Quercetin-3,4′-di-O-glucoside (3,4′-Qdg; ≥98.5%; Fig. 1A) and quercetin-4′-O-glucoside (4′-Qmg; ≥95%; Fig. 1B) were purchased from Extrasynthese (Genay, France). Quercetin 3-β-D-glucoside (3-Qmg; ≥98%; Fig. 1C) and rutin (≥95%; Fig. 1D) were purchased from Tauto Biotech Co., Ltd. (Shanghai, China). All the solvents and chemicals are of analytical grade and were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

HPLC analysis was conducted using an Agilent 1100 HPLC system with the G1315A module Diode Array Detector (Agilent Technologies, Inc., Santa Clara, CA, USA) and with Alltech^® Alltima^™ reversed-phase C18 HPLC columns (4.6×250 nm, 5 µm; Alltech Associates, Inc., Deerfield, IL, USA). The separation of quercetin glucosides was performed based on a previous method, but with a more highly concentrated solvent (30%) (19). The UV detection was monitored at a wavelength of 362 nm. The system was set to maintain a 5 min post-run for re-equilibration between injections.

3,4′-Qdg, 4′-Qmg and 3-Qmg were dissolved in methanol (1 mg/ml) to prepare standard solutions, which were stored at −20°C prior to use.

Onions (Allium cepa L.), Welsh onions (Allium fistulosum L.), garlic (Allium sativum) and Chinese onions (Allium chinense) were purchased from a local market. The outer skins of the onions and garlic were removed and the bulbs (10 g) were sliced into small pieces and ground in an electric mixer. The bulb juices were frozen at −80°C and lyophilized. Subsequently, the Chinese onions and welsh onions were divided into two parts, the stalk and the leaf. The Chinese onions and welsh onions were cleaned and cut into small pieces (5 g) and dried in a freeze drier, subsequent to being frozen at −80°C overnight. In total, five fractions of dried samples were taken to be analyzed.

The extraction of quercetin glucosides was performed according to a previous method (2). In total, ~1 g of lyophilized sample was extracted with 10 ml 80% aqueous methanol and sonicated for 20 min. The mixture was filtered through 0.45-µm filter paper using a Büchner funnel under reduced pressure. Subsequently, 10 ml 100% methanol was used to rinse the residue. The filtrates were pooled and mixed with 10 ml 100% methanol. The mixture was concentrated using a rotary evaporator at 60°C for 30 min. The remaining solvent (3 ml) was mixed with 5 ml 100% methanol to prepare a final volume of 10 ml with Milli-Q purified water (Merck Millipore, Darmstadt, Germany). The solution was filtered through a 0.45 µm polytetrafluoroethylene membrane filter and stored at −20°C prior to analysis.

The HepG2 cells (cat. no. HB-8065^™) were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). The HepG2 cell line was sub-cultured and routinely grown with Dulbecco's modified Eagle's medium supplemented with 10% FBS and 1% streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) and incubated at 37°C (5% CO₂ atmosphere). The complete growth medium was stored at −4°C prior to analysis.

The PC-3 cells (cat. no. CRL-1435^™; ATCC^®) were purchased from ATCC (USA) and cultured in Ham's F-12K medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in an atmosphere containing 5% CO₂. The HT-29 cells (cat. no., HTB-38^™) were purchased from the ATCC and cultured in Ham's F-12K medium at 37°C in an atmosphere containing 5% CO₂.

The effects of Allium cepa L. extracts and 3,4′-Qdg, 3-Qmg and 4′-Qmg on HepG2, PC-3 and HT-29 cell lines were determined in separate experiments. Cells were seeded at a density of 4,000 cells/well with complete growth medium in 96-well plates and allowed to incubate at 37°C for 24 h. Various concentrations of 3,4′-Qdg, 3-Qmg or 4′-Qmg in the growth medium were added into the wells, followed by 72 h incubation at 37°C. An MTT assay (Thermo Fisher Scientific, Inc.) was conducted according to the manufacturer's protocol, in order to determine cell viability. Subsequent to the incubation, 20% (v/v) MTT stock solution (5 mg/ml) was added to each well, followed by an additional 2-h incubation at 37°C. Subsequent to the removing and discarding of the supernatant, dimethyl sulfoxide was used to dissolve the converted formazan product with gentle agitation. Finally, a SpectraMax Plus 384 microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) was applied to determine the absorbance at 540 nm. The absorbance of the cells treated with quercetin glucosides was compared with that of the control cells to determine the relative cell viability.

