Use of a composition for the preparation of a medicament for the prevention and treatment of polycystic ovary syndrome

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of a composition in the preparation of a drug for the prevention and treatment of polycystic ovary syndrome (PCOS). The composition comprises bovine spleen peptide and adenosine; the mass ratio of bovine spleen peptide to adenosine is 1:(1-2.5). Bovine spleen peptide, as a bioactive polypeptide, significantly improves insulin resistance associated with PCOS by targeting and activating the PI3K-Akt signaling pathway and promoting insulin receptor substrate phosphorylation. Adenosine, as a natural bioactive substance, significantly reduces serum luteinizing hormone and testosterone levels in rats and increases follicle-stimulating hormone levels, thereby effectively alleviating hyperandrogenism symptoms such as hirsutism and acne in PCOS. Furthermore, this composition can significantly improve polycystic pathological changes in the ovarian tissue of model rats, reduce the number of cystic dilated follicles, restore the granulosa cell layer structure, alleviate ovarian stromal hyperplasia, and promote normal follicular development.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a composition in the preparation of a drug for the prevention and treatment of polycystic ovary syndrome. Background Technology

[0002] Polycystic ovary syndrome (PCOS) is one of the most common endocrine and metabolic disorders in women of reproductive age. Clinically, it mainly manifests as menstrual irregularities, hirsutism, acne, infertility, oily skin, and acanthosis nigricans, often accompanied by insulin resistance and obesity. Epidemiological data shows that the prevalence of PCOS in women of reproductive age is approximately 5-10%, with a prevalence of 6.05% in my country, accounting for 13.69% of infertile patients. In recent years, the incidence of this disease has been on the rise among young women, becoming a significant factor affecting women's reproductive health and quality of life.

[0003] The exact cause of PCOS is not yet fully understood, but it is generally believed to be the result of a combination of genetic, environmental, lifestyle, and psychosocial factors. Clinical treatment for PCOS mainly focuses on improving insulin resistance, correcting hyperandrogenism, and promoting ovulation. Common methods include drug therapy (such as oral contraceptives, progestins, metformin, etc.), lifestyle interventions, psychological support, surgery, and assisted reproductive technologies. However, existing drugs often have adverse reactions that may worsen metabolic disorders, and their overall treatment effectiveness is limited. Therefore, there is an urgent need to develop safer, more comprehensive, and sustainable new therapies.

[0004] Bovine spleen peptides are small-molecule bioactive peptides extracted from bovine spleen using enzymatic hydrolysis technology. They are rich in various amino acids and immunomodulatory components. Studies have shown that bovine spleen peptides have anti-inflammatory, immunomodulatory, and glucose-lipid metabolism-improving effects. Adenanthin is a natural diterpenoid compound extracted from plant glandular flowers. It has anti-androgenic effects and regulates estrogen receptor function. In addition, this component also has antioxidant and microcirculation-improving effects, which help optimize the local environment of the ovary and promote normal follicle development.

[0005] Against this background, this invention proposes for the first time to scientifically combine adenosine with bovine spleen peptides, aiming to provide a new prevention and treatment strategy that is more systematic and safer in improving insulin resistance and regulating hormone balance through multi-target and multi-pathway synergistic effects. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide an application of a composition in the preparation of a drug for the prevention and treatment of polycystic ovary syndrome, which has broad prospects.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The use of a composition in the preparation of a drug for the prevention and treatment of polycystic ovary syndrome, the composition comprising bovine spleen peptide and adenosine; wherein the mass ratio of bovine spleen peptide to adenosine is 1:(1-2.5).

[0008] Furthermore, the preparation method of the bovine spleen peptide includes the following steps: Water and protease were added to bovine spleen for enzymatic hydrolysis, followed by centrifugation and ultrafiltration membrane fractionation to obtain the bovine spleen peptide.

[0009] Furthermore, the mass ratio of bovine spleen, water, and protease is 1:(10-15):(0.002-0.004).

[0010] Furthermore, the protease is composed of papain, trypsin, and alkaline protease in a mass ratio of 1:(0.5-1):(0.5-0.8).

[0011] Furthermore, the enzymatic hydrolysis temperature is 50-60℃ and the time is 6-8h; the molecular weight cutoff of the ultrafiltration membrane is 5kDa and 3kDa, respectively.

[0012] Furthermore, the preparation method of the composition is as follows: Bovine spleen peptides, adenosine, and pharmaceutically acceptable excipients are mixed evenly to prepare the appropriate dosage form.

