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41[5]zhouxiao-wen. The Applicationof PEG on Optimization of Dosage Form[J]. Chinese Journal of Pharmaceuticals , 1995, 30 (12) : 7l3-714.
Cerebral metabonomics study on Parkinson's disease mice treated with extract
of Acanthopanax senticosus harms
Xu-zhao LIa, FangLUa*, Chang-feng LIU a, Ying ZHANG a, Guang-li YANb, Yu WANGa, Yu BAI
a, Na WANGa, Shu-min LIUa*
a: Institute of Traditional Chinese Medicine, Heilongjiang University of Chinese Medicine, Harbin 150040, PR China, b:College of Pharmacology,Heilongjiang University of Chinese Medicine, Harbin 150040, PR China Corresponding author:
Shu-min LIU:Tel: +86-451-87266988; Fax: +86-451-87266988; E-mail address: keji-liu@163.com; Postal address: Institute of Traditional Chinese Medicine, Heilongjiang University of Chinese Medicine, He Ping Road 24, Harbin 150040, PR China
FangLU;Tel: +86-451-87266829; Fax:+86-451-87266829; E-mail address:lufang_19790501@163.com; Postal address: Institute of Traditional Chinese Medicine, Heilongjiang University of Chinese Medicine, He Ping
Road 24, Harbin 150040, PR China
Abbreviations:BPI, base peak intensity; BV/h, bed volume/hour; CPT,carnitine O-palmitoyltransferase; DA, dopamine;EAS, extract ofAcanthopanaxsenticosusharms; MPTP-HCl, 1-Methyl-4-phenyl-1,2,3,6-tetrahydro -pyridineHydrochloride; MS, Mass spectrometry; NMR, nuclear magnetic resonance; PCA, principal componentsanalysis; PD, Parkinson's disease; PLS-DA, partial least squares-discriminate analysis; RSD, relative standarddeviations;TCM, traditional Chinese medicine;TH, tyrosine hydroxylase; UPLC-QTOF-MS, ultra-performanceliquid chromatography coupled with quadrupole time-of-flight mass spectrometry; VIP, variable importance of project;VLACD, very long-chain acyl-CoAdehydrogenase
Abstract
Extract ofAcanthopanaxsenticosusharms (EAS) has neuroprotectiveeffect on Parkinson's disease (PD)mice against dopaminergic neuronaldamage. However,studies of its anti-PD mechanism arechallenging, accounted for the complex pathophysiology of PD, and complexity of EAS with multipleconstituents acting on different metabolic pathways.In this study, metabonomics based on ultra-performanceliquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used toprofile the metabolic fingerprints ofcerebrumobtained from1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridineHydrochloride(MPTP-HCl)-induced PD model in mice with and withoutEAStreatment.Through partial least squares-discriminate analysis (PLS-DA), it was observed that metabolic perturbations induced byMPTPwere restoredafter treatment with EAS. Metabolites with significant changes inducedbyMPTP, including (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,were characterized as potential biomarkers involvedin the pathogenesis of PD. 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 thetyrosine metabolism,mitochondrial beta-oxidation of long chain saturated fatty acids, fatty acid metabolism,methionine metabolismand sphingolipid metabolism. This study indicated thatchanged metabolities can be certainly recovered by EAS, and the treatment of EAS can be connected with the regulation of related metabolic pathways.
Keywords :extract of Acanthopanaxsenticosus harms; Parkinson's disease;cerebral metabonomics; UPLC-QTOF-MS
1. Introduction
Parkinson's disease is a chronic neurological disorder. In ventral midbrain, particularly in substantianigra pathological features showthatdopaminergic neurons progressively degenerate, which causes a consequentreductionof dopamine(DA) levels in the striatum.The functions of acetylcholine neurons and dopaminergicneurons 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 qi-blood deficiency(Li et al., 2006; Zhang, 2006).
