A kind of abalone active peptide, composition, composite preparation for relieving dysmenorrhea and preparation method thereof

Abstract

This invention relates to an abalone active peptide, a composition, a compound preparation for relieving dysmenorrhea, and a method for preparing the same, belonging to the field of biopharmaceutical manufacturing. The amino acid sequence of the abalone active peptide is Tyr-Arg-Leu-Gly-Asp. The composition containing the abalone active peptide includes: abalone active peptide, dandelion, jujube, and rose. The abalone active peptide and plant extracts are mixed evenly according to a specific ratio to obtain the compound preparation for relieving dysmenorrhea. This invention uses a specific active peptide derived from abalone as its core, and achieves multi-target synergistic effects by combining it with extracts from medicinal and edible plants to regulate dysmenorrhea. It has the effects of being natural and safe, having multi-target synergistic effects, rapid onset of action, and effectively relieving the root cause of dysmenorrhea.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical manufacturing technology, specifically relating to an abalone active peptide, a composition, a compound preparation for relieving dysmenorrhea, and a method for preparing the same. Background Technology

[0002] Dysmenorrhea is a common condition of the female reproductive system, with a global incidence rate of 45-95% among women of reproductive age. Of these, 15-20% experience severe dysmenorrhea, significantly impacting their work, studies, and quality of life. The pathogenesis of dysmenorrhea is complex, but its core components are related to abnormal prostaglandin (PG) metabolism, uterine microcirculatory disturbances, and activated inflammatory responses. Specifically, during endometrial shedding, inflammatory factors (tumor necrosis factor-α and interleukin-6) increase, inducing enhanced cyclooxygenase-2 (COX-2) expression and promoting prostaglandin metabolism. Synthesis of substances such as PGE2 leads to excessive contraction of uterine smooth muscle, ischemia and hypoxia, resulting in spasmodic pain; uterine vasoconstriction leads to insufficient blood perfusion, further aggravating ischemia and hypoxia, forming a vicious cycle of "pain-ischemia"; at the same time, emotions such as anxiety and tension amplify pain perception through the hypothalamus-limbic system, exacerbating discomfort.

[0003] Currently, treatment options for dysmenorrhea have significant limitations: chemical drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., ibuprofen) have clear short-term analgesic effects, but long-term use can easily cause gastrointestinal damage and burden on liver and kidney function, and cannot alleviate or improve the fundamental mechanism of dysmenorrhea; opioids, due to their addictive nature, are only suitable for severe pain, limiting their clinical application. Traditional Chinese medicines for regulating menstruation, such as motherwort, require long-term decoction and administration, have a slow onset of action (usually requiring 2-3 consecutive cycles), and the single herb has limited target points. For example, the method for evaluating the efficacy and pharmacodynamic material basis of motherwort extract in animal models of dysmenorrhea disclosed in Chinese patent CN118846139A, through the construction of animal models, analyzes the efficacy and pharmacodynamic material basis of motherwort extract in simulated cold-coagulation and blood stasis type primary dysmenorrhea, revealing that motherwort extract can alleviate dysmenorrhea by improving relevant histopathological changes, verifying the in vivo efficacy of motherwort after administration. This technical solution relies on a single plant ingredient, lacks a clear target, and cannot specifically inhibit the COX-2 / PGE2 pathway, making it difficult to cover the multiple mechanisms of dysmenorrhea and limiting its therapeutic effect. Physical methods such as hot compresses and moxibustion can only act on local blood circulation, temporarily improving peripheral blood flow, but have no regulatory effect on core pathological aspects such as abnormal prostaglandin metabolism and the release of inflammatory factors. Moreover, the effect is greatly affected by the operation method and individual differences, resulting in poor stability. For example, a fast-acting dysmenorrhea patch disclosed in Chinese patent CN119367450A includes borneol, musk, saffron extract, white peony extract, angelica, frankincense extract, myrrh extract, motherwort extract, chuanxiong extract, artemisia extract, nano-magnetic powder, and far-infrared materials. It relieves dysmenorrhea by using a scientific ratio of multiple traditional Chinese medicine ingredients, modern extraction technology, and combination with physical therapy. This technical solution only relieves discomfort through local warming effects, does not design active ingredients targeting the core mechanisms of dysmenorrhea, and cannot fundamentally improve pain.

[0004] Furthermore, while some studies have focused on the bioactivity of abalone peptides, such as the Chinese patent CN109486892A which discloses an abalone peptide for enhancing immunity and its preparation method and application, the method includes: taking abalone meat, adding deionized water, and pulping to obtain a slurry containing crude protein; adding deionized water to the slurry to obtain a protein solution; adding protease, and enzymatically hydrolyzing to obtain crude abalone peptide. This technical solution utilizes protease to enzymatically hydrolyze abalone protein, and the resulting abalone peptide has the effect of enhancing immunity and stress resistance. This technical solution only focuses on the application of abalone peptides in enhancing immunity and stress resistance, and does not involve the field of dysmenorrhea regulation, nor does it explore the regulatory effects of abalone peptides on COX-2 activity, uterine microcirculation, or inflammatory factors, and is not related to the pathological mechanism of dysmenorrhea. Summary of the Invention

[0005] The purpose of this invention is to provide an abalone active peptide, a composition, a compound preparation for relieving dysmenorrhea, and a method for preparing the same. This invention uses specific active peptides derived from abalone as its core, and achieves multi-target synergistic effects by combining extracts from medicinal and edible plants to regulate dysmenorrhea. The prepared abalone active peptide, composition, and compound preparation for relieving dysmenorrhea are natural and safe, exhibit multi-target synergistic effects, rapid onset of action, and effectively alleviate the root cause of dysmenorrhea.

[0006] The technical solution adopted by this invention to solve the technical problem is as follows: This invention provides an abalone bioactive peptide with the amino acid sequence Tyr-Arg-Leu-Gly-Asp.

