Food Chemistry 152 (2014) 300–306

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Phenolic compounds from Origanum vulgare and their antioxidant and antiviral activities Xiao-Li Zhang a,b,1, Yu-Shan Guo a,b,1, Chun-Hua Wang a,b, Guo-Qiang Li a,b, Jiao-Jiao Xu a,b, Hau Yin Chung c,d, Wen-Cai Ye a,b, Yao-Lan Li a,b,⇑, Guo-Cai Wang a,b,⇑ a

Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 51032, China Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 51032, China School of Life Sciences, The Chinese University of Hong Kong, Hong Kong Special Administrative Region d Food and Nutritional Sciences Programme, The Chinese University of Hong Kong, Hong Kong Special Administrative Region b c

a r t i c l e

i n f o

Article history: Received 7 August 2013 Received in revised form 20 November 2013 Accepted 25 November 2013 Available online 1 December 2013 Keywords: Origanum vulgare Lamiaceae Phenolic compounds Antioxidant Antiviral

a b s t r a c t In the present study, six new phenolic compounds (1–6) along with five known ones were isolated from the ethanol extract of the whole plants of Origanum vulgare. The structures of the new compounds were identified on the basis of extensive spectroscopic analyses (UV, IR, NMR, and HRESIMS) and acid hydrolysis. Twenty-one phenolic compounds isolated from O. vulgare in our previous and present studies were evaluated for their in vitro antioxidant activity using 2,2-diphenyl-1-picryhydrazyl (DPPH) radical-scavenging and ferric-reducing antioxidant power (FRAP) assays; twelve of them including two new compounds exhibited significant antioxidant activity comparable to that of ascorbic acid. In addition, the antiviral effects against respiratory syncytial virus (RSV), Coxsackie virus B3 (CVB3) and herpes simplex virus type 1 (HSV-1) were tested by cytopathic effect (CPE) reduction assay. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Origanum vulgare (Lamiaceae) is a perennial herb distributed in Europe, North Africa, America and Asia (Kintzios, 2002). The herb is widely used as a spice in Western diets, and is also commonly used as a traditional medicine for the treatment of various diseases, such as cold, cough, and digestive disorders (Yin, Fretté, Christensen, & Grevsen, 2012). The plant is known for its powerful antimicrobial and antioxidant activities (Lemhadri, Zeggwagh, Maghrani, Jouad, & Eddouks, 2004), which may explain its use in traditional medicine. The antimicrobial activity of O. vulgare is due to its high content of volatile oils (Esen et al., 2007; Faleiro et al., 2005; Karakaya, El, Karagözlü, & Sahin, 2011). In addition, these volatile constituents significantly contribute to the aroma and flavour of the herb. The phenolic compounds including flavonoids and phenolic acids, another kind of abundant constituent in O. vulgare, are responsible for its antioxidant activity (Chou, Ding, Lin, Liang, & Liang, 2010; Ding, Chou, & Liang, 2010; Liang et al., 2012). Moreover, these phenolic antioxidants possess diverse biological ⇑ Corresponding authors at: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, China. Tel./fax: +86 20 8522 1559 (Y.-L. Li), +86 20 8522 3553 (G.-C. Wang). E-mail addresses: [email protected] (Y.-L. Li), [email protected] (G.-C. Wang). 1 These authors contributed equally to this work. 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.11.153

activities, for instance, anti-ulcer, anti-inflammatory, antidiabetic, antiviral, cytotoxic and antitumour (Saxena, Saxena, & Pradhan, 2012), and they are believed to be responsible for the health effects of O. vulgare. In this work, we report the isolation of six new phenolic compounds (1–6) (Fig. 1) from O. vulgare, along with five known ones: 2,5-dihydroxybenzoic acid (7) (Xie & Li, 2002), 3,4-dihydroxybenzoic acid (8) (Zhang, Fang, & Ye, 2008), rosmarinic acid (9) (Wu, Song, Zhao, & JIA, 2011a), origanoside (10) (Nakatani & Kikuzaki, 1987), and maltol 60 -O-(5-O-p-coumaroyl)-b-D-apiofuranosyl-b-Dglucopyranoside (11) (Li et al., 2008). The structures of the new compounds were identified with spectroscopic analyses and acid hydrolysis experiments. The phenolic compounds combining with the previously isolated ones E-caffeic acid (12), amburoside A (13), oresbiusin A (14), (+)-(R)-butyl rosmarinate (15), apigenin (16), apigenin 7-O-b-D-glucoside (17), luteolin (18), 6,7,40 -trihydroxyflavone (19), 5,7,30 ,40 -tetrahydroxy-8-C-p-hydroxybenzylflavone (20) and didymin (21) (Guo et al., 2012) were subjected to in vitro antioxidant evaluation with 2,2-diphenyl-1-picryhydrazyl (DPPH) radical-scavenging and ferric-reducing antioxidant power (FRAP) assays. Furthermore, their antiviral activities against respiratory syncytial virus (RSV), Coxsackie virus B3 (CVB3) and herpes simplex virus type 1 (HSV-1) were determined by cytopathic effect (CPE) assay.

