Earthworm protein composition for regulating ovarian function and improving climacteric syndrome as well as preparation method and application of earthworm protein composition

Abstract

The invention relates to the technical field of biological medicine, in particular to an earthworm protein composition for regulating ovarian function and improving climacteric syndrome and a preparation method and application thereof.The composition is prepared from, by mass, earthworm protein and cranberry extract, and the mass ratio of the earthworm protein to the cranberry extract is 1: (0.5-5). Or the active ingredients of the composition comprise earthworm protein, cranberry extract and carob bean extract according to the mass ratio of 1: (0.5-5): (0.5-5). The composition is simple in component design, and the problems of complex components, unknown mechanism, low bioavailability, poor long-term safety and the like in the prior art are effectively solved through multi-component synergy, so that the ovarian function is accurately regulated, and related symptoms of the climacteric syndrome are systematically improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an earthworm protein composition for regulating ovarian function and improving menopausal syndrome, as well as a preparation method and application thereof. Background Art

[0002] Ovarian dysfunction is the core pathological basis of perimenopausal syndrome (formerly known as "menopausal syndrome"). As a crucial female gonadal organ, the ovaries have both reproductive and endocrine functions: on the one hand, they maintain fertility through follicular development and ovulation; on the other hand, they secrete estrogen, progesterone, and a small amount of androgen, regulating female secondary sexual characteristics and the menstrual cycle, and protecting multiple systems, including the skeletal, cardiovascular, and nervous systems. Ovarian function in women naturally declines starting around age 35 (even earlier in some individuals), and this process exhibits staged characteristics. The essence of ovarian dysfunction is decreased estrogen secretion, a change that triggers systemic dysfunction across multiple systems through the "hormone-organ axis," resulting in the typical symptoms of perimenopausal syndrome. Specific manifestations include: vasomotor symptoms (such as hot flashes and night sweats, which occur in 75%-85% of women), neuropsychiatric symptoms (anxiety, depression, insomnia, and decreased concentration), genitourinary atrophy (vaginal dryness, dyspareunia, and frequent urination due to urethral mucosal atrophy), abnormal bone metabolism (accelerated bone loss and increased risk of osteoporosis), and changes in the skin and mucous membranes (dryness, decreased elasticity, and hair loss). Symptom severity varies from person to person and is related to factors such as genetics, lifestyle (such as smoking and lack of exercise), and nutritional status (calcium / vitamin D intake). In summary, the pathogenesis of perimenopausal syndrome can be summarized as a pathological chain of "ovarian dysfunction → decreased estrogen secretion → systemic multisystem dysfunction."

[0003] Based on this, clinical intervention strategies for perimenopause (such as hormone replacement therapy and lifestyle adjustments) essentially aim to alleviate symptoms and reduce long-term health risks (such as osteoporosis and cardiovascular disease) by supplementing estrogen or improving the body's adaptability to low estrogen levels. Current mainstream treatment options include hormone replacement therapy (HRT) and phytoestrogen supplementation (such as soy isoflavones). However, long-term HRT use may increase the risk of breast cancer and thrombosis, while phytoestrogens have issues such as low bioavailability and slow onset of action.

[0004] In recent years, traditional Chinese medicine (TCM) compound formulas have been increasingly used to improve menopausal symptoms. For example, Chinese invention patent CN104173897B discloses a TCM composition for treating menopausal syndrome caused by ovarian failure. The composition is composed of ingredients such as snow frog, Acanthopanax senticosus, Astragalus, Ginseng, Donkey-hide Gelatin, Angelica, Deer Placenta, Polygonum Multiflorum, Rehmannia Glutinosa, Rubus Idaeus, Placenta, Lycium Barbarum, Musk, Frankincense, Borneol, White Cardamom, Asparagus Codonopsis, Panax Ginseng, and Aquilaria Sinensis. The composition's complex and numerous ingredients make it difficult to clearly define the compatibility relationships between ingredients, potentially affecting the stability and reproducibility of its efficacy and hindering the elucidation of its pathway of action using modern pharmacological methods. Furthermore, the use of valuable or scarce ingredients such as snow frog (the oviduct of the forest frog), musk (a secretion from musk deer), deer placenta (the fetus of a deer), and placenta (the human placenta) in the formula hinders its widespread application. Chinese invention patent CN119548556A discloses a Herba Citri Reticulatae Extract for the prevention and treatment of ovarian dysfunction, as well as its preparation method and use. This Herba Citri Reticulatae Extract can slow the decline of estrogen and gonadotropin, addressing the lack of effective methods for preventing and treating ovarian dysfunction in the prior art and achieving the desired effect of slowing the decline of estrogen and gonadotropin. While this patent proposes using Herba Citri Reticulatae Extract to slow the decline of estrogen and gonadotropin, addressing the lack of effective methods, it still has potential shortcomings. For example, its composition is limited, involving only a single Herba Citri Reticulatae Extract, and lacks a multi-component combination design. Perimenopausal syndrome involves multi-system dysfunction (vasomotor, neuropsychiatric, and bone metabolism, among others). A single component may only target the single link of "slowing down estrogen decline" and cannot cover multiple targets and pathways through the synergistic effects of multiple components. This, in turn, limits the comprehensiveness and efficacy of improving systemic multi-system symptoms.

