Microbial Transformation of Licochalcones

Abstract

Microbial transformation of licochalcones B (1), C (2), D (3), and H (4) using the filamentous fungi Aspergillus niger and Mucor hiemalis was investigated. Fungal transformation of the licochalcones followed by chromatographic separations led to the isolation of ten new compounds 5–14, including one hydrogenated, three dihydroxylated, three expoxidized, and three glucosylated metabolites. Their structures were elucidated by combined analyses of UV, IR, MS, NMR, and CD spectroscopic data. Absolute configurations of the 2″,3″-diols in the three dihydroxylated metabolites were determined by ECD experiments according to the Snatzke’s method. The trans-cis isomerization was observed for the metabolites 7, 11, 13, and 14 as evidenced by the analysis of their ¹H-NMR spectra and HPLC chromatograms. This could be useful in better understanding of the trans-cis isomerization mechanism of retrochalcones. The fungal transformation described herein also provides an effective method to expand the structural diversity of retrochalcones for further biological studies.

1. Introduction

Licorice is one of the oldest and most commonly used herbal medicines in the world [1]. The name ‘licorice’ is derived from the dried roots of Glycyrrhiza species (Leguminosae family) and is the common name for these plants [2]. A variety of pharmacological activities have been described for licorice, and these bioactivities are attributed to the chemical constituents of licorice [3]. To date, more than 600 compounds have been isolated from licorice, and the major active constituents are saponins and flavonoids [4]. Among them, a series of retrochalcones, licochalcones A, B, C, D, E, G and the closely related compound echinatin, were identified from the roots of licorice [5]. Structurally, these retrochalcones belong to an unusual phenolic compound family and are distinguished from ordinary chalcones by the lack of a hydroxyl group at the C-2′ and C-6′ positions [6]. Several biological mechanism studies have proved that these compounds exhibited diverse biological effects such as anti-oxidant [7], antibacterial [8], anti-inflammatory [9,10], anticancer [11,12,13,14], osteogenesis [15], anti-hepatotoxic, antidiabetic and anti-allergic effects [16]. Moreover, the novel synthetic licochalcone F, a regioisomer of licochalcone E, showed in vivo anti-inflammatory and glucose intolerance effects without side effects [17]. Another synthetic licochalcone H, a regioisomer of licochalcone C, was found to exhibit anti-cancer activity by promoting apoptosis in human esophageal squamous cell carcinoma cells [18]. Even though promising biological studies of these small molecules were investigated, it is still essential to explore and generate more novel candidates for possible therapeutic uses [19].

Microbial transformation by filamentous fungi can be used as a feasible alternative to the conventional chemical methods when searching for new derivatives with potential biological properties [20]. These fungi could improve the regio- and stereo-selectivity of some chemical reactions [21]. The application of microbes for biotransformation of chalcones led to the formation of many novel derivatives by means of cyclization, hydroxylation, reduction, and dehydrogenation reactions [22,23,24]. However, to date few microbial transformation studies have been investigated directly on the series of retrochalcones isolated from licorice. In the present study, we report the microbial transformation of licochalcones B (LB, 1), C (LC, 2), D (LD, 3) and H (LH, 4) by the selected fungi Aspergillus niger and Mucor hiemalis.

2. Results and Discussion

2.1. Microbial Transformation of Licochalcones B (1), C (2), D (3) and H (4) by A. niger

Microbial transformation of LB (1) by A. niger produced the hydrogenated metabolite 5; microbial transformation of LC (2), LD (3) and LH (4) by A. niger furnished the corresponding dihydroxylated metabolites 6, 8, 10 and expoxidized metabolites 7, 9, 11, respectively (Scheme 1).

Metabolite 5 was obtained as a yellow amorphous powder. Its molecular formula was determined as C₁₆H₁₆O₅ by the positive [M + Na]⁺ peak at m/z 311.0896 (calcd for C₁₆O₁₆O₅Na, 311.0895) on the basis of its HRESIMS spectrum, which indicated that it was a dihydrogenerated derivative of 1. In the ¹H-NMR spectrum of 5, the trans-olefinic proton signals of H-α and H-β were replaced by two new methylene proton signals at δ_H 3.15 (2H, t, J = 7.4 Hz) and 2.87 (2H, t, J = 7.4 Hz). Consistently, ¹³C-NMR spectrum of 5 showed the absence of low field olefinic carbon signals and the presence of two new high field carbon signals at δ_C 39.2 and 25.1. Location of the hydrogenation was further determined to be at C-α and C-β based on the correlations of H-α (δ_H 3.15) and H-β (δ_H 2.87) with C=O (δ_C 200.0) in the HMBC spectrum of 5. Thus, coupound 5 was established to be α,β-dihydrolicochalcone B.

Microbial transformation of licochalcone C (2) by A. niger for three days produced metabolites 6 and 7. They were obtained as yellow amorphous powders. Metabolite 6 gave a molecular formula of C₂₁H₂₄O₆ from its HRESIMS, indicating that two oxygen atoms and two hydrogen atoms were inserted into the substrate 2. UV spectrum of 6 showed characteristic absorption maxima at 207 and 352 nm, indicating that the chalcone skeleton remained intact. The ¹H-NMR spectrum of 6 showed two doublet proton signals at δ_H 8.00 and 7.63 (each d, J = 15.6 Hz), which are characteristic of the E-configuration of the α,β-double bond in the chalcone derivatives. In comparison with the ¹H-NMR spectrum of 2, two proton signals H-1″ (δ_H 3.45, 2H, d, J = 6.9 Hz) and H-2” (δ_H 5.23, m, 1H) on the prenyl moiety disappeared, whereas three new proton signals δ_H 2.69 (1H, dd, J = 14.2, 10.3 Hz), 3.12 (1H, dd, J = 14.2, 2.0 Hz) and 3.65 (1H, dd, J = 10.3, 2.0 Hz) were observed in 6. It was suggested that two hydroxyl groups were newly introduced at C-2″ and C-3″. Dihydroxylation was also supported by ¹³C-NMR based on the presence of two new oxygen-bearing ¹³C signals at δ_C 72.6 (C-3″) and 78.9 (C-2″) instead of two olefinic carbon signals [24]. In HMBC spectrum, the proton signals at δ_H 2.69 (H-1″b) and 3.12 (H-1″a) were correlated with the carbon signal at δ_C 120.9 (C-3), and the proton signal at δ_H 3.65 (H-2″) was correlated with the carbon signal at δ_C 72.6 (C-3″). Absolute configuration of the 2″,3″-diols in 6 was determined using the ECD spectrum obtained by the Snatzke’s method [25,26,27]. Based on the empirical rule, induced CD (ICD) curve of the Mo-complex of 6 showed the negative Cotton effect at around 310 nm, indicating that the O-C-C-O dihedral angle in the favored conformation was negative for 6 (Figure 1). Therefore, the absolute configuration at C-2″ of 6 was assigned R. Based on these data, the structure of 6 was elucidated as (2″R)-2″,3″-dihydroxylicochalcone C.