TCMs are gaining more attention all over the world, due to their specifictheory and longhistorical clinical practiceAcanthopanaxsenticosus (Rupr. and Maxim.) Harms is a widelyused traditional Chinese herb. In the theory of TCM, it is described as following: Indications Hypofunctionof the spleen and the kidney marked by general weakness, lassitude, anorexia, aching of the loinsand knees; insomnia anddream-disturbed sleep (ChPC, 2010). In modern pharmacologicalresearches, wefound that Acanthopanaxsenticosus Harms haveneuroprotectivefeatures (Bocharov et al., 2010; Bocharov et al., 2008), stressprotectiveand adaptogenicactivities (Brekhman and Dardymov, 1969; EMEA/HMPC/102655/2007, 2008; Panossian and Wikman, 2010; WHO, 2004), and it was also applied to study on cranial andcerebraltraumas(Sandler, 1970a, b, 1972). Our previousstudyindicated that EAS can protectC57BL/6 mice against MPTP-induced dopaminergic neuronaldamage(Liu et al., 2012).However, its mechanism of anti-PD effect has not yet beenelucidated.
Metabonomicsis defined as 'the quantitative measurementof the dynamicmultiparametricmetabolicresponseoflivingsystemstopathophysiologicalstimuliorgeneticmo dification' (Nicholson et al., 1999), which can providevariation of whole metabolic networks for characterizing pathological states in animals and human,as well givingdiagnostic information and presenting mechanisticinsight 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 ofChinese herbal(Zhang et al., 2010).Mass spectrometry (MS) and nuclear magnetic resonance (NMR)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 gainedmore application due to the high resolution of chromatographicpeaks, increased analytic speed and sensitivity for complex mixtures (Plumb et al., 2002).In this study, cerebral metabonomics based on UPLC-QTOF-MSwas applied to investigate the metabolic profiles and potentialbiomarkers in a mice model of MPTPinduced PD after treatment of EAS, which may facilitateunderstanding the pathological changes of PD and anti-PD mechanism of EAS. To our knowledge, this study isthe first report of UPLC-QTOF-MS-based cerebralmetabonomicsmethod used to investigate the anti-PD effect of EAS.
2. Experimental
2.1. Chemicals and reagents
HPLC-grade acetonitrile was purchased from Thermo Fisher Scientific (USA).Purified water was produced by Milli-Q ultra-pure watersystem (Millipore, Billerica, USA). Formic acid (HPLC grade) waspurchasedfromDikma Technologies (USA). 1-Methyl-4-phenyl-1,2,3,6-
tetrahydropyridineHydrochloride (MPTP-HCl, CAS Number:23007854) was purchased from Sigma-Aldrich (USA).
2.2. Plant material and extraction
The herb (root and rhizome of Acanthopanaxsenticosus(Rupr.EtMaxim.)Harms) was derived from theirnatural origin, whichwas collected in Wuchang(N44° 39' ,E127°35') of Heilongjiangprovince, PR China. The voucher specimens (hlj-201003) of the herb wasauthenticated by Professor Ke Fu, Institute of Traditional ChineseMedicine, Heilongjiang University of
ChineseMedicine. EAS were prepared with Acanthopanaxsenticosus(Rupr. EtMaxim.) Harms that one gram of crude drugwas extracted 3 times with 10mL 80% ethanol for 2,2 and 2h,respectively,separatedand purified by AB-8macroporousadsorption resin (Polarity: Weak-polar, Particlediameter: 0.3-1.25 mm,Surface area: 480-520 m /g, Average pore diameter: 130-140 nm,Moisture content: 67.3% (Zhang et al., 2009)). The process for purification was as follows: the concentrationof sample solution was500g/L; adsorption flow rate was 2 BV/h (bed volume/hour); eluent was 30% ethanol; the dosage was 9 BV;elution flow rate was1 BV/h. The ethanol phase was evaporated under vacuum and thenoven dried at 60 °C togive the extracts. The drug-extract ratio was1.3% (Shao, 2011a, b).
