Article pubs.acs.org/JAFC
Potential Anti-inflammatory Steroidal Saponins from the Berries of Solanum nigrum L. (European Black Nightshade) Yihai Wang,†,‡,∥ Limin Xiang,†,‡,∥ Xiaomin Yi,†,‡ and Xiangjiu He*,†,‡ †
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China Guangdong Engineering Research Center for Lead Compounds & Drug Discovery, Guangzhou 510006, China
‡
S Supporting Information *
ABSTRACT: Solanum nigrum L. or European black nightshade (Solanum genus) is a common weed of crops and gardens. The berries and leaves of S. nigrum L. are consumed as food or vegetable in some regions and reported to possess a range of biological activities. In this study, nine new steroidal saponins, solanigrosides Y1−Y9 (1−6, 10−12), together with seven known congeners, were isolated from the berries of S. nigrum. Their potential inhibitory effects on nitric oxide (NO) and IL-6 and IL-1β production induced by lipopolysaccharide (LPS) in macrophages cell line RAW 264.7 were evaluated. Compound 1 exhibited significant inhibition on NO production with an IC50 value of 9.7 μM, and some compounds exhibited significant inhibition effects on the LPS-induced IL-6 and IL-1β production. These results suggest that the steroidal saponins from berries of S. nigrum demonstrated pronounced anti-inflammatory activity and might be explored as a healthy benefit agent. KEYWORDS: Solanum nigrum L., European black nightshade, steroidal saponins, anti-inflammatory activity
■
INTRODUCTION
the berries of this plant. In our continuing research seeking bioactive components from medicinal plants, fruits, and vegetables,15−17 steroidal saponins from the berries of S. nigrum were investigated. As a result, 16 steroidal saponins including 9 new compounds were obtained, and their potent antiinflammatory were also evaluated in vitro.
Solanum nigrum L. (European black nightshade), of the Solanum genus, is a common weed of crops, gardens, or waste areas in temperate, subtropical, and tropical environments.1 It is native to Eurasia and widely grown in China and India, but also spreads to the Americas, Australasia, and South Africa.2 As a highly variable taxon, the S. nigrum species were reported to have many varieties and forms. The berries of S. nigrum ripe color from green to dull black or purple-black also may be red for some strains.3 The ripe berries are sweet and salty and were reported to be used as a famine food in the 15th century in China. In India, the leaves and berries of this plant are commonly consumed as food or vegetable after cooking. In addition, some edible strains of S. nigrum are cultivated as a food crop, for both their fruit and leaves, in some regions of the world.4 S. nigrum has been reported to exhibit multiple biological effects. The whole plants are extensively used as Chinese folk medicine because of their antipyretic and diuretic activities. Some studies have shown that extracts of this plant have remarkable antitumor effects, including inhibiting the proliferation of cancer cells and inducing cancer cell death by apoptosis or autophagy, as well as antineoplastic effect against Sarcoma 180 in mice.5−9 Recent studies have revealed that the extract of S. nigrum had antioxidant,10 hepatoprotective,8 antihyperlipidemic, and antidiabetic activities.11,12 Previous phytochemical investigations on S. nigrum have led to the identification of steroidal saponins, phenolic compounds, and polysaccharides.6,13,14 As an important class of plant secondary metabolites, steroidal saponins have been reported to possess a series of biological activities such as anti-inflammatory, antitumor, platelet aggregation inhibition, antihypertensive, cholesterollowering, antifungal, and antiviral. However, most of these studies have focused on the whole plants, leaves, or ripe berries of S. nigrum; little is known about the bioactive components of © 2017 American Chemical Society
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MATERIALS AND METHODS
General Experimental Procedures. Optical rotations were measured with a P-1020 digital polarimeter (Jasco, Tokyo, Japan). UV spectra (200−400 nm) and IR spectra (4000−450 cm−1) were acquired with an UV−vis spectrophotometer (Pharmaspec UV-1700, Shimadzu, Kyoto, Japan) and a PerkinElmer Spectrum 100 FT-IR spectrometer (PerkinElmer Inc., Waltham, MA, USA), respectively. NMR spectra were acquired with a Bruker Avance III 500 MHz digital NMR spectrometer (Bruker, Switzerland). HR-ESI-MS were measured on an Acquity UPLC-Q-TOF Micro MS mass spectrometer (Waters Corp., Milford, MA, USA). Semipreparative HPLC was carried out on an HPXL solvent delivery system equipped with a refractive index detector (Gilson, Villier-le-Bel, France) and an ODS column (5C18MS-II, 10 × 250 mm, 5 μm, Nacalai Technologies Inc., Kyoto, Japan). A preparative column (COSMOSIL-Pack 5C18-AR-II, 20 × 250 mm, 5 μm, Nacalai Technologies Inc., Kyoto, Japan) was used for HPLC purification. D101 macroporus resin was the product of Xi’an Lanxiao Resin Corp. Ltd. (Xi’an, China). Silica gel (200−300 mesh) was obtained from Anhui Liangchen Silicon Material Co. Ltd. (Anhui, China). ODS (40−60 μm) used for column chromatography (CC) was the product of Merck KgaA (Darmstadt, Germany). Methanol used for HPLC was provided by Oceanpak Chemical Co. (Gothenburg, Sweden). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from HyClone Laboratories (Logan, UT, USA). Standard sugar used for GC analysis Received: Revised: Accepted: Published: 4262
March 2, 2017 May 5, 2017 May 9, 2017 May 9, 2017 DOI: 10.1021/acs.jafc.7b00985 J. Agric. Food Chem. 2017, 65, 4262−4272
Article
Journal of Agricultural and Food Chemistry Table 1. NMR Spectroscopic Data for Compounds 1−4 (1H, 500 MHz; 13C, 125 MHz) in Pyridine-d5 1 position
δC
1
37.4
2
30.1
3 4
77.8 35.0
5 6 7
45.0 29.2 32.5
8 9 10 11
34.5 53.8 36.1 31.8
12 13 14 15
79.6 47.4 55.2 32.5
16 17 18 19 20 21 22 23 24
82.5 63.1 11.6 12.5 38.6 15.2 110.7 78.4 31.5
25 26 27 3-O-Gal 1 2 3 4 5 6
34.5 181.1 16.7
2
δH (J in Hz) 1.46, 0.74, 1.98, 1.55, 3.87, 1.76, 1.33, 0.87, 1.13, 1.47, 0.74, 1.35, 0.61,
