JP2019156746A - Production method of triterpenoid high content seed extract and triterpenoid high content seed extract - Google Patents

Abstract

To provide a production method of a triterpenoid containing extract including at high density, triterpenoid such as limonoid that is simple and economical so that the method can be executed in an industrial scale.SOLUTION: The invention provides a production method of triterpenoid containing seed extract including following steps (1)-(3). Step (1) for subjecting seeds of citrus to extraction processing using a triterpenoid soluble solvent for acquiring a seed crude extraction liquid; step (2) for subjecting the seed crude extract to hydration processing and concentration processing for acquiring a seed crude extract; and step (3) for subjecting the seed crude extract to acid processing and salt addition processing for acquiring a triterpenoid containing seed extract as a first precipitate.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for producing a triterpenoid-rich seed extract using citrus seeds such as a seeker and a triterpenoid-rich seed extract.

As triterpenoids biosynthesized by citrus fruits, limonoids such as limonin, nomiline, echangin, and obaknon are known. Since limonoids generally have a bitter taste, portions containing a large amount of limonoids, such as pericarp and seeds, are removed when processing citrus fruits.

On the other hand, some limonoids have physiological activities such as an antitumor effect, an activity of detoxifying metabolic enzymes, an action to shorten sleep time during anesthesia, an analgesic action, and an action to improve serum lipid metabolism (see Patent Document 1). reference).

Among limonoids, a method for producing obacunone is known in Patent Document 2.

JP 2006-083150 A JP-A-6-199864

According to the method described in Patent Document 2, nobline is obtained by dissolving nomiline in acetonitrile and then subjecting the obtained solution to an alkali treatment. However, Patent Document 2 does not have a detailed disclosure of a method for isolating nomiline from citrus seeds.

For example, the total amount of limonoid contained in the citrus peel is about 0.01 wt%, which is a very small amount. There are few known methods for efficiently obtaining such a small amount of limonoid on an industrial scale.

Therefore, the present invention provides a method for producing a triterpenoid-containing extract containing a high concentration of triterpenoids such as limonoids, so that the invention can be carried out on an industrial scale. Is a problem to be solved.

As a result of intensive studies to solve the above-mentioned problems, the inventors of the present invention provide a crude seed extract obtained by subjecting citrus seeds to extraction treatment, followed by hydrolysis treatment, and then subjecting to acid treatment and salting treatment. Thus, it was found that a seed extract having a high content of triterpenoids such as nomiline and limonin can be obtained. Further, by subjecting the obtained seed extract to a treatment according to the water content of the seed extract, the content of either nomiline or limonin is large, and these amounts are 60 wt% or more of the whole. We succeeded in producing a high content of triterpenoids. The present invention has been completed based on such knowledge and success.

Therefore, according to one aspect of the present invention, the following methods [1] to [8] are provided.
[1] A method for producing a triterpenoid-containing seed extract comprising the following steps (1) to (3).
(1) A step of obtaining a crude seed extract by subjecting the citrus seeds to an extraction treatment using a triterpenoid-soluble solvent. (2) By subjecting the crude seed extract to a hydration treatment and a concentration treatment, Step (3) of obtaining an extract Step (2) of obtaining a triterpenoid-containing seed extract as a first precipitate by subjecting the crude seed extract to acid treatment and salting treatment [2] and the following step (4) and (5) The method according to [1].
(4) Step of obtaining a triterpenoid-containing seed extract and a second supernatant as a second precipitate by subjecting the first precipitate to a drying treatment and a washing treatment (5) The second supernatant The method according to [1], further comprising a step [3] of obtaining a triterpenoid-containing seed extract as a third precipitate by subjecting to a stationary treatment.
(6) Step of obtaining a triterpenoid-containing seed extract and a fourth supernatant as a fourth precipitate by subjecting the first precipitate to a washing treatment (7) Drying the fourth supernatant A step of obtaining a triterpenoid-containing seed extract and a fifth supernatant as a fifth precipitate by being subjected to a treatment, a solvent addition treatment and a stationary treatment [4], further comprising the following step (8): [3] The method described in 1.
(8) A step of obtaining a triterpenoid-containing seed extract as a sixth precipitate by subjecting the fifth precipitate to a washing treatment [5] The citrus fruits are citrus depressa , Citrus unshiu ( C. unshiu), Tachibana (C.tachibana), Koji (C.leiocarpa), Girimikan (C.tardiva), Jimikan (C.succosa), distichum (C.kinokuni), Kobenimikan (C.erythrosa), Sunki (C.sunki ), Chichuuka Lee Mandarin (C.deliciosa), King (C.nobilis), ponkan (C.retuculata), Dan Sea Tangerine (C.tangerina), Hanayu (C.hanayu) Mandarin orange (C.reticulata), Sanki (C.sunki), Stewartia Tachibana (C.nippokoreana), is at least one of citrus are selected from the group consisting of dekopon and Kiyomi, any one of [1] to [4] 2. The method according to item 1.
[6] The method according to any one of [1] to [5], wherein the triterpenoid is a limonoid.
[7] The method according to [6], wherein the limonoid is at least one limonoid selected from the group consisting of limonin, nomiline, nomylic acid, deacetylnomyrin, echangin, and obacnon.
[8] Any of [1] to [2] and [5] to [7], wherein the second precipitate is a triterpenoid-containing seed extract having a large limonin content relative to a nomiline content. The method according to claim 1.
[9] The fourth precipitate, the fifth precipitate, or the sixth precipitate is a triterpenoid-containing seed extract having a large nomiline content relative to a limonin content. [1] And the method according to any one of [3] to [7].

According to another one aspect | mode of this invention, the extract shown to the following [10]-[11] is provided.
[10] A triterpenoid-containing seed extract containing citrus seeds as a raw material and containing 1 to 90 wt% of a triterpenoid.
[11] A triterpenoid-containing seed extract containing 1 to 90 wt% of a triterpenoid obtained by the method according to any one of [1] to [9].

