Method for producing ellagic acid from young leaves of plants of genus camellia, family theaceae

Abstract

To provide a method capable of producing ellagic acid simply and quickly without requiring complicated steps as conventionally or expensive raw materials.SOLUTION: Young leaves of plants belonging to genus Camellia, family Theaceae, especially Camellia japonica, are subjected to extraction with a solvent comprising at least one selected from water and polar solvents, after which an alkali is added to the resulting extract so as to render it alkaline. Subsequently, the extract is processed at 50°C or above for a predetermined time, thereby enabling production of ellagic acid simply and quickly.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing ellagic acid from young leaves of the genus Camellia in the family Theaceae. [Background technology]

[0002] Ellagic acid is a type of polyphenol compound that is widely distributed in plants in nature. It is found in high amounts in pomegranates, strawberries, raspberries, cranberries, blackberries, walnuts, eucalyptus, and cod, and is known to be particularly abundant in blackberries.

[0003] Ellagic acid is known to have blood clotting effects, skin whitening effects, cholesterol and triglyceride lowering effects, and fat burning effects. Due to these effects, ellagic acid is also used as a component of functional foods with diet effects and blood flow improvement effects. As such, ellagic acid is a useful substance that can be widely used in the fields of cosmetics and foods, including functional foods. In addition, because it has antioxidant effects, it is also used as an antioxidant.

[0004] Ellagic acid has traditionally been produced by hydrolysis and oxidation of ellagitannin. However, it is known that the extraction efficiency of the raw material, tannin, is low. On the other hand, it has long been known that ellagic acid can be synthesized by oxidative coupling of gallic acid esters and subsequent acid treatment (Patent Document 1). A method for producing ellagic acid using guava leaf extract has also been disclosed. However, this production method requires heat treatment at high temperatures of 100°C or higher and special equipment, so it is difficult to say that ellagic acid can be produced easily (Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2002-205993 A [Patent Document 2] Patent Publication No. 2015-033375 Summary of the Invention [Problem to be solved by the invention]

[0006] However, the above-mentioned ellagitannins are often contained in medicinal plants such as Geranium herb, which are expensive and difficult to obtain in large quantities, making it difficult to industrially mass-produce ellagic acid from ellagitannins. Furthermore, the method for producing ellagic acid from gallic acid requires a step of first producing gallic acid using tannins as a raw material, and then a step of producing ellagic acid from the gallic acid obtained in this step, resulting in multiple steps in the production process and making it very complicated. Given this background, a method for easily and efficiently obtaining ellagic acid has been desired. [Means for solving the problem]

[0007] In light of these circumstances, the present inventors have conducted extensive research and found that an extract containing ellagic acid can be produced in a short time by extracting young leaves of the genus Camellia (Theaceae) with a solvent containing water and one or more polar solvents, then adding an alkali to make the extract alkaline, and further treating at a high temperature. Furthermore, in order to increase the purity of the ellagic acid obtained in this way, the alkali-treated extract can be neutralized to acidic conditions, and the precipitated solid can be redissolved in an alkaline aqueous solution such as sodium carbonate, and an acid can be added to this solution to return to acidic conditions to obtain a precipitate, thereby obtaining high-purity ellagic acid.

[0008] Specifically, the following applies: (1) A method for producing ellagic acid from young leaves of the genus Camellia of the family Theaceae, comprising: an extraction step of extracting young leaves of the genus Camellia of the family Theaceae using water or one or more solvents selected from highly polar organic solvents; and an alkali treatment step of adding an alkali to the extract and raising the temperature of the extract to 50°C or higher. (2) The method for producing ellagic acid according to (1), wherein the pH during the alkali treatment step is 7 to 11. (3) The method for producing ellagic acid according to (1) or (2), wherein the alkali is a bicarbonate or a carbonate. (4) The method for producing ellagic acid according to (1), wherein the alkali treatment is carried out for 1 hour or more. (5) The method for producing ellagic acid according to (4), wherein the pH during the alkali treatment step is 7 to 11. (6) The method for producing ellagic acid according to (4) or (5), wherein the alkali is a bicarbonate or a carbonate. (7) A preparation containing ellagic acid produced by the method described in (1). [Effects of the Invention]

[0009] The present invention makes it possible to easily produce ellagic acid by extracting young leaves of the genus Camellia in the family Theaceae with water and / or a highly polar organic solvent, followed by alkali treatment. DETAILED DESCRIPTION OF THE INVENTION

[0010] Specific examples of the Camellia genus (Camellia) of the Theaceae family used in the present invention include Camellia japonica, Camellia dulcis, Camellia japonica, Camellia sasanqua, Camellia yucca, Camellia japonica ...

