Diversity of Citrullus colocynthis (L.) Schrad Seeds Extracts: Detailed Chemical Profiling and Evaluation of Their Medicinal Properties

Seeds and fruits of Citrullus colocynthis have been reported to possess huge potential for the development of phytopharmaceuticals with a wide range of biological activities. Thus, in the current study, we are reporting the potential antimicrobial and anticancer properties of C. colocynthis seeds extracted with solvents of different polarities, including methanol (M.E.), hexane (H.E.), and chloroform (C.E.). Antimicrobial properties of C. colocynthis seeds extracts were evaluated on Gram-positive and Gram-negative bacteria, whereas, anticancer properties were tested on four different cell lines, including HepG2, DU145, Hela, and A549. All the extracts have demonstrated noteworthy antimicrobial activities with a minimum inhibitory concentration (MIC) ranging from 0.9–62.5 µg/mL against Klebsiella planticola and Staphylococcus aureus; meanwhile, they were found to be moderately active (MIC 62.5–250 µg/mL) against Escherichia coli and Micrococcus luteus strains. Hexane extracts have demonstrated the highest antimicrobial activity against K. planticola with an MIC value of 0.9 µg/mL, equivalent to that of the standard drug ciprofloxacin used as positive control in this study. For anticancer activity, all the extracts of C. colocynthis seeds were found to be active against all the tested cell lines (IC50 48.49–197.96 µg/mL) except for the chloroform extracts, which were found to be inactive against the HepG2 cell line. The hexane extract was found to possess the most prominent anticancer activity when compared to other extracts and has demonstrated the highest anticancer activity against the DU145 cell line with an IC50 value of 48.49 µg/mL. Furthermore, a detailed phytoconstituents analysis of all the extracts of C. colocynthis seeds were performed using GC–MS and GC–FID techniques. Altogether, 43 phytoconstituents were identified from the extracts of C. colocynthis seeds, among which 21, 12, and 16 components were identified from the H.E., C.E., and M.E. extracts, respectively. Monoterpenes (40.4%) and oxygenated monoterpenes (41.1%) were the most dominating chemical class of compounds from the hexane and chloroform extracts, respectively; whereas, in the methanolic extract, oxygenated aliphatic hydrocarbons (77.2%) were found to be the most dominating chemical class of compounds. To the best of our knowledge, all the phytoconstituents identified in this study are being reported for the first time from the C. colocynthis.


Introduction
Folk medicine has long been dependent on plants, which are considered as a crucial source of bioceuticals for the treatment and prevention of innumerable diseases for generations [1,2]. Albeit immense progress in modern medicine, a huge chunk of population across the globe, and particularly, people of a low-income category, are still dependent on natural product-based traditional methods of treatment for curing a variety of ailments [3,4]. The heavy use of these treatment methods is mainly derived by ancient knowledge, local credence, effectiveness, and low-cost [5]. Indeed, the recent emergence of pandemics has greatly renewed interests in the application of natural products, including plant-based materials and their compounds as nutraceuticals [6][7][8]. Traditionally, plant materials are dried,

Chemo-Profiling of Different Extracts
Bioactive secondary metabolites are important for the physiology of both plants and humans, as they protect them by acting as anti-oxidants against oxidative stress [38]. In this regards, numerous studies have been reported in the literature describing the important biological properties of secondary metabolites including anti-microbial and anti-cancer properties [39,40]. Thus, the chemical characterization analysis of phytoconstituents of different extracts of the seeds of C. colocynthis, which are extracted with solvents of varied polarities, including methanol (M.E.), hexane (H.E.), and chloroform (C.E.), is undertaken. Moreover, the biological potentials including the antibacterial and anticancer properties of each individual extract were also evaluated. The solvent extractions of the seeds of C. colocynthis from Saudi Arabia was carried out at room temperature using conventional percolation technique, as described in earlier reports [30], and is shown in Figure 1. C. colocynthis is an herbaceous desert plant consisting of perennial roots and vine-like stems [32]. It belongs to the family Cucurbitaceae, and it is native to the arid sandy areas of West Asia, Arabia, tropical Africa, and the Mediterranean [33]. This plant contains a battery of biologically active substances, including glycosides, flavonoids, alkaloids, fatty acids, and essential oils, etc. [34]. Different parts of C. colocynthis have long been used for treating various diseases, such as its fruit pulp (dried), which is effective in treating indigestion and gastroenteritis, while its fruit is known to possesses antioxidant, antimicrobial, and anti-inflammatory properties [35,36]. Moreover, the other pharmacological potential of the C. colocynthis include anti-diabetic, anthelmintic, analgesic, anti-allergic, and anticancerous properties, etc. [37]. Therefore, to investigate the effect of solvents on the biological potential of C. colocynthis, in this study, the plant material was extracted using different solvents, such as, methanol (M.E.), hexane (H.E.), and chloroform (C.E.). The phytoconstituents of each extract was determined separately using gas chromatographic methods. In addition, the antimicrobial and anticancer properties of each extract were assessed individually against several microorganisms and cell lines, respectively.

