Traditional Chinese medicine composition for treating semen nonliquefaction and prostatitis as well as preparation and application of traditional Chinese medicine composition

Abstract

The invention provides a traditional Chinese medicine composition for treating semen nonliquefaction and prostatitis as well as a preparation and application of the traditional Chinese medicine composition. The traditional Chinese medicine composition for treating non-liquefaction of semen comprises the following medicinal materials in parts by weight: 15-25 parts of semen plantaginis, 15-25 parts of lalang grass rhizome, 10-20 parts of Chinese fevervine herb, 8-12 parts of talc, 8-12 parts of ricepaperplant pith, 8-12 parts of dianthus superbus, 8-12 parts of polygonum aviculare, 13-18 parts of rhizoma smilacis glabrae, 15-25 parts of herba patriniae, 2-4 parts of rheum officinale, 8-12 parts of fructus gardeniae, 8-12 parts of liquorice and 4-6 parts of junci medulla. All the medicines are combined to achieve the effects of clearing away heat and dampness, removing dampness and heat and dredging collaterals, clearing heat and promoting diuresis, removing toxins and expelling turbidity and promoting blood circulation to remove blood stasis together so as to promote semen liquefaction, and the traditional Chinese medicine composition has a remarkable clinical curative effect on the non-liquefaction of semen. The clinical curative effect of the traditional Chinese medicine composition for treating the semen nonliquefaction is superior to that of levofloxacin, and the safety is higher. The traditional Chinese medicine composition disclosed by the invention can also obviously reduce inflammatory response of prostatitis, effectively reduce inflammatory factors in prostate tissues, obviously improve prostatitis and effectively treat type III chronic prostatitis.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to a traditional Chinese medicine composition for treating semen liquefaction disorder and prostatitis, and a preparation and application thereof. Background Art

[0002] The coagulation and liquefaction of semen is a natural physiological phenomenon that protects the normal movement and fertility of sperm. At room temperature, a normal male semen sample begins to liquefy within 10 to 30 minutes after being discharged from the body. If it still cannot liquefy or liquefies incompletely after more than 60 minutes, it is called semen liquefaction disorder. Liquefaction time is one of the important criteria for evaluating sperm quality, and abnormal semen liquefaction is one of the common causes of male infertility. Statistics from relevant literature in my country show that the incidence of semen liquefaction disorder accounts for about 2.51% to 42.65% of the causes of male infertility. Abnormal semen liquefaction will limit sperm activity and directly affect the conception process, thereby greatly reducing the conception rate.

[0003] Relevant studies in recent years have shown that semen liquefaction is not only a high incidence, but also has quite complex pathogenic factors. Western medicine has theories on the pathogenesis of this disease, such as the liquefaction factor theory, the coagulation factor theory, the endocrine disorder theory, and the reproductive tract infection theory. At present, semen liquefaction caused by chronic prostatitis, Ureaplasma urealyticum infection, chronic epididymitis, etc. is a hot topic of research. In a normal adult male's ejaculation volume, sperm only accounts for about 7%, and the rest is seminal plasma, of which 70% comes from the seminal vesicle, 20% comes from the prostate, and the remaining 3% comes from the bulbourethral gland. Once there is chronic prostatitis or mycoplasma or chlamydia infection, the internal environment of semen is affected, resulting in semen liquefaction. At present, Western medicine treatment for semen liquefaction mainly focuses on empirical treatments such as anti-inflammatory, antioxidant, vitamin and trace element supplementation, and there is no specific therapeutic drug, and there are shortcomings such as poor clinical effect, more side effects, and affected pregnancy rate.

[0004] Therefore, it is of great significance to study a traditional Chinese medicine formula for treating semen liquefaction disorder. Summary of the invention

[0005] In view of this, the present invention proposes a Chinese medicine composition for treating semen liquefaction disorder and its preparation and application, which has a significant effect in treating semen liquefaction disorder and is effective in treating chronic prostatitis.

[0006] The technical scheme of the present invention is achieved as follows: a traditional Chinese medicine composition for treating semen liquefaction disorder comprises the following medicinal materials in parts by weight: 15-25 parts of plantain seeds, 15-25 parts of Imperata rhizome, 10-20 parts of Caulis Patriniae, 8-12 parts of talc, 8-12 parts of tongcao, 8-12 parts of superbus, 8-12 parts of smilax glabra, 13-18 parts of smilax glabra, 15-25 parts of patrinia herb, 2-4 parts of rhubarb, 8-12 parts of gardenia, 8-12 parts of licorice and 4-6 parts of rush.

[0007] Preferably, the Chinese medicine composition for treating semen liquefaction disorder comprises the following medicinal materials in parts by weight: 18-22 parts of Plantago seeds, 18-22 parts of Imperata rhizome, 18-22 parts of Caulis Patriniae, 9-11 parts of talc, 9-11 parts of Smilax glabra, 9-11 parts of Dianthus superbus, 9-11 parts of Polygonum multiflorum, 14-16 parts of Smilax glabra, 18-22 parts of Patrinia herb, 2-4 parts of Rhubarb, 9-11 parts of Gardenia jasminoides, 9-11 parts of Licorice, and 4-6 parts of Juncus effusus.

[0008] More preferably, the Chinese medicine composition for treating semen liquefaction disorder comprises the following medicinal materials in parts by weight: 20 parts of Plantago seed, 20 parts of Imperata root, 15 parts of Caulis Patriniae, 10 parts of Talcum, 10 parts of Smilax glabra, 10 parts of Dianthus superbus, 10 parts of Polygonum multiflorum, 15 parts of Smilax glabra, 20 parts of Patrinia herb, 3 parts of Rhubarb, 10 parts of Gardenia jasminoides, 10 parts of Licorice, and 5 parts of Juncus effusus.

[0009] A traditional Chinese medicine preparation for treating semen liquefaction disorder comprises the traditional Chinese medicine composition described in any one of the present invention.

[0010] Preferably, the Chinese medicine composition preparation is a decoction, granules, pills, capsules, or oral liquid.

[0011] Use of the Chinese medicine composition described in any one of the present invention in preparing a medicine for treating semen liquefaction disorder and / or prostatitis.

[0012] Furthermore, the prostatitis is chronic prostatitis; further, the prostatitis is type III chronic prostatitis.

[0013] Modern pharmacological studies have shown that heat-clearing drugs can inhibit the body's inflammatory response, improve local inflammation and tissue damage, and have significant antibacterial effects, killing and inhibiting a variety of pathogenic microorganisms. Plantago also has anti-inflammatory, antibacterial, expectorant and antitussive effects, and immune regulation. Plantago polysaccharides can reduce the number of white blood cells, reduce the content of TNF-α and MDA in exudates, and the level of MDA in serum, and increase the level of SOD in exudates and serum. It exerts anti-inflammatory effects by inhibiting the exudation of inflammatory factors, eliminating free radicals, and anti-oxidation. Imperata root inhibits the activation of TLR4 / NFκB signaling pathway, reduces the release of inflammatory mediators, and regulates inflammatory responses. The iridoid components in the terpenoids of the terpenoids are the main active ingredients of the terpenoids that exert anti-inflammatory and analgesic effects, and have a good inhibitory effect on inflammation and pain in mice.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) The Chinese medicine composition of the present invention can effectively shorten the liquefaction time, improve the sperm concentration, increase the sperm motility, improve the semen quality, and significantly improve the sperm motility and the ratio of grade A sperm + grade B sperm. The clinical efficacy of the Chinese medicine composition of the present invention in treating semen liquefaction syndrome is better than levofloxacin, and it is safer.

