Termisoflavones A–C, Isoflavonoid Glycosides from Termite

Article pubs.acs.org/jnp

Termisoflavones A−C, Isoflavonoid Glycosides from TermiteAssociated Streptomyces sp. RB1 Hee Rae Kang,† Dahae Lee,† René Benndorf,‡ Won Hee Jung,§ Christine Beemelmanns,‡ Ki Sung Kang,# and Ki Hyun Kim*,† †

School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea Leibniz Institute for Natural Product Research and Infection Biology e.V., Hans-Knöll-Institute (HKI), Beutenbergstrasse 11a, 07745 Jena, Germany § Department of Systems Biotechnology, Chung-Ang University, Anseong, Gyeonggi-do 456-756, Republic of Korea # College of Korean Medicine, Gachon University, Seongnam 461-701, Republic of Korea ‡

S Supporting Information *

ABSTRACT: Three new isoflavonoid glycosides, termisoflavones A−C (1−3), and eight isoflavonoids (4−11) were isolated from termite-associated Streptomyces sp. RB1 recovered from the cuticle of the South African termite, Macrotermes natalensis. The structures of new compounds were determined by spectroscopic methods including 1D and 2D NMR and HR-MS analysis, as well as comparison of their NMR data with those of related isoflavonoid glycoside derivatives. The absolute configurations of the sugar moieties were clarified by chemical reactions. None of the isolates (1−11) displayed antifungal or antimicrobial activities (MICs > 100 μg/mL), whereas compounds 6 and 11 ameliorated cisplatininduced kidney cell damage to 80% of the control value at a cisplatin dose of 25 μM.

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produce antimicrobial secondary metabolites such as natalamycin A and microtermolides A and B.10,11 In an effort to extend earlier studies, we investigated the termite-associated isolate Streptomyces sp. RB1, which showed high antimicrobial activity in our screening assays. Herein, we report the isolation and structural determination of three new isoflavonoid glycosides, termisoflavones A (1), B (2), and C (3), along with eight known isoflavonoids from termite-associated Streptomyces sp. RB1, and describe their biological activities.

nsect-associated microorganisms have emerged as a source for the discovery of novel bioactive small molecules. Bacterial symbionts mediate ecologically important traits by producing small molecules. These molecules modulate interactions between the microbial producers and their relatives, allies, competitors, and hosts. Recent studies exploring the bioactive molecules of symbiotic interactions have reported several interesting metabolites including the depsipeptide dentigerumycin, which has selective antifungal activity and was isolated from the bacterial symbiont (Pseudonocardia sp.) of a fungus-growing ant (Apterostigma dentigerum);1,2 the polyene peroxide mycangimycin and polycyclic tetramate macrolactams, known as frontalamides A and B, isolated from the bacterial mutualist (Streptomyces sp.) of the southern pine beetle (Dendroctonus f rontalis);3−5 and the 26-membered polyene macrocyclic lactam called sceliphrolactam, from the bacterial symbiont (Streptomyces sp.) of the mud dauber (Sceliphron caementarium). 6 Recent studies by other groups have demonstrated that bacteria in the dung beetle ecosystem are also a promising source of novel organic compounds such as the cyclobutane-bearing macrocyclic lactam (tripartilactam), the dichlorinated indanone (tripartin), and novel branched cyclic peptides (coprisamides A and B).7−9 We have focused on Actinobacteria associated with the fungus-growing termite Macrotermes natalensis as part of our research into the metabolites of insect-associated bacteria. The termite-associated bacteria have previously been shown to © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Four termite-associated Actinomycetes were isolated from the cuticle (RB1 and RB2), termite gut (RB4), and fungus comb (RB3) of a M. natalensis worker collected in South Africa in 2015. Culture extracts of all strains were tested for antifungal and antibacterial activities. In particular, Streptomyces sp. RB1 showed significant activity against Staphylococcus aureus and weak activity against human-pathogenic Candida albicans. Therefore, Streptomyces sp. RB1 was grown in a preparative scale on 60 ISP-2 agar plates for 14 days. A chemical investigation of the MeOH extract of the culture led to the isolation of three new isoflavonoid glycosides, termisoflavones A (1), B (2), and C (3), along with eight known isoflavonoids (4−11). Received: August 10, 2016

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DOI: 10.1021/acs.jnatprod.6b00738 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

Figure 1. Key TOCSY (bold lines) and HMBC (arrows) correlations for compounds 1−3.

