CN117024512B - Anxiolytic active peptide, and preparation method and application thereof - Google Patents
Anxiolytic active peptide, and preparation method and application thereof Download PDFInfo
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- CN117024512B CN117024512B CN202310831148.9A CN202310831148A CN117024512B CN 117024512 B CN117024512 B CN 117024512B CN 202310831148 A CN202310831148 A CN 202310831148A CN 117024512 B CN117024512 B CN 117024512B
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- C—CHEMISTRY; METALLURGY
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- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A61P25/22—Anxiolytics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention belongs to the technical field of proteins, and relates to an anxiolytic active peptide, a preparation method and application thereof, wherein the sequence of the anxiolytic active peptide is LHPEWQQLVR. The anxiolytic peptide promotes the normalization of an HPA axis by enhancing the neurotrophic function, so that the time and the entering times of a mouse in an open arm of an elevated plus maze are obviously increased, and the anxiety behavior of the mouse is relieved. The invention also provides a preparation method and application of the anxiolytic active peptide.
Description
Technical Field
The invention belongs to the technical field of proteins, and particularly relates to an anxiolytic active peptide, a preparation method and application thereof.
Background
Anxiety refers to an abnormal response to a dangerous or stressful condition that accompanies our lives. However, as some stress increases, anxiety becomes anxiety, resulting in psychological and physical symptoms such as fear, fatigue, anxiety or agitation, palpitations, susceptibility to frightening and irritability, etc. About 1/4 of the people may or have had anxiety disorder based on the world mental health survey. The lifetime prevalence of anxiety disorder is about 14%. High prevalence, chronic and co-morbid characteristics lead to the world-guard organization to rank anxiety as the 9 th major health related disability cause.
Currently, psychological therapy and drug therapy are combined in the treatment of anxiety, and most of the drug therapy adopts drugs such as selective 5-hydroxytryptamine reuptake inhibitor (SSRIs), 5-hydroxytryptamine-norepinephrine reuptake inhibitor (SNRI) and Benzodiazepines (BDZ), but there are a plurality of side effects such as dry mouth, nausea, headache, somnolence, weight gain and the like, so that finding new safe alternatives is an effective means for treating anxiety.
The soybean peptide is an enzymolysis product of soybean protein, has low molecular weight and mostly consists of 3-20 amino acids, not only maintains the advantages of rich protein nutrition, balanced amino acids and the like, but also is easy to dissolve, absorb and utilize, and has biological activities of resisting oxidation, resisting inflammation, reducing blood pressure, regulating immunity and the like. In the aspect of nerve regulation, researchers at home and abroad find that the low molecular weight peptide can relieve and even treat mental diseases (depression, anxiety and bipolar disorder) and neurodegenerative diseases (cerebral ischemia, alzheimer's disease, parkinson's disease and the like). Therefore, the soybean peptide raw materials are utilized for separation, and key effective components are explored for nutrition supplementation so as to relieve anxiety, so that the soybean peptide has a great practical application value.
Disclosure of Invention
Accordingly, the present invention aims to provide an anxiolytic active peptide, a preparation method and application thereof, which aim to relieve anxiety through nutritional supplementation, avoid side effects caused by traditional medicines and actually help patients suffering from anxiety.
The object of the present invention and the solution of the technical problems thereof can be achieved by the following technical solutions.
In one aspect, the invention provides an anxiolytic active peptide having the amino acid sequence Leu-His-Pro-Glu-Trp-Gln-Gln-Leu-Val-Arg, or as shown in LHPEWQQLVR (SEQ ID NO: 1), and having a molecular weight of 1305.70Da.
In another aspect, the present invention provides a method for preparing the above anxiolytic active peptide, comprising the steps of:
1) carrying out in-vitro simulated digestion on soybean peptide, carrying out enzymolysis by using pepsin and pancreatin to obtain a soybean peptide digestion product, 2) carrying out in-vitro simulated absorption on the soybean peptide digestion product, separating by using a Caco-2 monolayer cell model to obtain an absorbed soybean peptide, and 3) carrying out peptide fragment identification on the absorbed soybean peptide to obtain the anxiolytic active peptide LHPEWQQLVR.