According to the method of one embodiment of the present invention, even if there is no complicated equipment or device, the amount of impurities is reduced by a simple process from citrus seeds, and the triterpenoid content is significantly increased. Inclusions can be obtained. Therefore, the method of one embodiment of the present invention is a method capable of easily and economically producing a high content of triterpenoids so that the method can be carried out on an industrial scale. In addition, according to the method of one embodiment of the present invention, a high content of triterpenoids having a large content of nomiline and / or limonin can be produced as desired.

According to the triterpenoid-containing seed extract of one aspect of the present invention, an antitumor action and an activity of a detoxifying metabolic enzyme comprising, as an active ingredient, a triterpenoid-containing extract itself or a purified triterpenoid obtained by purification from a triterpenoid-containing seed extract A wide variety of foods, drinks, pharmaceuticals, quasi-drugs, cosmetics and the like that are expected to have physiologically active effects such as action, sleep time shortening action during anesthesia, analgesic action, and serum lipid metabolism improving action can be produced.

FIG. 1 is a flowchart schematically illustrating the method according to the first aspect of the present invention. FIG. 2 is a flowchart schematically illustrating the method according to the second aspect of the present invention.

Hereinafter, although the method and the extract which are one aspect | mode of this invention are demonstrated, the technical scope of this invention is not limited only by the matter of this item, and as long as this invention achieves the objective. Various aspects can be taken.

The method of one aspect of the present invention relates to a method for producing a seed extract containing a high content of triterpenoids by sequentially subjecting citrus seeds to extraction treatment, water treatment, acid treatment, salting treatment, and the like. More specifically, the method of one embodiment of the present invention includes at least the following steps (1) to (3).
(1) A step of obtaining a crude seed extract by subjecting the citrus seeds to an extraction treatment using a triterpenoid-soluble solvent. (2) By subjecting the crude seed extract to a hydration treatment and a concentration treatment, Step of obtaining an extract (3) Step of obtaining a seed extract containing triterpenoid as a first precipitate by subjecting the crude seed extract to acid treatment and salting treatment

Triterpenoid-containing seed extract contains citrus seeds as a raw material and contains triterpenoids at a relatively high concentration.

The triterpenoid is not particularly limited as long as it is generally known. For example, a triterpenoid is a fat-soluble compound composed of 30 carbon atoms by combining 6 isoprene units composed of 5 carbons. It can be said that it is a compound group. The triterpenoid contained in the extract obtained by the method of one embodiment of the present invention is preferably a limonoid that is a cyclic compound having both a lactone ring and a furan ring, and limonin, nomiline, nomylic acid, deacetylnomyrin, and echan Gin and obacnone are more preferred, and limonin and nomiline are even more preferred.

Citrus is not particularly limited as long as it is a citrus plant containing a triterpenoid in its seed. As citrus fruits, for example, oranges such as quinotto, jaffa orange, joppa, navel orange, Valencia orange, Fukuhara orange, blood orange, bergamot; grapefruits such as orangelo, grapefruit; Shikuwasa, Citron, Sudachi, Daidai, Shinhime, Bushcan, Yuuko, Tangerine, Yuzu, Lime, Lemon and other perfumed citrus fruits; Cocktail fruit, Kawano Natsudaidai, yellow mandarin orange, bellows, Shonan gold, Sweety, Summer mandarin orange, Miscellaneous citrus fruits such as Hassaku, Haruka, Koharu, Hinata Summer; Aki no Shine (Decopon), Iyokan, Ehime Trial No. 28, Kiyomi, Saga Trial No. 34 (Decopon), Shion's Megumi, Shiranuhi (Dekopon), Setoka, Setomi, General Ki ( Tongols such as Kopon), Tankan, Harumi, Fertilizer (Dekopon), Marcot, Rei; Tangeros such as Agrifruit, Summer Fresh, Sweet Spring, Seminole, Tangero, Mineola; Buntans such as white birch and buntan (Zabong); Citrus unshiu such as Wenzhou mandarin orange, Otsu No.4, Calamancy, Ganpei, Kishu mandarin orange, Koji (citrus), Sakurajima mandarin orange, Tachibana, Fujinaka mandarin orange, Ponkan, mandarin orange; Ginseng species such as Karachi, Kumquase such as long-leaf kumquat, long-seed kumquat, Ningbo kumquat, Fuzhou kumquat, Hong Kong kumquat, and Marukin kumquat are preferred. Citrus of the ward and Citrus citrus More preferably Citrus depressa (Citrus depressa), satsuma mandarin (C.unshiu), Tachibana (C.tachibana), koji (C.leiocarpa), Girimikan (C.tardiva), Jimikan (C.succosa), distichum (C.Kinokuni ), Cobenimikan ( C. erythrosa ), Sunki ( C. sunki ), Cichlid Mandarin ( C. deliciosa ), King ( C. nobilis ), Ponkan ( C. retuculata ), Dancy tangerine ( C. tangerina C ) Hanayu ), Mandarin Orange ( C. reticulata ), Sanki ( C. sunki ), Kouraitachibana ( C. nippocoreana ), Shiranuhi and Kiyomi, and more preferably a seeker. Moreover, these citrus fruits should just be 1 type, or 2 or more types, and the hybrid of the citrus fruits mentioned as the said specific example may be sufficient.

Citrus seeds are seeds with flavedos (outer pericarp), albedo (medium pericarp), vascular bundles, oil vesicles, fruit hearts, carrots, flesh (sand). Although there may be, it is preferable that it is only a seed. The method for obtaining seeds from citrus fruits is not particularly limited, and examples thereof include known mechanical methods and manual methods.

Citrus seeds may be those immediately after harvesting, or may be subjected to pretreatment such as drying treatment, pulverization treatment, fragmentation treatment and combination treatment after harvesting. In the case where it takes time until the method of one embodiment of the present invention is used, it is preferably stored by a storage means commonly used by those skilled in the art such as low-temperature storage in order to prevent deterioration of citrus seeds.