[0011] Furthermore, the origin of the Camellia genus of the Theaceae family is not particularly limited as long as the effects of the invention are achieved. Camellia japonica grows in areas south of Honshu in Japan, including the Goto Islands and Izu Oshima.

[0012] The young leaves of camellia japonica used in the present invention are young leaves harvested within two months, preferably within one month, and more preferably within two weeks after sprouting from the leaf buds of the branches or shoots of the camellia japonica. The sprouting period of the young leaves of camellia japonica used in the present invention is not particularly limited as long as the effects of the invention are achieved, but it is preferable to use young leaves that sprout in spring (March to June). Even if the young leaves of camellia japonica used for extraction contain shoots or branches, there is no particular problem as long as the effects of the invention are achieved.

[0013] A characteristic of young leaves of Camellia japonica that can be used in the present invention is that their color ranges from yellow-green to bright green. Furthermore, the moisture content of young leaves that can be used in the present invention is 60% by weight or more, preferably 70% by weight or more. The moisture content can be measured by any common method, including a Karl Fischer moisture meter, a heat-drying moisture meter, and an infrared moisture meter. Furthermore, when young leaves of the genus Camellia (Theaceae) are dried at 60°C for 12 hours, the weight of the dried young leaves of the genus Camellia (Theaceae) is 40% by weight or less, more preferably 25% by weight or less. Furthermore, leaves that have grown to a certain extent (specifically, leaves that have grown for about six months) will weigh approximately 50% by weight or more after drying.

[0014] (Pretreatment) The young leaves of the genus Camellia of the family Theaceae used for this extraction can be used as is for producing ellagic acid by the method described in the present invention after harvesting, but can also be pre-treated as follows.

[0015] One method for processing young leaves is to dry them. Drying young leaves improves extraction efficiency and allows for long-term storage while maintaining the state of the young leaves immediately after harvesting. As a result, it becomes possible to produce large amounts of ellagic acid using large amounts of young leaves. The method for drying young leaves is not particularly limited as long as it achieves the effects of the invention. Specifically, young leaves can be dried by natural drying, sun drying, air drying, heat drying, hot air drying, vacuum drying, infrared drying, etc. These drying methods may also be combined. Among these, hot air drying is preferred. The temperature conditions for hot air drying are not particularly limited, but are specifically 50°C or higher, preferably 60°C or higher. The drying time is also not particularly limited, but is specifically 6 hours or longer, preferably 12 hours or longer. The weight of the young leaves after drying is preferably 40% by weight or less, more preferably 25% by weight or less, of the weight of the young leaves before drying.

[0016] The young leaves can also be refrigerated. By storing them in a refrigerator, after securing a certain amount of young leaves of the Theaceae family, a large amount of young leaves of the Theaceae family can be used for subsequent processes, allowing for the extraction of large amounts of ellagic acid. Furthermore, even if the harvesting location and the subsequent process location are different, the state of the young leaves of the Theaceae family can be maintained. The refrigeration conditions are not particularly limited as long as the effects of the invention are achieved, but specifically, the refrigeration temperature is 15°C or lower, preferably 10°C or lower, and more preferably 5°C or lower.

[0017] Thus, young leaves of the genus Camellia of the family Theaceae can be pretreated by drying, refrigeration, or a combination of these.

[0018] Furthermore, to improve extraction efficiency, it is preferable to finely chop or otherwise process young leaves of the genus Camellia of the family Theaceae before extraction from them, regardless of whether they are dried or refrigerated. Methods for finely chopping include, but are not limited to, pulverization using scissors, a grinder, or a mill such as a cutter mill, hammer mill, ball mill, or jet mill.

