1. Introduction
Xanthine oxidase (XOD) is a pivotal enzyme that facilitates the oxidation of xanthine/hypoxanthine to uric acid [
1]. It plays a crucial role in the biosynthesis of purine to uric acid in various organisms. Hyperuricaemia is a condition characterised by the accumulation of uric acid in the body, exceeding the capacity for excretion [
2]. Allopurinol is a pharmaceutical agent that has been granted clinical approval for the treatment of hyperuricaemia [
3]. The process of uric acid conversion can be interrupted by the inhibition of XOD activity. However, these drugs are associated with a number of adverse side effects, including allergic and hypersensitivity reactions, cardiovascular disease, and chronic liver disease [
4,
5]. It is, therefore, imperative to develop natural plant extracts that can inhibit XOD for the treatment of hyperuricaemia.
TZ is the dried branch of the persimmon tree (
Prunus persica (L.) Batsch), belonging to the genus Prunus and family Rosaceae. It has been demonstrated to have efficacy in the activation of blood circulation and the removal of blood stasis, as well as in the detoxification and killing of insects. It is frequently employed in the management of dysmenorrhoea, cardiac and abdominal discomfort, rheumatic arthralgia, and other ailments resulting from qi stagnation and blood stasis [
6,
7]. A number of studies have demonstrated that TZ contains a variety of compounds, including polyphenols, flavonoids, alkaloids, organic acids, and volatile oils [
8]. Among these, polyphenols and flavonoids have been identified as possessing particularly valuable pharmacological properties and significant potential for exploitation [
9,
10].
The current body of research on
Prunus persica (L.) Batsch has concentrated on the metabolomic and transcriptional differences observed in the peach’s leaves, flowers, and fruits [
11,
12,
13]. Additionally, studies have been conducted on the clinical applications of peach kernels in formulations, the identification of polysaccharides in peach gum, and the microbiology of the peach root zone [
14,
15,
16]. With regard to TZ, only a limited number of studies have reported the analysis of its constituent components [
8,
10], as well as the impact of TZ compost on microorganisms and its efficacy in the treatment of wilt [
17]. The extraction of total polyphenols from TZ and the undertaking of fingerprinting studies have yet to be reported in the literature.
Hyperuricemia will lead to rheumatic disease gout in the later stage, and traditional Chinese medicine believes that gout and hyperuricemia are the manifestations of phlegm and blood stasis. At present, the clinical treatment of hyperuricaemia is mostly related to the inhibition of xanthine oxidase, and the commonly used xanthine oxidase inhibitors and drugs include allopurinol, febuxostat, topiostat, etc. [
18], and there is still a large space for the research of designing and developing the inhibitors based on the main raw materials of Chinese herbs. TZ has the effect of promoting blood circulation and removing blood stasis and can be used to treat rheumatic joint pain diseases [
19]. Studies have shown that polyphenols can improve the symptoms of rheumatic diseases [
20]. However, it is unclear whether the polyphenols in TZ have the potential to inhibit XOD activity. Therefore, this study started with the total polyphenol components of TZ (TZ–TPC), determined the difference of total polyphenol content between different batches, analyzed the batch components by UPLC fingerprint, combining with the IC
50 value of its inhibitory effect on XOD, preliminarily screened out the component group that inhibited XOD in TZ.
The fingerprint of traditional Chinese medicine is an effective method for elucidating the relative relationship between the chemical components present in medicinal materials [
21,
22]. This method is an effective means of characterising, comprehensively evaluating, and controlling the internal quality of traditional Chinese medicine. Furthermore, chemometrics methods, including grey correlation analysis (GRA), bivariate correlation analysis (BCA), and orthogonal partial least squares discriminant analysis (OPLS-DA) [
23,
24,
25], are cross-applications of computer science and pharmacodynamics to study the spectrum–effect relationships of herbal medicines using multidimensional joint analysis. These analytical techniques are capable of elucidating the intricate interrelationships between multiple factors and variables [
23].
