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Article

Antimicrobial Lavandulylated Flavonoids from a Sponge-Derived Streptomyces sp. G248 in East Vietnam Sea

1
Advanced Center for Bioorganic Chemistry, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), Hanoi 11307, Vietnam
2
Faculty of Chemistry, Graduate University of Science and Technology, VAST, Hanoi 11307, Vietnam
3
Institute of Applied Science and Technology, University Industry of Vinh, Vinh 43115, Vietnam
4
Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA
5
Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60607, USA
*
Author to whom correspondence should be addressed.
Mar. Drugs 2019, 17(9), 529; https://doi.org/10.3390/md17090529
Received: 16 August 2019 / Revised: 30 August 2019 / Accepted: 6 September 2019 / Published: 10 September 2019

Abstract

:
Three new lavandulylated flavonoids, (2S,2″S)-6-lavandulyl-7,4′-dimethoxy-5,2′-dihydroxylflavanone (1), (2S,2″S)-6-lavandulyl-5,7,2′,4′-tetrahydroxylflavanone (2), and (2″S)-5′-lavandulyl-2′-methoxy-2,4,4′,6′-tetrahydroxylchalcone (3), along with seven known compounds 4–10 were isolated from culture broth of Streptomyces sp. G248. Their structures were established by spectroscopic data analysis, including 1D and 2D nuclear magnetic resonance (NMR), and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The absolute configurations of 1–3 were resolved by comparison of their experimental and calculated electronic circular dichroism spectra. Compounds 1–3 exhibited remarkable antimicrobial activity. Whereas, two known compounds 4 and 5 exhibited inhibitory activity against Mycobacterium tuberculosis H37Rv with minimum inhibitory concentration (MIC) values of 6.0 µg/mL and 11.1 µg/mL, respectively.

Graphical Abstract

1. Introduction

Marine sponges are known to harbor diverse microbial communities [1,2,3,4]. These marine-derived microorganisms have been a rich source for a multitude of biomedically relevant secondary metabolites [5,6,7]. Among actinomycetes, the genus Streptomyces is considered to be the most prolific producer of secondary metabolites for medical, agricultural and veterinary usage [8,9,10]. Previous reports showed that the Streptomyces genus has the ability to produce a variety of secondary metabolites with various activities, including antimicrobial, anticancer, and antiparasitic [11,12,13].
In search of bioactive metabolites from marine-derived actinomycetes, the Streptomyces sp. G248 (Figures S1–S3), from the sponge Halichondria panicea (Pallas, 1766) collected from the coast of Da Nang in Vietnam was selected as its extract displayed antimicrobial activity against pathogenic Enterococcus faecalis and Staphylococcus aureus strains. Herein, we report the isolation and structural characterization of three new lavanludylated flavonoids (13) and seven known compounds (410) from the fermentation broth of the Streptomyces sp. G248 (Figure 1). Furthermore, their antimicrobial and cytotoxic activity evaluations were also discussed.

