Original Papers
Metabonomic Study of the Effects of Acanthopanax senticosus on Peripheral System of Rats
Authors
Shuai-nan Zhang 1*, Xu-zhao Li 1*, Shu-min Liu 1, 2, Fang Lu 1
Affiliations
1 2
Key words " Acanthopanax senticosus l " Araliaceae l " metabonomic analysis l " potential intervention l targets " peripheral system l
received revised accepted
Nov. 11, 2014 March 8, 2015 March 10, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1545915 Published online April 29, 2015 Planta Med 2015; 81: 722–732 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Shu-min Liu Drug Safety Evaluation Center Heilongjiang University of Chinese Medicine He Ping Road 24 Harbin 150040 P. R. China Phone: + 86 4 51 82 19 32 78 [email protected] Correspondence Fang Lu Chinese Medicine Toxicological Laboratory Heilongjiang University of Chinese Medicine He Ping Road 24 Harbin 150040 P. R. China Phone: + 86 4 51 87 26 68 14 [email protected]
Chinese Medicine Toxicological Laboratory, Heilongjiang University of Chinese Medicine, Harbin, P. R. China Drug Safety Evaluation Center, Heilongjiang University of Chinese Medicine, Harbin, P. R. China
Abstract !
Acanthopanax senticosus is extensively used to treat various nervous and cerebrovascular diseases in traditional medicinal systems in China and Russia. Ultrahigh-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry coupled with pattern recognition methods was used to investigate the effects of A. senticosus on the peripheral system in rats. The analysis of possible pathways influenced by A. senticosus was performed with MetaboAnalyst and Cytoscape software. After treatment with A. senticosus, 21 modulated metabolites in heart tissue, 20 in liver tissue, 14 in spleen tissue, 17 in lung tissue, 16 in kidney tissue, and 12 in a serum sample were identified and considered potential biomarkers of A. senticosus treatments. The regulation of some endogenous metabolites by A. senticosus could be beneficial for the treatment of several peripheral system diseases, such as hypertension, cancer, and oxidative stress, etc. However, there were also some upregulated endogenous metabolites producing potential toxicity to the peripheral system. A metabonomic analysis revealed that protection and toxicity coexisted in the effects of A. senticosus on the peripheral sys-
Introduction !
Herbal medicines are composed of mixtures of compounds with complex pharmacological effects [1]. AS (Araliaceae) is also called “Ciwujia” in Chinese, and eleuthero or siberian ginseng in English. It is the herb used in traditional medicinal systems in China and Russia that has been primarily applied to the treatment of various nervous and cerebrovascular diseases, such as Par-
* Both of these authors contributed equally to this work.
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tem, which may be a practical guide for its safe use and beneficial to the expansion of its application.
Abbreviations !
AS:
Acanthopanax senticosus (Rupr. et Maxim.) Harms BPI: based peak intensity CNS: central nervous system NAFLD: nonalcoholic fatty liver disease NO: nitric oxide OPLS‑DA: orthogonal projection to latent structures discriminate analysis PCA: principal components analysis QC: quality control UHPLC-QTOF‑MS: ultrahigh performance liquid chromatography-quadrupole time-of-flightmass spectrometry VIP: variable importance in the projection VLCADD: very long chain acyl-CoA dehydrogenase deficiency Supporting information available online at http://www.thieme-connect.de/products
kinsonʼs disease, Alzheimerʼs disease, depression, mental fatigue, and transient global cerebral ischemia, etc. [2–8]. In the case of oral drugs acting on the CNS, both their bioactive ingredients and metabolites might be initially distributed in the peripheral system through blood circulation before reaching the site of action and produce potential effects including protection and toxicity. In addition, our previous studies have already reported metabolic processes and metabolites of some bioactive ingredients in the peripheral system [9–11]. However, few have investigated the effects of AS on the peripheral system, in particu-
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lar, its toxicity and side effects. Any medicine is used on the premise of safety or within the controllable range of toxicity. Therefore, understanding the potential interventions of AS on the peripheral system is crucial for its safe use and therapeutic potential exploitation. Metabonomics is a well-established method in systems biology, but it is still emerging for the study of herbal intervention [4, 12, 13]. Metabonomics provide a powerful platform for monitoring multiple endogenous metabolite levels simultaneously in the samples from cell lines [14], tissues [4], and body fluids [13, 15]. The change in the expression levels of endogenous metabolites can be used to characterize the therapeutic effects of herbal medicine [4] and assess their toxicity [13]. In this study, UHPLCQTOF‑MS coupled with pattern recognition methods was used to investigate the potential interventions of AS on the peripheral system in rats. An integrative analysis was used to identify the possible pathways and networks influenced by AS. From this research, we may discover the potential therapeutic or untoward effects of AS, which may be a practical guide for its safe use and beneficial to the expansion of its application scope.
Results !
