Metabonomics Approach to Comparing the Anti-stress Effects of Four

Subscriber access provided by Purdue University Libraries

Article

Metabonomics Approach to Comparing the Anti-stress Effects of Four Panax Ginseng Components in Rats Jingcheng Wang, Yuanlong Hou, Zhiying Jia, Xie Xie, Jiajian Liu, Yani Kang, Xin Wang, Xiaoyan Wang, and Wei Jia J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00559 • Publication Date (Web): 05 Jan 2018 Downloaded from http://pubs.acs.org on January 7, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Proteome Research

Metabonomics Approach to Comparing the Anti-stress Effects of Four Panax Ginseng Components in Rats

1†

1†

1

1

2

3

3

Jingcheng Wang , Yuanlong Hou , Zhiying Jia , Xie Xie , JiajianLiu , Yani Kang , Xin Wang , Xiaoyan Wang1*and Wei Jia*12 1

Ministry of Education Key Laboratory of Systems Biomedicine, Shanghai Center for Systems

Biomedicine, Shanghai Jiao Tong University, Shanghai, 200240, P. R. China 2

Shanghai Key Laboratory of Diabetes Mellitus and Center for Translational Medicine, Shanghai Jiao

Tong University Affiliated Sixth People’s Hospital, Shanghai, China 3

School of Biomedical Engineering, Bio-ID Center, Shanghai Jiao Tong University, Shanghai, 200240, P. R.

China

†

These authors contributed equally to this work.

*

To whom correspondence should be addressed;

Xiaoyan Wang at Ministry of Education Key Laboratory of Systems Biomedicine, Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China. Phone: 86-21-34207343;Fax: 86-21-34206059; E-mail: [email protected] Wei Jia at Ministry of Education Key Laboratory of Systems Biomedicine, Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China. Phone: 86-21-34207343;Fax: 86-21-34206059; E-mail: [email protected] 1

ACS Paragon Plus Environment

Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 25

ABSTRACT Different components of P. ginseng have different properties and medicinal effects. Metabonomics was a perspective approach to analyze global endogenous metabolites response to physiological and pathological process. In this study, untargeted-metabonomics method using GC/TOFMS combined with multivariate statistical techniques was applied to compare entire metabolite differences and the anti-stress variations among four components of P. ginseng, total ginsenosides (TG), panaxadiol (PD), panaxatriol (PT) and ginseng polysaccharide (PS), in Sprague Dawley rats. The results of metabolite analyzing showed that numerous urine metabolites involving neurotransmitters, amino acids, organic acids and gut microbiota metabolites, were changed after administration of the four components of P. ginseng, total ginsenosides (TG) had the least impact on urinary metabolites. The urinary metabolite profiling of these rats exposed to Acute combined stress (forced swimming and behavior restriction) demonstrated that the four Ginseng components attenuated urine metabolite changes involving gut microbiota metabolites, TCA cycle and energy metabolites, organic acids, to different degrees, while total ginsenosides (TG) improved most of the metabolites altered by stress.

Keywords:

Ginsenosides,

ginseng

polysaccharide,

metabonomics, rat, stress

2


panaxadiol,

panaxatriol,

Page 3 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Stress is an organism’s response to a stressor (stimulus), which leading to attack on stress hormones and related systema nervosum.1 Stressors are commonly present in our life, such as extreme temperature shift, toxins, Somatic and emotional trauma, and life pressure, which impact many physiological metabolic pathways inducing disorders and diseases, for instance, asthma, diabetes, gastrointestinal disorders,2 hypertension, depression, etc.3 Acute stress may result in multiple biological and metabolic variation, leading to rapid perturbation of the well established body system.4 Cold stress was reported to induced analgesia and opioid receptor activation in spinal cord.5, 6 Recent studies have shown that stress experiences play a crucial role in occurrence of depression.7 Panax ginseng is valuable Traditional Chinese Medicine widely distributed in many countries. Modern pharmacological investigation suggested that Panax ginseng possessed multiple activities, such as antioxidant, modulation metabolism of glucose and lipid, anti-inflammatory, and anti-tumor effects.8 Effective Panax Ginseng components include ginsenosides (panaxadiol saponins and

panaxatriol

saponins),

ginseng

polysaccharide,

polypeptide,

and

the former two were believed the main medicinal composition of Ginseng. 9-11 Our published metabonomics articles revealed that several kinds of stress produced systemic metabolites significantly variation with time.12 Furthermore, ginsenosides possessed defend action in resisting both acute and chronic stress.13, 14 This study was conducted as a subsequent research in comparison of anti-stress effects between four Ginseng components, total ginsenosides, panaxadiol, panaxatriol and Ginseng polysaccharide, in Sprague-Dawley (SD) rats. In the current study, a GC/TOFMS -based urinary metabonomic method was used to investigate the systemic responses to administration of the four Ginseng components. Furthermore the protective effects of the four Ginseng components in restoring metabolic network homeostasis were investigated and 3



compared on Acute combined stress (2h behavior restriction and forced swimming) induced rats.

