Yangyin Tongnao (YYTN) prescription is used as a traditional Chinese herbal formula, and it has antioxidant activity that mainly contributes in the treatment of cardiovascular and cerebrovascular diseases. However, the compounds related to its antioxidant activity are still unknown. In the present study, the fingerprints of YYTN extracts under different extraction conditions were obtained by high performance liquid chromatography (HPLC) to identify the common peaks to all the samples processed. A 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay and ferric reducing antioxidant power (FRAP) assay were carried out to evaluate the antioxidant activity of the extracts. Spectrum-effect relationship between HPLC fingerprints and antioxidant activity of YYTN was assessed by Pearson product-moment correlation coefficient (PPMCC) and multiple linear regression analysis (MLRA). The results showed that peaks 5, 6, 13, 15, and 24 of the fingerprints were closely connected to antioxidant activity. Five peaks were identified: vanillic acid (
Yangyin Tongnao prescription is a traditional Chinese herbal formula composed of six kinds of Chinese herbal medicines,
The herbal composition of YYTN simplified prescription.
Pharmaceutical name | Botanical name | Botanical family | Quantity (g) | Employed plant part |
---|---|---|---|---|
Scrophulariaceae | 1 | Tuberous root | ||
Leguminosae | 1 | Root | ||
Lamiaceae | 1.2 | Root | ||
Apiaceae | 0.67 | Root and rhizome |
Antioxidants play a crucial role in preventing various serious diseases caused by oxidative stress damage. At present, several methods have been applied to evaluate the antioxidant activity, including assays for ferric reducing antioxidant power (FRAP) [
Nowadays, spectrum-effect is used to study the relationship between components and efficacy of traditional Chinese medicines. For example, the main vasorelaxant components of rosemary have been found by using the chemical spectrum-effect relationship [
There are three major categories of data processing methods for spectrum-effect relationships, such as correlation analysis, cluster analysis, and regression analysis. Correlation analysis is a statistical method for studying the degree of closeness between variables [
In our study, HPLC fingerprints were combined with the data of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and ferric reducing antioxidant power (FRAP) assays to study the spectrum-effect relationship of YYTN using PPMC and MLRA. It lays the foundation for the pharmacokinetics substantial basis of YYTN.
HPLC analysis was performed using a Waters 2695 HPLC equipped with 2498 UV detector. The chromatographic separation was performed on a Hypersil BDS C18 column (4.6 mm × 300 mm, 5
The mixed reference standard solution was prepared by adding 3 mg of vanillic acid, 36 mg of puerarin, 2.4 mg of ferulic acid, 6 mg of daidzein, and 1.8 mg of formononetin in a 10 mL volumetric flask and dissolved in 50% methanol to obtain final concentrations of 0.3, 3.6, 0.24, 0.6, and 0.18 mg/mL, respectively. Then the solution was filtered through a 0.22
The four herbs were crushed into crude grains. Accurately weighed 1.00 g of
Independent variables and their levels.
Factors | Levels | ||
---|---|---|---|
1 | 2 | 3 | |
Temperature (°C) | 50 | 60 | 70 |
Extraction time (min) | 40 | 50 | 60 |
Solvent-to-material ratio (mL/g) | 6 | 7 | 8 |
Deviation | 1 | 2 | 3 |
The orthogonal table of sample solutions.
Run | Temperature (°C) | Extraction time (min) | Solvent-to-material ratio (mL/g) | Deviation |
---|---|---|---|---|
S1 | 1 | 1 | 1 | 1 |
S2 | 1 | 2 | 2 | 2 |
S3 | 1 | 3 | 3 | 3 |
S4 | 2 | 1 | 2 | 3 |
S5 | 2 | 2 | 3 | 1 |
S6 | 2 | 3 | 1 | 2 |
S7 | 3 | 1 | 3 | 2 |
S8 | 3 | 2 | 1 | 3 |
S9 | 3 | 3 | 2 | 1 |
Method precision was evaluated by five replicate injections of one sample, while repeatability was evaluated by five samples prepared independently from the same initial conditions. For the stability study, sample solutions were stored at room temperature and analyzed at 0, 2, 4, 8, 12, and 24 h, respectively.
The reference standard solutions were injected into the HPLC system, and the chromatographic peaks in the sample solutions were identified according to the retention time of the reference substances.
