JP6369207B2 - Method for producing nanoparticles based on natural product-derived components - Google Patents

Description

  The present invention relates to a method for producing nanoparticles comprising at least one animal protein selected from gallate catechins, gelatin, collagen, and degradation products thereof, and more specifically, optimization of the weight ratio of mixing these and The present invention relates to a method for producing nanoparticles having an average particle size of 100 nm or less by a simple mixing operation by selecting pH.

  In recent years, nanoparticulate technology for materials has been studied in various fields and is expected to be widely used. In particular, active investigations have been made in the pharmaceutical and cosmetic fields, and many reports have been issued.

  The drug field that has been especially studied so far has been the focus of attention on drug delivery systems (DDS) for providing pharmaceutical ingredients to target organs and tissues by nanoparticulation. For example, in cancer tissue, since angiogenesis is thriving, gaps are formed in blood vessels, and the use of nanoparticles has been considered for DDS and the like using the size of the gaps. Thereby, not only can the pharmaceutical ingredients be stably and efficiently transported, but also side effects can be reduced. For DDS in the pharmaceutical field, for example, base materials such as liposomes and polyethylene glycol have been studied (Non-patent Document 1). The size of nanoparticles suitable for use as an anticancer agent was 100 to 200 nm, and development at this size was active. However, since the production of nanoparticles based on these materials has used many components and solvents such as synthetic products, its use is limited to pharmaceuticals and is hardly used for foods.

  There are also reports on nanotechnology in the food sector. In the field of nano-particles in the food field, developments have been made with the aim of improving texture and taste, releasing flavors, improving solubility and transparency, absorbability and reactivity. However, as a matter of fact, nano-technology in the food field often includes micro-sizes close to nano-size, and sometimes included up to 100 μm. However, there is a report that the size at which absorption into the body is actually improved is 100 nm or less (Non-patent Document 2).

  So far, in the field of food and food additives, for example, methods for producing nanoparticles using chitosan (Patent Documents 1, 2, and 3) have been reported, and silica, nanoclay, liposome, platinum nanoparticles, etc. Has also been reported (Non-Patent Document 3). In the case of Patent Document 1, nanoparticles are obtained by a method in which chitosan is dissolved after being dissolved with an acid, and the average particle diameter is 800 nm to 3100 nm. In the case of Patent Document 2, particles of 100 nm or less are formed. However, in order to pass through the microchannel, a specialized apparatus is required for the production. In Patent Document 3, nanoparticles are obtained by mixing chitosan and tannin, but the particle diameter is not 100 nm or less. Furthermore, although these have been used for food as nanoparticles, they have strong properties as carriers and little attention has been paid to their own functionality.

  There is also a report on polyphenol encapsulation technology (Non-Patent Document 4). However, most of these reports are micro-scale and are considered to have different properties from nanoparticles.

  From the above, the prepared nanoparticles themselves have effective functionality, and the nanoparticle material is derived from natural products, has high safety, can be used for food, and has a size of 100 nm or less. Few particles have been reported.

  By the way, there are four types of gallate catechins mainly contained in natural products such as tea: epigallocatechin gallate (EGCg), epicatechin gallate (ECg), gallocatechin gallate (GCg), and catechin gallate (Cg). In addition, it has excellent functions such as an antioxidant action, an anti-obesity action, an anti-cancer action, an antibacterial action, and an anti-inflammatory action.

Among them, EGCg has attracted attention because of its large content, and many reports have been made. On the other hand, ECg has been reported to be particularly useful for ECg. For example, in methicillin-resistant Staphylococcus aureus, it has been reported that the effect of changing the cell wall by a synergistic effect with β-lactam antibiotics is superior to other gallate catechins (Non-Patent Document 5). In addition, the present inventors have found and reported that ECg is particularly useful as an antagonist of LOX-1 (lectin-like oxidized low density lipoprotein receptor) (Patent Document 4). Moreover, there is a report that ECg and ECg are more likely to interact with lipid bilayer membranes (Non-patent Document 6). In human tests, it has also been reported that ECg is more easily taken into blood (Non-patent Document 7).
From the above, it is clear that ECg, which is a gallate catechin, is a very useful compound.

