Extract containing kaempferol aglycone

Abstract

To provide a plant extract containing kaempferol aglycone, and a method for producing the same.SOLUTION: The plant extract contains kaempferol aglycone in an amount of ≥ 1mg / g expressed in terms of dried weight. The method for producing the extract comprises (1) extracting a plant raw material containing the kaempferol glycoside with a solvent and (2) hydrolyzing the extract obtained in the above (1).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention discloses technical matters relating to kaempferol aglycone and to a technique for improving the ability of plants to produce kaempferol. [Background technology]

[0002] Kaempferol is a substance with the following structural formula and is a type of natural flavonoid found in many edible plants, such as tea, broccoli, grapefruit, cabbage, kale, beans, arkroot, leek, tomato, strawberry, jasmine, Brussels sprouts, apple, quinoa, and horseradish.

[0003] [ka]

[0004] Kaempferol (hereinafter sometimes referred to as "KMP") has recently been reported to have physiological effects such as improving oxygen utilization efficiency in the body, inhibiting decline in exercise efficiency, and reducing fatigue (Patent Document 1). It has also been suggested that KMP may have anti-inflammatory and anti-cancer effects, and may be effective against various diseases such as metabolic syndrome (Non-Patent Document 1).

[0005] However, although kaempferol exerts its beneficial physiological effects in the form of aglycone, most of the kaempferol contained in plants exists as a glycoside, and therefore, even if the plant or its extract is ingested directly, the beneficial physiological effects of kaempferol are limited.

[0006] As methods for increasing the amount of polyphenols contained in plants, a cultivation method in which monocotyledonous cultivated plants such as leeks are irradiated with UV-B (Patent Document 2), a method in which plants are cultivated in warm water (Patent Document 3), and a method in which the activity of flavanone 3-hydroxylase is reduced using molecular genetic techniques (Patent Document 4) have been reported. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2019 / 044964 [Patent Document 2] Japanese Patent Application Laid-Open No. 2008-86272 [Patent Document 3] Japanese Patent Application Laid-Open No. 2017-18056 [Patent Document 4] Special Publication No. 2003-503032 [Non-patent literature]

[0008] [Non-Patent Document 1] Exp Ther Med 18:2759-2776,2019 Summary of the Invention [Problem to be solved by the invention]

[0009] Under these circumstances, one objective is to provide a means useful for utilizing the physiological effects of kaempferol, and another objective is to improve the ability of plants to produce kaempferol. [Means for solving the problem]

[0010] As a result of extensive research and studies, it has been discovered that certain plants contain large amounts of kaempferol, and that plant kaempferol glycosides can be efficiently converted to aglycones. It has also been discovered that kaempferol production ability is improved by cultivating plants under conditions of reduced oxygen concentration or oxygen partial pressure. Further research based on these findings has led to the provision of the following representative inventions.

[0011] Term A1 A plant extract containing 1 mg / g or more of kaempferol aglycone on a dry weight basis. Term A2 The plant extract according to Item A1, containing 30 mg / g or more of the kaempferol aglycone. Section A3 The plant extract according to Item 1A or A2, containing 100 mg / g or more of the kaempferol aglycone. Section A4 The plant extract according to any one of items A1 to A3, further containing quercetin in an amount of 0.1 mg / g or more on a dry weight basis. Section A5 The plant extract according to any one of Items A1 to A4, which is an extract of a plant of the Brassicaceae family. Section A6 The plant extract according to item A5, wherein the Brassicaceae plant is horseradish, kale, arugula, mustard greens, mizuna, turnip, daikon radish, broccoli, cabbage, radish sprouts, or bok choy. Section A7 The plant extract according to any one of Items A1 to A4, which is an extract of a plant of the family Theaceae. Section A8 The extract according to item A7, wherein the plant of the family Camellia is black tea, oolong tea, jasmine tea, green tea, roasted green tea, or sencha. Section A9 The plant extract according to any one of items A1 to A6, which is an extract of horseradish leaves. Section A10 A food, medicine, or cosmetic product containing the plant extract according to any one of items A1 to A9. Section A11 (1) Extracting plant materials containing kaempferol glycosides with a solvent; and (2) hydrolyzing the extract obtained in (1) above; A method for producing the plant extract according to any one of items A1 to A7, comprising: Section A12 Item 10. The method according to Item 9, wherein the hydrolysis treatment comprises treating the extract with an enzyme or a microorganism. Section A13 The method according to paragraph A11 or A12, wherein the hydrolysis treatment comprises acting an enzyme on the extract, and the enzyme comprises xylanase. Section A14 The method according to Item A13, wherein the enzyme further comprises β-glucosidase. Section A15 The method according to item A14, wherein the β-glucosidase is lactase.

[0012] Term B1 A method for producing a plant having an increased content of kaeperol and / or kaempferol glycoside per unit weight (converted to dry weight), comprising cultivating the plant under conditions of an oxygen concentration of 19% by volume or less or an oxygen partial pressure of 193 hPa or less. Term B2 A method for improving a plant's ability to produce kaempferol and / or kaempferol glycosides, comprising cultivating the plant under conditions of an oxygen concentration of 19% by volume or less or an oxygen partial pressure of 193 hPa or less. Term B3 A method for producing a plant with improved ability to produce kaempferol and / or kaempferol glycosides, comprising cultivating the plant under conditions of an oxygen concentration of 19% by volume or less or an oxygen partial pressure of 193 hPa or less. Section B4 The method according to any one of Items B1 to B3, wherein the condition is an oxygen concentration of 19% by volume or less. Section B5 The method according to any one of Items B1 to B3, wherein the condition is an oxygen partial pressure of 50 hPa or more and 193 hPa or less. Section B6 The method according to any one of Items B1 to B5, wherein the plant is a Brassicaceae plant. Section B7 The method according to Item B6, wherein the Brassicaceae plant is horseradish, kale, arugula, mustard greens, mizuna, turnip, radish, broccoli, cabbage, radish sprouts, or bok choy. Section B8 The method according to item B6 or B7, wherein the Brassicaceae plant is horseradish, radish, cabbage, or radish sprouts. Section B9 A plant obtained by the method according to any one of items B1 to B8. Section B10 Item 10. A method for producing an extract containing kaempferol and kaempferol glycosides, comprising extracting kaempferol and kaempferol glycosides from the plant according to Item 9. Section B11 (a) Extracting kaempferol and kaempferol glycosides from the plants described in paragraph B9; and (b) hydrolyzing the extract obtained in (a) above; A method for producing a kaempferol-containing extract, comprising: Section B12. The method according to any one of Items B1 to B8, which comprises providing L-tyrosine and / or L-phenylalanine to a plant.

[0013] Term C1 A method for increasing the efficiency of keperol production in a plant, comprising providing L-tyrosine and / or L-phenylalanine to a plant capable of producing keperol. Term C2 A method for increasing the kaempferol content in a plant, comprising providing L-tyrosine and / or L-phenylalanine to a plant capable of producing kaempferol. Term C3 A method for producing a plant with improved kaempferol production efficiency, comprising providing L-tyrosine and / or L-phenylalanine to a plant capable of producing kaempferol. Term C4 The method according to any one of Items C1 to C3, wherein the plant is a Brassicaceae plant. Section C5 The method according to item C4, wherein the Brassicaceae plant is horseradish, kale, arugula, mustard greens, mizuna, turnip, radish, broccoli, cabbage, radish sprouts, or bok choy. [Effects of the Invention]

[0014] In one embodiment, a plant extract containing a high content of kaempferol aglycone can be provided, and the physiological effects of kaempferol can be efficiently enjoyed. In another embodiment, plant-derived kaempferol glycoside can be efficiently converted into an aglycone.

