TW202043207A - Production method for composition containing isoxanthohumol - Google Patents

Abstract

The purpose of the present invention is to provide a production method or the like that enables obtaining of a composition containing isoxanthohumol with high isoxanthohumol yield and efficiency. The present invention pertains to a production method or the like that is for a composition containing isoxanthohumol, the method comprising: a conversion step for heating a first mixture that contains xanthohumol and a first solvent including water, and that has a pH of 8.0-13.5, to convert the xanthohumol into isoxanthohumol; a precipitate formation step for adjusting the pH of the mixture obtained in the conversion step and containing isoxanthohumol to acid to form a precipitate containing isoxanthohumol; and a precipitate collection step for collecting the precipitate containing isoxanthohumol.

Description

Manufacturing method of composition containing isoxanthohumol

The present invention relates to a manufacturing method of a composition containing isoxanthohumol. Furthermore, the present invention relates to a method of converting xanthohumol into isoxanthohumol.

Isoxanthohumol is a kind of polyphenols, and it has the effect of inhibiting bone density reduction and the like (Patent Document 1). Patent Document 1 describes a method of isomerizing a hop extract containing xanthohumol to obtain an isomerized hop extract containing isoxanthohumol. [Prior Technical Literature] [Patent Literature]

[Patent Document 1] International Publication No. 2004/089359

[The problem to be solved by the invention]

The method described in Patent Document 1 is to boil a 1N sodium hydroxide solution containing hop extracts for 1 hour to isomerize, then add hydrochloric acid, and filter the resulting solution to obtain a filtrate. Liquid partition extraction, and then repeated recrystallization, to obtain isoxanthohumol with a purity of more than 98%. However, this method still has room for improvement to increase the yield of isoxanthohumol.

The object of the present invention is to provide a method for producing isoxanthohumol-containing compositions with high isoxanthohumol yield and efficiently. [Means to solve the problem]

The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems and found that when xanthohumol is converted (isomerized) into isoxanthohumol, if it is boiled in a 1N sodium hydroxide solution (pH 13.8), the The generated isoxanthohumol decomposes and reduces the yield. Repeated studies have found that during the above conversion, if the pH of the mixture containing xanthohumol is heated to a specific range, the xanthohumol can be efficiently converted to isoxanthohumol, and isoxanthohumol can be inhibited. Decomposition of phenol. In addition, it was found that during the above conversion, if the pH of the mixture containing xanthohumol is adjusted to a specific range, xanthohumol can be efficiently converted to isoxanthohumol even at 10 to 50°C. When the temperature becomes 10~50℃, even if the pH is high, the decomposition of isoxanthohumol can be suppressed. Then, if the pH of the mixture containing the isoxanthohumol obtained as described above is adjusted to be acidic, the isoxanthohumol in the mixture can be efficiently precipitated, and isoxanthohumol can be obtained efficiently and in a high yield.

That is, the present invention relates to the production method of the following isoxanthohumol-containing composition, etc., but is not limited thereto. [1] A manufacturing method of a composition containing isoxanthohumol, comprising: heating a first mixture containing xanthohumol and a first solvent containing water and having a pH of 8.0 to 13.5 to convert the xanthohumol into Isoxanthohumol conversion step; the pH of the mixture containing isoxanthohumol obtained in the conversion step is adjusted to acidity to generate a precipitate containing isoxanthohumol; and the above-mentioned isoxanthohumol-containing precipitate is recovered The precipitation recovery step of precipitation. [2] A method for producing a composition containing isoxanthohumol, comprising: adjusting the pH of a second mixture containing xanthohumol and a second solvent containing water to 11.0 or higher, and heating the above-mentioned yellow humulol at 10-50°C A step of converting phenol to isoxanthohumol; adjusting the pH of the mixture containing isoxanthohumol obtained in the above conversion step to acidity to generate a precipitate containing isoxanthohumol; and recovering the above-mentioned isoxanthohumol containing The precipitation recovery step of the precipitation of Humicol. [3] The production method according to [1] or [2] above, wherein in the precipitation generation step, the pH of the mixture containing isoxanthohumol is adjusted to acidity, and salt or an aqueous solution of the salt is added to generate isoxanthohumol Precipitation of Humicol. [4] The production method according to the above [3], wherein the salt or an aqueous solution of the salt is added so that the concentration of the salt becomes 1 mM to 2M. [5] The production method according to any one of [1] to [4] above, wherein in the precipitation generation step, the pH of the mixture containing isoxanthohumol is adjusted to 1.0 to 4.0. [6] The manufacturing method of any one of [1] to [5] above, which further comprises washing the isoxanthohumol-containing precipitate recovered in the precipitation recovery step with a third solvent containing water Net steps. [7] The production method according to the above [1], wherein the first solvent is a mixture of water and a water-miscible solvent, or water. [8] The production method according to the above [2], wherein the second solvent is a mixture of water and a water-miscible solvent, or water. [9] The production method according to [7] or [8] above, wherein the water-miscible solvent is ethanol. [10] A method of converting xanthohumol to isoxanthohumol, which is to adjust the pH of a mixture containing xanthohumol and a water-containing solvent to 11.0 or more, and to convert the xanthohumol to isoxanthohumol at 10~50℃ Xanthohumol. [Effects of Invention]

According to the present invention, a composition containing isoxanthohumol can be efficiently obtained with a high yield of isoxanthohumol. According to the present invention, it is possible to provide a method for producing a composition containing isoxanthohumol with a high yield of isoxanthohumol, and the like.

The first aspect of the present invention is a method for producing an isoxanthohumol-containing composition (hereinafter also referred to as the first aspect of the production method), comprising: combining xanthohumol and a first solvent containing water, and the pH is The first mixture of 8.0~13.5 is heated to convert the above-mentioned xanthohumol into isoxanthohumol; the pH of the mixture containing the isoxanthohumol obtained in the above-mentioned conversion step is adjusted to acidity to produce isoxanthohumol The precipitation generating step of the precipitation; and the precipitation recovery step of recovering the precipitation containing isoxanthohumol. Xanthohumol is one of the polyphenols contained in hops (scientific name: Humulus lupulus).

