Traditional Chinese medicine formula with reproductive system radiation damage protection effect as well as preparation method and application of traditional Chinese medicine formula

Abstract

The invention discloses a traditional Chinese medicine formula with a reproductive system radiation damage protection effect as well as a preparation method and application of the traditional Chinese medicine formula. The traditional Chinese medicine formula comprises the following components in parts by mass: 15-30 parts of Chinese yam, 6-15 parts of dogwood, 10-30 parts of astragalus membranaceus, 10-30 parts of prepared rehmannia root, 10-15 parts of spina date seed, 6-15 parts of fructus psoraleae, 5-15 parts of fructus lycii and 3-9 parts of rhodiola rosea. In the traditional Chinese medicine formula provided by the invention, all the components have a synergistic effect, so that the injury of ionizing radiation to a reproductive system can be effectively relieved, the pathological change of testis tissues is remarkably improved, sperm quality parameters are improved, the apoptosis process of testis cells is inhibited, the injury caused by oxidative stress is repaired, and the DNA injury degree is reduced; therefore, the technical defects of high toxicity, high cost, insufficient protective effect on the reproductive system and the like of the existing radiation protective agent are overcome.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to a traditional Chinese medicine formula with protective effect against radiation damage to the reproductive system, its preparation method, and its application. Background Technology

[0002] Currently, commonly used radiation protectants for health damage caused by ionizing radiation include radionuclide absorbers, ovulation promoters, hematopoietic system protectants, and free radical scavengers. However, most of these drugs are chemically synthesized, resulting in significant toxic side effects, high treatment costs, and limited application. In particular, existing radiation protection strategies largely focus on protecting the hematopoietic or immune systems, while lacking safe and effective protective measures for the highly radiation-sensitive reproductive system. Although traditional Chinese medicine has conducted some research in radiation protection, it has largely focused on preliminary explorations of single herbs or their extracts, with limited research on compound formulas and unclear mechanisms of action. This has failed to develop a specific protective strategy for radiation damage to the reproductive system, thus failing to meet the urgent clinical need for highly effective and low-toxicity radiation protection drugs. Summary of the Invention

[0003] The main objective of this invention is to propose a traditional Chinese medicine formula with protective effects against radiation damage to the reproductive system, its preparation method, and its application. This aims to address the problems of high toxicity and cost of chemically synthesized radiation protection agents in the prior art, as well as the fact that existing research on traditional Chinese medicine mainly focuses on single herbs and lacks compound protective agents with clear mechanisms targeting the reproductive system.

[0004] To achieve the above objectives, the present invention provides a traditional Chinese medicine formula with protective effect against radiation damage to the reproductive system, comprising the following raw materials in parts by weight: 15-30 parts of Chinese yam, 6-15 parts of cornus officinalis, 10-30 parts of astragalus membranaceus, 10-30 parts of prepared rehmannia glutinosa, 10-15 parts of jujube seed, 6-15 parts of psoralea corylifolia, 5-15 parts of wolfberry, and 3-9 parts of rhodiola rosea.

[0005] In one embodiment, the traditional Chinese medicine formula includes the following raw materials in parts by weight: 30 parts of Chinese yam, 13 parts of cornus officinalis, 12 parts of astragalus membranaceus, 12 parts of prepared rehmannia glutinosa, 14 parts of jujube seed, 13 parts of psoralea corylifolia, 12 parts of wolfberry, and 9 parts of rhodiola rosea.

[0006] The present invention also provides a method for preparing any of the above-mentioned traditional Chinese medicine formulas, comprising the following steps:

[0007] S1. Weigh each component according to the weight ratio, add water and decoct to obtain the mother liquor; S2. The mother liquor is prepared into formulations of different concentrations to obtain a traditional Chinese medicine formula with protective effects against radiation damage to the reproductive system. This invention employs a simple and easy-to-operate preparation method that effectively extracts the active ingredients of traditional Chinese medicine, ensuring the stability and reproducibility of the efficacy.

[0008] This invention also provides the application of any of the above-mentioned traditional Chinese medicine formulas in the preparation of drugs for protecting the reproductive system from radiation damage. By applying these formulas to the preparation of radiation protection drugs, this invention provides a new and effective means for the clinical prevention and treatment of radiation-induced reproductive damage, and is particularly suitable for patients requiring radiotherapy or occupationally exposed populations.

[0009] In one embodiment, the drug for protecting the reproductive system from radiation damage can increase the testicular coefficient, epididymal coefficient, sperm density, sperm motility, and sperm count of the subject with radiation damage.

[0010] In one embodiment, the drug for protecting the reproductive system from radiation damage can reduce oxidative stress indicators in the testicular cells of the radiation-damaged subject, increase antioxidant enzyme activity, and reduce DNA damage and apoptosis.