Chemo-Profiling of Different Extracts
Bioactive secondary metabolites are important for the physiology of both plants and humans, as they protect them by acting as anti-oxidants against oxidative stress [38]. In this regards, numerous studies have been reported in the literature describing the important biological properties of secondary metabolites including anti-microbial and anticancer properties [39,40]. Thus, the chemical characterization analysis of phytoconstituents of different extracts of the seeds of C. colocynthis, which are extracted with solvents of varied polarities, including methanol (M.E.), hexane (H.E.), and chloroform (C.E.), is undertaken. Moreover, the biological potentials including the antibacterial and anticancer properties of each individual extract were also evaluated. The solvent extractions of the seeds of C. colocynthis from Saudi Arabia was carried out at room temperature using conventional percolation technique, as described in earlier reports [30], and is shown in Figure 1.  The extraction was carried out individually by using an initial 500 g of seeds of C. colocynthis in each solvent, which yielded 11.2 g, 50.0 g, and 70.0 g of seed extract in M.E., H.E., and C.E., respectively. Notably, the extraction processes have yielded an almost similar color (dark brown) of extracts in all the solvents; however, their amount was slightly Plants 2023, 12, 567 4 of 17 different due to the nature and quantity of the secondary metabolites extracted. For instance, C.E. extraction has resulted in higher yield, due to the high solubility of the long range of phytoconstituents including medium-polar and polar compounds in the chloroform solution. The chemo-profiling of all the extracts was carried out by GC-MS and GC-FID techniques, which has resulted in the recognition of a total of 21, 12, and 16 components from the H.E., C.E., and M.E. extracts, respectively. All the recognized phytochemicals generated from different extracts and their respective proportions are provided in the Table 1 based on the elution order of the compound from the column (HP-5MS). According to the results in the Table 1, the H.E. extract was dominated by monoterpene hydrocarbons; whereas, the C.E. and M.E. extracts contained oxygenated monoterpenes and oxygenated aliphatic hydrocarbons as major chemical class of compounds, respectively. Particularly, the M.E. extract consisted of 77.2% of oxygenated aliphatic hydrocarbons; meanwhile, H.E. and C.E. extracts contained an almost similar amount of monoterpene hydrocarbons, i.e., 40.4% and oxygenated monoterpene hydrocarbons, i.e., 41.1%, respectively. Apart from the major chemical classes of phytoconstituents, each individual extract contains different chemical categories of compounds as subsequent chemical classes. For example, besides monoterpene hydrocarbons, the H.E. extract consisted of oxygenated aliphatic hydrocarbons and aromatics in an almost similar percentage, i.e., 19.3 and 21.5%, respectively. In the case of the C.E. extract, the oxygenated aliphatic hydrocarbons were present at a distant second position, which was recorded at 27.3%. In addition to these, the C.E. extract also contains diterpenoids, oxygenated sesquiterpenes, aliphatic hydrocarbons, and aromatics; however, chemical classes of these compounds were present in minor amount (<10% each). On the other hand, the M.E. extract does not contain other chemical classes of compounds in large quantities; after their major class of compounds, the other classes of phytoconstituents in M.E. extract are present in minor quantity just below 20% of total constituents. The categories of chemical compounds include oxygenated monoterpenes (3%), aliphatic hydrocarbons (4%), diterpenoids (9.6%), and others (<1%).
It is noteworthy that all the major compounds found in all three different extracts of C. colocynthis seeds, such as α-pinene, thymol, 8,11-octadecadienoic acid methyl ester and others (Figure 3), have been found to be distinct to the plant species collected from Riyadh region in KSA. These phytoconstituents have not been found in C. colocynthis collected from other regions of the world, as shown in Table 2. For example, the plant collected from the city of Tangier in Morocco has demonstrated the presence of nonadienal (15.4%), linalool propanoate (14.3%), and 2,4-decadienal (7.8%) as major constituents [41]. Whereas, the Indian species of the C. colocynthis collected from different cities have shown the presence of 2-methyl,4-heptanone (48.0), 3-methyl,2-heptanone (12.9), n-hexadecanoic acid (12.4), and morphine (9.1) as dominant compounds [42,43]. Particularly, the three major compounds found in each separate extract, such as α-pinene, thymol, and 8,11octadecadienoic acid methyl ester in H.E., C.E., and M.E. extracts, respectively, have been known to possess excellent biological properties. These compounds are distinct to the plant species investigated in this study, and thus the seeds extract of the C. colocynthis collected from Riyadh are expected to demonstrate decent biological properties when compared to the same species gathered from other regions of the world.
