Extraction solvent containing a deep eutectic solvent and method for producing the same

Abstract

To enhance the safety of extracts using deep eutectic solvents, primarily when used in cosmetics and other products. [Solution] A method for producing an extract solution obtained by extracting components contained in a target substance, wherein an extraction solvent containing a deep eutectic solvent mixed with a hydrogen bond acceptor and a hydrogen bond donor is brought into contact with the target substance, thereby obtaining an extract solution in which the components in the target substance are dissolved in the extraction solvent. The hydrogen bond acceptor is an amino acid or a derivative thereof, and the hydrogen bond donor is one selected from sugars, organic acids, and alcohols.

Description

Technical Field

[0001] The present invention relates to an extraction solvent containing a deep eutectic solvent that can be used in, for example, cosmetics and quasi-drugs, and a method for producing the same.

Background Art

[0002] A deep eutectic solvent (DES) is a solvent that can become liquid near room temperature by mixing a compound that serves as a hydrogen bond donor and a compound that serves as a hydrogen bond acceptor at a certain ratio to cause a eutectic melting point depression. Deep eutectic solvents have characteristics similar to ionic liquids, exhibit high thermal stability and electrochemical stability, and dissolve substances that are insoluble in organic solvents such as lignin. In addition, since deep eutectic solvents can be adjusted simply by mixing a hydrogen bond acceptor and a hydrogen bond donor, they are easier to adjust than ionic liquids. Further, since deep eutectic solvents can use non-toxic and inexpensive natural-derived compounds such as sugars and amino acids as raw materials, there is a possibility that extraction solvents can be prepared safely, with low environmental impact, and at low cost. Therefore, in recent years, deep eutectic solvents have attracted attention as new environmentally friendly solvents.

[0003] For example, it has been reported that quercetin can be extracted from onion peels using a deep eutectic solvent composed of choline chloride and urea (Non-Patent Document 1). It has also been reported that lignin can be extracted from wood using a deep eutectic solvent composed of choline chloride and sugar (Non-Patent Document 2).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document

Non-Patent Document 2

[0005] Methods for extracting components from biological raw materials such as plants, animals, fungi, and algae generally involve the use of organic solvents such as ethanol and hexane. However, when extracting components using organic solvents harmful to the human body, such as hexane, there is a problem in that the organic solvent must be completely removed in order to use the extracted components in cosmetics or quasi-drugs. On the other hand, as shown in Non-Patent Documents 1 and 2, component extraction methods using deep eutectic solvents use choline chloride as a hydrogen bond acceptor and urea or sugar as a hydrogen bond donor, and are safer for the human body compared to methods using organic solvents such as hexane. However, choline chloride is an ingredient whose use in cosmetics is prohibited by cosmetic regulations in the EU and China, so there are still safety concerns regarding its use in cosmetics or quasi-drugs. In addition, choline chloride may increase the risk of skin irritation depending on the concentration, so it is essential to adjust the concentration used.

[0006] Therefore, the main objective of the present invention is to enhance the safety of extracting components using deep eutectic solvents, primarily for use in cosmetics or quasi-drugs. [Means for solving the problem]

[0007] The inventors of the present invention diligently studied means to solve the problems of the prior art described above, and as a result found a method to improve the safety of the extraction solvent by using compounds usable in cosmetics or quasi-drugs as hydrogen bond acceptors and hydrogen bond donors for preparing the deep eutectic solvent. Specifically, by using amino acids as hydrogen bond acceptors and sugars, organic acids, or alcohols as hydrogen bond donors, and performing component extraction from materials using deep eutectic solvents prepared with these specific combinations, the inventors found that lipid-soluble components and / or water-soluble components can be extracted. Based on this finding, the inventors realized that the problems of the prior art could be solved, and thus completed the present invention.

