CN117024575B - Acylated collagen, acylated collagen preparation and application thereof - Google Patents

Acylated collagen, acylated collagen preparation and application thereof Download PDF

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CN117024575B
CN117024575B CN202311241251.4A CN202311241251A CN117024575B CN 117024575 B CN117024575 B CN 117024575B CN 202311241251 A CN202311241251 A CN 202311241251A CN 117024575 B CN117024575 B CN 117024575B
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acylated collagen
anhydride
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彭新生
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Dongguan Collagen Biotechnology Co ltd
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Abstract

The invention relates to the technical field of natural active collagen modification, in particular to an acylated collagen, an acylated collagen preparation and application thereof, wherein the acylated collagen is obtained by modifying acid anhydride, the natural active collagen is dissolved in an acid solution, the pH is regulated, a certain amount of acid anhydride is added, stirring is required in the reaction process, the pH is regulated and a certain temperature is maintained, the stable environment is ensured in the reaction process, dialysis or ultrafiltration is carried out after the reaction is finished, and the acid anhydride modified acylated collagen is obtained after freeze drying and preservation. The acylated collagen preparation contains acylated collagen, and the acylated collagen preparation is used in preparing medicine or cosmetics for resisting inflammation, resisting oxidation, whitening skin and promoting collagen secretion. The acylated collagen prepared by the invention has good water solubility, retains the triple helix structure and biological activity of natural active collagen, has a certain potential for promoting cell proliferation and cell migration, and can be used for cell culture.

Description

Acylated collagen, acylated collagen preparation and application thereof
Technical Field
The invention relates to the technical field of natural active collagen modification, in particular to an acylated collagen, an acylated collagen preparation and application thereof.
Background
Collagen is a biopolymer protein with a special triple helix structure and widely distributed in tissues such as skin, bones, tendons and the like. The protein with the unique triple helix structure can promote cell growth and tissue repair, has high biocompatibility, biodegradability and other physical and chemical properties, and is widely applied to the fields of biomedical materials, drug delivery carriers, tissue engineering, cosmetics, foods and the like. The triple helix structure of collagen is the basis of its biological activity and physicochemical properties. However, in many cases of collagen application, the problem of use in neutral environment cannot be avoided, i.e., it is required that collagen exists stably in a solution state under neutral conditions, and at the same time, the natural triple helix structure of collagen is not altered or destroyed to retain good biological activity. But some chemical and physical modifications may disrupt some or all of the triple helix structure.
The Chinese patent publication No. CN114874316A discloses a water-soluble collagen and application thereof, the method utilizes the coding gene of the water-soluble collagen to be introduced into an expression host strain to obtain recombinant bacteria, cultures the recombinant bacteria and induces the protein to express, extracts and purifies the protein, and the method is realized through the induction of the coding group, has high operation difficulty and cost and is difficult to put into industrialization.
The Chinese patent publication No. CN105949477A discloses a water-soluble undenatured collagen and a preparation method thereof, wherein the method utilizes the reaction of polycarboxylic acid-NHS ester and natural collagen to prepare the water-soluble undenatured collagen, and the method is feasible, but the method for preparing the polycarboxylic acid-NHS ester is complex, and uses less organic solvent, and the collagen is gelled due to the excessively high esterification degree.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the invention aims to provide acylated collagen, an acylated collagen preparation and application thereof.
The aim of the invention is achieved by the following technical scheme: an acylated collagen, a method of preparing the acylated collagen, comprising the steps of:
(1) Dissolving the freeze-dried collagen in an acid solution, and then adjusting the pH to 8.5-9.5 with the alkali solution;
(2) Adding anhydride while stirring, regulating pH to 8.5-9.5 with alkali solution during reaction, maintaining the temperature at 20-30deg.C, and reacting for 2-4 hr;
(3) After the reaction is finished, collecting the solution, putting the solution into a dialysis bag, and dialyzing with deionized water for 3-7d, or purifying the solution by ultrafiltration for 2-8h;
(4) And (5) freeze-drying after purification to obtain the acid anhydride modified acylated collagen.
Preferably, in the step (1), the acid solution is acetic acid, hydrochloric acid, nitric acid, boric acid, phosphoric acid solution and combinations thereof with pH of 1.5-3.5, and the concentration of collagen is 1-5mg/mL.
Preferably, in the step (2), the acid anhydride is a C2-C20 linear alkyl acid anhydride, a branched alkyl acid anhydride, a cycloalkyl acid anhydride, an aromatic acid anhydride, or a combination thereof, and the mass ratio of the acid anhydride to the collagen is 1:0.2-1.0. Alternatively, the anhydride is selected from the group consisting of formic anhydride, acetic anhydride, propionic anhydride, butyric anhydride, isobutyric anhydride, valeric anhydride, caproic anhydride, heptanoic anhydride, caprylic anhydride, oxalic anhydride, lactic anhydride, acrylic anhydride, succinic anhydride, adipic anhydride, maleic anhydride, itaconic anhydride, citraconic anhydride, benzoic anhydride, phthalic anhydride, and combinations thereof. More preferably, the anhydride is at least one of oxalic anhydride, lactic anhydride, succinic anhydride, maleic anhydride, and itaconic anhydride.
Preferably, in the step (1) and the step (2), the alkaline solution is an alkali metal hydroxide, an alkaline salt of an organic or inorganic acid, an amine, an alkaline buffer, or a combination thereof. Alternatively, the alkali metal hydroxide is selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, and combinations thereof. The basic salt of an organic or inorganic acid is selected from the group consisting of sodium borate, sodium carbonate, sodium phosphate, potassium borate, potassium carbonate, potassium phosphate, sodium acetate, sodium citrate, and combinations thereof. The amine is selected from the group consisting of diethylamine, triethylamine, butylamine, 1, 2-ethylenediamine, triethanolamine, propylamine, dipropylamine, diethanolamine, monoethanolamine, isobutylamine, diisopropylamine, tert-butylamine, dibutylamine, diisobutylamine, tributylamine, pentylamine, dipentylamine, and combinations thereof. The buffer is selected from the group consisting of carbonate buffer, phosphate buffer, borate buffer, tris buffer, glycine buffer, and combinations thereof. More preferably, the alkali solution is at least one of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, triethylamine and sodium bicarbonate/sodium carbonate buffer solution.
