CN110200839A - The application of big rat-tail Type I collagen albumen - Google Patents

Abstract

The invention discloses the application of rat tail type I collagen. The rat tail type I collagen of the present invention is applied to the preparation of anti-wrinkle moisturizing and/or whitening products. The anti-wrinkle moisturizing and whitening product of the rat tail type I collagen of the present invention is more conducive to the migration of human skin fibroblasts, which proves that the product of the present invention is beneficial to the anti-wrinkle and moisturizing of the skin. The rat tail type I collagen of the present invention has an inhibitory effect on tyrosinase activity, which proves that it has a whitening effect. The rat tail type I collagen of the invention has the characteristics of safety and no side effects.

Description

Application of Rat Tail Type Ⅰ Collagen

technical field

The invention relates to the application of rat tail type I collagen, in particular to the application of rat tail type I collagen in the preparation of whitening and anti-wrinkle cosmetics and a cosmetic, belonging to the field of cosmetics.

Background technique

Rat tail type Ⅰ collagen (Collagen from rat tail, Type I) is prepared under aseptic conditions by the Birkedal-Hansen method, with a purity of over 95%, soluble in 0.006mol/L acetic acid. This product can be used for the coating of cell culture dishes, especially suitable for the cultivation of cells that are not easy to adhere to in ordinary cell culture dishes; it can also be used for the preparation of three-dimensional collagen gel, so that cells can grow in a simulated three-dimensional environment. It is widely used in cell culture.

Peony flower extract is rich in natural antioxidant active ingredients to protect the skin from exogenous free radical damage.

Propolis extract is rich in immune active factors, which can enhance the immunity of the skin and prevent skin infections.

Contents of the invention

The purpose of the present invention is to provide the application of rat tail type I collagen, specifically to provide an application of rat tail type I collagen in the preparation of whitening and anti-wrinkle cosmetics.

The rat tail type I collagen provided by the invention is used in the preparation of anti-wrinkle moisturizing and/or whitening products.

In the above application, the product includes at least one of food, cosmetics and health products.

The present invention also provides an anti-wrinkle moisturizing and/or whitening cosmetic, the active ingredient of which is the rat tail type I collagen.

In the present invention, the preparation method of the rat tail type I collagen comprises the following steps:

1. Wash the rat tail and soak it in 75% alcohol for 5 minutes;

2. Cut the tail, remove the fur, cut it into small pieces, and pull out the silver tail key;

3. Cut off the tail tendon and place it in a plate, soak it in PBS (pH 7.2-7.4, KH ₂ PO ₄ 2mM, Na ₂ HPO ₄ 8mM, NaCl 136mM, KCl 2.6mM);

4. Put all tail bonds in 0.05mol/L Tris-HCl, overnight at 4°C;

5. Suck off the Tris-HCl, and weigh the tail tendon (0.5-1 g);

6. Put the tail tendon in a plate, cut it into pieces, and transfer it to an Erlenmeyer flask;

7. Add 0.1% acetic acid solution at a ratio of 50 mL per gram of tail tendon;

8. Shake to disperse the tail tendon in the acetic acid solution, and place it at 4°C for a week;

9. Centrifuge, 4 degrees, 4000 rpm, 30 minutes;

10. Take the supernatant and pass it through a 300-mesh filter;

11. Add 100ml 0.14mol/L NaOH (5.6gNaOH dissolved in 1000ml water) to every 600ml of the above crude extract, centrifuge at 8000 rpm for 5min, discard the supernatant, and leave flocculent precipitate;

12. Place the flocculent precipitate in triple distilled water, and dissolve the precipitate with 0.1mol/L (1000:6) acetic acid.

The present invention also provides a cosmetic, which is made of the following components by mass: 2-3 parts of rat tail type I collagen; 0.5-1 part of peony flower extract; 0.6-1.2 parts of propolis extract; 3 parts of hydrogenated polydecene; 4 parts of caprylic capric triglyceride; 3 parts of simethicone; 1.2 parts of cetyl alcohol; 0.5 parts of behenyl alcohol; 4 parts of isotridecanyl isononanoate; 100 parts of water; 3 parts of glycerin; 3 parts of butanediol; 1 part of glyceryl stearic acid (SE); 1.5 parts of beheneth-20; 0.03 parts of disodium edetate; sodium acrylate sodium dimethyl taurate 0.5 parts of copolymer; 0.2 parts of preservative; 0.1 to 0.3 parts of essence.

In the present invention, the peony flower extract and the propolis extract are extracts known in the art or are extracted according to methods known in the art.

The present invention further provides a preparation method for the above-mentioned cosmetics, comprising the steps of: 1) mixing hydrogenated polydecene, caprylic triglyceride, simethicone, cetyl alcohol, behenyl alcohol and isotridecanyl isononanoate Mix and heat to obtain phase A;

2) Mix rat tail type Ⅰ collagen, peony flower extract water, glycerin, butylene glycol, glyceryl stearic acid, beheneth-20 and disodium edetate to obtain phase B, and then The phase A obtained in step 1) is added to the phase B, pre-emulsified and homogenized;

3) After the A phase and the B phase described in step 2) are treated, add sodium acrylate sodium acyl dimethyl taurate copolymer and mix;

4) Cool the system after the treatment in step 3), and then add preservatives and essence to homogenize to obtain the cosmetic.

In the above preparation method, in step 1), the heating is to 80°C;

In step 2), the time of the pre-emulsification is 5min, and the time of the homogenization is 3min;

In step 3), the system is cooled to 45°C;

In step 4), the homogenization time is 2 minutes.

