Method for separating F1-Cu ion neutral active peptide from sheep brain total white protein zymolyte and application of F1-Cu ion neutral active peptide

Abstract

The invention relates to a method for separating F1-Cu ion neutral active peptide from sheep brain total white protein zymolyte and application of the F1-Cu ion neutral active peptide. The method comprises the following steps: removing surface impurities and fresh blood from fresh sheep brain, performing low-temperature vacuum freeze drying, crushing and sieving to obtain sheep brain powder; degreasing with petroleum ether to obtain sheep brain degreased powder; sequentially carrying out distilled water extraction, centrifugation and freeze drying to obtain a sheep brain albumin crude extract, carrying out enzymolysis with trypsin, and carrying out freeze drying to obtain a sheep brain total albumin trypsin hydrolysate; cM-65OM and DEAE-650M cation and anion exchange resins are used for separation to obtain a neutral part, and then an HW-40F gel column is used for separation to prepare a neutral peptide F1 part combined with Cu ions, wherein the molecular weight of the neutral peptide F1 part is 4KDa. The method is simple in production process, environment-friendly, safe, low in cost, wide in raw material source, easy to obtain, low in price and safe. The obtained polypeptide component not only retains the biological activity of original substances in the sheep brain, but also turns waste into wealth and resources, makes full use of Xinjiang animal husbandry characteristic resources, and realizes high value-added utilization of animal husbandry resources.

Description

Technical Field

[0001] This invention relates to a method and its application for separating F1-Cu ion-bound neutral bioactive peptides from total albumin hydrolysate of sheep brain. Background Technology

[0002] Sheep brain is the brain marrow of goats or sheep, belonging to the Bovidae family. According to the Compendium of Materia Medica, sheep brain is sweet in taste, warm in nature, and slightly toxic. It enters the heart, liver, and kidney meridians and has the effects of nourishing the brain, moisturizing the skin, and removing chapped skin. Traditional Chinese medicine uses it to treat symptoms such as dizziness due to physical weakness, wind-cold entering the brain, persistent headaches, chapped skin, and muscle and bone injuries. Sheep brain protein is rich in essential amino acids such as lysine, phenylalanine, methionine, threonine, isoleucine, leucine, and valine. Glutamic acid is the most abundant, followed by aspartic acid. Sheep brain also contains abundant ascorbic acid, niacin, riboflavin, lecithin, thiamine, cerebrosides, protein, fat, calcium, phosphorus, and iron.

[0003] Currently, there is very little literature on sheep brain, and its development lacks a theoretical basis. Sheep brain and other parts are only used for food processing and feed, resulting in resource loss due to a lack of rational development and utilization. Research on sheep brain is beneficial for achieving high-value utilization of this resource. The rich nutritional value of sheep brain makes it a promising area for development. With an aging population and a rising incidence of brain diseases, cerebrovascular diseases, Alzheimer's disease, and other brain disorders have become common and prevalent diseases that seriously threaten human health, attracting widespread attention.

[0004] Alzheimer's disease (AD) is a common, progressive neurodegenerative disease, clinically characterized by progressive decline in memory, cognitive impairment, language and social functioning decline, short-term memory loss, primary progressive aphasia and mental confusion, and even personality changes and loss of daily living abilities. It places a tremendous burden on society and families. Due to limited understanding of its pathobiological molecular basis, there is still no effective treatment, making it a global health challenge.

[0005] China has a large elderly population and a rapid aging process, resulting in a high prevalence of chronic diseases among the elderly, with the prevalence trending towards doubling every five years. This disease has become a serious concern. Therefore, finding effective drugs to treat Alzheimer's disease has become a hot research topic in the medical field.

[0006] Hydrolysates of mammalian brain proteins have been clinically proven to treat some brain functional disorders. Animal organs and their corresponding human internal organs share many similarities in morphology, biochemical characteristics, tissue composition, and physiological function. Therefore, ancient Chinese medicine often treated diseases of one organ with those of the corresponding organ, a principle known as "treating the organ with the organ" and "like with like." Hydrolyzed brain protein drugs are among the most widely used biochemical drugs in recent years, holding an important position in related research fields and markets both domestically and internationally. As an active brain polypeptide extracted from healthy, fresh animal brain tissue through multi-enzyme hydrolysis, hydrolyzed brain protein contains various essential amino acids for the human brain, as well as important elements such as phospholipids, lecithin, and peptide nerve growth factors. It can act on the central nervous system in multiple ways, regulating and improving neuronal metabolism, promoting synapse formation, inducing neuronal differentiation, and further protecting nerve cells from damage caused by various ischemic and neurotoxic factors. It can cross the blood-brain barrier, promote the synthesis of proteins in the brain, affect the respiratory chain, have protective capabilities against hypoxia, improve energy metabolism in the brain, activate adenylate cyclase to catalyze other hormone systems, and provide neurotransmitters, peptide hormones, and coenzyme precursors.

[0007] Domestic and international research on brain protein hydrolysis primarily focuses on pig and bovine brains, while sheep brain represents a promising new research and development area. The purpose of this invention is to further refine experimental conditions and extraction procedures based on previous domestic and international research, and to accelerate the development of sheep brain protease hydrolysate peptides to obtain better, more abundant, and purer brain protease hydrolysate peptides. Therefore, further development and research of brain protein hydrolysate drugs is of great significance for the development of new anti-AD drugs.

[0008] A search revealed domestic reports on neutral bioactive peptides from sheep brain total albumin protein hydrolysate: Domestic and international research on brain protein hydrolysis mainly focuses on pig brain and bovine brain. Apart from the literature on enzymatic hydrolysis of peptides in sheep brain by Chang Fei et al., sheep brain research is a completely new research field, mainly focusing on antioxidant active peptides. Cu ion-binding acetylcholinesterase (AChE) inhibitory active peptides have not been isolated from sheep brain, bovine brain or pig brain, and have not been put into drug development and application.

