Citrullus colocynthis (L.) Schrad.: A Promising Pharmaceutical Resource for Multiple Diseases

Citrullus colocynthis (L.) Schrad. (Cucurbitaceae) is widely distributed in the desert areas of the world. The fruit bodies of C. colocynthis are recognized for their wide range of nutraceutical potential, as well as medicinal and pharmaceutical uses. The plant has been reported for various uses, such as asthma, bronchitis, cancer, colic, common cold, cough, diabetes, dysentery, and jaundice. The fruit has been extensively studied for its biological activities, which include insecticide, antitumor, and antidiabetic effects. Numerous bioactive compounds have been reported in its fruit bodies, such as essential oils, fatty acids, glycosides, alkaloids, and flavonoids. Of these, flavonoids or caffeic acid derivatives are the constituents associated with the inhibition of fungal or bacterial growth, whereas eudesmane sesquiterpenes or sesquiterpene lactones are most active against insects, mites, and nematodes. In this review, the scientific evidence for the biological activity of C. colocynthis against insecticide, cytotoxic, and antidiabetic effects is summarized.


Introduction
The family Cucurbitaceae, with about 123 genera and over 800 species, is found in the tropics or subtropics and is rare in temperate regions. This family of plants is generally frost-sensitive, intolerant to wet and poorly drained soils, and drought-tolerant [1,2]. The well-known members are bitter apples, cucumbers, gourds, pumpkins, and melons. Because of the increasing awareness of the health benefits of this family, their production has increased over time [3]. The genus Citrullus comprises annual or perennial herbaceous plants in the family Cucurbitaceae. This genus includes four species (C. rehmii De Winter, C. lanatus (Thunb.) Matsum. and Nakai, C. ecirrhosus Cogn., and C. colocynthis (L.) Schrad.) throughout tropical and South Africa, Southwest Asia, and the East Mediterranean region [4].
C. colocynthis (L.) Schrad., distributed in the desert areas of the world, including Sudan, Morocco, Jordan, Tunisia, and Pakistan, has nutraceutical and medicinal values [5][6][7][8][9][10][11][12]. The fruits are locally called Kattu Kattuvellari in Malayalam, colocynth/bitter in English, Pcitummatti in Tamil, Rakhal in Bengali, Anedri in Sanskrit, Indrayan in Hindi, and Hanjal in Urdu [2]. The plant is a traditional medicine used to treat asthma, jaundice, and diabetes. Recently, lots of studies have been conducted on its phytochemical compounds, pharmacology, and toxicology [13][14][15][16][17][18]. To date, there is no related review that focuses on the aspects of its insecticidal, antitumor, and antidiabetic effects. This review provides an overview of the habitat and insecticide, antitumor, and antidiabetic activities of C. colocynthis. Figure 1. The whole plant of C. colocynthis, including its fruits, as well as its geographical distribution (data from the Global Biodiversity Information Facility, "https://www.gbif.org/").

Traditional Uses
C. colocynthis is widely used in many parts of the world for a number of diseases including mastitis, cancer, joint pain, jaundice, bronchitis, asthma, leprosy, constipation, and diabetes [2,[5][6][7]. The clinical uses have been reported in indigenous systems of medicine in tropical and subtropical countries (Sudan, Morocco, Jordan, Tunisia, and Pakistan), which include its uses in gut disorders, such as gastroenteritis, dysentery, indigestion, and colic pain, as well as diabetes, wounds, toothache, cough, and the common cold [4,20,22,23].
In Saudi Arabia, much of the local population knows that the squeezed fruit extracts are used to elicit its purgative action, which can be achieved by treading on the fruits barefoot [5,24]. The population of Northeastern Morocco has used this plant since time immemorial to treat various cardiovascular system diseases [6]. In East Africa, seed tar is Figure 1. The whole plant of C. colocynthis, including its fruits, as well as its geographical distribution (data from the Global Biodiversity Information Facility, "https://www.gbif.org/").

