Article pubs.acs.org/JAFC
Flavonoids from the Pericarps of Litchi chinensis Qing Ma,‡,§ Haihui Xie,*,‡ Sha Li,† Ruifen Zhang,† Mingwei Zhang,† and Xiaoyi Wei‡ †
Sericulture and Agri-food Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510610, China Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China § University of Chinese Academy of Sciences, Beijing 100049, China ‡
S Supporting Information *
ABSTRACT: A new methylene-linked flavan-3-ol dimer, bis(8-epicatechinyl)methane (1), was isolated from the pericarps of Litchi chinensis Sonn. (Sapindaceae), together with dehydrodiepicatechin A (2), proanthocyanidin A1 (3), proanthocyanidin A2 (4), (−)-epicatechin (5), 8-(2-pyrrolidinone-5-yl)-(−)-epicatechin (6), (−)-epicatechin 8-C-β-D-glucopyranoside (7), naringenin 7-O-(2,6-di-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside (8), and rutin (9). It was the first report of compound 2 as a natural product and compounds 6−8 from this species. Compounds 1, 2, and 6−8 were evaluated for antioxidant activity. The ferric reducing antioxidant powers (FRAP) of compounds 1 and 6 were comparable to that of L-ascorbic acid, and the scavenging activities of compounds 1, 2, 6, and 7 toward 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and 2,2′-azinobis(3ethylbenzthiazoline-6-sulfonic acid) radical cations were more potent than those of L-ascorbic acid; compound 8 was weak in FRAP and DPPH assays. KEYWORDS: Litchi chinensis, pericarp, flavonoids, bis(8-epicatechinyl)methane, dehydrodiepicatechin A, antioxidant activity
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INTRODUCTION Litchi chinensis Sonn., an evergreen tree belonging to the Sapindaceae family, has been widely cultivated for fruit in tropical and subtropical areas of the world. Litchi fruit is popular in domestic and foreign markets because of its juicy and sweet aril and attractive red pericarp. The annual output of fresh fruit in China exceeds 1300 million kg, and the pericarp accounts for approximately 15% of the whole fruit in fresh weight.1 Litchi aril is consumed fresh or as processed products, while the pericarp is discarded as waste except that a small amount is used as a traditional medicine with hemostatic and antidysenteric functions.2 Phytochemical investigation of the pericarp is necessary to utilize it as a resource. Previous research on litchi pericarp was mainly focused on the characterization of phenolic constituents by thin layer chromatography (TLC) or high-performance liquid chromatography (HPLC) analyses, which indicated the presence of cyanidin 3,5-diglucoside, cyanidin 3-glucoside, cyanidin 3rutinoside, cyanidin 3-galactoside, pelargonidin 3,7-diglucoside, and malvidin 3-glucoside.3,4 However, with respect to the phenolics that were isolated from litchi pericarp in a pure state and structurally identified by spectroscopic methods, including nuclear magnetic resonance (NMR), there were only a few reports. The obtained compounds included rutin, cyanidin 3rutinoside, epicatechin, proanthocyanidins A2, B2, and B4, and epicatechin-(4β→8,2β→O→7)-epicatechin-(4β→8)-epicatechin.1,3,4 To identify phenolic constituents in litchi pericarp, further chemical investigation was conducted, and consequently, four dimers and three monomers of flavan-3-ol and the glycosides of a flavanone and a flavonol (Figure 1) were obtained. This study was undertaken to isolate and elucidate the structures of these flavonoids and determine their antioxidant activity. © 2014 American Chemical Society
MATERIALS AND METHODS
Chemicals and Reagents. 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), DPPH, and 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma-Aldrich (St. Louis, MO). L-Ascorbic acid was obtained from Shanghai Boao Biotech Co. (Shanghai, China). Silica gel (100−200 or 200−300 mesh) was purchased from Qingdao Haiyang Chemical Co. (Qingdao, China). Sephadex LH-20 was obtained from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). MCI gel CHP20P (75−150 μm) was obtained from Mitsubishi Chemical Corp. (Tokyo, Japan). Apparatus. Medium-pressure liquid chromatography (MPLC) was performed on an EZ Purifier (Lisure Science, Suzhou, China), and a 400 mm × 25 mm (inside diameter) (Shanghai Lisui E-Tech Co., Shanghai, China), 20−45 μm, Chromatorex RP-18 SMB100 column was used (Fuji Silysia Chemical, Kasugai, Japan). HPLC was conducted on a LC-6AD pump (Shimadzu, Kyoto, Japan) connected to a RID-10A refractive index detector (Shimadzu); a 250 mm × 4.6 mm (inside diameter) column was used for analysis, and a 250 mm × 20 mm (inside diameter), 5 μm, YMC-pack ODS-A column was used for preparation along with a 23 mm × 4 mm (inside diameter) guard column of the same material (YMC, Kyoto, Japan). 1H and 13C NMR spectra in addition to distortionless enhancement by polarization transfer (DEPT), 1H−1H correlated spectroscopy (COSY), heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC) were recorded on a Bruker DRX-400 instrument (Bruker BioSpin Gmbh, Rheinstetten, Germany) in CD3OD with residual peaks at δH 3.31 and δC 49.0 as references. Electrospray ionization mass spectrometry (ESI-MS) was conducted on a MDS SCIEX API 2000 LC-MS/MS apparatus (Applied Biosystems Inc., Foster City, CA). High-resolution (HR) ESI-MS was conducted on a Bruker maXis mass spectrometer (Bruker Received: Revised: Accepted: Published: 1073
October 6, 2013 January 20, 2014 January 21, 2014 January 21, 2014 dx.doi.org/10.1021/jf405750p | J. Agric. Food Chem. 2014, 62, 1073−1078
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Figure 1. Structures of compounds 1−9 from the pericarps of L. chinensis.
