New Triterpenoid Saponins from Green Vegetable Soya Beans and

Nov 29, 2017 - Department of Biotechnology, School of Life Science and Technology, Center for Informational Biology, University of Electronic Science ...
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Cite This: J. Agric. Food Chem. 2017, 65, 11065−11072

New Triterpenoid Saponins from Green Vegetable Soya Beans and Their Anti-Inflammatory Activities Xiuhua Lan,†,‡ Kejun Deng,† Jianping Zhao,§ Yiyi Chen,† Xuhui Xin,† Yanli Liu,‡ Ikhlas A. Khan,‡,§ Shilin Yang,† Taoyun Wang,*,‡,# and Qiongming Xu*,‡ †

Department of Biotechnology, School of Life Science and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu 610054, China ‡ College of Pharmaceutical Science, Soochow University, Suzhou 215123, China § National Center for Natural Products Research, Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677, United States # College of Chemical, Biological and Material Engineering, Suzhou Science and Technology University, Suzhou 215009, China S Supporting Information *

ABSTRACT: Ten compounds were isolated and identified from green vegetable soya beans, of which five are new triterpenoid saponins (1−5) and five are known compounds (6−10). The chemical structures of the five triterpenoid saponins (1−5) were elucidated to be 3β,24-dihydroxy-22β,30-epoxy-30-oxoolean-12-en 3-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl-(1 → 2)-β-D-glucuronopyranoside, 1; 3β,24-dihydroxy-22β,30-epoxy-30-oxoolean-12-en 3-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-(3″O-formyl)-galactopyranosyl-(1 → 2)-β-D-glucuronopyranoside, 2; 22-keto-3β,24-dihydroxy oleanane-12-ene 3-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-(3″-O-formyl)-galactopyranosyl-(1 → 2)-β-D-glucuronopyranoside, 3; 3β,22β,24-trihydroxy oxyolean18(19)-ene-29-acid 3-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 2)-β-D-glucuronopyranoside, 4; and punicanolic acid 3-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 2)-β-D-glucuronopyranoside, 5 from the spectroscopic data (IR, GTC/FID, HR-ESI-MS, and 1D and 2D NMR). The nitric oxide release inhibitions of compounds 1−10 in LPS-stimulated RAW264.7 cells were evaluated, and the data suggested that compounds 1, 2, and 5 might possess moderate anti-inflammatory activities, with IC50 values of 18.8, 16.1, and 13.2 μM, respectively. KEYWORDS: soya beans, Glycine max, triterpenoid saponin, structure elucidation, anti-inflammatory activities

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INTRODUCTION The soybean plant (Glycine max (L.) Merr., Fabaceae), one of the most important healthy foods and economic crops, is cultivated in many countries and regions. So far, following the United States, Brazil, and Argentina, China has ranked as the fourth most productive country in annual gross production of soybean.1−3 Green vegetable soya beans, known as maodou in China and edamame in Japan, are commonly consumed as snack foods and used in dishes in many Asian countries. According to previous studies, green vegetable soya beans are a cornucopia of plant proteins, essential amino acids, ω−3 fatty acids, and various micronutrients that could exert a series of physiological functions to humans and livestock.4−6 Recent studies indicated that isoflavones,7 saponins (soyasaponins),8 alkaloids,9 and steroids10 contributed to the diverse bioactivities of soybeans. Soyasaponins, the major ingredients of the hypocotyl of soybean,11,12 exhibit many biological activities, such as cholesterol-lowering,13 renin-inhibiting,14 hepatoprotective,15 anti-kidney-disease-progression,16 antioxidative,17,18 anti-inflammatory,19 and antitumor effects.20,21 Some soyasponins, specifically soyasaponin Ab, have shown potential anti-inflammatory activity and been the subject of intensive study. 22 In this study, the phytochemical investigation of saponins in green vegetable soya beans led to the isolation of five new soyasaponins 1−5 (Figure 1) along with five known ones, whose structural elucidation and antiinflammatory activities are reported herein. © 2017 American Chemical Society

MATERIALS AND METHODS

General. Optical rotations, IR spectra, and 1H and 13C NMR spectra were determined by a polarimeter (PerkinElmer Inc., Waltham, MA), a spectrometer (PerkinElmer Inc., Waltham, MA), and a 500 NMR facility (Varian Inc., Palo Alto, CA), respectively. The Q-TOF MS/MS system (Micromass Corporation, London, U.K.) was employed to detect the HR-ESI-MS data. Chromatography. Two chromatographic columns were used: column 1 (250 × 10.0 mm i.d., 5 μm, Kromsil C18, Akzo Nobel, Amsterdam, Holland) and column 2 (250 × 10.0 mm i.d., 5 μm, Cosmosil cholester, Nacalai Tesque, Inc., Kyoto, Japan). The analyses were performed on column 1 or 2, using the HPLC system (Shimadzu Corporation, Kyoto, Japan) with UV detection at 203 nm. Columns 3 (D101 macroporous resin column, 200 × 30 cm i.d.) and 4 (silica gel, 60−100 mesh, 20 × 12 cm i.d.) were used for preseparation. A glass MPLC column, 5 (460 mm × 26 mm i.d.), and an MPLC system including a C-650 pump (Büchi Corporation, Flawil, Switzerland) were used for purification. The silica gel (60−100 mesh) and the precoated silica gel, supplied by Qingdao Marine Chemical Factory Company, Ltd. (Qingdao, China), were employed for the raw-material purification. A 10% sulfuric acid ethanol solution was employed as the color-developing agent for TLC. A Sephadex LH-20 and D101 macroporous resin were supplied by GE Corporation (Piscataway, NJ) and Professor Xiao-ran Li Received: Revised: Accepted: Published: 11065

