JP7581625B2 - Method for decomposing glycosides and method for producing aglycones - Google Patents

Description

The present invention relates to a method for decomposing glycosides and a method for producing aglycones.

Glycosides are organic compounds in which sugars and aglycones (the non-sugar portion) are bound by glycosidic bonds. Glycosides and aglycones are naturally occurring organic compounds found in the flowers, leaves, roots, stems, fruits, seeds, etc. of various plants, including citrus fruits and beans. Aglycones have different characteristics and effects depending on the type, but among aglycones, polyphenols generally have strong antioxidant effects. For example, polymethoxyflavones, a type of polyphenol found in citrus fruits, are known to have antioxidant, anti-carcinogenic, antibacterial, antiviral, anti-allergic, melanin production inhibitory, and blood sugar inhibitory effects, and are expected to be used in a variety of applications such as medicines, health foods, and cosmetics.

As a method for producing flavonoids, a type of polyphenol, from citrus fruits, for example, a method is known in which flavonoids are extracted from citrus peels, etc., with an aqueous ethanol solution, and the extracted flavonoids are recovered from the solution (see, for example, Patent Document 1).

JP 2005-145824 A

However, conventional methods for producing polyphenols have the problem of low polyphenol yields. Therefore, there is a demand for the development of a production method that can improve the polyphenol yield.

For example, the peels of citrus fruits contain flavonoids, a type of polyphenol, as well as a larger amount of flavonoid glycosides. If these could be recovered as flavonoids, it would be possible to improve the yield of flavonoids. Glycosides are decomposed into sugars and aglycones by hydrolysis, and one method for decomposing flavonoid glycosides into flavonoids is to react the flavonoid glycosides with an acid such as hydrochloric acid. However, this method has problems in that the acid used may remain and be contaminated in the product, and there is also a risk of side reaction products being produced between the acid and the flavonoids.

Another method is to decompose the glycosides by subjecting the aqueous solution containing the glycosides to hydrothermal treatment until it reaches a subcritical state. In this case, the above problems do not occur because no acid or other substances are added.

Here, flavonoids and flavonoid glycosides are extracted from natural products such as citrus fruits and seaweed that contain flavonoids using a heated aqueous ethanol solution, and the aqueous solution containing glycosides to be subjected to hydrothermal treatment is an aqueous ethanol solution containing an extract of a natural product, or an aqueous solution obtained by drying the extract and then dissolving the dried solid in water at a predetermined concentration.

On the other hand, natural products from which flavonoids are extracted contain, in addition to flavonoids and flavonoid glycosides, sugars, water-soluble dietary fiber, amino acids, proteins, and organic acids such as citric acid and alginic acid, which are extracted at the same time as flavonoids and flavonoid glycosides are extracted. Therefore, the aqueous solution containing the above-mentioned glycosides also contains sugars, water-soluble dietary fiber, and organic acids, and when glycosides are decomposed in an acid or subcritical water region, sugars are produced from water-soluble dietary fiber and amino acids are produced from proteins as side reactions. Next, the caramel reaction, in which sugars are dehydrated and condensed, and the Maillard reaction, in which sugars and amino acids react, proceed simultaneously. At this time, organic acids act as catalysts for these two reactions, promoting the reactions.

Generally, sugars and amino acids are soluble in water but insoluble in organic solvents, but as their molecular weight increases through the caramel reaction and Maillard reaction, they become more hydrophobic and soluble in organic solvents, eventually becoming macromolecules that are insoluble in neither water nor organic solvents. When water-soluble glycosides are decomposed under acid or subcritical water conditions, flavonoids are almost insoluble in water and so precipitate out of the solution. For this reason, when the treatment liquid after glycoside decomposition is subjected to solid-liquid separation by filtration or centrifugation, a solid decomposition product containing flavonoids and compounds produced by the caramel reaction and Maillard reaction is obtained.

When flavonoids are extracted from the resulting solid decomposition product using an organic solvent in which flavonoids are soluble and then dried, the resulting solid extract contains a higher concentration of flavonoids than a primary extract obtained by extracting flavonoids from citrus peels, etc., using a heated aqueous ethanol solution, etc., and then drying the extract.