Table 1. 1H and 13C NMR Data of Compounds 1 and 6 in CD3OD 1 C/H 2 3 4 5 6 7 8 9 10 11 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″
δH (multiplet, J in Hz) 4.76 4.10 2.83 2.69
(2H, (2H, (2H, (2H,
br s) br dd, 4.7, 3.7) dd, 16.6, 4.7) dd, 16.6, 3.7)
5.96 (2H, s)
3.88 (2H, s) 6.96 (2H, br s)
6.73 (2H, br s) 6.73 (2H, br s)
6 δC (DEPT) 80.4 (CH) 67.2 (CH) 29.0 (CH2) 155.8 96.8 155.2 106.7 153.6 100.5 16.8 131.7 115.4 145.9 145.9 116.0 119.7
(C) (CH) (C) (C) (C) (C) (CH2) (C) (CH) (C) (C) (CH) (CH)
3″ 4″
δH (multiplet, J in Hz) 4.84 4.17 2.87 2.77
(br s) (ddd, 4.5, 3.0, 1.0) (dd, 16.7, 4.5) (ddd, 16.7, 3.0, 1.0)
6.00 (s)
6.98 (d, 2.0)
6.76 6.80 5.42 2.45 2.38 2.53 2.30
(d, 8.2) (dd, 8.2, 2.0) (dd, 9.2, 5.3) (m) (m) (m) (m)
δC 80.1 66.9 29.4 157.2 96.4 156.3 107.7 155.5 100.0 132.2 115.3 145.9 146.0 115.9 119.5 50.3 26.7 32.2 181.3
to afford ethyl acetate-soluble (109.35 g) and n-butanol-soluble (79.30 g) fractions after being taken to dryness under reduced pressure. The ethyl acetate-soluble fraction (107.35 g) was subjected to silica gel (2330 g, 100−200 mesh) column [545 mm × 120 mm (inside diameter)] chromatography and eluted with a chloroform (CHCl3)/ methanol (MeOH) mixture (v/v, 10:0, 38 L → 98:2, 24 L → 95:5, 19 L → 9:1, 33 L → 85:15, 38 L → 8:2, 25 L → 7:3, 25 L → 6:4, 9 L → 0:10, 9 L) to furnish fractions E1−E15 after they had been pooled according to their TLC profiles. Fraction E10 (10.7 g) was further subjected to silica gel (440 g, 200−300 mesh) column [720 mm × 44 mm (inside diameter)] chromatography and eluted with a CHCl3/ MeOH mixture (v/v, 95:5, 2.75 L → 9:1, 8.75 L → 85:15, 6.25 L) to yield fractions E10-1−E10-4. Fraction E10-4 was separated by MPLC and eluted with a MeOH/H2O mixture (v/v, 2:8, 3:7, 4:6, 5:5, 6:4, and 7:3, each 2.4 L) at a flow rate of 10 mL/min to produce fractions E104-1−10-4-4. Fractions E10-4-1 and E10-4-2 were individually separated by silica gel (200−300 mesh) column chromatography to afford compounds 1 (15 mg) and 5 (11 mg), respectively. Fraction
Daltonics Inc., Bremen, Germany). Optical rotation was acquired on a Perkin-Elmer (Waltham, MA) 343 polarimeter. Ultraviolet (UV) radiation was recorded on a Perkin-Elmer Lambda 650 UV−vis spectrophotometer (Perkin-Elmer). Infrared (IR) radiation was obtained on a WQF-520 FT-IR spectrometer (Beijing Rayleigh Analytic Instrument Co., Beijing, China). Plant Material. Fresh ripe litchi fruits, cv. ‘Heiye’, were collected from the Fruit Tree Research Institute, Guangdong Academy of Agricultural Sciences (Guangzhou, China) in June 2012, and the pericarps were manually separated from the fruits. Extraction and Isolation. The fresh pericarps (5.0 kg) were immersed in 85% aqueous ethanol, chopped with a multifood processor, transferred to flasks, and then heated at 50 °C for 2 h. Twenty liters of 85% aqueous ethanol was used each time, and the extraction was repeated three times. The solution was filtered and evaporated under vacuum to give an ethanolic extract (320 g). The extract (315 g) was dissolved in 2.0 L of water and then sequentially fractionated with ethyl acetate (1.5 L × 5) and n-butanol (1.2 L × 7) 1074
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Table 2. 1H and 13C NMR Data of Compound 2 in CD3OD C/H 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′
δH (multiplet, J in Hz) 3.82 4.45 2.83 2.65
(br s) (br d, 4.8) (br d, 16.5) (dd, 16.5, 4.8)
5.92 (d, 2.0) 5.94 (d, 2.0)
2.94 (d, 15.3) 2.83 (d, 15.3)
6.55 (s)
δC
C/H
72.9 65.1 25.0
2″ 3″ 4″
157.5 96.6 157.7 95.5 155.6 98.9 91.8 40.5
5″ 6″ 7″ 8″ 9″ 10″ 1‴ 2‴
95.5 193.8 113.6 164.7
3‴ 4‴ 5‴ 6‴
E10-4-3 was purified by preparative HPLC using a MeOH/H2O mixture (46:54, v/v) as the mobile phase at a flow rate of 5 mL/min to give compound 2 [retention time (tR) of 53.4 min, 25 mg]. Fraction E11 (13.88 g) was passed through a MCI gel column [259 mm × 26 mm (inside diameter)] for depigmentation, and the obtained MeOH eluate (7.20 g) was separated by MPLC and purified by preparative HPLC using an acetonitrile/H2O/acetic acid (AcOH) mixture (15:85:0.1, v/v/v) as the mobile phase at a flow rate of 5 mL/min to yield compounds 3 (tR of 60.7 min, 32 mg) and 4 (tR of 90.5 min, 30 mg). The n-butanol-soluble fraction (79.3 g) was subjected to silica gel (1310 g, 100−200 mesh) column [800 mm × 75 mm (inside diameter)] chromatography and eluted with a CHCl3/MeOH mixture (v/v, 9:1, 5.6 L → 85:15, 18.2 L → 8:2, 14.7 L → 7:3, 10.5 L → 0:10, 5.3 L) to furnish fractions B1−B7. Fraction B4 (12.4 g) was separated by silica gel (200−300 mesh) column chromatography, MPLC, and Sephadex LH-20 column [1215 mm × 14 mm (inside diameter)] chromatography that was eluted with MeOH to afford compound 6 (8 mg). Fraction B6 (2.8 g) was separated by MPLC to give fractions B61−B6-7. Fraction B6-1 was further separated by Sephadex LH-20 column chromatography and purified by preparative HPLC using a MeOH/H2O mixture (13:87, v/v) as the mobile phase at a flow rate of 5 mL/min to yield compound 7 (tR of 95.4 min, 4 mg). Fraction B6-6 was separated by Sephadex LH-20 column chromatography and purified by preparative HPLC using a MeOH/H2O/AcOH mixture (42:58:0.1, v/v/v) as the mobile phase at a flow rate of 5 mL/min to obtain compounds 8 (tR of 46.2 min, 15 mg) and 9 (tR of 44.5 min, 22 mg). Bis(8-epicatechinyl)methane (1). Off-white amorphous powder: [α]D20 −103.7 (c 0.138, MeOH); UV (MeOH) λmax (nm) (log ε) 281 (3.65); IR (KBr) νmax 3377, 1618, 1524, 1454, 1284, 1113 cm−1; HRESI-MS positive m/z 593.1651 [M + H]+ (calcd for C31H29O12+, 593.1654, error of 0.5 ppm), 615.1474 [M + Na]+ (calcd for C31H28O12Na+, 615.1473, error of −0.1 ppm); ESI-MS positive m/z 593 [M + H]+, 615 [M + Na]+, negative m/z 591 [M − H]−; 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) data in Table 1. Dehydrodiepicatechin A (2). Yellowish amorphous powder: [α]D20 373.8 (c 0.064, MeOH); ESI-MS positive m/z 577 [M + H]+, 599 [M + Na]+, negative m/z 575 [M − H]−; 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) data in Table 2. 8-(2-Pyrrolidinone-5-yl)-(−)-epicatechin (6). White amorphous powder: [α]D20 −14.5 (c 0.345, MeOH); ESI-MS positive m/z 374 [M + H]+, 396 [M + Na]+, negative m/z 372 [M − H]−, 408 [M + Cl]−; 1 H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) data in Table 1. FRAP Assay. Ferric reducing ability of the compounds was determined following the procedures as previously described.5 FRAP
δH (multiplet, J in Hz) 5.03 4.30 2.89 2.83
