Microbial Transformation and Biological Activities of the Prenylated Aromatic Compounds from Broussonetia kazinoki

Broussonetia kazinoki has been used as a traditional medicine for the treatment of burns and acne, and its extracts have been found to show tyrosinase inhibitory and anticancer activities. In this study, the tyrosinase inhibitory and cytotoxic activities of B. kazinoki were explored, leading to the isolation of kazinol C (1), kazinol E (2), kazinol F (3), broussonol N (4), and kazinol X (5), of which the compounds 4 and 5 have not been previously reported. Microbial transformation has been recognized as an efficient tool to generate more active metabolites. Microbial transformation of the major compounds 1 and 3 was conducted with Mucor hiemalis, where four glucosylated metabolites (6–9) were produced from 1, while one hydroxylated (10) and one glucosylated (11) metabolites were obtained from 3. Structures of the isolated metabolites were determined by extensive spectroscopic analyses. All compounds were evaluated for their tyrosinase inhibitory and cytotoxic activities. Compound 3 and its metabolites, kazinol Y (10) and kazinol F-4″-O-β-d-glucopyranoside (11), exhibited the most potent tyrosinase inhibitory activities with the IC50 values ranging from 0.71 to 3.36 µM. Meanwhile, none of the metabolites, except for kazinol C-2′,3″-di-O-β-d-glucopyranoside (7), showed moderate cytotoxic activities (IC50 17.80 to 24.22 µM) against A375P, B16F10 and B16F1 cell lines.


Introduction
Broussonetia kazinoki, a deciduous shrub tree belonging to the family Moraceae, is widely distributed throughout Korea, China, and Japan [1 -3]. Since ancient times, its leaves, branches, roots and fruits have been used for various therapeutic purposes including the amelioration of vision, and suppression of edema [1][2][3][4]. In addition, it has been traditionally used for dermatologic diseases such as burns and acne in Korea according to the Principles and Practice of Eastern Medicine, an encyclopedia of medical knowledge [5,6]. Previous biological investigations have demonstrated that B. kazinoki exhibited a variety of pharmacological effects, such as antioxidant, anti-inflammatory, anticancer, anti-allergic, anti-diabetic, and anti-hyperglycemic activities [2][3][4][5][6][7]. Moreover, the extract of B. kazinoki has been registered as a skin-whitening agent by the Korea Food and Drug Administration (KFDA) due to its potent tyrosinase inhibitory and anti-melanogenic effects [5,8]. Additionally, a series of prenylated polyphenols including kazinol F and broussonin C isolated from B. kazinoki have been reported to exhibit significant tyrosinase inhibitory effects [8].
Tyrosinase (EC 1.14.18.1), also known as catecholase or diphenol oxidase, is a multifunctional copper-containing enzyme and is widely distributed in plants, fungi, bacteria and animals [9,10]. It catalyzes the hydroxylation of L-tyrosine to L-DOPA (3,4-dihydroxy-L-phenylalanine) and the subsequent oxidation of L-DOPA to L-dopaquinone [10]. Then, the dopaquinone forms melanin through polymerization with a series of enzymatic and the dopaquinone forms melanin through polymerization with a series of enzymatic and nonenzymatic reactions [11,12]. Melanin is responsible for skin color and plays an important role in human skin, for example, the prevention of skin injury under normal physiological conditions [12]; however, an overproduction and accumulation of melanin can result in hyperpigmentary disorders of the skin, such as freckles, melasma, age spots, and melanoma [9]. More seriously, tyrosinase catalyzes the formation of neuromelanin, which is associated with neurodegenerative disorders like Parkinson's disease [10,11,13]. Therefore, inhibiting tyrosinase activity applies to the treatment of pigmentation disorders associated with melanin hyperpigmentation and some related diseases [9,14]. Though a number of well-known tyrosinase inhibitors, like arbutin, kojic acid, and hydroquinone, have been reported from natural or synthetic sources during the past few decades, their applications have been limited due to serious side effects such as dermatitis, cytotoxicity and hepatotoxicity [9,14]. Thus, it is desirable to find new tyrosinase inhibitors with improved safety.
Microbial transformation is known as a useful method to generate more active derivatives with minor structural modifications in bioactive substrates using the metabolic activities of microorganisms [15,16]. The transformation is accomplished by a series of enzymatic reactions including hydroxylation, oxidation, and glycosylation under mild conditions [16][17][18][19]. Furthermore, microbial transformation is considered to be an environmentally friendly tool and has been used successfully to produce pharmaceuticals from natural products [15,16].
In the present study, we isolated and characterized the constituents of the root barks of B. kazinoki with tyrosinase inhibitory properties based on the bioactivity-guided fractionation process (Figure 1). Further transformation of kazinols C (1) and F (3) was performed with Mucor hiemalis, which led to the isolation of five glucosylated and one oxidized metabolite (6)(7)(8)(9)(10)(11). All of the isolated metabolites were evaluated for their tyrosinase inhibitory and cytotoxic activities, and it was revealed that kazinol F-4′'-O-β-D-glucopyranoside (11) had the strongest anti-tyrosinase activity with no cytotoxic activity against melanoma cells.

