Bactericidal and Anti-Adhesive Properties of Culinary and Medicinal Plants Against Helicobacter Pylori

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PO Box 2345, Beijing 100023, China World J Gastroenterol 2005;11(47):7499-7507 www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327 [email protected] © 2005 The WJG Press and Elsevier Inc. All rights reserved. ELSEVIER

• Helicobacter pylori • Bactericidal and anti-adhesive properties of culinary and medicinal plants against Helicobacter pylori

Rachel O’Mahony, Huda Al-Khtheeri, Deepaka Weerasekera, Neluka Fernando, Dino Vaira, John Holton, Christelle Basset

Rachel O’Mahony, Huda Al-Khtheeri, John Holton, Centre for CONCLUSION: Several plants that were tested in our Infectious Diseases and International Health, Royal Free and Uni- study had bactericidal and/or anti-adhesive effects on versity College London Medical School, London, H pylori . Ingestion of the plants with anti-adhesive United Kingdom properties could therefore provide a potent alternative Deepeka Weerasekera, Department of Surgery, University of therapy for H pylori infection, which overcomes the Sri Jayewardenapura, Colombo, Sri Lanka problem of resistance associated with current antibiotic Neluka Fernando, Department of Microbiology, University of Sri Jayewardenapura, Colombo, Sri Lanka treatment. Dino Vaira, Department of Internal Medicine and Gastroenterol- ogy, University of Bologna, Bologna, Italy © 2005 The WJG Press and Elsevier Inc. All rights reserved. Christelle Basset, INSERM 0114, Physiopathologie des Mala- dies Inﬂ ammatoires Intestinales, CHU Lille, France Key words: H pylori ; Inhibition; Adhesion; Killing; Plants Supported by the European Union on EC contract QLK2- CT-2001-01216 (ADRI) O’Mahony R, Al-Khtheeri H, Weerasekera D, Fernando Correspondence to: Rachel O’Mahony, Centre for Infectious N, Vaira D, Holton J, Basset C. Bactericidal and anti- Diseases and International Health, Royal Free and University Col- adhesive properties of culinary and medicinal plants against lege London Medical School, Windeyer Building, 46 Cleveland Helicobacter pylori . World J Gastroenterol 2005; 11(47): Street, London, W1P 6DB, 7499-7507 United Kingdom. Telephone: +44-20-76799485 http://www.wjgnet.com/1007-9327/11/7499.asp Received: 2005-06-02 Accepted: 2005-04-28

INTRODUCTION Abstract Over half the human population is colonized by AIM: To investigate the bactericidal and anti-adhesive Helicobacter pylori (H pylori), a Gram-negative, microaerophilic properties of 25 plants against Helicobacter pylori bacterium. If untreated, infection is usually life-long and (H pylori ). leads to chronic active disease[1]. Although most infected people are asymptomatic, 5-10% of those infected with METHODS: Twenty-fi ve plants were boiled in water to this bacterium develop severe gastroduodenal diseases, produce aqueous extracts that simulate the effect of including gastric and duodenal ulcers, gastric lymphomas, cooking. The bactericidal activity of the extracts was and gastric adenocarcinomas[2]. assessed by a standard kill-curve with seven strains of The current and most effective treatment for peptic H pylori . The anti-adhesive property was assessed by ulcer disease is a triple therapy regimen consisting of the inhibition of binding of four strains of FITC-labeled a proton pump inhibitor, such as omeprazole, and two H pylori to stomach sections. antibiotics, clarithromycin and either amoxicillin or metronidazole. However, there is an increase that disturbs RESULTS: Of all the plants tested, eight plants, due to the prevalence of antibiotic resistance, which is including Bengal quince, nightshade, garlic, dill, black high in some areas of the world. Metronidazole resistance pepper, coriander, fenugreek and black tea, were found is more common than clarithromycin, the latter particularly to have no bactericidal effect on any of the isolates. Columbo weed, long pepper, parsley, tarragon, nutmeg, having an adverse effect on the eradication rate. Second yellow-berried nightshade, threadstem carpetweed, line therapies following failure of one of the initial sage and cinnamon had bactericidal activities against regimes include triple and quadruple regimens containing H pylori , but total inhibition of growth was not achieved antibiotics, such as levofl oxacin or furazolidone, or even in this study. Among the plants that killed H pylori , regimens containing five agents. Although the use of turmeric was the most efficient, followed by cumin, molecular methods can rapidly detect antibiotic resistance ginger, chilli, borage, black caraway, oregano and and host polymorphisms, which may lead to reduced [3,4] liquorice. Moreover, extracts of turmeric, borage and effi cacy of treatment and thus eradication failure , this is parsley were able to inhibit the adhesion of H pylori not a long-term solution to the rising trend of antibiotic strains to the stomach sections. resistance. 7500 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol December 21, 2005 Volume 11 Number 47