2. Results and Discussion

2.1. Isolation and Structural Elucidation

Culture broth solution (50 L) of Streptomyces sp. G248 was desalted by an Amberlite XAD-16 column, eluting with distilled water, then with methanol. The methanol solution was concentrated under reduced pressure. The crude extract (12.6 g) was purified by repeated open column chromatography to give compounds 110.
Compound 1 was isolated as yellow amorphous solid, and was optically active {[α]25D -5 (c 0.176, MeOH)}. Its positive HR-ESI mass spectrum (Figure S15) exhibited the proton adduct ion [M + H]+ at m/z 453.2270 (calcd. for C27H33O6, 453.2277) which, together with 13C NMR data, is consistent with the molecular formula of C27H32O6. The IR spectrum (Figure S16) indicated the absorption bands at νmax 3366 cm−1 (OH) and 1696 cm−1, (C=O) functionalities. In the 1H NMR spectrum (Figure S9), the presence of an ABX system at δH 6.49 (d, J = 2.3 Hz, H-3′), 6.47 (dd, J = 2.3, 8.5 Hz, H-5′) and 7.38 (d, J = 8.5 Hz, H-6′), and a singlet proton at δH 6.12 (s, H-8) was observed at the aromatic region. Additionally, the resonances of protons at δH 5.55 (H-2), 4.96 (H-4″), 4.52 (Ha-9″) and 4.59 (Hb-9″), two methoxy groups at δH 3.82 and 3.83, three singlet methyls at δH 1.48 (CH3-6″), 1.57 (CH3-7″) and 1.64 (CH3-10″) and seven aliphatic protons from 1.80 to 3.10 ppm were also noted. Analysis of the 13C NMR spectrum (Figure S10) with the aid of an HSQC experiment (Figure S12) revealed 27 carbon resonances for 1, including one ketone group (δC 193.6, C-4), ten sp2 quaternary carbons, five sp2 methines, one sp2 methylene, two sp3 methines, three sp3 methylenes, two methoxy groups and three methyls. Beside the aromatic ABX system, two other spin-spin coupling systems were revealed from the COSY spectrum (Figure S11): H-2 (δH 5.55)/CH2-3 (δH 2.67 and 2.89) (I), and CH2-1″ (δH 2.63)/H-2″ (δH 2.50)/CH2-3″ (δH 2.02)/H-4″ (δH 4.96) (II). The chemical shifts of carbons at δC 75.0 (C-2), 160.1 (C-2′), 159.0 (C-4′), 164.7 (C-5), 165.4 (C-7) and 161.9 (C-9) suggested their linkage to oxygen (Table 1). In the HMBC spectrum (Figure S13), cross-peaks of C-2″ (δC 48.2) with protons of CH3-10″ (δH 1.64) and CH2-9″ (δH 4.52 and 4.59), and those of C-8″ (δC 149.8) with protons of CH3-10″ indicated the linkage of the substructure II with the isopropenyl group through the C-2″/C-8″ bond. Additionally, HMBC correlations of C-4″ (δC 124.8) and C-5″ (δC 132.0) with protons of CH3-6″ (δH 1.48) and CH3-7″ (δH 1.57) defined the presence of a lavandulyl group in the structure of 1. The presence of the A-ring was confirmed by the HMBC cross-peaks of H-8 (δH 6.12) with C-6 (δC 109.6), C-7 (δC 165.4), C-9 (δC 161.9) and C-10 (δC 105.7). The aromatic ABX system (B-ring) was confirmed by HMBC correlations of H-3′ (δH 6.49) with C-1′ (δC 119.7), C-2′ (δC 160.1), C-4′ (δC 159.0) and C-5′ (δC 108.1). The connection of C-2 with the B-ring at C-1′ was revealed by cross-peaks of H-2 (δH 5.55) with C-1′ (δC 119.7) and C-6′ (δC 128.5). The spectral features of coupling systems I and the carbonyl group deduced from HMBC signals indicated structural similarity of 1 to flavanone compounds. Furthermore, the attachment of the lavandulyl group to the A-ring at C-6 via C-6/C-1″ linkage was revealed by HMBC cross-peaks of the protons of CH2-1″ (δH 2.63) with C-5 (δC 164.7), C-6 (δC 109.6) and C-7 (δC 165.4), and was supported by the correlations of the methoxy protons at C-7 with the protons of CH2-1″) in the ROESY spectrum of 1 (Figure S14). Finally, the two methoxy groups at δH 3.82 and 3.83 were attached to C-7 and C-4′ as indicated by their correlations in the HMBC spectrum (Figure 2). Complete analyses of 2D NMR spectra established the planar structure of 1 as 6-lavandulyl-7,4′-dimethoxy-5,2′-dihydroxylflavanone.
The 1D NMR spectra of compound 2 (Figures S18, S19 and S23) displayed resonances close to those of 1, except the absence of two methoxy groups. Complete analysis of 2D NMR spectra (Figures S20–S22 and S24) established the planar structure of 2 as 6-lavandulyl-5,7,2′,4′-tetrahydroxylflavanone (Figures S25 and S26), which shared the same planar structure with kushnol F, previously isolated from Sophora flavescens [14]. However, the absolute configuration of the chiral carbon C-2″ has not been determined. Moreover, compound 2 had an optical rotation activity of +3 (c 0.2, MeOH), while this value is -59.8 (c 0.82, MeOH) for kushnol F [14].