(1H, d, J = 2.0 Hz); 16 proton signals attributable to sugars at δH 5.58 (1H, br s), 5.55 (1H, d, J = 1.5 Hz), 4.26 (1H, t, J = 2.0 Hz), 4.04 (1H, m), 3.85 (1H, dd, J = 9.5, 3.5 Hz), 3.68 (1H, m), 3.61 (1H, m), 3.55 (2H, m, overlap), 3.50 (1H, t, J = 9.5 Hz), 1.28 (3H, d, J = 6.0 Hz), and 1.26 (3H, d, J = 6.0 Hz); and one methoxy group at δH 3.55 (3H, s). In particular, the aromatic proton signal at δH 8.22 (1H, br s) is indicative of H-2 in isoflavonoid core structures.12 The 13C NMR data showed the presence of 28 carbon signals composed of 15 carbons

Termisoflavone A (1) was obtained as a yellow gum with a negative specific rotation value ([α]25 D −111.0, MeOH). The molecular formula of 1 was deduced to be C28H32O13 from negative-ion HRESIMS, in addition to 1H and 13C NMR spectral data. Bands from hydroxy groups at 3384 cm−1 and a carbonyl group at 1692 cm−1 were present in the IR spectrum. The 1H NMR data showed the presence of seven aromatic proton signals at δH 8.22 (1H, br s), 7.54 (2H, d, J = 8.5 Hz), 7.18 (2H, d, J = 8.5 Hz), 6.74 (1H, d, J = 2.0 Hz), and 6.53 B

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Figure 2. Comparison of the protective effect of compounds 1−11 against cisplatin-induced damage in LLC-PK1 cells.

corresponding to the aglycone responsible for the isoflavonoid skeleton and 12 carbons for sugars, together with one methoxy carbon (δC 57.4). The NMR spectral data suggested that compound 1 was an isoflavonoid glycoside. The aglycone of the isoflavonoid moiety was predicted to be genistein based on a comparison of the spectroscopic data with previously reported values.12 This was confirmed by analysis of TOCSY and HMBC correlations (Figure 1). Sugar moieties were determined to be α-rhamnopyranoside (δC 100.1, 73.7, 72.2, 71.9, 71.5, 18.2) and 3-O-methyl-α-rhamnopyranoside (δC 99.8, 82.1, 72.8, 70.9, 68.2, 57.7, 18.2) by comparison with previously reported 13C NMR data for each sugar.13 The characteristic coupling constants of anomeric protons (broad singlet of H-1″ and J = 1.5 Hz of H-1‴) also suggested the presence of αrhamnopyranoside and 3-O-methyl-α-rhamnopyranoside, which was reconfirmed by analysis of TOCSY and HMBC experiments (Figure 1).

The positions of the two rhamnoses were established by an HMBC experiment in which correlations were observed between H-1″ (δH 5.58) and C-7 (δ 163.9) and between H1‴ (δH 5.55) and C-4′ (δC 158.0). Acid hydrolysis of compound 1 afforded the aglycone genistein (1a) and two rhamnoses, L-rhamnose ([α]25 D +9.3, H2O) and 3-O-methyl-L+10.5, H O). The aglycone genistein (1a) was rhamnose ([α]25 D 2 identified by LC/MS analysis, and the L-conformation of the two rhamnoses was confirmed by measurement of their specific rotation. Thus, the structure of 1 was determined to be genistein-4′-(3-O-methyl-α-L-rhamnopyranosyl)-7-α-L-rhamnopyranoside. Termisoflavone B (2) was obtained as a yellow gum with a positive specific rotation value ([α]25 D +11.2, MeOH). The molecular formula was deduced to be C29H34O13 from negativeion HRESIMS and 1H and 13C NMR spectral data. Bands for hydroxy groups at 3387 cm−1 and a carbonyl group at 1692 C