(br s) (br d, 3.9) (dd, 16.5, 3.9) (br d, 16.5)
δC 80.8 66.4 29.5
7.04 (br s)
166.5 91.7 167.0 104.7 156.2 103.6 131.2 115.1
6.81 (d, 8.0) 6.86 (br d, 8.0)
146.1 146.2 116.1 119.2
6.17 (s)
reagent was made freshly by mixing 300 mM acetate buffer (pH 3.6), a 10 mM TPTZ solution in 40 mM hydrochloric acid, and a 20 mM aqueous ferric chloride (FeCl3) solution in a 10:1:1 (v/v/v) ratio. The TPTZ solution was prepared on the same day. Test compounds were dissolved in methanol and diluted 2-fold to six concentrations. Twenty microliters of the compound solution and 180 μL of FRAP reagent were mixed in 96-well plates. L-Ascorbic acid was dissolved in methanol and used as a positive reference. Each treatment was conducted in quadruplicate. The plates were incubated at 37 °C for 30 min in the dark. The absorbance of the product (ferrous TPTZ complex) in each well was read at 595 nm using a Genios microplate reader (Tecan Group, Männedorf, Switzerland). One milliliter of ferrous sulfate (FeSO4) at six different concentrations and 1 mL of 10 mM TPTZ and 10 mL of 300 mM acetate buffer (pH 3.6) were used for a calibration curve. FRAP values were calculated and expressed as means ± the standard deviation (SD) in millimoles of Fe(II) per gram. DPPH Radical Scavenging Assay. The DPPH radical scavenging ability of the compounds was evaluated according to the method as previously described.5 DPPH was freshly prepared in methanol at a concentration of 0.1 mM. Test compounds were dissolved in methanol and diluted 2-fold to six concentrations. Twenty microliters of the compound solution and 180 μL of the DPPH solution were mixed in 96-well plates. L-Ascorbic acid was dissolved in methanol and used as a positive reference. The control contained methanol instead of the compound solution, and the blank contained methanol in place of the DPPH solution. Each treatment was conducted in quadruplicate. The plates were incubated at 37 °C for 30 min in the dark. The absorbance (OD) in each well was read at 515 nm on a Genios microplate reader (Tecan). The inhibitory rates of DPPH radicals were calculated according to the formula
inhibition (%) = [1 − (ODtreated − ODblank )/ODcontrol ] × 100 SC50 values (the concentrations required to scavenge 50% DPPH radicals present in the test solution) were calculated and expressed as means ± SD in micromolar. ABTS Radical Cation Decolorization Assay. The ABTS radical cation (ABTS•+) scavenging activity of the compounds was determined according to the method described previously.6,7 ABTS was dissolved in deionized water to a concentration of 7 mM. ABTS•+ was produced by reacting the ABTS solution with 2.45 mM (final concentration) potassium persulfate, and the mixture was kept at room temperature for 12−16 h in the dark before being used. The ABTS•+ solution was diluted with phosphate-buffered saline (PBS, pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm. Test compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted to 400, 200, 100, 50, 25, and 12.5 μg/mL. Ten microliters of the compound solution and 200 μL of the diluted ABTS•+ solution were mixed in 96-well 1075
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plates. L-Ascorbic acid was used as a positive reference. The control contained DMSO instead of the compound solution. Each treatment was conducted in triplicate. After a mixing time of 10 s and an incubation period of 6 min at 37 °C in the dark, the absorbance (OD) in each well was read at 415 nm on a Varioskan Flash microplate reader (Thermo Scientific). The inhibitory rates of ABTS•+ were calculated according to the formula
compounds were shown by coupling constants J2,3 and J4,3, which were 7.5 Hz and 8.8 or 5.4 Hz, respectively,8,10 rather different from those of compound 1. Compound 2 was deduced to have a molecular weight of 576 from the ESI-MS positive and negative ion peaks at m/z 577 [M + H]+, 599 [M + Na]+, and 575 [M − H]−. The 1H NMR (Table 2) spectrum exhibited three ABX-type aromatic protons at δ 7.04 (1H, br s, H-2‴), 6.81 (1H, d, J = 8.0 Hz, H-5‴), and 6.86 (1H, br d, J = 8.0 Hz, H-6‴), two meta-coupled aromatic protons at δ 5.92 (1H, d, J = 2.0 Hz, H-6) and 5.94 (1H, d, J = 2.0 Hz, H-8), two singlet aromatic protons at δ 6.55 (1H, H-5′) and 6.17 (1H, H-6″), four protons at δ 5.03 (1H, br s, H-2″), 4.45 (1H, br d, J = 4.8 Hz, H-3), 4.30 (1H, br d, J = 3.9 Hz, H3″), and 3.82 (1H, br s, H-2) connected to oxygenated carbons, and six aliphatic protons from δ 2.65 to 2.94. The 13C NMR (Table 2) spectrum displayed signals of a total of 30 carbons, including a carboxyl carbon at δ 193.8 (C-4′), four oxygenated carbons at δ 80.8 (C-2″), 72.9 (C-2), 66.4 (C-3″), and 65.1 (C3), and three aliphatic carbons at δ 40.5 (C-2′), 29.5 (C-4″), and 25.0 (C-4). Careful analysis of the HMBC spectrum (Figure 3) such as the correlations from H-2 to C-2′ and C-6′
inhibition (%) = (1 − ODtreated /ODcontrol ) × 100 IC50 values were calculated and expressed as means ± SD in micromolar.
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RESULTS AND DISCUSSION Compound 1 was obtained as an off-white amorphous powder and deduced to have the molecular formula C31H28O12 from the positive ESI-MS ion peaks at m/z 593 [M + H]+ and 615 [M + Na]+ as well as their HR-ESI-MS data. The IR spectrum displayed absorptions of the hydroxyl group at 3377 cm−1 and the phenyl moiety at 1618, 1523, and 1454 cm−1. The 1H NMR (Table 1) spectrum demonstrated eight aromatic protons at δ 6.73 (4H, br s), 6.96 (2H, br s), and 5.96 (2H, s), four protons at δ 4.76 (2H, br s, H-2) and 4.10 (2H, br dd, J = 4.7, 3.7 Hz, H-3) connected to oxygenated carbons, and four aliphatic protons at δ 2.83 (2H, dd, J = 16.6, 4.7 Hz, H-4) and 2.69 (2H, dd, J = 16.6, 3.7 Hz, H-4) that can be attributed to two methylenes. These signals suggested the presence of two epicatechinyl moieties in a symmetric state.5 Moreover, the 1H NMR spectrum exhibited a signal of two protons at δ 3.88 (s, H2-11) that were readily recognized for an additional methylene. The 13C NMR and DEPT (Table 1) spectra showed signals of 31 carbons, including a methylene carbon at δ 16.8 (C-11), among which the carbons at the same positions of two symmetric epicatechinyl moieties were overlapped. With the aid of the HSQC spectrum, all the protons were assigned to their corresponding carbons. In the HMBC spectrum (Figure 2), the correlations from H2-11 to δ 155.2 (C-7), 106.7 (C-8),
Figure 3. HMBC spectrum of compound 2.
