JP7148913B2 - Method for producing triterpenoid-rich seed extract and triterpenoid-rich seed extract - Google Patents

Description

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

As triterpenoids biosynthesized by citrus, limonoids such as limonin, nomirin, echangin and obakunon are known. Since limonoids generally have a bitter taste, parts rich in limonoids, such as peels and seeds, are removed when citrus fruits are processed.

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

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

Japanese Patent Application Laid-Open No. 2006-083150 JP-A-6-199864

According to the method described in Patent Document 2, obacunone is obtained by dissolving nomilin in acetonitrile and subjecting the resulting solution to alkali treatment. However, Patent Document 2 does not disclose a detailed method for isolating nomilin from citrus seeds.

Also, for example, the total amount of limonoids contained in citrus peels is about 0.01 wt %, which is a very small amount. Little is known of a method for efficiently obtaining such a trace amount of limonoid on an industrial scale.

Therefore, the present invention provides a simple and economical method for producing a triterpenoid-containing extract containing a high concentration of triterpenoids such as limonoids so that it can be implemented on an industrial scale. is the problem to be solved.

As a result of intensive studies to solve the above problems, the present inventors have found that a crude seed extract obtained by subjecting citrus seeds to extraction treatment is subjected to hydration treatment, and then to acid treatment and salting treatment. produced a seed extract with a high content of triterpenoids such as nomilin and limonin. Furthermore, by subjecting the obtained seed extract to a treatment according to the water content of the seed extract, the content of either nomirin or limonin is large, and these amounts account for 60 wt% or more of the total We succeeded in producing triterpenoid-rich content. The present invention is an invention completed based on such knowledge and successful examples.

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 subjecting citrus seeds to an extraction treatment using a triterpenoid-soluble solvent to obtain a seed crude extract (2) A step of subjecting the seed crude extract to a hydration treatment and a concentration treatment to obtain a seed crude extract a step of obtaining an extract (3) a step of subjecting the crude seed extract to an acid treatment and a salting treatment to obtain a triterpenoid-containing seed extract as a first precipitate [2] and further the following steps (4) and The method according to [1], including (5).
(4) subjecting the first precipitate to a drying treatment and a washing treatment to obtain a triterpenoid-containing seed extract and a second supernatant as a second precipitate; (5) the second supernatant; is subjected to a standing treatment to obtain a triterpenoid-containing seed extract as a third precipitate [3], further comprising the following steps (6) and (7).
(6) subjecting the first precipitate to a washing treatment to obtain a triterpenoid-containing seed extract and a fourth supernatant as a fourth precipitate; (7) drying the fourth supernatant; A step of obtaining a triterpenoid-containing seed extract and a fifth supernatant as a fifth precipitate by subjecting to treatment, solvent addition treatment and standing treatment [4] further comprising the following step (8) [3] The method described in .
(8) A step of subjecting the fifth precipitate to a washing treatment to obtain a triterpenoid-containing seed extract as the sixth precipitate [5] The citrus fruits include Citrus depressa , Unshu mandarin orange ( C. unshiu ), C. tachibana, C. leiocarpa, C. tardiva , C. succosa , C. kinokuni , C. erythrosa , C. sunki ), Mediterranean mandarin ( C.deliciosa ), King ( C.nobilis ), Ponkan ( C.retuculata ), Dancy tangerine ( C.tangerina ), Hanayu ( C.hanayu ), Mandarin orange ( C.reticulata ), Sanki ( The method according to any one of [1] to [4], wherein the citrus is at least one selected from the group consisting of C. sunki, C. nippokoreana , Shiranuhi and Kiyomi.
[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, nomirin, nomilic acid, deacetylnomirin, echangin and obakunon.
[8] Any of [1] to [2] and [5] to [7], wherein the second precipitate is a triterpenoid-containing seed extract having a higher limonin content than nomilin content or the method according to item 1.
[9] The fourth precipitate, the fifth precipitate, or the sixth precipitate is a triterpenoid-containing seed extract having a high nomirin content relative to the limonin content, [1] And the method according to any one of [3] to [7].

According to another aspect of the present invention, extracts shown in [10] to [11] below are provided.
[10] A triterpenoid-containing seed extract containing 1 to 90 wt% of triterpenoids, wherein the starting material is citrus seeds.
[11] A triterpenoid-containing seed extract containing 1 to 90 wt% of triterpenoids, obtained by the method according to any one of [1] to [9].

