Cosmetic composition for alleviating irritation and improving moisturization, containing enzymatically processed and low-temperature aged low-molecular-weight hyaluronic acid hydrolysate as active ingredient, and method for manufacturing hydrolysate thereof

Abstract

To provide a cosmetic composition for alleviating irritation and improving moisturization, containing enzymatically processed and low-temperature aged low-molecular-weight hyaluronic acid hydrolysate as an active ingredient, and a method for manufacturing hydrolysate thereof.SOLUTION: The present invention includes: a step (Step 1), which comprises mixing hyaluronic acid and mixed enzymes comprising proteinase and pectinase in distilled water, and conducting an enzymatic reaction to obtain mixed-enzyme-treated hyaluronic acid; and a step (Step 2), which comprises subjecting the mixed-enzyme-treated hyaluronic acid obtained in Step 1 to steam treatment, and subsequently to low-temperature aging treatment at 5°C to 20°C for 1 day to 5 days, wherein the low-molecular-weight hyaluronic acid prepared by the method of the present invention achieves a weight-average molecular weight (Mw) of 500 Da or less, resulting in significantly improved skin absorption rate and providing significantly superior effects in alleviating skin irritation and providing moisturization.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a cosmetic composition for alleviating irritation and improving moisture retention, which contains an enzyme-treated and low-temperature aged low molecular weight hyaluronic acid hydrolyzate as an active ingredient, and a method for producing the hydrolyzate. More specifically, when producing hyaluronic acid as an enzyme-treated and low-temperature aged hydrolyzate, a low molecular weight hyaluronic acid hydrolyzate with a weight average molecular weight (Mw) of 500 Da or less is obtained, and its histamine inhibitory ability, NO production inhibitory ability, and effect of improving moisture retention are confirmed.

Background Art

[0002] Hyaluronic acid is one of the glycosaminoglycans, exists in the extracellular matrix, is involved in maintaining the moisture of tissues, storing and diffusing cell growth factors and nutritional components, and is known to be synthesized by keratinocytes and fibroblasts. A decrease in hyaluronic acid in the skin is known to cause a lack of firmness and an increase in wrinkles in the skin.

[0003] Most of the hyaluronic acid produced by conventional methods is a high molecular polysaccharide with a high degree of polymerization of 500,000 daltons (500,000 Da = 500 kDa) or more, so it cannot pass through the skin. That is, if hyaluronic acid of 500,000 daltons or more is applied to the skin, due to its property of trying to absorb moisture in the air, it can prevent the evaporation of moisture from the skin, but it cannot penetrate the skin, so it stays on the surface of the skin and is easily washed away from the surface of the skin, making it difficult to sustain the moisturizing effect of the skin. For this reason, various attempts have been reported to reduce the molecular weight of hyaluronic acid.

[0004] As a prior art document for reducing the molecular weight of hyaluronic acid, Patent Document 1 relates to a method for producing a hyaluronic acid oligomer containing ultra-low molecular weight hyaluronic acid, and the production method discloses a method for producing low molecular weight hyaluronic acid by causing a deacetylation reaction. The composition is reported to have the effect of alleviating and improving wrinkles by promoting the activity of fibroblasts and the synthesis of collagen.

[0005] In addition, Patent Document 2 relates to a method for producing a low-molecular-weight product by irradiation with gamma rays and treatment with a hyaluronic acid-degrading enzyme, and provides a method for producing a low-molecular-weight hyaluronic acid hydrolyzate by irradiating gamma rays after treating a recombinant protein enzyme with hyaluronic acid. However, it is a method produced using a recombinant protein that uses chemicals or is somewhat complicated to purify, and there are some difficulties in industrial application.

[0006] In response to this, as a result of intensive research by the present inventors to maximize the performance of hyaluronic acid, when producing hyaluronic acid as a hydrolyzate by performing enzymatic treatment and low-temperature aging in parallel, it was found that the molecular weight of hyaluronic acid could be reduced and excellent effects of stimulating relaxation and moisturizing improvement could be provided, and thus the present invention was completed.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] The first object of the present invention is to provide a method for producing low-molecular-weight hyaluronic acid.

[0009] The second object of the present invention is to provide low-molecular-weight hyaluronic acid having a weight average molecular weight (Mw) of 500 Da or less produced by the above production method.