The molecular formula of the metabolite 7 was revealed as C₂₁H₂₂O₅ based on the HRESIMS ion peak at 377.1376 (calcd for C₂₁O₂₂O₅Na, 377.1365), indicating the addition of one oxygen atom compared to the substrate 2. Interestingly, the HPLC chromatogram of the purified 7 showed two peaks at t_R of 22.66 and 26.06 min. The corresponding UV λ_max of the two peaks were 240, 357 nm and 293 nm, respectively. Moreover, the ¹H-NMR peak pattern of 7 showed two sets of resonance signals in a ratio of approximately 1:0.3. They were clearly assignable to the suggested major (E)- and minor (Z)-isomers by observing the two sets of H-α and H-β proton signals including those at δ_H 7.64 (1H, d, J = 15.6 Hz) and 8.01 (1H, d, J = 15.6 Hz) in one set, and those at δ_H 6.55 (1H, d, J = 12.8 Hz) and 7.11 (1H, d, J = 12.8 Hz) in another. Assignment of the major trans isomer was confirmed by the presence of three new proton signals at δ_H 3.80 (1H, dd, J = 7.0, 5.0 Hz, H-2″), 3.04 (1H, dd, J = 17.1, 5.0 Hz, H-1″a) and 2.73 (1H, dd, J = 17.1, 7.0 Hz, H-1″b) coupled with the ¹³C-NMR signals at δ_c 68.3 (C-2″) and 25.8 (C-1″) based on the NMR analyses of 7. Location of the epoxidation of 7 was confirmed to be at C-2″ and C-3″ according to the HMBC correlation from H-1″a and H-1″b to the additional quaternary carbon signal at δ_c 77.3 (C-3″). Minor cis-form showed the same correlation patterns comparing with those of trans set signals in the 1D and 2D NMR spectra. Moreover, the assignments of the proton signals revealed general up-field shifts in the cis isomer as compared to those of the trans isomer in the ¹H-NMR spectrum. Therefore, the structure of 7 was confirmed as 2″, 3″-expoxylicochalcone C.

Microbial transformation of licochalcone D (3) by A. niger for three days produced the metabolites 8 and 9. Compounds 8 and 9 were obtained as yellow amorphous powders. The molecular formula of 8 (C₂₁H₂₄O₇) was compatible with the insertion of two oxygen and two hydrogen atoms to the substrate 3, which was supported by the analysis of its HRESIMS data (m/z 389.1603 [M + H]⁺, calcd for C₂₁H₂₅O₇, 389.1600). The ¹H-NMR and ¹³C-NMR spectroscopic data of 8 were almost identical with those of 3 except for resonances of the prenyl moiety. Presence of the prenyl moiety gave rise to the proton signals at δ_H 2.60 (1H, dd, J = 13.8, 10.4 Hz), 3.13 (1H, dd, J = 13.8, 1.3 Hz) and 3.66 (1H, dd, J = 10.4, 1.3 Hz), and two oxygen-bearing carbon signals at δ_C 78.1 and 72.5 instead of original olefinic signals. The locations of dihydroxylation were confirmed to be at C-2″ and C-3″ by the HMBC correlations from H-4″ and H-5″ to C-2″ and C-3″, and from H-1″ to C-2″ and C-3″. The absolute configuration at C-2″ was determined by comparison of their ICD spectra using the Snatzke’s method. The Mo-complex of compound 8 presented a negative Cotton effect at around 310 nm (Figure S35), indicating the R configuration at C-2″. Therefore, metabolite 8 was identified as (2″R)-2″,3″-dihydroxylicochalcone D.

Compound 9 had a molecular formula of C₂₁H₂₂O₆ on the basis of its HRESIMS data (m/z 371.1497 [M + H]⁺, calcd for C₂₁H₂₃O₆, 371.1495) which lacks H₂O moiety compared with that of 8. The NMR data of 9 were similar to those of 8, implying 9 possessed the same skeleton as 8. However, major differences in their ¹H-NMR spectra were observed in the resonances of δ_H 3.82 (1H, dd, J = 7.0, 5.0, H-2″), 3.11 (1H, dd, J = 16.8, 5.0, H-1″a), 2.82 (1H, dd, J = 16.8, 7.0, H-1″b), 1.36 (3H, s, H-4″), 1.31 (3H, s, H-5″), suggesting that the differences between 8 and 9 were at C-2″ and C-3″. In the HMBC spectrum, the H-1″a and H-1″b were coupled with the oxygen-bearing carbon signals at δ_C 68.6 (C-2″) indicating the epoxidation of the double bond in the prneyl group. Moreover, the proton signals H-4″ (δ_H 1.36) and H-5″ (δ_H 1.31) showed correlations with C-2″ and C-3″. Thus, metabolite 9 was identified as 2″,3″-expoxylicochalcone D.

Microbial transformation of licochalcone H (4) by A. niger for three days produced metabolites 10 and 11. The molecular formula of metabolite 10, C₂₁H₂₄O₆, obtained by the HRESIMS data (m/z 373.1652 [M + H]⁺, calcd for C₂₁H₂₅O₆, 373.1651), is consistent with dihydroxylated derivative of the substrate 4. The large coupling constant 15.6 Hz (J_{Hα –Hβ}) indicated that the E-configuration at the α,β-double bond remained the same in the compound 10. Compared to its substrate 4, the ¹H-NMR spectrum of 10 exhibited notable changes on a double bond in the prenyl group. Three new proton signals at δ_H 2.52 (1H, dd, J = 14.2, 10.4 Hz), 2.99 (1H, dd, J = 14.2, 1.7 Hz) and 3.61 (1H, dd, J = 10.4, 1.7 Hz) appeared in the ¹H NMR spectrum. Similar changes were also observed in the ¹³C-NMR spectrum, including two oxygenated carbon signals at δ_C 78.5 and δ_C 72.5. This suggested that 10 was a dihydroxylated derivative of 4. Locations of the two hydroxyl groups were confirmed to be at C-2″ and C-3″ by observation of HMBC correlations from H-4″/5″ (δ_H 1.26) to C-2″ (δ_C 78.5) and C-3″ (δ_C 72.5). On the basis of the ICD spectroscopic analysis, the absolute configuration at C-2″ was confirmed as R for the compound 10 (Figure S36). Therefore, structure of the compound 10 was elucidated as (2″R)-2”,3”-dihydroxylicochalcone H.