Compound 3 was isolated as yellow amorphous solid and optically active {[α]25D -1.8 (c 0.54, MeOH}. Its positive HR-ESI-MS (Figure S35) showed the proton adduct ion [M + H]+ at m/z 439.2117 (calcd. for C26H31O6, 439.2121), consistent with a molecular formula of C26H30O6. The 1D NMR spectra of 3 (Figures S28, S29 and S33) exhibited the resonances for the A- and B-ring systems, and the lavandulyl group as found in compounds 1 and 2. However, signals of a CH=CH system were observed in the 1D NMR spectra of 3 instead of resonances of the CH-2/CH2-3 spin system in the structures of 1 and 2.
Analysis of 2D NMR spectra of 3 (Figures S30–S32 and S34) confirmed the presence of the A and B-rings, and the lavandulyl substructure. Additionally, cross-peaks of H-β at δH 8.02 with C-2 (δC 160.3), C-6 (δC 131.6) and carbonyl carbon C-γ (δC 194.8) in the HMBC spectrum (Figure S32) indicated a chalcone skeleton for 3 (Figure S36). The methoxy group at C-2′ was revealed by an HMBC correlation of C-2′ at δC 162.3 with the methoxy protons at δH 3.91 (Figure 3). As in the case of compounds 1 and 2, the location of the lavandulyl group at C-5′ was established by the HMBC cross-peaks of C-4′ (δC 164.1), C-5′ (δC 108.9) and C-6′ (δC 166.6) with the protons of CH2-1″ (δH 2.65). The planar structure of 3 was finalized as 5′-lavandulyl-2′-methoxy-2,4,4′,6′-tetrahydroxylchalcone. This compound has the same planar structure as kuraridin, which was previously isolated from Albizzia julibrissin [15]. The optical rotation activity of kuraridin {[α]D20 +1.2 (c 0.025, MeOH)} [13] is opposite to that of compound 3 {[α]D25 –1.8 (c 0.54, MeOH)}, and the absolute configuration at C-2″ of kuraridin has not been reported.
Absolute configurations of the compounds 13 were resolved by comparison of their experimental and calculated electronic circular dichroism (ECD) spectra [16,17]. The ECD quantum chemical calculations were performed using the Gaussian 09 software [18]. To obtain minimum energy conformers, geometry optimization of each possible isomer of these compounds was conducted (Tables S1–S5 and Figures S4–S8). The calculated ECD spectra of compounds 13 were generated using the time-dependent density functional method at the B3LYP/LanL2DZ level. Since the circular dichroism (CD) spectrum of compound 1 (Figure S17) had a positive Cotton effect at 337 nm (Δε +3.6) and a negative Cotton effect at 291 nm (Δε −7.7), the S-configuration was thus assigned for carbon C-2 of 1 [19]. The ECD spectra of the two possible diastereomers (2S,2″S)-1 and (2S,2″R)-1 were generated. As shown in Figure 4, the ECD spectrum of (2S,2″S)-1 was similar as the experimental CD spectrum of 1, while an ECD spectrum of (2S,2″R)-1 displayed the opposite Cotton effect at 233 nm. The S-configuration was thus assigned for the chiral carbon C-2″ of 1. Similarly, the S-configuration at C-2 was determined for 2 by the observation of a positive Cotton effect at 315 nm (Δε +0.8) and a negative Cotton effect at 291 nm (Δε −5.9) in the CD spectrum of 2 (Figure S27). By comparison of the ECD spectra of the two diastereomers (2S,2″S)-2 and (2S,2″R)-2 with the experimental CD spectrum of 2 (Figure 5), the S-configuration was also assigned for C-2″ of 2. The ECD of (2″S)-3 was in good agreement with the experimental CD spectrum of 3 (Figure S37). This observation allowed determination the S-configuration for carbon C-2″ for 3 (Figure 6). Since the optical rotation activities of 2 and 3 are comparatively opposite with kushnol F and kuraridin, respectively, the absolute configuration at C-2″ of kushnol F and kuraridin would most likely be R, however further experiments are required to state this definitively.
Other known compounds, (2S,2″S)-6-lavandulyl-7-methoxy-5,2′,4′-trihydroxylflavanone (4) [20], 6-prenyl-4′-methoxy-5,7-dihydroxylflavanone (5) [21], norharman (β-carboline) (6) [22,23], cyclo-(Pro-Leu) (7) [24], cyclo-(Pro-Phe) (8) [25], cyclo-(Pro-Tyr) (9) [26], and adenine (10) [27] were also isolated and characterized from the culture broth of G248 strain. Their structures were established by spectroscopic data analysis and comparison with data reported in the literature.

