Cerebral metabonomics study on Parkinson's disease mice treated with extract of Acanthopanax senticosus harms

Phytomedicine 20 (2013) 1219–1229

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Cerebral metabonomics study on Parkinson’s disease mice treated with extract of Acanthopanax senticosus harms Xu-zhao Li a , Shuai-nan Zhang a , Fang Lu a,∗∗ , Chang-feng Liu a , Yu Wang a , Yu Bai a , Na Wang a , Shu-min Liu a,b,∗ a Chinese Medicine Toxicological Laboratory, Institute of Traditional Chinese Medicine, Heilongjiang University of Chinese Medicine, Harbin 150040, PR China b Drug Safety Evaluation Center, Heilongjiang University of Chinese Medicine, Harbin 150040, PR China

a r t i c l e

i n f o

Keywords: Acanthopanax senticosus harms Parkinson’s disease cerebral metabonomics UPLC-QTOF-MS

a b s t r a c t Extract of Acanthopanax senticosus harms (EAS) has neuroprotective effect on Parkinson’s disease (PD) mice against dopaminergic neuronal damage. However, studies of its anti-PD mechanism are challenging, owing to the complex pathophysiology of PD, and complexity of EAS with multiple constituents acting on different metabolic pathways. Here, we have investigated the metabolic profiles and potential biomarkers in a mice model of MPTP-induced PD after treatment of EAS. Metabonomics based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used to profile the metabolic fingerprints of mesencephalon obtained from 1Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine Hydrochloride (MPTP-HCl)-induced PD mice model with and without EAS treatment. Through partial least squares-discriminate analysis (PLS-DA), it was observed that metabolic perturbations induced by MPTP were restored after treatment with EAS. Metabolites with significant changes induced by MPTP, including L-dopa, 5 -methylthioadenosine, tetradecanoylcarnitine, phytosphingosine-1-P, Cer(d18:0/18:0), LysoPC(20:4(5Z,8Z,11Z,14Z)), L-palmitoyl -carnitine, tetracosanoylglycine, morphiceptin and stearoylcarnitine, were characterized as potential biomarkers involved in the pathogenesis of PD. The derivations of all those biomarkers can be regulated by EAS treatment except Cer(d18:0/18:0), LysoPC(20:4(5Z,8Z,11Z,14Z)), morphiceptin. The therapeutic effect of EAS on PD may involve in regulating the tyrosine metabolism, mitochondrial beta-oxidation of long chain saturated fatty acids, fatty acid metabolism, methionine metabolism, and sphingolipid metabolism. This study indicated that changed metabolites can be certainly recovered by EAS, and the treatment of EAS can be connected with the regulation of related metabolic pathways. © 2013 Elsevier GmbH. All rights reserved.

Introduction

Abbreviations: DA, dopamine; EAS, extract of Acanthopanax senticosus harms; MPTP-HCl, 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydro-pyridine Hydrochloride; MS, mass spectrometry; PCA, principal components analysis; PD, Parkinson’s disease; PLS-DA, partial least squares-discriminate analysis; TCM, traditional Chinese medicine; UPLC-QTOF-MS, ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry; VIP, variable importance of project; VLACD, very long-chain acyl-CoA dehydrogenase. ∗ Corresponding author at: Drug Safety Evaluation Center, Heilongjiang University of Chinese Medicine, He Ping Road 24, Harbin 150040, PR China. Tel.: +86 451 82193278; fax: +86 451 82193278. ∗∗ Corresponding author at: Chinese Medicine Toxicological Laboratory, Institute of Traditional Chinese Medicine, Heilongjiang University of Chinese Medicine, He Ping Road 24, Harbin 150040, PR China. Tel: +86 451 87266814; fax: +86 451 87266814. E-mail addresses: lufang [email protected] (F. Lu), [email protected] (S.-m. Liu). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.06.002

Parkinson’s disease is a chronic neurological disorder. In ventral midbrain, particularly in substantia nigra pathological features show that dopaminergic neurons progressively degenerate, which causes a consequent reduction of dopamine (DA) levels in the striatum. The functions of acetylcholine neurons and dopaminergic neurons in striatum are out of balance, which leads to PD. The patients have some characteristic symptoms, such as tremor, myotonia, and dyskinesia, etc. (Liu et al., 2012). The earliest description of PD was traced in the Yellow Emperor’s Internal Classic, a book written 2000 years ago. In traditional Chinese medicine (TCM), PD is termed as “shaking palsy”, a syndrome characterized by tremors, numbness and limpness and weakness of the four limbs, and the pathologic features of PD are liver-kidney Yin deficiency and qiblood deficiency (Li et al., 2013). TCMs are gaining more attention all over the world, due to their specific theory and long historical clinical practice.

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A

B

C

D

Figure 1. Molecular structure of (A) eleutheroside B, (B) eleutheroside E, (C) sesamin, and (D) isofraxidin.

