Chemical Profiling, Antioxidant, Antiproliferative, and Antibacterial Potentials of Chemically Characterized Extract of Citrullus colocynthis L. Seeds

Background: Citrullus colocynthis L. (C. colocynthis) is commonly known as colocynth. It belongs to the family Cucurbitaceae that is frequently used in alternative medicine in the north of Africa. The aim of the study: the present research was undertaken to investigate the chemical composition, antioxidant, antiproliferative, and antibacterial potentials of C. colocynthis seed extract. Material and methods: the chemical composition of C. colocynthis seed organic extract was characterized using gas chromatography/mass spectrometry (GC-MS). The antioxidant property was carried out using both β-carotene bleaching and DPPH assays. The antibacterial effect was effectuated using the agar disc diffusion method. The antiproliferative activity vs. human colorectal adenocarcinoma cell line (HT-29) and human breast adenocarcinoma cell line (MDA MB 231) were carried by WST-1 test. The chemical analysis showed the presence of interesting potentially bioactive compounds. The studied plant extract exhibited antioxidant potential with IC50 value of 2. 22 mg/mL (β-carotene bleaching) and 8.98 ± 0.619 mg/mL (DPPH). Concerning the antiproliferative activity, the seed extract was effective in MDA-MB-231 and HT-29 cancer cells with IC50 values 86.89 ± 3.395 and 242.1 ± 17.9 μg/mL, respectively, whilst the extract of Citrullus colocynthis seeds was non-toxic in healthy human dermal fibroblasts. Regarding the antibacterial test, the extract was effective in Gram-positive bacteria only. Conclusion: The outcome of this research indicated that the extracts from C. colocynthis seeds may compose a promising source with interesting compounds that can be used to fight cancer, free radicals damage, and bacterial infections.


Introduction
Plants have populated the planet for millions of years and have served humans and animals to meet their nutritional and medicinal requirements. Their uses have largely evolved with the discovery of their therapeutic properties [1]. Medicinal plants are defined as a vegetable that has at least a part (bark, leaves, roots, fruits) with medicinal properties [2]. Herbal medicine has been used as remedies in different forms (decoction, infusion, ingredients) to prevent and treat diseases. The development of aroma has started in ancient times for cosmetic purposes [3]. Aromatic and medicinal plants have been used in various dietary and therapeutic practices since prehistoric times [4].
Recently, people have returned to alternative medicine based on natural products due to its efficacy in the treatment and the prevention of diseases with negligible side effects. For example, the resistance of microbes to synthesized drugs is a new problem that has led to the use of plant derivatives [5]. The use of medicinal plants plays an important role in the health care system. It is estimated that 50 to 75% of people throughout the world use traditional medicine for medications [6]. It was reported that over 950 species have been used to cure a wide variety of illnesses in Morocco [7].
C. colocynthis belongs to the family Cucurbitaceae and has a large genetic diversity. It is a perennial plant that commonly spreads in tropical soil and can survive hard ecological conditions [8,9]. C. colocynthis fruit is recognized by a globular aspect, with different colors along with soft pulp and edible seeds [10]. Early civilizations have reported potential pharmacological activities of C. colocynthis. It has traditionally been used in the treatment of a bundle of diseases such as cough, constipation, leprosy, diabetes, asthma, and toothache [11,12]. In Mediterranean countries, C. colocynthis fruits have been used in the treatment of pulmonary and urinary infections [13]. C. colocynthis was observed early to exert potential hepatoprotective effects [14]. In Morocco, C. colocynthis seeds are commonly used as antidiabetic and antihypertension agents [15,16].
Phytochemical studies have reported that C. colocynthis contains different chemical families such as carbohydrates, flavonoids, alkaloids, and phenolic acids [17].
The nutritional composition of C. colocynthis seeds was investigated in earlier works by Milovanović and Pićurić-Jovanović (2005), who reported that the dry weight of seeds consisted of testa and kernel with 52.3% and 47.7%, respectively; the moisture content was found at 54.5%; the oil content ranged from 22.1-53.5%; the protein content was 21.8%; and the fatty acid content in the oilseed consisted of a majority of unsaturated fatty acid (77.4%), including linoleic (18:2) acid (62.2%) [18].
The current research work was undertaken to study the phytochemical composition, antioxidant, antibacterial, and antiproliferative potentials of C. colocynthis seeds since no previous work has investigated the pharmacological activities of seeds from C. colocynthis growing in the north of Morocco up to this date.

