The effect of ethyl acetate extract from persimmon leaves on Alzheimer's disease and its underlying mechanism

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The effect of ethyl acetate extract from persimmon leaves on Alzheimer’s disease and its underlying mechanism Q1

Shun-Wang Huang a,b, Wei Wang a, Meng-Yu Zhang c, Qing-Bo Liu a, Sheng-Yong Luo d, Ying Peng c, Bei Sun b, De-Ling Wu e,∗∗, 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 Anhui Institute of Food and Drug Control, Hefei 230022, China c School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China d China. Anhui Academy of Medical Sciences, Hefei, 230061, China e Anhui University of Chinese Medicine, Hefei 230012, China b

a r t i c l e

i n f o

Article history: Received 29 December 2015 Revised 3 March 2016 Accepted 19 March 2016 Available online xxx Keywords: Ethyl acetate extract of persimmon leaves Alzheimer’s disease Apoptosis JNK signaling pathway

a b s t r a c t Background: Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative disorders characterized by neuronal loss in the brain and cognitive impairment. AD is now considered to be the third major cause of death in developed countries, after cardiovascular disease and cancer. Persimmon leaves are used as a popular folk medicine to treat hypertension, angina and internal haemorrhage in Cyangbhina, and it has been reported that ethyl acetate extract of persimmon leaves (EAPL) displays a potential therapeutic effect on neurodegenerative diseases. Hypothesis/purpose: This study was designed to investigate the effects of EAPL on AD, to clarify the possible mechanism by which EAPL exerts its beneficial effects and prevents AD, and to determine the major constituents involved. Study design: AD model was established by bilateral injection of Aβ 1-42 into the hippocampus of rats. The cognitive performance was determined by the Morris water maze and step-down tests. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), apoptosis, total and phosphorylated c-Jun NH2 -terminal kinase (JNK/p-JNK), caspase-3, Bax and Bcl-2 were determined. In addition, a sensitive and reliable LC-QTOF-MS method was applied to identify the major compounds present in EAPL. Results: EAPL at doses of 20 0 mg/kg, 40 0 mg/kg could markedly reduce the latency, significantly increase the time in the first quadrant and number of the target crossing times in Morris water maze test, markedly increase the latency and reduce the number of errors in the step-down test, significantly inhibit the reductions in SOD and GSH-Px activities, and increase the level of MDA. In addition, EAPL treatment attenuated neuronal apoptosis in the hippocampus, reduced the expression of p-JNK, caspase-3, and the relative ratio of Bax/Bcl-2. Meanwhile, 32 constituents were identified by LC-QTOF-MS/MS assays. Conclusion: The results indicate that EAPL has a potent protective effect on cognitive deficits induced by Aβ in rats and this effect appears to be associated with the regulation of the antioxidative defense system and the mechanism of mitochondrial-mediated apoptosis. Furthermore, analysis of the LC-MS data suggests that flavonoids and triterpenoids may be responsible for the potential biological effects of EAPL. © 2016 Published by Elsevier GmbH.

Abbreviations: Aβ , amyloid-β peptide; Bax, Bcl-2 associated X protein; Bcl-2, B cell lymphoina/lewkmia-2; Caspase, cycteine asparticacid-specific protease; DTT, dithiothreitol; EAPL, ethyl acetate extract of persimmon leaves; ESI, electrospray ionization; GSH-Px, glutathione peroxidase; I/R, ischemia/reperfusion; JNK/p-JNK, total and phosphorylated c-Jun NH2 -terminal kinase; LC-MS, liquid chromatography coupled mass spectroscopy; LC-QTOF-MS, liquid chromatography coupled quadrupole time-of-flight mass spectroscopy; mapks, mitogen-activated protein kinases; MDA, malondialdehyde; MWM, morris water maze; SOD, superoxide dismutase;TEM, transmission electron microscopy; TUNEL, terminal deoxynucleotidyl transferase-mediated dutp-biotin nick end labeling assay. ∗ Corresponding author. Tel.: +86 24 23986088. fax: +86 24 23986510.

Introduction

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Alzheimer’s disease (AD), a chronic and progressive neurodegenerative disorder, is the main cause of dementia worldwide. It is characterized by cognitive impairment and behavioral dysfunction (De and Voet, 2012), and the pathogenesis of AD is associated

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∗∗

Corresponding author. Tel.: +86 551 68129066. fax: +86 551 68129066. E-mail addresses: [email protected] (D.-L. Wu), [email protected], [email protected] (S.-J. Song).

http://dx.doi.org/10.1016/j.phymed.2016.03.009 0944-7113/© 2016 Published by Elsevier GmbH.

