1. Introduction

Hyperuricemia (HUA) is a disorder resulting from the dysregulation of purine metabolism such that uric acid (UA) excretion becomes impaired, leading to the accumulation of abnormally elevated UA levels in the bloodstream [1]. HUA can lead to gout, which is an inflammatory disease resulting from excessive UA production and/or impaired UA excretion such that urate crystals are deposited in sites including the joint cartilage, synovium, and bursa [2]. Affected patients exhibited synovial birefringent sodium urate monohydrate crystals and clinical symptoms that can include acute arthritis and interstitial nephritis. HUA is an important risk factor for the development of gout [3]. At present, clinical strategies to lower serum UA levels include the use of allopurinol, febuxostat, and benbromarone [4]. While these compounds can be efficacious, they have the potential to lead to more frequent acute gout flares when initially used, and controversy persists regarding their cardiovascular safety profiles in gout patients [5].

Traditional Chinese medicine (TCM) is commonplace in China as an efficacious complementary strategy for the management of many diseases and disorders [6]. TCM practices are based on the principle of “Jun-Chen-Zuo-Shi” (“sovereign-minister-assistant-courier”), which emphasizes the complex clinical nature of most diseases through efforts to use multiple ingredients to interact with multiple targets [7, 8]. Paidu powder (PDP) is a TCM preparation composed of the herbs Fructus Mume (Wu Mei), Crataegi Fructus (Shan Zha), Anemarrhena asphodeloides Bunge (Zhi Mu), Chaenomeles sinensis (Mu Gua), Rhizoma Coptidis (Huang Lian), Radix Glycyrrhizae (Gan Cao), and Gypsum Fibrosuum (Shi Gao), which is a self-formulated recipe. However, previous studies have shown that Wu Mei [9], Shan Zha [10], Zhi Mu [11], Mu Gua [12], Huang Lian [13], Gan Cao [14], and Shi Gao [15] were widely used in many traditional Chinese medicine compound (TCMC), as main ingredients, for HUA treatment.

Network pharmacology has been extensively employed for the analysis of herbal compounds [16]. Here, a network pharmacology approach was used to screen for bioactive compounds present in the seven herbs that comprise PDP, allowing for the construction of an interaction network highlighting predicted associations between these compounds and genes associated with HUA. Enrichment analyses and molecular docking strategies were further employed to clarify the validity of the predicted interactions, allowing for the comprehensive assessment of the multifaceted biological and pharmacological interactions between PDP and HUA. Ultimately, this study led to the identification of quercetin, kaempferol, and naringenin as the primary bioactive compounds in PDP. Interestingly, the content of three main active substances was obvious increase after fermentation. In a subsequent clinical study, the beneficial effects of a fermented PDP formulation on HUA were found to be greater than those of an unfermented PDP decoction. Together, these results offer insight into the bioactive components of PDP while also supporting the advantageous effects of fermentation on the clinical efficacy of this TCM preparation.

2. Materials and Methods

3. Results

3.1. Identification of the Bioactive Components in PDP

Using the TCMSP and HERB databases, 122 bioactive ingredients in the seven herbs that comprise PDP and 1978 candidate targets of these compounds were identified with the input parameters for the ADME correlation model. After importing 1978 candidate targets into Cytoscape 3.7.2, the software preliminarily screened them and preliminarily displayed them as corresponding network diagrams. At the same time, the free targets with low correlation were deleted, and finally, the remaining targets were integrated to obtain the most critical 235 targets. The results were then visualized in Cytoscape 3.7.2 (Figure 1), revealing that the herbs “Gancao,” “Huanglian,” and “Zhimu” were associated with the largest numbers of active ingredients and candidate targets.

3.2. Identification of HUA-Related Targets of PDP

In total, 924 HUA-related targets were retrieved from the analyzed databases. The overlap between these targets and the drug-related targets identified above revealed 61 overlapping targets associated with PDP and HUA (Figure 2). Active ingredients associated with these common targets were retrieved from the PDP database, and Cytoscape 3.7.2 was used to establish a network diagram to inform molecular docking efforts.

