Application of sheep placenta extract in preparation of medicine for improving polycystic ovarian syndrome

Abstract

The invention discloses an application of a sheep placenta extract in preparation of a medicine for improving polycystic ovarian syndrome, and relates to the technical field of biological medicine, the medicine is used for improving endocrine disorder and abnormal inflammation caused by the polycystic ovarian syndrome, and particularly for reducing the plasma androgen level and increasing the plasma estrogen level and the plasma progestational hormone level; the level of an inflammatory factor TNF-alpha is reduced, and the level of an inflammatory factor IL-10 is increased. The sheep placenta extract can improve ovarian polycystic lesions caused by polycystic ovarian syndrome (PCOS) in a targeted manner, and experiments show that after intervention of the sheep placenta extract, the number of macroscopic cystic follicles in rat ovary is reduced, the weight of the ovary with abnormal weight gain is remarkably reduced, the cystic follicle ratio in ovarian histopathology detection is greatly reduced, and the sheep placenta extract has a good application prospect. The ratio of the number of the corpus luteum is closer to normal, and the pathological injury of the polycystic ovarian syndrome is repaired from the organ level.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically the application of sheep placental extract in the preparation of drugs to improve polycystic ovary syndrome. Background Technology

[0002] Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder with a prevalence of 5% to 10% in women of reproductive age. PCOS patients primarily present with metabolic abnormalities and other clinical features, including hyperandrogenism, hyperinsulinemia, obesity, dyslipidemia, insulin resistance, ovulation dysfunction, and menstrual irregularities. Furthermore, PCOS is often accompanied by chronic low-grade inflammation. Current treatments for PCOS mainly include lifestyle interventions, oral contraceptives, insulin sensitizers, and anti-androgen drugs. However, these treatments are prone to side effects such as weight gain, thrombosis risk, or liver damage, and their efficacy is unstable, making it difficult to fundamentally improve the pathological changes in the ovaries.

[0003] The placenta is a vital organ for exchange between the fetus and mother, rich in various bioactive components, including cytokines, growth factors, hormones, and peptides. Sheep placental extract (SPE) is a representative traditional Chinese medicine, containing abundant proteins and amino acids, peptides and growth factors, enzymes, vitamins and minerals, and immunomodulatory substances. Sheep placental extract is widely available, safe, and economical, and has been proven to possess various physiological effects, including anti-inflammatory, wound-healing-accelerating, antioxidant, and anti-aging effects. Studies have shown that sheep placental extract has broad biological effects and potential clinical application value in anti-inflammatory, antioxidant, immunomodulatory, and tissue-repairing fields. Therefore, the application of sheep placental extract in the preparation of drugs to improve polycystic ovary syndrome (PCOS) is proposed. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides the application of sheep placental extract in the preparation of drugs to improve polycystic ovary syndrome, thus solving the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: the application of sheep placental extract in the preparation of drugs to improve polycystic ovary syndrome.

[0006] Preferably, a letrozole-induced 6-week-old female SD rat model was used to demonstrate the application of the sheep placental extract in improving polycystic ovary syndrome.

[0007] Preferably, the sheep placenta extract is a freeze-dried sheep placenta powder.

[0008] Preferably, the drug is used to improve polycystic ovarian lesions and uterine atrophy caused by polycystic ovary syndrome, specifically by reducing the number of ovarian follicles, reducing the abnormally increased ovarian weight, and improving uterine atrophy and abnormal thickening of the endometrium.

[0009] Preferably, the drug is used to improve inflammatory disorders caused by polycystic ovary syndrome, specifically by reducing plasma pro-inflammatory factor TNF-α levels and increasing plasma anti-inflammatory factor IL-10 levels.

[0010] Preferably, the drug is used to improve metabolic disorders caused by polycystic ovary syndrome, specifically by increasing serum high-density lipoprotein cholesterol levels.

[0011] Preferably, the method of administration of the drug prepared from the sheep placental extract includes oral administration.

[0012] Preferably, when the sheep placenta extract is dissolved, the solvent includes sodium carboxymethyl cellulose.