MATERIALS AND METHODS Animal Handling and Sampling This study was carried out in conformity to the Chinese national legislation and local guidelines, and was operated at the Centre of Laboratory Animals, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, P. R. China. Male Sprague−Dawley (SD) rats of eight weeks old (200 ± 20 g) were bought from the Shanghai Laboratory Animal Co., moved respectively into stainless cages, given standard rat chow and tap water ad libunder the condition of 24 ± 1 °C(room temperature) and 45 ±15%(humidity). A 12 h on/off light cycle was utilized with lights beginning at 8 a.m. After two weeks of acclimation, rats were randomly grouped for further experiments. The experiment was executed in accordance with the experimental design shown in Figure 1. Rats were randomly grouped as follows (n=10): control group (C), model control group (M), total ginsenosides group (TG), panaxadiol group (PD), panaxatriol group (PT) and ginseng polysaccharide group (PS). Total ginsenosides, panaxadiol saponins, panaxatriol saponins and ginseng polysaccharide were purchased from Hunan Nuoz Biological technology Co.Ltd. Total ginsenosides, Ginseng polysaccharide, panaxadiol, panaxatriol saponins were dissolved in saline solution. Rats of TG, PD, PT and PS groups orally received TG (100 mg/kg), PD (50mg/kg), PT (50 mg/kg) and PS (200 mg/kg), respectively, while rats from the control and model group were given the same amount of saline solution once a day from day 1 to day 16. On day 15, all the rats except those in control group gain exposure to stress including Acute combined stress (Forced swimming at room temperature for 20 min and 2 h behavior restriction). Then each group's rats were put back to corresponding metabolic cages. For each rat, 24 hours urine samples were accumulated at day 0, 1, 7, 14, 16 as described in figure 1, and centrifuged at 6000 rpm for 10 min to discard precipitation. The resulting supernatants were 4


Page 4 of 25

Page 5 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


transferred into eppendorf tubes and stored at -80 °C refrigerator for further GC/TOFMS analysis. The related data was recorded in every experimental stage including the urine output and body weight of each rat.

GC/TOFMS Sample Preparation, Derivatization and Spectral Acquisition Urine samples preparation and related spectral acquisition were performed in line with previous published methods with few modiﬁcations.15 Briefly, a 10 μL urease (type C, 30 U/10 μL) was added into 50 μL aliquot of the urine supernatant and centrifuged. Two internal standards (0.3 mg/mL L-2-chlorophenylalanine in water 10 μL; 1 mg/mL heptadecanoic acid in methanol 10 μL) were spiked into the supernatant. The mixture was extracted with 200 μL methanol. An aliquot of the 200 μL supernatant was vacuum-dried up and residue was in following derivatization. Firstly, 80 μL methoxyamine, with 15 mg/mL in pyridine, was added to the residue, vortexed for 30 s, subsequently put in incubator at 30 °C for 90 min, and followed by 80 μL BSTFA (1%TMCS) for 60 min at 70 °C. Pooled samples were collected as QC to monitor batch reproducibility and the stability of GC/TOFMS，which were also treated through the above steps. The samples were run according to the order of “BLANK, C, M, TG, PT, PD, PS, C, M, BLANK, QC, BLANK”. A 1-µL aliquot of sample analysis was conducted on an Agilent 6890N gas chromatograph coupled to a Pegasus HT time-of-flight mass spectrometer (Leco Corporation, St Joseph, MI) with DB-5MS capillary column (30 m × 250 µm i.d., 0.25-µm film thickness, Agilent J&W Scientific, Folsom, U.S.A.) at a constant flow rate of 1.0 mL/min. Methoxyamine, HCl, bis (trimethylsilyl) - triﬂuoroacetamide (BSTFA, with 1% trimethyl chlorosilane, TMCS), and heptadecanoic acid were purchased from Sigma-Aldrich (St. Louis, MO). L-2-chlorophenylalanine was bought from Intechem Tech. Co. Ltd. (Shanghai, China).

Data treatment and Pattern Recognition Analysis The peak spectral from the GC/TOFMS were converted into NetCDF format by ChromaTOF software (v4.22，Leco Co.) and were directly input into online software processed by custom scripts in MATLAB (The MathWorks, Inc.). The baseline 5



correction, peak discrimination and alignment, internal standard exclusion, and normalization to the total sum of the spectral intensities with all the featured metabolites in each sample were conducted, according to previously published data pretreatment methods. The identification of metabolites was carried out by matching the mass fragments of the differential expressed metabolites with those of standard substance in commercially available databases including NIST, Wiley, NBS, and the self-established library with 70% similarity threshold. The accomplished data matrix included sample information of grouping, peak retention time, and peak area quant mass of each metabolic compound without noise and interference peaks. Then PCA (principal component analysis) and PLS-DA (partial least-squares discriminant analysis) were performed with SIMCA-p (13.0) software to identify differentially expressed compounds. When VIP (the variable importance in the projection) values were greater than 1.0, they were thought to be variables that contributed to differentiation between groups.