However, gallate type catechins EGCg, ECg, Cg and GCg have a bitter taste and astringency stronger than non-gallate catechins such as EGC and EC, although there is no great difference in bitterness and astringency between them. In particular, the bitterness and astringency threshold concentrations of EGCg and ECg are about three times lower (Non-patent Document 8).
Therefore, when adjusting green tea drinks or catechin-containing foods, the control of bitterness and astringency is a major problem for those skilled in the art.

  Thus, as a result of studies on the improvement of the bitterness and astringency of gallate catechins, the present inventors have heretofore found that the bitterness and unpleasantness of gallate catechins can be improved by using water-soluble collagen having a molecular weight of 4000 or more and water-soluble soybean dietary fiber. It has been found and reported that the taste is reduced (Patent Document 5). However, although the particle diameter of the obtained particles is not described under the conditions of Patent Document 2, it is about 130 nm or more as measured by the present inventors.

In addition, the inventors of the present invention have so far made a combination of gallate catechin and collagen with a lipase inhibitory action (Patent Document 6) and a high anti-obesity action with a composition further added with caffeine (Patent Document 7). It has been reported that such effects can be obtained.
From these reports, it is considered to be very useful to combine gallate catechin with collagen and gelatin.

  So far, there has been a report that nanoparticles were prepared for a mixture of catechin and gelatin containing EGCg (Non-patent Document 9), but the final concentrations of both catechin and gelatin are very limited to 0.1%. There is a description that the measurement result is unreliable because the final concentration is too thin at 0.05% catechin and 0.0125% gelatin under the condition that the average particle diameter is 100 nm or less. In addition, the average particle diameter is 152 nm or more under the condition that the content of both catechin-derived and gelatin-derived components is 0.1%. In order to efficiently take in the functional components gallate catechin and gelatin, preparation of nanoparticles at a higher concentration is required, but this cannot be achieved in Non-Patent Document 9.

In addition, a method of forming nanoparticles of EGCg and gelatin by layer-by-layer has been reported (Non-patent Document 10), but the manufacturing process is complicated, and MnCO ₃ and EDTA to be used cannot be used for foods. Hard to say safe. In addition, there is a report on nanoparticles prepared by coating gelatin on a layer-by-layer basis using polyanions and polycations and adsorbing EGCg (Non-Patent Document 11), but the manufacturing process is also complicated.

  From the above, the combination of gallate-type catechins and proteins, which are functional components, is useful, and although there are examples of producing these nanoparticles, each is under limited conditions and the safety of the materials used Therefore, it has been desired to produce nanoparticles having an average particle diameter of 100 nm or less that are more practical and can be used in foods by a simpler method.

Japanese Patent No. 0556200 JP 2009-090160 A US Pat. No. 8,642,088 JP 2012-111747 A JP 2013-000073 A JP 2013-082673 A Japanese Patent Application No. 2013-017762

Drug Delivery System 26-1, 2011 Research project commissioned by the Ministry of Agriculture, Forestry and Fisheries “Food Nanotechnology Project [Development of nanoscale processing and evaluation technology for food materials]” (created August 20, 2010) pages 61-63 Ministry of Health, Labor and Welfare “Study Report on Safety Measures for Nanomaterials” (March 31, 2009), pages 5-6 Pharmaceutics 2011, 3, 793-829 Microbiology 2007, 153, 2093-2103 Chemistry and Biology 2011, 49 (4), p243-249 Xenobiotica 2001, vol. 31, no. 12, 891-901 “Food Discoloration Chemistry”, p124-p125 J. et al. Agric. Food Chem. 2010, 58, 6728-6734. Journal of Colloid and Interface Science, 2009, 330, 276-283 ACSNANO, 2009, vol. 3, no. 7, 1877-1885

  Therefore, an object of the present invention is to provide a production method for producing nanoparticles derived from natural products using functional materials such as gallate catechins and animal proteins.

