Preparation method and application of pilose antler multi-effect growth factor

Abstract

The invention relates to a preparation method and application of a pilose antler multi-effect growth factor. The preparation method comprises the following steps: S1, obtaining a high expression region of the pilose antler multi-effect growth factor through targeted positioning; s2, carrying out precise separation on a high expression region; s3, preparing an extract from the separated cornu cervi pantotrichum tissue to obtain a primary target product; and S4, selecting a heparin affinity and ion exchange combined scheme for the primary target product to obtain the pilose antler multi-effect growth factor with the purity of more than 95%. The pilose antler multi-effect growth factor prepared on the basis of the method is applied to delaying brain aging. The pilose antler multi-effect growth factor prepared by the method can effectively delay brain aging and improve growth and functions of nerve cells, and has a good anti-aging effect.

Description

Technical Field

[0001] The invention relates to a preparation method of pilose antler pleiotropic growth factor and application thereof. Background Art

[0002] In recent years, with the intensification of global aging, brain aging has become a major issue affecting human health. Brain aging not only affects cognitive function but can also lead to the development of a variety of neurodegenerative diseases. Existing anti-aging drugs mostly focus on lifestyle improvements and nutritional supplements, however, effective treatments for brain aging remain scarce. Deer antler, a traditional Chinese medicinal herb rich in bioactive ingredients, has garnered increasing attention in recent years. Studies have shown that deer antler has the potential to promote cell growth, repair, and regeneration.

[0003] The active ingredients of deer antler velvet mainly include peptides, amino acids, minerals and vitamins. These ingredients have shown good effects in promoting blood circulation, enhancing immunity and resisting fatigue. Existing studies have shown that deer antler velvet extract can promote the growth and differentiation of nerve cells and improve nerve function. However, existing research has mostly focused on the mixed ingredient of deer antler velvet extract, resulting in unclear efficacy. Under the premise of unclear definition of the material basis of the efficacy, excessive reliance on the overall pharmacological evaluation of the mixed ingredients has led to significant cognitive blind spots in the explanation of the efficacy mechanism and a lack of systematic research and efficacy evaluation of certain peptides or classes. In addition, existing anti-aging drugs are mostly synthetic compounds with side effects and dependence problems. There is an urgent need to develop safer and more effective natural drugs.

[0004] Although deer antler is widely used in traditional medicine, its application in modern medicine still faces many challenges. The deer antler ingredients extracted and prepared using existing methods are not effective in treating brain aging. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a method for preparing a pleiotropic growth factor of pilose antler and its application.

[0006] A method for preparing velvet antler multifunctional growth factor comprises the following steps:

[0007] S1: Obtaining the high expression area of ​​velvet antler pleiotropic growth factor through targeted positioning;

[0008] S2: Accurately separate highly expressed regions;

[0009] S3: preparing an extract from the separated antler tissue to obtain a primary target product;

[0010] S4: For the primary target product, a combination of heparin affinity and ion exchange was selected to obtain velvet antler multifunctional growth factor with a purity greater than 95%.

[0011] In step S1, antler tissue is analyzed by spatial transcriptome sequencing to construct an antler spatial gene expression map, and the high expression area of ​​antler pleiotropic growth factor is located by spatial differential expression analysis, and the relative spatial coordinates of the high expression area during the growth period of antler are marked.

[0012] In step S2, laser microdissection is used to separate target area tissue based on the spatial coordinates.

[0013] In step S3, the tissue is freeze-dried under vacuum, and the freeze-dried powder is then extracted in distilled water at 4°C by low-temperature shaking. The extract is centrifuged, the supernatant is collected, sterilized through a 0.22 μm filter, and concentrated by ultrafiltration to enrich the primary pleiotropic growth factor mixture. The vacuum freeze-drying conditions for the tissue are: pre-freezing at -80°C, sublimation drying at -50°C, and a pressure of ≤10 Pa.

