Study on the Fingerprint Spectrum and the Spectrum-Effect Relationship of Analgesic and Anti-Inflammatory Effects of the Aqueous Extract from Dalbergia hancai Benth

Dalbergia hancai Benth. (D. hancai) is one of the most frequently utilized traditional Chinese medicine in Zhuang medicine. Simultaneously, it has been included in the “Quality Standard of Zhuang medicine in Guangxi Zhuang Autonomous Region (Vol. 2)” and possessed outstanding pharmacological effects. However, the pharmacodynamic material basis of D. hancai still remains unclear. In this study, the high-performance liquid chromatography (HPLC) method had been employed to establish the fingerprint of 10 batches of aqueous extract of D. hancai originated from different parts of China. At the same time, similarity evaluation, cluster analysis, and principal component analysis (PCA) had also been conducted to evaluate the common peaks. The acetic acid-induced writhing in mice had been employed as an analgesic model, and the carrageenan-induced toe swelling in mice was utilized as an anti-inflammatory model for pharmacodynamic experiments. The gray relational analysis (GRA) and partial least squares regression (PLSR) were applied to correlate the fingerprint and pharmacodynamic data to thoroughly examine its spectrum-effect relationship, whereby its analgesic and anti-inflammatory material basis had been comprehensively explored. The results revealed that the HPLC fingerprint of the aqueous extract of D. hancai had successfully identified 12 common peaks whereby two of which were further identified as protocatechuic acid and vitexin. Subsequently, through the analysis of GRA and PLSR, the chromatographic peaks that possess a critical correlation degree with the analgesic and anti-inflammatory effects of D. hancai had also been successfully discovered. Ultimately, the analgesic and anti-inflammatory effects of the 10 batches of D. hancai aqueous extract had been conclusively proved, and it was evidently indicated that these effects were attributable to the synergistic interactions between various components. Therefore, this study aims to serve as an effective analytical method for screening and predicting the effective substances of traditional Chinese medicine on the basis of the spectrum-effect relationship.