[0009] Patent application number 200610150892.9 describes the preparation of an injectable lyophilized brain protein hydrolysate from pig brain; patent application number 201110227333.4 describes the extraction of a brain protein hydrolysate solution from pig brain; patent application number 201210235771.X describes the preparation of a modified protein powder from pig brain and the preparation of brain protein hydrolysate using this protein powder; patent application number 201410319262.4 describes the preparation of a pure natural brain protein hydrolysate raw material from pig brain; patent application number 201410255066.5 describes the preparation of a lyophilized powder injection of brain protein hydrolysate from pig brain; patent application number 201510908547.6 describes the preparation of a pig brain protease-hydrolyzed brain polypeptide and small molecule brain peptide from pig brain; patent application number 201610547261.4... A composition containing pig brain extract was prepared from pig brain; Patent application number 201810920697.2, gangliosides were extracted and isolated from pig brain marrow; Patent application number 202010190496.9, an extract with strong antioxidant capacity was extracted from pig brain; Patent application number 202111361892.4, brain protein hydrolysate was prepared from pig brain, and a troxerutin brain protein hydrolysate injection was invented; Patent application number 201710460500.7, an extract was extracted from bovine pituitary gland; Patent application number 202410366072.1, a high-purity small molecule peptide was extracted from yak brain marrow; Patent application number 201310169143.0, an injectable preparation was prepared from brain protein hydrolysate isolated from animal brain. Summary of the Invention

[0010] The present invention aims to provide a method and application for isolating F1-Cu ion-neutral bioactive peptides from sheep brain total albumin hydrolysate. The method involves removing surface impurities and blood from fresh sheep brain, freeze-drying it under low-temperature vacuum, pulverizing and sieving to obtain sheep brain powder; defatting with petroleum ether to obtain defatted sheep brain powder; then sequentially extracting with distilled water, centrifuging, and freeze-drying to obtain crude sheep brain albumin extract; further hydrolyzing with trypsin and then freeze-drying to obtain sheep brain total albumin trypsin hydrolysate; separating the neutral fraction using CM-65OM and DEAE-650M cation and anion exchange resins; and then using an HW-40F gel column to prepare the Cu ion-bound neutral peptide F1 fraction with a molecular weight of 4 kDa. The F1 fraction yielded 73.37%, with a protein content of 44.91%, a Cu ion binding capacity of 70.49%, an ABTS free radical scavenging rate of 70.49%, an Fe ion binding capacity of 95.59%, and an acetylcholinesterase (AChE) inhibition rate of 50.92%. Twenty-two neutral polypeptides with molecular weights in the 4 kDa range from sheep brain total albumin trypsin hydrolysis were identified and screened by LC-MS / MS mass spectrometry. This method is simple, environmentally friendly, safe, and low-cost, utilizing readily available and inexpensive raw materials. This polypeptide fraction retains the biological activity of the original substances in sheep brain while also turning waste into treasure and resources, fully utilizing Xinjiang's unique livestock resources to achieve high-value-added utilization of livestock resources.

[0011] The method for separating F1-Cu ion-neutral bioactive peptides from total albumin hydrolysate of sheep brain according to the present invention is carried out according to the following steps: a. Select fresh sheep brains taken after slaughter, wash them with physiological saline to remove the outer membrane, surface bloodstains and impurities, freeze-dry them at -80℃ for 48 h to obtain freeze-dried sheep brain powder, and store them in a refrigerator at -20℃ for later use. b. After passing the freeze-dried sheep brain powder obtained in step a through a 40-mesh sieve, add petroleum ether at a material-to-liquid ratio of 1:30 and mechanically stir. Degrease the sheep brain powder three times at -4℃ for 8 hours each time. Let the defatted sheep brain powder stand for 12 hours, discard the supernatant, let the sediment settle, and place it in a ventilated area to dry for 48 hours to obtain defatted sheep brain powder. c. Add the defatted sheep brain powder obtained in step b to ultrapure water at a material-to-liquid ratio of 1:5 and pH 6±0.2. Perform ultrasonic extraction at room temperature for 60 min with auxiliary ultrasonication at 300W power, 10000r / min, and 4℃. Centrifuge for 10 min, discard the precipitate, take the supernatant, and then desalt it for 48 h at -4℃ using a 3500Da dialysis bag. Freeze-dry it for 48 h at 25 MPa and -80℃ to obtain freeze-dried powder of crude sheep brain total albumin extract. d. The lyophilized powder of crude total albumin of sheep brain obtained in step d was added to a 0.02 mol / L tris(hydroxymethyl)aminomethane hydrochloride solution, the pH was adjusted to 7.8 with 0.05M NaOH solution, and then 3% trypsin was added for enzymatic hydrolysis. The temperature was 37℃, the pH was adjusted every 30 minutes, the hydrolysis time was 4 h, and the degree of hydrolysis was 14.77%, thus obtaining the enzymatic hydrolysate. e. The enzymatic hydrolysate obtained in step d is inactivated at 95℃ for 8-10 minutes, centrifuged at 10000 r / min and 4℃ for 10 minutes, the supernatant is collected, and freeze-dried at 25 MPa and -80℃ for 48 hours to obtain sheep brain total albumin trypsin hydrolysate dry powder with a yield of 58.42% and a protein content of 55.6%. f. The sheep brain total albumin trypsin hydrolysate obtained in step e is separated and purified by cation exchange resin CM-650M, and eluted with NaCl at concentrations of 0.1M, 0.2M, 0.4M, 0.6M and 0.8M. The eluted portions and unbound portions of 0.1M, 0.2M, 0.4M, 0.6M and 0.8M NaCl are collected. g. The unconjugated fraction of the sheep brain total albumin trypsin hydrolysate obtained in step f is separated and purified by anion exchange resin DEAE-650M. It is eluted with NaCl at concentrations of 0.1M, 0.2M, 0.4M, 0.6M and 0.8M. The eluted fractions and neutral fractions of 0.1M, 0.2M, 0.4M, 0.6M and 0.8M NaCl are collected according to the elution gradient. h. Dissolve the neutral fraction of the sheep brain total albumin trypsin hydrolysate obtained in step f using a 20μm, 120 A solution. ° The protein was eluted and desalted using a C18 column with 75% ethanol. The ethanol was evaporated using a rotary evaporator at 40°C, and the product was freeze-dried at 25 MPa and -80°C for 48 h to obtain the neutral desalted fraction of sheep brain total albumin trypsin hydrolysate, with a yield of 29.5% and a protein content of 45.93%. i. The neutral desalted fraction of the sheep brain total albumin trypsin hydrolysate obtained in step h was separated using an HW-40F gel column (16mm x 10cm). Three column volumes were eluted with ultrapure water, and each fraction was collected. The fractions were then freeze-dried at 25 MPa and -80°C for 48 hours to obtain the neutral polypeptide fractions F1, F2, and F3 of the sheep brain total albumin trypsin hydrolysate. The antioxidant activity of the obtained F1, F2, and F3 fractions was then tested and copper ion-binding peptide components were screened. j. The F1 fraction obtained in step i was dissolved in 0.3 mol / L, pH 3.0 NaCl solution at a material-to-liquid ratio of 1:20 mg / mL, and CuSO4·5H2O was added at a mass ratio of 1:2. After chelation reaction at 37℃ for 60 min, the mixture was centrifuged at 10000 r / min for 5 min, and the supernatant was collected. The supernatant was then freeze-dried at 25 MPa and -80℃ for 48 h to obtain F1-Cu ion-binding active peptide with a molecular weight of 4 kDa. The obtained F1 fraction had a Cu ion binding capacity of 70.49%, an ABTS free radical scavenging rate of 96.96%, a hydroxyl (OH) free radical scavenging rate of 27.38%, an Fe ion binding capacity of 95.59%, a yield of 73.37%, and a protein content of 44.91%. It was named F1-Cu. The method describes the use of F1-Cu ion-active peptides from sheep brain total albumin hydrolysate in the preparation of antioxidant drugs.