3.3. PPI Analyses of HUA-Associated Targets of PDP

A PPI network was next established based on the overlapping targets associated with HUA and PDP, yielding a network with 46 nodes and 244 edges (Figure 3(a)). Core clusters within this network were obtained with the Cytoscape plugin (Figure 3(b)), revealing nine key targets with a degree value exceeding 90: IL-1B, TNF, IL-6, RELA, MAPK1, MAPK8, CASP3, TP53, and ESR1.

3.4. Functional Enrichment Analyses

The Metascape platform was next used for the GO and KEGG functional enrichment analyses of these 61 overlapping target genes, revealing 376, 36, and 62 BP, CC, and MF GO terms, respectively (Figure 4). Enriched BP terms included the response to xenobiotic stimulus, positive regulation of gene expression, and positive regulation of apoptotic process terms, whereas enriched CC terms included macromolecular complex, extracellular space, and extracellular region, and enriched MF terms included identical protein binding, enzyme binding, and protein binding. Moreover, 136 enriched KEGG pathways were identified, including the lipid and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, and Kaposi sarcoma-associated herpesvirus infection pathways (Figure 5).

3.5. Molecular Docking Analyses

These network pharmacology results were leveraged to screen the top three bioactive compounds (quercetin, kaempferol, and naringenin) for molecular docking with the IL-1B, TNF, IL-6, RELA, MAPK1, MAPK8, CASP3, TP53, and ESR1 targets. Docking results for these compounds and proteins are presented in Figure 6. These analyses revealed that the binding energy values for quercetin, kaempferol, and naringenin when interacting with these nine targets all exceeded those of the positive control drug allopurinol. Optimal docking images for particular receptor and ligand pairs are presented in Figure 7. Overall, quercetin and kaempferol were found to most strongly interact with ESR1, while naringenin was able to interact with IL-6.

3.6. HPLC Analyses

Through HPLC analyses, significant increases in the levels of quercetin, kaempferol, and naringenin by 308.96%, 1386.44%, and 719.21%, respectively, were detected following fermentation (Table 1 and Figure 8). These data support the ability of fermentation to significantly improve the bioactivity of these samples.

Table 1. The corresponding peak area of three active components in PDP decoction formulation and PDP fermentation formulation.

	PDP decoction formulation	Retention times (min)	PDP fermentation formulation	Retention times (min)	Peak area increase rate (%)
Quercetin	142.2505	31.205	581.7432	30.571	308.96
Kaempferol	34.9978	74.106	520.2222	72.687	1386.44
Naringenin	719.2587	36.559	5892.2422	35.870	719.21

3.7. Clinical Testing of PDP Preparations

Based on the overall changes in blood indices for patients that underwent treatment, the numbers of highly effective, effective, and ineffective treatment outcomes in the fermented PDP group were 53 (67.09%), 22 (27.85%), and 4 (5.06%), respectively, while the corresponding numbers in the PDP treatment group were 35 (47.30%), 20 (27.03%), and 19 (25.67%). These results are consistent with a clear increase in the number of cases in which PDP was highly effective following fermentation. The total effective rate was also 94.9% and 74.3% in the fermented and non-fermented PDP groups, respectively (Figure 9, p < 0.01). UA, CRE, and BUN levels declined following treatment in both groups (Table 2, p < 0.01). Moreover, UA levels trended lower in the fermented PDP group (39.8%) relative to the PDP group (29.2%), although the difference between groups was not significant. No apparent changes in CRE or BUN levels were detected between these two groups. A summary of adverse event incidence is presented in Table 3. Patients in the fermented PDP group presented with three total adverse events (including nausea and decreased appetite), as compared with six in the PDP group (including nausea, decreased appetite, and palpitations), for overall adverse event incidence rates of 0.33% and 0.81%, respectively.

Table 2. Comparisons of UA, CRE, and BUN levels in HUA patients prior to and following treatment.

