Actinoflavosides B–D, Flavonoid Type Glycosides from Tidal Mudflat-Derived Actinomyces

Three new secondary metabolites, actinoflavosides B–D (1–3), were discovered in the culture broth of two actinomycete strains (JML48 and JMS33) that were isolated from tidal mudflat sediment in Muan, Republic of Korea. The planar structures of the actinoflavosides were elucidated by MS, UV, and NMR analyses. The stereochemistry of an aminosugar, 2,3,6-trideoxy-3-amino-ribopyranoside in the actinoflavosides was determined by J-based configuration analysis using values obtained from DQF-COSY experiments and modified Mosher’s method. Actinoflavosides B–D (1–3) displayed antibacterial activity against Pseudomonas aeruginosa, and actinoflavoside D (3) significantly increased IL-2 production in mouse splenocytes.

In our search to discover new bioactive secondary metabolites from marine microorganisms, we isolated actinomycete strains from a tidal mudflat in Muan, Republic of Korea [10][11][12]. The tidal mudflat is greatly affected by extreme environmental changes, such as salinity, temperature, and water pressure, caused by tidal differences, and thus has high biodiversity, meeting our expectation of finding actinomycetes that produce various secondary metabolites. Metabolites of isolated strains were screened using liquid chromatography/mass spectrometry (LC/MS) analysis, and the Streptomyces sp. strains JML48 and JMS33 were identified to produce compounds containing a flavonoid moiety based on the characteristic ultraviolet (UV) and mass spectrometry (MS) data (UV λ max = 285, 235, and 215 nm, [M + Na] + m/z at 538, 518, and 480). Further scale-up of the culture of the strains was conducted, and three new compounds, namely actinoflavosides B, C, and D (1-3), were purified by high-performance liquid chromatography (HPLC).  Actinoflavoside C (2) was isolated as a yellow oil, and its molecular formula was determined to be C24H27NO8 based on HR-TOF-MS (m/z [M] + 457.1735 calculated for C24H27NO8, 457.1737) in combination with 1 H and 13 C NMR spectroscopic data ( Table 1). The 1 H NMR spectrum of (2) in DMSO showed features analogous to that of 1, except for the terminal structure of the 2-methyl-3-hydroxy-butyramide moiety in actinoflavoside B Actinoflavoside D (3) was obtained as a red oil, and its molecular formula was assigned as C 25 H 37 NO 9 by HR-TOF-MS (m/z [M] + 495.2447 calculated for C 25 H 37 NO 9 , 495.2468) in combination with 1 H and 13 C NMR data (Table 1). Although 1 and 3 had similar 1 H NMR spectra in DMSO, distinct differences in the chemical shifts of the aglycone moiety were found. Specifically, the chemical shift representing the aromatic ring (δ H 7.59, 7.43, and 7.38) disappeared from the spectrum of (3), while signals for methine (δ H 1.94), methylene (δ H 1.79 and 1.47), and methyl groups (δ H 0.92) were found. The emerged signals were connected by the COSY and TOCSY correlations from H 3 -4' (δ H 0.92) to H-3 (δ H 2.62) ( Figure 2). 495.2468) in combination with 1 H and 13 C NMR data (Table 1). Although 1 and 3 had similar 1 H NMR spectra in DMSO, distinct differences in the chemical shifts of the aglycone moiety were found. Specifically, the chemical shift representing the aromatic ring (δH 7.59, 7.43, and 7.38) disappeared from the spectrum of (3), while signals for methine (δH 1.94), methylene (δH 1.79 and 1.47), and methyl groups (δH 0.92) were found. The emerged signals were connected by the COSY and TOCSY correlations from H3-4' (δH 0.92) to H-3 (δH 2.62) ( Figure 2).   The relative configuration of the aminosugar in actinoflavoside B (1) was determined based on its 3 J HH values, DQF-COSY, and ROESY NMR spectroscopic data. The magnitude of 1 J CH (171 Hz) between C-1" and H-1" established the anomeric carbon at C-1" as an α-configuration. The coupling constants of aminosugar protons measured by DQF-COSY (J H3"H4" = 4.0 Hz and J H4"H5" = 9.0 Hz) indicated that H-3" was in equatorial position and H-4", H-5" were in axial. The ROESY correlations further supported these assignments ( Figure S31). To determine the relative configuration of 2-methyl-3-hydroxy-butyramide, Jbased configuration analysis coupled with methyl decoupling experiments was conducted. The observed large coupling constants of (J H2"'H3"' = 7.5 Hz) established the relative configurations of the stereogenic centers at C-2"' and C-3"' as anti. The absolute configuration of the secondary hydroxy groups at C-4"-OH and C-3"'-OH were determined by a modified version of Mosher's method by utilizing Rand S-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride (MTPA-Cl) and 1 H NMR analysis. The calculated ∆δ S-R values established the absolute configuration as 4"R and 3"'S ( Figure 3). Thus, the structure of actinoflavoside B (1) was defined as the stereoisomer of actinoflavoside A. Actinoflavosides (2) and (3) were assigned to have the same stereochemistry as that determined for 1 based on a careful comparison of chemical shifts, DQF-COSY, and Mosher's method. Stereogenic centers at C-2 of actinoflavosides B-D were confirmed as racemic.

