Application of Rubescensine A in the preparation of drugs for preventing or treating male oligoasthenospermia
Through the administration of Donglingcapromellin, the treatment problem of male oligospermia was solved, and the damage of testicles and semen was significantly improved. It provided an effective drug solution for oxidative damage of testicles and apoptosis of germ cells, with better Application prospects.
Patent Information
- Application Number
- CN202311271533.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-28
- Publication Date
- 2024-12-20
- Estimated Expiration
- 2043-09-28
AI Technical Summary
The prior art lacks effective drugs for the treatment or prevention of male oligospermia, especially for oligospermia caused by oxidative damage to the testicle and apoptosis of germ cells.
As the main ingredient, the dosage of lycopene is administered by oral, injection or infusion agent. It is used in ICR male mice. The dose is 10mg/kg body weight/day-20mg/kg body weight. It is continuously administered for 10-20 days, which is significantly Reverse the reproductive damage caused by Bologne and restore the normal structure and function of the testicles and semen.
Donglingcaoglycin significantly improves the testicular damage structure of mice in the Baixiaoan model group, improves the quality of the semen, enhances the sperm concentration and mobility rate, and has good development value and application prospects, providing new for the prevention and treatment of male oligospermia. drug regimen.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of pharmaceutical biotechnology, and in particular to the use of Rubescensine A in the preparation of a drug for preventing or treating male oligoasthenospermia. Background Art
[0002] Oligospermia is one of the most common causes of male infertility. The causes of oligospermia are complex and diverse, among which testicular oxidative damage and germ cell apoptosis are important causes of oligospermia. However, there is currently a lack of targeted treatment methods in clinical practice. Therefore, in order to prevent and treat oligospermia, it is urgent to explore drugs or programs that target this.
[0003] Oridonin, also known as oridonin and oridonin, is a kaurene diterpenoid compound extracted from the leaves of oridonin, a plant of the genus Oridonium of the Lamiaceae family. It is also the key component of oridonin to exert its medicinal effects. The molecular formula of oridonin is C 20 H 28 O6, molecular weight is 364.44, it is a light yellow crystalline powder; melting point is 248℃-250℃, slightly soluble in water, soluble in methanol, ethyl acetate, acetone, etc. Existing literature reports and clinical efficacy show that Oridonin has the effects of clearing heat and detoxifying, anti-inflammatory and analgesic, stomachic and blood circulation, and good anti-tumor activity. At present, studies have shown that Oridonin can improve the oxidative stress damage of endothelial cells in the acute phase of cerebral ischemia model mice, maintain the integrity of the blood-brain barrier, thereby reducing inflammatory infiltration and reducing brain damage. However, the efficacy of Oridonin in the prevention and treatment of male oligoasthenospermia is still unclear. Summary of the invention
[0004] The purpose of the present invention is to solve the shortcomings of the prior art and to propose the use of Rubescensine A in the preparation of drugs for preventing or treating male oligoasthenospermia. Rubescensine A can significantly reverse the male reproductive damage caused by busulfan, and Rubescensine A can be developed into a drug for preventing and treating male oligoasthenospermia.
[0005] In order to achieve the above object, the present invention adopts the following technical solutions:
[0006] Application of Rubescensine A in the preparation of a drug for preventing or treating male oligoasthenospermia.
[0007] Preferably, the general structural formula of Rubescensine A is:
[0008]
[0009] Preferably, the drug also includes a derivative drug with Rubescensine A as the main structure.
[0010] Preferably, the medicament is for use in humans or mammals.
[0011] Preferably, the drug is administered orally, by injection or infusion.
[0012] Preferably, the dosage of the drug for ICR male mice is 10 mg / kg body weight / day to 20 mg / kg body weight / day.
[0013] Preferably, the drug is administered to ICR male mice continuously for 10-20 days.
[0014] By adopting the above technical scheme: the oridonin in the present invention can vary widely in its product according to the individual conditions of the patient or animal, the route of administration and the dosage form. Generally speaking, the content of the product with oridonin as the active ingredient in the daily suitable dose of ICR male mice is usually 0.001 mg / kg-40 mg / kg body weight, preferably 10 mg / kg-20 mg / kg body weight. The dosage for other people and animals can be obtained according to the conversion formula of human and animal surface area. The above dosage can be administered in one dosage unit or divided into several dosage units, depending on the clinical experience of the doctor and the dosage regimen including the use of other treatment methods. Further, the present invention uses oridonin at a concentration of 10 mg / kg body weight for 0-20 days to determine the optimal administration time, preferably for 10 consecutive days. The above-mentioned administration days can be administered continuously or in stages, depending on the clinical experience of the doctor and the dosage regimen including the use of other treatment methods.