Resistance to antibiotics is not limited to H pylori and Lanka, Iran, and the Middle East. Moreover, we have used has been an increasing problem for many years. There is an in situ adhesion assay[23], with stomach tissues expressing therefore a constant need for new antimicrobial agents and either the Lewis a (Le a) or Lewis b (Le b) antigen, in order novel approaches to treatment, ideally preventing disease, to determine whether the plant extracts inhibit adhesion such as inhibition of adhesion or vaccination[5]. Plants by blocking the major H pylori adhesin BabA (which binds are known to be the source of phytochemicals which are to Le b) or have an effect on other adhesins. beneficial for health and could also prevent diseases[6]. Among these phytochemicals, two are of particular interest MATERIALS AND METHODS in the case of infectious diseases: antimicrobial and anti- H pylori isolates adhesive agents. H pylori NCTC 11637 and six fresh clinical strains from Numerous studies have been undertaken in order to Italian patients with peptic ulcer disease [obtained by one find antimicrobial agents from plants against organisms [7] of the authors (DV)] were used in the study. All strains ranging from viruses to protozoa . The major concern is were stored on beads at -80 ℃ until use. Bacteria were the validation in human beings with well-designed clinical grown for 2 d on Columbia agar plates supplemented with trials, and this has also been true for H pylori infection. 5% horse blood (Oxoid, UK) at 37 ℃ under microaerobic Several in vitro studies have looked at the effect of plant conditions. The number of bacterial cells was determined extracts on H pylori. Anti-microbial effects have been [8,9] [10] [11] [12] by obtaining viable counts of serial dilutions and reported for garlic , green tea , honey , thyme , some measurement of the optical absorbance at 600 nm Iranian plants[13] and the essential oils from several species [13] (Ultrospec II, LKB, UK) of a suspension of bacterial of mint . Some of these studies have been validated in cells to prepare a standard curve for H pylori. All studies animals and confi rmed the potential benefi t of using plants thereafter used the same concentration of organism based as the source of anti-microbial agents against H pylori. upon its absorbance. Although garlic and cinnamon have been tested in human [14] clinical trials with no signifi cant effect , a recent study has Fluorescent labeling of H pylori isolates shown that consumption of broccoli sprouts is associated 9 [15] Suspensions of 1×10 cells/mL were prepared from 1 mL with the eradication of H pylori in some patients , but carbonate buffer (0.15 mol/L NaCl/0.1 mol/L Na2CO3, more work needs to be done in determining the active pH 9.0). Five microliters of a 10 mg/mL fluorescein ingredients of broccoli as well as performing studies on a isothiocyanate (FITC, Isomer I, Sigma, UK) solution in larger number of patients. dimethyl sulfoxide (DMSO, Sigma, UK) was added to The search for anti-adhesive agents represents the each bacterial suspension and then incubated for 1 h with second alternative to antibiotic therapy, which has received continuous shaking. The suspensions were then washed less interest, although studies in animals and human beings three times with phosphate buffered saline (PBS, 8.0 g/L [16] have proved its potential as a new therapy . Successful NaCl; 1.21 g/L K2HPO4; 0.34 g/L KH2PO4, pH 7.4) inhibition of adhesion has been shown in vitro with containing 0.5 g/L Tween 20 (PBST; Sigma, UK), and [17] cranberry juice against H pylori and for the seaweed resuspended in PBS. Labeling was confirmed by using [18] [19] Cladosiphon fucoidan . Recently, Shibata et al. have fl ow cytometry (FACSCalibur, Becton Dickinson, UK). All demonstrated that Cladosiphon fucoidan inhibits adhesion incubation and washing steps were carried out in the dark of H pylori to porcine gastric mucin. By adding the plant at room temperature. to the drinking water of infected Mongolian gerbils, the prevalence of animals with infection was shown to be Plants [20] markedly reduced. Lengsfeld et al. have shown that Twenty-five plants were obtained from different sources adhesion of H pylori to human stomach sections was almost and are described in Table 1. Extracts from the plants were completely inhibited by pre-incubating H pylori with a fresh prepared as follows: for turmeric, ginger, fenugreek, cumin, juice preparation of the fruit of the okra plant [Abelmoschus fennel, coriander, and chilli, plants were boiled to 100 ℃ in [21] esculentus (L.) Moench]. Lengsfeld et al. have also sterile distilled water (100 mg/mL), fi ltered through sterile demonstrated that acidic high molecular weight galactans gauze, neutralized to pH 7.0 and then sterilized. Extracts from blackcurrant seeds could inhibit adhesion of H pylori from all the other plants were prepared as follows: plants to human gastric mucosa tissue sections. Moreover, Lee were boiled in sterile distilled water (100 mg/mL) for 10 [22] et al. have demonstrated the inhibition of H pylori min, allowed to cool and fi ltered through sterile fi lter paper adhesion by polysaccharide fractions of Panax ginseng and (Grade 1, Whatman, UK). Fresh garlic, black peppercorns Artemisia capillaris to a human gastric adenocarcinoma and cinnamon sticks were finely chopped before being epithelial cell line. boiled. All extracts were stored in the dark at -20 ℃ until As mentioned earlier, many plants have been shown to use. Before the experiments of adhesion, plant extracts kill microorganisms but rarely have been studied for their were fi ltered again (Grade 1 fi lter paper, Whatman, UK). anti-adhesive properties. We have therefore investigated both the bactericidal and anti-adhesive properties of Stomach sections 25 plants against H pylori. Sixteen of them have never H pylori-negative biopsies of human stomach were been tested before against H pylori and are plants that are obtained by one of the authors (DV), with the consent of frequently used in cooking as well as in medicine in Sri the Ethics Committee, St Orsola Hospital, Bologna. Two O’Mahony R et al. Plants against H pylori 7501