[0008] The first aspect of the present invention is a method for producing an extract solution obtained by extracting components contained in a target substance. The method for producing an extraction solvent according to the present invention includes an extraction step. In this extraction step, the components in the target substance are dissolved in the extraction solvent by bringing the extraction solvent, which contains a deep eutectic solvent mixed with a hydrogen bond acceptor and a hydrogen bond donor, into contact with the target substance. Before this extraction step, the method may include a step of mixing the hydrogen bond acceptor and the hydrogen bond donor to obtain a deep eutectic solvent (solvent preparation step). In the present invention, the hydrogen bond acceptor is an amino acid or a derivative thereof. The hydrogen bond donor is one selected from sugars, organic acids, and alcohols. The amino acids and their derivatives used as hydrogen bond acceptors and the sugars, organic acids, and alcohols used as hydrogen bond donors in the present invention are all compounds that can be safely used in cosmetics and quasi-drugs. Therefore, in this invention, a deep eutectic solvent, which is a mixture of these hydrogen bond acceptors and hydrogen bond donors, is used as a solvent for extracting components from a target substance, thereby obtaining an extraction solution in which the components in the target substance are dissolved in this solvent. As a result, this extraction solution can be incorporated directly into cosmetics or quasi-drugs, for example, without completely removing the extraction solvent from the solution.

[0009] In the present invention, the hydrogen bond acceptor is preferably proline, L-arginine, L-serine, or betaine. The hydrogen bond donor is preferably lactic acid, sucrose, xylitol, or 1,3-butylene glycol.

[0010] In the present invention, the deep eutectic solvent is preferably formed by 1) a combination of proline and 1,3-butylene glycol, 2) a combination of proline and lactic acid, 3) a combination of L-arginine and lactic acid, 4) a combination of L-serine and lactic acid, 5) a combination of betaine and 1,3-butylene glycol, 6) a combination of betaine and sucrose, or 7) a combination of betaine and xylitol. The deep eutectic solvent is particularly preferably formed by 1) a combination of proline and 1,3-butylene glycol or 7) a combination of betaine and xylitol.

[0011] In the present invention, it is preferable that the molar ratio of hydrogen bond acceptors to hydrogen bond donors constituting the extraction solvent is 3:1 to 1:6.

[0012] In the present invention, it is preferable that the extraction solvent further contains water.

[0013] In the present invention, the extraction solution is preferably one in which the lipid-soluble components in the target substance are dissolved.

[0014] In the present invention, the extraction solution is preferably a solution in which the water-soluble components in the target substance are dissolved.

[0015] The manufacturing method according to the present invention preferably further includes a filtration step to remove insoluble components from the extract solution by filtering the extract solution. It is preferable that the extract solution obtained through the filtration step, rather than the insoluble components removed in this filtration step, be used as a raw material for cosmetics or quasi-drugs.

[0016] The second aspect of the present invention relates to a method for manufacturing cosmetics or quasi-drugs (hereinafter also referred to as "cosmetics, etc."). The method for manufacturing cosmetics, etc. according to the present invention includes blending the extraction solution obtained by the method for manufacturing the extraction solution according to the first aspect described above as a raw material for cosmetics, etc.

[0017] The third aspect of the present invention relates to an extraction solvent. The extraction solvent according to the present invention includes a deep eutectic solvent in which a hydrogen bond acceptor and a hydrogen bond donor are mixed. The hydrogen bond acceptor is an amino acid or a derivative thereof. The hydrogen bond donor is any one selected from sugars, organic acids, and alcohols.

Advantages of the Invention

[0018] According to the present invention, it is possible to enhance the safety when mainly using the components extracted using the deep eutectic solvent for cosmetics or quasi-drugs.

Brief Description of the Drawings

[0019] [Figure 1] FIG. 1 is a sequence diagram showing a method for manufacturing an extraction solution according to an embodiment of the present invention. [Figure 2] FIG. 2 shows examples of compounds used as a hydrogen bond acceptor and a hydrogen bond donor. [Figure 3] FIG. 3 is a graph showing the DPA extraction rate and DHA extraction rate of Examples 1 to 11 and Comparative Examples 1 to 2. [[ID=二十六]] [Figure 4] FIG. 4 is a graph showing the total flavonoid yields of Examples 12 to 21 and Comparative Examples 3 to 5.

Modes for Carrying Out the Invention

[0020] Hereinafter, modes for carrying out the present invention will be described with reference to the drawings. The present invention is not limited to the modes described below, and also includes those appropriately modified by those skilled in the art within an obvious range from the following modes.

[0021] In the specification of the present application, "A to B" representing a numerical range means "A or more and B or less".

[0022] FIG. 1 shows each step of a method for producing an extraction solution according to an embodiment of the present invention. As shown in FIG. 1, this embodiment includes, in this order, a step of preparing a deep eutectic solvent (step S1), an extraction step (step S2), and a filtration step (step S3). By performing each of the above steps on a sample to be extracted, an extraction solution in which the active ingredient of the sample is dissolved in an extraction solvent is obtained.