In an embodiment of the method of the invention, step 1) comprises in particular the steps of:
1.1 Dissolving soybean peptide in water, adding simulated oral cavity liquid, oscillating for reaction to obtain soybean peptide oral cavity digestive liquid,
1.2 Adding simulated gastric juice into soybean peptide oral cavity digestive juice, regulating pH to 3.0, then adding pepsin, and oscillating for reaction to obtain soybean peptide gastric cavity digestive juice,
1.3 Adding simulated intestinal juice into soybean peptide gastric digestion liquid, adjusting pH to 7.0, adding pancreatin, oscillating for reaction to obtain soybean peptide intestinal digestion liquid, and
1.4 Freeze-drying the soybean peptide digestive juice to obtain digested soybean peptide powder, namely soybean peptide digestive products.
In an embodiment of the method of the invention, the simulated oral fluid comprises 15.1mMKCl、3.7mM KH2PO4、13.6mM NaHCO3、0.15mM MgCl2(H2O)6、0.06mM(NH4)2CO3、1.1mM HCl、1.5mM CaCl2(H2O)2.
In an embodiment of the method of the invention, the simulated gastric fluid comprises 6.9mM KCl、0.9mM KH2PO4、25mM NaHCO3、47.2mM NaCl、0.12mM MgCl2(H2O)6、0.5mM (NH4)2CO3、15.6mM HCl、0.15mM CaCl2(H2O)2.
In an embodiment of the method of the invention, the simulated intestinal fluid comprises 6.8mM KCl、0.8mM KH2PO4、85mM NaHCO3、38.4mM NaCl、0.33mM MgCl2(H2O)6、8.4mM HCl、0.6mM CaCl2(H2O)2.
In the embodiment of the method, in the step 1), pepsin and pancreatin are utilized for enzymolysis to carry out in-vitro simulated digestion on the soybean peptide, the addition amount of the pepsin can be 0.2-0.4% of the soybean peptide by mass fraction, the enzymolysis time can be 1-3h, and the enzymolysis temperature can be 35-40 ℃ and the pH value can be 2.5-3.0. In a specific embodiment of the invention, the pepsin may be added in an amount of 0.2%, 0.25%, 0.3%, 0.35% or 0.4% by mass of the soybean peptide, and the enzymolysis time may be 1h, 1.5h, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h, the enzymolysis temperature may be 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, and the pH may be 2.5, 2.6, 2.7, 2.8, 2.9, 3.0.
In an embodiment of the method of the present invention, pancreatin may be added in an amount of 0.02 to 0.04% by mass of the soybean peptide. The enzymolysis time can be 1-3h, the enzymolysis temperature can be 35-40 ℃, and the pH value is 7.0-7.5. In the specific proposal of the invention, the addition amount of pancreatin is 0.02 percent, 0.03 percent and 0.04 percent of the soybean peptide by mass percent, the enzymolysis time can be 1h, 1.5h, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h and 3h, the enzymolysis temperature can be 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃ and the pH can be 7.0, 7.1, 7.2, 7.3, 7.4 and 7.5.
In an embodiment of the method of the invention, the enzyme may be inactivated by a water bath at 95℃for 10min after the end of the enzymatic hydrolysis.
In an embodiment of the method of the invention, in step 2), the soybean peptide digest is dissolved in HBSS buffer and a Caco-2 monolayer of cells is inoculated, wherein the concentration of the soybean peptide digest in the HBSS buffer is 4mg/mL.
In an embodiment of the method of the present invention, in step 2), the soybean peptide digestion product is subjected to in vitro simulated absorption using a Caco-2 monolayer cell model, and the soybean peptide digestion product is separated into an absorbed soybean peptide and an unabsorbed soybean peptide. In a specific embodiment, the construction of a Caco-2 monolayer cell model is realized by culturing the Caco-2 cells for 21 days to stabilize the resistance value of the Caco-2 cells to 400-600 omega, and then the digested soybean peptide is separated into an absorbed soybean peptide and an unabsorbed soybean peptide through absorption for 1.5-2.5 hours. In particular embodiments, the Caco-2 monolayer cells can have a resistance value of 410, 420, 430, 440, 450, 480, 490, 500, 550, 560, 570, 580, 590, 600Ω.