[0010] The third object of the present invention is to provide a cosmetic composition for alleviating skin irritation and moisturizing containing low-molecular-weight hyaluronic acid produced by the above production method.

[0011] A fourth object of the present invention is to provide a cosmetic composition for relieving skin irritation and moisturizing, which contains low-molecular-weight hyaluronic acid with a weight-average molecular weight (Mw) of 500 Da or less.

Means for Solving the Problems

[0012] To achieve the above object, the present invention includes a step (step 1) of mixing hyaluronic acid and Proteinase and Pectinase as a mixed enzyme in distilled water, and performing an enzyme reaction to obtain hyaluronic acid treated with the mixed enzyme; After subjecting the hyaluronic acid treated with the mixed enzyme in step 1 to steaming treatment, a step (step 2) of low-temperature aging at 5 to 20 °C for 1 to 5 days is included, and a method for producing low-molecular-weight hyaluronic acid is provided.

[0013] In the production method according to the present invention, step 1 is a step of subjecting a hyaluronic acid raw material with a high weight-average molecular weight (Mw) to mixed enzyme treatment and hydrolysis. The weight-average molecular weight (Mw) of commercially available hyaluronic acid raw materials is approximately in the range of 1 million to 2 million Da.

[0014] The Proteinase in step 1 can be used alone or in combination of two or more of Flavourzyme, Protamex, α-chymotrypsin, subtilisin Carlsberg, thermolysin, papain, fungal protease, pepsin, etc.

[0015] The pectinase in the above step 1 can be used alone or in combination of two or more of Pectinex Ultra SP-L, Viscozyme L, Ultrazyme AFP, Pectinex Ultra AFP, Pectinex Ultra clear, α-herbzyme, etc.

[0016] Desirably, based on 1 part by weight of the pectinase, 1 - 5 parts by weight of proteinase can be used. Preferably, based on 1 part by weight of the pectinase, 2 - 4 parts by weight of proteinase can be used. More preferably, based on 1 part by weight of the pectinase, 3 parts by weight of proteinase can be used.

[0017] If it is outside the weight percentage of the above mixed enzyme, the problem that the hydrolysis efficacy for hyaluronic acid decreases may occur. Specifically, the weight average molecular weight (Mw) of hyaluronic acid may not reach 500 Da or less.

[0018] Also, in step 1, based on 100 parts by weight of hyaluronic acid, 2 - 6 parts by weight of the mixed enzyme can be used. Desirably, 3 - 5 parts by weight, and more desirably, 3.5 - 4.5 parts by weight can be used.

[0019] In the production method according to the present invention, the above step 2 is a step of subjecting the hydrolyzed hyaluronic acid to low-temperature aging treatment to further reduce its molecular weight.

[0020] Desirably, the steaming treatment in the above step 2 can be carried out at 70 - 90 °C for 2 - 4 hours, and the low-temperature aging can be carried out at 14 - 18 °C for 2 - 4 days.

[0021] The weight average molecular weight (Mw) of the low-molecular-weight hyaluronic acid produced by the production method according to the present invention is characterized by being 500 Da or less.

[0022] Further, the present invention provides a low-molecular-weight hyaluronic acid having a weight average molecular weight (Mw) of 500 Da or less produced by the above production method.

[0023] The low-molecular-weight hyaluronic acid having a weight average molecular weight (Mw) of 500 Da or less has a significantly improved skin absorption rate and remarkable effects on alleviating skin irritation and moisturizing efficacy.

[0024] Furthermore, the present invention provides a cosmetic composition for alleviating skin irritation and moisturizing containing the low-molecular-weight hyaluronic acid produced by the above production method.

[0025] Also, the present invention provides a cosmetic composition for alleviating skin irritation and moisturizing containing a low-molecular-weight hyaluronic acid having a weight average molecular weight (Mw) of 500 Da or less.

Effects of the Invention

[0026] The low-molecular-weight hyaluronic acid produced by the production method according to the present invention achieves a weight average molecular weight (Mw) of 500 Da or less, thereby significantly improving the skin absorption rate and having remarkable effects on alleviating skin irritation and moisturizing efficacy.

Brief Description of the Drawings

[0027]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0028] Hereinafter, the present invention will be described in detail.

[0029] [Cosmetic Composition]

[0030] The present invention provides a cosmetic composition containing an active substance.