m m m m m m m m m m m m m
1.82, m 1.47, m 3.48, dd (11.0, 4.5) 1.02, 2.00, 1.47, 4.94, 2.28, 1.17, 0.64, 2.96, 1.57,
overlapped m m dd (15.3, 7.0) dd (8.5, 6.6) s s m d (6.9)
4.71, 3.01, 1.91, 3.12,
m m m m
δC 37.3 30.2 77.7 35.1 45.2 29.3 29.8 35.7 48.1 35.8 32.8 71.7 46.3 48.5 32.8 82.3 54.0 17.8 12.5 37.7 15.7 110.5 78.1 31.5
3 δH (J in Hz)
1.47, 0.78, 1.94, 1.56, 3.84, 1.73, 1.34, 0.91, 1.14, 1.71, 1.56, 1.48, 1.29,
m m m m m m m m m m m m m
δC 36.9 29.9 77.3 34.9 44.7 28.9 32.1 34.4 55.7 36.6 38.2
1.57, m 0.87, m 3.93, m 2.01, 2.03, 1.39, 4.94, 3.19, 1.01, 0.66, 2.79, 1.33,
m overlapped m m dd (8.8, 6.9) s s p (6.8) d (6.9)
4.70, 3.03, 1.94, 3.13,
m m m m
213.0 56.3 55.9 32.0 81.0 54.4 16.6 12.0 38.2 14.7 110.6 78.0 31.4 34.4 181.1 16.7
4 δH (J in Hz)
1.27, 0.68, 1.95, 1.54, 3.86, 1.78, 1.32, 0.82, 1.15, 1.52, 0.73, 1.64, 0.84,
m m m m m m m m m m m m m
2.33, t (13.8) 2.18, dd (14.3, 4.9)
1.29, 2.02, 1.44, 4.80, 2.87, 1.15, 0.64, 2.69, 1.50,
m m m m dd (8.8, 6.6) s s m d (7.0)
4.68, 3.01, 1.93, 3.09,
m m m m
δC 37.0 30.1 77.4 35.0 44.8 28.9 32.1 34.5 55.8 36.6 38.2 213.1 56.3 56.0 32.1 81.0 54.5 16.6 12.0 38.2 14.7 110.6 78.1 31.4
m m m m m d (12.1) m m m m m m m
2.35, t (13.8) 2.20, dd (14.4, 5.0)
1.31, 2.04, 1.44, 4.81, 2.87, 1.16, 0.67, 2.71, 1.51,
m dd (12.2, 6.8) m m dd (8.7, 6.6) s s m d (6.9)
4.68, 3.02, 1.94, 3.09,
dd (8.7, 1.9) dd (16.9, 6.5) m m
1.19, d (7.2)
34.4 181.1 16.7
102.7 73.5 75.8 80.5 75.6 60.9
4.86, 4.41, 4.10, 4.59, 4.01, 4.68, 4.19,
d (7.6) m m d (2.7) m d (10.1) d (10.1)
102.6 73.5 75.8 80.5 75.5 60.9 4.18, m
4.84, 4.40, 4.10, 4.57, 4.00, 4.66,
d (7.6) m m m m m
102.7 73.5 75.8 80.5 75.6 60.9 4.21, m
4.86, 4.42, 4.13, 4.59, 4.02, 4.68,
d (7.6) m m m m m
102.7 73.6 75.9 81.4 75.5 60.9 4.24, m
4.88, 4.49, 4.12, 4.59, 4.03, 4.77,
d (7.7) m m m m m
Glc(1→4) 1 2 3 4 5 6
105.4 81.8 88.8 71.1 77.6 62.6
5.15, 4.37, 4.21, 3.84, 3.86, 4.54, 4.36,
d (7.9) m m m m m m
105.4 81.8 88.8 71.1 77.8 62.6
5.14, 4.37, 4.20, 3.84, 3.84, 4.52, 4.36,
d (7.9) m m m m m m
105.4 81.8 88.8 71.2 77.8 62.6
5.15, 4.38, 4.21, 3.84, 3.84, 4.54, 4.26,
d (7.9) m m m m m m
105.5 86.5 78.8 72.2 78.5 63.5
5.15, 4.17, 4.27, 3.97, 3.97, 4.63, 4.10,
d (7.8) m m m m d (10.6) m
Glc(1→2) 1 2 3 4 5 6
105.2 76.4 78.9 71.9 78.9 62.5
5.57, 4.05, 4.19, 4.14, 4.03, 4.57, 4.26,
d (7.7) m m m m m m
105.2 76.4 78.9 71.8 78.9 62.5
5.57, 4.06, 4.19, 4.14, 4.04, 4.55, 4.26,
d (7.7) m m m m m m
105.2 76.5 79.0 71.8 78.2 62.6
5.58, 4.07, 4.19, 4.15, 3.84, 4.50, 4.37,
d (7.7) m m m m m m
107.3 77.1 78.0 70.6 79.3 61.9
5.23, 4.06, 4.11, 4.23, 3.79, 4.57, 4.37,
d (7.5) m m m m m m
1.20, d (7.2)
1.20, d (7.2)
34.5 181.1 16.7
δH (J in Hz) 1.30, 0.70, 2.00, 1.59, 3.88, 1.80, 1.34, 0.84, 1.16, 1.54, 0.74, 1.65, 0.86,
1.21, d (7.1)
Glc(1→3)
4263
DOI: 10.1021/acs.jafc.7b00985 J. Agric. Food Chem. 2017, 65, 4262−4272
Article
Journal of Agricultural and Food Chemistry Table 1. continued 1 δC
position 1 2 3 4 5 6
104.8 75.6 78.2 71.1 78.9 63.3
2
δH (J in Hz) 5.30, 4.05, 4.13, 4.24, 4.03, 4.49, 4.00,
δC
d (7.9) m m m m m m
3 δH (J in Hz)
104.8 75.6 78.1 71.1 78.9 63.3
5.30, 4.05, 4.13, 4.24, 4.04, 4.48, 4.00,
d (7.8) m m m m m m
δC
4 δH (J in Hz)
104.8 76.5 78.0 71.1 79.0 63.3
5.31, 4.05, 4.13, 4.25, 4.05, 4.48, 4.01,
δC
δH (J in Hz)
d (7.8) m m m m m m
Table 2. NMR Spectroscopic Data for Compounds 5 and 6 (1H, 500 MHz; 13C, 125 MHz) in Pyridine-d5 5 position
δC
1
37.8
2
30.4
3 4 5 6 7
78.3 39.2 141.0 122.2 32.6
8 9 10 11
32.4 50.6 37.5 21.3
12
32.8
13 14 15
45.4 53.3 32.3
16 17 18 19 20 21 22 23 24
90.7 91.1 17.8 19.7 43.8 10.8 111.7 37.2 28.3
25 26
34.6 75.6
27
17.6
6
δH (J in Hz) 1.76, 0.96, 2.03, 1.85, 3.83, 2.74,
m m m m m m
5.26, 1.88, 1.52, 1.60, 0.97,
overlapped m m m m
1.59, 1.52, 2.17, 1.60,
m m m m
2.04, 2.17, 1.52, 4.77,
m m m t (7.1)
1.00, 1.09, 2.50, 1.38,
s s q (7.1) d (7.1)
2.06, 2.06, 1.69, 1.93, 3.95, 3.62, 0.99,
m m m m m dd (10.0, 6.5) d (6.6)
δC 37.8 30.5 78.4 39.3 141.1 122.2 32.7 32.0 50.7 37.4 21.4 40.2 41.1 56.9 32.8 81.4 64.2 16.8 19.7 41.0 16.8 111.0 37.5 28.7 34.5 75.7 3.55, m 17.8
5 δH (J in Hz) 1.71, 0.97, 2.06, 1.85, 3.86, 2.76,
m m m m m m
5.29, 1.84, 1.45, 1.54, 0.88,
brs m m m m
1.43, m 1.72, m 1.09, m 1.04, 2.00, 1.45, 4.93, 1.90, 0.89, 1.04, 2.23, 1.31,
m m m m m s s m d (6.7)
2.03, 2.03, 1.66, 1.90, 4.01, 3 0.98,
m m m m m d (6.6)
position
δC
1 2 3 4 5 6
100.6 78.1 78.0 78.4 77.1 61.5
1 2 3 4 5 6
102.3 72.9 73.1 74.4 69.8 18.9
1 2 3 4 5 6
102.0 82.4 72.9 74.7 70.0 18.6
1 2 3 4 5 6
107.6 76.0 78.8 71.8 78.6 63.1
1 2 79.1 4 5 6
6 δH (J in Hz)
3-O-Glc 4.93, 4.21, 4.21, 4.40, 3.66, 4.32, 4.26, Rha(1→2) 6.44, 4.85, 4.63, 4.38, 4.96, 1.76, Rha(1→4) 6.09, 4.66, 4.53, 4.21, 4.96, 1.59, Glc(1→2) 5.25, 4.10, 4.24, 4.09, 3.95, 4.56, 4.29,
26-O-Glc 105.2 75.5 4.24, m 72.0 78.9 63.1
4.82, 4.03, 78.8 4.24, 3.95, 4.56, 4.40,
δC
overlapped m m m d (9.4) m m
100.6 78.1 78.3 78.8 77.5 70.4
s m dd (9.3, 3.4) m m d (6.2)
102.3 72.9 73.1 74.4 69.8 19.0
s m dd (9.3, 3.4) m m d (6.1)
103.2 72.8 73.0 74.2 70.7 18.8
d (7.7) m m m m m m
105.8 75.5 78.8 71.9 77.2 61.6
d (7.7) m m m m m
δH (J in Hz) 3-O-Glc 4.94, 4.22, 4.22, 4.40, 4.04, 4.83, 4.33, Rha(1→2) 6.41, 4.84, 4.63, 4.35, 4.96, 1.76, Rha(1→4) 5.87, 4.69, 4.55, 4.35, 4.94, 1.62, Glc(1→6) 5.11, 4.03, 4.40, 4.16, 3.64, 4.21, 4.09,
26-O-Glc 105.1 75.3 4.23, m 71.9 78.7 63.0
m m m m m m m s m dd (9.3, 3.3) m m d (6.2) s brs dd (9.3, 3.3) m m d (6.2) overlapped m m m m m m
4.73, dd (7.8, 1.7) 3.95, m 4.24, 3.93, 4.51, 4.37,
m m m m
Extraction and Isolation. The dried unripe fruits of S. nigrum (6.0 kg) were pre-extracted with cyclohexane (2 × 30 L) for 2 h to remove lipids. Then the residue was refluxed three times with 70% MeOH (3 × 30 L) for 2 h and filtered. The filtrate was evaporated under vacuum to yield the liquid extract. The liquid extract was dispersed in hot distilled water (50 °C), and the suspension solution was applied to a D101 macroporus resin column (100 × 1300 mm), eluted with H2O (40 L), 10% MeOH (22 L), 30% MeOH (40 L), 50% MeOH (40 L), 70% MeOH (40 L), and MeOH (40 L) to get six fractions (A−F).