In a third aspect, the present invention provides an anxiolytic composition comprising an anxiolytic active peptide as described above. It will be appreciated by those skilled in the art that the composition may further comprise an active agent capable of enhancing anxiolytic activity of the peptide, or an active agent that synergistically increases anxiolytic activity, in combination with a pharmaceutically acceptable carrier or excipient.
In a fourth aspect of the invention, the invention provides the use of an anxiolytic active peptide as described above for the preparation of: 1) An emotion regulating product; and 2) anxiolytic drugs.
Compared with the prior art, the invention has the beneficial effects. The anxiolytic active peptide is obtained by carrying out enzymolysis, separation, identification and other processes on the soybean peptide, and has wide sources and easy operation and realization of the preparation process. Experimental evidence shows that the peptide can improve the behavior of mice in an elevated plus maze experiment, increase the time of the mice moving in the open arms and the times of entering the open arms, thereby relieving anxiety-like behaviors of the mice, and can also improve BDNF level, reduce CRH, ACTH and CORT level, enhance neurotrophic function, promote normalization of HPA axis, and thus relieve anxiety symptoms of the mice.
Drawings
FIG. 1 shows the change in resistance of Caco-2 monolayer cell model cultured for 21 days;
FIG. 2 is a graph showing the effect of various concentrations of digested soy peptide on Caco-2 cell viability;
FIG. 3 is a chromatogram of the digestion of soybean peptide at various times of absorption (A) the absorption of unabsorbed soybean peptide (B) the absorption of soybean peptide;
FIG. 4 is a diagram of an overhead plus maze apparatus and a mouse movement trace showing the effect of LHPEWQQLVR on mice in the overhead plus maze (A) time of mice in the open arm (B) number of times mice enter the open arm (C);
FIG. 5 is a graph showing the effect on BDNF levels in brain tissue of mice;
FIG. 6 is a graph showing the effect on (A) CRH, (B) ACTH and (C) CORT levels in mouse serum.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and detailed description, wherein it is to be understood that, on the premise of no conflict, the following embodiments or technical features may be arbitrarily combined to form new embodiments.
Example 1
This example provides anxiolytic peptides having the amino acid sequence Leu-His-Pro-Glu-Trp-Gln-Gln-Leu-Val-Arg, or LHPEWQQLVR, and a molecular weight of 1305.70Da. The preparation process of the anxiolytic active peptide comprises the following steps:
(1) Simulated oral fluid, simulated gastric fluid and simulated intestinal fluid were formulated according to the formulation provided by Brodkorb et al (Brodkorb,A.,Egger,L.,Alminger,M.,et al.,INFOGEST static in vitro simulation of gastrointestinal food digestion.Nature Protocols,2019,14(4),991-1014), as specifically shown in table 1.
Table 1 preparation of simulated oral fluid, simulated gastric fluid and simulated intestinal fluid
(2) 10G of soybean peptide is dissolved in 10mL of distilled water and uniformly mixed, 8mL of simulated oral liquid and 0.05mL of CaCl 2(H2O)2 solution are added, 1.95mL of distilled water is supplemented to make the system 20mL, and then the system is subjected to oscillation reaction at 37 ℃ for 2min, so as to obtain soybean peptide oral digestive juice. Then 16mL simulated gastric fluid and 0.01mL CaCl 2(H2O)2 solution are added into the soybean peptide oral cavity digestive fluid, the pH is regulated to 3.0 by using HCl, then 1mL pepsin is added to ensure that the final activity in the solution is 2000U/mL, distilled water is supplemented to ensure that the system is 40mL, and then the system is subjected to oscillation reaction for 2 hours at 37 ℃ to obtain the soybean peptide gastric digestion fluid. Finally, 16mL of simulated intestinal juice and 0.08mL of CaCl 2(H2O)2 solution are added into the soybean peptide gastric digestion liquid, the pH value is regulated to 7.0 by using NaOH, then 10mL of pancreatin solution is added to ensure that the final activity in the solution is 100U/mL, distilled water is supplemented to ensure that the system is 80mL, and then the system is subjected to oscillation reaction for 2 hours at 37 ℃ to obtain the soybean peptide gastric digestion liquid, namely the soybean peptide gastric digestion liquid. The digested soy peptide solution was stored at-40 ℃ and then freeze-dried for 48 hours to obtain a digested soy peptide powder.
