Bioassay-guided Isolation of Cytotoxic Fractions from Muntingia calabura Leaf

M. calabura L. or locally known as „Kerukup Siam“, belongs to the family Elaeocarpacea [1]. This plant is native to American continent and is widely cultivated in warm areas of Asian region, including Malaysia [2]. The leaf is used to provide relief from gastric ulcers and to reduce swelling of the prostate gland as reported in Peru folklore medicinal uses. The aim of the present study is to determine the in vitro cytotoxic activity of Muntingia calabura leaf against cancer (HL60 and MCF-7) and normal (WRL68) cell lines using MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] assay as described by Mosmann [3] but with slight modifications. The crude methanolic extract of M. calabura (MCME) was suspended in distilled water to afford an aqueous MeOH solution and then partitioned with petroleum ether and EtOAc to give petroleum ether, EtOAc and aqueous extracts. The EtOAc extract showed significant cytotoxic activity when tested against HL60 (IC₅₀=27.48±3.60µg/ml) and was further fractionated using vacuum liquid chromatography with gradient mixture of hexane-EtOAc (from 9:1 to 1:9) as solvent systems. Seven fractions were obtained (F1-F7), and subjected to cytotoxic activity against HL60, MCF7 and WRL68 cell lines. The IC₅₀ values of the M. calabura extracts and fractions are shown in Table 1. Fraction 5 tested against HL60 showed strong inhibition (IC₅₀=3.98±0.09µg/ml) as compared to the other cell lines as well as other fractions. Fraction 5 will be isolated further and the bioactive compounds responsible for the activity will be determined in the future study.

Table 1: IC₅₀ values of the M. calabura extracts and fractions

	HL60– IC50 (µg/ml)	MCF7 -IC50 (µg/ml)	WRL68 -IC50 (µg/ml)
MCME	30.90±4.73	36.56±5.40	>100
Partitions:
Petroleum ether	29.46±3.95	43.07±3.25	68.51±8.71
Ethyl acetate	27.48±3.60	40.72±6.18	76.57±5.32
Aqueous	>100	>100	>100
Fractions
F1	32.14±1.68	>100	>100
F2	35.89±3.69	35.65±4.41	43.93±6.06
F3	84.49±2.26	>100	37.07±4.66
F4	34.22±5.83	30.80±3.60	33.15±2.04
F5	3.98±0.09	35.47±5.44	32.29±4.23
F6	5.99±0.95	35.27±2.51	40.75±6.16
F7	28.13±3.59	42.16±4.34	36.60±1.29

Keywords: Muntingia calabura, Elaeocarpacea, cytotoxic, MTT assay

Acknowledgement: The authors wish to thank Faculty of Pharmacy, UiTM Malaysia for financial assistance.

References: [1]. Morton, J.F. (1987) Jamaica Cherry. In Fruits of Warm Climates. Miami: J.F. Morton; p.65

[2]. Chin WY (1989) A guide to the wayside trees of Singapore. BP Singapore Science Centre, pp: 145.

[3]. Mosmann T (1983) Journal of Immunol Methods 65: 55–63.