CN113249420A - Method for improving bioactivity of giant salamander peptide, giant salamander peptide glycosylation product and application - Google Patents
Method for improving bioactivity of giant salamander peptide, giant salamander peptide glycosylation product and application Download PDFInfo
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Abstract
The invention provides a method for improving bioactivity of giant salamander peptide, a giant salamander peptide glycosylation product and application, and relates to the technical field of peptide modification. According to the method, deacetylated hyaluronic acid is combined with giant salamander peptide by using transglutaminase to obtain a giant salamander peptide glycosylation product, so that the aims of improving the tyrosinase inhibition activity of the giant salamander peptide and promoting the growth of human skin fibroblasts are fulfilled. Meanwhile, the deacetylated hyaluronic acid modified giant salamander peptide has good affinity for human skin cells, and has wide application prospects in the fields of medicines and cosmetics.
Description
Technical Field
The invention belongs to the technical field of peptide modification, and particularly relates to a method for improving bioactivity of giant salamander peptide, a giant salamander peptide glycosylation product and application.
Background
The giant salamander peptide is a low molecular weight product obtained by hydrolyzing giant salamander protein with protease. The giant salamander peptide has strong antioxidant activity. The biological activity of the peptide can be improved by a modification method. For example, Xianchongrong, etc. utilizes transglutaminase to modify D-galactosamine to prepare the corn glycopeptide, so that the antioxidant activity of the corn peptide is obviously improved (Xianchongrong, Wangxiang, Jiangyuan, etc.. research on the anti-fatigue effect of D-galactosamine glycosylation modified corn peptide, China's Proc for grain and oil, 2021.02). The glycosylation modification of the peptide by the enzyme method has the advantages of strong reaction specificity, mild reaction conditions and safe application of reaction products.
Injection of small molecular hyaluronic acid and the like into the skin by an electronic syringe, i.e., hydro-phototherapy, is a micro-plastic treatment method that has prevailed in recent years. The mechanism is that small molecular hyaluronic acid is evenly injected into the dermis layer of the skin, and the effect of promoting the discharge of metabolites and pigments is achieved by utilizing the characteristic of combining the small molecular hyaluronic acid with water molecules, so that the effect of improving the texture of the skin is achieved. In contrast, low molecular weight hyaluronic acid exhibits greater bioactivity and is more easily penetrated into the dermis.
At present, reports of modifying giant salamander peptide by using small molecular hyaluronic acid so as to obtain the hyaluronic acid modified giant salamander peptide with better whitening and moisturizing effects are not found.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for improving bioactivity of a giant salamander peptide, a giant salamander peptide glycosylation product and an application thereof, wherein hyaluronic acid is used to modify the giant salamander peptide, so as to improve bioactivity of the giant salamander peptide and provide better affinity for skin cells of a human body.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for improving bioactivity of giant salamander peptide, which comprises the following steps: mixing deacetylated hyaluronic acid with the giant salamander peptide solution and transglutaminase for glycosylation reaction to obtain the giant salamander peptide glycosylation product.
Preferably, the method for preparing deacetylated hyaluronic acid comprises the following steps: mixing hyaluronic acid and chitin deacetylase for reaction for 12 hours, adding ethanol into a reaction system after the reaction is finished until the volume of the ethanol is 60-80% of the total volume of the system, centrifuging, drying and precipitating to obtain deacetylated hyaluronic acid.
Preferably, the hyaluronic acid has a relative molecular weight of less than 1X 105Da。
Preferably, the mass of the chitin deacetylase is 1% of the mass of the hyaluronic acid; the temperature of the mixing reaction is 45 ℃, and the pH value is 7.0-8.0.
Preferably, the mass ratio of the deacetylated hyaluronic acid to the giant salamander peptide in the giant salamander peptide solution is (1-2): 1;
the mass of the transglutaminase is 1% of the total mass of the deacetylated hyaluronic acid and the giant salamander peptide in the giant salamander peptide solution.
Preferably, after the glycosylation reaction, the method further comprises centrifugation, filtration, freezing and drying;
and the rotation speed of the centrifugation is 4000r/min, the time of the centrifugation is 15min, components with the molecular weight cutoff of not more than 300Da in the supernate are screened, and the components are frozen and then freeze-dried.
The invention also provides a giant salamander peptide glycosylation product prepared by the method and used for improving the bioactivity of the giant salamander peptide.
