Cosmetic composition for skin improvement comprising cross-linked hyaluronic acid

Abstract

The present invention relates to a composition for skin improvement in which two or more types of hyaluronic acids having different molecular weights are cross-linked. A composition comprising a cross-linked hyaluronic acid prepared by mixing, in a specific ratio, a first hyaluronic acid having a molecular weight of 1 kDa or more and less than 300 kDa, a second hyaluronic acid having a molecular weight of 300 kDa or more and less than 1,000 kDa, and a third hyaluronic acid having a molecular weight of 1,000-1,700 kDa, and then performing a cross-linking reaction was found to have excellent effects in terms of water absorption, cell regeneration ability, transdermal absorption rate, procollagen production ability, and fibronectin production ability, and thus is provided as a cosmetic composition having an excellent skin improvement effect.

Description

Cosmetic composition for skin improvement comprising cross-linked hyaluronic acid

[0001] The present invention relates to a composition for improving skin, in which two or more types of hyaluronic acids having different molecular weights are cross-linked.

[0002] Hyaluronic acid is a polysaccharide formed by the continuous and repeated bonding of N-acetyl-D-glycosamine and D-glucuronic acid. Hyaluronic acid is mainly distributed in connective tissue, epidermis, nerve cells, skin, and cartilage, but is also distributed in large quantities in eyes, synovial fluid, muscles, umbilical cord, etc. It is an important molecule that plays various roles in the body, such as maintaining moisture, preventing bacterial invasion in the human body, and lubricating to facilitate body movement. Due to its useful role, hyaluronic acid is also used as a pharmaceutical material such as artificial tears and as a material for functional cosmetics.

[0003] Hyaluronic acid has been developed as a functional cosmetic, pharmaceutical, and biomaterial due to its ability to promote collagen synthesis, improve skin density, and provide excellent moisturizing effects. However, due to its high viscosity and price, hyaluronic acid has been used in cosmetic formulations at low concentrations. The amount of hyaluronic acid used in most cosmetic formulations is so minute that it is difficult to expect any significant benefits from hyaluronic acid itself.

[0004] To maximize the effectiveness of hyaluronic acid, if the concentration exceeds 1%, the viscosity increases, causing skin tightness and other symptoms, significantly reducing the user experience. Furthermore, a film-like phenomenon remains on the skin surface over time after application, limiting its utility as a product. Therefore, the development of diverse methods to maximize the effectiveness of hyaluronic acid even at low concentrations is urgently needed.

[0005] Meanwhile, since hyaluronic acid is easily decomposed under acidic or alkaline conditions, its use is limited. Therefore, cross-linked hyaluronic acid has been proposed to improve structural stability. Cross-linked hyaluronic acid is a material in which hyaluronic acid is cross-linked and has good elasticity and a long retention period, so it has been developed for various purposes such as various fillers, adhesion inhibitors, drug delivery vehicles, and cell supports. Cross-linked hyaluronic acid can be manufactured in various sizes and shapes depending on the type of hyaluronic acid manufactured, and hyaluronic acid with an appropriate molecular weight is manufactured and used for each purpose.

[0006] The purpose of the present invention is to provide a cross-linked hyaluronic acid having the best skin improvement effect, a method for producing hyaluronic acid having excellent moisture absorption capacity, cell regeneration capacity, percutaneous absorption rate, procollagen production capacity, and fibronectin production capacity by combining two or more types of hyaluronic acid having different molecular weights, and a composition having excellent skin improvement effect produced thereby.

[0007] The present invention provides a cosmetic composition for improving skin, comprising cross-linked hyaluronic acid comprising a first hyaluronic acid having a molecular weight of 1 kDa or more and less than 300 kDa; a second hyaluronic acid having a molecular weight of 300 kDa or more and less than 1,000 kDa; and a third hyaluronic acid having a molecular weight of 1,000 kDa or more and less than 1,700 kDa.