[0005] Based on the above background, there is an urgent need to develop a composition with streamlined components and clear efficacy to accurately regulate ovarian function and systematically improve symptoms related to menopausal syndrome. Summary of the Invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a dragon protein composition for regulating ovarian function and improving menopausal syndrome, as well as its preparation method and application. The components of the composition are streamlined in design, and through the synergy of multiple components, it effectively solves the problems existing in the prior art such as "complex components, unclear mechanism, low bioavailability, and poor long-term safety", so as to accurately regulate ovarian function and systematically improve symptoms related to menopausal syndrome.

[0007] In order to achieve the above technical effects, the present invention adopts the following technical solutions: In a first aspect, the present invention provides an earthworm protein composition for regulating ovarian function and improving menopausal syndrome, wherein the active ingredients thereof include earthworm protein and cranberry extract in parts by mass, and the mass ratio of earthworm protein to cranberry extract is 1:(0.5-5).

[0008] Furthermore, the preparation method of the earth dragon protein is as follows: After the earthworm raw material is crushed, phosphate buffer with a pH of 7.0-8.5 is added at a material-liquid ratio of 1:8-12; Add compound protease and trypsin in sequence to carry out enzymatic hydrolysis; After enzymatic hydrolysis and enzyme inactivation, the product was separated by ultrafiltration membrane and active peptides with molecular weight of 8 kDa-30 kDa were collected.

[0009] Preferably, the enzymatic hydrolysis is a step-by-step enzymatic hydrolysis, comprising: The first stage of enzymatic hydrolysis: add 0.5%-1.5% of compound protease, hydrolyze at 37-40℃ for 2-4 hours; Second stage enzymatic hydrolysis: adjust the pH to 7.5-8.0, add 0.3%-1.0% trypsin, and continue hydrolysis at 35-40℃ for 1-3 hours; Furthermore, the preparation method of the cranberry extract is as follows: Ultrasonic-assisted ethanol extraction was used: 40%-70% ethanol was used as the solvent, and the solid-liquid ratio was 1:10-15 (w / v); Ultrasonic power 300-500 W, frequency 40 kHz, extraction temperature 40-50°C, time 30-60 minutes, repeated 2-3 times; The extracts were combined, concentrated under reduced pressure until there was no alcohol smell, passed through an AB-8 macroporous resin column, and gradient eluted with 30% and 50% ethanol in sequence. The 50% ethanol elution fraction was collected and freeze-dried to obtain the cranberry extract.

[0010] Preferably, the active ingredient of the earthworm protein composition further comprises carob extract, and the mass ratio of earthworm protein, cranberry extract and carob extract is 1:(0.5-5):(0.5-5).

[0011] Furthermore, the preparation method of the carob extract is as follows: Water extraction: Carob powder and ethanol solution are mixed at a ratio of 1:15-20 (w / v), and refluxed at 80-90℃ for 2 times, each time for 1-2 hours; Alcohol precipitation: Combine the extracts and concentrate to a relative density of 1.15-1.20 (60°C), add 95% ethanol to a final concentration of 60%-70%, let stand at 4°C for 12-24 hours, and centrifuge to obtain the precipitate; The precipitate is vacuum dried to obtain the carob extract.

[0012] In a second aspect, the present invention further provides a method for preparing the aforementioned earthworm protein composition, comprising the following steps: S1: Weigh earthworm protein, cranberry extract, and carob extract in a mass ratio of 1:(0.5-5):(0.5-5), respectively, and mix them evenly to obtain a mixture I; S2: adding clinically acceptable excipients to the aforementioned mixture I to prepare a composition for oral preparation.

[0013] Preferably, the oral preparation is any one of tablets, capsules, granules or oral liquids.

[0014] Most preferably, the oral preparation is a tablet, and the tablet further comprises any one of microcrystalline cellulose, cross-linked carboxymethyl cellulose sodium and magnesium stearate.

[0015] In a third aspect, the present invention further provides a use of the aforementioned earthworm protein composition in preparing a product having any of the following functions: a) Prevent or treat diseases related to ovarian failure in menopausal women; b) Improve vasomotor symptoms or neuropsychiatric symptoms in perimenopausal syndrome; c) As a functional food additive for maintaining or increasing bone density in women.

[0016] Compared with the prior art, the present invention has the following beneficial effects: First, the present invention provides a composition of earthworm protein for regulating ovarian function and improving menopausal syndrome. Its components are highly streamlined, reducing the complexity and ambiguity of traditional compound prescriptions due to the excessive number of medicinal ingredients. Furthermore, the components act synergistically across multiple targets to systematically intervene in multi-system dysfunction during perimenopause. Specifically, the composition includes: Earthworm protein active peptides (8-30 kDa): Through step-by-step enzymatic hydrolysis and ultrafiltration, the small molecular weight active peptide segments are precisely retained. They are easily absorbed and have high bioavailability. They can regulate hormone levels (slow down estrogen attenuation), improve local ovarian microcirculation (alleviate vasomotor symptoms such as hot flashes), and reduce neurooxidative damage through antioxidant effects (improve neuropsychiatric symptoms such as anxiety and insomnia). Cranberry extract (active ingredients such as proanthocyanidins): Ultrasound-assisted extraction and macroporous resin purification enriches anti-inflammatory and antioxidant components, which can improve genitourinary mucosal atrophy (relieving vaginal dryness, frequent urination, and urgency), and protect ovarian cells from oxidative damage. Carob extract: The water extraction and alcohol precipitation process retains high-purity estrogen-like active ingredients, which can directly or indirectly supplement estrogen deficiency, promote bone formation (prevent osteoporosis), and regulate sugar metabolism (assisting in improving perimenopausal metabolic disorders).

