阅读说明：本技术 绞股蓝皂苷及包含绞股蓝皂苷的药物组合物在卵巢储备和功能保护中的应用 (Application of gypenoside and pharmaceutical composition containing gypenoside in ovary reserve and function protection ) 是由 王世宣 张金金 陈骞 于 2020-12-17 设计创作，主要内容包括：本发明涉及生物医学和药物学技术领域,尤其涉及绞股蓝皂苷及包含绞股蓝皂苷的药物组合物在卵巢储备和功能保护中的应用。本发明通过化疗性卵巢损伤动物模型进行绞股蓝皂苷卵巢储备和功能的验证。研究发现,在化疗药物处理前后经过绞股蓝皂苷干预的小鼠相较于未经皂苷干预的小鼠而言,卵巢得到有效保护,其雌激素和孕酮分泌水平不下降,反而有所升高；产仔数也显著增加；卵泡形态良好,健康卵泡数量更多。本发明提出通过绞股蓝皂苷干预减轻化疗药物所致卵巢损伤,这对于化疗过程中的卵巢功能储备和保护有重要意义。(The invention relates to the technical field of biomedicine and pharmacology, in particular to application of gypenoside and a pharmaceutical composition containing the gypenoside in ovary reserve and function protection. The invention verifies the storage and function of the gypenoside ovary through a chemotherapeutic ovarian injury animal model. Researches show that compared with mice without saponin intervention, ovaries of the mice subjected to gypenoside intervention before and after chemotherapy drug treatment are effectively protected, and the secretion levels of estrogen and progesterone are not reduced but are increased; the litter size also increases significantly; the follicle morphology is good, and the number of healthy follicles is more. The invention provides the intervention and alleviation of ovarian injury caused by chemotherapeutic drugs through gypenoside, which has important significance for ovarian function storage and protection in the chemotherapeutic process.)

1. Application of gypenoside in ovary reserve and function protection is provided.

2. Use of gypenoside for resisting ovarian aging and/or preventing ovarian injury is provided.

3. Application of gypenoside in preparing medicine for resisting ovarian aging and/or preventing ovarian injury is provided.

4. The use of claim 2 or 3, wherein the ovarian senescence is genetically, environmentally, behaviorally, infection or immune-induced ovarian senescence; and/or the ovarian damage is genetic, environmental, behavioral, infection, or immune-induced ovarian damage.

5. The use of claim 3, wherein the ovarian senescence and the ovarian damage are both the result of a chemotherapeutic drug.

6. The use according to claim 5, wherein the chemotherapeutic agent is one or more of cyclophosphamide, cisplatin, paclitaxel or doxorubicin.

7. The use according to any one of claims 1 to 6, wherein the use is: inhibit the decrease of follicle number during the application of chemotherapy drugs, and/or inhibit the decrease of estrogen and progestogen secretion during the application of chemotherapy drugs.

8. A pharmaceutical composition for resisting ovarian aging and/or preventing ovarian damage, which contains gypenoside as an effective component.

9. The pharmaceutical composition of claim 8, wherein the content of gypenoside in the pharmaceutical composition is 60-80% by mass.

10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition is in the form of one or more of tablets, powders, granules, capsules, suppositories, ointments, creams, gels, inhalants, sprays, films, suspensions, troches, elixirs, syrups, water-in-oil or oil-in-water emulsions, oral liquids, or drop pills;

the pharmaceutical composition may further comprise any one or more of stabilizers, solubilizers, buffers, preservatives, thickeners, or other excipients, depending on the dosage form of the pharmaceutical composition.

Technical Field

The invention relates to the technical field of biomedicine and pharmacology, in particular to application of gypenoside and a pharmaceutical composition containing the gypenoside in ovary reserve and function protection.

Background

Ovarian function and fertility are important factors that affect the quality of life of women. The existing factors influencing the female ovarian function comprise: age, genetics, immunity, iatrogenicity, infectivity, environment, ethology, psychology, etc. The search for drugs for ovarian reserve and functional protection is more urgent and important. The iatrogenic ovarian damage caused by chemotherapy is common and urgently needed to be improved in clinic.

