Huyan-I formula attenuates renal senescence and fibrosis by inhibiting STAT3/NF-κB/NLRP3-driven SASP

Abstract

The Huyan-I formula (HY-I), a patented six‑herb traditional Chinese medicine used clinically at Jiangsu Provincial Hospital of TCM for early‑stage chronic kidney disease (CKD), has demonstrated therapeutic efficacy; however, its molecular mechanisms remain unclear. Given the central role of cellular senescence and fibrosis in CKD progression, particularly through the senescence‑associated secretory phenotype (SASP), this study investigated whether HY-I ameliorates renal senescence and fibrosis by modulating SASP‑related signaling. By UPLC‑Q‑TOF–MS analysis, 211 components were identified in HY‑I, and 29 prototype compounds plus 19 metabolites were detected in the serum of HY‑I‑treated mice. Network pharmacology and molecular docking predictions indicated interactions with the STAT3/NF‑κB/NLRP3/SASP axis. In naturally aged mice and in D‑galactose‑induced senescent human renal tubular (HK‑2) cells treated with HY-I or resveratrol (positive control), HY-I significantly reduced senescent cell burden and senescence markers, suppressed activation of STAT3, NF‑κB, and NLRP3, downregulated SASP expression, and improved tubular injury and renal fibrosis as assessed by Western blot, immunohistochemistry, multiplex immunofluorescence, and histopathological staining (HE, Masson, PAS). These findings demonstrate that HY-I alleviates renal aging and fibrosis by inhibiting the STAT3/NF‑κB/NLRP3 pathway and attenuating the SASP, thereby providing a mechanistic basis for its clinical application in CKD.

Graphical abstract

Introduction

Chronic kidney disease (CKD) represents a major global public health challenge, affecting approximately 10% of the world’s population [1]. Beyond its association with aging and morbidity, intrinsic renal aging processes significantly increase susceptibility to CKD progression in the elderly [2].

Renal fibrosis, characterized by excessive extracellular matrix (ECM) accumulation in glomeruli, tubulointerstitium, and vasculature, is a hallmark pathological feature of CKD that ultimately leads to end-stage kidney disease (ESKD) [3]. Substantial evidence implicates cellular senescence as a key driver of renal aging and fibrosis [4]. Senescent cells accumulate in aged and diseased kidneys, secreting a senescence-associated secretory phenotype (SASP) that includes proinflammatory cytokines (IL-1β, IL-6), chemokines (IL-8, MCP-1), growth factors (TGF-β, VEGF), and proteases. These SASP components collectively promote fibrosis by inducing tubular epithelial injury, fibroblast activation, ECM degradation, and fibrous deposition.

Transcriptional regulation of SASP involves complex signaling networks. NF-κB serves as a master regulator of inflammatory responses, upregulating proinflammatory genes and chemokine-mediated immune cell recruitment [5]. NLRP3 inflammasome assembly, triggered by NF-κB, activates caspase-1 to process IL-1β/IL-18 [6]. STAT3 synergizes with NF-κB to amplify SASP transcription through IL-6 and NLRP3 upregulation [7].

Resveratrol, a natural polyphenol with documented anti-senescence and anti-inflammatory effects, inhibits NF-κB and STAT3 signaling and NLRP3 activation [8]. Notably, it rescues renal function in aging models and CKD settings [9], serving as our positive control for evaluating HY-I’s effects on senescence.

Traditional Chinese Medicine (TCM) formulas like HY-I offer multi-component, multi-target therapeutic strategies. HY-I contains six herbs: Astragali Radix, Abelmoschi Corolla, Centellae Herba, Salviae Miltiorrhizae Radix et Rhizoma, Glycyrrhizae Radix et Rhizoma, and Cordyceps mycelia. This patented formula, derived from Jiangsu nephropathy therapies, significantly reduces serum creatinine/Cystatin-C and improves eGFR in stage 2–3 CKD patients.

In this study, we identified HY-I’s bioactive serum components and predicted targets via network pharmacology. Using naturally aged mice and D-galactose-induced tubular senescence models, we investigated HY-I’s renoprotective effects and SASP-related mechanisms.

Material and methods

Preparation of HY-I and drug-containing serum

HY-I contains six medicinal components: Astragalus membranaceus (15 g), Abelmoschus Manihot (15 g), Centella asiatica (15 g), Salvia miltiorrhiza (10 g), Glycyrrhiza uralensis (4 g), and Mortierella sp. mycelia powder (3.75 g). All crude materials were authenticated by a medicinal plant identification specialist at Jiangsu Provincial Hospital of Traditional Chinese Medicine.

This formula was patent-certified. According to the Chinese patent (ZL202311543505.8), the individual herbal components were mixed in the specified proportions, decocted in water, and concentrated to a density of 1.25 g/ml.

Based on a standard adult dose (60 kg body weight), the equivalent mouse dose was calculated as 2.7 g/kg/day after adjustment for body surface area differences (using a standard conversion factor). Experimental groups received either this equivalent dose or a double dose. HY-I was dissolved in distilled water and administered via intragastric gavage.

For serum preparation, 4-month-old male C57BL/6 mice were administered HY-I at a dose of 2.7 g/kg daily for 7 consecutive days. Blood was collected 1.5 h after the final administration. Serum was separated and heat-inactivated at 56°C for 30 min.

Animal study and dosing regimen

8 2-month-old and 32 18-month-old male C57BL/6 mice were purchased from Huachuang Sino Technology Co., Ltd. (Jiangsu, China). All animals were housed under specific pathogen-free conditions at Nanjing University of Chinese Medicine, with controlled temperature at 25 °C, humidity at 45%, and a 12-h light/dark cycle. The study protocol received approval from the Institutional Animal Ethics Committee of Nanjing University of Chinese Medicine under approval number 202411A022, with all procedures complying with animal research guidelines.

The eight 2-month-old mice served as the young control group. 18-month-old mice were randomly divided into four experimental groups:

Aging group: no treatment;

HY-I low-dose group receiving 2.7 g/kg/d HY-I;

HY-I high-dose group receiving 5.4 g/kg/d HY-I.

Resveratrol group receiving 40 mg/kg resveratrol (B20044, Orileaf, Shanghai).

All drugs were administered once daily via intragastric gavage for a period of two months. At the end of the intervention period, mice were anesthetized with 3% isoflurane for blood collection and then humanely euthanized by CO₂ inhalation. Serum, urine, and kidney tissue samples were collected from the mice.

Identification of HY-I by UPLC-Q-TOF–MS

UPLC method for qualitative analysis

Thermo-Obritrap-QE-MS was used for quality control and chemical component identification of HY-I and its drug-containing serum. Qualitative analysis was performed on ACQUITY UPLC I-Class and the ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 µm) was used in the system. The PDA detector has a scanning range of 210—240 nm and is model ACQUITY UPLC. The column temperature was set to 45 ℃, the flow rate was set to 0.35 mL/min, the injection volume was 5 μL, and water containing 0.1% formic acid (A) and acetonitrile (B) was used as the mobile phase. The optimal linear gradient elution conditions were shown in Table 1.

Table 1.

The mobile phase gradient

Time (min)	A% (0.1% aqueous formic acid)	B% (acetonitrile)
0	95	5
2	95	5
4	70	30
8	50	50
10	20	80
14	0	100
15	0	100
15.1	95	5
16	95	5

UPLC-MS method for qualitative analysis

The UPLC-MS/MS analysis was conducted on a Thermo-Obritrap-QE HF mass spectrometer equipped. A detection mode was ESI-Negative and Positive ion mode, and the mass spectrometer parameters were set as Table 2.

Table 2.

Mass parameters

MS parameter	ESI-negative	ESI-positive
Spray voltage (V)	3800	−3200
Capillary temperature (°C)	320	320
Aux gas heater temperature (℃)	350	350
Sheath gas flow rate (Arb)	35	35
Aux gas flow rate (Arb)	8	8
S-lens RF level	50	50
Mass range (m/z)	100–1500	100–1500
Full ms resolution	60,000	60,000
MS/MS resolution	15,000	15,000
NCE/stepped NCE	10, 20, 40	10, 20, 40

Data processing

The UHPLC-MS/MS mode was applied with an Orbitrap resolution of 60,000 for full-MS and 15,000 for dd-MS2. The raw data was analyzed by Xcalibur3.0 software. Prior to pattern recognition, the raw data were processed using the metabolomics software Progenesis QI v3.0 (Nonlinear Dynamics, Newcastle, UK) for baseline filtering, peak identification, integration, retention time correction, peak alignment, and normalization. Compound identification was performed based on accurate mass, MS/MS fragments, and isotopic distribution patterns, with qualitative analysis conducted using the TCM database. The Traditional Chinese Medicine (TCM) database is a specialized repository meticulously developed for plant specimens. It encompasses comprehensive information on more than 5,000 reference standards of TCM components. These reference standards were procured from esteemed suppliers, including Chengdu Le Meitian Pharmaceutical Technology Co., Ltd. and Shanghai Yuanye Biotechnology Co., Ltd.

Compounds are initially screened based on a mass deviation not exceeding 5 ppm and a database match score higher than 50%. Finally, compounds are verified through a comparison of the parent ions and secondary fragment ions in the raw data with those reported in the literature.

Cell culture and drug treatment

HK-2 cells were cultured in DMEM/F12 medium (L310KJ, Basal Media) supplemented with 10% fetal bovine serum (FBS, catalog 10,099—141C, Gibco) at 37 °C under 5% CO₂.

Cells were treated with varying concentrations of D-galactose (catalog D8310, Solarbio), HY-I-containing serum, or 10 μM resveratrol (catalog B20044, Orileaf) as the positive control group. Cell viability was assessed using CCK-8 Kit (BMU106-CN, Abbkine).

Western blot

Cellular and renal homogenates were lysed using RIPA buffer. Proteins were separated by electrophoresis on 4–12% SDS-PAGE gels and transferred to PVDF membranes (MIPVH0010, Millipore, USA). Membranes were incubated with appropriate primary and secondary antibodies, followed by detection with ECL reagent (P90720, Millipore USA) and visualization using a gel imaging system (LAS 4000, FUJIFILM, Japan).

For membrane reprobing, PVDF membranes were incubated with western blot stripping buffer (WB6500, New Cell & Molecular Biotech, Suzhou, China) at room temperature for 20 min. After PBST washing, membranes were re-incubated with antibodies and re-detected as described above. The list of antibodies is provided in Table 6.

Table 6.

The main antibodies used in the present study

Reagent	Manufacturer	Item no
Protein ladder (1—180 kDa)	Thermo Fisher Scientific	26616
Anti-Ki-67 (D3B5) Rabbit mAb	Cell Signaling Technology	9129S
Anti-p21 Rabbit pAb	Wanlei bio	WL0362
Anti-β-actin antibody	Abcam	ab179467
Anti-p16 Rabbit pAb	Abcam	ab51243
Anti-TGFβ1 Rabbit pAb	Abclona	A15103
Anti-IL-1B Rabbit pAb	Wanlei bio	WL02257
Anti-STAT3 Rabbit pAb	Wanlei bio	WL01836
Anti-p-STAT3 Rabbit pAb	Wanlei bio	WLP0624
Anti-NF-κB Rabbit pAb	Wanlei bio	WL01980
Anti- p-NF-κB Rabbit pAb	Wanlei bio	WL02169
Anti-NLRP3 Rabbit pAb	Abmart	p60622r3f
Anti-Caspase-1/Cleaved Caspase-1 Rabbit pAb	Wanlei bio	WL03450
Anti-IL18 Rabbit pAb	Wanlei bio	WL01127
Anti-FN/Fibronectin Rabbit pAb	Wanlei bio	WL00712a
Anti-Collagen1 Rabbit pAb	Abclonal-	A1352
Anti-Vimentin Rabbit pAb	Abcam	ab8978
α-smooth muscle actin rabbit mAb	Abclonal	A17910
Anti-rabbit IgG, HRP-linked Antibody	Cell signaling technology	7074S
Anti-mouse IgG, HRP-linked Antibody	Cell signaling technology	7076S

Renal histopathological staining

Paraffin-embedded mouse renal sections underwent xylene deparaffinization and ethanol gradient rehydration, followed by staining with H&E (G1120,Solarbio,China), PAS (G1280,Solarbio,China), and Masson’s trichrome (G1340, Solarbio,China) according to the manufacturer’s protocol for fibrosis assessment.

Immunofluorescence and immunohistochemistry

For IHC, antigen retrieval was performed in citrate or EDTA buffer. Endogenous peroxidase was quenched with H₂O₂, followed by BSA blocking. Primary antibodies were applied at 4°C overnight. After PBS washing, secondary antibodies were incubated for 1 h at room temperature. Signal detection used DAB.

For IF, tissues fixed in 4% PFA were permeabilized and subjected to multiplex staining using TSA Kit (Aifang Biological,Hunan,China). Nuclei were counterstained with DAPI. Images were acquired using a fluorescence microscope (Axio Vert A1, ZEISS).

