Use of bird's nest as a nerve growth factor sensitizer

Abstract

The present invention discloses the use of bird's nest as a nerve growth factor sensitizer. The enzymatically hydrolyzed bird's nest as a nerve growth factor sensitizer can increase the number of differentiated neurons, promote axon growth and / or increase the expression of neurofilament protein. The present invention reveals the relationship between enzymatically hydrolyzed bird's nest (EBN) and neuronal differentiation, explores new functions of EBN, expands the application range of EBN, and provides a new way to prevent and treat neurodegenerative diseases.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to the use of bird's nest as a nerve growth factor sensitizer. Background Art

[0002] Edible bird's nest is a functional food widely consumed in Asian countries, including Indonesia, Malaysia and Thailand. Bird's nest is considered a beneficial dietary supplement and therapeutic agent in traditional Chinese medicine. Bird's nest is reported to enhance immune function, slow down aging, and relieve asthma and cough symptoms. The bioactive components in bird's nest include proteins, sialic acid, glucosamine, amino acids, fatty acids, vitamins and minerals, which are considered to be beneficial to human health.

[0003] The process of neurogenesis involves the proliferation and differentiation of neural stem cells (NSCs) into new neurons. In the brain, neurogenesis is essential for neural plasticity, maintaining brain homeostasis, and cognitive function. Adult neurogenesis is affected by multiple factors, such as diet, alcohol, aging, oxidative stress, and neuroinflammation. Disorders in adult neurogenesis are associated with a variety of neurodegenerative diseases, including Alzheimer's disease, Huntington's disease, and Parkinson's disease, which are mainly characterized by progressive neuronal loss and behavioral and memory disorders. Current studies have shown that enhancing adult neurogenesis may improve cognitive dysfunction. Therefore, promoting neurogenesis using a specific diet may be an effective strategy to delay or prevent neurodegenerative diseases.

[0004] The PC12 pheochromocytoma cell line is a neural cell line derived from an adrenal tumor and has been widely used to study neuronal differentiation. PC12 cells can be induced by nerve growth factor (NGF) to differentiate into cells with a typical neuronal phenotype and extend axons. Therefore, PC12 cells are often used to study neuronal differentiation. Measuring the length of axons is an important evaluation indicator of neuronal differentiation and is widely used as a morphological marker of differentiation. In addition, neurofilament expression can also be used as a biomarker for PC12 cell differentiation.

[0005] Bird's nest can promote the intellectual development of infants and young children. However, there are currently no reports on the correlation between bird's nest and neuronal differentiation, and on bird's nest as a nerve growth factor sensitizer. Therefore, promoting neuronal differentiation through bird's nest is of great significance for the prevention and treatment of degenerative diseases and fully exploring the functions of bird's nest. Summary of the invention

[0006] In order to give full play to the efficacy of bird's nest, avoid the occurrence of neurological disorders, prevent and treat neurodegenerative diseases, and improve people's health level, the present invention provides the following technical solutions.

[0007] In a first aspect, the present invention provides a composition comprising enzymatically hydrolyzed bird's nest (EBN) and nerve growth factor.

[0008] Preferably, the enzymatically hydrolyzed bird's nest is the product of dry bird's nest hydrolyzed by acidic protease and alkaline protease.

[0009] Furthermore, the acidic protease is pepsin, and the alkaline protease is trypsin.

[0010] Preferably, the concentration of the enzymatically hydrolyzed bird's nest is 5-150 μg / mL, more preferably 10-100 μg / mL, for example 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL.

[0011] Preferably, the method for preparing enzymatically hydrolyzed bird's nest comprises the following steps:

[0012] (1) After the dried bird's nest is soaked, the solid-liquid ratio is adjusted, the water bath is heated, and impurities are removed to obtain a bird's nest solution;

[0013] (2) preheating the bird's nest solution by oscillation, adjusting the pH to acidic, adding acidic protease, and inactivating the enzyme after hydrolysis;

[0014] (3) adjusting the pH of the acidic protease hydrolyzate to alkaline, adding alkaline protease for hydrolysis, and inactivating the enzyme to obtain the enzymatically hydrolyzed bird's nest.

