Spectrum-Effect Relationship between HPLC Fingerprints and Antioxidant Activity of Yangyin Tongnao Prescription

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 (P5), puerarin (P7), ferulic acid (P13), daidzein (P21), and formononetin (P23). Our study successfully established the spectrum-effect relationship between HPLC fingerprints and antioxidant activity of YYTN, which provided a general method for establishing quality standards with a combination of chromatography and antioxidant activity.


Introduction
Yangyin Tongnao prescription is a traditional Chinese herbal formula composed of six kinds of Chinese herbal medicines, Radix Rehmanniae, Radix Astragali, Puerariae Lobatae Radix, Ligustici Chuanxiong Rhizoma, Dendrobii Caulis, and Hirudo. However, four kinds of these Chinese herbal medicines except Dendrobii Caulis and Hirudo have been studied as a simplified formula in a lot of research [1,2]. e proportions of herbal composition of Yangying Tongnao (YYTN) simplified prescription are shown in Table 1 [3]. e efficacy of YYTN has been evaluated by clinical trials and animal experiments. According to the Chinese medicine, it has the effects of nourishing yin and replenishing qi (deficiency of yin or qi is a kind of syndrome; deficiency of yin usually appears hot and bothered, and deficiency of qi has a feeling of weariness). In the Western medicine, it could promote blood circulation, especially in the treatment of cardiovascular and cerebrovascular diseases [4]. Previous researches have proved that YYTN had the functions of inhibiting neuronal apoptosis, improving haematological disorders [5], and is related with antioxidant and anti-inflammatory properties [6]. As a traditional Chinese herbal formula, YYTN can also improve the neurological deficits after focal cerebral ischemic infarction in rats [3]. At the same time, it was found that YYTN has protective effect on oxidative stress-induced cerebral ischemia injury in vivo through animal experiments [7]. However, there does not seem to be any report about the relationship between active ingredients and pharmaceutical efficacy of YYTN.
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 spectrumeffect relationship [19]. HPLC fingerprint and bioactivity verification were used to investigate the potential correlation between the chemical components and pharmaceutical effects [20,21]. It is a representative method to find the main active components in complex traditional Chinese herbal formulas based on the study of spectrum-activity relationship.
ere 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 [22], including grey relational analysis (GRA) and Pearson product-moment correlation coefficient (PPMCC). Cluster analysis is one of the important techniques in data mining and exploratory data analysis [23], including hierarchical clustering analysis (HCA) and nonhierarchical clustering analysis (N-HCA). Regression analysis refers to a statistical analysis method to determine the quantitative relationship between two or more variables [24], including multiple linear regression analysis (MLRA) and partial least squares (PLS). erefore, GRA, PPMCC, PLS, and MLRA are applied to study the spectrum-effect relationship in the correlation analysis between the components and the efficacy of Chinese formulas [25][26][27][28][29].
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. e chromatographic separation was performed on a Hypersil BDS C18 column (4.6 mm × 300 mm, 5 μm) maintained at 25°C. e mobile phase consisted of acetonitrile (A) and 0.5% formic acid water (B) with the following optimized gradient elution: 0 min: 2% A; 10 min: 10% A; 29 min: 12% A; 35 min: 15% A; 50 min: 25% A; 65 min: 60% A; 70 min: 90% A. e flow rate was 1.0 mL/min with an injection volume of 10 μL, and the detection wavelength was set at 280 nm.

Preparation of Sample Solutions.
e four herbs were crushed into crude grains. Accurately weighed 1.00 g of Radix Rehmanniae, 1.00 g of Radix Astragali, 1.20 g of Puerariae Lobatae Radix, and 0.67 g of Chuanxiong Rhizoma crude grains were mixed, and aqueous extracts were

Peak Identification and Evaluation of Fingerprints.
e 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. e fingerprints were matched automatically by the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine [30] (Version 2012A). en the reference fingerprint was formed by the system using the median method from the comparison of nine samples, and the similarity values between the reference and nine samples were calculated using this software.