Introduction
Dalbergia hancei Benth (D. hancai), a Zhuang medicine, is a plant of Dalbergia (Fabaceae), which is predominantly distributed in Guangxi, Guangdong, Guizhou, and various other places in China. Te roots and stems have been utilized as the medicinal parts of D. hancai [1]. As one of the commonly utilized folk prescriptions of the Guangxi Zhuang nationality, it possesses a long history of application and has demonstrated the efects of regulating qi, relieving pain, tendon-relaxing, and collateral-activating. At the same time, it has also been employed to treat lumbago, arthralgia, abdominal pain, and so on [2,3]. Modern pharmacological studies have demonstrated that Dalbergia L. f. plants possess excellent analgesic and anti-infammatory efects [4,5]. D. hancai is a plant of Dalbergia L. f. At present, there are relatively minimal reports that focused on its chemical constituents and pharmacological efects, as well as providing a quality analysis of D. hancai [6]. Te existing research only revealed a preliminary understanding of the chemical constituents such as tannins, favonoids, sterols, and triterpenoids in the stem bark of D. hancai [7]. As such, it is immensely challenging to accurately and efectively evaluate the quality of D. hancai. Hence, it is extremely vital to comprehensively investigate the quality of D. hancai from various diferent origins, so as to provide a certain reference for its development, utilization, and clinical use.
Nowadays, due to the absence of accurate and efective quality analysis as well as quality control methods, numerous traditional Chinese medicine which possess outstanding medicinal value, have not been fully developed and utilized. Te chromatogram or spectrum of the common peaks acquired by a specifc modern analysis technology following the correct treatment of Chinese herbal medicine or Chinese medicine preparation, which can clearly refect the chemical information of the plant, is referred to as the "fngerprint" of traditional Chinese medicine. Tis spectrum will be able to clearly reveal the population characteristics of each component and therefore able to accurately refect the population characteristics of the plant. However, due to the unavoidable complexity of the components that existed in traditional Chinese medicine and that its efcacy is the result of the coordination and comprehensive action of various components [8][9][10]. Te use of fngerprint cannot directly evaluate the efcacy of drugs, and only the chemical information obtained by fngerprint to standardize and control the quality of traditional Chinese medicine is not comprehensive. Tus, the safety and efcacy of drugs should also be selected as the relevant indicators of quality control. Simultaneously, performing pharmacodynamic studies to integrate the quality and efcacy of Chinese medicines can facilitate the clarifcation of the mechanism of efect [11]. Te spectrum-efect theory of traditional Chinese medicine will be able to efectively resolve the above problems. By associating traditional Chinese medicine's fngerprint with pharmacodynamics through chemometrics models, it signifes the development of a thorough assessment method incorporating chemical analysis and biological activity evaluation under the guideline of traditional Chinese medicine theory in order to efectively uncover the physical underpinnings of traditional Chinese medicine's efectiveness [12]. On the one hand, the "spectrumefect" research method will be able to make up for the shortcomings of the traditional Chinese medicine research model that focuses on component research and ignores pharmacodynamic research. On the other hand, it is able to efectively realize the organic combination of traditional Chinese medicine fngerprint and pharmacodynamic research, as well as supplement the necessary pharmacodynamic information of traditional Chinese medicine to the "spectrum" so as to successfully achieve the purpose of accurately predicting the efcacy of traditional Chinese medicine according to the "spectrum" and enhancing the consistency of "spectrum" and "efect" [13]. At present, the spectrum-efect relationship has been widely adopted in the study of the relationship between active components and the pharmacological efects of traditional Chinese medicine [14][15][16].
In our previous research, a hot plate test, acetic acid writhing test, xylene-induced ear swelling test in mice, and carrageenan-induced toe swelling test in mice had been employed to thoroughly investigate the analgesic and antiinfammatory efects of high, medium, and low doses of aqueous extract of D. hancai. It was discovered that the high and medium doses of aqueous extract of D. hancai possessed excellent analgesic and anti-infammatory efects. Terefore, in order to further evaluate the analgesic and antiinfammatory active substances of D. hancai, this experiment aims to establish the high-performance liquid chromatography (HPLC) fngerprint of D. hancai aqueous extract so as to comprehensively investigate the analgesic and anti-infammatory efects of D. hancai aqueous extract from diferent origins. Te data of fngerprint and pharmacodynamic indexes are correlated by gray relational analysis (GRA) and partial least squares regression (PLSR). Te relationship between the "spectrum" and "efect" of D. hancai had been analyzed, and the pharmacodynamic components related to analgesic and anti-infammatory efects were screened out, respectively. Subsequently, the pharmacodynamic basis of analgesic and anti-infammatory efects of D. hancai was preliminarily discussed in order to serve as an experimental basis for further exploration of analgesic and anti-infammatory pharmacodynamic substances, quality control research, and further development along with the utilization of D. hancai.

Preparation of Sample Solution for HPLC Fingerprint
Analysis. 2 g of D. hancai powder was extracted with 25 mL of distilled water in a plugged conical fask for 1 h. Subsequently, it was cooled to room temperature and distilled water was added to make up the original weight before being shaken and fltered. Te fltrate was centrifuged at a speed of 13 000 r·min −1 for 10 min, and the supernatant was utilized as the test solution for HPLC analysis.

Preparation of Reference Solution.
Appropriate amounts of protocatechuic acid and vitexin reference substance were precisely weighed, and methanol was used to prepare protocatechuic acid reference substance storage solution (0.0512 mg·mL −1 ) and vitexin reference substance storage solution (0.0485 mg·mL −1 ), respectively.

Samples Solution for Animal Experiments
Administration. Te fresh medicinal materials of D. hancai were dried in an oven at 60°C and crushed into powder. Te powder of D. hancai was repeatedly extracted with distilled water for 3 times. Te frst extraction was soaked in 10 times the amount of distilled water for 30 min before being heated and refuxed for 1 h. Te second and third extractions were directly refuxed with 8 times the amount of distilled water for 1 h. After each extraction, the extract was fltered and the fltrate concentrated with a rotary evaporator. Finally, an aqueous solution was acquired and stored in a refrigerator at 4°C. A total of 10 batches of D. hancai medicinal materials from diferent origins were prepared into extracts from the corresponding producing areas, according to the above methods. When the extract was utilized, distilled water was added to prepare a solution equivalent to 57.6 g/kg of crude drug as a sample solution for animal experiments.