Consequently, this study initially examined the methodology for the extraction of total polyphenols from TZ. A total of 21 batches of TZ–TPC were extracted, and their contents were determined. Subsequently, the optimal reaction conditions for XOD were determined, and the inhibitory effect of each batch on XOD was evaluated. UHPLC was employed to establish the batch characteristic fingerprints of TZ. Based on the fingerprint of TZ and its inhibitory effect on XOD, a comprehensive evaluation of the quality characteristics of TZ was conducted. This entails the utilisation of an array of techniques, encompassing spectrum–effect relationship analysis and molecular docking for the screening and verification of the component groups of TZ that inhibit XOD activity. The objective is to establish a more comprehensive and accurate quality evaluation system that can serve as a reference for its future development and utilisation.
3. Discussion
3.1. Determination of Total Polyphenol Content and XOD Activity of the Batch
Many studies have shown that TZ contains a large number of polyphenols. In this study, we investigated the optimal extraction conditions for its total polyphenols and determined the total polyphenol content in 21 batches of TZ. The top five batches of samples with the highest content were TZ–14, –11, –17, –6, and –13 (from highest to lowest). In addition, the results showed very little variation in content between batches, suggesting that the nature of the total polyphenol content of TZ is relatively stable among several different origins.
XOD catalyses the production of uric acid from xanthine and hypoxanthine [
30]. In clinical practice, effective drugs for the treatment of XOD–induced hyperuricaemia often produce side effects that seriously affect the prognosis of patients, which has led to an increasing interest in the extraction of anti–XOD-active components from plants and natural products [
31]. Under different conditions, the reaction rate of enzyme and substrate is different; therefore, in this study, the optimal reaction conditions for XOD were first determined, and the type of inhibition of XOD by TZ–TPC was designated as competitive inhibition. Subsequently, the magnitude of IC
50 values of 21 batches of TZ–TPC inhibiting XOD activity was determined. The results showed that the top five batches with the strongest inhibitory ability were TZ–15, –17, –1, –4, and –3, among which the inhibitory activity of batches TZ–15 and –17 was closest to that of the positive drug allopurinol. It is noteworthy that TZ–15 and TZ–1 demonstrated remarkable efficacy in XOD inhibitory activity yet exhibited relatively low levels of total polyphenol content. This observation suggests that the active ingredients in this batch of herbs, aside from the total polyphenol components, may possess inhibitory properties against XOD. TZ–17 demonstrated remarkable efficacy in both content determination and the inhibition of XOD activity, indicating a positive correlation between polyphenols and XOD activity inhibition in this particular batch.
3.2. Batch UHPLC Fingerprints
In order to gain greater insight into the compositional differences between batches, the samples were extracted under conditions that optimised the total polyphenol extraction. This allowed the establishment of batch fingerprints for use in similarity, CA and PCA. The UHPLC superimposed feature profiles demonstrated that the chemical compositions of the TZ from disparate origins exhibited a general similarity, albeit with slight variations in the content of the components. Furthermore, the fingerprint profiles of each batch of herbs demonstrated a high degree of similarity, with 16 common peaks being identified; F1 was chlorogenic acid, F2 was 4-O-Caffeoylquinic acid, F7 was hypericin, F10 was naringenin, and F13 was baicalin. Furthermore, the outcomes of the CA and PCA analyses indicated that samples TZ–1, –2, –5, –7, and –10 were categorised as Class I, whereas TZ–3, –4, –6, –8, and –9, along with samples 11 through 21, were classified as Class II. In comparison to the Class I samples, the Class II samples exhibited a relatively larger peak area in the fingerprint superposition mode, a higher colour block content in the heat map analysis, and a higher PCA score. The discrepancies observed may be attributed to the disparate geographical origins of the batches under consideration. Even when regionally similar batches are subjected to cluster analysis, they do not necessarily display identical behavioural patterns. To illustrate, the samples comprising batch TZ–1–7 were all sourced from Dangwu Town, situated within Guiyang City, Guizhou Province. The results of the clustering and scoring process indicate that TZ–3, TZ–4, and TZ–6 are classified as belonging to Class II, while the remaining samples are classified as belonging to Class I. Similarly, TZ–8 and TZ–10 are from Wangmo County, Xingyi City, Guizhou Province. However, TZ–8 is classified as belonging to Class II, while TZ–10 is classified as belonging to Class I.