[0019] (Crude extraction process of young leaves of the Camellia genus of the Theaceae family) To obtain ellagic acid from young leaves of the genus Camellia (Theaceae), first, necessary components are extracted from the young leaves using a solvent (hereinafter, this step is referred to as the "crude extraction step"). In this step, young leaves of the genus Camellia (Theaceae) can be used as they are, or young leaves that have been dried and / or refrigerated as described above can also be used. The solvent that can be used in this crude extraction step is not particularly limited as long as it has high polarity. Specifically, water, methanol, ethanol, phenol, ethylene glycol, butylene glycol, isopropanol, acetic acid, dimethyl sulfoxide, acetonitrile, etc. are preferred. Water, methanol, ethanol, ethylene glycol, and butylene glycol are preferred, and water, methanol, and ethanol are even more preferred. Two or more of these solvents may be used as long as the effects of the invention are achieved. The amount of solvent used in the crude extraction step is not particularly limited, but it is preferable to use a solvent in an amount 10 times or more by weight relative to the young leaves. When extracting using dried young leaves, it is also preferable to use a solvent in an amount 10 times or more by weight relative to the dried young leaves.

[0020] The temperature of the solvent during the crude extraction step is not particularly limited as long as the effects of the invention are achieved. Specifically, the temperature is preferably 40°C or higher, more preferably 50°C or higher, and even more preferably 60°C or higher.

[0021] The time for the crude extraction step is not particularly limited as long as the effects of the invention are achieved. Specifically, it is 1 hour or more, more preferably 2 hours or more, and even more preferably 3 hours or more.

[0022] The extraction method in the crude extraction step is not particularly limited. Specific examples include infusing young leaves in a solvent and stirring a solvent containing young leaves. In addition to these methods, decompression or pressurization may also be used as long as the effects of the invention are achieved.

[0023] Insoluble matter can be removed from the crude extract solution obtained in this crude extraction step of young leaves of the genus Camellia in the family Theaceae (hereinafter referred to as "crude young leaf extract") as needed. Removing the insoluble matter facilitates subsequent steps and makes it easier to produce products using the extract. Methods for removing insoluble matter include sieving through a sieve of a certain size, filtering using filter paper, gauze, mesh filters, etc., and using a centrifuge. These methods can also be combined.

[0024] (alkali treatment) Next, as the alkali treatment, an alkali is added to the crude young leaf extract to make the crude young leaf extract alkaline, and the solution is treated at high temperature.

[0025] The alkali used in this alkali treatment may be any substance that renders the crude young leaf extract alkaline. Specifically, the alkali is not particularly limited as long as it is such that, when the pH of the crude young leaf extract to which the alkali has been added is measured, the pH is 7 to 11, preferably 7 to 10, and more preferably 7 to 9. Specific alkalis include bicarbonates, carbonates, ammonium salts, amines, sodium hydroxide, calcium hydroxide, and oxides of alkali metals or alkaline earth metals, with bicarbonates, carbonates, and ammonia being preferred, and sodium bicarbonate being more preferred. It is also possible to use two or more of these alkalis.

[0026] When sodium bicarbonate is used as the alkali for the alkali treatment, the amount of sodium bicarbonate added is not particularly limited as long as the effects of the invention are achieved. Specifically, 1 mg or more of sodium bicarbonate can be added per gram of young leaves of the genus Camellia of the Theaceae family dried by the drying method described above, preferably 2 mg, and more preferably 4 mg. Furthermore, the proportion of sodium bicarbonate in the extract using dried young leaves is not particularly limited as long as the effects of the invention are achieved, but specifically, it is 0.5 wt. %, more preferably 1 wt. %, and even more preferably 2 wt. % of the extract.

[0027] The method for adding an alkali to the crude young leaf extract is not particularly limited, and specific examples include a method in which the alkali is added directly to the crude young leaf extract, and a method in which the alkali is dissolved in a solvent such as water in advance and the resulting solution is added to the crude young leaf extract.

[0028] The temperature of the alkali treatment may be 50°C or higher, preferably 60°C or higher, and more preferably 80°C or higher, as the temperature of the crude extract of young leaves to which the alkali has been added.

[0029] The alkali treatment time is not particularly limited as long as the effects of the present invention are achieved. Specifically, the alkali treatment time is 1 hour or more, preferably 2 hours or more, and more preferably 3 hours or more, at the above temperature.

