Antioxidant, antifungal, and aphicidal activity of the triterpenoids spinasterol and 22,23-dihydrospinasterol from leaves of Citrullus colocynthis L.

Terpenoids from natural plant sources are valuable for their diverse biological activities that have important roles in the medical and agrochemical industries. In this study, we assessed the antioxidant, antifungal, and aphicidal activities of a mixture of spinasterol and 22,23-dihydrospinasterol from the leaves of Citrullus colocynthis. We used 1,1-diphenyl-2-picrylhydrazyl (DPPH) to assess antioxidant activity, and we measured antifungal activity using mycelium growth inhibition assays with three pathogenic fungi, Magnaporthe grisea, Rhizoctonia solani, and Phytophthora infestans. Aphicidal activity against adults of Myzus persicae was determined using in vitro and in vivo assays. Spinasterol and 22,23-dihydrospinasterol exhibited moderate antioxidant activity, even at lower concentrations: 19.98% at 0.78 µg mL−1, 31.52% at 3.0 µg mL−1, 36.61% at 12.5 µg mL−1, and 49.76% at 50 µg mL−1. Spinasterol and 22,23-dihydrospinasterol showed reasonable levels of fungicidal activity toward R. solani and M. grisea, with EC50 values of 129.5 and 206.1 µg mL−1, respectively. The positive controls boscalid and carbendazim were highly effective against all fungi except boscalid for M. grisea (EC50 = 868 µg mL−1) and carbendazim for P. infestans (EC50 = 8721 µg mL−1). Significant insecticidal activity was observed in both residual and greenhouse assays, with LC50 values of 42.46, 54.86, and 180.9 µg mL−1 and 32.71, 42.46, and 173.8 µg mL−1 at 72, 48, and 24 h, respectively. The antioxidant activity of spinasterol and 22,23-dihydrospinasterol was strongly positively correlated with their antifungal and insecticidal activity. Spinasterol and 22,23-dihydrospinasterol therefore show good antioxidant and aphicidal activity with moderate fungicidal activity, making them suitable candidates for an alternative to synthetic agents.

Antifungal activity. Table 2 presents data on the fungicidal activity of spinasterol and 22,23-dihydrospinasterol, as well as two synthetic chemicals, boscalid and carbendazim, which served as positive controls. The EC 50 value for spinasterol and 22,23-dihydrospinasterol against R. solani was 129.56 µg mL −1 , demonstrating the activity of the mixture against this fungus. Its activity against M. grisea was moderate, with an EC 50 value of 206.09 µg mL −1 , but it provided negligible control of P. infestans, with an EC 50 value of 1093 µg mL −1 . Boscalid was highly effective against R. solani and P. infestans, with EC 50 values of 1.64 and 1.62 µg mL −1 , but it was not effective against M. grisea, for which its EC 50 value was 868 µg mL −1 . By contrast, carbendazim showed excellent results against M. grisea and R. solani, with EC 50 values < 0.78 µg mL −1 , but it was ineffective against P. infestans, with an EC 50 value of 8721.1 µg mL −1 .
Insecticidal activity. The data presented in Table 3 show the aphicidal activity of spinasterol and 22,23-dihydrospinasterol against the green peach aphid, M. persicae. In a residual assay in which adult aphids fed on individual, treated cabbage leaves in petri dishes, the highest mortality was observed after 72 h of exposure, with an LC 50 of 42.46 µg mL −1 , followed by 54.86 µg mL −1 at 48 h and 180.9 µg mL −1 at 24 h. Likewise, the highest mortality in a greenhouse assay was also recorded after 72 h, with an LC 50 of 32.71 µg mL −1 , followed by 42.46 µg mL −1 at 48 h and 173.8 µg mL −1 at 24 h. Mortality was therefore higher in the greenhouse assay than in the residual assay. The results presented in Table 4 show that after a prolonged exposure period of 72 h at a 50 µg mL −1 concentration, 63.3% mortality was observed in the greenhouse and 56.7% mortality in the residual assay. Higher mortality in the greenhouse than in the residual assay was also observed at 48 h (56.7% vs. 50%)  26.7%) at the 50 µg mL −1 concentration. Imidacloprid was used as the positive control at a rate of 0.0025 mL mL −1 water. It produced the highest rates of mortality after 72 h of exposure: 98.33% in the greenhouse assay and 96.67% in the residual assay. Likewise, Imidacloprid also produced significant mortality after 48 h of exposure: 91.67% in the greenhouse assay and 88.33% in the residual assay.