Plant Material and Extract Preparation
The plant was harvested in March 2015 from Morocco (Tangier city) and was authenticated by Dr. M. Bakkali before being deposited in the herbarium #LMB 06/04. Next, the fruits were washed with distilled water and dried at room temperature. Seeds were salvaged and ground before being macerated with hexane for 72 h. The whole mixture obtained was filtered using a Whatman filter and the extraction solvent was removed using a rotary set to 45 • C to obtain an oily paste. The obtained extract was meticulously saved at 4 • C until further use.

β-Carotene Bleaching Assay
The antioxidant activity was performed using bleaching of a beta-carotene assay. Briefly, 0.140 mg of β-carotene was solubilized in 0.70 mL of chloroform before being added to 200 µL of the β-carotene solution with 1.40 mg of linoleic acid and 14.00 mg of Tween 40. Thereafter, the solution was meticulously stirred with 3.5 mL of water before being added to the microtitration plates in 200 µL previously supplemented with 8 µL of different concentrations of the studied extracts (0.3125-10 mg/mL). The absorbance of each concentration was measured immediately using spectrophotometry at 470 nm. Afterward, samples were incubated at 50 • C for 120 min and the oxidation was assessed by reading the absorbance at 470 nm. BHT was used as a standard reference. The antioxidant property was given as an inhibition percentage [19].

DPPH Assay
The antioxidant effect test was done according to the previously reported protocols with slight modifications [19]. The plant extract and BHT (butylated hydroxytoluene) were tested with concentrations ranging from 1 to 14 mg/mL. Briefly, 150 µL of each concentration (1,2,4,6,8,10,12, and 14 mg/mL) were dropped in wells supplemented with 50 µL of previously prepared 1 mM DPPH solution. The prepared microplates were saved at an ambient temperature in darkness for 31 min. The reading of absorbance was effectuated at 517 nm. BHT was considered as a standard antioxidant product.
2.3. Antiprolifertaive Activity 2.3.1. Cell Culture MDA-MB-231 and HT-29 cancerous lines were used for screening the potential antiproliferative effect of C. colocynthis seed extract. Cells were obtained from UFR of pharmacy, Reims, France. The antiproliferative study was conducted according to protocols reported in earlier work [20].

Cell Viability Assay
The viability of cells was studied according to the assay as described in earlier work [21]. In this research work, the antiproliferative effect of C. colocynthis seed extract was investigated using MDA-MB-231 and HT-29 cells lines. These cancerous cells were kindly provided by Dr. L'Houcine Ouafik (laboratory of oncology-Marseille, France). A culture medium (MDEM) including 1% glutamine, 10% fetal calf serum, and 1% antibiotic (streptomycin/penicillin) was used to grow cells at 37 • C. For testing, cancerous cells were seeded on 96-well plates at a density of about 8000 cells per well. When the incubation period was finished, 10 µL of culture medium were replaced with an equivalent volume of C. colocynthis extract with concentrations ranging from 15.6 to 500 µg/mL. Next, the treated plates were incubated again for 72 h. Afterward, the WST-1 agent was added to the plates with further incubation for 4 h at 37 • C. For comparison purposes, the effect of C. colocynthis seed extract on healthy human dermal fibroblasts cultured under sub-similar conditions to those of cancer cells was investigated [22]. The reading of cell viability was conducted by using Wallac Victor X3 multiplate reader. In this protocol, non-treated cells were used as a negative control. The IC 50 value (concentration required for killing of 50% of the cell population) was calculated from plotting the inhibition percentage vs. concentrations (µg/mL).
The percentage of antiproliferative activity was done as follows: Cell death (%) = control OD − sample OD control OD * 100 The concentration responsible for 50% cell inhibition (IC50) was performed from the dose-response curve.