[0017] The above three ingredients synergistically cover multiple pathways such as "hormone regulation-anti-oxidation-anti-inflammation-bone metabolism", which are more comprehensive in terms of action mode than a single component, and the mechanism of action is easier to analyze than a complex compound.

[0018] At the same time, the composition provided by the present invention has significant optimization in the preparation process. For example, by combining step-by-step enzymatic hydrolysis with ultrafiltration membrane separation, 8-30 kDa active peptide segments are accurately obtained. Peptides in this molecular weight range are more easily absorbed by the intestine and avoid interference from macromolecular substances, ensuring the high activity of functional peptides. At the same time, in the preparation of cranberry extract, ultrasonic-assisted extraction is used to destroy the plant cell wall and accelerate the dissolution of active ingredients. Combined with AB-8 macroporous resin gradient elution (50% ethanol segment), core components such as proanthocyanidins are enriched in a targeted manner to improve the purity and biological activity of the extract. Finally, through the water extraction and alcohol precipitation process (60%-70% ethanol final concentration), D-inositol is retained in a targeted manner to avoid interference from impurities and ensure the efficient utilization of estrogen-like active ingredients. The improvements in the above preparation methods significantly improve the effectiveness and controllability of each component, and can effectively improve the bioavailability and stability of the active ingredients.

[0019] Finally, the composition provided by the present invention has a wide range of raw material sources, is safe and controllable; the form of the composition is convenient for preparation into various oral preparations such as tablets, capsules, granules or oral liquids to meet the needs of different groups of people; at the same time, it can be used as a functional food additive for daily health care, effectively expanding the application scenarios. DETAILED DESCRIPTION

[0020] The following examples are only used to more clearly illustrate the technical solutions of the present invention and are therefore only examples and are not intended to limit the scope of protection of the present invention. The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions made to the embodiments described below are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should all be included within the scope of the present invention.

[0021] To better illustrate the present invention, numerous specific details are provided in the following detailed description. It should be understood by those skilled in the art that the present invention can be practiced without certain specific details. In other embodiments, reagents, raw materials, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Unless otherwise specified, the units used in this specification are international standard units, and the numerical values and numerical ranges appearing in the present invention are all understood to include inevitable systematic errors in industrial production. At the same time, the experimental methods used in the following examples are all conventional methods; the materials, reagents or instruments used are all reagents and materials available from commercial channels if the manufacturer is not specified. In the examples, if specific conditions are not specified, they are all carried out according to conventional conditions or conditions recommended by the manufacturer. At the same time, the present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all common commercial products in the field of this technology. If not otherwise specified, the "ratios" referred to in the following examples are all ratios of mass fractions. Example 1

[0023] This embodiment provides an earthworm protein composition, the active ingredients of which are earthworm protein and cranberry extract in a mass ratio of 1:0.5.

[0024] Wherein, the preparation method of the earth dragon protein is as follows: A1: After grinding the earthworm raw material (dry), add pH 8.0 phosphate buffer at a material-liquid ratio of 1:10 to obtain a mixed system, and then perform enzymatic hydrolysis as follows: The first stage of enzymatic hydrolysis: enzymatic hydrolysis was performed using a composite protease, the addition amount of which was 1.0% (w / w) of the dry weight of the earthworm raw material, and hydrolysis was carried out at 38°C for 3 hours (the stirring speed was controlled at 200 rpm, and this stirring speed was used during the enzymatic hydrolysis process unless otherwise specified). The composite protease used in this embodiment was a mixture of subtilisin and papain in a mass ratio of 3:2 (hereinafter, the mass ratio is used unless otherwise specified).

[0025] Second stage enzymatic hydrolysis: adjust the pH to 7.0, add 1.0% trypsin (calculated based on the dry weight of the earthworm raw material), and continue hydrolysis at 37°C for 2 hours; A2: After the enzymatic hydrolysis is completed, heat at 90℃ for 10 min to inactivate the enzyme. First, filter the enzymatic hydrolysate after enzyme inactivation with a 30 kDa ultrafiltration membrane. The retained liquid is the macromolecules >30 kDa (discarded), and the permeate is the components <30 kDa. Then filter the permeate with an 8 kDa ultrafiltration membrane, take the retained material, and obtain the active peptide segment of 8-30 kDa, which is the target product. Collect it to obtain the active peptide segment with a molecular weight of 8 kDa-30 kDa, and freeze-dry it to obtain earthworm protein.

