KR102577433B1 - A production method of hyaluronic acid bead for cosmetic use - Google Patents

Abstract

The present invention is a method for producing hyaluronic acid beads, comprising: (a) adding hyaluronic acid or a salt thereof to an aqueous alkaline solution, adding a cross-linking agent, and reacting the homogeneously mixed reactants; (b) a cross-linking reaction step of stirring the reactant with oil to form a cross-linking covalent bond; (c) purifying the product obtained in the crosslinking reaction step; and (d) mixing the active ingredients.
According to the hyaluronic acid bead manufacturing method of the present invention, it is possible to manufacture hyaluronic acid spherical beads that are transparent, highly swellable, and have a uniform spherical shape. Such hyaluronic acid spherical beads have a transparent spherical shape and have a selected size, decomposition period, viscoelasticity, It exhibits a degree of swelling, has excellent biocompatibility, and is very stable against heat and enzymes.
According to the hyaluronic acid bead manufacturing method of the present invention, the stability of the active ingredient is increased, the skin beauty effect is maximized, and the user's purchase psychology is improved. It's about.

Description

Method for manufacturing hyaluronic acid beads for cosmetic use {A PRODUCTION METHOD OF HYALURONIC ACID BEAD FOR COSMETIC USE}

The present invention relates to a method of producing hyaluronic acid beads that are transparent, highly swellable, and have a uniform spherical shape. In detail, it relates to a method of manufacturing spherical beads containing active ingredients that can satisfy aesthetics to increase the stability of the active ingredients, maximize the skin care effect, and enhance the purchasing psychology of users.

The spherical beads of the present invention are transparent, have a high degree of swelling, and have a uniform spherical shape. By adjusting the ratio of oil and hyaluronic acid solution without using emulsifiers, surfactants, solubilizers, etc., beads of various sizes can be stably obtained. In order to maintain the spherical shape in the acquisition stage, methods such as adding synthetic polymers such as shell-forming materials such as PLGA and PCL, producing an insoluble precipitate through ionic bonding, or precipitating in an organic solvent are not used, and without additives. The spherical shape is maintained through a two-step crosslinking reaction in a hyaluronic acid solution dispersed in an oil phase. In addition, it relates to a manufacturing method by applying a two-step purification method and an active ingredient mixing step to remove impurities such as oil and unreacted substances and produce a transparent bead gel. The transparent spherical shape of the present invention having these characteristics can be used in various ways as a material for cosmetic formulations.

Hyaluronic acid (HA), Hyaluronan, (C14H20NNaO11)n (n>1000)) is a polymer that exists in living organisms and is a polysaccharide called glycosaminoglycan. Its structure consists of D-glucuronic acid and N-acetylglucosamine repeatedly linked through β-1,3 and β-1,4 bonds. In addition, it is a water-soluble substance, has very high viscosity and high elasticity characteristics, and is a wide range of straight-chain polysaccharides with a molecular weight ranging from 1,000 to 10,000,000 Da (daltons).

Hyaluronic acid has a strong moisturizing effect, has excellent lubricating properties under physical friction, and has desirable advantages in terms of efficacy and physical properties, such as a protective effect against invasion by bacteria, etc., so it has a wide range of applications for beauty and medical purposes. Have.

To develop such hyaluronic acid, biological tissue extraction methods or microbial culture methods are basically used, but recently, due to many disadvantages such as virus invasion, impurities, and inflammatory reactions, microorganisms that can control molecular weight and productivity and obtain high-quality raw materials are used rather than chicken comb extraction methods. Culture production methods are becoming popular.

In particular, the recent trend is that development uses are determined according to the molecular weight range of hyaluronic acid produced by controlling microbial culture. In other words, ultra-low molecular weight hyaluronic acid with an average molecular weight of 100,000 Da or less is mainly used for food or cosmetic purposes, and low molecular weight hyaluronic acid with an average molecular weight of 1 million Da is also used as a raw material for eye drops or for the development of derivatives, and hyaluronic acid with an average molecular weight of 3 to 4 million Da is used as a raw material for eye drops or derivatives. Hyaluronic acid has high utility as a raw material for knee joint injections.

In addition, its use as an ophthalmic surgical aid is increasing, and as an ultrapolymer in the body, it is attracting attention as a raw material for anti-adhesion agents.

However, hyaluronic acid is easily decomposed in vivo or under acid, alkali, etc. conditions, and has poor processability, which limits its development for various uses. Therefore, efforts are being made to develop materials that structurally stabilize hyaluronic acid. (Laurent, T.C. "The chemistry, biology and medical applications of hyaluronan and its derivatives" Portland Press Ltd, London, 1998).