As a pretreatment of citrus seeds, for example, citrus seeds can be made into dry powder by combining a drying treatment with a shredding treatment or a grinding treatment. At this time, the drying process and the pulverization process may be performed at the same time, or any of them may be performed first, but it is preferable to perform the pulverization process after the drying process is performed first for the convenience of work.

Although a drying process is not specifically limited, For example, the process etc. which dry so that the moisture content of a citrus seed may be 10 mass% (wt%) or less with respect to the whole quantity of this seed, Preferably it is 5 wt% or less. . The drying process is known to those skilled in the art such as a method of drying in the sun, hot air drying, hot air drying, high pressure steam drying, spray drying, reduced pressure drying, fluidized drying, electromagnetic wave drying, freeze drying using a dryer. This can be done by any method. Drying by heating can be performed at a temperature and time at which citrus seeds and triterpenoids are not altered by heating at, for example, 30 ° C to 140 ° C, preferably 40 ° C to 100 ° C.

The shredding process is not particularly limited, and examples thereof include a process of shredding citrus seeds so that the maximum length is 5 mm or less, preferably 3 mm or less. The shredding treatment is performed by any method commonly used by those skilled in the art, for example, using cutting tools such as scissors, knives and cutters, and shredding equipment and instruments such as food processors and slicers. Can be processed into coarsely chopped, chopped, sliced, etc. The shape of the citrus seed shredded product is not particularly limited, and examples thereof include a cubic shape, a rectangular parallelepiped shape, an elliptical shape, a circular shape, and an indefinite shape.

The pulverization treatment is not particularly limited.For example, pulverization of citrus fruit can be performed by any method commonly used by those skilled in the art using a pulverizing device or instrument such as a mixer, juicer, crusher, mill, blender, mass collider, or stone mill. The process which grind | pulverizes a seed is mentioned.

In the step (1), a crude seed extract is obtained by subjecting citrus seeds to an extraction treatment using a triterpenoid-soluble solvent.

The triterpenoid-soluble solvent is not particularly limited as long as it is a solvent that can dissolve the triterpenoid. For example, a solvent that is soluble in limonin or nomiline, specifically, an organic solvent such as ethanol or ethyl acetate, or these solvents. Examples thereof include a mixed solvent of an organic solvent and water, and preferred is hydrous ethanol which is a mixed solvent of ethanol and water. When water-containing ethanol is used as the triterpenoid-soluble solvent, for example, it is preferably 20 vol% (= 20% (v / v)) to 100 vol% ethanol. If the ethanol concentration is less than 20 vol%, the extraction efficiency tends to be poor. From the viewpoint of safety and industrialization in addition to extraction efficiency, the triterpenoid-soluble solvent is more preferably 60 vol% to 80 vol% ethanol.

The conditions for the extraction treatment are not particularly limited as long as the conditions are suitable for dissolving the triterpenoids in the citrus seeds in the solvent, depending on the state and amount of the citrus seeds, the type and amount of the triterpenoid-soluble solvent. It can be set appropriately. The conditions for the extraction treatment can be determined, for example, by appropriately sampling a treatment solution obtained from citrus seeds and a triterpenoid-soluble solvent and confirming the concentration of triterpenoid in the treatment solution.

As specific conditions for the extraction treatment, 20 to 100 vol% ethanol of 2 to 10 mass times with respect to citrus seeds is used, and it is 10 to 40 ° C, preferably around room temperature, for several hours to several days. However, the present invention is not limited to this. The content of triterpenoids in the crude seed extract is not particularly limited. For example, the content of limonin and / or nomiline contained in the concentrate of the crude seed extract when the crude seed extract is dried and concentrated. However, they are each 3 wt% or more, preferably 4 wt% or more, more preferably 5 wt% or more with respect to the total amount of the concentrate. However, triterpenoids in the crude seed extract tend to have a nomiline content higher than a limonin content. What is necessary is just to implement the measurement of the content of limonin and nomiline by the method as described in the Example mentioned later.

The crude seed extract contains residues derived from seeds. Therefore, from the viewpoint of the processing efficiency of the subsequent step (2), the seed crude extract obtained in the step (1) is subjected to a commonly known solid-liquid separation means such as centrifugation or filtration, and a seed residue is obtained. It is preferable to obtain as a liquid component from which is removed.

In the step (2), the seed crude extract is obtained by subjecting the crude seed extract obtained in the step (1) to a hydration treatment and a concentration treatment.

The hydration treatment is not particularly limited as long as the treatment is carried out under conditions such that triterpenoids in the crude seed extract are dissolved in water by adding water to the crude seed extract. .01-fold to 3-fold, preferably 0.1-fold to 1-fold water and a crude seed extract, and 10 ° C. to 40 ° C., preferably near room temperature, for several minutes to several hours to several days, It can be carried out by standing or stirring.

The concentration treatment is not particularly limited as long as it is a treatment carried out under conditions such that the liquid components in the seed crude extract are volatilized and reduced by a generally known concentration means, for example, evaporation concentration. The liquid component in the crude seed extract is volatilized by a known concentration means such as vacuum concentration. Although the degree of concentration is not particularly limited, for example, it is possible to reduce the volume to about 1/2 to 1/20 so that most of the liquid component contained in the crude seed extract is water. .

The crude seed extract obtained by subjecting the crude seed extract to a hydration treatment and a concentration treatment is preferably an emulsion that is difficult to separate by centrifugation.

In step (3), the seed extract obtained in step (2) is subjected to acid treatment and salting treatment to obtain a triterpenoid-containing seed extract as a first precipitate.

The acid treatment is not particularly limited as long as it is a treatment carried out under conditions that make the crude seed extract acidic. For example, the acid treatment is carried out using an acid capable of bringing the crude seed extract to near acidity, preferably to pH 1 to 3. Can do. The acid to be used is not particularly limited as long as it is an acid usually used for acid treatment, and examples thereof include hydrochloric acid, acetic acid, formic acid, citric acid, lactic acid, malic acid, sulfuric acid, phosphoric acid, citrus juice and extracts thereof. Inorganic acids and organic acids can be mentioned, and hydrochloric acid is preferred. The acid may be a mixture with a triterpenoid insoluble solvent such as water.

