KR20200000092A - Hydrogel for Skin Regeneration Comprising Stichopus japonicus Hydrolysates, and Fabricating Thereof - Google Patents

Abstract

A hydrogel for skin regeneration comprising a Stichopus japonicus hydrolysate according to an embodiment of the present invention comprises the Stichopus japonicus hydrolysate and a water-soluble polymer. By using the hydrogel for skin regeneration comprising the Stichopus japonicus hydrolysate as an active component and a manufacturing method thereof, it is possible to promote cell proliferation of human-derived fibroblasts and expression of collagen, which is a major component of skin, thereby being able to be used as a wound covering material and a medical engineering material for skin regeneration treatment.

Description

Hydrogel for skin regeneration including red sea ginseng hydrolyzate and its manufacturing method {Hydrogel for Skin Regeneration Comprising Stichopus japonicus Hydrolysates, and Fabricating Thereof}

The present invention relates to a hydrogel for skin regeneration comprising a red sea ginseng hydrolyzate as an active ingredient, and to a method for producing the same, and more specifically, to a water-soluble polymer after preparing a hydrolyzate from far-infrared dried Jeju red sea ginseng using a hydrolase. It relates to a method for producing a hydrogel for skin regeneration by encapsulating in.

Sea cucumber is a general term of sea cucumbers belonging to the equatorial animal sea cucumber river, and is divided into red sea cucumber, blue sea cucumber and black sea cucumber according to meat color. Sea cucumber is known to have various physiological activities such as hematopoietic action in pregnant women, teeth and skeletal formation, and normal contraction of muscles. It is a marine organism that is widely used as food and health food in East Asia. Sea cucumbers are reported to contain various active substances such as saponin, chondroitin sulfate, and holotoxin.In particular, sea cucumbers are remarkable for regeneration and removal of internal organs, even when parts of the body are cut. It has a playback function.

 Polyvinyl alcohol (PVA) is a water-soluble synthetic polymer having excellent biocompatibility, mechanical properties, non-toxicity, non-carcinogenicity, high water content and ease of manufacture, and is known as a material capable of various applications as a biomaterial. In particular, PVA is widely used as a drug carrier, wound dressings, implants, contact lenses, artificial organs and the like in the biomedical field. In addition, PVA has the advantage that it is possible to crosslink without chemical reaction through freezing-thawing, thereby avoiding the biological toxicity resulting from the chemical reaction.

Skin is the primary defense that protects internal organs from the external environment as a membrane that surrounds the outside of the body.

Skin is composed of epidermis, dermis, and subcutaneous tissue, which plays a role in maintaining body homeostasis such as body temperature control, metabolism of body, and excretion of waste, in addition to acting as a barrier to water retention, viral and fungal infection. The skin is the most frequently damaged tissue in our body, and it has a certain level of self-renewal ability to restore the function of the damaged tissue by closing the wound quickly for damage caused by external factors. As a result, the playability and speed are limited.

Therefore, the inventors of the present invention extracted hydrolysates derived from red sea ginseng dried with far-infrared rays and fused with PVA to produce hydrogels, and confirmed the present invention by confirming the cell proliferation effect on the human fibroblasts and the physical properties of the manufactured hydrogels. Completed.

KR 10-2014-0016461 A

Accordingly, it is an object of the present invention to provide a hydrogel and a method for producing the same, which enhances cell growth and collagen expression promoting effects by encapsulating red sea ginseng hydrolyzate in a water-soluble polymer.

The problem to be solved by the present invention is not limited to the problem (s) mentioned above, and other object (s) not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the hydrogel for skin regeneration comprising a red sea ginseng hydrolyzate according to an embodiment of the present invention is a red sea ginseng hydrolyzate; And a water-soluble polymer.

In one embodiment, the red sea ginseng hydrolyzate, based on 100% by weight of the hydrogel for skin regeneration, preferably comprises 0.25 to 1.5% by weight.

In one embodiment, the water-soluble polymer is preferably polyvinyl alcohol (Polyvinyl alcohol).