[0015] In a second aspect, the present invention provides use of the composition of the first aspect in the preparation of a drug for promoting nervous system function.

[0016] Preferably, the promoting nervous system function comprises at least one of the following:

[0017] (1) Promote neuronal differentiation.

[0018] (2) Increase the number of differentiated neurons.

[0019] (3) Promote axon growth.

[0020] (4) Increase the expression of neurofilament protein.

[0021] (5) Promote the repair of the nervous system after damage.

[0022] (6) Delaying neurodegenerative diseases such as Alzheimer's disease, Huntington's disease and Parkinson's disease.

[0023] (7) Stimulate the growth of motor nerves in patients with spinal cord injury.

[0024] Preferably, the enzymatically hydrolyzed bird's nest is used as a sensitizer of nerve growth factor (NGF).

[0025] Preferably, the neurons are differentiated from PC12 cells.

[0026] Preferably, the concentration of the enzymatically hydrolyzed bird's nest is 5-150 μg / mL, more preferably 10-100 μg / mL, for example 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL.

[0027] Preferably, the enzymatically hydrolyzed bird's nest is a product obtained by hydrolyzing dried bird's nest with acidic protease and alkaline protease;

[0028] Furthermore, the acidic protease is pepsin, and the alkaline protease is trypsin.

[0029] Preferably, the drug further comprises an excipient acceptable in drugs.

[0030] Furthermore, the auxiliary material in the medicine is selected from at least one of a disintegrant, a filler, a lubricant, a wetting agent, an emulsifier or a binder.

[0031] In a third aspect, the present invention provides the use of enzymatically hydrolyzed bird's nest (EBN) as a nerve growth factor (NGF) sensitizer.

[0032] Preferably, the use is for non-therapeutic purposes.

[0033] Preferably, the application includes at least one of the following:

[0034] (1) Promote neuronal differentiation.

[0035] (2) Increase the number of differentiated neurons.

[0036] (3) Promote axon growth.

[0037] (4) Increase the expression of neurofilament protein.

[0038] (5) Promote the repair of the nervous system after damage.

[0039] (6) Delaying neurodegenerative diseases such as Alzheimer's disease, Huntington's disease and Parkinson's disease.

[0040] (7) Stimulate the growth of motor nerves in patients with spinal cord injury.

[0041] Preferably, the neurons are differentiated from PC12 cells.

[0042] Preferably, the concentration of the enzymatically hydrolyzed bird's nest is 5-150 μg / mL, more preferably 10-100 μg / mL, for example 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL.

[0043] Preferably, the enzymatically hydrolyzed bird's nest is a product obtained by hydrolyzing dried bird's nest with acidic protease and alkaline protease;

[0044] Furthermore, the acidic protease is pepsin, and the alkaline protease is trypsin.

[0045] In a fourth aspect, the present invention provides a method for preparing enzymatically hydrolyzed bird's nest, comprising the following steps:

[0046] (1) After the dried bird's nest is soaked, the solid-liquid ratio is adjusted, the water bath is heated, and impurities are removed to obtain a bird's nest solution;

[0047] (2) preheating the bird's nest solution by oscillation, adjusting the pH to acidic, adding acidic protease, and inactivating the enzyme after hydrolysis;

[0048] (3) adjusting the pH of the acidic protease hydrolyzate to alkaline, adding alkaline protease for hydrolysis, and inactivating the enzyme to obtain the enzymatically hydrolyzed bird's nest.

[0049] Preferably, in step (1), the solvent for soaking the dried bird's nest is water, such as distilled water, purified water, sterilized water, or water for injection.

[0050] Preferably, the soaking time of dried bird's nest is 10-16 hours, for example: 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours.

