Herbal medicine composition for improving testicular dysfunction, and its manufacturing method and use

Abstract

To provide a herbal medicine composition for improving testicular dysfunction, to provide a method for manufacturing the same, and to provide uses thereof.SOLUTION: The present invention provides a herbal medicine composition comprising 10 to 40 parts of Codonopsis Root, 15 to 100 parts of Astragalus Root, 10 to 40 parts of Dioscorea Rhizome, 6 to 25 parts of Japanese Angelica Root, 6 to 40 parts of Atractylodes Rhizome, 10 to 30 parts of Poria Sclerotium, 10 to 30 parts of Peony Root, 6 to 15 parts of Psoralea corylifolia fruit, 6 to 20 parts of Cnidium Rhizome, 6 to 20 parts of Nelumbo nucifera seed, 6 to 20 parts of Euryale ferox Salisb, 3 to 10 parts of Amomum Xanthioides, 3 to 10 parts of Uncaria Hook, 6 to 20 parts of Fennel, 3 to 15 parts of Cinnamomum sieboldii, 6 to 10 parts of Evodia rutaecarpa, 6 to 15 parts of Piper nigrum, 6 to 15 parts of Zingiberis Rhizoma, 6 to 15 parts of Cyperus Rhizome, and 6 to 10 parts of Glycyrrhiza.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention belongs to the field of medical technology, specifically to a traditional Chinese medicine composition for improving testicular dysfunction and its preparation method and use. [Background technology]

[0002] Diabetes is one of the most important public health challenges today, and 90% of diabetic patients have type 2 diabetes mellitus (T2DM). Research has revealed that diabetes can cause sperm abnormalities (Kong ZL, Sudirman S, Hsu YC, et al. Fucoxanthin-Rich Brown Algae Extract Improves Male Reproductive Functionon Streptozotocin-Nicotinamide-Induced Diabetic Rat Model [J]. Int J Mol Sci, 2019, 20 (18).). About half of male diabetic patients have a 95% incidence of testicular dysfunction, and more than 50% of diabetic patients have infertility (Li Wenmei. Clinical study on serum reproductive hormones and semen quality in diabetic men [J]. New World of Diabetes, 2020, 23(05): 23-25.), and oligozoospermia patients account for about 75% of male infertile patients (Li Xinyang, Sun Zixue. Study on the effect of Yisheng Tongguang Boqi Fang on estrogen receptors in oligozoospermia rats based on network pharmacology and molecular docking [J]. Chinese Medicine, 2021, 43(11): 3216-3221.).

[0003] Because reproductive cells are sensitive to drugs, it is particularly important to find effective natural treatments with few side effects to improve and treat oligozoospermia caused by glucose and lipid metabolism disorders. Currently, oligozoospermia caused by type 2 diabetes has not received much attention, and Western medicine mainly treats it with surgery, blood sugar reduction and antioxidants, but this method has significant side effects. Chinese medicine has the advantages of individualization, good therapeutic effect and few side effects, but it has not yet formed an effective method to improve testicular dysfunction caused by type 2 diabetes accompanied by obesity. Summary of the Invention [Problem to be solved by the invention]

[0004] The objective of the present invention is to make up for the shortcomings of the prior art and provide a herbal medicine composition to improve hormone secretion, regulate the reproductive endocrine environment in male individuals, improve semen parameters, and improve testicular dysfunction. [Means for solving the problem]

[0005] In order to solve the above problems, the present invention provides a herbal medicine composition for improving testicular dysfunction, which contains as raw materials Doujinshi, Astragalus Root, Dioscorea Root, Angelica Root, Byakujutsu, Poria Cocos, Byakugan, Hokotsushi, Cnidium Root, Lens Root, Kenjitsu, Shanin, Soukou, Fennel, Cinnamon, Euonymus Root, Pepper, Ginger, Kokbushi, and Licorice.

[0006] Preferably, the raw materials based on parts by mass include 10 to 40 parts of Doujinshi, 15 to 100 parts of Astragalus Root, 10 to 40 parts of Dioscorea Root, 6 to 25 parts of Angelica Root, 6 to 40 parts of Atractylodes Root, 10 to 30 parts of Poria Cocos, 10 to 30 parts of Peony Root, 6 to 15 parts of Hokotsuka, 6 to 20 parts of Cnidium Root, 6 to 20 parts of Lotus Root, 6 to 20 parts of Kenjitsu Fruit, 3 to 10 parts of Shanin, 3 to 10 parts of Sophora Root, 6 to 20 parts of Fennel, 3 to 15 parts of Cinnamon, 6 to 10 parts of Euonymus Root, 6 to 15 parts of Pepper Root, 6 to 15 parts of Ginger, 6 to 15 parts of Japanese Kokubushi, and 6 to 10 parts of Licorice.

[0007] The present invention also provides a method for preparing a herbal composition for improving testicular dysfunction, which comprises mixing the ingredients as described in the above technical solutions and then extracting them.