Renal function assessment

Mouse serum and urine samples were centrifuged and analyzed using commercial kits (C035—2—1, C012—2—1, C013—2—1, C011—2—1; Jiancheng, Nanjing, China) for creatinine, uric acid, blood urea nitrogen, and urinary protein according to manufacturer’s instructions.

ELISA assays

SASP-related factors were quantified using ELISA kits: IL-6 (AF2163-A), TGFβ1 (AF2135-A), TNFα (AF2132-A), IL-1β (AF2040-A) from Aifang Biological, MMP-9(E-EL-M3052), GDF15(E-EL-M0604) from Elabscience Biological. Renal homogenates were incubated with detection reagents per protocol. After TMB substrate incubation at 37 °C for 15 min, reactions were stopped and absorbance measured at 450 nm.

SA-β-gal activity staining

Cryosections (8 μm) and cultured cells were stained using SA-β-gal staining kit (C0602, Beyotime, Shanghai, China) with nuclear fast red counterstain (G1320,Solarbio,Beijing,China) for tissue sections.

Establishment of a compound-target network

Collection of the targets

Targets of ingredients: The Canonical SMILES format of the HY-I ingredients was downlorded from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The potential targets for these ingredients were searched through the Swiss Target Prediction (http://swisstargetprediction.ch/).

Targets of kidney aging: kidney aging targets were identified using several databases, Gene Cards (https://www.genecards.org/), OMIM (https://omim.org/), Drug Bank (https://go.drugbank.com/), taking “kidney aging” as the search term. The targets gathered from these databases were subsequently merged, and any duplicate entries were eliminated. UniProt (https://www.uniprot.org) was employed to convert the targets into standardized Gene Symbols. Finally, the online Venn diagram website, Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/ venny/index.html), was utilized to visually represent a Venn diagram that described the intersection of HY-I and kidney aging. A compound-target network, based on HY-I compounds and their potential targets, was visualized using Cytoscape version 3.7.1.

Protein–protein interaction (PPI) network construction

The cross-target was uploaded to the STRING (https://www.string-db.org/) database, and the species “Homo sapiens” was selected. A protein–protein interaction (PPI) network was constructed with a high confidence level > 0.4. Subsequently, the network topology was analyzed and visualized with the aid of Cytoscape version 3.7.1.

GO and KEGG pathway enrichment

The intersected targets were entered into David (https://davidbioinformatics.nih.gov/) for biofunctional enrichment and pathway enrichment analysis. In accordance with the research topic, the top 30 results were selected based on their P-values to examine their main pathways and biological processes. Visualizations were generated using R software (v4.2.3).

Molecular docking

Molecular docking studies were performed to investigate the binding interactions between the main constituents of the HY-I formulation and disease-related targets. The three-dimensional structures of these compounds were retrieved from PubChem (https://www.ncbi.nlm.nih.gov/pccompound), and the corresponding protein structures of the targets were obtained from the Protein Data Bank (https://www.rcsb.org). Molecular docking was performed using AutoDock 4.2. After docking, the conformation exhibiting the lowest binding energy and the highest frequency of recurrent binding poses was selected as the optimal result. The selected complex was subsequently imported into PyMOL 3.1 and Discovery Studio 2021 for visualization and further structural analysis. The PDB ID corresponding to each docking target is listed in Table 7.

Table 7.

Molecular Docking

Target Gene	PBD ID	Ligand	PubChem ID	Binding Energy(kcal·mol-1)
STAS3	1BG1	Phloretin	4788	−3.66
		Luteolin	5280445	−5.19
		Pseudolaric acid C	6440704	−5.29
		18alpha-glycyrrhetinic acid	73398	−7.15
		Arjunolic acid	73641	−5.25
NLRP3	6NPY	Phloretin	4788	−5.18
		Luteolin	5280445	−5.99
		Pseudolaric acid C	6440704	−6.78
		18alpha-glycyrrhetinic acid	73398	−8.82
		Arjunolic acid	73641	−7.37
NFKB	1MY7	Phloretin	4788	−4.07
		Luteolin	5280445	−4.83
		Pseudolaric acid C	6440704	−5.59
		18alpha-glycyrrhetinic acid	73398	−7.16
		Arjunolic acid	73641	−5.46

Molecular Dynamics MD simulations were carried out with GROMACS 2022.3. The CHARMM36 force field described the proteins, and ligand parameters were derived from the CGenFF server. Each protein–ligand complex was centered in a 1.0‑nm cubic box filled with TIP3P water. NaCl was added to 0.15 M, and counterions neutralized the system.

Initial energy minimization used the steepest descent method (5000 steps, 0.01‑nm step size). The system was then equilibrated for 100 ps under NVT at 310 K (V‑rescale thermostat), followed by 100 ps under NPT at 1 bar (Parrinello–Rahman barostat). Bond lengths were constrained via LINCS. A 1.2‑nm cutoff was applied for non‑bonded interactions, and PME handled long‑range electrostatics.

Production runs lasted 100 ns with a 1 fs time step, saving coordinates every 10 ps. Each complex was simulated in triplicate. The four simulated complexes were: STAT3–18α‑glycyrrhetinic acid, NLRP3–18α‑glycyrrhetinic acid, NLRP3–arjunolic acid, and NF‑κB–pseudolaric acid C.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9.0. All values were expressed as mean ± standard errors of the means (SEMs). All the data were from at least three independent experiments. Differences among multiple groups were determined by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. Values were considered significantly different at p < 0.05.

Results

Major and serum-absorbed constituents of HY-I

The chemical constituents of HY-I (Fig. 1A, B and Table 3) and HY-I containing serum (Fig. 1E, F and Tables 4, 5) were analyzed using ultra-high performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry (Thermo Orbitrap QE). Based on the multistage mass spectrometry data of the samples and by referencing the natural product high-resolution mass spectrometry database, a total of 211 compounds were identified from HY-I, including 29 prototype components and 19 metabolites detected in serum (see Tables 6, 7). The following criteria were used to define blood‑absorbed components. A component was considered a blood‑absorbed prototype compound if it was present in either blank serum or drug‑containing serum, with a peak area ratio relative to blank serum ≥ FC (fold change threshold), or if the peak was undetectable in blank serum. A component detected in drug‑containing serum and verified by mass spectrometry as a metabolite of a prototype compound was designated as a blood‑absorbed metabolite.

Fig. 1.

Major and serum-absorbed constituents of HY-I. Typical total ion chromatograms (TIC) in both positive and negative ion modes. TIC chromatograms of HY-I in positive (A) and negative (B) ion modes. TIC chromatograms of Blank serum in positive (C) and negative (D) ion modes. TIC chromatograms of HY-I medicated serum in positive (E) and negative (F) ion modes

Table 3.