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O 7' 1'

1

O

HO HO

O

7

OH

OH

O 4

O

OH 2'

H 3CO

1''

HO 4''

5'

OH

7''

1

O O

HO OCH 3 HO

O

AcO

1' 4' 3'

OH

OH

OH

2

1'' O

O

O

OCH 3

OH

1''' O

O 7'

O 4

1

O

O

7

OAc

HO OH

HO

O

HO

OH OH

O HO O OH

1'' O

OH

1'''

3

O

OH

OR

O

4 R = CH 3 5R=H OH O

HO HO O

AcO

O

O

O

OCH 3

1'''

OH

HO

1''

OH

O

HO 6 Fig. 1. Chemical structures of compounds 1–6.

2. Materials and methods 2.1. General experimental procedures Melting points were obtained on an X-5 micro-melting point detector (Tech, Beijing, China). Optical rotation values were measured by a JASCO P-1020 polarimeter. UV spectra were recorded on a JASCO V-550 UV/VIS spectrophotometer (JASCO International Co., Ltd., Tokyo, Japan). IR spectra were measured on a JASCO FT/IR480 plus FT-IR spectrometer. HR-ESI-MS data were determined by an Agilent 6210 ESI/TOF mass spectrometer (Agilent, Santa Clara, CA). NMR spectra were carried out on a Bruker AV-400 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) using TMS as internal standard. A Waters 1525 pump and a 2487 dual k absorbance detector were used for analytical HPLC (Waters Corporation, Milford, MA) with a Cosmosil 5C18-MS-II column (4.6  250 mm). A Gilson 360 pump and a UV/VIS-152 detector were used for preparative HPLC with a Cosmosil 5C18-MS-II column (20  250 mm). Column chromatography was carried out on macroporous resin Diaion HP-20 (Mitsubishi Chemical Corporation, Tokyo, Japan), silica gel (200–300 mesh; Qingdao Marine Chemical Group Co., Ltd., Qingdao, China), ODS (50 lm, 120 Å; YMC, Kyoto, Japan) or Sephadex LH-20 (Pharmacia Biotech, Uppsala, Sweden). TLC was performed using precoated silica gel GF254 plates (Yantai Chemical Industry Research Institute, Yantai, China). L-ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO). 2.2. Plant material Whole plants of O. vulgare were collected in Guangzhou City, Guangdong Province of China, in September 2012 and authenticated by Prof. Guang-Xiong Zhou (College of Pharmacy, Jinan

University). A voucher specimen (No. 2012071113) was deposited in the Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou, China. 2.3. Extraction and isolation The air-dried whole plants of O. vulgare (10.0 kg) were powdered and percolated with 95% (V/V) ethanol solution at room temperature. The ethanol extract was concentrated in vacuo to yield a residue (240 g) which was suspended in water and partitioned with petroleum ether and ethyl acetate, respectively. The ethyl acetate-soluble fraction (112 g) was subjected to a silica gel column eluting with chloroform/methanol (CHCl3/MeOH) (100:0 ? 0:100, v/v) to afford 7 fractions (A–G). Fraction D (8 g) was further separated by silica gel column chromatography (2.5  80 cm, 200 g) with CHCl3/MeOH (98:2 ? 70:30, v/v) as well as monitored by TLC (CHCl3/MeOH, 80:20) to afford 10 subfractions D-1–10. Subfraction D-3 (1.8 g) was purified by Sephadex LH-20 column with CHCl3/MeOH (50:50, v/v) and preparative HPLC with methanol/water (MeOH/H2O) (60:40, v/v) to afford 3 (14 mg) and 9 (18 mg). Compounds 8 (21 mg) and 10 (24 mg) were separated from subfraction D-5 (2.1 g) by Sephadex LH-20 column (CHCl3/MeOH, 50:50, v/v) and preparative HPLC (MeOH/H2O, 60:40, v/v). Subfraction D-7 (1.1 g) was separated by preparative RP-HPLC (MeOH/H2O, 50:50, v/v) to afford compound 11 (40 mg). Similarly, fraction E (22 g) was subjected to a silica gel column eluting with CHCl3/MeOH (95:5 ? 70:30, v/v) to provide 12 subfractions E-1–12. Subfraction E-5 (3.0 g) was separated by a Sephadex LH-20 column (CHCl3/MeOH, 50:50, v/v) to give 4 (8 mg) and 7 (19 mg). Compounds 5 (5 mg) and 6 (10 mg) were isolated from subfraction E-7 (1.9 g) by an ODS column and preparative HPLC (MeOH/H2O, 60:40, v/v). Subfraction E-9 (10.8 g) was purified by an ODS column and preparative HPLC (MeOH/H2O, 40:60, v/v) to yield 1 (38 mg) and 2 (8 mg), respectively.