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cm−1 were present in the IR spectrum. The 1H NMR data were very similar to those of compound 1, with the only major difference being the presence of one additional methoxy group in 2 (δH 3.55 and δC 57.5). Detailed inspection of 2D NMR (TOCSY, HSQC, and HMBC) of 2 indicated the existence of two 3-O-methylrhamnoses, which were assigned as two 3-Omethyl-α-rhamnopyranosides on the basis of the small coupling constant of anomeric protons (J = 1.5 Hz of H-1″ and J = 2.0 Hz of H-1‴) for each and comparison with reported 13C NMR data.13 The positions of the rhamnoses were established to be C-7 and C-4′, respectively, by HMBC experiments, which showed correlations between H-1″ (δH 5.62) and C-7 (δ 164.2) and between H-1‴ (δH 5.55) and C-4′ (δC 157.9) (Figure 1). Acid hydrolysis of compound 2 afforded the aglycone genistein (2a) and a 3-O-methyl-L-rhamnose ([α]D25 +10.8, H2O). Genistein (2a) was identified by LC/MS analysis, and the Lconformation of the rhamnoses was confirmed by the observed specific rotation values. The complete structure of 2 was finally confirmed by analysis of 2D NMR (TOCSY, HSQC, and HMBC) data. Thus, the structure of 2 was determined to be genistein-4′,7-di-3-O-methyl-α-L-rhamnopyranoside. Termisoflavone C (3) was obtained as a yellow gum with a negative specific rotation value ([α]25 D −36.0, MeOH). The molecular formula of 3 was established to be C29H34O12 from negative-ion HRESIMS and 1H and 13C NMR spectral data. Bands for hydroxy groups at 3400 cm−1 and a carbonyl group at 1697 cm−1 were evident in the IR spectrum. The 1H NMR data of 3 were very similar to those of compound 2, with the only major difference being the absence of a hydroxy group at C-5 of the A-ring in 3 (δH 8.19, d, J = 9.0 Hz). Detailed inspection of 2D NMR (TOCSY, HSQC, and HMBC) data of 3 allowed us to confirm the substitution pattern of hydroxy groups of aromatic rings in the aglycone and the existence of two 3-Omethyl-α-rhamnopyranosides with small coupling constants of anomeric protons (J = 2.0 Hz of H-1″ and H-1‴). The position of the rhamnoses was established to be C-7 and C-4′ by HMBC experiments, which showed correlations between H-1″ (δH 5.69) and C-7 (δ 162.6) and between H-1‴ (δH 5.55) and C-4′ (δC 158.2) (Figure 1). Acid hydrolysis of compound 3 afforded the aglycone daidzein (3a) and a 3-O-methyl-L-rhamnose ([α]25 D +11.0, H2O). Daidzein (3a) was identified by LC/MS analysis, and the L-conformation of the rhamnonses was confirmed by the observed specific rotations. Thus, the structure of 3 was determined to be daidzein-4′,7-di-3-Omethyl-α-L-rhamnopyranoside. The known compounds were identified as daidzein (4),14 genistein-7-α-L-rhamnoside (5),15 daidzein-7-α-L-rhamnoside (6),16 6-O-methyl-7-O-α-L-rhamnopyranosyldaidzein (7),16 genistein-4′,7-di-α-L-rhamnoside (8),15 daidzein-4′,7-di-α-Lrhamnoside (9),15 genistin (10),17 and daidzin (11)17 by comparing the spectroscopic data with previously reported values and LC/MS analysis. This is the first report of 13C NMR data for compound 6 since the data had not previously been reported. It is unusual that Streptomyces bacteria produce isoflavonoids, but six isoflavonoids with β-galactosidase inhibiting activity were previously reported from the culture filtrate of Streptomyces xanthophaeus.15 The antifungal and antibacterial properties of all compounds were tested against two human-pathogenic bacteria, C. albicans and C. neoformans, the Gram-positive bacterium S. aureus, and the Gram-negative bacterium E. coli. The minimum inhibitory concentrations (MICs) of compounds 1−11 were determined to be >100 μg/mL, and they therefore could not account for

the observed antifungal and antibacterial activities of the crude extract. Interestingly, recent studies have shown that some flavonoids such as eupatilin, quercetin, and chalcones have a kidneyprotective effect.18−21 Widespread platinum-based anticancer drug therapies can cause renal damage and apoptotic kidney cell damage. The development of reno- and kidney-protective molecules is therefore urgently required. We additionally evaluated the kidney protective effects of compounds 1−11 in LLC-PK1 cells.22,23 The cell viability of LLC-PK1 cells treated with cisplatin was 60% of the control value, whereas cotreatments of these cells with compounds 2−6 and 8−11 resulted in significant protection against cisplatin-induced damage (Figure 2). Among them, compounds 6 and 11 exhibited a potent renoprotective effect by recovering cell viability to more than 80% of the control value at a cisplatin dose of 25 μM (Figure 2). The protection effects of compounds 6 and 11 on cisplatin-induced damage were stronger than that of N-acetylcysteine, a positive control (Figure 2).

