Preparation method for extracting active peptide (BAP) from cornu cervi pantotrichum and quality control thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine extraction and preparation, and discloses a preparation method and a quality control method for extracting active peptide from cornu cervi pantotrichum. Cornu cervi pantotrichum is a traditional rare traditional Chinese medicine, contains a plurality of growth factors and has high clinical application value, the active peptide liquid in the cornu cervi pantotrichum is obtained through separation by adopting an enzymolysis process, and the cornu cervi pantotrichum active peptide freeze-dried powder is obtained through freeze drying. And detecting the content of the insulin-like growth factor IGF-1 by adopting a double-site sandwich enzyme-linked immunosorbent assay as a quality control standard. The raw materials and the auxiliary materials involved in the method are all in the medicine-auxiliary level, safety is high, and meanwhile the method has the advantages of being easy to operate, low in cost and capable of achieving industrial production and preparation.

Description

Technical Field

[0001] The invention relates to a preparation method of active peptide (BAP) extracted from velvet antler and quality control thereof, belonging to the field of pharmaceutical preparations. Background Art

[0002] With the continuous advancement of science and technology, scientists have discovered that the most important active substance in the human body, active peptides, plays a key role in the process of human growth, metabolism, disease, aging and death. It is precisely because of the increase or decrease in its secretion in the body that humans have a cycle of infancy, childhood, adulthood, old age and death. Supplementing active peptides breaks this cycle of life, thereby achieving the magical effect of prolonging life and effectively delaying aging.

[0003] Humans and other animals contain active peptides, but their content is extremely low, and their secretion patterns vary at different growth stages. For example, the amount of active peptides secreted by normal humans is the highest during childhood, reaching its peak at the age of 18, and then decreasing at a rate of 1.5% per year. At the age of 30, the amount of secretion in the human body has dropped to 70% of that at the age of 18, and after the age of 60, it is less than 10% of that in childhood. Through supplementation, active peptides can stimulate the pituitary gland to resume its function of secreting hormones, thereby regulating the major secretory glands of the human body and allowing aging cells to regain vitality.

[0004] Studies have shown that the growth factors of deer are the closest to those of humans, and have received widespread attention in recent years. The sika deer and red deer, both medicinal and edible, are the medicinal parts of the cervidae family. They are a traditional precious Chinese medicine and tonic. Deer antlers are the young horns of male sika deer or red deer that have not yet ossified and are densely hairy. They contain a variety of peptide growth factors, including insulin-like growth factor-1 (IGF-1), which is an extremely important active factor. Its level disorder is closely related to diseases such as liver damage, insulin resistance and skeletal dysplasia; there is also epidermal growth factor EGF, which can strongly promote the division and growth of skin cells in very small amounts. In addition, it can also stimulate the synthesis and secretion of some extracellular macromolecules (such as hyaluronic acid and glycoproteins) and moisturize the skin. Therefore, it can be used as an additive for cosmetics and for facial plastic surgery to promote human skin metabolism and reduce skin deformities; it can also be used in medicine to treat skin trauma, postoperative wounds, bedsores, oral ulcers and gangrene, as well as dermatitis caused by radiotherapy, etc. It can accelerate the healing of wounds and ulcers. The polypeptide growth factors contained in deer antlers have great clinical application value.

[0005] At present, the quality of peptide products on the market varies greatly, and the publicity is even more varied. Truly active peptide products are rare. The fundamental reason is that the industry lacks corresponding quality standards, and concepts and markets are ahead of quality supervision, leading to chaos. How to ensure the quality of active peptide products, quality control methods are even more indispensable. Summary of the invention

[0006] The present invention aims to provide a preparation method of active peptides extracted from pilose antlers, and to provide a quality control method for determining the quality thereof. The pilose antler active peptides prepared by the method can be used as raw materials for preparations such as oral liquids and capsules.

[0007] The present invention provides a preparation method for extracting active peptides from velvet antlers and a quality control method thereof. The raw and auxiliary materials involved in the method are all of pharmaceutical auxiliary grade, safe and compliant, and can be industrially produced and prepared, and a quality control method is provided, which is technically mature and has a detection limit of 1 ng / ml.

[0008] A method for preparing active peptides extracted from velvet antlers: (1) Low temperature crushing: Place fresh antler or its products in a large-capacity mortar, add an appropriate amount of liquid nitrogen, and grind and crush.