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4-[[(20 ,50 -Dihydroxybenzoyl)oxy]methyl]phenyl O-b-D-glucopyranoside (1): white amorphous powder; ½a25 D +17.8 (c 0.18, MeOH); UV (MeOH) kmax: 205, 221, 263, 297 nm; IR (KBr) mmax: 3446, 2885, 2360, 1685, 1608 1518, 1314, 1084 cm1; 1H NMR (MeOD, 400 MHz) and 13C NMR (MeOD, 100 MHz), see Table 1; HRESIMS m/z 445.1101 [M+Na]+ (calcd for C20H22NaO10, 445.1105). 4-[[(30 ,40 -Dihydroxybenzoyl)oxy]methyl] phenyl O-b-D-[6-O(300 ,500 -dimethoxyl-400 -hydroxybenzoyl)] glucopyranoside (2): white amorphous powder; ½a25 D +32.5 (c 0.12, MeOH); UV (MeOH) kmax: 208, 220, 267, 295 nm; IR (KBr) mmax: 3413, 2358, 1698, 1608, 1516, 1227, 1114, 763 cm1; 1H NMR (MeOD, 400 MHz) and 13C NMR (MeOD, 100 MHz), see Table 1; HRESIMS m/z 625.1528 [M+Na]+ (calcd for C29H30NaO14, 625.1528). Acacetin 7-O-[400 0 -O-acetyl-b-D-apiofuransyl-(1 ? 3)]-b-D-xylopy ranoside (3): yellow amorphous powder; ½a25 D +81.0 (c 0.20, MeOH); UV (MeOH) kmax: 209, 265, 328 nm; IR (KBr) mmax: 3442, 2358, 1705, 1604, 1496, 1373, 1034 cm1; 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz), see Table 2; HRESIMS m/z 613.1525 [M+H]+ (calcd for C28H31O14, 613.1528). Acacetin 7-O-[6000 -O-acetyl-b-D-galactopyranosyl-(1 ? 3)]-b-Dxylopyranoside (4): yellow amorphous powder; ½a25 +27.0 (c D 0.20, MeOH); UV (MeOH) kmax: 205, 267, 330 nm; IR (KBr) mmax: 3414, 2360, 1701, 1606, 1512, 1081 cm1; 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz), see Table 2; HRESIMS m/z 621.1814 [M+H]+ (calcd for C29H33O15: 621.1814). Apigenin 7-O-[6000 -O-acetyl-b-D-galactopyranosyl-(1 ? 3)]-b-Dxylopyranoside (5): yellow amorphous powder; ½a25 D +4.1 (c 0.22, MeOH); UV (MeOH) kmax: 204, 268, 340 nm; IR (KBr) mmax: 3375, 2886, 1714, 1608, 1499, 1389, 1082 cm1; 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz), see Table 2; HRESIMS m/z 629.1478 [M+Na]+ (calcd for C28H30NaO15, 629.1477). Acacetin-7-O-[6000 -O-acetyl-b-D-galactopyranosyl-(1 ? 2)]-b25 D-glucopyranoside (6): yellow amorphous powder; ½aD +15.8 (c 0.12, MeOH); UV (MeOH) kmax: 205, 269, 326 nm; IR (KBr) mmax: 3414, 2360, 1701, 1606, 1599, 1399, 1081 cm1; 1H NMR (DMSOd6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz), see Table 2; HRESIMS m/z 651.1911 [M+H]+ (calcd for C30H35O16, 651.1919).

Table 1 H and 13C NMR data of compounds 1 and 2 (400 and 100 MHz, CD3OD, J in Hz).

1

No

1 dC

1 2, 6 3, 5 4 7 10 20 30 40 50 60 70 10 0 20 0 , 60 0 30 0 , 50 0 40 0 70 0 30 0 , 50 0 -OMe 10 0 0 20 0 0 30 0 0 40 0 0 50 0 0 60 0 0

157.3 116.3 129.7 129.8 65.4 120.6 150.6 115.5 122.0 145.1 116.3 165.6

100.4 73.3 76.7 69.8 77.1 60.8

2 dH

dC

7.05 (d, 8.6) 7.37 (d, 8.4) 5.16 (s)

6.81 (d, 8.3) 7.34 (dd, 8.3, 2.1) 7.38 (d, 2.1)

4.89 3.26 3.34 3.18 3.27 3.70 3.47

(d, 7.4) (m) (m) (m) (m) (m) (m)

159.0 117.9 130.7 132.0 67.1 122.9 117.6 146.3 152.0 116.1 123.9 168.3 121.5 108.7 149.1 142.4 168.0 57.1 102.3 75.0 78.1 72.3 75.8 65.4

