Pharmaceutical composition for treating prostatitis and preparation method thereof

Abstract

The invention provides a pharmaceutical composition for treating prostatitis and a preparation method thereof. The pharmaceutical composition is prepared from the following raw materials in parts by weight: 20 to 60 parts of palmatine, 20 to 60 parts of paeoniflorin, 10 to 30 parts of formononetin, 20 to 60 parts of astilbin and 10 to 40 parts of stigmasterol. In-vitro cell tests and in-vivo animal experiments prove that the pharmaceutical composition has the effect of relieving the chronic prostatitis reaction.

Description

Technical Field

[0001] This invention pertains to the field of traditional Chinese medicine and relates to a pharmaceutical composition that can be used to treat prostatitis and its preparation method. Background Technology

[0002] Prostatitis is a pathological change related to inflammation, immunity, and neuroendocrine factors caused by a variety of complex causes and triggers. Health statistics from the International Health Center show that 35% to 50% of adult men will experience prostatitis at some point in their lives, with a higher incidence in men under 50. Prostatitis is divided into two main categories: acute and chronic. Acute prostatitis often presents with symptoms such as fever, chills, perineal distending pain, lower abdominal pain, and painful urination. Chronic prostatitis clinical symptoms include prostate pain, urinary frequency, urgency, dysuria, urethral burning, and cloudy urine, accompanied by dizziness, tinnitus, insomnia, and even impotence and premature ejaculation. In Traditional Chinese Medicine (TCM), this disease falls under the categories of "seminal turbidity," "urinary tract infection," and "leukorrhea." Semen turbidity is characterized by abnormal urination and urethral discharge as the main symptom. Urinary tract infection manifests as frequent, short, and painful urination, dribbling urination, and lower abdominal cramping pain. The main pathogenesis is damp-heat stagnation, qi and blood stasis, yin deficiency with fire excess, or kidney yang deficiency, resulting in a deficiency in the root and an excess in the branch. Traditional Chinese medicine theory holds that the initial treatment of this disease should focus on clearing heat and promoting diuresis, while the later treatment should focus on nourishing kidney yin or warming and tonifying kidney yang, while also taking care of the liver and spleen.

[0003] Qianlieshutong capsules are a commonly used traditional Chinese medicine for treating prostatitis or benign prostatic hyperplasia (BPH). Its indications include chronic prostatitis and BPH due to damp-heat stagnation, with symptoms such as frequent urination, urgency, dribbling urination, perineal, lower abdominal, or lumbosacral distension or pain, and scrotal dampness. There are related patent applications for this traditional Chinese medicine: application number 02153461.6 discloses the formulation and preparation method of Qianlieshutong capsules, and application number 200410022067.1 discloses the dosage form and preparation method of the Qianlieshutong formulation. However, the Qianlieshutong formulation in these inventions consists of 13 medicinal materials, making its composition relatively complex. This results in high cost, difficulty in preparation, and difficulty in quality control. Due to the large number of medicinal materials and the extraction process involving mixing the medicinal materials with a certain concentration of ethanol or water, the concentration of effective substances is incomplete, leading to insufficient efficacy. The medication lacks precision, has a broad clinical application, and lacks research on the active ingredients of the medicinal materials. Therefore, it is particularly urgent to find a drug composition for the treatment of prostatitis that is well-formulated, highly pure, and precisely targeted for clinical use. Summary of the Invention

[0004] The technical solution of this invention is based on the simplified formula screening of Qianlieshutong capsules. Through extensive and meaningful experimental research, a more superior and refined formulation ratio has been obtained, aiming to overcome the aforementioned defects of Qianlieshutong capsules, reduce the difficulty of drug preparation process and quality control, and improve the accuracy and efficacy of clinical treatment. Specifically, the objective is to provide a pharmaceutical composition for treating prostatitis and its preparation method. The pharmaceutical composition includes five active ingredients in the formulation, as follows: A pharmaceutical composition for treating prostatitis, characterized in that the pharmaceutical composition comprises the following raw materials in the following weight ratios: 20-60 parts of berberine, 20-60 parts of paeoniflorin, 10-30 parts of gentianin, 20-60 parts of astilbene, and 10-30 parts of stigmasterol.