[0016] (2) The mechanism of the Chinese medicine composition of the present invention in treating semen liquefaction syndrome may be to reduce the concentrations of IL-1β, IL-6, IL-8, Fn, and PSA, and increase the concentrations of PAP and SEMG1, thereby shortening the semen liquefaction time and improving the semen quality.

[0017] (3) In the prescription of the present invention, Plantago seed, Imperata root and Cistanche deserticola are the main herbs. Plantago seed is sweet and cold, and is good at clearing heat, promoting diuresis and relieving stranguria; Imperata root can relieve stranguria, remove blood stasis and promote urination; Cistanche deserticola clears heat, detoxifies and resolves stagnation. The three herbs are combined as the main herbs, and have the effects of clearing heat, promoting diuresis and promoting diuresis, and resolving stagnation and removing blood stasis. Talc is light in taste and cold in nature, heavy and slippery in quality. Its lightness can penetrate dampness, its coldness can clear heat, its heaviness can descend, and its slipperiness can clear the orifices, so it can clear the water source; Herba Lycopodii can clear the heart fire, and remove dampness and heat, so that the dampness and heat evil can be removed from the urine; Herba Dianthus and Herba Polygoni Multiflori are both good at clearing heat, promoting diuresis and relieving stranguria; in addition, Rhizoma Smilacis Glabrae and Herba Patriniae can clear heat, dry dampness and remove toxicity, and together they are the assistant herbs, which strengthen the effect of clearing heat, promoting diuresis and relieving stranguria. Rhubarb can eliminate evil heat and remove dampness and heat from the stool. Gardenia is used as an adjuvant to clear the triple energizer and promote the flow of water, thus enhancing the functions of the main and auxiliary drugs in clearing heat, promoting diuresis and relieving stranguria; rhubarb can clear away pathogenic heat and remove dampness and heat from the stool. Licorice harmonizes the various drugs and can also clear heat, relieve urgency and relieve pain, which is the role of an adjuvant. Juncus is added to the decoction to enhance the function of promoting diuresis and relieving stranguria. The combination of various drugs can separate the clear and turbid, remove dampness and heat, and open the meridians, playing the functions of clearing heat and promoting diuresis, removing toxins and removing turbidity, and promoting blood circulation and removing blood stasis, so as to help the liquefaction of semen, and has a significant clinical effect on the treatment of semen liquefaction disorder.

[0018] (4) Studies have shown that the Chinese medicine composition of the present invention can also significantly reduce the inflammatory response of prostatitis, effectively reduce the inflammatory factors in prostate tissue, significantly improve prostatitis, and can effectively treat type III chronic prostatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Photos of the animal model construction process;

[0020] Figure 2 Photos of gavage treatment;

[0021] Figure 3 Photos of the prostate tissue sampling process;

[0022] Figure 4 The experimental results of the control group;

[0023] Figure 5 The experimental results of the high-dose group of the liquefaction-promoting decoction;

[0024] Figure 6 The experimental results of the medium-dose group of the liquefaction-promoting decoction;

[0025] Figure 7 The experimental results of the low-dose group of the liquefaction-promoting decoction;

[0026] Figure 8 Experimental results of the model group. DETAILED DESCRIPTION

[0027] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0028] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0029] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0030] Example 1

[0031] A traditional Chinese medicine composition for treating semen liquefaction disorder: composed of the following medicinal materials in parts by weight: 20 parts of plantain seeds, 20 parts of Imperata root, 15 parts of Caulis Patriniae, 10 parts of talc, 10 parts of tongcao, 10 parts of superbus, 10 parts of smilax glabra, 15 parts of rhubarb, 20 parts of patrinia, 3 parts of rhubarb, 10 parts of gardenia, 10 parts of liquorice and 5 parts of rush.

[0032] Example 2

[0033] A traditional Chinese medicine composition for treating semen liquefaction disorder: composed of the following medicinal materials in parts by weight: 15 parts of plantain seeds, 25 parts of Imperata root, 20 parts of Caulis Patriniae, 8 parts of talc, 12 parts of tongcao, 8 parts of superbus, 12 parts of smilax glabra, 13 parts of rhubarb, 15 parts of patrinia herb, 2 parts of rhubarb, 12 parts of gardenia, 8 parts of liquorice and 4 parts of rush.

[0034] Example 3

[0035] A traditional Chinese medicine composition for treating semen liquefaction disorder, comprising the following medicinal materials in parts by weight: 20 parts of plantain seeds, 20 parts of Imperata root, 10 parts of chicken manure, 12 parts of talc, 8 parts of tongcao, 12 parts of superbus, 8 parts of kelp, 18 parts of smilax glabra, 25 parts of patrinia herb, 4 parts of rhubarb, 8 parts of gardenia, 12 parts of liquorice and 6 parts of rush.

[0036] Test Example 1-Treatment of Semen Liquefaction

[0037] 1Clinical studies

[0038] 1.1 Case source

[0039] A total of 98 patients who met the diagnosis and inclusion criteria were selected from the National Medical Hall and the Outpatient Department of Surgery II of Hainan Provincial Hospital of Traditional Chinese Medicine from January 2021 to December 2022.

[0040] 1.2 Diagnostic criteria

[0041] 1.2.1 Criteria for semen liquefaction

[0042] Semen liquefaction syndrome refers to the fifth edition of WHO's "Manual for Laboratory Testing of Human Semen and Sperm-Cervical Mucus Interactions". That is, after the semen is discharged from the body, it is placed in a 37℃ water bath or incubator for more than 60 minutes and cannot be liquefied or is incompletely liquefied. The semen is viscous like jelly or even lumpy; and the person has not had sex for 3-7 days.

[0043] 1.2.2 Chronic bacterial prostatitis

[0044] The criteria were formulated with reference to the National Institutes of Health and the Chinese Guidelines for the Diagnosis and Treatment of Urological Diseases: history of recurrent urinary tract infections; NIH-CPSI pain symptom score ≥ 14 points; rectal examination showed prostate enlargement and tenderness; prostate fluid white blood cells > 10 / high-power field of view; prostate B-ultrasound showed unclear boundaries of prostate tissue structure;

[0045] 1.2.3 Ureaplasma urealyticum infection

[0046] The result of Uu test of semen is positive (take semen or urethral secretion for mycoplasma test)

[0047] 1.2.4 TCM syndrome diagnostic criteria

[0048] TCM diagnostic criteria: refer to "Chinese Medicine Andrology" and "Guidelines for Clinical Research of New Chinese Medicines for the Treatment of Male Infertility" to differentiate between damp-heat and endogenous turbid toxins.

[0049] Main symptoms:

[0050] ① The liquid is viscous like jelly, or even lumpy;

[0051] ② Urinary tract symptoms, with occasional white discharge;

[0052] ③ Discomfort in the lower abdomen, perineum, and testicles, or hematuria and hematospermia.