[0015] In another embodiment, it is possible to improve the ability of a plant to produce kaempferol and / or kaempferol glycosides. In this specification, kaempferol and kaempferol glycosides may be collectively referred to as kaempferol. [Brief explanation of the drawings]

[0016] [Figure 1] The following shows the results of examining the extraction conditions for KMP glycosides from plant materials. (A) shows the results of tests using water, 30% ethanol, 50% ethanol, and 70% ethanol as the extraction solvent. The solvent temperature was 70°C in all cases. (B) shows the results of measuring the effect of solvent temperature when 50% ethanol was used as the extraction solvent. [Figure 2] 1 shows the results of measuring the aglyconization rate of KMP glycosides by treating horseradish leaf extract or quinoa extract with β-glucosidase or α-glucosidase. [Figure 3] 1 shows the results of measuring the aglyconization rate of KMP glycosides when horseradish leaf extract was treated with xylanase at various concentrations. [Figure 4] 1 shows the results of measuring the aglyconization rate of KMP glycosides by treating extracts of horseradish leaves, black tea, radish leaves, quinoa, mustard greens, or kale with xylanase. [Figure 5] 1 shows the results of measuring the effects of pH and temperature on the aglyconation of KMP glycosides in horseradish leaf extract with xylanase. [Figure 6] 1 shows the results of measuring the products produced when KMP glycoside in a horseradish leaf extract was treated with xylanase and β-glucosidase. [Figure 7] The amounts of kaempferol and quercetin measured for radish sprouts grown at oxygen concentrations of 21 vol% or 17.5 vol% are shown. "KMP" is kaempferol, and "Que" is quercetin. The vertical axis represents the measured amount of KMP or Que (mg / g). [Figure 8]This shows the synthesis pathway for kaempferol in plants. "FLS" stands for flavonol synthase, and "F3'H" stands for flavonoid 3' hydroxylase. [Figure 9] The expression levels of the F3'H gene measured for radish sprouts and broccoli are shown. The open bars represent plants grown at an oxygen concentration of 21 vol%, and the solid bars represent plants grown at an oxygen concentration of 17.5 vol%. [Figure 10] The expression levels of the FLS gene measured for radish sprouts and broccoli are shown. The open bars represent plants grown at an oxygen concentration of 21 vol%, and the solid bars represent plants grown at an oxygen concentration of 17.5 vol%. [Figure 11] This shows the amount of kaempferol measured for horseradish grown under various oxygen concentration conditions. The horizontal axis represents oxygen concentration (volume %), and the vertical axis represents kaempferol content (mg / g). [Figure 12] This shows the leaf length measured for horseradish grown under various oxygen concentration conditions. The horizontal axis represents oxygen concentration (volume %), and the vertical axis represents leaf length (cm). [Figure 13] This shows the stem length measured for horseradish grown under various oxygen concentration conditions. The horizontal axis represents oxygen concentration (volume %), and the vertical axis represents stem length (cm). [Figure 14] The biosynthetic pathway from L-tyrosine or L-phenylalanine to kaempferol is shown. TAL is tyrosine ammonia lyase, PAL is phenylalanine ammonia lyase, C4H is cinnamic acid 4-hydroxylase, 4CL is 4-coumaric acid CoA ligase, CHS is chalcone synthase, CHI is chalcone isomerase, F3H is flavanone 3-hydroxylase, FLS is flavonol synthase, and F3'H is flavonoid 3'-hydroxylase. [Figure 15]The graph shows the kaempferol (KMP) content (top row) and quercetin (Qur) content measured for radish sprouts grown with an aqueous solution containing L-tyrosine and / or L-phenylalanine. The vertical axis shows the kaempferol or quercetin content, and the horizontal axis shows the concentration of L-tyrosine and / or L-phenylalanine in the aqueous solution. The open area indicates the case where an L-phenylalanine aqueous solution was used, the shaded area indicates the case where an L-tyrosine-containing aqueous solution was used, and the solid area indicates the case where an aqueous solution containing both L-phenylalanine and L-tyrosine was used. [Figure 16] The graph shows the kaempferol (KMP) content (top row) and quercetin (Qur) content measured for broccoli sprouts grown with an aqueous solution containing L-tyrosine and / or L-phenylalanine. The vertical axis shows the kaempferol or quercetin content, and the horizontal axis shows the concentration of L-tyrosine and / or L-phenylalanine in the aqueous solution. The open area indicates the case where an L-phenylalanine aqueous solution was used, the shaded area indicates the case where an L-tyrosine-containing aqueous solution was used, and the solid area indicates the case where an aqueous solution containing both L-phenylalanine and L-tyrosine was used. [Figure 17] The graph shows the kaempferol (KMP) content (top) and quercetin (Qur) content measured for radish sprouts grown with an aqueous solution containing L-tyrosine or L-phenylalanine. The vertical axis shows the kaempferol or quercetin content, and the horizontal axis shows the concentration of L-tyrosine and / or L-phenylalanine in the aqueous solution. The open bars represent the results of cultivation under conditions of an oxygen concentration of 21 vol%, and the closed bars represent the results of cultivation under conditions of an oxygen concentration of 17.5 vol%. DETAILED DESCRIPTION OF THE INVENTION

[0017] A. Plant extracts and their manufacturing methods A1.Food extract The plant extract preferably contains, for example, 1 mg / g to 500 mg / g, preferably 10 mg / g to 300 mg / g, 30 mg to 400 mg, and more preferably 100 mg to 200 mg of kaempferol aglycone, calculated on a dry weight basis. The upper and lower limits of the kaempferol aglycone content are not particularly limited; examples of lower limits include 1 mg / g, 5 mg / g, 10 mg / g, 30 mg, 50 mg / g, and 100 mg / g, and examples of upper limits include 400 mg / g, 300 mg / g, 250 mg / g, and 200 mg / g. A preferred range of the kaempferol aglycone content can be represented by any combination of the upper and lower limits. The quantification method for kaempferol aglycone in the plant extract is not particularly limited and can be performed by conventional methods, such as the method described in the Examples below. Kaempferol is preferably measured at a pH of 2 to 7.

[0018] By ingesting plant extracts that contain a high content of kaempferol aglycone, it is possible to improve the efficiency of oxygen utilization in the body, and enjoy effects such as preventing a decline in exercise efficiency, reducing fatigue, and preventing a decline in dynamic visual acuity.

[0019] The plant extract preferably contains quercetin (aglycone type) in addition to kaempferol aglycone. The quercetin content in the plant extract is, for example, 0.1 mg / g to 100 mg / g, preferably 1 mg / g to 30 mg / g, and more preferably 5 mg to 10 mg, calculated on a dry weight basis. The lower and upper limits are not particularly limited, but examples of lower limits include 0.1 mg / g, 0.5 mg / g, 1 mg / g, 2.5 mg / g, and 5 mg / g, and examples of upper limits include 100 mg / g, 50 mg / g, 30 mg / g, 20 mg / g, and 10 mg / g. A preferred range of quercetin content can be represented by any combination of the upper and lower limits. The method for quantifying quercetin in the plant extract is not particularly limited and can be measured by conventional methods, such as the method employed in the Examples described below.

[0020] In one embodiment, the content of aglycone quercetin in the plant extract is preferably 1 / 2000 or more, 1 / 1000 or more, 1 / 500 or more, 1 / 100 or more, or 1 / 50 or more of the content of aglycone kaempferol, calculated on a dry weight basis, and is preferably 1 / 10 or less, 1 / 20 or less, 1 / 50 or less, 1 / 100 or less, or 1 / 500 or less. These upper and lower limits can be combined arbitrarily.

[0021] By containing quercetin, the plant extract can effectively provide physiological effects such as antioxidant effect, anti-inflammatory effect, anti-arteriosclerotic effect, prevention of cerebrovascular disease, antitumor effect, antihypertensive effect, and / or vasorelaxant effect.