[0030] (Precipitation process) The alkali-treated crude young leaf extract can be used as is in products, etc., but by further adding an acid, ellagic acid can be precipitated, i.e., solidified. Precipitation, i.e., solidification, enables the ellagic acid produced by the method of the present invention to be stably stored for a long period of time. Here, "precipitation" refers to precipitating ellagic acid dissolved in the alkali-treated crude young leaf extract from the solution. The acid is added to the alkali-treated crude young leaf extract so that the pH of the solution becomes 2 to 7, preferably 3 to 6.5, and more preferably 4 to 6. The method for adding the acid is not particularly limited. It can also be added directly to the alkali-treated crude young leaf extract. Alternatively, the acid can be dissolved in a solvent and then added to the solution.

[0031] The acid that can be used in the precipitation step is not particularly limited as long as it can produce the effects of the present invention. Specific examples include citric acid, lactic acid, oxalic acid, phosphoric acid, acetic acid, malic acid, and hydrochloric acid. Preferred are citric acid, lactic acid, oxalic acid, phosphoric acid, and acetic acid, and more preferred are citric acid, lactic acid, and oxalic acid.

[0032] The temperature in the precipitation step is not particularly limited. Specifically, it can be performed at room temperature or below, preferably at 10°C or below, and more preferably at 5°C or below. The time for the precipitation step is not particularly limited, but is preferably 12 hours or more, more preferably 24 hours or more, even more preferably 48 hours or more, and even more preferably 120 hours or more. After the precipitation step, the solid matter can be filtered to separate the solid matter (hereinafter referred to as "camellia-derived ellagic acid solid matter").

[0033] The thus-obtained camellia-derived ellagic acid solid extracted from young leaves of the genus Camellia in the family Theaceae has a purity of 50% or more. The purity of ellagic acid can be further increased by adding the following process.

[0034] (Improved purity of ellagic acid) The purity of the ellagic acid obtained through the above steps can be increased by the following step: The camellia-derived ellagic acid solid product obtained after the precipitation step of the present invention is redissolved in an alkaline solution, and after the solid product is dissolved in the alkaline solvent, an acid is added to make the solution neutral or acidic. The ellagic acid obtained in this way (hereinafter referred to as "camellia-derived high-purity ellagic acid solid product") has a purity of 80% or more, preferably 90% or more.

[0035] The pH of the alkaline solution used in this step is not particularly limited as long as the effects of the invention are achieved, but specifically, it is 7.5 to 11, preferably 8 to 10. The solvent used in this step is not particularly limited as long as the effects of the invention are achieved, but purified water is preferred. Camellia-derived ellagic acid solids are dissolved in the alkaline solution thus prepared. It is also possible to disperse camellia-derived ellagic acid solids in purified water and then add an alkali to dissolve them. The alkali used in this step is not particularly limited as long as the effects of the invention are achieved, but includes bicarbonate, carbonate, ammonium salt, amine, sodium hydroxide, calcium hydroxide, and oxides of alkali metals or alkaline earth metals, preferably bicarbonate, carbonate, or ammonium salt, and more preferably carbonate.

[0036] The acid used in the next step is not particularly limited, but specific examples include citric acid, lactic acid, oxalic acid, phosphoric acid, acetic acid, malic acid, hydrochloric acid, etc. Preferred are citric acid, lactic acid, oxalic acid, phosphoric acid, and acetic acid, and more preferred are citric acid, lactic acid, and oxalic acid.

[0037] The ellagic acid thus obtained may be a salt of ellagic acid.

[0038] Ellagic acid thus produced from the genus Camellia of the Theaceae family can be used in a variety of products and applications.

[0039] Due to the functions of ellagic acid, it can be used in medicines, foods including functional foods, cosmetics, etc. Furthermore, the method of administration is not particularly limited.

[0040] The dosage form of ellagic acid-containing preparations is not particularly limited. Specific examples include solid oral preparations such as fine granules, powders, granules, tablets, and capsules, oral liquid preparations such as syrups and dry syrups that are dissolved when used, external preparations such as ointments, creams, external liquid patches, packs, emulsions, and lotions, and injections. Furthermore, in the food industry, the ellagic acid obtained by the present invention can be used in the form of confectioneries such as gum, tablets, candies, cookies, gummies, rice crackers, biscuits, and jellies, bread, noodles, cereals, jams, and seasonings.

[0041] In order to mold the ellagic acid obtained by the present invention into the above-mentioned dosage forms, various additives can be further added. Specific additives include excipients, sweeteners, colorants, preservatives, thickeners, stabilizers, gelling agents, binders, disintegrants, lubricants, antioxidants, color formers, bleaching agents, preservatives, antifungal agents (mold inhibitors), yeast food, gum bases, flavorings, acidulants, seasonings, emulsifiers, pH adjusters, alkaline water, leavening agents, nutritional fortifiers, etc.