[0007] The present invention also provides a composition containing the aforementioned abalone active peptide, comprising the following components in parts by weight: 8-10 portions of abalone active peptides; 15-35 parts of plant extract.

[0008] In a preferred embodiment, the plant extract comprises the following components by weight: 3-5 portions of dandelion; 6-8 portions of jujubes; 5-7 roses.

[0009] The present invention also provides a method for preparing a compound preparation for relieving dysmenorrhea from the said composition, comprising the following steps: Step 1: Fresh abalone muscle tissue is taken, washed, freeze-dried, and pulverized. Ethanol solution is added and stirred for extraction. The precipitate is collected by centrifugation, and deionized water is added to prepare a homogenate. 1-5% (by weight of abalone muscle tissue) of flavor protease is added to the homogenate, and enzymatic hydrolysis is carried out at 50-60℃ and pH 5.5-6.5 for 2-4 hours. The temperature is adjusted to 40-45℃ and pH to 7.0-7.5, and 3-5% (by weight of abalone muscle tissue) of complex protease is added, and enzymatic hydrolysis continues at a constant temperature for 4-6 hours. After enzyme inactivation, the mixture is cooled to room temperature, and the supernatant is collected by centrifugation. The supernatant is filtered, and the permeate is collected. The permeate is concentrated and desalted, and the retentate is collected. The retentate is then separated and purified sequentially using a DEAE-Sepharose FF weak anion exchange column and a Sephadex G-15 gel chromatography column, and the elution peaks are collected. The elution peaks are further purified using a C18 reversed-phase chromatography column. The purified components are freeze-dried to obtain abalone active peptides. Step 2: After washing, drying, and pulverizing the plant extract raw materials, rose powder is added to an ethanol solution, ultrasonically treated at room temperature, and filtered to obtain rose ethanol extract filtrate and residue. The residue is combined with dandelion powder and jujube powder to obtain a mixed powder. Deionized water is added to the mixed powder and stirred for extraction. The first low-temperature extract is collected by filtration, and deionized water is added to the residue for reflux extraction. The second high-temperature extract is collected by filtration. The rose ethanol extract filtrate, the first low-temperature extract, and the second high-temperature extract are combined. The combined extract is microfiltered, and the permeate is collected. After vacuum concentration and freeze-drying, the plant extract powder is obtained. Step 3: Mix abalone active peptides and plant extracts evenly according to the specified ratio to obtain a compound preparation for relieving menstrual cramps.

[0010] In a preferred embodiment, in step one, the complex protease is prepared by neutral protease and papain in a mass ratio of (1-4):(1-4).

[0011] In a preferred embodiment, the specific procedure for separating and purifying the retentate using a DEAE-Sepharose FF weak anion exchange column and a Sephadex G-15 gel chromatography column in step one is as follows: The retentate was diluted with 5-10 times deionized water and loaded onto a DEAE-Sepharose FF weak anion exchange column. Gradient elution was performed with Tris-HCl buffer containing 0-0.5M NaCl at a flow rate of 1.5-2.5 mL / min and a detection wavelength of 280 nm. The fraction eluted under the 0.1-0.2M NaCl gradient was collected, lyophilized, and reconstituted. The fraction was then separated and purified using a Sephadex G-15 gel chromatography column with deionized water as the elution solvent at a flow rate of 1.2-2.0 mL / min. The absorbance was detected at a wavelength of 280 nm, and the elution peak with a retention time of 11 min was collected.

[0012] In a preferred embodiment, the specific procedure for further purification of the elution peak using a C18 reversed-phase chromatography column in step one is as follows: Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid acetonitrile solution. The gradient elution program was: 0-15 min 5-20% B, 15-35 min 20-40% B, with a flow rate of 0.8-1.2 mL / min and a column temperature of 28-33℃. The fraction with a retention time of 21.5-22.5 min was collected.

[0013] In a preferred embodiment, in step two, 10-14 times the volume of deionized water is added to the mixed powder, and the mixture is stirred and extracted in a water bath at 55-65℃ for 1.0-1.5 hours. The first low-temperature extract is collected by filtration, and 8-12 times the volume of deionized water is added to the filter residue. The mixture is then refluxed at 80-90℃ for 1.5-2.5 hours, and the second high-temperature extract is collected by filtration.

[0014] In a preferred embodiment, in step three, the method for preparing the compound preparation for relieving dysmenorrhea into tablets is as follows: adding excipients to the preparation and obtaining tablets through a tableting process; the excipients include the following components by weight: 30-35 parts of maltodextrin, 12-15 parts of sodium carboxymethyl cellulose, and 6-8 parts of magnesium stearate.

[0015] In a preferred embodiment, in step three, the method for preparing the compound preparation for relieving dysmenorrhea into an oral liquid is as follows: dissolve the preparation in purified water, control the solid content at 15-20%, add 0.1-0.2% steviol glycosides to adjust the taste, and then fill the container to obtain the oral liquid.

[0016] The beneficial effects of this invention are: This invention obtains an abalone bioactive peptide from abalone protein through separation and purification by gel size exclusion chromatography and reversed-phase high-performance liquid chromatography. The peptide has the following amino acid sequence: Tyr-Arg-Leu-Gly-Asp, i.e., YRLGD. According to online databases BIOPEP and EROP-Moscow, this amino acid sequence is a novel small molecule bioactive peptide.

[0017] This invention utilizes isolated and purified abalone active peptides to prepare a composition and a compound preparation for relieving dysmenorrhea. Compared with existing technologies, this preparation has the following advantages: 1. Synergistic effect of multiple targets: The core components of the compound preparation prepared in this invention are abalone active peptides and extracts of medicinal and edible plants. The active peptides derived from abalone exert anti-inflammatory, antispasmodic, and prostaglandin synthesis-inhibiting effects through targeted action. Combined with a variety of medicinal and edible plant raw materials, it has a synergistic effect of anti-inflammatory, antispasmodic, and microcirculation-improving effects. It can not only quickly relieve pain, but also alleviate the root mechanism of dysmenorrhea, quickly relieve pain and break the vicious cycle of dysmenorrhea. The effect of relieving dysmenorrhea is significantly better than that of a single component.

