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Further dihydrochalcone C-glycosides from the fruit of Averrhoa carambola
LWT - Food Science and Technology 65 (2016) 604e609
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Further dihydrochalcone C-glycosides from the fruit of Averrhoa carambola Dan Yang a, c, Xuchao Jia a, c, Haihui Xie a, b, *, Xiaoyi Wei b a
Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China b Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China c University of Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 July 2015 Received in revised form 23 August 2015 Accepted 25 August 2015 Available online 28 August 2015
Further study on the chemical constituents present in the fruit of Averrhoa carambola L. (Oxalidaceae), commonly called star fruit or carambola, led to the isolation of six new dihydrochalcone C-glycosides, carambolasides E~J (1~6). Their structures were determined by spectroscopic and chemical methods, including low temperature nuclear magnetic resonance (NMR). Compounds 1~6 and two alkaline hydrolysates, 5a and 6a, exhibited more potent 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) radical cation (ABTS þ) scavenging activity with IC50 values ranging from 4.52 to 2.54 mM than L-ascorbic acid (14.21 mM). However, they were inactive in scavenging 1,1-diphenyl-2-picrylhydrazyl radical (DPPH ) and in ferric reducing antioxidant power (FRAP). © 2015 Elsevier Ltd. All rights reserved.
Keywords: Averrhoa carambola Star fruit Dihydrochalcone C-glycoside Low temperature NMR Antioxidant activity
1. Introduction
2. Materials and methods
Averrhoa carambola L. (Oxalidaceae) is a popular fruit in tropical and subtropical regions. The fruit acquires the common name of ‘star fruit’ from its star-shaped cross section when cut (O'Hare, 1993). Star fruit is yellow to bright orangey-yellow, waxy and glossy, oblong, very fleshy, sweet to acid sweet, crisp and juicy, and was found to be a good source of natural antioxidants which were attributed to its high contents of L-ascorbic acid (8~1426 mg) and total phenolics (118~5638 mg) per gram fresh weight (Lim, 2012). However, the phenolic compounds hitherto isolated from the fruit and structurally identified by spectroscopic methods were very few. Our previous study on the fruit clarified four dihydrochalcone C-glycosides, carambolasides A~D, and their potent ABTS þ scavenging activity (Yang, Xie, Jia, & Wei, 2015). The aim of this study was to further investigate this type of compounds under the guidance of ultraviolet (UV) absorbance at 225 and 280 nm.
2.1. General experimental procedures
* Corresponding author. South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China. E-mail address: [email protected] (H. Xie). http://dx.doi.org/10.1016/j.lwt.2015.08.061 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
Silica gel (100~200 mesh) was from Qingdao Haiyang Chemical Co. (Shandong, China). Sephadex LH-20 was from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Amberlite XAD-7HP macroporous resin, ABTS, DPPH, and 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) were from SigmaeAldrich (St. Louis, MO, USA). L-Ascorbic acid was from Shanghai Boao Biotech Co. (Shanghai, China). NMR spectra were measured on a Bruker DRX-400 or Ascend500 spectrometer (Bruker BioSpin Gmbh, Rheinstetten, Germany) in deuteromethanol (CD3OD) with its residual peaks of d 3.31 and 49.0 as references. Distortionless enhancement by polarization transfer (DEPT), 1He1H correlation spectroscopy (COSY), 13Ce1H COSY, hetero-nuclear single bond coherence (HSQC), and heteronuclear multiple bond correlation (HMBC) were obtained on the same NMR spectrometer. Preparative HPLC was conducted on a LC3000 set with a UV3000 scanning spectrophotometer detector (Beijing ChuangXin TongHeng Sci. & Tech. Co., Beijing, China) connected to a column of 250 mm length 20 mm inner diameter, YMC-Pack ODS-A, particle size 5 mm (YMC Co., Kyoto, Japan). Optical density (OD) values were read on a Genios microplate reader
D. Yang et al. / LWT - Food Science and Technology 65 (2016) 604e609
€nnedorf, Switzerland). The apparatuses used for (Tecan Group, Ma medium pressure liquid chromatography (MPLC), high resolution electrospray ionization mass spectrometry (HR-ESI-MS), ESI-MS, optical rotation (aD), and UV spectra were the same as previously described (Xu, Xie, & Wei, 2014).
605
2.5. Antioxidant activity evaluation Compounds 1~6, 5a, and 6a were evaluated for antioxidant ac tivity by ABTS þ scavenging assay, DPPH scavenging assay, and FRAP assay according to the methods as previously described (Ma et al., 2014; Zhou, Xie, Liang, & Wei, 2014).
2.2. Plant material 3. Result and discussion Fresh mature star fruits (sour in taste) were collected from South China Botanical Garden, Chinese Academy of Sciences (Guangzhou, Guangdong, China) in January 2011. The fruits were manually cut to pieces, sundried and followed by heating at 60 C for about 5 h, and then ground to powder.
Six new dihydrochalcone C-glycosides (1~6) (Fig. 1) were isolated from star fruit by means of methanol extraction and multiple separation techniques, including solvent fractionation, column chromatography, MPLC, and HPLC.
2.3. Extraction and isolation
3.1. Spectroscopic data of compounds
The fruit powder (6.8 kg) was extracted with methanol (MeOH) under reflux. The solution was evaporated under vacuum to give a brown extract (785 g). The extract was dissolved in water and sequentially partitioned with ethyl acetate and n-butanol to yield ethyl acetate-soluble (156.5 g) and n-butanol-soluble (443.5 g) fractions after being condensed under reduced pressure to dryness. The latter fraction was passed through an Amberlite XAD-7HP macroporous resin column eluted with water and then 90% ethanol to obtain an ethanol eluate (123.3 g). The eluate and the ethyl acetate-soluble fraction were merged (272.0 g) and subjected to silica gel column chromatography (CC) eluted with a mixture of chloroform (CHCl3)/MeOH to offer fractions 1~8. Fraction 6 (25.0 g) was subjected to silica gel CC eluted with CHCl3/MeOH to furnish fractions 6-1~6-13. Fraction 6-3 (1.1 g) was separated by MPLC and eluted with MeOH/H2O and purified by HPLC using MeOH/H2O (v/v, 65:35) as mobile phase at the flow rate of 5 mL/min to afford compounds 2 [retention time (tR) 78.2 min, 38.7 mg] and 3 (tR 49.1 min, 53.9 mg). Fraction 6-7 (2.6 g) was separated by MPLC and purified by HPLC using MeOH/H2O (v/v, 66:34) as mobile phase at 5 mL/min to yield compound 5 (tR 36.4 min, 42.0 mg). Fraction 6-10 (808 mg) was separated by MPLC and purified by HPLC using MeOH/H2O (v/v, 68:32) at 5 mL/min to obtain compound 6 (tR 45.3 min, 110.0 mg). Fraction 7 (23.5 g) was subjected to silica gel CC to offer fractions 7-1~7-10. Fraction 7-5 (1.2 g) was separated by MPLC eluted with MeOH/H2O to get fractions 7-5-1~7-5-3. Fraction 7-5-1 (68 mg) was purified by HPLC using MeOH/H2O (v/v, 58:42) as mobile phase at 5 mL/min to provide compound 4 (tR 20.8 min, 19 mg). Fraction 7-5-2 (62 mg) was separated by Sephadex LH-20 CC and silica gel CC to afford compound 1 (19 mg).