The second aspect of the present invention is a method of manufacturing a composition containing isoxanthohumol (hereinafter also referred to as the second aspect of the manufacturing method), including: at 10 ~ 50 ℃, containing xanthohumol and water The pH of the second mixture of two solvents is adjusted to 11.0 or higher, the conversion step of converting the xanthohumol into isoxanthohumol; the pH of the mixture containing the isoxanthohumol obtained in the conversion step is adjusted to acidity, and the resulting The precipitation generating step of the precipitation of isoxanthohumol; and the precipitation recovery step of recovering the precipitation containing isoxanthohumol.

In this manual, pH is the pH at 25°C. The pH can be measured with a commercially available pH meter.

First, the conversion steps in the manufacturing method of the first aspect of the present invention will be explained. The conversion step can also be referred to as an isomerization step. The first mixture used in the manufacturing method of the first aspect is a mixture containing xanthohumol and the first solvent and having a pH of 8.0 to 13.5. When xanthohumol is heated in a solvent with a pH of 8.0~13.5, xanthohumol can be efficiently converted (isomerized) into isoxanthohumol. Moreover, if it is the above pH, isoxanthohumol is not easily decomposed under heating, so isoxanthohumol can be obtained in a high yield.

The first solvent is a water-containing solvent, preferably a mixture of water and a water-miscible solvent, or water. Water-miscible solvents include, for example, methanol, ethanol, n-propanol, isopropanol, n-butanol, s-butanol, isobutanol, t-butanol and other monohydric alcohols with 1 to 4 carbon atoms; Ethylene glycol, propylene glycol, butylene glycol and other diols with carbon number 2~4; ketones such as acetone; cyclic ethers such as tetrahydrofuran; dimethylformamide, dimethylacetamide and other amide series Solvents; amine-based solvents such as dimethylamine; sulfite-based solvents such as dimethyl sulfide; nitrile solvents such as acetonitrile; organic acid solvents such as acetic acid, etc. Only one type of water-miscible solvent may be used, or two or more types may be used. Among them, monohydric alcohols with 1 to 4 carbons and diols with 2 to 4 carbons are preferred; ethanol is more preferred. Ethanol is preferred because it can be widely used in food and beverages.

As a mixture of water and a water-miscible solvent used as the first solvent, the concentration of the water-miscible solvent is preferably 30 vol% (v/v%) or less, more preferably 10 vol% or less. From the viewpoint of the conversion efficiency of xanthohumol to isoxanthohumol, the first solvent system with more water content is better, more preferably water.

The first mixture contains xanthohumol and the first solvent, and has a pH of 8.0 to 13.5, and may contain components other than these within a range that does not impair the effects of the present invention. For example, the first mixture may also contain hop-derived components other than xanthohumol (also called hop-derived substances). The first mixture may contain xanthohumol in raw materials derived from plants. The first mixture may be a solution or suspension obtained by dissolving or suspending xanthohumol in a solvent. The first mixture may be a mixture containing raw materials derived from plants containing xanthohumol.

The raw materials derived from plants containing xanthohumol include raw materials derived from hops. The raw material derived from hops containing xanthohumol, such as hop residue (also called used hops) obtained by supercritical carbon dioxide extraction of hops and hops; obtained by water extraction of used hops Residues, dried hops, dried hops, crushed dried hops, etc.

Xanthohumol can be obtained by solvent extraction of the above-mentioned raw materials derived from hops. The extraction solvent preferably uses a solvent containing a water-miscible solvent (preferably ethanol). For example, the raw material derived from hops is immersed in ethanol or an ethanol aqueous solution for extraction. After extraction, the raw material residues derived from hops are removed from the extract, and the resulting extract is concentrated or dried to obtain a hop extract containing xanthohumol. In addition, by separating or purifying the above-mentioned hop extract or its concentrate or dried product by chromatography, supercritical carbon dioxide extraction, etc., as required, a hop extract with more xanthohumol content can be obtained. In the present invention, the xanthohumol-containing hop extract obtained in this way can be used to prepare the first mixture. In addition, xanthohumol or hop extracts containing it are commercially available, and commercially available products can also be used. Commercially available hop extracts, for example, can be exemplified by extracting the pulverized hops with ethanol and then fractionating it by supercritical carbon dioxide extraction to increase the content of xanthohumol (for example, Xanthohumol-enriched hop product) (Trade name), Hopsteiner company), products containing about 80% by weight of xanthohumol (trade name Xantho-pure, Hopsteiner company).

The preparation method of the first mixture is not particularly limited, and it can be prepared by mixing xanthohumol or xanthohumol-containing plant-derived raw materials, solvents, and alkaline substances so that the pH becomes 8.0 to 13.5. The order of mixing these is not particularly limited. The solvent only needs to be water-containing, and it is preferable to use the above-mentioned water or a water-water miscible solvent. For the addition of alkaline substances, aqueous solutions of alkaline substances can also be used. The manufacturing method of the first aspect of the present invention may include a step of preparing the first mixture.

The alkaline substance is not particularly limited. For example, hydroxides of alkali metals or alkaline earth metals such as sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, etc.; sodium carbonate, potassium carbonate, magnesium carbonate, and dolomite can be used One or two or more of alkali metals or carbonates of alkaline earth metals are preferably sodium hydroxide. Alkaline substances, as long as the pH of the first mixture is used in an amount of 8.0~13.5, the usage amount can be set according to the type of alkaline substance. However, when considering the solid-liquid separation after the subsequent precipitation generation step, the alkaline substance is preferably dissolved in the mixture.