[0051] Preferably, the solid-liquid ratio in step (1) is 1:15-1:25, for example: 1:15, 1:17, 1:20, 1:23, 1:25.

[0052] Preferably, the temperature of the water bath in step (1) is ≥85°C, for example: 85°C, 87°C, 90°C, 92°C, 95°C, 97°C, 100°C.

[0053] Preferably, the water bath time in step (1) is 10-30 min, for example: 10 min, 15 min, 20 min, 25 min, 30 min.

[0054] Preferably, the preheating temperature in step (2) is 50-60°C, for example: 50°C, 53°C, 55°C, 57°C, 60°C.

[0055] Preferably, the oscillation preheating time in step (2) is 2.0-3.5 h, for example: 2.0 h, 2.5 h, 3.0 h, 3.5 h.

[0056] Preferably, the pH in step (2) is 1.5-2.5.

[0057] Preferably, the acidic protease in step (2) is pepsin.

[0058] Furthermore, the added amount of pepsin is 3500-5000U / mL, for example: 3000U / mL, 3500U / mL, 4000U / mL, 4500U / mL, 5000U / mL.

[0059] Preferably, the hydrolysis time of pepsin is 1.5-3 h, for example: 1.5 h, 2.0 h, 2.5 h, 3.0 h.

[0060] Preferably, the hydrolysis temperature of pepsin is 35-40°C.

[0061] Preferably, the pepsin is inactivated by a physical method, more preferably by high temperature treatment, and further preferably by boiling treatment.

[0062] Furthermore, the boiling time is 10-25 min, for example: 10 min, 15 min, 20 min, 25 min.

[0063] Preferably, the pH in step (3) is 7.2-8.0, for example: 7.2, 7.5, 7.8, 8.0.

[0064] Preferably, the alkaline protease in step (3) is trypsin.

[0065] Furthermore, the added amount of trypsin is 50-150U / mL, for example: 50U / mL, 60U / mL, 70U / mL, 80U / mL, 80U / mL, 90U / mL, 100U / mL, 110U / mL, 120U / mL, 130U / mL, 140U / mL, 150U / mL.

[0066] Preferably, the trypsin hydrolysis time is 1.5-3 h, for example: 1.5 h, 2.0 h, 2.5 h, 3.0 h.

[0067] Preferably, the hydrolysis temperature of the trypsin is 35-40°C.

[0068] Preferably, the trypsin is inactivated by a physical method, more preferably by high temperature treatment, and further preferably by boiling treatment.

[0069] Furthermore, the boiling time is 25-40 min, for example: 25 min, 30 min, 35 min, 40 min.

[0070] Preferably, step (3) further comprises cooling and freeze-drying the product after the enzyme is inactivated.

[0071] In a fifth aspect, the present invention provides an enzymatically hydrolyzed bird's nest (EBN) for promoting neuronal differentiation, wherein the enzymatically hydrolyzed bird's nest is prepared according to the preparation method described in the fourth aspect.

[0072] Beneficial effects of the present invention:

[0073] The present invention reveals the relationship between EBN, nerve growth factor and neuron differentiation, discovers the new function of EBN as a nerve growth factor sensitizer, and expands the application scope of EBN.

[0074] The EBN of the present invention can promote the differentiation of neurons, provide a new approach for preventing and treating neurodegenerative diseases, help promote the repair of the nervous system and delay neurodegenerative diseases, and is of great significance for improving people's health level. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 Shown are the effects of EBN on PC12 cell differentiation; (A) Microphotographs of PC12 cell differentiation on day 7 after administration, (B) the number of differentiated cells under different treatment conditions, (C) average axon length;

[0076] Figure 2 Shown are the effects of co-treatment of EBN and NGF on axon growth of PC12 cells; (A) Immunofluorescence images of cells on the 7th day under the conditions of 50 ng / mLNGF alone treatment, 1.5 ng / mLNGF and different concentrations of EBN (10 μg / mL, 50 μg / mL, 100 μg / mL) co-treatment, and 1.5 ng / mLNGF and SA co-treatment of PC12 cells, (B) the number of differentiated cells under different treatment conditions, (C) the average axon length;