(δ 164.7), H-3 to C-3′ (δ 95.5), H-5′ to C-1′ (δ 91.8), C-3′, and C-8″ (δ 104.7), and H-6″ to C-8″ led us to elucidate it as dehydrodiepicatechin A. This compound was originally obtained from (−)-epicatechin by biotransformation with crude extracellular enzymes of wood rotting fungi, Coriolus versicolor and Pycnoporus coccineus,11 and later from tea epicatechin by oxidation with peroxyl radicals generated by thermolysis of the initiator azo-bisisobutyrylnitrile.12 This was the first report of it as a natural product. Compound 6 was deduced to have a molecular weight of 373 from the ESI-MS ion peaks of m/z 374 [M + H]+, 396 [M + Na]+, 372 [M − H]−, and 408 [M + Cl]−, which indicated the presence of an odd-numbered nitrogen atom. The 1H and 13C NMR spectra showed signals readily assigned for an epicatechinyl moiety as compound 1 (Table 1). In the HMBC spectrum, the correlations from δ 5.42 (1H, dd, J = 9.2, 5.3 Hz, H-1″) to δ 156.3 (C-7), 107.7 (C-8), 155.5 (C-9), and 181.3 (C-4″), δ 2.45 and 2.38 (each 1H, m, H2-2″) to C-8 and C-4″, and δ 4.84 (1H, br s, H-2) to C-9 were observed, which suggested the presence of a pyrrolidin-2-one moiety13 and its connection to C-8. Hence, compound 6 was identified as 8-(2-pyrrolidinone-5-yl)-(−)-epicatechin. Jang et al.14 obtained a 2:1 mixture of two diastereomers of this compound
Figure 2. HMBC spectrum of compound 1.
and 153.6 (C-9), δ 4.76 (H-2) to C-9 and C-4 (δ 29.0), and δ 2.83 and 2.69 (H2-4) to C-9 and C-5 (δ 155.8) ascertained the connection of the methylene to C-8 of two epicatechinyl moieties. Consequently, compound 1 was determined to be bis(8-epicatechinyl)methane. Bis(8-catechinyl)methane was previously obtained from several plants such as Rhopalocnemis phalloides (Balanophoraceae)8 and Baccaurea ramif lora (Euphorbiaceae)9 and synthesized by hydrogenation of substituted aromatic aldehyde.10 The differences between these two 1076
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from the roots of Actinidia arguta for the first time, which showed two sets of 1H and 13C NMR data. However, compound 6 exhibited only one set of 1H and 13C NMR data, which were in good agreement with those of the major isomer. It was the first report of this compound as a single stereoisomer. The other known compounds were identified as proanthocyanidin A1 (3),15 proanthocyanidin A2 (4),15 (−)-epicatechin (5),16 (−)-epicatechin 8-C-β-D-glucopyranoside (7),17 naringenin 7-O-(2,6-di-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside (8),18 and rutin (9)19 by analyses of their 1H and 13C NMR and ESI-MS data in combination with the comparison of their data with the reported values. Among them, compounds 2 and 6−8 were reported from this species for the first time. Compounds 1, 2, and 6−8 were evaluated for antioxidant activity by FRAP, DPPH radical, and ABTS•+ scavenging assays. As shown in Table 3, the FRAP values of compounds 1
Funding
The research was financially supported by the National Nature Science Foundation of China (31171680), the National Basic Research Program of China (2013CB127106), and the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-EW-J-28). Notes
The authors declare no competing financial interest.
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Table 3. Antioxidant Activities of Compounds 1, 2, and 6− 8a compd 1 2 6 7 8 L-ascorbic acid a
FRAP (mmol/g) 10.6 3.1 8.3 4.5 0.5 11.6
± ± ± ± ± ±
DPPH (SC50, μM)
0.3 0.1 0.1 0.1 0.0 0.5
10.0 16.4 11.7 28.0 >100 35.6
± ± ± ±
0.4 0.9 0.1 0.9
± 0.3
ABTS (IC50, μM) 9.7 12.4 9.1 27.6 20.3 36.8
± ± ± ± ± ±
0.1 0.4 0.2 0.4 0.6 0.2
Values represent means ± SD.
and 6 were 10.6 ± 0.3 and 8.3 ± 0.1 mmol of Fe(II)/g, respectively, which were comparable to that of the positive reference, L-ascorbic acid (11.6 ± 0.5), and their IC50 values toward DPPH radicals and ABTS•+ were lower than those of three other test compounds and L-ascorbic acid. Compounds 2 and 7 exhibited potent DPPH radical and ABTS•+ scavenging activity and moderate FRAP values, while compound 8 was very weak in FRAP and DPPH radical scavenging assays. It was noteworthy that we reported the potent antioxidant activity of proanthocyanidin A1 (3), proanthocyanidin A2 (4), and (−)-epicatechin (5) from the seeds of L. chinensis by FRAP and DPPH radical scavenging assays in a previous report.5 In addition, the in vitro antioxidant properties of rutin (9), including strong DPPH radical scavenging activity and effective inhibition of lipid oxidation, were also reported previously.20 This study reveals the structures of nine flavonoids; five of them were reported from litchi for the first time. Bioassay results in this study and previous reports revealed that these flavonoids were important antioxidants present in litchi pericarps.
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ASSOCIATED CONTENT
S Supporting Information *
ESI-MS and NMR spectra of compounds 1, 2, and 6. This material is available free of charge via the Internet at http:// pubs.acs.org.
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REFERENCES
(1) Liu, L.; Xie, B. J.; Cao, S. Q.; Yang, E. N.; Xu, X. Y.; Guo, S. S. Atype procyanidins from Litchi chinensis pericarp with antioxidant activity. Food Chem. 2007, 105, 1446−1451. (2) State Administration of Traditional Chinese Medicine. Chinese Materia Medica; Shanghai Science & Technology Press: Shanghai, 1999; Vol. 13, pp 119. (3) Zhao, M. M.; Yang, B.; Wang, J. S.; Li, B. Z.; Jiang, Y. M. Identification of the major flavonoids from pericarp tissues of lychee fruit in relation to their antioxidant activities. Food Chem. 2006, 98, 539−544. (4) Sarni-Manchado, P.; Le Roux, E.; Le Guernevé, C.; Lozano, Y.; Cheynier, V. Phenolic composition of litchi fruit pericarp. J. Agric. Food Chem. 2000, 48, 5995−6002. (5) Xu, X. Y.; Xie, H. H.; Wang, Y. F.; Wei, X. Y. A-type proanthocyanidins from lychee seeds and their antioxidant and antiviral activities. J. Agric. Food Chem. 2010, 58, 11667−11672. (6) Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biol. Med. 1999, 26, 1231− 1237. (7) Dudonné, S.; Vitrac, X.; Coutière, P.; Woillez, M.; Mérillon, J. M. Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. J. Agric. Food Chem. 2009, 57, 1768−1774. (8) She, G. M.; Hu, J. B.; Zhang, Y. J.; Yang, C. R. Phenolic constituents from Rhopalocnemis phalloides with DPPH radical scavenging activity. Pharm. Biol. 2010, 48, 116−119. (9) Yang, X. W.; Wang, J. S.; Ma, Y. L.; Xiao, H. T.; Zuo, Y. Q.; Lin, H.; He, H. P.; Li, L.; Hao, X. J. Bioactive phenols from the leaves of Baccaurea ramif lora. Planta Med. 2007, 73, 1415−1417. (10) Boyer, F. D.; Ducrot, P. H. Hydrogenation of substituted aromatic aldehydes: Nucleophilic trapping of the reaction intermediate, application to the efficient synthesis of methylene linked flavanol dimers. Tetrahedron Lett. 2005, 46, 5177−5180. (11) Nishida, Y.; Mitsunaga, T. Chemical structures and GTase inhibitory effects of converted compounds from catechins by the extracellular enzymes of wood rotting fungi. Tennen Yuki Kagobutsu Toronkai Youshishu 1998, 40, 643−648. (12) Sang, S. M.; Tian, S. Y.; Wang, H.; Stark, R. E.; Rosen, R. T.; Yang, C. S.; Ho, C. T. Chemical studies of the antioxidant mechanism of tea catechins: Radical reaction products of epicatechin with peroxyl radicals. Bioorg. Med. Chem. 2003, 11, 3371−3378. (13) Staubmann, R.; Schubert-Zsilavecz, M.; Hiermann, A.; Kartnig, T. A complex of 5-hydroxypyrrolidin-2-one and pyrimidine-2,4-dione isolated from Jatropha curcas. Phytochemistry 1999, 50, 337−338. (14) Jang, D. S.; Lee, G. Y.; Lee, Y. M.; Kim, Y. S.; Sun, H.; Kim, D. H.; Kim, J. S. Flavan-3-ols having a γ-lactam from the roots of Actinidia arguta inhibit the formation of advanced glycation end products in vitro. Chem. Pharm. Bull. 2009, 57, 397−400. (15) Lou, H. X.; Yamazaki, Y.; Sasaki, T.; Uchida, M.; Tanaka, H.; Oka, S. A-type proanthocyanidins from peanut skins. Phytochemistry 1999, 51, 297−308. (16) Shi, J. Y.; Sun, J.; Wei, X. Y.; Shi, J.; Cheng, G. P.; Zhao, M. M.; Wang, J. S.; Yang, B.; Jiang, Y. M. Identificaion of (−)-epicatechin as the direct substrate for polyphenol oxidase from longan fruit pericarp. LWTFood Sci. Technol. 2008, 41, 1742−1747.