Univariate Statistical Analysis Besides the nonparametric test, classical one-way analysis of variance (ANOVA) was also applied to determining the statistical significance of the results. According to the thresholds on the fold change (ratio of arithmetic mean values between two groups) rank and p-values from ANOVA, the differential expressing metabolites achieved from above-mentioned multivariate statistical analyses were further tested by univariate statistical method. The threshold of p value was set to 0.05 and the threshold of fold change (FC) was set to > 1.2 or < 0.8 in this research.

RESULTS Metabolite variation induced by 4 groups of Ginseng components A total of 166 metabolite compounds were detected and indentified with information of the mass spectral databases and the standard substance library. Among these metabolites, 65 changed metabolites were associated with administration and anti-stress effects of the four ginseng components. PCA scores 6


Page 6 of 25

Page 7 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


plot was based on the data matrix of the control group and 4 Panax ginseng groups at the second week, as shown in Figure 2, metabolic states were clearly shown between the control group and 4 Panax ginseng groups at 2 week, and PLS-DA, a supervised pattern recognition method, was applied to show the metabolic distinction between control and Panax ginseng groups (Figure S1). From Table S1-4 and Figure 3 it can be seen that the impacts of all the four components on metabolites are raising the urine levels including amino acids, energy related metabolites. Metabolites associated with amino acid neurotransmitters including γ-Aminobutyrate (GABA), taurine, glutamate, valine and hypotaurine were increased in TG, PT, PD and PS groups. PD and PS have more effects on energy related metabolites while TG and PT group only affect the level of N-acetyl glucosamine. Besides, PT and PS can up-regulate the level of uracil and GABA. In addition, only PD can lead to the increase both of allantoin and urate. Finally, we can see that Phosphoenolpyruvate (PEP) had the opposite trend in PS and PD group-increased in PS group but decreased in PD group.

Metabolic variation induced by Acute combined stress and 4 Panax ginseng Components of Intervention PCA score plots by Acute combined stress generated from GC/TOFMS date was shown in Figure 4. As shown in Figure 4A, 4B, there were separations between 3M and 2M, 3M and 3C groups observed in the PCA scores plot. Thus, to characterize the major metabolites contributing to the variation, a supervised method, PLS-DA model was utilized and the scores plot in correlation with PCA model is given in Figure S2, S3. In Table S5, differential expressing metabolites were identified using VIP values and summarized. In Figure 3, metabolite differences at the different time points were shown in heatmap. As compared with Time-point 1 (administration of Ginseng components for one week), the metabolite impacts of two weeks administration were greater as seen more red and green highlights in the heatmap. Some amino acids, like L-alanine, 7



taurine, glutamate and hypotaurine, were up-regulated in samples of TG group. Most of the variation of metabolites induced by ginseng components was in the increase state. However, Acute combined stress made many metabolites go to the opposite direction as the relative contents of many metabolites, such as citrate, phenylacetate, benzoate, hippurate, were getting lower as compared to C group . As shown in Figure 4, the PCA scores plots revealed that clusters in metabolites induced by Acute combined stress in four ginseng components Intervention groups, especially in PT and TG group. The PLS-DA scores plot in correlation with PCA model is given in Figure S3. In Figure 5A, suberate, alanine and malonate increased and Benzoate declined in acute combined stress group while these metabolites all had no significant changes in four ginseng components Intervention groups with acute combined stress. What's more, 1,2-benzenediol, hippurate, malate and pantothenate had a falling trend and threitol had a rising trend in the acute combined stress group but these metabolites remained unchanged in TG, PS, PT groups after acute combined stress. Besides, 2-ketopentanedioate down-regulated and hydroxypyruvate up-regulated in acute combined stress group while these compounds stayed unchangeable in PD, TG, PT groups intervened with Acute combined stress. Additionally, there was an increase of galactosamine, citramalate and hexanoate and a decrease of β-D-galactose in M, PT or PD group but the four metabolites saw no obvious fluctuation in TG and PS group stimulated with acute combined stress, also seen in Table S5. Several differentially expressed metabolites were located in TCA cycle and the relevant pathways, as seen in Figure 5B. Almost all these metabolites changed by stress can be reversed in TG group, as none of the metabolites has both red and light blue arrows. Pyruvate related pathways seemed being solely impacted in PD group after stress including pyruvate, phosphoenolpyruvate (PEP) and glycine. These results suggested that pretreatment with TG, PT and PD may reverse metabolite variations induced by Acute combined stress, especially, in TG group, it has a better protective effect than other components of Panax ginseng. 8