The salting treatment is not particularly limited as long as the treatment is performed under the condition of mixing the crude seed extract and the salt. For example, the salting treatment may be performed by adding salt to the acid crude seed extract and mixing lightly. . The addition of the salt may be performed by adding the entire amount of salt at once, or by adding a small amount of salt in several portions. The salt to be used is not particularly limited as long as it is a salt usually used for salting treatment, and examples thereof include inorganic salts and organic salts such as sodium salt, potassium salt and calcium salt, and sodium chloride is preferable.

The amount of salt used is not particularly limited as long as triterpenoids are precipitated from the acidified crude seed extract. For example, when sodium chloride is used, 0.1 mg / kg of the crude seed extract is used. An amount such as ml to 10 mg / ml can be used. In addition, a salt can be used at once or divided into several times.

The conditions for the acid treatment and salting treatment are not particularly limited as long as triterpenoids are precipitated by subjecting the crude seed extract to these treatments. In the acid treatment and salting treatment, salting treatment may be performed after acid treatment, or acid treatment and salting treatment may be performed simultaneously. The crude seed extract is preferably allowed to stand for several seconds to several minutes to several hours after being subjected to acid treatment and salting treatment.

By subjecting the crude seed extract to acid treatment and salting treatment, a triterpenoid-containing seed extract can be obtained as the first precipitate. In order to separate the first precipitate from the liquid component, a generally known solid-liquid separation means such as centrifugation or filtration may be used. The first precipitate after the solid-liquid separation means is usually in a wet state containing water. Moreover, the liquid component separated from the first precipitate by the solid-liquid separation means is referred to as a first supernatant in this specification. The first supernatant preferably has a nomiline and limonin content below the lower limit of detection.

The method of the first aspect of the present invention, which is a specific method of the method of one aspect of the present invention, includes the following steps (4) and (5) in addition to steps (1) to (3). Including at least.
(4) Step of obtaining a triterpenoid-containing seed extract and a second supernatant as a second precipitate by subjecting the first precipitate to a drying treatment and a washing treatment (5) The second supernatant To obtain a triterpenoid-containing seed extract as a third precipitate by subjecting to a stationary treatment

In step (4), the first precipitate obtained in step (3) is subjected to a drying treatment and a washing treatment to obtain a triterpenoid-containing seed extract and a second supernatant as the second precipitate. .

The drying process in step (4) is performed by the above-described drying process, for example, so that the moisture content of the first precipitate is 5 wt% or less, preferably 1 wt% or less with respect to the total amount of the precipitate. . The drying means is not particularly limited, but lyophilization is preferred.

If the washing treatment in the step (4) is carried out so as to elute impurities other than the triterpenoid in the precipitate with a small amount of the triterpenoid poor solvent with respect to the first precipitate after the drying treatment, It is not limited. In the washing treatment, for example, a sufficiently small amount of ethanol with respect to the precipitate, preferably 0.5 ml to 1.5 ml of ethanol is added to 100 mg to 5,000 mg of the precipitate, both are mixed gently, Separation of the precipitate and the supernatant by centrifugation or the like can be carried out by repeating once to several times. The precipitate finally obtained by the washing treatment is the second precipitate. In addition, the supernatant separated from the precipitate by the washing treatment is collected each time the washing operation is repeated, and the sum of these is the second supernatant.

The content of triterpenoid in the second precipitate is not particularly limited. For example, the content of nomiline contained in the second precipitate is preferably 20 wt% or more with respect to the total amount of the precipitate. Is 25 wt% or more; the content of limonin contained in the second precipitate is 30 wt% or more, preferably 35 wt% or more based on the total amount of the precipitate; and / or The total content of nomiline and limonin contained in No. 2 precipitate is 50 wt% or more, preferably 60 wt% or more, more preferably 70 wt% or more with respect to the total amount of the precipitate.

The triterpenoid in the second precipitate tends to have a limonin content higher than that of nomiline. The fact that a second precipitate having a high limonin content ratio can be obtained from a crude seed extract having a high nomiline content ratio means that the method comprising steps (1) to (4) is excellent for obtaining a large amount of limonin. It shows that it is a method.

In the step (5), the second supernatant obtained in the step (4) is subjected to a stationary treatment to obtain a triterpenoid-containing seed extract as a third precipitate.

The standing treatment is not particularly limited as long as it is a conventionally known standing treatment. For example, the second supernatant is treated at 0 to 30 ° C., preferably at room temperature, without taking any special measures. It can be carried out by leaving it for several hours to several days until a sufficient amount of precipitation is confirmed. The precipitate thus confirmed is the third precipitate. In order to separate the third precipitate from the liquid component, a generally known solid-liquid separation means such as centrifugation or filtration may be used. The liquid component separated from the third precipitate is the third supernatant. If desired, the third precipitate can be removed by removing the solvent used in the washing treatment to obtain a dried third precipitate.

Although the content of triterpenoid in the third precipitate is not particularly limited, for example, the content of limonin contained in the third precipitate is preferably 20 wt% or more with respect to the total amount of the precipitate, Is 25 wt% or more; the content of nomiline contained in the third precipitate is 30 wt% or more, preferably 35 wt% or more based on the total amount of the precipitate; and / or The total content of nomiline and limonin contained in No. 3 precipitate is 50 wt% or more, preferably 60 wt% or more based on the total amount of the precipitate. Although the content of triterpenoid in the third supernatant is not particularly limited, for example, the content of limonin and nomiline contained in the third supernatant is about several wt% with respect to the total amount of the supernatant, respectively. It is.