Preferably, in the step (3), the molecular weight cut-off of the dialysis bag is 8000-14000.
The modification principle of the preparation method of the invention is as follows: the epsilon-amino group of the collagen side chain can lead succinic anhydride to open a ring, form an amide bond with the succinic anhydride, and change the other end of the succinic anhydride into a carboxyl group. The results show that the hydrophilic group of succinic anhydride is introduced into the collagen, and the long carbon chain lipophilic group is introduced, so that the collagen becomes a high molecular surfactant with bipolar groups, and the water solubility of the collagen is obviously improved.
Preferably, the acylated collagen has good water solubility, and retains the triple helix structure and biological activity of the naturally active collagen.
Preferably, the modification site of the acylated collagen is an N-terminal amino group and/or an epsilon-amino group.
Preferably, the grafting rate of the acylated collagen is 70-80%. The acylated collagen triple helix structure within the grafting ratio range has high integrity and good water solubility.
Preferably, the acylated collagen has an isoelectric point of 4.1-4.2.
The other object of the invention is achieved by the following technical scheme: an acylated collagen preparation comprising the acylated collagen described above.
The invention also aims at realizing the following technical scheme: use of an acylated collagen preparation in the preparation of an anti-inflammatory drug or cosmetic, or in the preparation of an antioxidant drug or cosmetic, or in the preparation of a whitening drug or cosmetic, or in the preparation of a drug or cosmetic that promotes collagen secretion.
Preferably, the acylated collagen preparation is prepared as a powder, microsphere, flake, fiber, fabric, pad, film, membrane, sponge, wound dressing, ointment or gel for medical or cosmetic applications.
The invention has the beneficial effects that: the acylated collagen has good water solubility, retains the triple helix structure and biological activity of natural active collagen, has certain potential for promoting cell proliferation and cell migration, and can be used for cell culture.
Compared with the prior art CN114874316A, the preparation method of the acylated collagen utilizes a chemical modification method to modify the natural active collagen, has a triple helix structure of the natural active collagen, and does not need to carry out coding group induction.
Compared with the prior art CN105949477A, the preparation method of the acylated collagen can control the proper grafting rate according to the using amount of the anhydride, does not use an organic solvent in use, and is safe and environment-friendly.
The acylated collagen preparation contains acylated collagen, so that the number of the zebra fish neutrophils is remarkably reduced, and the acylated collagen preparation has a relieving effect and can be used for preparing anti-inflammatory drugs or cosmetics; the fluorescence of zebra fish embryos can be remarkably reduced, which indicates that the acylated collagen preparation has an antioxidation effect and can be used for preparing antioxidation medicines or cosmetics; and has whitening effect on zebra fish embryo, and can be used for preparing whitening medicine or cosmetics; can also remarkably promote the secretion of the type I collagen, and can be used for preparing medicines or cosmetics for promoting the secretion of the collagen.
Drawings
FIG. 1 is a schematic representation of a lyophilized acylated collagen made in accordance with the present invention.
FIG. 2 is a CD spectrum of the acylated collagen prepared by the present invention and natural active collagen, wherein 1 is natural active collagen and 2 is acylated collagen.
FIG. 3 is an infrared spectrum of the acylated collagen prepared by the invention and natural active collagen, wherein 1 is natural active collagen and 2 is acylated collagen.
FIG. 4 is a SDS-PAGE diagram of acylated collagen prepared according to the present invention and native active collagen, wherein lanes 1-6 represent, respectively: protein markers, natural active collagen 1, natural active collagen 2, acylated collagen 1, acylated collagen 2, and acylated collagen 3.
FIG. 5 is an isoelectric point measurement of the acylated collagen produced by the present invention.
FIG. 6 is a graph showing the clarity of the acylated collagen produced by the present invention in solution at different pH's, wherein A is acylated collagen and B is natural active collagen.
FIG. 7 is a graph showing the comparison of the reaction between the acylated collagen produced by the present invention and a natural active collagen dye liquor.
FIG. 8 is a graph showing the experimental results of CCK-8 of the acylated collagen prepared by the present invention and natural active collagen (culture 1 d).
FIG. 9 is a graph showing the experimental results of CCK-8 of the acylated collagen prepared in the present invention and natural active collagen (culture 3 d).
FIG. 10 is a graph showing the cell scratch treatment of the acylated collagen and the natural active collagen prepared by the method, wherein A is the acylated collagen and B is the natural active collagen.
FIG. 11 is a graph showing the anti-inflammatory results of succinyl-end-deficient collagen solutions of the present invention on various groups of zebra fish.
FIG. 12 is a fluorescence microscopy image of zebra fish for each concentration group of succinyl-end-deficient collagen solutions of the present invention.
FIG. 13 is a graph showing a target region (dotted line region) for quantitative analysis of melanin in zebra fish according to the present invention.
FIG. 14 is a graph showing the relative cell viability of succinyl-end-deficient collagen solutions according to the present invention.
FIG. 15 is a morphological observation of succinyl-terminal collagen solutions of the present invention.
Detailed Description
The present invention is further described below with reference to examples and fig. 1-15 for the purpose of facilitating understanding of those skilled in the art, and the description of the embodiments is not intended to limit the invention.
The collagen adopted by the embodiment of the invention is cow leather collagen, the collagen is atelopeptide collagen, the succinic anhydride reaction site is N-terminal amino and epsilon-amino, and compared with the telopeptide collagen, the collagen has higher and more stable modification degree.
The reaction of the invention is carried out in alkaline environment, -NH 2 In a free state, the acid anhydride is easy to react with the collagen, and the reaction efficiency can be maximized. The applicant found that pH is an important factor affecting the acylation reaction, when pH is in an acidic environment, the degree of acylation is low, and the reaction solution is cloudy, when pH is in an alkaline environment, the degree of acylation is high, and the reaction solution is clear.
The dialysis referred to in the invention is a step of purifying the acylated collagen, and the purity of the acylated collagen is ensured to be higher before freeze-drying in order to remove unreacted complete succinic anhydride and byproducts as well as small molecule impurities.