Page 8 of 25

Page 9 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DISCUSSION Ginsenosides and ginseng polysaccharides are believed to be the main active constituents of Panax ginseng, responsible for decreasing oxidative stress, diminishing inflammation and antidepressant. Panaxadiol saponins contains a largest number of ginsenosides, including Rb1, Rb2, Rb3, Rc, Rd, Rh2, CK and Rg3, while Panaxatriol contains ginsenosides Rg1, Re, Rg2 and Rh1. Our previous research demonstrated that ginsenoside had prominent actions of resisting stress and external irritations.14, 16 As a follow-up investigation, we are now conducting this study to understand which kind of Ginseng's components has the main anti-stress effects in Acute combined stress rats. According to this issue, the prime objective of this research was two-fold: first, to define changed metabolites in response to the four components, total ginsenosides (TG), panaxadiol (PD), panaxatriol (PT) and ginseng polysaccharide (PS), of P. ginseng administration; second, to compare the anti-stress metabolic actions of the four components of P. ginseng that help to resist negative influences of acute combined stress on body, and discover the most effective intervention. Figure 3 and figure 5A indicated that TG markedly ameliorated Acute combined stress-induced changes, with the most black blocks, whereas PD and PT only partially reversed metabolite variations. PS possess little protective effect. Administration of Ginseng components mainly changed amino acids (neurotransmitters), energy metabolism and organic acids. Meanwhile, acute combined stress impact mainly changes the bacteria group, energy metabolism and organic acids. The main results were discussed as follows.

Variations of amino acids and neurotransmitter related metabolites induced by Ginseng components We characterized differentially expressed metabolites associated with amino acids, neurotransmitters and its metabolites detected in four administrated groups (Fgiure3, Table S1-4). First of all, inhibitory amino acids, 9



Page 10 of 25

including gamma-aminobutyric acid (GABA), taurine, alanine, valine and hypotaurine, respectively increased in various groups. Higher urinary GABA level was detected in samples of PT and PS group. Taurine, recognized as another vital inhibitory neurotransmitter with anti-anxiety properties, increased in urine in TG group.

17, 18

Alanine, was considered a potential inhibitory neurotransmitter18, and involved in lymphocyte reproduction and immunity regulation, which was significantly elevated in TG group. Xanthurenate, increased in PD group, is a metabolite from tryptophan and 5-HT catabolism19. Meanwhile, major excitatory amino acids, glutamate and aspartate, were increased in urine excretion of rats in TG group or PD group, which was in consistent with the function of ginsenosides on brain we reported previously. 14, 20

This experimental observation showed that more or less Ginseng's components

impact excretion of amino acid neurotransmitters as: PT and PS tended to effect inhibitory amino acids, PD increased excitatory amino acids, and TG had the action on both inhibitory and excitatory amino acids. Classical pharmacology research believed that Ginsenosides Rb1 (representative of PD) and Rg1 (representative of PT) had different, or even opposite effects on neurons and receptors in CNS.21, 22 Our results provide a possible reason for the difference. Other amino acids and relative metabolites were increased by different types of Ginsenosides in 14 d. It can be also found in Figure 3 that PT can increase levels of valine which is critical to response to stress, energy and muscle metabolism.23 And α-ketoisovalerate is a degradation product from valine, which elevated by PD (in Figure 3), indicating the impacts of ginsenosides in valine pathway. Pipecolate is mainly from the catabolism of dietary lysine by intestinal bacteria.24 All of them were increased in PD group. 4-Deoxyerythronate, a normal organic acid present in urine, metabolized from L-threonine, were up-regulated by acute combined stress and reversed by all the Ginseng components (Figure 5).25 It had been reported that arginine, glutamic acid, and aspartic acid was the major content of amino acids in Panax ginseng, while these amino acids were not always the significant variables in our study because of the different extraction procession of original crude herbal drugs.26 Hence the aforementioned varied amino acids just reflected the 10


Page 11 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pharmacologic effects of the four ginseng components.

Variations of metabolites in energy metabolism induced and prevented by Ginseng components Ginseng components had been recognized to be effective on metabolism of substances and energy, for example, ginsenosides and ginseng polysaccharide was proven to increase energy supplement.