(3) Caco-2 cells (from basic medical institute of China medical sciences) were cultured in a complete medium of MEM+20% FBS+1% NEAA+1% PS at 37℃with a culture humidity of 5% CO 2/95% air. Every two days, at a cell density of 80%, 0.25% TE was used for passaging at a ratio of 1:3. The effect of different concentrations of digested soybean peptide (0 mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL, 4mg/mL, 8 mg/mL) on Caco-2 cell viability was determined using the CCK-8 method and the results are shown in FIG. 2. In order to exclude the cytotoxic effect, the subsequent experiments were performed with the maximum concentration that did not affect the viability of Caco-2 cells, i.e., 4 mg/mL.
(4) Caco-2 cells were inoculated into a 12-well Transwell chamber, medium was changed every two days and transepithelial resistance was measured using a resistance meter, and the results were as shown in FIG. 1, and the cells were cultured for 21 days with a resistance value of 400 to 600Ω, thereby completing the construction of a Caco-2 monolayer cell model.
(5) Dissolving the digested soybean peptide powder obtained in the step (2) in HBSS buffer solution, wherein the concentration is 4mg/mL obtained in the step (3), taking 0.5mL of the digested soybean peptide powder to be connected into the AP side of the Caco-2 monolayer cell model constructed in the step (4), and connecting 1.5mL of HBSS buffer solution to be connected into the BL side. After absorption for 0.5h, 1h, 2h, 3h, 6h, 12h, 24h, the AP and BL side solutions were taken, respectively.
(6) The peptide content was determined by ultra-high pressure liquid chromatography. The chromatographic column is AdvanceBio Peptide Plus (2.1X105 mm,2.7 μm), the mobile phase A is acetonitrile, the mobile phase B is ultrapure water, and the gradient elution is carried out, wherein the elution program is 0-3min, and the elution program is 5% A;3-5min,5% -70% A;5-13min,70% -95% A;13-15min,95% A. The temperature is 25 ℃, the flow rate is 0.3mL/min, the sample injection amount is 3 mu L, and the detection wavelength is 220nm. The chromatograms of the soybean peptide absorbed at different absorption times are shown in figure 3, and after absorption for 2 hours, the soybean peptide is digested to basically complete most of transportation, and the peptide content is 0.25mg/mL. After 2h absorption, the solution collected on the AP side is unabsorbed soybean peptide, and the solution collected on the BL side is absorbed soybean peptide.
(7) And (3) peptide fragment identification is carried out on the absorbed soybean peptide in the step (5) by using liquid. Firstly, the sample is pretreated, soybean peptide absorbed for 2 hours is dissolved in ultrapure water, dithiothreitol solution is added to make the final concentration of the soybean peptide be 10mmol/L, and the soybean peptide is reduced for 1 hour in a water bath at 56 ℃. The iodoacetamide solution was added to a final concentration of 50mmol/L and reacted in the dark for 40min. Desalting was performed using a desalting column, and the solvent was evaporated in a vacuum centrifugal concentrator at 45 ℃. Then liquid chromatography detection is carried out, the nano-upgrading ultra-high performance liquid chromatograph Easy-nLC 1200 is used for detection, the capillary liquid chromatograph pre-column is ACCLAIM PEPMAP RPLC C (300 mu m i.d. x 5mm,5 mu m,) The analytical column was ACCLAIM PEPMAP RPLC C (50 μm i.d.×150mm,1.9 μm,/>)). Mobile phase a was 0.1% aqueous formic acid and B was 80% acetonitrile/0.1% aqueous formic acid. Gradient elution, wherein the elution program is 0-5min,4% -10% B;5-85min,10% -22% of B;85-110min,22% -40% B;110-115min,40% -95% B;115-120min,95% B. The flow rate is 600nL/min, and the sample injection amount is 5 mu L. Detection was performed using an electrospray-combined ion trap Orbitrap mass spectrometer with a spray voltage of 2.2kV and a capillary temperature of 270 ℃. Primary mass spectrometry parameters: resolution 70000, automatic gain control target 3×10 6, maximumIT ms, scanning range 300-1800m/z. Secondary mass spectrometry parameters: resolution 17500, automatic gain control target 1×10 5, maximum IT 50ms, topn 20, collision energy 28. Finally, database retrieval, using Byonic to retrieve soybean protein database from mass spectrum original file, protein modification to Carbamidomethyl (C) (fixed), oxidation (M) (variable); the enzyme is non-specific; the missing enzyme cutting site is 3; the primary mass spectrum error is 20ppm, and the secondary mass spectrum error is 0.02Da.