Identification of chemical constituents of HY-I based on Thermo-Obritrap-QE

No.	English name	RT	Formula	Calc. MW	Adducts	Error (ppm)	Theoretical Mass (m/z)	Experimental mass (m/z)	MS2 (m/z)	Score	Source
1	N6,N6,N6-Trimethyl-L-lysine	0.77	C9H20N2O2	188.1519	[M + H]+	−1.55	189.1598	189.1595	60.0813, 70.0656, 74.0718, 84.0812, 116.0707, 126.0916, 130.0861, 189.1346, 189.1593	53.7	/
2	Galactose 1-phosphate	0.77	C6H13O9P	260.0292	[M − H]−	−1.05	259.0224	259.0222	78.959, 96.9694, 259.0216	51.6	/
3	L-Histidine	0.79	C6H9N3O2	155.0689	[M − H]−	−1.01	154.0622	154.0620	93.0458, 94.9245, 96.9599, 110.0723, 137.0352, 154.0618	57.2	Glycyrrhiza uralensis Fisch、Cordycepssinensis、Astragalus membranaceus、Abelmoschus manihot
4	Maltopentaose	0.79	C30H52O26	828.2741	[M + FA-H]−	0.17	873.2729	873.2730	89.0241, 96.9599, 179.0554, 208.554, 383.1181, 827.2669	48.2	Astragalus membranaceus
5	D-mannitol	0.81	C6H14O6	182.0785	[M − H]−	−1.30	181.0718	181.0715	59.0137, 71.0138, 73.0294, 85.0294, 89.0244, 101.0243, 119.0348, 163.0609, 181.0715	57.3	Cordycepssinensis
6	L-Arginine	0.81	C6H14N4O2	174.1111	[M − H]−	−1.20	173.1044	173.1042	131.0824, 156.0775, 173.1052	57.7	Astragalus membranaceus, Cordycepssinensis
7	Stachyose	0.81	C24H42O21	666.2213	[M − H]−	0.20	711.2201	711.2202	89.0244, 179.056, 383.1195, 665.2175	56.7	Glycyrrhiza uralensis、Astragalus membranaceus
8	L-Asparagine	0.81	C4H8N2O3	132.0529	[M − H]−	−1.39	131.0462	131.0460	73.0293, 85.0294, 88.0400, 95.0251, 103.2722, 113.0239, 113.0354, 114.0196, 115.0033, 131.0452	55.8	Glycyrrhiza uralensis Fisch、Astragalus membranaceus
9	D-Tagatose	0.83	C6H12O6	180.0628	[M − H]−	−0.99	179.0561	179.0559	59.0137, 71.0137, 85.0293, 89.0244, 101.0243, 113.0242, 119.0349, 121.0437, 161.0455, 179.0551	55.1	Cordycepssinensis、Astragalus membranaceus
10	Turanose	0.85	C12H22O11	342.1157	[M − H]−	−2.08	387.1145	387.1137	89.0251, 119.034, 179.0566, 341.1084	58.2	Astragalus membranaceus、Salvia miltiorrhiza Bunge、Cordycepssinensis
11	Manninotriose	0.85	C18H32O16	504.1685	[M − H]−	−1.30	549.1673	549.1666	71.0138, 89.0244, 179.0558, 221.0664, 503.1614, 549.1666	57.8	Astragalus membranaceus、Salvia miltiorrhiza Bunge
12	L-Glutamic acid	0.87	C5H9NO4	147.0526	[M − H]−	−1.23	146.0459	146.0457	93.8274, 95.9757, 99.4594, 102.0558, 125.5103, 126.4884, 128.0351, 140.2054, 142.9211, 146.0453	55	Glycyrrhiza uralensis Fisch、Cordycepssinensis、Astragalus membranaceus
13	D-altrofurano-heptulose-3	0.88	C7H14O7	210.0734	[M – 2OH – H]–	−1.46	191.0561	191.0558	85.0295, 87.0087, 111.0087, 173.0089, 191.0211, 191.0559	55.6	/
14	Gluconic acid	0.88	C6H12O7	196.0578	[M − H]−	−1.19	195.0510	195.0508	75.0087, 87.0087, 99.0087, 129.0192, 159.0296, 177.0403, 195.0507	58.5	Astragalus membranaceus、Glycyrrhiza uralensis、Cordycepssinensis
15	Sucrose	0.88	C12H22O11	342.1157	[M + Na]+	−3.44	365.1055	365.1043	185.0416, 203.052, 365.1039	56.1	Glycyrrhiza uralensis、Astragalus membranaceus
16	Malic acid	0.94	C4H6O5	134.0210	[M − H]−	−1.41	133.0142	133.0141	71.0138, 72.9929, 115.0036, 133.014	52.6	Glycyrrhiza uralensis、Astragalus membranaceus、Abelmoschus manihot
17	L-Threonine	0.95	C4H9NO3	119.0577	[M + H]+	0.32	120.0655	120.0656	56.05, 74.0605, 102.0548, 120.0557, 120.0651	54.4	Glycyrrhiza uralensis、Astragalus membranaceus、Abelmoschus manihot、Salvia miltiorrhiza Bunge
18	DL-Proline	0.95	C5H9NO2	115.0628	[M + H]+	1.42	116.0706	116.0708	70.0656, 116.0706	67.4	Glycyrrhiza uralensis、Astragalus membranaceus、Abelmoschus manihot、Astragalus membranaceus
19	DL-Valine	0.95	C5H11NO2	117.0784	[M + H]+	0.75	118.0863	118.0863	58.0657, 59.0735, 72.0813, 118.0863	53.2	Cordycepssinensis
20	Cytosine	0.99	C4H5N3O	111.0427	[M + H]+	2.03	112.0505	112.0508	69.0453, 70.0656, 95.0243, 112.0506	53.6	Glycyrrhiza uralensis Fisch、Cordycepssinensis
21	Cytidine	0.99	C9H13N3O5	243.0850	[M + H]+	−2.46	244.0928	244.0922	70.0656, 112.0506, 244.0784	52.4	Cordycepssinensis
22	Valylalanine	1.01	C8H16N2O3	188.1155	[M + H]+	−2.01	189.1234	189.1230	84.0813, 118.0863, 126.0913, 129.0551, 130.0499, 130.0862, 143.1177, 144.1017, 172.0961, 189.1243	53.1	/
23	Citric acid	1.03	C6H8O7	192.0265	[M − H]−	−0.90	191.0197	191.0196	85.0294, 87.0087, 111.0086, 129.019, 191.0193	60.2	Astragalus membranaceus、Glycyrrhiza uralensis Fisch、Salvia miltiorrhiza Bunge、Abelmoschus manihot
24	Pinitol	1.05	C7H14O6	194.0785	[M + FA-H]−	−1.19	239.0773	239.0770	193.0703, 195.0288, 195.1377, 201.9223, 238.8911, 239.0168, 239.0230, 239.0551, 239.0626, 239.0770	53.9	Astragalus membranaceus
25	L-Pipecolic acid	1.05	C6H11NO2	129.0784	[M + H]+	−0.49	130.0863	130.0862	70.0656, 84.045, 84.0812, 130.0497, 130.086	51.8	/
26	DL-Pyroglutamic acid	1.11	C5H7NO3	129.0420	[M − H]−	−1.36	257.0779	257.0776	128.0352, 239.0666, 257.0766	53	Glycyrrhiza uralensis、Cordycepssinensis、Astragalus membranaceus
27	Cyclic AMP	1.16	C10H12N5O6P	329.0520	[M − H]−	−1.93	328.0452	328.0446	134.047, 328.0442	53.8	Glycyrrhiza uralensis、Cordycepssinensis、Astragalus membranaceus
28	5′-GMP	1.16	C10H14N5O8P	363.0575	[M − H]−	−1.49	362.0507	362.0502	78.9589, 150.0417, 211.0005, 362.05	56.8	Cordycepssinensis
29	Cyclic GMP	1.18	C10H12N5O7P	345.0469	[M − H]−	−2.41	344.0402	344.0393	133.0153, 150.0419, 344.0389	58.2	Cordycepssinensis
30	Uridine	1.22	C9H12N2O6	244.0690	[M − H]−	−1.42	243.0623	243.0619	82.0297, 110.0247, 111.0195, 115.0034, 128.0352, 140.035, 152.0353, 153.0303, 200.0563, 243.0621	73.2	Glycyrrhiza uralensis、Cordycepssinensis、Astragalus membranaceus、Abelmoschus manihot
31	N-Acetyl-L-glutamic acid	1.26	C7H11NO5	189.0632	[M − H]−	−0.23	188.0564	188.0564	100.0767, 102.0559, 116.0714, 126.0275, 126.0558, 128.0351, 144.0664, 146.0455, 170.0456, 188.0563	58.6	Cordycepssinensis、Astragalus membranaceus
32	Adenosine	1.27	C10H13N5O4	267.0962	[M + H]+	−2.75	268.1040	268.1033	136.0616, 268.1034	61.1	Astragalus membranaceus、Salvia miltiorrhiza Bunge、Cordycepssinensis
33	L-2-Hydroxyglutaric acid	1.36	C5H8O5	148.0366	[M − H]−	−1.07	147.0299	147.0297	75.0085, 85.0292, 87.0088, 103.0393, 129.0195, 147.0296	53.5	/
34	Cordycepin	1.4	C10H13N5O3	251.1013	[M + H]+	−2.48	252.1091	252.1085	136.0615, 252.1076	65	Cordycepssinensis
35	Methylmalonic acid	1.4	C4H6O4	118.0261	[M − H]−	−0.66	117.0193	117.0193	73.0295, 99.0087, 116.9284, 117.0192	57.3	/
36	Guanosine	1.46	C10H13N5O5	283.0911	[M − H]−	−1.31	282.0844	282.0840	133.015, 150.042, 282.0836	55.9	Cordycepssinensis、Astragalus membranaceus、Salvia miltiorrhiza Bunge
37	L-Leucine	1.51	C6H13NO2	131.0941	[M + H]+	−0.75	132.1019	132.1018	86.097	68	Glycyrrhiza uralensis、Cordycepssinensis、Astragalus membranaceus、Abelmoschus manihot、Salvia miltiorrhiza Bunge
38	2-Methylcitric acid	1.55	C7H10O7	206.0421	[M − H]−	−0.69	205.0354	205.0352	71.0501, 87.0087, 99.045, 101.0243, 111.0086, 125.0242, 187.0244, 205.035	60.7	/
39	Scandoside	1.67	C16H22O11	390.1157	[M 2OH – H]–	−1.22	371.0983	371.0979	135.045, 173.0452, 179.0348, 191.0559, 197.0456, 209.03, 353.0881, 371.0988	61.3	/
40	Gallic acid	1.8	C7H6O5	170.0210	[M − H]−	−0.44	169.0142	169.0142	125.0243, 169.014	64.7	Glycyrrhiza uralensis、Cordycepssinensis、Astragalus membranaceus、Abelmoschus manihot、Salvia miltiorrhiza Bunge
41	2′-O-Methyladenosine	1.94	C11H15N5O4	281.1119	[M + H]+	−2.16	282.1197	282.1191	136.0615, 282.1182	51.5	Cordycepssinensis
42	Benzaldehyde	2.28	C7H6O	106.0413	[M + H]+	2.87	107.0491	107.0494	53.0393, 79.0547, 91.0545, 95.0497, 107.049	52.3	Cordycepssinensis
43	L-Phenylalanine	2.32	C9H11NO2	165.0784	[M − H]−	−1.46	164.0717	164.0715	72.0098, 96.9601, 96.9691, 115.9205, 116.9283, 134.8943, 147.0449, 164.0338, 164.0712	57.2	Glycyrrhiza uralensis、Cordycepssinensis、Astragalus membranaceus、Salvia miltiorrhiza Bunge
44	N-(1-Deoxy-D-fructos-1—yl)-L-phenylalanine	2.36	C15H21NO7	327.1313	[M + H]+	−3.37	328.1391	328.1380	120.0808, 132.0806, 166.0859, 264.1223, 292.1171, 310.1275, 328.1378	67.9	Astragalus membranaceus
45	Ser-Leu	2.6	C9H18N2O4	218.1261	[M + H]+	−1.47	219.1339	219.1336	60.0449, 86.0968, 132.1017, 173.128, 201.122, 203.1425, 205.1583, 219.1116, 219.1333	54.5	/
46	Glycylleucine	2.6	C8H16N2O3	188.1155	[M + H]+	−0.38	189.1234	189.1233	75.0558, 84.0811, 86.0968, 98.0599, 118.0862, 129.0542, 132.1017, 143.1175, 144.0655, 189.1117	59.3	/
47	Ethylmalonic acid	2.63	C5H8O4	132.0417	[M − H]−	−1.22	131.0350	131.0348	85.0297, 87.0451, 113.0243, 131.0348	53.3	/
48	2,4,6-Trihydroxybenzoic acid	2.75	C7H6O5	170.0210	[M − H]−	-1.17	169.0142	169.0140	125.0244, 151.0034, 169.014	58.3	/
49	Glycylisoleucine	2.95	C8H16N2O3	188.1155	[M + H]+	−0.59	189.1234	189.1233	86.0968, 132.1017, 143.1175	57.5	/
50	Vanillic acid 4-beta-D-glucopyranoside	3.1	C14H18O9	330.0945	[M − H]−	−0.93	329.0878	329.0875	108.0216, 123.0451, 152.0111, 167.0348, 329.0875	50.8	Salvia miltiorrhiza Bunge
51	Aspartyl-Leucine	3.15	C10H18N2O5	246.1210	[M + H]+	−2.03	247.1289	247.1283	141.1020, 155.1175, 166.0860, 183.1124, 187.1068, 201.1228, 212.0916, 229.1177, 247.1084, 247.1293	58.2	/
52	Glutamylleucine	3.2	C11H20N2O5	260.1367	[M + H]+	−2.37	261.1445	261.1439	84.0447, 86.0969, 102.0553, 132.1019, 197.1281, 225.124, 243.1333, 261.1235	55.7	/
53	Succinyladenosine	3.2	C14H17N5O8	383.1072	[M + H]+	−3.23	384.1150	384.1137	162.0771, 234.0609, 252.072, 384.113	60.8	Astragalus membranaceus
54	Protocatechuic acid	3.29	C7H6O4	154.0261	[M − H]−	−0.98	153.0193	153.0192	109.0293, 153.0188	60.6	Glycyrrhiza uralensis、Astragalus membranaceus、Abelmoschus manihot、Salvia miltiorrhiza Bunge
55	Serylphenylalanine	3.62	C12H16N2O4	252.1105	[M + H]+	−0.98	253.1183	253.1180	60.045, 70.0657, 120.0808, 166.0859, 197.1169, 207.1123, 235.1064, 253.1189	61.4	/
56	Caftaric acid	3.71	C13H12O9	312.0476	[M − H]−	−0.50	311.0409	311.0407	135.0445, 149.0087, 179.0346, 311.0385	55.9	Abelmoschus manihot
57	Glycyl-Phenylalanine	3.73	C11H14N2O3	222.0999	[M + H]+	−1.29	223.1077	223.1074	70.0656, 72.0813, 86.0969, 120.0808, 166.0859, 177.1018, 205.0969, 223.1074	61.1	/
58	Isoleucyl-Valine	3.73	C11H22N2O3	230.1625	[M + H]+	−1.42	231.1703	231.1700	72.0813, 86.0968, 132.1016, 185.1644, 231.17	60.1	/
59	Neochlorogenic acid	3.75	C16H18O9	354.0945	[M − H]−	−0.92	353.0878	353.0875	135.045, 179.0348, 191.0559, 353.0875	70.7	Abelmoschus manihot、Salvia miltiorrhiza Bunge
60	Alanylphenylalanine	3.81	C12H16N2O3	236.1155	[M + H]+	−1.69	237.1234	237.1230	84.0812, 86.0969, 120.0807, 142.0494, 166.0859, 175.1225, 191.1177, 192.0650, 237.0911, 237.1236	63.4	/
61	Hydroxyphenyllactic acid	3.85	C9H10O4	182.0574	[M − H]−	−0.77	181.0506	181.0505	71.0138, 72.9930, 89.0244, 101.0243, 119.0501, 135.0450, 137.0241, 163.0399, 181.0147, 181.0506	59.4	Cordycepssinensis、、Salvia miltiorrhiza Bunge
62	10-Hydroxymajoroside	3.93	C17H24O11	404.1313	[2 M-H]−	0.66	807.2564	807.2570	807.2570	41.3	Abelmoschus manihot
63	Aspartylphenylalanine	3.94	C13H16N2O5	280.1054	[M + H]+	−1.19	281.1132	281.1129	86.0967, 88.0396, 120.0807, 136.0614, 162.0408, 166.0859, 235.1072, 246.0754, 263.1020, 281.1120	57.4	/
64	N-Acetyl-L-tyrosine	3.97	C11H13NO4	223.0839	[M − H]−	−0.91	222.0772	222.0770	58.0295, 107.0495, 163.0395, 178.0861, 179.0352, 180.0664, 205.0344, 222.077	56.9	/