[0023] As the sample, a biological tissue is used. The biological tissue is a raw material derived from a plant, alga, fungus, archaebacterium, or eubacterium in which cells have cell walls, or an animal in which cells do not have cell walls. In the case of a plant-derived raw material, a raw material derived from at least one of leaves, branches, trees, petals, stems, roots, pulp, fruit skins, and seeds of a specific plant is used as the sample. In the case of algae, unicellular or multicellular algal bodies such as green algae, yellow-green algae, red algae, brown algae, diatoms, Euglena, heterokont algae, dinoflagellates, etc., their dried products, extracts, crushed products, or mixtures thereof are used as raw materials. In the case of fungi, archaebacteria, or eubacteria, at least one of a culture, a cell body, spores, a mycelium, or hyphae is used as the sample. In the case of an animal-derived raw material, for example, soft tissues including skin, blood vessels, heart valves, corneas, amniotic membranes, dura mater, etc. derived from humans or heterologous mammals, organs including the heart, kidneys, liver, pancreas, brain, etc., or connective tissues including bones, cartilage, tendons, etc. are used as raw materials. The component extracted from the biological tissue may be a lipophilic component, a water-soluble component, or both of these.

[0024] Among them, it is preferable to use heterokont algae or a plant-derived raw material as the sample. Heterocytolians are heterotrophic algae that do not perform photosynthesis and grow primarily by taking in organic matter. Heterocytolians are known for their high DHA (docosahexaenoic acid) content and are attracting attention as ingredients for nutritional supplements and cosmetics. Heterocytolians, especially Aurantiochytrium, mainly contain lipid-soluble components such as DHA (docosahexaenoic acid), DPA (docosapentaenoic acid), squalene, and carotenoids (astaxanthin, β-carotene, etc.). Examples of heterocytolians include Aurantiochytrium, Pseudococcolithus, Biflagellates, Dictyocha, and Flagellates. Furthermore, as plant-derived raw materials, plants containing water-soluble components such as flavonoids and polyphenols are preferred. For example, Anastatica hierochuntica is a plant that inhabits desert areas such as Egypt and is known to contain flavonoids with antioxidant activity.

[0025] The deep eutectic solvent preparation step (step S1) is a step in which hydrogen bond acceptors and hydrogen bond donors are mixed to prepare the deep eutectic solvent. A deep eutectic solvent is a solvent obtained when low-melting-point solvent components interact via hydrogen bonds to form a eutectic mixture. A deep eutectic solvent is composed of multiple compounds and has the characteristic of having a lower melting point than the original compounds due to the hydrogen bonding ability of each compound. In particular, in this specification, a deep eutectic solvent means a solvent that becomes liquid at room temperature of 1 to 30°C when two or more compounds are mixed in a specific molar ratio, and exhibits a lower melting point than the original compounds.

[0026] In the present invention, in preparing the deep eutectic solvent, an amino acid or its derivative is selected as the hydrogen bond acceptor, and a sugar, organic acid, or alcohol is selected as the hydrogen bond donor. Examples of hydrogen bond acceptors are proline, L-arginine, L-serine, and betaine. Examples of hydrogen bond donors are lactic acid, sucrose, xylitol, or 1,3-butylene glycol. Furthermore, in preparing the deep eutectic solvent, water (purified water) may be added as a third component. When water is added as a third component, the range of molar ratios in which the deep eutectic solvent is formed by mixing the hydrogen bond acceptor and hydrogen bond donor can be broadened compared to when water is not added. In preparing a deep eutectic solvent, it is preferable to uniformly mix hydrogen bond acceptors and hydrogen bond donors and then heat and stir to form the deep eutectic solvent. The stirring temperature is preferably 60-100°C or 70-90°C, and particularly preferably 80-85°C. The stirring time is preferably 30 minutes to 5 hours or 1 to 3 hours, and particularly preferably 1.5 to 2.5 hours.

[0027] The first example of a combination of a hydrogen bond acceptor and a hydrogen bond donor is the combination of proline and 1,3-butylene glycol. In the first example, the molar ratio of proline to 1,3-butylene glycol is preferably 1:2 to 1:6, and more preferably 1:5 to 1:6.

[0028] The second example is a combination of proline and lactic acid. In the second example, the molar ratio of proline to lactic acid is preferably 3:1 to 1:3, and more preferably 1:2 to 1:3.