[0022] The plant extract preferably contains ferulic acid in addition to kaempferol aglycone. The content of ferulic acid in the plant extract, calculated on a dry weight basis, is preferably, for example, 0.1 mg / g or more, 0.2 mg / g or more, 0.3 mg / g or more, 0.4 mg / g or more, 0.5 mg / g or more, 0.6 mg / g or more, 0.7 mg / g or more, 0.8 mg / g or more, 0.9 mg / g or more, 1 mg / g or more, 1.1 mg / g or more, 1.2 mg / g or more, 1.3 mg / g or more, 1.4 mg / g or more, 1.5 mg / g or more, 1.6 mg / g or more, 1.7 mg / g or more, 1.8 mg / g or more, 1.9 mg / g or more, or 2 mg / g or more. There is no particular upper limit to the ferulic acid content, but it can be set to, for example, 10 mg / g, 9 mg / g, 8 mg / g, 7 mg / g, 6 mg / g, 5 mg / g, 4 mg / g, 3 mg / g, or 2 mg / g. The method for quantifying ferulic acid in a plant extract is not particularly limited, and it can be measured by a conventional method.

[0023] By containing ferulic acid, the plant extract can effectively provide physiological effects such as antioxidant and / or antitumor effects.

[0024] In one embodiment, the plant extract is preferably an extract of a Brassicaceae plant or a Theaceae plant. Examples of Brassicaceae plants include horseradish, kale, arugula, mustard greens, mizuna, turnip, radish, broccoli, cabbage, radish sprouts, and bok choy. Examples of Theaceae plants include black tea, oolong tea, jasmine tea, green tea, roasted green tea, and sencha. In one embodiment, the plant extract is preferably an extract of a plant described in the section "B. Method for improving kaempferol-producing ability and plants with improved kaempferol-producing ability" below.

[0025] In one embodiment, the plant extract may be from a plant other than the Brassicaceae and Theaceae families, such as saffron, gymnema, onion skins, chives, parsley, and cereals such as quinoa, Canario beans, and lentils.

[0026] The plant extract may be an extract of any part of a plant. For example, it may be an extract of the whole plant, leaves, stems, roots, and flowers of a plant, or any combination thereof. For example, when a Brassicaceae plant or a Theaceae plant is used as a raw material, it is preferable to use the leaves. For plants exemplified as plants other than the Brassicaceae and Theaceae families, it is preferable to use the leaves, except for onion.

[0027] In one embodiment, the plant extract is preferably an extract of horseradish, and more preferably an extract of horseradish leaves, since horseradish leaves contain a large amount of kaempferol glycosides.

[0028] The plant extract is preferably obtained by not only subjecting a plant raw material to extraction but also subjecting the plant raw material to a treatment for converting glycosides into aglycones. The plant extract can be obtained by any method, but is preferably obtained by the plant extract production method described below.

[0029] The above-mentioned plant extracts can be processed into any desired product, such as food, pharmaceutical, or cosmetic products containing the plant extract. When used for such purposes, the plant extract may be used as is after enzyme treatment, or may be purified before use. If necessary, the plant extract may be dried to form a dry solid for use. In order to improve the storage stability of the plant extract, it is desirable to solidify it by drying (including freeze-drying). Furthermore, the dried plant extract may be powdered if necessary.

[0030] When the plant extract is used as a food ingredient, the plant extract can be prepared in the form of, for example, granules, fine granules, capsules, tablets, powder, beverages (soft drinks, carbonated drinks, nutritional drinks, powdered drinks, fruit drinks, milk drinks, jelly drinks, etc.), dairy products, confectioneries (cookies, biscuits, chocolate confectioneries, chips, cakes, gum, candy, gummy candies, steamed buns, yokan, pudding, jelly, yogurt, ice cream, sherbet, etc.), butter, other solid foods, semi-solid foods, etc. Foods containing the plant extract can be general foods, as well as foods for specified health uses, foods with functional claims, dietary supplements, functional foods, foods for patients, etc.

[0031] The proportion of the plant extract in the food product can be appropriately determined depending on the type and purpose of the food product, the content of kaempferol, the age and gender of the person taking the food product, the expected effect, etc. For example, the plant extract is preferably contained in an amount of 2 mg or more, 10 mg or more, 50 mg or more, 100 mg or more, 500 mg or more, or 1 g or more per 100 g of food product, calculated on a dry weight basis, and is preferably contained in an amount of 50 g or less, 30 g or less, 10 g or less, 5 g or less, or 2 g or less. These lower and upper limits of the content can be arbitrarily combined. For example, the food product may contain, in dry weight terms, 2 mg to 50 g, 10 mg to 30 g, or 50 mg to 10 g of plant extract per 100 g of food product. Alternatively, for example, the content of kaempferol aglycone per 100 g of food, calculated on a dry weight basis, is preferably 0.1 mg or more, 0.5 mg or more, 1 mg or more, or 2 mg or more, and is preferably 1000 mg or less, 100 mg or less, 20 mg or less, 10 mg or less, or 5 mg or less. These lower and upper limits can be combined arbitrarily. For example, the food may contain, calculated on a dry weight basis, 0.1 mg to 1000 mg, 0.5 mg to 100 mg, or 1 mg to 20 mg of kaempferol aglycone per 100 g.

[0032] The daily intake of a food containing a plant extract varies depending on the age, weight, and purpose of intake of the consumer. For example, the daily intake of the plant extract can be adjusted to 2 mg or more, 10 mg or more, or 50 mg or more, and 200 mg or less, 150 mg or less, or 100 mg or less, calculated on a dry weight basis. Alternatively, the intake of kaempferol aglycone is preferably 0.1 mg or more, 1 mg or more, 2 mg or more, 3 mg or more, or 5 mg or more, and preferably 100 mg or less, 50 mg or less, 40 mg or less, 20 mg or less, or 12 mg or less. These lower and upper limits can be arbitrarily combined. For example, the daily intake of kaempferol aglycone can be adjusted to 0.1 mg or more and 100 mg or less, 1 mg or more and 50 mg or less, or 2 mg or more and 40 mg or less.

[0033] When using a plant extract as a pharmaceutical ingredient, the plant extract can be prepared into a pharmaceutical preparation in the form of, for example, tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, aerosols, patches, injections, or suppositories. The dosage of a pharmaceutical varies depending on the age, weight, symptoms, number of doses, and purpose of administration of the recipient and cannot be uniformly determined; however, for example, the daily dosage for an adult can be 0.1 mg / kg to 10 g / kg (body weight), preferably 1 mg to 5 g / kg (body weight), of the plant extract converted to dry weight.

[0034] When using a plant extract as a cosmetic ingredient, the plant extract is prepared in various desired forms, such as paste, mousse, gel, liquid, emulsion, suspension, cream, ointment, sheet, etc. Such cosmetics can be used as various cosmetic products, such as basic cosmetics such as emulsions, creams, lotions, oils, and packs; cleansing agents such as face washes, cleansers, and body washes; wiping agents; and detergents.

[0035] The blending ratio of the plant extract in the cosmetic product can be set appropriately depending on the type of cosmetic product, the content of kaempferol, the purpose of use, etc. For example, the plant extract may be added in an amount of 0.01 g to 95 g, preferably 0.1 g to 50 g, and more preferably 1 g to 5 g, in dry weight terms, per 100 g of the cosmetic product.

[0036] A2.Method of manufacturing food extracts The method for producing the above-mentioned plant extract is not particularly limited, but preferably includes extracting a plant material containing kaempferol glycoside with a solvent and subjecting the obtained extract to a hydrolysis treatment (aglycone treatment).

[0037] The plant raw material may be in a fresh state, or may be a dried product or a roughly dried product, but from the viewpoint of efficiently extracting kaempferol or its glycosides, it is preferable to use the raw material after crushing it by a conventional method. In one embodiment, it is preferable to use the plants described below in "B. Method for improving kaempferol-producing ability and plants with improved kaempferol-producing ability" as the plant raw material.