[0053] Secondary symptoms:

[0054] ①Thirst but no desire to drink, bitter taste in the mouth or sticky feeling in the mouth;

[0055] ② Dry or sticky stools, red or astringent urine, short, yellow or turbid urine;

[0056] ③ The tongue is red, with yellow tongue coating, either yellow and greasy or thin and yellow and greasy; the pulse is deep or slippery and rapid.

[0057] If the patient has any one of the main symptoms ① and ②-③, as well as any one of the secondary symptoms, the syndrome is diagnosed as downward movement of damp-heat and endogenous generation of turbid toxins.

[0058] 1.2.5 Case inclusion criteria

[0059] ① Meet the diagnostic criteria for semen liquefaction disorder;

[0060] ②Meet the diagnostic criteria for chronic bacterial prostatitis;

[0061] ③Meet the diagnostic criteria for Ureaplasma urealyticum infection;

[0062] ④ Male aged 22-45 years old;

[0063] ⑤ Have good compliance, voluntarily accept clinical trials and sign informed consent;

[0064] ⑥ No other drugs to improve semen liquefaction were taken within 3 months.

[0065] Applicants who meet items ①②④⑤⑥ or ①③④⑤⑥ and items ①-⑥ can be included.

[0066] 1.2.6 Case Exclusion Criteria

[0067] ① Patients who do not meet the above diagnostic criteria;

[0068] ② Those suffering from other diseases that affect semen liquefaction time;

[0069] ③ Those with organic lesions in reproductive organs;

[0070] ④ Those with severe cardiovascular and cerebrovascular diseases and diseases of important organs;

[0071] ⑤People who are allergic to this drug.

[0072] 1.2.7 Elimination criteria

[0073] ① Those who have never used the trial drug;

[0074] ②The case does not meet the inclusion criteria, or is found to not meet the diagnostic criteria after inclusion

[0075] ③ Those without any testing records;

[0076] ④ There are concurrent medications within the scope of the trial, which may affect the judgment of its effectiveness and safety;

[0077] 1.2.8 Shedding Standard

[0078] Patients who were included but failed to complete the clinical protocol were considered to have dropped out in the following circumstances:

[0079] ① The patient withdraws on his own initiative;

[0080] ② Lost to follow-up;

[0081] ③Poor compliance;

[0082] ④ In case of complications, the doctor shall order the patient to withdraw from the treatment.

[0083] 1.2.8 Test termination criteria

[0084] ① Safety issues such as severe allergic reactions or adverse reactions are found during the test;

[0085] ② The patient's condition continues to worsen or there are serious complications, and the attending physician determines that the subject should withdraw.

[0086] 1.3 Grouping and treatment plan

[0087] According to the random number table method, they were randomly divided into levofloxacin control group, liquefaction-promoting decoction treatment group, and levofloxacin combined with liquefaction-promoting decoction treatment group, 30 cases in each group.

[0088] Control group: Oral administration of levofloxacin lactate dispersible tablets (manufacturer: Sichuan Shenghe Pharmaceutical Co., Ltd., specification 0.1g x 24 tablets), 3 tablets at a time, orally, once a day;

[0089] Treatment group of the liquefaction-promoting decoction: 20g of Plantago seed, 20g of Imperata root, 15g of Cistanche deserticola, 10g of Talcum, 10g of Smilax glabra, 10g of Dianthus superbus, 10g of Polygonum multiflorum, 15g of Smilax glabra, 20g of Patrinia chinensis, 3g of Rhubarb, 10g of Gardenia jasminoides, 10g of Licorice, and 5g of Juncus effusus. Decoction method: add water to each dose until the raw herbs are covered, soak for 30 minutes, then turn to high heat and boil, then turn to low heat and boil for 20-30 minutes, and boil 150ml of the juice. Each dose is decocted 3 times, and the total juice is 300ml. Dosage: 1 dose per day, 2 times a day, 150ml each time, take warm after meals;

[0090] Levofloxacin combined with liquefaction-promoting decoction treatment group: liquefaction-promoting decoction was added to the control group;

[0091] The treatment course for the above three groups was 4 weeks, and routine semen examination, related inflammatory factors, liquefaction factors and coagulation factors were performed before and after treatment.

[0092] 1.3.1 Observation indicators

[0093] ① Vital signs: such as temperature (T), respiration (R), heart rate (P), blood pressure (BP);

[0094] ② Routine blood and urine examinations before and after treatment;

[0095] ③ Check liver and kidney function before and after treatment;

[0096] ④Observe the symptoms and signs of adverse reactions.

[0097] 1.3.2 Efficacy indicators

[0098] ① Observe the changes in routine semen parameters (semen liquefaction time, semen viscosity, semen pH value, sperm motility) before and after the three groups of drug treatment.

[0099] ② Detection of inflammatory factors, liquefaction factors, and coagulation factors in seminal plasma

[0100] The enzyme-linked immunosorbent assay was used to measure the concentrations of inflammatory factors IL-1β, TNF-α, IL-6, and IL-8; liquefaction factors PSA and PAP; and coagulation factors SEMG1 and Fn protein in the seminal plasma of the three groups of patients before and after treatment. The operation was performed by a dedicated person strictly according to the instructions of the kit.

[0101] 1.4 Efficacy evaluation criteria

[0102] Refer to the "Guidelines for Clinical Research of New Traditional Chinese Medicines for the Treatment of Male Infertility" and formulate standards in light of clinical conditions.

[0103] Cure: clinical symptoms disappear ≥ 95%, liquefaction time < 60 minutes, routine analysis is normal or basically normal;

[0104] Markedly effective: clinical symptoms improved by ≥70%, liquefaction time <60 minutes, and routine analysis was normal or basically normal;

[0105] Effective: clinical symptoms improved by ≥30%, liquefaction time>60 minutes, but significantly shorter than before, and routine analysis also significantly improved;

[0106] Ineffective: Clinical symptoms improved by <30%, and there was no significant change in various indicators after treatment.

[0107] 1.5 Experiments and Methods

[0108] 1.5.1 Drugs and reagents

[0109] ① Liquification-promoting decoction (Example 1): Plantago seed 20g, Imperata root 20g, Cistanche deserticola 15g, Talcum 10g, Smilax glabra 10g, Dianthus superbus 10g, Polygonum multiflorum 10g, Smilax glabra 15g, Patrinia 20g, Rhubarb 3g, Gardenia 10g, Licorice 10g, Juncus effusus 5g.

[0110] ② Levofloxacin lactate dispersible tablets (manufacturer: Sichuan Shenghe Pharmaceutical Co., Ltd., specification 0.1g x 24 tablets)

[0111] ③ Reagents used (see Table 1)

[0112] Table 1 Reagents used in the experiment

[0113] Instrument Name model Manufacturer Electric constant temperature blast drying oven DHG-9240A Shanghai Yuejin Medical Equipment Factory Pipette 20-200ul <![CDATA[ Finnpipette ]]> Ultrapure water instrument <![CDATA[ HB-RO / 60]]> <![CDATA[United States, Millipore > Conventional refrigerator BCD-189WDPV Haier, China Precision Constant Temperature Incubator BPH-9042 Shanghai, Yiheng Plate washer 988 plate washer <![CDATA[ Tianshi ]]> ELISA reader <![CDATA[ Rayto ,RT-6100]]> <![CDATA[ Rayto ]]> Biological Microscope CX31-12004 Olympus Fully automatic sperm quality analyzer CFT-9202 Jiangsu Ruiqi Life Science Instruments Electric constant temperature water thermostat H·SWX-600PS Shanghai Quanmai Scientific Instrument Co., Ltd.