[0012] The method describes the use of F1-Cu ion-active peptides from sheep brain total albumin hydrolysate in the preparation of acetylcholinesterase (AChE) inhibitors.

[0013] The method describes the pharmaceutical use of F1 and F1-Cu ion-active peptides obtained from sheep brain total albumin hydrolysate for depression, epilepsy, cerebral palsy, brain dysfunction, neurasthenia, cerebrovascular metabolic disorders, Parkinson's syndrome, and Alzheimer's disease.

[0014] The obtained F1-Cu ion-active peptide was used to determine the acetylcholinesterase (AChE) inhibition rate using the Ellman method, which was 50.92%.

[0015] This invention discloses a method and application for isolating F1-Cu ion-active peptides from sheep brain albumin hydrolysate. This method extracts and prepares acetylcholinesterase (AChE) inhibitory peptides with antioxidant activity and Cu ion binding from the neutral fraction of sheep brain total albumin hydrolysate. Currently, this method is not reported in the literature. This invention utilizes a traditional extraction and separation method, which simultaneously and efficiently and thoroughly removes fats and cholesterol from sheep brain, improving the drying and extraction efficiency and purity of sheep brain total albumin, increasing production efficiency suitable for industrial production. It also achieves the goal of simultaneously separating the active peptide fraction from the neutral fraction HW-40F of sheep brain total albumin hydrolysate; and systematically utilizes copper ion binding in the peptide fraction separated from the neutral fraction HW-40F of sheep brain total albumin hydrolysate, achieving the simplest and lowest-cost purification effect. All steps in the extraction process of this invention are operated under ambient temperature conditions, using conventional reagents and equipment, which minimizes the cost of the extraction process. The obtained product has no harmful solvent residues, no cytotoxicity, and is safe, environmentally friendly, and highly efficient. It also turns waste into treasure and makes full use of resources. The prepared peptides can be used in the fields of antioxidants and health foods.

[0016] This invention relates to a method and its application for isolating F1-Cu ion-neutral active peptides from sheep brain total albumin hydrolysate. The method involves removing surface impurities and blood from fresh sheep brain, freeze-drying it under low temperature and vacuum, pulverizing and sieving to obtain sheep brain powder; defatting with petroleum ether to obtain defatted sheep brain powder; then sequentially extracting with distilled water, centrifuging, and freeze-drying to obtain crude sheep brain total albumin extract; then hydrolyzing with trypsin and freeze-drying to obtain sheep brain total albumin trypsin hydrolysate with a degree of hydrolysis of 14.77%, a yield of 58.42%, and a protein content of 55.6%; separating and preparing neutral polypeptide fractions with antioxidant activity in the molecular weight range of 4 kDa using cation and anion exchange resins with a yield of 29.5% and a protein content of 45.93%; and finally separating the F1 polypeptide active fraction Cu using HW-40F gel. The ion binding capacity was 70.49%, the ABTS radical scavenging rate was 96.96%, the hydroxyl (OH) radical scavenging rate was 27.38%, the Fe ion binding capacity was 95.59%, the yield was 73.37%, the protein content was 44.91%, and the AchE inhibition rate was 50.92% (Table 1).

[0017]

[0018] When the sample concentration was between 0.5 and 0.063 mg / mL, it had virtually no toxicity to macrophages (RAW264.7), and the cell survival rate was above 90% (Figure 6). The polypeptide component was identified and screened by LC-MS / MS mass spectrometry, and 22 neutral polypeptides with molecular weights in the range of 4 kDa were obtained by trypsin hydrolysis of sheep brain total albumin, as shown in Table 2.

[0019]

[0020] Electron microscopy and elemental analysis results show that after Cu ions bind to the F1 site, the copper ion content is 0.08% ( Figure 8 Fluorescence spectroscopy (FS) results showed that the fluorescence intensity decreased after Cu ions bound to the F1 site. Figure 9 The ultraviolet (UV) spectroscopy results showed that the absorption intensity decreased after Cu ions were bound at the F1 site. Figure 10 ); FTIR spectroscopy results showed that after Cu ions bound at the F1 site, two new characteristic peaks appeared at wavelengths of 1104.27 and 614.81. Figure 11 Zeta-site detection results showed that the site increased after Cu ions bound to the F1 region. Figure 12 Circular dichroism (CD) spectroscopy results showed that the α and β secondary helical structures were altered after Cu ions bound to the F1 region. Figure 13 Thermogravimetric analysis results showed that after Cu ions bound to the F1 site, both the F1 and F1-Cu ion-bound neutral bioactive peptides exhibited a tertiary mass loss pattern. Figure 14 X-ray diffraction (XRD) results showed that after Cu ions bound to the F1 region, a sharp and strong small peak appeared at 2θ and 30°. F1-Cu existed in a semi-crystalline structure, indicating that the F1 peptide bound to F1-Cu ions. Figure 15 ).