The molecular formula of the metabolite 11 was determined as C₂₁H₂₂O₅ on the basis of the HRESIMS data (m/z 355.1544 [M + H]⁺, calculated for C₂₁H₂₃O₅, 355.1545) which was identical to that of 7. HPLC chromatography of the purified 11 showed two peaks at t_R of 17.15 and 19.56 min, respectively. UV λ_max of the two peaks were 267, 295 nm, and 276, 378 nm, respectively. Moreover, the ¹H NMR pattern of 11 showed two sets of resonances in the spectra of ¹H- and ¹³C-NMR. With the observations of two pairs of H-α and H-β proton signals at at δ_H 6.58 (1H, d, J = 12.8 Hz) and 7.00 (1H, d, J = 12.8 Hz), together with the signals at δ_H 7.70 (1H, d, J = 15.6 Hz) and 7.93 (1H, d, J = 15.6 Hz), it was revealed that 11 exists as a mixture of (Z)- and (E)-isomers. The integrals of (Z)-form and (E)-form (approximate ratio 0.3:1) indicated that the (E)-form was the major one. The NMR spectral data of the major trans-form showed three oxygenated proton signals at δ_H 3.67 (1H, dd, J = 7.5, 5.2 Hz, H-2″), 2.92 (1H, dd, J = 16.2, 5.2 Hz, H-1″a) and 2.60 (1H, dd, J = 16.2, 7.5 Hz, H-1″b) coupled with the signals at δ_c 68.4 (C-2″) and 30.6 (C-1″) on the basis of the combined analyses of ¹H-NMR, ¹³C-NMR and HSQC of 11, which indicated the epoxidation of the double bond between C-2″ and C-3″ of 4. Further, location of the epoxidation was deduced to be between C-2″ and C-3″ based on the HMBC correlations from H-1″a and H-1″b to the carbon signals at δ_c 68.4 (C-2″) and 113.3 (C-5). Assignment of the minor cis-form was made possible by observing the same correlation patterns as those of trans isomer signals in the 1D and 2D NMR spectra. Therefore, the structure of 11 was confirmed as 2″,3″-expoxylicochalcone H (11).

2.2. Microbial Transformation of Licochalcones B (1), C (2), D (3) and H (4) by M. hiemalis

Microbial transformation of LB (1), LC (2) and LH (4) by M. hiemalis yielded the corresponding glucosylated metabolites 12, 13 and 14, respectively (Scheme 2).

Microbial transformation of licochalcone B (1) by M. hiemalis for five days produced the metabolite 12. Compound 12 exhibited a molecular formula of C₂₂H₂₄O₁₀ on the basis of its HRESIMS (m/z 449.1449 [M + H]⁺, calcd for C₂₂H₂₅O₁₀, 449.1448), corresponding to the presence of an additional unit of C₆H₁₀O₅ to 1. The UV spectrum of 12 had λ_max at 208, 288 and 338 nm which is similar to that of 1. The large coupling constant of 15.8 Hz (J_Hα–Hβ) indicated that the E-configuration of the α,β-double bond remained the same in the compound 12. The ¹H- and ¹³C-NMR spectra of 12 showed characteristic signals of a sugar moiety. There were six carbon signals at δc 105.2, 77.7, 76.7, 74.4, 70.1, 61.2 and an anomeric proton signal at δ_H 4.74 (1H, d, J = 7.7 Hz). The other corresponding proton signals were assigned by the correlations in the spectrum of HSQC. Anomeric proton at δ_H 4.74 revealed that the glycoside linkage was formed between the anomeric hydroxyl group of the sugar moiety and the hydroxyl group of the C-3 (δ_H 138.7) of the aglycone based on their HMBC correlation. Acid hydrolysis of 12 afforded a d-glucose and the aglycone 1 which was identified by TLC and HPLC. All these evidences indicated that this sugar moiety was a β–d-glucopyranose. Therefore, the structure of 12 was assigned as licochalcone B 3-O-β-d-glucopyranoside.

Microbial transformation of licochalcone C (2) by M. hiemalis for three days produced the metabolite 13. Compound 13 was isolated as a yellow amorphous powder, with a molecular formula of C₂₇H₃₂O₉, which was determined by HRESIMS at m/z 501.2123 [M + H]⁺ (calcd for C₂₇H₃₃O₉, 501.2125). In the comparison of its ¹H- and ¹³C-NMR spectra with those of the substrate 2, compound 13 displayed NMR resonance signals for the presence of a sugar moiety, showing an anomeric proton signal at δ_H 5.02 (1H, d, J = 7.2 Hz). Complete assignments of the ¹H- and ¹³C-NMR signals of the sugar moiety were accomplished by HMBC and HSQC experiments. HMBC correlation from the anomeric proton signal at δ_H 5.02 (H-1’’’) to the carbon signal at δ_C 158.6 (C-4) confirmed the assignment of glycosylation at C-4. In the sugar analysis, d-glucopyranose was confirmed using TLC after acid hydrolysis of 13. All these indicated the presence of a β-d-glucopyranose unit. The aglycone moiety of the ¹H- and ¹³C-NMR spectra of the metabolite 13 displayed two sets of signals in the integral ratio of 1:0.2. Based on the observation of two pairs of H-α and H-β proton signals at δ_H 7.63 (1H, d, J = 15.7 Hz) and 7.93 (1H, d, J = 15.7 Hz), together with δ_H 6.58 (1H, d, J = 12.7 Hz) and 7.12 (1H, d, J = 12.7 Hz), 13 was clearly assigned as a mixture of (E)- and (Z)-isomers. On the basis of the above data and extensive 2D NMR experiments, structure of the compound 13 was assigned as licochalcone C 4-O-β-d-glucopyranoside.

Microbial transmformation of licochalcone H (4) by M. hiemalis for three days afforded metabolite 14. Metabolite 14 was isolated as a yellow amorphous powder, with a molecular formula of C₂₇H₃₂O₉, which was determined by HRESIMS at m/z 501.2122 [M + H]⁺ (calcd for C₂₇H₃₃O₉, 501.2125). In the ¹H-NMR spectrum of 14, the presence of a sugar moiety was deduced by the appearance of an anomeric doublet proton at δ_H 5.00 with the coupling constant of 7.3 Hz, which indicated the β-orientation of the sugar moiety. Based on the information of a set of hexose moiety signals (δ_c 100.9, 77.2, 77.0, 73.6, 70.3 and 61.4) in the ¹³C-NMR spectrum, it was indicated that 14 was a glucosylated product of 4. The identification was confirmed by acid hydrolysis and comparison with authentic glucose and the aglycone. The chemical shift of H-3 proton was significantly shifted downfield from δ_H 6.47 to 6.88, indicating that the glucosylation site was at 4-OH of the aglycone. Similarly to 13, the ¹H- and ¹³C-NMR supported the notion that the agalycone of 14 was an indivisible mixture of (Z)- and (E)-conformers. The aglycone part of the ¹H- and ¹³C-NMR of the metabolite 14 displayed two sets of signals in the integral ratio of 0.4:1. On the basis of the observations of two pairs of H-α and H-β proton signals at δ_H 6.36 (1H, d, J = 12.7 Hz) and 7.11 (1H, d, J = 12.7 Hz), together with δ_H 7.63 (1H, d, J = 16.2 Hz) and 7.99 (1H, d, J = 16.2 Hz), 14 was clearly assignable as a mixture of (Z)- and (E)-isomers. Based on these analyses, the structure of 14 was elucidated as licochalcone H 4-O-β-d-glucopyranoside.