However, when an aqueous solution containing flavonoids extracted from natural products is used as a raw material, the aqueous solution also contains organic acids, and therefore the higher the concentration of flavonoids extracted from natural products in the aqueous solution, the stronger the acidity becomes. In that case, the caramel reaction and Maillard reaction are promoted, and the amount of compounds produced by these reactions also increases. This causes problems such as a decrease in the concentration of flavonoids in the solid extract dried after organic solvent extraction, and a significant decrease in the yield of flavonoids, as the flavonoids are surrounded by compounds insoluble in water and organic solvents produced by the caramel reaction and Maillard reaction, making them difficult to extract with organic solvents.

The present invention has been made in consideration of the problems associated with the above-mentioned conventional techniques, and aims to provide a method for decomposing glycosides that can efficiently decompose glycosides into aglycones, and a method for producing aglycones that can produce aglycones in high yields with fewer impurities.

To achieve the above object, the present invention provides a method for decomposing glycosides, which includes a decomposition step of decomposing glycosides into aglycones by hydrothermal treatment of a reaction solution containing a raw material containing a glycoside and water, and the pH of the reaction solution is 4.5 or higher.

The above method allows glycosides to be efficiently decomposed into aglycones. Furthermore, by using this method, it is possible to produce aglycones in high yields with few impurities.

In the above method, the content of the glycoside in the reaction solution may be 0.05% by weight or more based on the total amount of the reaction solution. When the mass proportion of the glycoside is 0.05% by weight or more, the glycoside can be decomposed particularly efficiently.

In the above method, the glycoside may include sudachitin glycoside and/or demethoxysudachitin glycoside. According to the above method, sudachitin glycoside and demethoxysudachitin glycoside can be decomposed particularly efficiently.

In the above method, the glycoside may include quercetin glycoside. According to the above method, quercetin glycoside can be decomposed particularly efficiently.

In the above method, the hydrothermal treatment may be carried out under conditions of 110 to 300°C. A temperature within the above range can further promote the decomposition of glycosides.

The present invention also provides a method for producing an aglycone, comprising a decomposition step of decomposing a glycoside by the method of the present invention described above, and an extraction step of extracting an aglycone from the decomposition product obtained in the decomposition step. According to this production method, an aglycone can be produced efficiently at a high yield, with few impurities, and at low cost.

The present invention provides a method for decomposing glycosides that can efficiently decompose glycosides into aglycones without using acid, and a method for producing aglycones that can produce aglycones with a high yield and with fewer impurities.

The present invention will be described in detail below with reference to preferred embodiments. However, the present invention is not limited to the following embodiments.

In this specification, a numerical range indicated using "~" indicates a range including the numerical values before and after "~" as the minimum and maximum values, respectively. In the numerical ranges described in stages in this specification, the upper limit or lower limit of a numerical range in a certain stage can be arbitrarily combined with the upper limit or lower limit of a numerical range in another stage. In the numerical ranges described in this specification, the upper limit or lower limit of the numerical range may be replaced with a value shown in the examples. "A or B" may include either A or B, or may include both. Unless otherwise specified, the materials exemplified in this specification may be used alone or in combination of two or more types.

(Method of Decomposing Glycosides)
The glycoside decomposition method according to this embodiment includes a decomposition step of hydrothermally treating a reaction solution containing a raw material containing a glycoside and water to decompose the glycoside into an aglycone, and the pH of the reaction solution is 4.5 or higher.

A glycoside is a hydrophilic compound in which an aglycone and a sugar are bound by a glycosidic bond. The glycosides used in the present invention can be, but are not limited to, phenolic glycosides, coumarin glycosides, flavonoid glycosides, chalcone glycosides, anthocyanidin glycosides, anthraquinone glycosides, indole glycosides, and sphingoglycolipids.

The glycoside decomposition method according to this embodiment can also be applied to the acid hydrolysis of acid anhydrides and molecules having ester bonds in the molecule.

Flavonoids (aglycones) that are the source of flavonoid glycosides are aromatic compounds with a phenylchroman skeleton as a basic structure, and examples of such compounds include flavones, flavonols, flavanones, flavanonols, isoflavones, anthocyanins, flavanols, chalcones, and aurones. Among these, the flavonoid may be polymethoxyflavones, which are flavones, quercetin, which is a flavonol, or hesperitin (aglycone of hesperidin), which is a flavanone.

Examples of polymethoxyflavones include sudachitin, demethoxysudachitin, nobiletin, tangeretin, pentamethoxyflavone, tetramethoxyflavone, and heptamethoxyflavone. Among these, the polymethoxyflavone may be sudachitin or demethoxysudachitin.