Table 1 Plants used in the study

English name Sri Lankan name Scientiﬁ c name Part of the plant used Source Use

Bengal quince Belly Aegle marmelos Root Sri Lanka In cooking & medicine Black caraway (Europe)/Black Nigella sativa Seeds UK, Supermarket In cooking & medicine Cumin (USA) Black pepper Gammiris Piper nigrum Seed berries/fruit UK, Supermarket In cooking & medicine Black tea Thee Camellia sinensis Leaves and shoots Sri Lanka In cooking Borage (Starfl ower) Borago offi cinalis Flowers Iran In cooking & medicine Chilli Mirise Capsicum anunum Fruit Sri Lanka In cooking & medicine Cinnamon Curundu Cinnamomum verum Bark UK, Supermarket In cooking & medicine Columbo weed Veneval Coscinium fenestratum climbing root Sri Lanka In medicine Coriander Kottamalli Coriandrum sativum Seeds Sri Lanka In cooking & medicine Cumin (small cumin) Suduru Cuminum cyminum Seeds Sri Lanka In cooking & medicine Dilla Sududuru Anthum graveolens Leaves UK, Supermarket In cooking & medicine Fenugreek Mathe seeds Trigonella foenum-graecum Seeds Sri Lanka In cooking & medicine Garlica Sudulunu Allium sativum Bulb UK, Supermarket In cooking & medicine Ginger E'guru Zingiber offi cinale Rhizome Sri Lanka In cooking & medicine Liquorice Val'mee Glycyrrhiza glabra apofosa Stem Sri Lanka In cooking & medicine Long pepper Tiphili Piper longum Seeds Sri Lanka In cooking & medicine Nightshade Ela battu Solanum surattense Fruit and root Sri Lanka In medicine Nutmeg Sadikka Myristica fragans Kernel UK, Supermarket In cooking & medicine Oreganoa Oregano Origanum vulgare Leaves UK, Supermarket In cooking & medicine Parsleya Parsley Petroselinum crispum Leaves UK, Supermarket In cooking & medicine Sagea Minchi Salvia offi cinalis Leaves UK, Supermarket In cooking & medicine Tarragona Tarragon Artemisia dracunculoses/ Leaves UK, Supermarket In cooking & medicine Path dracunculus Threadstem carpetweed Paradagam Mollugo cerviana Seeds Sri Lanka In medicine Turmeric Kaha Curcuma longa Rhizome Sri Lanka In cooking & medicine Yellow-berried nightshade Katu val batu Solanum xanthocarpum Whole plant Sri Lanka In medicine

These plants were bought as both fresh and dried material. sets of formalin-ﬁ xed stomach biopsies were used: those These were incubated in gas jars under microaerophilic whose epithelial cells expressed the Lewis a blood group conditions using CampyPak (Oxoid, UK) for 3 d, and antigen (Le a stomach) and those expressing the Lewis colonies were counted (colony forming units per milliliter, b blood group antigen (Le b stomach). Five-micrometer CFU/mL). Plant extracts that killed 100% of H pylori thick sections of the stomach were cut using a Leica cells at 60 min (i.e. no colonies grew) were further tested SM2400 rocking microtome. Sections were collected at 0-, 15-, 30-, and 60-min intervals. All experiments were on polished glass slides coated with Vectabond (Vector performed three times. The effectiveness of the plants at Laboratories, UK). An antigen retrieval step was used by killing H pylori was expressed as percentage inhibition of boiling for 5 min in an 800-W microwave in plastic Coplin colony growth (i.e. percentage of bacteria killed) compared jars containing 15 mL of citrate buffer (pH 6.0, 100 mol/L to the control. sodium citrate tribasic dihydrate, Sigma, UK). The Lewis phenotype of the tissue was determined by standard Microscopic examination immunohistochemical staining using the DAKO EnVision In order to validate the inhibition of adhesion by the plant Plus HRP kit (DAKO, UK) with anti-Le a and anti-Le b extracts, any lytic effect of the extracts on the bacterial antibodies generously donated by Dr. J Bara, Hôpital St- cells was first examined. FITC-labelled and non-labelled Antoine, Paris, France. Both antibodies were supplied at bacteria (1×108 cells/mL) were centrifuged (9 000 g for a concentration between 1 and 5 μg/mL and diluted 1:10 3 min), re-suspended in 500 μL of undiluted plant extract for the anti-Le a antibodies and 1:50 for the anti-Le b (100 mg/mL) and incubated with continuous shaking for antibodies. 1 h at room temperature in the dark. After incubation, bacteria were centrifuged, washed in PBST and re- Viable colony count suspended in 500 μL sterile distilled water. One drop of Bactericidal activity of the plant extracts was determined the bacterial suspension was Gram stained. Using a Zeiss by a viable colony count. One hundred microliters of light microscope, the slides were observed for bacterial a suspension of 108 bacteria/mL was added to 900 μL lysis. All the plant extracts were examined, except long of plant extract for 60 min. The control consisted of pepper and threadstem carpetweed, which were not tested H pylori incubated with sterile distilled water. Serial due to their limited supply. The negative control consisted 10-fold dilutions were made, and 100 μL of each dilution of H pylori incubated with sterile distilled water and the was plated onto 50 g/L horse blood agar (Oxoid, UK). positive control of H pylori incubated with Puregene PCR 7502 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol December 21, 2005 Volume 11 Number 47

Table 2 Bactericidal activity of plants against Helicobacter pylori, determined using viable colony count. ‘% inhibition of growth’ indicates the percentage of bacteria that were killed by the plants, (i.e. the colonies did not grow), compared to the control.