Please cite this article as: S.-W. Huang et al., The effect of ethyl acetate extract from persimmon leaves on Alzheimer’s disease and its underlying mechanism, Phytomedicine (2016), http://dx.doi.org/10.1016/j.phymed.2016.03.009

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with the accumulation of intracellular neurofi brillary tangles and extracellular amyloid plaques (Treusch et al., 2011; Kerrigan and Randall, 2013). Aβ is a 39–43 amino acid peptide which is generated by transmembrane amyloid precursor protein. A wealth of data shows that Aβ is the main component of these plaques and is proposed to be a causative factor of AD. The accumulation of Aβ has a potent toxic effect on neuronal cells in both in vitro and in vivo model (Selkoe, 2002; Rajasekhar et al., 2015; Jaunmuktane et al., 2015). However, the precise mechanism for this is not completely understood. A great deal of evidence suggests that inflammatory responses and oxidative stress are the main factors of Aβ -induced neurotoxicity. In addition, accumulation of Aβ can change the expression of pro-apoptotic proteins containing activated caspase-3 and Bax, leading apoptotic cell death, followed by neuronal loss and this is one possible explanation for the progression of AD (Guglielmotto et al., 2014; Sachdeva and Chopra, 2015). Persimmon (Diospyros kaki Thunb.) is a plant which is native to China and widely distributed in tropics and subtropics of East Asia such as Japan and Korea. The persimmon leaves have long been used as a Chinese traditional medicine for the treatment of stroke and the syndrome of apoplexy in clinic to improve ischemia stroke, angina and internal hemorrhage, as well as in the treatment of paralysis, burns, frostbite, hemorrhage and constipation. It also has radical scavenging, neuroprotective, and atherosclerotic effects as well as exhibiting anti-thrombotic, anti-mutagenic, antiallergic and anti-histamine activities (Mallavadhani et al., 1998; Kotani et al., 20 0 0; Matsumotox et al., 20 02; Tanaka et al., 20 03; Sakanaka et al., 2005), and it is listed in part IV of the Chinese Pharmacopoeia 2015 edition. In addition, it has been reported that NaoXinQin, a protected drug of Traditional Chinese Medicine, the main component of which is the ethyl acetate extract of persimmon leaves (EAPL), can prevent neurodegenerative disease (Li, 2004). However, there is no the detailed knowledge about the pharmacological effects on AD and its possible mechanism of action. Therefore, the present study was designed to investigate protective effect of EAPL on Aβ 1-42 induced AD and its underlying mechanism. In addition, according to the data obtained from the LC-QTOF-MS analysis, 32 major compounds (14 flavonoids, 16 triterpenoids and 2 others) were identified as being present in the extract.

Materials and methods

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Drugs and reagents

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The persimmon leaves were collected in November, 2013 from Bengbu city of Anhui province in China. The sample was dried at room temperature and identified by Professor Jin-Cai Lu (the College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University) and deposited in the herbarium of Shenyang Pharmaceutical University (Voucher specimen number: 201312), China. The dried and milled the persimmon leaves (3.0 kg) were extracted with 30 l of 70% ethanol by refluxing twice at 90 °C two times (two periods each of 2 h). The filtrates were combined and concentrated to obtain a crude extract by removing the ethanol in a rotary evaporator at 60 °C. The obtained residue (450 g) was redissolved in water, decolorized and defatted with petroleum ether two times, then extracted twice with ethyl acetate followed by a further evaporation. The ethyl acetate fractions were dried under reduced pressure to yield 150 g of extract (EAPL). Solvents used as eluents were HPLC grade acetonitrile from Fisher Scientific (FairLawn, NJ, USA) and distilled water. Hyperoside, kaempferol, quercetin, myricetin, vitexin, trifolin, astragalin, barbinervic acid, 19α ,24-dihydroxy ursolic acid and pomolic acid were isolated by our lab in previous study.

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Human Aβ peptides (1–42) were obtained from Sigma Chemical Co. (St. Louis, MO) and were dissolved in sterile physiological saline to give a final concentration of 5.0 μg/μl. The kits of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA) were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). A TdT-mediated dUTP nick-end labeling (TUNEL) assay kit was purchased from Boster Biological Technolgy Co (Wuhan, China), and total and phosphorylated c-Jun NH2 -terminal kinase (JNK/p-JNK), caspase-3, Bax and Bcl-2 antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA), all other reagents were obtained from commercial sources.

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Animals

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A total of 60 male Sprague-Dawley rats weighing 250–300 g were provided by the Animal center of Anhui Medical University. All rats were housed at a room temperature of 22 ± 4 °C and maintained on a 12 h light/dark cycle. Animal care and experimental protocols were approved by the Animal Care Committee of Anhui Medical University animal care committee and conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication, 8th edition, 2011).