[0042] Furthermore, for products using ellagic acid, the extract obtained after the alkali treatment of the present invention may be used, or the precipitate obtained after the subsequent precipitation step may be used.

[0043] The amount of ellagic acid that can be contained in the product is not particularly limited. For example, it is 0.001 to 5.0% by weight, preferably 0.01 to 2.0% by weight, based on the total weight of the food. When ellagic acid is ingested as a functional food to prevent obesity, it can be contained in the product so that the amount is about 3 mg per day.

[0044] In addition, ellagic acid has antioxidant properties and can therefore be used as an additive as an antioxidant. [Example]

[0045] The present invention will now be described in more detail with reference to examples, but the present invention is not limited thereto.

[0046] (Pre-treatment of young leaves of Yabutsubaki) Approximately 1 kg of young leaves of Yabutsubaki grown on the Goto Islands, Nagasaki Prefecture, about two weeks after budding, were collected. The collected young leaves were dried by hot air at 60 - 70°C for 12 hours (using a Oki-type food dryer: manufactured by Oki Sangyo Co., Ltd.). The dried young leaves of Yabutsubaki were ground using a cutter mill (Konappi: manufactured by Labonext Co., Ltd.), and then sieved through a sieve with a 2 mm aperture to obtain dried young leaves of Yabutsubaki (hereinafter referred to as "dried young leaves"). Hereinafter, unless otherwise specified, this pre-treated dried young leaves were used in the following Examples and Test Examples.

[0047] (Method for analyzing ellagic acid) In the following Test Examples and Examples, unless otherwise mentioned, ellagic acid was quantified by the following method. Preparation of sample solution: 900 μL of methanol was added to 100 μL of the sample stock solution to obtain a sample solution. Preparation of ellagic acid standard solution: 10 mg of ellagic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was dispersed in purified water, 20 μL of saturated sodium carbonate solution was added, and after ellagic acid was dissolved, the volume was adjusted to 10 mL with purified water. Thus, a 1 mg / mL ellagic acid standard stock solution was prepared. Then, 900 μL of methanol was added to 100 μL of the ellagic acid standard stock solution to obtain a 0.1 mg / mL ellagic acid standard solution Internal standard: 10 mg of caffeic acid was dissolved in 10 mL of 20% acetonitrile, 100 μL of which was taken and adjusted by adding 900 μL of methanol. 500 μL each of the sample solution and the internal standard solution were mixed, and HPLC analysis was performed under the following HPLC conditions. Similarly, 500 μL each of the ellagic acid standard solution and the internal standard solution were mixed, and HPLC analysis was performed under the following conditions. Using the ratio of the sample solution to the internal standard and the ratio of the ellagic acid standard solution to the internal standard, the amount of ellagic acid in the sample solution, that is, the amount of ellagic acid in the extract of Yabutsubaki young leaves, was measured. <HPLC conditions> Column: CAOCELPAK C18 MGII (5 μm, 4.6 mm × 250 mm) Mobile phase: Acetonitrile / 0.2% phosphoric acid aqueous solution (20 / 80) Column temperature: room temperature Flow rate: 1.0mL / min Detector: Photodiode array (350 nm)

[0048] (Test Example 1-1) 2.5 g of dried young leaves were placed in 47.5 g of water, heated to 60°C, and extracted for 2 hours. The extract was then filtered (filter paper No. 131: manufactured by ADVANTEC) to obtain a crude young leaf extract. The water temperature of this extract was maintained at 60°C during the heating process, and 5 mL samples were taken 8, 24, and 48 hours after the start of the heating process. These sampled solutions were used as the original sample solutions.

[0049] (Test Examples 1-2 to 1-5) The test was carried out under the same conditions as in Test Example 1, except that the test conditions were changed to those shown in Table 1, and a sample stock solution was prepared.

[0050] The amounts of ellagic acid in the sample stock solutions of Camellia japonica prepared by the methods of Test Examples 1-1 to 1-5 were measured by HPLC. The results are shown in Table 1.

[0051] [Table 1]

[0052] As shown in Table 1, it was confirmed that ellagic acid was gradually produced when crude extract of young leaves of Camellia japonica was left at high temperatures. However, even after 48 hours of storage, the amount of ellagic acid produced was small.

