CN118557465A

CN118557465A - Composite collagen water photokinetic energy element and preparation method thereof

Info

Publication number: CN118557465A
Application number: CN202410699014.0A
Authority: CN
Inventors: 郑妹; 孙孟; 李泽
Original assignee: Shanghai Blueboat Biotechnology Co ltd
Current assignee: Shanghai Blueboat Biotechnology Co ltd
Priority date: 2024-05-31
Filing date: 2024-05-31
Publication date: 2024-08-30

Abstract

The application relates to a compound collagen water photokinetic energy element and a preparation method thereof, wherein the compound collagen water photokinetic energy element comprises the following raw materials: nucleotide compounds, titanium compositions, protein compositions, wherein the titanium compositions comprise titanium trisaccharide, yeast recombinant collagen peptides. The composite collagen water photodynamic element of the application is added with the peptide composition containing the titanium trisaccharide and the yeast recombinant collagen peptide, can increase the activity of collagen cells, and plays a role in improving dynamic wrinkles.

Description

Composite collagen water photokinetic energy element and preparation method thereof

Technical Field

The application belongs to the technical field of biology, and particularly relates to a composite collagen water photokinetic energy element and a preparation method thereof.

Background

The skin is the largest organ of human body, has the functions of preventing the loss of substances such as in-vivo moisture, electrolyte and the like, preventing the invasion of external harmful substances, keeping the stability of the in-vivo environment of the human body and the like, and is a target for common pursuit of people. Aging of the human body is a necessary rule for life development, and skin aging is a part of body aging. The factors of skin aging are numerous, mainly internal and external. Internal factors include oxidation of molecules within skin cells, saccharification of collagen, reduction of hormone secretion levels, etc.; external factors are mainly ultraviolet rays and environmental pollution, and particularly ultraviolet rays have a great influence on aging of skin.

At present, a plurality of products for improving skin aging are available, but some skin care products in the market are added with substances such as lead, mercury and the like or corticotropin stripping agents to damage normal skin physiological metabolism, so that the aim of improving skin aging cannot be achieved, and serious skin problems are easy to cause. Therefore, it is necessary to provide a novel formulation for skin anti-aging and anti-oxidation, and can safely and efficiently improve the comprehensive problem of skin, which is a problem to be solved in industry.

Disclosure of Invention

The present application aims to solve at least one of the technical problems existing in the prior art. Therefore, the application provides the compound collagen water photodynamic element and the preparation method thereof, and the compound collagen water photodynamic element can improve skin aging and does not cause skin problems.

According to a first aspect of the present application, there is provided a composite collagen water photodynamic element comprising the following raw materials: nucleotide compounds, titanium compositions, protein compositions, wherein the titanium compositions comprise titanium trisaccharide, yeast recombinant collagen peptides.

The compound collagen water photokinetic element provided by the embodiment of the application has at least the following beneficial effects: the compound collagen water photodynamic element is added with a peptide composition containing titanium trisaccharide and yeast recombinant collagen peptide, can increase the activity of collagen cells, and plays a role in improving dynamic wrinkles. Wherein, the tripeptide has the effects of resisting carbonylation, protecting collagen from being damaged by active carbon groups, promoting the growth of Sichuan collagen, resisting oxidation and resisting chemical reaction. Tripeptides are able to act on three major elements within the skin: glycosaminoglycans, elastin, collagen, and overall increase skin condition. And can directly solve the problems of collagen synthesis and loss and comprehensively improve the content of collagen. Yeast recombinant collagen peptides generally have the effect of preserving skin elasticity and skin shape, whether short-term smoothing of fine lines or long-term tightening of the skin. The yeast recombinant collagen peptide can exert the anti-aging effect of brightening eyes. Has effects in keeping moisture or nourishing, resisting aging, removing wrinkle, whitening skin, directly penetrating into dermis, supplementing lost collagen, recovering skin elasticity, promoting cell metabolism, and relieving cell aging. The yeast recombinant collagen peptide has the anti-alkalization effect and protects the collagen from being damaged by active carbon groups.

According to some embodiments of the application, the protein composition comprises fibronectin, thioredoxin, elastin, and metallothionein.