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EXPERIMENTAL SECTION

General Experimental Procedures. Specific rotations were measured on a Jasco P-1020 polarimeter. IR spectra were recorded on a Bruker IFS-66/S FT-IR spectrometer. UV spectra were acquired on an Agilent 8453 UV−visible spectrophotometer. HRESI mass spectra were recorded on a Waters UHPLC-QTOF Xevo G2-S mass spectrometer. LC/MS analysis was performed on an Agilent 1200 Series HPLC system equipped with a diode array detector and a 6130 Series ESI mass spectrometer using an analytical Kinetex C18 100 Å column (100 mm × 2.1 mm i.d., 5 μm). NMR spectra were recorded on a Bruker AVANCE III 700 NMR spectrometer operating at 700 MHz (1H) and 175 MHz (13C). Preparative HPLC used a Waters 1525 binary HPLC pump with a Waters 996 photodiode array detector. Semipreparative HPLC used a Shimadzu Prominence HPLC System with SPD-20A/20AV Series Prominence HPLC UV−vis detectors. Column chromatography was performed with silica gel 60 (Merck, 230−400 mesh) and RP-C18 silica gel (Merck, 230−400 mesh). Silica Waters Sep-Pak Vac 6 cm3 and C18 Waters Sep-Pak Vac 6 cm3 cartridges were also used for column chromatography. Merck precoated silica gel F254 plates and reversed-phase (RP)-18 F254s plates were used for TLC. Spots were detected on TLC under UV light or by heating after spraying with anisaldehyde−sulfuric acid. Isolation of Streptomyces spp. Material was collected from a Macrotermes natalensis nest in South Africa (Pretoria), placed into clean plastic bags, and processed within 1 day from collection. Samples (comb, gut, and cuticle) were mixed in sterile deionized water, and bacteria were isolated by plating the resulting suspensions on low nutrient media. Isolates with Actinobacteria-like morphology were transferred to ISP2 agar and subcultured until pure isolates were obtained. DNA Extraction and PCR Amplification. Actinobacteria were grown in nutrient-rich liquid media ISP2 for 5 to 7 days at 30 °C. Cells were harvested, and genomic DNA was extracted using the GenJet genomic DNA purification kit (Thermo Scientific, #K0721) following the manufacturer’s instructions with the following changes: (a) lysozyme treatment was extended to 40 min and (b) proteinase K treatment was extended to 40 min. DNA was quantified photometrically using a Nanodrop Lite spectrometer (Thermo Scientific) photometer. For phylogenetic studies, the 16 S rRNA gene was amplified using the primer set 1492R/27F.24,25 Amplification reactions were prepared in a 25 μL final reaction volume containing 7.25 μL of distilled water, 5 μL of HF buffer, 5 μL of each primer (2.5 μM), 0.5 μL of dNTPs (10 μM), 0.25 μL of Phusion High Fidelity DNA polymerase (New England Biolabs), and 2 μL of extracted DNA (template). PCR was performed under the following conditions: 98 °C/38 s, 32 cycles of 98 °C/30 s, 52 °C/45 s, 72 °C/1 min 20 s, and a D

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Table 1. 1H (700 MHz) and 13C NMR (175 HMz) Data of Termisoflavones A−C (1−3) in CD3ODa 2b

1

a

position

δC

2 3 4 5 6 7 8 9 10 1′ 2′, 6′ 3′, 5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 3″-OMe 3‴-OMe

155.8 124.9 182.4 159.5 95.8 163.9 101.1 162.4 108.1 126.2 131.6 117.6 158.0 100.1 71.9 72.2 73.7 71.5 18.2 99.8 68.2 82.1 72.8 70.9 18.2 57.7

δH (J in Hz)

δC

8.22 br s

155.5 124.6 182.5 159.2 95.6 164.2 100.8 162.2 108.0 126.1 131.5 117.3 157.9 99.5 67.8 81.8 72.5 71.2 18.2 99.4 67.8 81.8 72.5 70.4 18.2 57.5 57.5

6.74 d (2.0) 6.53 d (2.0)

7.54 d (8.5) 7.18 d (8.5) 5.58 4.04 3.85 3.50 3.61 1.28 5.55 4.26 3.55 3.68 3.55 1.26

br s m dd (9.5, 3.5) t (9.5) m d (6.0) d (1.5) t (2.0) m m m d (6.0)

3.55 s

Coupling constants (in Hz) are given in parentheses.