[0018] 2. High safety of natural ingredients: The compound preparation prepared by this invention uses all raw materials that are edible or of the same origin as food and medicine. Through scientific extraction and proportioning, it has no adverse reactions such as gastrointestinal damage or burden on liver and kidney function caused by chemical drugs, and is suitable for long-term daily treatment of dysmenorrhea. Detailed Implementation

[0019] In a first aspect, the present invention provides an abalone active peptide.

[0020] The present invention provides an abalone active peptide with the amino acid sequence Tyr-Arg-Leu-Gly-Asp.

[0021] In a second aspect, the present invention provides a composition containing the abalone active peptide.

[0022] The present invention provides a composition containing the aforementioned abalone active peptide, comprising the following components in parts by weight: 8-10 portions of abalone active peptides; 15-35 parts of plant extract.

[0023] According to the present invention, as a preferred embodiment, the plant extract comprises the following components by weight: 3-5 portions of dandelion; 6-8 portions of jujubes; 5-7 roses.

[0024] Thirdly, the present invention provides a method for preparing a compound preparation for relieving dysmenorrhea from the said composition.

[0025] The present invention provides a method for preparing a compound preparation for relieving dysmenorrhea from the aforementioned composition, which specifically includes the following steps: Step 1: Preparation of abalone bioactive peptides; (1) Take fresh abalone muscle tissue, remove the viscera and impurities, wash with deionized water at least 3 times, freeze dry in a freeze dryer at -80 to -85℃, pulverize and pass through a 70-90 mesh sieve, add 60% ethanol solution according to the ratio of abalone muscle tissue to ethanol solution (mass:volume) 1: (7-10), stir and extract at 40-50℃ for 0.5-1.5h, centrifuge at 4000-6000rpm for 8-15min, discard the supernatant (except for some fat, pigment and fishy substances), collect the precipitate, add deionized water according to the ratio of precipitate to deionized water (mass:volume) (1:10)-(1:15), stir to make a homogenate, and transfer to an enzymatic hydrolysis tank.

[0026] (2) Add 1-5% of the abalone muscle tissue mass of flavor protease to the homogenate and hydrolyze it at 50-60℃ and pH 5.5-6.5 for 2-4 hours. This step can hydrolyze long-chain proteins, produce more peptides of suitable length, and reduce bitterness.

[0027] (3) Adjust the system temperature to 40-45℃ and pH to 7.0-7.5, add 3-5% of the abalone muscle tissue mass of complex protease (prepared by neutral protease and papain in a mass ratio of (1-4): (1-4)) and continue constant temperature enzymatic hydrolysis for 4-6 hours; stir once every 1 hour during enzymatic hydrolysis, at a speed of 80-120 rpm, for 4-6 minutes each time.

[0028] (4) After the enzymatic hydrolysis is completed, heat the system to 90-95℃ and keep it at 8-15 min to inactivate the enzyme. After cooling to room temperature, centrifuge at 6000-10000 rpm for 12-20 min and collect the supernatant.

[0029] (5) Filter the supernatant with a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 3000-5000 Da at an operating pressure of 0.10-0.14 MPa and a temperature of 25-35℃, and collect the permeate; then concentrate and desalinate the permeate with a nanofiltration membrane with a molecular weight cutoff of 300-500 Da at an operating pressure of 0.5-1.5 MPa and a temperature of 25-35℃, and collect the retentate.

[0030] (6) Dilute the retentate with 5-10 times deionized water, load it onto a DEAE-Sepharose FF weak anion exchange column, and perform gradient elution with Tris-HCl buffer (pH 8.0) containing 0-0.5M NaCl at a flow rate of 1.5-2.5 mL / min and a detection wavelength of 280 nm. Collect the fraction eluted under the 0.1-0.2M NaCl gradient, freeze-dry and reconstitute, and separate and purify it using a Sephadex G-15 gel chromatography column with deionized water as the elution solvent at a flow rate of 1.2-2.0 mL / min. Detect the absorbance at a wavelength of 280 nm and collect the elution peak with a retention time of 11 min.

[0031] (7) The elution peak was further purified using a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm). Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid acetonitrile solution. The gradient elution program was: 0-15 min 5-20% B, 15-35 min 20-40% B, flow rate 0.8-1.2 mL / min, column temperature 28-33℃. The fraction with a retention time of 21.5-22.5 min was collected and freeze-dried to obtain abalone active peptides.

[0032] (8) Structural identification: The abalone bioactive peptide was analyzed by Edman degradation method and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to determine its amino acid sequence as Tyr-Arg-Leu-Gly-Asp, i.e., YRLGD, with a molecular weight of 631.7 Da. A search of the online databases BIOPEP and EROP-Moscow revealed that this amino acid sequence is a novel small molecule bioactive peptide with a purity ≥97.8%.