[0038] The extraction method is not particularly limited and can be carried out by a method commonly used in the fields of pharmaceutical science or food engineering. For example, extraction using water, or one or more organic solvents selected from the group consisting of ethanol, methanol, butanol, ether, ethyl acetate, and chloroform, or a mixture of these organic solvents with water, can be used. In one embodiment, extraction is preferably carried out using a 30 to 70% aqueous ethanol solution at 30 to 70°C. The pH during extraction is preferably 3 to 7.

[0039] The plant extract extracted with water or an organic solvent may be subjected to a purification treatment such as column purification or solid-liquid separation before being subjected to a hydrolysis treatment.

[0040] The means for hydrolysis (aglyconization) is not particularly limited, but can be carried out using an enzyme, a microorganism, an acid, and / or a base.

[0041] When an enzyme is used to convert the glycoside to aglycone, the enzyme to be used is preferably selected depending on the type of sugar constituting the kaempferol glycoside and the structure of the glycoside. Sugars constituting the kaempferol glycoside that can be contained in plant extracts include glucose, galactose, rhamnose, xylose, and combinations thereof (e.g., sophorose (glucose + glucose), rutinose (rhamnose + glucose), neohesperidose (rhamnose + glucose)). These sugars are O-glycosidically linked to the 3-, 5-, and / or 7-positions of kaempferol to form the glycoside.

[0042] The composition of kaempferol glycosides varies depending on the type of plant, and the relationship between specific plant species and the types of sugars that make up the glycosides is, for example, as shown below. Horseradish leaf: galactose + xylose Saffron: glucose; Tea leaves: glucose, galactose + rhamnose + glucose, xylose + rhamnose + glucose, galactose + glucose, glucose + rutinose (rhamnose + glucose); Kale: glucose + glucose Takana: glucose + glucose + glucose Arugula: glucose

[0043] Examples of enzymes suitable for converting kaempferol glycoside to aglycone include xylanase, lactase, glucosidase, arabinosidase, rhamnosidase, xylosidase, cellulase, hesperidinase, naringinase, glucuronidase, pectinase, galactosidase, amyloglucosidase, and amylase. One or more of these can be used in appropriate combination. For example, when converting kaempferol glycoside contained in horseradish leaf extract to aglycone, it is preferable to use xylanase, more preferably a combination of xylanase and β-glucosidase, and even more preferably a combination of xylanase and lactase.When converting kaempferol glycoside contained in extracts of kale, mustard greens, radish leaves, black tea, and quinoa to aglycone, it is preferable to use β-glucosidase.

[0044] In one embodiment, when xylanase and β-glucosidase (e.g., lactase) are used in combination for aglyconization, the amounts of xylanase and β-glucosidase used are not particularly limited. For example, from the viewpoint of efficient aglyconization, the weight ratio of xylanase:β-glucosidase is 10:0.5 to 10:10, preferably 10:2 to 10:4.

[0045] When kaempferol glycoside is converted into an aglycone using a microorganism, it is preferable to use a microorganism that produces the above-mentioned enzyme. Examples of such a microorganism include microorganisms of the genus Lactobacillus, Lactococcus, Aspergillus, Bacillus, Penicillium, Rhizopus, Rhizomucor, Talaromyces, Bifidobacterium, Mortierella, Cryptococcus, and Microbacterium. One or more of these can be used in combination as appropriate. For example, when converting kaempferol glycoside contained in horseradish leaf extract to an aglycone, the microorganism to be used is one or more species selected from the group consisting of L. pentosus, L. plantarum, L. casei, L. sakei, L. kefirgranum, L. brevis, and L. tucceti, and more preferably one or more species selected from the group consisting of L. pentosus, L. plantarum, L. casei, and L. tucceti.

[0046] It is preferable to convert glycosides to aglycones by adding a microorganism to a plant extract and culturing the microorganism under conditions suitable for growth. The conditions suitable for growth can be determined for each microorganism, and can be appropriately designed, for example, at 20 to 40°C and pH 4 to 7.

[0047] Acids that can be used to convert kaempferol glycoside to aglycone include, for example, one or more acids selected from the group consisting of hydrochloric acid, sulfuric acid, and nitric acid, or mixed solvents of these acids with one or more alcohols selected from the group consisting of ethanol, methanol, and butanol. The concentration of the acid is not particularly limited, but is, for example, 0.1 to 2N. The alcohol content of the mixed solvent is not particularly limited, but is, for example, 50 to 70%. The reaction temperature is, for example, 50 to 100°C, and the reaction time can be designed to be, for example, in the range of 0.5 to 24 hours.

[0048] Examples of bases that can be used to convert kaempferol glycoside to aglycone include one or more bases selected from the group consisting of sodium hydroxide and potassium hydroxide, or a mixed solvent of one of these bases with an alcohol selected from the group consisting of ethanol, methanol, and butanol. The concentration of the base is not particularly limited, but is, for example, 0.1 to 0.5 N. The alcohol content of the mixed solvent is not particularly limited, but is, for example, 50 to 70 v / v%. The reaction temperature is, for example, 50 to 100°C, and the reaction time can be designed to be, for example, in the range of 0.5 to 24 hours.

[0049] B. Method for improving kaempferol production ability and plants with improved kaempferol production ability The method for producing a plant having an increased content of kaeperol and / or kaempferol glycoside, the method for improving a plant's ability to produce kaempferol and / or kaempferol glycoside, and the method for producing a plant having an improved ability to produce kaempferol and / or kaempferol glycoside preferably comprise cultivating the plant under an acidity concentration lower than about 20.9% by volume, which is the acidity concentration in the natural environment, or under an oxygen partial pressure lower than about 213 hPa. The oxygen concentration in the environment in which the plant is cultivated can be measured using a commercially available oxygen concentration meter. Alternatively, it can be measured using a device for controlling the oxygen concentration or oxygen partial pressure in air, which will be described later.

[0050] An acidity concentration lower than about 20.9% by volume is preferably about 20% by volume or less, about 19.5% by volume or less, about 19% by volume or less, about 18.5% by volume or less, about 18% by volume or less, about 17.5% by volume or less, about 17% by volume or less, about 16.5% by volume or less, or about 16% by volume or less. The lower limit of the acidity concentration is not particularly limited as long as plants can grow therein, but can be set to, for example, 10% by volume or more, 11% by volume or more, 12% by volume or more, 13% by volume or more, 14% by volume or more, 15% by volume or more, or 16% by volume or more. These upper and lower limit values ​​can be combined arbitrarily.

[0051] The oxygen partial pressure lower than about 213 hPa is, for example, 203 hPa or less, 198 hPa or less, 193 hPa or less, 188 hPa or less, 183 hPa or less, 178 hPa or less, 173 hPa or less, 168 hPa or less, or preferably 163 Pa or less. The lower limit of the oxygen partial pressure is not particularly limited as long as plants can grow, but can be set to, for example, 50 hPa or more, 60 hPa or more, 70 hPa or more, 80 hPa or more, 90 hPa or more, 102 hPa or more, 112 hPa or more, 122 hPa or more, 132 hPa or more, 142 hPa or more, 152 hPa or more, or 163 hPa or more.

[0052] The method for controlling the oxygen concentration or oxygen partial pressure when cultivating plants as described above is arbitrary. For example, plants can be cultivated in an environment or facility or device in which plants can be cultivated while controlling the oxygen concentration or oxygen partial pressure within a certain range. For example, an agricultural greenhouse, a plant factory, or other container or cover in which plants can be cultivated can be used. As long as the oxygen concentration or oxygen partial pressure of the air in contact with the plants is within the above range, the oxygen concentration or oxygen partial pressure of the entire environment in which the plants are cultivated does not necessarily have to be within the above range.