In the manufacturing method of the first aspect of the present invention, the higher the content of xanthohumol in the solid content of the first mixture, the higher the content of isoxanthohumol in the obtained composition tends to be higher. The content of xanthohumol in the first mixture is not particularly limited. In one aspect, for example, the solid content is preferably 60% by weight or more, more preferably 70% by weight or more, and still more preferably 80% by weight. Above, it may be 99% by weight or less. By using such a first mixture to perform the conversion step and the precipitation generation step, a composition highly containing isoxanthohumol can be produced. Solid components refer to components other than solvents. In a preferred aspect of the present invention, the content of isoxanthohumol in the solid component can be obtained, which is higher than the content of xanthohumol in the solid component of the first mixture used, preferably higher than that of the first mixture used The xanthohumol content of the solid ingredients contains isoxanthohumol. In one aspect, it is preferable to use a hop extract with a xanthohumol content of more than 80% by weight to prepare the first mixture. In a preferred aspect of the present invention, for example, if the first mixture in which the content of xanthohumol in the solid content is 80% by weight or more is used for the conversion step and the precipitation generation step, the isoxanthohumol in the solid content can be obtained A composition containing isoxanthohumol with a content of more than 83% by weight. The content of xanthohumol in the solid content of the first mixture is not limited to the above, and may be 50% by weight or less in another aspect. In addition, the content of xanthohumol in the first mixture is not particularly limited. For example, it may be 0.0001% by weight or more, and it may be 5% by weight or less. The content of the solvent in the first mixture is, for example, preferably 90% by weight or more.

The pH of the first mixture is 8.0~13.5. From the viewpoint of the conversion efficiency of xanthohumol to isoxanthohumol and the yield of isoxanthohumol, the pH of the first mixture is preferably 9.0 or more, more preferably 10.0 or more, and preferably 13.0 or less, More preferably, it is 12.0 or less. The upper limit and lower limit can also be ranges formed by any combination. In one aspect, the pH of the first mixture is preferably 9.0 to 13.0, more preferably 10.0 to 13.0, and even more preferably 10.0 to 12.0.

In the manufacturing method of the first aspect, the above-mentioned first mixture is heated to convert xanthohumol into isoxanthohumol (conversion step). When heating the first mixture, reflux (heating reflux) may also be performed. From the viewpoint of conversion efficiency, the heating temperature is preferably 60°C or higher, more preferably 80°C or higher. From the viewpoint of equipment or component decomposition, the heating temperature is preferably 120°C or lower, more preferably 100°C or lower. In one aspect, the first mixture is preferably heated at 60 to 120°C, more preferably at 80 to 120°C, and even more preferably at 80 to 100°C. When performing reflux, the heating temperature preferably corresponds to the reflux temperature of the first solvent, and more preferably the reflux temperature is within the above-mentioned temperature range.

The heating time can be set according to the temperature, etc., and can be 1 minute to 5 hours, preferably 10 minutes to 3 hours, more preferably 30 minutes to 2 hours, and more preferably 30 minutes to 1 hour. Generally speaking, the higher the heating temperature, the more rapidly the conversion of xanthohumol to isoxanthohumol occurs. For example, when heating is performed at 80 to 120°C, preferably 80 to 100°C, the heating time is preferably 30 minutes to 2 hours, more preferably 30 minutes to 1 hour. During the conversion step, the mixture can also be stirred.

When the first mixture is heated, xanthohumol is converted to isoxanthohumol, and a mixture containing isoxanthohumol is obtained. The isoxanthohumol-containing mixture usually contains water derived from the first solvent. When the first mixture containing raw materials derived from plants containing xanthohumol is used for the above heating, the xanthohumol contained in the raw materials derived from plants can be extracted and combined with xanthohumol and isoxanthohumol. Conversion to obtain a mixture containing isoxanthohumol. When heating the first mixture containing plant-derived raw materials to perform the conversion step, it is preferable to remove the plant-derived raw material residues from the mixture containing isoxanthohumol before the precipitation generation step described later. For the removal of plant-derived raw material residues, well-known separation methods such as filtration and centrifugal separation can be used.

Next, the conversion step in the manufacturing method of the second aspect of the present invention will be explained. In the conversion step in the manufacturing method of the second aspect, the pH of the second mixture containing xanthohumol and the second solvent containing water is adjusted to 11.0 or higher, and the xanthohumol is converted into isotonic yellow at 10~50°C Humicol. By making the pH above 11.0, xanthohumol is efficiently converted to isoxanthohumol even at a temperature of 10-50°C. In addition, by setting the temperature to 10 to 50°C, isoxanthohumol is not easily decomposed even in a solvent with a high pH, and isoxanthohumol can be obtained in a high yield.

The second solvent is a water-containing solvent, preferably a mixture of water and a water miscible solvent, or water; more preferably, water. The water-miscible solvent is more preferably ethanol. The mixed liquid of water and water miscible solvent used as the second solvent and its preferred aspects are the same as the case of the first solvent. In the mixed liquid of water and a water-miscible solvent, the concentration of the water-miscible solvent is preferably 30 vol% (v/v%) or less, more preferably 10 vol% or less. From the viewpoint of the conversion efficiency of xanthohumol to isoxanthohumol, the second solvent system with more water content is better, more preferably water.

In a range that does not impair the effects of the present invention, the second mixture may contain components other than xanthohumol and the second solvent. For example, the second mixture may also contain hop-derived components other than xanthohumol (also referred to as hop-derived substances). The second mixture, like the first mixture, can use xanthohumol or a hop extract containing it. As the hop extract, those listed above can be used. In the second mixture, xanthohumol may also be contained in plant-derived raw materials. The second mixture may be a solution or suspension obtained by dissolving or suspending xanthohumol in a solvent. The second mixture may be a mixture containing raw materials derived from plants containing xanthohumol. The raw materials derived from plants containing xanthohumol include the above-mentioned ones.

