Hyaluronic acid powder

Abstract

The present invention relates to a hyaluronic acid powder containing hyaluronic acid particles, wherein the hyaluronic acid particles contain hyaluronic acid and / or its salt having an average molecular weight of 1,500,000 or more, and the content of hyaluronic acid particles having a particle diameter of 100 μm or less is 0 mass% to 35 mass% with respect to the total mass of the hyaluronic acid powder.

Description

Technology Field

[0001] The present invention relates to hyaluronic acid powder. Background Technology

[0002] Hyaluronic acid is widely distributed in biological tissues such as the tragus, umbilical cord, skin, cartilage, vitreous humor, and synovial fluid, and is widely used as an ingredient in, for example, cosmetics, pharmaceuticals, and food. In particular, hyaluronic acid is actively utilized as a raw material for medical products and cosmetics due to its high biocompatibility, gel swelling properties, and viscoelasticity.

[0003] Various technical means for improving the properties of hyaluronic acid have been reported. For example, Patent Document 1 discloses a powder composed of hyaluronic acid and / or its salt, having an average molecular weight of 200,000 or more and an average particle diameter of 50 to 500 μm. Patent Document 2 discloses a method for manufacturing hyaluronic acid powder and its salt, comprising a process of sequentially performing a dehydration treatment in which a water-soluble organic solvent is added to an aqueous solution containing hyaluronic acid and / or its salt to precipitate the hyaluronic acid and / or its salt and separate the precipitate from the mother liquor, a washing treatment in which the precipitate is washed with a 75 to 80 mass% aqueous solution of a water-soluble organic solvent, and a drying treatment in which the precipitate is dried after washing. Prior art literature

[0004] [Patent Document 1] Japanese Published Patent Application No. 2011-84588 [Patent Document 2] Japanese Published Patent Application No. 2009-256464 The problem to be solved

[0005] Cross-linked hyaluronic acid used for medical purposes (especially for cosmetic surgery) is prepared by dissolving high-molecular-weight hyaluronic acid powder, which is the raw material, at a high concentration (e.g., 2 mass% or more). However, when attempting to dissolve high-molecular-weight hyaluronic acid powder containing hyaluronic acid and / or its salt at a high concentration, lumps are likely to form (i.e., it takes time to completely dissolve). If a cross-linking reaction is performed while lumps are present, the viscosity of the obtained cross-linked hyaluronic acid may not be sufficiently developed.

[0006] The object of the present invention is to provide a hyaluronic acid powder containing hyaluronic acid and / or its salt having an average molecular weight of 1,500,000 or more, which can be dissolved in a short time even at high concentrations. means of solving the problem

[0007] The present invention relates, for example, to each of the following inventions.

[0008] [1] A hyaluronic acid powder containing hyaluronic acid particles, wherein the hyaluronic acid particles contain hyaluronic acid and / or its salt having an average molecular weight of 1,500,000 or more, and the content of hyaluronic acid particles having a particle diameter of 100 μm or less is 0 to 35 mass% based on the total mass of the hyaluronic acid powder.

[0009] [2] Hyaluronic acid powder described in [1], with a relative standard deviation of particle size distribution of 0.30% or less.

[0010] [3] Hyaluronic acid powder described in [1] or [2], wherein the content of hyaluronic acid particles having a particle diameter of 100 μm or less is 0 to 5 mass% based on the total mass of the hyaluronic acid powder.

[0011] [4] Hyaluronic acid powder described in any one of [1] to [3], having an average molecular weight of hyaluronic acid and / or its salt of 1,500,000 or more and 3,900,000 or less.