[0053] The oxygen concentration or oxygen partial pressure can be controlled by any means. For example, oxygen can be selectively removed from the environment in which the plants are grown, a gas other than oxygen (e.g., nitrogen or carbon dioxide) can be supplied to the environment in which the plants are grown, or air whose oxygen concentration or oxygen partial pressure has been controlled in advance can be supplied to the environment in which the plants are grown. Various devices for supplying air with a controlled oxygen concentration are known, and any of these can be selected and used as appropriate.

[0054] In one embodiment, cultivating a plant under an acidity concentration lower than about 20.9% by volume or an oxygen partial pressure lower than about 213 hPa preferably involves cultivating the plant while monitoring the oxygen concentration or oxygen partial pressure of the environment the plant is in. The monitoring frequency can be set appropriately, for example, within a range of 3 times per day to once per week, and specifically, can be set to 3 times per day, 2 times per day, 1 time per day, 1 time per 2 days, 1 time per 3 days, 1 time per 4 days, 1 time per 5 days, 1 time per 6 days, or 1 time per 7 days.

[0055] Plant cultivation conditions other than oxygen concentration or oxygen partial pressure can be appropriately designed within a range of conditions that allow plant growth, preferably conditions suitable for plant growth. For example, the light source may be natural light (sunlight) or an artificial light source, and irradiation with ultraviolet rays (e.g., UV-A, UV-B) or the like may or may not be performed. The cultivation method may be hydroponic culture (solid medium culture, hydroponics, spray culture) or conventional soil cultivation. When temperature conditions are controlled, they can be appropriately designed within a range of, for example, 10°C to 35°C or 15°C to 30°C. The location for cultivating plants is arbitrary, and in one embodiment, a location above sea level can be appropriately selected, for example, from 0 m to 1,000 m, 0 m to 800 m, or 0 m to 600 m. The concentration of gases other than oxygen (e.g., carbon dioxide) may or may not be controlled (reduced or increased) depending on the type of plant and the purpose.

[0056] In one embodiment, it is preferable to cultivate plants by providing them with L-tyrosine and / or L-phenylalanine. By providing L-tyrosine and / or L-phenylalanine, the biosynthesis of kaempferol in the plant can be promoted. The method of providing L-tyrosine and / or L-phenylalanine to plants is not particularly limited and can be any method. For example, L-tyrosine and / or L-phenylalanine can be dissolved in a solvent such as water and provided to the plant or the soil in which the plant grows, or L-tyrosine and / or L-phenylalanine can be added directly to the soil in which the plant grows. The amount of L-tyrosine and / or L-phenylalanine provided is not particularly limited and can be appropriately determined taking into account, for example, the type, size, growing environment, and growing conditions of the plant. When L-tyrosine and / or L-phenylalanine is dissolved in water and applied to plants, the concentration of L-tyrosine and / or L-phenylalanine in the aqueous solution can be, for example, 30 mg / L or more, 35 mg / L or more, 40 mg / L or more, 45 mg / L or more, 50 mg / L, 60 mg / L or more, 70 mg / L or more, 80 mg / L or more, 90 mg / L or more, 100 mg / L or more, 150 mg / L or more, 200 mg / L or more, 300 mg / L or more, 400 mg / L or more, or 500 mg / L or more. The upper limit of the concentration of L-tyrosine and / or L-phenylalanine in the aqueous solution can be any value, for example, 2000 mg / L or less, 1500 mg / L or less, 1000 mg / L or less, 500 mg / L or less, 400 mg / L or less, 300 mg / L or less, 200 mg / L or less, or 100 mg / L or less. These upper and lower concentration limits can be arbitrarily combined. L-tyrosine and / or L-phenylalanine may be applied to plants only once or continuously for a certain period of time. The certain period of time may be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, or 1 month or more, 1 year or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 4 weeks or less, 3 weeks or less, 2 weeks or less, or 1 week or less. These lower and upper limits can be combined in any manner.

[0057] The period for which the plant is cultivated under an acidity concentration lower than about 20.9% by volume or an oxygen partial pressure lower than about 213 hPa is arbitrary and can be set appropriately depending on the type of plant and the purpose, for example, from one day to several months. The period during which the plant is cultivated under an acidity concentration lower than about 20.9% by volume or an oxygen partial pressure lower than about 213 hPa during the plant growth process can also be arbitrary, and can be, for example, one or more periods selected from the group consisting of sowing, germination, the growing season, pre-harvest, post-harvest, and combinations thereof. In one embodiment, it is preferable to cultivate the plant under an acidity concentration lower than about 20.9% by volume or an oxygen partial pressure lower than about 213 hPa throughout the entire period from sowing to harvest.

[0058] The type of plant is not limited, but it is preferable that the plant has the ability to produce kaempferol. The plant capable of producing kaempferol may be a plant that inherently has the ability to produce kaempferol, or a plant in which the ability to produce kaempferol has been artificially acquired by genetic engineering techniques or the like. In one embodiment, the plant is preferably a Brassicaceae plant or a Theaceae plant, and more preferably a Brassicaceae plant.

[0059] Examples of Brassicaceae plants include horseradish, kale, arugula, mustard greens, mizuna, turnip, daikon radish, broccoli, cabbage, radish sprouts, and bok choy. In one embodiment, the Brassicaceae plant is preferably horseradish, daikon radish, cabbage, or radish sprouts. Examples of Theaceae plants include black tea, oolong tea, jasmine tea, green tea, roasted green tea, and green tea.

[0060] Examples of plants outside the Brassicaceae and Theaceae families that are capable of producing kaempferol include saffron, gymnema, onion skins, chives, parsley, and cereals such as quinoa, Canario beans, and lentils.

[0061] Plants cultivated by the above-mentioned method have a higher ability to produce kaempferol or kaempferol glycosides and / or a higher kaempferol or kaempferol glycoside content than plants cultivated under an acidity concentration of about 20.9% by volume, which is the acidity concentration in the natural environment, or under an oxygen partial pressure of about 213 hPa. Furthermore, it is preferable that plants cultivated by the above-mentioned method do not suffer from growth inhibition (e.g., reduced hypocotyl, reduced leaf area). The amount of kaempferol and / or kaempferol glycosides in a plant can be measured by the method employed in the Examples described below.

[0062] In one embodiment, there is provided a method for producing an extract containing kaempferol and kaempferol glycosides, which comprises extracting kaempferol and kaempferol glycosides from a plant cultivated by the above-mentioned method. By using a plant cultivated by the above-mentioned method as a raw material, an extract with a high content of kaempferol and / or kaempferol glycosides can be efficiently obtained.

[0063] The extraction method is not particularly limited and can be carried out by a method commonly used in the fields of pharmaceutical science or food engineering. For example, extraction using water, or one or more organic solvents selected from the group consisting of ethanol, methanol, butanol, ether, ethyl acetate, and chloroform, or a mixture of these organic solvents with water, can be used. In one embodiment, extraction is preferably carried out using a 30 to 70% aqueous ethanol solution at 30 to 70°C. The pH during extraction is preferably 3 to 7.

[0064] In one embodiment, there is provided a method for producing a kaempferol-containing extract, which comprises hydrolyzing kaempferol glycoside extracted from a plant cultivated by the above-described method. By using a plant cultivated by the above-described method as a raw material, an extract with a high kaempferol content can be efficiently obtained. The means for hydrolysis (aglycone formation) is not particularly limited, and can be carried out using an enzyme, a microorganism, an acid, and / or a base. For example, a kaempferol-containing extract can be obtained by the extraction method described in A above. Furthermore, the kaempferol-containing extract can be used for the uses, purposes, and forms described in A above.

