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Antimicrobial activity of Artemisia douglasiana and dehydroleucodine against Helicobacter pylori
Journal of Ethnopharmacology 124 (2009) 653–655
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Antimicrobial activity of Artemisia douglasiana and dehydroleucodine against Helicobacter pylori A.E. Vega a,∗ , G.H. Wendel b , A.O.M. Maria b , L. Pelzer b a b
Área Microbiología, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina Área Farmacología, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina
a r t i c l e
i n f o
Article history: Received 22 December 2008 Received in revised form 14 April 2009 Accepted 25 April 2009 Available online 5 May 2009 Keywords: Artemisia douglasiana Dehydroleucodine Antimicrobial activity Helicobacter pylori
a b s t r a c t Ethnopharmacological relevance: The increasing resistance of Helicobacter pylori to antibiotics demands the search for novel compounds from plant based sources. Artemisia douglasiana Besser is widely used in Cuyo region (Argentina) as folk medicine for the treatment of gastric ailments. Aim of study: Based on our previous studies that Artemisia douglasiana exert cytoprotective actions against ethanol-induced gastric mucosal injury we assayed the anti-Helicobacter pylori effect of the Artemisia douglasiana extract and its active compound, dehydroleucodine. Materials and methods: The in vitro anti-bacterial activity of Artemisia douglasiana extract and its active compound, dehydroleucodine were determined against one standard strain and six clinical isolates of Helicobacter pylori by using the agar dilution methods. Results: The results showed that both dehydroleucodine and Artemisia douglasiana extract had activity against the microorganism with MICs between 1–8 and 60–120 mg/L, respectively. Conclusions: Artemisia douglasiana may be a useful alternative treatment strategy principally in eradication of metronidazole and clarithromycin-resistant strain. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Helicobacter pylori colonizes human gastric mucous layer and has been regarded as a major cause of chronic gastritis and peptic ulcer with growing incidences worldwide, and is important in the pathogenesis of gastric cancer (Dunn et al., 1997). The eradication of Helicobacter pylori can therefore, contribute to the treatment and prevention of these gastroduodenal diseases (Kawakami et al., 2006). Many regimens are currently available based on triple therapy containing two antibiotics and a proton pump inhibitor; however Helicobacter pylori rapidly acquire resistance to some antibiotics. Probably the selection of resistant strains increases because antibiotic treatment of Helicobacter pylori involves drugs largely used for other kinds of infections, such as the use of metronidazole (MTZ) for anaerobic and parasitic infections (Dunn et al., 1997; Tankovic et al., 2000) or the use of clarithromycin (CLA) in respiratory tract infections (Kato et al., 2002). Indeed, Helicobacter pylori strains resistant to CLA and MTZ have been increasing (Torres et al., 2001; Alarcón et al., 2003). Therefore, a search for new antibacterial agents, both
Abbreviations: AE, aqueous extract; CLA, clarithromycin; DhL, dehydroleucodine; MIC, minimum inhibitory concentration; MTZ, metronidazole. ∗ Corresponding author. Fax: +54 02652 430224. E-mail address: [email protected] (A.E. Vega). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.04.051
highly effective and safe, that can serve as alternative solutions for the treatment of Helicobacter pylori is very important. Several investigators have documented that plants and plant products possess potent medicinal properties, suggesting that may be useful for specific medical conditions (Cos et al., 2006; Raju et al., 2007). The World Health Organization (WHO) estimates that 4 billion people (80% of the World’s population) use herbal medicines for some aspects of primary healthcare (Farnsworth et al., 1985). Many medicinal and food plants are used around the world for the treatment of gastritis, dyspepsia and peptic ulcers, and some plants species also have experimental antibacterial activity (Ankli et al., 2002; Stege et al., 2006; Park et al., 2008). In Argentina Artemisia douglasiana Besser (Asteraceae) leaves are used in folk medicine and is known by the vernacular name of “matico”. The popular use of the infusion prepared by boiling leaves of Artemisia douglasiana is as a cytoprotective agent against peptic ulcer, and is also used for the treatment of external sores (Ariza Espinar and Bonzan, 1992). The extract of Artemisia douglasiana prevents the gastric injury induced by absolute ethanol (Giordano et al., 1990). The main active cytoprotective principle of Artemisia douglasiana is dehydroleucodine (DhL), a sesquiterpene lactone of the guiainolide type (Fig. 1). The aim of this study was to evaluate the antibacterial activity of Artemisia douglasiana, as well as the active compound DhL against Helicobacter pylori strains.
