CN119792126A - A low molecular weight extracellular matrix active factor and its preparation method and use - Google Patents

Abstract

The invention belongs to the technical field of biological materials, and particularly relates to a low-molecular-weight extracellular matrix active element, a preparation method and application thereof. According to the invention, pepsin, hyaluronidase, collagenase, elastase and papain are used for enzymatic hydrolysis of cell matrix materials, so that the obtained extracellular matrix active element has the advantages of high yield and low molecular weight (Mw <1000 Da), the content ratio of Mw <500Da components can reach 71.35%, and the extracellular matrix active element can be widely applied to the field of beauty and skin care.

Description

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

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a low-molecular-weight extracellular matrix active element, a preparation method and application thereof.

Background

Extracellular matrix (Extracellular matrix, ECM) is secreted by cells and surrounds a complex network of structures distributed around cells, including various proteins, glycosaminoglycans, proteoglycans, and growth factors, among others. Besides structural support, various components in the extracellular matrix also have active sites, can be communicated with surrounding cells, participate in physiological activities such as cell proliferation, differentiation, migration and the like, and further play a role in maintaining tissue homeostasis. Acellular extracellular matrix (Decellularized extracellular matrix, dECM), abbreviated as acellular matrix, refers to extracellular matrix obtained by subjecting animal tissue to an acellular process. The research reports that the acellular matrix material can participate in and regulate extracellular matrix deposition, inflammatory reaction and the like, has bioactivity, and is applied to the field of skin repair of burns, cuts and the like. With the development of the cosmetic industry and the continuous and deep research of extracellular matrix foundation, more and more skin care products begin to be added with extracellular matrix components as functional components, thereby playing the roles of moisturizing, promoting collagen production, reducing skin inflammation and the like.

The main components of the acellular matrix material are collagen, hyaluronic acid, elastin, fibronectin, laminin, hyaluronic acid, chondroitin sulfate and other biological macromolecular materials, and the molecular weight of the acellular matrix material is far more than 1000 Da under the untreated condition. It is reported in the literature that 500Da is a dividing line for efficient transdermal of cosmetic materials, and that ingredients below 500Da have good transdermal effects. The technical guidelines for evaluating the safety of cosmetics published in 2021 in China indicate that if one or more structural units are chemically synthesized and linked through covalent bonds, a polymer with an average relative molecular mass of more than 1000 daltons and an oligomer content of less than 10% relative molecular mass of less than 1000 daltons and stable structure and properties (except raw materials with higher biological activity) can be used without consideration of transdermal absorption. Thus, the current industry accepted view of skin care materials is that materials generally below 1000 Da have transdermal effects. Natural dECM has excellent ability to promote repair and regeneration, which is greatly benefited by the fact that it contains abundant known or unknown active sites, can interact with various cells and factors, participate in and regulate various physiological activities, but the high molecular weight of the components limits its wide application in skin care products. It has been reported that ECM degradation produces a variety of peptide molecules that regulate cellular activity, which are collectively referred to as extracellular Matrix Activin (MATRIKINE), which are involved in tumor invasion processes by regulating tumor cell activity and angiogenesis. Thus, a method was established to reduce the molecular weight of the natural dECM components, perhaps enabling better application of dECM as a material to skin care products.

For degradation of biological macromolecules, there are currently mainly acid hydrolysis, alkali hydrolysis and enzymatic hydrolysis. Acid or base hydrolysis often results in loss of activity of the bioactive sample due to its severe reaction conditions (strong acid, strong base). The enzymolysis method is a common method for degrading the bioactive macromolecular material at present because of the characteristics of mildness, specificity and high efficiency. The main components of the acellular matrix material are collagen, hyaluronic acid, elastin, glycosaminoglycan and the like. The existing enzymolysis method uses single enzyme, and mainly aims at one or two components. Matrix metalloproteinases (Matrix metalloproteinases, MMPs) are a family of zinc-dependent endopeptidases capable of degrading almost all extracellular matrices, but are not used in large-scale production because of their difficult extraction, low yield and high cost, and are often used in scientific experiments such as biochemical molecules. To prepare low molecular weight collagen peptides, current practitioners utilize alkaline protease (patent CN 112899335A), complex flavourzyme and neutral protease (patent CN 104774896B), pepsin, collagenase in combination with chymotrypsin (patent CN 106722999B) to degrade collagen in tissues to obtain low molecular weight collagen peptides.

The acellular matrix is a multi-component material and contains bioactive components such as elastin, glycosaminoglycan and the like besides collagen. However, the existing enzymolysis method uses single enzyme, and the enzyme has specificity, so that only one or more components can be degraded, the rest components still exist in a biological macromolecule form, and the obtained extracellular matrix active element has generally higher molecular weight and does not have good transdermal effect. Therefore, there is a need to find a method for efficiently preparing low molecular weight (Mw <1000 Da) extracellular matrix active substances, which can better apply the extracellular matrix-free material to skin care products.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a low molecular weight extracellular matrix active element, a preparation method and application thereof.