AUTHOR INFORMATION
Corresponding Author
*Telephone: +86-20-37080950. Fax: +86-20-37252537. E-mail: [email protected]. 1077
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(17) Stark, T.; Hofmann, T. Application of a molecular sensory science approach to alkalized cocoa (Theobroma cacao): Structure determination and sensory activity of nonenzymatically C-glycosylated flavan-3-ols. J. Agric. Food Chem. 2006, 54, 9510−9521. (18) Kim, H. K.; Jeon, W. K.; Ko, B. S. Flavanone glycosides from Citrus junos and their anti-influenza virus activity. Planta Med. 2001, 67, 548−549. (19) Markham, K. R.; Ternai, B. 13C NMR of flavonoids. II: Flavonoids other than flavone and flavonol aglycones. Tetrahedron 1976, 32, 2607−2612. (20) Yang, J. X.; Guo, J.; Yuan, J. F. In vitro antioxidant properties of rutin. LWTFood Sci. Technol. 2008, 41, 1060−1066.
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Flavonoids from the Pericarps of Litchi chinensis Qing Ma,‡,§ Haihui Xie,*,‡ Sha Li,† Ruifen Zhang,† Mingwei Zhang,† and Xiaoyi Wei‡ †
Sericulture and Agri-food Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510610, China Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China § University of Chinese Academy of Sciences, Beijing 100049, China ‡
S Supporting Information *
ABSTRACT: A new methylene-linked flavan-3-ol dimer, bis(8-epicatechinyl)methane (1), was isolated from the pericarps of Litchi chinensis Sonn. (Sapindaceae), together with dehydrodiepicatechin A (2), proanthocyanidin A1 (3), proanthocyanidin A2 (4), (−)-epicatechin (5), 8-(2-pyrrolidinone-5-yl)-(−)-epicatechin (6), (−)-epicatechin 8-C-β-D-glucopyranoside (7), naringenin 7-O-(2,6-di-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside (8), and rutin (9). It was the first report of compound 2 as a natural product and compounds 6−8 from this species. Compounds 1, 2, and 6−8 were evaluated for antioxidant activity. The ferric reducing antioxidant powers (FRAP) of compounds 1 and 6 were comparable to that of L-ascorbic acid, and the scavenging activities of compounds 1, 2, 6, and 7 toward 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and 2,2′-azinobis(3ethylbenzthiazoline-6-sulfonic acid) radical cations were more potent than those of L-ascorbic acid; compound 8 was weak in FRAP and DPPH assays. KEYWORDS: Litchi chinensis, pericarp, flavonoids, bis(8-epicatechinyl)methane, dehydrodiepicatechin A, antioxidant activity
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INTRODUCTION Litchi chinensis Sonn., an evergreen tree belonging to the Sapindaceae family, has been widely cultivated for fruit in tropical and subtropical areas of the world. Litchi fruit is popular in domestic and foreign markets because of its juicy and sweet aril and attractive red pericarp. The annual output of fresh fruit in China exceeds 1300 million kg, and the pericarp accounts for approximately 15% of the whole fruit in fresh weight.1 Litchi aril is consumed fresh or as processed products, while the pericarp is discarded as waste except that a small amount is used as a traditional medicine with hemostatic and antidysenteric functions.2 Phytochemical investigation of the pericarp is necessary to utilize it as a resource. Previous research on litchi pericarp was mainly focused on the characterization of phenolic constituents by thin layer chromatography (TLC) or high-performance liquid chromatography (HPLC) analyses, which indicated the presence of cyanidin 3,5-diglucoside, cyanidin 3-glucoside, cyanidin 3rutinoside, cyanidin 3-galactoside, pelargonidin 3,7-diglucoside, and malvidin 3-glucoside.3,4 However, with respect to the phenolics that were isolated from litchi pericarp in a pure state and structurally identified by spectroscopic methods, including nuclear magnetic resonance (NMR), there were only a few reports. The obtained compounds included rutin, cyanidin 3rutinoside, epicatechin, proanthocyanidins A2, B2, and B4, and epicatechin-(4β→8,2β→O→7)-epicatechin-(4β→8)-epicatechin.1,3,4 To identify phenolic constituents in litchi pericarp, further chemical investigation was conducted, and consequently, four dimers and three monomers of flavan-3-ol and the glycosides of a flavanone and a flavonol (Figure 1) were obtained. This study was undertaken to isolate and elucidate the structures of these flavonoids and determine their antioxidant activity. © 2014 American Chemical Society
MATERIALS AND METHODS
Chemicals and Reagents. 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), DPPH, and 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma-Aldrich (St. Louis, MO). L-Ascorbic acid was obtained from Shanghai Boao Biotech Co. (Shanghai, China). Silica gel (100−200 or 200−300 mesh) was purchased from Qingdao Haiyang Chemical Co. (Qingdao, China). Sephadex LH-20 was obtained from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). MCI gel CHP20P (75−150 μm) was obtained from Mitsubishi Chemical Corp. (Tokyo, Japan). Apparatus. Medium-pressure liquid chromatography (MPLC) was performed on an EZ Purifier (Lisure Science, Suzhou, China), and a 400 mm × 25 mm (inside diameter) (Shanghai Lisui E-Tech Co., Shanghai, China), 20−45 μm, Chromatorex RP-18 SMB100 column was used (Fuji Silysia Chemical, Kasugai, Japan). HPLC was conducted on a LC-6AD pump (Shimadzu, Kyoto, Japan) connected to a RID-10A refractive index detector (Shimadzu); a 250 mm × 4.6 mm (inside diameter) column was used for analysis, and a 250 mm × 20 mm (inside diameter), 5 μm, YMC-pack ODS-A column was used for preparation along with a 23 mm × 4 mm (inside diameter) guard column of the same material (YMC, Kyoto, Japan). 1H and 13C NMR spectra in addition to distortionless enhancement by polarization transfer (DEPT), 1H−1H correlated spectroscopy (COSY), heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC) were recorded on a Bruker DRX-400 instrument (Bruker BioSpin Gmbh, Rheinstetten, Germany) in CD3OD with residual peaks at δH 3.31 and δC 49.0 as references. Electrospray ionization mass spectrometry (ESI-MS) was conducted on a MDS SCIEX API 2000 LC-MS/MS apparatus (Applied Biosystems Inc., Foster City, CA). High-resolution (HR) ESI-MS was conducted on a Bruker maXis mass spectrometer (Bruker Received: Revised: Accepted: Published: 1073
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Figure 1. Structures of compounds 1−9 from the pericarps of L. chinensis.