Generally, trans-chalcones are thermodynamically more stable than their corresponding cis isomers and most chalcones are thus isolated in trans form [28]. However, during the structure elucidation of the metabolites, the trans-cis isomerizations were observed by the analyses of the NMR spectra and HPLC chromatograms of metabolites 7, 11, 13, and 14 (Scheme 1 and Scheme 2). The (Z)-form observed in the structures of these metabolites might be considered as artefacts formed during incubation, sample processing, or isolation procedure. There might be interconversion between the (E)-form and its corresponding (Z)-form because it was detected again after separation of the peaks of corresponding (E)- and (Z)-forms. Recently, two dihydrobenzofuran congeners of licochalcone A isoated from the roots of Glycyrrhiza inflata were found to rapidly isomerize and yield trans and cis isomers when solutions were exposed to sunlight [29]. Additionally, one metabolite of licochalcone A produced by human liver microsomes was observed as a mixture of trans- and cis-forms [30]. Based on these evidences, it could be deduced that some substituents introduced in the B ring of the retrochalcones may affect the stability of trans-form. Therefore, further studies will be necessary to evaluate the mechanism of trans-cis isomerization. Also the thermodynamics of retrochalcone isomerization should be considered in biological evaluation

3. Conclusions

Fungal transformation of the four licochalcones 1–4 by A. niger and M. hiemalis resulted in the formation of ten different metabolites 5–14, including hydrogenated, dihydroxylated, expoxidized and glucosylated metabolites. Of them, the metabolites 5, 6, 8, 9, 10, and 12 were confirmed to be in (E)-form; whereas the metabolites 7, 11, 13, and 14 were confirmed as a mixture of (E)- and (Z)-isomers which were present in the integral ratios ranging from 1:0.3 to 1: 0.4 as evidenced by their ¹H-NMR spectra. Although further investigations such as thermodynamic stability sutdies on these isomers may be needed, it was concluded that 7, 11, 13, and 14 are present as interconverting mixtures of the major (E)- and minor (Z)-isomers.

Prenyl groups could affect the generation of the metabolites produced by A. niger and M. hiemalis (Figure 2). When prenyl groups were introduced in different positions of the retrochalcone backbone, it was observed that dihydroxylation or expoxidation of the corresponding substrates was preferentially performed on the prenyl side chains by A. niger. The position of glucosylation by M. hiemalis was preferred to be at the hydroxyl groups nearest to the prenyl groups. It was deduced that the flexibility of the prenyl substituents might have an effect on the regio-selectivity during the microbial transformation.

Microbial transformation is regarded as an efficient tool for the structural modification of bioactive natural and synthetic compounds [31]. Microbial transforamtion of the licochalcones can provide alternative approach to prepare the targeted licochaclcone derivatives under mild conditions by utilizing microbial system as biocatalysts.

4. Materials and Methods

4.1. General Experimental Procedures

Optical rotations were recorded with a 343 Plus polarimeter (Perkin Elmer, Waltham, MA, USA). UV spectra were recorded on a V-530 spectrophotometer (Jasco, Tokyo, Japan). IR spectra were obtained on a Jasco FT/IR 300-E spectrometer, and CD spectra were recorded on a Jasco J-815 CD spectrometer. NMR experiments were recorded using an Avance III 400 spectrometer (Bruker, Billerica, MA, USA) with TMS as the internal standard. HRESIMS were determined on Waters Synapt G2 QTOF (Waters, Milford, MA, USA). TLC was carried out on Merck silica gel F₂₅₄-precoated glass plates and RP-18 F₂₅₄s plates. Chromatography was performed on a Waters 1525 Binary HPLC pump connected to a 996 Photodiode Array (PDA) detector using Isco Allsphere ODS-2 (10 μm, 10 × 250 mm) and Nova-Pak C18 (4 μm, 3.9 × 150 mm) columns.

4.2. Preparation of Substrates

Licochalcones B (1) and D (3) were synthesized through acid-mediated Claisen-Schmidt condensation using 4-hydroxyacetophenone as a starting material [32]. And starting from 2,4-dihydroxybenzaldehyde, licochalcone C (2) and its regio-isomer licochalcone H (4) were synthesized by acid-mediated Claisen-Schmidt condensation [24]. The ¹H-NMR data of licochlacones B (1), C (2), D (3), and H (4) agreed with data in the literature data [24,32]. The purity of each substrate was determined to be above 95% by HPLC analysis.

Licochalcone B (1): ¹H NMR (DMSO-d₆, 400 MHz, δ in ppm, J in Hz) δ 8.02 (2H, d, J = 8.7, H-2′,6′), 7.86 (1H, d, J = 15.9, H-β), 7.66 (1H, d, J = 15.9, H-α), 7.33 (1H, d, J = 8.7, H-6), 6.89 (2H, d, J = 8.7, H-3′,5′), 6.64 (1H, d, J = 8.7, H-5), 3.78 (3H, s, 2-OMe).

Licochalcone C (2): ¹H NMR (CDCl₃, 400 MHz, δ in ppm, J in Hz) δ 8.02 (1H, d, J = 15.7, H-β), 7.99 (2H, d, J = 8.8, H-2′,6′), 7.51 (1H, d, J = 15.7, H-α), 7.47 (1H, d, J = 8.8, H-6), 6.96 (2H, d, J = 8.8, H-3′,5′), 6.70 (1H, d, J = 8.8, H-5), 5.23 (1H, m, H-2″), 3.74 (3H, s, 2-OMe), 3.45 (2H, d, J = 6.9, H-1″), 1.82 (3H, s, H-4″), 1.74 (3H, s, H-5″).

Licochalcone D (3): ¹H NMR (CD₃OD, 400 MHz, δ in ppm, J in Hz) δ 7.92 (1H, d, J = 15.7, H-β), 7.82 (1H, d, J = 8.7, H-6′), 7.80 (1H, s, H-2′), 7.60 (1H, d, J = 15.7, H-α), 7.18 (1H, d, J = 8.6, H-6), 6.85 (1H, d, J = 8.7, H-5′), 6.64 (1H, d, J = 8.6, H-5), 5.36 (1H, m, H-2″), 3.85 (3H, s, 2-OMe), 3.34 (2H, d, J = 7.4, H-1″), 1.76 (3H, s, H-4″), 1.74 (3H, s, H-5″).

Licochalcone H (4): ¹H NMR (CD₃OD, 400 MHz, δ in ppm, J in Hz) δ 7.99 (1H, d, J = 15.6, H-β), 7.95 (2H, d, J = 8.8, H-2′,6′), 7.55 (1H, d, J = 15.6, H-α), 7.39 (1H, s, H-6), 6.89 (2H, d, J = 8.8, H-3′,5′), 6.47 (1H, s, H-3), 5.32 (1H, m, H-2″), 3.87 (3H, s, 2-OMe), 3.26 (2H, d, J = 7.3, H-1″), 1.75 (3H, s, H-4″), 1.74 (3H, s, H-5″).

4.3. Microorganisms and Culture Media

All the microorganisms were obtained from the Korean Collection for Type Cultures (KCTC, Jeongeup, Korea) and Korean Culture Center of Microorganisms (KCCM, Seoul, Korea). Eight cultures were used for the preliminary screening process as listed below: Absidia coerulea KCTC 6936, Aspergillus fumigatus 6145, Cunninghamella elegans var. elegans 6992, Mortierella ramanniana var. angulispora 6137, Mucor hiemalis 26779, Penicillium chrysogenum 6933, Trichoderma koningii 6042, and Aspergillus niger KCCM 60332.

