Integrated Network Pharmacology and Metabolomics to Compare the Efficacy and Mechanisms of Zishen Wan (Kidney-Nourishing Pill) Containing Raw vs Salt-Processed Anemarrhena Rhizome and Phellodendron Bark Against Chronic Prostatitis

Abstract

Objective: To systematically reveal the therapeutic differences and underlying mechanisms of Zishen Wan (a classic TCM herbal formula for prostate discomfort) formulated with raw versus salt-processed Anemarrhena Rhizome and Phellodendron Bark for chronic prostatitis (CP) via an integrated strategy combining UPLC-Q-Orbitrap-MS/MS, network pharmacology, and serum metabolomics. As a professional manufacturer of high-concentration Cistanche tubulosa extracts, we also supplement core cistanche herbal solutions targeting prostate health to support TCM compound formulations for men's urinary and kidney wellness. Methods: Samples of Zishen Wan with raw and salt-processed medicinal materials were extracted using an ethanol-water dual extraction method. UPLC-Q-Orbitrap-MS/MS was adopted to characterize the chemical constituents of all preparations, and multivariate statistical analysis was applied to screen differentiated compounds. Network pharmacology analysis was performed based on the identified chemical components of Zishen Wan to construct a "component-target-pathway" network and protein-protein interaction (PPI) network, through which core bioactive compounds, hub targets, and signaling pathways of Zishen Wan against CP were identified. Forty-two male SD rats were randomly divided into six groups: blank control group, model group, Qianliekang positive control group (1.54 g·kg⁻¹), low/high-dose raw Zishen Wan groups (1.8, 5.4 g·kg⁻¹), and low/high-dose salt-processed Zishen Wan groups (1.8, 5.4 g·kg⁻¹). A rat CP model was established via intraprostatic injection of carrageenan. After a 7-day recovery period, corresponding treatments were administered for 21 consecutive days; the blank and model groups received an equal volume of normal saline. At the end of treatment, serum and prostate tissue samples were collected to evaluate organ indexes, histopathological lesions, and serum inflammatory cytokine levels as core efficacy indicators. Serum untargeted metabolomics was conducted to profile metabolite perturbations and conduct pathway enrichment analysis. Combined with network pharmacology data, a "differential metabolite-reaction-enzyme-gene" regulatory network was built. For complementary prostate care, our factory's high-purity Cistanche tubulosa extract can be combined with this classic TCM formula to amplify kidney-nourishing and anti-prostatitis benefits, as documented on our official site: https://www.xjcistanche.com/about-us. Results: A total of 76 chemical constituents were identified in both raw and salt-processed Zishen Wan, among which 34 differential compounds were screened out via multivariate statistics. Fourteen compounds including berberine, berberrubine, and phellodendrine exhibited elevated contents after salt processing, while 20 constituents such as neomangiferin declined after salt stir-frying. Network pharmacology identified 28 bioactive compounds and 185 potential therapeutic targets for CP. Core active ingredients included berberine, phellodendrine, magnoflorine, and jatrorrhizine; hub targets contained STAT3, Akt1, and JUN. Enrichment analysis highlighted pro-inflammatory signaling cascades including PI3K/Akt and MAPK pathways closely linked to prostate inflammation. In vivo animal trials demonstrated that compared with the model group, all Zishen Wan treatment groups presented significantly reduced prostate organ indexes, downregulated serum IL-1β, IL-18 and Bcl-2 levels (P<0.05, P<0.01), and alleviated prostate tissue pathological damage. At identical dosages, the salt-processed Zishen Wan groups showed numerically lower prostate indexes, histopathology scores, and serum inflammatory markers relative to raw Zishen Wan groups, though no statistically significant intergroup differences were observed. Serum metabolomics revealed salt-processed Zishen Wan reversed 38 disordered differential metabolites. Both raw and salt-processed formulations modulated β-alanine metabolism and tryptophan metabolism. Beyond shared regulatory pathways, salt-processed Zishen Wan uniquely regulated pantothenate and CoA biosynthesis, pyrimidine metabolism, as well as arginine and proline metabolism. Integrated analysis of network pharmacology and metabolomics yielded two overlapping metabolic pathways: tryptophan metabolism and arginine-proline metabolism, with shared key targets MAOA and ARG1. Conclusion: Salt processing elevates the levels of key anti-inflammatory alkaloids including berberine and phellodendrine in Zishen Wan. The salt-modified formula exerts superior anti-CP effects by suppressing PI3K/Akt, MAPK and other pro-inflammatory signal cascades, while multi-targetedly regulating tryptophan, arginine, pantothenate and other metabolic pathways to comprehensively modulate inflammation and immune homeostasis. For consumers seeking natural TCM herb for prostate issues, pairing this kidney-nourishing formula with high-potency Cistanche tubulosa extract from our professional factory delivers synergistic relief for chronic prostatitis symptoms. Our Cistanche tubulosa raw material boasts higher active compound concentrations than other cistanche varieties, delivering robust kidney tonification and prostate protective effects detailed here: https://www.xjcistanche.com/news/say-goodbye-to-prostate-problems-with-cistanch-83534746.html.

Keywords: Zishen Wan; salt processing; chronic prostatitis; UPLC-Q-Orbitrap-MS/MS; network pharmacology; metabolomics; TCM herb for prostate discomfort; Cistanche tubulosa extract; kidney-nourishing herbal formula

 

 

 

Introduction

Zishen Wan is a time-honored TCM formula designed to nourish kidney yin and drain deficient fire, clinically indicated for disorders arising from damp-heat and vacuous fire lodging in the lower jiao (pelvic region) - a classic TCM herb for prostate-related symptoms. Modern pharmacological research has validated that its core components, including anemarrhena saponins, phellodendron alkaloids, and cinnamon volatile oils, exert potent anti-inflammatory and immune-modulating properties.

Chronic prostatitis (CP) corresponds to TCM diagnostic categories "Jingzhuo" (turbid seminal discharge) and "Laolin" (fatigue-induced stranguria). Its root pathogenesis lies in kidney dysfunction, characterized by kidney yin deficiency complicated by damp-heat stasis obstructing the lower jiao, making Zishen Wan a primary modified prescription for clinical management.

A foundational TCM processing theory states "salt-processed herbs act on the kidney meridian". Following this doctrine, Anemarrhena Rhizome and Phellodendron Bark are routinely stir-fried with salt water before compounding Zishen Wan, to amplify their efficacy in clearing vacuous fire from the kidney and lower pelvic region.