Correlation of antioxidant activity with antifungal and insecticidal activities.
Pearson's correlation coefficients regarding the antioxidant activity of spinasterol and 22,23-dihydrospinasterol showed positive relationships at concentrations of 3.12 µg mL −1 and 0.78 µg mL −1 , which indicated that an increase in concentration of spinasterol and 22,23-dihydrospinasterol resulted in an increase in other values and showed significant (P < 0.05) results with antioxidant activities. Moreover, the antifungal activity towards M. grisea (B), R. solani (C), and Phytophthora (D), the insecticidal activity in the residual assay (E), and the insecticidal activity in the greenhouse assay (F) showed strong positive, significant (P < 0.01) relationships, as presented in Table 5.

Discussion
Medicinal plants are highly prized by humans for their wide variety of biologically active compounds that are used in the pharmaceutical and agricultural industries. These products show substantial potential as natural antioxidants and are also commonly used against various insects 25,26 . Citrullus colocynthis is a valuable source of antioxidant potential; for example, a butanol extract from C. colocynthis fruit showed an IC 50 value of 6 µg mL −1 , and an aqueous extract of fruit had an IC 50 value of 241.25 µg mL −1 . Antioxidant properties of C. colocynthis leaf and root extracts have also been documented: 45.9%, 39.81%, and 36.65% DPPH inhibition from hexane, aqueous and ethanol leaf extracts, respectively, and 29.12%, 35.51%, and 33.83% inhibition from root extracts 12 . The results of Benariba et al. 27 are also consistent with our findings; they reported inhibition of DPPH radicals by seed extracts of C. colocynthis with IC 50 values of 500, 580, and 350 µg mL −1 for aqueous, hydro-methanolic, and ethyl acetate extracts, respectively. Analysis of C. colocynthis extracts has revealed the presence of various biochemical compounds, including tannins, terpenoids, flavonoids, and coumarins, that may be responsible for the pronounced antioxidant effects and other biological activities of this plant 28 . Initial phytochemical screening of C. colocynthis revealed the presence of numerous  www.nature.com/scientificreports/ flavonoids and phenols and showed significant antioxidant activity: 88.8% from fruit extract with potential free radical scavenging consequences at a concentration of 2500 µg mL −14 . Phenolic and flavonoid contents were quantified in solvent extracts of C. colocynthis roots, leaves, and fruits to compare their antioxidant activities.
The total phenolic and flavonoid contents in leaf extracts were 3.07-18.6 mg g −1 and 0.51-13.9 mg g −1 of dry sample, respectively, followed by root and fruit extracts. Leaf ethanol extracts showed the highest antioxidant activity and DPPH radical scavenging activity compared with root and fruit extracts 29 . Chawech et al. 30 reported the antibacterial activity of the isolated compounds cucurbaticin E and glucocucurbaticin E from C. colocynthis against Bacillus cereus and Enterococcus faecalis. The minimum inhibitory concentrations (MIC) were 0.625 and 1.25 mg mL −1 , respectively. Moreover, all C. colocynthis extracts showed antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus, as well as antifungal activity against four Candidia species, Candida krusei, Candida glabrata, Candida parapsilosis, and Candida albicans 31 .
Plant extracts and essential oils contain secondary metabolites, including toxic phenolic, steroid, and terpenoid compounds that are stored in plant cells and show bio-pesticidal properties against pathogens and insect pests. Moreover, these compounds are easily biodegradable, reducing their ability to cause severe damage to humans and the environment [32][33][34] . Review of the literature shows several examples of plant products used for plant protection against a broad spectrum of pathogenic fungi. For instance, thymol and carvacrol have antifungal activity against Botrytis cinerea and Fusarium spp., and results indicate that these compounds could be employed independently as fungicidal agents against various phytopathogenic fungi 35 . The α-cadinol and t-muurolol compounds isolated from Calocedrus macrolepis exhibit significant fungicidal activity against Fusarium oxysporum and R. solani 36 . Methanol extract from the rhizome of Acorus gramineus contains numerous chemical compounds, such as caryophyllene, a-asarone, methyl isoeugenol, and isoasarone safrole, that show antifungal activity. In particular, asaronaldehyde (2,4,5-trimethoxybenzaldehyde) enabled complete control of Phytophthora infestans in potatoes and tomatoes and 75% control of R. solani 37 . Our findings on the antifungal Table 5. Correlation of the antioxidant activity of spinasterol and 22,23-dihydrospinasterol versus antifungal and insecticidal activities. A, antioxidant activity. B, antifungal activity against M. grisea. C, antifungal activity against R. solani. D, antifungal activity against Phytophthora. E, insecticidal activity in residual assay; F, insecticidal activity in greenhouse assay. *P < 0.05, **P < 0.01, significant Pearson's correlation coefficients.  