All the ingredients for microbial media, including dextrose, peptone, malt extract, yeast extract, and potato dextrose broth were purchased from Becton, Dickinson and Co. (Sparks, MD, USA). Two types of media were used in the fermentation experiments and are listed below: A. coerulea, A. fumigatus, A. niger, M. hiemalis, P. chrysogenum, and T. koningii were cultured on malt medium (malt extract 20 g/L, dextrose 20 g/L, peptone 1 g/L); C. elegans var. elegans and M. ramanniana var. angulispora were cultured on potato dextrose medium (24 g/L).

4.4. Procedure for Microbial Transformation

Microbial metabolism studies were carried out according to the standard two-stage procedure [21]. Briefly, the actively growing microbial cultures were inoculated in 250 mL Erlenmeyer flasks containing 50 mL of a suitable medium, and incubated with gentle agitation (200 rpm) at 25 °C in a temperature-controlled shaking incubator. The DMSO solutions (20 mg/mL, 100 μL) of the substrates (1, 2, 3 or 4) were added to each flask 24 h after inoculation, and further incubated at the same conditions for another 5 days. Sampling and TLC monitoring were performed at an interval of 24 h. UV (254 and 365 nm) and anisaldehyde-sulfuric acid reagent were used for identification of metabolites. Culture controls were carried out as a result of enzymatic activity, but not a consequence of degradation or non-metabolic changes.

Similarly, the preparative-scale fermentations were carried out in 500 mL flasks containing 150 mL of culture medium, and the selected microorganisms were pre-cultured under the culture conditions mentioned above for 24 h to obtain sufficient amounts for biotransformation. After that, 50 mg of 1, 80 mg of 2, 35 mg of 3 or 80 mg of 4 dissolved in DMSO (20 mg/mL) were used for preparative-scale fermentations. The other procedures and culture conditions were same as those of the screening experiments.

4.5. Extraction and Isolation of Metabolites

After incubation, the cultures of 1, 2, 3, or 4 were extracted with equal volume of EtOAc two times, and the organic layer was collected and concentrated. The EtOAc extract of 1 incubated with A. niger was subjected to reversed-phased HPLC with a gradient solvent system of 35% MeOH to 45% MeOH to afford 5 (5.8 mg, t_R = 10.92 min) at a flow rate of 1.0 mL/min. The EtOAc extract of 1 incubated with M. hiemalis was subjected to reversed-phased HPLC with an isocratic solvent system of 34% MeOH to afford 12 (4.0 mg, t_R = 8.79 min) at a flow rate of 1.0 mL/min. The EtOAc extract of 2 incubated with A. niger was subjected to reversed-phased HPLC with a gradient solvent system of 53% MeOH to 69% MeOH to afford 6 (4.6 mg, t_R = 22.17 min) and 7 (5.2 mg, t_R = 30.46 min) at a flow rate of 2.0 mL/min. The EtOAc extract of 2 incubated with M. hiemalis was subjected to reversed-phased HPLC with a gradient solvent system of 65% MeOH to 80% MeOH to afford 13 (5.0 mg, t_R = 12.94 min) at a flow rate of 2.0 mL/min. The EtOAc extract of 3 with incubated with A. niger was subjected to reversed-phased HPLC with a gradient solvent system of 58% MeOH to 65% MeOH to afford 8 (2.5 mg, t_R = 12.62 min) and 9 (3.2 mg, t_R = 20.08 min) at a flow rate of 2.0 mL/min. The EtOAc extract of 4 incubated with A. niger was subjected to reversed-phased HPLC with a gradient solvent system of 60% MeOH to 70% MeOH to afford 10 (4.3 mg, t_R = 14.40 min) and 11 (5.5 mg, t_R = 20.48 min) at a flow rate of 2.0 mL/min. The EtOAc extract of 4 incubated with M. hiemalis was subjected to reversed-phased HPLC with a gradient solvent system of 60% MeOH to 75% MeOH to afford 14 (5.0 mg, t_R = 16.51 min) at a flow rate of 2.0 mL/min.

4.6. Spectroscopic Data of Metabolites

α,β-Dihydrolicochalcone B (5): yellow powder; UV λ_max (MeOH): 208, 276 nm; IR ν_max: 3599, 3178, 1648, 1276, 1069, 798 cm⁻¹; HRESIMS m/z: 311.0896 [M + Na]⁺ (calcd for C₁₆H₁₆O₅Na, 311.0895); ¹H-NMR (CD₃OD, 400 MHz, δ in ppm, J in Hz) δ 7.89 (2H, d, J = 8.7, H-2′,6′), 6.84 (2H, d, J = 8.7, H-3′,5′), 6.52 (1H, d, J = 8.3, H-6) 6.49 (1H, d, J = 8.3, H-5), 3.80 (3H, s, 2-OMe), 3.15 (2H, t, J = 7.4, H-α), 2.87 (2H, t, J = 7.4, H-β); ¹³C-NMR (CD₃OD, 100 MHz): 200.0 (C=O), 162.3 (C-4′), 146.2 (C-2), 144.7 (C-4), 138.1 (C-3), 130.5 (C-2′,6′), 128.6 (C-1′), 125.2 (C-1), 119.1 (C-6), 114.9 (C-3′,5′), 110.5 (C-5), 59.5 (2-OMe), 39.2 (C-α), 25.1 (C-β).

(2″R)-2”,3”-Dihydroxylicochalcone C (6): yellow powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ +3.6° (c 0.10, MeOH); UV λ_max (MeOH): 207, 352 nm; IR ν_max: 3595, 3391, 1642, 1282, 1073, 808 cm⁻¹; HRESIMS m/z: 395.1472 [M + Na]⁺ (calcd for C₂₁H₂₄O₆Na, 395.1471); ¹H-NMR (CD₃OD, 400 MHz, δ in ppm, J in Hz) δ 8.00 (1H, d, J = 15.6, H-β), 7.99 (2H, d, J = 8.8, H-2′,6′), 7.65 (1H, d, J = 8.6, H-6), 7.63 (1H, d, J = 15.6, H-α), 6.90 (2H, d, J = 8.8, H-3′,5′), 6.72 (1H, d, J = 8.6, H-5), 3.80 (3H, s, 2-OMe), 3.65 (1H, dd, J = 10.3, 2.0, H-2″), 3.12 (1H, dd, J = 14.2, 2.0, H-1″a), 2.69 (1H, dd, J = 14.2, 10.3, H-1″b), 1.27 (6H, s, H-4″,5″); ¹³C-NMR (CD₃OD, 100 MHz, δ in ppm) 189.9 (C=O), 162.4 (C-4′), 160.1 (C-4), 160.1 (C-2), 139.6 (C-β), 130.8 (C-2′,6′), 129.8 (C-1′), 126.9 (C-α), 120.9 (C-1), 119.9(C-6), 119.1 (C-3), 115.0 (C-3′,5′), 112.5 (C-5), 78.9 (C-2″), 72.6 (C-3″), 61.9 (2-OMe), 25.9 (C-1″), 24.1 (C-5″), 23.8 (C-4″).

2”,3”-Epoxylicochalcone C (7, a mixture of trans and cis forms): yellow powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ +7.1° (c 0.20, MeOH); UV λ_max (MeOH): 208, 354 nm; IR ν_max: 3629, 3200, 1595, 1220, 1071, 814 cm⁻¹; HRESIMS m/z: 377.1376 [M + Na]⁺ (calcd for C₂₁H₂₂O₅Na, 377.1365).