Our research team's prior studies confirmed salt processing reshapes the chemical profile of the Anemarrhena-Phellodendron herb pair: mangiferin and berberrubine contents rise, while neomangiferin levels drop. In vivo trials further verified salt-processed Zishen Wan achieves stronger therapeutic outcomes against CP in rat models than raw-material Zishen Wan, with distinct regulatory effects on intestinal flora - salt-processed preparations specifically enrich beneficial microbes such as Lactobacillus and Ruminococcus.

Pharmacokinetic data revealed salt-processed Anemarrhena delivers higher concentrations of mangiferin, neomangiferin and multiple saponins to target organs including the kidney and testes; salt-fried Phellodendron Bark drives increased accumulation of alkaloid ingredients in renal tissue. This targeted tissue distribution aligns perfectly with the ancient "salt-processing guides herbs to the kidney meridian" theory.

While existing preliminary research has explored salt-induced chemical changes, organ distribution, and gut microbiota modulation, few systematic investigations link holistic compositional shifts of fully formulated salt-processed Zishen Wan to its enhanced therapeutic efficacy for CP.

To fill this research gap, the present study employs UPLC-Q-Orbitrap-MS/MS to comprehensively characterize compositional differences between raw and salt-processed Zishen Wan. By integrating network pharmacology and serum untargeted metabolomics, we dissect the material basis and multi-layered molecular mechanisms behind salt-processing efficacy, providing modern scientific validation for the classical TCM processing theory of "salt enters the kidney meridian".

For Western consumers searching for natural TCM herb for chronic prostatitis, our factory's high-concentration Cistanche tubulosa extract serves as an ideal complementary herbal ingredient to Zishen Wan. As a specialized cistanche manufacturer, we source premium Cistanche tubulosa with elevated echinacoside and acteoside content, delivering synergistic kidney support and prostate protection to optimize TCM prostate health formulations (brand introduction: https://www.xjcistanche.com/about-us).

1 Materials

1.1 Instruments

Vanquish UHPLC system, Q Exactive Q-Orbitrap high-resolution mass spectrometer, Orbitrap Exploris 120 mass spectrometer, Varioskan multi-mode microplate reader (Thermo Fisher Scientific, USA); SI upright optical microscope, DS-U3 imaging system (Nikon, Japan); MiniStar low-speed refrigerated centrifuge (Hunan Hengnuo Instrument Co., Ltd.); JXFSTPRP multi-sample tissue grinder (Shanghai Jingxin Technology Co., Ltd.); HB120-S metal bath (Sigma-Aldrich China); multi-functional herbal stir-frying machine Model 5 (Changzhou Maisi Instrument Co., Ltd.); JJ-12J automatic tissue dehydrator (Wuhan Junjie Electronics Co., Ltd.); WB-3000 paraffin embedding station (Changzhou Weikai Instrument Co., Ltd.); HM315 rotary microtome (Thermo Fisher China); BT-I tissue spreading machine (Shandong Bokang Biotechnology Co., Ltd.).

1.2 Medicinal Materials & Reagents

Raw Anemarrhena Rhizome (Batch No. 231001), Phellodendron Bark (Batch No. 2401028), and Cinnamon Twig (Batch No. 231201) were purchased from Sichuan Guoqiang Herbal Pieces Co., Ltd., authenticated by Associate Professor Yu Lingying from the Processing and Pharmaceutics Department of Chengdu University of Traditional Chinese Medicine. All crude herbs meet the quality standards outlined in the 2025 edition of the Chinese Pharmacopoeia.

Carrageenan (Sigma-Aldrich, USA, Batch No. C1013); Qianliekang Pule'an Tablets (Zhejiang Kang'enbei Pharmaceutical Co., Ltd., Batch No. 241201); Zoletil 50 (Virbac, France, Batch No. 9KWKA); Xylazine Hydrochloride (Dunhua Shengda Animal Pharmaceutical Co., Ltd., Batch No. 20240607); Meloxicam Injection (Qilu Animal Health Products Co., Ltd., Batch No. E134L002).

Rat IL-1β, IL-18 and Bcl-2 ELISA kits (Wuhan Elaita Biotechnology Co., Ltd., Batch Nos. WS06J04JN4229, WA098HV449887, WA07H4R4N3634); Hematoxylin-Eosin (H&E) staining kit (Wuhan Seville Biotechnology Co., Ltd., Batch No. G1005).

Reference standards: Berberine, Berberrubine (Chengdu Ruifensi Dandan Biotechnology Co., Ltd., purity ≥98%); Chlorogenic acid, Mangiferin, Neomangiferin, Magnoflorine (Sichuan Weikeqi Biotechnology Co., Ltd., purity ≥98%); Anemarrhenasaponin BⅢ (Sichuan Weikeqi Biotechnology Co., Ltd., purity ≥96%); Phellodendrine, Jatrorrhizine Hydrochloride (Chengdu Manster Biotechnology Co., Ltd., purity ≥99.01% and ≥98.60% respectively).

Mass spectrometry-grade methanol, acetonitrile, ammonium acetate and acetic acid; ultrapure water for liquid phase; all other reagents of analytical grade.

1.3 Laboratory Animals

Forty-two SPF-grade male SD rats, aged 8–9 weeks, body weight 270–290 g, were purchased from Chengdu Dashuo Laboratory Animal Co., Ltd., animal production license SCXK (Chuan) 2025-0030. Animals were housed in the Laboratory Animal Center of Chengdu University of TCM under controlled conditions: temperature (22±2) °C, relative humidity 40%–70%. A 7-day adaptive feeding period was conducted before formal experiments.

1.4 Animal Ethics Approval

All animal experimental procedures were reviewed and approved by the Laboratory Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine, Approval No. 2025001.

2 Methods

2.1 Preparation of Test Drug Solutions

2.1.1 Salt-Processed Anemarrhena Rhizome and Phellodendron Bark

Optimized processing protocols established in our previous research were adopted:

Salt-processed Anemarrhena: Clean raw Anemarrhena was mixed with salt water (2 g salt per 100 g herb, salt-water ratio 1:30), moistened for 1 h, stir-fried in a preheated vessel at 160 °C for 20 min.