The yields of the second precipitate and the third precipitate are not particularly limited. For example, the amount of the second precipitate may be 0.1 mg / g or more with respect to the total amount of citrus seeds. Preferably, it is 1.0 mg / g or more, more preferably 2.0 mg / g or more; and / or the amount of the third precipitate is based on the total amount of citrus seeds , 0.1 mg / g or more is preferable, 1.0 mg / g or more is more preferable, and 1.5 mg / g or more is more preferable.

The recovery rate of limonin and nomiline in the second precipitate and the third precipitate is not particularly limited. For example, the recovery rate of limonin is based on the content of limonin contained in the concentrate of the crude seed extract, It is preferably 40 wt% or more, more preferably 50 wt% or more, and even more preferably 60 wt% or more; and / or the recovery rate of nomilin is the nomilin contained in the concentrate of the crude seed extract Is preferably 30 wt% or more, more preferably 40 wt% or more, and further preferably 50 wt% or more.

The method of the second aspect of the present invention, which is a specific method of the method of one aspect of the present invention, includes the following steps (6) and (7) in addition to steps (1) to (3). Including at least.
(6) Step of obtaining a triterpenoid-containing seed extract and a fourth supernatant as a fourth precipitate by subjecting the first precipitate to a washing treatment (7) Drying the fourth supernatant A step of obtaining a triterpenoid-containing seed extract as a fifth precipitate by subjecting to a treatment, a solvent addition treatment and a stationary treatment

In addition to steps (1) to (3) and (6) to (7), the method of the third aspect of the present invention, which is a specific method of the method of one aspect of the present invention, further includes the following steps: (8) is included at least.
(8) A step of obtaining a triterpenoid-containing seed extract as the sixth precipitate by subjecting the fifth precipitate to a washing treatment.

In step (6), the first precipitate obtained in step (3) is subjected to a washing treatment to obtain a triterpenoid-containing seed extract and a fourth supernatant as a fourth precipitate.

If the washing treatment in the step (6) is carried out so as to elute impurities other than the triterpenoid in the precipitate using a small amount of the triterpenoid poor solvent with respect to the first precipitate in the wet state. It is not limited. In the washing treatment, for example, a sufficiently small amount of ethanol with respect to the precipitate, preferably 0.5 ml to 1.5 ml of ethanol is added to 100 mg to 5,000 mg of the precipitate, both are mixed gently, Separation of the precipitate and the supernatant by centrifugation or the like can be carried out by repeating once to several times. The precipitate finally obtained by the washing treatment is the fourth precipitate. In addition, the supernatant separated from the precipitate by the washing treatment is collected each time the washing operation is repeated, and the sum of these is the fourth supernatant.

Although the content of triterpenoid in the fourth precipitate is not particularly limited, for example, the content of nomiline contained in the fourth precipitate is preferably 30 wt% or more with respect to the total amount of the precipitate, Is 35 wt% or more, more preferably 40 wt% or more; the content of limonin contained in the fourth precipitate is 25 wt% or more, preferably 30 wt%, based on the total amount of the precipitate Or more, more preferably 35 wt% or more; and / or the total content of nomiline and limonin contained in the fourth precipitate is 60 wt% or more based on the total amount of the precipitate, Preferably it is 70 wt% or more, More preferably, it is 80 wt% or more.

The triterpenoid in the fourth precipitate tends to have a nomiline content higher than that of limonin. Therefore, it shows that the method including the steps (1) to (3) and (6) is an excellent method for obtaining a large amount of nomiline.

In the step (7), the fourth supernatant obtained in the step (6) is subjected to a drying treatment, a solvent addition treatment and a stationary treatment to obtain a triterpenoid-containing seed extract as a fifth precipitate.

Since the fourth supernatant is liquid, the drying process in step (7) is preferably a drying process in which the liquid evaporates, and more preferably a freeze-drying process.

The solvent addition treatment is not particularly limited as long as it is carried out so as to elute impurities other than triterpenoids in the dried product by using a small amount of triterpenoid poor solvent for the dried product of the fourth supernatant. In the solvent addition treatment, for example, a sufficiently small amount of ethanol, preferably 10 ml to 20 ml of ethanol is added to 100 mg to 1,000 mg of the dried product, and both are mixed. Can be implemented.

The standing treatment in the step (7) is not particularly limited as long as the standing treatment in the step (5) is performed. For example, the fourth supernatant subjected to the solvent addition treatment without taking any special means. Can be carried out by standing at 0 ° C. to 30 ° C., preferably at room temperature, for several hours to several days until a sufficient amount of precipitation is confirmed. The precipitate confirmed in this way is the fifth precipitate. In order to separate the fifth precipitate from the liquid component, a commonly known solid-liquid separation means such as centrifugation or filtration may be used. The liquid component separated from the fifth precipitate is the fifth supernatant.

In step (8), the fifth precipitate obtained in step (7) is subjected to a washing treatment to obtain a triterpenoid-containing seed extract as the sixth precipitate.

The cleaning process in the step (8) is not particularly limited as long as it is performed in the same manner as the cleaning process in the step (6). That is, in the washing treatment, for example, a sufficiently small amount of ethanol with respect to the precipitate, preferably 0.5 ml to 1.5 ml of ethanol is added to 100 mg to 5,000 mg of the precipitate, and both are mixed gently. Then, the separation of the precipitate and the supernatant by centrifugation or the like can be carried out by repeating one to several times. The precipitate finally obtained by the washing treatment is the sixth precipitate. In addition, the supernatant separated from the precipitate by the washing treatment is collected each time the washing operation is repeated, and the sum of these is the sixth supernatant.

The content of triterpenoid in the sixth precipitate is not particularly limited. For example, the content of limonin contained in the sixth precipitate is preferably 10 wt% or less with respect to the total amount of the precipitate. Is 5 wt% or less; and / or the content of nomiline contained in the sixth precipitate is 50 wt% or more, preferably 60 wt% or more based on the total amount of the precipitate, Preferably it is 70 wt% or more. The content of triterpenoid in the sixth supernatant is not particularly limited. For example, the content of limonin and nomiline contained in the sixth supernatant is about several wt% with respect to the total amount of the supernatant, respectively. It is.

