Three new isoflavonoid glycosides from the mangrove-derived actinomycete micromonospora aurantiaca 110B

Article abstract of DOI:10.3390/md17050294

The mangrove ecosystem is a rich resource for the discovery of actinomycetes with potential applications in pharmaceutical science. Besides the genus Streptomyces, Micromonospora is also a source of new bioactive agents. We screened Micromonospora from the rhizosphere soil of mangrove plants in Fujian province, China, and 51 strains were obtained. Among them, the extracts of 12 isolates inhibited the growth of human lung carcinoma A549 cells. Strain 110B exhibited better cytotoxic activity, and its bioactive constituents were investigated. Consequently, three new isoflavonoid glycosides, daidzein-40-(2-deoxy-a-l-fucopyranoside) (1), daidzein-7-(2-deoxy-a-l-fucopyranoside) (2), and daidzein-40,7-di-(2-deoxy-a-l-fucopyranoside) (3) were isolated from the fermentation broth of strain 110B. The structures of the new compounds were determined by spectroscopic methods, including 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESIMS). The result of medium-changing experiments implicated that these new compounds were microbial biotransformation products of strain M. aurantiaca 110B. The three compounds displayed moderate cytotoxic activity to the human lung carcinoma cell line A549, hepatocellular liver carcinoma cell line HepG2, and the human colon tumor cell line HCT116, whereas none of them showed antifungal or antibacterial activities.

Full text of DOI:10.3390/md17050294

marine drugs
Article
Three New Isoflavonoid Glycosides from the
Mangrove-Derived Actinomycete
Micromonospora aurantiaca 110B
Rui-Jun Wang 1, Shao-Yong Zhang 3, Yang-Hui Ye 1, Zhen Yu 3, Huan Qi 3, Hui Zhang 3
,
Zheng-Lian Xue 2, Ji-Dong Wang 2,3,* and Min Wu 1,
*
1
Ocean College, Zhejiang University, Zhoushan 316021, China; 15034663445@163.com (R.-J.W.);
yyhleslie@163.com (Y.-H.Y.)
College of Biochemical Engineering, Anhui Polytechnic University, Wuhu 241000, China;
xuezhen0851@sina.com
Zhejiang Key Laboratory of Antifungal Drugs, Zhejiang Hisun Pharmaceutical Co., Ltd., Taizhou 318000,
China; 1zhangshaoyong@163.com (S.-Y.Z.); yuzhen@hisunpharm.com (Z.Y.);
qihuan@hisunpharm.com (H.Q.); huizhang@hisunpharm.com (H.Z.)
2
3
*
Correspondence: jdwang@hisunpharm.com (J.-D.W.); wumin@zju.edu.cn (M.W.);
Tel.: +86-136-3400-5685 (J.-D.W.); +86-136-1651-0066 (M.W.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 4 May 2019; Accepted: 13 May 2019; Published: 17 May 2019
Abstract: The mangrove ecosystem is a rich resource for the discovery of actinomycetes with potential
applications in pharmaceutical science. Besides the genus Streptomyces, Micromonospora is also a
source of new bioactive agents. We screened Micromonospora from the rhizosphere soil of mangrove
plants in Fujian province, China, and 51 strains were obtained. Among them, the extracts of 12 isolates
inhibited the growth of human lung carcinoma A549 cells. Strain 110B exhibited better cytotoxic
activity, and its bioactive constituents were investigated. Consequently, three new isoflavonoid
glycosides, daidzein-40-(2-deoxy-
), and daidzein-40,7-di-(2-deoxy-
α
-l-fucopyranoside) ( ), daidzein-7-(2-deoxy-
-l-fucopyranoside) (3) were isolated from the fermentation broth
1
α-l-fucopyranoside)
(2
α
of strain 110B. The structures of the new compounds were determined by spectroscopic methods,
including 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization
mass spectrometry (HR-ESIMS). The result of medium-changing experiments implicated that these
new compounds were microbial biotransformation products of strain M. aurantiaca 110B. The three
compounds displayed moderate cytotoxic activity to the human lung carcinoma cell line A549,
hepatocellular liver carcinoma cell line HepG2, and the human colon tumor cell line HCT116, whereas
none of them showed antifungal or antibacterial activities.
Keywords: Micromonospora aurantiaca 110B; isoflavonoid glycosides; structure elucidation; cytotoxic
activity
1. Introduction
The mangrove ecosystem, distinguished by its land-to-ocean transition, is complex and special.
Mangroves thrive in a stressful environment of high salinity, high moisture, strong winds, and high
osmotic pressure [
numerous stresses and has developed unique metabolic pathways for the purpose of structural
ecological adaptability [ ]. Recent research has focused on the diversity of mangrove microorganisms
and their metabolic compounds [ ]. Among mangrove microorganisms, actinomycetes have gained
more attention due to their ability to produce impressive numbers of bioactive metabolites [ ]. There is
clear evidence that the mangrove ecosystem contains a large diversity of actinomycetes [ ]. As one of
1]. This environment breeds a distinctive microbial community that can tolerate
2,3
4–6
3
7
Mar. Drugs 2019, 17, 294; doi:10.3390/md17050294
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2019, 17, 294
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the genera of actinomycetes, the genus Micromonospora is an important prolific producer of secondary
metabolites [8,9].