dH

2.4. Acid hydrolysis and sugars analyses of 1–6 Compounds 1–6 (each 2.0 mg) were dissolved in 2 N HCl (10 mL) and heated at 80 °C for 2 h. The mixture was evaporated to dryness, and the residue was partitioned between dichloromethane and water. The aqueous phase was concentrated to furnish a monosaccharide residue. After drying under vacuum, anhydrous pyridine (1.0 mL) and L-cysteine methyl ester hydrochloride (4.0 mg) were added to the residue and the mixture was heated at 60 °C for 1 h. After the reaction mixture was evaporated to dryness, o-toyl isothiocyanate (10 lL) was then added, and the mixture was heated at 60 °C for 1 h. The reaction mixture was directly analysed by an Agilent 1260 HPLC system equipped with a photodiode array detector and a Capcell pak C18 column (4.6  250 mm, 5 lm) at 25 °C with isocratic elution of 25% CH3CN in 0.1% formic acid solution for 40 min at a flow rate of 0.8 mL/min. The injection volume was 10 lL and peaks were detected at 250 nm. The standards D-glucose, D-galactose, D-xylose, and D-apiose were treated by the same reaction and chromatographic conditions. As a result, D-apiose from the hydrolysate of 3, D-glucose from the hydrolysate of 1, 2 and 6, D-xylose from the hydrolysate of 3, 4 and 5, and then D-galactose from the hydrolysates of 4, 5 and 6 were detected with the same retention times as standard sugar derivatives. 2.5. DPPH free radical-scavenging activity The DPPH method was used to evaluated the antioxidant activity of the phenolic compounds (Gao, Zhang, Yang, Chen, & Jiang, 2008; Sun & Ho, 2005; Zhu et al., 2012). In a 96-well microplate, 100 lL of DPPH solution (200 lM in ethanol) were added to 100 lL of the test sample in ethanol at different concentrations ranging from 0 to 500 lM. The mixture was vortexed for 1 min and then incubated for 30 min in the dark at room temperature. The absorbance of the reaction mixtures was recorded at 517 nm using a multi-mode detection microplate reader (Synergy™2, Bio-Tek Instruments, Inc., Winooski, VT). The DPPH-scavenging activity was calculated by the following formula: % scavenging activity = 100  (Acontrol – Asample)/Acontrol, Acontrol = absorbance of control, Asample = absorbance of sample. SC50, the concentration of sample needed to scavenge 50% of DPPH radical, was obtained by plotting the DPPH-scavenging percentage of each sample against the sample concentration. Ascorbic acid was used as the positive control in this experiment.

7.03 (d, 8.6) 7.18 (d, 8.6)

2.6. FRAP assay

5.16 (s)

Ferric-reducing antioxidant power (FRAP) assay was performed according to the published procedures (Benzie & Strain, 1996; Xu, Xie, Wang, & Wei, 2010). This assay was based on the reduction of Fe3+-TPTZ, a colourless ferric 2,4,6-tripyridyl-s-triazine complex, to a blue-coloured Fe2+-TPTZ, which was read at 593 nm. The working FRAP reagent was freshly prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ solution in 40 mM HCl and 20 mM ferric chloride in H2O in the ratio of 10:1:1 (v/v/v). In the experiment, 20 lL of sample solution (100 lM in water) were added to 180 lL of FRAP reagent in a 96-well microplate. After the mixture was vortexed for 1 min and incubated for 4 min in the dark at room temperature, absorbance of the ferrous TPTZ complex was measured on a multi-mode detection microplate reader at 593 nm. Various concentrations (0.15–1.5 mM) of FeSO47H2O were used to establish a calibration curve. All samples were tested in triplicate. The assay results were expressed as the concentration of the test sample (lM) giving an absorbance increase equivalent to 1 mM Fe2+ solution.

7.47 (d)

6.85 (d, 8.0) 7.48 (dd, 8.0, 2.4)

7.33 (s)

3.83 4.93 3.81 3.51 3.51 3.79 4.73 4.42

(s) (d, 7.4) (m) (m) (m) (m) (m) (m)

303

X.-L. Zhang et al. / Food Chemistry 152 (2014) 300–306 Table 2 H and 13C NMR data of compounds 3–6 (400 and 100 MHz, DMSO-d6, J in Hz).

1

No

3 dC

2 3 4 5 6 7 8 9 10 10 20 , 60 30 , 50 40 40 -OMe 10 20 0 30 0 40 0 50 0

163.8 103.8 182.0 161.1 99.1 162.4 94.4 157.0 105.4 122.6 128.4 114.6 162.5 55.6 98.2 75.3 76.5 69.4 65.7

4 dH

dC

6.95 (s) 12.91 (s) 6.38 (d, 2.0) 6.81 (d, 2.0)

8.08 (br d, 8.8) 7.12 (br d, 8.8) 3.88 5.20 3.52 3.43 3.41 3.76 3.42

(s) (d, 7.6) (m) (m) (m) (m) (m)

163.9 103.8 182.1 161.1 99.6 162.5 94.7 157.0 105.5 122.7 128.5 114.6 162.7 55.6 98.7 75.4 82.7 68.8 65.6

5 dH

dC

6.95 (s) 12.91 (s) 6.38 (d, 2.0) 6.81 (d, 2.0)

8.08 (br d, 8.8) 7.12 (br d, 8.8) 3.88 5.24 3.52 3.42 3.41 3.76 3.42

6

(s) (d, 6.3) (m) (m) (m) (m) (m)

