Antioxidant, anticancer, apoptosis properties and chemical composition of black truffle Terfezia claveryi

Abstract

Desert truffles are seasonal and important edible fungi that grow wild in many countries around the world. Truffles are natural food sources that have significant compositions. In this work, the antioxidant, chemical composition, anticancer, and antiangiogenesis properties of the Terfezia claveryi truffle were investigated. Solvent extractions of the T. claveryi were evaluated for antioxidant activities using (DPPH, FRAP and ABTS methods). The extracts cytotoxicity on the cancer cell lines (HT29, MCF-7, PC3 and U-87 MG) was determined by MTT assay, while the anti-angiogenic efficacy was tested using ex-vivo assay. All extracts showed moderate anticancer activities against all cancer cells (p < 0.05). The hexane extract inhibited the brain cell line (U-87 MG) with an IC₅₀ of 50 μg/ml and significantly promoted cell apoptosis through the mitochondrial pathway and DNA fragmentation p < 0.001. The ethanol extract demonstrated potent antioxidants; DPPH, FRAP, and ABTS with an IC₅₀ value of 52, 48.5 and 64.7 μg/ml, respectively. In addition, the hexane and ethyl acetate extract significantly (p < 0.001) inhibited the sprouting of microvessels by 100% and 81.2%, at 100 μg/ml, respectively. The GC analysis of the most active extract (hexane) showed the presence of several potent phytochemicals such as stigmasterol, beta-Sitosterol, squalene, lupeol, octadecadienoic acid, and oleic acid.

Keywords: Angiogenesis; Antioxidant; Black truffle; Cancer; T. claveryi.

Figure 1

(a) Shows the effect of hexane extract of T. claveryi on mitochondrial membrane potential (ΔΨm) in U-87 MG cancer cells. The cells were incubated with the extract and then stained with Rhod123 to detect the ΔΨm. (A) Control untreated group; (B) THE; T. claveryi hexane extract at 50 μg/ml; (C) BA; butanolic acid (positive control) at 50 μg/ml; (b) fluorescence intensity of ΔΨm, evaluated by Rhod123, and calculated based on the number of cells that lost the mitochondrial membrane potential. Values represent 3 experiment means ± SD. ^∗∗∗p < 0.001 compared to the control group.

Figure 2

(a) Shows the effect of hexane extract of T. claveryi on nucleic morphology of U-87 MG cancer cells. Untreated and treated cells were stained with Hoechst 33342 to detect apoptotic morphology. (A) Control untreated cells. (B) Treated cells with 50 μg/ml of T. claveryi (C) Positive control, treated cells with 50 μg/ml of butanolic acid. Cells treated with the extract displayed characteristics of apoptosis with condensed and fragmented nuclei. Red arrows represent cells with DNA condensation. (b) Shows the Graphical percentage of apoptotic indices for U-87 MGcancer cells. The apoptotic index was expressed as a percentage ratio of number of apoptotic cells to the total number of cell in 10 different microscopic fields. Values are expressed as mean ± SD. ^∗∗∗p < 0.001 compared to the control group.

Figure 3

Shows images of the angiogenesis inhibitory effect of the T. claveryi extracts. C1; negative control, a DMSO treated ring shows a full growth of blood vessel. C2: hexane extract, treated ring showed 100% inhibition using 10 μg/ml. C3; ethyl acetate extract showed 81% inhibition of blood vessel using 10 μg/ml, C4; methanol extract showed 55% inhibition of blood vessel using 10 μg/ml, C5; ethanol extract showed 49% inhibition of blood vessel using 10 μg/ml, C6; water extract showed 41% inhibition of blood vessel using 10 μg/ml. C7 and C8 Suramine showed 100% inhibition of blood vessel using 10 μg/ml. ^∗∗∗p < 0.001 compared to the control group.

Figure 4

The total ion chromatograms (TIC) of the hexane extract of T. claveryi (the most active extract) showing the peaks of the main active compounds.