Monochlorodimethylsilane modified gelatin as well as preparation method and application thereof

Abstract

The invention provides monochlorodimethylsilane modified gelatin as well as a preparation method and application thereof, and belongs to the technical field of gelatin modification, monochlorodimethylsilane is adopted for modifying gelatin, the modified gelatin has the advantages of relatively high hydrophobicity and the like, the oxidation resistance is also remarkably improved, the scavenging rate of the modified gelatin to hydroxyl radicals is 90-95.0%, and the removal rate of the modified gelatin to hydroxyl radicals is 90-95.0%. The scavenging rate of DPPH free radicals is 80%-88.5%, and the scavenging rate of superoxide free radicals is 90%-95.3%; in addition, the modified gelatin has better external force resistance, the toughness and rigidity of the gelatin are obviously improved, cell migration can be promoted, and healing of skin wounds is accelerated.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to monochlorodimethylsilane modified gelatin and a preparation method and application thereof. Background Art

[0002] Gelatin is usually a partial hydrolysis product of collagen obtained from animal skin, bones, and connective tissue. Gelatin has excellent biocompatibility, can carry a large amount of water to moisturize the skin, can form a membrane structure on the skin surface, delay the loss of water in the body, maintain the moisture and luster of the skin, and has a certain anti-wrinkle effect; gelatin added to cosmetics can also form a network structure, which can effectively lock the active ingredients inside the network structure to prevent the loss of active ingredients; in addition, gelatin also has an antioxidant effect, which can remove some free radicals in the skin and delay skin aging.

[0003] Silicon is a mineral element necessary for human connective tissue. It is widely present in elastin and collagen in the form of organic silicon. Silicon mainly exists in the extracellular matrix and is the main component of glycosaminoglycans and related protein complexes in connective tissue. Exogenous organic silicon helps to rebuild connective tissue and promote the production of collagen. It also has good wettability and can improve the ductility and spreadability of cosmetics.

[0004] The invention patent application with publication number "CN107519035A" (application number: 201710766008.2) discloses the application of a gelatin-hydrolyzed gelatin compound in skin care products. After being added to cosmetics, the compound is more permeable to the skin due to the similarity between gelatin and protein structure. It can promote the synthesis of collagen in the skin, replenish skin collagen, and achieve the effect of skin health and restoration of elasticity. The invention patent with publication number "CN107513173A" (application number: 201710731344.3) discloses a method for preparing a silicone-modified collagen film. The method is to first completely dissolve animal collagen with acetic acid solution and prepare a collagen solution, remove bubbles and then cast it into a film, dry it to obtain a white transparent collagen film, and then place the prepared collagen film in an inorganic alkaline solution at room temperature and add silicone to react. After the reaction, rinse the surface and dry it to obtain a modified collagen film. The obtained collagen film greatly improves the hydrophobicity, basically eliminates the obstacles to the commercial application of collagen film, and avoids exogenous toxic chemicals from entering the collagen film and affecting its edibility.

[0005] Both of the above patents record the relevant compounding or modification methods of gelatin, which makes the modified gelatin have obvious improvements in skin care and other aspects, but there are still few records about the methods and applications of gelatin modification. In order to further explore more preparation methods and applications of modified gelatin, the present invention intends to modify gelatin using monochlorodimethylsilane to find more potential applications of modified gelatin in different fields. Summary of the invention

[0006] In view of the above problems, the present invention provides a monochlorodimethylsilane-modified gelatin and a preparation method and application thereof. The gelatin is modified by monochlorodimethylsilane. In addition to having advantages such as strong hydrophobicity, the modified gelatin also has significantly improved antioxidant properties. The scavenging rate of the modified gelatin for hydroxyl radicals is 90-95.0%, the scavenging rate of the modified gelatin for DPPH radicals is 80-88.5%, and the scavenging rate of the modified gelatin for superoxide radicals is 90-95.3%. In addition, the modified gelatin also has better resistance to external forces, its toughness and rigidity are significantly improved, and it can promote cell migration and accelerate the healing process of skin wounds.

[0007] The technical solution of the present invention is as follows:

[0008] A chlorodimethylsilane-modified gelatin, wherein the gelatin is modified by using chlorodimethylsilane, and the tensile strength of the chlorodimethylsilane-modified gelatin is 35 to 41.5 N·mm 2 , Young's modulus is 7-10.6 KPa, elongation at break is 30-37.3%, contact angle is 85-93°, scavenging rate for hydroxyl free radicals is 90-95.0%, scavenging rate for DPPH free radicals is 80-88.5%, scavenging rate for superoxide free radicals is 90-95.3%, and cell migration rate for epidermal cells is 40-45.2%.

[0009] Preferably, the infrared spectrum of the monochlorodimethylsilane-modified gelatin is at 1130 cm -1 The Si-O characteristic peak appeared, and the peak at 3110 cm -1 The amide II band of NH bond bending vibration at 1580 cm -1 The amide II band of NH bond bending vibration at 1490 cm -1 The amide III band of CN bond stretching vibration at 977 cm -1 The bending vibration peak of the CH bond.

[0010] The method for preparing monochlorodimethylsilane-modified gelatin comprises the following steps: using monochlorodimethylsilane to modify gelatin, and using tetrabutylammonium iodide and triethylamine as catalysts in the modification reaction to obtain monochlorodimethylsilane-modified gelatin.

