阅读说明：本技术 水苏糖在制备预防和/或治疗多囊卵巢综合征药物方面的新用途 (New application of stachyose in preparation of medicine for preventing and/or treating polycystic ovarian syndrome ) 是由 李天鹤 阴赪宏 刘瑞霞 高惠民 于 2020-12-07 设计创作，主要内容包括：本发明公开了水苏糖在制备预防和/或治疗多囊卵巢综合征药物方面的新用途,本发明首次将从天然植物中提取的水苏糖应用于多囊卵巢综合征相关的研究中,并首次证明了水苏糖可明显改善PCOS的排卵功能障碍,恢复大鼠的排卵功能。相对于目前常用的对症治疗的传统药物而言,水苏糖不但不会给人体造成任何副作用和产生依赖性,而且水苏糖又能很好地被肠道有益菌合理利用,对临床上治疗和/或辅助治疗PCOS具有十分重要的意义。(The invention discloses a new application of stachyose in preparing a medicine for preventing and/or treating polycystic ovarian syndrome. Compared with the conventional medicine commonly used for symptomatic treatment at present, the stachyose can not cause any side effect and dependence on a human body, and can be reasonably utilized by intestinal beneficial bacteria, thereby having very important significance for clinical treatment and/or auxiliary treatment of PCOS.)

1. A pharmaceutical composition comprising stachyose.

2. The pharmaceutical composition of claim 1, wherein stachyose is administered in a dose of 200 mg/kg;

preferably, the continuous use period of said stachyose is 12 days.

3. The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable carrier and/or adjuvant.

4. Application of stachyose in preparing medicine for preventing and/or treating polycystic ovary syndrome is provided.

5. The use of claim 4, wherein stachyose is administered in an amount of 200 mg/kg.

6. The use of claim 4, wherein the stachyose is administered for a period of 12 days.

7. A method for non-therapeutically reducing androgen levels, ameliorating polycystic ovarian changes, and restoring ovulation function in a mammal in vitro comprising administering stachyose to the mammal;

preferably, the stachyose is used in a dose of 200 mg/kg;

preferably, the continuous use period of said stachyose is 12 days.

8. The method of claim 7, wherein the method of administration comprises subcutaneous injection, intradermal injection, intramuscular injection, intraperitoneal injection, intravenous injection, oral administration, gastric lavage;

preferably, the method of administration is intragastric.

9. A system for screening candidate drugs for preventing and/or treating polycystic ovary syndrome is characterized by comprising stachyose, a test object and a non-human animal model.

10. The application of stachyose in a screening system of candidate drugs for preventing and/or treating polycystic ovary syndrome.

Technical Field

The invention relates to a new application of stachyose, in particular to a new application of stachyose in preparing a medicine for preventing and/or treating polycystic ovary syndrome.

Background

Stachyose (STA) is a tetrasaccharide with a structural formula of fructose-glucose-galactose and a molecular formula of C₂₄H₄₂O₂₁. The sugar is functional oligosaccharide, has no dependence, and has calorie of 1.5-2.4 kcal/g. It is refined and extracted from natural plants, the pure product is white powder, the taste is slightly sweet, the sweetness is 22 percent of that of cane sugar, the taste is pure, and no bad taste or peculiar smell exists. Can enter colon and be utilized by intestinal bacteria. Stachyose has obvious proliferation effect on beneficial bacteria such as bifidobacterium, lactobacillus and the like in gastrointestinal tract of human body, and can quickly improve the environment in the digestive tract of human body and regulate the balance of microecological bacteria. The stachyose can promote the formation of dominant bacteria of beneficial bacteria in the digestive tract, inhibit the production of putrefying bacteria such as clostridium aerogen and acidogenic bacillus, and the like, and also can produce a large amount of physiological active substances, regulate the pH value of the intestinal tract, kill pathogenic bacteria, inhibit the generation of putrefying products, inhibit the generation and absorption of endogenous carcinogens, decompose and derive multiple immune function factors, and the stachyose has the functions of improving the internal environment of the intestinal tract and improving the immunity of organisms.