[0015] Compared with the prior art, the present invention has the following beneficial effects:
[0016] 1. The oridonin of the present invention can be used to treat reproductive damage in mice caused by busulfan, significantly improve the testicular damage structure of busulfan model group mice, maintain the normal structure and physiological function of seminiferous tubules, and improve various indicators of semen.
[0017] 2. The oridonin of the present invention can treat male oligoasthenospermia and asthenospermia. Oridonin is expected to be used to prepare drugs for the prevention and treatment of oligoasthenospermia, and has good development value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is a schematic diagram of establishing a male oligoasthenospermia model in mice using busulfan in Example 1 of the present invention;
[0019] Figure 1 A: The size of mouse testicles (photographed); Figure 1 B: Testicular organ index and wet weight of bilateral testes; Normal (control group normal healthy mice), Busulfan (Busulfan); Figure 1 C: CASA results showed that the semen parameters of mice in the busulfan modeling group were significantly lower than those of normal healthy mice; Figure 1 D: PAS staining showed that the pathological structure of the testicular tissue of mice in the busulfan modeling group was destroyed, with fewer spermatogonia, supporting cells and long sperm; * P<0.05, ** P<0.01, *** P < 0.001 or **** P<0.0001 compared with the normal group, there was a significant difference.
[0020] Figure 2 This is a schematic diagram of the therapeutic effect of different concentrations of Rubescensine A administered for 30 days on the spermatogenesis of busulfan model group mice according to Example 2 of the present invention;
[0021] Figure 2 A: The size of testicular tissue (photographed); Figure 2 B: Testicular organ index and wet weight of bilateral testes; Normal (control group normal healthy mice), Busulfan (Busulfan), Oridonin (Russobacterium rubescens A); Figure 2 C: CASA test results showed that Rubescensine A could significantly improve semen quality indicators such as sperm concentration and sperm motility of mice in the model group; Figure 2 D: PAS staining showed that Rubescensine A significantly improved the pathological structure of the testicular tissue of the model group mice and restored spermatogenesis in the seminiferous tubules of the testes of mice. The optimal concentration of Rubescensine A was 10 mg / kg. * P<0.05, ** P<0.01, *** P < 0.001 or **** P<0.0001 compared with the normal group, there was a significant difference. # P<0.05, ## P<0.01, ### P < 0.001 or #### P<0.0001, compared with the busulfan model group, there was a significant difference.
[0022] Figure 3 The therapeutic effect of 10 mg / kg Rubescensine A administered for different days on busulfan model group mice according to Example 3 of the present invention;
[0023] Figure 3 A: The size of testicular tissue (photographed); Figure 3 B: Testicular organ index and wet weight of bilateral testes; Normal (control group normal healthy mice), Busulfan (Busulfan), Oridonin (Russobacterium rubescens A); Figure 3C: CASA test results showed that Rubescensine A could significantly improve semen quality indicators such as sperm concentration and sperm motility of mice in the model group; Figure 3 D: PAS staining showed that Rubescensine A significantly improved the pathological structure of the testicular tissue of the model group mice and restored spermatogenesis in the seminiferous tubules of the testes of the mice. The optimal treatment day for Rubescensine A was 10 days. * P<0.05, ** P<0.01, *** P < 0.001 or **** P<0.0001 compared with the normal group, there was a significant difference. # P<0.05, ## P<0.01, ### P < 0.001 or #### P<0.0001, compared with the busulfan model group, there was a significant difference. DETAILED DESCRIPTION
[0024] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, thereby making a clearer definition of the protection scope of the present invention. The embodiments described in the present invention are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention.
