Anti-leukemic activity of Wattakaka volubilis leaf extract against human myeloid leukemia cell lines

Abstract

Ethnopharmacological relevance: Wattakaka volubilis has been traditionally used in Ayurvedic medicine in India for treatment of several ailments such as bronchial asthma, inflammations, tumors, piles, leucoderma, application to boils, rat bite etc.

Aim of the study: The present study was designed to investigate anti-leukemic activity of the crude aqueous methanolic extract and to identify active compounds from the leaves of Wattakaka volubilis.

Materials and methods: The leaves of Wattakaka volubilis were extracted with aqueous methanol. Liquid-liquid fractionation of the crude methanolic extract with different organic solvents was done and the fractions were screened for in vitro anti-leukemic activity using different leukemic cell lines. The active fractions were then subjected to chromatographic separation for isolation of bioactive compounds. Structure of isolated compound was elucidated by spectroscopic methods. The in vitro anti-leukemic activities of different extracts of the leaves and isolated compound WVP were studied in U-937, HL-60 and K-562 cell-lines by using cell count, MTT [(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] and DNA laddering assays, flow-cytometric and confocal microscopic techniques.

Results: Kaempferol-3-O-[α-l-rhamnopyranosyl-(1→4)-O-α-l-rhamnopyranosyl-(1→6)-O]-β-d-glucopyranoside (WVP) was isolated from crude leaves extract of Wattakaka volubilis. Both the n-butanolic extract (WVB) of Wattakaka volubilis and its isolate WVP were found to be responsible for in vitro anti-leukemic activity. The IC(50) values of WVB were found be 120, 100 and 50(μg/ml) in U937, K562, and HL-60 cell lines, respectively. Whereas, the pure isolate WVP exhibited anti-leukemic activity with IC(50) values of 13.5, 10.8, and 13.2(μg/ml) in U937, K562, and HL-60 cell lines, respectively. The flow-cytometric analysis confirms that the cell cycle arrest occurs at G1 phase in case of U937 and K562 cell lines and G2/M phase in case of HL60 cell lines. Similarly both confocal microsocopic analysis and DNA laddering assay confirm the apoptosis and cell cycle arrests of leukemic cells.

Conclusion: The overall results provide evidence for the ethnopharmacological relevance for use of the plant Wattakaka volubilis in developing novel agents for the treatment of leukemia.