Evaluation of antibacterial and cytotoxic activity of Artemisia nilagirica and Murraya koenigii leaf extracts against mycobacteria and macrophages

BMC Complementary and Alternative Medicine

Avinash Sonawane¹  Rashmirekha Pati¹  Avinash Padhi¹  Soumitra Mohanty¹  Sumanta Kumar Naik¹ 

[1] School of Biotechnology, KIIT University, Campus-11, Bhubaneswar, Orissa 751024, India

关键词: Murraya koenigii;    Artemisia nilagirica;    Genotoxicity;    Cytotoxicity;    Antibacterial activity;    Mycobacteria;    Tuberculosis;   

Others  :  1220236
DOI  :  10.1186/1472-6882-14-87

 received in 2013-10-30, accepted in 2014-02-25,  发布年份 2014

【 摘 要 】

Background

Artemisia nilagirica (Asteraceae) and Murraya koenigii (Rutaceae) are widely distributed in eastern region of India. Leaves of Artemisia nilagirica plant are used to treat cold and cough by the local tribal population in east India. Murraya koenigii is an edible plant previously reported to have an antibacterial activity. Pathogenic strains of mycobacteria are resistant to most of the conventional antibiotics. Therefore, it is imperative to identify novel antimycobacterial molecules to treat mycobacterial infection.

Methods

In this study, ethanol, petroleum ether and water extracts of Artemisia nilagirica and Murraya koenigii were tested for antibacterial activity against Mycobacterium smegmatis and Mycobacterium bovis BCG in synergy with first line anti-tuberculosis (TB) drugs, and for cytotoxic activities on mouse macrophage RAW264.7 cells. Antibacterial activity was determined by colony forming unit (CFU) assay. Intracellular survival assay was performed by infecting RAW264.7 cells with M. smegmatis before and after treatment with plant extracts. Cytotoxity was checked by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay. Genotoxicity was studied by DAPI staining and COMET assay using mouse macrophage RAW264.7 cell line. Cell apoptosis was checked by Annexin-V/FITC dual staining method. Reactive oxygen species and nitric oxide production was checked by DCFH staining and Griess reagent, respectively.

Results

Ethanol extracts of A. nilagirica (IC₅₀ 300 μg/ml) and M. koenigii (IC₅₀ 400 μg/ml) were found to be more effective against Mycobacterium smegmatis as compared to petroleum ether and water extracts. M. koenigii extract showed maximum activity against M. bovis BCG in combination with a first line anti-TB drug rifampicin. M. koenigii leaf extract also exerted more cytototoxic (IC₅₀ 20 μg/ml), genotoxic and apoptosis in mouse macrophage RAW 264.7 cell line. Treatment of mouse macrophages with A. nilagirica extract increased intracellular killing of M. smegmatis by inducing production of reactive oxygen species and nitric oxide.

Conclusions

Ethanol extracts of A. nilagirica and M. koenigii were found to be more effective against mycobacteria. As compared to A. nilagirica, M. koenigii ethanol extract exhibited significant synergistic antibacterial activity against M. smegmatis and M. bovis BCG in combination with anti-tuberculosis drug rifampicin, and also showed increased cytotoxicity, DNA damage and apoptosis in mouse macrophages.