September 4, 2017 November 27, 2017 November 29, 2017 November 29, 2017 DOI: 10.1021/acs.jafc.7b04134 J. Agric. Food Chem. 2017, 65, 11065−11072

Article

Journal of Agricultural and Food Chemistry

Figure 1. Structures of compounds 1−10. fraction was further separated by semipreparative HPLC using column 1 and eluted with 70% MeOH at a flow of 2 min/mL to obtain compounds 3 (12 mg) and 10 (29 mg), whose retention times were 33.05 and 30.53 min, respectively. The fourth fraction (108 mg) was further separated by semipreparative HPLC using column 1 and eluted with 65% MeOH at 2 min/mL to give compound 7 (55 mg, tR of 42.15 min) and a mixture of compounds 1 and 2 (31 mg, tR of 44.38−48.33 min). This mixture was further isolated by semipreparative HPLC on column 2 and eluted with 60% MeOH to obtain compounds 1 (8.5 mg) and 2 (11.6 mg), whose retention times were 68.62 and 71.05 min, respectively. The 40% MeOH/CHCl3 eluate (1.7 g) was separated using an MPLC system with a reverse-phase ODS column and eluted with MeOH/H2O (40, 50, 60, 70, and 100%, each 500 mL) to afford 5 fractions, of which the third fraction (35 mg) was further isolated by semipreparative HPLC on column 1 and eluted with 65% MeOH at the flow of 2 min/mL, to give compounds 4 (8.1 mg, tR of 37.45 min) and 5 (12 mg, tR of 40.32 min). Compound 1. White, amorphous powder; [α]25 D −29.5 (c = 0.12, MeOH); IR (KBr) νmax: 3455, 2941, 1761, 1645, 1462, 1047, and 870 cm−1; 1H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectroscopic data: see Table 1; HR-ESI-MS (negative-ion mode) m/z: 923.4646 [M − H]− (C47H71O18, 923.4640). Compound 2. White, amorphous powder; [α]25 D −25.4 (c = 0.11, MeOH); IR (KBr) νmax: 3452, 2945, 2818, 2715, 1748, 1728, 1643, 1458, 1051, and 883 cm−1; 1H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectroscopic data: see Table 1; HR-ESI-MS (negative-ion mode) m/z: 981.4702 [M − H]− (C49H73O20, 981.4695). Compound 3. White, amorphous powder; [α]25 D −21.0 (c = 0.10, MeOH); IR (KBr) νmax: 3398, 2940, 2820, 2716, 1730, 1660, 1458, 1045, and 887 cm−1; 1H NMR (C5D5N, 500 MHz) and 13C NMR

(Soochow University), respectively. GC data were obtained by a GC14C system that included a flame ionization detector (FID, Shimadzu). The detailed analytical parameters were set as follows: a DB-5 column, 30 × 0.25 mm i.d., was used at a column temperature of 210 °C; the injector temperature was 270 °C, and the detector temperature was 300 °C. The DB-5 column was a gift from Suzhou Huitong Chromatography Technology Company, Ltd. (Suzhou, China). Materials. The fresh soya fruits were harvested in the Suzhou area (Jiangsu, China) during September 2012 and identified by Xiao-ran Li. In this study, the flakelets of the fresh fruits (5.0 kg) were dried in the sun. The voucher specimen was stored in the herbarium of Soochow University and labeled as No. 12-09-16-01. Extraction and Isolation Process. Sun-dried flakelets (1.2 kg) of the soya fruits were extracted two times by MeOH at 80 °C under reflux. Subsequently, the MeOH was recycled using the rotary evaporator to obtain a residue (225 g) that was sequentially extracted by petroleum ether, CHCl3, and EtOAc. Column 3 was employed to purify the remaining water fraction using an ethanol gradient (EtOH/H2O 0, 30, 60, and 90%, each 30 L). The dried 60/40% fraction (8 g) was purified on column 4, using MeOH/CHCl3 (10, 20, 30, 40, 50, 60, 70, and 100%, each 3.0 L). The dried MeOH/CHCl3 (20%, 1.2 g) was separated on an MPLC system with an ODS column, using MeOH/H2O (60, 70, 80, 90, and 100%, each 300 mL) to obtain five fractions. The second fraction (525 mg) was purified on column 1 using a semipreparative HPLC system with a mobile-phase (30% MeOH) flow rate of 2 min/mL to yield compounds 6 (113 mg), 8 (48 mg), and 9 (53 mg), whose retention times (tR) were 25.04, 29.47, and 26.82 min, respectively. The MeOH/CHCl3 (30%) fraction (0.9 g) was further purified by MPLC using a reverse-phase ODS column and eluted with MeOH/H2O (50, 60, 70, 80, and 100%, each 300 mL) to give five fractions. The third 11066