[0026] The preparation method of the cranberry extract is as follows: B1: Ultrasonic-assisted ethanol extraction: 60% ethanol as solvent, solid-liquid ratio 1:12 (w / v); B2: ultrasonic power 400 W, frequency 40 kHz, extraction temperature 45 °C, time 50 min, repeated 2 times; B3: Combine the extracts, concentrate under reduced pressure until there is no alcohol smell, pass through an AB-8 macroporous resin column, and elute with 30% and 50% ethanol gradients in sequence. Collect the 50% ethanol elution fraction and lyophilize to obtain the cranberry extract.

[0027] Based on the aforementioned earthworm protein and cranberry extract, the preparation method of the earthworm protein composition provided in this embodiment is: weigh the earthworm protein and cranberry extract according to the mass ratio, respectively, and mix them evenly to obtain the earthworm protein composition. Example 2

[0028] This embodiment provides a dragon protein composition, wherein the active ingredients are dragon protein and cranberry extract in a mass ratio of 1:1, wherein the preparation methods of the dragon protein and cranberry extract are the same as those in Example 1. The preparation method of the earthworm protein composition provided in this embodiment is as described in Example 1. Example 3

[0029] This embodiment provides a dragon protein composition, wherein the active ingredients are dragon protein and cranberry extract in a mass ratio of 1:3, wherein the preparation methods of the dragon protein and cranberry extract are the same as those in Example 1. The preparation method of the earthworm protein composition provided in this embodiment is as described in Example 1. Example 4

[0030] This embodiment provides a dragon protein composition, wherein the active ingredients are dragon protein and cranberry extract in a mass ratio of 1:5, wherein the preparation methods of the dragon protein and cranberry extract are the same as those in Example 1. The preparation method of the earthworm protein composition provided in this embodiment is as described in Example 1.

[0031] Comparative Example 1 This embodiment provides a ground dragon protein composition, the active ingredients of which are ground dragon protein and cranberry extract in a mass ratio of 1:3. The preparation method of the cranberry extract is the same as that of Example 1, while the preparation method of the ground dragon protein differs from the preparation method of the ground dragon protein in Example 3 in the following ways: The composite protease used in this embodiment is composed of subtilisin, papain and trypsin in a ratio of 3:1:1.

[0032] Comparative Example 2 This embodiment provides a composition of earthworm protein, which differs from Comparative Example 1 in that the preparation method of earthworm protein is different. Specifically, the composite protease used in this embodiment is a mixture of Bacillus licheniformis protease, Bacillus subtilisin, and papain in a ratio of 1:2:2.

[0033] Comparative Example 3 This embodiment provides a composition of earthworm protein, which differs from Comparative Example 1 in that the preparation method of earthworm protein is different. Specifically, the composite protease used in this embodiment is a mixture of Bacillus licheniformis protease, Bacillus subtilisin, papain and trypsin in a ratio of 1:2:1:1.

[0034] Comparative Example 4 This embodiment provides a composition of earthworm protein, which differs from Comparative Example 1 in that the preparation method of earthworm protein is different. Specifically, the composite protease used in this embodiment is a mixture of Bacillus licheniformis protease, papain and trypsin in a ratio of 3:1:1.

[0035] Comparative Example 5 This embodiment provides a earthworm protein composition, which differs from Comparative Example 1 in that the preparation method of the earthworm protein is different. Specifically, the composite protease used in this embodiment is a compound of Bacillus licheniformis protease, Bacillus subtilisin, papain, trypsin and bromelain in a ratio of 1:2:1:1:1.

[0036] Comparative Example 6 This embodiment provides a earthworm protein composition, which differs from Comparative Example 1 in that the preparation method of the earthworm protein is different. Specifically, the composite protease used in this embodiment is a compound of Bacillus licheniformis protease, Bacillus subtilisin, papain, trypsin, bromelain and elastase in a ratio of 1:2:1:1:1:1.

[0037] Comparative Example 7 This embodiment provides a composition of earthworm protein, which differs from Comparative Example 2 in that the preparation method of earthworm protein is different. Specifically, the composite protease used in this embodiment is a mixture of Bacillus licheniformis protease and Bacillus subtilisin in a ratio of 1:2.

[0038] Comparative Example 8 This embodiment provides a composition of earthworm protein, which differs from Comparative Example 2 in that the preparation method of earthworm protein is different. Specifically, the composite protease used in this embodiment is a mixture of Bacillus licheniformis protease and papain in a ratio of 3:2.

[0039] Comparative Example 9 This embodiment provides a composition of earthworm protein, which differs from Comparative Example 2 in that the preparation method of earthworm protein is different. Specifically, the composite protease used in this embodiment is a mixture of Bacillus licheniformis protease, Bacillus subtilisin, and bromelain in a ratio of 1:2:1.

[0040] Comparative Example 10 This embodiment provides a composition of earthworm protein, which differs from Comparative Example 2 in that the preparation method of earthworm protein is different. Specifically, the composite protease used in this embodiment is a mixture of Bacillus licheniformis protease, Bacillus subtilisin, and elastase in a ratio of 1:2:1.

[0041] Comparative Example 11 This example provides a composition of earthworm protein, which differs from Comparative Example 2 in that the preparation method of earthworm protein is different. Specifically, in this example, only the first stage enzymatic hydrolysis is performed, and the second stage enzymatic hydrolysis is not performed. After the first stage enzymatic hydrolysis, the active peptide segments of 8 kDa-30 kDa are directly collected.