【 授权许可】

   
2014 Naik et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Bishai JD, Bishai WR, Bishai DM: Heightened vulnerability to MDR-TB epidemics after controlling drug-susceptible TB. PLoS ONE 2010, 5(9):e12843.
• [2]Skrahina A, Hurevich H, Zalutskaya A, Sahalchyk E, Astrauko A, van Gemert W, Hoffner S, Rusovich V, Zignol M: Alarming levels of drug-resistant tuberculosis in Belarus: results of a survey in Minsk. Eur Respir J 2012, 39:1425-1431.
• [3]Ríos JL, Recio MC: Medicinal plants and antimicrobial activity. J Ethnopharmacol 2005, 100(1–2):80-84.
• [4]Kokoska L, Polesny Z, Rada V, Nepovim A, Vanek T: Screening of some Siberian medicinal plants for antimicrobial activity. J Ethnopharmacol 2002, 82(1):51-53.
• [5]Khan R, Islam B, Akram M, Shakil S, Ahmad A, Ali SM, Siddiqui M, Khan AU: Antimicrobial activity of five herbal extracts against multi drug resistant (MDR) strains of bacteria and fungus of clinical origin. Molecules 2009, 14(2):586-597.
• [6]Adeniyi BA, Groves MJ, Gangadharam PRJ: In vitro anti-mycobacterial activities of three species of Cola plant extracts (Sterculiaceae). Phytother Res 2004, 18(5):414-418.
• [7]Cowan MM: Plant products as antimicrobial agents. Clin Microbiol Rev 1999, 12(4):564-582.
• [8]Ojha AK, Baughn AD, Sambandan D, Hsu T, Trivelli X, Guerardel Y, Alahari A, Kremer L, Jacobs WR Jr, Hatfull GF: Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria. Mol Microbiol 2008, 69(1):164-174.
• [9]Ahameethunisa AR, Waheeta H: Antibacterial activity of Artemisia nilagrica leaf extracts against clinical and phytopathogenic bacteria. BMC Complementary & Alternative medicine 2010, 10:6. BioMed Central Full Text
• [10]Meléndez PA, Capriles VA: Antibacterial properties of tropical plants from Puerto Rico. Phytomedicine 2006, 13:272-276.
• [11]Nagappan T, Ramasamy P, Wahid ME, Segaran TC, Vairappan CS: Biological activity of carbazole alkaloids and essential Oil of Murraya koenigii against antibiotic resistant microbes and cancer cell lines. Molecules 2011, 16:9651-9664.
• [12]Luize PS, Tiuman TS, Morello LG, Maza PK, Nakamura TU, Filho BPD, Cortez DAG, de Mello JCP, Nakamura CV: Effects of medicinal plant extracts on growth of Leishmania (L.) amazonensis and Trypanosoma cruzi. Brazilian Journal of Pharmaceutical Sciences 2005, 41:85-94.
• [13]Sonawane A, Santos JC, Mishra BB, Jena P, Progida C, Sorensen OE, Gallo R, Appelberg R, Griffiths G: Cathelicidin is involved in the intracellular killing of mycobacteria in macrophages. Cell Microbiol 2011, 13(10):1601-1617.
• [14]Kuo CC, Liu TW, Chen LT, Shiah HS, Wu CM, Cheng YT, Pan WY, Liu JF, Chen KL, Yang YN, Chen SN, Chang JY: Combination of arsenic trioxide and BCNU synergistically triggers redox-mediated autophagic cell death in human solid tumors. Free Radic Biol Med 2011, 51(12):2195-2209.
• [15]Jena P, Mohanty S, Mohanty T, Kallert S, Morgelin M, Lindstrøm T, Borregaard N, Stenger S, Sonawane A, Sørensen OE: Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages. PLoS One 2012, 7(12):e50345.
• [16]Mohanty S, Jena P, Mehta R, Pati R, Banerjee B, Patil S, Sonawane A: Cationic antimicrobial peptides and biogenic silver nanoparticles kill mycobacteria without eliciting DNA damage and cytotoxicity in mouse macrophages. Antimicrob Agents Chemother 2013, 57(8):3688-3698.
• [17]Jena P, Mohanty S, Mallick R, Jacob B, Sonawane A: Toxicity and antibacterial assessment of chitosan-coated silver nanoparticles on human pathogens and macrophage cells. Int J Nanomedicine 2012, 7:1805-1818.
