Traditional Thai Medicines Inhibit Helicobacter Pylori in Vitro and in Vivo: Support for Ethnomedical Use
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Traditional Thai Medicines inhibit Helicobacter pylori in vitro and in vivo: Support for ethnomedical use Gail B. Mahady, Sutatip Bhamarapravati, Bolanle A. Adeniyi, Brian Doyle, Tracie Locklear, Christine Slover and Susan L. Pendland Research Abstract In Thailand, traditional plant-based medicines have always and can withstand the stomach’s environment by micro- been used to treat gastrointestinal ailments, including aerophilic growth capability and high urease activity (For- gastritis, peptic ulcer disease (PUD) and diarrhea. Since man & Graham 2004). Chronic HP infections are associ- Helicobacter pylori (HP) is an etiological agent of PUD, ated with the development numerous gastrointestinal dis- we have used an ethnomedical approach for screening orders, including dyspepsia, duodenal and gastric ulcer- plant extracts as potential treatments for HP infections, ation, gastric cancer, and mucosa-associated lymphoid including over 20 species from Thailand. International tissue (MALT) lymphoma (Breuer-Katschinski et al. 1999, Memoranda of Agreement were established between UIC Figueriredo et al. 2002, Forman et al. 2004). In 1994, the and Mahidol University in Thailand. Medicinal plants were International Agency for Research on Cancer classified collected, identified and extracted. Susceptibility testing HP as a human carcinogen, and a definite cause of gas- was performed with 15 HP strains using the agar dilution tric cancer (IARC 1994). procedure guidelines of the Clinical and Laboratory Stan- dards Institute. In vivo studies included evaluating bacte- According to statistics from the World Health Organiza- rial load, as well as acute and chronic inflammation in HP- tion, approximately 50% of the population in industrial- infected Mongolian gerbils. Extracts of Curcuma longa ized nations, and 80% of the populations in developing L. and Boesenbergia rotunda (L.) Mansf. significantly re- countries infected with HP (Perez-Perez et al. 1990). In duced HP-induced gastric lesions, as assessed both mac- Thailand, the incidence of HP infection is similar to other roscopically and microscopically in Mongolian gerbils. The countries in Southeast Asia, with HP antibodies present treatments reduced acute and/or chronic inflammation in in approximately 17.5% of children ages 5-9 years, but a prevention model of HP-induced gastritis. increasing to 75% in the 30- to 49-year-old group (Maha- chai et al. 2000). However, although there is a high preva- Introduction Although the incidence and mortality of gastric cancer has Correspondence declined in industrialized nations, it still remains the sec- ond leading cause of cancer deaths worldwide, particu- Gail B. Mahady, Bolanle A. Adeniyi, Brian Doyle, Tracie Lock- lear, Christine Slover, Susan L. Pendland, Department of Phar- larly in Southeast Asia (Jemal et al. 2002, Kneller et al. macy Practice, College of Pharmacy, University of Illinois at Chi- 1992). Globally, as many as 1 million people die each year cago, Chicago, IL 60612. U.S.A. of gastric cancer (Anon 2006). Gastric cancer evolves [email protected] through a multi-step mechanism in which the Gram-nega- tive bacterium Helicobacter pylori (HP) plays a major role Sutatip Bhamarapravati, Department of Nutrition, Thommasart (IARC 1994). University, Bangkok, THAILAND. Helicobacter pylori (HP) is a spiral or helical-shaped aero- Ethnobotany Research & Applications 4:159-165 (2006) bic bacillus that colonizes the gastric epithelial surface, http://hdl.handle.net/10125/249 160 Ethnobotany Research & Applications lence of HP infection in Thailand, the incidence of gastric zomes and 1 kg of dried turmeric rhizomes were obtained atrophic changes, intestinal metaplasia, and gastric can- from Jatujak market, Jatujak, Bangkok, Thailand. The cer are significantly lower than other countries (Atisook et collected plants were identified by Ms. Thaya Jenjittikul, al. 2003, Matsuhisa et al. 2003, Sriamporn et al. 2002). Department of Plant Science, Faculty of Science, Mahi- The reasons for this paradox are not well understood but dol University, Bangkok, Thailand. Herbarium specimens appear to be related to diet and life style (Bhamarapravati were deposited at Mahidol University, Faculty of Science. et al. 2003). The diet in Thailand includes a wide range of The outer surface of the rhizomes were cleaned with soap unique fruits, vegetables and spices, many of which are and water, the plant parts were cut into 1 cm slices and also used ethnomedically for the treatment of gastrointes- then dried in 550C hot air oven for 24-48 h. The dried rhi- tinal disorders (Murakami et al. 1994). zomes were then ground to a fine powder and extracted twice in an Erlenmeyer flask