Low-molecular-weight extracellular matrix active element as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological materials, and particularly relates to a low-molecular-weight extracellular matrix active element as well as a preparation method and application thereof. According to the invention, pepsin, hyaluronidase, collagenase, elastinase and papain are combined and used for carrying out enzymolysis on the cell matrix material, so that the obtained extracellular matrix active element has the advantages of high yield and low molecular weight (Mwlt: 1000 Da); the content ratio of 500Da components can reach 71.35%, and the product can be widely applied to the field of beauty and skin care.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to a low molecular weight extracellular matrix active element and a preparation method and application thereof. Background Art

[0002] Extracellular matrix (ECM) is a complex network structure secreted by cells and distributed around cells, including a variety of proteins, glycosaminoglycans, proteoglycans, and growth factors. In addition to structural support, various components in the extracellular matrix also have active sites, which can communicate with surrounding cells, participate in physiological activities such as cell proliferation, differentiation, and migration, and thus play a role in maintaining tissue homeostasis. Decellularized extracellular matrix (dECM), referred to as decellularized matrix, refers to the extracellular matrix obtained after animal tissue is treated by decellularization. Studies have reported that decellularized matrix materials can participate in and regulate extracellular matrix deposition, inflammatory response, etc., have biological activity, and have been applied to the field of skin repair such as burns and cuts. With the development of the cosmetics industry and the continuous deepening of basic research on extracellular matrix, more and more skin care products have begun to add components of extracellular matrix as functional ingredients to play a role in moisturizing, promoting collagen production, and reducing skin inflammation.

[0003] The main components of acellular matrix materials are biomacromolecules such as collagen, hyaluronic acid, elastin, fibronectin, laminin, hyaluronic acid, and chondroitin sulfate. Without treatment, the molecular weight is much greater than 1000Da. It has been reported in the literature that 500 Da is the dividing line for whether cosmetic raw materials can efficiently penetrate the skin, and ingredients below 500Da have good transdermal effects. The "Technical Guidelines for Cosmetic Safety Assessment" issued by my country in 2021 pointed out that if the chemically synthesized polymers (except raw materials with high biological activity) are composed of one or more structural units, linked by covalent bonds, with an average relative molecular mass greater than 1000 Daltons, and the content of oligomers with a relative molecular mass less than 1000 Daltons is less than 10%, and the structure and properties are stable, transdermal absorption can be ignored. Therefore, the currently recognized view in the skin care raw material industry is that, in general, raw materials below 1000 Da have a transdermal effect. Natural dECM has excellent pro-repair and pro-regeneration capabilities, which is largely due to its rich known or unknown active sites, which can interact with a variety of cells and factors, participate in and regulate a variety of physiological activities, but the high molecular weight of its components limits its wide application in skin care products. Studies have reported that after ECM degradation, it can produce a variety of peptide molecules that regulate cell activity. These molecules are collectively referred to as extracellular matrix active factors (Matrikine). Various extracellular matrix active factors participate in the tumor invasion process by regulating tumor cell activity and angiogenesis. Therefore, establishing a method to reduce the molecular weight of natural dECM components may enable the dECM material to be better applied in skin care products.

[0004] For the degradation of biomacromolecules, there are currently mainly acid hydrolysis, alkaline hydrolysis and enzymatic hydrolysis. Acid or alkaline hydrolysis often causes the loss of activity of biologically active samples due to its violent reaction conditions (strong acid, strong base). Enzymatic hydrolysis is currently a commonly used method for degrading bioactive macromolecular materials due to its mild, specific and efficient characteristics. The main components of decellularized matrix materials are collagen, hyaluronic acid, elastin, glycosaminoglycans, etc. The existing enzymatic hydrolysis methods use a single enzyme, mainly targeting one or two of the components. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that can degrade almost all extracellular matrices, but because of their difficulty in extraction, low yield and high price, they are often used in scientific research experiments such as biochemical molecules and have not been used in large-scale production. In order to prepare low molecular weight collagen peptides, some scholars currently use alkaline protease (patent CN112899335A), compound flavor protease and neutral protease (patent CN 104774896B), pepsin, collagenase and chymosin combination (patent CN 106722999B) to degrade collagen in tissues to obtain low molecular weight collagen peptides.

[0005] Decellularized matrix is ​​a multi-component material, which contains bioactive components such as elastin and glycosaminoglycans in addition to collagen. However, the existing enzymatic hydrolysis method uses a single enzyme, which is specific, so it can only degrade one or several components, and the remaining components still exist in the form of biomacromolecules. In addition, the molecular weight of the obtained extracellular matrix active factors is generally high, and there is no good transdermal effect. Therefore, it is urgent to find a method that can efficiently prepare low molecular weight (Mw < 1000 Da) extracellular matrix active factors, so as to better apply the decellularized extracellular matrix material to skin care products. Summary of the invention

[0006] In view of the problems of the prior art, the present invention provides a low molecular weight extracellular matrix active factor and a preparation method and use thereof.