[0025] Example 1: Establishment of a busulfan-induced oligospermia and asthenospermia model in mice
[0026] 1.1 Experimental methods
[0027] 1.1.1 Establishment of the oligospermia and asthenospermia model in busulfan-treated mice
[0028] For the first time, 8-week-old ICR male mice were purchased from the Experimental Animal Center of Nantong University and kept in the SPF animal room of the Experimental Animal Center of Nantong University. The animals were kept in a constant temperature of 23±2℃, humidity of 40±10%, and 12-hour day and night alternation environment. The feed used was ordinary irradiated feed (AIN-93). Intervention plan: 8-week-old ICR male mice were randomly divided into 2 groups (6 in each group), a single injection of 40 mg / kg body weight of busulfan as the model group, and wild-type ICR mice without any treatment as the healthy control group. Preparation of busulfan injection: weigh 100 mg of busulfan and dissolve it in DMSO at a concentration of 10 mg / mL, ready for use; a single intraperitoneal injection of 40 mg / kg of mouse body weight of busulfan injection was given, and the healthy control group was given a single intraperitoneal injection of the same amount of saline according to the mouse body weight. The above mice were free to drink distilled water and eat, and the mice were killed by cervical dislocation after 30 days. The body weight, testis size and weight of mice were tested, and the quality parameters of semen were tested by CASA and the tissue structure of testis was tested by PAS staining to evaluate the success of modeling.
[0029] 1.1.2 Macroscopic morphology observation
[0030] 30 days after the injection of busulfan, the mice were killed by cervical dislocation, and the weight of the mice was weighed and recorded. Then, the abdomen of the mice was disinfected with 75% alcohol, the testicles of the mice were removed, the surrounding fat tissue was peeled off, and the clean bilateral testicles of the mice were obtained. The testicles were photographed, weighed and recorded. The testicular organ index was calculated using the following formula: testicular organ index = testicular weight / body weight × 100%.
[0031] 1.1.3 Microscopic morphology of testis
[0032] The testicles of each group of mice were fixed in testis fixative overnight, dehydrated in graded ethanol-water series, and embedded in paraffin. The sections were cut into 5 μm thickness. The sections were then stained with glycogen (PAS) to detect the sugars in the testicular tissue, which can well show the spermatogenesis of each seminiferous tubule in the testicular tissue. Finally, the testicular tissue structure was observed under an optical microscope.
[0033] 1.1.4 CASA detection of sperm indicators
[0034] Under anesthesia, the epididymis of mice was obtained, and the bilateral epididymal tails were placed in 2 mL of HS (high salt solution) solution with pH = 7.4 preheated at 37°C for 20 minutes. The whole process was placed on a shaker and gently shaken to allow the sperm to swim out. 100 μL of the suspension was added to 900 μL of HS for dilution. The diluted sperm suspension was placed on a glass slide, and then the sperm concentration and motility were calculated using a computer-assisted sperm analysis (CASA) system to determine the quality of the semen.
[0035] 1.2 Experimental Results
[0036] The ICR mice used above were treated with a single intraperitoneal injection of 40 mg / kg busulfan, and the oligoasthenospermia model of mice was successfully induced. This is mainly reflected in the following: the macroscopic photos of the testicles of both mice showed that the testicular volume of busulfan-induced mice was significantly reduced compared with that of normal healthy mice, and the average testicular weight of busulfan-induced mice was only 31.2% of the average wet weight of the testicles of both sides of the normal healthy mice group. The testicular organ index was significantly reduced, and there was a significant difference in the t-test calculation analysis ( Figure 1 A and B). CASA results showed that the semen quality parameters of the busulfan modeling group were significantly lower than those of normal healthy mice, with the sperm concentration being only 12.4% of that of the normal healthy mouse group, and the sperm motility being reduced to 8.5% of that of the normal healthy mouse group ( Figure 1 C). PAS staining showed that the pathological structure of the testicular tissue of mice in the busulfan model group was significantly destroyed, and the number of spermatogenic cells, supporting cells and sperm in the seminiferous tubules was significantly reduced ( Figure 1 D).