38 , who showed that lactones, sesquiterpene, and triterpenes from Schinus molle fruits and leaves had antifungal potential against Alternaria alternata, Penicillium cyclopium, Aspergillus niger, Aspergillus flavus, Microsporum griseum, and Penicillium italicum. Similarly, the flavonoid 4′-methoxy-5,7-dihydroxyflavone 6-C-glucoside isolated from the stems and leaves of Aquilegia vulgaris showed antifungal activity against the mold A. niger 39 . The antimycotoxigenic and antifungal activity of alcohol and distilled water extracts of C. colocynthis were evaluated against A. flavus and Aspergillus ochraceus, and they showed excellent antifungal activity against A. ochraceus with good antiochratoxigenic activity in liquid medium, consistent with findings about the antifungal activity of the triterpenoids spinasterol and 22,23-dihydrospinasterol 40 . Activities of camphor, pulegone, and verbenone isolated from Myristica fragrans were assessed against the German cockroach Blattellea germanica, and these compounds showed LC 50 values of 0.07 mg cm −1 , 0.06 mg cm −1 , and 0.07 mg cm −1 , respectively 41 . Similarly, other compounds such as carvecol, eugenol, p-cymene, isoeugenol, and thymol displayed anti-adulticidal potential against B. germanica at a rate of 1 mg adult −142 . Likewise, spinasterol and 22,23-dihydrospinasterol exhibited medicinal and cytotoxic properties; these compounds were characterized in Bougainvillea spectabilis and exhibited marked inhibition of the enzyme xanthine oxidase, with an IC 50 value of 39.21 µM 16 . Our results on the toxicity of spinasterol and 22,23-dihydrospinasterol showed that they exhibited insecticidal activity and caused significant mortality of M. persicae. Similar outcomes were described by Torkey et al. 43 , who reported that 2-O-β-d-glucapyranosyl cucurbitacin E isolated from C. colocynthis showed toxicity against Aphis craccivora, causing substantial mortality with an LC 50 of 11,003 ppm. Moreover, 9-oxo-10,11-dehydroageraphorone isolated from Eupatorium adenophorum caused 73.33% mortality of Pseudoregma bambucicola at 2 mg mL −1 with a 6-h exposure. Moreover, 100% control of this pest was recorded at a similar concentration after one month of exposure in a field experiment 44 .
Contact toxicity of the new botanical insecticide Dayabon (SL 10%) was evaluated for different life stages of M. persicae. Its estimated LC 50 values for first, second, third, and fourth instar nymphs and adults were 3254, 3387, 4194, 3839, and 3508 ppm, respectively, and it did not leave residues 45 . Solanum incanum fruit sap extract at different concentrations showed some level of insecticidal and deterrent activity against green peach aphid 46 . The insecticidal and deterrent activity of Solanum incanum may be attributed to the presence of saponins, which alter feeding behavior and molting, causing death at different developmental stages [31][32][33] .
The efficacy of Xanthium strumarium, Tanacecetum parthenium, and Hypericum calycinum extracts towards M. persicae was assessed; they produced nymphal mortality of 89%, 88%, and 57%, respectively, and adult mortality of 12%, 82%, and 88% at the same concentration 47 . Similarly 48 , the leaf extracts of several plants were evaluated against M. persicae, and Ricinus communis extract was most toxic to M. persicae (553 ppm), followed by extracts of Robinia pseudoacacia (1150 ppm for a 24-h exposure) and Lantana camara (6660 ppm). Another study 49 reported that essential oil from F. vulgare caused significant mortality; this mortality was attributed to major compounds such as trans-anethole (67.9%) and fenchone (25.5%), with LC 50 = 0.6 2.4 mL L −1 and LC 90 = 2.4 mL L −1 , and the oil was safe for non-target organisms. These results are consistent with our results on mortality of M. persicae following application of spinasterol and 22,23-dihydrospinasterol.
Our results also showed that the antioxidant activity of spinasterol and 22,23-dihydrospinasterol was significantly correlated with antifungal and insecticidal activity.
Although multiple studies have investigated the antioxidant, antimicrobial, antifungal, and insecticidal activities of plant extracts, essential oils, and isolated compounds, such activities have not previously been evaluated for spinasterol and 22,23-dihydrospinasterol. Thus, this research represents the first investigation of their antioxidant and antifungal properties and extends previous findings on their aphicidal activity against adult M. persicae.