Antibacterial Activity
Bacterial strains were used in the current research work including Gram-positive Enterococcus faecalis 471, Listeria monocytogenes, and Staphylococcus aureus 476; and Gramnegative Pseudomonas aeruginosa, Escherichia coli and Salmonella typhimurium. Bacterial growth was carried out at 37 • C on solid Müeller-Hinton Agar medium and peptone liquid medium.
The antibacterial effect was evaluated using the disc diffusion method. Briefly, 100.00 µL of inoculum of each bacterial suspension was seeded in Petri dishes with 20 mL of MHA medium. After 5 min, a disk of sterile Whatman paper with 0.5 cm in diameter was impregnated with 20 µL of the solubilized extract before being deposited on the plate surface. Afterward, dishes were placed at room temperature for 1 h and incubated again at 37 • C for a further 24 h to be ready for reading.

Identification of Constituents by GC-MS Analysis
The identification of phytochemical compositions of the C. colocynthis seed extract was performed by GC-MS. Briefly, 1 µL was injected for analysis into gas chromatography (GC/MS) equipped with a Thermo Fischer capillary column directly coupled to the mass spectrometry system and a column with HP-5MS fused silica capillary (30 m × 250 µm). The analysis was performed under the following GC/MS conditions: initial temperature of 50 • C/2 min, speed of 11 • C/min to a final temperature of 200 • C, hold for 0 min, ramp of 6 • C/min to 240 • C, hold for 1 min, carrier gas; helium (1 mL/min). Solvent delay: 4.00 min; injection temperature: 280 • C; detection temperature: 250 • C; scan: 40 to 450 Da. The identification of the extract phytochemicals was carried out by comparing the retention indices with those of the references obtained from the databases along with the calculation of retention indices (RI) [23][24][25].

Statistical Analysis
Data obtained in the current research were expressed using the means of duplicate bioassays ± SD. The obtained significant difference was performed using a t-test. Statistically, a significant difference was considered when p < 0.05

Antiproliferative Effect of C. colocynthis Seeds
Generally, the findings showed that both cancerous cells HT-29 and MDA-MB231 were sensitive to hexane extract of C. colocynthis seeds. The IC 50 value of C. colocynthis seeds on MDA-MB231 cell lines was determined at 86.89 ± 3.395 µg/mL. Meanwhile, the IC 50 value in inhibiting HT-29 was determined at 242.1 ± 17.9 µg/mL. The MDA-MB231 cell lines found to be more sensitive to the plant seed extract when compared to HT-29 (p < 0.05) (Figures 1 and 2).
The antibacterial effect was evaluated using the disc diffusion method. Briefly, 100.00 μL of inoculum of each bacterial suspension was seeded in Petri dishes with 20 mL of MHA medium. After 5 min, a disk of sterile Whatman paper with 0.5 cm in diameter was impregnated with 20 μL of the solubilized extract before being deposited on the plate surface. Afterward, dishes were placed at room temperature for 1 h and incubated again at 37 °C for a further 24 h to be ready for reading.

Identification of Constituents by GC-MS Analysis
The identification of phytochemical compositions of the C. colocynthis seed extract was performed by GC-MS. Briefly, 1 μL was injected for analysis into gas chromatography (GC/MS) equipped with a Thermo Fischer capillary column directly coupled to the mass spectrometry system and a column with HP-5MS fused silica capillary (30 m × 250 μm). The analysis was performed under the following GC/MS conditions: initial temperature of 50 °C/2 min, speed of 11 °C/min to a final temperature of 200 °C, hold for 0 min, ramp of 6 °C/min to 240 °C, hold for 1 min, carrier gas; helium (1 mL/min). Solvent delay: 4.00 min; injection temperature: 280 °C; detection temperature: 250 °C; scan: 40 to 450 Da. The identification of the extract phytochemicals was carried out by comparing the retention indices with those of the references obtained from the databases along with the calculation of retention indices (RI) [23][24][25].

