Bioassay-guided isolation and characterization of active antiplasmodial compounds from Murraya koenigii extracts against Plasmodium falciparum and Plasmodium berghei.

Malaria is an overwhelming impact in the poorest countries in the world due to their prevalence, virulence and drug resistance ability. Currently, there is inadequate armoury of drugs for the treatment of malaria. This underscores the continuing need for the discovery and development of new effective and safe antimalarial drugs. To evaluate the in vitro and in vivo antimalarial activity of the leaf ethyl acetate extract of Murraya koenigii, bioassay-guided chromatographic fractionation was employed for the isolation and purification of antimalarial compounds. The in vitro antimalarial activity was assayed by the erythrocytic stages of chloroquine-sensitive strain of Plasmodium falciparum (3D7) in culture using the fluorescence-based SYBR Green I assay. The in vivo assay was done by administering mice infected with Plasmodium berghei (NK65) four consecutive daily doses of the extracts through oral route following Peter's 4-day curative standard test. The percentage suppression of parasitaemia was calculated for each dose level by comparing the parasitaemia in untreated control with those of treated mice. Cytotoxicity was determined against HeLa cells using MTT assay. Histopathology was studied in kidney, liver and spleen of isolated compound-treated Swiss albino mice. The leaf crude ethyl acetate extract of M. koenigii showed good in vitro antiplasmodial activity against P. falciparum. The in vivo test of the leaf crude ethyl acetate extract (600 mg/kg) showed reduced malaria parasitaemia by 86.6% against P. berghei in mice. Bioassay-guided fractionation of the leaf ethyl acetate extract of M. koenigii led to the isolation of two purified fractions C3B2 (2.84 g) and C3B4 (1.97 g). The purified fractions C3B2 and C3B4 were found to be active with IC50 values of 10.5 ± 0.8 and 8.25 ± 0.2 μg/mL against P. falciparum, and in vivo activity significantly reduced parasitaemia by 82.6 and 88.2% at 100 mg/kg/body weight on day 4 against P. berghei, respectively. The isolated fractions C3B2 and C3B4 were monitored by thin-layer chromatography until a single spot was obtained with R f values of 0.36 and 0.52, respectively. The pure compounds obtained in the present investigation were subjected to UV-visible spectroscopy, Fourier transformer infrared spectroscopy, 1D and 2D (1)H-Nuclear magnetic resonance (NMR), (13)C NMR, DEPT, COSY and Mass spectral analysis. Based on the spectral analysis, it is concluded that the isolated compounds were myristic acid (C3B2) and β-caryophyllene (C3B4). The cytotoxic effect of myristic acid and β-caryophyllene showed the TC50 values of >100 and 80.5 μg/mL, respectively against HeLa cell line. The histopathology study showed that protection against nephrotoxicity of kidney, hepatic damage of liver and splenocytes protection in spleen was achieved with the highest dose tested at 100 mg/kg/body weight. The present study provides evidence of antiplasmodial compounds from M. koenigii and is reported for the first time.