The present invention has the following advantages:

The rat tail type I collagen and the formula cosmetic prepared by the invention have anti-wrinkle, moisturizing and whitening effects, and are more conducive to the migration of human skin fibroblasts, which proves that the product of the invention is beneficial to skin repair, anti-wrinkle and moisturizing. The rat tail type I collagen of the present invention has an inhibitory effect on tyrosinase activity, which proves that it has a whitening effect. No toxic reaction is found in the acute skin irritation test, acute eye irritation test, skin phototoxicity test, skin allergy test, and mammalian cell chromosome aberration test of the rat tail type I collagen of the present invention. The oral acute toxicity test MTD of rat tail type Ⅰ collagen of the present invention is all greater than 15.0g/kgBW; genetic toxicity test: Ames test, mouse bone marrow polychromatic erythrocyte micronucleus test has no mutagenic effect; 90-day feeding test The results showed that there was no adverse effect on animal body weight, food utilization rate, hematology, blood biochemistry, viscera body ratio and histopathological examination. The rat tail type I collagen of the invention has the characteristics of safety and no side effects.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

In the following examples, the preparation method of rat tail type I collagen comprises the following steps:

1. Wash the rat tail and soak it in 75% alcohol for 5 minutes;

2. Cut the tail, remove the fur, cut it into small pieces, and pull out the silver tail key;

3. Cut off the tail tendon and place it in a plate, soak it in PBS (pH 7.2-7.4, KH ₂ PO ₄ 2mM, Na ₂ HPO ₄ 8mM, NaCl 136mM, KCl 2.6mM);

4. Put all tail bonds in 0.05mol/L Tris-HCl, overnight at 4°C;

5. Suck off the Tris-HCl, and weigh the tail tendon (0.5-1 g);

6. Put the tail tendon in a plate, cut it into pieces, and transfer it to an Erlenmeyer flask;

7. Add 0.1% acetic acid solution at a ratio of 50 mL per gram of tail tendon;

8. Shake to disperse the tail tendon in the acetic acid solution, and place it at 4°C for a week;

9. Centrifuge, 4 degrees, 4000 rpm, 30 minutes;

10. Take the supernatant and pass it through a 300-mesh filter;

11. Add 100ml 0.14mol/L NaOH (5.6gNaOH dissolved in 1000ml water) to every 600ml of the above crude extract, centrifuge at 8000 rpm for 5min, discard the supernatant, and leave flocculent precipitate;

12. Place the flocculent precipitate in triple distilled water, and dissolve the precipitate with 0.1mol/L (1000:6) acetic acid.

Example 1, Formula Cosmetics, Rat Tail Type Ⅰ Collagen Anti-wrinkle Moisturizing, Whitening Function Experiment

Formulated cosmetics: the active ingredients are rattail type I collagen, peony flower extract, and propolis extract, which have moisturizing, anti-wrinkle, and whitening effects. The specific formula is shown in Table 1.

Table 1

The preparation method of formula cosmetics specifically includes the following steps: adding each component in phase A into container A and heating to 80°C; simultaneously adding each component in phase B into container B and heating to 80°C; At the same time as the phase, add phase A, pre-emulsify for 5 minutes, and then homogenize for 3 minutes; while stirring, add the components of phase C; Mass for 2 minutes; continue to stir and cool down to room temperature.

1. Validation experiment of skin anti-wrinkle and moisturizing function of rat tail type Ⅰ collagen:

1. Materials and methods

1.1 Main instruments and equipment

The main equipment used in the experiment is shown in Table 2.

Table 2 List of main instruments and equipment used in the experiment

1.2 Main reagents

The main reagents are listed in Table 3.

Table 3 List of main detection reagents

1.3 Experimental method

1.3.1 Collagen plating:

Rat tail type Ⅰ collagen, bovine bone type Ⅰ collagen and fish skin protein were diluted to 0.1 mg/mL with sterile 0.006 mol/L acetic acid (0.36 g/L). Sterilize by filtration with a 0.22 μm microporous membrane on a clean bench, and then dispense into EP tubes. Lay the board the night before use - based on experience, place it on a sterilized ultra-clean bench overnight (preferably more than 18 hours, the time is too short for collagen to really attach), and dry it at a density of 5 μg/cm ² . Rinse twice with sterile PBS solution, then wash once with cell culture medium, and finally inoculate cells.

1.3.2 Cell passage and proliferation:

Use DMEM-H medium containing 10% fetal bovine serum by volume and 1% penicillin and streptomycin by volume, and put it in a cell culture box at 37°C and 5% CO ₂ by volume for cultivation . When the confluence rate of the cells reached more than 90% of the bottom area of the culture plate, they were digested with 0.25% trypsin and subcultured. The 3rd to 6th passage cells were used for follow-up research.

1.3.3 Scratch test:

Scratch test using an inverted microscope to observe cell growth and morphological changes. Scratch method to detect the effect of different collagens on the migration of human fibroblasts Human fibroblasts from the 3rd to 6th passages were inoculated in 12 wells embedded with rat tail type Ⅰ collagen, bovine bone type Ⅰ collagen, and fish skin protein In the plate, conventional culture, when the cell confluence rate reaches above 95%, discard the medium, starvation culture in serum-free medium for 12 to 16 hours; use a 10 microliter pipette tip, compare scratches with a ruler, and wash off with PBS Cells, the number of cells in the scratch was counted under a 10-fold microscope, and the number of cells in the scratch was measured again after 24 hours. Six fields of view were randomly selected for each group for subsequent statistical analysis. The experiment was repeated three times.

1.4 Statistical analysis All results were analyzed statistically using SPSS 13.0 software. Experimental results using The difference between groups was analyzed by one-way analysis of variance, and P<0.05 was considered statistically significant.