[0029] The third example is a combination of L-arginine and lactic acid. In the third example, the molar ratio of L-arginine to lactic acid is preferably 1:2 to 1:5, and more preferably 1:4 to 1:5.

[0030] The fourth example is a combination of L-serine and lactic acid. In the fourth example, the molar ratio of L-serine to lactic acid is preferably 1:2 to 1:5, and more preferably 1:4 to 1:5.

[0031] The fifth example is a combination of betaine and 1,3-butylene glycol. In the fifth example, the molar ratio of betaine to 1,3-butylene glycol is preferably 3:1 to 1:3, and more preferably 1:2 to 2:1.

[0032] The sixth example is a combination of betaine and sucrose. In the sixth example, the molar ratio of betaine to sucrose is preferably 5:1 to 1:1, and more preferably 3:1 to 1:1.

[0033] The seventh example is a combination of betaine and xylitol. In the seventh example, the molar ratio of betaine to xylitol is preferably 3:1 to 1:1, and more preferably 3:2 to 2:3.

[0034] The eighth example is a combination of betaine and lactic acid. In the eighth example, the molar ratio of betaine to lactic acid is 3:1 to 1:3, and it is more preferable to have a ratio of 1:2 to 2:1.

[0035] In addition to hydrogen bond donors and hydrogen bond acceptors, it is preferable to further add water as a third component to prepare a deep eutectic solvent. The ratio of the mixture of hydrogen bond donors and hydrogen bond acceptors to water is preferably 3:1 to 10:1 by weight, and particularly preferably 4:1 to 8:1 or 5:1 to 7:1.

[0036] The extraction step (step S2) is a process of immersing the sample in the deep eutectic solvent prepared in step S1 to extract components from the sample. This yields an extraction solution (before filtration) in which the active ingredients of the sample are dissolved in the deep eutectic solvent. Since the deep eutectic solvent is a solvent that is liquid at room temperature from 1 to 30°C, it can be used to extract components from a sample without requiring a high-temperature environment (e.g., above 80°C). However, in order to enhance the extraction capacity of the deep eutectic solvent, it is preferable to heat it to an extent that the individual compounds forming the deep eutectic solvent do not volatilize. The temperature of the deep eutectic solvent in the extraction step is preferably 35 to 90°C, and particularly preferably 40 to 80°C. Specifically, when extracting lipid-soluble components derived from algae or animals, the temperature is preferably 35 to 60°C, and particularly preferably 40 to 50°C. On the other hand, when extracting water-soluble components derived from plants, the temperature is preferably 60 to 90°C, and particularly preferably 70 to 85°C. Furthermore, the immersion time (extraction time) of the sample in the deep eutectic solvent is preferably 30 to 240 minutes, and particularly preferably 60 to 180 minutes or 90 to 120 minutes.

[0037] The filtration step (step S3) is a process of removing insoluble components such as extraction residue from the extraction solution obtained in step S2 by filtering it. In the filtration step, it is preferable to first separate the extraction residue using coarse filter paper immediately after the completion of extraction in step S2 (primary filtration). The mesh size of the filter paper used in this primary filtration should be, for example, 1 to 10 μm, and especially 5 to 8 μm. Next, the extraction solution is allowed to stand for about 12 to 48 hours to precipitate insoluble components in the extraction solution, and then the insoluble components are removed by suction filtration using a fine membrane filter (secondary filtration). It is also preferable to cool the extraction solution to 0 to 10°C, especially 1 to 6°C, during the standing period. For this secondary filtration, it is preferable to use a fine membrane filter with a mesh size of 0.3 to 1 μm, and especially 0.35 to 0.5 μm. Furthermore, the filtrate after secondary filtration is sterilized by filtration using a finer membrane filter and filled into a sterilized container (tertiary filtration). For tertiary filtration, it is preferable to use a membrane filter with an even finer mesh size of 0.1 to 0.5 μm, especially 0.15 to 0.25 μm. Note that tertiary filtration uses a membrane filter with a smaller mesh size than secondary filtration. Tertiary filtration is preferably performed in a clean environment such as a cleanroom or clean bench.

[0038] By going through steps S1 to S3 described above, an extract solution containing the deep eutectic solvent and the sample components, with insoluble components removed, is obtained. Since this extract solution uses components that are safe for use in cosmetics and quasi-drugs as a solvent, there is no need to heat or pressurize / depressurize it to remove the solvent. In other words, the extract solution containing the deep eutectic solvent and the sample components can be directly incorporated into cosmetics or quasi-drugs.

