Citrullus colocynthis (L.) Schrad: Chemical characterization, scavenging and cytotoxic activities

Abstract Citrullus colocynthis (L.) Schrad (C. colocynthis) called colocynth is a wild species that belongs to the family cucurbitaceae. The present research work aimed to study the phytochemical composition, cytotoxic and antioxidant activities of C. colocynthis seed extract. The chemical characterization of C. colocynthis seeds was effectuated using a gas chromatograph coupled to a mass spectrometer (GC-MS). The cytotoxic activity of C. colocynthis seed extract against breast cancer cell lines (MDA-MB-231) and colon cancer cell lines (HT-29) was assessed using the WST-1 bioassay. The antioxidant power was evaluated by the DPPH assay. The phytochemical characterization of C. colocynthis seed extract showed the richness of C. colocynthis seed extract in several families of bioactive compounds. Regarding the cytotoxic activity, the IC50 (the half-maximal inhibitory concentration) of C. colocynthis seed extract in inhibiting MDA-MB-231 and HT-29 were 170.34 and 132.31 µg/mL, respectively. The cancerous cell lines MDA-MB-231 seem to be more sensitive to C. colocynthis seed extract than HT-29 cell lines. C. colocynthis seed extract exhibited a strong antioxidant power with an IC50 value of 1.37 mg/mL. Insight into the results obtained, C. colocynthis seed extract may be used as a promising weapon to fight against cancer and free radicals’ damage.


Introduction
When traditional medicine has not become of great interest for being used in the treatment, doctors and health organizations strongly call for its application in the treatment of diseases because of its promising therapeutic effects. About 80% of the world population and more than 90% of those based in developing countries depend on herbal medicine for primary health care [1][2][3]. For many decades, medicinal plants have played an essential role in pharmacological research studies and drug realizations. Plants contain many active compounds, the great majority of which are derived from secondary metabolism. These constituents are used as therapeutic agents, as raw materials for drug synthesis, or as models for pharmacologically active compounds [4]. Plants possess very interesting biological properties which are attributed to their content in secondary metabolites like polyphenols, alkaloids, terpenes, and essential oils that are applied in various fields like medicine, pharmacy, cosmetology, and agriculture [5].
The knowledge of medicinal plants goes to be lost since the younger generations have not been interested in keeping the natural heritage [7]. As a result, substances developed in laboratories turn out to be more expensive to be used in the treatment [6]. This type of knowledge has a real cultural value and may ultimately lead to the development of new pharmaceutical drugs. The sustainable exploitation of medicinal plants could contribute not only to the preservation of a significant part of biological diversity but also to the improvement in the living conditions of local communities by developing the trade in medicinal plants or their derivatives [8].
C. colocynthis called colocynth is a wild species that belongs to the family cucurbitaceae and is considered to be one of the most genetically diverse plant groups [9]. C. colocynthis fruits have naturally globular aspects, yellow or red color with a potential of poisonous content [10].
The traditional use of C. colocynthis has been largely described in the earlier literature. The plant studied in this research work has been used in the treatment of several diseases comprising diabetes, asthma, constipation, toothache, leprosy, bronchitis, jaundice, joint pain, mastitis, and skin infections [11,12].
To the best of our knowledge, no previous literature has investigated the medicinal properties of C. colocynthis seeds; therefore, the present study was undertaken to screen the phytochemical composition, the cytotoxic effect, and the antioxidant activity of C. colocynthis seeds.

Plant material and extract preparation
The plant material was collected from the surrounding regions of Tangier city located in the north-west of Morocco. The plant was botanically identified by the taxonomist Pr M. Bakkali (Team of Research in Biotechnology and Biomolecular Genius -Faculty of Sciences and Techniques Tangier -Morocco) and the voucher specimen # LMB 06/04 has been deposited in the local herbarium. After drying the fruits at room temperature in the shade for ten days, seeds were salvaged and ground into a fine powder. One hundred milligrams of powder was extracted by maceration using ethanol for 72 h. Afterward, the mixture was filtered under reduced pressure using a rotary evaporator to obtain 2.4 g of crude extract and then kept at −20°C until further use [13].

Identification of bioactive constituents by GC-MS analysis
The analysis of C. colocynthis seed ethanolic extract was carried out using a GC-MS. The Clarus 580 chromatography apparatus equipped with a capillary column (5% phenyl, 95% methypolysiloxane) (30.0 MX 250 µm) and coupled to a mass spectrometer (Polaris Q) (EI 70 eV) was used in this assay. The carrier gas was helium at 1 mL/min. The split was 1/75 and the injection volume was 1 µL. The injection and detection temperatures were set to 250 and 280°C, respectively. The temperature of the furnace regulating the temperature of the column was programmed as follows: from 50°C to 200°C at a rate of 11°C/min, then from 200°C to 240°C at a rate of 6°C/min. The spectra of the major unknown compounds were compared to the spectrum of the known component in the NIST library [13].

Cell culture
Breast cancer cell lines (MDA-MB-231) and colon cancer cell lines (HT-29) were used to perform the current research work. The cancer cell lines were kindly provided by Dr H. Morjani, UFR Pharmacy, Reims, France. Cells were cultured in the DMEM (Dulbecco's Modified Eagle Medium) with the following supplements: Glutamine and fetal calf serum, and a mixture of streptomycin/penicillin was added at the rate of 1% to each. The cancerous cells were kept at 37°C with 5% CO 2 and 95% moisture for 24 h ( Figure 1).

