Active Flavonoid Components And Potential Mechanisms Of Aerial Parts Of Glycyrrhiza Uralensis For Chronic Prostatitis Based On Network Pharmacology And Experimental Validation
Nov 06, 2025
Abstract:
Objective: To determine the active ingredients and possible mechanisms of action of flavonoids from the aerial parts of Cistanche for chronic prostatitis. Methods: Ultra-performance liquid chromatography–quadrupole–electrostatic field Orbitrap high-resolution mass spectrometry (UPLC-Q-Exactive Orbitrap-MS) was used to identify the main flavonoid constituents in the alcoholic extract of the aerial parts of Cistanche. Network pharmacology and molecular docking were employed to predict core components and key targets related to chronic prostatitis. A rat model of chronic prostatitis was induced by carrageenan, and histopathological changes in rat prostates were observed by HE staining. Serum levels of DHT, IFN-γ, IgG, IL-6, PSA, and TNF-α were measured by ELISA to evaluate the therapeutic efficacy of the extract. Results: A total of 44 flavonoids were identified in the alcoholic extract. Network pharmacology indicated that core components, including glycyrrhizin, trichothecene isoflavones, prickly manzanita florifera, and glycyrrhizin (predicted as representative constituents), exert anti–chronic prostatitis effects by acting on key targets such as AKT1, TNF, VEGFA, EGFR, and MMP9, thereby regulating related signaling pathways. Molecular docking showed favorable binding affinities between core components and key targets. Animal experiments demonstrated that the alcoholic extract of the aerial parts of Cistanche produced a clear therapeutic effect on chronic prostatitis in rats. Conclusion: The anti–chronic prostatitis efficacy of the aerial parts of Cistanche is validated. Active components, including spiny aristolochicin, trichostatin isoflavones, and glycyrrhizin, may act via the PI3K–AKT signaling pathway to modulate EGFR, AKT1, and MMP9.
Keywords: aerial parts of Cistanche; chronic prostatitis; flavonoid; UPLC-Q-Exactive Orbitrap-MS; network pharmacology
cistanche benefits for prostate health
Introduction:
The traditional Chinese medicinal herb Cistanche (Cistanche spp.) comprises the dried medicinal material primarily utilized in TCM. It is an important bulk medicinal resource in China, with functions such as tonifying and supporting bodily functions, clearing heat and detoxifying, alleviating pain and harmonizing formulas, and it is commonly used as an adjunctive component in many multi-herb prescriptions. Market demand is high both domestically and internationally. The aerial parts, including stems and leaves, are abundant resources that await development and utilization.
Chronic prostatitis is a common urological disease among young and middle-aged men. Clinical symptoms include lower abdominal or pelvic pain, dysuria, and urinary frequency. Without timely treatment, it may lead to sexual dysfunction such as premature ejaculation and erectile dysfunction, seriously affecting men's physical and mental health.
Flavonoids are abundant in the aerial parts of Cistanche and show notable anti-inflammatory and antitumor activities. Prior studies have indicated that the aerial parts are rich in flavonoids with antitumor and anti-inflammatory effects. Enrichment of flavonoids from the aerial parts demonstrated good antioxidant activity in vitro. Pharmacodynamic research has also found that flavonoids from the aerial parts have preventive and therapeutic effects on chronic prostatitis.
In this study, UPLC-Q-Exactive Orbitrap-MS was used to profile flavonoids in the alcoholic extract of the aerial parts of Cistanche. Network pharmacology was applied to predict key targets and pathways for chronic prostatitis, molecular docking was used to infer mechanisms, and animal experiments were conducted to validate anti–chronic prostatitis activity, providing a reference for subsequent drug development and clinical application.
Materials
Animals
SPF-grade male SD rats (n = 24), 6–7 weeks old, body weight 230–250 g [Beijing Speifu Biotechnology Co., Ltd.; production license: SCXK (Jing) 2019-0010]. Animals were housed in the SPF animal facility at the Experimental Animal Center of Beijing University of Chinese Medicine. The protocol was approved by the institutional ethics committee (approval No.: BUCM-2023110204-4271).
Instruments
Ultimate 3000 UPLC system (Dionex, USA)
Thermo Q Exactive Plus high-resolution mass spectrometer (Thermo Fisher Scientific, USA)
KH500DE digital ultrasonic cleaner (Kunshan Hechuang)
FA2004N electronic balance (Beijing Sartorius)
H1650 low-speed refrigerated centrifuge (Changsha Xiangyi)
RE-5023 rotary evaporator (Shanghai Yarong)
Electronic temperature-controlled heating mantle (Tianjin Teste)
Multiskan MK3 microplate reader (Thermo Scientific, Shanghai)
B032 fluorescence inverted biological microscope (Nikon Instruments, Shanghai)
N5000 UV spectrophotometer (Shanghai Youke)
Reagents
Aerial parts of Cistanche (stems, leaves, and a small amount of pods) were harvested in September 2019 from Guazhou County, Jiuquan City, Gansu Province, and authenticated by Prof. Wang Wenquan (Institute of Medicinal Plant Development, CAMS) as Cistanche spp.
