Water-soluble walnut polypeptide capable of passing through blood-brain barrier, preparation method and application thereof

Abstract

The application discloses a water-soluble walnut polypeptide capable of penetrating through a blood-brain barrier and a preparation method and application thereof. The amino acid sequence of the water-soluble walnut polypeptide capable of penetrating through the blood-brain barrier is FGDNLKAGP. The water-soluble walnut polypeptide capable of penetrating through the blood-brain barrier is obtained through a solid-phase chemical synthesis method or extraction from walnut meal, and can be used for preparing brain nutrition supplement medicines, drugs or detection reagents capable of penetrating through the blood-brain barrier and probes. The water-soluble walnut polypeptide has the ability of penetrating through the blood-brain barrier, can penetrate through the blood-brain barrier to enter the brain to exert its functional role, and is used for preparing the drugs or detection reagents capable of penetrating through the blood-brain barrier and the positioning probes targeting brain tissues. The water-soluble walnut polypeptide has good targeting property and is safe and reliable.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide product technology, specifically relating to a water-soluble walnut polypeptide that crosses the blood-brain barrier and its preparation method and application. Background Technology

[0002] The blood-brain barrier (BBB) ​​restricts the entry of most drugs into the central nervous system, resulting in poor or even ineffective treatment of various brain diseases. Therefore, discovering effective substances that can cross the BBB will be beneficial for improving brain nutrition and preventing related central nervous system diseases. Currently, only some small molecules can cross the BBB; most substances, especially some large molecules, cannot cross it and require endogenous receptors to mediate their delivery into the brain. The cerebellum, as an important component of the human brain, plays a crucial role not only in motor coordination and balance but is also closely related to cognitive function, emotion regulation, and certain neurological diseases. However, our understanding of cerebellar function remains very limited, especially regarding the pathogenesis of cerebellar-related diseases and potential therapeutic targets, where much remains unknown. Furthermore, due to the high selectivity of the BBB, very few substances can effectively cross it and target the cerebellum. Existing research focuses primarily on lipid-soluble small molecules, while systematic exploration of the cross-barrier mechanisms of water-soluble peptides and other large molecules is lacking.

[0003] Walnuts are a widely cultivated oilseed crop in my country. Currently, walnut oil is typically obtained through pressing to develop high-value walnut products, leaving a large amount of walnut meal that is either used as animal feed or discarded, resulting in significant waste. The main component of walnut meal, protein, can be hydrolyzed into peptides with broad physiological activity. Walnuts are traditionally used for brain health. Patent CN 115109818A discloses a walnut peptide that can cross the blood-brain barrier, obtained through a two-step enzymatic hydrolysis of walnut meal. The resulting walnut peptide possesses unique permeability properties or contains active peptides with strong membrane permeability, exhibiting memory-improving effects and potential applications in the preparation of corresponding foods, nutritional supplements, or pharmaceuticals.

[0004] However, the walnut peptides obtained in the aforementioned patents are enzymatic hydrolysis products with relatively complex compositions. It is unknown which component(s) specifically exert their effect, and an excessive number of components may hinder the mediating effect. Whether walnut polypeptides can cross the blood-brain barrier to exert their nutritional and health benefits, or even their carrier or mediating effects, remains unclear. Therefore, in-depth research on walnut polypeptides is not only crucial for improving the utilization value of walnuts but also holds significant importance for the study of polypeptide compounds. Summary of the Invention

[0005] To address the above problems, this invention provides a water-soluble walnut polypeptide that crosses the blood-brain barrier, its preparation method, and its application. The water-soluble walnut polypeptide can cross the blood-brain barrier and can be used for brain nutrition supplementation and as a localization probe targeting brain tissue.

[0006] This invention is achieved through the following technical solution:

[0007] A water-soluble walnut polypeptide that crosses the blood-brain barrier, wherein the amino acid sequence of the water-soluble walnut polypeptide that crosses the blood-brain barrier is FGDNLKAGP.

[0008] In this invention, the water-soluble walnut polypeptide that crosses the blood-brain barrier is obtained by solid-phase chemical synthesis or by extraction from walnut meal.