[0077] Figure 3 The results show that EBN enhanced the expression of neurofilaments induced by NGF; PC12 cells were treated with different concentrations of EBN (μg / mL) for 7 days in the presence of trace amounts of NGF, and the protein expression of neurofilaments NF68, NF160 and NF200 was detected by Western blotting, and 50 ng / mL NGF was used as a positive control.

[0078] Figure 4The figure shows the effect of co-treatment of EBN and NGF on the phosphorylation of TrkA, ERK1 / 2, CREB and the protein expression of BDNF in PC12 cells; (A) TrkA, (B) p-CREB, (C) p-ERK1 / 2 and (D) BDNF;

[0079] Figure 5 Shown are the validation results of the signaling pathway of EBN neurogenic effects;

[0080] Figure 6 The potential mechanism by which EBN promotes NGF-induced PC12 cell differentiation, and the red arrows indicate the functions upregulated by EBN.

[0081] The data in the above figures are expressed as mean ± SEM (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences compared with the model group. DETAILED DESCRIPTION

[0082] The technical scheme of the present invention is clearly and completely described below in conjunction with the embodiments. The described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0083] In addition, unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by those skilled in the art to which the present invention relates.

[0084] The samples and reagents in the embodiment are as follows:

[0085] Dried bird's nest (produced in Malaysia, purchased from Beijing Xiaoxiandun Biotechnology Co., Ltd. (Beijing, China));

[0086] PC12 cells (CL-0412, Procell, Wuhan, China), RPMI-1640 medium (Procell, Wuhan, China);

[0087] Primary antibodies including β-III tubulin (ab18207, 1:1000) were purchased from Abcam;

[0088] Sialic acid (SA) standards were purchased from Beijing Solebow Technology Co., Ltd.;

[0089] Nerve growth factor (NGF) standard was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.;

[0090] 4% paraformaldehyde (P0099) and AlexaFluor 488-conjugated secondary antibodies were purchased from Beyotime;

[0091] TrkA receptor inhibitor (GW441756) (MedChemExpress, USA).

[0092] Example 1 Preparation of enzymatically hydrolyzed bird's nest (EBN) samples and SA

[0093] Soak 5 grams of dried bird's nest in distilled water for 12 hours. Then heat the soaked bird's nest in 95°C water at a solid-liquid ratio of 1:20 for 10-25 minutes. Take out 100mL of bird's nest solution, place it in an oscillator, and oscillate at 55°C and 50rpm for 3 hours. Adjust the pH to 2.0 with hydrochloric acid, and add pepsin (4000U / mL). After the sample is shaken for another 2 hours, boil it for 20 minutes to inactivate the enzyme. Then adjust the pH of the sample solution to 7.8, and add trypsin (100U / mL). Incubate the mixture at 37°C for 2 hours to simulate small intestine conditions. Then boil and inactivate the enzyme for 30 minutes to terminate the reaction, and cool and freeze-dry the digested sample for subsequent experiments.

[0094] The sialic acid (SA) standard was prepared into a 10 μg / mL standard solution for use.

[0095] Example 2 Effect of enzymatic hydrolysis of bird's nest (EBN) on neuronal differentiation

[0096] 2.1 Cell culture

[0097] Undifferentiated PC12 cells were seeded in RPMI-1640 medium supplemented with 15% horse serum, 6% fetal bovine serum and 1% penicillin / streptomycin, and then cultured at 5% CO 2 and 37° C., with fresh medium replaced every other day.

[0098] 2.2 Cell viability assay

[0099] PC12 cells were cultured at 5×10 3 The cells were inoculated into a 96-well plate at a density of 10 cells / well. After 24 hours of growth, the cells were treated with EBN for 24 hours, and a blank control of the medium without EBN was set up. Then 10 μL of CCK-8 solution was added to each well and incubated at 37°C in the dark for 2 hours.

