Secoiridoids and lignans from the leaves of Diospyros kaki Thunb. with antioxidant and neuroprotective activities

Journal of Functional Foods 24 (2016) 183–195

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Secoiridoids and lignans from the leaves of Diospyros kaki Thunb. with antioxidant and neuroprotective activities Shun-Wang Huang a,b, Jin-Wei Qiao c, Xue Sun a, Pin-Yi Gao d, Ling-Zhi Li a, Qing-Bo Liu a, Bei Sun b, De-Ling Wu c,**, Shao-Jiang Song a,* a Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China b Anhui Institute of Food and Drug Control, Hefei 230022, China c Anhui University of Chinese Medicine, Hefei 230012, China d College of Pharmaceutical and Biotechnology Engineering, Shenyang University of Chemical Technology, Shenyang 110142, China

A R T I C L E

I N F O

A B S T R A C T

Article history:

The leaves of persimmon (LP) have been used for centuries in China, Korea and Japan as a

Received 10 December 2015

delicate, pleasant beverage and an effective herbal remedy. Phytochemical investigations

Received in revised form 22 March

of LP have resulted in the discovery of two new secoiridoid glucosides, named persimmonoid

2016

A and B (1–2), together with ten known ones. Antioxidant and neuroprotective assays of

Accepted 28 March 2016

all compounds were carried out, and compounds (+)-medioresinol (5), (+)-pinoresinol (7),

Available online

(+)-pinoresinol-β-D-glucoside (10) and (−)-(7′S,8S,8′R)-4,4′-dihydroxy-3,3′,5,5′-tetramethoxy7′,9-epoxylignan-9′-ol-7-one (12) were found to exhibit significant activity in the ABTS radical

Keywords:

scavenging assay, while compounds (+)-syringaresinol (6), (+)-pinoresinol (7) and (+)-

Diospyros kaki Thunb

isolariciresinol (11) displayed stronger activity than the positive control in the FRAP assay.

Secoiridoids

In addition, some of these compounds showed statistically significant neuroprotective ac-

Lignans

tivities. The HPLC-QQQ-MS/MS method was successfully applied to quantify twelve compounds

Antioxidant activity

from LP and fruits of persimmon (FP). The bioactive studies supported LP potential for de-

Neuroprotective activity

velopment as a new antioxidant and neuroprotective functional food. © 2016 Elsevier Ltd. All rights reserved.

1.

Introduction

Oxidative stress plays a critical role in the pathogenesis of chronic neurodegenerative diseases, such as Alzheimer’s disease

(AD) (Kim et al., 2010; Shi, Xie, Yang, & Cheng, 2014). It is well known that functional foods rich in potential antioxidative and neuroprotective components may reduce the risk of this disease, and may be important tools for its prevention or postponement of onset. In the past decade, numerous studies have been

* Corresponding author. Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China. Tel.: +86 24 23986088; fax: +86 24 23986510. E-mail address: [email protected]; [email protected] (S.-J. Song). ** Corresponding author. Anhui University of Chinese Medicine, Hefei 230012, China. Tel.: +86 551 68129066; fax: +86 551 68129066. E-mail address: [email protected] (D.-L. Wu). http://dx.doi.org/10.1016/j.jff.2016.03.025 1756-4646/© 2016 Elsevier Ltd. All rights reserved.

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Journal of Functional Foods 24 (2016) 183–195

carried out to identify safe and effective antioxidative and neuroprotective agents (Geng, Chi, Dong, & Hu, 2015; Scherer & Godoy, 2009). Persimmon (Diospyros kaki Thunb.) is widely distributed in East Asia, and it has been reported that the leaves of persimmon are used as a health food (LP tea) to promote maternal health in Japan and Korea (Chen, Wei, Huang, & Sun, 2009b; Han et al., 2002), and they are also thought to be useful for the treatment of hypertension (Funayama & Hikino, 1979). In China, they are used as a plant source for manufacturing a variety of functional health food and cosmetics, such as LP tea, health protection tea, milky tea, other beverages, granules and freckle cream (Zheng & Lu, 2007). Some patents have described their use as a medicinal food or food additive that can improve patients’ physical condition by using extracts of LP for a long period. In addition, it was recorded in part IV of the 2015 edition of the Chinese Pharmacopoeia and used, among other things, as a folk remedy for ischaemic stroke, angina and internal haemorrhage (Chen, Wang, & Jia, 2009a; Kotani, Fujita, & Tanaka, 1999; Matsumoto et al., 2002; Xie, Xie, Xu, & Yang, 2015). More recently, many studies have demonstrated that extracts of LP have beneficial effects involving their antioxidant, cardio-protective, anti-inflammatory, anti-atherogenic, and antidiabetic activities (Funayama & Hikino, 1979; Sun, Zhang, Lu, Zhang, & Zhang, 2011; Xie et al., 2015), as well as their neuroprotective activity (Bei, Peng, Ma, & Xu, 2005; Bei et al., 2007, 2009). Previous phytochemical investigations of this plant have identified a variety of components, including flavonoids and terpenoids, and many other valuable constituents, such as vitamin C, polysaccharides, and polyphenols (Chen et al., 2002; Chen, Ren, & Yu, 2012; Chen et al., 2009b; Liu, Liu, Zhang, & Zhang, 2012; Thuong et al., 2008; Wang, Xu, Rasmussen, & Wang, 2011). To date, more than 80 compounds have been isolated from LP, and it has recently attracted increasing interest because of its important nutritional and medicinal effects. FP is a nutritionally beneficial fruit, which can be used to improve stomach, spleen, and intestinal conditions. It has been used for a long history on the prevention and treatment of aphtha, sore throat and insomnia in China (Chen, Fan, Yue, Wu, & Li, 2008). Previous studies on FP revealed a variety of pharmacological activities, such as antioxidative, hypocholesterolemic, antidiabetic, antitumour and multidrug resistance reversal activities, and effects on relieving alcoholism and reducing plasma lipid (Kawase et al., 2003; Lee, Chung, & Lee, 2006; Matsumoto, Watanabe, Ohya, & Yokoyama, 2006). These beneficial properties are considered to be related to numerous health-promoting compositions, including sugars, organic acids, amino acids, vitamin C, carotenoids, benzoic acid derivatives, flavonoids, terpenoids, polymerized flavan-3-ols procyanidins, tannins and minerals contained in this kind of fruit (Jiménez-Sánchez et al., 2015; Liu & Xie, 2001; Veberic, Jurhar, Mikulic-Petkovsek, Stampar, & Schmitzer, 2010). LP is an abundant natural resource in eastern Asia, and it has become more and more popular because of its use as a medicine and in the cosmetic and food industries. However, it has been estimated that, during the processing of the leaves, about 25% of it is turned into waste, which is simply burnt and discarded (Mallavadhani, Panda, & Rao, 2001). The abundance of LP and its potential uses have attracted our attention, and the use of LP as a potential source of novel antioxidant

and neuroprotective agents has been an ongoing project in our laboratory for several years. To the best of our knowledge, until now, few reports have described the presence of secoiridoid and lignan components of LP and FP (Chen et al., 2012). Reports of the antioxidant and neuroprotective activities of LP mainly focus on flavonoids (Bei et al., 2005, 2007, 2009). This present report describes a detailed chemical screening of LP extracts, and exhaustive chromatographic separation of the ethyl acetate extracts identified eight lignans and four secoiridoids (Fig. 1). We carried out the structural determination of two new secoiridoids, named persimmonoid A and B, based on a series of spectroscopic analyses (IR, UV, HR-ESIMS, 1D and 2D NMR), and examined the antioxidant and neuroprotective effects of compounds 1–12 against hydrogen peroxide (H2O2)-induced neuronal cell damage in human neuroblastoma SH-SY5Y cells. In addition, HPLC-QQQ-MS/MS method has been developed to allow for the quantification of compounds 1–12, in the extracts of LP and FP, which firstly revealed that the contents of the identified compositions between LP and FP were significantly different. The results obtained in this study will help in enhancing our knowledge of the bioactive components of LP and FP and their antioxidant and neuroprotective benefits to health in the food industry.