Method Validation of the HPLC Fingerprint.
Te HPLC fngerprint analysis method had been employed to efectively evaluate the precision, repeatability, and along with the stability of the sample by calculating the relative retention time of each common peak and the RSD value of the relative peak area with the peak 2 protocatechuic acid as the reference peak (S). Under the chromatographic conditions of paragraph "2.2", the precision was evaluated by continuous injection for 6 times, the repeatability was evaluated by parallel preparation of 6 sample solutions, and lastly the stability was evaluated by measuring and analyzing the samples at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h.

Study on Analgesic Efect.
Te KM male mice were randomly divided into twelve groups with ten mice in each. Group 1 acted as the model group (mice were given distilled water at the same volume), group 2 was the positive group (mice were given 0.03 g/kg rotundine solution at the same volume) while 3-12 were the medication administration groups (mice were given 57.6 g/kg aqueous extract of D. hancai from diferent origins (S1-S10)). After all the animals had experienced 3 days of the adaptation period, each group had been admitted with the corresponding liquid medicine, and the dosage volume of administration was 0.2 mL/10 g once a day for 7 days. Subsequently, one hour after the last administration, the mice in each individual group were intraperitoneally injected with 0.6% glacial acetic acid solution (injection volume of 0.1 mL/10 g). Te number of writhing in mice was observed within 15 minutes (with intraperitoneal depression, hip elevation, and hind limb extension as writhing criteria), and the corresponding analgesic inhibition rate was calculated. Te calculation formula is as follows: Inhibition rate of analgesia (%) � (average writhing times of model group − average writhing times in administration group)/average writhing times of model group × 100%.

Study on Anti-Infammatory Efect.
Te KM male mice were randomly divided into twelve groups with eight mice in each. Group 1 acted as the model group (mice were given distilled water at the same volume), group 2 served as the positive group (mice were given 0.006 g/kg dexamethasone acetate solution at the same volume), while groups 3-12 were the medication administration groups (mice were given 57.6 g/kg aqueous extract of D. hancai from diferent origins (S1-S10)). After the animals had experienced 3 days of the adaptation period, each group had been admitted with the corresponding liquid medicine, and the dosage volume of administration was 0.2 mL/10 g once a day for 7 days. One hour after the last administration, 40 μL 1% carrageenan was subcutaneously injected into the right hind toe of mice to induce infammation, and the left toe was treated as a reference. 30 min later, mice were cervically executed, and both feet were removed from the same part of the ankle joint, precisely weighed, and the diference in weight between the right and left hind feet of the same mouse was used to represent the swelling of the toe, and the swelling inhibition rate was successfully calculated. Te calculation formula is as follows: degree of toe swelling � right toe weight − left toe weight; swelling inhibition rate � (the swelling degree of the model group − the swelling degree of the administration group)/the swelling degree of the model group × 100%.

Data
Handling. Te fngerprint data of 10 batches of aqueous extracts of D. hancai from diferent origins were thoroughly evaluated for similarity by adopting the "Chinese traditional medicine chromatographic fngerprint similarity evaluation system (2012,1 Edition)." Te control fngerprint was generated by averaging the reference spectrum of sample S1 with a time window width of 0.1. After multipoint correction, the Mark peaks were automatically aligned to create a control fngerprint(R) with the median method. Subsequently, through the IBM SPSS Statistics 22.0 software, the 12 common peak areas of 10 batches of aqueous extract samples of D. hancai from diferent origins were systematically clustered by employing the method of between groups linkage, and the similarity of the samples was calculated by utilizing the square Euclidean distance as the measurement standard. Concurrently, the IBM SPSS Statistics 22.0 software had also been employed for PCA to efectively calculate the eigenvalue and variance contribution rate. Te IBM SPSS Statistics 22.0 software was utilized for the statistical analysis of animal experimental data. Te experimental data were expressed as x ± s. At the same time, an independent sample t-test was used to efectively compare the diferences between the administration group and the model group, and the result of P < 0.05 indicated that the diference had been statistically signifcant.
Te 12 common peak areas of the fngerprints of 10 batches of aqueous extract samples of D. hancai from diferent origins were determined as subsequence (X). Te inhibition rate of the aqueous extract of D. hancai on analgesia in mice and the inhibition rate of the swelling in mice were determined as parent sequence (Y), respectively. Te calculation method and steps were analyzed by gray correlation degree according to references [17][18][19][20]. Te peak area of each common peak in the fngerprints of 10 batches of aqueous extract of D. hancai was utilized as the independent variable X, and the inhibition rate of analgesic efect and toe swelling of aqueous extract of D. hancai in mice were taken as the dependent variable Y, respectively. Lastly, the SIMCA14.0 software was utilized for the PLSR analysis of the analgesic and anti-infammatory spectrum-efect relationship, respectively.