Structure Elucidation of Compounds from Broussonetia kazinoki
To isolate and identify the secondary metabolites of B. kazinoki for anti-tyrosinase activity, the EtOH extract of its root barks was investigated [8]. The CH2Cl2 fraction of the EtOH extract showed promising tyrosinase inhibitory effects, and the subsequent activityguided fractionation led to the isolation of five prenylated polyphenols 1-5 ( Figure 1).

Structure Elucidation of Compounds from Broussonetia kazinoki
To isolate and identify the secondary metabolites of B. kazinoki for anti-tyrosinase activity, the EtOH extract of its root barks was investigated [8]. The CH 2 Cl 2 fraction of the EtOH extract showed promising tyrosinase inhibitory effects, and the subsequent activity-guided fractionation led to the isolation of five prenylated polyphenols 1-5 ( Figure 1). Structures of the compounds were determined using the 1D-and 2D-NMR (nuclear magnetic resonance), including COSY (correlation spectroscopy), HSQC (heteronuclear single quantum correlation), and HMBC (heteronuclear multiple bond correlation), as well as mass spectroscopic data analyses. The three known compounds were identified and confirmed as kazinol C (1), kazinol E (2), and kazinol F (3) by comparison of their spectroscopic data with those reported in the literatures [8,20].

Microbial Transformation of Kazinols C and F by Mucor hiemalis
A total of 14 microbial cultures were screened to evaluate their ability to metabolize the isolated compounds 1 and 3 under a standard two-stage fermentation procedure [18,19]. Based on the analysis of TLC plates involving the substrates and culture controls, it was observed that Gliocladium deliquescens and Mucor hiemalis showed the ability to metabolize 1, and Alternaria alternata, Absidia coerulea, G. deliquescens and M. hiemalis showed the ability to metabolize 3 (Supplementary Table S1). Among the active strains, the fungus M. hiemalis was selected for preparative-scale fermentation studies since it exhibited the highest transformational capability towards 1 and 3. The subsequent transformation studies led to the production of five glucosylated and one oxidized metabolite (6)(7)(8)(9)(10)(11).
Compound 6 had a molecular formula of C 36 H 50 O 9 , as established by its HRFDMS peak at m/z 626.3439 [M] + (calcd. 626.3455), which was one glucose unit higher than that of 1, indicating that 6 was a glucosylated derivative of 1. This was supported by the occurrence of six new carbon signals in the 13 C NMR spectrum of 6, including five methine carbon resonances at δ C 103.4, 77.1, 75.8, 73.4, 69.7, and one methylene carbon at δ C 60.7. The corresponding proton signals at δ H 4.50 (1H) and 3.69-3.19 (6H) were observed in the 1 H NMR spectrum. All of these data were consistent with previous reports on the D-glucose moiety [19,24,25], and the glucose was determined to be in a β-configuration by the large coupling constant (7.1 Hz) of the anomeric proton signal at δ H 4.50 (H-1 ). The significantly downfield-shifted aromatic proton signal at δ H 6.81 (H-2 ) suggested the glucose moiety was attached to C-3 through an ether linkage. This was confirmed by a long-range correlation between the anomeric proton signal at δ H 4.50 (H-1 ) and carbon signal at δ C 143.1 (C-3 ) in the HMBC spectrum ( Figure 3). Thus, compound 6 was assigned to be kazinol C-3 -O-β-D-glucopyranoside.