[0012] [5] A method for selecting raw hyaluronic acid powder used in a method for manufacturing cross-linked hyaluronic acid and / or its salt, wherein the raw hyaluronic acid powder contains hyaluronic acid particles, and the hyaluronic acid particles contain hyaluronic acid and / or its salt having an average molecular weight of 1,500,000 or more, and the content ratio of hyaluronic acid particles having a particle diameter of 100 μm or less is 0 mass% or more and 35 mass% or less based on the total mass of the hyaluronic acid powder. Effects of the invention

[0013] According to the present invention, a hyaluronic acid powder containing hyaluronic acid and / or its salt having an average molecular weight of 1,500,000 or more and dissolving in a short time even at high concentrations can be provided. Specific details for implementing the invention

[0014] Embodiments of the present invention will be described in detail below. However, the present invention is not limited to the following embodiments.

[0015] <Features of the present invention>

[0016] Hyaluronic acid powder

[0017] The present invention provides a hyaluronic acid powder containing hyaluronic acid particles, wherein the hyaluronic acid particles contain hyaluronic acid and / or its salt having an average molecular weight of 1,500,000 or more, and the content of hyaluronic acid particles having a particle diameter of 100 μm or less is 0 mass% to 35 mass% based on the total mass of the hyaluronic acid powder.

[0018] Method for selecting raw hyaluronic acid powder used in the method for manufacturing cross-linked hyaluronic acid and / or its salt

[0019] The present invention relates to a method for selecting raw hyaluronic acid powder used in a method for manufacturing cross-linked hyaluronic acid and / or its salt, wherein the raw hyaluronic acid powder contains hyaluronic acid particles, and the hyaluronic acid particles contain hyaluronic acid and / or its salt having an average molecular weight of 1,500,000 or more, and the content ratio of hyaluronic acid particles having a particle diameter of 100 μm or less is 0 mass% to 35 mass% based on the total mass of the hyaluronic acid powder.

[0020] Hyaluronic Acid Powder

[0021] Hyaluronic acid powder is an aggregate of numerous hyaluronic acid particles.

[0022] Hyaluronic acid and / or its salts

[0023] "Hyaluronic acid" refers to a polysaccharide having one or more repeating units composed of glucuronic acid and N-acetylglucosamine disaccharides. "Salts of hyaluronic acid" are not particularly limited, but are preferably salts that are acceptable in food or medicine. Examples of salts of hyaluronic acid include sodium salts, potassium salts, calcium salts, zinc salts, magnesium salts, and ammonium salts.

[0024] <Average molecular weight>

[0025] The hyaluronic acid particles contain hyaluronic acid and / or its salt having an average molecular weight of 1,500,000 or more. The average molecular weight of the hyaluronic acid and / or its salt may be 1,500,000 or more, 1,600,000 or more, 1,700,000 or more, 1,800,000 or more, or 1,900,000 or more. The average molecular weight of the hyaluronic acid and / or its salt may be 3,900,000 or less, 3,500,000 or less, 3,000,000 or less, or 2,800,000 or less.

[0026] Method for Measuring Average Molecular Weight

[0027] In this specification, the “average molecular weight” of hyaluronic acid refers to the viscosity average molecular weight. The average molecular weight is calculated according to the method disclosed in the average molecular weight of “purified sodium hyaluronate” of the Japanese Pharmacopoeia (18th revision).

[0028] Specifically, the amount of hyaluronic acid powder was accurately measured such that the downward flow time of the solution obtained by dissolving hyaluronic acid powder in 100 mL of 0.2 mol / L sodium chloride test solution was 2.0 to 2.4 times that of the 0.2 mol / L sodium chloride test solution. Hyaluronic acid powder was dissolved in 0.2 mol / L sodium chloride test solution to prepare exactly 100 mL of sample solution (1). 16 mL, 12 mL, and 8 mL of sample solution (1) were accurately measured, and 0.2 mol / L sodium chloride test solution was added to each to prepare exactly 20 mL each of sample solution (2), sample solution (3), and sample solution (4).