[0031] The cosmetic composition may be in the form of, for example, a solution, gel, solid or anhydrous product, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, microemulsion, microcapsule, microgranulocyte or ionic (liposome), non-ionic vesicle dispersant. As more specific forms, it can be provided in the form of lotion, emulsion, cream, skin, lotion, beauty liquid, essence, emulsion, powder, cosmetic ointment, spray, gel, face pack, cleanser, soap, shampoo, conditioner, bath agent, detergent or concealer stick. It may also be produced in the form of a foam or an aerosol composition further containing a compressed propellant.

[0032] In addition, the cosmetic composition can contain, in addition to the active substance of the present invention, auxiliary agents commonly used in the cosmetic field, such as fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or non-ionic emulsifiers, fillers, sequestering agents and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic activators, lipid vesicles or any other components commonly used in cosmetics.

[0033] In the cosmetic composition containing the active substance of the present invention, the active substance of the present invention can be added to the commonly contained cosmetic composition in an amount of 0.1 to 50% by weight, preferably 1 to 10% by weight.

[0034] When the active substance of the present invention is used as a topical skin preparation, it can further contain auxiliary agents commonly used in the field of dermatology, such as fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or non-ionic emulsifiers, fillers, sequestering agents and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic activators, lipid vesicles or any other components commonly used in topical skin preparations. Also, these components can be introduced in amounts commonly used in the field of dermatology.

[0035] Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are merely for illustrating the present invention, and the content of the present invention is not limited by the following examples.

[0036] <Example 1> Production of a mixed enzyme and a hydrolyzate of hyaluronic acid treated by low-temperature aging (mixed enzyme protease and pectinase 3:1 by weight)

[0037] Step 1: Mixed enzyme treatment step

[0038] To 100 g of hyaluronic acid powder (200 mesh), as a mixed enzyme, 3 g of Flavourzyme (manufactured by Novozyme), which is a proteinase, and 1 g of Pectinex Ultra SP-L (manufactured by Novozyme), which is a pectinase, were mixed, 300 mL of water was added, and the mixture was reacted at 50 °C for 120 minutes.

[0039] Step 2: Low-temperature aging treatment step

[0040] The hyaluronic acid treated with the mixed enzyme prepared in Step 1 was first steamed at 80 °C for 3 hours and then aged at low temperature at 16 °C for 3 days.

[0041] To the hyaluronic acid hydrolyzate treated with the above-mentioned mixed enzyme and low-temperature aging, 3 L of purified water was added, and the mixture was stirred and extracted at 80 °C for 120 minutes, and then concentrated under reduced pressure and freeze-dried to obtain the hyaluronic acid hydrolyzate treated with the mixed enzyme and low-temperature aging.

[0042] <Example 2> Production of hyaluronic acid hydrolyzate treated with mixed enzyme and low-temperature aging (mixed enzyme proteinase and pectinase 2:1 weight%)

[0043] A hyaluronic acid hydrolyzate was obtained in the same manner as in Example 1, except that in Step 1 of Example 1, proteinase and pectinase were used at a weight ratio of 2:1 in a total weight of 4 g of the mixed enzyme.

[0044] <Example 3> Production of hyaluronic acid hydrolyzate treated with mixed enzyme and low-temperature aging (mixed enzyme proteinase and pectinase 4:1 weight%)

[0045] A hyaluronic acid hydrolyzate was obtained in the same manner as in Example 1, except that in Step 1 of Example 1, proteinase and pectinase were used at a weight ratio of 4:1 in a total weight of 4 g of the mixed enzyme.

[0046] <Example 4> Production of a mixed enzyme and a hydrolyzate of hyaluronic acid treated by low-temperature aging (mixed enzyme: protease and pectinase 1:1 by weight)

[0047] In Step 1 of Example 1, except that protease and pectinase were used at 1:1 by weight in a total of 4 g of the mixed enzyme, the procedure was the same as in Example 1 to obtain a hydrolyzate of hyaluronic acid.

[0048] <Example 5> Production of a mixed enzyme and a hydrolyzate of hyaluronic acid treated by low-temperature aging (mixed enzyme: protease and pectinase 5:1 by weight)

[0049] In Step 1 of Example 1, except that protease and pectinase were used at 5:1 by weight in a total of 4 g of the mixed enzyme, the procedure was the same as in Example 1 to obtain a hydrolyzate of hyaluronic acid.

