Antioxidant, Antifungal and Insecticidal Activity of Triterpenoids Spinasterol, 22,23-Dihydrospinasterol Isolated from Colocynthis (Citrullus colocynthis L.) Leaves.

Terpenoids from natural plants resources are valuable for diverse biological activities which exhibited important part in medical and agrochemicals industry. This study aimed to assess the antioxidant, antifungal and insecticidal activity of a mixture of Spinasterol, 22,23-dihydrospinasterol isolated from Citrullus colocynthis leaves. 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was used to assess the antioxidant activity whereas, antifungal activity was tested by mycelium growth inhibition assay on three pathogenic fungi Magnaporthe grisea, Rhizoctonia solani and Phytophthora infestans. Insecticidal activity against Brevicoryne brassicae was also determined by using In-vitro and In-vivo assays. The outcome of the study exposed that Spinasterol, 22, 23-dihydrospinasterol afforded prominent antioxidant activity even at lower concentrations i.e. 19.98, 31.52, 36.61 and 49.76% at 0.78, 3.0, 12.5 and 50µgmL -1 respectively. However, moderate fungicidal activity of Spinasterol, 22; 23-dihydrospinasterol was recorded as being EC 50 values 129.5 and 206.1µgmL -1 against R. solani and M. grisea respectively. On the other hand, Boscalid and Carbendazim being a positive control proved highly effective against all fungi except for M. grisea and P. infestans with EC 50 values 868 and 272109µgmL -1 respectively. The pronounced insecticidal activity of Spinasterol, 22,23-dihydrospinasterol was afforded via residual as well as greenhouse assay being LC 50 values as 42.46, 54.86, 180.9 µgmL -1 and 32.71, 42.46 and 173.8µgmL -1 at 72, 48 and 24 h respectively. The study concluded that isolated compound Spinasterol, 22,23-dihydrospinasterol possess promising antioxidant and aphicidal activity with moderate fungicidal activity which could be a suitable candidate as an alternative


Introduction
Natural plants are venerated source of phytochemical compounds responsible for biological activities and are employed in pharmacological as well as agrochemical industry. Although, synthetic chemicals are easily available source which are widely used as antioxidant, antimicrobial, antifungal and pesticidal purposes, however, their rigorous and continuous use has caused resistance development in pest and also poses harmful effects on human's health and the environmental concerns [1].
Study on phytochemical analysis from the leaves of Bryonies callus Rattler revealed that it possess ß-sit sterol, triterpens, spinasterol, 22, 23-dihydrospinasterol, glycosides and phenolic contents. Meanwhile, the extract from B. callus was found effective for the control of Aides aegypti larvae and this mortality may attributed to the existence of phenolic contents and spinasterol, 22,23-dihydrospinasterol. Moreover, larvicidal activity of the extract from Heliotropium indicum and Melothria maderaspatana was also reported [13]. The extract from the leaves of Mukia maderaspatana also possess potential antioxidant properties because of the presence of spinasterol, 22, 23-dihydrospinasterol, avonoid and phenolic contents [14]. It can also scavenge ABTS and DPPH radical molecules which also possess reducing power [15]. The pharmacological study of Bougainvillea spectabillis stems have shown that it has been used against hepatitis disease. It possess caffeic acid and Spinasterol, 22, 23-dihydrospinasterol which was used in herbal medicines against cancer hepatitis causing agents [16]. The leaves of Vitex negundo L. exhibited salicylic acid and 22,23-dihydro-α-spinasterol-β-d-glucoside showed repellency as well as toxicity properties against different strains of Tribolium castaneum [17].
Although, a little research has been made on separation and characterization of several biological compounds from natural plants resources including spinasterol, 22, 23-dihydrospinasterol but its isolation and identi cation from C. colocynthis and their consumption as antioxidant and antifungal activity was not appraised so far. Keeping in view the detailed literature reviewed signi cant biological activities of the compound, the current innovative work was assessed for the rst time for the evaluation of this biochemical compounds as antioxidant activities, antifungal activities against (Magnaporthe grisea, Rhizoctonia solani and Phytophthora infestans) and as insecticidal agent against Brevicoryne brassicae.