Our previous study has revealed that the effects of AS on the CNS had opposing consequences. It could not only provide neuroprotection but also produce some potential neurotoxicity [12]. This study mainly focuses on its effects on the peripheral system in the same treatment cycle and dosage. In addition, the general status of the animals during the treatment period was also observed in this study. Their food and water intake was normal, and their activity levels did not appear to be changed. No obvious health problems were encountered during the experiment. High reproducibility is crucial for any analytical protocols, especially for a metabonomic study that requires handling many samples. Reproducibility of the chromatography and MS was determined from five replicated analyses of the QC sample [4, 12]. The relative standard deviations of retention time and peak area of the QC sample were below 0.45% and 2.8 %, respectively. These results demonstrated the excellent stability and reproducibility of chromatographic separation and mass measurement during the whole sequence. The representative BPI chromatograms of tissues and serum samples from the control group and AS-treated groups are shown in " Fig. 1. Low molecular mass metabolites were well separated in l 12 min because of the small particles (less than 1.7 µm) of the UHPLC column. PCA was firstly carried out to investigate whether two groups can be separated and to find out their metabolic distinction. Then, OPLS‑DA was used to sharpen an already established (weak) separation between groups in PCA, separate samples into two blocks, and improve potential target discovery efforts. Score plots from OPLS‑DA showed an obvious separation between the control and AS-treated groups, which suggested that biochemical perturbation occurred significantly in AS-treat" Fig. 2). Combining the results of S and VIP plots ed groups (l " Fig. 3), the UHPLC/MS analysis platform from the OPLS‑DA (l provided the retention time, exact molecular mass, and MS/MS data for the structural identification of potential intervention targets. The analysis of control and AS-treated samples with MetaboAnalyst revealed differences between the two groups. Heatmap visualization of metabonomic data showed distinct segregation " Fig. 4). Finally, using VIP between control and AS-treated rats (l
values, 21 variables in heart tissue, 20 in liver tissue, 14 in spleen tissue, 17 in lung tissue, 16 in kidney tissue, and 12 in a serum sample were tentatively identified based on the metabolite identification strategy (Table 1S, Supporting Information). In order to illustrate how a given LC‑MS feature has been annotated, the procedure used for the identification of the ion [M + H]+ = 162.1163 at Rt = 0.38 is presented below. The assistant software available in the MassLynx i-FIT algorithm was used to determine the elemental composition for the ion. Using a mass tolerance of 5 mDa, C7H15NO3 was identified as the candidate molecular formula because of its high mass accuracy and low i-Fit value among the possible chemical formulas. The degree of unsaturation was calculated as 1, indicating that it might contain a double bond or a ring. The main fragment ions analyzed by MS/MS screening were m/z 104.0831, 103.0687, 102.1078, and 85.0500, which could correspond to a loss of -C2H3NO, -C3H7O, -CHNO2, and -C3H9O2, respectively. Finally, it was tentatively identified as L-carnitine by comparing the fragmentation pattern with the mass spectrum (PR100159) in the MassBank database. Metabolite identification confidence levels based on the criteria proposed by Creek et al. [16] are shown in Table 1S, Supporting Information. All of the potential intervention targets identified are also shown in this table. " Fig. 5) of By using an online database (HMDB) for classification (l the endogenous metabolites in these tissues and serum sample, 14–42 % were subgrouped as amino acids, peptides, and analogues, 25–65% belonged to lipids, and 6–14% were nucleosides, nucleotides, and analogues. From the “bio-function” distribution, the regulated metabolites mainly participated in the biological processes of energy metabolism, cell signaling, and membrane integrity/stability. In addition, these metabolites were primarily located in the extra cell or matrix, membrane, cytoplasm, and endoplasmic reticulum. Further analysis of pathways and networks influenced by AS was performed with MetaboAnalyst and Cytoscape software " Fig. 6). This analysis provided us additional valuable clues (l about the complex interactive link of the various identified metabolites for their commonly known interactive metabolite networks and for other cellular metabolic information as well. Metabolic pathway analysis revealed that the potential intervention targets in these tissues and serum sample are primarily responsible for sphingolipid metabolism, glycerophospholipid metabolism, amino acid degradation pathways, purine metabolism, etc.
Discussion !
To our knowledge, this is the first study using high-resolution UHPLC-QTOF‑MS to discover the potential intervention targets of AS and investigate their roles in the peripheral system of rats. The existence of interactions between drugs and metabolites suggests a potential way to discover drug targets. A promising approach in drug target discovery involves the integration of available metabolite data through mathematical modeling and data mining. The therapeutic potential of AS for the treatment of various CNS disorders has been discussed frequently [2–8], but the investigation of its effects on the peripheral system is challenging. Here, we provided a novel interesting insight into the molecular and cellular mechanisms of herbal medicine by disclosing the effects of AS on the metabolome changes upon stimulation of the peripheral system in rats. In the following sections, we
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Fig. 1 Based peak intensity chromatograms obtained from the positive ion UHPLC/MS analyses of heart, liver, spleen, lung, and kidney tissues, and serum sample from sampling on the 21st day. (Color figure available online only.)
briefly discuss some aspects of the identified metabolites that were found to be altered upon AS stimulation. In this study, AS enhanced the expression of S-adenosylmethionine in the heart, which may help to maintain DNA methylation in this tissue. DNA methylation is an essential epigenetic modification during mammalian development [17]. Maternal hypomethylation seems to be associated with offspring having congenital heart disease and Downʼs syndrome [18]. S-adenosylmethionine, the principal methyl donor in cytosine methylation, regulates the methylome dynamics during the process above [17].
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It has been observed that the L-carnitine level went down and the isobutyryl-L-carnitine expression was enhanced in the heart tissue. L-carnitine is an amino acid derivative, synthesized primarily from the amino acids lysine and methionine. It plays a critical role in fatty acid transport into the mitochondria and inhibits free radical generation, preventing the impairment of fatty acid betaoxidation in the mitochondria and protecting tissues from damage by repairing oxidized membrane lipids [19, 20]. The previous studies have shown that decreased myocardial L-carnitine levels may be related to the development of myocardial dysfunction and to chronic heart failure [20, 21]. In addition, patients with a
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OPLS‑DA score plots based on the metabolic profiling of heart, liver, spleen, lung, and kidney tissues, and serum sample. (Color figure available online
disturbed fatty acid oxidation or branched chain amino acid catabolism will accumulate abnormal acyl-CoA species, eventually leading to the accumulation of related unusual acylcarnitines [22]. Isobutyryl-L-carnitine, as a member of acylcarnitines family, is a product of the acyl-CoA dehydrogenases and associated with
VLCADD [22]. Patients with VLCADD who survive their initial presentation exhibit progressive cardiomyopathy and have a reported 75 % mortality rate in the first few years of life [23]. These results may suggest that we should pay more attention to the application of AS in patients with the abovementioned diseases.
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Fig. 3 Combination of S and VIP score plots constructed from OPLS‑DA of heart, liver, spleen, lung, and kidney tissues, and serum sample. The upregulations of ions by AS are present on the right hand quadrant, and the down-
In liver tissue, the levels of creatine and N-methylnicotinamide were upregulated by AS. Creatine is a naturally occurring metabolite responsible for maintaining ATP levels in tissues with high and rapidly fluctuating energy demands. One previous study has shown that an increase in the levels of creatine may prevent
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regulations of ions by AS are shown in the left hand quadrant. (Color figure available online only.)