164.3 103.1 182.0 161.1 99.5 162.6 94.7 156.9 105.4 121.0 128.6 116.0 161.4 98.7 75.4 82.7 68.8 65.6

dH

dC

6.95 (s) 12.91 (s) 6.38 (d, 2.0) 6.81 (d, 2.0)

8.08 (br d, 8.8) 7.12 (br d, 8.8)

5.24 3.52 3.42 3.41 3.76 3.42

(d, 6.3) (m) (m) (m) (m) (m)

60 0 000

163.8 103.1 182.0 161.1 99.6 162.5 94.9 156.9 105.4 122.7 128.4 114.6 162.9 55.6 98.3 83.0 75.6 69.1 77.0 60.5

1 20 0 0 30 0 0 40 0 0

108.2 76.7 77.2 73.8

50 0 0 60 0 0

67.0

3.98 (m) 3.73 (m) 3.92 (s)

Ac

169.9 20.3

1.82 (s)

5.38 (br s) 3.43 (m)

104.7 74.6 75.9 69.7 73.7 63.5 170.3 20.3

4.51 3.05 3.24 3.06

(d, 7.6) (m) (m) (m)

3.42 (m) 4.00 (m) 4.09 (m) 1.82 (s)

2.7. Cells and viruses Human larynx epidermoid carcinoma cell line (HEp-2, ATCC No. CCL-23), human cervical cancer cell line (HeLa, ATCC No.: CCL-2), and Africa green monkey kidney cell line (Vero, ATCC No.: CCL81) cells, which were kindly provided by Prof. Yi-Fei Wang of Guangzhou Biomedicine Research & Development Center, Jinan University, Guangzhou, China, were used for cytotoxic and antiviral studies. All the cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% foetal bovine serum (FBS), 25 lgmL1 gentamicin (Sigma) and 200 mM L-glutamine (Sigma). The test medium used for the cytotoxic and antiviral assays contained only 2% FBS. Virus stocks of human respiratory syncytial virus (RSV A2, ATCC No.: VR-1540 and RSV Long, ATCC No.: VR-26; purchased from Medicinal Virology Institute, Wuhan University, Wuhan, China), herpes simplex virus type 1 F strain (HSV-1, ATCC No.: VR733; kindly provided by Prof. Yi-Fei Wang), and the Coxsackie virus B3 Nancy strain (CVB3; kindly provided by Prof. Yi-Fei Wang) were prepared in HEp-2, Vero and HeLa cells, respectively. Ribavirin (Sigma) was used as the positive control in the anti-RSV and anti-CVB3 tests, while acyclovir (Sigma) was adopted in the antiHSV-1 experiment. The viral titres were detected by the cytopathic end-point assay and expressed as 50% tissue culture infective dose (TCID50).

104.7 74.5 75.9 69.7 73.7 63.5 170.3 20.3

4.51 3.05 3.24 3.06

(d, 7.6) (m) (m) (m)

3.42 (m) 3.99 (m) 4.09 (m) 1.82 (s)

104.7 74.6 75.9 69.7 73.7 63.6 170.3 20.3

dH 6.95 (s) 12.91 (s) 6.38 (d, 2.0) 6.80 (d, 2.0)

8.08 (br d, 8.8) 7.12 (br d, 8.8) 3.87 5.24 3.51 3.22 3.26 3.50 3.42 3.73 3.49 4.52 3.02 3.51 3.09

(s) (d, 6.3) (m) (m) (m) (m) (m) (m) (m) (d, 7.6) (m) (m) (m)

3.42 (m) 3.99 (m) 4.13 (m) 1.82 (s)

Briefly, 100 lL of twofold diluted samples were added into a 96-well microplate containing confluent cell monolayer, while the dilution medium without the sample was used as the control. All the cultures were incubated for 3 days. The morphology of cells was observed under a light microscope (Olympus Microscope DP70) daily. The CPE was scored against the control. The CPE scores were as follows: 0 = no CPE, 1 = 0–25% CPE, 2 = 25– 50% CPE, 3 = 50–75% CPE, 4 = 75–100% CPE. The maximal non-cytotoxic concentration (MNCC) was defined as the maximal concentration of a compound that did not exert a toxic effect and resulted in the presence of more than 90% viable cells (0% CPE). Then, the antiviral activities of the compound were tested in the starting concentration of NMCC as described subsequently. The tested samples (100 lL) in serial twofold dilutions and virus suspension (100 TCID50, 100 lL) were added simultaneously to one-day old confluent monolayers in 96-well microplates. Noninfected and infected cells both without the test sample were served as the cell and virus controls, respectively. The development of virus-induced CPE was monitored by light microscope, and scored. The concentration that reduced 50% of CPE in respect to virus control was defined as 50% inhibition concentration (IC50) for this assay. Ribavirin (Sigma, USA) was used as the positive control for both anti-RSV and anti-CVB3, while acyclovir (Sigma, USA) was the positive control for anti-HSV-1.

3. Results and discussion 2.8. Cytopathic effect (CPE) reduction assay 3.1. Identification of compounds 1–11 As described in our previous report (Wang et al., 2012), the antiviral activities of the compounds were evaluated by the CPE reduction assay. First, the cytotoxic activities of the compounds on HEp-2, HeLa and Vero cells were evaluated, respectively.