Antioxidant Activity
1,1-diphenyl-2-picrylhydrazyl (DPPH) is a steady free radicle molecule with properties of dark-colored crystalline powder commonly used in the laboratory research for antioxidant assay. It dissolved readily in methanol and recognized by absorption of color on spectrophotometer at wavelength of 517 nm.
Antioxidant molecules trap (scavenger) for other radical by the involvement of hydrogen particles, as it has violet color in the solution, and become colorless or pale yellow when neutralized and, thus, resulted in reduction of absorbance. Data obtained by DPPH inhibition (%) by scavenging action of free radical is revealed in (Table 1). Results revealed that at 50µgmL − 1 concentration maximum inhibition (%) afforded by a mixture of Spinasterol, 22,23-dihydrospinasterol was 49.46 followed by 36.61, 31.52 and 19.98 at 12.5, 3.0 and 0.78µgmL − 1 respectively. Results also demonstrated that inhibition % decreased signi cantly on decreasing concentration of the compound. Values are denoted as mean of the ve replicates ± standard error. Different letters given as superscript in the column are not signi cantly unlike according to (DMRT) at P = 0.05 level.

Antifungal Activity
The data on fungicidal activity offered by Spinasterol, 22,23-dihydrospinasterol, Boscalid and Carbendazim is presented in the (

Insecticidal Activity
The data presented in (Table 3)   The results presented in (Table 4) revealed that on prolonged exposure period of 72 h and at 50 mgmL − 1 concentration 63.3% and 56.7% mortality was observed via greenhouse and residual assay respectively. However, higher mortality i.e. 56.7% and 50% was also observed at 48 h at the same concentration via greenhouse and residual assay respectively. Whereas, 30 and 26.7% mortality was recorded at 24 h exposure via greenhouse and residual assay respectively at the same concentration.