[0007] An extracellular matrix active factor is obtained by enzymatic hydrolysis of decellularized matrix material with pepsin, hyaluronidase, collagenase, elastase and papain, and has a molecular weight of less than 1000 Da.

[0008] Preferably, its preparation method comprises the following steps: Step 1, reacting the decellularized matrix material with pepsin to obtain a digestive solution; the enzymatic activity of the pepsin is 5-200 U / mL; Step 2, reacting the digestion solution with collagenase to obtain an enzymatic solution 1; the enzymatic activity of the collagenase is 0.5-100 U / mL; Step 3, reacting the enzymatic solution 1 with hyaluronidase, elastase and papain to obtain an enzymatic solution 2; the enzymatic activity of the hyaluronidase is 0.005-1U / mL, the enzymatic activity of the elastase is 0.05-5U / mL, and the enzymatic activity of the papain is 0.5-500U / mL; Step 4, ultrafiltration and freeze-drying of the enzymatic hydrolysate 2 to obtain extracellular matrix active factors.

[0009] Preferably, the enzymatic activity of the pepsin is 55-65 U / mL; the enzymatic activity of the collagenase is 45-55 U / mL; the enzymatic activity of the hyaluronidase is 0.05-0.3 U / mL; the enzymatic activity of the elastase is 0.1-1 U / mL; and the enzymatic activity of the papain is 5-15 U / mL.

[0010] Preferably, in step 1, the reaction is carried out under the action of an acid, and the acid is selected from hydrochloric acid.

[0011] Preferably, in step 1, the reaction temperature is 15-37 °C, the rotation speed is 50-200 rpm, and the reaction time is 12-72 h; And / or, in step 2, the reaction temperature is 15-37°C, the rotation speed is 50-200 rpm, and the reaction time is 12-72 h.

[0012] Preferably, in step 3, the papain needs to be incubated in a chelating agent and a reducing agent before use; the chelating agent is selected from ethylenediaminetetraacetic acid; and the reducing agent is selected from cysteine.

[0013] Preferably, the incubation time is 10-30 min.

[0014] Preferably, in step 3, the reaction temperature is 15-37° C., the rotation speed is 50-200 rpm, and the reaction time is 4-48 h.

[0015] Preferably, in step 4, the ultrafiltration time is 20-60 min, and the ultrafiltration pressure is 1.8-3 bar.

[0016] The present invention also provides a method for preparing the above-mentioned extracellular matrix active factor, comprising the following steps: Step 1, reacting the decellularized matrix material with pepsin to obtain a digestive solution; the enzymatic activity of the pepsin is 5-200 U / mL; Step 2, reacting the digestion solution with collagenase to obtain an enzymatic solution 1; the enzymatic activity of the collagenase is 0.5-100 U / mL; Step 3, reacting the enzymatic solution 1 with hyaluronidase, elastase and papain to obtain an enzymatic solution 2; the enzymatic activity of the hyaluronidase is 0.005-1 U / mL, the enzymatic activity of the elastase is 0.05-5 U / mL, and the enzymatic activity of the papain is 0.5-500 U / mL; Step 4, ultrafiltration and freeze-drying of the enzymatic hydrolysate 2 to obtain extracellular matrix active factors.

[0017] The present invention also provides the use of the extracellular matrix active factor in preparing skin care products.

[0018] The present invention uses pepsin, hyaluronidase, collagenase, elastase and papain in combination for enzymatic decellularization of matrix materials, and the obtained extracellular matrix active element has the advantages of high yield and low molecular weight (Mw<1000 Da), and the content of Mw<500Da components can reach 71.35%, which has broad application prospects in the field of skin care products.

[0019] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0020] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is a flow chart of the preparation process of extracellular matrix active factor.

[0022] Figure 2 The GPC elution curves of the extracellular matrix active factor prepared in Example 1 and Control Example 1 are shown.

[0023] Figure 3 This is the GPC elution curve of the extracellular matrix active factor prepared in Control Example 2 and Control Example 3.

