Application of urolithin A in preparation of medicine or health food for delaying ovarian senescence

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of urolithin A in preparation of medicines or health-care foods for delaying ovarian aging. The invention proves that by supplementing urolithin A, the fertility of the senescent ovary can be improved, the estrus cycle of the senescent ovary can be shortened, the fibrosis level of a senescent ovary matrix can be reduced, the ovary reserve capacity can be improved, the quality of a senescent ovary oocyte can be improved, and the fertilization and embryonic development potential can be improved; it is proved that urolithin A can be used as an effective component for preparing the medicine or health food for delaying ovarian aging.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of urolithin A in preparing a drug or health food for delaying ovarian aging. Background Art

[0002] With the major changes in today's social economy and population structure, social problems caused by ovarian aging are becoming increasingly prominent. Due to changes in social and economic development, fertility concepts and career planning, women's marriage and childbearing age is being postponed. More and more women have missed the best childbearing age due to delayed childbearing and have to face the problem of ovarian aging. In the process of ovarian aging, ovarian function declines, and the number and quality of eggs decrease significantly, seriously affecting women's natural conception. Although assisted reproductive technology can help infertile women to a certain extent, its treatment effect is limited, and egg quality is the key factor in determining the success rate of assisted reproductive technology. Therefore, there is an urgent need to explore methods to improve the function of aging ovaries and improve egg quality.

[0003] At present, researchers are trying to explore measures to prevent and treat ovarian aging in various directions. Existing measures mainly include the following aspects: drug intervention, stem cell therapy, mitochondrial transplantation, ovarian in vitro activation, ovarian transplantation, artificial 3D ovary, etc. Looking at the above measures, there are some problems, such as uncertain efficacy, ethical issues, immature technology, and low patient acceptance. On the other hand, drug treatment is conservative but safe and has high public acceptance.

[0004] Urolithin A is the most important urolithin metabolite in the human body. It belongs to the benzocoumarin class of compounds and is metabolized by intestinal microorganisms in the body from dietary polyphenols ellagitannins and ellagic acid. Studies have shown that urolithin A plays an important role in the prevention and treatment of many diseases, such as cancer, cardiovascular disease, Alzheimer's disease, Parkinson's disease, diabetes, obesity, osteoarthritis, etc. At the same time, compared with precursor polyphenols and live bacterial preparations, urolithin A has the advantages of clear chemical structure, good absorption, metabolism, distribution and excretion. Animal and human experiments have shown that urolithin has high safety and good application value. There is currently no report on the effect of urolithin A in delaying ovarian aging. Summary of the invention

[0005] The purpose of the present invention is to provide the use of urolithin A in the preparation of drugs or health foods for delaying ovarian aging.

[0006] Based on the above purpose, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides the use of urolithin A in the preparation of a drug or health food for delaying ovarian aging, wherein the structural formula of urolithin A is:

[0008]

[0009] According to the above application, preferably, the manifestation of ovarian aging is decreased ovarian fertility.

[0010] According to the above application, preferably, the manifestation of ovarian aging is the prolongation of the estrous cycle of the aged ovaries.

[0011] According to the above application, preferably, the manifestation of ovarian aging is ovarian interstitial fibrosis.

[0012] According to the above application, preferably, the ovarian aging is manifested as a decrease in the ovarian's ability to reserve the total number of follicles, the number of primordial follicles and primary follicles, the number of secondary follicles and / or antral follicles in the ovary.

[0013] According to the above application, preferably, the manifestation of ovarian aging is a decrease in oocyte quality.

[0014] According to the above application, preferably, the manifestation of ovarian aging is a decrease in oocyte fertilization rate and embryo development potential; the embryo development potential evaluation indicators include 4-cell rate, morula rate and blastocyst formation rate.

[0015] The second aspect of the present invention provides a drug, characterized in that the drug contains urolithin A and a pharmaceutically acceptable carrier or excipient, and the structural formula of urolithin A is:

[0016]

[0017] According to the above-mentioned drugs, preferably, the carrier or excipient includes (but is not limited to): diluents, excipients such as lactose, sodium chloride, glucose, urea, starch, water, etc., fillers such as starch, sucrose, etc.; binders such as monosyrup, glucose solution, starch solution, cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; wetting agents such as glycerol; disintegrants such as dry starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate and sodium bicarbonate; absorption promoters quaternary ammonium compounds, sodium lauryl sulfate, etc.; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol, etc.; wetting agents such as glycerol, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin and soap clay, etc.; lubricants such as talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, etc.