Trans form of 7: ¹H-NMR (CD₃OD, 400 MHz, δ in ppm, J in Hz) δ 8.01 (1H, d, J = 15.6, H-β), 7.99 (2H, d, J = 8.8, H-2′,6′), 7.66 (1H, d, J = 8.6, H-6), 7.64 (1H, d, J = 15.6, H-α), 6.90 (2H, d, J = 8.8, H-3′,5′), 6.65 (1H, d, J = 8.6, H-5), 3.81 (3H, s, 2-OMe), 3.80 (1H, dd, J = 7.0, 5.0, H-2″), 3.04 (1H, dd, J = 17.1, 5.0, H-1″a), 2.73 (1H, dd, J = 17.1, 7.0, H-1″b), 1.35 (3H, s, H-4″), 1.30 (3H, s, H-5″); ¹³C-NMR (CD₃OD, 100 MHz, δ in ppm) 189.8 (C=O), 162.4 (C-4′), 159.4 (C-2), 156.9 (C-4), 138.9 (C-β), 130.9 (C-2′,6′), 129.8 (C-1′), 126.5 (C-α), 120.0 (C-1), 119.3(C-6), 115.0 (C-3′,5′), 114.2 (C-3), 113.6 (C-5), 77.3 (C-3″), 68.3 (C-2″), 60.8 (2-OMe), 25.8 (C-1″), 24.4 (C-4″), 20.0 (C-5″).

Cis form of 7: ¹H-NMR (CD₃OD, 400 MHz, δ in ppm, J in Hz) δ 7.82 (2H, d, J = 8.8, H-2′,6′), 7.11 (1H, d, J = 12.8, H-β), 7.06 (1H, d, J = 8.7, H-6), 6.76 (2H, d, J = 8.8, H-3′,5′), 6.55 (1H, d, J = 12.8, H-α), 6.36 (1H, d, J = 8.7, H-5), 3.73 (3H, s, 2-OMe), 3.70 (1H, dd, J = 7.2, 5.3, H-2″), 2.92 (1H, dd, J = 17.3, 5.5, H-1″a), 2.59 (1H, dd, J = 17.3, 7.2, H-1″b), 1.28 (3H, s, H-4″), 1.21 (3H, s, H-5″); ¹³C-NMR (CD₃OD, 100 MHz, δ in ppm) 194.7 (C=O), 162.5 (C-4′), 157.5 (C-2), 154.9 (C-4), 134.1 (C-β), 131.3 (C-2′,6′), 129.0 (C-1′), 128.6 (C-6), 125.1 (C-α), 120.6 (C-1), 114.8 (C-3′,5′), 113.6 (C-3), 112.2 (C-5), 76.8 (C-3″), 68.5 (C-2″), 60.1 (2-OMe), 25.8 (C-1″), 24.3 (C-4″), 19.7 (C-5″).

(2″R)-2”,3”-Dihydroxylicochalcone D (8): yellow powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ +5.1° (c 0.15, MeOH); UV λ_max (MeOH): 209, 363 nm; IR ν_max: 3627, 2924, 1591, 1264, 1063, 795 cm⁻¹; HRESIMS m/z: 389.1603 [M + H]⁺ (calcd for C₂₁H₂₅O₇, 389.1600); ¹H-NMR (CD₃OD, 400 MHz, δ in ppm, J in Hz) δ 8.00 (1H, d, J = 15.7, H-β), 7.97 (1H, d, J = 1.7, H-2′), 7.87 (1H, dd, J = 8.1, 1.7, H-6′), 7.67 (1H, d, J = 15.7, H-α), 7.26 (1H, d, J = 8.3, H-6), 6.90 (1H, d, J = 8.1, H-5′), 6.66 (1H, d, J = 8.3, H-5), 3.85 (3H, s, 2-OMe), 3.66 (1H, dd, J = 10.4, 1.3, H-2″), 3.13 (1H, dd, J = 13.8, 1.3, H-1″a), 2.60 (1H, dd, J = 13.8, 10.4, H-1″b), 1.26 (6H, s, H-4″,5″); ¹³C-NMR (CD₃OD, 100 MHz, δ in ppm) 190.2 (C=O), 160.7 (C-4′), 149.4 (C-4), 148.5 (C-2), 139.4 (C-β), 138.3 (C-3), 132.5 (C-2′), 129.8 (C-1′), 128.7 (C-6′), 126.9 (C-3′), 120.1 (C-1), 119.2 (C-α), 118.7 (C-6), 114.8 (C-5′), 111.3 (C-5), 78.1 (C-2″), 72.5 (C-3″), 60.4 (2-OMe), 32.5 (C-1″), 24.3 (C-5″), 23.7 (C-4″).

2”,3”-Epoxylicochalcone D (9): yellow powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ −2.4° (c 0.15, MeOH); UV λ_max (MeOH): 209, 363 nm; IR ν_max: 3625, 2924, 1575, 1258, 1059, 770 cm⁻¹; HRESIMS m/z: 371.1497 [M + H]⁺ (calcd for C₂₁H₂₃O₆, 371.1495); ¹H-NMR (CD₃OD, 400 MHz, δ in ppm, J in Hz) δ 7.89 (1H, d, J = 15.6, H-β), 7.87 (1H, d, J = 2.2, H-2′), 7.84 (1H, dd, J = 8.3, 2.2, H-6′), 7.64 (1H, d, J = 15.6, H-α), 7.24 (1H, d, J = 8.6, H-6), 6.87 (1H, d, J = 8.3, H-5′), 6.66 (1H, d, J = 8.6, H-5), 3.85 (3H, s, 2-OMe), 3.82 (1H, dd, J = 7.0, 5.0, H-2″), 3.11 (1H, dd, J = 16.8, 5.0, H-1″a), 2.82 (1H, dd, J = 16.8, 7.0, H-1″b), 1.36 (3H, s, H-4″), 1.31 (3H, s, H-5″); ¹³C-NMR (CD₃OD, 100 MHz, δ in ppm) 190.0 (C=O), 157.8 (C-4′), 149.5 (C-4), 148.6 (C-2), 139.8 (C-β), 138.3 (C-3), 131.1 (C-2′), 130.6 (C-1′), 128.3 (C-6′), 120.1 (C-3′), 120.0 (C-1), 119.1 (C-α), 118.9 (C-6), 116.8 (C-5′), 111.4 (C-5), 78.0 (C-3″), 68.6 (C-2″), 60.4 (2-OMe), 30.6 (C-1″), 24.5 (C-4″), 20.2 (C-5″),.