According to the method of one aspect of the present invention, triterpenoid-rich triterpenoid-rich triterpenoid-rich triterpenoid-rich triterpenoid-rich triterpenoid-rich triterpenoid-rich triterpenoid-rich triterpenoid seeds with reduced amount of contaminants by a simple process without complicated equipment or devices. Inclusions can be obtained. Therefore, the method of one aspect of the present invention is a method by which a triterpenoid-rich material can be produced simply and economically so that it can be implemented on an industrial scale. Moreover, according to the method of one aspect of the present invention, a triterpenoid-rich product having a high nomirin and/or limonin content can be produced as desired.

According to the triterpenoid-containing seed extract of one aspect of the present invention, the triterpenoid-containing extract itself or the purified triterpenoid obtained by purifying the triterpenoid-containing seed extract is used as an active ingredient for antitumor action and detoxification metabolic enzyme activity. It is possible to manufacture a wide variety of food and drink, pharmaceuticals, quasi-drugs, cosmetics, etc., which are expected to have physiological activities such as action, shortening of sleep time during anesthesia, analgesic action, improvement of serum lipid metabolism.

FIG. 1 is a flow chart outlining the method of the first aspect of the invention. FIG. 2 is a flow chart outlining the method of the second aspect of the invention.

Hereinafter, the details of the method and the extract, which are one aspect of the present invention, will be described. can take various aspects.

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

The triterpenoid-containing seed extract is made from citrus seeds and contains triterpenoids at a relatively high concentration.

Triterpenoids are not particularly limited as long as they are generally known. For example, triterpenoids are lipid-soluble triterpenoids composed of 30 carbon atoms composed of 6 isoprene units composed of 5 carbons. can be said to be a compound group of The triterpenoid contained in the extract obtained by the method of one embodiment of the present invention is preferably a limonoid, which is a cyclic compound having both a lactone ring and a furan ring, such as limonin, nomirin, nomilic acid, deacetylnomyrin, e-chan Gin and obacunone are more preferred, and limonin and nomirin are even more preferred.

Citrus fruits are not particularly limited as long as they are citrus plants containing triterpenoids in their seeds. As citrus fruits, for example, oranges such as Quinot, Jaffa Orange, Joppa, Navel Orange, Valencia Orange, Fukuhara Orange, Blood Orange, Bergamot; Orangelo, Grapefruit such as Grapefruit; Shikuwasa, Citron, Sudachi, Daidai, Niihime, Bushkan, Yukou, Citrus Citrus, Yuzu, Lime, Lemon and other aromatic citrus fruits; Miscellaneous citrus such as Hassaku, Haruka, Hime Koharu, Hyuga Natsu; Tangors such as Shiranuhi (Dekopon), Setoka, Setomi, Daishoki (Dekopon), Tankan, Harumi, Hi-no-Yu (Decopon), Markot, Reiko; Agrifruit, Summer Fresh, Sweet Spring, Seminole, Tangelo, Tangelos such as Minneola; Buntans such as Ansei citrus, Kawachi Bankan, Banpeiyu, and Buntan (pomelo); Satsuma mikan, Otsu 4, Calamansi, Kanpei, Kishu mikan, Koji (citrus fruits), Sakurajima mikan, Tachibana , Fujinaka mandarin oranges, ponkans, mandarin oranges and other mandarin oranges; Preferred are citrus fruits of the family Rutaceae, Citrus subfamily, Citrus genus, Late Citrus subgenus, and citrus fruits of the Rutaceae, Citrus subfamily, Citrus genus, Primary Citrus subtribe, Pomegranate Citrus, more preferably Citrus depressa , Unshu mandarin orange ( C. unshiu ), C. tachibana, C. leiocarpa, C. tardiva , C. succosa , C. kinokuni , C. erythrosa , C. sunki ), Mediterranean mandarin ( C.deliciosa ), King ( C.nobilis ), Ponkan ( C.retuculata ), Dancy tangerine ( C.tangerina ), Hanayu ( C.hanayu ), Mandarin orange ( C.reticulata ), Sanki ( C. sunki ), Korai Tachibana ( C. nippokoreana ), Shiranuhi and Kiyomi, and more preferably Shikwasa. In addition, one or more of these citrus fruits may be used, and hybrids of the citrus fruits mentioned as specific examples above may be used.

Citrus seeds, even if they are only seeds, have flavedo (exocarp), albedo (mesocarp), vascular bundles, oil sacs, fruit core, funnel, pulp (sand) attached to the seed. Although it may be present, it is preferably only seeds. The method for obtaining seeds from citrus fruits is not particularly limited, and examples thereof include known mechanical methods and manual methods.