An extracellular matrix active substance is prepared by subjecting acellular matrix material to enzymolysis with pepsin, hyaluronidase, collagenase, elastase, and papain, and has molecular weight less than 1000 Da.

Preferably, the preparation method comprises the following steps:

step 1, reacting a decellularized matrix material with pepsin to obtain digestive juice, wherein the enzyme activity of the pepsin is 5-200U/mL;

Step 2, reacting the digestion solution with collagenase to obtain enzymolysis solution 1, wherein the enzyme activity of the collagenase is 0.5-100U/mL;

Step 3, reacting the enzymolysis liquid 1 with hyaluronidase, elastase and papain to obtain enzymolysis liquid 2, wherein the enzyme activity of the hyaluronidase is 0.005-1U/mL, the enzyme activity of the elastase is 0.05-5U/mL, and the enzyme activity of the papain is 0.5-500U/mL;

And step 4, performing ultrafiltration and freeze-drying on the enzymolysis liquid 2 to obtain the extracellular matrix active element.

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

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

Preferably, in the step 1, the reaction temperature is 15-37 ℃, the rotation speed is 50-200 rpm, and the reaction time is 12-72 h;

And/or in the step 2, the reaction temperature is 15-37 ℃, the rotating speed is 50-200 rpm, and the reaction time is 12-72 h.

Preferably, in step 3, the papain is incubated with a chelating agent selected from ethylenediamine tetraacetic acid and a reducing agent selected from cysteine prior to use.

Preferably, the incubation time is 10-30 min.

Preferably, in step 3, the reaction temperature is 15-37 ℃, the rotation speed is 50-200 rpm, and the reaction time is 4-48 h.

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

The invention also provides a preparation method of the extracellular matrix active element, which comprises the following steps:

step 1, reacting a decellularized matrix material with pepsin to obtain digestive juice, wherein the enzyme activity of the pepsin is 5-200U/mL;

Step 2, reacting the digestion solution with collagenase to obtain enzymolysis solution 1, wherein the enzyme activity of the collagenase is 0.5-100U/mL;

Step 3, reacting the enzymolysis liquid 1 with hyaluronidase, elastase and papain to obtain enzymolysis liquid 2, wherein the enzyme activity of the hyaluronidase is 0.005-1U/mL, the enzyme activity of the elastase is 0.05-5U/mL, and the enzyme activity of the papain is 0.5-500U/mL;

And step 4, performing ultrafiltration and freeze-drying on the enzymolysis liquid 2 to obtain the extracellular matrix active element.

The invention also provides application of the extracellular matrix active element in preparing skin care products.

According to the invention, pepsin, hyaluronidase, collagenase, elastase and papain are used for enzymatic hydrolysis of cell matrix materials, so that the obtained extracellular matrix active element has the advantages of high yield and low molecular weight (Mw <1000 Da), the content ratio of Mw <500Da components can reach 71.35%, and the extracellular matrix active element has a wide application prospect in the field of skin care products.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 is a flow chart of a process for preparing extracellular matrix activin.

FIG. 2 shows GPC outflow curves of extracellular matrix active substances prepared in example 1 and comparative example 1.

FIG. 3 shows GPC outflow curves of extracellular matrix active substances prepared in comparative example 2 and comparative example 3.

FIG. 4 shows GPC outflow curves of extracellular matrix active substances prepared in comparative example 4 and comparative example 5.

Detailed Description

In the following examples and experimental examples, reagents and raw materials not specifically described are commercially available.

EXAMPLE 1 Process for the preparation of Low molecular weight extracellular matrix Agents

The method comprises the following steps:

(1) Weighing micronized decellularized matrix material 2.5 g, placing in a screw bottle of 1L, and then adding hydrochloric acid solution of 0.01M of 500 mL and pepsin of 2.5 g (to make pepsin activity in the solution be 60U/mL);

(2) Placing the screw bottle in a constant temperature oscillator, setting the program to be 30+/-1 ℃ and the rotating speed to be 120+/-5 rpm, and oscillating 72 h to obtain digestive juice;

(3) Regulating the pH of the digestive juice to 6.0+/-0.1, adding collagenase to make the final concentration of the digestive juice be 50U/mL, continuously placing the digestive juice in a constant-temperature oscillator, setting the procedure to be 30+/-1 ℃ and the rotating speed to be 120+/-5 rpm, and oscillating 12 h to obtain enzymolysis liquid 1;