[0008] Preferably, the mode of extraction comprises a water extraction.

[0009] Preferably, the method of water extraction comprises: The method for producing the herbal medicine composition includes mixing the mixture of raw materials with water in a mass to volume ratio of 136-500 g:400-1000 mL, steeping for 30 minutes, boiling over high heat, reducing heat to low heat and simmering for 40-60 minutes, and filtering.

[0010] The present invention also provides the use of the herbal composition according to the above technical solution in the manufacture of a product for improving testicular dysfunction.

[0011] Preferably, the product comprises a medicine or a food product; The testicular dysfunction includes testicular dysfunction caused by diabetes.

[0012] Preferably, said diabetes-induced testicular dysfunction comprises testicular dysfunction induced by type 2 diabetes associated with obesity.

[0013] The present invention also provides the use of the herbal composition according to the above technical solution in the manufacture of a product for improving testicular morphology and / or enhancing semen parameters.

[0014] The present invention also provides a drug for improving testicular dysfunction, comprising the herbal composition described in the above technical solution and pharma- ceutical acceptable additives. Effect of the Invention

[0015] The beneficial effects are: The present invention uses as raw materials Dojin, Astragalus Root, Dioscorea Root, Angelica Root, Atractylodes Root, Poria Coccus, Radix Radix, Cnidium Root, Lens Root, Kenjitsu, Shanin, Soukou, Fennel, Cinnamon, Euonymus Root, Pepper, Zingiber Root, Kokbushi, and Licorice to produce Chinese herbal medicines. Dojin, Astragalus Root, Dioscorea Root, Dioscorea Root, and Radix Radix are principal medicines that replenish qi and moisten yin fluid, produce fluids and increase blood, and replenish acquired conditions and regulate innate conditions. Atractylodes Root, Poria Coccus, Radix Radix, and Cnidium Root are subordinate medicines that dissolve dampness in the spleen and stomach, warm the kidneys, and replenish kidney yang. Dojin and Cnidium Root are subordinate medicines that dissolve dampness in the spleen and stomach, warm the kidneys, and replenish kidney yang. , Lotus Root, Shanin, Fennel, Cinnamon, Pepper, Ginger, Solanum Root, Euonymus Root, and Rhizome are auxiliary drugs, while Angelica Root and Cnidium Root improve blood flow and activate blood function, improve microcirculation, and restore sperm-producing function, Lotus Root purifies the heart fire, Cinnamon and Bupleurum pass Yang Qi, circulate Qi and blood, and warm the kidney to restore deficiency fire, Kidney Yang steams the kidney water and nourishes the yin fluid of the five organs, while Pepper, Shanin, Cinnamon Root, and Ginger smooth the circulation of Qi and blood, warm and tonify the body, strengthen the spleen Qi, and restore the transport and transformation of nutrients, and Licorice is an agent and harmonizes various drugs. This invention rationally selects raw materials and makes various drugs compatible with each other, and simultaneously cooks the three organs of the spleen, stomach, and kidney, restoring Qi, blood, and body fluids, providing a source of kidney essence transformation. Animal studies have revealed that the herbal medicine composition provided by the present invention can upregulate the expression of PI3K, p-Akt, and Bcl-2, improve testicular dysfunction, improve hormone secretion, regulate the reproductive endocrine environment in male individuals, and even improve semen parameters. [Brief description of the drawings]

[0016] In order to more clearly describe the embodiments of the present invention or the technical solutions of the prior art, the drawings used in the embodiments are briefly introduced below. [Figure 1] FIG. 1. Effect of different treatment methods on body weight in obese type 2 diabetic testicular dysfunction mice. [Diagram 2] FIG. 2 shows the effect of different treatment methods on blood glucose in obese mice with type 2 diabetes and testicular dysfunction. [Figure 3-1] Figure 3-1 shows the effect of the control group on testicular morphology in mice with testicular dysfunction due to type 2 diabetes accompanied by obesity (200x magnification). [Figure 3-2] FIG. 3-2 shows the effect of the model groups on the morphology of the testes of mice with testicular dysfunction due to type 2 diabetes accompanied by obesity (magnification: 200x). [Figure 3-3] Figure 3-3 shows the effect of the metformin group on testicular morphology in mice with testicular dysfunction due to type 2 diabetes accompanied by obesity (200x magnification). [Diagram 3-4] FIG. 3-4 shows the effect of a high dose group of the composition on the morphology of the testes of mice with testicular dysfunction due to type 2 diabetes accompanied by obesity (200x magnification). [Figure 3-5] Figures 3-5 show the effect of a low dose group of the composition on the morphology of the testes of mice with testicular dysfunction due to type 2 diabetes accompanied by obesity (200x magnification). [Figure 4] FIG. 4 shows the effect of different treatment methods on the expression of PI3K, p-Akt, and Bcl-2 proteins in the testes of mice with testicular dysfunction due to type 2 diabetes accompanied by obesity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention provides a herbal medicine composition for improving testicular dysfunction, which contains as raw materials Doujinshi, Astragalus Root, Dioscorea Root, Angelica Root, Byakujutsu, Poria Cocos, Byakugaku Root, Hokotsushi, Cnidium Root, Lens Root, Kenjitsu, Shanin, Soukou, Fennel, Cinnamon, Euonymus Root, Pepper, Ginger, Kokbushi, and Licorice.