[0011] This invention, based on the traditional Chinese medicine theory of "the kidneys storing essence and governing reproduction," and combined with the principles of TCM formulation, provides a set of TCM formulas for radiation protection against radiation damage to the reproductive system. The core of this formula is "strengthening the body's resistance and consolidating its foundation, tonifying the kidneys and replenishing essence," while also addressing issues such as removing blood stasis and detoxifying, invigorating qi and blood circulation, and calming the mind and spirit. It works from three dimensions: "replenishing raw materials," "unblocking channels," and "regulating and regulating," comprehensively repairing the radiation-damaged reproductive system, ensuring sufficient kidney essence and smooth blood circulation, ultimately restoring a certain degree of reproductive function. Using the TCM formula provided in this application, the synergistic effect of each component can effectively reduce the damage of ionizing radiation to the reproductive system, significantly improve testicular tissue pathology, enhance sperm quality parameters, inhibit testicular cell apoptosis, repair damage caused by oxidative stress, and reduce the degree of DNA damage. This overcomes the technical shortcomings of existing radiation protectants, such as high toxicity, high cost, and insufficient protection of the reproductive system. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0013] Figure 1 These are pathological sections of mouse testicular tissue from the control group, model group, low-dose group, medium-dose group, and high-dose group in the animal experiments of this invention. Figure 2 The diagram shows the results of testicular cell apoptosis in mice in the control group, model group, low-dose group, medium-dose group and high-dose group in the animal experiments of this invention. Figure 3 The diagram shows the results of the mouse testicular cell comet experiment in the control group, model group, low-dose group, medium-dose group and high-dose group in the animal experiments of this invention.

[0014] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, solution B, or a solution that simultaneously satisfies A and B. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, all raw materials in this application are commercially available.

[0016] Currently, commonly used radiation protectants for health damage caused by ionizing radiation include radionuclide absorbers, ovulation promoters, hematopoietic system protectants, and free radical scavengers. However, most of these drugs are chemically synthesized, resulting in significant toxic side effects, high treatment costs, and limited application. In particular, existing radiation protection strategies largely focus on protecting the hematopoietic or immune systems, while lacking safe and effective protective measures for the highly radiation-sensitive reproductive system. Although traditional Chinese medicine has conducted some research in radiation protection, it has largely focused on preliminary explorations of single herbs or their extracts, with limited research on compound formulas and unclear mechanisms of action. This has failed to develop a specific protective strategy for radiation damage to the reproductive system, thus failing to meet the urgent clinical need for highly effective and low-toxicity radiation protection drugs.

[0017] In view of this, the present invention provides a traditional Chinese medicine formula with protective effect against radiation damage to the reproductive system, comprising the following components in parts by weight: 15-30 parts of Chinese yam, 6-15 parts of cornus officinalis, 10-30 parts of astragalus membranaceus, 10-30 parts of prepared rehmannia glutinosa, 10-15 parts of jujube seed, 6-15 parts of psoralea corylifolia, 5-15 parts of wolfberry, and 3-9 parts of rhodiola rosea.

[0018] This invention is based on the traditional Chinese medicine theory of "the kidneys store essence and govern reproduction," and combines it with the principles of traditional Chinese medicine formulation to provide a set of radiation-protective traditional Chinese medicine formulas for radiation damage to the reproductive system. The formula focuses on "strengthening the body's resistance and consolidating its foundation, tonifying the kidneys and replenishing essence," while also addressing issues such as removing blood stasis and detoxifying, replenishing qi and promoting blood circulation, and calming the mind. It works from three dimensions: "replenishing raw materials," "unblocking channels," and "regulating and regulating," comprehensively repairing the radiation-damaged reproductive system, ensuring sufficient kidney essence and smooth blood circulation, ultimately restoring a certain degree of reproductive function. The specific effects of each ingredient are as follows: (1) Tonifying the kidneys and replenishing essence, strengthening the foundation of reproduction: Rehmannia glutinosa is the core medicinal material for tonifying the kidneys and replenishing essence, which can directly replenish the kidney essence depleted by radiation; Lycium barbarum tonifies liver and kidney yin, and Dioscorea opposita strengthens the spleen and benefits the kidneys. The three herbs, Rehmannia glutinosa, Lycium barbarum, and Dioscorea opposita, work together to repair damaged reproductive cells, serving as the material basis for restoring reproductive function. Psoralea corylifolia warms and tonifies kidney yang, while Cornus officinalis astringes and consolidates. The two herbs, one warming and one astringent, can not only "store" the replenished kidney essence, but also stimulate the driving force of kidney qi, thereby restoring the operation of the reproductive system. (2) Replenishing Qi and nourishing blood: Astragalus can greatly replenish vital energy. Qi can generate blood and promote blood circulation. It can promote the generation of Qi and blood in the reproductive system and promote the circulation of Qi and blood, so that nutrients can reach the reproductive organs smoothly. Rhodiola can invigorate blood and unblock meridians, specifically resolve local Qi and blood stagnation caused by radiation, improve the microcirculation of reproductive organs, and create a good environment for the repair of reproductive cells. (3) Calming the mind and regulating reproductive endocrine function: Sour jujube seed nourishes the heart yin and calms the mind, which can stabilize emotions and endocrine function, restore the "regulation center" of the reproductive system to normal, and indirectly assist in the repair of reproductive function.