[0050] <Comparative Example 1> Preparation of hyaluronic acid (untreated with mixed enzyme and untreated with low-temperature aging group)

[0051] Without the treatments in Steps 1 and 2 of Example 1, 3 L of purified water was added to 100 g of hyaluronic acid powder (200 mesh), stirred and extracted at 80 °C for 120 minutes, and then concentrated under reduced pressure and freeze-dried to obtain hyaluronic acid.

[0052] Specifically, 3 L of purified water was added to 100 g of hyaluronic acid powder (200 mesh), extracted at 80 °C for 120 minutes, and then concentrated under reduced pressure and freeze-dried to obtain hyaluronic acid.

[0053] <Comparative Example 2> Production of a hydrolyzate of hyaluronic acid treated with a mixed enzyme (untreated with low-temperature aging group)

[0054] Except for not performing Step 2 in Example 1, the procedure was the same as in Example 1 to produce a hydrolyzate of hyaluronic acid treated with a mixed enzyme.

[0055] Specifically, 100 g of hyaluronic acid powder (200 mesh) was mixed with 3 g of protease and 1 g of pectinase, 300 mL of water was added, and the mixture was reacted at 50°C for 120 minutes. The hyaluronic acid treated with the mixed enzyme was stirred and extracted at 80°C for 120 minutes after adding 3 L of purified water, and then concentrated under reduced pressure and freeze-dried to obtain a mixed enzyme-treated hyaluronic acid hydrolyzate.

[0056] <Comparative Example 3> Production of hyaluronic acid hydrolyzate treated with a single enzyme (single enzyme treatment and low-temperature aging untreated group)

[0057] A hyaluronic acid hydrolyzate treated with a single enzyme was produced in the same manner as in Example 1, except that 1 g of protease was used instead of the mixed enzyme in Step 1 of Example 1 and Step 2 was not carried out.

[0058] Specifically, 100 g of hyaluronic acid powder (200 mesh) was treated with 1 g of protease, 300 mL of water was added, and the mixture was reacted at 50°C for 120 minutes. 3 L of purified water was added to the hyaluronic acid treated with the single enzyme, and the mixture was stirred and extracted at 80°C for 120 minutes, and then concentrated under reduced pressure and freeze-dried to obtain a hyaluronic acid hydrolyzate treated with a single enzyme.

[0059] <Comparative Example 4> Production of low-temperature aged hyaluronic acid hydrolyzate (mixed enzyme untreated group)

[0060] A low-temperature aged hyaluronic acid hydrolyzate was produced in the same manner as in Example 1, except that Step 1 of Example 1 was not carried out.

[0061] Specifically, 100 g of hyaluronic acid powder (200 mesh) was first steamed at 80°C for 3 hours and aged at 16°C for 3 days. The low-temperature aged hyaluronic acid hydrolyzate was stirred and extracted at 80°C for 120 minutes after adding 3 L of purified water, and then concentrated under reduced pressure and freeze-dried to obtain a low-temperature aged hyaluronic acid hydrolyzate.

[0062] <Comparative Example 5> Production of hyaluronic acid hydrolyzate treated with protease alone (single enzyme treatment and low-temperature aging treatment group)

[0063] A hyaluronic acid hydrolyzate was obtained in the same manner as in Example 1, except that the treatment was performed with 4 g of proteinase alone instead of the mixed enzyme in Example 1.

[0064] <Comparative Example 6> Production of hyaluronic acid hydrolyzate treated with pectinase alone (single enzyme treatment and low-temperature aging treatment group)

[0065] A hyaluronic acid hydrolyzate was obtained in the same manner as in Example 1, except that the treatment was performed with 4 g of pectinase alone instead of the mixed enzyme in Example 1.

[0066] <Comparative Example 7> Production of hyaluronic acid hydrolyzate treated with a mixed enzyme and low-temperature aging treatment (mixed enzyme proteinase and pectinase 0.8:1 wt%)

[0067] A hyaluronic acid hydrolyzate was obtained in the same manner as in Example 1, except that proteinase and pectinase were used at a weight ratio of 0.8:1 in 4 g of the total weight of the mixed enzyme in Step 1 of Example 1.

[0068] <Comparative Example 8> Production of hyaluronic acid hydrolyzate treated with a mixed enzyme and low-temperature aging treatment (mixed enzyme proteinase and pectinase 5.2:1 wt%)

[0069] A hyaluronic acid hydrolyzate was obtained in the same manner as in Example 1, except that proteinase and pectinase were used at a weight ratio of 5.2:1 in 4 g of the total weight of the mixed enzyme in Step 1 of Example 1.