Microbial Transformation of Kazinols C and F by Mucor hiemalis
A total of 14 microbial cultures were screened to evaluate their ability to metabolize the isolated compounds 1 and 3 under a standard two-stage fermentation procedure [18,19]. Based on the analysis of TLC plates involving the substrates and culture controls, it was observed that Gliocladium deliquescens and Mucor hiemalis showed the ability to metabolize 1, and Alternaria alternata, Absidia coerulea, G. deliquescens and M. hiemalis showed the ability to metabolize 3 (Supplementary Table S1). Among the active strains, the fungus M. hiemalis was selected for preparative-scale fermentation studies since it exhibited the highest transformational capability towards 1 and 3. The subsequent transformation studies led to the production of five glucosylated and one oxidized metabolite (6)(7)(8)(9)(10)(11).
Compound 6 had a molecular formula of C36H50O9, as established by its HRFDMS peak at m/z 626.3439 [M] + (calcd. 626.3455), which was one glucose unit higher than that of 1, indicating that 6 was a glucosylated derivative of 1. This was supported by the occurrence of six new carbon signals in the 13 C NMR spectrum of 6, including five methine carbon resonances at δC 103.4, 77.1, 75.8, 73.4, 69.7, and one methylene carbon at δC 60.7. The corresponding proton signals at δH 4.50 (1H) and 3.69-3.19 (6H) were observed in the 1 H NMR spectrum. All of these data were consistent with previous reports on the D-glucose moiety [19,24,25], and the glucose was determined to be in a β-configuration by the large coupling constant (7.1 Hz) of the anomeric proton signal at δH 4.50 (H-1′''). The significantly downfield-shifted aromatic proton signal at δH 6.81 (H-2′') suggested the glucose moiety was attached to C-3′' through an ether linkage. This was confirmed by a longrange correlation between the anomeric proton signal at δH 4.50 (H-1′'') and carbon signal at δC 143.1 (C-3") in the HMBC spectrum ( Figure 3). Thus, compound 6 was assigned to be kazinol C-3′'-O-β-D-glucopyranoside. Compound 7 had a molecular formula of C42H60O14, as established by the peak at m/z 788.3997 [M] + (calcd. 788.3983) in its HRFDMS spectrum, which was one glucose unit higher than that of 6, indicating that two glucose units had been introduced into the molecule of 1. The additional proton signals at δH 4.64, 4.51 and 3.71-3.18 (12H), as well as the carbon signals at δC 103.5, 101.5, 77.1, 76.9, 76.8, 75.8, 73.5, 73.4, 69.7, 69.6, 60.7, and 60.7 observed in the 1 H and 13 C NMR spectra of 7, indicated the presence of two D-glucose residues [24,25]. The glucose units were identified as β-glucose by their large coupling constants (6.5 Hz and 6.6 Hz) of the anomeric proton signals δH 4.64 and 4.51, respectively. The significantly downfield-shifted aromatic proton signals at δH 6.60 (H-3′) and 6.82 (H-2′') suggested the two glucose moieties were linked to C-2′ and C-3′', respectively. These connections were confirmed by the long-range correlations between the anomeric proton Compound 7 had a molecular formula of C 42 H 60 O 14 , as established by the peak at m/z 788.3997 [M] + (calcd. 