Production of New Isoflavone Diglucosides from Glycosylation of 8-Hydroxydaidzein by Deinococcus geothermalis Amylosucrase

: 8-Hydroxydaidzein (8-OHDe) is a non-natural isoflavone polyphenol isolated from fermented soybean foods. 8-OHDe exhibits a wide range of pharmaceutical activities. However, both the poor solubility and instability of 8-OHDe limit its applications. To resolve the limitations of 8-OHDe, Deinococcus geothermalis amylosucrase (DgAS) has previously been used to glycosylate 8-OHDe to produce soluble and stable 8-OHDe-7- O - α -glucopyranoside (8-OHDe-7-G) in a 0.5 h reaction time. In this study, we aimed to use DgAS and an extended reaction time to produce 8-OHDe diglucosides. At least three 8-OHDe derivatives were produced after a 24 h reaction time, and two major products were successfully purified and identified as new compounds: 8-OHDe-7- O -[ α -glu-copyranosyl-(1 → 6)- α -glucopyranoside] (8-OHDe-7-G2) and 8-OHDe-7,4 ′ - O - α -diglucopyranoside (8-OHDe-7-G-4 ′ -G). 8-OHDe-7-G-4 ′ -G showed a 4619-fold greater aqueous solubility than 8-OHDe. In addition, over 92% of the 8-OHDe diglucosides were stable after 96 h, while only 10% of the 8-OHDe could be detected after being subjected to the same conditions. The two stable 8-OHDe diglucoside derivatives have the potential for pharmacological usage in the future.

However, certain drawbacks (e.g., low aqueous solubility and instability) limit the use of isoflavones in pharmaceuticals and cosmeceuticals [9]. One solution involves chemically or enzymatically glycosylating these molecules to improve their solubility and stability [10]. A comparison of these two methods showed that enzymatic glycosylation of flavonoids using glycosyltransferases (GTs) and glycoside hydrolases (GHs) offers more advantages than chemical methods [11]. GTs glycosylate flavonoids via a β-glycosidic linkage. In contrast, GHs glycosylate flavonoids via an α-glycosidic linkage. Both α-glycosidic and β-glycosidic flavonoids are more soluble than flavonoids [12,13]. However, GHs use cheaper sugars, such as starch, maltodextrin, maltose, and sucrose, as donors during glycosylation [14], whereas GTs use expensive uridine diphosphate-glucose (UDP-G) as the sugar donor. Therefore, GHs are generally preferred for the bioindustrial production of glycosylated molecules.

Enzymes and Chemicals
8-OHDe was purified following the biotransformation of daidzein by A. oryzae according to the method reported by Wu et al. [8]. Recombinant DgAS was produced by a recombinant E. coli (DE3) harboring the pETDuet-DgAS expression vector according to the method reported previously [13]. Soluble DgAS was then successfully purified using Ni 2+ chelate affinity chromatography, and the purity was confirmed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ( Figure S1). The specific sucrose hydrolysis activity of DgAS was determined to be 6.6 U/mg using a 3,5-dinitrosalicylic acid (DNS) solution and a previously described method [16]. Briefly, a reaction mixture containing 25 µg/mL DgAS with 25% (w/v) sucrose in 50 mM of phosphate buffer (PB) at pH 7 was incubated at 40 °C for 10 min. After the reaction was stopped by boiling, the resultant reducing sugars were estimated using the DNS method. One unit of DgAS activity was defined as the amount of DgAS that hydrolyzed sucrose into 1 µmol of fructose per min.

Biotransformation
The reaction mixture (1 mL) comprised 25 µg/mL DgAS, 1 mg/mL 8-OHDe, 10% (w/v) sucrose, and 50 mM PB at pH 7 and was incubated at 40 °C for 24 h, based on the previous study [13]. The reaction was stopped by adding an equal volume of methanol and analyzed using high-performance liquid chromatography (HPLC).
Conversion was calculated using the following formula and expressed as a percentage: (1-HPLC peak area of the residues of 8-OHDe divided by HPLC peak area of the initial 8-OHDe) × 100. The yield of each product was calculated using the following formula and expressed as a percentage: (HPLC peak area divided by HPLC peak area of the initial 8-OHDe) × 100.

HPLC Analysis
A combo system was used that consisted of an Agilent ® 1100 series HPLC system (Santa Clara, CA, USA) equipped with a gradient pump (Waters 600, Waters, Milford, MA, USA). The HPLC system was controlled via a PC workstation using Chromatography Data Station software (SISC, Scientific Information Service Co., LTD., Taipei, Taiwan). The stationary phase was a C18 column (Sharpsil H-C18, 5 µm, 4.6 i.d. × 250 mm, Sharpsil, Beijing, China), and the mobile phase was 1% acetic acid in water (A) and methanol (B). The elution condition was as follows: a linear gradient from 0 min with 40% B to 20 min with 70% B; isocratic elution from 20 min to 25 min with 70% B; a linear gradient from 25 min with 70% B to 28 min with 40% B; and isocratic elution from 28 min to 35 min with 40% B. The flow rate was 1 mL/min, the sample volume was 10 µL, and the detection condition was set at 254 nm.

