Application of plant extract composition in preparation of medicine for improving DNA damage of testicular spermatogenic cells

Abstract

The invention relates to the technical field of biological medicines, and discloses an application of a plant extract composition in preparation of a medicine for improving DNA damage of testicular spermatogenic cells, and the plant extract contains a herba epimedii extract and a rhizoma polygonati extract. The invention provides new application of the composition containing the herba epimedii extract and the rhizoma polygonati extract in preparation of the medicine for improving DNA damage of the testicular spermatogenic cells, the composition can remarkably improve the DNA damage of the testicular spermatogenic cells of mice caused by cyclophosphamide, recover the multiplication capacity of the mice, inhibit apoptosis of the mice and relieve spermatogenic function damage caused by cyclophosphamide, and the composition can be used for preparing the medicine for improving the DNA damage of the testicular spermatogenic cells of the mice. And the combined administration shows a synergistic effect instead of a simple additive effect, and the drug effect is obviously superior to that of any one of the epimedium extract and the rhizoma polygonati extract which are independently administered.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicines, and particularly relates to an application of a plant extract composition in preparation of a medicine for improving DNA damage of testicular spermatogenic cells. BACKGROUND

[0002] Normal spermatogenesis of men depends on normal development and maturation of spermatogenic cells at all levels in testicular seminiferous tubules, which originate from spermatogonial stem cells at the base of seminiferous tubules, and finally form sperm through differentiation into primary spermatocytes, secondary spermatocytes and spermatids. The normal spermatogenesis of men is maintained through precise regulation and mutual cooperation among spermatogenic cells at all levels. Recent studies have shown that DNA damage of spermatogenic cells is an important cause of spermatogenic dysfunction. Various factors such as ionizing radiation, environmental toxins, chemical drugs, chronic inflammation and oxidative stress can cause DNA base damage, double-strand breakage and chromatin remodeling of spermatogenic cells, and finally lead to reduction in sperm count and decrease in sperm motility, and spermatogenic dysfunction. Therefore, finding a medicine that can effectively improve DNA damage of spermatogenic cells is one of the key ways to improve spermatogenic dysfunction of men.

[0003] According to traditional Chinese medicine, deficiency of kidney yang (also known as kidney yang deficiency) is one of the common types of male infertility. Deficiency of kidney yang and decline of the fire of the gate of life cannot warm the sperm chamber, which leads to reduction in sperm quality. Therefore, kidney tonification and yang tonification are needed to warm the gate of life and nourish the sperm chamber. Epimedium is one of the most common tonics for kidney yang. However, simple and single yang tonification can cause hyperactivity of fire due to yin deficiency and imbalance between yin and yang. Therefore, kidney yin should be nourished while tonifying the kidney and yang. It is said that “a good tonic for yang must seek yang in yin, and then yang will be assisted by yin and be endless”. “Shizi Erxian Decoction” uses Epimedium and Herba Cynomorii as monarch drugs to warm and tonify the kidney yang, uses Cusparia as minister drugs to assist the monarch drugs, and uses six medicinal materials such as Fructus Lycii, Fructus Ligustri Lucidi, Rubus chingii Hu, Fructus Schisandrae Chinensis, Semen Plantaginis and Semen Vaccini to nourish kidney yin, benefit essence and marrow. This prescription effectively improves the sperm quality of patients with oligospermia due to deficiency of kidney yang by complementing yin and yang. “Tonic for Kidney and Blood-Activating Prescription” uses Epimedium, Semen Cannabis, Eucommia ulmoides Oliv. to tonify the kidney and benefit essence, and uses Radix Rehmanniae Preparata, Radix Angelicae Sinensis, Radix Codonopsis, etc. to nourish yin and benefit qi, so that essence is full and blood is unobstructed, which can effectively improve the sperm quality of patients with male infertility due to deficiency of the kidney and blood stasis. It can be seen that when Epimedium is used to treat deficiency of kidney yang in clinical practice of traditional Chinese medicine, it is particularly important to combine other medicinal materials to nourish kidney yin according to syndrome differentiation, which may have a better protective effect on spermatogenic function of men.

[0004] However, the existing medicines containing Epimedium for improving male reproductive function have complex components and mechanisms and high cost. At present, there is no report on the protective effect of a plant extract composition containing Epimedium extract and Rhizoma Polygonati extract on DNA damage of testicular spermatogenic cells. SUMMARY

[0005] The application aims to overcome the problems in the prior art and provide an application of a plant extract composition in the preparation of a medicine for improving DNA damage of testicular spermatogenic cells, which can significantly improve DNA damage of testicular spermatogenic cells in mice.

[0006] To achieve the above-mentioned purpose, the application provides an application of a plant extract composition in the preparation of a medicine for improving DNA damage of testicular spermatogenic cells, wherein the plant extract contains an Epimedium extract and a Polygonatum extract.

[0007] Preferably, the plant extract is a combination of the Epimedium extract and the Polygonatum extract.

[0008] Preferably, in the medicine, the Epimedium extract and the Polygonatum extract are both used in the form of granules, and the mass ratio of the use amount of the Epimedium granules to the Polygonatum granules is 1:7-10.

[0009] Preferably, the dose of the medicine is 5-5.6 g / kg / day, wherein the dose is calculated based on the total weight of the Epimedium granules and the Polygonatum granules.

[0010] Preferably, the Polygonatum extract is prepared by the following procedure: A1, mixing and soaking Polygonatum slices with water; A2, heating the mixed system obtained in step A1 in a water bath at 75-85 DEG C, condensing and refluxing extraction for 85-95 min, repeating the extraction for 2-3 times, collecting and combining the extract; A3, centrifuging, suction filtering the extract obtained in step A2, collecting the filtrate, and then performing vacuum concentration and freeze-drying.

[0011] Preferably, in step A1, the use amount ratio of the Polygonatum slices to water is 1 g:13-17 mL.

[0012] Preferably, in step A1, the soaking time is 25-35 min.

[0013] Preferably, in step A3, the centrifugation conditions include a rotation speed of 2800-3200 r / min and a time of 8-12 min.

[0014] Preferably, the Polygonatum extract is used in the form of granules.

[0015] Preferably, in 1 g of the Polygonatum granules, there are 75-80 mg of fructose.

[0016] Preferably, the Epimedium extract is prepared by the following procedure: B1, crushing Epimedium slices, and then mixing and soaking with 65-75% ethanol; B2, the soaking system obtained in step B1 is heated by water bath at 95-100 DEG C, and extracted by condensation reflux for 55-65 min, and the extraction is repeated for 1-2 times, and the extraction is collected and combined; B3, the extraction obtained in step B2 is centrifuged and filtered, and the filtrate is collected, and then concentrated under reduced pressure and freeze-dried.