[0033] Step 2: Prepare plant extracts; Dandelion, jujube, and rose raw materials were washed separately and dried at 55-65℃ for 1.5-2.5 hours, then pulverized through a 90-110 mesh sieve. Rose powder was taken and added to a 50% ethanol solution at a ratio of 1:(4-6) (mass:volume). The mixture was ultrasonically treated at room temperature (250-350W) for 10-20 minutes, filtered, and the resulting rose ethanol extract and residue were obtained. The residue was combined with dandelion and jujube powder to obtain a mixed powder. 10-14 times the volume of deionized water was added to the mixed powder, and the mixture was stirred and extracted in a 55-65℃ water bath for 1.0-1.5 hours. The first low-temperature extract was collected by filtration, and 8-12 times the volume of deionized water was added to the residue, and the mixture was extracted at 80-90℃. Reflux extraction for 1.5-2.5 h, filter and collect the secondary high-temperature extract; combine the rose alcohol extract filtrate, the first low-temperature extract, and the secondary high-temperature extract; the combined extract is first microfiltered through a 0.45 μm ceramic membrane at an operating pressure of 0.08-0.12 MPa and a temperature of 35-45 °C, the permeate is collected, and concentrated under reduced pressure at 55-65 °C and 0.05-0.12 MPa to obtain an extract paste, which is then freeze-dried in a freeze dryer to obtain plant extract powder, which is then sealed for later use.

[0034] Step 3: Prepare a compound preparation for relieving menstrual cramps; Weigh out abalone active peptides and plant extracts according to the formula, place them in a V-type mixer, and mix at 20-30 rpm for 35-45 minutes until they are evenly mixed to obtain a compound preparation for relieving menstrual cramps.

[0035] Step 4: Prepare into tablets or oral liquid; The method for preparing a compound preparation for relieving dysmenorrhea into tablets is as follows: add excipients to the preparation and obtain tablets through a tableting process; preferably, the excipients include the following components by weight: 30-35 parts of maltodextrin, 12-15 parts of sodium carboxymethyl cellulose, and 6-8 parts of magnesium stearate.

[0036] The method for preparing a compound preparation for relieving menstrual cramps into an oral liquid is as follows: dissolve the preparation in purified water, control the solid content at 15-20%, add 0.1-0.2% steviol glycosides to adjust the taste, and then fill the container to obtain the oral liquid.

[0037] This invention provides a compound preparation for relieving dysmenorrhea, which mainly has anti-inflammatory and antispasmodic effects, improves microcirculation, and relieves dysmenorrhea. Specifically, the compound preparation for relieving dysmenorrhea provided by this invention achieves the above-mentioned effects mainly through two synergistic mechanisms, as follows: 1. Synergistic anti-inflammatory and antispasmodic mechanism; The abalone active peptide (Tyr-Arg-Leu-Gly-Asp) prepared in this invention can directly inhibit COX-2 activity, reduce PGE2 synthesis, and block prostaglandin-mediated uterine smooth muscle contraction. At the same time, dandelion extract can directly inhibit the release of TNF-α and IL-6 inflammatory factors, reducing uterine inflammation from the source. The abalone active peptide and dandelion extract work together to form a dual anti-inflammatory advantage.

[0038] 2. Microcirculation improvement synergistic mechanism; Rose, as a traditional Chinese medicine, has the effects of promoting blood circulation, regulating qi, soothing the liver, and relieving depression. Rose extract contains active ingredients such as flavonoids and volatile oils, which can dilate blood vessels and improve blood circulation. At the same time, jujube extract contains cyclic adenosine monophosphate (cAMP), polysaccharides, and other components that can improve vascular permeability and promote blood circulation. Jujube is also rich in iron and vitamin C, which can promote hematopoietic function and improve anemia. The synergistic effect of rose extract and jujube extract can improve uterine microcirculation disorders and provide environmental support for inflammation repair and smooth muscle function stability.

[0039] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1: Preparation of a compound preparation for relieving dysmenorrhea Step 1: Preparation of abalone bioactive peptides; (1) Take 500g of fresh abalone muscle tissue, remove the viscera and impurities, wash it three times with deionized water, freeze dry it in a freeze dryer at -80℃, pulverize it through an 80-mesh sieve, add 60% ethanol solution according to the ratio of abalone muscle tissue to ethanol solution of 1:8 (mass:volume), stir and extract at 45℃ for 1h, centrifuge at 5000rpm for 10min, discard the supernatant (except for some fat, pigment and fishy substances), collect the precipitate, add deionized water according to the ratio of precipitate to deionized water of 1:12 (mass:volume), stir to make a homogenate, and transfer it to a 50L enzymatic hydrolysis tank.

[0041] (2) Add 2% of the flavor protease by weight of abalone muscle tissue to the homogenate and hydrolyze it at 50°C and pH 6.5 for 3 hours. This step can hydrolyze long-chain proteins, produce more peptides of suitable length, and reduce bitterness.

[0042] (3) Adjust the system temperature to 42℃ and pH to 7.0, add 3% of the abalone muscle tissue mass of complex protease (prepared by neutral protease and papain in a mass ratio of 1:1) and continue constant temperature enzymatic hydrolysis for 4 hours; stir once every 1 hour during enzymatic hydrolysis, at a speed of 100 rpm for 5 minutes each time.

[0043] (4) After the enzymatic hydrolysis is completed, the system is heated to 95°C and kept at 95°C for 10 min to inactivate the enzyme. After cooling to room temperature, it is centrifuged at 8000 rpm for 15 min and the supernatant is collected.

[0044] (5) Filter the supernatant with a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 5000 Da, operating at a pressure of 0.12 MPa and a temperature of 30°C, and collect the permeate; then concentrate and desalinate the permeate with a nanofiltration membrane with a molecular weight cutoff of 300 Da, operating at a pressure of 1.0 MPa and a temperature of 30°C, and collect the retentate.

[0045] (6) Dilute the retentate with 5 times deionized water, load it onto a DEAE-Sepharose FF weak anion exchange column, and perform gradient elution with Tris-HCl buffer (pH 8.0) containing 0-0.5M NaCl at a flow rate of 2.0 mL / min and a detection wavelength of 280 nm. Collect the fraction eluted under the 0.1-0.2M NaCl gradient, freeze-dry and reconstitute, and separate and purify it using a Sephadex G-15 gel chromatography column with deionized water as the elution solvent at a flow rate of 1.5 mL / min. Detect the absorbance at a wavelength of 280 nm and collect the elution peak with a retention time of 11 min.