3.3. Spectral Effect Correlation Analysis (SECA)
To better determine and analyse the pharmacodynamic effects of the ingredients contained in each batch on XOD, the results of the ‘spectra’ and ‘effects’ were fitted using spectral analysis. In the present study, 16 shared peaks in the TZ fingerprint profile were identified as exhibiting a high degree of correlation with XOD inhibitory activity, with correlation coefficients exceeding 0.2, using the GRA method. This indicates that TZ exerts its XOD inhibitory activity as a result of the synergistic effect of multiple components. To gain a more comprehensive understanding of the spectral-pharmacodynamic relationship, we also performed BCA and OPLS–DA analyses of the 16 shared peaks in the TZ fingerprints in combination with XOD pharmacodynamic indices. Pearson’s correlation coefficient in BCA demonstrated that F4 exhibited a markedly positive correlation with the inhibition of the pharmacodynamic activity of XOD, whereas F8 displayed a markedly negative correlation. Furthermore, the p-value analysis revealed that F4 and F8 exhibited a statistically significant correlation, indicating that F4 may play a pivotal role in enhancing the inhibitory effect of TZ on XOD activity, while F8 may exert an inhibitory effect. In the OPLS–DA analysis, components with a VIP value greater than 1 were identified, and the correlation coefficients were combined to determine the contribution of each peak to the pharmacodynamic index. The results demonstrated a positive correlation between peaks F2, 4, and 10 and a negative correlation between peaks 8, 9, 12, 13, and 16 in terms of XOD inhibition, indicating that the three peaks F2, 4, and 10 exert a significant influence on the inhibitory activity of XOD.
3.4. Molecular Docking Analysis
Combining the three spectral correlation methods, F2 (4–O–Caffeoylquinic acid), F4, and F10 (naringenin) were identified as positively correlated peaks significantly associated with the inhibition of XOD activity; among them, 4–O–Caffeoylquinic acid and naringin have been shown to have strong antioxidant activity [
32,
33,
34]. Higher levels of these peaks indicate stronger inhibition of XOD activity by TZ. The inhibitory effect of TZ on XOD activity was further verified by BCA and OPLS–DA, which demonstrated that this effect was the result of the synergistic effect of multiple compounds within TZ. In the meantime, the results demonstrated that not only components that enhance the inhibition of XOD activity but also components that attenuate the inhibition of XOD activity are present in TZ. Subsequently, the binding between the screened F2, F10, and XOD was verified using the molecular docking technique. The results demonstrated that the molecular binding energy scores of the binding conformations were low and that the interactions were more abundant and strong, indicating that the binding models exhibited good affinity and stability. Furthermore, the results indicate that F2 and F10 may inhibit XOD activity by binding to specific residue sites, including arginine, tryptophan, glutamic acid, serine, and threonine. The binding modes of F2 are more diverse than those of F10, including salt bridging and pi-stacking. Additionally, F2 has a greater number of amino acid residues, which may indicate that it binds to XOD in a more stable manner and has a stronger inhibitory effect.
Currently, there are few studies on the identification of chemical components of TZ. In subsequent experiments, we will consider combining mass spectrometry techniques to identify the components of shared peaks that have not yet been clarified in the fingerprints. Meanwhile, we will also combine the affinity ultrafiltration–ligand capture technique to obtain the XOD-bound components of TZ more accurately. We will explore the effect of TZ on the in vitro and in vivo activities of XOD and study in depth the expression of genes related to hyperuricaemia involved with XOD.
