Intervention Effects of Puerarin on Blood Stasis in Rats Revealed by a 1H NMR-Based Metabonomic Approach

Phytomedicine 22 (2015) 333–343

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Phytomedicine

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Intervention effects of puerarin on blood stasis in rats revealed by a 1H NMR-based metabonomic approach

Zhong Jie Zou a,∗, Zhong Hua Liu b, Meng Juan Gong a, Bin Han a, Shu Mei Wang a, Sheng Wang Liang a a School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou, People’s Republic of China b Experimental Animal Center, South China Agricultural University, Guangzhou, People’s Republic of China article info abstract

Article history: Puerarin possesses a wide spectrum of biological activities including ameliorating effects on blood stasis, but Received 25 July 2014 the definite mechanism of this effect is still not known. In this study, a 1H NMR-based plasma and urinary Revised 27 December 2014 metabonomic approach was applied to comprehensively and holistically investigate the therapeutic effects Accepted 5 January 2015 of puerarin on blood stasis and its underlying mechanisms. Puerarin was injected intraperitoneally once daily for consecutive 7 days. The blood stasis rat model was established by placing the rats in ice-cold water Keywords: during the time interval between two injections of adrenaline. With pattern recognition analysis, a clear Puerarin separation of blood stasis model group and healthy control group was achieved and puerarin pretreatment Blood stasis group was located much closer to the control group than the model group, which was consistent with results Metabonomics of hemorheology studies. 15 and 10 potential biomarkers associated with blood stasis in plasma and urine, Nuclear magnetic resonance respectively, which were mainly involved in energy metabolism, lipid and membrane metabolisms, amino acid metabolism and gut microbiota metabolism, were identified. Puerarin could prevent blood stasis through partially regulating the disturbed metabolic pathways. This work highlights that metabonomics is a valuable tool for studying the essence of blood stasis as well as evaluating the efficacy of the corresponding drug treatment. © 2015 Elsevier GmbH. All rights reserved.

Introduction pathogenesis of blood stasis is complicated, and thus further studies are necessary to characterize the precise underlying mechanisms. Puerarin, a daidzein-8-C-glucoside, is one of the key bioac- As a new omics technique, metabonomics was defined as quanti- tive constituents isolated from the root of Pueraria lobata (Willd.) tative measurement of time-related multiparametric metabolic re- Ohwi, which is well known as Gegen (Chinese name) in traditional sponse of multicellular systems to pathophysiological stimuli or Chinese medicine (TCM). As one of three major isoflavonoid com- generic modification (Nicholson et al. 1999). Recently, using ultra- pounds of Gegen, puerarin has drawn much attention for its vasodila- performance liquid chromatography coupled with mass spectrom- tory and cardioprotective activities, as well as inhibition of ischemia etry (UPLC/MS), the metabolic profiles of rats with blood stasis and reperfusion injury. Puerarin also shows antioxidative and anti- and the therapeutic effects of Xindi soft capsule (Zhao et al. 2008), inflammatory effects. Currently, the injection form of puerarin has Foshou powder (Huang et al. 2013) and Shaofu Zhuyu decoction (Su been approved by the State Food and Drug Administration in China et al. 2013) were investigated. As we all know, both nuclear magnetic for the complementary treatment of coronary heart disease, angina, resonance (NMR) spectroscopy and mass spectrometry (MS) (usu- myocardial infarction, etc. (Zhou et al. 2013). ally with a chromatographic separation step) are suitable techniques Blood stasis plays an important role in the pathogenesis and for metabonomic analysis but have different analytical strengths and development of many diseases such as angina pectoris and acute weaknesses and give complementary information. To the best of our myocardial infarction (Wang et al. 2014). Described in TCM theory knowledge, metabonomic study of blood stasis in rat model based on as a slowing or pooling of blood, it is often understood in biomedi- NMR has not been reported. cal terms in terms of hemorheological disorders (e.g., a rise in whole Previous studies revealed that puerarin could significantly de- blood and plasma viscosity) (Li et al. 2009). However, the molecular crease the whole blood viscosity, erythrocyte aggregation index, red blood cell deformation index and maximal platelet aggregation rate which increased in rats with blood stasis (Pan et al. 2003); but little ∗ Corresponding author. Tel.: +86 20 39352177; fax: +86 20 39352174. has been published on the response of rat biological systems to the E-mail address: [email protected] (Z.J. Zou). intake of puerarin. http://dx.doi.org/10.1016/j.phymed.2015.01.006 0944-7113/© 2015 Elsevier GmbH. All rights reserved. 334 Z.J. Zou et al. / Phytomedicine 22 (2015) 333–343