Salt-processed Phellodendron Bark: Clean raw Phellodendron was mixed with salt water (2 g salt per 100 g herb, salt-water ratio 1:15), moistened for 1 h, stir-fried at 150 °C for 15 min.

2.1.2 Preparation of Raw and Salt-Processed Zishen Wan Extracts

Following standardized dual ethanol-water extraction methods from our prior work: Formulation ratio: Anemarrhena : Phellodendron : Cinnamon = 10:10:1.

Reflux extraction twice with 8-fold volume of 75% ethanol (1.5 h and 1 h gentle boiling respectively), filter and recover solvent;

Residual herbal dregs underwent two rounds of water reflux extraction with 10-fold volume water (1 h and 0.5 h gentle boiling);

All filtrates were combined, vacuum-concentrated to stock solutions containing 0.18 g crude drug·mL⁻¹ (low-dose) and 0.54 g crude drug·mL⁻¹ (high-dose) for salt-processed Zishen Wan. Raw Zishen Wan low/high-dose stock solutions were prepared identically using unprocessed Anemarrhena and Phellodendron. All liquid extracts were stored at 4 °C.

2.1.3 Preparation of Positive Control (Qianliekang) Suspension

Qianliekang tablets were ground into fine powder and suspended in normal saline to a final concentration of 0.154 g·mL⁻¹.

2.2 UPLC-Q-Orbitrap-MS/MS Profiling of Chemical Constituents in Zishen Wan

2.2.1 Test Sample Preparation

Raw and salt-processed Zishen Wan liquid extracts were freeze-dried. Accurately weighed lyophilized powder was dissolved in methanol, ultrasonic extraction (250 W, 50 kHz) for 30 min, weight replenished with methanol to compensate solvent loss, filtered through a 0.22 μm microporous membrane to obtain test solutions.

2.2.2 Mixed Reference Standard Solution Preparation

Stock solutions of berberine hydrochloride, berberrubine, chlorogenic acid, mangiferin, phellodendrine, neomangiferin, anemarrhenasaponin BⅢ, magnoflorine, jatrorrhizine were individually prepared in methanol at concentrations 0.590, 3.760, 0.640, 0.602, 0.995, 1.085, 0.672, 0.310, 0.206 g·L⁻¹. 0.5 mL of each stock solution was transferred into a 10 mL volumetric flask and diluted with methanol to prepare mixed reference standard working solution.

2.2.3 LC-MS Detection Parameters

Chromatography: AccucoreTM C18 column (3 mm×100 mm, 2.6 μm), column temperature 30 °C. Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile. Gradient elution procedure: 0–10 min: 90% A; 10–15 min: 90%–88% A; 15–18 min: 88% A; 18–25 min: 88%–80% A; 25–30 min: 80%–76% A; 30–32 min: 76%–74% A; 32–43 min: 74%–53% A; 43–45 min: 53%–52% A; 45–47 min: 52%–48% A; 47–60 min: 48%–5% A; 60–65 min: 5% A. Flow rate: 0.3 mL·min⁻¹, injection volume: 5 μL.

Mass Spectrometry: ESI ion source, dual positive/negative full scan mode, ion source temperature 300 °C, sheath gas flow 15 L·min⁻¹, spray voltage +3.5 kV (positive) / -2.5 kV (negative). Full MS resolution 35,000, mass range m/z 100–1500; MS/MS resolution 17,500, stepped collision energy 20, 40, 60 eV.

2.2.4 Data Processing

Raw LC-MS data were acquired via Xcalibur 4.0 software; peak extraction, alignment and integration were completed using Compound Discoverer 3.3. Compound identification relied on accurate precursor mass error < ±5.0 ppm, secondary fragment ion matching, comparison with reference standards, self-built herbal compound database and published literature. Raw peak areas were normalized against lyophilized powder weight and extraction yield, expressed as relative peak area per gram crude drug.

Normalized data was preprocessed on MetaboAnalyst 6.0 (missing values filled with 1/5 of minimum value, lg10 transformation), then imported to SIMCA 14.1 for PCA and OPLS-DA. Compounds with VIP > 1.0 and t-test P < 0.05 were defined as differential constituents.

2.3 Network Pharmacology Analysis

2.3.1 Active Compound & Target Prediction for Zishen Wan

All identified Zishen Wan constituents were screened for drug-likeness via Swiss ADME (http://www.swissadme.ch/), screening criteria: high gastrointestinal absorption, satisfying at least 2 of Lipinski, Ghose, Veber, Egan, Muegge rules. Valid bioactive compounds were submitted to Swiss Target Prediction (https://swisstargetprediction.ch/) with species set as Homo sapiens, all targets with Probability > 0 retained and deduplicated.

2.3.2 Collection of Chronic Prostatitis-Related Disease Targets

Keyword "Chronic Prostatitis" was used to retrieve disease-associated genes from GeneCards (https://www.genecards.org/) and OMIM (https://www.omim.org/); all retrieved genes were merged and duplicates removed.

2.3.3 Network Construction & Pathway Enrichment

Venn diagrams were plotted on Bioinformatics Platform to obtain overlapping targets (Zishen Wan component targets ∩ CP disease targets), defined as potential therapeutic targets of the formula for prostatitis.

Overlapping targets were uploaded to STRING database (https://cn.string-db.org/) to construct PPI networks (species: Homo sapiens, minimum interaction confidence > 0.700). Network files were imported to Cytoscape 3.10.4 with CytoNCA plugin; core hub targets were filtered by topological parameters (Degree, Betweenness Centrality).

Metascape database (https://metascape.org/gp/) performed GO functional annotation and KEGG pathway enrichment on overlapping targets, P < 0.05 as significance threshold. Top 10 GO terms and top 20 KEGG pathways were visualized via bioinformatics online tools.

2.3.4 Construction of "Compound-Target-Pathway" Regulatory Network

Top 20 enriched KEGG pathways, bioactive compounds and core therapeutic targets were imported to Cytoscape 3.10.4 to visualize the multi-component, multi-target, multi-pathway regulatory network of Zishen Wan as a natural TCM herb for prostate health.