Discussion
Natural plants are God gifted treasures for humans which possess a widespread variety of biological compounds involved in pharmaceutical and agricultural industry. These products contain substantial potential as natural antioxidant and also commonly used against various insects [21,22].
Citrullus colocynthis is a valuable source of antioxidant potential such as butanol extract from C.  [12]. Results documented by Benariba et al. [23] are also in accordance with our ndings who reported inhibition of DPPH radical from seed extract of C. colocynthis being IC 50 values as 500, 580 and 350 µgmL − 1 via aqueous, hydro-methanolic and ethyl acetate extract respectively. The analysis of C. colocynthis extracts documented the existence of various biochemical compounds as tannins, terpenoids, avonoids and coumarins responsible for the pronounced antioxidant as well as other biological activities of this plant [24]. Initial screening for phytochemical of C. colocynthis revealed the existence of plenty of avonoids and phenols showed the signi cant antioxidant activity as 88.8% from fruit extract with potential free radical scavenging consequences at a concentration of 2500 µg mL − 1 [4]. The quanti cation of phenolic and avonoids contents from solvent extract of C. colocynthis roots, leaves and fruits extracts was evaluated to compare the antioxidant activities. The amounts of total phenolic and avonoids contents were (3.07-18.6 mgg − 1 and 0.51-13.9 mgg − 1 of dry sample respectively, followed by roots and fruits extract. Ethanol extract of leaves possessed the highest antioxidant activity as well as DPPH radical scavenging activities from roots and fruits extract [25]. In a study documented by Chawech et al. [26] reported the antibacterial activity of isolated compound Cucurbaticin E and Gluco-Cucurbaticin E from C. colocynthis against Bacillus cereus and Enterococcus faecalis. The minimum inhibitory concentration (MIC) values were 0.625 and 1.25 mgmL − 1 respectively. Moreover, all of the populations of C. colocynthis extract showed antibacterial activity against Pseudomonas aeruginosa and Escherichia coli, Enterococcus faecalis and Staphylococcus aureus and antifungal activity against four Candidia species i.e. Candida krusei, Candida glabrata, Candida parapsilosis and Candida albicans [27].
Extracts and essential oils from plant origin contain secondary metabolite; phenolic, steroid and terpenoids compounds which are toxic in nature and are stored in the plant cells and bears bio-pesticidal properties against pathogens and insect pests. Moreover, they are easily biodegradable, bene ting their existence without causing severe damage to the environment and humans [28][29][30]. Literature review showed that there are several examples of plant products used in plant protection measures as a broad spectrum of plant pathogenic fungi, for instance, thymol and carvacrol have antifungal activity against Botrytis cinerea and Fusarium spp. Results indicated that these compounds could be employed independently as fungicidal agents against various phytopathogenic fungi [31]. Besides, α-cadinol and Tmuurolol compounds isolated from the Calocedrus macrolepis exhibit signi cant fungicidal activity against Fusarium oxysporum and Rhizoctonia solani [32]. On the other hand, methanolic extract from the rhizome of Acorus gramineus comprises of numerous chemical compounds such as caryophyllene, aasarone, methyl isoeugenol, isoasarone safrole possessed antifungal activities however, asaronaldehyde (2,4,5-trimethoxybenzaldehyde) presented complete control of Phytophthora infestans in potatoes and tomatoes whereas, it showed 75% control of R. solani [33]. Our ndings on antifungal activity of triterpenoids (Spinasterol, 22,23-dihydrospinasterol) were supported by Quiroga et al. [34] that lactones, sesquiterpen and triterpenes from Schinus molle's fruits and leaves possessed antifungal potential against Alternaria alternate, Penicillium cyclopium, Aspergillus niger, Aspergillus avus Microsporum griseum and Penicillium italicum. Similarly, a avonoid 4'-methoxy-5,7-dihydroxy avone 6-C-glucoside isolated from the stem and leaves of Aquilegia vulgaris, presented its antifungal activity against mold A.
niger [35]. The antimycotoxigenic and antifungal activity of alcoholic and distilled water extracts of C. colocynthis were evaluated against Aspergillus avus and Aspergillus ochraceus and showed an excellent antifungal activity against A. ochraceus with good antiochratoxigenic power in the liquid medium which supported ndings about antifungal activity and triterpenoids spinasterol, 22,23dihydrospinasterol [36].
Activity of some of the biological compounds such as camphor, pulegone and verbenone which were isolated from Myristica fragrans was assessed against German cockroach Blattellea germanica with LC 50 values as 0.07 mgcm − 1 , 0.06 mgcm − 1 and 0.07 mgcm − 1 respectively [37]. Similarly, other isolated compounds like, carvecol, eugenol, p-cymene, isoeugenol and thymol had displayed anti-adulticidal potential at 1 mgadult − 1 against B. germanica [38]. Similarly, isolated compound Spinasterol, 22,23dihydrospinasterol exhibited medicinal and cytotoxic properties, moreover, the same was characterized from Bougainvillea spectabillis exibited sturdy inhibition of enzyme xanthine oxidase with IC 50 values as 39.21 µM [16]. Our results on toxicity of spinasterol, 22,23-dihydrospinasterol revealed that it exhibited potential insecticidal activity and caused signi cant mortality of B. brassicae. Similar outcomes were described by Torkey et al. [39] who reported activity of the 2-O-β-D-glucapyranosylcucurbitacin E isolated from C. colocynthis against Aphis craccivora with momentous mortality of this pest with LC 50 of 11,003 ppm. Moreover, insecticidal activity of isolated compound from Eupatorium adenophorum 9-oxo-10,11-dehydroageraphorone was appraised against Pseudoregma bambucicola exhibited mortality of 73.33% at 2 mgmL − 1 with 6 h exposure. Moreover, 100% control of this pest was recorded at the similar concentration at one month of post exposure in a eld experiment [40] Although, different studies had been conducted on extracts, essential oils and isolated compounds from natural plants as their antioxidant, antimicrobial, antifungal an insecticidal activities but such activities of Spinasterol, 22,23-dihydrospinasterol was not evaluated so for. Thus, this innovative research was performed for the rst time to investigate the antioxidant, antifungal and insecticidal properties of the isolated compound Spinasterol, 22,23-dihydrospinasterol .

Conclusions
The current investigations speci ed that Spinasterol, 22,23-dihydrospinasterol isolated from Citrullus colocynthis leaves exhibited pronounced antioxidant activities and insecticidal activity against Brevicoryne brassicae via residual and greenhouse assay as well as moderate antifungal activities against Magnaporthe grisea and Rhizoctonia solani. However, in comparison, greenhouse assay showed higher mortality of this pest. Based on the present ndings, Spinasterol, 22,23-dihydrospinasterol might be introduced as an antioxidant, antifungal and insecticidal purposes as an alternatives to synthetic chemical agents. However, more research is desirable on the isolation and characterization of other bioactive compounds for their evaluation as antioxidant, antifungal and insecticidal properties.