H pylori clinical strains Plant name NCTC strain 1 2 3 4 5 6

Turmeric % inhibition of growth 100 100 100 100 100 100 100 Minimum of time (min) 15 15 15 15 15 15 15 Ginger % inhibition of growth 100 100 100 100 100 100 100 Minimum of time (min) 30 30 30 30 30 30 30 Fenugreek % inhibition of growth 0 0 0 0 0 0 0 Minimum of time (min) 60 60 60 60 60 60 60 Cumin (small cumin) % inhibition of growth 100 100 100 100 100 100 100 Minimum of time (min) 30 30 30 30 30 30 30 Coriander % inhibition of growth 0 0 0 0 0 0 0 Minimum of time (min) 60 60 60 60 60 60 60 Chilli % inhibition of growth 100 100 100 100 100 100 100 Minimum of time (min) 30 30 30 30 30 30 30 Bengal quince % inhibition of growth 0 0 0 0 0 0 0 Minimum of time (min) 60 60 60 60 60 60 60 Columbo weed % inhibition of growth 99.9 99.9 99.9 99.5 99.8 99.9 99.8 Minimum of time (min) 60 60 60 60 60 60 60 Yellow-berried nightshade % inhibition of growth 99.9 99.7 99.5 99.9 99.9 99.9 99.9 Minimum of time (min) 60 60 60 60 60 60 60 Liquorice % inhibition of growth 100 100 100 100 100 100 100 Minimum of time (min) 60 60 60 60 60 60 30 Nightshade % inhibition of growth 0 0 0 0 0 0 0 Minimum of time (min) 60 60 60 60 60 60 60 Long pepper % inhibition of growth 99.9 99.9 100 99.3 99.7 99.9 99.9 Minimum of time (min) 60 60 60 60 60 60 60 Threadstem carpetweed % inhibition of growth 99.9 99.7 99.9 99.9 99.9 99.9 99.9 Minimum of time (min) 60 60 60 60 60 60 60 Garlic % inhibition of growth 0 0 0 0 0 0 0 Minimum of time (min) 60 60 60 60 60 60 60 Parsley % inhibition of growth 99.6 99.3 99.9 99.6 99.9 99.99 99.8 Minimum of time (min) 60 60 60 60 60 60 60 Dill % inhibition of growth 0 0 0 0 0 0 0 Minimum of time (min) 60 60 60 60 60 60 60 Sage % inhibition of growth 99.9 99.9 99.7 99.5 99.9 99.9 99.9 Minimum of time (min) 60 60 60 60 60 60 60 Oregano % inhibition of growth 100 100 100 100 100 100 100 Minimum of time (min) 30 30 30 30 60 30 30 Tarragon % inhibition of growth 96.9 99.4 99.2 99.9 99.9 99.9 99.9 Minimum of time (min) 60 60 60 60 60 60 60 Nutmeg % inhibition of growth 95.9 95.6 99.5 99.4 99.2 96.1 98.9 Minimum of time (min) 60 60 60 60 60 60 60 Black pepper % inhibition of growth 0 0 0 0 0 0 0 Minimum of time (min) 60 60 60 60 60 60 60 Cinnamon % inhibition of growth 100 100 100 100 99.9 99.9 100 Minimum of time (min) 60 30 30 30 60 60 60 Black caraway (Europe)/ % inhibition of growth 100 100 100 100 100 100 100 Black cumin (USA) Minimum of time (min) 60 60 60 60 60 60 60 Borage (Starﬂ ower) % inhibition of growth 100 100 100 100 100 100 100 Minimum of time (min) 60 60 60 60 60 60 60 Black tea % inhibition of growth 0 0 0 0 0 0 0 Minimum of time (min) 60 60 60 60 60 60 60 cell lysis solution (Flowgen, UK). The lysis experiments atmosphere with 200 μL of blocking buffer (PBST+20 were performed twice. g/L BSA) per slide. Next, the sections were washed three times, by placing in a slide rack in a glass trough Adhesion and adhesion-inhibition assay containing 350 mL of PBST, on a rotator for 10 min. Tissue sections were incubated for 30 min in a humidiﬁ ed Suspensions of FITC-H pylori were centrifuged and re- O’Mahony R et al. Plants against H pylori 7503

AB AB

CD CD

E Figure 2 Confocal images showing inhibition of H pylori adhesion by A: distilled water (control); B: turmeric (0.05 g/mL); C: borage (0.05 g/mL) and D: parsley (0.05 g/mL); to stomach sections expressing the Lewis b blood group antigen.

a good inhibition of adhesion, for all further inhibition experiments, all plants were used at this concentration. Inhibition of adhesion experiments were then performed on three clinical strains. All experiments were performed Figure 1 Bright-ﬁ eld images showing H pylori lysis experiment. A: negative control three times. (H pylori + distilled water); B: positive control (H pylori + cell lysis solution); C: H pylori + 0.05 g/mL turmeric; D: H pylori + 0.05 g/mL borage; E: H pylorii + Quantiﬁ cation of binding 0.05 g/mL parsley. Images taken using x100 objective. Sections were observed using a Laser Scanning Confocal Microscope (Zeiss: Axiovert 100 TV) with a ×20 Zeiss

8 LD-ACHR objective. The excitation wavelengths were 488 suspended in plant extract to give 1×10 bacteria/mL. nm (for FITC) and 568 nm (for PI). We used 522- and This was incubated with continuous shaking for 2 h, and 605-nm band-pass ﬁ lters to acquire FITC emission and PI then washed with the blocking buffer. For the controls, emission, respectively. Digital images (512×512 pixels) of bacteria were re-suspended in sterile distilled water. The the sections were captured using a Biorad Lasersharp 2000 bacterial suspension (200 μL) was then added to each Confocal Laser Scanning System. Images were converted of the stomach sections and incubated in a humidified to TIF files for processing. Two photographs showing atmosphere for 1 h. After incubation, slides were washed adjacent areas of the tissue were taken for each tissue in PBST as previously, and 200 μL of propidium iodide section. Using these digital images, the number of adherent (PI, 5 μg/mL, Sigma, UK) was added and each slide was H pylori were quantiﬁ ed as previously reported (Region of incubated for 3 min. Slides were then washed twice in Interest method with standard area method of counting) PBST, air-dried and mounted with Vectashield (V-1000, using Metamorph Image analysis software (version 4.5r, Vector Laboratories, UK). All incubation and washing Universal Imaging Corporation, USA)[23]. Using the Mann- steps were carried out in the dark at room temperature. Whitney (non-parametric) test, the difference between the Two tissue sections were used for each plant that was number of adherent cells for each plant was compared tested. statistically to the controls (0 μg/mL plant extract). All plant extracts were initially screened for inhibition of adhesion properties at a concentration of 50 mg/mL (diluted in sterile distilled water) and incubated RESULTS with H pylori strain 11 637 and Le b stomach sections. Bactericidal properties of plants For those plants which had an anti-adhesive effect, the Among the 25 plants tested for their bactericidal activity inhibition assay was repeated using Le a and Le b stomach against H pylori, eight showed no activity after 60 min of sections, and the plants were diluted to concentrations of 5, incubation, these were Bengal quince, nightshade, garlic, 10, and 50 mg/mL. Because 50 mg/mL was found to give dill, black pepper, coriander, fenugreek and tea (data 7504 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol December 21, 2005 Volume 11 Number 47

