Chemical Constituents of Apios americana Tubers and Their Inhibitory

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Article Cite This: J. Nat. Prod. 2018, 81, 1598−1603

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Chemical Constituents of Apios americana Tubers and Their Inhibitory Activities on Nitric Oxide Production in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages Young Hye Seo,†,‡,# Ju-Hyun Jeon,†,‡,# Miran Jeong,§ Seung Mok Ryu,⊥ Won Kyung Jeon,†,‡ Dae Sik Jang,§ Sang Hee Shim,∥ Dongho Lee,⊥ Jung-Hye Choi,§ and Jun Lee*,†,‡ †

Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon 34054, Republic of Korea Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology, Seoul 20792, Republic of Korea § Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea ⊥ Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea ∥ College of Pharmacy and Duksung IDC, Duksung Women’s University, Seoul 01369, Republic of Korea

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‡

S Supporting Information *

ABSTRACT: Apios americana is an important food crop producing edible tubers with high nutritional and medicinal values and is widely cultivated in many countries. Despite its usefulness, research on its secondary metabolites and biological activities has been limited. In the present study, a new coumaronochromone, (2R,3S)-3,7,4′-trihydroxy-5-methoxycoumaronochromone (1), and two new isoflavone glucosides, 7,2′,4′-trihydroxy-5-methoxyisoflavone-4′-O-β-D-glucopyranoside (3) and 5,7,4′trihydroxyisoflavone-7-O-β-D-gentiotrioside (5), were isolated from the tubers of A. americana via chromatographic separation. Seventeen known compounds (2, 4, and 6−20) were also obtained from this plant part. The chemical structures of 1, 3, and 5 were determined by the interpretation of spectroscopic data. The absolute structure of the new compound 1 was established from experimental and calculated electronic circular dichroism spectra. This is the first study to determine the absolute configuration of a 3-hydroxycoumaronochromone derivative. The potential anti-inflammatory activity of the 20 isolates obtained was evaluated by measuring their inhibitory effects on nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophages. Among the isolates, seven compounds (1, 3, 6−8, 15, and 20) showed substantial inhibition of nitric oxide production in RAW 264.7 cells, with the most active being compound 1 (IC50 value of 0.38 ± 0.04 μM). fatty acids, amino acids, carbohydrates, and starch in A. americana tubers.2−5 In pharmacological studies, it has been reported that A. americana is effective in reducing hypertension, hyperlipidemia, cancer, and diabetes.6−8 Several investigations on its secondary metabolites have indicated that isoflavone compounds isolated from this plant are

Apios americana Medik (American groundnut) is an edible tuber-producing leguminous plant native to North America. It is an economically and nutritionally important food crop and is now widely grown and consumed in many countries around the world.1 In Korea, this crop has been introduced recently and is currently being cultivated for edible and medicinal purposes. Edible tubers have been used as food sources by American Indians because of their high nutritional value and health benefits.2 Nutritional studies have reported the composition of © 2018 American Chemical Society and American Society of Pharmacognosy

Received: February 28, 2018 Published: June 22, 2018 1598

DOI: 10.1021/acs.jnatprod.8b00182 J. Nat. Prod. 2018, 81, 1598−1603

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Chart 1

associated with biological activities such as antioxidant, estrogen agonistic, α-glucosidase inhibitory, antiandrogenic, and soluble epoxide hydrolase inhibitory effects.9−13 A conjugated saponin has also been reported to occur in A. americana tubers.14 Nevertheless, there is still a comparative lack of information on the physiological effects and bioactive chemical constituents of A. americana. In particular, little is known about the secondary metabolites associated with the anti-inflammatory effects of this plant. Through the present study to discover new potential anti-inflammatory substances from A. americana, a new coumaronochromone (1) and two new isoflavone glucosides (3 and 5) along with 17 known compounds were isolated from the tubers of this plant. Herein, we report the results of the isolation and structural determination of the isolates, in addition to their biological evaluation in inhibiting nitric oxide (NO) production in a macrophage in vitro model.

