Antimycobacterial activity of fruit of Zanthoxylum acanthopodium DC against Mycobacterium smegmatis

Document Type : Original Research Article

Authors

1 Research Center for Biology - The Indonesian Institute of Sciences (LIPI). Jl. Raya Jakarta – Bogor Km 46, Cibinong 16911, Indonesia

2 Research Center for Chemsitry - The Indonesian Institute of Sciences (LIPI). Jl. Kw. Puspiptek, Muncul, Serpong, Kota Tangerang Selatan, Banten 15314, Indonesia

Abstract

Objective: Fruits of lemon pepper (Zanthoxylum acanthopodium DC., Rutaceae) have been traditionally used as a spice and in folk medicine for treatment of diarrhea and stomachache. Stomachache could be associated with mycobacterial infection. The present study was designed to investigate the activity of Z. acanthopodium fruits against a non-infectious Mycobacterium smegmatis and to identify the important phytochemical constituent that is toxic towards mycobacteria.
Materials and Methods: The broth microdilution method was used to determine the minimal inhibitory concentration (MIC) of ethyl acetate or hexane extract of green, young fruits of Z. acanthopodium. Effect of active extract (hexane) on cell membranee integrity was studied by measuring sodium and potassium leakage into extracelullar liquid using Atomic Absorbtion Spectrophotometer (AAS). Next, cell morphology was observedby using Scanning Microscope Electron (SEM).  Column chromatography was used for fractionation and purification of  hexane extract while the chemical structure of the active compound was determined using NMR technique. Rifampicin, an antimycobacterial compound, was used as positive control.
Results: Hexane extract was active against M. smegmatis with an MIC of 64 µg/ml. Plant extract at the concentration of 128 µg/ml caused ions leakage. Concentration of sodium in extracellular liquid of cells treated with plant extract was significantly higher than that of untreated cells. SEM observation revealed cell wall deformation in cultures treated with the extract. NMR spectroscopy analysis of the most active fraction revealed that the compound that exerted toxicity on M. smegmatis was geranyl acetate.
Conclusion: Geranyl acetate was an important constituent of Z. acanthopodium fruit that has antimycobacterial activity. Possibly, Z. acanthopodium fruit exert its toxic effects against M. smegmatisthrough damaging cell membrane.