Statistical Analysis
Data obtained in the current research were expressed using the means of duplicate bioassays ± SD. The obtained significant difference was performed using a t-test. Statistically, a significant difference was considered when p < 0.05

Antiproliferative Effect of C. colocynthis Seeds
Generally, the findings showed that both cancerous cells HT-29 and MDA-MB231 were sensitive to hexane extract of C. colocynthis seeds. The IC50 value of C. colocynthis seeds on MDA-MB231 cell lines was determined at 86.89 ± 3.395 μg/mL. Meanwhile, the IC50 value in inhibiting HT-29 was determined at 242.1 ± 17.9 μg/mL. The MDA-MB231 cell lines found to be more sensitive to the plant seed extract when compared to HT-29 (p < 0.05) (Figures 1 and 2).  MDA-MB-231 cell lines were found to be more vulnerable to C. colocynthis seed organic extract than HT-29. Meanwhile, the extract of C. colocynthis seeds showed safety in in normal human dermal fibroblasts. The observed difference in IC 50 values could be related to the difference in the treated cell lines (drug-resistant cell lines). The obtained findings in the current research conformed with a previous report, which showed that acetone pulp extract of C. colocynthis possessed cytotoxic effects on MCF-7, MDA-MB-231, and SiHa cancer lines [22,26]. In this sense, our hexane extract was majorly constituted of 2,4-dimethylhept-1-ene; (E)-hept-2-enal; 2-Pentenal, (E)-: 2,4,6-Trimethyloctane; Octane, 2,4,6-Trimethyl, and Undecane, which would be involved in the obtained results of antiproliferative activities; however, the C. colocynthis acetone extract reported in this literature is mainly constituted of oxalic acid; 2,2-Dimethyl-propyl 2,2-dimethyl-propanesulfinyl sulfone; cyclobutyl octadecyl ester; Octadecatrienal, which might be involved in the cytotoxic effect on MCF-7, MDA-MB-231, and SiHa cancer lines. Moreover, both extracts of acetone and hexane were active towards MDA-MB-231 cell lines, so we can confirm that solvents with different polarities can result in different bioactive compounds from C. colocynthis. MDA-MB-231 cell lines were found to be more vulnerable to C. colocynthis seed organic extract than HT-29. Meanwhile, the extract of C. colocynthis seeds showed safety in in normal human dermal fibroblasts. The observed difference in IC50 values could be related to the difference in the treated cell lines (drug-resistant cell lines). The obtained findings in the current research conformed with a previous report, which showed that acetone pulp extract of C. colocynthis possessed cytotoxic effects on MCF-7, MDA-MB-231, and SiHa cancer lines [22,26]. In this sense, our hexane extract was majorly constituted of 2,4dimethylhept-1-ene; (E)-hept-2-enal; 2-Pentenal, (E)-: 2,4,6-Trimethyloctane; Octane, 2,4,6-Trimethyl, and Undecane, which would be involved in the obtained results of antiproliferative activities; however, the C. colocynthis acetone extract reported in this literature is mainly constituted of oxalic acid; 2,2-Dimethyl-propyl 2,2-dimethyl-propanesulfinyl sulfone; cyclobutyl octadecyl ester; Octadecatrienal, which might be involved in the cytotoxic effect on MCF-7, MDA-MB-231, and SiHa cancer lines. Moreover, both extracts of acetone and hexane were active towards MDA-MB-231 cell lines, so we can confirm that solvents with different polarities can result in different bioactive compounds from C. colocynthis.

Antioxidant Effect
In the current research study, hexane extract of C. colocynthis was screened for potential antioxidant activity using β-carotene bleaching and DPPH assay. As reported in Figure 3, the C. colocynthis organic extract exhibited potent antioxidant activity with an IC50 value of 2.22 mg/mL in beta-carotene bleaching. The hexane extract of C. colocynthis seeds was also studied in terms of antioxidant activity using DPPH assay as presented in Figure  4. The antioxidant activity increases with increasing extract concentration (Figure 4). Therefore, the studied extract exhibited antioxidant properties in a dose-dependent man-

Antioxidant Effect
In the current research study, hexane extract of C. colocynthis was screened for potential antioxidant activity using β-carotene bleaching and DPPH assay. As reported in Figure 3, the C. colocynthis organic extract exhibited potent antioxidant activity with an IC 50 value of 2.22 mg/mL in beta-carotene bleaching. The hexane extract of C. colocynthis seeds was also studied in terms of antioxidant activity using DPPH assay as presented in Figure 4. The antioxidant activity increases with increasing extract concentration ( Figure 4). Therefore, the studied extract exhibited antioxidant properties in a dose-dependent manner. The IC 50 value of the organic studied extract was estimated at 8.98 ± 0.619 mg/mL.    The antioxidant activity of C. colocynthis seed extract effect was found more important. It is thus fitting that the present findings were in agreement with earlier work that reported free radical scavenging effects of C. colocynthis [27]. Many researchers have attributed the antioxidant property of C. colocynthis extract to the flavonoids content in its seeds and fruits, i.e., isosaponarin, isovitexin, and isoorientin3-O-methylether isolated from C. colocynthis possessed an important antioxidant activity with an IC50 value ranging from 5.62 × 10 −4 to 7.13 × 10 −2 mg/mL [28]. The reported findings in the current work were in accordance with earlier literature, which investigated the scavenging activity of C. colocynthis hexane extract [29].