2 results

2.1 Morphological observation of human fibroblasts Observe the cell morphology with an inverted microscope. After 24 hours of scratch experiment, the number of cells in the scratch of the six-hole plate treated with the product of the present invention is significantly greater than that of the cell culture plate treated with bovine bone type Ⅰ collagen and fish skin protein (P<0.05).

Table 4 Results of scratch experiment on embedded cells

*P<0.05 compared with rat tail type Ⅰ collagen of the present invention

3 Conclusion

Compared with bovine bone type I collagen and fish skin protein, the rat tail type I collagen of the present invention is more conducive to the migration of human skin fibroblasts, which proves that the rat tail type I collagen of the present invention is beneficial to skin repair.

2. Validation experiment of skin whitening function of rat tail type Ⅰ collagen:

1 Materials and methods

1.1 Main instruments and equipment

The main equipment used in the experiment. (See Table 5)

Table 5 List of main instruments and equipment used in the experiment

1.2 Main reagents

The main reagents are listed in Table 6.

Table 6 List of main detection reagents

1.3 Experimental method

1.3.1 Culture and treatment of mouse melanoma cells

The culture of mouse melanoma cells was carried out according to the cell culture guidelines provided by the National Experimental Cell Resource Sharing Platform, cultured in DMEM-H medium containing 10% FBS in a cell culture incubator at 37°C and 5% _CO2 , to be posted When the parietal cell density reaches about 90%, digest the cells with 0.05% Trypsin-EDTA, centrifuge at 500g, resuspend the cells in complete medium, passage at a ratio of 1:3, once every 3 days.

After the cells in the logarithmic growth phase were digested, the cells were counted, and 1×10 ³ , 1×10 ⁴ , and 1×10 ⁵ cells were inoculated in 96-well plates, 24-well plates, and 6-well plates, respectively, using normal medium After culturing for 24 hours, the reagents diluted to the final exposure concentration were added to the culture system. The final mass concentrations of rat tail type Ⅰ collagen, bovine bone type Ⅰ collagen and fish skin protein were 8 mg/mL, 4 mg/mL, and 2 mg/mL, respectively. 4 wells were set for each concentration of mL, 1mg/mL, 0.5mg/mL, and 0.25mg/mL, and the control group was incubated for 72 hours without adding rat tail type Ⅰ collagen. Carry out related experiments.

1.3.2 Cell proliferation inhibition rate

At the same time of inoculating the culture flask, inoculate the rest in 96-well cell culture plates, 7 groups, 5 wells in each group, 100 μL per well, and culture in a 37°C incubator; after culturing for 24 hours, add phosphate buffer solution to each well 0.5% MTT (5mg/mL) 20uL, cultivated in a 37°C incubator; after 4 hours, the culture was terminated, and the culture solution in the well was carefully aspirated. Add 150 μL of dimethyl sulfoxide to each well, shake on a shaker at low speed for 10 minutes, and fully dissolve the crystals. At the same time, the first well of each row is set as the zero well (medium, MTT, dimethyl sulfoxide). Measure the absorbance value of each well at OD570nm with a microplate reader; melanocyte proliferation inhibition rate=(1-average absorbance value of each concentration÷average absorbance value of the control group)×100%.

1.3.3 Determination of tyrosinase activity:

The final mass concentrations of rat tail type Ⅰ collagen, bovine bone type Ⅰ collagen and fish skin protein were 8 mg/mL, 4 mg/mL, 2 mg/mL, 1 mg/mL, 0.5 mg/mL and 0.25 mg/mL, respectively. Set 4 wells at one concentration, and in the control group, rat tail type Ⅰ collagen was not added, and incubated for 72 hours. The supernatant was discarded, washed twice with pH6.8 PBS, and 90 μL of PBS containing 1% (volume fraction) TritonX-100 was added to each well. Sonicate in an ice bath, add 10 μL of 1×10 ^-2 moL/L L-dopa to each well, incubate at 37°C for 60 min, use 490 nm wavelength for colorimetry, set the blank well to zero, and measure the absorbance value of each well. Inhibition rate of tyrosinase activity=(1—average absorbance value of each concentration÷average absorbance value of control group)×100%.

1.3.4 Determination of melanin content:

Mouse melanoma cells were seeded in three 6-well plates at a density of 1×105 cells/mL. After incubating in the carbon dioxide incubator for 24 hours, add rat tail type Ⅰ collagen, bovine bone type Ⅰ collagen and fish skin protein culture solution with the same concentration gradient, the concentrations are 8mg/mL, 4mg/mL, 2mg/mL, 1mg/mL, 0.5mg/mL, 0.25mg/mL, exposure for 3 days, discard the supernatant, wash with PBS, add 0.5mL trypsin to each well, constant volume and count the cells. Centrifuge the remaining cell suspension at 1500r/min for 10mln, discard the supernatant, add 200 microliters of distilled water and a mixture of ethanol and ether with a volume ratio of 1:1 (to remove some opaque substances), shake well, and place at room temperature for 15 minutes, 3000r/min min Centrifuge for 5 minutes. The supernatant was discarded, and 1 mL of 1 mol/L NaOH aqueous solution containing 10% (mass fraction) DMSO was added to the precipitate. Heat at 80° C. for 30 minutes (with a stopper), and select a wavelength of 490 nanometers to measure the absorbance value on an enzyme-linked immunosorbent assay instrument. Melanocyte growth rate=(drug well cell density ÷ control well cell density) × 100%, melanin synthesis inhibition rate=[1-(drug well absorbance value ÷ drug well cell density) ÷ (control well absorbance value ÷ control well cell density )] × 100%.