DOI: 10.1021/acs.jafc.7b04134 J. Agric. Food Chem. 2017, 65, 11065−11072

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Journal of Agricultural and Food Chemistry Table 1. NMR Spectroscopic Data for Compounds 1−5 (in C5D5N)a 1

2

δC

δH

δC

δH

δC

δH

δC

δH

δC

δH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

38.8 26.8 91.1 44.1 56.2 18.7 33.5 43.0 47.8 36.6 25.3 125.2 141.0 39.8 25.1 26.6 36.2 45.3 42.8 42.5 38.6 84.7 23.1 63.6

1.32m, 0.78m 2.19m, 1.87m 3.37dd (12.0, 4.5)

38.6 26.5 91.2 43.9 56.1 18.5 33.3 42.9 47.7 36.5 24.9 125.1 140.9 39.7 25.2 26.6 36.1 45.2 42.7 42.4 38.5 84.6 23.0 63.6

1.30m, 0.77m 2.14m, 1.80m 3.36m

39.2 26.2 91.8 44.5 56.7 17.4 34.8 40.5 48.5 37.1 23.9 124.7 142.5 42.7 27.3 28.0 47.3 48.5 47.3 34.8 51.6 216.4 23.6 64.2

1.38m, 0.85m 2.15m, 1.82m 3.38m

38.6 26.5 91.0 43.8 56.0 18.2 34.7 40.6 51.0 36.5 21.4 27.4 38.9 43.2 26.7 28.3 39.9 145.8 123.7 45.0 38.4 75.7 22.7 63.3

1.47m, 0.75m 2.20m, 1.85m 3.37dd (11.5, 3.5)

38.6 26.4 89.8 39.5 55.6 18.2 34.7 41.3 50.0 36.6 21.7 29.5 40.6 42.8 29.6 35.8 51.1 47.8 39.7 72.2 37.2 33.6 28.0 16.1

1.48m, 0.76m 2.18m, 1.80m 3.28dd (11.5, 4.5)

25 26 27 28 29 30 GlcA-1 2 3 4 5 6 Xyl-1 2 3 4 5 Gal-1 2 3 4 5 6 formyl Rha-1 2 3 4 5 6

15.9 16.9 25.1 23.7 20.8 180.5 105.6 78.2 76.8 74.1 77.7 172.8 102.0 77.7 75.8 70.7 67.1

number

a1

102.5 72.5 72.8 74.5 69.5 19.1

0.83m 1.32m, 1.24m 1.44m, 1.26m 1.52m 1.66m, 1.21m 5.10brs

1.66m, 1.21m 1.80m, 1.02m 2.11m 1.79m, 1.50m 1.95m, 2.25m 4.19dd (11.5, 5.5) 1.39s 4.25m 3.27d (11.5) 0.67s 0.8s 1.21s 0.97s 1.19s 4.93d (8.0) 4.53m 4.41mb 4.44mb 4.44mb

3

15.8 16.8 25.0 23.6 20.7 180.5 105.5 78.3 77.8 73.8 77.6 172.5

4

0.83m 1.54m, 1.22m 1.43m, 1.27m 1.52m 1.67m, 1.20m 5.10brs

1.67m, 1.21m 1.80m, 1.06m 2.11m 1.79m, 1.50m 1.96m, 2.25m 4.2m 1.39s 4.25m 3.28d (11.0) 0.66s 0.8s 1.20s 0.97s 1.18s 4.93d (8.0) 4.17m 4.23mb 4.42m 4.57m

16.5 17.4 26.2 21.7 32.6 26.2 106.1 79.0 77.0 74.4 78.3 173.0

0.85m 1.44m, 1.24m 1.43m, 1.28m 1.28m 1.51m, 1.21m 5.24brs

1.68, 1.36m 2.12m, 1.15m 2.35m 2.02m, 1.16m 2.57m, 2.14m 1.40s 4.27m 3.24m 0.69s 0.96s 1.28s 1.15s 0.96s 0.84s 4.93d (8.0) 4.35 4.05m 4.42m 4.60mb

5

16.7 15.9 14.9 18.7 179.2 28.3 105.1 78.2 76.5 73.7 77.4 170.2

0.73m 1.48m, 1.20m 1.39m, 1.21m 1.22m 1.43m, 1.16m 1.82m, 1.13m 2.41m 1.63m, 1.32m 1.68m, 0.96m

5.52s 2.92m, 1.90m 4.3dd (10.5, 3.5) 1.36s 4.23m 3.21d (11.5) 0.64s 1.00s 0.92s 1.36s 1.54s 4.93d (8.0) 4.55mb 4.53mb 4.40m 4.50mb