[0100] Optical density (OD) values ​​were measured at 450 nm using a microplate reader (Thermo, USA). Relative cell viability was expressed as a percentage of the control. Cell viability analysis was performed to determine the safe concentration range of EBN, and the results showed that EBN did not induce proliferation or death of PC12 cells in the concentration range of 0-1000 μg / mL.

[0101] 2.3 Determination of axonal growth of PC12 cells

[0102] Undifferentiated PC12 cells were cultured at 1×10 4 The density of cells / mL was inoculated into 12-well plates and incubated for 24 hours, and the plates were pre-coated with polylysine to enhance cell attachment. Then PC12 cells were set up in 6 groups, namely 10μg / mL EBN group, 50μg / mL EBN group, 100μg / mL EBN group, 10μg / mL SA group, 50μg / mLNGF group and blank control group, and the culture medium was changed every other day. On the 7th day of cell culture, PC12 cells were photographed using an optical microscope (Leica DM6B, Germany). Cells whose length of at least one axon exceeded the diameter of the corresponding cell body were judged as differentiated cells. The axon length of cells was analyzed using ImageJ software. At least 100 cells in three typical fields were randomly selected from each well, and the number of differentiated cells and the average length of axons were calculated.

[0103] like Figure 1 As shown in the figure, the cell differentiation rates of the 10μg / mL EBN group, the 50μg / mL EBN group, the 100μg / mL EBN group and the 10μg / mL SA group were all about 10%, which was not significantly different from the blank control group. However, the cell differentiation rate of the 50μg / mL NGF group reached more than 40%, which was significantly different from the other groups (p < 0.001). Compared with the blank control group, there was no significant change in the axon growth of PC12 cells between the 10μg / mL EBN group, the 50μg / mL EBN group, the 100μg / mL EBN group and the 10μg / mL SA group, indicating that the effect of EBN treatment alone in promoting cell axon growth was not obvious. However, compared with other groups, the axon growth of PC12 cells in the 50μg / mL NGF group was significant (p < 0.001), indicating that NGF can promote the axon growth of PC12 cells.

[0104] 2.4EBN enhances NGF-induced neurite outgrowth in PC12 cells

[0105] NGF is essential for the differentiation of PC12 cells, and its absence will greatly reduce the degree of differentiation of these cells. Axon length is an important evaluation index of neuronal differentiation. In order to study whether EBN can stimulate axon growth through NGF and reveal the effect of EBN on P12 cell differentiation, the following experiment was performed.

[0106] The detection indicators of cell differentiation are cell differentiation rate and cell axon length. When the length of at least one neurite exceeds the diameter of the corresponding cell body, the cell is classified as differentiated. The axon length of the cell is analyzed using Image J software. At least 100 cells from three typical fields were randomly selected from each well to calculate the number of differentiated cells and the average length of the axon.

[0107] Cell differentiation rate = number of differentiated cells / total number of cells in the selected area.

[0108] PC12 cells were divided into 7 groups, namely, blank group, 1.5ng / mL NGF group, 1.5ng / mL NGF+10μg / mL EBN group, 1.5ng / mLNGF+50μg / mL EBN group, 1.5ng / mLNGF+100μg / mL EBN group, 1.5ng / mLNGF+10μg / mL SA group, and 50ng / mLNGF group. The cultured PC12 cells were subjected to immunofluorescence staining, and images were captured using an Airyscan confocal microscope.