The genus Micromonospora, belonging to the family Micromonosporaceae, was proposed by
Ørskov [10]. Currently, this genus consists of more than 80 recognized species, according to the
List of Prokaryotic Names with Standing in Nomenclature (LPSN) [11]. About 20 new species
of Micromonospora have been isolated in the last five years. Many of them were isolated from
mangrove environments, such as Micromonospora sediminis CH3-3T [12], Micromonospora ovatispora
2701SIM06T [13], Micromonospora zhanjiangensis 2902at01T [14], Micromonospora sonneratiae 274745T [15],
and Micromonospora maritima D10-9-5T [16]. Meanwhile, some new metabolites with biological activities
have been isolated from the mangrove-derived Micromonospora strains. Micromonospora rifamycinica
AM105 produced rifamycin S and the geometric isomer of rifamycin S with antimicrobial activity against
methicillin-resistant Staphylococcus aureus (MRSA) [17]. Butremycin was isolated from Micromonospora
sp. K310 and displayed weak antibacterial activity against Gram-positive Staphylococcus aureus ATCC
25923 and Gram-negative Escherichia coli ATCC 25922 [18]. 3-Methyl-carboline from Micromonospora sp.
M2DG17 showed weak inhibitory activity on human colon carcinoma HCT 116 cell lines [19].
In our continuing study of mangrove actinomycetes, 51 strains of Micromonospora were isolated
from the rhizosphere soil of mangrove plants in Fujian province. The crude extracts obtained from
the isolates were evaluated for cytotoxic activity. Based on the cytotoxic activity, further chemical
investigations were performed on Micromonospora aurantiaca 110B. As a result, three new isoflavonoid
glycosides (1–3) were isolated from its fermentation broth. Herein, we describe the isolation, structure
elucidation, and cytotoxic activity of these three new compounds.
2. Results
2.1. Isolation and Screening of Strains for Cytotoxic Activities
Fifty-one strains belonging to the genus Micromonospora were isolated from mangrove soil samples.
The crude extracts of these isolates were examined for their cytotoxic activity. After primary screening,
12 strains showed cytotoxic activity to the human lung tumor cell line A549 (Figure 1). The 16S
rRNA genes of the 12 strains were sequenced and the sequences were blasted against GenBank
(https://www.ncbi.nlm.nih.gov/genbank/). The results showed that the isolates belonged to seven
different species (Figure 2) and that strain 110B was closely related to the type strain Micromonospora
aurantiaca ATCC 27029T (CP002162).
Due to the superior cytotoxic activity of strain 110B, further chemical investigations were performed
on this strain.
100 g/mL
20 g/mL
100
80
60
40
20
0
Q33 Q52 Q39 Q85 Q20-1 Q112 Q59 110B B40 B116 T36 T75
Figure 1. Cytotoxic activity of extracts obtained from 12 isolates against the human lung tumor cell
line A549 in vitro.

Mar. Drugs 2019, 17, 294
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Micromonospora wenchangensis 2602GPT1-05T (JQ768361)
57
39
Isolate Q39 (MK305978)
Isolate TA75 (MK3059780)
99
Micromonospora rifamycinica AM105T (AY561829)
Isolate T36 (MK305982)
90
65
Isolate QA85 (MK305975)
Micromonospora schwarzwaldensis HKI0641T (KC517406)
99
62
Isolate Q112 (MK305981)
Micromonospora aurantiaca ATCC 27029T (CP002162)
69
100
Isolate 110B (MH333275)
Isolate Q59 (MK305979)
Isolate B116 (MK305984)
70
Micromonospora terminaliae TMS7T (KX394339)
99
84
Isolate Q33 (MK305976)
Micromonospora tulbaghiae TVU1T (EU196562)
Micromonospora yasonensis DS3186T (JN989295)
Isolate Q63 (MK305985)
100
87
35
Isolate Q52 (MK305977)
Micromonospora citrea DSM 43903T (FMHZ01000002)
Isolate B40 (MK305983)
49
81
0.002
Figure 2. Neighbor-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences
showing relationships between the 12 active isolates and the type strains of the highest 16S rDNA
sequence similarity. Bootstrap values were based on 1000 replicates; only values
0.002 substitutions per nucleotide position.
≥
50% are shown. Bar:
2.2. Structural Elucidation
The strain 110B was grown in a preparative scale in 30 L of fermentation medium for 7 days.
A chemical investigation of its fermentation broth led to the isolation of three new isoflavonoid
glycosides (compounds 1–3).
Compound
1
was obtained as a white powder with a negative specific rotation value ([α]2D5−107,
EtOH) and UV (EtOH) λmax (log ε) 249 (4.49), 301 (4.10) nm. The molecular formula of 1 was established
to be C21H20O7 from positive-ion HR-ESIMS (Figure S1) and 13C NMR spectral (Figure S4) data (Table 1).
The IR spectrum (Figure S2) of
revealed carbonyl group absorption at 1628 cm−1 and hydroxy group
1
absorption at 3353 cm−1. The 1H NMR (Figure S3) data (Table 1) showed the characteristic isoflavone
signal for H-2 at δH 8.08 (1H, s); two sets of aromatic hydrogen peaks at δH 7.39 (2H, d, J = 8.7 Hz) and
δH 7.05 (2H, d, J = 8.7 Hz) corresponding to a 4-hydroxyphenyl moiety (B ring); three aromatic protons
corresponding to a 7-substituted A ring at δH 7.97 (1H, d, J = 9.0 Hz), 6.84 (1H, dd, J = 9.0, 2.4 Hz),
and δH 6.76 (1H, d, J = 2.4 Hz); and nine proton signals attributable to sugars at δH 5.62 (1H, br d,
J = 3.5 Hz), 4.09 (1H, ddd, J = 11.9, 5.0, 3.0 Hz), 3.89 (1H, q, J = 6.6 Hz), 3.55 (1H, br d, J = 3.0 Hz), 2.03
(1H, ddd, J = 12.8, 11.9, 3.6 Hz), 1.90 (1H, dd J = 12.8, 5.0 Hz), and δH 1.10 (3H, d, J = 6.6 Hz). The 13
NMR spectrum (Table 1) displayed 21 carbon resonances, including 15 carbons for the isoflavone
skeleton and 6 carbons for the sugar. The NMR spectral (Figure S6) data suggested that compound
was an isoflavone glycoside, and the isoflavone skeleton proved to be daidzein based on a comparison
of the spectroscopic data with previously reported values [20 21]. The 6-carbon resonances of sugar
can be categorized as 1 methyl (δC 18.4), 1 methylene (δC 32.09), 1 acetal (δC 97.6), and 3 methines
δC 72.1, 68.6, 66.7) by 13C NMR and distortionless enhancement by polarization transfer (DEPT,
C
1
,
(
Figure S5) experiments, indicating the presence of a 2,6-dideoxysugar moiety. The correlations of
H-1”/H2-2”/H-3”/H-4”/H-5”/H3-6” in the 1H–1H correlation spectroscopy (COSY) spectrum (Figure 3
and Figure S7) and the observed heteronuclear multiple bond correlation (HMBC) signals (Figure 3
and Figure S8) from H-1” to C-5” and H3-6” to C-4” further established the 2,6-dideoxysugar structure.