[0042] Comparative Example 12 This embodiment provides a composition of earthworm protein, which differs from Comparative Example 2 in that the preparation method of earthworm protein is different. Specifically, in this embodiment, only the second stage enzymatic hydrolysis is performed without the first stage enzymatic hydrolysis, and the active peptide segments of 8 kDa-30 kDa are directly collected after the second stage enzymatic hydrolysis.

[0043] It should be noted that the enzymes used in the present invention are described as follows: the activity of subtilisin is about 400,000 U / mg (Hubei Yuancheng Saichuang Technology Co., Ltd.); the enzymatic activity of papain is about 100,000 U / mg (Sichuan Lvcheng Biotechnology Co., Ltd.); the enzymatic activity of trypsin is 100,000 U / mg (Sichuan Lvcheng Biotechnology Co., Ltd.); the enzymatic activity of Bacillus licheniformis protease (Anhui Zhonghong Bioengineering Co., Ltd.) is about 200,000 U / mg; the enzymatic activity of bromelain is about 100,000 U / mg (Jiangxi Huayuyuan Biotechnology Co., Ltd.); and the enzymatic activity of elastase is about 100,000 U / mg (Jiangxi Huayuyuan Biotechnology Co., Ltd.).

[0044] Test Example 1 This test example tests the earthworm protein and earthworm protein composition prepared in Examples 1 to 4 and Comparative Examples 1 to 12, including: 1.1 Determination of Extraction Yield of Earthworm Protein (8 kDa-30 kDa) After two-step enzymatic hydrolysis, the earthworm raw material was separated by 30 kDa and 8 kDa ultrafiltration membranes, and the 8-30 kDa retentate was collected.

[0045] After freeze-drying, weigh and calculate the extraction rate. The calculation method is as follows: Extraction rate (%) = freeze-dried product mass (g) / total protein content of earthworm raw material (g) × 100%; The total protein content of earthworm raw materials was determined by precipitating protein with trichloroacetic acid (TCA) and then centrifuging the precipitate. The nitrogen content (containing only protein nitrogen) was determined and converted to protein content. Each experiment was performed independently three times, and the mean ± standard deviation was taken. The experimental results are shown in Table 1 (n=3).

[0046] 1.2 Evaluation of in vitro thrombolytic activity (fibrin plate method) Prepare agarose-fibrinogen mixture: dissolve 1.2% agarose + 0.1% fibrinogen (w / v) in PBS (pH 7.4), add thrombin (final concentration 2.5 U / mL), and quickly pour into the culture dish to form a fibrin plate after coagulation.

[0047] Then, the earthworm protein was dissolved in physiological saline to prepare a 2 mg / mL earthworm protein solution.

[0048] During the test, 50 μL of the prepared earthworm protein solution was taken with a pipette and spotted on a fibrin plate (6 parallel spots per group). 50 μL of normal saline was tested as a blank control. The plate with the test sample was incubated at 37°C for 18 hours. The fibrin was dissolved to form a transparent lysis zone. The area of the lysis zone (mm²) was measured using ImageJ software. The size of the lysis zone reflected the thrombolytic activity. The experimental results are shown in Table 1 (n=6).

[0049] Table 1 Comparison of extraction rate and in vitro thrombolytic activity of earthworm protein in each group (mean ± SD)

[0050] The above experimental results show that the extraction rate and in vitro thrombolytic activity of earthworm protein vary significantly under different experimental conditions, specifically: In Example Groups 1-4, as the mass ratio of earthworm protein to cranberry extract was adjusted, Example 3 (a 1:3 ratio) demonstrated the best overall effect. The earthworm protein extraction rate in this group (Example 3) was 75.0%, significantly higher than that in the other examples (Examples 1, 2, and 4), indicating that the 1:3 ratio resulted in better enzymatic hydrolysis efficiency and release of the target peptide (8-30 kDa). The in vitro thrombolytic activity of Example 3 was 192.5 mm², significantly higher than that of Example 1 (158.3 mm²) and Example 2 (172.6 mm²). These results demonstrate that the increased cranberry extract ratio (1:3) enhances plasminogen activator activity through the synergistic effect of polyphenols.

[0051] Among the comparative example groups 1-12, comparative example 2 (composite protease composition: Bacillus licheniformis protease + Bacillus subtilis protease + papain, 1:2:2) has the best comprehensive performance. The extraction rate of this group is as high as 82.3%, which is significantly better than all the examples and other comparative examples, indicating that the synergistic effect of Bacillus licheniformis protease and Bacillus subtilis protease can efficiently hydrolyze earthworm collagen fibers and release more small molecule active peptides; the thrombolytic activity of this group is 215.7 mm², which is 12.1% higher than that of Example 3 (192.5 mm²), indicating that the enzymatic hydrolysis effect of this group of composite proteases is better and significantly better than that of Example 3, Comparative Example 8, etc.

[0052] In addition, this experiment also found that the thrombolytic activities of Comparative Example 5 (containing bromelain) and Comparative Example 6 (containing elastase) were 208.1 mm² and 206.5 mm², respectively. Although significantly better than Example 3, they were still lower than Comparative Example 2, indicating that the addition of bromelain (targeting fibrin α chain) and elastase (targeting elastic fibers) failed to completely replace the multi-enzyme synergistic effect in Comparative Example 2.

