Method for producing low-molecular-weight hyaluronic acid

Abstract

To provide a method that is capable of reducing the molecular weight of high-molecular-weight hyaluronic acid as a starting material without employing sodium hypochlorite.SOLUTION: A method for producing low-molecular-weight hyaluronic acid includes a step of applying ultrasonic irradiation to a hyaluronic acid aqueous solution to reduce the molecular weight of hyaluronic acid present in the hyaluronic acid aqueous solution, wherein the pressure applied to the hyaluronic acid aqueous solution in the step is greater than atmospheric pressure.SELECTED DRAWING: None

Description

[Background technology]

[0001] Hyaluronic acid can be broadly divided into high molecular weight hyaluronic acid and low molecular weight hyaluronic acid. High molecular weight hyaluronic acid is naturally occurring hyaluronic acid with a high molecular weight (usually over 500,000). On the other hand, low molecular weight hyaluronic acid is artificially purified hyaluronic acid with a low molecular weight. Here, low molecular weight hyaluronic acid is a component with various medical applications. For example, low molecular weight hyaluronic acid is useful as an agent that increases polysaccharide angiogenesis, as an agent that inhibits inflammatory progression as a specific inhibitor of TNF, as an osteogenic agent, and as an antiviral agent.

[0002] Here, Patent Document 1 proposes a method for preparing low-molecular-weight hyaluronic acid, in which high-molecular-weight hyaluronic acid, which is the starting material, is simultaneously treated with sodium hypochlorite and ultrasound. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 2000-502141 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a method for reducing the molecular weight of the starting material, high molecular weight hyaluronic acid, at low cost. [Means for solving the problem]

[0005] The present invention is a method for producing low molecular weight hyaluronic acid, comprising the step of irradiating ultrasonic waves to the hyaluronic acid contained in the hyaluronic acid aqueous solution to make the hyaluronic acid contained in the hyaluronic acid aqueous solution low molecular weight, characterized in that in the step, the pressure applied to the hyaluronic acid aqueous solution is higher than atmospheric pressure.Here, the hyaluronic acid aqueous solution can be substantially free of sodium hypochlorite. [Effects of the Invention]

[0006] According to the present invention, it is possible to provide a method that can reduce the molecular weight of high molecular weight hyaluronic acid, which is the starting material, at low cost.Furthermore, according to the present invention, low molecular weight hyaluronic acid can be produced in large quantities and safely.Furthermore, according to the present invention, the composition containing low molecular weight hyaluronic acid obtained by the method of the present invention is less likely to be mixed with impurities than other means, and therefore has the effect of being excellent in product safety. [Brief explanation of the drawings]

[0007] [Figure 1] 1 is a block diagram of an ultrasonic irradiation device according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0008] Specific embodiments of the present invention will be described below. However, the present invention is not limited to the following specific embodiments. In the following, "A to B" means A or more and B or less.

[0009] This embodiment is the method for producing low molecular weight hyaluronic acid, comprising the step of irradiating ultrasonic waves to hyaluronic acid aqueous solution, and the hyaluronic acid contained in this hyaluronic acid aqueous solution is made low molecular weight, and the pressure that is applied to this hyaluronic acid aqueous solution in this step is higher than atmospheric pressure.Below, raw material, process and final product are explained in this order.

[0010] ≪Raw materials≫ (Hyaluronic acid) The hyaluronic acid that is raw material is not particularly limited, and can be hyaluronic acid, acetyl hyaluronic acid, the derivative of hyaluronic acid or acetyl hyaluronic acid, or their salts.The molecular weight of hyaluronic acid in this form varies depending on the number of repeating units and type, and is not particularly limited (usually more than 500,000).Here, the "molecular weight" referred to in this specification is the weight-average molecular weight determined by size exclusion chromatography.

[0011] (solvent) The solvent of hyaluronic acid is not particularly limited, as long as it is the liquid medium that is mainly composed of water.Here, "main component" means that, based on the total mass of the liquid medium, water is 50 mass% or more (for example, 55 mass% or more, 60 mass% or more, 65 mass% or more, 70 mass% or more, 75 mass% or more, 80 mass% or more, 85 mass% or more, 90 mass% or more, 92.5 mass% or more, 95 mass% or more, 97.5 mass% or more, 98 mass% or more, 99 mass% or more, 99.5 mass% or more).

[0012] (Other ingredients) Hyaluronic acid aqueous solution may contain other components than hyaluronic acid and liquid medium.But, it is preferable that hyaluronic acid aqueous solution does not substantially contain sodium hypochlorite.In addition, in this specification, "does not substantially contain" means that it is not added intentionally, and for example, the content of hyaluronic acid aqueous solution is 0.1 mass % or less (for example, 0.05 mass % or less, typically 0.01 mass % or less) based on the total mass of hyaluronic acid aqueous solution.