[0008] In addition, the present invention provides a method for preparing a cosmetic composition for improving skin, comprising the steps of preparing a hyaluronic acid mixture by dissolving a first hyaluronic acid having a molecular weight of 1 kDa or more and less than 300 kDa, a second hyaluronic acid having a molecular weight of 300 kDa or more and less than 1,000 kDa, and a third hyaluronic acid having a molecular weight of 1,000 kDa or more and less than 1,700 kDa in a sodium hydroxide solution; and the step of administering a crosslinking agent to the hyaluronic acid mixture to perform a crosslinking reaction.

[0009] According to the present invention, a composition comprising cross-linked hyaluronic acid prepared by mixing a first hyaluronic acid having a molecular weight of 1 kDa or more and less than 300 kDa, a second hyaluronic acid having a molecular weight of 300 kDa or more and less than 1,000 kDa, and a third hyaluronic acid having a molecular weight of 1,000 kDa or more and less than 1,700 kDa in a specific ratio and then subjecting the mixture to a cross-linking reaction has excellent effects in terms of moisture absorption capacity, cell regeneration capacity, percutaneous absorption rate, procollagen production capacity, and fibronectin production capacity, and thus can be provided as a cosmetic composition having excellent skin improvement effects.

[0010] The terms used in this specification have been selected from widely used, current terms, taking into account the functions of the present invention. However, these terms may vary depending on the intentions of those skilled in the art, precedents, the emergence of new technologies, etc. Furthermore, in certain cases, terms may be arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should not be defined simply as names, but rather based on their inherent meanings and the overall content of the present invention.

[0011] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0012]

[0013] Hereinafter, the present invention will be described in more detail.

[0014]

[0015] The present invention provides a cosmetic composition for improving skin, comprising cross-linked hyaluronic acid comprising a first hyaluronic acid having a molecular weight of 1 kDa or more and less than 300 kDa; a second hyaluronic acid having a molecular weight of 300 kDa or more and less than 1,000 kDa; and a third hyaluronic acid having a molecular weight of 1,000 kDa or more and less than 1,700 kDa.

[0016] The above cross-linked hyaluronic acid is cross-linked with first hyaluronic acid, second hyaluronic acid, and third hyaluronic acid in a weight % ratio of 1 to 4: 1: 1 to 4.

[0017] The above cross-linked hyaluronic acid is prepared by a cross-linking reaction in the presence of a cross-linking agent in an amount of 0.5 mol% to 5.0 mol%.

[0018] The crosslinking agent is 1,4-butandiol diglycidyl ether, 1,2-(bis(2,3-epoxypropoxy)ethylene, propylene glycol diglycidyl ether, polytetranethylene glycil diglycidyl ether, ethylene glycol diglycidyl ether (EGDGE), neopentyl glycoldiglydidiyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, 1.6-hexanediol diglycidyl ether, glycerol polyglycidyl ether. The crosslinking agent may be any one selected from among trimethylpropane polyglycidyl ether, pentaerythritol polyglycidyl ether, or sorbitol polyglycidyl ether, and preferably, the crosslinking agent is 1,4-butanediol diglycidyl ether.

[0019] The above skin improvement composition has excellent moisture absorption capacity, cell regeneration effect, percutaneous absorption rate, procollagen production capacity, and fibronectin production capacity, and thus has wrinkle improvement, moisturizing, and skin regeneration functions.

[0020] The above cosmetic composition may be formulated with one or more selected from the group consisting of skin, lotion, cream, serum, emulsion, essence, powder, foundation, spray, mask pack, sheet pack, sleeping pack, wash-off pack, and peel-off pack. The above cosmetic composition may be formulated by a conventional method.