Glycosphingolipids are formed by binding sugar and sphingosine via a glycosidic bond. An example of a glycosphingolipid is glucosylceramide.

The sugar that is the source of the glycoside is not particularly limited, and examples include known sugars that can be linked to an aglycone via a glycosidic bond to form the above-mentioned glycoside.

The raw material to be subjected to the glycoside decomposition process may contain other components besides glycosides. Examples of other components include aglycones, water-soluble dietary fiber, poorly soluble dietary fiber, sugars, proteins, organic acids, etc. The content of glycosides in the raw material is preferably 0.1 mass% or more, more preferably 0.25 to 30 mass%, and even more preferably 0.5 to 5 mass%, based on the total solid content of the raw material. When the raw material further contains aglycones, the content of glycosides is preferably 0.25 mass parts or more, more preferably 0.5 to 100 mass parts, and even more preferably 5 to 50 mass parts per 1 mass part of aglycone content.

Specific examples of the raw material that can be used include flowers, leaves, roots, stems, fruits, seeds, etc. of plants and seaweed. In particular, the peel contains a large amount of polymethoxyflavones and their glycosides, so the squeezed residue of citrus fruits can be suitably used. The raw material may be a dried powder obtained from citrus fruits, or a dried powder obtained from citrus peels. Examples of citrus fruits include sudachi, unshu mandarin oranges, ponkan, and shikuwasa. The citrus fruit may be sudachi, which contains a large amount of polymethoxyflavones such as sudachitin and demethoxysudachitin, and their glycosides.

The hydrothermal treatment can be carried out by sealing the raw materials together with water in a pressure-resistant sealed container and heating the sealed container at a temperature exceeding 100°C. By heating the reaction liquid containing the raw materials and water in the sealed container, the inside of the sealed container becomes a heated and pressurized environment, and hydrothermal treatment (hydrothermal synthesis) is carried out. The hydrothermal treatment may be carried out while stirring the reaction liquid. As the pressure-resistant sealed container, any known container that can be used for hydrothermal treatment can be used without any particular restrictions. From the viewpoint of obtaining high decomposition efficiency, the filling rate of the reaction liquid in the sealed container is preferably 20% by volume or more, and more preferably 40 to 80% by volume, based on the volume of the sealed container.

The content of glycosides in the reaction solution is not particularly limited, but is preferably 0.05% by mass or more, more preferably 5% by mass or more, and preferably 25% by mass or less, and more preferably 15% by mass or less, based on the total amount of the reaction solution. When the content of glycosides in the reaction solution is within the above range, the glycosides can be efficiently decomposed.

The reaction conditions for the hydrothermal treatment are not particularly limited, but can be, for example, 110 to 300°C and 0.5 to 20 hours. The reaction temperature is preferably 120 to 190°C, and more preferably 140 to 185°C. If the reaction temperature is 110°C or higher, the hydrothermal reaction tends to occur more smoothly, and if it is 300°C or lower, carbonization of the raw materials and aglycones tends not to progress, and the yield tends to be improved. The reaction time is preferably 0.5 to 20 hours, and more preferably 1 to 10 hours. If the reaction time is 0.5 hours or more, the reaction tends to proceed more easily, and if it is 20 hours or less, it tends to be easier to balance the reaction progress and costs.

The pressure inside the vessel during hydrothermal treatment may be equal to or higher than the saturated water vapor pressure corresponding to the above reaction temperature, but from the viewpoint of the pressure resistance of the apparatus, it is preferable for the pressure to be saturated water vapor pressure.

The pH of the reaction solution is 4.5 or higher, and is preferably 6.0 or higher, more preferably 8.0 or higher, and even more preferably 11.0 or higher, in order to reduce the amount of impurities contained in the glycoside decomposition product. The pH of the reaction solution can be adjusted by dissolving an acid or base in an aqueous solution of the raw material powder containing the specified glycoside. The acid and base used are preferably substances approved as food additives, and citric acid and sodium hydroxide can be used.

The adjustment of pH is also affected by the concentration of the raw powder. In particular, when using raw powder containing an acid, the pH may decrease as the concentration increases, and may fall below 4.5. However, even in this case, it is possible to obtain a high-concentration aglycone-containing powder at a high yield by adjusting the pH to 4.5 or higher with a base.

By performing hydrothermal treatment under the above conditions, glycosides can be efficiently decomposed into aglycones.

