Antioxidant Activity In Vitro Guided Screening and Identification of Flavonoids Antioxidants in the Extract from Tetrastigma hemsleyanum Diels et Gilg

Abstract

This study aimed to investigate the extract with high antioxidant activity of Tetrastigma hemsleyanum Diels et Gilg and identify the antioxidant components in vitro. α, α-Diphenyl-β-picrylhydrazyl (DPPH) radical assay, Trolox equivalent antioxidant capacity (TEAC) assay, ferric reducing antioxidant power (FRAP), and hydroxyl radical scavenging method were used to screen the extract with high antioxidant activity. The antioxidant capacity of the extracts was evaluated by the free radical scavenging ability of DPPH. The ability of extracts to scavenge 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radical was evaluated by TEAC assay. The FRAP method was used to evaluate the ability of extracts to reduce Fe³⁺. The ability to scavenge hydroxyl radicals produced by the interaction of hydrogen peroxide and Fe²⁺ was measured by monitoring the change in the absorbance of the reaction mixture at 536 nm. Then, high-performance liquid chromatography-DPPH (HPLC-DPPH) and HPLC-hydroxyl radical scavenging methods were used to screen the antioxidant components in the extract. The molecular weight of the above antioxidant components was investigated using the qualitative analytical method of high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC-Q-TOF LC/MS). Based on the concentrations of the samples (0.2-4 mg/mL), the DPPH free radical scavenging ability, ABTS+ free radical scavenging ability, hydroxyl free radical scavenging ability, and Fe³⁺ reducing ability of the ethyl acetate extract (EAE) were stronger than that of the crude extract (CE), petroleum ether extract (PEE), and n-butanol extract (BE). The EAE has higher antioxidant activity than CE, PEE, and BE. Six antioxidant components, rutin, quercetin, isoquercetin, astragalin, kaempferol, and kaempferol-3-o-rutoside, were identified in the EAE.

Figure 1

DPPH radical scavenging activity (%) of four extracts.  ^∗P < 0.05 vs. the blank group.

Figure 2

TEAC value (Trolox mg/g extract) of four extracts.  ^∗P < 0.05 vs. the blank group.

Figure 3

FRAP value (FeSO₄ mM/mg extract) of four extracts.  ^∗P < 0.05 vs. the blank group.

Figure 4

Hydroxyl radicals scavenging activity (%) of four extracts.  ^∗P < 0.05 vs. the blank group.

Figure 5

Chromatogram of antioxidant components in EAE (4 mg/mL) screened by HPLC. (a) Chromatogram of the sample without any added reagent. (b) Chromatogram of the sample with added DPPH. (c) Chromatogram of the sample with added hydroxyl radical.

Figure 6

Total ion current diagram of EAE (4 mg/mL).

Figure 7

Mass chromatogram of components in EAE (4 mg/mL). (a) Mass chromatogram of rutin. (b) Mass chromatogram of isoquercetin. (c) Mass chromatogram of kaempferol-3-o-rutoside. (d) Mass chromatogram of astragaloside. (e) Mass chromatogram of quercetin. (f) Mass chromatogram of kaempferol.

Figure 8

Chromatogram of standard reference and EAE (4 mg/mL). (a) Chromatogram of mixed standard reference. (b) Chromatogram of kaempferol-3-o-rutoside standard reference. (c) Chromatogram of EAE: 1, rutin; 2, isoquercetin; 3, astragaloside; 4, quercetin; 5, kaempferol; 6, kaempferol-3-o-rutoside.