27-29

Tricarboxylic acid cycle was

proven being disordered by physiological and psychological stress. The same as previous research, levels of citrate and malate in our results(Figure 5B) were deeply decreased in urine by acute combined stress.13 Nevertheless, citramalate level was elevated significantly in urine, as an analog of malate, indicating that the variation of malate level might be derived from the metabolites.30 Citrate was another TCA member decreased by stress, and both TG and PT had reversal effects on citrate and malate. However, from Figure 5B, many metabolites from outer circle pathways of TCA including metabolites from glycogenic amino acids, were found increased by stress and got improvements in four administration groups to certain degrees. Ginseng was used to prevent and treat metabolic syndrome, which might be associated with enhancing pancreatic β cell function and insulin secretion.31, 32

It can be found the level of 2-Ketohexanoic acid was increased in urine in TG

group (Figure 3). In agreement with previous findings, 2-Ketohexanoic acid is a potent insulin secretagogue, which can directly inhibits the K+ATP channel in pancreatic β cells.33 Furthermore, the rise of N-Acetyl glucosamine level was also found in TG and PT groups, whereas O-linked-beta-N-acetylglucosamine modification was reported potentially contributed to hyperglycemic-associated β cell gene expression.34 Our results suggest that TG and PT can act via beta-cell metabolism, which is different from other types Ginseng ingredients. In our results, pyruvate, D-ribose and hydroxypyruvate were up-regulated in PD group. Urate and allantoin, two products of purine metabolism in urine, were also found increasing in PD group. All of them indicated that PD might have a 11



different effect on body metabolism as compared with other three components.

Potential metabolic changes associated with gut microbiota metabolism Many metabolites, especially gut microbiota associated metabolites, were induced by acute combined stress and prevented by administrate Ginseng components (Figure 5 and Table S5). Figure 3 shows the comparison with control group, actually including two factors, Ginseng components administrations and stress. We have proven that stress could both affect endogenous and exogenous metabolism, 16 so in the discussion, we focus on results of Figure 5A. On the whole, TG is the most effective component as most black blocks beside the bright blocks of M group. It has been proposed widely that gut microbiota participate in the metabolism of aromatic amino acids. In our study, we observed that Acute combined stress down-regulated the levels of 1,2-benzenediol, benzoate and hippurate, associated with gut flora metabolism.35,

36

Taken together, such compounds are especially relevant given

evidence of microbiota disruption in severe acute stress. It was also observed that urinary excretion levels of pipecolate, 3-Ethylphenol, propionate, suberate and hydroxybutanoate, being considered to be the gut microbiota metabolites, altered in PD and (or) PS group.12, 37, 38 The result suggested that Panaxadiol Saponins and panax polysaccharide affected metabolism of intestinal flora. Our metabonomic results suggest that administrations beforehand might help to build up formidable resistibility in order that metabolites were not easily altered by stress. However, all the four components could not ameliorate the increased level of trans-2-Hexenedioic acid (a metabolite of adipic acid) by stress. Besides, administration of Ginsenosides was already proved to possess metabolic protective functions in cold-stressed rats.13 Compared with the results of our published articles, although stress ways and derivatization methods were not the same, many differentially expressed metabolites in this study, such as citrate, malate, valine, glycine, benzoate, were in the same changes, indicating the non-specificity of stress and robustness of our methods again. 12-14, 16 In conclusion, this study used a GC/TOFMS-based urinary metabonomic 12


Page 12 of 25

Page 13 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


approach to investigate the systemic responses to 4 components of P. ginseng and their anti-stress effects on Acute combined stress in rats. This global metabonomic analysis showed different impacts of administration of total ginsenosides (TG), panaxadiol (PD), panaxatriol (PT) and ginseng polysaccharide (PS) on rats, revealing the lowest metabolic influence of TG on normal rats. Furthermore, our results also indicated that perturbations of organic acids metabolism, aromatics degradation and gut microbiota homeostasis response to Acute Combined Stress, while TG possesses the best protective effect against the stress. Collectively, our results suggested that compared with other Ginseng's components, total ginsenosides possesses both the least impacts on normal urine metabolome and best protective effects of anti-stress.

SUPPORTING INFORMATION: The following files are available free of charge at ACS website http://pubs.acs.org: pr-2017-00559p-SI. Figure S1. PLS-DA sores plot based on the urine metabolic profiling of administration of PD, PS, PT, TG group in 2 weeks compared with control groups. Figure S2. PLS-DA sores plot based on the urine metabolic profiling of pre-exposure (2M) and post-exposure (3M), 3M and 3C (control group) to Acute combined stress rats in control group. Figure S3.PLS-DA sores plot based n the urine metabolic profiling of pre-exposure (2) and post-exposure (3) to Acute combined stress rats and pretreated with four administration groups. Table S1. Differential urine metabolites to be accountable for the separation between administration of TG group and Control Group after 2 weeks administration Table S2. Differential urine metabolites to be accountable for the separation between administration of PT group and Control Group after 2 weeks administration Table S3. Differential urine metabolites to be accountable for the separation between administration of PS group and Control Group after 2 weeks administration Table S4. Differential urine metabolites to be accountable for the separation between administration of PD group and Control Group after 2 weeks administration Table S5: Differential urine metabolites to be accountable for the separation between after and before Acute combined stress-induced in TG, PD, PS and PT administration group. 13



ACKNOWLEDGMENTS This work was financially supported by National Nature Science Foundation of China (30901997), Shanghai Jiao Tong University Biomedical Engineering Cross Research Foundation (YG2015MS15, YG2016MS40), Nanjing medical science and technology development program (YKK14096) and the National Basic Research Program of China (2012CB910102).

