Microbial conjugation studies of licochalcones and xanthohumol

Article abstract of DOI:10.3390/ijms22136893

Microbial conjugation studies of licochalcones (1–4) and xanthohumol (5) were performed by using the fungi Mucor hiemalis and Absidia coerulea. As a result, one new glucosylated metabolite was produced by M. hiemalis whereas four new and three known sulfated metabolites were obtained by transformation with A. coerulea. Chemical structures of all the metabolites were elucidated on the basis of 1D-, 2D-NMR and mass spectroscopic data analyses. These results could contribute to a better understanding of the metabolic fates of licochalcones and xanthohumol in mammalian systems. Although licochalcone A 4′-sulfate (7) showed less cytotoxic activity against human cancer cell lines compared to its substrate licochalcone A, its activity was fairly retained with the IC50 values in the range of 27.35–43.07 μM.

Full text of DOI:10.3390/ijms22136893

International Journal of
Molecular Sciences
Article
Microbial Conjugation Studies of Licochalcones
and Xanthohumol
Fubo Han, Yina Xiao and Ik-Soo Lee *
College of Pharmacy, Chonnam National University, Gwangju 61186, Korea; hanfubo0306@gmail.com (F.H.);
yogurtxiao@163.com (Y.X.)
* Correspondence: islee@chonnam.ac.kr; Tel.: +82-62-530-2932
Abstract: Microbial conjugation studies of licochalcones (1–4) and xanthohumol (5) were performed
by using the fungi Mucor hiemalis and Absidia coerulea. As a result, one new glucosylated metabolite
was produced by M. hiemalis whereas four new and three known sulfated metabolites were obtained
by transformation with A. coerulea. Chemical structures of all the metabolites were elucidated on
the basis of 1D-, 2D-NMR and mass spectroscopic data analyses. These results could contribute
to a better understanding of the metabolic fates of licochalcones and xanthohumol in mammalian
0
systems. Although licochalcone A 4 -sulfate (
7
) showed less cytotoxic activity against human cancer
cell lines compared to its substrate licochalcone A, its activity was fairly retained with the IC50 values
in the range of 27.35–43.07 µM.
Keywords: licochalcones; xanthohumol; sulfation; microbial conjugation
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
1. Introduction
Citation: Han, F.; Xiao, Y.; Lee, I.-S.
Microbial Conjugation Studies of
Licochalcones and Xanthohumol. Int.
J. Mol. Sci. 2021, 22, 6893. https://
doi.org/10.3390/ijms22136893
The licorice root, known as “Radix Glycyrrhizae”, has been used in traditional Chi-
nese medicines for centuries to treat respiratory infections, gastritis, tremors, and peptic
ulcers [
ing flavonoids, triterpene saponins, and alkaloids which are responsible for antidiabetic,
neuroprotective, anti-inflammatory, and other bioactive effects [ ]. Licochalcones, the sig-
nificant components of licorice flavonoids, including licochalcone A (LCA, ), licochalcone
(LCB, 2), licochalcone C (LCC, ), and so on, were reported to exhibit a variety of bioac-
tivities, and can be used in food and cosmetic industries [ ]. In recent years, licochalcones
have attracted more attention from research communities due to their anticancer potential
against different kinds of cancers, such as breast, lung, and gastric cancers [ ]. Although
1,2]. At present, there are hundreds of compounds isolated from licorice, includ-
2
Academic Editors:
Katerˇina Valentová and
Vladimír Krˇen
1
B
3
2
Received: 27 May 2021
Accepted: 24 June 2021
Published: 26 June 2021
2,3
the pharmacological activities of licochalcones have been extensively investigated, the
metabolic pathway of licochalcones in mammals remains largely unknown. It has been
reported that LCA can undergo both phase I and phase II metabolic processes in vitro
,
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
with the formation of oxygenated and glucuronidated metabolites [ ], and the in vivo
4,5
metabolism studies of LCA led to the determination of glucuronidated, N-acetyl-l-cysteine
conjugated, and sulfated metabolites [5,6].
In comparison to licochalcones, metabolism of xanthohumol (XN, 5) (Figure 1), a
prenylated chalcone from hops, was quite extensively examined and both in vitro and
in vivo studies have been conducted. The studies using human and rat liver microsomes re-
sulted in the identification of hydroxylated, cyclized, and glucuronidated metabolites [
In addition, sulfate conjugated metabolites of XN were observed after using sulfotrans-
ferases and detected in rats after administration of hop extract [10 11]. However, it seems
impossible to identify the conjugation position of the sulfate group by using the LC/MS
analysis method without a respective reference compound. It was reported that the phase
II enzymes UDP-glucuronosyltransferases and sulfotransferases were not only found in
liver but also in other tissues [12], suggesting that LCA and XN could be also metabolized
by other organs and tissues.
7–9].
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
,
Int. J. Mol. Sci. 2021, 22, 6893. https://doi.org/10.3390/ijms22136893
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Figure 1. Chemical structures of licochalcones A–C (1–3), H (4) and xanthohumol (5).
LCA and XN undergo complex biotransformation processes in mammalian systems
and they are mainly metabolized by phase II enzymes, although their interactions with
cytochrome P450 enzymes cannot be excluded. Polyphenolic substances are easy targets
for conjugation reactions and these are indeed the major biotransformation pathways of
licochalcones [13]. Therefore, it is more relevant to investigate the effects of metabolically
conjugated derivatives on human health rather than their parent compounds [14]. Gen-
erally, sulfate conjugation is considered as a detoxification pathway for endogenous and
exogenous phenolic compounds, as the conjugated derivatives are polar and water-soluble,
which facilitates their elimination from the body [15
that detoxification is not the only function of sulfation, and the biological activities of
compounds after sulfation can be retained, lowered, abolished, or even increased [14 17 18].
For instance, mangostin 3-sulfate exhibited stronger anti-mycobacterial activity against My-
cobacterium tuberculosis than -mangostin [19]. However, compared to the glucuronidated
,16]. Nevertheless, it is clear today
,
,
α
and methylated conjugates, the biological properties and cellular activities of sulfated
conjugates were least studied [14]. It is important to expand the structural diversity of
sulfated derivatives via chemical or biological methods for biological studies.