Plants 2023, 12, x FOR PEER REVIEW 6 of 17 21, 12, and 16 components were identified from the H.E., C.E., and M.E. extracts, respectively. Among these, the H.E. extract clearly stands out with maximum number of phytoconstituents. The total ion chromatogram of each extracts of the C. colocynthis seed extracts are given in Figure 2. The H.E. extract was mostly dominated by α-pinene (30.6) followed by o-methylacetophenone (10.8%), isopropyl butanoate (10.4%), and δ3-carene (5.1%), while the remaining compounds, such as p-xylene, pseudocumene, tetradecane, hexadecane, methyl hexadecanoate, ethyl hexadecanoate, and methyl oleate are all present in a minor quantity of less than 5%. In the case of the C.E. extract, the major compound was identified as thymol (37.2%), which is followed by 8,11-octadecadienoic acid methyl ester (13.0%), trans-ferruginyl acetate (8.1%), and β-ionol (4.8%). The minor components of the same extract include filifolide-A (3.9%), ethyl phenyl acetate (3.1%), 2E-decenal (3.4%), 8cedren-13-ol (2.8%), and tetracosane (2.1%). Whereas, the M.E. extract is mainly dominated by the 8,11-octadecadienoic acid methyl ester (28.6%) followed by the (Z)-9-octadecenoic acid methyl ester (20.4%), methyl hexadecanoate (18.3%), 6-ketoferruginol (9.6%), n-octadecanoic acid, methyl ester (7.4%), thymol (3.0%), and others, are present in an insignificant amount.    (12.4), and morphine (9.1) as dominant compounds [42,43]. Particularly, the three major compounds found in each separate extract, such as α-pinene, thymol, and 8,11-octadecadienoic acid methyl ester in H.E., C.E., and M.E. extracts, respectively, have been known to possess excellent biological properties. These compounds are distinct to the plant species investigated in this study, and thus the seeds extract of the C. colocynthis collected from Riyadh are expected to demonstrate decent biological properties when compared to the same species gathered from other regions of the world.     Typically, α-pinene is an important secondary metabolite, which is mainly found in essential oils from different plants, such as the Piper nigrum or Juniperus species [47]. α-pinene is a monoterpene, which consists of hydrophobic and volatile properties with fresh pine scent and woody flavour [48]. This compound has been known to possess excellent antimicrobial properties against various Gram-positive and Gram-negative bacterial strains, including the methicillin-resistant Staphylococcus aureus [49]. Additionally, α-pinene has been reported to demonstrate anticancer properties against the human ovarian cancer PA-1 cell line [50]. On the other hand, thymol, which is a monoterpene phenol mainly found in essential oils of the plants from Lamiaceae family (Thymus, Ocimum, Origanum, and Monarda genera) is also surprisingly found in the C.E. extract of the C. colocynthis seeds [51]. Mainly, the thymol-based plant species are used as flavouring and preservative agents and has also been recognized as "safe" (GRAS) or as approved food additives [52]. Thymol is known to possesses excellent anti-inflammatory, anti-microbial, anti-oxidant, and antifungal properties, besides being beneficial for the cardiovascular system [53]. The solvent-based variation in major constituents is not new. Indeed, plants demonstrate a huge difference in their phytochemical constituents, which are typically based on a variety of different factors, such as geographic location, genetic variations, ecological and environmental factors, etc. [39,54]. Similarly, different experimental conditions, such as the solvent, temperature, and pH of extraction processes also have serious effects on the quality and quantity of the phytomolecules. The difference in the major constituents may possibly have different synergistic interactions, which ultimately determine the biological activities of plant-based materials [55].
For example, in a recent study, the monoterpenes' thymol demonstrated direct antibacterial activity against the S. aureus IS-58 strain [56]. Additionally, thymol has also been recognized as anti-tumor agent, which is demonstrated in a recent study during the evaluation of the cell viability and apoptosis in U-87 cells treated with thymol at different concentrations. The half-maximal inhibitory concentration (IC 50 ) of thymol in the U-87 cells was 230 µM, while on a normal cell line it did not exhibit any cytotoxic effect at the same concentration [57]. Besides the two biologically active phytoconstituents, α-pinene and thymol, another major constituent, 8,11-octadecadienoic acid, as the methyl ester found in M.E. extract, has also demonstrated excellent biological properties in previously reported studies [58]. For example, the ethyl acetate extract of the seeds of Acacia nilotica Linn, which contained 11-Octadecenoic acid, methyl ester as major compound, has demonstrated excellent activities against the several tested microbes with zones of inhibition diameters ranging from 27-32 mm against Salmonela typhi, E. coli, Streptococcus feacalis, S. aureus, and so on [59].