In this study, a NMR-based metabolic profiling approach was ex- All data were presented as mean ± standard deviation (SD). Sta- plored to characterize the metabolic signature of blood stasis in rats. tistical analysis was carried out using one-way analysis of variance The protective effects of puerarin against the metabolic alteration (ANOVA) and Dunnett’s test. The values of P < 0.05 were considered were also investigated with this strategy. The finding of metabolic statistically significant. pathways will be helpful to understand the essence of blood stasis and the underlying mechanisms of puerarin treatment. Sample preparation and NMR measurements

Materials and methods For plasma samples, 50 μl of buffer solution (0.2 M Na2HPO4 and 0.2 M NaH2PO4,pH7.4)and50μlofD2O were added to 400 μlofeach Chemicals and reagents sample. For urine samples, 200 μlofbuffersolution(0.2MNa2HPO4 and 0.2 M NaH2PO4,pH7.4)wasmixedwith400μl urine to minimize Water was produced by a Milli-Q ultra-pure water system variations in the pH of the urine samples. The samples were allowed (Millipore, Bedford, MA, USA). Puerarin injection and adrenaline hy- to stand for 20 min prior to centrifugation at 4000 rpm for 10 min at drochloride injection were purchased from Guangzhou Baiyunshan 4 °C to remove any precipitates. Aliquots of the supernatant (500 μl) Tianxin Pharmaceutical Co., Ltd. (China) and Tianjin Jinyao Amino from each sample were transferred into 5 mm NMR tubes followed by Acid Co., Ltd. (China), respectively. Other materials, unless otherwise adding 50 μlofD2O containing 0.05% (w/v) of sodium 3-trimethylsilyl stated, were obtained from Sigma–Aldrich (St. Louis, MO, USA). [2,2,3,3-d4] propionate (TSP-d4). The TSP acted as a chemical shift ref- 1 erence (δ0), and the D2O provided a lock signal. All H NMR spectra Animal handling and sample collection were randomly measured at 298 K on a Bruker AVANCE III 500 MHz spectrometer (BrukerBiospin, Rheinstetten, Germany) operating at This study was carried out in strict accordance with the recom- 500.13 MHz 1H frequency. For plasma samples, the water-suppressed mendations in the Guide for the Care and Use of Laboratory Animals Carr–Purcell–Meibom–Gill (CPMG) spin-echo pulse sequence of the National Institutes of Health. All experimental procedures were (RD-90°-(τ -180°-τ )n-ACQ) with a total spin-echo delay (2nτ )of approved by the Committee on the Ethics of Animal Experiments of 100 ms was used to attenuate broad signals from proteins and lipopro- Guangdong Pharmaceutical University. teins. Sixty-four free induction decays (FIDs) were collected into 64k A total of 18 male Sprague-Dawley (SD) rats weighing 180 ± 10 g data points over a spectral width of 10,000 Hz with a relaxation de- were commercially obtained from the Experimental Animal Center of lay of 3 s and an acquisition time of 3.28 s. For urine samples, all 1H Sun Yat-Sen University (Guangdong, China), kept in plastic cages at a NMR spectra were collected using a standard 1D nuclear overhauser barrier system and provided with a certified standard rat chow and enhancement spectroscopy (NOESY)-presaturation pulse sequence. tap water ad libitum. Room temperature and humidity were regulated Sixty-four free induction decays (FIDs) were collected into 64k data at 22 ± 2 °Cand50± 10%, respectively. A 12/12-h light-dark cycle was points. Spectra were acquired with a spectral width of 10,000 Hz and set, with lights on at 8 a.m. After 7 days of acclimation, the animals an acquisition time of 3.28 s. Relaxation delay was set at 3 s. were transferred to individual metabolic cages and randomly divided into the following three groups with six rats per group: (1) healthy Data processing and multivariate statistical analysis control group, (2) blood stasis model group, (3) puerarin pretreatment group. The pretreatment group was injected intraperitoneally The obtained spectra of plasma and urine samples were processed with puerarin at a dose of 60 mg/kg (the weight of puerarin/body with a 0.3 Hz line-broadening factor prior to Fourier transforma- weight) once daily for consecutive 7 days. The dose level in this study tion and manually corrected for phase and baseline distortions using was set according to the literature (Pan et al. 2003). The control and MestReNova 6.1 software package (Mestrelab Research S.L, Santiago model groups received once daily the same volume of vehicle as the de Compostela, Spain). The NMR spectra of plasma were referenced to puerarin pretreatment group for 7 days. Blood stasis was induced methyl resonance of lactate (δ1.33). The integration was performed following established protocol: After the last dosing on day 7, rats over δ9.0–0.5 region with the bucket width set to 0.01. The region of in model and puerarin pretreatment groups were subcutaneously in- δ4.68–5.22 was removed to eliminate the effects of imperfect water jected with adrenaline at a dose of 0.8 mg/kg of body weight. After saturation. The NMR spectra of urine were referenced to the chemical 2 h, the rats were soaked in ice-cold water (0–2 °C) for 5 min keep- shift of TSP at δ0. The spectra in the region δ9.5–0.5 were integrated ing their heads outside surface, and then re-injected with adrenaline with the bucket width set to 0.01, leaving out the region of δ4.48–5.98, (0.8 mg/kg) subcutaneously at 2 h after the ice-bath (Li et al. 2009). which contained signals from residual water and urea. All remaining Rats in the control group only received an equal volume of saline with regions of the spectra were then normalized to the total integrated subcutaneous injection. Samples of 18 h urine were collected into ice- area of the spectra to reduce any significant concentration differences. cooled vessels containing 0.5 ml of 2% sodium azide during which rats The resulting dataset was imported into SIMCA-P 12.0 software fasted and were allowed free access to water. Then, blood was col- (Umetrics, Umea, Sweden) for multivariate data analysis after mean- lected from the retro-orbital venous sinus into heparinized (20 U/ml) centering and pareto scaling. The supervised partial least-squares dis- tubes after rats were anaesthetized with diethyl ether inhalation and criminant analysis (PLS-DA) was performed to achieve the maximum plasma was separated from blood by centrifugation at 4000 rpm for separation between samples and identify differential metabolites that 10 min at 4 °C. account for the separation between groups. To avoid overfitting of PLS-DA models, a default 7-fold cross-validation method was applied, Viscosity and hematocrit determination from which values of goodness of fit (R2Y) and predictability (Q2)were computed. In addition, model validation was also performed by 200 A total of 800 μl blood or plasma was used to determine the times permutation tests. Metabolites with VIP (variable importance whole blood viscosity at shear rats of 1, 50, 100 and 200 s−1 and in the projection) values ࣙ 1.0 were considered significant in this plasma viscosity at shear rate of 50 s−1 using a cone-plate viscome- study. Meanwhile, an independent sample t-test or Mann–Whitney ter (Model LG-R-80B, Steellex Co., China) maintained at 37 °C. After U-test was further used to validate those major contributing variables centrifugation at 12,000 rpm for 10 min with a microhematocrit from the PLS-DA models using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). centrifuge (Model TGL-12B, ShangHai Anting Scientific Instrument AvalueofP < 0.05 was considered statistically significant. Only those Factory, China), hematocrit was immediately measured in a capillary metabolites that meet the two criteria are eventually considered as tube. potential biomarkers. Z.J. Zou et al. / Phytomedicine 22 (2015) 333–343 335