Microbial transformation studies are regarded as one of the useful in vitro models to
mimic and predict the mammalian metabolism process of xenobiotic compounds [20]. In
addition, it allows the selective conversion of compounds into derivatives, which are diffi-
cult to obtain in chemical synthesis under mild conditions [21]. Such processes can provide
sufficient amounts of metabolites for structure elucidation and pharmacological activity
evaluation [21]. Khan and colleagues have reported that Absidia glauca [22], Aspergillus
alliaceus [23], Beauveria bassiana [24], Chaetomium cochlioides, Cunninghamella blakesleeana [23],
Cunninghamella echinulata [25], Mortierella zonata [23], and Mucor ramannianus [26] could
transform the flavones and flavanones to their corresponding sulfated metabolites. Thus,
biotransformation conducted by microorganisms could be used as a simple and efficient
method for obtaining not only phase I but also phase II metabolites of chalcones [20].
To the best of our knowledge, no metabolism studies of licochalcones B–D and H
were performed so far, except the study previously carried out in this lab [27]. Thus, in
order to identify the conjugated metabolites of licochalcones, further studies need to be
performed. In the present study, microbial transformation of licochalcones A–C, H and
xanthohumol (1–5) was carried out with Mucor hiemalis and Absidia coerulea, affording one
new glucosylated and four new sulfated metabolites, respectively. The conjugated metabo-
lites could be used as authentic reference standards in metabolic studies of licochalcones
and xanthohumol.

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2. Results and Discussion
2.1. Microbial Transformation of Licochalcone A and Xanthohumol by M. hiemalis
During our previous study, it was observed that licochalcones B, C, H could be
O-glucosylated by using the fungus M. hiemalis [27]. Here, the same process was per-
formed with LCA and XN, which led to the isolation of a new compound licochalcone
A 4-O-β-D-glucopyranoside (6 β-
) together with a known compound xanthohumol 40-O-
D-glucopyranoside [28]. Yin and colleagues discovered a phenolic glycosyltransferase
MhGT1 from M. hiemalis and applied the enzymatic approach to obtain a series of glycosy-
lated phenolic compounds [29]. Likewise, the O-glucosylation process observed here was
speculated to be catalyzed by the enzyme glycosyltransferase formed by M. hiemalis.
Compound
quasi-molecular ion at m/z 523.1943 [M+Na]+ (calcd. for C27H32O9Na, 523.1944), suggesting
the molecular formula of as C27H32O9, which was one glucose unit higher than that of
The presence of sugar moiety was further confirmed by comparison of its NMR spectra
with those of , which showed an anomeric proton signal at δH 5.01 (1H, d, J = 7.0 Hz) in
6 was isolated as an orange amorphous powder. HRESIMS of 6 exhibited a
6
1.
1
1H-NMR spectrum and a hexose carbon signals at δC (100.5, 73.8, 77.8, 70.7, 78, and 61.4) in
13C-NMR. The significant downfield-shifted proton signal of H-3 in ring B indicated that
the glucose moiety was attached to 4-OH. HMBC correlation from the anomeric proton
signal at δH 5.01 (H-1000) to the carbon signal at δC 159.4 (C-4) confirmed the assignment
of glycosylation at C-4 (Figure 2). The resonances of the glucose moiety were assigned by
HSQC and HMBC experiments (See Supplementary Materials). TLC comparison with the
authentic sample after acid hydrolysis of
be D-form [28
30]. The aglycone part of the 1H- and 13C-NMR spectra of
sets of signals in the integral ratio of around 1:1.3. Based on the two pairs of H-
proton signals at δH 7.67 (1H, d, J = 15.7 Hz) and 7.92 (1H, d, J = 15.7 Hz), together with δH
6.44 (1H, d, J = 12.8 Hz) and 7.03 (1H, d, J = 12.8 Hz), was clearly assigned as a mixture
6
led to the elucidation of the glucose moiety to
revealed two
and H-
,
6
α
β
6
of trans- and cis-isomers. Based on the above data and extensive 2D NMR experiments,
structure of the compound 6 was assigned as licochalcone A 4-O-β-D-glucopyranoside.
Figure 2. Microbial transformation of
by arrows.
1
by M. hiemalis. Selected HMBC correlations (1H
→
13C) of metabolite
6 are indicated
2.2. Microbial Transformation of Licochalcones A, B, C, H, and Xanthohumol by A. coerulea
Screening of licochalcones and XN ( ) with several microorganisms showed that
1–5
only A. coerulea was able to produce the highly polar metabolites, more polar than the
metabolites transformed by M. hiemalis. During the screening procedures, it was also
observed that by using more sulfate-containing media, the yield of sulfate-conjugated
metabolites could be increased. Thus, instead of using the original incubation medium
suggested by KCTC, the scale-up fermentation process was performed by using a modified
Czapek Dox media (dextrose 10 g/L, sodium nitrate 2 g/L, dipotassium phosphate 1 g/L,
magnesium sulfate 0.5 g/L, potassium chloride 0.5 g/L, ferrous sulfate 0.02 g/L). Isolation
of the metabolites from large-scale fermentations was processed successfully by using
EtOAc as extraction solvent.
Metabolite
maximum absorption bands at 266 and 387 nm, similar to those of substrate
HRESIMS spectra of
displayed a molecular ion peak at m/z 417.1024 [M-H]− and a
7
was isolated as a yellow amorphous powder. The UV spectrum showed
1
. The
7

Int. J. Mol. Sci. 2021, 22, 6893
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fragment ion peak at m/z 337.1449 [M-H-80]−, suggesting the presence of a sulfate group.
In addition, the isotopic mass peak at m/z 419.1087 [M-H+2]− indicated the presence of a
sulfur atom in the compound. The IR spectrum of
7 showed the presence of two strong
bands corresponding to the S=O (1251 cm−1) and C-O-S (1050 cm−1), supporting the
presence of a sulfate group [31]. The presence of the sulfate group was further supported
by the formation of white precipitate after treating the aqueous layer of acid-hydrolyzed
1
7
1
with BaCl2 [31]. Comparison of the H- and 13C-NMR spectroscopic data of
7
with
revealed the resonance signals being almost identical in both compounds, except for
the downfield shift of H-30/50, C-10, C-30/50 (0.44, 4.0, and 4.7 ppm, respectively) and
the upfield shift of C-40 (4.4 ppm), implying that the sulfate moiety was conjugated with
40-OH. These shifts were consistent with naringenin 40-sulfate, a transformed product of
naringenin, because the carbon directly attached to the sulfate ester and the carbons in the
meta-position are upfield-shifted, and the protons and carbons in ortho- and para-positions
to the sulfate group are downfield-shifted [18,
32]. The sulfate position at C-40 was further
confirmed by HMBC correlations of H-30/50 with C-10 and C-40 (Figure 3). Therefore, the
structure of 7 was identified as licochalcone A 40-sulfate.
