Abstract
Helicobacter pylori is the major etiologic agent of such gastric disorders as chronic active gastritis and gastric carcinoma. Over the past few years, the appearance of antibiotic-resistant bacteria has led to the development of better treatments, such as the use of natural products. This study evaluated the anti–H. pylori activity of 17 Mexican plants used mainly in the northwestern part of Mexico (Sonora) for the empirical treatment of gastrointestinal disorders. The anti–H. pylori activity of methanolic extracts of the plants was determined by using the broth microdilution method. The 50% minimum inhibitory concentrations ranged from less than 200 to 400 μg/mL for Castella tortuosa, Amphipterygium adstringens, Ibervillea sonorae, Pscalium decompositum, Krameria erecta, Selaginella lepidophylla, Pimpinella anisum, Marrubium vulgare, Ambrosia confertiflora, and Couterea latiflora and were greater than 800 μg/mL for Byophyllum pinnatum, Tecoma stans linnaeus, Kohleria deppena, Jatropha cuneata, Chenopodium ambrosoides, and Taxodium macronatum. Only Equisetum gigantum showed no activity against H. pylori. This study suggests the important role that these plants may have in the treatment of gastrointestinal disorders caused by H. pylori. The findings set the groundwork for further characterization and elucidation of the active compounds responsible for such activity.
Introduction
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Antibiotic treatment with a combination of 2 or 3 drugs has been successfully used to eradicate H. pylori infections. 3 However, infection recurs in some patients, and antibiotic resistance in strains all around the world continues to increase. 4 –6 Thus, it is important to seek new and effective anti–H. pylori drugs, and plants are a logical source of such new compounds. 7 For instance, anti–H. pylori activity has been reported in an extract of the lichen Cetraria islandica, in an aerial extract of Terminalia spinosa, and in Chinese green tea. 8 –10 In addition, a methanolic extract of the bark of Pteleopsis suberosa was shown to have anti–H. pylori activity and was used to treat gastric ulcers in some patients. 11
Of the 26,000 plant species identified in Mexico, an estimated 4,000 have some medicinal use. Among the plants used for gastrointestinal diseases in traditional Sonora medicine are Byophyllum pinnatum, Ambrosia confertiflora, Krameria erecta, Pscalium decompositum, Tecoma stans linnaeus, Amphipterygium adstringens, Kohleria deppena, Castella tortuosa, Coutarea latiflora, Chenopodium ambrosoides, Equisetum gigantum, Taxodium macronatum, Pimpinella anisum, Ibervillea sonorae, Marrubium vulgare, Jatropha cuneata and Selaginella lepidophylla.
We sought to evaluate the antibacterial activity of 17 native Mexican medicinal plants against 2 H. pylori strains (reference strain American Type Culture Collection [ATCC] 43504 and clinical isolate strain 25).
Materials and Methods
Plant material
Table 1 describes the plant species used in this study. They were selected on the basis of traditional medicinal uses against different gastrointestinal disorders. Dry samples of Byophyllum pinnatum, Ambrosia confertiflora, Krameria erecta, Pscalium decompositum, Tecoma stans linnaeus, Amphipterygium adstringens, Kohleria deppena, Castella tortuosa, Coutarea latiflora, Chenopodium ambrosoides, Equisetum gigantum, Taxodium macronatum, Pimpinella anisum, Ibervillea sonorae, Marrubium vulgare, Jatropha cuneata, and Selaginella lepidophylla were obtained from a local natural product store in Hermosillo, Sonora (northwestern Mexico). The anti–Helicobacter activity of Amphipterygium adstringens and its components has been previously reported 12 and was included in the present study for comparison.
Preparation of extracts
Plant extracts were prepared from dry plant samples, which were finely grounded with a Wiley mill (200 mesh) and extracted with methanol (1:10 w/v) at room temperature. Extractions were performed over a 10-day period, with brief manual shaking twice daily. The extracts were passed through filter paper, dried under reduced pressure at 45°C, and weighed. These “crude” methanolic extracts were dissolved in dimethyl sulfoxide (DMSO; Sigma Chemical Co., St. Louis, MO, USA) to a final concentration of 10 mg dry matter/mL. All extracts were stored in amber glass vials until use. 13
Microorganisms and growth conditions
Bacterial strains used in this study were as follows: H. pylori strain 25 (clinical isolate obtained from a gastric adenocarcinoma) and reference strain ATCC no. 43504 (provided by Dr. Humberto Astiazarán-García). The bacteria were grown in Brucella broth containing 10% horse serum and supplemented with antibiotics as reported elsewhere. 14 Before each assay, the bacteria were cultured for 4 days under microaerophilic conditions, then subcultured for 24 hours. The OD630 (optical density at 630 nm) was adjusted to 0.08, matching the turbidity of the 0.5 McFarland standard.