Extraction, Puri cation and Identi cation of Biochemical Compound
Extraction, separation, puri cation and identi cation of the puri ed compounds was achieved by solvents/cold extraction, various chromatographic techniques, mass spectrum and nuclear magnetic resonance (NMR) ( 1 H-NMR and 13 C-NMR) spectrum respectively as described in a recently published article of the author [11] and presented in the supplementary le.

Determination of Radical Scavenging Activity of DPPH
In order to assess the antioxidant action of Spinasterol, 22,23-dihydrospinasterol extracted by chromatographic techniques and identi ed by nuclear magnetic resonance analysis at various concentrations viz. 0.78, 3.00, 12.5 and 50 µg/mL accomplished in tween 20 (1% solution in distilled water), stable free radicals molecule 1,1-diphenyl-2-picrylhydrazyl (DPPH) (C 18 H 13 N 5 O 6 ) a dark colored crystalline powder was employed. In brief, into 3.5 mL freshly prepared DPPH solution (0.002 g 50 mL − 1 in HPLC grade methanol) and 0.25 mL of various concentration of puri ed compound prepared in methanol were added, shacked and left for incubation in darkness at 28 °C for half an hour. [12,41,42]. Consequently, absorbance was assessed at 517 nm by means of an absorbance micro-plate reader (SpectraMax Model No. 190, made in China and designed at USA) and inhibition percent of prepared 1, 1diphenyl-2 picrylhydrazyl solution was calculated on reducing of absorbance by using following Eq. (1). Conclusively, a lower absorbance degree validates higher radical scavenging activity.
Where: A blank = (absorbance of control treatment); A Sample = (absorbance of prepared samples).

Determination of Antifungal Activity
Antifungal activity of Spinasterol, 22,23-dihydrospinasterol against Magnaporthe grisea, Rhizoctonia solani) and Phytophthora infestans was evaluated in-vitro using radiated growing test on potatoes dextrose agar (PDA). Comercial synthetic fungicides, Boscalid and Carbendazim were used as positive control. Puri ed compound was placed in acetone to dissolve and then mixed with PDA to gain various concentrations with standard to lower concentration viz. 0.78, 3.00, 12.5 and 50 µgmL -1 . Then, the PDA with various concentrations was transferred into petri dishes (90 mm) diameter with 15 mL each in petri dish and then incubated along with 5 mm lumps of M. grisea, R. solani and P. infestans for each test compound and fungicides. The lumps of fungus were got by pressing at the corner of the mycelia colony from already prepared culture medium of PDA. After an incubation of one week at 25 °C, radius of mycelia growth were calculated at the inhibition percentages comparative to control (CK) with acetone (1%). All the teatmtments were replicated thrice and data was calculated adopting the standard method [43].

Determination of Insecticidal Activity
The in-vitro (residual) and in-vivo (greenhouse) insecticidal activity was assessed against cabbage aphid Brevicoryne brassicae. For residual assay, freshly cut cabbage leaves were dipped for 10 s in corresponding concentration and on drying, placed in glass petri dishes. Next, 10 wingless adult aphids were transferred on the leaves. Check (CK) was prepared in 1% solution of tween 20 deprived of puri ed compound and all petri dishes were incubated at room temperature, 60% relative humidity and 16:8 (Light: Dark) photoperiod for 72 h. For greenhouse assay, on clean and healthy plants of 5-7 true leaf stage, 10 wingless adult aphids were released. After one hour of releasing aphids and on complete settling of aphids on plants leaves, were sprayed with corresponding concentrations (2-3 showers; 10 mL) using hand sprayer. For control (CK) plants were sprayed with 1% solution of tween 20 then, treated plants along with check (CK) were placed in greenhouse for 72 h.
Mortality data for both in-vitro and in-vivo experiments was calculated regularly at 24, 48 and 72 h exposure period by examining the aphids using the binocular microscope. The individual's aphids were considered as dead who offered no response on needle stimulation.

Statistical Analysis
Analysis of variance (ANOVA) was used to analyze the data. Difference among the treatments was calculated at P