*2.4. Solubility, Stability, and Anti-Inflammatory Activity of 8-OHDe 7-*α*-glucoside.*

The aqueous solubility of 8-OHDe-7-*O*-α-glucoside was examined (Table 1). The results revealed that the maximum aqueous solubility of the 8-OHDe-7-*O*-α-glucoside is 3.92-fold, higher than that of 8-OHDe.

**Table 1.** Aqueous solubility of 8-OHDe and 8-OHDe-7-*O*-α-glucoside.


In addition, 8-OHDe is very unstable in the alkaline condition [19]. This property shortens the storage time of 8-OHDe in cosmetic or pharmaceutical products, and limits applications of 8-OHDe. Thus, the stability of the 8-OHDe-7-*O*-α-glucoside and 8-OHDe was compared (Figure 5). The half-time of 8-OHDe was 15.8 h, and only 6.8% of 8-OHDe remained in 50 mM of Tris buffer (pH 8.0) after 96-h incubation at 20 ◦C. However, 94.6% of 8-OHDe-7-*O*-α-glucoside still remained after 96-h incubation at the same condition. The half-time of 8-OHDe-7-*O*-α-glucoside was much longer than 96 h. Thus, 8-OHDe-7-*O*-α-glucoside is more than six-fold more stable than 8-OHDe in an alkaline solution.

**Figure 5.** Alkaline stability of 8-OHDe (open circle) and 8-OHDe-7-*O*-α-glucoside (closed square). A total of 1 mg/mL of the tested compound was dissolved in 50 mM of Tris buffer at pH 8.0, and stored at 20 ◦C for 96 h. During the storage time, samples were taken out for UPLC at the determined interval times. The mean (*n* = 3) is shown, and the standard deviations are represented by error bars.

Since the 8-OHDe-7-*O*-α-glucoside possesses much higher aqueous solubility and alkaline stability than those of 8-OHDe, and 8-OHDe was recently demonstrated with potent anti-inflammatory activity [15,16], the anti-inflammatory activity of 8-OHDe-7-*O*-α-glucoside and 8-OHDe was determined by the inhibition ability on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine macrophage RAW264.7 cells. Macrophages are involved in chronic inflammation, and once macrophages are elicited, an inflammatory mediator, such as NO, is produced. Thus, the NO level in the culture supernatant could be measured as an index of inflammatory mediators. The results of the anti-inflammatory assays indicated 8-OHDe-7-α-glucoside exhibited statistically significant and dose-dependent inhibitory activity with an IC50 value of 173.2 ± 12.9 μM (Figure 6a), while 8-OHDe showed potent anti-inflammatory activity with an IC50 value of 34.5 ± 5.3 μM (Figure 6c). In addition, the results of cell survival assay indicated that 100 ng/mL of LPS treatment would not induce statistically significant cell death as the false-positive signal and reduction of NO by the tested isoflavones was not due to the cytotoxicity of the isoflavones (Figure 6b,d).

**Figure 6.** *Cont.*

**Figure 6.** Effects of 8-OHDe-7-α-glucoside (**a**,**b**) and 8-OHDe (**c**,**d**) on the inhibition of LPS-induced NO production (**a**,**c**) and cell survival (**b**,**d**) in murine macrophage RAW264.7 cells. Cells were incubated with the indicated concentrations of isoflavone for 1 h before treatment with LPS (100 ng/mL) for 24 h. The amounts of NO were determined using the Griess reagent in the culture medium. Cell viability was determined with MTT assay. Each value indicates the mean ± standard deviation (SD), and is representative of the results obtained from four independent experiments. \* (*p* < 0.001) is statistically significantly different from the value for the cells treated with LPS treatment alone.