Polycystic ovary syndrome (PCOS) is a syndrome of endocrine-metabolic disorders that shows a high degree of clinical heterogeneity, and is the leading cause of anovulatory infertility in women of childbearing age. It is characterized by high androgens, anovulatory abnormalities and ovarian polycystic changes, often accompanied by insulin resistance. However, the pathogenesis of the disease is still unclear at present, and the treatment for the disease is mostly symptomatic treatment, including lifestyle change, androgen level reduction, and control treatment methods of improving metabolic abnormality, ovulation promotion and the like by using an insulin sensitizer, but the method of etiological treatment is lacked. Recently, related researches report that intestinal flora disturbance is an important risk factor of PCOS, and the researches show that the PCOS is closely related to intestinal flora and metabolites thereof (Qi X, Yun C, Sun L, et al. Gut microbial-double acid-interleukin-22 axis organic chemistry, 2019,25(8):1225 + 1233.), which provides an important reference for the research of the pathogenesis of the PCOS.

At present, no research on the application of stachyose in prevention and/or treatment of polycystic ovarian syndrome and the like exists. Aiming at the problems of great side effect on human body, dose dependence and the like of the existing medicine for preventing and treating polycystic ovarian syndrome, stachyose extracted from natural plants is applied to relevant research of polycystic ovarian syndrome, and the research finds that the stachyose has a protection effect on a PCOS rat model induced by DHEA, can obviously improve ovulation dysfunction of the PCOS rat model induced by DHEA and recover ovulation function of the rat.

Disclosure of Invention

The invention aims to provide a new application of stachyose in preparing a medicine for treating and/or preventing polycystic ovarian syndrome, so as to solve the problems that the existing medicine which is clinically used for symptomatic treatment of polycystic ovarian syndrome has large side effect on human bodies, dose dependence and the like.

The purpose of the invention is realized by the following technical scheme:

in a first aspect of the invention, a pharmaceutical composition is provided.

Further, the pharmaceutical composition comprises stachyose;

preferably, the stachyose is used in a dose of 200 mg/kg;

preferably, the continuous use period of said stachyose is 12 days.

Further, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier and/or an auxiliary material.

The stachyose can be used as an active ingredient to be directly prepared into various forms of pharmaceutical preparations with pharmaceutically acceptable carriers and/or auxiliary materials according to the conventional preparation process.

The "pharmaceutically acceptable carrier and/or adjuvant" as defined in the present invention refers to any carrier and/or adjuvant which can be administered to a patient together with stachyose according to the present invention, without destroying the pharmacological activity of stachyose according to the present invention.

Further, the pharmaceutically acceptable carriers and adjuvants used in the pharmaceutical composition of the present invention include (but are not limited to): ion exchangers, alumina, aluminum stearate, lecithin, galactized drug delivery systems (SEDDS), such as d α -tocopherol, polyethylene glycol 1000, succinate, surfactants used in pharmaceutical dosage forms, such as tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as potassium sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block copolymers, polyethylene glycol and lanolin. Cyclodextrins, such as alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin, or chemically modified derivatives, such as hydroxyalkyl cyclodextrins, including 2-and 3-hydroxypropyl-beta-cyclodextrin, or other solubilizing derivatives, may also be used to facilitate delivery of the compounds of the invention.

Modes of administration of the pharmaceutical compositions of the present invention include (but are not limited to): oral administration, parenteral administration, administration by inhalation spray, topical administration, rectal administration, nasal administration, buccal administration, vaginal administration, or administration by implanted reservoir devices, including subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intralesional, and intracranial injection or infusion techniques. Oral administration or injection administration is preferred.

The pharmaceutical compositions of the present invention may contain any of the usual non-toxic pharmaceutically acceptable carriers and/or adjuvants. In some cases, pharmaceutically acceptable acids, bases or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form in which it is administered.

The second aspect of the invention provides the application of stachyose in preparing a medicament for treating polycystic ovary syndrome.