[0046] (7) The elution peak was further purified using a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm). Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid acetonitrile solution. The gradient elution program was: 0-15 min 5-20% B, 15-35 min 20-40% B, flow rate 1.0 mL / min, column temperature 30℃. The fraction with a retention time of 21.5-22.5 min was collected and freeze-dried to obtain abalone active peptides.

[0047] (8) Structural identification: The abalone bioactive peptide was analyzed by Edman degradation method and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to determine its amino acid sequence as Tyr-Arg-Leu-Gly-Asp, i.e., YRLGD, with a molecular weight of 631.7 Da. A search of the online databases BIOPEP and EROP-Moscow revealed that this amino acid sequence is a novel small molecule bioactive peptide with a purity ≥97.8%.

[0048] Step 2: Prepare plant extracts; Four parts of dandelion, seven parts of jujube, and six parts of rose were washed and dried at 60℃ for 2 hours, then pulverized and passed through a 100-mesh sieve. Rose powder was added to a 50% ethanol solution at a ratio of 1:5 (mass:volume). The mixture was ultrasonically treated at room temperature (300W) for 15 minutes, filtered, and the resulting rose ethanol extract and residue were obtained. The residue was combined with dandelion and jujube powders to obtain a mixed powder. Twelve times the volume of deionized water was added to the mixed powder, and the mixture was stirred and extracted in a 60℃ water bath for 1 hour. The first low-temperature extract was collected by filtration. Ten times the volume of deionized water was added to the residue, and the mixture was refluxed at 85℃ for 2 hours. The second high-temperature extract was collected by filtration. The rose ethanol extract, the first low-temperature extract, and the second high-temperature extract were combined. The combined extract was first microfiltered through a 0.45μm ceramic membrane at an operating pressure of 0.1MPa and a temperature of 40℃. The permeate was collected and then filtered at 60℃ and 0.08MPa. The extract was concentrated under reduced pressure under controlled conditions, then freeze-dried in a freeze dryer to obtain plant extract powder, which was then sealed for later use.

[0049] Step 3: Prepare a compound preparation for relieving menstrual cramps; Weigh out 9 parts of abalone active peptide and 29 parts of plant extract according to the formula, place them in a V-type mixer, and mix at 25 rpm for 40 minutes until they are evenly mixed to obtain a compound preparation for relieving menstrual cramps.

[0050] Step 4: Prepare into tablets or oral liquid; The method for preparing a compound preparation for relieving dysmenorrhea into tablets is as follows: add excipients to the preparation (the mass ratio of excipients to preparation should be in the range of 1.07:1 ~ 2.52:1) and obtain tablets through a tableting process; preferably, the excipients include the following components by weight: 30 parts of maltodextrin, 15 parts of sodium carboxymethyl cellulose, and 8 parts of magnesium stearate.

[0051] The method for preparing a compound preparation for relieving menstrual cramps into an oral liquid is as follows: dissolve the preparation in purified water, control the solid content at 15-20%, add 0.2% steviol glycosides to adjust the taste, sterilize by high pressure steam at 121℃ for 15 minutes, and then fill into 10mL vials under aseptic conditions to obtain the oral liquid.

[0052] Experimental Example 1: In vitro recombinant human COX-2 enzyme activity inhibition experiment (1) Reagent and sample preparation; The abalone active peptide prepared in step one of Example 1 was dissolved in a reaction buffer (containing 50 mM Tris-HCl (pH 8.0) and 1 mM EDTA) to prepare a 10 mM stock solution; the compound preparation for relieving dysmenorrhea prepared in step three of Example 1 was dissolved in the same reaction buffer (containing 50 mM Tris-HCl (pH 8.0) and 1 mM EDTA) to prepare a 10 mM stock solution; celecoxib was dissolved in DMSO (final concentration ≤ 0.1%) to prepare a 10 mM stock solution.

[0053] Key reagents include: recombinant human COX-2 enzyme (Sigma, catalog number C2248), 1 mM heme stock solution (final concentration 5 μM), 10 mM arachidonic acid stock solution (final concentration 20 μM), PGE2 competitive ELISA kit (Wuhan E-EL-H0159), 100 mM SnCl2 stop solution, and Ellman's reagent containing 0.5 mM DTNB.

[0054] (2) Experimental grouping; Each group had 3 replicates. Before adding the arachidonic acid stock solution, the total volume of the system in each group was 200 μL. The specific groupings are as follows (heme iron was added to each group simultaneously at a final concentration of 5 μM, which together with other components constituted a 200 μL pre-incubation system): Background tube, add inactivated recombinant human COX-2 enzyme; Initial COX-2 activity tubes were then infused with recombinant human COX-2 enzyme. Celecoxib positive control tubes were prepared by adding celecoxib stock solution and recombinant human COX-2 enzyme, with a final celecoxib concentration of 0.1-50 μM. Pure peptide tubes of abalone active peptides were prepared by adding abalone active peptide stock solution and recombinant human COX-2 enzyme, resulting in a final concentration of abalone active peptides of 2-80 μM. Abalone active peptide equivalent concentration tubes were prepared by adding a compound preparation stock solution for relieving dysmenorrhea and recombinant human COX-2 enzyme. The final concentration of the compound preparation stock solution for relieving dysmenorrhea was 2-80 μM.

[0055] (3) Experimental treatment; Each group was pre-incubated at 37℃ for 10 min (to allow heme to fully bind with COX-2 enzyme to form a catalytically active holoenzyme), then arachidonic acid stock solution (final volume 210 μL) was added and incubated for 2 min. SnCl2 stop solution was then added and incubated at room temperature for 5 min to terminate the reaction. When using the PGE2 competitive ELISA kit for ELISA detection, the background tube solution was diluted 100-fold, and the solutions in the remaining sample tubes were diluted 2000-fold. After antigen-antibody binding (incubation at room temperature for 18 h), washing, polyglutamic acid-acetylcholinesterase (PG-AChE) was added and incubated for 1 h. Then, Ellman's reagent containing 0.5 mM DTNB was added and incubated in a dark room for 45 min. The OD value was measured at 405 nm.