[0114] 1.5.2 Instruments and equipment

[0115] ①Main instruments (see Table 2)

[0116] Table 2 Main instruments of the experiment

[0117]

[0118] 1.5.3 Methods

[0119] 1.5.3.1 Semen collection and routine analysis

[0120] ① Semen collection

[0121] After abstinence for 3-7 days, collect semen through masturbation into a clean and dry disposable wide-mouth plastic semen collection cup, then place it in a 37°C water bath to liquefy it and test it within 1 hour.

[0122] ② Routine analysis of semen

[0123] The fully automatic sperm treatment analyzer was used to perform routine analysis of semen, and the sperm liquefaction time, viscosity, pH value, and sperm motility were recorded. At the same time, the inflammatory factors, liquefaction factors, and coagulation factors in the seminal plasma were detected. The enzyme-linked immunosorbent assay was mainly used to measure the concentrations of inflammatory factors IL-1β, TNF-α, IL-6, and IL-8; liquefaction factors PSA and PAP; and coagulation factors SEMG1 and Fn protein in the seminal plasma of 90 patients in the three groups before and after treatment. The operation was performed by a dedicated person in strict accordance with the instructions of the kit. The specific steps are as follows:

[0124] The kit uses a double antibody sandwich enzyme-linked immunosorbent assay (ELISA). The seminal plasma sample to be tested, the standard substance, and the HRP-labeled detection antibody are added to the microwells pre-coated with the capture antibody, incubated and thoroughly washed. The substrate TMB is used for color development. TMB is converted into blue under the catalysis of peroxidase and into the final yellow under the action of acid. The absorbance (OD value) is measured at a wavelength of 450nm using an enzyme reader to calculate the sample concentration.

[0125] a. Dilution of standard: This kit provides one original standard, which users can dilute in a small test tube according to the following chart. See Table 3.

[0126] Table 3 Dilution of standard products

[0127] 20ng / L Standard No. 5 150μ1 of original standard is added to 150μ1 of standard diluent 10ng / L Standard No. 4 150μl of standard No. 5 was added to 150μl of standard diluent 5ng / L Standard No. 3 150μ1 of standard No. 4 was added to 150μ1 of standard diluent 2.5ng / L Standard No. 2 150μl of standard No. 3 was added to 150μl of standard diluent 1.25ng / L Standard No. 1 150μl of standard No. 2 was added to 150μl of standard diluent

[0128] b. Sample addition: Set up blank wells (blank control wells without adding samples and enzyme-labeled reagents, and the rest of the steps are the same), standard wells, and test sample wells. Accurately add 50μl of the standard sample to the enzyme-labeled plate, add 40μl of sample diluent to the test sample wells, and then add 10μl of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix.

[0129] c. Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes.

[0130] d. Liquid preparation: dilute the 30 times concentrated washing solution with 30 times distilled water for later use

[0131] e. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0132] f. Add enzyme: Add 50 μl of enzyme-labeled reagent to each well, except for the blank well.

[0133] g. Incubation: Same operation as 3.

[0134] h. Washing: Same operation as step 5.

[0135] i. Color development: first add 50μl of color developer A to each well, then add 50μl of color developer B, gently shake to mix, and develop the color at 37℃ in the dark for 10 minutes.

[0136] j. Termination: Add 50 μl of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0137] k. Determination: Use the blank well as zero setting, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The determination should be carried out within 15 minutes after adding the stop solution.

[0138] 1.6 Statistical analysis

[0139] GraphPad 9.0 software was used for statistical analysis, and the results are presented as mean ± standard deviation. The t-test was used for comparison between two groups, and ORDINARY ONE-WAY ANOVA was used for comparison among multiple groups. P < 0.05 was considered statistically significant.

[0140] 1.7 Ethical issues and solutions

[0141] After evaluating the benefits and risks of the study, we mainly took the following measures: all patients agreed, and the study was reviewed by the Ethics Committee of Hainan Provincial Hospital of Traditional Chinese Medicine, and the patients signed an informed consent form to avoid selecting patients with other diseases.

[0142] 1.8 Adverse Event Observation

[0143] The subjects are required to fill in the prepared adverse event record form truthfully during the trial, and record in detail the cause, time, severity and duration of the adverse event, as well as the effective measures taken and the outcome. When the consequences of the adverse event cannot be remedied and seriously affect the trial results, the subjects should withdraw from the trial on their own, and the trial subjects will be replaced in turn based on the previous visit order.

[0144] 2 Research results

[0145] 2.1 Included cases

[0146] A total of 96 cases were included in this study. After exclusion according to the exclusion criteria, a total of 90 effective cases were found. The semen routine parameters, efficacy, and concentrations of IL-1β, TNF-α, IL-6, IL-8, PSA, PAP, SEMG1, and Fn in seminal plasma of the 90 patients were analyzed. No abnormalities were found in the blood, urine routine, liver function, and kidney function of the 90 patients before and after treatment.

[0147] 2.2 Analysis of semen parameters

[0148] 2.2.1 Analysis of semen parameters before and after treatment in the three groups: oral liquefaction decoction group, oral levofloxacin tablet group, and oral liquefaction decoction + levofloxacin tablet group

[0149] After 4 weeks of oral treatment with levofloxacin, there was no significant difference in semen volume compared with before treatment in 30 patients with semen liquefaction disorder (P>0.05); liquefaction time was significantly shortened (P<0.01), sperm concentration was significantly increased (P<0.01), sperm motility was significantly increased (P<0.01), and grade A sperm + grade B sperm were significantly increased (P<0.01); after 4 weeks of oral treatment with levofloxacin, there was no significant difference in semen volume compared with before treatment (P>0.05); liquefaction time was shortened (P<0.01), sperm concentration was significantly increased (P<0.01), sperm motility was significantly increased (P<0.01), and grade A sperm + grade B sperm were significantly increased (P<0.01). There was no significant difference in sperm concentration (P>0.05), sperm motility was improved compared with before (P<0.01), and grade A sperm + grade B sperm were improved compared with before (P<0.01); 30 patients with semen liquefaction syndrome were treated with oral liquefaction-promoting decoction combined with levofloxacin for 4 weeks. There was no significant difference in semen volume compared with before treatment (P>0.05); liquefaction time was significantly shortened (P<0.01), sperm concentration was significantly improved (P<0.01), sperm motility was significantly improved (P<0.01), and grade A sperm + grade B sperm were significantly improved (P<0.01); the results are shown in Table 4.

[0150] Table 4 Comparative analysis of semen indexes before and after treatment in the groups of liquefaction-promoting decoction, levofloxacin tablets, and liquefaction-promoting decoction + levofloxacin tablets

[0151]

[0152] Note: “****” indicates P<0.0001, “***” indicates P<0.0002, “**” indicates P<0.0021, “*” indicates P<0.0332, and “ns” indicates that the comparison between the two groups was not significant.