[0013] Furthermore, the dosage form is an oral preparation.

[0014] Furthermore, the oral preparation is a granule, capsule, or tablet.

[0015] Furthermore, the excipients are selected from at least one of adhesives, disintegrants, and lubricants.

[0016] Furthermore, the adhesive is selected from corn starch, glucose, and cyclodextrin; the lubricant is selected from sodium stearoyl fumarate, magnesium stearate, talc, and sucrose fatty acid ester; and the disintegrant is sodium carboxymethyl starch or crospovidone.

[0017] Compared with the prior art, the main advantages of the present invention are as follows: This invention provides the application of a composition in the preparation of a drug for the prevention and treatment of polycystic ovary syndrome (PCOS). The composition comprises bovine spleen peptide and adenine, which synergistically enhance each other, providing an effective prevention and treatment strategy for PCOS by targeting multiple aspects, including improving insulin resistance and hyperandrogenemia. Specifically, bovine spleen peptide, as a bioactive polypeptide, significantly improves insulin resistance associated with PCOS by targeting and activating the PI3K-Akt signaling pathway and promoting phosphorylation of insulin receptor substrates. Adenine, as a natural bioactive substance, significantly reduces serum luteinizing hormone and testosterone levels in rats and increases follicle-stimulating hormone levels, thereby effectively alleviating hyperandrogenemia symptoms such as hirsutism and acne in PCOS. Furthermore, the composition of this invention can significantly improve polycystic pathological changes in the ovarian tissue of model rats, reduce the number of cystic dilated follicles, restore the granulosa cell layer structure, alleviate ovarian stromal hyperplasia, and promote normal follicular development. Attached Figure Description

[0018] Figure 1 PI3K mRNA expression level results; Figure 2 Pathological sections of rat ovarian tissue from each group. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to specific embodiments and accompanying drawings. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0020] In this invention, the protease is composed of papain, trypsin, and alkaline protease in a mass ratio of 1:0.6:0.7.

[0021] Example 1 A composition comprising bovine spleen peptide and adenosine in a mass ratio of 1:2.

[0022] The preparation method of the above-mentioned bovine spleen peptide is as follows: with the mass ratio of bovine spleen, water and protease of 1:12:0.003, water and protease are added to fresh bovine spleen, mixed evenly, heated to 55℃ for 7h for enzymatic hydrolysis, cooled to room temperature and centrifuged, and the supernatant is passed through ultrafiltration membranes with molecular weight cutoffs of 5kDa and 3kDa step by step to obtain bovine spleen peptide with a molecular weight of 3-5kDa.

[0023] The preparation method of the above composition is as follows: After mixing bovine spleen peptide and adenosine evenly, add 12% corn starch, 0.6% magnesium stearate and 5% sodium carboxymethyl starch by weight of the total drug, mix well and compress into tablets according to conventional tablet preparation process.

[0024] Example 2 A composition comprising bovine spleen peptide and adenosine in a mass ratio of 1:1.

[0025] The preparation method of the above-mentioned bovine spleen peptide is as follows: with the mass ratio of bovine spleen, water and protease of 1:10:0.002, water and protease are added to fresh bovine spleen, mixed evenly, heated to 50℃ for 8 hours for enzymatic hydrolysis, cooled to room temperature and centrifuged, and the supernatant is passed through ultrafiltration membranes with molecular weight cutoffs of 5kDa and 3kDa step by step to obtain bovine spleen peptide with a molecular weight of 3-5kDa.

[0026] The preparation method of the above composition is as follows: After mixing bovine spleen peptide and adenosine evenly, add 12% corn starch, 0.6% magnesium stearate and 5% sodium carboxymethyl starch by weight of the total drug, mix well and compress into tablets according to conventional tablet preparation process.

[0027] Example 3 A composition comprising bovine spleen peptide and adenosine in a mass ratio of 1:2.5.

[0028] The preparation method of the above-mentioned bovine spleen peptide is as follows: with the mass ratio of bovine spleen, water and protease of 1:15:0.004, water and protease are added to fresh bovine spleen, mixed evenly, heated to 60℃ for 6 hours for enzymatic hydrolysis, cooled to room temperature and centrifuged, and the supernatant is passed through ultrafiltration membranes with molecular weight cutoffs of 5kDa and 3kDa step by step to obtain bovine spleen peptide with a molecular weight of 3-5kDa.