[0019] The traditional Chinese medicine formula provided by this invention utilizes the synergistic effect of each component to effectively reduce the damage of ionizing radiation to the reproductive system, significantly improve the pathological changes of testicular tissue, enhance sperm quality parameters, inhibit the process of testicular cell apoptosis, repair damage caused by oxidative stress, and reduce the degree of DNA damage. This overcomes the technical defects of existing radiation protection agents, such as high toxicity, high cost, and insufficient protection of the reproductive system.

[0020] As an example, the mass fractions of Chinese yam can be 15, 16, 18, 20, 22, 24, 25, 26, 28, or 30 parts, or any one of the two values ​​mentioned above.

[0021] As an example, the mass fractions of Cornus officinalis can be 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 parts, or any of the two values ​​mentioned above.

[0022] As an example, the mass fractions of Astragalus membranaceus can be 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 parts, or any of the above two values.

[0023] As an example, the mass fractions of Rehmannia glutinosa can be 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 parts, or any of the above two values.

[0024] As an example, the mass fraction of jujube seed can be 10, 11, 12, 13, 14 or 15 parts, or any one of the two values ​​mentioned above.

[0025] As an example, the mass fractions of Psoralea corylifolia can be 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 parts, or any of the ranges between the two values ​​mentioned above.

[0026] As an example, the mass fraction of wolfberries can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 parts, or any one of the two values ​​above.

[0027] As an example, the mass fraction of Rhodiola rosea can be 3, 4, 5, 6, 7, 8 or 9 parts, or any one of the two values ​​mentioned above.

[0028] In an embodiment of the present invention, the traditional Chinese medicine formula comprises the following components in parts by weight: 30 parts of Chinese yam, 13 parts of cornus officinalis, 12 parts of astragalus membranaceus, 12 parts of rehmannia glutinosa, 14 parts of jujube seed, 13 parts of psoralea corylifolia, 12 parts of wolfberry, and 9 parts of rhodiola rosea. By employing this preferred ratio, the present invention further optimizes the synergistic effect of each component, demonstrating a more significant protective effect against radiation damage to the reproductive system in animal experiments.

[0029] The present invention also provides a method for preparing any of the above-mentioned traditional Chinese medicine formulas, comprising the following steps: S1. Weigh each component according to the weight ratio, add water and decoct to obtain the mother liquor; S2. The mother liquor is prepared into formulations of different concentrations to obtain a traditional Chinese medicine formula with protective effects against radiation damage to the reproductive system. This invention employs a simple and easy-to-operate preparation method that effectively extracts the active ingredients of traditional Chinese medicine, ensuring the stability and reproducibility of the efficacy.

[0030] In some embodiments of the present invention, the decoction is performed twice in step S1, with each decoction lasting 45 minutes. By controlling the number of decoctions and the time, the present invention ensures the full dissolution of active ingredients while avoiding over-decoction that could destroy the effective components, thus optimizing extraction efficiency.

[0031] This invention provides the application of any of the above-mentioned traditional Chinese medicine formulas in the preparation of drugs for protecting the reproductive system from radiation damage. By applying these formulas to the preparation of radiation protection drugs, this invention provides a new and effective means for the clinical prevention and treatment of radiation-induced reproductive damage, and is particularly suitable for patients undergoing radiotherapy or occupationally exposed populations.

[0032] In embodiments of the present invention, the drug for protecting the reproductive system from radiation damage can improve the testicular coefficient, epididymal coefficient, sperm density, sperm motility, and sperm count in subjects with radiation damage. By improving these key reproductive function indicators, the present invention demonstrates the comprehensive protective effect of this formulation on the male reproductive system, contributing to the restoration or maintenance of fertility.

[0033] In embodiments of the present invention, the drug for protecting the reproductive system from radiation damage can reduce oxidative stress indicators in testicular cells of radiation-damaged subjects, increase antioxidant enzyme activity, and reduce DNA damage and apoptosis. The present invention elucidates the radiation-protective effect of this formulation at the molecular mechanism level by regulating oxidative stress and apoptosis pathways, providing a solid pharmacological basis for its clinical application.

