Use of reyanning formulation in preparation of drug for preventing and treating prostatitis

Abstract

The use of a Reyanning formulation in the preparation of a drug for preventing and treating prostatitis. The Reyanning formulation is prepared from dandelion, giant knotweed rhizome, sonchi arvensis herba and herba scutellariae barbatae. The present application finds that the Reyanning formulation can reduce the contents of IL-6, IL-1β, NO and MDA in the serum of model rats, HE staining results show that the Reyanning formulation has certain relieving effect on the damage of prostate tissue, and it can be concluded from the molecular and tissue levels that the Reyanning formulation has certain prevention and treatment effect on chronic non-bacterial prostatitis.

Description

Application of Reyanning preparation in the preparation of drugs for the prevention and treatment of prostatitis

[0001] Cross-references to related applications

[0002] This application claims priority and benefit to Chinese Patent Application No. 202510026590.3, filed on January 8, 2025, entitled "Application of Reyanning Preparation in the Preparation of Drugs for the Prevention and Treatment of Prostatitis", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of traditional Chinese medicine technology, and in particular relates to the application of Reyanning preparation in the preparation of drugs for the prevention and treatment of chronic nonbacterial prostatitis. Background Technology

[0004] Nonbacterial prostatitis, also known as chronic nonbacterial prostatitis or chronic pelvic pain syndrome, is a common urinary tract disease, accounting for more than 90% of chronic prostatitis cases. This condition is characterized by long-term, recurrent pain or discomfort in the pelvic region, possibly accompanied by urinary symptoms and sexual dysfunction, severely impacting the patient's quality of life. The etiology of nonbacterial prostatitis is complex, possibly related to pathogen infection, urinary dysfunction, psychological factors, neuroendocrine abnormalities, and abnormal immune responses. Modern medicine treats this disease primarily with antibiotics, smooth muscle relaxants, anti-inflammatory drugs, analgesics, and anti-anxiety / antidepressant medications. However, long-term use of antibiotics has significant adverse reactions and easily leads to tolerance. Furthermore, the disease has a long course and is prone to recurrence, severely affecting the patient's quality of life.

[0005] Due to the side effects, patients' conditions are prone to recurrence and become chronic and difficult to cure. Traditional Chinese medicine (TCM) is based on syndrome differentiation and treatment. While treating chronic nonbacterial prostatitis, TCM can also protect organ function and slow down the progression of the disease. TCM treatment methods do not cause patients much pain, have few side effects, are convenient to use, and can fundamentally solve the problem. Therefore, there is a need to find a drug that can treat chronic nonbacterial prostatitis without the aforementioned side effects or with reduced side effects. Generally, Western medicines have more significant side effects, longer development cycles, and higher development costs. Finding a TCM that can prevent and treat chronic nonbacterial prostatitis is a good solution. Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of this invention is to provide an application of Reyanning preparation in the preparation of drugs for the prevention and treatment of chronic nonbacterial prostatitis. Reyanning preparation can reduce the content of IL-6, IL-1β, NO and MDA in the serum of model rats and improve the damage of chronic nonbacterial prostatitis to prostate tissue.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] This invention provides the application of a Reyanning preparation in the preparation of a drug for the prevention and treatment of chronic nonbacterial prostatitis, wherein the Reyanning preparation is made from dandelion, Polygonum cuspidatum, Patrinia scabiosaefolia and Scutellaria barbata.

[0009] Preferably, the Reyanning preparation is made from water extracts of dandelion, Polygonum cuspidatum, Patrinia scabra and Scutellaria barbata, with the weight ratio of dandelion, Polygonum cuspidatum, Patrinia scabra and Scutellaria barbata being 1-3:1-3:1-3:1.

[0010] Preferably, the weight ratio of dandelion, Japanese knotweed, valerian, and Scutellaria barbata is 2:2:2:1.

[0011] Preferably, the prostatitis is chronic nonbacterial prostatitis.

[0012] Preferably, the dosage form of the Reyanning preparation is a decoction, mixture, pill, oral liquid, tablet, capsule, granule, ointment, suppository or powder.

[0013] Preferably, the Reyanning preparation is selected from any one of Reyanning compound, Reyanning granules, Reyanning oral liquid, and Reyanning suppositories.

[0014] Preferably, the Reyaning preparation is administered to adults at a dose of 10ml-20ml per dose, and is administered three times a day.