[0017] Preferably, in step B1, the dosage ratio of the Epimedium slices to ethanol is 1g:8-12mL.

[0018] Preferably, in step B1, the soaking time is 25-35 min.

[0019] Preferably, in step B3, the centrifugation conditions include a rotation speed of 2800-3200 r / min and a time of 8-12 min.

[0020] Preferably, the Epimedium extract is used in the form of granules.

[0021] Preferably, in 1g of the Epimedium granules, there are 60-70 mg of total contents of Epimedoside A, Epimedoside B, Epimedoside C and Epimedium glycoside.

[0022] Preferably, the application of the plant extract composition in the preparation of the medicine for improving the DNA damage of the testicular spermatogenic cells induced by cyclophosphamide.

[0023] The application provides a new use of a composition containing Epimedium extract+Eleocharis tuberosa extract in the preparation of a medicine for improving the DNA damage of testicular spermatogenic cells, the composition can significantly improve the DNA damage of the testicular spermatogenic cells of mice caused by cyclophosphamide, restore the proliferation capacity of the testicular spermatogenic cells and inhibit the apoptosis of the testicular spermatogenic cells, relieve the damage of spermatogenic function caused by cyclophosphamide, and the combined administration shows a synergistic effect rather than a simple additive effect, and the drug efficacy is significantly better than that of either of the Epimedium extract and the Eleocharis tuberosa extract alone. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a result graph of the influence of each experimental group on the body weight, sperm quantity, sperm motility and sex organ weight of the mice; Figure 2 is a result graph of the influence of each experimental group on the testicular tissue morphology of the mice; Figure 3 is a result graph of the influence of each experimental group on the expression and location of the γH2AX protein in the testicular tissue of the mice; Figure 4 is a result graph of the influence of each experimental group on the expression and location of the PCNA protein in the testicular tissue of the mice; Figure 5 is a result graph of the influence of each experimental group on the expression and location of the Ki67 protein in the testicular tissue of the mice; Figure 6 is the result graph of the influence of each experimental group on the apoptosis of spermatogenic cells in the testis of mice. DETAILED DESCRIPTION

[0025] The specific embodiments of the present application are described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and explanatory and are not intended to limit the present application.

[0026] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges and the values are approximate values and are understood to be encompassed by the range or value they approximate. For ranges, the endpoints are included within the range unless specifically stated otherwise. For numerical values, the endpoints are included within the range unless specifically stated otherwise. For numerical ranges, the endpoints are included within the range unless specifically stated otherwise. The ranges and values are understood to encompass the endpoints.

[0027] The present application provides a use of a plant extract composition in the preparation of a drug for improving DNA damage of spermatogenic cells in testis, wherein the plant extract contains Epimedium extract and Polygonatum extract.

[0028] In a preferred embodiment, the plant extract is a combination of Epimedium extract and Polygonatum extract, and the combination of Epimedium + Polygonatum can significantly improve DNA damage of spermatogenic cells in testis, with low cost and simple operation.

[0029] In a preferred embodiment, in the drug, the Epimedium extract and the Polygonatum extract are both in the form of granules, and the mass ratio of the use amount of the Epimedium granules to the Polygonatum granules is 1:7-10. Under the above use amount relationship, the synergistic effect of the two can be better exerted, and the improvement of the combination on DNA damage of spermatogenic cells in testis is more significant.

[0030] In a preferred embodiment, the dose of the drug is 5-5.6 g / kg / day, wherein the drug is calculated based on the total weight of the Epimedium granules and the Polygonatum granules. Specifically, the dose of 5-5.6 g / kg / day means that each mouse needs to eat 5-5.6 g of the drug per day per 1 kg of body weight, wherein the drug is calculated based on the total weight of the Epimedium granules and the Polygonatum granules.

[0031] In a specific embodiment, the dose of the drug is: Epimedium granules 0.5-0.7 g / kg / day + Polygonatum granules 4.5-4.9 g / kg / day. That is, each mouse needs to eat 0.5-0.7 g of Epimedium granules and 4.5-4.9 g of Polygonatum granules per day per 1 kg of body weight.

[0032] In order to more fully extract the effective components in the Rhizoma Polygonati, while the operation is simple, in the preferred embodiment, the Rhizoma Polygonati extract is prepared according to the following procedure: A1, the Rhizoma Polygonati decoction pieces are mixed with water and soaked; A2, the mixed system obtained in step A1 is heated in a water bath at 75-85°C, and extracted by condensation reflux for 85-95 min, and the extraction is repeated for 2-3 times, and the extraction liquid is collected and combined; A3, the extraction liquid obtained in step A2 is centrifuged and filtered, the filtrate is collected, and then reduced pressure concentration and freeze-drying are performed.

[0033] In the preferred embodiment, in step A1, the ratio of the amount of Rhizoma Polygonati decoction pieces to water is 1g:13-17mL. Under the above solid-liquid ratio, not only can the dissolution of polysaccharides be high and the extraction rate be increased, but also the burden of subsequent concentration can be reduced and the efficiency of the process can be improved.

[0034] In the preparation process of the Rhizoma Polygonati extract, by the pretreatment of soaking in step A1, the extraction liquid can be gently immersed into the medicinal material, thereby improving the extraction rate. In the preferred embodiment, in step A1, the soaking time is 25-35 min, and specifically, for example, it can be 25 min, 26 min, 27 min, 28 min, 30 min, 32 min, 34 min or 35 min.

[0035] In the preparation process of the Rhizoma Polygonati extract, in step A2, the extraction is performed by heating in a water bath at 75-85°C and condensation reflux. Rhizoma Polygonati is rich in polysaccharides, and by extracting at 75-85°C, the extraction efficiency can be ensured, the destruction of the glycosidic bond in the main chain and side chain of polysaccharides can be avoided, and the normal molecular structure and biological activity of polysaccharides can be maintained.

[0036] In the preparation process of the Rhizoma Polygonati extract, in step A2, the number of repeated extractions is 2-3 times. In this way, the polysaccharides and other effective components in Rhizoma Polygonati can be effectively extracted, the cost can be saved, the burden of concentration can be reduced, and more impurities can be avoided.

[0037] In the preparation process of the Rhizoma Polygonati extract, in step A3, by centrifuging and filtering the extraction liquid, insoluble impurities in the extraction liquid can be efficiently removed. In specific implementation, step A3 includes: centrifuging the extraction liquid obtained in step A2, collecting the supernatant, filtering by a Buchner funnel under reduced pressure, collecting the filtrate, and then performing reduced pressure concentration and freeze-drying.

[0038] In the preferred embodiment, in step A3, the centrifugation conditions include a rotation speed of 2800-3200 r / min and a time of 8-12 min.