4. Materials and Methods
4.1. Reagents and Materials
In total, 21 batches of TZ were collected from different natural growth areas. Sample origins and batch numbers are shown in
Table S6. All samples were identified by Associate Professor Yang Ye of the Laboratory of Traditional Chinese Medicine and Pharmacognosy Laboratory, Guizhou University of Traditional Chinese Medicine, as dried branches of
Prunus persica (L.) Batsch, a plant of the genus Prunus, family Rosaceae.
Xanthine oxidase (X8020), xanthine (110F044, purity ≥ 98%), and allopurinol (SA5630, HPLC ≥ 98%) were purchased from Beijing Solabao Biotechnology Co. (Beijing, China); sodium carbonate anhydrous (CAS:497–19–8) was purchased from Tianjin Yongda Chemical Reagent Co. (Tianjing, China); folinol reagent (CAS:NONE6060) was purchased from Shanghai McLean Biochemical Technology Co. (Shanghai, China); sodium hydroxide (CAS:1310–73–2) was purchased from Chengdu Jinshan Chemical Reagent Co. (Chengdu, China); anhydrous ethanol (XK13–011–14001, AR) was purchased from Tianjin Fuyu Fine Chemical Co. (Tianjin, China); gallic acid (CAS:149–91–7) was purchased from Shanghai Ronghe Pharmaceutical Technology Development Co. (Shanghai, China); sodium chloride (CAS:7647–14–5) was purchased from Chongqing Chuandong Chemical (Group) Co. (Chongqing, China).
Reference standards of Chlorogenic acid, 4–O–Caffeoylquinic acid, hypericin, naringenin, and baicalin (purity ≥98%) were purchased from Chengdu Alfa Biotechnology Co., Ltd. (Chengdu, China); formic acid (K2215763) was obtained from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China); chromatographic-grade methanol and acetonitrile were provided from TEDIA. All other chemical reagents were of analytical grade.
4.2. Solutions Preparation
Preparation of test and control solutions: one gram of TZ powder was accurately weighed and placed in a 150 mL conical flask. Subsequently, 40 mL of a 60% ethanol solution was added and extracted by ultrasonication (60 W, 49 kHz) at 50 °C for 80 min. The resulting solution was then filtered to obtain the test solution. A 1 mg sample of gallic acid was weighed into a 10 mL volumetric flask, and distilled water was added. The solution was then subjected to ultrasonic waves to obtain a 0.1 mg/mL gallic acid control solution.
Colour Developer Preparation: weigh accurately 4 g of anhydrous sodium carbonate powder into a 20 mL volumetric flask. Add an appropriate volume of distilled water, sonicate until the solid is dissolved, then make up the volume with distilled water and shake well to obtain a 20% sodium carbonate solution (ready for use). The folinol reagent is ready for use.
XOD reaction test solution preparation: take 5.0 g of NaOH powder and put it in a 100 mL volumetric flask to obtain 5% NaOH solution; precision suck 13.70 mL of HCl solution with a concentration of 36.5%, put it in a 100 mL volumetric flask, and add distilled water to the scale to obtain 5% HCl solution; 1.8 mg of xanthine powder was measured and placed in a 10 mL volumetric flask with distilled water to obtain 180 mg/L xanthine solution configuration; pipette 16 μL of XOD stock solution (6250 U/L) in a 1 mL volumetric flask and add PBS solution to fix the volume to the scale line (the whole process of configuration was in an ice–water bath); that is, the 100 U/L XOD solution was obtained. Weigh 4 mg of allopurinol in a 10 mL volumetric flask and then fix the volume with 60% ethanol to obtain 0.400 mg/mL allopurinol mother liquor.
4.3. Colour Development Method
Refer to the method and modify [
35]. A total of 50 μL of the test solution was transferred into a 20 mL brown volumetric flask. Subsequently, 1 mL of folinol reagent and 2 mL of 20% sodium carbonate solution were added dropwise, and the reaction was allowed to proceed for a period of 3 min. Thereafter, the solution was diluted with distilled water. The reaction was then allowed to stand for a further 30 min, after which the absorbance was measured at 760 nm.
