Investigation of the anti-mycobacterial and cytotoxic effect of three medicinal plants used in the traditional treatment of tuberculosis in northern Mexico and the Southwest United States
Tuberculosis (TB) remains a major global public health threat. There is an urgent need for the development of new alternative treatments for the disease due to the increasing prevalence of drug and multidrug-resistant TB strains being detected around the world. The purpose of the in-vitro experimental pilot and main study was to investigate the mycobactericidal activity and cytotoxicity of three medicinal plants were "Gordolobo"(Pseudognaphalium canescens), "L. porteri (chuchupaste)" (Ligusticum Porteri ), and "Cuachalalate" (Amphiterygium adstringens) used in the traditional treatment of TB and other respiratory infections by the Tarahumara and other indigenous groups from northern Mexico and the Southwestern U.S. The working hypothesis was that L. porteri, A. adstringens and P. canescens would exhibit good in vitro killing activity of ≥50% against M. tuberculosis. It also was hypothesized that the three medicinal plants would have a cytotoxicity of ≤50% in human (U-937) cells. Fresh plants were obtained from plant vendors in local markets. Plant genus and species were verified by the UTEP Department of Biology herbarium. The three plants were washed with distilled water and machine dried at 40°C for 8 hours. Subsequently, they were each ground and macerated in 100% ethanol with a Soxhlet machine. The crude extract was evaporated to dryness at 45°C in a rotovapor machine to obtain the solid extract. Each solid extract was then diluted in distilled water. The high hydrophobicity of the A.adstringens plant also required an extra step which involved homogenization with dimetyl sulfoxide (DMSO). The three plant extracts next were filtered with a 0.22μm Millipore syringe filter and then separated in aliquots under refrigeration at −20°C prior to use in the experiments. The concentrations of the plant extracts that were prepared for use in the experiments were 100μg/ml, 200 μg/ml, 400 μg/ml, 800 μg/ml, 1600 μg/ml, and 3200 μg/ml. The first experiment assessed the mycobactericidal activity of each of the three extracts. A Middlebrook 7H11 agar medium with 0.5% glycerol was prepared. It was sterilized prior to adding a thermolabile OADC enrichment solution. Specific concentrations of each plant extract were then added and homogenized to the medium before solidification. All samples were prepared in triplicate. These were then transported to the El Paso-City County Tilman Laboratory were they were inoculated with a Mycobacterium tuberculosis H37Rv strain which had previously been tested for antibiotic drug sensitivity. The growth of bacterial colonies exposed to six concentrations of the three plant extracts were compared with the colony growth in control containing culture medium control. In addition, three first-line antibiotic drugs: isoniazid (1μg/ml), rifampin (1μg/ml), and ethambutol (5μg/ml) served as positive controls. Bacterial growth was evaluated after incubation at 20°C for 4–6 weeks. The Minimum Inhibitory Concentration (MIC) was defined as the lowest concentration required to inhibit ≥50% of M. tuberculosis growth. The second experiment evaluated the cytotoxicity of the plant extracts in human promonocyte U-937 cells, defined as the concentration killing 50% or more of the cells (LD50). The U-397 cells suspended in RPMI-1640 with 10% heat-inactivated fetal calf serum were cultured in the presence of 5% CO2 at 37°C. Subsequently, plant extracts previously dissolved in saline or DMSO were added in concentration ranging from 12 to 400μg/ml in relation with the culture media. Then, the cells were cultured for 48 hours. The cells were then pulsed during the last 18 hours of culture with 1 μCi of [3H]-thymidine. They were harvested on glass fiber filters. [3h]-thymidine incorporation was determined by liquid scintillation counting in a β-liquid scintillation counter (Perking Elmer Tri-Carb2900TR). All samples were prepared in triplicate. The results of this experiment are expressed as LD50. The results of the anti-mycobactericidal experiments revealed that all three plant extracts showed growth inhibition at low concentrations, however L. porteri (chuchupaste) inhibited more than 50% of the growth at the lowest used (5μg/ml). The results of the cytotoxicity experiment revealed 0% of cytotoxicity at the lowest concentration for all plants used. Specifically L. porteri (chuchupaste) was not found to be toxic to human cells until a concentration of 50–100μg/ml. In contrast, Gordolobo and A.adstringens showed significant cytotoxicity at concentrations as low as 12.5–25μg/ml and 25–50μg/ml, respectively. In conclusion these results suggest that L. porteri (chuchupaste), a traditional plant used by Tarahumara Indians to treat TB and other respiratory infections, may possess therapeutic value. Its potential warrants further investigated. Future in vivo studies should be conducted to further explain the antimycobactericidal action and cytotoxicity of L. porteri (chuchupaste).
Beltran, Oscar, "Investigation of the anti-mycobacterial and cytotoxic effect of three medicinal plants used in the traditional treatment of tuberculosis in northern Mexico and the Southwest United States" (2008). ETD Collection for University of Texas, El Paso. AAI1456752.