Table 1. 1H and 13C NMR Data of Compounds 1 and 6 in CD3OD 1 C/H 2 3 4 5 6 7 8 9 10 11 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″
δH (multiplet, J in Hz) 4.76 4.10 2.83 2.69
(2H, (2H, (2H, (2H,
br s) br dd, 4.7, 3.7) dd, 16.6, 4.7) dd, 16.6, 3.7)
5.96 (2H, s)
3.88 (2H, s) 6.96 (2H, br s)
6.73 (2H, br s) 6.73 (2H, br s)
6 δC (DEPT) 80.4 (CH) 67.2 (CH) 29.0 (CH2) 155.8 96.8 155.2 106.7 153.6 100.5 16.8 131.7 115.4 145.9 145.9 116.0 119.7
(C) (CH) (C) (C) (C) (C) (CH2) (C) (CH) (C) (C) (CH) (CH)
3″ 4″
δH (multiplet, J in Hz) 4.84 4.17 2.87 2.77
(br s) (ddd, 4.5, 3.0, 1.0) (dd, 16.7, 4.5) (ddd, 16.7, 3.0, 1.0)
6.00 (s)
6.98 (d, 2.0)
6.76 6.80 5.42 2.45 2.38 2.53 2.30
(d, 8.2) (dd, 8.2, 2.0) (dd, 9.2, 5.3) (m) (m) (m) (m)
δC 80.1 66.9 29.4 157.2 96.4 156.3 107.7 155.5 100.0 132.2 115.3 145.9 146.0 115.9 119.5 50.3 26.7 32.2 181.3
to afford ethyl acetate-soluble (109.35 g) and n-butanol-soluble (79.30 g) fractions after being taken to dryness under reduced pressure. The ethyl acetate-soluble fraction (107.35 g) was subjected to silica gel (2330 g, 100−200 mesh) column [545 mm × 120 mm (inside diameter)] chromatography and eluted with a chloroform (CHCl3)/ methanol (MeOH) mixture (v/v, 10:0, 38 L → 98:2, 24 L → 95:5, 19 L → 9:1, 33 L → 85:15, 38 L → 8:2, 25 L → 7:3, 25 L → 6:4, 9 L → 0:10, 9 L) to furnish fractions E1−E15 after they had been pooled according to their TLC profiles. Fraction E10 (10.7 g) was further subjected to silica gel (440 g, 200−300 mesh) column [720 mm × 44 mm (inside diameter)] chromatography and eluted with a CHCl3/ MeOH mixture (v/v, 95:5, 2.75 L → 9:1, 8.75 L → 85:15, 6.25 L) to yield fractions E10-1−E10-4. Fraction E10-4 was separated by MPLC and eluted with a MeOH/H2O mixture (v/v, 2:8, 3:7, 4:6, 5:5, 6:4, and 7:3, each 2.4 L) at a flow rate of 10 mL/min to produce fractions E104-1−10-4-4. Fractions E10-4-1 and E10-4-2 were individually separated by silica gel (200−300 mesh) column chromatography to afford compounds 1 (15 mg) and 5 (11 mg), respectively. Fraction
Daltonics Inc., Bremen, Germany). Optical rotation was acquired on a Perkin-Elmer (Waltham, MA) 343 polarimeter. Ultraviolet (UV) radiation was recorded on a Perkin-Elmer Lambda 650 UV−vis spectrophotometer (Perkin-Elmer). Infrared (IR) radiation was obtained on a WQF-520 FT-IR spectrometer (Beijing Rayleigh Analytic Instrument Co., Beijing, China). Plant Material. Fresh ripe litchi fruits, cv. ‘Heiye’, were collected from the Fruit Tree Research Institute, Guangdong Academy of Agricultural Sciences (Guangzhou, China) in June 2012, and the pericarps were manually separated from the fruits. Extraction and Isolation. The fresh pericarps (5.0 kg) were immersed in 85% aqueous ethanol, chopped with a multifood processor, transferred to flasks, and then heated at 50 °C for 2 h. Twenty liters of 85% aqueous ethanol was used each time, and the extraction was repeated three times. The solution was filtered and evaporated under vacuum to give an ethanolic extract (320 g). The extract (315 g) was dissolved in 2.0 L of water and then sequentially fractionated with ethyl acetate (1.5 L × 5) and n-butanol (1.2 L × 7) 1074
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Table 2. 1H and 13C NMR Data of Compound 2 in CD3OD C/H 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′
δH (multiplet, J in Hz) 3.82 4.45 2.83 2.65
(br s) (br d, 4.8) (br d, 16.5) (dd, 16.5, 4.8)
5.92 (d, 2.0) 5.94 (d, 2.0)
2.94 (d, 15.3) 2.83 (d, 15.3)
6.55 (s)
δC
C/H
72.9 65.1 25.0
2″ 3″ 4″
157.5 96.6 157.7 95.5 155.6 98.9 91.8 40.5
5″ 6″ 7″ 8″ 9″ 10″ 1‴ 2‴
95.5 193.8 113.6 164.7
3‴ 4‴ 5‴ 6‴
E10-4-3 was purified by preparative HPLC using a MeOH/H2O mixture (46:54, v/v) as the mobile phase at a flow rate of 5 mL/min to give compound 2 [retention time (tR) of 53.4 min, 25 mg]. Fraction E11 (13.88 g) was passed through a MCI gel column [259 mm × 26 mm (inside diameter)] for depigmentation, and the obtained MeOH eluate (7.20 g) was separated by MPLC and purified by preparative HPLC using an acetonitrile/H2O/acetic acid (AcOH) mixture (15:85:0.1, v/v/v) as the mobile phase at a flow rate of 5 mL/min to yield compounds 3 (tR of 60.7 min, 32 mg) and 4 (tR of 90.5 min, 30 mg). The n-butanol-soluble fraction (79.3 g) was subjected to silica gel (1310 g, 100−200 mesh) column [800 mm × 75 mm (inside diameter)] chromatography and eluted with a CHCl3/MeOH mixture (v/v, 9:1, 5.6 L → 85:15, 18.2 L → 8:2, 14.7 L → 7:3, 10.5 L → 0:10, 5.3 L) to furnish fractions B1−B7. Fraction B4 (12.4 g) was separated by silica gel (200−300 mesh) column chromatography, MPLC, and Sephadex LH-20 column [1215 mm × 14 mm (inside diameter)] chromatography that was eluted with MeOH to afford compound 6 (8 mg). Fraction B6 (2.8 g) was separated by MPLC to give fractions B61−B6-7. Fraction B6-1 was further separated by Sephadex LH-20 column chromatography and purified by preparative HPLC using a MeOH/H2O mixture (13:87, v/v) as the mobile phase at a flow rate of 5 mL/min to yield compound 7 (tR of 95.4 min, 4 mg). Fraction B6-6 was separated by Sephadex LH-20 column chromatography and purified by preparative HPLC using a MeOH/H2O/AcOH mixture (42:58:0.1, v/v/v) as the mobile phase at a flow rate of 5 mL/min to obtain compounds 8 (tR of 46.2 min, 15 mg) and 9 (tR of 44.5 min, 22 mg). Bis(8-epicatechinyl)methane (1). Off-white amorphous powder: [α]D20 −103.7 (c 0.138, MeOH); UV (MeOH) λmax (nm) (log ε) 281 (3.65); IR (KBr) νmax 3377, 1618, 1524, 1454, 1284, 1113 cm−1; HRESI-MS positive m/z 593.1651 [M + H]+ (calcd for C31H29O12+, 593.1654, error of 0.5 ppm), 615.1474 [M + Na]+ (calcd for C31H28O12Na+, 615.1473, error of −0.1 ppm); ESI-MS positive m/z 593 [M + H]+, 615 [M + Na]+, negative m/z 591 [M − H]−; 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) data in Table 1. Dehydrodiepicatechin A (2). Yellowish amorphous powder: [α]D20 373.8 (c 0.064, MeOH); ESI-MS positive m/z 577 [M + H]+, 599 [M + Na]+, negative m/z 575 [M − H]−; 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) data in Table 2. 8-(2-Pyrrolidinone-5-yl)-(−)-epicatechin (6). White amorphous powder: [α]D20 −14.5 (c 0.345, MeOH); ESI-MS positive m/z 374 [M + H]+, 396 [M + Na]+, negative m/z 372 [M − H]−, 408 [M + Cl]−; 1 H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) data in Table 1. FRAP Assay. Ferric reducing ability of the compounds was determined following the procedures as previously described.5 FRAP