(4) Adding hyaluronidase (0.2U/mL), elastase (0.6U/mL) and papain (10U/mL) into the enzymolysis liquid 1, incubating 30min in cysteine containing 1.1mM EDTA and 5.5 mM before use, continuously placing in a constant temperature oscillator, setting the procedure to 30+ -1 ℃ and the rotation speed to 120+ -5 rpm, and oscillating 12h to obtain enzymolysis liquid 2;

(5) Starting an ultrafiltration (10 kDa) device, performing ultrafiltration (2 bar,60 min) on the enzymolysis liquid 2 to remove enzymes and undissolved acellular matrix materials in the enzymolysis liquid, and collecting filtrate, namely extracellular matrix active substance purified matters;

(6) The extracellular matrix active substance purified product was lyophilized in a vacuum freeze dryer to obtain extracellular matrix active substance (FIG. 1).

In other preferred embodiments, high yields of extracellular matrix active ingredient with low molecular weight (Mw <1000 Da) are obtained when pepsin has an enzyme activity of 55U/mL or 65U/mL, collagenase has an enzyme activity of 45U/mL or 55U/mL, hyaluronidase has an enzyme activity of 0.05U/mL or 0.3U/mL, elastase has an enzyme activity of 0.1U/mL or 1U/mL, papain has an enzyme activity of 5U/mL or 15U/mL.

The following is a method for preparing a control sample.

Comparative example 1 Process for the preparation of extracellular matrix active Agents

The same preparation as in example 1 was carried out except that only pepsin was added and that hyaluronidase, collagenase, elastase and papain were not added.

Comparative example 2 preparation of extracellular matrix active Agents

The same preparation as in example 1 was carried out except that only pepsin and hyaluronidase were added and no collagenase, elastase, and papain were added.

Comparative example 3 preparation of extracellular matrix active Agents

The same preparation as in example 1 was carried out except that only pepsin, hyaluronidase and collagenase were added and that elastase and papain were not added.

Comparative example 4 preparation of extracellular matrix active Agents

The same preparation as in example 1 was carried out except that only pepsin, hyaluronidase, collagenase and elastase were added and papain was not added.

Comparative example 5 preparation of extracellular matrix active Agents

The same preparation as in example 1 was carried out except that only pepsin, hyaluronidase, elastase and papain were added and no collagenase was added. The concentrations used for each enzyme are shown in Table 1.

TABLE 1 enzyme concentration use in examples, comparative examples

Note that +represents addition of the enzyme, -represents absence of the enzyme

The technical scheme of the invention is further described through experiments.

Experimental example 1 yield of extracellular matrix Activity

1. Experimental method

The extracellular matrix activin products of each group were collected and the extracellular matrix activin yield of each group was calculated.

Y= ×100%

Y is the yield of extracellular matrix active agent product, M is the dry weight of extracellular matrix active agent product, and X is the dry weight of micronized decellularized matrix material.

2. Experimental results

As is clear from Table 2, the average yield of the extracellular matrix active substance product prepared by the multiple complex enzyme enzymolysis test protocol of the present invention (example 1) was 2.448.+ -. 0.007 g and the yield was 97.932%. The yields of the extracellular matrix active substance products of comparative example 1 (pepsin alone group), comparative example 2 (pepsin+hyaluronidase combination group), comparative example 3 (pepsin+hyaluronidase+collagenase combination group), comparative example 4 (pepsin+hyaluronidase+collagenase+elastase combination group), comparative 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, 1.535.+ -. 0.453 g, and the extracellular matrix active substance yields of comparative examples 1 to 5 were 13.675%, 36.091%, 86.188%, 93.808%, 61.399%, respectively. The total weight of the collected extracellular matrix active substances is in an upward trend along with the gradual increase of different enzymes serving as substrates, which shows that the ECM is thoroughly degraded, and a plurality of enzymes are needed to participate. The reason why the yield of comparative example 5 was lower than that of comparative example 4 is that the collagenase was not used in comparative example 5, and collagen whose triple helix structure was not destroyed in the ECM was precipitated at a pH near neutral, and was removed at the time of ultrafiltration. Therefore, the extracellular matrix active substance with high yield can be prepared by adopting multiple complex enzyme enzymolysis (pepsin, hyaluronidase, collagenase, elastase and papain) to remove the cell matrix material.

TABLE 2 statistical Table of extracellular matrix activin yield and yield

Experimental example 2 molecular weight distribution of extracellular matrix Activity product

1. Experimental method

The molecular weight distribution of the extracellular matrix active agent product was analyzed by gel permeation chromatography (Gel Permeation Chromatography, GPC) using the extracellular matrix active agent.