Antibacterial Properties
In order to test the antimicrobial efficacy of seeds' extracts of C. colocynthis, all the different extracts, including H.E., C.E., and M.E. extracts, were employed against both Gram-positive bacterial strains, such as S. aureus and M. luteus, and Gram-negative bacterial strains, such as K. planticola and E. coli, respectively. Whereas, ciprofloxacin, which is commonly prescribe as antibiotic, has been used as a control during this study. The extracts have delivered mixed results, i.e., the extracts were active against both Gram-positive and Gram-negative bacterial strains; however, neither of them demonstrated good activities against E. coli, which is Gram-negative bacteria. For instance, both H.E. and C.E. extracts were active against S. aureus and K. planticola, which are Gram-positive and Gram-negative bacteria, respectively. Whereas, the M.E. extract demonstrated antibacterial activity only against a single bacterial strain, i.e., K. planticola. Among these extracts, the H.E. extract has demonstrated superb antibacterial activity against K. planticola, which is almost comparable to the commercially available antibiotic. Meanwhile, the other extracts demonstrated very mild activities towards the tested strains.
The results further revealed that the C.E. extract exhibited mild activity against S. aureus with 7.8 µg/mL, but demonstrated excellent potential towards K. planticola, with 1.9 µg/mL. Meanwhile, the H.E. extract exhibited decent activity against S. aureus with 3.9 µg/mL; whereas, it demonstrated the best of all antibacterial activities against the K. planticola, with 0.9 µg/mL, which was equal to the activity of the commercially available antibiotic, i.e., ciprofloxacin (cf. Table 3). On the other hand, the M.E. was the least active extract among the tested materials, and demonstrated decent activity towards a single strain, which is 7.8 µg/mL towards the K. planticola. Notably, all the extracts studied have demonstrated very mild activity against the M. luteus and E. coli, except the slightly decent activity of the M.E. extract against M. luteus, with 62.2 µg/mL. Among all the extracts, the best antimicrobial activity was demonstrated by he H.E. extract against K. planticola, with 0.9 µg/mL, which is same as the commercially available antibiotic (ciprofloxacin). Similarly, the decent antibacterial activity of the H.E. extract was also observed against S. aureus, with 0.9 µg/mL. However, the same extract demonstrated very mild activity against the other two species of bacteria, namely M. leuteus and E. coli. These results are not surprising, as the H.E. extract contains high amount of α-pinene, which is already known to exhibit strong antibacterial activities against several bacterial strains, including K. planticola and S. aureus [60]. For example, the essential oil of Baccharis reticulata, which contains a high amount of α-pinene, demonstrated excellent antibacterial activities against S. aureus with a MIC value of 256 µg/mL; whereas, the same extract did not have a significant effect on E. coli and other bacteria [61]. α-pinene in a pure form demonstrates excellent antibacterial activities against a large number of bacterial strains; however, when present in the extract or essential oils together with other phytoconstituents, it selectively targets the bacterial species. This can be attributed to the presence of antagonist phytoconstituents, which may stop the action or effect of α-pinene. As in the H.E. extract, besides α-pinene, isopropyl butanoate, o-methylacetophenone, and many other phytochemicals are present, which may function as antagonist phytomolecules. Similar results were also obtained the in case of the C.E. extract, which consisted of thymol as the major constituent, and is already known to have demonstrated excellent antibacterial properties towards several strains [62]. For example, thymol-rich essential oils of Oliveria decumbens (Apiaceae) collected from different Iranian populations demonstrated high antibacterial properties against a variety of Gram-positive and Gram-negative bacteria. The essential oils obtained from Oliveria decumbens (Apiaceae) collected from the Behbahan region of Iran exhibited a MIC value of 1.0 µg/mL against S. aureus (Gram-positive), while the thymol-rich C.E. in this study demonstrated a near similar antibacterial property with a MIC value of 7.8 µg/mL [63]. The gas chromatographic-mass spectrometry analysis put in evidence four main volatile constituents, such as thymol (20.3-36.4%), However, very little has been published with regards to the biological properties of 8,11-Octadecadienoic acid, methyl ester, which is a major constituent of M.E. extract. This is also reflected in our study, where the M.E. extract has demonstrated the least antibacterial activities when compared to the other extracts.