Table 1 Effects of puerarin on whole blood and plasma viscosity in rats with blood stasis.

Group Whole blood viscosity (mPa·s) Plasma viscosity (mPa·s)

1s−1 50 s−1 100 s−1 200 s−1 50 s−1

Control 20.54 ± 1.37∗ 4.66 ± 0.29∗ 4.03 ± 0.31∗ 3.87 ± 0.29∗ 1.23 ± 0.08∗ Model 27.19 ± 3.06 5.63 ± 0.45 5.12 ± 0.68 4.64 ± 0.37 1.59 ± 0.19 Puerarin 22.26 ± 4.38∗ 4.92 ± 0.52∗ 4.38 ± 0.21∗ 4.15 ± 0.33∗ 1.32 ± 0.11∗

Data were presented as mean ± standard deviation (n = 6). Statistical analysis was performed by one-way ANOVA followed by Dunnett’s test. ∗ Compared with model group P < 0.05.

Results Table 2 Effects of puerarin on hematocrit and erythrocyte aggregation index (EAI) in rats with blood stasis. Effects of puerarin on hemorheological parameters in rats with blood stasis Group Hematocrit (%) EAI

Control 42.1 ± 1.6∗ 4.96 ± 0.38∗ The whole blood viscosity at all shear rates and plasma viscosity Model 51.7 ± 4.5 5.51 ± 0.42 significantly increased (P < 0.05) in the blood stasis model group com- Puerarin 44.9 ± 2.7∗ 5.02 ± 0.51∗ pared to those in the control group (Table 1), and were significantly Data were presented as mean ± standard devia- decreased in the puerarin pretreatment group relative to the model tion (n = 6). group (P < 0.05). Hematocrit was significantly higher (P < 0.05) in the Statistical analysis was performed by one-way model group than in the control group (Table 2). The rats pretreated ANOVA followed by Dunnett’s test. ∗ < with puerarin showed a decreased hematocrit (P < 0.05). Erythrocyte Compared with model group P 0.05. aggregation index (EAI) was calculated according to the equation EAI = ηL/ηH,whereηL was the value of whole blood viscosity at Analysis of NMR spectroscopy −1 low shear rate of 1 s and ηH was the value of whole blood viscosity − at the relatively high shear rate of 100 s 1 (Table 2). EAI in the model Typical 1H NMR spectra of rat plasma and urine were shown in group increased remarkably (P < 0.05) in comparison with that in the Figs. 1 and 2, respectively. The detected signals were assigned based control group. The administration of puerarin resulted in a decrease on matching the acquired NMR data (i.e., chemical shifts, coupling in EAI (P < 0.05), when compared with the model group. constant and multiplicity) to the reference spectra in the Human