NAFLD by stimulating fatty acid oxidation, which may be a potential and novel therapy for NAFLD in humans [24]. N-Methylnicotinamide was also upregulated by AS in liver tissue, which can mediate the efflux of cationic drugs to reduce their corresponding toxicity via regulating the activity of multidrug and toxin extru-
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Fig. 4 Heatmaps visualization for the heart, liver, spleen, lung, and kidney tissues, and serum sample. The heatmaps were constructed based on the potential intervention targets. Rows: samples; columns: metabolites. Color key indicates metabolite expression value, blue: lowest; red: highest. (Color figure available online only.)
sion (MATE1 and MATE2-K) [25]. In addition, the levels of two potentially toxic metabolites, 3-methylcrotonylglycine and glycocholic acid, were reduced by AS. The accumulation of 3-methylcrotonylglycine can induce oxidative damage by provoking lipid and protein oxidation [26]. Glycocholic acid is a sensitive indicator of hepatobiliary disease, whose upregulation is related to hepatocellular necrosis, edema of the portal tracts, and disruption of the limiting plate and parenchymal fibrosis [27]. Thus, we can observe that AS may produce the protective effects on liver tissue. The expressions of creatine and S-(2-carboxypropyl)-cysteamine were enhanced in spleen tissue. Creatine is the most popular supplement proposed to be an ergogenic aid as well, which increases lean body mass, muscular strength, and sprint power, and has the potential to act as a direct antioxidant against aque-
ous radical and reactive species ions [28]. Additionally, S-(2-carboxypropyl)-cysteamine can enhance the activity of Factor VIIEast Hartford, which is essential for procoagulant function [29]. From the results of this study, we observe that AS may exert the protection of spleen tissue through increasing the levels of these two metabolites. However, the upregulation of epinephrine by AS may produce potential toxicity to the spleen. The tissue is a dynamic reservoir of large platelets and retains one-third of body platelets in an exchangeable pool, which can be released in systemic circulation by epinephrine stimulation [30]. The large platelets are recognized as a strong predictor of impaired angiographic reperfusion and venous thromboembolism, whose upregulation is associated with larger myocardial infarction and increased development of
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Fig. 5 Pie charts depicting the classification of potential intervention targets of A. senticosus in heart, liver, spleen, lung, and kidney tissues, and serum sample. Representation of metabolites in terms of chemical taxonomy,
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bio-function, and cellular locations were based on the annotations of HMDB. (Color figure available online only.)
Fig. 6 Metabolic pathway analysis of potential intervention targets of A. senticosus. Ellipse nodes represent pathways, round rectangle nodes represent metabolites (red: upregulation, green: downregulation), and diamond nodes represent heart (pink), liver (green), spleen (blue), lung (yellow), and kidney (orange) tissues, and serum (purple) sample. Black edges represent
left ventricular failure or mural thrombus [30]. Therefore, interventions of AS on the spleen also need more attention. The upregulations of sphinganine, phytosphingosine, and dihydroceramide by AS may be harmful to lung tissue. These sphingolipid metabolites are generated intracellularly in response to tumor necrosis factor-α, which significantly increases lung permeability [31]. They might also participate in pulmonary edema and alveolar injury by altering a proteolipid complex that is essential for maintaining alveolar stability [31]. Interestingly, this potential toxicity in physiology may, in turn, contribute to the treatment of lung cancer, as shown in previous studies [32, 33]. Since this metabolite has cancer promoting effects [34], the inhibition of sumatriptan by AS is also beneficial for the exertion of this therapeutic potential. NO deficiency mediates oxidative stress in the kidney and is involved in the development of hypertension [35]. In the present study, AS increased the levels of citrulline in this tissue, which has antioxidant activity and acts as an NO precursor to increase NO production [35–37]. The upregulation of tetrahydropteridine by AS may enhance the function of citrulline, which is involved in oxidative stress as a regulator of NO synthase or as a direct radical scavenger [38]. The downregulation of epinephrine may also contribute to the therapeutic effects of AS on hypertension, which stimulates both the α- and β-adrenergic systems to cause vasoconstriction [39, 40]. In addition, cyclic AMP plays a central role in the regulation of both renin secretion and synthesis during kidney development, the distribution of which during this process is associated with branching of the renal arterioles [41].
the relationship between metabolites and pathways, red edges represent the possible relationship between metabolites and metabolites, and dashed edges describe the existence of metabolites in heart (pink), liver (green), spleen (blue), lung (yellow), and kidney (orange) tissues, and serum (purple) sample. (Color figure available online only.)
Thus, it can be seen that the effects of AS on this tissue is also worthy of further investigation. Pantetheine is hydrolyzed by pantetheinase into pantothenic acid and cysteamine to produce potent antioxidant activity [42]. Its upregulation may be conducive to the protection of AS on the circulatory system. In the serum sample, AS inhibited the expression of 11, 12, 15-THETA, which is upregulated in hypoxia, hypercholesterolemia, atherosclerosis, and anemia, etc., and, in turn, causes membrane hyperpolarization and relaxation to protect the circulatory system [43]. This may indicate that AS should be used cautiously in patients with the abovementioned diseases. In conclusion, the present study directly demonstrated that metabonomic analysis could be used for the investigation of potential interventions of AS on the peripheral system, which also provides an important reference basis for its safe use and therapeutic exploitation. According to our study, AS had protective effects on the peripheral system and showed potential toxicity at the same time. When using AS therapeutically, we should also pay attention to its regulatory effects on the metabolites producing toxicity. The findings were in line with our previous work done on the CNS [12]. In addition, since AS contains many bioactive ingredients [44], in-depth experimental studies are necessary to investigate the root cause of its dual effects on the peripheral system. The identification of the pharmacological actions of specific compounds would facilitate the safe application of this herb as a medicine in the clinic.
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Materials and Methods !
Chemicals and reagents HPLC grade acetonitrile was purchased from Thermo Fisher Scientific. Purified water was produced by a Milli-Q ultrapure water system (Millipore). Formic acid (HPLC grade) was purchased from Dikma Technologies). Leucine enkephalin was purchased from Sigma-Aldrich. All other reagents were of HPLC grade.