[0029] The preparation method of the above composition is as follows: After mixing bovine spleen peptide and adenosine evenly, add 12% corn starch, 0.6% magnesium stearate and 5% sodium carboxymethyl starch by weight of the total drug, mix well and compress into tablets according to conventional tablet preparation process.

[0030] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the molecular weight of bovine spleen peptide is 0-2 kDa.

[0031] The preparation method of bovine spleen peptide is as follows: with the mass ratio of bovine spleen, water and protease of 1:12:0.003, water and protease are added to fresh bovine spleen, mixed evenly, heated to 55℃ for 7h for enzymatic hydrolysis, cooled to room temperature and centrifuged, and the supernatant is passed through an ultrafiltration membrane with a molecular weight cutoff of 2kDa to obtain bovine spleen peptide with a molecular weight of 0-2kDa.

[0032] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that adenosine is omitted.

[0033] Experimental Example 1 Bovine spleen peptide and adenosine were added to DMEM complete medium at a mass ratio of 1:2.5 to obtain a final concentration of 50 μg / mL for bovine spleen peptide and 125 μg / mL for adenosine. The test solution was then prepared for use.

[0034] L929 fibroblasts in the logarithmic growth phase were harvested and their density adjusted to 1×10⁻⁶ cells using DMEM complete medium. 5 Cells were seeded at a density of 100 μL / well in 96-well plates and cultured at 37°C with 5% CO2 for 24 h. After cell attachment, the culture medium in each well was gently aspirated. 200 μL of the above-mentioned test solution was added to each well of the experimental group. A blank control group was set up, with only an equal volume of DMEM complete medium (without drugs or cell seeding). A negative control group was also set up, with an equal volume of DMEM complete medium (without drugs) added to each well. Each group had 6 replicates, and the cells were cultured for another 24 h. 10 μL of MTT reagent (5 mg / mL) was added to each well, and the cells were cultured for another 4 h. The supernatant was aspirated, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. The plates were shaken for 10 min to dissolve any crystals. The absorbance was measured at 490 nm using a microplate reader, and cell viability was calculated using the following formula: Cell viability (%) = [(OD value of experimental group - OD value of blank control group) / (OD value of negative control group - OD value of blank control group)] × 100%, the results are shown in Table 1.

[0035] Table 1. Effects of the drug of the present invention on the viability of L929 fibroblasts. As shown in Table 1, the drug of the present invention has no obvious cytotoxicity to L929 fibroblasts, and the cell viability is almost the same as that of the negative control group, indicating that bovine spleen peptide and adenosine in this concentration range can be safely used for subsequent related cell experiments.

[0036] Example of effect 1 (1) Establishment and grouping of PCOS-IR models Eighty 3-week-old female SPF-grade SD rats were housed in a temperature- and humidity-controlled environment with free access to food and water. After one week of acclimatization, experiments were conducted. Ten rats were randomly selected as the normal control group and injected with 0.2 mL of injectable sesame oil for 20 consecutive days. The remaining rats received subcutaneous injections of dehydroepiandrosterone (DHEA) 6 mg / 100 g + 0.2 mL of injectable sesame oil daily in the neck and back for 20 consecutive days. Vaginal exfoliative cytology was observed for 10 days. Rats with persistently keratinized vaginal epithelial cells were selected as successfully DHEA-induced PCOS animal models. Successful model rats were fasted at 8 PM on the first night, and fasting blood glucose and insulin were measured by collecting blood from the orbital vein at 8 AM the following morning. The insulin resistance index was calculated as: Rat insulin resistance index = fasting blood glucose × fasting insulin / 22.5. Rats with an insulin resistance index greater than 2.8 were considered successfully induced PCOS-IR model animals and continued in subsequent experiments, randomly divided into a model group, Example 1-3 groups, and Comparative Example 1-2 groups.

[0037] (2) Drug intervention Normal group: Administered an equal volume of normal saline by gavage for 28 days; Model group: administered an equal volume of physiological saline by gavage for 28 days; Example 1 group: The composition of Example 1 was administered by gavage at a dose of 50 mg / kg bovine spleen peptide + 100 mg / kg adenosine, once a day for 28 days; Example 2 group: The composition of Example 2 was administered by gavage at a dose of 50 mg / kg bovine spleen peptide + 50 mg / kg adenosine, once a day for 28 days; Example 3 group: The composition of Example 3 was administered by gavage at a dose of 50 mg / kg bovine spleen peptide + 125 mg / kg adenosine, once a day for 28 days; Comparative Example 1: The composition of Comparative Example 1 was administered by gavage at a dose of 50 mg / kg bovine spleen peptide + 100 mg / kg adenosine, once a day for 28 days. Comparative Example 2: The drug in Comparative Example 2 was administered by gavage at a dose of 50 mg / kg bovine spleen peptide once a day for 28 days.