[0005] Furthermore, the pharmaceutical composition comprises the following components in the indicated weight ratios: 40 parts berberine, 40 parts paeoniflorin, 20 parts gentianin, 40 parts astilbene, and 20 parts stigmasterol.

[0006] Inflammation has been known to humans for thousands of years. It is a highly dynamic process characterized by the immune system's initial protective response to foreign substances. The immune response is coordinated through a complex network of cells and molecules, with B lymphocytes and T lymphocytes being the main effector cells. Cytokines, as key signaling proteins, mediate the host's regulation of inflammation and the immune response, and mainly include interleukins (ILs), chemokines, growth factors, tumor necrosis factor-α (TNF-α), interferons, and colony-stimulating factors.

[0007] At the molecular level, multiple signaling pathways jointly regulate inflammation and immune responses, such as nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK), Janus kinase / signal transducer and activator of transcription (JAK / STAT), phosphatidylinositol 3-kinase / protein kinase B (PI3K / AKT), and the arachidonic acid pathway. These pathways coordinate the function of immune cells and the inflammatory process by influencing the activity of various signaling molecules.

[0008] Modern pharmacological studies have shown that chemical substances can significantly reduce prostate inflammation and macrophage infiltration by pharmacologically inhibiting the NF-κB and PI3K / AKT pathways and suppressing the activation of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome. Studies have shown that calycosin can significantly improve the pathological damage of prostate tissue in rats with chronic prostatitis (CP). Furthermore, calycosin can significantly downregulate the levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), TNF-α, immunoglobulin G (IgG), and secretory immunoglobulin A (SIgA). Further research revealed that verbascosin significantly inhibited oxidative stress in rats with chronic prostatitis and blocked the activation of the p38MAPK / NF-κB signaling pathway (Wang H, He L, Liu Z, et al. Calycosin protects against chronic prostatitis in rats via inhibition of the p38MAPK / NF-κB pathway. Open Med (Wars). 2023 Aug). 28;18(1):20230770); L-carnitine may alleviate the inflammatory response of chronic prostatitis / chronic pelvic pain syndrome and relieve oxidative stress symptoms by inhibiting the PI3K / Akt / FoxO1 signaling pathway (Zhang Qiang, Tang Qian, Su Xuzhen et al. Mechanism of action of L-carnitine in regulating CP / CPPS rats based on PI3K / Akt / FoxO1 signaling pathway [J]. Chinese Journal of Sexology, 2025(08):47-53); Frankincense-myrrh significantly reduced the expression level of IL-17 in rat serum and prostate tissue through multiple pathways such as NF-κB and PI3K-AKT; Berberine alleviates chronic prostatitis / chronic pelvic pain syndrome by modifying gut microbiome signaling. Asian J Androl. 2024 Sep 1;26(5):500-509.The ethnic medicine Kezimuke may reduce prostate inflammation and exert a therapeutic effect on chronic bacterial prostatitis (CBP) by inhibiting the release of pro-inflammatory factors IL-6 and TNF-α and regulating the phosphorylation expression of key proteins in the PI3K-AKT signaling pathway (Nurbahetti Houwati, Hu Mengying, Yelixati Danabek, et al. Exploring the mechanism of action of Kezimuke granules in the treatment of chronic bacterial prostatitis based on network pharmacology [J]. Bioresources, 2025, 47(4):379-388.). Sodium butyrate can inhibit oxidative stress and NLRP3 inflammasome activation, and alleviate EAP-related prostatitis and pelvic pain through the Nrf2 / HO-1 pathway (Hua X, Zhang J, Chen J, et al. Sodium butyrate alleviates experimental autoimmune prostatitis by inhibiting oxidative stress and NLRP3 inflammasome activation via the Nrf2 / HO-1 pathway. Prostate. 2024). May;84(7):666-681.