[0018] According to the above-mentioned medicine, preferably, the medicine is in oral dosage form.

[0019] The third aspect of the present invention provides a health food, characterized in that urolithin A is added to the health food, and the structural formula of urolithin A is:

[0020]

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention uses drugs on an aged ovarian mouse model, and experiments prove that supplementing urolithin A can improve the fertility of aged ovaries, shorten the estrous cycle of aged ovaries, reduce the level of matrix fibrosis of aged ovaries, delay the decline of aged ovarian reserve, improve the quality of aged ovarian oocytes and improve their fertilization and embryo development potential. It has high safety and good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results of the fertility test of the three groups of mice; Figure A is a representative picture of the litter size of the three groups of mice; Figure B is the statistical result of the average litter size of the three groups of mice; Figure C is the average cumulative litter size of each mouse in the three groups;

[0024] Figure 2 The following are the test results of the estrous cycle of three groups of mice; Figure A is the statistical result of the estrous cycle changes of some mice in the three groups; Figure B is the statistical result of the average estrous cycle of the three groups of mice;

[0025] Figure 3 The results of the detection of ovarian interstitial fibrosis deposition in three groups of mice; Figure A shows the Masson staining results of ovarian sections of three groups of mice, scale: a = 400 μm, b = 80 μm; Figure B shows the statistical results of the percentage of fibrosis area in the ovaries of three groups of mice;

[0026] Figure 4 The results of the ovarian reserve capacity test of the three groups of mice; Figure A shows the HE staining results of the paraffin sections of the ovaries of the three groups of mice and the representative pictures of follicles at all levels, scale: a = 400μm, b = 100μm, c = 25μm; Figures BE show the counting results of the total number of follicles, primordial follicles and primary follicles, secondary follicles, and antral follicles in the ovaries of the three groups of mice;

[0027] Figure 5 The results of the test on the quantity and quality of oocytes of three groups of mice; Figure A is a comparison of the ovaries of three groups of mice; Figure B is a comparison of the microscopic morphology of the spindles and chromosomes of oocytes of three groups of mice; Figure C is a comparison of the microscopic morphology of mature oocytes of three groups of mice; Figure D is the ovary / body weight ratio of three groups of mice; Figure E is the statistical result of the ovulation number of three groups of mice; Figure F is the statistical result of the abnormal rate of oocytes of three groups of mice; Figure G is the statistical result of the abnormal rate of spindles of oocytes of three groups of mice;

[0028] Figure 6Figure 1 shows the test results of oocyte fertilization rate and embryo development potential of three groups of mice; Figure A shows the microscopic morphological comparison of fertilized eggs of three groups of mice at different stages of development; Figure B shows the statistical results of fertilization rate of three groups of mice; Figure C shows the statistical results of 4-cell formation rate of three groups of mice; Figure D shows the statistical results of morula formation rate of three groups of mice; Figure E shows the statistical results of blastocyst formation rate of three groups. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below through embodiments in combination with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] This study used CD1 / ICR mice from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. as experimental animals, including female mice aged 8 weeks and 8 months and male mice aged 3 months. The daily feeding and management of mice included maintaining an ambient temperature of 22-25°C, a humidity of less than 50%, an artificial 12-h light-dark cycle, sufficient supply of pure water and food, and changing the bedding once a week. The experimental methods described in the following examples, unless otherwise specified, are conventional techniques in the art, or in accordance with the conditions recommended by the manufacturer; the reagents, materials and instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0031] Example 1

[0032] Forty 8-month-old CD1 / ICR female mice were randomly divided into two groups: the aged group (Aged) and the aged group supplemented with urolithin A (Aged+UA). The aged group supplemented with urolithin A was intragastrically administered with urolithin A at 50 mg / kg / d, and the aged group was intragastrically administered with an equal amount of a dissolving agent without urolithin A, which was a mixture of 5% VDMSO + 95% V corn oil.

[0033] After 12 weeks of oral administration of the two groups of mice, 20 8-week-old female mice of the same species were selected as the young control group for subsequent experiments. The three groups of mice were caged together for 20 consecutive weeks and the average number of litters per litter and the cumulative number of litters per female mouse were counted. Figure 1 .