2.

Materials and methods

2.1.

General

NMR spectra were recorded on a Bruker ARX-400 (1D) and ARX600 (2D) spectrometer with TMS as an internal standard in dimethyl sulphoxide-d6 (DMSO-d6). Mass spectra were determined on an HR-ESIMS: MicroTOF spectrometer (Bruker Co., Karlsruhe, Germany). Silica gel (200–300 mesh, Qingdao Marine Chemical Co., Qingdao, China), MCI gel (CHP20P, 75–150 µm, Mitsubishi Chemical Corporation, Tokyo, Japan), Sephadex LH20 (25–100 µm, Green Herbs Science and Technology Development Co., Ltd., Beijing, China), Polyamide (80–100 mesh, Qingdao Marine Chemical Co., Qingdao, China), and reversed-phase C18 silica gel (ODS 5 µm, Merck, Darmstadt, Germany) were used for column chromatography, and silica gel GF254 (Qingdao Marine Chemical Co., Qingdao, China) was used for TLC. In addition, ABTS (2,2′azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt), DPPH (1, 1′-diphenyl-2-picrylhydrazyl), FRAP (ferric reducing antioxidant power) and trolox as a standard were all purchased from Sigma–Aldrich (Steinheim, Germany) in order to screen the antioxidant activities of the isolated compounds. All solvents were of industrial purity and distilled prior to use.

2.2.

Plant materials

The persimmon leaves were collected from Bengbu city of Anhui Province, China, in November 2013. Samples were dried in the shade for a week and cut into pieces approximately 2 cm in width. The FP was purchased in January 2016 from a local market of Shenyang in China. They were identified according to the identification standard of the Pharmacopeia of the People’s Republic of China by Prof. Jincai Lu (Shenyang Pharmaceutical University). LP and FP voucher specimens (No.

Journal of Functional Foods 24 (2016) 183–195

185

Fig. 1 – Structures of the isolated compounds 1–12 from persimmon leaves.

201312 and No. 201601) were deposited at the College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University (Shenyang, China).

2.3.

Extraction and isolation

The dried and milled leaves (about 2 mm diameter) (50 kg) of persimmon were sequentially extracted with 95% (v/v) EtOH (600 L) and 50% (v/v) EtOH (500 L) by percolation for four days at room temperature. The extract solutions were collected and pooled, and concentrated under reduced pressure (−0.06 Mpa) at 72 °C to yield the extracts (4.5 kg). The residue was further suspended in H2O and successively extracted with petroleum ether (50 L × 3), EtOAc (50 L × 3) to afford 1.2 kg of petroleum ether fractions and 1.1 kg of EtOAc fractions. A part of the AcOEt extract (1.1 kg) was subjected to silica gel column chromatography, using a gradient of MeOH in CH2Cl2 (0:100, 5:95, 10:90, 20:80, 50:50, 100:0), to yield five fractions (Frs.1–Frs.5) based on thin-layer chromatography analysis. Frs.2 and Frs.3 were further chromatographed on Polyamide (80–100) with MeOH/H2O (0/100, 30/70, 60/40, 100/0) system to give four fractions (Frs.2-1-2-4)/(Frs.3-1-3-4). These subfractions Frs.2-1 (60 g) and Frs.3-1 (100 g) were subjected to medium-pressure liquid chromatography (MPLC) over Diaion

HP-20, eluting with a step gradient from 10 to 90% MeOH in H2O, to give Frs.2-1-1-Frs.2-1-3 and Frs.3-1-1-Frs.3-1-5. Frs.2-1-2 (22 g) and Frs.3-1-3 (26 g) were subjected to Sephadex LH-20 column to afford Frs.2-1-2-1-Frs.2-1-2-3 and Frs.3-1-3-1-Frs.3-1-3-3 respectively. The purification of Frs.2-1-2-2 (9 g) was achieved by preparative HPLC (detection at 210 nm, 6 mL/min) and semipreparative HPLC (detection at 210 nm, 2.5 mL/min), respectively, to yield compounds 5 (9.8 mg, tR 15 min), 6 (7.1 mg, tR 21 min), 7 (15.3 mg, tR 25 min), 8 (15.3 mg, tR 28 min) and 11 (15.1 mg, tR 33 min). Similarly, Fr.3-1-3-2 (15 g) was separated on preparative HPLC (refractive index detector (RI), 6 mL/min) and semipreparative HPLC (detection at 210 nm, 2.5 mL/min), respectively, to obtain compounds 10 (15.0 mg, tR 21 min), 2 (13.0 mg, tR 24 min), 1 (15.3 mg, tR 36 min), 4 (20.0 mg, tR 40 min) and 3 (2.6 mg, tR 45 min). Compounds 9 (51.0 mg, tR 21 min) and 12 (13.4 mg, tR 27 min) were obtained from Fr.2-1-1-2 (40% MeOH in H2O) by the above preparative HPLC method. The FP samples (1.0 kg) were randomly chosen and cut into thin slices (about 1 mm). The slices were lyophilized and the FP extract was prepared using the same extraction procedure as LP described previously. The resultant extract was used for determination of the contents of the identified 12 compounds.

186

2.4.

Journal of Functional Foods 24 (2016) 183–195

DPPH radical scavenging activity assay

The DPPH radical scavenging capacity of compounds 1–12 was determined according to Brand-Williams, Cuvelier, and Berset (1995). Sample solution was serially diluted to 10, 50, 100, and 200 µg/mL with methanol. In each reaction, the solutions were added to a 96-well plate, followed by 0.1 mL of 0.25 mM DPPH. The mixture plate was shaken vigorously and then immediately incubated at room temperature for 30 min. The absorbance of the reaction solution was determined using a Bio-Tek (Winooski, VT, USA) microplate reader at 517 nm. All samples, standards, and controls were run in triplicate. Trolox was used as a positive reference. The DPPH radical-scavenging activity in percentage of sample was calculated as follows:

S − SB ⎞ ⎛ DPPH scavenging activity (% ) = ⎜ 1 − ⎟ × 100% ⎝ C − CB ⎠ where S, SB, C and CB are the absorbances of the sample, the blank sample, the control and the blank control, respectively.

2.5.