3b δH (J in Hz) 8.23 br s

6.76 d (2.0) 6.55 d (2.0)

7.55 d (9.0) 7.18 d (9.0) 5.62 4.26 3.53 3.61 3.54 1.27 5.55 4.26 3.53 3.67 3.54 1.25 3.55 3.55

d (1.5) t (2.0) m m m d (6.0) d (2.0) t (2.0) m m m d (6.0) s s

δC 155.2 126.3 178.3 128.6 117.2 162.6 104.4 159.8 120.6 127.4 131.6 117.5 158.2 99.9 67.7 81.9 72.8 71.2 18.2 99.9 67.8 81.8 72.8 70.7 18.2 57.3 57.3

δH (J in Hz) 8.28 br s

8.19 d (9.0) 7.23 dd (9.0, 2.5) 7.31 d (2.5)

7.54 d (9.0) 7.18 d (9.0) 5.69 4.30 3.54 3.62 3.57 1.27 5.55 4.26 3.55 3.68 3.55 1.26 3.56 3.55

d (2.0) dd (3.0, 2.0) m m m d (6.0) d (2.0) t (2.0) m m m d (6.0) s s

b13

C NMR data were assigned on the basis of HSQC and HMBC experiments.

final extension of 72 °C/8 min. PCR products were visualized by agarose gel electrophoresis. PCR reactions were purified using a PCR purification kit (Thermo Scientific). DNA fragments were sequenced at GATC (Konstanz). Sequencing and Species Identification. Sequences were assessed for purity and mismatches using BioEdit.26−28 Obtained forward and reverse sequences of each strain were assembled with BioEdit and tested for chimeras using DECIPHER (http://decipher. cee.wisc.edu/FindChimerasOutputs.html). Resulting sequences were deposited in GenBank (accessions numbers: KX344915, KX344916, KX344917, KX344918, KX344919). A phylogenetic analysis was performed using the first hits obtained from BLASTn searches (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (first hits: GenBank accessions numbers KF830687.1, KJ781966.1, KX036626.1, and KP126261.1). Support for nodes on the phylogenetic tree was assessed by performing bootstrap analysis (1000 bootstrap replicates) using maximum likelihood under the T93+G model of sequence evolution, as well as by neighbor-joining using MEGA 6.0 (Supporting Information). Extraction and Isolation. Streptomyces sp. RB1 was grown on 60 ISP-2 agar plates (9 cm diameter) for 14 days at 30 °C. Agar was cut into squares, consolidated, and soaked overnight in MeOH. The MeOH phase was filtered and the solvent removed under reduced pressure. The resulting crude MeOH extract (20 g) was suspended in distilled water (250 mL) and then successively partitioned with hexane, CH2Cl2, ethyl acetate, and n-BuOH, yielding 0.06, 0.32, 0.35, and 1.57 g of residue, respectively. The CH2Cl2-soluble fraction (0.32 g) was separated over a silica Waters Sep-Pak vac 6 cm3 with CH2Cl2− MeOH (300:1 → 10:1 gradient) as an eluent to yield nine subfractions (M1−M9). Subfraction M7 (18 mg) was purified by semipreparative reversed-phase HPLC using a phenyl-hexyl column (Phenomenex, Luna, 250 × 10 mm, i.d., 5 μm) with 60% MeOH and a flow rate of 2 mL/min to yield compounds 2 (2.3 mg, tR = 30.0 min) and 3 (2.5 mg,