[0034] from Figure 1 As can be seen from A and B, compared with the young group, the average litter size of the old group decreased, while the litter size of the old group supplemented with urolithin A increased significantly; Figure 1As can be seen from C, the cumulative number of offspring per female mouse was the highest in the young control group, the lowest in the old control group, and the old group supplemented with urolithin A was in between the two. At the same time, the curve of the old group tended to be smooth after 13 weeks of cage combination, while the old group supplemented with urolithin A showed a step-like rise similar to that of the young control group, indicating that the mice in the old group gradually stopped reproducing, while the mice in the old group supplemented with urolithin A could still continue to reproduce.

[0035] Figure 1 The results showed that supplementation of urolithin A could improve the fertility of aged female mice.

[0036] Example 2

[0037] After 12 weeks of gavage operation in the elderly group and the elderly group supplemented with urolithin A, 10 mice were randomly selected from each of the elderly group, the elderly group supplemented with urolithin A, and the young control group without operation. Vaginal exfoliated cells were collected every morning for 18 consecutive days to make smears, and hematoxylin and eosin staining was performed to detect their estrous cycle. These 10 mice were still used in other subsequent experiments. The results are shown in Figure 2 .

[0038] from Figure 2 It can be seen that compared with the young group, the estrous cycle of the mice in the old group was irregular, and the cycle duration was significantly increased; while the estrous cycle of the mice in the old group supplemented with urolithin A was relatively regular, and the cycle duration was significantly shorter than that of the old group.

[0039] Figure 2 The results showed that supplementation of urolithin A could shorten the estrous cycle of aged female mice.

[0040] Example 3

[0041] After 12 weeks of oral gavage in the elderly group and the elderly group supplemented with urolithin A, three mice were randomly selected from each group, together with the young control group that did not undergo the operation. The ovaries of the three groups of mice were fixed in 4% formaldehyde and made into 5μm paraffin sections. After Masson staining, the deposition of ovarian interstitial fibers in the three groups of mice was observed. Figure 3 .

[0042] from Figure 3 It can be seen that the proportion of ovarian fibrosis in the aged control group was significantly higher than that in the young control group. After supplementing with urolithin A, the fibrosis in the ovaries of aged mice was significantly improved.

[0043] Figure 3 The results showed that supplementation of urolithin A could effectively reduce the level of ovarian fibrosis in aged female mice.

[0044] Example 4

[0045] The same method as in Example 3 was used to obtain paraffin sections of the ovaries of the three groups of mice. After staining with hematoxylin and eosin, the morphology of primordial follicles, primary follicles, secondary follicles, and antral follicles were observed under a microscope and counted. Figure 4 .

[0046] from Figure 4 It can be seen that during the process of ovarian aging, the number of follicles at each level decreases. The total number of follicles, primordial follicles, primary follicles, secondary follicles, and antral follicles in the ovaries of old mice supplemented with urolithin A are all greater than those of old mice not supplemented with urolithin A.

[0047] Figure 4 The results showed that urolithin A could improve the ovarian reserve capacity of aged female mice.

[0048] Example 5

[0049] Six mice randomly selected from each group of the aged group without urolithin A supplementation, the aged group supplemented with urolithin A, and the young control group were weighed and their ovaries were weighed to calculate the ovary / body weight ratio. The experimental method for observing the morphology of oocytes, their spindle morphology and chromosome arrangement under a microscope was as follows: 7.5 IU of pregnant mare serum gonadotropin (PMSG) was injected into the three groups of mice, and 7.5 IU of human chorionic gonadotropin (hCG) was injected 48 hours later. After 14 hours, the cumulus oocyte complexes (COCs) discharged into the ampulla of the oviduct were removed, and then 0.1% hyaluronidase was used to remove the granulosa cells around the oocytes. The morphology of the oocytes was observed and counted under a Zeiss laser confocal LSM880 microscope. The obtained oocytes were fixed in paraformaldehyde for 30 minutes, then permeabilized with 0.5% TritonX-100 for 20 minutes, blocked with 1% BSA for 1 hour, and incubated with 1:200 anti-α-tubulin monoclonal antibody at 4°C overnight. After washing with IVF solution three times, the oocytes were incubated with FITC-conjugated secondary antibody at room temperature for 1 hour, and then the nuclei were stained with propidium iodide (PI), transferred into 10μl antifade droplets, and the spindle morphology and chromosome arrangement were observed under a Zeiss laser confocal LSM880 microscope. The results are shown in Figure 5 .

