Anti-mycobacterial Activity of Extracts from Plants used in Mexico for the Treatment of Infectious Diseases
Samuel Estrada-Soto1, Valeria Arteaga-Gonzalez1, Patricia Castillo-Espana2, Gloria Maria Molina-Salinas3, Salvador Said-Fernandez3, Gabriel Navarrete-Vazquez1
1 Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Col. Chamilpa, 62209, Cuernavaca, Morelos, Mexico
2 Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Col. Chamilpa, 62209, Cuernavaca, Morelos, Mexico
3 División de Biología Celular y Molecular, Centro de Investigación Biomédica del Noreste, IMSS, San Luis Potosí y Dos de Abril, Colonia Independencia 64720, Monterrey, Nuevo León, Mexico
|Date of Submission||05-Jan-2009|
|Date of Decision||20-Jan-2009|
|Date of Acceptance||06-Feb-2009|
|Date of Web Publication||30-Dec-2009|
Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Col. Chamilpa, 62209, Cuernavaca, Morelos
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Twenty seven extracts from eight medicinal plants used in Morelos, Mexico, for the treatment of several infectious diseases were screened for their in vitro anti-Mycobacterium tuberculosis (anti-MTB) activity on H37Rv strain of Mycobacterium tuberculosis (MTB), using the microplate Atamar assay test. Extracts (hexane, dichloromethane and methanol) from wild specimen of Valerians edulis showed the most significant (p<0.05) activity against this strain (MICs values of 3.125, 25.0 and 50.0 μg/mL, respectively). On the other hand, hexanic and dichlorometanic extracts from in vitro-cultivated species of V. edulis also showed significantly activity against of M. tuberculosis (MICs values of 12.5 and 50.0 μg/mL, respectively). Most active extract (hexanic) obtained from V. edulis was also assayed against the drug-resistant strain (CIBIN 99), and the MIC value was 50 μmL. A qualitative TLC analysis of extracts from wild and in vitro-cultivated species of V. edutis showed that both content different chemical constituents. So, this last result explains the difference between anti-MTB activities of the extracts. Finally, dichloromethanic extracts from Lepechinia caulescens also showed a significantly activity against of M. tuberculosis (MIC 100 μglmL). In conclusion, V. edutis and L. caulescens hexanic extracts are sources for the isolation of new or known compounds that could be used as leads for development of potential anti-MTB drugs.
Keywords: Anti mycobacterial activity, Mycobacterium tuberculosis, plant extracts, Lepechinia caulescens, Valeriana edulis
|How to cite this article:|
Estrada-Soto S, Arteaga-Gonzalez V, Castillo-Espana P, Molina-Salinas GM, Said-Fernandez S, Navarrete-Vazquez G. Anti-mycobacterial Activity of Extracts from Plants used in Mexico for the Treatment of Infectious Diseases. Phcog Mag 2009;5:106-10
|How to cite this URL:|
Estrada-Soto S, Arteaga-Gonzalez V, Castillo-Espana P, Molina-Salinas GM, Said-Fernandez S, Navarrete-Vazquez G. Anti-mycobacterial Activity of Extracts from Plants used in Mexico for the Treatment of Infectious Diseases. Phcog Mag [serial online] 2009 [cited 2022 Jan 24];5:106-10. Available from: http://www.phcog.com/text.asp?2009/5/18/106/57966
| Introduction|| |
About one-third of the world's population infected and three million deaths per year are directly attributable to tuberculosis (TB). Currently, this health problem constitutes one of the primary causes of death or suffering cause in worldwide. Furthermore, multi-drug resistant strains of M. tuberculosis (MDR-MTB) as well as relationship between HIV/AIDS and TB have been main idea for the development of new anti-TB therapies  . According to World Health Organization reports, in 2003 there were 8.8 million new cases reported, killing 1.7 million of people worldwide. Based on a future trend, a total of 225 million new cases and 79 million deaths are expected from TB between 1998 and 2030  .