Antioxidant Activity Determination
2.3.1. DPPH Assay. DPPH radical scavenging activity was determined according to Zhou et al. [31] with slight modifications. Briefly, 100 µL of 0.4 mM DPPH anhydrous ethanol solution and 100 µL of each sample solution at various concentrations were added to the 96 microplate. After incubation for 30 min at room temperature in the dark, the absorbance of the mixture was measured at 517 nm (SpectraMax Plus384 Absorbance Microplate Reader, Molecular Devices, USA). DPPH radical scavenging activity was calculated according to the following equation: where A i was the absorbance of sample + DPPH; A j was the absorbance of sample + anhydrous ethanol; A 0 was the absorbance of anhydrous ethanol + DPPH.

FRAP Assay.
e total antioxidant activity was evaluated by FRAP assay [32].
e FRAP solution was prepared by mixing 1.50 mL of 300 mmol/L acetate buffer (pH � 3.

Pearson Product-Moment Correlation Coefficient.
e PPMCC is a statistical method to study the degree of closeness and direction of change between the two variables [33]. In this study, PPMCC was used to calculate the relationship between the peak area values of twenty-four common peaks to all the samples (P1 to P24) in HPLC fingerprints and the antioxidant activities.

Multiple Linear Regression Analysis.
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 [34] to analyze the peaks of HPLC fingerprints, IC 50 values of DPPH radical scavenging percentage, and the ferric reducing antioxidant power, respectively. erefore, characteristic peaks with significant correlation among multiple independent variables were selected.

Results of HPLC Fingerprints
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.

HPLC Fingerprints.
e HPLC fingerprints of the extracts of YYTN and their reference fingerprints are shown in Figure 1, and sample and mixed reference solution are shown in Figures 2 and 3, respectively. Twenty-four peaks with good separation and large peak areas were selected as common peaks to all the samples. e average retention time and peak areas of samples and the coefficient of variation (CV) of twenty-four peak areas are listed in Table 4. Values of CV were all more than 8.96%, which indicated that the content of each component of the samples under different extraction conditions varies greatly, especially the unknown compound represented by peak 2.

Quantitative Analysis of YYTN.
After comparing with the reference standard, five of the twenty-four common peaks were identified, which were vanillic acid (P5), puerarin (P7), ferulic acid (P13), daidzein (P21), and formononetin (P23). As shown in Table 5, quality control was performed on the contents of five compounds in nine samples.

Similarity Analysis of Fingerprints.
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 6.

DPPH Assay.
e IC 50 values of DPPH radical scavenging activity test are listed in Table 7.
e results revealed that nine extracts of YYTN solution exhibited the IC 50 values that appeared within the range of 3.023-4.396 mg/mL. As shown in Table 7, S5 exhibited the most DPPH radical scavenging activity.

FRAP Assay.
According to the FRAP kit, the absorbance value of the FeSO 4 standard was plotted on the abscissa, and the concentration corresponding to each absorbance value was plotted on the ordinate. e regression equation was as follows: Y � 3.4887X − 0.0291, R 2 � 0.9994. e results revealed that the nine extracts of YYTN solution varied from 5.331 to 8.245 mmol/L equivalent to FeSO 4 . As shown in Table 7, S9 exhibited the highest ferric reducing antioxidant power.

Pearson Product-Moment Correlation Coefficient.
PPMCC analysis is performed with peak areas of twentyfour peaks as independent variables and IC 50

Discussion
YYTN is composed of Radix Rehmanniae, Radix Astragali, Puerariae Lobatae Radix, and Chuanxiong Rhizoma. Radix Rehmanniae had been found to have anticancer effects through promoting the activation of natural killer cells and dendritic cells [35,36]. Radix Astragali has antitumor activity in vitro and in vivo [37]. It also exerts pharmacological activities on the cardiovascular, immune, respiratory, and hepatic systems [38]. Puerariae Lobatae Radix has been