Preparation of part of the reagents: 0.01mol/L dilute hydrochloric acid solution with pH 2.0: measuring 0.833mL of concentrated hydrochloric acid with mass fraction of 37% and adding water to fix volume to 1000mL; carbonate buffer at ph 9.6: to 1000mL of purified water, 1.59g of sodium carbonate and 2.93g of sodium bicarbonate were added, and the mixture was dissolved and mixed well.
Example 1
A preparation method of acylated collagen comprises dissolving natural active collagen in acetic acid solution with pH of 3.0 to obtain collagen solution with concentration of 3 mg/mL; then maintaining the temperature at 25 ℃ all the time, gradually adding 1000mg of succinic anhydride into 500mL of collagen solution, stirring and reacting for 3 hours, using sodium hydroxide solution to keep the pH value of the reaction solution at 9.0 all the time during the reaction, and dialyzing for 3d by using deionized water; freeze drying to obtain acylated collagen.
Example two
The present embodiment is different from the second embodiment in that: the natural active collagen was dissolved in a dilute hydrochloric acid solution having a pH of 2.0, and the pH was adjusted using a sodium bicarbonate/sodium carbonate buffer solution, and the obtained acylated collagen was more stable by the same procedure as in example 1.
Example III
A preparation method of acylated collagen comprises dissolving natural active collagen in hydrochloric acid solution with pH of 2.0 to obtain collagen solution with concentration of 2 mg/mL; then, maintaining the temperature at 28 ℃ all the time, gradually adding 760mg of succinic anhydride into 500mL of collagen solution, stirring and reacting for 3 hours, and during the reaction, using sodium bicarbonate/sodium carbonate buffer solution to keep the pH value of the reaction solution at 9.0 all the time, and then using deionized water to dialyze for 3 days; and freeze-drying to obtain acylated collagen, wherein the freeze-dried sample is shown in figure 1. The grafting rate of the prepared acylated collagen is 74.65 percent through detection.
Comparative example one
The present embodiment is different from the third embodiment in that: 1140mg of succinic anhydride was dissolved in 500mL of collagen solution. The grafting rate of the prepared acylated collagen is 93.25% by detection, and the different amounts of succinic anhydride used in the example result in different grafting rates.
Example IV
A preparation method of acylated collagen comprises dissolving natural active collagen in nitric acid solution with pH of 1.5 to obtain collagen solution with concentration of 1 mg/mL; then maintaining the temperature at 20 ℃ all the time, gradually adding 350mg of oxalic anhydride into 500mL of collagen solution, stirring and reacting for 2h, and during the reaction, keeping the pH value of the reaction solution at 8.5 all the time by using potassium hydroxide solution, and dialyzing for 4d by using deionized water, or purifying by ultrafiltration for 3h; freeze drying to obtain acylated collagen.
Example five
A preparation method of acylated collagen comprises dissolving natural active collagen in boric acid solution with pH of 2.5 to obtain collagen solution with concentration of 4 mg/mL; then maintaining the temperature at 22 ℃ all the time, gradually adding 1500mg of maleic anhydride into 500mL of collagen solution, stirring and reacting for 2.5h, and during the reaction, using sodium carbonate solution to keep the pH value of the reaction solution at 8.8 all the time, and then dialyzing for 5d by using deionized water, or purifying by ultrafiltration for 5h; freeze drying to obtain acylated collagen.
Example six
A preparation method of acylated collagen comprises dissolving natural active collagen in phosphoric acid solution with pH of 3.5 to obtain collagen solution with concentration of 5 mg/mL; then maintaining the temperature at 30 ℃ all the time, gradually adding 1850mg of itaconic anhydride into 500mL of collagen solution, stirring and reacting for 4h, and during the reaction, keeping the pH value of the reaction solution at 9.0 all the time by using triethylamine solution, and dialyzing for 6d by using deionized water, or purifying by ultrafiltration for 7h; freeze drying to obtain acylated collagen.
1. Characterization of the acylated collagen produced by the present invention:
1. CD characterization: the natural active collagen and the acylated collagen prepared in the third embodiment (substantially, the same detection results are obtained in other embodiments) are subjected to circular dichroism chromatography comparison characterization, the result is shown in fig. 2, a circular dichroism chromatography mark 1 curve is the natural active collagen extracted by an acid enzyme combination method, a mark 2 curve is the acylated collagen prepared by the invention, and as can be seen from fig. 2, the natural active collagen and the acylated collagen prepared by the invention have a negative peak at 190nm and a positive peak at 220nm, which indicates that the acylated collagen prepared by the invention still has a unique triple helix structure of the collagen.
2. IR characterization: the result is shown in figure 3, and the result shows that the acylated collagen prepared by the invention still maintains the characteristic peaks of five functional groups, namely amide A, amide B, amide I, amide II and amide III, respectively, and maintains the triple helix structure of the collagen. The collagen from different sources is folded in different modes, different intramolecular and intermolecular hydrogen bond interactions are generated, and the wave number position of the absorption peak is influenced by the strength of the hydrogen bond. Amide I, amide II and amide III are closely related to the peptide chain skeleton structure and indicate the condition of the triple helix structure of the collagen.
3. And (3) electrophoresis detection: the results are shown in FIG. 4, which shows that lanes 1-6 represent, respectively: protein markers, natural active collagen 1, natural active collagen 2, acylated collagen 1, acylated collagen 2, acylated collagen 3; as can be seen from fig. 2, the acylated collagen prepared by the present invention consists of two α1 chains and one α2 chain and β chain, as in the naturally active collagen. It also shows that acylation does not disrupt the unique triple helix structure of the native active collagen. In addition, the bands of acylated collagen prepared by the invention migrate upward, indicating that the reaction of a portion of the collagen molecules with the anhydride increases a portion of the molecular weight.
4. Isoelectric point detection: as shown in FIG. 5, the isoelectric point of the acylated collagen prepared by the invention is 4.2, and compared with the neutral isoelectric point of the natural active collagen, the isoelectric point is reduced, so that the water solubility under the far neutral condition can be obviously improved.
5. And (3) dissolution detection: as shown in fig. 6, when comparing the collagen solution with the acylated collagen solution at different pH, it can be observed that the acylated collagen prepared by the present invention is still clear and transparent in the neutral pH solution, but the natural active collagen is cloudy in the neutral pH solution.