In the HMBC experiment, the correlation between the anomeric proton (H-1”) and the carbon at δC
158.3 (C-40) confirmed the attachment of the sugar moiety to the phenolic 40-O-atom. From the detailed
1
analysis of the H NMR spectrum, the small coupling constant of H-1” (δH 5.62 br d, J = 3.5 Hz)
revealed that the sugar is α-glycosidically linked to the phenolic oxygen atom [22]. The signal patterns

Mar. Drugs 2019, 17, 294
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of H-3” (δH 4.09 ddd, J = 11.9, 5.0, 3.0 Hz) and H-4” (δH 3.55 br d, J = 3.0 Hz) showed equatorial 3”-OH
and axial 4”-OH groups. Further, signals at H-4” exhibited 3.0 coupling, which could be assigned to
the axial H-5” and axial H-3”, respectively. This 2,6-dideoxysugar was assumed to be 2-deoxy-l-fucose
on the basis of 1H and 13C NMR data [23
of compound
identified by HPLC analysis. The retention time of the aglycone moiety of
,
24] and the NOESY experiment (Figure S9). Acid hydrolysis
afforded the aglycone daidzein and a 2-deoxy-l-fucose. The aglycone fraction was
was in good agreement
1
1
with that of daidzein (Figure S28). The sugar fraction liberated by acid hydrolysis was treated with
l-cysteine methyl ester, followed by reaction with O-tolylisothiocyanate. By reversed-phase HPLC
analysis, the identical retention time of the liberated sugar derivative with that of the 2-deoxy-l-fucose
derivative indicated that the sugar moiety of 1 was 2-deoxy-fucose (Figure S29). Consequently, the
structure of 1 was elucidated to be daidzein-40-(2-deoxy-α-l-fucopyranoside) (Figures 3 and 4).
Compound
EtOH). The molecular formula of
(Figure S10) and 13C NMR spectral data (Table 1). Bands for hydroxy group at 3357 cm−1 and the
carbonyl group at 1634 cm−1 were evident in the IR spectrum (Figure S11). In the 1H NMR spectrum
2
was obtained as a white powder with a negative specific rotation value ([α]2D5−112,
2 was established as C21H20O7 from positive-ion HR-ESIMS
(Table 1) of 2, the characteristic H-2 signal of an isoflavone was exhibited at δH 8.12 (1H, s), and the
4-hydroxyphenyl nature of the B ring was deduced from the signals at δH 7.31 (2H, d, J = 9.0 Hz) and
δH 6.78 (2H, d, J = 9.0 Hz). Three aromatic proton signals were observed at δH 8.06 (1H, d, J = 9.0 Hz),
7.18 d (1H, d, J = 2.3 Hz), and δH 7.09 (1H, dd, J = 9.0, 2.3 Hz), indicating that the A ring was substituted
at C-7. An anomeric proton signal at δH 5.78 d (1H, br d, J = 3.6 Hz) also appeared in the 1H NMR
spectrum of 2. Comparison of the NMR data (Figures S12–S18) of 2 with those of 1 revealed close
similarities, except for the substituted pattern of the 2,6-dideoxysugar moiety. The observed HMBC
correlation (Figure 3) from the anomeric proton H-1” to C-7 (δC 163.1) confirmed the position of the
2,6-dideoxysugar. The 2,6-dideoxysugar was deduced to be 2-deoxy-l-fucose according to HPLC
analysis of the corresponding O-tolylthiocarbamate derivative of the liberated sugar, as described
above. Thus, the structure of 2 was identified as daidzein-7-(2-deoxy-α-l-fucopyranoside) (Figure 3).
Table 1. 1H and 13C NMR (100 MHz) data of compounds 1−3 in CD3OD.