[0053] Finally, the extraction rates (58.4% and 52.7%) and thrombolytic activities (140.2 mm² and 125.6 mm²) of Comparative Example 11 (first-stage enzymatic hydrolysis only) and Comparative Example 12 (second-stage enzymatic hydrolysis only) were significantly lower than those of the other groups, verifying the necessity of the two-step enzymatic hydrolysis process. The possible reasons are that the first-stage enzymatic hydrolysis (complex protease) is responsible for the initial degradation of macromolecules, and the second stage (trypsin) further releases active peptides. This theory needs further verification.

[0054] In summary, Example 3 (1:3 ratio) performed optimally among the examples, demonstrating that the mass ratio of earthworm protein to cranberry extract plays a key role in thrombolytic activity. Comparative Example 2 (Bacillus licheniformis protease complex protease), through the synergistic action of multiple enzymes, significantly surpassed all other groups in both extraction efficiency and thrombolytic activity. Optimization of the enzymatic hydrolysis process (introduction of Bacillus licheniformis protease) is the core strategy for enhancing functionality. This demonstrates that the two-step enzymatic hydrolysis process (complex protease → trypsin) and the refined design of the complex protease ratio are crucial factors in enhancing the functional activity of earthworm protein.

[0055] Example 5 The pharmacological activity of carob extract is primarily reflected in regulating metabolism, improving endocrine-related diseases, and combating inflammation. Its core active ingredients include D-chiro-inositol (DCI) and polysaccharides. D-chiro-inositol, one of the key active ingredients in carob extract, has an insulin-sensitizing effect, helping to lower blood sugar levels. DCI also has significant benefits for ovulation and menstrual cycle restoration in patients with polycystic ovary syndrome (PCOS). Clinical trials have shown that after taking carob extract for 6-8 weeks, 86% of obese and 60% of lean PCOS patients were able to ovulate spontaneously. Furthermore, after 6-15 months of use, 62.5% of women with menstrual disorders experienced effective menstrual cycle restoration.

[0056] Therefore, based on the research on carob extract in the prior art, the inventors further combined carob extract with the earthworm protein and cranberry extract prepared in the aforementioned comparative example 2 to observe whether there is a synergistic effect between the above components. The specific experimental design is as follows: First, this embodiment provides a method for preparing carob extract, which is specifically prepared according to the following method: C1: Water extraction: Dried carob (from the seeds of the Mediterranean carob tree (Ceratonia siliqua)) was ground into a powder, passed through an 80-100 mesh sieve, and mixed with 40% ethanol solution at a ratio of 1:18 (w / v). Extraction was performed under reflux at 90°C twice, each time for 2 hours.

[0057] C2: Alcohol precipitation: The combined extracts were concentrated to a relative density of 1.15-1.20 (60°C), and 95% ethanol was added to a final concentration of 65%. The mixture was allowed to stand at 4°C for 24 hours, and the precipitate was collected by centrifugation.

[0058] C3: vacuum drying the precipitate to obtain carob extract; Secondly, the present invention provides multiple compositions, and tests the pharmacological activities of the multiple compositions, including: 5.1 Experimental Grouping Animal selection: SPF female SD rats (12 weeks old) were randomly divided into 9 groups (n=10), including: Normal control group (sham operation, not ovariectomized); Model group (OVX surgery, no treatment); positive control group (OVX + estradiol, 0.1 mg / kg / d); control group 1 (OVX + carob extract 400 mg / kg / d); Control group 2 (OVX + earthworm protein 400 mg / kg / d, prepared by the method in Comparative Example 2); 3 control groups (OVX + cranberry extract 400 mg / kg / d, prepared by the method of Comparative Example 2); Composition group 1 (OVX + earthworm protein and cranberry extract at a mass ratio of 1:3 as active ingredients, total dosage 400 mg / kg / d); Composition group 2 (OVX + earthworm protein and carob extract in a mass ratio of 1:1 as active ingredients, total dosage 400 mg / kg / d); Composition group 3 (OVX + cranberry extract and carob extract at a mass ratio of 3:1 as active ingredients, total dose 400 mg / kg / d); Composition group 4 (OVX + earthworm protein, cranberry extract, and carob extract in a mass ratio of 1:3:1 as active ingredients; total administration dose of 400 mg / kg / d); Except for the normal control group, all other groups underwent bilateral ovariectomy (OVX). The modeling method is not described here. The drug was administered orally starting on day 4 after modeling, once daily, for 28 weeks (at a dose of 400 mg / kg / d).

[0059] 5.2 Experimental Results and Analysis One-way ANOVA was used to analyze the data in this experiment, and Tukey's HSD test was used to compare multiple groups. Compared with the model group: *p<0.05, **p<0.01.

[0060] (1) Detection of behavioral indicators related to perimenopausal symptoms: Estrogen deficiency after OVX surgery reduces the synthesis of neurotransmitters such as serotonin (5-HT) and norepinephrine (NE) in the brain, leading to anxiety- and depression-like behaviors (decrease in the proportion of time spent in the open arms and increase in the immobility period in the model group). If the proportion of time spent in the open arms increases and the immobility period decreases after intervention, it suggests that the drug may alleviate mood disorders by increasing E2 levels, improving oxidative stress, or regulating neurotransmitters (such as 5-HT).