[0039] In the preparation process of the Rhizoma Polygonati extract, in step A3, the filtrate is concentrated under reduced pressure. Compared with the traditional boiling concentration, the reduced pressure concentration has the advantages of low temperature, rapidness and safety, which is extremely beneficial to the heat-sensitive components and can protect the bioactivity and chemical structure of the effective components.

[0040] In the preparation process of the Rhizoma Polygonati extract, in step A3, the freeze-drying method is used, which is carried out at low temperature and can avoid the damage to the activity of the effective components. The freeze-dried product is loose and porous, and the drying effect is much higher than that of the traditional drying, which is beneficial to the long-term preservation of the sample.

[0041] In the application of the present application, the use form of the Rhizoma Polygonati extract is not limited. In order to facilitate storage and use, in the preferred embodiment, the Rhizoma Polygonati extract is used in the form of granules. Specifically, the Rhizoma Polygonati extract is granulated according to the conventional wet granulation method in the art to obtain Rhizoma Polygonati granules.

[0042] In some embodiments, the Rhizoma Polygonati granules are prepared according to the following process: the Rhizoma Polygonati extract is stirred with malt dextrin, 90% ethanol is sprayed during the stirring process, and then the mixture is ground, sieved and dried.

[0043] In the preferred embodiment, the mass ratio of the Rhizoma Polygonati extract to malt dextrin is 100:110-130.

[0044] In the specific embodiment, the Rhizoma Polygonati extract is slowly and batchwise mixed with malt dextrin.

[0045] In the specific embodiment, the 90% ethanol is sprayed in multiple times and small amounts.

[0046] In the specific embodiment, the sieving is sieving through a 10-20 mesh sieve.

[0047] In the specific embodiment, the drying conditions include a temperature of 55-65°C and a time of 0.5-1.5h.

[0048] Further preferably, 75-80mg of fructose is contained in every 1g of Rhizoma Polygonati granules.

[0049] In some embodiments, 1-2g of Rhizoma Polygonati decoction pieces are equivalent to 1g of Rhizoma Polygonati granules. That is, 1-2g of Rhizoma Polygonati decoction pieces are used to prepare 1g of Rhizoma Polygonati granules.

[0050] In the preferred embodiment, the Epimedium extract is prepared according to the following process: B1, the Epimedium decoction pieces are crushed, then mixed with 65-75% ethanol and soaked; B2, the soaking system obtained in step B1 is heated by a 95-100℃ water bath, and extracted by condensation reflux for 55-65 min, and the extraction is repeated for 1-2 times, and the extraction liquid is collected and combined; B3, the extraction liquid obtained in step B2 is centrifuged and filtered, and the filtrate is collected, and then subjected to vacuum concentration and freeze-drying.

[0051] For the main effective components in Epimedium, such as icariin, hypoiicariin and total flavonoids of Epimedium, pure water is not conducive to the extraction of flavonoids, and more than 90% high-concentration ethanol is not conducive to the extraction of glycosides containing hydrophilic sugar groups. In the present application, 65-75% ethanol is used to extract Epimedium, which can more effectively extract the effective components in Epimedium. In this paper, "65-75%" in 65-75% ethanol refers to the volume fraction.

[0052] In the preferred embodiment, in step B1, the ratio of the use amount of Epimedium decoction pieces to ethanol is 1g:8-12mL, which can better dissolve the effective components in Epimedium and has a lower subsequent concentration burden under the above solid-liquid ratio condition.

[0053] In the preferred embodiment, in step B1, the soaking time is 25-35 min.

[0054] In the preparation process of the Epimedium extract, in step B2, 95-100℃ water bath heating and condensation reflux extraction are used, and the effective components such as icariin have good thermal stability, and the extraction rate is high under the above extraction temperature.

[0055] In the preparation process of the Epimedium extract, in step B3, the filtrate is subjected to vacuum concentration and freeze-drying, and the drying effect of vacuum concentration and freeze-drying is much higher than that of drying and spray drying. The water content of the sample after freeze-drying is between 1-3%, and low-temperature drying is better for the protection of the biological activity of the extract and is beneficial to long-term preservation.

[0056] In the preferred embodiment, in step B3, the centrifugation conditions include a rotation speed of 2800-3200 r / min and a time of 8-12 min.

[0057] In the application of the present application, the use form of the Epimedium extract is not limited. In order to facilitate storage and use, in the preferred embodiment, the Epimedium extract is used in the form of granules. Specifically, the Epimedium extract is prepared into granules according to the conventional wet granulation method in the art to obtain Epimedium granules.

[0058] In some embodiments, the Epimedium granules are prepared by the following procedure: mixing Epimedium extract with starch dextrin, spraying 70% ethanol during the mixing process, grinding uniformly, then sieving and drying. Preferably, the mass ratio of the use amount of the Epimedium extract to the starch dextrin is 100:85~95.

[0059] In preferred embodiments, 60~70 mg of total of the contents of Epimedin A, Epimedin B, Epimedin C and Icaritin are contained in 1 g of the Epimedium granules.

[0060] In some embodiments, 1 g of the Epimedium granules is equivalent to 4~6 g of Epimedium decoction pieces. That is, 1 g of the Epimedium granules is prepared from 4~6 g of Epimedium decoction pieces.

[0061] Cyclophosphamide (CTX) is a biological alkylating agent, which is metabolically activated in the liver by cytochrome P450 enzymes to generate active metabolites such as phosphoramide mustard and acrolein. These metabolites can cause DNA molecule breakage in cells. In experimental studies, CTX is often used to induce DNA damage in spermatogenic cells of testis. In the application described in the present application, specifically, the application of the plant extract composition in the preparation of a medicine for improving CTX-induced DNA damage in spermatogenic cells of testis.

[0062] The present application will be described in detail below by way of examples, but the scope of protection of the present application is not limited thereto. In the following examples, the experimental methods are all conventional methods in the art unless otherwise specified. The experimental materials used in the following examples are all commercially available products unless otherwise specified.

[0063] Example 1 Preparation of total extract of Rhizoma Polygonati: Take 300 g of Rhizoma Polygonati decoction pieces, crush, add 4500 mL of distilled water, soak in a 10000 mL round-bottom flask for 30 min, then heat and reflux extract under water bath at 80 ℃ for 90 min, filter with gauze, collect the filtrate, repeat the extraction of the residue twice in the same way; after the end, combine the three filtrates, centrifuge at 3000 r / min for 10 min, collect the supernatant, filter under reduced pressure with a Buchner funnel, collect the filtrate, reduce pressure and concentrate, freeze-dry to obtain 106.2 g of total extract of Rhizoma Polygonati, with an extraction rate of 35.4%.

[0064] Wet granulation: take 100 g of dried total extract of Rhizoma Polygonati, slowly and in batches mix with 120 g of malt dextrin in a mortar to stir uniformly, spray 90% ethanol in small amounts in multiple times, grind uniformly until "hand kneading into a ball, light pressing and scattering", pass through a 16-mesh sieve, and dry at 60 ℃ for 1 h to obtain Rhizoma Polygonati granules.