Traditional Uses
C. colocynthis is widely used in many parts of the world for a number of diseases including mastitis, cancer, joint pain, jaundice, bronchitis, asthma, leprosy, constipation, and diabetes [2,[5][6][7]. The clinical uses have been reported in indigenous systems of medicine in tropical and subtropical countries (Sudan, Morocco, Jordan, Tunisia, and Pakistan), which include its uses in gut disorders, such as gastroenteritis, dysentery, indigestion, and colic pain, as well as diabetes, wounds, toothache, cough, and the common cold [4,20,22,23].
In Saudi Arabia, much of the local population knows that the squeezed fruit extracts are used to elicit its purgative action, which can be achieved by treading on the fruits barefoot [5,24]. The population of Northeastern Morocco has used this plant since time immemorial to treat various cardiovascular system diseases [6]. In East Africa, seed tar is applied to the skin by nomads. But, the digestion of this fruit results in acute toxic colitis, bloody diarrhea, and changes in the colon [25]. In southern Punjab, Pakistan, the dry powder of the fruits mixed with jaggery is used in a physic drench ball [7,11,19]. In Jordan, swallowing fresh seeds, locally known as Handal, is used to treat various health issues, including its use as an abortive agent, a cathartic, a diuretic, as well as arthritis and rheumatism [8,12,26]. In southern Tunisia, C. colocynthis is a useful medicine for gout, arthritis, and inflammatory disorders. But, overdosing on the plant's immature fruit is a hazard. Intoxication manifests via cerebral congestions, hypothermia, delirium, gastrointestinal irritations, and colitis [9]. In the Sariska region, the fruits are used to treat fever, general sickness, and obstructive stomachache [27]. In Eastern, Central, and Southern Iran, the fruit is recognized as an antiepileptic, abortifacient, hair growth promoter, analgesic, antidiabetic, and purgative. Adverse events (i.e., vomiting, hematochezia, diarrhea, and colic) have been associated with the use of this plant [1,28]. In Israel, the seed oils and fruits of C. colocynthis have been used as a laxative [2].
Cucurbitacins are the main constituents of this species. The serial compounds are bitter-tasting, mainly tetracyclic, highly oxygenated, derived from skeletons [19-(10→9β)abeo-10α-lanost-5-en]. There are 12 classes of cucurbitacins according to their structure, but not all of them are present in C. colocynthis. Yoshikawa and Zheng et al. [4,32] systematically reported the cucurbitacins (triterpenoids and their glycosides) from this species (such as cucurbitacins A-L, and cucurbitacin E 2-O-β-D-glucopyranoside). Among these cucurbitacins, cucurbitacin E is the main component in C. colocynthis fruit pulp, while compound 10 is detected as the principal cucurbitacin of the fruits [29]. Besides cucurbitacins, preliminary phytochemical screening of this species shows the presence of alkaloids, flavonoids, and phenolic acids ( Figure 2). Twelve alkaloids, including quinoline, nicotinamide, uracil, 2-hydroxyquinoline, 2-methylquinoline, 4-hydroxyquinoline, 4methylquinoline, 6-hydroxyquinoline, 6-methylquinoline, 7, 8-benzoquinoline, 8-hydroxyquinoline, and 8-methylquinoline, were detected in C. colocynthis fruits [36]. Among these, 4-methylquinoline is an effective natural insecticide for weevils in grain storage and the management of spider mites. Apart from the principal constituents, volatile compounds, ketones, epoxy compounds, hydrocarbons [39], and fatty acids [40] are also detected in C. colocynthis.  C. colocynthis is used for insecticidal activities in many countries [37,[41][42][43][44]. In the results of Ahmed et al., the leaf extract of C. colocynthis was exceptional at controlling Brevicoryne brassicae L. (cabbage aphid). Cucurbitacin E [45] and spinasterol [14], isolated from this species, show strong insecticidal effects against Aphis craccivora. Chawech et al. [46] reported that the ethyl acetate and pure compounds (compounds 5 and 10) showed significant larvicidal activities against Galba truncatula (mollusc gastropod) with the deterioration rate exceeding 89.2% and with no toxic effects against associated fauna (Melanoides tuberculate, Aromia moshata, Hydrophilus triangularis, and Athous haemorhoidalis). Elazab et al. [47] indicated that the methanol extracts of C. colocynthis were active against Toxoplasma gondii (an Apicomplexa intracellular protozoan) with an IC 50 of 22.86 µg/mL. In Pakistan, the fruits, in combination with common or black salt, are used to treat lice infestation [48] and helminthiasis [7]. The methanol extract displays potent antimalarial activity against multidrug-resistant and chloroquine-sensitive Plasmodium falciparum strains, with no toxicity (IC 50 = 6.9 and 2.01 µg/mL, respectively) [17]. The nano-extracts of C. colocynthis are efficient against Trichomonas vaginalis and safer than the drug metronidazole [49]. 7,8-Benzoquinoline isolated from fruit bodies is most effective against Tetranychus urticae. Regardless of the application method, quinoline and its structural analogs show insecticidal activities (Sitophilus zeamais and S. oryzae) [36]. Ponsankar et al. [50] reported the screening of cucurbitacin E against different larval instars and analyzed the antifeedant activity using a choice-based test. Petroleum ether shows larvicidal activity against Aedes aegypti L. and  Figure 3 briefly summarizes the medicinal sites or pure compounds with insecticide activity in C. colocynthis, and its detailed insecticide information for different species is listed in Table 2. significant larvicidal activities against Galba truncatula (mollusc gastropod) with the deterioration rate exceeding 89.2% and with no toxic effects against associated fauna (Melanoides tuberculate, Aromia moshata, Hydrophilus triangularis, and Athous haemorhoidalis). Elazab et al. [47] indicated that the methanol extracts of C. colocynthis were active against Toxoplasma gondii (an Apicomplexa intracellular protozoan) with an IC50 of 22.86 µg/mL. In Pakistan, the fruits, in combination with common or black salt, are used to treat lice infestation [48] and helminthiasis [7]. The methanol extract displays potent antimalarial activity against multidrug-resistant and chloroquine-sensitive Plasmodium falciparum strains, with no toxicity (IC50 = 6.9 and 2.01 µg/mL, respectively) [17]. The nano-extracts of C. colocynthis are efficient against Trichomonas vaginalis and safer than the drug metronidazole [49]. 7,8-Benzoquinoline isolated from fruit bodies is most effective against Tetranychus urticae. Regardless of the application method, quinoline and its structural analogs show insecticidal activities (Sitophilus zeamais and S. oryzae) [36]. Ponsankar et al. [50] reported the screening of cucurbitacin E against different larval instars and analyzed the antifeedant activity using a choice-based test. Petroleum ether shows larvicidal activity against Aedes aegypti L. and Culex quinquefasciatus Say with LC50 values of 74.57 and 88.24 ppm, respectively [41,51,52].  Figure 3 briefly summarizes the medicinal sites or pure compounds with insecticide activity in C. colocynthis, and its detailed insecticide information for different species is listed in Table 2.