2.2. Biological Activity

Compounds 110 were evaluated for antibacterial activity against Enterococcus faecalis (ATCC13124), Staphylococcus aureus (ATCC25923), Bacillus cereus (ATCC13245), Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), Salmonella enterica (ATCC12228), antiyeast activity against Candida albicans (ATCC1023), and in a separate experiment for anti-TB activity against Mycobacterium tuberculosis H37Rv. Compounds 13 exhibited antimicrobial activity, 2 exhibiting the most potent activity profile of the group. Compound 3 seems to have a broad spectrum of antimicrobial activity as it was active against all tested microbial strains (with the exception of M. tuberculosis H37Rv). Except against E. coli, compounds 1 and 2 showed significant inhibition activity against the remaining tested strains. The two known compounds 4 and 5 exhibited IC90 values of 6.0 and 11.1 µg/mL against M. tuberculosis H37Rv, respectively. This is the first report of the anti-TB activity for these two compounds (Table 2). Since, compound 4 exhibited anti-TB activity while 1 and 2 had no effect against M. tuberculosis, the methoxy group at C-7 and hydroxyl functionality at C-4′ seem to be critical for the anti-TB activity of these compounds. Cytotoxicity evaluation indicated that compounds 4 and 5 had inhibition against four tested cancer cell lines, KB, Hep-G2, Lu-1 and MCF-7 with IC50 values from 2.0 to 14.6 µg/mL (Table 3). Other compounds had weak or no cytotoxicity against these four tested cancer cell lines.

3. Materials and Methods

3.1. General Experimental Procedures

Optical rotations were recorded on a Polax-2L polarimeter in MeOH. HR-ESIMS were recorded on a FT-ICR 910-MS TQFTMS-7 T mass spectrometer. CD spectra were taken on a Chirascan CD spectrometer. IR spectra were recorded on a Nicolet Impact 410 FT-IR spectrometer, and NMR spectra on a Bruker AM500 MHz spectrometer operating at 125.76 MHz for 13C NMR, and at 500.13 MHz for 1H NMR. 1H chemical shifts were referenced to CD3OD at δ 3.31 ppm, while the 13C chemical shifts were referenced to the central peak at δ 49 ppm. For HMBC experiments the delay (1/2J) was 70 ms.

3.2. Actinomycete Material

The actinomycete strain G248 was obtained from the Halichondria panicea (Pallas, 1766) sponge (sponge taxonomy was identified by Prof. Do Cong Thung, Institute of Marine Environment and Resources—Vietnam Academy of Science and Technology), collected in Son-Tra island (Da Nang)—Vietnam in August 2016. A voucher specimen was deposited at the Institute of Marine Environment and Resources, Vietnam Academy of Science and Technology (VAST).
The strain G248 was isolated from above-mentioned sponge sample. The taxonomy of the strain G248 was identified by using 16S rRNA gene sequence analysis. Its gene sequence was registered with GenBank access code MG917690 (Figure S3). On the basis of morphological and phylogenetic evidence (Figures S1 and S2), the actinomycete strain G248 was assigned to the genus Streptomyces.

3.3. Fermentation and Extraction

Strain G248 was activated and inoculated into 1 L of A1 medium pH 7.0 comprising starch (5.0 g), yeast extract (2.0 g), peptone (1.0 g) and artificial sea salt (30.0 g) in 1.0 L of distilled water. After 7 days of incubation at 28 °C with agitation, the culture broth was used to inoculate the fermentation in 50 L of high-nutrient medium A1+ (soluble starch: 10 g/L; yeast extract: 4 g/L; peptone: 2 g/L; instant ocean: 30 g/L; CaCO3: 1 g/L; agar: 15 g/L; add water to 1 L, pH 7.0). The fermentation was incubated at 28 °C with agitation of 200 rpm and harvested on the tenth day.