65	4-Carboxycarbostyril	4.07	C10H7NO3	189.0420	[M + H]+	−0.73	190.0499	190.0497	144.1017, 162.0545, 190.0495	59.5	/
66	3,4-Dihydroxybenzaldehyde	4.09	C7H6O3	138.0311	[M + H]+	0.20	139.0390	139.0390	65.039, 93.0336, 111.0442, 121.0282, 139.0387	57.1	Astragalus membranaceus
67	Cryptochlorogenic acid	4.17	C16H18O9	354.0945	[M − H]−	−1.49	353.0878	353.0873	135.045, 173.0452, 179.0348, 191.0559, 353.0874	65.6	Salvia miltiorrhiza Bunge、Abelmoschus manihot
68	Cis-Ferulic acid 4-O-beta-D-glucopyranoside	4.34	C16H20O9	356.1102	[M − H]−	−0.93	355.1035	355.1031	149.0605, 173.0450, 175.0513, 178.0267, 191.0195, 191.0556, 193.0496, 209.0300, 224.0675, 355.0953	55.9	/
69	Myricetin 3—o-beta-d-xylopyranosyl(1—2)-beta-D-glucopyranoside	4.42	C26H28O17	612.1321	[M − H]−	−0.32	611.1254	611.1252	271.0248, 316.022, 611.125	75.2	/
70	Cynarin	4.46	C25H24O12	516.1262	[M − H]−	−1.13	515.1195	515.1189	135.045, 161.0244, 179.0348, 191.0559, 335.0768, 353.0874, 515.119	69.1	/
71	3-O-caffeoylshikimic acid	4.48	C16H16O8	336.0840	[M − H]−	−0.61	335.0772	335.0770	93.0342, 111.0438, 135.0449, 155.0347, 161.0241, 173.045, 179.0347, 335.0775	63	/
72	3-O-p-coumaroylquinic acid	4.48	C16H18O8	338.0996	[M − H]−	−0.88	337.0929	337.0926	93.0348, 163.04, 173.0453, 191.0559, 337.0925	71.1	/
73	Luteolin-3′,7—di-glucuronide	4.5	C27H26O18	638.1114	[M − H]−	0.36	637.1046	637.1049	285.0397, 317.0295, 351.056, 461.0722, 479.0787, 637.1017	52.9	/
74	Salvianic acid c	4.5	C18H18O9	378.0945	[M − H]−	−0.93	377.0878	377.0875	72.993, 135.0451, 137.0243, 161.0241, 178.0508, 179.0345, 197.0454, 359.0769, 377.0931	65.7	Salvia miltiorrhiza Bunge
75	Schaftoside	4.52	C26H28O14	564.1474	[M + H]+	−1.07	565.1552	565.1546	451.1017, 457.1102, 469.1122, 481.1120, 493.1120, 499.1225, 511.1221, 529.1326, 547.1434, 565.1542	63.1	/
76	Quercetin 3-gentiobioside	4.54	C27H30O17	626.1478	[M + H]+	−1.84	627.1556	627.1544	85.0288, 303.049, 317.0651, 319.043, 465.1018, 481.0961	63.2	/
77	5,7-Dihydroxy-4-oxo-4H-chromene-2-carboxylic acid	4.56	C10H6O6	222.0159	[M − H]−	−0.50	221.0092	221.0091	133.0294, 177.0191, 177.055, 221.009	58	/
78	Quercetin 3-sambubioside	4.6	C26H28O16	596.1372	[M − H]−	0.00	595.1305	595.1305	271.0246, 300.027, 595.1301	60.4	Abelmoschus manihot
79	Doryphornine	4.62	C11H11NO3	205.0733	[M + FA-H]−	−0.98	250.0721	250.0719	88.0403, 115.0036, 132.0301, 135.0449, 250.0717	62	/
80	3-Feruloylquinic acid	4.62	C17H20O9	368.1102	[M − H]−	−1.32	367.1035	367.1030	93.0345, 134.0371, 173.0452, 191.0558, 193.0503, 367.1032	67.7	Salvia miltiorrhiza Bunge
81	(E/Z)-ferulic acid	4.62	C10H10O4	194.0574	[M -H2O + H] +	−0.35	177.0546	177.0546	76.0510, 89.0389, 117.0336, 118.0499, 145.0282, 149.0230, 149.0594, 160.0715, 163.0385, 177.0540	56.8	Astragalus membranaceus
82	Rutin	4.72	C27H30O16	610.1528	[M + H]+	0.59	611.1607	611.1610	85.0288, 303.049, 465.1019	72.3	Astragalus membranaceus、Abelmoschus manihot
83	Aloe-emodin-8-O-beta-D-glucopyranoside	4.79	C21H20O10	432.1051	[M − H]−	−1.41	431.0984	431.0978	113.0244, 139.1761, 153.0183, 175.0242, 208.4511, 208.4739, 209.3472, 255.0657, 431.0968	45.8	Astragalus membranaceus
84	Liquiritigenin-7—o-apiosyl(1—2)-glucoside	4.79	C26H30O13	550.1681	[M − H]−	−0.76	549.1614	549.1609	135.0086, 255.0661, 549.1613	61	Astragalus membranaceus
85	Taxifolin 7-O-rhamnoside	4.8	C21H22O11	450.1157	[M + H-H2O] +	−2.12	433.1129	433.1120	313.0695, 323.0909, 337.0698, 349.0700, 361.0684, 367.0800, 379.0804, 397.0909, 415.1018, 433.1120	54.2	/
86	Calycosin-7-O-beta-D-glucoside	4.82	C22H22O10	446.1207	[M + H]+	−3.29	447.1286	447.1271	285.0751, 447.1275	70.1	Astragalus membranaceus
87	Neoliquiritin	4.84	C21H22O9	418.1258	[M + H]+	−2.70	419.1337	419.1325	137.0229, 147.0437, 257.0802	69.6	Glycyrrhiza uralensis
88	Apigenin 7-O-(2G-rhamnosyl)gentiobioside	4.85	C33H40O19	740.2158	[M – 2OH – H]−	−0.42	721.1985	721.1982	353.0658, 383.0759, 457.1135, 463.0882, 721.1985	63.2	/
89	( +)-3,4′,5,7-Flaventetrol	4.86	C15H14O5	274.0836	[M + H-H2O] +	−2.00	257.0808	257.0803	137.0231, 147.0438, 257.0804	57.5	/
90	Liquiritin	4.87	C21H22O9	418.1258	[M − H]−	−1.78	417.1191	417.1184	119.0500, 135.0086, 153.0191, 175.0399, 179.0360, 197.0455, 255.0661, 373.0926, 417.0763, 417.1194	60.1	Glycyrrhiza uralensis
91	3-Coumaric acid	4.87	C9H8O3	164.0468	[M − H]−	−1.3	163.0401	163.0399	119.0501, 163.0399	57.3	/
92	Suberic acid	4.89	C8H14O4	174.0887	[M − H]−	−1.49	173.0819	173.0817	111.0814, 129.092, 173.0818	57.8	/
93	3,4-Dimethoxybenzyl alcohol	4.96	C9H12O3	168.0781	[M + NH4]+	−1.43	186.1125	186.1122	83.086, 111.0808	40.4	/
94	Quercetin 3-O-malonylglucoside	4.97	C24H22O15	550.0953	[M − H]−	−0.61	549.0886	549.0883	135.0086, 153.0191, 255.0658, 271.0244, 300.0271, 505.0982	69.3	Abelmoschus manihot
95	Vanillin	4.98	C8H8O3	152.0468	[M + H]+	−1.13	153.0546	153.0544	95.0860, 97.0649, 107.0858, 109.1015, 110.0364, 111.0442, 125.0597, 134.0596, 135.1166, 153.0543	57.8	Astragalus membranaceus、Glycyrrhiza uralensis Fisch、Salvia miltiorrhiza Bunge
96	Methyl protocatechuate	4.99	C8H8O4	168.0417	[M − H]−	−1.29	167.0350	167.0348	152.0114, 167.0348	69.5	/
97	Nepitrin	5.01	C22H22O12	478.1106	[M − H − H2O]−	−0.47	459.0933	459.0931	459.0931	42.5	/
98	1,3-Dicaffeoylquinic acid	5.01	C25H24O12	516.1262	2[M − H]-	−0.67	1031.2463	1031.2456	173.0453, 179.0347, 191.0559, 353.0875, 515.1187, 1031.2421	60.7	/
99	Breynioside a	5.01	C19H20O9	392.1102	[M – H] – 2OH	−1.45	373.0929	373.0923	72.993, 175.0398, 179.0347, 197.0453, 257.0826, 329.1031, 373.093	63.7	/
100	Isochlorogenic acid C	5.02	C25H24O12	516.1262	[M + H]+	−2.31	517.1341	517.1329	163.0386, 499.1227	59.4	Salvia miltiorrhiza Bunge、Abelmoschus manihot
101	Kaempferol 3-O-D-galactoside	5.03	C21H20O11	448.1000	[M − H]−	−1.49	447.0933	447.0926	255.0295, 284.0323, 447.0927	60.4	/
102	kaempferol-O-glucuronide	5.03	C21H18O12	462.0793	[M − H]−	−1.03	461.0725	461.0721	113.0244, 175.0245, 285.04, 461.0719	62.1	/
103	Trifolin	5.04	C21H20O11	448.1000	[M + H]+	−2.01	449.1078	449.1069	287.0544, 288.0575, 303.049	63.2	/
104	Gossypetin	5.09	C15H10O8	318.0370	[M − H]−	−4.54	317.0303	317.0288	139.0035, 166.9983, 317.0297	58.5	Abelmoschus manihot
105	Hibifolin	5.1	C21H18O14	494.0691	[M − H]−	−1.11	493.0624	493.0618	295.0602, 317.0298, 493.0532, 493.062	72.1	/
106	Astilbin	5.12	C21H22O11	450.1157	[M + H-H2O] +	−2.27	433.1129	433.1119	271.0594, 272.063, 433.1078	54.5	/
107	Salvianolic acid E	5.12	C36H30O16	718.1528	[M + Na]+	−1.93	741.1427	741.1412	70.0667, 86.0967, 345.0362, 363.0482, 381.0581, 543.0893, 561.0999, 741.1422	60.4	Salvia miltiorrhiza Bunge
108	Hydrangetin	5.12	C10H8O4	192.0417	[M + H]+	−1.23	193.0495	193.0493	81.0703, 133.0281, 133.1008, 193.0492	63.1	/
109	Salvianolic acid H	5.22	C27H22O12	538.1106	[M − H]−	−1.42	537.1038	537.1031	135.0449, 159.0444, 179.0348, 185.0242, 197.0443, 295.0608, 313.0703, 339.0503, 493.1133, 537.1030	74.4	Salvia miltiorrhiza Bunge
110	Spiraeoside	5.26	C21H20O12	464.0949	[M + H]+	−2.28	465.1028	465.1017	85.0288, 303.049, 465.1019	59.9	Abelmoschus manihot
111	Rosmarinic	5.26	C18H16O8	360.0840	[M − H]−	−1.52	359.0772	359.0767	72.993, 135.045, 161.0242, 179.0348, 197.0453, 359.0772	67.8	Astragalus membranaceus、Salvia miltiorrhiza Bunge
112	Licraside	5.28	C26H30O13	550.1681	[M − H]−	−0.82	549.1614	549.1609	119.05, 135.0086, 153.0191, 255.0661, 549.1614	66.7	Astragalus membranaceus
113	Isoliquiritin apioside	5.32	C26H30O13	550.1681	[M + H]+	−2.54	551.1759	551.1745	137.023, 147.0437, 257.0802, 303.049, 419.1327	65.3	Astragalus membranaceus
114	Salvianolic B	5.32	C36H30O16	718.1528	[M + NH4]+	−1.77	736.1872	736.1859	135.044, 139.0388, 163.0388, 181.0493, 277.0486, 295.059, 323.0541, 521.1069	66.4	Salvia miltiorrhiza Bunge
115	m-Methoxyphenol	5.32	C7H8O2	124.0519	[M + H – H2O]+	1.67	107.0492	107.0493	107.0493	41	/
116	5-Methoxyindoleacetate	5.34	C11H11NO3	205.0733	[M − H]−	−0.85	204.0666	204.0664	117.0708, 130.0668, 160.0765, 204.0662	71	/
117	Lithospermic acid	5.35	C27H22O12	538.1106	[M + H – H2O]+	−2.30	521.1078	521.1066	203.0340, 249.0542, 267.0640, 277.0487, 281.0439, 295.0596, 323.0543, 341.0644, 493.1131, 521.1086	56.4	Salvia miltiorrhiza Bunge
118	Salvianolic acid B	5.37	C36H30O16	718.1528	[M − H]−	0.08	717.1461	717.1462	185.024, 277.0501, 279.0291, 293.0456, 295.0606, 321.0399, 339.0504, 519.0926, 717.1456	67.3	Salvia miltiorrhiza Bunge
119	Asiaticoside B	5.37	C48H78O20	974.5081	[M + Na]+	−1.82	957.5053	957.5036	433.3095, 439.3193, 451.3198, 469.3303, 471.1704, 487.3404, 597.3752, 633.3982, 649.3926, 795.4539	55.9	Centella asiatica (L.) Urban
120	Beta-D-Glucopyranoside, 2-((benzoyloxy)methyl)-4-hydroxyphenyl	5.39	C20H22O9	406.1258	[M + Na]+	−1.64	429.1156	429.1149	429.1149	52.7	/
121	Isololiolide	5.41	C11H16O3	196.1094	[M + H]+	−1.67	197.1172	197.1169	107.0857, 133.101, 135.1166, 161.0958, 179.1061, 197.1152	62.8	/
122	6″-O-Acetylglycitin	5.44	C24H24O11	488.1313	[M + H]+	−2.75	489.1391	489.1378	285.0751, 489.1368	55.2	Astragalus membranaceus
123	Asiaticoside B	5.49	C48H78O20	974.5081	[M − H]−	−0.03	973.5008	973.5008	101.0242, 161.0453, 247.0811, 469.1555, 503.3367, 973.5008	63.9	Centella asiatica (L.) Urban
124	Isoliquiritin	5.51	C21H22O9	418.1258	[M − H]−	−1.17	417.1191	417.1186	119.0498, 135.0086, 148.0163, 153.019, 175.0399, 254.0582, 255.0661, 417.1176, 417.1257	64	Glycyrrhiza uralensis Fisch
125	Ononin	5.56	C22H22O9	430.1258	[M + H]+	−2.19	431.1337	431.1327	269.0803, 431.1286	62.5	Astragalus membranaceus
126	Oroxin A	5.57	C21H20O10	432.1051	[M − H]−	−1.54	431.0984	431.0977		56
127	Licorice glycoside A	5.57	C36H38O16	726.2154	[M − H]−	0.38	725.2087	725.2090	119.0498, 134.037, 135.0085, 153.0188, 175.0398, 193.0499, 255.0663, 531.1516, 549.1627, 725.2092	67.8	Glycyrrhiza uralensis Fisch
128	Isoononin	5.59	C22H22O9	430.1258	[M + FA – H]−	−0.90	475.1246	475.1242	252.0423, 267.066, 475.124	76.2	Astragalus membranaceus
129	Indole-3-acetic acid	5.61	C10H9NO2	175.0628	[M + H]+	−2.14	176.0706	176.0702	130.065, 149.0228, 176.0702	58.7	/
130	Salvianolic acid A	5.65	C26H22O10	494.1207	[M – H]–	−0.22	493.1140	493.1139	135.0452, 185.0242, 197.0451, 295.0608, 313.0713, 317.0298, 493.0486, 493.0592, 493.1143, 493.1212	56.2	Salvia miltiorrhiza Bunge
131	Scheffoleoside A	5.75	C48H78O19	958.5132	[M + H]+	−1.89	959.5210	959.5192	407.3304, 435.3241, 453.3352, 471.1745, 471.3463, 489.3565, 635.4145, 651.4111, 797.4689, 813.4622	61.3	Centella asiatica (L.) Urban
132	Madecassoside	5.78	C48H78O19	958.5132	[M − H]−	0.64	1003.5119	1003.5125	101.0243, 161.0455, 247.0822, 469.1559, 487.3423, 957.5062, 1003.5131	64.5	/
133	Licochalcone B	5.82	C16H14O5	286.0836	[M − H]−	−0.88	285.0768	285.0766	150.032, 270.0531, 285.0417, 285.0759	61.4	/
134	Lactiflorin	5.85	C23H26O10	462.1520	[M + NH4]+	−2.42	480.1865	480.1853	167.07, 301.1063, 463.1584	53.7	Astragalus membranaceus
135	3-O-a-Laminaribiosylplatycodigenin methyl ester	5.9	C43H70O17	858.4608	[M − H]−	0.66	857.4540	857.4546	193.0504, 323.0979, 487.3424, 811.45, 857.2621, 857.4526	65.8	/
136	7-O-Methylluteolin-6-C-beta-glucoside	5.94	C16H14O7	318.0734	[M – 2 OH – H]−	−0.94	299.0561	299.0558	256.0374, 269.0453, 284.0324, 284.0689, 299.0189, 299.0556	62	Astragalus membranaceus
