Application of Reiyanning preparation in preparation of medicine for preventing and treating prostatitis

Abstract

The invention discloses application of a Reiyanning preparation in preparation of a medicine for preventing and treating prostatitis. The Reiyanning preparation is prepared from dandelion, polygonum cuspidatum, patrinia villosa and sculellaria barbata. The application finds that the Reiyanning preparation can reduce the contents of IL-6, IL-1beta, NO and MDA in serum of a model rat, an HE staining result shows that the Reiyanning preparation has a certain improvement effect on damage of prostate tissues, and the Reiyanning preparation has a certain prevention and treatment effect on chronic abacterial prostatitis from molecular and tissue levels.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to the application of a Reyanning preparation in preparing medicines for preventing and treating chronic nonbacterial prostatitis. Background Art

[0002] Nonbacterial prostatitis, also known as chronic nonbacterial prostatitis or chronic pelvic pain syndrome, is a common urinary system disease, accounting for more than 90% of chronic prostatitis. This condition is characterized by long-term, recurrent pain or discomfort in the pelvic area, which may be accompanied by urination symptoms and sexual dysfunction, seriously affecting the patient's quality of life. The etiology of nonbacterial prostatitis is complex and may be related to pathogen infection, urination dysfunction, mental and psychological factors, neuroendocrine abnormalities, abnormal immune response and other factors. Modern medicine mainly treats this disease with antibacterial, smooth muscle relaxation, anti-inflammatory, analgesic, anti-anxiety and depression, etc., but long-term use of antibiotics has large adverse reactions and is prone to tolerance. In addition, the course of this disease is long and prone to repeated attacks, which seriously affects the patient's quality of life.

[0003] Under the influence of side effects, the patient's condition is prone to recurrence and protracted. Traditional Chinese medicine is based on dialectical treatment. While treating chronic non-bacterial prostatitis, traditional Chinese medicine can also protect organ function and delay the progression of chronic non-bacterial prostatitis. Traditional Chinese medicine treatment methods will not cause too much pain to patients, do not have too many side effects, are very convenient to use, and can fundamentally solve the problem. Therefore, it is necessary to find a drug that can treat chronic non-bacterial prostatitis without the above side effects or with reduced side effects. Generally, Western medicine has relatively large side effects, a long development cycle, and high development costs. Finding a traditional Chinese medicine that can prevent and treat chronic non-bacterial prostatitis is a good solution. Summary of the invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to provide a use of a Reyanning preparation in the preparation of a drug for preventing and treating chronic non-bacterial prostatitis. The Reyanning preparation can reduce the contents of IL-6, IL-1β, NO, and MDA in the serum of model rats and improve the damage of chronic non-bacterial prostatitis to prostate tissue.

[0005] The objective of the present invention is achieved through the following technical solutions: The invention provides an application of a Reyanning preparation in preparing a medicine for preventing and treating chronic nonbacterial prostatitis. The Reyanning preparation is prepared from dandelion, polygonum cuspidatum, Patrinia serrata and Scutellaria barbata.

[0006] Preferably, the Reyanning preparation is prepared from water extracts of dandelion, Polygonum cuspidatum, Patrinia serrata and Scutellaria barbata, and the weight ratio of dandelion, Polygonum cuspidatum, Patrinia serrata and Scutellaria barbata is 1-3:1-3:1-3:1.

[0007] Preferably, the weight ratio of dandelion, knotweed, patrinia chinensis and scutellaria barbata is 2:2:2:1.

[0008] Preferably, the prostatitis is chronic non-bacterial prostatitis.

[0009] Preferably, the dosage form of the Reyanning preparation is a decoction, a mixture, a pill, an oral solution, a tablet, a capsule, a granule, an ointment, a suppository or a powder.

[0010] Preferably, the Reyanning preparation is selected from any one of Reyanning mixture, Reyanning granules, Reyanning oral liquid and Reyanning suppository.

[0011] Preferably, the Reyanning preparation is administered at a dosage of 10 ml to 20 ml per time for adults, and is administered three times a day.

[0012] Preferably, the preparation method of the Reyanning preparation comprises: extracting dandelion, polygonum cuspidatum, Patrinia serrata and Scutellaria barbata by decocting in water, filtering the extract and centrifuging it, adding stevioside and ethylparaben to the supernatant, and boiling it to obtain the Reyanning preparation.