4.4. Plotting of Standard Curve and Determination of TZ–TPC Content
Take 0.2, 0.3, 0.4, 0.5, and 0.6 mL of the gallic acid standard solution, as described in
Section 4.2, and transfer to a 10 mL volumetric flask. The colour should be developed in accordance with the methodology outlined in
Section 4.3, after which the absorbance value should be measured. The standard curve is generated by linear regression, with absorbance as the vertical coordinate and mass concentration as the horizontal coordinate.
Take 50 μL of TZ test solution from solution
Section 4.2 and transfer it to a 20 mL volumetric flask. The colour should be developed in accordance with the methodology outlined in
Section 4.3, after which the absorbance value should be measured. This value should then be substituted into the standard curve in order to calculate the concentration of polyphenol compounds present in the sample. Finally, the total polyphenol yield of TZ can be calculated using the following formula:
where
W is the TZ weighing volume (g);
N is the dilution of TZ extract;
C is the total polyphenol concentration (mg/mL);
V is the sampling volume (mL).
4.5. Optimisation of Total Polyphenol Extraction Conditions and Determination of TPC in 21 TZ Batches
The fixed conditions for the initial one-way investigation of TZ–TPC extraction were as follows: ethanol concentration of 50%, volume of 25 mL, extraction temperature of 50 °C, ultrasonic extraction time of 50 min, ultrasonic frequency of 49 KHz, and ultrasonic power of 60 W. The selected extraction methods were ultrasonic and reflux; the extraction solvents were methanol and ethanol; the solvent concentration was 40–80%; and the material–liquid ratio was 1:30–1:50 g/mL; the extraction time was 50–90 min, which was used as a one-way experiment to design the influencing factors for the controlled variable method. The TZ sample solution (TZ–1) was prepared accurately according to the sample solution preparation method under ‘
Section 4.2’, the polyphenols content was determined according to the method under ‘
Section 4.3 and
Section 4.4’, and the optimal extraction conditions were selected based on the total polyphenol content extracted.
After screening the optimal extraction conditions, 21 batches of TZ–TPC samples were prepared according to the conditions, and the differences in polyphenol content between the batches were calculated and compared.
4.6. Screening of Optimal Reaction Conditions for XOD
In references [
31,
36,
37], the XOD activity condition screening system was set up as follows: 400 μL of PBS solution was added to a 1.5 mL centrifuge tube, followed by the addition of 80 μL of XOD solution. The contents were then shaken well and incubated at a constant temperature of 25 °C for 15 min. Subsequently, 320 μL of xanthine solution was added, and the mixture was shaken well once more. The assay was conducted at 290 nm every 50 s for a total of six times. The impact of varying XOD concentrations (25–400 U/L), substrate concentrations (100–220 mg/L), pH levels of PBS solutions (6.0–8.5), and incubation temperatures (15–35 °C) on XOD activity was examined by maintaining a consistent volume of solvent, incubation time, and assay time.
4.7. A Study of the Type of Inhibition of XOD
The method of reference [
31] was modified as follows: the fixed reaction volume was maintained, the enzyme concentration was 200 U/L, the incubation time was 15 min, the pH was 7.5, and the temperature was 30 degrees Celsius. A volume of 80 μL of TZ (TZ–1) extract at concentrations of 0.090, 0.018, 0.036, and 0.049 mg/mL was taken, and 320 μL of substrate xanthine solution at concentrations of 12.5, 25, 37.5, and 50 mg/L was added. Subsequently, 320 μL of PBS buffer was added, and finally, 80 μL of XOD. Using the inverse of the reaction rate of the enzyme as the vertical coordinate and the inverse of the concentration of the substrate xanthine as the horizontal coordinate, the Lineweaver–Burk double inverse method was used to determine the type of inhibition of XOD by the herbs of the peach branch.
