*4.7. Determination of Anti-Inflammatory Activity*

The murine macrophage RAW 264.7 cell line was maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/L streptomycin, and 100 IU/mL penicillin at 37 ◦C in a 5% CO2 atmosphere. The RAW 264.7 cells were seeded at a density of <sup>5</sup> <sup>×</sup> 105 cells/well in 24-well plates, and incubated for 12 h at 37 ◦C and 5% CO2. Different concentrations of the tested isoflavones were added. After 1-h treatment, the cells were stimulated with 100 ng/mL of LPS for 24 h. Culture supernatants (equal volumes) were mixed with Greiss reagent at room temperature for 10 min, and then the absorbance was measured at the wavelength 540 nm using a microplate reader (Sunrise, Tecan, Männedorf, Switzerland), as described previously [15]. This analysis was performed in tetraplicate. Relative inhibition of NO production was calculated with the equation: Relative inhibition (%) = [(OD570 with LPS only–OD570 with both LPS and isoflavones)/(OD570 with LPS only–OD570 without LPS or isoflavone)] × 100%. An IC50 value means a concentration of the drug that exhibited 50% of inhibition.

## **5. Conclusions**

8-OHDe-7-α-glucoside is successfully produced from *O*-glucosylation of 8-OHDe with recombinant DgAS of *Deinococcus geothermalis* with a cheap and abundant renewable substrate, sucrose, as a sugar donor. The isoflavone glucoside is more soluble and stable than those of 8-OHDe in working buffers. The long half-life of 8-OHDe-7-α-glucoside maintains moderate anti-inflammatory activity, and could be used for industrial applications in the future.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1420-3049/24/12/2236/s1, Table S1. nmR spectroscopic data for compound (**1**) (in DMSO-d6; 700 MHz); Figure S1. The 1H-NMR (700 MHz, DMSO-d6) spectrum of compound (**1**); Figure S2. The 13C-NMR (176 MHz, DMSO-d6) spectrum of compound (**1**); Figure S3. The DEPT-90 and DEPT-135 (176 MHz, DMSO-d6) spectra of compound (**1**); Figure S4. The HSQC (700 MHz, DMSO-d6) spectrum of compound (**1**); Figure S5. The HMBC (700 MHz, DMSO-d6) spectrum of compound (**1**); Figure S6. The H-H COSY (700 MHz, DMSO-d6) spectrum of compound (**1**); Figure S7. The H-H NOESY (700 MHz, DMSO-d6) spectrum of compound (**1**); Figure S8. The key HMBC (blue arrows) and NOESY (pink arrow) correlations of compound (**1**).

**Author Contributions:** Conceptualization: T.-S.C.; data curation: T.-S.C. and S.-Y.Y.; methodology: T.-S.C., Y.-H.K. and C.-M.C.; project administration: T.-S.C.; writing—original draft: T.-S.C., T.-Y.W., and C.M.C.; writing—review and editing: T.-Y.W., J.-Y.W., and C.-M.C.

**Funding:** This research was financially supported by grants from the National Scientific Council of Taiwan (project no. MOST 107-2622-E-024-002-CC3).

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**

1. Franke, A.A.; Custer, L.J.; Cerna, C.M.; Narala, K.K. Quantitation of phytoestrogens in legumes by HPLC. *J. Agric. Food Chem.* **1994**, *42*, 1905–1913. [CrossRef]


**Sample Availability:** 1 mg of 8-OHDe-7-α-glucoside for each request is available from the authors.

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