137	Liquiritigenin	5.96	C15H12O4	256.0730	[M − H]−	0.49	255.0663	255.0664	119.05, 135.0086, 153.0191, 255.0661	61	Glycyrrhiza uralensis Fisch
138	Isomucronulatol 7-O-glucoside	5.96	C23H28O10	464.1677	[M − H]−	−1.07	463.1616	463.1616	135.0447, 161.0238, 197.0451, 286.0828, 301.1077, 311.0555, 333.0247, 347.1852, 463.1616	61.6	Astragalus membranaceus
139	Isoliquiritigenin	5.98	C15H12O4	256.0730	[M + H]+	−2.34	257.0808	257.0802	137.023, 138.0264, 147.0433, 148.0471, 155.1064, 257.0802	65.3	Glycyrrhiza uralensis Fisch
140	Luteolin	6	C15H10O6	286.0472	[M + H]+	−2.17	287.0550	287.0544	121.0285, 137.0231, 151.0388, 245.0803, 287.0535	45.6	Glycyrrhiza uralensis Fisch、Centella asiatica (L.) Urban
141	Sebacic acid	6.02	C10H18O4	202.1200	[M − H]−	−0.68	201.1132	201.1131	89.0246, 116.9284, 139.1126, 183.1018, 201.1129	66.2	/
142	Lithospermic acid	6.05	C27H22O12	538.1106	[M – H] – 2OH	−0.60	519.0933	519.0930	109.0295, 185.0245, 277.0505, 279.03, 293.0457, 295.062, 321.0399, 339.0508, 519.0927	69.7	Salvia miltiorrhiza Bunge
143	Calycosin	6.17	C16H12O5	284.0679	[M − H]−	−0.77	283.0612	283.0610	268.0374, 283.0607	63.5	Astragalus membranaceus、Glycyrrhiza uralensis Fisch
144	Syringaresinol	6.24	C22H26O8	418.1622	[M + H – H2O]+	−3.52	401.1594	401.1580	315.0855, 323.1257, 330.1090, 339.1208, 343.1151, 351.1218, 369.1295, 371.1465, 383.1478, 401.1588	60.7	/
145	Ixerisoside D	6.34	C21H28O8	408.1779	[M − H]−	3.71	407.1711	407.1727	407.1727	54.1	/
146	Isosalvianolic acid c	6.35	C26H20O10	492.1051	[M + H]+	−2.41	493.1129	493.1117	139.0387, 181.0488, 225.0536, 249.0536, 253.0477, 267.0646, 277.0485, 295.0592, 493.1134	68.5	Salvia miltiorrhiza Bunge
147	Phloretin	6.51	C15H14O5	274.0836	[M − H]−	−0.61	273.0768	273.0767	273.0767	40.9	/
148	Echinatin	6.72	C16H14O4	270.0887	[M + H]+	−2.93	271.0965	271.0957	107.0494, 121.0284, 123.044, 177.0543, 229.0856, 271.0592, 271.0954	57.1	Glycyrrhiza uralensis
149	Naringenin	6.72	C15H12O5	272.0679	[M − H]−	−1.44	271.0612	271.0608	109.029, 135.0087, 135.0448, 153.019, 165.0185, 211.1334, 271.0598	47	Astragalus membranaceus、Glycyrrhiza uralensis Fisch
150	Bernardioside A	6.96	C36H58O11	666.3974	M + NH4	−1.85	684.4318	684.4305	325.1103, 359.0235, 405.3170, 429.0883, 433.3112, 451.3189, 469.3296, 487.3392, 586.5322, 669.2875	52.3	/
151	5-hydroxy-7,8-dimethoxy-6-methyl-3-(3′,4′-dihydroxybenzyl)chroman-4-one	6.97	C19H20O7	360.1204	[M – H2O – H]−	−2.51	341.1030	341.1022	89.0243, 119.0349, 179.0557, 253.1231, 297.1127, 341.1059	53.9	/
152	Pseudolaric Acid C	7.03	C21H26O7	390.1673	[M − H]−	1.95	389.1606	389.1613	389.1613	40.3	/
153	Isorhamnetin	7.07	C16H12O7	316.0578	[M − H]−	−0.73	315.0510	315.0508	109.0293, 300.0272, 315.0508	63.6	Glycyrrhiza uralensis Fisch
154	4-Methoxymedicarpin	7.09	C17H16O5	300.0992	[M − H]−	−1.69	299.0925	299.0920	269.0815, 284.0324, 299.0552, 299.0916	54.3	Astragalus membranaceus
155	Asiaticoside D	7.09	C48H78O18	942.5183	[M + FA – H]−	1.49	987.5170	987.5184	101.0244, 125.0243, 143.0345, 161.0454, 247.0832, 367.1236, 469.1557, 471.3481, 941.5105, 987.5128	62.4	/
156	Tectorigenin	7.13	C16H12O6	300.0628	[M − H]−	−1.34	299.0561	299.0557	271.0252, 284.0323, 299.0183, 299.0549	56.6	Astragalus membranaceus
157	Astragaloside VI	7.22	C47H78O19	946.5132	[M + FA − H]−	−0.46	991.5120	991.5115	945.5055, 991.5023	62.3	/
158	Traumatic acid	7.3	C12H20O4	228.1356	[M − H]−	−0.92	227.1289	227.1287	165.1287, 181.0714, 183.1385, 184.1423, 227.1287	69.6	/
159	Licorice saponin G2	7.31	C42H62O17	838.3982	[M + H]+	−1.38	839.4060	839.4048	141.0179, 451.3198, 469.3304, 487.3407, 645.3594, 663.3655, 839.4042	73.7	Glycyrrhiza uralensis
160	pinocembrin	7.42	C15H12O4	256.0730	[M − H]−	−0.97	255.0663	255.0660	119.0501, 135.0087, 153.0192, 255.0662	65.6	Glycyrrhiza uralensis Fisch
161	Isoliquiritigenin	7.44	C15H12O4	256.0730	[M + H]+	−2.27	257.0808	257.0803	137.0231, 147.0439, 257.0802	57	Astragalus membranaceus
162	Marmin	7.47	C19H24O5	332.1618	[M − H]−	−1.99	331.1551	331.1544	239.1438, 240.1466, 287.2012, 288.1676, 330.2366	42.8	/
163	Corosolic acid	7.67	C30H48O4	472.3547	[M + H – H2O]+	−2.85	455.3519	455.3506	109.1012, 123.117, 125.0964, 141.127, 143.1065, 297.2193, 419.3294, 437.3412, 455.225, 455.3501	51.6	Astragalus membranaceus
164	Astragaloside III	7.67	C41H68O14	784.4604	[M + FA – H]−	−0.08	829.4591	829.4590	829.4592	55.7	Astragalus membranaceus
165	Astragaloside IV	7.69	C41H68O14	784.4604	[M + H]+	−1.39	785.4682	785.4671	86.0966, 124.9996, 125.0961, 143.1065, 184.073, 437.3419, 455.3485	50	Astragalus membranaceus
166	Formononetin	7.7	C16H12O4	268.0730	[M − H]−	−0.00	267.0663	267.0663	252.0425, 267.0659	51.4	Astragalus membranaceus
167	Glycyrrhizic acid	7.77	C42H62O16	822.4032	[M + H]+	−0.97	823.4111	823.4103	453.3353, 471.346, 647.3774	68.6	Glycyrrhiza uralensis Fisch
168	Alpha-Glycyrrhizin	7.78	C42H62O16	822.4032	[M − H]−	0.59	821.3965	821.3970	113.0243, 821.3904	66.4	/
169	Dihydroactinidiolide	8.06	C11H16O2	180.1145	[M + H]+	−1.40	181.1223	181.1221	107.0857, 111.0441, 135.1166, 163.1113, 181.1218	60.7	/
170	Soyasaponin Ba	8.12	C48H78O19	958.5132	[M + H]+	−2.71	959.5210	959.5184	405.3513, 423.3613, 441.3716, 501.1389, 581.3779, 599.3934, 617.4045, 635.4154, 797.4703, 959.5205	65.3	Centella asiatica (L.) Urban
171	Isomucronulatol	8.14	C17H18O5	302.1149	[M + H]+	−2.61	303.1227	303.1219	123.044, 133.0646, 149.059, 161.0595, 167.07, 181.0852, 193.0854, 303.0493	57.1	Astragalus membranaceus
172	Sandosaponin B	8.23	C48H76O19	956.4975	[M + H]+	−1.75	957.5054	957.5037	455.3502, 599.3959, 605.4351, 613.3696, 617.3990, 631.3812, 635.4091, 649.3883, 811.4476, 957.5079	66.2	/
173	Glicoricone	8.25	C21H20O6	368.1254	[M + H]+	−2.65	369.1333	369.1323	169.0494, 189.0906, 199.0749, 213.0883, 215.1062, 271.0597, 285.0745, 313.0697, 369.1301	71.5	Glycyrrhiza uralensis
174	Sinensetin	8.25	C20H20O7	372.1204	[M + H]+	−2.90	373.1282	373.1271	312.0972, 343.0803, 357.0961, 373.1268	60.1	Glycyrrhiza uralensis Fisch
175	Licorice-saponin H2	8.27	C42H62O16	822.4032	[M + H]+	−1.13	823.4111	823.4101	453.3353, 471.3472, 647.3771	65.3	Glycyrrhiza uralensis
176	Soyasaponin Bb	8.33	C48H78O18	942.5183	[M + H]+	−1.49	943.5261	943.5247	441.3715, 581.3784, 599.3939, 605.4382, 617.4047, 635.4142, 781.4689, 797.4675, 943.5126, 943.5311	61.5	Astragalus membranaceus
177	Soyasaponin III	8.33	C42H68O14	796.4604	[M + H]+	−1.09	797.4682	797.4673	247.2049, 383.3297, 405.3498, 423.3611, 441.3716, 581.3814, 599.3936, 617.4048, 635.4114, 797.4686	60.7	Astragalus membranaceus
178	TMC-58B	8.56	C25H26N2O3	402.1938	[M—H]-	−1.70	401.1871	401.1864	120.0453, 146.0609, 267.1139, 280.1339, 401.1867	61.4	/
179	Medicarpin	8.56	C16H14O4	270.0887	[M + H]+	−2.00	271.0965	271.0959	123.0441, 137.0596, 161.0592, 271.0956	51.4	Astragalus membranaceus、 Glycyrrhiza uralensis Fisch
180	Pulsatilla saponin D	8.64	C47H76O17	912.5077	[M + H]+	−1.48	913.5155	913.5142	323.0973, 405.3507, 423.3611, 441.3716, 599.3931, 605.4415, 617.4045, 635.4149, 767.4562, 781.4749	44.8	Astragalus membranaceus
181	Acacetin	8.77	C16H12O5	284.0679	[M − H]−	−1.14	283.0612	283.0609	268.0377, 283.0266, 283.0607	59.7	Glycyrrhiza uralensis、Astragalus membranaceus、Abelmoschus manihot
182	Rishitin	8.83	C14H22O2	222.1614	[M + FA – H]−	−1.55	267.1603	267.1598	223.1702, 267.0663, 267.1598	59
183	AstragalosideII	8.91	C43H70O15	826.4709	[M + FA – H]−	0.22	871.4697	871.4699	112.9856, 871.4694	59.5	Astragalus membranaceus
184	Paris saponin VII	8.97	C51H82O21	1030.5343	[M + H]+	−3.61	1031.5421	1031.5384	1031.5384	43.5	/
185	14alpha-hydroxy Sprengerinin C	9	C44H70O17	870.4608	[M − H]−	−0.1	869.4540	869.4539	869.4539	40.4	/
186	Dehydrosoyasaponin I	9.07	C48H76O18	940.5026	[M + H]+	−1.24	941.5104	941.5093	309.1171, 421.3458, 439.3557, 457.3661, 597.3780, 603.4197, 615.3898, 633.4066, 795.4581, 941.5150	66	/
187	Glycycoumarin	9.08	C21H20O6	368.1254	[M − H]−	−1.84	367.1187	367.1180	139.0398, 297.0396, 309.0399, 367.1191	59.1	Glycyrrhiza uralensis Fisch
188	Glyasperin C	9.22	C21H24O5	356.1618	[M + H]+	−3.01	357.1697	357.1686	165.0541, 179.0690, 221.1171, 235.1313, 289.1072, 294.1197, 301.1062, 312.1293, 357.1621, 357.1681	63.8	Glycyrrhiza uralensis
189	Artepillin A	9.27	C19H24O4	316.1669	[M − H]−	−1.94	315.1602	315.1596	225.1642, 271.1699, 315.1595	57	/
190	Arjunolic acid	9.39	C30H48O5	488.3496	[M + FA – H]−	−0.67	533.3484	533.3480	487.3423, 533.3499	66.5	Centella asiatica (L.) Urban
191	Angenomalin	9.53	C14H12O3	228.0781	[M + H]+	−1.56	229.0859	229.0856	175.039, 187.0388, 229.0854	62.4
192	Trijuganone B	9.54	C18H16O3	280.1094	[M + FA – H]−	−1.89	325.1082	325.1076	281.1179, 325.1076	68	Salvia miltiorrhiza Bunge
193	Astragaloside I	9.57	C45H72O16	868.4815	M + Na	−1.26	891.4713	891.4702	711.4077, 891.4701	50.6	Astragalus membranaceus
194	Hederagenin	9.59	C30H48O4	472.3547	[M + H – H2O]+	-2.19	455.3519	455.3509	109.1015, 123.1169, 125.0959, 141.1271, 143.1064, 297.2205, 419.3307, 437.341, 455.351	63.2	Astragalus membranaceus
195	Danshenxinkun A	9.6	C18H16O4	296.1043	[M − H]−	−1.65	295.0976	295.0971	237.0918, 265.087, 267.1019, 295.0968	63.3	Salvia miltiorrhiza Bunge
196	Licoisoflavone A	9.62	C20H18O6	354.1098	[M − H]−	−1.56	353.1031	353.1025	125.0242, 353.1025	62.5	Glycyrrhiza uralensis Fisch
197	Neoglycyrol	9.83	C21H18O6	366.1098	[M − H]−	−2.83	365.1031	365.1020	295.0237, 297.1132, 307.0244, 365.102	56.5	Glycyrrhiza uralensis
198	3-Hydroxydodecanoic acid	9.87	C12H24O3	216.1720	[M − H]−	−1.26	215.1653	215.1650	59.0137, 116.9287, 160.9347, 215.1292, 215.1647	46.3	/
199	Beta-Kudinlactone	9.89	C30H46O5	486.3340	[M − H]−	−0.96	485.3272	485.3268	485.3277	50.7	Abelmoschus manihot
200	Dihydrotanshinone I	10.19	C18H14O3	278.0937	[M + H]+	−1.76	279.1016	279.1011	149.0231, 233.0957, 261.0905, 279.1009	62.5	Salvia miltiorrhiza Bunge
201	21-Hydroxygypsogenin	10.27	C30H46O5	486.3340	[M − H]−	−1.11	485.3272	485.3267	485.3275	53.7	Glycyrrhiza uralensis
202	9,10-Dihydroxystearic acid	10.39	C18H36O4	316.2608	[M − H]−	−1.89	315.2541	315.2535	315.2535	52.6	/
203	Neocryptotanshinone	10.44	C19H22O4	314.1513	[M + H]+	−2.50	315.1591	315.1583	251.1427, 279.1374, 297.1476, 315.1581	59.7	Salvia miltiorrhiza Bunge
204	( ±)9-HODE	10.81	C18H32O3	296.2346	[M − H]−	−1.80	295.2279	295.2273	277.2161, 295.2272	70.5	Astragalus membranaceus
205	Tanshinone I	10.89	C18H12O3	276.0781	[M + H]+	−1.97	277.0859	277.0854	249.0905, 277.0853	62.6	Salvia miltiorrhiza Bunge
206	Diosgenin glucoside	11.22	C33H52O8	576.3656	[M − H]−	0.45	577.3735	577.3738	577.3738	52
207	Licorice-saponin H2_qt	11.46	C30H46O4	470.3391	[M + H]+	−1.89	471.3469	471.3460	471.3466	59.1	/
208	18alpha-Glycyrrhetinic acid	11.49	C30H46O4	470.3391	[M − H]−	−1.58	469.3323	469.3316	469.0264, 469.3314	55.6	Glycyrrhiza uralensis Fisch
209	Tanshinone IIA	11.67	C19H18O3	294.1250	[M + H]+	−1.97	295.1329	295.1323	249.1268, 277.1215, 295.1322	61.9	Salvia miltiorrhiza Bunge
210	3-Hydroxypalmitic acid	12.22	C16H32O3	272.2346	[M − H]−	−1.06	271.2279	271.2276	225.2221, 271.2273	66.7	/
211	Docosa-4,7,10,13,16,19-hexaenoic acid	12.52	C22H32O2	328.2397	[M − H]−	−1.4	327.2330	327.2325	229.1957, 283.2427, 327.2324	50.6	/