[0021] This invention discloses a method and application for isolating F1-Cu ion-bound neutral bioactive peptides from sheep brain total albumin protein hydrolysate. The Cu ion-bound bioactive peptides obtained by this method have high purity, low cost, require less solvent, are simple to operate, have short extraction time, and high reproducibility. The preparation method is easy to scale up, has high yield, is pollution-free, and utilizes abundant medicinal resources. This provides technical support for fully utilizing sheep brain resources, avoids the waste of Xinjiang's abundant sheep brain resources, and provides high-quality resources and technical guarantees for the full utilization of Xinjiang's abundant and inexpensive resources and the development of products with increased added value and technological content. It also lays the foundation for developing new sheep brain drugs. Attached Figure Description

[0022] Figure 1 These are SDS-PAGE gel electrophoresis images of the various parts separated in this invention; Figure 2 This is a diagram illustrating the antioxidant activity of the F1-Cu ion-neutral active peptide of the present invention. Figure 3 This is a gel separation diagram using HW-40F gel in this invention; Figure 4This is an SDS-PAGE gel electrophoresis image of the F1 site and F1-Cu ion neutral active peptide separated by HW-40F gel according to the present invention. Figure 5 This is a molecular weight distribution diagram of the F1 site and the F1-Cu ion neutral active peptide of the present invention; Figure 6 This is a diagram illustrating the anti-inflammatory activity of the F1-Cu ion-neutral active peptide of the present invention. Figure 7 This is an RT-HPLC C8 column separation chromatogram of the F1-Cu ion neutral bioactive peptide of this invention; Figure 8 Electron microscopy and elemental analysis diagrams of the F1 site and the F1-Cu ion neutral active peptide of the present invention; Figure 9 This is a fluorescence spectrum (FS) detection diagram of the F1 site and the F1-Cu ion neutral active peptide of the present invention; Figure 10 This is a UV spectrum of the F1 site and the F1-Cu ion neutral active peptide of the present invention. Figure 11 The image shows the Fourier Transform Infrared (FTIR) spectra of the F1 site and the F1-Cu ion neutral bioactive peptide of this invention. Figure 12 This is a zeta-site detection diagram of the F1 site and the F1-Cu ion neutral active peptide of the present invention; Figure 13 This is a circular dichroism (CD) spectrum of the F1 site and the F1-Cu ion neutral active peptide of the present invention. Figure 14 This is a thermogravimetric analysis (TGA) result of the F1 site and the F1-Cu ion-neutral bioactive peptide of the present invention. Figure 15 This is an X-ray diffraction spectrum of the F1 site and the F1-Cu ion neutral active peptide of the present invention. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments given are not limited to the present invention. Example 1

[0024] a. Select fresh sheep brains taken after slaughter, wash them with physiological saline to remove the outer membrane, surface bloodstains and impurities, freeze-dry them at -80℃ for 48 h to obtain freeze-dried sheep brain powder, and store them in a refrigerator at -20℃ for later use. b. After passing the freeze-dried sheep brain powder obtained in step a through a 40-mesh sieve, add petroleum ether at a material-to-liquid ratio of 1:30 and mechanically stir. Degrease the sheep brain powder three times at -4℃ for 8 hours each time. Let the defatted sheep brain powder stand for 12 hours, discard the supernatant, let the sediment settle, and place it in a ventilated area to dry for 48 hours to obtain defatted sheep brain powder. c. Add the defatted sheep brain powder obtained in step b to ultrapure water at a material-to-liquid ratio of 1:5 and pH 6±0.2. Perform ultrasonic extraction at room temperature for 60 min with auxiliary ultrasonication at 300W power, 10000r / min, and 4℃. Centrifuge for 10 min, discard the precipitate, take the supernatant, and then desalt it for 48 h at -4℃ using a 3500Da dialysis bag. Freeze-dry it at 25 MPa and -80℃ for 48 h to obtain the freeze-dried powder of crude sheep brain total albumin extract. d. Add the lyophilized powder of crude total albumin of sheep brain obtained in step d to a 0.02 mol / L tris(hydroxymethyl)aminomethane hydrochloride solution, adjust the pH to 7.8 with 0.05 M NaOH solution, and then add 3% trypsin by mass for enzymatic hydrolysis. The temperature is 37℃, the pH is adjusted every 30 minutes, the enzymatic hydrolysis time is 4 h, and the degree of enzymatic hydrolysis is 14.77%, to obtain the enzymatic hydrolysate. e. The enzymatic hydrolysate obtained in step d is inactivated at 95℃ for 8-10 minutes, centrifuged at 10000 r / min and 4℃ for 10 minutes, the supernatant is collected, and freeze-dried at 25 MPa and -80℃ for 48 hours to obtain sheep brain total albumin trypsin hydrolysate dry powder with a yield of 58.42% and a protein content of 55.6%. f. The sheep brain total albumin trypsin hydrolysate obtained in step e is separated and purified by cation exchange resin CM-650M, and eluted with NaCl at concentrations of 0.1M, 0.2M, 0.4M, 0.6M and 0.8M. The eluted portions and unbound portions of 0.1M, 0.2M, 0.4M, 0.6M and 0.8M NaCl are collected. g. The unconjugated fraction of the sheep brain total albumin trypsin hydrolysate obtained in step f is separated and purified using anion exchange resin DEAE-650M. It is eluted with NaCl at concentrations of 0.1M, 0.2M, 0.4M, 0.6M and 0.8M, and the eluted fractions and neutral fractions are collected according to the elution gradient. h. Dissolve the neutral fraction of the sheep brain total albumin trypsin hydrolysate obtained in step f using a 20μm, 120 A solution. °The protein was eluted and desalted using a C18 column with 75% ethanol. The ethanol was evaporated using a rotary evaporator at 40°C, and then freeze-dried at 25 MPa and -80°C for 48 h to obtain the neutral desalted fraction of sheep brain total albumin trypsin hydrolysate, with a yield of 29.5% and a protein content of 45.93%. i. The neutral fractions of the sheep brain total albumin trypsin hydrolysate obtained in step h were separated using an HW-40F gel column (16mm x 10cm). Three column volumes were eluted with ultrapure water, and each fraction was collected. The fractions were then freeze-dried at 25 MPa and -80°C for 48 hours to obtain the neutral polypeptide fractions F1, F2, and F3 of the sheep brain total albumin trypsin hydrolysate. The antioxidant activity of the obtained F1, F2, and F3 fractions was tested, and copper ion-binding peptide components were screened. j. The F1 fraction obtained in step i was dissolved in 0.3 mol / L, pH 3.0 NaCl solution at a material-to-liquid ratio of 1:20 mg / mL, and CuSO4·5H2O was added at a mass ratio of 1:2. After chelation reaction at 37℃ for 60 min, the mixture was centrifuged at 10000 r / min for 5 min, and the supernatant was collected. The supernatant was then freeze-dried at 25 MPa and -80℃ for 48 h to obtain F1-Cu ion-bound neutral active peptide with a molecular weight of 4 kDa. The obtained F1 fraction had a Cu ion binding capacity of 70.49%, an ABTS free radical scavenging rate of 96.96%, a hydroxyl (OH) free radical scavenging rate of 27.38%, an Fe ion binding capacity of 95.59%, a yield of 73.37%, and a protein content of 44.91%. It was named F1-Cu. k. The obtained F1-Cu ions were combined with neutral active peptides, and the inhibition rate of acetylcholinesterase (AChE) was determined by the Ellman method. The inhibition rate of acetylcholinesterase (AChE) was 50.92% (Table 3). Table 3. Acetylcholinesterase (AChE) inhibition rate Example 2

