An additive for resisting cow ovarian aging and its application

Abstract

The invention discloses an additive for resisting cow ovarian aging and its application. The additive is made of a composition of anthocyanidins, glycyrrhizic acid and wolfberry glycopeptide. When the composition is used in clinical veterinary treatment of cow ovarian aging, the role of primary cow ovarian granulosa cells is clarified through transcription, which is mainly involved in regulating programmed cell death, Ras protein signal transduction, tissue development, cell serine, tryptophan kinase activity, MAPK signaling pathway, PI3K-AKT signaling pathway, and laser confocal results show that the composition can significantly reduce the production of mitochondrial reactive oxygen; at the same time, the therapeutic effect on the persistent corpus luteum of the main cow is achieved by improving the intestinal flora structure, and more specifically, the colony is significantly changed upstream at the genus and species level; B-ultrasound results reveal that for a persistent corpus luteum cow, more large follicles can be detected on the ovaries after one month of gavage with the additive composition.

Description

Technical Field

[0001] The invention relates to the technical field of prevention and treatment of cow ovarian aging, and specifically to an additive for resisting cow ovarian aging and application thereof, and specifically to the application of the additive for resisting cow ovarian aging made from a composition of anthocyanidin, glycyrrhetinic acid and lycium barbarum glycopeptide. Background Art

[0002] In animals, ovarian aging is mostly limited to experimental mice, while in humans, there are more studies. Ovarian aging can have serious negative effects on the reproductive health of female animals, leading to infertility, and is often accompanied by the occurrence of chronic diseases. However, the physiological and pathological mechanisms of ovarian aging are still unclear. In recent years, the role of long noncoding RNAs (lncRNAs) in age-related diseases has attracted the attention of researchers [GrammatikakisI, Panda AC, Abdelmohsen K, et al. Long noncoding RNAs (lncRNAs) and themolecular hallmarks of aging [J]. Aging (Albany NY), 2014. 6(12): 992-1009.].

[0003] Grape seed proanthocyanidins (GSPs) are widely present in grape branches, leaves, peels, pulp and seeds, and are composed of different numbers of flavanol monomers. Studies have shown that GSPs can upregulate the level of interleukin-12 (IL-12) in rat serum, promote the activation of CD4+T cells, and improve the body's immune function. GSPs can promote the proliferation of rat spleen lymphocytes cultured in vitro, and have the effect of improving cellular antioxidant and cellular immunity. At present, research on the application of GSPs in ruminants mainly focuses on the body's antioxidant capacity and the regulation of the rumen environment, but there is no relevant research on anti-aging of cow ovaries.

[0004] Glycyrrhizic acid is an effective active substance extracted from licorice. It can increase the feed intake and immune function of animals and enhance disease resistance. It has become a hot topic in current research. However, there are few reports on the growth performance of ruminants by adding glycyrrhizic acid to their diets. Zhao Zhengwei et al. studied that glycyrrhizic acid significantly increased the serum MDA content of Tan sheep compared with other groups [Zhao Zhengwei, Zhao Tao, Yin Ji, et al. Effects of Chinese herbal medicine additives on growth, immune function and meat quality of Tan sheep [J]. China Animal Husbandry Journal, 2020, 56(11): 150-153.].

[0005] Lyciumbar barum polysaccharide (LBP) is the main active ingredient of Lycium barbarum biological activity, with the characteristics of no drug resistance, no residue, safety, simple manufacturing process, etc. Studies have shown that LBP has antioxidant, anti-inflammatory, neuroprotective, hypoglycemic and lipid-lowering, and immunomodulatory functions. LBP can induce NK cell activation, enhance NK cell cytotoxicity, and can induce the phenotype and functional maturation of dendritic cells through Notch signaling, and promote the activity of dendritic cell-mediated cytotoxic T lymphocytes. Zhang Haiyan et al. found that adding 50 mg / kg LBP to the diet of Hy-Line brown laying hens can significantly increase the weight of the thymus and bursa of Fabricius, increase the content of albumin and lysozyme in serum, and promote the proliferation of CD3+T lymphocytes in the blood. It can be seen that Lycium barbarum polysaccharide has the clinical effect of improving the body's immune function. Lycium barbarum glycopeptide (LbGp) is a glycoconjugate with stronger immune activity obtained by further separation and purification from LBP. Zhao Zhengwei's research showed that the SOD activity of the Lycium barbarum polysaccharide group was significantly lower than that of the glycyrrhizic acid group.