[0009] In this invention, the water-soluble walnut polypeptide that crosses the blood-brain barrier is used in the preparation of brain nutritional supplements.

[0010] In this invention, the water-soluble walnut polypeptide that crosses the blood-brain barrier is used in the preparation of drugs or diagnostic reagents that cross the blood-brain barrier.

[0011] In this invention, the water-soluble walnut polypeptide that crosses the blood-brain barrier is used in the preparation of probes.

[0012] Furthermore, the probe is a localization probe that targets brain tissue.

[0013] Furthermore, the water-soluble walnut polypeptide that crosses the blood-brain barrier is coupled with a fluorescent group.

[0014] Furthermore, the fluorescent group is one of Cy5, Cy7, and ICG.

[0015] Beneficial effects achieved by the present invention

[0016] The water-soluble walnut polypeptide provided by this invention has the ability to cross the blood-brain barrier, and can enter the brain to exert its function. The water-soluble walnut polypeptide is derived from walnuts, can be artificially synthesized, and has high safety.

[0017] The ability of water-soluble walnut polypeptides to cross the blood-brain barrier in this invention enables them to be used in the study of localization probes targeting brain tissue and in the preparation of drugs or detection reagents that cross the blood-brain barrier, thereby accelerating the study of brain nutrition or disease pathogenesis and facilitating the development of cerebellar nutritional products or drug formulations. Attached Figure Description

[0018] Figure 1 In vivo fluorescence imaging of mice injected with crude walnut peptide-Cy7;

[0019] Figure 2 This is a molecular sieve segmentation diagram of crude walnut peptides.

[0020] Figure 3 In vivo fluorescence imaging of mice after tail vein injection of crude walnut peptides separated by molecular sieve.

[0021] Figure 4 Prepare a liquid chromatogram for molecular sieve 3;

[0022] Figure 5 In vivo fluorescence imaging of each of the three components of the molecular sieve;

[0023] Figure 6 The total ion chromatogram of peak 9 in Example 3;

[0024] Figure 7 The HPLC chromatogram of FGDNLKAGP;

[0025] Figure 8 The mass spectrum of FGDNLKAGP;

[0026] Figure 9 HPLC purification chromatogram of FGDNLKAGP-Cy7 conjugate;

[0027] Figure 10 Mass spectrum of FGDNLKAGP-Cy7 conjugate;

[0028] Figure 11 Fluorescence imaging of mouse brains containing FGDNLKAGP-Cy7 conjugate and Cy7;

[0029] Figure 12 Predict the binding target of FGDNLKAGP for Swisstargetprediction. Detailed Implementation

[0030] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0031] Example 1

[0032] (1) Crush the walnut meal to obtain walnut powder;

[0033] (2) Weigh 10 g of walnut powder, add 90 ml of water, add 1% of commercially available plant protein hydrolysis complex enzyme (Nanning Pangbo Bioengineering Co., Ltd.), put it in a water bath constant temperature shaker, keep it at 50℃ for 6 h, centrifuge at 8000 r / min for 10 min, take the supernatant, evaporate it at 80℃ and 100 r / min, and freeze-dry the resulting extract to obtain crude walnut peptide;

[0034] (3) Prepare an aqueous solution of crude walnut peptide at 13.3 mg / ml, adjust the pH to 8 with NaOH, centrifuge at 4℃ and 18000 r / min for 5 min, and take the supernatant for later use;

[0035] (4) Accurately weigh 1 mg Cy7 (Sulfo-Cy7SE, Beijing Fubaike) and dissolve it in 50 μl of dimethyl sulfoxide (DMSO). Add 150 μl of the supernatant from step (3), mix well, and react overnight at 4°C in the dark with shaking to obtain crude walnut peptide-Cy7 labeled solution. The blank aqueous solution without crude walnut peptide is treated in the same way to obtain the blank Cy7 labeled solution.