The citrus seeds may be freshly harvested or may be harvested and then subjected to pretreatments such as drying, pulverization, shredding, and combinations thereof. When it takes a long time to apply the method of one aspect of the present invention, it is preferable to store the citrus seeds by a storage means commonly used by those skilled in the art, such as cold storage, in order to prevent deterioration of the citrus seeds.

As a pretreatment of citrus seeds, for example, citrus seeds can be dried and pulverized by combining a drying treatment with a shredding treatment or a pulverizing treatment. At this time, the drying treatment and the pulverizing treatment may be carried out simultaneously, or either one may be carried out first, but it is preferable to carry out the pulverizing treatment after the drying treatment in order to simplify the work.

The drying treatment is not particularly limited, but includes, for example, drying such that the moisture content of the citrus seeds is 10% by mass (wt%) or less, preferably 5 wt% or less, relative to the total amount of the seeds. . The drying treatment is, for example, a method of drying in the sun, hot air drying using a dryer, warm air drying, high pressure steam drying, spray drying, reduced pressure drying, fluidized drying, electromagnetic wave drying, freeze drying, etc. known to those skilled in the art. It can be done by any method. Drying by heating can be carried out at, for example, 30° C. to 140° C., preferably 40° C. to 100° C., at a temperature and time at which the citrus seeds and triterpenoids are not degraded by heating.

The shredding treatment is not particularly limited, but includes, for example, a treatment of shredding citrus seeds so that the maximum length is 5 mm or less, preferably 3 mm or less. Shredding is performed by any method commonly used by those skilled in the art, for example, using scissors, cutlery such as kitchen knives and cutters, and shredding equipment and instruments such as food processors and slicers. Citrus seeds can be roughly chopped, minced, or sliced. The shape of the shredded citrus seeds is not particularly limited, and examples thereof include cubic, rectangular parallelepiped, elliptical, circular and irregular shapes.

The crushing treatment is not particularly limited, but for example, using mixers, juicers, crushers, mills, blenders, masscolloiders, millstones, and other crushing equipment and instruments, any method commonly used by those skilled in the art can be used to crush citrus fruits. A treatment of crushing the seeds can be mentioned.

In step (1), citrus seeds are subjected to an extraction treatment using a triterpenoid-soluble solvent to obtain a crude seed extract.

The triterpenoid-soluble solvent is not particularly limited as long as it is a solvent capable of dissolving triterpenoids. A mixed solvent of an organic solvent and water can be used, and hydrous ethanol, which is a mixed solvent of ethanol and water, is preferred. When water-containing ethanol is used as the triterpenoid-soluble solvent, it is preferably 20 vol% (=20% (v/v)) to 100 vol% ethanol, for example. If the ethanol concentration is less than 20 vol %, the extraction efficiency tends to be poor. From the viewpoint of safety and industrialization as well as 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 they are conditions suitable for dissolving the triterpenoids in the citrus seeds in the solvent. Can be set as appropriate. Conditions for 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 triterpenoids in the treatment solution.

As specific conditions for the extraction treatment, using 20 vol% to 100 vol% ethanol, which is 2 to 10 times the mass of citrus seeds, at 10 ° C. to 40 ° C., preferably around room temperature, for several hours to several days. and the like, but are not limited to these conditions. Although the content of triterpenoids in the crude seed extract is not particularly limited, for example, the content of limonin and/or nomilin contained in the concentrate of the crude seed extract when the crude seed extract is subjected to drying and concentration treatment are each 3 wt % or more, preferably 4 wt % or more, more preferably 5 wt % or more, relative to the total weight of the concentrate. However, triterpenoids in the crude seed extract tend to contain more nomirin than limonin. The content of limonin and nomirin may be measured by the method described in the examples below.

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

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

The hydration treatment is not particularly limited as long as it is performed under conditions such that the triterpenoids in the crude seed extract are dissolved in water by adding water to the crude seed extract. .01 to 3 times, preferably 0.1 to 1 times, of water and crude seed extract are brought into contact with each other at 10° C. to 40° C., preferably around 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 the crude seed extract is subjected to a commonly known concentration method under conditions such that the liquid component in the crude seed extract volatilizes and the volume is reduced, for example, evaporative concentration. It can be carried out by volatilizing the liquid component in the crude seed extract by known concentration means such as concentration under reduced pressure. Although the degree of concentration is not particularly limited, for example, the liquid component contained in the crude seed extract is mostly water so that the volume is reduced to about 1/2 to 1/20. .

