Abstract
BackgroundArtemisia 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.MethodsIn 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.ResultsEthanol 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.ConclusionsEthanol 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.
Highlights
Artemisia nilagirica (Asteraceae) and Murraya koenigii (Rutaceae) are widely distributed in eastern region of India
multi-drug resistant (MDR)-TB is resistant to the major first line anti-TB drugs isoniazid and rifampicin, while XDR-TB is resistant to any fluoroquinolone, and at least one of three injectable second-line drugs, in addition to isoniazid and rifampicin
Antibacterial activity of plant extracts Several plant leaf extracts, Eupatorium triplinerve, Nyctanthes arbortristis, Azadirachta indica, Barringtonia acutangula, Achyranthes aspera, Moringa oleifera, Artemisia nilagrica, Murraya koenigii, Lantana camara, Mentha spicata were screened for anti-mycobacterial activity
Summary
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. Though for several decades TB was successfully treated with a combination of antibiotics, current vaccine and chemotherapeutic measures are limited in their efficacy and are failing to prevent spread of the disease. This problem is further worsened due to the emergence of multi-drug resistant (MDR) and extremely drug-resistant (XDR) Mtb strains that has made most of the front line drugs ineffective, too often impossible, to cure fatal infections [2]. It is important to identify and develop new molecules that can overcome the limitations of the present drugs and perturb development of antibiotic resistance by mycobacteria
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