[0153] 2.2.2 Comparative analysis of semen parameters before and after treatment among the oral liquefaction decoction group, the oral levofloxacin tablet group, and the oral liquefaction decoction + levofloxacin tablet group

[0154] After 4 weeks of treatment with oral liquefaction-promoting decoction, levofloxacin tablets, and liquefaction-promoting decoction plus levofloxacin tablets, 30 patients in each group were treated with the liquefaction-promoting decoction, levofloxacin tablets, and liquefaction-promoting decoction plus levofloxacin tablets, as shown in Table 5. After the treatment, there was no significant difference in semen volume, liquefaction time, and sperm concentration between the two groups (P>0.5), and the therapeutic effects were consistent. In terms of sperm motility and A-grade sperm + B-grade sperm, the liquefaction-promoting decoction group had a better improvement effect than the levofloxacin group (P<0.005). Compared with the oral levofloxacin group, there was no significant difference in semen volume, liquefaction time, and sperm concentration between the oral liquefaction-promoting decoction + levofloxacin tablets group (P>0.5), and the therapeutic effects were consistent; while the oral liquefaction-promoting decoction combined with levofloxacin tablets group could more significantly improve sperm motility and the ratio of grade A sperm + grade B sperm, with significant differences (P<0.05); compared with the oral liquefaction-promoting decoction group and the oral liquefaction-promoting decoction combined with levofloxacin tablets group, there was no significant difference in semen volume, liquefaction time, sperm concentration, sperm motility, and grade A sperm + grade B sperm (P>0.5).

[0155] Table 5 Comparative analysis of semen parameters after treatment among the three groups of liquefaction-promoting decoction, levofloxacin tablets, and liquefaction-promoting decoction + levofloxacin tablets

[0156]

[0157]

[0158] 2.3 Efficacy determination

[0159] 2.3.1 Clinical efficacy of 90 patients with semen liquefaction disorder in each group after 4 weeks of treatment

[0160] ① Clinical efficacy of 30 patients with semen liquefaction disorder treated with oral liquefaction-promoting decoction for 4 weeks: 12 cases were cured, 8 cases were markedly effective, 7 cases were effective, 3 cases were ineffective, and the total effective rate was 90.00%; (see Table 6)

[0161] ② Clinical efficacy of 30 patients with semen liquefaction disorder treated with oral levofloxacin tablets for 4 weeks: 10 cases were cured, 7 cases were markedly effective, 9 cases were effective, 4 cases were ineffective, and the total effective rate was 86.67%; (see Table 6)

[0162] ③ Clinical efficacy of 30 patients with semen liquefaction disorder treated with oral liquefaction-promoting decoction + levofloxacin tablets for 4 weeks: 13 cases were cured, 7 cases were markedly effective, 8 cases were effective, 2 cases were ineffective, and the total effective rate was 93.33%; (see Table 6)

[0163] Table 6 Clinical efficacy of 90 patients with semen liquefaction disorder in each group after 4 weeks of treatment

[0164]

[0165] 2.4 Determination of IL-1β, TNF-α, IL-6, IL-8, SEMG1, Fn, PSA, and PAP in seminal plasma

[0166] 2.4.1 Comparative analysis of seminal plasma indices in groups A, B, and C before and after treatment

[0167] Thirty patients in each group were treated with liquefaction-promoting decoction (group A), levofloxacin tablets (group B), and liquefaction-promoting decoction + levofloxacin tablets (group C) for 4 weeks. The results are shown in Table 7. Compared with those before treatment, the levels of IL-1β, IL-6, IL-8, Fn, and PSA in seminal plasma in the three groups after treatment were significantly decreased (P<0.0001), and PAP and SEMG1 were significantly increased (P<0.0001). At the same time, TNF-α was significantly increased in the liquefaction-promoting decoction group and the levofloxacin tablet group, and was significantly decreased in the liquefaction-promoting decoction group and the levofloxacin tablet combined treatment group (P<0.0001). The differences were statistically significant.

[0168] Table 7 Comparative analysis of seminal plasma indexes in groups A, B and C before and after treatment

[0169]

[0170] 2.4.2 Comparative analysis of concentration changes of seminal plasma indicators among groups A, B, and C after treatment

[0171] Thirty patients in each group were treated with oral liquefaction-promoting decoction (Group A), levofloxacin tablets (Group B), and liquefaction-promoting decoction plus levofloxacin tablets (Group C) for 4 weeks. The changes in the concentrations of seminal plasma factors in each group were calculated to clarify the differences between the liquefaction-promoting decoction and the combined treatment with liquefaction-promoting decoction plus levofloxacin tablets compared with the traditional Western medicine levofloxacin tablets treatment. The results are shown in Table 8.

[0172] From 2.4.1, it can be seen that the treatment with liquefaction-promoting decoction, levofloxacin tablets, and liquefaction-promoting decoction combined with levofloxacin tablets can significantly reduce the concentrations of inflammatory factors IL-1β, IL-6, IL-8, liquefaction factor PSA, and coagulation factor Fn in seminal plasma; and significantly increase the coagulation factor SEMG1 and liquefaction factor PAP.

[0173] Compared with the levofloxacin tablets group, the liquefaction-promoting decoction group and the liquefaction-promoting decoction combined with levofloxacin tablets group could reduce the concentrations of inflammatory factors IL-1β, IL-6, IL-8, liquefaction factor PSA, and coagulation factor Fn in seminal plasma samples to a greater extent, and increase the concentrations of coagulation factor SEMG1 and liquefaction factor PAP to a greater extent; the differences were significant (P<0.0001).

[0174] Compared with the liquefaction-promoting decoction group, the liquefaction-promoting decoction combined with levofloxacin tablets group could reduce the concentrations of inflammatory factors IL-1β, IL-6, IL-8, liquefaction factor PSA, and coagulation factor Fn in seminal plasma samples to a greater extent, and increase the concentrations of coagulation factor SEMG1 and liquefaction factor PAP to a greater extent; the differences were significant (P<0.0001).

[0175] Table 8 Comparative analysis of concentrations of seminal plasma indicators among groups A, B and C after treatment

[0176]

[0177] 2.5 Adverse Reactions

[0178] During the treatment, one patient in the levofloxacin group experienced gastrointestinal discomfort, which was relieved by rest or diet adjustment. No other adverse reactions were observed.

[0179] 3 Conclusion

[0180] 3.1 This study conducted a randomized controlled clinical trial on 90 cases and found that the liquefaction-promoting decoction has significant therapeutic effects on improving sperm concentration, liquefaction time, sperm motility, and grade A sperm + grade B sperm, with a total effective rate of 90%. If levofloxacin tablets are added at the same time, the total effective rate reaches 93.33%, and compared with the control group, sperm motility and the ratio of grade A sperm + grade B sperm are more significantly improved. No adverse reactions occurred during the medication process, and no abnormalities were found in blood and urine routine tests and liver and kidney function monitoring. This study shows that:

[0181] 3.1.1 Liquefaction-promoting decoction can effectively shorten the liquefaction time, increase sperm concentration, and increase sperm motility and improve semen quality. If levofloxacin is added to enhance anti-inflammatory effects, the improvement of sperm motility and the ratio of grade A sperm to grade B sperm will be more obvious.

[0182] 3.1.2 Liquefaction-promoting decoction is highly safe in treating patients with semen liquefaction syndrome.