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Experimental protocol

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The rats were randomly divided into the following 6 groups: a sham group (normal saline), a model group (Aβ 1-42 + normal saline), a positive group (Aβ 1-42 + intragastric administration of 1.5 mg/kg/day donepezil), a low-dose EAPL group (Aβ 1-42 + intragastric administration of 100 mg/kg/day EAPL), a middle-dose EAPL group (Aβ 1-42 + intragastric administration of 200 mg/kg/day EAPL), and a high-dose EAPL group (Aβ 1-42 + intragastric administration of 400 mg/kg/day EAPL). Rats were anesthetized by intraperitoneal injection (ip) of chloral hydrate (300 mg/kg) and placed into a stereotaxic apparatus. After exposure of the occipital bone, two microliters of Aβ 1-42 (5 μg/μl, 0.2 μl/min) was injected into the hippocampus CA1 region of rats according to the atlas of Paxinos and Watson (1986) in which bregma is A-3.0 mm, L-2.0 mm, and the dura is V-3.5 mm. Rats in the sham group were injected the same volume of saline at the same site. After surgery, the rats received intragastric administration once a day for 30 days (Wei et al., 2014).

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Morris water maze test

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On the 30th day after surgery, the cognitive function of the rats was measured using the Morris water maze test as previously described (Lakshmi et al., 2015). The Morris water maze was consisted of a circular pool (100 cm in diameter, 40 cm in height), containing water (23 ± 1 °C), which was made opaque by the addition of ink. A hidden escape platform (15 cm diameter) was submerged below the water surface and placed inside the pool at the midpoint of one quadrant. In a pre-learning phase, the rats were released into the water and given 90 s to mount the platform. If the rat failed to find the platform within the given time, it was gently guided to the platform and allowed to stay there for 15 s. Each rat was trained per day for five consecutive days and the latency to escape from the water maze was recorded. After 24 h of 5th day, a probe test was conducted to evaluate the memory consolidation. In the probe test, the platform was removed, and the rats were released on the opposite site of the platform quadrant (the target quadrant) and allowed to swim freely for 120 s. The time spent in the target quadrant and the number of crossings of the platform site was used to represent the degree of memory consolidation.

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Step-down test

HPLC-QTOF-MS/MS analysis and structural identification of EAPL

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Two days after the Morris water maze test, the step-down test was performed according to the previously described method (Figueiró et al., 2011). Before the experiments, each rat was gently placed in the box and allowed to adapt for 3 min. When each rat stepped down and placed all its paws on the grid floor, shocks were delivered for 15 s and the rats would jump onto the platform to avoid the electric shock. After a 24 h interval, each rat was placed on the platform again. The latency to step down on the grid for the first time and the errors that resulted in a shock within 3 min were taken as a measurement of the learning and memory performance. After the step-down test, the rats were sacrificed by giving them an overdose of 3% sodium pentobarbital (150 mg/kg, i.v.), and their brains were immediately removed.

MS data were recorded using the HPLC-QTOF-MS/MS system (Bruker, Germany) with an electrospray ionization (ESI) source in negative ion mode. The parameters of ESI-MS were set as follows: capillary voltage, +3800 V; nebulizer gas pressure, 1.2 bar; dry gas flow rate, 8.0 l/min; and temperature, 180 °C. Mass spectra were recorded across the range m/z 50–1500. The data were analyzed using Bruker Daltonics Data Analysis 3.4 software. Chromatographic analysis was performed on an Agilent Technologies 1200 Series System (Agilent, USA) equipped with an Agilent Technologies G1376A binary pump, Agilent Technologies G1367B autosampler and an Agilent Technologies G1316A column oven. A Dikma Diamonsil C18 column (4.6 × 200 mm, 5 μm, Dikma, USA) was used. The mobile phases were composed of acetonitrile (A) and water with 0.1% formic acid (B) using a multistep gradient elution of 22% A at 0–10 min, 22%-30% A at 10–20 min, 30–45% A at 20–22 min, 45–90% A at 22–50 min with the flow rate kept at 0.8 ml/min. The sample volume injected was set at 10 μl.

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Statistical analysis

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The experimental results are presented as means ± SD. Statistical significance of the differences between groups was evaluated by one-way analysis of variance (ANOVA) followed by the Duncan test. Value of p < 0.05 was considered statistically significant.

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Measurement of the activities of SOD, GSH-Px and levels of MDA The left brain of each rat was removed and the hippocampus was separated from the cerebral cortex. Half of the left hippocampus were rapidly homogenized in ice-cold saline and centrifuged at 30 0 0 rpm at 4 °C for 15 min. The activities of SOD, and GSH-Px and the levels of MDA in the supernatant were measured using commercially available assay kits according to the procedures described by the manufacturers.