Further, the dosage of the stachyose is 200 mg/kg;

preferably, the continuous use period of said stachyose is 12 days.

In a third aspect of the invention, there is provided a method for non-therapeutically reducing androgen levels in a mammal in vitro, ameliorating changes in ovarian polycystic properties, and restoring ovulation function in a mammal.

Further, the method comprises administering stachyose to the mammal;

preferably, the stachyose is used in a dose of 200 mg/kg;

preferably, the continuous use period of said stachyose is 12 days.

Further, the methods of administration include subcutaneous injection, intradermal injection, intramuscular injection, intraperitoneal injection, intravenous injection, oral administration, gastric lavage;

preferably, the method of administration is intragastric.

Further, the androgens include (but are not limited to): dehydroepiandrosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone sulfate, testosterone.

In an embodiment of the invention, the androgen is preferably testosterone, the indicator of ovarian polycystic change comprises the number of corpus luteum, the number of cystic follicles in ovarian tissue, and the indicator of ovulation function comprises the estrus cycle.

Further, the mammal includes a female mammal;

preferably, the mammal comprises a mammal with polycystic ovary syndrome;

preferably, the mammal comprises a human or non-human mammal;

more preferably, the non-human mammal comprises a rodent, such as a mouse, rat.

In an embodiment of the invention, the non-human mammal is preferably a rat.

The fourth aspect of the invention provides a system for screening candidate drugs for preventing and/or treating polycystic ovarian syndrome, which is characterized by comprising stachyose, a test object and a non-human animal model.

In the embodiment of the present invention, preferably, the stachyose-treated non-human animal model is a positive control group, the test-substance-treated non-human animal model is a test group, and the sesame oil-treated non-human animal model is a negative control group.

Further, the non-human animal model is a non-human mammalian model;

preferably, the non-human mammal comprises a female mammal;

preferably, the female mammal comprises a female mammal suffering from polycystic ovary syndrome;

more preferably, the female mammal includes a rodent, such as a mouse, rat;

most preferably, the female mammal is a rat.

Further, the treatment method comprises subcutaneous injection, intradermal injection, intramuscular injection, intraperitoneal injection, intravenous injection, oral administration and gastric lavage;

preferably, the treatment method is a gavage method.

Furthermore, the system also comprises the steps of respectively detecting androgen level, ovarian polycystic change condition and ovulation function recovery condition of the test group, the positive control group and the negative control group, and comparing the experimental results of the groups.

Further, the androgens include (but are not limited to): dehydroepiandrosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone sulfate, testosterone.

In an embodiment of the invention, the androgen is preferably testosterone.

Further, the testosterone includes (but is not limited to): testosterone in mammalian serum, testosterone in mammalian blood.

In an embodiment of the invention, the testosterone is preferably testosterone in mammalian serum.

Further, the detection indexes of the ovarian polycystic change comprise the number of corpus luteum and the number of cystic follicles in ovarian tissues.

Further, the detection index of ovulation function includes an estrus cycle.

In the present example, the estrus cycle was examined by taking vaginal cell smears of mammals at the same time every afternoon for 10 consecutive days, observing the cell morphology after staining, and recording the analysis.

Further, the staining method includes (but is not limited to): giemsa staining, papanicolaou staining, shore staining, swiss staining.

Preferably, the staining method is preferably a swiss stain. Vaginal cell smears were stained with swiss stain (leageene, 1029a20) in the examples of the invention.

In the embodiment of the invention, the number of corpus luteum and cystic follicle in the ovarian tissue is detected by HE staining the mammalian ovarian tissue and recording the analysis.

Further, the method for detecting androgen includes (but is not limited to): enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (FIA), Radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), time-resolved fluorescence assay (TRFIA).

In the embodiment of the present invention, the detection method of androgen is preferably enzyme-linked immunosorbent assay (ELISA).

Further, if the degree of decrease in androgen level, the degree of improvement in ovarian polycystic change and/or the degree of recovery of ovulation function of the test group test substance in the non-human animal model is significantly higher than that of the negative control group, it is suggested that the test compound is a candidate for the prevention and/or treatment of polycystic ovarian syndrome.