[0183] 3.1.3 This study also measured the levels of IL-1β, TNF-α, IL-6, IL-8, SEMG1, Fn, PSA, and PAP in seminal plasma. The double antibody sandwich enzyme-linked immunosorbent assay was used to detect each index and perform statistical analysis. The results showed that: within the group, compared with before treatment, the levels of IL-1β, IL-6, IL-8, Fn, and PSA in seminal plasma of the three groups were significantly reduced, and PAP and SEMG1 were significantly increased after treatment; at the same time, TNF-α was significantly increased in the liquefaction-promoting decoction group and the levofloxacin tablets group, and was significantly decreased in the liquefaction-promoting decoction group and the levofloxacin tablets combined treatment group, and the differences were statistically significant. Comparison of concentration changes among the three groups showed that the liquefaction-promoting decoction group and the liquefaction-promoting decoction combined with levofloxacin tablets group could reduce the concentrations of inflammatory factors IL-1β, IL-6, IL-8, liquefaction factor PSA, and coagulation factor Fn in seminal plasma samples to a greater extent, and increase the concentrations of coagulation factor SEMG1 and liquefaction factor PAP to a greater extent; the difference was significant; compared with the liquefaction-promoting decoction group, the liquefaction-promoting decoction combined with levofloxacin tablets group could reduce the concentrations of inflammatory factors IL-1β, IL-6, IL-8, liquefaction factor PSA, and coagulation factor Fn in seminal plasma samples to a greater extent, and increase the concentrations of coagulation factor SEMG1 and liquefaction factor PAP to a greater extent. The results showed that the mechanism of liquefaction-promoting decoction in treating semen liquefaction may be to reduce the concentrations of IL-1β, IL-6, IL-8, Fn, and PSA, and increase the concentrations of PAP and SEMG1, thereby shortening the semen liquefaction time and improving semen quality.

[0184] Comparative Study

[0185] Comparative Example 1

[0186] The main difference from Example 1 is that the radix scutellariae is replaced by Phellodendron amurense (Phellodendron amurense clears heat and removes dampness). A Chinese medicine composition for treating semen liquefaction disorder: composed of the following medicinal materials in parts by weight: 15 parts of Plantago, 25 parts of Imperatae Rhizoma, 20 parts of Phellodendron amurense, 8 parts of Talcum, 12 parts of Thunbergia scoparia, 8 parts of Dianthus superbus, 12 parts of Polygonum multiflorum, 13 parts of Smilax glabra, 15 parts of Patrinia scabra, 2 parts of Rhubarb, 12 parts of Gardenia jasminoides, 8 parts of Licorice, and 4 parts of Juncus effusus.

[0187] Comparative Example 2

[0188] The main difference from Example 1 is that Dianthus superbus is replaced by Gentiana scabra (Gentiana scabra clears away heat and dampness). A Chinese medicine composition for treating semen liquefaction syndrome: composed of the following medicinal materials in parts by weight: 20 parts of Plantago asiatica, 20 parts of Imperata cylindrica, 15 parts of Caulis datura, 10 parts of Talcum, 10 parts of Tongcao, 10 parts of Gentiana scabra, 10 parts of Polygonum multiflorum, 15 parts of Smilax glabra, 20 parts of Patrinia scabra, 3 parts of Rhubarb, 10 parts of Gardenia jasminoides, 10 parts of Licorice, and 5 parts of Juncus effusus.

[0189] Comparative Example 3

[0190] The main difference from Example 1 is that Patriniae Herba is replaced by Sophora flavescens (Sophora flavescens clears heat, dries dampness and removes toxins). A Chinese medicine composition for treating semen liquefaction syndrome: composed of the following medicinal materials in parts by weight: 20 parts of Plantago, 20 parts of Imperata cylindrica, 15 parts of Caulis datura, 10 parts of Talcum, 10 parts of Thunbergia scoparia, 10 parts of Dianthus superbus, 10 parts of Polygonum multiflorum, 15 parts of Smilax glabra, 20 parts of Sophora flavescens, 3 parts of Rhubarb, 10 parts of Gardenia jasminoides, 10 parts of Licorice, and 5 parts of Juncus effusus.

[0191] Comparative Example 4

[0192] The main difference from Example 1 is that rhubarb is replaced by scutellaria baicalensis (scutellaria baicalensis relieves heat). A Chinese medicine composition for treating semen liquefaction syndrome: composed of the following medicinal materials in parts by weight: 20 parts of plantain seeds, 20 parts of Imperata root, 15 parts of Caulis Patriniae, 10 parts of talc, 10 parts of tongcao, 10 parts of Dianthus superbus, 10 parts of Polygonum multiflorum, 15 parts of Smilax glabra, 20 parts of Patrinia scabra, 3 parts of Scutellaria baicalensis, 10 parts of Gardenia jasminoides, 10 parts of licorice, and 5 parts of Juncus effusus.

[0193] Comparative Example 5

[0194] The main difference from Example 1 is that Gardenia is replaced by Acorus calamus (Acorus calamus clears heat and promotes diuresis). A Chinese medicine composition for treating semen liquefaction syndrome: composed of the following medicinal materials in parts by weight: 20 parts of Plantago, 20 parts of Imperata cylindrica, 15 parts of Caulis Patriniae, 10 parts of Talc, 10 parts of Thunbergia scoparia, 10 parts of Dianthus superbus, 10 parts of Polygonum multiflorum, 15 parts of Smilax glabra, 20 parts of Patrinia scabra, 3 parts of Rhubarb, 10 parts of Acorus calamus, 10 parts of Licorice, and 5 parts of Juncus effusus.

[0195] The use of the Chinese medicines in Comparative Examples 1-5 has certain therapeutic effects, but the improvement effects on increasing sperm concentration, shortening liquefaction time, and improving sperm motility are significantly lower than those in Example 1.

[0196] Test Example 2-Treatment of Type III Chronic Prostatitis

[0197] 1. Construction of prostatitis rat model and drug intervention

[0198] 1 Experimental Materials and Instruments

[0199] 1.1 Experimental animals

[0200] Thirty 8-week-old male SD rats weighing about 240 g were provided by Chongqing Enswell Biotechnology Co., Ltd. All rats were raised under the same conditions, with a temperature of 23 ± 2 °C, a humidity of 35-60%, and a 12-h day-night alternation in the animal room.

[0201] 1.2 Main instruments and reagents

[0202] Table 9 Main instruments

[0203]

[0204]

[0205] Table 10 Main reagents

[0206]

[0207] 1.3 Preparation of main reagents

[0208] (1) Grind the liquefaction soup granules into fine powder

[0209] Formula of liquefaction-promoting soup (Example 1): 20g of Plantago seed, 20g of Imperata root, 15g of Caulis Patriniae, 10g of Talc, 10g of Thunbergia 10g, 10g of Dianthus superbus, 10g of Polygonum multiflorum, 15g of Smilax glabra, 20g of Patrinia chinensis, 3g of Rhubarb, 10g of Gardenia jasminoides, 10g of Liquorice, and 5g of Juncus effusus. Grind the medicinal materials of liquefaction-promoting soup into fine powder.

[0210] ① High-dose liquefaction-promoting decoction group (3.78 g / kg): 6.804 g of liquefaction-promoting decoction powder was dissolved in 30 mL of warm water.

[0211] ② Medium-dose group of liquefaction-promoting decoction (1.89 g / kg): dissolve 3.402 g of liquefaction-promoting decoction powder in 30 mL of warm water.

[0212] ③ Low-dose group of liquefaction-promoting decoction (0.945 g / kg): dissolve 1.701 g of liquefaction-promoting decoction powder in 30 mL of warm water.