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Apoptosis assay

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The right brain was removed and the hippocampus was separated from cerebral cortex, and five samples of hippocampus in each group were immersion-fixed in 4% paraformaldehyde, and the TUNEL method was used to detect the apoptotic cells according to the manufacturer’s recommended protocol (Liu et al., 2013). The number of positive neurons was measured in three adjacent×400 microscopic images and the mean percentage of apoptotic cells was counted relative to normal cells. The remains of the hippocampus samples in each group were prepared for transmission electron microscopy as previously described (Du et al., 2013). In brief, the hippocampus was fixed with 2.5% glutaraldehyde for 12 h at 4 °C followed by full rinsing with PBS, and then the samples were dehydrated in ethanol and embedded in Epon 812. Finally, the samples were cut into serial sections (50 nm) using an ultramicrotome. The ultrastructure of the hippocampus was observed by transmission electron microscopy (JEM-20 0 0EX, JEOL, Tokyo, Japan).

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Western blotting

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The total proteins in the left brain tissues of rat were extracted with the method previously described (Ma et al., 2014). Briefly, the cerebral cortex and hippocampus tissue were homogenized and lsyed with RIPA lysis buffer (1 mM sodium orthovanadate, 1% Triton X-100, 50 mM Tris HCl, 1 mM glycerophosphate, 150 mM NaCl, 1 mM DTT and protease inhibitor) for 30 min at 4 °C. Then, the suspension was centrifuged at 12,0 0 0 rpm for 10 min at 4 °C. Samples involving 60 mg protein were separated on 12% polyacrylamide gel and electrically transferred to PVDF membrane. The membranes were blocked with buffer containing 2% nonfat dry milk in TBST followed by incubation overnight at 4 °C with the primary antibodies of JNK or Caspase-3 or Bax or Bcl-2. Thereafter, the membranes were washed three times with 0.1% Tween-20 for 15 min and then incubated with appropriate secondary antibody for 1 h at 37 °C. The relative density of the protein bands was measured by densitometry of radioautograph films.

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Results

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Effect of EAPL on the learning and memory ability of AD rats undergoing the Morris water maze (MWM) test

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The Morris water maze test was used to assess the spatial learning and memory ability of the rats. As shown in Fig. 1A, compared with the sham group, the rats in the model group spent markedly more time to find the platform during the 5 d of training, and the time in the target (first) quadrant and number of target crossing times decreased significantly (p < 0.01) (Fig. 1B and 1C, Supplementary Table S1). However, treatment with EAPL 20 0 mg/kg and 40 0 mg/kg markedly reduced the latency, and significantly increased the time in the first quadrant and number of the target crossings compared with the model group (p < 0.01). Donepezil (1.5 mg/kg) had a similar effect as that in the EAPL (200 mg/kg and 400 mg/kg) group (p < 0.01 vs. the model group) (Supplementary Table S2).

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Step-down test

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The step-down test was used to evaluate the effect of EAPL on memory ability. As shown in Fig. 2A, the latency of rats in the model group (78.70 ± 19.56) was shortened significantly compared with that in the sham group (139.10 ± 20.78) (Supplementary Table S3). Treatment with EAPL 200 mg/kg and 400 mg/kg markedly increased the latency from 78.70 ± 19.56 in the model group to 101.50 ± 24.90, 114.40 ± 26.09, respectively (p < 0.05 or p < 0.01) while, compared with the sham group (2.20 ± 0.63), the number of errors significantly increased in model group (7.40 ± 2.01) (Fig. 2B, Supplementary Table S3). Treatment with EAPL 200 mg/kg and 400 mg/kg markedly reduced the number of errors from 7.40 ± 2.01 in the model group to 5.50 ± 1.72 and 5.30 ± 1.42, respectively (p < 0.05 or p < 0.01). Donepezil (1.5 mg/kg) had a similar effect to that in EAPL (400 mg/kg) group (p < 0.01 vs. the model group) (Supplementary Table S3).

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Q2

Fig. 1. Effects of EAPL on the Aβ 1-42 -induced cognitive impairment in the MWM test.

Fig. 2. Effects of EAPL on the Aβ 1-42 -induced cognitive impairment in the step-down test.

