A walnut-derived peptide capable of penetrating the blood-brain barrier to improve learning and memory and its application

Abstract

The present invention discloses a walnut-derived peptide that can penetrate the blood-brain barrier to improve learning and memory. Its amino acid sequence is Thr-Trp-Leu-Pro-Tyr-Pro-Arg (TWLPYPR). The present invention also discloses its application in the preparation of drugs for neuroprotection and improvement of learning and memory. The walnut-derived peptide TWLPYPR of the present invention has high antioxidant activity, can penetrate the blood-brain barrier of the body, directly enter the brain and act on the central nervous system, protect nerve cells from oxidative damage, and improve the learning and memory of the body. The walnut-derived peptide TWLPYPR of the present invention belongs to a natural-derived peptide, has the characteristics of high activity, high bioavailability and high safety, can be applied to the preparation of drugs for neuroprotection and improvement of learning and memory, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a walnut-derived peptide capable of penetrating the blood-brain barrier to improve learning and memory, and its application in the preparation of drugs for neuroprotection and improvement of learning and memory. Background Art

[0002] Oxidative stress is that the endogenous antioxidant system in the body cannot effectively scavenge the accumulated free radicals, resulting in an imbalance between the antioxidant and oxidation systems, and is considered an important factor in aging and diseases. As the most metabolically active organ in the body, the brain tissue is extremely vulnerable to oxidative stress, and oxidative damage increases with age. Excessive reactive oxygen species (ROS) cause oxidative damage to cellular biomolecules, induce neuroinflammation and apoptosis, cause DNA breaks, protein denaturation and membrane phospholipid peroxidation, lead to abnormal synaptic plasticity and neuronal damage, and ultimately result in decreased memory and cognitive ability. In severe cases, it gradually develops into dementia. Research has confirmed that neuroprotective factors can significantly improve the learning and memory levels of model animals by increasing the activity of antioxidant enzymes to scavenge excessive ROS. Therefore, targeting the maintenance of the body's oxidative defense balance can be an effective strategy for neuroprotection and improvement of learning and memory decline.

[0003] With the aggravation of the aging of the world's population, the occurrence and development of oxidative stress have been exacerbated. Central nervous system (CNS) diseases such as Alzheimer's disease, Parkinson's disease, anxiety, depression, epilepsy, amyotrophic lateral sclerosis, etc. have become the second largest category of diseases threatening human life. The blood-brain barrier is mainly composed of brain endothelial cells, astrocytes and pericytes, and plays a crucial role in maintaining the physiological stability of the brain tissue and protecting the CNS from harmful substances in the blood stream. The physiological basis such as high electrical resistance, high efflux and low vesicle transport between brain endothelial cells hinders the entry of almost all macromolecules and 98% of small molecules into the CNS, which is a difficult problem that needs to be solved urgently in the development of central nerve function factors. Therefore, overcoming the blood-brain barrier and targeting the delivery to the central nervous system is the primary problem to be solved to improve the bioavailability of natural-source antioxidant active peptides and achieve the neuroprotective function of brain tissue.

[0004] It is currently believed that food intake is an important source of exogenous antioxidants, and the dietary intervention effects of bioactive peptides as functional nutrients in chronic diseases have been widely reported. Antioxidant active peptides have a wide range of sources, are safe and effective, have strong biological activities, low drug resistance, can directly enter cells to exert biological activities, and show considerable therapeutic potential in oxidative stress-related diseases. As a by-product after walnut oil extraction, walnut meal has a protein content as high as 52%, making it an important source for obtaining antioxidant active peptides. The research of this invention shows that the derivative peptide TWLPYPR of walnut antioxidant active peptide TWLPLPR can penetrate the blood-brain barrier, protect nerve cells from oxidative damage, improve learning and memory, and can be used as an ingredient in drug formulations to play an important role in the pharmaceutical field. Summary of the Invention

[0005] The purpose of this invention is to provide a walnut-derived peptide that can penetrate the blood-brain barrier and improve learning and memory, and its application in the preparation of drugs for neuroprotection and improvement of learning and memory. It belongs to natural-derived peptides, has high safety, and has functions of high antioxidant activity, being able to penetrate the blood-brain barrier, protecting nerve cells from oxidative damage, and improving learning and memory.