[0065] C. The method for increasing the efficiency of kaeperol production in a plant, the method for increasing the kaempferol content in a plant, and the method for producing a plant with increased kaempferol production preferably include providing L-tyrosine and / or L-phenylalanine to the plant. Here, the type of plant, the method for providing L-tyrosine and / or L-phenylalanine to the plant, etc. are the same as those described in B above. [Example]

[0066] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0067] A1. Measuring the amount of KMP for each material The total KMP content of each plant material was measured using the following procedure. After milling each material, 0.8 g of the ground material was taken and 40 mL of 70% ethanol was added. The material was then suspended in a Polytron homogenizer at 20,000 rpm at room temperature for 1 minute. 1 mL of the suspension was dispensed into a glass test tube and weighed. 1 mL of 2N hydrochloric acid was added, mixed using a vortex mixer, and heated in a heat block at 100°C for 20 minutes. After heating, the suspension was cooled on ice for at least 5 minutes. After cooling, 5 mL of hexane was added and the mixture was shaken horizontally 20 times. The mixture was centrifuged in a refrigerated centrifuge at 3,000 rpm at 4°C for 5 minutes, and the upper layer was discarded. 5 mL of ethyl acetate was added and the mixture was shaken horizontally at 250 rpm at room temperature for 10 minutes. The mixture was centrifuged in a large refrigerated centrifuge at 3,000 rpm at 4°C for 5 minutes, and the upper layer was collected in a glass test tube for drying. 5 mL of ethyl acetate was added to the lower layer, and the mixture was shaken horizontally at room temperature at 250 rpm for 10 minutes. After centrifugation at 3,000 rpm at 4°C for 5 minutes, the upper layer was collected in a glass test tube for drying. The collected upper layer was dried at 60°C using a nitrogen blower. After drying, 1 mL of 70% aqueous ethanol was added, and the mixture was sonicated for 1 minute and dissolved by vortexing. The solution was passed through a 0.45 μm filter and then subjected to HPLC analysis for KMP aglycone analysis.

[0068] Furthermore, because the horseradish leaf raw material suspension, extract, and aglycone solution contain few contaminants, the following simple method was used to measure (1) the total KMP amount or (2) the KMP aglycone amount. (1) Measurement of total KMP content (acid hydrolysis method): 1 mL of sample solution was dispensed into a glass test tube and weighed. 1 mL of 2N hydrochloric acid was added, mixed using a vortex, and then heated in a heat block at 100°C for 20 minutes. After heating, the solution was cooled on ice for at least 5 minutes. After cooling, 960 μL of 2N sodium hydroxide solution was added and mixed to adjust the pH to 4-7. The entire neutralization solution was transferred to a 10 mL volumetric flask using a Pasteur pipette, and the solution was made to volume with 70% ethanol solution and mixed. After passing through a 0.45 μm filter, KMP aglycone analysis was performed using HPLC to determine the total KMP content. (2) Measurement of KMP aglycone content: 1 mL of the sample solution was dispensed and weighed into a 10 mL volumetric flask, and the volume was adjusted to 10 mL with 70% ethanol solution and mixed. After passing through a 0.45 μm filter, KMP aglycone was analyzed by HPLC to determine the KMP aglycone content.

[0069] As shown in Table 1 below, high total KMP contents were found in Brassica plants such as horseradish leaves, kale, arugula, takana (green mustard greens), and bonito leaf; Theaceae plants such as black tea leaves, oolong tea, jasmine tea, green tea, and roasted green tea; and grains such as quinoa, Canario beans, and lentils.

[0070] [Table 1]

[0071] A2.Examination of extraction conditions for KMP glycosides The extraction solvent and extraction temperature conditions for the extraction of KMP glycosides from plants were investigated. 75 g of water or 30–70% ethanol solution was added to 1.5 g of horseradish leaves as the extraction solvent, and two extraction procedures were performed at 70°C. 75 g of 50% ethanol solution was added to 1.5 g of horseradish leaves as the extraction solvent, and two extraction procedures were performed at 30–70°C. The total amount of the resulting extract was weighed, and the KMP aglycone content was measured as described above in "(2) KMP Aglycone Content Measurement." The extraction recovery rate was calculated by setting the sum of the KMP content in the extraction residue and the extract as 100%. Figure 1 shows the extraction recovery rate of KMP under each condition. As shown in Figure 1, KMP glycosides from horseradish leaves could be recovered by hot water extraction or 30–70% water-ethanol extraction, and it was confirmed that extraction temperatures of 30–70°C were optimal.

[0072] A3. Aglyconization of KMP glycosides A3-1. Aglyconization test 1 (aglyconization by β-glucosidase) Hot water extracts of horseradish leaves or quinoa were prepared and adjusted to a KMP aglycone concentration of 50 μg / mL. To the prepared reaction solution, β-glucosidase (aromase H2, Amano Enzyme), β-glucosidase (Sumiteam BGA, Shin-Nippon Chemical Industry Co., Ltd.), and α-glucosidase (Amano Enzyme) were added to a final concentration of 500 μg / mL. The mixture was incubated at 60°C for 60 minutes, and the KMP aglycone concentration was measured by HPLC. The aglycone conversion rate is shown in Figure 2, with the KMP aglycone concentration at acid hydrolysis set at 100%. Aglycone conversion of quinoa extracts was not performed using enzymes other than β-glucosidase (aromase H2).

[0073] As shown in Figure 2, when β-glucosidase (aromase H2) was used, approximately 75% of the KMP glycosides in quinoa extract were aglyconized, but only 2-5% of the KMP glycosides in horseradish leaf extract were aglyconized. Similar results were confirmed in tests using another type of β-glucosidase (Sumizyme BGA). Furthermore, no aglyconization was observed with α-glucosidase.

[0074] A3-2. Aglyconization test 2 (aglyconization by xylanase) A hot water extract of horseradish leaves was prepared and adjusted to 50 μg / mL of KMP aglycone and pH 5.0. Amano Hemicellulase 90 was added to the prepared reaction solution at final concentrations of 250, 500, 1000, and 1500 μg / mL, and the mixture was allowed to react at 50°C for 5 hours. The KMP aglycone concentration was then measured by HPLC. The aglycone conversion rate is shown relative to the KMP aglycone concentration at the time of acid hydrolysis (100%).

[0075] As shown in Figure 3, when a hot water extract of horseradish leaves was treated with xylanase (hemicellulase "Amano" 90, Amano Enzyme Co., Ltd.), 5 hours of reaction was required to achieve 70% aglyconization with 250 μg / mL of enzyme, whereas over 90% aglyconization was achieved in 4 hours with 500 μg / mL of enzyme. 1000 μg / mL of enzyme achieved 100% aglyconization in 2 hours, and 1500 μg / mL of enzyme achieved over 100% aglyconization in 1 hour. These results demonstrate that xylanase is effective in aglyconizing KMP glycosides in horseradish leaf extract.

[0076] A3-3. Aglyconization of KMP glycosides in various plant extracts by xylanase Hot water extracts of kale, mustard greens, quinoa, radish leaves, black tea leaves, and horseradish leaves were prepared and adjusted to 25 μg / mL of KMP aglycone and pH 5.0. Xylanase (hemicellulase "Amano" 90) was added to the reaction mixture at a final concentration of 1250 μg / mL, and the mixture was incubated at 50°C for 6 hours. The KMP aglycone concentration was measured by HPLC. The aglycone conversion rate is shown relative to the KMP aglycone concentration at the time of acid hydrolysis (100%).

[0077] As shown in Figure 4, it was found that almost no aglycone formation occurred when quinoa extract was treated with xylanase. It was confirmed that extracts of black tea leaves, radish leaves, mustard greens, and kale were aglycone-converted by xylanase more efficiently than quinoa extract.