In the manufacturing method of the second aspect, the method of adjusting the pH of the second mixture is not particularly limited, as long as the pH of the mixture containing xanthohumol and the second solvent is 11.0 or more. For example, the pH can be adjusted after the mixture containing xanthohumol and the second solvent is prepared, or the mixture preparation and pH adjustment can be performed at the same time. The method of adjusting the pH of the second mixture, for example, can include a method of mixing xanthohumol and a solvent, and then mixing an alkaline substance therein; a method of mixing xanthohumol, an alkaline substance and a solvent; The method of mixing the raw materials of plants and solvents, and mixing them with alkaline substances; the method of mixing raw materials derived from plants containing xanthohumol, alkaline substances and solvents, etc. The solvent only needs to be one that contains water in the resulting mixture, and it is preferable to use the above-mentioned water or a water-water miscible solvent. For the addition of alkaline substances, aqueous solutions of alkaline substances can also be used. The alkaline substance and its preferred aspect are the same as those in the first aspect of the manufacturing method, and sodium hydroxide is preferred. The alkaline substance may be used in such an amount that the pH of the second mixture becomes 11.0 or more.

In the production method of the second aspect of the present invention, the higher the content of xanthohumol in the solid content of the second mixture, the higher the content of isoxanthohumol in the obtained composition tends to be higher. The content of xanthohumol in the second mixture is not particularly limited. In one aspect, for example, the solid content is preferably 60% by weight or more, more preferably 70% by weight or more, and may be 99% by weight or less . In a preferred aspect of the present invention, the content of isoxanthohumol in the solid component can be obtained, which is more than the content of xanthohumol in the solid component of the second mixture used, preferably higher than that of the second mixture used The xanthohumol content of the solid content is a composition containing isoxanthohumol. In a preferred aspect, when the second mixture having a xanthohumol content in the solid content of 80% by weight or more is used for the conversion step and the precipitation generation step, the isoxanthohumol content in the solid content can be obtained, for example, 81% by weight Above, preferably 83% by weight or more of the composition containing isoxanthohumol. In one aspect, it is preferable to use a hop extract with a xanthohumol content of 80% by weight or more to prepare the second mixture. The xanthohumol content in the solid content of the second mixture is not limited to the above, and in another aspect, it may be 50% by weight or less. In addition, the content of xanthohumol in the second mixture is not particularly limited. For example, it may be 0.0001% by weight or more, and may be 5% by weight or less. The content of the solvent in the second mixture is, for example, preferably 90% by weight or more.

In the conversion step of the second aspect of the manufacturing method, from the viewpoint of the conversion efficiency of xanthohumol to isoxanthohumol, the pH of the second mixture is preferably 11.5 or higher, more preferably 12.0 or higher, and even more preferably 12.5 or more, particularly preferably 12.8 or more. The pH of the second mixture is 14.0 or less, preferably 13.8 or less. In one aspect, the pH of the second mixture is preferably 11.5 to 14.0, more preferably 12.0 to 14.0, still more preferably 12.5 to 13.8, particularly preferably 12.8 to 13.8.

In the production method of the second aspect, from the viewpoint of the yield of isoxanthohumol, the temperature in the conversion step is preferably 20°C or higher, more preferably 25°C or higher, and still more preferably 40°C or higher, and, Preferably it is 45 degrees C or less. In one aspect, the temperature in the conversion step is preferably 20-50°C, more preferably 25-50°C, still more preferably 40-50°C, and still more preferably 40-45°C.

The time of the conversion step is not particularly limited. After the pH is adjusted, it is preferably 1 minute or more, more preferably 5 minutes or more, still more preferably 10 minutes or more, and preferably 2 hours or less, more preferably 1 hour or less . The conversion (isomerization) rate of isoxanthohumol increases as the pH becomes higher. In one aspect, for example, when the pH of the second mixture is 12.8-13.8, the time of the conversion step is preferably 5-20 minutes. When the second mixture has a pH of 11.5 to 12.5, the time for the conversion step is preferably 30 minutes to 1 hour. During the conversion step, the mixture can also be stirred.

By making the pH of the second mixture 11.0 or higher, xanthohumol is converted to isoxanthohumol, and a mixture containing isoxanthohumol is obtained. The isoxanthohumol-containing mixture obtained in the conversion step usually contains water derived from the second solvent. When using the second mixture containing xanthohumol-containing raw materials for the conversion step, the xanthohumol contained in the plant-derived raw materials can be extracted, and the xanthohumol-to-isoxanthohumol Conversion to obtain a mixture containing isoxanthohumol. When the second mixture containing plant-derived raw materials is used for the conversion step, it is preferable to remove the plant-derived raw material residue from the mixture containing isoxanthohumol before the precipitation generation step described later. The above-mentioned well-known separation methods can be used to remove residues of raw materials derived from plants.

In the manufacturing method of the first aspect and the manufacturing method of the second aspect of the present invention, a mixture containing isoxanthohumol is obtained through the above conversion step. Hereinafter, the manufacturing method of the first aspect and the manufacturing method of the second aspect of the present invention are collectively referred to as the manufacturing method of the present invention. In the production method of the present invention, by adjusting the pH of the xanthohumol-containing mixture obtained in the conversion step to acidity, a precipitate containing isoxanthohumol can be generated (precipitation generation step). In the precipitation generation step, from the viewpoint of increasing the yield of isoxanthohumol, the temperature of the mixture containing isoxanthohumol is preferably 50°C or lower, more preferably 10-50°C, and still more preferably 10-30°C, More preferably, it is 15~27℃, particularly preferably 15~25℃.

In the precipitation generation step, as long as the pH of the mixture containing isoxanthohumol is adjusted to acidity, from the viewpoint of increasing the yield of isoxanthohumol, the pH is preferably adjusted to 1.0 to 4.0, more preferably to 1.5 to 2.5 . The pH can be adjusted by adding acid to the mixture. Acid can also be added to acid solutions.

Examples of acids include inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, and nitric acid; organic acids such as citric acid, acetic acid, butyric acid, lactic acid, succinic acid, maleic acid, malic acid, and oxalic acid. These can be used singly or in combination of two or more. Preferably, it is an inorganic acid; more preferably, it is hydrochloric acid and phosphoric acid.