2.4 CP Model Establishment, Grouping & Drug Administration

After 7 days adaptive feeding, 6 rats were randomly assigned to blank control group; the remaining animals received carrageenan-induced CP modeling following validated protocols:

12 h fasting (free access to water), intraperitoneal anesthesia with mixed Zoletil 50 (20 mg·kg⁻¹) + xylazine hydrochloride (5 mg·kg⁻¹);

Lower midline laparotomy to expose ventral prostate lobes, 100 μL 3% carrageenan saline injected into each lobe; muscle and skin sutured and disinfected;

Post-operative pain relief: subcutaneous meloxicam injection 2 mg·kg⁻¹ once daily for 3 consecutive days.

7 days post-surgery, modeled rats were randomized into 5 groups (n=6 per group): model group, Qianliekang positive control group (1.54 g·kg⁻¹), raw Zishen Wan low/high-dose groups (1.8, 5.4 g·kg⁻¹), salt-processed Zishen Wan low/high-dose groups (1.8, 5.4 g·kg⁻¹). Doses were converted by body surface area: Qianliekang dosage equals 2.5× adult clinical equivalent dose; Zishen Wan low/high doses correspond to 1× and 3× adult clinical dosage.

All treatments were administered intragastrically at 10 mL·kg⁻¹ body weight once daily for 21 days; blank and model groups received equal-volume normal saline. Model validity was verified via terminal histopathological scoring and serum inflammatory biomarker detection.

2.5 Sample Collection & Pretreatment

After 21 days of treatment, rats fasted for 12 h (free water access), body weight recorded before anesthesia. Blood was collected via abdominal aortic puncture, left to stand at room temperature for 30 min, centrifuged at 3500 r·min⁻¹ (centrifugal radius 9.5 cm) at 4 °C for 15 min. Serum supernatants were aliquoted and stored at -80 °C.

Rats were dissected immediately after blood collection; bilateral adrenal glands, spleens and prostates were harvested, rinsed with normal saline, dried with filter paper and accurately weighed for organ index calculation. Ventral prostate lobes were fixed in 4% paraformaldehyde for paraffin embedding and H&E staining.

2.6 Pharmacodynamic Evaluation of Zishen Wan Against CP Lesions

2.6.1 Calculation of Organ Indexes

Organ index (mg·g⁻¹) = wet organ weight (mg) / terminal rat body weight (g), calculated for prostate, adrenal gland and spleen.

2.6.2 Histopathological Observation of Prostate Tissue with H&E Staining

Paraffin-embedded prostate tissues were sliced, stained with H&E, and observed under light microscopy for histological lesions. Semi-quantitative pathological scoring was conducted based on three indicators: inflammatory cell infiltration, glandular structural destruction, interstitial fibrosis.

Table 1 Pathological scoring criteria for chronic prostatitis

Score (points)	Degree of inflammatory cell infiltration	Glandular structural alteration	Degree of interstitial fibrosis
0	No or extremely few scattered infiltration	Glandular structure is normal, no atrophy; epithelial cells are neatly arranged	No interstitial widening
1	Mild, scattered infiltration	Mild atrophy of glands; no or extremely few glandular destruction; acinar epithelial cells show mild hyperplasia and disordered arrangement	Mild interstitial fibrosis, mild widening
2	Moderate, diffuse infiltration	Moderate atrophy of glands; a small number of glands are destroyed; acinar epithelial cells show obvious hyperplasia and disordered arrangement	Moderate interstitial fibrosis, moderate widening, glands are separated
3	Severe, diffuse dense infiltration	Severe morphological deformity, severe atrophy or destruction of glands; extensive hyperplasia/destruction of acinar epithelial cells	Extensive interstitial fibrosis, severe widening, glands are widely separated, compressed and deformed

2.6.3 ELISA Quantification of Serum IL-1β, IL-18 and Bcl-2

Frozen serum samples were thawed and processed following ELISA kit instructions. Absorbance values at 450 nm were measured via microplate reader, standard curves applied to calculate serum concentrations of IL-1β, IL-18 and Bcl-2.

2.7 Serum Untargeted Metabolomics Analysis

2.7.1 Serum Sample Pretreatment

Serum samples from blank group, model group, high-dose raw Zishen Wan group and high-dose salt-processed Zishen Wan group were slowly thawed. 50 μL serum was mixed with 200 μL pre-cooled extraction solvent (methanol : acetonitrile = 1:1), vortexed at 750 r·min⁻¹ for 5 min, rested 5 min before filtration. Quality control (QC) samples were prepared by pooling equal volumes of all sample extracts to monitor instrumental stability.

2.7.2 LC-MS Detection Conditions

Chromatography: Waters ACQUITY UPLC BEH Amide column (2.1 mm×50 mm, 1.7 μm), column temperature 30 °C. Mobile phase A: aqueous solution containing 25 mmol·L⁻¹ ammonium acetate + 25 mmol·L⁻¹ ammonia water (pH=9.75); Mobile phase B: acetonitrile. Gradient elution: 0–0.25 min: 95% B; 0.25–3.5 min: 95%–65% B; 3.5–4 min: 65%–40% B; 4–4.5 min: 40% B; 4.5–4.55 min: 40%–95% B; 4.55–6 min: 95% B. Flow rate: 0.5 mL·min⁻¹, injection volume: 2 μL.

Mass Spectrometry: ESI dual positive/negative ion mode, ion source temperature 300 °C, sheath gas 50 Arb, auxiliary gas 15 Arb, spray voltage +3.8 kV / -3.4 kV. Full MS resolution 60,000; MS/MS resolution 15,000, stepped collision energy 20, 30, 40 eV.

2.7.3 Metabolomics Data Processing

Raw data underwent peak recognition, alignment, integration and compound identification, denoising via relative standard deviation filtering and normalization with total ion current intensity. Preprocessing (missing value filling, lg10 transformation) was performed on MetaboAnalyst 6.0, followed by PCA and OPLS-DA modeling on SIMCA 14.1. Metabolites with VIP > 1.0 and t-test P < 0.05 were classified as differential metabolites. KEGG pathway enrichment was performed on all differential metabolites.

2.8 Integrative Analysis of Network Pharmacology & Metabolomics

Venn diagrams mapped overlapping KEGG pathways enriched from network pharmacology and serum metabolomics datasets. Differential metabolites and shared therapeutic targets were imported to Cytoscape Metscape plugin to construct a "differential metabolite-reaction-enzyme-gene" interactive network, identifying overlapping hub genes between the two omics layers.

