Neuroprotective Effects of 1,2-Diarylpropane ... - ACS Publications

Shenyang Pharmaceutical University, Shenyang 110016, People's Republic of China. § Chinese People's Liberation Army 210 Hospital, Dalian 116021, ...
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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Neuroprotective Effects of 1,2-Diarylpropane Type Phenylpropanoid Enantiomers from Red Raspberry against H2O2‑Induced Oxidative Stress in Human Neuroblastoma SH-SY5Y Cells Le Zhou,†,⊥ Guo-Dong Yao,†,⊥ Xiao-Yu Song,† Jie Wang,† Bin Lin,‡ Xiao-Bo Wang,§ Xiao-Xiao Huang,*,†,§ and Shao-Jiang Song*,† †

School of Traditional Chinese Materia Medica, Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China ‡ School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China § Chinese People’s Liberation Army 210 Hospital, Dalian 116021, People’s Republic of China S Supporting Information *

ABSTRACT: Red raspberry (Rubus idaeus L.) is an edible fruit-producing species belonging to the Rosaceae family. In our search for the health-promoting constituents from this fruit, four pairs of enantiomeric phenylpropanoids (1a/1b−4a/4b), including three new compounds (1a and 2a/2b), were isolated from red raspberry. Their structures were elucidated by a combination of the extensive NMR spectroscopic data analyses, high-resolution electrospray ionization mass spectrometry and comparison between the experimental measurements of electronic circular dichroism (ECD) and calculated ECD spectra by time-dependent density functional theory (TDDFT). In addition, their neuroprotective effects against H2O2-induced oxidative stress in human neuroblastoma SH-SY5Y cells were investigated, and the results showed enantioselectivity, in which that 3a exhibited noticeable neuroprotective activity, while its enatiomer 3b exhibited no obvious protective effect. Further study demonstrated that 3a could selectively inhibit the apoptosis induction and reactive oxygen species (ROS) accumulation by enhancing the activity of catalase (CAT) in H2O2-treated human neuroblastoma SH-SY5Y cells. These findings shed much light on a better understanding of the neuroprotective effects of these enantiomers and provide new insights into developing better treatment of neurodegenerative diseases in the future. KEYWORDS: oxidative stress, red raspberry, enantiomers, SH-SY5Y, apoptosis, catalase

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INTRODUCTION Oxidative stress, caused by an imbalance between antioxidants defense system and excessive production of prooxidants, can mediate the pathogenesis of neurodegenerative disorders.1 Increased reactive oxygen species (ROS) can damage protein, lipid, and nucleic acids, decrease the levels of endogenous antioxidant glutathione, and reduce the activities of various antioxidant enzymes, such as superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase, and eventually lead to various diseases including inflammation, cancer, and cardiovascular diseases as well as aging.2 Natural antioxidants derived from fruits and vegetables can protect the human body from free radicals and ROS effects and retard the progression of many chronic diseases as well as lipid oxidative rancidity.3 Therefore, the development and utilization of more effective antioxidants are highly desired. Red raspberry (Rubus ideaus L.), a member of the Rosaceae family, is grown primarily for its edible fruit-producing species.4 This species is distributed over most of the continents and mainly cultivated in Eastern Europe, Eastern Asia, and North America, with a continuously increasing world production.5 There is a long history of using fresh or frozen red raspberry in making desserts and a variety of processed food such as jams, wines, jellies, confectionery products, and yogurts.6 Like many other fruits, red raspberry has additional health benefits because © XXXX American Chemical Society

it is rich in polyphenolic compounds such as anthocyanins, flavonoids, and phenolic acids, which are all natural antioxidants and may provide protection against various human diseases caused by oxidative stress.7−9 During our continuing search for structurally diverse metabolites with antioxidant activities from red raspberry, four pairs of enantiomeric phenylpropanoids (1a/1b−4a/4b, Figure 1) including three new compounds (1a and 2a/2b) were isolated.10,11 It is generally known that enantiomers abound in natural plants and are difficult to separate in the past years by traditional methods. Different enantiomers of a chiral drug possess the same physicochemical properties. However, they usually exhibit significant differences in bioactivity, toxicity, excretion, metabolism, and effects on beneficial and nontarget organisms.12,13 Additionally, there are few comprehensive pharmacological mechanism studies on enantioselective effects of natural products, especially in the field of food chemistry. Therefore, it is essential to evaluate the enantiomers in terms of their underlying mechanisms of bioactivity. Herein, the enantioselective neuroprotective effects against H2O2-induced Received: Revised: Accepted: Published: A

September 25, 2017 December 6, 2017 December 7, 2017 December 7, 2017 DOI: 10.1021/acs.jafc.7b04430 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of compounds 1a/1b−4a/4b.