Carambolaside E (1): yellowish amorphous powder; [a]20 D þ24.7 (c 1.14, MeOH); UV (MeOH) lmax nm (log ε) 227 (4.60) and 287 (4.58); ESI-MS m/z 566 [M þ H]þ and 589 [M þ Na]þ; HR-ESI-MS m/ z 567.2069 [M þ H]þ (calcd for C27H35Oþ 13, 567.2072, error 0.6 ppm) and 589.1894 [M þ Na]þ (calcd for C27H34NaOþ 13, 589.1892, error 0.5 ppm); 1H NMR (400 MHz) and 13C NMR (100 MHz) data in CD3OD, see Table 1. Carambolaside F (2): yellowish amorphous powder; [a]20 D 103.6 (c 1.0, MeOH); UV (MeOH) lmax nm (log ε) 223 (4.63) and 280 (4.62); ESI-MS m/z 719 [M þ Na]þ and 695 [M H]; HR-ESI-MS m/z 697.2492 [M þ H]þ (calcd for C36H41Oþ 14, 697.2491, error 0.2 ppm) and 719.2317 [M þ Na]þ (calcd for C36H40NaOþ 14, 719.2310, error 0.9 ppm); 1H NMR (500 MHz) and 13C NMR (125 MHz) data in CD3OD, see Table 1. Carambolaside G (3): yellowish amorphous powder; [a]20 D 54.9 (c 0.18, MeOH); UV (MeOH) lmax nm (log ε) 223 (4.61) and 281 (4.65); ESI-MS m/z 697 [M þ H]þ, 719 [M þ Na]þ, and 695 [M H]; HR-ESI-MS m/z 697.2482 [M þ H]þ (calcd for C36H41Oþ 14, 697.2491, error 1.2 ppm) and 719.2310 [M þ Na]þ (calcd for C36H40NaOþ 14, 719.2310, error 0 ppm); 1H NMR (400 MHz) and 13C NMR (100 MHz) data in CD3OD, see Table 1. Carambolaside H (4): yellowish amorphous powder; [a]20 D 124.4 (c 0.50, MeOH); UV (MeOH) lmax nm (log ε) 227 (4.46) and 288 (4.48); ESI-MS m/z 735 [M þ Na]þ; HR-ESI-MS m/z 713.2448 [M þ H]þ (calcd for C36H41Oþ 15, 713.2440, error 1.1 ppm) and 735.2275 [M þ Na]þ (calcd for C36H40NaOþ 15, 735.2259, error 2.1 ppm); 1H NMR (400 MHz) and 13C NMR (100 MHz) data in CD3OD, see Table 1. Carambolaside I (5): yellowish amorphous powder; [a]20 D 132.4 (c 0.82, MeOH); UV (MeOH) lmax nm (log ε) 223 (4.50) and 282 (4.54); ESI-MS m/z 851 [M þ Na]þ, 827 [M H], and 863 [M þ Cl]; HR-ESI-MS m/z 829.2925 [M þ H]þ (calcd for C41H49Oþ 18, 829.2913, error 1.5 ppm) and 851.2752 [M þ Na]þ (calcd for C41H48NaOþ 18, 851.2733, error 2.2 ppm); 1H NMR (500 MHz) and 13C NMR (125 MHz) data in CD3OD, see Table 2. Carambolaside Ia (5a): yellowish amorphous powder; [a]20 D e105.0 (c 0.58, MeOH); UV (MeOH) lmax nm (log ε) 225 (4.29) and 287 (4.14); HR-ESI-MS m/z 721.2322 [M þ Na]þ (calcd for 1 13 C32H42NaOþ 17, 721.2314, error 0.7 ppm); H NMR (500 MHz) and C NMR (125 MHz) in CD3OD, see Table 2. Carambolaside J (6): yellowish amorphous powder; [a]20 D 141.4 (c 0.65, MeOH); UV (MeOH) lmax nm (log ε) 223 (4.50) and 280 (4.63); ESI-MS m/z 997 [M þ Na]þ and 973 [M H]; HR-ESI-MS m/ z 975.3485 [M þ H]þ (calcd for C47H59Oþ 22, 975.3493, error 0.8 ppm) and 997.3317 [M þ Na]þ (calcd for C47H58NaOþ 22, 997.3312, error 0.5 ppm); 1H NMR (500 MHz) and 13C NMR (125 MHz) data in CD3OD, see Table 2. Carambolaside Ja (6a): yellowish amorphous powder; [a]20 D 23.0 (c 0.54, MeOH); UV (MeOH) lmax nm (log ε) 215 (4.24) and 281 (4.11);
2.4. Alkaline hydrolysis of compounds 2~6 Compound 2 (19 mg) was dissolved in 2.5 mL of 0.15 M aqueous sodium hydroxide and stirred at 30 C for 4 h. The solution was neutralised with 0.15 M aqueous hydrochloride and fractionated with n-butanol (3 mL 3). The n-butanol layer was combined and condensed under vacuum to give a residue, which was purified by HPLC using acetonitrile (MeCN)/H2O/formic acid (v/v/v, 30:70:0.1) as mobile phase at 5 mL/min to yield a product (tR 69.8 min, 7 mg), of which the 1H and 13C NMR, ESI-MS, and [a]D data were identical to compound 1. Through similar procedures, a new product 5a (tR 109 min, 4.9 mg) was purified from the hydrolytic solution of compound 5 by HPLC using MeCN/H2O/formic acid (v/v/v, 27:73:0.1) as mobile phase at 5 mL/min, and a new product 6a (tR 53 min, 11.0 mg) was obtained from the hydrolytic solution of compound 6 by HPLC using MeCN/H2O/formic acid (v/v/v, 25:75:0.1) as mobile phase at 5 mL/min. Compound 1 was identified in the hydrolytic solutions of compounds 2~4 by HPLC analysis in comparison with the authentic sample.
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D. Yang et al. / LWT - Food Science and Technology 65 (2016) 604e609
Fig. 1. Structures of compounds 1~6 and two hydrolytic products 5a and 6a.
Table 1 1 H and 13C NMR data of compounds 1~4 in CD3OD. H/C
1
2
dH (J in Hz) 1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 100 200 300 400 500 600 1000 2000 3000 4000 5000 6000 1000 0 2000 0 3000 0 4000 0 5000 0 6000 0 7000 0 8000 0 9000 0
7.04 (d, 8.5) 6.69 (d, 8.5) 6.69 7.04 2.85 3.29
(d, 8.5) (d, 8.5) (2H, dd, 8.4, 7.2) (2H, dd, 8.4, 7.2)
5.89 (s) 4.85 4.18 3.68 3.97 3.77 1.29 4.53 3.56 3.49 3.60 3.66 1.27
(d, 9.6) (t, 9.6) (dd, 9.6, 2.8) (d, 2.8) (q, 6.4) (3H, d, 6.4) (d, 7.5) (dd, 9.7, 7.5) (dd, 9.7, 3.3) (d, 3.3) (q, 6.4) (3H, d, 6.4)
dC (DEPT) 133.9 130.3 116.1 156.4 116.1 130.3 31.4 47.5 206.7 105.5 164.5 104.8 164.5 96.1 164.5 75.8 70.3 85.8 72.8 75.8 17.3 105.9 72.8 74.7 72.9 71.9 16.9
(C) (CH) (CH) (C) (CH) (CH) (CH2) (CH2) (C) (C) (C) (C) (C) (CH) (C) (CH) (CH) (CH) (CH) (CH) (CH3) (CH) (CH) (CH) (CH) (CH) (CH3)
3
dH (J in Hz) 7.05/6.90 (br s) 6.66 (br s) 6.66 (br s) 7.05/6.90 (br s) 2.87/2.58 (2H, br s) 3.27/3.11 (2H, br s)
5.83 (br s) 5.08 (d, 9.2) 5.83/5.71 (br s) 4.04 (dd, 7.3, 3.0) 4.03 (d, 3.0) 3.89 (q, 6.3) 1.35 (3H, d, 6.3) 4.40 (d, 7.6) 3.47 (dd, 9.8, 7.6) 3.39 (dd, 9.8, 3.3) 3.57 (d, 3.3) 3.63 (q, 6.5) 1.26 (3H, d, 6.5) 7.50 7.37 7.37 7.37 7.50 7.52 6.33
(br s) (br s) (br s) (br s) (br s) (d, 15.9) (d, 15.9)
dC 133.8 130.2 116.1 156.2 116.1 130.2 31.2 47.4 206.6 104.8 164.8 103.0 163.6 96.0 164.8 74.1 71.8 82.2 73.1 76.3 17.2 105.7 72.1 74.5 72.7 71.8 16.9 135.6 129.1 129.9 131.4 129.9 129.1 146.1 118.7 167.9
dH (J in Hz) 7.05/6.89 (br s) 6.65 (br s) 6.65 (br s) 7.05/6.89 (br s) 2.85/2.65 (2H, br s) 3.28/3.13 (2H, br s)
5.85 (br s) 5.02 (d, 9.9) 5.92/5.70 (br s) 3.94 (br d, 9.6) 3.99 (d, 2.9) 3.85 (q, 6.4) 1.32 (3H, d, 6.4) 4.36 (d, 7.0) 3.46 (dd, 9.5, 7.0) 3.42 (dd, 9.5, 3.1) 3.58 (d, 3.1) 3.61 (q, 6.4) 1.26 (3H, d, 6.4) 7.50/7.37 (br s) 7.20 (br s) 7.20 (br s) 7.20 (br) 7.50/7.37 (br s) 6.78 (d, 12.5) 5.83 (d, 12.5)
4
dC 133.9 130.3 116.0 156.2 116.0 130.3 31.1 47.2 206.7 105.0 165.4 103.3 163.6 96.1 165.0 74.0 71.9 82.1 73.1 76.4 17.2 105.9 72.1 74.8 72.9 71.9 16.9 135.9 128.9 130.9 129.9 130.9 128.9 144.0 120.1 166.8
dH (J in Hz) 7.06/6.92 (br s) 6.67 (br s) 6.67 (br s) 7.05/6.92 (br s) 2.86/2.59 (2H, br s) 3.28/3.11 (2H, br s)
5.83 (br s) 5.07 (d, 9.7) 5.83/5.70 (br s) 4.00 (dd, 9.6, 2.8) 4.01 (d, 2.8) 3.85 (q, 6.3) 1.33 (3H, d, 6.3) 4.39 (d, 7.6) 3.48 (dd, 9.7, 7.6) 3.40 (dd, 9.7, 3.2) 3.56 (d, 3.2) 3.59 (q, 6.3) 1.25 (3H, d, 6.3) 7.36/7.23 (d, 8.5) 6.78 (d, 8.5) 6.78 (d, 8.5) 7.36/7.23 (d, 8.5) 7.45 (d, 16.0) 6.15 (d, 16.0)
dC 133.9 130.3 116.1 156.2 116.1 130.3 31.4 47.5 206.7 104.9 165.3 103.0 163.6 96.0 165.3 74.1 72.1 82.1 73.1 76.3 17.2 105.7 72.1 74.6 72.8 71.8 16.9 127.1 131.3 116.7 161.0 116.7 131.3 146.5 115.0 168.6
d: chemical shift in ppm; J: coupling constant; s: singlet; br s: broad singlet; d: doublet; br d: broad doublet; dd: double doublet; t: triplet; q: quartet; m: multiplet.
