Rapid Identification and Isolation of Inhibitors of Rat Lens Aldose Reductase and Antioxidant in Maackia amurensis

Abstract

Oxidative stress and aldose reductase activity have been implicated in the development of diabetic complications. In this study, the antioxidant and aldose reductase (AR) inhibitory effects of Maackia amurensis (MA) were investigated. The ethyl acetate fraction of the MA extract showed the highest inhibitory activity in antioxidant and rat lens AR (RLAR). To identify and isolate the active components in the ethyl acetate fraction of the MA extract, high-speed countercurrent chromatography and Sephadex LH-20 column chromatography were performed and guided by an offline HPLC-ABTS assay and HPLC microfractionation AR assay. Four antioxidants, namely, piceatannol (IC₅₀ = 6.73 μM), resveratrol (IC₅₀ = 11.05 μM), trans-ferulic acid (IC₅₀ = 13.51 μM), and chlorogenic acid (IC₅₀ = 27.23 μM), and six AR inhibitors, namely, chlorogenic acid (IC₅₀ = 4.2 μM), tectoridin (IC₅₀ = 50.4 μM), genistein (IC₅₀ = 57.1 μM), formononetin (IC₅₀ = 69.2 μM), resveratrol (IC₅₀ = 117.6 μM), and daidzein (IC₅₀ = 151.9 μM), were isolated and identified. The screening results of the offline HPLC-ABTS assay and HPLC microfractionation AR assay matched the activity of isolated compounds. Thus, MA is potentially valuable for antioxidant and AR inhibitor discovery and efficient drug design for the prevention and treatment of diabetic complications.

Figure 1

Screening test from the ethyl acetate fraction of Maackia amurensis by offline HPLC. The peak before reaction is the upper line and the peak after reaction is the inverse line in each chromatography. (a) The result of ABTS-offline HPLC for antioxidants. (b) The result of HPLC microfractionation for RLAR inhibition.

Figure 2

HPLC analysis of ethyl acetate fractions from Maackia amurensis by an n-hexane-EtOAc-methanol-water (2 : 8 : 1 : 9, v/v) solvent system divided into two layers. (a) EtOAc fraction of Maackia amurensis. (b) The lower layer is included in methanol-water solution. (c) The upper layer is included in n-hexane-EtOAc solution.

Figure 3

HSCCC separation of active compounds from an ethyl acetate fraction of Maackia amurensis. (a) The upper layer from an ethyl acetate fraction of Maackia amurensis from an HSCCC separation solvent system: n-hexane-EtOAc-methanol-water (2.5 : 7.5 : 5 : 5, v/v); flow rate, 4.0 mL/min; revolution speed, 500 rpm; sample size, 2.0 g; injection volume, 40 mL; detection wavelength, 254 nm. (b) HPLC analysis of isolated compounds by an HSCCC system; peak 5, piceatannol; peak 6, resveratrol; peak 7, daidzein; peak 8, genistein; peak 9, formononetin.

Figure 4

The lower layer of ethyl acetate soluble fraction from Maackia amurensis was isolated with Sephadex LH-20 and isolated compounds were monitored by HPLC analysis at 254 nm. (a) The lower layer of EtOAc fraction from M. amurensis. (b) Isolated compounds; peak 1, chlorogenic acid; peak 2, formononetin-7-O-β-D-glucosyl [1–6] glucoside; peak 3, trans-ferulic acid; peak 4, tectoridin.

Figure 5

Chemical structure of compounds identified from the ethyl acetate fraction of Maackia amurensis.