Ultrasound-, subcritical water- and ultrasound assisted subcritical water-derived Tartary buckwheat polyphenols show superior antioxidant activity and cytotoxicity in human liver carcinoma cells

Abstract

The effects of ultrasound-assisted (UAE), subcritical water (SWE) and ultrasound assisted-subcritical water (UA-SWE) treatments on tartary buckwheat polyphenol yield, composition, antioxidant activity and cytotoxicity in human liver carcinoma cells were studied. Folin Ciocalteu assay was used to measure total free phenol content (TFPC), and ABTS, DPPH, FRAP and TEAC assays were used to measure antioxidant activity (AA). Polyphenol characterization was done by LC-MS and cell antioxidant activity (CAA) and cytotoxicity were done using the 2,2′-Azobis-(2-amidinopropane) dihydrochloride [ABAP] and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide [MTT] assays respectively. The highest polyphenol yield was obtained by SWE (53.3 mg g−1), followed by UA-SWE (31.8 mg g−1), UAE (19.3 mg g−1) and HWE (4.2 mg g−1). Also, SWE had the highest TFPC (7.9 mgGAE/gdw). UAE and UA-SWE showed no differences with TFPC being 6.6 and 6.8 mgGAE/gdw, respectively. The control method (HWE) had the largest number of phenolic compounds identified (25), followed by UAE, SWE and UA-SWE which had 20, 13 and 11 phenolics respectively. Beside phenolic acids, all treatments extracted a number of flavonoids such as flavan-3-ols (catechin-7-O-glucoside, epigallocatechin-3-gallate, epigallocatechin, epicatechin), flavonols (kaempferol-3-O-glucoside, kaempferol, kaempferol-3-rutinoside, rutin, quercetin, quercetin-3-O-glucuronide hyperin), flavones (vitexin, isovitexin, orientin, isoorientin) and anthocyanins (cyanidin-3-O-rutinoside, Cyanidin 3-O-galactoside, cyanidin-3-O-glucoside). SWE gave the highest AA for all tests. However, the AA of those obtained by UAE and UA-SWE did not vary (P < 0.05), but were higher than HWE. Different extracts had best AA at different concentrations (HWE, 300; UAE, 250; SWE, 150; UA-SWE, 200 μg/mL). The IC50 of AA were 270.8 ± 21.3, 198.1 ± 16.0, 97.9 ± 13.5, and 150.4 ± 12.8 μg/mL, respectively for HWE, UAE, SWE and UA-SWE. Generally, SWE and UA-SWE showed the highest cytotoxic activities, followed by UAE, with HWE being the lowest. IC50 of cytotoxicity were 76.1 ± 3.3, 79.5 ± 7.0 and 92.6 ± 4.9 µg/mL for SWE, UA-SWE and UAE, respectively. SWE is a promising method for polyphenol extraction and its combination with ultrasound should be optimized for high yield and conservation of bioactivity.