2.3. Animals
30 male C57BL/6 mouse (2 months old, 18-22 g, Inbred Mice)were purchased from Yi-SiLaboratoryAnimal Technology Co.,Ltd (China). They were allowed at least 1 week to adapt to theirenvironment before used forexperiments. Animals were housed 5per cage (320mm*180 mm*160 mm) under a normal 12-h/12-hlight/dark schedule with the lights on at 07:00 a.m. They werehoused at room temperature (23±2°C) with relative humidity(55±5%), and givena standard chow and water ad libitum for theduration of the study. The ethicalapproval for the experiment wasfollowed by the Legislation on the Protection of Animals Used forExperimentPurposes (Directive 86/609/EEC).
2.4. PD mice and drug administration
Mice was 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 dayfor 5 days(Liu et al., 2012). Control group received equal volumesaline (20mL/kg i.p) once a day for 5 days. FromDay 6, MPTP+EAS groupwas orally administrated with EASfor 20 days, and dose amountedto 45.5 mg/kgdaily. Control group and MPTP model group were orally administrated with equal volume saline(20 mL/kg daily)for 20 days.
2.5.Sample collection and preparation
On the 26th day, all mice of each group weredecapitated. Cerebrumwas weighed accurately and immediately frozen inliquid nitrogen and stored at -80 °C until used.Fordetermination,cerebrumtissue blocks were thawed on ice. Once thawed, ice-cold saline (3 mLsaline / 0.5g 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 5mLmethanol for 2 min andcentrifugedat 15,000 Xg for 15 min at 4 °C.The upper organic layer wastransferred into another tube,evaporated to dryness undernitrogen stream and the residue dissolved with700 ^Lmethanoland vortexed for 1 min.The mixture wascentrifuged at 4,000 Xg for 5 min at 4 °C, and the supernatant was filteredandstored at -80 °CforUPLC/MS analysis.
2.6. UPLC conditions
Waters AcquityTMUPLC (consisting of a vacuum degasser,autosampler, a binary pump, photodiode arraydetectorand oven)was equipped with ACQUITYUPLCTMHSS T3 column (100 mm*2.1 mm,i.d. 1.8^m, WatersCorp, Milford, USA). The analytical column was maintained at a temperature of 40 °C and the mobilephase was composed of acetonitrile (A)and water (B) each containing 0.1% formic acid. A solvent gradientsystem was used: 2-100% A for 0-15 min. The flow rate was0.4mL/min.Injection volume was 5^L.
2.7. MS conditions
MS analysis was performed on Q-TOF analyzer in SYNAPT HDMSsystem (Waters Corporation, Milford, MA,USA) in positive ionmode,using the following parameters: capillary voltage, 1000 V; sample cone voltage, 40 V; source temperature, 110°C; desolvationtemperature, 350°C; desolvationgas flow, 750 L/h; cone gas flow,20L/h.
2.8. Data analysis
The raw data were analyzed using the MicromassMarkerLynx 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 detectedpeaks from each data file in the data set.
The main parameterswere set as Mlows:retention time range 0-12.5 min, mass range 50-1000 amu, mass tolerance 0.01, minimum intensity 1%, masswindow 0.05, retention time window 0.20, and noise eliminationlevel 6. The resulting data were analyzed byprincipal componentsanalysis (PCA) and partial least squares-discriminate analysis (PLS-DA) using MarkerLynx XS software (version 4.1, Waters Corporation, USA).Student's t-test was used for statistical analysis to evaluate thesignificant difference of potentialbiomarkers (SPSS 18.0 (SPSS Inc., Chicago, IL)).
3. Results
3.1. Method development and validation
Metabolic profiling of cerebral samples was acquired usingUPLC/TOF-MS/MS system in the positive ion mode. Thebase peak intensity(BPI) chromatograms ofcerebral samplesfrom control group, MPTP group, and EAS-treated group are shown in Figure 1 (A, B and C).The average peak basewidth of 4s was set for thisseparation, which generated a seriesof peaks with retention time and m/z pairs (tR_m/z pair) as variables.