Antibacterial Activity
The antibacterial effect of the tested extract varied as a function of the target microorganism. The studied extract did not affect Gram-negative bacterial strains whilst the hexane extract generated a clear growth inhibition zone on E. faecalis 471 strains with a diameter of inhibition zone reaching 3 mm (Gram-positive). However, S. aureus as a Gram-positive was not sensitive to the C. colocynthis extract.
Bacterial infections have presented a great challenge because of the eventual resistance of bacteria to modern drugs, hence the development of alternative drugs remains one of the most effective solutions to mitigate the extensions of bacterial infections. Desiring to contribute to the bacterial infection palliation, we tested C. colocynthis for its potential antibacterial activity in the current study. In this sense, the tested seed extract of C. colocynthis was active on some positive bacterial strains such as E. faecalis 471 and L. monocytogenes. However, no effect was observed on Gram-negative bacteria nor S. aureus. These findings were in agreement with previously reported studies, which highlighted a poor effect of C. colocynthis ethanolic extract on Gram-negative bacteria [30]. However, the aqueous and acetone extracts of C. colocynthis were active on both Gram-positive and The antioxidant activity of C. colocynthis seed extract effect was found more important. It is thus fitting that the present findings were in agreement with earlier work that reported free radical scavenging effects of C. colocynthis [27]. Many researchers have attributed the antioxidant property of C. colocynthis extract to the flavonoids content in its seeds and fruits, i.e., isosaponarin, isovitexin, and isoorientin3-O-methylether isolated from C. colocynthis possessed an important antioxidant activity with an IC 50 value ranging from 5.62 × 10 −4 to 7.13 × 10 −2 mg/mL [28]. The reported findings in the current work were in accordance with earlier literature, which investigated the scavenging activity of C. colocynthis hexane extract [29].

Antibacterial Activity
The antibacterial effect of the tested extract varied as a function of the target microorganism. The studied extract did not affect Gram-negative bacterial strains whilst the hexane extract generated a clear growth inhibition zone on E. faecalis 471 strains with a diameter of inhibition zone reaching 3 mm (Gram-positive). However, S. aureus as a Gram-positive was not sensitive to the C. colocynthis extract.
Bacterial infections have presented a great challenge because of the eventual resistance of bacteria to modern drugs, hence the development of alternative drugs remains one of the most effective solutions to mitigate the extensions of bacterial infections. Desiring to contribute to the bacterial infection palliation, we tested C. colocynthis for its potential antibacterial activity in the current study. In this sense, the tested seed extract of C. colocynthis was active on some positive bacterial strains such as E. faecalis 471 and L. monocytogenes. However, no effect was observed on Gram-negative bacteria nor S. aureus. These findings were in agreement with previously reported studies, which highlighted a poor effect of C. colocynthis ethanolic extract on Gram-negative bacteria [30]. However, the aqueous and acetone extracts of C. colocynthis were active on both Gram-positive and Gram-negative bacteria, as reported in earlier data [31]. In this sense, the differences can be attributed to solvent polarities used for extraction. The antibacterial activity could probably due to the presence of cucurbitacin molecules since the cucurbitacin E was effective against M. tuberculosis H37Rvat [32].