Each sample solution and each standard solution was analyzed with the HPLC optimized condition to obtain the HPLC fingerprints. The fingerprints were matched automatically by the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine [
DPPH radical scavenging activity was determined according to Zhou et al. [
The total antioxidant activity was evaluated by FRAP assay [
The PPMCC is a statistical method to study the degree of closeness and direction of change between the two variables [
To eliminate the problem of multicollinearity that may exist among multiple independent variables, this study used the stepwise regression module in multiple linear regression in SPSS statistical software [
The IC50 was calculated using the following URL:
Method validation for HPLC fingerprint results showed that precision of the same sample solution was within the range of 0.19–1.37% for relative retention time and 0.75–2.67% for the average peak area of the common peaks to all the samples. Repeatability of the experiment appeared within the range of 0.15–1.04% for the relative retention time and 0.57–2.48% for relative peak area of common peaks. Sample stability was 0.11–1.32% for relative retention time and 0.69–2.74% for relative peak area of the common peaks to all the samples. All results indicated that the method of HPLC analysis was valid and satisfactory.
The HPLC fingerprints of the extracts of YYTN and their reference fingerprints are shown in Figure
HPLC fingerprints of YYTN and their reference (the detection wavelength was set at 280 nm, and the time is expressed in minutes).
The HPLC fingerprint of YYTN.
The HPLC fingerprint of mixed reference solution. Vanillic acid (
The peak area of each common peak to all the samples.
Peak no. | Compounds | Mean peak area of each common peak ( | CV (%) | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Control | |||||||||||||
4.35 | Unknown | 58.239 | 1598.968 | 1434.003 | 2242.623 | 1847.072 | 1928.97 | 1514.767 | 1870.101 | 1977.346 | 1608.01 | 37.13 | |
13.874 | Unknown | 894.038 | 124.294 | 106.241 | 153.788 | 127.161 | 149.786 | 112.926 | 141.261 | 149.642 | 217.682 | 110.10 | |
14.459 | Unknown | 158.758 | 485.685 | 410.196 | 602.872 | 477.105 | 570.061 | 429.368 | 537.156 | 520.098 | 465.7 | 26.48 | |
17.721 | Unknown | 2097.543 | 1797.045 | 1551.842 | 2169.958 | 1794.198 | 2087.783 | 1494.875 | 1850.784 | 1858.046 | 1622.726 | 12.73 | |
20.252 | Vanillic acid | 188.243 | 199.638 | 160.548 | 267.092 | 216.213 | 258.9 | 190.743 | 241.708 | 237.757 | 196.955 | 15.98 | |
23.059 | Unknown | 98.432 | 268.577 | 247.529 | 487.828 | 425.278 | 529.692 | 497.381 | 759.812 | 755.699 | 452.248 | 46.47 | |
23.891 | Puerarin | 12356.807 | 11137.137 | 9685.376 | 12278.256 | 10341.828 | 12285.794 | 9020.642 | 10978.242 | 10895.806 | 10997.765 | 10.16 | |
26.192 | Unknown | 3068.374 | 2724.584 | 2365.726 | 3034.727 | 2546.809 | 3021.683 | 2213.586 | 2713.51 | 2681.596 | 2472.933 | 10.92 | |
27.737 | Unknown | 2692.924 | 2497.148 | 2104.842 | 2746.072 | 2280.202 | 2812.948 | 2077.906 | 2645.69 | 2564.506 | 2491.36 | 10.39 | |
33.463 | Unknown | 451.595 | 431.389 | 415.448 | 893.588 | 757.391 | 812.497 | 767.16 | 924.589 | 988.359 | 715.78 | 29.60 | |
37.292 | Unknown | 55.412 | 208.42 | 138.712 | 237.401 | 195.599 | 230.8 | 189.65 | 238.356 | 233.033 | 191.932 | 29.67 | |
38.998 | Unknown | 118.244 | 134.402 | 218.107 | 382.948 | 320.283 | 391.466 | 352.623 | 444.97 | 479.304 | 315.816 | 39.00 | |