Figure 3. Microbial transformation of
by arrows.
1
by A. coerulea. Selected HMBC correlations (1H
→
13C) of metabolite
7 are indicated
Metabolite
8 was obtained as a mixture with 7 8, the
. In the 1H-NMR spectrum of
significant downfield-shifted signal at δH 7.51 (H-3) was observed, indicating that the
sulfate group was attached to 4-OH. Two sets of proton resonance signals were observed
for
8, which is similar to the glucosylated metabolite
6. Thus, the structure of
8
was
identified as trans- and cis-isomers of licochalcone A 4-sulfate.
Metabolite
9 was obtained as a yellow solid. The HRESIMS spectra of 9 exhibited
a molecu−lar ion peak at m/z 365.0336 [M-H]− and a fragment ion peak at m/z 285.0767
[M-H-80] , suggesting the presence of a sulfate group. The 1H-NMR data of
9 were similar
to
2
, except for the H-6 signal, which was downfield-shifted by 0.33 ppm. Comparison
of 13C-NMR data of
9
and substrate showed that the C-3 signal was upfield-shifted
2
by 5.7 ppm, and the signals of C-2, C-4, and C-6 were downfield-shifted by 4.1, 3.2, and
5.0 ppm, respectively [33]. All of these suggested that the sulfate group was attached to
3-OH, and this was further confirmed by the HMBC correlations between H-5 and C-1/3
(Figure 4). Thus, the structure of 9 was identified as licochalcone B 3-sulfate.
Metabolite 10 was obtained as a mixture with 9
. In the 1H-NMR spectrum of 10, the
significant downfield-shifted signal at δH 7.22 (H-5) was observed, indicating that the
sulfate group was attached to 4-OH. This was supported by the deshielding of C-3 and
C-5 carbons, which are ortho to the sulfate site, compared to those of substrate
2 in the

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13C NMR of 10 by 3.0 and 5.2 ppm, respectively. Furthermore, the para carbon at C-1
in 10 was deshielded by 4.0 ppm, while the ipso carbon at C-4 position was shielded by
7.7 ppm [33]. Based on the above analysis, the structure of 10 was identified as licochalcone
B 4-sulfate. The resonances of 10 were completely assigned by HSQC and HMBC spectra
(See Supplementary Materials).
Figure 4. Microbial transformation of
indicated by arrows.
2
by A. coerulea. Selected HMBC correlations (1H
→
13C) of metabolites
9 and 10 are
Metabolite 11 was obtained as a yellow solid. The HRESIMS spectra of 11 exhibited
a molecular ion peak at m/z 417.1010 [M-H]− and a fragment ion peak at m/z 337.1438
[M-H-80]−, suggesting the presence of a sulfate group. Two sets of signals in the integral
ratio of around 1:1.1 were observed in the 1H- and 13C-NMR spectra of 11. It was assigned
as a mixture of trans- and cis-isomers according to the two pairs of H-α and H-β proton
signals at δH 7.73 (1H, d, J = 15.9 Hz) and 8.01 (1H, d, J = 15.9 Hz), together with δH 6.65
(1H, d, J = 12.9 Hz) and 7.10 (1H, d, J = 12.9 Hz). Comparison of the 1H-NMR spectrum
of LCC sulfate 11 with the parent compound 3 clearly showed a strong difference in the
chemical shift for H-5 in ring B, whereas the chemical shifts of protons in ring A were
only slightly changed, suggesting that the sulfate moiety was attached to 4-OH. This was
supported by the significant upfield-shifted signal of C-4 and downfield-shifted signals
of C-1, C-3, and C-5 in the 13C-NMR spectra of 11 compared with those of
3 [33]. The
HMBC correlations between H-6 and C-4, C-2 further confirmed the attachment at C4-OH
(Figure 5). Thus, the structure of 11 was identified as licochalcone C 4-sulfate.
Figure 5. Microbial transformation of
are indicated by arrows.
3
–5
by A. coerulea. Selected HMBC correlations (1H
→
13C) of metabolites 11 and 12

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Metabolite 12 was obtained as a yellow solid. The HRESIMS spectra of 12 exhibited
a molecular ion peak at m/z 417.0974 [M-H]− and a fragment ion peak at m/z 96.9881,
suggesting the presence of a sulfate group. Two sets of signals in the integral ratio of
around 1:1.2 were observed in the 1H- and 13C-NMR spectra of 12. It was assigned as a
mixture of trans- and cis-isomers, according to the two pairs of H-α and H-β proton signals
at δH 7.68 (1H, d, J = 15.8 Hz) and 7.98 (1H, d, J = 15.8 Hz), together with δH 6.43 (1H, d,
J = 12.8 Hz) and 7.12 (1H, d, J = 12.8 Hz). Comparison of the 1H-NMR spectrum of LCH
sulfate 12 with the parent compound 4 clearly showed a strong difference in the chemical
shift for H-3 in ring B, whereas the chemical shifts of protons in ring A were only slightly
changed, suggesting that the sulfate moiety was attached to 4-OH. This was supported by
the significant upfield-shifted signal of C-4 and downfield-shifted signals of C-1, C-3, and
C-5 in the 13C-NMR spectra of 12 compared with those of
4 [34]. Thus, the structure of 12
was identified as licochalcone H 4-sulfate. The resonances of 12 were completely assigned
by HSQC and HMBC spectra (See Supplementary Materials).
Metabolite 13 was isolated as a yellow amorphous powder. The HRESIMS spectra
of 13 displayed a molecular ion peak at m/z 433.1092 [M-H]− and a fragment ion peak at
m/z 353.1355 [M-H-80]−, suggesting the presence of a sulfate group. Comparison of the
1H- and 13C-NMR spectroscopic data of 13 with
5 indicated almost identical resonances
for both compounds, except for the downfield-shift of H-50, C-10, C-30, C-50 (0.88, 3.2, 6.0,
0
and 4.7 ppm, respectively) and the upfield-shift of C-4 (5.8 ppm), implying that the sulfate
moiety was conjugated with 40-OH [35]. Therefore, the structure of 13 was identified as
xanthohumol 40-sulfate.
It has been reported that chalcones can undergo trans to cis transformation in solution
when light exposure is available. The presence of a free hydroxyl group at the C-4 position
of chalcones inhibits photoisomerization between the trans- and cis-isomers, as the hydroxyl
group at C-4 position enables keto-enol tautomerization and free rotation around the
bond, which allows the rapid conversion of cis chalcones to the stable trans-isomers [
The electron-donating group at C-4 position is expected to weaken the -double bond of
chalcones through electron-delocalization effects (Figure 6), thus the electron-withdrawing
effects carried out by the glucose and sulfate moieties of 11, and 12 may quench
α,β-
4
].