D. Yang et al. / LWT - Food Science and Technology 65 (2016) 604e609
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Table 2 1 H and 13C NMR data of compounds 5, 5a, 6, and 6a in CD3OD. H/C
5
5a
dH (J in Hz) 1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 100 200 300 400 500 600 1000 2000 3000 4000 5000 6000 1000 0 2000 0 3000 0 4000 0 5000 0 6000 0 7000 0 8000 0 9000 0 A1 A2 A3 A4 A5 R1 R2 R3 R4 R5 R6
6.87 (br s) 6.60 (d, 8.0) 6.60 (d, 8.0) 6.87 (br s) 2.73/2.64 (2H, br s) 3.37/3.10 (2H, br s)
6.17 (s) 5.09 5.54 3.86 3.81 3.83 1.33
(d, 8.8) (br s) (dd, 9.6, 3.2) (d, 3.2) (q, 6.4) (3H, d, 6.4)
7.52 7.41 7.41 7.41 7.52 7.53 6.29
(br s) (br s) (br s) (brs) (br s) (d, 16.0) (d, 16.0)
5.74 4.17 4.11 3.96 3.64 3.79 3.95 3.35 3.24 3.41 2.88 0.77
(d, 1.5) (br d, 4.5) (dd, 7.7, 4.5) (m) (dd, 12.2, 4.3) (dd, 12.2, 2.2) (br s) (m) (br d, 9.5) (dd, 9.5, 7.5) (br s) (3H, br s)
dC 133.9 130.4 116.2 156.5 116.2 130.4 30.2 47.5 206.1 105.3 165.0 106.1 164.6 96.1 161.7 73.8 73.1 74.5 73.4 76.8 17.2
135.8 129.2 130.1 131.5 130.1 129.2 146.0 118.9 167.6 107.1 92.9 76.2 84.4 61.9 105.6 72.7 72.1 74.8 71.7 16.9
6
dH (J in Hz) 7.09 (d, 8.1) 6.71 (d, 8.1) 6.71 7.09 2.91 3.47 3.30
(d, 8.1) (d, 8.1) (2H, t, 7.5) (dd, 9.9, 7.5) (m)
6.23 (s) 4.82 4.06 3.56 3.72 3.74 1.27
5.79 4.29 4.16 4.02 3.67 3.80 4.17 3.49 3.37 3.30 3.24 1.00
(d, 9.6) (t, 9.6) (dd, 9.6, 2.2) (d, 2.2) (q, 6.3) (3H, d, 6.3)
(d, 1.2) (dd, 4.0, 1.2) (dd, 7.5, 4.0) (m) (dd, 12.3, 4.8) (dd, 12.3, 2.6) (br s) (m) (dd, 9.7, 3.2) (m) (q, 6.4) (3H, d, 6.4)
dC 133.9 130.5 116.3 156.6 116.3 130.5 30.5 47.5 206.3 106.4 165.6 106.4 164.7 96.0 161.3 75.8 71.1 76.6 73.4 76.6 17.2
107.1 92.3 76.2 85.3 62.2 105.4 72.8 72.0 74.9 72.1 16.7
HR-ESI-MS m/z 867.2899 [M þ Na]þ (calcd for C38H52NaOþ 21, 867.2893, error 0.6 ppm); 1H NMR (500 MHz) and 13C NMR (125 MHz) data in CD3OD, see Table 2. 3.2. Structure determination Compound 1 was determined to have the molecular formula C27 H34O13 based on its HR-ESI-MS and NMR data. The 1H and 13C NMR (Table 1) spectra exhibited proton and carbon signals assignable for a dihydrochalcone skeleton (Qin & Liu, 2003) and two sugar moieties. Analysis of the 13Ce1H COSY spectrum revealed the direct connections of carbons to protons. According to their d (chemical shift) and J (coupling constant) values as well as the mutual correlations between ortho-coupled protons in the 1He1H COSY spectrum, both sugar moieties were determined to be b-fucopyranosyls (Cazarolli et al., 2012). In the HMBC spectrum, the correlations from H-100 to C-30 and C-20 /40 clarified the direct connection of C-100 to C-30 . In addition, the correlations from H-1000 to C-300 and
6a
dH (J in Hz) 6.88 (br s) 6.60 (d, 7.9) 6.60 (d, 7.9) 6.88 (br s) 2.74/2.65 (2H, br s) 3.36/3.09 (2H, br s)
6.18 (s) 5.14 5.70 4.04 4.02 3.91 1.34 4.38 3.47 3.38 3.56 3.62 1.26
(d, 9.1) (br s) (dd, 9.0, 2.7) (d, 2.7) (q, 6.4) (3H, d, 6.4) (d, 7.6) (dd, 9.7, 7.6) (dd, 9.7, 3.3) (d, 3.3) (q, 6.4) (3H, d, 6.4)
7.50 7.41 7.41 7.41 7.50 7.51 6.27
(br s) (br s) (br s) (br s) (br s) (d, 16.0) (d, 16.0)
5.74 4.18 4.12 3.96 3.64 3.79 3.97 3.40 3.25 3.42 2.91 0.80
(br s) (dd, 4.5, 1.1) (dd, 7.8, 4.5) (m) (dd, 12.4, 5.2) (br d, 12.4) (br s) (m) (br d, 9.6) (dd, 9.6, 7.8) (br s) (3H, d, 5.0)
dC 134.0 130.5 116.3 156.6 116.3 130.5 30.2 47.5 206.2 106.0 165.2 106.0 164.8 96.2 161.8 73.9 71.8 82.5 73.3 76.5 17.2 105.7 72.3 74.7 72.9 72.0 16.9 135.8 129.3 130.1 131.5 130.1 129.3 146.0 119.0 167.6 107.1 92.9 76.3 84.5 62.0 105.2 72.8 72.2 74.9 71.8 16.9
dH (J in Hz) 7.09 (d, 8.3) 6.70 (d, 8.3) 6.70 7.09 2.92 3.47 3.37
(d, 8.3) (d, 8.3) (2H, dd, 12.5, 6.6) (dd, 9.6, 6.6) (dd, 9.6, 3.2)
6.22 (s) 4.88 4.23 3.67 3.98 3.78 1.29 4.52 3.56 3.47 3.61 3.66 1.27
(d, 9.3) (t, 9.3) (m) (d, 2.4) (q, 6.2) (3H, d, 6.2) (d, 7.5) (dd, 9.2, 7.5) (dd, 9.2, 2.7) (d, 2.7) (q, 6.2) (3H, d, 6.2)
5.80 4.29 4.16 4.02 3.67 3.81 4.16 3.49 3.37 3.30 3.23 0.98
(br s) (dd, 5.0, 1.5) (dd, 7.6, 5.0) (m) (m) (dd, 12.4, 2.7) (br s) (m) (dd, 9.7, 3.3) (m) (q, 6.3) (3H, d, 6.4)
dC 134.0 130.5 116.3 156.6 116.3 130.5 30.5 47.6 206.3 106.3 165.6 106.9 164.7 96.0 161.4 75.8 70.0 86.2 72.9 75.5 17.3 106.0 72.0 74.8 72.9 72.0 16.9
107.1 92.4 76.5 85.2 62.1 105.5 72.8 72.8 74.9 72.1 16.6
H-300 to C-1000 revealed the connection of C-1000 to C-300 via an oxygen bridge. In view of the absolute configuration of all the fucosyl moieties in carambolasides A~D from star fruit to be D-type (Yang et al., 2015), compound 1 was hence identified as phloretin 30 -C(3-O-b-D-fucopyranosyl)-b-D-fucopyranoside, and trivially named carambolaside E. The HR-ESI-MS and NMR data of compound 2 led us to assign C36 H40O14 as its molecular formula. The 1H and 13C NMR (Table 1) spectra showed signals assignable for a dihydrochalcone skeleton, two b-fucopyranosyl moieties, and a trans-cinnamoyl unit (Yang et al., 2015). Analysis of the 1He1H COSY, 13Ce1H COSY, and HMBC spectra led to the full assignment of its 1H and 13C NMR data. The change pattern of d values at C-100 (1.7 ppm), C-200 (þ1.5), and C-300 (3.6) due to the esterification in comparison to 1 clarified the connection of cinnamoyl unit to C-200 (Xie & Yoshikawa, 2013). In the 1H NMR spectrum measured at low temperature, some protons such as H-2/6, H-3/5, and H-200 , which appeared as broad singlets in the 1H NMR spectrum measured at normal temperature, were
608
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turned into clear splits at the ratio of 5:4 (Fig. 2), suggesting the coexistence of two rotamers at the ratio of 5:4 in this situation. The two rotamers were conformational variants resulting from restricted rotation around the single bond between C-9 and C-10 re zou, due to steric hindrance of the cinnamoyl moiety (Camargo, Fe Tinoco, Kaiser, & Costa, 2012; Yang et al., 2015). In the HMBC spectrum measured at low temperature, the correlation from H-200 to C-90000 was clearly observed, which confirmed the connection of cinnamoyl unit to C-200 . Further, alkaline hydrolysis of 2 gave 1. Therefore, compound 2 was identified as phloretin 30 -C-(2-O-transcinnamoyl-3-O-b-D-fucopyranosyl)-b-D-fucopyranoside, and trivially named carambolaside F. Compound 3 was determined to have the same molecular formula as compound 2. Comparison of the 1H and 13C NMR (Table 1) spectra of these two compounds revealed their difference to be cinnamoyl moiety. Two olefinic protons in 3 were at d 6.78 (1H, d, J ¼ 12.5 Hz, H-70000 ) and 5.83 (1H, d, J ¼ 12.5 Hz, H-80000 ), which were typical of a cis configuration (Hiradate, Morita, Sugie, Fujii, & Harada, 2004). Moreover, alkaline hydrolysis of 3 afforded 1. Thus, compound 3 was identified as phloretin 30 -C-(2-O-cis-cinnamoyl-3-O-b-D-fucopyranosyl)-b-D-fucopyranoside, and trivially named carambolaside G. Compound 4 was determined to have the molecular formula C36H40O15, an oxygen atom more than compound 2. The 1H NMR spectrum (Table 1) exhibited signals of four ortho-coupled aromatic protons H-20000 /60000 and H-30000 /50000 , and two olefinic protons at d 7.45 (1H each, d, J ¼ 16.0 Hz, H-70000 ) and 6.13 (1H, d, J ¼ 16.0 Hz, H-80000 ), which were characteristic of a trans-coumaroyl moiety (Xu, Xie, Wu, & Wei, 2013). Alkaline hydrolysis of 4 yielded 1.