[0053] (Example 1-1) 2.5 g of dried young leaves were placed in 47.5 g of water, heated to 60°C, and extracted for 2 hours. The crude young leaf extract thus extracted was filtered. 50 mg of sodium bicarbonate (manufactured by Yoneyama Pharmaceutical Co., Ltd.) (equivalent to 0.1% by weight of the total extract) was added to this extract, and an alkali treatment was performed. The alkali treatment temperature was 80°C. 5 mL samples were taken 1 hour, 2 hours, 4 hours, and 8 hours after alkali treatment. 20 μL of saturated aqueous sodium carbonate solution was added to the sampling solution to prepare the original sample solution.

[0054] (Examples 1-2 to 1-3) The test conditions were as shown in Table 2. The test was carried out under the same conditions as in Example 1-1, except that the amount of sodium bicarbonate added for the alkali treatment was changed, and a sample stock solution was prepared.

[0055] The amount of ellagic acid in the sample stock solution of Camellia japonica was measured by HPLC using the methods of Examples 1-1 to 1-4. The results are shown in Table 2.

[0056] [Table 2]

[0057] As shown in Table 2, ellagic acid could be obtained in a few hours by maintaining alkaline conditions at a high temperature of 80°C. Furthermore, the amount of ellagic acid extracted increased significantly by alkaline treatment. It was also confirmed that increasing the amount of sodium bicarbonate used in alkaline treatment increased the amount of ellagic acid extracted from young Camellia japonica leaves.

[0058] Example 2-1 Four grams of dried young leaves were placed in 76 grams of purified water and extracted for one hour while maintaining the solvent temperature at 40°C. This extract was then filtered to obtain a crude young leaf extract. 40 mL of this extract was added with 150 μL of 10% aqueous sodium bicarbonate solution and subjected to alkaline treatment at 80°C for two hours. The sample was then returned to room temperature, and 1.33 mL of 10% aqueous citric acid solution was added to the solution. The solution was then stored at 4°C for six days. After storage, the mixture was thoroughly dispersed, and 10 mL of the mixture was centrifuged and the supernatant was discarded. 10 mL of purified water was added to the resulting precipitate, which was then dispersed. 33 μL of 10% aqueous sodium carbonate solution was then added to dissolve the precipitate. This solution was then filtered to obtain the original sample solution.

[0059] (Examples 2-2 to 2-12) The test was carried out under the same conditions as in Example 2-1, except that the extraction temperature and extraction time were changed as shown in Table 3, and the sample stock solution was prepared.

[0060] The amounts of ellagic acid in the sample stock solutions of Camellia japonica prepared by the methods of Examples 2-1 to 2-12 were measured by HPLC. The results are shown in Table 3.

[0061] [Table 3]

[0062] As shown in Table 3, when the extraction temperature and extraction time from young Camellia leaves were changed, the amount of ellagic acid increased the higher the extraction temperature and the longer the extraction time. Furthermore, sufficient ellagic acid was extracted even with an extraction temperature of 40°C and an extraction time of 1 hour.

[0063] (Example 3-1) 2.5 g of dried young leaves were placed in 47.5 g of water, heated to 60°C, and extracted for 2 hours. The crude young leaf extract thus extracted was filtered. 40 mg (equivalent to 0.1%) of sodium bicarbonate was added to 40 mL of the filtrate, and the solution was heated to 60°C for alkaline treatment. The alkaline-treated solution was sampled 2 and 4 hours after the start of alkaline treatment. The sampled solution was used as the original sample solution.

[0064] (Examples 3-2 to 3-4) The test conditions were as shown in Table 4. The test was carried out under the same conditions as in Example 3-1, except that the amount of sodium bicarbonate added in the alkali treatment and the temperature of the alkali treatment were changed, and a sample stock solution was prepared.

[0065] The amounts of ellagic acid in the sample stock solutions of Camellia japonica prepared by the methods of Examples 3-1 to 3-4 were measured by HPLC. The results are shown in Table 4.

[0066] [Table 4]

[0067] As shown in Table 4, it was confirmed that the amount of ellagic acid extracted from young Camellia japonica leaves increased with the amount of sodium bicarbonate, the temperature of the alkaline treatment, and the time of the alkaline treatment.