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A.E. Vega et al. / Journal of Ethnopharmacology 124 (2009) 653–655
with 7% horse blood under the same microaerophilic atmosphere mentioned above (5% O2 , 10% CO2 , and 85% N2 ) at 37 ◦ C for 48 h. The bacterial suspension in Mueller–Hinton broth was adjusted to a final concentration of a McFarland No. 0.5 standard, 2 L of the adjusted inoculum was delivered to the agar plates. After 72 h of incubation under the microaerophilic atmosphere (5% O2 , 10% CO2 , and 85% N2 ) at 37 ◦ C, the MIC of CLA, MTZ, DhL, and Artemisia douglasiana extract was determined. The MIC was recorded as the lowest concentration of drugs inhibiting visible growth. Resistance was defined as the CLA MIC being ≥2 mg/L and MTZ MIC being ≥8 mg/L (NCCLS, 2000; Glupczynski et al., 2001). All experiments were conducted in duplicate. Fig. 1. Structure of dehydroleucodine, isolated from Artemisia douglasiana.
3. Results and discussion 2. Materials and methods 2.1. Extraction and purification of DhL Artemisia douglasiana was collected in the mountains of the province of San Luis, Argentina, and a voucher specimen was deposited in the Herbarium of the Universidad Nacional of San Luis (UNSL No. 55). The powdered, dried aerial part of Artemisia douglasiana (10 g) were extracted by infusion with distilled water (1 × 100 mL). The infusion was filtered after 20 min to obtain the extract. The air-dried material of the aerial parts (580 g) was soaked in chloroform at room temperature. The extracts were evaporated in vacuum and dissolved in 95% ethanol. After addition of 4% aqueous lead tetraacetate solution, the aqueous cloudy solution was filtered through a celite pad, and the filtrate was concentrated under vacuum. The mixture was extracted three times with chloroform and the solution was concentrated under vacuum. The final residue was chromatographed in a medium-pressure chromatography system using 1:9 EtAcO/hexane as eluent (stationary phase: Silica gel 60G). DhL (100% purity, 1 g) was identified by 1 H or 13 C nuclear magnetic resonance, mass spectrometry, or melting point analysis, and its structure was identical to that cited by other authors (Giordano et al., 1990). 2.2. Antimicrobial activity 2.2.1. Bacterial strains The reference strain of Helicobacter pylori (NCTC 11638) obtained from Microbiology Service of the Hospital Universitario de la Princesa, Madrid, Spain, and six clinical isolates obtained from gastric antral biopsy specimens were used for this study. Helicobacter pylori strains were grown in Mueller–Hinton agar supplemented with 7% horse blood under a microaerophilic atmosphere (5% O2 , 10% CO2 , and 85% N2 ) at 37 ◦ C for 7–10 days and the identification was based on microaerophilic growth requirement, morphology, Gram’s stain, and oxidase, catalase and urease reaction. Strains were stored in trypticase soy broth supplemented with 20% glycerol at −80 ◦ C until use. 2.2.2. Determination of minimum inhibitory concentration The antibacterial activity was assayed by agar dilution method, according to National Committee for Clinical Laboratory Standards guidelines (NCCLS, 2000) using Mueller–Hinton agar supplemented with 7% horse blood. Serial dilutions of the CLA (Abbott Laboratories, Argentina) and MTZ (Sigma Chemical Co., St. Louis, MO) ranging from 128 to 0.008 mg/L were used as a control in the susceptibility test. DhL was dissolved in distilled water and added to Mueller–Hinton agar supplemented with 7% horse blood at concentrations ranging from 0.008 to 128 mg/L. Artemisia douglasiana extract was tested in the range of 40 at 150 mg/L. The bacteria were subcultured on Mueller–Hinton agar supplemented
