Evaluation of the anticancer activity and fatty acids composition of “Handal” (Citrullus colocynthis L.) seed oil, a desert plant from south Jordan

Abstract Background The chemical composition of Handal (Citrullus colocynthis L.) seed oil cultivated in Jordan deserts was characterized, and its bioactivity was evaluated. Methods The oil was extracted from the grinded seeds in 500 ml Soxhlet extractor for 24 hr using n‐hexane, and the recovered fatty acids were methylated with methanolic‐HCL. The fatty acid methyl esters (FAMEs) composition was analyzed using GC‐MS and GC‐FID. The anticancer activity associated with the oil was assessed against colon cancer cell lines (Caco‐2 and HCT‐116) and compared to its cytotoxicity on the human skin fibroblast. Multivariate analysis was used to determine relationship of the fatty acid composition with that of the anticancer activity. Results The results demonstrated that fatty acid composition of Citrullus colocynthis seed oil chiefly contains Linoleic acid, denoted as C18:2n6 (75%), followed by Palmitic acid C16:0 (8%), Stearic acid C18:0 (5%), and Oleic acid C18:1n9 (9%). It is demonstrated as an excellent source of essential fatty acids omega‐6 (e.g., Linoleic acid), whereas omega‐3 (e.g., α‐Linolenic acid) and hydroxy polyunsaturated fatty acids are found at small level. Interestingly, the oil exhibited reasonable anticancer effects against colorectal cancer cell lines with IC50 values varying between 4 and 7 mg/ml. The correlation test revealed a relationship between the fatty acid composition and the effectiveness on treatments. Conclusions Handal plant from Jordan appears to have very high level of Linoleic acid compared to other oils measured in different geographic locations and that there appears to be some anticancer activities associated with the fatty acid content of Handal seed oil.

Medicinal plants are currently of great interest as natural sources for new anticancer agents due to their potent antioxidant capacity, antimutagenic properties, low side effects, low cost, and being easily accessible (Chekroun et al., 2015;De Martel et al., 2012;Hussain et al., 2014;Kim et al., 2015;Shokrzadeh et al., 2010). Phytochemical studies have shown that Citrullus colocynthis (L.) Schrad plant is a rich source of flavonoids and alkaloids with proven anticancer activities (Patal & Krishnamurthy, 2013).
Vegetable oils are known as natural products that contain fatty acids of different saturation levels (Nesma et al., 2010). They also contain triglycerides, diglycerides, monoglycerides, and vitamins (A, D, E, and K) in small amounts (Emmanuel & Mudiakeoghene, 2008).
Nonetheless, fatty acids are often classified into long-chain fatty acids, with more than 14 carbon atoms and higher melting points, and short-chain fatty acids with lower than six carbon atoms and lower melting points (Fasina et al., 2006).
In Jordan, Citrullus colocynthis (Handal) is creeping plant growing in arid deserts located in southern and eastern parts of Jordan.
Handal crops cost harvesting and extracting are highly economic relative to other vegetable oils. The present study aims to characterize the chemical composition of fatty acids in Handal seed oil cultivated in Jordan deserts and assess the potential use of this oil as anticancer agent against colon cancer cells.

| Seed material
Handal fruits were collected from desert areas in south Jordan Seeds were deshelled and dried at room temperature overnight. The dried Handal seeds were ground using an electric blender. Seed oil was then extracted at 25°C using a Soxhlet extractor to determine total fat content. No approval/permission to collect the plant/fruit samples was required.

F I G U R E 1 Location map showing the Handal fruits distribution in south
Jordan (adopted from Al-Hwaiti et al. 2015). Al-Hwaiti, M., Araf, K, Harara, M. (2015). Removal of heavy metals from waste phosphogypsum materials using polyethylene glycol and polyvinyl alcohol methods. Arabian Journal of chemistry, 12, 3141-3150