Figure 1. Key HMBC (→) correlations of compounds 1, 3, and 5.

oxygenated methine (δC 111.0), five aromatic carbons (δC 94.9, 96.8, 97.3, 109.2, 125.6), an sp3 oxygenated quaternary carbon (δC 80.3), and seven sp2 quaternary carbons (δC 103.5, 117.9, 159.9, 160.0, 160.7, 161.7, 164.7), were observed in the 13 C NMR spectrum, and their presence was supported by the 2D-NMR data measured (HSQC and HMBC). In the HMBC spectrum, H-2 showed long-range coupling with C-2′ in the B ring and exhibited correlations with C-3, C-4, C-9, and C-1′. In addition, HMBC cross-peaks between the hydroxy group at C3 and its adjacent carbons (C-1′, C-2, C-3, and C-4) were observed, indicating the position of the hydroxy group (Figure 1). From these 1D- and 2D-NMR data, compound 1 was determined to be 3,7,4′-trihydroxy-5-methoxycoumaronochromone, although a compound with the same skeleton has been isolated recently from A. americana, but its configuration was not determined. This substance showed a great difference in optical rotation value ([α]20D +44.0, MeOH) when compared to compound 1 ([α]22D −184.6, MeOH), confirming that 1 has a new structure.13 A few other coumaronochromone analogues bearing a hydroxy group at the C-3 position have been reported from several plant

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RESULTS AND DISCUSSION Compound 1 was obtained in the form of a white powder, and its molecular formula was determined as C16H12O7 from the HRESIMS peak at m/z 315.0500 [M − H]− (calcd for C16H11O7 315.0505). The 1H NMR spectrum revealed signals for two characteristic meta-coupled aromatic protons at δH 6.03 (1H, d, J = 2.0 Hz, H-8) and 6.11 (1H, d, J = 2.0 Hz, H-6) in the A ring, an oxygenated methine at δH 6.17 (1H, s, H-2), and a methoxy group at δH 3.69 (3H, s, MeO-5). Three aromatic protons in the 2′,4′-O-substituted B ring were also observed at δH 6.27 (1H, d, J = 2.0 Hz, H-3′), 6.34 (1H, dd, J = 7.8, 2.0 Hz, H-5′), and 6.99 (1H, d, J = 7.8 Hz, H-6′) in the 1H NMR spectrum, in which the B ring system was confirmed by longrange coupling between H-6′ and C-3 (δC 80.3) in the HMBC spectrum (Figure 1). Three hydroxy groups also appeared at δH 6.64 (1H, s, OH-3), 9.79 (1H, s, OH-4′), and 10.78 (1H, br s, OH-7) in the 1H NMR spectrum. Sixteen carbon signals, a carbonyl group (δC 185.7), a methoxy group (δC 55.8), an 1599

DOI: 10.1021/acs.jnatprod.8b00182 J. Nat. Prod. 2018, 81, 1598−1603

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Figure 2. Calculated and experimental ECD spectra of compound 1.