High reproducibility is crucial for any analytical protocols, especiallyfor metabonomics study which requires handling many samples.Reproducibility of the chromatography and MS was determined fromfive replicatedanalyses of the same cerebral sample. The relative standarddeviations (RSD) of retention timeand peak area are below 0.51% and 3.1%, respectively. These resultsdemonstrated the excellent stability and reproducibility of chromatographic separation and mass measurement during the whole sequence.
A
Figure 1.BPI chromatograms obtained from the positive ion UPLC-MS analyses of (A)control group, (B)MPTP groupand (C) (MPTP+EAS) group that sampling on the 26th day.
3.2. Multivariate analysis of UPLC/MS data PCA was firstly carried out toinvestigate whether two groups can be separated and to find outtheir metabolicdistinction. Then PLS-DA, a supervised multivariable statistical methodto sharpen an already established (weak) separation betweengroups of observations plotted in PCA was performed. The variables responsible for differentiating MPTP group and controlgroup were selected as potential biomarkers of progress of diseases byvariable importance of project (VIP) statistics.
In order to gain an overview of the micecerebralmetabolic profiling, here, PCA was used in the subsequentUPLC Q-TOF/MS data analysis.PLS-DA wasperformed and the results shown in Figure2(A and B) and 3, whichindicated that the metabolic profile of MPTP model group deviatedfrom the control group, suggesting that significant biochemicalchanges induced by MPTP.The metabolic profile of EAS treatedgroup fairly differedfrom the MPTP group and close to controlgroupin Figure3, indicating the deviations induced by MPTP were significantlyimproved after treatment of EAS.
Scores Comp[1] vs. Comp[2] colored by Sample Group (original)
3.3. Potential biomarkers responsible for the PD inducedby MPTP and the anti-PD effect of
EAS
Corresponding VIP statistics of PLS-DA and S-plots were usedto extract the important variables responsible for the differentiation. The VIP value calculated by MarkerLynx XS software signifiesthe influence of metaboliteion on the classification. A VIP value>1 means that variables have above average influence on theclassification.S-plot is a tool for visualizing covariance and correlation between the metabolites and the modeled class, thoseions far from the origin contributing to the clustering significantly. As shown in Figure2 B, S-plots based on cerebral metabolicprofiles indicated 10 variables representing individual metabolites as biomarker candidate ionswith retention time and m/zpairs 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 arelist in Table 1.Those contributedsignificantly to differentiate the clustering of MPTP group fromthat of control group, could be considered as potential biomarkersresponsible for derivations of metabolic profile induced by MPTP.Their structures were tentatively identified based on accuratemass measurements via UPLC-TOF-MS, by analysis of accuratemolecular weight and the MS/MS spectra. 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 weretentatively 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).
Figure 4. The ion intensity of potential biomarkers in different groups. The corresponding markers represented to the Table 1. a Change trend compared with control group. b Change trend compared with MPTP model group.
Here, we take the ion of m/z198.0376as 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 accuratemolecular weight of the quasi-molecular ion was m/z 198.0376,suggested a molecular formula with [C9H12NO4]+.Candidates are obtained in searching molecularweight 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 candidateswith molecular formula of [C9H12NO4]+, which are described as 3-hydroxy-2-methylpyridine-4,5-dicarboxylate, L-dopa, DL-dopa, metanephrine, and phosphoguanidinoacetaterespectively.The fragment ion at m/z 182.0859 (C9H12NOs),m/z 168.0853 (C9H12O3), m/z 153.0792 (CgHuNO2),and m/z 107.0674(C7H9N) from the ion at m/z198.0376 was generated from the loss of 16 (O), 30 (NO), 45 (CHO2) and 90 (C2H2O4), respectively.Compared to3-hydroxy-2-methylpyridine-4,5-dicarboxylate, DL-dopa, metanephrine, and phosphoguanidinoacetate, the fragmentation mode ofL-dopawas matched more analogously to the observation of fragment ion in MS2spectrum of m/z198.0376.By comparing thefragmentation pattern with the mass spectrumin HMDB(http://www.hmdb.ca/),this metabolite wastentatively identified asL-dopa.