[0018] The raw materials for producing the herbal medicine composition described in the present invention include Doujinshi, and the mass number of the Doujinshi is preferably 10 to 40 parts, more preferably 15 to 35 parts, even more preferably 20 to 30 parts, and most preferably 25 parts.

[0019] The raw materials for producing the herbal medicine composition described in the present invention include Astragalus Root, and the number of parts by mass of Astragalus Root based on the number of parts by mass of Astragalus Root is preferably 15 to 100 parts, more preferably 20 to 80 parts, even more preferably 30 to 60 parts, and most preferably 40 to 50 parts.

[0020] The raw materials for manufacturing the herbal medicine composition described in the present invention include Dioscorea Rhizome, and the number of parts by mass of the Dioscorea Rhizome is preferably 10 to 40 parts, more preferably 15 to 35 parts, even more preferably 20 to 30 parts, and most preferably 25 parts, based on the number of parts by mass of the Doujinshi.

[0021] The raw materials for manufacturing the herbal medicine composition described in the present invention include Radix Paeoniae Radix, and the number of parts by mass of the Radix Paeoniae Radix based on the number of parts by mass of the Radix Paeoniae Radix is ​​preferably 10 to 30 parts, more preferably 15 to 25 parts, and even more preferably 20 parts.

[0022] In this invention, Doujinshi, Astragalus Root, Dioscorea Rhizome, and Radix White are all used as the main medicines, replenishing qi, moistening yin fluid, producing body fluids and increasing blood, thereby replenishing the acquired condition and adjusting the innate condition.

[0023] The raw materials for manufacturing the herbal medicine composition described in the present invention include Byaedochrosite, and the number of parts by mass of the Byaedochrosite based on the number of parts by mass of the Doujinshi is preferably 6 to 40 parts, more preferably 10 to 35 parts, even more preferably 20 to 30 parts, and most preferably 25 parts.

[0024] The raw materials for producing the herbal medicine composition according to the present invention include Poria Cocos Rhizome, and the number of parts by mass of the Poria Cocos Rhizome based on the number of parts by mass of the Doujinshi is preferably 10 to 30 parts, more preferably 15 to 25 parts, and even more preferably 20 parts.

[0025] The raw materials for producing the herbal medicine composition described in the present invention include Kenjitsu, and the number of parts by mass of the Kenjitsu based on the number of parts by mass of the Doujinshi is preferably 6 to 20 parts, more preferably 8 to 15 parts, and even more preferably 10 parts.

[0026] The raw material for producing the herbal composition according to the present invention includes Hokotsushi, and the number of parts by mass of the Hokotsushi is preferably 6 to 15 parts, more preferably 8 to 10 parts, based on the number of parts by mass of the Doujin.

[0027] In the present invention, Byaku Atractylodes Root, Poria Cocos Root, Kenjitsu Root, and Hokotsuji Root are all used as subordinate medicines to eliminate dampness in the spleen and stomach, warm the kidneys, and replenish kidney yang.

[0028] The raw materials for producing the herbal medicine composition described in the present invention include Dongqi, and the number of parts by mass of the Dongqi is preferably 6 to 25 parts, more preferably 8 to 20 parts, and even more preferably 10 to 15 parts, based on the number of parts by mass of the Dongzin.

[0029] The raw materials for manufacturing the herbal medicine composition described in the present invention include Cnidium officinalis, and the number of parts by mass of the Cnidium officinalis based on the number of parts by mass of the Doujinshi is preferably 6 to 20 parts, more preferably 8 to 15 parts, and even more preferably 10 parts.

[0030] The raw materials for producing the herbal medicine composition described in the present invention include Lotus Root, and the number of parts by mass of the Lotus Root, based on the number of parts by mass of the Doujinshi, is preferably 6 to 20 parts, more preferably 8 to 15 parts, and even more preferably 10 parts.

[0031] The raw material for producing the herbal composition according to the present invention contains Shanin, and the number of parts by mass of the Shanin is preferably 3 to 10 parts, more preferably 5 to 8 parts, based on the number of parts by mass of the Doujinshi.

[0032] The raw material for producing the herbal medicine composition according to the present invention includes fennel, and the number of parts by mass of the fennel based on the number of parts by mass of the Doujinshi is preferably 6 to 20 parts, more preferably 8 to 15 parts, and even more preferably 10 parts. The fennel according to the present invention is preferably Fennel Saussurea.