[0009] (2) Enzymatic hydrolysis Weigh 100g~200g of crushed velvet antler powder, add 3600~7200ml of production water, prepare a solution, use pharmaceutical excipients pepsin and trypsin for combined enzymatic hydrolysis, add 5-15U / g pepsin for enzymatic hydrolysis, adjust the pH to 2-2.5 with 0.1mol / LHCL, heat the mixing tank to 40-50℃, start stirring for 2-4h; adjust the pH to 7.5-8.5 with 0.1mol / LNaOH, keep the temperature at 40-50℃, and stir for 2-4h. After the enzymatic hydrolysis is completed, the temperature of the mixing tank is raised to 100℃, heated for 20-30min to inactivate the enzyme, turn off the heating and return to room temperature, and let it stand for 4-6h.

[0010] (3) Freeze-dry the supernatant, add lyophilizing protective agent in the ratio of 1.5-3.5:15-35, stir evenly, transfer to a plate and place in a -20℃ low-temperature refrigerator for pre-freezing for 4 hours, and then transfer to a vacuum freeze drying box for low-temperature drying into powder.

[0011] A quality control method for extracting active peptides from velvet antlers The velvet antler active peptide obtained by this preparation process is rich in natural factors, such as insulin-like growth factor IGF-1, growth hormone HGH, epidermal growth factor EGF, nerve growth factor NGF, fibroblast growth factor FGF, growth-promoting releasing factor GHRH, etc. One of the ingredients is selected to detect its content as the standard for quality control. Since insulin-like growth factor IGF-1 has been studied the most, the content of IGF-1 in velvet antler active peptide freeze-dried powder is selected as the standard for quality control.

[0012] The content of insulin-like growth factor IGF-1 was determined by double-site sandwich enzyme-linked immunosorbent assay (ELISA) to determine the level of deer insulin-like growth factor 1 (IGF-1) in the sample. Standards, samples to be tested, and HRP-labeled deer insulin-like growth factor 1 (IGF-1) antibodies were added to the enzyme-labeled wells pre-coated with deer insulin-like growth factor 1 (IGF-1) antibodies. After incubation and washing, unbound components were removed, and then substrates A and B were added to produce blue, which was converted into the final yellow under the action of acid. The depth of the color is positively correlated with the concentration of deer insulin-like growth factor 1 (IGF-1) in the sample.

[0013] Different concentration gradients of deer insulin-like growth factor 1 (IGF-1) standard were set and the standard curve was prepared by the same method. Specific embodiments

[0014] A method for preparing active peptides extracted from velvet antlers: (1) Low temperature crushing: Place fresh antler or its products in a large-capacity mortar, add an appropriate amount of liquid nitrogen, and grind and crush.

[0015] (2) Enzymatic hydrolysis Weigh 100g of crushed velvet antler powder, add 3600ml of production water, prepare a solution, use pharmaceutical excipients pepsin and trypsin for combined enzymatic hydrolysis, add 5U / g pepsin for enzymatic hydrolysis, adjust the pH to 2.5 with 0.1mol / LHCL, heat the mixing tank to 45℃, and stir for 2h; adjust the pH to 7.5 with 0.1mol / LNaOH, keep the temperature at 45℃, and stir for 2h. After the enzymatic hydrolysis is completed, the temperature of the mixing tank is raised to 100℃, heated for 30min to inactivate the enzyme, turn off the heating and return to room temperature, and let it stand for 6h.

[0016] (3) Freeze-dry the supernatant, add lyophilizing agent in a ratio of 1.5:15, stir evenly, transfer to a plate and place in a -20℃ low-temperature refrigerator for pre-freezing for 4 hours, and then transfer to a vacuum freeze drying oven for low-temperature drying into powder.

[0017] A quality control method for extracting active peptides from velvet antlers The microplate was coated with purified IGF-1 antibody to prepare a solid-phase antibody. IGF-1 was added to the microwells coated with the monoclonal antibody in sequence, and then combined with HRP-labeled IGF-1 antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB was added for color development. TMB was converted into blue under the catalysis of HRP enzyme, and converted into the final yellow under the action of acid. The depth of color is positively correlated with the IGF-1 in the sample. The absorbance (OD value) was measured at a wavelength of 450nm using an enzyme reader, and the concentration of IGF-1 in the sample was calculated using the standard curve.

[0018] Steps: Prepare working solutions of the standard at 160 μg / L, 80 μg / L, 40 μg / L, 20 μg / L, and 10 μg / L.

[0019] Add samples: Set up blank wells (blank control wells without adding samples and enzyme-labeled reagents, and the rest of the steps are the same), standard wells, and test sample wells. Accurately add 50μl of the standard sample to the enzyme-labeled plate, add 40μl of sample diluent to the test sample wells, and then add 10μl of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix.