α,β
6, 8,
the electron-donating capacity of the O-atom at C-4 [36]. As a result, glucosylation and
sulfation of C4-OH raise the barrier and make the cis-trans chalcone isomerization a slow
kinetically distinct process [36]. As demonstrated with the isolated metabolites 6, 11, and
12, the ratio of cis- to trans-isomers was found to be >1 in the NMR solvent, suggesting
that the cis isomer produced here is structurally stable under the observed conditions [37].
Such observations are quite remarkable in this case as it is commonly known that trans
chalcones are more stable than their cis-isomers.
Figure 6. The proposed mechanism of trans-cis isomerization.

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The conjugation reactions are widely known as major metabolic pathways for the
detoxification of xenobiotic compounds [15]. The conjugated derivatives are more polar
and water-soluble and therefore more easily excreted from the body through urine or
feces [14,38]. However, an increasing amount of evidence suggests that detoxification
is not the only role of sulfation in the organisms. Sulfation might change the biological
activities of numerous compounds, and some of the sulfated compounds work therapeu-
tically against cancer, diabetes, and various metabolic diseases [18,39]. A demonstration
of parallelism between mammalian and microbial systems, conjugation reaction is of
considerable importance for the drug metabolism investigations. However, sulfation in
microbial systems is rare and reports on this topic are limited. So far, in addition to the list
of microorganisms reported by Khan [22–26], several fungi including Absidia coerulea [40],
Colletotrichum gloeosporioides [19], Cunninghamella elegans [32], Gliocladium deliquescens [41],
Mucor hiemalis [42], Mucor sp. [43], Neosartorya spathulata [19], Streptomyces fulvissimus,
Syncephalastrum racemosum [21], and Trichothecium roseum [44] were reported to perform
sulfation of some phenolic compounds.
Unlike the complicated sulfation procedure of 8-prenylnaringein, which requires
a two-step process with the use of a phosphate buffer [21], we obtained the sulfated
metabolites of licochalcones and xanthohumol (
A. coerulea. The metabolite was previously identified as a phase II sulfate conjugate
of LCA during the metabolism studies using human hepatocytes [ ]. Results obtained
7–13) by directly using the cultures of
7
5
from this study showed that A. coerulea is capable of sulfation of chalcones and exhibits
parallels between microbial and mammalian metabolism not only in phase I but also
in phase II metabolism [21,31]. It is widely known that the phase II sulfation reactions
are mediated by the enzyme sulfotransferases, which catalyze the transfer of sulfonate
group from the active sulfate to the substrates containing hydroxyl groups [45]. Thus, the
sulfate metabolites of licochalcones and xanthohumol were supposed to be produced by
the enzyme sulfotransferase from A. coeralea.
To demonstrate the biological activity of compounds 7, 13 and their aglycones (1, 5),
the cytotoxic activities against three different human cancer cell lines were evaluated by
using MTT assay (Table 1). Although, as expected, the sulfated metabolites were less active
than their aglycones, they still exhibited moderate cytotoxic activities. The glucosylated
metabolites displayed weak cytotoxic activity with IC50 > 100 µM. From the results obtained
(Table 1 and Table S1), it was observed that all of the test compounds, except LCC, exhibited
higher cytotoxic activities against A375P cells than MCF-7 and A549 cells. LCC showed
stronger activity against MCF-7 cells than A549 cells, which was supported by the result
reported by Zheng and colleagues [46]. Zheng and colleagues observed that LCB (2)
exhibited weak cytotoxic activity against MCF-7 cells with IC50 > 80 µM after 24 and 48 h
treatment [47], which was similar to our result (Supporting Table S1). All the compounds
tested displayed much stronger cytotoxic activities than LCB, indicating that the prenyl
group plays a significant role in the cytotoxic activity of these compounds against the tested
cancer cell lines. However, Shim and colleagues have reported that LCB exhibited moderate
activities against A375 and A431 skin cancer cells (IC50 13.7 and 19.1
and showed stronger cytotoxic activity against HSC4 oral squamous cell carcinoma cells
(IC50 13 M) than LCA (IC50 20.42 M), LCC (IC50 27.1 M), and LCH (IC50 14.4 M) after
48 h treatment [49 52]. Though LCA and XN exhibited good activity in most cases, they
µM, respectively) [48],
µ
µ
µ
µ
–
were found to show low cytotoxic and apoptotic activity against LNCaP prostate cancer
cells [53]. Thus, the cytotoxic activity of compounds against different types of cancer cell
lines should be evaluated on a case-by-case basis.

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Table 1. Cytotoxic activities of compounds against cancer cell lines.
Cell Lines (IC50, µM)
Compound
A375P
MCF-7
A549
1
5
7
12.86 ± 3.42
11.02 ± 2.09
27.35 ± 1.44
41.04 ± 1.61
9.86 ± 0.57
19.16 ± 0.65
23.11 ± 1.36
30.85 ± 4.58
81.27 ± 3.05
5.12 ± 0.44
18.14 ± 1.26
26.93 ± 1.56
43.07 ± 1.63
99.40 ± 1.99
4.01 ± 0.78
13
DZ 1
1
Used as positive control.
3. Materials and Methods
3.1. Materials and Microorganisms
Licochalcone A ( ) and demethylzeylasteral (DZ) were purchased from Biopurify
Phytochemicals Ltd. (Chengdu, China) and confirmed by 1H-NMR. Licochalcones B, C, H
and xanthohumol ( ) were prepared using previously reported methods [27 28]. All the
1
2
–5
,
microorganisms were obtained from the Korean Collection for Type Cultures (KCTC). All of
the ingredients for microbial media including dextrose, peptone, malt extract, yeast extract,
and potato dextrose broth were purchased from Becton, Dickinson and Company (Sparks,
MD, USA). Dulbecco’s Modified Eagle Medium (DMEM) and Antibiotic-Antimycotic were
purchased from Gibco (Invitrogen, Carlsbad, CA, USA); fetal bovine serum (FBS) was
purchased from Welgene Inc. (Gyeongsan-si, Korea). Phosphate-buffered saline (PBS)
tablets were purchased from Takara Korea Biomedical Inc. (Seoul, Korea) and thiazolyl
blue tetrazolium bromide (MTT) was from Thermo Fisher Scientific (Waltham, MA, USA).
