Water-soluble walnut polypeptide passing through blood brain barrier as well as preparation method and application of water-soluble walnut polypeptide

Abstract

The invention discloses a water-soluble walnut polypeptide passing through a blood brain barrier and a preparation method and application thereof.The amino acid sequence of the water-soluble walnut polypeptide passing through the blood brain barrier is FGDNLKAGP, and the water-soluble walnut polypeptide passing through the blood brain barrier is obtained through a solid-phase chemical synthesis method or extracted from walnut meal. The compound can be used in preparation of brain nutrition supplement drugs or health care products, and preparation of blood-brain barrier-crossing drugs or detection reagents and probes. The water-soluble walnut polypeptide has the capacity of penetrating through the blood-brain barrier, can penetrate through the blood-brain barrier to enter the brain to exert the function of the blood-brain barrier, is used for preparing a medicine or a detection reagent across the blood-brain barrier and a positioning probe for targeting brain tissue, and is good in targeting property, safe and reliable.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide products, and particularly relates to a water-soluble walnut polypeptide that passes through the blood-brain barrier and a preparation method and application thereof. Background Art

[0002] The blood-brain barrier (BBB) ​​restricts most drugs from entering the central nervous system, making the treatment of various brain diseases ineffective or even ineffective. Therefore, the discovery of effective cross-brain barrier substances will be beneficial to improving brain nutrition and preventing and treating related central nervous system diseases. At present, only some small molecules can cross the blood-brain barrier, and most substances, especially some large molecules, cannot cross the blood-brain barrier. They need to be mediated by endogenous receptors to deliver large molecules (including drugs, probes, etc.) into the brain. As an important part of the human brain, the cerebellum not only plays a key role in motor coordination and balance, but is also closely related to cognitive function, emotional regulation and certain neurological diseases. However, the current understanding of cerebellar function is still very limited, especially in the study of the pathogenesis and potential therapeutic targets of cerebellar-related diseases. At the same time, due to the high selectivity of the blood-brain barrier, there are very few substances that can effectively pass through the blood-brain barrier and target the cerebellum. Existing studies have mostly focused on lipid-soluble small molecules, while there is still a lack of systematic exploration of the cross-barrier mechanism of large molecules such as water-soluble peptides.

[0003] Walnut is an oil crop widely planted in my country. In order to develop high-value walnut products, walnut oil is currently obtained by pressing oil, and a large amount of walnut meal is left as animal feed or discarded, causing great waste. The main component protein in walnut meal can be hydrolyzed into polypeptides with a wide range of physiological activities. Walnuts have traditional uses for brain health. Patent CN 115109818A discloses a walnut peptide that can penetrate the blood-brain barrier. The walnut peptide obtained by two-step enzymatic hydrolysis of walnut meal has special permeability, or contains active peptides with strong membrane permeability, has the effect of improving memory, and can be used in the preparation of corresponding foods, nutritional supplements or drugs. However, the walnut peptides obtained in the above patents are enzymatic products with relatively complex ingredients. It is unknown which specific ingredients play a role, and too many ingredients are not conducive to the mediation effect. It is not clear whether the protein peptides in walnuts can pass through the blood-brain barrier to play their nutritional and health care or even carrier or mediating role. Therefore, in-depth research and development of walnut peptides can not only improve the utilization value of walnuts, but also have important significance for the research of polypeptide compounds. Summary of the invention

[0004] In view of the above problems, the present invention provides a water-soluble walnut polypeptide that passes through the blood-brain barrier and an application of its preparation method. The water-soluble walnut polypeptide can pass through the blood-brain barrier and can be used for brain nutritional supplementation and as a positioning probe targeting brain tissue.

[0005] The present invention is achieved through the following technical solutions: A water-soluble walnut polypeptide that passes through the blood-brain barrier, wherein the amino acid sequence of the water-soluble walnut polypeptide that passes through the blood-brain barrier is FGDNLKAGP.

[0006] In the present invention, the water-soluble walnut polypeptide that passes through the blood-brain barrier is obtained by solid phase chemical synthesis or extracted from walnut meal.

[0007] In the present invention, the water-soluble walnut polypeptide that passes through the blood-brain barrier is used in the preparation of brain nutritional supplement medicines or health products.