In one embodiment, the red sea ginseng hydrolyzate is preferably hydrolyzed using the Flavorzyme (Flavourzyme).

In one embodiment, the pore size of the hydrogel is preferably 3 ~ 30 ㎛.

Method for producing a hydrogel for skin regeneration comprising a red sea ginseng hydrolyzate according to an embodiment of the present invention, (a) adding a water-soluble polymer to a solvent and heating to prepare a polymer aqueous solution; (b) adding a red sea ginseng hydrolyzate to the aqueous polymer solution to prepare a mixture; And (c) freezing and thawing the mixture to prepare a hydrogel; characterized in that it comprises a.

In one embodiment, the water-soluble polymer is preferably polyvinyl alcohol (Polyvinyl alcohol).

In one embodiment, the aqueous polymer solution and red sea ginseng hydrolyzate of step (b) is preferably included in a weight ratio of 1: 0.5 to 2.

In one embodiment, the freezing and thawing of the step (c) is preferably performed 2 to 4 times.

In one embodiment, the red sea ginseng hydrolyzate, Far-infrared drying red sea ginseng; Hydrolyzing the dried red sea ginseng with a proteolytic enzyme; And centrifuging and desalting the hydrolyzed red sea ginseng to obtain a hydrolyzate.

In one embodiment, the hydrolysis is preferably hydrolyzed using Flavorzyme as a proteolytic enzyme.

By using the hydrogel for skin regeneration comprising the red sea ginseng hydrolyzate according to the present invention as an active ingredient and a method for producing the same, skin regeneration of human-derived fibroblasts and the expression of collagen, which is a major component of skin, can be promoted. There is an advantage that can be used as a therapeutic wound dressing and medical engineering material.

1 is a flow chart showing a method for producing a hydrogel for skin regeneration comprising a red sea ginseng hydrolyzate as an active ingredient according to an embodiment of the present invention.
Figure 2 shows a schematic diagram of the hydrogel manufacturing process according to an embodiment of the present invention.
Figure 3 shows the drug release capacity according to the concentration of the hydrogel according to an embodiment of the present invention.
Figure 4 shows the swelling ratio of the hydrogel according to an embodiment of the present invention.
5 is a result of measuring the storage modulus of the hydrogel according to an embodiment of the present invention with a rheometer.
Figure 6 shows the results of analyzing the hydrogel according to an embodiment of the present invention by a scanning microscope (SEM).
Figure 7 shows the cell growth rate of the hydrogel according to an embodiment of the present invention.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Advantages and features of the present invention, and a method of achieving the same will be apparent with reference to the embodiments described below in detail with reference to the accompanying drawings.

However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and are commonly used in the art. It is provided to fully inform the person of knowledge of the scope of the invention. Moreover, the invention is only defined by the scope of the claims.

Furthermore, in the following description of the present invention, if it is determined that related related technologies and the like may obscure the gist of the present invention, detailed description thereof will be omitted.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Advantages and features of the present invention, and a method of achieving the same will be apparent with reference to the embodiments described below in detail with reference to the accompanying drawings.

However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and are commonly used in the art. It is provided to fully inform the person of knowledge of the scope of the invention. Moreover, the invention is only defined by the scope of the claims.

Furthermore, in the following description of the present invention, if it is determined that related related technologies and the like may obscure the gist of the present invention, detailed description thereof will be omitted.

The present invention relates to a hydrogel for skin regeneration comprising a red sea ginseng hydrolyzate as an active ingredient, and a method for preparing the same. The cell proliferation efficacy was confirmed for the normal human dermal fibroblasts (NHDF) from the hydrogel prepared by encapsulation in the hydrogel, and the water absorption rate, storage modulus, surface change, etc. of the physical properties of the hydrogel were confirmed. When the red sea ginseng hydrolyzate was encapsulated in the polymer, it was confirmed that the pore size of the surface was increased and the water absorption was improved.

The present invention provides a hydrogel for skin regeneration comprising a red sea ginseng hydrolyzate and a water-soluble polymer.