16.4 16.4 14.9 179.0 18.8 30.6 105.1 78.3 77.1 73.4 78.5 172.4

0.68m 1.34m, 1.23m 1.42m, 1.25m 1.24m 1.39m, 1.13m 1.31m, 1.17m 2.69m 1.95m, 1.23m 2.32m, 1.55m 2.42m 2.43d (3.5) 2.05m, 1.81m 2.30m, 1.97m 1.30s 1.03s 0.67s 0.97s 1.00s 1.34d (10.0) 1.39s 5.01d (8.0) 4.48mb 4.58m 4.40mb 4.58m

5.51d (7.0) 4.58m 4.00 4.00mb 4.17m, 3.57m

6.14(brs) 4.73(brs) 4.64m 4.29mb 4.92m 1.74d (6.0)

101.7 77.7 77.6 71.2 76.4 61.6 164.2 102.4 72.4 72.6 74.3 69.4 19.0

5.68d (8.0) 4.45mb 4.57m 4.33mb 4.04m 4.34m,b 4.26m 8.59s 6.16(brs) 4.69(brs) 4.63m 4.27mb 4.96m 1.73d (6.5)

102.4 77.3 78.4 71.7 77.2 62.2 164.8 103.0 73.0 73.3 75.0 70.0 19.6

5.69d (8.0) 4.53m 4.47mb 4.35mb 3.90m 4.36o, 4.27m 8.59s 6.18(brs) 4.74(brs) 4.63mb 4.29mb 4.93m 1.74d (7.0)

101.5 77.3 76.5 70.9 76.4 61.4

5.72d (8.0) 4.50mb 4.53mb 4.35m 4.05m 4.36m, 4.28m

101.8 78.4 79.2 72.6 77.5 63.2

5.80d (8.5) 4.26m 4.19m 4.02m 3.81m 4.45, 4.26m

102.1 72.2 72.5 74.1 69.1 18.7

6.22(brs) 4.75(brs) 4.66m 4.29mb 4.95m 1.73d (6.0)

101.8 72.2 72.5 74.1 69.2 18.8

6.35(brs) 4.72(brs) 4.66m 4.30mb 5.01mb 1.77d (6.0)

H NMR at 500 MHz and 13C NMR at 125 MHz in DMSO-d6. bSignals overlapped. Compound 4. White, amorphous powder; [α]25 D −23.2 (c = 0.11, MeOH); IR (KBr) νmax: 3380, 2940, 1658, 1465, 1052, and 884 cm−1;

(C5D5N, 125 MHz) spectroscopic data: see Table 1; HR-ESI-MS (positive-ion mode) m/z: 969.5059 [M + H]+ (C49H77O19, 969.5059). 11067

DOI: 10.1021/acs.jafc.7b04134 J. Agric. Food Chem. 2017, 65, 11065−11072

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Journal of Agricultural and Food Chemistry

Figure 2. Key HMBC and NOESY correlations for compounds 1−5. reported.23 In this study, the GC retention times of the standard monosaccharides (i.e., D-glucuronic acid, D-glucuronic acid methyl ester, D-galactose, D-xylose, and L-arabinose) were 19.58, 24.35, 25.55, 31.98, and 33.00 min, respectively. MTT Assay for Cell Viability. An MTT (Sigma-Aldrich, St. Louis, MO) assay was employed to detect the cytotoxicity of compounds 1− 10. Briefly, RAW264.7 macrophage cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were planted into 96-well plates at densities of 105 cells/well and subsequently treated with the isolated saponins for 24 h. This was followed by MTT treatment (0.5 mg/mL) for 3 h at 37 °C. UV-absorbance values were detected at 570 nm. Determination of Nitric Oxide Production by the Griess Assay. The inhibitory activities of compounds 1−10 on NO production in lipopolysaccharide (LPS)-induced RAW264.7 mouse macrophages

1

H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectroscopic data: see Table 1; HR-ESI-MS (negative-ion mode) m/z: 971.4815 [M − H]− (C48H75O20, 971.4852). Compound 5. White, amorphous powder; [α]25 D −22.1 (c = 0.10, MeOH); IR (KBr) νmax: 3414, 2942, 1663, 1447, 1039, and 887 cm−1; 1 H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectroscopic data: see Table 1; HR-ESI-MS (negative-ion mode) m/z: 957.5027 [M − H]− (C48H77O19, 957.5059). Acid-Hydrolysis and Sugar Analyses of the Five New Triterpenoid Saponins (1−5). The procedures of the acid-hydrolysis and sugar analyses of the five new triterpenoid saponins (1−5) were performed according to the published literature.23 The detailed process of acid hydrolysis and the GC conditions were the same as those 11068

DOI: 10.1021/acs.jafc.7b04134 J. Agric. Food Chem. 2017, 65, 11065−11072

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Journal of Agricultural and Food Chemistry were evaluated using a previously reported method.24 Briefly, RAW264.7 cells were planted into 24-well plates at densities of 5 × 105 cells/well and then incubated with or without 1 μg/mL LPS and with certain dosages of the various indicated compounds for 24 h. Nitrite levels (NO) in the culture media were determined by the Griess assay (Beyotime Institute of Biotechnology, Haimen, China). Statistical Analysis. All data were presented as means ± SD. Oneway analysis of variance (one-way ANOVA), performed using GraphPad Prism 6.0 software, was employed to evaluate the significances (p < 0.05) of the intergroup differences.