[0070] <Experimental Example 1> Cytotoxicity test: WST assay

[0071] To confirm the presence or absence of irritation to cells using the hyaluronic acid samples produced in Example 1 and Comparative Examples 1 to 4, a cytotoxicity experiment was conducted using macrophages (RAW264.7).

[0072] RAW264.7 cells, a type of macrophage, were counted identically at 1.0×10 4 cells / well and dispensed into a 24-well plate using DMEM medium containing 1% penicillin / streptomycin and 10% FBS (Fetal bovine serum). After that, they were cultured for 24 hours under conditions of 37°C and 5% CO 2 2. The hyaluronic acid samples produced in Example 1 and Comparative Examples 1 to 4 were mixed with the medium for each concentration (0%, 0.5%, 1%, 2%, 4%) and added to each well at 1 mL per well. Then, they were reacted for 24 hours in an incubator under conditions of 37°C and 5% CO 2 5. After that, only the supernatant of each well was separately collected. Then, WST-1 assay solution (ez-cytox) was added to each well and reacted in the incubator for 2 hours. Subsequently, the absorbance was measured at 450 nm using an ELISA reader machine.

[0073] The cell viability was calculated using the following formula 1 in comparison with the treatment group without sample treatment (untreated group).

[0074] [Formula 1] Cell viability (%) = (Absorbance of sample-treated group / Absorbance of sample-untreated group) × 100

[0075] Figure 1 is a graph showing the results of the cytotoxicity experiment of the hyaluronic acid samples produced in Example 1 and Comparative Examples 1 to 4.

[0076] As shown in Figure 1, it was confirmed that none of the hyaluronic acid samples produced in Example 1 and Comparative Examples 1 to 4 showed cytotoxicity and they can be used as safe materials for cosmetics.

[0077] <Experimental Example 2> Stimulation Relief Test: Histamine Inhibitory Activity

[0078] β-Hexosaminidase is a substance that constitutes the granules of mast cells. The histamine secretion due to mast cell degranulation is proportional to the release amount of β-hexosaminidase. Therefore, the degranulation and histamine secretion inhibitory effects of the Panax japonicus Torr. saponin fraction were confirmed by measuring the release amount of β-hexosaminidase in RBL-2H3 cells, a rat mast cell line. After suspending RBL-2H3 cells in DMEM containing 10% FBS, 2×10 5 cells per well were dispensed into a 24-well plate, and then sensitized with 0.5 μg / mL of LDNP-IgE per well and cultured overnight in a 5% CO 2 incubator. Thereafter, the cells in each well were washed twice with Siraganian buffer (119 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.4 mM MgCl 2 , 25 mM HEPES, 40 mM NaOH, 1 mM CaCl 2 , 0.1% BSA, pH 7.2), then allowed to react completely with Siraganian buffer at 37°C for 10 minutes, and reacted for another 10 minutes after adding the test substance. Thereafter, the cells were treated with an antigen (DNP-BSA, 10 μg / mL) at 37°C for 30 minutes to be in a degranulated state, then left on ice for 10 minutes to terminate the reaction, 20 μL of the supernatant was taken and transferred to a 96-well plate, and then 1 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide was added and cultured for 1 hour, and then a stop solution (0.1 M Na 2 CO 3 / NaHCO 3 ) was added, and then measured and quantified by ELISA at an absorbance of 405 nm.

[0079] Figure 2 is a graph showing the results of the histamine inhibitory activity of the hyaluronic acid samples produced in Example 1 and Comparative Examples 1 to 4.

[0080] As shown in Figure 2, it can be seen that the histamine inhibitory activity of the hyaluronic acid sample produced in Example 1 is significantly superior to that of Comparative Examples 1 to 4, and it was found that the histamine inhibitory activity improves in a concentration-dependent manner up to a treatment concentration of 2.0%.

[0081] <Experimental Example 3> Anti-inflammatory efficacy test: Inhibitory activity of NO (Nitric oxide) generation

[0082] In order to measure the anti-inflammatory activity of the hyaluronic acid samples produced in Example 1 and Comparative Examples 1 to 4, an experiment was conducted to measure the concentration of NO generated by an inflammation-induced reaction.