Keywords

Main Subjects


Broxmeyer, L, Sosnowska D, Miltner, E., Chacón, O, Wagner, D., McGarvey, J., Barletta, R.G., Bermudez, L.E., 2002. Killing of Mycobacterium avium and Mycobacterium tuberculosis by a Mycobacteriophage Delivered by a Nonvirulent Mycobacterium: A Model for Phage Therapy of Intracellular Bacterial Pathogens. J Infect Dis, 186:1155-1160
Burke KA, Patel A, Jayaratnam A, Thiruppathy K and Snooks S. 2014. Diagnosing abdominal tuberculosis in the acute abdomen. ‎Int J Surg, 12: 494-499
Campen RL, Ackerley DF, Cook GM. and O'Toole RF. 2015. Development of a Mycobacterium smegmatis transposon mutant array for characterising the mechanism of action of tuberculosis drugs: Findings with isoniazid and its structural analogues. Tuberculosis, 95:432-439
Chakraborti AK and   Gulhane R. 2003.  Perchloric acid adsorbed on silica gel as a new, highly efficient, and versatile catalyst for acetylation of phenols, thiols, alcohols, and amines. Chem Commun, 1896-1897
Ding, LW, Lai CC, Lee LN, and Hsueh PR. 2006. Abdominal nontuberculous mycobacterial infection in a university hospital in Taiwan from 1997 to 2003. J Formos Med Assoc. 105:370-376
El Baz AF and Shetaia YM. 2005. Evaluation of Different Assays for the Activity of Yeast Killer Toxin. Int J Agr Biol, 7: 1003 - 1006
Fan S Li DF, Wang DC, Fleming J, Zhang H, Zhou Y, Zhou L, Zhoud J,  Chen T, Chen G, Zhang XE, and Bi L. 2015. Structure and function of Mycobacterium smegmatis 7-keto-8-aminopelargonic acid (KAPA) synthase.  Int J Biochem Cell B, 58:71-80
Figueroa D, Guzman N, and Isache C. 2016. Tuberculous Enteritis: A Rare Complication of Miliary Tuberculosis.  Case Reports in Infectious Diseases. Article ID 6949834, 3 pages http://dx.doi.org/10.1155/2016/6949834.
Gupta S, Pawaria S, Lu C,  Hade MD, Singh C, Yeh, SR, and Dikshit KL. 2012. An unconventional hexacoordinated flavohemoglobin from Mycobacterium tuberculosis. J Biol Chem, 287: 16435–16446.
Merali N, Chandak P, Doddi S, Sinha P. 2015. Non-healing gastro-duodenal ulcer: A rare presentation of primary abdominal tuberculosis.Int J Surg Case Rep, 6: 8-11
Moodley S,  Koorbanally NA, Moodley T, Ramjugernath D, and Pillay M. 2014. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay is a rapid, cheap, screening test for the in vitro anti-tuberculous activity of chalcones. J. Microbiol Methods,  104: 72–78
Nazzaro F, Fratianni F, De Martino L, Coppola R, and De Feo V. 2013. Effect of Essential Oils on Pathogenic Bacteria. Pharmaceuticals, 6: 1451-1474.
Parhusip A, Yasni S,  and Elisabeth Y.2003. Kajian metode ekstraksi andaliman (Zanthoxylum acanthopodium DC.) terhadap mikroba patogen dan perusak pangan. J Ilmu Tekno Pangan, 1: 112-123
Piccaro G, Pietraforte D, Giannoni F, Mustazzolu A, and Fattorinia L. 2014. Rifampin Induces Hydroxyl Radical Formation in Mycobacterium tuberculosis.    Antimicrob Agents Chemother, 58: 7527-7533
Saravanakumar, DEM, Folb PI, Campbell BW and Smith P. 2008.. Antimycobacterial Activity of the Red Alga Polysiphonia virgata.  Pharm Biol,  46: 254-260.
Singh, B., Jain M, Singh SV, Dhama K, Aseri GK, Jain N, Datta M, Kumar N, Yadav P, Jayaraman S, Gupta S, Chaubey, KK and Sohal JS. 2015. Plants as Future Source of Anti-Mycobacterial Molecules and Armour for Fighting Drug Resistance. J Anim VetAdv, 10: 443-460.
Staudinger TR, Redl RB, and Glasgow BJ. 2014. Antibacterial activity of rifamycins for M. smegmatis with comparison of oxidation and binding to tear lipocalin.  Biochim Biophys Acta, Proteins Proteomics,1844:750-758
Suriyanarayanan B, Shanmugam K, and Santosh, RS. 2013. Synthetic quercetin inhibits mycobacterial growth possibly by interacting with DNA gyrase. Rom Biotechnol Lett, 18: 8587-8593.
Trombetta D, Castelli F, Sarpietro MG, Venuti V, Cristani M, Daniele C, Saija A,  Mazzanti G, and Bisignano G. 2005. Mechanisms of Antibacterial Action of Three Monoterpenes. Antimicrob Agents Chemother, 49: 474–2478
Vik A, James A, and Gundersen LL. 2007. Screening of Terpenes and Derivatives for Antimycobacterial Activity; Identification of Geranylgeraniol and Geranylgeranyl Acetate as Potent Inhibitors of Mycobacterium tuberculosis in vitro. Planta Medica, 73: 1410-1412
Yanti, Pramudito TE, Nuriasari N, and Juliana K. 2011. Lemon Pepper Fruit Extract (Zanthoxylum acanthopodium DC.) Suppresses the Expression of Inflammatory Mediators in Lipopolysaccharide-Induced Macrophages In Vitro. Am J Biochem Biotechnol, 7: 190-195
Zhang P, Minardi LM, Kuenstner JT, and Kruzelock R. 2015. Mycobacterium avium subspecies hominissuis in Crohn's disease: a case report.  Gastroenterol Rep, 1–4