[0006] The research on the above three substances is currently mostly limited to the extraction and function of monomers, while no relevant personnel have conducted research on the function and application of the combination of the three substances. Summary of the invention

[0007] One purpose of the present invention is to determine the antioxidant mechanism of the three-substance composition of anthocyanidin, glycyrrhetinic acid and lycium barbarum glycopeptide on primary ovarian granulosa cells of cows, which is mainly involved in multiple signal pathways, and has stronger functions than the single addition of each substance. It is finally determined that the three compositions can more significantly reduce the production of mitochondrial reactive oxygen species compared with a single substance. The present invention combines anthocyanidin, glycyrrhetinic acid and lycium barbarum glycopeptide to study its mechanism of action in anti-aging of cow ovaries, and also studies the application of additives composed of these three substances, which is the first time. Ovarian aging is a complex biological process in which multiple factors inside and outside the body interact and gradually accumulate, and its speed is earlier than other organs of the body. The present invention proposes for the first time that in the process of cow ovarian aging, there are three substance compositions related to it that participate in regulating programmed cell death, Ras protein signal transduction, tissue development, cell serine, tryptophan kinase activity, MAPK signaling pathway, and PI3K-AKT signaling pathway.

[0008] The second purpose of the present invention is to propose for the first time that the therapeutic effect on the cow itself is to improve the intestinal flora structure, more specifically, to cause significant changes in the upstream genus and species level of the colony and increase the species abundance of the intestinal flora.

[0009] The third purpose of the present invention is to provide certain theoretical basis and experimental data for original drugs and / or feed additives for treating ovarian aging in cows.

[0010] Ovarian insufficiency and aging occur frequently in cow production, resulting in a shortened production period of cows. The cow follicles are continuously consumed with age. Female reproductive stem cells have the ability to differentiate into oocytes and continuously renew themselves, so they can replenish the primordial follicle pool to delay aging. Anthocyanidins, glycyrrhetinic acid and wolfberry glycopeptide monomers all have certain antioxidant, anti-inflammatory, immune-enhancing and aging-delaying functions. The present invention prepares the above three monomers into a composition, and studies the effects of the composition on the ovarian granulosa cells of the host animal cow and the host animal, especially the participation of the composition in regulating programmed cell death, Ras protein signal transduction, tissue development, cell serine, tryptophan kinase activity, MAPK signaling pathway, PI3K-AKT signaling pathway, etc.; at the same time, experimental research on the regulation of intestinal flora in the host animal is also carried out.

[0011] The present invention verifies the effect of different ratios of the above three substance compositions on improving the ovarian function of aged ovaries and normal cows, and finally determines that anthocyanin (200 mg / kg·d) + glycyrrhizic acid (400 mg / kg·d) + wolfberry glycopeptide (200 mg / kg·d) is the best ratio of the composition.

[0012] The present invention conducts a comparative study on the combination of anthocyanins, glycyrrhizic acid and lycium barbarum glycopeptide and the three single substances through cell conversion rate experiments, tissue section experiments, cell antioxidant experiments, and studies on changes in the intestinal microbial flora structure and colony genus level in primary animals (ovarian aging cows screened by B-ultrasound).

[0013] The research results of the present invention show that compared with the three single substances, the combination has stronger efficacy in the cell conversion rate experiment; the combination has more spleen germinal centers under a certain field of view in the tissue section experiment; in the antioxidant study, the final GO clustering analysis results show: pairwise comparison analysis, the molecular functions of the differentially expressed genes are mainly involved in metabolic processes, biological regulation, cellular processes, etc.; the cellular components of the differentially expressed genes are mainly concentrated in membranes and organelles; biological process analysis reveals that the differentially expressed genes are mainly involved in binding; KEGG signaling pathway reveals that the combination up-regulates the differentially expressed genes and is mainly involved in signal transduction, endocrine system, etc.; the single substance down-regulates the differentially expressed genes and is mainly involved in signal transduction, endocrine system, immune system, etc.; laser confocal microscopy results show that the combination can significantly reduce the production of mitochondrial reactive oxygen species.

[0014] In the present invention, in the study of changes in the intestinal microbial flora structure and colony genus and species level of the main body animals (B-ultrasound screening of ovarian aging cows), the development of follicles and corpus luteum were monitored, blood was collected to measure ovarian hormones, and feces were collected to measure intestinal microbial flora, and the effect results of the combination and the single substance were compared. The results showed that the colonies of the combination test group changed significantly at the upstream genus and species level, and the species abundance of the intestinal flora increased, which was significantly different from that of the single substance. The B-ultrasound results revealed that in the cows with persistent corpus luteum, more large follicles could be detected in the ovaries after one month of gavage with the combination.

[0015] For female animals, there are two main ways to consume the primordial follicle bank: cyclical ovulation and programmed apoptosis. In the second stage of embryonic development, a large number of primordial follicles die, and there are only 1-2 million follicles at birth, and 300,000-400,000 follicles at the reproductive period. The hormones P4, estrogen 2 (E2), inhibin B (INHB), anti-Mullerian hormone (AMH) and progesterone P4 have significant morphological effects on the endometrium stimulated by estrogen in the body, and are necessary for maintaining pregnancy. Estradiol E2 is one of the estrogens and one of the main sex hormones secreted by the ovaries. It plays an important role in maintaining the reproductive function and secondary sexual characteristics of female animals. Serum inhibin B is a heterodimeric protein hormone secreted by the granulosa cells of the female animal's ovaries. AMH generally refers to anti-Mullerian hormone. AMH examination can reliably and quickly evaluate the ovarian reserve function, and know the number of antral eggs that may be formed in the female animal's ovaries, thereby knowing the reproductive capacity of the female animal. These hormone levels are directly correlated with the ovarian aging function. During the ovarian aging process, the levels of P4, E2, INHB and AMH decrease. The above hormones are relatively sensitive indicators for evaluating ovarian reserve function.