[0050] Figure 5 A and D show that the ovary / body weight ratio of the elderly group is lower than that of the young group, while the ovary / body weight ratio of the elderly group supplemented with urolithin A is larger than that of the elderly group; Figure 5Figures C and E show that the number of ovulations in the old group decreased compared with the young group, while the number of ovulations in the old group supplemented with urolithin A increased compared with the old group; Figures C and F show that the abnormality rate of oocytes (oocyte fragmentation and division) increased in the old group compared with the young group, while the abnormality rate of oocytes in the old group supplemented with urolithin A decreased; Figure 5 B and G show that the rate of spindle abnormalities (multiple-stage spindles, irregular spindles, abnormal chromosome arrangement) in the aged group was significantly increased compared with the young group, while the rate of spindle abnormalities in the aged group supplemented with urolithin A was significantly reduced.

[0051] Figure 5 The results showed that supplementation with urolithin A could increase the number of ovulations and improve the quality of oocytes in aged female mice.

[0052] Example 6

[0053] The COCs obtained in Example 5 were fertilized in vitro with sperm of 3-month-old male mice treated with in vitro capacitation in IVF droplets for 6 hours, and then the fertilized eggs were transferred to KSOM culture droplets, washed and transferred to new KSOM droplets, and cultured for another 6 hours. The pronuclear formation was observed, and the fertilization rate (2-cell), 4-cell rate, morula rate and blastocyst rate were counted. The results are shown in Figure 6 .

[0054] from Figure 6 It can be seen that compared with the young group, the fertilization rate, 4-cell rate, morula rate, and blastocyst formation rate of the oocytes in the old group were significantly reduced. The fertilization rate, 4-cell rate, and morula rate of the oocytes in the old group of mice supplemented with urolithin A were increased by 21.6%, 18.5%, and 11.7% respectively compared with the old group. In terms of blastocyst formation rate, the old group supplemented with urolithin A was significantly higher than the old control group of the same age.

[0055] Figure 6 The results showed that supplementation with urolithin A could increase the oocyte fertilization rate, 4-cell rate, morula rate and blastocyst formation rate.

[0056] In summary, the present invention proves that supplementing urolithin A can improve the fertility of aging ovaries, shorten the estrous cycle of aging ovaries, reduce the level of stromal fibrosis of aging ovaries, delay the decline of aging ovarian reserve, improve the quality of aging ovarian oocytes and improve their fertilization and embryonic development potential. This shows that urolithin A can be used as an active ingredient for the preparation of drugs that delay ovarian aging. At the same time, studies have shown that oral urolithin A has high safety and can be added to food for ovarian health care. The role of the above embodiment is to illustrate the substantive content of the present invention, but it is not intended to limit the scope of protection of the present invention. It should be understood by those of ordinary skill in the art that the technical solution of the present invention can be modified or equivalently replaced without departing from the essence and scope of protection of the technical solution of the present invention.

Claims

1. The use of urolithin A in the preparation of a drug or health food for delaying ovarian aging, wherein the structural formula of urolithin A is: 。 2. The use according to claim 1, characterized in that: The ovarian aging is manifested as decreased ovarian fertility.

3. The use according to claim 1, characterized in that: The ovarian aging is manifested by the prolonged estrous cycle of the aged ovaries.

4. The use according to claim 1, characterized in that: The manifestation of ovarian aging is ovarian interstitial fibrosis.

5. The use according to claim 1, characterized in that: The ovarian aging is manifested in the decreased ability of the ovary to reserve the total number of follicles, the number of primordial follicles and primary follicles, the number of secondary follicles and / or antral follicles in the ovary.

6. The use according to claim 1, characterized in that: The ovarian aging is manifested by the decline of oocyte quality.

7. The use according to claim 1, characterized in that: The ovarian aging is manifested by a decrease in the oocyte fertilization rate and embryo development potential; the embryo development potential evaluation indicators include the 4-cell rate, the morula rate and the blastocyst formation rate.

8. A drug, characterized in that The drug is made of an active ingredient, urolithin A, and a pharmaceutically acceptable carrier or excipient. The structural formula of urolithin A is: 。 9. The drug according to claim 8, characterized in that the drug is in oral dosage form.

10. A health food, characterized in that: The health food is added with urolithin A, and the structural formula of urolithin A is: 。

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