Three New Isoflavonoid Glycosides from the Mangrove-Derived Actinomycete Micromonospora aurantiaca 110B

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-4′-(2-deoxy-α-l-fucopyranoside) (1), daidzein-7-(2-deoxy-α-l-fucopyranoside) (2), and daidzein-4′,7-di-(2-deoxy-α-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.


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 27029 T (CP002162).
Due to the superior cytotoxic activity of strain 110B, further chemical investigations were performed on this strain. diversity of actinomycetes [7]. As one of the genera of actinomycetes, the genus Micromonospora is an important prolific producer of secondary metabolites [8,9].
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)(2)(3) were isolated from its fermentation broth. Herein, we describe the isolation, structure elucidation, and cytotoxic activity of these three new compounds.

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 27029 T (CP002162).

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).

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 2 was obtained as a white powder with a negative specific rotation value ([α] 25 D −112, EtOH). The molecular formula of 2 was established as C 21 H 20 O 7 from positive-ion HR-ESIMS ( Figure S10) and 13 C 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 1 H NMR spectrum (Table 1)  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 (δ C 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).   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  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   Figure S19) and 13 C NMR spectral data ( Table 1). The IR spectrum ( Figure S20) ), indicating that the isoflavone aglycone of 3 was identical to 1 and 2. Two sugar moieties were evident from the anomeric proton signals at δ H 5.84 (1H, br d, J = 3.5 Hz) and δ H 5.69 (1H, br d, J = 3.5 Hz) in the 1 H NMR spectrum. The 1 H and 13 C NMR spectral ( Figures S21-S23) data of 3 were similar to those of 1 and 2, except for an extra 2,6-dideoxysugar moiety. The HMBC ( Figure S26) correlations from H-1 to C-4 and H-1 to C-7 revealed that the two 2,6-dideoxysugar moieties were located at C-4 and C-7, respectively. The sugar moieties of 3 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, the structure of 3 was determined to be daidzein-4 ,7-di-(2-deoxy-α-l-fucopyranoside), as shown in Figure 3.
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.
Mar. Drugs 2019, 17, x 7 of 13 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.

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 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.

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 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.

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.
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.
. . Figure 6. Alignment of the deduced amino acid sequence of AXH94677.

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. 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.

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 NaHCO 3, 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 CH 3 CN/H 2 O (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 1-3 gave peaks at 22.34, 22.38, and 22.46 min, respectively, while the t R values for standard daidzein were observed at 22.47 min, suggesting that the aglycone moieties of 1-3 were daidzein. The aqueous fraction was evaporated in vacuo and the sugar residue was dissolved in pyridine (2 mL) containing 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 µm, 250 × 4.6 mm inner diameter; 0.8 mL/min −1 ; 250 nm) eluting with CH 3

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% CO 2 incubator. The adherent cells of the logarithmic growth stage were digested and seeded in a 96-well culture plate at a density of 1 × 10 4 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 IC 50 values. Doxorubicin was used as a positive control, and cell solutions containing 0.5% DMSO were tested as a negative control.

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.