[0015] Preferably, the preparation method of the Reyanning preparation includes: extracting dandelion, Polygonum cuspidatum, Patrinia scabiosaefolia and Scutellaria barbata by decoction with water, filtering the extract and centrifuging it, adding stevioside and ethylparaben to the supernatant, boiling it, and then obtaining the Reyanning preparation.

[0016] In this invention, the prevention and treatment include "prevention" and / or "treatment," wherein "treatment" refers to the process of eliminating the cause or lesion by directly targeting a living human or animal body. Beneficial or desired clinical outcomes of the treatment include, but are not limited to, symptom relief, disease severity reduction, disease stability (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable.

[0017] In this invention, the objects of prevention and control include those who already have a condition or disease, as well as those who are susceptible to developing a condition or disease. When terms such as mitigation, relief, weakening, moderate, and alleviation are mentioned, their meanings also include elimination, disappearance, and non-occurrence.

[0018] In this invention, the target of prevention and treatment refers to an organism receiving treatment with the traditional Chinese medicine described in this invention, including mammals or non-mammals receiving treatment for diseases or symptoms. Exemplary mammals include bovids, equines, sheep, suidae, canines, felines, rodents, and primates. Preferably, the mammal is a human. In this embodiment, a non-mammalian animal is used as an example to verify the efficacy; specifically, the non-mammalian animal is a zebrafish.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This application found that the Reyaning preparation can reduce the levels of IL-6, IL-1β, NO, and MDA in the serum of model rats. HE staining results showed that the Reyaning preparation has a certain ameliorative effect on prostate tissue damage. From the molecular and tissue levels, it can be concluded that the Reyaning preparation has a certain preventive and therapeutic effect on prostatitis, especially chronic nonbacterial prostatitis. Attached Figure Description

[0021] Figure 1 shows the comparison of HE staining results of prostate tissue from rats in the control group and the model group (HE staining, scale bar = 200 μm);

[0022] Figure 2 shows the effects of the Reyanning preparation on body weight and organ index in rats with chronic nonbacterial prostatitis (compared to the control group). ### P<0.001; compared with the model group: * P<0.05, *** P<0.001); Among them: A. Effect of Reyanning preparation on body weight of prostatitis rats; B. Effect of Reyanning preparation on prostate index of prostatitis rats; C. Effect of Reyanning preparation on thymus index of prostatitis rats; D. Effect of Reyanning preparation on spleen index of prostatitis rats; E. Effect of Reyanning preparation on liver index of prostatitis rats; F. Effect of Reyanning preparation on kidney index of prostatitis rats;

[0023] Figure 3 shows the effect of the Reyanning preparation on serum inflammatory factors in rats with chronic nonbacterial prostatitis (compared to the blank group). ## P<0.01, compared with the model group: *P<0.05); Among them: A. Effect of Reyanning preparation on serum inflammatory factor TNF-α in prostatitis rats; B. Effect of Reyanning preparation on serum inflammatory factor IL-6 in prostatitis rats; C. Effect of Reyanning preparation on serum inflammatory factor IL-1β in prostatitis rats; D. Effect of Reyanning preparation on serum inflammatory factor iNOS in prostatitis rats; E. Effect of Reyanning preparation on serum inflammatory factor NO in prostatitis rats; F. Effect of Reyanning preparation on serum inflammatory factor MDA in prostatitis rats; G. Effect of Reyanning preparation on serum inflammatory factor SOD in prostatitis rats;

[0024] Figure 4 shows the effect of Reyanning preparation on the histopathology of prostate tissue in rats with chronic nonbacterial prostatitis (HE staining, scale bar = 200 μm);

[0025] Figure 5 shows the effects of Reyaning preparation on body weight and organ indices in normal rats (compared with the control group: ###P<0.001; compared with the model group: *P<0.05, ***P<0.001); where: A. effect of Reyaning preparation on body weight in normal rats; B. effect of Reyaning preparation on thymus index in normal rats; C. effect of Reyaning preparation on spleen index in normal rats; D. effect of Reyaning preparation on liver index in normal rats; E. effect of Reyaning preparation on kidney index in normal rats.

[0026] Figure 6 shows the effects of the Reyanning preparation on the organs and tissues of normal rats (HE staining, scale bar for thymus, spleen, and liver = 200 μm; scale bar for kidney = 50 μm). Detailed Implementation

[0027] This invention provides an application of the Reyanning preparation in the preparation of a drug for the prevention and treatment of chronic nonbacterial prostatitis. The Reyanning preparation is made from dandelion, Polygonum cuspidatum, Patrinia scabiosaefolia, and Scutellaria barbata, with a weight ratio of 1-3:1-3:1-3:1. The Reyanning preparation is selected from any one of Reyanning compound, Reyanning granules, Reyanning oral liquid, and Reyanning suppositories, preferably Reyanning compound. The Reyanning preparation is administered to adults at a dosage of 10-20 ml / time, three times daily. The preparation method of the Reyanning preparation includes extracting the raw materials by decoction with water, filtering the extract, centrifuging, adding stevioside and ethylparaben to the supernatant, and boiling to obtain the traditional Chinese medicine.