In one aspect, in the precipitation generation step, it is preferable to adjust the pH of the isoxanthohumol-containing mixture to acidity, and to add salt or an aqueous solution of the salt to generate isoxanthohumol-containing precipitation. When salt or salt aqueous solution is added, the yield of isoxanthohumol is more improved. The order of pH adjustment and the addition of the salt or the salt aqueous solution is not particularly limited, and either of them can be carried out first, or they can be carried out simultaneously. It is preferable to add salt or an aqueous solution of salt after adjusting the pH.

As the salt, an inorganic salt or an organic salt can be used. Inorganic salts include sodium chloride, potassium chloride, lithium chloride, sodium bromide, potassium bromide, lithium bromide, sodium iodide, potassium iodide, lithium iodide and other alkali metal halides; calcium chloride, magnesium chloride, bromine Halides of alkaline earth metals such as calcium chloride and magnesium bromide; sulfates of sodium sulfate, potassium sulfate, magnesium sulfate, ammonium sulfate, etc. Examples of organic salts include organic acid salts. Examples of the organic acid salt include trisodium citrate. One type of salt may be used, or two or more types may be used in combination. The salt is preferably a water-soluble salt. When using water-soluble salt, the salt can be removed from the precipitation by performing the washing step described later, and the content of isoxanthohumol can be increased. The salt is more preferably sodium chloride and potassium chloride; and even more preferably sodium chloride.

The salt or the aqueous solution of the salt is preferably added so that the concentration of the salt becomes 1mM to 2M. When the above amount of salt is added, the yield of isoxanthohumol increases. The above-mentioned salt or the aqueous solution of the salt is preferably added so that the concentration of the salt becomes 10mM to 1.8M, more preferably 10mM to 100mM. The concentration of the above-mentioned salt is the concentration of the added salt.

After the pH is adjusted to be acidic, the time for the precipitation to be formed is not particularly limited and can be set appropriately. In one aspect, it is preferably 0.1 minutes to 24 hours, more preferably 10 minutes to 24 hours. After the pH is adjusted to be acidic, in order to make isoxanthohumol easy to precipitate, it is preferable to make the mixture at 15-25°C for a certain time (for example, 10 minutes to 24 hours, preferably 30 minutes to 1 hour).

The precipitate formed as described above contains isoxanthohumol. The produced precipitate containing isoxanthohumol is recovered (precipitation recovery step). The precipitation can be recovered by well-known methods such as filtration and centrifugal separation. The recovered precipitate can be used as a composition containing isoxanthohumol. The solvent can also be removed from the precipitation as desired to obtain a powder containing isoxanthohumol. The solvent can be removed by a well-known method such as vacuum drying.

The manufacturing method of the present invention may include steps other than the above in a range that does not impair the effects of the present invention. In the production method of the present invention, it is preferable to perform a step of washing the recovered isoxanthohumol-containing precipitate with a third solvent containing water (washing step). The isoxanthohumol content in the obtained isoxanthohumol-containing composition can be increased by washing the above-mentioned precipitate with a water-containing solvent. In other words, the purity of isoxanthohumol can be improved. The third solvent used for washing is not particularly limited, and is preferably water or an aqueous ethanol solution, more preferably water. The amount of the third solvent used is, for example, preferably 10 times or more, more preferably 50 times or more, preferably 500 times or less, more preferably 300 times or less in weight ratio with respect to precipitation. The amount of the third solvent used is, for example, preferably 10 to 500 times by weight, and more preferably 50 to 300 times in terms of weight ratio relative to precipitation.

The washing only needs to contact the precipitate with the third solvent. The precipitate after washing can be used as a composition containing isoxanthohumol. In one aspect, the solvent is preferably removed from the precipitation after washing. The solvent can be removed by a well-known method such as vacuum drying. The isoxanthohumol-containing powder obtained in this way can be used as the isoxanthohumol-containing composition of the present invention.

The isoxanthohumol-containing composition obtained by the present invention preferably contains isoxanthohumol at 60% by weight or more, more preferably 70 to 99% by weight. In this way, a composition with a high isoxanthohumol content can be used as a high-purity isoxanthohumol. The content of isoxanthohumol can be determined using high-speed liquid chromatography (HPLC). The measurement conditions can be those described in the examples. The isoxanthohumol-containing composition obtained by the present invention can be widely used, for example, in the production of foods and beverages such as health foods, pharmaceuticals, and such raw materials. In one aspect, the method for producing the isoxanthohumol-containing composition of the present invention can be used as a method for producing high-purity isoxanthohumol.

The present invention also includes the following method of converting xanthohumol into isoxanthohumol (hereinafter also referred to as the conversion method of the present invention). A method for converting xanthohumol into isoxanthohumol, which is to adjust the pH of a mixture containing xanthohumol and a water-containing solvent to above 11.0, and convert the xanthohumol into isoxanthohumol at 10~50℃ . If the conversion method of the present invention is used, xanthohumol can be efficiently converted into isoxanthohumol. In addition, the decomposition of isoxanthohumol produced can be suppressed. The water-containing solvent and its preferred aspect are the same as the second solvent in the second aspect of the manufacturing method. The preferred aspect of the conversion method of the present invention is the same as the preferred aspect of the conversion step in the manufacturing method of the second aspect of the present invention described above. The isoxanthohumol-containing mixture obtained by the conversion method of the present invention can also be subjected to the above precipitation generation step and the like. In one aspect, the isoxanthohumol can be purified from the isoxanthohumol-containing mixture by performing the above-mentioned precipitation generation step and recovery step, as well as the desired washing step. [Example]

The following examples illustrate the present invention in more detail, but do not limit the scope of the present invention. When there is no special indication in the examples,% means% by weight.