[0011] Preferably, the preparation method specifically comprises the following steps:

[0012] (1) preparing a 3-5% wt gelatin solution, allowing it to stand at room temperature, placing the gelatin solution in a 42-48° C. water bath and stirring for 10-15 min, and adjusting the pH to 9-10;

[0013] (2) adding tetrabutylammonium iodide to the gelatin solution of step (1) under oxygen-free conditions to obtain reactant 1;

[0014] (3) dissolving monochlorodimethylsilane in dimethyl sulfoxide to obtain a monochlorodimethylsilane solution, adding the monochlorodimethylsilane solution to reactant 1, and mixing thoroughly to obtain reactant 2;

[0015] (4) preparing a mixed solvent of dimethyl sulfoxide and water in a ratio of 5 to 7:4 to 5 (v / v), dissolving triethylamine in the mixed solvent to obtain a triethylamine solution, adding the triethylamine solution to reactant 2, and stirring the reaction at 42 to 48° C. for 40 to 50 hours to obtain reactant 3;

[0016] (5) Pour the reactant 3 into a mold and dry it in an oven at 40-45°C under normal pressure, then rinse it with acetone and dry it again to obtain monochlorodimethylsilane-modified gelatin.

[0017] Preferably, the standing time at room temperature in step (1) is more than 1 hour;

[0018] Preferably, the method for isolating oxygen in step (2) is: continuously passing nitrogen into the gelatin solution of step (1) for 10 minutes, then adding tetrabutylammonium iodide, and continuing to pass nitrogen for 30 minutes.

[0019] Preferably, the weight ratio of gelatin, tetrabutylammonium iodide, monochlorodimethylsilane and triethylamine is 30-40:1:5:1.

[0020] The application of monochlorodimethylsilane modified gelatin in improving the tensile strength of gelatin.

[0021] The application of monochlorodimethylsilane modified gelatin in promoting wound healing.

[0022] The application of monochlorodimethylsilane modified gelatin in improving the antioxidant property of gelatin.

[0023] The application of monochlorodimethylsilane modified gelatin in improving the hydrophobicity of gelatin.

[0024] Application of the monochlorodimethylsilane modified gelatin in the preparation of skin care products.

[0025] Preferably, the skin care products include lotions, cosmetic milks, moisturizing creams, hand creams, foot creams, bath products, shampoo products, and hair care products, and the dosage forms of the skin care products include water-based solutions, lotions, creams, gels, and facial mask products.

[0026] Beneficial effects:

[0027] The invention provides monochlorodimethylsilane-modified gelatin and a preparation method and application thereof. Monochlorodimethylsilane is used to modify gelatin. In addition to having a strong hydrophobic advantage, the modified gelatin also has significantly improved antioxidant properties. The scavenging rate of the modified gelatin for hydroxyl free radicals is 90-95.0%, the scavenging rate of the modified gelatin for DPPH free radicals is 80-88.5%, and the scavenging rate of the modified gelatin for superoxide free radicals is 90-95.3%. In addition, the modified gelatin also has better resistance to external forces, and its toughness and rigidity are significantly improved. It can promote cell migration and accelerate the healing of skin wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The infrared spectra of gelatin before modification, monochlorodimethylsilane and monochlorodimethylsilane modified gelatin are shown in Figure 1.

[0029] Figure 2 The results of hydrogen spectrum test of gelatin before modification, monochlorodimethylsilane and monochlorodimethylsilane modified gelatin;

[0030] Figure 3 The stress-strain curves of gelatin before modification and gelatin modified with monochlorodimethylsilane;

[0031] Figure 4 The cytotoxicity test results of gelatin before modification and gelatin modified with monochlorodimethylsilane are shown in Figure 2.

[0032] Figure 5 The results are the test results of scavenging rate of hydroxyl radicals by gelatin before modification and gelatin modified with monochlorodimethylsilane;

[0033] Figure 6 The results are the test results of DPPH free radical scavenging rate of gelatin before modification and gelatin modified with monochlorodimethylsilane;

[0034] Figure 7 The results are the test results of superoxide radical scavenging rate of gelatin before modification and gelatin modified with monochlorodimethylsilane;

[0035] Figure 8 The photos of cell scratches at 0h and 24h;

[0036] Fig. 9 is the cell migration rate of gelatin before modification and gelatin modified with monochlorodimethylsilane at 24 h;

[0037] Fig.10 To test the stability of moisturizing cream in centrifugal experiment;

[0038] Fig.11 To test the heat resistance, cold resistance, and alternating heat and cold resistance of moisturizing creams;

[0039] Fig.12 A patch test to evaluate the safety of moisturizing creams;

[0040] Fig.13 The results of the experiment on the moisturizing effect of moisturizing cream on the skin;

[0041] Fig.14 The results of the experiment on the effect of moisturizing cream on skin elasticity;

[0042] Fig.15 The improvement results of skin roughness after using moisturizing cream for 28 days;

[0043] Fig.16 The improvement results of skin roughness after using moisturizing cream for 14 days; DETAILED DESCRIPTION

[0044] The following is explained in conjunction with specific embodiments:

[0045] Source of experimental materials:

[0046] CCK-8 detection solution: purchased from Shanghai Weihuan Biotechnology Co., Ltd.;

[0047] Human skin fibroblasts: purchased from Pronocell Biotechnology Co., Ltd.

[0048] Hacat cells: purchased from Abiwei Biotechnology;

[0049] Electronic universal tensile testing machine: purchased from Xinte Testing Machine Co., Ltd.;

[0050] Contact angle measuring instrument: purchased from Shengding Precision Instrument Co., Ltd.

[0051] Microplate reader: purchased from Refudi Biomedical (Shanghai) Co., Ltd.

[0052] Coreometer CM825 skin moisture content test probe: purchased from Shanghai Tusen Vision Technology Co., Ltd.;

[0053] Cutometer MPA580 instrument probe: purchased from Shanghai Tusen Vision Technology Co., Ltd.;

[0054] Skin surface texture testing system: purchased from Shanghai Tusen Vision Technology Co., Ltd.;

[0055] VisioScan VC 20plus: purchased from Shanghai Tusen Vision Technology Co., Ltd.