1a
2b
3b
Position
δc
δH (J in Hz)
δc
δH (J in Hz)
δc
δH (J in Hz)
2
3
4
5
6
7
8
9
154.9
125.6
178
8.08 s
154.9
126.2
178.1
128.2
117.2
163.1
104.4
159.4
119.8
124.1
131.4
116.3
158.8
98.2
8.12 s
155.2
125.9
177.9
128.2
117.2
163.1
104.4
159.3
119.7
125.9
131.3
117.4
158.3
97.6
8.21 s
128.5
116.5
164.7
103.3
159.8
118.2
126.7
131.3
117.5
158.3
97.6
7.97 d (9.0)
6.84 dd (9.0, 2.4)
8.06 d (9.0)
7.09 dd (9.0, 2.3)
8.12 d (8.9)
7.15 dd (8.9, 2.2)
6.76 d (2.4)
7.18 d (2.3)
7.25 d (2.2)
10
10
20, 60
30, 50
40
7.39 d (8.7)
7.05 d (8.7)
7.31 d (9.0)
6.78 d (9.0)
7.47 d (8.7)
7.13 d (8.7)
1”
2”
5.62 br d (3.5)
2.03 ddd (12.8, 11.9, 3.6)
1.90 dd (12.8, 5.0)
4.09 ddd (11.9, 5.0, 3.0)
3.55 br d (3.0)
5.78 br d (3.6)
2.07 ddd (13.2,12.0, 3.6)
1.96 dd (13.2, 5.0)
4.09 ddd (12.0, 5.0, 3.0)
3.57 br d (3.0)
5.69 br d (3.5)
2.08 ddd (12.8, 12.0, 3.5)
1.91 dd (12.8, 5.1)
4.16 ddd (12.0, 5.1, 3.1)
3.62 m
32.1
32.9
33.2
3”
4”
5”
66.7
72.1
68.6
18.4
66.6
71.9
69.3
17.2
66.7
71.9
69.3
17.2
98.2
32.9
3.89 q (6.6)
1.10 d (6.6)
3.86 q (6.6)
1.12 d (6.6)
3.91 m
1.18 d (6.2)
6”
1000
2000
5.84 br d (3.5)
2.12 ddd (13.1, 12.0, 3.5)
1.98 dd (13.1, 5.1)
4.13 ddd (12.0, 5.1, 3.1)
3.62 m
3000
4000
5000
6000
66.5
72.1
68.6
17.2
3.95 m
1.16 d (6.2)
a 1H (600 MHz); b 1H (400 MHz).

Mar. Drugs 2019, 17, 294
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1
2
3
Figure 3. The structures and key 1H–1H COSY and HMBC correlations for compounds 1–3.
Figure 4. The key NOESY correlations for compound 1.
Compound
EtOH). The molecular formula of
(Figure S19) and 13C NMR spectral data (Table 1). The IR spectrum (Figure S20) revealed carbonyl
absorption at 1650 cm−1 and hydroxy absorption at 3381 cm−1. The 1H NMR spectrum of
(Table 1)
3
was obtained as a white powder with a negative specific rotation value ([α]2D5−27.0,
3 was deduced to be C27H30O10 from positive-ion HR-ESIMS
3

Mar. Drugs 2019, 17, 294
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showed eight aromatic proton signals at δH 8.21 (1H, s), 8.12 (1H, d, J = 8.9 Hz), 7.47 (2H, d, J = 8.7 Hz),
7.25 (2H, d, J = 2.2 Hz), 7.15 (1H, dd, J = 8.9, 2.2 Hz), and δH 7.13 (1H, d, J = 8.7 Hz), indicating that the
isoflavone aglycone of
3
was identical to
1
and
2
. Two sugar moieties were evident from the anomeric
1
proton signals at δH 5.84 (1H, br d, J = 3.5 Hz) and δH 5.69 (1H, br d, J = 3.5 Hz) in the H NMR
spectrum. The 1H and 13C NMR spectral (Figures S21–S23) data of
, except for an extra 2,6-dideoxysugar moiety. The HMBC (Figure S26) correlations from H-100 to
C-40 and H-1000 to C-7 revealed that the two 2,6-dideoxysugar moieties were located at C-40 and C-7,
respectively. The sugar moieties of were determined by HPLC analysis of the O-tolylthiocarbamate
derivative of the liberated sugar from acid hydrolysis and a standard 2-deoxy-l-fucose derivative. So,
3 were similar to those of 1 and
2
3
the structure of
Figure 3.
3 α-l-fucopyranoside), as shown in
was determined to be daidzein-40,7-di-(2-deoxy-
Isoflavonoids have been frequently isolated from bacterial and fungal cultures, but their
biosynthetic origin has not been unveiled in all cases. Not all of the isoflavonoids were synthesized
‘de novo’. Ndejouong and Hertweck found that Streptomyces mirabilis could transform isoflavones
into hydroxylated and reduced derivatives [25]. In order to clarify whether compounds 1–3 were
synthesized ‘de novo’, we carried out a series of experiments. We prepared different media (media 1–4)
to explore whether the ingredients in the fermentation medium will affect the production of compounds
1–3, and we mainly focused on the medium that lacked soybean cake or peptone. We found that the
three new compounds were only detected in medium containing soybean cake. Then, when daidzein
was added to the medium lacking soybean cake (giving medium 5), we could observe formation of
these new compounds (Figure 5). These results indicated that M. aurantiaca 110B could transform plant
daidzein into fucosylated derivatives.
Figure 5. HPLC analysis of the formation of compounds 1–3 in different media, with a mobile phase of
CH3CN/H2O (25:75, v/v), flow rate of 1.5 mL/min−1, and detection wavelength at 254 nm.
2.3. Analysis of Deoxy-Sugar Glycosyltransferases
The genome of the strain 110B was sequenced and analyzed. We focused on whether there were
any deoxy-sugar-related enzymes in strain M. aurantiaca 110B because the sugar moieties of the three
new isoflavonoid glycosides were 2,6-dideoxysugars. Deoxy-sugar glycosyltransferases are interesting
enzymes because of their important role in many natural product drugs. Glycosyltransferase-related
protein sequences of the M. aurantiaca 110B were aligned with protein sequences of the UniProt
database [26
,27], and one protein (AXH94677.1) similar to AknK was found (Figure 6). AknK is an
l-2-deoxy-l-fucose transferase found in Streptomyces galilaeus, which catalyzes the addition of the

Mar. Drugs 2019, 17, 294
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second sugar to aclacinomycin A [28]. The genome information of strain 110B also showed that the
sugar moieties of the three new isoflavonoid glycosides might be 2-deoxy-fucose. This conclusion was
consistent with the results of the structural analysis.