100 parsley. For sections expressing the Le b antigen, inhibition rates were 62.3%, 59.5%, and 48.5% for turmeric, borage 75 and parsley, respectively. The differences between the number of adherent bacteria compared to the control 50 (0 μg/mL) were: not quite significant (P = 0.07) for turmeric; significant (P<0.05) for borage; and not % Inhibition 25 signiﬁ cant (P>0.05) for parsley. There was no signiﬁ cant difference between the number of adherent bacteria (to

0 Le a or Le b stomach sections) when comparing each of Turmeric Borage Parsley the plant-treatments, except for adhesion of bacteria to Le a sections where there was a highly signifi cant difference Figure 3 Inhibition of H pylori adhesion by turmeric, borage and parsley to (P<0.001) between turmeric and parsley. stomach sections expressing either the Lewis a or Lewis b blood group antigen. Experiments were performed three times using four strains of H pylori. Mean percentage of inhibition is shown for each plant. Gray bar, Lewis a stomach; Black bar, Lewis b stomach. DISCUSSION Investigations into plant materials as alternative sources of antimicrobials have become more common over the past not shown). The other 17 plants had activity (results are few years, due to the increased rate of development of summarized in Table 2). Turmeric was the most effi cient in antibiotic resistant organisms. New strategies to combat killing the seven strains within 15 min. Ginger, cumin, and infection are also being sought and one such strategy is the use of ‘anti-adhesive’ molecules, targeting the primary step chilli were able to kill all the strains within 30 min, whereas [5] liquorice, oregano, black caraway, and borage killed the of infection - adhesion of the organism to host tissue . In strains within 60 min. Cinnamon had a bactericidal activity this study, we therefore examined both the anti-microbial against H pylori, although the seven bacterial strains and anti-adhesive properties of 25 plant extracts against differed in their sensitivity. Moreover, the bacterial colonies H pylori, obtained simply by boiling, as would occur during were very small, refl ecting some resistance to cinnamon. the normal cooking process. Finally, the following plants showed a bactericidal Of the eight plants that were not able to kill H pylori activity against H pylori but without achieving a complete after 60 min of incubation (Bengal quince, nightshade, inhibition of growth (i.e., killing all the colonies) within garlic, dill, black pepper, coriander, fenugreek, and black 60 min: columbo weed, yellow-berried nightshade, long tea), two had been tested previously against H pylori. Several studies have shown that garlic does kill H pylori pepper, threadstem carpetweed, sage, tarragon, nutmeg, [8,9] and parsley. Small colonies were seen with yellow-berried in vitro and in vivo . It is probably due to the boiling nightshade, threadstem carpetweed, and sage. method of extraction that this effect was not observed in this study, since boiling has been shown to reduce the [24,25] Morphological studies inhibitory activity of garlic against H pylori . Fenugreek sprouts have been shown to have high anti-microbial None of the 23 plant extracts that were tested caused activity against H pylori[26]. In the present study, fenugreek lysis of H pylori cells, as the organisms that remained were appeared inactive but the seeds were examined rather than spiral and intact like the negative control. Bacterial cells sprouts, which may account for the difference in results. incubated with Puregene PCR cell lysis solution were lysed Green tea catechins have previously been reported to have and no intact spiral shaped bacteria could be observed antibacterial effect against H pylori, which was confi rmed (Figure 1). [27] in Mongolian gerbils . Moreover, Chinese tea has been shown to reduce H pylori infection in patients[28]. In this Anti-adhesive properties of plants study, black tea was tested, but no bactericidal activity Of the 23 plants screened for anti-adhesive properties, against H pylori was observed. turmeric, borage, and fresh parsley (50 mg/mL) were Columbo weed, long pepper, parsley, tarragon, nutmeg, found to inhibit adhesion of H pylori 11637 to the stomach yellow-berried nightshade, threadstem carpetweed, sage, sections expressing the Le b antigen (Figure 2). Further and cinnamon had bactericidal activity against H pylori, but testing of these three plant extracts using H pylori 11637 total inhibition of growth was not achieved within 60 min. and three clinical isolates confi rmed that they were able to Moreover, a reduced colony size was observed with some inhibit H pylori adhesion to stomach sections expressing of them, such as yellow-berried nightshade, threadstem either the Le b antigen or the Le a antigen (Figure 3). carpetweed, sage, and cinnamon. Nutmeg[29], tarragon[30], Mean inhibition rates of the four strains to the stomach and cinnamon[31] have previously been reported to inhibit sections expressing the Le a antigen were 61.9%, 61.1%, the growth of H pylori in vitro. When tested in clinical and 33.9% for turmeric, borage and parsley, respectively. trials, however, cinnamon was found to be ineffective at The differences between the number of adherent eradicating H pylori[32]. bacteria compared to the controls (0 μg/mL) were: highly Among the plants that killed H pylori, turmeric was significant (P<0.01) for turmeric; not quite significant the most efficient, followed by cumin, ginger, and chilli. (P = 0.07) for borage; and not significant (P>0.05) for These results confi rmed what has been published before O’Mahony R et al. Plants against H pylori 7505 regarding turmeric[33], chilli[34], and ginger[35], but cumin has to the stomach - an alternative strategy to bactericidal not been previously tested. Other plants, such as borage, compounds. Because inhibition of adhesion works on the black caraway, and oregano, which have never been tested principle of sterically blocking bacteria from attaching before against H pylori, were also found to have bactericidal to host tissue, the