Acanthopanax senticosus (Rupr. and Maxim.) Harms is a widely used traditional Chinese herb. In the theory of TCM, it is described as following: Indications hypofunction of the spleen and the kidney marked by general weakness, lassitude, anorexia, aching of the loins and knees; insomnia and dream-disturbed sleep (ChPC, 2010). In modern pharmacological researches, we found that Acanthopanax senticosus Harms have neuroprotective features (Bocharov et al., 2010; Bocharov et al., 2008), stressprotective and adaptogenic activities (Brekhman and Dardymov, 1969; EMEA/HMPC/102655/2007, 2008; Panossian and Wikman, 2010; WHO, 2004), and it was also applied to study on cranial and cerebral traumas (Sandler, 1970a, b, 1972). The major chemical constituents of Acanthopanax senticosus Harms contain eleutheroside B, eleutheroside E, sesamin, and isofraxidin, etc. (Fig. 1). Eleutheroside B could protect PC12 cells from damage induced by MPP+ (Dong Y, 2011). Eleutheroside B, eleutheroside E and isofraxidin showed obvious protective effects against A␤ (25-35)-induced atrophies of axons and dendrites(Bai et al., 2011). Sesamin has a preventive effect on behavioral dysfunction in rotenone-induced rat (Fujikawa et al., 2005). Our previous study has indicated that EAS can protect C57BL/6 mice against MPTP-induced dopaminergic neuronal damage (Liu et al., 2012). However, its mechanism of anti-PD effect has not yet been elucidated. Metabonomics is defined as ‘the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification’ (Nicholson et al., 1999), which can provide variation of whole metabolic networks for characterizing pathological states in animals and human, as well giving diagnostic information and presenting mechanistic insight into the biochemical effects of the toxins and drugs (Coen et al., 2008; Dai et al., 2010). The application of metabolomics for TCM will facilitate the understanding of the intrinsic quality of TCM syndromes and the evaluation of the therapeutic effects of

Chinese herbal (Zhang et al., 2010). Mass spectrometry (MS) and nuclear magnetic resonance spectroscopy are two analytical tools commonly used in metabonomics studies (Coen et al., 2008; Dai et al., 2010; Griffin et al., 2000; Zheng et al., 2010). In the MS -based metabonomics, UPLC-QTOF-MS has gained more application due to the high resolution of chromatographic peaks, increased analytic speed and sensitivity for complex mixtures (Plumb et al., 2002). In this study, cerebral metabonomics based on UPLC-QTOF-MS was applied to investigate the metabolic profiles and potential biomarkers in a mice model of MPTP-induced PD after treatment of EAS, which may facilitate understanding the pathological changes of PD and anti-PD mechanism of EAS. Methods Chemicals and reagents 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine Hydrochloride (MPTP-HCl, CAS Number: 23007854) was purchased from Sigma-Aldrich (USA). HPLC-grade acetonitrile was purchased from Thermo Fisher Scientific (USA). Purified water was produced by Milli-Q ultra-pure water system (Millipore, Billerica, USA). Formic acid (HPLC grade) was purchased from Dikma Technologies (USA). Leucine encephalin was purchased from Sigma-Aldrich (USA). All other reagents were HPLC grade. Plant material and extraction The herb (root and rhizome of Acanthopanax senticosus (Rupr. Et Maxim.) Harms) was derived from their natural origin, which was collected in Wuchang (N44◦ 39 , E127◦ 35 ) of Heilongjiang province, PR China. The voucher specimen (hlj-201003) of the herb was authenticated by Professor Ke Fu, Institute of Traditional Chinese

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Medicine, Heilongjiang University of Chinese Medicine. EAS was prepared with Acanthopanax senticosus (Rupr. Et Maxim.) Harms that one gram of crude drug was extracted 3 times with 10 ml 80% ethanol for 2, 2 and 2 h, respectively, separated and purified by AB-8 macroporous adsorption resin (Polarity: Weak-polar, Particle diameter: 0.3–1.25 mm, Surface area: 480–520 m2 /g, Average pore diameter: 130–140 nm, Moisture content: 67.3% (Zhang et al., 2009). The process for purification was as follows: the concentration of sample solution was 500 g/l; adsorption flow rate was 2 bed volumes/hour; eluent was 30% ethanol; the dosage was 9 bed volumes; elution flow rate was 1 bed volume/hour. The ethanol phase was evaporated under vacuum and then oven dried at 60 ◦ C to give the extracts. The drug-extract ratio was 1.3% (Liu et al., 2012). The contents of eleutheroside B and eleutheroside E in EAS were 7.63 ± 0.34% (w/w) and 10.90 ± 0.22% (w/w) respectively. Animals 30 male C57BL/6 mouse (2 months old, 18-22 g, Inbred Mice) were purchased from Yi-Si Laboratory Animal Technology Co., Ltd (China). They were allowed at least 1 week to adapt to their environment before used for experiments. Animals were housed 5 per cage (320 mm × 180 mm × 160 mm) under a normal 12-h/12h light/dark schedule with the lights on at 07:00 a.m. They were housed at room temperature (23 ± 2 ◦ C) with relative humidity (55 ± 5%), and given a standard chow and water ad libitum for the duration of the study. The ethical approval for the experiment was followed by the Legislation on the Protection of Animals Used for Experiment Purposes (Directive 86/609/EEC). PD mice and drug administration Mice were randomly divided into control group, MPTP model group, and EAS treated group with MPTP (MPTP+EAS group). MPTP model group and MPTP+EAS group received MPTP-HCl (30 mg/kg i.p) once a day for 5 days (Liu et al., 2012). Control group received equal volume saline (20 mL/kg i.p) once a day for 5 days. From Day 6, MPTP+EAS group was orally administrated with EAS for 20 days, and dose amounted to 45.5 mg/kg daily. Control group and MPTP model group were orally administrated with equal volume saline (20 ml/kg daily) for 20 days.