AXH94677.1
A0A1C4KH77
AknK
A0A1M5X8V1
A0A2G7A0A5
Consensus
MRVLFTTIAAPTHLHAQVPLAWALRAAGHDVRVASQPDLTEAITGAGLTAVAVGPPLGAD
MRVLFVTLAAATHLHAQVPVAWALRAAGHDVCVASHPDVVDDIVRSGLTPVPVGEPMDIG
MKVLFTTFAAKSHMHAQVPLAWALQTAGHEVRIASQPDLAEDITRTGLTAVCVGEPLLLE
MKVLFTTFAAKSHMHAQVPLAWALQTAGHEVRIASQPDLAEDITRTGLTAVCVGEPLLLE
MKVLFTTFAAKSHMHAQVPLAWALQTAGHEVRIASQPDLAEDITRTGLTAVCVGEPLLLE
60
60
60
60
60
m vlf t aa h haqvp awal agh v as pd
i
glt v vg p
AXH94677.1
A0A1C4KH77
AknK
A0A1M5X8V1
A0A2G7A0A5
Consensus
RWSQDVVEEVAERWEFTGDELWGPGGRMRWELFDLCELRRQRLTYDYVQSVLTAWTSLDH
RWADVPPLTKEELQDET..NIWDARGGVDWELFDLCELRPEKLDYAYMQAVFSAWVGASF
EQMQRVNEGLGDDAEIM..ESQAEAG......MDMTETRPEMLTWDHVLGVFTSMTAMAF
EQMQRVNEGLGDDAEIM..ESQAEAG......MDMTETRPEMLTWDHVLGVFTSMTAMAF
EQMQRVNEGLGDDAEIM..ESQAEAG......MDMTETRPEMLTWDHVLGVFTSMTAMAF
120
118
112
112
112
g
d e r
l
v
AXH94677.1
A0A1C4KH77
AknK
A0A1M5X8V1
A0A2G7A0A5
Consensus
QLSCSAGMIDDLVAFTQDWRPDLVVRDPVTFAGSVAAIASGAAHARLLFGLDVFGHMRST
QEISSPRVMDDLVGFARQWRPDLIIWDPLTFAAPVAAVAVGAAHARMMFGFDVIGRMRQT
QNSCPERMIDDVVAFAREWQPDLIVWDTLSFAGPVAAQVTGAAHARLLFGLDLLGRMRET
QNSCPERMIDDLVAFSREWQPDLVIWDTLSFAGPVAAQVTGAAHARLLFGLDLLGRMRET
QNSCPERMIDDLVAFSREWQPDLVIWDTLSFAGPVAAQVTGAAHARLLFGLDLLGRMRET
180
178
172
172
172
q
dd v f
w pdl
d
fa vaa
gaahar fg d g mr t
AXH94677.1
A0A1C4KH77
AknK
A0A1M5X8V1
A0A2G7A0A5
Consensus
YLELLRGRVPALRDDPLQEWLGDVLARHGHPFTEQAVLGQFTIDPVPASLRLPVDHHYLP
YRAALADRPAALREDPFEEWLGTALARYGGGFDEQAVLGQWTIDTVPSSMRLEVDQLYVP
FLDLQEERLPEQRDDPLREWLTWTLGRYGAEFEEEVAVGQWTVDPVPPSMRFPVKQPFVP
FLDLQAERLPEQRDDPLREWLTWTLGRYGAEFEEEVAVGQWTIDPVPPSMRFPVKQPFVP
FLDLQAERLPEQRDDPLREWLTWTLGRYGAEFEEEVAVGQWTIDPVPPSMRFPVKQPFVP
240
238
232
232
232
r
r dp ewl
l r g f e
gq t d vp s r v
p
AXH94677.1
A0A1C4KH77
AknK
A0A1M5X8V1
A0A2G7A0A5
Consensus
MRYTPYNGPATVPDWLREPPTRPRVCLTLGRSFRETLGGDRTAVTELLDAVATLDVDVVA
MRYTPYNGRSTVEPWLWHPPERRRVCLTLGSSFRELMGGDRADIGELLRAVADLDVEVIA
LRYIPYNGQAVIPDWLHEPPKKRRVCLTLGVAHREVLDGDRASIGELVEALAELDVEVVA
LRYIPYNGQAVIPQWLHEPPKKRRVCLTLGVAHREVLDGDRASIGELVGALAELDVEVVA
LRYIPYNGQAVIPQWLHEPPKKRRVCLTLGVAHREVLDGDRASIGELVGALAELDVEVVA
300
298
292
292
292
ry pyng
wl pp
rvcltlg
re
gdr
el a a ldv v a
AXH94677.1
A0A1C4KH77
AknK
A0A1M5X8V1
A0A2G7A0A5
Consensus
TLSNSQIPPGYPIPDNVRTVEFVNLDVLLPTCAAIIHHGGSGTSQTALIHGVPQVIVPAV
TLNTDQLDSLPPLPDNVRAVDFVPLDALLPSCSAIIHHSGSGTTQTALAHGVPQVIVPTN
TLNEKQLAG.MELPDNVRAVDFVPLNALLPTCSAVIHHGGSGTFQTALAHGVPQLIVPDM
TLNAKQLAG.LELPDNVRAVDFVPLNALLPTCSAVIHHGGSGTFQTALAHGVPQLIVPDM
TLNAKQLAG.LELPDNVRAVDFVPLNALLPTCSAVIHHGGSGTFQTALAHGVPQLIVPDM
360
358
351
351
351
tl
q
pdnvr v fv l llp c a ihh gsgt qtal hgvpq ivp
AXH94677.1
A0A1C4KH77
AknK
A0A1M5X8V1
A0A2G7A0A5
Consensus
LLDNHLKSSRVAEAGAALCG.SAEKCTARELRALLSRVLEESSFAAAAARLRVEMLAAPA
MMDNVLKAQRVEAAGAGLHVRDTAALTADSLRALLLRVLDEPSFAAKAAAVRREMLAAPA
VWDTIHKAKQLERFGAGLYLHDVDNYTAQDLRDHLLRLLEEPSFAENCARIRREMVGTPS
VWDTIHKAKQLEKFGAGLYLHDVDHYTAQDLREHVLRLLEEPSFAENCARIRREMVGTPS
VWDTIHKAKQLEKFGAGLYLHDVDHYTAQDLREHVLRLLEEPSFAENCARIRREMVGTPS
419
418
411
411
411
d
k
ga l
ta lr
r l e sfa
a r em
p
AXH94677.1
A0A1C4KH77
AknK
A0A1M5X8V1
A0A2G7A0A5
Consensus
PAELVPALRRLTRALR..............