[0068] (Example 4-1) 2.5 g of dried young leaves were added to 47.5 g of water and heated to 40°C for 2 hours. The crude young leaf extract thus extracted was filtered. 200 μL of 10% aqueous sodium bicarbonate solution was added to 40 mL of the filtrate, and the solution was heated to 60°C for alkaline treatment. The alkaline-treated solution was sampled 2 and 3 hours after the start of alkaline treatment. 1.33 mL of 10% aqueous citric acid solution was added to the sampled extract. The extract thus obtained was refrigerated at 4°C for 5 days. The refrigerated extract was then centrifuged (5,000 rpm, 5 minutes) to obtain a precipitate. 10 mL of purified water was added to the precipitate, and the mixture was thoroughly stirred to disperse the precipitate. 33 μL of 10% aqueous sodium carbonate solution was added to dissolve the precipitate. The solution thus prepared was filtered to obtain the original sample solution.

[0069] (Examples 4-2 to 4-8 and Test Examples 4-1 to 4-4) The test was carried out under the same conditions as in Example 4-1, except that the extraction temperature and alkali treatment temperature were changed as shown in Table 5, and the sample stock solution was prepared.

[0070] The amounts of ellagic acid in the sample stock solutions of Camellia japonica prepared by the methods of Examples 4-1 to 4-8 and Test Examples 4-1 to 4-4 were measured by HPLC. The results are shown in Table 5.

[0071] [Table 5]

[0072] As shown in Table 5, the extraction of ellagic acid from young leaves of Camellia japonica was insufficient at an alkali treatment temperature of 40°C, but was sufficient at 60°C and 80°C. Furthermore, at alkali treatment temperatures of 60°C and 80°C, sufficient ellagic acid could be obtained even after one hour.

[0073] (Example 5-1) Five grams of dried young leaves were added to 95 grams of purified water and extracted at 40°C for two hours. The extract was then filtered. 400 μL of 10% sodium bicarbonate solution was added to 80 mL of the resulting filtrate, and alkaline treatment was performed at 80°C. 20 mL of each aliquot was taken 1, 2, and 3 hours after the start of alkaline treatment. Each sample was returned to room temperature, and 500 μL of 10% citric acid solution was added. The samples thus prepared were stored at 4°C for six days. The refrigerated extract was then centrifuged (5,000 rpm, 5 minutes) to obtain a precipitate. 20 mL of purified water was added to the precipitate, and the mixture was thoroughly stirred to disperse the precipitate. 66 μL of 10% sodium carbonate solution was then added to dissolve the precipitate. The solution thus prepared was filtered to obtain the original sample solution.

[0074] (Examples 5-2 to 5-11) The test conditions were as shown in Table 6. The test was carried out under the same conditions as in Example 5-1, except that the solvent and extraction temperature during extraction were changed, and the sample stock solution was prepared.

[0075] The amounts of ellagic acid in the sample stock solutions of Camellia japonica prepared by the methods of Examples 5-1 to 5-11 were measured by HPLC. The results are shown in Table 6.

[0076] [Table 6]

[0077] As shown in Table 6, when the solvent in the crude extraction step was extracted with an aqueous solution containing water and / or a polar solvent and then alkaline treatment was performed at 80°C under alkaline conditions, the amount of ellagic acid extracted was determined regardless of the type of extraction solvent used in the crude extraction step.

[0078] (Example 6-1) 20 g of young leaves from Camellia japonica, harvested on April 5, 2023, within one month of sprouting, were collected and dried with hot air at 60-70°C for 12 hours. 18 mL of purified water was added to 2 g of dried young leaves, and the mixture was extracted at 60°C for 2 hours. The crude extract of young leaves thus extracted was cooled and then filtered. 200 μL of 10% aqueous sodium bicarbonate solution was added to 10 mL of this filtrate, and the mixture was subjected to alkaline treatment at 80°C for 2 hours. After 2 hours, the solution was returned to room temperature. The solution thus obtained was used as the sample stock solution.

[0079] (Examples 6-2 to 6-4, Test Examples 6-1 to 6-4) The test was carried out under the same conditions as in Example 6-1, except that leaves of Camellia japonica collected at the times shown in Table 7 were used, and a sample stock solution was prepared.

[0080] The amounts of ellagic acid in the sample stock solutions of Camellia japonica prepared by the methods of Examples 6-1 to 6-4 and Test Examples 6-1 to 6-4 were measured by HPLC. The results are shown in Table 7.