ABTS radical cation scavenging activity assay

The antioxidant capacity of compounds 1–12 was also evaluated using an improved ABTS•+ decolourization assay (Alasalvar et al., 2009). A 7 mM ABTS ammonium was dissolved in water and mixed with 2.45 mM potassium persulfate and the mixture was allowed to stand in the dark at room temperature for 16 h before use. The ABTS•+ solution was diluted with ethanol to an absorbance of 0.7 ± 0.02 at 734 nm. Then, an ethanolic solution (100 µL) of the samples or trolox standard at various concentrations (10, 50, 100 and 200 µg/mL) was mixed with 150 µL diluted ABTS•+ solution. After reaction at room temperature for 30 min, the absorbance was read at 734 nm using a Bio-Tek microplate reader. The capability to scavenge ABTS•+ was calculated using the formula given below:

heat-inactivated FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C with 5% CO2. Cells were cultured in 96-well plates at a density of 1 × 104 cells/well in 200 µL for 24 h. Cells were incubated with test compounds (25, 50 and 100 µM) for 12 h. To induce an oxidative stress, 100 µM H2O2 freshly prepared was added to the cells and incubated at 37 °C for 1.5 h. Cell viability was determined colourimetrically using MTT assay (Tian et al., 2015). Then 20 µL of the MTT solution (5 mg/mL) was added into each well. After incubation for 4 h at 37 °C, the cells were finally lysed with 150 µL of DMSO. The absorbance was read at 490 nm with a microplate reader (ELX 800, BioTek). The value for cell viability was expressed as the percentage of the control value. The results were expressed as means ± SD of the indicated numbers from three independent experiments. Statistical analysis was performed by one-way analysis of variance (ANOVA) and Student’s Dunnett test using the SPSS statistical software (version 19 for Windows). P values below 0.05 were considered statistically significant.

2.8.

HPLC-QQQ-MS/MS analysis of LP and FP

All samples were analysed by using an Agilent 1260 HPLC system (Agilent Technologies Inc., Santa Clara, USA) equipped with a Dikma Diamonsil C18 column (4.6 mm × 250 mm, 5 µm). The column temperature was maintained at 25 °C. The mobile phase used consisted of acetonitrile containing 0.1% formic acid (A) and water containing 0.1% formic acid (B), and using gradient elution of 20% A at 0–2 min, 20%–50% A at 2–22 min, 50%– 20% A at 22–23 min, and 20% A at 23–28 min, with the flow rate kept at 0.8 mL/min. The sample volume injected was set at 10 µL. LC-MS/MS data of samples were collected by using an AB5500 Triple Quadrupole mass spectrometer with an electrospray interface (ESI) operated in the positive ion mode using the following settings: ion spray voltage: −4500 V; temperature: 650 °C; curtain gas: 35 psi; ion source gas 1: 50 psi; ion source gas 2: 50 psi.

S − SB ⎞ ⎛ ABTS+ scavenging activity (% ) = ⎜ 1 − ⎟ × 100% ⎝ C − CB ⎠ where S, SB, C and CB are the absorbances of the sample, the blank sample, the control and the blank control, respectively. Tests were performed in triplicate.

2.6.

Ferric reducing antioxidant power (FRAP) assay

The FRAP reagent was made freshly by mixing 300 mM acetate buffer, 10 mM TPTZ solution in 40 mM hydrochloric acid, and 20 mM aqueous FeCl3 solution in the ratio of 10:1:1 (v/v/v) (Xu, Xie, Wang, & Wei, 2010). Test compounds were dissolved in methanol to serial concentrations, and 20 µL of the solution and 180 µL of FRAP reagent were transferred to a 96-microplate and each concentration was set in quadruplicate. Trolox was used as positive control. The plate was incubated at 37 °C for 30 min in the dark. The absorbance of the resultant coloured product in each well was read at 595 nm using a microplate reader.

2.7.

Neuroprotective activity assay

Human neuroblastoma SH-SY5Y cells were cultured in 100 mm dishes and grown in DMEM supplemented with 10%

3.

Results and discussion

3.1.

Phytochemical investigation

Compound 1 was obtained as colourless oil. Its molecular formula was found to be C26H32O14, based on NMR data and its HR-ESIMS ion at m/z 591.1667 [M+Na]+ (calcd 591.1690), with 11 degrees of unsaturation. Its UV spectrum showed a peak absorbance at λmax 278 nm, and the IR spectrum showed absorption bands for hydroxyl (3420 cm−1) and carbonyl (1721 and 1634 cm−1) groups. In the 1H NMR spectrum (Table 1), characteristic signals due to an olefinic proton (H-3) appeared at δH 7.58 (1H, s) as a singlet, an acetalcarbinol proton (H-1) as a singlet at δH 6.04 (1H, s), a trisubstituted vinyl proton (H-8) as a doublet at δH 6.26 (1H, d, J = 0.7 Hz), and one methylene group, one methine group, and two methoxyl groups were assigned to the secoiridoidic aglycone moiety (He et al., 2001). The doublet at δH 4.66 was attributed to the anomeric proton of the glucose moiety. In addition, the characteristic aromatic protons of AA′BB′ spin systems at δH 7.01 (2H, d, J = 8.4 Hz, H-2″, 6″) and δH 6.67 (2H, d, J = 8.4 Hz, H-3″, 5″) showed J values of 8.4 Hz together with two methylene groups,

Journal of Functional Foods 24 (2016) 183–195

187

Table 1 – 1H (400 MHz) and 13C (100 MHz) NMR data of compounds 1, 2 (DMSO-d6). NO.

1 δH

1 2 3 4 5 6 7 8 9 10 11 10′ 11′ 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″

2 δC

δH

δC

6.04 s

91.7

6.04 s

91.7

7.58 s

153.0 107.6 30.9 39.4

7.59 s

153.0 107.6 30.9 39.5

4.80 dd (8.4, 5.0) 2.65 dd (14.0, 5.0) 2.59 dd (14.0, 8.5) 6.26 d (0.7)

3.65 s 3.67 s 4.66 d (7.8) 3.12 m 3.20 m 3.09 m 3.21 m 3.61 3.45 7.01 d (8.4) 6.67 d (8.4) 6.67 d (8.4) 7.01 d (8.4) 4.09 ddd (14.5, 9.0, 5.6) 4.02 dt (10.7, 7.2) 2.71 t (7.2)

170.1 117.5 147.1 165.4 166.8 51.5 51.4 99.1 73.2 77.4 69.9 76.3 61.1 127.7 129.7 115.3 155.9 115.3 129.7 65.0 33.3

4.80 dd (8.3, 5.0) 2.66 m 2.58 m 6.26 d (0.5)

3.68 s 3.65 s 4.66 d (7.7) 3.10–3.20 m 3.21 m 3.19 m 3.10 m 3.62 3.45 6.46 dd (8.0, 2.0) 6.62 d (8.0)

6.59 d (2.0) 4.08 dd (8.3, 5.0) 4.01 d (6.8) 2.65 t (6.3)

170.1 117.9 146.6 165.5 165.8 51.4 51.3 99.1 73.2 77.5 69.9 76.4 61.1 128.4 119.5 115.6 145.1 143.7 116.2 65.1 33.6

Assignments were based on HSQC and HMBC experiments.