Compound 1 was isolated as amorphous powder. The molecular formula was established to be C20H22O10 by its HRESIMS m/z 445.1101 [M+Na]+ (calcd for C20H22O10Na, 445.1105). The IR

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spectrum indicated the presence of hydroxyl (3446 cm1), carbonyl (1684 cm1) and aromatic ring (1608, 1518 cm1). The 1H NMR spectrum showed the presence of a 1,4-disubstituted benzene moiety [dH 7.05 (2H, d, J = 8.6 Hz) and 7.38 (2H, d, J = 8.4 Hz)], a 1,2,4-trisubstituted benzene moiety [dH 6.81 (1H, d, J = 8.3 Hz), 7.34 (1H, dd, J = 8.3, 2.1 Hz) and 7.38 (1H, d, J = 2.1 Hz)], and an anomeric proton of sugar unit [dH 4.93 (1H, d, J = 7.4 Hz)] (Table 1). The above data indicated that 1 had two benzene rings and one sugar. Comparison of the NMR data of 1 with those of amburoside A (13) (José et al., 1999) revealed that they were very similar except for the differences of the ring B. The NMR data of ring B in 1 were identical to those of 2,5-dihydroxybenzoic acid (7), suggesting ring B had two hydroxyls at C-20 and C-50 positions in 1, respectively. It was also confirmed by the 1 H–1H COSY and HMBC correlations (Fig. 2). Acid hydrolysis of 1 afforded D-glucose, which was identified by reversed-phase HPLC after conversion of the sugar to a thiocarbamoyl-thiazolidine derivative. (Tanaka, Nakashima, Ueda, Tomii, & Kouno, 2007). The J value of the anomeric proton indicated the configuration of glucose was b. So 1 was determined to be 4-[[(20 ,50 -dihydroxybenzoyl)oxy]methyl] phenyl-O-b-D-glucopyranoside. The molecular formula of compound 2 was determined as C29H30O14 on the basis of HRESIMS m/z 625.1528 [M+Na]+ (calcd for C29H30O14Na, 625.1528). The presence of hydroxyl (3413 cm1), carbonyl (1698 cm1), and aromatic ring (1608, 1518 cm1) were suggested by the IR spectrum. The NMR data of 2 were also similar to those of amburoside A (José et al., 1999) except for the presence of the signals of an extra 1,3,4,5-tetrasubstituted benzoyl (dC 121.5, 108.7  2, 149.1  2, 142.4, 168.0) and two extra methoxy groups (dC 57.1  2) in 2. The HMBC correlations (Fig. 2) between dH 4.73 (H-6000 ) and dC 168.0 (C-700 ) suggested the benzoyl was connected to C-60 00 through an ester bond. The positions of methoxy groups were elucidated by the HMBC correlations between dH 3.83 (H-3a00 , H-5a00 ) and dC 149.1 (C-300 , C-500 ). HPLC analysis after acid hydrolysis and derivatisation indicated the presence of D-glucose in 2 (Tanaka et al., 2007). Therefore, the structure of 2 was determined to be 4-[[(30 ,40 -dihydroxybenzoyl)oxy]methyl]phenyl-O-b-D-[6-O-(300 ,500 -dimethoxyl-400 -hydroxybenzoyl)] glucopyranoside. The HRESIMS of compound 3 showed an [M+H]+ ion peak at m/z 613.1525 (calcd for C28H31O14, 613.1528) for the molecular formula of C28H30O14. The IR spectrum showed a hydroxyl group at 3442 cm1, carbonyl group at 1705 cm1, and aromatic ring at 1604, 1496 cm1. The signals of one methoxyl [dH 3.88 (3H, s)], two anomeric protons [dH 5.20 (1H, d, J = 7.6 Hz), 5.38 (1H, br s)] and seven aromatic protons (dH 6.38–8.08) were observed in the 1 H NMR spectrum. The 13C NMR spectrum exhibited fifteen