[0036] Nude mice were anesthetized by intraperitoneal injection of 300 μL (215 mg / kg) of 2% sodium pentobarbital. Then, 175 μL (0.5 mg / g) of physiological saline, Cy7 blank labeling solution, and crude walnut peptide-Cy7 labeling solution were injected via tail vein. The mice were placed prone in the recording chamber of a small animal multispectral in vivo imaging system. Excitation wavelengths of 700–770 nm were used to record fluorescence emission within the animals after the injection of the labeling solutions. The results are as follows: Figure 1 As shown, Figure 1 The top, middle, and bottom images are in vivo fluorescence images of mice injected with saline, Cy7, and crude walnut peptide-Cy7, respectively; by Figure 1 It was found that the fluorescence intensity in the brains of mice injected with crude walnut peptide was significantly increased.

[0037] Example 2

[0038] Crude walnut peptides were prepared according to the method in Example 1. The crude walnut peptides were dissolved in 0.02M PBS (pH 7.2), filtered through a 0.22µm filter membrane, and loaded onto a molecular sieve at a flow rate of 3ml / min. The sample was then separated into five segments (molecular sieve 1, 2, 3, 4, and 5) using a molecular sieve (200PE, 30×2.6cm). The molecular sieve segmentation diagram of the crude walnut peptides is shown below. Figure 2 As shown, the five isolated product segments were labeled with Cy7 (denoted as molecular sieve 1-Cy7, molecular sieve 2-Cy7, molecular sieve 3-Cy7, molecular sieve 4-Cy7, and molecular sieve 5-Cy7, respectively), and injected into the tail vein of mice. Using Cy7 and blank (physiological saline) as controls, in vivo fluorescence imaging images of each segment of crude walnut peptide after tail vein injection were recorded. The results are as follows. Figure 3As 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), molecular sieve 3 was selected for liquid-phase separation because of its relatively high fluorescence intensity selectivity in the cerebellar region of the brain.

[0039] Example 3

[0040] Example 1: The crude walnut peptide fraction (molecular sieve 3) separated by molecular sieve was subjected to liquid phase separation. The preparative liquid chromatogram is shown below. Figure 4 As shown, by Figure 4 Nine peaks were separated. These nine peaks (phases 1, 2, 3, 4, 5, 6, 7, 8, and 9) were labeled with Cy7 and injected via the tail vein of mice. The in vivo fluorescence imaging is shown below. Figure 5 As shown, from left to right, the samples 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, and blank (physiological saline). Based on the relatively good peak separation, the sample with peak 9 was selected for liquid chromatography-mass spectrometry analysis.

[0041] Example 4

[0042] The total ion chromatogram of peak 9 in Example 3 is as follows: Figure 6 As shown, 76 peptide information was obtained by matching the database using 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 the representative peptide with the amino acid sequence of SEQ ID NO.1: FGDNLKAGP was selected for chemical synthesis.

[0043] Example 5

[0044] Example 4 obtained SEQ ID NO.1: FGDNLKAGP, which was synthesized by solid-state synthesis, specifically as follows:

[0045] (1) Weigh 100 mg Rink Amide Resin (0.01 mmol scale), place it in a reaction flask, add 3 mL DMF (dimethylformamide), soak for 30 minutes to allow it to fully swell, then add 3 mL of deprotection solution (20% piperidine DMF solution), shake the reaction for 10 minutes, and then wash the resin 3 times with DMF, 3 mL each time, to ensure the removal of Fmoc groups and residual reagents;

[0046] (2) Couple 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 for 1 hour, and then wash the resin 3 times with DMF, 3 mL each time; repeat the above amino acid coupling steps, coupling each amino acid in the order of Gly (G), Ala (A), Lys (Boc) (K), Leu (L), Asn (Trt) (N), Asp (OtBu) (D), Gly (G), Phe (F);

[0047] (3) Mix the resin with 3 mL of cutting solution (95% TFA (trifluoroacetic acid) + 2.5% H2O + 2.5% TIS (volume ratio)) to cut the peptide from the resin. Shake the reaction for 2 hours, filter the reaction solution, collect the filtrate and add 10 times the volume of cold ether to precipitate the peptide. Centrifuge to precipitate, remove the supernatant, wash once with cold ether and dry to obtain crude peptide;