3.4. Isolation and Purification

The fermentation broth (50 L) was passed through a XAD-16 column (10 kg XAD-16). The column was washed with distilled water (70 L), followed by eluting with methanol (80 L). The methanol solution was concentrated under reduced pressure. The crude extract (12.6 g) was purified by column chromatography (CC) on silica gel, eluted with CH2Cl2/EtOH gradient to give 11 fractions. Fraction F4 (1.23 g) was separated by CC on Sephadex LH-20 (CH2Cl2/MeOH: 9/1), providing 4 subfractions. Subfraction F4.3 (90 mg) was purified by CC on silica gel (0 to 50% MeOH in CH2Cl2) to furnish compounds 7 (5 mg). Fraction F5 (1.72 g) was subjected to CC on silica gel (0 to 100% MeOH in CH2Cl2), leading to four subfractions. Subfraction F5.3 (305 mg) was separated by CC on silica gel, eluted with a CH2Cl2/acetone gradient, providing 4 sub-subfractions. Sub-subfraction F5.3.4 was chromatographed on silica gel, eluted with EtOAc/MeOH gradient, giving compounds 6 (5 mg) and 8 (15 mg). Fraction F6 (550 mg) was purified by CC on silica gel (0 to 100% MeOH in CH2Cl2) providing 4 subfractions. Subfraction F6.4 (1.16 g) was separated by CC on Sephadex LH-20 (MeOH/CH2Cl2: 9/1), followed by preparative thin-layer chromatography (TLC) (CH2Cl2/MeOH: 8.5/1.5) to furnish compounds 9 (3.9 mg). Fraction 9 (360 mg) was separated by CC on Sephadex LH-20 (MeOH/CH2Cl2: 9/1), leading to 7 subfractions. Subfraction F9.5 (35 mg) was purified by CC on silica gel (0 to 100% MeOH in EtOAc), followed by preparative TLC (EtOAc/MeOH: 8/2) to furnish compound 10 (5.9 mg). Fraction 10 (1.62 g) was separated by CC on silica gel (0 to 100% MeOH in CH2Cl2), furnishing six subfractions. Subfraction F10.2 (55 mg) was subjected to CC on Sephadex LH-20 (MeOH/CH2Cl2: 9/1), followed by preparative TLC (CH2Cl2/acetone: 7/3) to yield compounds 1 (2.5 mg) and 5 (3.7 mg). Subfraction F10.4 (40 mg) was chromatographed on Sephadex LH-20 column (MeOH/CH2Cl2: 9/1), affording compounds 2 (3.0 mg) and 3 (6.0 mg). Finally, subfraction F10.5 (70 mg) was subjected to CC on Sephadex LH-20 (MeOH/CH2Cl2: 9/1), followed by preparative TLC (CH2Cl2/acetone: 7/3) to afford compound 4 (3.9 mg).

3.5. Spectral Data

(2S,2″S)-6-lavandulyl-7,4′-dimethoxy-5,2′-dihydroxylflavanone (1)—amorphous yellow solid; [α]D25 -5 (c 0.176, MeOH); Rf: 0.7 (CH2Cl2-MeOH, 8.5:1.5); IR (KBr): 3366, 2916, 1696, 1497, 1457, 1411, 1376, 1278, 1151, 1093, 974, 886 cm−1; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz), Table 1; CD (MeOH) nm (Δε): 205 (−10.2), 225 (+12.8), 259 (+0.7), 291 (−7.7), 337 (+3.6); HR-ESI-MS m/z 453.2270 [M + H]+ (calcd. for C27H33O6, 453.2277).
(2S,2″S)-6-lavandulyl-5,7,2′,4′-tetrahydroxylflavanone (2)—amorphous yellow solid; [α]D25 +3 (c 0.2, MeOH); Rf: 0.45 (CH2Cl2-MeOH, 8.5:1.5); IR (KBr): 3261, 2961, 2921, 1631, 1604, 1514, 1439, 1382, 1296, 1162, 1083, 974, 887 cm−1; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz), Table 1; CD (MeOH) nm (Δε): 204 (−6.5), 220 (+8.4), 258 (+0.6), 291 (−5.9), 315 (+0.8); HR-ESI-MS m/z 425.1960 [M + H]+ (calcd. for C25H29O6, 425.1964).
(2″S)-5′-lavandulyl-2′-methoxy-2,4,4′,6′-tetrahydroxylchalcone (3)—amorphous yellow solid; [α]D25 -1.8 (c 0.54, MeOH); Rf: 0.33 (CH2Cl2-MeOH, 8.5:1.5); IR (KBr): 3695, 2925, 2854, 1605, 1546, 1449, 1233, 1141, 1104 cm−1; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz), Table 1; CD (MeOH) nm (Δε): 212 (+0.9), 259 (+0.5), 291 (−0.2), 319 (+0.06); HR-ESI-MS m/z 439.2117 [M + H]+ (calcd. for C26H31O6, 439.2121).