• [18]Rastogi RP, Singh SP, Häder DP, Sinha RP: Detection of reactive oxygen species (ROS) by the oxidant-sensing probe 2′,7′-dichlorodihydrofluorescein diacetate in the cyanobacterium Anabaena variabilis PCC 7937. Biochem Biophys Res Commun 2010, 397(3):603-607.
• [19]Cogliati M, Roverselli A, Boelaert JR, Taramelli D, Lombardi L, Viviani MA: Development of an in vitro macrophage system to assess Penicillium marneffei growth and susceptibility to nitric oxide. Infect Immun 1997, 65(1):279-284.
• [20]Mikusová K, Slayden RA, Besra GS, Brennan PJ: Biogenesis of the mycobacterial cell wall and the site of action of ethambutol. Antimicrob Agents Chemother 1995, 39(11):2484-2489.
• [21]Viveiros M, Leandro C, Amaral L: Mycobacterial efflux pumps and chemotherapeutic implications. Int J Antimicrob Agents 2003, 22(3):274-278.
• [22]Sonawane A, Mohanty S, Jagannathan L, Bekolay A, Banerjee S: Role of glycans and glycoproteins in disease development by Mycobacterium tuberculosis. Crit Rev Microbiol 2012, 38(3):250-266.
• [23]Kureel SP, Kapil RS, Popli SP: Terpenoid alkaloids from Murraya koenigii Spreng.-II. The constitution of cyclomahanimbine, bicyclomahanimbine & mahanimbidine. Tetrahedrone Letters 1969, 44:3857-3862.
• [24]Handral HK, Pandith A, Shruthi SD: Review on murraya koenigii: multipotential medicinal plant. Asian Journal of Pharmaceutical and Clinical Research 2012, 5:5-14.
• [25]Mandal S, Nayak A, Kar M, Banerjee SK, Das A, Upadhyay SN, Singh RK, Banerji A, Banerji J: Antidiarrhoeal activity of carbazole alkaloids from Murraya koenigii Spreng (Rutaceae) seeds. Fitoterapia 2010, 81(1):72-74.
• [26]Tembhurne SV, Sakarkar DM: Beneficial effects of EthanolicExtract of murraya koenigii Linn. Leaves in cognitive deficit aged mice involving possible anticholinesterase and cholesterol lowering mechanism. International Journal of PharmTech Research 2010, 2(1):181-188.
• [27]Banerji A, Luthriya DL, Kokate SD: Toxicity of capillin, the insecticidal principle of Artemisia nilagirica Clarke. Indian J Exp Biol 1990, 28:588-589.
• [28]Shafi PM, Nambiar MKG, Clery RA, Sarma YR, Veena SS: Composition and antifungal activity of the oil of Artemisia nilagirica (Clarke) Pamp. JEOR 2004, 16:377-379.
• [29]Bishayee K, Paul A, Ghosh S, Sikdar S, Mukherjee A, Biswas R, Boujedaini N, Khuda-Bukhsh : Condurango-glycoside-A fraction of Gonolobus condurango induces DNA damage associated senescence and apoptosis via ROS-dependent p53 signalling pathway in HeLa cells. Mol Cell Biochem 2013, 382(1-2):173-183.
• [30]Lv L, Zheng L, Dong D, Xu L, Yin L, Xu Y, Qi Y, Han X, Peng J: Dioscin, a natural steroid saponin, induces apoptosis and DNA damage through reactive oxygen species: a potential new drug for treatment of glioblastoma multiforme. Food Chem Toxicol 2013, 59:657-669.
• [31]Zhang J: Transcriptome analysis reveals novel entry mechanisms and a central role of SRC in host defense during high multiplicity mycobacterial infection. PLoS ONE 2013, 8(6):01-09.
• [32]Li JM, Li N, Zhu DY, Wan LG, He YL, Yang C: Isocitrate lyase from mycobacterium tuberculosis promotes survival of mycobacterium smegmatis within macrophage by suppressing cell apoptosis. Chin Med J 2008, 121(12):114-119.
• [33]Spelman K, Burns J, Nichols D, Winters N, Ottersberg S, Tenborg M: Modulation of cytokine expression by traditional medicines: a review of herbal immunomodulators. Altern Med Rev 2006, 11(2):128-150.