Anticancer Properties
Besides antibacterial properties, the seed extracts of C. colocynthis were also evaluated for their potential anticancer properties, which is explored against a variety of cell lines, including HepG2 (hepatic cancer cells), DU145 (prostate cancer cells), Hela (cervical cancer cells), and A549 (human lung cancer cells). During this study, doxorubicin, a prescription anticancer drug, was employed as a control, which is a commercially available anticancer drug (cf. Table 4). All the studied extracts have demonstrated diverse anticancer activities against different cell lines, which are provided in Table 4. When compared to the controlled drug used in this study, which has shown IC 50 (µg/mL) of less than one (<1) against all the studied cell lines, neither of the extracts has demonstrated the activity, which is close to the value of doxorubicin. The M.E. extract has exhibited IC 50 values of 126.6, 91.9, 99.9, 70.1 µg/mL against HepG2, DU145, Hela, and A549, respectively. Whereas, the H.E. and C.E. extracts have demonstrated the IC 50 values of 177.0 and no activity, and 48.4 and 53.3, 197.2 and 83.8, 82.9 and 154.0 µg/mL against the same cell lines, respectively. However, the IC 50 values are insignificant when compared to the controlled drug, and, according to a reported study on the extensive screening of several extracts from a variety of plants, a plant extract is usually considered to possess in vitro cytotoxic activity when the IC 50 (concentration that causes a 50% cell kill) value is less than 20 µg/mL for the extract [64]. Taking this into account, only the H.E. and C.E. extract has demonstrated very mild 48.4 and 53.3 µg/mL, respectively, against a single cell line, namely, DU145. Nevertheless, the major constituents of these extracts, including α-Pinene and thymol, have been reported to demonstrate considerable anticancer properties against a battery of cell lines. For example, pine needle oil from the crude extract of pine needles, which consists of large amount of α-Pinene, has exhibited considerable inhibitory effect on hepatoma carcinoma BEL-7402 cells, with an inhibitory rate of 79.3% in vitro and 69.1% in vivo [65]. Similarly, the crude extract of Trachyspermum ammi consisting of thymol in large amount has also shown potential cytotoxic activity in the breast cancer cell line MCF-7. The MTT assay demonstrated that the IC 50 values of thymol on MCF-7 cells for 48 h and 72 h were 54 and 62 µg/mL, respectively [66]. These values are close to the IC 50 values of the C.E. extract of C. colocynthis seeds (53.2 µg/mL), which has thymol as major constituents.
Despite the mild anticancer properties of all the studied extracts against different cancer cell lines, the data still demonstrate a clear trend for the selection of extracts for the activity guided isolation of phytomolecules, which is essential in the quest of finding biologically active phytoconstituents [67]. In this regard, no prior reports on the comprehensive analysis of the anticancer properties of the seed extracts of C. colocynthis in different solvents with varying polarities have been reported in the literature. However, few reports have appeared on the anticancer potential of the essential oils of the seed of C. colocynthis, which have demonstrated reasonable anticancer properties towards colorectal cancer cell lines, with IC 50 values varying between 4 and 7 mg/mL [68]. Meanwhile, the other studies have reported that the seed and pulp extracts (extracted using a single solvent) of the fruit of C. colocynthis were effective against various cancer cell lines [69]. However, in this study, we have employed three different solvents with varying polarities to isolate the extracts of the C. colocynthis seeds, which have delivered notable results with different major constituents in a different solvent extract. Similar to the antibacterial results, the M.E. extract with 8,11-Octadecadienoic acid, methyl ester as major constituent has not demonstrated decent anticancer activities with the IC 50 values of more 75 µg/mL against almost all the cell lines studied.