Although different types of anti-TB agents are available in world market for the treatment of this disease, there is a growing interest in herb remedies due unspecific side effects associated with synthetic therapeutics agents  . Because of natural products derived scaffolds are therapeutic templates for the design of new therapeutic drugs using medicinal chemistry and computer-assisted design techniques, they have had a remarkable impact on the treatment of TB in comparison with classical FDA-approved drugs such as rifampin, kanamycin and cycloserine. Anti- M. tuberculosis (anti-MTB) compounds isolated from natural sources as plants, fungi and marine organisms have been found with different skeleton chemical forms and conformations  .
In Mexico, a large number of plants have been used empirically in traditional medicine for the treatment of different diseases, including TB , [5,],,,, . Furthermore, it exist a great background of research in natural products that has allow found new drugs with potential therapeutic uses. In this context, the main goal of current research was to evaluate anti-MTB effect of eight Mexican medicinal plants used for the treatment of different diseases [Table 1]. Species selected for the study included Valeriana edulis (wild and in vitro-cultivated specimens), Cochlospermum vitifolium, Tournefortia hartwegiana, Lepechinia caulescens, Laelia autumnalis, Cordia morelosana, Lepidium virginicum and Verbena carolina  . The species were mainly selected for their ethnomedicinal uses in Mexican traditional medicine against parasitic infections. An in vitro assay was performed to proof anti-MTB activity using H37Rv strain, a drug-sensitive MTB strain, and drug-resistant strain (CIBIN 99)" by "An in vitro assay was performed to evaluate anti-MTB activity using a drug sensitive and drug resistant M. tuberculosis strains.
| Materials and Methods|| |
Plant collection was carried out between July 2002 and October 2006 from their natural habitat in different regions of Morelos, Mexico. Plants were collected and identified by Dr. P. Castillo-Espana and R. Ramirez and all voucher specimens were deposited at "Centro de Educacion Ambiental e Investigacion Sierra de Huautla" HUMO-Herbarium, Cuernavaca, Morelos, Mexico. Different parts of collected plants were obtained and treated individually. An in vitro-generated specimen of V. edutis developed from a wild specimen was also assayed  . The details of medicinal plants features and their acquisition code number are listed in [Table 1].
Preparation of extracts
Air-dried plant material (100 g from each species) were ground into powder and extracted exhaustively by maceration at room temperature with hexane, dichloromethane and methanol, respectively. Each solvent was replaced every 72 h during 3 times to allow a better extraction of metabolites. After a filtration process, the extracts were concentrated in vacuo at 40 °C, and the percentage yields determined.
Anti-Mycobacterium tuberculosis assay.
The activity of plant extracts against M. tuberculosis strains was tested using the microplate Alamar Blue assay (MABA) modified by Molina-Salinas and coworkers  . The strain of M. tuberculosis used for this assay were H 37 Rv (ATTC 27294), a strain sensitive at five first-line anti-TB drugs (streptomycin, isoniazid, rifampin, ethambutol and pyrazinamide) and drug-resistant strain (CIBIN 99), resistant to above anti-TB agents.
All organic extracts for anti-MTB bioassay were prepared at a concentration of 4 mg/mL in 100% of DMSO. The concentrations for plant extracts ranged from 100 to 0.195 μg/mL. Rifampin and Ofloxacin (Sigma-Aldrich Co., St. Louis, MO, USA) were used as positive controls with concentrations ranging from 2.000 to 0.062 Rg of rifampin/mL and 16 to 0.50 Rg of ofloxacin/mL. All assays were performed in duplicate.
Qualitative thin layer chromatography of the extracts from V. edul is
Qualitative TLC analysis of V. edutis extracts (in vitro-cultivated and wild specimens) was carried out on Silica gel 60 F 254 (Merck®), using a dichloromethane:ethyl acetate (98:2) mixture as mobile phase. 50 pL of each extract (having a concentration of 1mg/mL) were added in the origin of the TLC plate.
| Results and Discussion|| |
All plants investigated in this opportunity, except V. edutis (wild), were mainly selected by their antiinfection properties into Mexican folklore medicine [Table 1]. The criterion to select screened plant species was their antimicrobial and anti-parasitic uses. This selection ensure to find potential natural sources for isolate new or known compounds that would be therapeutic agents in the treatment of this complex disease that, until this moment, remain killing people in worldwide.