tR = 17.3 min). Subfractions M8 (11 mg) and M9 (12 mg) were combined and then purified by the same HPLC system with a flow rate of 2 mL/min, using a gradient solvent program (water [A], methanol [B]: 0−30 min, 50% B; 30−50 min, 55% B) to furnish compound 1 (2.8 mg, tR = 50.1 min). The EtOAc-soluble fraction (0.35 g) was applied to a Sephadex LH-20 column (50% → 100% MeOH) for chromatographic separation to give 14 subfractions (H1− H14). Subfraction H7 (6 mg) was purified by semipreparative reversed-phase HPLC (54% MeOH) using a phenyl-hexyl column with a flow rate of 2 mL/min to yield compound 4 (0.8 mg, tR = 22.1 min). Subfraction H10 (13 mg) was purified by the same HPLC system using a gradient program (water [A], methanol [B]: 0−23 min, 42% B; 23−40 min, 47% B) to give compounds 10 (0.8 mg, tR = 21.6 min) and 11 (1.3 mg, tR = 13.3 min). Subfraction H11 (10 mg) was further purified by the same HPLC system with 34% MeOH to obtain compound 9 (1.6 mg, tR = 12.4 min). Finally, subfraction H14 (20 mg) was purified by the same HPLC system using a gradient program (water [A], methanol [B]: 0−25 min, 44% B; 25−30 min, 44% → 53% B; 30−55 min, 53% → 60% B) to yield compounds 5 (1.1 mg, tR = 35.0 min), 6 (1.4 mg, tR = 22.7 min), 7 (1.2 mg, tR = 25.1 min), and 8 (1.0 mg, tR = 29.7 min). Termisoflavone A (1): yellow gum; [α]25 D −111.0 (c 0.03, MeOH); IR (KBr) νmax 3384, 2943, 2830, 1692, 1617, 1033 cm−1; UV (MeOH) λ max (log ε) 202 (3.21), 260 (4.20), 324 (0.50) nm; 1H (700 MHz) and 13C NMR (175 MHz), see Table 1; negative HRESIMS m/z 575.1764 [M − H]− (calcd for C28H31O13, 575.1765). Termisoflavone B (2): yellow gum; [α]25 D +11.2 (c 0.03, MeOH); IR (KBr) νmax 3387, 2942, 2830, 1692, 1617, 1033 cm−1; UV (MeOH) λmax (log ε) 198 (2.48), 260 (3.10), 324 (0.33) nm; 1H (700 MHz) and 13C NMR (175 MHz), see Table 1; negative HRESIMS m/z 589.1929 [M − H]− (calcd for C29H33O13, 589.1921). Termisoflavone C (3): yellow gum; [α]25 D −36.0 (c 0.04, MeOH); IR (KBr) νmax 3400, 2946, 2832, 1697, 1618, 1033 cm−1; UV (MeOH) E

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λmax (log ε) 198 (3.20), 250 (3.30), 296 (1.10) nm; 1H (700 MHz) and 13C NMR (175 MHz), see Table 1; negative HRESIMS m/z 619.2021 [M + HCOO−]− (calcd for C30H35O14, 619.2027). Daidzein-7-α-L-rhamnoside (6): yellow gum; 1H NMR (700 MHz, CD3OD) δ 8.23 (1H, s, H-2), 8.18 (1H, d, J = 8.9 Hz, H-5), 7.41 (2H, d, J = 8.6 Hz, H-2′, 6′), 7.29 (1H, d, J = 2.2 Hz, H-8), 7.21 (1H, dd, J = 8.9, 2.3 Hz, H-6), 6.88 (2H, d, J = 8.6 Hz, H-3′, 5′), 5.65 (1H, d, J = 1.3 Hz, H-1″), 4.08 (1H, dd, J = 3.4, 1.8 Hz, H-2″), 3.88 (1H, dd, J = 9.5, 3.5 Hz, H-3″), 3.62 (1H, dd, J = 9.5, 6.2 Hz, H-5″), 3.52 (1H, t, J = 9.5 Hz, H-4″), 1.28 (3H, d, J = 6.0 Hz, H-6″); 13C NMR (175 MHz, CD3OD) δ 178.2 (C-4), 162.5 (C-7), 159.4 (C-9), 159.1 (C-4′), 155.2 (C-2), 131.6 (C-2′, 6′), 128.6 (C-5), 126.4 (C-1′), 124.2 (C-3), 120.3 (C-10), 116.4 (C-3′, 5′), 117.1 (C-6), 104.7 (C-8), 100.2 (C-1″), 73.7 (C-4″), 