[0056] Embodiment 1:

[0057] Preparation of Gelatin Modified with Monochlorodimethylsilane

[0058] The specific steps are as follows:

[0059] (1) Weigh 5.00 g of gelatin into a 250 mL three-necked flask, add 95 mL of water to prepare a 5% wt gelatin solution, let stand at room temperature for 1 hour, place the three-necked flask containing the gelatin solution in a 45°C water bath and stir for 10 minutes, and adjust the pH to 9.0;

[0060] (2) nitrogen was continuously introduced into the gelatin solution of step (1) for 10 min, and then 0.13 g of tetrabutylammonium iodide was added, and nitrogen was continued to be introduced for 30 min to obtain reactant 1;

[0061] (3) dissolving 0.70 g of monochlorodimethylsilane in 5 mL of dimethyl sulfoxide to obtain a monochlorodimethylsilane solution, transferring the monochlorodimethylsilane solution to a constant pressure dropping funnel, and adding dropwise to reactant 1 at a rate of 1 d / 2 s to obtain reactant 2;

[0062] (4) 6 mL of dimethyl sulfoxide and 4 mL of deionized water were prepared into a mixed solvent, 1.30 g of triethylamine was dissolved in the mixed solvent to obtain a triethylamine solution, the triethylamine solution was transferred to a constant pressure dropping funnel, and the solution was added dropwise to reactant 2 at a rate of 1 d / 2 s, and the reaction was stirred at 45 ° C for 48 h to obtain reactant 3;

[0063] (5) The reactant 3 was poured into a polytetrafluoroethylene mold and dried in an oven at 45°C under normal pressure, then rinsed with acetone and dried again to obtain monochlorodimethylsilane-modified gelatin.

[0064] The gelatin before modification, monochlorodimethylsilane and the monochlorodimethylsilane modified gelatin obtained above were subjected to infrared spectroscopy detection. The results are as follows: Figure 1 shown by Figure 1 It can be obtained that monochlorodimethylsilane modified gelatin (monochlorogelatin) is at 1130cm -1 The Si-O characteristic peak appeared, and the 2160cm -1 、595cm -1 The Si-H bond and Cl-Si bond at 3110 cm-1 disappeared. In addition, monochlorodimethylsilane-modified gelatin (monochlorogelatin) appeared at 3110 cm-1. -1 The amide II band of NH bond bending vibration at 1580 cm -1 The amide II band of NH bond bending vibration at 1490 cm -1The amide III band of CN bond stretching vibration at 977 cm -1 The bending vibration peak of the CH bond.

[0065] The gelatin before modification, monochlorodimethylsilane and the monochlorodimethylsilane modified gelatin obtained above were subjected to hydrogen spectrum detection. The results are as follows: Figure 2 shown by Figure 2 It can be seen that compared with the gelatin before modification, monochlorodimethylsilane-modified gelatin (monochlorogelatin) has a new chemical shift. The peaks a and b near 3.2ppm and 1.2ppm are both proton peaks of methyl, which indicates that monochlorodimethylsilane-modified gelatin has been successfully synthesized.

[0066] Experimental Example 1:

[0067] Analysis of Mechanical Properties of Gelatin Modified by Monochlorodimethylsilane

[0068] 0.05 g of the gelatin before modification (G) and the monochlorodimethylsilane-modified gelatin (1cl-G) prepared in Example 1 were weighed and placed in 1 mL of deionized water, and a 5% wt gelatin solution was prepared at 45° C. and a stirring speed of 200 rad / s. Each gelatin solution was placed at 4° C. to accelerate the formation of gelatin gel;

[0069] After the gelatin gel is formed, the gel is made into a dumbbell shape with a diameter of 60 mm and a length of 10 mm. The tensile strength, Young's modulus and elongation at break of the gelatin are tested on an electronic universal tensile testing machine at 25°C. The weight sensor of the electronic universal tensile testing machine is 200N and the crosshead speed is 5mm / min.

[0070] The calculation formula of tensile strength is: P = F / S; where: P is tensile strength (N·mm 2 ), F is the load force when the gel breaks (N), S is the cross-sectional area of ​​the gel (mm 2 );

[0071] The calculation formula of Young's modulus is: E = σ / ε; where: E is Young's modulus (KPa), σ is tensile stress (KPa), and ε is tensile strain (%);

[0072] The calculation formula of elongation at break is: e=(L a -L0) / L0; where: e is the elongation at break (%), L0 is the initial length of the gel, L a is the elongation of the gel when it breaks.

[0073] The stress-strain curves of each gelatin sample are shown in Figure 3 The tensile strength, Young's modulus and elongation at break are shown in Table 1 below:

[0074] Table 1. Test results of tensile strength, Young's modulus and elongation at break of each gelatin sample

[0075] Sample name <![CDATA[P(N·mm 2 )]]> E(KPa) e(％) Gelatin before modification (G) 36.2 9.4 26.5 Monochlorodimethylsilane modified gelatin (1cl-G) 41.5 10.6 37.3

[0076] Depend on Figure 3 As shown in Table 1, the tensile strength of monochlorodimethylsilane-modified gelatin increased by 14.6% compared with the gelatin before modification, the Young's modulus increased by 12.7% compared with the gelatin before modification, and the elongation at break increased by 40.7% compared with the gelatin before modification; this shows that monochlorodimethylsilane-modified gelatin has better resistance to external forces, and its toughness and rigidity are improved accordingly.