which indicated the presence of a para-substituted phenylethyl group. The 13C-NMR spectrum (Table 1) showed a carboxyl carbon at δC 170.1 (C-7), two methoxy groups (δC 51.5, C-10′; δC 51.4, C-11′) supposedly connected to carboxyl groups (δC 165.4, C-10; δC 166.8, C-11), two groups of olefinic carbons at δC 153.0 (C-3), 107.6 (C4), 117.5 (C-8), 147.1 (C-9), δC 127.7 (C-1′′), 129.7 (C-2′′, 6′′), 115.3 (C-3′′, 5′′), 155.9 (C-4′′), and an acetal carbon at δC 91.7 (C-1), three methylenes δC 65.0 (C-7′′), 39.4 (C-6), 33.3 (C-8′′), one methine δC 30.9 (C-5), and six glycoside signals (δC 99.1, 73.2, 77.4, 69.9, 76.3, 61.1). Further confirmation of the planar skeleton structure of 1 was obtained from the HMBC investigation (Fig. 2). The HMBC spectrum exhibited correlations from δH 7.58 (1H, s, H-3) to C-5 (δC 30.9) and C-11 (δC 166.8), δH 4.80 (1H, dd, J = 8.4, 5.0 Hz, H-5) to C-6 (δC 39.4) and C-7 (δC 170.1), δH 6.26 (1H, d, J = 0.7 Hz, H-8) to C-1 (δC 91.7) and C-5 (δC 30.9), a methoxyl group (δH 3.65, 10′OCH3) to C-10 (δH 165.4), as well as protons of a methoxyl group (δH 3.67, 11′-OCH3) to C-11 (δH 166.8), indicating the planar structure of the secoiridoidic skeleton. A long range correlation between H-7′′ (δH 4.09, 4.02) and C-7 (δH 170.1) indicated that the phenethyl group in compound 1 was conjugated at C-7. Furthermore, the glycosidic site was established unambiguously by an HMBC investigation in which a long-range correlation between δH 6.04 (H-1) and δC 99.1 (C-1′) was observed (Fig. 2). The NOESY spectrum of 1 revealed an NOE enhancement between H-1 (δH 6.04) and H2-6 (δH 2.65), as shown in Fig. 2. This indicates that

Fig. 2 – Key HMBC (C-H) and NOESY correlations (H-H) of 1 and 2.

H-1 and H-6 are on the α-face as found in compound 1. In the sugar part, an anomeric proton signal at δH 4.66 (1H, d, J = 7.8 Hz, H-1′) and the 13C NMR signals of sugar showed the presence of a β-glucopyranosyl moiety (Table 1). This evidence led us to formulate the structure of compound 1 as shown, it was determined to be (2β, 3E, 4α), 3-(2-methoxy-2-oxoethylidene)-2-(β-Dglucopyranosyloxy)-3,4-dihydro-5-(methoxycarbonyl)-2H-pyran4-acetic acid, 4-[2-(4-hydroxyphenyl)ethyl]ester, and given the trivial name persimmonoid A. Compound 2 was obtained as colourless oil, with a molecular formula of C26H32O15 as determined by the HR-ESIMS ion at m/z 607.1651 [M+Na]+ (calcd 607.1639) with 11 degrees of unsaturation. The absorption maximum was at 282 nm in the UV spectrum. The IR spectrum showed absorption bands for hydroxyl (3424 cm−1) and carbonyl (1721 and 1634 cm−1) groups. The 1H and 13C NMR (Table 1) chemical shift assignments were made from HSQC, COSY and HMBC spectra. Analysis of 1H and 13C NMR spectra indicated that the data of compound 2 were similar to those of compound 1 except for a set of signals for aromatic protons with the ABX system at δH 6.62 (1H, d, J = 8.0 Hz), δH 6.46 (1H, dd, J = 8.0, 2.0 Hz) and δH 6.59 (1H, d, J = 2.0 Hz). By comparison of the 13C NMR spectrum of compound 2 with that of compound 1, the chemical shift assignable to the C-5″ of 2 shifted downfield at δC 143.7, while ortho C-6″ and C-4″ shifted upfield at δC 116.2 and δC 145.1, respectively. This information indicated that the phenyl ring in the phenethyl moiety of compound 2 was 3, 4-substituted. An HMBC (Fig. 2) correlation peak was observed between H-7″ (δH 4.08 and δH 4.01) and C-7 (δC 170.1), which indicated that this 3,4-dihydroxyphenethyl group was connected to the aglycone at C-7. The result of the NOESY investigation of compound 2 was identical with that of 1, suggesting that H-1 and H-6 are on the α-face as exhibited by compound 2 and the structure of 2 was elucidated as shown; it was determined as (2β, 3E, 4α), 3-(2-methoxy-2-oxoethylidene)-2-(β-D-glucopyranosyloxy)3,4-dihydro-5-(methoxycarbonyl)-2H-pyran-4-acetic acid, 4-[2(3,4-dihydroxyphenyl)ethyl]ester, and given the trivial name persimmonoid B. Ten known compounds (Fig. 1) from the leaves of persimmon were identified as ligustroside (3) (He et al., 2001), oleuropein (4) (Kwak, Kang, Roh, Choi, & Zee, 2009), (+)medioresinol (5) (Sribuhom, Sriphana, Thongsri, & Yenjai, 2015), (+)-syringaresinol (6) (Verma, Gangwar, Sahai, Nath, & Singh, 2015), (+)-pinoresinol (7) (Sribuhom et al., 2015), (+)-medioresinol

188

Journal of Functional Foods 24 (2016) 183–195

Table 2 – Antioxidant activity of compounds 1–12 and trolox. Experiment

Trolox

1

2

3

4

5

6

7

8

9

10

11

12

DPPH (IC50, µg/mL) ABTS (IC50, µg/mL) FRAP (0.75 mg/mL)

11.9 11.9 86.9

– 18.3 4.3

102.6 22.9 73.9

– 15.1 –

31.2 11.8 32.7

37.5 10.7 86.7

49.7 13.2 98.5

–

– 23.7 48.8

104.2 18 73.5

–

109.9 19 95.1

40.4 8.6 72.8

3.6 72.1

4′′ and an independent double bond at positions C-8, 9. Previous studies have demonstrated that LP possesses free radicals scavenging activity (Han et al., 2002; Sakanaka, Tachibana, & Okada, 2005). Han et al. (2002) reported the IC50 value of methanol extract of LP was 0.11 mg/mL against DPPH radical. Flavonoids from LP possessed scavenging activity of DPPH radical (EC50, 96.36 ± 2.63 µg/mL), superoxide anion (41.58 ± 0.21 µg/mL) and hydroxyl radical (70.64 ± 3.22 µg/mL), reducing power and iron chelating activity (Sun et al., 2011). In comparison with these extracts from LP, the result in this present study is consistent with the previous results reported on LP.Therefore, the 3 models may be useful tools for evaluating the antioxidant capacity of secoiridoids and lignans isolated from LP. This present study on the characterization of 12 compounds in LP is in progress to build the connection between antioxidant activity and chemical composition.

monoglucoside (8) (Deyama, Ikawa, & Nishibe, 1985), (+)syringaresinol-β-D-glucoside (9) (Lami, Kadota, Kikuchi, & Momose, 1991), (+)-pinoresinol-β-D-glucoside (10) (Liu, Xu, & Wang, 1998), (+)-isolariciresinol (11) (Zhao, Shao, & Li, 2009), and (−)-(7′S,8S,8′R)-4,4′-dihydroxy-3,3′,5,5′-tetramethoxy-7′,9epoxylignan-9′-ol-7-one (12) (Xiong et al., 2011), respectively, by comparison of their physical and spectroscopic data with values reported in the literature.

3.2.