[0006] This invention adopts the following technical solutions:

[0007] In the first aspect of this invention, a walnut-derived peptide that can penetrate the blood-brain barrier and improve learning and memory is provided, and its amino acid sequence is Thr-Trp-Leu-Pro-Tyr-Pro-Arg (TWLPYPR).

[0008] In the second aspect of this invention, the application of the above-mentioned walnut-derived peptide in the preparation of drugs for neuroprotection and improvement of learning and memory is provided.

[0009] The beneficial effects of this invention are as follows:

[0010] Using walnut peptide TWLPLPR as a template, after amino acid substitution, the derivative peptide TWLPYPR with better antioxidant activity and better learning and memory improvement activity is obtained through in vitro BBB model and antioxidant activity screening. The walnut-derived peptide TWLPYPR of this invention has high antioxidant activity, can penetrate the blood-brain barrier of the body, directly enter the brain and act on the central nervous system, protect nerve cells from oxidative damage, and improve the learning and memory of the body. The walnut-derived peptide TWLPYPR of this invention belongs to natural-derived peptides, has the characteristics of high activity, high bioavailability, and high safety, can be applied to the preparation of drugs for neuroprotection and improvement of learning and memory, and has good application prospects. Description of the Drawings

[0011] Figure 1Graphs showing the ABTS and ORAC assay results of walnut peptide TWLPLPR and walnut-derived peptide TWLPYPR; a shows the ABTS assay results; b shows the ORAC assay results.

[0012] Figure 2 Graph showing the protective effect of walnut peptide TWLPLPR and walnut-derived peptide TWLPYPR on H2O2-damaged HT22 cells.

[0013] Figure 3 Graph showing the imaging results of walnut-derived peptide TWLPYPR in the mouse brain; a shows the imaging graph of TWLPYPR in the brain after intragastric administration to mice; b shows the imaging graph of TWLPYPR in the brain after tail vein injection to mice.

[0014] Figure 4 Graph of the Morris water maze experiment in mice; a shows the swimming path graph of mice in the location navigation experiment; b shows the bar graph of escape latency; c shows the bar graph of the number of times each group of mice crossed the original platform in the spatial exploration experiment. Specific implementation mode

[0015] The present invention will be further explained below in conjunction with embodiments and drawings, but the embodiments do not limit the present invention in any way.

[0016] The walnut-derived peptide TWLPYPR was obtained by amino acid substitution based on the walnut peptide TWLPLPR and screening through an in vitro BBB model and antioxidant activity. The walnut peptide TWLPLPR and walnut-derived peptide TWLPYPR involved in the following examples were directly synthesized by a commercial company. For the specific synthesis method, see the reference (Huang Bei. Research progress in solid-phase synthesis of polypeptides [J]. Henan Chemical Industry, 2013, 30(01): 28-30+58.).

[0017] Example 1: Determination of the antioxidant activity of walnut-derived peptide TWLPYPR

[0018] 1) Determination of ABTS radical scavenging ability: An ABTS radical stock solution was prepared by mixing 7 mmol / L ABTS aqueous solution and 2.49 mmol / L potassium persulfate aqueous solution at a volume ratio of 1:1 and allowing it to stand in the dark at 4 °C for 12 h. When in use, the concentration was adjusted with 5 mmol / L PBS at pH 7.4 to make the absorbance of the ABTS radical stock solution 0.700 ± 0.002 at a wavelength of 734 nm to obtain the ABTS radical working solution. Take 10 μL of the aqueous solution of walnut-derived peptide TWLPYPR with a concentration of 100 μM, 10 μL of the aqueous solution of walnut peptide TWLPLPR with a concentration of 100 μM, and 10 μL of the aqueous solution of glutathione (GSH) with a concentration of 100 μM. Among them, the 100 μM GSH aqueous solution was used as the control group, and they were respectively added to a black 96-well plate, and then 190 μL of the ABTS radical working solution was added respectively. After reacting accurately for 6 min, the absorbance value (A1) was measured at a wavelength of 734 nm using a microplate reader. The clearance rate calculation formula is as follows:

[0019]

[0020] In the formula: A0 is the absorbance value when distilled water is used to replace the aqueous solution of walnut-derived peptide or walnut peptide or GSH aqueous solution in the ABTS radical working solution (without adding walnut-derived peptide, walnut peptide or GSH); A1 is the absorbance value when the aqueous solution of walnut-derived peptide or walnut peptide or GSH aqueous solution is added to the ABTS radical working solution; A2 is the absorbance value of the solution using 5 mmol / L PBS solution to replace the ABTS radical working solution (adding walnut-derived peptide, walnut peptide or GSH).