By comparison of the ion intensity of potential biomarkersbetween MPTP group and control group, tenmetaboliteswere up-regulated by MPTP stimulus (Figure4). After treated by EAS, the groupshowed the tendency to correct the derivations of1, 2, 3, 4, 7, 8 and 10.
4. Discussion
4.1. Tyrosine Metabolism(Figure 5)
L-dopais 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-dopaderived fromL-tyrosine by tyrosine hydroxylase (TH). Our previousstudyindicated that the DAlevels of striatum in MPTP group wassignificantly lower than control group(Liu et al., 2012), and the concentration of L-dopa in MPTP groupincreased significantly compared to control group in this study.These results may indicate that MPTP inhibited L-dopafrom convertinginto DA.Down-regulation of L-dopa by EAS indicated the EAS treatment could facilitateL-dopato convert into DA in MPTP-induced PD mice, andthe therapeutic effects of EASmay base on the regulation of the levels ofL-dopa and DA in tryptophan metabolism.
4.2. Mitochondrial Beta-Oxidation of Long Chain Saturated Fatty Acids and Fatty acid Metabolism (Figure 5)
Stearoylcarnitine and L-palmitoylcarnitine are derived from mitochondrial beta-oxidation of long chain saturated fatty acids and fatty acid metabolismrespectively.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 (CPT) 2(Wang et al., 2007).L-palmitoylcarnitine reversed the inhibition mediated by L-carnitineandincreasedcaspase activity, which can induceapoptosis.The caspase activity may be regulated in part by the balance of L-carnitine and L-palmitoylcarnitine(Mutomba et al., 2000).The concentration ofstearoylcarnitine and L-palmitoylcarnitinein MPTP groupincreased significantly compared to control group in this study, which indicated that MPTP may induce caspase activityindirectly, and cause mitochondrial neurodegeneration in PD.Down-regulation ofstearoylcarnitine and L-palmitoylcarnitineby EAS indicated the EAS treatment couldfacilitatestearoylcarnitineto convert into L-carnitine and reducethe level of L-palmitoylcarnitine.Thus EAS mayprotect the dopaminergicneurons in PD mice against apoptosis induced by caspase.
Palmityl-CoAis a fatty acid coenzyme derivative which plays a key role in fatty acid oxidation andbiosynthesis.Palmityl-CoA is converted into tetradecanoyl-CoA by very long-chain acyl-CoAdehydrogenase (VLACD).Tetradecanoylcarnitineis one of the main biochemical markers forVLACDdeficiency, which affectspalmityl-CoAconversion.The concentration of tetradecanoylcarnitine in MPTP groupincreased 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 dysfunctionof 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.
deficiency in urine
4.3.Methionine Metabolism (Figure 5)
5'-methylthioadenosine is a crucial step in the methioninemetabolism, which is theprecursor 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-methionineappeared in PD (Muller et al., 2001), and the concentration of 5'-methylthioadenosine in MPTP groupincreased 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 facilitate5'-methylthioadenosineto convert into L-methioninein MPTP-induced PD mice, andtherapeutic effects of EASmay base on the regulation of the levels of5'-methylthioadenosine and L-methionine in methioninemetabolism.
4.4.SphingolipidMetabolism (Figure 5) Phytosphingosine-1-P is an intermediate in sphingolipid metabolism pathway, which is the phosphate of phytosphingosine.Phytosphingosine exerted strong cytotoxiceffects, modulated the Caenorhabditiselegans muscarinicacetylcholine receptor-mediated signaltransduction pathway and induced cell death(Lee et al., 2001).The concentration ofphytosphingosine-1-Pin MPTP groupincreased significantly, which indicated that MPTP could induce cell death through increasing the level of phytosphingosine-1-P, and facilitatingitto convert into phytosphingosine. Down-regulation of phytosphingosine-1-P by EAS indicated the EAS treatment could inhibit phytosphingosine-l-Pfrom convertinginto phytosphingosine and protect cell against apoptosis.