6. And (3) dyeing detection: the results of the detection of natural active collagen, acylated collagen and gelatin by using sirius red are shown in fig. 7, and the results show that both the natural active collagen and the acylated collagen have fiber floccules, while blank groups and gelatin groups have no fiber floccules, so that the acylated collagen prepared by the invention has a complete triple helix structure similar to the natural active collagen.
2. Drug test of the acylated collagen prepared by the invention
The acylated collagen prepared in example III (and in essence, other examples gave the same test results) was dissolved as a drug in a culture medium for relevant cell experiments such as CCK-8 and cell scoring experiments.
1. The specific experimental procedure of CCK-8 is as follows:
(1) CCK-8 experiments were performed using the fibroblast cell line L929;
(2) L929 in the flask was digested into 96-well plates, and the cell density required for each well of the 96-well plates was determined according to the number of days of culture. (cell density is about 5X 10) 3 -1×10 4 );
(3) Culturing L929 cells in an incubator for 1d, and then culturing L929 cells in the incubator for 1-7d by taking acylated collagen solutions with different concentrations as medicines;
(4) After enough culture days, removing the supernatant, adding 100uL of CCK-8 solution into each hole, placing into an incubator for 2 hours, and measuring by using an enzyme-labeled instrument;
(5) According to analysis of the measurement results (see fig. 8-9), the acylated collagen-cultured L929 cells were not significantly different from the basal medium and the complete medium-cultured cells, demonstrating the potential of the acylated collagen to culture the cells.
2. The procedure for cell scratch experiments was as follows:
(1) cell scratch experiments with a migration competent fibroblast line L929
(2) L929 cells in the flask were seeded into 6-well plates with a cell density of about 5X 10 per well 5 -8×10 5 Post-plating culture was carried out overnight.
(3) Observing the cell density of each hole, ensuring no gap basically between cells, namely, scraping the original culture solution after scraping by using a 10 mu L gun head (straight line is drawn by using iron) and cleaning the culture solution for 3 times by using PBS.
(4) The acylated collagen and the collagen mixed culture solution are added into each hole, the mixture is carefully shaken uniformly, and the mixture is photographed every 12 hours and cultured in an incubator. As shown in FIG. 10, the acylated collagen prepared by the invention has a certain potential for promoting cell proliferation and cell migration, and can be used for cell culture.
3. Application of acylated collagen preparation of the invention
The application of the acylated collagen preparation in preparing anti-inflammatory drugs or cosmetics, or in preparing antioxidant drugs or cosmetics, or in preparing whitening drugs or cosmetics, or in preparing drugs or cosmetics for promoting collagen secretion is disclosed. The acylated collagen preparation is prepared as a powder, microsphere, flake, fiber, fabric, pad, membrane, septum, sponge, wound dressing, ointment or gel for medical or cosmetic applications.
The acylated collagen preparation adopted in the embodiment of the invention is concentrated solution of acylated collagen, wherein the mass concentration of the acylated collagen is 2mg/mL, and the specific name is succinyl end-missing collagen solution. Of course, other effective components of medicines (such as traditional Chinese medicine extracts for bacteriostasis, whitening and the like), other medicines (Chinese patent medicines or western medicines) or auxiliary materials (such as auxiliary agents of surfactants and the like) and the like can be added into the acylated collagen preparation according to the requirements.
A. Anti-inflammatory test
1. Principle of
Inflammation is a reaction of the immune system to tissue injury and infection and is mainly characterized by the accumulation of leukocytes (granulocytes, macrophages) around the infected tissue. The immune system of zebra fish is very similar to mammals. When a wound occurs, neutrophils and macrophages respond to traumatic inflammation almost simultaneously, the migration speed of the neutrophils is high, the neutrophils are firstly recruited to the damaged part, and then the macrophages reach the damaged part. After several hours, the inflammation began to subside, and macrophages and neutrophils left the injury site. Copper sulfate damages the nerve dome of the zebra fish (the peripheral organ of the lateral line device of the body surface of the zebra fish), causes death of the nerve dome cells, and neutrophils of the zebra fish generate immune response, gather around the nerve dome and phagocytize the dead cells.
Zebra fish CuSO 4 The inflammation model is formed by CuSO 4 Model of induced acute inflammation. Copper is a functional component of the innate immune system, and actively regulates inflammatory response by inducing oxidative stress through Reactive Oxygen Species (ROS) and the like. CuSO in young and adult zebra fish 4 Stimulation can cause zebra fish immune cells to migrate to nerve hills rapidly, thus CuSO 4 Exposure is often used to induce and mimic inflammatory features. Unlike physical injury and infectious agent methods, copper as an inflammatory agent can be handled using non-invasive methods. Some anti-inflammatory drugs can inhibit migration of immune cells to the nerve dome, and can reflux the immune cells, and the anti-inflammatory activity of the drugs can be evaluated by using the model. I.e. adding CuSO 4 Inducing immune response of inflammatory cells in transgenic zebra fish (such as mpo, lyz, mpeg and other strains), adding the test sample, analyzing the inflammatory cell number of the inflammatory site (nerve dome site behind the colonisation hole) under a fluorescence microscope, and comparing with a model group so as to reflect the relieving efficacy of the test sample.
2. Experimental materials and apparatus
2.1 test materials: zebra fish (Danio rerio): the neutral grain transgenic strain Tg (MPO: GFP) and F1 generation are purchased from the national zebra fish resource center, and 2 pairs of non-Fl parent fish are bred and spawned after being bred and cultured by Bode research (Guangzhou) biotechnology limited company, and the spawning batch is 202307180.
2.2 test equipment: zebra fish farming systems (Hua Xi organisms), thermostated incubators (Yamato), pipette guns (thermo scientific), fluorescence microscopy (SOPTOP), mini vortexing.
3. Test method
3.1H-Buffer solution preparation: 7.000g of a sodium chloride, 0.400g of b sodium bicarbonate, 0.100g of c potassium chloride and 0.235g of d calcium chloride are weighed; the four reagents were added sequentially into a 2L beaker and the volume was set to 2000mL with ultrapure water.
3.2NOEC concentration exploration
The test adopts a method of a DECT 236 fish acute toxicity test, and the maximum safe concentration of the zebra fish fed in the test is screened out by performing zebra fish embryo 72h acute toxicity detection. Test set-up blank (H-Buffer solution), sample concentration set-up is shown in Table 1, with 1 well per concentration set-up. The specific operation steps are as follows:
1) Succinyl-terminated collagen solutions were diluted to 0.625%, 1.250%, 2.500%, 5.000%, 10.000%, 20.000% by volume (V/V) and test substances were prepared at different concentrations.
