Earthworm protein composition for regulating ovarian function to improve menopausal syndrome and preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a diplommatin composition for regulating ovary function and improving menopausal syndrome, a preparation method and application thereof, active ingredients of the composition include diplommatin and cranberry extract by mass fraction, and the mass ratio of diplommatin to cranberry extract is 1:(0.5-5), or active ingredients of the composition are composed of diplommatin, cranberry extract and carob extract according to a mass ratio of 1:(0.5-5):(0.5-5). The composition has a simple component design, effectively solves the problems of "complex components, unknown mechanism, low bioavailability and poor long-term safety" in the prior art through multi-component synergy, and precisely regulates ovary function and systematically improves menopausal syndrome related symptoms.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an earthworm protein composition for regulating ovarian function and improving menopausal syndrome, as well as its preparation method and application. Background Technology

[0002] Ovarian dysfunction is the core pathological basis for perimenopausal syndrome (formerly known as "menopausal syndrome"). The ovary, as an important female gonad, has dual reproductive and endocrine functions: on the one hand, it maintains fertility through follicle development and ovulation; on the other hand, it secretes estrogen, progesterone, and a small amount of androgens, regulating female secondary sexual characteristics and the menstrual cycle, and playing a protective role in the functions of multiple systems, including the skeletal, cardiovascular, and nervous systems. Ovarian function naturally declines from around age 35 (in some individuals it may decline earlier), a process characterized by stages. The essence of ovarian dysfunction is a decrease in estrogen secretion. This change triggers systemic dysfunction through the "hormone-organ axis," leading to the typical symptoms of perimenopausal syndrome. Specifically, it manifests as: vasomotor symptoms (such as hot flashes and night sweats, which occur in 75%-85% of women), neuropsychiatric symptoms (anxiety, depression, insomnia, and decreased attention), atrophy of the reproductive and urinary systems (vaginal dryness, painful intercourse, and urinary frequency and urgency 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). The severity of symptoms varies from person to person and is related to factors such as genetics, lifestyle (e.g., smoking, insufficient exercise), and nutritional status (calcium / vitamin D intake). In summary, the pathogenesis of perimenopausal syndrome can be summarized as a pathological chain of "ovarian function decline → decreased estrogen secretion → systemic multi-system dysfunction."

[0003] Therefore, clinical intervention strategies for perimenopause (such as hormone replacement therapy and lifestyle modifications) 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 ability to adapt to a low estrogen state. Currently, mainstream treatment options include hormone replacement therapy (HRT) and phytoestrogens supplementation (such as soy isoflavones). However, long-term use of HRT may increase the risk of breast cancer and thrombosis; while phytoestrogens have problems such as low bioavailability and slow onset of action.

[0004] In recent years, traditional Chinese medicine (TCM) compound prescriptions 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 dysfunction. This composition consists of ingredients such as snow frog (a type of frog), Acanthopanax senticosus, Astragalus membranaceus, ginseng, donkey-hide gelatin, Angelica sinensis, deer placenta, Polygonum multiflorum, Rehmannia glutinosa, raspberry, placenta, wolfberry, musk, frankincense, borneol, cardamom, asparagus, ginseng, and agarwood. The complex and numerous ingredients make it difficult to define the compatibility of the herbs, potentially affecting the stability and reproducibility of the therapeutic effect and hindering the analysis of its mechanism of action using modern pharmacological methods. Furthermore, the use of expensive or rare herbs such as snow frog (ovipositor of the forest frog), musk (secretions of musk deer), deer placenta (fetus of the deer family), and placenta (human placenta) makes large-scale application of this composition difficult. Chinese invention patent CN119548556A discloses a *Potentilla fruticosa* extract for the prevention and treatment of ovarian dysfunction, its preparation method, and its application. This *Potentilla fruticosa* extract can slow down the decline of estrogen and gonadotropins, solving the problem of the lack of effective methods for preventing and treating ovarian dysfunction in the prior art, and achieving the effect of slowing down the decline of estrogen and gonadotropins. While this patent proposes to slow down the decline of estrogen and gonadotropins through *Potentilla fruticosa* extract, solving the problem of "lack of effective methods," it still has the following potential shortcomings. For example, its composition is singular, involving only a single *Potentilla fruticosa* extract, without a multi-component combination design. Perimenopausal syndrome involves multiple systemic dysfunctions (vasomotor, neuropsychiatric, skeletal metabolism, etc.). A single component may only target the single aspect of "slowing down estrogen decline," and cannot cover multiple targets and pathways through the synergistic effect of multiple components, thus limiting the comprehensiveness and efficacy in improving systemic multi-system symptoms.

[0005] Against this backdrop, there is an urgent need to develop a composition with simplified components and clearly defined efficacy to precisely regulate ovarian function and systematically improve menopausal syndrome-related symptoms. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide an earthworm protein composition for regulating ovarian function and improving menopausal syndrome, as well as its preparation method and application. This composition features a simplified component design and, through the synergistic effect of multiple components, effectively solves the problems of "complex components, unclear mechanisms, low bioavailability, and poor long-term safety" existing in the prior art, thereby precisely regulating ovarian function and systematically improving menopausal syndrome-related symptoms.

[0007] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides an earthworm protein composition for regulating ovarian function and improving menopausal syndrome, wherein the active ingredients, by mass parts, include earthworm protein and cranberry extract, and the mass ratio of earthworm protein to cranberry extract is 1:(0.5-5).