protons at δH 6.52 (1H, d, J = 1.2 Hz, H-6) and 6.828 (1H, overlapped, H-8), and an isoflavone characteristic singlet at δH 8.37 (1H, s, H-2) were observed in the 1H NMR spectrum of 5. Signals of three hexose units including those of three anomeric protons [δH 4.17 (1H, d, J = 7.6 Hz); δC 103.4 (C1⁗), δH 4.21 (1H, d, J = 7.6 Hz); δC 103.7 (C-1‴), and δH 5.10 (1H, d, J = 7.6 Hz); δC 99.6 (C-1″)] were observed in the 1D-NMR (1H and 13C NMR) spectra. These spectroscopic data indicated that compound 5 is composed of an aglycone, 5,7,4′-trihydroxyisoflavone, and three glucopyranose units with a β configuration supported by the larger coupling constants (J = 7.6 Hz) of the anomeric proton signals.19 Each 1H and 13C NMR signal of the three glucopyranose units was assigned by 2D-NMR data interpretation including 1H−1H COSY, HSQC, and HMBC. The absolute configuration of each glucopyranose unit was determined by UHPLC analysis (at tR 3.451 min) of L-cysteine methyl ester and o-tolyl isothiocyanate derivatives, as described above. The linkages between each glucopyranose unit were confirmed by HMBC correlation peaks between H1‴ (δH 4.21) and C-6″ (δC 69.2) and between H-1⁗ (δH 4.17) and C-6‴ (δC 68.9), indicating the sugar moiety to be gentiotrioside. The connection of this sugar unit with the aglycone was also confirmed from the HMBC cross-peak between H-1″ (δH 5.10) and C-7 (δC 162.9) (Figure 1). From the above evidence, the structure of compound 5 was established as 5,7,4′-trihydroxyisoflavone-7-O-β-D-gentiotrioside. Additionally, 17 known compounds were identified as lupinalbin A (2),20 2′-hydroxygenistein-4′-O-β-D-glucopyranoside (4),18 2′-hydroxy-5-methoxygenistein-7,4′-O-β-D-diglucopyranoside (6),12 2′-hydroxygenistein-7,4′-O-β-D-diglucopyranoside (7),12 2′-hydroxygenistein-7-O-β-D-gentibioside (8),12 genistein-7-O-β-D-gentibioside (9),12 2′-hydroxy-5-methoxygenistein-7-O-β-D-glucopyranoside (10),12 5-methoxygenistein7-O-β-D-glucopyranoside (11),12 5,3′-dimethoxygenistein-7-Oβ-D-glucopyranoside (12),12 2′-hydroxygenistein-7-O-β-D-glucopyranoside (13),12 genistin (14),21 barpisoflavone A (15),10 7,4′-dihydroxy-5-methoxyisoflavone (16),10 gerontoisoflavone A (17),10 2′-hydroxygenistein (18),22 genistein (19),23 and allantoin (20).24 Compounds 2, 4, and 20 were isolated from A. americana for the first time. The potential anti-inflammatory activity of the isolates was evaluated against lipopolysaccharide (LPS)-stimulated NO production in the RAW 264.7 macrophages. Two new compounds (1 and 3) exhibited potent inhibitory activities against NO production with IC50 values of 0.38 ± 0.04 and 0.47 ± 0.07 μM, respectively (Table 1 and Figure 3). Compounds 6−8, 15, and 20 also showed inhibitory effects

sources, but their stereostructures have not been reported.15−17 This is the first study to determine the absolute configuration of a 3-hydroxycoumaronochromone derivative. The absolute configuration at the C-2 and C-3 positions of 1 was determined through comparison between the experimental and calculated electronic circular dichroism (ECD) spectra. The experimental ECD spectrum of 1 matched the calculated ECD spectrum of the structure with a 2R/3S configuration (Figure 2). Thus, compound 1 was established as (2R,3S)3,7,4′-trihydroxy-5-methoxycoumaronochromone. Compound 3 was obtained as a white powder with an elemental formula of C22H22O11, as observed from the HRESIMS peak at m/z 463.1250 [M + H]+ (calcd for C22H23O11 463.1240). The 1H NMR data of 3 showed signals for a 2′,4′-O-substituted phenyl ring and meta-coupled aromatic protons, similar to compound 1. Signals for a characteristic singlet [δH 7.96; δC 151.9 (C-2)] of isoflavone, a methoxy group (δH 3.79; δC 55.9), two hydroxy groups (δH 9.39/10.72, OH-2′/OH-7), and a glucopyranose unit were also evident in the 1D-NMR (1H and 13C NMR) spectra. Based on these spectroscopic data, this compound was proposed to be an isoflavone glucoside. The 2′,4′-O-substituted B ring system was confirmed by the HMBC cross-peaks of H-6′ (δH 7.02) with C-3 (δC 122.8) and OH-2′ (δH 9.39) with C-1′/C-2′/C3′ (δ C 113.6, 156.3, and 104.1). HMBC long-range correlations between a methoxy group and C-5 (δC 161.0) and between anomeric proton H-1″ (δH 4.81) and C-4′ (δC 158.2) indicated that the methoxy group and the sugar unit are connected to positions C-5 and C-4′, respectively (Figure 1). The NMR data of compound 3 were similar to those of compound 4,18 except for the presence of an additional methoxy group of 3. The β-linkage of the glucopyranosyl unit was proven by the coupling constant (J value) of the anomeric proton signal (δH 4.81, d, J = 7.0 Hz),19 and the absolute configuration of the glucopyranose unit was determined by acid hydrolysis and UHPLC analysis of L-cysteine methyl ester and o-tolyl isothiocyanate derivatives. The peaks of standard glucopyranosyl derivatives were recorded at tR 3.102 (L-glucose derivative) and 3.451 (D-glucose derivative) min, respectively, and the derivative of 3 was observed at tR 3.462 min. Thus, compound 3 was determined as 7,2′,4′-trihydroxy-5-methoxyisoflavone-4′-O-β-D-glucopyranoside. Compound 5 was isolated in the form of a white powder with the molecular formula C33H40O20, as determined by the HRESIMS ion at m/z 757.2169 [M + H]+ (calcd for C33H41O20 757.2191). Signals for two symmetric protons at δH 6.831 (2H, d, J = 8.8 Hz, H-2′/H-6′) and 7.40 (2H, d, J = 8.8 Hz, H-3′/H-5′) in the B ring, two meta-coupled aromatic 1600