Biological Evaluation
In our endeavor to find new bioactive natural products, among a wide range of untargeted bioactivity tests, actinoflavosides B-D (1-3) showed antibacterial activities and immunomodulatory activity. First, actinoflavosides B-D were measured for growth inhibition activity against pathogenic bacteria, Bacillus subtilis ATCC 6051, Pseudomonas aeruginosa KCTC 22073, Escherichia coli ATCC 11775, and Erwinia rhapontici ATCC 29283. Actinoflavoside B and D inhibited the growth of P. aeruginosa with minimum inhibitory concentration (MIC) values of 0.29 and 0.30 mM, respectively. Actinoflavoside B showed antibacterial activity against B. subtilis with a MIC value of 0.14 mM. Additionally, to study the immunomodulatory activities of actinoflavosides B-D, interleukin-2 (IL-2) cytokine levels were measured in splenocytes isolated from mice. IL-2, a cytokine produced by Th1-type cells, is a T cell growth factor essential for T cell proliferation and differentiation [13,14]. Splenocytes were pretreated with actinoflavosides B-D (100 µg/mL) for 1 h and then concanavalin A (ConA; a T cell activator: 1 µg/mL) for 72 h. As shown in Figure 4, actinoflavoside D significantly increased the IL-2 cytokine production from splenocytes with ConA treatment. Next, we tested the effects of actinoflavoside D on the production of interleukin-4 (IL-4), a cytokine produced by Th2-type cells; this cytokine can produce many responses, some of which are associated with allergy and asthma [15]. Splenocytes were treated with actinoflavoside D (10, 30, or 100 µg/mL) and induced with ConA (1 µg/mL) for 72 h. IL-2 and IL-4 cytokines produced in the cell supernatants were detected by sandwich enzyme-linked immunosorbent assay (ELISA). As shown in Figure 5, actinoflavoside D significantly increased the IL-2 cytokine in ConA-induced splenocytes in a dose-dependent manner. By contrast, actinoflavoside D decreased the IL-4 cytokine production in ConA-induced splenocytes. Taken together, these results suggest that actinoflavoside D plays an important role in the T cell immune response by increasing primary T cell activation and IL-2 cytokine production without Th2-type cell activation. We investigated the effects of actinoflavoside D on splenocyte proliferation in the absence or presence of ConA. Splenocytes isolated from BALB/c mice spleens were treated with actinoflavoside D. As a result, the proliferation of splenocytes was significantly increased by actinoflavoside D (3, 10, 30, or 100 µg/mL) without ( Figure 6A) or with ( Figure 6B) ConA (1 µg/mL). ginosa KCTC 22073, Escherichia coli ATCC 11775, and Erwinia rhapontici ATCC 29283. Actinoflavoside B and D inhibited the growth of P. aeruginosa with minimum inhibitory concentration (MIC) values of 0.29 and 0.30 mM, respectively. Actinoflavoside B showed antibacterial activity against B. subtilis with a MIC value of 0.14 mM. Additionally, to study the immunomodulatory activities of actinoflavosides B-D, interleukin-2 (IL-2) cytokine levels were measured in splenocytes isolated from mice. IL-2, a cytokine produced by Th1type cells, is a T cell growth factor essential for T cell proliferation and differentiation [13,14]. Splenocytes were pretreated with actinoflavosides B-D (100 µg/mL) for 1 h and then concanavalin A (ConA; a T cell activator: 1 µg/mL) for 72 h. As shown in Figure 4, actinoflavoside D significantly increased the IL-2 cytokine production from splenocytes with ConA treatment. Next, we tested the effects of actinoflavoside D on the production of interleukin-4 (IL-4), a cytokine produced by Th2-type cells; this cytokine can produce many responses, some of which are associated with allergy and asthma [15,16]. Splenocytes were treated with actinoflavoside D (10, 30, or 100 µg/mL) and induced with ConA (1 µg/mL) for 72 h. IL-2 and IL-4 cytokines produced in the cell supernatants were detected by sandwich enzyme-linked immunosorbent assay (ELISA). As shown in Figure 5, actinoflavoside D significantly increased the IL-2 cytokine in ConA-induced splenocytes in a dose-dependent manner. By contrast, actinoflavoside D decreased the IL-4 cytokine production in ConA-induced splenocytes. Taken together, these results suggest that actinoflavoside D plays an important role in the T cell immune response by increasing primary T cell activation and IL-2 cytokine production without Th2-type cell activation. We investigated the effects of actinoflavoside D on splenocyte proliferation in the absence or presence of ConA. Splenocytes isolated from BALB/c mice spleens were treated with actinoflavoside D. As a result, the proliferation of splenocytes was significantly increased by actinoflavoside D (3, 10, 30, or 100 µg/mL) without ( Figure 6A) or with ( Figure 6B) ConA (1 µg/mL).   The absorbance was read at 450 nm using an ELISA microplate reader. Data were expressed as mean ± SEM. +++ p < 0.001 compared with the non-treated group. *** p < 0.001 compared with the ConA treated group. Figure 6. Effects of actinoflavoside D on splenocytes proliferation. Splenocytes isolated from BALB/c female mice (7 weeks) were treated with actinoflavoside D (3, 10, 30, or 100 µg/mL) in the (A) absence or (B) presence of ConA for 72 h, and then MTS reagents were added for 1 h. The absorbance was read at 490 nm using an ELISA microplate reader. Data were expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the non-treated group. + p < 0.05, ++ p < 0.01, +++ p < 0.001 compared with the ConA treated group.