2 results:

2.1 Proliferation of mouse melanoma cells

Rat tail type Ⅰ collagen, bovine bone type Ⅰ collagen and fish skin protein had no inhibitory effect on the proliferation of mouse melanoma cells. See Table 7.

Table 7 Determination of mouse melanoma cell proliferation inhibition rate

2.4 Effects of rat tail type Ⅰ collagen, bovine bone type Ⅰ collagen and fish skin protein on tyrosinase activity:

Each dose group of rat tail type Ⅰ collagen, bovine bone type Ⅰ collagen and fish skin protein has obvious inhibitory effect on tyrosinase activity of mouse melanoma cells, showing a dose-response relationship. When the dose was greater than or equal to 0.5 mg/mL, at the same dose level, the inhibition rate of bovine bone type Ⅰ collagen and fish skin protein on tyrosinase activity was significantly lower than that of rat tail type Ⅰ collagen. See Table 8.

The impact of table 8 tyrosinase activity

* means P<0.05 compared with this product at the same concentration

2.5 Effect of rat tail type Ⅰ collagen, bovine bone type Ⅰ collagen and fish skin protein embedding on melanin synthesis

Each dose group of rat tail type Ⅰ collagen, bovine bone type Ⅰ collagen and fish skin protein has a significant inhibitory effect on the melanin level of mouse melanoma cells, showing a dose-response relationship. When the dose is greater than or equal to 0.5mg/ml, at the same dose level, the inhibition rate of bovine bone type Ⅰ collagen and fish skin protein on tyrosinase activity is significantly lower than that of rat tail type Ⅰ collagen. See Table 9.

Table 9 Effect of rat tail type Ⅰ collagen on melanin

* means P<0.05 compared with this product at the same concentration

Conclusion: Compared with rat tail type Ⅰ collagen, bovine bone type Ⅰ collagen and fish skin protein, the whitening effect is better.

Embodiment 2, skin, eyes, genotoxicity test of rat tail type Ⅰ collagen

1. Acute skin irritation test

Carry out the following experiments according to "Hygienic Standards for Cosmetics" (2007 edition):

Materials and methods:

1. Test substance: rat tail type Ⅰ collagen (prepared with the formula and method in Example 1)

2. Animals and breeding environment: New Zealand rabbits, common grade, 4 in total, weighing 2-3kg. Beijing Jinmuyang Experimental Animal Breeding Co., Ltd. [license number: SCXK (Beijing) 2015-0005]. Animal breeding location: animal room of China Center for Disease Control (permit number: SYXK (Beijing) 2014-0043); the temperature of the animal room is 20-25° C., and the humidity is 40-50% RH. Animal feed was purchased from Beijing Huafukang Biotechnology Co., Ltd. (permit number: SCXK (Beijing) 2014-0008), and drinking water was purified water.

3. Test method: About 24 hours before the test, cut off the hair on both sides of the back and spine of the animal, and the left and right sides of the hair removal range are about 3cm×3cm. Take about 0.5 of the rat tail type Ⅰ collagen of the test substance and apply it directly on the skin of 2.5cm×2.5cm, then cover it with two layers of gauze and a layer of cellophane, and then fix it with non-irritating adhesive tape and bandage. The skin on the other side served as a control. The application time is h. Then wash to remove residual test substance. 1h, 24h, 48h, 72h and the 4th, 5th, and 6th day after removing the test substance, observe the skin reaction of the application site, according to the standard in "Skin Irritation/Corrosion Test" in "Hygienic Standards for Cosmetics" (2007 Edition) Carry out the skin reaction score, and judge the intensity of skin irritation according to the highest integral mean value at each observation time point of 24, 48 and 72 hours according to the standard.

Test results: as shown in Table 10.

Table 10 Acute skin irritation test results of rat tail type Ⅰ collagen to rabbits

As can be seen from the results in Table 10, the test substance is non-irritating to the acute skin irritation of rabbits.

2. Acute eye irritation test

Carry out the following experiments according to "Hygienic Standards for Cosmetics" (2007 edition):

Materials and methods:

1. Test substance: rat tail type Ⅰ collagen.

2. Animals and breeding environment: New Zealand rabbits, ordinary grade, Beijing Jinmuyang Experimental Animal Breeding Co., Ltd. [license number: SCXK (Beijing) 2015-0005], a total of 3 rabbits. The body weight is 2-3kg. Animal breeding location: animal room of China Center for Disease Control (permit number: SYXK (Beijing) 2014-0043); the temperature of the animal room is 20-25° C., and the humidity is 40-50% RH. Animal feed was purchased from Beijing Huafukang Biotechnology Co., Ltd. (permit number: SCXK (Beijing) 2014-0008), and drinking water was purified water.

3. Test method: The eyes of the rabbits were checked 24 hours before the test, and all the selected animals had no symptoms of eye irritation, corneal defects and conjunctival damage. During the test, about 0.1ml of the test substance was dripped into the conjunctival sacs of the right eyes of 3 rabbits, and the upper and lower eyelids were passively closed for 1 second, and rinsed with a sufficient amount of normal saline at a faster flow rate for 30 seconds in the 4th second. The left eye was treated in the same way as its own control. Animal eyes were inspected at 1h, 24h, 48h, 72h and 4th and 7th days after exposure, and the eye irritation reaction scores were scored according to the standard in "Acute Eye Irritation/Corrosion Test" in "Hygienic Standards for Cosmetics" (2007 Edition) , according to the standard for eye irritation classification.

Test results: as shown in Table 11.

Table 11 Test substance to rabbit acute eye irritation test result 30s flushing

From the results in Table 11, it can be seen that the test substance rat tail type Ⅰ collagen is non-irritating to rabbits in the acute eye irritation test.