Cytotoxic Activity
In the professional literature, researchers have reported the antitumor activity of extracts and isolates from this species. Among them, antiproliferative effects have been observed via signaling pathways, including apoptotic pathways (inhibiting STAT3 function and increasing caspase-3) [54][55][56][57][58]. The plant extract increases the number of apoptotic cells and the proportion of sub-G1 cells [59]. The extract also promotes DNA damage in breast cancer cells via the ATM/CHK2/p53 signaling pathway ( Figure 4). And, the antitumor activity of this species is achieved through cell cycle arrest [60]. The plant extract holds significant antitumor activity through the regulation of lipid metabolism (ELOVL2, ACSL5, HMGCLL1, and FASN) [61].  Ayyad et al. [23,62] reported that compounds 5 and 11 have potent inhibitory antitumor activities on HepG2, with IC50 values of 3.5 and 2.8 nmol/mL, respectively. The compounds also prolonged the normalization of the biochemical parameters, life span, and survival times of experimental mice. Two researchers showed cucurbitacin E and its analogs present significant cytotoxic activity against human colon cancer cell lines (HL-60, Caco-2, and HT29) [34,35]. An immunoblot analysis by Saeed et al. [63] highlighted that cucurbitacin E targets epidermal growth factor receptors (EGFRs). In a cell cycle analysis, compounds 2 and 10 resulted in the accumulation of breast cell lines (MDA-MB-231) at the G2/M phase [64,65]. In addition to cucurbitacin, linoleic acid, when compared to other oils, exhibits significant antitumor effects against colorectal cancer cells with IC50 values between 4 and 7 mg/mL [66]. Details of the cytotoxic activity of extracts or pure compounds from C. colocynthis are summarized in Table 3. Table 3. Cytotoxic activity of extracts or pure compounds from C. colocynthis.