O

OH

O

OH H 3CO

O

O

HO HO

HO

O

OH

aromatic carbons at dC 94.4–182.0, one carbonyl group at dC 169.9, one methoxyl at dC 55.6 and two anomeric carbons at dC 98.2 and 108.2. All the above data indicated that 3 was a flavone glycoside which had two sugars. The aglycone of 3 was established to be acacetin by comparing the NMR data with those published in the literature (Wu et al., 2011b). Acid hydrolysis of 3 afforded D-xylose and D-apiose which were identified by HPLC (Tanaka et al., 2007). The sequence and linkage positions of sugar moiety were subsequently deduced from HMBC correlations between H-100 (dH 5.20) and C-7 (dC 162.4), as well as between H-400 (dH 3.73) and C-1000 (dC 108.2) (Fig. 2). The correlation between H-40 0 (dH 3.73) and C-60000 (dC 169.9) indicated the acetyl was connected to the hydroxyl of C-400 in apiose. In addition, the HMBC correlation between H-40 a (dH 3.88) and C-40 (dC 162.5) implied the methoxyl was located at C-40 position (Fig. 2). Hence, the structure of 3 was determined to be acacetin 7-O-[4000 -O-acetyl-b-D-apiofuransyl-(1 ? 3)]b-D-xylopyranoside. Compound 4 showed an [M+H]+ ion peak at m/z 621.1814 (calcd for C29H33O15, 621.1814) in the HRESIMS spectrum, consistent with the molecular formula of C29H32O15. The 1H and 13C NMR spectra of 4 were similar to those of 3, except that 4 had the signals of a galactose (dC 104.7, 74.6, 75.9, 69.7, 73.7, 63.5) and 3 had the signals of an apiose (dC 108.2, 76.7, 77.2, 73.8, 67.0). HPLC analysis of the derivatives of the acid hydrolysates of 4 revealed the presence of D-xylose and D-galactose (Tanaka et al., 2007). The sequence and linkage positions of sugar moieties were deduced from the HMBC correlation from H-1000 to C-300 , from H-6000 to a carbonyl (dC 170.3), and from H-100 to C-7 (Fig. 2). So the structure of 4 was determined to be acacetin 7-O-[6000 -O-acetyl-b-D-galactopyranosyl-(1 ? 3)]-b-D-xylopyranoside. Compound 5 was isolated as yellow amorphous powder. Its molecular formula was determined as C28H30O15 by the HRESIMS at m/z 629.1478 [M+Na]+ (calcd for C28H30O15Na, 629.1477). Based on detailed analysis of its NMR spectra as well as the comparison of the NMR data with those of compound 4, it was found that they were very similar, except that 5 did not have the signal of a methoxyl at C-40 position. The whole structure was confirmed by the 1 H–1H COSY and HMBC spectra. HPLC method analysis also confirmed the presence of D-xylose and D-galactose (Tanaka et al., 2007). Consequently, 5 was determined to be apigenin 7-O-[6000 O-acetyl-b-D-galactopyranosyl-(1 ? 3)]-b-D-xylopyranoside. The molecular formula of 6 was established as C30H34O16 by HRESIMS at m/z 651.1911 [M+H]+ (calcd for C30H35O16, 651.1919). The 1H and 13C NMR data were similar to those of 4, except that 6 had the signals of a glucose (dC 98.3, 83.6, 75.6, 69.1, 77.0, 60.5), but 4 had the signals of xylose (dC 98.7, 75.4, 82.7, 68.8, 65.6), suggesting the xylose in 4 was substituted by glucose

OH

O O

HO OCH 3 HO

O O

O

OH

OH

1

OH 2

O AcO

O

O HO

O

O

OH OH

OH OH

O

O

OCH 3

OAc HO HO

O HO O

O

O OH OH

OH 4

3 Fig. 2. Key HMBC correlations of compounds 1–4.

O

OCH 3

X.-L. Zhang et al. / Food Chemistry 152 (2014) 300–306

in 6. Acid hydrolysis of 6 gave D-glucose and D-galactose, which were identified by HPLC (Tanaka et al., 2007). The sequence and linkage positions of sugar moieties were deduced from the HMBC correlation of H-100 0 /C-200 , H-60 00 /carbonyl (dC 170.3), and H-100 /C-7. Obviously, the structure of 6 was determined to be acacetin-7-O[6000 -O-acetyl-b-D-galactopyranosyl-(1 ? 2)]-b-D-glucopyranoside. Five known phenolic compounds were identified as 2,5-dihydroxybenzoic acid (7) (Xie & Li, 2002), 3, 4-dihydroxybenzoic acid (8) (Zhang et al., 2008), rosmarinic acid (9) (Wu et al., 2011a), origanoside (10) (Nakatani & Kikuzaki, 1987), and maltol 60 -O-(5-O-pcoumaroyl)-b-D-apiofuranosyl-b-D-glucopyranoside (11) (Li et al., 2008) by comparing their physical and spectroscopic data with those reported in the literature. 3.2. Antioxidant activities In the past decade, polyphenol-rich foods and herbs have received particular attention due to their various biological effects including antioxidant activity. The phenolic compounds from O. vulgare have been reported to possess strong antioxidant activity, most of which have phenolic moieties including 3,4-dihydroxyphenyl, 4-(b-D-glucopyranosyloxy)benzyl alcohol (gastrodin) and 3-(3,4-dihydroxyphenyl)lactic acid (danshensu). (Chou et al., 2010; Ding et al., 2010; Kikuzani & Nakatani, 1989; Liang et al., 2012; Liu et al., 2012). Therefore, twenty-one phenolic compounds isolated from O. vulgare in our previous and present studies were tested for their antioxidant activities using DPPH radical-scavenging and FRAP assays. The DPPH radical-scavenging assay is a simple and sensitive method used to evaluate the antioxidant activity of chemical substances (Huang, Ou, & Prior, 2005). As shown in Table 3, twelve phenolic compounds (1, 2, 7–9, 12–15, 18, 19) exhibited potent DPPH radical-scavenging activities with SC50 values ranging from 16.7 ± 1.1 to 221.8 ± 49.0 lM, comparable to that of ascorbic acid (SC50 = 37.7 ± 0.7 lM). Compounds 9 and 15, which have two dansensu moieties, showed the strongest radical scavenging activities,