[0009] In addition to the compounds mentioned above, Prostatin has also been reported to alleviate the inflammatory response of chronic prostatitis. Literature review shows that Prostatin capsules have a good protective effect on rats with chronic nonbacterial prostatitis. Its mechanism may be related to the inhibition of PI3K / Akt / NF-κB signaling pathway protein expression and inflammatory mediator secretion (Li Bo, Huang Zhuangzhuang, Xu Gang, et al. Exploring the effect of Prostatin capsules on rats with chronic nonbacterial prostatitis based on the PI3K / Akt / NF-κB pathway [J]. Chinese Traditional and Herbal Drugs, 2023, 45(7):2174-2179). NF-κB, PI3K / AKT, and MAPK signaling pathways may be the main pathways through which Prostatin capsules treat chronic prostatitis (Liu Shengjing, Gao Qinghe, Wang Fu, et al. Exploring the molecular mechanism of Prostatin capsules in treating chronic prostatitis based on network pharmacology and molecular docking [J]. Chinese Journal of Integrated Traditional and Western Medicine, 2020(7):805-810).

[0010] In the formula of Qianlie Shutong, Phellodendron bark and red peony root are the principal herbs, Sparganium rhizome, Smilax glabra rhizome, and Verbena officinalis rhizome are the assistant herbs, Saxifraga stolonifera rhizome, Portulaca oleracea rhizome, Alisma plantago-aquatica rhizome, Ligusticum striatum rhizome, Achyranthes bidentata rhizome, and Bupleurum chinense rhizome are the adjuvant herbs, and Glycyrrhiza uralensis rhizome is the guiding herb.

[0011] Phellodendron bark is the dried bark of the Phellodendron amurense tree (Rutaceae family), commonly known as "Chuan Huangbai" (Sichuan Phellodendron). The dried bark of Phellodendron chinense is used medicinally as "Guan Huangbai," with functions including clearing heat and dampness, purging fire and eliminating steaming heat, and detoxifying and healing sores. The chemical components isolated from these two medicinal materials mainly include alkaloids, limonenes, phenolic acids, terpenes, phenylpropanoids, volatile components, and trace elements. Existing technologies have disclosed the isolation of 25 alkaloid compounds from these two medicinal materials, including berberine, purslane, and palmatine. Palmatine has a wide range of pharmacological effects, including anticancer, antioxidant, anti-inflammatory, neuroprotective, antibacterial, antiviral, and lipid-regulating effects. It has a certain inhibitory effect on the growth and metabolism of Staphylococcus aureus, and also inhibits Gram-positive bacteria such as Escherichia coli, Candida albicans, and Shigella dysenteriae, as well as certain fungi, and has a certain inhibitory effect on molds. Modern pharmacological studies have shown that berberine reduces tissue inflammation by inhibiting the activation of the NLRP3 inflammasome and NF-κB signaling, thereby reducing the production of inflammatory factors. It also relaxes the smooth muscle of the prostate and urethra by non-competitively antagonizing α1 receptors, reducing urethral pressure and relieving urinary obstruction and pain.

[0012] Red peony root (Paeoniflorin) is the dried root of the peony plant (Paeonia lactiflora), belonging to the Ranunculaceae family. Its main functions are clearing heat and cooling the blood, dispersing blood stasis and relieving pain. The chemical components of the medicinal material include glycosides, tannins, and volatile oils. These include pinane monoterpenes and glycosides, such as paeoniflorin, oxypaeoniflorin, and benzoylpaeoniflorin; lactone monoterpenes and glycosides, such as paeoniflorin lactone and paeoniflorin glycoside; and tannins, such as 1,2,3,4,6-pentagalloylglucose and the corresponding hexagalloylglucose and heptagalloylglucose. The volatile oil mainly contains benzoic acid, paeonol, and other alcohols and phenolic components. Paeoniflorin, in particular, has the effects of dilating coronary arteries, increasing coronary blood flow, counteracting acute myocardial ischemia, inhibiting platelet aggregation, and lowering blood pressure. It also possesses various pharmacological activities such as sedation, analgesia, anti-inflammation, anti-ulceration, antipyresis, antispasmodicity, and hypoglycemia. Studies on its anti-inflammatory effects have shown that paeoniflorin improves lipopolysaccharide (LPS)-induced inflammatory responses and pain symptoms in mice by synergistically inhibiting NF-κB transcriptional activity and NLRP3 inflammasome activation. Paeoniflorin also exhibits anti-inflammatory effects on LPS-stimulated human colon adenocarcinoma cells (Caco-2), significantly inhibiting LPS-induced expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), TNF-α, IL-6, and matrix metalloproteinase-9 (MMP-9), and suppressing the activation of the NF-κB signaling pathway in LPS-stimulated Caco-2 cells.

