CN111420023B - Complex containing type I collagen and hyaluronic acid, preparation and application - Google Patents
Complex containing type I collagen and hyaluronic acid, preparation and application Download PDFInfo
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Abstract
The invention provides a compound containing type I collagen and hyaluronic acid, which is characterized in that the compound comprises type I collagen, hyaluronic acid and balancing substances; wherein the balancing substance comprises NaH 2 PO 4 ‑Na 2 HPO 4 A system, a Tris-HCl system, a Tris-maleic acid system or a HEPES system, comprising NaCl and/or KCl; the pH of the complex is 6-8, and the concentration of NaCl and/or KCl is 0.2-0.9 wt%. The invention also provides a preparation method of the compound. The compound of the invention can be mutually dissolved in the proportion of type I collagen and hyaluronic acid in a wider range within the physiological pH value range, and the system is stable, and can not generate precipitation or denaturation for a long time; whileAnd the osmotic pressure and the tension of the compound are suitable for physiological systems. The compound provided by the invention can effectively promote the secretion of collagen fibers by epidermal cells, inhibit inflammation and effectively promote the repair of skin tissues. The preparation method of the compound containing the type I collagen and the hyaluronic acid is simple, low in cost and easy for mass production.
Description
Technical Field
The invention belongs to the biomedical field, and in particular relates to a compound containing type I collagen and hyaluronic acid, a preparation method of the compound, and application of the compound in preparation of a medicine for repairing skin injury or cosmetics for repairing skin injury.
Background
Skin lesions are defects and loss of skin tissue structure and function caused by multiple factors in vivo and in vitro. Skin lesions can be classified as minor lesions and heavier lesions. In general, minor lesions heal by themselves, but heavier lesions do not heal by themselves well.
The metabolic balance of cells is the basis of skin tissue remodeling and repair, and exogenously added active monomers can improve the microenvironment of damaged skin tissues, promote the anabolism of nutrients required by skin tissue metabolism, and are vital to accelerating the skin regeneration and repair process.
Collagen and Hyaluronic Acid (HA) are the most important components of the cell secretory matrix in skin tissue. Collagen in natural skin tissues forms a fiber network, hyaluronic acid is inlaid and integrated into a tissue structure and endows physiological functions, and the hyaluronic acid is synergistic in biological action, so that cell migration, differentiation and proliferation metabolic activity are promoted. The extracellular important matrix components such as collagen, hyaluronic acid and the like are subjected to physical and chemical property optimization and recombination, and are very suitable for skin repair and regeneration under the microenvironment required by local skin healing.
HA exists in the body in its acid form, whereas existing extraction methods are usually obtained in the form of its salts due to process limitations; collagen and HA in salt form are oppositely charged, and polymer is easily formed in a liquid state to generate precipitation, so that research and application of the collagen-HA bionic composite repair material are greatly limited. In order to expand the application of the collagen-HA bionic composite repair material, the prior methods reported in the patent literature comprise the design of isolating the collagen-HA bionic composite repair material from the collagen-HA bionic composite repair material (201310099817.4); there are also methods of mixing the two by physical or chemical means, such as electrochemical treatment to remove sodium salt from HA (20090041033. X), but the process is relatively complex; the modified hydrophobic HA is self-crosslinked with collagen to form a hydrogel three-dimensional scaffold (201711462799.6), and the modified HA can influence the exertion of the functional activity of the collagen.
In addition, the isoelectric point of collagen is about ph7.4, and when the solution is near neutral, collagen is unstable and is likely to precipitate. However, the neutral environment is the metabolically optimal pH in the cell.
Disclosure of Invention
Aiming at the main problems existing in the prior art, the invention provides a compound which is prepared by dissolving type I collagen and hyaluronic acid and keeps the activities of the type I collagen and the hyaluronic acid, and the compound is simple to prepare, low in cost and stable in system.
Accordingly, in one aspect, the present invention provides a complex comprising type I collagen and hyaluronic acid, characterized in that the complex comprises type I collagen, hyaluronic acid and a balancing substance; wherein the balancing substance comprises NaH 2 PO 4 -Na 2 HPO 4 A system, a Tris-HCl system, a Tris-maleic acid system or a HEPES system, comprising NaCl and/or KCl; the pH of the complex is 6-8.
In another aspect, the invention provides the use of the complex in the manufacture of a medicament for the repair of skin lesions; or the complex is used in cosmetics for repairing skin damage.
In another aspect, the present invention provides a method of preparing a complex comprising type I collagen and hyaluronic acid, wherein the method comprises the steps of:
a. preparing a balancing substance: naH is added to water 2 PO 4 -Na 2 HPO 4 Substances of the system, substances of the Tris-HCl system, substances of the Tris-maleic acid system or substances of the HEPES system, and NaCl and/or KCl;
b. adding type I collagen and hyaluronic acid into the balance liquid to obtain a compound containing type I collagen and hyaluronic acid, wherein the pH value of the compound is 6-8.