[0016] In summary: The present invention proposes for the first time that an additive made of a three-substance composition of anthocyanidin, glycyrrhetinic acid and wolfberry glycopeptide can resist cow ovarian aging and its new application, suggesting that the new application mechanism of anthocyanidin + glycyrrhetinic acid + wolfberry glycopeptide in clinical veterinary treatment of cow ovarian aging is to increase the number of germinal centers in cow spleen, and more specifically, the antioxidant effect of the three-substance composition on primary ovarian granulosa cells of cows is mainly achieved by participating in the regulation of programmed cell death, Ras protein signaling, tissue development, cell serine, tryptophan kinase activity, MAPK signaling pathway, and PI3K-AKT signaling pathway, and finally determines that the composition can significantly reduce the production of mitochondrial reactive oxygen; At the same time, it also reveals that the therapeutic mechanism of the three-substance composition on the persistent corpus luteum of the main animal cow is to improve the intestinal flora structure, more specifically, to significantly change the colony upstream at the genus and species level, and increase the species abundance of the intestinal flora, which is quite different from a single substance. The B-ultrasound results reveal that for persistent corpus luteum cows, more large follicles can be detected on the ovaries after one month of oral administration of the composition additive.

[0017] Beneficial Effects

[0018] 1. The present invention proposes for the first time that an additive for resisting cow ovarian aging is made from a combination of anthocyanidins, glycyrrhetinic acid and lycium barbarum glycopeptide.

[0019] 2. The present invention discloses for the first time a new application of the composition of "anthocyanidins + glycyrrhizic acid + wolfberry glycopeptide" in the clinical treatment of ovarian aging in cows, which is to increase the number of germinal centers in the spleen; more specifically, it discloses the antioxidant mechanism of the composition on primary ovarian granulosa cells in cows, which is mainly involved in regulating programmed cell death, Ras protein signal transduction, tissue development, cell serine, tryptophan kinase activity, MAPK signaling pathway, and PI3K-AKT signaling pathway, and finally determines that the composition can significantly reduce the production of mitochondrial reactive oxygen species.

[0020] 3. The present invention reveals for the first time that the therapeutic effect of the "anthocyanidin + glycyrrhizic acid + wolfberry glycopeptide" composition on cows is through improving the intestinal flora structure, more specifically, causing significant changes in the upstream genus and species level of the colonies, thereby increasing the species abundance of the intestinal flora, which is significantly different from using a single substance.

[0021] 4. The B-ultrasound results of the composition administered orally to cattle with persistent corpus luteum revealed that after one month of medication, more large follicles could be detected on the ovaries of the cattle with persistent corpus luteum, and estrogen P4, estrogen 2 (E2), inhibin B (INHB), and anti-Mullerian hormone (AMH) all increased, indicating that the anthocyanin + glycyrrhizic acid + wolfberry glycopeptide composition has the effect of restoring ovarian function, because the ovaries mainly secrete these hormones. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A graph showing the effects of the combination and single substance in Example 2 on bovine spleen tissue sections.

[0023] Figure 2-1 Effects of the combination and single substances in Example 3 on primary ovarian granulosa cells: heat map of gene expression among the four groups.

[0024] Figure 2-2 Effects of the composition and single substance in Example 3 on primary ovarian granulosa cells: GO cluster analysis diagram.

[0025] Figure 2-3 Effects of the combination and single substance on primary ovarian granulosa cells in Example 3: KEGG signaling pathway diagram of differentially upregulated genes in group D and other groups.

[0026] Figure 2-4 Effects of the combination and single substance on primary ovarian granulosa cells in Example 3: KEGG signaling pathway diagram of differentially down-regulated genes in group D and other groups.

[0027] Figure 2-5 Effects of the composition and single substance in Example 3 on primary ovarian granulosa cells: GO functional analysis diagram.

[0028] Figure 2-6 Effects of the composition and single substance in Example 3 on primary ovarian granulosa cells: KEGG functional analysis diagram.

[0029] Figure 2-7 Effects of the combination and single substance in Example 3 on primary ovarian granulosa cells: laser confocal microscopy results.

[0030] Figure 3-1 Effects of the combination and single substances in Example 4 on the persistent corpus luteum in cows: Estrogen P4 levels.

[0031] Figure 3-2 Effect of the combination and single substances in Example 4 on persistent corpus luteum in cows: Figure 4 of inhibin B levels.

[0032] Figure 3-3 Effects of the combination and single substances in Example 4 on persistent corpus luteum in cows: heat map of species abundance clustering at the genus level.

[0033] Figure 3-4 B-ultrasound images of the ovaries of cattle in Group A in Example 4 before and after treatment with anthocyanins (200 mg / kg•d).