General Experimental Procedures
General rotations were measured by a PerkinElmer Model 343 plus polarimeter (Waltham, MA, USA) with a 1.0 cm cell. UV spectra were recorded using an Agilent Technologies 1260 Series Infinity II LC system (Agilent Technologies, Santa Clara, CA, USA). Infrared (IR) spectra were measured with a PerkinElmer spectrum 400 FT-IR and FT-NIR spectrometer (Waltham, MA, USA). 1 H, 13 C, and 2D NMR spectra were acquired on a Varian Unity INOVA 600 MHz spectrometer at the Korea Basic Science Institute (KBSI) at Gwangju and on a Bruker Avance II 800 MHz NMR spectrometer (Bruker, Billerica, MA, USA) at the KBSI at Ochang. Electrospray ionization (ESI) low-resolution LC/MS data were acquired on an Agilent G6125B MSD system coupled with an Agilent Technologies 1260 Series Infinity II LC system using a Phenomenex Luna reversed-phase C18 column (100 × 4.6 mm, 5 µm). High-Resolution TOF Mass Spectrum Field Desorption ion source data were acquired using a JMS-T200GC (Jeol, Akishima, Tokyo) at the Chonnam National University Cooperative Center for Research Facilities (CCRF). High-resolution electrospray ionization (HR-ESI) mass spectra were obtained using an Agilent Technologies 1290 Series HPLC coupled to an Agilent 6530 iFunnel Q-TOF LC/MS system (Agilent Technologies, Santa Clara, CA, USA). Figure 6. Effects of actinoflavoside D on splenocytes proliferation. Splenocytes isolated from BALB/c female mice (7 weeks) were treated with actinoflavoside D (3, 10, 30, or 100 µg/mL) in the (A) absence or (B) presence of ConA for 72 h, and then MTS reagents were added for 1 h. The absorbance was read at 490 nm using an ELISA microplate reader. Data were expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the non-treated group. + p < 0.05, ++ p < 0.01, +++ p < 0.001 compared with the ConA treated group.