PRDIVPVLERLTALGRSQAVPELPDTPWAV
PNDIVPLLEKLTAEHRRDRGARGTVRGEQ.
PNDIVPLLEKLAAEHRGARDSRSAVRGER.
PNDIVPLLEKLAAEHREARYSRSAVRGER.
435
448
440
440
440
p
vp l l
r
Figure 6. Alignment of the deduced amino acid sequence of AXH94677.1 in strain 110B with different
2-deoxyfucosyltransferases: A0A1C4KH77 (SCD60843.1) from Streptomyces sp. DvalAA-19, AknK
(AAF70102.1) from Streptomyces galilaeus, A0A1M5X8V1 (SHH96196.1) from Streptomyces sp. 3214.6,
and A0A2G7A0A5 (PIG12760.1) from Streptomyces sp. 1121.2.
2.4. Biological Activity
Compounds 1–3 were tested for their activity against the proliferation of the human hepatocellular
liver carcinoma cell line HepG2, human lung tumor cell line A549, and human colon tumor cell line
HCT116. The results (Table 2) showed that the three compounds exhibited moderate cytotoxic activity
against the three cell lines.

Mar. Drugs 2019, 17, 294
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Table 2. Cytotoxic activity of compounds 1–3 against selected human tumor cell lines.
IC50 (µg/mL)
Compounds
A549
HepG2
HCT116
1
2
3
17.55
16.95
14.79
1.02
52.71
50.90
44.42
0.86
16.00
15.40
13.48
0.91
Doxorubicin
The antifungal and antibacterial properties of the three compounds were tested against three
human pathogens: the fungus Candida albicans, the Gram-positive bacterium methicillin-resistant
S. aureus, and the Gram-negative bacterium E. coli. The minimum inhibitory concentrations (MICs) of
compounds 1–3 were determined to be >10 mg/mL, so the new compounds had no activity against
these tested pathogens.
3. Materials and Methods
3.1. General Experimental Procedures
Optical rotation was measured on a Perkin-Elmer 341 polarimeter (Perkin-Elmer, Suzhou, China).
IR spectra were recorded on a Nicolet Magna FT-IR 750 spectrometer (Nicolet, Tokyo, Japan). UV spectra
were recorded on a Varian CARY 300 BIO spectrophotometer (Varian, Cary, NC, USA). The HR-ESIMS
spectra were taken on a Q-TOF Micro LC-MS-MS mass spectrometer (Waters Co, Milford, MA,
USA). NMR spectra were measured with a Bruker DRX-400 (400 MHz for 1H and 100 MHz for 13C)
spectrometer (Bruker, Rheinstetten, Germany). HPLC separation was performed on semipreparative
HPLC (Agilent 1100, Zorbax SB-C18, 5
µ
m, 250
×
9.4 mm inner diameter; 1.5 mL/min; 220 nm; Agilent,
Palo Alto, CA, USA). Column chromatography was performed with commercial silica gel (Qing Dao
Hai Yang Chemical Group Co., 100–200 and 200–300 mesh). Silica gel plates HSGF254 (Yantai Chemical
Industry Research Institute, Yantai, China) were used for thin-layer chromatography (TLC).
3.2. Isolation and Identification of Strain
Mangrove soils were collected from Zhangzhou (24◦200, 117◦450), Fujian province, China.
◦
The samples were placed into clean plastic bags and stored at 4 C prior to isolation. Samples
were dried and mixed with sterile deionized water, and actinobacteria were isolated by plating the
resulting suspensions on four different selective isolation media: M6 (raffinose–histidine medium),
M8 (glycerol asparagine agar medium), HV (humic acid–vitamin agar), and GP (glucose–tryptone
agar medium). All of the media were supplemented with 25 mg/L of nalidixic acid and 50 mg/L
of nystatin. Also, 10% supernatant of the strain Micrococcus luteus JCM 21373T, which contained
resuscitation-promoting factor (rpf), was added to each medium. The strain M. luteus JCM 21373T was
inoculated in LMM (lactate minimal medium) at a rate of 5% (v/v) and incubated at 30 ◦C for 48 h until
the end of the logarithmic growth phase [29]. Then, the culture supernatant was collected following
centrifugation at 10,000 rpm and sterilized by filtration through a 0.22-µm filter. After incubation,
isolates with Micromonospora-like morphology were transferred to ISP medium 2 (yeast extract-malt
extract agar) and incubated for 7–10 days at 28 ◦C.