signals for one xylopyranosyl group, which replaced those of the glucuronopyranosyl group located at the C-2 of the glucuronopyranosyl methyl ester in the structure of albiziasaponin A. Thus, compound 1 was identified as 3β,24-dihydroxy-22β,30epoxy-30-oxoolean-12-en 3-O-α-L-rhamnopyranosyl-(1 → 2)-βD-xylopyranosyl-(1 → 2)-β-D-glucuronopyranoside. Compound 2, a white, amorphous powder, has a molecular formula of C49H74O20 according to its [M − H]− data. In terms of its IR data, a hydroxyl group (3452 cm−1), γ-lactone group (1748 cm−1), and formyl group (2818, 2715, 1728 cm−1) existed in compound 2. The 13C NMR spectrum data exhibited 48 carbon peaks, of which 18 were ascribed to three sugar moieties, and the remaining 30 were ascribed to a triterpene skeleton. The aglycone NMR data of compound 2 were identical to those of compound 1. As shown in Table 1, three protons (δH 4.93, 1H, d, J = 8.0 Hz; 5.68, 1H, d, J = 8.0 Hz; and 6.16, 1H, brs) were correlated with three carbons (δC 105.5, 101.7, and 102.4), respectively, indicating the presence of glucuronic acid, galactose, and rhamnose moieties. The types of sugars in compound 2 were identified as L-rhamnose, D-galactose, and D-glucuronic acid by the acid-hydrolysis assay. The NMR data of the glycosidic chain of compound 2 were similar to those of soyasaponin Bh (compound 7), with exception of data indicating an additional formyl group. This additional formyl group was determined to be located at the C-3 of the galactopyranosyl group, according to the HMBC correlations between the carbon at δC 77.6 (C-3 of galactose) and the proton at δH 8.59 (H-1 of the formyl group) and between the carbon at δC 164.2 (C-1 of the formyl group) and the proton at δH 4.57 (H-3 of galactose). Thus, compound 2 was identified as 3β,24-dihydroxy-22β,30-epoxy-30-oxoolean12-en 3-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-(3″-O-formyl)galactopyranosyl-(1 → 2)-β-D-glucuronopyranoside. Compound 3, isolated as a white, amorphous powder, was deduced by HR-ESI-MS data to possess a molecular formula of C49H76O19. In the 1H NMR spectrum data, seven tertiary methyl groups (δH 1.40, 3H, s; 1.28, 3H, s; 1.15, 3H, s; 0.96, 3H, s; 0.96, 3H, s; 0.84, 3H, s; and 0.69, 3H, s), an olefinic proton (δH 5.24, 1H, m), a hydroxymethylene group (δH 4.27, 3.24, 2H, m), and a methine group (δH 3.38, 1H, m) were observed. In addition, as shown in the HSQC spectrum, three anomeric protons (δH 4.93, 1H, d, J = 8.0 Hz; 5.69, 1H, d, J = 8.0 Hz; and δ 6.18, 1H, brs) showed correlations with three carbons at δC 106.1, 102.4, and 103.0, respectively, indicating the existence of three sugar units in compound 3. The sugar residues of 3 were identified as Lrhamnose, D-galactose, and D-glucuronic acid using the acidhydrolysis assay. The 1H and 13C NMR data of 3 were identical to those of dehydrosoyasaponin I (compound 10),31 except for the presence of data indicating an additional formyl group in 3. The downfield shift of the C-3 signal of galactose (+1.8 ppm) at δC 78.4 suggested that the additional formyl group was linked to the C-3 of the galactopyranosyl group, which was further confirmed by the HMBC correlations of the C-3 of galactose (δC 78.4) to the H-1 of the formyl group (δH 8.59) and the C-1 of the formyl group (δC 164.8) to the H-3 of galactose (δH 4.47). Thus, compound 3 was identified as 3-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-(3″-O-formyl)-galactopyranosyl-(1 → 2)-β-D-glucuronopyranoside. Compound 4, a white, amorphous powder, had a molecular formula of C48H76O20 according to the [M − H]− data. In the 1H NMR spectrum, the signals of six tertiary methyl groups (δH 1.54, 3H, s; 1.36, 3H, s; 1.36, 3H, s; 1.00, 3H, s; 0.92, 3H, s; and 0.64, 3H, s), a hydroxymethylene group (δH 4.23, 1H, m; 3.21, 1H, d, J = 11.5 Hz), an olefinic proton (δH 5.52, 1H, brs), and two