Fig. 1. Representative 1H NMR spectra of rat plasma. Control (A), model (B) and puerarin pretreatment (C) groups. Keys: 1, Isoleucine; 2, valine; 3, lysine; 4, alanine; 5, glutamate; = = 6, pyruvate; 7, glutamine; 8, creatine; 9, choline; 10, phosphocholine; 11, trimethylamine N-oxide; 12, VLDL/LDL –CH3; 13, VLDL/LDL –CH2–; 14, Lipid CHCH2CH ; 15, Lipid CH=CH. 336 Z.J. Zou et al. / Phytomedicine 22 (2015) 333–343

Fig. 2. Representative 1H NMR spectra of rat urine. Control (A), model (B) and puerarin pretreatment (C) groups. Keys: 1, N-acetyl glycoprotein; 2, succinate; 3, 2-oxoglutarate; 4, citrate; 5, dimethylamine; 6, taurine; 7, glycine; 8, sarcosine; 9, phenylacetylglycine; 10, hippurate.

Metabolome Database version 3.0 and previous reports (Lindon et al. (P < 0.05), 15 and 10 endogenous metabolites related to blood sta- 1999; Nicholson et al. 1995), as well as with the aid of several two- sis were identified as potential biomarkers in plasma and urine, dimensional NMR experiments including correlation spectroscopy respectively (Fig. 4 and Table 3). As compared with the control = = (COSY) and total correlation spectroscopy (TOCSY). group, VLDL/LDL –CH3, VLDL/LDL –CH2–, Lipid CHCH2CH , Lipid CH=CH and trimethylamine N-oxide were significantly decreased, while isoleucine, valine, lysine, alanine, glutamate, glutamine, pyru- Metabolic variation in rats induced by blood stasis vate, creatine, choline and phosphocholine increased in the plasma of the model group. Urine obtained from rats with blood stasis The PLS-DA scores plots constructed with NMR spectral data from contained higher level of taurine and lower levels of N-acetyl rat plasma and urine samples were utilized to depict the general glycoprotein, succinate, 2-oxoglutarate, citrate, dimethylamine, variation between control and model groups (Fig. 3A and B). The glycine, sarcosine, phenylacetylglycine and hippurate. model group was clearly separated from the control group suggesting that plasma and urine metabolic profiles of rats with blood stasis were significantly changed compared with those of healthy controls. Influence of puerarin on metabolic pattern of blood stasis rat model The model parameters were as follows: R2Y = 0.94, Q2 = 0.87 for plasma; R2Y = 0.99, Q2 = 0.98 for urine. In general, excellent mod- The PLS-DA scores plots derived from 1H NMR spectra of plasma els were obtained when values of R2Y and Q2 were above 0.8 (Xuan and urine samples in the control, model and puerarin pretreatment et al. 2011). Furthermore, the robustness of these PLS-DA classifica- groups were illustrated in Fig. 5. The modeling parameters were tion models was assessed by 200-times permutation tests. The R2 and R2Y = 0.98, Q2 = 0.90 (Fig. 5A); R2Y = 0.97, Q2 = 0.89 (Fig. 5B); Q2 values derived from the permuted data were lower than the origi- R2Y = 0.99, Q2 = 0.89 (Fig. 5C) and R2Y = 0.99, Q2 = 0.91 (Fig. 5D), nal ones and all the blue regression lines of the Q2-points intersected which indicated the model had good ability of prediction and reliabil- the vertical axis below zero, indicating the validation of these PLS-DA ity. It could be clearly observed that the puerarin pretreatment group models (Fig. 3C and D). was much closer to the control group than the model group. The results suggested that puerarin could inhibit the pathological process Identification of discriminatory metabolites associated with blood stasis of blood stasis and effectively normalize the metabolic perturbation in rats induced by blood stasis. Moreover, puerarin could significantly Selected according to the VIP values from the PLS-DA models attenuate the alterations of 13 biomarkers at different degrees (except (VIP ࣙ 1) and the P values from univariate statistical analysis valine and glutamate) in the plasma of blood stasis rats. Meanwhile, Z.J. Zou et al. / Phytomedicine 22 (2015) 333–343 337