[0077] Experimental Example 2:

[0078] Analysis of Hydrophobicity of Gelatin Modified by Monochlorodimethylsilane

[0079] 0.05 g of the gelatin before modification and the monochlorodimethylsilane-modified gelatin prepared in Example 1 were weighed and placed in 1 mL of deionized water, and a 5% wt gelatin solution was prepared at 45° C. and a stirring speed of 200 rad / s; the gelatin solution was dropped onto the ex vivo pig skin with a rubber-tipped dropper and spread flat, and then dried naturally to form a smooth film, i.e., the gelatin sample; the gelatin sample was placed in a contact angle meter to measure the contact angle (θ); the experimental results are shown in Table 2 below:

[0080] Table 2. Contact angle measurement results of various gelatin samples

[0081] Sample name Number of experiments θ(°) Gelatin before modification 6 75±2 Monochlorodimethylsilane modified gelatin 6 90±3

[0082] It can be seen from Table 2 that the contact angle of monochlorodimethylsilane-modified gelatin is 15° higher than that of gelatin before modification, which indicates that monochlorodimethylsilane-modified gelatin has better hydrophobicity. The enhancement of the hydrophobicity of gelatin can make it have better peelability when used as some skin contact materials.

[0083] Experimental Example 3:

[0084] Biocompatibility experiment of monochlorodimethylsilane-modified gelatin

[0085] Cytotoxicity experiments simulate the growth environment of organisms in vitro and can screen the cytotoxicity of samples in a relatively short period of time. They are an important basis for conducting other cell experiments. Cell Counting Kit-8 (CCK-8) reagent can be used to quickly and easily identify the toxic effects of analytical substances. The working principle is as follows: it contains WTS-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt], whose molecular structure contains multiple nitro (-NO2) and aromatic sulfonic acid (-SO3H) groups. These groups have strong light absorption and undergo photolysis to produce active substances such as free radicals or ions. These active substances further undergo redox reactions and are reduced to yellow formazan products by dehydrogenases in cells under the action of 1-Methoxy PMS. The amount of the product is proportional to the survival rate. The yellow formazan product is characterized by high water solubility and special absorption at 450nm. The cytotoxicity of the substance to be tested can be determined by detecting its absorbance.

[0086] This experiment uses human skin fibroblasts as a cell model, monochlorodimethylsilane-modified gelatin as a test sample, and uses the CCK-8 method for determination. The specific steps are as follows:

[0087] (1) Human skin fibroblasts of appropriate density were selected, digested with trypsin to prepare a cell suspension, and inoculated into a 96-well plate. The volume of the suspension in each well was controlled to be 100 μL, and the number of cells in the suspension was 6 to 8 × 10 3 As the sample group, a blank control group was set up at the same time. No cells were added to the blank control group, and only the same volume of cell suspension was added to each well. The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 24 hours.

[0088] (2) After the cells in the 96-well plate in step (1) are firmly attached to the wall, monochlorodimethylsilane-modified gelatin is added to the sample group and the blank control group, respectively, so that the final concentration of monochlorodimethylsilane-modified gelatin in each well is 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL and 100 μg / mL, respectively, and three replicate wells are set for each concentration; the 96-well plate is incubated in a 37° C., 5% CO2 incubator for 24 h;

[0089] (3) After the incubation is completed, the 96-well plate is taken out of the incubator, 10 μL of CCK-8 detection solution is added to each well, and the plate is incubated in a 37°C, 5% CO2 incubator for 3 h. After the incubation is completed, the absorbance of the liquid in the plate is measured at 450 nm.

[0090] (4) Result analysis: The cell viability was calculated using the measured absorbance. The calculation formula is as follows:

[0091] Cell survival rate = A x / A0×100%; where A x represents the absorbance of the blank control group at 450 nm, and A0 represents the absorbance of the sample group at 450 nm.

[0092] The results of cell survival rate under different concentration gradients are as follows Figure 4 shown by Figure 4 It can be obtained that the survival rate of human skin fibroblasts under monochlorodimethylsilane-modified gelatin is close to 100%, which indicates that the monochlorodimethylsilane-modified gelatin product has reached the implantable level.

[0093] Experimental Example 4:

[0094] Antioxidant Experiment of Gelatin Modified by Monochlorodimethylsilane

[0095] 1. Hydroxyl free radical scavenging ability

[0096] Using Fenton reaction to generate hydroxyl radicals: H2O2+Fe 2+ =OH - +Fe 3+ +H2O; salicylic acid is added to the reaction system, and the hydroxyl radicals generated by the Fenton reaction react with salicylic acid to generate 2,3-dihydroxybenzoic acid with special absorption at 510nm; if a test substance with the function of scavenging hydroxyl radicals is added to the reaction system, the generation of hydroxyl radicals will be reduced, thereby reducing the amount of 2,3-dihydroxybenzoic acid generated accordingly; this experimental example uses a fixed reaction time method to measure the absorbance at 510nm to determine the scavenging effect of monochlorodimethylsilane-modified gelatin on hydroxyl radicals. The specific steps are as follows:

[0097] First, 7 mL of 100 μg / mL pre-modified gelatin (gelatin) and 7 mL of monochlorodimethylsilane-modified gelatin (silanized modified gelatin) were added to different test tubes, and then 1 mL of 9 mmol / L salicylic acid, 9 mmol / L FeSO4 and 8.8 mmol / L H2O2 were added to each test tube in turn. The reaction was carried out at a constant temperature of 37°C for 30 minutes to obtain the reaction product. The absorbance of the reaction product at 510 nm was measured by a UV-visible spectrophotometer, and the hydroxyl radical scavenging rate was calculated according to the following formula:

[0098] Hydroxyl radical scavenging rate = [1-(A x -A x0 ) / A0]×100%; where: A x represents the absorbance of the reaction system containing gelatin sample, salicylic acid, FeSO4, and H2O2 at 510nm, A x0represents the absorbance of the reaction system containing gelatin sample, salicylic acid, FeSO4 and deionized water at 510 nm, and A0 represents the absorbance of the reaction system containing salicylic acid, FeSO4, H2O2 and deionized water at 510 nm.