[0024] Figure 4 This is the GPC elution curve of the extracellular matrix active factor prepared in Control Example 4 and Control Example 5. DETAILED DESCRIPTION

[0025] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0026] Example 1 Preparation method of low molecular weight extracellular matrix active factor The following steps are involved: (1) Weigh 2.5 g of micronized decellularized matrix material and place it in a 1 L screw-cap bottle. Then add 500 mL of 0.01 M hydrochloric acid solution and 2.5 g of pepsin (so that the pepsin activity in the solution is 60 U / mL); (2) Place the screw-cap bottle in a constant temperature oscillator, set the program to 30±1°C, speed 120±5 rpm, oscillate for 72 hours to obtain the digestion solution; (3) Adjust the pH of the digestion solution to 6.0±0.1, add collagenase to a final concentration of 50 U / mL, continue to place in a constant temperature oscillator, set the program to 30±1°C, speed 120±5 rpm, oscillate for 12 h, and obtain enzymatic solution 1; (4) Add hyaluronidase (0.2 U / mL), elastase (0.6 U / mL), and papain (10 U / mL, incubated in 1.1 mM EDTA and 5.5 mM cysteine ​​for 30 min before use) to the above enzymatic solution 1, and continue to place it in a constant temperature oscillator, set the program to 30±1°C, speed 120±5 rpm, and oscillate for 12 h to obtain enzymatic solution 2; (5) starting the ultrafiltration (10 kDa) device, ultrafiltering the enzymatic hydrolysate 2 (2 bar, 60 min) to remove the enzyme and undissolved decellularized matrix material in the enzymatic hydrolysate, and collecting the filtrate, i.e., the purified extracellular matrix active factor; (6) The purified extracellular matrix active factor is placed in a vacuum freeze dryer and freeze-dried to obtain the extracellular matrix active factor ( Figure 1 ).

[0027] In other preferred embodiments, when the enzymatic activity of pepsin is 55 U / mL or 65 U / mL, the enzymatic activity of collagenase is 45 U / mL or 55 U / mL, the enzymatic activity of hyaluronidase is 0.05 U / mL or 0.3 U / mL, the enzymatic activity of elastase is 0.1 U / mL or 1 U / mL, and the enzymatic activity of papain is 5 U / mL or 15 U / mL, high yield, low molecular weight (Mw < 1000Da) extracellular matrix active factors can also be obtained.

[0028] The following is the preparation method of the control sample.

[0029] Comparative Example 1 Preparation method of extracellular matrix active factor The preparation method is the same as that of Example 1, except that only pepsin is added, and hyaluronidase, collagenase, elastase, and papain are not added.

[0030] Comparative Example 2 Preparation Method of Extracellular Matrix Active Agent The preparation method is the same as that of Example 1, except that only pepsin and hyaluronidase are added, and collagenase, elastase and papain are not added.

[0031] Comparative Example 3 Preparation method of extracellular matrix active factor The preparation method is the same as that of Example 1, except that only pepsin, hyaluronidase and collagenase are added, and elastase and papain are not added.

[0032] Comparative Example 4 Preparation Method of Extracellular Matrix Active Factor The preparation method is the same as that of Example 1, except that only pepsin, hyaluronidase, collagenase and elastase are added, and papain is not added.

[0033] Comparative Example 5 Preparation method of extracellular matrix active factor The preparation method is the same as that of Example 1, except that only pepsin, hyaluronidase, elastase and papain are added, and collagenase is not added. The concentration of each enzyme used is shown in Table 1.

[0034] Table 1 Enzyme concentrations used in Examples and Control Examples Note: + represents the addition of the enzyme, - represents the absence of the enzyme The technical solution of the present invention is further illustrated by experiments below.

[0035] Experimental Example 1 Yield of Extracellular Matrix Active Factors 1. Experimental Methods The extracellular matrix active factor products of each group were collected and the yield of extracellular matrix active factor of each group was calculated.

[0036] Y= ×100% Y is the yield of the extracellular matrix active factor product; M is the dry weight of the extracellular matrix active factor product; and X is the dry weight of the micronized decellularized matrix material.

[0037] 2. Experimental Results As shown in Table 2, the average yield of the extracellular matrix active factor product prepared by the multiple complex enzyme enzymatic hydrolysis test scheme of the present invention (Example 1) is 2.448±0.007 g; the yield is 97.932%. The yields of the extracellular matrix active factor products in Control Example 1 (pepsin alone), Control Example 2 (pepsin + hyaluronidase combination group), Control Example 3 (pepsin + hyaluronidase + collagenase combination group), Control Example 4 (pepsin + hyaluronidase + collagenase + elastase combination group), and Control Example 5 (pepsin + hyaluronidase + elastase + papain combination group) were 0.342±0.036 g, 0.902±0.044 g, 2.155±0.106 g, 2.345±0.034 g, and 1.535±0.453 g, respectively; the yields of the extracellular matrix active factor in Control Examples 1-5 were 13.675%, 36.091%, 86.188%, 93.808%, and 61.399%, respectively. As the enzymes with different substrates gradually increase, the weight of the collected extracellular matrix active element is on an upward trend as a whole, indicating that the thorough degradation of the composite material of ECM requires the participation of multiple enzymes. Compared with Reference Example 4, the yield of Reference Example 5 is reduced. This is because Reference Example 5 does not use collagenase. When the pH is close to neutral, the collagen with undestroyed triple helical structure in ECM will precipitate and be removed during ultrafiltration. It can be seen that the present invention uses multiple composite enzymes to hydrolyze (pepsin + hyaluronidase + collagenase + elastase + papain) acellular matrix materials to prepare high-yield extracellular matrix active elements.