[0034] Figure 3-5 Ultrasound images of the ovaries of cattle in Group B before and after treatment with glycyrrhetinic acid (400 mg / kg•d) in Example 4.

[0035] Figure 3-6 B-ultrasound images of the ovaries of cattle in Group C in Example 4 before and after treatment with Lycium barbarum glycopeptide (200 mg / kg•d).

[0036] Figure 3-7 B-ultrasound images of the ovaries of Group D in Example 4 before and after administration of the bovine anthocyanin combination (200 mg / kg•d) + glycyrrhizic acid (400 mg / kg•d) + wolfberry glycopeptide (200 mg / kg•d). DETAILED DESCRIPTION

[0037] The technical solution is further described below in conjunction with specific embodiments.

[0038] In the specific implementation of the present invention, the experimental animals and drugs used are from:

[0039] Beef cattle from a beef cattle farm in Ningxia;

[0040] Proanthocyanidins were extracted from wine grapes and purchased from Nanjing Zelang Co., Ltd.;

[0041] Lycium barbarum glycopeptide was extracted from Ningxia wolfberry, and Lycium barbarum glycopeptide was purchased from Ningxia Tianren Lycium barbarum Biotechnology Co., Ltd.;

[0042] Glycyrrhizic acid was extracted from licorice herbs, and glycyrrhetinic acid was purchased from Gansu Panzhi Pharmaceutical Co., Ltd.

[0043] 1. The present invention first investigated the effects of anthocyanidins (150 mg / mg / kg•d, 200 mg / kg•d, 250 mg / kg•d); glycyrrhetinic acid (350 mg / kg•d, 400 mg / kg•d, 450 mg / kg•d); Lycium barbarum glycopeptide (150 mg / kg•d, 200 mg / kg•d, 250 mg / kg•d) and a blank control group on the A490nm value of peripheral blood lymphocytes in ovarian aging cows on the 7th, 14th, 21st and 28th days after the first administration, and screened out the optimal dosages of the three substances, which are A. anthocyanidins (200 mg / kg•d); B. glycyrrhetinic acid (400 mg / kg•d); and C. Lycium barbarum glycopeptide (200 mg / kg•d).

[0044] 2. The present invention also investigated the effects and mechanisms of four groups of substances, namely A. anthocyanidins (200 mg / kg•d); B. glycyrrhetinic acid (400 mg / kg•d); C. wolfberry glycopeptide (200 mg / kg•d); and D. anthocyanidins (200 mg / kg•d) + glycyrrhetinic acid (400 mg / kg•d) + wolfberry glycopeptide (200 mg / kg•d), in combating ovarian aging in cows.

[0045] 1) The experiment selected 40 cows with similar body condition (body condition score 5-6), parity 2, and B-ultrasound examination showed ovarian inactivity. They were randomly divided into 4 groups: Group A, Group B, Group C and Group D, with 10 cows in each group. The dosing regimens of each group were: A. anthocyanidin (200 mg / kg·d); B. glycyrrhetinic acid (400 mg / kg·d); C. Lycium barbarum glycopeptide (200 mg / kg·d); D. anthocyanidin (200 mg / kg·d) + glycyrrhetinic acid (400 mg / kg·d) + Lycium barbarum glycopeptide (200 mg / kg·d). The administration was continued for 31 days. Administration method: oral administration.

[0046] (1) On the 7th, 14th, 21st and 28th day after the first administration, 2 cows were randomly selected from each group, and blood was collected to measure the A490nm value of peripheral blood lymphocytes. Then, the cows were slaughtered, and their spleens were taken to prepare paraffin sections, which were then stained with HE and examined under a microscope. The test results were analyzed.

[0047] (2) After the first administration, the ovaries were examined by B-ultrasound every three days, and attention was paid to the development of follicles and corpus luteum. On the 3rd, 7th, 11th, 15th, 19th, 23rd, 27th and 31st days after administration, 2 cows were randomly selected from each group for blood sampling. The serum was collected and tested by Elisa for serum E2, P4, Inhibit B, AMH and other related indicators. The test results were analyzed.

[0048] (3) On the 3rd, 7th, 11th, 15th, 19th, 23rd, 27th and 31st days after the first administration, the intestinal contents were collected through the rectum. The collected intestinal contents were divided into four groups according to the dosing regimen (A. anthocyanidins (200 mg / kg•d); B. glycyrrhetinic acid (400 mg / kg•d); C. Lycium barbarum glycopeptide (200 mg / kg•d); D. anthocyanidins (200 mg / kg•d) + glycyrrhetinic acid (400 mg / kg•d) + Lycium barbarum glycopeptide (200 mg / kg•d) for the determination of intestinal microbial flora.

[0049] 2) A healthy cow that had not been given any medication was selected for the experiment. Its ovaries were taken to isolate and culture ovarian granulosa cells in vitro, and transcriptome sequencing was performed. The experimental results were analyzed. The anti-aging effects and mechanisms of three single substances, A. anthocyanidins (200 mg), B. glycyrrhetinic acid (400 mg), and C. Lycium barbarum glycopeptide (200 mg), and the combination of D. anthocyanidins (200 mg) + glycyrrhetinic acid (400 mg) + Lycium barbarum glycopeptide (200 mg), on cow ovarian granulosa cells were investigated.