The compound containing the type I collagen and the hyaluronic acid provided by the invention can be mutually dissolved in any proportion within the physiological pH value range, and the system is stable and can not be precipitated after being placed for a long time; also in a preferred manner, the osmotic pressure and tonicity of the complex are compatible with physiological systems.
The compound containing the type I collagen and the HA provided by the invention can effectively promote the epidermal cells to secrete collagen fibers and matrixes, inhibit inflammatory allergy and anti-aging active ingredients, regulate and control the cell metabolism activity, and can effectively promote the skin tissue repair and restore the blood circulation.
The preparation method of the compound containing the type I collagen and the hyaluronic acid is very simple, low in cost and easy for mass production.
Drawings
Fig. 1: SDS-PAGE electrophoresis of type I collagen prepared in example 1 and supplied by Sigma company;
fig. 2: characteristic absorption peaks for type i collagen prepared in example 1;
fig. 3A: characteristic absorption peaks of the mixed solution prepared in comparative example 1; fig. 3B: characteristic absorption peaks of the gel prepared in example 2;
fig. 4A: characteristic absorption peaks of the mixed solution prepared in comparative example 2; fig. 4B: characteristic absorption peaks of the gel solution prepared in example 3;
fig. 5A: characteristic absorption peaks of the mixed solution prepared in comparative example 3; fig. 5B: characteristic absorption peaks of the gel prepared in example 4;
FIG. 6A is a graph of mRNA expression of Col1- α1 in the control, addition of CI and HA, addition of CI, HA and SAC groups; FIG. 6B is a graph of mRNA expression of Col17- α1 in the control, addition of CI and HA, addition of CI, HA and SAC groups; FIG. 6C is a graph of mRNA expression of iNOS in the control group, the addition of CI and HA groups, the addition of CI, HA and SAC groups; FIG. 6D is a graph showing the expression of mRNA of LIF in the control, CI and HA added, CI, HA and SAC added groups; 6E is a graph of mRNA expression of MMP-1 in the control, addition of CI and HA, addition of CI, HA and SAC groups;
FIG. 7A is a front face condition treated with a human-like collagen repair dressing applied after laser treatment of acne; FIG. 7B is a facial condition at 2.5 days of application of a human-like collagen repair dressing; FIG. 7C is a facial view of a person at 0.5 days when the gel of the present invention is applied; FIG. 7D is a facial view of a 1.5 day application of a gel solution of the present invention; FIG. 7E is a facial view of a person 2.5 days after application of a gel solution of the present invention;
FIG. 8A is an illustration of the condition of acne prior to the use of the gel of the present invention; FIG. 8B is an acne condition at 2.5 days of use of the gel solution of the present invention;
FIG. 9A is a graph showing the condition of scar formation prior to use of the gel solution of the present invention; FIG. 9B shows the condition of scar 2 weeks after the gel solution of the present invention was applied.
Detailed Description
The invention provides a compound containing type I collagen and hyaluronic acid, which is characterized in that the compound comprises type I collagen, hyaluronic acid and balancing substances; wherein the balancing substance comprises NaH 2 PO 4 -Na 2 HPO 4 A system, a Tris-HCl system, a Tris-maleic acid system or a HEPES system, comprising NaCl and/or KCl; the pH of the complex is 6-8.
In order to obtain isotonicity, the concentration of NaCl and/or KCl is preferably 0.2-0.9% by weight.
In the composite provided by the invention, the type I collagen and the hyaluronic acid are mutually soluble in a wide range of proportions, and therefore, the weight proportion thereof is not particularly limited.
In order to more closely approximate the composition of type I collagen and hyaluronic acid in cartilage tissue, it is preferable that the weight ratio of type I collagen and hyaluronic acid is 1 to 20:10-1; more preferably 1 to 5:5-1.
The concentration of the type I collagen and hyaluronic acid is not particularly limited, but in order to effectively supplement the type I collagen and hyaluronic acid, the concentration of the type I collagen is preferably 0.5 to 8mg/ml, preferably 1 to 5mg/ml; the concentration of hyaluronic acid is 0.1-8mg/ml, more preferably 0.5-5mg/ml.
To provide a closer approach to physiologyComplex of the system, in a preferred embodiment of the invention, the balancing substance comprises NaH 2 PO 4 -Na 2 HPO 4 System and NaCl; wherein in the complex, naH 2 PO 4 The concentration of (C) is 0.04-0.7 wt%, na 2 HPO 4 The concentration of (2) is 0.1-0.9 wt%, and the concentration of NaCl is 0.3-0.8 wt%; preferably, naH 2 PO 4 Concentration of 0.08-0.64 wt%, naH 2 PO 4 The concentration of (C) is 0.2-0.8 wt% and the concentration of NaCl is 0.4-0.5 wt%.
In order to further improve the stability of the compound containing the type I collagen and the hyaluronic acid, the balance substance further contains one or more of glucose, 1,3 propylene glycol and tween 80.
In a preferred embodiment, the balance comprises glucose, 1,3 propanediol, and tween 80; preferably, the concentration of glucose is 0.1-10 wt%, preferably 1-5 wt%; the concentration of said 1,3 propanediol is 0.5-3 wt%, preferably 0.5-2 wt%; the concentration of tween 80 is 0.1-1 wt.%, preferably 0.1-0.5 wt.%.