  As a result of intensive studies on nanoparticles that can be used in foods, the present inventors have found that gallate catechins and animal proteins can be mixed with gallate catechins and animal proteins under a very simple method. And succeeded in producing nanoparticles having an average particle diameter of 100 nm or less made of a raw material derived from a natural product. In addition, the present inventors have discovered a surprising phenomenon in which the molecular weight of gelatin and collagen and the particle diameter of the nanoparticles are in opposite phases, and have completed the present invention.

The gist of the present invention is as follows.
[1] An average particle diameter of 10 to 100 nm, a method for producing nanoparticles based on a natural product-derived component,
A step of dissolving and / or dispersing a gallate-type catechin or a composition containing a gallate-type catechin in water or a water-containing solvent or an organic solvent to produce a gallate-type catechin-containing solution or dispersion;
A step of dissolving and / or swelling at least one animal protein selected from gelatin, collagen, and degradation products thereof in water, a water-containing solvent or an organic solvent to produce an animal protein-containing solution or a swelling solution; And the gallate-type catechin-containing solution or dispersion and the animal protein-containing solution or swelling liquid, wherein (a) the solid content of the gallate-type catechin (b) the weight of the solid content of the animal protein is 0.07 ≦ ( b) / (a) were mixed in a ≦ 8.0, to form a nanoparticle have a step of preparing a nanoparticle-containing solution in a mixed solution was adjusted to PH1.0～4.0,
The mixed liquid has a solid content derived from gallate catechin or a composition containing gallate catechin of 0.1% by weight or more, and derived from at least one animal protein selected from gelatin, collagen, and degradation products thereof. characterized in that it contains a solid content of 0.1 wt% or more, a method of manufacturing a nanoparticle consisting of gallate catechins and animal proteins,
[2] a method of manufacturing the animal the average molecular weight of the protein is 39000 or more [1] The nanoparticles described,
[3] The method for producing nanoparticles according to [1] or [2] , wherein the animal protein is an animal protein other than porcine bone-derived protein and cow bone-derived protein,
[4] Analysis setting of the nanoparticle-containing liquid adjusted to a solid content value of 0.2 to 1.0% by weight using a zeta potential / nanoparticle diameter measurement system (“DelsaMax PRO” manufactured by Beckman Coulter, Inc.) The method according to any one of [1] to [3] above, wherein the absolute value of the zeta potential obtained using water as a water is 10 mV or more.

  Nanoparticles obtained in the present invention are made of raw materials derived from natural products such as gallate catechins and animal proteins, and are expected to have excellent health functions derived from gallate catechins and animal proteins. For example, the present inventors have previously reported a lipase inhibitory composition using collagen and gallate catechin, but the nanoparticles obtained by the present invention are expected to retain their functions. The

FIG. 1 is a graph showing the relationship between the average molecular weight (x) of animal protein calculated in Example 4 and the average particle diameter (y) of nanoparticles. It can be seen that the larger the average molecular weight of the animal protein, the smaller the particle size.

  Hereinafter, the present invention will be described in detail.

The method for producing nanoparticles of the present invention is a method for producing nanoparticles based on a natural product-derived component having an average particle diameter of 10 to 100 nm,
A step of preparing a gallate catechin-containing solution or dispersion by dissolving or dispersing a gallate catechin or a composition containing a gallate catechin in water, a water-containing solvent or an organic solvent,
A step of dissolving or swelling at least one animal protein selected from gelatin, collagen, and a degradation product thereof in water, a water-containing solvent or an organic solvent to produce an animal protein-containing solution or a swelling solution; and The gallate-type catechin-containing solution or dispersion and the animal protein-containing solution or swelling liquid are divided into (a) solid content of gallate-type catechin (b) solid content of animal protein is 0.07 ≦ (b) / (A) It is mixed so that it may become <= 8.0, and it has the process of forming a nanoparticle in the liquid mixture adjusted to pH1.0-4.0, and producing a nanoparticle containing liquid.