Table 1. 1H NMR (400 MHz) and 13C NMR (100 MHz) for Compounds 1 and 2 (δ in ppm) 1a δH

position 1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7-OCH3 3′-OCH3 a

7.92 (d, J = 8.8 Hz) 6.78 (d, J = 8.8 Hz) 6.78 (d, J = 8.8 Hz) 7.92 (d, J = 8.8 Hz) 4.74 (dd, J = 8.7, 5.2 Hz) 4.24 (dd, J = 10.7, 8.7 Hz) 3.71 (dd, J = 10.7, 5.2 Hz) 6.78 (br. s)

6.62 (d, J = 8.1 Hz) 6.65 (br. d, J = 8.1 Hz) 3.80 (s)

2 δC 129.8 132.4 116.3 164.1 116.3 132.4 199.7 56.2 65.5 129.7 116.5 149.2 146.9 112.7 122.2 56.3

δHb 6.84 (d, J = 8.4 Hz) 6.56 (d, J = 8.4 Hz) 6.56 6.84 4.27 2.88 3.75

(d, J = 8.4 Hz) (d, J = 8.4 Hz) (d, J = 8.3 Hz) (m) (m)

6.47 (overlapped)

6.49 6.37 3.05 3.60

(d, J = 8.0 Hz) (dd, J = 8.0, 1.6 Hz) (s) (s)

δ Cb 130.2 128.6 114.4 156.3 114.4 128.6 84.1 53.7 62.5 131.5 113.3 146.6 144.5 114.7 121.4 55.8 55.4

δH (CDCl3) 6.93 (d, J = 8.5 Hz) 6.67 (d, J = 8.5 Hz) 6.67 6.93 4.33 3.02 4.16 3.85

(d, J = 8.5 Hz) (d, J = 8.5 Hz) (d, J = 9.4 Hz) (m) (dd, J = 11.1, 8.0 Hz) (dd, J = 11.1, 4.3 Hz)

6.33 (d, J = 1.8 Hz)

6.73 6.49 3.22 3.71

(d, J = 8.1 Hz) (dd, J = 8.1, 1.8 Hz) (s) (s)

Recorded in CD3OD. bRecorded in DMSO-d6. 6AD high-pressure pump with an SPD-20A ultraviolet−visible (UV− vis) light absorbance detector. The Chiralpak AD-H and Chiralpak IC columns (4.6 × 250 mm2, 5 μm, Daicel Polymer Ltd., Tokyo, Japan) were used in the HPLC system. The organic solvents were distilled prior to the separation process. Plant Material. The fruits of red raspberry were collected in June 2015 from Shenyang, Liaoning province, PR China. Botanical authentication was performed by Professor Jin-Cai Lu (Department of Natural Products Chemistry, Shenyang Pharmaceutical University, PR China), and a voucher specimen has been deposited in the Herbarium of Shenyang Pharmaceutical University (voucher number 20150601). Extraction and Isolation. Air-dried fruits of red raspberry (20 kg) were chopped and soaked in 70% aqueous ethanol at room temperature (3 × 50 L) three times (3 h each). The solvent was evaporated under reduced pressure at 50 °C to afford a crude extract (1750 g). The extract was chromatographed on D101 macroporous resin eluting with a step gradient of EtOH-H2O (2:8, 6:4, 9:1, 10:0) to yield four corresponding fractions (I−IV). Fraction III portion was separated using a silica gel column eluting with an increasing gradient of CH2Cl2-MeOH to afford seven subfractions (IIIa to IIIg). Fraction IIIb (26.5 g) was further submitted to separation over a YMC gel ODS-A column using EtOH-H2O (from 1:9 to 7:3) to obtain six fractions (A−F) on the basis of HPLC analysis. Among them, fractions A (4.1 g) was chromatographed on silica gel eluted with CH2Cl2MeOH (from 9:1 to 1:1) to afford five subfractions (A1−A5) on the basis of silica gel TLC analysis. Then subfraction A1 was separated

oxidative stress and the possible mechanisms were investigated in human neuroblastoma SH-SY5Y cells.

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MATERIALS AND METHODS

General Experimental Procedures. Optical rotations were measured on a JASCO DIP-370 digital polarimeter (JASCO Analytical Instrumentals, Easton, MD, USA) at room temperature. The UV and IR spectra were recorded on a Shimadzu UV-1700 spectrophotometer (Shimadzu, Kyoto, Japan) and Bruker IFS-55 spectrometer (Bruker Co., Karlsruhe, Germany) with KBr pellets, respectively. Electronic circular dichroism (ECD) experiments were performed on a MOS 450 spectrometer (Bio-Logic Science Instruments, Seyssinet-Pariset, France). The NMR spectra were recorded on a Bruker ARX-400 MHz and a Bruker AV-600 MHz spectrometers (Bruker, Karlsruhe, Germany) with TMS as an internal standard. The high-resolution electrospray ionization mass spectrometry (HRESIMS) data were obtained with an Agilent G6520 Q-TOF spectrometer (Santa Clara, CA, USA). Silica gel column chromatography was carried out over silica gel 60 (100−200 mesh, 200−300 mesh, Qingdao Marine Chemical, Ltd., Qingdao, China). Column chromatography was performed with silica gel 60 (100−200 mesh, 200−300 mesh, Qingdao Marine Chemical, Ltd., Qingdao, China), D101 macroporous resin (26−60 mesh, Cangzhou Bon Adsorber Technology Co., Ltd., Cangzhou, China), and YMC gel ODS-A (50 μm, YMC Co. Ltd., Kyoto, Japan). Semipreparative high-performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) system consisted of a LCB