It should be noted that fibronectin can be linked to cellular fibers and proteoglycans, and has an adhesive effect. In addition, the substances have nonspecific immunoregulatory effect, and are helpful for phagocytes to phagocytose gram-positive bacteria; and can promote cell migration: promoting migration, binding and differentiation of cells with hyaluronic acid, collagen heparin and the like; can participate in hemostasis and blood coagulation; in addition, it also has growth factor effect: is beneficial to wound healing; meanwhile, the metabolic rate can be enhanced: the adherence rate and the confluence rate of cells can be effectively improved, the time required by confluence of the cells can be effectively shortened, and the good morphological structure of the cells can be maintained; and promote hair growth.

According to some embodiments of the application, the fibronectin, thioredoxin, elastin, and metallothionein are each (10-30) parts by mass.

According to some embodiments of the application, the weight portions of the titanium trisodium and the yeast recombinant collagen peptide are respectively (10-20).

According to some embodiments of the application, the feedstock further comprises (10-20) parts by weight of reduced coenzyme I.

It is understood that the reduced coenzyme I NADH (Nicotinamide adenine dinucleotide) is a chemical substance, is a reduced form of nicotinamide adenine dinucleotide, and belongs to reduced coenzyme I. The reduced coenzyme I has antioxidant, mitochondrial function improving, and photodamage relieving effects; can maintain and repair epidermis, promote collagen synthesis, reduce collagen injury, further effectively prevent skin aging, and reduce wrinkles; also has effects in inhibiting melanin synthesis (whitening), and is a skin care component with multiple skills

According to some embodiments of the application, the raw materials further comprise (10-30) parts by weight of vitamin H.

It should be noted that vitamin H, also called biotin, is a cofactor in an enzyme system involved in carboxylation and decarboxylation processes in the metabolism of the body, and can participate in carboxylation, gluconeogenesis, and protein synthesis. Thus, biotin is a substance necessary for life, epithelial tissue growth and maintenance, and reproduction. In addition, biotin plays an important role in carbohydrate, fatty acid, protein and nucleic acid metabolism in the body. In many cases, people pay more attention to the nutrition and health care functions, and at present, biotin is widely considered to have positive effects on maintaining the normal functions of skin, nails and hair, and the lack of biotin causes the skin symptoms such as hair thinning, loss of luster, white hair, alopecia, dermatitis, eczema and the like. Nevertheless, biotin is very widely available (in many foods including egg yolk, nuts, beans, fish and fruits), and thus, it is desirable to have a diet that is free of additional supplements. However, it has also been shown that diabetes, pregnancy and malnutrition can lead to biotin deficiency, when appropriate exogenous supplementation is required.

According to some embodiments of the application, the raw materials further comprise 50-150kDa hyaluronic acid, glycine and physiological saline for injection.

According to some embodiments of the present application, the parts by weight of the 50-150kDa hyaluronic acid, glycine and physiological saline for injection are (1-10) parts: (10-30 parts of: (50-90) parts.

According to some embodiments of the application, the nucleotide compound comprises 10-90ppm nicotinamide adenine dinucleotide.

It is understood that NAD+ (Nicotinamide adenine dinucleotide) Chinese, collectively referred to as "nicotinamide adenine dinucleotide", coenzyme I for short, is a proton-transferring (precisely hydrogen ion) coenzyme that occurs in multiple metabolic reactions of cells. NAD+ is a coenzyme for dehydrogenases, such as Alcohol Dehydrogenase (ADH), for oxidizing ethanol. It plays an irreplaceable role in glycolysis, gluconeogenesis, tricarboxylic acid cycle and respiratory chain.

According to a second aspect of the present application, there is provided a method for preparing a composite collagen water photodynamic element, comprising the steps of: dissolving a nucleotide compound, a titanium composition, a protein composition, coenzyme I, vitamin H and glycine in normal saline for injection according to a certain proportion, stirring to completely dissolve the substances, then adding 50-150kDa hyaluronic acid according to a certain proportion, continuously stirring to dissolve the substances, and sterilizing to obtain the compound collagen water photokinetic energy element.

It will be appreciated that the sterilization conditions described above are radiation sterilization at a dose of 10-25 KGy.