The average particle size of the nanoparticles produced in the present invention is 10 to 100 nm, and preferably 10 to 90 nm, more preferably 20 to 80 nm, from the viewpoint of good absorbability into the body and manufacturability. In particular, the thickness is preferably 30 to 70 nm, and more preferably 30 to 60 nm.
The average particle diameter of the nanoparticles can be measured with a zeta potential / nanoparticle diameter measurement system (“DelsaMax PRO” manufactured by Beckman Coulter, Inc.) as described in the Examples below.

  The natural product-derived component in the present invention indicates that both the gallate catechin or the composition containing gallate catechin and the animal protein, which are raw materials, are derived from natural products. In addition, when using a reagent etc. as said raw material, the reagent should just be derived from a natural product.

(Galate-type catechin-containing solution or dispersion preparation process)
In the method for producing nanoparticles of the present invention, a gallate catechin-containing solution or dispersion is prepared by dissolving or dispersing the gallate catechin or the composition containing a gallate catechin in water, a hydrous solvent, or an organic solvent.

  Examples of the gallate catechin used in the present invention include EGCg, ECg, GCg, and Cg. The gallate catechin may be a non-polymer or a polymer, and they may be mixed or used alone. From the viewpoint of efficient particle formation, it is preferable to contain EGCg and / or ECg.

  Moreover, as a composition containing the gallate type catechin used by this invention, the tea extract, coffee extract, etc. which contain the said gallate type catechin are mentioned, for example. From the viewpoint of particle production efficiency, the gallate catechin content in the composition is preferably 20% by weight or more, more preferably 30% by weight or more, and more preferably 60% by weight or more. .

  The organic solvent used as the solvent is not particularly limited as long as it is miscible with water, but it is preferable to select a solvent suitable for the intended use of the obtained nanoparticles, for example, glycerin, propylene as food Examples of the pharmaceutical include methanol, acetone, dimethyl sulfoxide and the like in addition to the above. The hydrous solvent used as the solvent refers to a mixed solvent of the organic solvent and water.

  The means for dissolving or dispersing is not particularly limited as long as it is a known means. For example, gallate catechin or a composition containing gallate catechin can be dissolved or dispersed by adding and mixing with the solvent. Further, when dissolving or dispersing, it is preferable to adjust the temperature of the solvent to 20 to 90 ° C. from the viewpoint of solubility of the gallate catechin, but there is no particular limitation as long as it dissolves or disperses.

  The solid content value of the composition containing gallate catechin or gallate catechin in the gallate catechin-containing solution or dispersion is 0.1 to 24 weight from the viewpoint of efficiently producing nanoparticles having an average particle size of 100 nm or less. % Is preferred. More preferably, it is preferably 0.1 to 20% by weight, but is not particularly limited as long as desired nanoparticles can be produced.

(Animal protein-containing solution or swelling liquid preparation process)
In the method for producing nanoparticles of the present invention, the animal protein is dissolved or swollen in water, a water-containing solvent or an organic solvent to prepare an animal protein-containing solution or swelling liquid.

The animal protein used in the present invention may be gelatin, collagen peptide, or a degradation product thereof capable of forming coacervate with gallate catechin. Animal protein can be derived from pigs, fish, chickens, etc., and genetically modified organisms. These derived proteins can be used alone or in combination of two or more. Also good. In addition, although animal protein derived from bovine bone or pork bone is partially formed with particles of 100 nm or less, its average particle diameter exceeds 100 nm, and is difficult to use in the present invention.
However, even an animal protein containing a protein derived from bovine bone or pork bone can be used without particular limitation as long as nanoparticles having an average particle diameter of 100 nm or less can be produced.

  The animal protein used in the present invention preferably has a molecular weight of 39000 or more, more preferably so that the average particle diameter of the obtained nanoparticles is 100 nm from the particle diameter graph obtained when prepared using gelatin, for example. 50,000 or more, more preferably 100,000 or more.

