Metabolite Profile and In Vitro Beneficial Effects of Black Garlic (Allium sativum L.) Polar Extract

Abstract

Over the centuries, humans have traditionally used garlic (Allium sativum L.) as a food ingredient (spice) and remedy for many diseases. To confirm this, many extensive studies recognized the therapeutic effects of garlic bulbs. More recently, black garlic (BG), made by heat-ageing white garlic bulbs, has increased its popularity in cuisine and traditional medicine around the world, but there is still limited information on its composition and potential beneficial effects. In this study, the metabolite profile of methanol extract of BG (BGE) was determined by high-performance liquid chromatography coupled to tandem mass spectrometry in high-resolution mode. Results allowed to establish that BGE major components were sulfur derivatives, saccharides, peptides, organic acids, a phenylpropanoid derivative, saponins, and compounds typical of glycerophospholipid metabolism. Characterization of the BGE action in cancer cells revealed that antioxidant, metabolic, and hepatoprotective effects occur upon treatment as well as induction of maturation of acute myeloid leukemia cells. These results are interesting from the impact point of view of BG consumption as a functional food for potential prevention of metabolic and tumor diseases.

Keywords: LC-ESI/LTQOrbitrap/MS/MS; black garlic; cancer cells; cellular bioactivity; methanol extract; secondary metabolites.

Figure 1

LC-ESI/HRMS chromatogram (base peak intensity, negative ion mode) of black garlic methanol extract (BGE).

Figure 2

Effects of black garlic extract on cell viability and oxidative stress. In the upper panel, the inhibitory effects of different concentrations of BGE (0–2.5 mg/mL) on HepG2 and U937 cells were evaluated by MTT assay and expressed as percentage of inhibition compared to control (HepG2 and U937 cells without BGE treatment). After 24 h of BGE treatment, the IC₅₀ were 0.8 mg/mL and 2 mg/mL on HepG2 and U937, respectively. In the central panel, the effect of BGE on free nitric oxide (NO) level evaluated in the intercellular medium of BGE-treated HepG2 and U937 (p = 0.0038) cells was reported. In the bottom panel, lipid peroxidation in cytosol was evaluated by TBARS assay in BGE-treated HepG2 (p = 0.0089) and U937 cells. Data were mean ± SD (n = 3).

Figure 3

BGE reduces neutral lipid accumulation in HepG2 cells exposed to high glucose. (A) ORO staining microscopy at 20X magnification revealed a decrease of lipid droplets in cytoplasm of BGE-treated HG-HepG2 cells compared to untreated control cells (CTR); (B) lipid droplets decrease in cytoplasm of BGE-treated HG-HepG2 cells was more clearly revealed by ORO staining microscopy at 40X magnification.

Figure 4

Black garlic methanol extract (BGE) induces proliferative block in hematological cancer cells. Images are representative of microscopic analysis of the indicated cell lines (U937 and NB4) after 48 h of treatment with 2.5 and 5.0 mg/mL BGE.

Figure 5

Black garlic methanol extract (BGE) did not restore the apoptotic program in U937 cells, as compared to untreated cells (Ctr). (A) Western blot of the indicated proteins after 2.5 mg/mL BGE treatment for 24 h on U937 cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as loading control. (B) Western blot of the indicated protein after 2.5 mg/mL BGE treatment for 24 h on U937 cells. Extracellular signal-regulated protein kinases 1 and 2 (ERKs 1/2) as loading control.

Figure 6

Black garlic methanol extract (BGE) induced hematological cancer cell differentiation. FACS analysis of CD11c (left) and CD14 (right) expression in U937 cells upon treatment with 1.25 and 2.50 mg/mL BGE for 24 h, as compared to untreated cells (Ctr). Error bars: standard deviation from independent experiments in duplicate. Isotype control (ctr) as negative control.