Cytotoxic and immunopotentiating effects of ethanolic extract of Nigella sativa L. seeds

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Abstract

In-vitro cytotoxic screening of extracts of Nigella sativa L. seeds (Ranunculaceae) indicated cytotoxicity in the ethyl-acetate fraction (EAF) against different classes of cancer cell lines, P388, Molt4, Wehi 164, LL/2, Hep G2, SW620 and J82, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The ethyl-acetate column chromatographic fraction (CC-5) showed selectivity against Hep G2, Molt4, and LL/2. CC-5 was relatively non-toxic against human umbilical cord endothelial cells at 50 μg/ml. CC-5 had no stimulatory effect on mouse splenocytes as such. CC-5 and water fraction, however, enhanced the proliferative response in the presence of ConA (3 μg/ml), but not LPS (1 and 6 μg/ml). These data indicate that CC-5 possesses a potent cytotoxic effect as well as a potentiating effect on the cellular immune response. The mechanism whereby it produces this needs to be resolved.

Introduction

Nigella sativa L., commonly known as black cumin seed, belongs to the botanical family of Ranunculaceae. It has been used in many Middle Eastern countries as a natural remedy for 2000 years. N. sativa seeds are commonly eaten alone or in combination with honey and in many food preparations. The seeds are believed to have carminative, stimulatory and diaphoretic properties and are used in the treatment of bronchial asthma and eczema (Boulos, 1983). Recent pharmacological investigations of the seed extract reveal a wide spectrum of activities including anti-inflammatory (Houghton et al., 1995), anti-bacterial, anti-fungal and anti-helminthic (Agarwal et al., 1949). The effects of the black seeds have also been evaluated in clinical and animal studies (Salomi and Panikkar, 1989, Nair et al., 1991). Most of the activities have been attributed to the volatile oil obtained from the blackseed preparation. Thymoquinone and dithymoquinone, the main active components of the volatile oil, were shown to be cytotoxic to both parental and multidrug resistant tumor cells in-vitro (David et al., 1998). Nigellone, the carbonyl polymer of thymoquinone, in relatively low concentrations, was effective in inhibiting histamine release from mast cells in-vitro (Chakravarty, 1993). The active principles of N. sativa seeds containing fatty acids were found to completely inhibit the Ehrlich ascites carcinoma in mice (Salomi et al., 1991, Salomi et al., 1992). Pharmacological actions of the alkaloids isolated from N. sativa seeds viz. nigellidine, nigellimine, and nigellicine have not been reported (Atta-ur-Rahman and Sohail Malik, 1985, Atta-ur-Rahman et al., 1992, Atta-ur-Rahman et al., 1995). No further studies on the anti-tumor activities of N. sativa have been recorded.

In the present study, we investigated the in-vitro cytotoxic effect of the ethanolic extract of N. sativa seeds and its fractions against a variety of cancer cell lines as well as their effects on isolated mouse splenocytes.

Section snippets

Preparation of extract

N. sativa seeds, 1 kg, purchased from the local market, were authenticated by Dr Ruth Kiew, and stored as a voucher specimen (BT3, Herbarium, Singapore Botanical Garden). The seeds were washed, dried, and crushed to a powder with an electric microniser. Three hundred grams of the powder were extracted with 90% ethanol at room temperature until exhaustion. The extract was centrifuged at 10 000×g for 15 min to remove residual solid debris. The clear supernatant was then concentrated under reduced

Statistical analysis

The significance of differences between control and treated test was analyzed using Student’s t-test. Differences with P values <0.05 were considered to be statistically significant.

Results

In vitro screening of the extract of N. sativa seeds indicated that only the ethyl-acetate fraction appeared to be cytotoxic against the tumor cells. A dose-dependent cytotoxic effect of this fraction was obsened for all cell lines used (Fig. 1 and Fig. 2). The ED50 values of this fraction against Molt4, P388, J82, Wehi 164, LL/2, SW620, and Hep G2 were 12, 17, 22, 14, 16, 18, and 11 μg/ml, respectively. At the concentrations of 50 and 100 μg/ml the EAF completely killed all cell types tested.

Discussion

This in-vitro study was undertaken to demonstrate the effects of N. sativa seed extract on different classes of human cancer cells and to determine whether the extract has immunomodulatory properties. Cytotoxic assay was carried out using two leukemic cell lines and five solid tumor cell lines. The ethyl-acetate fraction exhibited significant growth inhibition of all cell lines used. The ED50 values of the extract showed increased sensitivity towards Hep G2, LL/2 and Molt4 cell lines compared

Acknowledgements

The authors are grateful to the National University of Singapore for the research grant (RP 960329) and Research Studentship awarded to S. Muthu kumara swamy.

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