Abstract
Flavonoids are plant secondary metabolites that are well known for their health-promoting properties as nutraceuticals in diets. Bioavailability and biological activities of flavonoids vary among the individual subclasses with different patterns of substitution, inclusive of glycosylation, to their basic structures. Many flavonoids exist as glycosides in plants. This study investigated the possibility of glycosylation of flavonoids through biotransformation using filamentous fungi as whole-cell biocatalysts. Microbial transformations of ten flavonoids (four flavones, four flavonols, a flavanone, and an aurone) were performed in cultures of Mucor hiemalis KCTC 26779. As a result, a flavonoid glycoside was obtained which has not been described previously. The chemical structure of this product was elucidated as 6,2′-dimethoxyflavonol-3-O-β-
Keywords
Flavonoids, found in fruits, vegetables, grains, roots, flowers, tea, and wine, have been shown to possess biological activities that vary due to the diversity of their phenolic structures. 1 -3 Reported pharmacological activities of flavonoids include antioxidant, 4,5 anti-inflammatory, 6 cardioprotective, 7 anticancer, 8,9 antiallergic, 10 and antidiabetic effects. 11,12 Because of the health-promoting and pharmacological effects of flavonoids, these compounds are utilized in food technology, as supplements, and by the pharmaceutical industries. 13 However, most flavonoids are unsatisfactory in terms of water solubility and stability in certain media, resulting in low absorption. 14 To overcome these problems, the glycosylation or hydroxylation of flavonoids is a well-known method that can improve solubility, intracellular and intercellular transport, chemical stability, and bioactivity. 7,14 -17 The solubility and stability of flavonoids can be improved by structural modification using chemical methods, biocatalysts, and biotransformation. 18,19
Microbial biotransformation is extensively used to transform many compounds, including hydrocarbons and pharmaceutical substances. 20 It has the potential to generate chemoselective, regioselective, and stereoselective compounds under milder conditions than those used in chemical synthesis. 21 Microbial biotransformation has been used for hydroxylation, dehydroxylation, O-methylation, O-demethylation, glycosylation, deglycosylation, dehydrogenation, and hydrogenation. 22,23 Therefore, it is able to produce novel bioactive compounds and enhance physicochemical and pharmacological properties. 24 Microbial biotransformation also has the advantage of being cost-effective, environmentally friendly, producing high yields and significant amounts of metabolites. 25
In our previous study on Coreopsis lanceolata, several types of flavonoids were isolated as flavone, flavonol, flavanone, chalcones, and aurone.
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As part of our search for new derivatives and/or enhancers of bioactivity and bioavailability through microbial transformation, we performed biotransformations of isolates

Biotransformation of 8 by Mucor hiemalis KCTC 26779.
Results and Discussion
Compound

Key heteronuclear multiple bond correlation (→), correlation spectroscopy (▬), and nuclear Overhauser effect spectroscopy (↔) correlations of compound 11.
In this study, the microbial transformation was attempted for a number of compounds with hydroxyl and/or methoxyl groups at diverse positions in flavone and flavonol structures. Among them, only compound
Many natural compounds are discovered in nature in the form of glycosides and they show a broad spectrum of benefits for human health. Almost all natural flavonoids are present in plants as either their O-glycosides or C-glycosides. These glycosylated flavonoids have been reported to have greater bioactivity, solubility, stability, and/or bioavailability than their aglycones. For example, the solubility of 5-O-α-
Conclusions
In conclusion, microbial transformation studies of 10 compounds with flavonoid structures with different substituents were carried out with M. hiemalis, and only 6,2′-dimethoxy-flavonol (
Experimental
General
The measurement of optical rotations was conducted on a JASCO DIP-1000 polarimeter (JASCO Co., Tokyo, Japan). One-dimensional (1D) and 2-dimensional (2D)-NMR spectra were obtained on a JNM-ECA 500 MHz NMR instrument (JEOL Ltd., Tokyo, Japan) with tetramethylsilane used as the internal standard. HR-ESI-MS were obtained on a Waters SYNAPT G2 mass spectrometer (Waters, Milford, MA, USA). Thin-layer chromatographic (TLC) analysis was performed on Kieselgel 60 F254 (Merck, Darmstadt, Germany) and Kieselgel 60 RP-18-F254S (Merck, Darmstadt, Germany). Analytical plates were visualized under ultraviolet light (254 and 365 nm) and sprayed with 10% (v/v) sulfuric acid, followed by heating at 180 °C for 2 minutes. Column chromatography (CC) was conducted using silica gel (70, 230 mesh, Merck, Darmstadt, Germany), RP-18 (YMC gel ODS-A, 12 nm, S-75 μm, YMC Co., Tokyo, Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences, Uppsala, Sweden).
Materials and Microorganism
Luteolin (
Screening Procedures
Microbial biotransformation studies were performed according to the standard two-stage procedure.
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The actively growing microbial cultures were inoculated in 250 mL flasks containing 50 mL of malt medium and incubated with gentle agitation (200 rpm) at 25 °C in a temperature-controlled shaking incubator. After inoculation for 24 hours, the dimethylsulfoxide (DMSO) solutions (1.5 mg/200 µL) of
Scale-Up Fermentations of 8
Preparative scale-up fermentations were carried out in three 500 mL flasks each containing 150 mL of malt medium, and each 15 mg of
6,2′-Dimethoxyflavonol-3-O-β-D -Glucopyranoside (11)
Brown solid.
[α]D: +28.5◦ (c 0.01, CH3OH).
1H NMR (DMSO-d 6, 500 MHz) δH 7.70 (1H, dd, J = 7.6, 1.7 Hz, H-6′), 7.59 (1H, d, J = 9.2 Hz, H-8), 7.48 (1H, br t, J = 8.3 Hz, H-4′), 7.45 (1H, d, J = 3.0 Hz, H-5), 7.38 (1H, dd, J = 9.2, 3.0 Hz, H-7), 7.12 (1H, dd, J = 8.3, 1.0 Hz, H-3′), 7.00 (1H, td, J = 7.6, 1.0 Hz, H-5′), 5.22 (1H, d, J = 7.8 Hz, H-1″), 3.53 (1H, d, J = 11.3 Hz, H-6″b), 3.35 (1H, d, J = 11.3 Hz, H-6″a), 3.12 (1H, m, H-3″), 2.98 (2H, m, H-4″, 5″), 2.86 (1H, m, H-2″), 3.86 (3H, s, 6-OCH3), 3.74 (3H,s, 2′-OCH3).
13C NMR (DMSO-d 6 , 125 MHz) δC 173.7 (C-4), 157.7 (C-2′), 157.3 (C-2), 156.9 (C-6), 150.5(C-9), 137.2(C-3), 132.7(C-6′), 132.4(C-4′), 124.8 (C-10), 124.2 (C-7), 120.6 (C-8), 120.4 (C-5′), 120.0 (C-1′), 112.1 (C-3′), 104.9 (C-5), 101.4 (C-1″), 77.8 (C-5″), 76.9 (C-3″), 74.5 (C-2″), 70.2 (C-4″), 61.4 (C-6″), 56.3 (6-OCH3), 56.2 (2′-OCH3)
HR-ESI-MS: m/z 483.1448 [M + Na]+ (calcd. for C23H26O10Na, 483.1267).
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Grants from Radiation Technology R&D program (No. 2017M2A2A6A05018541) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning.