[0029] For the sample solutions (1) to (4) described above, the time taken for a certain amount of liquid to flow down through a capillary at 30°C ± 0.1°C is measured using a Ubbelohde type viscometer with a downward flow time of 200 to 300 seconds for a 0.2 mol / L sodium chloride test solution by the general test method viscosity measurement method (viscosity measurement by capillary viscometer), and the specific viscosity (Equation (1)) and the reduction viscosity at each concentration (Equation (2)) are calculated. A graph is plotted with the reduction viscosity on the vertical axis and the concentration of the converted dry matter of hyaluronic acid (g / 100 mL) on the horizontal axis, and the infinite viscosity is obtained at the intersection point of the vertical axis and the straight line connecting each point. The average molecular weight is calculated from the infinite viscosity obtained here by Equation (3).

[0030] Specific viscosity = {(Time required for sample solution to flow downward (seconds)) / (Time required for 0.2 mol / L sodium chloride solution to flow downward (seconds))} -1 ···(1)

[0031] Reducing viscosity (dL / g) = Specific viscosity / (Concentration of the product relative to the converted dry matter (g / 100 mL))···(2)

[0032]

[0033] <Content of hyaluronic acid particles with a particle diameter of 100 μm or less>

[0034] In the hyaluronic acid powder, the content of hyaluronic acid particles having a particle diameter of 100 μm or less is 35 mass% or less based on the total mass of the hyaluronic acid powder, and may be 30 mass% or less, 25 mass% or less, 20 mass% or less, 15 mass% or less, 10 mass% or less, 5 mass% or less, 3 mass% or less, 2 mass% or less, or 1 mass% or less in terms of the short dissolution time at high concentration. In the hyaluronic acid powder, the content of hyaluronic acid particles having a particle diameter of 100 μm or less is 0 mass% or more based on the total mass of the hyaluronic acid powder, and may be 0 mass%.

[0035] <Content of hyaluronic acid particles with a particle diameter of 155 μm or less>

[0036] In the hyaluronic acid powder, the content of hyaluronic acid particles having a particle diameter of 155 μm or less may be 55 mass% or less, 50 mass% or less, 45 mass% or less, 40 mass% or less, 35 mass% or less, 30 mass% or less, 25 mass% or less, 20 mass% or less, 15 mass% or less, 10 mass% or less, 5 mass% or less, 3 mass%, 2 mass% or less, or 1 mass% or less, based on the total mass of the hyaluronic acid powder, in terms of high concentration and short dissolution time. In the hyaluronic acid powder, the content of hyaluronic acid particles having a particle diameter of 155 μm or less may be 0 mass% or more and 0 mass% based on the total mass of the hyaluronic acid powder.

[0037] <Content of hyaluronic acid particles with a particle diameter of 190 μm or less>

[0038] In the hyaluronic acid powder, the content of hyaluronic acid particles having a particle diameter of 190 μm or less may be 65 mass% or less, 60 mass% or less, 55 mass% or less, 50 mass% or less, 45 mass% or less, 40 mass% or less, 35 mass% or less, 30 mass% or less, 25 mass% or less, 20 mass% or less, 15 mass% or less, 10 mass% or less, 5 mass% or less, 3 mass% or less, or 2 mass% or less, based on the total mass of the hyaluronic acid powder. In the hyaluronic acid powder, the content of hyaluronic acid particles having a particle diameter of 190 μm or less may be 0 mass% or more and 0.5 mass% or more based on the total mass of the hyaluronic acid powder.

[0039] Methods for Measuring Particle Size Distribution

[0040] The content of hyaluronic acid particles with a particle diameter of 100 μm or less, hyaluronic acid particles with a particle diameter of 155 μm or less, and hyaluronic acid particles with a particle diameter of 190 μm or less is measured using a laser diffraction particle size analyzer. Specific measurement conditions are the same as those described in the examples below.

[0041] Relative standard deviation of particle size distribution

[0042] The relative standard deviation of the particle size distribution may be 0.30% or less, 0.28% or less, 0.25% or less, 0.20% or less, 0.15% or less, 0.10% or less, or 0.05% or less, given that the dissolution time at high concentrations is short. The relative standard deviation of the particle size distribution may be 0.01% or more, 0.02% or more, or 0.03% or more. The relative standard deviation of the particle size distribution refers to the value calculated by the method shown below.