[0021] Specifically, the cosmetic composition can be prepared as a general emulsified formulation and a solubilized formulation. For example, the cosmetic composition can be formulated as a toner such as a flexible toner or a nourishing toner; an emulsion such as a facial lotion or a body lotion; a cream such as a nourishing cream, a moisturizing cream, or an eye cream; an essence; a cosmetic ointment; a spray; a gel; a pack; a sunscreen; a makeup base; a foundation such as a liquid type, a solid type, or a spray type; a powder; a makeup remover such as a cleansing cream, a cleansing lotion, or a cleansing oil; or a cleanser such as a cleansing foam, soap, or a body wash, but is not limited thereto. In addition, the cosmetic composition can be formulated as a skin external preparation such as an ointment, a patch, a gel, a cream, or a spray, but is not limited thereto.

[0022] In addition to the essential ingredients, other ingredients may be appropriately blended into each formulation of the above cosmetic composition, within a range that does not impede the purpose of the present invention, depending on the type of formulation or intended use.

[0023] The above cosmetic composition may typically include an acceptable carrier, such as oil, water, surfactant, moisturizer, lower alcohol, thickener, chelating agent, pigment, preservative, fragrance, etc., but is not limited thereto.

[0024] The above acceptable carriers may vary depending on the formulation. For example, when formulated as an ointment, paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, or extracts thereof may be used as carrier components.

[0025] When the above cosmetic composition is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or extracts thereof may be used as a carrier component, and in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane, butane or dimethyl ether may be further included.

[0026] When the above cosmetic composition is formulated as a solution or emulsion, a solvent, a solubilizer, or an emulsifier can be used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, propylene glycol, 1,3-butylglycol oil can be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, glycerol fatty acid ester, polyethylene glycol, or fatty acid ester of sorbitan can be used.

[0027] When the above cosmetic composition is formulated as a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. can be used as a carrier component.

[0028] When the above cosmetic composition is formulated as soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. can be used as carrier components.

[0029] The above cosmetic composition may additionally contain adjuvants commonly used in the field of cosmetology or dermatology, such as fatty substances, organic solvents, solubilizers, thickeners, gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestering agents, chelating agents, preservatives, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, and any other ingredients commonly used in cosmetics, depending on the quality or function of the final product.

[0030] However, the above-mentioned auxiliary agent and its mixing ratio can be appropriately selected so as not to affect the desirable properties of the cosmetic composition according to the present invention.

[0031] In addition, the present invention provides a method for preparing a cosmetic composition for improving skin, comprising the steps of preparing a hyaluronic acid mixture by dissolving a first hyaluronic acid having a molecular weight of 1 kDa or more and less than 300 kDa, a second hyaluronic acid having a molecular weight of 300 kDa or more and less than 1,000 kDa, and a third hyaluronic acid having a molecular weight of 1,000 kDa or more and less than 1,700 kDa in a sodium hydroxide solution; and the step of administering a crosslinking agent to the hyaluronic acid mixture to perform a crosslinking reaction.

[0032] The above cross-linking reaction is carried out by administering 0.5 mol% to 5.0 mol% of a cross-linking agent and at 10°C to 40°C.

[0033] Hereinafter, to aid understanding of the present invention, experimental examples and examples will be described in detail. However, the following experimental examples and examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental examples and examples of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0034]

[0035] Experimental Example 1. Selection of hyaluronic acid composition ratio

[0036] Hyaluronic acids of various molecular weight ranges were prepared and classified into first, second, and third hyaluronic acids. Low-molecular-weight hyaluronic acids having a molecular weight of 1 kDa or more and less than 300 kDa were classified as first hyaluronic acids. Medium-molecular-weight hyaluronic acids having a molecular weight of 300 kDa or more and less than 1,000 kDa were classified as second hyaluronic acids. High-molecular-weight hyaluronic acids having a molecular weight of 1,000 kDa or more and less than 1,700 kDa were classified as third hyaluronic acids. The three classified hyaluronic acids were dissolved in sodium hydroxide solutions at respective ratios as shown in Table 1 to prepare preparation examples 1 to 22.