(Method for producing aglycone)
The method for producing an aglycone according to the present embodiment includes a decomposition step of decomposing a glycoside and an extraction step of extracting an aglycone from the decomposition product obtained in the decomposition step. The decomposition step is a step of decomposing a glycoside by the glycoside decomposition method according to the present embodiment described above.

In the extraction step, aglycone is extracted from the decomposition product obtained in the decomposition step. In addition to aglycone, the decomposition product contains sugar, glycosides that remain undecomposed, water-soluble and poorly soluble cellulose, and their decomposition products. Here, aglycone is hydrophobic, whereas sugar, glycosides, water-soluble cellulose, and their decomposition products are hydrophilic. Therefore, the components that are insoluble in the aqueous solution after the hydrothermal treatment contain aglycone at high concentrations, and the aglycone can be concentrated by separating the aqueous solution and the insoluble matter after the hydrothermal treatment. In addition, the water-insoluble matter can be further dissolved in a solvent that dissolves aglycone, such as ethanol, ethyl acetate, hexane, toluene, etc., and a mixed solvent thereof, and the insoluble matter is removed by filtration, etc., to further extract and purify the aglycone. Thereafter, the filtrate can be dried to obtain a high concentration of aglycone.

The above method allows aglycone to be produced efficiently with a high yield and few impurities. The aglycone produced by the production method of this embodiment may be polymethoxyflavone, or may be sudachitin and/or demethoxysudachitin. The production method of this embodiment is suitable for producing polymethoxyflavone, particularly sudachitin and demethoxysudachitin, and can greatly improve the yield.

The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples.

Example 1
2 g of powdered sudachi peel extract (manufactured by Ikeda Yakuso Co., Ltd.) with a sudachitin content of 1000 ppm by mass and a glycoside-derived sudachitin content of 9000 ppm by mass was dissolved/dispersed in 50 g of ultrapure water, and sodium hydroxide aqueous solution powder (manufactured by Wako Pure Chemical Industries, Ltd.) was further added to obtain a reaction solution. The pH of the obtained reaction solution was measured with a pH meter and found to be 5.0. This reaction solution was placed in a Teflon (registered trademark) container with a capacity of 100 ml, and then placed in a hot air circulation autoclave (manufactured by Ashida Manufacturing Co., Ltd.) with a tank volume of 2 ^m3 , and the solution was subjected to glycoside decomposition treatment at 180°C for 1 hour. The decomposition treatment was performed by supplying saturated steam at 180°C from a boiler into the tank (pressure vessel) of the autoclave, and adjusting the supply amount of steam and the pressure valve so that the pressure inside the tank was 1 MPa, which is the saturated steam pressure of water at 180°C. The pressure in the tank after the decomposition treatment was 0.9 MPa, and the temperature in the tank was 180 ° C. The autoclave was naturally cooled for 10 minutes until the pressure in the tank was 0.7 MPa and the temperature in the tank was 165 ° C. After natural cooling, the valve was opened, and compressed air at a pressure of 1 MPa was sent into the tank using a compressor attached to the device. Since the pressure in the tank immediately after the compressed air was sent exceeded 1 MPa, compressed air was introduced into the tank while maintaining a pressure not lower than 0.75 MPa by manually opening and closing the exhaust valve, and cooling with compressed air was started. During cooling, cooling was performed while appropriately lowering the pressure in the tank so as not to fall below the saturated water vapor pressure at that time. Two hours after the start of cooling with compressed air, the temperature of the solution fell below 100 ° C. (the saturated water vapor pressure of the aqueous dispersion fell below normal pressure (0.1 MPa)), so the lid of the tank was opened, the container was removed, and the tank was naturally cooled to normal temperature (25 ° C.). After cooling, the glycoside decomposition products that had precipitated and adhered to the inner surface of the container were removed using a medicine spoon.

Next, the solution and solids in the container were filtered under reduced pressure using a hydrophilic PTFE membrane filter (Omnipore 0.2 μm JG (Merck Millipore, product name)) with a 0.2 μm mesh size and a diaphragm pump. The solids obtained were dried in an oven at 120°C for 5 hours to obtain a powdered glycoside decomposition product. The glycoside decomposition product was then adjusted to a 5% dispersion in ethanol, treated under reflux at 60°C for 1 hour, and filtered under reduced pressure using a hydrophilic PTFE membrane filter (Omnipore 0.2 μm JG (Merck Millipore, product name)) with a 0.2 μm mesh size and a diaphragm pump. The resulting solution was vacuum dried using a diaphragm pump at 60°C to obtain 0.16 g of powdered flavonoid concentrated powder 1.