[0033] The raw material for producing the herbal medicine composition according to the present invention contains Cinnamomum lucidum, and the number of parts by mass of the Cinnamomum lucidum is preferably 3 to 15 parts, more preferably 8 to 10 parts, based on the number of parts by mass of the Doujinshi.

[0034] The raw materials for producing the herbal medicine composition according to the present invention include pepper, and the number of parts by mass of the pepper is preferably 6 to 15 parts, more preferably 8 to 10 parts, based on the number of parts by mass of the Doujinshi.

[0035] The raw material for producing the herbal medicine composition according to the present invention contains Zingiber officinale, and the number of parts by mass of Zingiber officinale based on the number of parts by mass of Doujinshi is preferably 6 to 15 parts, more preferably 8 to 10 parts.

[0036] The raw material for producing the herbal composition according to the present invention contains Sophora Root, and the number of parts by mass of the Sophora Root is preferably 3 to 10 parts, more preferably 5 to 9 parts, based on the number of parts by mass of the Doujinshi.

[0037] The raw material for producing the herbal composition according to the present invention includes Evodia Fruit, and the mass parts of the Evodia Fruit are preferably 6 to 10 parts, more preferably 8 to 10 parts, based on the mass parts of the Doujinshi.

[0038] The raw material for producing the herbal composition according to the present invention includes Magnolia Root, and the mass parts of Magnolia Root based on the mass parts of Doujinshi is preferably 6 to 15 parts, more preferably 8 to 15 parts.

[0039] In the present invention, Angelica Root, Cnidium Root, Lotus Root, Shanin, Fennel, Cinnamon, Pepper, Zingiber officinale, Sophora Root, Euonymus Root, and Oriental Green Tea are all auxiliary drugs, and Angelica Root and Cnidium Root improve blood flow and invigorate blood function, improve microcirculation, and restore sperm-producing function, Oriental Green Tea purifies the heart fire, Cinnamon and Oriental Green Tea distribute yang qi, circulate qi and blood, and warm the kidneys to restore deficiency fire, Kidney Yang steams the kidney water and nourishes the yin fluids of the five internal organs, and Pepper, Shanin, Fennel, and Zingiber officinale improve the circulation of qi and blood, warm and tonify the body, strengthen the spleen qi, and restore the transportation and transformation of nutrients.

[0040] The raw material for producing the herbal medicine composition according to the present invention contains licorice, and the number of parts by mass of the licorice is preferably 6 to 10 parts, more preferably 8 parts, based on the number of parts by mass of the Doujinshi.

[0041] In the present invention, licorice is used as a herbal medicine to harmonize various medicines.

[0042] This invention rationally selects raw materials and makes the various medicines compatible with each other, simultaneously treating the three organs of spleen, stomach and kidney, restoring qi, blood and body fluids, and providing the source of kidney essence transformation.

[0043] The present invention also preferably provides a method for preparing a herbal composition for improving testicular dysfunction, which comprises mixing the raw materials described in the above technical solutions and then extracting them.

[0044] In the present invention, the raw materials described in the technical solution are mixed and then extracted. In the present invention, the extraction method preferably includes water extraction.

[0045] In the present invention, the method of water extraction preferably includes mixing the mixture of the raw materials for producing the herbal medicine composition with water in a mass to volume ratio of 136-500g:400-1000mL, steeping for 30 minutes, boiling on high heat, then reducing to low heat and simmering for 40-60 minutes, filtering, and obtaining an extract, that is, the herbal medicine composition described in the present invention. The mass to volume ratio of the mixture and water described in the present invention is preferably 140-450g:450-900mL, more preferably 200-400g:500-800mL, even more preferably 250-350g:600-700mL, and most preferably 300g:650mL.

[0046] The present invention preferably also provides the use of the herbal composition according to the above technical solution in the manufacture of a product for improving testicular dysfunction.

[0047] In the present invention, the product preferably includes a medicine or food, and the testicular dysfunction preferably includes testicular dysfunction caused by diabetes, and more preferably includes testicular dysfunction caused by type 2 diabetes.

[0048] In mouse test, the herbal composition provided by the present invention can upregulate the expression of PI3K, p-Akt, Bcl-2 in mouse body, significantly increase superoxide dismutase, glutathione peroxidase, and downregulate malondialdehyde, effectively improve the oxidative stress level in male animal body, inhibit the apoptosis of reproductive cells caused by the increase of ROS level, and the interference of oxidative stress on spermatogenesis process, thereby increasing the testosterone level and improving semen parameters in obese type 2 diabetes mice.The herbal composition provided by the present invention can also significantly increase glucose intake, improve insulin sensitivity, and the effect is close to that of metformin, and has a certain improvement effect on type 2 diabetes.

[0049] The present invention also provides the use of the herbal composition according to the above technical solution in the manufacture of a product for improving testicular morphology and / or enhancing semen parameters.