All clinical isolates were CLA resistant and one strain was MTZ resistant. Reference strain and two clinical isolates were inhibited at 60 mg/L of the extract of Artemisia douglasiana (EA). Complete inhibition in vitro was achieved by 150 mg/L of EA, however 71.4% (5/7) of strains was inhibited at 90 mg/L of EA. DhL showed activity against all strains tested with MICs ranging from 1 to 8 mg/L. Reference strain and one clinical isolate showed lowest inhibition with DhL, 2 and 1 mg/L, respectively (Table 1). The eradication of Helicobacter pylori is seen as important in accelerating the healing and preventing the relapse of gastrointestinal diseases, principally peptic ulcer. However the primary impediments to successful treatment are lack of compliance with the drug regimens for undesirable side effects, contraindications, significant costs and development of antibiotic-resistant Helicobacter pylori strains (Ali et al., 2005). An increase of resistance to clarithromycin was observed in the Helicobacter pylori strains both children and adults (Vega et al., 2003). Since prehistoric times, people have used natural resources for medicinal purposes. The folk medicine of Argentina employs many plants to counteract diverse diseases such as catarrh, bronchitis, pneumonia, ulcers and diarrhoeas (Tousarkissian, 1980). Artemisia douglasiana is widely utilized in the Cuyo region (Argentina), as a folk medicine for its beneficial effects in patients with gastroduodenal disorders. DhL increases the synthesis of gastric mucosal glycoproteins (Guardia et al., 1994), confirmed by histological studies in gastric and duodenal mucous (Piezzi et al., 1995). It prevents the formation of the gastric mucous lesions induced by absolute ethanol and by other necrotizing agents (Giordano et al., 1990; Penissi et al., 1998). The mechanism of the protective action of DhL remains unknown but it seems to be related to PGE (Maria et al., 1998). On the other hands, recurrent ulcer disease after peptic ulcer perforation mainly occurs in patients with Helicobacter pylori infection, and all patients with perforated peptic ulcer should be treated. Helicobacter pylori-induced nuclear factor NF-B activation, and the subsequent release of interleukin (IL)-8 plays an important role
Table 1 In vitro antibacterial activity (mg/L) of clarithromycin (CLA), metronidazole (MTZ), dehydroleucodine (DhL) and Artemisia douglasiana Besser extract (AE) against Helicobacter pylori. Helicobacter pylori strains
NCTC 11638 HP781 HP786 HP788 HP789 HP795 HP796
MIC (mg/L) DhL
AE
CLA
MTZ
2 4 1 8 8 4 2
60 90 60 120 120 90 60
0.125 8 8 4 8 8 4
0.5 16 1 2 1 2 1
A.E. Vega et al. / Journal of Ethnopharmacology 124 (2009) 653–655
in gastric mucosal inflammation response induced by Helicobacter pylori infection (Aihara et al., 1997; Naumann, 2001; Lai et al., 2008) Artemisia douglasiana and DhL activities against Helicobacter pylori may relate to the anti-inflammatory effect. Previous findings in our laboratory have shown that DhL may prevent or reverse the inflammation process in rats. DhL inhibited both chronic and acute adjuvant carrageenan-induced inflammation phases, being most effective in the chronic phase (Guardia et al., 2003). An anti-inflammatory effect has previously been observed in other sesquiterpene lactones (Hall et al., 1979). Recently, several Asteraceae (Artemisia ludoviciana ssp. mexicana, Polymia maculata) have been identified as potent inhibitors of the transcription factor NFB, a central mediator of the inflammatory response (Bork et al., 1997). Extracts from Mexican Indian medicinal plants used in traditional indigenous medicine for inflammation treatment contain sesquiterpene lactones, which specifically inhibit transcription factor NF-B (Ankli et al., 2002). Band-shift experiments with total HeLa cell proteins revealed that DhL prevents NF-B induction (Wendel et al., 2002). Furthermore, Helicobacter pylori infection induces active inflammation with neutrophilic infiltrations as well as production of oxygen free radicals (Chang et al., 2004). The data reported in previous work indicate that the extract of Artemisia douglasiana and DhL have an antioxidant capacity (Maria et al., 2000). Natural products, either as pure compounds or as standardized plant extracts, provide unlimited opportunities for new drug leads because of the unmatched availability of chemical diversity (Cos et al., 2006). Results obtained in this study indicate that Artemisia douglasiana extract and its active compound DhL, have significant antimicrobial properties against Helicobacter pylori, and it could be a promising native herb