and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as well as indomethacin were provided by Sigma Chemical Co. (St. Louis, MO, USA). Plant Material. The unripe green fruits of S. nigrum were collected from the suburbs of Shenyang (Liaoning, China) and dried at room temperature in the shade. A voucher specimen (no. GDPU-NPR2013002SN) has been deposited in the School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, China. 4264
DOI: 10.1021/acs.jafc.7b00985 J. Agric. Food Chem. 2017, 65, 4262−4272
Article
Journal of Agricultural and Food Chemistry Table 3. NMR Spectroscopic Data for Compounds 10−12 (1H, 500 MHz; 13C, 125 MHz) in Pyridine-d5 10
11
12
position
δC
1
37.5
1.50, m
37.4
1.49, m
37.4
1.49, m
2
30.2
0.77, m 2.03, m 1.59, m
30.2
0.77, m 2.01, m 1.57, m
30.1
0.77, m 2.01, m 1.57, m
3 4
77.7 35.1
5 6 7
45.0 29.2 32.7
8
35.5
9 10 11
54.7 36.1 21.6
12
40.5
13 14
41.4 56.7
15
32.7
16
81.4
17 18
64.3 17.1
19 20 21
12.6 41.0 16.8
22 23 24
111.0 37.5 28.7
25 26
27
1 2 3 4 5
3.92, 1.76, 1.32, 0.87, 1.08, 1.48, 0.75, 1.37,
m m m m m m m m
0.47, m 1.39, 1.19, 1.70, 1.04,
m m m m
1.00, m 2.00, m 1.41, m 4.94, dd (14.2, 6.8) 1.91, m 0.86, s 0.61, s 2.22, m 1.33, d (6.8)
2.04, m 2.04, m 1.68, m 34.5 1.91, m 75.7 4.01, m 3.55, dd (9.5, 6.1) 17.8 0.97, d (6.6) 3-O-Gal 102.7 4.89, d (7.6) 73.5 4.43, m 75.9 4.12, m 80.5 4.60, m 75.5 4.04, m
δH (J in Hz)
10
δH (J in Hz)
δC
77.7 35.1
3.89, 1.76, 1.32, 0.86, 1.07, 1.50, 0.75, 1.38,
44.9 29.2 32.7 35.5 54.7 36.1 21.5
0.47, m 1.35, 1.18, 1.70, 1.03,
40.5 41.4 56.6
2.00, m 1.41, m 4.93, dd (15.0, 7.2) 1.90, m 0.88, s
81.4
64.2 17.1 12.6 41.1 16.7
0.61, s 2.22, m 1.31, d (6.8)
110.9 37.6 28.5 34.6 75.4 3.59, m
102.7 73.4 75.8 80.2 75.6
m m m m
1.00, m
32.6
17.8
m m m m m m m m
2.01, 2.02, 1.62, 1.96, 3.86,
m m m m m
1.05, d (6.4) 3-O-Gal 4.88, d (7.6) 4.41, m 4.13, m 4.60, m 4.02, m
δH (J in Hz)
δC
77.7 35.1
3.89, 1.76, 1.34, 0.86, 1.08, 1.49, 0.75, 1.37,
44.9 29.2 32.6 35.5 54.7 36.1 21.5
0.47, m 1.36, 1.19, 1.69, 1.03,
40.5 41.4 56.6
2.01, m 1.57, m 4.93, dd (14.5, 7.2) 1.90, m 0.86, s
81.4
64.3 17.0 12.6 40.9 16.8
0.61, s 2.21, m 1.32, d (6.7)
110.9 37.4 28.7 34.5 75.6 3.54, m
102.7 73.4 75.8 80.2 75.6
m m m m
0.99, m
32.6
17.7
m m m m m m m m
2.02, 2.03, 1.66, 1.90, 4.00,
m m m m m
0.96, d (6.7) 3-O-Gal 4.87, d (7.3) 4.40, m 4.12, m 4.60, m 4.02, m
The 50% MeOH elute (F, 160g) was subjected to silica gel CC (200− 300 mesh, 3 kg, 100 × 1200 mm) and eluted gradiently with CHCl3/ MeOH (10:1 to 0:1, v/v) to give 18 fractions (F1−F18). Fraction F12 (590.0 mg) was further purified by an ODS column and eluted gradiently with MeOH/H2O (1:9 to 6:4, v/v) to yield five subfractions (F12-1−F12-5). Then subfraction F12-3 (87.6 mg) was further separated using semipreparative HPLC (3.0 mL/min, 45% MeOH in water) to give compound 4 (17.5 mg, tR = 22.8 min). Fraction F13 (20.7 g) was purified by an ODS column eluted agradiently with
11
position
δC
δH (J in Hz)
6
60.9
4.69, m
δC 60.9
12
δH (J in Hz)
δC
4.69, t 60.9 (9.5) 4.24, m 4.23, m Glc(1→4) Glc(1→4) Glc(1→4) 1 105.4 5.16, d 105.4 5.19, d 105.4 (7.8) (7.8) 2 81.8 4.38, m 81.6 4.42, m 81.6 3 88.8 4.21, m 87.0 4.17, m 87.0 4 71.1 3.85, m 70.7 3.82, m 70.7 5 77.8 4.05, m 78.9 3.89, m 77.8 6 70.4 4.83, m 62.7 4.56, m 70.4 4.33, m 4.35, m Glc(1→2) Glc(1→2) 1 105.2 5.58, d 105.1 5.56, d 105.1 (7.8) (7.3) 2 76.5 4.06, m 76.5 4.07, m 76.4 3 79.0 4.19, m 79.0 4.10, m 79.0 4 71.9 4.16, m 71.6 4.19, m 71.8 5 78.7 3.93, m 78.0 3.88, m 78.7 6 62.6 4.54, m 63.2 4.51, m 63.2 4.37, m 4.05, m Glc(1→3) Xyl(1→3) 1 104.8 5.31, d 105.2 5.23, d 105.2 (8.0) (7.7) 2 75.6 4.05, m 75.3 3.96, m 75.3 3 78.2 4.12, m 77.9 4.10, m 78.0 4 71.2 4.24, m 71.0 4.10, m 71.0
5 6
79.0 63.3
4.05, m 4.50, m
67.6
4.23, m 3.67, t (10.4)
4.01, m Glc(1→6) 105.8 5.11, d (7.8) 75.5 4.05, m 78.8 4.24, m 71.9 4.24, m 77.5 3.93, m 62.6 4.54, m 4.26, m 26-O-Glc
26-O-Glc 103.4 4.84, d (7.7) 84.4 4.14, m 78.2 4.32, m 71.2 4.22, m 78.3 3.88, m 62.8 4.51, m 4.35, m Glc′(1→2)
1
105.1
106.8
2 3
75.3 78.8
4 5 6
71.9 78.7 63.0
1 2 3 4 5 6
4.74, d (7.8) 3.96, m 4.24, m 4.16, 3.93, 4.50, 4.37,
m m m m
77.2 78.5 71.7 79.0 62.8
5.28, d (7.7) 4.10, m 4.23, m 4.29, 3.96, 4.51, 4.42,
m m m m
67.6
105.7 75.4 78.7 71.2 77.4 62.7
105.1
δH (J in Hz) 4.68, m 4.21, m 5.18, d (7.8) 4.41, m 4.16, m 3.81, t (9.3) 3.90, m 4.82, t (9.7) 4.32, m Glc(1→2) 5.55, d (7.2) 4.05, m 4.07, m 4.16, m 3.90, m 4.50, m 4.04, m Xyl(1→3) 5.22, d (7.8) 3.94, m 4.09, m 4.09, m
4.22, m 3.66, t (10.5)
Glc(1→6) 5.09, overlapped 4.12, m 4.22, m 4.11, m 3.89, m 4.56, m 4.36, m 26-O-Glc
4.72, d (7.7)
75.3 78.6
3.94, m 4.22, m
71.8 78.9 62.9
4.22, 3.90, 4.50, 4.36,
m m m m
MeOH/H2O (1:9 to 7:3, v/v) to afford nine fractions (F13-1−F13-9). Compounds 8 (148.1 mg, tR = 25.1 min), 9 (80.5 mg, tR = 38.2 min), and 16 (70.3 mg, tR = 59.7 min) were obtained by preparative HPLC (8.0 mL/min, 60% MeOH in water, v/v) from the subfraction F13-8 (1.39 g). Fraction F14 (8.1 g) was applied to ODS CC and eluted gradiently with MeOH/H2O (1:9 to 7:3, v/v) to afford 11 fractions (F14-1−F14-11). Compound 1 (110.0 mg, tR = 27.0 min) was obtained by semipreparative HPLC (2.5 mL/min, 45% MeOH in water, v/v) from subfraction F14-6 (338.6 mg). Subfraction F14-7 4265
DOI: 10.1021/acs.jafc.7b00985 J. Agric. Food Chem. 2017, 65, 4262−4272