[0109] like Figure 2 As shown, 1.5 ng / mL NGF alone failed to significantly induce PC12 cell differentiation ( Figure 2 A, B), while the combination of 1.5ng / mL NGF and EBN can promote cell differentiation. The differentiation rates of PC12 cells treated with 10μg / ml, 50μg / ml and 100μg / ml EBN and NGF were 22.8%, 31.2% and 42.7%, respectively, which were significantly higher than those of cells cultured with the control medium containing 1.5ng / ml NGF (13.8%, p<0.05) ( Figure 2 A, B), and the increase was dose-dependent. Figure 2 C shows that when the concentration of EBN used in combination with NGF increased from 10 μg / mL to 100 μg / L, the average axon length increased. When the EBN concentration was 50 μg / mL, the difference was significant compared with the control group (p<0.05), and when the EBN concentration was 100 μg / mL, the difference was extremely significant compared with the control group (p<0.001). The increasing trend of axon length was consistent with the increasing trend of the percentage of cell differentiation.

[0110] The expression of neurofilament protein in PC12 cells was determined according to the method in 2.3. Figure 3 .

[0111] Depend on Figure 3It can be seen that the expression levels of NF68, NF160, and NF200 have increased. Compared with the control group, the expression levels of the three neurofilament proteins did not change significantly when cells were treated with 1.5ng / mL EBN alone and NGF. However, when cells were co-treated with 10μg / mL EBN and NGF, the expression levels of NF160 and NF200 increased significantly by 38.2% and 34.5%, respectively (p<0.05). In addition, in cells co-treated with 50μg / mL and 100μg / mL EBN and NGF, the levels of the three neurofilament proteins increased by 2-3 times (p<0.05), indicating that EBN can act as a sensitizer of NGF to enhance the differentiation-inducing effect of low-concentration NGF on PC12 cells.

[0112] 2.5 Pathway of EBN-induced PC12 cell differentiation

[0113] NGF triggers neuronal differentiation and enhances cell viability by activating TrkA receptors in PC12 cells. To reveal how EBN affects PC12 cell differentiation, changes in the TrkA signaling pathway were examined.

[0114] like Figure 4 As shown in the figure, except for the blank control group, the phosphorylation of TrkA in PC12 cells treated with 1.5ng / mLNGF alone was minimal. After co-treatment with 1.5ng / mLNGF and EBN, the phosphorylation of TrkA increased significantly, which was nearly 2 times that of NGF alone (p<0.05) ( Figure 4 A). In addition, compared with the control group treated with 1.5 ng / mL NGF, the phosphorylation of TrkA in the SA group (sialic acid group) was also significantly increased (p<0.05) ( Figure 4 A).

[0115] The expression of TrkA downstream effectors, extracellular signal-regulated kinases (Erk1 / 2) and cAMP response element binding protein (CREB), was detected. The results showed that the group treated with NGF and EBN showed a 3-fold increase in ERK1 / 2 phosphorylation and a 2-fold increase in CREB phosphorylation compared with the control group treated with 1.5 ng / ml NGF (p<0.05) ( Figure 4 B, C). In addition, the SA group showed a significant increase in the phosphorylation levels of ERK1 / 2 and CREB (p<0.05) ( Figure 4 B, C). This indicates that EBN can regulate the activation of ERK1 / 2 and the phosphorylation of CREB in the nucleus. In addition, phosphorylated CREB (p-CREB) can induce the expression of brain-derived neurotrophic factor (BDNF), which is essential for neurogenesis and neuronal differentiation. Compared with the control group, BDNF in EBN was upregulated by 44.8% (p<0.05) ( Figure 4 D). Figure 4 The results showed that EBN enhanced NGF-induced TrkA phosphorylation, which in turn activated CREB through the ERK1 / 2 signaling cascade, ultimately leading to a significant increase in BDNF expression. EBN induced the differentiation of PC12 cells through the TrkA-ERK1 / 2-CREB-BDNF signaling pathway.

[0116] Effects of 2.6EBN on the TrkA-ERK1 / 2-CREB-BDNF signaling pathway in PC12 cell differentiation

[0117] GW441756 was dissolved in dimethyl sulfoxide and prepared as a 10 mM stock solution at -20 °C in the dark.