[0065] It is calculated that 1 g of Rhizoma Polygonati granules is equivalent to 1.3 g of Rhizoma Polygonati decoction pieces.

[0066] The test showed that the characteristic spectrum of the product met the requirements, and each 1 g of Huangjing granules contained 78 mg of fructose.

[0067] Example 2 Preparation of total extract of Herba Epimedii: Take 300 g of Herba Epimedii decoction pieces, crush, add 3000 mL of 70% ethanol, soak in a 5000 mL round-bottom flask for 30 min, then heat and condense by refluxing at 100 ℃ for 60 min, filter with gauze, and collect the filtrate. The filter residue is repeatedly extracted once in the same way. After the end, centrifuge the combined filtrate at 3000 r / min for 10 min, collect the supernatant, filter under reduced pressure with a Buchner funnel, collect the filtrate, reduce pressure and concentrate, and freeze-dry to obtain 32.4 g of total extract of Herba Epimedii, with an extraction rate of 10.8%.

[0068] Wet granulation: take 100 g of dried Herba Epimedii extract, grind thoroughly with 90 g of starch dextrin in a mortar in multiple times, spray 70% ethanol in multiple times and small amounts, grind uniformly, until the soft material "can be kneaded into a ball and scattered by light pressing", pass through a 16-mesh sieve, and dry at 60 ℃ for 1 h to obtain Herba Epimedii granules.

[0069] According to the calculation, each 1 g of Herba Epimedii granules is equivalent to 5 g of Herba Epimedii decoction pieces.

[0070] The test showed that the characteristic spectrum of the product met the requirements, and each 1 g of Herba Epimedii granules contained total flavonoids 205 mg (calculated as icariin), and a total of 64 mg of chomilaidin A, chomilaidin B, chomilaidin C and icariin per 1 g of granules.

[0071] Test Example 1. Experimental materials 1.1. Experimental animals 65 8-week-old specific pathogen free (SPF) level BALB / c male mice were purchased and raised in the Experimental Animal Center of Three Gorges University, with experimental animal production license number SCXK (E) 2022-0012 and experimental animal quality certificate number No. 42010200010107. During the feeding process, the animals were allowed to eat and drink freely, the feeding temperature was 20-25 ℃, the relative humidity was 55%-60%, and the lighting system was automatically controlled for 12 h of light and dark alternation. The animal experiment was approved by the Experimental Animal Welfare and Ethics Review Committee of Three Gorges University with approval number 2024031J1, and the experimental unit used license number SYXK (E) 2022-0061.

[0072] 1.2. Experimental drugs Cyclophosphamide for injection (Guojingzhengzi H32020857, product batch number 23122225) was purchased from Jiangsu Hengrui Medicine Co., Ltd. The specification was 0.2 g (calculated as C7H 15 Cl2N2O2P). After dissolving cyclophosphamide for injection with a small amount of 0.9% NaCl solution, it was diluted to 40 mL. The concentration of the cyclophosphamide solution was 5 mg / mL, and it was stored in a 4 ℃ refrigerator in the dark.

[0073] Huangjing granules were prepared in Example 1; Yiyangyao granules were prepared in Example 2.

[0074] After preliminary pre-experiment, the average weight of mice was 25 g, and the average daily free feeding of each mouse was 3 g of feed.

[0075] According to the provisions of the Chinese Pharmacopoeia 2020 edition, combined with the drug dose conversion coefficient of adults and mice, the dose of Yiyangyao decoction pieces for mice was determined to be 1 g / kg / day, which was the low dose of Yiyangyao decoction pieces. At the same time, 3 g / kg / day was defined as the high dose of Yiyangyao decoction pieces. Therefore, in the feed of the Yiyangyao low-dose group, 0.025 g of Yiyangyao decoction pieces should be contained in every 3 g of feed; in the feed of the Yiyangyao high-dose group, 0.075 g of Yiyangyao decoction pieces should be contained in every 3 g of feed. Since 1 g of Yiyangyao granules is equivalent to 5 g of Yiyangyao decoction pieces, it is equivalent to that the drug-containing feed of Yiyangyao low-dose contains 16.67 g of Yiyangyao granules per 10 kg of feed; the drug-containing feed of Yiyangyao high-dose contains 50 g of Yiyangyao granules per 10 kg of feed.

[0076] Similarly, after conversion, the dose of Huangjing decoction pieces for mice was determined to be 2 g / kg / day, which was the low dose of Huangjing decoction pieces. At the same time, 6 g / kg / day was defined as the high dose of Huangjing decoction pieces. Therefore, in the feed of the Huangjing low-dose group, 0.05 g of Huangjing decoction pieces should be contained in every 3 g of feed; in the feed of the Huangjing high-dose group, 0.15 g of Huangjing decoction pieces should be contained in every 3 g of feed. Since 1 g of Huangjing granules is equivalent to 1.3 g of Huangjing decoction pieces, it is equivalent to that the drug-containing feed of Huangjing low-dose contains 128.21 g of Huangjing granules per 10 kg of feed; the drug-containing feed of Huangjing high-dose contains 384.62 g of Huangjing granules per 10 kg of feed.

[0077] Huangjing granules and Yiyangyao granules were made into drug-containing feed by Wuhan Wanqianjiaxing Biological Technology Co., Ltd. The experimental animal feed production license number was SCXK (E) 2021-0011, and the execution standard was GB 14924.3-2010. The formula was as follows: Yiyangyao drug-containing feed: 0.050 kg Yiyangyao granules + 9.950 kg mouse maintenance feed; Huangqi-containing feed: 0.385 kg of Huangqi granules + 9.615 kg of mouse maintenance feed; Low-dose Epimedium + Huangqi-containing feed: 0.017 kg of Epimedium granules + 0.128 kg of Huangqi granules + 9.855 kg of mouse maintenance feed; High-dose Epimedium + Huangqi-containing feed: 0.050 kg of Epimedium granules + 0.385 kg of Huangqi granules + 9.565 kg of mouse maintenance feed.