[0013] Sparganium rhizome, the dried tuber of *Sparganium stoloniferum*, a plant in the family Sparganaceae, has functions such as relieving indigestion, alleviating pain, and promoting blood circulation. Modern research shows that Sparganium rhizome mainly contains phenolic compounds, alkaloids, saponins, and organic acids. Phenolic components, including flavonoids, are the main and active ingredients. Among them, formononetin is one of the aforementioned flavonoid components, possessing various pharmacological effects such as cardiovascular protection, anti-tumor activity, anti-inflammation, and anti-oxidation. It exerts its anti-inflammatory effect by downregulating overactivated COX2 / PGE2, NF-κB, MAPK, and JAK-STAT pathways, thereby inhibiting the release of cytokines and chemokines and reducing the production of NLRP3 inflammasomes. Studies have explored the anxiolytic effect of formononetin on chronic inflammatory pain in C57BL / 6 male mice. The results showed that administration of formononetin inhibited the NF-κB signaling pathway and microglial activation in mice with chronic inflammatory pain, thereby alleviating anxiety symptoms.

[0014] Smilax glabra is the dried rhizome of *Smilax glabra*, a plant in the Liliaceae family. Its functions include detoxification, dampness removal, and joint mobility promotion. Its chemical components include flavonoids and flavonoid glycosides, saponins, organic acids, and volatile oils. Flavonoids and flavonoid derivatives include astilbin, smilax glabra glycoside, astilbin, and quercetin. Astilbin, a type of flavonoid compound, has a wide range of pharmacological effects, with its core mechanism being anti-inflammatory and antioxidant activity. Astilbin significantly reduces the levels of various cytokines, such as IL-6, IL-1β, PGE2, and MCP-1, by inhibiting the NF-κB signaling pathway. Studies have shown that astilbin, as an anti-inflammatory drug for treating rheumatoid arthritis, works by regulating the production of inflammatory factors and inhibiting the expression of key molecules in the Toll-like receptor-mediated NF-κB signaling pathway.

[0015] Verbena officinalis is the dried aerial part of the plant Verbena officinalis, belonging to the Verbenaceae family. Its functions include promoting blood circulation and removing blood stasis, detoxification, diuresis, jaundice relief, and malaria treatment. Its chemical components include iridoids, organic acids, flavonoids, sterols, and phenylpropanoid glycosides. Among them, stigmasterol, a type of phytosterol, possesses various pharmacological effects such as anti-inflammatory, antioxidant, anticancer, and cholesterol-lowering properties. In recent years, clinical studies have confirmed that phytosterols can inhibit prostatic hyperplasia and improve bladder contraction, significantly improving symptoms such as urinary frequency, nocturnal urinary frequency, bladder urgency, and urinary obstruction. Other studies have shown that stigmasterol treatment can improve cognitive dysfunction in Alzheimer's disease model mice and reduce the content of β-amyloid protein 42 (Aβ42) in the cerebral cortex and hippocampus. It exerts its inhibitory effect on neuroinflammation by reducing the level of pro-inflammatory cytokines and inhibiting microglial cell activation.

[0016] The present invention also provides a method for preparing the above-mentioned pharmaceutical composition, the method comprising the following steps: The pharmaceutical composition is obtained by mixing berberine, paeoniflorin, astilbene, and stigmasterol.

[0017] Furthermore, the berberine, paeoniflorin, gentiopicrin, astilbene, and stigmasterol can be obtained by extracting Chinese medicinal materials using conventional extraction methods in the field, or can be purchased directly from commercially available products.

[0018] The present invention provides the use of the pharmaceutical composition described herein in the preparation of a medicament for the treatment of chronic prostatitis.

[0019] Furthermore, the present invention provides the use of the pharmaceutical composition described herein in the preparation of a medicament for treating chronic nonbacterial prostatitis.