The composition of quercetin glucosides in the genus Allium was analyzed by HPLC and identified by mass spectrometry. Quercetin glucoside compounds were well separated by HPLC. The respective content of quercetin glucosides obtained from the methanolic extraction of Allium cepa L. was obtained by the HPLC analysis. The results reveal that 3-Qmg, 4′-Qmg and 3,4′-Qdg are the major quercetin glucosides present in Allium cepa L., which is consistent with previous studies (1–3). Other Allium varieties contained various amounts of quercetin glucosides (Table I). The composition of quercetin glucosides in genus Allium is presented in Table II.

The effects of quercetin glucosides on cancer cell lines varied depending on the composition of quercetin glucosides in the vegetable extracts. The effects of Allium cepa L. extract on HepG2, HT-29 and PC-3 cancer cell lines were studied. The results revealed that the Allium cepa L. extract did not exhibit marked effects on cancer cell lines. However, individual quercetin glucosides exhibited significant effects (P<0.05) on HepG2 (Fig. 2), PC-3 (Fig. 3) and HT-29 cancer cell lines (Fig. 4) subsequent to 72 h of incubation. The present findings suggest that 4′-Qmg exhibited higher cytotoxic effects, compared with the other quercetins tested in the present study.

Allium cepa L. extract contained a higher level of quercetin glucosides, as quantified by HPLC analysis (Table II). Varied compositions of quercetin glucosides among the Allium genus may contribute to the variations in the anticancer properties of the extracts. Previous studies revealed the anticancer effects of quercetin glucosides in in vitro studies (15,20,21). The present results indicate that quercetin glucosides are potent in HepG2, HT-29 and PC-3 cancer cell lines. The results are concordant with previous studies, which demonstrated that the Allium genus contains quercetins that exhibit health benefits in humans (8–9,11). The major extractable quercetin glucosides in the Allium cepa L. were 3,4′-Qdg and 4′-Qmg, and 3′-Qmg was also detected in this species. The composition analysis of Allium cepa L is presented in Table I, which indicates that 3,4′-Qdg is the major phenolic compound present in onions (20,21). Certain quercetin glucosides may be cross-linked to cell-wall polysaccharides in the form of dimers that may be attributed to the quercetin glucosides level in the fraction obtained from different genus Allium (20,21).

Treatment of various cancer cell lines with Allium extracts was performed. The content of quercetin glucosides extracted from Allium samples differed. The anticancer effect of quercetin glucosides was observed in different cancer cell lines with varied effects. The anti-proliferative effects of quercetin glucosides, obtained from onion extracts, on HepG2 subsequent to 72-h incubation was studied (Figs. 2–4). The results are concordant with previous studies (22,23), which suggested that Quercetin glucosides in food products have been reported to exhibit chemopreventive activities. The lowest IC₅₀ value for onion extracts in cancer cell lines has been observed for those extracts with the highest presence of quercetin glucosides (22,23). This indicates that quercetin glucosides may be the most potent bioactive compound in onion extracts. The findings of the present study also suggested that 4′-Qmg exhibits higher anti-proliferative activities, compared with other compounds in onion extracts, and only bioactive quercetin glucosides may exhibit inhibitory effects on cancer cells. The primary phenolic compounds in the methanolic extract were quercetin glucosides, including 3,4′-Qdg and 4′-Qmg (Table II). The results suggest that 3,4′-Qdg was an effective inhibitor on cell growth of HepG2, PC-3 and HT-29 cells. Quercetins have been reported to exhibit anti-proliferative activity towards cancer cells in vitro and in vivo (15,20,21,23). Quercetin glucosides are considered to act as antioxidants in the genus Allium (22,23). The combination of quercetin glucosides may contribute to the anti-proliferative activity of onion extracts on cancer cells (20–23). Among the quercetin glucosides present in onions, 4′-Qmg has been demonstrated to exhibit the most antioxidant activity in cancer cell lines (23). In addition, quercetin glucosides may function as apoptosis inducers in human cancer cells with inhibitory effects on proliferation (23).