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

In step (3), the crude 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 the treatment is carried out under conditions that make the crude seed extract acidic. can. The acid to be used is not particularly limited as long as it is an acid that is usually used for acid treatment. Examples include inorganic acids and organic acids, 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 it is a treatment performed under conditions where the crude seed extract and salt are mixed. For example, it can be performed by adding salt to the acidified crude seed extract and lightly mixing. . The addition of salt may be carried out by adding the whole amount of salt at once, or may be carried out by adding small amounts of salt in portions. The salt to be used is not particularly limited as long as it is a salt that is commonly used for salting treatment, and examples thereof include inorganic salts such as sodium salts, potassium salts, calcium salts, and organic salts, with sodium chloride being preferred.

The amount of salt used is not particularly limited as long as it is an amount that allows triterpenoids to precipitate from the acidified crude seed extract. For example, when sodium chloride is used, it is 0.1 mg / Amounts from ml to 10 mg/ml can be used. In addition, the salt can be used once or divided into several times.

Other conditions for the acid treatment and salting treatment are not particularly limited as long as triterpenoids are precipitated by subjecting the seed crude extract to these treatments. The acid treatment and the salting treatment may be performed after the acid treatment, or the acid treatment and the 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. If it is desired to separate the first precipitate from the liquid component, generally known solid-liquid separation means such as centrifugation and filtration may be used. The first precipitate after the solid-liquid separation means is usually in a wet state containing water. Also, the liquid component separated from the first precipitate by the solid-liquid separation means is herein referred to as the first supernatant. The first supernatant preferably contains nomilin and limonin 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, further includes steps (4) and (5) below in addition to steps (1) to (3). At least include.
(4) subjecting the first precipitate to a drying treatment and a washing treatment to obtain a triterpenoid-containing seed extract and a second supernatant as a second precipitate; (5) the second supernatant; to a static treatment to obtain a triterpenoid-containing seed extract as a third precipitate

In step (4), the first precipitate obtained in step (3) is dried and washed to obtain a triterpenoid-containing seed extract and a second supernatant as second precipitates. .

The drying treatment in step (4) is performed by the above-described drying treatment, 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. . Drying means is not particularly limited, but freeze-drying is preferred.

The washing treatment in step (4) is performed on the first precipitate that has undergone the drying treatment using a small amount of triterpenoid poor solvent so as to elute impurities other than triterpenoids in the precipitate, especially Not limited. For the washing treatment, for example, a sufficiently small amount of ethanol relative 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, the two are gently mixed, and then Separation of the precipitate and the supernatant by centrifugation or the like can be repeated 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 collected supernatant is the second supernatant.

Although the content of triterpenoids in the second precipitate is not particularly limited, for example, the content of nomirin 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, relative to the total amount of the precipitate; and/or the second The total content of nomilin and limonin contained in the precipitate of 2 is 50 wt% or more, preferably 60 wt% or more, more preferably 70 wt% or more, relative to the total weight of the precipitate.

The triterpenoids in the second precipitate tend to contain more limonin than nomirin. The fact that a second precipitate with a high limonin content can be obtained from a seed crude extract with a high nomilin content ratio means that the method including steps (1) to (4) is excellent for obtaining a large amount of limonin. method.

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

The stationary treatment is not particularly limited as long as it is a commonly known stationary treatment. It can be carried out by leaving for several hours to several days until a sufficient amount of precipitation is confirmed. The precipitate thus confirmed is the third precipitate. If it is desired 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. The third precipitate is optionally obtained by removing the solvent used in the washing treatment to obtain a dried third precipitate.

The content of triterpenoids in the third precipitate is not particularly limited, but 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 nomirin contained in the third precipitate is 30 wt% or more, preferably 35 wt% or more, relative to the total amount of the precipitate; and/or The total content of nomilin and limonin contained in the precipitate of 3 is 50 wt% or more, preferably 60 wt% or more, relative to the total weight of the precipitate. The content of triterpenoids in the third supernatant is not particularly limited. is.

Although 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 1.0 mg/g or more, more preferably 2.0 mg/g or more; and/or the amount of the third precipitate relative to the total amount of citrus seeds , preferably 0.1 mg/g or more, more preferably 1.0 mg/g or more, and even more preferably 1.5 mg/g or more.