The sequence information of the peptide fragment of the soybean peptide after 2h absorption is shown in table 2, and the soybean peptide with potential anxiolytic activity is selected according to the characteristics of low molecular weight, ultraviolet absorption capacity (A 280), hydrophobicity, stability, leu, tyr, pro, trp and other amino acids of the anxiolytic peptide fragment. LHPEWQQLVR has low molecular weight, ultraviolet absorption capability, stability, and contains 4 key amino acids such as leucine, proline, tryptophan, etc., and the score is top. Thus, the present peptide fragment was selected for subsequent experiments.
TABLE 2 peptide fragment analysis of soybean peptide after 2h absorption
Experimental example 1
30 Male C57BL/6J mice of 6-7 weeks of age were randomly grouped and subsequently dosed, in the following manner:
1) Blank group: n=10, physiological saline for intraperitoneal injection
2) Diazepam group: n=10, 1mg/kg diazepam was intraperitoneally injected
3) LHPEWQQLVR groups: n=10, 1mg/KG LHPEWQQLVR of intraperitoneal injection
An overhead plus maze (EPM) experiment was performed 30min after dosing. EPM is one of the most widely used animal models of anxiety, and mainly uses the conflict between mouse exploration and avoidance to evaluate anxiety behavior. The EPM consists of two open arms and two closed arms, and the two open arms and the two closed arms are mutually perpendicular and cross. Each arm is 30cm long and 5cm wide, the closed arm is provided with a fence with the height of 15cm, a camera is arranged above the joint of the open arm and the closed arm, the behavior of the mice in the EPM is recorded, and the whole maze is arranged on a bracket 40cm away from the ground. Before the experiment, the EPM device is confirmed to be clean and tasteless, and the mice are transported to a behavioural laboratory in advance and are adapted to the environment for about 3 hours. The mice were placed in the junction of the open arm and the closed arm facing the open arm, the activity of the mice in the EPM was recorded for a total time of 5min, and the mice were returned to the cage after the experiment was completed. The EPM experimental setup was wiped with 75% ethanol.
Experimental results as shown in fig. 4, the time and number of times in the EPM arm of mice injected intraperitoneally with diazepam and LHPEWQQLVR increased significantly, and the movement trace was also concentrated in the arm.
Experimental example 2
Brain-derived neurotrophic factor (BDNF) is involved in neuronal growth and proliferation, synaptic neurotransmission and neuroplasticity. Anxiety inhibits BDNF expression. The hypothalamic-pituitary-adrenal axis is mainly responsible for maintaining the response to homeostasis and stress, the hypothalamus synthesizes and secretes Corticotropin Releasing Hormone (CRH), which promotes the release of corticotropin (ACTH) by the pituitary, which in turn acts on the adrenal cortex to synthesize Corticosterone (CORT). The pressure will result in the CORT always being at a higher level.
Immediately after the end of the elevated plus maze experiment, the mice were dissected and brain tissue and serum were obtained. The BDNF content in the brain tissue of the mice and the CRH, ACTH, CORT content in the serum are determined according to the instructions of the BDNF kit of the allied organism and the CRH, ACTH, CORT kit of the Wuhan Yillert organism. Results as shown in fig. 5 and 6, mice with lower BDNF content, higher CRH, ACTH and CORT content, decreased neurotrophic function and disorder of HPA axis produced anxiety behavior. LHPEWQQLVR can significantly increase BDNF level, significantly reduce CRH, ACTH and CORT levels, enhance the neurotrophic function of mice, and normalize HPA axis, thereby relieving anxiety-like behavior of mice.