DOI: 10.1021/acs.jnatprod.8b00182 J. Nat. Prod. 2018, 81, 1598−1603

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μm F5 100 Å LC column (50 × 2.1 mm inner diameter, Phenomenex, Le Pecq, France). Flash chromatography (FC) was performed using the Biotage Isolera One Spektra flash purification system, with SNAP Ultra C18 (400, 120, and 30 g) and SNAP Ultra (25 and 10 g) prepacked cartridges (Biotage, Uppsala, Sweden). Diaion HP-20 (Supelco, Bellefonte, PA, USA) and Sephadex LH-20 (MilliporeSigma, St. Louis, MO, USA) were also used for FC using manually packed Biotage SNAP dry load cartridges (340, 100, and 10 g scales). TLC was performed on precoated silica gel 60 F254 and RP-18 F254S plates (Merck, Darmstadt, Germany). Plant Material. Fresh tubers of A. americana were purchased from a local farm (Sacheon, Republic of Korea) in April 2015 and identified by Dr. Go Ya Choi (K-herb Research Center, Korea Institute of Oriental Medicine, Republic of Korea). A voucher specimen (Apam5) was deposited at the Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Republic of Korea. Extraction and Isolation. Dried tubers of A. americana (8.5 kg) were powdered and extracted with 70% EtOH (2 × 85 L) at 40 °C for 15 h by maceration, following which the extract solution was filtered and concentrated in vacuo to yield the total extract (1.7 kg, 20.6%). The 70% EtOH extract (300.0 g) was suspended in distilled water and partitioned sequentially using organic solvents to yield n-hexane- (1.2 g), EtOAc- (4.9 g), n-BuOH- (28.0 g), and water-soluble (263.3 g) fractions. The EtOAc-soluble extract (4.9 g) was fractionated by a flash chromatography system (FCS) using a SNAP Ultra C18 cartridge (400 g, water−MeOH, 95:5−60:40) to provide 29 subfractions (F01−F29). F07 (47.1 mg) was further fractionated using a FCS (SNAP Ultra C18 cartridge 120 g, water−MeOH, 90:10) to obtain four subfractions (F0701−F0704). Compound 1 (2.8 mg) was then separated from F0702 (8.8 mg) using a FCS (SNAP Ultra cartridge 10 g × 3, CHCl3−MeOH−water, 90:10:1). F10 (47.3 mg) was subjected to FC (SNAP Ultra C18 cartridge 120 g × 2, water−MeOH, 90:10−70:30) to produce five subfractions (F1001−F1005). Compound 10 (6.9 mg) was obtained from F1003 (8.5 mg) by crystallization. Chromatographic separation of F11 (85.5 mg) was also performed using a FCS (SNAP Ultra C18 cartridge 120 g × 2, water−MeOH, 90:10−70:30) to obtain compound 13 (22.0 mg). F12 (68.0 mg) was fractionated by FC (SNAP Ultra C18 cartridge 120 g × 2, water−MeOH, 90:10−70:30) to provide seven subfractions (F1201−F1207). Compound 11 (5.5 mg) was obtained by crystallization from F1202 (8.8 mg). Subfraction F1204 (8.8 mg) was fractionated using a FCS (SNAP Ultra cartridge 10 g × 3, CHCl3−MeOH−water, 90:10:1) to produce compound 12 (2.0 mg). Fractionation of F19 (176.1 mg) was also performed using a FCS (SNAP Ultra C18 cartridge 120 g × 2, water−MeOH, 100:0−50:50) to yield nine subfractions (F1901−F1909). Compound 15 (37.6 mg) was obtained by crystallization from F1908 (49.0 mg). F20 (290.3 mg) was subjected to FC (SNAP Ultra C18 cartridge 120 g, water− MeOH, 100:0−50:50) to yield 15 subfractions (F2001−F2015).