[0025] Polyacrylamide gel electrophoresis (SDS-PAGE): The molecular weight range and separation and purification effect of the samples obtained in Example 1 were subjected to 16% polyacrylamide gel electrophoresis (SDS-PAGE) vertical electrophoresis: The compositions of the separating and stacking gels are shown in Table 4; Sample buffer: 0.5 mL 0.5 mol / L pH 6.8 Tris-HCl solution, 2 mL 10% sodium dodecyl sulfate (SDS), 1 mL glycerol, 0.5 mL β-mercaptoethanol, 1 mL ultrapure water, and a trace amount of bromophenol blue; Electrophoresis conditions: Sample concentration 5 mg / mL, sample volume 40 µL, heating for 10 min before sample loading, initial injection voltage of 50 V as standard, and voltage increased to 75 V after the sample is concentrated into a single line in the stacking gel; Electrophoresis buffer: 25 mM Tris, 192 mM glycine, 0.1% sodium dodecyl sulfate (SDS), pH 8.3; Fixative: 20% (v / v) ethanol, 10% (v / v) acetic acid; Coomassie brilliant blue staining solution: 0.6 Coomassie Brilliant Blue R 250 was dissolved in 200 mL of decolorizing solution; the decolorizing solution consisted of ethanol and acetic acid in a volume ratio of 25:8. Table 4 Series of Diluted Bovine Serum Albumin (BSA) Standards Example 3

[0026] Protein content determination: Protein concentration was determined using the bovine serum albumin (BCA) method (Arcan and Yemenicioğlu, 2010) (the relevant procedures were performed according to the BCA kit instructions). Standard curve construction: According to Table 5, the bovine serum albumin (BSA) protein standard from the kit was taken, diluted with water, and shaken well. 25 µL of each tube was placed in a 96-well plate, and 175 µL of BCA working solution (BCA reagent A: BCA reagent B = 50:1) was added to each well. After mixing, the plate was placed in a 37 ℃ incubator. After 30 min, the absorbance was measured at 562 nm. The absorbance was measured three times and the average value was taken. The standard curve was plotted with protein concentration on the x-axis and absorbance on the y-axis. Table 5 Series of diluted bovine serum albumin (BSA) standards

[0027] Sample determination: Accurately weigh 1.0 mg of sample and dissolve it in 1 mL of distilled water. Centrifuge at 6000 r / min for 3 min, take the supernatant, and perform the following operations according to the requirements of the BCA protein detection kit. Read the absorbance at 562 nm using an ELISA reader. The protein content is calculated by averaging three parallel measurements. Example 4

[0028] Calculation of extraction rate in Example 1: The extraction rate of the enzymatic hydrolysate was calculated according to equation (1); (1) In the equation m Se It is the mass (mg) of the extract after separation, m Sd It is the mass (mg) of the extract before separation. Example 5

[0029] Determination of the degree of enzymatic hydrolysis in Example 1: Mix 10 µL of hydrolysate with 1.0 mL of o-phthalaldehyde (OPA) reagent, shake at room temperature for 2 min, centrifuge the mixture for 15 min at 4500 r / min; read the absorbance of each component at 340 nm using a spectrophotometer; use distilled water as a blank after 2 min; calculate the degree of enzymatic hydrolysis (DH %) according to formulas 2 and 3: (2) (3)

[0030] Example 6 Isolation and purification of the neutral fraction from total albumin hydrolysate of sheep brain: The neutral fraction of the sheep brain total albumin trypsin hydrolysate obtained in Example 1 was separated using an HW-40F gel column (16 mm × 10 cm). The sample loading amount was 30 mg, prepared to a concentration of 30 mg / mL. Three column volumes of ultrapure water were eluted at a flow rate of 0.2 mL / min, with 6 mL collected from each tube. The concentration was maintained at 25 MPa, and fractions F1, F2, and F3 were collected. The fractions were then freeze-dried at -80℃ for 48 h to obtain lyophilized powders with yields of 73.37%, 3.79%, and 3.42%, and protein contents of 44.91%, 55.9%, and 24.17%, respectively.

[0031] Example 7 Molecular weight distribution analysis: The chromatographic apparatus used a TSKgel G2000SWXL column (30 cm × 7.8 mm, 5 μm) and a TSKgel guard column SWXL (4 cm × 6 mm, 12 μm) for protection. Isocratic elution was performed at a flow rate of 0.6 mL / min for 40 min, with the column maintained at 25 °C. The injection volume was 20 μL, and the detection wavelength was 220 nm. The mobile phase was 0.15 mol / L phosphate buffer (PBS) at pH 7.0. The standards included egg white protein (chicken), myoglobin (horse heart), aprotinin (bovine lung), neurotensin, and angiotensin II (human), with molecular weights of 45,000, 17,000, 6,700, 1,700, and 1,000 Da, respectively.