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   4 Journal of Analytical Methods in Chemistry shown to be effective in the treatment of hypertension, cerebral ischemia, and diabetes [39][40][41]. Chuanxiong Rhizoma has a protective effect on diabetic nephropathy and has anticancer and antioxidant pharmacological activities [42,43]. erefore, YYTN, as the traditional Chinese herbal formula that combines these four Chinese medicines, has    been extensively applied in the treatment of brain injury [3]. However, there are few reports on the HPLC fingerprint and pharmacological efficacy of YYTN. From Figure 1 and Table 4, there were certain differences in the peak areas between the nine samples under different extraction conditions. However, as shown in Table 6, the similarity value of nine sample fingerprints and reference fingerprints ranged from 0.907 to 0.999, which indicated that the quality of the YYTN composed of Radix Rehmanniae, Radix Astragali, Puerariae Lobatae Radix, and Chuanxiong Rhizoma was relatively consistent and stable. We identified five compounds in YYTN by comparing Figures 2 and 3: P5 was vanillic acid, P7 was puerarin, P13 was ferulic acid, P21 was daidzein, and P23 was formononetin. Subsequently, we carried out the quantitative determination of these compounds' content.
e results are shown in Table 5: the contents of known components in YYTN were puerarin > formononetin > daidzein > vanillic acid > ferulic acid, indicating that puerarin was the most abundant in YYTN. e result of this experiment was consistent with that of previous research of our laboratory.
As shown in Table 7, DPPH radical scavenging percentage and FRAP values can reflect the antioxidant activity [44]. e results showed that YYTN had a good antioxidant activity. e literature showed that the antioxidant activity increased with the decrease of IC 50 of DPPH radical scavenging percentage and the increase of FRAP values [45,46]. According to the PPMCC shown in Table 8, P4, P6, P7, P8, P9, P10, P12, P16, P21, P23, and P24 are positively correlated with DPPH scavenging percentage and FRAP values. P6, P10, and P12 were negatively correlated with IC 50 of DPPH radical scavenging percentage, while these peaks were positively correlated with FRAP. e order of correlation between peaks and IC 50 of DPPH radical scavenging percentage followed P12 > P10 > P6, and FRAP values followed P6 > P12 > P10. MLRA could eliminate the problem of multicollinearity that may exist among multiple independent variables. erefore, MLRA analysis showed fewer peaks related to antioxidant activity than PPMCC. By MLRA, P5, P6, P9, P13, P15, P16, P21, and P24 were related to IC 50 of DPPH radical scavenging percentage and FRAP values. Among them, P5, P6, P13, P15, and P24 were negatively correlated with IC 50 of DPPH radical scavenging percentage, and P5, P6, P13, and P24 were positively correlated with FRAP, which meant that P5, P6, P13, P15, and P24 may have significant antioxidant activity. P5 and P13 were identified by HPLC as vanillic acid and ferulic acid, but P6, P15, and P24 were not identified in this study. erefore,     we will further identify these three unknown components and prove whether these five peaks have a greater contribution to the antioxidant activity.
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 [47,48]. In this study, the fingerprints of YYTN were established, and no related reports were found in the literature. It was concluded that this study laid a foundation for the establishment of the fingerprint of YYTN. Subsequently, the antioxidant activity of YYTN was tested, which showed that YYTN had a certain extent of antioxidant activity and provided a certain therapeutic idea for the treatment of cardiovascular and cerebrovascular diseases because YYTN has been shown to be effective for cerebral ischemia and cardiac ischemia in animal experiments [49,50]. Finally, the components of YYTN related to antioxidant activity were determined by correlation analysis, which may be significant to the research of the therapeutic material basis of YYTN.
Mathematical modeling is used in spectrum-effect relationship of traditional Chinese medicines [34]. For example, the two methods (PPMCC and MLRA) we used in this work indicated that both could establish the spectral correlation model to determine the chromatographic peaks which are closely related to the antioxidant activity. e two antioxidant methods mentioned in this work were also used in a lot of research [51][52][53]. e results showed that the methods were reliable for the determination of antioxidant activity in vitro. Of course, we will verify this result further in both cell and animal experiments in vivo. Our study not only established the spectrum-effect relationship, but also quantitative analysis of the active components in YYTN compared to other traditional Chinese medicine spectrumeffect correlations [20,34].
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.

Conclusion
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. e 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. e results showed that the similarity values of nine samples were more than 0.907, and puerarin was the most abundant chemical constituent in YYTN.
e 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. is 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.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.