Fig. 3. Distinct metabolic proﬁles between model and control groups. Clear separation between control group ( black squares) and model group ( red triangles) was achieved as shown in PLS-DA scores plots derived from 1H NMR spectra of plasma (A) and urine (B) samples. The corresponding validation plots (C and D) based on 200 times permutation tests demonstrated the robustness of the PLS-DA models. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

among the 10 potential biomarkers associated with blood stasis in rat ing of the underlying mechanisms of blood stasis as well as treatments urine, changes of 8 biomarkers including N-acetyl glycoprotein, suc- (Li et al. 2009; Pan et al. 2003). cinate, 2-oxoglutarate, citrate, glycine, sarcosine, phenylacetylglycine Whole blood viscosity is the reflection of intrinsic resistance of and hippurate were significantly reduced by puerarin (Fig. 4 and blood to flow in vessels. In our experiment, whole blood viscosity, Table 3). which increased significantly at all shear rates in the blood stasis model group, was decreased in the puerarin pretreatment group compared to the model group (Table 1). This indicated that blood stasis Discussion induced a hyperviscosity of blood and puerarin could reduce this induction, thereby decreasing the intrinsic resistance of blood flow and It is one of classical methods of establishing blood stasis to place ameliorating tissue perfusion. As we know, red blood cells (RBCs), the rats in ice-cold water during the time interval between two injec- platelets and plasma influence whole blood viscosity. RBCs are one tions of adrenaline. As an acute-stress, injecting adrenaline could proof important factors affecting whole blood viscosity, since RBCs ac- duce the hemorheological disorders in various forms, such as blood count for almost 50% of blood volume and constitute the majority hypercoagulability and a rise of whole blood viscosity (Thrall and Lip of the cellular content in blood. In the present study, hematocrit 2005). Rapidly decreased skin blood flow could be detected when the (the RBC volume in blood) was significantly decreased in puerarin subjects were exposed to ice-cold water (Shibahara et al. 1996). These pretreatment group compared to the model group, suggesting that data suggested that injection of adrenaline combined with exposure the amelioration effect of puerarin on whole blood viscosity might to ice-cold water might induce blood stasis, resulting in hemorheo- be partly due to the hematocrit decrease (Table 2). The results in logical abnormalities (Tables 1 and 2). This animal model mimicked Table 2 also indicated that puerarin could efficiently lower the ery- the pathological state of blood stasis to some extent in patients and throcyte aggregation index. Whole blood viscosity at a low shear contributed greatly to important advances in the current understand- rate reflected RBC aggregation, since the fluid shear forces were low 338 Z.J. Zou et al. / Phytomedicine 22 (2015) 333–343

Fig. 4. Changes in the relative signal intensities of potential biomarkers associated with blood stasis in rat plasma (A and B) and urine (C). Date were expressed as mean ± standard deviation (n = 6). One-way ANOVA was used to determine the signiﬁcance of difference. Compared to model group, ∗∗ and ∗ represented P < 0.01 and P < 0.05, respectively.

enough to allow red cells to form rouleaux or rouleaux–rouleaux com- only the glucose aerobic oxidation but also the major pathways for plexes (Wen et al. 2000). The decrease in the erythrocyte aggregation fat and amino acid metabolisms. Therefore, our results demonstrated index suggested that the reduction in whole blood viscosity at a low the inhibition of energy production through aerobic respiration in- shear rate in rats pretreated with puerarin might be related to the duced by blood stasis (Fig. 6). Moreover, creatine/phosphocreatine inhibition of RBC aggregation. Plasma viscosity also plays an impor- (Cr/PCr) in plasma and glycine in urine were significantly up- or tant role in whole blood viscosity and our results demonstrated that down-regulated in rats with blood stasis, respectively. It is well pretreatment with puerarin in blood stasis rats was able to prevent known that the transfer of the amidino group of arginine to glycine the increase in plasma viscosity (Table 2), which suggested that the to yield l-ornithine and guanidinoacetate (GAA), which is catalyzed decrease in whole blood viscosity was mediated, at least partly, via by l-arginine:glycine amidinotransferase, represents the first of two the inhibition of an increase in plasma viscosity by puerarin. steps in the biosynthesis of Cr. GAA is then methylated at the Blood is a bodily fluid in animals that delivers necessary substances amidino group to give Cr by the action of S-adenosyl-l-methionine:N- such as nutrients and oxygen to the cells and transports metabolic guanidinoacetate methyltransferase. Cr and PCr are in rapid exchange waste products away from those same cells. Blood stasis, i.e. the devia the reversible transphosphorylation reaction catalyzed by enzyme crease of blood flow velocity, disturbs the normal blood flow, and creatine kinase (CK) (Wyss and Kaddurah-Daouk 2000). The CK/PCr/Cr this implies that oxygen transport may be inhibited in such state, system plays an essential role in the normal energy metabolism of tis- resulting in the disturbance of energy metabolism. Decreased levels sues that have high, fluctuating energy demands such as muscle and of succinate, 2-oxoglutarate and citrate in urine together with in- brain, because it acts as a buffer for the adenosine triphosphate (ATP) creased pyruvate level in plasma were observed in the model group concentration. For this reason, Cr is a popular supplement among as compared with the control group in the present study (Fig. 4 and sprinters and other athletes who rely on short bursts of energy. In ad- Table 3). Pyruvate, the product of glycolysis, represents an important dition, the presence of CK in blood plasma is indicative of tissue dam- junction point in carbohydrate catabolism under aerobic and anaer- age and is used in the diagnosis of myocardial infarction (Schlattner obic conditions. Citrate, 2-oxoglutarate and succinate are key inter- et al. 2006). As mentioned above, TCA cycle activity was suppressed mediate products of tricarboxylic acid (TCA) cycle which involves not in rats with blood stasis, and so, on the basis of our findings, it is Z.J. Zou et al. / Phytomedicine 22 (2015) 333–343 339