Plant material and extraction The crude drug is the root and rhizome of AS and was collected in Wuchang of Heilongjiang Province, P. R. China (N44°39′, E127° 35′; no specific permissions were required for these locations and activities; the field studies did not involve endangered or protected species). A voucher specimen (hlj-201003) of the herb was authenticated by Professor Ke Fu, Institute of Traditional Chinese Medicine, Heilongjiang University of Chinese Medicine. The preparation of extracts has been shown in our previous studies [2, 4, 12, 45]. The contents of eleutheroside B and eleutheroside E in AS were 7.63 ± 0.34 % (w/w) and 10.90 ± 0.22 % (w/w), respectively [2, 45]. The same batch of extract has also been analyzed in our previous studies [2, 12, 45], and the chromatographic profile is shown in Fig. 1S, Supporting Information.
Animals and drug administration Experimental procedures were in accordance with the Legislation on the Protection of Animals Used for Experiment Purposes (directive 86/609/EEC) and were approved by the Institutional Animal Care Committee (date of approval: September 20, 2013, approval No. 20 130 916). All efforts were made to minimize animal suffering and to reduce the number of animals used. The condition of the animals was monitored twice daily during the feeding and experiment period. Male Sprague-Dawley rats (1 year old, ~ 250 g) were obtained from the Drug Safety Evaluation Center (Heilongjiang University of Chinese Medicine) and randomly divided into control and AS-treated groups (10 each). According to our own recent study, the average daily dose of AS per rat was 31.6 mg/kg body weight [12]. The AS-treated group was orally administrated with AS once a day for 20 days, and the control group received an equal volume saline once a day for 20 days.
Sample collection and preparation All operations were carried out under sterile conditions. On the 21st day, all animals were sacrificed, and blood was collected from the abdominal aorta. Hearts, livers, spleens, lungs, and kidneys were collected immediately after blood was drawn, and the tissues were washed with saline buffer and stored at − 80 °C until used. Blood was centrifuged at 900 × g for 10 min, and the serum was collected and stored at − 80 °C. Frozen tissue samples were homogenized with 10 volumes of ice-cold methanol for 2 min in an ice bath, and thawed serum samples were vortex-mixed vigorously with 10 volumes of ice-cold methanol for 3 min. The tissue homogenate and serum mixture were centrifuged twice at 13 000 × g for 15 min at 4 °C. The supernatants were transferred into Eppendorf tubes and stored at − 80 °C for UHPLC/MS analysis.
UHPLC conditions Waters Acquity™ UHPLC (consisting of a vacuum degasser, autosampler, binary pump, photodiode array detector, and oven) was equipped with an ACQUITY UPLC® BEH C18 column (2.1 mm × 50 mm, i. d. 1.7 µm, Waters Corp). The analytical column was maintained at a temperature of 40 °C and the mobile phase was
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composed of acetonitrile (A) and water (B), each containing 0.1 % formic acid. A solvent gradient system was used: 2–100% A for 16 min. The flow rate was 0.4 mL/min and the injection volume was 2 µL. The eluent was introduced to the MS directly without a split.
MS conditions MS analysis was performed on a Q‑TOF analyzer in the SYNAPT HDMS system (Waters Corporation) in the positive ion mode, using the following parameters: capillary voltage, 1500 V; sample cone voltage, 100 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 from m/z 100–1500. All the data were acquired using an independent reference lock mass via the LockSpray™ interface to ensure accuracy and reproducibility during the MS analysis. Leucine enkephalin was used as the reference ion for the positive ion mode ([M + H]+ = 556.2771) at a concentration of 1 ng/mL under a flow rate of 30 µL/min. The data were collected in the centroid mode, and the LockSpray frequency was set at 15 s and was averaged over five scans for correction.
Multivariate data analysis The raw data were analyzed using MassLynx V4.1 and MarkerLynx software (Waters). The intensity of each ion was normalized with respect to the total ion count to generate a data matrix that consisted of the retention time, m/z value, and the normalized peak area. The multivariate data matrix was analyzed by EZinfo software (Waters). The unsupervised segregation was checked by PCA using pareto-scaled data. With OPLS‑DA, various metabolites could be identified as being responsible for the separation between the control and AS groups and were therefore viewed as differentiating metabolites. Potential intervention targets of interest were extracted from the combining S and VIP plots that were constructed from OPLS‑DA, and intervention targets were chosen based on their VIP statistics. A VIP value > 1 means that variables have above average influence on the classification. Studentʼs t-test was used for statistical analysis to evaluate the significant difference of potential intervention targets. Statistical significance was accepted if p < 0.05. The heatmap, implemented in MetaboAnalyst tool and commonly used for unsupervised clustering, was constructed based on the potential intervention targets.
Potential intervention targets identification and metabolic pathway analysis Exact molecular mass data from redundant m/z peaks corresponding to the formation of different parent and product ions were first used to help confirm the metabolite molecular mass. MS/MS data analysis highlights neutral losses or product ions, which are characteristic of metabolite groups and can serve to discriminate between database hits. The MassFragment™ application manager (Waters MassLynx v4.1) was used to facilitate the MS/MS fragment ion analysis process by way of chemically intelligent peak-matching algorithms. Databases such as HMDB (http://www.hmdb.ca/) and MassBank (http://www.massbank. jp/) were used for confirmation. Metabolic pathway analysis was performed with MetaboAnalyst Pathway Analysis (http://www. metaboanalyst.ca/Metabo-Analyst/) and Cytoscape software (version 3.1.0) based on database sources including KEGG (http://www.genome.jp/kegg/), SMPDB (http://www.smpdb.ca/
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), and HMDB to identify the affected metabolic pathways analysis and visualization.
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Supporting information The UHPLC‑TOF/MS BPI profile of A. senticosus and the metabolite identification confidence levels are available as Supporting Information.
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Acknowledgements !
This article is supported by the National Natural Science Foundation of China (81 270 056), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20 132 327 110 009), the New Century Excellent Talents Program of Higher Education of Heilongjiang Province (1254-NCET‑020), the outstanding talents cultivation fund of Heilongjiang University of Chinese Medicine (2013jc01), and the outstanding innovative talent support programs of Heilongjiang University of Chinese Medicine.
Conflict of Interest
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The authors declare that they have no conflict of interest.