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Effect of EAPL on activities of SOD, GSH-Px and levels of MDA in the hippocampus As shown in Fig. 3B and 3C, the SOD and GSH-Px activities in the rat hippocampus in the model group (131.86 ± 28.27, 58.63 ± 17.37) decreased significantly compared with that in the sham group (212.39 ± 32.13, 118.47 ± 23.59) (Supplementary Table S4). Treatment with EAPL 200 mg/kg and 400 mg/kg clearly inhibited the decreases in SOD and GSH-Px activi-

ties, and the activities of SOD and GSH-px were increased to 179.19 ± 29.20, 186.72 ± 28.42 and 86.44 ± 19.86, 91.02 ± 20.70, respectively (p < 0.05) (Supplementary Table S4). Also, the MDA level in the model group (12.67 ± 1.52) was markedly increased compared with the sham group (3.57 ± 0.81). Treatment with EAPL 20 0 mg/kg and 40 0 mg/kg significantly reduced the MDA level to 7.91 ± 1.84 and 7.8 ± 61.82, respectively (p < 0.05) (Fig. 3A, Supplementary Table S4).

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Fig. 3. Effect of EAPL on activities of SOD, GSH-Px and levels of MDA in the hippocampus.

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Effect of EAPL on neuronal apoptosis in the hippocampus

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The TUNEL assay was used to detect neuronal apoptosis in the hippocampus. As shown in Fig. 4A and 4B, the number of TUNEL-positive neurons in the hippocampus of model group rat (10.00 ± 1.58) increased significantly compared with that in sham group (1.60 ± 0.55) (Supplementary Table S5). Treatment with EAPL 20 0 mg/kg and 40 0 mg/kg markedly attenuated neuronal apoptosis, and the number of TUNEL-positive neurons was reduced to 5.80 ± 1.48 and 5.40 ± 1.14, respectively (p < 0.05) (Supplementary Table S5). Transmission electron microscopy (TEM) was used to investigate the ultrastructural changes in the hippocampus. As shown in Fig. 5a, TEM examination revealed that in the sham group, the hippocampal neurons contained a complete nuclear membrane, well-distributed chromatin, continuous endoplasmic reticulum and compact mitochondrial matrices. However, the hippocampal neurons in the model group exhibited clear pyknosis, nuclear invagination, karyorrhexis and increased numbers of lysosomes. Furthermore, the mitochondria had degenerated and were vacuolated (Fig. 5b). Treatment with EAPL 200 mg/kg and 400 mg/kg clearly reversed these changes (Fig. 5c and 5d, Supplementary Table S5).

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Fig. 4. Effect of EAPL on neuronal apoptosis in the hippocampus.

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Fig. 5. Effect of EAPL on the ultrastructural change of the hippocampus.

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Effect of EAPL on activation of JNK

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Western blotting was used to examine the expression of total and phosphorylated (activated) JNK (p-JNK). As shown in Fig. 6A and 6B, compared with the sham group, Aβ 1-42 significantly increased the expression of p-JNK in the model group (p < 0.01). Treatment with EAPL 200 mg/kg and 400 mg/kg clearly reduced the expression of p-JNK from 2.23 ± 0.29 in the model group to 1.69 ± 0.28 and 1.62 ± 0.23, respectively (p < 0.05) (Supplementary Table S6).

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Effect of EAPL on expression of Bax and Bcl-2

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Western blotting was used to examine the expression of Bax and Bcl-2. As shown in Fig. 7A and 7B, Aβ 1-42 markedly increased the expression of Bax and reduced the expression of Bcl2 in the model group. The relative ratio of Bax/Bcl-2 was significantly increased in model group compared with that in the sham group (p < 0.01). Treatment with EAPL 200 mg/kg and 400 mg/kg markedly inhibited the increases in the relative ratio of Bax/Bcl-2. The relative ratio was reduced from 2.12 ± 0.25 in the model group

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to 1.72 ± 0.28 and 1.68 ± 0.31, respectively (p < 0.05) (Supplementary Table S7).

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Effect of EAPL on expression of Caspase-3

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Western blotting was used to examine the expression of caspase-3. As shown in Fig. 8A and 8B, the result also showed that Aβ 1-42 caused an increase in caspase-3 expression in the model group (p < 0.01 vs. sham group). Treatment with EAPL 200 mg/kg and 400 mg/kg markedly reduced the expression of caspase-3 from 2.30 ± 0.30 in the model group to 1.74 ± 0.27 and 1.70 ± 0.31, respectively (p < 0.05) (Supplementary Table S8).

