Article pubs.acs.org/JAFC
New Triterpenoid Saponins from Ilex cornuta and Their Protective Effects against H2O2‑Induced Myocardial Cell Injury Shanshan Li,† Jianping Zhao,‡ Yanli Liu,† Zhong Chen,† Qiongming Xu,*,†,‡ Ikhlas A. Khan,†,‡ and Shilin Yang† †
College of Pharmaceutical Science, Soochow University, Suzhou 215123, China National Center for Natural Products Research, and Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677, United States
‡
S Supporting Information *
ABSTRACT: Five new triterpenoid saponins, 1−5, together with 10 known ones, 6−15 were isolated from the aerial parts of Ilex cornuta. The structures of compounds 1−5 were determined as 3β-O-α-L-arabinopyranosyl-19α,23-dihydroxy-20α-urs-12-en28-oic acid 28-O-β-D-glucopyranosyl ester, 1; 3β-O-β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl-19-hydroxy-20α-urs-12-en28-oic acid 28-O-β-D-glucopyranosyl ester, 2; 19α,23-dihydroxyurs-12-en-28-oic acid 3β-O-β-D-glucuronopyranoside-6-O-methyl ester, 3; 19α,23-dihydroxyurs-12-en-28-oic acid 3β-O-[β-D-glucuronopyranoside-6-O-methyl ester]-28-O-β-D-glucopyranosyl ester, 4; and 3β-O-[α-L-arabinopyranosyl-(1→2)-β-D-glucuronic acid]-oleanolic acid 28-O-β-D-glucopyranosyl ester, 5, on the basis of spectroscopic analyses (IR, ESI-MS, HR-ESI-MS, 1D and 2D NMR) and chemical reactions. Protective effects of compounds 1−15 were tested against H2O2-induced H9c2 cardiomyocyte injury, and the data showed that compounds 1, 4, 6, and 13 had significant cell-protective effects. No significant DPPH radical scavenging activity was observed for compounds 1−15. KEYWORDS: Ilex cornuta, Aquifoliaceae, triterpenoid saponins, structure identification, cardiomyocyte oxidative injury, DPPH radical scavenging activity
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INTRODUCTION Ilex cornuta Lindl. et Paxt. (Aquifoliaceae family) is an evergreen shrub with dense foliage. Its leaves are the major source of “Ku-Ding-Cha”, a popular herbal tea consumed in China and other countries.1 The tea is widely used for its health benefits, such as relief of sore throat and weight loss.2 According to the Traditional Chinese Medicine theory, the leaves of I. cornuta, which are bitter and cool-natured, can invigorate the liver and kidney and can expel “wind” and “heat”. It has been used to cure weakness of waist, knees, arthrodynia, head-wind, acute conjunctivitis, toothache, and urticaria.3 The results from modern pharmacology research showed that the plant had many therapeutic effects, such as increase of coronary blood flow4 and anticancer5 and antibacterial properties.6 Previous phytochemical investigation on the Ilex genus led to the isolation of triterpenes, triterpenoid saponins,7 flavones, flavonoid glycosides,8 polyphenols,9 and fatty acids.10 Cardiovascular disease is one of the major causes of deaths worldwide. Oxidative stress is considered as a causative factor in the pathogenesis of cardiomyocyte ischemic injury. Activation of oxidative stress leads to a variety of intracellular signaling events that ultimately cause dysfunction of the endothelium, proliferation of vascular smooth muscle cells, expression of proinflammatory genes, and reconstruction of the extracellular matrix.11 Growing evidence has suggested that the intake of foods rich in antioxidants can result in a lower risk of cardiovascular disease.12 In our effort to develop the extract of I. cornuta as a herbal dietary supplement for the prevention of coronary artery disease, a phytochemical investigation of its aerial parts was undertaken, which led to the isolation of 5 new © 2013 American Chemical Society
triterpenoid saponins, 1−5 (Figure 1), along with 10 known ones. In this paper, we report the isolation and structural elucidation of the new compounds, as well as the protective effects of the isolates against cardiomyocyte injury induced by H2O2. In addition, their radical scavenging activity was evaluated by DPPH assay.