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Metabonomic Study of the Effects of Acanthopanax senticosus on Peripheral System of Rats
Authors
Shuai-nan Zhang 1*, Xu-zhao Li 1*, Shu-min Liu 1, 2, Fang Lu 1
Affiliations
1 2
Key words " Acanthopanax senticosus l " Araliaceae l " metabonomic analysis l " potential intervention l targets " peripheral system l
received revised accepted
Nov. 11, 2014 March 8, 2015 March 10, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1545915 Published online April 29, 2015 Planta Med 2015; 81: 722–732 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Shu-min Liu Drug Safety Evaluation Center Heilongjiang University of Chinese Medicine He Ping Road 24 Harbin 150040 P. R. China Phone: + 86 4 51 82 19 32 78 [email protected] Correspondence Fang Lu Chinese Medicine Toxicological Laboratory Heilongjiang University of Chinese Medicine He Ping Road 24 Harbin 150040 P. R. China Phone: + 86 4 51 87 26 68 14 [email protected]
Chinese Medicine Toxicological Laboratory, Heilongjiang University of Chinese Medicine, Harbin, P. R. China Drug Safety Evaluation Center, Heilongjiang University of Chinese Medicine, Harbin, P. R. China
Abstract !
Acanthopanax senticosus is extensively used to treat various nervous and cerebrovascular diseases in traditional medicinal systems in China and Russia. Ultrahigh-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry coupled with pattern recognition methods was used to investigate the effects of A. senticosus on the peripheral system in rats. The analysis of possible pathways influenced by A. senticosus was performed with MetaboAnalyst and Cytoscape software. After treatment with A. senticosus, 21 modulated metabolites in heart tissue, 20 in liver tissue, 14 in spleen tissue, 17 in lung tissue, 16 in kidney tissue, and 12 in a serum sample were identified and considered potential biomarkers of A. senticosus treatments. The regulation of some endogenous metabolites by A. senticosus could be beneficial for the treatment of several peripheral system diseases, such as hypertension, cancer, and oxidative stress, etc. However, there were also some upregulated endogenous metabolites producing potential toxicity to the peripheral system. A metabonomic analysis revealed that protection and toxicity coexisted in the effects of A. senticosus on the peripheral sys-
Introduction !
Herbal medicines are composed of mixtures of compounds with complex pharmacological effects [1]. AS (Araliaceae) is also called “Ciwujia” in Chinese, and eleuthero or siberian ginseng in English. It is the herb used in traditional medicinal systems in China and Russia that has been primarily applied to the treatment of various nervous and cerebrovascular diseases, such as Par-
* Both of these authors contributed equally to this work.
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tem, which may be a practical guide for its safe use and beneficial to the expansion of its application.
Abbreviations !
AS:
Acanthopanax senticosus (Rupr. et Maxim.) Harms BPI: based peak intensity CNS: central nervous system NAFLD: nonalcoholic fatty liver disease NO: nitric oxide OPLS‑DA: orthogonal projection to latent structures discriminate analysis PCA: principal components analysis QC: quality control UHPLC-QTOF‑MS: ultrahigh performance liquid chromatography-quadrupole time-of-flightmass spectrometry VIP: variable importance in the projection VLCADD: very long chain acyl-CoA dehydrogenase deficiency Supporting information available online at http://www.thieme-connect.de/products
kinsonʼs disease, Alzheimerʼs disease, depression, mental fatigue, and transient global cerebral ischemia, etc. [2–8]. In the case of oral drugs acting on the CNS, both their bioactive ingredients and metabolites might be initially distributed in the peripheral system through blood circulation before reaching the site of action and produce potential effects including protection and toxicity. In addition, our previous studies have already reported metabolic processes and metabolites of some bioactive ingredients in the peripheral system [9–11]. However, few have investigated the effects of AS on the peripheral system, in particu-
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lar, its toxicity and side effects. Any medicine is used on the premise of safety or within the controllable range of toxicity. Therefore, understanding the potential interventions of AS on the peripheral system is crucial for its safe use and therapeutic potential exploitation. Metabonomics is a well-established method in systems biology, but it is still emerging for the study of herbal intervention [4, 12, 13]. Metabonomics provide a powerful platform for monitoring multiple endogenous metabolite levels simultaneously in the samples from cell lines [14], tissues [4], and body fluids [13, 15]. The change in the expression levels of endogenous metabolites can be used to characterize the therapeutic effects of herbal medicine [4] and assess their toxicity [13]. In this study, UHPLCQTOF‑MS coupled with pattern recognition methods was used to investigate the potential interventions of AS on the peripheral system in rats. An integrative analysis was used to identify the possible pathways and networks influenced by AS. From this research, we may discover the potential therapeutic or untoward effects of AS, which may be a practical guide for its safe use and beneficial to the expansion of its application scope.
Results !