[0083] RAW264.7 cells, a type of macrophage, were counted identically at 1.0×10 4 cells / well and dispensed into a 24-well plate using DMEM medium containing 1% penicillin / streptomycin and 10% FBS (Fetal bovine serum), and then cultured under conditions of 37°C and 5% CO 2 for 24 hours. After mixing the hyaluronic acid samples produced in Example 1 and Comparative Examples 1 to 4 with the medium at a concentration of 50 μg / mL in the cells cultured for 24 hours, 1 mL was added to each well and reacted in an incubator under conditions of 37°C and 5% CO 2 for 24 hours. At this time, LPS (Lipo poly saccharide), an inflammation-inducing factor that expresses NO, was treated together at a concentration of 1 μg / mL and reacted under conditions of 37°C and 5% CO 2 for 24 hours. Then, only the supernatant of each well was separately taken, and then 100 mL of the culture solution was collected in a 96-well plate using a NO detection kit for each well. After adding 50 μL of Griess reagent A (N-1-naphthylethylenediamine (NEDHC)) and 50 μL of Griess reagent B (sulfanilamide) respectively, the mixture was reacted for 10 minutes, and then the absorbance was measured at 540 nm using an ELISA plate reader.

[0084] Figure 3 is a graph showing the results of the NO (Nitric oxide) generation inhibitory ability of the hyaluronic acid samples produced in Example 1 and Comparative Examples 1 to 4.

[0085] As shown in Fig. 3, it was found that the ability to inhibit NO production was remarkably excellent in the hyaluronic acid samples produced in Comparative Examples 1 to 4 and Example 1.

[0086] <Experimental Example 4> Moisture retention improvement test: Clinical evaluation of the efficacy of improving the moisture retention of 20 layers of the skin

[0087] In order to measure the effect of improving moisture retention on the hyaluronic acid samples produced in Example 1 and Comparative Examples 1 to 4, a clinical evaluation was carried out.

[0088] After diluting the hyaluronic acid samples produced in the Example and Comparative Examples 1 to 4 to a concentration of 50 (μg / mL), the samples are applied to the forearms 10 cm away from both wrists of the subjects. The moisture on the skin surface is measured using Epsilon E100, and the amount of moisture in the skin is measured by repeating the stripping by applying the same pressure for 2 seconds using Scotch Magic Invisible Tape (manufactured by 3M).

[0089] Fig. 4 is a graph showing the rate of increase in skin moisture by treatment with the hyaluronic acid sample produced in Example 1 and the control sample (Comparative Example 1).

[0090] As shown in Fig. 4, it was found that the moisture retention effect was excellent up to 20 layers of the skin in the treatment group with the hyaluronic acid sample produced in Example 1 compared to the control sample (Comparative Example 1).

[0091] <Experimental Example 5> Evaluation of the molecular weight of hyaluronic acid

[0092] The molecular weights of the hyaluronic acid hydrolysates obtained in Comparative Examples 1 to 8 and Examples 1 to 5 were measured and compared.

[0093] The molecular weight of the mixed enzyme of Example 1 and the hyaluronic acid hydrolysate treated by low-temperature aging was requested for molecular weight confirmation test analysis from the Gyeonggi Institute of Economic Chemistry (GBSA), and the results are shown in Figs. 5 and 6.

[0094] For the molecular weight measurement method, 10 mg / mL of DHB (2,5-dihydroxybenzoic acid) was added to a 0.1% TFA / ACN (1:1, v / v) solvent to prepare a matrix solution. 2 μL of the sample of Example 1 was directly mixed with 2 μL of the matrix solution on a MALDI target and dried under vacuum. As the molecular weight measurement equipment, autoflex maX (manufactured by Bruker Daltonics) was used. The measurement equipment settings are as follows.

[0095] 1. Instrument control: Flex Control 3.4 (manufactured by Bruker Daltonics) 2. Analysis mode: Linear mode 3. Polarity: Positive 4. Detection: m / z 100 - 100,000 5. Laser repetition rate: 2,000 Hz 6. Number of shots: 500 shots 7. Deflection: On, 100 Da 8. Voltage

[0096]

Table 1

[0097] Figure 5 is a spectral image of measuring the molecular weight of the hyaluronic acid hydrolyzate of Example 1.