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MATERIALS AND METHODS
General. The specific optical rotation values were determined with a Perkin-Elmer model 241 polarimeter (Perkin-Elmer Inc., Waltham, MA, USA). IR spectra were determined on a Perkin-Elmer 983 G spectrometer. 1H and 13C NMR and 2D NMR spectra were recorded on a Varian Inova 500 spectrometer (Varian Inc., Palo Alto, CA, USA) in C5D5N using tetramethylsilane (TMS) as the internal standard. HRESI-MS spectra were determined on a Micromass Q-TOF2 spectrometer (Micromass Corp., London, UK). Chromatography. HPLC analysis and purification were performed with an Agilent Zorbax SB-C18 semipreparative HPLC column (250 × 9.4 mm i.d., 5 μm, Agilent Corp., Palo Alto, CA, USA) on a Shimadzu HPLC system composed of an LC-20AT pump with an SPD-20A detector (Shimadzu Corp., Kyoto, Japan), and the wavelength for detection was 203 nm. Medium-pressure liquid chromatography (MPLC) purification was performed on a Büchi flash chromatography system composed of a C-650 pump and a flash column (460 × 26 mm i.d., Büchi Corp., Flawil, Switzerland). Silica gel (60−100 mesh) used for column chromatography and precoated silica gel for TLC plates were purchased from Qingdao Marine Chemical Received: Revised: Accepted: Published: 488
October 16, 2013 December 27, 2013 December 29, 2013 December 29, 2013 dx.doi.org/10.1021/jf4046667 | J. Agric. Food Chem. 2014, 62, 488−496
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Figure 1. Structures of compounds 1−15. 100:0, each 500 mL) to afford five fractions. Fraction 2 (125 mg) was separated by semipreparative HPLC to yield compounds 7 (25 mg, tR 35.27 min), 8 (8 mg, tR 38.28 min), and 9 (33 mg, tR 41.32 min). The CHCl3/MeOH (80:20) eluate (4.9 g) yielded five fractions after separation by MPLC. Fraction 3 (90 mg) was further separated by using semipreparative HPLC to yield compounds 1 (9 mg, tR 60.27 min) and 10 (19 mg, tR 63.19 min). From fraction 4 (1500−2000 mL, 180 mg) compounds 3 (40 mg, tR 70.15 min) and 4 (8 mg, tR 73.23 min) were obtained after it was subjected to the semipreparative HPLC separation. In a similar way, the CHCl3/MeOH (70:30) eluate (6.7 g) from the vacuum chromatography was first separated into six fractions by MPLC over the ODS column, and then these fractions were further separated by using semipreparative HPLC to yield compounds 6 (20 mg), 12 (55 mg), 13 (10 mg), 14 (9 mg), and 15 (14 mg). Moreover, compounds 2 (10 mg), 5 (17 mg), and 11 (36 mg) were obtained from the CHCl3/MeOH (60:40) eluate (4.8 g) after MPLC and semipreparative HPLC separations as described above. 3β-O-α-L-Arabinopyranosyl-19α,23-dihydroxy-20α-urs-12-en-28oic acid 28-O-β-D-glucopyranosyl ester (1): white amorphous powder; [α]25 D −9.5 (c 0.12, MeOH); IR (KBr) νmax 3385 (OH), 2943, 1745 (CO), 1692, 1641, 1460, 1050 (COC), 876 cm−1; 1 H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectroscopic data, see Tables 1 and 2; HR-ESI-MS (positive ion mode) m/z 805.4303 ([M + Na]+; calcd for C41H66O14Na, 805.4350). 3β-O-β-D-Glucopyranosyl-(1→2)-α-L-arabinopyranosyl-19-hydroxy-20α-urs-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester (2): white amorphous powder; [α]25 D −15.4 (c 0.15, MeOH); IR (KBr) νmax
Factory (Qingdao, China). A 10% sulfuric acid alcohol solution was used for TLC colorization. Sephadex LH-20 for column chromatography was purchased from GE Corp. (Piscataway, NJ, USA). D101 macroporous resin for column chromatography was purchased from Xi’an Sunresin New Materials Co. Ltd. (Xi’an, China). GC analyses were conducted on a GC-14C (Shimadzu Corp.) instrument with a flame ionization detector (FID), and the analytical conditions were as follows: DB-5 column (i.d. = 0.25 μm, length = 30 m, Suzhou Huitong Chromatography Technology Co., Ltd., Suzhou, China); column temperature at 210 °C; injector temperature at 270 °C; detector temperature at 300 °C. Materials. The aerial parts of I. cornuta were collected from Liuan City of Anhui province, in April 2011, and were authenticated by Professor Xiao-ran Li. A voucher specimen (04-21-01-11) was deposited in the herbarium of the College of Pharmaceutical Science, Soochow University. Extraction and Isolation. The dried plant material (10 kg) was crushed into powder and extracted twice with 50% EtOH at 80 °C under reflux. The solvent was subsequently removed under reduced pressure to yield a residue (325 g), which was then dissolved in 30 L of distilled water and passed through D101 macroporous resin column (200 × 30 cm i.d.), eluted with a gradient of aqueous EtOH (EtOH/ H2O 0, 30, 60, 90%). The 90% EtOH eluate (30 g) was further vacuum chromatographed on a silica gel (60−100 mesh) column (30 × 18 cm i.d.), eluted with a gradient of CHCl3/MeOH (90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 0:100, each 6.0 L). The CHCl3/ MeOH (90:10) eluate (1.5 g) was then separated by MPLC over an ODS column, eluted with MeOH/H2O (60:40, 70:30, 80:20, 90:10, 489
dx.doi.org/10.1021/jf4046667 | J. Agric. Food Chem. 2014, 62, 488−496
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Table 1. NMR Spectroscopic Data for Aglycone Moieties of Compounds 1−5 (in C5D5N) 1
a
no.