Drawings

The application is further described with reference to the accompanying drawings and examples, in which:

FIG. 1 is a graph showing the observations of the acute systemic toxicity test in example 7 of the present application;

FIG. 2 is a graph showing the observation of acute systemic toxicity test in example 7 of the present application;

FIG. 3 is a graph showing individual body weight analysis of animals at day 28 of implantation in example 8 of the present application;

FIG. 4 is a graph showing the analysis of the change in the weight of immune organs of the mice at day 28 of implantation in example 8 of the present application;

FIG. 5 is a summary chart of the thymus histopathological examination of the 28 day mice implanted in example 8 of the present application;

FIG. 6 is a negative control group chart (100X) in example 8 of the present application;

FIG. 7 is a chart of the low dose group (100X) in example 8 of the present application;

FIG. 8 is a graph of the medium dose group (100X) in example 8 of the present application;

FIG. 9 is a high dose panel (100X) for example 8 of the present application;

FIG. 10 is a graph (100X) of the positive control group in example 8 of the present application;

FIG. 11 is a graph of an oral response scoring system according to example 9 of the present application;

FIG. 12 is a graph showing the results of the experiment in example 9 of the present application;

FIG. 13 is a graph showing the response scoring system for the intradermal stimulus test in example 10 of the present application;

FIG. 14 is a graph showing the results of the experiment in example 10 of the present application;

FIG. 15 is a graph showing the plate efficiency and relative survival (4 h exposure) without the metabolic activation system (-S9) in example 11 of the present application;

FIG. 16 is a graph showing the plate efficiency and relative survival (4 h exposure) with the metabolic activation system (-S9) in example 11 of the present application;

FIG. 17 is a graph showing the plate efficiency and relative survival (24 h contact) without the metabolic activation system (-S9) in example 11 of the present application.

Detailed Description

The conception and the technical effects produced by the present application will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present application. It is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present application based on the embodiments of the present application.

In the description of the present application, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Skin aging is a part of the aging of the body. The factors of skin aging are numerous, mainly internal and external. Internal factors include oxidation of molecules within skin cells, saccharification of collagen, reduction of hormone secretion levels, etc.; external factors are mainly ultraviolet rays and environmental pollution, and particularly ultraviolet rays have a great influence on aging of skin. In addition, skin is a collective protective barrier for humans, the first line of defense for humans against external "aggressors," and comprises epidermis and dermis, where dermis provides elastic support, epidermis provides protection, and is an important component of the "skin barrier. The epidermal barrier refers to a layer of natural protective film of the skin, including the sebaceous membrane and the stratum corneum. Wherein, the sebum membrane is weak acid, the water-oil ratio is 7:3, when the skin barrier is damaged, the horny layer can not prevent water from being scattered, a great deal of water is lost in the skin, pollution and microorganisms can enter hair follicles, sebaceous glands in the hair follicles are stimulated, excessive secretion of sebum is caused, various skin inflammations are caused, various skin diseases and skin are sensitive, even the aging of the skin is accelerated, and unless the barrier is restored, a plurality of moisturizing components and nutritional components are useless. For people with sensitive skin, the repair of skin barrier is particularly important, and meanwhile, the skin barrier of sensitive skin is very fragile, and the improper use of skin care products can accelerate skin sensitivity, so that the usable range of skin care products for people with sensitive skin is suddenly reduced, and malignant circulation is formed. Therefore, when the anti-aging skin care product is used, the anti-aging skin care product is safe and does not cause sensitive problems to skin, and is one of key problems to be solved urgently.

Specific embodiments of the present application are described in detail below.

Example 1

The raw materials of the first component are selected according to the following formula (parts by weight): NAD+ (nicotinamide adenine dinucleotide) (10 ppm), tripeptide (1 part), yeast recombinant collagen peptide (1 part), fibronectin (10 parts), thioredoxin (10 parts), elastin (10 parts), metallothionein (10 parts), coenzyme 1 (1 part), glycine (10 parts), vitamin H (10 parts). The above-mentioned first component raw material was dissolved in 50 parts of physiological saline for injection, and stirred to completely dissolve the raw material, thereby obtaining a first solution. Then adding 50-150kDa hyaluronic acid (1 part) into the first solution, stirring to completely dissolve, and finally sterilizing the dissolved solution to obtain the compound collagen water photodynamic element.

Example 2

The raw materials of the first component are selected according to the following formula (parts by weight): NAD+ (nicotinamide adenine dinucleotide) (90 ppm), tripeptide (20 parts), yeast recombinant collagen peptide (20 parts), fibronectin (30 parts), thioredoxin (30 parts), elastin (30 parts), metallothionein (30 parts), coenzyme 1 (20 parts), glycine (30 parts), vitamin H (30 parts). The above-mentioned first component raw material was dissolved in 90 parts of physiological saline for injection, and stirred to completely dissolve the raw material, thereby obtaining a first solution. Then adding 50-150kDa hyaluronic acid (10 parts) into the first solution, stirring to completely dissolve, and finally sterilizing the dissolved solution to obtain the compound collagen water photodynamic element.