Group	PDP group	Fermentation-PDP group
Cases	74	79
UA level
Before treatment	561.6 ± 56.4	597.3 ± 64.7
After treatment	397.5 ± 42.4 ∗∗	359.8 ± 39.8 ∗∗
CRE level
Before treatment	55.19 ± 9.1	55.57 ± 10.13
After treatment	48.08 ± 8.14 ∗∗	52.16 ± 10.43 ∗
BUN level
Before treatment	5.36 ± 0.73	5.14 ± 0.94
After treatment	4.67 ± 0.60 ∗∗	4.77 ± 0.86 ∗∗

• Note: Relative to before treatment levels.
•  ^∗p < 0.05.
•  ^∗∗p < 0.01.

Table 3. Adverse event incidence and details for treated patients.

Adverse reactions	PDP group	Fermentation PDP group
Nausea	3	2
Decreased appetite	2	1
Palpitations	1	0
Incidence of adverse reaction	0.81	0.33

4. Discussion

HUA and gout incidence rates have been rising globally in recent years. The purine compounds involved in the development of gout include both endogenous and exogenous types, with exogenous purines being those generated through the breakdown of food, which is thought to be an important contributor to elevated blood UA levels [24]. Dietary modification, metabolic adjustments, and the regulation of purinergic compound production thus represent promising approaches to preventing or treating HUA and gout. TCM treatment strategies have been shown to effectively protect against HUA in a range of mouse model systems through the inhibition of XOD activity, the regulation of urate transporters, and the induction of antioxidant and anti-inflammatory activity [25, 26].

PDP is a TCM preparation comprising Wu Mei, Shan Zha, Zhi Mu, Mu Gua, Huang Lian, Gan Cao, and Shi Gao. Per the “Jun-Chen-Zuo-Shi” principle, Shan Zha is employed for the elimination of food accumulation, the promotion of blood circulation, and the dispersal of blood stasis [27], making it vital for protecting against greasy meat accumulation such that it is considered a “principal drug.” Mu Gua relaxes the tendons, activates collaterals, and dissipates dampness and stomach issues [28]. Wu mei can alleviate gastric distress and quench thirst, improving principal drug efficacy [29]. Huang Lian, Shi Gao, and Zhi Mu are also commonly used for their ability to clear heat and toxic materials, purge fire, and eliminate dampness [30, 31]. These five herbs are considered as the “ministerial drugs” in this preparation. Gan Cao is capable of coordinating drug actions, relaxing spasming, and alleviating pain [32], and it is thus considered an “assistant drug.” In the clinical trial performed herein, PDP exhibited promising therapeutic efficacy in HUA patients, although the material basis remained uncertain.

A network pharmacology strategy was employed in an effort to better understand how PDP may protect against HUA. Using the TCMPS database, active compounds of interest were identified, including quercetin, kaempferol, and naringenin, all of which exhibit demonstrable activity in prior studies. For example, quercetin is able to lower UA levels in vivo while increasing catalase and superoxide dismutase levels and lowering MDA, TNF-α, and IL-1β levels, supporting the notions that its beneficial effects are related to antioxidant and anti-inflammatory activity [33]. Molecular docking studies indicated that kaempferol is capable of binding xanthine oxidase primarily via hydrogen binding, interfering with substrate interactions and thereby inhibiting XO [34]. Naringen intake may also provide benefits in HUA through the promotion of renal UA excretion and the attenuation of inflammation through reductions in inflammatory cytokine production [35]. These results suggest that these compounds may be integral to the therapeutic benefits of PDP treatment in HUA, providing a basis for further work. Moreover, the 61 overlapping targets associated with both HUA and PDP included targets with high degree values in a PPI network, including IL1B, TNF, IL6, RELA, MAPK1, MAPK8, CASP3, TP53, and ESR1.