Commonly known methods for producing beads composed of cross-linked hyaluronic acid include mixing chemical and physical methods. Methods for separating the beads include sprinkling them through a fine nozzle or lipidizing and encapsulating the outside. Methods for dispersing solubilized biodegradable polymers using surfactants and then obtaining them through solvent extraction and evaporation are known.

In order to increase the uniformity of particles by physical methods, it is affected by the shape of the impeller and the stirring speed, but in most cases, a high stirring speed must be maintained. The method using a fine nozzle creates an appearance similar to a pomegranate fruit and does not maintain a perfect spherical shape. If a spherical bead is obtained through lipidizing and encapsulating the outside, it is broken by physical external force, and the liquid inside flows out, and the solvent When using the extraction and evaporation method, surfactants and organic solvents that are harmful to the human body are inevitably used, so complete removal of the final product is necessary.

Other common methods of producing beads composed of cross-linked hyaluronic acid include using an excessive amount of a cationic material to form an insoluble precipitate through ionic bonding with anionic hyaluronic acid, or using an excessive amount of a cross-linking agent to create an insoluble form. Dry and physically form particles, or bind a hydrophobic substance to the carboxyl group of hyaluronic acid and dissolve it in an organic solvent, then form microbeads using an emulsifier in two different solvents where the phases are separated, and then use solvent extraction, etc. to form microbeads. A method to retrieve them is emerging. For example, U.S. Patent Nos. 6,066,340 and 6,039,970 use hyaluronic acid derivatives synthesized by combining ethyl alcohol or benzyl alcohol with the carboxyl group of hyaluronic acid. This substance is insoluble in water but soluble in organic solvents such as dimethyl sulfoxide. There are examples of solid-phase microbeads produced using such hydrophobic hyaluronic acid derivatives by emulsion solvent extraction.

In addition, research is being conducted on manufacturing beads with chitosan and using them as a drug delivery medium (S. T. Lim, G. P. Martin, D. J. Berry and M. B. Brown, Journal of Controlled Release, 2-3, 66(2000), 281-292, S. T. Lim, B. Forbes, D. J. Berry, G. P. Martin and M. B. Brown, International Journal of Pharmaceutics, 1, 231 (2002), 73-82).

In addition, in literature related to the production of hyaluronic acid beads cross-linked by 1,3-butadiene diepoxide, a mixture of hyaluronic acid and vegetable oil is stirred in an aqueous alkaline solution, and then 1,3-butadiene diepoxide is added to perform a cross-linking reaction. By applying an excess of 0.4 mL of 3-butandiene diepoxide (about 800 mol% compared to the cross-linking reaction site) to 100 mg of ronic acid, the swelling (%) was around 500%, so it was too hard and had an uneven shape, and an excessive amount of cross-linking agent was used. Due to this, it has limitations in application fields (Polymer (Korea), Vol.29, No.5, pp445-450, 2005).

In addition, a method of producing hyaluronic acid microbeads is made by dropping drops of an aqueous hyaluronic acid solution through a nozzle under the pressure of compressed air through a microbead generator into a stirring cross-linking solution, using divinyl sulfone as a cross-linking agent. Problems with safety may arise, problems such as uneven shape and poor uniformity between particles, and the need to use expensive equipment exist (Biomaterials Research (2009) 13(3): 105-108, Biomaterials Research (2010) 14(4): 157-160, Chun et al , Biomaterials Research (2016) 20:24, microbeads based on polymer polysaccharides and their manufacturing method (Domestic Patent Registration No. 1005489650000 (2006.01.26)).

In addition, this method of producing solid bead-shaped emulsion beads by dropping them into a frozen refrigerant at -10℃ to -200℃ contains a large number of ingredients and does not require a purification process, which limits its use in applications (bead-shaped emulsion by low-temperature cooling) Bead manufacturing method and obtained bead-shaped emulsion beads, domestic patent registration number 1015532330000 (2015.09.09).

In addition, in the literature related to the production of hyaluronic acid beads cross-linked by BDDE, after dissolving hyaluronic acid in an aqueous alkaline solution overnight at 10°C to lower the molecular weight, an excess amount of BDDE (20 mg of BDDE per 100 mg of hyaluronic acid, approx. After adding olive oil (about 40 mol%), stirring reaction with olive oil, recovery through filer, and purification with 70% acetone aqueous solution, distilled water (DW), and PBS, the product has a high chemical strain of close to 20 to 30%. With a low swelling degree of 18.99 ~ 23.02, it is too hard and has an uneven shape, showing limitations in its use in application fields ( ACS Omega 2019, 4, 9, 13834-13844, Hyaluronic Acid-Based Hybrid Hydrogel Microspheres with Enhanced Structural Stability and High Injectability).