The inventor finds that the balance substance can provide the pH value under the physiological environment in the compound containing the type I collagen and the hyaluronic acid, HAs solubilization and dissolution-assisting effects, and is beneficial to the mutual dissolution of the type I collagen and HA in any proportion; meanwhile, the liquid isotonicity and osmotic pressure can be effectively maintained, and the method is very suitable for direct application of physiological systems.
In order to further improve the skin repair effect, the inventor of the application researches a large number of traditional Chinese medicine components, and unexpectedly discovers that the salvia miltiorrhiza extract is added into the compound containing the type I collagen and the hyaluronic acid, so that the compound can protect and improve the growth microenvironment of chondrocytes, and simultaneously has the effects of stimulating physiological metabolism and secretion activities of cells, regulating and controlling the signal channel activity to promote the regeneration of epidermal cells, inhibiting the expression of activation injury inflammatory genes and induced apoptosis key factors iNOS, inhibiting the expression of interleukin LIF genes involved in mediating inflammatory responses and stimulating acute inflammatory protein synthesis and the like, thereby effectively improving inflammation and anaphylactic reactions while further improving the repair effect.
Therefore, the compound containing the type I collagen and the hyaluronic acid provided by the invention also contains the salvia miltiorrhiza extract.
In order to further improve the restoration effect, the content of the active ingredient in the salvia miltiorrhiza extract is 50 to 99 weight percent, preferably 60 to 90 weight percent.
In order to further enhance the healing effect, the concentration of the active ingredient is 3-200. Mu.g/ml, preferably 7-100. Mu.g/ml, more preferably 10-50. Mu.g/ml.
In order to further maintain the activity of the active ingredient of the red sage root and to allow the drug to be slowly released, the red sage root extract is present in the form of microcapsules in the complex. The microcapsule can be prepared by the prior art, preferably, the microcapsule is prepared by wrapping water-soluble materials such as chitosan, gelatin, alginate, methylcellulose and the like as capsule materials, wherein the capsule center is the extract of the red sage root. The microcapsule can overcome the problem that the water-soluble effective components of the red sage root are easy to reduce the moisture absorption activity, and has a slow release effect.
Preferably, the red sage root extract is a water-soluble extract.
Preferably, the complex further comprises a gel matrix adjuvant; preferably, the concentration of the gel matrix auxiliary material is 0.1-3 wt%. The gel matrix includes, but is not limited to, one or more of cellulose derivatives, carbomers, alginates, tragacanth, and gelatin.
The compound provided by the invention can be used for preparing medicines for repairing skin injury; or the complex is used in cosmetics for repairing skin damage.
The skin lesion includes: trauma caused by various external factors: skin injuries caused by, for example, burns, scalds, frostbites, sunburns, cuts, etc.; pressure sores, e.g., bedsores, protective against pressure injuries; scar; skin allergies, for example, seasonal skin allergies; red swelling; acne; flushing; laser treatment of postoperative redness and inflammation.
The invention also provides a pharmaceutical composition for repairing damaged skin, which comprises the composition containing the type I collagen and hyaluronic acid. The pharmaceutical compositions may also include other ingredients, such as antibiotics, as the case may be. The pharmaceutical compositions may also include a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers can be, for example, fillers, fragrances, flavoring agents, coloring agents, wetting agents, excipients, surfactants.
The pharmaceutical compositions provided by the present invention may be prepared according to methods known in the art and formulated into any dosage form suitable for human or animal use.
The content of the complex comprising type I collagen and hyaluronic acid in the pharmaceutical complex provided by the invention is generally 0.1-95% by weight, or can be adjusted accordingly by a person skilled in the art according to different applications.
In the pharmaceutical composition provided by the invention, the pharmaceutical composition can be gel, dressing (for example, non-woven fabric and dressing pad are used as a bearing) and the like.
The invention also provides that the pharmaceutical composition can be used as an injection or as a tissue engineering implantation material. The invention also provides a cosmetic for repairing damaged skin, which can be facial cleanser, cream, emulsion, facial mask (can be a facial mask containing non-woven fabrics or cotton fibers or a facial mask without non-woven fabrics), dressing and the like.
The content of the complex comprising type I collagen and hyaluronic acid in the cosmetic provided by the invention is generally 0.1 to 95% by weight, or can be adjusted accordingly by one skilled in the art according to different applications.
The present invention also provides a method for preparing a complex comprising type I collagen and hyaluronic acid, wherein the method comprises the steps of:
a. preparing a balancing substance: naH is added to water 2 PO 4 -Na 2 HPO 4 Substances of the system, substances of the Tris-HCl system, substances of the Tris-maleic acid system or substances of the HEPES system, and NaCl and/or KCl;
b. adding type I collagen and hyaluronic acid into the balance liquid to obtain a compound containing type I collagen and hyaluronic acid, wherein the pH value of the compound is 6-8;
in a preferred embodiment, the concentration of NaCl and/or KCl is 0.2-0.9% by weight.