| Oil extraction
The oil was extracted from the grinded seeds in 500 ml Soxhlet extraction for 24 hr using n-hexane. The extract was filtered and transferred to a rotary evaporator (LabTech, Germany) to remove n-hexane under vacuum at 40°C. The raw oil was cloudy yellow color and contained some suspected impurities, such as water, and coextracted compounds. Therefore, it was refined by a set of pretreatment processes. These processes included adding silica gel (5 g per 100 ml oil) to the oil to remove any coagulated and gummy compounds. The mixture (oil and silica gel) was stored at 4°C for 120 min and then centrifuged at 3,500 rpm for 5 min. The oil layer was poured into separatory funnel and washed with distilled water (5 ml water:100 ml oil) for 15 min. After that, the oil was heated to 105°C for 30 min to remove any water and residues. Finally, the oil was bubbled (dried) with a stream of N 2 -gas (grad 5). The refined oil was then stored for physical, chemical, and biological analysis.

| Fatty acids analysis using GC/MS and GC/FID
Handal seed oil fatty acids were esterified to fatty acid methyl esters (FAMEs). A 300 μl of the seed oil was spiked with internal standard (30 μl of C18:0 alkane, 1 mg/ml in n-hexane). Esterification process was performed using 500 μl of 1.5N methanolic-HCl (Sigma-Aldrich).
Then, the content was vortexed and incubated (60°C) for 30 min.
Following incubation, distilled water (0.8 ml) was added to the cooled samples, and the FAMEs extracted 3 times with 500 μl hexane. The combined fractions were dried under N 2 and resuspended in n-hexane (300 μl).
The FAMEs composition was analyzed using GC-MS and GC-FID. GC-MS analysis of Handal seed oil fatty acids was performed using a Trace 1,300 gas chromatography (GC) coupled to a TSQ 8,000 Triple Quadrupole mass spectrometer (ThermoScientific).
Methylated fatty acids were separated using a BPX70 high resolution column (10 m × 0.1 mm ID × 0.2 μm) (Canadian Life Science, ON, Canada) using helium as the carrier gas at a flow rate of 1 ml/ min. A split mode (1:15) injection was applied with 1 μL volume injection of each sample using a Tri-plus auto-sampler (Thermo Scientific). The oven temperature program was set as follow: the initial oven temperature of 50°C (held for 0.75 min), then programmed to increase at 40°C/min to 155°C, then increased at 6°C/min to 210°C, and then increased at 15°C/min to 250°C (held for 2 min), total time: 17 min.
GC-FID analysis of Handal seed oil fatty acids was conducted using a Trace 1,300 gas chromatography coupled to a Flame Ionization Detector (Thermo Fisher Scientific). Methylated fatty acids were separated based on the same GC parameters as de-

| Anticancer activity
Normal skin fibroblasts were cultured in DMEM/F12 (Dulbecco's modified essential medium/Ham's 12 nutrient mixture, Gibco). Note: Values (% by weight composition) represent means ± standard errors. The fatty acid was detected in form of fatty acid methyl ester (FAME).
The components with the C number before the colon represent total number of carbons, while the numbers after the colon represent the total number of double bonds, n-represent the position of the first double bond counting from the methyl end or omega end (e.g., C18:3n3 = omega 3linolenic acid or α-linolenic acid).
In vitro assessment of the oil antiproliferative activity was carried out using the Promega CellTiter 96 ® AQueous Non-Radioactive Cell Proliferation (MTS) assay to evaluate the number of viable cells in media (Promega 2005). The oil was applied to test wells at concentrations of 5, 10, 20, and 50 mg/ml and incubated at 37ºC with 5% CO 2 for exposure period of 48 hr. After the completion of the incubation time, the MTS mixture (20 μl/well) was employed. A microplate enzyme-linked immuno-assay (ELISA) reader was utilized to read absorbance of the formazan product at 492 nm. Each point was performed in triplicates (Bardaweel et al., 2015).

| Statistical analysis
The chemical parameters measured included lipid analysis of the seed oil, and IC 50 against the cancer cell lines. All measurements were made in four replications. Effects of the treatments on the chemical parameters were done using one-way analysis of variance (ANOVA) and means separated using Fisher's LSD at α = 0.05.    Kamalakar et al. (2015). minor levels, that is, n3-PUFA and OH-PUFA were shown as 0.39% and 0.94%, respectively, in Table 1.

