Comparison of anti-oxidant activities of seventy herbs that have been used in Korean traditional medicine

Abstract

Many herbs have been used as therapeutics in Korean traditional medicine. In view of their clinical indications, anti-oxidant activity may contribute to their pharmacological effects. However, anti-oxidant information on these plants has not been available. In this study, seventy herbs which have been used in Korean traditional medicine were selected and screened for anti-oxidant activity using their water extracts. The anti-oxidant activity was assessed by their ability to inhibit three oxidation reactions; luminol/Fenton reagent, 2, 7-dichlorodihydrofluorescein (DCHF)/Fenton reagent and DCHF/peroxynitrite. In each assay, 70 herbs were divided into two groups; anti-oxidant group which inhibited the respective oxidation reaction and was majority (about 60 herbs), and pro-oxidant group which enhanced the oxidation reaction but was minority (more or less 10 herbs). When the herbs were listed in the order of their anti-oxidant strength, the orders obtained from each assay were found to be quite similar. The upper top rankers (more or less 10 herbs) in each assay showed strong activity compared to the others. The uppermost rankers in each assay were Rubus coreanus Miquel/ Rubus schizostylus, Schisandra chinensis Baillon/ Schizandra chinensis and Terminalia chebula Retzius/ Terminalia chebula. Of the pro-oxidant herbs, about 4-5 herbs were strongly pro-oxidant, which enhanced the control oxidation reactions to 150-300%. But the meaning of this observation is not known since few of them in one assay were also anti-oxidant in other assays. The results obtained in the present study may serve as information for understanding pharmacological effects of these herbs and developing new drugs from them.

Keywords: Anti-oxidants; Fenton reagent; chemiluminescence; herbs; peroxynitrite.

Fig. 1

Anti-oxidant activities of herbs used in Korean traditional medicine assessed by inhibition of chemiluminescence emitting from luminol/Fenton reagent reaction. Water extracts of seventy herbs were prepared and each of water extracts was assayed by measuring the inhibition of chemiluminscence produced from luminol on its oxidation by Fenton reaction. Luminol (10 mM) was mixed with 30 mM H₂O₂, 0.5 mM FeCl₂ and phosphate, pH 7.4 in the absence or presence of various volumes of each water extract. Total volume was 2 ml and reaction was started by adding H₂O₂ last and allowed at 37℃. After 10 min, chemiluminescence was measured using a chemiluminescence analyzer. Of the 70 herbs, 60 were shown to inhibit the chemiluminescence. The anti-oxidant activity was expressed by a reciprocal of the volume of the water extract required to inhibit the control chemiluminescence observed in the absence of water extract to 50% (1/50% inhibitory volume; 1/IV₅₀). 1/IV₅₀ of 60 herbs are presented in this figure in the order of their magnitude. Numbers given at each herb are the serial numbers shown in Table 1.

Fig. 2

Pro- oxidant activities of herbs used in Korean traditional medicine assessed by stimulation of chemiluminescence emitting from luminol/Fenton reagent reaction. The experimental conditions were the same as in Fig. 1. Of 70 herbs, 10 shown in the figure stimulated the chemiluminescence. The results are CPM (count per minute) of chemiluminescence by water extracts of each herb. Numbers given at each herb are the serial numbers shown in Table 1.

Fig. 3

Anti-oxidant activities of herbs used in Korean traditional medicine assessed by inhibition of fluorescence emitting from DCHF/Fenton reagent reaction. The anti-oxidant activities of 70 herbs were assessed using oxidation of DCHF by Fenton reagent. DCHF (50 µM) was mixed with 60 mM H₂O₂, 0.75 mM FeCl₂ and PBS, pH 7.4 in the absence or presence of each water extract (5 µl) in 96 well plates. Total volume was 200 µl and reaction was started by adding 60 mM H₂O₂ last, allowed at 37℃ for 10 min and then fluorescence was measured. Of the 70 herbs, 59 were shown to inhibit the fluorescence. The anti-oxidant activity was expressed by % inhibition of the control fluorescence [(control fluorescence-experimental fluorescence)/control fluorescence×100]. Numbers given at each herb are the serial numbers shown in Table 1.

Fig. 4

Herbs which stimulated fluorescence emitting from DCHF/Fenton reagent reaction. The experimental conditions were the same as in Fig. 3. Of the 70 herbs, 11 were shown to stimulate the fluorescence. The results are % stimulation by each herb. Numbers given at each herb are the serial numbers shown in Table 1.

Fig. 5

Anti-oxidant activities of herbs used in Korean traditional medicine assessed by inhibition of fluorescence emitting from DCHF/peroxynitrite. The anti-oxidant activities of the 70 herbs were assessed using oxidation of DCHF by peroxynitrite. DCHF (0.5 mM), sodium peroxynitrite (0.5 mM) and , sodium phosphate buffer (0.3 M) were incubated in the absence or presence of aech water extract (5 µl) in 96-well plates at 37℃ for 10 min. Total volume was 200 µl and reaction was started by adding sodium peroxynitrite and then fluorescence was measured using a spectrofluorimeter. Of the 70 herbs, 59 were shown to inhibit the fluorescence. The anti-oxidant activity was expressed by % inhibition of the control fluorescence [(control fluorescence-experimental fluorescence)/control fluorescence×100]. Numbers given at each herb are the serial numbers shown in Table 1.

Fig. 6

Herbs which stimulated fluorescence emitting from DCHF/peroxynitrite reaction. The experimental conditions were the same as in Fig. 5. Of the 70 herbs, 2 were shown to stimulate the fluorescence. The results are % stimulation by each herb. Numbers given at each herb are the serial numbers shown in Table 1.