[0008] In the present invention, the water-soluble walnut polypeptide that passes through the blood-brain barrier is used in the preparation of drugs or detection reagents that cross the blood-brain barrier.

[0009] In the present invention, the water-soluble walnut polypeptide that passes through the blood-brain barrier is used in the preparation of probes.

[0010] Furthermore, the probe is a positioning probe targeting brain tissue.

[0011] Furthermore, the water-soluble walnut polypeptide that passes through the blood-brain barrier is coupled with a fluorescent group.

[0012] Furthermore, the fluorescent group is one of Cy5, Cy7 and ICG.

[0013] Beneficial effects of the present invention The water-soluble walnut polypeptide provided by the present invention has the ability to penetrate the blood-brain barrier and can enter the brain to exert its functional effects. The water-soluble walnut polypeptide is derived from walnuts, can be artificially synthesized, and has high safety. The ability of the water-soluble walnut polypeptide in the present invention to pass through the blood-brain barrier enables it to be used in the study of positioning probes targeting brain tissue and the preparation of drugs or detection reagents that cross the blood-brain barrier, etc., which accelerates the study of brain nutrition supplementation or disease pathogenesis and is conducive to the development of cerebellar nutrition products or pharmaceutical preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the in vivo fluorescence imaging of mice injected with crude walnut peptide-Cy7; Figure 2 This is the molecular sieve segmentation diagram of the crude walnut peptide; Figure 3This is the in vivo fluorescence imaging of crude walnut peptides separated by molecular sieves after injection into the mouse tail vein; Figure 4 Prepare liquid chromatogram for molecular sieve 3; Figure 5 This is the in vivo fluorescence imaging of the three components of the molecular sieve; Figure 6 It is the total ion current chromatogram of peak component No. 9 in Example 3; Figure 7 is the HPLC chart of FGDNLKAGP; Figure 8 is the mass spectrum of FGDNLKAGP; Fig. 9 This is the HPLC purification diagram of FGDNLKAGP-Cy7 conjugate; Fig.10 is the mass spectrum of FGDNLKAGP-Cy7 conjugate; Fig.11 Fluorescence imaging of FGDNLKAGP-Cy7 conjugate and Cy7 in mouse brain; Fig.12 Predict FGDNLKAGP binding targets for Swisstargetprediction. DETAILED DESCRIPTION

[0015] The present invention is further explained below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0016] Example 1 (1) Grinding walnut meal to obtain walnut powder; (2) Weigh 10 g of walnut powder, add 90 ml of water, add 1% of commercially available plant protein hydrolyzing complex enzyme (Nanning Pangbo Bioengineering Co., Ltd.), put it in a water bath constant temperature oscillator, keep it at 50°C for 6 h, 8000 r / min, centrifuge it for 10 min, take the supernatant, and rotary evaporate it at 80°C and 100 r / min. The obtained extract is frozen and dried to obtain the crude walnut peptide; (3) The crude walnut peptide was prepared into a 13.3 mg / ml aqueous solution, adjusted to pH 8 with NaOH, centrifuged at 4°C and 18,000 r / min for 5 min, and the supernatant was taken for later use; (4) Accurately weigh 1 mg of Cy7 (Sulfo-Cy7SE, Beijing Fubaike) and dissolve it in 50 μl of dimethyl sulfoxide (DMSO). Add 150 μl of the supernatant in step (3), mix well, and react overnight at 4°C in the dark with shaking to obtain the crude walnut peptide-Cy7 labeling solution. The blank aqueous solution without adding the crude walnut peptide is treated in the same way to obtain the Cy7 blank labeling solution. The nude mice were anesthetized by intraperitoneal injection of 2% sodium pentobarbital 300ul (215 mg / kg), and physiological saline, Cy7 blank labeling solution, and walnut peptide crude-Cy7 labeling solution 175μl (0.5 mg / g) were injected through the tail vein. The nude mice were placed in a prone position in the recording dark box of the small animal multispectral in vivo imaging system, and the excitation wavelength was detected at 700~770 nm. The imaging of the fluorescence emitted in the animal body after the injection of the labeling solution was recorded. The results are shown in Figure 1 As shown, Figure 1 The upper, middle and lower pictures are the in vivo fluorescence images of mice injected with saline, Cy7 and crude walnut peptide-Cy7 respectively; Figure 1 It can be seen that the fluorescence intensity of the brain of mice injected with crude walnut peptide was significantly increased.

