Method for producing oleanolic acid-containing extract

Abstract

To provide a method for producing an oleanolic acid-containing extract which enables easy production of an oleanolic acid-containing extract from grape pomace without the need of using high-concentration organic solvents.SOLUTION: This method includes: a first step of preparing a mixed solution containing a natural deep eutectic solvent with sugar beet-derived betaine as the hydrogen bond acceptor and grape pomace; and a second step of deriving an extract containing an oleanolic acid from the mixed solution prepared in the first step.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing an extract containing oleanolic acid. More specifically, the present invention relates to a method for producing an extract containing oleanolic acid, which can easily obtain an extract containing oleanolic acid from grape pomace without using a high-concentration organic solvent. [Background technology]

[0002] Wine pumice, which is made from grape pulp and pomace (e.g., grape skins and seeds residues) produced during wine brewing, is known to be rich in oleanolic acid, a triterpenoid natural organic acid. It has been reported that oleanolic acid has biological activities such as antioxidant effects and inhibitory effects on the proliferation of dental caries bacteria. In addition, natural oleanolic acid obtained from wine pumice is sometimes used in cosmetics.

[0003] Conventionally, a method for extracting oleanolic acid from grape pomace such as wine pumice has been proposed (see, for example, Patent Document 1). Patent Document 1 discloses, as a method for extracting triterpenes from grape skins, a technique for extracting the raw grape skins with a 90% ethanol solution and then filtering the obtained extract. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2022-020257 Summary of the Invention [Problem to be solved by the invention]

[0005] However, in the conventional extraction method described in Patent Document 1, an organic solvent such as ethanol or acetone is generally used as an extraction solvent at a high concentration of 80% or more. Therefore, there is a strong demand for the development of a method for easily extracting oleanolic acid from grape pomace without using a high-concentration organic solvent as an extraction solvent.

[0006] The present invention has been made in consideration of the problems of the prior art. The present invention provides a method for producing an oleanolic acid-containing extract that can easily obtain an oleanolic acid-containing extract from grape pomace without using a high-concentration organic solvent. [Means for solving the problem]

[0007] According to the present invention, there is provided a method for producing an oleanolic acid-containing extract as shown below.

[0008] [1] A method for producing an extract containing oleanolic acid, comprising: a first step of obtaining a mixture containing a natural deep eutectic solvent having sugar beet-derived betaine as a hydrogen bond acceptor and grape pomace; and a second step of obtaining an extract containing oleanolic acid from the mixture obtained in the first step.

[0009] [2] A method for producing the oleanolic acid-containing extract described in [1] above, wherein at least one selected from the group consisting of L-lactic acid, glycerol, sucrose, xylitol, and 1,3-butylene glycol is used as a hydrogen bond donor for the natural deep eutectic solvent.

[0010] [3] A method for producing an oleanolic acid-containing extract according to [1] or [2], wherein the grape pomace is the pomace produced from grape pulp and skin during wine brewing.

[0011] [4] The method for producing an oleanolic acid-containing extract according to [1] or [2], wherein the second step includes stirring the mixture obtained in the first step, centrifuging the stirred mixture, and filtering the separated supernatant and precipitate.

[0012] [5] A method for producing an oleanolic acid-containing extract described in [1] or [2] above, wherein the extract obtained in the second step is used as a cosmetic raw material. Effect of the Invention

[0013] According to the method for producing an oleanolic acid-containing extract of the present invention, an extract containing oleanolic acid can be easily obtained from grape pomace without using a high-concentration organic solvent. In addition, the method for producing an oleanolic acid-containing extract of the present invention can achieve an extraction rate of oleanolic acid that is comparable to that of a conventional method using a high-concentration organic solvent. Furthermore, the method for producing an oleanolic acid-containing extract of the present invention uses a natural deep eutectic solvent in which both the hydrogen bond acceptor and the hydrogen bond donor are natural products as a deep eutectic solvent for extracting oleanolic acid, so that the extract containing oleanolic acid can be used as it is, for example, as a cosmetic raw material. In particular, all of the components contained in the natural deep eutectic solvent in which betaine derived from sugar beet is used as a hydrogen bond acceptor are substances that are commonly used as cosmetic raw materials, and the extract can be used extremely suitably as a cosmetic raw material. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The following describes the embodiments of the present invention, but the present invention is not limited to the following embodiments. Therefore, it should be understood that the scope of the present invention includes any modifications and improvements to the following embodiments based on the ordinary knowledge of a person skilled in the art without departing from the spirit of the present invention.