[0078] 1.3. Experimental reagents Phospho-Histone H2AX-S139 Rabbit mAb (Cat No: AP0687) was purchased from Wuhan Abmole Biotechnology Co., Ltd; PCNA Recombinant Rabbit mAb (Cat No: S0B2253), Anti-Ki67 antibody (Cat No: ab16667), Alexa Fluor 594-conjugated AffiniPure Donkey Anti-Rabbit IgG (H+L) (Cat No: 711-585-152) were purchased from Shanghai Youningwei Biotechnology Co., Ltd; YF594 TUNEL Apoptosis Kit (Cat No: T6014) was purchased from Suzhou Youyilan Di Biological Technology Co., Ltd; Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (Cat No: KR0011) was purchased from Wuhan Kerui Biological Technology Co., Ltd; DAB Staining Solution (20x) (Cat No: DAB-0031) was purchased from Fuzhou Mayxin Biological Technology Development Co., Ltd; Hematoxylin Staining Solution (Cat No: G1004), Hematoxylin Differentiation Solution (Cat No: G1039), Hematoxylin Blue Solution (Cat No: G1040), Testicular Fixative (Cat No: G1121), Anti-Fluorescence Quenching Mounting Medium (Cat No: G1401) were purchased from Wuhan Saivier Biological Technology Co., Ltd; Urea (Cat No: 30191228), Neutral Gum (Cat No: 10004160), Xylene (Cat No: 10023418), Anhydrous Ethanol (Cat No: 10009218), 95% Ethanol (Cat No: 10009164), Trisodium Citrate Dihydrate (Cat No: 10019418) were purchased from China National Pharmaceutical Group Chemical Reagent Co., Ltd; Tween 20 (Cat No: MB2483) was purchased from Dalian Melin Biological Technology Co., Ltd; Tris-Hydroxymethyl Amino Methane (Cat No: T8060), Ethylenediaminetetraacetic Acid (Cat No: E8040), Eosin Y (Cat No: E8090), Triton X-100 (Cat No: T8200) were purchased from Beijing Solaybao Technology Co., Ltd; DAPI (Cat No: C1002), Bovine Serum Albumin (Cat No: ST023) were purchased from Shanghai Biyun Tian Biological Technology Co., Ltd.

[0079] 1.4. Experimental instruments The TP1020 automatic table type tissue dehydrator and the HistoCore Arcadia H type paraffin embedding machine were purchased from Leica Microsystems (Shanghai) Trading Co., Ltd.; the DP260 type automatic HE staining machine was purchased from Shenzhen Dako Medical Technology Co., Ltd.; the YGQ-3128D type paraffin section machine was purchased from Xiaogan Yaguang Medical Electronic Technology Co., Ltd.; the YD-AB type biological tissue baking and slicing machine was purchased from Jinhua Yide Medical Equipment Co., Ltd.; the IRX60 type automatic inverted fluorescence microscope was purchased from Ningbo Shunyu Instrument Co., Ltd.; the SY-BJ10L type ultrapure water machine was purchased from Chengdu Shenyuan Technology Co., Ltd.; and the EasyScan type digital section scanning and application system was purchased from the Macoody Industrial Group Co., Ltd.

[0080] 2. Experimental methods and experimental results 2.1. Animal grouping and data processing (1) Animal grouping and treatment After 65 SPF level 8-week-old BALB / c male mice were adaptively fed in the Experimental Animal Center of Three Gorges University for 1 week, they were randomly divided into a normal control group (Normal), a model group (Model), an Epimedium group (EB, 0.6 g / kg / day of Epimedium granules), a Polygonatum group (PC, 4.6 g / kg / day of Polygonatum granules), a low-dose Epimedium + Polygonatum composition group (EBL+PCL, the drug dosage was 1.7 g / kg / day, specifically: 0.2 g / kg / day of Epimedium granules + 1.5 g / kg / day of Polygonatum granules), and a high-dose Epimedium + Polygonatum composition group (EBH+PCH, the drug dosage was 5.2 g / kg / day, specifically: 0.6 g / kg / day of Epimedium granules + 4.6 g / kg / day of Polygonatum granules). Taking 0.6 g / kg / day of Epimedium granules as an example, it means that each mouse needs to eat 0.6 g of Epimedium granules per day per 1 kg of body weight.

[0081] Every 2 days, the Model group, the EB group, the PC group, the EBL+PCL group, and the EBH+PCH group of mice were intraperitoneally injected with a cyclophosphamide solution (concentration of 5 mg / mL, the average body weight of mice was 25 g, and each mouse was injected with 0.25 mL) at a dose of 50 mg / kg. The Normal group was intraperitoneally injected with 0.25 mL of a 0.9% NaCl solution at the same time. This lasted for 30 days.

[0082] During the modeling period, the Normal group and the Model group of mice freely ate mouse maintenance feed every day, the EB group of mice freely ate Epimedium medicated feed every day, the PC group of mice freely ate Polygonatum medicated feed every day, the EBL+PCL group of mice freely ate low-dose Epimedium + Polygonatum composition medicated feed every day, and the EBH+PCH group of mice freely ate high-dose Epimedium + Polygonatum composition medicated feed every day. This lasted for 30 days.

[0083] (2) Data analysis and statistics Statistical analysis of experimental data was performed using GraphPad Prism 8, and the data were expressed as mean ± standard error (SEM). One-way ANOVA was used to determine the differences between groups, and Tukey's test was used for multiple comparisons. P < 0.05 was considered statistically significant. The Q value between combined drugs and single drugs was calculated using the Kim Jung-geun Q value method. Q > 1.15 indicates that the drug combination has a synergistic effect, 0.85 ≤ Q ≤ 1.15 indicates that the drug combination has an additive effect, and Q < 0.85 indicates that the drug combination has an antagonistic effect.

[0084] 2.2, Effects of each group on body weight, sperm count, sperm motility, and reproductive organ weight in mice Test method: (1) Animal sampling and reproductive organ weighing After modeling, all mice were fasted but not watered for 12 h. After weighing, each mouse was intraperitoneally injected with 0.1 mL of 10% urea solution at a concentration of 10 g of mouse body weight. The mice were anesthetized, and blood was taken from the eyeball. The mice were sacrificed by cervical dislocation, and the testes, epididymides, and seminal vesicles were quickly isolated. The reproductive organs were weighed, and the organ index was calculated. Organ index = organ weight / body weight × 100%.

[0085] (2) Sperm quality analysis Both epididymides were immediately placed into an ep tube containing 1 mL of 0.9% NaCl solution. The tissue was cut and crushed, and the sperm suspension was obtained by incubating in a 37°C incubator for 5 min. 10 μL of sperm suspension was mixed with 10 μL of eosin solution, and 10 μL of the stained solution was dropped onto a glass slide. The number of live and dead sperm was counted under a microscope, and the sperm motility was calculated. 20 μL of sperm suspension was mixed with 480 μL of 0.9% NaCl solution, and the sperm count was calculated using a cell counting plate. Sperm count (per mouse) = average count of four quadrants × 10 4 × 25.

[0086] The test results are shown in Figure 1 Figure A: mouse body weight; B: sperm count; C: sperm motility; D: testis weight; E: testis index; F: epididymis weight; G: epididymis index; H: seminal vesicle weight; I: seminal vesicle index. Compared with the Normal group, ** P <0.01, *** P <0.001; compared with the Model group,# P <0.05, ## P <0.01, ### P <0.001.