[0037] Example 2: Exploring the most suitable concentration of Rubescensine A for preventing and treating reproductive damage of busulfan in mice
[0038] 2.1 Experimental methods:
[0039] 2.1.1 Raising mice and intraperitoneal administration of Rubescensine A
[0040] Eight-week-old ICR male mice were purchased from the Experimental Animal Center of Nantong University and were kept in the SPF animal room of the Experimental Animal Center of Nantong University. The animals were kept in a constant temperature of 23±2℃, humidity of 40±10%, and 12-hour day and night alternation environment. The feed used was ordinary irradiated feed (AIN-93). Intervention scheme: Eight-week-old ICR male mice were randomly divided into 5 groups (5 mice in each group), including the model group, intervention group (5mg / kg oridonin group, 10mg / kg oridonin group, 20mg / kg oridonin group), and wild-type ICR mice without any treatment as the healthy control group. Preparation of busulfan injection: Weigh 100 mg of busulfan and dissolve it in DMSO at a concentration of 10 mg / mL, and prepare it immediately; a single intraperitoneal injection of 40 mg / kg of mouse body weight of busulfan injection was given, and the healthy control group was given a single intraperitoneal injection of the same amount of saline according to the mouse body weight. On the second day of modeling, the mice in the intervention group were intraperitoneally injected with different concentrations of oridonin solution (5 mg / kg oridonin group, 10 mg / kg oridonin group, and 20 mg / kg oridonin group), which was prepared with normal saline 1 hour before the experiment; the mice in the healthy control group and the model group were intraperitoneally injected with the same amount of normal saline. The drug was administered once a day at a fixed time, and the intraperitoneal injection was continued for 30 days. The mice were then killed by cervical dislocation.
[0041] 2.1.2 Processing of testicular tissue samples
[0042] The mice were weighed and their testicles were weighed, photographed, and paraffin sections were prepared from the testicles. At the same time, sperm was obtained from the epididymis of the mice (same as in Experimental Method 1.1.4), and the quality parameters of mouse semen were tested by CASA to evaluate the effect of Rubescensine A on busulfan-induced reproductive damage in mice.
[0043] 2.2 Experimental Results
[0044] Experiments in mice showed that Rubescensine A could partially improve the reduced testicular volume of mice in the model group. The testicles of mice in the Rubescensine A intervention group were significantly fuller and plumper than those in the model group ( Figure 2 A); the testicular organ index and bilateral testicular wet weight were also significantly higher than those in the busulfan model group; intraperitoneal injection of 5 mg / kg, 10 mg / kg, and 20 mg / kg body weight of oridonin was used for treatment, and it was found that 5 mg / kg body weight of oridonin restored the average bilateral testicular wet weight of the model group mice from 31.2% of the average bilateral testicular wet weight of the normal healthy group mice to 41.1%; it was found that the average testicular organ weight index of the model group mice was restored from 33.6% of the average testicular organ weight index of the normal healthy group mice to 50.1%, 39.8%, and 40% ( Figure 2 B). PAS staining showed that Rubescensine A could significantly improve the pathological structure of the testicular tissue of the model group mice and restore spermatogenesis in the seminiferous tubules of the testes of the mice. The CASA test results showed that Rubescensine A significantly improved the semen quality of the model group mice: intraperitoneal injection of 5 mg / kg body weight, 10 mg / kg body weight and 20 mg / kg body weight of Rubescensine A restored the average sperm concentration of the model group mice from 9.9% of the average sperm concentration of the normal healthy group mice to 58.6%, 85.1% and 75.5%, respectively; and restored the average sperm motility of the model group mice from 8.5% of the average sperm motility of the normal healthy group mice to 55%, 87.5% and 61.8%, respectively. Figure 2 C). PAS staining revealed that the types of spermatogenic cells in the seminiferous tubules of the testes of mice in the Rubescensine A treatment group were abundant, but a large number of spermatogenic cells in the seminiferous tubules of the testes of mice in the busulfan model group were lost, and there were fewer long sperm ( Figure 2 D).
[0045] Example 3: Exploring the optimal administration days for Rubescensine A to prevent and treat reproductive damage of busulfan in mice
[0046] 3.1 Experimental methods:
[0047] 3.1.1. Raising mice and intraperitoneal administration of Rubescensine A
[0048] Eight-week-old ICR male mice were purchased from the Experimental Animal Center of Nantong University and kept in the SPF animal room of the Experimental Animal Center of Nantong University. ICR animals were kept in a constant temperature of 23±2℃, humidity of 40±10%, and 12-hour day and night alternation environment. The feed used was ordinary irradiated feed (AIN-93). Intervention scheme: Eight-week-old ICR male mice were randomly divided into 5 groups (5 mice in each group), the model group intervention group (10 mg / kg body weight of oridonin was continuously administered for 5 days, 10 days, and 20 days respectively), and the wild-type ICR mice without any treatment were the healthy control group. Preparation of busulfan injection: Weigh 100 mg of busulfan and dissolve it in DMSO at a concentration of 10 mg / mL, and prepare it for use immediately; a single intraperitoneal injection of 40 mg / kg mouse body weight of busulfan injection was given, and the healthy control group was given a single intraperitoneal injection of the same amount of normal saline according to the mouse body weight. On the second day of modeling, the intervention group mice and the oridonin control group mice were intraperitoneally injected with oridonin, which was prepared with normal saline 1 hour before the experiment; the healthy control group and the model group mice were intraperitoneally injected with an equal amount of normal saline. The drug was administered once a day at a fixed time, and the intraperitoneal injection was continuous. The above mice were free to drink distilled water and eat, and the mice were killed by cervical dislocation on the 31st day.