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each containing 0.1% formic acid. A solvent gradient system was used: 2-100% A for 0-15 min. The flow rate was 0.4 ml/min. Injection volume was 5 ␮l. The eluent was introduced to the MS directly without a split. MS conditions MS analysis was performed on Q-TOF analyzer in SYNAPT HDMS system (Waters Corporation, Milford, MA, USA) in positive ion mode, using the following parameters: capillary voltage, 1000 V; sample cone voltage, 40 V; source temperature, 110 ◦ C; desolvation temperature, 350 ◦ C; desolvation gas flow, 750 l/h; cone gas flow, 20 l/h. MS data were collected in the full scan mode. All the data were acquired using an independent reference lock mass via the LockSprayTM interface to ensure accuracy and reproducibility during the MS analysis. Leucine enkaphalin was used as the reference compound (positive ion mode ([M+H]+ =556.2771) and [M-H]- =554.2615) at a concentration of 0.2 ng/ml under a flow rate of 100 ␮l·min−1 . The data were collected in the centroid mode, and the LockSpray frequency set at 15 s and averaged over 10 scans for correction. Data analysis The raw data were analyzed using the Micromass MarkerLynx Applications Manager version 4.1, this allowed deconvolution, alignment and data reduction to give a list of mass and retention time pairs with corresponding intensities for all the detected peaks from each data file in the data set. The main parameters were set as follows: retention time range 0-12.5 min, mass range 50-1000 amu, mass tolerance 0.01, minimum intensity 1%, mass window 0.05, retention time window 0.20, and noise elimination level 6. The resulting data were analyzed by principal components analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) using MarkerLynx XS software (version 4.1, Waters Corporation, USA). Statistical analysis data were presented as mean ± standard deviation (¯x ± s). Student’s t-test was used for statistical analysis to evaluate the significant difference of potential biomarkers (SPSS 18.0 (SPSS Inc., Chicago, IL)). Statistical significance was accepted if p < 0.05. Results

Sample collection and preparation Method development and validation On the 26th day, all mice of each group were decapitated. Mesencephalon was weighed accurately and immediately frozen in liquid nitrogen and stored at -80 ◦ C until used. For determination, mesencephalic tissue blocks were thawed on ice. Once thawed, ice-cold saline (2 ml saline/100 mg sample) was added and homogenized with a tissue homogenizer for 2 min in iced bath. Aliquots of 1 ml homogenate was suctioned, vortex-mixed with 5 ml methanol for 2 min and centrifuged at 15,000 × g for 15 min at 4 ◦ C. The upper organic layer was transferred into another tube, evaporated to dryness under nitrogen stream and the residue dissolved with 700 ␮l methanol and vortexed for 1 min. The mixture was centrifuged at 4,000 × g for 5 min at 4 ◦ C, and the supernatant was filtered and stored at -80 ◦ C for UPLC/MS analysis. UPLC conditions Waters AcquityTM UPLC (consisting of a vacuum degasser, autosampler, a binary pump, photodiode array detector and oven) was equipped with ACQUITY UPLCTM HSS T3 column (100 mm × 2.1 mm, i.d. 1.8 ␮m, Waters Corp, Milford, USA). The analytical column was maintained at a temperature of 40 ◦ C and the mobile phases was composed of acetonitrile (A) and water (B)

Metabolic profiling of mesencephalic samples was acquired using UPLC/TOF-MS/MS system in the positive ion mode. The base peak intensity chromatograms of mesencephalic samples from control group, MPTP group, and EAS-treated group are shown in Fig. 2 (A, B and C). The average peak base width of 4s was set for this separation, which generated a series of peaks with retention time and m/z pairs (tR m/z pair) as variables. High reproducibility is crucial for any analytical protocols, especially for metabonomics study which requires handling many samples. Reproducibility of the chromatography and MS was determined from five replicated analyses of the same mesencephalic sample. The relative standard deviations of retention time and peak area are below 0.51% and 3.1%, respectively. These results demonstrated the excellent stability and reproducibility of chromatographic separation and mass measurement during the whole sequence. Multivariate analysis of UPLC/MS data PCA was firstly carried out to investigate whether two groups can be separated and to find out their metabolic distinction. Then

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Figure 2. Base peak intensity chromatograms obtained from the positive ion UPLC-MS analyses of (A) control group, (B) MPTP group and (C) (MPTP+EAS) group that sampling on the 26th day.