14


Page 14 of 25

Page 15 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Reference 1.

Vaccarino, V.; Bremner, J. D., Stress response and the metabolic syndrome. Cardiology 2005, 11,

(Part 2), 1. 2.

McEwen, B. S.; Stellar, E., Stress and the individual: mechanisms leading to disease. Archives of

internal medicine 1993, 153, (18), 2093-2101. 3.

Teague, C. R.; Dhabhar, F. S.; Barton, R. H.; Beckwith-Hall, B.; Powell, J.; Cobain, M.; Singer, B.;

McEwen, B. S.; Lindon, J. C.; Nicholson, J. K., Metabonomic Studies on the Physiological Effects of Acute and Chronic Psychological Stress in Sprague− Dawley Rats. Journal of proteome research 2007, 6, (6), 2080-2093. 4.

Pacak, K.; Palkovits, M.; Kopin, I. J.; Goldstein, D. S., Stress-induced norepinephrine release in the

hypothalamic paraventricular nucleus and pituitary-adrenocortical and sympathoadrenal activity: in vivo microdialysis studies. Frontiers in neuroendocrinology 1995, 16, (2), 89-150. 5.

Killian, P.; Holmes, B. B.; Takemori, A.; Portoghese, P.; Fujimoto, J., Cold water swim stress-and

delta-2 opioid-induced analgesia are modulated by spinal gamma-aminobutyric acidA receptors. Journal of Pharmacology and Experimental Therapeutics 1995, 274, (2), 730-734. 6.

Briggs, S. L.; Rech, R. H.; Sawyer, D. C., Kappa antinociceptive activity of spiradoline in the

cold-water tail-flick assay in rats. Pharmacology Biochemistry and Behavior 1998, 60, (2), 467-472. 7.

Joca, S. R. L.; Padovan, C. M.; Guimarães, F. S., Estresse, depressão e hipocampo Stress,

depression and the hippocampus. Rev Bras Psiquiatr 2003, 25, (Supl II), 46-51. 8.

Kiefer, D.; Pantuso, T., Panax ginseng. American family physician 2003, 68, (8), 1539-1542.

9.

Jia, L.; Zhao, Y., Current evaluation of the millennium phytomedicine-ginseng (I): etymology,

pharmacognosy, phytochemistry, market and regulations. Current medicinal chemistry 2009, 16, (19), 2475-2484. 10. Jang, D. J.; Lee, M. S.; Shin, B. C.; Lee, Y. C.; Ernst, E., Red ginseng for treating erectile dysfunction: a systematic review. British journal of clinical pharmacology 2008, 66, (4), 444-450. 11. Lee, M. S.; Yang, E. J.; Kim, J.-I.; Ernst, E., Ginseng for cognitive function in Alzheimer's disease: a systematic review. Journal of Alzheimer's Disease 2009, 18, (2), 339-344. 12. Wang, X.; Zhao, T.; Qiu, Y.; Su, M.; Jiang, T.; Zhou, M.; Zhao, A.; Jia, W., Metabonomics approach to understanding acute and chronic stress in rat models. Journal of Proteome Research 2009, 8, (5), 2511-2518. 13. Wang, X.; Su, M.; Qiu, Y.; Ni, Y.; Zhao, T.; Zhou, M.; Zhao, A.; Yang, S.; Zhao, L.; Jia, W., Metabolic regulatory network alterations in response to acute cold stress and ginsenoside intervention. Journal of proteome research 2007, 6, (9), 3449-3455. 14. Wang, X.; Zeng, C.; Lin, J.; Chen, T.; Zhao, T.; Jia, Z.; Xie, X.; Qiu, Y.; Su, M.; Jiang, T., Metabonomics approach to assessing the modulatory effects of St John’s wort, ginsenosides, and clomipramine in experimental depression. Journal of proteome research 2012, 11, (12), 6223-6230. 15. Cheng, Y.; Xie, G.; Chen, T.; Qiu, Y.; Zou, X.; Zheng, M.; Tan, B.; Feng, B.; Dong, T.; He, P.; Zhao, L.; Zhao, A.; Xu, L. X.; Zhang, Y.; Jia, W., Distinct urinary metabolic profile of human colorectal cancer. J Proteome Res 2012, 11, (2), 1354-63. 16. Zhang, Z.; Wang, X.; Wang, J.; Jia, Z.; Liu, Y.; Xie, X.; Wang, C.; Jia, W., Metabonomics Approach to Assessing the Metabolism Variation and Endoexogenous Metabolic Interaction of Ginsenosides in Cold Stress Rats. Journal of proteome research 2016, 15, (6), 1842-1852. 17. Singh, K.; Trivedi, R.; Haridas, S.; Manda, K.; Khushu, S., Study of neurometabolic and behavioral 15