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LC-MS fingerprint and identification of major peaks in EAPL

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There were more than 32 major peaks in the LC-MS fingerprint chromatogram of EAPL (Fig. 9.). Hyperoside, kaempferol, quercetin, myricetin, vitexin, trifolin, astragalin, barbinervic acid, 19α ,24-dihydroxy ursolic acid and pomolic acid in EAPL were identified by LC-MS assays according to their m/z and retention time

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Rt (min)

Experimental [M–H]− (m/z)

Theoretical [M–H]− (m/z)

1 2∗ 3 4∗ 5 6 7∗ 8∗ 9∗ 10∗ 11 12∗ 13∗ 14 15 16∗ , a 17∗

5.8 6.9 9.0 9.4 10.8 11.8 12.2 12.8 13.0 14.7 15.7 22.9 26.2 26.9 27.1 27.9 28.3

431.0962 479.0819 463.0865 615.0978 447.0917 447.0925 477.1017 477.1021 599.1038 417.0836 317.0289 599.1035 695.4012 331.0461 301.0355 487.3453 499.3086

431.0973 479.0820 463.0871 615.0981 447.0922 447.0922 477.1039 477.1039 599.1042 417.0827 317.0303 599.1042 695.4012 331.0459 301.0354 487.3429 499.3065

18 19∗ , b 20 21∗

35.2 31.0 32.4 34.0

285.0406 485.3656 487.3447 501.3246

285.0405 485.3636 487.3429 501.3222

22∗ , a 23∗ , b 24 25∗ , b

35.0 36.6 37.1 37.6

487.3448 485.3616 487.3451 485.3619

26∗ , c

38.6

27∗ , c

Formula

|Error| (ppm)

Reference

C21 H20 O10 C21 H20 O13 C21 H20 O12 C28 H24 O16 C21 H20 O11 C21 H20 O11 C22 H22 O12 C22 H22 O12 C28 H24 O15 C20 H18 O10 C15 H10 O8 C28 H24 O15 C37 H60 O12 C16 H12 O8 C15 H10 O7 C30 H48 O5 C30 H44 O6

2.6 0.2 1.3 0.5 1.1 0.7 4.5 3.8 0.7 2.2 4.4 1.2 0.1 0.6 0.3 4.9 4.2

Chen et al., 2005 Chen et al., 2009 Chou, 1984 Kawakami et al., 2011 Chou, 1984 Chou, 1984 Chen et al., 2005 Markhma et al., 1978 Kawakami et al., 2011

Kaempferol Uknown Barbinervic acid 3α ,19α -dihydroxyurs-12-en-24,28-dioic acid

C15 H10 O6 C31 H50 O4 C30 H48 O5 C30 H46 O6

0.4 4.1 3.7 4.8

487.3429 485.3636 487.3429 485.3636

Unkown Unkown 19α ,24-dihydroxy ursolic acid Unkown

C30 H48 O5 C31 H50 O4 C30 H48 O5 C31 H50 O4

3.9 4.1 4.5 3.5

471.3491

471.3480

Unkown

C30 H48 O4

2.3

39.0

471.3503

471.3480

Unkown

C30 H48 O4

4.9

28∗ , c

39.6

471.3497

471.3480

Unkown

C30 H48 O4

3.7

∗,d

40.9

425.3808

425.3789

Unkown

C30 H50 O

4.5

42.6

471.3482

471.3480

Pomolic acid

C30 H48 O4

0.4

31∗ , c

43.1

471.3477

471.3480

Unkown

C30 H48 O4

0.7

∗,c

47.9

471.3497

471.3480

Unkown

C30 H48 O4

3.5

29 30

32

a b c d

Vitexin Myricetin-3-O-glucopyranoside Hyperoside Quercetin-3-O-galloylglucoside Astragalin Trifolin Isorhamnetin-3-β -D-glucopyranoside Isorhamnetin-3-β -D-galactoside Kaempferol-3-O-galloylglucoside Salvianolic acid D Myricetin Kaempferol-3-O-galloylgalactoside 3-O-β -D-glucopyranosylplaty codigenin methylester Q3 Annulatin Quercetin Unkown 3α ,19α -dihydroxyurs-12,20 (30)-dien-24,28-dioic acid

Chen et al., 2009 Kawakami et al., 2011 Chen et al., 2007 Chou, 1984 Fan and He, 2006 Phuong et al., 2008 Phuong et al., 2008 Chou et al.,1984 Mallavadhani et al., 2001 Chen et al., 2005 Phuong et al., 2008 Fan and He, 2006 Phuong et al., 2008 Mallavadhani et al., 2001 Chen et al., 20 0 0 Mallavadhani et al., 2001 Phuong et al., 2008 Mallavadhani et al., 2001 Fan and He, 2006 Phuong et al., 2008 Mallavadhani et al., 2001 Fan and He, 2006 Phuong et al., 2008 Mallavadhani et al., 2001 Fan and He, 2006 Chen et al., 2005 Higa et al., 1998 Mahato and Kundu, 1994 Phuong et al., 2008 Mallavadhani et al., 2001 Fan and He, 2006 Phuong et al., 2008 Mallavadhani et al., 2001 Fan and He, 2006

No confirmation in comparison with authentic standards, and according to reference literature. belongs to one of rotungenic acid, 3α ,24,29-trihydroxyolean-12-en-28-oic acid or spathodic acid. belongs to one of maslinic acid methyl ester, or pomolic acid methyl ester. belongs to one of corsolic acid, 24-hydroxy ursolic acid, 24-hydroxy-3-epi-ursolic acid or 24-hydroxy-3-epi-oleanolic acid. belongs to one of friedelin or lupeol.