Currently, the incidence of cancer is still increasing year by year. With the rapid development of medical technology, the survival rate of cancer patients is remarkably improved, and female tumor surviving patients face the quality of life problem concerning life dignity: ovarian function and fertility.

Chemotherapeutic ovarian lesions are a major cause of ovarian function and loss of fertility in cancer surviving patients. With the younger patients with cancer, the higher incidence rate and the higher cure rate of cancer, more and more female patients suffer from the pain of the reduction of ovarian function and even menopause infertility after receiving chemotherapy. Ovarian function and fertility are among the important reasons affecting the quality of life of women. Meanwhile, after menopause of women, the estrogen level in the body is reduced, the incidence rate of estrogen-related aging-related diseases such as cardiovascular diseases, osteoporosis and the like is remarkably increased, and premature menopause caused by chemotherapy drugs can have serious adverse effects on the work and life of the women after the women enter the middle-aged and the elderly. Therefore, the survival quality of the female tumor survivors needs to be improved urgently.

The existing ovarian function protection measures mainly comprise embryo, oocyte and ovarian tissue cryopreservation, GnRHa and the like. However, most of the measures are invasive operation, the application range is narrow, chemotherapy can be delayed, and the effect is controversial; and is mainly suitable for fertility protection and can not maintain the ovarian function for a long time. The drug-induced prevention strategy has great potential, but the research and development of new drugs are time-consuming and labor-consuming, and the trend is to search potential drugs from old drugs or natural plants.

The gynostemma pentaphylla extract is named as 'southern ginseng' and is a 'medicinal and edible' substance. The main active ingredient of the medicine is gypenoside which is completely the same as ginsenoside in four types. The gypenoside has multiple physiological functions of resisting oxidation, resisting cancer, regulating immunity, etc.

Disclosure of Invention

In order to solve the problems in the prior art, the gypenoside and the pharmaceutical composition containing the gypenoside are applied to ovary storage and function protection. According to the invention, the damage of ovaries caused by various environmental factors is reduced by the gypenoside, so that the storage and the function of the ovaries can be effectively protected, and the damage to the ovaries is reduced.

In a first aspect, the invention provides the use of gypenoside in ovarian reserve and functional protection.

The invention further provides application of the gypenoside in resisting ovarian aging and/or preventing ovarian injury.

The invention further provides application of the gypenoside in medicaments for resisting ovarian aging and/or preventing ovarian injury.

Further, the ovarian senescence is genetically, environmentally, behaviorally, infection, or immune-induced ovarian senescence; and/or the ovarian damage is genetic, environmental, behavioral, infection, or immune-induced ovarian damage.

Further, both the ovarian senescence and the ovarian damage are caused by chemotherapeutic drugs.

Further, the chemotherapeutic drug is one or more of cyclophosphamide, cisplatin, paclitaxel or doxorubicin.

Further, the application is as follows: inhibit the decrease of follicle number during the application of chemotherapy drugs, and inhibit the decrease of secretion of estrogen and progestogen during the application of chemotherapy drugs.

In a second aspect, the present invention provides a pharmaceutical composition for combating ovarian aging and/or preventing ovarian damage, which comprises gypenoside as an active ingredient.

Further, the content of the gypenoside in the pharmaceutical composition is 60-80% by mass ratio.

Further, the dosage form of the pharmaceutical composition is one or more of tablets, powders, granules, capsules, suppositories, ointments, creams, gels, inhalants, sprays, films, suspensions, lozenges, elixirs, syrups, water-in-oil or oil-in-water emulsions, oral liquids or dropping pills;

the pharmaceutical composition may further comprise any one or more of stabilizers, solubilizers, buffers, preservatives, thickeners, or other excipients, depending on the dosage form of the pharmaceutical composition.