[0013] This invention provides the application of sheep placental extract in the preparation of drugs to improve polycystic ovary syndrome, which has the following beneficial effects:

[0014] The sheep placental extract of this invention contains a variety of bioactive components. Sheep placental extract can prevent and improve polycystic ovary syndrome by improving or alleviating pathological damage to the ovaries and uterus, improving abnormal blood lipid disorders and inflammatory responses. This invention uses animal-derived natural active medicine rich in sheep placental extract to assist or replace traditional drug treatments, such as contraceptives, insulin sensitizers, and anti-androgen drugs. Sheep placental extract is safe, effective, economical, suitable for long-term use, and has significant preventive and therapeutic effects. Attached Figure Description

[0015] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a diagram of the rat experiment design in an embodiment of the present invention;

[0017] Figure 2 These are the ovarian morphology and ovarian weight detection results in this embodiment of the invention;

[0018] Figure 3 The results of ovarian tissue pathology examination in this embodiment of the invention;

[0019] Figure 4 These are the uterine morphology, uterine weight, and pathological examination results in the embodiments of the present invention;

[0020] Figure 5 These are the results of plasma lipid level detection in rats in each group in this embodiment of the invention;

[0021] Figure 6 The results of plasma inflammatory factor level detection in each group of rats in this embodiment of the invention are shown. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] This invention provides the application of sheep placental extract in the preparation of drugs for improving polycystic ovary syndrome. To achieve the above objective, this invention is implemented through the following technical solution:

[0024] Using a letrozole-induced 6-week-old female SD rat model, the application of sheep placental extract in improving polycystic ovary syndrome was demonstrated.

[0025] The medication is used to improve polycystic ovarian changes and uterine atrophy caused by polycystic ovary syndrome, specifically by reducing the number of cystic or degenerated follicles in the ovaries, reducing the abnormally heavy ovarian weight, and restoring the uterine morphology, pathology, and abnormal uterine weight changes.

[0026] The medication is used to improve metabolic disorders caused by polycystic ovary syndrome, specifically by increasing serum high-density lipoprotein cholesterol levels.

[0027] The medication is used to improve the inflammatory disorders caused by polycystic ovary syndrome, specifically by reducing the level of plasma inflammatory factor TNF-α and increasing the level of anti-inflammatory factor IL-10.

[0028] The sheep placenta extract is specifically a freeze-dried sheep placenta powder extract.

[0029] Pharmaceutical formulations of drugs include tablets, soft capsules, and oral preparations.

[0030] Drugs prepared from sheep placental extracts can be administered orally.

[0031] When dissolving sheep placenta extract, the solvent includes sodium carboxymethyl cellulose.

[0032] Example 1

[0033] Experimental design for studying the effects of sheep placental extract on PCOS and preparation of a rat model of polycystic ovary syndrome.

[0034] 1. Laboratory animals and model making

[0035] Twenty-one 6-week-old female SPF-grade SD rats, weighing 130–150g, were purchased from the Experimental Animal Center of Ningxia Medical University. After a week of acclimatization, they were used for experiments. The housing environment met SPF standards. The rats were housed in an SPF-grade animal room with environmental conditions of 22–25℃ and 50%–60% humidity, with a 12-hour light-dark cycle (light time 8:00–20:00). They were given free access to standard rat food (containing 20%–22% protein and 4%–5% fat) and free access to sterilized purified water. After a week of acclimatization, the rats' activity, diet, and defecation were observed daily. Once no abnormalities were found, the rat model was established.

[0036] Modeling drug: letrozole (purity ≥98%), an aromatase inhibitor, can induce PCOS-like hyperandrogenemia, polycystic ovarian changes and ovulation disorders in rats by inhibiting estrogen synthesis;

[0037] Reagent preparation: Weigh the corresponding mass of lexiconazole according to the dosage of "lexiconazole 1 mg / kg / d" and administer it by gavage for 4 weeks. Record the rat's weight every 3-4 days during this period (weighing at a fixed time each week) and plot the weight change curve. Adjust the gavage volume according to the weight change (ensuring that the lexiconazole dose remains at 1 mg / kg / d). At the same time, observe the rat's estrous behavior (monitored by vaginal smear method; the normal estrous cycle of female rats is 4-5 days, while PCOS model rats will show estrous cycle disorder or persistent anestrus).

[0038] Vaginal smears from rats showed no typical estrous cycle for more than 10 consecutive days (characterized by persistent keratinocytes or leukocytes, lacking the normal proestrus-estrus-metaestrus-diestrus transition), thus completing the establishment of a rat polycystic ovary syndrome model.

[0039] 2. Experimental Procedure

[0040] 2.1. The experimental group rats were divided into groups and subjected to drug intervention (see Appendix). Figure 1 )

[0041] Example 2

[0042] Ovarian morphology and ovarian weight testing

[0043] Appearance and morphology assessment: Observe the number of follicles on the surface of the ovaries of rats in each group (to determine the degree of polycystic ovaries), the overall fullness of the ovaries, and the volume and thickness of the uterine wall (to determine whether atrophy has occurred).