3. Skin phototoxicity test

Carry out the following experiments according to "Hygienic Standards for Cosmetics" (2007 edition):

Materials and methods:

1. Test substance: rat tail type Ⅰ collagen

2. Positive substance: 0.005% 8-methoxypsoralen.

3. Animals and breeding environment: Albino guinea pigs, ordinary grade, Beijing Jinmuyang Experimental Animal Breeding Co., Ltd. [license number: SCXK (Beijing) 2015-0005], 6 in total. The body weight is 220-260g for females and 230-280g for males. Animal breeding location: animal room of China Center for Disease Control (permit number: SYXK (Beijing) 2014-0043); the temperature of the animal room is 20-25° C., and the humidity is 40-50% RH. Animal feed was purchased from Beijing Huafukang Biotechnology Co., Ltd. (permit number: SCXK (Beijing) 2014-0008), and drinking water was purified water.

4. Test method: UV light source: a black light tube with a diameter of 38mm, a power of 40W, and a wavelength of 350-400nm, produced by GE in the United States. UVB<0.1J/cm ² .

18-24 hours before the test, prepare 4 pieces (numbered 1, 2, 3, 4) on both sides of the guinea pig's spine for hair removal. The hair removal area of each piece is about 2cm × 2cm. on the right. During the experiment, the animals were fixed on a special wooden board, and about 0.2 g of the test substance was applied to the depilated areas 1 and 2, and no test substance was applied to the 3 and 4 areas. After 30 minutes of applying the test substance, cover areas 1 and 3 with aluminum foil and fix them with adhesive tape, and irradiate areas 2 and 4 with a UV light source. The skin reaction was observed at 1, 24, 48 and 72 hours after the irradiation, and the reaction score of each animal was judged according to the standard in "Skin Phototoxicity Test" in "Hygienic Standards for Cosmetics" (2007 edition).

The average light intensity: 2.57mW/cm ² ; the irradiation time is calculated as 65min according to the following formula.

Experimental results: as shown in Table 12.

Table 12 Test substance to guinea pig skin phototoxicity test result

Note: 1, 2, 3, and 4 are the test areas shown in Part II of "Hygienic Standards for Cosmetics" (2007), Part VII, Skin Phototoxicity Test.

Table 13 Positives are to guinea pig skin phototoxicity test result

Note: 1, 2, 3, and 4 are the test areas shown in Part II of "Hygienic Standards for Cosmetics" (2007), Part VII, Skin Phototoxicity Test. The test period of the positive control group was from February 1, 2018 to March 10, 2019.

From the above experimental results, it can be known that the phototoxicity test of the test substance rat tail type I collagen on guinea pig skin was negative.

4. Skin allergy test

Materials and methods:

1. Cell line: Chinese hamster ovary (CHO) cell line.

2. Metabolic activation system: a mixture prepared by combining rat liver homogenate induced by phenobarbital sodium and β-naphthoflavone and adding corresponding cofactors was used as the in vitro metabolic activation system (S ₉ mix).

3. Test substance: Rat tail type Ⅰ collagen was diluted with serum-free DMEM culture solution and used for the test.

4. Test method:

(1) Culture medium: 10% fetal bovine serum and 100 IU/ml penicillin and streptomycin were added to DMEM culture medium.

(2) Determination of the highest final concentration: the test set up a test substance group and a negative control group. CHO cells were inoculated in a culture dish with a diameter of 6 cm at a seeding density of 1.2×10 ⁶ /dish. After culturing for 24 hours at 37°C and 5% CO ₂ , the culture medium in the culture dish was sucked off, and different concentrations of test substances and Serum-free culture solution, metabolic activation group was added with S ₉ mix at the same time, placed in the incubator for 3 hours, the culture solution containing the test substance was discarded, the cells were washed with D-hanks solution for 3 times, and 10% fetal bovine The serum culture solution was continued to be cultured for 24 hours, and only the culture solution was added to the negative control group (S ₉ was added to the metabolic activation group at the same time). Observe the growth of the cells in each plate under an inverted microscope, and determine the highest final concentration of the test substance during the test according to the change in the degree of cell coverage.

(3) Chromosomal aberration test: the test was carried out with and without the metabolic activation system. Set up test substance group, negative control group and positive control group. According to the preliminary test results, the final concentrations of the test substance groups were set at 250, 125 and 62.5 μg/mL (-S ₉ ), and 2000, 1000 and 500 μg/mL (+S ₉ ). The test substances were added according to the same treatment method as the pre-test, the medium was changed after 3 hours of action, and the cells were harvested after continuing to culture for 24 hours. Colchicine was added 4 hours before harvest to a final concentration of 1.0 μg/mL. Digestion, hypotonicity, fixation, slide preparation and Giemsa staining were performed according to conventional methods. For each test substance group, negative control group and positive control group, 100 well-dispersed metaphase cells were selected for chromosome aberration analysis. Record the type and number of chromosomal structural aberrations, and calculate the chromosomal aberration rate.

Statistical analysis: χ ² test was used to compare the chromosomal aberration rate of cells in each test group with the negative control group to determine whether there was a significant difference.

test results:

Table 14 In vitro mammalian cell chromosome aberration test results (-S ₉ )

*P<0.01

Table 15 In vitro mammalian cell chromosome aberration test results (+S ₉ )

*P<0.01

From the above experimental results, it can be seen that under the experimental conditions, the sample in the in vitro cultured mammalian Chinese hamster ovary cell (CHO) chromosome aberration detection system, compared with the negative control group, no matter whether the activation system was added, the rat tail Type Ⅰ collagen did not cause a significant increase in the chromosomal aberration rate, so it did not exhibit mutagenicity in this test system.