In a preferred embodiment, the weight ratio of type I collagen to hyaluronic acid in the prepared complex containing type I collagen and hyaluronic acid is not particularly limited, and is preferably 1 to 10:10-1; more preferably 1 to 5:5-1.
In a preferred embodiment, the concentration of type I collagen in the prepared complex comprising type I collagen and hyaluronic acid is 0.5-8mg/ml, preferably 1-5mg/ml; the concentration of hyaluronic acid is 0.1-8mg/ml, preferably 0.5-5mg/ml.
In a preferred embodiment of the present invention, the balancing substance comprises NaH 2 PO4-Na 2 HPO 4 System and NaCl; and in the prepared complex containing type I collagen and hyaluronic acid, naH 2 PO 4 The concentration of (C) is 0.04-0.7 wt%, na 2 HPO 4 The concentration of (2) is 0.1-0.9 wt%, and the concentration of NaCl is 0.3-0.8 wt%; preferably, naH 2 PO 4 Concentration of 0.08-0.5 wt%, naH 2 PO 4 The concentration of (C) is 0.3-0.8 wt%, and the concentration of NaCl is 0.4-0.5 wt%.
In a preferred embodiment of the present invention, the balance substance further comprises one or more of glucose, 1,3 propanediol and tween 80.
In a preferred embodiment, the balance comprises glucose, 1,3 propanediol and tween 80 in the preparation of a complex comprising type I collagen and hyaluronic acid; preferably, the concentration of glucose is 0.1-10 wt%, preferably 1-5 wt%; the concentration of the 1,3 propylene glycol is 0.5-3mg/ml, preferably 0.5-2mg/ml; the concentration of Tween 80 is 0.1-1mg/ml, preferably 0.1-0.5mg/ml.
In a preferred embodiment, the method of preparing a complex comprising type I collagen and hyaluronic acid according to the present invention further comprises adding a microcapsule encapsulating the extract of red sage root to the complex comprising type I collagen and hyaluronic acid obtained in step b.
The microcapsules may be prepared using techniques known in the art, preferably, the microcapsules are prepared by encapsulation using a spray drying process; preferably chitosan is used as the capsule wall material.
Both type I collagen and hyaluronic acid are commercially available.
Examples
Example 1: preparation of collagen and physical and chemical property detection
Pretreatment: cutting tendon of beef tendon, cleaning, washing with physiological saline, encapsulating, and passing Co 60 And (5) sterilizing. The remaining steps are carried out under aseptic conditions.
Preparation of high purity type i collagen solution: proper amount of 0.5M acetic acid is added into the crushed beef tendon, pepsin is added (1 gram of pepsin is added into 20g of beef tendon), enzymolysis is carried out for 24-48 hours at 6 ℃, and 15mmol/LEDTA is used for stopping the enzymolysis. The supernatant was collected by centrifugation as a type I collagen solution, and this procedure was performed at 4 ℃.
Purifying: adding collagen solution and 10% NaCl according to the volume of 1:1, salting out, and standing overnight. Centrifuging to obtain precipitate, dissolving with 0.5M acetic acid, and dialyzing in deionized water to pH 5 to obtain high-purity type I collagen solution (+.10mg/ml).
Standard control product: sigmaI collagen product C9301, 0.6mg was weighed, added with 120. Mu.L of 0.5M acetic acid, and slowly shaken to dissolve it, and prepared into a 5mg/ml solution. 10. Mu.L of the solution was added to the equilibration solution.
Type I collagen quality assay (see chinese pharmaceutical industry standard YY0954-2015 annex B hybrid protein assay method): preparing 6% separating gel and 5% concentrating gel by adopting a Tris-glycine discontinuous separating system. Taking 10mg/ml of type I collagen solution and 10 mu L of Sigma standard control solution, adding 90 mu L of double distilled water for dilution, adding 2 xLoding buffer according to a proportion, mixing uniformly, and carrying out boiling water bath for 10min; 15. Mu.L of supernatant was centrifuged at 12000Xg and 5min for SDS-PAGE. Conditions are as follows: 60V,20min;120V,1h 25min (instrument model: bio-Rad, powerPac Universal), stained with Coomassie Brilliant blue R-250 staining solution, and scanned gel imaging (Umax, powerLook,2100 XL-USB) to give high purity characteristic type I collagen bands. The left lane in FIG. 1 is the molecular weight standard, and the left two lanes are the type I collagen prepared as described above; the right lane is sigma type I collagen.
2 mu L of sample is taken, and scanned by using an ultra-trace nucleic acid protein detector (Nanodrop 2000), the wavelength is 200nm-800nm, and the readings of collagen characteristic peaks 217 and 230nm are 1.592 and 1.397 (figure 2) respectively, so that the collagen type I characteristic requirement is met.
Subsequent experiments were performed using the type I collagen prepared in example 1.