[0099] The results of hydroxyl radical scavenging rate were as follows: Figure 5 shown by Figure 5 It can be seen that the hydroxyl radical scavenging rates of gelatin before modification and monochlorodimethylsilane-modified gelatin at a concentration of 12 mg / mL reached 91.5% and 95.0%, respectively, which indicates that monochlorodimethylsilane-modified gelatin has a more prominent scavenging effect on hydroxyl radicals.

[0100] 2. DPPH free radical scavenging ability

[0101] The principle of DPPH free radical scavenging method: DPPH is a free radical that is not easy to change. Its color in methanol solution or ethanol solution is dark purple, and it has the maximum absorption peak at 520nm. The ability to scavenge DPPH is actually a test of the antioxidant capacity of the sample to be tested: antioxidants can donate H, so that DPPH becomes DPPH-H, the color of the solution disappears or becomes lighter, and the antioxidant capacity of the sample to be tested can be analyzed by measuring its absorbance; the ability of the test substance to scavenge DPPH free radicals can be evaluated by the change in the absorbance of the DPPH free radical before and after the addition of the test substance. The greater the change in absorbance, the stronger the antioxidant capacity of the sample.

[0102] This experiment was conducted in a 96-well plate, and sample group 1, sample group 2 and control group were set up at the same time. Each group had three replicate wells. Sample group 1 was as follows: gelatin (gelatin) before modification of the test substance was dissolved in 180 μL of distilled water, and 90 μL of 0.1 mM DPPH solution was added to make the final gelatin concentrations of 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / ml, 10 mg / mL, and 12 mg / mL; sample group 2 was as follows: the test substance was monochlorodimethylsilane-modified gelatin (silanized modified gelatin), and other conditions were the same as sample group 1; the control group was as follows: 180 μL of distilled water and 90 μL of 0.1 mM DPPH solution were used. After mixing the components, the 96-well plate was placed on a shaker to avoid light for 15 minutes, and the value at 520 nm, i.e. OD, was measured using an enzyme marker. 520 .

[0103] The calculation formula of DPPH free radical scavenging rate is: DPPH free radical scavenging rate = [(A0-A x ) / A0]×100%; where A0 represents the absorbance OD of the control group 520 , A x The absorbance OD of the representative sample group 520 .

[0104] DPPH free radical scavenging rate test results are as follows Figure 6 shown by Figure 6 It can be seen that at a concentration of 12 mg / mL, the scavenging rates of unmodified gelatin and monochlorodimethylsilane-modified gelatin on DPPH free radicals reached 53.7% and 88.5%, respectively, and the higher the concentration, the higher the scavenging rate, which indicates that the scavenging capacity of monochlorodimethylsilane-modified gelatin on DPPH free radicals is greatly improved.

[0105] 3. Superoxide free radical scavenging ability

[0106] Under weak alkaline conditions, pyrogallol is easily self-oxidized, and the intermediate products in the self-oxidation process are colored, and the intermediate products have a special absorption peak at 320nm; therefore, by measuring the change in absorbance, the rate of self-oxidation of pyrogallol can be evaluated; if the substance to be tested has antioxidant ability, the output of the intermediate products of pyrogallol that are easily self-oxidized will decrease, which will cause the absorbance of the solution to change. This principle can be used to evaluate the antioxidant ability of the substance to be tested.

[0107] In this experiment, gelatin before modification (gelatin) and monochlorodimethylsilane-modified gelatin (silanized modified gelatin) were used as samples to evaluate their antioxidant capacity. The specific operation steps are as follows:

[0108] (1) Zero adjustment solution: Place 600 μL of 0.1 M Tris-HCl buffer (pH = 8.2) and 600 μL of distilled water in a centrifuge tube, mix well, and place in a 25°C water bath for 20 min;

[0109] (2) Preparation of Solution I: 600 μL of 0.1 M Tris-HCl buffer (pH = 8.2) solution and 580 μL of distilled water were mixed and added to the samples so that the final concentrations of the samples were 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / ml, 10 mg / mL, and 12 mg / mL; the control group was 1200 μL of distilled water;

[0110] (3) Preparation of Solution II: 20 μL of 5 mM pyrogallol solution;

[0111] (4) Under 37°C water bath conditions, quickly mix solution I and solution II and let stand for 10 min to obtain a reaction solution. Measure the absorbance of the reaction solution at 325 nm (OD 325 ), during the determination process, the first time was recorded as 0min, and then recorded again after an interval of 5min to calculate the superoxide free radical scavenging rate of the sample;

[0112] The calculation formula of superoxide free radical scavenging rate is: Superoxide free radical scavenging rate = [(ΔA0-ΔA x) / ΔA0]×100%; where ΔA0 represents the absorbance difference of the control group; ΔA x It indicates the absorbance difference after adding the sample.

[0113] The results of superoxide free radical scavenging rate test are as follows Figure 7 shown by Figure 7 It can be seen that the superoxide radical scavenging rates of unmodified gelatin and monochlorodimethylsilane-modified gelatin at a concentration of 12 mg / mL reached 70.4% and 95.3%, respectively, and the higher the concentration, the higher the scavenging rate, which indicates that the superoxide radical scavenging ability of monochlorodimethylsilane-modified gelatin has also been greatly improved.