Table 3 Potential biomarkers associated with blood stasis in rat plasma and urine.

Metabolite Chemical shift (ppm)a VIPb FCc Controld Puerarind

Plasma ∗∗ ∗ VLDL/LDL –CH3 0.87(m) 5.07 0.76 ↑ ↑ ∗∗ ∗∗ VLDL/LDL –CH2– 1.28(m) 7.83 0.49 ↑ ↑ = = ∗∗ ∗∗ Lipid CHCH2CH 2.75(m) 1.55 0.74 ↑ ↑ Lipid CH=CH 5.30(m) 2.72 0.65 ↑∗∗ ↑∗∗ Isoleucine 0.94(t), 1.01(d), 1.96(m), 3.67(d) 1.30 1.35 ↓∗∗ ↓∗∗ Valine 0.99(d), 1.04(d) 1.61 1.21 ↓∗ – Lysine 1.47(m), 1.90(m), 3.02(m) 1.21 1.39 ↓∗∗ ↓∗ Alanine 1.48(d), 3.77(q) 2.22 1.46 ↓∗∗ ↓∗∗ Glutamate 2.08(m), 2.35(m), 3.75(m) 1.09 1.20 ↓∗∗ – Glutamine 2.45(m), 3.77(m) 2.72 1.39 ↓∗∗ ↓∗ Pyruvate 2.37(s) 1.56 1.96 ↓∗∗ ↓∗ Creatine/phosphocreatine 3.04(s), 3.93(s) 2.23 1.94 ↓∗∗ ↓∗ Choline 3.20(s), 3.51(t), 4.05(t) 1.96 1.27 ↓∗∗ ↓∗∗ Phosphocholine 3.22(s), 3.61(t), 4.21(t) 2.89 1.37 ↓∗∗ ↓∗ Trimethylamine N-oxide 3.27(s) 2.21 0.83 ↑∗ ↑∗ Urine N-acetyl glycoprotein 2.02(s) 2.36 0.48 ↑∗∗ ↑∗ Succinate 2.39(s) 3.73 0.28 ↑∗∗ ↑∗∗ 2-Oxoglutarate 2.43(t), 3.00(t) 2.78 0.31 ↑∗∗ ↑∗∗ Citrate 2.52(d), 2.65(d) 3.49 0.27 ↑∗∗ ↑∗ Dimethylamine 2.71(s) 1.03 0.71 ↑∗ – Taurine 3.23(t), 3.39(t) 2.63 1.85 ↓∗∗ – Glycine 3.56(s) 1.22 0.79 ↑∗ ↑∗ Sarcosine 3.58(s) 1.01 0.74 ↑∗ ↑∗ Phenylacetylglycine 3.68(s), 3.75(d), 7.35(m), 7.46(t) 1.77 0.76 ↑∗ ↑∗ Hippurate 3.97(d), 7.55(t), 7.68(t), 7.86(d) 1.92 0.35 ↑∗∗ ↑∗∗

a Letters in parentheses indicate the peak multiplicities: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. b VIP was obtained from PLS-DA model (Fig. 3). c Fold change (FC) was calculated as the ratio of the mean metabolite levels between model and control groups. FC with a value >1 indicates a relatively higher concentration while a value <1 means a relatively lower concentration present in model group as compared to the controls. d Compared to model group: ↑ indicates relative increase in signal while ↓ indicates relative decrease in signal. ∗∗ and ∗ represent P < 0.01 and P < 0.05, respectively, whereas – denotes no statistically signiﬁcant difference.