Chemical Analysis of C. colocynhtis Seed Extract
The finding of chemical analysis of C. colocynthis seed extract revealed the presence of many interesting compounds majorly consisting of 2,4-Nonadienal, Tetradecane, hexadecane, pentadecane, cinnamic acid derivaties, Linalool, and butylated hydroxyanisole ( Figure 5; Table 1). to have antioxidant power in previous work [45].
Our extract was generally rich in compounds belonging to alkanes, hydrocarbons, phenolic, and fatty acids. The presence of these compounds in our C. colocynthis seeds extract is probably correlated to its antioxidant, antiproliferative, and antibacterial effects. The identified compounds can react individually or in synergy and even with potential potentiation effects [46].    to have antioxidant power in previous work [45]. Our extract was generally rich in compounds belonging to alkanes, hydrocarbons, phenolic, and fatty acids. The presence of these compounds in our C. colocynthis seeds extract is probably correlated to its antioxidant, antiproliferative, and antibacterial effects. The identified compounds can react individually or in synergy and even with potential potentiation effects [46].  antioxidant and antimicrobial properties [43,44]. Butylated hydroxyanisol is also reported to have antioxidant power in previous work [45]. Our extract was generally rich in compounds belonging to alkanes, hydrocarbons, phenolic, and fatty acids. The presence of these compounds in our C. colocynthis seeds extract is probably correlated to its antioxidant, antiproliferative, and antibacterial effects. The identified compounds can react individually or in synergy and even with potential potentiation effects [46].

Conclusions
The present study evidenced the phytochemical composition, antioxidant, antibacterial, and acute toxicity testing of C. colocynthis extract. The studied plant extract possesses an interesting chemical composition with antioxidant, antiproliferative, and antibacterial properties. Therefore, C. colocynthis was used with the hope to contribute to the development of effective drugs against cancer and free-radical-related diseases, alongside bacterial infections.

Conclusions
The present study evidenced the phytochemical composition, antioxidant, antibacterial, and acute toxicity testing of C. colocynthis extract. The studied plant extract possesses an interesting chemical composition with antioxidant, antiproliferative, and antibacterial properties. Therefore, C. colocynthis was used with the hope to contribute to the development of effective drugs against cancer and free-radical-related diseases, alongside bacterial infections.

Conclusions
The present study evidenced the phytochemical composition, antioxidant, antibacterial, and acute toxicity testing of C. colocynthis extract. The studied plant extract possesses an interesting chemical composition with antioxidant, antiproliferative, and antibacterial properties. Therefore, C. colocynthis was used with the hope to contribute to the development of effective drugs against cancer and free-radical-related diseases, alongside bacterial infections.

Conclusions
The present study evidenced the phytochemical composition, antioxidant, antibacterial, and acute toxicity testing of C. colocynthis extract. The studied plant extract possesses an interesting chemical composition with antioxidant, antiproliferative, and antibacterial properties. Therefore, C. colocynthis was used with the hope to contribute to the development of effective drugs against cancer and free-radical-related diseases, alongside bacterial infections.  The pharmacological activities investigated in this study are frequently related to the identified compounds in the C. colocynthis seed extract since the chemical analysis showed many potentially bioactive compounds with pharmacological activities, including antioxidant and antibacterial activities as reported in earlier works [30][31][32][33][34][35]. In this sense, Jasmonic acid played a crucial role in defense against insects, pathogenic microorganisms, kin aging, as well cancer development [36,37]. 2,4-Nonadienal is previously reported to possess antifungal, antidiarrheal, and antioxidant activities [38][39][40]. Tetradecane and hexadecane as well as pentadecane are alkanes possessing antifungal and antibacterial effects [41]. Cinnamic acid occurs naturally in flora. Cinnamic acid derivatives are reported to possess a wide spectrum of biological activities including antioxidant and anticancer activities [42]. Linalool is one of the major compounds in the C. colocynthis seed extract, with antioxidant and antimicrobial properties [43,44]. Butylated hydroxyanisol is also reported to have antioxidant power in previous work [45].
Our extract was generally rich in compounds belonging to alkanes, hydrocarbons, phenolic, and fatty acids. The presence of these compounds in our C. colocynthis seeds extract is probably correlated to its antioxidant, antiproliferative, and antibacterial effects. The identified compounds can react individually or in synergy and even with potential potentiation effects [46].

Conclusions
The present study evidenced the phytochemical composition, antioxidant, antibacterial, and acute toxicity testing of C. colocynthis extract. The studied plant extract possesses an interesting chemical composition with antioxidant, antiproliferative, and antibacterial properties. Therefore, C. colocynthis was used with the hope to contribute to the development of effective drugs against cancer and free-radical-related diseases, alongside bacterial infections.