[0015] [Method for producing oleanolic acid-containing extract] One embodiment of the method for producing an oleanolic acid-containing extract of the present invention is a method for extracting oleanolic acid from the residue remaining after squeezing grapes (hereinafter referred to as "grape pomace") to obtain an extract containing the extracted oleanolic acid. Oleanolic acid is a type of triterpenoid natural organic acid and is represented by the following structural formula (1).

[0016] [ka]

[0017] The method for producing an oleanolic acid-containing extract of this embodiment (hereinafter also simply referred to as the "production method of this embodiment") includes the first and second steps shown below. The first step is a step of obtaining a mixture containing a natural deep eutectic solvent having sugar beet-derived betaine as a hydrogen bond acceptor and grape pomace. The second step is a step of obtaining an extract containing oleanolic acid from the mixture obtained in the first step (i.e., a mixture containing the above-mentioned natural deep eutectic solvent and grape pomace).

[0018] According to the manufacturing method of this embodiment, an extract containing oleanolic acid can be easily obtained from grape pomace without using a high-concentration organic solvent. In addition, the manufacturing method of this embodiment can achieve an extraction rate of oleanolic acid that is comparable to that of the conventional method using a high-concentration organic solvent. The first and second steps of the manufacturing method of this embodiment will be described in more detail below.

[0019] The first step is to obtain a mixture containing a natural deep eutectic solvent with betaine derived from sugar beet as a hydrogen bond acceptor (HBA) and grape pomace. Here, a deep eutectic solvent (DES) is a solvent (eutectic mixture) obtained by mixing two or more solid substances having the properties of a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA) in a certain molar ratio. That is, a deep eutectic solvent is a solvent that is liquefied at room temperature to less than 100°C by mixing a hydrogen bond donor and a hydrogen bond acceptor, which causes a phenomenon called eutectic. Note that a deep eutectic solvent is a solvent with similar properties to an ionic liquid, but is a solvent that is liquefied by a phenomenon called eutectic, that is, by mixing two or more solid substances, which causes a eutectic melting point depression, and is completely different from an ionic liquid.

[0020] In the manufacturing method of this embodiment, the deep eutectic solvent is a natural deep eutectic solvent made from natural organic resources, and in particular, a deep eutectic solvent using beet-derived betaine as a hydrogen bond acceptor is used. Sugar beet is also called beet, and is a crop cultivated for the purpose of producing beet sugar, for example. And, this sugar beet contains betaine. Betaine has positive and negative charges at non-adjacent positions in the same molecule, and the positively charged atom is not bonded to dissociable hydrogen, and the molecule as a whole is a compound (inner salt) that does not have a charge. Thus, betaine is a zwitterionic substance contained in sugar beet, and may be used as a seasoning in food additives and as a moisturizing agent in cosmetic raw materials. In particular, sugar beet contains a compound represented by the following structural formula (2) as betaine. Betaine represented by the following structural formula (2) is also called trimethylglycine, and is a type of natural amino acid.

[0021] [ka]

[0022] There is no particular limitation on the hydrogen bond donor of the natural deep eutectic solvent, and it is sufficient that it forms a eutectic with betaine as the hydrogen bond acceptor. For example, a carboxylic acid or an alcohol can be used as the hydrogen bond donor. For example, an example of the carboxylic acid is L-lactic acid shown in the following structural formula (3). In addition, an example of the alcohol is glycerol shown in the following structural formula (4), sucrose shown in the following structural formula (5), xylitol shown in the following structural formula (6), and 1,3-butylene glycol shown in the following structural formula (7).

