Polyphenols from the sagebrush Artemisia tridentata ssp. tridentata affect the redox state of cultured hepatocytes by direct and indirect mechanisms

Abstract

Basin big sagebrush (Artemisia tridentata Nutt. ssp. tridentata (Asteraceae)), is a widespread North American shrub which produces a variety of polyphenolic compounds. Although sagebrush has been used as a traditional remedy by natives and settlers to the region, the polyphenols in Artemisia tridentata ssp. tridentata have not been highly investigated for their bioactive properties. To determine whether these polyphenols affect the intracellular redox state, we measured their ability to neutralize radicals in vitro and in a human liver carcinoma cell line (HepG2), and their effects on intracellular glutathione levels. Extracts from Artemisia tridentata ssp. tridentata decreased the oxidation of 2'7'-dichlorofluorescin in vitro and in cultured cells. Cells treated with polyphenolic extracts showed increased levels of glutathione in a time and dose-dependent manner. Approximately 48 polyphenolic compounds were distinguishable in extracts, by HPLC/UV absorbance detection. Mass spectroscopy was used to identify thirteen compounds as aesculin, aesculetin, apigenin, apigenin-7-O-glucoside, axillarin, casticin, chlorogenic acid, isoscopoletin, kaempferol, luteolin, methyl-axillarin, quercetin, and scopoletin. These results indicate that polyphenols produced in Artemisia tridentata ssp. tridentata affect the redox state of living cells by multiple mechanisms.

Keywords: Artemisia tridentata; antioxidant; glutathione; polyphenol; sagebrush.

Figure 1.

Peroxyl radical scavenging capacity of methanol extracts of sagebrush. Higher PSC units indicate stronger antioxidant activity. PSC values for commercially obtained quercetin were measured under the same conditions. Results indicate the mean + s.d. of two experiments. * indicates PSC differs from methanol control at p < 0.05.

Figure 2.

Effect of sagebrush extract on levels of reactive oxygen species in HepG2 cells. Cells were pretreated with either SBE or commercially obtained quercetin for the indicated times then the levels of intracellular ROS determined. Values represent the percent of intracellular ROS as compared to control cells treated with methanol alone. Results indicate the mean + s.d. of three experiments. * indicates ROS differs from control at p < 0.05.

Figure 3.

Effect of sagebrush extract on total cellular levels of glutathione in HepG2 cells. (a) Increasing doses of SBE were applied to cells for 24 hours. C + V indicates cells treated with methanol alone. Results indicate the mean + s.d. of six experiments. * indicates value differs from C + V at p < 0.05. (b) Cells were treated with 10 nM SBE, 100 μM SBE, or methanol alone (C + V) for the times indicated. Results indicate the mean + s.d. of four experiments. * indicates value differs from C + V at p < 0.05.

Figure 4.

Separation of compounds in SBE by analytical reverse phase HPLC. Detection of eluting compounds was by absorbance at 280 nm.

Figure 5.

Fractionation of SBE by preparative-scale HPLC. Eluting compounds were detected by absorbance at 280 nm. Eluate was collected in pools I (2.5–12.5min), II (12.5–22.5min), III (22.5–42.5 min), IV (42.5–62.5 min), and V (62.5–67.5min).

Figure 6.

Antioxidant and bioactive effects of SBE fractionated by HPLC. (a) Peroxyl radical scavenging capacity (PSC) of reconstituted HPLC eluate pools I-V. Equal volumes of reconstituted pools were used in the assay. Results indicate the mean + s.d. of two experiments. *indicates value is different than methanol control at p < 0.05. (b) Effect of reconstituted HPLC eluate pools I-V on glutathione levels in HepG2 cells. Cells were treated with reconstituted HPLC fractions at a dilution which was equivalent to that used to give 100 μM of the original SBE, an equal volume of methanol (cv), or left untreated (ctr) for 12 hours. Results indicate the mean ± s.d. of five experiments. *indicates value different from cv at p < 0.05.