Method Validation.
Te results of the precision test showed that the RSD value of the relative retention time of common peaks was 0.05%∼0.17%, and the RSD value of relative peak area was 1.26%∼2.73% (n � 6), indicating that the precision of the instrument was good. Te repeatability test results showed that the RSD values of the relative retention time of the common peaks were 0.03%∼0.51%, and the RSD values of the relative peak area were 1.32%∼2.99% (n � 6), indicating that the method had good repeatability. Te results of the stability test showed that the RSD value of the relative retention time of common peaks was 0.08%∼ 0.29%, and the RSD value of the relative peak area was 0.2%∼ 2.76%, indicating that the test solution had good stability within 24 h.

HPLC Fingerprint.
Te HPLC fngerprints of 10 batches of aqueous extracts of D. hancai from diferent origins had been successfully established, and 12 common peaks were determined. Trough the reference substance comparison method, peak 2 was successfully identifed as protocatechuic acid, and peak 11 was identifed as vitexin. Te standard substance utilized for identifcation, inspection, and content determination is referred to as the reference substance. Peak 2 (protocatechuic acid), among them, exhibited a steady peak shape, excellent peak form and separation, a moderate retention time, and no tailing phenomenon. Terefore, it was adopted as reference peak for fngerprint research. Te RSD of the relative retention time of the common peaks of the fngerprints of 10 batches of D. hancai aqueous extract was calculated to be 0.05%∼0.30%; thus, it indicates that the chemical components of the 10 batches of samples were relatively stable and consistent. Te relative peak area's RSD value, however, was 13.57%∼86.43% and fuctuated signifcantly, refecting that the number of common components in D. hancai samples from various origins was quite diferent. Te diference in content may be afected by climate, ecological environment, and various factors [21,22], As the growth and quality of various traditional Chinese medicines are afected by ecological conditions, once the ecological environment changes, the content, color, and smell of traditional Chinese medicines will also change [23]. Concurrently, the harvest season and time of traditional Chinese medicine are also immensely close related to the quality of medicinal materials. Terefore, the content of active ingredients in the medicinal part of plants is diferent according to their growing stages [23,24].
Te chromatograms of 10 batches of aqueous extract of D. hancai from diferent origins are present in Figure 1, the reference fngerprint in Figure 2, and the HPLC chromatogram of the reference solution in Figure 3. Te similarity evaluation results are present in Table 2. Te similarity of fngerprints of these batches of D. hancai aqueous extract was greater than 0.98, except for S2 and S3. It indicated that the samples of D. hancai aqueous were of a high degree of similarity and the overall quality of the samples was generally stable. Te lower similarity of the fngerprints of S2 and S3 may be attributed to the diferences in the contents of D. hancai herbs from these two producing areas due to diferent growing environment, climate, and seasonal factors [21][22][23][24]. In general, it conforms with the 0.9 fngerprint similarity criteria [25,26].  40  38  36  34  32  30  28  26  24  22  20  18  16  14  12  10  8  6  4  2  0   0  5  10  15  20  25  30  35  40  45  50  55  60  65  70  75  80  85  90