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RESULTS AND DISCUSSION By sequentially using D101 resins and ODS-column chromatography, the MeOH extract was separated to afford five new compounds (1−5) and five known ones: oxytrogenin 3β-O-α-Lrhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-Dglucuronopyranoside, 6,25 soyasaponin Bh, 7,26 soyasaponin Bb, 8,27 complogenin 3β-O-α-L-rhamnopyranosyl-(1 → 2)-β-Dgalactopyranosyl-(1 → 2)-β-D-glucuronopyranoside, 9,28 and dehydresoyasaponin I, 1029 (Figure 1). Compound 1 was a white, amorphous powder with the formula C47H72O18, according to its HR-ESI-MS [M − H]− data. Its IR data at 1760 and 1640 cm−1 indicated the existence of γlactone and an olefin group on compound 1, and an oligoglycoside structure was inferred by the data at 3453 and 1047 cm−1. From the 1H NMR spectrum, six tertiary methyl groups (δH 1.39, 3H, s; 1.21, 3H, s; δH 1.19, 3H, s; 0.97, 3H, s; δH 0.80, 3H, s; and 0.67, 3H, s) were labeled as methyls C 23, 27, 29, 28, 26, and 25, respectively. In addition, the characteristic proton signals of an olefinic proton (δH 5.10 1H, m), a hydroxymethylene group (δH 4.25 1H, m and δH 3.27 1H, d, J = 11.5 Hz), and two oxygenated methine groups (δH 3.37 1H, J = 12.0, 4.5 Hz and δH 4.19 1H, J = 11.5, 5.5 Hz) were observed in the 1H NMR spectrum. A total of 47 carbon resonances appeared in the 13C NMR spectrum, of which 30 were ascribed to a triterpene skeleton, and the remaining 17 were ascribed to three sugar moieties. The three sugar residues were confirmed by the three anomeric-proton signals (δH 4.93, 1H, d, J = 8.0 Hz; δH 5.51,1H, d, J = 7.0 Hz; and δH 6.14 1H, brs), which were further identified as the H-1’s of glucuronic acid, xylose, and rhamnose, respectively. In the HSQC spectrum (Table 1), these anomeric-proton signals showed correlations with carbons at δC 105.6 (C-1 of glucuronic acid), 102.0 (C-1 of xylose), and 102.5 (C-1 of rhamnose), respectively. The types of sugar residues on compound 1 were identified as L-rhamnose, D-xylose, and D-glucuronic acid using the acid-hydrolysis assay. As shown in Figure 2, the HMBC correlation between the proton at δH 4.93 (H-1 of glucuronic acid) and the carbon at δC 91.1 (C-3 of aglycone) displayed that the glucuronic acid group was linked to the C-3 of aglycone. The HMBC correlations between the proton at δH 5.51 (H-1 of xylose) and the carbon at δC 78.2 (C-2 of glucuronic acid) and between the proton at δH 6.14 (H-1 of rhamnose) and the carbon at δC 77.7 (C-2 of xylose) indicated the linkage type of the sugar moieties of compound 1 were α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl-(1 → 2)β-D-glucuronopyranosyl. In terms of their JH‑1, H‑2 coupling constants, the β-anomeric configurations of the glucuronopyranosyl and xylopyranosyl were determined, and the α-anomeric configuration of the rhamnopyranosyl unit was elucidated by the NOESY correlation between the protons at δH 6.14 (H-1 of rhamnose) and δH 1.74 (H-6 of rhamnose) (Figure 2). The 1H and 13C NMR spectra data of compound 1 were similar to those of albiziasaponin A, a triterpenoid glycoside isolated from the stems of Albizia myriophylla,30 except for the existence of the 11069

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11070

28.6 ± 2.34 93.8 ± 7.79 33.5 ± 2.16 90.3 ± 7.47 26.7 ± 1.49 103.7 ± 8.62 27.4 ± 1.68 96.1 ± 9.87 a

Cell viability was measured at the IC50 values corresponding to the inhibition of NO. * indicates p < 0.05

>50 85.7 ± 5.15* 13.2 ± 1.43 96.8 ± 6.92 >50 81.4 ± 5.71* 16.1 ± 1.51 95.2 ± 6.65 18.8 ± 1.62 97.6 ± 8.81 IC50 for inhibition of NO production (μM) cell viabilitya(% of cell survival)