[0020] The technical solution provided by this invention has the following beneficial technical effects: The five active ingredients are derived from the five herbs in Qianlieshutong capsules: Phellodendron bark, red peony root, Sparganium rhizome, Smilax glabra rhizome, and Verbena officinalis. These herbs have compatibility relationships in traditional Chinese medicine. Through extensive experimental research, a scientific and effective formula has been developed. Modern pharmacological studies have found that berberine, paeoniflorin, astilbene, and stigmasterol have anti-inflammatory activities. The resulting drug composition can exert anti-inflammatory effects through multiple mechanisms of action, relieving chronic prostatitis, improving medication accuracy, and reducing dosage.

[0021] In vitro cell experiments confirmed that, within a non-cytotoxic concentration range, the pharmaceutical composition formed by the five active ingredients significantly inhibited the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in RAW 264.7 macrophages induced by lipopolysaccharide (LPS). In vivo animal experiments showed that the composition significantly inhibited the inflammatory response in rats with chronic prostatitis in a concentration-dependent manner and effectively reduced the expression levels of TNF-α, interleukin-1β (IL-1β), and IL-6 in rats, with better effects than Qianlieshutong capsules, thus achieving targeted treatment for chronic prostatitis. Attached Figure Description

[0022] Figure 1 The survival status of mouse mononuclear macrophage leukemia cell lines (RAW264.7) in groups 1-9 was compared with that of the control group. Figure 2 The results of nitric oxide production measurements were obtained for the control group, the LPS group, and groups 1-9. Figure 3 The results of the determination of the production of inflammatory factor TNF-α in groups 1-9 were used as the control group and LPS group. Figure 4The results of the measurement of the production of inflammatory factor IL-6 in groups 1-9 were used as the control group and LPS group. Figure 5 The results of measuring the production of the inflammatory factor TNF-α in rats in the control group, model group, compound group, low-dose group, medium-dose group, and high-dose group are as follows: Figure 6 The results of measuring the production of the inflammatory factor IL-6 in rats in the control group, model group, compound group, low-dose group, medium-dose group, and high-dose group are as follows: Figure 7 The results of measuring the production of the inflammatory factor IL-1β in rats in the control group, model group, compound group, low-dose group, medium-dose group, and high-dose group are as follows: Detailed Implementation

[0023] To make the objectives and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0024] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art. Example

[0025] A pharmaceutical composition for treating prostatitis, characterized in that the pharmaceutical composition comprises the following raw materials in the indicated weight ratios: 40 mg of berberine, 40 mg of paeoniflorin, 20 mg of gentianin, 40 mg of astilbin, and 20 mg of stigmasterol. Example

[0026] A pharmaceutical composition for treating prostatitis, characterized in that the pharmaceutical composition comprises the following raw materials in the following weight ratios: 20 mg of berberine, 20 mg of paeoniflorin, 10 mg of gentianin, 20 mg of astilbin, and 10 mg of stigmasterol. Example

[0027] A pharmaceutical composition for treating prostatitis, characterized in that the pharmaceutical composition comprises the following raw materials in the following weight ratios: 60 mg of berberine, 60 mg of paeoniflorin, 30 mg of gentianin, 60 mg of astilbin, and 30 mg of stigmasterol. Example

[0028] After mixing the raw materials of Example 1, they are thoroughly mixed with an appropriate amount of dextrin and sucrose powder (flavoring agent); a soft mass is prepared with an appropriate amount of ethanol, granulated by passing through a 14-mesh sieve, dried and granulated; and packaged into granules containing 0.16g of active ingredient per bag. Example

[0029] In Example 2, all raw materials were mixed in a three-dimensional mixer for 30 minutes to ensure uniformity. After passing through a 100-mesh sieve, 10% of the total raw material mass of microcrystalline cellulose was added as a filler, and 1% of magnesium stearate was added as a lubricant. The mixture was then mixed for another 15 minutes. The mixture was then filled into No. 0 hollow capsule shells using a fully automatic capsule filling machine. Each capsule contained 300 mg of contents. Finally, a quality inspection was performed, and the capsules were packaged in aluminum-plastic blister packs. Experimental Example 1. Evaluation of in vitro anti-inflammatory effects 1.1 Instruments, reagents and materials The mouse mononuclear macrophage leukemia cell line (RAW264.7) was commercially available. DMEM medium and fetal bovine serum (FBS) were obtained from Gibco (Thermo Fisher Scientific). Thiazol blue (MTT), lipopolysaccharide (LPS), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich. Gliese reagents were obtained from Shanghai Bosheng Biotechnology. The microplate reader (model SMR16.1) was purchased from Wuhan Youersheng Life Science Equipment Co., Ltd., and the ELISA kit was obtained from Thermo Fisher Scientific.