The triterpenoid in the sixth precipitate has a tendency that the content of nomiline is higher than that of limonin and can substantially contain only nomiline. Therefore, the method including the steps (1) to (3) and (6) to (8) indicates that it is an excellent method for obtaining a large amount of nomiline.

The yields of the fourth precipitate and the sixth precipitate are not particularly limited. For example, the amount of the fourth precipitate may be 0.1 mg / g or more with respect to the total amount of citrus seeds. Preferably, it is 1.0 mg / g or more, more preferably 2.0 mg / g or more; and / or the amount of the sixth precipitate is based on the total amount of citrus seeds , 0.1 mg / g or more is preferable, and 0.5 mg / g or more is more preferable.

The recovery rate of limonin and nomiline in the fourth precipitate and the sixth precipitate is not particularly limited. For example, the recovery rate of limonin is based on the content of limonin contained in the concentrate of the crude seed extract, It is preferably 10 wt% or more, more preferably 20 wt% or more, and even more preferably 30 wt% or more; and / or the recovery rate of nomilin is nomilin contained in the concentrate of the crude seed extract Is preferably 30 wt% or more, more preferably 40 wt% or more, and further preferably 50 wt% or more.

According to the method of one embodiment of the present invention, as the first precipitate, the second precipitate, the third precipitate, the fourth precipitate, the fifth precipitate, and / or the sixth precipitate, A triterpenoid-containing seed extract containing a high concentration of triterpenoids such as nomiline is obtained.

For example, when the triterpenoid-containing seed extract is used as a raw material for an oral composition or an external composition, it is preferably washed with water or the like. The triterpenoid-containing seed extract may be subjected to further purification steps. For example, a mixture of a triterpenoid-containing seed extract collected and washed with water and a triterpenoid-soluble solvent (good solvent) is subjected to solid-liquid separation, and then crystallized using a triterpenoid-insoluble solvent (poor solvent). A reduced triterpenoid-containing seed extract can be obtained.

In the method of one embodiment of the present invention, various processes and operations can be added before or after the above-described process or during the process as long as the problem of the present invention can be solved.

Hereinafter, as a specific embodiment of the method of one embodiment of the present invention, a method for producing a triterpenoid-containing seed extract containing citrus seeds as a raw material and containing a high concentration of triterpenoids will be described with reference to FIGS. As will be described, the production method of the present invention is not limited to the following.

Citrus seeds 1 that has been dried and shredded or ground with scissors or a mixer are added to 10 vol% to 80 vol% ethanol, which is 5 times the volume of the seeds, and 10 to 30 ° C for 2 to 4 days. The crude seed extract 50 is obtained by subjecting it to the extraction process 10 carried out in step (b). Next, the crude seed extract 50 is subjected to a hydrolyzing / concentration treatment 11 that is concentrated after using 0.5 times the volume of water relative to the seed crude extract, and ethanol is volatilized to reduce the volume to 0.2 times. Thus, a crude seed extract 51 of the emulsion is obtained.

After adjusting the pH to 1 to 3 by adding an inorganic acid to the crude seed extract 51, 0.1 to 0.2 times the inorganic salt is added to the crude seed extract, and for several seconds to several minutes. After performing acid, salting, and standing treatment 12 that are allowed to stand for several hours, a first precipitate 52 and a first supernatant 53 in a wet state are obtained by centrifugation.

As shown in FIG. 1, in the method of the first aspect of the present invention, the first precipitate 52 is dried so as to remove moisture from the precipitate, and then ethanol is added to the obtained dried product. It is subjected to a drying / washing process 20 in which light shaking and centrifugation are repeated multiple times for washing. After the washing, a second precipitate 60 is obtained, and the second supernatant 61 is obtained by collecting the supernatant for each washing. The second precipitate 60 is preferably a triterpenoid-containing seed extract having a large limonin content relative to the nomiline content.

The second supernatant 61 is subjected to a stationary treatment 21 that is allowed to stand for several minutes to several hours to several days. The third precipitate 62 and the third supernatant 63 are obtained by centrifuging after the stationary treatment.

As shown in FIG. 2, the first precipitate 52 is subjected to a washing process 30 in which ethanol is added, lightly shaken and centrifuged after repeated multiple times in a wet state. After the washing, a fourth precipitate 70 is obtained, and the supernatant for each washing is collected to obtain a fourth supernatant 71. The fourth precipitate 70 is preferably a triterpenoid-containing seed extract having a large nomiline content relative to the limonin content.

The fourth supernatant 71 is dried so as to remove water, and then a small amount of a triterpenoid insoluble solvent (poor solvent) is added to and mixed with the obtained dried product, and then for several minutes to several hours to several days. It is subjected to drying, solvent addition, and stationary treatment 31 for standing. The solution after the treatment 31 is centrifuged to obtain a fifth precipitate 72 and a fifth supernatant 73. The fifth precipitate 72 is subjected to a washing treatment 32 by repeating washing with ethanol and centrifugation a plurality of times to obtain a sixth precipitate 74 and a sixth supernatant 75. The sixth precipitate is preferably a triterpenoid-containing seed extract having a very large nomiline content relative to the limonin content, and is preferably a triterpenoid-containing seed extract substantially free of limonin. More preferred.

The triterpenoid-containing seed extract produced by the method of one aspect of the present invention can be included in the present invention as another aspect. That is, the triterpenoid-containing seed extract of the present invention is not particularly limited as long as it is a composition derived from citrus seeds and containing a high concentration of triterpenoids as compared with the prior art, for example, an oral composition Or from the point of the cost at the time of the product manufacture by applying as a raw material of an external composition, content of the triterpenoid per dry mass of this extract is 1 wt% or more as a minimum, for example, Preferably it is 40 wt% More preferably, it is 50 wt% or more, more preferably 60 wt% or more, still more preferably 70 wt% or more; the upper limit is 100 wt% or less, preferably 90 wt% or less. The triterpenoid-containing seed extract of one embodiment of the present invention is obtained by the method of one embodiment of the present invention.