Materials and methods
Collection of materials. Leaf samples of C. colocynthis (Cucurbitales: Cucurbitaceae), also known locally as tumba, were collected from a desert area of Punjab Province, Pakistan (29° 59′ 34″ N, 73° 15′ 13″ E) during 2019. The collected plant samples were identified as (Colocynthis) C. colocynthis by Dr. Dilbar Hussain Entomologist and Hafiz Naveed Ramzan Agronomist at the Entomological Research Institute, Ayub Agriculture Research Institute, Faisalabad, Pakistan. However, a voucher specimen of this material was not deposited because of the lack of an available herbarium. As this plant grows widely in vast, uncultivated desert regions and is partially used on a commercial basis, no permissions or licenses were required for sample collection.
Pure colonies of three pathogenic fungi, rice blast (M. grisea), sheath blight (R. solani), and Phytophthora (P. infestans), were obtained from Department of Pesticides Science, College of Plant Protection, Shenyang Agricultural University, Shenyang, China. The green peach aphids were collected from peach plants and were sustained on cabbage plants grown in a greenhouse at 20 ± 5 °C and 45 ± 5% relative humidity (RH) with a 16 h light/8 h dark photoperiod.
Extraction, purification, and identification of biochemical compounds. Extraction, separation, purification, and identification of the purified compounds were performed by solvent/cold extraction, various chromatographic techniques, mass spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy ( 1 H-NMR and 13 C-NMR), respectively, as previously documented by the author 11  www.nature.com/scientificreports/ purified compounds prepared in methanol were added to 3.5 mL freshly prepared DPPH solution (0.002 g 50 mL −1 in HPLC grade methanol); the mixtures were shaken and incubated in darkness at 28 °C for 30 min. Subsequently, their absorbances were measured at 517 nm with a microplate reader (SpectraMax 190, Molecular Devices, made in China and designed in the USA), and percent inhibition of the prepared DPPH solution was calculated based on the decrease in absorbance using Eq. (1). A lower absorbance value indicated higher radical scavenging activity.
where A blank is the (absorbance of the control treatment) and A sample is the (absorbance of the prepared sample).
Determination of antifungal activity. The antifungal activity of spinasterol and 22,23-dihydrospinasterol against M. grisea, R. solani, and P. infestans was evaluated in vitro by radial growth tests on potato dextrose agar (PDA). The commercial synthetic fungicides boscalid and carbendazim were used as positive controls.
The purified compound was dissolved in acetone and then mixed with PDA to obtain various concentrations (0.78, 3.0, 12.5, and 50 µg mL −1 ). The prepared PDA was transferred into 90-mm petri dishes (15 mL per dish) and inoculated with 5-mm pieces of M. grisea, R. solani, or P. infestans. The pieces of fungus were obtained by pressing at the corner of a mycelial colony already growing on PDA medium. After incubation for one week at 25 °C, the radius of mycelial growth was used to calculate the inhibition percentage of each chemical treatment relative to the 1% acetone control (CK). All treatments were replicated three times, and data were analyzed by standard methods.
Determination of aphicidal activity. Aphicidal activity against the green peach aphid M. persicae was assessed in vitro (residual) and in vivo (greenhouse). For the residual assay, freshly cut cabbage leaves were dipped for 10 s in various concentrations of the tested compounds, dried, and placed in glass petri dishes. Next, 10 adult wingless aphids were transferred onto the leaves. The check (CK) was prepared using a 1% Tween 20 solution with no additional compounds, and all petri dishes were incubated at room temperature and 60% RH with a 16 h light/8 h dark photoperiod for 72 h. For the greenhouse assay, 10 adult wingless aphids were released on clean and healthy plants at the 5-7 true leaf stage. One hour after release, when the aphids had completely settled on the plant leaves, the plants were sprayed with various concentrations of compounds (2-3 sprays; 10 mL each) using a hand sprayer. Imidacloprid 25% WP (wettable powder) was used as the positive control at a rate of 0.0025 mL mL −1 of water, and control (CK) plants were sprayed with a 1% Tween 20 solution. Treated plants, positive controls, and CKs were placed in a greenhouse for 72 h. Mortality data for the in vitro and in vivo experiments were collected after 24, 48, and 72 h of exposure by examining the aphids using a stereomicroscope. Individual aphids were considered to be dead if they made no response to needle stimulation.