[0087] The results of Figure 1A show that the body weight of mice in the model group was significantly decreased (P<0.001) compared with the control group. The body weight of mice in the Epimedium group (P<0.01) and the Rhizoma Polygonati group (P<0.01) was significantly increased compared with the model group. Similarly, the body weight of mice in the combination drug group was increased in a dose-dependent manner compared with the model group, and the body weight of mice in the high-dose combination drug group was significantly increased (P<0.001).

[0088] Figure 1 The results of Figures 1B and 1C show that the sperm count (P<0.001) and viability (P<0.001) of the model group were significantly decreased compared with the control group. The sperm count (P<0.05) and viability (P<0.001) of the Rhizoma Polygonati group were significantly increased compared with the model group. Similarly, the sperm count and viability were increased in a dose-dependent manner compared with the model group, and the sperm count (P<0.01) and viability (P<0.001) of the high-dose combination drug group were significantly increased. In addition, the sperm count (P<0.01) and viability (P<0.01) of the high-dose combination drug group were higher than those of the Epimedium group. The Q value of the sperm viability of the combination drug group compared with the Epimedium and Rhizoma Polygonati groups was 1.23, and the Q value of the sperm count was 1.24, indicating that the combination drug had a synergistic effect on improving sperm quality.

[0089] The results of Figures 1D, 1E, 1H, and 1I show that the testis weight (P<0.001), testis index (P<0.001), seminal vesicle weight (P<0.01), and seminal vesicle index (P<0.001) of the model group were significantly decreased compared with the control group. The testis weight of the high-dose combination drug group was significantly increased (P<0.05) compared with the model group.

[0090] 2.3, Effect of each group on the morphology of testicular tissue of mice HE staining was used to observe the morphology of testicular tissue. After the testicular tissue was fixed with testicular fixative, it was dehydrated in an automatic dehydration machine. After completion, it was embedded with paraffin. The 4 μm paraffin sections prepared by the pathological paraffin section machine were spread on pathological microscope glass slides in 40 °C double distilled water, and then adhered to the pathological microscope glass slides and baked at 60 °C for 5 h. The sections were stained in the automatic HE staining machine according to the set program. After staining, neutral balsam was added for sealing, and then dried. The sections were observed and photographed under a microscope.

[0091] The results of HE staining of paraffin sections of testicular tissue are shown in Figure 2.

[0092] Figure 2 The results show that the testis seminiferous tubules of the control group mice are full of morphology, the cells in the seminiferous tubules are arranged closely and orderly, the basal part can be seen oval or triangular pyramid-shaped Sertoli cells, 5-8 layers of various spermatogenic cells can be seen from the base to the near chamber, which constitutes a complete spermatogenic epithelium. Compared with the control group, part of the seminiferous tubules in the model group appear atrophy, the arrangement of spermatogenic cells is loose and disorder, the number of cells is significantly reduced, the thickness of spermatogenic epithelium is thinned, only 2-3 layers of spermatogenic cells can be seen from the base to the near chamber in part of the region. Compared with the model group, the Epimedium group can only slightly improve the structure of spermatogenic epithelium, there are still only 2-4 layers of spermatogenic cells in part of the seminiferous tubules, while the cell arrangement of the Rhizoma Polygonati group is more closely, 3-5 layers of spermatogenic cells can be seen in part of the seminiferous tubules. Similarly, compared with the model group, with the increase of the dose of Epimedium + Rhizoma Polygonati combination drug, the morphology of seminiferous tubules gradually recovers, the arrangement of spermatogenic cells gradually becomes close, the number of cells and the thickness of spermatogenic epithelium also gradually increase, 5-6 layers of spermatogenic cells can be seen in part of the seminiferous tubules in the high-dose group of Epimedium + Rhizoma Polygonati combination drug. Compared with the Epimedium and Rhizoma Polygonati single drug groups, the improvement effect of the high-dose group of Epimedium + Rhizoma Polygonati combination drug is more significant. This shows that the improvement effect of high-dose Epimedium + Rhizoma Polygonati combination on the morphological damage of testis tissue caused by cyclophosphamide in mice is better than that of Epimedium and Rhizoma Polygonati single drug administration.

[0093] 2.4, the influence of each group on the expression and location of γH2AX protein in mouse testis γH2AX is considered as a marker of DNA damage.

[0094] Immunofluorescence detection of γH2AX protein expression and localization: The testicular tissue sections were baked in a 60 ℃ oven for 30 min, then immersed in xylene (30 min), anhydrous ethanol (10 min), 95% ethanol (10 min), 80% ethanol (10 min), 70% ethanol (10 min), and double distilled water (10 min) for dewaxing and rehydration, respectively. Then, the sections were immersed in a sodium citrate buffer (containing 10 mM sodium citrate, 0.05% Tween-20, pH=6.0) for high-pressure repair for 10 min, naturally cooled to room temperature, and PBS-immersed for 3 times (5 min each time). The sections were permeabilized with 0.3% Triton for 30 min, PBS-immersed for 3 times (5 min each time), and 5% BSA solution was added dropwise for room temperature blocking for 60 min. The blocking solution was discarded, and γH2AX antibody (diluted with 1% BSA at a ratio of 1:1000) was added dropwise for 4 ℃ refrigerator incubation for 12-14 h. Then, the sections were rewarmed at room temperature for 20 min, the antibody was discarded, PBS was immersed for 6 times (5 min each time), fluorescent secondary antibody Alexa Fluor 594-conjugated AffiniPure Donkey Anti-Rabbit IgG (H+L) (diluted with 1% BSA at a ratio of 1:200) was added dropwise, and incubated at room temperature in the dark for 60 min. Then, PBS was immersed for 6 times (5 min each time), DAPI working solution was added dropwise for staining for 10 min, PBS was immersed for 6 times (5 min each time), and anti-fluorescence quenching agent was added dropwise for mounting, and the dark box was stored. The observation and photography were performed under a fluorescence microscope.

[0095] The test results of immunofluorescence are shown in Figure 3 .

[0096] The results of FIG. 3 show that the control group mice have little γH2AX expression on the testicular spermatogonia (white arrows), and a small amount of expression on the primary spermatocytes due to meiosis. Compared with the control group, the model group mice have significantly increased γH2AX expression on the testicular spermatogonia, and to a certain extent, the primary spermatocytes also have increased expression. Compared with the model group, the γH2AX expression on the testicular spermatogonia and primary spermatocytes of the Epimedium group is reduced, and the γH2AX expression of the Polygonatum group is reduced more obviously. Similarly, compared with the model group, the γH2AX expression in the testicular spermatogonia and primary spermatocytes gradually decreases with the increase of the Epimedium+Polygonatum combination drug concentration. Compared with the Epimedium group and the Polygonatum group alone, the γH2AX expression of the high-dose Epimedium+Polygonatum combination drug group decreases more obviously. This indicates that the high-dose Epimedium+Polygonatum combination drug has a better improvement effect on the DNA damage of the testicular spermatogenic cells of the mice caused by cyclophosphamide than the Epimedium and Polygonatum drugs alone.