likelihood of resistance developing in effects. Finally, the previous reported bactericidal effect of organisms, which occurs when the organism is killed, is liquorice has been confi rmed[36,37]. less likely. As well as looking at their bactericidal activity, all these Although the majority of individuals colonized by plants were also tested for their anti-adhesive effects, using H pylori are asymptomatic, a proportion of patients develop an in situ adhesion assay. Stomach tissues expressing either peptic ulcers (duodenal and gastric), and an even smaller the Le a or Le b antigen were used. This was in order to proportion develop gastric cancer. Globally, H pylori is the determine whether the plant extracts inhibit adhesion by major cause of gastric cancer and has been classifi ed as a blocking the major H pylori adhesin BabA, which binds Class I carcinogen by the WHO. Some plants are known to Le b, or have an effect on other adhesins. Most studies to have anti-ulcerogenic and anti-cancer effects. Most of have not attempted to determine which adhesin is being the plants tested here, which have bactericidal and/or blocked, and this may be important because combinations anti-adhesive properties, also have anti-ulcerogenic or [38,39] [40,41] of plants targeting several different adhesins may provide anti-cancer effects, for example, turmeric , ginger , [42,43] [44] [45] [46] a more potent therapeutic treatment. cumin , borage , liquorice , and parsley . Chilli has Extracts of turmeric, borage, and parsley were able to recently been shown to have detrimental effects on the [47] inhibit the adhesion of H pylori strains to both Le a and gastric mucosa . The anti-cancer and anti-ulcerogenic Le b stomach sections. Turmeric was the most effective at effects of ginger, cumin, liquorice, parsley, turmeric, and inhibiting H pylori adhesion to both Le a and Le b stomach borage combined with their bactericidal and anti-adhesive sections, followed by borage and fresh parsley. From these properties, as shown in this study, suggests that ingestion results, it seems that both turmeric and borage could of these six plants could have important therapeutic inhibit adhesins other than BabA, because the percentage implications for patients with H pylori-induced peptic of inhibition was almost exactly the same for Le a and Le ulcer disease or gastric cancer. As turmeric killed 100% b stomach sections. Parsley possibly inhibited BabA as well of organisms within 15 min, it could therefore be a useful as other adhesins, because inhibition occurred on Le a and anti-Helicobacter agent in vivo, because, if given orally, it Le b stomach sections, but was higher for Le b stomach would be able to kill H pylori despite the short amount of sections, indicating that it had a greater effect at inhibiting time that it would remain in the stomach during digestion. BabA. The morphological studies demonstrated that the Moreover, these plants could be used in combination with antibiotics, possibly increasing the success of eradication, reduction in adhesion caused by these plant extracts was [48] due to the true inhibition of adhesion, but not because as has been shown in vitro for cranberry juice . the plants caused the bacteria to lyse. Tarragon is the Most studies have used plant extracts that are obtained only plant in this study that has been tested previously by chemical processing, for example, ethanol extracts. for its anti-adhesive effects, where it was found to inhibit In developing countries where antibiotics are less freely H pylori adhesion to human gastric adenocarcinoma available, such processing methods of plants would cells[22]. However, in the present study, tarragon failed to be impractical and/or expensive. This present study is inhibit H pylori adhesion to human stomach sections. This therefore of importance because it has demonstrated that discrepancy is probably due to the differences between several plant extracts are effective against H pylori and are using cell-lines and whole-tissue. obtained simply by boiling the plants. Such a method may Plants contain multiple organic components including provide a treatment that is simple, relatively inexpensive phenols, quinones, flavones, tannins, terpenoids, and and could be incorporated into the normal diet of the alkaloids all of which are known to have bactericidal patient, which is highly favorable. Herbal treatments are effects[7]. These substances are also water-soluble and usually unregulated and the safety of plants consumed is often unknown. The plants used and shown to be effective therefore very likely to be present in the plant extracts in this study are already commonly consumed, and thus produced in this study, and are likely candidates they are already known to be safe. responsible for the killing effect of the extracts on H pylori. Although the cidal and anti-adhesive effects of ginger, Plants also contain many water-soluble proteins, lectins cumin, liquorice, parsley, turmeric, and borage have been and carbohydrates which may bind specifically to sugar shown in vitro, further studies are needed to be carried residues, polysaccharides, glycoproteins or glycolipids, such out to investigate their effects in vivo, to see whether the as the adhesins present on the cell surface of H pylori. In extracts are able to remain effective despite the harsh the event of such interactions occurring, the result would process of digestion. be to block the availability of the adhesin to its receptor and hence prevent adhesion of the bacterium to the stomach sections. ACKNOWLEDGMENTS Despite the success of many plant materials at killing We are grateful to Martin Avedian for the idea of testing organisms, resistance can develop to these too, as shown borage and for its supply and Ms A. Al-Dossary (School in this study. The present work is of importance because of Hygiene and Tropical Medicine, UK) for providing plant extracts were shown to inhibit adhesion of H pylori black caraway. 7506 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol December 21, 2005 Volume 11 Number 47