[0118] Undifferentiated PC12 cells were pre-incubated with complete medium containing 100 nM inhibitor for 1 hour, and PC12 cells were pre-incubated with complete medium without GW441756. Then the cells were divided into 10 groups, namely blank group, blank group + GW441756 group, 1.5 ng / mL LNGF group, 1.5 ng / mL NGF + GW441756 group, 1.5 ng / mL NGF + 100 μg / mL EBN co-treatment group, 1.5 ng / mL NGF + 100 μg / mL EBN + GW441756 co-treatment group, 1.5 ng / mL LNGF + 10 μg / mL SA group, 1.5 ng / mL NGF + 10 μg / mL SA + GW441756 co-treatment group, 50 ng / mL NGF group, 50 ng / mL LNGF + GW441756 group. After PC12 cells were cultured for 48 hours, the expression level of neurofilaments was determined according to the method in 2.3.

[0119] like Figure 5 As shown, the levels of all three neurofilament proteins were significantly decreased after administration of GW441756 (p<0.05), indicating that EBN promotes neuronal differentiation by upregulating TrkA, thereby enhancing the sensitivity of cells to low concentrations of NGF.

[0120] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A composition, characterized in that The composition consists of enzymatically hydrolyzed bird's nest and nerve growth factor; The concentration of the enzymatically hydrolyzed bird's nest in the composition is 10-100 μg / mL, and the concentration of the nerve growth factor is 1.5 ng / mL; The preparation method of the enzymatically hydrolyzed bird's nest comprises the following steps: (1) After the dried bird's nest is soaked, the solid-liquid ratio is adjusted to 1:15-1:25, heated in a water bath at a temperature ≥85°C for 10-30 minutes, and impurities are removed to obtain a bird's nest solution; (2) preheating the bird's nest solution at 50-60° C. for 2.0-3.5 h, adjusting the pH to 1.5-2.5, adding pepsin, hydrolyzing at 35-40° C. for 1.5-3 h, and then boiling to inactivate the enzyme; (3) The pH of the pepsin hydrolyzate is adjusted to 7.2-8.0, trypsin is added for hydrolysis, and the hydrolysis is carried out at 35-40° C. for 1.5-3 h, followed by boiling to inactivate the enzyme, thereby obtaining the enzymatically hydrolyzed bird's nest.

2. The use of enzymatically hydrolyzed bird's nest in the preparation of a nerve growth factor sensitizer, characterized in that: The enzymatically hydrolyzed bird's nest is a product obtained by hydrolyzing dried bird's nest with acidic protease and alkaline protease; The enzymatic hydrolysis of bird's nest promotes the neuronal differentiation effect of nerve growth factor; The concentration of the enzymatically hydrolyzed bird's nest is 10-100 μg / mL, and the concentration of the nerve growth factor is 1.5 ng / mL; The preparation method of the enzymatically hydrolyzed bird's nest comprises the following steps: (1) After the dried bird's nest is soaked, the solid-liquid ratio is adjusted to 1:15-1:25, heated in a water bath at a temperature ≥85°C for 10-30 minutes, and impurities are removed to obtain a bird's nest solution; (2) preheating the bird's nest solution at 50-60° C. for 2.0-3.5 h, adjusting the pH to 1.5-2.5, adding pepsin, hydrolyzing at 35-40° C. for 1.5-3 h, and then boiling to inactivate the enzyme; (3) The pH of the pepsin hydrolyzate is adjusted to 7.2-8.0, trypsin is added for hydrolysis, and the hydrolysis is carried out at 35-40° C. for 1.5-3 h, followed by boiling to inactivate the enzyme, thereby obtaining the enzymatically hydrolyzed bird's nest.

3. The use according to claim 2, characterized in that: The promoting neuronal differentiation comprises at least one of the following: (1) Increase the number of differentiated neurons; (2) Promote axon growth; (3) Increase the expression of neurofilament protein.

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