72.3 (C-3″), 71.9 (C-2″), 71.4 (C-5″), 18.2 (C-6″); ESIMS m/z 423.1 [M + Na]+. Acid Hydrolysis of 1−3 and Sugar Analysis. Compounds 1 (1.8 mg), 2 (1.3 mg), and 3 (1.5 mg) were refluxed with 1 mL of 2 N HCl for 2 h at 90 °C. The hydrolysate was extracted with EtOAc, and the organic layer was evaporated in vacuo to yield the aglycones genistein (1a, 0.7 mg; 2a, 0.4 mg) and daidzein (3a, 0.6 mg) as a yellow gum. Each of them was identified by LC/MS analysis. Each aqueous phase was neutralized through an Amberlite IRA-67 column to give the respective sugar fraction. Each fraction was subjected separately to column chromatography using a C18 silica gel Waters Sep-Pak Vac 6 cm3 (MeCN−H2O, 8:1) to yield L-rhamnopyranose and 3-O-methyl-L-rhamnopyranose. The specific rotations of the sugars obtained from the aqueous residues were as follows: [α]25 D +9.3 (c 0.05, H2O) of L-rhamnopyranose from 1, [α]25 D +10.5 (c 0.03, H2O) of 3-O-methyl-L-rhamnopyranose from 1, [α]25 D +10.8 (c 0.04, H2O) of 3-O-methyl-L-rhamnopyranose from 2, and [α]25 D +11.0 (c 0.04, H2O) of 3-O-methyl-L-rhamnopyranose from 3. Determination of Antifungal and Antibacterial Activity. To estimate the antifungal activity of the molecules against Candida albicans (SC5314) and Cryptococcus neoformans (H99), minimal inhibitory concentrations were determined according to the approved standard M27-A3 of the Clinical and Laboratory Standards Institute (CLSI).29,30 Antibacterial activity against Escherichia coli (DH10B) and Staphylococcus aureus (NCTC 8325-4) was determined according to the European Committee for Antimicrobial Susceptibility Testing (EUCAST) method.31,32 Streptomycin sulfate and fluconazole were used as positive controls for antibacterial and antifungal activities, respectively. Renoprotective Effect against Cisplatin-Induced Kidney Cell Damage. The renoprotective effect of the compounds against cisplatin-induced renal cell damage was evaluated using LLC-PK1 cells.22,23 LLC-PK1 (pig kidney epithelium, CL-101) cells were purchased from the American Type Culture Collection (Rockville, MD, USA) and cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 1% penicillin/ streptomycin, and 4 mM L-glutamine at 37 °C with 5% CO2 in air. Cells were seeded in 96-well culture plates at 1 × 104 cells per/well and allowed to adhere for 2 h. Thereafter, the test sample and/or 25 μM cisplatin as the radical donor were added to the culture medium and incubated for 24 h. Subsequently, the medium containing the test sample and/or radical donor was removed, and cells were incubated in serum-free medium (90 μL/well) and Ez-Cytox reagent (10 μL/well) for 2 h at 37 °C. Cell viability was measured by absorbance at 450 nm using a microplate reader (PowerWave XS; Bio-Tek Instruments, Winooski, VT, USA). N-Acetyl cysteine was used as a positive control.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-31-290-7700. Fax: +82-31-290-7730. E-mail: [email protected]. ORCID