The red sea ginseng hydrolyzate, based on 100% by weight of the hydrogel, may include 0.25 to 1.5% by weight, preferably 1% by weight.

When the content of the red sea ginseng hydrolyzate is less than 0.25% by weight, the size of the pores in the hydrogel is small, the improved water absorption and storage elasticity is difficult to improve, there is a problem that the proliferation effect of the cell is low, the content of the red sea ginseng hydrolyzate is If it exceeds 1.5% by weight, the pore size of the water-soluble polymer is too large, there is a problem that the mechanical properties are falling, the above range is preferred.

The water-soluble polymer may be a polyvinyl alcohol, and if the water-soluble polymer capable of producing a hydrogel by encapsulating red sea ginseng hydrolyzate, the type is not limited.

The red sea ginseng hydrolyzate is far-infrared dried red sea ginseng pepsin, trypsin, α-chymotrypsin, papain, alcalase, nutrase and flavoza It may be hydrolyzed with at least one hydrolase selected from the group consisting of Flavorzyme, preferably hydrolyzed with Flavozyme.

Flavozyme is extracted from the fungus and is used for hydrolysis of proteins. The enzyme has both endoprotease and exoprotease activity, and the optimum temperature of the enzyme activity is 50 ° C.

The pore size of the hydrogel may be 3 ~ 30 ㎛, if the pore size of the hydrogel is less than 3 ㎛ there is a problem that the water absorption rate and storage modulus falls, if the pore size exceeds 30 ㎛ mechanical mechanical The above range is preferable because there is a problem of inferior properties.

1 is a flow chart showing a method for producing a hydrogel for skin regeneration comprising a red sea ginseng hydrolyzate as an active ingredient according to an embodiment of the present invention.

Hereinafter, with reference to Figure 1 will be described in detail step by step a method for producing a hydrogel for skin regeneration comprising the red sea ginseng hydrolyzate according to the present invention as an active ingredient.

Method for producing a hydrogel for skin regeneration comprising a red sea ginseng hydrolyzate as an active ingredient according to an embodiment of the present invention, (a) step of preparing a polymer aqueous solution by adding a water-soluble polymer to a solvent and heating (S100); (B) adding a red sea ginseng hydrolyzate to the aqueous polymer solution to prepare a mixture (S200); And (c) freezing and thawing the mixture to prepare a hydrogel (S300).

In the method for producing a hydrogel for skin regeneration comprising red sea ginseng hydrolyzate according to an embodiment of the present invention as an active ingredient, step (a) (S100), a water-soluble polymer is added to a solvent and heated to prepare a polymer aqueous solution. Manufacturing step.

The polymer aqueous solution may be prepared in 2-15% aqueous polymer solution, preferably 10% aqueous solution, and the heating temperature is not limited.

The water-soluble polymer may be polyvinyl alcohol (PVA).

The solvent may be distilled water, and any kind of solvent may be used as long as it can dissolve the PVA.

In the method for producing a hydrogel for skin regeneration comprising red sea ginseng hydrolyzate according to an embodiment of the present invention as an active ingredient, step (b) (S200), adding the red sea ginseng hydrolyzate to the aqueous polymer solution to prepare a mixture Manufacturing step.

The red sea ginseng hydrolyzate may be added to distilled water to prepare 0.5 to 2% red sea ginseng hydrolyzate aqueous solution and then added to the aqueous polymer solution.

The aqueous polymer solution and red sea ginseng hydrolyzate of step (b) (S200) may be included in a weight ratio of 1: 0.5 to 2, preferably in a 1: 1 weight ratio.

The method may further include preparing the red sea ginseng hydrolyzate before preparing the mixture of step (b) (S200).

The method for producing the red sea ginseng hydrolyzate, the step of drying the red sea ginseng infrared; Hydrolyzing the far-infrared dried red sea ginseng with a proteolytic enzyme; And obtaining a hydrolyzate for centrifuging and desalting the hydrolyzed red sea ginseng.