The blood UA in HUA exceeds the normal level, leading to inflammatory factor enrichment (such as IL1B, TNF, and IL6). Meanwhile, several studies have shown that quercetin, kaempferol, and naringenin have anti-inflammatory effects in many diseases [36–38]. These researches supported our findings. On the other hand, previous research studies have indicated that RELA, MAPK1, MAPK8, CASP3, TP53, and ESR1 signal pathways involved in the development of HUA-induced inflammatory pathological processes [39–41]. Quercetin, as a flavonol drug, not only represents a potential therapeutic agent for obesity and obesity-induced inflammation by regulation of MAPK signaling [42] but also prevents LPS-induced increases of the gene expressions of programmed cell death factors CASP3 [43]. Others studies also shown that kaempferol was associated with the regulation of inflammation by regulating critical targets such as MAPK1, MAPK8, and RELA [44], and the core targets of naringenin in the treatment of nonalcoholic fatty liver disease are TP53, CASP3, AKT1, RELA, ESR1, MAPK1, and TNF [45]. Taken together, the mechanism of PDP on HUA may be the combined effects of three main bioactive compounds on several targets and signal multipathways.

Functional enrichment analyses revealed that these targets were associated with CC terms such as macromolecular complex, extracellular space, and extracellular region, MF terms including protein binding, enzyme binding, and protein binding, and BP terms including response to xenobiotic stimulus, positive regulation of gene expression and positive regulation of the apoptotic process. Molecular docking results also provided support for the predicted interactions.

Quality control is an important guarantee for improving drug efficacy [46]. Fermentation is an extremely important approach to the processing of medicinal herbs in TCM practices, with the microbe-mediated fermentation of these herbs under appropriate environmental conditions leading to the enhancement of active compound content together with reductions in the levels of toxicants present in these samples [47].

After probiotics fermentation, the enzymatic hydrolysis of TCM matrices enables easier products the active compounds as well as release less toxicity of the preparations derived [48]. In addition, the effects of some flavonoid compounds can be obviously influenced during probiotics fermentation, which can change their chemical structure and alter their kinetic parameters, such as bioactivity, bioavailability, and absorption. As an important species, Aspergillus oryza has been widely used in food industry fields, which has been used as production organisms in industrial fermentations for the production of various substances. In the present study, PDP was fermented using Aspergillus oryza AS3.042, and the results indicated that fermentation treatment significantly increased the levels of the top bioactive compounds quercetin, kaempferol, and naringenin by 308.96%, 1386.44%, and 719.21%, respectively. This suggests that fermentation is likely to increase bioactivity and to thereby protect against adverse events. This was confirmed in the subsequent clinical trial. While UA levels were significantly decreased in both the PDP and fermented PDP groups among HUA patients (p < 0.01), a higher overall effective rate was observed in the fermented PDP group relative to the PDP group (94.9% vs. 74.3%, p < 0.01). The rates of decreases in UA levels in these two respective groups were 32.4% (fermented PDP group) and 29.9% (unfermented PDP group). Although we have not compared the contents of toxicity substances between PDP and fermented PDP, the clinical trial has shown that the adverse event rates were decreased from 0.81% to 0.33% after fermentation treatment.

5. Conclusion

In summary, guided by the Chinese principle of “Jun-Chen-Zuo-Shi” and studies focused on clinical efficacy, a network pharmacology approach was herein used to screen out quercetin, kaempferol, and naringenin as key bioactive components of PDP that were then used to establish a network highlighting their interactions with HUA-related target genes, informing enrichment and molecular docking analyses. The levels of these three compounds were also markedly increased by fermentation. When the therapeutic benefits of fermented and nonfermented PDP were compared in patients with HUA, the fermented products were confirmed to exhibit superior efficacy coupled with a lower risk of adverse events. While the network pharmacology results offer potential insight into the underlying mechanisms, future experimental work will be necessary to confirm the predicted interactions.

Conflicts of Interest

The authors declare no conflicts of interest.

Author Contributions

Sun Yan and Wu Tong contributed equally to this work.

Funding

This work was supported by the “Yantai Science and Technology Development Plan Project” (no. 2024YT06000320).