No. Active Ingredients
In Vitro/In Vivo Result References Significant cytotoxic activity in Ayyad et al. [23,62] reported that compounds 5 and 11 have potent inhibitory antitumor activities on HepG2, with IC 50 values of 3.5 and 2.8 nmol/mL, respectively. The compounds also prolonged the normalization of the biochemical parameters, life span, and survival times of experimental mice. Two researchers showed cucurbitacin E and its analogs present significant cytotoxic activity against human colon cancer cell lines (HL-60, Caco-2, and HT29) [34,35]. An immunoblot analysis by Saeed et al. [63] highlighted that cucurbitacin E targets epidermal growth factor receptors (EGFRs). In a cell cycle analysis, compounds 2 and 10 resulted in the accumulation of breast cell lines (MDA-MB-231) at the G2/M phase [64,65]. In addition to cucurbitacin, linoleic acid, when compared to other oils, exhibits significant antitumor effects against colorectal cancer cells with IC 50 values between 4 and 7 mg/mL [66]. Details of the cytotoxic activity of extracts or pure compounds from C. colocynthis are summarized in Table 3.

Antidiabetic Activity
Diabetic diseases have side effects (peripheral vascular disease, stroke, nephropathy, neuropathy, and retinopathy) [6,16,24,[68][69][70]. The fruit extracts possess insulin-enhancing activity [71]. C. colocynthis could directly reduce the formation of glycated hemoglobin (HbA1c) [13]. Benariba et al. [72,73] reported that a concentration-response correlation was observed with fruit extracts in the modulation of the insulin secretory response to D-glucose. The fruit extracts could lead to an increase in epididymal fat weight and a lesser decrease in body weight [74]. The ethanolic extract of the seeds has antioxidant and DPPH decolorization potential. It also exhibited a time-dependent decrease in blood glucose levels [75]. C. colocynthis seeds display a direct effect on endocrine pancreatic B cells [76].
Diabetes mellitus causes serious complications affecting multiple organs, and the literature reports the positive effects of C. coliformis on diabetes complications. Aqueous extracts of C. colocynthis ameliorate the toxic effects of streptozotocin. Oral administration of the plant extract reduced the plasma levels of aspartate aminotransferase (AST) and lactic dehydrogenase (LDH) significantly [77]. The fruit had a positive effect on the treatment of diabetic neuropathy, decreasing the number of demyelinated and degenerated nerve fibers [78]. The literature also showed the protective effects against cognitive impairments [79], pancreatic β-cell mass [80], liver/kidney [81], and diabetic neuropathic pain [82]. The antidiabetic activity of C. coliformis is summarized in Figure 5 and detailed in Table 4.
Molecules 2023, 28, x FOR PEER REVIEW 10 of 16 of diabetic neuropathy, decreasing the number of demyelinated and degenerated nerve fibers [78]. The literature also showed the protective effects against cognitive impairments [79], pancreatic β-cell mass [80], liver/kidney [81], and diabetic neuropathic pain [82]. The antidiabetic activity of C. coliformis is summarized in Figure 5 and detailed in Table 4.  2

Fruit extracts Hemoglobin
Increasing time reduced the formation of HbA1c and, thus, inhibited the production of glycated proteins [13] 3 Saponin extract Rabbit Direct hypoglycemic agent [25] 4

Fruit extracts Rat
Lowered glycemia in short-and long-term experiments and during an oral glucose tolerance test (OGTT), lowered serum triglyceride concentration, prevention of a progressive increase in serum cholesterol concentration, increase in epididymal fat weight and lesser decrease in body weight [74] 6

Seed extracts Rat
Glucose homeostasis in rats injected with the β-cytotoxic agent and glucose-stimulated insulin secretion from rat-isolated pancreatic islets [73]   Fruit extracts Rat Glucose-stimulated insulin release [72] 5

Fruit extracts Rat
Lowered glycemia in short-and long-term experiments and during an oral glucose tolerance test (OGTT), lowered serum triglyceride concentration, prevention of a progressive increase in serum cholesterol concentration, increase in epididymal fat weight and lesser decrease in body weight [74] 6

Seed extracts Rat
Glucose homeostasis in rats injected with the β-cytotoxic agent and glucose-stimulated insulin secretion from rat-isolated pancreatic islets [73] 7

Seed extracts Rat
Decolorized DPPH, possessed antioxidant potential, decrease in serum glucose levels, and time-dependent decrease in blood glucose levels [75] 8 Seed extracts Rat Protective effect against pancreatic β-cell mass [80] 14