[0017] Example 2 According to the method of Example 1, a crude walnut peptide was prepared. The crude walnut peptide was dissolved in 0.02M PBS (pH 7.2), filtered through a 0.22um filter membrane, loaded at a flow rate of 3ml / min, and separated into 5 sections (molecular sieve 1, molecular sieve 2, molecular sieve 3, molecular sieve 4 and molecular sieve 5) through a molecular sieve (200PE, 30×2.6cm). The molecular sieve segmentation diagram of the crude walnut peptide is shown in FIG. Figure 2 As shown in the figure, the five separated products were labeled with Cy7 (respectively denoted as molecular sieve 1-Cy7, molecular sieve 2-Cy7, molecular sieve 3-Cy7, molecular sieve 4-Cy7 and molecular sieve 5-Cy7), and injected into the tail vein of mice. Cy7 and blank (normal saline) were used as controls, and the in vivo fluorescence imaging of each segment of walnut peptide crude product after mouse tail vein injection was recorded. The results are shown in the figure. Figure 3 As shown (1. Molecular sieve 1- Cy7, 2. Molecular sieve 2- Cy7, 3. Molecular sieve 3- Cy7, 4. Molecular sieve 4- Cy7, 5. Molecular sieve 5- Cy7, 6. Cy7, 7. Blank), due to the relatively high selectivity of the fluorescence intensity of molecular sieve 3 in the cerebellum region of the brain, it was screened out for preparative liquid phase separation.

[0018] Example 3 Example 1 The third section walnut peptide crude product (molecular sieve 3) separated by molecular sieve was subjected to liquid phase separation, and its preparative liquid chromatogram is as follows: Figure 4 As shown by Figure 4It can be seen that 9 peaks were separated. The 9 peak components (liquid phase 1, liquid phase 2, liquid phase 3, liquid phase 4, liquid phase 5, liquid phase 6, liquid phase 7, liquid phase 8 and liquid phase 9) were labeled with Cy7 and injected into the mouse tail vein. The in vivo fluorescence imaging is shown in Figure 2. Figure 5 As shown, from left to right are liquid phase 1- Cy7, liquid phase 2- Cy7, liquid phase 3- Cy7, liquid phase 4- Cy7, liquid phase 5- Cy7, liquid phase 6- Cy7, liquid phase 7- Cy7, liquid phase 8- Cy7, liquid phase 9- Cy7, mixed sample (molecular sieve 3)-Cy7, Cy7, blank (normal saline). From the perspective of relatively good peak separation, the sample of peak 9 was selected for liquid-mass separation analysis.

[0019] Example 4 The total ion current chromatogram of peak component No. 9 in Example 3 is as follows Figure 6 As shown, 76 peptide information were obtained by matching the database with the software PEAKSStudio10.6; the stability, hydrophilicity and other physicochemical properties of the above peptide sequences were analyzed by the online tool ProtParam provided by ExPASy (Expert Protein Analysis System), and a representative peptide with an amino acid sequence of SEQ ID NO.1: FGDNLKAGP was initially screened out for chemical synthesis.