PLS-DA, a supervised multivariable statistical method to sharpen an already established (weak) separation between groups of observations plotted in PCA was performed. The variables responsible for differentiating MPTP group and control group were selected as potential biomarkers of progress of diseases by variable importance of project (VIP) statistics. In order to gain an overview of the mice mesencephalic metabolic profiling, here, PCA was used in the subsequent UPLC Q-TOF/MS data analysis. PLS-DA was performed and the results shown in Fig. 3A and 3C, which indicated that the metabolic profile of MPTP model group deviated from that of control group, suggesting that significant biochemical changes were induced by MPTP. The metabolic profile of EAS treated group fairly differed from that of MPTP group and close to that of control group in Fig. 3C, indicating the deviations induced by MPTP were significantly improved after treatment of EAS. Potential biomarkers responsible for the PD induced by MPTP and the anti-PD effect of EAS Corresponding VIP statistics of PLS-DA and S-plots were used to extract the important variables responsible for the differentiation. The VIP value calculated by MarkerLynx XS software signifies the influence of metabolite ion on the classification. A VIP value>1 means that variables have above average influence on the classification. S-plot is a tool for visualizing covariance and

correlation between the metabolites and the modeled class, those ions far from the origin contributing to the clustering significantly. As shown in Fig. 3B, S-plots based on mesencephalic metabolic profiles indicated 10 variables representing individual metabolites as biomarker candidate ions with retention time and m/z pairs of (1) 0.65 198.0376, (2) 2.38 298.0974, (3) 9.32 372.3108, (4) 9.64 398.3274, (5) 10.13 568.3401, (6) 10.14 544.3404, (7) 10.38 400.3422, (8) 10.65 426.3577, (9) 10.93 522.3559, (10) 11.37 428.3721. Their VIP values are list in Table 1. Those contributed significatly to differentiate the clustering of MPTP group from that of control group, could be considered as potential biomarkers responsible for derivations of metabolic profile induced by MPTP. Their structures were tentatively identified based on accurate mass measurements via UPLC-TOF-MS, by analysis of accurate molecular weight and the MS/MS spectra. The MassFragmentTM application manager (Waters MassLynx v4.1, Waters) was used to facilitate the MS/MS fragment ion analysis process by way of chemically intelligent peak-matching algorithms. Database such as HMDB (http://www.hmdb.ca/), MassBank (http://www.massbank.jp/) and KEGG (http://www.genome.jp/ kegg/) were used for confirmation. Consequently, ten biomarker candidate ions were tentatively identified as (1) L-dopa, (2) 5 -methylthioadenosine, (3) tetradecanoylcarnitine, (4) phytosphingosine-1-P, (5) Cer(d18:0/18:0), (6) LysoPC(20:4(5Z,8Z,11Z,14Z)), (7) L-palmitoylcarnitine, (8) tetracosanoylglycine, (9) morphiceptin, (10) stearoylcarnitine (Table 1).

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Figure 3. (A) PLS-DA score plot based on the mesencephalic metabolic profiling of the control (䊉) and MPTP model () mice on 26th day by Markerlynx XS software (n = 10 in each group). A clearly separation between the model group and control group was obtained, indicating that the mesencephalic metabolic pattern was significantly changed with the process of MPTP. (B) PLS-DA S-plot based on mesencephalic profiling of the control and MPTP model mice on 26th day by Markerlynx XS software (n = 10 in each group). 10 variables far from the origin contributed significantly to differentiate the clustering of MPTP model group from that of control group and were considered as potential biomarkers. (C) PLS-DA scores plot derived from the integrated MS data of mesencephalic samples obtained from control group (䊉), MPTP group () and (MPTP+EAS) group () on 26th day Markerlynx XS software (n = 10 in each group).

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Table 1 Metabolites selected as biomarkers characterized in mesencephalic profile (n = 10 in each group). tR (min)

VIP

m/z [M+H]+

Formula

MS/MS

Losses

Metabolites

1

0.65

2.5452

198.0376

C9 H11 NO4

182.0858

-O

L-Dopa

C11 H15 N5 O3 S

168.0860 153.0798 107.0674 281.0583

-NO -CHO2 - C2 H2 O4 -CH5

5 -Methylthioadenosine

C21 H41 NO4

254.0712 230.0884 222.0753 170.0514 162.0416 153.9929 136.0558 85.0290 314.2695

-C2 H4 O -C2 N2 O -C2 H6 NS -C4 H6 N3 O2 -C5 H12 O2 S -C5 H12 N4 O -C6 H4 N5 O -C7 H11 N5 OS -C3 H6 O

Tetradecanoylcarnitine

C18 H40 NO6 P

313.2556 286.2746 253.2250 211.2062 197.0814 161.1027 85.0351 67.0596 337.2382

-C3 H7 O -C4 H6 O2 -C5 H13 NO2 -C7 H15 NO3 -C11 H29 N -C14 H27 O -C17 H37 NO2 -C16 H35 NO4 -C2 H5 O2

Phytosphingosine-1-P

2

3

4

2.38

9.32

9.64

3.0358

4.3487

3.4470

298.0974

372.3108

398.3274

Proposed structure

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No.