[0050] Embodiment 1:

[0051] Screen the optimal dosage of three substances: anthocyanins, glycyrrhizic acid and wolfberry glycopeptide.

[0052] Specifically, the effects of anthocyanins (150 mg / kg•d, 200 mg / kg•d, 250 mg / kg•d); glycyrrhizic acid (350 mg / kg•d, 400 mg / kg•d, 450 mg / kg•d); wolfberry glycopeptide (150 mg / kg•d, 200 mg / kg•d, 250 mg / kg•d) and a blank control group on the A490nm value of peripheral blood lymphocytes in cows with ovarian aging on the 7th, 14th, 21st and 28th days after the first administration were investigated. A comparative study on the effects of the blank control group and the low-dose wolfberry glycopeptide (150 mg / kg•d) group on the transformation of peripheral blood lymphocytes was also conducted to screen out the optimal dosage of these three substances. The experiment selected 40 cows with similar body condition (body condition score 5-6), two parities, and ovarian quiescence under B-ultrasound examination. They were randomly divided into 4 groups, with 10 cows in each group. On the 7th, 14th, 21st and 28th day after the first administration, blood was collected from each group to measure the A490nm value of peripheral blood lymphocytes. The measurement results are shown in Tables 1, 2, 3 and 4.

[0053] Table 1 Effect of anthocyanin dosage on peripheral blood lymphocyte transformation (A 490nm)

[0054] .

[0055] Table 2 Effect of glycyrrhetinic acid dosage on peripheral blood lymphocyte transformation (A 490nm)

[0056] .

[0057] Table 3 Effect of Lycium barbarum glycopeptide dosage on peripheral blood lymphocyte transformation (A 490nm)

[0058] .

[0059] Table 4 Effects of blank control group and lowest dose of Lycium barbarum glycopeptide on peripheral blood lymphocyte transformation (A 490nm)

[0060] .

[0061] As can be seen from Table 1, on days 7, 14, 21 and 28 after the first administration, the A490nm values ​​of peripheral blood lymphocytes in the 200 mg / kg•d anthocyanidin group were significantly higher than those in the other groups.

[0062] As can be seen from Table 2, on days 7, 14, 21 and 28 after the first administration, the A490nm values ​​of peripheral blood lymphocytes in the 400 mg / kg•d glycyrrhetinic acid group were significantly higher than those in the other groups.

[0063] As can be seen from Table 3, on the 7th, 14th, 21st and 28th days after the first administration, the A490nm values ​​of peripheral blood lymphocytes in the 200 mg / kg•d Lycium barbarum glycopeptide group were significantly higher than those in the other groups.

[0064] As can be seen from Table 4, on the 7th, 14th, 21st and 28th days after the first administration, the A490nm values ​​of peripheral blood lymphocytes in the blank control group were lower than those in the low-dose (150 mg / kg•d) Lycium barbarum glycopeptide group.

[0065] It can be seen from this that the optimal dosages of these three substances are A. anthocyanins (200 mg / kg•d); B. glycyrrhizic acid (400 mg / kg•d); C. Lycium barbarum glycopeptide (200 mg / kg•d).

[0066] Embodiment 2:

[0067] Based on Example 1, this example uses the optimal dosage of three single substances, anthocyanin (200 mg / kg•d), glycyrrhetinic acid (400 mg / kg•d) and lycium barbarum glycopeptide (200 mg / kg•d), and a combination of the three substances, anthocyanin (200 mg / kg•d) + glycyrrhetinic acid (400 mg / kg•d) + lycium barbarum glycopeptide (200 mg / kg•d), to investigate the effects of the four groups of substances on the transformation of peripheral blood lymphocytes of cows and on the spleen of cows.

[0068] The experiment selected 40 cows with similar body condition (body condition score 5-6), 2 parities, and B-ultrasound examination showed ovarian inactivity. They were randomly divided into 4 groups: Group A, Group B, Group C and Group D, with 10 cows in each group. The dosing regimens of each group were: A. anthocyanidin (200 mg / kg•d); B. glycyrrhetinic acid (400 mg / kg•d); C. Lycium barbarum glycopeptide (200 mg / kg•d); D. anthocyanidin (200 mg / kg•d) + glycyrrhetinic acid (400 mg / kg•d) + Lycium barbarum glycopeptide (200 mg / kg•d); continuous administration for 31 days. Dosage method: oral administration.

[0069] Sources of experimental animals and drugs:

[0070] Beef cattle from a beef cattle farm in Ningxia. Lycium barbarum glycopeptide was purchased from Ningxia Tianren Lycium barbarum Biotechnology Co., Ltd., anthocyanin was purchased from Nanjing Zelang Co., Ltd., and glycyrrhizic acid was purchased from Gansu Panzhi Pharmaceutical Co., Ltd. The experiment selected 40 cattle with similar body condition (body condition score 5-6), 2 parities, and B-ultrasound examination of ovarian quiescence, and randomly divided into 4 groups, 10 cattle in each group. On the 7th, 14th, 21st and 28th days after the first administration, 2 cattle were randomly selected from each group, blood was collected, and their spleens were taken after slaughter.