[0114] Experimental Example 5:

[0115] Cell scratch test of monochlorodimethylsilane-modified gelatin

[0116] The cell scratch assay is an experimental technique that studies cell migration, repair ability and cell-to-cell interaction by scratching a cell monolayer and capturing images periodically through a time-lapse microscope. The basic principle is to artificially create a blank area on a fused cell monolayer, called a "scratch", and the cells at the edge of the scratch will gradually move into the blank area to heal the "scratch". Images are captured periodically during the cell migration process, and the cell migration ability can be judged by measuring the scratch spacing at different time points and calculating the difference. Because the cell scratch assay is similar to the in vitro wound healing process, it is also called the wound healing assay, and can be used to observe the effects of exogenous factors such as drugs and genes on cell migration, repair and interaction.

[0117] Exogenous sodium hyaluronate can promote the proliferation and differentiation of epidermal cells and has a certain repair effect on skin damage. It can promote skin cell growth and wound healing, and restore the damaged area to health. Therefore, it is widely used as a skin wound healing and regeneration aid, drug carrier, etc.

[0118] The specific steps of this experiment are as follows:

[0119] (1) Use a marker pen to evenly draw horizontal lines on the back of the 6-well plate, with the intervals between the lines being 0.5 to 1 cm, across the wells, with at least 5 lines across each well;

[0120] (2) Hacat cells (human immortalized epidermal cells) cultured to 80-90% confluence were prepared into a cell suspension and 5×10 5 Add the amount of cells to the well plate, and place the well plate in a 5% CO2, 37°C cell constant temperature incubator for culture;

[0121] (3) When the cultured cells reach a confluence of more than 95%, use a 200 μL pipette tip to make a straight scratch on the bottom of the well plate perpendicular to the horizontal line, and take a photo of the scratch at 0 h;

[0122] (4) 100 μg / mL sample solutions were added to the well plate, respectively. The sample solutions were gelatin solution before modification (gelatin), monochlorodimethylsilane-modified gelatin (silanized modified gelatin) solution and sodium hyaluronate solution. The blank control group was a cell culture medium of the same volume. After culturing for 24 h, the cell scratch healing was recorded by taking photos and the cell migration rate was calculated.

[0123] The cell migration rate is calculated as follows: M = (S0-S t ) / S0; where S0 represents the initial scratch area, S t Represents the scratch area at time t.

[0124] The cell scratch photos at 0h and 24h are shown in Figure 8 As shown, the calculation results of cell migration rate at 24h are as follows Fig. 9 shown by Fig. 9 It can be seen that both the sample group and the control group had corresponding degrees of cell migration behavior at 24 hours, and the migration rate of the sample group was higher than that of the blank control group; from the perspective of cell migration rate, the migration rates of monochlorodimethylsilane-modified gelatin, gelatin before modification, sodium hyaluronate, and blank control group were 45.2%, 24.1%, 36.8%, and 18.6%, respectively, which shows that monochlorodimethylsilane-modified gelatin can promote cell migration faster than gelatin before modification, and its effect exceeds that of sodium hyaluronate, and it is expected to be applied to the field of wound repair technology to promote the healing process of wounds.

[0125] Embodiment 2:

[0126] Application of monochlorodimethylsilane modified gelatin in skin care products

[0127] 1. Preparation of moisturizing cream:

[0128] In this example, monochlorodimethylsilane-modified gelatin is applied in a moisturizing cream to study the efficacy of monochlorodimethylsilane-modified gelatin in skin care products.

[0129] First, four formulas were used to prepare moisturizing creams respectively, and the specific components were shown in Table 3 below:

[0130] Table 3. Components of moisturizing cream

[0131]

[0132]

[0133] Moisturizing creams were prepared according to the four formulas in Table 3. The specific steps are as follows:

[0134] (1) Accurately weigh phase A, disperse it evenly, and heat it to 80°C to obtain component 1;

[0135] (2) Accurately weigh phase C and heat it to 80°C to obtain component 2;

[0136] (3) Add component 1 to component 2 under homogenization, maintain the temperature at 75-80°C, and homogenize for 3 minutes;

[0137] (4) Add phase B, homogenize for 2 minutes, and cool down under stirring;

[0138] (5) When the temperature drops to 65°C, add phase E, stir evenly, and continue cooling while stirring;

[0139] (6) When the temperature drops to 45°C, add phase D and stir evenly, then add phase F (do not add phase F for formula 1) and continue stirring evenly to obtain a moisturizing cream.

[0140] 2. Determination of the safety properties of facial cream

[0141] (1) pH value evaluation:

[0142] Using the direct measurement method, first put an appropriate amount of cream samples in formulas 1 to 4 into a beaker at room temperature for use. After calibrating the pH meter, rinse the electrode with distilled water and dry it with a paper towel; slowly put the electrode into the cream sample, wait for the reading to stabilize, and accurately record the value to 0.1; after recording the data, clean the electrode and continue measuring; measure a total of 5 times and take the average value.

[0143] After testing, the pH value of the four formulas of moisturizing creams was 6.5, which is suitable for human skin.

[0144] (2) Stability evaluation

[0145] ① Centrifugation experiment: At room temperature, samples of formulas 1 to 4 were placed in a high-speed centrifuge and centrifuged for 30 min at 500 rpm / min, 1000 rpm / min, 2000 rpm / min, and 3000 rpm / min, respectively. The blank group was not centrifuged. The experimental results of formula 3 cream are as follows: Fig.10 As shown, the creams prepared by formulas 1 to 4 did not show any precipitation, stratification, discoloration, or obvious change in hardness after high-speed centrifugation, and were able to maintain a stable shape.

[0146] ② Heat resistance test: Take two equal amounts of samples from formulas 1 to 4, place one at room temperature and the other in a 40°C constant temperature box. After 10 days, take it out and place it at room temperature for more than 8 hours. Then compare it with the sample at room temperature to observe whether the sample in the high temperature environment has changes in color, precipitation, stratification or hardness. The experimental results of formula 3 cream are as follows: Fig.11 As shown in Figure A, the left side shows the sample at room temperature.