Table 1. Inhibitory Effects of Compounds Isolated from the Tubers of A. americana on NO Production in LPSStimulated RAW 264.7 Macrophages compound 1 3 6 7 8 15 20 NILb

IC50 (μM)a 0.38 0.47 0.42 0.71 0.60 0.60 0.50 1.55

± ± ± ± ± ± ± ±

0.04 0.07 0.05 0.02 0.21 0.10 0.03 0.03

a The IC50 value is defined as the concentration that results in a 50% decrease in the production of NO. The IC50 values of the other compounds tested (2, 4, 5, 9−14, and 16−19) were higher than 1 μM. The values represent the means of the results from three independent experiments with similar patterns. bL-N6-(1-Iminoethyl)lysine (NIL) was used as the positive control.

with IC50 values ranging from 0.42 to 0.71 μM (Table 1). Seven compounds (1, 3, 6−8, 15, and 20) exhibited inhibitory effects at nontoxic concentrations (Supporting Information). The structure−activity relationships of the active compounds depended on the presence of specific functional groups in each molecular structure. In particular, all three potent compounds (1, 3, and 6) have a 2′,4′-O-substituted phenyl ring and a 5methoxy group in their structures. Although there are some exceptions, these results suggest that these two structural combinations may be associated with anti-inflammatory activity for compounds with both isoflavone and coumaronochromone skeletons.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-2000 polarimeter (JASCO, Easton, MD, USA) with a 100 mm microcell. UV spectra were recorded on a Mecasys Optizen Pop spectrometer (Mecasys, Daejeon, Korea), and ECD spectra were obtained using a JASCO J-1100 spectropolarimeter. NMR spectra were run on an Agilent DD2 600 MHz FT NMR (Agilent Technologies, Santa Clara, CA, USA) and a Bruker magnet system 800/45 ASCEND NMR (Bruker, Billerica, MA, USA) spectrometer using tetramethylsilane as an internal standard. HRESIMS data were obtained using a Waters Q-TOF micromass spectrometer (Waters, Milford, MA, USA). Analytical UHPLC was carried out on an Agilent 1290 Infinity II system with a Kinetex 1.7

Figure 3. Effects of compounds 1 (A) and 3 (B) isolated from the tubers of A. americana on NO production in LPS-stimulated RAW 264.7 macropharges. Cells were pretreated with the indicated concentrations of the isolates for 1 h and then stimulated with LPS (1 μg/mL) for 24 h. The NO levels in the culture medium were measured by the Griess assay. NIL (iNOS inhibitor, 10 μM) was used as a positive control to inhibit NO production. Values represent the means ± SD of three independent experiments. #p < 0.05 vs the control. *p < 0.05 vs LPS-stimulated group. CON, control; NIL, L-N6-(1-iminoethyl)lysine. 1601