δC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
38.9 26.3 82.0 43.6 47.7 18.5 33.2 40.6 48.0 37.1 24.2 127.8 139.0 42.2 29.4 26.9 48.4 47.3 73.5 43.0 24.8 32.0 64.6 13.7 16.2 17.7 24.4 177.2 29.8 16.4
2 δH
1.16m, 1.69m 2.12m, 2.34m 4.34m 1.78m 1.46m, 1.75m 1.50m, 1.80m 2.01m 2.00m, 2.13m 5.58m
1.28m, 2.53m 2.12m, 3.28m 3.26s 2.02m 1.27m, 2.66m 1.98m, 2.18m 3.78d (11.5), 4.34m 1.02s 1.08s 1.30s 1.72s 1.45s 1.05d (7.5)
3
4
5
δC
δH
δC
δH
δC
δH
δC
δH
38.9 26.6 89.0 39.6 56.0 18.8 33.6 40.6 47.9 37.1 24.1 127.8 138.9 42.3 29.3 26.9 48.5 47.3 73.5 43.0 24.8 32.0 28.3 16.9 15.8 17.6 24.4 177.1 29.9 16.2
0.96m, 1.60m 1.92m, 2.16m 3.27dd (5.0, 15.0a)
38.8 26.2 82.3 43.6 47.6 18.4 33.3 40.5 47.9 36.9 24.1 128.1 140.1 42.2 29.5 26.5 48.4 54.7 72.8 42.3 27.1 38.7 64.4 13.7 16.2 17.4 24.8 180.8 27.2 16.9
1.01m, 1.56m 2.04m, 2.26m 4.36m
38.9 26.2 82.4 43.6 47.6 18.4 33.3 40.7 47.9 37.0 24.2 128.6 139.4 42.2 29.4 26.2 48.7 54.6 72.8 42.8 26.8 37.8 64.5 13.7 16.3 17.6 24.7 177.1 27.1 16.8
1.00m, 1.58m 2.06m, 2.28m 4.36m
38.8 26.8 89.2 39.7 55.9 18.6 33.3 40.0 48.1 37.0 23.5 123.0 144.3 42.3 28.4 23.9 47.1 41.9 46.3 30.9 34.1 32.7 28.1 16.6 15.7 17.6 26.3 176.6 33.3 23.8
0.90m, 1.44m 1.94m, 2.32m 3.37dd (4.0, 11.5)
0.83d (15.0) 1.40m, 1.53m 1.55m, 1.65m 1.85m 2.11m 5.59m
1.35m, 2.56m 2.17m, 3.31m 3.27s 2.03m 1.28m, 2.68m 1.98m, 2.18m 1.28s 1.13s 0.99s 1.28s 1.78s 1.47s 1.07d (10.0)
1.76m 1.45m, 1.79m 1.42m, 1.80m 1.97m 2.11m 5.66m
1.30m, 2.37m 2.08m, 3.17m 3.12s 1.56m 1.41m, 2.14m 2.14m, 2.22m 3.78m, 4.39m 1.01s 0.98s 1.17s 1.77s 1.50s 1.19d (6.5)
1.76m 1.43m, 1.76m 1.49m, 1.81m 1.98m 2.08m, 2.12m 5.62m
1.47m, 2.52m 2.01m, 3.15m 3.00s 1.44m 1.29m, 1.50m 1.93m, 2.13m 3.78m, 4.39m 1.03s 1.06s 1.28s 1.72s 1.46s 1.14d (5.0)
0.80d (11.5) 1.39m, 1.56m 1.39m, 1.52m 1.68m 2.11m 5.49m
1.26m, 2.41m 1.95m, 3.24m 1.31m, 1.83m 1.18m, 1.44m 1.82m, 1.86m 1.37s 1.17s 0.88s 1.16s 1.35s 0.98s 0.95s
Data in parentheses are J values (in hertz).
