Terfezia boudieri: A Desert Truffle With Anticancer and Immunomodulatory Activities

Abstract

Desert truffles have high nutritional value and grow wild in the Mediterranean basin and Western Asia. Although, many studies were performed to evaluate truffles nutritious values and phytochemical composition, studies are limited to evaluate their anticancer and/ or immunomodulatory effects. Our study was conducted to evaluate the anticancer and immunomodulatory effects of Terfezia boudieri (desert truffle). Different solvent extracts were prepared from the truffle and MTT assay was used to measure their anticancer activity against cancer cell lines (T47D, MCF-7, MDA-MB231, HCT-116, and Hela). Total phenolic content in each extract was determined by using Folin-Ciocalteu reagent and qualitative phytochemical screening was performed using standard methods. The degree of apoptosis induction (using caspase 3 assay) and vascular endothelial growth factor expression were detected using standard kits. Also, ELISA was used to measure levels of IFN-γ, IL-2, IL-4, and IL-10 secreted by splenocytes after treatment with the extracts. The effect of the extracts on splenocytes proliferation was measured using MTT assay. Macrophage function was evaluated using nitro blue tetrazolium assay and pinocytosis function was evaluated using neutral red method. Terpenoids, phytosterols, and carbohydrates were present in all the solvent extracts, while tannins, alkaloids and flavonoids were detected only in aqueous/methanol and aqueous extracts. The highest total phenolic content was observed in aqueous and aqueous methanol extracts. The growth of cancer cell lines was inhibited by T. boudieri extracts in a dose dependent manner. N-hexane extract was the most potent against most cell lines. Aqueous/methanol extract showed high apoptosis induction and angiogenesis suppression effects. An increase in TH1 cytokines (IFN-γ, IL-2) level and a decrease in TH2 cytokine (IL-4) level were evident after lymphocytes stimulation by aqueous/methanol, n-hexane and ethyl acetate extracts of T. boudieri. Ethyl acetate extract of T. boudieri were the most potent extracts to stimulate lymphocytes proliferation while all other extracts showed moderate stimulation. Aqueous/methanol extract was the most active extract to stimulate phagocytosis. Ethyl acetate extract was the most active extract to stimulate pinocytosis. The use of T. boudieri provides variable health benefits. N-hexane, ethyl acetate, and aqueous/methanol extracts exhibited anticancer activities and are potent stimulators of innate and acquired immunity. Further testing is needed to identify the biologically active compounds and detect them quantitatively using GC-MS analysis.

Keywords: anticancer; functional food; fungi; immunomodulatory; truffles.

Figure 1

(A) Antiproliferative activity of aqueous/methanol extract of T. boudieri on MCF-7, T47D, MDA-MB231, HCT-116, Hela and Vero cell lines. (B) Antiproliferative activity of aqueous extract of T. boudieri on MCF-7, T47D, MDA-MB231, HCT-116, Hela and Vero cell lines. (C) Antiproliferative activity of n-hexane extract of T. boudieri on MCF-7, T47D, MDA-MB231, HCT-116, Hela and Vero cell lines. (D) Antiproliferative activity of ethyl acetate extract of T. boudieri on MCF-7, T47D, MDA-MB231, HCT-116, Hela and Vero cell lines. Results are expressed as means of three independent experiments (bars) ± SEM (lines).

Figure 2

Equivalent of gallic acid in mg per 1 ml of aqueous/methanol, aqueous, n-hexane, and ethyl acetate extracts of T. boudieri in different concentrations. Results are expressed as means of three independent experiments (bars) ± SEM (lines).

Figure 3

The effect of T. boudieri aqueous/methanol extract (at a concentration of 12.5 mg/ml) and doxorubicin (250 nM) on VEGF (vascular endothelial growth factor) expression (pg/ml) in T47D cells. Results are expressed as means of three independent experiments (bars) ± SEM (lines). The asterisks represent significant difference compared with the negative control (*P < 0.05); compared with doxorubicin treatment (^#P < 0.05).

Figure 4

The effect of T. boudieri aqueous/methanol extract (at a concentration of 25 mg/ml) and doxorubicin (250 nM) on caspase 3 activity expressed by number of folds of negative control. Results are expressed as means of 3 independent experiments (bars) ± SEM (lines). The asterisks represent significant difference compared with the negative control (*P < 0.05); compared with doxorubicin treatment (^#P < 0.05).

Figure 5

The effect of T. boudieri different extracts (at a concentration of 25 mg/ml) on the expression pattern of various cytokines (pg/ml). Results are expressed as means of three independent experiments (bars) ± SEM (lines).

Figure 6

(A) The effect of T. boudieri different extracts at different concentrations on the proliferation of splenic lymphocytes in the presence of Con A (5 μg/ml). (B) The effect of T. boudieri different extracts at different concentrations on the proliferation of splenic lymphocytes in the presence of LPS (4 μg/ml). Results are expressed as means of three independent experiments (bars) ± SEM (lines).

Figure 7

The effect of T. boudieri different extracts at different concentrations on the proliferation of splenic lymphocytes in absence of mitogens. Results are expressed as means of three independent experiments (bars) ± SEM (lines).

Figure 8

In vitro phagocytic assay using nitro blue tetrazolium (NBT) reduction test of peritoneal macrophage treated with various concentrations of T. boudieri extracts for 48 h. Results are expressed as means of three independent experiments (bars) ± SEM (lines).

Figure 9

The effect of T. boudieri different extracts on macrophage pinocytosis. Results are expressed as means of three independent experiments (bars) ± SEM (lines).