Specifically, the red sea ginseng may be removed by removing the intestine of the red sea ginseng and washing it cleanly before far-infrared drying, and description thereof is omitted since it is a known method.

In addition, the red sea ginseng may further comprise the step of drying after the infrared drying, the grinding method, time, temperature and particle size of the pulverized red sea ginseng powder is not limited.

Far-infrared drying the red sea ginseng may be performed using a far-infrared dryer for 4 to 8 hours at 40 ~ 60 ℃.

Next, the step of hydrolyzing the far-infrared dried red sea ginseng with a proteolytic enzyme is carried out by mixing the far-infrared red sea ginseng and hydrolase in a weight ratio of 1: 0.01 to 0.1 to 30 to 50 ℃ for 6 to 24 hours Can be.

If the weight ratio of the hydrolase is less than 0.01, the hydrolysis efficiency is lowered, and if it exceeds 0.1, the hydrolysis rate may be good but economically undesirable. In addition, in order to increase the hydrolysis efficiency, hydrolysis may be performed in a stirrer at 1,000 to 1,500 rpm.

The hydrolysis may be carried out at a pH of 6 to 8, the pH conditions may be adjusted to the pH conditions by adding 1 M NaOH or HCl as the optimum conditions of the hydrolase.

The hydrolase is Pepsin, Trypsin, α-chymotrypsin, Papain, Alcalase, Neutrase and Flavozyme. It may be at least one selected from the group consisting of, preferably Flavorzyme (Flavourzyme).

Centrifuging and desalting the hydrolyzed red sea ginseng may centrifuge the hydrolyzed red sea ginseng at 5,000 to 10,000 rpm for 20 to 40 minutes at 4 to 10 ° C., and desalting the centrifuged red sea ginseng.

The desalting may be a reverse osmosis membrane as a process for removing the salt.

This desalting process is well known in the art to which this technique belongs, so detailed description thereof will be omitted. In addition, it is natural that other methods other than the reverse osmosis membrane may be used in the desalting process.

In the method for producing a hydrogel for skin regeneration comprising red sea ginseng hydrolyzate according to an embodiment of the present invention as an active ingredient, step (c) (S300), freezing and thawing the mixture to prepare a hydrogel Step.

Freezing and thawing of the step (c) (S300) may be performed 2 to 4 times, preferably 3 times.

The temperature for freezing the mixture of step (c) (S300) may be -90 ~ -70 ℃, preferably 80 ℃. The temperature for thawing the mixture may be 20 ~ 35 ℃, preferably 25 ℃.

The step (c) (S300) may further include a step of freeze-drying, it may be performed for 36 to 72 hours at 3 ~ 5 mTorr conditions using a freeze dryer of -70 ~ -50 ℃ temperature.

If the pressure is less than 3 mTorr, the freeze-drying time is increased, if the pressure exceeds 5 mTorr, the time to increase the vacuum pressure is increased, and the equipment price is increased by using a material with a solid material capable of raising the vacuum pressure.

Hereinafter, the present invention will be described in more detail with reference to specific examples and experimental examples.

(Example)

Preparation of Red Sea Ginseng Hydrolyzate

Jeju red sea ginseng was washed, and then far-infrared dried for 3 days using a far-infrared dryer, and then sufficiently ground using a grinder to prepare a powder.

1 kg of the prepared red sea ginseng and 10 g of the hydrolase flavozyme were mixed and hydrolyzed at 37 ° C. for 12 hours. At this time, pH was 7.

Next, the hydrolyzed red sea ginseng was centrifuged at 8,000 rpm for 20 minutes at 4 ° C, and salts were removed using a reverse osmosis membrane to prepare a hydrolyzate.

Preparation of hydrogel for skin regeneration comprising red sea ginseng hydrolyzate as an active ingredient

Figure 2 shows a schematic diagram of the manufacturing and hydrogel process according to an embodiment of the present invention.