[0030] 1.2 Experimental Principle Upon LPS stimulation, RAW264.7 cells activate signaling pathways such as NF-κB and MAPK, initiating the transcription and translation of a series of inflammation-related genes, thereby synthesizing and releasing large amounts of key pro-inflammatory mediators such as IL-1β, IL-6, TNF-α, and NO. This makes LPS-stimulated RAW264.7 cells a reliable model for studying inflammatory response mechanisms and screening anti-inflammatory drugs.

[0031] 1.3 Cell Culture and Processing RAW264.7 cells were cultured in DMEM medium containing 10% heat-inactivated fetal bovine serum and 1% penicillin / streptomycin solution in a constant temperature and humidity incubator at 37°C and 5% CO2. After 2–3 days of culture to the logarithmic growth phase, the cells were used for subsequent experiments. Cells were treated with different concentrations of drug combinations, followed by LPS stimulation and continued culture for a certain period.

[0032] 1.4 Experimental Grouping The experiment was divided into eleven groups: a control group (administered in DMEM medium), an LPS group (administered with 0.1 μg / mL lipopolysaccharide), and a drug administration group consisting of nine subgroups: groups 1-9. Groups 1-2 were single-component drugs, group 3 was a two-component drug, groups 4-6 were three-component drugs, and groups 7-9 were five-component drugs. The specific drug concentrations in each group are as follows: Group 1: Berberine 40 μg / mL; Group 2: Paeoniflorin 40 μg / mL; Group 3: berberine 40 μg / mL, paeoniflorin 40 μg / mL; Group 4: berberine 40 μg / mL, paeoniflorin 40 μg / mL, gentianin 20 μg / mL; Group 5: berberine 40 μg / mL, paeoniflorin 40 μg / mL, astilbene 40 μg / mL; Group 6: berberine 40 μg / mL, paeoniflorin 40 μg / mL, stigmasterol 20 μg / mL; Group 7: berberine 20 μg / mL, paeoniflorin 20 μg / mL, gentianin 10 μg / mL, astilbene 20 μg / mL, stigmasterol 10 μg / mL; Group 8: berberine 40 μg / mL, paeoniflorin 40 μg / mL, gentianin 20 μg / mL, astilbene 40 μg / mL, stigmasterol 20 μg / mL; Group 9: berberine 60 μg / mL, paeoniflorin 60 μg / mL, gentianin 30 μg / mL, astilbene 60 μg / mL, stigmasterol 30 μg / mL; 1.5 Cell viability assay The cytotoxicity of the drug combination to RAW264.7 cells was evaluated using the MTT assay. The experimental procedure was as follows: RAW264.7 cells were seeded in 96-well plates and cultured at 37°C for 12 h, followed by replacement with fresh culture medium. At 37°C, the control group received no drug, while the other groups were treated with the appropriate drug concentrations. Each group had six parallel wells. After culturing for 24 h, 20 μL of 0.5 mg / mL MTT solution was added to each well, and the cells were cultured for another 4 h. The culture medium was discarded, and 150 μL of DMSO was added to dissolve the formazan precipitate within the cells. The absorbance of each well at 570 nm was measured using a microplate reader.

[0033] 1.6 Detection of Nitric Oxide Formation RAW264.7 cells were cultured at a rate of 2 × 10⁻⁶. 5Cells were seeded at a density of cells / mL. Cells in the drug-treated group were pre-treated with the corresponding drug concentration for 2 h, while the LPS group received no treatment. Both groups were then stimulated with 0.1 μg / mL LPS and cultured for 24 h. Immediately after treatment, 100 μL of cell culture supernatant was collected and mixed with an equal volume of Griess reagent, which was reacted at room temperature for 10 min. The Griess reagent consisted of a 5% phosphate solution containing 0.1% α-naphthylamine and 1% p-aminobenzenesulfonic acid. Based on the Griess reaction, the nitrite content accumulated in the culture medium was measured as an indicator of nitric oxide (NO) production. The absorbance of the reaction product at 525 nm was measured using a microplate reader, and the nitrite concentration in the sample was calculated based on the sodium nitrite standard curve.