[0013] In the present invention, the prevention and treatment includes "prevention" and / or "treatment", wherein the "treatment" refers to the process of eliminating the cause or focus of the disease with a living human or animal body as the direct implementation object. The beneficial or desired clinical results of the treatment include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no deterioration), delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete), whether detectable or undetectable.

[0014] In the present invention, the objects of prevention and treatment include objects that have suffered from the disease or illness and objects that are susceptible to the disease or illness. When the terms such as alleviation, mitigation, attenuation, alleviation and relief are mentioned, their meanings also include elimination, disappearance and non-occurrence.

[0015] In the invention, the object of prevention and treatment refers to an organism receiving treatment with the Chinese medicine of the present invention, including a mammal or non-mammal receiving treatment for a disease or condition, and the mammal exemplarily includes bovines, equines, ovines, swine, canines, felines, rodents, and primates. Preferably, the mammal is a human. In the embodiment, the efficacy is verified by taking a non-mammalian animal as an example, and the non-mammal is specifically a zebrafish.

[0016] Compared with the prior art, the beneficial effects of the present invention are: The present application found that the Reyanning preparation can reduce the content 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 improvement effect on the damage of prostate tissue. From the molecular and tissue levels, it can be concluded that the Reyanning preparation has a certain preventive and therapeutic effect on prostatitis, especially chronic non-bacterial prostatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Comparison of HE staining results of prostate tissues between the control group and the model group (HE staining, scale bar = 200 μm); Figure 2 The effect of Reyanning preparation on body weight and organ indexes of rats with chronic nonbacterial prostatitis (compared with the blank 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 rats with prostatitis; B. Effect of Reyanning preparation on prostate index of rats with prostatitis; C. Effect of Reyanning preparation on thymus index of rats with prostatitis; D. Effect of Reyanning preparation on spleen index of rats with prostatitis; E. Effect of Reyanning preparation on liver index of rats with prostatitis; F. Effect of Reyanning preparation on kidney index of rats with prostatitis; Figure 3 Effects of Reyanning preparation on inflammatory factors in serum of rats with chronic nonbacterial prostatitis (compared with the blank group: ## P< 0.01, compared with the model group: * P< 0.05); Among them: A. The effect of Reyanning preparation on the inflammatory factor TNF-α in the serum of rats with prostatitis; B. The effect of Reyanning preparation on the inflammatory factor IL-6 in the serum of rats with prostatitis; C. The effect of Reyanning preparation on the inflammatory factor IL-1β in the serum of rats with prostatitis; D. The effect of Reyanning preparation on the inflammatory factor iNOS in the serum of rats with prostatitis; E. The effect of Reyanning preparation on the inflammatory factor NO in the serum of rats with prostatitis; F. The effect of Reyanning preparation on the inflammatory factor MDA in the serum of rats with prostatitis; G. The effect of Reyanning preparation on the inflammatory factor SOD in the serum of rats with prostatitis; Figure 4 The effect of Reyanning preparation on prostate tissue pathology in rats with chronic nonbacterial prostatitis (HE staining, scale bar = 200 μm); Figure 5The effects of Reyanning preparation on the body weight and organ indexes of normal rats (compared with the blank group: ### P<0.001; compared with the model group: *P< 0.05, ***P< 0.001); Among them: A. is the effect of Reyanning preparation on the body weight of normal rats; B. is the effect of Reyanning preparation on the thymus index of normal rats; C. is the effect of Reyanning preparation on the spleen index of normal rats; D. is the effect of Reyanning preparation on the liver index of normal rats; E. is the effect of Reyanning preparation on the kidney index of normal rats; Figure 6 This is the effect of Reyanning preparation on normal rat organ tissues (HE staining, thymus, spleen, liver scale = 200μm; kidney scale = 50μm). DETAILED DESCRIPTION

[0018] The embodiment of the present invention provides an application of a Reyanning preparation in the preparation of a drug for preventing and treating chronic non-bacterial prostatitis, wherein the Reyanning preparation is made of dandelion, Polygonum cuspidatum, Patrinia sutchuenensis and Scutellaria barbata, and the weight ratio of dandelion, Polygonum cuspidatum, Patrinia sutchuenensis and Scutellaria barbata is 1-3:1-3:1-3:1. The Reyanning preparation is selected from any one of Reyanning mixture, Reyanning granules, Reyanning oral liquid and Reyanning suppository, preferably Reyanning mixture, and the Reyanning preparation is administered at a dosage of 10ml-20ml / time for adults, and is administered three times a day. The preparation method of the Reyanning preparation comprises extracting the raw materials by decoction, filtering the extract and centrifuging, adding stevioside and ethylparaben to the supernatant, and boiling to obtain the traditional Chinese medicine.