[0038] Table 2 Statistics of extracellular matrix active factor production and yield Experimental Example 2 Molecular Weight Distribution of Extracellular Matrix Active Factor Products 1. Experimental Methods The extracellular matrix active factor was taken and the molecular weight distribution of the extracellular matrix active factor product was analyzed by gel permeation chromatography (GPC).

[0039] 2. Experimental Results Depend on Figure 2-4 As shown in Table 3, the maximum molecular weight of the extracellular matrix active factors prepared in Example 1 and Control Examples 1-5 is less than 15 kDa, and the proportion of components with Mw < 10 kDa is higher than 99%, indicating that the ultrafiltration device is effective in removing components with Mw > 10 kDa and various enzymes in the enzymatic hydrolyzate. In the extracellular matrix active factors prepared in Example 1 and Control Examples 1-5, the components with Mw<1000 Da accounted for 91.98%, 86.58%, 68.41%, 86.39%, 88.11% and 68.30%, respectively; the components with Mw<500Da accounted for 71.35%, 68.92%, 48.09%, 64.94%, 67.59% and 45.40%, respectively. The above results show that in the extracellular matrix active factors prepared in Example 1, the contents of the components with Mw<1000 Da and Mw<500Da are the highest, and the content of the components with Mw<500Da accounts for up to 71.35%, indicating that it is easier to obtain low molecular weight extracellular matrix active factors by enzymatic hydrolysis of ECM using the preparation method of the present invention.

[0040] Table 3 Statistical table of molecular weight distribution of extracellular matrix active factor products Experimental Example 3 Proportion and yield of extracellular matrix active factor components with different molecular weights 1. Experimental Methods The extracellular matrix active factor was taken, and the molecular weight distribution of the extracellular matrix active factor product was analyzed by gel permeation chromatography (GPC), and the yield of the extracellular matrix active factor products with different molecular weights was calculated.

[0041] The calculation method is: y= ×100% y is the yield of a certain molecular weight extracellular matrix active factor product; A is the ratio of a certain molecular weight extracellular matrix active factor to the extracellular matrix active factor product; M is the dry weight of the extracellular matrix active factor product; and X is the dry weight of the micronized decellularized matrix material.

[0042] 2. Experimental Results As can be seen from Table 4, increasing the use of hyaluronidase (Control Example 2 vs Control Example 1), the yields of the components with 500 Da < Mw < 1000 Da, 200 Da < Mw < 500 Da, and Mw < 200 Da in the extracellular matrix activin product all increased. The increased components may be the products of the enzymatic hydrolysis of glycosaminoglycans such as hyaluronic acid and chondroitin sulfate by hyaluronidase. Increasing the use of collagenase (Control Example 3 vs Control Example 2), the yields of the components with 500 Da < Mw < 1000 Da, 200 Da < Mw < 500 Da, and Mw < 200 Da increased. The increased components may be the products of the enzymatic hydrolysis of collagen by collagenase, namely collagen peptides. Increasing the use of elastase (Control Example 4 vs Control Example 3), the yields of the components with 200 Da < Mw < 500 Da and Mw < 200 Da increased. The increased components may be the products of the enzymatic hydrolysis of denatured collagen, elastin, and fibrin by elastase. Increasing the use of papain (Example 1 vs Control Example 4), the yields of the components with 500 Da < Mw < 1000 Da, 200 Da < Mw < 500 Da, and Mw < 200 Da increased. The increased components may be the products of the enzymatic hydrolysis of gelatin and elastin by papain. Compared with Control Example 3, Control Example 4, and Example 1, the yields of the components with 200 Da < Mw < 500 Da and Mw < 200 Da in Control Example 5 decreased. This is because collagenase was not used in Control Example 5, the triple-helix structure of collagen was not destroyed, and further degradation did not occur. Therefore, the yield of the low-molecular-weight (Mw < 1000 Da) components decreased. Compared with Control Example 1, the increase in the product yields of Control Examples 2-5 and Example 1 may be due to the action of these enzymes on the corresponding substrates, and the substrates were enzymatically hydrolyzed and the molecular weight decreased to be able to pass through the ultrafiltration membrane used in the present invention and be collected. From the above results, it can be seen that when increasing the enzymes with different action substrates, the yield of the low-molecular-weight (Mw < 1000 Da) extracellular matrix activin components increases, and collagenase is necessary for preparing extracellular matrix activin products with high yield and low molecular weight.

