Helicobacter pylori is a spiral Gram-negative bacterium associated with inflammation of the gastric mucosa, peptic ulcer, and gastric adenocarcinoma; it is one of the leading causes of cancer death worldwide. Treatment prescribed to patients with gastric ulcer has failed in many cases mainly due to antibiotic resistance and important side effects such as taste disturbances, vaginal candidiasis, and pseudomembranous colitis. Interest in botanical medicine is increasing as a viable alternative to the traditional one. Plant leaves of the Asteraceae family are used to treat diarrhea, ulcers and rheumatism. The aim of this study was to evaluate the activity of aqueous and methanolic extracts from plants of the Asteraceae family against H. pylori growth in vitro, using the colorimetric tetrazolium bromide (MTT) reduction assay. We observed that methanolic extracts from plants of the Asteraceae family showed up to 82% H. pylori growth inhibition (MIC at 500 µg/mL).The results of the present study contribute to the body of knowledge of medicinal plants with antimicrobial potential, particularly against H. pylori

de la Garza Ramos, Myriam

Espinosa Ramos, David

Gomez Flores, Ricardo

Quintanilla Licea, Ramiro

Tamez Guerra, Patricia

English

Revista Mexicana de Estomatología

Revista Mexicana de EstomatologíaVol. 2 No. 2  Enero - Junio 2015ISSN: 2007-9052http://remexesto.comInvestigación original 12Inhibition of Helicobacter pylori growth by an Asteraceae family plant methanol extract “Inhibición del crecimiento de Helicobacter pylori por un extractode metanol de una planta de la familia Asteraceae” David Espinosa-Ramos1, Ricardo Gomez-Flores1,  Patricia Tamez-Guerra1, Ramiro Quintanilla-Licea1, Myriam de la Garza-Ramos21 Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León. Ave. Universidad s/n. San Nicolás de los Garza, N. L. México. 66450.2 Facultad de Odontología / Centro de Investigación y Desarrollo en Ciencias de la Salud, Universidad Autónoma de Nuevo León. Calle Dr. Carlos    Canseco y Ave. Gonzalitos s/n, Colonia Mitras Centro, Monterrey, Nuevo León, 64460, México.Abstract:Helicobacter pylori is a spiral Gram-negative bacterium associated with inflammation of the gastric mucosa,peptic ulcer, and gastric adenocarcinoma; it is one of the leading causes of cancer death worldwide. Treatmentprescribed to patients with gastric ulcer has failed in many cases mainly due to antibiotic resistance and importantside effects such as taste disturbances, vaginal candidiasis, and pseudomembranous colitis. Interest in botanicalmedicine is increasing as a viable alternative to the traditional one. Plant leaves of the Asteraceae family areused to treat diarrhea, ulcers and rheumatism. The aim of this study was to evaluate the activity of aqueous andmethanolic extracts from plants of the Asteraceae family against H. pylori growth in vitro, using the colorimetrictetrazolium bromide (MTT) reduction assay. We observed that methanolic extracts from plants of the Asteraceaefamily showed up to 82% H. pylori growth inhibition (MIC at 500 μg/mL).The results of the present study contributeto the body of knowledge  of medicinal plants with antimicrobial potential, particularly against H. pylori.Keywords: Helicobacter pylori ● Asteraceae family plant ● Methanolic extracts● Aqueous extractsRevista Mexicana de EstomatologíaVol. 