5

7

10.14

10.38

3.2552

4.2238

6.7454

568.3401

544.3404

400.3422

C36 H73 NO3

-C3 H12 NO -CH10 NO3 P -C3 H13 O3 P -C2 H9 O6 P -C7 H20 O4 P -C11 H24 O4 P -C13 H30 O4 P -C12 H29 NO6 P -C19 H41 NO

Cer(d18:0/18:0)

C28 H50 NO7 P

227.1426 201.1729 184.0901 145.1210 91.0699 58.0782 527.3359

-C24 H53 -C25 H51 O -C27 H60 -C28 H57 NO -C33 H65 O -C32 H64 O3 -HO

LysoPC(20:4(5Z,8Z,11Z,14Z))

C23 H45 NO4

487.2699 428.2566 328.1671 186.0892 167.1072 86.0970 356.3529

-C4 H9 -C6 H12 O2 -C12 H24 O3 -C19 H37 NO3 P -C18 H36 NO5 P C23 H39 O7 P -CO2

L-Palmitoylcarnitine

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6

10.13

320.1753 283.2273 270.2069 238.2499 199.1579 147.1259 117.0790 84.0968 269.2563

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Table 1 (Continued) No.

8

10

10.65

10.93

11.37

VIP

6.0677

6.7938

5.3097

m/z [M+H]+

426.3577

522.3559

428.3721

Formula

MS/MS

Losses

Metabolites

C26 H51 NO3

337.2601 240.2086 145.1103 109.0742 85.0372 368.3234

-C3 H11 O -C8 H18 NO2 -C16 H31 O2 -C16 H37 NO3 -C19 H41 NO2 -C4 H10

Tetracosanoylglycine

C28 H35 N5 O5

324.2903 309.2966 157.0657 86.1012 504.2387

-C6 H14 O -C6 H13 O2 -C19 H41 -C20 H38 NO3 -H4 N

Morphiceptin

C25 H49 NO4

464.2549 259.1321 185.0926 166.0868 71.0792 325.3345

-CH2 N2 O -C14 H19 N2 O3 -C20 H23 N3 O2 -C19 H24 N4 O3 -C24 H27 N4 O5 -C4 H7 O3

Stearoylcarnitine

312.3083 269.1627 195.1021 167.0708 128.0733

-C5 H10 NO2 -C11 H27 -C14 H35 NO -C16 H39 NO -C19 H40 O2

Proposed structure

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9

tR (min)

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Here, we take the ion of m/z 198.0376 as an example to illustrate the biomarker identification process. Firstly, the accurate mass of the ion was obtained from UPLC Q-TOF/MS in ESI+ modes. The accurate molecular weight of the quasi-molecular ion was m/z 198.0376, suggested a molecular formula with [C9 H12 NO4 ]+ . Candidates are obtained in searching molecular weight at 198.0376 Da (Positive mode, MW tolerance ± 0.05 Da) from database such as HMDB (http://www.hmdb.ca/). As a result, there are five candidates with molecular formula of [C9 H12 NO4 ]+ , which are described as 3-hydroxy-2-methylpyridine-4,5-dicarboxylate, L-dopa, DLdopa, metanephrine, and phosphoguanidinoacetate respectively. The fragment ion at m/z 182.0859 (C9 H12 NO3 ), m/z 168.0853 (C9 H12 O3 ), m/z 153.0792 (C8 H11 NO2 ), and m/z 107.0674 (C7 H9 N) from the ion at m/z 198.0376 was generated from the loss of 16 (O), 30 (NO), 45 (CHO2 ) and 90 (C2 H2 O4 ), respectively. Compared to 3-hydroxy-2-methylpyridine-4,5-dicarboxylate, DL-dopa, metanephrine, and phosphoguanidinoacetate, the fragmentation mode of L-dopa was matched more analogously to the observation of fragment ion in MS2 spectrum of m/z 198.0376. By comparing the fragmentation pattern with the mass spectrum in HMDB (http://www.hmdb.ca/), this metabolite was tentatively identified as L-dopa. By comparison of the ion intensity of potential biomarkers between MPTP group and control group, ten metabolites were upregulated by MPTP stimulus (Fig. 4). After treated by EAS, the group showed the tendency to correct the derivations of 1, 2, 3, 4, 7, 8 and 10.

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facilitate stearoylcarnitine to convert into L-carnitine and reduce the level of L-palmitoylcarnitine. Thus EAS may protect the dopaminergic neurons in PD mice against apoptosis induced by caspase. Palmityl-CoA is a fatty acid coenzyme derivative which plays a key role in fatty acid oxidation and biosynthesis. Palmityl-CoA is converted into tetradecanoyl-CoA by very long-chain acyl-CoA dehydrogenase (VLACD). Tetradecanoylcarnitine is one of the main biochemical markers for VLACD deficiency, which affects palmitylCoA conversion. The concentration of tetradecanoylcarnitine in MPTP group increased significantly compared to control group, which was consistent with the increased level of tetradecanoylcarnitine in the VLACD deficiency (Costa et al., 1997). Down-regulation of tetradecanoylcarnitine by EAS indicated the EAS treatment could recover the dysfunction of VLACD in MPTP-induced PD mice, the therapeutic effects of EAS may base on the regulation of the dysfunction of VLACD in mitochondrial beta-oxidation of long chain saturated fatty acids and fatty acid metabolism. Regulation of genes expression associated with lipid metabolism by Acanthopanax senticosus harms has been recently described, which is in line with the results obtained in the present study (Panossian et al., 2013). Acanthopanax senticosus harms has an influence on transcriptional control of metabolic regulation both on the cellular level and the level of the whole organism. The adaptogenic effects of Acanthopanax senticosus harms on gene expression profiles in neuroglial cells mainly affected energy metabolism and central nervous system functions. Methionine Metabolism (Fig. 5)

