Elsevier

Food Chemistry

Volume 114, Issue 4, 15 June 2009, Pages 1351-1358
Food Chemistry

Apoptosis-mediated proliferation inhibition of human colon cancer cells by volatile principles of Citrus aurantifolia

https://doi.org/10.1016/j.foodchem.2008.11.033Get rights and content

Abstract

Fruits of Citrus aurantifolia were subjected to hydro-distillation using Clevenger type apparatus to obtain volatile oil. Chemical composition of volatile oil was analysed by GC–MS. Twenty-two compounds representing more than 89.5% of the volatile oil were identified. d-limonene (30.13%) and d-dihydrocarvone (30.47%) were found to be the major compounds in the lime volatile oil. This oil showed 78% inhibition of human colon cancer cells (SW-480) with 100 μg/ml concentration at 48 h. Lime volatile oil showed DNA fragmentation and induction of caspase-3 up to 1.8 and 2- folds after 24 h and 48 h, respectively, which may be due to the involvement of apoptosis. Analysis of apoptosis-related protein expression further confirmed apoptosis induction by lime volatile oil. The above results suggested that lime volatile oil has potential benefits in colon cancer prevention. This is the first report, showing the possible mechanism of antiproliferative effect of lime volatile oil for the prevention of colon cancer in cell culture models.

Introduction

Colon cancer is one of the most prevalent cancers throughout the world and especially in the western countries. This is continuously increasing worldwide due to rapid changes in dietary pattern and preferences. Many epidemiological studies indicated that western-style diet, primarily, the consumption of red meat, is positively associated with a high colon cancer incidence (Abeysinghe et al., 2007). Continuous efforts are being made for search of novel source of bioactive compounds to prevent colon carcinoma. In this direction, bioactive compounds of natural origin, particularly from a dietary source, are gaining significance. In the recent years, citrus has gained importance due to their ability to provide multitude health benefits not only from vitamin-C but also from other bioactive compounds. Our group has been involved with citrus research over a decade, mainly focusing on the identification of a wide array of novel bioactive compounds such as flavonoids, phenolics, limonoids and phytosterols (Jayaprakasha et al., 2006, Jayaprakasha et al., 2007, Raman et al., 2004). Other investigations from our laboratory have also provided evidences for health-promoting properties of these compounds, such as in vitro antioxidant activity, prevention of cancer, in animal and cell culture studies (Jayaprakasha et al., 2008, Jayaprakasha et al., 2007, Jayaprakasha et al., 2008). Our research has also demonstrated that citrus bioactive compounds are capable of inhibiting human neuroblastoma and colon cancer cells (Poulose, Harris, & Patil, 2006). An animal study has revealed that freeze–dried grapefruit juice powder (13.7 g/kg) and its bioactive compounds naringin and limonin (200 mg/kg) are capable of inhibiting aberrant crypts through the suppression of cyclo-oxygenase-2 and inducible nitric oxide synthase (iNOS) in azoxymethane-treated animals (Vanamala et al., 2006).

Lime [Citrus aurantifolia (Christm.) Swingle] is a polyembryonic species with greenish yellow, smooth surfaced, thin-skinned fruits, and solid core at maturity with highly acidic juice. Limes are cultivated across the globe mostly in hot subtropical or tropical regions such as Southern Florida in USA, India, Mexico, Egypt, and West Indies (Morton, 1987). Both juice and volatile oils are the major commercial products of lime, and volatile oil is widely used as a flavouring agent in beverages and food products (Chamblee, Karelitz, Radford, & Clark, 1997). Pharmaceutical industries also use lime volatile oil as a flavouring agent in syrups and suspensions (Porta, Reverehon, & Barth, 1997). In perfumery, lime volatile oils have been used as a base for many compositions, which have a higher market value per pound than other citrus varieties such as orange, grapefruit, or tangerine volatile oils. Some of the reported chemical compositions of lime volatile oil consists of d-limonene, α-terpineol, 4-terpineol, 1,4-cineole, 1,8-cineole, p-cymene, β-pinene, β-bisabolene, citral, geranial and neral (Ranganna, Govindrajan, & Ramana, 1983). Recently, some more additional flavour compounds such as neryl acetate, α-bergamotene, valencene and germacrene-D have been reported (Veriotti & Sacks, 2001). Kaffir lime (Citrus hystrix) volatile oil has been demonstrated to reduce blood pressure and relieve depression in human studies (Hongratanaworakit & Buchbauer, 2007), which provides strong evidence on potent health benefits of citrus volatile oils. The above studies have demonstrated that citrus fruits and their bioactive compounds have a significant role in human disease prevention. However, the antiproliferative activity of lime volatile oil has never been explored. In this context, study was conducted to analyse the chemical composition of the lime volatile oil by GC–MS. Further, potential of antiproliferative mechanism on cultured human adenocarcinoma cells has been studied for the first time.

Section snippets

Chemicals and reagents

All the chemicals used were of analytical grade and procured from Sigma (St. Louis, MO, USA). d-Limonene, β-linalool, α-terpineol, C8–C20 n-alkanes and α-pinene were obtained from Sigma (St. Louis, MO, USA). Reagents and media used for cell culture studies were of molecular biology grade and purchased from Hyclone (Logan, UT, USA) and Mediatech Inc. (Manassas, VA, USA). Caspase-3 kit was purchased from BD Pharmingen (San Jose, CA, USA). All the reagents for immunoblotting were from Bio-Rad,

Results and discussion

Lime volatiles are well known for their application in pharmaceuticals and cosmetics. However, there are no reports on health benefits; this may be due to the lack of scientific evidences. In this context, the present study is an attempt to understand the chemical composition of lime volatile oil and, possible mechanism of inhibition of colon carcinoma using cell culture model has been demonstrated for the first time.

Conclusions

Twenty-two volatile components were identified from Citrus aurantifolia using GC–MS. The major compounds were found to be d-limonene, d-dihydrocarvone, verbena, β-linalool, α-terpinol, trans-α-bergamotene. For the first time, we have demonstrated the principles of volatile oil that can induce apoptosis-mediated cells death in human colon adenocarcinoma cells. Induction of apoptosis by lime volatile oils was supported with evidence of DNA fragmentation, elevated caspase-3 content and expression

Acknowledgements

This project was funded by the USDA-Designing Foods for Health through the Vegetable & Fruit Improvement Center, TAES 06-118409. First author would like to acknowledge Honourable Vice-chancellor, University of Agriculture Sciences, Dharwad, India, for providing the opportunity to conduct research at the VFIC, Texas A&M University, College Station, TX, USA.

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