The use of the triterpenoid-containing seed extract of the present invention is not particularly limited. For example, physiology such as antitumor action, activity of detoxifying metabolic enzyme, action of shortening sleep time during anesthesia, analgesic action, and serum lipid metabolism improving action of triterpenoids. In anticipation of activity, the composition can be used as a raw material for each composition such as an oral composition such as a food or drink or a pharmaceutical, or an external composition such as a cosmetic, or the composition itself. The content of triterpenoid in the triterpenoid-containing seed extract of the present invention can be measured by the method described in Examples described later.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples, and the present invention can take various modes as long as the problems of the present invention can be solved. .

[Example 1. Method for measuring nomilin and limonin]
The content (mg) of nomiline and limonin was measured using a COSMOSIL 5C18-AR-2 column as a column and MeOH: H ₂ O = 20: 80 to 100: 0 as a mobile phase, a flow rate of 1 ml / min, and a measurement wavelength of 215. With a setting of 0 nm, the measurement was carried out by a calibration curve method as a peak having a retention time of about 16 minutes for limonin and about 17 minutes for nomiline.

[Example 2. Method for producing seeker seed extract with high content of nomiline and limonin (1)]
10.0 g of dried shikwasa seeds were roughly chopped with scissors. The shredded seeds were added to 50 mL of 80 vol% ethanol and subjected to an extraction treatment at room temperature for 3 days to obtain a rough extract of seeker seeds. The crude extract of the sequoccer seed was subjected to drying and concentration treatment to obtain 583.8 mg of a concentrate of the sequoccer seed crude extract. The contents of nomiline and limonin in the concentrate were measured and found to be 34.9 mg (6.0 wt%) and 34.5 mg (5.9 wt%), respectively.

50 ml of Sequoccer seed crude extract was added to 20 ml of water and subjected to concentration treatment. When ethanol was volatilized, the concentration treatment was terminated to obtain a crude seed extract. The obtained seed crude extract was an emulsion and was not separated into layers by centrifugation.

To 15 ml of the obtained crude seed extract, hydrochloric acid was added to adjust the pH to 1 to 3, and then 50 mg to 100 mg of NaCl was added and shaken lightly to obtain a crude extract mixture. The obtained crude extract mixture was allowed to stand for 5 minutes and then centrifuged to obtain a first supernatant and a first precipitate. Among these, when the content of nomiline and limonin was measured for the first supernatant, both substances were below the lower limit of detection.

The first precipitate was subjected to a lyophilization treatment to obtain a lyophilized product from which moisture was removed. 165 mg of the obtained lyophilized product was washed twice with 1 ml of ethanol. That is, after adding ethanol to the lyophilized product, the precipitate and the supernatant were obtained by gently shaking and centrifuging. The same operation was repeated once more for this precipitate to obtain 36.6 mg of the second precipitate, and the supernatant was further collected to obtain the second supernatant. The content of nomiline and limonin in the second precipitate was measured and found to be 11.1 mg (30.2 wt%) and 16.0 mg (43.8 wt%), respectively. That is, it was possible to obtain a seeker seed extract containing 74% or more of triterpenoids such as nomiline and limonin.

The second supernatant was allowed to stand for one day and then centrifuged to obtain 22.3 mg of the third precipitate and 106.0 mg of the third supernatant. The contents of nomiline and limonin in the third precipitate were measured and found to be 8.6 mg (38.7 wt%) and 5.8 mg (26.1 wt%), respectively. Moreover, when the content of nomiline and limonin in the third supernatant was measured, they were 3.7 mg (3.5 wt%) and 2.3 mg (2.2 wt%), respectively.

From the above results, 19.7 mg and 21.8 mg of nomiline and limonin were obtained as the second precipitate and the third precipitate, respectively. Since the contents of nomiline and limonin in the sequoccer seed crude extract were 33.1 mg and 32.5 mg, respectively, based on this, the content of nomiline and limonin in the second precipitate and the third precipitate The recoveries were 60% and 67%, respectively.

In addition, it was a surprising result that the content of limonin in the second precipitate was higher than that of nomilin in view of the fact that the content of the sequoccer seed crude extract was lower in limonin. Therefore, it was found that this method is an excellent method for obtaining a large amount of limonin.

[Example 3. Method for producing seeker seed extract containing high content of nomiline and limonin (2)]
300.0 g of dried seeds of seekercer were coarsely crushed with a mixer. The crushed seed was added to 1 L of 80 vol% ethanol and subjected to an extraction treatment at room temperature for 3 days to obtain a sequoccer seed crude extract. The concentrate of the sequoccer seed crude extract was obtained by subjecting the sequoccer seed crude extract to drying and concentration treatment, and the contents of nomiline and limonin were measured and found to be about 1,200 mg and 1,000 mg, respectively. .

1 L of Sequoccer seed crude extract was added to 300 ml of water and subjected to a concentration treatment. When ethanol was volatilized, the concentration treatment was terminated to obtain 150 ml of a crude seed extract. The obtained seed crude extract was an emulsion and was not separated into layers by centrifugation.

To the obtained crude seed extract 50 ml × 3 times (total 150 ml), hydrochloric acid was added to adjust the pH to 1 to 3, and then 150 mg of NaCl was added in three portions, and lightly shaken to roughly An extract mixture was obtained. The obtained crude extract mixture was allowed to stand for 5 minutes and then centrifuged to obtain a first supernatant and a first precipitate. Of these, the contents of nomiline and limonin were measured for the supernatant, and both substances were below the lower limit of detection.