Example 3

The raw materials of the first component are selected according to the following formula (parts by weight): NAD+ (nicotinamide adenine dinucleotide) (50 ppm), tripeptide (10 parts), yeast recombinant collagen peptide (10 parts), fibronectin (20 parts), thioredoxin (20 parts), elastin (20 parts), metallothionein (20 parts), coenzyme 1 (10 parts), glycine (20 parts), vitamin H (20 parts). The above-mentioned first component raw material was dissolved in 70 parts of physiological saline for injection, and stirred to completely dissolve the raw material, thereby obtaining a first solution. Then adding 50-150kDa hyaluronic acid (5 parts) into the first solution, stirring to completely dissolve, and finally sterilizing the dissolved solution to obtain the compound collagen water photodynamic element.

Example 4

The raw materials of the first component are selected according to the following formula (parts by weight): NAD+ (nicotinamide adenine dinucleotide) (30 ppm), tripeptide (5 parts), yeast recombinant collagen peptide (5 parts), fibronectin (15 parts), thioredoxin (15 parts), elastin (15 parts), metallothionein (15 parts), coenzyme 1 (5 parts), glycine (15 parts), vitamin H (15 parts). The above-mentioned first component raw material was dissolved in 60 parts of physiological saline for injection, and stirred to completely dissolve the raw material, thereby obtaining a first solution. And adding 50-150kDa hyaluronic acid (3 parts) into the first solution, stirring to completely dissolve, and finally sterilizing the dissolved solution to obtain the compound collagen water photodynamic element.

Example 5

The raw materials of the first component are selected according to the following formula (parts by weight): NAD+ (nicotinamide adenine dinucleotide) (70 ppm), tripeptide (15 parts), yeast recombinant collagen peptide (15 parts), fibronectin (25 parts), thioredoxin (25 parts), elastin (25 parts), metallothionein (25 parts), coenzyme 1 (15 parts), glycine (25 parts), vitamin H (25 parts). The above-mentioned first component raw material was dissolved in 80 parts of physiological saline for injection, and stirred to completely dissolve the raw material, thereby obtaining a first solution. Then adding 50-150kDa hyaluronic acid (7 parts) into the first solution, stirring to completely dissolve, and finally sterilizing the dissolved solution to obtain the compound collagen water photodynamic element.

Example 6

The anti-aging test data of examples 1-3 are described using the collagen neogenesis ratio after 28 days, see the following table:

example group	Formulation of	Collagen regeneration ratio after 28 days
			Example 1	Example 1 formulation	The same ratio is increased by 16 percent, and the medium effect is judged
Example 2	Example 2 formulation	The same ratio is increased by 15 percent, and the medium effect is judged
			Example 3	Example 3 formulation	The same ratio is increased by 25 percent, and the judgment is high-efficiency
Comparative example 1	Comparative example 1 formulation	The same ratio is increased by 2 percent, and the judgment is low

The first component raw materials were selected according to the following formulation (parts by mass) of comparative example 1: NAD+ (nicotinamide adenine dinucleotide) (70 ppm), fibronectin (25 parts), thioredoxin (25 parts), elastin (25 parts), metallothionein (25 parts), coenzyme 1 (15 parts), glycine (25 parts), vitamin H (25 parts). The above-mentioned first component raw material was dissolved in 80 parts of physiological saline for injection, and stirred to completely dissolve the raw material, thereby obtaining a first solution. Then adding 50-150kDa hyaluronic acid (7 parts) into the first solution, stirring to completely dissolve, and finally sterilizing the dissolved solution to obtain the compound collagen water photodynamic element.

Example 7

Acute systemic toxicity test

Test system: animal species-mice. Animal strain: ICR. Sex of animals: and (5) male. Week-old: and (5) early adult. Body weight range: the body weight at the beginning of the test was 17g to 22g. The adaptation time is as follows: for at least 5 days. Number of animals: 10. Marking method, dyeing method.

First, the mice were placed in the holder. Then, the test sample and the blank were injected by the intraperitoneal route at a dose of 50mL/kg ³, and animal weights were measured and recorded before the test sample (the complex collagen photokinetic proteins of examples 1 to 5 were taken as test groups 1 to 5, respectively) and the blank were injected. After injection, the animals were observed for immediate biological response and the general status, toxicity and number of dead animals were observed and recorded at 4h, 24h, 48h and 72h for the test and control animals, and the animal weights were weighed and recorded at 24h, 48h and 72 h.