[0034] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0035] Example 1 A traditional Chinese medicine formula with protective effect against radiation damage to the reproductive system includes 27g of Chinese yam, 9g of cornus officinalis, 18g of astragalus membranaceus, 25g of rehmannia glutinosa, 12g of jujube seed, 8g of psoralea corylifolia, 10g of wolfberry, and 4g of rhodiola rosea.

[0036] Examples 2 to 10 The components of Examples 2 to 10 are similar to those of Example 1, except that the amounts of each component are different. Please refer to Table 1 for details.

[0037] Table 1 shows the herbal formulation ratios for Examples 1-10 (unit: g).

[0038] Animal experiments 1. Statistical methods: SPSS 18.0 statistical software was used for variance analysis. The data results are expressed as mean ± standard deviation (±s). One-way ANOVA was used to compare differences between groups. P < 0.05 was considered statistically significant.

[0039] 2. Preparation methods of traditional Chinese medicine prescriptions: The preparation method of the traditional Chinese medicine formulas with protective effects against radiation damage to the reproductive system listed in Table 1 above includes the following steps: (1) Weigh each ingredient according to the weight ratio, add water to cover the surface of the medicine by 1-3 cm, decoct for the first time for 45 minutes, filter to obtain the dregs and the first filtrate; (2) Add water to the dregs until it covers the surface of the herbs by 1-3 cm, and decoct for 45 minutes. Then filter to obtain the second filtrate. (3) Mix the first filtrate and the second filtrate, heat and concentrate to a final volume of 100 mL to obtain concentrated mother liquor.

[0040] 3. Feasibility experiment on drug efficacy: 3.1 Grouping of experimental animals: One hundred and twenty healthy four-week-old ICR mice were randomly divided into 12 groups of 10 mice each, according to their body weight. The specific groupings were as follows: control group (gated with the same volume of 0.9% saline solution as the drug by gavage); model group (gated with the same volume of 0.9% saline solution as the drug by gavage); and experimental group (gated with the traditional Chinese medicine formulas from Examples 1-10, prepared according to the corresponding raw material ratios in Examples 1-10, to a concentration of 0.28 g crude drug / mL concentrated mother liquor, and administered by gavage at a concentration of 10 g crude drug / kg body weight). The mice in the 12 groups were administered the drugs continuously for 14 days.

[0041] 3.2 Establishment of the radiation damage model: On day 7 of drug administration, mice were placed in plexiglass boxes while awake to restrict their movement. Except for the control group, all other groups were treated with... 60 A single whole-body irradiation with a Coγ source was performed at a dose of 6.5 Gy and a dose rate of 1.0 Gy / min to establish a radiation damage model. Drug administration continued for 7 days post-irradiation.

[0042] 3.3 Detection of reproductive hormone levels, sperm count, and sperm abnormality rate: 3.3.1 Reproductive hormone level detection: On day 7 post-irradiation, blood was collected from the orbital sinus of mice in each group and added to EDTA-2Na anticoagulant tubes to obtain mouse serum samples. Following the instructions of the testosterone and estradiol enzyme-linked immunosorbent assay (ELISA) kit, the levels of sex hormones (testosterone and estradiol) in the serum were detected using an ELISA reader. The test results are shown in Table 1.

[0043] 3.3.2 Sperm count and deformity rate detection: On day 7 post-irradiation, mice were euthanized by cervical dislocation after blood collection. The abdominal cavity was opened, both epididymis were separated, fat and mesentery were removed, and the intact epididymal tail was extracted. The epididymal tail was placed in 2 mL of preheated 37°C physiological saline, the tail was cut into small pieces, and the suspension was mixed by pipetting. The suspension was allowed to stand for 3–5 minutes to allow sperm to become free, forming a sperm suspension. 10 μL of the sperm suspension was taken, mixed, and added dropwise to a hemocytometer for counting. The sperm concentration in the epididymis of each mouse was calculated based on the counting results. The test results are shown in Table 1.

[0044] A small amount of the mixed sperm suspension was placed on a glass slide and subjected to morphological analysis under an optical microscope. First, an area with a clear background and dispersed sperm was selected under low magnification (10×), then the morphology of each sperm was observed under high magnification (40×). At least 200 intact sperm were examined per mouse, with a total of ≥1000 sperm examined per group. Abnormalities were classified according to the following criteria: 1) Head deformities: no hook, banana-shaped, fat head, amorphous, two heads; 2) Tail deformities: folded tail, double tail, broken tail.

[0045] The sperm abnormality rate was calculated using the following formula: Sperm abnormality rate (%) = Number of abnormal sperm / Total number of sperm × 100%. The test results for sperm abnormality rate are shown in Table 1.

[0046] Table 1 Results of reproductive hormone levels, sperm count, and sperm abnormality rate.