[0050] The present invention also provides a drug for improving testicular dysfunction, comprising the herbal composition described in the above technical solution and pharma- ceutical acceptable additives.

[0051] In the present invention, the dosage form of the drug preferably includes any of aqueous preparations, granules, and freeze-dried preparations. In the present invention, there is no strict limitation on the type of the pharma- ceutically acceptable additives, and they may be generally selected according to the dosage form of the drug.

[0052] In order to further explain the present invention, the herbal medicine composition for improving testicular dysfunction provided by the present invention and its preparation method and use are described in detail below with reference to the drawings and examples, but they should not be construed as limitations on the scope of protection of the present invention.

[0053] Example 1 A method for producing a herbal medicine composition for improving testicular dysfunction, comprising the following steps: (1) Preparation of ingredients: 20g of Dojin, 30g of Astragalus, 20g of Dioscorea, 20g of Angelica, 10g of Atractylodes Root, 10g of Poria Cocos, 10g of Peony Root, 10g of Cnidium, 10g of Lotus Root, 10g of Celastrus orbiculatus, 10g of Celastrus Root, 5g of Shannen, 10g of Sophora Root, 20g of Fennel, 10g of Cinnamon, 10g of Euonymus Root, 10g of White Pepper, 10g of Ginger, 10g of Japanese Kokubushi, and 10g of Licorice.

[0054] (2) Place all the ingredients from step (1) in a clay pot, add 400 mL of water, soak for 30 minutes, bring to a boil over high heat, then reduce to low heat and simmer for 40 to 60 minutes. After filtering, about 200 mL of a herbal medicine composition was obtained.

[0055] (Usage example 1) 1.Material 1.1 Test animals Thirty 4-week-old SPF C57BL / 6J male mice, weighing 20±2 g, were provided by the Experimental Animal Center of Ningxia Medical University, with the Experimental Animal Qualification Certificate Number of 10752309202100163.

[0056] 1.2 Test conditions The test was conducted in the SPF laboratory of the Test Animal Center of Ningxia Medical University, with a temperature of 22-23°C, humidity of 50-60%, a 12-hour day-night rotation, free access to food and water, and a quiet environment. The laboratory environment complies with clean-level animal breeding conditions.

[0057] 1.3 Drugs The herbal medicine composition obtained in Example 1, high-fat feed (purchased from Research Diets, Inc., USA, catalog number D12492), and metformin hydrochloride tablets (China-Mexico Shanghai Shiguibao Pharmaceutical Co., Ltd., specification 0.5g).

[0058] 1.4 Main Reagents PI3K, AKT, p-AKT, and Bcl-2 antibodies were all purchased from Cell Signaling Technology, USA, and testosterone, superoxide dismutase, malondialdehyde, and glutathione peroxidase kits were all purchased from Elabsicience.

[0059] 2. Method 2.1 Animal grouping and model establishment The test animals were kept for one week until they adapted, and then randomly divided into a control group (Con), a model group (T2DM), a high-dose composition group (YPBS-H), a low-dose composition group (YPBS-L), and a metformin group (Metformin, Met), with six animals in each group. The six animals in the control group (Con) were fed a normal diet, and the remaining treated groups were fed a 60% high-fat diet. Body weights were recorded once every seven days during the period, and the results are shown in Figure 1A.

[0060] As can be seen from A of Figure 1, the mice in the model group, high-dose composition group, low-dose composition group and metformin group obviously gained weight and were significantly obese. During the test, the random blood glucose levels of the model mice exceeded 11.1 mmol / L, the blood glucose detected by OGTT and ITT were significantly higher than that of the control group, the sperm count, sperm motility and viable sperm rate were significantly lower than that of the control group, and the abnormal sperm rate was significantly higher than that of the control group, indicating that a type 2 diabetes mouse model with obesity was successfully created.

[0061] 2.2 Administration Method After the mice in each treatment group in step 2.1 were raised for 16 weeks, the models were successfully created, and the mice in each treatment group were administered treatments, specifically as follows: A control group (Con) receives physiological saline. The model group (T2DM) will be administered saline. The high dose composition group (YPBS-H) was administered the herbal composition obtained in Example 1 at a dose of 4 g / kg / day / rat. The low dose composition group (YPBS-L) was administered the herbal composition obtained in Example 1 at a dose of 2 g / kg / day / rat. Metformin group (Met) was administered at a dose of 200 mg / kg / day / rat.

[0062] During the administration period, the mice were fed with general feed and allowed to drink water normally. Changes in the mice's body weight were observed and analyzed using statistical software GraphPad Prism 8.4. The test results were expressed as ratios / percentages (%), and measurement data as mean values ​​± standard deviations. Intergroup comparisons were performed using one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference. The results are shown in Table 1 and Figure 1B. [Table 1] Note: Compared with the control group, * indicates P<0.01; compared with the model group, △ indicates P<0.01; compared with the metformin group, ▲ indicates P<0.05; the same applies below.