[0037] First hyaluronic acid Second hyaluronic acid Third hyaluronic acid Manufacturing example 1111 Manufacturing example 2211 Manufacturing example 3121 Manufacturing example 4112 Manufacturing example 5221 Manufacturing example 6212 Manufacturing example 7122 Manufacturing example 8411 Manufacturing example 9611 Manufacturing example 10811 Manufacturing example 111011 Manufacturing example 12141 Manufacturing example 13161 Manufacturing example 14181 Manufacturing example 151101 Manufacturing example 16114 Manufacturing example 17116 Manufacturing example 18118 Manufacturing example 191110 Manufacturing example 201 Manufacturing example 211 Manufacturing example 221

[0038]

[0039] For each manufacturing example, the cross-linking agent 1,4-butanediol diglycidyl ether (BDDE) was administered equally, and the cross-linking reaction was performed at 35°C. The hyaluronic acid after the cross-linking reaction was placed in a phosphate-buffered saline (PBS) buffer solution to remove any remaining cross-linking agent and neutralize. The appropriate ratio of cross-linked hyaluronic acid for each manufacturing example was determined by evaluating the water absorption capacity (%) and cell regeneration efficacy (%).

[0040]

[0041] Experimental Example 1-1. Moisture Absorption Evaluation (Moisturizing Functionality)

[0042] In vitro water absorption capacity was evaluated. Each composition (1 g) and 10 ml of phosphate buffer were added to a graduated test tube, and the mixture was stirred for 5 minutes. The mixture was left to stand at 37°C for 48 hours, and 100 μl of Coomassie Brilliant Blue solution was added to confirm layer separation and color differences. Using layer separation as the boundary, the volume of the hydrogel settled in the lower layer was confirmed, and the water absorption capacity (%) of each composition was derived using the ratio of the height increased by swelling (area under the Coomassie Brilliant Blue dye) to the height of the initial gel.

[0043]

[0044] Experimental Example 1-2. Cell Regeneration Effect (Skin Regeneration Functionality)

[0045] The cell regeneration effect was evaluated in vitro. Human keratinocytes (HaCaT) were seeded in 24-well plates at a density of 5 × 10 4The cells were dispensed at a concentration of 1 cell / well and cultured overnight in a CO2 incubator. Afterwards, the cells were scraped using a 200 μl pipette tip, and serum-free cell medium containing 1 mg / ml of each cross-linked hyaluronic acid complex was added to each well, and cell migration was measured for 48 hours. The scraped area of ​​each well was photographed using a microscope (Nikon Instruments Inc., Japan), and the same area was photographed again after 48 hours of culture, and the cell regeneration ability (%) was derived as the ratio of the cell migration area before and after treatment with the composition to the untreated group.

[0046] Moisture content (%) Cell regeneration capacity (%) Manufacturing example 156.162.4 Manufacturing example 261.263.8 Manufacturing example 363.858.4 Manufacturing example 462.460.8 Manufacturing example 564.263.1 Manufacturing example 689.585.8 Manufacturing example 781.661.5 Manufacturing example 860.868.4 Manufacturing example 952.568.1 Manufacturing example 1058.465.1 Manufacturing example 1162.160.5 Manufacturing example 1259.459.4 Manufacturing example 1358.772.6 Manufacturing example 1461.470.1 Manufacturing example 1556.456.7 Manufacturing example 1660.764.8 Manufacturing example Manufacturing example 1756.867.8 Manufacturing example 1860.454.4 Manufacturing example 1960.462.4 Manufacturing example 2046.550.9 Manufacturing example 2152.458.4 Manufacturing example 2249.546.5

[0047]

[0048] As shown in Table 2 above, when the first hyaluronic acid, the second hyaluronic acid, and the third hyaluronic acid were mixed and manufactured according to the ratio, it was confirmed that the water absorption capacity and cell regeneration capacity were superior to each single hyaluronic acid, and among them, in particular, the water absorption capacity and cell regeneration capacity were shown to be the best in Manufacturing Example 6 mixed at a ratio of 2:1:2.

