Quantitative Analysis and Biological Efficacies regarding the Neuroprotective and Antineuroinflammatory Actions of the Herbal Formula Jodeungsan in HT22 Hippocampal Cells and BV-2 Microglia

Abstract

Jodeungsan (JDS) is a traditional herbal formula that comprises seven medicinal herbs and is broadly utilized to treat hypertension, dementia, and headache. However, the effects of JDS and its herbal components on neurodegenerative diseases have not been reported. We examined the inhibitory effects of JDS and its seven components on neuronal cell death and inflammation using HT22 hippocampal cells and BV-2 microglia, respectively. Among its seven herbal components, Uncaria sinensis (US), Chrysanthemum morifolium (CM), Zingiber officinale (ZO), Pinellia ternata (PT), Citrus unshiu (CU), and Poria cocos (PC) exhibited significant neuroprotective effects in HT22 cells. In BV-2 cells, JDS significantly suppressed the production of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), indicating the antineuroinflammatory activity of JDS. In addition, the herbal extracts from ZO, Panax ginseng (PG), PT, CU, and PC exhibited inhibitory effects on the inflammatory response in microglia. These data imply that the JDS effect on neurodegeneration occurs via coordination among its seven components. To establish a quality control for JDS, a simultaneous analysis using five standard compounds identified hesperidin (37.892 ± 1.228 mg/g) as the most abundant phytochemical of JDS. Further investigation of the combinatorial activities of two or more standard compounds will be necessary to verify their antineurodegenerative regulatory mechanisms.

Figure 1

Chemical structures of the five standard compounds of JDS.

Figure 2

HPLC chromatograms of the 70% ethanol extract of JDS (a) and a standard mixture (b) at 236 nm and 263 nm. Narirutin (1), hesperidin (2), luteolin (3), apigenin (4), and 6-gingerol (5).

Figure 3

Neuroprotective effect of JDS and its herbal components in H₂O₂-treated HT22 cells. Cells were seeded on 96-well plates and cotreated with various concentrations of JDS and its seven components, and H₂O₂ (250 μM) for 6 h. Cell viability was assessed using the CCK-8 assay. Results are shown for JDS (a), US (b), CM (c), ZO (d), PG (e), PT (f), CU (g), and PC (h). The results are expressed as the mean ± SEM of three independent experiments. ^###P < 0.001 versus vehicle control cells, ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 versus H₂O₂-treated cells.

Figure 4

Effects of JDS and its herbal components on TNF-α production in LPS-stimulated BV-2 cells. Cells were pretreated with various concentrations of JDS and its seven components for 2 h and then stimulated with LPS (1 μg/mL) for an additional 22 h. TNF-α production was determined using an ELISA kit. Results are shown for JDS (a), US (b), CM (c), ZO (d), PG (e), PT (f), CU (g), and PC (h). The results are expressed as the mean ± SEM of three independent experiments. ^###P < 0.001 versus vehicle control cells, ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 versus LPS-treated cells.

Figure 5

Effects of JDS and its herbal components on IL-6 production in LPS-stimulated BV-2 cells. Cells were pretreated with various concentrations of JDS and its seven components for 2 h and then stimulated with LPS (1 μg/mL) for an additional 22 h. IL-6 production was determined using an ELISA kit. Results are shown for JDS (a), US (b), CM (c), ZO (d), PG (e), PT (f), CU (g), and PC (h). The results are expressed as the mean ± SEM of three independent experiments. ^###P < 0.001 versus vehicle control cells, ^∗P < 0.05 and ^∗∗∗P < 0.001 versus LPS-treated cells.