Results of anti-MTB evaluations and minimal inhibitory concentration (MIC) values are shown in [Table 2]. From the plant species evaluated, only V. edulis ssp. procera (wild and in vitro-cultivated specimens) and L. caulescens showed considerable anti-MTB activity. Hexanic extracts of V. edulis rhizomes from both wild and in vitro-cultivated specimens were the most potent samples showing MIC values of 3.125 and 12.5 μg/mL, respectively. In addition, dichloromethane extracts from both specimens showed MICs values of 25 and 50 μg/mL, respectively. Finally, methanolic extract of wild V. edulis showed a MIC value of 50 μg/mL. Thus, hexanic extract from V. edulis wild specimen was more active than in vitro-cultivated specimen for the evaluation. These results could be related with metabolic contents of each specimen since active compound might be overproduced in wild species more than in vitro-cultivated species. In order to test this hypothesis in a qualitatively manner, we did TLC analysis of extracts obtained from both types of V. edulis [Figure 1]. This analysis showed that only hexanic extracts have similar phytochemicat profile, while dichloromethanic and methanotic extracts of both specimens have different types or less quantity of active compounds.
On the other hand, we decided to evaluate the more active extract (hexanic) obtained from V. edulis against the drug-resistant strain (MDR) CIBIN 99, a clinical isolate of Mycobacterium tuberculosis resistant to all five first-time antituberculosis drugs. The MIC value determined was 50μg/mL. Our results support the potential for identifying new compounds effective against MDR strains because we identified a plant species that has acceptable antituberculosis activity against both the sensitive and the resistant strains. In addition, there are some reports about isolation of sesquiterpenoid, monoterpenoid and iridoid scaffolds from genus Valeriana that could be involved in biological effect for this evaluation  .Previously studies about some species of Valeriana have demonstrated the useful in traditional medicine as sedative agents such as V. officinalis, V. wallichii, V. fauriei and V. angustifalia. This biological activity is related to the presence of valepotriates and other terpenoids derivates  . However, Gu and coworkers  reported that above-ground biomass and hexanic and dichloromethane extracts from V. laxiflora roots possess further anti-MTB activity besides having sedative effect. Bio-guided fractionation of these extracts led the isolation of active compounds as the new iridolactone [(4R, 5R, 7S,8S, 9S) - 7-hyd roxt-8-hydroxymethyl-4-methylperhydrocyclopenta[c]pyran-1-one] and the new lignan [(+)-1-hydroxy-2,b-bis-epi-pinoresinol] as well as betutinic acid, betulin, ursolic acid, oleanolic acid and other triterpenoid derivates, with MIC values that ranged among 15.5 and 127μg/mL  . Despite these results, hexanic extracts of both wild and in vitro-cultivated V. edulis showed better inhibitory effect for MTB being more potent than V. laxiflora.
Finally, hexanic extracts of L. caulescens flowers and roots also showed a relevant anti-MTB activity with MIC 100μg/mL. In addition, it's important to mention that Avila-Acevedo and colleagues  demonstrated that essential oil constituted with borneol, camphor and trans-caryophyllene possessed a significantly anti-Vibrio cholerae activity with MIC 4μg/mL.