PVA was dissolved in distilled water at 60 ° C. for 12 hours to prepare a 10 wt% PVA aqueous solution, and red sea ginseng hydrolysate was dissolved in distilled water to prepare 0.5, 1, and 2 wt% aqueous red sea ginseng hydrolysates, respectively.

Three 0.5, 1, and 2 wt% red sea ginseng hydrolyzate aqueous solutions were mixed with the prepared 10 wt% PVA aqueous solution, frozen at -80 ° C. for 3 hours, and thawed at 25 ° C. three times, and then -70 ° C. Lyophilization produced a hydrogel.

Experimental Example

Figure 3 shows the drug release capacity according to the concentration of the hydrogel according to an embodiment of the present invention.

3 is PVA / SC-I (PVA 5% + Red Sea Ginseng 0.25%), PVA / SC-II (PVA 5% + Red Sea Ginseng 0.5%), and PVA / SC-III (PVA 5) according to an embodiment of the present invention. % + Red sea ginseng 1%) After the drug was added to the hydrogel was measured drug release capacity.

PVA / SC-I (PVA 5% + Red Sea Ginseng 0.25%), PVA / SC-II (PVA 5% + Red Sea Ginseng 0.5%), and PVA / SC-III (PVA 5% + Red Sea Ginseng 1%) hydrogels were initially All showed a high drug release capacity, in particular, PVA / SC-III (PVA 5% + red sea ginseng 1%) can be seen that the drug release ability is more excellent.

Figure 4 shows the swelling ratio of the hydrogel according to an embodiment of the present invention.

Figure 4 shows the swelling ratio when the maximum swelling by weighing each hydrogel in distilled water by time

4, PVA / SC-I (PVA 5% + Red Sea Ginseng 0.25%), PVA / SC-II (PVA 5% + Red Sea Ginseng 0.5%), and PVA / SC-III (PVA 5% + red sea ginseng 1%) hydrogel absorbed more than five times its own weight was able to confirm that the high swelling ratio of more than 500%.

5 is a result of measuring the storage modulus of the hydrogel according to an embodiment of the present invention with a rheometer.

Figure 5 was obtained in a frequency sweep mode (Frequency sweep mode) using a rheometer dynamic analyzer RDA II rheometer (elastic measurement of the hydrogel according to an embodiment of the present invention).

Referring to Figure 5, as the frequency is increased, PVA / SC-I (PVA 5% + Red Sea Ginseng 0.25%), PVA / SC-II (PVA 5% + Red Sea Ginseng 0.5) prepared according to an embodiment according to the present invention %), And the storage modulus of PVA / SC-III (PVA 5% + red sea ginseng 1%) hydrogel is similar to the storage modulus of PVA.

Figure 6 shows the results of analyzing the hydrogel according to an embodiment of the present invention by a scanning microscope (SEM).

6, PVA / SC-I (PVA 5% + Red Sea Ginseng 0.25%), PVA / SC-II (PVA 5% + Red Sea Ginseng 0.5%), and PVA / Among the SC-III (PVA 5% + Red Sea Ginseng 1%) hydrogels, in particular PVA / SC-III (PVA 5% + Red Sea Ginseng 1%) hydrogels can be seen that the largest pore size, this result is the water absorption rate and storage The results of modulus can be supported.

Figure 7 shows the cell growth rate of the hydrogel according to an embodiment of the present invention.

PVA / SC-I (PVA 5% + Red Sea Ginseng 0.25%), PVA / SC-II (PVA 5% + Red Sea Ginseng 0.5%), and PVA / SC-III (PVA 5) prepared according to one embodiment according to the present invention % + Red sea ginseng 1%) Experiments were conducted to confirm the cell proliferation efficacy of the normal human dermal fibroblasts (NHDF) of the hydrogel.