788.3983) in its HRFDMS spectrum, which was one glucose unit higher than that of 6, indicating that two glucose units had been introduced into the molecule of 1. The additional proton signals at δ H 4.64, 4.51 and 3.71-3.18 (12H), as well as the carbon signals at δ C 103.5, 101.5, 77.1, 76.9, 76.8, 75.8, 73.5, 73.4, 69.7, 69.6, 60.7, and 60.7 observed in the 1 H and 13 C NMR spectra of 7, indicated the presence of two D-glucose residues [24,25]. The glucose units were identified as β-glucose by their large coupling constants (6.5 Hz and 6.6 Hz) of the anomeric proton signals δ H 4.64 and 4.51, respectively. The significantly downfield-shifted aromatic proton signals at δ H 6.60 (H-3 ) and 6.82 (H-2 ) suggested the two glucose moieties were linked to C-2 and C-3 , respectively. These connections were confirmed by the long-range correlations between the anomeric proton signal at δ H 4.51 (H-1 ) and the carbon signal at δ C 142.9 (C-3 ), as well as between δ H 4.64 (H-1 ) and δ C 154.1 (C-2 ) in the HMBC spectrum of 7 ( Figure 3). Thus, compound 7 was assigned to be kazinol C-2 ,3 -di-O-β-D-glucopyranoside.
Compound 8 had a molecular formula of C 36 H 50 O 9 , as deduced from the peak at m/z 626.3469 [M] + (calcd. 626.3455) in its HRFDMS spectrum, which was one glucose unit higher than that of 1, indicating that 8 was also a monoglucosylated derivative of 1. The additional proton signals at δ H 4.42 and 3.64-3.15 (6H), as well as the carbon signals at δ C 105.9, 77.3, 76.2, 74.1, 69.5, and 60.8 in the 1 H and 13 C NMR spectra of 8, indicated the presence of a D-glucose residue. The coupling constant (7.6 Hz) of anomeric proton (δ H 4.42) in the 1 H NMR spectrum of 8 indicated a β-configuration of this glucose moiety. The significantly downfield-shifted aromatic carbon signals at δ C 147.0 (C-3 ) and 133.9 (C-5 ) suggested this glucose moiety was attached to C-4 , and it was confirmed by the correlation between H-1 and C-4 in the HMBC spectrum of 8 ( Figure 3). Thus, compound 8 was assigned to be kazinol C-4 -O-β-D-glucopyranoside. The highly downfield-shifted oxymethine proton signal at δ H 4.58 (1H, t, J = 9.1 Hz) together with the corresponding carbon signal at δ C 89.3 indicated that a dihydrobenzofuran group was formed from the prenyl group substituted on ring B [26]. The spectroscopic data of 10 were quite similar to those of kazinol V [22], except for the signal of the methoxy group, which was supposed to be located at the C-9 position. The location of the additional methoxy group was confirmed by the cross-peak of the proton signal at δ H 3.16 (3H, s) and carbon signal at δ C 77.5 in its HMBC spectrum ( Figure 4). The absolute configuration of compound 10 at C-8 was considered to be R based on its negative specific rotation ([α] 20 D −12.38 • ) by comparison with the reported data of related compounds which had a dihydrofuran group in their structures [18,22,23]. Based on the above analyses, compound 10 was assigned to be 6 -(3-methylbut-2-enyl)-4 ,5 -[(R)-2-(1-methoxy-1-methylethyl)]dihydrofuranyl-2 ,4 ,3 -trihydroxydiphenylpropane, and was named kazinol Y. signal at δH 4.51 (H-1′'') and the carbon signal at δC 142.9 (C-3"), as well as between δH 4.64 (H-1′''') and δC 154.1 (C-2′) in the HMBC spectrum of 7 ( Figure 3). Thus, compound 7 was assigned to be kazinol C-2′,3′'-di-O-β-D-glucopyranoside. Compound 8 had a molecular formula of C36H50O9, as deduced from the peak at m/z 626.3469 [M] + (calcd. 626.3455) in its HRFDMS spectrum, which was one glucose unit higher than that of 1, indicating that 8 was also a monoglucosylated derivative of 1. The additional proton signals at δH 4.42 and 3.64-3.15 (6H), as well as the carbon signals at δC 105.9, 77.3, 76.2, 74.1, 69.5, and 60.8 in the 1 H and 13 C NMR spectra of 8, indicated the presence of a D-glucose residue. The coupling constant (7.6 Hz) of anomeric proton (δH 4.42) in the 1 H NMR spectrum of 8 indicated a β-configuration of this glucose moiety. The significantly downfield-shifted aromatic carbon signals at δC 147.0 (C-3′') and 133.9 (C-5′') suggested this glucose moiety was attached to C-4′', and it was confirmed by the correlation between H-1′'' and C-4′' in the HMBC spectrum of 8 ( Figure 3). Thus, compound 8 was assigned to be kazinol C-4′'-O-β-D-glucopyranoside.