[0023] [ka]

[0024] [ka]

[0025] [ka]

[0026] [ka]

[0027] [ka]

[0028] There is no particular limitation on the method for preparing the natural deep eutectic solvent, but for example, the following method can be mentioned. First, prepare the hydrogen bond acceptor and hydrogen bond donor to be used. Next, place the prepared hydrogen bond acceptor and hydrogen bond donor in a container such as a recovery flask, heat to a temperature equal to or higher than the eutectic point of the deep eutectic solvent, and stir. In this way, a deep eutectic solvent corresponding to the hydrogen bond acceptor and hydrogen bond donor used can be prepared.

[0029] In the first step, grape pomace is added to the natural deep eutectic solvent described above to obtain a mixed liquid containing them. The grape pomace may be any residue remaining after squeezing grapes, and may be, for example, powdered grape pomace. In particular, wine pomace, which is made of grape pulp and skins produced during wine brewing, can be preferably used as the grape pomace.

[0030] There are no particular limitations on the blending ratio of the natural deep eutectic solvent and the grape pomace in the first step.

[0031] The mixture obtained in the first step may further contain other solvents in addition to the natural deep eutectic solvent and grape pomace described above. For example, in the first step, a solvent other than the natural deep eutectic solvent may be further added to the mixture. Such a solvent is preferably an organic solvent capable of extracting oleanolic acid from grape pomace and a substance that does not adversely affect the human body. For example, such a solvent may be alcohol such as ethanol or 1,3-butylene glycol. For example, 1,3-butylene glycol has been conventionally used as an extraction solvent for cosmetic raw materials. Even if the mixture thus further contains other solvents, it is more preferable that these other solvents are substances that have been conventionally used for cosmetic raw materials, for example. The mixture obtained in the first step may further contain other liquids such as water. The range of the mixing ratio of the natural deep eutectic solvent to the other solvent is "9:1 to 1:1", more preferably "3:1 to 1:1", and even more preferably "2:1 to 1:1".

[0032] By carrying out the above-mentioned first step, a mixture containing a natural deep eutectic solvent and grape pomace is obtained. Then, in the next second step, an extract containing oleanolic acid is obtained from the obtained mixture. That is, the second step is a step of extracting oleanolic acid from grape pomace into a natural deep eutectic solvent as an extract.

[0033] In the second step, the extraction temperature is not particularly limited as long as it is within a range in which oleanolic acid can be efficiently extracted from grape pomace. The extraction method may be natural extraction, i.e., static extraction, but from the viewpoint of efficiently extracting oleanolic acid, known extraction methods such as stirring extraction and centrifugation extraction can be used. Two or more of known extraction methods such as stirring extraction and centrifugation extraction may be used in combination. For example, the second step may include an operation of stirring the mixed liquid obtained in the first step, centrifuging the stirred mixed liquid, and filtering the supernatant and precipitate separated by centrifugation. The stirring of the mixed liquid is not particularly limited, but can be performed for 2 hours at a temperature of 40°C.

[0034] The second step as described above allows an extract containing oleanolic acid to be obtained. In this manner, the manufacturing method of the present embodiment uses a natural deep eutectic solvent in which both the hydrogen bond acceptor and the hydrogen bond donor are natural products as the deep eutectic solvent for extracting oleanolic acid, and therefore the extract containing oleanolic acid can be used as it is, for example, as a cosmetic raw material. That is, all of the components contained in the natural deep eutectic solvent with betaine as the hydrogen bond acceptor are substances that are commonly used as cosmetic raw materials, and the extract can be used extremely suitably as a cosmetic raw material. In addition, since betaine derived from sugar beet is used as the hydrogen bond acceptor of the natural deep eutectic solvent, the obtained extract is a cosmetic raw material with high added value. EXAMPLES

[0035] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0036] [Preparation of natural deep eutectic solvent] Five natural deep eutectic solvents were prepared by fixing the hydrogen bond acceptor to betaine derived from sugar beet, a natural amino acid, and using L-lactic acid, glycerol, sucrose, xylitol, and 1,3-butylene glycol as hydrogen bond donors. The natural deep eutectic solvents were prepared by placing the hydrogen bond acceptor and hydrogen bond donor in an eggplant flask, heating and stirring them in the eggplant flask.