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Identity

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Please cite this article as: S.-W. Huang et al., The effect of ethyl acetate extract from persimmon leaves on Alzheimer’s disease and its

underlying mechanism, Phytomedicine (2016), http://dx.doi.org/10.1016/j.phymed.2016.03.009

Table 1 Identification of the chemical constituents of EAPL by HPLC-QTOF-MS in negative ion mode.

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Fig. 6. Effect of EAPL on the activation of JNK.

Fig. 7. Effect of EAPL on expression of Bax and Bcl-2.

Fig. 8. Effect of EAPL on expression of Caspase-3.

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(Fig. 9. and Table 1). Also, other compounds were tentatively characterized based on a comparison of the accurate mass (within an error of 5 ppm) and fragment information with those reported in the literatures (Table 1). Discussion Persimmon leaves are used traditionally for many medicinal purposes in China, including the treatment of frostbite, paralysis,

burns, hypertension, angina and internal haemorrhage (Xie et al., 2015). It has been reported that EAPL has neuroprotective effects against ischemia/reperfusion (I/R) damage, excitotoxic injury and hypoxia-reoxygen injury due to improving the redox imbalance and inhibiting apoptosis (Sun et al., 2011; Bei et al., 2004). In this study, we examined the effects of EALP on AD. Aβ is considered to be the main factor that leads to the development of AD, which can impair memory and produce pathological changes in the brain. Therefore, we used intrahippocampal

Please cite this article as: S.-W. Huang et al., The effect of ethyl acetate extract from persimmon leaves on Alzheimer’s disease and its underlying mechanism, Phytomedicine (2016), http://dx.doi.org/10.1016/j.phymed.2016.03.009

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Fig. 9. Base peak chromatograms (BPC) of the main components in EAPL.

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injection of Aβ 1-42 to establish the AD rat model. In the Morris water maze test, the results obtained showed that Aβ 1-42 could significantly increase the time to find the platform, reduce the time in the first quadrant and number of the target crossings in the model group. However, EAPL markedly reduced the latency, significantly increased the time in the first quadrant and the number of the target crossings. In the step-down test, injection of Aβ 1-42 significantly increased the number of errors and shortened the latency. Also, EAPL could markedly increase the latency and reduce the number of errors. The observed improvement in the memory function demonstrates that EAPL has a neuroprotective effect against Aβ 1-42 -induced memory impairment. Recent evidence has suggested that oxidative stress plays a key role in Aβ -induced cellular damage and, consequently, contributes to the pathophysiology of AD (Butterfield et al., 2013; Swomley et al., 2014). SOD and GSH-Px are two important endogenous antioxidants which can reduce oxidative stress and prevent lipid peroxidation (Da et al., 2013). GSH-Px activity is also considered to be a first indicator for oxidative stress in the brain. MDA is the product of lipid oxidation, and indirectly reflects the extent of oxidative stress-induced injury (Klaunig and Kamendulis, 2004). In this study, Aβ 1-42 -treated rats exhibited reduced SOD and GSHPx activity, while the MDA level was increased. However, EAPL increased SOD and GSH-Px activities while reducing the MDA level. This result indicates that EAPL has potent antioxidant activity. Donepezil is a potent acetylcholinesterase (AChE) inhibitor and has positive actions on animal and human cognitive functions (Ginani et al., 2011). Several studies reported the neuroprotective effects of donepezil are mediated through the inhibition of GSK-3 activity via the activation of Akt (Noh et al., 2009), ameliorating the mitochondrial swelling and reducing ATP level on Aβ 1-42 injured mitochondria (Ye et al., 2015). In tau-opathy mouse model, it also could inhibite inflammatory gene expression (Yoshiyama et al., 2010). In our research, donepezil didn’t exhibit significantly positive effects in SOD, GSH-Px and MDA experiments, which showed the effect of donepezil on AD might not be associated with antioxidant and were not contradictory to previous research. Apoptosis is another important factor in the pathogenesis of AD, and it has been reported to be associated with oxidative stress and the mechanism governing central cholinergic system dysfunction (Chen et al., 2011; Chai et al., 2014). Aβ peptide deposition can induce neuronal apoptosis (Cies´ lik et al., 2015; Thangnipon et al., 2012) and, in the present study, we found that the number of TUNEL-positive neurons significantly increased in the hippocampus of Aβ 1-42 -injected rats. Also, EAPL could markedly reduce the number of TUNEL-positive neurons and attenuate the ultrastructural damage to the hippocampus neurons, which suggests that EAPL is able to suppress Aβ -induced neuronal apoptosis. JNK is one member of the MAPKs family (Mitogen-activated protein kinases), which plays a crucial role in maintaining cell homeostasis and controls many cellular processes, including cell transformation, differentiation and apoptosis (Kanaji et al., 2013; Yuan et al.,