To best of our knowledge, no previous research has been developed about anti-tuberculosis properties of all plant species assayed here. Bio-guided fractionations of active extracts are in progress in order to isolate and characterize the lead compounds.
| Acknowledgments|| |
This research was supported by CONACyT (SEP-2003C02-43440/280) and , PROMEP-SEP and IMSS FOFOI (2005/1/1/021) Arteaga-Gonzalez (Bachelor in pharmacy) is grateful to CONACyT for the scholarship grant (SEP-2003-C02-43440/280).
| References|| |
|1.||A.C. Giddens, HIM. Boshoff, S.G. Franzblau, C.E. Barry and B. R. Copp. Antimycobacterial natural products: synthesis and preliminary biological evaluation of the oxazole-containing alkaloid texaline. Tetrahedron Lett 46: 7355-7357 (2005). |
|2.||R.P. Tangallapally, R.E. Lee, A.J. Lenaerts and R.E. Lee. Synthesis of new and potent analogues of anti-tuberculosis agent 5-nitro-furan-2-carboxylic acid 4-(4-benzyl-piperazin1-yl)-benzylamide with improved bioavailability. Bioorg and Med Chern Lett 16: 2584-2589 (2006). [PUBMED] |
|3.||G.F. Pauli, R.J. Case, T. Inui, Y. Wang, S. Cho, N. Fischer and S.G. Franzblau. New perspectives on natural products in TB drug research. Life Sci 78: 485-494 (2005). |
|4.||R. Encarnacion, M. Virgen and N.Ochoa. Antimicrobial activity of medicinal plants from Baja California Sur (Mexico). Pharm Biol 36 33-43 (1998). |
|5.||Jimenez-Arellanes, M. Meckes, R. Ramirez, J. Torres and J. Luna-Herrera. Activity against multidrug-resistant Mycobacterium tuberculosis in Mexican plants used to treat respiratory diseases. Phytotherapy Res 17: 903-908 (2003). |
|6.||G.M. Molina-Salinas, M.C. Ramos-Guerra, J. Vargas Villarreal, B.D. Mata-Cardenas, P. Becerril-Montes and S. Said-Fernandez. Bactericidal activity of organic extracts from Fluorensia cernua DC against strains of Mycobacterium tuberculosis. Arch Med Res 37: 45-49 (2006). |
|7.||G.M. Molina-Salinas, A. Perez-Lopez, P. Becerril-Montes, R. Salazar-Aranda, S. Said-Fernandez and N.W. Torres. Evaluation of the flora of Northern Mexico for in vitro antimicrobial and antituberculosis activity. J Ethnophatmacol 109: 435-441 (2007). |
|8.||Monroy-Ortiz and P. Castillo.. Plantas medicinales utilizadas en el estado de Morelos. Centro de Investigaciones Biologicas, Universidad Autonoma del Estado de Morelos, Cuernavaca, Mexico, 2nd Ed. (2007). |
|9.||Rivero-Cruz, L. Acevedo, J.A. Guerrero, S. Martinez, R. Bye, R. Pereda-Miranda, S. Franzblau, B.N. Timmerman and R. Mata. Antimycobacterial agents from selected Mexican medicinal plants. J Pharm Pharmacol 57:1117-1126 (2005). |
|10.||G. Rojas, J. Levaro, J. Tortoriello and V. Navarro. Antimicrobial evaluation of certain plants used in Mexican traditional medicine for the treatment of respiratory diseases. JEthnopharmacot74:97-101 (2001). |
|11.||P. Castillo, J. Marquez, A. Rubluo, G. Hernandez and M. Lara. Plant regeneration from callus and suspension cultures from Valeriana edulis ssp. procera via simultaneous o ganogenesis and somatic embryogenesis. Plant Sciences 151:115-119 (2000). |
|12.||E.A. Carlini. Plants and the central nervous system. Pharm Biochem Behav75: 501-512 (2003) |
|13.||J.Q. Gu, Y. Wang, S.G. Franzblau, G. Montenegro, D. Yang and B.N. Timmermann. Antitubercular constituents of Valeriana laxiflora. Planta Med 70: 509-514 (2004). |
|14.||J. G. Avila-Acevedo, J.L. Munoz-Lopez, A., Martinez Cortes, A.M. Garcia-Bores, G. Martinez-Cortes and I. Penalosa-Castro. In vitro anti-Vibrio cholerae activity of essential oil from Lepechinia caulescens. Fitoterapia, 76: 104-107 (2005). |
[Table 1], [Table 2]