Embodiment 3, rat tail type Ⅰ collagen toxicological test

1 Materials and methods

1.1 Sample: Rat tail type Ⅰ collagen is a colorless transparent jelly-like colloid.

1.2 Experimental animals and feeding conditions: Animals: SD rats (clean grade, certificate number: SYXK (Beijing) 2014-0004) provided by Beijing Huafukang Biotechnology Co., Ltd. were selected. Breeding place: animal room of China Center for Disease Control (permit number: SYXK (Beijing) 2014-0043); feed: purchased from Beijing Huafukang Biotechnology Co., Ltd. (permit number: SCXK (Beijing) 2014-0004).

1.3 Main instruments and reagents: BECKMAN GS15R centrifuge (608112); Hitachi 7080 automatic biochemical analyzer (060817); BECKMAN COULTER A ^c .T diff2 ^TM blood analyzer (060816); NIKON biological microscope (608105); Automatic dehydrator (630125), TEK-CC/TEK-EC automatic embedding machine (630122), E0106 semi-automatic paraffin slicer (630121), ST5010 automatic dyeing machine (630127), SCA-5600 automatic sealing machine ( 630126). Coulter electrolyte balance solution and Coulter hemolysis reagent were purchased from Beckman Coulter Experimental System (Suzhou) Co., Ltd.; blood biochemical detection kits were purchased from Zhongsheng Beikong Biotechnology Co., Ltd.

1.4 Experimental method:

1.4.1 Rat acute toxicity test: the maximum tolerated dose method (MTD) was used. 20 healthy SD rats (10 male and 10 each) were selected for the experiment. Rats weighed 180-210 grams. 45g test substance was dissolved to 180ml (concentration is 0.25g/ml) with distilled water, carried out intragastric administration with 20ml/kgBW intragastric administration, 3 times a day, namely the acute toxicity dose was 15g/kg BW, observed continuously for 14 days after intragastric administration. sky. Animal poisoning performance and death were recorded.

1.4.2 Genotoxicity test:

1.4.2.1 Ames test: Four test strains of Salmonella typhimurium TA97, TA98, TA100 and TA102, which have been identified and meet the requirements, are used for the test. S-9 prepared from rat liver homogenate induced by polychlorinated biphenyls (PCB) was used as the activation system. According to the toxicity determination result, test is set 0.313, 0.625, 1.250, 2.500, 5.000mg/dish 5 doses, after 1g test substance is sterilized with ultraviolet ray, is dissolved in aseptic water and is settled to 20ml as highest dose (5.0mg/dish concentration ), and the remaining doses were diluted 2-fold with sterile water, and the sample volume was 100 μL/dish. At the same time set up untreated control, solvent control and positive control dishes. Add 0.1ml test strain enrichment solution, 0.1ml test substance solution and 0.5ml S-9 mixed solution (when metabolic activation is required) to the top agar, mix well and pour it on the bottom medium plate. Incubate at 37°C for 48 hours, and count the number of reverted colonies per dish. If the number of reverted colonies of the test substance is more than 2 times the number of colonies of the solvent control, and there is a dose-response relationship, it is regarded as positive. The whole test was repeated under the same conditions.

1.4.2.2 Mice bone marrow polychromatic erythrocyte micronucleus test: The test was carried out by oral gavage twice at intervals of 24 hours. Fifty mice with a body weight of 25-30 grams were selected and randomly divided into 5 groups according to body weight, with 10 mice in each group, half male and half male. Weigh 20g of the test substance and dissolve it in distilled water to 80ml as the high dose, and then dilute the medium and low doses by 2 times. They are 1.88, 3.75, 7.50g/kgBW respectively. Cyclophosphamide at a dose of 40mg/kgBW was used as a positive control, and distilled water was used as a negative control. Six hours after the last administration of tail type Ⅰ collagen, the animals were sacrificed by cervical dislocation, and the sternal bone marrow was diluted with calf serum for smears, fixed with methanol, and stained with Giemsa. Under a biological microscope, count the number of polychromatic erythrocytes (PCE) in 200 red blood cells (RBC) per animal, and calculate its proportion; count 1000 polychromatic erythrocytes per animal, and the incidence of micronuclei It is calculated by the PCE percentage per thousand containing micronuclei, and statistically processed.

1.4.3 90-day feeding test: Select 80 weaned rats weighing 60-90g and divide them into 4 groups at random, namely the control group and 3 test substance groups. Weigh 20g of the test substance and dissolve it in distilled water to 80ml as the high dose (being the maximum use concentration), and the medium and low doses are sequentially diluted by 2 times, and the gavage volume is 10ml/kgBW. That is, the high, medium and low doses were 0.63, 1.25, 2.50 g/kgBW respectively, and the control group was given distilled water by intragastric administration. There were 20 animals in each group, half male and half male. The animals were fed in a single cage, free to eat and drink, and the rats were weighed twice a week, and the amount of gavage was adjusted according to the body weight. Food intake, leftover food, and sprinkled food were recorded twice a week, and observed continuously for 90 days. At the end of the experiment, blood was collected to measure various hematology, blood biochemical indicators, and histopathological examinations.

1.4.3.1 Observation indicators:

1.4.3.1.1 Animal general performance, body weight, food utilization

1.4.3.1.2 Blood routine and biochemical indicators: white blood cell count and its classification, red blood cell count, hemoglobin, platelet count; aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, urea nitrogen, creatinine, cholesterol, triglyceride, blood sugar, Total Protein, Albumin.