Our previous study has revealed that the effects of AS on the CNS had opposing consequences. It could not only provide neuroprotection but also produce some potential neurotoxicity [12]. This study mainly focuses on its effects on the peripheral system in the same treatment cycle and dosage. In addition, the general status of the animals during the treatment period was also observed in this study. Their food and water intake was normal, and their activity levels did not appear to be changed. No obvious health problems were encountered during the experiment. High reproducibility is crucial for any analytical protocols, especially for a metabonomic study that requires handling many samples. Reproducibility of the chromatography and MS was determined from five replicated analyses of the QC sample [4, 12]. The relative standard deviations of retention time and peak area of the QC sample were below 0.45% and 2.8 %, respectively. These results demonstrated the excellent stability and reproducibility of chromatographic separation and mass measurement during the whole sequence. The representative BPI chromatograms of tissues and serum samples from the control group and AS-treated groups are shown in " Fig. 1. Low molecular mass metabolites were well separated in l 12 min because of the small particles (less than 1.7 µm) of the UHPLC column. PCA was firstly carried out to investigate whether two groups can be separated and to find out their metabolic distinction. Then, OPLS‑DA was used to sharpen an already established (weak) separation between groups in PCA, separate samples into two blocks, and improve potential target discovery efforts. Score plots from OPLS‑DA showed an obvious separation between the control and AS-treated groups, which suggested that biochemical perturbation occurred significantly in AS-treat" Fig. 2). Combining the results of S and VIP plots ed groups (l " Fig. 3), the UHPLC/MS analysis platform from the OPLS‑DA (l provided the retention time, exact molecular mass, and MS/MS data for the structural identification of potential intervention targets. The analysis of control and AS-treated samples with MetaboAnalyst revealed differences between the two groups. Heatmap visualization of metabonomic data showed distinct segregation " Fig. 4). Finally, using VIP between control and AS-treated rats (l
values, 21 variables in heart tissue, 20 in liver tissue, 14 in spleen tissue, 17 in lung tissue, 16 in kidney tissue, and 12 in a serum sample were tentatively identified based on the metabolite identification strategy (Table 1S, Supporting Information). In order to illustrate how a given LC‑MS feature has been annotated, the procedure used for the identification of the ion [M + H]+ = 162.1163 at Rt = 0.38 is presented below. The assistant software available in the MassLynx i-FIT algorithm was used to determine the elemental composition for the ion. Using a mass tolerance of 5 mDa, C7H15NO3 was identified as the candidate molecular formula because of its high mass accuracy and low i-Fit value among the possible chemical formulas. The degree of unsaturation was calculated as 1, indicating that it might contain a double bond or a ring. The main fragment ions analyzed by MS/MS screening were m/z 104.0831, 103.0687, 102.1078, and 85.0500, which could correspond to a loss of -C2H3NO, -C3H7O, -CHNO2, and -C3H9O2, respectively. Finally, it was tentatively identified as L-carnitine by comparing the fragmentation pattern with the mass spectrum (PR100159) in the MassBank database. Metabolite identification confidence levels based on the criteria proposed by Creek et al. [16] are shown in Table 1S, Supporting Information. All of the potential intervention targets identified are also shown in this table. " Fig. 5) of By using an online database (HMDB) for classification (l the endogenous metabolites in these tissues and serum sample, 14–42 % were subgrouped as amino acids, peptides, and analogues, 25–65% belonged to lipids, and 6–14% were nucleosides, nucleotides, and analogues. From the “bio-function” distribution, the regulated metabolites mainly participated in the biological processes of energy metabolism, cell signaling, and membrane integrity/stability. In addition, these metabolites were primarily located in the extra cell or matrix, membrane, cytoplasm, and endoplasmic reticulum. Further analysis of pathways and networks influenced by AS was performed with MetaboAnalyst and Cytoscape software " Fig. 6). This analysis provided us additional valuable clues (l about the complex interactive link of the various identified metabolites for their commonly known interactive metabolite networks and for other cellular metabolic information as well. Metabolic pathway analysis revealed that the potential intervention targets in these tissues and serum sample are primarily responsible for sphingolipid metabolism, glycerophospholipid metabolism, amino acid degradation pathways, purine metabolism, etc.
Discussion !
To our knowledge, this is the first study using high-resolution UHPLC-QTOF‑MS to discover the potential intervention targets of AS and investigate their roles in the peripheral system of rats. The existence of interactions between drugs and metabolites suggests a potential way to discover drug targets. A promising approach in drug target discovery involves the integration of available metabolite data through mathematical modeling and data mining. The therapeutic potential of AS for the treatment of various CNS disorders has been discussed frequently [2–8], but the investigation of its effects on the peripheral system is challenging. Here, we provided a novel interesting insight into the molecular and cellular mechanisms of herbal medicine by disclosing the effects of AS on the metabolome changes upon stimulation of the peripheral system in rats. In the following sections, we
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Fig. 1 Based peak intensity chromatograms obtained from the positive ion UHPLC/MS analyses of heart, liver, spleen, lung, and kidney tissues, and serum sample from sampling on the 21st day. (Color figure available online only.)
briefly discuss some aspects of the identified metabolites that were found to be altered upon AS stimulation. In this study, AS enhanced the expression of S-adenosylmethionine in the heart, which may help to maintain DNA methylation in this tissue. DNA methylation is an essential epigenetic modification during mammalian development [17]. Maternal hypomethylation seems to be associated with offspring having congenital heart disease and Downʼs syndrome [18]. S-adenosylmethionine, the principal methyl donor in cytosine methylation, regulates the methylome dynamics during the process above [17].
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It has been observed that the L-carnitine level went down and the isobutyryl-L-carnitine expression was enhanced in the heart tissue. L-carnitine is an amino acid derivative, synthesized primarily from the amino acids lysine and methionine. It plays a critical role in fatty acid transport into the mitochondria and inhibits free radical generation, preventing the impairment of fatty acid betaoxidation in the mitochondria and protecting tissues from damage by repairing oxidized membrane lipids [19, 20]. The previous studies have shown that decreased myocardial L-carnitine levels may be related to the development of myocardial dysfunction and to chronic heart failure [20, 21]. In addition, patients with a
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OPLS‑DA score plots based on the metabolic profiling of heart, liver, spleen, lung, and kidney tissues, and serum sample. (Color figure available online
disturbed fatty acid oxidation or branched chain amino acid catabolism will accumulate abnormal acyl-CoA species, eventually leading to the accumulation of related unusual acylcarnitines [22]. Isobutyryl-L-carnitine, as a member of acylcarnitines family, is a product of the acyl-CoA dehydrogenases and associated with
VLCADD [22]. Patients with VLCADD who survive their initial presentation exhibit progressive cardiomyopathy and have a reported 75 % mortality rate in the first few years of life [23]. These results may suggest that we should pay more attention to the application of AS in patients with the abovementioned diseases.
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Fig. 3 Combination of S and VIP score plots constructed from OPLS‑DA of heart, liver, spleen, lung, and kidney tissues, and serum sample. The upregulations of ions by AS are present on the right hand quadrant, and the down-
In liver tissue, the levels of creatine and N-methylnicotinamide were upregulated by AS. Creatine is a naturally occurring metabolite responsible for maintaining ATP levels in tissues with high and rapidly fluctuating energy demands. One previous study has shown that an increase in the levels of creatine may prevent
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regulations of ions by AS are shown in the left hand quadrant. (Color figure available online only.)