[0034] 1.7 Detection of Inflammatory Factor Production Following existing research methods, the levels of TNF-α and IL-6 in the culture medium were detected using an enzyme-linked immunosorbent assay (ELISA) kit. The simplified procedure is as follows: RAW264.7 cells were seeded in 24-well plates and pretreated with the appropriate drug concentration for 2 h, followed by stimulation with LPS for 20 h. Immediately after treatment, 100 μL of cell culture supernatant was collected and centrifuged at 12000×g for 3 min to remove particulate matter. The concentrations of TNF-α and IL-6 in each sample were detected according to the kit instructions.

[0035] 1.8 Experimental Results The viability of RAW264.7 cells after 24 hours of treatment with the drug composition of this invention was detected using the MTT assay. The results showed that the single-component, two-component, three-component, and five-component drugs had almost no effect on the viability of RAW264.7 cells, and the difference was not statistically significant compared with the control group. Figure 1 Therefore, it can be concluded that the five-component pharmaceutical composition provided by this invention has extremely low cytotoxicity.

[0036] The effects of single-component, two-component, three-component, and five-component drugs on LPS-induced NO production in RAW264.7 cells were investigated by measuring the accumulation of nitrite in the culture medium. The results showed that LPS stimulation significantly increased NO concentration. Compared with the LPS group, the single-component, two-component, three-component, and five-component drugs all inhibited LPS-induced NO production to varying degrees, with statistically significant differences. Different doses of the five-component drug showed better inhibitory effects on LPS-induced NO production than the single-component, two-component, and three-component drugs, with statistically significant differences. Furthermore, the five-component drug composition of this invention can significantly inhibit LPS-induced NO production in a dose-dependent manner. Figure 2 .

[0037] The effects of single-component, two-component, three-component, and five-component drugs on LPS-induced production of inflammatory cytokines TNF-α and IL-6 in RAW264.7 cells were also investigated. Compared with the LPS group, the single-component, two-component, three-component, and five-component drugs all inhibited LPS-induced TNF-α and IL-6 production to varying degrees, with statistically significant differences. Different doses of the five-component drug showed better inhibitory effects on LPS-induced TNF-α and IL-6 production, superior to the single-component, two-component, and three-component drugs, with statistically significant differences. Furthermore, the five-component drug composition of this invention can significantly inhibit LPS-induced TNF-α and IL-6 production in a dose-dependent manner, see [see details]. Figure 3 and Figure 4 .

[0038] 2. Evaluation of anti-inflammatory effects in vivo 2.1 Laboratory Animals Male SD rats, 6-8 weeks old, commercially available. The rats were housed in a temperature-controlled environment with a 12-hour light / 12-hour dark cycle and humidity maintained at 60%-65%, with free access to food and water.

[0039] 2.2 Experimental Grouping Male SD rats were randomly divided into 5 groups, with 10 rats in each group: control group, model group, compound group (administered with Prostatin Capsules), low-dose group, medium-dose group, and high-dose group. The dosages were as follows: Control group: 50 μl of physiological saline was injected into the left and right ventral lobes of the prostate of rats, once each.

[0040] Compound group: The adult daily clinical dose of Prostatin Capsules is 3.6 g / 60 kg / d. Based on body weight conversion, the equivalent dose for rats = human dose (mg / kg) × conversion factor, which is 6.3 (refer to Zhang Fumei, Zhao Yu, and Wang Rui. "Experimental Guide to Animal Physiology" [M]. Beijing: China Light Industry Press, 2023.). Therefore, the dosage for rats is 378 mg / kg. Accurately weigh the calculated drug powder and add it to 20% β-cyclodextrin saline to prepare a solvent.