[0056] (4) Data processing; The OD value (B0) of the initial active COX-2 tube (CIA) after subtracting the blank was controlled between 0.3 and 0.8 AU. The inhibition rate was calculated using the formula I% = [(CIA - Csample) / CIA] × 100%, where CIA is the measured value of the initial active COX-2 tube and Csample is the measured value of other sample tubes. A dose-response curve was plotted against the logarithm of the inhibition rate and concentration. The IC50 was calculated using the probability unit regression method in SPSS 26.0. 50 The data are expressed as “mean±SD” and an independent samples t-test was performed.

[0057] (5) Experimental results; All groups showed concentration-dependent inhibition of recombinant human COX-2 enzyme activity: the IC50 of the celecoxib positive control tube... 50 The concentration was 5.2 ± 0.3 μM; the IC50 of the abalone active peptide pure peptide tube was 45 μM. 50 The effective concentration (IC50) of abalone active peptide was 12.8 ± 0.5 μM, with an inhibition rate of 82.3%. The IC50 of the compound preparation used to relieve dysmenorrhea was 45 μM. 50 The concentration was 13.1 ± 0.4 μM, with an inhibition rate of 84.7%; the IC50 values ​​of the two test samples were [missing information]. 50There was no significant difference (P>0.05), indicating that abalone active peptides are the core active ingredients that inhibit the activity of recombinant human COX-2 enzyme, and the preparation process can effectively preserve its biological activity.

[0058] Experimental Example 2: LPS-stimulated endometrial epithelial cell inflammatory factor inhibition experiment Rat endometrial epithelial cells were used, and cells in the logarithmic growth phase were seeded into 6-well cell culture plates. (cells / well), after the cells fused, the medium was replaced with FBS-free DMEM / F12 medium for starvation treatment for 12 hours, and then replaced with DMEM / F12 medium containing 15% FBS.

[0059] Five groups were then set up (three replicates per group): blank control group, model control group (LPS 100 ng / mL), dandelion group (dandelion 200 μg / mL), abalone active peptide group (abalone active peptide 200 μg / mL), and abalone active peptide + dandelion group (abalone active peptide and dandelion each 200 μg / mL). The dandelion group, abalone active peptide group, and abalone active peptide + dandelion group were pretreated with the corresponding drugs for 2 h. The blank control group was then given an equal volume of culture medium (15% FBS in DMEM / F12 medium), while the model control group and each drug group were given 100 ng / mL LPS. All groups were incubated at 37℃ and 5%... After 12 hours of stimulation in the incubator, the cell culture supernatant and cells were collected. For testing, the concentrations of TNF-α, IL-6, CXCL8, and PGE2 in the supernatant were measured according to the ELISA kit instructions, and the expression of COX-2 protein in the cells was detected using Western blot.

[0060] The experimental results showed that, compared with the blank control group, the concentrations of all inflammatory factors and the expression of COX-2 protein in the model control group were significantly increased (P < 0.01); compared with the model control group, the concentrations of all drug groups were significantly reduced (P < 0.05 or P < 0.01): the dandelion group showed an inhibition rate of 42.5% and 45.8% against TNF-α and IL-6, respectively; the abalone active peptide group showed an inhibition rate of 70.2%, 76.7%, and 62.5% against TNF-α, IL-6, and PGE2, respectively, and the expression of COX-2 protein was reduced by 58.3% compared with the model control group (P < 0.01). This confirms that abalone active peptides can inhibit COX-2... The combination of abalone active peptides and dandelion reduced PGE2 synthesis. The inhibition rates of TNF-α, IL-6, CXCL8, and PGE2 in the abalone active peptide + dandelion group increased to 85.6%, 84.3%, 78.6%, and 68.5%, respectively, and were all significantly higher than those in the single-drug groups (P < 0.05). The TNF-α concentration in the abalone active peptide + dandelion group (24.3 ± 3.5 pg / mL) was not significantly different from that in the blank control group (23.5 ± 3.1 pg / mL), thus confirming that the combination of abalone active peptides and dandelion can significantly enhance anti-inflammatory activity and synergistically inhibit the release of inflammatory factors.

[0061] Table 1 Indicators of Cellular Inflammatory Factor Inhibition

[0062] Note: Compared with the model control group, .

[0063] Experimental Example 3: Study on the effect of improving microcirculation disorders (zebrafish experiment) 210 wild-type AB strain zebrafish (4 dpf) were randomly selected and reared in a six-well plate at 28℃ in standard fish culture water with a light-dark cycle of 14h:10h. Six groups were set up (30 zebrafish per well, 3 replicates per group): blank control group, model control group (ponatinib 2μM), positive control group (ponatinib 2μM + acetylsalicylic acid 0.03mg / mL), abalone active peptide group (abalone active peptide 0.1mg / mL + ponatinib 2μM), abalone active peptide + rose group (abalone active peptide 0.1mg / mL + rose extract 0.1mg / mL + ponatinib 2μM), and abalone active peptide + rose + jujube group (abalone active peptide 0.1mg / mL + rose extract 0.1mg / mL + jujube extract 0.1mg / mL + ponatinib 2μM). Each well had a volume of 3mL. The positive control group and each drug group were pretreated with the corresponding drug for 2 hours. Then, except for the blank control group, all other groups were treated with ponatinib (final concentration 2 μM) to induce microcirculatory disturbance. All groups were treated together in a constant temperature incubator at 28℃ for 18 hours.