[0009] Furthermore, the method for preparing the earthworm protein is as follows:

[0010] After crushing the earthworm raw material, add phosphate buffer solution with pH 7.0-8.5 at a material-to-liquid ratio of 1:8-12;

[0011] Add the complex protease and trypsin in sequence for enzymatic hydrolysis;

[0012] After enzymatic hydrolysis and enzyme inactivation, the active peptides with molecular weights of 8 kDa-30 kDa were separated by ultrafiltration membrane and collected.

[0013] Preferably, the enzymatic hydrolysis is a stepwise enzymatic hydrolysis, comprising:

[0014] First stage of enzymatic hydrolysis: Add 0.5%-1.5% of the compound protease and hydrolyze at 37-40℃ for 2-4 hours;

[0015] 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;

[0016] Furthermore, the preparation method of the cranberry extract is as follows:

[0017] Ultrasonic-assisted ethanol extraction was employed, using 40%-70% ethanol as the solvent, with a material-to-liquid ratio of 1:10-15 (w / v).

[0018] Ultrasonic power 300-500 W, frequency 40 kHz, extraction temperature 40-50℃, time 30-60 minutes, repeated 2-3 times;

[0019] The extracts were combined and concentrated under reduced pressure until no alcohol odor remained. The extracts were then passed through an AB-8 macroporous resin column and eluted sequentially with 30% and 50% ethanol. The 50% ethanol eluent was collected and freeze-dried to obtain cranberry extract.

[0020] Preferably, the active ingredients of the earthworm protein composition further include carob extract, and the mass ratio of earthworm protein, cranberry extract and carob extract is 1:(0.5-5):(0.5-5).

[0021] Furthermore, the preparation method of the carob extract is as follows:

[0022] Water extraction: Carob powder and ethanol solution are mixed at a ratio of 1:15-20 (w / v), and refluxed at 80-90℃ twice, each time for 1-2 hours;

[0023] Alcohol precipitation: Combine the extracts and concentrate to a relative density of 1.15-1.20 (60℃), add 95% ethanol to a final concentration of 60%-70%, let stand at 4℃ for 12-24 hours, and centrifuge to collect the precipitate;

[0024] The precipitate was dried under vacuum to obtain carob extract.

[0025] Secondly, the present invention also provides a method for preparing the aforementioned earthworm protein composition, comprising the following steps:

[0026] S1: Weigh earthworm protein, cranberry extract and carob extract according to the mass ratio of 1:(0.5-5):(0.5-5), and mix them evenly to obtain mixture I;

[0027] S2: Add clinically acceptable excipients to the aforementioned mixture I to prepare an oral formulation.

[0028] Preferably, the oral preparation is any one of tablets, capsules, granules, or oral liquid.

[0029] Most preferably, the oral formulation is a tablet, and the tablet further comprises any one of microcrystalline cellulose, croscarmellose sodium, and magnesium stearate.

[0030] Thirdly, the present invention also provides the use of the aforementioned earthworm protein composition in the preparation of a medicament having any of the following functions:

[0031] a) Improves menopausal symptoms;

[0032] b) Improve vasomotor or neuropsychiatric symptoms in perimenopausal syndrome.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] First, the earthworm protein composition provided by this invention for regulating ovarian function and improving menopausal syndrome has a highly simplified composition, reducing the complexity and mechanistic ambiguity caused by the excessive number of ingredients in traditional compound prescriptions. Simultaneously, through multi-target synergistic effects, the various components can systematically intervene in multi-system functional disorders during perimenopause. Specifically, it includes:

[0035] Earthworm protein active peptides (8-30 kDa): Through stepwise enzymatic hydrolysis and ultrafiltration, small molecular weight active peptides are precisely preserved, which are easily absorbed and have high bioavailability. They can regulate hormone levels (slow down estrogen decline), improve local microcirculation in the ovary (relieve vasomotor symptoms such as hot flashes), and reduce neurooxidative damage through antioxidant effects (improve neuropsychiatric symptoms such as anxiety and insomnia).

[0036] Cranberry extract (active ingredients such as proanthocyanidins): Through ultrasonic-assisted extraction and macroporous resin purification, anti-inflammatory and antioxidant components are enriched, which can improve atrophy of the mucous membrane of the reproductive and urinary system (relieve vaginal dryness, urinary frequency and urgency) and protect ovarian cells from oxidative damage.

[0037] Carob extract: Through water extraction and alcohol precipitation, high-purity estrogen-like active ingredients are preserved. It can directly or indirectly supplement estrogen deficiency, promote bone formation (prevent osteoporosis), and regulate glucose metabolism (help improve perimenopausal metabolic disorders).

[0038] The above three components work synergistically to cover multiple pathways, including hormone regulation, antioxidation, anti-inflammation, and bone metabolism. Compared with single components, their mechanisms of action are more comprehensive, and compared with complex compound formulas, their mechanisms of action are easier to elucidate.

[0039] Meanwhile, the composition provided by this invention features significantly optimized preparation processes. For example, stepwise enzymatic hydrolysis combined with ultrafiltration membrane separation precisely obtains 8-30 kDa active peptides. Peptides in this molecular weight range are more easily absorbed by the intestines and avoid interference from large molecules, ensuring high activity of the functional peptides. Furthermore, in the preparation of cranberry extract, ultrasonic-assisted extraction is used to disrupt plant cell walls and accelerate the dissolution of active ingredients. Combined with gradient elution using AB-8 macroporous resin (50% ethanol fraction), core components such as proanthocyanidins are selectively enriched, improving the purity and bioactivity of the extract. Finally, a water extraction and alcohol precipitation process (60%-70% ethanol final concentration) specifically retains D-inositol, avoiding impurities and ensuring efficient utilization of estrogen-like active ingredients. These improvements in preparation methods significantly enhance the effectiveness and controllability of each component, effectively improving the bioavailability and stability of active ingredients.