Seed extracts Rat
Reduction in aspartate aminotransferase (AST) and lactic dehydrogenase (LDH) and increase in blood levels of gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP) [77] 2.5. Clinical Study C. colocynthis could have systemic therapeutic effects on type II diabetic patients (40 patients aged 45-65) through dermal absorption. Experiments showed that the extract reduced insulin secretion and blood glucose (BG) levels. It also decreased serum urea levels, but there was no significant change in micro-albuminuria, hepatic enzymes, lipid profiles, creatinine levels, and other related indices [83]. Barghamdi et al. showed that consumption of C. colocynthis extracts in the intervention group significantly reduced mean glycosylated hemoglobin and fasting blood glucose levels and did not show any side effects (≤125 mg/day). These results indicate that the aqueous extracts had hypoglycemic effects on patients with diabetes, which was associated with their saponins and glycosidic components [84]. In the research of Huseini et al. [85], a clinical experiment was conducted on 50 type II diabetic patients for 2 months. In the research of Li's group, thirty-two type II diabetes patients (ages from 30 to 60) were arranged for this research and distributed into four groups. Capsules of different C. colocynthis extracts were given to patients twice a day for 30 days (1 g per day dosage) and investigated for cholesterol, triglyceride, and glucose levels. C. colocynthis reduced HDL, TGL, cholesterol, and glucose levels by 5, 6, 6, and 35 percent, respectively. From a clinical experiment, it was concluded that powdered C. colocynthis possessed good antidiabetic features [86].

Toxicity
The fruit of C. colocynthis has been used as a traditional medicine, mostly for mastitis, cancer, joint pain, jaundice, bronchitis, asthma, leprosy, constipation, and diabetes. The ingestion of this fruit, however, may have many undesired effects. The biblical story of non-fatal accidental poisoning (described in The Book of 2 Kings) is a related report of the medical toxicology of C. colocynthis in the Old Testament [87]. Feeding a mixture of Nerium oleander and C. colocynthis caused more marked effects and the death of rats [21]. In 1985, a 37-year-old Saudi man was admitted to a local hospital (Riyadh Armed Forces Hospital) with one episode of vomiting, colicky abdominal pain, and severe bloody diarrhea after he had drunk the fruit extracts of C. colocynthis for self-medication for indigestion. He had fresh bleeding from his rectum, and an examination revealed extreme tenderness and slight tenderness in the lower abdomen [5]. These manifestations may be accompanied by transudate in serous cavities, epicardial fat, gelatinization of renal tissue, and enterohepato-nephrotoxicity [88].

Materials and Methods
This literature review used published scientific materials collected from the Web of Science ® and PubMed ® databases without restriction regarding the year of publication and includes literature published through July 2023. The search term used was "Citrullus colocynthis (L.) Schrad.". The chemical names agree with the original references.

Conclusions and Future Perspectives
C. colocynthis is a valuable cucurbit plant and is widely distributed in desert regions of the world. Despite its high dietary value, C. colocynthis is not widely known. In our review, we systematically reviewed the research on this traditional medicine and summarized the related data on the phytochemical structure. Phytochemical studies of the species have resulted in 75 components. Of these, cucurbitacins have previously been reported as the main constituent of this species.
As mentioned above, C. colocynthis contains a variety of chemical components, both non-volatile and volatile. Systematic purification and identification of chemical compositions in C. colocynthis are important. LC-MS and GC-MS is currently the most commonly used characterization technique for quickly and systematically identifying possible nonvolatile and volatile components in plants [100][101][102]. However, for co-eluting compounds with similar spectra, an equivocal identification may be obtained. In recent years, some new technologies, such as gas chromatography ion mobility spectrometry (GC-IMS), have been able to provide the retention time of analytes in GC columns and the separation time of ionized compounds in IMS drift tubes, as well as the amount of each ionized compound reaching the detector in the IMS drift tube, which can significantly improve the identification of co-eluting compounds with similar spectra [103]. Therefore, the combination of LC/GC and IMS may be a good solution for the systematic identification of plants with complex chemical compositions, such as C. colocynthis.
Author Contributions: Investigation and data curation, X.C., M.Q., and R.C.; writing-original draft preparation, X.C.; writing-review and editing, Q.Z. and G.C.; visualization, Y.J. and A.W.; supervision and project administration, B.L. and W.R.; funding acquisition, A.W. and W.R. All authors have read and agreed to the published version of the manuscript.