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NaoShuanTong capsule (NSTC) has been commonly used to treat blood circulation disorders and neurological dysfunction against cerebral ischemia in China. In this study, using an acute blood stasis rat model, canonical correlation analysis between chemical high performance liquid chromatography (HPLC) fingerprint and in vivo biotests was conducted to discover and identify the bioactive compounds responsible for NSTC’s protective effects on hemorheological disorders. Four independent effect components were extracted from the 12 originally detected parameters. Three bioactive compounds were found to have significant contribution in two or more effect components against hemorheological disorders. Typhaneoside could influence the erythrocyte aggregation and coagulation function. Isorhamnetin-3-O-neohesperidoside had impacts on the erythrocyte aggregation, erythrocyte deformability and platelet aggregation. Benzoyl-oxypaeoniflorin was associated with erythrocyte aggregation, coagulation function and platelet aggregation. The findings could offer useful references for NSTC’s quality control and clinical practice.
| Abbreviations | ||
| Adr | = | adrenaline |
| ASP | = | aspirin |
| APTT | = | activated partial thromboplastin time |
| CCA | = | canonical correlation analysis |
| EAI | = | erythrocyte aggregation index |
| EDI | = | erythrocyte deformability index |
| EEI | = | erythrocyte electrophoresis index |
| HCT | = | haematokrit |
| HPLC | = | high performance liquid chromatography |
| MPAR | = | maximum platelet aggregation rate |
| NSTC | = | NaoShuanTong Capsule |
| PCA | = | principal component analysis |
| PT | = | Pollen Typhae |
| PT | = | prothrombin time |
| PV | = | plasma viscosity |
| RC | = | Radix Curcumae |
| RG | = | Rhizome Gastrodiae |
| RPR | = | Radix Paeoniae Rubra |
| RR | = | Radix Rhapontici |
| TCM | = | traditional Chinese medicine |
| WBV | = | whole blood viscosit |
Introduction
Plants have been used not only for nutritional purposes, but also to meet personal and social needs, e.g. curing diseases. Plants include a lot of phytochemicals that are important for human health [Citation1–4]. NaoShuanTong capsule (NSTC), a Chinese herbal formula (CHF) composed of Pollen Typhae (PT, pollen of Typha angustifolia L.), Radix Paeoniae Rubra (RPR, root of Paeonia Lactiflora Pall.), Rhizome Gastrodiae (RG, root of Gastrodia elata Bl.), Radix Rhapontici [RR, root of Rhaponticum unifloroum (L.) DC.], and Radix Curcumae (RC, root of Curcuma wenyujin Y.H. Chen & C. Ling), has been demonstrated to have neuroprotective effects against cerebral ischemia [Citation5]. As a medicine, NSTC is commonly used to treat blood circulation disorders and neurological dysfunction. However, what the main bioactive compounds are and how they perform the effects remain unknown until now. Fingerprint techniques have been widely used for the quality control of traditional CHF [Citation6]. Fingerprint methods can clarify the chemical compounds efficiently, but it is still difficult to discover and identify the bioactive compounds because of the disconnection with corresponding biotests. Therefore, based on the canonical correlation analysis (CCA) between chemical high performance liquid chromatography (HPLC) fingerprints and in vivo biotests, this study was, thus, designed to discover bioactive compounds in NSTC which have positive effects on hemorheological disorders.
In addition to neuronal apoptosis, hemorheology disorders also play an important role in the pathogenesis and development of stroke. Hemorheological disturbances affect >40% of ischemic stroke (IS) cases [Citation7]. The decreased cerebral blood flow has been proved to be relevant to the elevated fibrinogen concentration, plasma viscosity (PV), whole blood viscosity (WBV) and erythrocyte aggregability [Citation7]. Blood stasis, a traditional Chinese medicine (TCM) term used to reflect the blood circulation disorders, is an indicator of hemorheological abnormalities and is characterized by the decrease of blood flow velocity [Citation8]. Blood stasis has been reported as the major brain syndrome element in 324 patients with ischemic cerebral apoplexy [Citation9]. The promotion of blood circulation could improve hemorheological disorders [Citation10]; various TCM and extracts have positive effects on blood stasis syndrome [Citation11]. For NSTC, our previous work has demonstrated the protective effects on hemorheological disorders and energy metabolisms in blood stasis syndrome [Citation12]. Therefore, in this study, CCA between chemical HPLC fingerprint and in vivo biotests was further conducted to find the corresponding bioactive compounds responsible for NSTC’s positive effects.
Materials and methods
Experimental instruments and reagents
Ultimate 3000 Dual-Gradient Analytical LC System (Dionex, USA). 1200SL RRLC-6410 QQQ mass spectrometer (Agilent, USA) equipped with electrospray ionization (ESI) source. Numerical control ultrasonic cleaning machine (KQ-250DE, Kunshan Ultrasonic Instrument, China). BP211D Electronic analytical balance (Sartorius, Germany). Ultra-low temperature freezer (BCD-568W, Haier, China). Refrigerated centrifuge (5430R, Eppendorf, Germany). Platelet aggregation analyzer (LBY-NJ4, Beijing Precil, China). Full-automatic blood coagulation analyzer (STA Compact, Stago, USA). Full-automatic self-cleaning haemorheology analyzer (LBY-N6B, Beijing Precil, China). Ultimate AQ-C18 column (150 mm × 4.6 mm, 3 μm, Agilent, USA). Ultimate XB-C18 column (150 mm × 3 mm, 3 μm, Agilent, USA). Solvents were of HPLC grade. Adrenaline (Adr) Injection (Shanghai Harvest Pharmaceutical Co., Ltd., Batch No. 20150609). Aspirin (Asp) (Jilin-Luwang pharmaceutical Co., Ltd., Batch No. BTA7WH2).
Preparation of NSTC
NSTC was defined as the mixture of PT (pollen of T. angustifolia L.), RPR (root of Paeonia Lactiflora Pall.), RG (root of Gastrodia elata Bl.), RR [root of Rhaponticum unifloroum (L.) DC.], Radix Curcumae (RC, root of Curcuma wenyujin Y.H. Chen & C. Ling) in a ratio of 33:24:10:14:19. NSTC was prepared through precision processes of multiple soaking, filtering and evaporating. RC was soaked in five times the volume of ethanol (80%, v/v) for 12 h, followed by two rounds of reflux extraction, each time for 1 h, mixing extracts, filtering and gathering residue individually. RPR was refluxed with five times the volume of ethanol (70%, v/v) two times, each time for 1 h, followed by mixing extracts, filtering, recovering ethanol and concentrating to obtain soft extract. PT, RG, RR and RC residue were mixed and then soaked in 10 times the volume of warm water (80 °C) for 2 h, followed by decocting two times, each time for 1 h, mixing extracts, filtering, mixing with RC extract, recovering ethanol, concentrating to soft extract and mixing with RPR soft extract to obtain NSTC. The plant species were identified by Prof. Wen-bo Liao, Sun Yat-Sen University. The voucher specimens were deposited in our laboratory.
Preparation of nine NSTC samples based on uniform design
Based on the original herb proportion of NSTC (PT:RPR:RG:RR:RC = 33:24:10:14:19), the five factors–nine levels uniform design method () was used to adjust the proportions of the five herbs, thus to prepare nine NSTC samples with herb content differences according to the standard process above.
Table 1. Adjusted formulations of NSTC samples based on uniform design.
HPLC and RRLC/MS/MS analysis
NSTC samples were analyzed with the HPLC system (Dionex, USA), consisting of DGP-3600SD Pump, SRD-3600 Degasser, WPS-3000SL Autosampler, TCC-3000RS Column Compartment, DAD-3000 Diode Array Detector. The original data were calculated and processed with Chromeleon 6.8 Software. The HPLC methods were as follows [Citation13]: the column was Ultimate AQ-C18 (150 mm ×4.6 mm, 3 μm); the mobile phase consisted of acetonitrile (A), tetrahydrofuran (B) and 0.05% phosphoric acid (C); the linear gradient elution program was set to be 2–20% A, 0–10% B from 0–70 min; the flow rate was 1.1 mL/min with UV absorbance detection at 254 nm (before 53 min) and 275 nm (after 53 min); the injected volume was 2 mL; the temperature was set at 20 °C; the calculated theoretical plate number on the paeoniflorin peak should be equal to or more than 6000. In the RRLC/MS/MS analysis, the column was Ultimate XB-C18 (150 mm ×3 mm, 3 μm) and the other LC methods were the same as above. The LC–MS worked in a full scan mode and the mass range was set at m/z 100–1000 in both positive and negative ion modes. The conditions of the mass spectrometer were as follows [Citation14]: ion source gas1 55 psi; ion source gas2 55 psi; curtain gas 35 psi; temperature 550 °C; ion spray voltage floating 5500 V; collision energy 40 V; collision energy spread 20 V and declustering potential 80 V. Nitrogen was used as nebulizer and auxiliary gas. Typhaneoside, isorhamnetin-3-O-neohesperidoside, paeoniflorin and β-ecdysterone were identified by comparison of the retention time and UV spectra with those of authentic compounds. The 10 identified compounds [Citation13,Citation14] and their peak areas in the HPLC fingerprint of nine NSTC samples are shown in .
Table 2. Peak areas of 10 compounds in the HPLC fingerprint of NSTC samples.
Cluster analysis of nine NSTC samples
Ten compounds’ peak areas were used to conduct cluster analysis in SPSS 19.0. The cluster method was between-groups linkage, the distance calculation method was Euclidean, and the rescaled distance cluster combine was 5.
Animal treatments
One hundred and twenty Sprague Dawley male rats, specific pathogen free, 180–220 g, were purchased from Guangdong Medical Laboratory Animal Center (SCXK-(Yue) 2013-0002). The rats were kept in plastic cages at 22 ± 2 °C with relative humidity of 50–65%, and fed with standard laboratory diet and water on a 12 h light/dark cycle. The rats were acclimatized to the new environment for a week. The animal welfare and experimental procedures were in accordance with the guidelines of the U.S. Institutional Animal Care and Use Committees for (National Research Council of USA, 1996) and related ethical regulations of Sun Yat-sen University. The rats were randomly divided into 12 groups with 10 in each. Group 1 was the control: rats were given normal saline at the same volume. Group 2 was the model: rats were given normal saline at the same volume. Group 3 was model + aspirin (ASP): rats were given 100 mg/(kg/d) ASP; Groups 4–12 were groups with administration of nine NSCT samples: rats were given 1600 mg/(kg/d) for each NSTC sample. The human equivalent dosage for NSTC in clinic is 400 mg/kg. The NSTC sample dosage was translated and calculated to crude herb as 7.62 g/(kg/d) (average yield 21%, w/w, dried extracts/crude herbs). All treatments were performed by gavage once daily.
Experimental modelling
After the 10th drug treatment, the rats in Groups 2–13 were subcutaneously injected Adr (0.8 mg/kg); the rats in Group 1 were injected normal saline at the same volume. After 2 h, the rats in Groups 2–13 were kept in ice water (0–2 °C) for 5 min, and subcutaneously reinjected Adr (0.8 mg/kg) after 2 h. Then, the rats were fasted for 12 h before blood samples were collected.
Blood samples collection
Rats were anesthetized with chloral hydrate (10%, 0.35 mL/100 g). Blood was drawn from the abdominal aortas and then collected into plastic tubes with 3.8% sodium citrate (citrate/blood: 1/9, v/v). Plasma was obtained at 3820 r/min, 20 °C, 15 min. Then, 0.9 mL blood was put directly into LBY-N6B to determine the erythrocyte aggregation index (EAI), the erythrocyte electrophoresis index (EEI) and the erythrocyte deformability index (EDI), the haematokrit (HCT) and the WBV at 5, 30, 50, 150 and 200/s shear rates. Samples of 0.5 mL plasma were read in a STA Compact blood coagulation analyzer to detect the activated partial thromboplastin time (APTT), the prothrombin time (PT) and the PV. Platelet-rich plasma (PRP) was prepared (500 r/min, 20 °C, 10 min). Platelet-poor plasma (PPP) was prepared (3000 r/min, 20 °C, another 10 min). Each PRP sample was counted for platelets and was standardized (∼4 × 105/mL) by adjusting the PRP with autologous PPP. The aggregation response was measured by the changes in light transmission of the platelet suspension. Platelet aggregation was induced by the addition of adenosine diphosphate (ADP) at a final concentration of 5 μmol/L, and the changes in the light transmission were recorded. The maximum platelet aggregation rate (MPAR) was determined using LBY-NJ4. All experiments were completed within 3 h after blood collection ().
Table 3. Effects of nine NSTC samples on haemorheological parameters.
CCA between compounds and bioactivity
Principal component analysis
The 12 detected parameters were simplified and classified into four components through the principal component analysis (PCA) module in SPSS 19.0. The four components explained 96.122% of the total variance of parameters (). The rotated component matrix between parameters and components is shown in , which suggested the clinical meaning of the four components. X1 represented the erythrocyte aggregation. X2 represented erythrocyte deformability. X3 represented coagulation function. X4 represented the platelet aggregation. The component scores were calculated and regarded as new dependent variables, as shown in .
Table 4. Total variance explained by components X1–X4.
Table 5. Rotated component matrix.
Table 6. Scores of components.
Canonical correlation analysis
The 10 identified peak areas in the HPLC fingerprint were regarded as a group of independent variables. The four components’ scores were regarded as four groups of dependent variables. CCA was then conducted through the function “CANONCORR” in MATLAB 2010a. The canonical correlation coefficients (CCCs) and their Chi-square test coefficient (χ2) were calculated to evaluate the CCA model (). The established CCA model is shown in . The canonical loadings of compounds towards the effect components were calculated to assess the effect contribution (; [Citation15]).
Table 7. Chi-square test results.
Table 8. CCA model between compounds and effects.
Table 9. Corelation between parameters and components.
Statistical analysis
The data are expressed as mean values with standard deviation (±SD) and evaluated by one-way analysis of variance (ANOVA). P-values of <0.05 or 0.01 presented statistical significance.
Results and discussion
Based on our previous work, 10 compounds were identified or characterized using HPLC and RRLC-MS/MS [Citation13,Citation14]. Their peak areas are shown in . The HPLC fingerprints of NSTC samples are shown in . The cluster analysis results for the NSTC samples are shown in . Nine NSTC samples could be divided into seven categories: S4 and S5 belonged to one class, S8 and S9 belonged to another class, and the rest of the samples each represented a separate class. The results indicated that nine NSTC samples had chemical differences in the compounds.
Figure 1. HPLC fingerprints of NSTC samples.
Figure 2. Cluster analysis of NSTC samples.
The results from nine NSTC samples based on the 12 parameters are shown in . WBV reflects the intrinsic resistance of blood to flow in vessels. Abnormalities in WBV are associated with increased cardiovascular risk, and reduced blood fluidity can lead to tissue ischemia more rapidly in atherosclerotic diseases [Citation16]. Numerous studies have documented that the WBV rises in stroke patients [Citation17–19]. Coagulation function parameters including PT, APTT, PV and MPAR, which could reflect the type and concentration of the proteins and platelets in the plasma, also influence the hemorheology. WBV in the model group significantly increased at all shear rates. After treatment, WBV significantly decreased in NSTC5 at both low and high shear rates. In the NSTC1, NSTC2, NSTC4, NSTC7 and ASP groups, WBV significantly decreased at low shear rates. In the model group, EEI and EAI were significantly high, whereas EDI significantly decreased. In the NSTC3 and NSTC7 groups, EEI and EAI significantly decreased, whereas EDI significantly increased. Except for the NSTC6 group, EAI significantly decreased. EDI significantly increased only in groups NSTC3, NSTC7 and NSTC9. The model group had significantly low PT and APTT, whereas the NSTC5, NSTC7, NSTC8 and NSTC9 groups had significantly high APTT. The model group had significantly high PV and MPAR. After treatment, PV in the NSTC4 and NSTC5 groups significantly decreased. MPAR in NSTC1, NSTC4, NSTC5 and ASP groups significantly decreased. As shown in –Citation6, to simplify the effect criterion, four independent effect components were extracted from the 12 original parameters by PCA. The most important parameter was erythrocyte aggregation, which indicates the WBV at a low rate. Next came the erythrocyte deformability, which indicates the WBV at a high rate. The third and fourth effect parameter reflected the coagulation function and platelet aggregation.
Based on the correlation analysis between these 10 compounds and four simplified parameters, the bioactive compounds in NSTC were clarified using the CCA model. As shown in , the first three sets of canonical variables were selected with the coefficients close to 1, whereas the coefficient of the fourth set of canonical variables was relatively small. The P-value of the first and the second set of canonical variables was <0.01. Although the P-value of the third set of canonical variables was 0.075, the third set of variables still passed the test because of the importance of the third set of canonical variables in real life. As shown in , X1, X2, X3, X4 represented four effect parameters and y1–y10 represented 10 compounds; the correlation in these first three sets of canonical variables indicated that Vi and Wi (i = 1,2,3) in each set of canonical variables had a significant linear relationship. As shown in , computational results suggested that isorhamnetin-3-O-neohesperidoside played a crucial role in the aggregation of erythrocytes; typhaneoside and benzoyl-oxypaeoniflorin also had positive effects. β-Ecdysterone played a crucial role in erythrocyte deformability; isorhamnetin-3-O-neohesperidoside, paeoniflorin and gastrodin also had positive effects. Regarding the coagulation function, typhaneoside played a crucial role; followed by benzoyl-oxypaeoniflorin and galloyl-paeoniflorin, which also had positive effects. Regarding PV and MPAR, benzoyl-oxypaeoniflorin played a crucial role; isorhamnetin-3-O-neohesperidoside, gallic acid and ajugasterone-C also had positive effects. Typhaneoside, isorhamnetin-3-O-neohesperidoside and benzoyl-oxypaeoniflorin were associated with more than one effect and could thus be regarded as the core bioactive compounds against hemorheological disorders. It has been reported that isorhamnetin-3-O-neohesperidoside and typhaneoside have close association with antioxidant capacity [Citation20]. Benzoyl-oxypaeoniflorin might have anti-inflammatory and anti-oxidant effects [Citation21].
Both typhaneoside and isorhamnetin-3-O-neohesperidoside belong to PT [Citation22], which indicated that PT was the main ingredient that contributed to the effect of the NSTC formulation, corresponding to the term ‘monarch’ in the TCM theory. Benzoyl-oxypaeoniflorin belongs to RPR [Citation23], which suggested that RPR also played an important role in the NSTC formulation, corresponding to the term ‘ministerial’ in the TCM theory. Therefore, these results, to some extent, proved that the ‘monarch’ drug and the ‘ministerial’ drug played important roles in the whole prescription and demonstrated the rationality of the formula compatibility.
Conclusions
This study identified three active compounds of NSTC, based on CCA between the chemical HPLC fingerprint and in vivo biotests, that had close relation with the improvement of hemorheological disorders. Typhaneoside could influence the erythrocyte aggregation and coagulation function; isorhamnetin-3-O-neohesperidoside had impacts on the erythrocyte aggregation, erythrocyte deformability and platelet aggregation; and benzoyl-oxypaeoniflorin was associated with the erythrocyte aggregation, coagulation function and platelet aggregation. Our findings could offer useful references for NSTC’s quality control and clinical application. They might also provide important guidance for drug discovery.
Disclosure statement
All the authors involved in this research have no competing interests in the submission of this manuscript. Chao-feng Long works as a researcher in Guangdong Zhongsheng Pharmaceutical Co., Ltd. All the materials exclusively for our experiments did not represent any commercial company’s interests. There was no financial support from any commercial companies. There are no patents, products in development, or marketed products to declare. This information does not alter our adherence to any of the Biotechnology & Biotechnological Equipment policies on sharing data and materials.
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References
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