[0047] Note: Compared with the control group, #: P < 0.05; ##: P < 0.01; compared with the model group, :P<0.05; :P<0.01.

[0048] Table 1 shows that, compared with the control group, the model group had significant differences in reproductive hormone levels, sperm count, and abnormality rate (P<0.01), indicating successful model establishment. Furthermore, compared with the model group, the reproductive hormone levels and sperm count of all 10 formulations in Examples 1-10 recovered, and the abnormality rate decreased. Specifically, regarding sperm count and abnormality rate, the formulations in Examples 3, 6, 9, and 10 showed significant differences (P<0.01), while the formulations in Examples 1 and 5 showed differences (P<0.05). Regarding reproductive hormone levels, the formulations in Examples 3 and 6 showed significant differences in testosterone recovery (P<0.01), as did the formulations in Examples 1 and 9 (P<0.05); the formulations in Examples 3 and 9 showed significant differences in estradiol recovery (P<0.01). Furthermore, the results in Table 1 also show that the mice treated with the formulation of Example 3 by gavage showed the most comprehensive recovery in reproductive hormone levels, sperm count, and abnormality rate, and the most significant protective effect.

[0049] 3.4 Acute toxicity test: Sixty healthy ICR mice, half male and half female, aged 6-8 weeks (males weighing 20-24g and females 18-22g), were randomly divided into low, medium, and high-dose groups (sex and male mice were housed separately). The drug was prepared according to the formulation in Example 3 and the dosages in Table 2. Each mouse was given 0.8mL once daily. Mice were fed a standard diet for 15 days, and their survival and mortality rates were recorded. The results of the acute toxicity test in mice are shown in Table 3, where "g crude drug / kg" indicates the amount of raw medicinal material (crude drug) administered per kilogram of test animal body weight.

[0050] Table 2 Dosage for low, medium, and high dose groups

[0051] Table 3 Results of acute toxicity test in mice

[0052] The high dose of 80 g / kg in Table 2 is equivalent to 41.7 times the adult clinical dose (1.92 g / kg), which far exceeds the requirement for low-toxicity drugs to reach the clinical dose in the "Technical Guidelines for Single-Dose Toxicity Studies of Drugs", and can fully reflect safety.

[0053] The results in Table 3 show that the mortality rate of mice given low and medium doses was 0 within 15 days, while the mortality rate of mice given high doses was 5.26% within 15 days.

[0054] 4. Test on the protective effect against radiation damage to the mouse reproductive system: 4.1 Grouping of experimental animals: Fifty healthy 4-week-old ICR mice were randomly divided into 5 groups of 10 mice each according to their body weight. The 5 groups are as follows: control group, model group, low-dose group, medium-dose group and high-dose group.

[0055] 4.2 Drug preparation and administration: Prepare the formula according to the proportions in Example 3. Weigh the medicinal materials according to the prescription ratio, decoct in water and concentrate to 100mL of mother liquor, then dilute with distilled water to the appropriate concentrations. The concentrations of the mother liquor are as follows: 115g of crude drug yields 100mL of mother liquor, that is, 1mL of mother liquor = 1.15g of crude drug.

[0056] The administration was as follows: the control group was administered the same volume of the drug to 0.9% saline via gavage daily; the model group was administered the same volume of the drug to 0.9% saline via gavage daily; the low-dose, medium-dose, and high-dose groups were administered the traditional Chinese medicine formula from Example 3 daily, with the drug concentrations for the low, medium, and high-dose groups being 0.288 g / mL, 0.575 g / mL, and 1.15 g / mL, respectively. The crude drug dosages for the low-dose, medium-dose, and high-dose groups were 0.058, 0.115, and 0.23 g crude drug / 20 g body weight, respectively, with a daily gavage volume of 0.1 mL / (10 g·bw). Mice in each group were administered the drug for 14 consecutive days.

[0057] 4.3 Establishment of the radiation damage model: On day 7 of drug administration, mice were placed in plexiglass boxes while awake to restrict their movement. Except for the control group, all other groups were treated with... 60 A single whole-body irradiation with a Coγ source was performed at a dose of 6.5 Gy and a dose rate of 1.0 Gy / min to establish a radiation damage model. Drug administration continued for 7 days post-irradiation.

[0058] 4.4 Detection indicators: The reproductive system is one of the most sensitive targets for radiation damage. This study investigates the effects of ionizing radiation on the reproductive system by measuring indicators such as testicular coefficient, epididymal coefficient, sperm density, grade A sperm count, grade A+B sperm count, testicular pathological morphology, apoptosis, DNA damage, and testicular cell oxidative damage.