7.5 95.0

Antioxidant activity

All 12 compounds were tested for their antioxidant activity in a DPPH and ABTS radical scavenging and FRAP assay. The IC50 values and FRAP values of compounds (1–12) are shown in Table 2.The assay involved a comparison with trolox (IC50 = 11.9, 11.9 µg/mL in the DPPH and ABTS assay), which was used as a positive control. Compounds 5, 7, 10 and 12 displayed stronger activities compared with the positive control in the ABTS radical scavenging assay (Fig. 3). Also, the clearance (ABTS) of compounds 1–3, 8, 9 and 11 showed a concentration dependence of 15–23 µg/mL. Compounds 6, 7 and 11 displayed stronger activities compared with the positive control in the FRAP assay (Fig. 4). The different antioxidant activities of the test compounds could be due to their structural differences. Compounds 5, 6 and 11 with a lignan skeleton were more active than compounds 8 and 9, suggesting that the presence of more phenolic hydroxyl groups might increase the antioxidant activity. Compound 4 was more active than the other monoterpenes due to the presence of two phenolic hydroxyl groups at positions C-3′′,

3.3. Protective effect of the 11 compounds against H2O2-induced injury Human SH-SY5Y neuroblastoma cells is a dopaminergic neuronal cell line that is widely used as a suitable in vitro model for chronic neurodegenerative diseases compared to primary neurons (Wang, Zhu, Zhang, Zhou, & Zhu, 2015). In order to discover potentially neuroprotective agents from natural products, the protective activity of compounds 1, 3–12 against neuroblastoma SH-SY5Y cell injury induced by H2O2 was tested. Compounds 1, 3, 4 and 12 exhibited statistically significant neuroprotective effects (Table 3). However, a higher concentration

120

DPPH ABTS

100

IC50-μg/mL

80

60

40

20

* 0

# Trolox c-1

# c-2

c-3

c-4

c-5

c-6

#

#

c-7

c-8

#

*

*

c-9 c-10 c-11 c-12

Fig. 3 – DPPH and ABTS radical scavenging activity of compounds 1–12 and trolox.

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*

100

*

*

* 80

60

40

20

0

## Trolox c-1

c-2

c-3

c-4

c-5

c-6

c-7

c-8

c-9 c-10 c-11 c-12

Fig. 4 – FRAP assay activity of compounds 1–12 and trolox.

of 5 and 9 showed cytotoxic effects, while a lower concentration had weak protective activity, comparable with that of the standard compound Vitamin E. The neuroprotective data and the structural characteristics of the 11 compounds were unable to confirm any clear structure–activity relationships. Similar results were reported in the previous study. Flavonoids from LP were reported to exhibit significant neuroprotective effects against hydrogen peroxide (H2O2)-induced injury of NG108-15 cells (Bei et al., 2005). Five triterpenoid compounds from LP suppress stimulus-induced superoxide generation in human neutrophils in a concentration-dependent manner (Chen et al., 2002). These findings show that further research into LP and its constituents would be valuable, as these neuroprotective compounds might provide useful bioactive molecules.

3.4.

Characteristic data of compounds

Persimmonoid A (1): a colourless oil; UV (MeOH) λmax (logε): 278 (4.20); IR (KBr) Vmax: 3420, 2919, 2850, 1721, 1634, 1515, 1438, 1383,

1348, 1262, 1229, 1171, 1102, 1076, 957, 878, 831, 769, 620 and 558 cm −1 ; HR-ESIMS at m/z 591.1667 [M+Na] + (calcd for C26H32O14Na, 591.1690); 1H and 13C NMR (see Table 1). Persimmonoid B (2): a colourless oil; UV (MeOH) λmax (logε): 282 (4.22); IR (KBr) Vmax: 3424, 2920, 2850, 1721, 1634, 1522, 1440, 1384, 1350, 1284, 1172, 1102, 1076, 958, 928, 878, 770, 619 and 576 cm −1 ; HR-ESIMS at m/z 607.1651 [M+Na] + (calcd for C26H32O15Na, 607.1639); 1H and 13C NMR (see Table 1). Ligustroside (3): white amorphous powder; 1H NMR (400 MHz, DMSO) δH 7.52 (1H, s, H-3), 7.02 (2H, d, J = 8.4 Hz, H-2′′, 6′′), 6.68 (2H, d, J = 8.4 Hz, H-3′′, 5′′), 5.95 (1H, q, J = 6.7 Hz, H-8), 5.87 (1H, s, H-1), 4.66 (1H, d, J = 7.8 Hz, Glc -1′), 4.14 (1H, dt, J = 10.7, 7.0 Hz, H1- 7″), 4.06 (1H, dt, J = 10.7, 7.2 Hz, H2- 7″), 3.84 (1H, dt, J = 10.8, 5.4 Hz, H- 5), 3.65 (3H, s, 11′-OCH3), 3.47-3.09 (6H, m, Glc-2′-6′), 2.75 (2H, t, J = 7.2 Hz, H-8″), 2.63 (1H, dd, J = 14.4, 4.1 Hz, H1-6), 2.41 (1H, dd, J = 14.4, 9.3 Hz, H2-6), 1.62 (3H, dd, H-10). 13C NMR (100 MHz, DMSO) δC 170.7 (C-7), 166.2 (C-11), 155.9 (C-4′′), 153.4 (C-3), 129.8 (C-2′′, 6′′), 129.1 (C-9), 127.8 (C-1′′), 123.0 (C-8), 115.1 (C-3′′, 5′′), 107.7 (C-4), 99.0 (C-1′), 92.9 (C-1), 77.4 (C-3′), 76.3

Table 3 – Protection of compounds 1, 3−11 against neuroblastoma SH-SY5Y cells injury induced by H2O2 (M ± SD). NO.

1 3 4 5 6 7 8 9 10 11 12

CON

100 ± 2.73 100 ± 2.73 100 ± 2.73 100 ± 0.82 100 ± 0.82 100 ± 0.82 100 ± 0.82 100 ± 2.73 100 ± 2.73 100 ± 0.82 100 ± 2.73

Negative (H2O2)

52.68 ± 4.64 52.68 ± 4.64### 52.68 ± 4.64### 51.82 ± 3.47### 51.82 ± 3.47### 51.82 ± 3.47### 51.82 ± 3.47### 52.68 ± 4.64### 52.68 ± 4.64### 51.82 ± 3.47### 52.68 ± 4.64### ###

Comp. + H2O2 50 µΜ

25 µΜ

12.5 µΜ

58.35 ± 5.75* 53.41 ± 1.54 52.12 ± 2.49 39.53 ± 2.22*** 30.47 ± 2.19 62.48 ± 1.91 49.91 ± 0.47 14.01 ± 0.52*** 54.07 ± 2.14 48.00 ± 2.16 49.96 ± 6.16

61.43 ± 2.59** 61.01 ± 2.55** 56.03 ± 2.50** 55.53 ± 3.47 55.69 ± 1.61* 61.90 ± 4.15 53.84 ± 1.85 14.17 ± 1.13*** 52.58 ± 1.82 53.09 ± 1.41 58.89 ± 1.66*

58.02 ± 2.46* 58.35 ± 3.26* 56.07 ± 5.18* 57.18 ± 5.30* 55.69 ± 7.81 58.71 ± 5.47 55.98 ± 4.48 51.74 ± 4.76 54.61 ± 4.76 56.11 ± 3.54 58.19 ± 0.83*

All data were shown as mean ± SD of three separate experiments. ### P < 0.001, *P < 0.05, **P < 0.01, ***P < 0.001 compared with negative control.