Table 4.

Analysis and identification of prototype compounds in mouse plasma of HY-I based on UPLC-Q-TOF–MS

No.	No. (HY-I)	English name	RT	Formula	Calc. MW	Adducts	Error (ppm)	Theoretical mass (m/z)	Experimental mass (m/z)	MS2 (m/z)
P1	24	Pinitol	1.05	C7H14O6	194.0785	[M + FA-H]−	−1.19	239.0773	239.0770	193.0703, 195.0288, 195.1377, 201.9223, 238.8911, 239.0168, 239.0230, 239.0551, 239.0626, 239.0770
P2	45	Ser-Leu	2.6	C9H18N2O4	218.1261	[M + H]+	−1.47	219.1339	219.1336	60.0449, 86.0968, 132.1017, 173.128, 201.122, 203.1425, 205.1583, 219.1116, 219.1333
P3	79	Doryphornine	4.62	C11H11NO3	205.0733	[M + FA-H]−	−0.98	250.0721	250.0719	88.0403, 115.0036, 132.0301, 135.0449, 250.0717
P4	83	Aloe-emodin-8-O-beta-D-glucopyranoside	4.79	C21H20O10	432.1051	[M − H]−	−1.41	431.0984	431.0978	113.0244, 139.1761, 153.0183, 175.0242, 208.4511, 208.4739, 209.3472, 255.0657, 431.0968
P5	85	Taxifolin 7-O-rhamnoside	4.8	C21H22O11	450.1157	[M + H-H2O] +	−2.12	433.1129	433.1120	313.0695, 323.0909, 337.0698, 349.0700, 361.0684, 367.0800, 379.0804, 397.0909, 415.1018, 433.1120
P6	89	( +)-3,4′,5,7-Flaventetrol	4.86	C15H14O5	274.0836	[M + H-H2O] +	−2.00	257.0808	257.0803	137.0231, 147.0438, 257.0804
P7	93	3,4-Dimethoxybenzyl alcohol	4.96	C9H12O3	168.0781	[M + NH4]+	−1.43	186.1125	186.1122	83.086, 111.0808
P8	97	Nepitrin	5.01	C22H22O12	478.1106	[M − H − H2O]−	−0.47	459.0933	459.0931	459.0931
P9	101	Kaempferol 3-O-D-galactoside	5.03	C21H20O11	448.1000	[M − H]−	−1.49	447.0933	447.0926	255.0295, 284.0323, 447.0927
P10	103	Trifolin	5.04	C21H20O11	448.1000	[M + H]+	−2.01	449.1078	449.1069	287.0544, 288.0575, 303.049
P11	106	Astilbin	5.12	C21H22O11	450.1157	[M + H-H2O] +	−2.27	433.1129	433.1119	271.0594, 272.063, 433.1078
P12	116	5-Methoxyindoleacetate	5.34	C11H11NO3	205.0733	[M − H]−	−0.85	204.0666	204.0664	117.0708, 130.0668, 160.0765, 204.0662
P13	123	Asiaticoside B	5.49	C48H78O20	974.5081	[M − H]−	−0.03	973.5008	973.5008	101.0242, 161.0453, 247.0811, 469.1555, 503.3367, 973.5008
P14	126	Oroxin A	5.57	C21H20O10	432.1051	[M − H]−	−1.54	431.0984	431.0977	431.0977
P15	151	5-hydroxy-7,8-dimethoxy-6-methyl-3-(3′,4′-dihydroxybenzyl)chroman-4-one	6.97	C19H20O7	360.1204	[M—H2O—H]−	−2.51	341.1030	341.1022	89.0243, 119.0349, 179.0557, 253.1231, 297.1127, 341.1059
P16	152	Pseudolaric acid C	7.03	C21H26O7	390.1673	[M − H]−	1.95	389.1606	389.1613	389.1613
P17	162	Marmin	7.47	C19H24O5	332.1618	[M − H]−	−1.99	331.1551	331.1544	239.1438, 240.1466, 287.2012, 288.1676, 330.2366
P18	168	Alpha-Glycyrrhizin	7.78	C42H62O16	822.4032	[M − H]−	0.59	821.3965	821.3970	113.0243, 821.3904
P19	182	Rishitin	8.83	C14H22O2	222.1614	[M + FA—H]−	−1.55	267.1603	267.1598	223.1702, 267.0663, 267.1598
P20	189	Artepillin A	9.27	C19H24O4	316.1669	[M − H]−	−1.94	315.1602	315.1596	225.1642, 271.1699, 315.1595
P21	190	Arjunolic acid	9.39	C30H48O5	488.3496	[M + FA—H]−	−0.67	533.3484	533.3480	487.3423, 533.3499
P22	198	3-Hydroxydodecanoic acid	9.87	C12H24O3	216.1720	[M − H]−	−1.26	215.1653	215.1650	59.0137, 116.9287, 160.9347, 215.1292, 215.1647
P23	199	Beta-Kudinlactone	9.89	C30H46O5	486.3340	[M − H]−	−0.96	485.3272	485.3268	485.3277
P24	201	21-Hydroxygypsogenin	10.27	C30H46O5	486.3340	[M − H]−	−1.11	485.3272	485.3267	485.3275
P25	202	9,10-Dihydroxystearic acid	10.39	C18H36O4	316.2608	[M − H]−	−1.89	315.2541	315.2535	315.2535
P26	204	Neocryptotanshinone	10.44	C19H22O4	314.1513	[M + H]+	−2.50	315.1591	315.1583	251.1427, 279.1374, 297.1476, 315.1581
P27	206	Diosgenin glucoside	11.22	C33H52O8	576.3656	[M − H]−	0.45	577.3735	577.3738	577.3738
P28	207	Licorice-saponin H2_qt	11.46	C30H46O4	470.3391	[M + H]+	−1.89	471.3469	471.3460	471.3466
P29	208	18alpha-Glycyrrhetinic acid	11.49	C30H46O4	470.3391	[M − H]−	−1.58	469.3323	469.3316	469.0264, 469.3314

Table 5.