[0147] ③ Cold resistance test: Take two equal amounts of samples from formulas 1 to 4, place one at room temperature and the other in a -20℃ refrigerator. After 10 days, take it out and place it at room temperature for more than 8 hours. Then compare it with the sample at room temperature to observe whether the sample in the low temperature environment has changes in color, precipitation, stratification, hardness, etc. The experimental results of formula 3 cream are as follows: Fig.11 As shown in Figure B, the left side shows the sample at room temperature.

[0148] ④ Hot and cold alternation experiment: Take two equal amounts of samples from formulas 1 to 4, place one at room temperature, and place the other in a 40°C constant temperature box and a 20°C refrigerator, alternate between hot and cold for 24 hours, for a total of 10 days, then take it out and place it at room temperature for more than 8 hours, and then compare it with the sample at room temperature to observe whether the samples in the hot and cold alternation environment have changes in color, precipitation, stratification, hardness, etc.; the experimental results of formula 3 cream are as follows Fig.11 As shown in Figure C, the left side shows the sample at room temperature.

[0149] After the cold resistance, heat resistance and hot and cold alternating tests, the creams prepared by the above formulas 1 to 4 did not show any precipitation, stratification, discoloration, or significant change in hardness, and were able to maintain a stable shape, indicating that the creams have good stability.

[0150] 3. Safety evaluation of facial cream

[0151] There are many types of skin care products with complex and diverse ingredients. In order to ensure their safety for human use and avoid skin allergies or other phenomena, product safety must be evaluated. Currently, there are two commonly used methods: human trial experiments and patch tests.

[0152] Patch test is the most commonly used evaluation method for the safety of cosmetic raw materials and cosmetics. This experiment uses human patch test to evaluate the safety of face cream. First, 15 subjects are selected. All of them need to participate voluntarily, have no history of skin allergies, are in good health, and have not participated in other skin care efficacy evaluations in the past three months; then put an appropriate amount of moisturizing cream into the standard patch test chamber, and slowly stick the patch tester rich in the test substance on the inside of the subject's arm for 12 hours to maintain the release of product efficacy; after the end, remove the patch tester and observe the changes in the skin on the inside of the subject's arm at 30 minutes (b), 12 hours (c) and 24 hours (d). The results are as follows: Fig.12 Finally, the skin closure type patch test reaction results were observed and recorded according to the scoring criteria in Table 4, and the results are shown in Table 5:

[0153] Table 4. Patch test scoring criteria

[0154]

[0155]

[0156] Table 5. Patch test reaction results Depend on Fig.12 As shown in Table 8, none of the 15 subjects had adverse reactions, which shows that the safety of the moisturizing creams prepared by Formulas 1 to 4 meets the requirements.

[0157] 4. Evaluation of the efficacy of facial cream

[0158] The creams prepared by formulas 1 to 4 were applied to the skin surface of the subjects to further test the efficacy of the creams. The specific steps were as follows:

[0159] (1) Skin moisture content test: In this operation, the Coreometer CM825 skin moisture content test probe is used for testing. The probe test principle is based on the capacitance method. The specific method is as follows: first, disinfect the instrument probe with 75% alcohol and debug the equipment. Open the software, and point the probe vertically downward and lightly press it on the test site of the subject. Take 3 points in the same area and finally take the average value, which is the moisture content of the stratum corneum. The larger the value, the higher the skin moisture content.

[0160] Moisturizing cream moisturizes the skin Fig.13 shown by Fig.13 It can be seen that the water content of the stratum corneum of the skin at the 6th hour increased by 6.6%, 24.8%, 39.1% and 48.2% respectively compared with the 0th hour; this shows that the addition of monochlorodimethylsilane-modified gelatin and tripeptide can increase the water content of the stratum corneum of the skin, and the modified gelatin has excellent moisturizing effect.

[0161] (2) Skin elasticity test: Skin elasticity is an important indicator for measuring skin condition, among which the content and arrangement of collagen and elastic fibers play a key role; during the aging process, the elastic fibers arranged in a circular pattern and the filaments arranged in a vertical pattern are lost, and the skin begins to sag, wrinkle, and lose elasticity. By testing skin elasticity, the degree of skin aging can be reflected to a certain extent. This operation is based on the principle of suction and stretching, using the Cutometer MPA580 instrument probe test, and by analyzing the relationship curve between the length of skin stretching and time, the parameters representing the skin elasticity index are obtained, and finally the skin elasticity values ​​R2 and R5 are recorded; the larger the R2 and R5 values, the better the skin elasticity;

[0162] How does moisturizing cream affect skin elasticity? Fig.14 As shown in Figures A to D, Figures A, B, C, and D represent Formula 1, Formula 2, Formula 3, and Formula 4 respectively; Fig.14 It can be seen that when using the creams of formula 3 and formula 4, the R2 and R5 values ​​showed an upward trend compared with the 0th hour, and the difference was significant (**P<0.01); the R2 value increased by 12.1% and 19.1%, respectively. The R5 value increased by 10.7% and 11.2%, respectively; this shows that the addition of monochlorodimethylsilane-modified gelatin can significantly improve skin elasticity.

[0163] (3) Skin roughness test: Roughness measures the degree of skin dryness. The skin surface texture test system is used to capture skin surface images and use the skin surface analysis software SELS to analyze the skin surface grayscale value to obtain the test parameter Ser. The specific method is as follows: establish a test project, use VisioScan VC 20plus to take photos of the test area, record the skin roughness data, and export the captured images; the smaller the Ser value, the lower the degree of skin roughness;

[0164] The experimental results of moisturizing cream improving skin roughness are as follows Fig.15 , 16 shown by Fig.15 It can be seen that after using the creams of Formula 3 and Formula 4 for 28 days, the skin roughness decreased and there was a significant difference; Fig.16 It can be seen that the facial roughness decreased by 11.6% and 16.3% after using the creams of Formula 3 and Formula 4 for 14 days, respectively; this shows that the addition of monochlorodimethylsilane-modified gelatin can significantly improve skin roughness and effectively reduce wrinkles.