3.2. General Experimental Procedures
The 1H- and 13C-NMR spectra were obtained in DMSO-d6 or CD3OD on a Bruker
Avance III HD 400 spectrometer at 400 and 100 MHz, respectively, using TMS as an internal
standard. The chemical shift values (δ) are reported in ppm units, and the coupling
constants (J) are in Hz. IR spectra were obtained on a JASCO FT/IR-300E spectrometer
(Jasco, Tokyo, Japan). UV spectra were measured with a JASCO V-530 spectrophotometer
(Jasco, Tokyo, Japan). HRESIMS was determined on a Synapt G2 mass spectrometer (Waters,
Milford, MA, USA). TLC analyses were carried out on precoated silica gel 60 F254 plates
(Merck, Darmstadt, Germany). The developing system used was chloroform:methanol
(5:1, v/v) solution, and visualization of the TLC plates was performed using anisaldehyde-
H2SO4 spray reagent. For column chromatography, the adsorbent used was Sephadex LH-
20 from GE Healthcare (Chicago, IL, USA). HPLC was performed on a Waters 1525 Binary
HPLC Pump (Waters Corp., Milford, MA, USA) connected to a Waters 996 Photodiode
Array detector using Zorbax SB-CN (9.4
2.0 mL/min.
×
250 mm) with MeOH:H2O at a flow rate of
3.3. Screening Procedures
In the screening studies, 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. Then,
100 µL of DMSO solution (20 mg/mL) of substrates 1–5 was added to each flask 24 h after
inoculation, and further incubated at the same condition for another 7 days. 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.
3.4. Biotransformation of 1 by M. hiemalis KCTC 26779
Scale-up fermentations were carried out under the same temperature-controlled shak-
ing condition with ten 500 mL Erlenmeyer flasks, each containing 125 mL malt medium,

Int. J. Mol. Sci. 2021, 22, 6893
9 of 14
and 60 mg licochalcone A dissolved in DMSO was distributed evenly between flasks.
After incubation for 4 days, the microbial culture broth was extracted with EtOAc (1.5 L)
three times, and the organic layers were combined and concentrated in vacuo. The EtOAc
extract (265 mg) was chromatographed by semi-preparative reversed-phase HPLC with
60% MeOH as mobile phase at 2 mL/min to afford metabolites 6 (43 mg).
Licochalcone A 4-O-β-D-glucopyranoside (6)
Orange amorphous powder. IR (KBr) ν
max cm−1: 3387, 2962, 1597, 1506, 1281, 1164,
1031. HRESIMS: m/z 523.1943 [M+Na]+ (calcd for C27H32O9Na, 523.1944), 501.2120 [M+H]+
(calcd for C27H33O9, 501.2125).
1
Trans form: UV (MeOH)
λ
(log
ε
) nm: 242 (0.93), 315 (1.23), 372 (1.97). H-NMR
max
(DMSO-d6, 400 MHz)
(1H, d, J = 15.7 Hz, H-
δ
8.00 (2H, d, J = 8.0 Hz, H-20/60), 7.92 (1H, d, J = 15.7 Hz, H-
), 7.59 (1H, s, H-6), 6.90 (2H, d, J = 8.0 Hz, H-3 /5 ), 6.89 (1H, s, H-3),
β), 7.67
0
0
α
6.34 (1H, dd, J = 10.7
,
17.3 Hz, H-200), 5.01 (1H, d, J = 7.0 Hz, H-1000), 5.01 (1H, dd, J = 1.2
,
17.30H00 z, H-300a), 4.97 (1H, dd, J = 1.2
,
10.7 Hz, H-300b), 3.90 (3H, s, -OCH3), 3.77 (1H, m,
H-6000 a), 3.44 (1H, m, H-6000b), 3.38 (1H, 0m0 , H-2000), 3.32 (2H, m, H-3000/5000), 3.13 (1H, m,
H-4 ), 1.50 (3H, s, H-4 ), 1.49 (3H, s, H-5 ). 13C-NMR (DMSO-d6, 100 MHz)
δ 187.7 (C=O),
00
162.8 (C-40), 159.4 (C-4), 158.7 (C-2), 148.1 (C-200), 138.6 (C- ), 131.4 (C-20/60), 129.7 (C-10),
β
128.7 (C-5), 127.5 (C-6), 119.7 (C-α
), 115.9 (C-1), 115.9 (C-30/50), 110.7 (C-300), 100.5 (C-1000),
100.0 (C-3), 78.0 (C-5000), 77.8 (C-3000), 73.8 (C-2000), 70.7 (C-4000), 61.4 (C-6000), 56.1 (-OCH3),
40.2 (C-100), 28.2 (C-400), 27.2 (C-500).
1
Cis form: UV (MeOH)
λ
max
(log
ε
0
) nm: 260 (1.69), 294 (1.63). H-NMR (DMSO-d6,
0
400 MHz
)
δ
7.76 (2H, d, J = 7.9 Hz, H-2 /6 ), 7.09 (1H, s, H-6), 7.03 (1H, d, J = 12.8 Hz, H-
β),
6.78 (2H, d, J = 7.9 Hz, H-30/50), 6.76 (1H, s, H-3), 6.44 (1H, d, J = 12.8 Hz, H-
α), 6.02 (1H,
dd,0J0 = 10.6, 17.5 Hz, H-200), 4.90 (1H, d, J = 7.2 Hz, H-1000), 4.79 (1H, dd, J = 1.2, 17.5 Hz,
00
000
H-3 a), 4.76 (1H, dd, J = 1.2, 10.6 Hz, H-3 b), 3.77 (1H, m, H-6 a), 3.76 (3H, s, -OCH3), 3.44
000
000
000 000
000
(1H, m, H-6 b), 3.41 (1H, m, H-5 ), 3.32 (2H, m, H-2 /3 ), 3.13 (1H, m, H-4 ), 1.24 (3H,
s, H-400), 1.19 (3H, s, H-500). 13C-NMR (DMSO-d6, 100 MHz) 193.8 (C=O), 163.0 (C-40),
157.6 (C-4), 156.7 (C-2), 148.0 (C-200), 132.0 (C- ), 131.7 (C-20/60), 129.1 (C-6), 128.7 (C-10),
δ
β
127.3 (C-5), 125.8 (C-α
), 116.5 (C-1), 115.8 (C-30/50), 110.3 (C-300), 100.5 (C-1000), 99.5 (C-3),
77.9 (C-5000), 77.8 (C-3000), 73.8 (C-2000), 70.7 (C-4000), 61.4 (C-6000), 55.8 (-OCH3), 40.0 (C-100),
27.6 (C-400), 27.0 (C-500).