Fibroblasts from neonatal foreskin tissue were placed in a 24-well plate at 2 × 10 ³ cells per well, and then cultured in DMEM medium containing 10% FBS (fetal bovine serum) and 1% antibiotic-antimycotic for 24 hours. On one side, the hydrogel and PVA samples were added to the culture medium at a concentration of 200 μg / ml, and only the culture medium was added to the control medium. After incubation for 1 day, the MTT assay was performed to determine the proliferation of the cells. In MTT assay, 0.1 ml of MTT solution was added to cultured cells, incubated for 4 hours, culture medium was removed, and 0.5 ml of DMSO was added thereto. It was. The absorbance of each of the hydrogels, PVA and the control group was measured, and the cell proliferation rate was calculated using the absorbance.

Referring to FIG. 7, PVA / SC-I (PVA 5% + Red Sea Ginseng 0.25%), PVA / SC-II (PVA 5% + Red Sea Ginseng 0.5%), and PVA / SC-III (PVA 5% +) Red sea ginseng 1%) was confirmed that the high cell growth rate of the hydrogel, in particular, PVA / SC-III (PVA 5% + red sea ginseng 1%) was found to have a superior cell proliferation effect.

So far it has been described with respect to a specific embodiment of the hydrogel for skin regeneration comprising the red sea ginseng hydrolyzate according to an embodiment of the present invention as an active ingredient and a method for producing the same, within the scope not departing from the scope of the present invention It is obvious that the embodiment can be modified.

Therefore, the scope of the present invention should not be limited to the described embodiments, but should be determined not only by the claims below but also by the equivalents of the claims.

In other words, the foregoing embodiments are to be understood in all respects as illustrative and not restrictive, the scope of the invention being indicated by the following claims rather than the detailed description, and the meaning and scope of the claims and All changes or modifications derived from the equivalent concept should be construed as being included in the scope of the present invention.

S100: preparing a polymer aqueous solution
S200: step of preparing the mixture
S300: step of preparing a hydrogel

Claims (11)

In the hydrogel for skin regeneration,
Red sea ginseng hydrolyzate; And
Hydrogel for skin regeneration comprising a water-soluble polymer.
The method of claim 1,
The red sea ginseng hydrolyzate,
Based on the skin regeneration hydrogel 100% by weight,
Hydrogel for skin regeneration, characterized in that it comprises 0.25 to 1.5% by weight.
The method of claim 1,
The water-soluble polymer is a hydrogel for skin regeneration, characterized in that the polyvinyl alcohol (Polyvinyl alcohol).
The method of claim 1,
The red sea ginseng hydrolyzate,
Hydrogel for skin regeneration, characterized in that hydrolyzed using Flavozyme (Flavourzyme).
The method of claim 1,
The pore size of the hydrogel is a hydrogel for skin regeneration, characterized in that 3 ~ 30 ㎛.
(a) adding a water-soluble polymer to a solvent and heating to prepare an aqueous polymer solution;
(b) adding a red sea ginseng hydrolyzate to the aqueous polymer solution to prepare a mixture; And
(C) freezing and thawing the mixture to prepare a hydrogel; a method for producing a hydrogel for skin regeneration, characterized in that it comprises a.
The method of claim 6,
The water-soluble polymer is a method of producing a hydrogel for skin regeneration, characterized in that the polyvinyl alcohol (Polyvinyl alcohol).
The method of claim 6,
The method of preparing a hydrogel for skin regeneration, characterized in that (b) the aqueous polymer solution and red sea ginseng hydrolyzate is included in a weight ratio of 1: 0.5 to 2.
The method of claim 6,
The freezing and thawing of the step (c) is a method for producing a hydrogel for skin regeneration, characterized in that is performed 2 to 4 times.
The method of claim 6,
The red sea ginseng hydrolyzate,
Far-infrared drying red sea ginseng;
Hydrolyzing the dried red sea ginseng with a proteolytic enzyme; And
Centrifuging and desalting the hydrolyzed red sea ginseng to obtain a hydrolyzate; The method of producing a hydrogel for skin regeneration characterized in that it comprises a.
The method of claim 10,
The hydrolysis is,
Method for producing a hydrogel for skin regeneration, characterized in that the hydrolysis using flavozyme as a protein hydrolase (Flavourzyme).

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