All isolates were grown in marine broth 2216 (MB, difco) medium for 5 days at 30 ◦C. Genomic
DNA was extracted using the Bacteria Genomic DNA Extraction Kit (DongSheng Biotech) according
to the manufacturer’s instructions. For phylogenetic studies, the 16S rRNA gene was amplified
using two universal primers: 27F (5’-GAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-AGAAAGG
AGGTGATCCAGCC-3’) [30]. Amplification reactions were prepared in a 25-
containing 11 L of distilled water, 12.5 L of PCR SuperMix, 1 L of each primer (2.5
0.5 L of extracted DNA (template). PCR was performed under the following conditions: 30 cycles
of 94 ◦C/5 min, 4 ◦C/30 s, 55 ◦C/30 s, 72 ◦C/75 s, and a final extension of 72 ◦C/10 min. PCR products
µL final reaction volume
µ
µ
µ
µM), and
µ

Mar. Drugs 2019, 17, 294
9 of 12
were detected by agarose gel electrophoresis. The obtained sequence of each strain was assembled
with DNASTAR SeqMan (LaserGene, Madison, WI). The 16S rRNA gene sequence was analyzed
using the ExTaxon-e service [31] and the BLASTN program (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
For phylogenetic analysis, multiple sequence alignment was accomplished via the CLUSTAL W
program of the MEGA 5 package [32]. The strain 110B was identified as M. aurantiaca because its 16S
rRNA sequence (accession no: MH333275 in the GenBank) exhibited a high sequence similarity of
100% with that of M. aurantiaca ATCC 27029T (accession no: CP002162).
3.3. Preparation of Crude Extracts
All strains were inoculated into 250-mL Erlenmeyer flasks containing 30 mL of MB medium and
incubated at 28 ◦C for 7 days while shaking at 200 rpm. After cultivation, 60 mL of methanol was
added to each of the cultures. After 24 h, the mixture was filtered and concentrated under vacuum.
The extracts were dissolved in dimethyl sulfoxide for cytotoxic evaluation.
3.4. Fermentation and Extraction of Strain 110B
Strain 110B was inoculated into a 1000-mL flask containing 250 mL of seed culture medium
consisting of (in %) malt extract (1.0), glucose (0.4), yeast extract (0.4), and CaCO3 (0.2), with a pH of
7.2–7.4. Incubation was carried out at 28 ◦C for 3 days on a rotary shaker operating at 250 rpm. Then,
a 5.0% seed culture broth was cultured in a 50-L fermenter (containing 30 L of fermentation medium)
at 28 ◦C for 7 days. The fermentation medium was a modified 2216 medium which contained (per liter
distilled water): soybean cake (20 g), maltodextrin (10 g), peptone (5.0 g), yeast extract (10 g), glucose
(10 g), NaCl (19.45 g), MgCl2
·
6H2O (12.6 g), MgSO4 7H2O (6.64 g), CaCl2 (1.8 g), KCl (0.55 g), NaHCO3
·
(0.16 g), H3BO3 (22.0 mg), KBr (0.08 g), Na2SiO3 (4.0 mg), NaF (2.4 mg), NH4NO3 (1.6 mg), Na2PO4
(8.0 mg), SrCl2·6H2O (57 mg), and ferric citrate (0.1 g).
The final 30 L of fermentation broth was centrifuged to separate the mycelial cake and supernatant.
The mycelial cake was washed with water (3 L) and subsequently extracted with MeOH (3 L).
The supernatant and the wash water were passed through a Diaion HP-20 resin column (Mitsubushi
Chemical Co, Ltd., Tokyo, Japan) and eluted with 95% EtOH. The MeOH extract and the EtOH
◦
eluents were evaporated under reduced pressure to yield a mixture (25 g) at 50 C. The mixture
was chromatographed on a silica gel column (Qingdao Haiyang Chemical Group, Qingdao, China;
100–200 mesh) and successively eluted with CHCl3/MeOH (100:0, 98:2, 95:5, 90:10, 85:15, 80:20, 70:30,
60:40, and 50:50 v/v) to give six fractions (Fr.1–6) based on the TLC profiles. After the Fr.3 was
concentrated in vacuo, the material was subjected to a Sephadex LH-20 gel column (GE Healthcare,
Glies, UK), eluted with CHCl3/MeOH (1:1, v/v), and detected using TLC to give three fractions
(Fr.3-1–3-3). Fr.3-2 was further analyzed and purified by semipreparative reversed-phase HPLC
eluting with CH3CN/H2O (30:70, v/v) to give compounds 1 (tR 15.5 min, 12.2 mg) and 2 (tR 13.5 min,
10.1 mg). Fr.4 was subjected to a Sephadex LH-20 column eluted with CHCl3/MeOH (1:1, v/v) and
detected by TLC to give three subfractions (Fr.4-1–4-3). Fr.4-2 was further separated by semipreparative
reversed-phase HPLC eluting with CH3CN/H2O (30:70, v/v) to give compound
3 (tR 10.6 min, 8.9 mg).
Compound
1
: white powder; [α]2D5−107 (c 0.04, EtOH); IR (KBr) νmax 3663, 3553, 2984, 2905, 1628, 1407,
1049 cm−1; UV (EtOH) λmax (log ) 249 (4.49), 301 (4.10) nm; 1H (400 MHz) and 13C NMR (100 MHz)
ε
data, see Table 2; positive HR-ESIMS m/z 385.1286 [M + H]+ (calcd. for C21H21O7, 385.1282).
Compound
2
: white powder; [α]2D5−112 (c 0.04, EtOH); IR (KBr) νmax 3357, 2984, 2838, 1634, 1408,
1023 cm−1; UV (MeOH) λmax (log ) 249 (4.18), 262 (4.23), 305 (3.76) nm; 1H (400 MHz) and 13C NMR
ε
(100 MHz) data, see Table 2; positive HR-ESIMS m/z 385.1287 [M + H]+ (calcd. for C21H21O7, 385.1282).