[0048] (4) Using a reverse-phase C18 column, gradient elution (0-100% ACN in H2O, containing 0.1% TFA), monitoring wavelength 214 nm, collecting the target peak, and lyophilizing to obtain a white powder, which is the water-soluble walnut polypeptide with the amino acid sequence FGDNLKAGP; the HPLC chromatogram of FGDNLKAGP is shown below. Figure 7 As shown, the mass spectrum is as follows Figure 8 As shown, its molecular weight purity is 97.33%, and its mass-to-nucleus ratio is [M+H]. + It has a value of 918.45 and a molecular weight of 917.45.

[0049] Example 5

[0050] (1) Prepare a 13.3 mg / ml aqueous solution of the water-soluble walnut polypeptide FGDNLKAGP obtained in Example 4, adjust the pH to 8.0, centrifuge at 13000 rpm and 4℃ for 5 min, and take the supernatant for later use;

[0051] (2) Weigh 1 mg of Cy7, add 50 μL of DMSO to dissolve it, then add 150 μL of the supernatant from step (1), mix slowly for 2 h to obtain a reaction solution containing FGDNLKAGP-Cy7 conjugate;

[0052] (3) The reaction solution was separated by HPLC using a COSMOSIL packed column (4.6 mm × 250 mm, 5 µm); the mobile phase was acetonitrile (A) and water (B) containing 0.1% TFA, with a gradient elution program of 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 chromatogram of the separation and purification of the FGDNLKAGP-Cy7 conjugate is shown below. Figure 9 As shown; where red line: FGDNLKAGP; green line: Cy7; blue line: coupling mixture, where the arrow points to FGDNLKAGP-Cy7 coupling, which is collected, concentrated and dried separately, and its structural formula is shown below;

[0053]

[0054] The mass spectrum of the FGDNLKAGP-Cy7 conjugate is shown below. Figure 10 As shown, the mass-to-nucleus ratio [M+H]+ is 1583.5, and the molecular weight is 1582.5.

[0055] (4) After lyophilizing the FGDNLKAGP-Cy7 conjugate, it was dissolved in water and injected into the tail vein of mice. The images were then observed using a small animal in vivo imaging system. (To eliminate fluorescence interference from external brain tissues and peripheral blood, brain tissue (mainly the cerebrum and cerebellum, excluding the olfactory bulb and medulla oblongata) was removed, and the fluorescence intensity was compared. The brain fluorescence images are shown below.) Figure 11 As shown; in the left image: 1. physiological saline; 2. Cy7, fluorescence intensity decreases from the center of the brain outwards (black indicates the highlighted area); in the right image: 1. physiological saline; 2. FGDNLKAGP-Cy7 conjugate, fluorescence intensity decreases from the cerebellum towards the cerebrum (yellow indicates the highlighted area). Note: 1 is the dorsal side of the brain tissue; 2 and 3 are midsagittal sections of the brain tissue: cut along the midline of the brain tissue, dividing the brain tissue into left and right halves. The upper part of the image shows the anterior side of the brain tissue, and the lower part shows the posterior side of the brain tissue. Figure 11It can be seen that, compared to the diffusion of Cy7 fluorescence intensity from the center of the whole brain to the outer edge, the fluorescence intensity center of the sample with increased FGDNLKAGP shifted to the lower cerebellum. This shows that the presence of FGDNLKAGP altered the distribution of Cy7 in the mouse brain, allowing it to cross the blood-brain barrier and accumulate relatively in the cerebellum of the mouse brain.

[0056] Example 6

[0057] (1) The water-soluble walnut polypeptide FGDNLKAGP is represented as the chemical molecular structure string form of SMILES:

[0058] 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 of FGDNLKAGP has been verified as correct using the online tool PubChem Sketcher. The structure is shown below. Selecting the database using Swisstargetprediction as an example, and inputting the SMILES expression for FGDNLKAGP, Swisstargetprediction predicts the binding target of FGDNLKAGP as follows. Figure 12 As shown, 102 potential binding targets were predicted among the reported human targets.