[0013] <Process> The manufacturing method according to the present embodiment includes a step of irradiating an aqueous solution of hyaluronic acid with ultrasonic waves using an ultrasonic irradiation device. This step will be described in detail below.

[0014] (Device) 1, the ultrasonic irradiation device 10 includes a storage tank 1, which is a container for irradiating an aqueous hyaluronic acid solution with ultrasonic waves, and can circulate the degassed and pressurized aqueous hyaluronic acid solution in the storage tank 1. In detail, the ultrasonic irradiation device 10 further includes a degassing tank 31 for degassing the aqueous hyaluronic acid solution, a circulation pump 34 for circulating the aqueous hyaluronic acid solution, a cooler 35, which is a heat exchanger for controlling the aqueous hyaluronic acid solution to a predetermined temperature, a pressurizing device 36 for pressurizing the aqueous hyaluronic acid solution, and a tank 37 for holding the aqueous hyaluronic acid solution.

[0015] The degassing tank 31 can be of any type, as long as it can degas the hyaluronic acid aqueous solution. For example, a common type using hollow fibers is acceptable, but a tank with high degassing capacity is preferable. For example, the degassing tank 31 can be a type that performs both vacuuming and ultrasonic irradiation. In this case, a roughly cylindrical container is provided, and a liquid phase using a liquid medium and a gas phase for vacuuming are formed in the closed space inside. A vacuum pump 33 is attached to the upper side of the cylindrical container to communicate with the gas phase, and an ultrasonic vibrator 32 is attached to the lower side to irradiate the liquid phase with ultrasonic waves. This allows the degassing tank 31 to irradiate the hyaluronic acid aqueous solution with ultrasonic waves while evacuating it. Furthermore, by generating cavitation in the hyaluronic acid aqueous solution, the release of gas dissolved in the hyaluronic acid aqueous solution into the gas phase can be promoted. With this configuration, the degassing tank 31 has high degassing capacity. In addition, a discharge port 39 for discharging the hyaluronic acid aqueous solution to the outside of the ultrasonic irradiation device 10 is provided in the pipe for discharging the hyaluronic acid aqueous solution from the degassing tank 31 .

[0016] The circulation pump 34 circulates the hyaluronic acid aqueous solution degassed in the degassing tank 31 inside the ultrasonic irradiation device 10 by sending it to the cooler 35. As will be described later, the circulation pump 34 may also circulate the hyaluronic acid aqueous solution discharged from the storage tank 1 to the cooler 35.

[0017] The cooler 35 can control the temperature of the hyaluronic acid aqueous solution supplied to the storage tank 1 at a predetermined temperature. By keeping the temperature of the hyaluronic acid aqueous solution low and stable, the energy of the cavities generated by ultrasonic waves in the storage tank 1 can be kept high and stable. It is also preferable to perform heat exchange between the inlet and outlet paths of the hyaluronic acid aqueous solution to the cooler 35 to improve the heat exchange speed.

[0018] The pressurizing device 36 is connected to the pipeline between the cooler 35 and the reservoir 1 and can apply a predetermined pressure to the hyaluronic acid aqueous solution circulating in a liquid-tight state. For example, a pneumatic-hydraulic pressure converter equipped with an air-hydraulic pressure converter cylinder with gas and liquid phases that converts air pressure to hydraulic pressure can be used as the pressurizing device 36. The air-hydraulic pressure converter cylinder is a vertically placed cylinder approximately 50-100 mm in diameter and 200-300 mm long. It can be powered by compressed air, which is relatively readily available in factories, and pressure adjustment is easy. In this case, it is preferable to make the connecting path from the pressurizing device 36 to the pipeline thin and long, thereby preventing the hyaluronic acid aqueous solution with dissolved air in the air-hydraulic pressure converter cylinder from flowing into the reservoir 1. A pump can also be used as the pressurizing device, but a pressure regulator such as a pressure reducing valve is required.

[0019] The tank 37 stores the aqueous hyaluronic acid solution under atmospheric pressure to be circulated within the ultrasonic irradiation device 10. The tank 37 is piped so that it can receive the aqueous hyaluronic acid solution from the storage tank 1 or the cooler 35 and supply the aqueous hyaluronic acid solution to the degassing tank 31, and is connected to a liquid supply port 38 that receives the aqueous hyaluronic acid solution from outside the ultrasonic irradiation device 10.

[0020] (ultrasound) The frequency of the ultrasonic waves applied in this step is not particularly limited and may be, for example, 19.0 KHz to 25 KHz. The ultrasonic power density of the ultrasonic waves applied in this step is not particularly limited and may be, for example, 100 W / L or more. The duration of ultrasonic wave application is not particularly limited and may be, for example, 60 to 240 minutes.