2 No. 2   Enero - Junio 2015Inhibition of Helicobacter pylori growth by anAsteraceae family plant methanol extractISSN: 2007-9052http://remexesto.com13INTRODUCTIONH. pylori infects and colonizes the human stomach in 50% of the world’s population (Hongying et al.,2014). Chronic infection in the human stomach is characterized by chronic inflammation. The develop-ment of gastric adenocarcinoma, particularly of the intestinal type, is preceded by the development ofchronic gastritis, atrophic gastritis, intestinal metaplasia, and dysplasia. In developing countries, 70 to90% of the population becomes infected before 10 years of age and more than 80% of adults and 50%of children are colonized by H. pylori compared with 30% of adults and 10% of children in developedcountries (Dunn et al., 1997, Dye et al., 2002). Researchers have mentioned the oral cavity as the mainreservoir of intragastric H. pylori and it is likely that oral health is directly related to infection or reinfectionwith H. pylori (Cover et al., 1995). H. pylori has been detected in saliva, plaque and periodontal diseasepatients by PCR, culture, urease test; this organism can promote the development of lesions of the oralmucosa, particularly recurring ulcer disease and lesions of the oral mucosa (Rlgglo and Lennon, 1999;Nguyen et al., 1993). In México, seroprevalence of H. pylori is higher than 50% (Alvarado-Esquivelet al., 2013). The first-line treatment option for H. pylori consists of a 7 to 10 days regimen with proton-pump inhibitor (PPI), plus amoxicillin, and clarithromycin (Alahdab et al., 2014).The use of clarithromycinincreases resistance to H. pylori (Wu et al., 2014). Treatment prescribed to patients with gastric ulcerfail in many cases mainly due to antibiotic resistance, in addition to induced side effects. Interest in bo-tanical medicine has increased in recent years, physicians and people seem to show preference forproducts that contain “natural extracts” instead of products based on “synthetic” substances (Borcherset al., 2000, Torrado-Truiti et al., 2003) The first report of plants  against H. pylori was made in 1991(Cassel-Beraud et al.,1991). There are reports of methanol and aqueous extracts of Mexican plantsthat have inhibitory effect against H. pylori (Castillo-Juárez et al., 2009).The plant compounds includingpolyphenols, flavonoids, quinones, coumarins, terpenoids and alkaloids.The  anti-H. pylori action me-chanism, including inhibition of enzymatic(urease, DNA gyrase, dihydrofolate reductase, N-acetyltrans-ferase, and myeloperoxidase) and anti-adhesion activities, high redox potential andhydrophilic/hydrophobic natures of compounds (Wang, 2014). The aim of this study was to evaluateaqueous and  methanol plants extracts using the MTT tetrazolium reduction assay as a viability test toassess direct effects of these plant extracts against H. pylori growth in vitro.Materials and MethodsFive grams of each sample were used to prepare the extracts. For aqueous extracts, the plants wereboiled for 10 min with 80 mL of purified water filtered and then lyophilized in freeze dry system (LAB-CONCO Corp., Kansas City, MI) for about four hours. Once the sample was obtained the amount ofextracts was calculated. In the case of methanol extracts the material was extracted for 72 hours in 80mL of methanol. After filtration the extracts were evaporated in a Speed Vac (Milford, MA). The plantsscreened in the present study were Persea americana Mill (Lauraceae), Pachycereus marginatus (DC.)Britton & Rose, a plant of the Asteraceae family (its use is in the process of patenting), Ibervillea sonoraeGreen, and Phoenix dactylifera Linn.Revista Mexicana de EstomatologíaVol. 2 No. 2   Enero - Junio  2015Inhibition of Helicobacter pylori growth by anAsteraceae family plant methanol extract      ISSN: 2007-9052http://remexesto.com14Bacterial strain and culture conditionsH. pylori standard strain ATCC 43504 was grown on Brucella broth for a day at 37°C. The  strain was identifiedby Gram staining morphology and biochemical positive tests for catalase and urease.Minimum inhibitory concentration (MIC) determinationsThe aqueous and methanol extracts were tested by MTT tetrazolium reduction assay in 96-flat well microplates.The extracts were dissolved in Brucella broth to obtain a final concentration 7.8, 15.6, 31.2, 62.5,125, 250, and500 μg/mL. A volume of 50 μL of H. pylori (2.5x105 bacteria/mL) was placed in the plate wells and incubated.The MIC was