[0040] Finally, the composition provided by the present invention has widely available and safe raw materials; the composition is easy to prepare into various oral formulations such as tablets, capsules, granules or oral liquids to meet the needs of different groups of people. Detailed Implementation

[0041] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used 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 to the embodiments described below are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

[0042] To better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art will understand that the present invention can be practiced without certain specific details. In other embodiments, reagents, raw materials, methods, means, apparatus, and steps well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production. Furthermore, the experimental methods used in the following embodiments are conventional methods; materials, reagents, or instruments whose manufacturers are not specified are commercially available; conditions not specifically specified in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer; and this invention does not limit the source of raw materials used. Unless otherwise specified, all raw materials used in this invention are commercially available products commonly found in this technical field. Unless otherwise specified, "ratios" in the following embodiments refer to ratios of parts by mass.

[0044] Example 1

[0045] 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.

[0046] The preparation method of this earthworm protein is as follows:

[0047] A1: After pulverizing the earthworm raw material (dry), add it to a phosphate buffer solution with pH 8.0 at a material-to-liquid ratio of 1:10 to obtain a mixed system, and then perform enzymatic hydrolysis, specifically as follows:

[0048] The first stage of enzymatic hydrolysis: Enzymatic hydrolysis was carried out using a compound protease, which was added at 1.0% (w / w) of the dry weight of the earthworm raw material. Hydrolysis was carried out at 38°C for 3 hours (with the stirring speed controlled at 200 rpm; unless otherwise specified, this stirring speed will be used throughout the enzymatic hydrolysis process). The compound protease used in this example was composed of Bacillus subtilis protease and papain in a mass ratio of 3:2 (unless otherwise specified, all ratios are mass ratios).

[0049] Second stage of enzymatic hydrolysis: Adjust the pH to 7.0, add 1.0% trypsin (calculated based on the dry weight of earthworm raw material), and continue hydrolysis at 37°C for 2 hours;

[0050] A2: After enzymatic hydrolysis, heat at 90℃ for 10 min to inactivate the enzyme. First, filter the enzyme-inactivated hydrolysate through a 30 kDa ultrafiltration membrane. The retentate is a macromolecule with a molecular weight >30 kDa (discarded), and the permeate is a component with a molecular weight <30 kDa. Then, filter the permeate through an 8 kDa ultrafiltration membrane, collect the retentate, and obtain an active peptide fragment with a molecular weight of 8-30 kDa, which is the target product. Collect it to obtain an active peptide fragment with a molecular weight of 8 kDa-30 kDa. Freeze-dry it to obtain earthworm protein.

[0051] The preparation method of this cranberry extract is as follows:

[0052] B1: Ultrasonic-assisted ethanol extraction: using 60% ethanol as solvent, with a material-to-liquid ratio of 1:12 (w / v).

[0053] B2: Ultrasonic power 400W, frequency 40 kHz, extraction temperature 45 ℃, time 50 minutes, repeated twice;

[0054] B3: Combine the extracts, concentrate under reduced pressure until no alcohol odor remains, pass through an AB-8 macroporous resin column, and elute sequentially with 30% and 50% ethanol. Collect the 50% ethanol eluent and freeze-dry to obtain cranberry extract.

[0055] Based on the aforementioned earthworm protein and cranberry extract, the preparation method of the earthworm protein composition provided in this embodiment is as follows: weigh the earthworm protein and cranberry extract according to the mass ratio, and mix them evenly to obtain the earthworm protein composition.

[0056] Example 2

[0057] This embodiment provides an earthworm protein composition, the active ingredients of which are earthworm protein and cranberry extract in a mass ratio of 1:1. The preparation methods of earthworm protein and cranberry extract are the same as in Example 1.

[0058] The preparation method of the earthworm protein composition provided in this embodiment is the same as that in Example 1.

[0059] Example 3

[0060] This embodiment provides an earthworm protein composition, the active ingredients of which are earthworm protein and cranberry extract in a mass ratio of 1:3. The preparation methods of earthworm protein and cranberry extract are the same as in Example 1.

[0061] The preparation method of the earthworm protein composition provided in this embodiment is the same as that in Example 1.

[0062] Example 4

[0063] This embodiment provides an earthworm protein composition, the active ingredients of which are earthworm protein and cranberry extract in a mass ratio of 1:5. The preparation methods of earthworm protein and cranberry extract are the same as in Example 1.

[0064] The preparation method of the earthworm protein composition provided in this embodiment is the same as that in Example 1.

[0065] Comparative Example 1

[0066] This embodiment presents an earthworm protein composition, the active ingredients of which are earthworm protein and cranberry extract in a mass ratio of 1:3. The preparation method of the cranberry extract is the same as in Example 1, while the preparation method of the earthworm protein differs from that in Example 3 in the following ways:

[0067] The complex protease used in this embodiment is composed of subtilisin, papain and trypsin in a ratio of 3:1:1.