[0049] 3.1.2 Processing of testicular tissue samples
[0050] The mice were weighed and their testicles were weighed, photographed, and paraffin sections were prepared from the testicles. At the same time, sperm was obtained from the epididymis of the mice (same as in Experimental Method 1.1.4), and the quality parameters of mouse semen were tested by CASA to evaluate the effect of Rubescensine A on busulfan-induced reproductive damage in mice.
[0051] 3.2 Experimental Results
[0052] Experiments in mice showed that different days of administration of Rubescensine A had different effects on improving the testicular volume of busulfan-induced mice, and the mice that received continuous intraperitoneal injection of Rubescensine A for 10 days had the best improvement effect ( Figure 3 A). The testicular organ index was significantly higher than that of the busulfan model group; continuous administration of 10 mg / kg body weight of Rubescensine A for 5, 10, and 20 days restored the average bilateral testicular wet weight of the mice in the model group from 31.8% of the average bilateral testicular wet weight of the mice in the normal healthy group to 78.6%, 63.4%, and 49.3%, respectively; and restored the average testicular organ weight index of the mice in the model group from 30.8% of the average testicular organ weight index of the mice in the normal healthy group to 83.2%, 67.1%, and 53.7%, respectively. Figure 3B). CASA test results showed that continuous intraperitoneal injection of 10 mg / kg body weight of Rubescensine A for 10 days can significantly improve the semen quality of the model group mice: continuous administration of 10 mg / kg body weight of Rubescensine A for 5 days, 10 days, and 20 days can restore the average sperm concentration of the model group mice from 11.8% of the average sperm concentration of the normal healthy group mice to 40.7%, 102.1%, and 86.1%, respectively; the average sperm motility of the model group mice was restored from 24.1% of the average sperm motility of the normal healthy group mice to 66.23%, 105.2%, and 67.4%, respectively; it can be noted that the continuous intraperitoneal injection of 10 mg / kg body weight of Rubescensine A for 10 days had the most obvious improvement effect on the above indicators ( Figure 3 C). PAS staining showed that Rubescensine A could significantly improve the pathological structure of the testicular tissue of the model group mice and restore spermatogenesis in the seminiferous tubules of the testes of the mice. Continuous intraperitoneal injection of 10 mg / kg of Rubescensine A for 10 days had the most significant effect on the restoration of the pathological structure of the testicular tissue of the intervention group mice ( Figure 3 D).
[0053] In summary, the oridonin of the present invention can improve the reduced testicular volume of busulfan model group mice, significantly improve the pathological structure of testicular tissue of model group mice, significantly restore spermatogenesis of testicular seminiferous tubules of mice, and significantly improve sperm concentration, sperm motility, etc. of model group mice. Therefore, oridonin is expected to be developed into a drug for preventing and treating male oligoasthenospermia.
[0054] The description and practice disclosed in the present invention are easy to think and understand for ordinary technicians in the technical field, and several improvements and modifications can be made without departing from the principles of the present invention. Therefore, modifications or improvements made without departing from the spirit of the present invention should also be regarded as the protection scope of the present invention.
Claims
1. Use of Rubescensine A in the preparation of a drug for preventing or treating male oligoasthenospermia, characterized in that: The general structural formula of Rubescensine A is: ; The drug is used for humans or mammals; The drug is administered orally, by injection or infusion.
2. The use according to claim 1, characterized in that: The dosage of the drug for ICR male mice is 10 mg / kg body weight / day to 20 mg / kg body weight / day.
3. The use according to claim 1, characterized in that: The drug is administered to ICR male mice for 10-20 consecutive days.