[0063] As can be seen from Table 1 and Figure 1B, when treated on the 16th week of model creation, the weight area under the curve of the mice in the model group, metformin group, composition high dose group and low dose group was significantly increased compared with the control group (P<0.05), but there was no significant difference between the model group, metformin group, composition high dose group and composition low dose group (P>0.05). Compared with the model group, the weight and area under the curve of the mice in the metformin group, composition high dose group and low dose group were significantly decreased, and the metformin group was lower than the composition high dose group and low dose group (P<0.05). On the 30th day of treatment, the body weight of the mice in the model group was significantly higher than that of the control group, and the body weights of the metformin group, the high-dose composition group and the low-dose composition group were significantly lower than those of the model group, and the body weights of the mice in the metformin group were lower than those of the low-dose composition group (P<0.05), but there was no statistically significant difference from the high-dose composition group (P>0.05). The herbal medicine composition provided by the present invention can improve the body weight of mice with testicular dysfunction due to type 2 diabetes accompanied by obesity.

[0064] 2.3 After administration of each treatment group in step 2.2 for 30 days, the following detections were performed. 2.3.1 Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) In the OGTT test, the mice were fasted for 15 hours, then weighed, and fasting plasma glucose (FPG) was measured. The amount of glucose used was then calculated from the mouse weight, and the changes in blood glucose 15, 30, 60, and 120 minutes after forced oral administration were detected and analyzed using statistical software GraphPad Prism 8.4. The counting data of the test results were recorded as ratio / percentage (%), and the measurement data were recorded as mean ± standard deviation. Intergroup comparisons were performed using one-way analysis of variance, and a statistically significant difference was indicated when P<0.05. The results are shown in Table 2 and Figure 2A.

[0065] In the ITT test, the mice were fasted for 5 hours, then weighed, and the amount of insulin injected was calculated. FPG was measured, and insulin was intraperitoneally injected. The changes in blood glucose 15 minutes, 30 minutes, 60 minutes, and 120 minutes after insulin injection were detected, and analyzed using statistical software GraphPad Prism 8.4. The counting data of the test results were recorded as ratio / percentage (%), and the measurement data were recorded as mean ± standard deviation. Inter-group comparison was performed using one-way analysis of variance, and a statistically significant difference was detected when P<0.05. The results are shown in Table 2 and Figure 2B. [Table 2]

[0066] As can be seen from Table 2 and Figure 2, in the oral glucose tolerance test, blood glucose was detected 15 minutes, 30 minutes, 60 minutes, and 120 minutes after FPG and forced oral administration, respectively, and the blood glucose level of the model group was significantly higher than that of the control group, the glucose intake of the high-dose composition group and the metformin group was significantly improved, and the OGTT-AUC of the mice in the model group was significantly increased (P<0.05), indicating the establishment of a diabetic state, and compared with the model group, the OGTT-AUC of the metformin group and the high-dose composition group was significantly decreased (P<0.05), indicating an increase in glucose intake. In the insulin resistance test, blood glucose was detected 15 minutes, 30 minutes, 60 minutes, and 120 minutes after FPG and insulin injection, respectively, and the results were similar, but the high-dose composition group was superior to the metformin group (P<0.05). The herbal medicine composition of the present invention can improve insulin sensitivity and increase glucose intake. Furthermore, compared with the model group, the fasting blood glucose levels of the mice in the high dose composition group and metformin group were significantly reduced (P<0.05).

[0067] 2.3.2 Detection of testicular morphology Testicular tissues from the mice were collected and stained with HE staining, and the results are shown in Figures 3-1 to 3-5.

[0068] As can be seen from Figures 3-1 to 3-5, the testicular tissue of the mice in the control group (Figure 3-1) had a clear structure, and the germ cells at each level in the seminiferous tubules were arranged in an orderly manner, and spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, etc. were observed in the basement membrane, and mature spermatozoa were observed in the cavity. The testicular tissue of the mice in the model group (Figure 3-2) had a disordered structure, and the germ cells at each level were arranged in a disordered manner and their numbers were significantly reduced, and large cavities appeared in the cavity. Compared with the model group, the testicular morphology of the mice in the metformin group (Figure 3-3), high-dose composition group (Figure 3-4), and low-dose composition group (Figure 3-5) was significantly improved, the number of germ cells at each level was significantly increased, and the luminal structure of the seminiferous tubules was significantly improved.