[0068] Comparative Example 2

[0069] This embodiment presents a lump worm protein composition, which differs from Comparative Example 1 in that the lump worm protein is prepared using a different method. Specifically, the complex protease used in this embodiment is composed of Bacillus licheniformis protease, Bacillus subtilis protease, and papain in a 1:2:2 ratio.

[0070] Comparative Example 3

[0071] This embodiment presents a lichen protein composition, which differs from Comparative Example 1 in that the lichen protein is prepared using a different method. Specifically, the complex protease used in this embodiment is composed of Bacillus licheniformis protease, Bacillus subtilis protease, papain, and trypsin in a ratio of 1:2:1:1.

[0072] Comparative Example 4

[0073] This embodiment presents a lichen protein composition, which differs from Comparative Example 1 in that the lichen protein is prepared using a different method. Specifically, the complex protease used in this embodiment is composed of Bacillus licheniformis protease, papain, and trypsin in a 3:1:1 ratio.

[0074] Comparative Example 5

[0075] This embodiment presents a composition of earthworm protein, which differs from Comparative Example 1 in that the preparation method of earthworm protein is different. Specifically, the complex protease used in this embodiment is composed of Bacillus licheniformis protease, Bacillus subtilis protease, papain, trypsin, and bromelain in a ratio of 1:2:1:1:1.

[0076] Comparative Example 6

[0077] This embodiment presents a lichen protein composition, which differs from Comparative Example 1 in that the lichen protein is prepared using a different method. Specifically, the complex protease used in this embodiment is composed of Bacillus licheniformis protease, Bacillus subtilis protease, papain, trypsin, bromelain, and elastase in a ratio of 1:2:1:1:1:1.

[0078] Comparative Example 7

[0079] This embodiment presents a lump worm protein composition, which differs from Comparative Example 2 in that the lump worm protein is prepared using a different method. Specifically, the complex protease used in this embodiment is a mixture of Bacillus licheniformis protease and Bacillus subtilis protease in a 1:2 ratio.

[0080] Comparative Example 8

[0081] This embodiment presents a type of earthworm protein composition, which differs from Comparative Example 2 in that the earthworm protein is prepared using a different method. Specifically, the complex protease used in this embodiment is a mixture of Bacillus licheniformis protease and papain in a 3:2 ratio.

[0082] Comparative Example 9

[0083] This embodiment presents a type of earthworm protein composition, which differs from Comparative Example 2 in that the earthworm protein is prepared using a different method. Specifically, the complex protease used in this embodiment is composed of Bacillus licheniformis protease, Bacillus subtilis protease, and bromelain in a 1:2:1 ratio.

[0084] Comparative Example 10

[0085] This embodiment presents a lump worm protein composition, which differs from Comparative Example 2 in that the lump worm protein is prepared using a different method. Specifically, the complex protease used in this embodiment is composed of Bacillus licheniformis protease, Bacillus subtilis protease, and elastase in a 1:2:1 ratio.

[0086] Comparative Example 11

[0087] This embodiment describes a type of earthworm protein composition, which differs from Comparative Example 2 in that the earthworm protein is prepared using a different method. Specifically, in this embodiment, only the first stage of enzymatic hydrolysis is performed, without the second stage of enzymatic hydrolysis. After the first stage of enzymatic hydrolysis, the active peptides of 8 kDa-30 kDa are directly collected.

[0088] Comparative Example 12

[0089] This embodiment describes a type of earthworm protein composition, which differs from Comparative Example 2 in that the earthworm protein is prepared using a different method. Specifically, in this embodiment, only the second-stage enzymatic hydrolysis is performed, without the first-stage enzymatic hydrolysis. After the second-stage enzymatic hydrolysis, the active peptides of 8 kDa-30 kDa are directly collected.

[0090] It should be noted that the enzymes used in this invention are described as follows: Bacillus subtilis protease activity is approximately 400,000 U / mg (Hubei Yuancheng Saichuang Technology Co., Ltd.); papain activity is approximately 100,000 U / mg (Sichuan Lvcheng Biotechnology Co., Ltd.); trypsin activity is 100,000 U / mg (Sichuan Lvcheng Biotechnology Co., Ltd.); Bacillus licheniformis protease (Anhui Zhonghong Bioengineering Co., Ltd.) activity is approximately 200,000 U / mg; bromelain activity is approximately 100,000 U / mg (Jiangxi Huayuyuan Biotechnology Co., Ltd.); and elastase activity is approximately 100,000 U / mg (Jiangxi Huayuyuan Biotechnology Co., Ltd.).

[0091] Experimental Example 1

[0092] This test example examines the earthworm protein and earthworm protein compositions prepared in Examples 1-4 and Comparative Examples 1-12, including:

[0093] 1.1 Determination of earthworm protein extraction rate (8 kDa-30 kDa)

[0094] After two-step enzymatic hydrolysis, the earthworm raw material is separated by passing it through 30 kDa and 8 kDa ultrafiltration membranes in sequence, and the 8-30 kDa retentate is collected.

[0095] After freeze-drying, weigh the product and calculate the extraction rate. The calculation method is as follows:

[0096] Extraction rate (%) = mass of freeze-dried product (g) / total protein content of earthworm raw material (g) × 100%;

[0097] The method for determining the total protein content of earthworm raw material is as follows: protein is precipitated with trichloroacetic acid (TCA), centrifuged, and the nitrogen content (containing only protein nitrogen) in the precipitate is determined and converted into protein content. Each group of experiments is performed independently 3 times, and the mean ± standard deviation is taken. The experimental results are shown in Table 1 (n=3).