3395 (OH), 2948, 1742 (CO), 1643, 1454, 1056 (COC), 886 cm−1; 1H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectroscopic data, see Tables 1 and 2; HR-ESI-MS (positive ion mode) m/z:951.4887 ([M + Na]+; calcd for C47H76O18Na, 951.4929). 19α,23-Dihydroxyurs-12-en-28-oic acid 3β-O-β-D-glucuronopyranoside-6-O-methyl ester (3): white amorphous powder; [α]25 D −11.0 (c 0.12, MeOH); IR (KBr) νmax 3382 (OH), 2942, 1740 (CO), 1693, 1644, 1455, 1046 (COC), 887 cm−1; 1H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectroscopic data, see Tables 1 and 2; HR-ESI-MS (negative ion mode) m/z 677.3869 ([M − H]−; calcd for C37H57O11, 677.3901). 19α,23-Dihydroxyurs-12-en-28-oic acid 3β-O-[β-D-glucuronopyranoside-6-O-methyl ester]-28-O-β-D-glucopyranosyl ester (4): white amorphous powder; [α]25 D −13.2 (c 0.10, MeOH); IR (KBr) νmax 3381 (OH), 2946, 1741 (CO), 1648, 1462, 1057 (COC), 882 cm−1; 1 H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectroscopic data, see Tables 1 and 2; HR-ESI-MS (positive ion mode) m/z 863.4405 ([M + Na]+; calcd for C43H68O16Na, 863.4405). 3β-O-[α-L-Arabinopyranosyl-(1→2)-β-D-glucuronic acid]-oleanolic acid 28-O-β-D-glucopyranosyl ester (5): white amorphous powder; [α]25 D −12.1 (c 0.10, MeOH); IR (KBr) νmax 3384 (OH), 2949, 1743 (CO), 1693, 1644, 1449, 1038 (COC), 889 cm−1; 1 H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectroscopic data, see Tables 1 and 2; HR-ESI-MS (positive ion mode) m/z 949.4729 ([M + Na]+; calcd for C48H78O16Na 949.4772). Acid Hydrolysis of Compounds 1−5. A solution of each compound (4 mg) dissolved in 1 N HCl (dioxane/H2O, 1:1, 2.0 mL) was heated to 90 °C for 2 h. After cooling, the reaction mixture was neutralized with silver carbonate (10 mg), and the solvent was
evaporated to dryness under N2 and then extracted with CHCl3 and H2O. The aqueous layer was concentrated to dryness, the residue was dissolved in dry pyridine (0.2 mL), and then L-cysteine methyl ester hydrochloride in dry pyridine (0.06 M, 0.2 mL) was added. The mixture was reacted at 60 °C for 2 h, and 0.2 mL of (trimethylsilyl)imidazole dissolved in dry pyridine was added. After being held at 60 °C for another 2 h, the reactant was partitioned with n-hexane and H2O (0.1 mL, each). The n-hexane fraction was subjected to gas chromatographic analysis. The sugar residues in compounds 1−5 were identified by comparison with the standard references: D-glucose (tR 10.49 min), L-glucose (tR 11.10 min), D-glucuronic acid (tR 9.21 min), L-glucuronic acid (tR 9.66 min), D-arabinose (tR 8.52 min), and Larabinose (tR 8.33 min).13 Assay for Testing the Protective Effects against Myocardial Cell Injury Induced by H2O2. H9c2 cells (1 ×105/well; Cell Bank of the Chinese Academy of Sciences Shanghai, China) were seeded into 96-well plates. After incubation with different concentrations of compounds 1−15 (12.5−200 μM) for 12 h, the cells were treated with 200 μM H2O2 for 24 h.14,15 The cells in the control groups were treated with the same volume of phosphate-buffered saline (PBS). Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which is based on the reduction of MTT by the mitochondrial dehydrogenase of intact cells to a purple formazan product. The quantity of formazan was determined by measuring the absorbance at 540 nm using an ELISA plate reader. The percentage cell viability was calculated as a ratio of the optical density (OD) value of the sample to the OD value of the control.16 All experiments were done three times. DPPH Radical Scavenging Capacity Assay. DPPH free radical scavenging activity of fractions and compounds was determined in 490
dx.doi.org/10.1021/jf4046667 | J. Agric. Food Chem. 2014, 62, 488−496
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Table 2. NMR Spectroscopic Data for Sugar Moieties of Compounds 1−5 (in C5D5N) 1 no. C3−O− Ara-1 2 3 4 5 Glc-1′ 2′ 3′ 4′ 5′ 6′ GlcA-1 2 3 4 5 6 methyl-1 C28−O− Glc-1 2 3 4 5 6 a
2
3
δC
δH
δC
δH