2) Plating and sample adding: 10 fertilized eggs of 24hpf, which were consistently healthy in development, were randomly taken from each of the 24-well plates, wherein 2mL of H-Buffer solution was added to one well, and sample solutions of different concentrations were added to the other well (see Table 1).
3) And (3) detection: culturing the fertilized eggs of the zebra fish in the 2) in a temperature incubator at 28 ℃ until 72hpf and observing the malformation and death of the zebra fish embryo under an optical microscope. The concentration group at which no malformations of the zebra fish embryos of 72+2hpf occurred was determined as NOEC concentration.
4) The zebra fish malformation rate calculation formula:
3.3 formal test
3.3.1 method for preparing test solution
Mother liquor: taking 1mL of succinyl end-lacking collagen solution, adding 9mL of H-Buffer solution, and putting into a vortex instrument for vortex oscillation dissolution to obtain mother solution with the theoretical concentration of 10.00%. It is ready for use.
Test solution: the mother solution was diluted with H-Buffer solution to obtain working solutions with theoretical mass concentrations of 0.313%, 0.625% and 1.250% for ready use.
3.3.2 test procedure
10 zebra fish embryos developed to 72hpf were randomly allocated to each well in a 24-well plate, and 3 duplicate wells were made for each concentration. The H-Buffer solution is NC (blank control) group; the 5 μm dexamethasone solution was the PC (positive control) group; the 20. Mu.M copper sulfate solution is a molding agent solution and is a Model group; and groups of samples of different concentrations. The Buffer water was removed from the 24 well plate without damaging the embryos, then 2mL of SAM in dexamethasone was added rapidly to the PC group, 2m1 of sample solution at different concentrations was added to each well of the sample group, and 2mL of H-Buffer solution was added to the NC group and the Model group. Incubate in a biochemical incubator at (28.5 th.) ℃for 120min. After 120min incubation, the solution in the 24 well plate was removed without damaging the zebra fish embryos, 2mL of H-Buffet buffer solution was rapidly added to the NC group, 2mL of modeling agent solution was added to the Model group, and 2mL of modeling agent solution was added to the PC group and the sample group. The plate was covered and incubated in a biochemical incubator at (28.5 th.) ℃for 120min.
At least 5 animals were randomly selected from each group of normal phenotype and behavior zebra fish after incubation, fixed with 4% methylcellulose, photographed under a fluorescence microscope, with the zebra fish side facing down, head to left, and body kept horizontal.
After photographing is completed, selecting the part of the young zebra fish after the colonization hole, and counting the green fluorescence quantity by using IPP software, thereby counting the quantity of inflammatory cells.
4. Test results
4.1NOEL concentration exploration
TABLE 1 succinyl end-of-absence collagen solution 72h acute toxicity test deformity results summary table
4.2 anti-inflammatory results for each group of zebra fish are shown in detail in FIG. 11.
4.3 statistical analysis and processing of data
TABLE 2 summary of the results of zebra fish neutrophil counts
Remarks: when statistical analysis is performed using the single-factor variance, the significance of the Model group is represented by # with P < 0.05 being represented by #, P < 0.01 being represented by #, and P < 0.001 being represented by # # as compared to the NC group. The significance of the Model group was expressed as P < 0.05, P < 0.01, and P < 0.001, respectively, compared to the PC group and the sample group.
From the above table, the number of zebra fish neutrophils was significantly increased (P < 0.001) in the Model group compared with the NC group, while the number of zebra fish neutrophils was significantly decreased (P < 0.001) in the PC group compared with the Model group, indicating that the experiment was effective; the number of the zebra fish neutrophil granulocyte is extremely obviously reduced (P is less than 0.001) compared with the Model group, and the succinyl-end lack collagen solution-0.313%, the succinyl-end lack collagen solution-0.625% and the succinyl-end lack collagen solution-1.250% show that the succinyl-end lack collagen solution has a relieving effect at the concentration of 0.313%, 0.625% and 1.250%.
B. Antioxidation test
1. Principle of
Oxidative stress refers to the fact that when the body is subjected to various stimuli, the balance of the oxidation system and the antioxidation system in the body is broken, so that high-activity molecules such as reactive oxygen species (Reactive Oxygen Species, ROS) are excessively produced, and the oxidation degree exceeds the oxide scavenging capacity, thereby leading toThe state of cytoblast can be reflected by detecting ROS levels in the body to reflect whether the compound induces oxidative stress in the body. Under normal conditions, ROS in the body are in a dynamic balance state, and normal metabolism and immune function in the body are maintained. ROS are normal metabolites in zebra fish and can be used as specific dye H 2 DCFDA detection, zebra fish were treated with 4-tert-butyl hydroperoxide to create oxidative stress damage to zebra fish, resulting in a zebra fish peroxidation model. Adding cosmetics or cosmetic raw materials based on the model, and using H 2 The DCFDA method detects the antioxidant capacity of cosmetics or cosmetic raw materials.
2. Test materials and apparatus
2.1 test materials
Wild type AB-line zebra fish (Danio rerio), F1 generation is purchased from the national zebra fish resource center, and after being bred and cultured by Bode research (Guangzhou) biotechnology limited company, 2 pairs of non-F1 generation parent fish are bred and spawned, and spawning batch is 20230725.
2.2 test apparatus
Zebra fish culture system (Hua Xi organisms), incubator (Yamato), pipette (thereto scientific), stereo microscope (nikon), mini vortex.
3. Test method
Preparation of 3.1H-Buffer solution
7.000g of sodium chloride, 0.400g of sodium bicarbonate, 0.100g of potassium chloride and 0.235g of calcium chloride are weighed and dissolved in 2L of ultrapure water to prepare the chemical, wherein the pH value is 6.5-8.5, and the chemicals are all of analytical grade.
3.2NOEC concentration studies were identical to anti-inflammatory assays.
3.3 formal experiments
3.3.1 method for preparing test solution
Mother liquor: taking 1mL of succinyl end-lacking collagen solution, adding 9mL of H-B buffer solution, and putting into a vortex instrument for vortex oscillation dissolution to obtain mother solution with the theoretical concentration of 10.00 percent, and preparing for use.