Consequently, compound 4 was identified as phloretin 30 -C-(2-Otrans-coumaroyl-3-O-b-D-fucopyranosyl)-b-D-fucopyranoside, and trivially named carambolaside H. Compound 5 was assigned the molecular formula C41H48O18 based on its NMR and HR-ESI-MS data. The 1H and 13C NMR (Table 2) spectra demonstrated signals readily assignable for a dihydrochalcone skeleton, a b-fucopyranosyl moiety, and a transcinnamoyl unit. Alkaline hydrolysis of 5 gave a new product 5a. Excluding the carbons of dihydrochalcone and fucosyl moieties, there remained eleven carbons in the 13C NMR spectrum of 5a (Table 2). With the aid of the HSQC spectrum, the protons directly connected to these carbons were assigned, among which the signals of an a-rhamonosyl moiety with anomeric proton at d 4.17 (1H, br s, H-R1) could be easily recognized. Analysis of the 1He1H COSY spectrum clarified the remained six protons to be H-A1, H-A2, H-A3, H-A4, and H2-A5, to which directly connected five carbons were C-A1, C-A2, C-A3, C-A4, and C-A5, which suggested the presence of an a-arabinofuranosyl moiety (Ma et al., 2001). The HMBC correlations from H-100 to C-30 , C-20 , and C-40 confirmed the direct connection of fucosyl moiety to C-30 . Moreover, the correlations from H-R1 to C-A2 and H-A2 to C-R1 proved the connection of rhamonsyl moiety to C-A2, and the correlation from H-1A to C-60 revealed the two-sugar moiety's connection to C-60 via an oxygen bridge. Based on the above evidence, compound 5a was determined to be phloretin 30 -C-b-D-fucopyranosyl-60 -O-a-L-rhamnopyranosyl-(1/2)-a-L-arabinofuranoside. Further, the change pattern of d values at C-100 (2.0 ppm), C-200 (þ2.0), and C-300 (2.1) of 5 in comparison with 5a ascertained the connection of cinnamoyl unit to C-200 (Xie & Yoshikawa, 2013). Therefore, compound 5
Fig. 2. Comparison of 1H NMR spectra of compound 2 measured at 25 C (lower) and 25 C (upper).
D. Yang et al. / LWT - Food Science and Technology 65 (2016) 604e609
distinguishing dominant mechanisms for different antioxidants (Shahidi & Zhong, 2015).
Table 3 Antioxidant activity of compounds 1~6, 5a, and 6a. Compound
ABTS (IC50, mM)
1 2 3 4 5 6 5a 6a L-Ascorbic acid
4.52 4.12 3.43 2.54 3.27 2.95 3.46 3.76 14.21
± ± ± ± ± ± ± ± ±
0.12 0.05 0.15 0.02 0.06 0.03 0.14 0.05 0.23
609
DPPH (IC50, mM)
FRAP (mmol/g)
>100 >100 >100 >100 >100 >100 >100 >100 27.62 ± 1.74
0.31 0.16 0.26 0.98 0.18 1.48 0.02 0.16 14.96
± ± ± ± ± ± ± ± ±
0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.38
The values represent means ± standard deviation (SD) (n ¼ 3).
was identified as phloretin 30 -C-(2-O-trans-cinnamoyl)-b-D-fucopyranosyl-60 -O-a-L-rhamnopryanosyl-(1/2)-a-L-arabinofuranoside, and trivially named carambolaside I. The HR-ESI-MS and NMR data of compound 6 led us to assign C47 H58O22 as its molecular formula, C6H10O4 more than compound 5. Comparison of the 1H and 13C NMR (Table 2) of compounds 5, 6, and 1 revealed the presence of an additional b-fucopryanosyl moiety than 5 and its direct connection to C-300 , which was confirmed by the HMBC correlation from H-1000 to C-300 . Alkaline hydrolysis of 6 afforded 6a. Thus, compound 6 was identified as phloretin 30 -C-(2-O-trans-cinnamoyl-3-O-b-D-fucopyranosyl)-b-Dfucopyranosyl-60 -O-a-L-rhamnopyransyl-(1/2)-a-L-arabinofuranoside, and trivially named carambolaside J. 3.3. Antioxidant activity As shown in Table 3, compounds 1~6, 5a, and 6a exhibited more potent ABTS þ scavenging activity with half maximal inhibitory concentration (IC50) values ranging from 4.52 to 2.52 mM than the positive control, L-ascorbic acid (14.21 mM). However, their IC50 values in DPPH scavenging assay were over 100 mM and FRAP values were much lower than the positive control, which suggested that they were weak or inactive in these two assays. Comparison of the IC50 values of the obtained eleven dihydrochalcone C-glycosides, carambolasides A ~ J, from star fruit reported here and in a previous paper (Yang et al., 2015) and two alkaline hydrolytic products 5a and 6a towards ABTS þ reveals that the addition of sugar moieties or a cinnamoyl (coumaroyl) unit to phloretin 30 -C-bD-fucopyranosyl has no obvious influence on the activity. ABTS þ scavenging assay is applicable to the study of both watersoluble and lipid-soluble antioxidants, pure compounds, and food extracts (Re et al., 1999). It was reported that the antioxidant ca pacity detected by ABTS þ scavenging assay was significantly higher for fruits, vegetables, and beverages compared to that by DPPH scavenging assay (Floegel, Kim, Chung, Koo, & Chun, 2011). Our results were consistent with the rule likely because these compounds possessed two or more sugar moieties and so had high solubility in aqueous solution. As a result, their radical scavenging capacities were better reflected by ABTS þ scavenging assay than by DPPH scavenging assay. The FRAP assay has poor correlation with other antioxidant measurements. It is therefore suggested that this assay could be used in combination with other methods in
4. Conclusions This study revealed the structures of six new dihydrochalcone Cglycosides from the fruit of Averrhoa carambola and two new alkaline hydrolytic products and their potent ABTS þ scavenging activity. Acknowledgements We are grateful for the financial support of National Basic Research Program of China (2013CB127106). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.lwt.2015.08.061. References re zou, J. P., Tinoco, L. W., Kaiser, C. R., & Costa, S. S. (2012). Camargo, L. M. M., Fe Flavonoids from Mimosa xanthocentra (Leguminosae: Mimosoideae) and molecular modelling studies for isovitexin-200 -O-a-l-rhamnopyranoside rotamers. Phytochemistry Letters, 5, 427e431. Cazarolli, L. H., Kappel, V. D., Pereira, D. F., Moresco, H. H., Brighente, I. M. C., Pizzolatti, M. G., et al. (2012). Anti-hyperglycemic action of apigenin-6-C-bfucopyranoside from Averrhoa carambola. Fitoterapia, 83, 1176e1183. Floegel, A., Kim, D. O., Chung, S. J., Koo, S. I., & Chun, O. K. (2011). Comparison of ABTS/DPPH assays to measure antioxidant capacity in popular antioxidant-rich US foods. Journal of Food Composition and Analysis, 24, 1043e1048. Hiradate, S., Morita, S., Sugie, H., Fujii, Y., & Harada, J. (2004). Phytotoxic cis-cinnamoyl glucosides from Spiraea thunbergii. Phytochemistry, 65, 731e739. Lim, T. K. (2012). Edible Medicinal and Non-medicinal Plants. In Fruits (Vol. 1, pp. 454e464). Springer. Ma, S. J., Mizutani, M., Hiratake, J., Hayashi, K., Yagi, K., Watanabe, N., et al. (2001). Substrate specificity of b-primeverosidase, a key enzyme in aroma formation during oolong tea and black tea manufacturing. Bioscience Biotechnology & Biochemistry, 65, 2719e2729. Ma, Q., Xie, H. H., Li, S., Zhang, R. F., Zhang, M. W., & Wei, X. Y. (2014). Flavonoids from the pericarps of Litchi chinensis. Journal of Agricultural and Food Chemistry, 62, 1073e1078. O'Hare, T. J. (1993). Postharvest physiology and storage of carambola (starfruit): a review. Postharvest Biology and Technology, 2, 257e267. Qin, D., & Liu, J. K. (2003). A new sweet dihydrochalcone-glucoside from leaves of Lithocarpus pachyphyllus (Kurz) Rehd. (Fagaceae). Zeitschrift fure Naturforschung C. Journal of Biosciences, 58, 759e761. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology & Medicine, 26, 1231e1237. Shahidi, F., & Zhong, Y. (2015). Measurement of antioxidant activity. Journal of Functional Foods. http://dx.doi.org/10.1016/j.jff.2015.01.047. Xie, H. H., & Yoshikawa, M. (2013). Further acylated flavonol bisdesmosides from Sinocrassula indica. Journal of Natural Products Research, 15, 885e890. Xu, X. Y., Xie, H. H., & Wei, X. Y. (2014). Jasmonoid glucosides, sesquiterpenes and coumarins from the fruit of Clausena lansium. LWT e Food Science and Technology, 59, 65e69. Xu, Q. L., Xie, H. H., Wu, P., & Wei, X. Y. (2013). Flavonoids from the capitula of Eriocaulon australe. Food Chemistry, 139, 149e154. Yang, D., Xie, H. H., Jia, X. C., & Wei, X. Y. (2015). Flavonoid C-glycosides from star fruit and their antioxidant activity. Journal of Functional Foods, 16, 204e210. Zhou, W. T., Xie, H. H., Xu, X. Y., Liang, Y. G., & Wei, X. Y. (2014). Phenolic constituents from Isodon lophanthoides var. graciliflorus and their antioxidant and antibacterial activities. Journal of Functional Foods, 6, 492e498.