[0098] 1.2 Evaluation of in vitro thrombolytic activity (fibrin plate method)

[0099] Prepare the 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), quickly pour into a culture dish, and allow to solidify to form a fibrin plate.

[0100] Then, the earthworm protein was dissolved in physiological saline to prepare a 2 mg / mL earthworm protein solution.

[0101] During the test, 50 μL of earthworm protein solution prepared by pipetting was applied to fibrin plates (6 parallel spots per group). 50 μL of physiological saline was used as a blank control. The plates with the test sample were incubated at 37°C for 18 hours. The fibrin dissolved to form a transparent thrombolytic zone. The area of ​​the thrombolytic zone (mm²) was measured using ImageJ software. The size of the thrombolytic zone area reflects the thrombolytic activity. The experimental results are shown in Table 1 (n=6).

[0102] Table 1. Comparison of extraction rate and in vitro thrombolytic activity of earthworm protein in each group (mean ± standard deviation)

[0103] Group Earthworm protein extraction rate (%) In vitro thrombolytic activity (dissolution zone area, mm²) Example 1 65.2 ± 1.3 158.3 ± 4.2 Example 2 68.7 ± 1.1 172.6 ± 3.8 Example 3 75.0 ± 1.2 192.5 ± 3.1 Example 4 70.1 ± 1.2 185.7 ± 4.0 Comparative Example 1 71.5 ± 1.0 198.4 ± 2.9 Comparative Example 2 82.3 ± 0.9 215.7 ± 2.5 Comparative Example 3 73.8 ± 1.1 207.2 ± 3.0 Comparative Example 4 69.4 ± 1.4 188.6 ± 3.7 Comparative Example 5 74.2 ± 0.9 208.1 ± 2.8 Comparative Example 6 72.9 ± 1.0 206.5 ± 3.2 Comparative Example 7 74.8 ± 0.9 205.3 ± 2.7 Comparative Example 8 73.5 ± 1.0 200.6 ± 3.3 Comparative Example 9 74.3 ± 1.0 203.8 ± 3.0 Comparative Example 10 72.1 ± 1.2 196.5 ± 3.5 Comparative Example 11 58.4 ± 1.5 140.2 ± 4.8 Comparative Example 12 52.7 ± 1.8 125.6 ± 5.1

[0104] The above experimental results indicate that the extraction rate and in vitro thrombolytic activity of earthworm protein vary significantly under different experimental conditions, specifically:

[0105] In Examples 1-4, with the adjustment of the mass ratio of earthworm protein to cranberry extract, Example 3 (1:3 ratio) showed the best overall effect. In the earthworm protein preparation method of this group (Example 3), the earthworm protein extraction rate was 75.0%, which was significantly higher than that of other examples (Examples 1, 2, and 4), indicating that the enzymatic hydrolysis efficiency and the release effect of the target peptide (8-30 kDa) were better at the 1:3 ratio. The in vitro thrombolytic activity of the Example 3 group was 192.5 mm², which was significantly improved compared with Example 1 (158.3 mm²) and Example 2 (172.6 mm²). This experimental result shows that the increase of the cranberry extract ratio (1:3) enhanced the plasminogen activation ability through the synergistic effect of polyphenols.

[0106] Among comparative examples 1-12, comparative example 2 (composite protease composition: Bacillus licheniformis protease + Bacillus subtilis + papain, 1:2:2) showed the best overall performance, with an extraction rate as high as 82.3%, significantly better than all examples and other comparative examples. This indicates 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 was 215.7 mm², which was 12.1% higher than that of example 3 (192.5 mm²), indicating that the enzymatic hydrolysis effect of this composite protease was better and significantly better than example 3, comparative example 8, etc.

[0107] Furthermore, 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 these were significantly better than those of Example 3, they were still lower than those of Comparative Example 2. This indicates that the addition of bromelain (targeting the α-chain of fibrous protein) and elastase (targeting elastic fibers) failed to completely replace the synergistic effect of the multiple enzymes in Comparative Example 2.

[0108] Finally, the extraction rates (58.4%, 52.7%) and thrombolytic activities (140.2 mm², 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 other groups, verifying the necessity of the two-step enzymatic hydrolysis process. The possible reason is that the first-stage enzymatic hydrolysis (complex protease) is responsible for the initial degradation of macromolecules, while the second stage (trypsin) further releases active peptides. This theory needs further verification.

[0109] In summary, Example 3 (1:3 ratio) performed best among the examples, demonstrating that the mass ratio of earthworm protein to cranberry extract has a crucial impact on thrombolytic activity. Comparative Example 2 (Bacillus licheniformis protease complex protease), through the synergistic effect of multiple enzymes, significantly outperformed all groups in both extraction rate and thrombolytic activity. The optimization of its enzymatic hydrolysis process (introduction of Bacillus licheniformis protease) is the core strategy for enhancing function. This proves that the refined design of the two-step enzymatic hydrolysis process (complex protease → trypsin) and the ratio of complex protease types are important factors influencing the functional activity of earthworm protein.