3.5. Biotransformation of 1–5 by A. coerulea KCTC 6936
Scale-up fermentations were carried out under the same temperature-controlled shak-
ing condition with five 500 mL Erlenmeyer flasks, each containing 125 mL suitable media,
and 30 mg licochalcone A dissolved in DMSO was distributed evenly between flasks. After
incubation for 5 days, the microbial culture broth was extracted with EtOAc (0.75 L) four
times, and the organic layers were combined and concentrated in vacuo. The EtOAc extract
(396 mg) from A. coerulea culture broth was chromatographed by semi-preparative reversed-
phase HPLC with 55% MeOH as mobile phase at 2 mL/min to give metabolite
7 (14.86 mg)
and a mixture of and (2.55 mg, 1:1.5). A similar scale-up process was performed for
7
8
licochalcones B (24 mg), C (12 mg), H (24 mg) and xanthohumol (30 mg). The EtOAc extract
of LCB (50.84 mg) was chromatographed using Sephadex LH-20 eluted with methanol to
give metabolite
9 (1.58 mg) and three fractions, fraction 2 was subjected to reversed-phase
HPLC with 70% MeOH as mobile phase at 2 mL/min to afford a mixture of metabolites
9
and 10 (6.85 mg, 3:1). The EtOAc extract of LCC (31.92 mg) was chromatographed using
Sephadex LH-20 eluted with methanol to give metabolite 11 (2.23 mg). The EtOAc extract
of LCH (50.73 mg) was chromatographed using Sephadex LH-20 eluted with methanol
to give metabolite 12 (2.25 mg) and four fractions, fractions 2 and 3 were subjected to
reversed-phase HPLC with 70% MeOH as mobile phase at 2 mL/min to afford metabolite
12 (3.80 mg). The EtOAc extract of XN (265 mg) was subjected to reversed-phase HPLC
with 70% MeOH as mobile phase at 2 mL/min to afford metabolite 13 (5.2 mg).

Int. J. Mol. Sci. 2021, 22, 6893
10 of 14
3.5.1. Licochalcone A 40-Sulfate (7)
Yellow amorphous powder. IR (neat)
ν
max cm−1: 3503, 2924, 1646, 1599, 1557, 1251,
1212, 1050, 866, 842. UV (MeOH)
λ
(log
ε
) nm: 265 (1.01), 384 (1.78). HRESIMS: m/z
max
417.1024 [M-H]− (calcd for C21H21O7S, 417.1008). H-NMR (DMSO-d6, 400 MHz)
δ
8.02
1
(2H, d, J = 8.4 Hz, H-20/60), 7.92 (1H, d, J = 15.6 Hz, H-
β
), 7.59 (1H, d, J = 15.6 Hz, H- ),
α
7.54 (1H, s,0H0 -6), 7.32 (2H, d, J = 8.4 Hz, H-30/50), 6.54 (1H, s, H-3), 6.25 (1H, dd, J = 10,
00
00 00
17.6 Hz H-2 ), 4.94 (2H, br d, J = 14.4 Hz, H-3 ), 3.83 (3H, s, -OCH3), 1.45 (6H, s, H-4 /5 ).
13C-NMR (DMSO-d6, 100 MHz)
δ
188.5 (C=O), 160.5 (C-4), 158.9 (C-2), 157.8 (C-40), 148.0
(C-200), 140.0 (C-
β
), 133.3 (C-10), 130.1 (C-20/60), 128.5 (C-6), 127.1 (C-5), 120.0 (C-30/50),
118.3 (C-α), 113.9 (C-1), 110.5 (C-300), 100.4(C-3), 56.0 (-OCH3), 40.0 (C-100), 27.5 (C-400/500).
3.5.2. Licochalcone A 4-Sulfate (8)
Yellow amorphous powder. Trans form: UV (MeOH)
λ
max (log
ε
) nm: 318 (0.52), 361
1
(0.64). H-NMR (CD3OD, 400 MHz)
δ 8.00 (1H, d, J = 15.6 Hz, H-β), 7.96 (2H, d, J = 8.4
Hz, H-20/60), 7.58 (1H, s, H-6), 7.57 (1H, d, J = 15.6 Hz, H-
α), 7.51 (1H, s, H-3), 6.89 (2H,
d, J = 8.4 Hz, H-30/50), 6.26 (1H, dd, J = 10.5, 17.4 Hz, H-200), 5.05 (1H, dd, J = 1.3, 17.4 Hz,
H-300a), 5.04 (1H, dd, J = 1.3, 10.5 Hz, H-300b), 3.94 (3H, s, -OCH3), 1.53 (6H, s, H-400/500).
1
Cis form: UV (MeOH)
λ
max (log
ε
) nm: 259 (0.32), 288 (0.27). H-NMR (CD3OD, 400 MHz)
δ
7.78 (2H, d, J = 8.7 Hz, H-20/60), 07.33 (1H, s, H-6), 7.16 (1H, d, J = 12.8 Hz, H-
β), 7.04 (1H,
0
s, H-3), 6.72 (2H, d, J = 8.7 Hz, H-3 /5 ), 6.36 (1H, d, J = 12.8 Hz, H-α), 5.92 (1H, dd, J = 10.7,
17.4 Hz, H-200), 4.79 (1H, dd, J = 1.4, 10.7 Hz, H-300b), 4.76 (1H, dd, J = 1.4, 17.4 Hz, H-300a),
3.79 (3H, s, -OCH3), 1.25 (6H, s, H-400/500).
3.5.3. Licochalcone B 3-Sulfate (9)
Yellow amorphous powder. IR (neat) ν
max cm−1: 3451, 2929, 1646, 1592, 1258, 1049,
866. UV (MeOH)
(calcd for C16H13O8S, 365.0331). H-NMR (CD3OD, 400 MHz)
λ
max (log
ε
) nm: 242 (0.62), 348 (1.46). HRESIMS: m/z 365.0366 [M-H]−
1
δ
7.99 (1H, d, J = 15.6 Hz,
), 7.57 (1H, d, J = 8.8
H-
β
), 7.99 (2H, d, J = 8.8 Hz, H-20/60), 07.66 (1H, d, J = 15.6 Hz, H-
α
0
Hz, H-6), 6.89 (2H, d, J = 8.8 Hz, H-3 /5 ), 6.74 (1H, d, J = 8.8 Hz, H-5), 4.01 (3H, s, -OCH3).