106.9 73.3 74.9 69.8 67.2
5.06d (7.0a) 4.49m 4.14m 4.32m 4.35m, 3.79m
104.9 81.1 73.5 68.4 65.0 106.1 76.5 78.3 71.7 78.3 62.7
5.03d (7.0) 4.66m 4.43m 4.44m 4.36m, 3.84m 5.26d (9.5) 4.16m 4.26m 4.39m 3.88m 4.50m
δC
106.6 75.6 78.0 73.3 77.4 171.0 52.1 95.9 74.2 79.1 71.1 79.4 62.2
6.43d (8.0) 4.29m 4.37m 4.46m 4.12m 4.62m
95.9 74.2 79.1 71.3 79.3 62.4
4 δH
5.31d (8.0) 4.15m 4.23m 4.52m 4.54m 3.77s
6.43d (10.0) 4.30m 4.37m 4.45m 4.12m 4.50m
δC
5 δH
106.5 75.6 78.0 73.3 77.4 170.9 52.1 96.0 74.2 79.1 71.4 79.3 62.5
5.31d (10.0) 4.16m 4.23m 4.53m 4.54m
δC
δH
106.9 73.9 74.4 69.3 67.2
5.28d (6.0) 4.66m 4.28m 4.39m 4.47m, 3.87d (11.5)
105.5 83.7 77.7 73.4 77.3 173.1
5.09d (5.5) 4.27m 4.43m 4.61m 4.62m
3.78s 6.39d (10.0) 4.30m 4.37m 4.44m 4.12m 4.54m, 4.48m
95.9 74.3 79.1 71.2 79.5 62.3
6.43d (12.5) 4.28m 4.35m 4.43m 4.11m 4.53m, 4.48m
Data in parentheses are J values (in hertz). Ara refers to arabinose, GlcA refers to gluconic acid, and Glc refers to glucose.
triplicate according to the method proposed by Hatano.17 In brief, 100 μL of tested fraction or compound at final concentrations (5−200 μM) in methanol was added to 100 μL of a 0.5 mM DPPH radical solution in methanol. The mixture was shaken vigorously and allowed to stand for 30 min in the dark. Vitamin E was considered as a positive control. A freshly prepared DPPH solution exhibits a deep purple color with absorption maximum at 517 nm with a spectrophotometer. The radical scavenging rate of free radical DPPH in percent (I%) was calculated in the following way:
Compound 1 was obtained as a white powder. The molecular formula of C41H66O14 was determined on the basis of its HR-ESI-MS [M + Na]+ ion peak at m/z 805.4303 (calcd 805.4350). The 1H NMR spectrum showed singlet resonances of five methyl groups at δ 1.72 (3H, s, Me-27), 1.45 (3H, s, Me29), 1.30 (3H, s, Me-26), 1.08 (3H, s, Me-25), 1.02 (3H, s, Me24) and one doublet methyl signal at δ 1.05 (3H, d, J = 7.5 Hz, Me-30), an olefinic proton signal at δ 5.58 (1H, m, H-12), and signals of a hydroxymethylene group at δ 4.34 (1H, m, H-23) and 3.78 (1H, d, J = 11.5 Hz, H-23), as well as a signal at δ 4.34 (1H, m, H-3). The 13C NMR data, the downfield shift of C-3, and the upfield shift of C-28 suggested the presence of a bisdesmosidic saponin aglycone. The 1H NMR data also indicated the presence of two sugar residues, which was inferred by the two anomeric proton signals at δ 5.06 (1H, d, J = 7.0 Hz, H-1 of arabinose) and δ 6.43 (1H, d, J = 8.0 Hz, H-1 of glucose), which correlated with δC 106.9 (C-1 of arabinose), 95.9 (C-1 of glucose), respectively, in the HSQC spectrum (Table 2). The types of sugar residues obtained from the acid hydrolysis of 1 were identified as L-arabinose and D-glucose by the GC analysis. The glycosidic side chain was located at C-28 of the aglycone, revealed by the HMBC correlation between δH 6.43 (H-1 of glucose) and δC 177.2 (C-28 of aglycone) (Figure 2). The β configuration of the glucopyranosyl group was confirmed on the basis of the large 3JH‑1,H‑2 coupling constant. The location of another glycosidic unit, α-L-arabinopyranosyl, was determined to be attached at C-3, evidenced by the HMBC correlation between δH 4.34 (H-3 of aglycone) and δC 106.9 (C-1 of arabinose), as well as correlation between δH 5.06 (H-1 of arabinose) and δC 82.0 (C-3 of aglycone). The configuration of the arabinopyranosyl group was assigned as α on the basis of
I % = (100 × [1 − (A sample − A blank )/Acontrol ] Acontrol is the absorbance of the control reaction (containing all reagents except the test compound), Asample is the absorbance of the test compound, and Ablank is the absorbance of blank sample. Statistical Analysis. Data were expressed as the mean ± SD and evaluated using one-way ANOVA. The post hoc test was done with Student’s Dunnett test. p < 0.05 was considered statistically significant. Calculations were performed using the SPSS 16.0 statistical package.