  The hydrous solvent used as the solvent refers to an organic solvent miscible with water. The organic solvent is not particularly limited as long as it is miscible with water, but it is preferable to select a solvent suitable for the intended use of the obtained nanoparticles, for example, glycerin, propylene glycol, ethanol as food. Examples of pharmaceuticals include methanol, acetone, dimethyl sulfoxide and the like in addition to the above.

The means for dissolving or swelling is not particularly limited as long as it is a known means. For example, the animal protein can be dissolved or swollen by adding and mixing with the solvent.
In addition, swelling means adding water, a hydrous solvent, or an organic solvent to animal protein, and making it gelatinous.
Moreover, when making it melt | dissolve or swell, it is preferable to adjust the temperature of the said solvent to 20-90 degreeC from a viewpoint of making it melt | dissolve or swell efficiently.

The solid content value of the animal protein in the animal protein-containing solution or the swelling solution is preferably 0.1 to 19% by weight from the viewpoint of efficiently producing nanoparticles having an average particle size of 100 nm or less, More preferably, it is 0.1 to 10% by weight, but is not particularly limited as long as desired nanoparticles can be produced.
When gelatin is used, if the solid content value is 20% by weight or more, it becomes difficult to handle due to an increase in the viscosity of the liquid.

(Nanoparticle-containing liquid preparation process)
In the method for producing nanoparticles of the present invention, the gallate-type catechin-containing solution or dispersion and the animal protein-containing solution or swelling liquid are (a) solid content of gallate-type catechin (b) solid content of animal protein. The nanoparticle-containing liquid is formed by mixing nanoparticles so that the weight of the mixture becomes 0.07 ≦ (b) / (a) ≦ 8.0 and adjusting the pH to 1.0 to 4.0. Is made.

  The method for mixing the gallate-type catechin-containing solution or dispersion and the animal protein-containing solution or swelling liquid in this step is only required to be uniformly mixed, and the gallate-type catechin-containing solution that is allowed to stand still Or a method of adding the animal protein-containing liquid or swelling liquid to the dispersion, a reverse addition method, a method of adding while stirring, a method of adding while homogenizing, a method of previously mixing water with each liquid, etc. Can be used, but there is no particular limitation.

  In this step, the conditions such as the temperature at the time of mixing may be any conditions that do not cause a significant change in the components and can be uniformly mixed, and may be any temperature suitable for the components to be used. For example, in the case of gelatin, when the temperature is low, the viscosity of the solution increases, and when the concentration is as high as several percent or more, it becomes difficult to mix uniformly. Furthermore, in the case of high temperature, since a change of a component becomes easy to occur, 50-80 degreeC is more preferable, More preferably, 50-70 degreeC is good.

In this step, as the amount of the gallate-type catechin-containing solution or dispersion and the animal protein-containing solution or swelling solution,
(A) Solid content of gallate type catechin (b) Weight of animal protein solid content is adjusted to be 0.07 ≦ (b) / (a) ≦ 8.0.

  When the weight ratio exceeds 8.0, some particles of 100 nm or less are generated, but the average particle diameter exceeds 100 nm. The same applies if the weight ratio is less than 0.07. As for the weight ratio, the upper limit value is preferably 7.0 or less.

  Moreover, the pH of the liquid mixture of the gallate catechin-containing solution or dispersion and the animal protein-containing liquid or swelling liquid is 1.0 to 4.0, and more preferably 1.5 to 3.5. . More preferably, 1.5-3.1 is good. If the pH is too lower than 1.0, the nanoparticles are dissolved or the particle size is increased. There are few reports of adjusting the particle size of the nanoparticles at such a low pH. On the other hand, if the pH is higher than 4.0, particles are temporarily formed, but aggregation and precipitation are likely to occur. On the other hand, when the pH is 7 or higher, the stability of the gallate catechin is decreased, so that efficient nanoparticles cannot be formed.