NAFLD by stimulating fatty acid oxidation, which may be a potential and novel therapy for NAFLD in humans [24]. N-Methylnicotinamide was also upregulated by AS in liver tissue, which can mediate the efflux of cationic drugs to reduce their corresponding toxicity via regulating the activity of multidrug and toxin extru-
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Fig. 4 Heatmaps visualization for the heart, liver, spleen, lung, and kidney tissues, and serum sample. The heatmaps were constructed based on the potential intervention targets. Rows: samples; columns: metabolites. Color key indicates metabolite expression value, blue: lowest; red: highest. (Color figure available online only.)
sion (MATE1 and MATE2-K) [25]. In addition, the levels of two potentially toxic metabolites, 3-methylcrotonylglycine and glycocholic acid, were reduced by AS. The accumulation of 3-methylcrotonylglycine can induce oxidative damage by provoking lipid and protein oxidation [26]. Glycocholic acid is a sensitive indicator of hepatobiliary disease, whose upregulation is related to hepatocellular necrosis, edema of the portal tracts, and disruption of the limiting plate and parenchymal fibrosis [27]. Thus, we can observe that AS may produce the protective effects on liver tissue. The expressions of creatine and S-(2-carboxypropyl)-cysteamine were enhanced in spleen tissue. Creatine is the most popular supplement proposed to be an ergogenic aid as well, which increases lean body mass, muscular strength, and sprint power, and has the potential to act as a direct antioxidant against aque-
ous radical and reactive species ions [28]. Additionally, S-(2-carboxypropyl)-cysteamine can enhance the activity of Factor VIIEast Hartford, which is essential for procoagulant function [29]. From the results of this study, we observe that AS may exert the protection of spleen tissue through increasing the levels of these two metabolites. However, the upregulation of epinephrine by AS may produce potential toxicity to the spleen. The tissue is a dynamic reservoir of large platelets and retains one-third of body platelets in an exchangeable pool, which can be released in systemic circulation by epinephrine stimulation [30]. The large platelets are recognized as a strong predictor of impaired angiographic reperfusion and venous thromboembolism, whose upregulation is associated with larger myocardial infarction and increased development of
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Fig. 5 Pie charts depicting the classification of potential intervention targets of A. senticosus in heart, liver, spleen, lung, and kidney tissues, and serum sample. Representation of metabolites in terms of chemical taxonomy,
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bio-function, and cellular locations were based on the annotations of HMDB. (Color figure available online only.)
Fig. 6 Metabolic pathway analysis of potential intervention targets of A. senticosus. Ellipse nodes represent pathways, round rectangle nodes represent metabolites (red: upregulation, green: downregulation), and diamond nodes represent heart (pink), liver (green), spleen (blue), lung (yellow), and kidney (orange) tissues, and serum (purple) sample. Black edges represent
left ventricular failure or mural thrombus [30]. Therefore, interventions of AS on the spleen also need more attention. The upregulations of sphinganine, phytosphingosine, and dihydroceramide by AS may be harmful to lung tissue. These sphingolipid metabolites are generated intracellularly in response to tumor necrosis factor-α, which significantly increases lung permeability [31]. They might also participate in pulmonary edema and alveolar injury by altering a proteolipid complex that is essential for maintaining alveolar stability [31]. Interestingly, this potential toxicity in physiology may, in turn, contribute to the treatment of lung cancer, as shown in previous studies [32, 33]. Since this metabolite has cancer promoting effects [34], the inhibition of sumatriptan by AS is also beneficial for the exertion of this therapeutic potential. NO deficiency mediates oxidative stress in the kidney and is involved in the development of hypertension [35]. In the present study, AS increased the levels of citrulline in this tissue, which has antioxidant activity and acts as an NO precursor to increase NO production [35–37]. The upregulation of tetrahydropteridine by AS may enhance the function of citrulline, which is involved in oxidative stress as a regulator of NO synthase or as a direct radical scavenger [38]. The downregulation of epinephrine may also contribute to the therapeutic effects of AS on hypertension, which stimulates both the α- and β-adrenergic systems to cause vasoconstriction [39, 40]. In addition, cyclic AMP plays a central role in the regulation of both renin secretion and synthesis during kidney development, the distribution of which during this process is associated with branching of the renal arterioles [41].
the relationship between metabolites and pathways, red edges represent the possible relationship between metabolites and metabolites, and dashed edges describe the existence of metabolites in heart (pink), liver (green), spleen (blue), lung (yellow), and kidney (orange) tissues, and serum (purple) sample. (Color figure available online only.)
Thus, it can be seen that the effects of AS on this tissue is also worthy of further investigation. Pantetheine is hydrolyzed by pantetheinase into pantothenic acid and cysteamine to produce potent antioxidant activity [42]. Its upregulation may be conducive to the protection of AS on the circulatory system. In the serum sample, AS inhibited the expression of 11, 12, 15-THETA, which is upregulated in hypoxia, hypercholesterolemia, atherosclerosis, and anemia, etc., and, in turn, causes membrane hyperpolarization and relaxation to protect the circulatory system [43]. This may indicate that AS should be used cautiously in patients with the abovementioned diseases. In conclusion, the present study directly demonstrated that metabonomic analysis could be used for the investigation of potential interventions of AS on the peripheral system, which also provides an important reference basis for its safe use and therapeutic exploitation. According to our study, AS had protective effects on the peripheral system and showed potential toxicity at the same time. When using AS therapeutically, we should also pay attention to its regulatory effects on the metabolites producing toxicity. The findings were in line with our previous work done on the CNS [12]. In addition, since AS contains many bioactive ingredients [44], in-depth experimental studies are necessary to investigate the root cause of its dual effects on the peripheral system. The identification of the pharmacological actions of specific compounds would facilitate the safe application of this herb as a medicine in the clinic.
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Materials and Methods !
Chemicals and reagents HPLC grade acetonitrile was purchased from Thermo Fisher Scientific. Purified water was produced by a Milli-Q ultrapure water system (Millipore). Formic acid (HPLC grade) was purchased from Dikma Technologies). Leucine enkephalin was purchased from Sigma-Aldrich. All other reagents were of HPLC grade.