The invention also provides an application of the giant salamander peptide glycosylated product prepared by the method or the application of the giant salamander peptide glycosylated product in preparing whitening, moisturizing and antioxidant reagents.
Preferably, the agent comprises a food, a pharmaceutical or a cosmetic.
The invention also provides a reagent with whitening, moisturizing and antioxidation effects, and the effective components of the reagent are selected from the giant salamander peptide glycosylation product prepared by the method or the giant salamander peptide glycosylation product.
The invention provides a method for improving bioactivity of giant salamander peptide, wherein deacetylated hyaluronic acid is combined with the giant salamander peptide by using transglutaminase to obtain a giant salamander peptide glycosylation product, so that the aims of improving tyrosinase activity inhibition of the giant salamander peptide and promoting growth of human skin fibroblasts are fulfilled. Meanwhile, the deacetylated hyaluronic acid modified giant salamander peptide has good affinity for human skin cells, and has wide application prospects in the fields of medicines and cosmetics.
In the embodiment of the invention, the giant salamander peptide glycosylation products with different concentrations are used for co-incubation with human skin fibroblasts, and the result shows that the human skin fibroblasts are remarkably increased along with the increase of the concentration of the giant salamander peptide glycosylation products, namely, the giant salamander peptide glycosylation products can remarkably promote the proliferation of the human skin fibroblasts; meanwhile, as the concentration of the giant salamander peptide glycosylation product is increased, the tyrosinase inhibition rate is increased, and free radicals can be effectively eliminated, so that the giant salamander peptide glycosylation product can be applied to the fields of food, medicines and cosmetics.
Drawings
FIG. 1 is an infrared spectrum of a giant salamander peptide modified by low molecular weight hyaluronic acid according to the invention;
FIG. 2 is a graph showing the effect of low molecular weight hyaluronic acid modified giant salamander peptide on human skin fibroblasts.
Detailed Description
The invention provides a method for improving bioactivity of giant salamander peptide, which comprises the following steps: mixing deacetylated hyaluronic acid with the giant salamander peptide solution and transglutaminase for glycosylation reaction to obtain the giant salamander peptide glycosylation product.
The method for preparing deacetylated hyaluronic acid of the invention preferably comprises the following steps: mixing hyaluronic acid and chitin deacetylase for reaction for 12h, adding ethanol into a reaction system after the reaction is finished until the volume of the ethanol is 60-80% of the total volume of the system, centrifuging, drying and precipitating to obtain deacetylateHyaluronic acid of the group. The hyaluronic acid of the invention is preferably a hyaluronic acid with a small molecular weight, and the relative molecular weight is preferably less than 1 x 105Da. The mass of the chitin deacetylase of the present invention is preferably 1% of the mass of the hyaluronic acid. The temperature of the mixing reaction is preferably 45 ℃, the pH value is preferably 7.0-8.0, and under the condition, the relative activity of the chitin deacetylase to the colloidal chitin reaches 111.2%. The source of the chitin deacetylase is not particularly limited in the present invention, and in the examples, it is preferably prepared by the method of CN111172141A (patent name: A chitin deacetylase, Applicant: Tianjin scientific and technological university).
When the glycosylation reaction is carried out, the deacetylated hyaluronic acid is preferably added into the giant salamander peptide solution, the pH value is adjusted to 8.0, then transglutaminase is added, and the mass ratio of the deacetylated hyaluronic acid to the giant salamander peptide in the giant salamander peptide solution is preferably (1-2): 1; and the mass of the transglutaminase is preferably 1% of the total mass of the deacetylated hyaluronic acid and the giant salamander peptide in the giant salamander peptide solution. After the glycosylation reaction system is prepared, the film is preferably sealed and put into a constant temperature water bath oscillator at 44 ℃ to start glycosylation reaction for 7 hours. After the glycosylation reaction is finished, cooling reaction liquid to room temperature, preferably, centrifuging, filtering, freezing and drying; the rotation speed of the centrifugation is preferably 4000r/min, the time of the centrifugation is preferably 15min, components with the molecular weight cutoff of not more than 300Da in the supernate are screened, and the components are frozen and then freeze-dried. The invention preferably passes the supernatant fluid obtained after through a nanofiltration membrane with the molecular weight cutoff of 300Da, freezes the filtered solution in a refrigerator, freezes and dries to obtain the giant salamander peptide glycosylation product (also called as the low molecular weight hyaluronic acid modified giant salamander peptide).