[0028] I. Research Content

[0029] 1.1 Materials

[0030] 1.1.1 Medicines

[0031] The Reyanning preparation is prepared according to the following method: Take 372g of dandelion, 372g of Polygonum cuspidatum, 372g of Patrinia scabiosaefolia, and 186g of Scutellaria barbata, add water and decoct twice. For the first decoction, add 8 times the amount of water and decoct for 2 hours. For the second decoction, add 5 times the amount of water and decoct for 1 hour. Filter the decoction, concentrate the filtrate under reduced pressure, combine the concentrates, centrifuge, filter, add 1.5g of stevioside and 0.5g of ethylparaben, heat to boiling, and make 1000ml.

[0032] Celecoxib capsules (batch number: 8149393) were purchased from Pfizer Pharmaceuticals Co., Ltd. 84 clean-grade male SD rats, weighing (170±10) g, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

[0033] 1.1.2 Reagents

[0034] Carrageenan (Product No.: S30559-25g) Shanghai Yuanye Biotechnology Co., Ltd.

[0035] Anhydrous ethanol, xylene, neutral resin (batch numbers: 100092683, 10023418, 10004160), Sinopharm Chemical Reagent Co., Ltd.

[0036] Hematoxylin-Eosin (H&E) High-Resolution Constant Staining Kit (Batch No.: G1076), Universal Tissue Fixative (Batch No.: G1101), and Environmentally Friendly Dewaxing Solution (Batch No.: G1128) are from Servicebio.

[0037] The following ELISA kits were purchased from Jiangsu Enzyme Immunoassay Co., Ltd.: Rat Tumor Necrosis Factor α (TNF-α) ELISA Kit, Rat Interleukin 6 (IL-6) ELISA Kit, Rat Interleukin 1β (IL-1β) ELISA Kit, Rat Inducible Nitric Oxide Synthase (iNOS) ELISA Kit, Rat Nitric Oxide (NO) ELISA Kit, Rat Malondialdehyde (MDA) ELISA Kit, and Rat Superoxide Dismutase (SOD) ELISA Kit (catalog numbers: MM-0180R1, MM-0190R1, MM-0047R1, MM-0889R1, MM-20607R1, MM-0385R1, MM-0386R1).

[0038] 1.1.3 Instruments

[0039] Thermo AN GO 1510 full-wavelength microplate reader, Thermo Scientific Forma 900 Series -80℃ ultra-low temperature freezer, cryostat, Thermo Fisher Scientific (China) Co., Ltd.; upright optical microscope and imaging system, Nikon, Japan; ME204 / 02 electronic balance, Leica Biosysterms Nussloch GmbH; pathology slicer, Leica Instruments Shanghai Co., Ltd.; dehydrator, DIAPATH; embedding machine and freezing stage, Wuhan Junjie Electronics Co., Ltd.; tissue spreader, Kedi Instruments, Jinhua City, Zhejiang Province; oven, Leiborui Instruments & Equipment, Tianjin.

[0040] 1.2 Methods

[0041] 1.2.1 Establishment and Evaluation Methods of a Rat Model of Chronic Nonbacterial Prostatitis

[0042] Twelve rats were placed in each group. After seven days of acclimatization, the rats were anesthetized by intraperitoneal injection of 3 mg / ml sodium pentobarbital at a dose of 1 mL / 100g, according to their body weight. After anesthesia, two injections of 50 μL of 3% carrageenan were made into the left and right ventral lobes of the prostate through a midline abdominal incision under sterile conditions. On the twenty-first day, three control rats and three model rats were sacrificed, and prostate tissue was harvested from the ventral side for HE staining and histological observation to determine the success of the model establishment.

[0043] Starting on the fourteenth day post-surgery, for twenty-one consecutive days, rats in the control and model groups were administered physiological saline by gavage at noon, while the intervention group was administered the corresponding medication by gavage. The Reyaning preparation was administered at the clinical dosage of 10-20 ml twice daily, converted to rat dosages: low-dose Reyaning (6.67 ml / kg, 2 ml), medium-dose Reyaning (13.33 ml / kg, 4 ml), and high-dose Reyaning (20.00 ml / kg, 6 ml). Celecoxib was administered (0.035 g / kg). On the forty-second day, the spleen, liver, kidneys, prostate, and thymus were weighed and analyzed. Blood was collected from the abdominal aorta, centrifuged, and serum was stored at -80℃ for later use.