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Further dihydrochalcone C-glycosides from the fruit of Averrhoa carambola Dan Yang a, c, Xuchao Jia a, c, Haihui Xie a, b, *, Xiaoyi Wei b a
Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China b Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China c University of Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 July 2015 Received in revised form 23 August 2015 Accepted 25 August 2015 Available online 28 August 2015
Further study on the chemical constituents present in the fruit of Averrhoa carambola L. (Oxalidaceae), commonly called star fruit or carambola, led to the isolation of six new dihydrochalcone C-glycosides, carambolasides E~J (1~6). Their structures were determined by spectroscopic and chemical methods, including low temperature nuclear magnetic resonance (NMR). Compounds 1~6 and two alkaline hydrolysates, 5a and 6a, exhibited more potent 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) radical cation (ABTS þ) scavenging activity with IC50 values ranging from 4.52 to 2.54 mM than L-ascorbic acid (14.21 mM). However, they were inactive in scavenging 1,1-diphenyl-2-picrylhydrazyl radical (DPPH ) and in ferric reducing antioxidant power (FRAP). © 2015 Elsevier Ltd. All rights reserved.
Keywords: Averrhoa carambola Star fruit Dihydrochalcone C-glycoside Low temperature NMR Antioxidant activity
1. Introduction
2. Materials and methods
Averrhoa carambola L. (Oxalidaceae) is a popular fruit in tropical and subtropical regions. The fruit acquires the common name of ‘star fruit’ from its star-shaped cross section when cut (O'Hare, 1993). Star fruit is yellow to bright orangey-yellow, waxy and glossy, oblong, very fleshy, sweet to acid sweet, crisp and juicy, and was found to be a good source of natural antioxidants which were attributed to its high contents of L-ascorbic acid (8~1426 mg) and total phenolics (118~5638 mg) per gram fresh weight (Lim, 2012). However, the phenolic compounds hitherto isolated from the fruit and structurally identified by spectroscopic methods were very few. Our previous study on the fruit clarified four dihydrochalcone C-glycosides, carambolasides A~D, and their potent ABTS þ scavenging activity (Yang, Xie, Jia, & Wei, 2015). The aim of this study was to further investigate this type of compounds under the guidance of ultraviolet (UV) absorbance at 225 and 280 nm.
2.1. General experimental procedures
* Corresponding author. South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China. E-mail address: [email protected] (H. Xie). http://dx.doi.org/10.1016/j.lwt.2015.08.061 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
Silica gel (100~200 mesh) was from Qingdao Haiyang Chemical Co. (Shandong, China). Sephadex LH-20 was from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Amberlite XAD-7HP macroporous resin, ABTS, DPPH, and 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) were from SigmaeAldrich (St. Louis, MO, USA). L-Ascorbic acid was from Shanghai Boao Biotech Co. (Shanghai, China). NMR spectra were measured on a Bruker DRX-400 or Ascend500 spectrometer (Bruker BioSpin Gmbh, Rheinstetten, Germany) in deuteromethanol (CD3OD) with its residual peaks of d 3.31 and 49.0 as references. Distortionless enhancement by polarization transfer (DEPT), 1He1H correlation spectroscopy (COSY), 13Ce1H COSY, hetero-nuclear single bond coherence (HSQC), and heteronuclear multiple bond correlation (HMBC) were obtained on the same NMR spectrometer. Preparative HPLC was conducted on a LC3000 set with a UV3000 scanning spectrophotometer detector (Beijing ChuangXin TongHeng Sci. & Tech. Co., Beijing, China) connected to a column of 250 mm length 20 mm inner diameter, YMC-Pack ODS-A, particle size 5 mm (YMC Co., Kyoto, Japan). Optical density (OD) values were read on a Genios microplate reader
D. Yang et al. / LWT - Food Science and Technology 65 (2016) 604e609
€nnedorf, Switzerland). The apparatuses used for (Tecan Group, Ma medium pressure liquid chromatography (MPLC), high resolution electrospray ionization mass spectrometry (HR-ESI-MS), ESI-MS, optical rotation (aD), and UV spectra were the same as previously described (Xu, Xie, & Wei, 2014).
605
2.5. Antioxidant activity evaluation Compounds 1~6, 5a, and 6a were evaluated for antioxidant ac tivity by ABTS þ scavenging assay, DPPH scavenging assay, and FRAP assay according to the methods as previously described (Ma et al., 2014; Zhou, Xie, Liang, & Wei, 2014).
2.2. Plant material 3. Result and discussion Fresh mature star fruits (sour in taste) were collected from South China Botanical Garden, Chinese Academy of Sciences (Guangzhou, Guangdong, China) in January 2011. The fruits were manually cut to pieces, sundried and followed by heating at 60 C for about 5 h, and then ground to powder.
Six new dihydrochalcone C-glycosides (1~6) (Fig. 1) were isolated from star fruit by means of methanol extraction and multiple separation techniques, including solvent fractionation, column chromatography, MPLC, and HPLC.
2.3. Extraction and isolation
3.1. Spectroscopic data of compounds
The fruit powder (6.8 kg) was extracted with methanol (MeOH) under reflux. The solution was evaporated under vacuum to give a brown extract (785 g). The extract was dissolved in water and sequentially partitioned with ethyl acetate and n-butanol to yield ethyl acetate-soluble (156.5 g) and n-butanol-soluble (443.5 g) fractions after being condensed under reduced pressure to dryness. The latter fraction was passed through an Amberlite XAD-7HP macroporous resin column eluted with water and then 90% ethanol to obtain an ethanol eluate (123.3 g). The eluate and the ethyl acetate-soluble fraction were merged (272.0 g) and subjected to silica gel column chromatography (CC) eluted with a mixture of chloroform (CHCl3)/MeOH to offer fractions 1~8. Fraction 6 (25.0 g) was subjected to silica gel CC eluted with CHCl3/MeOH to furnish fractions 6-1~6-13. Fraction 6-3 (1.1 g) was separated by MPLC and eluted with MeOH/H2O and purified by HPLC using MeOH/H2O (v/v, 65:35) as mobile phase at the flow rate of 5 mL/min to afford compounds 2 [retention time (tR) 78.2 min, 38.7 mg] and 3 (tR 49.1 min, 53.9 mg). Fraction 6-7 (2.6 g) was separated by MPLC and purified by HPLC using MeOH/H2O (v/v, 66:34) as mobile phase at 5 mL/min to yield compound 5 (tR 36.4 min, 42.0 mg). Fraction 6-10 (808 mg) was separated by MPLC and purified by HPLC using MeOH/H2O (v/v, 68:32) at 5 mL/min to obtain compound 6 (tR 45.3 min, 110.0 mg). Fraction 7 (23.5 g) was subjected to silica gel CC to offer fractions 7-1~7-10. Fraction 7-5 (1.2 g) was separated by MPLC eluted with MeOH/H2O to get fractions 7-5-1~7-5-3. Fraction 7-5-1 (68 mg) was purified by HPLC using MeOH/H2O (v/v, 58:42) as mobile phase at 5 mL/min to provide compound 4 (tR 20.8 min, 19 mg). Fraction 7-5-2 (62 mg) was separated by Sephadex LH-20 CC and silica gel CC to afford compound 1 (19 mg).