2015). Furthermore, previous studies have shown that the activation of JNK is involved in Aβ -induced toxicity and is associated with the transcriptional regulation of members of the Bcl-2 family (Fan et al., 20 0 0). Bcl-2 family proteins are key regulators of apoptosis including pro-apoptotic proteins such as Bax, Bad and Bim, and anti-apoptotic proteins such as Bcl-2 and Bcl-xl (Siddiqui et al., 2015; Wang et al., 2013). The ratio of Bcl-2 to Bax determines whether cells are able to escape apoptosis (Vela et al., 2013; Zhang et al., 2015). Bcl-2 is an inhibitor of apoptosis protein and it exhibits anti-apoptotic effects, which are predominantly localized on the outer mitochondrial membrane. In contrast, Bax has an opposite effect to Bcl-2, which is predominantly localized in the cytosol. Following apoptotic stimulation, Bax exposes the N and C termini and then inserts its C-terminus into the outer mitochondrial membrane. Consequently, the release of cytochrome c is triggered, which causes the activation of caspase-9 and caspase3 (Singla and Dhawan, 2014). As a family of cysteine proteases, caspase-3 is a potent effector for triggering apoptosis, and is the terminal caspase in mitochondrial-dependent apoptosis pathways (Snigdha et al., 2012; Chu et al., 2014). In this study, we found that injection of Aβ 1-42 caused an increase in the expression of phosphorylated JNK and caspase-3 and the ratio of Bax/Bcl-2. In contrast, EAPL can significantly attenuate these changes. These results indicate that the anti-apoptotic effect of EAPL may be achieved by modulating the JNK/caspase-3 signaling pathway. The phytochemical fingerprinting of EAPL was carried out by LC-ESI-MS. Flavonoids and triterpenoids were found to be the major constituents of EAPL. However, there have been no reports of the effect of flavonoids and triterpenoids from persimmon leaves on AD. The main therapeutic components of NaoXingQing are flavonoids, which have been widely used to treat the apoplexy syndrome for many years (Cai et al., 2001). Previous studies have demonstrated that NaoXingQing could protect hippocampal neurons from glutamate-induced excitotoxic injury as well as cortical neurons from hypoxia-induced through its antioxidative activity (Bei et al., 2007). In addition, the triterpenoid compounds isolated from leaves of Diospyros kaki also have effect of suppressing N-formyl-methionyl-leucylphenylalanine (fMLP)-induced superoxide generation (Chen et al., 2002). The reactive oxygen species (ROS) oxidative stress was closely related to the common pathological mechanism in AD. Therefore, we speculated the EAPL has protective effect on AD. The present study have also verified the above hypothesis. Although these results suggest that EAPL has potential as a neuroprotective agent, more studies are needed for clinical use to cure AD.

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In conclusion, this study provides evidence that EAPL is able to increase cognitive function, which may be associated with the regulation of the antioxidative defense system and the mechanism of mitochondrial-mediated apoptosis. EAPL is, therefore, a potentially important agent for treating AD. LC-ESI-MS fingerprinting of

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EAPL showed that the extract consists mainly of flavonoids and triterpenoids. However, its molecular mechanisms of action, its active constituents, and whether these active constituents correspond to the fingerprint chromatogram in our results remain unknown. Therefore, more detailed studies are required to answer these questions.

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Conflict of interest

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The authors declare that there are no conflicts of interest.

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Uncited References

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G. Chen et al., 2009, Fan and He, 2006, Mahato and Kundu, 1994, Cai and Yang, 2001

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Acknowledgments

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This work was supported by Key Laboratory of Structure-Based Drug Design & Discovery (Shenyang Pharmaceutical University), Ministry of Education. Financial support from the National Natural Science Foundation of China (81373925, 81573319), and the Foundation (LT2015027) from the Project of Innovation Team are gratefully acknowledged.

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Supplementary materials

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Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.03.009.

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