Claims (6)
1. An anxiolytic active peptide, characterized in that the peptide has the amino acid sequence LHPEWQQLVR and the molecular weight 1305.70Da.
2. A process for preparing an anxiolytic active peptide according to claim 1, comprising the steps of:
1) Performing in vitro simulated digestion on soybean peptide, performing enzymolysis by pepsin and pancreatin to obtain soybean peptide digestion product,
2) Performing in vitro simulated absorption on the soybean peptide digestion product, separating by using a Caco-2 monolayer cell model to obtain absorption soybean peptide, and
3) Peptide fragment identification is carried out on the absorption soybean peptide to obtain the anxiolytic active peptide LHPEWQQLVR,
Wherein, the step 1) specifically comprises the following steps:
1.1 Dissolving the soybean peptide in water, adding simulated oral cavity liquid, and carrying out oscillation reaction to obtain soybean peptide oral cavity digestive juice,
1.2 Adding simulated gastric juice into the soybean peptide oral cavity digestive juice, regulating the pH value to 3.0, then adding pepsin, and carrying out oscillation reaction to obtain the soybean peptide gastric cavity digestive juice,
1.3 Adding simulated intestinal juice into the soybean peptide gastric digestion liquid, regulating the pH to 7.0, then adding pancreatin, carrying out oscillation reaction to obtain the soybean peptide intestinal digestion liquid, and
1.4 Freeze-drying the soybean peptide digestive juice to obtain digested soybean peptide powder, namely the soybean peptide digestive product,
Wherein the simulated oral fluid comprises 15.1mM KCl、3.7mM KH2PO4、13.6mM NaHCO3、0.15mM MgCl2(H2O)6、0.06mM(NH4)2CO3、1.1mM HCl、1.5mM CaCl2(H2O)2,
The simulated gastric fluid comprises 6.9mM KCl、0.9mM KH2PO4、25mMNaHCO3、47.2mMNaCl、0.12mM MgCl2(H2O)6、0.5mM(NH4)2CO3、15.6mM HCl、0.15mM CaCl2(H2O)2,
The simulated intestinal fluid comprises 6.8mM KCl、0.8mM KH2PO4、85mMNaHCO3、38.4mMNaCl、0.33mMMgCl2(H2O)6、8.4mM HCl、0.6mM CaCl2(H2O)2, in the step 1), the addition amount of pepsin is 0.2-0.4% of the soybean peptide by mass fraction, the addition amount of pancreatin is 0.02-0.04% of the soybean peptide by mass fraction,
In the step 1), the enzymolysis time of the pepsin is 1-3h, the enzymolysis temperature is 35-40 ℃, and the pH is 2.5-3.0; the pancreatin enzymolysis time is 1-3h, the enzymolysis temperature is 35-40deg.C, pH is 7.0-7.5, the enzyme is inactivated in water bath at 95deg.C for 10min after the enzymolysis is completed, and
In step 2), the soybean peptide digestion product is dissolved in HBSS buffer and Caco-2 monolayer cells are inoculated, wherein the concentration of the soybean peptide digestion product in HBSS buffer is 4mg/mL.
3. The method for producing anxiolytic active peptide according to claim 2, wherein the Caco-2 monolayer cell has a resistance value of 400 to 600 Ω.
4. A process for the preparation of an anxiolytic active peptide according to claim 3, characterized in that in step 2) the absorption lasts for 1.5-2.5h.
5. An anxiolytic composition comprising the anxiolytic peptide of claim 1.
6. Use of an anxiolytic active peptide according to claim 1 for the preparation of an anxiolytic drug.
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| CN107868124A (en) * | 2017-11-13 | 2018-04-03 | 江苏大学 | A kind of zeins anti-inflammatory polypeptide and preparation method thereof |
| CN112056453A (en) * | 2020-08-31 | 2020-12-11 | 华南理工大学 | Aromatic amino acid-rich sleep improvement zymolyte and preparation method thereof |
| CN115925795A (en) * | 2022-11-07 | 2023-04-07 | 北京工商大学 | Selenium-rich peptide with high antioxidant activity and preparation method and application thereof |
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