Carambolaside E (1): yellowish amorphous powder; [a]20 D þ24.7 (c 1.14, MeOH); UV (MeOH) lmax nm (log ε) 227 (4.60) and 287 (4.58); ESI-MS m/z 566 [M þ H]þ and 589 [M þ Na]þ; HR-ESI-MS m/ z 567.2069 [M þ H]þ (calcd for C27H35Oþ 13, 567.2072, error 0.6 ppm) and 589.1894 [M þ Na]þ (calcd for C27H34NaOþ 13, 589.1892, error 0.5 ppm); 1H NMR (400 MHz) and 13C NMR (100 MHz) data in CD3OD, see Table 1. Carambolaside F (2): yellowish amorphous powder; [a]20 D 103.6 (c 1.0, MeOH); UV (MeOH) lmax nm (log ε) 223 (4.63) and 280 (4.62); ESI-MS m/z 719 [M þ Na]þ and 695 [M H]; HR-ESI-MS m/z 697.2492 [M þ H]þ (calcd for C36H41Oþ 14, 697.2491, error 0.2 ppm) and 719.2317 [M þ Na]þ (calcd for C36H40NaOþ 14, 719.2310, error 0.9 ppm); 1H NMR (500 MHz) and 13C NMR (125 MHz) data in CD3OD, see Table 1. Carambolaside G (3): yellowish amorphous powder; [a]20 D 54.9 (c 0.18, MeOH); UV (MeOH) lmax nm (log ε) 223 (4.61) and 281 (4.65); ESI-MS m/z 697 [M þ H]þ, 719 [M þ Na]þ, and 695 [M H]; HR-ESI-MS m/z 697.2482 [M þ H]þ (calcd for C36H41Oþ 14, 697.2491, error 1.2 ppm) and 719.2310 [M þ Na]þ (calcd for C36H40NaOþ 14, 719.2310, error 0 ppm); 1H NMR (400 MHz) and 13C NMR (100 MHz) data in CD3OD, see Table 1. Carambolaside H (4): yellowish amorphous powder; [a]20 D 124.4 (c 0.50, MeOH); UV (MeOH) lmax nm (log ε) 227 (4.46) and 288 (4.48); ESI-MS m/z 735 [M þ Na]þ; HR-ESI-MS m/z 713.2448 [M þ H]þ (calcd for C36H41Oþ 15, 713.2440, error 1.1 ppm) and 735.2275 [M þ Na]þ (calcd for C36H40NaOþ 15, 735.2259, error 2.1 ppm); 1H NMR (400 MHz) and 13C NMR (100 MHz) data in CD3OD, see Table 1. Carambolaside I (5): yellowish amorphous powder; [a]20 D 132.4 (c 0.82, MeOH); UV (MeOH) lmax nm (log ε) 223 (4.50) and 282 (4.54); ESI-MS m/z 851 [M þ Na]þ, 827 [M H], and 863 [M þ Cl]; HR-ESI-MS m/z 829.2925 [M þ H]þ (calcd for C41H49Oþ 18, 829.2913, error 1.5 ppm) and 851.2752 [M þ Na]þ (calcd for C41H48NaOþ 18, 851.2733, error 2.2 ppm); 1H NMR (500 MHz) and 13C NMR (125 MHz) data in CD3OD, see Table 2. Carambolaside Ia (5a): yellowish amorphous powder; [a]20 D e105.0 (c 0.58, MeOH); UV (MeOH) lmax nm (log ε) 225 (4.29) and 287 (4.14); HR-ESI-MS m/z 721.2322 [M þ Na]þ (calcd for 1 13 C32H42NaOþ 17, 721.2314, error 0.7 ppm); H NMR (500 MHz) and C NMR (125 MHz) in CD3OD, see Table 2. Carambolaside J (6): yellowish amorphous powder; [a]20 D 141.4 (c 0.65, MeOH); UV (MeOH) lmax nm (log ε) 223 (4.50) and 280 (4.63); ESI-MS m/z 997 [M þ Na]þ and 973 [M H]; HR-ESI-MS m/ z 975.3485 [M þ H]þ (calcd for C47H59Oþ 22, 975.3493, error 0.8 ppm) and 997.3317 [M þ Na]þ (calcd for C47H58NaOþ 22, 997.3312, error 0.5 ppm); 1H NMR (500 MHz) and 13C NMR (125 MHz) data in CD3OD, see Table 2. Carambolaside Ja (6a): yellowish amorphous powder; [a]20 D 23.0 (c 0.54, MeOH); UV (MeOH) lmax nm (log ε) 215 (4.24) and 281 (4.11);
2.4. Alkaline hydrolysis of compounds 2~6 Compound 2 (19 mg) was dissolved in 2.5 mL of 0.15 M aqueous sodium hydroxide and stirred at 30 C for 4 h. The solution was neutralised with 0.15 M aqueous hydrochloride and fractionated with n-butanol (3 mL 3). The n-butanol layer was combined and condensed under vacuum to give a residue, which was purified by HPLC using acetonitrile (MeCN)/H2O/formic acid (v/v/v, 30:70:0.1) as mobile phase at 5 mL/min to yield a product (tR 69.8 min, 7 mg), of which the 1H and 13C NMR, ESI-MS, and [a]D data were identical to compound 1. Through similar procedures, a new product 5a (tR 109 min, 4.9 mg) was purified from the hydrolytic solution of compound 5 by HPLC using MeCN/H2O/formic acid (v/v/v, 27:73:0.1) as mobile phase at 5 mL/min, and a new product 6a (tR 53 min, 11.0 mg) was obtained from the hydrolytic solution of compound 6 by HPLC using MeCN/H2O/formic acid (v/v/v, 25:75:0.1) as mobile phase at 5 mL/min. Compound 1 was identified in the hydrolytic solutions of compounds 2~4 by HPLC analysis in comparison with the authentic sample.
606
D. Yang et al. / LWT - Food Science and Technology 65 (2016) 604e609
Fig. 1. Structures of compounds 1~6 and two hydrolytic products 5a and 6a.
Table 1 1 H and 13C NMR data of compounds 1~4 in CD3OD. H/C
1
2
dH (J in Hz) 1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 100 200 300 400 500 600 1000 2000 3000 4000 5000 6000 1000 0 2000 0 3000 0 4000 0 5000 0 6000 0 7000 0 8000 0 9000 0
7.04 (d, 8.5) 6.69 (d, 8.5) 6.69 7.04 2.85 3.29
(d, 8.5) (d, 8.5) (2H, dd, 8.4, 7.2) (2H, dd, 8.4, 7.2)
5.89 (s) 4.85 4.18 3.68 3.97 3.77 1.29 4.53 3.56 3.49 3.60 3.66 1.27
(d, 9.6) (t, 9.6) (dd, 9.6, 2.8) (d, 2.8) (q, 6.4) (3H, d, 6.4) (d, 7.5) (dd, 9.7, 7.5) (dd, 9.7, 3.3) (d, 3.3) (q, 6.4) (3H, d, 6.4)
dC (DEPT) 133.9 130.3 116.1 156.4 116.1 130.3 31.4 47.5 206.7 105.5 164.5 104.8 164.5 96.1 164.5 75.8 70.3 85.8 72.8 75.8 17.3 105.9 72.8 74.7 72.9 71.9 16.9
(C) (CH) (CH) (C) (CH) (CH) (CH2) (CH2) (C) (C) (C) (C) (C) (CH) (C) (CH) (CH) (CH) (CH) (CH) (CH3) (CH) (CH) (CH) (CH) (CH) (CH3)
3
dH (J in Hz) 7.05/6.90 (br s) 6.66 (br s) 6.66 (br s) 7.05/6.90 (br s) 2.87/2.58 (2H, br s) 3.27/3.11 (2H, br s)
5.83 (br s) 5.08 (d, 9.2) 5.83/5.71 (br s) 4.04 (dd, 7.3, 3.0) 4.03 (d, 3.0) 3.89 (q, 6.3) 1.35 (3H, d, 6.3) 4.40 (d, 7.6) 3.47 (dd, 9.8, 7.6) 3.39 (dd, 9.8, 3.3) 3.57 (d, 3.3) 3.63 (q, 6.5) 1.26 (3H, d, 6.5) 7.50 7.37 7.37 7.37 7.50 7.52 6.33
(br s) (br s) (br s) (br s) (br s) (d, 15.9) (d, 15.9)
dC 133.8 130.2 116.1 156.2 116.1 130.2 31.2 47.4 206.6 104.8 164.8 103.0 163.6 96.0 164.8 74.1 71.8 82.2 73.1 76.3 17.2 105.7 72.1 74.5 72.7 71.8 16.9 135.6 129.1 129.9 131.4 129.9 129.1 146.1 118.7 167.9
dH (J in Hz) 7.05/6.89 (br s) 6.65 (br s) 6.65 (br s) 7.05/6.89 (br s) 2.85/2.65 (2H, br s) 3.28/3.13 (2H, br s)
5.85 (br s) 5.02 (d, 9.9) 5.92/5.70 (br s) 3.94 (br d, 9.6) 3.99 (d, 2.9) 3.85 (q, 6.4) 1.32 (3H, d, 6.4) 4.36 (d, 7.0) 3.46 (dd, 9.5, 7.0) 3.42 (dd, 9.5, 3.1) 3.58 (d, 3.1) 3.61 (q, 6.4) 1.26 (3H, d, 6.4) 7.50/7.37 (br s) 7.20 (br s) 7.20 (br s) 7.20 (br) 7.50/7.37 (br s) 6.78 (d, 12.5) 5.83 (d, 12.5)
4
dC 133.9 130.3 116.0 156.2 116.0 130.3 31.1 47.2 206.7 105.0 165.4 103.3 163.6 96.1 165.0 74.0 71.9 82.1 73.1 76.4 17.2 105.9 72.1 74.8 72.9 71.9 16.9 135.9 128.9 130.9 129.9 130.9 128.9 144.0 120.1 166.8
dH (J in Hz) 7.06/6.92 (br s) 6.67 (br s) 6.67 (br s) 7.05/6.92 (br s) 2.86/2.59 (2H, br s) 3.28/3.11 (2H, br s)
5.83 (br s) 5.07 (d, 9.7) 5.83/5.70 (br s) 4.00 (dd, 9.6, 2.8) 4.01 (d, 2.8) 3.85 (q, 6.3) 1.33 (3H, d, 6.3) 4.39 (d, 7.6) 3.48 (dd, 9.7, 7.6) 3.40 (dd, 9.7, 3.2) 3.56 (d, 3.2) 3.59 (q, 6.3) 1.25 (3H, d, 6.3) 7.36/7.23 (d, 8.5) 6.78 (d, 8.5) 6.78 (d, 8.5) 7.36/7.23 (d, 8.5) 7.45 (d, 16.0) 6.15 (d, 16.0)
dC 133.9 130.3 116.1 156.2 116.1 130.3 31.4 47.5 206.7 104.9 165.3 103.0 163.6 96.0 165.3 74.1 72.1 82.1 73.1 76.3 17.2 105.7 72.1 74.6 72.8 71.8 16.9 127.1 131.3 116.7 161.0 116.7 131.3 146.5 115.0 168.6
d: chemical shift in ppm; J: coupling constant; s: singlet; br s: broad singlet; d: doublet; br d: broad doublet; dd: double doublet; t: triplet; q: quartet; m: multiplet.