[0039] Examples of cosmetic and quasi-drug formulations that may incorporate the extract solution include lotions, creams, emulsions, gels, aerosols, essences, masks, cleansers, bath products, foundations, powders, lipsticks, ointments, and poultices. In addition, this extract solution can also be incorporated into products such as soaps, body washes, facial cleansers, shampoos, conditioners, treatments, and toothpaste. [Examples]

[0040] [1. Combinations and molar ratios of hydrogen bond acceptors and hydrogen bond donors] Betaine, L-serine, L-arginine, or proline were used as hydrogen bond acceptors, and lactic acid, sucrose, or 1,3-butylene glycol (1,3-BG) were used as hydrogen bond donors (Figure 2). After mixing the hydrogen bond acceptors and hydrogen bond donors in a round-bottom flask in a predetermined molar ratio, the mixture was first stirred at 80°C for 2 hours without adding water to confirm whether a deep eutectic solvent could be formed. Then, water was added to the mixture so that the weight ratio of the hydrogen bond acceptor / donor mixture to water was 6:1, and the mixture was stirred at 80°C for 2 hours to confirm whether a deep eutectic solvent could be formed again. The results of confirming deep eutectic solvent formation for an example of hydrogen bond acceptor / donor combination are shown in Table 1 below.

[0041] [Table 1]

[0042] The definition and confirmation method for deep eutectic solvents are as follows. In Table 1, samples that formed deep eutectic solvents are indicated with ○, and samples that did not form deep eutectic solvents are indicated with ×. (definition) A deep eutectic solvent is a solvent that becomes liquid at room temperature (1-30 degrees Celsius) when two or more compounds are mixed in a specific molar ratio, and exhibits a melting point lower than the melting points of the individual compounds. (How to check) Based on the above definition, the deep eutectic solvent was defined as a transparent, liquid substance at a temperature much lower than the melting points of the hydrogen bond acceptor and hydrogen bond donor used (at least 20°C lower than each melting point, except for those containing 1,3-BG, which were at least 80°C lower). The individual melting points of the processed materials used as hydrogen bond acceptors and hydrogen bond donors are shown in Table 2 below.

[0043] [Table 2]

[0044] From Table 1 above, the range of combinations and molar ratios of hydrogen bond acceptors and hydrogen bond donors that form deep eutectic solvents was determined. Furthermore, it was confirmed that when water was added as a third component, the range of molar ratios that form deep eutectic solvents was broadened compared to when water was not added.

[0045] (Without adding water) In the combination of proline and 1,3-butylene glycol, a deep eutectic solvent was formed when the molar ratio of 1,3-butylene glycol was 5 or higher. Similarly, in the combination of proline and lactic acid, a deep eutectic solvent was formed when the molar ratio of lactic acid was 2 or higher. Furthermore, in the combinations of L-arginine and lactic acid, and L-serine and lactic acid, a deep eutectic solvent was formed when the molar ratio of lactic acid was 4 or higher.

[0046] (When water is added) In the combination of proline and 1,3-butylene glycol, a deep eutectic solvent was formed when the molar ratio of 1,3-butylene glycol was 2 or higher. Furthermore, in the combinations of proline:lactic acid, L-arginine:lactic acid, and L-serine:lactic acid, all the molar ratios tested in this study also resulted in the formation of a deep eutectic solvent.

[0047] [2. Extraction of DPA and DHA from Aurantiochytrium using a deep eutectic solvent] Examples 1-11 and Comparative Examples 1-2 involved extraction experiments targeting Aurantiochytrium. Aurantiochytrium is a type of microalgae and is known to be rich in omega-6 fatty acids DPA (docosapentaenoic acid) and omega-3 fatty acids DHA (docosahexaenoic acid). Here, we confirmed the combinations and molar ratios that form deep eutectic solvents and extracted DPA and DHA from Aurantiochytrium using deep eutectic solvents.

[0048] In Examples 1-11, 0.1 g of aurantiochytrium powder was mixed with 1 mL of deep eutectic solvent and 100 μL of methyl nonadecanate, an internal standard, and stirred at 40°C for 2 hours. Then, 3 mL of Et2O (diethyl ether) was added, and after centrifugation, the deep eutectic solvent was removed from the Et2O layer obtained by back extraction to obtain an Et2O layer containing lipid-soluble components. The fatty acids contained in this layer were methylated using a fatty acid methylation kit, dissolved in ethanol, and the unsaturated fatty acids were quantified by GC-MS.