[0110] Example 5

[0111] The pharmacological activities of carob extract are mainly reflected in regulating metabolism, improving endocrine-related diseases, and anti-inflammation. Its core active ingredients include D-chiroinositol (DCI) and polysaccharides. D-chiroinositol is one of the key active ingredients of carob extract, which has an insulin-sensitizing effect and helps lower blood sugar levels. At the same time, DCI has a significant effect on the ovulation function and menstrual cycle recovery of patients with polycystic ovary syndrome (PCOS): clinical trials showed that after 6-8 weeks of taking carob extract, 86% and 60% of obese and underweight PCOS patients, respectively, were able to ovulate spontaneously; after 6-15 months of taking it, 62.5% of women with menstrual cycle disorders experienced effective restoration of their menstrual cycle.

[0112] Therefore, based on existing research on carob extract, the inventors further combined carob extract with earthworm protein and cranberry extract prepared in Comparative Example 2 to observe whether there is a synergistic effect among the above components. The specific experimental design is as follows:

[0113] First, this embodiment provides a method for preparing carob extract, specifically prepared according to the following method:

[0114] C1: Water extraction: Take dried carob seeds (from the Mediterranean carob tree (Ceratonia siliqua)) and grind them into powder. Pass the powder through an 80-100 mesh sieve and mix it with 40% ethanol solution at a ratio of 1:18 (w / v). Reflux and extract twice at 90℃, 2 hours each time.

[0115] C2: Alcohol precipitation: Combine the extracts and concentrate to a relative density of 1.15-1.20 (60℃), add 95% ethanol to a final concentration of 65%, let stand at 4℃ for 24 hours, and centrifuge to collect the precipitate.

[0116] C3: The precipitate was dried under vacuum to obtain carob extract;

[0117] Secondly, the present invention provides multiple compositions and tests the pharmacological activities of these multiple compositions, including:

[0118] 5.1 Experimental Grouping

[0119] Animal selection: SPF-grade female SD rats (12 weeks old) were randomly divided into 9 groups (n=10), including:

[0120] Normal control group (sham surgery, ovaries not removed);

[0121] Model group (OVX surgery, no treatment);

[0122] Positive control group (OVX + estradiol, 0.1 mg / kg / d);

[0123] Control group 1 (OVX + carob extract 400 mg / kg / d);

[0124] Control group 2 (OVX + earthworm protein 400 mg / kg / d, prepared by the method in Comparative Example 2);

[0125] Control group 3 (OVX + cranberry extract 400 mg / kg / d, prepared by the method in Comparative Example 2);

[0126] Composition 1 (OVX + with earthworm protein and cranberry extract as active substances in a mass ratio of 1:3, total dose 400 mg / kg / d).

[0127] Composition 2 (OVX + with earthworm protein and carob extract in a mass ratio of 1:1 as active substances, total dose 400 mg / kg / d).

[0128] Composition 3 groups (OVX + with cranberry extract and carob extract in a mass ratio of 3:1 as active substances, total dose 400 mg / kg / d);

[0129] Composition 4 groups (OVX + with earthworm protein, cranberry extract and carob extract in a mass ratio of 1:3:1 as active substances; total dose 400 mg / kg / d);

[0130] Except for the normal control group, the other groups underwent bilateral oophorectomy (OVX), and the modeling method will not be described here. Gavage administration began on day 4 after modeling, once daily for 28 weeks (dose of 400 mg / kg / day).

[0131] 5.2 Experimental Results and Analysis

[0132] Data analysis in this experiment used one-way ANOVA, and Tukey's HSD test was used for comparisons among multiple groups. Compared with the model group: *p<0.05, **p<0.01.

[0133] (1) Detection of behavioral indicators related to perimenopausal symptoms:

[0134] Postoperative estrogen deficiency after open-arm surgery reduces the synthesis of neurotransmitters such as serotonin (5-HT) and norepinephrine (NE) in the brain, leading to anxiety and depression-like behaviors (a decreased proportion of open-arm time and prolonged immobile time in the model group). If the proportion of open-arm time increases and the immobile time decreases after intervention, it suggests that the medication may alleviate mood disorders by increasing E2 levels, improving oxidative stress, or regulating neurotransmitters (such as 5-HT).

[0135] In the elevated cross maze, a lower percentage of time spent in open-arm positions indicates a lower willingness among rats to explore open areas (due to anxiety-induced avoidance of exposed environments), reflecting common perimenopausal anxiety symptoms. In the forced swimming test, a longer period of immobility indicates that rats have given up actively struggling ("behavioral despair"), reflecting perimenopausal depressive-like behaviors. The experimental results are shown in Table 2.

[0136] Table 2. Results of detection of various behavioral indicators related to perimenopausal symptoms in rats

[0137] Group Percentage of time spent with arms open (%) Stationary time (s) normal control group 42.3 ± 3.1 85.2 ± 6.8 Model group 18.5 ± 2.7 162.4 ± 10.5 Positive control group 38.6 ± 2.9** 92.7 ± 7.2** Control group 1 24.7 ± 2.5* 140.1 ± 9.3* Control group 2 22.8 ± 2.2* 148.6 ± 8.7* Control group 3 26.4 ± 2.8* 135.9 ± 8.9** Composition 1 30.2 ± 2.6** 120.3 ± 7.5** Composition 2 28.9 ± 2.3** 125.8 ± 8.1** Composition 3 34.1 ± 3.0** 110.5 ± 7.9** Composition 4 (three components) 39.8 ± 3.2** 95.3 ± 6.5**

[0138] The above experimental results show that the open-arm time ratio and immobility time of composition 4 are close to those of the positive control group (estradiol), and significantly better than all single-component and two-component compositions. This suggests that the three-component combination can more effectively improve anxiety and depression-like behaviors by regulating the synergistic effects of neurotransmitters (5-HT / NE) and oxidative stress.