[0071] 1. Blood sampling to determine the A490nm value of peripheral blood lymphocytes. For the cows in Group A, Group B, Group C and Group D, the dosing regimens for each group were: A. anthocyanidins (200 mg / kg•d); B. glycyrrhetinic acid (400 mg / kg•d); C. Lycium barbarum glycopeptide (200 mg / kg•d); D. anthocyanidins (200 mg / kg•d) + glycyrrhetinic acid (400 mg / kg•d) + Lycium barbarum glycopeptide (200 mg / kg•d). On the 7th, 14th, 21st and 28th days after the first administration, 2 cows were randomly selected from each group, blood was collected, and the A490nm value of peripheral blood lymphocytes was determined.

[0072] The results are shown in Table 5.

[0073] Table 5 Effects of the combination and single substance on transformation of peripheral blood lymphocytes (A490nm)

[0074] .

[0075] As shown in Table 5, on the 7th day, the A490nm value of peripheral blood lymphocytes in group D was significantly higher than that in group A (P < 0.05); on the 14th day, the A490nm value of peripheral blood lymphocytes in group D was extremely significantly higher than that in other groups (P < 0.01); on the 21st day, the A490nm value of peripheral blood lymphocytes in group D was extremely significantly higher than that in other groups (P < 0.01); on the 28th day, the A490nm value of peripheral blood lymphocytes in group D was extremely significantly higher than that in other groups (P < 0.01).

[0076] 2. Paraffin sections were prepared from the spleen and then stained with HE and examined under a microscope to investigate the effects of three single substances, anthocyanin (200 mg / kg•d), glycyrrhetinic acid (400 mg / kg•d) and C. lycium barbarum glycopeptide (200 mg / kg•d), as well as the combination of anthocyanin (200 mg / kg•d) + glycyrrhetinic acid (400 mg / kg•d) + lycium barbarum glycopeptide (200 mg / kg•d) on the spleen of cows.

[0077] On the 7th, 14th, 21st and 28th days after the first administration, the spleens of two cows randomly selected from each group were completely placed in 10% NBF and stored at room temperature for 3 days. Paraffin sections were prepared according to routine procedures and then HE stained and examined under a microscope. The results of microscopy are shown in Figure 1 .Depend on Figure 1 It can be seen that on the 7th day after the first administration, the lymphocytes of each group were evenly distributed, and there was no significant difference between the groups (P>0.05). Figure 1 On the 14th day, the number of germinal centers in group D was about 25-30; the number of germinal centers in groups A, B, and C was about 20; the number of germinal centers in group D was about 40-50, which was significantly higher than that in groups A, B, and C (P < 0.05). Figure 1On the 21st day, the number of germinal centers in group D was about 30-40; the number of germinal centers in the other groups was about 25-30, which was significantly lower than that in group D (P < 0.05), and the germinal centers were smaller and the splenic sinuses were widened. On the 28th day, the number of germinal centers in group D was about 30-40, and there was no significant difference between the groups (P > 0.05). Figure 1 .

[0078] Embodiment 3:

[0079] This example investigates the anti-aging effects and mechanisms of three single substances, A. anthocyanidins (200 mg), B. glycyrrhetinic acid (400 mg) and C. lycium barbarum glycopeptide (200 mg), and a combination of D. anthocyanidins (200 mg) + glycyrrhetinic acid (400 mg) + lycium barbarum glycopeptide (200 mg) on ​​bovine ovarian granulosa cells.

[0080] In this example, a healthy cow that had not been given any medication was randomly selected, and its ovaries were collected after being slaughtered for the experiment.

[0081] The collected ovaries were used to isolate and culture ovarian granulosa cells in vitro, and transcriptome sequencing was performed to analyze the experimental results. The specific experimental steps are as follows:

[0082] 1. Cultivation of Granulosa Cells (GCs)

[0083] 1.1 Preparation of GCs culture medium

[0084] Prepare 40 ml GCs culture medium (37.6 ml MEM-α culture medium, add 400 µl triple antibody (100×), 2 ml FBS), filter and sterilize, and store at 4°C until use.

[0085] 1.2 Cultivation of GCs

[0086] (1) Take 8 small culture dishes, add 3 ml of PBS solution to each dish and number them ①-⑧. Add 100 µl of tertiary antibody (100×) to dish ①, and add 30 µl of tertiary antibody (100×) to dishes ②-⑧. Shake horizontally to mix.

[0087] (2) Aseptically obtain the ovarian tissue, quickly separate the ovaries, place them in dish No. 1 and rinse them, and then place them in dishes No. 2-6 in sequence and rinse them.

[0088] (3) Place the rinsed ovarian tissue evenly in dishes No. 7-8. Under a stereomicroscope, use a 1 ml sterile syringe to quickly puncture the ovarian tissue to release the granulosa cells.