[0038] (3) Observation indicators 1. After the last administration, rats in each group were fasted overnight. The rats were sacrificed the following day, blood was collected from the heart, adipose tissue was dissected, and both ovaries were removed. One ovary was weighed, and total RNA was extracted from the ovarian tissue of the other ovary using the TRIzol method. The expression level of PI3K mRNA was detected using a reverse transcription cDNA kit and real-time quantitative PCR. Primer sequences are shown in Table 2. -ΔΔCt The method calculates relative expression; see results below. Figure 1 And Table 3.

[0039] 2. Blood was collected and centrifuged at 2500 r / min for 20 min to obtain serum for later use. A portion of the serum was used to determine the levels of testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) using radioimmunoassay. The remaining serum was used to determine fasting blood glucose and fasting insulin levels, and the insulin resistance index of rats was calculated. The results are shown in Table 4.

[0040] 3. Ovarian tissues from rats in each group were collected, fixed in 4% paraformaldehyde solution, routinely embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), dehydrated, cleared, and mounted. Pathological changes in the ovarian tissues were observed under a light microscope; results are shown below. Figure 2 .

[0041] Table 2 Primer Sequences Table 3. Effects of each drug group on ovarian quality and PI3K mRNA levels Table 4. Effects of each drug group on hormones and insulin resistance index in rats. As shown in Table 3, the ovarian mass of rats in the model group increased compared to the normal group. Compared to the model group, the ovarian mass of rats decreased after using the drugs in Examples 1-3 and Comparative Examples 1-2. Experimental Conclusion: The pharmaceutical composition of the present invention alleviates the symptoms of polycystic ovary syndrome (PCOS) by reducing the ovarian mass of rats with PCOS.

[0042] From Table 3, Table 4 and Figure 1 As can be seen, compared with the normal group, the expression level of PI3K mRNA in the ovarian tissue of rats in the model group was decreased, and the insulin resistance index was increased. Compared with the model group, after using the drugs of Examples 1-3 and Comparative Examples 1-2, the expression level of PI3K mRNA in the ovarian tissue of rats increased, and the insulin resistance index decreased. The effect of Example 1 was the best, and the effect of Comparative Example 1 was the worst. Experimental conclusion: The pharmaceutical composition of the present invention can significantly improve the insulin resistance status of patients with polycystic ovary syndrome by targeting and activating the PI3K-Akt signaling pathway and promoting the phosphorylation of insulin receptor substrates.

[0043] Table 4 also shows that, compared to the normal group, the serum T and LH levels in the model group rats were increased, while the FSH level was decreased. Compared to the model group, after using the drugs in Examples 1-3 and Comparative Examples 1-2, the serum T and LH levels in rats were decreased, while the FSH level increased. Example 1 showed the best effect, while Comparative Example 2 showed the worst effect. Experimental conclusion: The pharmaceutical composition of the present invention can significantly reduce the serum T and LH levels in rats and increase the FSH level, thereby effectively alleviating hyperandrogen symptoms such as hirsutism and acne in polycystic ovary syndrome.

[0044] Depend on Figure 2 It was found that the ovaries of the normal group rats had intact structures, with follicles at different developmental stages (primordial follicles, primary follicles, secondary follicles, and mature follicles). The follicles were regularly shaped, with intact and tightly arranged granulosa cells, and no obvious cystic dilatation of follicles. The ovarian stroma was evenly distributed without significant proliferation, exhibiting typical histological characteristics of a healthy ovary. Compared with the normal group, the ovarian tissue of the model group rats showed typical polycystic pathological changes: a large number of cystic dilatation follicles (thinned follicle walls, cavities filled with cystic fluid), a significantly reduced and disordered arrangement of the granulosa cell layer, and even detachment of the follicles; significant proliferation and fibrosis of the ovarian stroma, with no normal mature follicles observed, consistent with the pathological characteristics of a polycystic ovary syndrome model. After drug intervention, the ovarian pathological changes in the rats of Examples 1-3 were significantly improved: the number of cystic dilatation follicles was significantly reduced, and the follicle wall structure was restored; the arrangement of the granulosa cell layer tended to be intact, and the number of layers increased; ovarian stroma proliferation was reduced, and follicles at different developmental stages were visible, and follicle development disorders were significantly alleviated. In Comparative Example 1, the ovaries of rats still showed numerous cystic dilated follicles, a thinned granulosa cell layer, and significant ovarian stroma hyperplasia. In Comparative Example 2, the ovaries of rats showed numerous cystic dilated follicles, an incomplete granulosa cell layer, significant ovarian stroma hyperplasia, and poor follicular development. These results indicate that the composition of this invention can effectively improve the polycystic pathological changes in the ovaries of rats with polycystic ovary syndrome and promote normal follicular development. The synergistic combination of bovine spleen peptide with a specific molecular weight and adenosine is the key factor in achieving this effect.