treatment for patients with gastric ulcer caused by Helicobacter pylori. Although evidence on efficacy of the tested plant has been obtained, further investigations are necessary prior to their recommendation for use as safe and effective agent. Acknowledgements We thank Dr. Patricia Gomez for supplying biopsies, Abbott laboratories for providing clarithromycin. This work was supported by funds of C. y T. Projects 8802, 9303 and 8504 from the Universidad Nacional of San Luis, Argentine. References Aihara, M., Tsuchimoto, D., Takizawa, H., Azuma, A., Wakebe, H., Ohmoto, Y., Imagawa, K., Kikuchi, M., Mukaida, N., Matsushima, K., 1997. Mechanisms involved in Helicobacter pylori-induced interleukin-8 production by a gastric cancer cell line, MKN45. Infection and Immunity 65, 3218–3224. Alarcón, T., Vega, A.E., Domingo, D., Martínez, M.J., López-Brea, M., 2003. Clarithromycin resistance among Helicobacter pylori strains isolated from children: prevalence and study of mechanism of resistance by PCR-restriction fragment length polymorphism analysis. Journal of Clinical Microbiology 41 (1), 486–499. Ali, S.M., Khan, A., Ahmed, I., Musaddiq, M., Ahmed, K., Polasa, H., VenkateswarRao, L., Habibullah, C.H., Sechi, L., Ahmed, N., 2005. Antimicrobial activities of eugenol and cinnamaldehyde against the human gastric pathogen Helicobacter pylori. Annals Clinical. Microbiology and Antimicrobial 4, 1–7. Ankli, A., Heinrich, M., Bork, P., Wolfram, L., Bauerfeind, P., Brun, R., Schimd, C., Weiss, C., Bruggisser, R., Gertsch, J., Wasescha, M., Sticher, O., 2002. Yucatec Mayan medicinal plants: evaluation based on indigenous uses. Journal of Ethnopharmacology 79, 43–52. Ariza Espinar, L., Bonzan, N., 1992. El matico de la región de Cuyo (Argentina). Acta Farmaceutica Bonaerense 11, 139–145. Bork, P.M., Lienhard Schmitz, M., Kuhnt, M., Escher, C., Heinrich, M., 1997. Sesquiterpene lactone containing Mexican Indian medicinal plants and pure
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sesquiterpene lactones as potent inhibitors of transcription factor NF-B. Federation of European Biochemical Societies Letters 402, 85–90. Chang, C.-S., Chen, W.-N., Lin, H.-H., Wu, C.-C., Wang, C.-J., 2004. Increased oxidative DNA damage, inducible nitric oxide synthase, nuclear factor B expression and enhanced antiapoptosis-related proteins in H. pylori-infected non-cardiac gastric adenocarcinoma. World Journal of Gastroenterology 10 (15), 2232–2240. Cos, P., Vlietinck, A.J., Vanden Berghe, D., Maes, L., 2006. Anti-infective potential of natural products: how to develop a stronger in vitro ‘proof-of concept’. Journal of Ethnopharmacology 106, 290–302. Dunn, B.E., Cohen, H., Blaser, M.B., 1997. Helicobacter pylori. Clinical Microbiology Reviews 40, 720–741. Farnsworth, N.R., Akelere, O., Bingel, A.S., Soejarta, D.D., Eno, Z., 1985. Medicinal plants in therapy. Bulletin of the World Health Organization 63, 965–981. Giordano, O., Guerreiro, E., Pestchanker, M., Guzmán, J., Pastor, D., Guardia, T., 1990. The gastric cytoprotective effect of several sesquiterpene lactones. Journal of Natural Products 53, 803–809. Glupczynski, Y., Mégraud, F., López-Brea, M., Andersen, L.P., 2001. European multicentre survey of in vitro antimicrobial resistance in Helicobacter pylori. European Journal of Clinical Microbiology & Infectious Diseases 20, 820–823. Guardia, T., Juarez, A.O., Guerreiro, E., Guzmán, J.A., Pelzer, L.E., 2003. Antiinflammatory activity and effect on gastric secretion of dehydroleucodine isolated from Artemisia douglasiana. Journal of Ethnopharmacology 88 (2–3), 195–198. Guardia, T., Guzmán, J., Pestchanker, M., Guerreiro, E., Giordano, O., 1994. Mucus synthesis and sulfhydryl groups in cytoprotection mediated by dehydroleucodine, a sesquiterpene lactone. Journal of Natural Products 57, 507–509. Hall, I.H., Lee, K.H., Starnes, C.O., Sumida, Y., Wu, R.Y., Waddell, T.G., Cochran, J.W., Gerhart, K.G., 1979. Anti-inflammatory activity of sesquiterpene lactones and related compounds. Journal of Pharmaceutical Sciences 68 (5), 537–541. Kato, S., Fujimura, S., Udagawa, H., Shimizu, T., Maisawa, S., Ozawa, K., Iinuma, K., 2002. Antibiotic