[0165] Embodiment 2:

[0166] Different from Example 1, the weight ratio of gelatin, tetrabutylammonium iodide, monochlorodimethylsilane and triethylamine in Example 2 is 37:1:5:1; the other steps are the same as Example 1; and finally monochlorodimethylsilane-modified gelatin is obtained.

[0167] Embodiment 3:

[0168] The difference from Example 1 is that in step (1) of this Example 3, a 4% wt gelatin solution is prepared, and after standing at room temperature for 1 hour, the three-necked flask containing the gelatin solution is placed in a 45°C water bath and stirred for 10 minutes, and the pH is adjusted to 10; the other steps are the same as in Example 1; and finally monochlorodimethylsilane-modified gelatin is obtained.

[0169] Embodiment 4:

[0170] Different from Example 1, in step (4) of this Example 4, 5 mL of dimethyl sulfoxide and 5 mL of deionized water are prepared into a mixed solvent, 1.30 g of triethylamine is dissolved in the mixed solvent to obtain a triethylamine solution, the triethylamine solution is transferred to a constant pressure dropping funnel, and added dropwise to reactant 2 at a rate of 1d / 2s, and the reaction is stirred at 46°C for 45h to obtain reactant 3; the other steps are the same as in Example 1; and finally monochlorodimethylsilane-modified gelatin is obtained.

[0171] In summary, the present invention uses monochlorodimethylsilane to modify gelatin. In addition to the advantages of strong hydrophobicity, antioxidant properties, and moisturizing properties, the modified gelatin also has better resistance to external forces, can promote cell migration, and accelerate the healing of skin wounds.

Claims

1. A chlorodimethylsilane-modified gelatin, characterized in that: Gelatin is modified by using monochlorodimethylsilane, and the tensile strength of the monochlorodimethylsilane-modified gelatin is 35 to 41.5 N·mm 2 , Young's modulus is 7-10.6 KPa, elongation at break is 30-37.3%, contact angle is 85-93°, scavenging rate for hydroxyl free radicals is 90-95.0%, scavenging rate for DPPH free radicals is 80-88.5%, scavenging rate for superoxide free radicals is 90-95.3%, and cell migration rate for epidermal cells is 40-45.2%.

2. The monochlorodimethylsilane-modified gelatin according to claim 1, characterized in that The infrared spectrum of the monochlorodimethylsilane-modified gelatin is at 1130 cm -1 The Si-O characteristic peak appeared, and the peak at 3110 cm -1 The amide II band of NH bond bending vibration at 1580 cm -1 The amide II band of NH bond bending vibration at 1490 cm -1 The amide III band of CN bond stretching vibration at 977 cm -1 The bending vibration peak of the CH bond.

3. The method for preparing monochlorodimethylsilane-modified gelatin according to claim 1, characterized in that: The method comprises the following steps: using monochlorodimethylsilane to modify gelatin, and using tetrabutylammonium iodide and triethylamine as catalysts in the modification reaction to obtain monochlorodimethylsilane modified gelatin.

4. The method according to claim 3, characterized in that The specific steps include: (1) preparing a 3-5% wt gelatin solution, allowing it to stand at room temperature, placing the gelatin solution in a 42-48° C. water bath and stirring for 10-15 min, and adjusting the pH to 9-10; (2) adding tetrabutylammonium iodide to the gelatin solution of step (1) under oxygen-free conditions to obtain reactant 1; (3) dissolving monochlorodimethylsilane in dimethyl sulfoxide to obtain a monochlorodimethylsilane solution, adding the monochlorodimethylsilane solution to reactant 1, and mixing thoroughly to obtain reactant 2; (4) preparing a mixed solvent of dimethyl sulfoxide and water in a ratio of 5 to 7:4 to 5 (v / v), dissolving triethylamine in the mixed solvent to obtain a triethylamine solution, adding the triethylamine solution to reactant 2, and stirring the reaction at 42 to 48° C. for 40 to 50 hours to obtain reactant 3; (5) Pour the reactant 3 into a mold and dry it in an oven at 40-45° C. under normal pressure, then rinse it with acetone and dry it again to obtain monochlorodimethylsilane-modified gelatin; Preferably, the standing time at room temperature in step (1) is more than 1 hour; Preferably, the method for isolating oxygen in step (2) is: continuously passing nitrogen into the gelatin solution of step (1) for 10 minutes, then adding tetrabutylammonium iodide, and continuing to pass nitrogen for 30 minutes.

5. The method according to claim 3, characterized in that The weight ratio of the gelatin, tetrabutylammonium iodide, monochlorodimethylsilane and triethylamine is 30-40:1:5:

1.

6. Use of the monochlorodimethylsilane modified gelatin according to claim 1 in improving the tensile strength of gelatin.

7. Use of the monochlorodimethylsilane modified gelatin according to claim 1 in promoting wound healing.

8. Use of the monochlorodimethylsilane-modified gelatin according to claim 1 in improving the antioxidant property of gelatin.

9. Use of the monochlorodimethylsilane modified gelatin according to claim 1 in improving the hydrophobicity of gelatin.

10. Use of the monochlorodimethylsilane-modified gelatin according to claim 1 in the preparation of skin care products; preferably, the skin care products include lotions, cosmetic milks, moisturizing creams, hand creams, foot creams, bath products, shampoo products, and hair care products, and the dosage forms of the skin care products include water solutions, emulsions, creams, gels, and facial masks.

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