[0213] Note: Each group was given drugs according to the above method. The gavage volume for a 300 g rat was 2.5 mL each time, twice a day, morning and evening.

[0214] (2) Preparation of estradiol benzoate 0.25 mg / kg: Dissolve 1.8 mg of estradiol benzoate in 4.8 ml of olive oil (the subcutaneous injection volume for a 300 g rat is 0.2 mL).

[0215] (3) 3% sodium pentobarbital: Dissolve 3 g of sodium pentobarbital powder in about 60 mL of ultrapure water, make up to 100 mL with ultrapure water, and store at room temperature away from light.

[0216] (4) 4% paraformaldehyde solution: accurately weigh 40 g of paraformaldehyde using an electronic analytical balance, add 700 mL of ultrapure water to dissolve it, and then add ultrapure water to make up to 1000 mL.

[0217] 2 Experimental steps

[0218] 2.1 Animal Adaptive Feeding

[0219] The animal room had a 12-h day-night cycle, and rats were allowed to drink water and eat freely. The temperature was 23-25°C, and the rats entered the experiment after one week of adaptive feeding.

[0220] 2.2 Animal grouping

[0221] Thirty experimental animals were randomly divided into a control group, a model group, a low-dose liquefaction-promoting decoction group, a medium-dose liquefaction-promoting decoction group, and a high-dose liquefaction-promoting decoction group, with 6 animals in each group.

[0222] 2.3 Animal model construction

[0223] Rats were anesthetized by intraperitoneal injection of 50 mg / kg sodium pentobarbital, and then laid flat on the operating table. A skin incision of about 4 cm was made from the scrotal suture line at the lowest end of the scrotum to the midline of the abdomen. Use the index and middle fingers of the left hand to push one side of the scrotum backwards, so that the testicle and the tunica vaginalis protrude through the incision, and tighten the tunica vaginalis around the testicle. Fix the testicle, cut the tunica vaginalis, and remove the testicle from the incision. Hold the testicle with the left hand, clamp the epididymal tail ligament with hemostatic forceps in the right hand, and tear the epididymal tail ligament from the epididymal tail. Tear the mesentery with the right hand, and continue to pull the testicle with the left hand to completely expose the spermatic cord. Then, ligate and cut the spermatic cord, remove the testicle with surgical scissors, and then suture the tunica vaginalis and skin. From the second day, rats were subcutaneously injected with 0.25 mg / kg of estradiol benzoate on the back for 30 consecutive days, and the control group was injected with an equal amount of normal saline. Photos of the animal model construction process are shown in the figure below. Figure 1 shown.

[0224] 2.4 Administration

[0225] Rats were treated with three doses of liquefaction-promoting decoction (e.g. Figure 2 The model group and the control group were gavaged with the same dose of normal saline once every 2 days for 14 days. After 14 days, the mice were killed and their blood was collected for subsequent detection.

[0226] 3 materials

[0227] After the rats were anesthetized, the abdominal cavity was opened to collect blood, and prostate tissue was taken for subsequent testing, such as Figure 3 shown.

[0228] 2. HE staining to detect the pathological morphology of prostate tissue

[0229] 1 Experimental Principle

[0230] Hematoxylin-eosin staining, referred to as HE staining, is one of the commonly used staining methods in paraffin section technology. Hematoxylin staining solution is alkaline, which mainly makes the chromatin in the cell nucleus and the ribosomes in the cytoplasm purple-blue; eosin is an acidic dye, which mainly makes the components in the cytoplasm and extracellular matrix red. HE staining is the most basic and widely used technical method in the teaching and scientific research of histology, embryology, and pathology.

[0231] 2 Experimental Materials and Instruments

[0232] 2.1 Experimental Materials

[0233] Rat prostate tissue.

[0234] 2.2 Experimental instruments and reagents

[0235] Table 11 Main instruments

[0236]

[0237]

[0238] Table 12 Main reagents

[0239]

[0240] 3 Experimental methods

[0241] 3.1 Working fluid configuration

[0242] (1) Fixative: 4% paraformaldehyde (prepared in PBS), 4 g paraformaldehyde, 80 mL PBS, heat to 60°C, stir continuously until completely dissolved, adjust volume to 100 mL, and store at RT.

[0243] (2) 1% hydrochloric acid-alcohol differentiation solution: Add 1 mL of concentrated hydrochloric acid to 99 mL of 70% ethanol.

[0244] (3) 75% ethanol: Take 75 mL of anhydrous ethanol and dilute to 100 mL with deionized water (the same applies to 80% ethanol, 90% ethanol, and 95% ethanol).

[0245] 3.2 Staining procedure

[0246] 3.2.1 Preparation of tissue paraffin

[0247] (1) After obtaining the sample, wash it with PBS and fix it with 4% paraformaldehyde.

[0248] (2) Dehydration: Soak the tissue in 70% ethanol for 5 h, 80% ethanol for 2 h, 90% ethanol for 2 h, 100% ethanol I for 1 h, 100% ethanol II for 1 h, and 100% ethanol for 1 h.

[0249] (3) Transparency: Soak the dehydrated tissue in xylene I and xylene II for 30 min respectively.

[0250] (4) Paraffinization: Soak the transparentized tissue in melted paraffin I for 1 hour and in melted paraffin II for 2 hours.

[0251] (5) Embedding.

[0252] (6) Sectioning: Use a microtome to cut the tissue in the paraffin block into 2.5 μm thick slices and spread them flat on anti-shedding glass slides.

[0253] (7) Slide baking: Place the slices on a 55°C slide baking machine so that the tissue slices are tightly attached to the anti-slip glass slide.

[0254] 3.2.2 Dewaxing and rehydration of paraffin sections

[0255] (1) Dewaxing: Soak the paraffin sections in xylene I, xylene II, and 1 / 2 xylene for 10 min each.

[0256] (2) Rehydration: Soak the dewaxed paraffin sections in 100% ethanol I, 100% ethanol II, 95% ethanol I, 95% ethanol II, 85% ethanol, and 75% ethanol for 5 min each.

[0257] (3) Wash with double distilled water three times, 2 min each time.

[0258] 3.2.3 HE staining

[0259] (1) Stain with hematoxylin stain for 5 minutes.

[0260] (2) Rinse off excess staining solution with tap water for about 5 minutes.

[0261] (3) Rinse again with distilled water (a few seconds is enough).

[0262] (4) Use 1% hydrochloric acid alcohol to decolorize and remove excess hematoxylin staining solution in the cytoplasm.

[0263] (5) Stain with eosin solution for 2 minutes.

[0264] (6) Rinse off excess dye with tap water for about 1 min.

[0265] (7) Wash again with distilled water.

[0266] 3.2.4 Dehydration and sealing

[0267] (1) Dehydrate in 95% ethanol for 2 min.

[0268] (2) Dehydrate with 100% anhydrous ethanol for 2 min.

[0269] (3) Xylene transparent for 5 minutes.

[0270] (4) Replace with fresh xylene and clear for another 5 minutes.

[0271] (5) Seal with neutral resin.

[0272] (6) Under microscopic examination, the cell nucleus is blue and the cytoplasm is red or pink.

[0273] 4 Image acquisition

[0274] The slices were imaged using a Mshot MF53 microscope produced by Guangzhou Mingmei Optoelectronics Technology Co., Ltd.