[0045] As shown above, bovine spleen peptide, as a bioactive polypeptide, significantly improves insulin resistance in patients with polycystic ovary syndrome (PCOS) by targeting and activating the PI3K-Akt signaling pathway and promoting phosphorylation of insulin receptor substrates. Adenine, as a natural bioactive substance, significantly reduces serum luteinizing hormone (LH) and testosterone levels in rats while increasing follicle-stimulating hormone (FSH) levels, thereby effectively alleviating hyperandrogenism symptoms such as hirsutism and acne in PCOS. Simultaneously, the composition of this invention can significantly improve polycystic pathological changes in the ovarian tissue of model rats, reduce the number of cystic dilated follicles, restore the granulosa cell layer structure, alleviate ovarian stromal hyperplasia, and promote normal follicular development. The synergistic effect of these two ingredients, targeting multiple aspects such as improving insulin resistance and hyperandrogenemia, provides an effective prevention and treatment strategy for PCOS.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. The use of a composition in the preparation of a drug for the prevention and treatment of polycystic ovary syndrome, characterized in that, The composition comprises bovine spleen peptide and adenosine; the mass ratio of bovine spleen peptide to adenosine is 1:(1-2.5).

2. The use of the composition according to claim 1 in the preparation of a medicament for the prevention and treatment of polycystic ovary syndrome, characterized in that, The preparation method of the bovine spleen peptide includes the following steps: Water and protease were added to bovine spleen for enzymatic hydrolysis, followed by centrifugation and ultrafiltration membrane fractionation to obtain the bovine spleen peptide.

3. The use of the composition according to claim 2 in the preparation of a medicament for the prevention and treatment of polycystic ovary syndrome, characterized in that, The mass ratio of bovine spleen, water, and protease is 1:(10-15):(0.002-0.004).

4. The use of the composition according to claim 3 in the preparation of a medicament for the prevention and treatment of polycystic ovary syndrome, characterized in that, The protease is composed of papain, trypsin, and alkaline protease in a mass ratio of 1:(0.5-1):(0.5-0.8).

5. The use of the composition according to claim 2 in the preparation of a medicament for the prevention and treatment of polycystic ovary syndrome, characterized in that, The enzymatic hydrolysis is performed at a temperature of 50-60℃ for 6-8 hours; the ultrafiltration membrane has a molecular weight cutoff of 5kDa and 3kDa, respectively.

6. The use of the composition according to claim 1 in the preparation of a medicament for the prevention and treatment of polycystic ovary syndrome, characterized in that, The composition is prepared as follows: Bovine spleen peptides, adenosine, and pharmaceutically acceptable excipients are mixed evenly to prepare the appropriate dosage form.

7. The use of the composition according to claim 6 in the preparation of a medicament for the prevention and treatment of polycystic ovary syndrome, characterized in that, The dosage form is an oral preparation.

8. The use of the composition according to claim 7 in the preparation of a medicament for the prevention and treatment of polycystic ovary syndrome, characterized in that, The oral preparation is a granule, capsule, or tablet.

9. The use of the composition according to claim 6 in the preparation of a medicament for the prevention and treatment of polycystic ovary syndrome, characterized in that, The excipients are selected from at least one of adhesives, disintegrants, and lubricants.

10. The use of the composition according to claim 9 in the preparation of a medicament for the prevention and treatment of polycystic ovary syndrome, characterized in that, The adhesive is selected from corn starch, glucose, and cyclodextrin; the lubricant is selected from sodium stearoyl fumarate, magnesium stearate, talc, and sucrose fatty acid ester; and the disintegrant is sodium carboxymethyl starch or crospovidone.

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