Further, if the ratio of the degree of androgen level reduction, ovarian polycystic change improvement and/or ovulation function recovery of the test object of the test group to the degree of androgen level reduction, ovarian polycystic change improvement and/or ovulation function recovery of the non-human animal model of stachyose of the positive control group is more than or equal to 80%, the test object is suggested to be a candidate drug for preventing and/or treating polycystic ovarian syndrome.

The fifth aspect of the invention provides the application of stachyose in a candidate drug screening system for preventing and/or treating polycystic ovary syndrome.

Further, the screening system is the system according to the fourth aspect of the present invention.

The invention has the advantages and beneficial effects that:

the invention firstly applies the stachyose extracted from natural plants to the research related to the polycystic ovarian syndrome, and firstly proves that the stachyose can obviously improve the ovulation dysfunction of PCOS and recover the ovulation function of rats. Compared with the conventional medicine commonly used for symptomatic treatment at present, the stachyose can not cause any side effect and dependence on a human body, and the stachyose serving as oligosaccharide can be reasonably utilized by intestinal beneficial bacteria well, promotes the self rapid proliferation and further reduces the population quantity of various harmful bacteria.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is an observation of the estrous cycle of rats in each group, wherein, A is a graph: graph of body weight change of rats during molding, graph B: oil + Saline group, Panel C: DHEA + Saline group, Panel D: DHEA + STA group;

fig. 2 is a graph showing the results of HE staining of rat ovarian tissue, in which, a graph: oil + Saline group, Panel B: DHEA + Saline group, panel C: DHEA + STA group, # ovarian corpus luteum, # cystic follicles;

fig. 3 is a statistical plot of the number of cystic follicles, the number of corpus luteum and the level of testosterone in serum in rat ovarian tissue, wherein panel a: number of cystic follicles, panel B: number of corpus luteum, panel C: testosterone levels in serum, P <0.05 compared to Oil + salt group, # 0.05 compared to DHEA + salt group.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

EXAMPLE 1 construction of animal models

3 weeks old SD rats, divided into three groups: control group, PCOS model group, stachyose treatment group, each group 9-11. The construction method of each group of rat models is as follows:

(1) control group (Oil + salt group): 200 mu L of sesame oil is injected subcutaneously, and after 21 days of continuous injection, the normal saline is perfused continuously for 12 days.

(2) PCOS model group (DHEA + salt group): dehydroepiandrosterone (DHEA) (Solarbio, D8950)6mg/100g, 200. mu.L was injected subcutaneously, and after 21 consecutive days, normal saline was gavaged for 12 consecutive days.

(3) Stachyose treatment group (DHEA + STA group): dehydroandrosterone (DHEA) (6mg/100g, 200. mu.L) was injected subcutaneously, and gavage of stachyose (TCI, S0397) (200mg/kg) was administered for 12 consecutive days after 21 consecutive days of injection.

The weight was measured once per week during molding. On day 31 of molding, three groups of experimental animals were divided into 2 groups, and 3 rats in each group were smeared with vaginal cells at the same time every afternoon for 10 consecutive days, and the morphology of the cells was observed after staining with the Leanene's staining solution (LeAGENE, 1029A 20). Blood and ovarian tissue samples were taken after anaesthesia in the remaining rats.

Example 2 Observation of the estrous cycle in rats, Observation of the morphology of ovarian tissue and measurement analysis of serum hormones 1, Observation of the estrous cycle in rats

Rats are animals with a perennial estrus and have a relatively stable sexual cycle, which is divided into: the typical change of the vaginal mucosa of each period is generated in the prophase estrus (P), the anaphase estrus (E), the anaphase estrus (M) and the anaphase estrus (D), and the period can be judged according to the cytological characteristics of the vaginal cell smear. In this example, 3 rats in three experimental animals of a control group (Oil + salt group), a PCOS model group (DHEA + salt group), and a stachyose treatment group (DHEA + STA group) were smeared with vaginal cells at the same time every afternoon for 10 consecutive days, stained with a lawy staining solution (leageee, 1029a20) and then observed for cell morphology, and the results of the experiments were recorded and counted, respectively.