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RESULTS AND DISCUSSION The 50% EtOH extract of the aerial parts of I. cornuta was separated by repeated D101 resin CC and ODS CC to afford 5 new triterpenoid saponins, 1−5, as well as 10 known ones, which were identified as 19α-hydroxyurs-12-en-28-oic acid 3βO-α-L-arabinopyranosyl-(1→2)-β-D-glucuronopyranoside-6-Omethylester, 6;18 siaresinolic acid, 7;19 hederagenin-28-β-Dglucopyranoside, 8;20 hederacolchiside A, 9;21 ilexolic acid A, 10;22 3β,23-dihydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester, 11;23 cynarasaponin E, 12;24 cynarasaponin E methyl ester, 13;24 mateside, 14;25 and ilekudinoside D, 15,26 (Figure 1). 491
dx.doi.org/10.1021/jf4046667 | J. Agric. Food Chem. 2014, 62, 488−496
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Figure 2. Key HMBC correlations for 1−5.
the NOESY correlations between δ 5.06 (H-1 of arabinose) and δ 4.14 (H-3 of arabinose), 4.32 (H-4 of arabinose) (Figure 3). The 1H and 13C NMR spectral data of 1 were similar to those of 3β-O-α-L-arabinopyranosyl-19α,23-dihydroxyurs-12-en-28oic acid 28-O-β-D-glucopyranosyl ester,26 but showed differences for C-18 and C-22. Significant upfield shifts of the C-18 signal (−7.2 ppm) at δ 47.3 and C-22 signal (−5.9 ppm) at δ 32.0 were observed for compound 1, which could be explained as the results of the γ-effect of the 30-β (axial) methyl group instead of a 30-a (equatorial) methyl group in the 3β,23dihydroxyurs-12-en-28-oic acid moiety.27 The β orientation of Me-30 was also confirmed by the NOESY correlations of Me30 (δH 1.05) to both Me-29 (δH 1.45) and H-18 (δH 3.26) (Figure 3). The orientation of H-20 was determined to be α on the basis of the NOE information. Thus, compound 1 was identified as 3β-O-α-L-arabinopyranosyl-19α,23-dihydroxy-20αurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester. Compound 2 was obtained as a white powder. The molecular formula was determined as C47H76O18 on the basis of its HR-ESI-MS [M + Na]+ ion peak at m/z 951.4887 (calcd
951.4929). The 1H NMR spectrum showed singlet resonances of six tertiary methyl groups at δ 1.78 (3H, s, Me-27), 1.47 (3H, s, Me-29), 1.28 (3H, s, Me-26), 1.28 (3H, s, Me-23), 1.13 (3H, s, Me-24), and 0.99 (3H, s, Me-25), a doublet methyl signal at δ 1.07 (3H, d, J = 10.0 Hz, Me-30), and an olefinic proton signal at δ 5.59 (1H, m, H-12), as well as a signal at δ 3.27 (1H, dd, J = 15.0, 5.0 Hz, H-3). The 13C NMR data and the downfield shift of C-3 and the upfield shift of C-28 indicated the presence of a bisdesmosidic saponin aglycone. The 1 H NMR spectroscopic data also indicated the presence of three sugar residues, which was inferred by three anomeric proton signals at δ 5.03 (1H, d, J = 7.0 Hz, H-1 of arabinose), δ 5.26 (1H, d, J = 9.5 Hz, H-1′ of glucose 1′), and δ 6.43 (1H, d, J = 10.0 Hz, H-1 of glucose 1), correlated with δC 104.9 (C-1 of arabinose), 106.1 (C-1′ of glucose 1′), and 95.9 (C-1 of glucose 1), respectively, in the HSQC spectrum (Table 2). The sugar residues were identified as L-arabinose and D-glucose by the GC analysis. The 1H and 13C NMR data of 2 were similar to those of brevicuspisaponin 328 except for the presence of additional signals attributed to one glucose moiety. The location of this 492
dx.doi.org/10.1021/jf4046667 | J. Agric. Food Chem. 2014, 62, 488−496
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Figure 3. Key NOESY correlations for 1−5.