alterations in rodent model of mild traumatic brain injury: a pilot study. NMR in Biomedicine 2016, 29, (12), 1748-1758. 18. da Silva Francisco, E.; Guedes, R. C. A., Neonatal taurine and alanine modulate anxiety-like behavior and decelerate cortical spreading depression in rats previously suckled under different litter sizes. Amino acids 2015, 47, (11), 2437-2445. 19. Takeuchi, F.; Tsubouchi, R.; Shibata, Y., Effect of tryptophan metabolites on the activities of rat liver pyridoxal kinase and pyridoxamine 5-phosphate oxidase in vitro. The Biochemical journal 1985, 227, (2), 537-44. 20. Liu, X. J.; Li, Z. Y.; Li, Z. F.; Gao, X. X.; Zhou, Y. Z.; Sun, H. F.; Zhang, L. Z.; Guo, X. Q.; Du, G. H.; Qin, X. M., Urinary metabonomic study using a CUMS rat model of depression. Magnetic Resonance in Chemistry 2012, 50, (3), 187-192. 21. Takagi, K.; Saito, H.; Nabata, H., Pharmacological studies of Panax ginseng root: estimation of pharmacological actions of Panax ginseng root. The Japanese Journal of Pharmacology 1972, 22, (2), 245-259. 22. Li, N.; Zhou, L.; Li, W.; Liu, Y.; Wang, J.; He, P., Protective effects of ginsenosides Rg1 and Rb1 on an Alzheimer's disease mouse model: a metabolomics study. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 2015, 985, 54-61. 23. Gatt, J. M.; Nemeroff, C. B.; Schofield, P. R.; Paul, R. H.; Clark, C. R.; Gordon, E.; Williams, L. M., Early life stress combined with serotonin 3A receptor and brain-derived neurotrophic factor valine 66 to methionine genotypes impacts emotional brain and arousal correlates of risk for depression. Biological psychiatry 2010, 68, (9), 818-24. 24. Hallen, A.; Jamie, J. F.; Cooper, A. J., Lysine metabolism in mammalian brain: an update on the importance of recent discoveries. Amino Acids 2013, 45, (6), 1249-72. 25. Kassel, D.; Martin, M.; Schall, W.; Sweeley, C., Urinary metabolites of L‐threonine in type 1 diabetes determined by combined gas chromatography/chemical ionization mass spectrometry. Biological Mass Spectrometry 1986, 13, (10), 535-540. 26. Wan, J. Y.; Fan, Y.; Yu, Q. T.; Ge, Y. Z.; Yan, C. P.; Alolga, R. N.; Li, P.; Ma, Z. H.; Qi, L. W., Integrated evaluation of malonyl ginsenosides, amino acids and polysaccharides in fresh and processed ginseng. Journal of pharmaceutical and biomedical analysis 2015, 107, 89-97. 27. Xiao, H.; Tan, C.; Yang, G.; Dou, D., The effect of red ginseng and ginseng leaves on the substance and energy metabolism in hypothyroidism rats. Journal of ginseng research 2017, 41, (4), 556-565. 28. Wang, J. R.; Zhou, H.; Yi, X. Q.; Jiang, Z. H.; Liu, L., Total ginsenosides of Radix Ginseng modulates tricarboxylic acid cycle protein expression to enhance cardiac energy metabolism in ischemic rat heart tissues. Molecules 2012, 17, (11), 12746-57. 29. Li, X. T.; Chen, R.; Jin, L. M.; Chen, H. Y., Regulation on energy metabolism and protection on mitochondria of Panax ginseng polysaccharide. The American journal of Chinese medicine 2009, 37, (6), 1139-52. 30. Shaw, W.; Kassen, E.; Chaves, E., Increased urinary excretion of analogs of Krebs cycle metabolites and arabinose in two brothers with autistic features. Clinical chemistry 1995, 41, (8), 1094-1104. 31. Yin, J.; Zhang, H.; Ye, J., Traditional chinese medicine in treatment of metabolic syndrome. Endocrine, metabolic & immune disorders drug targets 2008, 8, (2), 99-111. 32. Luo, J. Z.; Kim, J. W.; Luo, L., EFFECTS OF GINSENG AND ITS FOUR PURIFED GINSENOSIDES (Rb2, Re, Rg1, Rd) ON HUMAN PANCREATIC ISLET β CELL IN VITRO. European journal pharmaceutical and medical research 2016, 3, (1), 110. 16