δH (multiplet, J in Hz) 5.03 4.30 2.89 2.83
(br s) (br d, 3.9) (dd, 16.5, 3.9) (br d, 16.5)
δC 80.8 66.4 29.5
7.04 (br s)
166.5 91.7 167.0 104.7 156.2 103.6 131.2 115.1
6.81 (d, 8.0) 6.86 (br d, 8.0)
146.1 146.2 116.1 119.2
6.17 (s)
reagent was made freshly by mixing 300 mM acetate buffer (pH 3.6), a 10 mM TPTZ solution in 40 mM hydrochloric acid, and a 20 mM aqueous ferric chloride (FeCl3) solution in a 10:1:1 (v/v/v) ratio. The TPTZ solution was prepared on the same day. Test compounds were dissolved in methanol and diluted 2-fold to six concentrations. Twenty microliters of the compound solution and 180 μL of FRAP reagent were mixed in 96-well plates. L-Ascorbic acid was dissolved in methanol and used as a positive reference. Each treatment was conducted in quadruplicate. The plates were incubated at 37 °C for 30 min in the dark. The absorbance of the product (ferrous TPTZ complex) in each well was read at 595 nm using a Genios microplate reader (Tecan Group, Männedorf, Switzerland). One milliliter of ferrous sulfate (FeSO4) at six different concentrations and 1 mL of 10 mM TPTZ and 10 mL of 300 mM acetate buffer (pH 3.6) were used for a calibration curve. FRAP values were calculated and expressed as means ± the standard deviation (SD) in millimoles of Fe(II) per gram. DPPH Radical Scavenging Assay. The DPPH radical scavenging ability of the compounds was evaluated according to the method as previously described.5 DPPH was freshly prepared in methanol at a concentration of 0.1 mM. Test compounds were dissolved in methanol and diluted 2-fold to six concentrations. Twenty microliters of the compound solution and 180 μL of the DPPH solution were mixed in 96-well plates. L-Ascorbic acid was dissolved in methanol and used as a positive reference. The control contained methanol instead of the compound solution, and the blank contained methanol in place of the DPPH solution. Each treatment was conducted in quadruplicate. The plates were incubated at 37 °C for 30 min in the dark. The absorbance (OD) in each well was read at 515 nm on a Genios microplate reader (Tecan). The inhibitory rates of DPPH radicals were calculated according to the formula
inhibition (%) = [1 − (ODtreated − ODblank )/ODcontrol ] × 100 SC50 values (the concentrations required to scavenge 50% DPPH radicals present in the test solution) were calculated and expressed as means ± SD in micromolar. ABTS Radical Cation Decolorization Assay. The ABTS radical cation (ABTS•+) scavenging activity of the compounds was determined according to the method described previously.6,7 ABTS was dissolved in deionized water to a concentration of 7 mM. ABTS•+ was produced by reacting the ABTS solution with 2.45 mM (final concentration) potassium persulfate, and the mixture was kept at room temperature for 12−16 h in the dark before being used. The ABTS•+ solution was diluted with phosphate-buffered saline (PBS, pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm. Test compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted to 400, 200, 100, 50, 25, and 12.5 μg/mL. Ten microliters of the compound solution and 200 μL of the diluted ABTS•+ solution were mixed in 96-well 1075
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plates. L-Ascorbic acid was used as a positive reference. The control contained DMSO instead of the compound solution. Each treatment was conducted in triplicate. After a mixing time of 10 s and an incubation period of 6 min at 37 °C in the dark, the absorbance (OD) in each well was read at 415 nm on a Varioskan Flash microplate reader (Thermo Scientific). The inhibitory rates of ABTS•+ were calculated according to the formula
compounds were shown by coupling constants J2,3 and J4,3, which were 7.5 Hz and 8.8 or 5.4 Hz, respectively,8,10 rather different from those of compound 1. Compound 2 was deduced to have a molecular weight of 576 from the ESI-MS positive and negative ion peaks at m/z 577 [M + H]+, 599 [M + Na]+, and 575 [M − H]−. The 1H NMR (Table 2) spectrum exhibited three ABX-type aromatic protons at δ 7.04 (1H, br s, H-2‴), 6.81 (1H, d, J = 8.0 Hz, H-5‴), and 6.86 (1H, br d, J = 8.0 Hz, H-6‴), two meta-coupled aromatic protons at δ 5.92 (1H, d, J = 2.0 Hz, H-6) and 5.94 (1H, d, J = 2.0 Hz, H-8), two singlet aromatic protons at δ 6.55 (1H, H-5′) and 6.17 (1H, H-6″), four protons at δ 5.03 (1H, br s, H-2″), 4.45 (1H, br d, J = 4.8 Hz, H-3), 4.30 (1H, br d, J = 3.9 Hz, H3″), and 3.82 (1H, br s, H-2) connected to oxygenated carbons, and six aliphatic protons from δ 2.65 to 2.94. The 13C NMR (Table 2) spectrum displayed signals of a total of 30 carbons, including a carboxyl carbon at δ 193.8 (C-4′), four oxygenated carbons at δ 80.8 (C-2″), 72.9 (C-2), 66.4 (C-3″), and 65.1 (C3), and three aliphatic carbons at δ 40.5 (C-2′), 29.5 (C-4″), and 25.0 (C-4). Careful analysis of the HMBC spectrum (Figure 3) such as the correlations from H-2 to C-2′ and C-6′
inhibition (%) = (1 − ODtreated /ODcontrol ) × 100 IC50 values were calculated and expressed as means ± SD in micromolar.
