Comprehensive evaluation of the antioxidant capacity of Perilla frutescens leaves extract and isolation of free radical scavengers using step-wise HSCCC guided by DPPH-HPLC

Figures & data

Figure 1. Evaluation of Perilla frutescens antioxidant activity. (A) Hydroxyl radical scavenging activity of the fractions isolated from P. frutescens. The concentration of the fractions was 33.33 μg/mL. (B) Hydrogen peroxide scavenging activity of the fractions isolated from P. frutescens. The concentration of the fractions was 200 μg/mL. (C) Ferrous ion chelating activity of the fractions isolated from P. frutescens. The concentration of the fractions was 200 μg/mL. (D) Nitric oxide scavenging activity of the fractions isolated from P. frutescens. The concentration of the fractions was 66.67 μg/mL. The absorbance values were converted to scavenging effects (%). (E) Total antioxidant capacity of the fractions isolated from P. frutescens (expressed as equivalents of ascorbic acid). (F) Ferric reducing/antioxidant power of the fractions isolated from P. frutescens. (G) ABTS scavenging activity of the fractions isolated from P. frutescens. (H) DPPH scavenging activity of the fractions isolated from P. frutescens. Na is not active.

Table 1. Correlation between free radical scavenging and different antioxidant activities of the different fractions of PF.

	HO∙^a	H2O2^b	FIC^c	TAC^d	NO^e	ABTS	DPPH	FRAP^f
HO∙	1.00
H2O2	0.70	1.00
FIC	−0.92	−0.80	1.00
TAC	0.14	0.45	−0.51	1.00
NO∙	−0.21	−0.04	0.09	0.37	1.00
ABTS	0.21	−0.24	−0.28	0.19	−0.51	1.00
DPPH	0.28	−0.25	−0.29	0.08	−0.53	0.99	1.00
FRAP	−0.02	0.46	−0.19	0.62	0.83	−0.02	−0.68	1.00

^aHydroxyl radical scavenging activity.

^bHydrogen peroxide scavenging activity.

^cFerrous ion-chelating activity.

^dTotal antioxidant capacity.

^eNitric oxide scavenging activity.

^fFerrous reducing antioxidant activity.

Figure 2. HPLC-UV (red line) and DPPH-HPLC-UV (blue line) analyses of the EtOAc fraction of the Perilla frutescens leaves. The full chromatogram (A) and extracted peaks for compounds 1–11 (B–K). HPLC conditions: column, Eclipse SB-C18 Rapid Resolution column (150 × 4.6 mm, 5 μm, Agilent); mobile phase, consisted of A (0.1% trifluoroacetic acid in water) and B (methanol), which was programmed as follows: 0–15 min, 5–50% B, 15–23 min, 50–100% B; flow rate, 0.7 mL/min; UV wavelength, 254 nm; column temperature, 30°C.

Figure 3. Separation of antioxidants from Perilla frutescens using step-wise HSCCC. (A) Phase diagram for the n-Hex:EtOAc:n-BuOH solvent system; (B) step-wise elution chromatogram of the HSCCC; (C) HPLC analysis of the isolated compounds; (D) structures of compounds from P. frutescens; (E) the content of each compound in ethyl acetate fraction of P. frutescens leaves extract.

Figure 4. (A) DPPH scavenging activity of isolated compounds from Perilla frutescens. (B) Antioxidant activity of isolated compounds from the P. frutescens leaves in the FRAP assay. (C) FRAP assay of different concentrations of the four highly active compounds. Na is not active.