[0059]

[0060] (2) Prioritize the analysis of water-soluble walnut peptides FGDNLKAGP that have a high probability of relevance and are close to the research direction, in order to discover new targets and potential activity development directions. For example:

[0061] 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 primary area of ​​lesion. However, studies have shown that the neural networks in the cerebellum may be affected by the accumulation of Aβ peptides or downstream neurotoxicity. If water-soluble walnut peptides and their optimized derivatives have the ability to bind to BACE1, it is possible to investigate whether they affect cerebellar function by regulating Aβ production. Cerebellar atrophy (such as spinocerebellar ataxia) may involve protein metabolism disorders, and inhibiting BACE1 activity may indirectly improve cerebellar functions related to motor coordination and memory.

[0062] 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, presenting intracellular antigens (such as viral and tumor antigens) to CD8+ T cells; HLA-A is an important component of immune checkpoints and tumor immunity. Immune-mediated cerebellar diseases include cerebellar ataxia with antibody syndrome (PCA), a cerebellar degenerative disease caused by autoimmunity, which may involve abnormal immune responses mediated by HLA-A; multiple sclerosis (MS), in which the cerebellar white matter is often affected, HLA-A participates in the immune process of MS; in addition, in cerebellar diseases (especially neuroinflammatory diseases), overexpression or abnormal expression of HLA-A may mediate immune attack. If peptides and their optimized derivatives can bind to HLA-A, they may modulate immune responses, and be used to suppress abnormal immune responses or treat cerebellar inflammatory diseases. In addition to human targets, there are target databases for other species, and these databases are constantly being updated and expanded, with potential targets and effects still to be explored.

[0063] As brain science research deepens, the water-soluble walnut polypeptide FGDNLKAGP can be used as a model probe tool for studying the mechanism by which polypeptides cross the blood-brain barrier and target the cerebellum. This will help advance research on brain nutrition or disease treatment and facilitate the development of cerebellar nutritional products or drug formulations.

Claims

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

2. A method for preparing the water-soluble walnut polypeptide that crosses the blood-brain barrier as described in claim 1, characterized in that, The water-soluble walnut polypeptide that crosses the blood-brain barrier is obtained through solid-phase chemical synthesis or extraction from walnut meal; wherein, the method for extracting the water-soluble walnut polypeptide from walnut meal includes: The walnut meal is crushed to obtain walnut powder; Weigh 10g of walnut powder, add 90ml of water, add 1% of commercially available plant protein hydrolysis complex enzyme, place in a water bath constant temperature shaker, keep at 50℃ for 6h, centrifuge at 8000r / min for 10min, take the supernatant, evaporate at 80℃ and 100r / min, and freeze-dry the obtained extract to obtain crude walnut peptide. The crude walnut peptide was dissolved in 0.02M, pH 7.2 PBS, filtered through a 0.22µm filter membrane, loaded at a flow rate of 3ml / min, and separated into 5 products by passing through a 200PE, 30×2.6cm molecular sieve. The component with relatively high fluorescence intensity in the cerebellum region of the brain was selected to obtain the third crude walnut peptide. The crude walnut peptide in the third stage was subjected to liquid phase separation, and nine peak components were separated. The component with the relatively good separation angle was selected to obtain peak component number 9. The components of peak 9 were detected by total ion chromatography and analyzed by physicochemical properties of peptide sequences, and water-soluble walnut polypeptides were obtained through screening.

3. The application of the water-soluble walnut polypeptide that crosses the blood-brain barrier as described in claim 1 in the preparation of a probe, wherein the probe is used as a delivery carrier for polypeptides across the blood-brain barrier, and the water-soluble walnut polypeptide that crosses the blood-brain barrier is prepared by coupling with a fluorescent group to obtain the probe.

4. The application according to claim 3, characterized in that, The probe described is a localization probe that targets cerebellar tissue.

5. The application according to claim 3, characterized in that, The fluorescent group is one of Cy5, Cy7, and ICG.

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