28.3 ± 1.97 93.4 ± 9.38

2 1 compounds

Table 2. Anti-Inflammatory Activities of Compounds 1−10a

3

4

5

6

7

8

9

10

hexadecadrol

oxygenated methine groups (δH 3.37, 1H, J = 11.5, 3.5 Hz; 4.30, 1H, J = 10.5, 3.5 Hz) were observed. The 13C NMR spectrum exhibited 48 carbon signals, of which 18 were attributed to three sugar moieties, and the remaining 30 were attributed to a triterpene skeleton. In addition, three anomeric-proton signals (δH 4.93, 1H, d, J = 8.0 Hz; δH 5.72, 1H, d, J = 8.0 Hz; and δH 6.22, 1H, brs; Table 1) showed correlations with three carbons (δC 105.1, 101.5, and 102.1, respectively) in the HSQC spectrum. The types of sugar residues of 4 were identified as L-rhamnose, Dgalactose, and D-glucuronic acid by the acid-hydrolysis assay. The 1 H and 13C NMR data of 4 were similar to those of sophoraflavoside II,32 except for the significant downfield shifts of the C-18 signal (+101.0 ppm) at δC 145.7 and the C-19 signal (+82.2 ppm) at δC 123.7, accompanied by significant upfield shifts of the C-12 signal (−100.4 ppm) at δC 22.7 and the C-13 signal (−105.4 ppm) at δC 38.9, on account of the movement of the double bond from C-12 and C-13 in sophoraflavoside II to C18 and C-19 in compound 4. The assignment of the double bond location to C-18 and C-19 was also confirmed by the HMBC correlations of H-19 at δH 5.52 with C-29 at δC 179.2 and C-30 at δC 28.3. Thus, compound 4 was identified as 3β,22β,24trihydroxy oxyolean-18(19)-ene-29-acid 3-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 2)-β-D-glucuronopyranoside. Compound 5 was isolated as a white, amorphous powder with a molecular formula of C48H78O19 based on its HR-ESI-MS [M − H]− data. In its 1H NMR spectrum, signals of six tertiary methyl groups (δH 1.39, 3H, s; 1.30, 3H, s; 1.03, 3H, s; 0.97, 3H, s; 1.00, 3H, s; and 0.67, 3H, s), a doublet methyl group (δH 1.34, 3H, d, J = 10.0 Hz), and a methine group (δH 3.28, 1H, dd, J = 11.5, 4.5 Hz) were observed. The three anomeric-proton signals (δH 5.01, 1H, d, J = 8.0 Hz; 5.80, 1H, d, J = 8.5 Hz; and 6.35, 1H, brs) showed HSQC (Table 1) correlations with three carbons at δC 105.1, 101.8, and 101.8, respectively, indicating the existence of three sugar moieties. The sugar residues of compound 5 were identified as L-rhamnose, D-galactose, and D-glucuronic acid by the acid-hydrolysis assay. The 1H and 13C NMR data of the aglycone of compound 5 were similar to those of punicanolic acid,33except for the downfield shift of the C-3 signal (+11.6 ppm) at δC 89.8, due to the glycosidation shift of the additional glycosidic chain linked to the C-3 of the aglycone in compound 5. The location of the glycosidic chain was also deduced from the HMBC correlation between the proton at δH 5.01 (H-1 of glucuronic acid) and the carbon at δC 89.8 (C-3 of aglycone). The NMR data of the glycosidic chain of compound 5 were same as those of compound 4, suggesting the linkage of the glycosidic chain was α-L-rhamnopyranosyl-(1 → 2)-β-D-galactopyranosyl(1 → 2)-β-D-glucuronopyranosyl. Therefore, compound 5 was identify as punicanolic acid 3-O-α-L-rhamnopyranosyl-(1 → 2)β-D-galactopyranosyl-(1 → 2)-β-D-glucuronopyranoside. Anti-Inflammatory Activities. Soyasaponins isolated from soybean were reported to have significant therapeutic effects on many inflammation-related diseases, such as acute lung injuries,22 hepatitis,33 and diabetic nephropathy.34 Until now, only crude extracts of soyasaponins and a few soyasaponins were confirmed to contribute anti-inflammatory properties.35 In this study, the anti-inflammatory activities of compounds 1−10 were investigated by an assay of NO release using LPS-stimulated RAW264.7 cells. As shown in Table 2, no cytotoxicity was observed in any of the treated RAW264.7 cells. Compounds 1, 2, and 5 displayed moderate anti-inflammatory activities, with IC50 values of 18.8, 16.1, and 13.2 μM, respectively, compared with that of hexadecadrol (IC50 = 0.41 μM), which was used as the

0.41 ± 0.03 

Journal of Agricultural and Food Chemistry

DOI: 10.1021/acs.jafc.7b04134 J. Agric. Food Chem. 2017, 65, 11065−11072

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Journal of Agricultural and Food Chemistry positive control. The activities of compounds 3, 7, 8,35 9, and 10 were weak. Compounds 4 and 6 showed no anti-inflammatory activity. This could be the result of the presence of carboxylic acid in their structures, as compared with other, active soyasaponins,36,37 but further studies are needed for confirmation. Nevertheless, the findings from this study provide evidence that soyasaponins could possess anti-inflammatory activities with lower cytotoxicities in macrophage-mediated inflammatory responses.