Analysis and identification of metabolites in mouse plasma of HY-I based on UPLC-Q-TOF–MS

No.	Metabolites	RT(min)	Formula	Calc. MW	Adducts	Mass Error (ppm)	m/z	Score	Parent compound	Transformations
M1	10-Hydroxymajoroside_M1	2.28	C10H12O9S	308.0196	[M + FA-H]−	3.99	353.0196	48.8	10-Hydroxymajoroside	Deglycosidation, hydrolysis, sulfation
M2	5,7-Dihydroxy-4-oxo-4H-chromene-2-carboxylic acid_M2	3.81	C17H16O13	428.0585	[2 M-H]−	0.47	855.1113	43	5,7-Dihydroxy-4-oxo-4H-chromene-2-carboxylic acid	Hydroxylation, glucuronidation, methylation
M3	5,7-Dihydroxy-4-oxo-4H-chromene-2-carboxylic acid_M1	3.83	C10H6O7	238.0108	[M + FA-H]−	1.43	283.0099	41.9	5,7-Dihydroxy-4-oxo-4H-chromene-2-carboxylic acid	Hydroxylation
M4	3-Coumaric acid_M1	4.28	C9H8O6S	244.0036	[M − H]−	−1.10	242.9966	41.1	3-Coumaric acid	Sulfation
M5	3,4-Dihydroxybenzaldehyde_M1	4.41	C7H6O2	122.0362	[M-H2O-H]−	2.54	105.0338	57.1	3,4-Dihydroxybenzaldehyde	Dehydroxylation
M6	Beta-D-Glucopyranoside, 2-((benzoyloxy)methyl)-4-hydroxyphenyl_M1	4.42	C9H9NO3	179.0577	[M − H]−	−0.99	178.0508	54.4	Beta-D-Glucopyranoside, 2-((benzoyloxy)methyl)-4-hydroxyphenyl	Hydrolysis, glycination
M7	m-Methoxyphenol_M1	4.73	C7H8O5S	204.0087	[M − H]−	−0.53	203.0019	53.9	m-Methoxyphenol	Sulfation
M8	Luteolin_M1	4.75	C21H18O15S	542.0361	[M − H]−	−0.71	541.0290	54.3	Luteolin	Glucuronidation, sulfation
M9	Methyl protocatechuate_M1	4.93	C9H10O7S	262.0142	[M − H]−	−1.41	261.0071	54.5	Methyl protocatechuate	Methylation, sulfation
M10	Indoleacetic acid_M1	4.99	C11H11NO4	221.0682	[M-H2O-H]−	−0.60	202.0508	40.8	Indoleacetic acid	Hydroxylation, hydroxylation, methylation
M11	5-hydroxy-7,8-dimethoxy-6-methyl-3-(3′,4′-dihydroxybenzyl)chroman-4-one_M1	6.17	C19H22O8	378.1309	[M + FA-H]−	−0.96	423.1293	49.4	5-hydroxy-7,8-dimethoxy-6-methyl-3-(3′,4′-dihydroxybenzyl)chroman-4-one	Reduction, hydroxylation
M12	Phloretin_M1	6.25	C15H14O8S	354.0404	[M − H]−	−1.40	353.0332	59.6	Phloretin	Sulfation
M13	Ixerisoside D_M1	7.45	C15H16O3	244.1094	[M − H]−	−1.36	243.1023	44.2	Ixerisoside D	Deglycosidation, oxidation
M14	14alpha-hydroxy Sprengerinin C_M1	9.39	C27H42O5	446.3027	[M − H]−	−0.71	445.2956	55	14alpha-hydroxy Sprengerinin C	Deglycosidation, hydroxylation
M15	Paris saponin VII_M1	9.74	C27H42O5	446.3027	[M + H]+	−1.88	447.3097	56	Paris saponin VII	Deglycosidation, hydroxylation
M16	3-Hydroxypalmitic acid_M2	9.79	C16H30O4	286.2139	[M-H2O-H]−	−0.76	267.1964	46.8	3-Hydroxypalmitic acid	Oxidation, hydroxylation
M17	Docosa-4,7,10,13,16,19-hexaenoic acid_M1	10.73	C22H32O3	344.2346	[M − H]−	−1.55	343.2273	45.5	Docosa-4,7,10,13,16,19-hexaenoic acid	Hydroxylation
M18	3-Hydroxypalmitic acid_M1	10.77	C16H30O4	286.2138	[M-H2O-H]−	−1.27	267.1962	42.5	3-Hydroxypalmitic acid	Oxidation, hydroxylation
M19	Docosa-4,7,10,13,16,19-hexaenoic acid_M2	11	C22H32O3	344.2346	[M − H]−	−1.53	343.2273	53.8	Docosa-4,7,10,13,16,19-hexaenoic acid	Hydroxylation

HY-I ameliorates renal senescence and pathological injury in aged mice

HY-I efficacy was evaluated through renal function parameters, senescence biomarkers, and histopathological analysis. Intervention with HY-I significantly reduced serum urea nitrogen, creatinine, uric acid, and urinary protein levels in aged mice (Fig. 2A–D), indicating improved age-related renal dysfunction.

Fig. 2.

HY-I Ameliorates renal senescence and pathological injury in aged mice. A–D Analysis of renal function parameters (creatinine, BUN, urinary protein, uric acid) across experimental groups. n = 6. One-way ANOVA was used. * indicates comparison with the aging model group; *p < 0.05, **p < 0.01, ***p < 0.001; ns indicates no significant difference. E SA-β-gal staining of renal tissues. Scale bars, 100 μm. F, G Renal histopathology assessed by H&E and PAS staining. Scale bars, 100 μm. H Immunofluorescent detection of proliferation marker Ki67. Scale bars, 50 μm. I–K Representative immunoblots of p21 and p16 expression. n = 3. One-way ANOVA was used. * indicates comparison with the aging model group; *p < 0.05, **p < 0.01, ***p < 0.001; ns indicates no significant difference

SA-β-gal staining demonstrated markedly decreased senescent cells in renal tubules following HY-I treatment (Fig. 2E). Histological assessment revealed that HY-I alleviated age-induced tubular pathologies including vacuolar degeneration, tubular atrophy, and brush border loss, as evidenced by H&E and PAS staining (Fig. 2F, G).

Furthermore, HY-I treatment restored the proliferation marker Ki67 (Fig. 2H) while suppressing senescence markers p21 and p16 (Fig. 2I–K). Collectively, these findings demonstrate HY-I effectively mitigates renal senescence in aged mice.

Network pharmacology analysis of HY-I on renal senescence

48 bioactive components of HY-I detectable in serum were selected. After integration and deduplication of the corresponding targets, a total of 681 unique proteins were identified. Venn analysis revealed 420 overlapping targets shared by HY-I and renal aging (Fig. 3A). Following serum-adsorbed compound attribution, a “herb–compound–target–disease” network was constructed (Fig. 3B).

Fig. 3.

Network pharmacology analysis of HY-I on renal senescence. A Venn diagram of HY-I targets versus kidney aging (KA)-related genes. B HY-I-KA pharmacological network of compound-target interactions. C Core target PPI network after screening. D Top 30 genes in PPI network ranked by degree centrality. E GO enrichment analysis. F KEGG pathway enrichment

The 420 common targets were further analyzed using the STRING database for protein–protein interactions (PPI). Topological analysis in Cytoscape 3.7.1 was performed based on parameters of betweenness centrality, closeness centrality, and degree. Using the median degree as a threshold, 65 core targets were selected for PPI network visualization, where node color and size were scaled according to degree values (Fig. 3C). The top 30 targets, ranked by degree, included IL-6, STAT3, and IL-1β, which are known to play essential roles in inflammation-associated senescence.

Enrichment analysis of the top 30 GO terms ranked by P-value revealed significant enrichment in biological processes such as chromatin remodeling and protein phosphorylation. The main cellular components were the cytoplasm and receptor complexes, while the predominant molecular functions involved protein and enzyme binding (Fig. 3E). Integrating the top 30 key targets identified as functionally important, KEGG pathway analysis further indicated that the JAK-STAT, NOD-like receptor, and NF-κB signaling pathways were prominently involved in the aging process (Fig. 3F).

HY-I attenuates SASP expression in aged renal tubules

Renal tubular epithelial cells, being the most abundant and metabolically active renal cells, are highly susceptible to senescence. SASP—comprising proinflammatory cytokines, chemokines, and growth factors—drives localized inflammation and fibrosis. Network pharmacology predicted HY-I targets SASP components (IL-6, TGFβ1, IL-1β, MMP9) and inflammatory regulators (NF-κB, STAT3, NLRP3), suggesting its anti-senescence mechanism.

To validate HY-I’s effects on tubular SASP, drug-containing serum was applied to senescent tubular cells. SASP expression was assessed via IHC, WB, and ELISA in murine renal tissues and cells.

CCK-8 assays identified 200 mM D-galactose as optimal for HK-2 senescence induction (Fig. 4A). Drug-containing serum (5%-10%) restored cellular viability (Fig. 4B). Subsequent experiments used 5% and 10% concentrations. HY-I-containing serum reduced SA-β-gal-positive cells (Fig. 4C). WB/ELISA analyses of cell lysates, supernatants, and renal homogenates demonstrated HY-I-mediated suppression of SASP factors (Fig. 4D–M). IHC confirmed decreased TGF-β1, IL-6, and IL-1β expression in HY-I-treated kidneys (Fig. 4N). These results indicate HY-I concurrently mitigates tubular senescence and renal SASP.

Fig. 4.

HY-I attenuates SASP expression in aged renal tubules. A, B CCK-8 assay: Cytotoxicity of D-gal in HK-2 cells and rescue by HY-I-containing serum. (n = 6). C SA-β-Gal staining of senescent cells. Scale bars, 100 μm. D–F WB analysis of TGF-β1 and IL-1β expression in D-gal-treated HK-2 cells with HY-I-containing serum intervention. (n = 3). G IL-6 levels in cell supernatants by ELISA. (n = 3). H–M Renal tissue SASP factors (TNF-α, IL-1β, IL-6, TGF-β1) quantified by ELISA. (n = 6). One-way ANOVA was used. * indicates comparison with the aging model group; *p < 0.05, **p < 0.01, ***p < 0.001; ns indicates no significant difference. N Representative immunohistochemical images of renal SASP factors (IL-1β, IL-6, TGF-β1). Scale bars, 100 μm

HY-I attenuates tubular inflammation and senescence via STAT3/NF-κB/NLRP3 axis

Integrated network pharmacology and experimental validation suggested HY-I modulates inflammation through STAT3-NF-κB-NLRP3 signaling and downstream effectors (CASP1, IL-18). Western blotting demonstrated HY-I significantly reduced phosphorylation of NF-κB and STAT3, while downregulating NLRP3, CASP1, and IL-18 protein expression in renal tissues (Fig. 5A, B).

Fig. 5.

HY-I attenuates tubular inflammation and senescence via STAT3/NF-κB/NLRP3 Axis. A, B Western blot analysis of STAT3, p-STAT3, NF-κB, p-NF-κB, NLRP3, CASP1, and IL-18 expression in renal tissues. n = 3. One-way ANOVA was used. * indicates comparison with the aging model group; *p < 0.05, **p < 0.01, ***p < 0.001; ns indicates no significant difference. C Multiplex immunofluorescence of renal sections: p-NF-κB (red), p-STAT3 (yellow), NLRP3 (green), DAPI (blue). Scale bars, 50 μm. D Immunofluorescence of CASP1 (red) and IL-18 (green). Scale bars, 50 μm

Multiplex immunofluorescence revealed enhanced nuclear translocation of p-NF-κB and p-STAT3 with concomitant upregulation of NLRP3 and co-expression of CASP1/IL-18 during renal aging. HY-I intervention attenuated these alterations (Fig. 5C, D). These findings indicate HY-I suppresses SASP-associated inflammation by inhibiting STAT3/NF-κB/NLRP3 signaling and downstream CASP1/IL-18 activation.

HY-I attenuates renal fibrosis in aged mice

Senescent cells promote extracellular matrix deposition through SASP-mediated chemokines and inflammatory factors, contributing to fibrotic progression in aging kidneys.

Masson’s trichrome staining revealed reduced collagen deposition in HY-I-treated kidneys (Fig. 6A). Sirius red staining under polarized light demonstrated decreased type I and III collagen accumulation (Fig. 6B). Immunohistochemistry showed attenuated fibronectin diffusion in tubulointerstitial areas (Fig. 6C). Western blotting confirmed downregulation of type I collagen and EMT/fibrosis markers (vimentin, α-SMA) (Fig. 6E).

Fig. 6.

HY-I attenuates renal fibrosis in aged mice. A, B Masson’s trichrome and Sirius Red staining demonstrating renal collagen deposition. Scale bars, 100 μm. C Representative immunohistochemistry of fibronectin (FN) in renal tissues. Scale bars, 50 μm. D–G Western blot analysis of collagen I, vimentin, and α-SMA expression. (n = 3). One-way ANOVA was used. * indicates comparison with the aging model group; *p < 0.05, **p < 0.01, ***p < 0.001; ns indicates no significant difference

Molecular docking analysis of the interaction between HY-I-derived serum components and potential targets

Key serum-absorbed compounds identified through network pharmacology included arjunolic acid, phloretin, 18alpha-glycyrrhetinic acid, pseudolaric acid C, and luteolin.