[0037] Example 1 In Example 1, a powdered wine pomace was prepared as grape pomace, and oleanolic acid was extracted from the wine pomace to produce an extract containing oleanolic acid. Specifically, 500 μL of a natural deep eutectic solvent using L-lactic acid as a hydrogen bond donor and 500 μL of ethanol were added to 0.1 g of powdered wine pomace, and these were mixed to obtain a mixed solution. Next, the obtained mixed solution was stirred at 40° C. for 2 hours, and then centrifuged for 1 minute. Next, the supernatant and precipitate of the mixed solution separated by centrifugation were filtered to obtain the extract.

[0038] The extract thus obtained was subjected to detection and quantitative analysis of oleanolic acid using reversed-phase column chromatography. From the quantitative analysis results, the extraction rate (%) of oleanolic acid from the raw material wine pumice was calculated. The results are shown in Table 1.

[0039] [Table 1]

[0040] (Examples 2 to 5) An extract containing oleanolic acid was produced in the same manner as in Example 1, except that the natural deep eutectic solvent used was changed as shown in Table 1. The extraction rate (%) of oleanolic acid was determined for each of the obtained extracts. The results are shown in Table 1.

[0041] Comparative Example 1 An extract containing oleanolic acid was produced in the same manner as in Example 1, except that water was used as the extraction solvent. The extraction rate (%) of oleanolic acid was calculated for each of the obtained extracts. The results are shown in Table 1.

[0042] Comparative Example 2 An extract containing oleanolic acid was produced in the same manner as in Example 1, except that 1,3-butylene glycol was used as the extraction solvent. The extraction rate (%) of oleanolic acid was calculated for each of the obtained extracts. The results are shown in Table 1.

[0043] (result) As shown in Table 1, Examples 1 to 5, in which an extract containing oleanolic acid was produced using a natural deep eutectic solvent as the extraction solvent, had a higher extraction rate of oleanolic acid than Comparative Example 1, in which water was used as the extraction solvent. In particular, Example 5, which used a natural deep eutectic solvent with betaine as the hydrogen bond acceptor and xylitol as the hydrogen bond donor, was able to extract oleanolic acid from wine pumice with the highest efficiency (extraction rate of 0.25%). This extraction rate was comparable to that of 1,3-butylene glycol (Comparative Example 2), which is frequently used as an extraction solvent for cosmetic raw materials. [Industrial Applicability]

[0044] The method for producing an extract containing oleanolic acid according to the present invention can be used as a method for producing an extract containing oleanolic acid from grape pomace. In particular, the obtained extract can be used as a cosmetic raw material.

Claims

1. A first step of obtaining a mixed solution containing a natural deep eutectic solvent having sugar beet-derived betaine as a hydrogen bond acceptor and grape pomace; A second step of obtaining an extract containing oleanolic acid from the mixture obtained in the first step; The method for producing an extract containing oleanolic acid comprises the steps of:

2. The method for producing an oleanolic acid-containing extract according to claim 1, wherein at least one selected from the group consisting of L-lactic acid, glycerol, sucrose, xylitol, and 1,3-butylene glycol is used as a hydrogen bond donor of the natural deep eutectic solvent.

3. The method for producing an oleanolic acid-containing extract according to claim 1 or 2, wherein the grape pomace is grape pulp and skin produced during wine brewing.

4. The method for producing an oleanolic acid-containing extract according to claim 1 or 2, wherein the second step includes stirring the mixture obtained in the first step, centrifuging the stirred mixture, and filtering the separated supernatant and precipitate.

5. The method for producing an oleanolic acid-containing extract according to claim 1 or 2, wherein the extract obtained in the second step is used as a cosmetic raw material.

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