Antimicrobial assay
In vitro antibacterial studies were carried out by using the broth microdilution method as described elsewhere, 15 with some modifications. Briefly, 100 μL of a 108 colony-forming units/mL bacterial suspension was inoculated into each well of a flat 96-well microplate (Costar, Corning, NY, USA) containing 100 μL of different concentrations of plant extracts (serial dilutions at final concentrations of 0–1500 μg/mL) in Mueller–Hinton broth. The methanolic extracts were first dissolved in DMSO and then diluted in sterile broth. Each antibacterial test also included wells containing the culture media plus DMSO in order to obtain a control of the solvent antibacterial effect. Bacterial cultures were incubated at 37°C, and plates were read at 630 nm in an enzyme-linked immunosorbent assay microplate reader (Benchmark Microplate Reader, Bio-Rad, Hercules, CA, USA) at 24-hour intervals, up to 96 hours. The minimal inhibitory concentration (MIC50) was defined as the lowest methanolic extract concentration that inhibited at least 50% of the bacterial growth after incubation at 37°C for 24 hours. Control experiments showed that a final concentration of DMSO (0.2%–0.8%) did not affect the bacterial growth.
Results
To determine the antibacterial activity of methanolic extracts from 17 plants from northwest Mexico (Table 1), we evaluated the effects of these plants on the growth of H. pylori, a human gastric pathogen associated with the development of gastric cancer. In general, the methanolic extracts of the plants tested exhibited broad-spectrum antimicrobial activity against both H. pylori strains, which was more evident at 96 hours after treatment. At that time, the growth of H. pylori was assessed by means of an OD630 measurement in comparison with extract-free cultures, using the microplate dilution method.
The results shown in Table 2 indicate that the Castella tortuosa, Ibervillea sonorae, and Ambrosia confertiflora extracts were the most efficient (MIC50<200 μg/mL) bacterial growth inhibitors, followed by extracts from Amphipterygium adstringens, Krameria erecta, Selaginella lepidophyla, and Couterea latiflora (MIC50 at 400 μg/mL). The rest of the extracts, with the exception of Equisetum giganteum (which showed no activity against the pathogen), required concentrations higher than 800 μg/mL to inhibit the growth of both H. pylori strains.
Results are shown as the MIC50 obtained from 3 independent repetitions.
MIC50, lowest methanolic extract concentration that inhibited at least 50% of bacterial growth; ND, not detected with concentrations tested.
Furthermore, methanolic extracts of Castella tortuosa, Ibervillea sonorae, and Ambrosia confertiflora showed the most effective antibacterial activity on H. pylori growth because they fully inhibited growth of both strains at a concentration of 200 μg/mL (100% inhibition). Extracts of Pimpinella anisum and Pscalium decompositum only inhibited the growth of H. pylori strain 25 at the same degree, indicating a bactericidal activity against the human pathogen.
Discussion
In traditional medicine, the use of plants to treat different health ailments has evolved through trial and error; within Sonora's tribes, their use for gastrointestinal disorders has been reported. 16
Some research groups have evaluated antibacterial activities of different natural sources, such as garlic (Allium sativum), cranberry (Vaccinium macrocarpon), and oregano (Origanum vulgare) aqueous extracts, with different inhibitory effects on bacterial growth, indicating the possible role of flavonoids in such activity. 17 Furthermore, antibacterial activity of Sonoran propolis against gram-negative bacteria has been shown. 15 Thus, our present study evaluated 17 native Mexican medicinal plants, traditionally used for some gastrointestinal disorders, as inhibitors of H. pylori growth. We focused mainly on Sonora's flora at the northwestern region of Mexico.
We compared our results with those of studies that assessed other species with proven anti–H. pylori activity because we found no previous reports for most of the plants studied here (other than Amphipterygium adstringens). For instance, Wang and Huang studied herbs from Taiwanese folk medicinal plants and reported MIC values greater than 1 mg/mL, 18 superior to those reported here. Stege et al. evaluated cold extract, infusion, and decoction preparations of Larrea divaricata against 7 H. pylori strains; the average MIC value of 60 μg/mL 19 falls within the range for extracts with high anti–Helicobacter activity in our study (<200 μg/mL).
The activity reported here for Amphipterygium adstringens is equivalent to that previously reported; 12 that study showed the role of anacardic acids to be relevant.
Overall, H. pylori strain 43504 was more susceptible to the plant extracts tested than was the 25 strain, even though both strains present similar genotypic backgrounds. On the other hand, the 25 strain has been further characterized and shown to bind several compounds, such as lectins, 20 that could ultimately interact with plant compounds. Once the active compounds have been identified and characterized, their interaction with H. pylori will be better understood.
Footnotes
Acknowledgments
The present work was supported by the Mexican Council for Science and Technology (CONACyT, grant number 90355). This work was presented in part at the XVI Italo–Latino American Congress on Ethnomedicine. Special thanks to Carmen Escobar-Landaverde and Mónica L. Arteaga-Symonds for sample processing.
Author Disclosure Statement
No competing financial interests exist.