Molecular docking using AutoDock demonstrated interactions between these compounds and core targets (NF-κB, STAT3, NLRP3). Among 15 docking configurations (Fig. 7A), multiple exhibited binding energies < -5 kcal/mol, indicating strong binding affinity. Structural visualizations generated in PyMOL (Fig. 7B–D) provide molecular insights into HY-I-mediated suppression of SASP-associated senescence and fibrosis via STAT3/NF-κB/NLRP3 signaling.

Fig. 7.

Molecular docking analysis of the interaction between HY-I-derived serum components and potential targets. A Binding affinity heatmap of five compounds against three targets. B Molecular docking visualization for NF-κB. C Molecular docking visualization for STAT3. D Molecular docking visualization for NLRP3. E Backbone root mean square deviation (RMSD) of four representative ligand-target complexes over 100 ns molecular dynamics (MD) simulation, evaluating the overall conformational stability of the complexes relative to the initial docking structure. F Radius of gyration (Rg) of the four complexes during 100 ns MD simulation, assessing the compactness and folding state of the protein tertiary structure. G Solvent accessible surface area (SASA) of the four complexes during 100 ns MD simulation, reflecting the solvent exposure level of the protein structure. H Time-dependent changes in the number of intermolecular hydrogen bonds between the target protein and its corresponding ligand during 100 ns MD simulation, characterizing the stability of ligand–protein binding interactions. I Root mean square fluctuation (RMSF) of each amino acid residue of the target protein in the four complexes, quantifying the flexibility of individual protein residues during the simulation. J 2D Gibbs free energy landscape (GEL) and 3D surface visualization of the four complexes, identifying the thermodynamically stable conformation basins of the ligand-bound proteins

We next performed 100 ns all-atom molecular dynamics (MD) simulations on four compound-target complexes with optimal docking affinity (STAT3—18alpha-glycyrrhetinic acid, NLRP3—18alpha-glycyrrhetinic acid, NLRP3-arjunolic acid, NF-κB-pseudolaric acid C) to verify their dynamic binding stability under near-physiological conditions (Fig. 7E–J).

Backbone RMSD analysis (Fig. 7E) confirmed all four complexes reached conformational equilibrium within the simulation period, with no persistent structural drift. The NF-κB-pseudolaric acid C and STAT3—18alpha-glycyrrhetinic acid complexes presented the narrowest fluctuation ranges, indicating superior overall conformational stability.

Consistent with RMSD results, the radius of gyration (Rg, Fig. 7F) and solvent accessible surface area (SASA, Fig. 7G) of all complexes remained stable throughout the simulation, verifying that the target proteins maintained a compact folded state without global unfolding.

Intermolecular hydrogen bond detection (Fig. 7H) showed the NLRP3-arjunolic acid complex maintained the most abundant and continuous hydrogen bond interactions between ligand and protein, while the other three complexes also exhibited sustained hydrogen bond formation as the core driving force for stable ligand anchoring.

RMSF analysis (Fig. 7I) revealed low fluctuation amplitudes of residues within the ligand-binding pocket in all complexes, indicating that ligand binding rigidified the binding region and stabilized the local protein conformation.

Gibbs free energy landscape (GEL) analysis (Fig. 7J) identified well-defined, concentrated low-energy conformational basins for all four complexes, confirming the thermodynamic stability of the ligand-bound states.

Collectively, these MD data validated the stable binding of HY-I’s core active components to their targets at the dynamic molecular level, further supporting the mechanism of HY-I regulating STAT3/NF-κB/NLRP3 signaling to alleviate SASP-related senescence and fibrosis.

Discussion

The kidneys, responsible for concentrating and excreting metabolites, drugs, and toxins, exhibit high metabolic activity that renders them particularly vulnerable to aging [2]. Structural alterations during renal aging—including tubular atrophy and interstitial expansion—significantly increase susceptibility to CKD and accelerate its progression in the elderly [4]. As primary functional units of the kidney, tubular epithelial cells experience diminished regenerative capacity with advancing age and pathological stress [10]. This accumulation of senescent cells ultimately drives renal functional decline. Critically, senescent cells in aged and CKD kidneys establish a pro-inflammatory, pro-fibrotic microenvironment through SASP secretion [11]. Consequently, targeted modulation of SASP-mediated inflammatory/fibrotic mechanisms represents a crucial therapeutic strategy for age-related CKD. Building on our prior clinical evidence demonstrating HY-I-mediated improvement of renal function in stage 2—3 CKD patients [12], the current study establishes that clinically equivalent HY-I doses ameliorate renal aging and functional deterioration in murine models.

Notably, NF-κB serves as the central transcriptional regulator rapidly activated by senescence stimuli, where it directly controls core SASP components including IL-6 and IL-8 [13]. STAT3 further amplifies and sustains SASP transcription through synergistic cooperation with NF-κB [14]. Downstream of this axis, the NLRP3 inflammasome functions as a critical effector: NF-κB/STAT3 signaling induces NLRP3 and pro-IL-1β transcription, while subsequent NLRP3 activation by damage-associated molecular patterns from senescent cells catalyzes proteolytic maturation of highly inflammatory SASP components IL-1β and IL-18—key mediators that potentiate inflammatory/fibrotic cascades [15]. The STAT3/NF-κB/NLRP3 signaling circuit thus forms a self-reinforcing loop that governs SASP initiation, amplification, and maintenance, making it an optimal therapeutic target for age-related renal decline.

Traditional Chinese Medicine (TCM) demonstrates significant efficacy in modulating aging processes and renal pathologies through the polypharmacological actions inherent to multi-component formulations. Astragalus membranaceus extracts enhance physical function in aging murine models by reversing age-related functional decline [16]. Abelmoschus manihot flavonoids mitigate oxidative stress in D-galactose-induced senescence through reducing reactive oxygen species generation [17]. Centella asiatica contributes triterpenoid compounds such as asiaticoside that exert therapeutic effects across diverse age-related conditions including chronic inflammation, skin photoaging, and neurodegenerative disorders [18, 19], while Salvia miltiorrhiza synergizes with Astragalus to potentiate nephroprotective effects via combinatorial regulation [20, 21]. Glycyrrhiza uralensis serves dual roles as both a harmonizing agent and bioactive source with documented anti-inflammatory and antiviral properties [22, 23]. Cultured Cordyceps mycelia (Mortierella sp.)—derived from artificial fermentation of fungal strains isolated from caterpillar larvae—demonstrate established efficacy in clinical renal disease management [24].

In this study, network pharmacology screening identified several key serum-absorbed constituents from HY-I herbal components.

Resveratrol, which has beneficial effects on both inflammation and aging, was selected as a positive control. In our in vivo experiments, high-dose HY‑I intervention showed effects similar to resveratrol in restoring renal function (serum creatinine, uric acid, urinary protein) and reducing the number of senescent kidney cells in aged mice. In terms of improving blood urea nitrogen levels and certain aging molecular markers (Ki67, p16), high-dose HY‑I was superior to resveratrol. Moreover, HE staining revealed that HY‑I was more effective than resveratrol in preserving renal tubular morphology. In our in vitro experiments, HY‑I and resveratrol exhibited comparable effects on improving senescence-associated secretory phenotype (SASP) factors such as inflammatory cytokines; for IL‑18 and other inflammatory factors, HY‑I was slightly more effective than resveratrol alone. These findings suggest that HY‑I not only alleviates aging of the kidney and renal tubular epithelial cells but also has greater potential for improving renal function.

We discovered that HY-I significantly modulates the STAT3/NF-κB/NLRP3-SASP signaling axis, which is critical in renal senescence, with experimental evidence confirming this activity. Molecular docking analysis indicated that five representative compounds derived from the five herbal components of the HY-I formulation interact with STAT3, NF-κB, and NLRP3 through distinct binding modes. For example, Arjunolic acid binding to NF-κB may alter p65 spatial conformation and affect its phosphorylation. Meanwhile, luteolin and other components bind to the pTyr705 pocket in STAT3’s SH2 domain, which could hinder phosphorylation-dependent dimerization and nuclear translocation. NLRP3 assembly may be disrupted by 18α-glycyrrhetinic acid and similar compounds, potentially blocking downstream inflammatory activation. These findings, together with cellular assays and molecular dynamics simulations, suggest a polypharmacological basis for HY-I. This multi-target synergistic mechanism may more effectively block SASP production while conferring additional therapeutic benefits beyond SASP inhibition and improved safety, distinguishing HY-I’s efficacy from single-agent anti-aging drugs such as resveratrol.

HY-I exerts a significant modulatory effect on renal cellular senescence, and these findings provide preliminary mechanistic insights from a multi-target pharmacological perspective. Specifically, HY-I not only effectively suppresses the secretion of senescence-associated secretory phenotype (SASP) factors by renal tubular epithelial cells, but also simultaneously regulates upstream inflammatory triggers. Compared with a single target drug such as resveratrol, which acts on a limited signal pathway, HY-I has advantages in the improvement of some renal function indicators and the protection of renal tubules. Although resveratrol, as a multi‑target inhibitor, provided evidence supporting the inhibitory effect of HY‑I on STAT3/NLRP3, a limitation of this study is the lack of specific STAT3 or NLRP3 inhibitors. Therefore, the specificity of the therapeutic mechanism by which HY‑I targets STAT3/NLRP3 has not been fully established. In summary, the coordinated regulation of the STAT3/NF-κB/NLRP3–SASP axis by HY-I alleviates inflammation and mitigates fibrosis associated with cellular aging, highlighting its potential as a promising therapeutic candidate for the prevention of age-related renal functional decline.

Conclusion

This study demonstrates that HY-I attenuates renal senescence and fibrosis in both in vivo aging models and in vitro senescence systems by inhibiting the STAT3/NF-κB/NLRP3-SASP axis. These protective effects are mediated through constituent-driven suppression of STAT3 phosphorylation, NF-κB nuclear translocation, and NLRP3 inflammasome assembly. Collectively, our findings provide a mechanistic basis for employing HY-I in clinical management of early-stage CKD to protect against age-related renal deterioration, while advancing its therapeutic potential for further development.

Abbreviations

CKD

Chronic kidney disease

ECM

Extracellular matrix

SASP

Senescence-associated secretory phenotype

ESKD

End⁃stage kidney disease

KA

Kidney aging

HK-2

Human renal tubular cells-2

Res

Resveratrol

CCK-8

Cell counting kit-8

ELISA

Enzyme-linked immunosorbent assay

H&E

Hematoxylin–eosin staining

PAS

Periodic acid-Schiff staining

SA-β-gal

Senescence-associated ß-galactosidase

PPI

Protein–protein interaction

KEGG

Kyoto encyclopedia of genes and genomes

TCMSP

Traditional Chinese medicine systems pharmacology database and analysis platform

D-gal

D-Galactose

Ki67

Marker of proliferation Ki-67

TGFβ1

Transforming growth factor-beta 1

IL-1β

Interleukin-1β

IL6

Interleukin-6

IL18

Interleukin-18

STAT3

Signal transducer and activator of transcription 3

NF-κB

Nuclear factor kappa-B

NLRP3

NOD-like receptor family pyrin domain containing 3

CASP1

Caspase-1

α-SMA

α-Smooth muscle actin

Author contributions

Kaizhi Wen: Writing—review & editing, Writing—original draft, Validation, Conceptualization. Xiaofan Yin: Formal analysis, Investigation, Conceptualization, Writing—original draft, Funding acquisition. Lingling Sun: Formal analysis, Investigation, Validation. Kena Yu: Investigation, Methodology. Liyan Huang: Investigation. Qin Song: Validation. Buhui Liu: Funding acquisition, Supervision. Xinyuan Cui: Formal analysis. Yue Tu: Writing—review & editing, Funding acquisition, Supervision. Weiming He: Writing—review & editing, Funding acquisition, Supervision, Conceptualization.

Funding

This work was supported by the National Natural Science Foundation of China (Grant numbers: 821742494, 82174472); the Natural Science Foundation of Jiangsu Province (BK20241042); Jiangsu Province Traditional Chinese Medicine Technology Development Plan (QN202331); the Postgraduate Research and Practice Innovation Program of Jiangsu Province (KYCX25_2301); Jiangsu Provincial Medical Innovation Center (NO.202215).

Data availability

Data will be made available on request.

Declarations

Conflict of interest

The authors report no conflicts of interest in this work.

Ethical approval

The study protocol received approval from the Institutional Animal Ethics Committee of Nanjing University of Chinese Medicine under approval number 202411A022, with all procedures complying with animal research guidelines.