39.844 | Ferulic acid | 537.599 | 227.175 | 341.181 | 481.695 | 367.531 | 450.176 | 324.529 | 400.558 | 437.673 | 396.457 | 22.17 | |
44.968 | Unknown | 53.301 | 210.321 | 153.735 | 280.818 | 177.932 | 210.875 | 185.905 | 233.502 | 270.171 | 174.027 | 33.13 | |
46.175 | Unknown | 119.763 | 155.493 | 123.31 | 252.22 | 161.781 | 170.748 | 149.025 | 186.218 | 221.438 | 171.111 | 23.92 | |
46.762 | Unknown | 318.118 | 175.192 | 120.313 | 183.771 | 131.151 | 157.711 | 134.175 | 161.501 | 147.948 | 169.987 | 32.89 | |
47.891 | Unknown | 567.238 | 1400.02 | 1197.264 | 1678.034 | 1391.706 | 1645.648 | 1264.935 | 1542.522 | 1496.576 | 1290.745 | 23.53 | |
48.897 | Unknown | 102.963 | 171.029 | 160.95 | 196.841 | 163.064 | 193.266 | 142.509 | 174.395 | 174.906 | 152.996 | 16.49 | |
50.891 | Unknown | 541.894 | 225.149 | 194.53 | 275.116 | 230.743 | 267.933 | 190.102 | 258.261 | 244.444 | 209.586 | 38.67 | |
52.074 | Unknown | 480.788 | 483.885 | 403.028 | 540.935 | 453.536 | 531.823 | 424.461 | 547.367 | 501.954 | 431.888 | 10.64 | |
56.112 | Daidzein | 1632.799 | 617.779 | 593.706 | 561.935 | 520.579 | 618.128 | 268.638 | 354.741 | 442.295 | 623.4 | 60.14 | |
58.608 | Unknown | 232.051 | 245.562 | 204.692 | 333.709 | 276.466 | 323.545 | 212.518 | 301.423 | 304.541 | 244.717 | 17.31 | |
64.372 | Formononetin | 218.719 | 179.679 | 178.056 | 174.758 | 174.97 | 173.96 | 174.36 | 173.419 | 171.243 | 155.605 | 8.96 | |
68.493 | Unknown | 200.638 | 237.3 | 214.887 | 266.736 | 211.177 | 245.117 | 209.784 | 207.319 | 239.623 | 225.842 | 9.26 |
CV (%) =
After comparing with the reference standard, five of the twenty-four common peaks were identified, which were vanillic acid (
The content of each group sample.
Compounds | Standard curve ( | The content of each group sample (mg/mL) | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
Vanillic acid | 0.059 | 0.056 | 0.048 | 0.069 | 0.059 | 0.068 | 0.054 | 0.064 | 0.063 | |
Puerarin | 2.397 | 2.230 | 1.940 | 2.458 | 2.071 | 2.460 | 1.807 | 2.198 | 2.182 | |
Ferulic acid | 0.024 | 0.021 | 0.018 | 0.028 | 0.013 | 0.026 | 0.017 | 0.022 | 0.025 | |
Daidzein | 0.124 | 0.130 | 0.124 | 0.116 | 0.106 | 0.130 | 0.043 | 0.064 | 0.086 | |
Formononetin | 0.155 | 0.163 | 0.162 | 0.159 | 0.159 | 0.158 | 0.158 | 0.157 | 0.155 |
Each sample fingerprint of YYTN was analyzed by comparing with the reference fingerprint to evaluate the similarity among these samples. Results of similarity values between the reference and the nine samples are shown in Table
Sample similarity analysis.
Control | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|
1 | 0.907 | 0.951 | 0.942 | 0.944 | 0.947 | 0.943 | 0.942 | 0.941 | 0.957 | |
0.907 | 1 | 0.956 | 0.954 | 0.954 | 0.955 | 0.953 | 0.952 | 0.952 | 0.964 | |
0.951 | 0.956 | 1 | 0.998 | 0.999 | 0.999 | 0.997 | 0.996 | 0.996 | 0.998 | |
0.942 | 0.954 | 0.998 | 1 | 1 | 0.999 | 0.999 | 0.998 | 0.998 | 0.998 | |
0.944 | 0.954 | 0.999 | 1 | 1 | 1 | 0.999 | 0.999 | 0.999 | 0.998 | |
0.947 | 0.955 | 0.999 | 0.999 | 1 | 1 | 0.999 | 0.999 | 0.998 | 0.999 | |
0.943 | 0.953 | 0.997 | 0.999 | 0.999 | 0.999 | 1 | 1 | 1 | 0.998 | |
0.942 | 0.952 | 0.996 | 0.998 | 0.999 | 0.999 | 1 | 1 | 1 | 0.997 | |
0.941 | 0.952 | 0.996 | 0.998 | 0.999 | 0.998 | 1 | 1 | 1 | 0.997 | |
Control | 0.957 | 0.964 | 0.998 | 0.998 | 0.998 | 0.999 | 0.998 | 0.997 | 0.997 | 1 |
This analysis was carried out with the software “Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine.”