[0097] 2.5 Effects of each group on the expression and localization of PCNA protein in mouse testes PCNA begins to be synthesized in the G1 phase of cells and reaches its peak in the S phase. It directly participates in DNA replication and is gradually degraded in the G2 and M phases. However, due to its long half-life of up to 20 hours, it can still be detected in large quantities in cells in the G2 and M phases. Its expression is extremely low in cells in the G0 phase. It is one of the commonly used markers reflecting cell proliferation capacity.

[0098] Immunohistochemical detection of PCNA protein expression and localization: Dewaxing and rehydration were performed as described in section 2.4. The sections were then immersed in Tris-EDTA buffer (containing 10 mM Tris, 1 mM EDTA, and 0.05% Tween-20, pH 9.0) for microwave retrieval for 10 min. After natural cooling to room temperature, the sections were washed three times with PBS (5 min each time), inactivated by adding 3% H2O2 for 10 min, washed three times with PBS (5 min each time), and blocked with 5% BSA solution at room temperature for 60 min. The blocking solution was discarded, and PCNA antibody (diluted 1% BSA at a ratio of 1:8000) was added and incubated at 4 ℃ for 12-14 h. The sections were then incubated at room temperature for 20 min, the antibodies were discarded, and the sections were washed 6 times (5 min each time) with PBS. Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (diluted 1% BSA at a ratio of 1:200) was added, and the sections were incubated at room temperature for 60 min. The sections were then washed 6 times (5 min each time) with PBS. DAB staining solution was added for 17 s, followed by PBS washing. Hematoxylin staining solution was added for 35 s, followed by PBS washing. Hematoxylin differentiation solution was added for 10 s, followed by PBS washing. Hematoxylin blueing solution was added for 3 s, followed by PBS washing. After staining, the sections were sequentially immersed in 70% ethanol (5 min), 80% ethanol (5 min), 95% ethanol (5 min), anhydrous ethanol (5 min), and xylene (5 min) for dehydration and permeabilization. Neutral resin was added for mounting, and the sections were air-dried. The sections were then observed and photographed under a microscope. ImageJ software was used to perform semi-quantitative analysis of the immunohistochemical results by analyzing the mean optical density (MOD) and the percentage of positive area (Area %).

[0099] Test results are as follows Figure 4 As shown in the figure, compared to the Normal group, ** P <0.01, *** P <0.001; compared to the Model group, # P <0.05,## P <0.01, ### P <0.001; n=3.

[0100] Figure 4 The results showed that in the control group, PCNA was mainly expressed in mouse testicular spermatogonia and primary spermatocytes during promeiosis. Compared with the control group, the PCNA-positive area in the model group ( P <0.001), average optical density ( P <0.01) all significantly decreased. Compared with the model group, both Epimedium and Polygonatum increased the positive area and mean optical density of PCNA, and the increase in PCNA positive area in the Polygonatum group was significant ( P <0.05). Compared with the model group, with the increase of the dose of the Epimedium + Polygonatum combination drug, the positive area and mean optical density of PCNA both increased in a dose-dependent manner, and the positive area of ​​the high-dose group of Epimedium + Polygonatum combination drug ( P <0.001), average optical density ( P The increase in PCNA positive area (<0.01) was statistically significant. Compared with the Epimedium group, the high-dose Epimedium + Polygonatum combination drug group showed a significantly larger PCNA positive area (<0.01). P <0.05) and average optical density ( P <0.05) were all significantly increased; compared with the Polygonatum group, the PCNA positive area in the high-dose group of Epimedium + Polygonatum combination drug was significantly increased ( P <0.05) Significantly increased. Compared with the administration of Epimedium and Polygonatum alone, the Q value of the positive area of ​​the combination of Epimedium and Polygonatum was 1.31 and the Q value of the average optical density was 1.35. This indicates that the high-dose administration of the combination of Epimedium and Polygonatum has a more significant effect on upregulating PCNA protein expression and has a synergistic effect.

[0101] 2.6 Effects of each group on the expression and localization of Ki67 protein in mouse testes Ki67 is only found in cells in the active cell cycle (G1-M phase) and is not expressed in quiescent cells (G0 phase). Due to its short half-life and high detection sensitivity, it is often used to detect cell proliferation activity.

[0102] Immunohistochemical method for detecting Ki67 protein expression and localization: follow the steps in 2.4, then immerse the section in Tris-EDTA buffer (containing 10 mM Tris, 1 mM EDTA, 0.05% Tween-20, pH = 9.0) for microwave repair for 15 min, naturally cool to room temperature, PBS immersion for 3 times (5 min / time), 0.3% Triton permeabilization for 30 min, PBS immersion for 3 times (5 min / time), drop 3% H2O2 for 10 min, PBS immersion for 3 times (5 min / time) after dropping 5% BSA solution for 60 min at room temperature. Discard the blocking solution, drop Ki67 antibody (diluted with 1% BSA at a ratio of 1:100), incubate in the refrigerator at 4 ℃ for 16-18 h. Then warm the section at room temperature for 20 min, discard the antibody, PBS immersion for 6 times (5 min / time) after dropping the secondary antibody Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (diluted with 1% BSA at a ratio of 1:200), incubate at room temperature for 60 min. Again, immerse in PBS for 6 times (5 min / time). Drop DAB color developing working solution for color development for 90 s, drop hematoxylin staining solution for nuclear staining for 35 s, drop hematoxylin differentiation solution for differentiation for 10 s, drop hematoxylin return blue solution for return blue for 3 s, and then immerse in PBS. After staining, immerse the section in 70% ethanol (5 min), 80% ethanol (5 min), 95% ethanol (5 min), anhydrous ethanol (5 min), and xylene (5 min) for dehydration and permeabilization, drop neutral gum for mounting, dry, and observe and take pictures under a microscope. Use Image J software to analyze the mean optical density value (MOD) and the percentage of positive area (Area %) for semi-quantitative analysis of immunohistochemical results.

[0103] Semi-quantitative analysis of the percentage of positive expression area and the mean optical density value of Ki67 in mouse testis tissue was performed using Image J software, and the test results are shown in Figure 5 Compared with the Normal group, *** P <0.001; compared with the Model group, # P <0.05, ## P <0.01, ### P <0.001; n = 3.