The amount of xanthohumol and isoxanthohumol in the sample was determined by high-speed liquid chromatography (HPLC). ＜HPLC system＞ High-pressure gradient pump: LC-30AD×2 units ((share) Shimadzu Corporation) Autosampler: SIL-30AC ((share) Shimadzu Corporation) Column oven: CTO-20AC ((share) Shimadzu Corporation) Photodiode array detector: SPD-M20A ((share) Shimadzu Corporation) <HPLC measurement conditions> Column: YMC-Triart C18, 2.1×150mm ((share) YMC) Mobile phase: liquid A: distilled water containing 0.1vol% formic acid, liquid B: acetonitrile containing 0.1vol% formic acid Gradient conditions (liquid B concentration (%) is vol%): 0-6 minutes to maintain 50% liquid B, 6-8 minutes to linearly dissolve B liquid until 100%, 8-15 minutes to maintain B liquid 100% , 15~16 minutes to return B liquid to 50%, 16~20 minutes to balance. Column temperature: 40℃ Flow rate: 0.2mL/min Injection volume: 2μL Detection wavelength: isoxanthohumol UV286nm, xanthohumol UV367nm When the sample is a powder, the sample is dissolved in a 20 vol% acetonitrile aqueous solution to make 0.02 mg/mL, a sample for HPLC measurement is prepared, and analyzed under the above conditions to determine the amount of isoxanthohumol and isoxanthohumol. The sample of the solution is directly analyzed under the above conditions.

The pH (25°C) was measured with LAQUA (F-74BW) (product name, manufactured by HORIBA).

＜Reference example 1＞ (Sample preparation) 1.0 g of a hop extract (product name Xantho-pure manufactured by Hopsteiner) containing about 80% xanthohumol was put into a conical flask, and 100 mL of a 30 mM sodium hydroxide aqueous solution was added. The pH of the resulting xanthohumol solution was 10.2.

(isomerisation) Add a stir bar, and react the xanthohumol solution for 2 hours at 60℃, 80℃ or reflux conditions (98~100℃) for isomerization (conversion from xanthohumol to isoxanthohumol). Furthermore, at this time, time sampling was performed to determine the concentration of isoxanthohumol and xanthohumol in the sample.

When the total amount (ppm) of xanthohumol (XN) and isoxanthohumol (IX) contained in the sample taken as 100%, the proportion (%), IX of XN (ppm) in the sample The ratio (%) of (ppm) is used as the conversion rate. ppm is ppm by weight.

Figure 1A, Figure 1B, and Figure 1C show the conversion (isomerization) of xanthohumol at pH 10.2 at 60°C, 80°C or reflux conditions (98~100°C) (Figure 1A is 60°C) , Figure 1B is 80°C, Figure 1C is the reflux condition). In Figure 1A, Figure 1B and Figure 1C, the black circle (●) is xanthohumol and the white circle (○) is isoxanthohumol. In addition, the ratio of XN and IX contained in the sample before isomerization is 100% (IX is 0%). The ratio (conversion rate) of XN and IX contained in the sample after heating for 2 hours. When heated at 60°C, XN is 63%, IX is 37%, and heated at 80°C, XN is 8%, IX It is 92%. When heating at reflux temperature, XN is 6% and IX is 94%. In addition, the total concentration of xanthohumol and isoxanthohumol in the sample after the reaction was divided by the concentration of xanthohumol given (sample before isomerization), and the decomposition rate was calculated by the following formula . In all conditions, the decomposition rate did not reach 1.0%. Decomposition rate (%)=100-100×(Total concentration of XN and IX after reaction)/(Concentration of XN given)

＜Refer to Example 2＞ (Sample preparation) 0.5 g of hop extract containing about 70% of xanthohumol was put into a three-necked flask, and 50 mL of sodium hydroxide aqueous solution with pH 10.2 to 13.7 was added to obtain xanthohumol solution. In more detail, an aqueous sodium hydroxide solution of pH 10.2 (NaOH 30 mM), pH 11.7 (NaOH 50 mM), pH 12.9 (NaOH 100 mM), pH 13.4 (NaOH 250 mM), or pH 13.7 (NaOH 500 mM) was used.

(isomerisation) The xanthohumol solution was brought into a reflux state (98-100°C) in an oil bath, and after heating for 1.5 hours, sampling was performed. Measure the concentration (ppm) of xanthohumol and isoxanthohumol, divide the total concentration of xanthohumol and isoxanthohumol after the reaction by the concentration of xanthohumol administered, and obtain the decomposition in the same manner as in Reference Example 1. rate. The results are shown in Table 1. It can be seen that when the pH exceeds 13.5 under the above heating, xanthohumol and isoxanthohumol are significantly decomposed.

In the same manner as in Reference Example 1, the ratio (conversion rate) of isoxanthohumol to the total of isoxanthohumol and xanthohumol was determined. The ratio (conversion rate) of isoxanthohumol in the above heated sample is 92% for pH 10.2, 93% for pH 11.7, 89% for pH 12.9, 95% for pH 13.4, and pH 13.7. 91%.

<Comparative example 1> (Sample preparation) 1.0 g of a hop extract (product name Xantho-pure manufactured by Hopsteiner) containing about 80% xanthohumol was dissolved in 10 mL of a 100 vol% ethanol aqueous solution to obtain a xanthohumol solution.

(isomerisation) Put 10 mL of 1M sodium hydroxide aqueous solution and 80 mL of distilled water into a three-necked flask, and put it in a reflux state in an oil bath. The xanthohumol solution (pH of the mixture: 13.8) was mixed, and heated under reflux conditions for 200 minutes for isomerization. In addition, at this time, time sampling was performed to measure the concentrations (ppm) of xanthohumol and isoxanthohumol. The results are shown in Figure 3.

Figure 3 is a graph showing the changes in the concentration of xanthohumol and isoxanthohumol in the sample when the xanthohumol is converted by heating and refluxing at pH 13.8. The black circle is xanthohumol, and the white circle is isoxanthohumol. The total concentration of xanthohumol and isoxanthohumol is indicated by "×". When heating at pH 13.8 during conversion, the isoxanthohumol produced decreases over time. The total concentration of xanthohumol and isoxanthohumol after heating at pH 13.8 for 200 minutes was divided by the concentration of xanthohumol administered, and the decomposition rate was determined in the same manner as in Reference Example 1. The decomposition rate is 56%.