Plant Material
Entire aerial parts of the C. colocynthis grown in the region of Taif, a city in the Mecca Province of southwest Saudi Arabia, were procured in May 2020. Identifications of C. colocynthis were authenticated by Dr. Rajakrishnan Rajagopal from the herbarium division of King Saud University. A specimen sample (24,531) of C. colocynthis is retained in the herbarium division of the King Saud University.

Preparation of C. colocynthis Seeds Extracts
Procured aerial parts of C. colocynthis were air-dried at room temperature. The fruits, leaves, and stem of the plant were separated and subjected to drying separately until a constant weight was achieved. From the fruits of C. colocynthis, the seeds were carefully removed and then ground using a grinder. The resultant C. colocynthis seeds (500 g) were first percolated with n-hexane (550 mL) three times at room temperature. After n-hexane extraction, the marc was again subjected to extraction three times with CHCl 3 (550 mL). Finally, the same extraction process was repeated using the residual marc with methanol (550 mL) for three times at room temperature. Notably, each time, the extraction process was carried out for 3 days for all the solvents employed. The resultant n-hexane, chloroform, and methanol extracts of C. colocynthis seeds were separately dried under a vacuum at 40 • C until the solvents were completely removed using a Buchi rotary evaporating system (Rotavapor R-215, Buchi, Flawil, Switzerland) equipped with a vacuum controller (V-850) and vacuum pump (V-700). These separately dried n-hexane, CHCl 3 , and methanol extracts were used for the screening of anticancer and antimicrobial activities, as well as for the GC analysis.

GC and GC-MS Analysis of C. colocynthis Seeds Extracts
In order to identify the chemical constituents of the extracts of C. colocynthis seeds, the dried extracts of n-hexane and CHCl 3 extracts were dissolved in diethyl ether, whereas the methanol extract was dissolved in methanol and subjected to GC-FID and GC-MS analyses. The GC-MS system was equipped with stationary phase columns (HP-5MS) employing the same method, as described previously [70]. Detailed methodology is given in Supplementary Materials (Scheme S1). The identified constituents from n-hexane, CHCl 3 , and methanol extracts of C. colocynthis seeds and their relative percentages are provided in Table 1, and the constituents are listed according to their elution order on the HP-5MS column.