[0019] 1. Research Content

[0020] 1.1 Materials

[0021] 1.1.1 Medicines The Reyanning preparation is prepared according to the following preparation method: take 372g of dandelion, 372g of polygonum cuspidatum, 372g of northern patrinia, and 186g of bark twig, add water and boil twice, add 8 times the water for the first time and boil for 2 hours, add 5 times the water for the second time and boil for 1 hour, filter the decoction, concentrate the filtrate under reduced pressure, combine the concentrate, centrifuge, filter, add 1.5g of stevioside and 0.5g of ethyl hydroxybenzoate, heat to boiling, and make it into 1000ml.

[0022] 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.

[0023] 1.1.2 Reagents Carrageenan (Cat. No. S30559-25 g) Shanghai Yuanye Biotechnology Co., Ltd.; Anhydrous ethanol, xylene, neutral gum (Batch No.: 100092683, 10023418, 10004160) Sinopharm Chemical Reagent Co., Ltd.; Hematoxylin-eosin (H&E) high-definition constant staining kit (lot number: G1076), universal tissue fixative (lot number: G1101), environmentally friendly dewaxing solution (lot number: G1128), Servicebio company.

[0024] Rat tumor necrosis factor α (TNF-α) ELISA research kit, rat interleukin 6 (IL-6) ELISA research kit, rat interleukin 1β (IL-1β) ELISA research kit, rat inducible nitric oxide synthase (iNOS) ELISA research kit, rat nitric oxide (NO) ELISA research kit, rat malondialdehyde (MDA) ELISA research kit, rat superoxide dismutase (SOD) ELISA research kit (Cat. No.: MM-0180R1, MM-0190R1, MM-0047R1, MM-0889R1, MM-20607R1, MM-0385R1, MM-0386R1) were all purchased from Jiangsu ELISA Industrial Co., Ltd.

[0025] 1.1.3 Instruments Thermo an GO 1510 full-wavelength microplate reader, Thermo Scientific Forma 900 Series-80℃ ultra-low temperature refrigerator, and frozen section machine are from Thermo Fisher Scientific (China) Co., Ltd.; upright optical microscope and imaging system are from Nikon, Japan; ME204 / 02 electronic balance is from Leica BiosystermsNussloch GmbH; pathological section machine is from Shanghai Leica Instrument Co., Ltd.; dehydrator is from DIAPATH; embedding machine and freezing table are from Wuhan Junjie Electronics Co., Ltd.; tissue spreader is from Cody Instrument in Jinhua City, Zhejiang Province; and oven is from Tianjin Lepore Instrument Equipment.

[0026] 1.2 Methods

[0027] 1.2.1 Establishment and evaluation methods of chronic nonbacterial prostatitis rat model There were 12 rats in each group. After seven days of adaptive feeding, the rats were anesthetized by intraperitoneal injection of 3mg / ml sodium pentobarbital at a dose of 1 mL / 100 g according to their body weight. After the rats were anesthetized, a midline incision was made in the abdomen under sterile conditions, and two injections of 3% carrageenan were injected into the left and right ventral lobes of the prostate, totaling 50μL. On the 21st day, three rats in the control group and three rats in the model group were killed, and the prostate tissues were taken from the ventral side for HE staining and tissue morphology observation to determine whether the model was successfully established.

[0028] Starting from the fourteenth day after surgery, rats in the control group and model group were gavaged with normal saline at noon every day for twenty-one consecutive days, and the intervention group was gavaged with the corresponding drugs. The Reyanning preparation was administered 2-4 times a day at a clinical dose of 10-20 ml at a time, which was converted into the dose used in rats, namely, the Reyanning preparation low-dose group (6.67 ml / kg, 2 ml), the Reyanning preparation medium-dose group (13.33 ml / kg, 4 ml), and the Reyanning preparation high-dose group (20.00 ml / kg, 6 ml). Celecoxib group (0.035 g / kg). On the forty-second day, the spleen, liver, kidney, prostate, and thymus were weighed and analyzed, and blood was collected from the abdominal aorta and centrifuged to obtain serum for storage at -80°C.