DOI: 10.1021/acs.jafc.7b04430 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry over RP-HPLC using a mobile phase of MeOH-H2O (22:78, v/v) to obtain five subfractions (A1−1 to A1−5). Consequently, subfraction A1−3 was purified by the semipreparative HPLC (MeOH-H2O, 18:82, v/v, 3.5 mL/min) over a YMC C18 column (4.6 × 250 mm2, 5 μm) to afford compounds 1 (18.3 mg) and 4 (6.1 mg). Compound 3 (9.6 mg) was isolated from subfraction A1−4 by a semiprearative HPLC column with MeCN-H2O (12:88, v/v). Subfraction B1 was also purified by RP-HPLC with the mobile phase MeOH/H2O (45:55, v/v) to obtain compound 2 (4.2 mg). Chiral resolution of 1 and 3 were performed on a Daicel Chiralpak AD-H column (eluted with n-hexane-2-propanol, v/v, 4:1, flow rate 0.8 mL/min) to obtain 1a (4.7 mg), 1b (5.0 mg) and 3a (4.4 mg), 3b (4.5 mg), respectively. Compounds 4a (2.6 mg) and 4b (2.6 mg) were successfully separated using the same Chiralpak AD-H column as 1 and 3 eluting with n-hexane-2-propanol (v/v, 5:1, flow rate 0.8 mL/min). 2 (9.5 mg) was eluted by Daicel chiralpak IC column (n-hexane-2-propanol, v/v, 4:1, flow rate 0.7 mL/min) to give 2a (1.8 mg) and 2b (1.7 mg). (1): colorless oil; [α]20 D + 0.3 (c 0.08, MeOH); UV (MeOH) λmax (log ε), 216 (2.32), 283 (0.66); IR (KBr) υmax, 3420, 1633, 1437, 1407, 1384, 1317, 1272, 1020, 953, and 709 cm−1; HRESIMS at m/z 311.0912 [M + Na]+ (calcd for C16H16NaO5, 311.0895); 1H and 13C NMR, see Table 1. (1a): [α]20 D −27.7 (c 0.10, MeOH); ECD (MeOH) λmax (Δε) 234 (+27.88), 269 (−32.64), 300 (+36.61) nm. (1b): [α]20 D + 26.5 (c 0.10, MeOH); ECD (MeOH) λmax (Δε) 236 (−29.53), 263 (+34.73), 301 (−39.38) nm. (2): colorless oil; [α]20 D + 0.5 (c 0.11, MeOH); UV (MeOH) λmax (log ε), 226 (2.36), 278 (0.74); IR (KBr) υmax, 3433, 2925, 1632, 1517, 1403, 1384, 1271, 1127, 1009, 833, 704, and 619 cm−1; HRESIMS at m/z 327.1210 (calcd for C17H20NaO5, 327.1208); 1H and 13C NMR, see Table 1. (2a): [α]20 D + 32.7 (c 0.10, MeOH); ECD (MeOH) λmax (Δε) 233 (−24.72), 251 (+0.65), 283 (−5.10) nm. (2b): [α]20 D − 35.4 (c 0.11, MeOH); ECD (MeOH) λmax (Δε) 233 (+23.70), 252 (+0.54), 284 (+5.29) nm. ECD Calculations. The absolute configurations of compounds 1 and 2 were determined by using density functional theory (DFT) calculations and carried out with the Gaussian 09 program package.14 First, the conformational searches were performed using the MMFF94S force field to obtain energy minimization by CONFLEX.15 Next, those conformations whose energy was no more 3 kcal/mol higher than the lowest energy (24 conformations for compound 1 and 36 conformations for compound 2) were optimized at the B3LYP/631G(d) level. They were checked by frequency calculation and resulted in no imaginary frequencies. The ECD of the conformers were then calculated by the TDDFT method at the B3LYP/6-311+ +G(2d,p) level with the CPCM model in methanol solution. The calculated ECD curve was generated using SpecDis 1.51.16 Cell Culture. Human neuroblastoma SH-SY5Y cells (ATCC, Manassas, USA) were cultured in DMEM medium (Hyclone, Logan, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Gaithersburg, USA) in a humidified atmosphere containing 5% CO2 at 37 °C. Logarithmically growing cells were used in all the experiments. Cell Viability Assay. SH-SY5Y cells were pretreated with various of concentrations (25, 50, 100 μM) tested compounds for 1 h before H2O2 (200 μM) treatment for another 4 h. Then 20 μL of MTT (5 mg/mL) was added to each well for 4 h, and the crystals were dissolved in DMSO. Optical density at 490 nm was determined by using a plate microreader (Thermo, Waltham, USA). The cell viability was calculated as follows:

Cell viability (%) =

A490,sample − A490,blank A490,control − A490,blank

mL) for 15 min at room temperature. Apoptotic cells, which exhibited condensed and fragmented nuclei, were identified by a fluorescence microscope (Olympus, Tokyo, Japan). Annexin V-FITC/PI Staining. Annexin V-FITC and PI double staining were applied to evaluate apoptotic ratio according the manufacturer’s instructions. The treated cells were stained with Annexin V-FITC followed by PI at room temperature for 15 min. To quantify apoptotic ratio, cell samples were analyzed by a FACScan flow cytometry (Becton Dickinson, Franklin Lakes, USA). Western Blot Analysis. Cells were collected and lysed in wholecell RIPA lysis buffer (Beyotime, Shanghai, China). The protein content of the supernatant was examined using BCA Protein Assay Kit (Beyotime, Shanghai, China). After denaturation with boiling water for 10 min, the lysates were separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto a Millipore Immobilon-P Transfer Membrane (Millipore, Billerica, USA). The membranes were soaked in 5% skim milk and then incubated with primary polyclonal antibodies overnight, followed by incubation with the corresponding horseradish peroxidase (HRP)conjugated secondary antibodies. The blots were visualized using the SuperSignal West Pico Chemiluminescent Substrate. The protein bands were quantified with the ImageJ software. Fluorescent Microscopy of ROS Production. Cell samples in a 24-well plate were treated with 200 μM H2O2 in the presence or absence of 3, 3a, or 3b for 4 h. The cells were rinsed with PBS twice and then incubated with 10 μM H2DCF-DA at 37 °C for 20 min. After incubation, the cells were observed by fluorescent microscopy. Catalase (CAT) Assay. The intracellular CAT activity was measured using the CAT Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Cell samples were lysed, and the CAT activity was expressed in terms of the protein content for signal normalization. Statistical Analysis. All results and data were confirmed in at least three separate experiments. Data are expressed as means ± SD. Statistical comparisons were analyzed by student’s t-test using GraphPad Prism. P < 0.05 was considered statistically significant.

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RESULTS AND DISCUSSION Compound 1 was isolated as a colorless oil. The molecular formula was established as C16H16O5 on the basis of its HRESIMS ion at m/z 311.0912 [M + Na]+ (calcd 311.0895), indicating the presence of 9 degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl group (3420 cm−1), conjugated carbonyl group (1633 cm−1), and phenyl (1437, 1407, and 1384 cm−1) group. The 1H NMR data (Table 1) of 1 indicated the presence of a 1,3,4-trisubstituted benzene system [δH 6.78 (1H, br. s, H-2′), 6.65 (1H, br. d, J = 8.1, H6′), and 6.62 (1H, d, J = 8.1 Hz, H-5′)], a symmetric 1,4disubstituted benzene system [δH 7.92 (2H, d, J = 8.8 Hz, H2,6), 6.78 (2H, d, J = 8.8 Hz, H-3,5)], a methine group at δH 4.74 (1H, m, H-8), an oxygenated methylene group [δH 4.24 (1H, dd, J = 10.7, 8.7 Hz, H-9a), 3.71 (1H, dd, J = 10.7, 5.2 Hz, H-9b)], and a methoxyl groups at δH 3.80 (3H, s). The 13C NMR data displayed 16 carbon signals including a ketone carbonyl at δC 199.7 (C-7). The HMBC correlations (Figure S1.6, Supporting Information) of H-2,6/C-7 and H-9/C-7 indicated that a ketone carbonyl (δC 199.7) is linked to C-7. Additionally, HMBC correlations of H-8/C-1′, C-2′/6′ and H9/C-1′ required the direct linkage of C-8 to C-1′. From all the above evidence, the chemical structure of 1 was established as 3-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(4-hydroxyphenyl)-propan-1-one. The absence of any Cotton effect in the ECD spectrum and optical rotation indicated compound 1 was a pair of racemic mixture. The subsequent chiral resolution led to the isolation of the enantiomers 1a and 1b, which showed anticipated mirror

× 100 (1)

Observation of Morphological Changes. In the absence or presence of 3, 3a, or 3b, cells were treated with 200 μM H2O2 for 4 h. The cellular morphology was examined under a phase contrast microscope. Apoptotic morphology was studied by staining with Hoechst 33258 (Beyotime, Shanghai, China), a fluorescent DNAbinding dye. Cell samples were stained with Hoechst 33258 (5 μg/ C

DOI: 10.1021/acs.jafc.7b04430 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. Calculated and experimental ECD spectra of 1a/1b and 2a/2b.