Discussion Tyrosine Metabolism (Fig. 5) L-dopa is used for the treatment of PD and the immediate precursor of DA, which can be taken orally and crosses the blood-brain barrier and converted into DA. In the cerebrum, L-dopa derived from L-tyrosine by tyrosine hydroxylase. Our previous study indicated that the DA levels of striatum in MPTP group was significantly lower than control group (Liu et al., 2012), and the concentration of L-dopa in MPTP group increased significantly compared to control group in this study. These results may indicate that MPTP inhibited L-dopa from converting into DA. Down-regulation of L-dopa by EAS indicated the EAS treatment could facilitate L-dopa to convert into DA in MPTP-induced PD mice, and the therapeutic effects of EAS may base on the regulation of the levels of L-dopa and DA in tryptophan metabolism. Mitochondrial Beta-Oxidation of Long Chain Saturated Fatty Acids and Fatty acid Metabolism (Fig. 5) Stearoylcarnitine and L-palmitoylcarnitine are derived from mitochondrial beta-oxidation of long chain saturated fatty acids and fatty acid metabolism respectively. L-carnitine plays an integral role in attenuating the brain injury associated with mitochondrial neurodegenerative disorders such as PD, which is derived from stearoylcarnitine by carnitine O-palmitoyltransferase 2 (Wang et al., 2007). L-palmitoylcarnitine reversed the inhibition mediated by L-carnitine and increased caspase activity, which can induce apoptosis. The caspase activity may be regulated in part by the balance of L-carnitine and L-palmitoylcarnitine (Mutomba et al., 2000). The concentration of stearoylcarnitine and L-palmitoylcarnitine in MPTP group increased significantly compared to control group in this study, which indicated that MPTP may induce caspase activity indirectly, and cause mitochondrial neurodegeneration in PD. Down-regulation of stearoylcarnitine and L-palmitoylcarnitine by EAS indicated the EAS treatment could

5 -methylthioadenosine is a crucial step in the methionine metabolism, which is the precursor of L-methionine. 5 -methylthioadenosine has been shown to influence regulation of gene expression, proliferation, differentiation and apoptosis (Ansorena et al., 2002). A significant decrease of L-methionine appeared in PD (Muller et al., 2001), and the concentration of 5 -methylthioadenosine in MPTP group increased significantly compared to control group in this study. These results may indicate that MPTP inhibited 5 -methylthioadenosine from converting into L-methionine. Down-regulation of 5 methylthioadenosine by EAS indicated the EAS treatment could facilitate 5 -methylthioadenosine to convert into L-methionine in MPTP-induced PD mice, and therapeutic effects of EAS may base on the regulation of the levels of 5 -methylthioadenosine and Lmethionine in methionine metabolism. Sphingolipid Metabolism (Fig. 5) Phytosphingosine-1-P is an intermediate in sphingolipid metabolism pathway, which is the phosphate of phytosphingosine. Phytosphingosine exerted strong cytotoxic effects, modulated the Caenorhabditis elegans muscarinic acetylcholine receptor-mediated signal transduction pathway and induced cell death (Lee et al., 2001). The concentration of phytosphingosine-1-P in MPTP group increased significantly, which indicated that MPTP could induce cell death through increasing the level of phytosphingosine-1-P, and facilitating it to convert into phytosphingosine. Down-regulation of phytosphingosine-1-P by EAS indicated the EAS treatment could inhibit phytosphingosine-1-P from converting into phytosphingosine and protect cell against apoptosis. Other Biochemical Metabolism (Fig. 5) In MPTP group, the concentration of tetracosanoylglycine, Cer(d18:0/18:0), morphiceptin and LysoPC(20:4(5Z,8Z,11Z,14Z)) increased significantly compared to control group, which indicate that MPTP could reduce the levels of tetracosanoylglycine,

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Figure 4. The ion intensity of potential biomarkers in different groups. Data represent the mean ± SE of each group (n = 10 in each group). a Change trend compared with control group, p<0.05. b Change trend compared with MPTP model group, p<0.05.

Figure 5. The perturbed metabolic pathways in response to MPTP modeling and EAS treatment. The levels of potential biomarkers in MPTP group compared to normal control group were labeled with (↓) down-regulated and (↑) up-regulated. () Metabolites in abnormal could be regulated by EAS; CPT 2, carnitine O-palmitoyltransferase 2; MPTP, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine Hydrochloride; TH, tyrosine hydroxylase; VLACD, very long-chain acyl-CoA dehydrogenase.) The effects of MPTP and EAS on DA were published in reference (Liu et al., 2012).