The observation content is shown in fig. 1, and the observation result is shown in fig. 2. According to the results of the acute systemic toxicity test shown in FIG. 2, the compound collagen water photokinetic element prepared in examples 1-5 has no acute systemic toxicity reaction.

Example 8

Immunotoxicity test

Weighing animals: all animals were weighed prior to subcutaneous injection and once weekly after injection (injection of the complex collagen water photokinetic element prepared according to example 1). On day 28 of injection, the following operations were performed, respectively:

Hematology detection: taking eyeball blood into EDTA anticoagulation tube, mixing well, measuring the following indexes in hemocyte analyzer: hemoglobin, red blood cells, platelets, white blood cell count.

Immunoglobulin (lgE, igG, igM) and complement C3 concentration assay: blood is collected from eyeballs and put into an EP tube, the blood is centrifuged, serum is collected, the serum is stored at 20 ℃ after sub-packaging, an enzyme-linked immunosorbent (Enzyme Linked Immunosorbent Assay, ELISA) test is carried out according to the instruction of a kit, and the concentration of immunoglobulin (lgE, igG, igM) and the concentration of complement C3 and the concentration of inflammatory factors (TNF-a and IL-6) in the serum are detected.

[ As a result ]

[ IgG ] Total IgG levels in serum were measured by ELISA. On day 28 of the sample implantation, the serum IgG concentrations in the low, medium and high dose groups were not significantly different (p > 0.05) from those in the negative control group, while the IgG concentrations in the positive control group were significantly higher than those in the negative control group (p 0.01).

[ IgM ] Total IgM levels in serum were detected by ELISA. On day 28, there was no significant difference in IgM concentration in the serum of the low, medium and high dose groups compared to the negative control group (p > 0.05), and the positive control group serum IgM level was significantly higher than the negative control group (p < 0.01).

[ TNF-a ] levels of TNF-a in serum were measured by ELISA. On day 28, the serum TNF-a levels were significantly higher in the positive control than in the negative control (p < 0.01), with no significant difference p0.05 between the low, medium and high dose serum levels.

[ IL-6 ] the serum IL-6 level was measured by ELISA. On day 28 of the test sample implantation, there was no significant difference (p > 0.05) in the IL-6 content in serum of the low, medium and high dose groups compared to the negative control group; in addition, the serum IL-6 level of the positive control group was significantly higher than that of the negative control group (p < 0.01).

Test results

In this test, the sample to be tested (the compound collagen prepared in example 1) was subcutaneously injected into BALB/C mice via the back at three doses, i.e., low, medium and high, and the immunotoxicity index of the mice was measured at day 28 of injection. As can be seen from the experimental data (fig. 3-10), the mice were subcutaneously injected with the test samples, the weight gain of each experimental group was normal, the spleen, thymus weight and organ coefficients of each dose group of the test samples were not statistically different (p > 0.05) from those of the negative control group, and microscopic examination of thymus tissue revealed that no significant histopathological changes were found in the low, medium, high dose groups and the positive control group of the test samples as compared with the negative control group. The blood cell parameters (hemoglobin, red blood cell count, white blood cell count and platelets) were not significantly changed (p > 0.05) in the high, medium and low dose groups compared to the negative control group. In the mouse humoral immune response detection, the serum IgE, igG, igM and complement C3 contents of the low, medium and high dose groups of the detected samples are close to the level of a negative control group, and no statistical difference (p > 0.05) exists; in the specific cellular immune response detection, compared with the negative control group, the detected sample has no obvious effect on the cellular immunity of mice after being implanted for 28 days, and does not cause obvious cell-mediated immune response. The detection of inflammatory factors shows that the content of TNF-a and IL-6 in serum implanted by the tested samples in each dose group is not significantly different from that of the negative control group (p > 0.05). In addition, there was no significant change in the T cell number, helper T cell number, suppressor T lymphocyte number, activated T lymphocyte number, B lymphocyte number, NK and NKT cell percentages for each dose group of the samples tested compared to the negative control group in each subpopulation classification of mouse lymphocytes (p > 0.05). Compared with the negative control group, the spleen and thymus weights and organ coefficients, humoral immune response, specific cell immune response and mouse lymphocyte subgroup analysis of the positive control group mice are all significantly different (p < 0.01), which indicates that the test system operates normally. Under the test condition, the comprehensive evaluation of the immune toxicity test result shows that compared with a negative control group, the low-medium and high-dose treatment of the tested sample for 28 days has no adverse effect on the functions of the BALB/C mouse immune system.