Test solution: and diluting the mother solution by using an H-Buffer solution to obtain working solution with the theoretical mass concentration of 1.250 percent, and preparing the working solution for use at present.
3.3.2 test procedure
10 fertilized 24hpf zebra fish embryos of developmental health were randomly assigned to each well in 24 well plates, 30 tails per group (C3 parallel duplicate wells). 1mmol/L of 4-tert-butyl hydroperoxide solution was of the Model group; the H-Buffer solution is NC (blank control) group; the 20 mug/mL glutathione solution is PC (positive control) group; and 3 groups of sample solutions of different concentrations. The H-Buffer solution in the 24-well plate was removed without damaging the embryos, and then 0.2mL of 1 mmol/L4-t-butyl hydroperoxide solution was rapidly added to the Model group; adding 0.2mL of H-Buffer solution into the NC group; adding a mixed solution of 0.2mL of 4-t-butyl hydroperoxide and 20. Mu.g/mL of glutathione solution to the PC group; to the sample set was added 0.2mL of a mixed solution of 4-t-butylhydroperoxide and sample solutions of different concentrations. Incubate to 72 Shi 1hpf in a 28 Shi 1℃incubator.
After incubation, the 24-well plate was blotted without damaging the zebra fish embryos and 2m1 of 20 μmo1/L of specific dye H was added to each well 2 DCFDA was placed in a 28-bar incubator at 1℃for 1h.
Adding specific dyes H 2 After the DCFDA solution incubation is finished, the fish is placed under a fluorescence microscope to be excited for 15min, then the juvenile fish is washed by an H-Buffer solution for 3 times, and finally the fish embryo is placed sideways (with the head facing left). And photographing the fish embryo under a fluorescence microscope according to the uniform photographing parameters.
After photographing is completed, fluorescence intensity is analyzed by image J software, thereby counting ROS signal intensity.
4. Test results
4.1NOEC concentration studies were identical to anti-inflammatory assays.
4.2 oxidation resistance results of each group of zebra fish are shown in figure 12.
4.3 statistical analysis and processing of data
TABLE 3 summary of the results of fluorescence intensity (% of Model group) for each group of zebra fish embryos
Remarks: when statistical analysis is performed using the single-factor variance, the significance of the Model group is represented by # with P < 0.05 being represented by #, P < 0.01 being represented by #, and P < 0.001 being represented by # # as compared to the NC group. The significance of the Model group was expressed as P < 0.05, P < 0.01, and P < 0.001, respectively, compared to the PC group and the sample group.
As can be seen from the table above, the fluorescence of the zebra fish embryos is significantly increased (P < 0.001) in the Model group compared with the NC group, and significantly decreased (P < 0.01) in the PC group compared with the Model group, which indicates that the experiment is effective; the fluorescence of zebra fish embryos is extremely significantly reduced (P < 0.001) compared with the Model group by 1.250% of succinyl-end-deficiency collagen solution, which indicates that the succinyl-end-deficiency collagen solution has an antioxidation effect at the concentration of 1.250%.
C. Whitening experiment
1. Principle of
Zebra fish embryos are transparent throughout the body at the beginning of development, and melanin begins to grow from retinal epithelial cells when the embryo develops for 24 hours. Pigment cells originate from neural crest cells differentiated from the dorsal ectoderm and then proliferate, migrate, differentiate into pigment blast cells. The test agent can be used for inhibiting melanin formation of zebra fish. The test substance group incubated at constant temperature to the end point is compared with the blank control group, the intensity of melanin signal of the head of the zebra fish is expressed by an Optical Density (OD), the melanin generation amount of the test substance is calculated, and the whitening efficacy of the cosmetic product or raw material is evaluated.
2. Test organism and test apparatus
2.1 test organisms
The school name: zebra fish (Danio rerio)
Strain: wild type AB strain (national zebra fish resource center)
The source is as follows: f1 generation is purchased from the national zebra fish resource center, and 2 pairs of non-F1 generation parent fishes are bred and spawned after being bred and cultured by Bode research (Guangzhou) biotechnology limited company, and spawning batch is 20230721.
2.2 test apparatus
Zebra fish culture system (Hua Xi organisms), incubator (Yamato), pipette (tllermo scientific), stereo microscope (nikon), mini vortex.
3. Test method
3.1 preparation of standard dilution water: the self-prepared standard dilution water is adopted, and the following solutions are respectively prepared by ultrapure water: a. 11.76g/L of calcium chloride dihydrate, 4.93g/L of magnesium sulfate heptahydrate, 2.52g/L of sodium bicarbonate and 0.22g/L of potassium chloride are taken out, 25mL of each of the above four solutions are mixed and diluted to 1L with ultrapure water.
3.2 preparation of test solution for test substance
Mother liquor: taking 1mL of succinyl end-lacking collagen solution, adding 9mL of H-Buffer solution, and putting into a vortex instrument for vortex oscillation dissolution to obtain mother solution with the theoretical concentration of 10.00 percent, and preparing the mother solution for use.
Test solution: the mother solution was diluted with H-Buffer solution to obtain working solutions with theoretical mass concentrations of 0.313%, 0.625% and 1.250% for ready use.
3.3NOEC concentration studies were identical to anti-inflammatory assays.
3.4 formal experiments
3.4.1 test procedure
Randomly selecting and placing 10 normal 6hpf zebra fish embryos in each hole of a 24-hole plate, removing standard dilution water in the 24-hole plate under the condition of not damaging the embryos, rapidly adding 2mL of test solution of a test object with corresponding concentration, blank control (standard dilution water) and positive control (alpha-Xiong Guopu with the concentration of 9 mg/mL) into each hole, fully and uniformly mixing, covering a culture plate panel, wrapping with aluminum foil paper, and incubating in a biochemical incubator at the temperature of (28.5 th.) ℃ for 72 hours in a dark place to reach an incubation end point. Each group was run in 3 replicates and the highest sample concentration was not higher than the NOEC.