Cer(d18:0/18:0), morphiceptin and LysoPC(20:4(5Z,8Z,11Z, 14Z)). However, EAS did not improve metabolic disturbance of Cer(d18:0/18:0), morphiceptin and Lyso -PC(20:4(5Z,8Z,11Z,14Z)) induced by MPTP. Conclusions A UPLC-QTOF-MS-based cerebral metabonomics method has been established and used to evaluate the anti-PD efficacy and mechanism of EAS on a PD model of mice induced by MPTP.

Pattern recognition with multivariate statistical analysis allowed the metabolic profile of MPTP-induced PD group clearly separated from control group, and that of EAS group was close to the control group after 20 days treatment. 10 metabolites with significant changes in MPTP group were considered as potential biomarkers to MPTP-induced PD and characterized to be (1) L-Dopa, (2) 5 -methylthioadenosine, (3) tetradecanoylcarnitine, (4) phytosphingosine-1-P, (5) Cer(d18:0/18:0), (6) LysoPC(20:4(5Z,8Z,11Z,14Z)), (7) L-palmitoylcarnitine, (8) tetracosanoylglycine, (9) morphiceptin and (10) stearoylcarnitine,

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respectively. The derivations of all those biomarkers can be regulated by EAS treatment except (5) Cer(d18:0/18:0), (6) LysoPC(20:4(5Z,8Z,11Z,14Z)), (9) morphiceptin, which suggested that the therapeutic effect of EAS on PD may involve in regulating the tyrosine metabolism, mitochondrial beta-oxidation of long chain saturated fatty acids, fatty acid metabolism, methionine metabolism and sphingolipid metabolism. This study indicated that changed metabolites can be certainly recovered by EAS, and the treatment of EAS can be connected with the regulation of related metabolic pathways, which will provide better understanding of the anti-PD mechanism of EAS in clinical use. Disclosures The authors declare that they have no conflict of interest. Acknowledgments This article is supported by the National Natural Science Foundation of China (81270056), the National Natural Science Foundation of Youth Science Fund (30901974), and the outstanding innovative talent support programs of Heilongjiang University of Chinese Medicine. References Ansorena, E., Garcia-Trevijano, E.R., Martinez-Chantar, M.L., Huang, Z.Z., Chen, L., Mato, J.M., Iraburu, M., Lu, S.C., Avila, M.A., 2002. S-adenosylmethionine and methylthioadenosine are antiapoptotic in cultured rat hepatocytes but proapoptotic in human hepatoma cells. Hepatology 35, 274–280. Bai, Y., Tohda, C., Zhu, S., Hattori, M., Komatsu, K., 2011. Active components from Siberian ginseng (Eleutherococcus senticosus) for protection of amyloid beta(25-35)-induced neuritic atrophy in cultured rat cortical neurons. J Nat Med 65, 417–423. Bocharov, E.V., Ivanova-Smolenskaya, I.A., Poleshchuk, V.V., Kucheryanu, V.G., Il’enko, V.A., Bocharova, O.A., 2010. Therapeutic efficacy of the neuroprotective plant adaptogen in neurodegenerative disease (Parkinson’s disease as an example). Bull Exp Biol Med 149, 682–684. Bocharov, E.V., Kucherianu, V.G., Bocharova, O.A., Karpova, R.V., 2008. [Neuroprotective features of phytoadaptogens]. Vestn Ross Akad Med Nauk, 47–50. Brekhman, I.I., Dardymov, I.V., 1969. New substances of plant origin which increase nonspecific resistance. Annu Rev Pharmacol 9, 419–430. ChPC, 2010. Radix et, Rhizoma seu Caulis Acanthopanacis Senticosi (Ciwaujia). In: Pharmacopoeia of the People’s Republic of China, Vol.1. CHINA MEDICAL SCIENCE PRESS, Beijing, China, pp. 192. Coen, M., Holmes, E., Lindon, J.C., Nicholson, J.K., 2008. NMR-based metabolic profiling and metabonomic approaches to problems in molecular toxicology. Chem Res Toxicol 21, 9–27. Costa, C.G., Struys, E.A., Bootsma, A., ten Brink, H.J., Dorland, L., Tavares de Almeida, I., Duran, M., Jakobs, C., 1997. Quantitative analysis of plasma acylcarnitines using gas chromatography chemical ionization mass fragmentography. J Lipid Res 38, 173–182. Dai, Y., Li, Z., Xue, L., Dou, C., Zhou, Y., Zhang, L., Qin, X., 2010. Metabolomics study on the anti-depression effect of xiaoyaosan on rat model of chronic unpredictable mild stress. J Ethnopharmacol 128, 482–489. Dong Y, L.S., AN LF, Lu F, Tang B, Zhou SH, 2011. The effect of Eleutheroside B on ERK1/2 of MPP+-induced PC12 cells. J Mol Diagn Ther 3, 155–158.