glucose residue was determined at C-2 of the arabinose moiety, inferred by the downfield shift of C-2 of arabinose and HMBC correlation between δH 5.26 (H-1′ of glucose 1′) and δC 81.1 (C-2 of arabinose). Thus, the structure of compound 2 was established as 3β-O-β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl-19-hydroxy-20α-urs-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester. Compound 3 was obtained as a white powder. The negative HR-ESI-MS of 3 showed a quasi-molecular ion peak at m/z 677.3869 ([M − H]−, calcd 677.3901), indicating the molecular formula of C37H58O11. The 1H NMR spectrum showing singlet resonances of five tertiary methyl groups at δ 1.77 (3H, s, Me27), 1.50 (3H, s, Me-29), 1.17 (3H, s, Me-26), 1.01 (3H, s, Me24), 0.98 (3H, s, Me-25), a doublet methyl signal at δ 1.19 (3H, d, J = 6.5 Hz) corresponding to Me-30, an olefinic proton at δ 5.66 (1H, m, H-12), and hydroxymethylene group signals at δ 4.39, 3.78 (2H, m, H-23), as well as a proton signal at δ 4.36 (1H, m, H-3), suggested it contained an aglycone as 19α,23dihydroxyurs-12-en-28-oic acid.29 An anomeric proton was observed at δH 5.31 (1H, d, J = 8.0 Hz, H-1 of glucuronic acid), which showed HSQC correlation to the anomeric carbon at δC
106.6 (C-1 of glucuronic acid), indicating the presence of a sugar unit in the structure of compound 3. The sugar residue obtained from the acid hydrolysis of 3 was identified as Dglucuronic acid by the GC analysis. The 1H and 13C NMR data of 3 were very similar to those of ilexasoside A,30 except that it had an additional hydroxyl group. The downfield shift of C-23 signal (+36.3 ppm) at δC 64.4 suggested that the additional hydroxyl group was located at C-23, which was confirmed by the HMBC correlations of C-23 (δC 64.4) to Me-24 (δH 1.01) and H-3 (δH 4.36). Thus, compound 3 was identified as 19α,23-dihydroxyurs-12-en-28-oic acid 3β-O-β-D-glucuronopyranoside-6-O-methyl ester. Compound 4 had a molecular formula of C43H68O16, which showed a quasi-molecular ion peak ([M + Na]+) at m/z 863.4405 (calcd 863.4405). The signals of two anomeric protons at δH 5.31 (1H, d, J = 10.0 Hz, H-1 of glucuronic acid) and 6.39 (1H, d, J = 10.0 Hz, H-1 of glucose), which correlated to the two anomeric carbon signals at δC 106.5 (C-1 of glucuronic acid) and δC 96.0 (C-1 of glucose), respectively, in the HSQC spectrum, suggested the presence of two sugar residues. The sugar residues were identified as D-glucuronic acid 493
dx.doi.org/10.1021/jf4046667 | J. Agric. Food Chem. 2014, 62, 488−496
Journal of Agricultural and Food Chemistry
Article
Table 3. Protective Effects of Triterpenoidal Saponins from Ilex cornuta on H2O2-Induced H9c2 Cell Injury concentrationa compd 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 vitamin Eb a
0 μM 69.78 63.25 65.10 63.25 63.25 69.78 63.82 70.28 69.70 68.74 69.22 59.85 69.70 69.78 70.28 64.36
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.82 1.25 2.45 1.25 1.25 2.82 3.94 2.78 5.48 3.86 4.18 1.18 5.48 2.82 2.78 2.18
12.5 μM 65.33 54.41 68.91 65.86 60.38 74.39 68.33 67.74 63.06 63.53 63.54 62.76 70.06 66.47 68.99 65.39
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
25 μM
1.65 1.80 5.99 1.17 2.52 3.06 6.45 4.92 5.86 2.35 3.63 4.83 3.26 5.50 4.15 2.33
73.38 62.85 67.95 74.42 61.04 78.01 68.84 62.27 67.55 69.75 65.74 69.02 75.40 69.88 67.81 67.25
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.27 2.64 2.39 1.39** 1.53 4.58 7.13 2.43 6.95 2.98 5.28 3.07 2.42 3.22 4.91 1.94
50 μM 77.78 65.22 65.53 80.79 68.28 82.00 64.70 63.87 60.61 69.80 63.86 68.27 83.23 65.65 66.94 68.17
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
100 μM
0.43* 2.10 3.55 2.61** 3.44 5.59 1.50 3.44 0.82 1.63 5.12 4.52 3.95 4.72 1.17 2.06
79.22 62.02 65.70 86.54 62.57 85.44 62.93 64.21 67.87 65.95 71.37 60.47 88.42 65.79 68.12 75.84
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.55** 2.89 3.72 3.76** 0.28 1.08* 4.39 4.95 1.21 4.52 0.49 2.98 4.71 5.69 3.42 1.88*
200 μM 83.84 62.61 63.29 91.29 65.46 87.77 69.19 64.27 66.99 62.54 72.32 66.21 89.34 67.61 69.95 81.49
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.44** 1.06 4.08 4.60** 3.62 3.02* 5.40 2.75 1.54 3.10 2.91 5.90 5.86* 6.81 1.01 1.96**
*, p < 0.05, and **, p < 0.01, vs 0 μM group. bPositive control.