At least 18 zebra fish are randomly selected from the zebra fish with normal phenotype and behavior after incubation, fixed by 4% methyl cellulose, observed and photographed under a stereoscopic microscope, the zebra fish should face left, the abdomen face down and the body keeps horizontal when photographing, photographing of all the zebra fish should be completed under the same instrument and environmental conditions, and the positions of the zebra fish should be kept consistent.
After photographing is completed, the obtained zebra fish pictures are analyzed by using image analysis software, and the selected quantitative analysis target area is from the edge of the zebra fish head (except the eye part) to the tangent part with the yolk sac. Quantitative analysis of target area is shown in fig. 13. The intensity of melanin signal in the head of zebra fish is expressed as Optical Density (OD), and 15 pieces of effective data are taken from each group.
4. Test results
4.1NOEC concentration search results were the same as in the anti-inflammatory test.
4.2 hatchability, survival rate and total survival number of control groups
Table 4 number of hatching and survival tables (n=30) of zebra fish treated with succinyl-end-lacking collagen solution
The survival rate of fertilized eggs in NC (blank (dilution water) control) group is 100%, and the survival rate after successful hatching is more than 80% or the survival rate after hatching is more than 90%.
The survival rate of fertilized eggs in a PC (positive (9 mg/mL alpha-Xiong Guopu) control) group is 100%, and the survival rate after successful hatching is more than 80% or the survival rate after hatching is more than 90% according with requirements.
4.3 statistical analysis and processing of data
TABLE 5 summary of the values of the optical density of the melanin from each group of zebra fish and the p value from the blank group (p.ltoreq.0.05)
Group of experiments PC 0.313% 0.625% 1.250%
NC <0.001 *** 0.132 0.987 <0.001 ***
As can be seen from the above table, the blank control group has a very significant difference from the positive control, and the test system is considered to be established; the blank control group has a significant difference from succinyl-terminated collagen solution-1.250%, and the experiment proves that the succinyl-terminated collagen solution has a whitening effect on zebra fish embryos at the concentration of 1.250%.
D. Test for promoting collagen secretion
1. Principle of
During the skin aging process, the structure and function of the extracellular matrix (extracellular matrix, ECM) of the dermis layer are altered in a number of ways. Collagen fibers, elastin fibers and proteoglycans are important components of ECM that impart tensile strength, elasticity and hydration capability, respectively, to the skin. Of these, the lunar cross-hair proteins and elastin are mainly produced by fibroblasts in the dermis. Dermal fibroblasts synthesize type I procollagen in cells, secrete the procollagen out of the cells, separate terminal peptides under the action of terminal procollagen peptidase to form I-type collagen, and polymerize the type I collagen to form collagen fibers. The collagen fibers are aligned parallel to the skin surface, which gives the skin a high tensile strength and prevents tearing due to excessive stretching. Whereas elastin fibers are arranged in a sparsely distributed sub-epidermal network, providing good elasticity to the skin.
The human dermal fibroblasts can be used as a cell model for researching the improvement of the collagen I content of cosmetics, and whether the test substance is effective in promoting the collagen synthesis or not can be evaluated by measuring the up-regulation rate of the collagen I content after the administration of a blank control and the test substance. The method for measuring the content of the type I collagen adopts an enzyme-linked immunosorbent assay (ELISA) method, wherein the specific principle is that after the type I collagen is specifically combined with a collagen antibody coated on an ELISA plate, the type I collagen is combined with an anti-type I collagen antibody with a substrate label, a colored product is generated after the substrate is catalyzed by enzyme, the content of the type I collagen is positively correlated with the color depth of the colored product, and the optical density (OD value) is measured at the wavelength of 450nm by using an ELISA instrument, so that the content of the type I collagen is calculated.
Elastase has the ability to degrade various proteins such as collagen and elastin. Degradation of elastin in skin tissue by elastase is closely related to the skin aging process, and thus, it is one of the important ways to delay skin aging to counter the degradation of elastin by elastase and restore skin elasticity. Porcine pancreatic elastase inhibition assay was performed on succinyl-tri-alanine p-nitroaniline (AAAPAN) substrate using uv spectroscopy. Elastase hydrolysis resulted in the release of paranitroaniline, yielding a measured absorbance at 41 Onm.
2. Test materials and apparatus
2.1 test materials
Human dermal fibroblasts, cell lines were derived from Zhejiang Meisen cell technologies.
Fetal bovine serum (FBS, procall), DMEM high sugar medium (DMEM, procall), dimethyl sulfoxide (DMSO, microphone), phosphate buffer (PBS, procall), thiazole blue (MTT, sigma), transforming growth factor beta-induced protein 1 (TGF-beta 1, siuobilio logical) human type I collagen (ColI) kit (built in south tokyo).
2.2 test apparatus
Super clean bench (safe in Suzhou), inverted microscope (Mingmei), carbon dioxide incubator (Memmert), pipette (Eppendorf), cell waste liquid pump (its Linbell), enzyme-labeled instrument (BioTelc), 96-well plate (Shuohua), ultra low temperature preservation box (sea).
3. Test method
3.1 toxicity detection based on human dermal fibroblasts
The test adopts MTT method to detect cell activity, and the maximum safe concentration of cell sample is screened. The test was run with a blank (medium) and zeroed wells (PBS), the sample concentrations were set to 12.500%, 6.250%, 3.125%, 1.563%, 0.781%, 0.391%, 0.195%, 0.098% by volume (V/V) and 3 replicate wells were set for each concentration. The specific operation steps are as follows:
(1) Inoculating: taking cells in logarithmic growth phase, inoculating to 96-well plate, placing the culture plate at 37deg.C, and 5% CO 2 Incubating and culturing for 18-24h in an incubator.
(2) And preparing test objects with different concentrations according to concentration settings in a table worker.
(3) Sample feeding: the supernatant was discarded after 18-24h of cell growth, medium containing different concentrations of the test substance was added and the plates were placed at 37℃with 5% CO 2 Incubating and culturing for 18-24h in an incubator.
(4) And (3) detection: after 18-24h of cell culture, the supernatant was discarded, and the formulated and filtered MTT (0.5 mg/mL) was added, gently mixed, and incubated at 37℃for 4h in the absence of light. After the incubation, the supernatant was discarded, 100. Mu.L of DMSO was added to each well, and the wells were shaken for 10min, and the OD was read with an ELISA reader 490nm Values.