[0059] 4.4.1 Testicular coefficient and epididymal coefficient detection: The final body weight of mice in each group was recorded. The testes and epididymis were removed and weighed. The testicular coefficient and epididymal coefficient were calculated using the following formulas: Testicular coefficient (mg / 10g) = mouse testicular mass (mg) / mouse final body weight (10g); Epididymal coefficient (mg / 10g) = mouse epididymal mass (mg) / mouse final body weight (10g). The test results are shown in Table 4.

[0060] Table 4. Changes in body weight and testicular and epididymal coefficients of mice in each group.

[0061] Note: Compared with the control group, #: P < 0.05; ##: P < 0.01; compared with the model group, P < 0.05; P < 0.01.

[0062] Radiation damage often causes organ atrophy in mice, manifested as a decrease in their coefficients. The testes and epididymis are important sex organs; if they atrophy or lose weight, the hormones secreted by these organs will decrease accordingly, potentially leading to sexual dysfunction. Table 4 shows that compared to the control group, the testicular and epididymal coefficients of irradiated mice decreased, with significant differences in decrease among the model group, low-dose group, and high-dose group (P<0.01). Compared to the model group, the low-dose group showed an increase (P<0.05), while the testicular and epididymal coefficients of mice in the medium-dose and high-dose groups recovered significantly (P<0.01). The final body weight of the medium-dose group showed a significant increase (P<0.01). Preliminary findings indicate that the formulation provided in this invention has a certain protective effect on the reproductive function of radiation-damaged mice.

[0063] 4.4.2 Semen quality testing: The intact epididymal tail was removed and placed in 2 mL of preheated 37°C physiological saline. The tail was then cut into small pieces and thoroughly shaken to allow sperm to swim out. The mixture was incubated in a constant temperature incubator for 15 minutes, filtered through a 200-mesh filter, and sperm count and motility were performed. The number of Grade A sperm (i.e., morphologically normal, with good motility, capable of rapid linear movement at a speed ≥25 μm / s) and Grade B sperm (i.e., morphologically normal, capable of linear movement, but slower than Grade A sperm) was determined. The test results are shown in Table 5.

[0064] Table 5 Effects on mouse semen quality

[0065] Note: Compared with the control group, #: P < 0.05; ##: P < 0.01; compared with the model group, P < 0.05; P < 0.01.

[0066] Sperm density, grade A sperm count, and grade A+B sperm count in mice are important indicators for assessing their fertility. Table 5 shows that compared with the control group, the model group mice had decreased sperm density and sperm count (P<0.01); compared with the model group, the sperm density, grade A sperm count, and grade A+B sperm count in all three dosage groups increased significantly (P<0.01, P<0.05). These results confirm that the formulation provided by this invention has a certain protective effect against radiation-induced sperm damage.

[0067] 4.4.3 Effects on oxidative damage to mouse testicular cells: Testicular tissue was washed with PBS (pH 7.4) buffer and homogenized. The homogenate was then centrifuged at 3000 rpm for 15 min. The supernatant was collected, and superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, reactive oxygen species (ROS) level, and total antioxidant capacity (T-AOC) were measured using a kit from Nanjing Jiancheng Biotechnology Institute. The test results are shown in Table 6.

[0068] Table 6 Effects of SOD, MDA, ROS and T-AOC levels in mouse testes

[0069] Note: Compared with the control group, #: P < 0.05; ##: P < 0.01; compared with the model group, P < 0.05; P < 0.01.

[0070] The activity level of SOD reflects the body's ability to scavenge free radicals, while the level of T-AOC in tissues directly reflects the body's overall antioxidant capacity. MDA reflects the degree of free radical damage to the body's cells. ROS is the main mediator of oxidative stress damage and can react with polyunsaturated fatty acids on the sperm membrane surface to produce MDA. Therefore, the ROS content can reflect the degree of oxidative stress damage to sperm.

[0071] As shown in Table 6, compared with the control group, the SOD activity and T-AOC of testicular cells in the model group mice were significantly decreased (P<0.01), while the MDA and ROS contents were significantly increased (P<0.01). Compared with the model group, the SOD activity and T-AOC contents in the testicular tissue of the three drug-treated groups were significantly restored and increased (P<0.01, P<0.05), while the MDA and ROS contents were significantly decreased (P<0.01, P<0.05). These results indicate that the formulation provided by this invention can alleviate radiation-induced oxidative damage by repairing the antioxidant enzyme system, improve the ability of mice to scavenge free radicals, and thus protect cell structure and function.