Example 9

Oral mucosa irritation test

The test is carried out by evaluating the potential of the test sample for leading golden mice to generate oral tissue stimulation reaction under test conditions and adopting the method recommended in GB/T16886.10-2017 medical appliance biological evaluation, part 10, stimulation and skin sensitization test. The prepared complex collagen water photokinetic element test sample of example 1 was directly infused into the cheek at 0.1m 1/time/and the other side cheek pouch was not placed as a control and contacted for 5min. The above steps were repeated 1 time per hour for a total of 4 hours. 24h after the last contact, the cheek sacs were visually observed, the mice were sacrificed without pain, and tissue samples of representative parts of the frequency sacs were taken and fixed in a suitable fixing solution for histological evaluation. Under the test conditions, the test results show that the test sample has no oral mucosa irritation reaction.

The method comprises the following specific steps: 1. animals were anesthetized with an appropriate anesthetic, and after the cheek pouch was turned over and flushed with normal saline, the presence or absence of abnormality was checked. 2. The prepared compound collagen water photokinetic element test sample in the example 1 is directly infused into cheeks at a rate of 0.1/time, and the other side cheeks are not placed as a sample to serve as a control, and the control animals are subjected to parallel test and contact for 5min. After contact, the cheek pouch was flushed with physiological saline, and the above procedure was repeated 1 hour for 4 hours without contaminating the other side of the pouch, and the cheeks were inspected after each contact and before repeated placement. Cheek sacs were visually observed 24h after the last contact and scored by the oral response scoring system (fig. 11). 4. Finally, the animals are sacrificed without pain, and tissue samples of representative parts of the cheeks are taken and placed into a proper fixing solution for fixation, and then histological evaluation is carried out.

The test result is shown in figure 12, and the result shows that the composite collagen water photokinetic element prepared by the method has no oral mucosa irritation reaction.

Example 10

Intradermal stimulation test

The animal species is rabbit. Animal strain New Zealand albino rabbit. Sex of animals, male. Body weight range: body weight at test >2.0kg. The adaptation time is as follows: for at least 5 days. Number of animals: 3. The marking method comprises the following steps: and (5) marking a cage label.

The steps are as follows: 1. the day before the test, the fur on both sides of the back and the spine of the animal is thoroughly removed. 2. The exposed skin was cleaned with 75% (V) ethanol and 0.2ml of the prepared complex collagen hydrodynamic photokinetic test sample of example 1 was injected intradermally at five points on one side of each post-free, and likewise, a blank was injected on the other side of the spine of each rabbit. 3. Immediate response at the injection site was observed immediately after injection, and the status of each injection site was recorded at 24h, 48h and 72h after injection, and the tissue response scores for each injection site for each observation period were recorded as per the scoring system (fig. 13), and the test results were recorded.

Test results (fig. 14):

The score for each animal test sample or placebo was calculated by adding the total erythema and edema scores for 24h, 48h, 72h, respectively, for each animal test sample or placebo, after 72h scoring, divided by 15[3 (scoring time point) 5 (test sample placebo injection point) ]. The total average score for each test sample and corresponding blank was calculated by dividing the sum of the 3 animal scores by 3. The final score for the test sample was obtained by subtracting the placebo score from the test sample score. If the final score of the test sample is not greater than 1.0, the test requirements are met. In any observation period, if the average reaction of the test sample is suspected to be larger than that of the blank control reaction, 3 other test-free samples should be taken, and if the final score of the test sample is not larger than 1.0, the test sample meets the test requirement. Under the test conditions, the test results showed no intradermal reaction of the test samples.

Example 11

Biological genotoxicity (test of Compound collagen Water photokinetic element prepared in example 1)

1. The well-grown cells were collected by the contact treatment, and the cell density was adjusted to 1X 0/mL (to ensure that the final cell concentration was 5X 10/mL). Group S9 (no metabolic activation system) 10mL of cell suspension was mixed with 2mL of test or control sample and 1mLPBS added to a final volume of 20mL of serum-containing medium; with group S9 (with metabolism activation System) 10mL of cell fluid was mixed with 2mL of test or control sample and mIS parts of the mixture and added to a final volume of 20L in serum-containing medium. Placing the above mixed solutions in 37C shaking culture for 4h, wherein the shaking frequency is 70-80 r/min. If the S9-free group is negative after 4 hours of contact, the time is prolonged to 24 hours.