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 controlgroup were labeled with (I) 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-tetrahydropyridineHydrochloride; 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). A
Table 1. Metabolites selected as biomarkers characterized in cerebral profile and their change trends (n=10 in each group)._
No. tR (min)
VIP m/z [M+H]+
Formula MS/MS
Losses
Meta bolite
s
Proposed structure
1
0.65
2.545 2
198.03 76
C9H11NO4
182.0858 168.0860 153.0798 107.0674
-O -NO -CHO2 C2H2O4
L-Dopa
2 2.38
3 9.32
3.035 298.09 C11H15N5 281.0583 -CH5 5'-
8 74 O3S 254.0712 -C2H4O Meth
230.0884 -C2N2O ylthio
222.0753 -C2H6NS adeno
170.0514 -C4H6N3O2 sine
162.0416 -C5H12O2S
153.9929 -C5H12N4O
136.0558 -C6H4N5O
85.0290 -
C7H11N5OS
4.348 C21H41NO 314.2695 -C3H6O Tetra
7 372.31 4 313.2556 -C3H7O decan
08 286.2746 -C4H6O2 oylca
253.2250 -C5H13NO2 rnitin
211.2062 -C7H15NO3 e
197.0814 -C11H29N
161.1027 -C14H27O
85.0351 -C17H37NO2
67.0596 -C16H35NO4
398.32 C18H40NO 337.2382 -C2H5O2 Phyto
3.447 74 6P 320.1753 -C3H12NO sphin
0 283.2273 -CH10NO3P gosin
270.2069 -C3H13O3P e-1-P
238.2499 -C2H9O6P
199.1579 -C7H20O4P
147.1259 -C11H24O4P
117.0790 -C13H30O4P
84.0968 -
C12H29NO6 P P
3.255 568.34 C36H73NO 269.2563 -C19H41NO Cer(d
2 01 3 227.1426 -C24H53 18:0/
201.1729 -C25H51O 18:0)
184.0901 -C27H60
145.1210 -C28H57NO
91.0699 -C33H65O
58.0782 -C32H64O3
26
HO OH
4 9.64
5 10.13
6 10.14
544.34 04
C28H50NO
7P
4.223 В
527.3359 4В7.2699 42В.2566 328.1671 1В6.0В92 167.1072 В6.0970
-HO -C4H9 -C6H12O2
-C12H24O3
C19H37NO3 P
C1BH36NO5 P
C23H39O7P
Lyso PC(2 0:4(5 Z,8Z, 11Z,1 4Z))
7 10.38
6.745 4
В 10.65
6.067 7
400.34 22
C23H45NO
426.35 77
C26H51NO
356.3529 337.2601 240.20В6 145.1103 109.0742 В5.0372
36В.3234 324.2903 309.2966 157.0657 В6.1012
-CO2
-C3H11O -CbHIBN02 -C16H31O2 -C16H37NO3 -C19H41NO2
-C4H10 -C6H14O -C6H13O2 -C19H41 -C20H3BNO3
L-Palmi toylca rnitin e
9 10.93
522.35 6.793 59 В
10 11.37
C28H35N5 O5
504.23В7 464.2549 259.1321 1В5.0926 166.0В6В 71.0792
-H4N -CH2N2O
C14H19N2O3
C20H23N3O2
Morp hicept in
C19H24N4O3
C24H27N4O5
5.309 42В.37 C25H49NO 325.3345 -C4H7O3 Stear
7 21 4 312.3083 -C5H10NO2 oylca
269.1627 -C11H27 rnitin
195.1021 -C14H35NO e
167.0708 -C16H39NO
128.0733 -C19H40O2
4
3
4.5. Other BiochemicalMetabolism (Figure 5)
In MPTP group, the concentration oftetracosanoylglycine, Cer(d18:0/18:0), morphiceptin andLysoPC(20:4(5Z,8Z,11Z,14Z))increased significantlycompared to control group, which indicate that MPTP could reduce the levels of tetracosanoylglycine, Cer(d18:0/18:0), morphiceptin andLysoPC(20:4(5Z,8Z,11Z,14Z)).However, EAS did not improve metabolic disturbance ofCer(d18:0/18:0), morphiceptin andLyso -PC(20:4(5Z,8Z,11Z,14Z))induced by MPTP. 5. Conclusions
A UPLC-QTOF-MS-based cerebralmetabonomics method has beenestablished and used for the first time to evaluate the anti-PD efficacy and mechanism of EAS on a PD modelof mice induced by MPTP.Pattern recognition with multivariatestatistical analysis allowed the metabolic profile of MPTP-induced PD group clearly separated from controlgroup, and thatof 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 PDand 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, 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 thetyrosine metabolism,mitochondrial beta-oxidation of long chain saturated fatty acids, fatty acid metabolism,methionine metabolismand sphingolipid metabolism. This study indicated thatchanged metabolities can be certainly recovered by EAS, andthe treatment of EAS can be connected with the regulation of related metabolic pathways, which will provide better understanding of theanti-PD mechanism of EAS in clinical use.