[0021] 2) Determination of oxygen radical absorbance capacity (ORAC): In a black 96-well plate, add 25 μL of 75 mmol / L PBS buffer at pH 7.4, 25 μL of Trolox standard solution (concentrations are 12.5 μM, 25 μM, 50 μM, and 100 μM respectively, dissolved in 75 mmol / L PBS buffer at pH 7.4), or 25 μL of PBS solution of walnut-derived peptide, or 25 μL of PBS solution of walnut peptide, or 25 μL of PBS solution of GSH (the concentrations of PBS solutions of walnut-derived peptide, walnut peptide, and GSH are all 100 μM, and all are dissolved in 75 mmol / L PBS buffer at pH 7.4). Set three parallel wells for each sample. Place the black 96-well plate in a microplate reader and shake for 5 s, then incubate at 37 °C for 10 min. Use a multi-channel pipette to add 150 μL of 63 mmol / L sodium fluorescein solution (prepared with 75 mmol / L PBS buffer at pH 7.4) to each well simultaneously, shake for 5 s, and incubate at 37 °C for 20 min. Use a multi-channel pipette to quickly add 25 μL of 6 mmol / L 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH, freshly prepared with 75 mmol / L PBS buffer at pH 7.4) to each well. Set up two control groups at the same time. One is the +AAPH control group, in which the Trolox standard solution or the PBS solution of walnut-derived peptide or the PBS solution of walnut peptide or the PBS solution of GSH is replaced with an equal volume of 75 mmol / L PBS buffer at pH 7.4. The other is the -AAPH control group, in which the Trolox standard solution, the PBS solution of walnut-derived peptide or the PBS solution of walnut peptide or the PBS solution of GSH is replaced with an equal volume of 75 mmol / L PBS buffer at pH 7.4, and at the same time AAPH is also replaced with an equal volume of 75 mmol / L PBS buffer at pH 7.4. Both groups are placed in a microplate reader and shaken for 5 s (set three parallel wells for each group). Set the excitation wavelength at (485 ± 20) nm and the emission wavelength at (530 ± 20) nm. Keep the whole system at 37 °C and measure the fluorescence intensity every 2 min until the measured fluorescence intensity reaches 5% of the initial fluorescence intensity (set 60 cycles in this experiment). The relative fluorescence intensity is obtained by comparing the fluorescence intensity of each well at different time points measured in the experiment with the fluorescence intensity at the initial time (the initial fluorescence intensity value is set to 1). The area under the fluorescence decay curve (AUC) is calculated using the approximate integral method:

[0022] AUC = 0.5 × [2 × (f0 + f1… + f n-1 + f n ) - f0 - f n × Δt

[0023] In the formula, f nThe relative fluorescence intensity at the nth measurement point; △t is the time interval of 2 min between adjacent time points.

[0024] Using the Trolox concentration as the abscissa and the AUC trolox value as the ordinate, plot the Trolox standard curve.

[0025] The measurement results are expressed as ORAC values. The ORAC values of the test samples are expressed in μmol TE / g or μmol TE / mL (indicating how many milligrams of the antioxidant capacity of the standard product is represented by the antioxidant capacity of each gram or milliliter of the sample).

[0026] ORAC value = [(AUC 样品 - AUC 空白 ) / (AUC Trolox - AUC 空白 )] × C Trolox / C 样品 .

[0027] Among them, AUC 样品 is the result of the PBS solution of walnut-derived peptide or the PBS solution of walnut peptide or the PBS solution of GSH; AUC 空白 is the result of the +AAPH control group; AUC Trolox is the result of adding the Trolox standard solution (concentration 100 μM); C Trolox is the molar concentration of Trolox (100 μM); C 样品 is the molar concentration of walnut-derived peptide or walnut peptide or GSH, which is 100 μM. Among them, the Trolox standard curve is y = 0.0924x + 39.144, R 2 = 0.996, the abscissa x is the Trolox molar concentration (12.5 μM, 25 μM, 50 μM, and 100 μM), and the ordinate y is AUC Trolox .