4.8. Determination of XOD Inhibitory Activity of TZ-TPC Batch
In total, 0.7, 0.8, 0.9, 1.0, and 1.1 mL of each batch of TZ-TPC extract was taken in a 5 mL volumetric flask, respectively, and was fixed and set aside. Referring to the literature [
36], the experiment was divided into four treatment groups, and the samples were spiked according to
Table 8. PBS, XOD solution, and different mass concentrations of TZ extracts were added sequentially to the reaction system, and after the addition of the samples was completed, the samples were incubated in a metal bath for 15 min, and then xanthine solution was added and mixed homogeneously.
The reaction was timed from the addition of xanthine solution for 5 min, and the absorbance A was measured at 290 nm to calculate the inhibition rate. Take the allopurinol original solution under
Section 4.2, dilute it to 0.008, 0.016, 0.032, 0.064, and 0.128 mg/mL, add samples according to
Table 8, replace the TZ extract with allopurinol solution, and perform tests, measurements, and calculations according to the above steps.
The calculation formula was as follows:
where
A1–
A2 is the sample group, and
A3–
A4 is the blank control group. The raw drug concentration of TZ extract was plotted against the inhibition rate, and the raw drug concentration corresponding to 50% inhibition was calculated as the semi–inhibitory concentration (IC
50) using GraphPad Prism 6.01 non-linear fitting.
4.9. UHPLC Fingerprinting of 21 Batches of TZ-TPC Extracts
4.9.1. Chromatographic Conditions
UHPLC analyses were carried out on an Agilent Ultra-High Performance Liquid Chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a binary solvent delivery pump, an autosampler, and a diode array detector. Samples were drawn and passed through a 0.22 μm microporous filter membrane. Subsequently, the injection volume of the UHPLC system was set at 2 μL, and the separation was performed on an Agilent Eclipse Plus C18 RRHD (P.N. 959757–902, 2.1 × 50 mm, 1.8 μm) column with the separation temperature set at 25 °C, the flow rate set at 0.2 mL/min, and the detection wavelength at 280 nm. The mobile phases employed were a 0.01% aqueous solution of formic acid (A) and a 0.01% acetonitrile solution of formic acid (B). Gradient elution routines were 0–5 min, 9–12% B; 5–9 min, 12–20% B; 9–20 min, 20–46% B.
4.9.2. Preparation of Standard Solution and Sample Solutions
For the preparation of analytical samples, 1.0 mg of chlorogenic acid, 4–O–Caffeoylquinic acid, hypericin, naringenin, and baicalin were accurately weighed and dissolved in 50% methanol to form a 1 mg/mL control solution; TZ samples of 21 batches were extracted from the TZ–TPC solution according to the method of preparation in ‘
Section 4.2’ and evaporated and concentrated to obtain a dry paste, which was dissolved in 50% methanol to prepare analytical samples. All samples were filtered through a 0.22 µm microporous membrane.
4.9.3. Fingerprinting and Methodological Validation
The TZ sample solution (TZ–1) was prepared according to the method under ‘
Section 4.2’. The relative peak area (RPA) and relative retention time (RRT) of the common peaks were used as the detection indexes, and the RSD values were calculated for the methodological validation of the UHPLC fingerprints. Then, the sample solution (TZ–1) was repeatedly injected 6 times for precision analysis. Similarly, six samples of the same batch of sample solution (TZ–1) were prepared separately post-injection and evaluated for reproducibility. The same test solution was injected 0, 2, 4, 8, 12, and 24 h after sample preparation and tested under the chromatographic conditions under ‘
Section 4.9.1’ to assess its stability. The RSD value of <3% [
38] calculated from the methodological validation indicates that the analytical method is stable and reliable.