[0044] 1.2.2 Effects of Reyanning preparation on body weight and organ index in prostatitis rats

[0045] On the 42nd day after modeling, each group of animals was anesthetized after the last administration of the drug, and blood was drawn from the abdominal aorta. The spleen, liver, kidneys, prostate, and thymus tissues were then dissected and weighed. Organ index calculation.

[0046] 1.2.3 Effects of Reyanning preparation on serum inflammatory factors in prostatitis rats

[0047] After extracting blood from rats, serum was obtained by centrifugation at 3500 rpm for 10 minutes. The levels of TNF-α, IL-6, IL-1β, iNOS, NO, MDA, and SOD in rat serum were detected according to the kit instructions from Jiangsu Enzyme Immunoassay Co., Ltd.

[0048] 1.2.4 Effects of Reyanning preparation on prostate tissue pathology in rats with prostatitis

[0049] Rat prostate tissue was extracted and fixed in paraformaldehyde for histological analysis. The rat prostate tissue was cleaned, embedded in paraffin, and sectioned using a microtome. Sections were sequentially immersed in environmentally friendly dewaxing solution I for 20 min, environmentally friendly dewaxing solution II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, followed by rinsing with pure water. Frozen sections were thawed and fixed, then pretreated with high-resolution constant staining solution for 1 min. Sections were then stained with hematoxylin for 3-5 min, rinsed with pure water, differentiated with differentiation solution, rinsed with pure water, stained with eosin for 15 s, and then blued with a blueing solution. After rinsing, the sections were dehydrated with ethanol in a gradient from low to high concentrations, cleared with xylene, mounted with neutral resin, and the pathological changes in the rat prostate were observed under a light microscope. Images were acquired and analyzed.

[0050] 1.2.5 Safety Evaluation of Reyanning Mixture

[0051] This experiment added a high-dose negative control of Reyanning preparation. Except for surgical modeling, the treatment in this group was the same as that in the high-dose Reyanning group (i.e., Reyanning mixture was administered by gavage at a dose of 20.00 ml / kg) to evaluate the safety of Reyanning mixture.

[0052] 1.2.6 Statistical Methods

[0053] All data are expressed as the mean of at least three independent experiments. Means were compared using one-way variance analysis and Tukey analysis. Statistical analysis was performed using GraphPad Prism Version 5. A p-value < 0.05 was considered statistically significant.

[0054] II. Research Results

[0055] 2.1 Evaluation methods for rat models of chronic nonbacterial prostatitis

[0056] In the control group, the prostate tissue of rats showed clear structure, regular lumens, and no obvious pathological changes. In the nonbacterial prostatitis model group, the prostate tissue exhibited vasodilation, glandular epithelial hyperplasia with cuboidal, columnar, tall columnar, and pseudostratified structures, varying degrees of interstitial edema, dilated lumens containing secretions, loose interstitium, and chronic inflammatory cell infiltration. The results are shown in Figure 1.

[0057] 2.2 Effects of Reyanning preparation on body weight and organ index in prostatitis rats

[0058] During the experimental period, the body weight of rats in the control group was generally higher than that of rats in the other groups. After 32 days, the body weight of rats in the high-dose Reyaning group was higher than that of all other groups except the control group (Figure 2A). Except for differences in prostate index and liver index between the model group and the control group (Figures 2B and 2E), no differences were found in other organ indices. At the organ level, the prostatitis surgical model was successfully established. The index trends showed that Reyaning preparation had some improvement on prostate tissue enlargement, but the difference was not significant. This indicates that Reyaning preparation may have a certain ameliorative effect on chronic nonbacterial prostatitis in rats and may have some impact on liver tissue (N=6 for each group).

[0059] 2.3 Effects of Reyanning preparation on serum inflammatory factors in prostatitis rats

[0060] As shown in Figure 3, compared with the control group, the levels of IL-6, IL-1β, NO, and MDA in the serum of rats in the model group differed (Figures 3B, 3C, 3E, and 3F). The levels of these inflammatory factors in each dose group of Reyaning showed a decreasing trend compared with the model group, and the differences were not significant. However, the effects on the levels of TNF-α, iNOS, and SOD in the serum were not obvious (Figures 3A, 3D, and 3G). These results indicate that Reyaning can reduce most inflammatory factors in the serum of rats with chronic nonbacterial prostatitis, suggesting that Reyaning has an anti-inflammatory effect and can improve chronic nonbacterial prostatitis in rats to some extent (N=3 for each group).