arabinose, D-glucuronic acid, and D-glucose by GC analysis. The arabinose group was located at the C-2 of the glucuronic acid unit, evidenced by the HMBC correlation between δH 5.28 (H1 of arabinose) and δC 83.7 (C-2 of glucuronic acid). The β anomeric configuration of the glucopyranosyl unit was established on the basis of the observation of the large 3 JH‑1, H‑2 coupling constant. The anomeric configuration of the arabinopyranosyl unit was assigned to be α, deduced from the NOESY correlations among the signals at δH 5.28 (H-1 of arabinose), δH 4.28 (H-3 of arabinose), and δH 4.39 (H-4 of arabinose) (Figure 3). Therefore, compound 5 was identified as 3β-O-[α-L-arabinopyranosyl-(1→2)-β-D-glucuronic acid]-oleanolic acid 28-O-β-D-glucopyranosyl ester. Protective Effects of the Isolates against H2O2Induced Myocardial Cell Injury. Cardiovascular diseases may be caused by intracellular oxidative damage to biomolecules via reactive oxygen species (ROS).32 A number of studies showed that antioxidants reduced oxidative stressmediated cellular injury.33 Among ROS, H2O2 readily crosses cellular membranes and gives rise to the highly reactive hydroxyl radical, which is the most reactive and induces severe damage to adjacent biomolecules.34 The H2O2-mediated oxidative injury resulted in a significant decrease of mitochondrial respiration, glutathione, and protein thiol content and in an increased level of GSSG.35 H2O2 has been used frequently in models of oxidative stress in H9c2 cardiomyocytes to investigate protective function.36,37 The I. cornuta extract was reported to have the protective effect on myocardial ischemia.38 In this study, the protective effects of compounds 1−15 against H2O2-induced myocardial cell injury were tested. As shown in Table 3, compounds 1, 4, 6, and 13 (12.5−200 μM) significantly increased the viability of H9c2 induced by H2O2 in a dose-dependent manner, ranging from the concentrations at 12.5 to 200 μM. Compound 4 was shown to be the most efficient among the isolates. It increased the cell viability at a low concentration of 25 μM and was more efficient than vitamin E, which was used as the positive control. Compounds 1, 6, and 13 showed moderate protective effects. They significantly improved cell viability at a concentration of 200 μM. Previous studies have indicated that vitamin E inhibits peroxidative damage associated with ischemia and reperfu-
Table 4. Maximal Scavenging Rate of Compounds 1−15 against DPPH in Vitro compda 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 vitamin E
scavenging rate (200 μM) 21.06 11.30 8.49 17.62 15.34 28.85 18.32 16.94 20.53 8.13 9.72 10.03 21.43 23.58 11.86 92.74
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.98 0.34 2.53 1.19 3.29 2.13 2.42 0.53 0.38 3.64 1.23 5.11 2.73 3.18 3.32 0.70
a For compounds 1−15, the test concentrations ranged from 5 to 200 μM; for vitamin E, the test concentration was 200 μM.
and D-glucose by the hydrolysis and GC analysis. The 1H and 13 C NMR data of 4 were similar to those of 3 except for the presence of the additional signals corresponding to one glucose group. This glucose unit was attached to C-28 of the aglycone, evidenced by the downfield shift of C-28 and the HMBC correlation between δH 6.39 (H-1 of glucose) and δC 177.1 (C28 of aglycone) (Figure 2). Both the glucopyranosyl and glucuronopyranosyl units existed in the form of β anomeric configuration, based on the observation of the large 3JH‑1, H‑2 coupling constants. Thus, compound 4 was identified as 19α,23-dihydroxyurs-12-en-28-oic acid 3β-O-[β-D-glucuronopyranoside-6-O-methyl ester]-28-O-β-D-glucopyranosyl ester. Compound 5 had a molecular formula of C47H74O18 on the basis of its HR-ESI-MS [M + Na]+ ion peak at m/z 949.4729 (calcd 949.4772). The 1H and 13C NMR data of 5 were similar to those of silphioside G31 except for the presence of additional signals corresponding to one arabinose group. Compound 5 contained three sugar residues, which were identified as L494
dx.doi.org/10.1021/jf4046667 | J. Agric. Food Chem. 2014, 62, 488−496
Journal of Agricultural and Food Chemistry
Article
sion.39,40 The triterpenoid saponins contained in I. cornuta might play a similar preventive role. Further studies are warranted to explore their potential. DPPH Radical Scavenging Capacity Assay. The antioxidant activity bioassay of compounds 1−15 was tested by DPPH radical scavenging capacity assay. The maximal scavenging rates of compounds 1−15 are shown in Table 4; the scavenging rates of compounds 1−15 were