Purification and Identification of the Biotransformation Metabolites
To purify the biotransformation metabolites, the biotransformation reaction described above was scaled up to 30 mL. After the reaction, purification was conducted using previously described methods [13]. Briefly, the mixture was filtered through a 0.2 µm nylon membrane, and the filtrate was injected into a preparative YoungLin HPLC system (YL9100, YL Instrument, Gyeonggi-do, Korea) equipped with a preparative C18 reversedphase column (Inertsil, 10 µm, 20.0 i.d. × 250 mm, ODS 3, GL Sciences, Eindhoven, Netherlands) to separate the biotransformation products. The operational conditions for the preparative HPLC were the same as those used for the analytical HPLC. The fractions corresponding to the metabolite peaks identified during the HPLC analysis were collected, condensed under a vacuum, and then lyophilized. Finally, 3.3, 12.8, and 6.7 mg of compounds (1), (2), and (3), respectively, were obtained from the 30 mL reaction, and the compound structures were confirmed with mass spectral analysis and nuclear magnetic resonance (NMR). The mass spectral analysis was performed using a Finnigan LCQ Duo mass spectrometer (ThermoQuest Corp., San Jose, CA, USA) with electrospray ionization (ESI). 1 H-and 13 C-NMR, distortionless enhancement by polarization transfer (DEPT), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond connectivity (HMBC), correlation spectroscopy (COSY), and nuclear Overhauser effect spectroscopy (NOESY) spectra were recorded using a Bruker AV-700 NMR spectrometer (Bruker Corp., Billerica, MA, USA) at ambient temperature. Standard pulse sequences and parameters were used for the NMR experiments, and all chemical shifts were reported in parts per million (ppm, δ).

Determination of Solubility and Stability
Aqueous solubility and stability were determined using previously described methods [13]. For the aqueous solubility assay, the tested compound was vortexed in deionized H2O for 1 h at 25 °C. The mixture was analyzed using HPLC. For the stability assay, the test compound stock solution (100 mg/mL in dimethyl sulfoxide) was diluted 100-fold to 1 mg/mL in 50 mM Tris buffer at pH 8.0. Then, aliquots were taken for HPLC analysis at the determined time intervals.

Biotransformation of 8-OHDe by DgAS over 24 h
Rha et al. (2019) indicated that DgAS can glycosylate an isoflavone glucoside (daidzin) to form daidzin glucosides after a 24 h reaction time [16]. Our previous study also revealed that DgAS can glycosylate 8-OHDe to form an isoflavone glucoside (8-OHDe-7-G) within 0.5 h. Therefore, this study was designed to determine whether 8-OHDe-7-G could be glycosylated by DgAS over a 24 h period. The reaction mixtures were analyzed using HPLC. Figures 1 and 2 show the HPLC analysis and time course for the 8-OHDe derivatives formed by DgAS after various reaction times. The results demonstrate that 8-OHDe was glycosylated by DgAS to form 8-OHDe-7-G after 0.5 h. The production of 8-OHDe-7-G was at its maximum after 1 h and then decreased gradually ( Figure 2). The other three products, compounds (1) to (3), also appeared after 1 h (Figure 2). Throughout the time course experiment, the amount of compound (1) was low and remained steady and stable. However, the large amounts of compounds (2) and (3) gradually increased from 1 h onwards. These results reveal that 8-OHDe-7-G can be glycosylated further by DgAS, which is consistent with the activity of DgAS on daidzin [16]. In contrast, no 8-OHDe glucoside was produced when heat-inactivated DgAS was used in the control reaction ( Figure S2).
In addition to its low solubility, 8-OHDe is unstable in alkaline solutions-a property that limits its application [9]. Thus, the stability of the two 8-OHDe diglucosides was also studied. Table 2 shows that over 92% of the 8-OHDe diglucosides remained in 50 mM Tris buffer (pH 8.0) after a 96 h incubation at 25 °C. By contrast, only 10% of 8-OHDe remained under the same conditions. The stabilities of the two 8-OHDe diglucosides were equal to that of the 8-OHDe monoglucoside (8-OHDe-7-G) [13]. The results indicate that 8-OHDe glucosides are stable in alkaline solutions because of the modification of the 7,8-ortho-diphenol structure of 8-OHDe. Some studies have indicated that ortho-hydroxyl substitutions, whether on the B-or A-ring, are the most important feature influencing the antioxidant activity of flavonoid compounds [21,22]. Therefore, the high antioxidant activity of 8-OHDe may be due to the 7,8-ortho-dihydroxyl groups in the A-ring [1], although this conformation would lead to 8-OHDe being unstable. Contrastingly, in the 8-OHDe glucosides, the ortho-hydroxyl groups were glycosylated, and the ortho-dihydroxyl groups disappeared. For this reason, both 8-OHDe diglucosides were found to be considerably more stable than 8-OHDe in solution. In short, the glycosylation of 8-OHDe increases its stability in alkaline conditions, and the two novel 8-OHDe diglucosides could be utilized in future bioindustrial activities.  [13].
A recent study indicated that in vitro enzymatically O-glycosylated flavonoids could be deglycosylated and reverted to their parent (pre-glycosylation) compounds under intestinal conditions [23]. In other studies, in vitro enzymatically O-glycosylated flavonoids reverted to their parent molecules under in vitro [23] or in vivo [24] fecal fermentation conditions, in which a variety of bacterial species metabolize sugars of flavonoid glycosides, such as glucose, that serve as easily accessible energy sources. On the other hand, in vitro enzymatically glycosylated flavonoids possess higher solubility and stability than their parent molecules. Thus, glycosylation is viewed as an attractive feature that could be exploited to enhance bioavailability and improve the cellular absorption of consumed flavonoids [19,20]. Therefore, the findings of these reports indicate that, upon oral ingestion, the two 8-OHDe glucosides identified in this study might also be digested to produce 8-OHDe, which could then be absorbed and exert its wide range of pharmaceutical activities. Moreover, the two stable and highly water-soluble 8-OHDe glucosides might possess higher bioavailability than 8-OHDe, as found in previous studies.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in the article or supplementary material.

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