2. Experimental results

As can be seen from FIGS. 2-4 and Table 3, the maximum molecular weight of the extracellular matrix active substances prepared in example 1 and comparative examples 1-5 is less than 15 kDa, and the ratio of Mw <10 kDa components is higher than 99%, which indicates that the ultrafiltration device has better effect of removing Mw >10 kDa components and various enzymes in the enzymatic hydrolysate. The results of the extracellular matrix active substances prepared in example 1 and comparative examples 1-5, wherein the Mw <1000 Da component accounts for 91.98%, 86.58%, 68.41%, 86.39%, 88.11% and 68.30% respectively, and the Mw <500Da component accounts for 71.35%, 68.92%, 48.09%, 64.94%, 67.59% and 45.40% respectively, show that the extracellular matrix active substances prepared in example 1 have the highest Mw <1000 Da and Mw <500Da components, and the Mw <500Da component accounts for 71.35% respectively, so that the extracellular matrix active substances with low molecular weight can be obtained by enzymolysis of the ECM by the preparation method of the invention.

TABLE 3 statistical table of molecular weight distribution of extracellular matrix activin products

Experimental example 3 ratios of extracellular matrix Activity components of different molecular weights and yields thereof

1. Experimental method

The molecular weight distribution of the extracellular matrix activin product was analyzed by gel permeation chromatography (Gel Permeation Chromatography, GPC) and the yields of the extracellular matrix activin products of different molecular weights were calculated.

The calculation method is that y=×100%

Y is the yield of the extracellular matrix active element product with certain molecular weight, A is the proportion of the extracellular matrix active element with certain molecular weight to the extracellular matrix active element product, M is the dry weight of the extracellular matrix active element product, and X is the dry weight of the micronized acellular matrix material.

2. Experimental results

As can be seen from Table 4, the use of hyaluronidase was increased (comparative example 2 vs, comparative example 1), and the yields of both 500 Da<Mw<1000 Da,200 Da<Mw<500 Da and Mw <200 and Da components in the extracellular matrix active substance product were increased, and the increased components were probably the products of 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), increased yields of 500 Da<Mw<1000 Da,200 Da<Mw<500 Da and Mw <200 Da components, which may be the products of collagenase enzymatic hydrolysis of collagen, i.e. collagen peptides. Increased elastase use (control example 4 vs control example 3), increased yields of components 200 Da < Mw <500 Da and Mw <200 Da, the increased components being possibly products of elastase enzymatic denaturation of collagen, elastin, fibrin. Increased papain use (example 1 vs control 4), increased yields of 500 Da<Mw<1000 Da,200 Da<Mw<500 Da and Mw <200 Da components, which may be the products of papain enzymatic gelatin, elastin. The yields of the 200 Da < Mw <500 Da and Mw <200 Da components of comparative example 5 were reduced compared to comparative example 3, comparative example 4, example 1, since the collagenase was not used in comparative example 5, the collagen triple helix structure was not destroyed, no further degradation occurred, and thus the yield of the low molecular weight (Mw <1000 Da) component was reduced. The increase in product yields of comparative examples 2-5 and example 1, as compared to comparative example 1, may be due to the action of these enzymes on the corresponding substrates, which are enzymatically degraded and have a molecular weight reduced to be able to be collected by the ultrafiltration membranes used in the present invention. From the above results, it is clear that when increasing the amount of enzyme acting on different substrates, the yield of the low molecular weight (Mw <1000 Da) extracellular matrix active ingredient increases, and collagenase is necessary for preparing the extracellular matrix active ingredient product in high yield and low molecular weight.

TABLE 4 ratio of extracellular matrix Activity component to different molecular weights and yield thereof

In conclusion, the invention uses pepsin, hyaluronidase, collagenase, elastase and papain in combination for enzymatic hydrolysis of cell matrix materials, and the obtained extracellular matrix active substance has the advantages of high yield and low molecular weight (Mw <1000 Da), and the content ratio of Mw <500Da components can reach 71.35%, thus having wide application prospect in the fields of skin care products and medical science.

Claims (10)

1. An extracellular matrix active factor, characterized in that it is obtained by enzymatic hydrolysis of acellular matrix material with pepsin, hyaluronidase, collagenase, elastase, and papain, and its molecular weight is less than 1000 Da; the preparation method of the extracellular matrix active factor 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 55-65U/mL; the enzymatic activity of the collagenase is 45-55U/mL; the enzymatic activity of the hyaluronidase is 0.05-0.3U/mL; the enzymatic activity of the elastase is 0.1-1U/mL; and the enzymatic activity of the papain is 5-15U/mL. 3. The extracellular matrix active agent according to claim 1, characterized in that in step 1, the reaction is carried out under the action of an acid selected from hydrochloric acid. 4. The extracellular matrix active agent 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 agent 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 agent 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 agent according to claim 1, characterized in that in step 4, the ultrafiltration time is 20-60 minutes 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 it 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 0.005-1 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.