Example 2: preparation of the composite of the present application (pH 6)
a. Preparation of balance mass (90 ml): adding NaH into deionized water 2 PO 4 0.643g，Na 2 HPO 4 0.189g; naCl 0.47g, glucose 0.1g,1,3 propanediol 0.5ml, tween 80 0.1ml.
b. 10mg/ml type I collagen solution (10 ml) was measured.
HA 800mg was dissolved in 40ml of the equilibrated material prepared in a, HA concentration 20mg/ml.
d. Mixing steps a, b, c to obtain 100ml of a complex comprising collagen type I and HA, the pH of the complex being 6, naH 2 PO 4 Is about 0.643 wt%, na 2 HPO 4 About 0.189 wt.%, naCl about 0.47 wt.%, glucose about 0.1 wt.%, 1,3 propanediol about 0.5ml/ml, tween 80 about 0.1ml/ml, collagen type I about 1mg/ml, and HA about 8mg/ml.
Example 3: preparation of the complexes of the present application (pH 7)
a. Formulation balance (70 ml) material: adding NaH into deionized water 2 PO 4 0.322g，Na 2 HPO 4 0.566g,NaCl 0.45g glucose 2g,1,3 propanediol 1.5ml, tween 80 0.5ml.
b. 30ml of type I collagen liquid of 10mg/ml is measured.
HA 200mg was dissolved in 20ml of the balance of a formulation with HA concentration of 10mg/ml.
d. Mixing steps a, b, c to obtain 100ml of a complex containing type I collagen and hyaluronic acid, the pH of the complex being 7, naH 2 PO 4 Is about 0.322 wt.%; na (Na) 2 HPO 4 About 0.566 wt%, naCl about 0.44 wt%, glucose about 5 wt%, 1,3 propanediol about 1.5ml/ml, tween 80 about 0.5ml/ml, hyaluronic acid about 2mg/ml, and type I collagen about 3mg/ml.
Example 4: preparation of the complexes of the present application (pH 8)
a. Preparation of balance mass (20 ml): adding NaH into deionized water 2 PO 4 Is 0.04g; na (Na) 2 HPO 4 0.897g; naCl 0.42g, glucose 4g,1,3 propylene glycol 3ml, tween 80 0.98ml.
b. 80ml of type I collagen liquid of 10mg/ml is measured.
HA 50mg was dissolved in 5ml of balance material prepared in a, HA concentration 10mg/ml.
d. Mixing steps a, b, c to obtain 100ml of a complex comprising type I collagen and hyaluronic acid, the complex having a pH of 8, naH 2 PO 4 Is about 0.04 wt.%; na (Na) 2 HPO 4 Is about 0.897 wt.%; the concentration of NaCl was about 0.42 wt%, the concentration of glucose was about 4 wt%, the concentration of 1,3 propanediol was 3ml/ml, the concentration of Tween 80 was 0.98ml/ml, the concentration of hyaluronic acid was 0.5mg/ml, and the concentration of type I collagen was 8mg/ml.
Comparative example 1: preparation of a Low concentration collagen and HA mixture
A type I collagen solution was prepared as in example 1 at a concentration of 2mg/ml.
HA solution was prepared with PBS at a concentration of 16mg/ml, pH 6.
Mixing the solutions prepared in steps a and b at 1:1, i.e. according to a final concentration of hyaluronic acid of 8 after mixing
The concentration of type I collagen was 1mg/ml, and a mixed solution (precipitate was formed) containing type I collagen and hyaluronic acid was obtained, and the pH of the mixed solution was 6.
Comparative example 2: preparation of a Medium concentration collagen and HA mixture
A high concentration type I collagen solution was prepared as in example 1 at a concentration of 6mg/ml.
HA solution was prepared at a concentration of 4mg/ml in PBS.
Mixing the solutions prepared in the steps a and b at a ratio of 1:1, namely adding the solution according to the final concentration of hyaluronic acid of 2mg/ml and the concentration of type I collagen of 3mg/ml after mixing, so as to obtain a mixed solution (with precipitation generation) containing type I collagen and hyaluronic acid, wherein the pH of the mixed solution is 7.
Comparative example 3: preparation of a high concentration collagen and HA mixture (pH 8)
A high concentration type I collagen solution was prepared as in example 1 at a concentration of 16mg/ml.
HA solution was prepared at a concentration of 1mg/ml with PBS.
Mixing the solutions prepared in steps a and b at 1:1, i.e. adding hyaluronic acid at a final concentration of 0.5mg/ml and collagen type I at a concentration of 8mg/ml after mixing, to obtain a mixed solution (with precipitate formation) containing collagen type I and hyaluronic acid, the pH of the mixed solution being 8.
Physical properties test 1: detection of type I collagen compatibility with HA stability (pH 6)
The complex prepared in example 2 and the low concentration mixed solution prepared in comparative example 1 were centrifuged at 12000Xg for 10min at 4℃respectively, and the supernatant was sucked for use. Respectively taking 2 μl of samples for detection, continuously scanning with an ultra-trace nucleic acid protein detector (Nanodrop 2000) at 200nm-800nm, and reading the type I collagen characteristic peak at 230nm to find that the characteristic peak in the supernatant liquid of the mixed solution is basically disappeared at 230 nm; the characteristic peaks of the composite gel solution of the invention do not change significantly before and after centrifugation (figure 3).