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(C-5′), 73.3 (C-2′), 69.9 (C-4′), 65.1 (C-7″), 61.1 (C-6′), 51.2 (C-11′), 39.5 (C-6), 33.5 (C-8″), 30.1 (C-5), 12.9 (C-10). Oleuropein (4): white amorphous powder; 1H NMR (400 MHz, DMSO) δH 7.52 (1H, s, H-3), 6.65 (1H, d, J = 8.0 Hz, H-5′′), 6.62 (1H, d, J = 2.0 Hz, H-2′′), 6.48 (1H, dd, J = 8.0, 2.0 Hz, H-6′′), 5.97 (1H, q, J = 6.8 Hz, H-8), 5.88 (1H, s, H-1), 4.66 (1H, d, J = 7.7 Hz, glc1′), 4.12 (1H, dd, J = 10.6, 7.2 Hz, H1-7″), 4.05 (1H, dd, J = 10.7, 7.3 Hz, H2-7″), 3.86 (1H, dd, J = 9.1, 4.2 Hz, H-5), 3.68 (3H, s, 11′-OCH3), 3.43-3.05 (6H, m, Glc -2′-6′), 2.75 (2H, t, J = 7.1 Hz, H-8″), 2.63 (1H, dd, J = 14.4, 6.1 Hz, H1-6), 2.41 (1H, dd, J = 14.4, 9.3 Hz, H2-6), 1.63 (3H, dd, H-10). 13C NMR (100 MHz, DMSO) δC 170.7 (C-7), 166.3 (C-11), 153.5 (C-3), 145.13 (C-3′′), 143.8 (C-4′′), 129.2 (C-9), 128.5 (C-1′′), 123.1 (C-8), 119.6 (C-6′′), 116.2 (C-2′′), 115.6 (C-5′′), 107.7 (C-4), 99.1 (C-1′), 93.0 (C-1), 77.5 (C-3′), 76.4 (C-5′), 73.2 (C-2′), 69.9 (C-4′), 65.1 (C-7″), 61.1 (C-6′), 51.2 (C-11′), 39.5 (C-6), 33.6 (C-8″), 30.2 (C-5), 12.9 (C-10). (+)-Medioresinol (5): colourless oil; 1H NMR (400 MHz, DMSO) δH 8.92 (1H, s, 4-OH), 8.27 (1H, s, 4′-OH), 6.90 (1H, d, J = 1.5 Hz, H-2′), 6.77 (1H, dd, J = 8.2, 1.5 Hz, H-6′), 6.73 (1H, d, J = 8.2 Hz, H-5′), 6.61 (2H, s, H-2, 6), 4.62 (1H, s, H- 7′), 4.61 (1H, s, H-7), 4.15 (2H, dd, J = 15.4, 8.5 Hz, H-9, 9′), 3.77 (3H, s, 3′-OCH3), 3.75 (6H, s, 3, 5-OCH3), 3.73 (2H, d, J = 3.3 Hz, H-9, 9′), 3.05 (2H, m, H-8, 8′). 13C NMR (100 MHz, DMSO) δC 147.9 (C-3, 5), 147.5 (C-3′), 145.9 (C4′), 134.8 (C-4), 132.2 (C-1′), 131.4 (C-1), 118.6 (C-6′), 115.1 (C5′), 110.4 (C-2′), 103.6 (C-2, 6), 85.4 (C-7), 85.1 (C-7′), 71.0 (C-9′), 70.90 (C-9), 56.0 (C-3, 5-OCH3), 55.6 (C-3′-OCH3), 53.7 (C-8′), 53.5 (C-8). (+)-Syringaresinol (6): colourless oil; 1H NMR (400 MHz, DMSO) δH 8.28 (2H, br. s, 4, 4′-OH), 6.61 (4H, s, H-2, 6, 2′, 6′), 4.63 (2H, d, J = 3.9 Hz, H-7, 7′), 4.17 (2H, m, H-9, 9′), 3.78 (2H, m, H-9, 9′), 3.76 (12H, s, 3, 5, 3′, 5′-OCH3), 3.06 (2H, m, H-8, 8′). 13C NMR (150 MHz, DMSO) δC 148.4 (C-3, 5, 3′, 5′), 134.2 (C-4, 4′), 131.8 (C-1, 1′), 103.1 (C-2, 6, 2′, 6′), 85.8 (C-7, 7′), 71.6 (C-9, 9′), 56.9 (C-3, 5, 3′, 5′OCH3), 54.1 (C-8, 8′). (+)-Pinoresinol (7): colourless oil; 1H NMR (400 MHz, DMSO) δH 8.93 (2H, s, 4, 4′-OH), 6.90 (2H, d, J = 1.5 Hz, H-2, 2′), 6.76 (2H, dd, J = 8.2, 1.5 Hz, H-6, 6′), 6.74 (2H, d, J = 8.2 Hz, H-5, 5′), 4.62 (2H, d, J = 4.1 Hz, H-7, 7′), 4.12 (2H, dd, J = 8.8, 6.8 Hz, H-9, 9′), 3.77 (6H, s, 3, 3′-OCH3), 3.73 (2H, dd, J = 9.1, 3.4 Hz, H-9, 9′), 3.04 (2H, m, H-8, 8′). 13C NMR (100 MHz, DMSO) δC 147.5 (C-3, 3′), 145.9 (C-4, 4′), 132.2 (C-1, 1′), 118.6 (C-6, 6′), 115.1 (C-5, 5′), 110.4 (C-2, 2′), 85.2 (C-7, 7′), 70.9 (C-9, 9′), 55.6 (C-3, 3′-OCH3), 53.6 (C-8, 8′). (+)-Medioresinol monoglucoside (8): a white powder, 1H NMR (400 MHz, DMSO) δH 8.95 (1H, br. s, 4′-OH), 6.90 (1H, d, J = 1.5 Hz, H-2′), 6.77 (1H, dd, J = 8.2, 1.5 Hz, H-6′), 6.73 (1H, d, J = 8.2 Hz, H-5′), 6.66 (2H, s, H-2, 6), 4.89 (1H, m, Glc-1′′), 4.67 (1H, d, J = 4.6 Hz, H-7′), 4.62 (1H, d, J = 4.7 Hz, H-7), 4.17 (2H, dt, J = 9.0, 6.6 Hz, H-9, 9′), 3.79 (2H, dd, J = 7.9, 2.9 Hz, H-9, 9′), 3.76 (9H, s, 3, 5, 3′OCH3), 3.60-3.08 (6H, m, Glc-2′′-6′′), 3.05 (2H, m, H-8, 8′). 13CNMR (100 MHz, DMSO) δC 152.6 (C-3, 5), 147.5 (C-3′), 145.9 (C-4′), 137.1 (C-4), 133.7 (C-1′), 132.2 (C-1), 118.7 (C-6′), 115.1 (C5′), 110.4 (C-2′), 104.2 (C-2, 6), 102.7 (C-1′′), 85.1 (C-7, 7′), 77.2 (C5′′), 76.5 (C-3′′), 74.2 (C-2′′), 71.2 (C-9), 71.0 (C-9′), 69.9 (C-4′′), 60.9 (C-6′′), 56.4 (C-3, 5-OCH3), 55.6 (C-3′-OCH3), 53.7 (C-8′), 53.5 (C-8). (+)-Syringaresinol-β-D-glucoside (9): colourless oil; 1H NMR (400 MHz, DMSO) δH 8.27 (1H, br. s, 4′-OH), 6.67 (2H, s, H-2, 6), 6.61 (2H, s, H-2′, 6′), 4.90 (1H, d, J = 4.5 Hz, Glc -1′′), 4.68 (1H, d, J = 3.9 Hz, H-7), 4.63 (1H, d, J = 3.9 Hz, H-7′), 4.19 (2H, m, H-9, 9′), 3.81 (2H, d, J = 10.0 Hz, H-9, 9′), 3.77 (6H, s, 3, 5-OCH3), 3.76 (6H, s, 3′, 5′-OCH3), 3.61-3.09 (6H, m, Glc-2′′-6′′), 3.06 (2H, m, H-8, 8′).