[0061] 2.4 Effects of Reyanning Compound on Prostate Histopathology in Rats with Prostatitis

[0062] HE staining was performed on the prostate tissue of rats with prostatitis. Histopathological analysis (Figure 4) revealed that the control group had no inflammatory cell infiltration; the model group showed glandular epithelial hyperplasia, interstitial edema, and chronic inflammatory cell infiltration; in the low, medium, and high dose groups of Reyanning compound, interstitial edema gradually decreased, cell lumen decreased, and chronic inflammatory cells and secretions gradually decreased with increasing drug concentration, which may be directly proportional to the drug dose; the positive control group showed corresponding relief of the above manifestations compared with the model group.

[0063] 2.5 Safety Evaluation Results of Reyanning Mixture

[0064] 2.5.1 Results of body weight and organ index in the safe group of Reyanning Mixture

[0065] During the experimental period, there was a difference in body weight between the control group and the safety evaluation group, and after 20 days, the mean body weight of the rats in the safety evaluation group remained lower than that of the control group. Except for differences in spleen and liver indices between the safety evaluation group and the control group (Figures 5C and 5D), no differences were observed in other organ indices. This indicates that the Reyaning preparation may have some effect on the spleen and liver of normal rats (N=6 for each group).

[0066] 2.5.2 HE staining results of organs in the safe group of Reyanning preparation

[0067] As shown in Figure 6, the thymus, spleen, liver, and kidney tissues of rats in both the control and safety groups were stained with hematoxylin and eosin (HE) to observe any abnormal changes. The results showed that the thymus in both the control and safety groups was structurally intact, with a clear boundary between the cortex and medulla, and dense cortical cells. The spleen in both the control and safety groups showed a clear boundary between the red and white pulp, no obvious congestion of the splenic sinuses, and clear splenic corpuscle structure. The liver in both the control and safety groups had intact tissue structure, normal hepatocyte morphology, no inflammatory cell infiltration, and hepatocytes arranged radially outward from the central vein. The kidneys in both the control and safety groups had clear tissue structure, with intact and regularly arranged glomeruli and tubules, no abnormalities in the glomerular basement membrane, no obvious fibrosis in the tubular structure, normal Bowman's capsule volume and lumen size, and normal proximal convoluted tubular epithelial cell structure. In conclusion, the Reyanning preparation did not cause organic damage to the thymus, spleen, liver, or kidneys of normal rats.

[0068] III. Research Conclusions

[0069] This study found that the Reyanning preparation could reduce the levels of IL-6, IL-1β, NO, and MDA in the serum of model rats. HE staining results showed that the Reyanning preparation had a certain ameliorative effect on prostate tissue damage. From both molecular and tissue perspectives, it can be concluded that the Reyanning preparation may have a certain preventive and therapeutic effect on chronic nonbacterial prostatitis.

[0070] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of Reyanning preparation in the preparation of drugs for the prevention and treatment of prostatitis, wherein the Reyanning preparation is made from dandelion, Polygonum cuspidatum, Patrinia scabiosaefolia and Scutellaria barbata.

2. Use according to claim 1, characterized in that, The Reyanning preparation is made from the water extracts of dandelion, Polygonum cuspidatum, Patrinia scabiosaefolia and Scutellaria barbata, with the weight ratio of dandelion, Polygonum cuspidatum, Patrinia scabiosaefolia and Scutellaria barbata being 1-3:1-3:1-3:

1.

3. Use according to claim 2, characterized in that, The weight ratio of dandelion, Japanese knotweed, valerian, and Scutellaria barbata is 2:2:2:

1.

4. Use according to claim 3, characterized in that, The prostatitis mentioned is chronic nonbacterial prostatitis.

5. The application according to any one of claims 1 to 4, characterized in that, The dosage forms of the Reyanning preparation are decoction, mixture, pill, oral liquid, tablet, capsule, granule, ointment, suppository or powder.

6. The application according to claim 5, characterized in that, The preparation method of the Reyanning preparation includes: extracting dandelion, Polygonum cuspidatum, Patrinia scabiosaefolia and Scutellaria barbata by decoction with water, filtering the extract and centrifuging it, adding stevioside and ethylparaben to the supernatant, and boiling it to obtain the Reyanning preparation.

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