The pharmaceutical compositions provided by the present invention may be administered by any suitable route, for example parenterally in the form of injections, or orally in the form of: for example, tablets, capsules, powders, granules, lozenges, dragees, pills, troches, aqueous or anhydrous solutions, suspensions, water-in-oil or oil-in-water emulsions, elixirs or syrups. For parenteral administration, the pharmaceutical composition may be prepared as a powder, suspension, emulsion, sterile injectable solution or dispersion containing sterile powder. The pharmaceutical composition is also provided as a long acting injection. Other suitable administration forms of the pharmaceutical composition are: suppositories, sprays, ointments, creams, gels, inhalants and skin patches. The pharmaceutical composition may be prepared in any of the forms listed above using any method known in the art.

The pharmaceutical composition provided by the invention is administered to a subject in a manner acceptable to the subject mammal, such as injection, oral administration, nasal spray, gavage, rectal, vaginal, and the like. The present invention is preferably intragastric.

Wherein the mammalian subject includes, but is not limited to, rat, mouse, non-human primate, human, dog, cat, horse, cow, sheep, pig, goat. Preferably human or mouse.

According to the prepared dosage form, the pharmaceutical composition provided by the invention can be prepared by mixing various carriers, such as a stabilizer, a solubilizer, a buffer, a preservative, a thickening agent or other excipients in corresponding forms to obtain the pharmaceutical composition with various dosage forms.

The invention also provides a medicine box which comprises the medicine composition and an instruction for use.

The invention has the following beneficial effects:

the study of the invention finds that the gypenoside has the capacity of protecting the ovarian reserve and the ovarian function, and the verification is carried out by constructing a chemotherapeutic ovarian injury mouse model, and the gypenoside is found to be capable of effectively inhibiting the decrease of the number of follicles and the decrease of progestogen in the ovarian injury process caused by chemotherapeutic drugs, so that the gypenoside can be prepared into preparations in various forms and applied to the protection of the ovarian reserve and the ovarian function.

Drawings

FIG. 1 shows the body weight record and ovarian coefficient test results of mice during administration provided by the present invention; wherein A is the change condition of the body weight of the mouse, B is the detection result of the ovarian coefficient of the mouse, NC in A and B is a blank control group, CTX is a simple modeling group, G-L is a low-dose gypenoside group, and G-H is a high-dose gypenoside group;

FIG. 2 shows the result of detecting the endocrine function of mouse ovary provided by the present invention; wherein A is an estrus cycle statistical chart, B is a percentage of regular estrus cycles, C is a serum estrogen level, and D is a serum progestogen level;

FIG. 3 shows the result of the test of the mouse reproductive function provided by the present invention; wherein A is serum estrogen level and B is serum progestin level;

FIG. 4 shows the counting result of the follicles of the mouse under the optical microscope provided by the present invention; wherein A is an ovary HE staining pattern, and B is the result of counting follicles at each stage.

Detailed Description

The invention discloses a gynostemma pentaphylla medicinal composition, a preparation method thereof and application thereof in ovarian function protection. Ovarian damage caused by chemotherapy is a common and accepted ovarian damage model in experimental research, and a treatment mode based on the model research has reference significance for treating multiple ovarian damage factors. Specifically, a certain dose of CTX solution is given to a tested mammal body to induce and generate chemotherapeutic ovarian damage, a tested mammal body containing an effective dose of the gypenoside medicinal composition and a simple control group is given with normal saline, and the ovarian function of the tested mammal body is detected after continuous administration for a period of time.

Definition and use of terms of the invention:

gypenoside: is the main component of the traditional Chinese medicine gynostemma pentaphylla, is a general term of saponin substances separated from the gynostemma pentaphylla, and has the effects of resisting oxidation, resisting cancer, regulating immunity, resisting inflammation and the like.

Chemotherapy: is the abbreviation of chemical drug therapy, and achieves the purpose of treatment by using chemical therapeutic drugs to kill cancer cells. Clinically, chemotherapeutic drugs also have different types, and can be classified into alkylating agents, antimetabolites, antibiotics, antitumor drugs, plant antitumor drugs, hormone antitumor drugs, miscellaneous drugs and the like according to different action mechanisms. Most patients need to be treated by more than two chemotherapeutics in combination, including oral chemotherapeutics, intravenous chemotherapeutics and the like, and in addition, part of patients can also be infused with the drugs through arteries in an interventional mode.