[0029] 1.2.2 Effects of Reyanning preparation on body weight and organ indexes in rats with prostatitis On the 22nd day after modeling, the animals in each group were anesthetized after the last administration and blood was collected from the abdominal aorta. The spleen, liver, kidney, prostate and thymus tissues were removed and weighed. Calculation of organ index 1.2.3 Effect of Reyanning preparation on inflammatory factors in serum of rats with prostatitis The blood of the rats was taken out and centrifuged to obtain serum at a speed of 3500 rpm for 10 minutes. According to the instructions of the kit of Jiangsu Enzyme Absorption Industry Co., Ltd., the contents of TNF-α, IL-6, IL-1β, iNOS, NO, MDA and SOD in the serum of the rats were detected.

[0030] 1.2.4 Effect of Reyanning Preparation on Prostate Tissue Pathology in Rats with Prostatitis The prostate tissue of the rat was removed and fixed in paraformaldehyde for animal tissue section analysis. The prostate tissue of the rat was cleaned, embedded in paraffin, and sliced ​​by a slicer. The slices were placed in environmentally friendly dewaxing solution I for 20 minutes, environmentally friendly dewaxing solution II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and 75% alcohol for 5 minutes, and then rinsed with pure water. After the frozen sections were rewarmed and fixed, they were placed in high-definition constant staining pretreatment solution for 1 minute. Then the sections were stained with hematoxylin staining solution for 3-5 minutes, rinsed with pure water, differentiated with differentiation solution, rinsed with pure water, and then stained with eosin for 15 seconds, and blued with blueing solution. After rinsing, ethanol was dehydrated from low to high gradients, and finally xylene was used to make it transparent, and the sections were sealed with neutral resin. The pathological changes of the rat joints were observed under a light microscope, and images were collected for analysis.

[0031] 1.2.5 Safety evaluation of Reyanning mixture A high-dose negative control of the Reyanning preparation was added in this experiment. Except for the surgical modeling, the treatment of this group was the same as that of the high-dose Reyanning group (i.e., intragastric administration of 20.00 ml / kg of Reyanning mixture) to evaluate the safety of the Reyanning mixture.

[0032] 1.2.6 Statistical methods All data are presented as the mean of at least 3 independent experiments. One-way analysis of variance and Tukey analysis were used for comparison of means. GraphPad Prism Version 5 was used for statistical analysis. P < 0.05 was considered statistically significant.

[0033] 2. Research Results

[0034] 2.1 Evaluation method of chronic nonbacterial prostatitis rat model The prostate tissue structure of the rats in the control group was clear, the glandular cavity was regular, and no obvious pathological changes were observed. The prostate tissue of the rats in the non-bacterial prostatitis model group showed vascular dilation, glandular epithelial hyperplasia, and cubic, columnar, high columnar, pseudostratified and other manifestations. The interstitial edema to varying degrees, the glandular cavity was dilated, there were secretions in the cavity, the interstitial was loose, and there was chronic inflammatory cell infiltration. The results are shown in Figure 1 .

[0035] 2.2 Effects of Reyanning preparation on body weight and organ indexes in rats with prostatitis

[0036] During the experimental period, the weight of rats in the control group was generally higher than that of rats in the other groups. After 32 days, the weight of rats in the high-dose Reyanning group was higher than that of rats in the other groups except the control group ( Figure 2 A). In addition, there were differences in prostate index and liver index between the model group and the control group ( Figure 2 B, 2E), there was no difference in the indexes of other organs. From the organ level, it can be seen that the surgical model of prostatitis was successful. From the index trend, it can be seen that the Reyanning preparation has a certain improvement on prostate tissue enlargement, but the difference is not significant. This shows that the Reyanning preparation may have a certain improvement effect on chronic non-bacterial prostatitis in rats and have a certain impact on liver tissue (N=6 in each group).

