In Vitro Sprouted Plantlets of Citrullus colocynthis (L.) Schrad Shown to Possess Interesting Levels of Cucurbitacins and Other Bioactives against Pathogenic Fungi

Abstract

Cucurbitacins, structurally different triterpenes mainly found in the members of Cucurbitaceae, possess a vast pharmacological potential. Genus Cucurbita, Cucumis, and Citrullus are affluent in these bioactive compounds, and, amongst them, Citrullus colocynthis (L.) Schrad. is widely exploited in folk medicine, since a huge number of diseases are successfully treated with organic and aqueous extracts obtained from different organs and tissues of the plant. The well-known pharmacological activities of such species have been attributed to its peculiar composition, which includes cucurbitacins and other bioactive molecules; thus, owing to its high importance as a valuable natural resource for pharmaceuticals and nutraceuticals, C. colocynthis propagation and multiplication protocols are considered significant, but the exploitation of its phytochemical potential is limited by the restricted cultivation conditions and the low rate of seed germination in the natural environment; in fact, the assessment of accumulation rate of specific phytochemicals under controlled conditions is still missing. Axenically sprouted plantlets obtained without the use of culture media or the addition of hormones have been evaluated here for the production of bioactive compounds and relevant bioactive features. Our results proved that derived organic extracts contain cucurbitacins and other bioactives, show antioxidant potential, and exert activity against some pathogenic fungi (Candida krusei, C. albicans, C. parapsilosis, C. glabrata, and Aspergillus flavus), supporting the feasibility of a methodology intended to scale-up cultivation of this species as a source of pharmaceutically interesting compounds, achievable from plantlets cultivated under laboratory conditions.

Keywords: Aspergillus flavus; Candida spp.; Citrullus colocynthis (L.) Schrad; anti-mycotoxigenic activity; antifungal compounds; cucurbitacins; in vitro sprouted plantlets.

Figure 1

Flowchart of C. colocynthis plantlet cultivation. Manually decoated seeds, previously sorted for viability, were soaked and led to germination in sterilized, wetted cotton wool, then transferred into open-top boxes for liquid cultivation until the stage of 6–7 leaves. Credit for icons: noun-plant-seeds-5083968 by Black, noun-plants-1731784 by BGBOXXX Design and noun-hydroponic-1890773 by Wahyu Hadi from “The Noun Project” (https://thenounproject.com (accessed on 17 July 2022)).

Figure 2

Total Phenolic Content of chloroform (CHL) and ethanol (EtOH) extracts of C. colocynthis plantlets. Values are expressed as mg of gallic acid equivalents (GAE)/g of extract ± S.D. (vertical bars; p < 0.005).

Figure 3

Negative LC-MS base peak chromatograms of C. colocynthis extracts. (A): ethanol-extracted seed tegument (Teg-EtOH); (B): adult leaves extracted in chloroform (Leaf-CHL); (C): ethanol-extracted plantlets from in vitro cultures (PL-EtOH); (D): chloroform-extracted plantlets from in vitro cultures (PL-CHL). Numbers indicate the following metabolites: (1) Cucurbitacin E 2-O-glucoside; (2) Cucurbitacin E; (3) Apigenin-6-C-glucoside; (4) Isoorientin 3′-O-methyl ether; (5) Cucurbitacin I 2-O-glucoside; (6) Benzyl alcohol hexosyl pentoside; (7) Luteolin-C-hexoside; (8) Methyl apigenin-C-hexoside derivative; (9) Hydroxy-methyl-methoxyflavone-C-hexoside derivative; (10) Cucurbitacin B formic acid adduct.

Figure 4

Main metabolite classes detected in C. colocynthis extracts. Cake graphs represent the percentages with respect to the total LC-MS signal of the different metabolite classes in: ethanol-extracted seed tegument (Teg-EtOH, (A), ethanol-extracted plantlets from in vitro cultures (PL-EtOH, (B); adult leaves extracted in chloroform (Leaf-CHL, (C)); chloroform-extracted plantlets from in vitro cultures (PL-CHL, (D)).

Figure 5

Anti-aflatoxigenic activity of chloroform (PL-CHL) and ethanol (PL-EtOH) extracts of C. colocynthis plantlets, mature leaf (Leaf-CHL), and seed coat (Teg-EtOH) at increasing concentrations. Values are means of four replicates and are expressed as inhibition percentage with respect to control ± S.D. Different superscript letters indicate statistically significant differences (p < 0.005).