(2″R)-2”,3”-Dihydroxylicochalcone H (10): yellow powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ +2.2° (c 0.20, MeOH); UV λ_max (MeOH): 208, 379 nm; IR ν_max: 3422, 2930, 1647, 1290, 1063, 836 cm⁻¹; HRESIMS m/z: 373.1652 [M + H]⁺ (calcd for C₂₁H₂₅O₆, 373.1651); ¹H-NMR (CD₃OD, 400 MHz, δ in ppm, J in Hz) δ 8.06 (1H, d, J = 15.6, H-β), 7.99 (2H, d, J = 8.8, H-2′,6′), 7.63 (1H, d, J = 15.6, H-α), 7.58 (1H, s, H-6), 6.90 (2H, d, J = 8.8, H-3′,5′), 6.49 (1H, s, H-3), 3.87 (3H, s, 2-OMe), 3.61 (1H, dd, J = 10.4, 1.7, H-2″), 2.99 (1H, dd, J = 14.2, 1.7, H-1″a), 2.52 (1H, dd, J = 14.2, 10.4, H-1″b), 1.26 (6H, s, H-4″,5″); ¹³C-NMR (CD₃OD, 100 MHz, δ in ppm) 190.2 (C=O), 162.2 (C-4′), 159.9 (C-4), 159.3 (C-2), 140.0 (C-β), 131.6 (C-6), 130.7 (C-2′,6′), 130.1 (C-1′), 119.4 (C-5), 117.8 (C-α), 115.1 (C-1), 114.9 (C-3′,5′), 98.6 (C-3), 78.5 (C-2″), 72.5 (C-3″), 54.8 (2-OMe), 31.8 (C-1″), 24.3 (C-4″), 23.7 (C-5″).

2”,3”-Epoxylicochalcone H (11): yellow powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ +1.5° (c 0.14, MeOH); UV λ_max (MeOH): 208, 292, 371 nm; IR ν_max: 3680, 2926, 1593, 1284, 1057, 840 cm⁻¹; HRESIMS m/z: 355.1544 [M + H]⁺ (calcd for C₂₁H₂₃O₅, 355.1545);

Trans form of 11: ¹H-NMR (DMSO-d₆, 400 MHz, δ in ppm, J in Hz) δ 8.03 (2H, d, J = 8.6, H-2′,6′), 7.93 (1H, d, J = 15.6, H-β), 7.72 (1H, s, H-6), 7.70 (1H, d, J = 15.6, H-α), 6.90 (2H, d, J = 8.6, H-3′,5′), 6.44 (1H, s, H-3), 3.82 (3H, s, 2-OMe), 3.67 (1H, dd, J = 7.5, 5.2, H-2″), 2.92 (1H, dd, J = 16.2, 5.2, H-1″a), 2.60 (1H, dd, J = 16.2, 7.5, H-1″b), 1.30 (3H, s, H-4″), 1.21 (3H, s, H-5″); ¹³C-NMR (DMSO-d₆, 100 MHz, δ in ppm) 187.6 (C=O), 162.3 (C-4′), 158.6 (C-2), 156.9 (C-4), 138.0 (C-β), 131.3 (C-2′,6′), 130.3 (C-6), 130.0 (C-1′), 119.0 (C-α), 116.1 (C-1), 115.8 (C-3′,5′), 113.3 (C-5), 100.8 (C-3), 78.5 (C-3″), 68.4 (C-2″), 56.2 (2-OMe), 30.6 (C-1″), 26.1 (C-4″), 21.3 (C-5″);

Cis form of 11: ¹H-NMR (DMSO-d₆, 400 MHz, δ in ppm, J in Hz) δ 7.77 (2H, d, J = 8.6, H-2′,6′), 7.14 (1H, s, H-6), 7.00 (1H, d, J = 12.8, H-β), 6.81 (2H, d, J = 8.6, H-3′,5′), 6.58 (1H, d, J = 12.8, H-α), 6.30 (1H, s, H-3), 3.65 (3H, s, 2-OMe), 3.56 (1H, dd, J = 7.2, 5.2, H-2″), 2.67 (1H, dd, J = 16.0, 5.2, H-1″a), 2.39 (1H, dd, J = 16.0, 7.7, H-1″b), 1.24 (3H, s, H-4″), 1.12 (3H, s, H-5″); ¹³C-NMR (DMSO-d₆, 100 MHz, δ in ppm) 192.3 (C=O), 162.3 (C-4′), 157.1 (C-2), 155.1 (C-4), 133.9 (C-β), 131.9 (C-6), 131.4 (C-2′,6′), 129.5 (C-1′), 124.8 (C-α), 116.9 (C-1), 115.6 (C-3′,5′), 111.7 (C-5), 99.5 (C-3), 78.0 (C-3″), 68.4 (C-2″), 55.8 (2-OMe), 30.6 (C-1″), 26.0 (C-4″), 20.9 (C-5″).

Licochalcone B 3-O-β-d-glucopyranoside (12): yellow powder; UV λ_max (MeOH): 208, 288, 338 nm; IR ν_max: 3568, 3448, 1641, 1000, 786 cm⁻¹; HRESIMS m/z: 449.1449 [M + H]⁺ (calcd for C₂₂H₂₅O₁₀, 449.1448); ¹H- NMR (DMSO-d₆, 400 MHz, δ in ppm, J in Hz) δ 8.00 (2H, d, J = 8.8, H-2′,6′), 7.82 (1H, d, J = 15.8, H-β), 7.66 (1H, d, J = 15.8, H-α), 7.63 (1H, d, J = 8.8, H-6), 6.88 (2H, d, J = 8.8, H-3′,5′), 6.66 (1H, d, J = 8.8, H-5), 4.74 (1H, d, J = 7.7, H-1″), 3.89 (3H, s, 2-OMe), 3.65 (1H, dd, J = 11.7, 1.7, H-6″a), 3.49 (1H, m, H-6″b), 3.34 (1H, m, H-2″), 3.26 (1H, m, H-5″), 3.20 (1H, m, H-4″), 3.19 (1H, m, H-3″). ¹³C-NMR (CD3OD, 400 MHz): 187.7 (C=O), 162.4 (C-4′), 153.9 (C-2), 153.9 (C-4), 138.7 (C-3), 138.1 (C-β), 131.4 (C-2′,6′), 129.9 (C-1′), 124.6 (C-6), 119.9 (C-α), 119.8 (C-1), 115.8 (C-3′,5′), 113.4 (C-5), 105.2 (C-1″), 77.7 (C-3″), 76.7 (C-5″), 74.4 (C-2″), 70.1 (C-4″), 62.6 (2-OMe), 61.2 (C-6″).

Licochalcone C 4-O-β-d-glucopyranoside (13): yellow powder; UV λ_max (MeOH): 208, 343 nm; IR ν_max: 3655, 3328, 1590, 1224, 1072, 838 cm⁻¹; HRESIMS m/z: 501.2123 [M + H]⁺ (calcd for C₂₇H₃₃O₉, 501.2125).