C-NMR (150 MHz, DMSO) δC 152.7 (C-3, 5), 147.9 (C-3′, 5′), 137.2 (C-1), 134.9 (C-4′), 133.7 (C-4), 131.4 (C-1′), 104.2 (C-2, 6), 103.7 (C-2′, 6′), 102.7 (C-1′′), 85.4 (C-7), 85.1 (C-7′), 77.2 (C-5′′), 76.5 (C3′′), 74.2 (C-2′′), 71.3 (C-9′), 71.2 (C-9), 69.9 (C-4′′), 60.9 (C-6′′), 56.5 (C-3, 5-OCH3), 56.0 (C-3′, 5′-OCH3), 53.7 (C-8′), 53.6 (C-8). (+)-Pinoresinol-β-D-glucoside (10): colourless oil; 1H NMR (400 MHz, DMSO) δH 8.91 (1H, br. s, 4-OH), 7.05 (1H, d, J = 8.4 Hz, H-5), 6.95 (1H, d, J = 1.5 Hz, H-2), 6.89 (1H, d, J = 1.5 Hz, H-2′), 6.87 (1H, dd, J = 8.4, 1.5 Hz, H-6), 6.75 (1H, dd, J = 8.1, 1.5 Hz, H-6′), 6.72 (1H, d, J = 8.1 Hz, H-5′), 4.89 (1H, d, J = 6.6 Hz, Glc -1′′), 4.67 (1H, d, J = 3.8 Hz, H-7), 4.61 (1H, d, J = 4.0 Hz, H-7′), 4.16 (4H, m, H-9, 9′), 3.81 (2H, m, H-9, 9′), 3.77 (3H, s, 3-OCH3), 3.75 (3H, s, 3′-OCH3), 3.67-3.10 (6H, m, Glc-2′′-6′′), 3.06 (2H, m, H-8, 8′). 13C NMR (100 MHz, DMSO) δC 148.9 (C-3), 147.5 (C-3′), 145.9 (C-4), 145.8 (C-4′), 135.2 (C-1), 132.2 (C-1′), 118.6 (C-6), 118.1 (C-6′), 115.3 (C-5), 115.1 (C-5′), 110.6 (C-2), 110.4 (C-2′), 100.2 (C-1′′), 85.2 (C7′), 84.9 (C-7), 77.0 (C-5′′), 76.9 (C-3′′), 73.2 (C-2′′), 71.0 (C-9′), 70.9 (C-9), 69.7 (C-4′′), 60.7 (C-6′′), 55.7 (C-3-OCH3), 55.6 (C-3′), 53.7 (C-8), 53.6 (C-8′). (+)-Isolariciresinol (11): colourless oil; 1H NMR (400 MHz, DMSO). δH 6.72 (1H, dd, J = 7.9 Hz, H-5′), 6.66 (1H, br. s, H-2′), 6.62 (1H, s, H-2), 6.52 (1H, br. d, J = 7.9 Hz, H-6′), 6.12 (1H, s, H-5), 3.75 (1H, br. d, J = 10.5 Hz, H-7′), 3.71 (3H, s, 3-OCH3), 3.68 (3H, s, 3′OCH3), 3.58 (1H, s, H-9), 3.48 (2H, s, H-9, 9′), 3.19 (1H, m, H-9′), 2.68 (2H, m, H-7), 1.85 (1H, m, H-8), 1.65 (1H, s, H-8′). 13C NMR (100 MHz, DMSO) δC 147.3 (C-3′), 145.5 (C-3), 144.7 (C-4), 144.1 (C-4′), 137.1 (C-1′), 132.7 (C-6), 127.2 (C-1), 121.5 (C-6′), 116.3 (C5), 115.3 (C-5′), 113.3 (C-2′), 111.8 (C-2), 63.6 (C-9), 59.8 (C-9′), 55.7 (C-3-OCH3), 55.5 (C-3′-OCH3), 45.9 (C-7′, 8′), 38.1 (C-8), 32.3 (C-7). (−)-(7′S,8S,8′R)-4,4′-dihydroxy-3,3′,5,5′-tetramethoxy-7′,9epoxylignan-9′-ol-7-one (12): colourless oil; 1H NMR (400 MHz, DMSO). δH 9.60 (1H, br. s, 4-OH), 8.29 (1H, brs, 4′-OH), 7.31 (2H, s, H-2, 6), 6.64 (2H, s, H-2′, 6′), 4.55 (1H, d, J = 7.9 Hz, H-7′), 4.21 (1H, dd, J = 12.9, 7.2 Hz, H-8), 4.12 (1H, t, J = 8.3 Hz, H1-9), 4.06 (1H, m, H2-9), 3.85 (6H, s, 3, 5-OCH3), 3.74 (6H, s, 3′, 5′-OCH3), 3.53 (2H, m, H-9′), 2.49 (1H, m, H-8′). 13C NMR (100 MHz, DMSO) δC 197.2 (C-7), 147.8 (C-3, 5), 147.6 (C-3′, 5′), 141.1 (C-4), 134.9 (C4′), 131.8 (C-1′), 126.7 (C-1), 106.4 (C-2, 6), 104.0 (C-2′, 6′), 83.0 (C7′), 69.8 (C-9), 59.9 (C-9′), 56.1 (C-3, 5-OCH 3 ), 56.0 (C-3′, 5′-OCH3), 53.4 (C-8′), 48.6 (C-8). 13

3.5.

Quantification

Quantitation is particularly important for quality control of functional food. Fan and He (2006) developed a simple HPLC method to simultaneously quantify three bioactive triterpene acids in LP. Six kinds of phenolics in FP were identified and quantified by HPLC using a flow rate gradient elution system (Chen et al., 2008). However, until now, multi-component analysis and comparison of LP and FP are very limited, with few reports regarding quality control. In this study, we tested the mixed standard solution of four secoiridoids and eight lignans (Fig. 5 and Table 4). However, only eight compounds in LP and seven compounds in FP were found by comparing the mass spectra and retention times with that of reference standards in MRM (multiple reaction monitor) mode (Fig. 6). The content of compound 9 was highest in both LP and FP, and it was 50.0 and 6.3 µg/g, respectively. The contents of secoiridoids in LP, including compounds 2, 4, 3 and 1, were 9.3, 12.5, 7.1 and 2.5 µg/g, respectively, and in FP were 0.7, 0.5, 0.6

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Fig. 5 – EIC (extract ion chromatogram) of mixed standard solution of four secoiridoids and eight lignans in MRM (multiple reaction monitor) mode.