Compound
3
: white powder; [α]2D5−27 (c 0.04, EtOH); IR (KBr) νmax 3381, 2987, 1650, 1450, 1407,
1022 cm−1; UV (MeOH) λmax (log ) 249 (4.16), 259 (4.23), 305(3.81) nm; 1H (400 MHz) and 13C NMR
ε
(100 MHz) data, see Table 2; positive HR-ESIMS m/z 515.1912 [M + H]+ (calcd. for C27H31O10, 515.1912).

Mar. Drugs 2019, 17, 294
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3.5. Determination of Aglycone Moieties and Sugars Configuration
Compounds
1 (2.0 mg), 2 (2.0 mg), and 3 (1.5 mg) were refluxed with 2 mL of 2 N HCl and heated
for 2 h at 80 ◦C. After being neutralized with NaHCO3, the hydrolysate was extracted with EtOAc to
separate the organic and aqueous layers. The organic layer was dried in vacuo and subjected to column
chromatography using a Zorbax B-C18 column, mobile phase of CH3CN/H2O (30:70, v/v), flow rate of
1.5 mL/min−1, and detection wavelength at 254 nm. HPLC analysis of the aglycone-containing fractions
of
were observed at 22.47 min, suggesting that the aglycone moieties of
fraction was evaporated in vacuo and the sugar residue was dissolved in pyridine (2 mL) containing
1–
3
gave peaks at 22.34, 22.38, and 22.46 min, respectively, while the tR values for standard daidzein
1–3
were daidzein. The aqueous
l-cysteine methyl ester hydrochloride (1.0 mg), followed by heating at 60 ◦C for 1 h. A 25-
µL solution
of O-tolylisothiocyanate was added to the mixture, which was heated at 60 ◦C for a further 1 h [33].
Likewise, the O-tolylthiocarbamate derivative of 2-deoxy-l-fucose was prepared according to the
method described above. Then, the O-tolylthiocarbamate derivatives were analyzed by reversed-phase
HPLC (Amethyst C18-H, 5
CH3CN/H2O (30:70, v/v). Under these conditions, the derivative of standard sugar gave a peak at tR
(min) = 25.06, while O-tolylthiocarbamate derivatives of the liberated sugars of showed peaks at
µm, 250
×
4.6 mm inner diameter; 0.8 mL/min−1; 250 nm) eluting with
1–3
25.40, 25.02, and 25.02 min, respectively. The sugar moieties of the three new compounds (1–3) were
identified as 2-deoxy-l-fucose.
3.6. Biological Assays
The antimicrobial activity of the three compounds against pathogenic fungi C. albicans, pathogenic
bacteria methicillin-resistant S. aureus, and E. coli was investigated with the minimum inhibitory
concentration (MIC) method recommended by the Clinical and Laboratory Standards Institute [34].
Gentamicin (an antibacterial antibiotic) and amphotericin B (an antifungal antibiotic) were used as a
positive control.
The cytotoxicity of the three compounds was assayed in vitro against the human lung carcinoma
cell line A549, hepatocellular liver carcinoma cell line HepG2, and the human colon tumor cell line
HCT116 by the cell counting kit-8 (CCK8) colorimetric method. The cell lines were cultured in
Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% calf serum at 37 ◦C for 4 h in a 5%
CO2 incubator. The adherent cells of the logarithmic growth stage were digested and seeded in a
96-well culture plate at a density of 1
×
104 cells per/well. Test samples and control were added to the
medium and incubated for 48 h. Then, the cell counting kit-8 (CCK-8, Dojindo, Kumamoto, Japan)
reagent was added to the medium and incubated for 3 h. Cell viability was measured by absorbance at
450 nm using a SpectraMax M5 microplate reader (Molecular Devices Inc., Sunnyvale, CA, USA) [35].
The inhibitory rate of cell proliferation was expressed as IC50 values. Doxorubicin was used as a
positive control, and cell solutions containing 0.5% DMSO were tested as a negative control.
4. Conclusions
The study was designed to isolate mangrove-derived Micromonospora strains and explore their
bioactive metabolites. Fifty-one strains belonging to the genus Micromonospora were isolated, and the
crude extracts of 12 isolates showed cytotoxic activity against the human lung carcinoma cell line
A549. Furthermore, a chemical investigation was carried out on the strain M. aurantiaca 110B. This
investigation led to the isolation of three new isoflavonoid glycosides (compounds 1–3). The structures
of the new compounds were determined by NMR, HR-ESIMS, acid hydrolysis, and HPLC analysis on
the O-tolylthiocarbamate derivatives of sugar moieties. Moreover, the three new compounds were the
result of biotransformation by the strain M. aurantiaca 110B. The three compounds showed moderate
activity against the human lung carcinoma cell line A549, hepatocellular liver carcinoma cell line
HepG2, and the human colon tumor cell line HCT116.

Mar. Drugs 2019, 17, 294
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Supplementary Materials: The following are available online http://www.mdpi.com/1660-3397/17/5/294/s1:
HR-ESIMS and NMR spectra (Figures S1–S29) of compounds 1–3, as well as other supporting data.
Author Contributions: R.-J.W. isolated strain 110B, new compounds, and prepared the manuscript. Y.-H.Y. and
H.Q. tested the biological activities. S.-Y.Z. performed the spectroscopic data analysis. Z.Y. undertook the genome
analysis. H.Z. and Z.-L.X. designed the experiments. J.-D.W. and M.W. determined the final structures of new
compounds and guided the manuscript writing.
Funding: This work was supported by the China Ocean Mineral Resources R&D Association (grant No.
DY135-E2-2-05).
Acknowledgments: The authors thank Yu Liu (Life Science Institute of Zhejiang University) for performing
NMR spectrometry.
Conflicts of Interest: The authors declare no conflict of interest.
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