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Figure 1. Structures of the isolated compounds 1−16. min), 11 (48.6 mg, tR = 47.9 min), and 12 (39.8 mg, tR = 44.5 min), respectively. Solanigroside Y1 (1): white amorphous powder; [α]14 D −27.5 (c 0.50, MeOH); UV (MeOH) λmax (log ε), 254 (1.18) nm; IR νmax (KBr) cm−1, 3427 (OH), 2932 (CH), 1759 (CO), 1636, 1454, 1074, 891; 1H and 13C NMR data, see Table 1; HR-ESI-MS m/z 1128.5492 [M + NH4]+ (calcd for C51H86NO26, [M + NH4]+, 1128.5438). Solanigroside Y2 (2): white amorphous powder; [α]14 D −30.9 (c 0.50, MeOH); UV (MeOH) λmax (log ε), 248 (1.18) nm; IR νmax (KBr) cm−1, 3411 (OH), 2932 (CH), 1760 (CO), 1454, 1074, 882; 1 H and 13C NMR data, see Table 1; HR-ESI-MS m/z 1128.5497 [M + NH4]+ (calcd for C51H86NO26, [M + NH4]+, 1128.5438). Solanigroside Y3 (3): white amorphous powder; [α]14 D −13.6 (c 0.50, MeOH); UV (MeOH) λmax (log ε), 278 (0.10) nm; IR νmax (KBr) cm−1, 3412 (OH), 2930 (CH), 1764 (CO), 1705 (CO),
(631.2 mg) was further applied to semipreparative HPLC (2.8 mL/ min, 45% MeOH in water, v/v) to give compounds 2 (49.1 mg, tR = 61.1 min) and 3 (96.7 mg, tR = 54.6 min). Compounds 5 (117.7 mg, tR = 51.9 min), 6 (79.2 mg, tR = 24.2 min), 13 (160.6 mg, tR = 63.6 min), 14 (131.5 mg, tR = 56.6 min), and 15 (55.6 mg, tR = 53.0 min) were separated from fraction F15 by ODS column and semipreparative HPLC (3.0 mL/min, 50% MeOH in water, v/v). Fraction F16 (18.2 g) was subjected to ODS column and eluted with a gradient of MeOH/H2O (1:9 to 7:3, v/v) to obtain eight fractions (F16-1−F168). A half part of subfraction F16-6 (1.2 g) was further purified by preparative HPLC eluted with 60% MeOH in water (v/v) at a flow rate of 8.0 mL/min to afford three fractions (F16-6-1−F16-6-3). Subfraction F16-6-1 was further purified by semipreparative HPLC eluted with 50% MeOH in water (v/v) at a flow rate of 2.5 mL/min to yield compounds 7 (13.1 mg, tR = 38.5 min), 10 (20.8 mg, tR = 36.4 4266
DOI: 10.1021/acs.jafc.7b00985 J. Agric. Food Chem. 2017, 65, 4262−4272
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Journal of Agricultural and Food Chemistry
(δC 11.6, 12.5, 15.2, and 16.7), four oxygenated methines at δC 77.8, 78.4, 79.6, and 82.5, and one ester carbonyl carbon at δC 181.1. The NMR data of the aglycone moiety of 1 were similar to those of solanigroside D.20 The major difference was the downshift of the C-12 from 40.2 to 79.6, indicating the linkage of a hydroxyl group at C-12. This hypothesis was further confirmed by 2D NMR data. The 1H−1H COSY spectrum revealed that the oxymethine proton δH 3.48 (1H, dd, J = 11.0, 4.5 Hz, H-12) was coupled with two methylene protons δH 1.82 (1H, m, H-11) and 1.47 (1H, m, H-11), and one methine proton δH 0.61 (1H, m, H-9) was coupled to H-11. These findings confirmed the location of the hydroxyl group at C-12. The key ROESY (Figure 2) correlations between H-12α and H-
1453, 1072, 892; 1H and 13C NMR data, see Table 1; HR-ESI-MS m/z 1126.5308 [M + NH4]+ (calcd for C51H84NO26, [M + NH4]+, 1126.5282). Solanigroside Y4 (4): white amorphous powder; IR νmax (KBr) cm−1, 3401 (OH), 2931 (CH), 1774 (CO), 1706 (CO), 1454, 1070, 892; 1H and 13C NMR data, see Table 1; HR-ESI-MS m/z 964.4782 [M + NH4]+ (calcd for C45H74NO21, [M + NH4]+, 964.4753). Solanigroside Y5 (5): white amorphous powder; [α]14 D −64.6 (c 0.50, MeOH); UV (MeOH) λmax (log ε), 254 (0.51) nm; IR νmax (KBr) cm−1, 3400 (OH), 2934 (CH), 1662, 1454, 1075, 1043, 912; 1H and 13C NMR data, see Table 2; HR-ESI-MS m/z 1244.6339 [M + NH4]+ (calcd for C57H98NO28, [M + NH4]+, 1244.6275). Solanigroside Y6 (6): white amorphous powder; [α]14 D −73.7 (c 0.50, MeOH); IR νmax (KBr) cm−1, 3401 (OH), 2936 (CH), 1382, 1046, 911; 1H and 13C NMR data, see Table 2; HR-ESI-MS m/z 1228.6338 [M + NH4]+ (calcd for C57H98NO27, [M + NH4]+, 1228.6326). Solanigroside Y7 (10): white amorphous powder; [α]14 D −8.9 (c 0.50, MeOH); UV (MeOH) λmax (log ε), 254 (1.35) nm; IR νmax (KBr) cm−1, 3411 (OH), 2932 (CH), 1384, 1074, 894; 1H and 13C NMR data, see Table 3; HR-ESI-MS m/z 1424.6948 [M + NH4]+ (calcd for C63H110NO34, [M + NH4]+, 1424.6909). Solanigroside Y8 (11): white amorphous powder; [α]14 D −35.9 (c 0.50, MeOH); IR νmax (KBr) cm−1, 3400 (OH), 2930 (CH), 1383, 1074, 893; 1H and 13C NMR data, see Table 3; HR-ESI-MS m/z 1394.6846 [M + NH4]+ (calcd for C62H108NO33, [M + NH4]+, 1394.6804). Solanigroside Y9 (12): white amorphous powder; [α]14 D −45.9 (c 0.50, MeOH); IR νmax (KBr) cm−1, 3401 (OH), 2931 (CH), 1380, 1071, 895; 1H and 13C NMR data, see Table 3; HR-ESI-MS m/z 1394.6815 [M + NH4]+ (calcd for C62H108NO33, [M + NH4]+, 1394.6804). Acid Hydrolysis and GC Analysis. Compounds 1−6 and 10−12 (1−2 mg) were hydrolyzed in 5 mL of 2 M HCl at 90 °C for 2−8 h. The sugars obtained from the hydrolysates were analyzed by GC as previously described.18 Measurement of Anti-inflammatory Activity. In vitro antiinflammatory activities of the steroidal saponins isolated from S. nigrum were performed by measuring the accumulation of nitrite in the culture supernatant using the Griess reagent, as previously described.18,19 Levels of IL-6 and IL-1β in the culture supernatant were quantified by enzyme-linked immunosorbent assay (ELISA) according to their manufacturer’s protocol. Indomethacin was selected as a positive control. Statistical Analysis. Date are presented as the mean ± SD. Analyses were performed using Graphpad Prism 5 software with oneway ANOVA followed by Dunnett’s test. Differences with p values 7.0 Hz). The linkages of the sugar chain were further confirmed by the following HMBC correlations: H-1 (δH 5.30) of glucosyl III with C-3 (δC 88.8) of glucosyl I, H-1 (δH 5.57) of glucosyl II with C-2 (δC 81.8) of glucosyl I, H-1 (δH 5.15) of glucosyl I with C-4 (δC 80.5) of galactosyl, and H-1 (δH 4.86) of galactosyl with C-3 (δC 77.8) of the aglycone. The 13C NMR chemical shifts of the sugar moiety of 1 were identical to the reported data.20,21 From the above evidence, compound 1 was identified as (25R)-5αspirostan-3β,12β,23α-triol-26-one-3-O-β-D-glucopyranosyl-(1→ 2)-[β-D-glucopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-βD-galactopyranoside and named solanigroside Y1.