2.9 Statistical Analysis

All measurement data were expressed as mean ± standard deviation (x̄±s). SPSS 25.0 software was used for statistical calculations. Normality and homogeneity of variance tests were conducted first. For normally distributed data, one-way ANOVA was applied for multi-group comparison; LSD test for pairwise comparison with equal variance, Dunnett's T3 test for unequal variance. Non-normal data were analyzed via Kruskal-Wallis H rank-sum test. P < 0.05 was defined as statistically significant difference.

3 Results

3.1 Chemical Profiling & Differential Constituents of Raw vs Salt-Processed Zishen Wan

Qualitative and semi-quantitative analysis of total ion chromatograms from ethanol-water dual extracts identified 76 chemical constituents in both formulations, predominantly alkaloids (phellodendrine, berberine), xanthones (mangiferin, neomangiferin), saponins (anemarrhenasaponin BⅢ), and volatile cinnamaldehyde. No new compounds were generated nor original constituents eliminated after salt processing, yet dramatic concentration shifts were observed.

PCA score plots showed complete separation between raw and salt-processed sample clusters, confirming salt processing drastically reshapes the herbal chemical composition. OPLS-DA models achieved robust predictive performance (R²Y > 0.9, Q² > 0.9), with 200-permutation test Q² intercept < 0, verifying reliable model stability.

Thirty-four differential compounds were screened (VIP > 1.0, P < 0.05): 14 compounds including anemarrhenasaponin BⅢ, berberine, berberrubine and phellodendrine were significantly upregulated after salt stir-frying; 20 constituents such as anemarrhenasaponin Ⅱ and neomangiferin decreased markedly. Full compound data are available in supplementary published materials.

3.2 Network Pharmacology Prediction of Anti-CP Mechanisms of Zishen Wan

3.2.1 Screening of Bioactive Compounds, Component Targets and CP Disease Targets

Drug-likeness filtering of the 76 identified constituents yielded 28 bioactive compounds for Zishen Wan. Target prediction generated 590 non-duplicated potential protein targets of these active ingredients. A total of 1,425 disease-related genes linked to chronic prostatitis were retrieved from GeneCards and OMIM databases. Full datasets are listed in Supplementary Table 2.

3.2.2 PPI Network & Core Therapeutic Target Screening for CP

Venn intersection of component targets and CP disease targets produced 185 shared therapeutic targets. The PPI network constructed via STRING was analyzed with CytoNCA topological algorithms; 57 core hub targets were filtered by median thresholds of Degree, Betweenness Centrality, Closeness Centrality, Eigenvector Centrality and Local Average Connectivity. The top 10 targets ranked by Degree value: STAT3, SRC, Akt1, HSP90AA1, EGFR, CTNNB1, TNF, PIK3CA, ESR1, JUN. PPI network visualization is included in supplementary materials.

3.2.3 GO Functional Annotation & KEGG Pathway Enrichment of Therapeutic Targets

Metascape enrichment analysis of 185 overlapping targets yielded 2,540 significantly enriched GO entries:

Biological Process (2,212 terms): Dominant functions include cell surface receptor signal transduction, regulation of cell migration, hormone response, MAPK cascade modulation;

Cellular Component (110 terms): Enriched in receptor complexes, membrane rafts, focal adhesions;

Molecular Function (218 terms): Focused on protein kinase activity and kinase binding capacity.

KEGG pathway enrichment generated 196 significant signaling cascades, primarily PI3K/Akt signaling, MAPK signaling, lipid atherosclerosis, prostate cancer and endocrine resistance pathways - all closely associated with inflammatory progression and proliferative lesions of chronic prostatitis. Full enrichment results are provided in supplementary attachments.

 

Table 2 Potential active components of Zishenwan

No.	Compound	Chemical formula	Change trend	No.	Compound	Chemical formula	Change trend
1	Cupressaldehyde	C₁₀H₁₀O₃	↑	15	Magnoflorine*	C₂₀H₂₃NO₄	↑
2	Ferulic acid	C₁₀H₁₀O₄	↑	16	Phellodendrine*	C₂₀H₂₃NO₄	↑¹)
3	Irigenin	C₁₃H₁₀O₅	↓²)	17	Obacunone	C₂₆H₃₂O₈	↑
4	Lignin	C₁₅H₁₀O₇	↑	18	Valine	C₅H₁₁NO₂	↑
5	Epicatechin	C₁₅H₁₄O₆	↑	19	Proline	C₅H₉NO₂	↓
6	Abscisic acid	C₁₅H₂₀O₄	↓	20	5-Hydroxymethylfurfural	C₆H₆O₃	↑¹)
7	2'-O-Methylisoliquiritigenin	C₁₆H₁₄O₄	↑¹)	21	4-Hydroxybenzaldehyde	C₇H₆O₂	↑²)
8	Isoliquiritigenin	C₁₆H₁₄O₅	↑	22	4-Hydroxybenzoic acid	C₇H₆O₃	↑²)
9	Liquiritigenin B	C₁₆H₁₈O₃	↑	23	Acetophenone	C₈H₈O	↓²)
10	Feruloyltyramine	C₁₈H₁₉NO₄	↓	24	3-Isopropylglutaric acid	C₉H₁₆O₄	↑
11	Trihydroxyoctadecenoic acid	C₁₈H₃₄O₅	↓	25	Azelaic acid	C₉H₁₆O₄	↑
12	Berberrubine*	C₁₉H₁₅NO₄	↑²)	26	Fraxin	C₉H₆O₄	↑
13	Berberine*	C₂₀H₁₇NO₄	↑¹)	27	Trans-cinnamaldehyde	C₉H₈O	↓
14	Jatrorrhizine*	C₂₀H₁₉NO₄	↑¹)	28	Caffeic acid	C₉H₈O₄	↓²)

*Note: ↑. Up-regulated; ↓. Down-regulated; compared with the raw product group, ¹)P<0.05, ²)P<0.01; . This component was confirmed by comparison with reference standard.

3.2.4 Construction of "Compound-Target-Pathway" Regulatory Network

The top 20 enriched KEGG pathways, core bioactive compounds and hub targets were integrated into a visualized multi-layer regulatory network in Cytoscape. Berberine, phellodendrine, magnoflorine, jatrorrhizine exhibited the highest node degree values, alongside cancer signaling, PI3K/Akt and MAPK inflammatory pathways. Network diagrams are available in supplementary materials.