conceivable to suggest that synthesis of Cr was accelerated and Meanwhile, the metabolite profiling of plasma showed that three CK/PCr/Cr system provided an alternative energy source (Fig. 6). essential amino acid including isoleucine, valine and lysine increased The up-regulation of succinate, 2-oxoglutarate, citrate and glycine significantly in model rats (Fig. 4 and Table 3), indicating blood sta- as well as down-regulation of pyruvate and Cr/PCr was present in sis induced amino acid metabolism disturbance. Protein damage by the puerarin pretreatment group compared with those in the model oxidation is implicated in protein misfolding. Misfolded proteins are group, indicating that puerarin was able to efficaciously ameliorate degraded by proteasome to ensure the high quality of intracellular the altered energy metabolism (Fig. 4 and Table 3). proteins (Goldberg 2003). The elevated levels of amino acids sug- In this study, blood stasis caused perturbation of lipid metabolism gested protein degradation induced by oxidative stress. Amino acids as evidenced by the significantly reduced levels of lipid signals in NMR are quantitatively the most important source of ammonia, which has spectra of plasma of the model group, which was consistent with a a direct neurotoxic effect on the central nervous system (CNS) and previous study (Wang et al. 2011). Puerarin could greatly inhibit the must be transported to the liver for its eventual conversion to urea decrease of these metabolites, suggesting its protective action on lipid or to the kidney where it can be used in the excretion of protons. metabolism (Fig. 4 and Table 3). Although ammonia is constantly produced in the tissues, it is present Reduced activity of superoxide dismutase (SOD) and raised level at very low levels in blood. This is due both to the rapid removal of of malondialdehyde (MDA) (Dong et al. 1996) in serum demonstrated blood ammonia by the liver, and the fact that many tissues, particu- oxidative stress might be one of the most important pathogenesis larly muscle, release amino acid nitrogen in the form of glutamine or of blood stasis. Oxidative stress was able to produce cellular mem- alanine, rather than as free ammonia. The ATP-requiring formation brane lipid peroxidation, lipid–protein interaction alteration, enzyme of glutamine from glutamate and ammonia by glutamine synthetase inactivation and DNA breakage, and in the end, to cause cell injury, occurs primarily in the muscle and liver, but is also important in the apoptosis or necrosis (Liu et al. 2010). The increased levels of choline CNS where it is the major mechanism for the removal of ammonia containing metabolites, choline and phosphocholine, were detected in the brain. Glutamine, the amide of glutamate, provides a nontoxic in plasma of the blood stasis model group (Fig. 4 and Table 3). Choline storage and transport form of ammonia. In tissues where glycoly- is a constituent of cell membranes and lipoprotein phospholipids, and sis is active (e.g. muscle), the glucose–alanine cycle is primarily used plays an important role in the integrity of cell membranes and lipid to remove toxic ammonia. Alanine, the α-amino acid analog of the metabolism (Zeisel 1981). The increased choline levels have been as- α-keto acid pyruvate, is most commonly produced by the reductive sociated with drug induced disruption of cellular membrane (Griffin amination of pyruvate via alanine transaminase. This reversible re- et al. 2001). In the current study, the elevation of choline and phospho- action involves the interconversion of alanine and pyruvate, coupled choline was likely caused by oxidative damage to cellular membrane to the interconversion of α-ketoglutarate and glutamate (Felig 1973). structure. Thus, the altered levels of glutamine, pyruvate, alanine and glutamate 340 Z.J. Zou et al. / Phytomedicine 22 (2015) 333–343

Fig. 5. Visualization of intervention effects of puerarin on blood stasis in rats. The PLS-DA scores plots generated from 1H NMR spectra of plasma (A and C) and urine (B and D) samples showed that puerarin pretreatment group (• green dots) was completely separated from model group ( red triangles) and located much closer to control group ( black squares) than model group. This indicated that administration of puerarin could effectively attenuate the metabolic perturbation in rats induced by blood stasis. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