Figure 3. Neuroprotective effects of compounds against H2O2-induced injury of SH-SY5Y cells. (A) Cytotoxic effect of the tested compounds (25, 50, 100 μM) in SH-SY5Y cells using MTT assay. (B) In the presence or absence of the tested compounds at different concentrations (25, 50, 100 μM), MTT assay was used to determine the cell viability after H2O2 (200 μM) treatment for 4 h. ∗P < 0.05 versus H2O2-treated group; #P < 0.05 was considered statistically significant when compared with its enantiomer.

chemical structure of 2 was then identified as 2-(4-hydroxy-3methoxyphenyl)-1-(4-hydroxyphenyl)-1-methoxy-propan-3-ol. The relative configuration was established on the basis of interpretation of 1H−1H coupling constants. A coupling constant of 9.4 Hz (in CDCl3) between H-7 and H-8 implied a threo configuration of these two protons.18 Its NMR data and optical rotation ([α]20 D 0.3) were the same as those of a compound isolated from the fruits of Juglans mandshurica with absolute configuration undetermined.14 Similarly, compound 2 was subjected to a Daicel Chiralpak IC chiral column to check its racemic nature, and 2a and 2b were successfully obtained. The calculated ECD curve of (7S,8R)-2 at the B3LYP/6-311+ +G(2d,p) level with the CPCM model in methanol solution matched very well with the experimental ECD curve of 2a (Figure 2), suggesting the (7S,8R) absolute configuration for 2a. Thus, 2a and 2b were assigned as (7S,8R)-2-(4-hydroxy-3methoxyphenyl)-1-(4-hydroxyphenyl)-1-methoxy-propan-3-ol and (7R,8S)-2-(4-hydroxy-3-methoxyphenyl)-1-(4-hydroxyphenyl)-1-methoxy-propan-3-ol.

image-like ECD curves (Figure 2). Their absolute configurations were determined by comparison of its experimental and calculated ECD spectra at the B3LYP/6-311++G(2d,p) level with the CPCM model in methanol solution. On the basis of the comparison, the absolute configurations of 1a and 1b were assigned as (S)-3-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(4-hydroxyphenyl)propan-1-one and (R)-3-hydroxy-2-(4hydroxy-3-methoxyphenyl)-1-(4-hydroxyphenyl)propan-1one,17 respectively. Compound 2 was obtained as a colorless oil, and its molecular formula was assigned as C17H20O5 from its 13C NMR spectroscopic data and the HRESIMS [M + Na]+ ion peak at m/z 327.1210 (calcd for C17H20NaO5, 327.1208). Detailed comparison of the NMR data (Tables 1) of 2 and 1 indicated that the two compounds were related structurally. The main difference between the two compounds was that the ketone carbonyl group (δC 199.7) in 1 was reduced and then replaced by a methoxy moiety in 2, which was verified by the HMBC correlations of H-7/C-1, C-2, C-6, C-9, C-1′, and H-8/C-1, C7, C-9, C-1′, C-2′ (Figure S2.7, Supporting Information). The D

DOI: 10.1021/acs.jafc.7b04430 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 4. Effects of 3, 3a, and 3b (50 μM) on morphological changes of H2O2-treated SH-SY5Y cells. Upper, cellular morphological changes were observed by phase contrast microscopy; lower, the nuclear changes were examined by Hoechst 33258 staining. Arrows: apoptotic cells. Scale bar: 50 μm.

Figure 5. Compound 3a was prone to attenuate apoptosis of H2O2-treated SH-SY5Y cells. SH-SY5Y cells were pretreated with 3, 3a, and 3b (50 μM) and then treated with H2O2 for 4 h. Flow cytometry was applied to determine the apoptotic ratio after Annexin V-FITC/PI staining. The percentage of apoptotic cells was calculated in the bar chart. ∗P < 0.05 versus control group; #P < 0.05 versus H2O2-treated group.

oxidative damage to evaluate neurotoxicity and neuroprotection.24 First, MTT assay was applied to determine whether these compounds have cytotoxic effects on cells. The results showed that there was no obvious cytotoxicity on the SH-SY5Y cells at 25, 50, and 100 μM, respectively (Figure 3A). Therefore, the cells were pretreated with these compounds (25, 50, 100 μM) before H2O2 treatment, and the cell viability was examined using MTT assay. As shown in Figure 3B, the results showed that the cell viability of SH-SY5Y cells was suppressed at 58.91% by H2O2 (200 μM) treatment. Interestingly, we found that pairs of phenylpropanoid enantiomers exerted distinctive neuroprotective effects on H2O2-treated SH-SY5Y cells. The cell viability was restored to 55.64% and 65.17% (by 1a and 1b

Upon comparing their measured spectroscopic data analysis with values reported in the literature, the known compounds were identified as (S)-3-hydroxy-1,2-bis(4-hydroxy-3-methoxyphenyl)-1-propanone (3a),19 (R)-3-hydroxy-1,2-bis(4-hydroxy-3-methoxyphenyl)-1-propanone (3b),20 (1S,2R)-1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol (4a),21 and (1R,2S)-1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol (4b).22 Neuroprotective Activity Study in Vitro. Hydrogen peroxide (H2O2), a major ROS contributor, is implicated in the progression of many neurodegenerative diseases.23 Therefore, H2O2-induced cytotoxicity in neuroblastoma cell line SH-SY5Y has been extensively regarded as a cultured cell model of E

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Figure 6. Effects of 3, 3a, and 3b (50 μM) on the expression of PARP in SH-SY5Y cells. (A) Expression of PARP and its cleavage were detected by Western blot analysis and β-actin was used as a loading control. (B) Band densities of proteins were quantified by ImageJ software. ∗P < 0.05 versus control group; #P < 0.05 versus H2O2-treated group.