Compound 9 had a molecular formula of C42H60O14, as established by the peak at m/z 788.4016 [M] + (calcd. 788.3983) in its HRFDMS spectrum, which was one glucose unit higher than that of 8, indicating that two glucose units had been introduced into the molecule of 1. The additional proton signals at δH 4.85, 4.65 and 3.69-3.02 (12H), as well as the carbon signals at δC 103.5, 102.3, 77.1, 77.1, 76.5, 75.8, 74.3, 73.5, 70.0, 69.8, 61.1, and 60.7 in the 1 H and 13 C NMR spectra of 9, indicated the presence of two D-glucose residues. The coupling constants (7.4 Hz and 7.6 Hz) of the anomeric protons (δH 4.85 and 4.64) in the 1 H NMR spectrum of 9 indicated the β-configuration of these two glucose moieties. The aromatic proton signals of ring A at δH 6.70 (s, H-6′) and 6.29 (s, H-3′) were quite similar to those of compound 1, and together with the significantly downfield-shifted aromatic proton signal at δH 6.93 (H-2′') suggested that these two glucose moieties should be linked to C-3′' and C-4′'. These connections were confirmed by the cross-peaks of H-1′'' and C-3′' as well as H-1′''' and C-4′' in the HMBC spectrum of 10 ( Figure 3). Based on the above analyses, compound 9 was assigned to be kazinol C-3′',4′'-di-O-β-D-glucopyranoside.
Compound 10 showed a [M] + peak at m/z 426.2414 (calcd. for C26H34O5, 426.2406) in its HRFDMS spectrum, which established a molecular formula of C26H34O5. Significant differences were observed in the 1 H and 13 C NMR spectra of 10 compared with those of 3. The highly downfield-shifted oxymethine proton signal at δH 4.58 (1H, t, J = 9.1 Hz) together with the corresponding carbon signal at δC 89.3 indicated that a dihydrobenzofuran group was formed from the prenyl group substituted on ring B [26]. The spectroscopic data of 10 were quite similar to those of kazinol V [22], except for the signal of the methoxy group, which was supposed to be located at the C-9′' position. The location of the additional methoxy group was confirmed by the cross-peak of the proton signal at δH 3.16 (3H, s) and carbon signal at δC 77.5 in its HMBC spectrum ( Figure 4). The absolute configuration of compound 10 at C-8′' was considered to be R based on its negative specific rotation ([α] -12.38°) by comparison with the reported data of related compounds which had a dihydrofuran group in their structures [18,22,23]. Based on the above analyses, compound 10 was assigned to be 6′'-(3-methylbut-2-enyl)-4′',5′'-[(R)-2-(1-methoxy-1-methylethyl)]dihydrofuranyl-2′,4′,3′'-trihydroxydiphenylpropane, and was named kazinol Y.  Compound 11 had a molecular formula of C 31 H 42 O 9 , as deduced from the peak at m/z 558.2839 [M] + (calcd. 558.2829) in its HRFDMS spectrum, which was one glucose unit higher than that of 3, indicating that 11 was also a monoglucosylated derivative of 3. The additional proton signals at δ H 4.43 and 3.65-3.16 (6H), as well as the carbon signals at δ C 105.9, 77.3, 76.2, 74.1, 69.6, and 60.8 in the 1 H and 13 C NMR spectra of 11, indicated the presence of a D-glucose residue. The coupling constant (7.7 Hz) of the anomeric proton (δ H 4.43) in the 1 H NMR spectrum of 11 indicated a β-configuration of this glucose moiety. The significantly downfield-shifted aromatic carbon signals at δ C 147.1 (C-3 ) and 133.9 (C-5 ) suggested this glucose moiety was attached to C-4 , and it was confirmed by the correlation between H-1 and C-4 in the HMBC spectrum of 11 ( Figure 3). Thus, compound 11 was assigned to be kazinol C-4 -O-β-D-glucopyranoside.