[0014] In step S4, the primary target product is passed through a heparin column and eluted with gradually increasing salt concentrations (0.1 → 1.0 M NaCl). PTN is enriched in the 0.6-0.8 M NaCl range. The product is then passed through an SP cation exchange column, bound with a pH 7.0 buffer, and eluted with a pH gradient or salt gradient to separate PTN from the heparin column elution fraction. Finally, the product is subjected to Superdex 200 gel filtration, separated with PBS buffer, and freeze-dried for storage.

[0015] The antler pleiotropic growth factor prepared based on the above method is used in delaying brain aging.

[0016] The present invention has the beneficial effect of effectively delaying brain aging, improving the growth and function of nerve cells, and exhibiting excellent anti-aging effects. Experiments have shown that treated aging mice show a significant reduction in apoptosis in brain tissue and improved neurological function, demonstrating the significant potential of velvet antler pleiotropic growth factor in delaying brain aging. Furthermore, the use of a natural drug reduces the risk of side effects and dependency, meeting the demand for safe and effective anti-aging drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the spatial omics map of velvet antler tissue. DETAILED DESCRIPTION

[0018] The method for preparing the velvet antler pleiotropic growth factor of the present invention comprises the following steps:

[0019] S1: Obtain the high expression area of ​​velvet antler pleiotropic growth factor through targeted positioning. Specifically including the following steps: S11: Spatial transcriptome sequencing (Visium platform) analyzes the velvet antler growth center and constructs a spatial gene expression map. S12: Locate the high expression area of ​​velvet antler pleiotropic growth factor through spatial differential expression analysis. S13: Mark the spatial coordinates of the high expression area (coordinate accuracy ±50μm). Figure 1 As shown, through the gene expression matrix, PCA was used to reduce the dimension of the matrix data, and then the UMAP algorithm was used for clustering. Through analysis, it was found that the yellow area was the high expression area of ​​antler pleiotrophic growth factor, while the expression content of antler pleiotrophic growth factor in other areas of antler was low.

[0020] S2: Precisely isolate high-expression regions. Specifically, the following steps are included: S21: Based on spatial coordinates, separate the target region tissue using laser microdissection (LCM). S22: Vacuum freeze-dry the tissue (pre-freeze at -80°C, sublimate and dry at -50°C, pressure ≤10Pa). S23: Grind the freeze-dried material into powder, and extract the freeze-dried powder in 4°C distilled water by low-temperature shaking (100 rpm, time ≤2h).

[0021] S3: Prepare an extract from the separated antler tissue to obtain the primary target product. After centrifugation of the extract from S2, the supernatant is removed, sterilized through a 0.22 μm filter, and concentrated by ultrafiltration to enrich the primary target product. The tissue is vacuum freeze-dried under the following conditions: pre-freezing at -80°C, sublimation drying at -50°C, and pressure ≤10 Pa.

[0022] S4: The primary target product is passed through a heparin column, eluted with gradually increasing salt concentrations (0.1 → 1.0 M NaCl). PTN is enriched in the 0.6-0.8 M NaCl range. The product is then passed through an SP cation exchange column, bound using a pH 7.0 buffer, and eluted using a pH or salt gradient to separate PTN from the heparin column elution fraction. Finally, the product is subjected to Superdex 200 gel filtration, separated in PBS buffer, and lyophilized for storage.

[0023] Experimental verification:

[0024] First, culture neuroblasts and glial cells in vitro

[0025] After obtaining high-purity velvet antler pleiotropic growth factor through the above steps, in vitro experiments were conducted to evaluate its effect on nerve cells. The specific steps are as follows:

[0026] Select appropriate neuroblasts and glial cells for culture, use DMEM medium, add appropriate amount of fetal bovine serum and antibiotics, and maintain the cells in an environment of 37°C and 5% CO2.

[0027] Prepare a cell aging model and treat cells with H2O2 or other inducers to simulate the aging state.

[0028] After the cells reached the senescent state, 100 ng / ml of velvet antler pleiotropic growth factor was administered to observe its effects on cell apoptosis and proliferation.