General Experimental Procedures
General rotations were measured by a PerkinElmer Model 343 plus polarimeter (Waltham, MA, USA) with a 1.0 cm cell. UV spectra were recorded using an Agilent Technologies 1260 Series Infinity II LC system (Agilent Technologies, Santa Clara, CA, USA). Infrared (IR) spectra were measured with a PerkinElmer spectrum 400 FT-IR and FT-NIR spectrometer (Waltham, MA, USA). 1 H, 13 C, and 2D NMR spectra were acquired on a Varian Unity INOVA 600 MHz spectrometer at the Korea Basic Science Institute (KBSI) at Gwangju and on a Bruker Avance II 800 MHz NMR spectrometer (Bruker, Billerica, MA, USA) at the KBSI at Ochang. Electrospray ionization (ESI) low-resolution LC/MS data were acquired on an Agilent G6125B MSD system coupled with an Agilent Technologies 1260 Series Infinity II LC system using a Phenomenex Luna reversed-phase C 18 column (100 × 4.6 mm, 5 µm). High-Resolution TOF Mass Spectrum Field Desorption ion source data were acquired using a JMS-T200GC (Jeol, Akishima, Tokyo) at the Chonnam National University Cooperative Center for Research Facilities (CCRF). High-resolution electrospray ionization (HR-ESI) mass spectra were obtained using an Agilent Technologies 1290 Series HPLC coupled to an Agilent 6530 iFunnel Q-TOF LC/MS system (Agilent Technologies, Santa Clara, CA, USA).

Bacterial Isolation
The strains JML48 and JMS33 were isolated in August 2020 from a tidal mudflat sample in Muan, Republic of Korea. Dried sediment (2 g) was diluted in 4 mL of sterilized seawater, and the mixture was spread onto actinomycete isolation medium. The JML48 strain was isolated by culture on A1 agar medium (18 g agar, 25 mg cycloheximide, and 10 mg of nalidixic acid in 1 L seawater), and JMS33 was isolated by culture on TWYE agar medium (0.25 g of yeast extract, 0.5 g K 2 HPO 4 , 18 g agar, 25 mg cycloheximide, and 10 mg of nalidixic acid in 1 L seawater). The JML48 and JMS 33 strain were the most closely related to Streptomyces althioticus (99.9% identity, accession #LN864578), and Streptomyces sanglieri (99.0% identity, accession #AB735535) based on 16S rDNA sequencing analysis data.

Cultivation and Extraction
The JML48 was cultivated in 50 mL of YEME medium (3 g of yeast extract, 3 g of malt extract, 5 g peptone, 2 g soytone, and 10 g glucose in 1 L seawater) in a 100 mL Erlenmeyer flask. After the strains were cultivated at 25 • C for 3 days at 190 rpm on a rotary shaker, 3.5 mL of the culture was inoculated into a 500 mL Erlenmeyer flask containing 150 mL of YEME medium and shaken for 2 days. Then, 20 mL of the culture was inoculated into 1 L of YEME medium in a 2.5 L Ultra Yield flask. JML48 (8 L) was incubated at 25 • C and 190 rpm has isobutyl alkylated chromone structure modified from a flavonoid structure, which is a very unique structure that has not been reported in natural products derived from microorganisms and plants. This study not only draws attention to the discovery of natural products after a very long time but is also important in reporting the new biological activity of the metabolites. In recent medicine, which emphasizes the treatment and prevention of infectious diseases worldwide, our research on marine microbial natural products suggests an effort to discover immunomodulatory therapeutic compounds.

Patents
This section is not mandatory but may be added if there are patents resulting from the work reported in this manuscript.