The invention also provides the giant salamander peptide glycosylation product for improving the bioactivity of the giant salamander peptide, which is prepared by the method, and the product is powdery, easy to dissolve in water and insoluble in ethanol.
The infrared spectrogram of the giant salamander peptide glycosylation product (low-molecular-weight hyaluronic acid modified giant salamander peptide) is shown in figure 1, and is 3428cm-1The peaks are OH stretching vibration peak and NH stretching peak; 1774cm-1The peak is C ═ O stretching peak; 1408cm-1Peak is COO-Symmetrical telescopic peaks; 618cm-1The peak is an amide characteristic peak; the low molecular weight hyaluronic acid modified giant salamander peptide has higher bioactivity than that of the giant salamander peptide, can promote the remarkable increase of human skin fibroblasts, remarkably inhibit the activity of tyrosinase, remarkably improve the antioxidation effect of the tyrosinase, and can be used for preparing foods, medicines or cosmetics with whitening, moisturizing and antioxidation functions.
The invention also provides an application of the giant salamander peptide glycosylated product prepared by the method or the application of the giant salamander peptide glycosylated product in preparing whitening, moisturizing and antioxidant reagents.
The agent of the present invention preferably comprises a food, a pharmaceutical or a cosmetic.
The invention also provides a reagent with whitening, moisturizing and antioxidation effects, and the effective components of the reagent are selected from the giant salamander peptide glycosylation product prepared by the method or the giant salamander peptide glycosylation product.
The method for improving bioactivity of giant salamander peptide, the giant salamander peptide glycosylation product and the application provided by the invention are described in detail in the following with reference to the examples, but they should not be construed as limiting the scope of the invention.
Example 1
1g of low molecular weight hyaluronic acid (relative molecular weight less than 1X 10)5Da) at 45 deg.C, pH7.0, adding chitin deacetylase with a substrate mass of 1% to react for 12 h. After the reaction is finished, adding ethanol solution to 70% (V/V), centrifuging at 3000rpm for 20min to obtain precipitate, and lyophilizing to obtain deacetylated hyaluronic acid.
2. Deacetylated hyaluronic acid is added into 100ml of a giant salamander peptide solution (giant salamander peptide powder + water, the giant salamander peptide powder is purchased from Jinchi giant salamander biotechnologies Co., Ltd., Zhang Jia, China) with the concentration of 0.05g/ml, wherein the mass ratio of the giant salamander peptide to the deacetylated hyaluronic acid is 1: 2. After the pH value is adjusted to 8.0, transglutaminase with the mass ratio of 1% is added, and the membrane is sealed and put into a constant temperature water bath oscillator with the temperature of 44 ℃ to start the glycosylation reaction for 7 hours. And after the reaction is finished, cooling the reaction solution to room temperature, centrifuging at 4000r/min for 15min, taking the supernatant, passing the supernatant through a nanofiltration membrane with the molecular weight cutoff of 300Da, freezing the concentrated solution in a refrigerator, and freeze-drying to obtain the small-molecular-weight hyaluronic acid modified giant salamander peptide.
Example 2
Taking human skin fibroblast in logarithmic growth phase, digesting with trypsin to obtain cell density of 4 × 104Suspension of one/ml. Inoculating 100 μ l cell suspension into 96-well plate, placing at 37 deg.C and 5% CO2Culturing in an incubator. When the adherent growth of the cells is good and the cell density reaches about 50%, the low-molecular-weight hyaluronic acid modified giant salamander peptide with different concentrations prepared in the example 1 is added respectively for co-incubation. Mu.l of MTS was added to each well over 48h and after mixing, co-incubation in the incubator was continued for 3.5 h. The absorbance value of 450nm is measured by using an enzyme-labeling instrument, the cell survival rate is calculated, and the result is shown in table 1 and figure 2, the human skin fibroblasts are obviously increased along with the increase of the concentration of the low molecular weight hyaluronic acid modified giant salamander peptide, which shows that the low molecular weight hyaluronic acid modified giant salamander peptide provided by the embodiment of the invention can obviously promote the proliferation effect of the human skin fibroblasts.
Percent cell survival ═ 100% (OD low molecular weight hyaluronic acid modified giant salamander peptide group/OD blank control group) ×, formula I.