[0037] 2.3 Effect of Reyanning preparation on inflammatory factors in serum of rats with prostatitis like Figure 3 As shown, compared with the control group, the levels of IL-6, IL-1β, NO, and MDA in the serum of rats in the model group were different ( Figure 3 B, 3C, 3E, 3F), compared with the model group, the levels of the above inflammatory factors in the Reyanning groups at each dose showed a decreasing trend and there were differences; however, the effects on the levels of TNF-α, iNOS, and SOD in serum were not obvious ( Figure 3A, 3D, 3G). The results showed that Reyanning preparation could reduce most of the inflammatory factors in the serum of rats with chronic nonbacterial prostatitis, indicating that Reyanning preparation had anti-inflammatory effect and could improve chronic nonbacterial prostatitis in rats to a certain extent (N=3 in each group).

[0038] 2.4 Effect of Reyanning Mixture on Prostate Tissue Pathology in Rats with Prostatitis

[0039] The prostate tissues of rats with prostatitis were stained with HE and the histopathological results were analyzed ( Figure 4 ) found that there was no inflammatory cell infiltration in the control group; glandular epithelial hyperplasia, interstitial edema and chronic inflammatory cell infiltration were observed in the model group; in the low-, medium- and high-dose groups of Reyanning mixture, as the drug concentration increased, interstitial edema gradually alleviated, the cell cavity decreased, and the chronic inflammatory cells and secretions gradually decreased, which may be positively correlated with the drug dose; the above manifestations were correspondingly alleviated in the positive drug group compared with the model group.

[0040] 2.5 Safety evaluation results of Reyanning mixture

[0041] 2.5.1 Results of body weight and organ index in the safety group of Reyanning mixture There was a difference in body weight between the control group and the safety evaluation group during the experimental period, and the average body weight of the rats in the safety evaluation group continued to be lower than that of the rats in the control group after 20 days. Figure 5 C, 5D), no difference was found in the indexes of other organs. This indicates that the Reyanning preparation may have a certain impact on the spleen and liver of normal rats (N=6 in each group).

[0042] 2.5.2 HE staining results of organs in the safety group of Reyanning preparation like Figure 6 As shown in the figure, the thymus, spleen, liver and kidney tissues of the rats in the control group and the safety group were stained with HE to observe whether there were any abnormal changes. The results showed that the thymus of the control group and the safety group were intact in structure, with clear boundaries between the cortex and medulla, and dense cortical cells; the spleen of the control group and the safety group had obvious boundaries between the red pulp and white pulp, no obvious congestion of the splenic sinusoids, and clear splenic body structure; the liver of the control group and the safety group had intact tissue structure, normal hepatocyte morphology, no inflammatory cell infiltration, and hepatocytes were arranged radially around the central vein; the kidney of the control group and the safety group had clear tissue structure, complete and regular arrangement of glomeruli and renal tubules, no abnormalities in the glomerular basement membrane, no obvious fibrosis in the renal tubule structure, normal volume and cystic cavity of the renal capsule, and normal structure of proximal tubular epithelial cells. In summary, the Reyanning preparation did not cause organic damage to the thymus, spleen, liver and kidney of normal rats.

[0043] 3. Research Conclusion This study found that Reyanning preparation can reduce the levels of IL-6, IL-1β, NO, and MDA in the serum of model rats. HE staining results showed that Reyanning mixture had a certain improvement effect on prostate tissue damage. From the molecular and tissue levels, it can be concluded that Reyanning preparation may have a certain preventive and therapeutic effect on chronic non-bacterial prostatitis.

[0044] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of the Reyanning preparation in the preparation of medicines for preventing and treating prostatitis, wherein the Reyanning preparation is made from dandelion, polygonum cuspidatum, Patrinia scabra and Scutellaria barbata.

2. The use according to claim 1, characterized in that: The Reyanning preparation is prepared from water extracts of dandelion, polygonum cuspidatum, patrinia chinensis and Scutellaria barbata, and the weight ratio of dandelion, polygonum cuspidatum, patrinia chinensis and Scutellaria barbata is 1-3:1-3:1-3:

1.

3. The use according to claim 2, characterized in that: The weight ratio of dandelion, knotweed, patrinia cuspidatum and scutellaria barbata is 2:2:2:

1.

4. The use according to claim 3, characterized in that: The prostatitis is chronic non-bacterial prostatitis.

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

6. The use according to claim 5, characterized in that: The preparation method of the Reyanning preparation comprises: extracting dandelion, Polygonum cuspidatum, Patrinia serrata and Scutellaria barbata by decocting in water, filtering the extract and centrifuging, adding stevioside and ethylparaben to the supernatant, and boiling to obtain the Reyanning preparation.

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