containing 95% ethanol (2 x Ethnobotanical searches and reviews Traditional Thai 500 ml) at room temperature on a rotary shaker for 24 h. medical texts, as well as the Napralert database for plants Each extract was filtered through Whatman no. 1 filter pa- used in Thailand for the treatment of gastrointestinal and per and the resultant filtrate was concentrated to dryness HP infections, led to a list of over 50 plant species, of under reduced pressure. The extracts were then defatted which 20 (as 25 plant parts) were collected in Thailand for with 500 ml of hexane and the resulting defatted extracts testing. The plant parts were extracted and the extracts were concentrated to dryness under reduced pressure. tested for anti-HP activity in vitro, of which 12 extracts The concentrated extracts were stored in coded sterile bo- were shown to be active in vitro (13). Here, we report new rosilicate glass vials under desiccation at -20ºC. A 10.0 mg in vitro and in vivo data on two Thai plants from the Zingib- sample of each plant extract was used for the in vitro sus- eraceae, namely finger-root (Boesenbergia rotunda (L.) ceptibility testing against the various HP strains. For the Mansf.) and turmeric (Curcuma longa L.), both of which animal studies, 100 grams of the extracts were prepared. are used in Thailand for the treatment of gastrointestinal ailments, including peptic ulcer disease (Mahady 2005). In Vitro susceptibility Testing Susceptibility testing was performed using the agar dilu- Materials and Methods tion procedure guidelines of The National Committee for Clinical Laboratory Standards (NCCLS 2005). The plant Reviews of Traditional Thai medical texts and the Napralert extract were dissolved using a minimal volume of solvent. database for plants used in Thailand for the treatment of The solvents used to dissolve the extracts were dimethyl gastrointestinal and HP infections were performed as de- sulfoxide (DMSO), ethanol, or sterile distilled water. Ster- scribed (Bhamarapravati et al. 2003). These searches led ile distilled water was used for further dilutions (serial) of to a list of over 50 plant species from Thailand, of which the dissolved plant extracts. Final concentrations of the the results of 20 were previously reported (Bhamarapra- extracts tested included 100, 50, 25, and 12.5 µg/ml for vati et al. 2003)(Figure 1). Two plant species were select- each sample. The in vitro HP susceptibility tests were per- ed for further in vitro and in vivo testing: Boesenbergia formed exactly as previously described (Bhamarapravati rotunda (finger-root) andCucurma longa (turmeric) based et al. 2003, Mahady et al. 2003a,b). A total of five cagA+ on their use in Traditional Thai medicine for the treatment strains of HP were tested, rodent adapted strain B128, of gastrointestinal ailments. M23-3, GTD7-13, G1-1, SS1 and the American Type Cul- ture Collection (ATCC) (Rockville, MD) strain (#43504) of The rhizomes of these plants were collected in Thailand HP were used in the susceptibility testing. Clinical isolates after obtaining a Memorandum of Agreement between the were obtained from Dr. Rick Peek, Division of Gastroen- University of Illinois at Chicago and Mahidol University, terology, Vanderbilt University, Nashville, TN. The identi- Bangkok, Thailand. Five hundred grams of finger-root rhi- ficationNA of each organism has been confirmed by Gram stain appearance and a positive urease test. Staphylococ- 12% cus aureusMIC ATCC > 29213100 wasmcg included in the susceptibility 20% 40% testing of the plant extracts and amoxicillin for validation of results.M IC=100m cg 12% 12% 4% An inoculumM IC=50m of each isolate cg was prepared by suspending the organism in 4.5 ml of sterile tryptic soy broth and ad- justing Mthe IC=25mturbidity to that cg of a 2.0 McFarland Standard us- ing a spectrophotometer at 625 nm. This density produces Figure 1. Results of 25 Thai plant NA a suspension of approximately 1 x 106 CFU/ml of HP. The MIC > 100 mcg MIC=12.5mcg samples tested against Helicobacter organisms were inoculated onto agar plates containing the M IC=100m cg pylori. plant extracts via a 32-prong inoculating device. The de- M IC=50m cg vice delivers 8 µl per spot resulting in a final inoculum of M IC=25m cg approximately 1 x 104 CFU/spot. After the spots dried, the MIC=12.5mcg http://hdl.handle.net/10125/249 Mahady et al. - Traditional Thai Medicines inhibit Helicobacter pylori 161 plates were incubated at 370C in 10% CO2 and examined Six weeks post-infection, all animals were sacrificed un- for growth after 5 days. All procedures were performed der CO2 euthanasia and their stomachs were resected, in duplicate. The minimum inhibitory concentration (MIC) opened along the greater curvature, and washed twice was determined for each plant extract. The MIC is defined with saline. The macroscopic gastric lesions