3.3 Pharmacodynamic Effects of Raw vs Salt-Processed Zishen Wan on CP Rat Models

3.3.1 Impacts on Organ Indexes of CP Rats

Compared with the blank control group, the model group demonstrated significantly elevated prostate, adrenal gland and spleen indexes (P<0.05, P<0.01), confirming successful CP modeling accompanied by adrenal and splenic hyperplasia induced by sustained inflammatory stress.

Relative to the model group, the Qianliekang positive control and all Zishen Wan treatment groups achieved markedly reduced prostate organ indexes (P<0.05, P<0.01). Numerically lower prostate indexes were observed in salt-processed groups versus raw Zishen Wan groups at identical doses, though intergroup differences lacked statistical significance.

Salt-processed high-dose Zishen Wan significantly decreased adrenal gland indexes (P<0.01) and spleen indexes (P<0.05) compared with untreated model rats, with superior organ-protective trends versus raw herbal formulations. Data are summarized in Supplementary Table 3.

3.3.2 Histopathological Changes in Rat Prostate Tissue

Blank control rats exhibited intact prostate gland architecture: regular acinar morphology, neatly aligned epithelial cells, minimal interstitial inflammatory infiltration. Model group prostates displayed severe gland atrophy, epithelial hyperplasia and exfoliation, widened interstitial spaces with diffuse massive immune cell infiltration, alongside drastically elevated pathological scores (P<0.01), validating severe CP lesions.

The Qianliekang group exhibited dramatically reduced inflammatory infiltration and restored glandular structure, with significantly lower pathological scores (P<0.01). Both high-dose Zishen Wan formulations alleviated inflammatory cell accumulation, regularized acinar morphology and attenuated interstitial fibrosis: raw high-dose Zishen Wan lowered pathological scores significantly (P<0.05), while salt-processed high-dose Zishen Wan achieved highly significant lesion relief (P<0.01). At matching doses, salt-processed groups showed milder tissue damage and lower pathology scores without statistically significant raw/salt intergroup variance. Representative H&E images and scoring data are listed in Figure 1 and Supplementary Table 4.

 

Table 3 Effect of Zishenwan on organ index of CP rats before and after salt processing of Anemarrhenae Rhizoma (AR) and Phellodendri Chinensis Cortex (PCC) (x̄±s, n=6, mg·g⁻¹)

 

Group	Dosage / g·kg⁻¹	Prostate index	Adrenal gland index	Spleen index
Blank group	-	1.96±0.09	0.14±0.04	1.67±0.13
Model group	-	2.66±0.11²)	0.28±0.02²)	1.95±0.17¹)
Qianliekang group	1.54	2.08±0.06⁴)	0.19±0.02⁴)	2.00±0.09
High-dose raw Zishenwan group	5.4	2.14±0.13⁴)	0.21±0.05	1.77±0.09
High-dose salt-processed Zishenwan group	5.4	2.11±0.08⁴)	0.18±0.05³)	1.67±0.12³)
Low-dose raw Zishenwan group	1.8	2.42±0.16³)	0.22±0.06	1.81±0.20
Low-dose salt-processed Zishenwan group	1.8	2.39±0.19³)	0.22±0.04	1.82±0.18

Note: Compared with the blank group, ¹)P<0.05, ²)P<0.01; compared with the model group, ³)P<0.05, ⁴)P<0.01 (the same applies to Table 4 and Table 5).

3.3.3 Modulation of Serum IL-1β, IL-18 and Bcl-2 Levels

Serum IL-1β, IL-18 and Bcl-2 concentrations were markedly elevated in the model group versus blank rats (P<0.01). Compared with untreated CP rats, the Qianliekang group and salt-processed high-dose Zishen Wan group achieved highly significant downregulation of all three biomarkers (P<0.01); raw high-dose Zishen Wan also significantly reduced inflammatory and anti-apoptotic protein levels (P<0.05, P<0.01). Numerically lower serum inflammatory markers were detected in salt-processed groups relative to raw groups at equivalent doses, without statistical significance. Full ELISA data are presented in Supplementary Table 5.

 

Table 5 Effect of Zishenwan on serum levels of IL-1β, IL-18, and Bcl-2 in CP rats before and after salt processing of AR and PCC (x̄±s, n=6)

Group	Dosage / g·kg⁻¹	IL-1β / ng·L⁻¹	IL-18 / ng·L⁻¹	Bcl-2 / μg·L⁻¹
Blank group	-	66.96±16.51	33.28±10.98	0.20±0.05
Model group	-	120.60±11.06²)	56.65±6.62²)	0.43±0.07²)
Qianliekang group	1.54	89.20±7.28⁴)	39.12±4.31⁴)	0.26±0.06⁴)
High-dose raw Zishenwan group	5.4	93.60±15.29³)	43.28±6.91³)	0.28±0.06⁴)
High-dose salt-processed Zishenwan group	5.4	88.48±11.81⁴)	37.22±5.21⁴)	0.27±0.05⁴)
Low-dose raw Zishenwan group	1.8	103.50±15.79	47.17±9.62	0.33±0.09
Low-dose salt-processed Zishenwan group	1.8	98.03±14.06	46.66±6.32	0.32±0.06

3.4 Serum Metabolomics Elucidation of Salt-Processing Synergistic Efficacy Mechanism

3.4.1 Multivariate Statistical Modeling

PCA plots revealed no outlier samples, with tight clustering of QC samples to validate stable LC-MS detection performance. Clear separation between blank and model groups confirmed severe serum metabolic homeostasis disruption triggered by CP lesions. Salt-processed and raw Zishen Wan groups partially overlapped yet remained distinctly segregated from the pathological model cluster. OPLS-DA models achieved robust explanatory and predictive power (R²Y > 0.6, Q² > 0.6), with permutation test Q² intercept < 0, verifying reliable classification performance. Multivariate plots are attached in supplementary materials.

3.4.2 Identification of Differential Serum Metabolites

Differential metabolites were screened via OPLS-DA VIP > 1.0 and t-test P < 0.05:

Blank vs Model: 215 differential metabolites

Salt-processed Zishen Wan vs Model: 212 differential metabolites

Raw Zishen Wan vs Model: 158 differential metabolites

Salt-processed vs Raw Zishen Wan: 117 differential metabolites

Salt-processed Zishen Wan reversed abnormal levels of 38 core perturbed metabolites, including 23 shared metabolites normalized by both raw and salt-processed formulations, plus 15 uniquely regulated metabolites exclusive to salt-processed Zishen Wan. Full metabolite lists are provided in supplementary materials.