  The pH is not particularly limited as long as it is a usable acid depending on the intended use of the nanoparticles. For example, citric acid, ascorbic acid, gluconic acid, carboxylic acid, tartaric acid, succinic acid, acetic acid or phthalic acid, organic acids such as trifluoroacetic acid, inorganic acids such as hydrochloric acid, perchloric acid, carbonic acid, or buffers, However, it is not limited to these. When the obtained nanoparticles are used for pharmaceuticals, cosmetics, foods, etc., it is preferable to select an acid suitable for each use application.

  In order to adjust the pH of the mixed solution, the pH of the gallate catechin-containing solution or dispersion and the animal protein-containing solution or swelling solution may be adjusted in advance. By adjusting the pH in advance as described above, the pH of the mixed solution can be adjusted to a range of 1.0 to 4.0 even by mixing the gallate catechin-containing solution or dispersion and the animal protein-containing solution or swelling solution. Can be adjusted.

  The gallate catechin and the animal protein form a coacervate in the mixed solution whose pH is adjusted to the range of 1.0 to 4.0 as described above, and nanoparticles having an average particle size of 10 to 100 nm are formed in the coacervate. Occurs.

In the mixed solution, from the viewpoint of efficiently producing nanoparticles, the solid content derived from gallate catechin or a composition containing gallate catechin is 0.1% by weight or more, gelatin, collagen, and degradation products thereof. It is preferable to contain at least 0.1% by weight of a solid content derived from at least one animal protein selected from The total amount of solid content derived from the gallate catechin or the composition containing gallate catechin and solid content derived from animal protein is more preferably 0.28% by weight or more, and further preferably 1.0% by weight or more. 1.8% by weight or more is most preferable.
In addition, you may adjust so that it may become a desired density | concentration at the time of mixing with the said gallate type catechin containing solution or dispersion, and the said animal protein containing liquid or swelling liquid, and you may concentrate after producing a nanoparticle.

  The nanoparticle-containing liquid obtained as described above may be subjected to ultrafiltration, dialysis and the like. If dialysis is performed, it is easy to separate non-particulate components. Examples of the ultrafiltration membrane include a pencil-type UF membrane (manufactured by Asahi Kasei), and examples of the dialysis membrane include SnakeSkin (manufactured by Pierce). There is no particular limitation as long as ultrafiltration and dialysis can be performed without losing nanoparticles.

The nanoparticles obtained in the present invention are excellent in stability. For example, a method of measuring the zeta potential on the nanoparticle surface is known as an index indicating the stability of the nanoparticle, and it can be said that the greater the absolute value of this zeta potential, the better the stability. For example, as the nanoparticles obtained in the present invention, a nanoparticle-containing liquid adjusted to a solid content value of 0.2 to 1.0% by weight is used as a zeta potential / nanoparticle diameter measurement system (manufactured by Beckman Coulter, Inc. It is preferable that the absolute value of the zeta potential obtained using DelsaMax PRO ") and the analysis setting as water is 10 mV or more.
In addition, the solvent of the nanoparticle-containing liquid at the time of measurement may be any of water, a hydrous solvent, and an organic solvent, but is preferably water or a hydrous solvent from the viewpoint of hardly causing measurement errors.

  The nanoparticle obtained by this invention may be mix | blended with food-drinks, when produced on the conditions which can be utilized for a foodstuff. It does not specifically limit as food-drinks, For example, a drink, alcoholic beverage, jelly, confectionery, functional food, health food, health-oriented food, etc. are mentioned. In consideration of preservability, portability, ease of ingestion and the like, confectionery is preferable, and among confectionery, hard candy, soft candy, gummy candy, tablet, chewing gum and the like are preferable.

  When the nanoparticles are blended in a food or drink, the content of the nanoparticles in the food or drink may be an amount that can be expected to have a physiological activity effect. Usually, it is preferable to determine the blending amount so that 10 to 10000 mg, more preferably 100 to 3000 mg can be taken per day. For example, 5 to 50% by weight is preferable for solid foods, and 0.01 to 10% by weight for liquid foods such as beverages.