[0089] (4) Add 2 ml of culture medium to dishes ⑦-⑧, pipette to mix the released granular cells, transfer the cell suspension to a 10 ml centrifuge tube, and centrifuge at 2000 rpm for 5 min.

[0090] (5) Discard the supernatant and add an appropriate amount of GCs culture medium according to the number of cells and the requirements for the plate. Pipette thoroughly to resuspend the cells, plate them, and culture them in a 37°C, 5% CO2 incubator.

[0091] (6) 4-12 hours after inoculation, wash the cells twice with PBS and replace with fresh culture medium. Change the medium every other day.

[0092] 1.3 Passaging of GCs

[0093] (1) Use a pipette to remove the culture medium, add an appropriate amount of PBS to each well to wash the cells and then discard.

[0094] (2) Add an appropriate amount of 0.5% trypsin to each well and place in a 37°C cell culture incubator for 3 min. Add an equal volume of MEM-α culture medium containing 10% FBS to terminate trypsin digestion.

[0095] (3) Use a pipette to blow the cells thoroughly to detach them from the bottom of the culture plate. Transfer the cell suspension to a centrifuge tube and centrifuge at 2000 rpm for 5 minutes.

[0096] (4) Use a pipette to carefully remove the supernatant from the centrifuge tube, add an appropriate amount of GCs culture medium, blow and resuspend the cells, add the cell suspension to the culture plate, and culture in a 37°C, 5% CO2 incubator. Change the medium every other day.

[0097] 1.4 After GCs were stable after passage, four cells A, B, C, and D were separated. Hydrogen peroxide (H2O2) was added to each cell for oxidative modeling. After the cells detached from the cell wall, the corresponding drugs A. anthocyanidin (200 mg), B. glycyrrhetinic acid (400 mg), C. Lycium barbarum glycopeptide (200 mg), and D. anthocyanidin (200 mg) + glycyrrhetinic acid (400 mg) + Lycium barbarum glycopeptide (200 mg) were added.

[0098] 1.5 After the final termination of the culture, transcriptome sequencing was performed.

[0099] 2. Result analysis:

[0100] The effects of anthocyanidins (200 mg) + glycyrrhetinic acid (400 mg) + lycium barbarum glycopeptide (200 mg) combination and anthocyanidins (200 mg), glycyrrhetinic acid (400 mg), and lycium barbarum glycopeptide (200 mg) single substances on primary ovarian granulosa cells are shown in Figure 2-1 , 2-2 , 2-3, 2-4, 2-5, 2-6, 2-7. The specific analysis is as follows:

[0101] The heat map results revealed that there were differences in gene expression among the four groups, see Figure 2-1 .

[0102] The results of GO cluster analysis showed that: the molecular functions of differentially expressed genes were mainly involved in metabolic processes, biological regulation, cellular processes, etc.; the cellular components of differentially expressed genes were mainly concentrated in membranes, organelles, etc.; the biological process analysis revealed that differentially expressed genes were mainly involved in binding, see Figure 2-2 .

[0103] KEGG signaling pathway revealed that the up-regulated genes between group D and other groups were mainly involved in signal transduction, endocrine system, etc. Figure 2-3 .

[0104] KEGG signaling pathway revealed that the down-regulated genes between group D and other groups were mainly involved in signal transduction, endocrine system, immune system, etc. Figure 2-4 .

[0105] GO functional analysis revealed that the differentially expressed genes in group D and other groups were mainly involved in regulating programmed cell death, Ras protein signal transduction, etc. Figure 2-5 .

[0106] KEGG functional analysis revealed that the differentially expressed genes between group B and group C were mainly involved in the MAPK signaling pathway, PI3K-AKT signaling pathway, etc. Figure 2-6 .

[0107] Laser confocal microscopy results showed that group D significantly reduced the production of mitochondrial reactive oxygen species compared with other groups. Figure 2-7 .

[0108] Embodiment 4:

[0109] This example studies the effects of three monomers and one composition on the ovarian aging of the main animal cow by investigating the effects of three monomers and one composition on the main animal cow with persistent corpus luteum and its mechanism. The experiment selected 40 cows with similar body condition (body condition score 5-6), 2 parities, and ovarian quiescence by B-ultrasound examination, and randomly divided them into 4 groups, 10 cows in each group. The 4 groups of dosing regimens were A. anthocyanidin (200 mg / kg•d); B. glycyrrhetinic acid (400 mg / kg•d); C. Lycium barbarum glycopeptide (200 mg / kg•d); D. anthocyanidin (200 mg / kg•d) + glycyrrhetinic acid (400 mg / kg•d) + Lycium barbarum glycopeptide (200 mg / kg•d).