[0072] 4.4.4 Testicular histopathological examination: Testicular tissue was fixed in 4% paraformaldehyde for 24 hours, then dehydrated using a gradient method, cleared in xylene for 20 minutes, and then embedded in paraffin. The paraffin sections were baked in a 60°C oven for later use. The sections were then dewaxed in xylene I and xylene II for 10 minutes each. Next, they were sequentially immersed in anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 minutes each, followed by 5 minutes of distilled water to remove alcohol. Hematoxylin staining was performed for 8 minutes, followed by differentiation in 1% hydrochloric acid ethanol for 30 seconds, and then rinsed with running water. Eosin staining was performed for 3 minutes. The tissue was then dehydrated in 95% ethanol and anhydrous ethanol for 5 minutes each. Clearing was performed in xylene I and xylene II for 5 minutes each, followed by mounting. The tissue was observed and photographed under a light microscope. The results are as follows: Figure 1 As shown.

[0073] Figure 1 HE staining results indicated that the testicular cells of the model group mice showed significant spermatocyte vacuolation, with marked cell shedding and relaxation. The thickness of the seminiferous epithelium in the treatment groups was significantly increased. Specifically, the cell shedding and relaxation were significantly improved in the medium-dose group, while the seminiferous tubule diameter was significantly increased in the high-dose group. These results demonstrate that the formulation provided by this invention can alleviate pathological morphological damage to testicular tissue, thereby exerting an anti-radiation effect.

[0074] 4.4.5 Flow cytometry detection of testicular cell apoptosis: Testicular tissue was collected and washed in pre-chilled PBS (pH 7.4), then rolled through a 70 μm nylon mesh to prepare a single-cell suspension. The cell suspension was centrifuged at 1500 rpm for 8–10 min, and the supernatant was discarded. 1 mL of Tris-NH4Cl buffer (pH 7.4) was added, and the cells were incubated at room temperature for 5 min to lyse the red blood cells. 10 mL of PBS was added to terminate lysis. After centrifugation, the cell pellet was washed once more with PBS, and the centrifugation process was repeated, discarding the supernatant. The cells were resuspended in PBS, and the cell density was adjusted to 5 × 10⁶ cells / mL. 5 Cells / 100μL, protected from light for staining. Strictly follow the antibody instructions, diluting the fluorescent antibody (Annexin V-FITC) and adding propidium iodide for sample staining. Wash and resuspend in PBS before flow cytometry analysis. Flow cytometry results are shown below. Figure 2 As shown.

[0075] The percentage of apoptotic cells in the model group was 66.67% ± 1.37%, while that in the control group was 11.7% ± 0.81%. Compared with the control group, the percentage of apoptotic testicular cells in the model group was significantly higher (P < 0.01), indicating that the radiation dose selected in this experiment can induce testicular cell apoptosis. Compared with the model group, the percentage of apoptotic cells in each dose group was significantly reduced (P < 0.01). The apoptosis rates in the low, medium, and high concentration groups of the formulation were 44.31% ± 1.22%, 25.48% ± 0.74%, and 34.27% ± 1.93%, respectively. These results show that the formulation provided by this invention can significantly reduce radiation-induced testicular cell apoptosis and exert its anti-radiation effect by inhibiting cell apoptosis.

[0076] 4.4.6 Comet assay for detecting the degree of DNA damage in testicular cells: Based on the fact that the formulation provided by this invention can significantly reduce radiation-induced testicular cell apoptosis, the mechanism was further analyzed. DNA is an important target of radiation damage. When exposed to radiation, DNA undergoes cross-linking, single-strand breaks, and double-strand breaks, which can lead to cell apoptosis. Comet assays can sensitively predict the damaging effects of test substances on the cell nucleus and DNA bases. By measuring the DNA migration length, tail DNA content, and olive tail momentum (OTM) of testicular cells, the degree of DNA damage in testicular cells was analyzed, thereby determining the correlation between the dosage of the formulation provided by this invention and the DNA damage effect.