Cluster Analysis.
Te clustering analysis utilizes statistical methods to categorize unknown samples based on their variable characteristics in terms of similarity and is broadly applied in fngerprinting to classify diferent samples based on shared peaks, with the predominant clustering indicators being correlation coefcients and distances [27]. Te results of the cluster analysis are displayed in Figure 4. In the fgure, the X-axis represents the distance, while the Y-axis represents the spectrum number. Te closer the distance is, the greater the similarity of the spectrum is, and the higher the reliability of the fngerprint spectrum is. Te typical distances in the literature available are 5 [16], 10 [28], 15 [29], and so on. Te results of the fngerprint clustering analysis in this experiment remained identical regardless of whether the distance selected was 5 or 10. Consequently, 10 batches of D. hancai aqueous extract samples from various producing regions can be categorized into 3 groups when the selection distance is 10, whereby cluster 1 is producing areas S5, S6, and S7; cluster 2 is producing areas S2 and S3; and cluster 3 is producing areas S1, S4, S8, S9, and S10. Te 12 common peak areas of 10 batches of D. hancai from various origins functioned as the initial data for the cluster analysis, and the peak area represented the content of a chemical components. Te results were grouped into three categories, demonstrating that samples from various production areas possessed varying contents. Te diference in content between these 10 origins may be due to a variety of environmental conditions, which include local soil, the season, climate, the time of harvest, the development stage, and the level of maturity [21][22][23][24].  Table 3. At the same time, by observing the gravel diagram of the frst two principal components ( Figure 5), it can be observed that the frst two principal components are steeper and the other components are relatively gentle. Tus, the frst two principal component factors are selected for evaluation. Depending on the load's absolute value, the fundamental component's contribution may vary. Table 4 demonstrates that the principal component 1 primarily represents information from the chromatographic peaks 1, 2, and 6-12, while the principal component 2 primarily refects information from the chromatographic peaks 3, 4, and 5. According to the scoring diagram of PCA (Figure 6), the 10 batches of aqueous extract samples of D. hancai could be divided into 3 categories with S5, S6, and S7 were divided into one category, S2, and S3 were divided into another category, and S1, S4, S8, S9, and S10 were divided into the last category. Tis was consistent with the results of the cluster analysis.

Animal Experimental Results.
In our previous experiment, the analgesic and anti-infammatory efects of a high dose (57.6 g/kg), a middle dose (28.8 g/kg), and a low dose (7.2 g/kg) of aqueous extract of D. hancai collected from Nanning, Guangxi, were investigated. Te results revealed that the high-dose group (57.6 g/kg) of aqueous extracts of D. hancai from diferent origins exhibited the most excellent analgesic and anti-infammatory efects in mice. Terefore, the most efective dose of 57.6 g/kg crude drug was selected as the administration dose for mice. Te results of the analgesic test demonstrated that the 10 batches of aqueous extract of D. hancai from diferent origins could efectively relieve the pain symptoms of mice induced by acetic acid, and its analgesic efect was superior as compared to that of the positive group. Te results are presented in Table 5. Te results of the anti-infammatory experiment revealed that the 10 batches of aqueous extract of D. hancai from diferent origins could reduce the toe swelling degree of mice and inhibit the infammation caused by carrageenan. Te corresponding values are presented in Table 6.

Results of Spectral-Efect Relationship Analysis.
Te analysis methods of the spectrum-efect relationship of traditional Chinese medicine predominantly comprise gray relational analysis (GRA), bivariate correlation analysis (BCA), principal component analysis (PCA), partial least squares regression analysis (PLSR), multiple linear regression (MLR), canonical correlation analysis (CCA), artifcial neural network (ANN) analysis, and so on [31]. GRA is employed to quantitatively describe the correlation between factors or things by correlation degree. Hence, it is able to refect the contribution of fngerprint chromatographic peak components to drug efcacy thereby rendering it to be suitable for atlases with numerous factors and minimal information. At the same time, it is mainly able to analyze the correlation between each component and drug efcacy but is unable to efectively analyze the positive and negative efect trend of each component and drug efcacy [32]. Te PLSR analysis can refect the magnitude of the positive or negative contribution between each component Journal of Analytical Methods in Chemistry and the drug efect. However, it is unable to efectively acquire the correlation between the component and the drug efect [33]. As a result, this method is employed to enable the two to complement each other and integrated applications, in order to be able to analyze the fngerprint and efcacy of the aqueous extract of D. hancai analgesic and antiinfammatory spectrum-efect relationship more accurately.