resistance of Helicobacter pylori strains in Japanese children. Journal of Clinical Microbiology 40, 649–653. Kawakami, Y., Oana, K., Hayama, M., Ota, H., Takeuchi, M., Miyashita, K., Matsuzawa, T., Kanaya, K., 2006. In vitro bactericidal activities of Japanese rice-fluid against Helicobacter pylori strains. International Journal of Medical Sciences 3, 112–116. Lai, C.H., Fang, S.H., Rao, Y.K., Geethangilio, M., Tang, C.H., Lin, Y.J., Hung, C.H., Wang, W.C., Tzeng, Y.M., 2008. Inhibition of Helicobacter pylori-induced inflammation in human gastric epithelial AGS cells by Phyllanthus urinaria extracts. Journal of Ethnopharmacology 118, 522–526. Maria, A.O.M., Repetto, M., Llesuy, S., Giordano, O., Guzmán, J.A., Guerreiro, E., 2000. Antioxidant activity of extract and dehydroleucodine from Artemisia douglasiana Besser. Phytotherapy Research 14, 558–560. Maria, A., Franchi, G., Gimmeno, M., Guzmán, J., Giordano, O., Guerreiro, E., 1998. Gastric cytoprotective activity of dehydroleucodine in rats. Role of prostaglandins. Biological & Pharmaceutical Bulletin 21 (4), 335–338. National Committee for Clinical Laboratory Standards, 2000. Approved Standard M100-S10. MIC Testing Supplemental Tables. National Committee for Clinical Laboratory Standards, Wayne, PA. Naumann, M., 2001. Host cell signalling in Helicobacter pylori infection. International Journal of Medical Microbiology 291, 299–305. Park, S.W., Oh, T.Y., Kim, Y.S., Sim, H., Park, S.J., Jang, E.J., Park, J.S., Baik, H.W., Hahm, K.B., 2008. Artemisia asiatica extracts protect against ethanol-induced injury in gastric mucosa rats. Journal of Gastroenterology and Hepatology 23, 976–984. Penissi, A.B., Fogal, T.H., Guzmán, J.A., Piezzi, R.S., 1998. Gastroduodenal mucosal protection induced by dehydroleucodine. Mucus secretion and role of monoamines. Digestive Diseases and Sciences 43 (4), 791–798. Piezzi, R., Guzmán, J., Guardia, T., Pestchanker, M., Guerreiro, E., Giordano, O., 1995. Dehydroleucodine prevents ethanol-induced necrosis in the mice duodenal mucosa. A histological study. Biocell 19 (1), 27–33. Raju, G., Arvind, S., Sanjay, M.J., 2007. Indian medicinal plants as a source of antimycobacterial agents. Journal of Ethnopharmacology 110, 200–234. Stege, P.W., Davicino, R.C., Vega, A.E., Casali, Y., Correa, S., Micalizzi, B., 2006. Antimicrobial activity of aqueous extracts of Larrea divaricata Cav (jarilla) against Helicobacter pylori. Phytomedicine 13, 724–727. Tankovic, J., Lamarque, D., Delchier, J.C., Soussy, C.J., Labigne, A., Jenks, P., 2000. 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Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Antimicrobial activity of Artemisia douglasiana and dehydroleucodine against Helicobacter pylori A.E. Vega a,∗ , G.H. Wendel b , A.O.M. Maria b , L. Pelzer b a b
Área Microbiología, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina Área Farmacología, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina
a r t i c l e
i n f o
Article history: Received 22 December 2008 Received in revised form 14 April 2009 Accepted 25 April 2009 Available online 5 May 2009 Keywords: Artemisia douglasiana Dehydroleucodine Antimicrobial activity Helicobacter pylori
a b s t r a c t Ethnopharmacological relevance: The increasing resistance of Helicobacter pylori to antibiotics demands the search for novel compounds from plant based sources. Artemisia douglasiana Besser is widely used in Cuyo region (Argentina) as folk medicine for the treatment of gastric ailments. Aim of study: Based on our previous studies that Artemisia douglasiana exert cytoprotective actions against ethanol-induced gastric mucosal injury we assayed the anti-Helicobacter pylori effect of the Artemisia douglasiana extract and its active compound, dehydroleucodine. Materials and methods: The in vitro anti-bacterial activity of Artemisia douglasiana extract and its active compound, dehydroleucodine were determined against one standard strain and six clinical isolates of Helicobacter pylori by using the agar dilution methods. Results: The results showed that both dehydroleucodine and Artemisia douglasiana extract had activity against the microorganism with MICs between 1–8 and 60–120 mg/L, respectively. Conclusions: Artemisia douglasiana may be a useful alternative treatment strategy principally in eradication of metronidazole and clarithromycin-resistant strain. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Helicobacter pylori colonizes human gastric mucous layer and has been regarded as a major cause of chronic gastritis and peptic ulcer with growing incidences worldwide, and is important in the pathogenesis of gastric cancer (Dunn et al., 1997). The eradication of Helicobacter pylori can therefore, contribute to the treatment and prevention of these gastroduodenal diseases (Kawakami et al., 2006). Many regimens are currently available based on triple therapy containing two antibiotics and a proton pump inhibitor; however Helicobacter pylori rapidly acquire resistance to some antibiotics. Probably the selection of resistant strains increases because antibiotic treatment of Helicobacter pylori involves drugs largely used for other kinds of infections, such as the use of metronidazole (MTZ) for anaerobic and parasitic infections (Dunn et al., 1997; Tankovic et al., 2000) or the use of clarithromycin (CLA) in respiratory tract infections (Kato et al., 2002). Indeed, Helicobacter pylori strains resistant to CLA and MTZ have been increasing (Torres et al., 2001; Alarcón et al., 2003). Therefore, a search for new antibacterial agents, both
Abbreviations: AE, aqueous extract; CLA, clarithromycin; DhL, dehydroleucodine; MIC, minimum inhibitory concentration; MTZ, metronidazole. ∗ Corresponding author. Fax: +54 02652 430224. E-mail address: [email protected] (A.E. Vega). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.04.051
highly effective and safe, that can serve as alternative solutions for the treatment of Helicobacter pylori is very important. Several investigators have documented that plants and plant products possess potent medicinal properties, suggesting that may be useful for specific medical conditions (Cos et al., 2006; Raju et al., 2007). The World Health Organization (WHO) estimates that 4 billion people (80% of the World’s population) use herbal medicines for some aspects of primary healthcare (Farnsworth et al., 1985). Many medicinal and food plants are used around the world for the treatment of gastritis, dyspepsia and peptic ulcers, and some plants species also have experimental antibacterial activity (Ankli et al., 2002; Stege et al., 2006; Park et al., 2008). In Argentina Artemisia douglasiana Besser (Asteraceae) leaves are used in folk medicine and is known by the vernacular name of “matico”. The popular use of the infusion prepared by boiling leaves of Artemisia douglasiana is as a cytoprotective agent against peptic ulcer, and is also used for the treatment of external sores (Ariza Espinar and Bonzan, 1992). The extract of Artemisia douglasiana prevents the gastric injury induced by absolute ethanol (Giordano et al., 1990). The main active cytoprotective principle of Artemisia douglasiana is dehydroleucodine (DhL), a sesquiterpene lactone of the guiainolide type (Fig. 1). The aim of this study was to evaluate the antibacterial activity of Artemisia douglasiana, as well as the active compound DhL against Helicobacter pylori strains.
654
A.E. Vega et al. / Journal of Ethnopharmacology 124 (2009) 653–655
with 7% horse blood under the same microaerophilic atmosphere mentioned above (5% O2 , 10% CO2 , and 85% N2 ) at 37 ◦ C for 48 h. The bacterial suspension in Mueller–Hinton broth was adjusted to a final concentration of a McFarland No. 0.5 standard, 2 L of the adjusted inoculum was delivered to the agar plates. After 72 h of incubation under the microaerophilic atmosphere (5% O2 , 10% CO2 , and 85% N2 ) at 37 ◦ C, the MIC of CLA, MTZ, DhL, and Artemisia douglasiana extract was determined. The MIC was recorded as the lowest concentration of drugs inhibiting visible growth. Resistance was defined as the CLA MIC being ≥2 mg/L and MTZ MIC being ≥8 mg/L (NCCLS, 2000; Glupczynski et al., 2001). All experiments were conducted in duplicate. Fig. 1. Structure of dehydroleucodine, isolated from Artemisia douglasiana.