■
RESULTS AND DISCUSSION Structure Elucidation. The 70% methanol extract of the green fruits of S. nigrum was successively chromatographed on D101 macroporus resin, silica gel, ODS, and finally purified by preparative HPLC to afford 16 steroidal glycosides, including 9 new compounds, solanigrosides Y1−Y9 (1−6, 10−12). Their chemical structures are shown in Figure 1. Compound 1 was obtained as white amorphous powder, [α]14 D −27.5 (c 0.50, MeOH). Its molecular formula was deduced as C51H82O26 on the basis of positive-ion HR-ESI-MS at m/z 1128.5492 [M + NH4]+ (calcd for C51H86NO26 [M + NH4]+, 1128.5438) and its 13C and DEPT NMR data (Table 1). Acid hydrolysis and GC analysis of 1 gave D-galactose and Dglucose. The 1H NMR spectrum of 1 displayed four typical steroidal methyl signals at δH 1.57 (3H, d, J = 6.9 Hz, Me-21), 1.19 (3H, d, J = 7.2 Hz, Me-27), 1.17 (3H, s, Me-18), and 0.64 (3H, s, Me-19). The 13C NMR spectrum showed a characteristic spirostanol carbon signal at δC 110.7 (C-22), four methyls 4267
DOI: 10.1021/acs.jafc.7b00985 J. Agric. Food Chem. 2017, 65, 4262−4272
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Journal of Agricultural and Food Chemistry Compound 2 was obtained as a white amorphous powder, [α]14 D −30.9 (c 0.50, MeOH). The HR-ESI-MS gave a positive ion at m/z 1128.5497 [M + NH4]+ (calcd for C51H86NO26, 1128.5438), consistent with the molecular formula C51H82O26, the same as 1. The 1H and 13C NMR data of 2 were similar to those of 1 except for the chemical shifts at C-9, C-11, C-12, C13, and C-14 positions in the C ring of the aglycone. The upfield chemical shifts of C-9 (ΔδC = −5.7), C-12 (ΔδC = −7.9), C-13 (ΔδC = −1.1), and C-14 (ΔδC = −6.7) and the downfield chemical shift C-11 (ΔδC = 1.0) of compound 2 compared with compound 1 sugggested the α-orientation of the hydroxyl group at C-12. This was further confirmed by the presence of any ROESY correlation between H-12β and H-18β/ H-20β. Therefore, the structure of 2 was elucidated as (25R)5α-spirostan-3β,12α,23α-triol-26-one-3-O-β-D-glucopyranosyl(1→2)-[β-D-glucopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→ 4)-β-D-galactopyranoside and named solanigroside Y2. Compound 3 was obtained as a white amorphous powder, [α]14 D −13.6 (c 0.50, MeOH). The molecular formula was deduced as C51H80O26 by using HR-ESI-MS and 13C NMR. Comparison of the NMR spectra of the sugar moieties of 3 with those of 1 and 2 indicated that 3 possessed the same sugar chains as 1 and 2. The 13C NMR data for the aglycone of 3 exhibited considerable similarity to 1 and 2 except for the carbon signals around C-12. Comparison of the aglycone of 3 to the literature suggested the presence of a carbonyl group at C-12, which caused the similar chemical shifts of the carbons at C-11, C-12, and C-13 positions.22−24 This was further verified by the HMBC correlations from C-12 (δC 213.0) to Me-18 (δH 1.15), H-17 (δH 2.87), H-14 (δH 1.29), and H-11 (δH 2.18 and 2.33) (Figure 3). On the basis of the above evidence, the structure of 3 was established as (25R)-5α-spirostan-3β,23αdiol-12,26-dione-3-O-β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside and named solanigroside Y3. Compound 4 was obtained as a white amorphous powder. The HR-ESI-MS of 4 gave the ion at m/z 964.4782 [M + NH4]+, and its molecular formula was deduced as C45H70O21 according to the analysis of 13C NMR and DEPT spectra. From a comparison of 1H and 13C NMR data of 4 (Table 1) with those of 3, it was apparent that 4 contained the same aglycone as 3, except for a little difference in the saccharide chains attached at C-3 of the aglycone. Comparison of 1H and 13C NMR spectroscopic data for the sugar moiety of 4 with those of reported compound (25R)-5α-spirostan-3β-ol-12-one-3-O-β-Dglucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside22 suggested that they contained the same sugar chain. The linkages of the sugar residues were further confirmed from the following HMBC correlations: H-1 (δH 5.23) of glucosyl III with C-2 (δC 86.5) of glucosyl I, H-1 (δH 5.15) of glucosyl I with C-4 (δC 81.4) of galactosyl, and H-1 (δH 4.88) of galactosyl with C-3 (δC 77.4) of the aglycone. Therefore, 4 was formulated as (25R)-5α-spirostan-3β,23αdiol-12,26-dione-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside and named solanigroside Y4. Compound 5 was obtained as a white amorphous powder, and its molecular formula was determined as C57H94O28 by the HR-ESI-MS peak at m/z 1244.6339 [M + NH4]+ (calcd for C57H98NO28 [M + NH4]+, 1244.6275), showing a positive reaction to the Ehrlich reagent. Acid hydrolysis and GC analysis of 5 revealed the presence of D-glucose and L-rhamnose. The 1 H NMR spectrum of 5 showed two tertiary methyl groups at
Figure 3. Selected 1H−1H COSY and HMBC correlations of compounds 3, 5, 6, and 11.