[0110] On the 3rd, 7th, 11th, 15th, 19th, 23rd, 27th and 31st days after the first administration, blood was collected from 2 cows in each group and their intestinal contents were collected through the rectum. The collected blood and intestinal contents were divided into four groups according to the administration regimen (A. anthocyanidins (200 mg / kg•d); B. glycyrrhetinic acid (400 mg / kg•d); C. Lycium barbarum glycopeptide (200 mg / kg•d); D. anthocyanidins (200 mg / kg•d) + glycyrrhetinic acid (400 mg / kg•d) + Lycium barbarum glycopeptide (200 mg / kg•d). Blood was collected for the determination of estrogen (P4), inhibin B (INHB) and other related indicators; the collected intestinal contents were used to determine the intestinal microbial flora. At the same time, for each group of cows, after the first administration, the ovaries were examined by B-ultrasound every three days to pay attention to the development of follicles and corpus luteum. The test results were analyzed.

[0111] The specific detection methods and results are as follows:

[0112] 1. Methods

[0113] 1.1 Serum collection and related index detection

[0114] On days 3, 7, 11, 15, 19, 23, 27 and 31 after the first administration, serum (or plasma) was collected and ELISA was used to detect relevant indicators such as estrogen (P4) and inhibin B (INHB) levels.

[0115] 1.2 Ultrasound examination of follicle development and estrous cycle recovery

[0116] After the first administration, the ovaries of the four groups of cattle were examined by B-ultrasound every three days to pay attention to the development of follicles and corpus luteum.

[0117] 1.3 On the 3rd, 7th, 11th, 15th, 19th, 23rd, 27th and 31st days after the first administration, the intestinal contents (feces) collected should be promptly stored at -80°C for intestinal flora testing.

[0118] 2. Results

[0119] The test results of the effects of the combination D. anthocyanidins (200 mg / kg•d) + glycyrrhetinic acid (400 mg / kg•d) + lycium barbarum glycopeptide (200 mg / kg•d) and the single substances A. anthocyanidins (200 mg / kg•d), B. glycyrrhetinic acid (400 mg / kg•d), and C. lycium barbarum glycopeptide (200 mg / kg•d) on the persistent corpus luteum of cows in the main body are shown in the table. Figure 3-1 , 3-2 , 3-3, 3-4, 3-5, 3-6, 3-7.

[0120] The specific analysis is as follows:

[0121] Compared with other groups, the level of estrogen (P4) in group D was significantly increased. Figure 3-1 .

[0122] Compared with other groups, the level of inhibin B (INHB) in group D was significantly increased. Figure 3-2 .

[0123] The results of the intestinal flora test on the 23rd day of administration are as follows: The genus-level species abundance clustering heat map of group D compared with other groups is shown in Figure 3-3 ; The heat map results revealed that the species abundance of the intestinal flora in group D increased, which was significantly different from that in other groups.

[0124] Ultrasound results revealed that the selected cows had persistent corpora lutea and almost no follicles could be detected. After less than 1 month of treatment, a relatively large number of large follicles could be detected on the ovaries of group D, while only a small number of large follicles could be detected in groups A, B, and C.

[0125] B-ultrasound images of the ovaries of group A cows before and after treatment with anthocyanins (200 mg / kg•d) Figure 3-4 ;

[0126] B-ultrasound images of the ovaries of cattle in group B before and after treatment with glycyrrhetinic acid (400 mg / kg•d) are shown in Figure 3-5 ;

[0127] B-ultrasound images of the ovaries of the cattle in group C before and after treatment with Lycium barbarum glycopeptide (200 mg / kg•d) are shown in Figure 3-6 ;

[0128] B-ultrasound images of the ovaries of group D before and after treatment with the combination of anthocyanins (200 mg / kg•d) + glycyrrhetinic acid (400 mg / kg•d) + wolfberry glycopeptide (200 mg / kg•d) are shown in the figure. Figure 3-7 .

[0129] In summary, oral administration of the composition can significantly change the colonies at the genus and species level, increase the species abundance of the intestinal flora, and have a greater difference compared with oral administration of a single substance. Ultrasound results revealed that in cattle with persistent corpus luteum, after less than one month of medication treatment, more large follicles could be detected on the ovaries of group D cattle, and estrogen (P4) and inhibin B (INHB) increased. The increase in these hormone levels suggests that oral administration of the composition has a better effect in restoring ovarian function.

[0130] The above embodiments are only preferred embodiments of the present invention, and the embodiments of the present invention are not limited to the above embodiments. It should be pointed out that ordinary technicians in this technical field can design many other modified or optimized implementations without departing from the technical principles of the present invention. These modified or optimized implementations will fall within the scope of the present application.

Claims

1. Use of a composition consisting of anthocyanidins, glycyrrhetinic acid and lycium barbarum glycopeptide in the preparation of a drug for treating ovarian aging in cows, characterized in that: The dosage of the composition is anthocyanidin 200 mg / kg•d+glycyrrhetinic acid 400 mg / kg•d+lycium barbarum glycopeptide 200 mg / kg•d.

2. The use of a composition consisting of anthocyanidins, glycyrrhetinic acid and lycium barbarum glycopeptide in the preparation of a feed additive for treating ovarian aging in cows, characterized in that: The dosage of the composition is 200 mg / kg•d of anthocyanidins + 400 mg / kg•d of glycyrrhetinic acid + 200 mg / kg•d of lycium barbarum glycopeptide.

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