[0032] Example 8 The antioxidant activity of the F1-Cu ion-neutral active peptide obtained in Example 1 was determined: 1) ABTS free radical scavenging activity: The obtained sample was dissolved in ultrapure water at pH 6 ± 0.5 to prepare a sample solution with a concentration of 2 mg / mL. 20 μL of the solution was added to 180 μL of freshly prepared ABTS radical solution in a 96-well plate, and the experiment was repeated three times in parallel. The solution was mixed and reacted at room temperature for 6 min. Vitamin C was used as a positive control, and the absorbance was measured at a wavelength of 734 nm. The ABTS radical scavenging rate was calculated according to the following formula (4): (4) Where: A0 is the absorbance of the system without sample; Ai is the absorbance of the system with sample; Aj is the absorbance of the control group containing only sample; 2) Hydroxyl (OH) radical scavenging activity: Mix 1.0 mL of 1.865 mM 1,10-phenanthroline solution, 1.0 mL of 1.865 mM FeSO4 solution, and 2 mL of 2 mg / mL sample in a test tube. Initiate the reaction by adding 1.0 mL of 0.03% H2O2. Incubate the reaction mixture in a water bath at 37 °C for 30 min. Measure the absorbance of the mixture at 520 nm. Use vitamin C as a positive control and a mixture without H2O2 as a blank. Calculate the OH radical scavenging rate using the following formula (5): (5) Where: A0 is the absorbance of the system without sample; Ai is the absorbance of the system with sample; Aj is the absorbance of the control group containing only sample;

[0033] 3) Iron (Fe) ion chelating ability (1) Preparation of phenanthridine reagent: Accurately measure 61.55 mg of phenanthridine solid powder, dissolve it in distilled water, and make up to 25 mL in a volumetric flask to prepare a phenanthridine solution with a concentration of 5 mmol / L.

[0034] (2) Ferrous chloride solution: Accurately measure 25.4 mg of ferrous chloride, dissolve it in distilled water, and make up to 100 mL in a volumetric flask to prepare a ferrous chloride solution with a concentration of 2 mmol / L.

[0035] (3) Prepare each enzymatically hydrolyzed polypeptide component with a concentration of 2 mg / mL. Take 250 μL of sample solutions of different concentrations, add 25 μL of ferrous chloride solution and 50 μL of phenanthroxazine solution, mix well, react at room temperature for 5 min, and measure the absorbance at 562 nm. Use distilled water instead of sample solution as a blank. The ferrous ion chelating capacity is calculated according to the following formula (6): (6) In the formula, Ai, Aj, and A0 are the absorbances of the sample group and the blank group, respectively;

[0036] 4) Copper (Cu) ion chelating ability (1) Catechol Purple-Cu 2+ Preparation: Dissolve catechol purple (0.04 mM) and copper sulfate (0.08 mM) in acetic acid buffer solution (50 mM, pH 6). (Preparation method: Weigh 0.68 g of sodium acetate, dissolve in water, and bring the volume to 100 mL; measure 0.3 mL of acetic acid, dilute with water to 100 mL; mix the two in proportion and test the pH.) (2) Prepare stock solutions of each polypeptide sample with a concentration of 4 mg / mL, take 500 μL of sample solutions of different concentrations, and add 500 μL of catechol purple-Cu. 2+ The solution was mixed and reacted at room temperature for 10 min. The absorbance was measured at 623 nm. Distilled water was used as a blank instead of the sample solution. The copper ion chelating ability was calculated according to the following formula (7): (7) In the formula, Ai, Aj, and A0 are the absorbances of the sample group and the blank group, respectively;

[0037] Ai — Sample group = Sample + Catechol Violet-Cu 2+ Complex Aj – Control group = Sample + Ferrous chloride + Acetic acid buffer solution (instead of catechol violet-Cu) 2+ (complex) A0 – Blank group = Water (replacing the sample group) + Catechol Violet - Cu 2+ Complex.

[0038] Example 9 Acetylcholinesterase (AChE) inhibition rate assay: The acetylcholinesterase (AchE) inhibitory activity of the samples was determined by the Ellman method (Ellman et al. 1961). The reaction system consisted of 60 μL of tris(hydroxymethyl)aminomethane hydrochloride buffer (Tris-HCl) (pH = 8.0, 0.01 M), 20 μL of sample solution, 10 μL of 5,5-dioxanone (2-nitrobenzoic acid) (DTNB) (stock solution concentration 3 mM), and 20 μL of acetylcholinesterase (AchE) stock solution concentration 1 U / mL. The mixture was incubated at 37℃ for 10 min, 10 μL of thioacetylcholine iodide (ATCI) was added, and the mixture was incubated at 37℃ for 15 min. Then, 80 μL of 1% sodium dodecyl sulfate (SDS) solution was added to stop the reaction. Finally, the absorbance was measured at 412 nm using a 96-well microplate reader, and the acetylcholinesterase inhibition rate was calculated using formula (8). (8) In the formula, A is the microplate reader reading; the control group uses 20 μL PBS instead of the sample, and the blank group uses 20 μL PBS instead of ATCI.