δH 1.00 (3H, s, Me-18) and 1.09 (3H, s, Me-19), four secondary methyl groups at δH 0.99 (3H, d, J = 6.6 Hz, Me-27), 1.38 (3H, d, J = 7.1 Hz, Me-21), 1.59 (3H, d, J = 6.1 Hz, MeRha II), and 1.76 (3H, d, J = 6.2 Hz, Me-Rha I), and an olefinic methine group at δH 5.26 (1H, brs, H-6), as well as five anomeric protons at δH 4.82 (1H, d, J = 7.7 Hz, H-1-Glc), 4.93 (1H, overlapped, H-1-Glc I), 5.25 (1H, d, J = 7.7 Hz, H-1-Glc II), 6.09 (1H, s, H-1-Rha II), and 6.44 (1H, s, H-1-Rha I). The 13 C NMR spectrum of the aglycone of 5 exhibited a characteristic furostanol carbon signal at δC 111.7 (C-22), two olefinic carbons at δC 141.0 (C-5) and 122.2 (C-6), one oxygenated quaternary carbon at δC 91.1 (C-17), one oxygenated methane at δC 90.7 (C-16), and five anomeric carbons. The 1H NMR data, the acetal carbon signal at δC 111.7, and positive reaction to Ehrlich’s test revealed that 5 was a 22-hydroxyfurostanol saponin with five monosaccharides.25 By comparison of the 13C NMR data (Table 2) of 5 with those of the reported compound ((25R)-spirostan-5(6)-en-3β,17αdiol-3-O-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→ 4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside),20 it exhibited similarity with some variation at the F ring and additional signals for a hexose. These indicated the structure of the aglycone moiety of 5 to be (25R)-furost-5(6)-en3β,17α,22α,26-tetraol. The 25R stereochemistry was inferred by the observed difference (Δab = δa − δb = 0.33) of the 1H NMR chemical shifts between the H2-26 geminal protons, which was consistent with that of 25R furostane-type steroidal saponins (Δab < 0.48 for 25R; Δab > 0.57 for 25S).26−28 The sugar units were further verified in the HMBC experiment (Figure 3) by the correlations between H-1 (δH 5.25) of glucosyl II and C-2 (δC 82.4) of rhamnosyl II, H-1 (δH 6.09) of 4268
DOI: 10.1021/acs.jafc.7b00985 J. Agric. Food Chem. 2017, 65, 4262−4272
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Journal of Agricultural and Food Chemistry
3β,22α,26-triol-3-O-β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-[β-D-glucopyranosyl-(1→6)] -β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside and named solanigroside Y7. The molecular formula of 11 was inferred as C62H104O33 due to the appearance of a [M + NH4]+ ion at m/z 1394.6846 in the HR-ESI-MS and the 13C NMR data. Acid hydrolysis and GC analysis of 11 gave D-glucose, D-xylose, and D-galactose. The 13C NMR data of the aglycone moiety and the sugar moiety were considerably similar to those of the known compound uttroside B (compound 16),21 except for a little difference in the sugar chain at C-26 of the aglycone. The identification of the monosaccharides was made by combination of DEPT, 1H−1H COSY, HSQC, HMBC, and TOCSY spectra. The linkage of the sugar chain at C-26 was defined by HMBC correlations (Figure 3) between H-1 (δH 5.28) of the terminal glucosyl and C-2 (δC 84.4) of the inner glycosyl and H-1 (δH 4.84) of the inner glucosyl and C-26 (δC 75.4) of the aglycone. Therefore, compound 11 was assigned to be (25R)26-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-5α-furost3β,22α,26-triol-3-O-β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside and named solanigroside Y8. Compound 12 was obtained as a white amorphous powder, with the same molecular formula of C62H104O33 as for 11 by HR-ESI-MS (m/z 1394.6815 [M + NH4]+). Acid hydrolysis of 12 also gave D-glucose, D-xylose, and D-galactose as 11. From a comparison of the NMR data of 12 with those of 11, it was apparent that 12 contained the same aglycone as 11, except for a little difference in the saccharide chains attached at C-3 and C-26 of the aglycone. The identity of the monosaccharides and the linkage of the sugar residues were carried out by comprehensive analysis of DEPT, 1H−1H COSY, HSQC, HMBC, and TOCSY spectra. The sugar chain at C-3 was established as β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl(1→3)]-[β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-(1→ 4)-β- D -galactopyranosyl due to the HMBC long-range correlations between H-1 of glucose II (δH 5.55) and C-2 (δC 81.6) of glucose I, H-1 (δH 5.22) of xylose and C-3 (δC 87.0) of glucose I, H-1 (δH 5.09) of glucose III and C-6 (δC 70.4) of glucose I, H-1 (δH 5.18) of glucose I and C-4 (δC 80.2) of galactose, and H-1 (δH 4.87) of galactose and C-3 (δC 77.7) of the aglycone. The remaining glucose was determined to be linked to the C-26 position by the HMBC correlation between H-1 of the glucose (δH 4.72) and C-26 (δC 75.6) of the aglycone. Thus, compound 12 was elucidated as (25R)-26-O-βD-glucopyranosyl-5α-furost-3β,22α,26-triol-3-O-β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-[β-D-glucopyranosyl(1→6)]-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside and named solanigroside Y9. Seven known compounds were identified by comparison of the NMR data with reported data as (25R)-26-O-β-Dglucopyranosylfurost-5(6)-ene-3β,22α,26-triol-3-O-β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside (7),30,31 (25R)-26-O-β-Dglucopyranosylfurost-5(6)-ene-3β,22α,26-triol-3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranoside (8),32 (25R)-26-O-β-D-glucopyranosylfurost-5(6)ene-16α-methoxy-3β,26-diol-3-O-α-L-rhamnopyranosyl-(1→ 2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranoside (9),33 (25R)-26-O-β-D-glucopyranosyl-5α-furost-3β,22α,26-triol-3-Oβ-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-β-Dglucopyranosyl-(1→4)-β-D-galactopyranoside (13),21 (25S)-26O-β-D-glucopyranosyl-5α-furost-3β,22α,26-triol-3-O-β-D-gluco-
rhamnosyl II and C-4 (δC 78.4) of glucosyl I, H-1 (δH 6.44) of rhamnosyl I and C-2 (δC 78.1) of glucosyl I, and H-1 (δH 4.93) of glucosyl I and C-3 (δC 78.3) of the aglycone. The long-range correlation between H-1 (δH 4.82) of the other glucose and C26 (δC 75.6) of the aglycone indicated that the additional glucose was attached at C-26 of the furostanol skeleton. On the basis of the above evidence, the structure of 5 was deduced as (25R)-26-O-β-D-glucopyranosylfurost-5(6)-en-3β,17α,22α,26tetraol-3-O-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl(1→4)-[α- L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside and named solanigroside Y5. Compound 6 exhibited a molecular formula of C57H94O27 as determined by HR-ESI-MS (positive ion mode) at m/z 1228.6338 [M + NH4]+ (calcd for C57H98NO27 [M + NH4]+, 1228.6326). Acid hydrolysis and GC analysis of 6 also gave Dglucose and L-rhamnose. The 1H NMR data of 6 contained two tertiary methyl groups at δH 0.89 (3H, s, Me-18) and 1.04 (3H, s, Me-19), four secondary methyl groups at δH 0.98 (3H, d, J = 6.6 Hz, Me-27), 1.31 (3H, d, J = 6.7 Hz, Me-21), 1.62 (3H, d, J = 6.2 Hz, Me-Rha II), and 1.76 (3H, d, J = 6.2 Hz, Me-Rha I), an olefinic methine group at δH 5.29 (1H, brs, H-6), and five anomeric protons at δH 4.73 (1H, d, J = 7.8 Hz, H-1-Glc), 4.94 (1H, overlapped, H-1-Glc I), 5.11 (1H, overlapped, H-1-Glc II), 5.87 (1H, s, H-1-Rha II), and 6.41 (1H, s, H-1-Rha I). The 13 C NMR spectrum of 6 displayed a characteristic furostanol carbon signal at δC 111.0 (C-22), two olefinic carbons at δC 141.1 (C-5) and 122.2 (C-6), and five anomeric carbons at δC 100.6 (C-1-Glc I), 102.3 (C-1-Rha I), 103.2 (C-1-Rha II), 105.1 (C-1-Glc), and 105.8 (C-1-Glc II). The 13C NMR data of the aglycone moiety and the sugar moiety were closely similar to those of protodioscin,29 except for the glycosidation shift of C-6 (+8.9 ppm) of the glucosyl attached at C-3 of the aglycone and additional signals for a hexose. This significant glycosidation shift indicated that the additional glucosyl was linked to the C-6 of the inner glucosyl. The attachments of sugar chain were further confirmed by the HMBC spectrum (Figure 3). Therefore, the structure of 6 was identified as (25R)-26-O-β-D-glucopyranosylfurost-5(6)-en-3β,22α,26-triol3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→ 4)]-[β- D-glucopyranosyl-(1→6)]-β- D -glucopyranoside and named solanigroside Y6. Compound 10 was obtained as a white amorphous powder, showing a positive reaction to the Ehrlich reagent. The molecular formula was determined as C63H106O34 according to the positive-ion HR-ESI-MS peak at m/z 1424.6948 [M + NH4]+ (calcd for C63H110NO34 [M + NH4]+, 1424.6909) and its 13C and DEPT NMR data. On acid hydrolysis, compound 10 liberated D-galactose and D-glucose. A comparison of the NMR data (Table 3) of 10 to those of known compound 13 ((25R)-26-O-β-D-glucopyranosyl-5α-furost-3β,22α,26-triol-3O-β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-βD-glucopyranosyl-(1→4)-β-D-galactopyranoside) showed the NMR data of those two compounds were almost the same apart from the signals of the sugar chain linked to the C-3 position of the aglycone and additional signals for a hexose. Comparison of 13C NMR data of the sugar moiety at C-3 with the reported data indicated the additional glucosyl was determined to be attached to C-6 of glucosyl I by a downfield shift of C-6 of the glucosyl I from δC 62.7 in 13 to δC 70.4 in 10.21 This linkage was further verified by the cross peak between H-1 (δH 5.11) of glucosyl IV and C-6 (δC 70.4) of glucosyl I in the HMBC spectrum of 10. Thus, compound 10 was elucidated as (25R)-26-O-β-D-glucopyranosyl-5α-furost4269
DOI: 10.1021/acs.jafc.7b00985 J. Agric. Food Chem. 2017, 65, 4262−4272
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Journal of Agricultural and Food Chemistry Table 4. Inhibitory Effects of Isolated Compounds from S. nigrum on NO Production Induced by LPS in RAW 264.7 Macrophagesa compound IC50 (μM) a
1
2
3
5
6
9
11
12
indomethacinb
9.7 ± 0.3
17.8 ± 1.5
14.0 ± 0.4
38.3 ± 2.2
41.0 ± 3.9
22.1 ± 7.2
48.5 ± 3.5
44.0 ± 0.8
47.4 ± 4.5
Values are presented as means ± SD (n = 3). bPositive control.