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(10) Laos, S.; Caimari, A.; Crescenti, A.; Lakkis, J.; Puiggròs, F.; Arola, L.; del Bas, J. M. Long-term intake of soyabean phytosterols lowers serum TAG and NEFA concentrations, increases bile acid synthesis and protects against fatty liver development in dyslipidaemic hamsters. Br. J. Nutr. 2014, 112, 663−673. (11) Ireland, P. A.; Dziedzic, S. Z.; Kearsley, M. W. Saponin content of soya and some commercial soya products by means of high-performance liquid chromatography of the sapogenins. J. Sci. Food Agric. 1986, 37, 694−698. (12) Shiraiwa, M.; Harada, K.; Okubo, K. Composition and content of saponins in soybean seed according to variety, cultivation year and maturity. Agric. Biol. Chem. 1991, 55, 323−331. (13) Lee, S. O.; Simons, A. L.; Murphy, P. A.; Hendrich, S. Soyasaponins lowered plasma cholesterol and increased fecal bile acids in female golden Syrian hamsters. Exp. Biol. Med. 2005, 230, 472− 478. (14) Takahashi, S.; Hori, K.; Shinbo, M.; Hiwatashi, K.; Gotoh, T.; Yamada, S. Isolation of human renin inhibitor from soybean: soyasaponin I is the novel human renin inhibitor in soybean. Biosci., Biotechnol., Biochem. 2008, 72, 3232−3236. (15) Kinjo, J.; Imagire, M.; Udayama, M.; Arao, T.; Nohara, T. Structure-hepatoprotective relationships study of soyasaponins I-IV having soyasapogenol B as aglycone. Planta Med. 1998, 64, 233−236. (16) Philbrick, D. J.; Bureau, D. P.; Collins, F. W.; Holub, B. J. Evidence that soyasaponin Bb retards disease progression in a murine model of polycystic kidney disease. Kidney Int. 2003, 63, 1230−1239. (17) Yoshikoshi, M.; Yoshiki, Y.; Okubo, K.; Seto, J.; Sasaki, Y. Prevention of hydrogen peroxide damage by soybean saponins to mouse fibroblasts. Planta Med. 1996, 62, 252−255. (18) Ishii, Y.; Tanizawa, H. Effects of soyasaponins on lipid peroxidation through the secretion of thyroid hormones. Biol. Pharm. Bull. 2006, 29, 1759−1763. (19) Lee, I. A.; Park, Y. J.; Yeo, H. K.; Han, M. J.; Kim, D. H. Soyasaponin I attenuates TNBS induced colitis in mice by inhibiting NF-kB pathway. J. Agric. Food Chem. 2010, 58, 10929−10934. (20) Zhang, W.; Popovich, D. G. Group B oleanane triterpenoid extract containing soyasaponins I and III from soy flour induces apoptosis in Hep-G2 cells. J. Agric. Food Chem. 2010, 58, 5315−5319. (21) Kerwin, S. M. Soy saponins and the anticancer effects of soybeans and soy based foods. Curr. Med. Chem.: Anti-Cancer Agents 2004, 4, 263−272. (22) Lin, J.; Cheng, Y. W.; Wang, T.; Tang, L. H.; Sun, Y.; Lu, X. Y.; Yu, H. M. Soyasaponin Ab inhibits lipopolysaccharide-induced acute lung injury in mice. Int. Immunopharmacol. 2016, 30, 121−128. (23) Li, X.; Zhao, J. P.; Peng, C. P.; Chen, Z.; Liu, Y. L.; Xu, Q. M.; Khan, I. A.; Yang, S. L. Cytotoxic triterpenoid glycosides from the roots of Camellia oleifera. Planta Med. 2014, 80, 590−598. (24) Gao, H. W.; Sun, W.; Zhao, J. P.; Wu, X. X.; Lu, J. J.; Chen, X. P.; Xu, Q. M.; Khan, I. A.; Yang, S. L. Tanshinones and diethyl blechnics with anti-inflammatory and anti-cancer activities from Salvia miltiorrhiza Bunge. Sci. Rep. 2016, 6, 33720. (25) Cui, B.; Kinjo, J.; Nohara, T. Triterpene glycosides from the bark of Robinia pseudo-acacia L. II. Chem. Pharm. Bull. 1993, 41, 553−556. (26) Ali, Z.; Khan, S. I.; Khan, I. A. Soyasaponin Bh, a triterpene saponin containing a unique hemiacetal-functional five-membered ring from Glycine max (soybeans). Planta Med. 2009, 75, 371−374. (27) Zha, L. Y.; Mao, L. M.; Lu, X. C.; Deng, H.; Ye, J. F.; Chu, X. W.; Sun, S. X.; Luo, H. J. Anti-inflammatory effect of soyasaponins through suppressing nitric oxide production in LPS-stimulated RAW 264.7 cells by attenuation of NF-κB-mediated nitric oxide synthase expression. Bioorg. Med. Chem. Lett. 2011, 21, 2415−2418. (28) Cui, B.; Inoue, J.; Takeshita, T.; Kinjo, J.; Nohara, T. Triterpene glycosides from the seeds of Astragalus sinicus L. Chem. Pharm. Bull. 1992, 40, 3330−3333. (29) Miyao, H.; Sakai, Y.; Takeshita, T.; Kinjo, J.; Nohara, T. Triterpene saponins from Abrus cantoniensis (Leguminosae). I. Isolatioin and characterization of four new saponins and a new sapogenol. Chem. Pharm. Bull. 1996, 44, 1222−1227.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b04134. Spectroscopic data for compounds 1−5 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: 86-512-69561421, Fax: 86-512-65882089, E-mail: [email protected] (Q.X.). *Tel.: 86-512-65882080, E-mail: [email protected] (T.W.). ORCID

Qiongming Xu: 0000-0001-6145-5509 Funding

This work was financially supported by the Science and Technology Project of Sichuan Province (2017NZ0006). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors sincerely appreciate Dr. Jon Parcher at the University of Mississippi for reviewing the manuscript and providing the valuable suggestions.

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REFERENCES

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