Cell viability assay
Cell viability assay was performed using the WST-1 assay, according to the methods described in the earlier data [13]. Briefly, during the exponential growth of cells, MDA-MB-231and colon HT-29 were initially seeded on 96-well microplates. The C. colocynthis extract was applied at concentrations that ranged from 7.81 to 250 µg/mL. At the end of the treatment period, 10 µL of the medium was replaced with 10 µL of the WST-1 reagent in each well and then the plates were incubated for further time. Cell viability was evaluated by reading the absorbance at 450 nm using a multiplate reader. The percentage of cytotoxicity was calculated using the following equation: In the present study, the concentration giving 50% cell growth inhibition (IC 50 ) was calculated from the dose-response curve obtained by plotting the percentage of inhibition vs concentrations (µg/mL).

Antioxidant activity
The antioxidant activity was conducted, according to the earlier found protocols [14]. C. colocynthis extract was tested in different concentrations that ranged from 0.313 to 10 mg/mL. Briefly, each concentration was dropped in a microplate with 96 wells and then 50 µL of 1 mM DPPH was added to each well. The microplates were placed at room temperature for 30 min under dark conditions and then the absorbance was read at 517 nm. BHT (butylated hydroxytoluene) was used as a positive control. The percentage of free radical inhibition was calculated as follows: IC 50 (the required concentration for inducing 50% inhibition of free radical) was calculated from the graph.

Statistical analysis
Data were expressed as the means of duplicate assays ± SD (standard deviation). The significant difference between the means was calculated using the Student's t-test. Statistically, a significant difference was considered, when P < 0.05.
Ethical approval: The conducted research work is not related to either human or animal use.

Gas chromatography-mass spectrometry analysis
The results of C. colocynthis seed extract showed the presence of interesting bioactive molecules ( Figure 2; Table 1).

Antioxidant effect
C. colocynthis ethanolic extract showed a promising free radical inhibition in dose-dependent concentration. The IC 50 value (the required concentration for inhibiting 50% of free radicals) generated by C. colocynthis ethanolic extract was determined at 4.56 mg/mL by the time the BHT, which

Cytotoxic effect of C. colocynthis seeds
The

Discussion
Cancer is a major burden worldwide, although the modern medicine has succeeded to limit its impact by involving large therapeutic categories, resistance to classical modern therapeutic agents continues to be a major challenge in cancer therapies. It is worth searching for new effective agents with no or few secondary effects for cancer treatment which is highly appreciated. In this context, natural products have continued to receive increasing attention due to their potential preventive and therapeutic effects vs cancer. The role of medicinal plants in cancer treatment has been widely investigated [15]. As earlier described, the products derived from plants are good sources for bioactive compounds' discovery as well as drug conception and development [16,17].
The traditional use of the currently studied plant in North African alternative medicine has been documented in the earlier data, which showed the use of C. colocynthis for the treatment of diabetes and hypertension [18].
In the present research work, we investigated the chemical composition, antioxidant and cytotoxic activities of C. colocynthis ethanolic extract. Regarding the antioxidant activity, the DPPH scavenging method was used to screen this activity. The findings obtained showed that the ethanolic extract possesses a promising antioxidant power with an IC 50 value of 4.56 mg/mL. These results are highly justified by the presence of bioactive compounds in the plant extract characterized with a GC-MS (Table 1), which showed the presence of polyphenols and flavonoids as responsible agents for the antioxidant activity [19]. Isoorientin 3-O-methylether, isosaponarin, and isovitexin isolated from C. colocynthis possess a strong antioxidant power with an IC 50 value ranging from 5.62 × 10 −4 to 7.13 × 10 −2 mg/mL [20]. The antioxidant power of C. colocynthis is in consent with those reported in the earlier data [21]. It was also revealed that the antioxidant activity is attributed to the cucurbitacin glycoside content in the ethanolic extract, as shown in the earlier literature [22].  In the current research work, we also investigated the toxic effects of C. colocynthis on both HT-29 and MDA-MB-231 cell lines. The results obtained showed that the studied extract possesses interesting cytotoxic effects with IC 50 values of 170.34 and 132.31 µg/mL, respectively. These findings were in agreement with the earlier data which showed an important antiproliferative effect on human breast cancer cells induced by cucurbitacin, isolated from the leaves of C. colocynthis [22]. The cytotoxic effects of the fruit pulp of C. colocynthis against cancer breast cell lines MCF-7 were studied [23]. Some chemical compounds revealed in the current extract such as ethylbenzene and tetrachloroethylene could determine the cytotoxic effect of C. colocynthis on HT-29 and MDA-MB-231 cell lines as to whether their effects are due to a single molecule or due to a potential synergy between them, and therefore further studies that may lead to determining the responsible compounds for the current activities are still appreciated.

Conclusion
The present research work provides interesting data about the chemical characterization, antioxidant and cytotoxic effects of C. colocynthis ethanolic extract. Based on the results obtained, C. colocynthis seed extract may be used as a promising weapon to fight cancer and free radicals' damage.