Results of GRA.
Te writhing inhibition rate and foot swelling inhibition rate of the 10 batches of D. hancai were utilized as the reference sequence, and the peak area of the common peaks of 10 batches of D. hancai was taken as the comparison sequence. Te gray correlation degree is calculated after data averaging whereby the greater the correlation degree, the greater the contribution of the compounds that correspond to the peak and the efcacy of D. hancai [34].     Te results of GRA are presented in Table 7. Te correlation between the chromatographic peaks of D. hancai aqueous extract to the inhibition rate of analgesia in mice was from peak 1 > 8 > 2 > 9 > 7 > 10 > 5 > 11>4 > 6>12 > 3 in descending order, and the correlation between all peaks except peak 3 was larger than 0.7. Te correlation between the chromatographic peaks of D. hancai aqueous extract and swelling inhibition rate in mice was from peak 1 > 8>2 > 9 > 7 > 5>10 > 11>4 > 6>12 > 3 in descending order, and the correlation between all peaks except peak 3 was larger than 0.7. Te results indicated that the compounds represented by the peaks, except peak 3, were signifcantly correlated with the analgesic and inhibitory efects of D. hancai.  Note. Compared with the model group: * P < 0.05; * * P < 0.01. Note. Compared with the model group: * P < 0.05; * * P < 0.01.

Results of PLSR.
Te variable importance in projection (VIP) values is indicative of the magnitude of the role of each independent variable in the overall analysis system. Terefore, when the VIP value of the independent variable is >1.0, the independent variable is deemed to exert a signifcant contribution to the dependent variable [34]. Te correlation coefcient and variable importance obtained by regression analysis of the fngerprint of aqueous extract of D. hancai and analgesic efect are presented in Table 8 and Figures 7 and 8. Te results revealed that X1, X2, and X6∼X12 were all positively correlated with analgesic efect. Te VIP values of X1, X2, X6, and X8∼X12 were all greater than 1, and the order was X8 > X9 > X11 > X10 > X12 > X6 > X1 > X2, indicating that the above chromatographic peaks exhibited had a signifcant efect on the analgesic efect of mice. When the content increased, the analgesic ability of the aqueous extract of D. hancai was signifcantly enhanced.
Te correlation coefcient and variable importance obtained by regression analysis of fngerprint and antiinfammatory efect of aqueous extract of D. hancai are demonstrated in Table 9 and Figures 9 and 10. It can be observed that X1, X2, and X6∼X12 were positively correlated with the anti-infammatory efect. Te VIP values of X2, X6, and X8∼X12 were all greater than 1, and the order was X8 > X2 > X9 > X10 > X11 > X12 > X6. Hence, it indicated that the above chromatographic peaks exhibited a signifcant efect on the anti-infammatory efect of mice.

Comprehensive Analysis.
Te GRA and PLSR were combined to analyzed, and the gray correlation degree >0.7, the partial least squares regression coefcient was positive, and the VIP value was greater than 1 as the chromatographic peak screening conditions. Eventually, it was discovered that the main components of the analgesic efect of the aqueous extract of D. hancai were peaks 1, 2, 6, 8, 9, 10, 11, and 12; the key elements of the anti-infammatory efect of the aqueous extract of D. hancai were peaks 2, 6, 8, 9, 10, 11, and 12.