Page 16 of 25

Page 17 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


33. Heissig, H.; Urban, K. A.; Hastedt, K.; Zünkler, B. J.; Panten, U., Mechanism of the insulin-releasing action of α-ketoisocaproate and related α-keto acid anions. Molecular pharmacology 2005, 68, (4), 1097-1105. 34. Durning, S. P.; Flanagan-Steet, H.; Prasad, N.; Wells, L., O-Linked beta-N-acetylglucosamine (O-GlcNAc) Acts as a Glucose Sensor to Epigenetically Regulate the Insulin Gene in Pancreatic Beta Cells. The Journal of biological chemistry 2016, 291, (5), 2107-18. 35. Ou, K.; Sarnoski, P.; Schneider, K. R.; Song, K.; Khoo, C.; Gu, L., Microbial catabolism of procyanidins by human gut microbiota. Molecular nutrition & food research 2014, 58, (11), 2196-2205. 36. Zheng, S.; Yu, M.; Lu, X.; Huo, T.; Ge, L.; Yang, J.; Wu, C.; Li, F., Urinary metabonomic study on biochemical changes in chronic unpredictable mild stress model of depression. Clinica Chimica Acta 2010, 411, (3), 204-209. 37. Goudarzi, M.; Mak, T. D.; Jacobs, J. P.; Moon, B.-H.; Strawn, S. J.; Braun, J.; Brenner, D. J.; Fornace Jr, A. J.; Li, H.-H., An Integrated Multi-Omic Approach to Assess Radiation Injury on the Host-Microbiome Axis. Radiation Research 2016, 186, (3), 219-234. 38. Ying, H.; Tao, S.; Wang, J.; Ma, W.; Chen, K.; Wang, X.; Ouyang, P., Expanding metabolic pathway for de novo biosynthesis of the chiral pharmaceutical intermediate L-pipecolic acid in Escherichia coli. Microbial cell factories 2017, 16, (1), 52.

17



Legends of Figures Figure 1. Timeline of experimental design Figure 2. PCA sores plot based on the urine metabolic profiling of administration of PD, PS, PT, TG group for 2 weeks (at time point 2) compared with C (Control) group. Figure 3. Heatmap of fold change (administration group/control group) of differentially expressed metabolite contents in urine samples of M, TG, PD, PS and PT groups at time point 1, 2, 3 Figure 4. PCA sores plot of stress and anti-stress effect of ginseng components, urine metabolic profiling of A: pre and post exposure Acute Combined Stress (2M vs. 3M); B: post-exposure Acute Combined Stress groups of rats (3M vs.3C); C-F: pre and post exposure Acute Combined Stress of pretreated with 4 group components of Panax ginseng. Figure 5. Anti-stress metabolic regulation effects of the four Ginseng components, Heatmap (A) and metabolic pathways (B) of fold change (after / before stress) of differentially expressed metabolite contents in urine samples of TG, PD, PS and PT groups at time point 2 and 3

18


Page 18 of 25

Page 19 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1

19



Figure 2

20


Page 20 of 25

Page 21 of 25

Pyrocatechol 3-Ethylphenol 3-Hydroxybenzeneacetate Phenylacetate Benzoate Hippurate D-Pinitol Indoleacetate Phenylacetylglycine Propionate Suberate 4-Deoxyerythronate L-Alanine Taurine a-Ketoisovalerate Pipecolate γ-Aminobutyrate L-Aspartate L-Tyrosine Glutamate Kynurenate Hypotaurine 1-Methylhistidine N-acetyl-lysine L-Threonine Valine Xanthurenate cis-Aconitic acid Pyruvate D-Lactate D-Ribose Galactinol N-Acetyl glucosamine Phosphoenolpyruvate Citrate D-Galactose Malate Hydroxypyruvate Glycerate Maleate 2-Ethylhydracrylate Citramalate Isocaproate 5-Hydroxyhexanoate Oxalate Heptadecanoate Pantothenate Hydroxybutanoate Hexanoate Malonate Citraconate Glutarate Urate Glyceraldehyde Uracil Allantoin Ethanolamine Galactosamine Sorbitol Myoinositol N-Acetyl-5-hydroxytryptamine Putrescine D-Threitol Formamide 2-Ketohexanoate

3PS/3C

3PD /3C

3PT /3C

3M/3C 3TG/3C

2PD /2C 2PS/2C

2M/2C 2TG/2C 2PT /2C

1PS/1C

1PD /1C

1PT /1C

Figure 3. 1M/1C 1TG/1C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Gut microbiota metabolites

Amino acids

Energy metabolites

Organic acids

Others FC FC>2 1.2

Metabonomics Approach to Comparing the Anti-stress Effects of Four

Recommend Documents