13C-NMR (CD3OD, 100 MHz)
δ
189.8 (C=O), 162.4 (C-40), 154.1 (C-2), 154.0 (C-4), 139.0
(C-β α), 115.0
), 134.0 (C-3), 130.9 (C-20/60), 129.8 (C-10), 125.1 (C-6), 120.7 (C-1), 119.7 (C-
(C-30/50), 113.1 (C-5), 61.1 (-OCH3).
3.5.4. Licochalcone B 4-Sulfate (10)
UV (MeOH)
λ
max (log
ε
) nm: 242 (0.82), 345 (1.99). HRESIMS: m/z 365.0336 [M-H]−
1
(calcd for C16H13O8S, 365.0331). H-NMR (CD3OD, 400 MHz)
δ
8.02 (1H, d, J = 15.7 Hz,
), 7.33 (1H, d, J = 8.8
H-β
), 8.01 (2H, d, J = 8.7 Hz, H-20/60), 7.73 (1H, d, J = 15.7 Hz, 0H- 0
α
Hz, H-6), 7.21 (1H, d, J = 8.8 Hz, H-5), 6.90 (2H, d, J = 8.7 Hz, H-3 /5 ), 3.90 (3H, s, -OCH3).
13C-NMR (CD3OD, 100 MHz)
δ
189.6 (C=O), 162.5 (C-40), 148.7 (C-2), 143.1 (C-4), 143.0
(C-3), 138.4 (C-β α), 117.9 (C-5), 117.4
), 131.0 (C-20/60), 129.6 (C-10), 125.5 (C-1), 121.7 (C-
(C-6), 115.1 (C-30/50), 61.3 (-OCH3).
3.5.5. Licochalcone C 4-Sulfate (11)
Yellow amorphous powder. IR (neat) ν
max cm−1: 3503, 2925, 1646, 1599, 1557, 1250,
1212, 1050, 866. UV (MeOH)
λ
max (log
ε
) nm: 327 (1.82). HRESIMS: m/z 417.1010 [M-H]−
1
(calcd for C21H21O7S, 417.1008). Trans form: H-NMR (CD3OD, 400 MHz)
δ
8.03 (2H, d,
J = 8.7 Hz, H-20/60), 8.01 (1H, d, J = 15.9 Hz, H-
β
), 7.73 (1H, d, J = 15.9 Hz0, H0-
α), 7.72 (1H,
d, J = 8.8 Hz, H-6), 7.43 (1H, d, J = 8.7 Hz, H-5), 6.90 (2H, d, J = 8.7 Hz, H-3 /5 ), 5.26 (1H, t,
00
00
00
J = 7.1 Hz, H-2 ), 3.77 (3H, s, -OCH3), 3.49 (2H, d, J = 7.1 Hz, H-1 ), 1.81 (3H, s, H-4 ), 1.67
(3H, s, H-500). 13C-NMR (CD3OD, 100 MHz) 189.6 (C=O), 162.5 (C-40), 159.0 (C-2), 150.5
δ
(C-4), 138.6 (C-
(C-1), 122.6 (C-2 ), 121.5 (C-
(C-100), 16.7 (C-500).
β
0)0, 131.0 (C-20/60), 130.8 (C-300), 129.6 (C-10), 128.8 (C-3), 125.4 (C-6), 124.7
α
), 117.4 (C-5), 115.1 (C-30/50), 61.8 (-OCH3), 24.5 (C-400), 23.2

Int. J. Mol. Sci. 2021, 22, 6893
11 of 14
1
Cis form: H-NMR (CD3OD, 400 MHz)
δ
7.84 (2H, d, J = 8.8 Hz, H-20/60), 7.13 (1H,
d, J = 8.7 Hz, H-5), 7.10 (1H, d, J = 12.9 Hz, H-
β
), 7.07 (1H, d, J = 8.7 Hz, H-6), 6.76 (2H, d,
α
J = 8.8 Hz, H-30/50), 6.65 (1H, d, J = 12.9 Hz, H-
), 5.14 (1H, t, J = 6.8 Hz, H-200), 3.71 (3H,
s, -OCH3), 3.39 (2H, d, J = 6.8 Hz, H-100), 1.76 (3H, s, H-400), 1.65 (3H, s, H-500). 13C-NMR
(CD3OD, 100 MHz)
δ
194.4 (C=O), 162.6 (C-40), 157.4 (C-2), 151.9 (C-4), 133.5 (C-
β), 131.3
(C-20/60), 130.5 (C-300), 128.3 (C-10), 127.8 (C-3), 127.4 (C-6), 126.9 (C-
α), 125.7 (C-1), 122.8
(C-200), 116.5 (C-5), 114.9 (C-30/50), 60.8 (-OCH3), 24.5 (C-400), 23.1 (C-100), 16.7 (C-500).
3.5.6. Licochalcone H 4-Sulfate (12)
Yellow amorphous powder. IR (neat)
1249, 1212, 1050, 866. UV (MeOH) (log
ν
max cm−1: 3436, 2959, 2925, 1646, 1599, 1557,
) nm: 306 (1.87), 359 (1.54). HRESIMS: m/z
λ
ε
max
−
417.0974 [M-H] (calcd for C21H21O7S, 417.1008). Trans form 1H-NMR (CD3OD, 400 MHz)
δ
7.98 (1H, d, J = 15.8 Hz, H-
β
), 7.97 (2H, d, J = 8.9 Hz, H-20/60), 7.680(1H, d, J = 15.8 Hz,
0
H-α
), 7.49 (1H, s, H-6), 7.26 (1H, s, H-3), 6.89 (2H, d, J = 8.9 Hz, H-3 /5 ), 5.35 (1H, t, J = 7.4
00
00
00
Hz, H-2 ), 3.93 (3H, s, -OCH3), 3.42 (2H, d, J = 7.4 Hz, H-1 ), 1.76 (3H, s, H-4 ), 1.75 (3H, s,
H-500). 13C-NMR (CD3OD, 100 MHz)
δ
191.8 (C=O), 163.7 (C-40), 159.2 (C-2), 154.6 (C-040),
140.8 (C-
β
), 134.4 (C-300), 132.3 (C-20/60), 131.2 (C-6), 130.7 (C-10), 126.9 (C-5), 123.8 (C-2 ),
), 121.3 (C-1), 116.4 (C-30/50), 106.1 (C-3), 56.4 (-OCH3), 28.8 (C-100), 25.9 (C-400),
122.2 (C-
α
18.0 (C-500).