Figure 7. Compound 3a downregulated ROS levels of SH-SY5Y cells. In the absence or presence of 3, 3a, and 3b (50 μM), SH-SY5Y cells were incubated with 200 μM H2O2. Intracellular ROS generation was observed by fluorescence microscopy with H2DCF-DA staining. Average fluorescence intensity values were quantified by ImageJ software. Scale bar: 50 μm. ∗P < 0.05 versus control group; #P < 0.05 versus H2O2-treated group.

at 100 μM), 73.29% and 56.48% (by 3a and 3b at 50 μM), and 58.01% and 66.62% (by 4a and 4b at 100 μM), respectively. However, only 3a and 3b (50 μM) had a statistic difference (P < 0.05). The results indicated that 3a had more noticeable protective effect than 3b, and it was chosen to study the underlying mechanism in the subsequent experiment. Some studies have reported that pairs of enantiomers had distinctive effects on the neuroprotective or antioxidant activities. For example, pairs of alkaloid enantiomers, isolated from the stems of Clausena lansium, exhibited different neuroprotective activities.25 Paradoxically, in some cases, both enantiomers resulted in comparable activities. Pairs of flavonol enantiomers from the leaves of Uncaria rhynchophylla showed similar antioxidant activities.26 However, the detailed mechanisms of the distinctive or comparable bioactivities of enantiomers need to be further investigated. Apoptosis Study. To further investigate the different neuroprotective effects between enantiomers, the same concentration (50 μM) of compounds 3a and 3b as well as their racemate (3) was incubated with H2O2 (200 μM) in SH-

SY5Y cells. The results from morphological observation showed that 3a significantly suppressed the typical characteristics of apoptosis including marked cell shrinkage and blebbing (Figure 4, upper panel). Similar results were produced after Hoechst 33258 staining, as 3a could decrease nuclear shrinkage, chromatin condensation, and fragmentation induced by H2O2 (Figure 4, lower panel). However, 3 and 3b had no obvious effects on H2O2-induced apoptosis in SH-SY5Y cells. Annexin V-FITC/PI doubling staining is an established assay to quantify apoptotic cells.27 As illustrated in Figure 5, about 22.69% SH-SY5Y cells underwent apoptosis when treatment with 200 μM H2O2. However, only 3a could significantly decrease the percentage of apoptotic cells induced by H2O2. To further illustrate the underlying mechanisms of neuroprotection provided by 3a, the expression of apoptosisassociated protein PARP (poly(ADP-ribose) polymerase) in SH-SY5Y cells was detected by a Western blotting assay. PARP, an important enzyme for DNA repairing, is inactivated by caspase cleavage when cells experiencing apoptosis.28 The results from Western blot analysis indicated that H2O2 F

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promoted the cleavage of PARP, while 3a obviously reversed this change (Figure 6A,B). Overall, the above results demonstrated that only optically pure compound 3a could protect SH-SY5Y cells from apoptosis, while its enantiomer 3b or racemate 3 had no neuroprotective effects. ROS Generation. ROS present an important regulatory factor in cellular signaling pathways of cancer cells.29 However, excessive ROS generation contributes to irreversible cellular injuries including DNA damage and apoptosis.30 As shown in Figure 7, H2O2-treated cells increased ROS generation using fluorescent microscopy after H2DCF-DA staining. 3a could significantly decrease the DCF fluorescence intensity of H2O2treated SH-SY5Y cells, while 3 and 3b did not decrease the intensity. Catalase (CAT) Activity. Catalase (CAT), an important antioxidant enzyme in living tissue, mainly protect the cells from oxidative damage induced by H2O2.31,32 To evaluate the effects of 3, 3a, and 3b on CAT activity, a commercial kit was applied. The results suggested that H2O2 dramatically decreased the activity of CAT, 3a had a better promoting effect than 3 and 3b (Figure 8). Hence, the CAT enzyme may be a key factor for the difference about the enantioselective effect between 3a and 3b.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +86-24-23986088. Fax: +86-24-23986088. *E-mail: [email protected]. Phone: +86-24-23986510. Fax: +86-24-23986510. ORCID

Shao-Jiang Song: 0000-0002-9074-2467 Author Contributions ⊥

L.Z. and G.-D.Y. contributed equally to this work.

