Cytotoxic and apoptotic activity of essential oil from Ocimum viride towards COLO 205 cells

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Abstract

We investigated the apoptosis inducing effect of essential oil (EO) from aerial parts of Ocimum viride in human colorectal adenocarcinoma cells (COLO 205 cell line). The COLO 205 cells were exposed to 0.0125–0.1 μl/ml of EO for 24, 48 and 72 h. Growth inhibition was determined by sulphorhodamine B (SRB) assay. Double staining with acridine orange and ethidium bromide for nuclear changes was performed. Cell cycle analysis and change in mitochondrial membrane potential was quantified by flow cytometry. Subsequently, using annexin V/PI assay, the proportion of cells actively undergoing apoptosis was determined. Changes in DNA were observed by DNA ladder assay. Eventually the surface morphology of apoptotic cells was studied by scanning electron microscopy. EO is cytotoxic to COLO 205 cells in dose and time-dependent manner, as is evident by SRB assay. This observed cell death was due to apoptosis, as established by annexin V/PI assay, DNA ladder formation and scanning electron microscopy. Our results reveal that EO has apoptosis inducing effect against COLO 205 cells in vitro and is a promising candidate for further anti-cancer study.

Introduction

Colon cancer is rapidly rising in Asia. The incidence in many Asian countries is in fact on par with the west. It is defined as any malignant neoplasm arising from the inner lining of the colonic epithelium. The occurrence of colon cancer is strongly related to age, with 90% of the cases arising in people who are 50 years or older (Rajamanickam and Agarwal, 2008). Most colorectal cancers take approximately 8–10 years to develop from an adenomatous polyp into an invasive cancer. It is now the third most common malignant disease in both men and women in Asia (Sung, 2007). Conventional treatments such as surgical resection, radiation therapy and chemotherapy are not still satisfactory and prevention of this disease or at least stopping it at its inception is important (Kim et al., 2005). To relieve certain symptoms of cancer and to alleviate the side effects, which come with the use of conventional treatments, the medical world has now turned to complementary therapies for assistance. Ethno-traditional use of plant-derived natural products such as essential oils has been a major source for discovery of potential medicinal agents (Schwartsmann et al., 2000).

Essential oils are known for their antiseptic, antimicrobial, analgesic, anti-inflammatory properties. They are also used in embalment, preservation of foods and as sedative, spasmolytic and locally anesthesic remedies. The application of essential oils in the anti-cancer therapy may appear unconventional however their easy availability, pleasant aroma and low or insignificant toxicity make them more attractive candidates for the long term treatment of various chronic ailments (Kumar et al., 2008). Therefore, there exists high hope for effective treatment of different cancers by systematic screening of a variety of essential oil based natural products.

The genus Ocimum (Lamiaceae, formerly Labiatae) ranks among the most important aromatic medicinal plants from the time of old civilization. Being a polymorphic group of economically useful herbs, it forms a rich source of many naturally occurring essential oils and aroma chemicals that are of great value in perfumery, flavouring and pharmaceutical industry (Lans, 2006). Ocimum viride, the fever plant of Sierra Leone, is native of Africa and finds an important place in the indigenous system of medicine. Preparations from the whole plant and leaves of O. viride are used as anticonvulsant, to stop diarrhoea, treatment of catarrh and bronchitis (Gill, 1988). Hot aqueous extract of O. viride possess anti bacterial properties (Isu, 2005). Hot leaves infusion is taken with milk and sugar as febrifuge and diaphoretic. Leaf decoction is effective against fever and cough. The plant has been introduced at Indian Institute of Integrative Medicine (formerly RRL), Jammu, India and a genetically improved species was produced in view of diverse industrial uses and increasing demand of essential oils and their isolates. The EO derived from O. viride is a mixture of terpenes and one of the main compounds of this oil is a natural terpenoid thymol. Thymol exhibits multiple biological activities including anti-inflammatory, immunomodulating, antioxidant, antibacterial, antifungal and free radical scavenging properties (Bukovska et al., 2007). Many terpenes are known to be use in the treatment or prophylaxis of cancer (Myers et al., 1997). Therefore we focused our study on the cytotoxicity and apoptotic activities of this essential oil on COLO 205 human colon adenocarcinoma cells.

Section snippets

Chemicals

RPMI-1640, trypsin, EDTA, gentamycin, penicillin, 5-fluorouracil (5-FU), doxorubicin, mitomycin-c, camptothecin, propidium iodide (PI), sulforhodamine B (SRB), phosphate buffer saline (PBS), agarose, ethidium bromide, sodium dodecyl sulphate (SDS), proteinase-K, DNase-free RNase A, dimethyl sulfoxide (DMSO), Rhodamine-123 (Rh-123) and foetal bovine serum (FBS) were purchased from Sigma Chemical Co., USA. Annexin V – FITC kit was purchased from Axxora, San Diego, CA. Other reagents and chemicals

Chemical composition of the essential oils

The EO obtained by hydro-distillation from whole plant of O. viride was qualitatively and quantitatively analysed by GC–MS. The major constituents of essential oil are thymol, β-caryophyllene oxide, α-pinene, geranyl acetate and 4-terpineol which constitutes 95% of the essential oil. Data is shown in Table 1.

Cytotoxicity of EO on COLO 205 cancer cells

The effects of EO on COLO 205 cells as assessed by the colorimetric SRB assay with different concentrations (0.0125–0.1 μl/ml) are shown in Fig. 1. A dose and time dependent inhibition by EO

Discussion

In recent years, considerable attention has been focused on identifying naturally occurring substances capable of inhibiting, retarding, or reversing the process of multistage carcinogenesis. Plant essential oils as antioxidants are believed to reduce the risk of cancer (Bennani et al., 2007). Because of the great number of constituents, essential oils seem to have no specific cellular targets. As typical lipophiles, they pass through the cytoplasmic membrane, disrupt the structure of their

Conclusion

In conclusion EO exhibited a cytotoxic effect on human colon cancer cells COLO 205, causes DNA damage, increase mitochondrial membrane permeability and eventually induce apoptosis. The mechanistic link between these events remains to be determined. However, the present data point to the importance of EO as a potential candidate for colon cancer treatment.

Conflict of interest

The authors declare that there is no conflict of interest.

Acknowledgements

Madhunika Sharma and Satyam Kumar Agrawal would like to thank Council for Scientific and Industrial Research (CSIR), New Delhi, India, for Senior Research Fellowships.

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