3. Results and discussion 2. Materials and methods 2.1. Extraction and purification of DhL Artemisia douglasiana was collected in the mountains of the province of San Luis, Argentina, and a voucher specimen was deposited in the Herbarium of the Universidad Nacional of San Luis (UNSL No. 55). The powdered, dried aerial part of Artemisia douglasiana (10 g) were extracted by infusion with distilled water (1 × 100 mL). The infusion was filtered after 20 min to obtain the extract. The air-dried material of the aerial parts (580 g) was soaked in chloroform at room temperature. The extracts were evaporated in vacuum and dissolved in 95% ethanol. After addition of 4% aqueous lead tetraacetate solution, the aqueous cloudy solution was filtered through a celite pad, and the filtrate was concentrated under vacuum. The mixture was extracted three times with chloroform and the solution was concentrated under vacuum. The final residue was chromatographed in a medium-pressure chromatography system using 1:9 EtAcO/hexane as eluent (stationary phase: Silica gel 60G). DhL (100% purity, 1 g) was identified by 1 H or 13 C nuclear magnetic resonance, mass spectrometry, or melting point analysis, and its structure was identical to that cited by other authors (Giordano et al., 1990). 2.2. Antimicrobial activity 2.2.1. Bacterial strains The reference strain of Helicobacter pylori (NCTC 11638) obtained from Microbiology Service of the Hospital Universitario de la Princesa, Madrid, Spain, and six clinical isolates obtained from gastric antral biopsy specimens were used for this study. Helicobacter pylori strains were grown in Mueller–Hinton agar supplemented with 7% horse blood under a microaerophilic atmosphere (5% O2 , 10% CO2 , and 85% N2 ) at 37 ◦ C for 7–10 days and the identification was based on microaerophilic growth requirement, morphology, Gram’s stain, and oxidase, catalase and urease reaction. Strains were stored in trypticase soy broth supplemented with 20% glycerol at −80 ◦ C until use. 2.2.2. Determination of minimum inhibitory concentration The antibacterial activity was assayed by agar dilution method, according to National Committee for Clinical Laboratory Standards guidelines (NCCLS, 2000) using Mueller–Hinton agar supplemented with 7% horse blood. Serial dilutions of the CLA (Abbott Laboratories, Argentina) and MTZ (Sigma Chemical Co., St. Louis, MO) ranging from 128 to 0.008 mg/L were used as a control in the susceptibility test. DhL was dissolved in distilled water and added to Mueller–Hinton agar supplemented with 7% horse blood at concentrations ranging from 0.008 to 128 mg/L. Artemisia douglasiana extract was tested in the range of 40 at 150 mg/L. The bacteria were subcultured on Mueller–Hinton agar supplemented
All clinical isolates were CLA resistant and one strain was MTZ resistant. Reference strain and two clinical isolates were inhibited at 60 mg/L of the extract of Artemisia douglasiana (EA). Complete inhibition in vitro was achieved by 150 mg/L of EA, however 71.4% (5/7) of strains was inhibited at 90 mg/L of EA. DhL showed activity against all strains tested with MICs ranging from 1 to 8 mg/L. Reference strain and one clinical isolate showed lowest inhibition with DhL, 2 and 1 mg/L, respectively (Table 1). The eradication of Helicobacter pylori is seen as important in accelerating the healing and preventing the relapse of gastrointestinal diseases, principally peptic ulcer. However the primary impediments to successful treatment are lack of compliance with the drug regimens for undesirable side effects, contraindications, significant costs and development of antibiotic-resistant Helicobacter pylori strains (Ali et al., 2005). An increase of resistance to clarithromycin was observed in the Helicobacter pylori strains both children and adults (Vega et al., 2003). Since prehistoric times, people have used natural resources for medicinal purposes. The folk medicine of Argentina employs many plants to counteract diverse diseases such as catarrh, bronchitis, pneumonia, ulcers and diarrhoeas (Tousarkissian, 1980). Artemisia douglasiana is widely utilized in the Cuyo region (Argentina), as a folk medicine for its beneficial effects in patients with gastroduodenal disorders. DhL increases the synthesis of gastric mucosal glycoproteins (Guardia et al., 1994), confirmed by histological studies in gastric and duodenal mucous (Piezzi et al., 1995). It prevents the formation of the gastric mucous lesions induced by absolute ethanol and by other necrotizing agents (Giordano et al., 1990; Penissi et al., 1998). The mechanism of the protective action of DhL remains unknown but it seems to be related to PGE (Maria et al., 1998). On the other hands, recurrent ulcer disease after peptic ulcer perforation mainly occurs in patients with Helicobacter pylori infection, and all patients with perforated peptic ulcer should be treated. Helicobacter pylori-induced nuclear factor NF-B activation, and the subsequent release of interleukin (IL)-8 plays an important role