[0077] Testicular tissue was collected as a sample and placed in 3 mL of dispersing solution (Hank's equilibration solution containing 20 mmol / L EDTA-2Na and 10% DMSO, pH 7.2). The tissue was minced to release cells, and the sample was filtered through a 40 μm filter to obtain a single-cell suspension, which was then stored on ice. 30 μL of the cell suspension (concentration 1×10⁻⁶) was then taken. 6Mix 140 μL of LMP agarose with the prepared suspension (cells / mL). Heat 1% normal melting point agarose to 55 °C, drop 100 μL onto a ground glass slide, cover with a coverslip, and cool at 4 °C for 10 min. Remove the coverslip, drop 70 μL of the prepared suspension (cells-LMP agarose mixture) onto the first layer of agarose, prepare two replicates for each sample, and cool at 4 °C. Remove the coverslip and immerse the sample in lysis buffer (1 M Tris-HCl + 0.5 M EDTA + 10% SDS + 5 M NaCl + ddH2O) at 4 °C for 1 h, then wash twice with cooled PBS, 5 min each time. Place the slide in alkaline electrophoresis buffer (12.0 g NaOH and 0.37 g EDTA-2Na mixed and diluted to 1 L with deionized water, pH 13) pre-cooled to 4 °C for 40 min to unwind. Pre-cooled alkaline electrophoresis buffer was added to a horizontal electrophoresis tank, and electrophoresis was performed at 4°C (20 min, 25 V, 300 mA). After electrophoresis, the slides were washed twice with PBS for 5 min each time, and then placed in neutralization solution (48.46 g Tris-base dissolved in 800 mL of water, 6 mol / L HCl added dropwise to adjust the pH to 7.5, and then deionized water was added to bring the volume to 1 L) for 15 min, and incubated overnight at 4°C. PI (1:1000) was used for staining. 75 μL of staining solution was evenly dropped onto the gel surface, and the slides were placed in a dark box and stained at room temperature in the dark for 15 min. Excess staining solution was removed with a pipette, and the slides were immersed in distilled water and gently rinsed twice for 2 min each time. The slides were then removed and allowed to air dry at room temperature. Observation and photography were performed using a fluorescence microscope (excitation wavelength 530 nm). One hundred cells were randomly selected and analyzed using CASP comet analysis software. The comet tail length (hereinafter referred to as "tail length"), tail DNA percentage (hereinafter referred to as "tail DNA%)", and tail momentum were statistically analyzed for each group. The results of the mouse testicular cell comet experiment are as follows: Figure 3 As shown in Table 7.

[0078] Table 7 Effects on DNA damage in mouse testicular cells

[0079] Note: Compared with the control group, # : P <0.05; ## : P <0.01; compared with the model group, : P <0.05; : P <0.01.

[0080] Figure 3The results in Table 7 show that, compared with the control group, the DNA damage in the irradiated mice was significant. In particular, the comet tail DNA%, tail length, and tail momentum of the testicular cells of the model group and the low-dose group were significantly increased (P<0.01), indicating that radiation can severely damage the DNA structure of mouse testicular cells and cause DNA damage. Compared with the model group, the comet tail DNA%, tail length, and tail momentum of the mouse testicular cells of the low-dose, medium-dose, and high-dose groups were all reduced, and the reduction was more significant in the medium-dose and high-dose groups (P<0.01). The above results show that the formulation provided by this invention has a protective effect against radiation-induced DNA damage in mouse testicular cells.

[0081] In summary, radiation causes a decrease in the testicular and epididymal coefficients in mice, testicular tissue lesions, testicular cell apoptosis, DNA breaks, oxidative damage, and abnormal sperm quality. The formulation provided in this invention exerts its anti-radiation effect through methods such as tonifying the spleen and stomach, tonifying the kidneys and astringing essence, nourishing kidney yin, and replenishing essence. It protects against radiation-induced reproductive damage in mice from the perspectives of pathological tissue, cell apoptosis, sperm quality, and DNA damage.

[0082] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A traditional Chinese medicine formula with protective effect against radiation damage to the reproductive system, characterized in that, The traditional Chinese medicine formula includes the following ingredients in parts by weight: 15-30 parts of Chinese yam, 6-15 parts of cornus officinalis, 10-30 parts of astragalus membranaceus, 10-30 parts of prepared rehmannia glutinosa, 10-15 parts of jujube seed, 6-15 parts of psoralea corylifolia, 5-15 parts of wolfberry, and 3-9 parts of rhodiola rosea.

2. The traditional Chinese medicine formula according to claim 1, characterized in that, The traditional Chinese medicine formula includes the following ingredients in parts by weight: 30 parts of Chinese yam, 13 parts of cornus officinalis, 12 parts of astragalus membranaceus, 12 parts of prepared rehmannia glutinosa, 14 parts of jujube seed, 13 parts of psoralea corylifolia, 12 parts of wolfberry, and 9 parts of rhodiola rosea.

3. A method for preparing the traditional Chinese medicine formula according to claim 1 or 2, characterized in that, The method includes the following steps: S1. Obtain the raw materials according to the raw material ratio of claim 1 or 2, and then boil them with water to obtain the mother liquor; S2. Prepare preparations of different concentrations from the mother liquor to obtain the traditional Chinese medicine formula with the protective effect against radiation damage to the reproductive system.

4. The use of the traditional Chinese medicine formula according to claim 1 or 2 in the preparation of a medicament for protecting the reproductive system from radiation damage.

5. The application as described in claim 4, characterized in that, The drug can increase the testicular coefficient, epididymal coefficient, sperm density, sperm motility, and sperm count in radiation-damaged subjects.

6. The application as described in claim 4, characterized in that, The drug can reduce oxidative stress indicators in testicular cells of radiation-damaged subjects, increase antioxidant enzyme activity, and reduce DNA damage and apoptosis.

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