Calculation of Linear Retention Indices (LRIs)
LRI values of chemical constituents of C. colocynthis seeds extracts were determined following a previously reported method [70], and they are listed in Table 1. The detailed methodology is provided in Supplementary Materials (Scheme S2).

Identification of Volatile Chemical Components
Identification of the chemical constituents of C. colocynthis seeds extracts were carried out through an analysis on a HP-5MS column, as described previously [70]. Detailed methodology for the identification of chemical constituents is provided in the Supplementary Materials (Scheme S3) [71][72][73]. GC-MS chromatograms for the identified constituents of n-hexane, chloroform, and methanol extracts of C. colocynthis seeds on the HP-5MS column are given in Figure 2. 3.7. Evaluation of Antimicrobial and Anticancer Activity 3.7.1. Antimicrobial Activity Antimicrobial activity of the C. colocynthis seeds extracts was examined using the well diffusion method [74] towards a panel of four pathogenic bacterial strains, including Staphylococcus aureus MTCC 96, Micrococcus luteus MTCC 2470, Escherichia coli MTCC 739, and Klebsiella planticola MTCC 530. The four pathogenic reference strains were spread on the surface of the Mueller-Hinton agar Petri plates with 0.1 mL of previously prepared microbial suspensions individually containing 1.0 × 10 7 CFU/mL (equal to 0.5 McFarland standard). Using a cork borer, the wells of the 6.0 mm diameter were prepared in the media plates, and the prepared test extracts at a dosage range of 250-0.48 µg/well were added in each well under sterile conditions in a laminar air flow chamber. The standard antibiotic solution of Ciprofloxacin at a dose range of 250-0.48 µg/well and the well containing dimethyl sulfoxide (DMSO) served as positive and negative controls, respectively. The plates were incubated for 24 h at 37 • C, and the well containing the least concentration showing the inhibition zone was considered as the minimum inhibitory concentration (MIC). All experiments were carried out in duplicates and mean values are represented.

Anticancer Activity
Cytotoxicity of C. colocynthis seeds extracts was assessed against the human lung adenocarcinoma cell line (A549), human hepatocarcinoma cell line (HepG2), human cervical cancer cell line (HeLa), and human prostate cancer cell line (DU145) using the MTT assay [75]. Briefly, 1 × 10 4 exponentially growing cells were seeded into each 96-well plate (counted by Trypan blue exclusion dye method) and allowed to grow until 60-70% confluence; then, different concentrations of test extracts were added to the culture medium along with negative (DMSO) and positive controls (Doxorubicin). The plates were incubated for 48 h in a CO 2 incubator at 37 • C with a 90% humidified atmosphere and 5% CO 2 . Then, the media of the wells were replaced with 90 µL of fresh serum-free media and 10 µL of MTT (5 mg/mL of PBS), and the plates were incubated at 37 • C for 2 h. The media was discarded and allowed to dry for 30 min. Later, 100 µL of DMSO was added in each well to dissolve the purple formazan crystals, and the absorbance was recorded at 570 nm using Spectra Max plus 384 UV-Visible plate reader (Molecular Devices, Sunnyvale, CA, USA). Each test compound was examined at various concentrations in triplicate, and the results are expressed as a mean with standard deviation (mean ± SD), (n = 3). One-way ANOVA and Dunnett's post-comparison test were used to analyse the data for significant differences (test vs. control). The statistical significance for the experiment was set at p < 0.05.

Conclusions
In this study, the effect of the polarity of the extraction solvents on the phytochemical contents and biological potential of the extracts of the seeds of C. colocynthis were explored. To do this, three different solvents, including M.E., C.E., and H.E., were selected to isolate the phytoconstituents of the studied plant material. The contents of all the studied extracts were vastly different with respect to their major components, and the H.E. and C.E. extracts demonstrated α-pinene and thymol as their major constituents, respectively; whereas, the M.E. extract demonstrated the presence of 8,11-octadecadienoic acid, methyl ester in a large quantity. Out of all the extracts, the H.E. and C.E. extracts clearly stood out in terms of their major constituents and their biological potential. Particularly, the H.E. extract, consisting of