3.3 g of the first precipitate was washed 3 times with 1 ml of ethanol. That is, after adding ethanol to the first precipitate, the precipitate and the supernatant were obtained by gently shaking and centrifuging. The same operation was further repeated twice for this precipitate to obtain 901.4 mg of the fourth precipitate, and the supernatant was further collected to obtain the fourth supernatant. At this time, in order to dissolve nomiline and precipitate limonin, the first precipitate was used in a water-containing state without being dried. The contents of nomiline and limonin in the fourth precipitate were measured and found to be 394.5 mg (43.8 wt%) and 336.5 mg (37.3 wt%), respectively. That is, a seeker seed extract containing 81% or more of a triterpenoid such as nomiline and limonin could be obtained.

The fourth supernatant was subjected to lyophilization to remove water. A mixed solution of 2.4 g of the obtained lyophilized product and 15 ml of ethanol was allowed to stand for 3 days, and then centrifuged to obtain a fifth precipitate and a fifth supernatant. The fifth precipitate was washed with ethanol and centrifuged three times to obtain 339.8 mg of the sixth precipitate and 2,100 mg of the sixth supernatant. The content of nomiline and limonin in the sixth precipitate was measured and found to be 257.0 mg (75.6 wt%) and 0 mg (0 wt%), respectively. Further, the content of nomiline and limonin in the sixth supernatant was measured and found to be 45.5 mg (2.2 wt%) and 6.1 mg (0.3 wt%), respectively.

From the above results, 651.5 mg and 336.5 mg of nomiline and limonin were obtained as the fourth precipitate and the sixth precipitate, respectively. Since the contents of nomiline and limonin in the Sikuwasa seed crude extract were 1,200 mg and 1,000 mg, respectively, on the basis of this, the contents of nomiline and limonin in the fourth precipitate and the sixth precipitate The recoveries were 54% and 34%, respectively.

In this method, a fourth precipitate and a sixth precipitate having a large content of nomilin could be obtained. In particular, it was a very surprising result that the sixth precipitate contained nomilin and little limonin. Therefore, it was found that this method is an excellent method for obtaining a large amount of nomiline.

The present invention is useful in the fields of foods and drinks, pharmaceuticals, quasi drugs, cosmetics, etc., and in particular, antitumor compositions, compositions for detoxifying metabolic enzyme activity, compositions for reducing sleep time during anesthesia, analgesia This is useful in that it can produce a composition for improving serum lipid metabolism, a composition for improving serum lipid metabolism, or a raw material for these compositions.

DESCRIPTION OF SYMBOLS 1 Citrus seed 2 Limonin-rich seed extract 3 Nomiline-rich seed extract 10 Extraction treatment 11 Hydrolysis treatment and concentration treatment 12 Acid treatment, salting treatment and static treatment 20 Drying treatment and washing treatment 21 Static treatment 30 washing treatment 31 Drying treatment, solvent addition treatment and stationary treatment 32 Washing treatment 50 Seed crude extract 51 Seed crude extract 52 First precipitate 53 First supernatant 60 Second precipitate 61 Second supernatant 62 Second Third precipitate 63 third supernatant 70 fourth precipitate 71 fourth supernatant 72 fifth precipitate 73 fifth supernatant 74 sixth precipitate 75 sixth supernatant

Claims (11)

A method for producing a triterpenoid-containing seed extract comprising the following steps (1) to (3).
(1) A step of obtaining a crude seed extract by subjecting the citrus seeds to an extraction treatment using a triterpenoid-soluble solvent. (2) By subjecting the crude seed extract to a hydration treatment and a concentration treatment, Step of obtaining an extract (3) Step of obtaining a seed extract containing triterpenoid as a first precipitate by subjecting the crude seed extract to acid treatment and salting treatment The method according to claim 1, further comprising the following steps (4) and (5).
(4) Step of obtaining a triterpenoid-containing seed extract and a second supernatant as a second precipitate by subjecting the first precipitate to a drying treatment and a washing treatment (5) The second supernatant To obtain a triterpenoid-containing seed extract as a third precipitate by subjecting to a stationary treatment The method according to claim 1, further comprising the following steps (6) and (7).
(6) Step of obtaining a triterpenoid-containing seed extract and a fourth supernatant as a fourth precipitate by subjecting the first precipitate to a washing treatment (7) Drying the fourth supernatant A step of obtaining a triterpenoid-containing seed extract as a fifth precipitate by subjecting to a treatment, a solvent addition treatment and a stationary treatment The method according to claim 3, further comprising the following step (8):
(8) A step of obtaining a triterpenoid-containing seed extract as the sixth precipitate by subjecting the fifth precipitate to a washing treatment. The citrus, Citrus depressa (Citrus depressa), satsuma mandarin (C.unshiu), Tachibana (C.tachibana), koji (C.leiocarpa), Girimikan (C.tardiva), Jimikan (C.succosa), distichum (C. kinokuni), Kobenimikan (C.erythrosa), Sunki (C.sunki), Chichuuka Lee Mandarin (C.deliciosa), King (C.nobilis), ponkan (C.retuculata), Dan Sea Tangerine (C.tangerina), Hanayu ( C.hanayu), mandarin orange (C.reticulata), Sanki (C.sunki), Stewartia Tachibana (C.nippokore The method of any one of Claims 1-4 which is at least 1 sort (s) of citrus fruit chosen from the group which consists of ana ), Shiranuhi, and Kiyomi. The method according to claim 1, wherein the triterpenoid is a limonoid. The method according to claim 6, wherein the limonoid is at least one limonoid selected from the group consisting of limonin, nomiline, nomylic acid, deacetylnomyrin, echangin and obacnon. The method according to any one of claims 1 to 2 and 5 to 7, wherein the second precipitate is a triterpenoid-containing seed extract having a large limonin content relative to a nomiline content. The fourth precipitate, the fifth precipitate, or the sixth precipitate is a triterpenoid-containing seed extract having a large nomiline content relative to a limonin content. 8. The method according to any one of items 7. A triterpenoid-containing seed extract containing citrus seeds as a raw material and containing 1 to 90 wt% of a triterpenoid. The triterpenoid containing seed extract obtained by the method of any one of Claims 1-9 containing 1-90 wt% triterpenoid.