  Further, since the nanoparticles obtained in the present invention are considered to be excellent in safety, not only for humans, but also for non-human animals such as rats, mice, guinea pigs, rabbits, sheep, pigs, You may mix | blend with therapeutic agents or feed, such as a mammal such as a cow, a horse, a cat, a dog, a monkey, a chimpanzee, a bird, an amphibian, a reptile. As feed, for example, livestock feed used for sheep, pigs, cattle, horses, chickens, etc., feed for small animals used for rabbits, rats, mice, etc., feed for seafood used for eel, Thailand, yellowtail, shrimp, etc., dogs, Pet foods used for cats, small birds, squirrels, etc. are listed.

  EXAMPLES Next, although this invention is demonstrated in detail based on an Example, this invention is not limited only to this Example.

(Example 1) Examination of nanoparticle production using gallate catechin and animal protein G fine powder (derived from pork skin), APH-250 (derived from pig skin), FGL-250 (derived from fish), GBL-250 (derived from pork bone) ), A330 (derived from bovine bone), # 250 (derived from bovine bone) (all manufactured by Nitta Gelatin Co., Ltd.), chicken gelatin (derived from chicken, manufactured by Nippon Ham Co., Ltd.) and other animal proteins having an average molecular weight of 39000 or more. 90 g of each of seven animal protein-containing aqueous solutions prepared by dissolving 1 g in 50 ° C. water was prepared.
On the other hand, seven 10 g of gallate catechin-containing aqueous solutions were prepared by dissolving 0.18 g of green tea extract (gallate catechin (hereinafter referred to as G catechin) content 67 wt%) in water at 50 ° C.
Next, 10 g of a gallate-type catechin-containing aqueous solution was added to 90 g of the seven animal protein-containing aqueous solutions and mixed together. It was. The average particle size and zeta potential of the obtained liquid particles were measured with a zeta potential / nanoparticle size measurement system (“DelsaMax PRO” manufactured by Beckman Coulter, Inc.).
Moreover, the comparative product using the green tea extract Sanphenon XLB-100 (made by Taiyo Kagaku Co.) which does not contain G catechin was also produced. The results are shown in Table 1.
The zeta potential and average particle size were measured using water as the analytical setting.

From the results in Table 1, when green tea extract and G fine powder containing no G catechin are used, nanoparticles are not formed, whereas animal protein and G catechin derived from pig skin, fish, chicken, etc. It can be seen that nanoparticles having a size of 100 nm or less can be produced by mixing with. When animal protein derived from pig skin, fish, chicken, etc. is used, the absolute value of zeta potential is 19-46 mV, which is superior to animal protein derived from pork bone and cow bone. I understood it.
On the other hand, when animal proteins derived from pork bone and cow bone were used, the average particle diameter of the particles exceeded 100 nm for unknown reasons.

(Example 2) Examination of the ratio of animal protein to G catechin In order to examine the ratio of animal protein to G catechin in the preparation of nanoparticles, only the ratio of gelatin to G catechin to be mixed was changed, and the same procedure as in Example 1 was followed. The average particle size was measured by the method. G fine powder was used as gelatin. The results are shown in Table 2. The units of the numerical values of “G differential” and “G catechin” in the table are both weight%.

  From the results in Table 2, the ratio ((b) / (a)) between the solid content (b) of animal protein and the solid content (a) of G catechin is 100 nm or less within the range of 0.07 to 7.0. It can be seen that the nanoparticles are formed.

(Example 3) Examination of pH In order to study the pH in the production of nanoparticles, nanoparticles were produced by the method according to Example 1, and then the mixture was mixed with citric acid, sodium hydrogen carbonate, sodium hydroxide. The average particle size was measured by adjusting the pH. G fine powder was used as gelatin, and the weight ratio of the solid content of animal protein and the solid content of G catechin was 0.83. The results are shown in Table 3.