DOI: 10.1021/acs.jnatprod.8b00182 J. Nat. Prod. 2018, 81, 1598−1603

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Chromatographic separation of F2013 (67.0 mg) was also performed by a FCS using manually packed Sephadex LH-20 (100 g scale, water−MeOH, 90:10−30:70) and SNAP Ultra C18 (30 g, water− MeOH, 70:30) cartridges to afford compound 18 (43.7 mg). Compounds 16 (12.6 mg) and 17 (17.8 mg) were obtained from F22 (64.5 mg) by a FCS (SNAP Ultra C18 cartridge 120 g, water− MeOH, 60:40−50:50). Compounds 19 (20.8 mg) and 2 (6.1 mg) were obtained from F26 (67.1 mg) and F28 (80.6 mg), respectively, by a FCS (SNAP Ultra C18 cartridge 120 g × 2, water−MeOH, 60:40−50:50). Compound 14 (35.2 mg) was obtained from F17 (40.2 mg) by precipitation. The BuOH-soluble extract (25.0 g) was subjected to FC with a manually packed Diaion HP-20 cartridge (340 g scale, water−MeOH, 100:0−0:100) to afford 14 pooled fractions (B01−B14). Compounds 4 (17.6 mg) and 20 (70.6 mg) were obtained from fractions B04 (83.4 mg) and B11 (1.2 g), respectively, by precipitation. Fraction B08 (2.1 g) was fractionated using a FCS (SNAP Ultra C18 cartridge 120 g × 2, water−MeOH, 90:10−60:40) to produce 18 subfractions (B0801−B0818). B0803 (44.0 mg) was fractionated using a FCS with manually packed Sephadex LH-20 (10 g scale × 4, water−MeOH, 30:70) and SNAP Ultra (25 g, CHCl3−MeOH−water, 90:10:1) cartridges to give compound 6 (2.8 mg). Compounds 7 (16.0 mg) and 8 (98.9 mg) were obtained from fractions B0804 (16.0 mg) and B0807 (127.4 mg), respectively, by precipitation. Compound 3 (4.3 mg), along with seven subfractions (B081201−B081207), was obtained from B0812 (48.1 mg) by a FCS (SNAP Ultra C18 cartridge 30 g, water−MeOH, 75:25). Repeated chromatographic isolation of fraction B081207 (25.2 mg) was carried out by a FCS using manually packed Sephadex LH-20 (100 g scale × 2, water−MeOH, 75:25) and SNAP Ultra C18 (30 g × 2, water−MeOH, 75:25) cartridges to generate compound 5 (4.6 mg). Fractionation of F0813 (340.2 mg) was performed by a FCS using a SNAP Ultra C18 cartridge (120 g, water−MeOH, 75:25−65:35) to afford compound 9 (6.3 mg). (2R,3S)-3,7,4′-Trihydroxy-5-methoxycoumaronochromone (1): white powder; [α]22D −184.6 