Ki Hyun Kim: 0000-0002-5285-9138 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, & Future Planning (2015R1C1A1A02037383). We are grateful for financial support from the German National Academy of Sciences Leopoldina (LPDS 2011-2) and the Daimler Benz Foundation to C.B. We thank S. Otani and M. Poulsen (University of Copenhagen) for providing the fungal isolates and their help throughout the preparation of the manuscript. We also thank Z. W. de Beer, M. J. Wingfield, and the staff and students at the Forestry and Agricultural Biotechnology Institute, University of Pretoria, for hosting field work and the Oerlemans family (Mookgophong) for permission to sample colonies on their farm.

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REFERENCES

(1) Oh, D. C.; Poulsen, M.; Currie, C. R.; Clardy, J. Nat. Chem. Biol. 2009, 5, 391−393. (2) Currie, C. R.; Scott, J. A.; Summerbell, R. C.; Malloch, D. Nature 1999, 398, 701−704. (3) Scott, J. J.; Oh, D.-C.; Yuceer, M. C.; Klepzig, K. D.; Clardy, J.; Currie, C. R. Science 2008, 322, 63. (4) Blodgett, J. A. V.; Oh, D.-C.; Cao, S.; Currie, C. R.; Clardy, J. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 11692−11697. (5) Oh, D.-C.; Scott, J. J.; Currie, C. R.; Clardy, J. Org. Lett. 2009, 11, 633−636. (6) Oh, D.-C.; Poulsen, M.; Currie, C. R.; Clardy, J. Org. Lett. 2011, 13, 752−755. (7) Park, S.-H.; Moon, K.; Bang, H.-S.; Kim, S.-H.; Kim, D.-G.; Oh, K.-B.; Shin, J.; Oh, D.-C. Org. Lett. 2012, 14, 1258−1261. (8) Kim, S.-H.; Kwon, S. H.; Park, S.-H.; Lee, J. K.; Bang, H.-S.; Nam, S.-J.; Kwon, H. C.; Shin, J.; Oh, D.-C. Org. Lett. 2013, 15, 1834−1837. (9) Um, S.; Park, S. H.; Kim, J.; Park, H. J.; Ko, K.; Bang, H.-S.; Lee, S. K.; Shin, J.; Oh, D.-C. Org. Lett. 2015, 17, 1272−1275. (10) Kim, K. H.; Ramadhar, T. R.; Beemelmanns, C.; Cao, S.; Poulsen, M.; Currie, C. R.; Clardy, J. Chem. Sci. 2014, 5, 4333−4338. (11) Carr, G.; Poulsen, M.; Klassen, J. L.; Hou, Y.; Wyche, T. P.; Bugni, T. S.; Currie, C. R.; Clardy, J. Org. Lett. 2012, 14, 2822−2855. (12) Hosny, M.; Rosazza, J. P. N. J. Nat. Prod. 1999, 62, 1609−1612. (13) Popper, Z. A.; Sadler, I. H.; Fry, S. C. Biochem. Syst. Ecol. 2004, 32, 279−289. (14) Kim, H. J.; Lee, D. H.; Hwang, Y. Y.; Lee, K. S.; Lee, J. S. J. Agric. Food Chem. 2005, 53, 5882−5888. (15) Hazato, T.; Naganawa, H.; Kumagai, M.; Aoyagi, T.; Umezawa, H. J. Antibiot. 1979, 32, 217−222. (16) Cheenpracha, S.; Zhang, H.; Mar, A. M.; Foss, A. P.; Foo, S. H.; Lai, N. S.; Jee, J. M.; Seow, H. F.; Ho, C. C.; Chang, L. C. J. Nat. Prod. 2009, 72, 1520−1523. (17) Maharik, N. A.; Botting, N. P. Eur. J. Org. Chem. 2008, 33, 5622−5629. (18) Kuhlmann, M. K.; Horsch, E.; Burkhardt, G.; Wagner, M.; Köhler, H. Arch. Toxicol. 1998, 72, 536−540. (19) Lee, D.; Kim, K. H.; Moon, S. W.; Lee, H.; Kang, K. S.; Lee, J. W. Bioorg. Med. Chem. Lett. 2015, 25, 1929−1932.

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(20) Park, J. Y.; Lee, D.; Jang, H. J.; Jang, D. S.; Kwon, H. C.; Kim, K. H.; Kim, S. N.; Hwang, G. S.; Kang, K. S.; Eom, D. W. Evid. Based. Complement. Alternat. Med. 2015, 2015, 483980. (21) Kim, Y.-J.; Kim, T.-W.; Seo, C.-S.; Park, S.-R.; Ha, H.; Shin, H.K.; Jung, J.-Y. Nat. Prod. Sci. 2014, 20, 251−257. (22) Han, M.-S.; Han, I.-H.; Lee, D.; An, J. M.; Kim, S.-N.; Shin, M.S.; Yamabe, N.; Hwang, G. S.; Yoo, H. H.; Choi, S.-J.; Kang, K. S.; Jang, H.-J. J. Ginseng Res. 2016, 40, 135−140. (23) Kang, H. R.; Lee, D.; Eom, H. J.; Lee, S. R.; Lee, K. R.; Kang, K. S.; Kim, K. H. J. Funct. Foods 2016, 20, 358−368. (24) Schumann, P. J. Basic Microbiol. 1991, 31, 479−480. (25) Visser, A. A.; Nobre, T.; Currie, C. R.; Aanen, D. K.; Poulsen, M. Microb. Ecol. 2012, 63, 975−985. (26) Hall, B. G. Mol. Biol. Evol. 2013, 30, 1229−1235. (27) Hall, T. A. Nucleic Acids Symp. Ser. 1999, 41, 95−98. (28) Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. Mol. Biol. Evol. 2013, 30, 2725−2729. (29) CLSI, 2002. Clinical and Laboratory Standards Institute, Wayne, PA. (30) Lee, D. G.; Lee, A. Y.; Kim, S. J.; Jung, Y. S.; Lee, D. H.; Cho, E. J.; Lee, S. Nat. Prod. Sci. 2014, 20, 107−112. (31) EUCAST, 2015. European Committee for Antimicrobial Susceptibility Testing. Available from http://www.eucast.org/ast_of_ bacteria/disk_diffusion_methodology/. (32) Lee, J.; Lee, D.; Choi, H.; Kim, H. H.; Kim, H.; Hwang, J. S.; Lee, D. G.; Kim, J. I. BMB Rep. 2014, 47, 625−630.

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