[0043] Relative standard deviation (%) = Standard deviation / Average particle diameter (micrometers) × 100

[0044] The standard deviation and average particle diameter are measured by a laser diffraction particle size analyzer.

[0045] In the hyaluronic acid powder, the content of hyaluronic acid particles having a particle diameter of 1 mm or more may be 5 mass% or less, 4 mass% or less, 3 mass% or less, 2 mass% or less, or 1 mass% or less, based on the total mass of the hyaluronic acid powder, in terms of the short dissolution time at high concentration. In the hyaluronic acid powder, the content of hyaluronic acid particles having a particle diameter of 1 mm or less may be 95 mass% or more, 96 mass% or more, 97 mass% or more, 98 mass% or more, or 99 mass% or more, based on the total mass of the hyaluronic acid powder.

[0046] <Origin of Hyaluronic Acid and / or Its Salts>

[0047] Hyaluronic acid and / or its salts may be extracted from natural products such as animals (e.g., living tissues such as a rooster's cocoon, umbilical cord, skin, synovial fluid), obtained by culturing microorganisms, animal cells, or plant cells (e.g., through a fermentation method using bacteria such as streptococci), or synthesized chemically or enzymatically.

[0048] Method for manufacturing hyaluronic acid powder

[0049] The method for producing hyaluronic acid powder according to the present invention is, for example, a method for producing hyaluronic acid powder containing hyaluronic acid particles having an average molecular weight of 1,500,000 or more and / or a salt thereof, wherein the content of hyaluronic acid particles having a particle diameter of 100 μm or less is controlled. A conventional method may be used except for controlling the content of hyaluronic acid particles having a particle diameter of 100 μm or less. For example, the hyaluronic acid powder according to the present invention may be produced through a sieve extraction method or a microbial fermentation method.

[0050] Method for Extracting the Crown

[0051] Heat treatment is applied to the sieve. The heat-treated sieve is made into a paste and alkali-treated. Next, a proteolytic enzyme is added to the alkali-treated product of the sieve to protease-treat the product. After deodorizing and decolorizing the obtained protease-treated product by adding activated carbon, the product is filtered. Sodium chloride is dissolved in the obtained filtrate, ethanol is added thereto to precipitate hyaluronic acid, and the precipitate is collected. Subsequently, hydrated ethanol with an ethanol concentration of about 80 to 95 volume% is added to this precipitate, and the precipitate is collected by washing with a homogenizer. Hyaluronic acid powder can be obtained by repeating this washing with hydrated ethanol about 2 to 10 times and drying the fractionated precipitate.

[0052] Microbial Fermentation Method

[0053] Activated carbon is added to a culture medium of a microorganism of the genus Streptococcus (Streptococcus Zooepidemicus) that produces hyaluronic acid to deodorize and decolorize the mixture, and then filtered. Sodium chloride is dissolved in the obtained filtrate, and then ethanol is added to precipitate hyaluronic acid, and the precipitate is collected. After that, hydrated ethanol with an ethanol concentration of about 80 to 95 volume% is added to the precipitate, and the precipitate is washed with a homogenizer and collected. This washing with hydrated ethanol is repeated about 2 to 10 times, and the fractionated precipitate is dried to obtain hyaluronic acid powder.

[0054] Hyaluronic acid solution

[0055] The hyaluronic acid solution contains the aforementioned hyaluronic acid powder and a solvent. In the hyaluronic acid solution, the hyaluronic acid powder is dissolved in the solvent. If no insoluble substances are identified through visual inspection, it is determined that the hyaluronic acid powder is dissolved in the solvent.

[0056] Solvent of Hyaluronic Acid Solution

[0057] The solvent of the hyaluronic acid solution may be, for example, water. The hyaluronic acid solution may contain inorganic salts along with the solvent. Examples of inorganic salts include sodium hydroxide, sodium chloride, and phosphates.