The hydroxyproline method is used for measuring the concentration of collagen (refer to the detection method of collagen A in the appendix A of Chinese medical industry standard YY 0954-2015): the kit is operated according to the specification of a Nanjing finished hydroxyproline detection kit (A030-2-1). Sucking 0.25ml of centrifugal supernatant of the two samples treated in the step a, adding 0.5ml of hydrolysate in the kit, uniformly mixing, and hydrolyzing in a boiling water bath for 20min; the pH was adjusted to 65, and the supernatant was centrifuged according to the procedure, absorbance was measured at 550nm and compared with a standard to calculate the type I collagen content (Table 1).
Sodium hyaluronate content was determined by glucuronic acid method (see method a of appendix a of standard YY0308-2004 of chinese pharmaceutical industry): and (3) absorbing 200 mu l of the centrifugal supernatant fluid of the two samples treated in the a and 200 mu l of glucuronic acid standard solution respectively, putting the two samples into an ice water bath, slowly adding 1ml of precooled 0.025M sodium tetraborate sulfuric acid, shaking uniformly, boiling the mixture in the boiling water bath for 20min, taking out the mixture, cooling the mixture to room temperature, adding 40 mu l of precooled 0.1% carbazole ethanol solution, shaking uniformly fully, and putting the mixture at the room temperature for 2h. The absorbance of each standard tube and sample tube at 550nm was measured by a spectrophotometer. The glucuronic acid content in the sample tube was calculated using a standard curve and the sodium hyaluronate mass concentration in the sample was converted according to the formula (table 1).
The results show that the concentration of the HA and the collagen are obviously reduced after the mixed solution group is centrifuged and compared with the mixed solution group before centrifugation; but the concentration of HA and collagen of the compound prepared by 2 in seawater before and after centrifugation are not significantly different, which shows that the balance substance HAs good stability and the function of promoting compatibility.
TABLE 1 detection and analysis of Low concentration type I collagen and HA stability compatibility experimental proteins at pH 6
Note that: * Significant differences in
Physical properties test 2: detection of type I collagen compatibility with HA stability (pH 7)
The compound prepared in example 3 and the medium-concentration mixed solution prepared in comparative example 2 were centrifuged at 12000Xg for 10min at 4℃respectively, and the supernatant was sucked for use. Respectively taking 2 μl of samples for detection, continuously scanning with ultra-trace nucleic acid protein detector (Nanodrop 2000) at 200nm-800nm, and reading the type I collagen characteristic peak at 230nm to find that the characteristic peak at 230nm in the supernatant is basically disappeared; while the characteristic peaks of the complexes of the invention did not change significantly before and after centrifugation (FIG. 4).
The remaining steps are identical to b-d in performance test 1, and the results show that the balance of the invention (pH 7) has good stability and a miscibility promoting effect (Table 2).
TABLE 2 detection and analysis of medium concentration type I collagen and HA stable compatibility experimental protein at pH7
Note that: * Significant differences in
Physical properties test 3: detection of type I collagen compatibility with HA stability (pH 8)
The complex prepared in example 4 and the high-concentration mixed solution prepared in comparative example 3 were centrifuged at 12000Xg for 10min at 4℃respectively, and the supernatant was sucked for use. Respectively taking 2 μl of samples for detection, continuously scanning with ultra-trace nucleic acid protein detector (Nanodrop 2000) at 200nm-800nm, and reading the type I collagen characteristic peak at 230nm to find that the characteristic peak at 230nm in the supernatant is basically disappeared; while the characteristic peaks of the complexes of the invention did not change significantly before and after centrifugation (FIG. 5).
The remaining steps are identical to b-d in performance test 1, and the results show that the balance of the invention (pH 8) has good stability and a miscibility promoting effect (Table 3).
TABLE 3 detection and analysis of high concentration type I collagen at pH8 and HA stability compatibility test protein
Note that: * Significant differences in
Physical property test 4: storage stability detection of type I collagen and HA complex
After the compound of type I collagen and HA (pH 7) prepared in the performance test 2 is stored for 12 weeks, the concentration of the collagen is measured by a hydroxyproline method, the content of sodium hyaluronate is measured by a glucuronic acid method, and the result shows that the content and pH of the type I collagen and HA are unchanged after the compound is placed for 12 weeks, so that the balance substance HAs good stability.
TABLE 4 pH 7I collagen and HA gel stability test for 12 weeks
Performance test 5: effect of the complexes of the invention on the phenotype of epithelial cells
Type I collagen-coated culture plates: the type I collagen complex (pH 7) prepared in example 3 was added to a 24-well culture plate at 0.3ml per well and dried in an incubator at 37℃for 2 days to form a film. 1ml of DMEM-F12 medium was added just before use and equilibrated for further use.