[0275] 5 Experimental results and analysis

[0276] The experimental results of the control group are as follows Figure 4 As shown, the prostate gland lumen is enlarged; the glandular epithelial cells are arranged neatly; pink dye is seen in the glandular lumen, forming prostate condensate; the interstitium is loose, and a small number of inflammatory cells are seen.

[0277] The experimental results of the high-dose group of the liquefaction-promoting decoction are as follows Figure 5 As shown, the lumen of some prostate glands is slightly atrophied; the glandular epithelial cells are arranged basically neatly, and occasionally epithelial cells are seen falling off into the lumen; pink dye is seen in the glandular lumen, and prostate condensates are partially formed; the interstitium is loose, and inflammatory cell infiltration is seen.

[0278] The experimental results of the medium dose group of the liquefaction-promoting decoction are as follows Figure 6 As shown, the prostate gland lumen is significantly atrophied; some glandular epithelial cells are disordered and the cell layer is thickened; the pink dye in the glandular lumen is reduced, and occasionally desquamated epithelial cells are seen; fibrosis and inflammatory cell infiltration are seen in the interstitium and around the gland.

[0279] The experimental results of the low-dose group of the liquefaction-promoting decoction are as follows Figure 7 As shown in the figure, the lumen of the prostate gland is greatly atrophied or even blocked; the glandular epithelial cells are arranged in disorder, the cell layer is thickened, and there is shedding; the pink dye in the glandular lumen is reduced, and there are a small number of shedding epithelial cells; fibrosis and inflammatory cell infiltration are seen in the interstitium and around the gland.

[0280] The experimental results of the model group are as follows Figure 8 As shown, the prostate gland lumen is atrophic and occluded as a whole; pink dye is occasionally seen in part of the lumen; the glandular epithelial cells are disorderly arranged, the cell layer is thickened, aggregated into clusters, and the lumen is blocked; a large amount of fibrous tissue hyperplasia and inflammatory cell infiltration are seen in the interstitium and around the lumen.

[0281] 3. ELISA to detect the levels of IL-1β, IL-2, IL-6, IL-17A, and TNF-α in prostate tissue

[0282] 1. General Introduction of Experimental Methods

[0283] Enzyme-linked immunosorbent assay (ELISA) refers to a qualitative and quantitative detection method of immune response by binding soluble antigens or antibodies to solid phase carriers such as polystyrene and utilizing the specific binding of antigens and antibodies.

[0284] 2 Experimental Materials and Instruments

[0285] 2.1 Experimental Materials

[0286] Rat prostate tissue was processed as follows:

[0287] (1) Rinse the tissue with pre-cooled PBS (0.01 M, pH 7.4) to remove blood or impurities remaining on the surface;

[0288] (2) Weigh the tissue block and then cut it into as small pieces as possible to ensure thorough homogenization;

[0289] (3) Add an appropriate amount of pre-cooled PBS (m:v = 1:9) and homogenize thoroughly with a glass homogenizer on ice or in an ice bath;

[0290] (4) The homogenate was pipetted into a centrifuge tube and centrifuged at 5000 × g for 5–10 min at 4 °C. The supernatant was collected for subsequent testing.

[0291] 2.2 Instruments and reagents

[0292] Table 13 Main instruments

[0293]

[0294] Table 14 Main reagents

[0295]

[0296]

[0297] 3. Detection steps

[0298] (1) Equilibrate the test kit at room temperature;

[0299] (2) Setting standard wells, zero value wells, blank wells, and sample wells. Add 50 μL of standard of different concentrations to each standard well, add 50 μL of sample diluent to the zero value well, add nothing to the blank well, and add 50 μL of the sample to be tested to the sample well;

[0300] (3) Add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to the standard wells, 0 value wells, and sample wells except the blank wells. Cover the reaction plate with a sealing film and incubate in a 37°C water bath or incubator for 60 min.

[0301] (4) Remove the sealing film, discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 20 seconds, shake off the washing solution, pat dry on absorbent paper, and repeat this process 5 times;

[0302] (5) Mix substrate A and B in a 1:1 ratio and add 100 μL of the substrate mixture to all wells. Cover the reaction plate with a sealing film and incubate in a 37°C water bath or incubator for 15 min.

[0303] (6) Add 50 μL of stop solution to all wells and read the absorbance (OD value) of each well on a microplate reader.

[0304] 4 Results Analysis

[0305] Table 15 Comparison of the contents of IL-1β, IL-2, IL-6, IL-17A and TNF-α in prostate tissue of each group

[0306]

[0307] Note: *P<0.05, there is a significant difference compared with the control group; **P<0.01, there is a very significant difference compared with the control group.

[0308] Conclusion: The liquefaction-promoting decoction (Example 1) of the present invention significantly reduces the inflammatory response of prostatitis, effectively reduces the inflammatory factors in prostate tissue, and significantly improves prostatitis.

[0309] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Chinese medicine composition for treating semen liquefaction disorder and prostatitis, characterized in that: The invention comprises the following medicinal materials in parts by weight: 15-25 parts of plantain seeds, 15-25 parts of Imperata rhizome, 10-20 parts of Caulis Patriniae, 8-12 parts of talc, 8-12 parts of tongcao, 8-12 parts of superbus, 8-12 parts of smilax glabra, 13-18 parts of smilax glabra, 15-25 parts of patrinia herb, 2-4 parts of rhubarb, 8-12 parts of gardenia, 8-12 parts of licorice and 4-6 parts of rush.

2. The Chinese medicine composition according to claim 1, characterized in that: The invention comprises the following medicinal materials in parts by weight: 18-22 parts of plantain seeds, 18-22 parts of Imperata rhizome, 18-22 parts of Caulis Patriniae, 9-11 parts of talc, 9-11 parts of tongcao, 9-11 parts of superbus, 9-11 parts of smilax china, 14-16 parts of smilax glabra, 18-22 parts of patrinia herb, 2-4 parts of rhubarb, 9-11 parts of gardenia, 9-11 parts of licorice and 4-6 parts of rush.

3. The Chinese medicine composition according to claim 2, characterized in that: The invention comprises the following medicinal materials in parts by weight: 20 parts of plantain seeds, 20 parts of Imperata rhizome, 15 parts of Cistanche deserticola, 10 parts of talc, 10 parts of tongcao, 10 parts of superbus, 10 parts of smilax china, 15 parts of smilax glabra, 20 parts of patrinia herb, 3 parts of rhubarb, 10 parts of gardenia, 10 parts of licorice and 5 parts of rush.

4. A Chinese medicine preparation for treating semen liquefaction disorder and prostatitis, characterized in that: A Chinese medicine composition comprising any one of claims 1 to 3.

5. The Chinese medicine preparation according to claim 3, characterized in that: The Chinese medicine composition preparation is a decoction, granules, pills, capsules, and oral liquid.

6. Use of the Chinese medicine composition according to any one of claims 1 to 3 in the preparation of a medicament for treating semen liquefaction disorder and / or prostatitis.

7. The use according to claim 6, characterized in that: The application of the traditional Chinese medicine composition in preparing medicine for treating chronic prostatitis.

8. The use according to claim 6, characterized in that: The application of the traditional Chinese medicine composition in preparing medicine for treating type III chronic prostatitis.

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