[0028] As Figure 1 shown, when the concentration of walnut-derived peptide TWLPYPR is 100 μM, its ABTS scavenging rate reaches 29.71%, significantly higher than 10.35% of GSH and also significantly higher than 18.75% of walnut peptide TWLPLPR; the ORAC value reaches 1727.36 μmol TE / g, significantly higher than 917.66 μmol TE / g of GSH and even more significantly higher than 752.05 μmol TE / g of walnut peptide TWLPLPR. The above results indicate that the walnut-derived peptide TWLPYPR described in the present invention has high antioxidant activity.

[0029] Example 2: Determination of the protective ability of walnut-derived peptide TWLPYPR against HT22 cells

[0030] HT22 cells in the logarithmic growth phase were collected, and cell suspensions were prepared using DMEM complete medium containing 10% fetal bovine serum + 1% penicillin / streptomycin. The cells were seeded into 96-well plates at a density of 5×10 4 cells / well and divided into a blank group, a model group, and an active peptide sample group, and then cultured in an incubator with 5% CO2 at 37°C. Among them, 100 μL of DMEM complete medium was added to 5 wells in the 96-well plate. After 24 h of culture, the culture medium was replaced with 100 μL of fresh DMEM complete medium, which served as the blank group (Control). 100 μL of DMEM complete medium was added to 5 wells in the 96-well plate. After 24 h of culture, it was replaced with 100 μL of DMEM complete medium containing 800 μM H2O2 and treated for 2 h, which served as the model group (Model). 100 μL of DMEM complete medium containing 100 μM of walnut-derived peptide TWLPYPR or 100 μL of 100 μM walnut peptide TWLPLPR was added to 5 wells in the 96-well plate. After 24 h of culture, it was replaced with 100 μL of DMEM complete medium containing 800 μM H2O2 and treated for 2 h, which served as the active peptide sample group (TWLPYPR or TWLPLPR).

[0031] After the culture was completed, the culture medium in each well was discarded. After washing three times with DMEM complete medium, 100 mL of DMEM complete medium was added to each well, and then 10 μL of 5 mg / mL MTT solution (prepared freshly with DMEM complete medium) was added and allowed to act for 4 h (in an incubator with 5% CO2 at 37°C). Subsequently, the culture medium of the blank group, the model group, and the active peptide sample group was discarded, and then 150 μL of DMSO solution was added to each well. The mixture was immediately shaken at 37°C for 10 min on an oscillator to fully dissolve the purple crystalline substance. The absorbance value of each well was measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the average value of 5 wells in each group was taken. The cell viability calculation formula is as follows:

[0032] Cell viability (%) = (A1 / A0) × 100%

[0033] In the formula: A0: absorbance value of the blank group; A1: absorbance value of the active peptide sample group or the model group;

[0034] As Figure 2 shown, the cell viability of the blank group (Control) was 100%, the cell viability of the model group (Model) was 56.42%, and the active peptide sample group (TWLPYPR) could increase the cell viability of HT22 cells induced by 800 μM H2O2 oxidative damage from 56.42% to 87.36%. Moreover, the effect of the active peptide sample group (TWLPYPR) was significantly higher than that of the active peptide sample group (TWLPLPR). The above results indicate that the walnut-derived peptide TWLPYPR described in the present invention has a neuroprotective function.

[0035] Example 3: Determination of the blood-brain barrier penetration ability of walnut-derived peptide TWLPYPR in mice

[0036] Jiangsu Jitai Peptide Industry Technology Co., Ltd. synthesized TWLPYPR labeled with rhodamine B (RB-TWLPYPR) by solid-phase synthesis. The rhodamine B labeling does not affect the function and properties of the peptide. A 10 mg / mL RB-TWLPYPR solution was prepared using sterile normal saline. After intragastric administration (150 mg / kg body weight) to mice for 4, 8, and 12 h, the mice were sacrificed and the brain tissues were completely removed and imaged using a small animal in vivo imaging system. After tail vein injection (50 mg / kg body weight) to mice for 1, 2, 4, 8, and 12 h, the mice were sacrificed and the brain tissues were completely removed and imaged using a small animal in vivo imaging system.