[0061] In the elevated plus maze, a lower percentage of time spent in the open arms indicates a lower willingness to explore the open area (due to anxiety-induced avoidance of the exposed environment), reflecting anxiety symptoms common during perimenopause. In the forced swim test, longer periods of immobility indicate a rat's cessation of active struggle ("behavioral despair"), reflecting depressive-like behaviors associated with perimenopause. The results are shown in Table 2.

[0062] Table 2 Test results of behavioral indicators related to perimenopausal symptoms in various rats

[0063] The above experimental results show that the proportion of arm open time and immobility time of composition 4 are close to those of the positive control group (estradiol), and are significantly better than all single-component and two-component compositions, suggesting that the three components are more effective in improving anxiety and depressive-like behaviors by regulating the synergistic effects of neurotransmitters (5-HT / NE) and oxidative stress.

[0064] (2) Serum TNF-α, IL-6 levels, E2, FSH, and LH detection TNF-α (tumor necrosis factor-α) and IL-6 (interleukin-6) are classic proinflammatory cytokines. Estrogen deficiency after OVX surgery activates inflammatory pathways such as NF-κB, leading to increased secretion of TNF-α and IL-6 (significantly increased in the model group), reflecting the local and systemic chronic inflammatory state of the ovary.

[0065] E2, secreted by granulosa cells in the ovarian follicles, is a core indicator of ovarian function. Ovaries are removed after OVX surgery, disrupting E2 production (E2 levels were significantly reduced in the model group). FSH and LH, secreted by the pituitary gland, are negatively regulated by E2. When E2 decreases, the pituitary loses its inhibitory effect, leading to increased FSH and LH secretion (FSH and LH levels were significantly elevated in the model group), creating the perimenopausal hormonal profile of "low E2 and high FSH / LH ratios." If E2 levels increase and FSH / LH ratios decrease after intervention, it indicates that the medication is effectively improving ovarian function.

[0066] The detection method is as follows: after the experiment, 2-3 mL of blood is collected from the orbital venous plexus, and the blood is allowed to stand at 4°C for 30 minutes, then centrifuged at 3000 r / min for 15 minutes to separate the serum. The serum is then tested using an enzyme-linked immunosorbent assay (ELISA) kit. At the same time, based on the obtained serum samples, E2, FSH, and LH are further tested using a chemiluminescence immunoassay (CLIA): using a fully automatic chemiluminescence immunoassay instrument (such as Roche Cobas e411), the hormone concentrations in the serum are detected (E2 unit: pg / mL; FSH, LH unit: mIU / mL) through specific antibody-antigen reactions combined with chemiluminescent markers. The experimental results are shown in Table 3.

[0067] Table 3 Serum TNF-α, IL-6 levels, E2, FSH, and LH test results of rats in each group

[0068] Combination 4 significantly reduced proinflammatory cytokines (TNF-α, IL-6), increased E2, and decreased FSH / LH, outperforming all control groups and the two-component combination (p < 0.01). The three-component combination may restore ovarian-pituitary axis function by synergistically regulating inflammatory pathways (such as NF-κB) and hormonal feedback mechanisms.

[0069] (3) Oxidative stress level detection: SOD (superoxide dismutase) is a key antioxidant enzyme that can remove O2 - Malondialdehyde (MDA) is the end product of lipid peroxidation and reflects the degree of free radical damage to cell membranes. Decreased SOD activity and elevated MDA in the model group suggest severe oxidative stress damage in ovarian tissue (estrogen deficiency leads to a decrease in antioxidant defense system function). Post-intervention decreases in TNF-α and IL-6, increased SOD, and decreased MDA suggest anti-inflammatory and antioxidant effects.

[0070] Ovarian tissue was immediately removed after the rats were sacrificed, and blood stains were washed away with physiological saline. The tissue was then dried with filter paper and weighed. Nine volumes of pre-cooled physiological saline (0.9% NaCl) were added and homogenized in an ice bath. The supernatant was collected by centrifugation and the SOD activity was detected by the xanthine oxidase method. The MDA content was detected by the thiobarbituric acid (TBA) method. The experimental results are shown in Table 4.

[0071] Table 4 Results of oxidative stress level detection in ovarian tissue homogenates of rats in each group

[0072] The SOD activity of composition 4 returned to near normal levels, and the MDA content was significantly lower than that of all control groups and the two-component composition (p<0.01), suggesting that earthworm protein, cranberry extract and carob extract synergistically alleviated oxidative damage through multiple targets.

[0073] The above experimental results show that composition 4 (earth dragon protein: cranberry: carob = 1:3:1) exhibits significant synergistic effects in all indicators.