It can be seen from the above that when xanthohumol is heated at pH 9.0~13.5, xanthohumol can be converted (isomerized) into isoxanthohumol and the decomposition of isoxanthohumol can be inhibited.

<Example 1> (Sample preparation) 1.0 g of hop extract containing about 70% of xanthohumol was put into an Erlenmeyer flask, and 100 mL of 40 mM sodium hydroxide aqueous solution was added. The pH of the resulting xanthohumol solution was 11.0. The ratio of XN and IX in the sample before isomerization is 100% (IX is 0%).

(isomerisation) A stir bar was added, and the xanthohumol solution was reacted at 50°C for 1.5 hours. In addition, at this time, sampling was performed over time, the concentration of isoxanthohumol and xanthohumol in the sample was measured, and the conversion rate was determined in the same manner as in Reference Example 1.

The results are shown in Figure 2. Figure 2 is a graph showing the conversion rate of xanthohumol at pH 11.0 and 50°C. In Figure 2, the black circles are xanthohumol and the white circles are isoxanthohumol. The decomposition rate was calculated in the same way as in Reference Example 1, and the decomposition rate was less than 1.0%.

<Examples 2 to 6 and Comparative Examples 2 to 3> (Sample preparation) 1.0 g of a hop extract containing about 70% xanthohumol was put into an Erlenmeyer flask, and pH 9.8 (Comparative Example 2), pH 10.2 (Comparative Example 3), pH 11.7 (Example 2), pH 12. 9 (Example 3), pH 13.4 (Example 4), pH 13.7 (Example 5), or pH 13.8 (Example 6) 100 mL of sodium hydroxide aqueous solution.

(isomerisation) A stirring bar was added, and the xanthohumol solution was reacted at room temperature (25°C) for 1 hour. The concentration of isoxanthohumol and xanthohumol after the reaction was measured, and the ratio (conversion rate) of isoxanthohumol to the total of isoxanthohumol and xanthohumol was determined in the same manner as in Reference Example 1. The conversion rate (proportion of isoxanthohumol) (%) is shown in Table 2. The conversion from xanthohumol to isoxanthohumol is carried out at pH 11.7~13.8 at room temperature. At pH 11.7, the ratio of isoxanthohumol (conversion rate) is 71% at a reaction time of 30 minutes. At pH 12.9, the ratio of isoxanthohumol is more than 90% in the reaction time of 10 minutes. At pH 13.4~13.8, the ratio of isoxanthohumol is more than 90% within 5 minutes of reaction time. After obtaining the decomposition rate in the same way as in Reference Example 1, the decomposition rate under all conditions of Examples 2 to 6 (pH 11.7 to 13.8) did not reach 1.0%.

It can be seen that even if the pH is above 11.0 and below 50°C, xanthohumol can be converted to isoxanthohumol and the decomposition of isoxanthohumol can be suppressed.

<Example 7> (Sample preparation) 20.0 g of a hop extract (product name Xantho-pure manufactured by Hopsteiner) containing about 80% of xanthohumol was dissolved in 200 mL of ethanol to obtain a xanthohumol solution.

(isomerisation) 600 mL of 100 mM sodium hydroxide aqueous solution and 1200 mL of distilled water were put into a three-necked flask, and a mantle heater (manufactured by Dake Electric Co., Ltd.) was used to make it reflux. The above-mentioned xanthohumol solution was mixed with the solution, and heated at pH 10.2 for 1 hour under reflux conditions for isomerization (conversion of xanthohumol to isoxanthohumol).

(Precipitation generation) The isomerized mixture was cooled to 25°C, 60 mL of 1.0 M hydrochloric acid was added thereto, and the pH was adjusted to 2.0 to precipitate a precipitate. After that, 200 g of NaCl was added to obtain further precipitation. The concentration of NaCl added was 1.7M. In order to recover the obtained precipitate, after filtering with a Büchner funnel, the precipitate was washed with 1 L of distilled water and dried under vacuum to obtain 18.9 g of isoxanthohumol-containing powder. After analyzing the obtained isoxanthohumol-containing powder by HPLC, the isoxanthohumol purity (isoxanthohumol content) was 81%. The yield of isoxanthohumol was 93%. The yield is the converted value when the weight of xanthohumol contained in the hop extract used is 100%.

<Example 8> (Sample preparation) 1.0 g of a hop extract (product name Xantho-pure manufactured by Hopsteiner) containing about 80% of xanthohumol was dissolved in 10 mL of ethanol to obtain a xanthohumol solution.

(isomerisation) 30 mL of 100 mM sodium hydroxide aqueous solution and 60 mL of distilled water were put into a three-necked flask, and the flask was refluxed in an oil bath. The above-mentioned xanthohumol solution (pH of the mixture: 10.2) was mixed with this solution, and heated under reflux conditions for 1.5 hours for isomerization.

(Precipitation generation) The isomerized mixture was cooled to 25° C., and 232 μL of 75% phosphoric acid was added to adjust the pH to 2.0. Afterwards, in order to recover the obtained precipitate, after filtering with a Buchner funnel, the precipitate was washed with 200 mL of distilled water and dried in vacuum to obtain 0.8 g of powder containing isoxanthohumol. After analyzing the obtained isoxanthohumol-containing powder by HPLC, the purity of isoxanthohumol was 90%. The yield of isoxanthohumol was 90%.

<Example 9> (Sample preparation) 1.0 g of a hop extract (product name Xantho-pure manufactured by Hopsteiner) containing about 80% xanthohumol was put into a three-necked flask, and 100 mL of a 30 mM sodium hydroxide aqueous solution was added. The pH of the resulting xanthohumol solution was 10.2.