[0049]

[0050] Experimental Example 2. Evaluation of Crosslinking Agent Dosage

[0051] The most suitable cross-linking agent dosage was evaluated by analyzing the water absorption capacity (%), cell regeneration capacity (%), and percutaneous absorption rate (%) by varying the amount of cross-linking agent. Manufacturing Example 6 was performed by mixing the first hyaluronic acid, the second hyaluronic acid, and the third hyaluronic acid in a ratio of 2:1:2, and 1,4-butanediol diglycidyl ether was used as the cross-linking agent. The amount of cross-linking agent was 0.5 mol% (Example 1), 1.0 mol% (Example 2), 2.0 mol% (Example 3), 3.0 mol% (Example 4), or 5.0 mol% (Example 5).

[0052]

[0053] Experimental Example 2-1. Transdermal absorption rate (%) (moisturizing functionality)

[0054] According to the "Guidelines for Skin Absorption Tests in Living Beings", Franz diffusion cell (effective area: 0.64 cm) 2 , volume of the receptor chamber: 5 ml) equipment (Logan Instruments, New Jersey, USA) was used to conduct a skin permeation test. The receptor phase used in the skin permeation test was phosphate buffered saline (PBS) with a pH of 7.4, and the epidermal layer of human skin (1.5 cm x 1.5 cm) was placed over the receptor chamber with the stratum corneum facing upward, covered with a donor chamber, and fixed with a clamp. The receptor phase was filled into a Franz diffusion cell and maintained at 32°C. 100 mg of the crosslinked complex solution of each condition was constantly applied to the donor site to conduct a permeation test. After 24 hours of conducting the permeation test, the receptor phase was collected, and the content of glucuronic acid in the aqueous solution was quantified as a converted value, and the percutaneous absorption rate (%) was derived.

[0055] Water absorption capacity (%) Cell regeneration capacity (%) Transdermal absorption rate (%) Example 176.964.663.1 Example 281.269.069.1 Example 392.185.879.9 Example 473.865.172.4 Example 568.466.356.8

[0056]

[0057] As shown in Table 3 above, Example 3, in which the crosslinking agent was added at 2 mol%, showed the best water absorption capacity (%), cell regeneration capacity (%), and percutaneous absorption rate (%).

[0058]

[0059] Experimental Example 3. Skin Improvement Functional Evaluation (Wrinkle Improvement Functionality)

[0060] In order to evaluate the functionality of the composition for skin improvement, cell regeneration ability (%), type 1 procollagen production ability (%), and fibronectin production ability (%) were analyzed. The evaluation was performed using Example 3, in which the first hyaluronic acid, the second hyaluronic acid, and the third hyaluronic acid were mixed in a ratio of 2: 1: 2 and a cross-linking agent was administered at 2 mol%. For the functionality evaluation, Comparative Example 1, in which the first hyaluronic acid, the second hyaluronic acid, and the third hyaluronic acid were simply mixed in a ratio of 2: 1: 2, was used. Human-derived fibroblast cell line (CCD-986SK cells) were seeded in a 48-well plate at 5 X 10 4The cells were seeded at a concentration of 10 cells / well and cultured overnight in a 37°C, 5% CO2 incubator using DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS. After 24 hours of culture in serum-free DMEM, the cells were treated with each of Example 3 or Comparative Example 1, and the cultured supernatant was collected after 24 hours. 5 ng / mL of TGF-β was used as a positive control. The amount of collagen in the collected medium was measured using an ELISA Kit (Procollagen type I-peptide EIA kit, TAKARA), and the amount of fibronectin was measured using an ELISA Kit (Fibronectin ELISA Kit, Invitrogen).

