Diversity in Nutrition and Bioactivity of Momordica cochinchinensis

Momordica cochinchinensis (Cucurbitaceae) aril is the richest source of the carotenoids of all known fruits with high anticancer activity. The plant is restricted to South East Asia with diverse morphology and genetic variations. The effect of varietal variations on carotenoids (lycopene and β-carotene) and anticancer activity was investigated to determine cultivars important for agricultural development. M. cochinchinensis aril samples were collected from 17 provinces within Thailand and Vietnam and compared with samples from Australia. Lycopene and β-carotene was quantified using HPLC and cytotoxicity on melanoma (MM418C1, D24) and breast cancer (MCF7) cells were assessed using CCK 8 assay. The greatest lycopene content was from Central Vietnam (6.3 mg/g) and greatest β-carotene was from Northern Vietnam (5.7 mg/g). Cytotoxicity was greatest from Northern Vietnam, with 70% melanoma cell death. The best varieties possessing the highest nutritional and bioactivity identified in this study will be incorporated into future breeding and crop improvement programs. Keywords— Momordica cochinchinensis; melanoma, breast cancer; carotenoids; lycopene; β-carotene.


I. INTRODUCTION
Momordica cochinchinensis (Spreng.) is distributed across South East Asia with the vernacular names gấc in Vietnam, Mak kao in Laos, fahk khao in Thailand and Bhat kerala in India [1,2]. The species is diverse with genetic and phenotypic variations [3,4]. The fruit is the richest source of lycopene and β-carotene of all known fruits and vegetables [5]. These carotenoids are bio-active and contribute to a variety of health benefits including antioxidant, anticancer, cardio-protective and anti-inflammatory effects [6][7][8]. Increasing breast cancer and melanoma incidence and mortality rates has lead to improved public health policies and awareness [9,10]. Plant derived medicines have less or no toxicity on normal cells and used as chemotherapeutic or chemo-preventive supplements [11]. Water extracts of the fruit aril has anticancer activity against colon cancer by inducing necrosis [12] but the effects on other cancers such as melanoma and breast is unknown. Therefore the aim of this study was to elucidate the effect of varietal variations on carotenoids and anticancer properties of M. cochinchinensis from Australia, Thailand and Vietnam.

II. MATERIALS AND METHODS
Plants were collected from rural and metropolitan provinces as previously detailed [4] and prepared for HPLC or cell culture assays.
A. Extraction 1) Crude hexane extract for HPLC: Carotenoids were extracted using a method described previously [13]. The aril (2 g) was placed in a vessel protected from sunlight and mixed with 100 mL of extraction solvent (hexane/acetone/ethanol: 50:25:25 v/v/v) [13]. The mixture was ultra-sonicated (Unisonics, Australia) for 30 min and then 15 mL of distilled water was added to enhance phase separation whereby the upper organic layer contained carotenoids and the bottom water layer contained cell debris. The upper organic layer was evaporated to dryness and the residue was dissolved in THF/acetonitrile/methanol: 50:25:25 v/v/v to a final volume of 4 mL for HPLC analysis.
2) Crude water extract for cell culture: 15 M. cochinchinensis aril samples from different provinces were used to extract. The frozen sample (2 g) was placed in a vessel, protected from sunlight and mixed with 100 mL of deionised water. The mixture was sonicated for 30 min, filtered and evaporated to dryness. The crude extract was dissolved in milliQ water and mixed using water-bath sonicator (Unisonics, Australia) for 10 min and filter sterilised using 0.1 µm filters and diluted to 1 mg/ml in sterile water.

B. HPLC analysis of carotenoids
Carotenoid extracts were analysed via reversed phase HPLC using solvents and isocratic conditions as described previously [13]. Analysis was performed using an isocratic HPLC method (90% CH3CN/H2O) on an Alltech Alltima HP C18 (250 x 4.6) 5 μ column at a flow rate of 1.0 mL/min. HPLC analyses were performed on a Dionex P680 solvent delivery system equipped with a PDA100 UV detector (operated using "Chromeleon" software). The column temperature was 30°C and UV detection was at 465 nm. Concentrations of lycopene and β-carotene were quantified by reference to commercial standards of lycopene and βcarotene (Sigma Chemical, St. Louis, USA).