Other reagents: Ulatan (Shandong Keyuan Biochemical Co., batch C15887472); carrageenan (Beijing Solarbio, batch 20220601); Qianliekang Pule'an tablets (Zhejiang Conba Pharmaceutical); sodium carboxymethyl cellulose (CMC-Na; Shanghai YuanYe, batch S14016); ELISA kits for rat DHT, IFN-γ, IgG, IL-6, PSA, and TNF-α (Shanghai Enzyme-linked Biotechnology); Multiskan MK3 microplate reader (Thermo Scientific); tissue fixative (Guangzhou Shuopu, batch 23257838); ethanol (Beijing Chemical Works, analytical grade); methanol, formic acid, acetonitrile (Fisher Scientific, MS grade); water (Wahaha Group). Reference standards (purity > 98%, Shanghai YuanYe): liquiritigenin, puerarin, isoorientin II, naringenin, isoquercitrin, isoliquiritin, ononin, isoquercitrin, quercetin, astragalin, kaempferol, formononetin.
Methods and Results
2.1 Chemical profiling of the alcoholic extract from aerial parts of Cistanche
2.1.1 Sample preparation
Appropriate amounts of aerial parts were extracted with 70% ethanol. The extract was concentrated to dryness. The residue was ultrasonically dissolved in water for 20 min, centrifuged at 3000 r·min−1 for 15 min, and the supernatant was concentrated to dryness. An appropriate amount of the dried sample was placed in a 5 mL volumetric flask and brought to volume with methanol to obtain the test solution.
2.1.2 Chromatographic and mass spectrometric conditions
UPLC conditions: ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm); mobile phase A, acetonitrile; mobile phase B, 0.5% formic acid in water; gradient elution (0–20 min, 5%–20% A; 20–70 min, 20%–30% A); flow rate 0.2 mL·min−1; injection volume 2 μL; column temperature 30°C.
MS conditions: HESI source; positive and negative ion modes; full-scan acquisition; spray voltage +3.2 kV (positive) and −3.0 kV (negative); capillary temperature 320°C; sheath gas 40 arb; auxiliary gas 15 arb; auxiliary heater 350°C; MS resolution 70,000; MS/MS resolution 17,500; scan range m/z 100–1500.
Cistanche Specs List
2.1.3 Component identification
The aerial-part sample was analyzed by UPLC-Q-Exactive Orbitrap-MS to obtain TICs (Figure 1). Data were processed with Thermo Xcalibur and compared with component libraries and literature, identifying 44 flavonoids in total (Table 1).
2.2 Network pharmacology
Identified constituents were imported into the TCMSP 2.3 database (http://tcmspw.com/tcmsp.php). Based on oral bioavailability (OB) ≥ 30.00% and drug-likeness (DL) ≥ 0.18, 19 active components were screened. Predicted targets of active components and disease targets for chronic prostatitis were intersected, yielding 88 common targets. STRING 11.0 (http://string-db.org) was used to construct the PPI network. Core targets ranked by degree included AKT1, TNF, VEGFA, EGFR, SRC, ESR1, PTGS2, MMP9, BCL2L1, and MMP2. GO and KEGG enrichment (DAVID 6.8, http://david.ncifcrf.gov) indicated biological processes such as positive regulation of protein kinase B, negative regulation of apoptosis, and positive regulation of MAPK activity; cellular components including extracellular region, macromolecular complexes, and receptor complexes; molecular functions including tyrosine kinase, serine kinase, and threonine kinase activities. KEGG pathways were enriched in pathways in cancer, PI3K–AKT signaling, prostate cancer, and resistance to EGFR tyrosine kinase inhibitors. Cytoscape 3.7.1 was used to construct the "active component–core target–pathway" network (Figure 2). The top 10 components by degree were liquiritigenin, spinacetin (representative flavone), isovitexin, chrysoeriol, santin, kaempferol, schaftoside, luteolin, ononin, and formononetin.
Molecular docking was performed pairwise between the 10 core components and the key targets from the PPI analysis. Molecular structures of core components were downloaded from TCMSP 2.3 (mol2 format), and 3D structures of key targets were obtained from the PDB (http://www.rscb.org/) in pdb format. Autodock was used for docking of each ligand–protein pair. Binding energies < −20.92 kJ·mol−1 indicated favorable binding; the lower the energy, the better the affinity. The best-scoring target–ligand pairs are listed in Table 2. Representative docking complexes with five targets were visualized in PyMol (Figure 3).
2.3 In vivo validation
A rat model of chronic prostatitis was established using carrageenan. HE staining revealed histopathological lesions characteristic of chronic prostatitis in model animals, whereas treatment with the alcoholic extract of the aerial parts of Cistanche improved glandular architecture and reduced inflammatory infiltration. ELISA showed that, compared with the model group, treatment groups exhibited regulation of serum DHT, IFN-γ, IgG, IL-6, PSA, and TNF-α toward normal levels, indicating therapeutic efficacy.
Discussion
Comprehensive analysis integrating high-resolution MS-based profiling, network pharmacology, molecular docking, and animal validation supports that flavonoids from the aerial parts of Cistanche exert anti–chronic prostatitis effects. The putative mechanisms involve modulation of PI3K–AKT and related inflammation- and proliferation-associated pathways and key targets such as EGFR, AKT1, and MMP9. The results provide a scientific basis for the development and clinical application of aerial-part flavonoid preparations from Cistanche in the management of chronic prostatitis.
Conclusion
Flavonoids from the aerial parts of Cistanche demonstrate therapeutic potential against chronic prostatitis, likely via PI3K–AKT pathway regulation and interaction with EGFR, AKT1, and MMP9. Integration of chemical profiling, network pharmacology, molecular docking, and animal experiments corroborates their efficacy and elucidates plausible mechanisms.