[0078] A3-4. Aglyconization by combination of enzymes A hot water extract of horseradish leaves was prepared and adjusted to 50 μg / mL of KMP aglycone and pH 5.0. Xylanase (hemicellulase "Amano" 90) was added to the adjusted reaction solution at the final concentration shown in the table below. Lactase or β-glucosidase (aromase) was then added at the final concentrations shown in the table below, and the reaction was carried out at 50°C for 4 hours. The KMP aglycone concentration was measured by HPLC, and the effect of combining multiple enzymes was confirmed. The aglycone conversion rate is shown relative to the KMP aglycone concentration at the time of acid hydrolysis (100%).

[0079] As shown in Table 2, it was found that the efficiency of aglyconation was synergistically increased by combining xylanase with lactase or β-glucosidase.

[0080] [Table 2]

[0081] A3-5. Optimal conditions for aglyconization by xylanase A hot water extract of horseradish leaves was prepared and suspended at 50 μg / mL of KMP aglycone. The suspension was adjusted to pH 3–6.5, and xylanase (hemicellulase "Amano" 90) was added to a final concentration of 500 μg / mL. The mixture was then incubated at 50°C for 30 minutes to examine pH dependency. Additionally, xylanase (hemicellulase "Amano" 90) was added to a hot water extract of horseradish leaves adjusted to pH 5.0 at a final concentration of 500 μg / mL. The mixture was then incubated at 30–70°C for 30 minutes. The KMP aglycone concentration was measured by HPLC to confirm the optimal conditions for the enzymatic reaction. The aglycone conversion rate is shown relative to the KMP aglycone concentration during acid hydrolysis (100%). As shown in Figure 5, the optimal activity of xylanase was confirmed to be between pH 4 and 6, and the optimal activity temperature was around 50°C.

[0082] A3-6. Analysis of aglyconation A hot water extract of horseradish leaves was prepared and adjusted to 50 μg / mL of KMP aglycone and pH 5.0. Xylanase (hemicellulase "Amano" 90) was added to the prepared reaction solution at a final concentration of 1000 μg / mL, and the mixture was allowed to react at 50°C for 30, 60, and 120 minutes. The HPLC analysis chart was then observed to confirm the presence or absence of horseradish intermediate peaks.

[0083] As shown in Figure 6, it was confirmed that the aglyconization of horseradish leaf-derived KMP glycoside by xylanase occurs via an intermediate. Based on the results of 2-1, 2-2, and 2-4 above, it is thought that when horseradish leaf KMP glycoside is aglyconized by xylanase and β-glucosidase (lactase), an intermediate is first produced by the action of xylanase, and then aglycon-type KMP is produced by the action of β-glucosidase on the intermediate.

[0084] A4. Aglyconization using microorganisms A hot water extract of horseradish leaves was adjusted to a concentration of 50 μg / mL and inoculated with 188 bacterial strains isolated from food. After inoculation, the mixture was aerobically cultured at 37°C. After 24 and 120 hours, the KMP aglycone concentration was measured by HPLC to determine whether horseradish KMP glycosides could be converted to KMP aglycones by microbial culture. As shown in Table 3, lactic acid bacteria isolated from fermented products such as mibuna pickles, shibazuke pickles, Chinese cabbage pickles, kimchi, fermented tea, fermented pork, fermented fish, and sake koji (rice koji), including Lactobacillus plantarum, L. brevis, L. tucceti, L. sakei, L. pentosus, L. casei, and L. kefigranum, were able to efficiently convert horseradish leaf-derived KMP glycosides to aglycones with a conversion rate of over 95% within 24 hours.

[0085] [Table 3]

[0086] A5.Component analysis A 50% ethanol extract of horseradish leaves was concentrated under reduced pressure to remove the ethanol and adjusted to 3 mg / mL KMP aglycone and pH 5.0. Xylanase (hemicellulase "Amano" 90) and lactase were added to the reaction mixture at a final concentration of 3 mg / mL and 0.9 mg / mL, respectively, and the mixture was incubated at 50°C for 2 hours. The enzymes were then inactivated by heating at 105°C for 15 minutes and then lyophilized. Compounds were extracted from the resulting enzyme-treated horseradish leaf extract sample using methanol. The extract was then filtered and a spin column was used to remove insoluble matter and low-polarity components. A mock sample was also prepared by performing the same extraction procedure. The mock sample was prepared to confirm and eliminate background noise during sample preparation and LC-MS analysis. Each extract was subjected to LC-MS analysis for metabolomic analysis.

[0087] As a result, as shown in Table 4 below, it was found that the contents of quercetin and ferulic acid, as well as the aglycone type KMP, were significantly increased by the enzyme treatment. Quercetin is known to have physiological effects such as antioxidant, anti-inflammatory, anti-arteriosclerotic, prevention of cerebrovascular disease, antitumor, antihypertensive, and vasorelaxant effects. Ferulic acid is known to have physiological effects such as antioxidant and antitumor effects.

[0088] [Table 4]

[0089] B1. Measurement of KMP glycoside concentration in radish sprouts grown under low-oxygen conditions B1-1. Radish sprout cultivation The radish sprouts were sown in water-soaked absorbent cotton so that the seeds did not overlap. They were cultivated for one week in an environment where the oxygen concentration was controlled at 21.0 vol% or 17.5 vol% using an oxygen controller ProOx110 and a chamber (Kyodo International). The oxygen concentration was controlled (reduced) by filling the chamber with nitrogen.

[0090] B1-2. Measurement of kaempferol and quercetin The amounts of kaempferol and quercetin were measured after hydrolysis of all glycosides and conversion to their aglycones. The procedure was as follows: After milling each sample, 2.0 g of the ground material was weighed, 40 mL of 70% ethanol was added, and the mixture was suspended in a Polytron homogenizer at 20,000 rpm at room temperature for 1 minute. 1 mL of the suspension was dispensed into a glass test tube, 1 mL of 2N hydrochloric acid was added, mixed using a vortex mixer, and heated in a heat block at 100°C for 30 minutes. After heating, the mixture was cooled on ice for at least 5 minutes. After cooling, 5 mL of hexane was added and the mixture was shaken horizontally 20 times. After centrifugation at 3,000 rpm at 4°C for 5 minutes in a refrigerated centrifuge, the upper layer was discarded. 5 mL of ethyl acetate was added, and the mixture was shaken horizontally at 250 rpm at room temperature for 10 minutes. After centrifugation at 3,000 rpm at 4°C for 5 minutes in a large refrigerated centrifuge, the upper layer was collected in a glass test tube for drying. 5 mL of ethyl acetate was added to the lower layer, and the mixture was shaken horizontally at 250 rpm at room temperature for 10 minutes. After centrifugation at 3,000 rpm at 4°C for 5 minutes, the upper layer was collected in a glass test tube for drying. The collected upper layer was dried at 60°C using a nitrogen blower. After drying, 1 mL of 70% aqueous ethanol was added, sonicated for 1 minute, and dissolved by vortexing. The solution was passed through a 0.45 μm filter and subjected to HPLC analysis for KMP aglycone analysis, and the KMP aglycone content was determined.

[0091] B1-3.Results Figure 7 shows the amounts of kaempferol and quercetin measured for radish sprouts grown at oxygen concentrations of 21 vol% and 17.5 vol%. The KMP content in radish sprouts grown at an oxygen concentration of 17.5 vol% was 0.207 ± 0.005 mg / g (mean ± standard deviation, hereinafter the same), a significant increase of approximately twofold compared to the KMP content of radish sprouts grown under conventional cultivation conditions at an oxygen concentration of 21 vol% (0.096 ± 0.002 mg / g). On the other hand, the quercetin contents in radish sprouts grown under low-oxygen and conventional conditions were 0.02 ± 0.00 mg / g and 0.02 ± 0.01 mg / g, respectively, showing no significant effect of low-oxygen cultivation. These results confirm that reducing the oxygen concentration during cultivation increases kaempferol production.