Funding

This study was supported by the Project of Innovation Team (LT2015027) of Liaoning of P. R. China. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Song, S. L.; Xiao, X. Y.; Guo, D.; Mo, L. Q.; Bu, C.; Ye, W. B.; Den, Q. W.; Liu, S. T.; Yang, X. X. Protective effects of Paeoniflorin against AOPP-induced oxidative injury in HUVECs by blocking the ROS-HIF-1α/VEGF pathway. Phytomedicine 2017, 34, 115−126. (2) Zhang, Y.; Wu, Y.; Tashiro, S.; Onodera, S.; Ikejima, T. Reactive oxygen species contribute to oridonin-induced apoptosis and autophagy in human cervical carcinoma hela cells. Acta Pharmacol. Sin. 2011, 32, 1266−1275. (3) Yonny, M. E.; Rodríguez Torresi, A. O.; Cuyamendous, C.; Reversat, G.; Oger, C.; Galano, J. M.; Durand, T.; Vigor, C.; Nazareno, M. O. Thermal stress in melon plants: phytoprostanes and phytofurans as oxidative stress biomarkers and the effect of antioxidant supplementation. J. Agric. Food Chem. 2016, 64, 8296−8304. (4) Wang, S. Y.; Chen, C. T.; Wang, C. Y. The influence of light and maturity on fruit quality and flavonoid content of red raspberries. Food Chem. 2009, 112, 676−684. (5) Giovanelli, G.; Limbo, S.; Buratti, S. Effects of new packaging solutions on physico-chemical, nutritional and aromatic characteristics of red raspberries (Rubus idaeus L.) in postharvest storage. Postharvest Biol. Technol. 2014, 98, 72−81. (6) Vladisavljević, G. T.; Vukosavljević, P.; Veljović, M. S. Clarification of red raspberry juice using microfiltration with gas backwashing: A viable strategy to maximize permeate flux and minimize a loss of anthocyanins. Food Bioprod. Process. 2013, 91, 473−480. (7) Ludwig, I. A.; Mena, P.; Calani, L.; Borges, G.; Pereira-Caro, G.; Bresciani, L.; Del Rio, D.; Lean, M. E. J.; Crozier, A. New insights into the bioavailability of red raspberry anthocyanins and ellagitannins. Free Radical Biol. Med. 2015, 89, 758−769. (8) Chen, W.; Su, H.; Xu, Y.; Bao, T.; Zheng, X. D. Protective effect of wild raspberry (Rubus hirsutus Thunb.) extract against acrylamideinduced oxidative damage is potentiated after simulated gastrointestinal digestion. Food Chem. 2016, 196, 943−952. (9) Nowak, A.; Sójka, M.; Klewicka, E.; Lipińska, L.; Klewicki, R.; Kolodziejczyk, K. Ellagitannins from Rubus idaeus L. exert geno- and cytotoxic effects against the human colon adenocarcinoma cell line Caco-2. J. Agric. Food Chem. 2017, 65, 2947−2955. (10) Xu, Y.; Li, L. Z.; Cong, Q.; Wang, W.; Qi, X. L.; Peng, Y.; Song, S. J. Bioactive lignans and flavones with in vitro antioxidant and neuroprotective properties from Rubus idaeus rhizome. J. Funct. Foods 2017, 32, 160−169. (11) Zhou, L.; Lou, L. L.; Wang, W.; Lin, B.; Chen, J. N.; Wang, X. B.; Huang, X. X.; Song, S. J. Enantiomeric 8-O-4′ type neolignans from red raspberry as potential inhibitors of β-amyloid aggregation. J. Funct. Foods 2017, 37, 322−329. (12) Liu, H.; Xia, Y.; Cai, W.; Zhang, Y. N.; Zhang, X. Q.; Du, S. T. Enantioselective oxidative stress and oxidative damage caused by Racand S-metolachlor to Scenedesmus obliquus. Chemosphere 2017, 173, 22−30.

Figure 8. Effects of 3, 3a, and 3b (50 μM) on the activity of CAT in SH-SY5Y cells. The commercial kit was applied to examine the activity of CAT. ∗P < 0.05 versus control group; #P < 0.05 versus H2O2-treated group.

In conclusion, four pairs of enantiomeric 1,2-diarylpropane type phenylpropanoids (1a/1b−4a/4b), including three new phenylpropanoids (1a and 2a/2b), were isolated from the fruit of R. ideaus, and their chemical structures and absolute configurations were determined by NMR, HRESIMS, and calculated and experimental ECD. Biologically, the protective effects of these compounds against H2O2-indcued oxidative injury in SH-SY5Y cells and the possible mechanism were investigated. Collectively, these findings suggested that despite having the same chemical structure, the opposite absolute configurations for 3a and 3b resulted in different effects on H2O2-induced SH-SY5Y cell damage.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b04430. HRESIMS, UV, IR, and 1D, 2D-NMR spectra; ECD spectra of compounds 1a/1b-4a/4b (PDF) G

DOI: 10.1021/acs.jafc.7b04430 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

of the p38 MAPK/ROS pathway. Mol. Cell. Biochem. 2016, 415, 145− 155. (31) Chelikani, P.; Fita, I.; Loewen, P. C. Diversity of structures and properties among catalases. Cell. Mol. Life Sci. 2004, 61, 192−208. (32) Imlay, J. A. Pathways of oxidative damage. Annu. Rev. Microbiol. 2003, 57, 395−418.