[0039] Example 10 The anti-inflammatory activity of the F1-Cu ion-neutral active peptide from sheep brain total albumin protein hydrolysate prepared in Example 1 of this invention at the cellular level was verified by experiments. The experimental methods and results are as follows: The toxic effect of F1-Cu ion neutral active peptide isolated from sheep brain total albumin protein hydrolysate on macrophages (RAW264.7): The effect of F1-Cu ion neutral active peptide isolated from sheep brain total albumin protein hydrolysate on the proliferation ability of macrophages (RAW264.7) was determined by MTT assay. Logarithmically growing macrophages (RAW264.7) were counted using a cell counter and seeded into 96-well plates at a density of 1 × 10⁴ cells / well, 100 μL per well, and cultured at 37℃ for 24 h. The supernatant was discarded, and 0.063 mg / mL, 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL of the cytotoxic peptide were added. F1-Cu ion neutral active peptide solution and lipopolysaccharide (LPS) (1 μg / mL) were separated from the total albumin protein hydrolysate of sheep brain. Three replicates were set for each concentration. The culture medium was used as a blank control. The culture was carried out at 37℃ for 24 h. Then, 100 μL of thiazolyl blue (MTT) solution was added to each well and the culture was continued in an incubator for 3-4 h. After that, the supernatant was aspirated and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. The mixture was shaken at low speed for 10 min to fully dissolve the crystals. The absorbance (OD) at 570 nm was detected by an enzyme-linked immunosorbent assay (ELISA) reader. The inhibition rate of the sample on the growth of macrophages (RAW264.7) was then calculated. The calculation formula is (9): (9) When the sample concentration was between 0.5 and 0.063 mg / mL, it showed virtually no toxicity to macrophages (RAW264.7), with cell viability exceeding 90%. Figure 6 As shown; Effect of F1-Cu ion neutral bioactive peptides from sheep brain total albumin hydrolysate on NO secretion by macrophages (RAW264.7): Logarithmic growth phase cells were collected, cells were counted, cell suspensions were diluted with culture medium, and the cell suspension concentration was adjusted to 2 x 10⁻⁶. 6 Cells / mL were seeded into 96-well plates, 100 μL per well, and cultured at 37℃ for 24 h. The supernatant was discarded. F1-Cu ion neutral bioactive peptide sample solutions with concentrations of 0.063 mg / mL, 0.125 mg / mL, 0.25 mg / mL and 0.5 mg / mL were added to the experimental groups, with 3-6 replicates for each concentration. The positive control group was the cell group with culture medium, and the negative control group was the cell group with lipopolysaccharide (LPS) solution (1 μg / mL). All cells were cultured at 37℃ for 24 h. The effect of F1-Cu ion-neutral bioactive peptides from sheep brain total albumin protein hydrolysate on NO secretion by macrophages (RAW264.7) was detected using Griess reagent. Aspirate 50 µL of cell culture supernatant per well into a 96-well plate, add 50 µL of Griess Reagent I, then add 50 µL of Griess Reagent II and vortex to mix. Measure the absorbance at 540 nm using a microplate reader. Simultaneously, plot standard curves by diluting different concentrations (0, 1, 2, 5, 10, 20, 40, 60, 100 μM) of standards with complete culture medium, establish a regression equation, and calculate the NO content in the cell supernatant based on the regression equation. The experimental results are attached. Figure 5 As shown; by Figure 5 It is evident that the F1-Cu ion-neutral bioactive peptides of sheep brain total albumin protein hydrolysate can inhibit lipopolysaccharide (LPS)-induced NO secretion by RAW264.7 macrophages in a dose-dependent manner, and the IC50 of NO inhibition is [not specified]. 50 It is 3.4.

[0040] Example 11 The F1-Cu ion-neutral bioactive peptides of sheep brain total albumin protein hydrolysate were further purified using a semi-preparative RP-HPLC column. The separation system and conditions are as follows: High-performance liquid chromatography (HPLC) system: Shimadzu P680; Column specifications: 5C8-MS Pached Column (COSMOSIL, 10ID × 250 mm, 5 µ m,); Temperature: 30℃; Injection volume: 100μL; Flow rate: 2 mL / min; UV spectroscopy detection wavelength: 214 nm and 280 nm; Mobile phase: A: 0.1% trifluoroacetic acid (TFA); B: acetonitrile; Elution conditions: Gradient elution, equilibrate with 5% B solution before loading, elute with 5% B solution for 10 min after loading, then elute with 5-90% B solution for 10 min, elute with 90-95% B solution for 5 min, continue eluting with 95% B solution for 15 min, elute with 95-5% B solution for 5 min, elute with 5% B solution for 15 min; Analysis time: 60 min.

[0041] Example 12 LC-MS / MS mass spectrometry identification: The F1-Cu ion neutral bioactive peptides from sheep brain total albumin protein hydrolysate were separated by capillary high performance liquid chromatography and then analyzed by mass spectrometry using a QExactive HF-X mass spectrometer (Thermo Fisher). The analysis time was 60 min, and the detection mode was positive ion. The mass-charge ratio of the peptides and peptide fragments was collected using the following method: 10 fragment spectra were collected after each full scan (MS2 scan). The raw mass spectrometry test files were retrieved from the relevant database using MaxQuant 1.5.5.1 software, and finally the protein identification and quantification results were obtained.

[0042] Example 13 Electron microscopy (SEM) and elemental analysis (EDX) F1 and F1-Cu ion-neutral active peptide samples were attached to double-sided adhesive tape on a sample holder and placed in an ion sputtering instrument to deposit a conductive platinum film. The surface morphology of the samples was observed using a scanning electron microscope (ZEISS SUPRA 55VP) analysis system under an accelerating voltage of 20 kV (SEM), and images were taken at magnifications of 500-50000×. Elemental analysis of the peptide samples was performed using an energy-density X-ray (EDX) instrument (Bruker Nano GmbH, Germany).

[0043] Example 14 Fluorescence spectroscopy (FS) analysis The conformational changes of F1 and F1-Cu ion-neutral bioactive peptide samples were evaluated using a fluorescence spectrophotometer. The sample was prepared as an aqueous solution with a concentration of 1 mg / mL, and the fluorescence spectrum of the emission wavelength from 300 to 500 nm was recorded at an excitation wavelength of 280 nm.

[0044] Example 15 Ultraviolet (UV) spectroscopy analysis Aqueous solutions of F1 and F1-Cu ion neutral active peptides with a concentration of 1 mg / mL were prepared, and the full-wavelength scan absorption spectra in the range of 190-400 nm were measured using a UV spectrophotometer.

[0045] Example 16 FTIR spectroscopy analysis 1 mg of lyophilized F1 and F1-Cu ion-neutral bioactive peptide samples were mixed with 160 mg of dry KBr and compressed into uniform, transparent sheets. The samples were then analyzed using an FTIR spectrophotometer at 4000–500 cm⁻¹. -1 Within the specified range, FTIR spectra of F1 and F1-Cu ion-bound neutral bioactive peptide samples were recorded at a resolution of 4.0 cm⁻¹. -1 .

[0046] Example 17 Zeta potential: Aqueous solutions of F1 and F1-Cu ion-neutral bioactive peptides were prepared at a concentration of 1 mg / mL. The zeta potential of the peptide samples was measured using a zeta potential meter. All samples were measured three times in this experiment, and the results are expressed as the average value.