3.6. Antimicrobial Activity Assay

Antimicrobial assays were carried out using E. coli (ATCC25922), P. aeruginosa (ATCC27853), S. enterica (ATCC12228), E. faecalis (ATCC13124), S. aureus (ATCC25923), B. cereus (ATCC13245), and C. albicans (ATCC1023). Stock solutions of samples were prepared in DMSO, and the antimicrobial assays were carried out in 96-well microtiter plates against the microbial strains (5 × 105 CFU/mL) using a modification of the published method [28]. After incubation for 24 h at 37 °C, the absorbance at 650 nm was measured using a microplate reader. Streptomycin and nystatin were used as reference compounds.

3.7. Anti-Mycobacterial Activity Assay

M. tuberculosis H37Rv (ATCC 27294) was purchased from American Type Culture Collection (ATCC). These strains were cultured to late log phase in Middlebrook 7H9 broth supplemented with 0.2% (vol/vol) glycerol, 0.05% Tween™ 80, and 10% (v/v) oleic acid-albumin-dextrose-catalase (OADC). The culture was harvested and resuspended in phosphate-buffered saline. Suspensions were then filtered through 8 μm filter membranes and frozen at ‒80 °C. Prior to use of bacterial stocks for the anti -TB assay, colony-forming units (CFUs) were determined by plating on 7H11 agar media. The MIC is defined here as the lowest concentration resulting in ≥90% growth inhibition of the bacteria relative to untreated controls. MIC against replicating M. tuberculosis was measured by the Microplate Alamar Blue Assay (MABA) in 7H12 media [29].

3.8. Cytotocicity Assay

An MTT assay was used to determine the cytotoxic activity of compounds 110 with human cancer cell lines (KB, LU-1, Hep-G2 and MCF-7) acquired from the American Type Culture Collection (ATCC, Manassas, VA) using a modification of the published method [30]. Cells were cultured in medium RPMI 1640 supplemented with 10% FBS (fetal bovine serum) under a humidified atmosphere of 5% CO2 at 37 °C. Compounds 110 were dissolved in DMSO at a concentration of 20 mg/mL. A series of dilutions for each compound was prepared to final concentrations of 128, 32, 8, 2 and 0.5 mg/mL. Samples (100 µL) of the complexes with different concentrations were added to the wells on 96-well plates. Cells were separated with trypsin and ethylenediaminetetraacetic acid (EDTA), and seeded in each well with 3 × 104 cells per well. An MTT solution (20 µL, 4 mg/mL) of phosphate buffer saline (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 per L) was added to each well after being incubated for 48 h. The cells were further incubated for 4 h and a purple formazan precipitate was formed, which was separated by centrifugation. The precipitate was dissolved by adding DMSO (100 µL) to each well. The optical density of the solution was determined by a plate reader (TECAN) at 540 nm. The inhibition ratio was achieved on the basis of the optical densities from the calculation of three replicate tests.