(13) Zhang, W.; Cheng, C.; Chen, L.; Di, S. S.; Liu, C. X.; Diao, J. L.; Zhou, Z. Q. Enantioselective toxic effects of cyproconazole enantiomers against Chlorella pyrenoidosa. Chemosphere 2016, 159, 50−57. (14) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, revision B.01; Gaussian, Inc.: Wallingford, CT, 2009. (15) Jagannadh, B.; Reddy, S. S.; Thangavelu, R. P. Conformational preferences of 1,4,7-trithiacyclononane: a molecular mechanics and density functional theory study. J. Mol. Model. 2004, 10, 55−59. (16) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Bringmann, G. Specdis: quantifying the comparison of calculated and experimental electronic circular dichroism spectra. Chirality 2013, 25, 243−249. (17) Park, S.; Kim, N.; Yoo, G.; Kim, S. N.; Kwon, H. J.; Jung, K.; Oh, D. C.; Lee, Y. H.; Kim, S. H. Phenolics and neolignans isolated from the fruits of juglans mandshurica maxim. and their effects on lipolysis in adipocytes. Phytochemistry 2017, 137, 87−93. (18) Hsiao, J. J.; Chiang, H. C. Lignans from the wood of Aralia bipinnata. Phytochemistry 1995, 39, 899−902. (19) Naoto, H.; Tetsushi, K.; Masahiro, S.; Tomotaka, Y. Metabolism of a diarylpropane type lignin model compound by Pseudomonas sp. TMY1009. Mokuzai Gakkaishi. 1989, 35, 348−355. (20) Wu, T. S.; Yeh, J. H.; Wu, P. L. The heartwood constituents of Tetradium glabrifolium. Phytochemistry 1995, 40, 121−124. (21) Li, X.; Cao, W.; Shen, Y.; Li, N.; Dong, X. P.; Wang, K. J.; Cheng, Y. X. Antioxidant compounds from Rosa laevigata fruits. Food Chem. 2012, 130, 575−580. (22) Jiang, M. M.; Gao, H.; Dai, Y.; Zhang, X.; Wang, N. L.; Yao, X. S. Phenylpropanoid and lignan derivatives from Antiaris toxicaria and their effects on proliferation and differentiation of an osteoblast-like cell line. Planta Med. 2009, 75, 340−345. (23) Yan, M. H.; Wang, X. L.; Zhu, X. W. Mitochondrial defects and oxidative stress in Alzheimer disease and Parkinson disease. Free Radical Biol. Med. 2013, 62, 90−101. (24) Huang, C.; Lin, Y.; Su, H.; Ye, D. Forsythiaside protects against hydrogen peroxide-induced oxidative stress and apoptosis in PC12 cell. Neurochem. Res. 2015, 40, 27−35. (25) Liu, J.; Du, Y. Q.; Li, C. J.; Li, L.; Chen, F. Y.; Yang, J. Z.; Chen, N. H.; Zhang, D. M. Alkaloids from the stems of Clausena lansium and their neuroprotective activity. RSC Adv. 2017, 7, 35417−35425. (26) Li, R. X.; Cheng, J. T.; Jiao, M. J.; Li, L.; Guo, C.; Chen, S.; Liu, A. New phenylpropanoid-substituted flavan-3-ols and flavonols from the leaves of Uncaria rhynchophylla. Fitoterapia 2017, 116, 17−23. (27) Pietkiewicz, S.; Schmidt, J. H.; Lavrik, I. N. Quantification of apoptosis and necroptosis at the single cell level by a combination of Imaging Flow Cytometry with classical AnnexinV/propidium iodide staining. J. Immunol. Methods 2015, 423, 99−103. (28) Doetsch, M.; Gluch, A.; Poznanovic, G.; Bode, J.; Vidakovic, M. YY1-binding sites provide central switch functions in the PARP-1 gene expression network. PLoS One 2012, 7, e44125. (29) Sun, A.; Liu, J.; Pang, S.; Lin, J.; Xu, R. Two novel phenanthraquinones with anti-cancer activity isolated from Bletilla striata. Bioorg. Bioorg. Med. Chem. Lett. 2016, 26, 2375−2379. (30) Kang, N.; Jian, J. F.; Cao, S. J.; Zhang, Q.; Mao, Y. W.; Huang, Y. Y.; Peng, Y. F.; Qiu, F.; Gao, X. M. Physalin A induces G2/M phase cell cycle arrest in human non-small cell lung cancer cells: involvement H

DOI: 10.1021/acs.jafc.7b04430 J. Agric. Food Chem. XXXX, XXX, XXX−XXX