(5) Relative cell viability calculation formula:
3.2 determination of collagen type I based on human dermal fibroblasts
3.2.1 cell administration treatments
(1) Cell plating: 200. Mu.L of cell suspension was added to each well (96 well plate) and placed in CO 2 In an incubator at 37℃with 5% CO 2 Culturing for 20-24 hr.
(2) Preparing liquid: test and positive controls were formulated according to table 2.
Table 6 test grouping and concentration setting
(3) Administration: will be formulatedThe samples are respectively added into the corresponding positions of a 96-well plate for drug administration, the culture plate is placed at 37 ℃ and 5 percent CO 2 Culturing was continued under the conditions for 24 hours.
(4) Cell supernatant collection: after the incubation, 200. Mu.L of cell culture supernatant was collected per well in a 1.5mL sterile centrifuge tube and stored in an ultra-low temperature refrigerator at-80 ℃.
(5) ELISA detection: ELISA detection is required to be carried out according to the instruction of the human type I collagen ELISA kit. Before formal detection, the dilution ratio is required to be set, a pre-experiment is carried out, and the dilution ratio of an ELISA detection experimental group is determined through fumbling, so that the detection value is ensured to fall in the range of a standard curve.
(6) And (3) data processing: various data obtained in the test were processed and plotted by Excel software. Statistical analysis was performed using SPSS17.0, and group comparisons were performed using one-way analysis of variance (ANOVA), with significant differences being judged when P < 0.05, and with very significant differences being judged when P < 0.01.
4. Test results
4.1 toxicity test results based on human dermal fibroblasts
The cytotoxicity test results are shown in Table 7, the relative cell viability change trend is shown in FIG. 14, and the morphological observation results of the cells are shown in FIG. 15.
TABLE 7 human dermal fibroblast cytotoxicity assay results
Cell efficacy test concentration selection criteria:
(1) Relative cell viability > 90%, no significant difference (P > 0.05) compared to negative control group;
(2) No obvious change in cell morphology compared to the negative control group;
based on the cytotoxicity test results and the cell morphology observation results, the administration concentration on human dermal fibroblasts was finally selected to be 6.250%.
4.2 measurement of collagen type I of human dermal fibroblast
The measurement was performed according to the procedure 3.2 of the test method, and the measurement of the type I collagen content of human dermal fibroblasts was performed as follows.
TABLE 8 measurement results of human dermal fibroblast type I collagen
As can be seen from the above table, compared with NC group, PC (TGF-group) group can obviously improve the secretion amount (P < 0.05) of type I collagen of human dermal fibroblasts at a treatment concentration of 1OOng/mL, which indicates that the test system is established. The succinyl-terminated collagen solution has a tendency to significantly promote the secretion of type I collagen (P < 0.05) at an experimental concentration of 6.250%, and the secretion amount of type I collagen is up-regulated by 0.994 times than that of NC group.
In conclusion, the acylated collagen preparation contains acylated collagen, so that the number of the zebra fish neutrophils can be remarkably reduced, and the acylated collagen preparation has a relieving effect and can be used for preparing anti-inflammatory drugs or cosmetics; the fluorescence of zebra fish embryos can be remarkably reduced, which indicates that the acylated collagen preparation has an antioxidation effect and can be used for preparing antioxidation medicines or cosmetics; and has whitening effect on zebra fish embryo, and can be used for preparing whitening medicine or cosmetics; can also remarkably promote the secretion of the type I collagen, and can be used for preparing medicines or cosmetics for promoting the secretion of the collagen.
The above embodiments are preferred embodiments of the present invention, and besides, the present invention may be implemented in other ways, and any obvious substitution is within the scope of the present invention without departing from the concept of the present invention.

Claims (8)

1. An application of acylated collagen, wherein the preparation method of the acylated collagen comprises the following steps:
(1) Dissolving the freeze-dried collagen in an acid solution, and then adjusting the pH to 8.5-9.5 with the alkali solution;
(2) Adding anhydride while stirring, regulating pH to 8.5-9.5 with alkali solution during reaction, maintaining the temperature at 20-30deg.C, and reacting for 2-4 hr;
(3) After the reaction is finished, collecting the solution, putting the solution into a dialysis bag, and dialyzing with deionized water for 3-7d, or purifying the solution by ultrafiltration for 2-8h;
(4) Freeze drying is carried out after purification is finished, and the acid anhydride modified acylated collagen is obtained;
the method is characterized in that: the grafting rate of the acylated collagen is 70-80%, and the acylated collagen is used for preparing anti-inflammatory drugs or cosmetics, antioxidant drugs or cosmetics, whitening drugs or cosmetics, or drugs or cosmetics for promoting collagen secretion.
2. Use of an acylated collagen according to claim 1, wherein: in the step (1), the acid solution is acetic acid, hydrochloric acid, nitric acid, boric acid, phosphoric acid solution and a combination thereof, wherein the pH value of the acetic acid, the hydrochloric acid, the nitric acid, the boric acid and the phosphoric acid is 1.5-3.5.
3. Use of an acylated collagen according to claim 1, wherein: in the step (2), the anhydride is C2-C20 linear chain alkyl anhydride, branched chain alkyl anhydride, cycloalkyl anhydride, aromatic anhydride and the combination thereof, and the mass ratio of the anhydride to the collagen is 1:0.2-1.0.
4. Use of an acylated collagen according to claim 1, wherein: in the step (1) and the step (2), the alkali solution is alkali metal hydroxide, alkaline salt of organic or inorganic acid, amine, alkaline buffer solution and combination thereof; in the step (3), the molecular weight cut-off of the dialysis bag is 8000-14000.
5. Use of an acylated collagen according to claim 1, wherein: the acylated collagen has good water solubility, and the triple helix structure and biological activity of the natural active collagen are maintained.
6. Use of an acylated collagen according to claim 1, wherein: the modification site of the acylated collagen is N-terminal amino and/or epsilon-amino; the grafting rate of the acylated collagen is 70% -80%.
7. Use of an acylated collagen according to claim 1, wherein: the isoelectric point of the acylated collagen is 4.1-4.2.
8. Use of an acylated collagen according to claim 1, wherein: the acylated collagen is prepared as a powder, microsphere, flake, fiber, fabric, pad, membrane, septum, sponge, wound dressing, ointment, or gel for medical or cosmetic applications.
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