Supplementary Materials

The following are available online at https://www.mdpi.com/1660-3397/17/9/529/s1, Figure S1: Morphological appearance of strain G248, Figure S2: Gel electrophoresis of PCR product, Figure S3: 16S rRNA gene sequence of Streptomyces sp. G248 on GenBank, Figure S4: Optimized conformers of (2S,2″R)-1, Figure S5: Optimized conformers of (2S,2″S)-1, Figure S6: Optimized conformers of (2S,2″R)-2, Figure S7: Optimized conformers of (2S,2″S)-2, Figure S8: Optimized conformers of (2″S)-3, Figure S9: 1H NMR spectrum of 1 (500 MHz, CD3OD), Figure S10: 13C NMR spectrum of 1 (125 MHz, CD3OD), Figure S11: 1H-1H COSY spectrum of 1 (500 MHz, CD3OD), Figure S12: HSQC spectrum of 1 (1H: 500 MHz, 13C: 125 MHz, CD3OD), Figure S13: HMBC spectrum of 1 (1H: 500 MHz, 13C: 125 MHz, CD3OD), Figure S14: ROESY spectrum of 1 (500 MHz, CD3OD), Figure S15: HR-ESI-MS of 1, Figure S16: IR spectrum of 1, Figure S17: CD spectrum of 1, Figure S18: 1H NMR spectrum of 2 (500 MHz, CD3OD), Figure S19: 13C NMR spectrum of 2 (125 MHz, CD3OD), Figure S20: 1H-1H COSY spectrum of 2 (500 MHz, CD3OD), Figure S21: HSQC spectrum of 2 (1H: 500 MHz, 13C: 125 MHz, CD3OD), Figure S22: HMBC spectrum of 2 (1H: 500 MHz, 13C: 125 MHz, CD3OD), Figure S23: 1H NMR spectrum of 2 (500 MHz, DMSO-d6), Figure S24: ROESY spectrum of 2 (500 MHz, DMSO-d6), Figure S25: HR-ESI-MS of 2, Figure S26: IR spectrum of 2, Figure S27: CD spectrum of 2, Figure S28: 1H NMR spectrum of 3 (500 MHz, CD3OD), Figure S29: 13C NMR spectrum of 3 (125 MHz, CD3OD), Figure S30: 1H-1H COSY spectrum of 3 (500 MHz, CD3OD), Figure S31: HSQC spectrum of 3 (1H: 500 MHz, 13C: 125 MHz, CD3OD), Figure S32: HMBC spectrum of 3 (1H: 500 MHz, 13C: 125 MHz, CD3OD), Figure S33: 1H-NMR spectrum of 3 (500 MHz, DMSO-d6), Figure S34: ROESY spectrum of 3 (500 MHz, DMSO-d6), Figure S35: HR-ESI-MS of 3, Figure S36: IR spectrum of 3, Figure S37: CD spectrum of 3, Table S1. Conformational population (%) for the most stable conformers of (2S,2″R)-1, Table S2. Conformational population (%) for the most stable conformers of (2S,2″S)-1, Table S3. Conformational population (%) for the most stable conformers of (2S,2″R)-2, Table S4. Conformational population (%) for the most stable conformers of (2S,2″S)-2, Table S5: Conformational population (%) for the most stable conformers of (2″S)-3.

Author Contributions

V.C.P., B.T.M., V.M.C. and H.D.T.M., contributed to the co design of the study and wrote the manuscript. D.D.C., T.T.V.T, D.T.T and V.N.V. performed experiments and analyzed data. Q.V.T., M.A.N. and T.T.D. did the isolation and identification of the actinomyces. H.M.L. performed the antimicrobial activity assay. S.C., R.M. and G.S. performed anti-TB assay. All authors contributed to the manuscript revision, and read and approved the submitted version.