  From the results in Table 3, it can be seen that nanoparticles having an average particle diameter of 100 nm or less can be obtained by adjusting the pH of the mixed solution to 1.9 to 4.0.

(Example 4) Examination of molecular weight In order to study the molecular weight of proteins in nanoparticle production, three types of collagen peptides as animal proteins (1) Trade name: HBC-P20, Nitta Gelatin Co., Ltd., molecular weight 20000, ( 2) Product name: SCP-5200, Nitta Gelatin Co., Ltd., molecular weight 5000, (3) Product name: SCP-2000, Nitta Gelatin Co., Ltd., molecular weight 2000 and G derivative (average molecular weight 100000) Three types of nanoparticles were prepared by a method according to Example 1. The weight ratio of the solid content of animal protein and the solid content of G catechin was 0.83. The results are shown in FIG.

From the results shown in FIG. 1, it can be seen that the average molecular weight of the animal protein and the average particle diameter of the nanoparticles are in the opposite phase (correlation coefficient 0.9926), and the following formula was found. It was. Subsequently, the average molecular weight which forms a particle | grain of 100 nm or less was computed.
Formula: Average particle size = (447955 x average molecular weight) ^-0.7952
And when the average particle diameter of 100 nm of nanoparticles was substituted into the above formula, the average molecular weight of animal protein was calculated to be 39000. Therefore, nanoparticles having an average molecular weight of 39000 or more were used to obtain nanoparticles having a size of 100 nm or less. It can be seen that it can be formed.

(Example 5) Evaluation of absorbability of nanoparticles An absorbency evaluation test using rats was performed for the evaluation of absorbability of nanoparticles.
A 7-week-old rat is administered with the present invention product using the G fine powder obtained in Example 1 and gallate-type catechin alone, and blood is collected from the tail at 1, 2, 3, 5 and 7 hours to produce plasma. did. The obtained plasma was deconjugated with Grucuronodase (manufactured by Sigma) and Sulfatase (manufactured by Sigma), and the catechin concentration was measured by LC-MS. The catechins to be measured were EGCg and ECg. As a result, an improvement in absorbability was observed in the product of the present invention.

Claims (4)

An average particle diameter is 10 to 100 nm, a method for producing nanoparticles based on a natural product-derived component,
A step of preparing a gallate catechin-containing solution or dispersion by dissolving or dispersing a gallate catechin or a composition containing a gallate catechin in water, a water-containing solvent or an organic solvent,
A step of dissolving or swelling at least one animal protein selected from gelatin, collagen, and a degradation product thereof in water, a water-containing solvent or an organic solvent to produce an animal protein-containing solution or a swelling solution; and The gallate-type catechin-containing solution or dispersion and the animal protein-containing solution or swelling liquid are divided into (a) solid content of gallate-type catechin (b) solid content of animal protein is 0.07 ≦ (b) were mixed so that /(A)≦8.0, by forming nanoparticles have a step of preparing a nanoparticle-containing solution in a mixed solution was adjusted to PH1.0～4.0,
The mixed liquid has a solid content derived from gallate catechin or a composition containing gallate catechin of 0.1% by weight or more, and derived from at least one animal protein selected from gelatin, collagen, and degradation products thereof. A method for producing nanoparticles comprising gallate-type catechins and animal protein, characterized by containing 0.1 wt% or more of a solid content of . Method for producing an average molecular weight of nanoparticles of claim 1 Symbol placement is 39000 or more of the animal protein. The method for producing nanoparticles according to claim 1 or 2 , wherein the animal protein is an animal protein other than porcine bone-derived protein and cow bone-derived protein. Using a zeta potential / nanoparticle diameter measurement system (manufactured by Beckman Coulter, Inc., “DelsaMax PRO”), the nanoparticle-containing liquid adjusted to a solid content value of 0.2 to 1.0 wt% is used as the analysis setting. The method for producing nanoparticles according to any one of claims 1 to 3 , wherein the absolute value of the obtained zeta potential is 10 mV or more.

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