[0069] 2.3.3 Analysis of semen parameters Unilateral epididymis was collected from each treatment group, placed in a 12-well plate, diluted with 500μL of saline, thoroughly cut with surgical scissors, incubated in a 37℃ incubator, filtered through a 200 mesh filter, and 20μL of the filtrate was added to a cell counting plate. The sperm count was counted under a microscope, and the viable sperm rate, sperm motility, and abnormality rate were counted by eosin-nigrosin and Diff-Quick staining, respectively. The results were analyzed using the statistical software GraphPad Prism 8.4. The counting data of the test results were reported as ratio / percentage (%), and the measurement data were reported as mean ± standard deviation. Intergroup comparison was performed using one-way ANOVA, and a statistically significant difference was detected when P<0.05. The results are shown in Tables 3 and 4. [Table 3]

[0070] As can be seen from Table 3, compared with the control group, the testis index, epididymis index, and seminal vesicle index of the mice in the model group were significantly decreased (P<0.01), and compared with the model group, the testis index, epididymis index, and seminal vesicle index of the high-dose and low-dose composition groups were significantly increased (P<0.05). Regarding semen parameters, compared with the control group, the sperm count, viable sperm rate, and sperm motility of the mice in the model group were significantly decreased, and the abnormal sperm rate was significantly increased (P<0.01), and compared with the model group, this situation was significantly reversed in the high-dose composition group (P<0.05), but the sperm motility of the high-dose composition group was higher than that of the metformin group (P<0.05). The herbal medicine composition provided by the present invention can improve the sperm count of obese type 2 diabetic mice, significantly increase sperm count and vitality, reduce the rate of malformation, effectively improve reproductive dysfunction in male diabetic mice, and improve semen parameters, with the effect being particularly significant in the high-dose group.

[0071] 2.3.4 Detection of oxidative stress parameters and testosterone The levels of superoxide dismutase (T-SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) in serum were detected by colorimetric method, and the levels of testosterone (T) in serum were detected by ELISA method. All were performed according to the instructions, and the data was analyzed using statistical software GraphPad Prism 8.4. The counting data of the test results were recorded as ratio / percentage (%), and the measurement data were recorded as mean value ± standard deviation. Inter-group comparison was performed by one-way analysis of variance, and a statistically significant difference was indicated when P<0.05. The results are shown in Table 4. [Table 4]

[0072] PI3K / AKT plays an important role in regulating various biological processes, such as insulin signaling, apoptosis, proliferation, differentiation, and intermediary metabolism. PI3K / AKT is a classical insulin signaling pathway and is also an important target for the treatment of T2DM. In diabetes, impaired PI3K / AKT pathway is thought to be the main cause of insulin resistance due to increased reactive oxygen species (ROS) levels, and long-term hyperglycemia leads to the production of excessive ROS, which causes oxidative stress, promotes the death of germ cells, and interferes with the spermatogenesis process. Studies have revealed that oxidative stress is one of the most important pathogenic mechanisms of sperm dysfunction, and testosterone is crucial for spermatogenesis, and diabetes can reduce serum testosterone levels.

[0073] As can be seen from Table 4, compared with the control group, the serum superoxide dismutase, glutathione peroxidase levels of mice in the model group were significantly decreased, and the malondialdehyde level was significantly increased (P<0.01); compared with the model group, the serum superoxide dismutase, glutathione peroxidase levels of mice in the high-dose composition group were significantly increased, and the malondialdehyde level of mice in the high-dose composition group was significantly decreased (P<0.05). The herbal composition provided by the present invention can improve the serum superoxide dismutase and glutathione peroxidase content of mice with testicular dysfunction due to obesity-associated type 2 diabetes mellitus, reduce the content of malondialdehyde, and improve the oxidative stress level of mice with testicular dysfunction due to obesity-associated type 2 diabetes mellitus.

[0074] The testosterone levels of mice in the model group were lower than those in the control group, and the testosterone levels of the high-dose composition group were significantly higher than those in the model group (P<0.01) and superior to those in the metformin group (P<0.05). After treatment with the herbal medicine composition of the present invention, the testosterone levels of obese type 2 diabetes mice are increased.

[0075] 2.3.5 Detection of PI3K, p-AKT, and Bcl-2 expression in testis by Western Blot Add an appropriate amount of protein sample to the concentrated gel in the electrophoresis tank, electrophoresed, transferred, blocked with skim milk, washed the membrane, added primary antibody (PI3K (rabbit antibody), AKT (rabbit antibody), p-AKT (rabbit antibody), Bcl-2 (rabbit antibody), β-actin (β-actin) (mouse antibody) all diluted at 1:1000), kept in a refrigerator at 4°C overnight, washed the membrane the next day, blocked with secondary antibody (mouse secondary antibody (1:5000) and rabbit secondary antibody (1:2000)), washed the membrane, developed, analyzed the gray scale, analyzed the data using statistical software GraphPad Prism 8.4, recorded the counting data of the test results as ratio / percentage (%), and recorded the measurement data as mean ± standard deviation, performed one-way analysis of variance for intergroup comparison, P<0.05 was considered to be statistically significant, and the results are shown in Table 5 and Figure 4. [Table 5]

[0076] PI3K / AKT is involved in oligozoospermia, and PI3K is believed to be a key regulator in inducing sperm motility, hyperactivation, and acrosome reaction. PI3K / AKT is involved in multiple stages of male reproduction, including regulating the hypothalamic-pituitary-gonadal (HPG) axis during spermatogenesis, and regulating the proliferation and differentiation of spermatogonia and somatic cells. Inhibition of PI3K activity leads to an apoptotic cascade of sperm, characterized by a rapid loss of motility. AKT plays an important role in controlling cell survival and apoptosis, and is also involved in the regulation of spermatogenesis and sperm motility. Bcl-2, a downstream gene of AKT, regulates the cell cycle to promote cell proliferation, inhibits apoptosis, and maintains sperm motility.