[0098] Figure 6 is a table organizing the peak information in the spectrum of measuring the molecular weight of the hyaluronic acid hydrolyzate of Example 1. The gray cells indicate the matrix peak molecular weight.

[0099] As shown in Figures 5 and 6, it was confirmed that the weight-average molecular weight excluding the matrix peak was 368.3 Da.

[0100] In the same manner as the molecular weight measurement method of Example 1, the measurement results of the molecular weights of the allonic acids obtained in Examples 2 - 5 and Comparative Examples 1 - 8 are shown in Table 1 and Table 2 below, respectively.

[0101]

Table 2

[0102]

Table 3

[0103] As shown in Table 1 above, it was found that when the weight percentage of the protease and pectinase mixed enzyme is used in the range of 1 to 5:1, the hyaluronic acid hydrolysis rate is remarkably high.

[0104] As shown in Table 2 above, Comparative Example 1 and Comparative Example 4 are samples that differ only in whether or not they are subjected to low-temperature aging treatment. By comparing these, it was found that there is a hydrolysis effect due to low-temperature aging treatment. Such a result can also be understood by comparing Comparative Example 2 and Example 1. Further, Comparative Example 5 and Comparative Example 6 are samples treated with a single enzyme, and when compared with Examples 1 to 5, it was found that the hydrolysis rate is remarkably improved in the sample treated with a single enzyme compared to the sample treated with a mixed enzyme.

[0105] Manufacturing example of cosmetics

[0106] The active substance according to the present invention can be produced in many forms of cosmetics depending on the purpose. The following exemplifies the production methods of some cosmetics containing the active substance according to the present invention as an active ingredient, but the present invention is not limited thereto.

[0107] <Production Example 1 of Cosmetics> Production of Softening Lotion

[0108]

Table 4

[0109] <Production Example of Cosmetics> Production of Nutritional Cream

[0110]

Table 5

[0111] So far, the present invention has been well described mainly with reference to the desirable embodiments. Those having ordinary knowledge in the technical field to which the present invention pertains will probably be able to understand that the present invention can be embodied in a modified form within the scope not departing from the essence of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is defined not by the foregoing description but by the claims particularly, and all differences within the scope equivalent thereto should be construed to be within the scope of the present invention.

Claims

1. Step 1: mixing hyaluronic acid with distilled water and mixed enzymes, proteinase and pectinase, to obtain mixed enzyme-treated hyaluronic acid through an enzymatic reaction; and (2) steaming the mixed enzyme-treated hyaluronic acid in step 1 and then aging it at low temperature at 5 to 20°C for 1 to 5 days (step 2). A method for producing low molecular weight hyaluronic acid.

2. The method for producing low molecular weight hyaluronic acid according to claim 1, wherein the proteinase in step 1 is Flavourzyme, Protamex, α-chymotrypsin, subtilisin Carlsberg, thermolysin, papain, fungal protease, or pepsin.

3. 2. The method of claim 1, wherein the pectinase in step 1 is Pectinex Ultra SP-L, Viscozyme L, Ultrazyme AFP, Pectinex Ultra AFP, Pectinex Ultra clear, or α-herbzyme.

4. The method for producing low molecular weight hyaluronic acid according to claim 1, wherein the mixed enzyme comprises 1 to 5 parts by weight of proteinase based on 1 part by weight of pectinase.

5. The method for producing low molecular weight hyaluronic acid according to claim 4, wherein the mixed enzyme comprises 2 to 4 parts by weight of proteinase based on 1 part by weight of pectinase.

6. The method for producing low molecular weight hyaluronic acid according to claim 1, wherein the steaming treatment in step 2 is carried out at 70-90°C for 2-4 hours, and the low temperature aging is carried out at 14-18°C for 2-4 days.

7. 2. The method for producing low molecular weight hyaluronic acid according to claim 1, wherein the weight average molecular weight (Mw) of the hyaluronic acid produced by the method for producing low molecular weight hyaluronic acid is 500 Da or less.

8. A low molecular weight hyaluronic acid having an average molecular weight (Mw) of 500 Da or less produced by the production method described in claim 1.

9. A cosmetic composition for reducing skin irritation and moisturizing the skin, comprising a low molecular weight hyaluronic acid produced by the method according to claim 1.

10. A cosmetic composition for alleviating skin irritation and moisturizing the skin, comprising low molecular weight hyaluronic acid having a weight average molecular weight (Mw) of 500 Da or less.

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