[0020] Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes.

[0021] Solution preparation: dilute the 30 times concentrated washing solution with 30 times distilled water for later use Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0022] Add enzyme: Add 50 μl of enzyme labeling reagent to each well, except for the blank well.

[0023] Incubation: Same as step 3.

[0024] Washing: Same operation as step 5.

[0025] Color development: first add 50μl of color developer A to each well, then add 50μl of color developer B, gently shake to mix, and develop the color at 37℃ in the dark for 10 minutes.

[0026] 10. Stop: Add 50 μl of stop solution to each well to stop the reaction (the blue color immediately turns to yellow).

[0027] 11. Determination: Use blank as zero and measure the absorbance (OD value) of each well in sequence at 450nm. The determination should be carried out within 15 minutes after adding the stop solution.

[0028] 12. Calculation Draw a standard curve on the coordinate paper with the concentration of the standard as the horizontal axis and the OD value as the vertical axis. Find the corresponding concentration from the standard curve based on the OD value of the sample; then multiply it by the dilution factor; or use the concentration of the standard and the OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor to get the actual concentration of the sample.

[0029] The standard curve was obtained: y=0.0045x+0.1658. After testing, this batch of velvet antler active peptides contained IGF-165ng / ml.

Claims

1. The present invention aims to provide a preparation method of active peptides extracted from pilose antlers, and to provide a quality control method for determining the quality thereof. The active peptides of pilose antlers prepared by the method can be used as raw materials for preparations such as oral liquids and capsules.

2. The present invention provides a preparation method for extracting active peptides from velvet antlers and a quality control method thereof. The raw and auxiliary materials involved in the method are all of pharmaceutical grade, safe and compliant, and can be industrially produced and prepared. A quality control method is provided, which is technically mature and has a detection limit of 1 ng / ml.

3. A method for preparing active peptides extracted from velvet antlers: (1) Low temperature crushing: Place fresh antler or its products in a large-capacity mortar, add an appropriate amount of liquid nitrogen, and grind and crush.

4. (2) Enzymatic hydrolysis Weigh 100g~200g of crushed velvet antler powder, add 3600~7200ml of production water, prepare a solution, use pharmaceutical excipients pepsin and trypsin for combined enzymatic hydrolysis, add 5-15U / g pepsin for enzymatic hydrolysis, adjust the pH to 2-2.5 with 0.1mol / LHCL, heat the mixing tank to 40-50℃, and stir for 2-4h; adjust the pH to 7.5-8.5 with 0.1mol / LNaOH, keep the temperature at 40-50℃, and stir for 2-4h. After the enzymatic hydrolysis is completed, raise the temperature of the mixing tank to 100℃, heat for 20-30min, inactivate the enzyme, turn off the heating and return to room temperature, and let it stand for 4-6h.

5. (3) Freeze-dry the supernatant, add lyophilizing agent in the ratio of 1.5-3.5:15-35, stir evenly, transfer to a plate and place in a -20℃ low-temperature refrigerator for pre-freezing for 4 hours, then transfer to a vacuum freeze drying oven for low-temperature drying into powder.

6. A quality control method for extracting active peptides from velvet antlers The velvet antler active peptide obtained by this preparation process is rich in natural factors, such as insulin-like growth factor IGF-1, growth hormone HGH, epidermal growth factor EGF, nerve growth factor NGF, fibroblast growth factor FGF, growth-promoting releasing factor GHRH, etc. One of the ingredients is selected to detect its content as the standard for quality control. Since insulin-like growth factor IGF-1 has been studied the most, the content of IGF-1 in velvet antler active peptide freeze-dried powder is selected as the standard for quality control.

7. Determination of insulin-like growth factor IGF-1 content, using a two-site sandwich enzyme-linked immunosorbent assay (ELISA) to determine the level of deer insulin-like growth factor 1 (IGF-1) in the sample. Add the standard, the sample to be tested and the HRP-labeled deer insulin-like growth factor 1 (IGF-1) antibody to the enzyme-labeled well pre-coated with the deer insulin-like growth factor 1 (IGF-1) antibody. After incubation and washing, remove the unbound components, and then add substrates A and B to produce blue, which is converted into the final yellow under the action of acid. The depth of the color is positively correlated with the concentration of deer insulin-like growth factor 1 (IGF-1) in the sample.

8. Use the deer insulin-like growth factor 1 (IGF-1) standard to set different concentration gradients and prepare a standard curve using the same method.