2. Expression: the above mixtures were centrifuged at 200g for 5min, the supernatant was removed, and the cells were washed twice with serum-free RPMI1640 medium. The cells were resuspended in RPMI1640 medium to a cell density of 3X0 cells/mL and incubated at 37C for 2d. Cell density was checked at 24h and adjusted to 3x 10/mL.

3. Preparing each group of micro-pore plates PE (plating effect of 1 d: plating cell liquid after the grafting is taken out is diluted to 8 cells/mL by a 1640 culture medium containing 20% serum in a gradient way, inoculating 96-pore plates, adding 0.2mI (namely, the average cell inoculation number of each pore is 1.6) into each pore, inoculating two plates PE (plating efficiency of 2 d) in each dose, after the 2d expression culture is finished, taking a proper amount of cell liquid, inoculating 96-pore plates according to a PE plate preparation method, inoculating two plates with each dose, namely, a TFT antagonizing plate, namely, after the 2d expression culture is finished, taking a proper cell suspension, adjusting the cell density to 1x 10/mL by a 1640 culture medium containing 20% serum, adding TFT (final concentration is 3 g/mL), uniformly mixing, inoculating 96-pore plates, adding 0.2mL (namely, the average cell inoculation number of each pore is 2000), inoculating two plates with each dose, placing all plates in a carbon dioxide box 37C 10 d-12 d.4, counting the diameter of colonies, and a microscopic colony with a small diameter (diameter: 1.4 d) of the micro-cell colony is counted, and the diameter is counted by visual observation, and the diameter of each colony is small, and the colony diameter is 4 d is small, and the colony diameter is counted continuously (1: small diameter is small, and the diameter is 4, and the diameter is small).

Conclusion of the test

Under the above test conditions, the test results of FIGS. 15-17 demonstrate that the test samples were non-reproductive genotoxic.

While the embodiments of the present application have been described in detail with reference to the specific embodiments, the present application is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present application within the knowledge of those skilled in the art. Furthermore, embodiments of the application and features of the embodiments may be combined with each other without conflict.

Claims

1. The composite collagen water photodynamic element is characterized by comprising the following raw materials: nucleotide compounds, titanium compositions, protein compositions, wherein the titanium compositions comprise titanium trisaccharide, yeast recombinant collagen peptides.

2. The composite collagen water photodynamic element of claim 1 wherein the protein composition comprises fibronectin, thioredoxin, elastin and metallothionein.

3. The compound collagen water photodynamic element according to claim 2, wherein the fibronectin, thioredoxin, elastin and metallothionein are respectively (10-30) parts by mass.

4. The composite collagen water photodynamic element according to claim 1, wherein the weight portions of the titanium trisaccharide and the yeast recombinant collagen peptide are respectively (10-20).

5. The compound collagen water photodynamic element according to any one of claims 1 to 4, wherein the raw material further comprises (10-20) parts by mass of reduced coenzyme I.

6. The compound collagen water photodynamic element of any one of claims 1 to 4 wherein the raw material further comprises (10-30) parts by mass of vitamin H.

7. The compound collagen water photodynamic element of any one of claims 1 to 4 wherein the raw material further comprises 50-150KDa hyaluronic acid, glycine, physiological saline for injection.

8. The compound collagen water photodynamic element according to claim 7, wherein the parts by mass of the 50-150kDa hyaluronic acid, glycine and physiological saline for injection are (1-10) parts: (10-30 parts of: (50-90) parts.

9. The complex collagen water photodynamic element of any one of claims 1 to 4 wherein the nucleotide compound comprises 10-90ppm nicotinamide adenine dinucleotide.

10. The preparation method of the composite collagen water photodynamic element is characterized by comprising the following steps of: dissolving a nucleotide compound, a titanium composition, a protein composition, coenzyme 1, vitamin H and glycine in normal saline for injection according to a certain proportion, stirring to completely dissolve the substances, then adding 50-150kDa hyaluronic acid according to a certain proportion, continuously stirring to dissolve the substances, and sterilizing to obtain the compound collagen water photokinetic energy element.