[0044] Weight quantification analysis: Accurately weigh the ovaries (total weight of both sides) and uterus to quantitatively assess the degree of pathological damage to the organs (abnormal ovarian weight gain, uterine atrophy and weight loss).

[0045] Specific testing methods

[0046] Sample collection

[0047] After 4 weeks of intervention, the rats were euthanized, and the abdominal cavity was quickly opened to separate the bilateral ovaries from the intact uterus and remove the surrounding fat, fascia and other connective tissues.

[0048] Gently rinse the surface of the ovary / uterus with 0.9% saline solution to remove blood and impurities, blot dry with sterile filter paper, and place in a sterile culture dish.

[0049] Visual observation and recording:

[0050] Under natural light (or under a 10× stereomicroscope), observe and record the number of follicles on the surface of the ovary of each rat in each group (counted as "follicles / both ovaries", with follicles ≥2mm in diameter considered as abnormal follicles) and the color of the ovary (normal is light pink, pathological is dark purple).

[0051] Observe the morphology of the uterus: a normal uterus is full and has a thick wall, while an atrophied uterus is smaller and has a thin wall. Record the incidence of uterine atrophy in each group. Take standardized photos of the ovary / uterus in each group (under the same lighting and at the same distance) for subsequent comparative analysis.

[0052] Weight measurement:

[0053] Using an electronic analytical balance with an accuracy of 0.001 g, the total weight of both ovaries and the weight of the uterus of each rat were weighed. The raw data were recorded, and the mean ± standard deviation (x ± s) of the ovarian and uterine weights for each group were calculated for statistical analysis between groups (see Appendix). Figure 2-4 ).

[0054] Example 3

[0055] Ovarian histopathological examination

[0056] Ovarian tissue: Count the number of cystic follicles (to assess abnormal follicular development) and the number of corpora lutea (to assess ovulation function) to determine the extent of ovarian pathological damage repair. Uterine tissue: Measure endometrial thickness (to assess abnormal endometrial proliferation) to determine the degree of improvement in uterine pathological condition.

[0057] Specific testing methods

[0058] Sample fixation and processing: Half of the separated ovarian tissue (1 cm from each side) and uterine tissue (1 cm from the middle section) were taken and fixed in 4% paraformaldehyde solution for 24 hours (the volume of the fixative solution was more than 10 times the volume of the tissue). After multi-stage dehydration (70% ethanol for 2 h → 80% ethanol for 2 h → 90% ethanol for 1 h → 95% ethanol for 1 h → 100% ethanol I for 30 min → 100% ethanol II for 30 min), xylene clearing (I for 30 min → II for 30 min), and paraffin embedding (immersion in paraffin at 60℃ for 3 h), 4 μm thick serial sections were prepared.

[0059] HE staining: Dewaxing sections (xylene I 10 min → xylene II 10 min) → gradient rehydration (100% ethanol I 5 min → 100% ethanol II 5 min → 95% ethanol 5 min → 90% ethanol 5 min → 80% ethanol 5 min → 70% ethanol 5 min → distilled water 5 min), staining with hematoxylin for 5 min → rinsing with tap water for 10 min (blueing back) → staining with 0.5% eosin for 2 min → rapid rinsing with distilled water → gradient dehydration again, clearing, and mounting with neutral resin.

[0060] Microscopic observation and data acquisition:

[0061] Ovarian sections were observed using an optical microscope (400× magnification). Five non-overlapping fields of view were randomly selected, and the number of cystic follicles (follicular cavity diameter ≥50μm and granulosa cell layer ≤2 layers) and corpus luteum in each field of view were counted. The average value of each group was calculated.

[0062] Endometrial thickness in uterine slices was measured using image analysis software (5 sites were randomly measured, and the average value was taken), with units of μm (see Appendix). Figure 3 ).

[0063] Example 4

[0064] Rat body weight and blood lipid level detection

[0065] Test content

[0066] The levels of total cholesterol (CHO), triglycerides (TG), low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL) in the serum of rats in each group were measured to assess the degree of improvement of lipid metabolism disorders by sheep placental extract in a rat model of polycystic ovary syndrome.

[0067] Specific testing methods

[0068] serum treatment

[0069] Take rat serum frozen at -80℃, thaw at room temperature, centrifuge at 12000rpm for 5min at 4℃ to remove the precipitate, and collect the clear serum from the supernatant for later use.