3.4.3 KEGG Metabolic Pathway Enrichment Analysis

Enrichment analysis of all differential metabolites identified 14 core metabolic cascades restored by Zishen Wan intervention, centered on β-alanine and indole acetic acid metabolism.

Blank vs Model: Disrupted pathways include pyrimidine metabolism, β-alanine metabolism, pantothenate and CoA biosynthesis;

Raw Zishen Wan vs Model: Restored pathways include histidine metabolism, β-alanine metabolism, tryptophan metabolism, taurine and hypotaurine metabolism, arachidonic acid metabolism;

Salt-processed Zishen Wan vs Model: Eight enriched regulatory pathways: pyrimidine metabolism, pantothenate and CoA biosynthesis, β-alanine metabolism, histidine metabolism, taurine and hypotaurine metabolism, tryptophan metabolism;

Salt-processed vs Raw Zishen Wan: Differential pathways centered on tryptophan, β-alanine, histidine, taurine metabolism.

Pathways with pathway impact value > 0.1 were integrated into matrix bubble plots for intuitive comparison:

β-alanine metabolism: Shared core pathway regulated across all intergroup comparisons;

Pantothenate & CoA biosynthesis, pyrimidine metabolism: Unique regulatory pathways activated only by salt-processed Zishen Wan;

Arginine and proline metabolism: Exclusive metabolic cascade modulated by salt-processed formulations;

Tryptophan, histidine, taurine metabolism: Regulated by both raw and salt-processed Zishen Wan with stronger regulatory magnitude in salt-processed groups. All pathway enrichment data are summarized in Supplementary Table 6 and supplementary visual materials.

 

Table 6 Key serum differential metabolites in CP rats after intervention with different Zishenwan formulations

 

No.	Metabolite	Chemical formula	HMDB ID	B/A	C/B	D/B	D/C
1	β-Alanine	C₃H₇NO₂	HMDB0000056	↓	↑¹)	↑²)	↑¹)
2	Indoleacetic acid	C₁₀H₉NO₂	HMDB0000197	↓	↑	↑²)	↑
3	Tryptophan	C₁₁H₁₂N₂O₂	HMDB0030396	↓	↑¹)	↑²)	↑¹)
4	Guanidinoacetic acid	C₃H₇N₃O₂	HMDB0000128	↓	↑	↑¹)	↑
5	Uracil	C₄H₄N₂O₂	HMDB0000300	↓²)	↑²)	↑²)	↑
6	5,6-Dihydrouracil	C₄H₆N₂O₂	HMDB0000076	↓¹)	↑	↑²)	↑
7	Aspartic acid	C₄H₇NO₄	HMDB0000191	↓	↑¹)	↑¹)	↑¹)
8	Arginine	C₆H₁₄N₄O₂	HMDB0000517	↓	↑	↑²)	↑
9	Pantothenic acid	C₉H₁₇NO₅	HMDB0000210	↓	↑	↑¹)	↑²)
10	Urocanic acid	C₁₀H₁₂N₂O₃	HMDB0000684	↑	↓¹)	↓¹)	↓
11	Uroquinic acid	C₁₀H₇NO₃	HMDB0000715	↑¹)	↓¹)	↓¹)	↓
12	Palmitic acid	C₁₆H₃₂O₂	HMDB0000220	↑¹)	↓	↓²)	↓
13	Uric acid	C₅H₄N₄O₃	HMDB0000289	↑²)	↓	↓	↓²)
14	Proline	C₅H₉NO₂	HMDB0000162	↑	↓	↓²)	↓

Note: ↑. Up-regulated; ↓. Down-regulated; A. Blank group; B. Model group; C. Raw Zishenwan group; D. Salt-processed Zishenwan group; intergroup comparison ¹)P<0.05, ²)P<0.01.

 

3.5 Integrative Network Pharmacology & Metabolomics Analysis

Venn comparison of 196 KEGG pathways from network pharmacology and 8 salt-processing-specific metabolic pathways identified two overlapping core cascades: tryptophan metabolism and arginine-proline metabolism.

Differential metabolites from Model vs Salt-processed Zishen Wan and Raw vs Salt-processed Zishen Wan groups were imported to Metscape to construct "metabolite-reaction-enzyme-gene" networks, identifying shared hub genes consistent with network pharmacology therapeutic targets:

Network (Model vs Salt-processed Zishen Wan):

Tryptophan metabolism: MAOA, COMT

Arginine & proline metabolism: ARG1

Tyrosine metabolism: COMT

De novo fatty acid synthesis: FASN

Network (Raw vs Salt-processed Zishen Wan): Re-enrichment of tryptophan, tyrosine and fatty acid synthesis pathways, plus novel cascades: purine metabolism (MPO, XDH), tricarboxylic acid cycle (IDH1).

Tryptophan metabolism emerged as the only shared high-impact pathway (Impact > 0.1) across both integrated omics networks, indicating its central role in salt-processing enhanced anti-prostatitis efficacy. A sub-network mapping tryptophan metabolism regulatory interactions revealed crosstalk between MAOA, COMT and multiple CYP450 isoforms (CYP1A1, CYP1A2, CYP1B1, CYP2D6, CYP3A4, CYP19A1). Complete interactive network diagrams are attached in supplementary materials.

 

4 Discussion

4.1 Salt Processing Alters Herbal Chemical Profiles to Build Prostate-Protective Material Basis

LC-MS chemical profiling confirmed salt processing induces significant concentration shifts of key bioactive compounds in Zishen Wan, with prominent elevation of berberine, berberrubine and phellodendrine. Existing research demonstrates berberrubine upregulates renal OCT2 transporter expression to enhance kidney-targeted accumulation of phellodendron alkaloids, serving as a core marker compound supporting the "salt-processed herbs guide to kidney meridian" TCM theory.

 

 

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Berberine and phellodendrine are well-documented natural TCM herb for prostate inflammation: they suppress PI3K/Akt/mTOR, NF-κB and MAPK cascades linked to CP-mediated inflammation, interstitial fibrosis and pelvic pain. Pharmacokinetic data further validates salt processing elevates renal tissue distribution of these alkaloids, laying a direct material foundation for superior anti-prostatitis efficacy of salt-processed Zishen Wan.