[0043] Table 4 Proportion and Yield of Extracellular Matrix Activin Components with Different Molecular Weights In summary, the present invention combines the use of pepsin, hyaluronidase, collagenase, elastase, and papain for enzymatic digestion of decellularized matrix materials. The obtained extracellular matrix activin has the advantages of high yield and low molecular weight (Mw < 1000 Da), and the content proportion of the components with Mw < 500 Da can reach 71.35%, showing broad application prospects in the fields of skin care products and medical aesthetics.

Claims

1. An extracellular matrix active factor, characterized in that The decellularized matrix material is obtained by enzymolysis with pepsin, hyaluronidase, collagenase, elastase and papain, and the molecular weight thereof is less than 1000 Da. The preparation method of the extracellular matrix active element comprises the following steps: Step 1, reacting the decellularized matrix material with pepsin to obtain a digestive solution; the enzymatic activity of the pepsin is 5-200 U / mL; Step 2, reacting the digestion solution with collagenase to obtain an enzymatic solution 1; the enzymatic activity of the collagenase is 0.5-100 U / mL; Step 3, reacting the enzymatic solution 1 with hyaluronidase, elastase and papain to obtain an enzymatic solution 2; the enzymatic activity of the hyaluronidase is 0.005-1U / mL, the enzymatic activity of the elastase is 0.05-5U / mL, and the enzymatic activity of the papain is 0.5-500U / mL; Step 4, ultrafiltration and freeze-drying of the enzymatic hydrolysate 2 to obtain extracellular matrix active factors.

2. The extracellular matrix active agent according to claim 1, characterized in that The enzymatic activity of the pepsin is 55-65 U / mL; the enzymatic activity of the collagenase is 45-55 U / mL; the enzymatic activity of the hyaluronidase is 0.05-0.3 U / mL; the enzymatic activity of the elastase is 0.1-1 U / mL; and the enzymatic activity of the papain is 5-15 U / mL.

3. The extracellular matrix active factor according to claim 1, characterized in that In step 1, the reaction is carried out under the action of an acid, and the acid is selected from hydrochloric acid.

4. The extracellular matrix active factor according to claim 1, characterized in that In step 1, the reaction temperature is 15-37 °C, the rotation speed is 50-200 rpm, and the reaction time is 12-72 h; And / or, in step 2, the reaction temperature is 15-37°C, the rotation speed is 50-200 rpm, and the reaction time is 12-72h.

5. The extracellular matrix active factor according to claim 1, characterized in that In step 3, the papain needs to be incubated in a chelating agent and a reducing agent before use; the chelating agent is selected from ethylenediaminetetraacetic acid; and the reducing agent is selected from cysteine.

6. The extracellular matrix active factor according to claim 5, characterized in that The incubation time is 10-30 min.

7. The extracellular matrix active factor according to claim 1, characterized in that In step 3, the reaction temperature is 15-37°C, the rotation speed is 50-200 rpm, and the reaction time is 4-48 h.

8. The extracellular matrix active factor according to claim 1, characterized in that In step 4, the ultrafiltration time is 20-60 min, and the ultrafiltration pressure is 1.8-3 bar.

9. The method for preparing the extracellular matrix active agent according to any one of claims 1 to 8, characterized in that: The steps include: Step 1, reacting the decellularized matrix material with pepsin to obtain a digestive solution; the enzymatic activity of the pepsin is 5-200 U / mL; Step 2, reacting the digestion solution with collagenase to obtain an enzymatic solution 1; the enzymatic activity of the collagenase is 0.5-100 U / mL; Step 3, reacting the enzymatic solution 1 with hyaluronidase, elastase and papain to obtain an enzymatic solution 2; the enzymatic activity of the hyaluronidase is 0.005-1 U / mL, the enzymatic activity of the elastase is 0.05-5 U / mL, and the enzymatic activity of the papain is 0.5-500 U / mL; Step 4, ultrafiltration and freeze-drying of the enzymatic hydrolysate 2 to obtain extracellular matrix active factors.

10. Use of the extracellular matrix active agent according to any one of claims 1 to 8 in the preparation of skin care products.

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