[0070] Direct method (selective inhibition method) detection operation

[0071] Reagent and sample preparation:

[0072] Remove reagent 1 (selective inhibitor, used to inhibit the reactivity of low-density lipoprotein and very low-density lipoprotein), reagent 2 (enzyme reagent, containing HDL-specific reactive enzyme), and HDL standards (concentration gradient: 0.5, 1.0, 1.5, 2.0, 2.5 mmol / L) from the -20℃ freezer and place them at room temperature (20-25℃) for 30 minutes to ensure that the reagent components are fully dissolved and the temperature is consistent.

[0073] Take a clean 96-well reaction plate and label the blank wells, standard wells, and sample wells (corresponding to the control group CON, model group MOD, and model intervention group MOD-SP, respectively). Set up 3 replicates for each group of samples to reduce detection errors.

[0074] Sample addition and incubation:

[0075] Blank well: Add 20 μL of 1% carboxymethyl cellulose (1% CMC, consistent with the drug delivery solvent);

[0076] Standard wells: Add 20 μL of HDL standard at different concentrations respectively;

[0077] Sample wells: Add 20 μL of the treated rat serum from each group.

[0078] Add 100 μL of reagent 1 (selective inhibitor) to each well, gently shake the 96-well reaction plate for 10 seconds to mix the liquid thoroughly, and incubate in a 37°C water bath for 5 minutes to achieve the inhibition of non-HDL lipoprotein activity.

[0079] After incubation, add 100 μL of reagent 2 (enzyme reagent) to each well, shake to mix for 10 seconds, and incubate in a 37°C water bath for 10 minutes to allow the enzyme to fully react with HDL to generate a colored product.

[0080] Washing and absorbance testing:

[0081] After incubation, discard the reaction solution in the wells and slowly rinse each well with the washing solution provided with the kit, soaking for 30 seconds each time, for a total of 5 washes. After washing, invert the reaction plate onto absorbent paper and pat it dry to avoid residual washing solution in the wells affecting the detection results.

[0082] The absorbance (OD value) of each well was measured at a wavelength of 546 nm using a biochemical analyzer (or ELISA reader). The OD value of the blank well was used as the reference for zeroing, and the OD values ​​of the standard well and the sample well were recorded.

[0083] Concentration calculation:

[0084] With the concentration of HDL standard as the x-axis and the OD value of the corresponding standard well as the y-axis, a standard curve was fitted using linear regression to obtain the regression equation (y=ax+b, where a is the slope and b is the intercept).

[0085] Substitute the OD values ​​of each group of sample wells into the standard curve regression equation to calculate the actual concentration of HDL in the serum of each group of rats (see attached figure). Figure 5 ).

[0086] Example 5

[0087] Plasma inflammatory factor detection (ELISA method)

[0088] Test content

[0089] The levels of interleukin-6 (IL-6), interleukin-17A (IL-17A), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in the plasma of rats in each group were measured to assess the degree of improvement in inflammatory disorders.

[0090] Specific testing methods

[0091] Plasma processing: Same as "Plasma sex hormone detection". Take the plasma sample frozen at -80℃, thaw at room temperature, centrifuge at 12000rpm for 5min at 4℃, and take the supernatant for detection (to remove precipitate impurities).

[0092] ELISA detection procedure (using the rat TNF-α kit as an example):

[0093] Add 100 μL of standard (concentration gradient: 0, 10, 20, 40, 80, 160 pg / mL) or sample to each well of the ELISA plate and incubate at 37°C for 90 min.

[0094] Washing: After discarding the liquid, wash 5 times with detergent and pat dry;

[0095] Add detection antibody: Add 100 μL of biotin-labeled detection antibody to each well, incubate at 37°C for 60 min, and wash repeatedly;

[0096] Add enzyme conjugate: Add 100 μL of horseradish peroxidase-labeled streptavidin to each well, incubate at 37°C for 30 min, and wash repeatedly;

[0097] Colorimetric analysis and readings: Same as "Plasma Sex Hormone Detection". Measure the OD value at a wavelength of 450nm, and calculate the plasma TNF-α and IL-10 concentrations based on the standard curve (see attached). Figure 6 ).