REFERENCES Helicobacter pylori adhesion to human gastric adenocarcinoma epithelial cells by acidic polysaccharides from Artemisia 1 Achtman M and Suerbaum S. Helicobacter pylori: Molecular capillaris and Panax ginseng. Planta Med 2004; 70: 615-619 and Cellular Biology. Horizon scientifi c, Norfolk 2001; 61 23 O’Mahony R, Basset C, Holton J, Vaira D, Roitt I. Comparison 2 Montecucco C, Rappuoli R. Living dangerously: how Helico- of image analysis software packages in the assessment of ad- bacter pylori survives in the human stomach. Nat Rev Mol Cell hesion of microorganisms to mucosal epithelium using confo- Biol 2001; 2: 457-466 cal laser scanning microscopy. J Microbiol Methods 2005; 61: 3 Basset C, Holton J, Gatta L, Ricci C, Bernabucci V, Liuzzi G, 105-126 Vaira D. Helicobacter pylori infection: anything new should we 24 Canizares P, Gracia I, Gomez LA, Garcia A, Martin De Argila C, know? Aliment Pharmacol Ther 2004; 20 Suppl 2: 31-41 Boixeda D, de Rafael L. Thermal degradation of allicin in gar- 4 Megraud F. H pylori antibiotic resistance: prevalence, impor- lic extracts and its implication on the inhibition of the in-vitro tance, and advances in testing. Gut 2004; 53: 1374-1384 growth of Helicobacter pylori. Biotechnol Prog 2004; 20: 32-37 5 Basset C, Holton J, O’Mahony R, Roitt I. Innate immunity and 25 Canizares P, Gracia I, Gomez LA, Martin de Argila C, Boixeda pathogen-host interaction. Vaccine 2003; 21 Suppl 2: S12-S23 D, Garcia A, de Rafael L. Allyl-thiosulfi nates, the bacteriostatic 6 Lampe JW. Health effects of vegetables and fruit: assessing compounds of garlic against Helicobacter pylori. Biotechnol mechanisms of action in human experimental studies. Am J Prog 2004; 20: 397-401 Clin Nutr 1999; 70: 475S-490S 26 Randhir R, Lin YT, Shetty K. Phenolics, their antioxidant and 7 Cowan MM. Plant products as antimicrobial agents. Clin Mi- antimicrobial activity in dark germinated fenugreek sprouts crobiol Rev 1999; 12: 564-582 in response to peptide and phytochemical elicitors. Asia Pac J 8 Jonkers D, van den Broek E, van Dooren I, Thijs C, Dorant E, Clin Nutr 2004; 13: 295-307 Hageman G, Stobberingh E. Antibacterial effect of garlic and 27 Takabayashi F, Harada N, Yamada M, Murohisa B, Oguni I. omeprazole on Helicobacter pylori. J Antimicrob Chemother Inhibitory effect of green tea catechins in combination with su- 1999; 43: 837-839 cralfate on Helicobacter pylori infection in Mongolian gerbils. 9 Iimuro M, Shibata H, Kawamori T, Matsumoto T, Arakawa J Gastroenterol 2004; 39: 61-63 T, Sugimura T, Wakabayashi K. Suppressive effects of garlic 28 Yee YK, Koo MW, Szeto ML. Chinese tea consumption and extract on Helicobacter pylori-induced gastritis in Mongolian lower risk of Helicobacter infection. J Gastroenterol Hepatol gerbils. Cancer Lett 2002; 187: 61-68 2002; 17: 552-555 10 Matsubara S, Shibata H, Ishikawa F, Yokokura T, Takahashi 29 Bhamarapravati S, Pendland SL, Mahady GB. Extracts of spice M, Sugimura T, Wakabayashi K. Suppression of Helicobacter and food plants from Thai traditional medicine inhibit the pylori-induced gastritis by green tea extract in Mongolian ger- growth of the human carcinogen Helicobacter pylori. In Vivo bils. Biochem Biophys Res Commun 2003; 310: 715-719 2003; 17: 541-544 11 al Somal N, Coley KE, Molan PC, Hancock BM. Susceptibility 30 Wang YC, Huang TL. Screening of anti-Helicobacter pylori of Helicobacter pylori to the antibacterial activity of manuka herbs deriving from Taiwanese folk medicinal plants. FEMS honey. J R Soc Med 1994; 87: 9-12 Immunol Med Microbiol 2005; 43: 295-300 12 Tabak M, Armon R, Potasman I, Neeman I. In vitro inhibition 31 Tabak M, Armon R, Neeman I. Cinnamon extracts’ inhibi- of Helicobacter pylori by extracts of thyme. J Appl Bacteriol tory effect on Helicobacter pylori. J Ethnopharmacol 1999; 67: 1996; 80: 667-672 269-277 13 Nariman F, Eftekhar F, Habibi Z, Falsafi T. Anti-Helicobacter 32 Nir Y, Potasman I, Stermer E, Tabak M, Neeman I. Controlled pylori activities of six Iranian plants. Helicobacter 2004; 9: trial of the effect of cinnamon extract on Helicobacter pylori. 146-151 Helicobacter 2000; 5: 94-97 14 Martin KW, Ernst E. Herbal medicines for treatment of bacte- 33 Mahady GB, Pendland SL, Yun G, Lu ZZ. Turmeric (Curcuma rial infections: a review of controlled clinical trials. J Antimicrob longa) and curcumin inhibit the growth of Helicobacter pylori, Chemother 2003; 51: 241-246 a group 1 carcinogen. Anticancer Res 2002; 22: 4179-4181 15 Galan MV, Kishan AA, Silverman AL. Oral broccoli sprouts 34 Jones NL, Shabib S, Sherman PM. Capsaicin as an inhibitor of for the treatment of Helicobacter pylori infection: a prelimi- the growth of the gastric pathogen Helicobacter pylori. FEMS nary report. Dig Dis Sci 2004; 49: 1088-1090 Microbiol Lett 1997; 146: 223-227 16 Ofek I, Hasty DL, Sharon N. Anti-adhesion therapy of bacte- 35 Mahady GB, Pendland SL, Yun GS, Lu ZZ, Stoia A. Ginger rial diseases: prospects and problems. FEMS Immunol Med (Zingiber officinale Roscoe) and the gingerols inhibit the Microbiol 2003; 38: 181-191 growth of Cag A+ strains of Helicobacter pylori. Anticancer 17 Burger O, Ofek I, Tabak M, Weiss EI, Sharon N, Neeman I. Res 2003; 23: 3699-3702 A high molecular mass constituent of cranberry juice inhibits 36 Fukai T, Marumo A, Kaitou K, Kanda T, Terada S, Nomura T. helicobacter pylori adhesion to human gastric mucus. FEMS Anti-Helicobacter pylori fl avonoids from licorice extract. Life Immunol Med Microbiol 2000; 29: 295-301 Sci 2002; 71: 1449-1463 18 Shibata H, KimuraTakagi I, Nagaoka M, Hashimoto S, Sawa- 37 Krausse R, Bielenberg J, Blaschek W, Ullmann U. In vitro anti- da H, Ueyama S, Yokokura T. Inhibitory effect of Cladosiphon Helicobacter pylori activity of Extractum liquiritiae, glycyr- fucoidan on the adhesion of Helicobacter pylori to human gas- rhizin and its metabolites. J Antimicrob Chemother 2004; 54: tric cells. J Nutr Sci Vitaminol (Tokyo) 1999; 45: 325-336 243-246 19 Shibata H, Iimuro M, Uchiya N, Kawamori T, Nagaoka M, 38 Azuine MA, Bhide SV. Chemopreventive effect of turmeric Ueyama S, Hashimoto S, Yokokura T, Sugimura T, Wak- against stomach and skin tumors induced by chemical car- abayashi K. Preventive effects of Cladosiphon fucoidan against cinogens in Swiss mice. Nutr Cancer 1992; 17: 77-83 Helicobacter pylori infection in Mongolian gerbils. Helicobacter 39 Huang MT, Lou YR, Ma W, Newmark HL, Reuhl KR, Conney 2003; 8: 59-65 AH. Inhibitory effects of dietary curcumin on forestomach, 20 Lengsfeld C, Titgemeyer F, Faller G, Hensel A. Glycosyl- duodenal, and colon carcinogenesis in mice. Cancer Res 1994; ated compounds from okra inhibit adhesion of Helicobacter 54: 5841-5847 pylori to human gastric mucosa. J Agric Food Chem 2004; 52: 40 Yamahara J, Mochizuki M, Rong HQ, Matsuda H, Fujimura H. 1495-1503 The anti-ulcer effect in rats of ginger constituents. J Ethnophar- 21 Lengsfeld C, Deters A, Faller G, Hensel A. High molecular macol 1988; 23: 299-304 weight polysaccharides from black currant seeds inhibit adhe- 41 Yoshikawa M, Yamaguchi S, Kunimi K, Matsuda H, Okuno Y, sion of Helicobacter pylori to human gastric mucosa. Planta Yamahara J, Murakami N. Stomachic principles in ginger. III. Med 2004; 70: 620-626 An anti-ulcer principle, 6-gingesulfonic acid, and three mono- 22 Lee JH, Park EK, Uhm CS, Chung MS, Kim KH. Inhibition of acyldigalactosylglycerols, gingerglycolipids A, B, and C, from O’Mahony R et al. Plants against H pylori 7507