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 214 (4.18), 292 (4.03) nm; ECD (c 0.3 mM, CH3CN) λmax (Δε) 211 (+19.9), 239 (−2.9), 262 (+5.1), 292 (−19.9) nm; 1H NMR (DMSO-d6, 800 MHz) δ 3.69 (3H, s, MeO-5), 6.03 (1H, d, J = 2.0 Hz, H-8), 6.11 (1H, d, J = 2.0 Hz, H-6), 6.17 (1H, s, H-2), 6.27 (1H, d, J = 2.0 Hz, H-3′), 6.34 (1H, dd, J = 7.8, 2.0 Hz, H-5′), 6.64 (1H, s, OH-3), 6.99 (1H, d, J = 7.8 Hz, H-6′), 9.79 (1H, s, OH-4′), 10.78 (1H, br s, OH-7); 13C NMR (DMSO-d6, 200 MHz) δ 55.8 (CH3, MeO-5), 80.3 (C, C-3), 94.9 (CH, C-6), 96.8 (CH, C-8), 97.3 (CH, C-3′), 103.5 (C, C-10), 109.2 (CH, C-5′), 111.0 (CH, C-2), 117.9 (C, C-1′), 125.6 (CH, C-6′), 159.9 (C, C-9), 160.0 (C, C-2′), 160.7 (C, C-4′), 161.7 (C, C-5), 164.7 (C, C-7), 185.8 (C, C-4); HRESIMS (negative) m/z 315.0500 [M − H]− (calcd for C16H11O7 315.0505). 7,2′,4′-Trihydroxy-5-methoxyisoflavone-4′-O-β-D-glucopyranoside (3): white powder; [α]22D −64.8 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 214 (4.37), 257 (4.36) nm; 1H NMR (DMSO-d6, 600 MHz) δ 3.19 (1H, m, H-4″), 3.22 (1H, m, H-2″), 3.27 (1H, overlapped, H-5″), 3.28 (1H, overlapped, H-3″), 3.49 (1H, m, H6″b), 3.68 (1H, d, J = 7.7 Hz, H-6″a), 3.79 (3H, s, MeO-5), 4.81 (1H, d, J = 7.0 Hz, H-1″), 6.41 (1H, s, H-8), 6.39 (1H, s, H-6), 6.52 (1H, d, J = 7.6 Hz, H-5′), 6.55 (1H, s, H-3′), 7.02 (1H, d, J = 7.6 Hz, H6′), 7.96 (1H, s, H-2), 9.39 (1H, s, OH-2′), 10.72 (1H, br s, OH-7); 13 C NMR (DMSO-d6, 150 MHz) δ 55.9 (CH3, MeO-5), 60.6 (CH2, C-6″), 69.6 (CH, C-4″), 73.2 (CH, C-2″), 76.6 (CH, C-5″), 77.0 (CH, C-3″), 94.7 (CH, C-8), 96.5 (CH, C-6), 100.4 (CH, C-1″), 104.1 (CH, C-3′), 106.7 (CH, C-5′), 107.7 (C, C-10), 113.6 (C, C1′), 122.8 (C, C-3), 131.9 (CH, C-6′), 151.9 (CH, C-2), 156.3 (C, C2′), 158.2 (C, C-4′), 159.1 (C, C-9), 161.0 (C, C-5), 162.4 (C, C-7), 174.2 (C, C-4); HRESIMS (positive) m/z 463.1250 [M + H]+ (calcd for C22H23O11 463.1240). 5,7,4′-Trihydroxyisoflavone-7-O-β-D-gentiotrioside (5): white powder; [α]22D −37.6 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 210 (4.56), 262 (4.55) nm; 1H NMR (DMSO-d6, 600 MHz) δ 2.97 (1H, m, H-2⁗), 3.02 (1H, m, H-2‴), 3.045 (1H, m, H-4⁗), 3.047 (1H, m, H-5⁗), 3.107 (1H, m, H-3⁗), 3.129 (1H, m, H-3‴), 3.136