[0098] Observation results as follows Figure 2-6 As shown, we can conclude that:

[0099] 1. Ovarian and uterine morphology: In the control group (CON), the ovaries were smooth and light pink, and the uterus was full; in the model group (MOD), a large number of dark purple abnormal vesicles with a diameter ≥2mm were visible on the surface of the ovaries, and the uterus was significantly atrophied; in the intervention group (MOD-SP), the number of abnormal vesicles in the ovaries was significantly reduced, the color returned to light pink, and the fullness of the uterus was significantly improved.

[0100] 2. Histopathology: In the control group, follicular development was normal, with a mean number of corpora lutea of ​​6 per field. In the model group, follicular development was impaired, with a mean number of corpora lutea decreasing to 2 per field and a mean number of cystic follicles increasing to 11 per field. In the intervention group, the mean number of corpora lutea recovered to 9 per field, and the mean number of cystic follicles decreased to 3 per field, indicating a significant recovery in ovulation function. The endometrium in the model group was significantly thinner, while the endometrial thickness in the intervention group was close to the normal level of the control group.

[0101] 3. Lipid Metabolism: The body weight change curves showed that, starting 15 days after the sheep placenta extract intervention, the weight gain in the intervention group was significantly less than that in the PCOS model group. Serum high-density lipoprotein (HDL) levels in the model group were not significantly different from those in the control group; however, HDL levels in the sheep placenta extract intervention group were significantly elevated, approaching the normal levels of the control group, indicating that sheep placenta extract can effectively improve body weight and lipid metabolism disorders in PCOS model rats.

[0102] 4. Inflammatory factor levels: The pro-inflammatory factor TNF-α level was significantly increased in the model group (10.77 pg / mL vs. 8.37 pg / mL in the control group), while the anti-inflammatory factor IL-10 level was significantly decreased (3.05 pg / mL vs. 6.57 pg / mL in the control group). In the intervention group, the TNF-α level decreased to 8.59 pg / mL, and the IL-10 level significantly increased to 8.20 pg / mL, indicating a significant improvement in the inflammatory disorder.

[0103] As can be seen from the above embodiments, the sheep placenta extract of the present invention has good improvement and therapeutic effects on ovarian pathological damage, uterine pathological damage, weight and lipid metabolism disorders, and abnormal inflammatory disorders caused by polycystic ovary syndrome. It is expected to be used as a drug for the prevention and treatment of polycystic ovary syndrome and has good application prospects.

[0104] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of sheep placenta extract in the preparation of a medicine for improving polycystic ovary syndrome.

2. The application of the sheep placental extract according to claim 1 in the preparation of a drug for improving polycystic ovary syndrome, characterized in that: The sheep placenta extract is specifically a freeze-dried powder of sheep placenta.

3. The use of the sheep placental extract according to claim 1 in the preparation of a drug for improving polycystic ovary syndrome, characterized in that: The medicine is used for improving polycystic ovary syndrome caused by polycystic ovary disease, specifically for reducing the number of abnormal follicular cysts and the weight of the abnormally enlarged ovary. 4.The use of the sheep placenta extract according to claim 1 in the preparation of a drug for improving polycystic ovary syndrome, characterized in that: The medicine is used for improving endocrine disorders caused by polycystic ovary syndrome, specifically for reducing the level of plasma androgen, increasing the level of plasma estrogen and plasma progesterone.

5. The use of the sheep placental extract according to claim 1 in the preparation of a drug for improving polycystic ovary syndrome, characterized in that: The medicine is used for improving inflammatory disorders caused by polycystic ovary syndrome, specifically for reducing the level of plasma pro-inflammatory factor TNF-α and increasing the level of anti-inflammatory factor (IL-10). 6.The use of the sheep placenta extract according to claim 1 in the preparation of a drug for improving polycystic ovary syndrome, characterized in that: The medicine is used for improving metabolic disorders caused by polycystic ovary syndrome, specifically for increasing the level of serum high-density lipoprotein cholesterol. 7.The use of the sheep placenta extract according to claim 1 in the preparation of a drug for improving polycystic ovary syndrome, characterized in that: According to mammals other than humans, the dose of the sheep placenta extract used as the medicine is 1 mg / kg / d; according to human adults, the dose of the sheep placenta extract used as the medicine is 0.5-1 mg / kg / d. 8.The use of the sheep placenta extract according to claim 1 in the preparation of a drug for improving polycystic ovary syndrome, characterized in that: The use mode of the medicine prepared from the sheep placenta extract includes oral administration.

9. Use of the sheep placenta extract according to claim 1 in the preparation of a drug for improving polycystic ovary syndrome, characterized in that, When the sheep placenta extract is dissolved, the solvent includes sodium carboxymethyl cellulose.

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