Phenotypic control experiments of epidermal cells (human keratinocytes, hacat cell line):
the experimental components are as follows: the control group without drug, the collagen type I (CI) +HA-treated group, and the collagen type I (CI) +HA+radix Salviae Miltiorrhizae extract (radix Salviae Miltiorrhizae active ingredient (Salvia active components, SAC)) treated group, are three groups. Hacat cells were seeded in 24-well plates, wherein the cells of type I Collagen (CI) +ha-treated groups, and type I Collagen (CI) +ha+sac-treated groups were seeded in the above-described type I collagen-coated 24-well plates; cell inoculation density is 3 ten thousand per hole, MEM culture medium containing 10% serum and 1% diabody is added, the radix salvianolic acid B extract is dissolved by PBS, and the effective concentration of salvianolic acid B is preferably 20 mug/ml; three samples were run at 37℃with 5% CO 2 Culturing for 24 hours under the condition.
Fluorescence quantitative qPCR detection, analysis and test of 5 genes are Col 1-alpha 1, col 17-alpha 1, iNOS, LIF and MMP-1 respectively: meanwhile, the GAPDH assay was used as a reference gene for differential expression analysis (FIG. 6). The results show that: (1) expression of the forward phenotype gene Col1- α1, which promotes proliferation and differentiation of cells: compared with the control group, the expression of Col 1-alpha 1 of the Collagen I (CI) +HA group and the expression of Col 1-alpha 1 of the Collagen I (CI) +HA+SAC group are obviously up-regulated, and the difference is obvious; of these, the type I Collagen (CI) +ha+sac group showed the best effect (fig. 6A). (2) Expression of gene Col17, which mediates adhesion of keratinocytes to basement membrane and serves as a bridge for intracellular and extracellular communication, and for alleviating skin aging: compared with the control group, the expression of Col17 in the group consisting of type I Collagen (CI) +HA and type I Collagen (CI) +HA+SAC is obviously up-regulated, and the difference is extremely obvious; of these, the type I Collagen (CI) +ha+sac group showed the best effect (fig. 6B). (3) Activation of the expression of the injury inflammatory genes and the key factors of apoptosis, iNOS: the expression of iNOS in the group I Collagen (CI) +HA and the group I Collagen (CI) +HA+SAC is significantly up-regulated, and the difference is extremely significant, compared with the control group; wherein the collagen type I (CI) +ha+sac group shows the best effect (fig. 6C); (4) the expression of the interleukin LIF gene involved in mediating inflammatory response and stimulating acute inflammatory protein synthesis shows a significant down-regulation trend along with the increase of metabolic activity after adding a growth regulating factor, and compared with a control group, the expression of iNOS of the type I Collagen (CI) +HA group and the type I Collagen (CI) +HA+SAC group is significantly up-regulated, and the difference is extremely significant; of these, the type I Collagen (CI) +ha+sac group showed the best effect (fig. 6D). (5) Involved in the cleavage of cell surface receptors, releasing apoptotic ligands and inactivating chemokines/cytokines, resulting in disrupted MMP-1 expression by the cell: MMP-1 was statistically significantly down-regulated in both the collagen type I (CI) +HA group and the collagen type I (CI) +HA+SAC group compared to the control group, wherein MMP-1 expression in the collagen type I (CI) +HA+SAC group was nearly absent, showing the best effect. The results show that the complex provided by the invention has multiple regulation effects of improving the growth microenvironment of human keratinocytes, protecting the metabolic balance of the cells and the like (figure 6E).
The following experiments will detect the effect of the compound containing the type I collagen and the hyaluronic acid in the aspects of improving red swelling, acne, scars and the like
The gel used in the following experiments was prepared by the following method: 99% by weight of the gel obtained in example 3 was homogeneously mixed with 1% by weight of hydroxymethyl propyl cellulose.
Performance test 6:
case of enrollment subjects (1 volunteer): patients with facial redness, inflammation and allergy symptoms on the day after surgery with laser treatment.
The experimental process comprises the following steps: at night on the day of acne treatment with laser, facial skin is cleaned with running water by conventional method, and wiped dry. An appropriate amount of "human-like collagen repair dressing" (Simarouba Biotechnology Co., ltd.) was extruded, and the dressing was applied to the affected part skin uniformly, once a day in the morning and at night, for 2.5 days, with the patient feeling of redness removal and insignificant anti-inflammatory effects, see FIGS. 7A and 7B. FIG. 7A is a front face condition coated with a "human-like collagen repair dressing"; FIG. 7B is a facial condition at 2.5 days of application of a "human-like collagen repair dressing";
the patient is then coated with the gel solution of the present invention in the same manner as the "human-like collagen repair dressing". FIG. 7C shows the facial situation at 0.5 days with the gel of the present invention, and FIG. 7D shows the facial situation at 1.5 days with the gel of the present invention; FIG. 7D is a 2.5 day face condition using the gel of the present invention. The results show that: the gel liquid can obviously eliminate red swelling and inflammation after laser operation.
Performance test 7:
enrolled subject case (10 volunteers): acne in puberty, facial acne, pimple, pustule, and redness.