Funding

This research was financially supported by the Survey of Marine Resources and Environmental Management Program of Vietnam (Program 47, Grant number VAST.ĐA47.12/16-19), and by the Fogarty International Center of the National Institutes of Health under Award Number D43TW010530. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Health.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Structures of compounds 110.
Figure 1. Structures of compounds 110.
Marinedrugs 17 00529 g001
Figure 2. Key HMBC correlations of 1.
Figure 2. Key HMBC correlations of 1.
Marinedrugs 17 00529 g002
Figure 3. Selected HMBC correlations of 3.
Figure 3. Selected HMBC correlations of 3.
Marinedrugs 17 00529 g003
Figure 4. Calculated ECD and experimental CD spectra of 1.
Figure 4. Calculated ECD and experimental CD spectra of 1.
Marinedrugs 17 00529 g004
Figure 5. Calculated ECD and experimental CD spectra of 2.
Figure 5. Calculated ECD and experimental CD spectra of 2.
Marinedrugs 17 00529 g005
Figure 6. Calculated ECD and experimental CD spectra of 3.
Figure 6. Calculated ECD and experimental CD spectra of 3.
Marinedrugs 17 00529 g006
Table 1. NMR data for compounds 13 (CD3OD, 1H: 500 MHz, 13C: 125 MHz).
Table 1. NMR data for compounds 13 (CD3OD, 1H: 500 MHz, 13C: 125 MHz).
123
NδCδH mult. (J in Hz)δCδH mult. (J in Hz)δCδH mult. (J in Hz)
1----116.3-
275.05.55 dd (2.5, 13.5)75.85.58 dd (3.0, 13.0)160.3-
345.42.89 dd (13.5, 17.0)
2.67 dd (2.5, 17.0)
43.32.99 dd (13.0, 17.0)
2.75 dd (3.0, 17.0)
103.76.35 br.s
4193.6-198.9-162.4-
5164.7-162.6-109.06.36 dd (2.0, 8.0)
6109.6-108.7-131.67.41 d (8.0)
7165.4-167.0---
893.66.12 s96.45.93 s--
9161.9-163.2---
10105.7-103.2---
1′119.7-118.4-106.5-
2′160.1-156.7-162.3-
3′99.86.49 d (2.3)103.46.36 d (2.5)91.66.02 s
4′159.0-159.6-164.1-
5′108.16.47 dd (2.3, 8.5)107.76.37 dd (2.0, 8.5)108.9-
6′128.57.38 d (8.5)128.67.32 d (8.5)166.6-
1″28.22.63 m28.02.60 m28.22.65 m
2″48.22.50 m48.42.49 m48.02.57 m
3″32.32.02 m32.32.01 m32.42.09 m
4″124.84.96 t (5.5)124.84.99 t (5.5)125.05.06 td (1.0, 6.5)
5″132.0-132.0-131.8-
6″17.81.48 s17.81.50 s17.91.58 s
7″25.81.57 s25.81.59 s25.91.65 s
8″149.8-149.8-149.9-
9″111.24.52 s
4.59 s
111.14.54 br s
4.60 br s
111.14.61 br s
4.55 br s
10″19.11.64 s19.21.65 s19.11.72 s
OMe55.93.82 s----
OMe55.93.83 s--56.13.91
α----125.47.95 d (16.0)
β----139.88.02 d (16.0)
γ----194.8-
Table 2. Antimicrobial activities of compounds 110 (IC90: μg/mL).
Table 2. Antimicrobial activities of compounds 110 (IC90: μg/mL).
Compd.E. faecalisS. aureusB. cereusE. coliP. aeruginosaS. entericaC. albicansM. tuberculosis
1888>256881648.0
2111>256181>50
388848816>50
43232161283232326.0
5>256>256>256>256>256>256>25611.1
6>256>256>256128>256>256>256>50
7>256>256>256>256>256>256>256>50
8>256>256>256>256>256>256>256>50
9>256>256>256>256>256>256>256>50
1032ntnt128ntnt64>50
Strep25625612832256128ntnt
Cyclohexntntntntntnt32nt
Strep: Streptomycin; Cyclohex: Cyclohexamide; nt: not tested.
Table 3. Cytotoxic activity of compounds 110 (IC50: μg/mL).
Table 3. Cytotoxic activity of compounds 110 (IC50: μg/mL).
CompdKBHep-G2Lu-1MCF-7
159.732.080.071.7
2118.4>128>128>128
3>128>128>128>128
44.82.274.014.5
52.02.04.811.8
647.378.471.560.0
7>12856.0>128>128
8>128>128>128>128
9>128>128>128>128
Ellipticine0.3 ± 0.050.3 ± 0.050.4 ± 0.050.5 ± 0.05

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Cao, D.D.; Trinh, T.T.V.; Doan Thi Mai, H.; Vu, V.N.; Le, H.M.; Vu Thi, Q.; Nguyen, M.A.; Duong, T.T.; Tran, D.T.; Chau, V.M.; Ma, R.; Shetye, G.; Cho, S.; Murphy, B.T.; Pham, V.C. Antimicrobial Lavandulylated Flavonoids from a Sponge-Derived Streptomyces sp. G248 in East Vietnam Sea. Mar. Drugs 2019, 17, 529. https://doi.org/10.3390/md17090529

AMA Style

Cao DD, Trinh TTV, Doan Thi Mai H, Vu VN, Le HM, Vu Thi Q, Nguyen MA, Duong TT, Tran DT, Chau VM, Ma R, Shetye G, Cho S, Murphy BT, Pham VC. Antimicrobial Lavandulylated Flavonoids from a Sponge-Derived Streptomyces sp. G248 in East Vietnam Sea. Marine Drugs. 2019; 17(9):529. https://doi.org/10.3390/md17090529

Chicago/Turabian Style

Cao, Duc Danh, Thi Thanh Van Trinh, Huong Doan Thi Mai, Van Nam Vu, Hong Minh Le, Quyen Vu Thi, Mai Anh Nguyen, Thu Trang Duong, Dang Thach Tran, Van Minh Chau, Rui Ma, Gauri Shetye, Sanghyun Cho, Brian T. Murphy, and Van Cuong Pham. 2019. "Antimicrobial Lavandulylated Flavonoids from a Sponge-Derived Streptomyces sp. G248 in East Vietnam Sea" Marine Drugs 17, no. 9: 529. https://doi.org/10.3390/md17090529

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