1.4.3.1.3 Visceral body ratio: the animals were sacrificed at the end of the experiment, and the liver, kidney, spleen, heart, thymus and testis were weighed to calculate the corresponding visceral body ratio (calculated in 100 grams of body weight).

1.4.3.1.4 Histopathology: gross observation and histopathological examination by anatomy. When the general examination of the animals in each dose group did not find obvious lesions and abnormal changes in biochemical indicators, only the animals in the high-dose group and the control group were examined for heart, adrenal gland, thymus, liver, kidney, stomach and duodenum, Pathological observation of spleen, testis or ovary.

1.4.3.2 Statistical method: The mean and standard deviation of the data are calculated by SPSS10.0, and statistical analysis is carried out.

2 results

2.1 Rat acute toxicity test: It can be seen from Tables 1 and 6 that female and male rats were given 15.0g/kgBW rat tail type Ⅰ collagen by oral gavage. There was no death, and the results showed that the MTD of the test substance to female and male rats was greater than 15.0g/kgBW.

Table 16 rat tail type Ⅰ collagen rat acute toxicity test results

gender Initial weight (g) Final weight (g) MTD (g/kgBW) female 194.6±5.8(185-206) 230.2±10.1(215-246) >15 male 196.2±6.8(182-210) 294.6±17.0(259-319) >15

2.2 Genetic toxicity test:

2.2.1 Ames test: It can be seen from Table 17 and Table 18 that the number of back-changing colonies in the control dish is within the normal range, and the number of back-changing colonies in each dosage group of rat tail type Ⅰ collagen does not exceed 2 times the number of solvent control colonies, nor does it Dose-response relationship, so for the four test strains of Salmonella typhimurium TA97, TA98, TA100, TA102, when adding or not adding S-9, there was no mutagenic effect on rat tail type Ⅰ collagen.

Table 17 The results of the first Ames test of rat tail type Ⅰ collagen

Note: The above results are the average value of three dishes

Table 18 Results of the second Ames test of rat tail type Ⅰ collagen

Note: The above results are the average value of three dishes.

2.2.2 Mouse bone marrow polychromatic erythrocyte micronucleus test: It can be seen from Table 19 that the percentage of polychromatic erythrocytes (PCE) in each test substance dosage group is not less than 20% of the negative control, indicating that the test substance is under the test dose. No cytotoxicity; no matter male or female mouse cyclophosphamide positive control group micronucleus incidence rate was significantly higher than negative control group and each dose group of test substance (Poisson distribution test P<0.01), while each dose of test substance There was no significant difference between the control group and the negative control group (P>0.05). It shows that the test substance has no mutagenic effect on mouse somatic chromosomes.

Table 19 Effect of rat tail type Ⅰ collagen on the incidence of bone marrow micronuclei in mice

Note: ^a : show that compared with each test substance treatment group, Poisson distribution statistics P>0.05;

^b : show that compared with each test substance treatment group and negative control group, Poisson distribution statistics P<0.01.

2.3 90-day feeding test:

2.3.1 Growth status and food utilization rate:

Animals in each group moved and grew normally, and their coats were thick and shiny. It can be seen from Table 20 that rat tail type Ⅰ collagen was given to rats by intragastric administration for 90 days with doses of 0.63, 1.25, and 2.50 g/kgBW. There was no significant difference between groups (P>0.05).

Table 20 Effect of rat tail type Ⅰ collagen feeding test on rat body weight in 90 days

2.3.2 Blood routine and blood biochemical indicators:

It can be seen from Table 21-22 that the rat tail type Ⅰ collagen of 0.63, 1.25, 2.50g/kgBW dosage was given to rats by intragastric administration for 90 days. There was no significant difference between groups (P>0.05).

Table 21 Effect of 90-day feeding test of rat tail type Ⅰ collagen on rat mid-term blood routine

Table 22 Effect of 90-day feeding test of rat tail type Ⅰ collagen on blood routine of rats

It can be seen from Table 23-24 that the rat tail type Ⅰ collagen of 0.63, 1.25, 2.50g/kgBW dosage was given to rats by intragastric administration for 90 days, and compared with the control group, the blood biochemical indicators of each test substance dosage group were compared with those of the control group. There was no significant difference between them (P>0.05).

Table 23 Effect of rat tail type Ⅰ collagen 90-day feeding test on rat mid-term blood biochemical indicators

Table 24 Effect of 90-day feeding test of rat tail type Ⅰ collagen on blood biochemical indicators in rats

2.3.3 Gross pathological and histological examination: It can be seen from Table 25 that the rat tail type Ⅰ collagen at doses of 0.63, 1.25, and 2.50 g/kg BW was given to rats by intragastric administration for 90 days, and the body weight, There was no significant difference in the weight of liver, kidney, spleen and testis and the viscera ratio of each organ compared with the control group (P>0.05).

Table 25 Effect of 90-day feeding test of rat tail type Ⅰ collagen on rat organ weight and viscera body ratio