[0077] As can be seen from Table 5 and Figure 4, compared with the control group, the expression of PI3K, p-Akt, Bcl-2 in the model group was significantly down-regulated (P<0.05), and after treatment with the herbal composition, the expression of PI3K, p-Akt, Bcl-2 was significantly up-regulated (P<0.05). The herbal composition provided by the present invention can improve the expression of PI3K, p-Akt, Bcl-2 genes and inhibit the apoptosis of germ cells in the case of testicular dysfunction caused by type 2 diabetes accompanied by obesity.

[0078] As can be seen from the above, the herbal medicine composition provided by the present invention can improve testicular morphology and semen parameters in mice and ameliorate testicular dysfunction by promoting glucose intake, improving insulin sensitivity, and promoting the expression of PI3K, p-Akt, and Bcl-2 genes.

[0079] Although the above embodiments describe the present invention in detail, they are only some embodiments of the present invention, not all of the embodiments. Based on these embodiments, other embodiments can be obtained without inventive step, and all of these embodiments fall within the protection scope of the present invention.

Claims

1. A herbal medicine composition for improving testicular dysfunction caused by type 2 diabetes accompanied by obesity, comprising: A herbal medicine composition characterized in that the raw materials, based on parts by mass, are 10-40 parts of Doujinshi, 15-100 parts of Astragalus Root, 10-40 parts of Dioscorea Root, 6-25 parts of Angelica Root, 6-40 parts of Atractylodes Root, 10-30 parts of Poria Root, 10-30 parts of Peony Root, 6-15 parts of Hokotsuka, 6-20 parts of Cnidium Root, 6-20 parts of Lens Root, 6-20 parts of Kenjitsu, 3-10 parts of Shanin, 3-10 parts of Sophora Root, 6-20 parts of Fennel, 3-15 parts of Cinnamon, 6-10 parts of Euonymus Root, 6-15 parts of Pepper Root, 6-15 parts of Zingiber Root, 6-15 parts of Corylus Officinalis, and 6-10 parts of Licorice.

2. The herbal medicine composition according to claim 1, characterized in that the raw materials for producing the herbal medicine composition, based on parts by mass, are 20 parts of Doujinshi, 30 parts of Astragalus Root, 20 parts of Dioscorea Rhizome, 20 parts of Angelica Root, 10 parts of Atractylodes Root, 10 parts of Poria Cocos, 10 parts of Peony Root, 10 parts of Cnidium Root, 10 parts of Lens Root, 10 parts of Celastrus Orbiculatus, 10 parts of Spiraea Root, 5 parts of Shanin, 10 parts of Sophora Root, 20 parts of Fennel, 10 parts of Cinnamon, 10 parts of Euonymus Salicornithum, 10 parts of White Pepper, 10 parts of Zingiber officinale, 10 parts of Corynebacterium Root, and 10 parts of Licorice.

3. A method for producing a herbal medicine composition for improving testicular dysfunction caused by type 2 diabetes accompanied by obesity, comprising mixing the raw materials described in claim 1 or 2 and then extracting the mixture.

4. The method of claim 3, wherein the extraction method comprises water extraction.

5. The method of water extraction comprises: The method according to claim 3, comprising mixing the mixture of the raw materials according to claim 1 or 2 with water in a mass to volume ratio of 136-500 g:400-1000 mL, steeping for 30 minutes, boiling on high heat, reducing heat to low heat and simmering for 40-60 minutes, and filtering.

6. Use of the herbal composition according to claim 1 or 2 or the herbal composition obtained by the method according to any one of claims 3 to 5 in the manufacture of a medicine for improving testicular dysfunction.

7. 7. The use according to claim 6, characterized in that the testicular dysfunction includes testicular dysfunction caused by diabetes.

8. 8. The use according to claim 7, characterized in that the testicular dysfunction caused by diabetes includes testicular dysfunction induced by type 2 diabetes associated with obesity.

9. Use of the herbal composition according to claim 1 or 2 or the herbal composition obtained by the manufacturing method according to any one of claims 3 to 5 in the manufacture of a medicine for improving testicular morphology and / or enhancing semen parameters.

10. A drug for improving testicular dysfunction, comprising the herbal composition according to claim 1 or 2 and a pharma- ceutical acceptable additive.

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