Example 2
The same procedure as in Example 1 was repeated, except that the amount of sodium hydroxide powder added was changed and a reaction liquid with a pH of 7.0 was used, to obtain 0.15 g of a powdered flavonoid-enriched powder 2.

Example 3
The same procedure as in Example 1 was repeated, except that the amount of sodium hydroxide powder added was changed and a reaction liquid with a pH of 10.0 was used, to obtain 0.12 g of a powdered flavonoid-enriched powder 3.

Example 4
The same procedure as in Example 1 was repeated, except that the amount of sodium hydroxide powder added was changed and a reaction liquid with a pH of 13.5 was used, to obtain 0.08 g of a powdered flavonoid-enriched powder 4.

Example 5
Onion skin powder (Awajishima onion powder, Shizen Kenkou Co., Ltd.) was dispersed in a mixed solution of ultrapure water and ethanol (mixture ratio: ultrapure water:ethanol = 50:50) to a concentration of 5 wt% to obtain a dispersion of onion skin powder. The obtained dispersion was heated at 60 ° C for 3 hours to extract quercetin aglycone and quercetin glycoside contained in the onion skin powder. After extraction, solid-liquid separation was performed using a centrifuge at 10,000 rpm for 10 minutes, and the supernatant was separated and dried in an oven to obtain a dry powder. The obtained dry powder was pulverized in an agate mortar to obtain an onion skin extract powder containing 7 mass % quercetin aglycone and 1 mass % quercetin glycoside.

1 g of the obtained onion skin extract powder was dissolved/dispersed in 50 g of ultrapure water, and 0.25 g of powdered flavonoid concentrated powder 5 was obtained in the same manner as in Example 1, except that a solution to which sodium hydroxide powder was added was used as the reaction liquid. The pH of the reaction liquid was 5.0.

Example 6
0.2 g of soybean-derived glucosylceramide powder (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved/dispersed in 50 g of ultrapure water, and sodium hydroxide powder was added to the solution used as the reaction solution, and 0.18 g of powdered ceramide concentrated powder 6 was obtained in the same manner as in Example 1. The pH of the reaction solution was 6.0.

(Comparative Example 1)
Except for using as a reaction solution a solution prepared by dissolving/dispersing 2 g of sudachi peel extract powder in 50 g of ultrapure water, the procedure was repeated as in Example 1 to obtain 0.15 g of powdered glycoside decomposition sample 7. The pH of the reaction solution was 3.9.

<Measurement of aglycone concentration>
The concentration of sudachitin, quercetin or ceramide aglycone in the concentrated powder obtained in each Example and Comparative Example was measured by the following method. First, 0.1 g of sample was dissolved/dispersed in ethanol so that the dilution ratio was 500 times, and filtered through a PTFE filter with a pore size of 0.1 μm to obtain an ethanol solution. The components of this ethanol solution were analyzed by high performance liquid chromatography (HPLC). Calibration curves were prepared using commercially available standard purified samples of each flavonoid and standard purified samples of ceramide aglycone as standard substances, and the concentrations of sudachitin, quercetin and ceramide aglycone in the concentrated powder were estimated using the calibration curves. The HPLC device used was a "Chrome Master" manufactured by Hitachi High-Tech. The results are summarized in Table 1.

As shown in Table 1, the aglycone concentrations in the concentrated powders obtained in Examples 1 to 6 were all higher than those in the raw powder, and were also higher than those in Comparative Example 1, which had a pH of 3.9.

Claims (4)

The method includes a decomposition step of hydrothermally treating a reaction solution containing a raw material containing a glycoside and water to decompose the glycoside into an aglycone,
The pH of the reaction solution is 4.5 or more,
The glycoside includes at least one selected from the group consisting of sudachitin glycoside, demethoxysudachitin glycoside, and quercetin glycoside,
The method for decomposing a glycoside, wherein the hydrothermal treatment time is 0.5 to 20 hours. The decomposition method according to claim 1 , wherein the content of the glycoside in the reaction solution is 0.05% by mass or more based on the total amount of the reaction solution. The decomposition method according to claim 1 or 2, wherein the hydrothermal treatment is carried out at a temperature of 110 to 300°C. A decomposition step of decomposing a glycoside by the method according to any one of claims 1 to 3;
an extraction step of extracting aglycone from the degradation product obtained in the decomposition step;
A method for producing an aglycone, comprising: