Several effective constituents, such as vanillin, ferulic acid, senkyunolide I, senkyunolide H, coniferyl ferulate, Z-ligustilide, butylphthalide, senkyunolide A, and levistilide A, are unstable and possess mutual transformation relationships in Chuanxiong Rhizoma (CR). Traditional Chinese medicine mainly involves decoction, and the content of effective constituents and antiplatelet aggregation bioactivity (AAB) in CR may vary with different decoction time (10 min, 20 min, 30 min, 40 min, 50 min, and 60 min). Here, we showed that coniferyl ferulate and levistilide A were detected in CR material, but not in the decoction. The effective components possessed transformation and degradation in CR decoction of different times. The effective components and the strength of AAB at 10 and 20 minutes were the strongest, followed by 30–50 minutes, and 60 minutes were the weakest by analysis of SIMCA-PLS in CR decoction of different times. In the Pearson correlation analysis, there were correlations (
Chuanxiong Rhizoma (CR), the dried rhizome of
Conversion relation of nine effective constituents.
To date, no study on the determination of multiconstituents and AAB in CR decoctions at different decoction times has been conducted. In this study, the ultraperformance liquid chromatography (UPLC) method was utilized to quantitatively determine the content of nine constituents (vanillin, ferulic acid, senkyunolide I, senkyunolide H, coniferyl ferulate, Z-ligustilide, butylphthalide, senkyunolide A, and levistilide A) in CR materials or decoctions at different decoction times, and the AAB was determined simultaneously. In different decoction time periods (10 min, 20 min, 30 min, 40 min, 50 min, and 60 min), the difference in effective constituents was analyzed by SIMCA-PLS and cluster analysis, and the difference of AAB was analyzed by Student–Newman–Keuls. The correlation between effective constituents and AAB was assessed using SIMCA-PLS, Pearson correlation analysis, and stepwise regression analysis. This study conducted a basic assessment of using CR in TCM.
Acetonitrile (HPLC grade) was obtained from Fisher Corporation (Waltham, MA, USA). Adenosine-5-two sodium phosphate (C10H13N5Na2O10P2) was purchased from Sigma-Aldrich Co (St. Louis, MO, USA). Sodium ferulic acid was obtained from Shanghai Yuan Ye Biotechnology Co., Ltd. (Shanghai, China). Glacial acetic acid, dimethyl sulfoxide (DMSO), and three sodium citrate (two water) C6H5Na3O7·2(H2O) were of analytical grade and acquired from Guangzhou Chemical Reagent Factory (Guangzhou, China). Sodium chloride injection (0.9%, W/V) was purchased from Sichuan Cologne Pharmaceutical Co., Ltd. (Chengdu, China). CR decoction pieces (Product Batch No. 180802731) were manufactured by Kangmei Pharmaceutical Co., Ltd. (Guangdong, China) and deposited in the traditional Chinese Medicine Laboratory of Puning Production Base of Kangmei Pharmaceutical. Standards of vanillin, ferulic acid, senkyunolide I, senkyunolide H, coniferyl ferulate, Z-ligustilide, butylphthalide, senkyunolide A, and levistilide A (purities ≥ 98% by HPLC) were purchased from Chengdu Pufei De Biotech Co., Ltd. (Chengdu, China). Japanese big-eared white rabbits, male, weighing about 2.5 kg, were provided by Chengdu Dashuo Biotechnology Co., Ltd. (Chengdu, China).
BSA224S Precision electronic balance was purchased from Beijing Sartorius Scientific Instrument Co., Ltd. (Beijing, China). ZNHW-II Intelligent digital display electric hearting set was purchased from Gongyi Yuhua Instrument Co., Ltd. (Tianjin, China). Frontier™ 5000 Multi Pro multifunction centrifuge was purchased from OHAUS Company (Pine Brook, USA); RE-2000B rotatory evaporator was purchased from Shanghai Yarong Biochemical Instrument Factory (Shanghai, China). SC-2000 platelet aggregation instrument was purchased from Beijing Succeeder Technology Development Co., Ltd. (Beijing, China). KQ-500VDE double-frequency digital ultrasonic cleaning instrument was purchased from Kunshan Ultrasonic Instrument Co., Ltd. (Kunshan, China). UPLC was performed with an Agilent 1290 Infinity II system (Agilent, Palo Alto, CA, USA) equipped with binary pump solvent management system, online degasser, and autosampler.
The column was an Agilent Eclipse Plus C18 column (1.8
Each dried material was pulverized to 50 mesh. Approximately 0.5 g of pulverized powder was accurately weighed and then extracted with 50 mL methanol by ultrasound extraction (300 W of efficiency, 45 kHz of frequency) for 1 h, cooled to room temperature, and the final solution volume was adjusted to 50 mL with methanol.
Each dried material was pulverized to 50 mesh. Approximately 0.5 g of pulverized powder was accurately weighed and then extracted with 50 mL pure water by decocting (100°C) for 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min, cooling to room temperature, and the final solution volume was adjusted to 50 mL with pure water. The solution was centrifuged at 6,000 r/min for 20 min. The supernatant was collected and passed through a filter (0.22
The test substance solutions are citrate three sodium anticoagulant (3.2%, W/V), adenosine-5-two sodium phosphate solution (C10H13N5Na2O10P2; final concentration: 10
Each dried material was pulverized to 50 mesh. Approximately 0.5 g of pulverized powder was placed into each of the 10 conical flasks, to which 50 mL of pure water was added. The solution was weighed and decocted for 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min, to complement weightlessness. The extract was filtered with a gauze, centrifuged, and then subjected to vacuum filtration with a filter paper. The extract was concentrated to about 5 mL by decompressing (85°C, 15 min) and then transferred to 10 mL volumetric flasks. Pure water was added to volume and then shaken well. Approximately 1 mL of the extract was placed in a 10 mL volumetric flask, mixed with 1.5 mL of DMSO solution to dissolve, and then physiological saline was added to volume and shaken well. The solution was transferred to a centrifuge tube and centrifuged at 6,000 rpm for 20 min, and then the supernatant was collected.
Heart blood of the normal rabbits was collected and mixed with trisodium citrate solution as anticoagulant. The ratio of trisodium citrate solution and blood was 1 : 9. The anticoagulant and blood were mixed thoroughly by gently inverting the centrifuge tube.
The mixture was centrifuged at 800 rpm for 10 min twice. The upper plasma, which was the platelet-rich plasma (PRP), was collected. The lower layer of blood was collected from the first centrifugation and then centrifuged at 3,500 rpm for 10 min. The upper layer of the plasma was platelet-poor plasma (PPP).
Approximately 280
The platelet aggregation instrument was preheated to 37°C, and then 10
The results of effective constituents were analyzed by SIMCA-PLS and cluster analysis. The AAB results were expressed as the mean ± SD (standard deviation). The data were compared by one-way ANOVA followed by Student–Newman–Keuls with SPSS 19.0 software (Palo Alto, CA, USA). The differences were considered statistically significant when the different subsets in the subset of alpha = 0.05, conversely, the same subset was no significant difference. The correlation between constituents and AAB was analyzed by Pearson correlation analysis and stepwise regression analysis. In the Pearson correlation analysis, the differences were considered statistically significant when
Development of the calibration curves: calibration curves were developed from the chromatographic peak area relative to the weights of vanillin, ferulic acid, senkyunolide I, senkyunolide H, coniferyl ferulate, Z-ligustilide, butylphthalide, senkyunolide A, and levistilide A. And limit of detection (LOD, S/N = 3) and limit of quantification (LOQ, S/N = 10) were calculated. The results are shown in Table
Calibration curves for 9 ingredients.
Compound | Linear |
|
Range ( |
LOD (ng/ml) | LQD (ng/ml) |
---|---|---|---|---|---|
Vanillin |
|
0.9997 | 0.09656–1.4484 | 4.37 | 13.52 |
Ferulic acid |
|
0.9999 | 0.06784–1.0176 | 3.29 | 9.98 |
Senkyunolide I |
|
1.0000 | 0.0904–1.356 | 4.31 | 12.90 |
Senkyunolide H |
|
1.0000 | 0.0596–0.894 | 2.10 | 6.71 |
Coniferyl ferulate |
|
1.0000 | 0.1507–2.2608 | 2.31 | 6.98 |
Senkyunolide A |
|
0.9999 | 0.18224–2.7336 | 0.41 | 1.38 |
Butylphthalide |
|
0.9999 | 0.01119–1.791 | 1.22 | 4.01 |
Z-Ligustilide |
|
1.0000 | 0.2312–3.468 | 1.47 | 5.10 |
Levistilide A |
|
1.0000 | 0.07944–1.1916 | 0.75 | 2.49 |
The accuracy, repeatability, and stability (12 h) were evaluated by the peak areas of the nine constituents, with six samples in parallel, and they were expressed as RSD (%) within 5%. The result is shown in Table
UPLC method validation parameters for the nine components.
Compound | Precision (RSD) (%) | Stability (RSD) (%) | Reproducibility (RSD) (%) |
---|---|---|---|
Vanillin | 0.32 | 0.33 | 0.34 |
Ferulic acid | 0.35 | 0.35 | 0.15 |
Senkyunolide I | 0.23 | 0.39 | 0.18 |
Senkyunolide H | 0.34 | 0.34 | 0.34 |
Coniferyl ferulate | 0.20 | 0.20 | 0.09 |
Senkyunolide A | 0.13 | 0.10 | 0.09 |
Butylphthalide | 0.15 | 0.16 | 0.16 |
Z-Ligustilide | 0.22 | 0.25 | 0.12 |
Levistilide A | 0.26 | 0.19 | 0.24 |
When CR was extracted by the water in the extraction solvent, coniferyl ferulate can be hydrolyzed into ferulic acid and coniferol. At the same time, phthalides in CR are unstable and are easy to be degraded and transformed in the heating process. Therefore, in order to retain the nine active ingredients of CR to the greatest extent and which represent the ingredients contained of CR, 100% methanol was chosen as the extraction solvent, and ultrasonic extraction for 60 minutes was used to represent the nine active ingredients in CR [
Chromatogram of samples and standard reference compounds of CR. (a) Chromatogram of samples in CR at various decoction times. S: methanol extraction; S1: 10 min; S2: 20 min; S3: 30 min; S4: 40 min; S5: 50 min; S6: 60 min. (b) Chromatogram of nine standard reference compounds. 1. Vanillin. 2. Ferulic acid. 3. Senkyunolide I. 4. Senkyunolide H. 5. Coniferyl ferulate. 6. Senkyunolide A. 7. Butylphthalide. 8. Z-Ligustilide. 9. Levistilide A.
(a) Changes in vanillin, ferulic acid, senkyunolide I, senkyunolide H, and butylphthalide contents. (b) Changes in coniferyl ferulate, senkyunolide A, and Z-ligustilide contents.
Contents of nine constituents at different decoction times (mg/g,
|
Vanillin (%) | Ferulic acid (%) | Senkyunolide I (%) | Senkyunolide H (%) | Coniferyl ferulate (%) | Senkyunolide A (%) | Butylphthalide (%) | Z-Ligustilide (%) | Levistilide A (%) |
---|---|---|---|---|---|---|---|---|---|
0 | 0.14 ± 0.69 | 1.34 ± 0.79 | 1.54 ± 0.42 | 0.28 ± 0.44 | 5.94 ± 1.32 | 31.47 ± 1.38 | 0.47 ± 1.36 | 14.47 ± 1.21 | 0.02 ± 1.27 |
10 | 0.13 ± 1.22 | 4.19 ± 1.35 | 1.73 ± 0.81 | 0.37 ± 0.38 | 0.00 | 13.61 ± 1.21 | 0.16 ± 1.38 | 1.03 ± 0.98 | 0.00 |
20 | 0.12 ± 1.31 | 4.25 ± 0.77 | 1.74 ± 0.38 | 0.37 ± 0.29 | 0.00 | 10.64 ± 0.98 | 0.12 ± 1.69 | 0.44 ± 1.35 | 0.00 |
30 | 0.12 ± 1.65 | 3.97 ± 0.28 | 1.69 ± 1.12 | 0.35 ± 0.87 | 0.00 | 6.00 ± 0.77 | 0.08 ± 1.47 | 0.00 | 0.00 |
40 | 0.12 ± 1.69 | 3.96 ± 0.54 | 1.70 ± 1.21 | 0.34 ± 0.78 | 0.00 | 5.95 ± 1.26 | 0.08 ± 2.19 | 0.00 | 0.00 |
50 | 0.12 ± 0.89 | 3.84 ± 0.39 | 1.71 ± 1.33 | 0.34 ± 0.98 | 0.00 | 6.00 ± 1.39 | 0.08 ± 1.32 | 0.00 | 0.00 |
60 | 0.12 ± 1.27 | 3.12 ± 0.41 | 1.40 ± 1.29 | 0.28 ± 1.22 | 0.00 | 5.01 ± 1.27 | 0.08 ± 1.18 | 0.00 | 0.00 |
Trace amounts of coniferyl ferulate and levistilide A were undetectable at 10 min, indicating that these might have been degraded or not dissolved. Previous reports have shown that conifer ferulate readily hydrolyzes during decoction, and thus conifer ferulate degrades within 0–10 min. Levistilide A is a dimer of Z-ligustilide, and its chemical structure is more stable than Z-ligustilide, but Z-ligustilide content was higher at 0 min, and the degradation rate was higher within 0–10 minutes. Levistilide A was undetectable at 10 min, indicating that it might not have been dissolved. Based on the above results, the main constituents of CR were divided into two parts with different decoction times: the relative stable constituents and the unstable constituents. For the relative stable constituents, namely, ferulic acid, senkyunolide I, senkyunolide H, and vanillin, if we take the traditional decoction time 30 to 40 minutes as standard, then these four constituents were stable within 40 min during CR decoction and complied with the requirements of traditional decoction time. Ferulic acid, senkyunolide I, and senkyunolide H are active substances in CR, whereas vanillin requires further studies to confirm whether it is indeed an effective constituent. The unstable constituents included coniferyl ferulate, Z-ligustilide, senkyunolide A, butylphthalide, and levistilide A. The contents of these five constituents decreased or dissolved during decocting. The content of coniferyl ferulate was 0, but it could have undergone hydrolysis into ferulic acid during decoction, indicating that coniferyl ferulate is an indirect effective constituent. The dissolution rate of Z-ligustilide was low or easily degraded in water, but it can oxidize to senkyunolide I and senkyunolide H. However, as the oral bioavailability of Z-ligustilide was extremely low, the main metabolites
The differences of effective components were comprehensively analyzed in 10–60 minutes of decocting time by SIMCA-PCA. Because the contents of coniferous ferulate and levistilide A were 0 and the difference was small with the contents of vanillin, the other effective components were carried out by SIMCA-PCA analysis and cluster analysis. The result of SIMCA-PCA indicated that the first two principal components were selected,
Results of statistical analysis by SIMCA-PCA and cluster analysis: (a) score scatter plot; (b) dendrogram.
Based on the results of antiplatelet aggregation, the calculated platelet inhibitory rate (PIR) was 10 min (47.94 ± 1.90%) > 20 min (47.63 ± 0.68%) > 30 min (42.38 ± 1.99%) > 40 min (39.20 ± 1.93%) > 50 min (38.93 ± 2.60%) > 60 min (24.76 ± 3.56%) (Figure
Platelet inhibition ratio of CR using different decoction times (
One-way analysis of variance (ANOVA) was performed, and Student–Newman–Keuls (SNK) was selected. In the statistical analysis of SNK, there was no significant difference between the same subset and there was significant difference between the different subsets in the subset of alpha = 0.05. In SPSS 19.0 statistical software, SNK in one-way ANOVA was selected to compare the differences of AAB in 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min. The result indicated that there were significant differences (
ANOVA.
Source | Sum of squares | df | Mean square |
|
Sig. |
---|---|---|---|---|---|
Group | 0.108 | 5 | 0.022 | 41.711 | 0.000 |
Total | 0.114 | 17 |
Student–Newman–Keulsa.
Group |
|
Subset for alpha = 0.05 | ||
---|---|---|---|---|
1 | 2 | 3 | ||
60 min | 3 | 0.248 | ||
50 min | 3 | 0.389 | ||
40 min | 3 | 0.392 | ||
30 min | 3 | 0.424 | ||
20 min | 3 | 0.476 | ||
10 min | 3 | 0.479 | ||
Sig. | 1.000 | 0.194 | 0.873 |
Based on results of SIMCA-PCA, ferulic acid, senkyunolide I, senkyunolide H, Z-ligustilide, butylphthalide, and senkyunolide A as independent variable (
Results of statistical analysis by SIMCA-PLS: (a) dot plot; (b) loading scatter plot.
The correlations of two variables could be studied by Pearson correlation analysis. In the decoction of CR for 10–60 minutes, vanillin, ferulic acid, senkyunolide I, senkyunolide H, senkyunolide A, butylphthalide, Z-ligustilide, and AAB were selected to conduct Pearson correlation analysis (Table
Correlation coefficients of Pearson correlation analysis.
Variables | Vanillin | Ferulic acid | Senkyunolide I | Senkyunolide H | Senkyunolide A | Butylphthalide | Z-Ligustilide | AAB |
---|---|---|---|---|---|---|---|---|
Vanillin | — | −0.540 | −0.395 | −0.466 | 0.669 | 0.795 | 0.859 | −0.482 |
Ferulic acid | — | — | 0.976 |
0.972 |
−0.385 | −0.548 | −0.564 | 0.965 |
Senkyunolide I | — | — | — | 0.982 |
−0.364 | −0.503 | −0.499 | 0.973 |
Senkyunolide H | — | — | — | — | −0.319 | −0.483 | −0.495 | 0.999 |
Senkyunolide A | — | — | — | — | — | 0.974 |
0.947 |
−0.313 |
Butylphthalide | — | — | — | — | — | — | 0.993 |
−0.481 |
Z-Ligustilide | — | — | — | — | — | — | — | −0.497 |
AAB | — | — | — | — | — | — | — | — |
The relationship may be multivariate linear regression between effective components and AAB; i.e., multicomponent was correlated with AAB at the same time. According to the results of stepwise regression analysis, the
Model summary.
Model |
|
|
Adjusted |
Std. error of the estimate |
---|---|---|---|---|
1 | 1.000a | 1 | 1 | 0.0010433 |
aPredictors: (constant), senkyunolide H, and senkyunolide I.
ANOVA.
Model | Sum of squares | Df | Mean square |
|
Sig. | |
---|---|---|---|---|---|---|
1 | Regression | 0.036 | 2 | 0.018 | 16575.747 | 0.000a |
Residual | 0 | 3 | 0 | |||
Total | 0.036 | 5 |
aPredictors: (constant), senkyunolide H, and senkyunolide I.
Coefficientsa.
Variables | Unstandardized coefficients | Standardized coefficients |
|
Sig. | |
---|---|---|---|---|---|
|
Std. error | Beta | |||
(Constant) | −0.422 | 0.007 | −63.728 | 0 | |
Senkyunolide H | 3.044 | 0.045 | 1.187 | 67.199 | 0 |
Senkyunolide I | −0.13 | 0.012 | −0.199 | −11.244 | 0.002 |
aDependent variable: AAB.
Excluded variablesa.
Model | Beta |
|
Sig. | Partial correlation | Collinearity statistics tolerance |
---|---|---|---|---|---|
Vanillin | 0.006a | 0.799 | 0.508 | 0.492 | 0.622 |
Ferulic acid | 0.011a | 0.183 | 0.872 | 0.128 | 0.012 |
Senkyunolide A | −0.001a | −0.091 | 0.936 | −0.064 | 0.195 |
Butylphthalide | 0.001a | 0.1 | 0.929 | 0.071 | 0.264 |
Z-Ligustilide | 0.003a | 0.229 | 0.84 | 0.16 | 0.302 |
aPredictors: (constant), senkyunolide H, and senkyunolide I.
The contents of effective ingredients are different in CR at different times. With the prolongation of decoction time, effective ingredients possess transformation and degradation, and AAB decreases. The changes of effective ingredients are consistent with AAB in CR decoction: 10 minutes, 20 minutes > 30 minutes, 40 minutes, and 50 minutes > 60 minutes. There are synergistic relationships between effective ingredients; meanwhile, effective ingredients and AAB are correlated. In the analysis of correlation between multicomponent and AAB, senkyunolide I, senkyunolide H, and AAB showed a linear relationship, senkyunolide H was positively correlated, senkyunolide I was negatively correlated, and the other effective ingredients were not correlated with AAB.
The data used to support the findings of this study are available from the corresponding author upon request.
Linming Chen is the co-first author.
The authors declare that there are no conflicts of interest.