Tyrosinase Inhibitory Activity
All the compounds were investigated for their tyrosinase inhibitory effects using L-tyrosine as the substrate. Kojic acid, a well-known tyrosinase inhibitor currently used in cosmetics as a skin-whitening agent, was used as a positive control. Metabolite 11 exhibited the most potent inhibitory effect against tyrosinase (IC 50 , 0.71 µM), followed by its parent compound 3 and metabolite 10, with IC 50 values of 2.12 and 3.36 µM, respectively (Table 1). Compound 4, which also exhibited a stronger inhibitory effect than the kojic acid, showed moderate activity with an IC 50 value of 24.11 µM. All of the other compounds, which have a 1,1-dimethylallyl group in their A-ring, were considered to be inactive, as their IC 50 values were over 80 µM. Moreover, it has been reported that kazinol F and broussonin C have exhibited much stronger tyrosinase inhibition than the ring A-prenylated 1,3diphenylpropanes [8], and similar results were observed in studies reporting the flavonoids as tyrosinase inhibitors from Broussonetia papyrifera [27]. The above analyses indicated that prenylation in the ring A of the compounds with a 1,3-diphenylpropane skeleton might weaken their tyrosinase inhibition activities.

Cytotoxic Activity
To evaluate the anticancer potential of compounds 1-11, an MTT assay was used to determine the cell viability of human melanoma (A375P) and murine melanoma (B16F10 and B16F1) cell lines following a 24 h treatment with compounds 1-11. From the results shown in Table 1, it was observed that compounds 1-4 and metabolite 7 displayed potent cytotoxic effects against all cancer cell lines tested. Compounds 5 and 10 exhibited quite weak cytotoxicity compared to compounds 1 and 3, suggesting that cyclization between the prenyl moiety and the adjacent phenolic hydroxyl group might decrease the cytotoxic activity. This was similar to the result that kazinol R, possessing a pyran ring in its structure, exhibited weaker cytotoxic effects than kazinol Q [28]. In addition, metabolite 7, which had a glucose moiety in the ring A of its structure, showed much stronger activities than the other glucosylated metabolites, suggesting that O-glycosylation of 1,3-diphenylpropanes in the ring A might enhance their cytotoxic activities.

