Rapid identification of aldose reductase inhibitory compounds from Perilla frutescens

Abstract

The ethyl acetate (EtOAc) soluble fraction of methanol extracts of Perilla frutescens (P. frutescens) inhibits aldose reductase (AR), the key enzyme in the polyol pathway. Our investigation of inhibitory compounds from the EtOAc soluble fraction of P. frutescens was followed by identification of the inhibitory compounds by a combination of HPLC microfractionation and a 96-well enzyme assay. This allowed the biological activities to be efficiently matched with selected HPLC peaks. Structural analyses of the active compounds were performed by LC-MS(n). The main AR inhibiting compounds were tentatively identified as chlorogenic acid and rosmarinic acid by LC-MS(n). A two-step high speed counter current chromatography (HSCCC) isolation method was developed with a solvent system of n-hexane-ethyl acetate-methanol-water at 1.5:5:1:5, v/v and 3:7:5:5, v/v. The chemical structures of the isolated compounds were determined by (1)H- and (13)C-nuclear magnetic resonance spectrometry (NMR). The main compounds inhibiting AR in the EtOAc fraction of methanol extracts of P. frutescens were identified as chlorogenic acid (2) (IC50 = 3.16 μ M), rosmarinic acid (4) (IC50 = 2.77 μ M), luteolin (5) (IC50 = 6.34 μ M), and methyl rosmarinic acid (6) (IC50 = 4.03 μ M).

Figure 1

HPLC chromatogram (a) and active profile (b) of the HPLC microfractionated of EtOAc soluble fraction of P. frutescens for rAR inhibition in 96-well plate.

Figure 2

HSCCC separation of the EtOAc soluble fraction from the leaves of P. frutescens. Solvent system: n-hexane-ethyl acetate-methanol-water (1.5 : 5 : 1 : 5, v/v); flow-rate, 5.0 mL/min; revolution speed, 400 rpm; sample size, 4.0 g; injection volume, 50 mL; detection wavelength, 280 nm; stationary phase retention, 56%. Peaks I, II, III, and IV in the HSCCC chromatogram correspond to compounds 3, 1, 2, and 4, respectively.

Figure 3

HSCCC chromatogram of P. frutescens after HSCCC separation. Solvent system: n-hexane-ethyl acetate-methanol-water (3 : 7 : 5 : 5, v/v); flow rate, 5.0 mL/min; revolution speed, 400 rpm; sample size, 1.2 g; injection volume, 50 mL; detection wavelength, 280 nm; stationary phase retention, 53%. Peaks V and VI in the HSCCC chromatogram correspond to compounds 6 and 5, respectively.

Figure 4

HPLC analysis of compounds 1–6 separated by HSCCC. Conditions: column, Eclipse SB-C18 Rapid Resolution column (150 × 4.6 mm, 5 μm, Agilent); column temperature, 30°C; mobile phase, 0.1% TFA (solvent A) and acetonitrile (solvent B); HPLC analysis, linear gradient from 7.5 to 55% B (0–40 min). Flow rate, 0.7 mL/min; detection, photodiode array detector; injection volume, 10 μL.

Figure 5

Main correlation HMBC for 6.