[0104] Figure 5The results showed that in the control group, Ki67 was mainly expressed in mouse testicular spermatogonia and primary spermatocytes in prophase of meiosis. Compared with the control group, the Ki67 positive area (P<0.001) and the average optical density (P<0.001) of the model group were significantly reduced. Compared with the model group, the Ki67 positive area of the Rhizoma Polygonati group (P<0.05) was significantly increased. In addition, compared with the model group, the Ki67 positive area and the average optical density were increased in a dose-dependent manner with the increase of the dosage of the Epimedium + Rhizoma Polygonati combination drug, and the positive area (P<0.001) and the average optical density value (P<0.01) of the high-dose group of the Epimedium + Rhizoma Polygonati combination drug were significantly increased. Compared with the Epimedium group, the Ki67 positive area of the high-dose group of the Epimedium + Rhizoma Polygonati combination drug (P<0.05) was significantly increased. The Q value of the positive area of the Epimedium + Rhizoma Polygonati combination drug was 1.17, and the Q value of the average optical density was 1.18, which indicated that the high-dose administration of the Epimedium + Rhizoma Polygonati combination drug could more significantly up-regulate the expression of Ki67 protein, and had a synergistic effect.

[0105] 2.7, the influence of each group on the apoptosis of mouse testicular spermatogenic cells TUNEL fluorescence method was used to detect the apoptosis of testicular spermatogenic cells: the dewaxing and rehydration were the same as in step 2.4, then 40 μg / mL of proteinase K was added dropwise and incubated at 37 ℃ for 30 min, PBS was washed 3 times (5 min each time), TUNEL reaction solution (TdT enzyme and TUNEL Reaction Buffer) was added dropwise and incubated at 37 ℃ in the dark for 60 min, the TUNEL reaction solution was discarded, PBS was washed 6 times (5 min each time), DAPI working solution was added dropwise and stained for 10 min, PBS was washed 6 times (5 min each time), anti-fluorescence quencher was added dropwise for mounting, and the dark box was stored. Under the fluorescence microscope, the average number of TUNEL positive cells in the seminiferous tubules was observed, photographed, and counted.

[0106] Test results Figure 6 As shown in the figure, compared with the Normal group, *** P <0.001; compared with the Model group, # P <0.05, ### P <0.001; n=3.

[0107] Figure 6 The results showed that compared with the control group, the number of apoptotic cells in the testis of the model group was significantly increased (P<0.001) P<0.001), co-localization with DAPI revealed that TUNEL-positive cells were mainly spermatogonia, with some primary spermatocytes. Compared with the model group, Epimedium reduced the number of apoptotic cells, but the difference was not statistically significant. P >0.05), while the number of apoptotic cells was reduced in the Polygonatum group, and the difference was statistically significant ( P <0.05). Compared with the model group, the number of apoptotic cells decreased in a dose-dependent manner with increasing dose of the Epimedium + Polygonatum combination drug, and the difference was statistically significant in the high-dose group of Epimedium + Polygonatum combination drug. P <0.001). Compared with the administration of Epimedium and Polygonatum alone, the high-dose group of Epimedium + Polygonatum combination had fewer apoptotic cells, with a Q value of 1.16. This indicates that the high-dose administration of Epimedium + Polygonatum combination has a more significant effect on improving cyclophosphamide-induced apoptosis of testicular spermatogenic cells and has a synergistic effect.

[0108] Based on the above test results, it can be seen that high-dose administration of the combination of Epimedium and Polygonatum can significantly improve the DNA damage of mouse testicular spermatogenic cells induced by cyclophosphamide, restore their proliferation capacity, inhibit their apoptosis, and alleviate the damage to spermatogenic function caused by cyclophosphamide. Moreover, the combined administration showed a synergistic effect, and its efficacy was better than either Epimedium or Polygonatum alone.

[0109] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. Use of a plant extract composition in the manufacture of a medicament for ameliorating DNA damage in spermatogenic cells of the testis, wherein, The plant extract contains Epimedium extract and Rhizoma Polygonati extract.

2. Use according to claim 1, characterized in that, The plant extract is a combination of Epimedium extract and Rhizoma Polygonati extract.

3. Use according to claim 1 or 2, characterized in that, In the medicine, the Epimedium extract and the Rhizoma Polygonati extract are both in the form of granules, and the mass ratio of the use amount of the Epimedium granules to the Rhizoma Polygonati granules is 1:7-10. Preferably, the dose of the medicine is 5-5.6 g / kg / day, wherein the total weight of the Epimedium granules and the Rhizoma Polygonati granules is used as the basis.

4. Use according to any one of claims 1 to 3, characterized in that, The Rhizoma Polygonati extract is prepared according to the following procedure: A1, mixing Rhizoma Polygonati decoction pieces with water and then soaking; A2, heating the mixed system obtained in step A1 in a water bath at 75-85 DEG C, condensing and refluxing extraction for 85-95 min, repeating the extraction for 2-3 times, collecting and combining the extract; A3, centrifuging the extract obtained in step A2, vacuum filtering, collecting the filtrate, and then vacuum concentrating and freeze-drying.

5. Use according to claim 4, characterized in that, In step A1, the use amount ratio of the Rhizoma Polygonati decoction pieces to water is 1g:13-17 mL; Preferably, in step A1, the soaking time is 25-35 min. Preferably, in step A3, the centrifuging conditions include a rotation speed of 2800-3200 r / min and a time of 8-12 min.

6. The use according to any one of claims 1 to 5, characterized in that, The Rhizoma Polygonati extract is used in the form of granules; Preferably, in 1 g of Rhizoma Polygonati granules, there are 75-80 mg of fructose.

7. The use according to any one of claims 1 to 3, characterized in that, The Epimedium extract is prepared according to the following procedure: B1, crushing Epimedium decoction pieces, then mixing with 65-75% ethanol and soaking; B2, heating the soaking system obtained in step B1 in a water bath at 95-100 DEG C, condensing and refluxing extraction for 55-65 min, repeating the extraction for 1-2 times, collecting and combining the extract; B3, centrifuging the extract obtained in step B2, vacuum filtering, collecting the filtrate, and then vacuum concentrating and freeze-drying.

8. Use according to claim 7, characterized in that, In step B1, the use amount ratio of the Epimedium decoction pieces to ethanol is 1g:8-12 mL; Preferably, in step B1, the soaking time is 25-35 min. Preferably, in step B3, the centrifuging conditions include a rotation speed of 2800-3200 r / min and a time of 8-12 min.

9. The use according to any one of claims 1 to 8, characterized in that, The Epimedium extract is used in the form of granules; Preferably, in 1 g of Epimedium granules, there are 60-70 mg of total of epimedium A, epimedium B, epimedium C and icariin.

10. The use according to claim 1, characterized in that, Specifically, the application of the plant extract composition in the preparation of a medicine for improving the DNA damage of spermatogenic cells induced by cyclophosphamide.

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