[0081] [Table 7]

[0082] As shown in Table 7, when young leaves of the Camellia genus of the Theaceae family collected within one month of sprouting were extracted and treated with alkali, ellagic acid was able to be extracted.

[0083] Example 7 10 kg of dried young leaves were added to 185 kg of purified water and extracted at 60°C for 2 hours. The extracted solution was filtered (filter paper No. 131 (manufactured by ADVANTEC)). The residue after filtration was washed with 5 kg of purified water and combined with the previous filtrate to obtain a crude ellagic acid extract. 3.3 kg of 10% aqueous sodium bicarbonate solution was added and left at 80°C for 2 hours. After cooling, 1.65 g of citric acid was added and left at 4°C for 6 days. The precipitate formed upon cooling was then filtered, and the remaining solid was dried at 60°C for 12 hours (Daiki food dryer, manufactured by Daiki Sangyo) to obtain 500 g of camellia-derived ellagic acid solids.

[0084] Example 8 200 g of the camellia-derived ellagic acid solids produced in Example 7 were dispersed in 50 L of water, and 270 g of a 10% aqueous sodium carbonate solution was added. After thorough stirring, the mixture was filtered (filter paper No. 131 (manufactured by ADVANTEC)). 200 g of citric acid was added to the filtrate, and the mixture was cooled at 4°C for 3 days. After cooling, the resulting precipitate was collected by centrifugation (5,000 rpm, 5 min). The amount of the thus-obtained high-purity camellia-derived ellagic acid solids was approximately 80 g.

[0085] Confirmation of the purity of camellia-derived ellagic acid solids and high-purity camellia-derived ellagic acid solids 10 mg of the camellia-derived ellagic acid solid prepared in Example 7 was dispersed in 5 mL of purified water, 40 μL of saturated aqueous sodium carbonate solution was added, and after confirming that the solid was completely dissolved, purified water was added to make the total volume 10 mL, which was used as a sample solution of camellia-derived ellagic acid solid.

[0086] 10 mg of the high-purity ellagic acid solids derived from camellia prepared in Example 8 was dispersed in 5 mL of purified water, 40 μL of saturated aqueous sodium carbonate solution was added, and after confirming that the solids were completely dissolved, purified water was added to make the total volume 10 mL. This was used as a sample solution of high-purity ellagic acid solids derived from camellia.

[0087] The internal standard described above was added to each sample solution prepared in this way, and HPLC was performed under the conditions described above. The results were used to measure the quantitative values ​​using an ellagic acid standard solution prepared in the same way as the standard.

[0088] [Table 8]

[0089] As shown in Table 8, the method of the present invention made it possible to obtain ellagic acid with few impurities. [Industrial Applicability]

[0090] According to the present invention, a method for producing highly pure ellagic acid from young camellia leaves is provided. The young camellia leaves are extracted with water or a polar solvent aqueous solution, followed by the addition of bicarbonate or carbonate to make the mixture weakly alkaline and allowing it to stand under heating. This method allows for the production of ellagic acid in a shorter time and more easily than conventional methods. Furthermore, ellagic acid with improved purity can be produced by dissolving the resulting precipitate with carbonate, removing insoluble matter, and adding citric acid to the filtrate to acidify it. The ellagic acid produced in this manner can be incorporated into foods, beverages, and cosmetics.

Claims

1. A method for producing ellagic acid from young leaves of the genus Camellia of the family Theaceae, comprising: An extraction step of young leaves of the genus Camellia of the family Theaceae using water or one or more solvents selected from highly polar organic solvents; An alkaline treatment process in which alkali is added to the extract and the temperature of the extract is raised to 50°C or higher. A manufacturing method comprising the steps of:

2. The method for producing ellagic acid according to claim 1, wherein the pH during the alkali treatment is 7 to 11.

3. 3. The method for producing ellagic acid according to claim 1 or 2, wherein the alkali is a bicarbonate or a carbonate.

4. 2. The method for producing ellagic acid according to claim 1, wherein the alkali treatment time is 1 hour or more.

5. The method for producing ellagic acid according to claim 4, wherein the pH during the alkali treatment is 7 to 11.

6. 6. The method for producing ellagic acid according to claim 4 or 5, wherein the alkali is a bicarbonate or a carbonate.

7. A preparation containing ellagic acid produced by the method described in claim 1.

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