[0058] Content of hyaluronic acid and / or its salts

[0059] The content of hyaluronic acid and its salt in the hyaluronic acid solution (concentration of hyaluronic acid) may be 2 mass% or more, 3 mass% or more, 4 mass% or more, 5 mass% or more, 6 mass% or more, 7 mass% or more, 8 mass% or more, 9 mass% or more, 10 mass% or more, 11 mass% or more, 12 mass% or more, 13 mass% or more, or 14 mass% or more, and may be 20 mass% or less, or 18 mass% or less.

[0060] Uses of Hyaluronic Acid Solution

[0061] The hyaluronic acid solution of the present invention can be used as a raw material for manufacturing cross-linked hyaluronic acid. Cross-linked hyaluronic acid can be used for medical purposes (particularly for cosmetic surgery), etc.

[0062] The present invention will be explained in more detail below based on examples and the like. However, the present invention is not limited to the following examples. Hereinafter, "%" means "mass%".

[0063] Preparation of Hyaluronic Acid Powder

[0064] Hyaluronic acid powder of Example 1 with an average molecular weight of 1,900,000 was prepared by the microbial fermentation method described above. The concentration of ethanol used in preparation was 80% to 95%, and the number of times the precipitate was washed was 10.

[0065] The hyaluronic acid of Example 1 was ground to obtain the hyaluronic acid powder of Comparative Example 5. The hyaluronic acid powder of Example 1 and the hyaluronic acid powder of Comparative Example 5 were mixed according to the mass ratio of Table 1 below to obtain the hyaluronic acid powder of Example 2 and the hyaluronic acid powders of Comparative Examples 1 to 4.

[0066]

[0067] The measurement results for the particle ratios of Examples 1 and 2 and Comparative Examples 1 to 4 are shown in Table 2.

[0068] Methods for Measuring Particle Size Distribution

[0069] The particle size distribution was measured using a laser diffraction particle size analyzer SALD-2200 (Shimadzu Corporation) under the following conditions.

[0070] Detection of diffraction-scattered light

[0071] Average frequency: 64 times

[0072] Number of measurements: 1 time

[0073] Measurement interval: 2 seconds

[0074] Measured absorbance range

[0075] Maximum value: 0.2000

[0076] Minimum value: 0.0100

[0077] Blank area / Measurement area

[0078] Maximum allowed variation for blank measurement: 20

[0079] Optimal measurement range (max): 1500

[0080] Optimal measurement range (minimum): 700

[0081] Dispersion solvent: ethanol

[0082] Dispersant: None

[0083] Dispersion method: Ultrasonic dispersion

[0084] Pump speed: 6.0

[0085] Internal ultrasound examination time: 10 seconds

[0086] <Test 1: Relationship between Particle Size Distribution and Dissolution Time>

[0087] (Method to check solubility)

[0088] After adding 20 mL of a 1% NaOH aqueous solution to a 50 mL capacity beaker, the hyaluronic acid powder of the example or comparative example was added while stirring with a magnetic stirrer (manufactured by Tokyo Glass Machinery Co., Ltd., model number F-601, dial scale 4). The time from the time the hyaluronic acid powder was added until the hyaluronic acid powder was completely dissolved was measured visually and recorded as the dissolution time. The content of hyaluronic acid (concentration of hyaluronic acid) in the hyaluronic acid solution was set to 10 mass% relative to the total amount of the hyaluronic acid solution.

[0089] (result)

[0090] The measurement results of the dissolution time of the hyaluronic acid powders of Examples 1 to 2 and Comparative Examples 1 to 5 are shown.

[0091]

[0092] It was confirmed that hyaluronic acid powder with a ratio of 35 mass% or less of hyaluronic acid particles of 100 μm or less can be dissolved in a short time even at high concentrations (hyaluronic acid concentration: 10 mass%).

[0093] <Test 2: Effect of Molecular Weight>

[0094] Hyaluronic acid powder of Example 3 was prepared having an average molecular weight of 2,760,000 and the same particle size distribution as Example 1. The dissolution time of the hyaluronic acid powder of Example 3 was measured using the same method as in Test 1. The results are shown in Table 3.