[0064] Ten zebrafish were randomly selected from each group during the test. The blood flow velocity in their tail veins was measured using a cardiac blood flow analysis system. The results showed that the average blood flow velocity in the ponatinib-induced model control group (409 μm / s) was significantly lower than that in the blank control group (1380 μm / s) (P < 0.001), while the average blood flow velocity in the positive control group was 1143 μm / s, significantly higher than that in the model control group (P < 0.001), indicating successful model establishment. The average blood flow velocities in the zebrafish of each drug group were 1123 μm / s, 1165 μm / s, and 1206 μm / s, respectively, significantly higher than those in the model control group (P < 0.001), showing a dose-dependent trend. This confirms that the abalone active peptide + rose + jujube group has a significant effect on improving microcirculation disorders and is significantly better than the positive control drug acetylsalicylic acid (P < 0.05), almost restoring to normal levels.

[0065] Table 2. Effects of each component on improving blood flow velocity in zebrafish with microcirculatory disorders (n=10)

[0066] Note: Compared with the model control group, .

[0067] Experiment Example 4: Validation Experiment of Synergistic Efficacy of Compound Preparations for Relieving Dysmenorrhea (Animal Model) 1. Grouping and processing methods; Sixty SPF-grade female SD rats, weighing 200-220g and aged 6-8 weeks, were acclimatized for one week (temperature 22±2℃, humidity 50±5%, 12h light-dark cycle). Except for the blank control group, all other groups received subcutaneous injections of diethylstilbestrol (0.2mg / kg) on ​​days 1-3 of the experiment to enhance uterine sensitivity to oxytocin; on day 14 (after the intervention), oxytocin (2U / kg) was injected intraperitoneally to induce uterine smooth muscle spasm, establishing a primary dysmenorrhea model. Relevant indicators were observed and recorded after oxytocin injection. The 60 SD rats were randomly divided into 6 groups of 10 each. The grouping and intervention methods for each group are shown in Table 3.

[0068] Table 3 Grouping and Intervention Methods for Each Group

[0069] 2. Detection indicators and methods; Uterine smooth muscle spasm index: After injection of oxytocin, rats were immediately anesthetized, uterine tissue was separated and placed in Krebs solution at 37℃. The frequency (times / 10min) and average amplitude (mV) of uterine contractions within 30min were recorded using a biosignal acquisition system, and contractile vitality (contraction amplitude × frequency) was calculated.

[0070] Inflammation and prostaglandin-related indicators: Uterine tissue homogenate was collected, and the contents of TNF-α and IL-6 in the homogenate were detected by ELISA kit. COX-2 activity was detected by COX-2 activity assay kit, and PGE2 content was detected by PGE2 assay kit.

[0071] Uterine microcirculation indicators: 30 minutes before oxytocin injection, the blood perfusion volume (PU value) of rat uterine tissue was detected by laser Doppler flowmeter; venous blood was collected and the erythrocyte deformability index was detected by erythrocyte deformability meter.

[0072] Pain behavioral indicators: The number of writhing movements of rats within 30 minutes after injection of oxytocin was recorded (coordinated movements of abdominal concavity, hind limb extension, and hip elevation were counted as 1 writhing movement); the pain threshold of rats before and after intervention was detected by the hot plate test (55±0.5℃) (the time from the rat's foot contacting the hot plate to the appearance of licking the foot or raising the leg, unit: s, if it exceeds 60s, it is counted as 60s).

[0073] 3. Test results; (1) Improvement effect on uterine smooth muscle spasm; As shown in Table 4, the frequency, average amplitude, and contractile activity of uterine contractions in the model control group were significantly higher than those in the blank control group (P<0.01), indicating that the dysmenorrhea model was successfully established. All intervention groups significantly improved uterine spasm (P<0.05 or P<0.01). The uterine contractile activity of the abalone active peptide group, the abalone active peptide + dandelion group, and the complete group significantly decreased. Among them, the complete group showed the best improvement, with a uterine contraction frequency of only 4.5±0.6 times / 10min, an average contraction amplitude of 2.5±0.4mV, and a contractile activity reduced to 11.3±2.1, significantly better than the ibuprofen group and the abalone active peptide group. P <0.01).

[0074] Table 4. Indicators of uterine smooth muscle spasm (n=10)

[0075] Note: Compared with the model control group, .

[0076] (2) Improvement effect of inflammation and prostaglandin-related indicators; As shown in Table 5, the activities of TNF-α, IL-6, COX-2, and PGE2 in the uterine tissue of the model control group were significantly higher than those in the blank control group (P<0.01). The ibuprofen group significantly reduced the above indicators (P<0.01). Among the intervention groups, the abalone active peptide + dandelion group significantly inhibited the synthesis of inflammatory factors and PGE2 (P<0.01). The whole group showed the most significant improvement, with TNF-α and IL-6 decreasing to 10.8±1.1 pg / mg protein and 7.3±1.0 pg / mg protein, respectively. The COX-2 activity (0.37±0.05 U / mg protein) and PGE2 (14.2±2.1 pg / mg protein) were close to the levels of the blank control group and significantly better than those of the ibuprofen group (P<0.01).

[0077] Table 5 Inflammation and Prostaglandin Indicators (n=10)

[0078] Note: Compared with the model control group, .

[0079] (3) Improvement of uterine microcirculation and pain behavior; As shown in Table 5, the uterine blood perfusion and erythrocyte deformability index of the model control group were significantly lower than those of the blank control group, while the number of writhing movements was significantly increased and the pain threshold was significantly decreased (P<0.01). The abalone active peptide group had a weaker effect on improving microcirculation and pain than the ibuprofen group; the number of writhing movements in the abalone active peptide + dandelion group decreased to 10.5±1.8 times / 30min, and the pain threshold increased to 42.3±4.1s; the uterine blood perfusion of the whole group reached 172.5±15.8PU, the erythrocyte deformability index was 0.81±0.06, the number of writhing movements was only 8.4±1.5 times / 30min, the pain threshold was 52.3±4.5s, and the microcirculation indicators were close to those of the blank control group. The improvement in pain behavior was significantly better than that of the model control group (P<0.01).