Table 3 Antioxidant activity of compounds 1–21 from O. vulgare (n = 3). Compounds

DPPH SC50 (lM)a

FRAP value (lM)b

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

27.8 ± 0.2 43.7 ± 1.8 >500c >500 >500 >500 38.4 ± 2.9 42.3 ± 3.3 17.5 ± 1.1 >500 >500 39.0 ± 1.3 22.5 ± 0.8 43.8 ± 3.5 16.7 ± 1.1 >500 221.8 ± 49.0 36.5 ± 1.0 63.2 ± 3.2 >500 >500 37.7 ± 0.7

372 ± 5 358 ± 38 n.d.d n.d. n.d. n.d. 426 ± 4 363 ± 22 143 ± 4 n.d. n.d. 278 ± 25 288 ± 22 420 ± 6 202 ± 8 n.d. n.d. 768 ± 102 1156 ± 78 n.d. n.d. 460 ± 15

a SC50 is the concentration of sample needed to scavenge 50% of DPPH radical; data are represented as mean ± SD. b The FRAP value is expressed as the concentration of the test sample (lM) giving an absorbance increase equivalent to 1 mM Fe2+ solution; data are represented as mean ± SD. c The SC50 value of sample is higher than 500 lM. d n.d. Not detectable.

305

with SC50 values of 17.5 ± 1.1 lM and 16.7 ± 1.1 lM, respectively. The scavenging action of the new compound 1 was also higher than that of ascorbic acid, and this may due to its 3,4-dihydroxyphenyl and gastrodin moieties. New compound 2 also exhibited potent radical-scavenging activity, with SC50 value of 43.7 ± 1.8 lM. Moreover, the radical-scavenging activity of phenolic compounds is also ascribed to the hydroxyl groups substituted on the aromatic ring (Graf, 1992). In fact, hydrogen atoms of the phenolic hydroxyl groups can be donated to stabilise the free radicals, and phenolic compounds with more phenolic hydroxyl groups show stronger radical scavenging activity (Shahidi & Wanasundara, 1992). In the present study, FRAP assay was adopted to evaluate the antioxidant activity of phenolic compounds as well as the free radical-scavenging assay. The reduction power of the test compounds is a significant indicator of their potential antioxidant activity (Meir, Karrner, Akiri, & Hadas, 1995). As shown in Table 3, the FRAP assay gave similar results to the DPPH radical-scavenging activity assay. Compounds 9 and 15 showed the highest ferric reducing power as well, and FRAP values were 143 ± 4 lM and 201 ± 8 lM, respectively, while the FRAP value of the positive control (ascorbic acid) was 460 ± 15 lM. Besides, the FRAP values of the new compounds 1 and 2 were 372 ± 5 lM and 358 ± 38 lM, respectively. 3.3. Antiviral activities In the previous studies, we have found some phenolic compounds including caffeoylquinic acids, flavans and flavone C-glycosides from natural medicines exhibited potent antiviral activities (Li, But, & Ooi, 2005; Li, Leung, Yao, Ooi, & Ooi, 2006; Wang et al., 2011b). Therefore, the phenolic compounds isolated from O. vulgare were also subjected to in vitro antiviral evaluation against RSV, CVB3 and HSV-1 with CPE reduction assay. However, most of the compounds did not show inhibitory activities against RSV, HSV-1 and CVB3 at their MNCC. Only compound 16 showed moderate to weak inhibitory activity against RSV Long strain with IC50 value of 23.1 lM, and compound 3 exhibited weak activity against RSV A strain with IC50 value of 81.7 lM. Compounds 6 and 7 also exhibited weak effects against HSV-1 with IC50 values of 38.5 and 32.7 lM, respectively, while the IC50 value of the positive control acyclovir was 0.6 lM. 4. Conclusion In summary, phytochemical analysis of the ethanol extract of O. vulgare resulted in the isolation and characterisation of six new and five known phenolic compounds. Their structures were elucidated by chemical and spectroscopic analyses, including 1D, 2D NMR and HRESI-MS. Biological studies disclosed that twelve phenolic compounds (1, 2, 7–9, 12–15, 18, 19) from this plant exhibited potent antioxidant activity but only a few compounds showed weak antiviral activity. The result of our chemical investigation further revealed the chemical composition of O. vulgare, and the biological investigation of these compounds also confirmed that the antioxidant activity of O. vulgare was due to the abundant phenolic compounds in the plant, and that this herb was beneficial to human health. Acknowledgements This work was supported financially by the Fundamental Research Funds for the Central Universities (21612417), the National Natural Science Foundation (Nos. 81273390, 81202429, 81072535) and Program of the Pearl River Young Talents of Science and Technology in Guangzhou, China.

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