Trans form of 13: ¹H-NMR (CD₃OD, 400 MHz, δ in ppm, J in Hz) δ 8.02 (2H, d, J = 8.8, H-2′,6′), 7.93 (1H, d, J = 15.7, H-β), 7.72 (1H, d, J = 8.8, H-6), 7.63 (1H, d, J = 15.7, H-α), 6.91 (2H, d, J = 8.8, H-3′,5′), 7.05 (1H, d, J = 8.8, H-5), 5.26 (1H, m, H-2″), 5.02 (1H, d, J = 7.2, H-1’’’), 3.91 (1H, dd, J = 11.9, 1.7, H-6’’’a), 3.75 (3H, s, 2-OMe), 3.72 (1H, m, H-6’’’b), 3.52-3.46 (1H, H-2’’’), 3.50-3.44 (2H, m, H-3’’’,5’’’), 3.49-3.42 (1H, m, H-4’’’), 3.45 (2H, overlapped, H-1″), 1.80 (3H, s, H-4″), 1.67 (3H, s, H-5″); ¹³C-NMR (CD₃OD, 100 MHz, δ in ppm) 189.9 (C=O), 162.5 (C-4′), 159.0 (C-2), 158.6 (C-4), 139.0 (C-β), 131.0 (C-2′,6′), 130.9 (C-3″), 129.6 (C-1′), 126.4 (C-6), 125.0 (C-3), 122.8 (C-2″), 122.4 (C-1), 120.5 (C-α), 115.2 (C-3′,5′), 111.1 (C-5), 100.5 (C-1’’’), 76.8 (C-3’’’), 76.6 (C-5’’’), 73.6 (C-2’’’), 69.9 (C-4’’’), 61.8 (2-OMe), 61.1 (C-6’’’), 24.6 (C-5″), 22.6 (C-1″), 16.8 (C-4″);

Cis form of 13: ¹H-NMR (CD₃OD, 400 MHz, δ in ppm, J in Hz) δ 7.83 (2H, d, J = 8.8, H-2′,6′), 7.12 (1H, d, J = 12.7, H-β), 7.10 (1H, d, J = 8.8, H-6), 6.78 (2H, d, J = 8.8, H-3′,5′), 6.77 (1H, d, J = 8.8, H-5), 6.58 (1H, d, J = 12.7, H-α), 5.14 (1H, m, H-2″), 4.86 (1H, overlapped, H-1’’’), 3.85 (1H, m, H-6’’’a), 3.72 (1H, m, H-6’’’b), 3.70 (3H, s, 2-OMe), 3.52-3.46 (1H, H-2’’’), 3.50-3.44 (2H, m, H-3’’’,5’’’), 3.49-3.42 (1H, m, H-4’’’), 3.48 (2H, overlapped, H-1″), 1.75 (3H, s, H-4″), 1.64 (3H, s, H-5″); ¹³C-NMR (CD₃OD, 100 MHz, δ in ppm) 194.9 (C=O), 162.6 (C-4′), 157.2 (C-2), 156.9 (C-4), 133.8 (C-β), 131.5 (C-2′,6′), 130.5 (C-3″), 128.8 (C-1′), 128.2 (C-6), 126.1 (C-α), 124.4 (C-3), 123.3 (C-1), 123.0 (C-2″), 114.9 (C-3′,5′), 110.1 (C-5), 100.7 (C-1’’’), 76.9 (C-3’’’), 76.6 (C-5’’’), 73.6 (C-2’’’), 69.9 (C-4’’’), 61.1 (C-6’’’), 60.9 (2-OMe), 24.6 (C-5″), 22.5 (C-1″), 16.8 (C-4″).

Licochalcone H 4-O-β-d-glucopyranoside (14): yellow powder; UV λ_max (MeOH): 207, 287, 358 nm; IR ν_max: 3723, 3160, 1602, 1227, 1064, 854 cm⁻¹; HRESIMS m/z: 501.2122 [M + H]⁺ (calcd for C₂₇H₃₃O₉, 501.2125).

Trans form of 14: ¹H-NMR (CD₃OD, 400 MHz, δ in ppm, J in Hz) δ 7.99 (1H, d, J = 16.2, H-β), 7.95 (2H, d, J = 8.8, H-2′,6′), 7.63 (1H, d, J = 16.2, H-α), 7.45 (1H, s, H-6), 6.90 (2H, d, J = 8.8, H-3′,5′), 6.88 (1H, s, H-3), 5.34 (1H, m, H-2″), 5.00 (1H, d, J = 7.3, H-1’’’), 3.92 (1H, overlapped, H-6’’’a), 3.92 (3H, s, 2-OMe), 3.66 (1H, m, H-6’’’b), 3.52-3.46 (1H, m, H-2’’’), 3.49-3.35 (1H, m, H-3’’’, 5’’’), 3.35-3.33 (1H, m, H-4’’’), 3.34 (2H, overlapped, H-1″), 1.74 (6H, s, H-4″, 5″); ¹³C-NMR (CD₃OD, 100 MHz, δ in ppm) 190.2 (C=O), 162.3 (C-4′), 158.7 (C-2), 158.6 (C-4), 139.6 (C-β), 131.8 (C-3″), 130.8 (C-2′,6′), 129.9 (C-1′), 129.6 (C-6), 123.2 (C-5), 122.7 (C-2″), 119.5 (C-α), 117.3 (C-1), 115.0 (C-3′,5′), 100.9 (C-1’’’), 99.0 (C-3), 77.2 (C-3’’’), 77.0 (C-5’’’), 73.6 (C-2’’’), 70.3 (C-4’’’), 61.4 (C-6’’’), 55.0 (2-OMe), 27.3 (C-1″), 24.6 (C-4″), 16.6 (C-5″);

Cis form of 14: ¹H-NMR (CD₃OD, 400 MHz, δ in ppm, J in Hz) δ 7.80 (2H, d, J = 8.7, H-2′,6′), 7.11 (1H, d, J = 12.7, H-β), 6.88 (1H, s, H-6), 6.76 (1H, s, H-3), 6.73 (2H, d, J = 8.7, H-3′,5′), 6.36 (1H, d, J = 12.7, H-α), 5.40 (1H, m, H-2″), 4.85 (1H, overlapped, H-1’’’), 3.92 (1H, overlapped, H-6’’’a), 3.76 (3H, s, 2-OMe), 3.66 (1H, m, H-6’’’b), 3.52-3.46 (1H, m, H-2’’’), 3.49-3.35 (1H, m, H-3’’’, 5’’’), 3.35-3.33 (1H, m, H-4’’’), 3.13 (2H, overlapped, H-1″), 1.63 (3H, s, H-4″), 1.55 (3H, s, H-5″); ¹³C-NMR (CD₃OD, 100 MHz, δ in ppm) 196.0 (C=O), 162.5 (C-4′), 156.7 (C-4), 156.4 (C-2), 133.0 (C-β), 131.9 (C-3″), 131.6 (C-2′,6′), 130.6 (C-6), 128.6 (C-1′), 124.9 (C-α), 122.2 (C-2″), 122.0 (C-5), 118.2 (C-1), 114.8 (C-3′,5′), 101.2 (C-1’’’), 98.8 (C-3), 77.1 (C-3’’’), 76.9 (C-5’’’), 73.6 (C-2’’’), 70.2 (C-4’’’), 61.3 (C-6’’’), 54.7 (2-OMe), 26.6 (C-1″), 24.5 (C-4″), 16.4 (C-5″).

4.7. Acid Hydrolysis

Each solution of metabolites 12–14 (each 1 mg) in 2N HCl was heated for 2 h. After cooling, the reaction mixture was neutralized and partitioned with EtOAc. The organic and aqueous extracts were analyzed by HPLC and TLC, respectively. The monosaccharide of each metabolite was confirmed to be d-glucose by comparing its R_f value with that of authentic d-glucose on TLC plate and the aglycone of each metabolite was confirmed by comparing the retention times with those of aglycones (1, 2, 4) on HPLC.