(Fig. 4 and Table 3) in the model group might reflect disorders of attenuated the alterations of choline, TMAO, PAG and hippurate, re- glucose–alanine cycle and formation of glutamine, and could be used flecting its protective action on gut microbiota metabolism. as an index of disturbance in amino acid metabolism caused by blood In addition, the metabolic pathway analysis (MetPA) with Metabo- stasis. Furthermore, taurine is an antioxidant and an organic osmolyte Analyst (www.metaboanalyst.ca/MetaboAnalyst) was applied to ex- capable of protecting and stabilizing cells (Waterfield et al. 1991). The plore the most relevant pathways involved in blood stasis. Based on increase in urinary taurine (Fig. 4 and Table 3) might be attributed to the impact value greater than 0.1, seven disturbed metabolic path- an intrinsic self-defense against the oxidative damage triggered by ways including taurine and hypotaurine metabolism, glycine, serine blood stasis. and threonine metabolism, valine, leucine and isoleucine biosynthe- Puerarin could restore the increased levels of choline, phospho- sis, glyoxylate and dicarboxylate metabolism, TCA cycle, alanine, as- choline, isoleucine, lysine, glutamine, alanine and pyruvate, mani- partate and glutamate metabolism and pyruvate metabolism were festing its ability to rectify the disturbance of membrane and amino revealed (Fig. 7 and Table 4). And these pathways might denote their acid metabolisms mainly induced by oxidative stress. And this was in potential as the targeted pathways of puerarin against blood stasis. good agreement with the antioxidant activity of puerarin (Zhou et al. In conclusion, a metabonomic approach based on NMR tech- 2013). nique was developed to investigate the specific physiopathologic The elevated choline level in plasma of the model group was state of blood stasis in rats and to reveal the intervention mecha- accompanied by a decrease in trimethylamine N-oxide (TMAO) in nisms of puerarin. 15 and 10 potential biomarkers associated with plasma and dimethylamine (DMA) in urine (Fig. 4 and Table 3). It is blood stasis in plasma and urine, respectively, primarily involved in well known that breakdown of choline by gut microbiota leads to energy metabolism, lipid and membrane metabolisms, amino acid the formation of trimethylamine (TMA), which is subsequently ei- metabolism and gut microbiota metabolism, were identified. These ther oxidized into TMAO via the flavine monooxygenase system or potential metabolites appeared to have diagnostic and/or prognos- decomposed to DMA prior to excretion (Smith et al. 1994). So, it was tic values for blood stasis, which deserved to be further investigated. plausible to expect that blood stasis induced a disturbance to gut Consistent with results of hemorheology studies, puerarin could re- microbiotal colonies in rats (Fig. 6). Furthermore, the precursors of verse the pathological process of blood stasis through regulating the phenylacetylglycine (PAG) and hippurate are also produced by gut disturbed metabolic pathways. This proof-of-concept study indicated bacteria (Wei et al. 2009), thus, evidence for the possible damaged that the metabonomic strategy based on NMR was a promising tool to gut microbiota could also be supported by the observed decreased search potential biomarkers related to blood stasis and to dissect the urinary PAG and hippurate (Fig. 4 and Table 3). Puerarin effectively underlying efficacy and mechanisms of drugs in treating blood stasis. Z.J. Zou et al. / Phytomedicine 22 (2015) 333–343 341

Fig. 6. Disrupted metabolic pathways related to blood stasis. ↑ represents signiﬁcant up-regulations of metabolites in the model group compared with the control group, whereas ↓ indicates down-regulations. Metabolites observed in this study were shown in bold type.

Table 4 Results of ingenuity pathway analysis with MetPA.

Pathway name Total Hits Raw P −log(P) Impact

Taurine and hypotaurine metabolism 8 1 0.114 2.1715 0.43 Glycine, serine and threonine metabolism 32 5 7.05E-05 9.56 0.35 Valine, leucine and isoleucine biosynthesis 11 2 0.010839 4.5246 0.33 Glyoxylate and dicarboxylate metabolism 16 1 0.21556 1.5345 0.29 TCA cycle 20 4 0.000155 8.7742 0.22 Alanine, aspartate and glutamate metabolism 24 4 0.000326 8.0281 0.21 Pyruvate metabolism 22 1 0.28434 1.2576 0.19 Glycolysis or gluconeogenesis 26 1 0.32697 1.1179 0.10 Glycerophospholipid metabolism 30 2 0.072277 2.6273 0.07 Primary bile acid biosynthesis 46 2 0.14922 1.9023 0.06 Cysteine and methionine metabolism 28 1 0.34736 1.0574 0.02 Arginine and proline metabolism 44 3 0.025849 3.6555 0.01 Glutathione metabolism 26 1 0.32697 1.1179 0.01 d-Glutamine and d-glutamate metabolism 5 3 2.85E-05 10.467 0 Butanoate metabolism 20 3 0.002806 5.8761 0 Nitrogen metabolism 9 2 0.007224 4.9303 0 Aminoacyl-tRNA biosynthesis 67 4 0.015413 4.1726 0 Biotin metabolism 5 1 0.072784 2.6203 0 Cyanoamino acid metabolism 6 1 0.086722 2.4451 0 Methane metabolism 9 1 0.12735 2.0608 0 Pantothenate and CoA biosynthesis 15 1 0.2035 1.5921 0 Propanoate metabolism 20 1 0.26208 1.3391 0 Porphyrin and chlorophyll metabolism 27 1 0.33724 1.087 0 Valine, leucine and isoleucine degradation 38 1 0.44081 0.81915 0 Pyrimidine metabolism 41 1 0.46626 0.76302 0 Purine metabolism 68 1 0.65069 0.42973 0

Total is the total number of compounds in the pathway; Hits is the actually matched number from the user uploaded data; raw P is the original P value calculated from the enrichment analysis; impact is the pathway impact value calculated from pathway topology analysis. 342 Z.J. Zou et al. / Phytomedicine 22 (2015) 333–343

Fig. 7. Summary of pathway analysis with MetPA. (a) Taurine and hypotaurine metabolism; (b) glycine, serine and threonine metabolism; (c) valine, leucine and isoleucine biosynthesis; (d) glyoxylate and dicarboxylate metabolism; (e) TCA cycle; (f) alanine, aspartate and glutamate metabolism; (g) pyruvate metabolism.

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