori activity of GGL and its chemical constituents even against clarithromycin, amoxicillin, and metronidazole resistant strains. O’Mahony et al. (2005) further showed no bactericidal activity of CSL and TFGL in their study which is similar with our results, both possessing MBC value of ≥500 ␮g/ml in almost all of the strains. Mahady et al. (2005) reported in vitro anti-Helicobacter pylori effect of PAL with MIC value of 100 ␮g/ml and our results also revealed similar activity of PAL with MBC value range of 125–250 ␮g/ml in most of the strains. The same study further demonstrated the inhibitory activity of AML, FVM, and OML with MIC value of 50 ␮g/ml while that of CCL with 100 ␮g/ml. The results obtained from our study exhibited weak bactericidal activity of AML and FVM with MBC value of 500 ␮g/ml while OML and CCL has MBC value of 250–500 ␮g/ml. Moreover, Stamatis et al. (2003) also documented the inhibitory activity of OML (MIC; 0.625–5.00 mg/ml) while no activity was seen with FVM even at >5.00 mg/ml. One of the most striking features of Helicobacter pylori is its great genetic diversity and a high degree of interstrain genetic variation is observed in the Helicobacter pylori population worldwide (Reuse and Bereswill, 2007). To assess whether these DNA disparities can affect the bactericidal activity of plant extracts, we compared the MBC values of the four most active extracts (CAR, MPM, MFHA, and PSCL) against three Japanese Helicobacter pylori strains. The results obtained revealed no such difference in the MBC values between Pakistani and Japanese strains which further strengthen the antiHelicobacter pylori activities of these extracts even in geographically diverged strains. Although, eradication of Helicobacter pylori by ingestion of these medicinal plants is controversial in clinical settings (Graham et al., 1999) but it is plausible that they may repress the pathogenecity of Helicobacter pylori by various mechanisms. Curcumin and capsaicin, isolated from plants, have been reported to suppress IL-8 expression and NF-␬B activation in gastric epithelial cells/Helicobacter pylori coculture experiments (Foryst-Ludwig et al., 2004; Lee et al., 2007). Furthermore, Paraschos et al. (2007) hypothesized that habitual long-term consumption of mastic gum extract may be effective in moderating Helicobacter pylori colonization in the stomach. Recently, a clinical study comprising of curcumin-based one week triple therapy failed to eradicate Helicobacter pylori, however, significant improvement of dyspeptic symptoms and reduction of gastric inflammatory response has been demonstrated even after

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two months of the therapy (Mario et al., 2007). Although authors of the above mentioned study hypothesized the activation of the vanilloid receptor type 1 (TRPV1) by curcumin for this prolonged beneficial effects, still there is a desperate need to explore the underlying mechanisms behind these propitious consequences of medicinal plants and their constituents. In conclusion, we reported herein potential in vitro antiHelicobacter pylori activity of medicinal plants from Pakistan that are used to cure GI disorders. As Helicobacter pylori is widely accepted to be the causative agent for numerous GI diseases including dyspepsia, the findings of our study may partially validate the use of these herbs in gastric diseases but further in vivo and clinical studies are required to scientifically justify the rationale of their traditional use. Moreover, these medicinal herbs may afford lead compounds which could be beneficial for future drug development especially against resistant Helicobacter pylori strains. References Azhar, I., Mazhar, F., 2003. Spices as Medicine. Research Institute of Indusyunic Medicine, Pakistan, p. 223. Bae, E.A., Han, M.J., Kim, N.J., Kim, D.H., 1998. Anti-Helicobacter pylori activity of herbal medicines. Biological and Pharmaceutical Bulletin 21, 990–992. Bhamarapravati, S., Pendland, S.L., Mahady, G.B., 2003. Extracts of spice and food plants from Thai traditional medicine inhibit the growth of the human carcinogen Helicobacter pylori. In vivo 17, 541–544. Chung, A.Y.F., Chow, P.K.H., Yu, W.K., Ho, J.M., Chan, H.S., Wong, W.K., Spp, K.C., 2001. Prevalence of Helicobacter pylori in gastric cancer in South-east Asian population by 14C-urea breath test. ANZ Journal of Surgery 71, 574–576. Covacci, A., Telford, J.L., Giudice, G.D., Parsonnet, J., Rappuoli, R., 1999. Helicobacter pylori virulence and genetic geography. Science 284, 1328–1333. Foryst-Ludwig, A., Neumann, M., Schneider-Brachert, W., Naumann, M., 2004. Curcumin blocks NF-␬B and the motogenic response in Helicobacter pylori-infected epithelial cells. Biochemical and Biophysical Research Communications 316, 1065–1072. Fukai, T., Marumo, A., Kaitou, K., Kanda, T., Terada, S., Nomura, T., 2002. AntiHelicobacter pylori flavonoids from licorice extract. Life Sciences 71, 1449–1463. Graham, D.Y., Anderson, S.Y., Lang, T., 1999. Garlic or jalapeno peppers for treatment of Helicobacter pylori infection. American Journal of Gastroenterology 94, 1200–1202. Hentschel, E., Brandstatter, G., Dragsics, B., Hirschil, A.M., Nemec, H., Schutze, K., Taufer, M., Wurzer, H., 1993. Effect of ranitidine and amoxicillin plus metronidazole on the eradication of Helicobacter pylori and the recurrence of duodenal ulcer. The New England Journal of Medicine 328, 308–312. IARC Working Group on the evaluation of carcinogenic risks to humans, Schistosomes, Liver flukes and Helicobater pylori. Infections with Helicobacter pylori 1994. IARC monographs on the evaluation of carcinogenic risks to humans. International Agency fro Research on Cancer, Lyon, France, pp 177–201. Krausse, R., Bielenberg, J., Blaschek, W., Ullmann, U., 2004. In vitro anti-Helicobacter pylori activity of Extractum liquiritiae, glycyrrhizin and its metabolites. Journal of Antimicrobial Chemotherapy 54, 243–246. Kumar, V.P., Chauhan, N.S., Padh, H., Rajani, M., 2006. Search for antibacterial and antifungal agents selected from Indian medicinal plants. Journal of Ethnopharmacology 107, 182–188. Lee, I.O., Lee, K.H., Pyo, J.H., Kim, J.H., Choi, Y.J., Lee, Y.C., 2007. Anti-inflammatory effect of capsaicin in Helicobacter pylori-infected gastric epithelial cells. Helicobacter 12, 510–517. Lind, T., Megraud, F., Unge, P., Bayerdorffer, E., O’morain, C., Spiller, R., et al., 1999. The MACH2 study: role of omeprazole in eradication of Helicobacter pylori with 1-week triple therapies. Gastroenterology 116, 248–253. Mahady, G.B., Pendland, S.L., Stoia, A., Hamill, F.A., Fabricant, D., Dietz, B.M., Chadwick, L.R., 2005. In vitro susceptibility of Helicobacter pylori to botanical extracts used traditionally for the treatment of gastrointestinal disorders. Phytotherapy Research 19, 988–991. Mahady, G.B., Pendland, S.L., Yun, G., Lu, Z.Z., 2002. Turmeric (Curcuma longa) and curcumin inhibit the growth of Helicobacter pylori, a group 1 carcinogen. Anticancer Research 22, 4179–4181. Malekzadeh, F., Ehsanifar, H., Shahamat, M., Levin, M., Colwell, R.R., 2001. Antibacterial activity of black myroblan (Terminalia chebula Retz) against Helicobacter pylori. International Journal of Antimicrobial Agents 18, 85–88.

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