determined using the MTT reduction assay, being the lowest concentration of the extracts in theplate with no bacterial growth. Before extracts were incubated, we added 15 μL of MTT  and incubated for 15min, then  80 μL of DMSO were added to dissolve the formazan crystals. The absorbances were measured in amicroplate reader at 570 nm (DTX 800/880 Multimode Detectors, Fullerton, CA). All the experiments were per-formed in triplicate and repeated at least three times. Tetracyclin was used as a positive control.ResultsMethanolic and aqueous extracts of 5 different plants were tested in vitro for their anti H. pylori activity. Noaqueous extract of the 5 plants showed in vitro effect against H.pylori ATCC 43504. Only methanolic  extractsfrom plants of the Asteraceae family showed inhibitory effect against H. pylori. Such results are shown in Figure1. The vehicle control did not affect H. pylori viability.Figure 1. Inhibitory effect of methanol plant leaves extract of a plant of the Asteraceae family.Revista Mexicana de EstomatologíaVol. 2 No. 2   Enero - Junio  2015Inhibition of Helicobacter pylori growth by anAsteraceae family plant methanol extract  ISSN: 2007-9052http://remexesto.com15As seen in Figure 1, the Asteraceae plant methanolic extracts showed up to 82.3% growth inhibition of H. pyloriATCC 43504 at a concentration of 500 μg/mL (p<0.05), and the growth inhibition activity was in a concentration-dependent manner.DiscussionThe beneficial medicinal effects of plant materials typically result from the combinations of secondary productspresent in the plant. In plants, these compounds are mostly secondary metabolites such as alkaloids, steroids,tannins, and phenol compounds, which are synthesized and deposited in specific parts or in all parts of the plant(Parekh et al., 2005).Although our aqueous extracts of Persea americana Mill (Lauraceae), Pachycereus marginatus (DC.) Britton &Rose, a plant of the Asteraceae family, Ibervillea sonorae Green, and Phoenix dactylifera Linn did not affect H.pylori viability, authors such as Castillo et al., 2013, reported anti-Helicobacter pylori activity from aqueous extractsof Persea americana Mill with a minimum inhibitory concentration (MIC) > 1000 μg/mL tested with the agar dilutionmethod.Methanolic extracts of plants used in this work have previously been tested against  H. pylori. The MIC of Ibervilleasonorae Green against H. pylori is reported of 200 to 400 μg/mL (Robles-Zepeda et al., 2011). Also, methanolextract of Persea americana Mill has been reported as active with a MIC <7.5 μg/mL tested with the broth dilutionmethod (Castillo et al., 2013).Asteraceae plants have been reported to contain essential oils, flavonoids (Wollenweber et al., 1981), amongother substances, and possess antimicrobial and antitumor activities.Our Asteraceae plant methanolic extracts showed a growth inhibition of H. pylori ATCC 43504 at a concentrationof 500μg/mL, and the activity was in a concentration-dependent manner. However the results obtained in this re-search can not be compared with others due to the bioassay employed. The active concentrations used in thepresent study can be considered high, nevertheless there are active components which can inhibit the growth ofthe bacteria.The MTT reduction assay was standardized in order to test the activity anti-Helicobacter pylori of plants. It ispossible that during the extraction method or when the plants were boiled some trace amounts of compoundsevaporated.Conclusions:The methanolic extracts from a plant of the Asteraceae family showed up to 82.3% H. pylori growth inhibition ata concentration of 500μg/mL, which warrants further studies on diverse in vitro and in vivo parameters. Revista Mexicana de EstomatologíaVol. 