TABLE 1 Effect of low molecular weight hyaluronic acid modified giant salamander peptide on cell survival
Example 3
The reagents shown in table 2 are prepared to measure the tyrosinase inhibition rate, the measured tyrosinase inhibition rate is shown in table 3, and the low-molecular-weight hyaluronic acid modified giant salamander peptide has obvious tyrosinase activity inhibition.
Tyrosinase inhibition rate ═ a [ ("a3-A4)/(A1-A2)]X 100%, formula II.
TABLE 2 tyrosinase inhibition rate determination method
TABLE 3 tyrosinase inhibition rates for different samples
Example 4
Free radical scavenging ability of low molecular weight hyaluronic acid modified giant salamander peptide prepared in examples
Experimental groups: preparing sample solutions with different concentrations (0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL and 1.0mg/mL) of the low-molecular-weight hyaluronic acid modified giant salamander peptide obtained in example 1 by using deionized water, respectively taking 2mL of the sample solutions with different concentrations into a test tube, adding 2mL of the prepared DPPH solution (0.004g of DPPH powder is added into a 50mL volumetric flask, the volume is determined by using 95% ethanol, and the mixture is stored at 4 ℃ in a dark place), uniformly mixing, and keeping the mixture at room temperature for 30min in a dark place. Centrifuging at 10000rpm for 10min, collecting supernatant, and measuring absorbance A at 517nmjSimultaneously measuring the absorbance A of 2ml of different sample solutions added with 2ml of 95% ethanoliAbsorbance A of 2ml DPPH plus 2ml distilled Water0。
DPPH radical scavenging ratio (%) - [ A [ ]0-(Aj-Ai)]/A0X 100%, formula III;
in the formula: a. the0Absorbance values for the blank control; a. thejThe light absorption value of the sample liquid; a. theiIs the absorbance of itself.
The control sample is small molecular weight hyaluronic acid (relative molecular weight less than 1 × 10)5Da) and giant salamander peptides.
The results are shown in table 4, the low molecular weight hyaluronic acid modified giant salamander peptide has a higher DPPH free radical clearance rate and a higher antioxidant activity than the giant salamander peptide or the low molecular weight hyaluronic acid.
TABLE 4 DPPH radical scavenging ratio of different samples
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for improving bioactivity of giant salamander peptide is characterized by comprising the following steps: mixing deacetylated hyaluronic acid with the giant salamander peptide solution and transglutaminase for glycosylation reaction to obtain the giant salamander peptide glycosylation product.
2. The method of claim 1, wherein the deacetylated hyaluronic acid is prepared by a method comprising: mixing hyaluronic acid and chitin deacetylase for reaction for 12 hours, adding ethanol into a reaction system after the reaction is finished until the volume of the ethanol is 60-80% of the total volume of the system, centrifuging, drying and precipitating to obtain deacetylated hyaluronic acid.
3. The method of claim 2, wherein the hyaluronic acid has a relative molecular weight of less than 1 x 105Da。
4. The method of claim 2, wherein the mass of the chitin deacetylase is 1% of the mass of the hyaluronic acid; the temperature of the mixing reaction is 45 ℃, and the pH value is 7.0-8.0.
5. The method according to claim 1, wherein the mass ratio of the deacetylated hyaluronic acid to the giant salamander peptide in the giant salamander peptide solution is (1-2): 1;
the mass of the transglutaminase is 1% of the total mass of the deacetylated hyaluronic acid and the giant salamander peptide in the giant salamander peptide solution.
6. The method of claim 1, further comprising centrifugation, filtration, and drying after freezing after the glycosylation reaction;
and the rotation speed of the centrifugation is 4000r/min, the time of the centrifugation is 15min, components with the molecular weight cutoff of not more than 300Da in the supernate are screened, and the components are frozen and then freeze-dried.
7. The giant salamander peptide glycosylation product capable of improving the bioactivity of the giant salamander peptide, prepared by the method of any one of claims 1 to 6.
8. The giant salamander peptide glycosylation product prepared by the method of any one of claims 1 to 6 or the application of the giant salamander peptide glycosylation product of claim 7 in preparing whitening, moisturizing and antioxidant agents.
9. Use according to claim 8, wherein the agent comprises a food, a pharmaceutical or a cosmetic.
10. An agent with whitening, moisturizing and antioxidant effects, wherein the active ingredients of the agent are selected from giant salamander peptide glycosylation products prepared by the method of any one of claims 1 to 6 or giant salamander peptide glycosylation products of claim 7.
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