Bactericidal effect of selected spices, medicinal plants and tea on Helicobacter pylori strains from Sri Lanka

: The bactericidal activity of 21 plant extracts on Helicobacter pylori was investigated. Plants were boiled in water to produce aqueous extracts. Bactericidal activity of the extracts was assessed by a standard kill-curve using five strains of H. pylori isolated from Sri Lanka and the NCTC 11637 strain. Among the plants that showed bactericidal activity for H.pylori, turmeric and ginger were the most efficient followed by chilli and black tea. Nutmeg, liquorice, cinnamon, Colombo weed, yellow-berried nightshade, threadstem carpetweed, sage, parsley, long pepper, and cumin also showed bactericidal activity against H. pylori. These could serve as potent alternative therapies for H. pylori infection, avoiding the problem of resistance associated with current antibiotic treatment.


INTRODUCTION
Helicobacter pylori is a strict human pathogen which has colonized half the world population 1 . If untreated it can lead to chronic active disease and gastric malignancy. According to current recommendations, treatment of infection by H. pylori includes triple therapy inclusive of a proton pump inhibitor.
Although antibiotic resistance of H. pylori is not known in Sri Lanka, it is reported to be increasing world over. The need therefore arises to find an alternative with anti microbial properties. Studies conducted mainly in the developed countries have demonstrated inhibition of H. pylori by extracts of ginger, black tea, garlic, thyme, and mint 26 .The overall data for the prevalence of H pylori in Sri Lanka differ from 3-70 % and seems to correspond more to that of an industrialized country 78 .
The reason for the low prevalence of H. pylori in Sri Lanka is an enigma. One of the reasons for the low prevalence may be the Sri Lankan diet. Rice is the staple food in a Sri Lankan traditional diet with a significant contribution from bread, pulses and vegetables. Spices and chilli are essential and popular additives to food. Tea is consumed in plenty.
A study in the UK used Italian strains of H. pylori to test the bactericidal activity of spices, tea and medicinal plants. The results of the study demonstrated inhibitory action in some of the tested plants extracts 6 .
The genetic composition of H. pylori strains differ in various geographic regions of the world 6 and this study was designed to determine the inhibitory effects of certain spices, medicinal plants and tea against H. pylori strains isolated from Sri Lanka.

METHODS AND MATERIALS
Isolation of H. pylori: H. pylori NCTC 11637 and five strains of H. pylori isolated from patients presenting with peptic ulcer disease were included in the study. All strains were stored on beads at -80°C until tests were performed. Bacteria were grown on Colombia blood agar (oxoid) plates supplemented with 5% sheep blood at 37°C under micro aerophilic conditions. Preparation of extracts: 10 g of each powdered substance (refer to Table 1) was dissolved in 100 mL of distilled water (100 mg/mL) and was boiled for 20 min. The extracts were filtered through sterile gauze and pH neutralized. Finally the extracts were sterilized by autoclaving. All extracts were stored in the dark at -20 °C until use.

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Viable colony count: Bactericidal activities of the extracts were determined by viable colony counts. One hundred micro liters (p. L) of bacterial suspension (5xl0 8 ) from the NCTC strain and the five isolates were added to 900 p. L of each of the test extracts and incubated for 60 min in gas jars under microaerophilic conditions. The control consisted of H. pylori incubated with sterile normal saline. At the end of 60 min, 100 uL of this 1/10 dilution was inoculated on to Colombia agar plates and incubated for 3 d under microaerophilic conditions. The colony count was determined for each of the extracts (colony forming units per milliliter, cfu/mL). Plant extracts that inhibited growth of H. pylori (i.e. no colonies grew) were further tested. As described above, 1 in 10 dilutions were prepared for all plant extracts and incubated for varying time intervals e.g. 0,15,30, and 60 min. 100 p. L of this 1/10 dilution was then inoculated on to Colombia blood agar (oxoid) plates and incubated for 3 d under microaerophilic conditions. All experiments were performed three times. The effectiveness of the extracts in killing H. pylori was expressed as inhibition of colony growth.

RESULTS
Of the 21 plant extracts tested for bactericidal activity against H. pylori, 7 (fenugreek, coriander, bengal quince, nightshade, garlic, black pepper, fennel) did not show inhibition after 60 minutes of incubation under microaerophilic condition (Table 1).
Turmeric and ginger were the most efficient, with an ability to kill all 6 strains within 15 minutes. Chilli & black tea were able to kill all 6 strains within 60 minutes. Incomplete bactericidal activity were observed for 10 extracts (Table 2)

DISCUSSION
In this study we have demonstrated the bactericidal properties of 21 plant extracts against H. pylori. The extracts were obtained simply by boiling selected parts of plants in water. The current treatment for H. pylori infection with combination of antibiotics, is expensive and associated with adverse side effects. Further, the development of resistance in H. pylori to antibiotics is also a problem.  In this study, fenugreek appeared inactive where the seeds were tested rather than sprouts, which may account for the difference in results, as compared to the European study*. In this study, only one Sri Lankan strain showed complete inhibition with cumin extract within one hour. However, in a study done in U.K., cumin extract was able to kill all strains within 30 minutes 6 . This difference is interesting and should be studied further.
In this study, black tea and chilli extracts killed all the strains within one hour, but in the U.K. study* no bactericidal activity against H. pylori was seen with black tea. The difference in these results may be due to different genotypes available in different geographic regions of the world. Green tea catechins have previously been reported to have antibacterial effect against H. pylori which was confirmed in Mongolian gerbils 3 .
The plant extracts of nutmeg, cinnamon, columbo weed, yellow-berried nightshade, liquorice, long pepper, threadstem carpetweed, sage, parsley & cumin showed bactericidal activity against H. pylori but did not achieve complete inhibition within 60 minute.
As in the U.K. study* we observed a reduced colony size with some spices such as sage, cinnamon, nutmeg, yellow-berried nightshade and threadstem carpetweed.
Extractsofturmericandgingerwerethemost efficient and killed H. pylori. These results have been also shown by a previous study done in the UK*. However, it is the first time that anti H. pylori properties of spices have been demonstrated against strains isolated from Sri Lanka. These plant extracts can be used in combination with antibiotics, possibly increasing the success of eradication, as has been shown in vitro for cranberry juice 12