It can be seen from the above Table 25 that no abnormalities were found in any organs by gross anatomy. The results of histological examination of rat liver, kidney, stomach, duodenum, spleen, testis (or ovary) viscera are as follows: liver: normal structure can be clearly identified, liver lobule is arranged neatly, and liver cells are centered on the central vein as the axis The heart was in a roughly radial direction; most of the liver cells were normal in shape, and some of the liver cells showed mild watery degeneration; 1 female animal in the control group (1/20) and 1 female animal in the 2.50g/kgBW dose group (1/20 20) Focal necrosis of liver cells can be seen. This lesion often occurs in experimental animals. Kidney: The structure can be clearly identified, the nephrons are evenly distributed, the structure of glomeruli and renal tubules is normal, no interstitial cell proliferation, inflammatory cell exudation, glomerular atrophy and other lesions are seen; some renal convoluted epithelial cells are watery transsexual. Heart: The tissue structure can be identified, consisting of the myocardium, endocardium, and epicardium. Myocardial fibers are staggered, multinucleated, branched striated muscle cells with a small amount of connective tissue and blood vessels in between. Spleen: The tissue structure of the spleen is easy to identify. The capsule stretches to the deep layer to form a trabecular septum of connective tissue. Blood vessels run through it. The white pulp and red pulp are normal. Splenic sinuses are occasionally slightly dilated and congested. The above-mentioned changes in liver, kidney, heart and spleen are common lesions in experimental animals, and there is no difference between the control group and the high-dose group, so it is considered to have nothing to do with the test substance. Stomach: The structure of the gastric wall is easy to identify, and the epithelium of the mucosa is normal. There is no hemorrhage, necrosis, inflammatory exudation, or changes in the composition of glandular cells. Duodenum: Each layer of mucosa, submucosa, muscular layer and serosa can be clearly identified, the structure and cells of each layer are normal, and there is no hemorrhage, necrosis and inflammatory cell infiltration in the mucosa. Testis: each component is easy to identify, the seminiferous tubule boundary membrane is clear, primary spermatocytes, secondary spermatocytes, spermatids and spermatozoa can be seen, no hemorrhage, necrosis and abnormal development of spermatocytes are seen. Ovary: The structural composition is easy to identify. Follicles of different development stages (primordial follicles, primary follicles, secondary follicles, and mature follicles) and corpus luteum can be seen in the cortex. Abnormal development of follicles and pathological changes such as hemorrhage and inflammation in the tissue are not seen. Thymus: The surface is covered with a thin layer of connective tissue, and the normal structure of thymus tissue can be seen below, which is divided into two parts: cortex and medulla. Occasionally, a small amount of adipocytes are mixed in the thymus tissue. No obvious pathological changes were seen. There was no significant difference between the control group and the experimental group. Adrenal gland: The surface is covered with a thin layer of connective tissue, and the normal structure of the adrenal gland tissue can be seen below, which is divided into two parts: the cortex on the surface and the medulla in the center. No obvious pathological changes were seen. There were no significant pathological changes in liver, kidney, spleen, stomach, duodenum, testis or ovary, heart, thymus and adrenal gland.

3 Summary:

3.1 Acute toxicity test: rat tail type Ⅰ collagen has acute oral toxicity to female and male rats, and the MTD is greater than 15.0g/kgBW.

3.2 Genetic toxicity test: Ames test and mouse bone marrow polychromatic erythrocyte micronucleus test did not show mutagenic effect of rat tail type Ⅰ collagen.

3.3 90-day feeding test: 0.63, 1.25, 2.50 g/kg BW doses of rat tail type Ⅰ collagen were given to rats by gavage for 90 days. Compared with the control group, the body weight, weight gain and food utilization rate of the animals in the dosage group had no significant difference (P>0.05). Compared with the control group in each dose group of hematological detection index, there was no significant difference in the blood routine index of the male and female test substance dosage groups compared with the control group (P>0.05); There was no significant difference in the blood biochemical indexes of the drug dosage groups (P>0.05); the weights of heart, thymus, liver, kidney, spleen and testis and the viscera ratio of each organ in each drug dosage group were not significantly different from those of the control group. Sexual differences (P>0.05); pathological indicators also did not see abnormal changes caused by the test substance. Therefore, no adverse effects of the test substance on animal body weight, food utilization rate, hematology, blood biochemistry, visceral body ratio and histopathological examination were found.

Claims (5)

1. The application of rat tail type Ⅰ collagen in the preparation of anti-wrinkle moisturizing and/or whitening products. 2. The application according to claim 1, wherein the product includes at least one of food, cosmetics and health products. 3. A cosmetic, characterized in that: the cosmetic comprises the following components by mass: 2 to 3 parts of rat tail type I collagen; 0.5 to 1 part of peony flower extract; 0.6 to 1.2 parts of propolis extract ; 3 parts hydrogenated polydecene; 4 parts caprylic capric triglyceride; 3 parts simethicone; 1.2 parts cetyl alcohol; 0.5 parts behenyl alcohol; 4 parts isotridecanyl isononanoate; 100 parts water ; 3 parts of glycerin; 3 parts of butanediol; 1 part of glyceryl stearic acid (SE); 1.5 parts of beheneth-20; 0.03 parts of disodium edetate; 0.5 parts of sodium copolymer; 0.2 parts of preservatives; 0.1 to 0.3 parts of essence. 4. the preparation method of cosmetics in claim 3, comprises the steps: 1) hydrogenated polydecene, caprylic triglyceride, simethicone, cetyl alcohol, behenyl alcohol and isotridecyl isononanoate Mix and heat to obtain phase A; 2) Mix rat tail type Ⅰ collagen, peony flower extract water, glycerin, butylene glycol, glyceryl stearic acid, beheneth-20 and disodium edetate to obtain phase B, and then The phase A obtained in step 1) is added to the phase B, pre-emulsified and homogenized; 3) After the A phase and the B phase described in step 2) are treated, add sodium acrylate sodium acyl dimethyl taurate copolymer and mix; 4) Cool the system after the treatment in step 3), and then add preservatives and essence to homogenize to obtain the cosmetic. 5. The preparation method according to claim 4, characterized in that: in step 1), the heating is to 80°C; In step 2), the time of the pre-emulsification is 5min, and the time of the homogenization is 3min; In step 3), the system is cooled to 45°C; In step 4), the homogenization time is 2 minutes.