D. Yang et al. / LWT - Food Science and Technology 65 (2016) 604e609
607
Table 2 1 H and 13C NMR data of compounds 5, 5a, 6, and 6a in CD3OD. H/C
5
5a
dH (J in Hz) 1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 100 200 300 400 500 600 1000 2000 3000 4000 5000 6000 1000 0 2000 0 3000 0 4000 0 5000 0 6000 0 7000 0 8000 0 9000 0 A1 A2 A3 A4 A5 R1 R2 R3 R4 R5 R6
6.87 (br s) 6.60 (d, 8.0) 6.60 (d, 8.0) 6.87 (br s) 2.73/2.64 (2H, br s) 3.37/3.10 (2H, br s)
6.17 (s) 5.09 5.54 3.86 3.81 3.83 1.33
(d, 8.8) (br s) (dd, 9.6, 3.2) (d, 3.2) (q, 6.4) (3H, d, 6.4)
7.52 7.41 7.41 7.41 7.52 7.53 6.29
(br s) (br s) (br s) (brs) (br s) (d, 16.0) (d, 16.0)
5.74 4.17 4.11 3.96 3.64 3.79 3.95 3.35 3.24 3.41 2.88 0.77
(d, 1.5) (br d, 4.5) (dd, 7.7, 4.5) (m) (dd, 12.2, 4.3) (dd, 12.2, 2.2) (br s) (m) (br d, 9.5) (dd, 9.5, 7.5) (br s) (3H, br s)
dC 133.9 130.4 116.2 156.5 116.2 130.4 30.2 47.5 206.1 105.3 165.0 106.1 164.6 96.1 161.7 73.8 73.1 74.5 73.4 76.8 17.2
135.8 129.2 130.1 131.5 130.1 129.2 146.0 118.9 167.6 107.1 92.9 76.2 84.4 61.9 105.6 72.7 72.1 74.8 71.7 16.9
6
dH (J in Hz) 7.09 (d, 8.1) 6.71 (d, 8.1) 6.71 7.09 2.91 3.47 3.30
(d, 8.1) (d, 8.1) (2H, t, 7.5) (dd, 9.9, 7.5) (m)
6.23 (s) 4.82 4.06 3.56 3.72 3.74 1.27
5.79 4.29 4.16 4.02 3.67 3.80 4.17 3.49 3.37 3.30 3.24 1.00
(d, 9.6) (t, 9.6) (dd, 9.6, 2.2) (d, 2.2) (q, 6.3) (3H, d, 6.3)
(d, 1.2) (dd, 4.0, 1.2) (dd, 7.5, 4.0) (m) (dd, 12.3, 4.8) (dd, 12.3, 2.6) (br s) (m) (dd, 9.7, 3.2) (m) (q, 6.4) (3H, d, 6.4)
dC 133.9 130.5 116.3 156.6 116.3 130.5 30.5 47.5 206.3 106.4 165.6 106.4 164.7 96.0 161.3 75.8 71.1 76.6 73.4 76.6 17.2
107.1 92.3 76.2 85.3 62.2 105.4 72.8 72.0 74.9 72.1 16.7
HR-ESI-MS m/z 867.2899 [M þ Na]þ (calcd for C38H52NaOþ 21, 867.2893, error 0.6 ppm); 1H NMR (500 MHz) and 13C NMR (125 MHz) data in CD3OD, see Table 2. 3.2. Structure determination Compound 1 was determined to have the molecular formula C27 H34O13 based on its HR-ESI-MS and NMR data. The 1H and 13C NMR (Table 1) spectra exhibited proton and carbon signals assignable for a dihydrochalcone skeleton (Qin & Liu, 2003) and two sugar moieties. Analysis of the 13Ce1H COSY spectrum revealed the direct connections of carbons to protons. According to their d (chemical shift) and J (coupling constant) values as well as the mutual correlations between ortho-coupled protons in the 1He1H COSY spectrum, both sugar moieties were determined to be b-fucopyranosyls (Cazarolli et al., 2012). In the HMBC spectrum, the correlations from H-100 to C-30 and C-20 /40 clarified the direct connection of C-100 to C-30 . In addition, the correlations from H-1000 to C-300 and
6a
dH (J in Hz) 6.88 (br s) 6.60 (d, 7.9) 6.60 (d, 7.9) 6.88 (br s) 2.74/2.65 (2H, br s) 3.36/3.09 (2H, br s)
6.18 (s) 5.14 5.70 4.04 4.02 3.91 1.34 4.38 3.47 3.38 3.56 3.62 1.26
(d, 9.1) (br s) (dd, 9.0, 2.7) (d, 2.7) (q, 6.4) (3H, d, 6.4) (d, 7.6) (dd, 9.7, 7.6) (dd, 9.7, 3.3) (d, 3.3) (q, 6.4) (3H, d, 6.4)
7.50 7.41 7.41 7.41 7.50 7.51 6.27
(br s) (br s) (br s) (br s) (br s) (d, 16.0) (d, 16.0)
5.74 4.18 4.12 3.96 3.64 3.79 3.97 3.40 3.25 3.42 2.91 0.80
(br s) (dd, 4.5, 1.1) (dd, 7.8, 4.5) (m) (dd, 12.4, 5.2) (br d, 12.4) (br s) (m) (br d, 9.6) (dd, 9.6, 7.8) (br s) (3H, d, 5.0)
dC 134.0 130.5 116.3 156.6 116.3 130.5 30.2 47.5 206.2 106.0 165.2 106.0 164.8 96.2 161.8 73.9 71.8 82.5 73.3 76.5 17.2 105.7 72.3 74.7 72.9 72.0 16.9 135.8 129.3 130.1 131.5 130.1 129.3 146.0 119.0 167.6 107.1 92.9 76.3 84.5 62.0 105.2 72.8 72.2 74.9 71.8 16.9
dH (J in Hz) 7.09 (d, 8.3) 6.70 (d, 8.3) 6.70 7.09 2.92 3.47 3.37
(d, 8.3) (d, 8.3) (2H, dd, 12.5, 6.6) (dd, 9.6, 6.6) (dd, 9.6, 3.2)
6.22 (s) 4.88 4.23 3.67 3.98 3.78 1.29 4.52 3.56 3.47 3.61 3.66 1.27
(d, 9.3) (t, 9.3) (m) (d, 2.4) (q, 6.2) (3H, d, 6.2) (d, 7.5) (dd, 9.2, 7.5) (dd, 9.2, 2.7) (d, 2.7) (q, 6.2) (3H, d, 6.2)
5.80 4.29 4.16 4.02 3.67 3.81 4.16 3.49 3.37 3.30 3.23 0.98
(br s) (dd, 5.0, 1.5) (dd, 7.6, 5.0) (m) (m) (dd, 12.4, 2.7) (br s) (m) (dd, 9.7, 3.3) (m) (q, 6.3) (3H, d, 6.4)
dC 134.0 130.5 116.3 156.6 116.3 130.5 30.5 47.6 206.3 106.3 165.6 106.9 164.7 96.0 161.4 75.8 70.0 86.2 72.9 75.5 17.3 106.0 72.0 74.8 72.9 72.0 16.9
107.1 92.4 76.5 85.2 62.1 105.5 72.8 72.8 74.9 72.1 16.6
H-300 to C-1000 revealed the connection of C-1000 to C-300 via an oxygen bridge. In view of the absolute configuration of all the fucosyl moieties in carambolasides A~D from star fruit to be D-type (Yang et al., 2015), compound 1 was hence identified as phloretin 30 -C(3-O-b-D-fucopyranosyl)-b-D-fucopyranoside, and trivially named carambolaside E. The HR-ESI-MS and NMR data of compound 2 led us to assign C36 H40O14 as its molecular formula. The 1H and 13C NMR (Table 1) spectra showed signals assignable for a dihydrochalcone skeleton, two b-fucopyranosyl moieties, and a trans-cinnamoyl unit (Yang et al., 2015). Analysis of the 1He1H COSY, 13Ce1H COSY, and HMBC spectra led to the full assignment of its 1H and 13C NMR data. The change pattern of d values at C-100 (1.7 ppm), C-200 (þ1.5), and C-300 (3.6) due to the esterification in comparison to 1 clarified the connection of cinnamoyl unit to C-200 (Xie & Yoshikawa, 2013). In the 1H NMR spectrum measured at low temperature, some protons such as H-2/6, H-3/5, and H-200 , which appeared as broad singlets in the 1H NMR spectrum measured at normal temperature, were
608
D. Yang et al. / LWT - Food Science and Technology 65 (2016) 604e609
turned into clear splits at the ratio of 5:4 (Fig. 2), suggesting the coexistence of two rotamers at the ratio of 5:4 in this situation. The two rotamers were conformational variants resulting from restricted rotation around the single bond between C-9 and C-10 re zou, due to steric hindrance of the cinnamoyl moiety (Camargo, Fe Tinoco, Kaiser, & Costa, 2012; Yang et al., 2015). In the HMBC spectrum measured at low temperature, the correlation from H-200 to C-90000 was clearly observed, which confirmed the connection of cinnamoyl unit to C-200 . Further, alkaline hydrolysis of 2 gave 1. Therefore, compound 2 was identified as phloretin 30 -C-(2-O-transcinnamoyl-3-O-b-D-fucopyranosyl)-b-D-fucopyranoside, and trivially named carambolaside F. Compound 3 was determined to have the same molecular formula as compound 2. Comparison of the 1H and 13C NMR (Table 1) spectra of these two compounds revealed their difference to be cinnamoyl moiety. Two olefinic protons in 3 were at d 6.78 (1H, d, J ¼ 12.5 Hz, H-70000 ) and 5.83 (1H, d, J ¼ 12.5 Hz, H-80000 ), which were typical of a cis configuration (Hiradate, Morita, Sugie, Fujii, & Harada, 2004). Moreover, alkaline hydrolysis of 3 afforded 1. Thus, compound 3 was identified as phloretin 30 -C-(2-O-cis-cinnamoyl-3-O-b-D-fucopyranosyl)-b-D-fucopyranoside, and trivially named carambolaside G. Compound 4 was determined to have the molecular formula C36H40O15, an oxygen atom more than compound 2. The 1H NMR spectrum (Table 1) exhibited signals of four ortho-coupled aromatic protons H-20000 /60000 and H-30000 /50000 , and two olefinic protons at d 7.45 (1H each, d, J ¼ 16.0 Hz, H-70000 ) and 6.13 (1H, d, J ¼ 16.0 Hz, H-80000 ), which were characteristic of a trans-coumaroyl moiety (Xu, Xie, Wu, & Wei, 2013). Alkaline hydrolysis of 4 yielded 1.