[0049] In Example 1, proline was mixed as a hydrogen bond acceptor and 1,3-butylene glycol as a hydrogen bond donor in a molar ratio of 1:2. Water was then added to this mixture in a weight ratio of 6:1, and the mixture was stirred at 80°C for 2 hours to prepare a deep eutectic solvent. Aurantiochytrium extraction was performed using this deep eutectic solvent according to the procedure described above.

[0050] In Example 2, the molar ratio of Example 1 was changed to 1:3, and aurantiochytrium extraction was performed using the same procedure as in Example 1.

[0051] In Example 3, the molar ratio of Example 1 was changed to 1:4, and aurantiochytrium extraction was performed using the same procedure as in Example 1.

[0052] In Example 4, the molar ratio of Example 1 was changed to 1:5, and aurantiochytrium extraction was performed using the same procedure as in Example 1.

[0053] In Example 5, the molar ratio of Example 1 was changed to 1:6, and aurantiochytrium extraction was performed using the same procedure as in Example 1.

[0054] In Example 6, proline was mixed as a hydrogen bond acceptor and lactic acid as a hydrogen bond donor in a molar ratio of 2:1. Water was then added to this mixture in a weight ratio of 6:1, and the mixture was stirred at 80°C for 2 hours to prepare a deep eutectic solvent. Aurantiochytrium extraction was performed using this deep eutectic solvent in the same procedure as in Example 1.

[0055] In Example 7, L-arginine was mixed as a hydrogen bond acceptor and lactic acid as a hydrogen bond donor in a molar ratio of 1:5. Water was then added to this mixture in a weight ratio of 6:1, and the mixture was stirred at 80°C for 2 hours to prepare a deep eutectic solvent. Aurantiochytrium extraction was performed using this deep eutectic solvent in the same procedure as in Example 1.

[0056] In Example 8, L-serine was mixed as a hydrogen bond acceptor and lactic acid as a hydrogen bond donor in a molar ratio of 1:5. Water was then added to this mixture in a weight ratio of 6:1, and the mixture was stirred at 80°C for 2 hours to prepare a deep eutectic solvent. Aurantiochytrium extraction was performed using this deep eutectic solvent in the same procedure as in Example 1.

[0057] In Example 9, betaine was mixed as a hydrogen bond acceptor and 1,3-butylene glycol as a hydrogen bond donor in a molar ratio of 1:1. Water was then added to these mixtures in a weight ratio of 6:1, and the mixture was stirred at 80°C for 2 hours to prepare a deep eutectic solvent. Aurantiochytrium extraction was performed using this deep eutectic solvent in the same procedure as in Example 1.

[0058] In Example 10, betaine was mixed as a hydrogen bond acceptor and lactic acid as a hydrogen bond donor in a molar ratio of 1:1. Water was then added to these mixtures in a weight ratio of 6:1, and the mixture was stirred at 80°C for 2 hours to prepare a deep eutectic solvent. Aurantiochytrium extraction was performed using this deep eutectic solvent in the same procedure as in Example 1.

[0059] In Example 11, betaine was mixed as a hydrogen bond acceptor and sucrose as a hydrogen bond donor in a molar ratio of 2:1. Water was then added to this mixture in a weight ratio of 6:1, and the mixture was stirred at 80°C for 2 hours to prepare a deep eutectic solvent. Aurantiochytrium extraction was performed using this deep eutectic solvent in the same procedure as in Example 1.

[0060] In Comparative Example 1, aurantiochytrium extraction was performed using a 50 vol% ethanol solution in the same procedure as in Example 1.

[0061] In Comparative Example 2, aurantiochytrium extraction was performed using hexane in the same procedure as in Example 1.

[0062] The quantitative results of the DPA extraction rate (%), DHA extraction rate (%), and viscosity (mPa·s) for each example and comparative example are shown in Table 3 below. Figure 3 shows graphs of the DPA and DHA extraction rates for each of Examples 1-11 and each comparative example.