[0061] Cell regeneration ability (%) Type 1 procollagen production ability (%) Fibronectin production ability (%) Example 387.78 1.96 5.3 Comparative example 160.95 0.13 1.7

[0062]

[0063] As shown in Table 4 above, Example 3 was found to be superior to Comparative Example 1 in cell regeneration ability (%), procollagen production ability (%), and fibronectin production ability (%).

[0064]

[0065] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0066] Numerical ranges are inclusive of the values ​​defined in the ranges above. Any maximum numerical limitation given throughout this specification includes any lower numerical limitation, as if that lower numerical limitation were explicitly stated. Any minimum numerical limitation given throughout this specification includes any higher numerical limitation, as if that higher numerical limitation were explicitly stated. Any numerical limitation given throughout this specification will include any better numerical range within the broader numerical range, as if that narrower numerical limitation were explicitly stated.

Claims

A first hyaluronic acid having a molecular weight of 1.1 kDa or more and less than 300 kDa; A second hyaluronic acid having a molecular weight of 300 kDa or more and less than 1,000 kDa; and A cosmetic composition for improving skin, comprising cross-linked hyaluronic acid with a third hyaluronic acid having a molecular weight of 1,000 kDa or more and 1,700 kDa or less.

2. A cosmetic composition for improving skin, characterized in that in the first paragraph, the cross-linked hyaluronic acid is cross-linked with first hyaluronic acid, second hyaluronic acid, and third hyaluronic acid in a weight % ratio of 1 to 4: 1: 1 to 4.

3. A cosmetic composition for improving skin, characterized in that the cross-linked hyaluronic acid in the first paragraph is prepared by a cross-linking reaction in the presence of a cross-linking agent in an amount of 0.5 mol% to 5.0 mol%.

4. In the third paragraph, the crosslinking agent is 1,4-butandiol diglycidyl ether, 1,2-(bis(2,3-epoxypropoxy)ethylene, propylene glycol diglycidyl ether, polytetranethylene glycil diglycidyl ether, ethylene glycol diglycidyl ether (EGDGE), neopentyl glycoldiglydidiyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, 1.6-hexanediol diglycidyl ether, A cosmetic composition for improving skin, characterized by comprising any one selected from among glycerol polyglycidyl ether, trimethylpropane polyglycidyl ether, pentaerythritol polyglycidyl ether, and sorbitol polyglycidyl ether.

5. A cosmetic composition for skin improvement, characterized in that the composition for skin improvement in paragraph 1 has wrinkle improvement, moisturizing, and skin regeneration functions. A step of preparing a hyaluronic acid mixture by dissolving a first hyaluronic acid having a molecular weight of 6.1 kDa or more and less than 300 kDa, a second hyaluronic acid having a molecular weight of 300 kDa or more and less than 1,000 kDa, and a third hyaluronic acid having a molecular weight of 1,000 kDa or more and less than 1,700 kDa in a sodium hydroxide solution; and A method for producing a cosmetic composition for improving skin, comprising a step of inducing a cross-linking reaction by adding a cross-linking agent to the above hyaluronic acid mixture.

7. In the 6th paragraph, the crosslinking agent is 1,4-butandiol diglycidyl ether, 1,2-(bis(2,3-epoxypropoxy)ethylene, propylene glycol diglycidyl ether, polytetranethylene glycil diglycidyl ether, ethylene glycol diglycidyl ether (EGDGE), neopentyl glycoldiglydidiyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, 1.6-hexanediol diglycidyl ether, A method for producing a cosmetic composition for improving skin, characterized in that the composition comprises at least one selected from among glycerol polyglycidyl ether, trimethylpropane polyglycidyl ether, pentaerythritol polyglycidyl ether, and sorbitol polyglycidyl ether.

8. A method for producing a cosmetic composition for skin improvement, characterized in that in paragraph 6, the crosslinking reaction is carried out by administering a crosslinking agent in an amount of 0.5 mol% to 5.0 mol% and at a temperature of 10°C to 50°C.