[0139] (2) Serum TNF-α, IL-6 levels, E2, FSH, and LH detection

[0140] TNF-α (tumor necrosis factor-α) and IL-6 (interleukin-6) are classic pro-inflammatory cytokines. Estrogen deficiency after OVX surgery activates inflammatory pathways such as NF-κB, leading to increased secretion of TNF-α and IL-6 (significantly elevated in the model group), reflecting the local and systemic chronic inflammatory state of the ovary.

[0141] E2, secreted by the granulosa cells of ovarian follicles, is a core indicator of ovarian function. After OVX surgery, the ovary is removed, interrupting E2 synthesis (E2 levels were significantly lower in the model group). FSH and LH are secreted by the pituitary gland and are regulated by negative feedback from E2: when E2 decreases, the pituitary gland loses its inhibitory effect, leading to increased FSH and LH secretion (FSH and LH levels were significantly higher in the model group), resulting in the perimenopausal hormonal characteristic of "low E2 and high FSH / LH". If E2 increases and the FSH / LH ratio decreases after intervention, it indicates that the medication can effectively improve ovarian function.

[0142] The detection method was as follows: After the experiment, 2-3 mL of blood was collected from the orbital venous plexus, incubated at 4℃ for 30 min, and then centrifuged at 3000 r / min for 15 min to separate the serum. The serum was then detected using an enzyme-linked immunosorbent assay (ELISA) kit. Simultaneously, based on the obtained serum samples, E2, FSH, and LH were further detected using chemiluminescent immunoassay (CLIA): A fully automated chemiluminescent immunoassay analyzer (such as Roche Cobas e411) was used to detect the concentration of hormones in the serum (E2 unit: pg / mL; FSH and LH units: mIU / mL) through a specific antibody-antigen reaction combined with chemiluminescent markers. The experimental results are shown in Table 3.

[0143] Table 3. Detection results of serum TNF-α, IL-6, E2, FSH, and LH levels in rats of each group.

[0144] Group TNF-α (pg / mL) IL-6 (pg / mL) E2 (pg / mL) FSH (mIU / mL) LH (mIU / mL) normal control group 12.5 ± 1.2 8.3 ± 0.9 32.4 ± 2.5 6.8 ± 0.7 5.2 ± 0.6 Model group 38.7 ± 3.5 26.4 ± 2.1 9.6 ± 1.1 24.3 ± 2.2 18.9 ± 1.8 Positive control group 15.3 ± 1.5** 10.1 ± 1.0** 28.7 ± 2.3** 8.1 ± 0.8** 6.5 ± 0.7** Control group 1 30.2 ± 2.8* 20.5 ± 1.9* 14.2 ± 1.3* 18.6 ± 1.7* 14.3 ± 1.5* Control group 2 32.8 ± 3.0* 22.1 ± 2.0* 12.8 ± 1.2* 19.7 ± 1.8* 15.1 ± 1.6* Control group 3 28.4 ± 2.6* 18.7 ± 1.7** 15.6 ± 1.4** 17.2 ± 1.6** 13.8 ± 1.4** Composition 1 22.6 ± 2.1** 14.3 ± 1.3** 18.9 ± 1.7** 13.5 ± 1.3** 11.2 ± 1.2** Composition 2 25.1 ± 2.3** 16.8 ± 1.5** 17.5 ± 1.6** 14.8 ± 1.4** 12.4 ± 1.3** Composition 3 19.4 ± 1.8** 12.6 ± 1.2** 22.3 ± 2.0** 10.9 ± 1.1** 9.8 ± 1.0** Composition 4 14.9 ± 1.4** 9.8 ± 0.9** 26.5 ± 2.4** 9.2 ± 0.9** 7.6 ± 0.8**

[0145] Composition 4 significantly reduced pro-inflammatory factors (TNF-α, IL-6), increased E2, and decreased FSH / LH, with better effects than all control groups and the two-component composition (p<0.01). The three components may restore ovarian-pituitary axis function by synergistically regulating inflammatory pathways (such as NF-κB) and hormonal feedback mechanisms.

[0146] (3) Detection of oxidative stress level:

[0147] SOD (superoxide dismutase) is a key antioxidant enzyme that can scavenge O2. - MDA (malondialdehyde) is an end product of lipid peroxidation, reflecting the degree of free radical damage to cell membranes. The model group showed decreased SOD activity and increased MDA, indicating severe oxidative stress damage to ovarian tissue (estrogen deficiency leading to decreased antioxidant defense system function). After intervention, decreased TNF-α and IL-6, increased SOD, and decreased MDA indicate that the drug has anti-inflammatory and antioxidant effects.

[0148] Ovarian tissue was removed immediately after rats were euthanized. Blood was rinsed off with physiological saline, the tissue was blotted dry with filter paper, weighed, and 9 times the volume of pre-cooled physiological saline (0.9% NaCl) was added. The mixture was homogenized in an ice bath, centrifuged, and the supernatant was collected. SOD activity was detected using the xanthine oxidase method, and MDA content was detected using the thiobarbituric acid (TBA) method. The experimental results are shown in Table 4.