CTX: cyclophosphamide is a nitrogen mustard derivative which enters the human body and is hydrolyzed by an excessive amount of phosphoramidase or phosphatase present in the liver or tumor to become activated phosphoramide.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

The application method comprises the following steps:

s1, preparing a chemotherapeutic ovarian injury mouse model:

under the condition of keeping out of the sun, preparing CTX solution with the concentration of 10mg/ml by using normal saline, and fully mixing and dissolving; weighing the weight of the mice, and calculating the injection amount of each mouse according to the amount of 120mg/kg given by CTX;

fixing the mouse by the left hand, sucking corresponding amount of CTX solution by the right hand, and injecting into the mouse by the abdominal cavity; the blank control group is given with a corresponding amount of normal saline, and the rest process is the same as the model establishment;

s2, drug treatment:

before chemotherapy, the stomach is perfused to administer 100mg/kg of gypenoside in mice with low dose of gypenoside (G-L group) and 400mg/kg of gypenoside in mice with high dose of gypenoside (G-H group) for 14 days, after chemotherapy, the corresponding dose of gypenoside is continuously administered for 14 days, and the blank control group and the mice with a single model group are administered with normal saline with the same volume.

S3, general condition and drug safety:

the body weight of the mice was measured and recorded every two days during the administration period; after intervention is finished, the mice are sacrificed in an estrus period, important organs are taken for weighing and photographing, and an ovarian index is calculated; the results are shown in FIG. 1: when the body weight of mice in the CTX and G-L, G-H groups was reduced after the CTX intervention on day 14, as seen in the graph A in FIG. 1, the body weight of mice in the G-L and G-H groups was eventually higher than that in the CTX group; the ovarian index of mice in groups G-L and G-H was significantly higher than that in group CTX, as seen in FIG. 1B.

S4, ovarian endocrine function detection:

dipping the vaginal secretion of the mouse with normal saline for ten consecutive days to carry out estrus cycle detection; detecting the level of the female progestogen in the serum of the mouse by using an Elisa kit; the results are shown in FIG. 2: as can be seen from A in FIG. 2, the estrous cycles of the G-L and G-H groups of mice are more regular. Further, as shown in fig. 2B, the ratio of the estrous cycle regularity obtained by statistics is higher in the mice treated with the gypenoside. As can be seen from C and D in FIG. 2, estrogen and progesterone levels were not decreased but increased in the mice treated with gypenoside, and the increased levels were higher in the G-H group compared to the G-L group.

S5, reproductive function detection:

reserving 5 mice in each group for cage combination, independently putting each female mouse and one male mouse together during the cage combination period, taking out the male mouse after 10 days, and observing whether the female mouse is pregnant or not and the litter size after 20 days; every 30 days is a period, and 6 periods are carried out; the results are shown in fig. 3, with significant reductions in litter size and average litter size in the CTX group of mice. The average farrowing frequency of mice treated by the gypenoside is not obviously reduced, and the litter size is obviously higher than that of the CTX group.

S6, ovarian reserve function test:

fixing mouse ovary with formalin, sending into continuous section, taking one ovary section at intervals of 80 μm for HE staining, and counting all levels of follicles under an optical microscope; as shown in FIG. 4, A in FIG. 4 shows that the ovarian sections of the mice in the NC group, G-L group and G-H group are observed mainly in the primordial follicles and the growing follicles under the optical microscope, while the ovarian sections of the mice in the CTX group are observed mainly in the atretic follicles. B in fig. 4 shows that the gynosaponin-treated mice had significantly higher numbers of primordial and growing follicles than the CTX group, while the number of atretic follicles was decreased. (PMF is primordial follicle; PF is primary follicle, SF is secondary follicle, ANF is antral follicle, the three are collectively called growing follicle; ATF is atretic follicle)

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.