[0080] Table 6. Uterine microcirculation and pain behavior indicators (n=10)

[0081] Note: Compared with the model control group, .

[0082] The above experimental results show that the whole combination group (abalone active peptide + plant extract), which is a compound preparation for relieving dysmenorrhea, showed significant improvement in uterine smooth muscle spasm, inflammatory factor expression, prostaglandin level, uterine microcirculation and pain behavior indicators. In terms of comprehensively improving uterine spasm, inflammation, microcirculation and pain behavior, it is more effective than ibuprofen group and abalone active peptide group, demonstrating the advantages of multi-target synergistic treatment.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. However, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An abalone bioactive peptide, characterized in that, The amino acid sequence of the abalone active peptide is Tyr-Arg-Leu-Gly-Asp.

2. A composition containing the abalone active peptide according to claim 1, characterized in that, It includes the following components by weight: 8-10 portions of abalone active peptides; 15-35 parts of plant extract.

3. The composition according to claim 2, characterized in that, The plant extract comprises the following components by weight: 3-5 portions of dandelion; 6-8 portions of jujubes; 5-7 roses.

4. A method for preparing a compound preparation for relieving dysmenorrhea from the composition of claim 2, characterized in that, Includes the following steps: Step 1: Take fresh abalone muscle tissue, wash, freeze-dry, and pulverize it. Add ethanol solution and stir to extract. Centrifuge to collect the precipitate and add deionized water to make a homogenate. Add 1-5% of the abalone muscle tissue mass of flavor protease to the homogenate and enzymatically hydrolyze at 50-60℃ and pH 5.5-6.5 for 2-4 hours. Adjust the temperature to 40-45℃ and pH to 7.0-7.5, add 3-5% of the abalone muscle tissue mass of complex protease and continue enzymatic hydrolysis at a constant temperature for 4-6 hours. After enzyme inactivation, cool to room temperature and centrifuge to collect the supernatant. Filter the supernatant and collect the permeate. Concentrate and desalt the permeate and collect the retentate. Separate and purify the retentate sequentially using a DEAE-Sepharose FF weak anion exchange column and a Sephadex G-15 gel chromatography column, and collect the elution peaks. The elution peak was further purified using a C18 reversed-phase column, and the purified component was freeze-dried to obtain abalone active peptides. Step 2: After washing, drying, and pulverizing the plant extract raw materials, rose powder is added to an ethanol solution, ultrasonically treated at room temperature, and filtered to obtain rose ethanol extract filtrate and residue. The residue is combined with dandelion powder and jujube powder to obtain a mixed powder. Deionized water is added to the mixed powder and stirred for extraction. The first low-temperature extract is collected by filtration, and deionized water is added to the residue for reflux extraction. The second high-temperature extract is collected by filtration. The rose ethanol extract filtrate, the first low-temperature extract, and the second high-temperature extract are combined. The combined extract is microfiltered, and the permeate is collected. After vacuum concentration and freeze-drying, the plant extract powder is obtained. Step 3: Mix abalone active peptides and plant extracts evenly according to the specified ratio to obtain a compound preparation for relieving menstrual cramps.

5. The preparation method according to claim 4, characterized in that, In step one, the complex protease is prepared by neutral protease and papain in a mass ratio of (1-4):(1-4).

6. The preparation method according to claim 4, characterized in that, In step one, the specific procedure for separating and purifying the retentate using a DEAE-Sepharose FF weak anion exchange column and a Sephadex G-15 gel chromatography column is as follows: The retentate was diluted with 5-10 times deionized water and loaded onto a DEAE-Sepharose FF weak anion exchange column. Gradient elution was performed with Tris-HCl buffer containing 0-0.5M NaCl at a flow rate of 1.5-2.5 mL / min and a detection wavelength of 280 nm. The fraction eluted under the 0.1-0.2M NaCl gradient was collected, lyophilized, and reconstituted. The fraction was then separated and purified using a Sephadex G-15 gel chromatography column with deionized water as the elution solvent at a flow rate of 1.2-2.0 mL / min. The absorbance was detected at a wavelength of 280 nm, and the elution peak with a retention time of 11 min was collected.

7. The preparation method according to claim 4, characterized in that, In step one, the specific procedure for further purification of the elution peak using a C18 reversed-phase column is as follows: Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid acetonitrile solution. The gradient elution program was: 0-15 min 5-20% B, 15-35 min 20-40% B, with a flow rate of 0.8-1.2 mL / min and a column temperature of 28-33℃. The fraction with a retention time of 21.5-22.5 min was collected.

8. The preparation method according to claim 4, characterized in that, In step two, add 10-14 times the volume of deionized water to the mixed powder, stir and extract in a water bath at 55-65℃ for 1.0-1.5h, filter and collect the first low-temperature extract, add 8-12 times the volume of deionized water to the filter residue, reflux and extract at 80-90℃ for 1.5-2.5h, filter and collect the second high-temperature extract.

9. The preparation method according to claim 4, characterized in that, In step three, the method for preparing the compound preparation for relieving dysmenorrhea into tablets is as follows: add excipients to the preparation and obtain tablets through a tableting process; the excipients include the following components by weight: 30-35 parts of maltodextrin, 12-15 parts of sodium carboxymethyl cellulose, and 6-8 parts of magnesium stearate.

10. The preparation method according to claim 4, characterized in that, In step three, the method for preparing the compound preparation for relieving dysmenorrhea into an oral liquid is as follows: dissolve the preparation in purified water, control the solid content at 15-20%, add 0.1-0.2% steviol glycosides to adjust the taste, and then fill the container to obtain the oral liquid.

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