C. Cytotoxicity assay on melanoma and breast cancer cells
Cytotoxicity of cancer cells were determined by CCK-8 assay using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technology, Gaithersburg, MD). The melanoma (MM418C1, D24) and breast cancer (MCF7) cells were seeded in 96 well plates containing 10% FBS DMEM and RPMI (Invitrogen) media at 5000 cells/well and 3000 cells/well, respectively. Cells were permitted to adhere for 24 h at 37 °C in 5%CO2 and then treated with 1 mg/ml of water extract. Concurrent control cells were cultured in medium only. After treatment, all plates were incubated for 72h. Cytotoxicity assay was determined after incubation in 37°C for 2 h with CCK-8 reagents and the absorbance was measured spectrophotometrically at 450 nm using CLARIOstar® High Performance Monochromator Multimode Microplate Reader (BMG LABTECH). Data was presented as proportional viability (%) by comparing the treated cells with the untreated cells (control), where the viability was 100% in untreated cells. All tests and analyses were duplicated.

D. Statistical analysis
Quantification of lycopene and β-carotene from HPLC was analysed using Minitab statistical software (version 16). For normally distributed data, a one-way analysis of variance (ANOVA) with least significant differences (LSD) from Fishers test was conducted using the equation: where t was 2-tailed values at P=0.05; MSE=least square error from ANOVA, n = replicate number. Not normally distributed data was transformed or analysed using Kruskal Wallis method. Statistical values of P≤0.05 were considered as significantly different

A. Carotenoid content
The carotenoid content of crude extracts in this study was highly variable, ranging from 0.88 to 6.30 mg/g for lycopene and 0.25 to 5.70 mg/g for β-carotene (Fig. 1). The highest lycopene content was observed in samples collected from Lam Ha (6.3 mg/g) and Lam Dong (6.20 mg/g) provinces of Central Vietnam, three times greater than previously reported for the same species [14] and 200 times greater than hydroponic tomatoes [15]. The highest β-carotene content was from Hoa Binh (5.70 mg/g) province in Northern Vietnam, almost eight times greater than previously reported from the same species [14] and 54 times greater than carrots [17].
The nutritional superiority of cultivars from specific provinces in Central and Northern Vietnam may possibly be an adaptation mechanism to increase cellular and metabolic protection for survival at higher altitudes and suboptimal temperatures as observed in other economically valuable fruits [18][19][20]. What these mechanisms are and how it influences the carotenoid synthesis pathway in M. cochinchinensis are unknown and unraveling the mystery could improve our understanding on plant responses to environmental stress. Highlighting these regions of interest that contain valuable superior varieties is the first step for conservation of the existing populations to facilitate a sustainable and consistent high quality product as an alternative functional food that shadows even tomatoes and carrots commonly associated with high nutritional value.

B. Anti-cancer activity
The cytotoxicity of crude plant extracts on cancer cells were variable and significantly affected by varietal differences ranging from 0-71% loss in cell viability (Fig. 2). Extracts specifically from Northern and Central Vietnam significantly reduced cancer cell viability and subsequently cell growth, by up to 70% relative to controls and was suggestive of anticancer activity. This dramatic reduction in cell viability might be through necrotic [12] or apoptotic cellular pathways [21,22] which will be confirmed in future studies with the analysis of morphology and biochemical tests.
The greatest cytotoxicity was from Northern Vietnam with 60% and 71% mortality on breast cancer and melanoma cells, respectively (Fig. 2). These same samples also produced the highest β-carotene which was unlikely to be the bioactive compound since β-carotene is water insoluble [23] and possibly might be due a protein as reported in a previous studies in M. cochinchinensis aril and seeds [12,24,25]. The responsible compound for the cytotoxicity observed in this study from the aril has not been studied before and further fractionation and purification is necessary to determine the bioactive protein is the same.

IV. CONCLUSIONS
The carotenoid content and anticancer potential in the aril of M. cochinchinensis was highly variable and likely an expression of genetic and phenotypic differences. This study achieved the aims in that 1) important cultivars were identified from existing wild and cultivated populations, 2) regions that produced highly nutritious and bioactive fruits were identified for replication in agriculture, specifically Central and Northern Vietnam, and 3) the extent of variation in carotenoids and bioactivity of aril compared to common fruits and vegetables. This study provided vital information on collection sites of M. cochinchinensis which possess the highest nutritional and bioactivity for future breeding and crop improvement.