[0020] Example 5 SEQ ID NO.1: FGDNLKAGP obtained in Example 4 was synthesized by solid phase synthesis, specifically: (1) Weigh 100 mg of Rink Amide Resin (0.01 mmol scale) and place it in a reaction bottle. Add 3 mL of DMF (dimethylformamide) and soak for 30 minutes to allow it to swell completely. Then add 3 mL of deprotection solution (20% piperidine in DMF solution) and shake for 10 minutes. Then wash the resin with DMF 3 times, 3 mL each time, to ensure that the Fmoc group and residual reagents are removed. (2) Coupling of amino acids: weigh 0.04 mmol Fmoc-Pro (P) (dissolved in 1 mL DMF), 0.04 mmol DIC (N,N'-diisopropylcarbodiimide) and 0.04 mmol Oxyma Pure, add the coupling solution to the resin for coupling, shake the reaction for 1 hour, and then wash the resin 3 times with DMF, 3 mL each time; repeat the above amino acid coupling steps, and couple each amino acid in the order of Gly (G), Ala (A), Lys (Boc) (K), Leu (L), Asn (Trt) (N), Asp (OtBu) (D), Gly (G), and Phe (F); (3) Mix the resin with 3 mL of cutting solution (95% TFA (trifluoroacetic acid) + 2.5% H 2 O + 2.5% TIS (volume ratio)) to cut the polypeptide from the resin, shake and react for 2 hours, filter the reaction solution, collect the filtrate and add 10 times the volume of cold ether to precipitate the polypeptide, centrifuge the precipitation, remove the supernatant, wash it again with cold ether once and dry it to obtain the crude polypeptide; (4) Use a reverse phase C18 column and gradient elution (0-100% ACN in H 2 O, containing 0.1% TFA), monitoring wavelength 214 nm, collecting the target peak, freeze-drying to obtain a white powder, which is a water-soluble walnut polypeptide with an amino acid sequence of FGDNLKAGP; the HPLC chart of FGDNLKAGP is shown in Figure 7 As shown in the mass spectrum Figure 8 As shown, it can be seen that its molecular weight purity is 97.33%, and the mass-to-nuclear ratio [M+H] + The molecular weight is 918.45 and 917.45.

[0021] Example 5 (1) The water-soluble walnut polypeptide FGDNLKAGP prepared in Example 4 was prepared into a 13.3 mg / ml aqueous solution, the pH was adjusted to 8.0, and centrifuged at 13000 rpm and 4°C for 5 min, and the supernatant was collected for later use; (2) Weigh 1 mg of Cy7, add 50 ul of DMSO to dissolve it, then add 150 ul of the supernatant in step (1), and slowly mix for 2 h to obtain a reaction solution containing FGDNLKAGP-Cy7 conjugate; (3) The reaction solution was separated by HPLC using a COSMOSIL column (4.6 mm × 250 mm, 5 µm); the mobile phase was acetonitrile (A) and water (B) solution containing 0.1% TFA, and the gradient elution program was 0.0 min: 15% solvent A, 85% solvent B; 25.0 min: 40% solvent A, 60% solvent B; 25.1 min: 100% solvent A, 0% solvent B; 30.0 min, stop; the detection wavelength was 220 nm, the flow rate was 1.0 ml / min, and the injection volume was 20 µl. The HPLC chart of the separation and purification of FGDNLKAGP-Cy7 conjugate is shown in Figure 2. Fig. 9 As shown; where the red line: FGDNLKAGP; the green line: Cy7; the blue line: the coupling mixture, where the arrow points to the FGDNLKAGP-Cy7 coupling, which is collected separately, concentrated and dried, and the structural formula is shown below; The mass spectrum of FGDNLKAGP-Cy7 conjugate is shown in Fig.10 As shown, the mass-to-nuclear ratio [M+H]+ is 1583.5 and the molecular weight is 1582.5.

[0022] (4) The FGDNLKAGP-Cy7 conjugate was freeze-dried, dissolved in water, and injected into the mouse tail vein. The conjugate was then observed using a small animal in vivo imaging device (to eliminate the interference of fluorescence from tissues outside the brain and peripheral blood, brain tissue (mainly the cerebrum and cerebellum, olfactory bulb and medulla oblongata) was removed to compare fluorescence intensity. The brain fluorescence image is shown in Figure 2. Fig.11 As shown; among them, the left picture: 1. Normal saline; 2. Cy7, the fluorescence intensity decreases from the center of the brain to the outside (black is the highlighted area); the right picture: 1. Normal saline; 2. FGDNLKAGP-Cy7 conjugate, the fluorescence intensity decreases from the cerebellum to the cerebrum (yellow is the highlighted area), Note: 1 is the dorsal side of the brain tissue; 2 and 3 are the midsagittal sections of the brain tissue: cut along the midline of the brain tissue to divide the brain tissue into left and right halves. The top of the picture is the front side of the brain tissue, and the bottom is the back side of the brain tissue. Fig.11 It can be seen that compared with the diffusion of Cy7 fluorescence intensity from the center of the whole brain to the outside, the center of fluorescence intensity of the sample with FGDNLKAGP moved to the cerebellum below. It can be seen that the presence of FGDNLKAGP changed the distribution of Cy7 in the mouse brain, which can pass through the blood-brain barrier and be relatively enriched in the cerebellum of the mouse brain.