[0063] [Table 3]

[0064] The quantitative and measurement methods for the DPA extraction rate (%), DHA extraction rate (%), and viscosity shown in Table 3 above are as follows. (Method for quantifying DPA / DHA extraction rate) DPA and DHA in the extracted solution were quantified using the internal standard method by GC-MS (Shimadzu; GCMS-QP2010 SE) under the following measurement conditions, and the extraction rate was calculated using the following formula. TIFF2026076977000005.tif20166 · Column: Agilent DB-WAX (0.25mm id x 30m) Carrier gas flow rate: Helium, 1.74 mL / min Column temperature: 150℃ → 2.5℃ / min → 250℃ (Method for measuring the viscosity of deep eutectic solvents) The viscosity of the prepared deep eutectic solvent was measured using a vibrating viscometer (Viscometer TVB-10, Toki Sangyo) and a small sample adapter (THM-11, Toki Sangyo).

[0065] As shown in Table 3 and Figure 3, Examples 1 to 11 all successfully extracted DPA and DHA from aurantiochytrium. Furthermore, each example demonstrated sufficiently practical extraction rates compared to conventionally used extraction solvents such as 50 vol% ethanol solution (Comparative Example 1) and hexane (Comparative Example 2). In particular, Examples 1 to 5 showed higher extraction rates compared to Comparative Examples 1 and 2.

[0066] [3. Flavonoid extraction from anzange using deep eutectic solvents] Examples 12-21 and Comparative Examples 3-5 involved extraction experiments using dried powder of Anastatica hierochuntica. Anastatica hierochuntica is a plant that inhabits desert regions such as Egypt and is known to contain flavonoids with antioxidant activity. Here, total flavonoids were extracted from Anastatica hierochuntica using various deep eutectic solvents (DES), and the results were compared with conventional solvents. The quantitative results of the total flavonoid yield [mg / g-DW] for each example and comparative example are shown in Table 4 below.

[0067] [Table 4]

[0068] (Extraction method) 0.1 g of dried Anzanju powder was mixed with 1 mL of the solvent shown in Table 4 above, and the mixture was stirred at 80°C for 2 hours. After stirring, the solids were removed by centrifugation, and the supernatant was collected as the extract. 0.1 mL of this extract was taken and diluted with 0.9 mL of water (10-fold dilution) to prepare the analytical sample.

[0069] (Method for determining total flavonoids) The total flavonoid content of the obtained extract was quantified using the aluminum chloride colorimetric method (AlCl3 method). 0.1 mL of the extraction solution was diluted 10-fold with water to prepare the analytical sample. 0.5 mL of water was added to this analytical sample, and a 5% sodium nitrite (NaNO2) solution was added and allowed to stand for 5 minutes. Subsequently, a 10% AlCl3 solution was added and allowed to stand for 6 minutes. Then, 0.3 mL of 1N sodium hydroxide (1N NaOH) and 0.1 mL of water were added to stop the reaction. After filtering the mixture, the absorbance at a wavelength of 510 nm was measured using a UV-Vis spectrophotometer. This quantitative method was based on Chang et al. (2002) “Estimation of total flavonoid content in propolis by two complementary colorimetric methods”, Journal of Food and Drug Analysis, 10(3), 178-182. Using quercetin as a standard substance, the total flavonoid yield per 1 g of dried anzange powder (mg QE / g - DW (dry weight); QE = Quercetin Equivalent) was calculated from the total flavonoid amount calculated based on the calibration curve using the following formula. TIFF2026076977000007.tif14166

[0070] (Examples and Comparative Examples) In Example 12, proline was mixed as a hydrogen bond acceptor and lactic acid as a hydrogen bond donor in a molar ratio of 2:1. Water was added to this mixture in a weight ratio of 6:1, and the mixture was stirred at 80°C for 2 hours to prepare a deep eutectic solvent. Extraction from the dried anzange powder was performed using this deep eutectic solvent according to the extraction method described above.

[0071] In Example 13, proline was used as a hydrogen bond acceptor and 1,3-butylene glycol was used as a hydrogen bond donor, mixed in a molar ratio of 1:5. A deep eutectic solvent was prepared in the same manner as in Example 12, and extraction was performed.

[0072] In Example 14, betaine was used as the hydrogen bond acceptor and sucrose as the hydrogen bond donor, mixed in a molar ratio of 2:1, and extraction was performed in the same manner as in Example 12.

[0073] In Example 15, arginine was used as a hydrogen bond acceptor and lactic acid as a hydrogen bond donor in a molar ratio of 1:5, and extraction was performed in the same manner as in Example 12.

[0074] In Example 16, betaine was used as a hydrogen bond acceptor and 1,3-butylene glycol was used as a hydrogen bond donor, mixed in a molar ratio of 1:1, and extraction was performed in the same manner as in Example 12.