Disclosure statement
None of the authors have potential or actual conflicts ofinterest, and all the authors have seen the manuscript beforesubmission.
The ethicalapproval for the experiment wasfollowed by the Legislation on the Protection of Animals Used forExperimentPurposes (Directive 86/609/EEC). Acknowledgements
This article is supported by NationalNatural Science Foundation of China (81270056) and NationalNatural Science Foundation of Youth Science Fund (30901974). References
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The clinical chemistry and metabonomic profiles of the efficacy of a Chinese traditional prescription Tianqi Jiangtang Capsule on fat emulsion and alloxan-
induced type 2 diabetes mullitus rat model
Shuxiang Zhanga'b, Hui Suna, Yimin Niu a, Guozheng Jiaoa, Kun Yang a, Xijun Wanga' *
aDepartmentofPharmacognosy,bInstitute of Traditional Chinese Medicine, HeilongjiangUniversityofChineseMedicine,No.24HepingRoad,Harbin,China 150040
Abstract
In this research, clinical chemistrycombined with ultra-performance liquid chromatography/ electrospray ionization high definition mass spectrometry (UPLC/ESI-HDMS) were used to analysis urinary samples from an improved rat model of type 2 diabetes mellitus (T2DM) induced by daily ig constant dosage of fat emulsion for 10 days and twice ip low dosage of alloxan. The indexes of clinical chemistry and the metabonomic profiles of PCA illustrated the panorama of T2DM disorders, and proved the success of this kind of T2DM rat model as well as the efficacy of Tianqi Jiangtang Capsule to redress metabolite disorders.
Keywords: Metabonomics; rat model for type 2 diabetes mellitus; Tianqi Jiangtang Capsule;
1.Introduction
Type 2 diabetes mellitus (T2DM), characterized as a kind of multiple, complex molecular disorders, is one of the most prevalent metabolic diseases [1]. Lots of animal models have been developed for better understanding T2DM, including genetically spontaneous models and experimentally nonspontaneous ones [2], of which model evaluation methods mostly focus on clinical chemistry and histopathology.
Metabonomics as a systemic biological approach for studying profiles of small-molecule metabolites in vivo can provide us information on disease processes at several stages[3]. It tries to fully interpret the influences of diseases or drugs on organisms through metabolite variations [4-5]. The mtabonomic data can be obtained by mass spectrometry coupled with gas chromatography (GC-MS) or liquid chromatography (LC-MS) [6], and nuclear magnetic resonances (NMR) [7], and finally give out the information of small-molecule metabolites in vivo that will reflect the internal overall changes in state of disease.
Nowadays, people pay more and more attention to herbal drugs in the role of diabetes prevention and therapy. Tianqi Jiangtang(TQJT) Capsule, licensed as a Grade V New Drugs in China, is a traditional Chinese materia medica preparation. The bulk drug is aqueous extraction of