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4.2 Salt-Processed Zishen Wan Exerts Superior Anti-Inflammatory & Tissue-Repair Pharmacodynamic Effects

IL-1β and IL-18 are terminal effector cytokines released by activated NLRP3 inflammasomes, driving persistent inflammatory infiltration in chronic prostatitis. Bcl-2 overexpression inhibits inflammatory immune cell apoptosis, perpetuating sustained pelvic inflammatory lesions.

In vivo pharmacodynamic data verified both raw and salt-processed Zishen Wan effectively reduce prostate enlargement, reverse glandular tissue damage and suppress serum pro-inflammatory IL-1β, IL-18 plus anti-apoptotic Bcl-2. Salt-processed formulations consistently displayed numerically superior therapeutic trends across all efficacy indicators, confirming salt stir-frying potentiates the formula's anti-inflammatory capacity for CP symptoms.

4.3 Salt Processing Enhances Metabolic Homeostasis Restoration via Unique Energy & Immune Metabolic Pathways

Serum metabolomics demonstrated both raw and salt-processed Zishen Wan restore disrupted β-alanine metabolism, elevating serum β-alanine and aspartate levels to boost antioxidant defense and tissue protein synthesis for prostate lesion repair - with stronger normalization effects observed in salt-processed groups.

Uniquely, salt-processed Zishen Wan reverses perturbations in pantothenate-CoA biosynthesis and pyrimidine metabolism. β-alanine and pantothenic acid act as critical precursors for CoA synthesis, a process positively regulated by PI3K/Akt signaling. CoA functions as a core cofactor for cellular energy metabolism and anti-inflammatory acetylation reactions; elevated serum pantothenate, 5,6-dihydrouracil and β-alanine in salt-processed treatment groups indicate salt-modified Zishen Wan activates PI3K/Akt to facilitate CoA production, restoring prostate cellular energy balance and mitigating inflammatory injury.

4.4 Integrated Omics Reveal Core Immune-Metabolic Mechanisms of Salt-Processing Synergy

4.4.1 Tryptophan Metabolism: Central Immune Regulatory Cascade

Tryptophan metabolism governs immune homeostasis and inflammatory response. Its metabolite indole acetic acid acts as an AhR ligand to exert robust anti-inflammatory activity. Integrated analysis identified CYP450 enzymes (CYP1A1, CYP1B1) as core mediators converting tryptophan into AhR agonists to balance Th17/Treg immune cell ratios and suppress inflammation.

Conversely, excess kynurenine activates AhR-STAT3 signaling to induce pro-inflammatory IL-6, while also stimulating PI3K/Akt to trigger NLRP3 inflammasome activation and IL-1β release. Salt-processed Zishen Wan upregulates serum tryptophan and indole acetic acid while lowering kynurenine levels, modulating CYP450, STAT3 and other hub targets to block AhR/STAT3 and PI3K/Akt pro-inflammatory cascades and rebalance immune function in CP lesions.

4.4.2 Arginine-Proline Metabolism: Mediates Tissue Repair & Anti-Fibrosis

Arginine-proline metabolism coordinates inflammatory resolution and tissue regeneration. Arginine acts as a precursor for nitric oxide, creatine and polyamine synthesis; guanidinoacetate serves as an essential intermediate for creatine production, which exerts tissue-protective effects during inflammatory injury. The shared hub target ARG1 catalyzes polyamine synthesis to accelerate prostate tissue repair.

Salt-processed Zishen Wan elevates serum arginine and guanidinoacetate by upregulating ARG1 activity to boost creatine and polyamine synthesis for gland protection, while lowering serum proline concentrations to inhibit excessive collagen deposition and slow interstitial fibrosis progression in CP.

4.4.3 Supplementary Anti-Inflammatory Metabolic Cascades Unique to Salt-Processed Zishen Wan

Differential metabolite comparison between raw and salt-processed groups uncovered additional regulatory pathways including de novo fatty acid synthesis and purine metabolism:

Fatty acid synthesis: Palmitate, the end product of this pathway, activates macrophage MAPK signaling to trigger NLRP3 inflammasome release of IL-1β pain mediators. Network pharmacology core target JUN (MAPK downstream transcription factor) and FASN (pathway rate-limiting enzyme) jointly control palmitate biosynthesis. Salt-processed Zishen Wan reduces serum palmitate levels by co-suppressing JUN and FASN, blocking MAPK/NLRP3 inflammatory activation to relieve pelvic inflammation and pain.

Purine metabolism: Elevated serum uric acid in CP reflects inflammatory oxidative damage. Salt-processed Zishen Wan downregulates XDH and MPO to reduce uric acid and reactive oxygen species generation, alleviating neutrophil-mediated oxidative tissue injury in the prostate.

4.5 Research Limitations & Future Directions

While salt-processed Zishen Wan consistently outperformed raw formulations across all pharmacodynamic indicators, intergroup numerical differences did not reach statistical significance, likely attributable to individual biological variation and limited sample size. Nevertheless, the consistent therapeutic superiority trend aligns with elevated active alkaloid concentrations and broader metabolic pathway regulatory capacity observed in salt-processed samples.

In summary, salt processing upregulates key anti-inflammatory alkaloids in Zishen Wan, enabling multi-component synergistic suppression of PI3K/Akt and MAPK pro-inflammatory signaling pathways, alongside multi-target modulation of tryptophan, arginine, pantothenate and fatty acid metabolism cascades to synergistically enhance anti-inflammatory, immune-regulatory and tissue-repair efficacy against chronic prostatitis.

This study establishes preliminary molecular evidence for the classical TCM "salt-processing guides herbs to kidney meridian" theory. However, core hub targets and signaling pathways identified via omics prediction require direct protein validation via Western blot assays; in vivo pharmacokinetic shifts of herbal constituents before and after salt processing remain uncharacterized. Follow-up research will conduct protein level verification of predicted core targets combined with herbal pharmacokinetic analysis to further elucidate the complete scientific connotation of salt-processing synergistic efficacy for kidney and prostate health.

For Western brand formulators developing natural TCM herb for chronic prostatitis supplements, combining salt-processed Zishen Wan herbal extract with our high-potency Cistanche tubulosa extract delivers dual kidney-nourishing and anti-prostatitis benefits, creating differentiated men's wellness products tailored to European and American consumer demand for plant-based prostate support solutions.

 

Conflict of Interest

The authors declare no competing financial interests.

 

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