4.9.4. Batch Fingerprint Analysis
Twenty-one batches of TZ-TPC samples were prepared according to the sample preparation method in ‘
Section 4.2’, and the chromatograms were obtained under the chromatographic conditions in ‘
Section 4.9.1’. The chromatograms of the 21 batches of TZ obtained from the UHPLC analysis system were imported into the “Chromatographic Fingerprint Similarity Evaluation System for Traditional Chinese Medicines” (2012 version). S1 was set as the control chromatogram with a time width of 0.5, which was generated according to the median, and the control chromatogram R and the common pattern superposition were obtained by multi-point correction and automatic matching for the calculation of chromatogram similarity. After obtaining the common peaks of each batch in the matching mode, the relative retention time and relative peak area were calculated, and the peak area data of chromatographic fingerprint profiles were Z–score standardised using SPSS 26.0 software and used for CA and PCA [
27].
4.10. Spectrum–Effect Relationship
In the present study, the spectral relationship between the shared peaks of chromatographic fingerprints and the XOD inhibitory activity of TZ–TPC was investigated using GRA, OPLS–DA, and BCA. The common characteristic peak areas of 16 common peaks were designed as the comparison sequence using the XOD inhibition activity index as the reference sequence. Due to the inconsistency of dimensions between the values of the sequences to be tested, it may lead to unreliable evaluation results, which directly affects the correctness of the conclusions. Therefore, the raw data were pre-processed and normalised by Z–score using SPSS 26.0 software before the spectrum–effect relationship analysis of GRA, OPLS-DA, and BCA.
In the GRA, the reference sequences, comparison sequences, and absolute difference sequences were represented by {X
0 (n)}, {X
i (n)}, and Δ
0i(k) (i representing the number of samples), respectively. Designing the k as evaluation index sequence (k = 1, 2, …, n) to obtain the evaluation sequence {X
i k} (i = 2; n = 19, in this study), the reference sequences and comparison sequences were recorded as {X
0 (k)} and {X
i (k)} when the n = k (k representing peak), and the gray relational coefficients (η(k)) and gray relational grade(r) for each common peak were calculated at the same time according to the expression as follows [
39]:
where M and n are the optimal value and the worst value in the absolute difference sequence, and
ρ is the resolution coefficient, which is usually chosen to be 0.5.
BCA was used to test whether the data were normally distributed using SPSS 26.0 software, Pearson’s parameters were selected for bivariate correlation test, and OPLS–DA was used to carry out orthogonal analyses of the relationship between the common peak data and the inhibition of XOD activity using SIMCA 14.1 software.
4.12. Statistical Analysis
In this study, the weighing of a sample of TZ and the determination of flavonoid content and antioxidant activity were repeated operations in parallel three times and averaged, and data were presented in the form of mean ± standard deviations (X ± SD). The collection, processing, and analysis of data were performed using the software SPSS 26.0 and Simca 14.1, and GraphPad 6.01 were applied to make figures.
5. Conclusions
In summary, in this study, the total polyphenol extraction method of TZ was optimised using a one-factor controlled variable method, and the optimal reaction conditions for XOD were investigated. The total polyphenol content of 21 batches of TZ and their IC50 values for the inhibition of XOD activity were also determined. In addition, the fingerprint of TZ was established by UHPLC, and sixteen common peaks were identified, of which five were recognised, and the similarity of each batch was greater than 0.9. In order to investigate the relationship between the components and the efficacy of the drug, mathematical correlation models, GRA, BCA, and OPLS–DA analyses were used to demonstrate that the inhibition of XOD by TZ is a synergistic effect of the components, and the more significant components were F2, F4, and F10. Molecular docking was used to fit the identified F2 and F10 components to the XOD proteins, and the results showed that the binding energies between the two were less than −7.5 kcal/mol, and the binding modes were diverse, including hydrogen bonding, hydrophobic forces, salt bridges, and so on. Meanwhile, the binding amino acid residue sites of the two are abundant, including arginine, serine, threonine, etc., indicating that the binding conformation of F2 and F10 small molecules to XOD protein is stable and has good affinity. It is speculated that small molecules may inhibit XOD activity through these amino acid residue sites. This study provides a reference for finding natural inhibitors of XOD and provides a theoretical basis for the development and utilisation of TZ medicinal materials.