and 0.3 µg/g, respectively. The lignans, including compounds 12, 9, 8 and 10 were 25.0, 50.0, 10.0 and 0.03 µg/g in LP, and in FP were 2.7, 6.3, 5.0 and 0.0 µg/g (Table 5). The other four compounds including compounds 5, 7, 6 and 11 could not be detected simultaneously with the eight analytes above in the crude extracts of LP and FP, as their concentrations were lower than LODs (limit of detection) of MRM mode by HPLC-QQQ-MS/MS (15, 30, 35, 135 ng/g, respectively). Compound 10 could not be detected in FP, as its concentration was lower than the LOD of 25 ng/g. For the further quantitative analysis of undetected compounds (5–7, 11), the SRM (single reaction monitor) mode was used to improve the sensitivity of HPLC-QQQ-MS/MS and to increase the LODs of analytes. In SRM mode, only one Q1/Q3 channel was set for each assay to

determine the single compounds. In this case, the contents of the other four compounds (5–7, 11) in LP have been determined (Fig. 7 and Table 5). However, components 5–7, 10 and 11 were not found in FP. As a result, the contents of the seven components of LP and FP were similar, but they were more abundant in the LP. The results indicate that the proposed method which contains MRM and SRM mode is reliable for the rapid analysis and quantitation of a group of ingredients present in LP and FP and applicable in the differentiation of complex samples that share similar chemical constituents. Previous studies have shown that the LP and FP possess antioxidant effects (Chen et al., 2008; Sun et al., 2011). Chen et al. (2008) reported that the content of total phenolics of the FP

Table 4 – MS parameters for 12 target compounds. Peak

Compound

tR (min)

Q1/Q3 (m/z)

DP (volts)

CE (volts)

1 2 3 4 5 6 7 8 9 10 11 12

(+)-Medioresinol monoglucoside (8) (+)-Syringaresinol-β-D-glucoside (9) Persimmonoid B (2) (−)-(7′S,8S,8′R)-4,4′-dihydroxy-3,3′,5,5′-tetramethoxy-7′,9-epoxylignan-9′-ol-7-one (12) Oleuropein (4) Ligustroside (3) Persimmonoid A (1) (+)-Medioresinol (5) (+)-Pinoresinol-β-D-glucoside (10) (+)-Pinoresinol (7) (+)-Syringaresinol (6) (+)-Isolariciresinol (11)

11.64 11.71 13.79 14.59 14.66 16.33 16.52 11.19 12.02 12.05 12.01 12.28

573/410 603/440 607/445 457/232 563/401 547/385 591/429 411/136 543/309 381/217 417/387 359/344

64 147 138 116 139 107 127 141 93 83 −87 −104

42 46 40 30 38 36 38 40 35 26 −36 −40

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Fig. 6 – EIC (extract ion chromatogram) of eight identified compounds from FP (A1) (B1) and LP (A2) (B2) (C) in MRM (multiple reaction monitor) mode.

and the antioxidant activity against ABTS/DPPH radicals were significantly higher (P < 0.05) than those of grape, apple, and tomato. Some researchers have indicated that FP is one of the most bioactive fruits, and particular components of FP are more active (Achiva, Hibasami, Katsuzaki, Imai, & Komiya, 1997; Uchida et al., 1989). Among the phenolic compounds, gallic acid was found to be a strong antioxidant, antimutagenic and anticarcinogenic agent, a high content in FP is especially important (Gunckel et al., 1998). Sugar is an important factor contributing to the internal quality and taste of FP, and carotenoids are a clear visual influence on the consumer preferences (Veberic et al., 2010). FP tannins are 20 times more potent than vitamin E in terms of antioxidation, which can prolong life and reduce the incidence of stroke in hypertensive rats (Jung et al., 2005). However, literatures on secoiridoids and lignans of LP and FP are scarce. This study indicated that minor secoiridoid and lignan constituents may contribute to bioactivities of LP

and FP, and enhanced the knowledge about the minor active components, and the antioxidant and neuroprotective effects of LP and FP.

4.

Conclusions

The occurrence of secoiridoids (1–4) from persimmon leaf was observed for the first time. Meanwhile, antioxidant activity of all the twelve isolates was elucidated by three different methods, as well as neuroprotective evaluation of them using human SH-SY5Y cells. There was an obvious relationship between the structures and their antioxidant activity. Among them compounds persimmonoid A (1), ligustroside (3), oleuropein (4) and (−)-(7′S,8S,8′R)-4,4′-dihydroxy-3,3′,5,5′tetramethoxy-7′,9-epoxylignan-9′-ol-7-one (12) also showed

Fig. 7 – EIC (extract ion chromatogram) of four identified compounds from LP in SRM (single reaction monitor) mode.

10.0 50.0 9.3 25.0 12.5 7.1 2.5 0.01 0.03 0.02 0.02 0.03 2–64 6.25–200 1–32 6.25–200 2–64 1–32 1–32 0.2–6.4 0.5–16 0.2–6.4 0.2–6.4 0.5–16

5.0 6.3 0.7 2.7 0.5 0.6 0.3 – – – – –

Contents in LP (µg/g) Ranges (ng/ml)

Contents in FP (µg/g)

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significant neuroprotective activity at concentrations of 12.5 and 25 µΜ. Furthermore, the established HPLC-QQQ-MS/MS method was applied for the simultaneous determination of eight compounds from LP and seven compounds from FP, including four secoiridoids (1–4), and four lignans (8–10, 12), and the trace constituents including compounds (5–7, 11) from LP could be quantified by SRM mode alone. The structural features, the contents, the diverse biological and pharmacological properties of the bioactive constituents are particularly attractive to us engaged in the research of functional food. Further investigations may be worthwhile to illustrate the chemodiversity, quantity and biological significance of bioactive compounds.

Conflict of interest

0.9995 0.9998 0.9996 0.9998 0.9991 0.9990 0.9995 0.9991 0.9992 0.9997 0.9994 0.9998 Y = 2.52E + 3x + 5.26E + 3 Y = 2.59E + 3x + 1.68E + 4 Y = 9.38E + 3x + 2.08E + 4 Y = 2.27E + 3x + 1.79E + 4 Y = 5.28E + 3x + 3.07E + 4 Y = 9.93E + 3x + 1.84E + 4 Y = 4.42E + 4x + 2.65E + 4 Y = 2.81E + 4x + 1.01E + 4 Y = 9.43E + 4x + 1.85E + 4 Y = 1.32E + 4x + 2.55E + 4 Y = 9.48E + 4x + 4.56E + 4 Y = 1.21E + 4x + 3.33E + 4 (+)-Medioresinol monoglucoside (8) (+)-Syringaresinol-β-D-glucoside (9) Persimmonoid B (2) (−)-(7′S,8S,8′R)-4,4′-dihydroxy-3,3′,5,5′-tetramethoxy-7′,9-epoxylignan-9′-ol-7-one (12) Oleuropein (4) Ligustroside (3) Persimmonoid A (1) (+)-Medioresinol (5) (+)-Pinoresinol-β-D-glucoside (10) (+)-Pinoresinol (7) (+)-Syringaresinol (6) (+)-Isolariciresinol (11)

Calibration curves Compounds

Table 5 – Calibration and quantitation of twelve identified compounds in LP and FP.

Correlations (r2)

The authors declare that there are no conflicts of interest.

Acknowledgements This work was supported by the Key Laboratory of StructureBased Drug Design & Discovery (Shenyang Pharmaceutical University), Ministry of Education. This study received financial support from the National Natural Science Foundation of China (81373925, 81573319), and the Foundation (LT2015027) from the Project of Innovation Team is gratefully acknowledged. The authors thank Li W. and Sha Y. of Shenyang Pharmaceutical University for recording NMR spectra.

Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.jff.2016.03.025.

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