In addition, a cross-linking reaction is performed with a group of ligands consisting of alendronate (ALD), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) as a cross-linking agent to produce cross-linked hyaluronic acid, which is then dissolved and dropped onto polyvalent cations to form nano / This is a method of manufacturing microbeads, but its use in applications is limited due to safety and stability issues (Method for manufacturing hyaluronic acid microbeads and uses of the hyaluronic acid microbeads, Domestic Patent Registration No. 1021085520000 (2020.04. 29)).

Meanwhile, as an example of a method of manufacturing bead-type cosmetics using hyaluronic acid as a raw material, hyaluronic acid is mixed with hydrophilic polymers and low molecules, dropped into liquid nitrogen to produce spherical solids, and freeze-dried to improve the purchasing psychology of cosmetic users. There is a porous spherical freeze-dried formulation that can be improved (Method for manufacturing porous spherical freeze-dried products and cosmetic compositions using the same, domestic patent registration number 1016784740000 (2016.11.16)).

In addition, there is a method of producing beads through ionic bonding by dropping a mixed solution of alginic acid and hyaluronic acid into a solution of calcium, magnesium, barium, etc. (Mask beads and their manufacturing method, domestic patent registration number 1021624530000 (2020.09.25)).

In addition, in order to provide users with visual enjoyment, aesthetic sensibility, and the pleasure of makeup, an emulsion is prepared with a liquid anionic material at a temperature below 10°C, and a cationic high-frequency material is added to form emulsion beads through ionic bonding. There is a method for manufacturing (emulsion beads and their manufacturing method, domestic patent registration number 1016696530000 (2016.10.29)).

However, hyaluronic acid is a material with a fairly high hydrophilicity, and there have been limitations in producing transparent, highly swellable, but uniform spherical beads using the generally known method. Hyaluronic acid in aqueous solution in the oil phase has a very high hydrophilicity, so it is difficult to remove moisture by solvent evaporation, and when organic solvent is added by solvent extraction, the particles tend to clump together, forming beads of uniform size. It could not be manufactured. In addition, the method for producing hyaluronic acid beads known to date is to use an excessive amount of a cationic material to form an insoluble precipitate through ionic bonding with anionic hyaluronic acid, or to make an insoluble precipitate using an excessive amount of cross-linking agent and then dry it to physically form it. These were produced by granulating them into particles, or by combining a hydrophobic substance with the carboxyl group of hyaluronic acid and dissolving it in an organic solvent, followed by solvent extraction. The particles were quite small in size, had a non-uniform shape, and had a low degree of swelling in an aqueous solution. As it is a very hard and opaque particle form, it has shown limitations in application fields.

Due to the unique material properties of hyaluronic acid as described above, it was not possible to manufacture beads with a uniform spherical shape and transparency, but with a very high degree of swelling in an aqueous solution, using known manufacturing methods.

In addition, it was not possible to manufacture bead-type aqueous cosmetic formulations other than ionic bonding or freeze-drying methods.

Therefore, the purpose of the present invention is to provide hyaluronic acid spherical beads that are transparent, have a high degree of swelling, and have a uniform spherical shape that can solve the problems of the prior art.

In other words, the purpose of the present invention is to control the uniform spherical shape and size of hyaluronic acid spherical beads through cross-linking, which have high swelling properties in aqueous solutions and excellent physical stability, thereby providing excellent biocompatibility in various environments inside and outside the body, so that they can be used as medical devices. , to provide spherical hyaluronic acid beads that are transparent, highly swellable, and uniformly spherical, which can be usefully used in the pharmaceutical, cosmetics, and food industries.

Another object of the present invention is to provide a method for manufacturing such hyaluronic acid spherical beads through a highly efficient two-step reaction process using a small amount of cross-linking agent to form a sphere, a two-step purification process, and an active ingredient mixing step. It is done. This includes existing methods of lipidizing and encapsulating the outside of a spherical bead, using emulsifiers and surfactants, using an excessive amount of cross-linking agent to create an insoluble form and drying it to physically particleize, and hydrophobic materials. Since it does not involve phase separation after combining and dissolving in an organic solvent, it is suitable for providing transparent, high-purity hyaluronic acid spherical beads.