[0092] B2. Effect of low-oxygen cultivation on the phenol biosynthetic pathway We investigated the effects of low-oxygen cultivation on the phenol biosynthetic pathway. Specifically, we measured the expression levels of flavonol synthase (FLS), which is involved in the synthesis of kaempferol from dihydrokaempferol and quercetin from dihydroquercetin, and flavonoid 3'-hydroxylase (F3'H), which is involved in the synthesis of dihydrokaempferol from dihydrohercetin, using the following procedure. Figure 8 shows the involvement of FLS in the synthesis of kaempferol from dihydrokaempferol and dihydroquercetin from quercetin, as well as the involvement of F3'H in the synthesis of dihydrokaempferol from dihydrohercetin.

[0093] B2-1. How to grow radish sprouts and broccoli The radish sprouts and broccoli were sown in moistened cotton so that the seeds did not overlap. They were grown for one week in an environment where the oxygen concentration was controlled at 21.0 vol% or 17.5 vol% using an oxygen controller ProOx110 and a chamber (Kyodo International). The oxygen concentration was controlled (reduced) by filling the chamber with nitrogen.

[0094] B2-2. Measurement of gene expression levels Each sample (whole plant) was frozen in liquid nitrogen and crushed with a mortar and pestle. 100 mg of the crushed material was weighed. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). The total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermofisher, Waltham, MA). PCR was performed using TaqMan Gene Expression Assays [flavonoid 3'-hydroxylase (F3'H)), flavonol synthase (FLS), and ACTIN] and TaqMan Fast Advanced Master Mix (Thermofisher) in a QuantStudio system. TM 3 The analysis was performed using a Real Time PCR System (Thermofisher). Actin was used as an endogenous control for quantification.

[0095] B2-3.Results The results of measuring the expression levels of the F3'H gene are shown in Figure 9, and the results of measuring the expression levels of the FLS gene are shown in Figure 10. When the F3'H gene expression level during normal cultivation at an oxygen concentration of 21 vol% is set to 1, we confirmed that the kaempferol content increased. When cultivated at an oxygen concentration of 17.5 vol%, we confirmed that the F3'H gene expression level was significantly reduced in both radish sprouts and broccoli. Furthermore, we confirmed that the expression level of FLS was significantly increased in both radish sprouts and broccoli when grown at an oxygen concentration of 17.5 vol% compared to when grown at an oxygen concentration of 21 vol%. These results suggest that when grown under low-oxygen conditions, the metabolic reaction from dihydrokaempferol to kaempferol is activated and the synthesis of other substances from dihydrokaempferol is suppressed in kaempferol-producing plants such as Brassicaceae.

[0096] B3. Effect of low-oxygen cultivation on horseradish KMP production B3-1. Cultivation of horseradish Horseradish rhizomes were sown in soil. They were cultivated for one week in an environment where the oxygen concentration was controlled at 21.0 vol%, 18.5 vol%, 17.5 vol%, or 16.5 vol% using an oxygen controller ProOx110 and a chamber (Kyodo International). The oxygen concentration was controlled (reduced) by filling the chamber with nitrogen.

[0097] B3-2. How to measure leaf length and petiole length Leaves and petioles were cut from each sample and measured using a ruler.

[0098] B3-3. Results The amount of kaempferol measured in horseradish leaves grown under each oxygen concentration condition using the same method as in 1-2 above is shown in Figure 11. It was confirmed that the amount of kaempferol produced increased as the oxygen concentration decreased during cultivation. As shown in Figures 12 and 13, limiting the oxygen concentration did not have a significant effect on leaf length or stem length.

[0099] C. Stimulation of KMP synthesis by the addition of phenylalanine and tyrosine C-1. Biosynthetic pathway from phenylalanine or tyrosine to KMP L-tyrosine and L-phenylalanine are indirect raw materials for kaempferol biosynthesis. As shown in Figure 14, L-tyrosine can be converted to kaempferol via p-coumaric acid, 4-coumaroyl-CoA, naringenin chalcone, naringenin, and dihydrokaempferol. L-phenylalanine can also be converted to kaempferol via cinnamic acid, p-coumaric acid, 4-coumaroyl-CoA, naringenin chalcone, naringenin, and dihydrokaempferol.

[0100] C-2. Effect of L-tyrosine or L-phenylalanine addition 1 Aqueous solutions containing L-phenylalanine at concentrations of 25, 50, 100, 200, or 400 mg / L, L-tyrosine at concentrations of 25, 50, 100, 200, or 400 mg / L, and both L-tyrosine and L-phenylalanine at concentrations of 25, 50, 100, 200, or 400 mg / L were prepared. Radish sprouts and broccoli sprouts were used as test plants. Each test plant was grown in the dark under an oxygen concentration of 21 vol% and given 10 ml of one of the solutions daily for 7 days. After 7 days, the kaempferol and quercetin contents of each test plant were measured in the same manner as in B1-2 above. The results for the radish sprouts are shown in Figure 15, and the results for the broccoli sprouts are shown in Figure 16. It was confirmed that the kaempferol content increased in both radish sprouts and broccoli sprouts when L-tyrosine and / or L-phenylalanine were added. L-tyrosine and / or L-phenylalanine had no significant effect on the quercetin content in radish sprouts. Furthermore, quercetin was not detected in broccoli sprouts, regardless of the addition of L-tyrosine and L-phenylalanine. Furthermore, no clear effect was observed on plant growth (total length and weight) when L-tyrosine and / or L-phenylalanine was added.

[0101] C-3. Effect of L-tyrosine or L-phenylalanine supplementation 2 Aqueous solutions containing L-phenylalanine at concentrations of 25 or 50 mg / L and L-tyrosine at concentrations of 25 or 50 mg / L were prepared. Radish sprouts were used as test plants. They were given 10 mL / day of either of these solutions for 7 days and grown in the dark under oxygen concentrations of 21 vol% or 17.5 vol%. After 7 days, the kaempferol and quercetin contents were measured as described above in B1-2. As shown in Figure 17, the addition of L-tyrosine or L-phenylalanine increased the kaempferol content under both oxygen concentrations. The increase in kaempferol content due to the addition of L-tyrosine or L-phenylalanine was significantly greater under low oxygen concentrations (17.5 vol%). This indicates that the addition of L-tyrosine or L-phenylalanine is more effective when grown under low oxygen concentrations. The addition of L-tyrosine or L-phenylalanine had no clear effect on plant growth (total length and weight).

Claims

1. A plant extract containing 100 mg / g or more of kaempferol aglycone and 0.1 mg / g or more of quercetin on a dry weight basis, The aforementioned plants are kale, arugula, mustard greens, mizuna, turnip, radish, broccoli, cabbage, daikon radish sprouts, bok choy, black tea, oolong tea, jasmine tea, green tea, roasted green tea, or sencha. plant extract.

2. (1) Extracting a plant material containing kaempferol glycoside with a solvent, and (2) The extract obtained in (1) above is subjected to decomposition by microorganisms. Includes, The aforementioned plants are horseradish, kale, arugula, mustard greens, mizuna, turnip, radish, broccoli, cabbage, daikon radish sprouts, bok choy, black tea, oolong tea, jasmine tea, green tea, roasted green tea, or sencha, The microorganism is one or more microorganisms selected from Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus tucceti, Lactobacillus sakei, Lactobacillus pentosus, Lactobacillus casei, and Lactobacillus kefigranum. A method for producing plant extracts.

3. The method according to claim 2, wherein the plant is a Brassicaceae plant.

4. The method according to claim 2 or 3, wherein the plant is horseradish.

5. The method according to Claims 2 to 4, wherein the microorganism produces xylanase.

6. The method according to claim 5, wherein the microorganism further produces β-glucosidase.

7. The method according to claim 6, wherein the β-glucosidase is lactase.

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