[0074] Example 6 This embodiment further provides a tablet containing earthworm protein composition I based on the composition 4 of the aforementioned embodiment 5. The specific preparation method is as follows: (1) Mixing and granulation Weigh 50 g of mixture 4 (earth dragon protein: cranberry: carob = 1:3:1), fillers (microcrystalline cellulose 150 g, lactose 100 g), and 2 / 3 of the disintegrant (crospovidone 20 g) into a high-efficiency mixing granulator and mix at low speed for 15 minutes. Then, slowly add 5% HPMC aqueous solution to prepare a soft material, and pass the soft material through an 18-mesh sieve to prepare wet granules. (2): Drying and granulation The wet granules are placed in a boiling dryer and dried at 50°C (to avoid high temperature denaturation of earthworm protein) until the moisture content is no more than 4% (detected by a moisture meter). The dried granules are sieved through a 20-mesh sieve to remove fine powder and lumps to ensure the fluidity of the granules.

[0075] (3): Total mixing and tableting The whole granules and the remaining 1 / 3 disintegrant (10 g of cross-linked polyvinylpyrrolidone), lubricant (5 g of magnesium stearate), and flavoring agent (10 g of oligofructose) were added to a three-dimensional mixer, mixed at a low speed for 18 minutes, and then tableted to obtain earthworm protein composition tablets.

[0076] The above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art will appreciate that the technical solutions of the present invention may be modified or replaced with equivalents without departing from the spirit and scope of the technical solutions of the present invention, and such modifications or equivalents shall be encompassed by the claims of the present invention. Any techniques, shapes, and structures not described in detail herein are well known.

Claims

1. A composition of earthworm protein for regulating ovarian function and improving menopausal syndrome, characterized in that: Its active ingredients, calculated by weight, include earthworm protein and cranberry extract, and the mass ratio of earthworm protein to cranberry extract is 1:(0.5-5).

2. The earthworm protein composition for regulating ovarian function and improving menopausal syndrome according to claim 1, characterized in that: The preparation method of the earth dragon protein is as follows: After the earthworm raw material is crushed, phosphate buffer with a pH of 7.0-8.5 is added at a material-liquid ratio of 1:8-12; Add compound protease and trypsin in sequence to carry out enzymatic hydrolysis; After enzymatic hydrolysis and enzyme inactivation, the product was separated by ultrafiltration membrane and active peptides with molecular weight of 8 kDa-30 kDa were collected.

3. The earthworm protein composition for regulating ovarian function and improving menopausal syndrome according to claim 1, characterized in that: The enzymatic hydrolysis is a step-by-step enzymatic hydrolysis, comprising: The first stage of enzymatic hydrolysis: add 0.5%-1.5% of compound protease, hydrolyze at 37-40℃ for 2-4 hours; The second stage of enzymatic hydrolysis: adjust the pH to 7.5-8.0, add 0.3%-1.0% trypsin, and continue hydrolysis at 35-40℃ for 1-3 hours.

4. The earthworm protein composition for regulating ovarian function and improving menopausal syndrome according to claim 1, characterized in that: The preparation method of the cranberry extract is as follows: Ultrasonic-assisted ethanol extraction was used: 40%-70% ethanol was used as the solvent, and the solid-liquid ratio was 1:10-15 (w / v); Ultrasonic power 300-500 W, frequency 40 kHz, extraction temperature 40-50°C, time 30-60 minutes, repeated 2-3 times; The extracts were combined, concentrated under reduced pressure until there was no alcohol smell, passed through an AB-8 macroporous resin column, and gradient eluted with 30% and 50% ethanol in sequence. The 50% ethanol elution fraction was collected and freeze-dried to obtain the cranberry extract.

5. The earthworm protein composition for regulating ovarian function and improving menopausal syndrome according to claim 1, characterized in that: Its active ingredients also include carob extract, and the mass ratio of earthworm protein, cranberry extract and carob extract is 1:(0.5-5):(0.5-5).

6. The earthworm protein composition for regulating ovarian function and improving menopausal syndrome according to claim 5, characterized in that: The preparation method of the carob extract is as follows: Water extraction: Carob powder and ethanol solution are mixed at a ratio of 1:15-20 (w / v), and refluxed at 80-90℃ for 2 times, each time for 1-2 hours; Alcohol precipitation: Combine the extracts and concentrate to a relative density of 1.15-1.20 (60°C), add 95% ethanol to a final concentration of 60%-70%, let stand at 4°C for 12-24 hours, and centrifuge to obtain the precipitate; The precipitate is vacuum dried to obtain the carob extract.

7. The method for preparing a earthworm protein composition for regulating ovarian function and improving menopausal syndrome according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1: Weigh earthworm protein, cranberry extract, and carob extract in a mass ratio of 1:(0.5-5):(0.5-5), respectively, and mix them evenly to obtain a mixture I; S2: adding clinically acceptable excipients to the aforementioned mixture I to prepare a composition for oral preparation.

8. The preparation method according to claim 7, wherein The oral preparation is any one of tablets, capsules, granules or oral liquids.

9. The preparation method according to claim 7, wherein The tablet further comprises any one of microcrystalline cellulose, croscarmellose sodium and magnesium stearate.

10. Use of the earthworm protein composition for regulating ovarian function and improving menopausal syndrome according to any one of claims 1 to 6 in the preparation of a product having any of the following functions: a) Prevent or treat diseases related to ovarian failure in menopausal women; b) Improve vasomotor symptoms or neuropsychiatric symptoms in perimenopausal syndrome.

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