In order to achieve this goal, the present inventors, after extensive research and numerous experiments, adjusted the size of the beads through the relative ratio of hyaluronic acid solution and oil without using emulsifiers or surfactants, and used a small amount of cross-linking agent. It was manufactured through highly efficient chemical bonding through a step cross-linking reaction, a two-step purification process, and a mixing step of active ingredients. The hyaluronic acid spherical beads not only have a transparent spherical shape with high purity and uniform and selective size, but also have the characteristic of not decomposing even under sterilization conditions at 121 degrees Celsius, and are confirmed to have excellent biocompatibility, viscoelasticity, swelling, and stability. This led to completion of the present invention.

In order to solve the above technical problem, the present invention is a method for producing hyaluronic acid beads, comprising: (a) adding hyaluronic acid or a salt thereof to an aqueous alkaline solution, adding a cross-linking agent, and reacting the homogeneously mixed reactants; (b) a cross-linking reaction step of stirring the reactant with oil to form a cross-linking covalent bond; (c) purifying the product obtained in the crosslinking reaction step; and (d) mixing the obtained product with an active ingredient. It provides a method for producing homogeneous spherical hyaluronic acid beads.

As an embodiment of the present invention, a manufacturing method is provided wherein hyaluronic acid beads contain effective ingredients such as moisturizing, whitening, and wrinkle improvement.

As another embodiment of the present invention, the production method of the present invention is characterized by not using emulsifiers, chemical surfactants, and chemical solubilizers.

As another embodiment of the present invention, the production method of the present invention is characterized in that in step (a), the cross-linking agent is added in an amount of 1% to 30 equivalent% based on the repeating unit of hyaluronic acid or a salt thereof.

If the cross-linking agent is added in less than 1 equivalent % based on the repeating unit of hyaluronic acid or its salt, it has the disadvantage of being easily decomposed and unable to maintain its shape. If added in more than 30 equivalent %, the use of a high cross-linking agent causes The frequency of side effects increases and it is also undesirable in terms of ease of use due to its hard properties with low swelling.

As another embodiment of the present invention, the concentration of hyaluronic acid or its salt in the reactant in step (a) may be 1 to 30% by weight, and more preferably 5 to 20% by weight, based on the 0.1 to 10 N aqueous alkaline solution. do.

If the concentration of hyaluronic acid or salt is less than 1% by weight relative to an aqueous alkaline solution of 0.1 to 10 N, it is undesirable because crosslinking does not work properly and it is difficult to maintain the shape. If it exceeds 30% by weight, the concentration is too high, making homogeneous mixing difficult and resulting in unevenness. This is undesirable because a reaction occurs and too high crosslinking results in hard properties.

As another embodiment of the present invention, step (a) may be performed at a reaction temperature of 5 to 50°C and a reaction time in the range of 30 minutes to 24 hours.

As another embodiment of the present invention, step (b) may be performed at a reaction temperature of 5 to 50°C, a reaction time of 1 hour to 96 hours, and a stirring speed of 100 rpm to 2,000 rpm. desirable.

As another embodiment of the present invention, in step (b), the weight ratio of the alkaline aqueous solution of hyaluronic acid or its salt to which a crosslinking agent is added and the oil may be 1:1 to 1:9.

After covalent cross-linking in step (c), various types of organic solvents are used to precipitate the cross-linked product to remove remaining moisture, and the cross-linked hyaluronic acid beads are recovered by washing several times to remove oil. .

In another embodiment of the present invention, the crosslinking agent is butanediol diglycidyl ether (1,4-butandiol diglycidyl ether, BDDE), ethylene glycol diglycidyl ether (EGDGE), and hexane die. All diglycidyl ether (1,6-hexanediol diglycidyl ether), propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, poly polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycerol Diglycerol polyglycidyl ether, glycerol polyglycidyl ether, tri-methylpropane polyglycidyl ether, bisepoxypropoxyethylene (1,2-(bis(2, One or more selected from the group consisting of 3-epoxypropoxy)ethylene), pentaerythritol polyglycidyl ether, and sorbitol polyglycidyl ether can be used.

In another embodiment of the present invention, the oil is mineral oil, medium chain triglycerides, menhaden oil, linseed oil, coconut oil. ), cod oil, fish oil, soya bean oil, whale oil, cotton seed oil, sesame seed oil, peanut oil , olive oil, lard oil, palm oil, corn oil, rapeseed oil, bone oil, china wood oil and One or more selected from the group consisting of castor oil may be used.