Table 1 In vitro antibacterial activity (mg/L) of clarithromycin (CLA), metronidazole (MTZ), dehydroleucodine (DhL) and Artemisia douglasiana Besser extract (AE) against Helicobacter pylori. Helicobacter pylori strains
NCTC 11638 HP781 HP786 HP788 HP789 HP795 HP796
MIC (mg/L) DhL
AE
CLA
MTZ
2 4 1 8 8 4 2
60 90 60 120 120 90 60
0.125 8 8 4 8 8 4
0.5 16 1 2 1 2 1
A.E. Vega et al. / Journal of Ethnopharmacology 124 (2009) 653–655
in gastric mucosal inflammation response induced by Helicobacter pylori infection (Aihara et al., 1997; Naumann, 2001; Lai et al., 2008) Artemisia douglasiana and DhL activities against Helicobacter pylori may relate to the anti-inflammatory effect. Previous findings in our laboratory have shown that DhL may prevent or reverse the inflammation process in rats. DhL inhibited both chronic and acute adjuvant carrageenan-induced inflammation phases, being most effective in the chronic phase (Guardia et al., 2003). An anti-inflammatory effect has previously been observed in other sesquiterpene lactones (Hall et al., 1979). Recently, several Asteraceae (Artemisia ludoviciana ssp. mexicana, Polymia maculata) have been identified as potent inhibitors of the transcription factor NFB, a central mediator of the inflammatory response (Bork et al., 1997). Extracts from Mexican Indian medicinal plants used in traditional indigenous medicine for inflammation treatment contain sesquiterpene lactones, which specifically inhibit transcription factor NF-B (Ankli et al., 2002). Band-shift experiments with total HeLa cell proteins revealed that DhL prevents NF-B induction (Wendel et al., 2002). Furthermore, Helicobacter pylori infection induces active inflammation with neutrophilic infiltrations as well as production of oxygen free radicals (Chang et al., 2004). The data reported in previous work indicate that the extract of Artemisia douglasiana and DhL have an antioxidant capacity (Maria et al., 2000). Natural products, either as pure compounds or as standardized plant extracts, provide unlimited opportunities for new drug leads because of the unmatched availability of chemical diversity (Cos et al., 2006). Results obtained in this study indicate that Artemisia douglasiana extract and its active compound DhL, have significant antimicrobial properties against Helicobacter pylori, and it could be a promising native herb treatment for patients with gastric ulcer caused by Helicobacter pylori. Although evidence on efficacy of the tested plant has been obtained, further investigations are necessary prior to their recommendation for use as safe and effective agent. Acknowledgements We thank Dr. Patricia Gomez for supplying biopsies, Abbott laboratories for providing clarithromycin. This work was supported by funds of C. y T. Projects 8802, 9303 and 8504 from the Universidad Nacional of San Luis, Argentine. References Aihara, M., Tsuchimoto, D., Takizawa, H., Azuma, A., Wakebe, H., Ohmoto, Y., Imagawa, K., Kikuchi, M., Mukaida, N., Matsushima, K., 1997. Mechanisms involved in Helicobacter pylori-induced interleukin-8 production by a gastric cancer cell line, MKN45. Infection and Immunity 65, 3218–3224. Alarcón, T., Vega, A.E., Domingo, D., Martínez, M.J., López-Brea, M., 2003. Clarithromycin resistance among Helicobacter pylori strains isolated from children: prevalence and study of mechanism of resistance by PCR-restriction fragment length polymorphism analysis. Journal of Clinical Microbiology 41 (1), 486–499. Ali, S.M., Khan, A., Ahmed, I., Musaddiq, M., Ahmed, K., Polasa, H., VenkateswarRao, L., Habibullah, C.H., Sechi, L., Ahmed, N., 2005. Antimicrobial activities of eugenol and cinnamaldehyde against the human gastric pathogen Helicobacter pylori. Annals Clinical. Microbiology and Antimicrobial 4, 1–7. Ankli, A., Heinrich, M., Bork, P., Wolfram, L., Bauerfeind, P., Brun, R., Schimd, C., Weiss, C., Bruggisser, R., Gertsch, J., Wasescha, M., Sticher, O., 2002. Yucatec Mayan medicinal plants: evaluation based on indigenous uses. Journal of Ethnopharmacology 79, 43–52. Ariza Espinar, L., Bonzan, N., 1992. El matico de la región de Cuyo (Argentina). Acta Farmaceutica Bonaerense 11, 139–145. Bork, P.M., Lienhard Schmitz, M., Kuhnt, M., Escher, C., Heinrich, M., 1997. Sesquiterpene lactone containing Mexican Indian medicinal plants and pure
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