Discussion
In the early stage of the experiment, the separation efects of diferent chromatographic columns (Termo ODS-2 HYPERSIL, Agilent ZORBAX SB-Aq, Waters Atlantis C18) were comprehensively investigated. Te baseline, resolution, signal intensity, and peak number of the three chromatograms were also thoroughly analyzed. Te results revealed that the Waters Atlantis C18 column could efectively separate the main compounds with a more excellent peak shape. Simultaneously, it could also refect more other chemical components in the aqueous extract of D. hancai. In addition, the diferent mobile phases (methanol-water, methanol-0.1% phosphoric acid, acetonitrile-water, acetonitrile-0.1% phosphoric acid), wavelength (220 nm, 240 nm, 260 nm, 280 nm, 300 nm, and 320 nm), fow rate (0.8 mL/min and 1 mL/min), column temperature (35°C and 40°C) were comprehensively investigated. Finally, by integrating with the peak signal intensity, peak number, resolution and baseline stability of the chromatogram, the most optimal chromatographic conditions for the fngerprint of the aqueous extract of D. hancai were successfully determined. Table 7: GRA results of 12 common peaks in D. hancai cochinchinensis and analgesic and anti-infammatory efects.   Te correlation between HPLC fngerprint and analgesic and anti-infammatory efects of aqueous extract of D. hancai from diferent origins was analyzed by GRA and PLSR. Te GRA revealed that the correlation degree between each chromatographic peak of the aqueous extract of D. hancai and the inhibition rate of analgesia and toe swelling in mice were all >0.6, and the correlation degree of peaks 1, 2, 5, 7, 8, 9, and 10 were all >0.8, indicating that the chemical components represented by each chromatographic peak were correlated with the pharmacodynamic indexes, and the analgesic and anti-infammatory efects of D. hancai might be the result of the combined action of multiple components. Te PLSR revealed that peaks 1, 2, and 6∼12 were positively correlated with analgesic efect while peaks 2, 6, and 8∼12 were positively correlated with antiinfammatory efect and that the VIP values were all greater than 1. Terefore, according to the results of GRA and PLSR, No. 1, 2, 6, and 8∼12 chromatographic peaks of the aqueous extract of D. hancai contributed signifcantly to the analgesic efect, which might serve as the material basis of the analgesic efect. Peaks 2, 6, and 8∼12 of D. hancai aqueous extract exhibited a higher degree of contribution to the anti-infammatory efect, possibly accounting for the material basis of the anti-infammatory efect of the D. hancai aqueous extract. Protocatechuic acid is at peak 2 and vitexin is at peak 11, among others. It was discovered that protocatechuic acid [35,36] and vitexin [37,38] both exhibited excellent analgesic efects and obvious antiinfammatory efects, which could inhibit the production of infammatory mediators and infammatory factors as well as slow down the infammatory response. It can be observed that protocatechuic acid and vitexin may be one of the potential analgesic and anti-infammatory active ingredients of D. hancai. However, the remaining characteristic peaks have not been successfully identifed. Terefore, in the follow-up study, the structure of unknown chromatographic peaks should be identifed by Q-TOF or LC-MS/MS to further clarify the active components related to analgesic and anti-infammation in the aqueous extract of D. hancai.

Conclusions
In this research, 12 similar peaks and the HPLC fngerprints of 10 batches of D. hancai aqueous extract were successfully established. Peaks 2 and 11 were identifed as protocatechuic acid and vitexin, respectively, through comparison to reference chemicals. Te evaluation of fngerprint similarity yielded data that indicated that exception of S2 and S3, all other producing areas' fngerprint similarity was larger than 0.98. Te results of cluster analysis and PCA were consistent,    and 10 batches of medicinal materials were clustered into 3 categories, indicating that the method can be adopted for the quality evaluation of the aqueous extract of D. hancai.
In this experiment, based on the quantitative analysis of HPLC fngerprint characteristic peaks as well as the analgesic and anti-infammatory data of aqueous extract of D. hancai from 10 diferent origins, the correlation between the analgesic and anti-infammatory spectrum of D. hancai was obtained by GRA and PLSR. Te results revealed that peaks 1, 2, 6, 8, 9, 10, 11, and 12 in the aqueous extract of D. hancai were the chromatographic peaks that contributed signifcantly to the analgesic efect, and peaks 2, 6, 8, 9, 10, 11, and 12 were the chromatographic peaks that contributed substantially to the anti-infammatory efect. Te compounds may be the material basis for the analgesic and antiinfammatory efects of the aqueous extract of D. hancai. Terefore, this experiment is able to serve as the basis for further in-depth exploration of the analgesic and antiinfammatory substances and quality evaluation of D. hancai.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that there are no conficts of interest regarding to the publication of this paper.