The IC50 values of DPPH radical scavenging activity test are listed in Table
The results of antioxidant activities (
Sample no. | DPPH | FRAP |
---|---|---|
3.775 ± 0.017 | 6.328 ± 0.025 | |
4.396 ± 0.024 | 6.170 ± 0.034 | |
3.290 ± 0.018 | 5.331 ± 0.029 | |
3.694 ± 0.011 | 7.242 ± 0.022 | |
3.023 ± 0.016 | 6.003 ± 0.019 | |
3.637 ± 0.016 | 7.762 ± 0.022 | |
3.307 ± 0.017 | 7.345 ± 0.025 | |
3.296 ± 0.022 | 7.959 ± 0.033 | |
3.116 ± 0.017 | 8.245 ± 0.032 |
According to the FRAP kit, the absorbance value of the FeSO4 standard was plotted on the abscissa, and the concentration corresponding to each absorbance value was plotted on the ordinate. The regression equation was as follows:
PPMCC analysis is performed with peak areas of twenty-four peaks as independent variables and IC50 values of DPPH radical scavenging percentage and FRAP values as dependent variables. Results of PPMCC are shown in Table
PPMCC between the twenty-four common peaks of YYTN and antioxidant activities.
Factor | Peak number | |||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
IC50 (mg/mL) | −0.2435 | 0.2430 | −0.1080 | 0.3392 | −0.0309 | −0.4989 | 0.4827 | 0.4651 | 0.3832 | −0.5174 | −0.1174 | −0.6136 | −0.2102 | −0.1070 | −0.1094 | 0.4606 | −0.1174 | −0.0125 | 0.2710 | 0.2299 | 0.3774 | −0.0647 | 0.3425 | 0.3301 |
FeSO4 (mmol/L) | −0.2473 | −0.1771 | −0.4057 | −0.5325 | −0.2092 | 0.5277 | −0.6005 | −0.5876 | −0.3878 | 0.3466 | 0.0211 | 0.3863 | −0.1669 | −0.0786 | −0.1410 | −0.4685 | −0.3621 | −0.6388 | −0.3650 | −0.2388 | −0.7714 | −0.2389 | −0.5031 | −0.6329 |
Using SPSS software, the peak areas of twenty-four common peaks were selected as independent variables and expressed as
YYTN is composed of
From Figure
As shown in Table
Because of the complexity of the components of traditional Chinese medicines, the chemical fingerprint cannot accurately evaluate the related information between the components and their efficacy [
Mathematical modeling is used in spectrum-effect relationship of traditional Chinese medicines [
In general, the main advantage of this research is that the relationship between HPLC fingerprints and the antioxidant activity of YYTN was established. Main antioxidant components of YYTN are further determined and would contribute to the quality standards of YYTN and to the study of the mechanism of other antioxidant components.
In the present study, we established the spectrum-effect relationship between the HPLC fingerprints and scavenging activity for DPPH and the total antioxidant activity for FRAP of YYTN. The HPLC analysis was used to build fingerprints of YYTN which contained up to twenty-four common peaks, and the similarity values of these fingerprints were evaluated by similarity analysis. The results showed that the similarity values of nine samples were more than 0.907, and puerarin was the most abundant chemical constituent in YYTN. The results of the spectrum-effect relationship indicate that peaks 5, 6, 13, 15, and 24 may be the main components responsible for the antioxidant activity of YYTN. This study shows that the fingerprinting method has been validated; it could provide a reliable and practical method for the consistency of quality of traditional Chinese medicine and other herbal preparations.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare no conflicts of interest.
Yangyang Zhang and Li Yu contributed equally to this work. Yangyang Zhang performed the experiments. Li Yu designed the experiments and drafted the manuscript. Xixi Zhao analyzed the data. Haofang Wan and Yu He helped to revise the manuscript. Haitong Wan and Jiehong Yang conceived the study and revised the manuscript.
This research was funded by the National Natural Science Foundation of China (Grant nos. 81904083, 81973560, and 81630105), key projects of the Natural Science Foundation of Zhejiang Province (Grant no. LZ18H270001), and the Foundation of Zhejiang Chinese Medical University (Grant no. 2018ZJ11).