Zingiberis Rhizoma originating in Taiwan. Chem Pharm Bull macol 1994; 46: 148-149 (Tokyo) 1994; 42: 1226-1230 46 Al-Howiriny T, Al-Sohaibani M, El-Tahir K, Rafatullah S. 42 Aruna K, Sivaramakrishnan VM. Anticarcinogenic effects of Prevention of experimentally-induced gastric ulcers in rats by some Indian plant products. Food Chem Toxicol 1992; 30: 953-956 an ethanolic extract of “Parsley” Petroselinum crispum. Am J 43 Gagandeep, Dhanalakshmi S, Mendiz E, Rao AR, Kale RK. Chin Med 2003; 31: 699-711 Chemopreventive effects of Cuminum cyminum in chemically 47 Lopez-Carrillo L, Lopez-Cervantes M, Robles-Diaz G, induced forestomach and uterine cervix tumors in murine Ramirez-Espitia A, Mohar-Betancourt A, Meneses-Garcia A, model systems. Nutr Cancer 2003; 47: 171-180 Lopez-Vidal Y, Blair A. Capsaicin consumption, Helicobacter 44 Gonzalez CA, Sanz JM, Marcos G, Pita S, Brullet E, Saigi E, pylori positivity and gastric cancer in Mexico. Int J Cancer Badia A, Agudo A, Riboli E. Borage consumption as a possible 2003; 106: 277-282 gastric cancer protective factor. Cancer Epidemiol Biomarkers 48 Shmuely H, Burger O, Neeman I, Yahav J, Samra Z, Niv Y, Prev 1993; 2: 157-158 Sharon N, Weiss E, Athamna A, Tabak M, Ofek I. Susceptibil- 45 Dehpour AR, Zolfaghari ME, Samadian T, Vahedi Y. The ity of Helicobacter pylori isolates to the antiadhesion activity protective effect of liquorice components and their derivatives of a high-molecular-weight constituent of cranberry. Diagn against gastric ulcer induced by aspirin in rats. J Pharm Phar- Microbiol Infect Dis 2004; 50: 231-235

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