In another embodiment of the present invention, the purification step includes (a) recovering the hyaluronic acid bead gel that has undergone the crosslinking reaction step; (b) a first purification step of washing the recovered spherical gel using at least one selected from distilled water, an acidic solution, an aqueous solution containing an acid salt, and PBS; (c) a second purification step of washing the hyaluronic acid bead gel that has undergone the first purification step with an aqueous organic solvent solution; and (d) separating and recovering the hyaluronic acid beads obtained in the second purification step and mixing the active ingredients.

Hyaluronic acid or its salt cross-linked product, which has been cross-linked to a certain level in the purification step, is washed several times in step (b) with distilled water, an acidic solution, an aqueous solution with added salt, and PBS to remove unreacted cross-linking agent and hydroxide ions.

After precipitating the bead gel purified in step (b) again in an organic solvent, in step (c), it was swollen in an organic solvent aqueous solution for a certain period of time, and then washed several times with an organic solvent to remove the oil, unreacted crosslinking agent, and Remove impurities.

Finally, in step (d), the active ingredients are mixed to prepare hyaluronic acid beads.

As another embodiment of the present invention, the organic solvent may be one or more selected from DMSO, DMF, acetonitrile, methanol, THF, acetone, aqueous acetone solution, and alcohols having 2 to 6 carbon atoms.

As another embodiment of the present invention, the aqueous organic solvent solution used in the swelling step preferably has a weight ratio of distilled water to the organic solvent of 9:1 to 1:9.

As another embodiment of the present invention, it is preferable to include ingredients used for moisturizing, whitening, and wrinkle improvement in the step of mixing the active ingredients.

As described above, when manufacturing hyaluronic acid spherical beads, the size and physical properties of the hyaluronic acid spherical bead gel can be adjusted by controlling the types and relative ratios of various types of oils and aqueous solutions and oils without using an emulsifier in the oil phase.

In addition, through a two-step crosslinking reaction, not only can high crosslinking efficiency and yield be obtained while using a small amount of crosslinking agent, but also the transparency and physical properties of hyaluronic acid spherical beads can be controlled through a two-step purification method.

The hyaluronic acid spherical beads produced through the present invention have a transparent spherical shape, exhibit a selected size, decomposition period, and viscoelasticity, have excellent biocompatibility, and are very stable against heat and enzymes.

Figure 1 is a view showing transparent, spherical hyaluronic acid beads swollen in a moisturizing, whitening, and wrinkle-improving active ingredient solution in a cosmetic container.
Figure 2 is a view showing transparent, spherical hyaluronic acid beads swollen in a moisturizing, whitening, and wrinkle-improving active ingredient solution in a container.
Figure 3 is a view showing transparent, spherical hyaluronic acid beads swollen in a moisturizing, whitening, and wrinkle-improving active ingredient solution in a container.
Figure 4 is a view using an optical microscope to show that hyaluronic acid beads, which are transparent and have a spherical shape, are swollen in a solution of moisturizing, whitening, and wrinkle-improving active ingredients.

In the present invention, when manufacturing hyaluronic acid spherical beads, the bead size is controlled by the type and relative ratio of the hyaluronic acid solution and oil without using an emulsifier in the oil phase, and chemically combined with a small amount of cross-linking agent (polyfunctional epoxide) having two or more epoxy reactors. Provided is a bound hyaluronic acid spherical bead gel and a method for manufacturing the same.

The size of the hyaluronic acid spherical beads is not particularly limited, but the average diameter of the fine particles is preferably 100 to 5,000 um. The application range can be varied depending on the size of the hyaluronic acid spherical bead gel.

In the present invention, the term hyaluronic acid briefly expresses the form of the acid and its salt. The hyaluronic acid salt includes sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, and cobalt hyaluronic acid. It includes both inorganic salts such as tetrabutylammonium hyaluronic acid and organic salts such as tetrabutylammonium hyaluronic acid.

In order to form hyaluronic acid spherical beads that are transparent, highly swellable, and uniform, and have various physical properties and biocompatibility, the molecular weight of the hyaluronic acid or its salt is not particularly limited, and is preferably 100,000 to 5,000,000 daltons. .

The production method according to the present invention includes a first reaction step in which hyaluronic acid or its salt is added to an aqueous alkaline solution and a small amount of cross-linking agent is added at the same time and mixed uniformly to react, and a second reaction step is carried out by dispersing the reaction mixture on various types of oils. Cross-linking reaction step, a first purification step in which the reactants that have undergone this reaction are recovered and purified with distilled water, an acidic solution, or an aqueous solution with added salts and PBS to remove unreacted substances and impurities, and a second purification step in which the reactants that have undergone this reaction are purified with an aqueous organic solvent solution. Includes steps. It also includes the step of mixing active ingredients.

The hyaluronic acid spherical beads of the present invention may further contain other ingredients within the range that do not impair the effect of the present invention.

As described above, the hyaluronic acid used in this specification is a concept that includes both the acid form and its salt, and therefore, in the production method of the present invention, “hyaluronic acid aqueous solution” refers to an aqueous solution of hyaluronic acid, an aqueous solution of a hyaluronic acid salt, and It contains both a mixed aqueous solution of hyaluronic acid and hyaluronic acid salt.

The alkaline reagent used to prepare the aqueous alkaline solution dissolving the hyaluronic acid is not particularly limited, but preferred examples include reagents such as sodium hydroxide, potassium hydroxide, and aqueous ammonia. The concentration of the alkaline aqueous solution is not particularly limited, and a concentration of 0.1 to 10N is preferable for dissolution and crosslinking reaction of hyaluronic acid.

The cross-linking agent reacts with the alcohol group of hyaluronic acid or its salt to form an ether bond, thereby forming a cross-linking between hyaluronic acid or its salt. It is known that the reaction between an epoxy functional group and alcohol proceeds at high temperature and in an alkaline state, and as the reaction progresses, it is attached to hyaluronic acid or its salt, so no by-products are generated. However, if the reaction efficiency is low, unreacted epoxy functional groups among the various epoxy functional groups of the cross-linking agent bound to hyaluronic acid or its salt remain in the hyaluronic acid or its salt, and this remaining amount of unreacted epoxy functional groups may be used with in vivo materials in the future. There is a risk of reaction, so additional reactions and processes are required to minimize this or to hydrolyze the epoxy functional group and convert it into an inert functional group. Additionally, when an excessive amount of cross-linking agent is added, there is a very high risk that unreacted cross-linking agent will remain in the cross-linking material of hyaluronic acid or its salt. Therefore, in order to minimize this risk, it is highly desirable not only from the viewpoint of biocompatibility of hyaluronic acid spherical beads to induce a highly efficient reaction using an appropriate amount of cross-linking agent as described in the present invention, but also from the viewpoint of manufacturing process efficiency.

The aqueous solution of hyaluronic acid or its salt, in which the cross-linking agent is uniformly mixed, is mixed with various types of oils, then stirred appropriately and subjected to a cross-linking reaction at a certain temperature. The alkaline aqueous solution of hyaluronic acid or its salt dispersed in the oil phase gradually changes to a high concentration as the solvent evaporates. As a result, the actual reaction concentration increases and the efficiency of the reaction can be maximized. By using a small amount of cross-linking agent, hyaluronic acid with excellent physical properties can be obtained. Spherical beads can be manufactured. Therefore, in the hyaluronic acid cross-linking reaction process described above, the cross-linking reaction takes place at a relatively high concentration as the cross-linking agent is mixed at a low concentration and the solvent evaporates. Therefore, there are advantages when a low concentration of the cross-linking agent is used and there are advantages when a high concentration of the cross-linking agent is used. You can take advantage of both at the same time. This method of manufacturing hyaluronic acid spherical beads can not only produce hyaluronic acid spherical beads that are sturdy and have special physical properties, but can also have cross-linking reaction efficiency.

In addition, when mixing the aqueous solution of hyaluronic acid or its salt containing the cross-linking agent with various types of oil, the mixing time and the ratio of oil to aqueous solution can be adjusted to adjust the physical properties of hyaluronic acid spherical beads.

In the absence of an emulsifier, the size of the hyaluronic acid spherical beads is controlled through the stirring speed, type of oil, and the ratio of oil to aqueous solution. As the viscosity of the oil phase increases, the size of the hyaluronic acid spherical beads becomes smaller. It is showing. This appears to be because the alkaline aqueous solution of hyaluronic acid and its salt, which has a relatively higher viscosity than the oil phase, is dispersed more smoothly as the difference in viscosity between the two phases is reduced.

Additionally, the size of the beads becomes smaller as the stirring speed increases and the viscosity of the alkaline aqueous solution of hyaluronic acid and its salt decreases.

After the covalent crosslinking, an organic solvent containing an alcohol having 2 to 6 carbon atoms or an aqueous alcohol solution such as DMSO, DMF, acetonitrile, methanol, THF, acetone, acetone aqueous solution, and ethanol can be used.

Through various experiments, the present inventors observed that the physical properties of the spherical hyaluronic acid beads prepared vary depending on the characteristics of the alkaline aqueous solution of hyaluronic acid or its salt mixed with the various organic solvents and oil phase described above, and that the polarity index of the organic solvent is that of distilled water. It was found that the closer to , the transparency of the hyaluronic acid spherical beads increased and the complex viscosity decreased, suggesting a method for producing hyaluronic acid spherical beads with various physical properties.

The temperature during the crosslinking reaction of the mixture is not particularly limited, but is preferably 5°C to 50°C, and more preferably 15 to 40°C. In addition, the duration for the creation of crosslinks in the reaction solution is not particularly limited, but there is a problem that the longer the reaction time, the lower the physical viscoelasticity of hyaluronic acid spherical beads prepared by hydrolyzing hyaluronic acid under alkaline conditions, so the reaction The time is preferably 30 minutes to 24 hours in the first stage and 1 hour to 96 hours in the second stage.

After the crosslinking reaction of hyaluronic acid or its salt, the hyaluronic acid spherical bead product is free of oil using an organic solvent. The organic solvent may be an alcohol or aqueous alcohol solution having 2 to 6 carbon atoms, such as diethyl ether, DMSO, DMF, acetonitrile, methanol, THF, acetone, aqueous acetone solution, and ethanol.

The hyaluronic acid spherical bead product from which the oil has been removed is washed several times in distilled water, an acidic solution, an aqueous solution with added salt, and PBS to remove unreacted cross-linking agents and hydroxide ions.

By using an aqueous solution containing the salt, the washing efficiency of unreacted substances can be increased. In this case, aqueous sodium chloride solution, aqueous phosphoric acid solution, and aqueous sodium chloride solution containing phosphate are most suitable.

The purified hyaluronic acid spherical beads are prepared by mixing active ingredients such as moisturizing, whitening, and wrinkle improvement, or recovered in a precipitated state in an organic solvent or aqueous organic solvent solution, dried through nitrogen, air, heat, and reduced pressure, and used for application purposes. Accordingly, hyaluronic acid spherical beads can be manufactured by separating them according to shape or size and re-swelling them in a solution of active ingredients for moisturizing, whitening, and wrinkle improvement.

The organic solvent used for the precipitation of the hyaluronic acid spherical bead gel may be an alcohol or an aqueous alcohol solution having 2 to 6 carbon atoms, such as DMSO, DMF, acetonitrile, methanol, THF, acetone, acetone aqueous solution, and ethanol.

Hyaluronic acid or its salt cross-linked material prepared by the present invention can be separated and/or purified in various ways by conventional methods known in the art, representative examples of which include distillation (distillation under atmospheric pressure and reduced pressure distillation). (including), recrystallization, column chromatography, ion exchange chromatography, gel chromatography, affinity chromatography, thin layer chromatography, phase separation, solvent extraction, dialysis, washing, etc. The separation and/purification may be performed after each process or after a series of reactions when manufacturing hyaluronic acid derivative microbeads.

The hyaluronic acid spherical beads of the present invention increase the stability of the active ingredients, maximize the skin beauty effect, and the spherical beads containing active ingredients that can satisfy the aesthetic sense to enhance the user's purchasing psychology are various cosmetic materials. It can be used as.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples only illustrate the present invention, and the content of the present invention is not limited by the following examples.

Example

[Example 1] Preparation of hyaluronic acid spherical beads

Sodium hyaluronate (molecular weight: 1,000,000) at a concentration of 50 mg/ml was added to 0.25 N 200mL NaOH solution, and 0.3024 g of butanediol diglycidyl ether (BDDE) was added at the same time (the ratio of BDDE to the hyaluronic acid repeating unit) was added at a concentration of 50 mg/ml. Equivalence ratio: 6 mol%). After mixing using a self-powered centrifugal mixer for 30 minutes, the first stage reaction was performed at 40°C for 1 hour. The above reactant was dropped onto 200 g of MCT oil. A two-step reaction was performed at 40°C for 24 hours while stirring at a speed of 700 rpm.

After 24 hours, 400 ml of isopropanol was slowly added to recover the hyaluronic acid beads dispersed in the mixed solution. Washed three times with isopropanol. The recovered hyaluronic acid beads were washed 5 times with 20L of PBS each. Add 5L of isopropanol to the washed hyaluronic acid beads to cause precipitation. Isopropanol replacement was repeated three times. Add 1.5L of 30% isopropanol to the precipitate and allow it to swell for 1 hour. After swelling, isopropanol replacement was repeated three times and recovered.

Some of the washed hyaluronic acid spherical beads contained glycerin at a concentration of 10 mg/mL, 1,3 butylene glycol at a concentration of 30 mg/mL, 1,2 hexanediol at a concentration of 20 mg/mL, niacinamide at a concentration of 20 mg/mL, and 0.4 mg/mL. After swelling in a 10L active ingredient solution with a high concentration of adenosine, hyaluronic acid spherical beads were manufactured, and some of the other hyaluronic acid spherical beads were re-swelled in an organic solvent precipitation process, nitrogen gas drying process, fractionation process, and active ingredient solution. , hyaluronic acid spherical beads were prepared.

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Claims (13)

Contains effective ingredients for moisturizing, whitening, and wrinkle improvement, and has an average diameter of 100 to 5,000um. A method for producing hyaluronic acid beads for cosmetic use, comprising:
(a) a reaction step of adding hyaluronic acid or its salt to an aqueous alkaline solution, adding a crosslinking agent, and reacting the homogeneously mixed reactants;
(b) a cross-linking reaction step of forming a cross-linking covalent bond by stirring the reactant with oil without using an emulsifier;
(c) purifying the product obtained in the crosslinking reaction step; and
(d) mixing the obtained product with active ingredients for moisturizing, whitening and wrinkle improvement,
A method for producing homogeneous spherical hyaluronic acid beads in which step (c) includes the following detailed processes.
(i) recovering the hyaluronic acid beads that have undergone the crosslinking reaction step;
(ii) a first purification step of washing the recovered spherical beads using one or more selected from distilled water, an acidic solution, an aqueous solution containing an acid salt, and PBS;
(iii) a precipitation step of precipitating the hyaluronic acid beads that have undergone the first purification step in an organic solvent;
(iv) a second purification step of swelling the hyaluronic acid beads that have undergone the precipitation step in an aqueous organic solvent solution, followed by washing several times with an organic solvent to remove oil, unreacted cross-linking agent, and impurities; and
(v) Separating and recovering the hyaluronic acid beads obtained in the second purification step.
delete According to paragraph 1,
In step (a), the cross-linking agent is added in an amount of 1 to 30 equivalent% based on the repeating unit of hyaluronic acid or a salt thereof.
According to paragraph 1,
In step (a), the concentration of hyaluronic acid or its salt in the reactant is 0.1 to 0.1.
Method for producing 1 to 30% by weight hyaluronic acid beads based on a 10 N aqueous alkaline solution.
According to paragraph 1,
Step (a) is a hyaluronic acid bead manufacturing method performed at a reaction temperature of 5 to 50°C and a reaction time of 30 minutes to 24 hours.
According to paragraph 1,
Step (b) is a hyaluronic acid bead manufacturing method performed at a reaction temperature of 5 to 50°C and a reaction time of 1 hour to 96 hours.
According to paragraph 1,
In step (b), the weight ratio of the alkaline aqueous solution of hyaluronic acid or its salt to which a crosslinking agent is added and the oil is 1:1 to 1:9.
delete According to paragraph 1,
The crosslinking agent is butanediol diglycidyl ether (1,4-butandiol diglycidyl ether, BDDE), ethylene glycol diglycidyl ether (EGDGE), and hexanediol diglycidyl ether (1, 6-hexanediol diglycidyl ether), propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytedramethylene glycol diglycidyl ether Diglycerol (polytetramethylene glycol diglycidyl ether), neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, Glycerol polyglycidyl ether, tri-methylpropane polyglycidyl ether, bisepoxypropoxyethylene (1,2-(bis(2,3-epoxypropoxy)ethylene), penta A method of manufacturing at least one hyaluronic acid bead selected from the group consisting of erythritol polyglycidyl ether (pentaerythritol polyglycidyl ether) and sorbitol polyglycidyl ether.
According to paragraph 1,
The oil includes mineral oil, medium chain triglycerides, menhaden oil, linseed oil, coconut oil, cod oil, fish oil, soya bean oil, whale oil, cotton seed oil, sesame seed oil, peanut oil, olive oil, Consists of lard oil, palm oil, corn oil, rapeseed oil, bone oil, china wood oil and castor oil. A method of manufacturing one or more hyaluronic acid beads selected from the group.
delete According to paragraph 1,
The organic solvent is one or more selected from DMSO, DMF, acetonitrile, methanol, THF, acetone, acetone aqueous solution, and alcohols having 2 to 6 carbon atoms.
According to paragraph 1,
A method of producing hyaluronic acid beads in which the aqueous organic solvent solution used in the swelling step has a weight ratio of distilled water to the organic solvent of 9:1 to 1:9.