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RESULTS AND DISCUSSION Compound 1 was obtained as an off-white amorphous powder and deduced to have the molecular formula C31H28O12 from the positive ESI-MS ion peaks at m/z 593 [M + H]+ and 615 [M + Na]+ as well as their HR-ESI-MS data. The IR spectrum displayed absorptions of the hydroxyl group at 3377 cm−1 and the phenyl moiety at 1618, 1523, and 1454 cm−1. The 1H NMR (Table 1) spectrum demonstrated eight aromatic protons at δ 6.73 (4H, br s), 6.96 (2H, br s), and 5.96 (2H, s), four protons at δ 4.76 (2H, br s, H-2) and 4.10 (2H, br dd, J = 4.7, 3.7 Hz, H-3) connected to oxygenated carbons, and four aliphatic protons at δ 2.83 (2H, dd, J = 16.6, 4.7 Hz, H-4) and 2.69 (2H, dd, J = 16.6, 3.7 Hz, H-4) that can be attributed to two methylenes. These signals suggested the presence of two epicatechinyl moieties in a symmetric state.5 Moreover, the 1H NMR spectrum exhibited a signal of two protons at δ 3.88 (s, H2-11) that were readily recognized for an additional methylene. The 13C NMR and DEPT (Table 1) spectra showed signals of 31 carbons, including a methylene carbon at δ 16.8 (C-11), among which the carbons at the same positions of two symmetric epicatechinyl moieties were overlapped. With the aid of the HSQC spectrum, all the protons were assigned to their corresponding carbons. In the HMBC spectrum (Figure 2), the correlations from H2-11 to δ 155.2 (C-7), 106.7 (C-8),
Figure 3. HMBC spectrum of compound 2.
(δ 164.7), H-3 to C-3′ (δ 95.5), H-5′ to C-1′ (δ 91.8), C-3′, and C-8″ (δ 104.7), and H-6″ to C-8″ led us to elucidate it as dehydrodiepicatechin A. This compound was originally obtained from (−)-epicatechin by biotransformation with crude extracellular enzymes of wood rotting fungi, Coriolus versicolor and Pycnoporus coccineus,11 and later from tea epicatechin by oxidation with peroxyl radicals generated by thermolysis of the initiator azo-bisisobutyrylnitrile.12 This was the first report of it as a natural product. Compound 6 was deduced to have a molecular weight of 373 from the ESI-MS ion peaks of m/z 374 [M + H]+, 396 [M + Na]+, 372 [M − H]−, and 408 [M + Cl]−, which indicated the presence of an odd-numbered nitrogen atom. The 1H and 13C NMR spectra showed signals readily assigned for an epicatechinyl moiety as compound 1 (Table 1). In the HMBC spectrum, the correlations from δ 5.42 (1H, dd, J = 9.2, 5.3 Hz, H-1″) to δ 156.3 (C-7), 107.7 (C-8), 155.5 (C-9), and 181.3 (C-4″), δ 2.45 and 2.38 (each 1H, m, H2-2″) to C-8 and C-4″, and δ 4.84 (1H, br s, H-2) to C-9 were observed, which suggested the presence of a pyrrolidin-2-one moiety13 and its connection to C-8. Hence, compound 6 was identified as 8-(2-pyrrolidinone-5-yl)-(−)-epicatechin. Jang et al.14 obtained a 2:1 mixture of two diastereomers of this compound
Figure 2. HMBC spectrum of compound 1.
and 153.6 (C-9), δ 4.76 (H-2) to C-9 and C-4 (δ 29.0), and δ 2.83 and 2.69 (H2-4) to C-9 and C-5 (δ 155.8) ascertained the connection of the methylene to C-8 of two epicatechinyl moieties. Consequently, compound 1 was determined to be bis(8-epicatechinyl)methane. Bis(8-catechinyl)methane was previously obtained from several plants such as Rhopalocnemis phalloides (Balanophoraceae)8 and Baccaurea ramif lora (Euphorbiaceae)9 and synthesized by hydrogenation of substituted aromatic aldehyde.10 The differences between these two 1076
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from the roots of Actinidia arguta for the first time, which showed two sets of 1H and 13C NMR data. However, compound 6 exhibited only one set of 1H and 13C NMR data, which were in good agreement with those of the major isomer. It was the first report of this compound as a single stereoisomer. The other known compounds were identified as proanthocyanidin A1 (3),15 proanthocyanidin A2 (4),15 (−)-epicatechin (5),16 (−)-epicatechin 8-C-β-D-glucopyranoside (7),17 naringenin 7-O-(2,6-di-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside (8),18 and rutin (9)19 by analyses of their 1H and 13C NMR and ESI-MS data in combination with the comparison of their data with the reported values. Among them, compounds 2 and 6−8 were reported from this species for the first time. Compounds 1, 2, and 6−8 were evaluated for antioxidant activity by FRAP, DPPH radical, and ABTS•+ scavenging assays. As shown in Table 3, the FRAP values of compounds 1
Funding
The research was financially supported by the National Nature Science Foundation of China (31171680), the National Basic Research Program of China (2013CB127106), and the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-EW-J-28). Notes
The authors declare no competing financial interest.
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Table 3. Antioxidant Activities of Compounds 1, 2, and 6− 8a compd 1 2 6 7 8 L-ascorbic acid a
FRAP (mmol/g) 10.6 3.1 8.3 4.5 0.5 11.6
± ± ± ± ± ±
DPPH (SC50, μM)
0.3 0.1 0.1 0.1 0.0 0.5
10.0 16.4 11.7 28.0 >100 35.6
± ± ± ±
0.4 0.9 0.1 0.9
± 0.3
ABTS (IC50, μM) 9.7 12.4 9.1 27.6 20.3 36.8
± ± ± ± ± ±
0.1 0.4 0.2 0.4 0.6 0.2
Values represent means ± SD.
and 6 were 10.6 ± 0.3 and 8.3 ± 0.1 mmol of Fe(II)/g, respectively, which were comparable to that of the positive reference, L-ascorbic acid (11.6 ± 0.5), and their IC50 values toward DPPH radicals and ABTS•+ were lower than those of three other test compounds and L-ascorbic acid. Compounds 2 and 7 exhibited potent DPPH radical and ABTS•+ scavenging activity and moderate FRAP values, while compound 8 was very weak in FRAP and DPPH radical scavenging assays. It was noteworthy that we reported the potent antioxidant activity of proanthocyanidin A1 (3), proanthocyanidin A2 (4), and (−)-epicatechin (5) from the seeds of L. chinensis by FRAP and DPPH radical scavenging assays in a previous report.5 In addition, the in vitro antioxidant properties of rutin (9), including strong DPPH radical scavenging activity and effective inhibition of lipid oxidation, were also reported previously.20 This study reveals the structures of nine flavonoids; five of them were reported from litchi for the first time. Bioassay results in this study and previous reports revealed that these flavonoids were important antioxidants present in litchi pericarps.
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ASSOCIATED CONTENT
S Supporting Information *
ESI-MS and NMR spectra of compounds 1, 2, and 6. This material is available free of charge via the Internet at http:// pubs.acs.org.
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REFERENCES
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