[0041] Low-dose group: 20 mg / kg of berberine, 20 mg / kg of paeoniflorin, 10 mg / kg of astilbene, 20 mg / kg of stigmasterol, and 10 mg / kg of stigmasterol were weighed in sequence, mixed thoroughly, and then a solvent was prepared by adding 20% ​​β-cyclodextrin in physiological saline.

[0042] Medium-dose group: 40 mg / kg of berberine, 40 mg / kg of paeoniflorin, 20 mg / kg of astilbene, 40 mg / kg of phytosterol, and 20 mg / kg of stigmasterol. The above active ingredients were weighed in sequence, mixed thoroughly, and then added to physiological saline containing 20% ​​β-cyclodextrin to prepare a solvent.

[0043] High-dose group: 60 mg / kg of berberine, 60 mg / kg of paeoniflorin, 30 mg / kg of astilbene, 60 mg / kg of phytosterol, and 30 mg / kg of stigmasterol. The above active ingredients were weighed in sequence, mixed thoroughly, and then a solvent was prepared by adding 20% ​​β-cyclodextrin in physiological saline.

[0044] 2.3 Establishment of a chronic prostatitis (CP) model After anesthetizing rats with ether, the abdominal skin was incised to expose the prostate. 50 μl of 3% carrageenan solution was injected once into each of the left and right ventral lobes of the prostate to establish a chronic prostatitis model. Seven days after modeling, light microscopy revealed thickening of the prostate capsule, disappearance of mucosal folds within the lumen, destruction of acini, swollen and blurred interstitium, abundant glial fiber proliferation, congestion, and inflammatory cell infiltration, indicating successful model establishment. Except for the control group, all other groups were modeled using the same method, and then administered drugs according to the experimental groupings in section 2.2. After four weeks of continuous drug administration, the rats were sacrificed by cervical dislocation. Prostate tissue was then dissected and collected from each group of rats.

[0045] 2.4 Detection of inflammatory factor levels Processed prostate tissue was collected, homogenized thoroughly using a tissue homogenizer, and centrifuged to collect the supernatant. The supernatant sample was added to 96-well plates for culture, with horseradish peroxidase (HRP)-labeled binding reagent added to each well. Following strict adherence to the kit instructions, an ELISA kit was used to detect the levels of inflammatory factors in the samples, including interleukin-1β (IL-1β), IL-6, and TNF-α. The absorbance of each well was measured at 450 nm using the ELISA reader, according to the manufacturer's instructions. The experiment was independently repeated three times.

[0046] 2.5 Experimental Results The levels of inflammatory factors in the prostate tissue of a rat model of chronic prostatitis were detected using ELISA. The results showed that the expression levels of TNF-α, IL-1β, and IL-6 in the prostate tissue of the model group were significantly higher than those in the control group, with statistically significant differences. Compared with the model group, in the compound group, low, medium, and high doses of the five-component drug combination significantly inhibited the upregulation of the expression of the above-mentioned inflammatory factors, with statistically significant differences. Compared with the compound group, the medium and high doses of the five-component drug combination showed better inhibitory effects on the above-mentioned inflammatory factors, with statistically significant differences. Figure 5-7 The above results indicate that the pharmaceutical composition of the present invention can inhibit the inflammatory response in rats with chronic prostatitis.

Claims

1. A pharmaceutical composition for treating prostatitis, characterized in that, The pharmaceutical composition comprises the following raw materials in the following weight ratios: 20-60 parts of berberine, 20-60 parts of paeoniflorin, 10-30 parts of gentianin, 20-60 parts of astilbene, and 10-30 parts of stigmasterol.

2. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition comprises the following components in the indicated weight ratios: 40 parts berberine, 40 parts paeoniflorin, 20 parts gentianin, 40 parts astilbin, and 20 parts stigmasterol.

3. The method for preparing the pharmaceutical composition according to any one of claims 1-2, characterized in that, The preparation method includes the following steps: mixing berberine, paeoniflorin, gentianin, astilbene, and stigmasterol to obtain the composition.

4. Use of the pharmaceutical composition according to any one of claims 1-2 in the preparation of a medicament for treating chronic prostatitis.

5. Use of the pharmaceutical composition according to any one of claims 1-2 in the preparation of a medicament for treating chronic nonbacterial prostatitis.

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