[0029] The cell survival rate, apoptosis rate and proliferation ability were quantitatively analyzed by flow cytometry and cell counting plate.

[0030] Then, a D-galactose-induced aging mouse model was constructed

[0031] A D-galactose-induced aging mouse model was constructed to evaluate the anti-aging effects of velvet antler pleiotropic growth factor in vivo. The specific steps are as follows:

[0032] Select mice of appropriate age (such as 6-week-old C57BL / 6 mice) and randomly divide them into experimental and control groups.

[0033] The mice in the experimental group were injected intraperitoneally with D-galactose (100 mg / kg) to establish an aging model and were treated with the prepared antler pleiotropic growth factor.

[0034] The experimental period was set at 90 days, during which the behavioral changes and physiological indicators of the mice were regularly observed.

[0035] After the treatment, the brain tissues of the mice were taken for histological evaluation, and the apoptosis of brain tissue cells was assessed by HE staining and TUNEL method.

[0036] The cell apoptosis rate was quantitatively analyzed by microscopic observation and image analysis software to evaluate the protective effect of antler pleiotropic growth factor on the brain tissue of aging mice.

[0037] Through the above-mentioned embodiments, the present invention provides a natural medicine based on velvet antler pleiotropic growth factor, which can effectively delay brain aging, improve the growth and function of nerve cells, and has excellent anti-aging effects. Research results show that apoptosis in brain tissue of aged mice treated with velvet antler pleiotropic growth factor is significantly reduced, and neurological function is improved, indicating that velvet antler pleiotropic growth factor has significant potential in delaying brain aging.

Claims

1. A method for preparing velvet antler multifunctional growth factor, characterized in that: It includes the following steps: S1: Obtaining the high expression area of ​​velvet antler pleiotropic growth factor through targeted positioning; S2: Accurately separate highly expressed regions; S3: preparing an extract from the separated antler tissue to obtain a primary target product; S4: For the primary target product, a combination of heparin affinity and ion exchange was selected to obtain velvet antler multifunctional growth factor with a purity greater than 95%.

2. The method for preparing a pleiotropic growth factor of pilose antler according to claim 1, wherein: In step S1, antler tissue is analyzed by spatial transcriptome sequencing to construct an antler spatial gene expression map, and the high expression area of ​​antler pleiotropic growth factor is located by spatial differential expression analysis, and the relative spatial coordinates of the high expression area during the growth period of antler are marked.

3. The method for preparing a pleiotropic growth factor of pilose antler according to claim 1, wherein: In step S2, laser microdissection is used to separate target area tissue based on the spatial coordinates.

4. The method for preparing a pleiotropic growth factor of pilose antler according to claim 1, wherein: In step S3, the tissue is freeze-dried in a vacuum environment, and the freeze-dried powder is then extracted in distilled water at 4°C by low-temperature shaking. The extract is centrifuged, the supernatant is taken, sterilized through a 0.22 μm filter membrane, and concentrated by ultrafiltration to enrich the primary target product.

5. The method for preparing a pleiotropic growth factor of pilose antler according to claim 4, characterized in that: The conditions for vacuum freeze-drying of tissues were: pre-freezing at -80°C, sublimation drying at -50°C, and pressure ≤10Pa.

6. The method for preparing a pleiotropic growth factor of pilose antler according to claim 1, wherein: In step S4, the primary target product is passed through a heparin column and eluted with gradually increasing salt concentrations, and PTN is enriched in the range of 0.6-0.8 M NaCl. The product is further passed through an SP cation exchange column, bound with a pH 7.0 buffer, and eluted with a pH gradient or salt gradient to separate PTN from the heparin column elution fraction. Finally, the product is subjected to Superdex 200 gel filtration, separated with PBS buffer, and freeze-dried for storage.

7. The velvet antler pleiotropic growth factor prepared according to any one of claims 1 to 6, characterized in that: Its application in delaying brain aging.

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