2. Ovarian tissue HE staining

Fresh ovarian tissue was fixed in 4% paraformaldehyde for 24h prior to HE staining. Washing the obtained material in sterilized and precooled PBS, washing blood as thoroughly as possible to avoid the influence of red blood cells on the result when observing slices, dehydrating the material for 1h by 50 percent and 70 percent alcohol respectively, preserving the sample in 70 percent alcohol at 4 ℃ until embedding paraffin, taking out tissue blocks from 70 percent alcohol, dehydrating the tissue blocks in 80 percent, 95 percent and 100 percent alcohol for 1h respectively, and clearing the tissue blocks in 100 percent alcohol/dimethylbenzene (1:1) for 30 min; and then the tissue blocks are transparent in xylene, the specific time is determined according to the size of the tissue blocks, the tissue blocks are preferably completely transparent, the embedding machine can directly receive the heated and filtered paraffin liquid, and the tissue blocks are respectively wax-permeated in xylene/paraffin (1:1), paraffin and paraffin for 2 hours. The method comprises the steps of firstly spreading paraffin with a certain thickness at the bottom of an embedding box, placing the embedding box on a hot table, placing tissues into the hot table by using forceps, adjusting the direction, placing the embedding box on a cold table, slightly solidifying the bottom, continuously pouring the paraffin, placing a slicing support, freezing the cold table, pouring the paraffin, and freezing until the whole paraffin block is molded. And (5) placing the paraffin blocks on a cooling table, and taking out the paraffin blocks by using the slicing support after the paraffin is completely solidified. And (4) tightly fixing the slicing knife rest, and then clamping the wax block on the slicer fixing device by using the slicing support. When slicing, the thickness is adjusted to 5 μm, the wax sheet is cut into pieces, the wax sheet is shot by a small tweezers on the right hand, the wax sheet is gently separated along the blade edge by a brush pen on the left hand, the cut surface faces downwards, the slices are placed into water with the temperature of 42 ℃, after flattening, the continuous wax sheets are gently separated by the tweezers, and then the continuous wax sheets are fished up by a glass slide, dried and placed into a slice box. Baking the slices at 37 ℃ overnight, dewaxing, performing HE dyeing, and recording the experimental results.

3. Assay for serum hormones

ELISA method for detecting testosterone level in rat serum

Respectively anaesthetizing experimental animals in a control group (Oil + Saline group), a PCOS model group (DHEA + Saline group) and a stachyose treatment group (DHEA + STA group), taking blood samples, measuring the serum testosterone level in the blood of rats in each group by adopting an ELISA method, and recording and counting experimental results.

4. Results of the experiment

The results show that the body weight of the rats continuously increased during the molding, and there was no difference in the midbody weight among the three groups of rats (see fig. 1A). The rats in the Oil + salt group have regular estrus cycles (see figure 1B), the rats in the DHEA + salt treatment group have abnormal estrus cycles, the postestrus/episodic period (M/D) is about 3-4 days (see figure 1C), and the relatively regular estrus cycles (see figure 1D) can be recovered to a certain extent after the stachyose treatment, which shows that the stachyose can improve the ovulation dysfunction of the rats induced by DHEA and recover the ovulation function of the rats.

Ovarian tissue HE staining results show that the number of corpus luteum is reduced and the number of cystic follicles is increased in the ovarian tissue of rats in DHEA + Saline group, and the number of corpus luteum is increased and the number of cystic follicles is reduced in the ovarian tissue of rats treated by stachyose (see FIGS. 2A-C and FIG. 3A, B). In contrast to the Oil + Saline group rats, the levels of testosterone in the serum of rats were significantly up-regulated by DHEA + Saline treatment, and decreased and returned to normal levels by stachyose treatment (see FIG. 3C). The stachyose is proved to have a protective effect on DHEA-induced PCOS rats.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.