The using method comprises the following steps: the facial skin is cleaned by flowing water and wiped dry by a conventional method. The gel liquid of the invention is uniformly smeared on the skin of an affected part once in the morning and evening. After 1 day of use, acne was significantly reduced in all volunteers. After 2.5 days of use, the effect is very remarkable. FIG. 8A is an illustration of acne condition in one patient prior to use of the gel of the present invention; fig. 8B is an acne condition at 2.5 days of use of the gel of the present invention.
Performance test 8:
case of selected observation object (1): laser removal of nevus from the hands, and 4 months after surgery, a hypertrophic scar is formed.
The using method comprises the following steps: the cleaning liquid washes hands and dries. The gel liquid of the invention is uniformly applied to the skin of an affected part for 1 time per day. FIG. 9A is a graph showing the condition of scar formation prior to use of the gel solution of the present invention; FIG. 9B shows the condition of scar 2 weeks after the gel solution of the present invention was applied. The results show that: the gel liquid can obviously reduce scars, and the originally raised granulation tissues become flat, so that pigmentation is obviously reduced.
Performance test 9:
enrolled observations (20 volunteers): wearing protective articles such as masks and goggles for a long time, the face is damaged under pressure, or allergic manifestations such as redness, pimple or itching.
The using method comprises the following steps: before sleeping, the facial skin is cleaned by flowing water and wiped dry. The gel liquid of the invention is uniformly applied to the skin of an affected part. The next morning, all volunteers had significantly disappeared symptoms of redness, pimple or itching.
Claims (9)
1. A complex comprising type I collagen and hyaluronic acid, wherein the complex consists of type I collagen, hyaluronic acid and a balancing substance; wherein the balancing substance consists of: 1. NaH (NaH) 2 PO4-Na 2 HPO 4 A system, a Tris-HCl system, a Tris-maleic acid system or a HEPES system; 2. glucose, 1,3 propanediol, and tween 80;3. NaCl and/or KCl; the pH of the compound is 6-8, wherein the concentration of glucose is 0.1-10 wt%, the concentration of 1,3 propanediol is 0.5-3 wt%, and the concentration of tween 80 is 0.1-1 wt%; the concentration of NaCl and/or KCl is 0.2-0.9 wt%;
wherein, the type I collagen is prepared according to the following method: adding 0.5M acetic acid into crushed tendon of beef, and adding pepsin for enzymolysis for 24-48 hours, wherein the process is carried out at 4 ℃; centrifuging to collect supernatant collagen solution, adding collagen solution and 10% NaCl according to volume of 1:1, salting out, and standing overnight; the precipitate was obtained by centrifugation, dissolved in 0.5M acetic acid, and dialyzed against deionized water to pH 5.
2. The compound of claim 1, wherein the weight ratio of type I collagen to hyaluronic acid is from 1 to 20:10-1.
3. The complex of claim 1, wherein the concentration of type I collagen is 0.5-8mg/ml and the concentration of hyaluronic acid is 0.1-8 mg/ml.
4. The complex of claim 1, wherein the concentration of type I collagen is 1-5mg/ml and the concentration of hyaluronic acid is 0.5-5mg/ml.
5. The composite of claim 1, wherein the balancing substance consists of: naH (NaH) 2 PO 4 -Na 2 HPO 4 System, glucose, 1,3 propanediol and tween 80, and NaCl; wherein in the complex, naH 2 PO 4 The concentration of (C) is 0.04-0.7 wt%, na 2 HPO 4 The concentration of (2) is 0.1-0.9 wt%; the concentration of the glucose is 1-5 wt%, the concentration of the 1,3 propanediol is 0.5-2 wt%, and the concentration of the tween 80 is 0.1-0.5 wt%; the concentration of NaCl is 0.3-0.8 wt%.
6. The complex of claim 5, wherein NaH 2 PO 4 Concentration of 0.08-0.64 wt%, naH 2 PO 4 The concentration of (C) is 0.2-0.8 wt% and the concentration of NaCl is 0.4-0.5 wt%.
7. Use of a complex according to any one of claims 1-6 for the manufacture of a medicament for the repair of skin lesions; or the use of said complex in the preparation of a cosmetic for repairing skin lesions.
8. The use of claim 7, wherein the skin injury comprises a wound, skin allergy, redness, acne, flushing, and scar repair.
9. A method of preparing the type I collagen and hyaluronic acid containing complex of claim 1, wherein the method comprises the steps of:
a. preparing a balancing substance: naH is added to water 2 PO 4 -Na 2 HPO 4 The material of the system, the material of the Tris-HCl system, the material of the Tris-maleic acid system or the material of the HEPES system; 2. glucose, 1,3 propanediol, and tween 80;3. NaCl and/or KCl; the concentration of the glucose is 0.1-10 wt%, the concentration of the 1, 3-propanediol is 0.5-3 wt%, and the concentration of the tween 80 is 0.1-1 wt%;
b. adding type I collagen and hyaluronic acid into the balance liquid to obtain a compound containing type I collagen and hyaluronic acid, wherein the pH of the compound is 6-8, and the concentration of NaCl and/or KCl is 0.2-0.9 wt%.
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