2 No. 2   Enero - Junio 2015Inhibition of Helicobacter pylori growth by anAsteraceae family plant methanol extract  ISSN: 2007-9052http://remexesto.com16ReferencesHongying F, Xianbo W, Fang Y, Yang B, Beiguo L. 2014.Oral Immunization with Recombinant Lactobacillus aci-dophilus Expressing the Adhesin Hp0410 of Helicobacter pylori Induces Mucosal and Systemic Immune Res-ponses Clin.Vaccine Immunol. 21(2):126-132.Dunn B, Cohen H, Blaser M. 1997. Helicobacter pylori. Clin. Microbiol. Rev., 10(4); 720-741.Dye B, Kruszon-Moran D, McQuillan G. 2002. The relationship between periodontal disease attributes and He-licobacter pylori infection among adults in the United States. Am. J. Public Health., 92(11); 1809-1815.Cover T, GlupczynskiI Y,  Lage A, Burette A, Tummuru M, Perez-Perez  G,Blaser M. 1995. Serologic Detectionof Infection with cagA1 Helicobacter pylori Strains. Journal of Clinical Microbiology., 33(6); 1496–1500.Riggio M, Lennon A. 1999. Identification by PCR of Helicobacter pylori in subgingival plaque of adult periodontitispatients. J. Med. Microbiol.  48 (1 999), 3 17-322.Nguyen A, Engstrand L, Genta R, Graham D,  El-Zaatari F. 1993. Detection of Helicobacter pylori in dental plaqueby reverse transcription-polymerase chain reaction. J Clin Microbiol. 31 (1993); 783–787.Alvarado-Esquivel C, Hernandez-Tinoco J, Sanchez-Anguiano L, Ramos-Nevarez A, Cerrillo-Soto S, Saenz-Soto L. 2013. High Seroprevalence of Helicobacter Pylori Infection in Inmates: A Case Control Study in a NorthernMexican City. Gastroenterology Research, 6(6); 227-232.Alahdab Y, Kalayci C. 2014. Helicobacter pylori: Management in 2013.World J Gastroenterol, 20(18); 5302-5307.Wu J, Wang S, Lee Y, Yamaoka Y, Graham D, Jan C, Wu D. 2014. Detection of genotypic clarithromycin-resistantHelicobacter pylori by string tests.World J Gastroenterol., 20(12): 3343-3349.Borchers A, Keen C, Stern J, Gershwin M.2000. Inflammation and native American medicine: the role of botani-cals. Am. J. Clin. Nutr., 72(2); 339-47.Torrado-Truiti M, Sarragiotto M, Abreu Filho B, Vataru-Nakamura C, Dias Filho B. 2003. In Vitro antibacterial ac-tivity of a 7-O-β-D-glucopyranosyl-nutanocoumarin from Chaptalia nutans (Asteraceae). Mem. Inst. OswaldoCruz., 98 (2);283-286. Cassel-Beraud A, Le Jan J, Mouden J, Andriantsoa M. Andriantsiferana R 1991. Preliminary study of the preva-lence of Helicobacter pylori in Tananarive, Madagascar and the antibacterial activity in vitro of 13 Malagasy me-dicinal plants on this germ. Archives de l’Institut Pasteur de Madagascar   59: 9-23.Castillo-Juárez I, González V, Jaime-Aguilar H,  Martínez G, Linares E, Bye R, Romero I. 2009.Anti-Helicobacterpylori activity of plants used in Mexican traditional medicine for gastrointestinal disorders. J.  Ethnopharmacol.,122(2); 402 -405.Wang Y. 2014. Medicinal plants and H. pylori-induced diseases. World J. Gastroenterol., 20(30): 10368-10382Potential Antibacterial Activity. Turk J. Biol. 29; 203-210.Revista Mexicana de EstomatologíaVol. 2 No. 2   Enero - Junio 2015Inhibition of Helicobacter pylori growth by anAsteraceae family plant methanol extract ISSN: 2007-9052http://remexesto.com17Parekh J, Jadeja D, Chanda S 2005. Efficacy of Aqueous and Methanol Extracts of Some Medicinal Plants forPotential Antibacterial Activity. Turk J. Biol. 29; 203-210.Robles-Zepeda R, Velázquez-Contreras C, Garibay-Escobar A, Gálvez-Ruiz J, Ruiz-Bustos E. 2011. Antimicrobialactivity of Northwestern Mexican plants against Helicobacter pylori. J Med Food. 14:1280–1283.Wollenweber E, Dietz V 1981.Occurrence and distribution of free flavonoid aglycones in plants. Phytochemistry20; 869–932.Autor de correspondencia:David Espinosa Ramos.alonsoespinosa81@hotmail.comArtículo recibido: 12 de Mayo 2015.Artículo aprobado para publicación:26 de Junio de 2015.

Inhibition of Helicobacter pylori growth by an Asteraceae family plant methanol extract.

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Inhibition of Helicobacter pylori growth by an Asteraceae family plant methanol extract.

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