Plant material and extraction The crude drug is the root and rhizome of AS and was collected in Wuchang of Heilongjiang Province, P. R. China (N44°39′, E127° 35′; no specific permissions were required for these locations and activities; the field studies did not involve endangered or protected species). A voucher specimen (hlj-201003) of the herb was authenticated by Professor Ke Fu, Institute of Traditional Chinese Medicine, Heilongjiang University of Chinese Medicine. The preparation of extracts has been shown in our previous studies [2, 4, 12, 45]. The contents of eleutheroside B and eleutheroside E in AS were 7.63 ± 0.34 % (w/w) and 10.90 ± 0.22 % (w/w), respectively [2, 45]. The same batch of extract has also been analyzed in our previous studies [2, 12, 45], and the chromatographic profile is shown in Fig. 1S, Supporting Information.
Animals and drug administration Experimental procedures were in accordance with the Legislation on the Protection of Animals Used for Experiment Purposes (directive 86/609/EEC) and were approved by the Institutional Animal Care Committee (date of approval: September 20, 2013, approval No. 20 130 916). All efforts were made to minimize animal suffering and to reduce the number of animals used. The condition of the animals was monitored twice daily during the feeding and experiment period. Male Sprague-Dawley rats (1 year old, ~ 250 g) were obtained from the Drug Safety Evaluation Center (Heilongjiang University of Chinese Medicine) and randomly divided into control and AS-treated groups (10 each). According to our own recent study, the average daily dose of AS per rat was 31.6 mg/kg body weight [12]. The AS-treated group was orally administrated with AS once a day for 20 days, and the control group received an equal volume saline once a day for 20 days.
Sample collection and preparation All operations were carried out under sterile conditions. On the 21st day, all animals were sacrificed, and blood was collected from the abdominal aorta. Hearts, livers, spleens, lungs, and kidneys were collected immediately after blood was drawn, and the tissues were washed with saline buffer and stored at − 80 °C until used. Blood was centrifuged at 900 × g for 10 min, and the serum was collected and stored at − 80 °C. Frozen tissue samples were homogenized with 10 volumes of ice-cold methanol for 2 min in an ice bath, and thawed serum samples were vortex-mixed vigorously with 10 volumes of ice-cold methanol for 3 min. The tissue homogenate and serum mixture were centrifuged twice at 13 000 × g for 15 min at 4 °C. The supernatants were transferred into Eppendorf tubes and stored at − 80 °C for UHPLC/MS analysis.
UHPLC conditions Waters Acquity™ UHPLC (consisting of a vacuum degasser, autosampler, binary pump, photodiode array detector, and oven) was equipped with an ACQUITY UPLC® BEH C18 column (2.1 mm × 50 mm, i. d. 1.7 µm, Waters Corp). The analytical column was maintained at a temperature of 40 °C and the mobile phase was
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composed of acetonitrile (A) and water (B), each containing 0.1 % formic acid. A solvent gradient system was used: 2–100% A for 16 min. The flow rate was 0.4 mL/min and the injection volume was 2 µL. The eluent was introduced to the MS directly without a split.
MS conditions MS analysis was performed on a Q‑TOF analyzer in the SYNAPT HDMS system (Waters Corporation) in the positive ion mode, using the following parameters: capillary voltage, 1500 V; sample cone voltage, 100 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 from m/z 100–1500. All the data were acquired using an independent reference lock mass via the LockSpray™ interface to ensure accuracy and reproducibility during the MS analysis. Leucine enkephalin was used as the reference ion for the positive ion mode ([M + H]+ = 556.2771) at a concentration of 1 ng/mL under a flow rate of 30 µL/min. The data were collected in the centroid mode, and the LockSpray frequency was set at 15 s and was averaged over five scans for correction.
Multivariate data analysis The raw data were analyzed using MassLynx V4.1 and MarkerLynx software (Waters). The intensity of each ion was normalized with respect to the total ion count to generate a data matrix that consisted of the retention time, m/z value, and the normalized peak area. The multivariate data matrix was analyzed by EZinfo software (Waters). The unsupervised segregation was checked by PCA using pareto-scaled data. With OPLS‑DA, various metabolites could be identified as being responsible for the separation between the control and AS groups and were therefore viewed as differentiating metabolites. Potential intervention targets of interest were extracted from the combining S and VIP plots that were constructed from OPLS‑DA, and intervention targets were chosen based on their VIP statistics. A VIP value > 1 means that variables have above average influence on the classification. Studentʼs t-test was used for statistical analysis to evaluate the significant difference of potential intervention targets. Statistical significance was accepted if p < 0.05. The heatmap, implemented in MetaboAnalyst tool and commonly used for unsupervised clustering, was constructed based on the potential intervention targets.
Potential intervention targets identification and metabolic pathway analysis Exact molecular mass data from redundant m/z peaks corresponding to the formation of different parent and product ions were first used to help confirm the metabolite molecular mass. MS/MS data analysis highlights neutral losses or product ions, which are characteristic of metabolite groups and can serve to discriminate between database hits. The MassFragment™ application manager (Waters MassLynx v4.1) was used to facilitate the MS/MS fragment ion analysis process by way of chemically intelligent peak-matching algorithms. Databases such as HMDB (http://www.hmdb.ca/) and MassBank (http://www.massbank. jp/) were used for confirmation. Metabolic pathway analysis was performed with MetaboAnalyst Pathway Analysis (http://www. metaboanalyst.ca/Metabo-Analyst/) and Cytoscape software (version 3.1.0) based on database sources including KEGG (http://www.genome.jp/kegg/), SMPDB (http://www.smpdb.ca/
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), and HMDB to identify the affected metabolic pathways analysis and visualization.
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Supporting information The UHPLC‑TOF/MS BPI profile of A. senticosus and the metabolite identification confidence levels are available as Supporting Information.
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Acknowledgements !
This article is supported by the National Natural Science Foundation of China (81 270 056), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20 132 327 110 009), the New Century Excellent Talents Program of Higher Education of Heilongjiang Province (1254-NCET‑020), the outstanding talents cultivation fund of Heilongjiang University of Chinese Medicine (2013jc01), and the outstanding innovative talent support programs of Heilongjiang University of Chinese Medicine.
Conflict of Interest
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The authors declare that they have no conflict of interest.
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