[0075] In Example 17, betaine was used as a hydrogen bond acceptor and lactic acid as a hydrogen bond donor in a molar ratio of 1:1, and extraction was performed in the same manner as in Example 12.

[0076] In Example 18, betaine was used as a hydrogen bond acceptor and xylitol as a hydrogen bond donor in a molar ratio of 3:2, and extraction was performed in the same manner as in Example 12.

[0077] In Example 19, serine was used as a hydrogen bond acceptor and lactic acid as a hydrogen bond donor in a molar ratio of 1:5, and extraction was performed in the same manner as in Example 12.

[0078] In Comparative Example 3, methanol was used as the solvent, and extraction was performed from the dried anzange powder in the same manner as in Example 12, following the procedure of the extraction method described above.

[0079] In Comparative Example 4, ethanol was used as the solvent, and extraction was performed in the same manner as in Example 12.

[0080] In Comparative Example 5, 1,3-butylene glycol was used as the solvent, and extraction was performed in the same manner as in Example 12.

[0081] (Results of the implementation) For each of Examples 12-21 and Comparative Examples 3-5, the total amount of flavonoids in the extracted solution was measured by the aluminum chloride colorimetric method (AlCl3 method, according to Chang et al., 2002). The results are shown in Table 4 and Figure 4. The values ​​are the mean and standard deviation (SD) of three measurements, and the unit is Quercetin Equivalent (mg QE / g-DW).

[0082] (Consideration) In the examples, several DES systems outperformed conventional solvents. In particular, the betaine / xylitol system (Bet / Xyl) showed the highest yield, averaging 170.9 mg QE / g-DW. Furthermore, the proline / butylene glycol system (Pro / BG) and the betaine / butylene glycol system (Bet / BG) also showed high yields exceeding 100 mg QE / g-DW. These results clearly demonstrate that flavonoids can be extracted from anzange with high efficiency by using deep eutectic solvents, and the betaine / xylitol system, in particular, can be considered the most effective combination.

[0083] In this specification, embodiments and examples of the present invention have been described with reference to the drawings in order to express the content of the present invention. However, the present invention is not limited to the above embodiments and examples, and includes modifications and improvements that are obvious to those skilled in the art based on the matters described in this specification.

Claims

1. A method for producing an extract solution obtained by extracting components contained in a target object, The process includes an extraction step of obtaining an extract solution in which components in the target substance are dissolved in the extraction solvent by bringing an extraction solvent containing a deep eutectic solvent mixed with hydrogen bond acceptors and hydrogen bond donors into contact with the target substance. The hydrogen bond acceptor is an amino acid or a derivative thereof. The hydrogen bond donor is one selected from sugars, organic acids, and alcohols. Method for producing an extraction solution.

2. The hydrogen bond acceptor is proline, L-arginine, L-serine, or betaine. The hydrogen bond donor is lactic acid, sucrose, xylitol, or 1,3-butylene glycol. The manufacturing method according to claim 1.

3. The molar ratio of hydrogen bond acceptors to hydrogen bond donors constituting the extraction solvent is 3:1 to 1:

6. The manufacturing method according to claim 1.

4. The deep eutectic solvent is A combination of proline and 1,3-butylene glycol. The combination of proline and lactic acid, The combination of L-arginine and lactic acid, A combination of L-serine and lactic acid, A combination of betaine and 1,3-butylene glycol. A combination of betaine and sucrose, or A combination of betaine and xylitol It was formed by The manufacturing method according to claim 1.

5. The extraction solvent further contains water. The manufacturing method according to claim 1.

6. The aforementioned extraction solution is a solution in which the lipid-soluble components in the object are dissolved. The manufacturing method according to claim 1.

7. The aforementioned extraction solution is a solution in which the water-soluble components in the target substance are dissolved. The manufacturing method according to claim 1.

8. The process further includes a filtration step to remove insoluble components from the extraction solution by filtering the extraction solution. The manufacturing method according to claim 1.

9. A method for producing a cosmetic or quasi-drug containing an extract solution obtained by the manufacturing method described in claim 1.

10. An extraction solvent comprising a deep eutectic solvent in which hydrogen bond acceptors and hydrogen bond donors are mixed, The hydrogen bond acceptor is an amino acid or a derivative thereof. The hydrogen bond donor is one selected from sugars, organic acids, and alcohols. Extraction solvent.

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