(isomerisation) Bring the xanthohumol solution to a reflux state (98-100°C) in an oil bath, and perform isomerization for 1 hour.

(Precipitation generation) The isomerized mixture was cooled to 25°C, and 232 μL of 75% by weight phosphoric acid was added thereto to adjust the pH to 2.0. After that, in order to recover the obtained precipitate, after filtering with a Buchner funnel, the precipitate was washed with 200 mL of distilled water and dried in vacuum to obtain 0.89 g of powder containing isoxanthohumol. After analyzing the obtained isoxanthohumol-containing powder by HPLC, the purity of isoxanthohumol was 83%. The yield of isoxanthohumol was 92%.

<Comparative Example 4> (Sample preparation) 1.0 g of a hop extract (product name Xantho-pure manufactured by Hopsteiner) containing about 80% of xanthohumol was dissolved in 10 mL of ethanol to obtain a xanthohumol solution.

(isomerisation) 30 mL of 100 mM sodium hydroxide aqueous solution and 60 mL of distilled water were put into a three-necked flask, and the flask was refluxed in an oil bath. The xanthohumol solution (pH of the mixture: 10.2) was mixed, and heated for 1 hour to perform isomerization.

(Precipitation generation) Cool the isomerized mixture to 25°C, filter it with a Buchner funnel if it is not neutralized (add acid), and wash the precipitate with 200 mL of distilled water. The components of the filtrate and the washing liquid were analyzed to explore how much xanthohumol was eluted. As a result, it was found that 75% of isoxanthohumol was eluted in the filtrate in terms of weight with respect to the xanthohumol fed.

From Examples 7 to 9, it can be seen that after isomerizing xanthohumol, by adjusting the pH to acidity, the produced isoxanthohumol can be efficiently precipitated. In Comparative Example 4 where the pH did not become acidic after isomerization, most of the isoxanthohumol was present in the solution, so most of the isoxanthohumol could not be recovered. After converting xanthohumol to isoxanthohumol, by making the pH acidic, isoxanthohumol can be recovered as a precipitate in a high yield.

<Example 10> (Sample preparation) 5.0 g of a hop extract (product name Xantho-Flav_New manufactured by Hopsteiner) containing about 90% of xanthohumol was put into a two-necked flask, and 500 mL of a 30 mM sodium hydroxide aqueous solution was added. The pH of the resulting xanthohumol solution was 10.2.

(isomerisation) The xanthohumol solution was brought into a reflux state (120°C) in an oil bath, and isomerization was performed for 1 hour.

(Precipitation generation) The isomerized mixture was cooled to 25° C., 75% by weight phosphoric acid was added thereto to adjust the pH to 2.2, and the precipitate was precipitated. After that, 0.3 g of NaCl was added while stirring, and a precipitate was further obtained. The concentration of NaCl added was 10 mM. In order to recover the obtained precipitate, after suction filtration with a Buchner funnel, the precipitate was washed with 500 mL of distilled water and dried in vacuum to obtain 4.85 g of powder containing isoxanthohumol. After analyzing the obtained isoxanthohumol-containing powder by HPLC, the isoxanthohumol purity (isoxanthohumol content) was 87%. The yield of isoxanthohumol was 93%.

[Figure 1] Figure 1A, Figure 1B, and Figure 1C show the conversion (isomerization) yellow rot at pH 10.2 at 60°C (Figure 1A), 80°C (Figure 1B) or reflux conditions (Figure 1C) Phenol conversion rate graph. [Fig. 2] Fig. 2 is a graph showing the conversion rate when converting xanthohumol at pH 11.0 and 50°C. [Figure 3] Figure 3 is a graph showing the changes in the concentration of xanthohumol and isoxanthohumol in the sample when the xanthohumol is converted by heating and refluxing at pH 13.8.

Claims (10)

A manufacturing method of a composition containing isoxanthohumol, comprising: heating a first mixture containing xanthohumol and a first solvent containing water and having a pH of 8.0 to 13.5 to convert the xanthohumol into isoxanthohumol Phenol conversion steps; The pH of the mixture containing the isoxanthohumol obtained in the aforementioned conversion step is adjusted to be acidic to generate a precipitate containing isoxanthohumol; and The precipitation recovery step of recovering the precipitation containing isoxanthohumol. A method for producing a composition containing isoxanthohumol, which comprises: adjusting the pH of a second mixture containing xanthohumol and a second solvent containing water to 11.0 or more, and converting the xanthohumol into xanthohumol at 10~50°C Conversion steps of isoxanthohumol; The pH of the mixture containing the isoxanthohumol obtained in the aforementioned conversion step is adjusted to be acidic to generate a precipitate containing isoxanthohumol; and The precipitation recovery step of recovering the precipitation containing isoxanthohumol. According to the manufacturing method of claim 1 or 2, wherein in the precipitation generation step, the pH of the mixture containing isoxanthohumol is adjusted to acidity, and salt or an aqueous solution of the salt is added to generate isoxanthohumol-containing precipitation. The manufacturing method of claim 3, wherein the salt or an aqueous solution of the salt is added so that the concentration of the salt is 1 mM to 2M. According to the manufacturing method of any one of claims 1 to 4, wherein in the precipitation generation step, the pH of the mixture containing isoxanthohumol is adjusted to 1.0 to 4.0. The manufacturing method according to any one of claims 1 to 5, further comprising a washing step of washing the isoxanthohumol-containing precipitate recovered in the precipitation recovery step with a third solvent containing water. According to the manufacturing method of claim 1, wherein the aforementioned first solvent is a mixture of water and a water miscible solvent, or water. According to the manufacturing method of claim 2, wherein the aforementioned second solvent is a mixture of water and a water-miscible solvent, or water. The manufacturing method of claim 7 or 8, wherein the water-miscible solvent is ethanol. A method for converting xanthohumol into isoxanthohumol, which is to adjust the pH of a mixture containing xanthohumol and a water-containing solvent to above 11.0, and convert the xanthohumol into isoxanthohumol at 10~50℃ .

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