Figure 4. Inhibitory effects of compounds 1−6, 9, 10, and 12 on the LPS-induced IL-6 and IL-1β production in RAW 264.7 macrophages. Cells were treated with LPS (1.0 μg/mL) and different concentrations (12.5, 25.0 μM) of the test compounds for 24 h. Levels of IL-6 and IL-1β in the culture supernatant were quantified by ELISA. Data are presented as the mean ± SD (n = 2). (∗) p < 0.05 and (∗∗) p < 0.01 are compared with the LPS-induced group.
9.7 μM, comparable to that of the positive control. Compounds 2, 3, 5, 6, 9, 11, and 12 exhibited moderate inhibition with IC50 values between 14.0 and 48.5 μM. Other compounds showed IC50 values >50 μM and were considered to be inactive. ELISA was also performed to determine the inhibitory effects of the isolated steroidal saponins on pro-inflammatory cytokines IL-6 and IL-1β. As shown in Figure 4, compounds 1, 3, and 5 exhibited significant inhibition effects on the LPSinduced IL-6 production at a concentration of 25.0 μM, and compounds 1−5 and 12 significantly inhibited the production of IL-1β at a concentration of 25.0 μM. As an important class of natural products found in many food and medicinal plants, steroidal saponins are believed to have a variety of biological activities, such as cytotoxic, hemolytic, antiplatelet aggregation, anti-inflammatory, and antibacterial activities.35 In this study, steroidal saponins isolated from the weed plant S. nigrum were demonstrated to exert potential NO production inhibitory activities, and the effects were sensitive to the change of their structures either in the sugar chain or in the aglycone part. For example, compound 4 showed weak activity (IC50 > 50 μM), whereas compounds 1−3 showed strong inhibition (IC50 = 9.7−14.0 μM), indicating that the introduction of a β-D-glucose to the C-3 position of the inner glucose (Glc I of S4 in Figure 1) increased the inhibitory
pyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside (14),21 (25R)-26-O-β-Dglucopyranosyl-22α-methoxy-5α-furost-3β,26-diol-3-O-β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside (15),21 and uttroside B ((25R)-26-O-β-D-glucopyranosyl-5α-furost-3β,22α,26-triol-3O-β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-Dglucopyranosyl-(1→4)-β-D-galactopyranoside) (16).21 Compounds 7, 8, and 9 are reported for the first time from S. nigrum, to our knowledge. Anti-inflammatory Activities. Nitric oxide is a signaling molecule that plays an important role in the pathogenesis of inflammation. Inhibitors of NO production represent potential therapeutic agents for inflammatory diseases.34 Compounds 1− 16 were tested for their inhibitory activities against NO production induced by LPS in RAW 264.7 macrophages (Table 4). Cell viability was examined by the MTT method to define whether inhibitory activities of NO production of these compounds were due to their cytotoxicity.18 No obvious cytotoxicity against RAW 264.7 macrophage cells treated with these compounds at their effective concentrations was observed. Indomethacin, with an IC50 value of 47.4 μM, was selected as the positive control. Compound 1 exhibited significant inhibition on NO production with an IC50 value of 4270
DOI: 10.1021/acs.jafc.7b00985 J. Agric. Food Chem. 2017, 65, 4262−4272
Article
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activity significantly. A similar phenomenon was observed in compounds 11 and 16, which possess the same sugar chain linked to C-3 position; attachment of a β-D-glucose to C-2 position of the C-26 β-D-glucose (Glc of S1 in Figure 1) led to increased inhibition (compound 11, IC50 = 48.5 μM). However, compared to compounds 13, 14, and 15 (IC50 > 50 μM), neither a β-D-glucose introduced to the C-6 position of the inner glucose (Glc I of S5 in Figure 1) (compound 10, IC50 > 50 μM) nor a β-D-xylose instead of β-D-glucose incorporated to the C-3 position of the inner glucose (Glc I of S5 in Figure 1) (compound 16, IC50 > 50 μM) improved the inhibition. Intriguingly, when a β-D-glucose was introduced to the C-6 position of the inner glucose (Glc I of S5 in Figure 1) and a βD-xylose instead of β-D-glucose was introduced to the C-3 position of the inner glucose (Glc I of S5 in Figure 1) at the same time, a moderate inhibitory effect was observed in compound 12 (IC50 = 44.0 μM). On the other hand, the functionalization of the aglycone also affected the inhibitory effects of these steroidal saponins. For instance, compound 8 showed weak inhibition (IC50 > 50 μM), whereas reduction of the hydroxyl group at the C-22 position and connection of a methoxyl group to the C-16 position in compound 9 led to a significant improvement of the inhibitory effects (compound 9, IC50 = 22.1 μM). These results are in good agreement with the literature and provide important evidence regarding the influence of the variation in the sugar moiety as well as the functionalization of the aglycone on the NO inhibitory activity of steroidal saponin.36,37 S. nigrum is a common weed of crops and gardens. Ripe berries and cooked leaves of some edible strains are consumed as food in some locales and believed to possess serious health benefits such as antioxidant activity, antidiabetes activity, and hepatoprotection. In this research, steroidal saponins isolated from the unripe fruits of S. nigrum showed potential antiinflammatory activity by inhibiting the production of NO and pro-inflammatory cytokines IL-6 and IL-1β induced by LPS in RAW 264.7 macrophages. The mechanisms of action of these saponins in the inflammation prevention and the structure− activity relationship are worth further investigation. Results of this study could be helpful to better utilize this weed plant as a healthy food or natural source of chemopreventive agents for inflammation-related diseases.
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This research was supported by a project of the Guangdong Natural Science Foundation (No. 2014A030313588) and the Guangdong Provincial Department of Science and Technology (2015A020211027). Notes
The authors declare no competing financial interest.
