A powerful on line ABTS+-CE-DAD method to screen and quantify major antioxidants for quality control of Shuxuening Injection

Abstract

A novel method of on-line 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)-Capillary Electrophoresis-Diode Array Detector (on-line ABTS⁺-CE-DAD) was developed to screen the major antioxidants from complex herbal medicines. ABTS⁺, one of well-known oxygen free radicals was firstly integrated into the capillary. For simultaneously detecting and separating ABTS⁺ and chemical components of herb medicines, some conditions were optimized. The on-line ABTS⁺-CE-DAD method has successfully been used to screen the main antioxidants from Shuxuening injection (SI), an herbal medicines injection. Under the optimum conditions, nine ingredients of SI including clitorin, rutin, isoquercitrin, Quercetin-3-O-D-glucosyl]-(1-2)-L-rhamnoside, kaempferol-3-O-rutinoside, kaempferol-7-O-β-D-glucopyranoside, apigenin-7-O-Glucoside, quercetin-3-O-[2-O-(6-O-p-hydroxyl-E-coumaroyl)-D-glucosyl]-(1-2)-L-rhamnoside, 3-O-{2-O-[6-O-(p-hydroxyl-E-coumaroyl)-glucosyl]}-(1-2) rhamnosyl kaempfero were separated and identified as the major antioxidants. There is a linear relationship between the total amount of major antioxidants and total antioxidative activity of SI with a linear correlation coefficient of 0.9456. All the Relative standard deviations of recovery, precision and stability were below 7.5%. Based on these results, these nine ingredients could be selected as combinatorial markers to evaluate quality control of SI. It was concluded that on-line ABTS⁺-CE-DAD method was a simple, reliable and powerful tool to screen and quantify active ingredients for evaluating quality of herbal medicines.

Figure 1

Effects of parameters on the migration time and resolution of fourteen (including ABTS⁺) peaks (n = 3).

Figure 2

(a) Capillary electropherograms of SI: SI on-line mixed with H₂O and on-line mixed with ABTS⁺ (n = 3). The blue one is the electrophoretogram of mixtrue of H₂O and SI, and the red one is the electrophoretogram of mixture of ABTS⁺ and SI. (b) Capillary electropherograms of standard mixture of thirteen compounds and SI (n = 3). The blue one is the electrophoretogram of SI, and the red one is the electrophoretogram of standards (1 Clitorin, 2 Rutin,3 Isoquercitrin, 4 Quercetin-3-O-D-glucosyl]-(1-2)-L-rhamnoside,5 Kaempferol-3-O-rutinoside,6 Narcissoside,7 Kaempferol-7-O-β-D-glucopyranoside,8 Apigenin-7-O-Glucoside, 9 Quercetin-3-O-[2-O-(6-O-p-hydroxyl-E-coumaroyl)-D-glucosyl]-(1-2)-L-rhamnoside, 10 Quercetin,11, 3-O-{2-O-[6-O-(p-hydroxyl-E-coumaroyl)-glucosyl]-(1-2)rhamnosyl kaempferol, 12 Kaempferol,13 Isorhamnetin).

Figure 3

(a) Capillary electropherograms of ABTS⁺: ABTS⁺ solution; on-line mixed with SI (n = 3). The blue one is the electrophoretogram of ABTS^+, and the red one is the electrophoretogram of mixture. (b) Relationship of the total quantitative and antioxidant activity of SI.

Figure 4

The diagram of the steps of the experiment procedure.