Cis form 1H-NMR (CD3OD, 400 MHz)
J = 12.8 Hz, H-
), 7.06 (1H, s, H-3), 6.93 (1H, s, H-6), 6.76 (2H, d, J = 8.9 Hz, H-30/50), 6.43
(1H, d, J = 12.8 Hz, H-
), 5.01 (1H, t, J = 7.4 Hz, H-200), 3.76 (3H, s, -OCH3), 3.17 (2H, d,
J = 7.4 Hz, H-100), 1.63 (3H, s, H-400), 1.55 (3H, s, H-500). 13C-NMR (CD3OD, 100 MHz)
197.1 (C=O), 164.0 (C-40), 157.1 (C-2), 152.8 (C-4), 133.9 (C- ), 133.5 (C-300), 132.9 (C-20/60),
δ
7.81 (2H, d, J = 8.9 Hz, H-20/60), 7.12 (1H, d,
β
α
δ
β
131.9 (C-6), 129.9 (C-10), 127.4 (C- ), 126.6 (C-5), 123.4 (C-200), 122.2 (C-1), 116.2 (C-30/50),
α
105.6 (C-3), 56.0 (-OCH3), 28.1 (C-100), 25.9 (C-400), 17.7 (C-500).
3.5.7. Xanthohumol 40-Sulfate (13)
Yellow amorphous powder. IR (neat)
1213, 1051, 867. UV (MeOH) (log
ν
max cm−1: 3447, 2924, 1645, 1598, 1559, 1241,
λ
max
ε
) nm: 297 (1.01, sh), 362 (2.18). HRESIMS: m/z
433.1092 [M-H]− (calcd. for C21H21O8S, 433.0957). H-NMR (CD3OD, 400 MHz)
δ
7.79
1
(1H, d, J = 15.8 Hz, H-
β
), 7.72 (1H, d, J = 15.8 Hz, H-
α
), 7.52 (2H, d, J = 8.5 Hz, H-2/6),
6.89 (1H, s, H-50), 6.83 (2H, d, J = 8.5 Hz, H-3/5), 5.23 (1H, t, J = 7.3 Hz, H-200), 3.95 (3H,
s, -OCH3), 3.36 (2H, d, J = 7.4 Hz, H-100), 1.79 (3H, s, H-400), 1.65 (3H, s, H-500). 13C-NMR
(CD3OD, 100 MHz) δ 193.8 (C=O), 163.4 (C0-20), 159.9 (C-60), 159.8 (C-4), 156.5 (C-40), 143.0
00 00
(C-β), 130.4 (C-3 ), 130.1 (C-2/6), 126.8 (C-1 ), 124.2 (C-α), 122.2 (C-2 ), 115.5 (C-3/5), 113.9
(C-30), 108.3 (C-10), 94.9 (C-50), 55.1 (-OCH3), 24.6 (C-400), 21.7 (C-100), 16.7 (C-500).
3.6. Cytotoxic Activity Evaluation
The cytotoxic activities of the compounds LCA, XN, and their metabolites against
A375P (human melanoma), MCF-7 (human breast adenocarcinoma), and A549 (human
lung carcinoma) cancer cell lines were performed by using MTT assay, which is based
on the reduction of MTT to formazan by mitochondrial dehydrogenase. Briefly, the cells
(5~6 × 103/100 µL medium) were treated with each compound and incubated for 24 h
(A375P, MCF-7) and 48 h (A549) at 37 ◦C. After the MTT reagent (0.5 mg/mL) was added
to the well and incubated for another 4 h, the supernatant was then removed and the
MTT-formazan was dissolved in 100 µL DMSO. The amount of formed formazan was
quantified by measuring the absorbance at 490 nm using a microplate reader. Demethylzey-
lasteral (DZ) was used as a positive control and DMSO was used as a negative control in
all experiments.
3.7. Hydrolysis of Compounds 6–13
A solution of compound
6 in 1 N HCl was heated for 2 h. After cooling, the reaction
mixture was neutralized and partitioned between EtOAc and H2O. The EtOAc and H2O ex-

Int. J. Mol. Sci. 2021, 22, 6893
12 of 14
tracts were analyzed by TLC, respectively. By comparing the TLC with those of compound
1
and D-glucose, the agylcone of
confirmed to be D-glucose [29].
Compounds 13 were dissolved in MeOH and mixed with 3% HCl at room tempera-
ture, respectively. Then MeOH was evaporated, and the aglycone extracted with EtOAc.
The residue was analyzed by TLC and HPLC by direct comparison with substrates
6 was found to be that of 1 and the monosaccharide was
7
–
1–5.
When BaCl2 was added to the aqueous layer, a white precipitate was formed which proved
the presence of BaSO4 [31].
4. Conclusions
In the present study, nine conjugated metabolites of licochalcones and xanthohumol
were obtained by using the fungi M. hiemalis and A. coerulea. Among them, the glucosylated
metabolite of LCA (
been reported. The glucoside and sulfate moieties substituted at C4-OH were thought to
be exhibited as the cis-trans isomerization barriers, which led to the metabolites ( 11 and
6), and the sulfated metabolites of LCB, LCC, LCH (9–12) have never
6,
12) existing as a mixture of trans- and cis-isomers with the cis form as major ones. By using
the fungus A. coerulea, it was possible to obtain the sulfated compounds of licochalcones
A-C, H and xanthohumol. The sulfation reaction is one of the major phase II metabolic
pathways in mammalian metabolism of xenobiotics, thus these sulfated metabolites are
of significant interest, for they can be used as the reference standards for the detection
and identification of the metabolic products of 1–5 that occur in mammalian systems.
Comparison studies of the cytotoxic activities of the parent compounds and their sulfated
metabolites against human cancer cell lines showed that sulfation of LCA and XN reduced
but did not inactivate the cytotoxic activity.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/ijms22136893/s1, Figures S1–S29: 1D, 2D NMR and HRESIMS spectra of 6–13.
Author Contributions: Conceptualization, I.-S.L.; methodology, F.H. and I.-S.L.; validation, F.H.;
formal analysis, I.-S.L.; investigation, F.H., Y.X. and I.-S.L.; resources, F.H.; data curation, F.H. and Y.X.;
writing—original draft preparation, F.H.; writing—review and editing, I.-S.L. and Y.X.; visualization,
F.H.; supervision, I.-S.L.; project administration, I.-S.L.; funding acquisition, I.-S.L. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was supported by the Basic Science Research Program through the National
Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2019R1I1A3A01043084).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors are grateful for the NMR experimental supports of the Center for
Research Facilities, Chonnam National University, and for the HRESIMS analyses of the Korea Basic
Science Institute (KBSI).
Conflicts of Interest: The authors declare no conflict of interest.
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