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(1) Wang, F. Z.; Tang, J. Flora of China; Science Publishing House: Beijing, China, 1980; Vol. 67, pp 76−79. (2) Jimoh, F. O.; Adedapo, A. A.; Afolayan, A. J. Comparison of the nutritional value and biological activities of the acetone, methanol and water extracts of the leaves of Solanum nigrum and Leonotis leonorus. Food Chem. Toxicol. 2010, 48, 964−971. (3) Venkateswarlu, J.; Krishna Rao, M. Inheritance of fruit colour in the Solanum nigrum complex. Proc. Indian Acad. Sci. Sect. B 1971, 74, 137−141. (4) Irving, M. The Forager Handbook: A Guide to the Edible Plants of Britain; Ebury Press, 2009; pp 28−32. (5) Yang, M.; Hsu, L.; Peng, C.; Shi, Y.; Wu, C.; Wang, C. Polyphenol-rich extracts from Solanum nigrum attenuated PKC αmediated migration and invasion of hepatocellular carcinoma cells. J. Agric. Food Chem. 2010, 58, 5806−5814. (6) Ding, X.; Zhu, F.; Yang, Y.; Li, M. Purification, antitumor activity in vitro of steroidal glycoalkaloids from black nightshade (Solanum nigrum L.). Food Chem. 2013, 141, 1181−1186. (7) Huang, H.-C.; Syu, K. Y.; Lin, J. K. Chemical composition of Solanum nigrum Linn extract and induction of autophagy by leaf water extract and its major flavonoids in AU565 breast cancer cells. J. Agric. Food Chem. 2010, 58, 8699−8708. (8) Hsu, J. D.; Kao, S. H.; Tu, C. C.; Li, Y. J.; Wang, C. J. Solanum nigrum L. extract inhibits 2-acetylaminofluorene-induced hepatocarcinogenesis through overexpression of glutathione S-transferase and antioxidant enzymes. J. Agric. Food Chem. 2009, 57, 8628−8634. (9) Jeong, J. B.; Jeong, H. J.; Park, J. H.; Lee, S. H.; Lee, J. R.; Lee, H. K.; Chung, G. Y.; Choi, J. D.; De Lumen, B. O. Cancer-preventive peptide lunasin from Solanum nigrum L. inhibits acetylation of core histones H3 and H4 and phosphorylation of retinoblastoma protein (Rb). J. Agric. Food Chem. 2007, 55, 10707−10713. (10) Aly, Y. S.; Shallan, M. A. Antioxidant properties of wild Solanum nigrum ripe fruit. Planta Med. 2011, 77, 1367−1367. (11) Hou, T. H.; Chung, J. P.; Chen, S. S.; Chang, T. L. Antioxidation and antiglycation of 95% ethanolic extracts prepared from the leaves of black nightshade (Solanum nigrum). Food Sci. Biotechnol. 2013, 22, 839−844. (12) Sohrabipour, S.; Kharazmi, F.; Soltani, N.; Kamalinejad, M. Effect of the administration of Solanum nigrum fruit on blood glucose, lipid profiles, and sensitivity of the vascular mesenteric bed to phenylephrine in streptozotocin-induced diabetic rats. Med. Sci. Monit. Basic Res. 2013, 19, 133−40. (13) Zhou, X.; He, X.; Wang, G.; Gao, H.; Zhou, G.; Ye, W.; Yao, X. Steroidal saponins from Solanum nigrum. J. Nat. Prod. 2006, 69, 1158− 1163. (14) Lin, T.; Fan, M.; Wang, S.; Huang, J. Identification of the Solanum nigrum extract component involved in controlling cabbage black leaf spot disease. J. Agric. Food Chem. 2011, 59, 1667−1672. (15) Qiao, A. M.; Wang, Y. H.; Xiang, L. M.; Zhang, Z. X.; He, X. J. Novel triterpenoids isolated from hawthorn berries functioned as antioxidant and antiproliferative activities. J. Funct. Foods 2015, 13, 308−313. (16) Wang, Y. H.; Xiang, L. M.; Wang, C. H.; Tang, C.; He, X. J. Antidiabetic and antioxidant effects and phytochemicals of mulberry fruit (Morus alba L.) polyphenol enhanced extract. PLoS One 2013, 8, e71144. (17) He, X. J.; Liu, R. H. Triterpenoids isolated from apple peels have potent antiproliferative activity and may be partially responsible for apple’s anticancer activity. J. Agric. Food Chem. 2007, 55, 4366−4370.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00985.
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Original spectra of new compounds 1−6 and 10−12, including UV, IR, 1H and 13C NMR, 2D-NMR, and HRESI-MS (PDF)
AUTHOR INFORMATION
Corresponding Author
*(X.H.) Phone/fax: +86 20 3935 2132. E-mail: hexiangjiu@ 163.com. ORCID
Xiangjiu He: 0000-0002-1631-7657 Author Contributions ∥
REFERENCES
Y.W. and L.X. contributed equally to this work. 4271
DOI: 10.1021/acs.jafc.7b00985 J. Agric. Food Chem. 2017, 65, 4262−4272
Article
Journal of Agricultural and Food Chemistry (18) Xiang, L. M.; Wang, Y. H.; Yi, X. M.; He, X. J. Antiproliferative and anti-inflammatory furostanol saponins from the rhizomes of Tupistra chinensis. Steroids 2016, 116, 28−37. (19) Thambiraj, S. R.; Phillips, M.; Koyyalamudi, S. R.; Reddy, N. Antioxidant activities and characterisation of polysaccharides isolated from the seeds of Lupinus angustifolius. Ind. Crops Prod. 2015, 74, 950− 956. (20) Zhou, X. L.; He, X. J.; Wang, G. H.; Gao, H.; Zhou, G. X.; Ye, W. C.; Yao, X. S. Steroidal saponins from Solanum nigrum. J. Nat. Prod. 2006, 69, 1158−1163. (21) Zhou, X. L.; He, X. J.; Zhou, G. X.; Ye, W. C.; Yao, X. S. Steroidal glycosides from Solanum nigrum. Chin. Tradit. Herbal Drugs 2006, 37, 1618−1621. (22) Yokosuka, A.; Mimaki, Y. Steroidal saponins from the whole plants of Agave utahensis and their cytotoxic activity. Phytochemistry 2009, 70, 807−815. (23) Su, L.; Chen, G.; Feng, S.; Wang, W.; Li, Z.; Chen, H.; Liu, Y.; Pei, Y. Steroidal saponins from Tribulus terrestris. Steroids 2009, 74, 399−403. (24) Jin, J.; Liu, X.; Yang, C. Three new hecogenin glycosides from fermented leaves of Agave americana. J. Asian Nat. Prod. Res. 2003, 5, 95−103. (25) Yokosuka, A.; Suzuki, T.; Tatsuno, S.; Mimaki, Y. Steroidal glycosides from the underground parts of Yucca glauca and their cytotoxic activities. Phytochemistry 2014, 101, 109−115. (26) Agrawal, P. K. Dependence of 1H NMR chemical shifts of geminal protons of glycosyloxy methylene (H-2-26) on the orientation of the 27-methyl group of furostane-type steroidal saponins. Magn. Reson. Chem. 2004, 42, 990−993. (27) Agrawal, P. K. Assigning stereodiversity of the 27-Me group of furostane-type steroidal saponins via NMR chemical shifts. Steroids 2005, 70, 715−724. (28) Challinor, V. L.; Piacente, S.; De Voss, J. J. NMR assignment of the absolute configuration of C-25 in furostanol steroidal saponins. Steroids 2012, 77, 602−608. (29) Ikeda, T.; Tsumagari, H.; Okawa, M.; Nohara, T. Pregnane- and furostane-type oligoglycosides from the seeds of Allium tuberosum. Chem. Pharm. Bull. 2004, 52, 142−145. (30) Janeczko, Z.; Jansson, P. E.; Sendra, J. A new steroidal saponin from Polygonatum of f icinale. Planta Med. 1987, 53, 52−54. (31) Chen, H. F.; Wang, N. L.; Yao, X. S. Studies on bioactive steroidal saponins of Allium macrostemon Bunge. Chin. J. Med. Chem. 2005, 15, 142−147. (32) Chen, Y.; Yin, J.; Fu, W. Novel medical usage of yam rhizome dioscoreae septemlobae of Chinese traditional medicine and method for preparing its extractive. Patent CN1415364 (A), 2003. (33) Ono, M.; Kakiuchi, T.; Ebisawa, H.; Shiono, Y.; Nakamura, T.; Kai, T.; Ikeda, T.; Miyashita, H.; Yoshimitsu, H.; Nohara, T. Steroidal glycosides from the fruits of Solanum viarum. Chem. Pharm. Bull. 2009, 57, 632−635. (34) Sharma, J. N.; Al-Omran, A.; Parvathy, S. S. Role of nitric oxide in inflammatory diseases. Inflammopharmacology 2007, 15, 252−259. (35) Sparg, S. G.; Light, M. E.; van Staden, J. Biological activities and distribution of plant saponins. J. Ethnopharmacol. 2004, 94, 219−243. (36) Gonzalez, M.; Zamilpa, A.; Marquina, S.; Navarro, V.; Alvarez, L. Antimycotic spirostanol saponins from Solanum hispidum leaves and their structure-activity relationships. J. Nat. Prod. 2004, 67, 938−941. (37) Wang, Y. H.; Li, C.; Xiang, L. M.; Huang, W. J.; He, X. J. Spirostanol saponins from Chinese onion (Allium chinense) exert pronounced anti-inflammatory and anti-proliferative activities. J. Funct. Foods 2016, 25, 208−219.
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