(1H, m, H-4‴), 3.17 (1H, m, H-4″), 3.28 (1H, m, H-2″), 3.30 (1H, overlapped, H-5‴), 3.33 (1H, overlapped, H-3″), 3.44 (1H, m, H6⁗b), 3.59 (1H, dd, J = 11.2, 7.1 Hz, H-6‴b), 3.65 (2H, overlapped, H-6″b and H-6⁗a), 3.79 (1H, t, J = 8.5 Hz, H-5″), 3.94 (1H, d, J = 11.2 Hz, H-6″a), 4.01 (1H, d, J = 10.6 Hz, H-6‴a), 4.17 (1H, d, J = 7.6 Hz, H-1⁗), 4.21 (1H, d, J = 7.6 Hz, H-1‴), 5.10 (1H, d, J = 7.6 Hz, H-1″), 6.52 (1H, d, J = 1.2 Hz, H-6), 6.828 (1H, overlapped, H8), 6.831 (2H, d, J = 8.8 Hz, H-2′ and H-6′), 7.40 (2H, d, J = 8.8 Hz, H-3′ and H-5′), 8.37 (1H, s, H-2), 9.63 (1H, br s, OH-4′), 12.9 (1H, br s, OH-5); 13C NMR (DMSO-d6, 150 MHz) δ 61.0 (CH2, C-6⁗), 68.9 (CH2, C-6‴), 69.2 (CH2, C-6″), 69.9 (CH, C-4″), 70.0 (CH, C4⁗), 70.1 (CH, C-4‴), 73.2 (CH, C-2″), 73.4 (CH, C-2⁗), 73.5 (CH, C-2‴), 75.2 (CH, C-5″), 75.4 (CH, C-5‴), 76.3 (CH, C-3″), 76.6 (CH, C-3‴), 76.7 (CH, C-3⁗), 76.8 (CH, C-5⁗), 94.7 (CH, C8), 99.6 (CH, C-1″), 99.7 (CH, C-6), 103.4 (CH, C-1⁗), 103.7 (CH, C-1‴), 106.1 (C, C-10), 115.1 (2CH, C-2′ and 6′), 121.0 (C, C-3), 122.5 (C, C-1′), 130.1 (2CH, C-3′ and 5′), 154.4 (CH, C-2), 157.3 (C, C-9), 157.5 (C, C-4′), 161.5 (C, C-5), 162.9 (C, C-7), 180.5 (C, C-4); HRESIMS (positive) m/z 757.2169 [M + H]+ (calcd for C33H41O20 757.2191). Acid Hydrolysis and Sugar Identification. The absolute configuration of the sugar moiety of compounds 3 and 5 was determined by the method of Tanaka et al.25 The compounds (0.5 mg) were hydrolyzed with 0.5 M HCl (0.1 mL) for 2 h at 95 °C and neutralized with Amberlite IRA400 (MilliporeSigma). The residue was evaporated in vacuo and then dissolved in pyridine (0.1 mL) containing L-cysteine methyl ester (0.5 mg, MilliporeSigma), which was heated at 60 °C for reaction. After 1 h, o-tolyl isothiocyanate (5 μL, MilliporeSigma) was added to the mixture and further heated at 60 °C for 1 h. The standard L-glucose and D-glucose were reacted via the same method. Subsequently, analytical UHPLC was carried out to analyze the reaction mixture on a Kinetex 1.7 μm F5 100 Å LC column at 35 °C with isocratic elution (water−CH3CN−H3PO4, 75:25:0.1) for 10 min at a flow rate of 0.2 mL/min. Peaks were detected at 250 nm. The absolute configuration of glucose in compounds 3 and 5 was confirmed by comparing their retention times to those of standard derivatives recorded at tR 3.102 min (Lglucose derivative) and 3.451 min (D-glucose derivative). Computational Methods. Three-dimensional models of compound 1 were built using the Chem3D software package, and conformer distribution was performed by the Merck Molecular Force Field (MMFF) as implemented in the Spartan’14 software (Wavefunction, Inc., Irvine, CA, USA). The selected conformers were optimized at the density functional theory [B3LYP functional/631+G(d,p) basis set] level, and ECD calculations were carried out at the time-dependent density functional theory (CAM-B3LYP/SVP basis set) level with a conductor-like polarizable continuum solvent model in CH3CN by the Gaussian 09 software package (Gaussian, Inc., Wallingford, CT, USA). The calculated ECD spectra were converted to a half-bandwidth of 0.3 eV by the SpecDis 1.64 software (University of Wuerzburg, Wuerzburg, Germany), and ECD curves of the conformers were weighted by the Boltzmann distribution. After UV correction, the calculated ECD spectra were compared with the experimental ECD spectrum. Determination of NO Production Inhibitory Effects. The inhibitory activities of the isolates on NO production were evaluated in LPS-stimulated RAW 264.7 cells, which were plated at a density of 0.9 × 105 cells/mL in a 96-well plate and incubated for 24 h. The accumulated nitrite, an indicator of NO production, in the culture medium was measured according to the Griess reagent. The detailed experimental method was the same as described in a previous contribution.26 MTT Assay. Cell viability studies were carried out by the MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; MilliporeSigma) assay, as previously described.26 1602

DOI: 10.1021/acs.jnatprod.8b00182 J. Nat. Prod. 2018, 81, 1598−1603

Journal of Natural Products

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Article

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00182.

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NMR and HRESIMS spectra for the new compounds 1, 3, and 5 and cell viability of compounds 1, 3, 6−8, 15, and 20 (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +82-42-868-9341. Fax: +82-42-861-5800. E-mail: [email protected] (J. Lee). ORCID

Dae Sik Jang: 0000-0001-5472-5232 Sang Hee Shim: 0000-0002-0134-0598 Jung-Hye Choi: 0000-0001-5178-6814 Author Contributions #

Y. H. Seo and J.-H. Jeon contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Research Council of Science & Technology (NST) grant by the Korean government (MSIP) (Nos. CRC-15-04-KIST, G15120, G16230, G17290, K15312).

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DOI: 10.1021/acs.jnatprod.8b00182 J. Nat. Prod. 2018, 81, 1598−1603