Consequently, compound 4 was identified as phloretin 30 -C-(2-Otrans-coumaroyl-3-O-b-D-fucopyranosyl)-b-D-fucopyranoside, and trivially named carambolaside H. Compound 5 was assigned the molecular formula C41H48O18 based on its NMR and HR-ESI-MS data. The 1H and 13C NMR (Table 2) spectra demonstrated signals readily assignable for a dihydrochalcone skeleton, a b-fucopyranosyl moiety, and a transcinnamoyl unit. Alkaline hydrolysis of 5 gave a new product 5a. Excluding the carbons of dihydrochalcone and fucosyl moieties, there remained eleven carbons in the 13C NMR spectrum of 5a (Table 2). With the aid of the HSQC spectrum, the protons directly connected to these carbons were assigned, among which the signals of an a-rhamonosyl moiety with anomeric proton at d 4.17 (1H, br s, H-R1) could be easily recognized. Analysis of the 1He1H COSY spectrum clarified the remained six protons to be H-A1, H-A2, H-A3, H-A4, and H2-A5, to which directly connected five carbons were C-A1, C-A2, C-A3, C-A4, and C-A5, which suggested the presence of an a-arabinofuranosyl moiety (Ma et al., 2001). The HMBC correlations from H-100 to C-30 , C-20 , and C-40 confirmed the direct connection of fucosyl moiety to C-30 . Moreover, the correlations from H-R1 to C-A2 and H-A2 to C-R1 proved the connection of rhamonsyl moiety to C-A2, and the correlation from H-1A to C-60 revealed the two-sugar moiety's connection to C-60 via an oxygen bridge. Based on the above evidence, compound 5a was determined to be phloretin 30 -C-b-D-fucopyranosyl-60 -O-a-L-rhamnopyranosyl-(1/2)-a-L-arabinofuranoside. Further, the change pattern of d values at C-100 (2.0 ppm), C-200 (þ2.0), and C-300 (2.1) of 5 in comparison with 5a ascertained the connection of cinnamoyl unit to C-200 (Xie & Yoshikawa, 2013). Therefore, compound 5
Fig. 2. Comparison of 1H NMR spectra of compound 2 measured at 25 C (lower) and 25 C (upper).
D. Yang et al. / LWT - Food Science and Technology 65 (2016) 604e609
distinguishing dominant mechanisms for different antioxidants (Shahidi & Zhong, 2015).
Table 3 Antioxidant activity of compounds 1~6, 5a, and 6a. Compound
ABTS (IC50, mM)
1 2 3 4 5 6 5a 6a L-Ascorbic acid
4.52 4.12 3.43 2.54 3.27 2.95 3.46 3.76 14.21
± ± ± ± ± ± ± ± ±
0.12 0.05 0.15 0.02 0.06 0.03 0.14 0.05 0.23
609
DPPH (IC50, mM)
FRAP (mmol/g)
>100 >100 >100 >100 >100 >100 >100 >100 27.62 ± 1.74
0.31 0.16 0.26 0.98 0.18 1.48 0.02 0.16 14.96
± ± ± ± ± ± ± ± ±
0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.38
The values represent means ± standard deviation (SD) (n ¼ 3).
was identified as phloretin 30 -C-(2-O-trans-cinnamoyl)-b-D-fucopyranosyl-60 -O-a-L-rhamnopryanosyl-(1/2)-a-L-arabinofuranoside, and trivially named carambolaside I. The HR-ESI-MS and NMR data of compound 6 led us to assign C47 H58O22 as its molecular formula, C6H10O4 more than compound 5. Comparison of the 1H and 13C NMR (Table 2) of compounds 5, 6, and 1 revealed the presence of an additional b-fucopryanosyl moiety than 5 and its direct connection to C-300 , which was confirmed by the HMBC correlation from H-1000 to C-300 . Alkaline hydrolysis of 6 afforded 6a. Thus, compound 6 was identified as phloretin 30 -C-(2-O-trans-cinnamoyl-3-O-b-D-fucopyranosyl)-b-Dfucopyranosyl-60 -O-a-L-rhamnopyransyl-(1/2)-a-L-arabinofuranoside, and trivially named carambolaside J. 3.3. Antioxidant activity As shown in Table 3, compounds 1~6, 5a, and 6a exhibited more potent ABTS þ scavenging activity with half maximal inhibitory concentration (IC50) values ranging from 4.52 to 2.52 mM than the positive control, L-ascorbic acid (14.21 mM). However, their IC50 values in DPPH scavenging assay were over 100 mM and FRAP values were much lower than the positive control, which suggested that they were weak or inactive in these two assays. Comparison of the IC50 values of the obtained eleven dihydrochalcone C-glycosides, carambolasides A ~ J, from star fruit reported here and in a previous paper (Yang et al., 2015) and two alkaline hydrolytic products 5a and 6a towards ABTS þ reveals that the addition of sugar moieties or a cinnamoyl (coumaroyl) unit to phloretin 30 -C-bD-fucopyranosyl has no obvious influence on the activity. ABTS þ scavenging assay is applicable to the study of both watersoluble and lipid-soluble antioxidants, pure compounds, and food extracts (Re et al., 1999). It was reported that the antioxidant ca pacity detected by ABTS þ scavenging assay was significantly higher for fruits, vegetables, and beverages compared to that by DPPH scavenging assay (Floegel, Kim, Chung, Koo, & Chun, 2011). Our results were consistent with the rule likely because these compounds possessed two or more sugar moieties and so had high solubility in aqueous solution. As a result, their radical scavenging capacities were better reflected by ABTS þ scavenging assay than by DPPH scavenging assay. The FRAP assay has poor correlation with other antioxidant measurements. It is therefore suggested that this assay could be used in combination with other methods in
4. Conclusions This study revealed the structures of six new dihydrochalcone Cglycosides from the fruit of Averrhoa carambola and two new alkaline hydrolytic products and their potent ABTS þ scavenging activity. Acknowledgements We are grateful for the financial support of National Basic Research Program of China (2013CB127106). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.lwt.2015.08.061. References re zou, J. P., Tinoco, L. W., Kaiser, C. R., & Costa, S. S. (2012). Camargo, L. M. M., Fe Flavonoids from Mimosa xanthocentra (Leguminosae: Mimosoideae) and molecular modelling studies for isovitexin-200 -O-a-l-rhamnopyranoside rotamers. Phytochemistry Letters, 5, 427e431. Cazarolli, L. H., Kappel, V. D., Pereira, D. F., Moresco, H. H., Brighente, I. M. C., Pizzolatti, M. G., et al. (2012). Anti-hyperglycemic action of apigenin-6-C-bfucopyranoside from Averrhoa carambola. Fitoterapia, 83, 1176e1183. Floegel, A., Kim, D. O., Chung, S. J., Koo, S. I., & Chun, O. K. (2011). Comparison of ABTS/DPPH assays to measure antioxidant capacity in popular antioxidant-rich US foods. Journal of Food Composition and Analysis, 24, 1043e1048. Hiradate, S., Morita, S., Sugie, H., Fujii, Y., & Harada, J. (2004). Phytotoxic cis-cinnamoyl glucosides from Spiraea thunbergii. Phytochemistry, 65, 731e739. Lim, T. K. (2012). Edible Medicinal and Non-medicinal Plants. In Fruits (Vol. 1, pp. 454e464). Springer. Ma, S. J., Mizutani, M., Hiratake, J., Hayashi, K., Yagi, K., Watanabe, N., et al. (2001). Substrate specificity of b-primeverosidase, a key enzyme in aroma formation during oolong tea and black tea manufacturing. Bioscience Biotechnology & Biochemistry, 65, 2719e2729. Ma, Q., Xie, H. H., Li, S., Zhang, R. F., Zhang, M. W., & Wei, X. Y. (2014). Flavonoids from the pericarps of Litchi chinensis. Journal of Agricultural and Food Chemistry, 62, 1073e1078. O'Hare, T. J. (1993). Postharvest physiology and storage of carambola (starfruit): a review. Postharvest Biology and Technology, 2, 257e267. Qin, D., & Liu, J. K. (2003). A new sweet dihydrochalcone-glucoside from leaves of Lithocarpus pachyphyllus (Kurz) Rehd. (Fagaceae). Zeitschrift fure Naturforschung C. Journal of Biosciences, 58, 759e761. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology & Medicine, 26, 1231e1237. Shahidi, F., & Zhong, Y. (2015). Measurement of antioxidant activity. Journal of Functional Foods. http://dx.doi.org/10.1016/j.jff.2015.01.047. Xie, H. H., & Yoshikawa, M. (2013). Further acylated flavonol bisdesmosides from Sinocrassula indica. Journal of Natural Products Research, 15, 885e890. Xu, X. Y., Xie, H. H., & Wei, X. Y. (2014). Jasmonoid glucosides, sesquiterpenes and coumarins from the fruit of Clausena lansium. LWT e Food Science and Technology, 59, 65e69. Xu, Q. L., Xie, H. H., Wu, P., & Wei, X. Y. (2013). Flavonoids from the capitula of Eriocaulon australe. Food Chemistry, 139, 149e154. Yang, D., Xie, H. H., Jia, X. C., & Wei, X. Y. (2015). Flavonoid C-glycosides from star fruit and their antioxidant activity. Journal of Functional Foods, 16, 204e210. Zhou, W. T., Xie, H. H., Xu, X. Y., Liang, Y. G., & Wei, X. Y. (2014). Phenolic constituents from Isodon lophanthoides var. graciliflorus and their antioxidant and antibacterial activities. Journal of Functional Foods, 6, 492e498.