Correlation of antioxidant activity with antifungal and insecticidal activity. The correlations
between antioxidant activity of spinasterol and 22,23-dihydrospinasterol and antifungal activity (rice blast, sheath blight, and Phytophthora) and insecticidal activity were calculated using IBM-SPSS statistics version 25.0 and assessed at the P < 0.05 significance level. Statistical analysis. Data were analyzed by analysis of variance (ANOVA), and differences among treatments were assessed using Duncan's multiple range test (DMRT) at the P = 0.05 level using IBM-SPSS statistics version 25.0. Probability analysis was performed for the calculation of LC 50 values using the EPA Probit analysis program version 1.5. Inhibition ratio and EC 50 values were obtained using Log-Probit analysis.

Statement of compliance.
For experimental research, plants leaves were collected from wild habitat following institutional, national, and international guidelines and legislation. As the plant Citrullus colocynthis is wildly grown on vast uncultivated desert area and partially used on commercial basis so, no permissions or licenses was required for the collection of samples.

Conclusions
Spinasterol and 22,23-dihydrospinasterol from C. colocynthis leaves showed moderate antioxidant activity, significant aphicidal activity against M. persicae in residual and greenhouse assays, and moderate antifungal activity against M. grisea and R. solani. Insect mortality was higher in the greenhouse assay than in the residual assay. The antioxidant activity of spinasterol and 22,23-dihydrospinasterol was strongly positively correlated with antifungal and insecticidal activity. Based on these findings, spinasterol and 22,23-dihydrospinasterol could be used for antioxidant, antifungal, and insecticidal purposes as an alternative to synthetic chemical agents. However, more research is needed on the isolation and characterization of other bioactive compounds and their evaluation as antioxidant, antifungal, and insecticidal agents.

Data availability
The data that support the findings of this study are available in the manuscript.