[0149] Table 4. Results of oxidative stress level detection in rat ovarian tissue homogenates of each group

[0150] Group SOD activity (U / mg protein) MDA content (nmol / mg protein) normal control group 56.3 ± 4.2 2.8 ± 0.3 Model group 28.7 ± 2.5 6.9 ± 0.6 Positive control group 50.1 ± 3.8** 3.1 ± 0.3** Control group 1 35.2 ± 3.0* 5.2 ± 0.5* Control group 2 33.8 ± 2.8* 5.6 ± 0.5* Control group 3 38.6 ± 3.2** 4.7 ± 0.4** Composition 1 42.5 ± 3.5** 4.1 ± 0.4** Composition 2 40.3 ± 3.3** 4.3 ± 0.4** Composition 3 46.2 ± 3.7** 3.6 ± 0.3** Composition 4 53.7 ± 4.0** 3.0 ± 0.3**

[0151] The SOD activity of composition 4 was restored 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 reduce oxidative damage through multiple targets.

[0152] The above experimental results show that composition 4 (earthworm protein: cranberry: carob = 1:3:1) exhibits a significant synergistic effect in all indicators.

[0153] Example 6

[0154] Based on composition 4 of Example 5 above, this embodiment further provides a tablet containing earthworm protein composition I, and the specific preparation method is as follows:

[0155] (1) Mixing and granulation

[0156] Weigh 50g of mixture 4 (earthworm protein: cranberry: carob = 1:3:1), filler (150g microcrystalline cellulose, 100g lactose), and 2 / 3 of the disintegrant (20g crospovidone) and add them to a high-efficiency mixing granulator. Mix at low speed for 15 minutes, then slowly add 5% HPMC aqueous solution to make a soft mass. Pass the soft mass through an 18-mesh sieve to make wet granules.

[0157] (2): Drying and granulation

[0158] The wet granules are placed in a fluidized bed dryer and dried at 50°C (to avoid denaturation of earthworm protein at high temperatures) until the moisture content is no higher than 4% (as measured by a moisture meter). The dried granules are then sieved through a 20-mesh sieve to remove fine powder and clumps, ensuring granule flowability.

[0159] (3): Total mixture and tableting

[0160] The granulated granules, along with the remaining 1 / 3 disintegrant (10g crospovidone), lubricant (5g magnesium stearate), and flavoring agent (10g fructooligosaccharide), are added to a three-dimensional mixer and mixed at low speed for 18 minutes. Then, the mixture is compressed to obtain earthworm protein composition tablets.

[0161] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A composition of earthworm protein for regulating ovarian function and improving menopausal syndrome, characterized in that, Its active ingredients, by mass parts, include earthworm protein, cranberry extract and carob extract, with the mass ratio of earthworm protein, cranberry extract and carob extract being 1:(0.5-5):(0.5-5); The method for preparing the earthworm protein is as follows: After crushing the earthworm raw material, add phosphate buffer solution with pH 7.0-8.5 at a material-to-liquid ratio of 1:8-12; Complex protease and trypsin were added sequentially for enzymatic hydrolysis; after enzymatic hydrolysis and enzyme inactivation, the active peptides with molecular weights of 8 kDa-30 kDa were separated by ultrafiltration membrane and collected. The enzymatic hydrolysis is a stepwise enzymatic hydrolysis, including: First stage of enzymatic hydrolysis: Add 0.5%-1.5% of the compound protease and hydrolyze at 37-40℃ for 2-4 hours; 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; The preparation method of the cranberry extract is as follows: Ultrasonic-assisted ethanol extraction was performed using 40%-70% ethanol as the solvent, with a material-to-liquid ratio of 1:10-15 (w / v). Ultrasonic power 300-500 W, frequency 40 kHz, extraction temperature 40-50℃, time 30-60 minutes, repeated 2-3 times; Combine the extracts, concentrate under reduced pressure until no alcohol odor remains, pass through an AB-8 macroporous resin column, and elute sequentially with 30% and 50% ethanol. Collect the 50% ethanol eluent and freeze-dry to obtain cranberry extract. The method for preparing the carob extract is as follows: Water extraction: Carob powder and ethanol solution are mixed at a w / v ratio of 1:15-20, and extracted twice by reflux at 80-90℃, each time for 1-2 hours; Alcohol precipitation: Combine the extracts at 60℃ and concentrate to a relative density of 1.15-1.20, add 95% ethanol to a final concentration of 60%-70%, let stand at 4℃ for 12-24 hours, and centrifuge to collect the precipitate; The precipitate was dried under vacuum to obtain carob extract.

2. The method for preparing the earthworm protein composition for regulating ovarian function and improving menopausal syndrome as described in claim 1, characterized in that, Includes the following steps: S1: Weigh earthworm protein, cranberry extract and carob extract according to the mass ratio of 1:(0.5-5):(0.5-5), and mix them evenly to obtain mixture I; S2: Add clinically acceptable excipients to the aforementioned mixture I to prepare an oral formulation.

3. The preparation method according to claim 2, characterized in that, The oral preparation is any one of tablets, capsules, granules, or oral liquid.

4. The preparation method according to claim 3, characterized in that, The tablets also include any one of microcrystalline cellulose, croscarmellose sodium cellulose, and magnesium stearate.

5. The use of the earthworm protein composition for regulating ovarian function and improving menopausal syndrome as described in claim 1, or the earthworm protein composition for regulating ovarian function and improving menopausal syndrome prepared by any one of claims 2-4, in the preparation of a medicament having any of the following functions: a) Improves menopausal symptoms; b) Improve vasomotor or neuropsychiatric symptoms in perimenopausal syndrome.