General Experimental Procedures
The NMR spectra were recorded in CDCl 3 , acetone-d 6 or DMSO-d 6

Materials and Microorganisms
The root barks of Broussonetia kazinoki were collected and identified by Eden farm in Jeonju, Korea, in August 2020, and a voucher specimen has been deposited at the Two types of media were used in the screening experiments and are listed below: A. coerulea, A. alternata, A. fumigatus, M. hiemalis, P. chrysogenum, and T. koningii were incubated on a malt medium (malt extract 20 g/L, dextrose 20 g/L, and peptone 1 g/L). Other microbes were cultured on a potato dextrose medium (24 g/L).

Isolation of Active Compounds from Broussonetia kazinoki
The root barks (650 g) of B. kazinoki were extracted with 94% ethanol (3 × 6 L) under sonication at room temperature. The combined ethanol extract was concentrated under reduced pressure, which was suspended in water and successively partitioned using hexane, dichloromethane (CH 2 Cl 2 ), ethyl acetate (EtOAc), and butanol. The CH 2 Cl 2 extract was subjected to silica gel column chromatography, using hexane:EtOAc mixtures to give sixteen fractions. Fraction 12 was then chromatographed using Sephadex LH-20 and eluted with methanol to give three subfractions. Subfraction 12-2 was purified by a C 18 HPLC column with a methanol:water gradient elution system (75→80%) to afford compounds 1 (110 mg), 2 (8 mg), and 5 (4 mg). Fraction 14 was applied to Sephadex LH-20 column chromatography and eluted with methanol to give four subfractions. Subfraction 14-3 was further purified by semi-preparative HPLC with a methanol:water gradient elution system (75→80%) to afford compounds 3 (24 mg) and 4 (3 mg). Kazinol

Microbial Screening Procedures
The culture fermentation was carried out according to the usual two-stage procedure [24][25][26]. In the screening studies, the actively growing microbial cultures were inoculated in 250 mL flasks containing 50 mL of media and incubated in a temperature-controlled shaking incubator with gentle agitation (200 rpm) at 25 • C for one day. Then 100 µL of the prepared ethanol solution (10 mg/mL) of each substrate was added to the flask and further incubated for another seven days under the same condition. Sampling and TLC monitoring were performed at an interval of 24 h. Culture controls consisted of fermentation cultures in which the microorganisms were grown without the addition of substrates.

Scale-Up Fermentation of 1 and 3 with Mucor hiemalis
Scale-up fermentation was carried out with M. hiemalis using 500 mL flasks each containing 150 mL of malt media and 5 mg of compound 1 (total of 110 mg) under the same temperature-controlled shaking conditions for five days. After fermentation, the microbial cultures were extracted with the same volume of EtOAc three times and then the combined organic layers were concentrated in vacuo. The EtOAc extract of kazinol C (1) was separated by semi-preparative HPLC using isocratic 83% MeOH to afford metabolites 6 (4.6 mg), 7 (7.8 mg), 8 (3.5 mg), and 9 (4.5 mg). A similar fermentation process was performed for compound 3 (65 mg in total), which was incubated for three days. The yielded EtOAc extract was subjected to a C 18 HPLC column using isocratic 77% MeOH under a flow rate of 2 mL/min to afford metabolites 10 (2.5 mg) and 11 (3.

Conclusions
The activity-guided fractionation of the root barks of B. kazinoki led to the isolation of five compounds, including two previously unreported prenylated polyphenols 4 and 5.
The subsequent microbial transformation studies on the two major compounds, kazinol C (1) and kazinol F (3), resulted in the production of five glucosylated and one oxidized metabolite (6)(7)(8)(9)(10)(11). All the obtained compounds were evaluated for their tyrosinase inhibitory and cytotoxic activities. Compounds 3, 4, 10 and 11, which lack a prenyl group in the ring A of their structures, exhibited potent tyrosinase inhibitory activities, with glucosylated metabolite 11 being the most active. Although all the metabolites showed weaker cytotoxic activities than their parent compounds, the compound 8, which had a glucose moiety in its structure, exhibited a moderate activity against the cancer cell lines tested. All of these indicated that glycosylation plays a role in the biological activities of the prenylated polyphenols from B. kazinoki.