| RE SULTS AND D ISCUSS I ON
The results from the current study are in close agreement with previous literature reports on fatty acids composition of Citrullus colocynthis L. seed oil from different origins (Ashish et al., 2010;Kamalakar et al., 2015;Kulkarni et al., 2012;Solomon et al., 2010;Zohara et al., 1999) (Table 2). It was found that seed oil is composed of four major fatty acids: palmitic, stearic, oleic, and linoleic acids. Of the four major fatty acids, linoleic acid is the most prevalent with the value ranging between 55.90% and 74.77%, with the most prominent level found in Jordan seed oil (74.77%). Palmitic acid (C16:0) ranged from 8.35% in oil from Jordan and compared to 11.70% in oil from Nagpur and was the predominant saturated fatty acid. The composition of total saturated fatty acids (Palmitic C16:0 and Stearic C18:0) and unsaturated fatty acid (Oleic and Linoleic) contents of the oil was reported between 13.71-21.40% and 77.50-83.81%, respectively (Table 2). Due to the different origin of the Citrullus colocynthis seed and an effect of laboratory variation, the lowest saturated fatty acids were seen from the oil produced in Jordan (this study) and the highest were reported from the group in Nagpur, India. In contrast, Jordan's Handal oil contains the highest unsaturated content (83.81%) as compared to the lowest (77.5%) in the oil obtained from Nagpur (Kulkarni et al., 2012).

| Cytotoxicity
Cell growth of cancer cells (Caco-2 and HCT-116) and normal skin fibroblast cells were assessed with the MTS assay after 48h exposure period. The IC 50 values, described as the concentration at which 50% of cell growth is inhibited, are presented in Table 3. Notably, there was statistically significant difference between cancer cell growth in wells treated with the examined oil, relative to the growth of normal skin fibroblasts treated with same concentration of the oil. The results indicated that cancer cell viability was considerably affected after 48 hr exposure relative to the human fibroblasts upon treatment with the examined oil, using the MTS assay at concentration points up to 50 mg/ml, suggesting a reasonable anticancer activity and safety profile against the human skin fibroblasts (Bardaweel et al., 2013). However, health complications such as colic, diarrhea, vomiting, and liver impairment have been frequently reported with the use of C. colocynthis (Jouad et al., 2001). Oil 7 ± 0.9 * 4 ± 0.3 * 88 ± 6 * Values are expressed as mean ± SD (n = 9).

F I G U R E 3
Biplot showing relationships between the fatty acid composition of Citrullus colocynthis L. seed oil from Jordan and suppression of cellular growth (IC 50 ) in 2 cancer cell lines and normal skin fibroblasts shown to have significant correlation with the levels of α-Linolenic acid C18:3n3 and Arachidic acid C20:0 (value in bold shown in Table 4).

| CON CLUS IONS
The Overall, these findings indicate that Handal plant from Jordan has very high level of linoleic acid compared to other oils measured in different geographic locations and that there appears to be some anticancer activities associated with the fatty acid profile of Handel seed oil. Further studies using isolated constituents instead of the whole extract should be carried out to better understand the relationship between Handel seed oil composition and potential anticancer activity.

ACK N OWLED G M ENTS
The authors thank Eng. Njoud Al-Habahbeh for providing the Handal plants used in this study and for assistance during laboratory work.

CO N FLI C T S O F I NTE R E S T S
Not applicable.

AUTH O R S' CO NTR I B UTI O N S
MA, RT, and SB conceived and designed the experiments; EA, GA, BB, and TP performed the experiments; TP, SB, RT, and MA analyzed the data; MA, SB, AT, and TP wrote the paper; MA and SB supervised the project. All authors read and approved.

E TH I C A L A PPROVA L
Not applicable.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed in the current study are available from the corresponding author on reasonable request.

O RCI D
Sanaa K. Bardaweel https://orcid.org/0000-0002-4823-0708  Values in bold correspond for each variable to the factor for which the squared cosine is the largest.

R E FE R E N C E S
TA B L E 4 Squared cosines of the variables showing the correlations of fatty acid composition and the three cell lines treatment efficiency. (move Table S-1 to  Supplementary)