[0021] (pressure) The pressure that is applied to hyaluronic acid aqueous solution is not particularly limited as long as it is higher than atmospheric pressure (for example, standard atmospheric pressure=101325 Pascal), and is preferably 0.01MPa or higher, 0.015MPa or higher, 0.02MPa or higher, 0.025MPa or higher, 0.5MPa or lower, 0.25MPa or lower, 0.15MPa or lower, 0.1MPa or lower, 0.075MPa or lower, 0.05MPa or lower, higher than atmospheric pressure.When ultrasonic wave is applied under pressure higher than atmospheric pressure, it can generate the powerful shock wave that can realize the degree of breaking down hyaluronic acid into smaller molecules.

[0022] (dissolved oxygen content) The dissolved oxygen content of hyaluronic acid aqueous solution is preferably 1.5mg / L or less.When the dissolved oxygen content is within the suitable range, in the cavitation phenomenon that occurs due to ultrasonic vibration, the shape of the cavity that is generated by negative pressure becomes closer to spherical.Therefore, when this cavity is compressed and disappears, it can generate a higher shock wave.As a result, it can efficiently decompose hyaluronic acid into low molecular weight.

[0023] (temperature) The temperature (water temperature) of the hyaluronic acid aqueous solution is preferably 4°C to 9°C. Within this range, a stronger shock wave can be generated when the cavities generated by the cavitation phenomenon are compressed and disappear. As a result, the molecular weight of the hyaluronic acid can be efficiently reduced.

[0024] Final product The low molecular weight hyaluronic acid obtained by the production method of this embodiment is not particularly limited as long as it has a lower molecular weight than the raw material high molecular weight hyaluronic acid, for example, a molecular weight of 300,000 or less, 100,000 or less, 30,000 or less, 10,000 or less, 3,000 or less, or 1,000 or less. [Example]

[0025] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0026] Using the same hyaluronic acid aqueous solution as the starting material, the molecular weight of hyaluronic acid was measured under the following conditions. XL The analysis was carried out by size exclusion chromatography using a column. Specifically, the standard solution {Shodex Standard P-82 (Showa Denko K.K.)} and the test solution were injected into a high-performance liquid chromatograph using a size exclusion column. The results were analyzed using a 480II Data Station GPC program (System Instruments Co., Ltd.). The high-performance liquid chromatograph operating conditions were as follows: Model: Shodex GPC-101 Detector: Parallax refractometer RI-71S (Showa Denko K.K.) Column: TSKgel GMPW XL Two columns {φ7.8mm x 300mm (Tosoh Corporation)} connected together Column temperature: 40℃ Mobile phase: 0.1 mol / l sodium nitrate solution Flow rate: 1.0ml / min Injection volume: 100μl

[0027] (Test Solution / Example 1) Ultrasonic frequency: 20KHz Ultrasonic bath diameter: 100φ Ultrasonic power output: 160W Ultrasonic power density: 128W / l Temperature: 6℃ Dissolved oxygen content: 0.8 mg / l Ultrasonic irradiation time: 6 hours Pressure: atmospheric pressure + 0.05 MPa (Test Solution / Example 2) Same as Example 1 except that the pressure was atmospheric pressure + 0.15 MPa (Test Solution / Comparative Example 1: Sample 1) No ultrasound (Test Solution / Comparative Example 2: Sample 2) Same as Example except that the pressure was atmospheric pressure

[0028] The results are shown in Table 1 (sample 1, sample 2), Table 2 (Example 1) and Table 3 (Example 2). As is clear from these results, when the water pressure in the ultrasonic bath is increased and made higher than atmospheric pressure, hyaluronic acid is decomposed and low molecular weight hyaluronic acid is produced (Examples 1 and 2 in Table 2 and Table 3). Furthermore, when ultrasonic waves are applied at atmospheric pressure, there is no change in molecular weight (sample 1 and sample 2 in Table 1).

[0029] [Table 1]

[0030] [Table 2]

[0031] [Table 3] [Industrial Applicability]

[0032] The present invention is useful in the fields of cosmetics, quasi-drugs, pharmaceuticals, supplements, foods, etc., which utilize low-molecular-weight hyaluronic acid.

Claims

1. A method for producing low-molecular-weight hyaluronic acid, comprising the step of irradiating an aqueous solution of hyaluronic acid with ultrasound to reduce the molecular weight of the hyaluronic acid contained in the aqueous solution of hyaluronic acid, In the above step, the pressure applied to the aqueous hyaluronic acid solution is higher than atmospheric pressure. A manufacturing method characterized by:

2. The method according to claim 1 , wherein in the step, the pressure is applied to the aqueous hyaluronic acid solution while the aqueous hyaluronic acid solution is in a liquid-tight state.

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