[0047] Example 18 Circular dichroism (CD) The secondary structures of F1 and F1-Cu ion-neutral active peptides were acquired using a Chirascan CD spectrometer. A 0.5 mg / mL peptide sample was prepared using 50 mM pH 7.0 phosphate buffer. The CD spectra of the sample were acquired in the range of 190-260 nm. The spectral scans were performed with phosphate buffer as the background. Each sample was scanned three times. After averaging and smoothing, the CD spectra were analyzed using the K2D3 online tool.

[0048] Example 19 Thermogravimetric analysis Thermogravimetric analysis (TG), differential thermogravimetric analysis (DTG), and differential scanning calorimetry (DSC) were performed using a thermogravimetric analyzer (STA449F3, Netzsch, Germany). After the F1 and F1-Cu ion-bound neutral active peptides were placed on a stable plate, they were heated from 30 °C to 600 °C at a nitrogen flow rate of 50 mL / min at 20 °C / min.

[0049] Example 20 X-ray diffraction spectroscopy analysis: The crystal structures of F1 and F1-Cu ion-neutral bioactive peptides were determined using X-ray powder diffraction (D8 Advance, Bruker, Germany). The detection was performed at room temperature, with a 2θ range of 5–80 °C, a step size of 0.01°, and a step time of 0.1 s / step.

Claims

1. A method for isolating F1-Cu ion-neutral bioactive peptides from sheep brain total albumin protein hydrolysate, characterized in that, Follow these steps: a. Select fresh sheep brains taken after slaughter, wash them with physiological saline to remove the outer membrane, surface bloodstains and impurities, freeze-dry them at -80℃ for 48 h to obtain freeze-dried sheep brain powder, and store them in a refrigerator at -20℃ for later use. b. After passing the freeze-dried sheep brain powder obtained in step a through a 40-mesh sieve, add petroleum ether at a material-to-liquid ratio of 1:30 and mechanically stir. Degrease the sheep brain powder three times at -4℃ for 8 hours each time. Let the defatted sheep brain powder stand for 12 hours, discard the supernatant, let the sediment settle, and place it in a ventilated area to dry for 48 hours to obtain defatted sheep brain powder. c. Add the defatted sheep brain powder obtained in step b to ultrapure water at a material-to-liquid ratio of 1:5 and pH 6±0.

2. Perform ultrasonic extraction at room temperature for 60 min with auxiliary ultrasonication at 300W power, 10000r / min, and 4℃. Centrifuge for 10 min, discard the precipitate, take the supernatant, and then desalt it for 48 h at -4℃ using a 3500Da dialysis bag. Freeze-dry it at 25 MPa and -80℃ for 48 h to obtain the freeze-dried powder of crude sheep brain total albumin extract. d. Add the lyophilized powder of crude total albumin of sheep brain obtained in step d to a 0.02 mol / L tris(hydroxymethyl)aminomethane hydrochloride solution, adjust the pH to 7.8 with 0.05 M NaOH solution, and then add 3% trypsin by mass for enzymatic hydrolysis. The temperature is 37℃, the pH is adjusted every 30 minutes, the enzymatic hydrolysis time is 4 h, and the degree of enzymatic hydrolysis is 14.77%, to obtain the enzymatic hydrolysate. e. The enzyme hydrolysate obtained in step d is inactivated at 95℃ for 8-10 minutes, centrifuged at 10000 r / min and 4℃ for 10 minutes, the supernatant is collected, and freeze-dried at 25 MPa and -80℃ for 48 hours to obtain sheep brain total albumin trypsin hydrolysate dry powder. f. The sheep brain total albumin trypsin hydrolysate obtained in step e is separated and purified by cation exchange resin CM-650M, and eluted with NaCl at concentrations of 0.1M, 0.2M, 0.4M, 0.6M and 0.8M. The eluted portions and unbound portions of 0.1M, 0.2M, 0.4M, 0.6M and 0.8M NaCl are collected. g. The unconjugated fraction of the sheep brain total albumin trypsin hydrolysate obtained in step f is separated and purified using anion exchange resin DEAE-650M. It is eluted with NaCl at concentrations of 0.1M, 0.2M, 0.4M, 0.6M and 0.8M, and the eluted fractions and neutral fractions are collected according to the elution gradient. h. Dissolve the neutral fraction of the sheep brain total albumin trypsin hydrolysate obtained in step f using a 20μm, 120 A solution. ° The C18 column was used for elution and desalting with 75% ethanol. The ethanol was evaporated by rotary evaporator at 40℃ and then freeze-dried at 25 MPa and -80℃ for 48 h to obtain the neutral desalted fraction of sheep brain total albumin trypsin hydrolysate. i. The neutral desalted fraction of the sheep brain total albumin trypsin hydrolysate obtained in step h was separated using an HW-40F gel column (16mm x 10cm). Three column volumes were eluted with ultrapure water, and each fraction was collected. The fractions were then freeze-dried at 25 MPa and -80℃ for 48 hours to obtain the neutral polypeptide fractions F1, F2, and F3 of the sheep brain total albumin trypsin hydrolysate. The antioxidant activity of the obtained F1, F2, and F3 fractions was then tested and copper ion-binding peptide components were screened. j. Dissolve the F1 fraction obtained in step i in 0.3 mol / L, pH 3.0 NaCl solution at a material-to-liquid ratio of 1:20 mg / mL, and add CuSO4·5H2O at a mass ratio of 1:

2. After chelation reaction at 37℃ for 60 min, centrifuge at 10000 r / min for 5 min, take the supernatant, and freeze-dry at 25 MPa and -80℃ for 48 h to obtain an F1-Cu ion-bound neutral active peptide with a molecular weight of 4 kDa, named F1-Cu.

2. The use of the F1-Cu ion neutral active peptide obtained from sheep brain total albumin hydrolysate by the method according to claim 1 in the preparation of antioxidant drugs.

3. The use of the F1-Cu ion neutral active peptide obtained from sheep brain total albumin hydrolysate by the method described in claim 1 in the preparation of AChE inhibitory drugs.

4. The use of the F1 and F1-Cu ion-bound neutral bioactive peptides obtained from the total albumin hydrolysate of sheep brain obtained by the method described in claim 1 for pharmaceutical applications in treating depression, epilepsy, cerebral palsy, brain dysfunction, neurasthenia, cerebrovascular metabolic disorders, Parkinson's syndrome, and Alzheimer's disease.

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