[0023] Example 6 (1) The water-soluble walnut polypeptide FGDNLKAGP is expressed as a chemical molecular structure string in the form of SMILES: N[C@@H](Cc1ccccc1)C(=O)NCC(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N1[C@@H](CCC1)C(=O)O. The structure was verified to be correct by the online tool PubChem Sketcher. The structural formula of FGDNLKAGP is shown below. Select the database taking Swisstargetprediction as an example, input the SMILES expression of FGDNLKAGP, and Swisstargetprediction predicts the binding target of FGDNLKAGP as follows: Fig.12 As shown, 102 possible binding targets were predicted among the reported human targets.

[0024] (2) Prioritize the analysis of water-soluble walnut peptide FGDNLKAGP targets with high probability and close to the research direction to discover new targets and potential activity development directions. For example: 1) Beta-secretase 1 (BACE1), a membrane-bound aspartic protease, is involved in the pathological process of Alzheimer's disease (AD). BACE1 is expressed in the cerebellum. Although it is not the main area of ​​lesions, studies have shown that the neural network in the cerebellum may be affected by Aβ peptide accumulation or downstream neurotoxicity; if water-soluble walnut peptides and their optimized derivatives have the ability to bind to BACE1, it can be studied whether they affect cerebellar function by regulating the production of Aβ; cerebellar atrophy (such as spinocerebellar ataxia) may involve protein metabolism disorders, and inhibiting the activity of BACE1 may indirectly improve the functions related to motor coordination and memory in the cerebellum.

[0025] 2) HLA class I histocompatibility antigen A-3 (HLA-A), a subtype of human leukocyte antigen (HLA) class I molecules, participates in antigen presentation in the immune system and presents intracellular antigens (such as viruses and tumor antigens) to CD8+ T cells; HLA-A is an important component of immune checkpoints and tumor immunity. Immune-mediated cerebellar diseases include paraneoplastic cerebellar degeneration (PCA), a cerebellar degeneration caused by autoimmunity, which may involve abnormal immune responses mediated by HLA-A; multiple sclerosis (MS), cerebellar white matter is often affected, and HLA-A is involved in the immune process of MS; in addition, in cerebellar diseases (especially neuroinflammatory diseases), excessive or abnormal expression of HLA-A may mediate immune attacks. If the peptide and its optimized derivatives can bind to HLA-A, it may regulate the immune response and be used to inhibit abnormal immune responses or treat cerebellar inflammatory diseases. In addition to human targets, there are target databases for other species. Moreover, these databases are constantly being supplemented and updated, and there is still potential for research and exploration of targets and efficacy.

[0026] As brain science research deepens, the water-soluble walnut polypeptide FGDNLKAGP will be used as a model probe tool for studying the mechanisms of polypeptides crossing the blood-brain barrier and targeting the cerebellum, promoting research on brain nutrition or disease treatment, and facilitating the development of cerebellar nutrition products or pharmaceutical preparations.

Claims

1. A water-soluble walnut polypeptide that passes through the blood-brain barrier, characterized in that: The amino acid sequence of the water-soluble walnut polypeptide that passes through the blood-brain barrier is FGDNLKAGP.

2. A method for preparing a water-soluble walnut polypeptide that passes through the blood-brain barrier according to claim 1, characterized in that: The water-soluble walnut polypeptide that passes through the blood-brain barrier is obtained by solid phase chemical synthesis or extracted from walnut meal.

3. Use of the water-soluble walnut polypeptide that passes through the blood-brain barrier according to claim 1 in the preparation of brain nutritional supplements or health products.

4. Use of the water-soluble walnut polypeptide that passes through the blood-brain barrier according to claim 1 in the preparation of drugs or detection reagents that pass through the blood-brain barrier.

5. Use of the water-soluble walnut polypeptide that passes through the blood-brain barrier according to claim 1 in preparing a probe.

6. The use according to claim 5, characterized in that: The probe is a positioning probe targeting cerebellar tissue.

7. The use according to claim 5, characterized in that: The water-soluble walnut polypeptide that passes through the blood-brain barrier is coupled with a fluorescent group to prepare a probe.

8. The use according to claim 7, characterized in that: The fluorescent group is one of Cy5, Cy7 and ICG.

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