[0037] As Figure 3 shown, after intragastric administration and tail vein injection of RB-TWLPYPR to mice, it can cross the blood-brain barrier and be detected in the brain. The content of the brain-derived peptide was the highest 4 h after intragastric administration, and the fluorescence signal could still be detected in the brain tissue at 12 h. The content of the brain-derived peptide was the highest 1 h after tail vein injection, and the fluorescence signal could still be detected in the brain tissue at 12 h. The above results indicate that the walnut-derived peptide TWLPYPR described in the present invention can cross the blood-brain barrier after administration through different routes.

[0038] Example 4: Improvement of learning and memory in D-galactose-induced memory impairment model mice by walnut-derived peptide TWLPYPR

[0039] The mouse studies were approved by the relevant department and followed the Guidelines for the Care and Use of Laboratory Animals issued by the European Commission. Male C57BL / 6N mice (20 - 25 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Benxi, China). The mice were housed at a stable temperature of (20 ± 2°C), exposed to 12 hours of light / dark conditions each, and had free access to food and water. The mice were randomly divided into 4 groups (n = 10): (i) The mice were gavaged with normal saline (100 ml per day) for 60 days, and at the same time, normal saline (100 ml per day) was injected subcutaneously into the nape of the neck for 60 days (blank group, Control); (ii) The mice were gavaged with normal saline (100 ml per day) for 60 days, and at the same time, D-galactose (500 mg / kg body weight per day, prepared with normal saline) was injected subcutaneously into the nape of the neck for 60 days (model group, Model); (iii) The mice were gavaged with VC (30 mg / kg body weight per day, prepared with normal saline) for 60 days, and at the same time, D-galactose (500 mg / kg body weight per day, prepared with normal saline) was injected subcutaneously into the nape of the neck for 60 days (positive group, Positive); (iv) The mice were gavaged with walnut-derived peptide TWLPYPR (30 mg / kg body weight per day, prepared with normal saline) for 60 days, and at the same time, D-galactose (500 mg / kg body weight per day, prepared with normal saline) was injected subcutaneously into the nape of the neck for 60 days (walnut-derived peptide TWLPYPR group).

[0040] Morris water maze behavioral experiment

[0041] (1) Place navigation experiment

[0042] The diameter of the circular pool of the water maze was 1200 mm, and warm water at 22 ± 2°C was injected into it. The platform was fixed 1 cm below the water surface in the northeast quadrant of the maze. The mice were gently placed into the water from the opposite side of the platform, the monitoring time was set to 120 s, and the computer image acquisition system recorded the swimming path images of the mice. The time taken for the mice to find the platform within 120 s was recorded as the escape latency. The swimming path of the mice from the entrance to the hidden platform was recorded by the computer image acquisition system ( Figure 4 a), the escape latency ( Figure 4 b). Compared with the blank group (Control), the swimming paths of the mice in the model group (Model) were chaotic and the escape latency was long, indicating that the mice in the model group had memory impairment. Compared with the model group, the swimming paths of the mice in the walnut-derived peptide TWLPYRP group were regular and the escape latency was significantly shortened (from 60.15 s to 23.01 s), indicating that the memory impairment of the mice was significantly improved. This shows that the walnut-derived peptide TWLPYRP described in the present invention has the effect of improving the learning and memory ability of D-galactose-induced memory impairment model mice.

[0043] (2) Spatial exploration experiment

[0044] After the positioning and heading experiment was completed, the platform placed underwater was taken out. The mice were gently placed opposite the original platform, and the computer image acquisition system recorded the swimming paths of the mice while timing for 90 s. According to the computer image acquisition system, the number of times the mice crossed the original platform within 90 s was analyzed and calculated ( Figure 4 c). Compared with the blank group (Control), the number of times the mice in the model group (Model) crossed the original platform was significantly reduced, indicating that the mice in the model group had memory impairment. Compared with the model group (Model), the number of times the mice in the walnut-derived peptide TWLPYRP group crossed the original platform increased significantly (from 1.65 times to 5.49 times), indicating that the walnut-derived peptide TWLPYRP described in the present invention has the effect of improving the learning and memory ability of mice in the D-galactose-induced memory impairment model.

Claims

1. A walnut-derived peptide that can penetrate the blood-brain barrier to improve learning and memory, characterized in that, Its amino acid sequence is Thr-Trp-Leu-Pro-Tyr-Pro-Arg.

2. Use of the walnut-derived peptide according to claim 1 in the preparation of a medicament for improving learning and memory.

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