[0095]

[0096] Even when the average molecular weight was high, hyaluronic acid powder with a ratio of 35 mass% or less of hyaluronic acid particles of 100 μm or less could be dissolved in a short time at high concentrations.

[0097] <Test 3: Effect of Hyaluronic Acid Concentration>

[0098] The hyaluronic acid powders of Example 1 and Comparative Example 5 were dissolved in a 1% sodium hydroxide solution to have the hyaluronic acid concentrations shown in Table 4. The time until the hyaluronic acid powder was dissolved is shown in Table 4.

[0099]

[0100] Even when the concentration of hyaluronic acid in the hyaluronic acid solution was high, hyaluronic acid powder having a ratio of 35 mass% or less of hyaluronic acid particles of 100 μm or less could be dissolved in a short time at a high concentration.

Claims

Claim 1 A hyaluronic acid powder containing hyaluronic acid particles, wherein the hyaluronic acid particles contain hyaluronic acid and / or a salt thereof having an average molecular weight of 1.5 million or more, and the content ratio of the hyaluronic acid particles having a particle diameter of 100 μm or less is 0 mass% or more and 35 mass% or less based on the total mass of the hyaluronic acid powder, and the relative standard deviation of the particle size distribution is 0.30% or less. Claim 2 In claim 1, a hyaluronic acid powder having a relative standard deviation of particle size distribution of 0.30% or less. Claim 3 A hyaluronic acid powder according to claim 1 or 2, wherein the content ratio of the hyaluronic acid particles having a particle diameter of 100 μm or less is 0 mass% or more and 30 mass% or less based on the total mass of the hyaluronic acid powder. Claim 4 A hyaluronic acid powder according to claim 1 or 2, wherein the content ratio of the hyaluronic acid particles having a particle diameter of 100 μm or less is 0 mass% or more and 5 mass% or less based on the total mass of the hyaluronic acid powder. Claim 5 Hyaluronic acid powder according to claim 1 or 2, wherein the average molecular weight of the hyaluronic acid and / or its salt is 1.6 million or more. Claim 6 Hyaluronic acid powder according to claim 1 or 2, wherein the average molecular weight of the hyaluronic acid and / or its salt is 1.7 million or more. Claim 7 Hyaluronic acid powder according to claim 1 or 2, wherein the average molecular weight of the hyaluronic acid and / or its salt is 1.8 million or more. Claim 8 Hyaluronic acid powder according to claim 1 or 2, wherein the average molecular weight of the hyaluronic acid and / or its salt is 1.9 million or more. Claim 9 Hyaluronic acid powder according to claim 1 or 2, wherein the average molecular weight of the hyaluronic acid and / or its salt is 2.76 million or more. Claim 10 Hyaluronic acid powder according to claim 1 or 2, wherein the average molecular weight of the hyaluronic acid and / or its salt is 1.5 million or more and 3.9 million or less. Claim 11 A hyaluronic acid powder according to claim 1 or 2, wherein the content ratio of the hyaluronic acid particles having a particle diameter of 155 μm or less is 0 mass% or more and 55 mass% or less based on the total mass of the hyaluronic acid powder. Claim 12 A hyaluronic acid powder according to claim 1 or 2, wherein the content ratio of the hyaluronic acid particles having a particle diameter of 190 μm or less is 0 mass% or more and 60 mass% or less based on the total mass of the hyaluronic acid powder. Claim 13 A method for selecting raw hyaluronic acid powder used in a method for manufacturing cross-linked hyaluronic acid and / or its salt, wherein the raw hyaluronic acid powder contains hyaluronic acid particles, and the hyaluronic acid particles contain hyaluronic acid and / or its salt having an average molecular weight of 1.5 million or more, and the content ratio of the hyaluronic acid particles having a particle diameter of 100 μm or less is 0 mass% or more and 35 mass% or less based on the total mass of the hyaluronic acid powder.

Images