1229

EMEA/HMPC/102655/2007, 2008. Reflection Paper on the Adaptogenic Concept. European Medicines Agency, London. Fujikawa, T., Kanada, N., Shimada, A., Ogata, M., Suzuki, I., Hayashi, I., Nakashima, K., 2005. Effect of sesamin in Acanthopanax senticosus HARMS on behavioral dysfunction in rotenone-induced parkinsonian rats. Biol Pharm Bull 28, 169–172. Griffin, J.L., Walker, L.A., Garrod, S., Holmes, E., Shore, R.F., Nicholson, J.K., Zhang, A., Sun, H., Wang, Z., Sun, W., Wang, P., Wang, X., 2000. NMR spectroscopy based metabonomic studies on the comparative biochemistry of the kidney and urine of the bank vole (Clethrionomys glareolus), wood mouse (Apodemus sylvaticus), white toothed shrew (Crocidura suaveolens) and the laboratory rat. Lee, J.S., Min, D.S., Park, C., Park, C.S., Cho, N.J., 2001. Phytosphingosine and C2-phytoceramide induce cell death and inhibit carbachol-stimulated phospholipase D activation in Chinese hamster ovary cells expressing the Caenorhabditis elegans muscarinic acetylcholine receptor. FEBS Lett 499, 82–86. Li, X.Z., Zhang, S.N., Liu, S.M., Lu, F., 2013. Recent advances in herbal medicines treating Parkinson’s disease. Fitoterapia 84, 273–285. Liu, S.M., Li, X.Z., Huo, Y., Lu, F., 2012. Protective effect of extract of Acanthopanax senticosus Harms on dopaminergic neurons in Parkinson’s disease mice. Phytomedicine 19, 631–638. Muller, T., Woitalla, D., Hauptmann, B., Fowler, B., Kuhn, W., 2001. Decrease of methionine and S-adenosylmethionine and increase of homocysteine in treated patients with Parkinson’s disease. Neurosci Lett 308, 54–56. Mutomba, M.C., Yuan, H., Konyavko, M., Adachi, S., Yokoyama, C.B., Esser, V., McGarry, J.D., Babior, B.M., Gottlieb, R.A., 2000. Regulation of the activity of caspases by L-carnitine and palmitoylcarnitine. FEBS Lett 478, 19–25. Nicholson, J.K., Lindon, J.C., Holmes, E., 1999. ‘Metabonomics’: understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica 29, 1181–1189. Panossian, A., Hamm, R., Kadioglu, O., Wikman, G., Efferth, T., 2013. Synergy and Antagonism of Active Constituents of ADAPT-232 on Transcriptional Level of Metabolic Regulation of Isolated Neuroglial Cells. Frontiers in neuroscience 7, 16. Panossian, A., Wikman, G., 2010. Effects of Adaptogens on the Central Nervous System and the Molecular Mechanisms Associated with Their Stress-Protective Activity. pharmaceuticals 3, 188–224. Plumb, R.S., Stumpf, C.L., Gorenstein, M.V., Castro-Perez, J.M., Dear, G.J., Anthony, M., Sweatman, B.C., Connor, S.C., Haselden, J.N., 2002. Metabonomics: the use of electrospray mass spectrometry coupled to reversed-phase liquid chromatography shows potential for the screening of rat urine in drug development. Rapid Commun Mass Spectrom 16, 1991–1996. Sandler, B.I., 1970a. The influence of Eleutherococcus extracts on several neurosomatovegetative disturbances in patients with acute cranial and cerebral traumas. Medicines of the Soviet Far East 10, 78–81. Sandler, B.I., 1970b. Some data about application of an Eleutherococcus extract at acute craniocereberal trauma. Medicines of the Soviet Far East 10, 74–77. Sandler, B.I., 1972. Influence of Eleutherococcus extract on cerebral blood circulation with cranial and cerebral traumas (Rheoencephalography data). Medicines of the Soviet Far East 11, 109–113. Wang, C., Sadovova, N., Ali, H.K., Duhart, H.M., Fu, X., Zou, X., Patterson, T.A., Binienda, Z.K., Virmani, A., Paule, M.G., Slikker Jr., W., Ali, S.F., 2007. L-carnitine protects neurons from 1-methyl-4-phenylpyridinium-induced neuronal apoptosis in rat forebrain culture. Neuroscience 144, 46–55. WHO, 2004. Radix Eleutherococci. In: WHO Monographs on selected medicinal plants,vol.2. WHO, Geneva, pp. 83–96. Zhang, A., Sun, H., Wang, Z., Sun, W., Wang, P., Wang, X., 2010. Metabolomics: towards understanding traditional Chinese medicine. Planta Med 76, 2026–2035. Zhang, Z.F., Liu, Y., Luo, P., Zhang, H., 2009. Separation and purification of two flavone glucuronides from Erigeron multiradiatus (Lindl.) Benth with macroporous resins. J Biomed Biotechnol 2009, 875629. Zheng, S., Yu, M., Lu, X., Huo, T., Ge, L., Yang, J., Wu, C., Li, F., 2010. Urinary metabonomic study on biochemical changes in chronic unpredictable mild stress model of depression. Clin Chim Acta 411, 204–209.