The recovery rates of limonin and nomirin in the second precipitate and the third precipitate are not particularly limited. It is preferably 40 wt% or more, more preferably 50 wt% or more, and even more preferably 60 wt% or more; is preferably 30 wt% or more, more preferably 40 wt% or more, and even more preferably 50 wt% or more, based on the content of

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, further includes steps (6) and (7) below in addition to steps (1) to (3). At least include.
(6) subjecting the first precipitate to a washing treatment to obtain a triterpenoid-containing seed extract and a fourth supernatant as a fourth precipitate; (7) drying the fourth supernatant; obtaining a triterpenoid-containing seed extract as a fifth precipitate by subjecting it to treatment, solvent addition treatment and standing treatment;

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 in addition to steps (1) to (3) and (6) to (7) (8) is included at least.
(8) a step of obtaining a triterpenoid-containing seed extract as a sixth precipitate by subjecting the fifth precipitate to a washing treatment;

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

Especially if the washing treatment in step (6) is performed on the wet first precipitate using a small amount of triterpenoid poor solvent so as to elute impurities other than triterpenoids in the precipitate. Not limited. For the washing treatment, for example, a sufficiently small amount of ethanol relative 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, the two are gently mixed, and then Separation of the precipitate and the supernatant by centrifugation or the like can be repeated 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 collected supernatant is the fourth supernatant.

The content of triterpenoids in the fourth precipitate is not particularly limited, but for example, the content of nomirin 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%, relative to the total amount of the precipitate or more, more preferably 35 wt% or more; and/or the total content of nomirin and limonin contained in the fourth precipitate is 60 wt% or more with respect to the total amount of the precipitate, It is preferably 70 wt % or more, more preferably 80 wt % or more.

The triterpenoids in the fourth precipitate tend to contain more nomirin than limonin. Therefore, the method comprising steps (1)-(3) and (6) is an excellent method for obtaining large amounts of nomirin.

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

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

The solvent addition treatment is not particularly limited as long as it is carried out on the dried product of the fourth supernatant using a small amount of triterpenoid poor solvent so as to elute impurities other than triterpenoids in the dried product. Solvent addition treatment, for example, a sufficiently small amount of ethanol relative to the dry matter of the fourth supernatant, preferably 100 mg to 1,000 mg of the dry matter, add ethanol 10 ml to 20 ml, and mix both It can be implemented by

The stationary treatment in step (7) is not particularly limited as long as it is performed in the same manner as the stationary treatment in step (5). For example, the fourth supernatant that has been subjected to solvent addition treatment without taking special measures can be dried 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 thus confirmed is the fifth precipitate. If it is desired to separate the fifth 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 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 a sixth precipitate.

The cleaning treatment in step (8) is not particularly limited as long as it is performed in the same manner as the cleaning treatment in step (6). That is, the washing treatment is performed, for example, by adding a sufficiently small amount of ethanol to the precipitate, preferably 0.5 ml to 1.5 ml of ethanol to 100 mg to 5,000 mg of the precipitate, and lightly mixing the two. Then, separation of the precipitate and the supernatant by centrifugation or the like can be repeated 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 collected supernatant is the sixth supernatant.

Although the content of triterpenoids 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 nomirin contained in the sixth precipitate is 50 wt% or more, preferably 60 wt% or more, relative to the total amount of the precipitate, and more Preferably, it is 70 wt% or more. The content of triterpenoids in the sixth supernatant is not particularly limited. is.

The triterpenoids in the sixth precipitate tend to contain more nomirin than limonin, and may contain substantially only nomirin. Therefore, the method comprising steps (1)-(3) and (6)-(8) is shown to be an excellent method for obtaining large amounts of nomirin.

The yields of the fourth precipitate and the sixth precipitate are not particularly limited, but 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 1.0 mg/g or more, more preferably 2.0 mg/g or more; and/or the amount of the sixth precipitate relative to the total amount of citrus seeds , preferably 0.1 mg/g or more, more preferably 0.5 mg/g or more.

The recovery rates of limonin and nomirin in the fourth precipitate and the sixth precipitate are not particularly limited. It is preferably 10 wt% or more, more preferably 20 wt% or more, and even more preferably 30 wt% or more; is preferably 30 wt% or more, more preferably 40 wt% or more, and even more preferably 50 wt% or more, based on the content of

According to the method of one aspect of the present invention, limonin or A triterpenoid-containing seed extract containing a high concentration of triterpenoids such as nomilin is obtained.

The triterpenoid-containing seed extract is preferably washed with water or the like, for example, when used as a raw material for an oral composition or a composition for external use. 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) to remove impurities. A reduced triterpenoid-containing seed extract can be obtained.

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

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

Citrus seeds 1 dried and chopped or pulverized with scissors or a mixer are added to 10 vol% to 80 vol% ethanol that is 5 times the volume of the seeds, at 10 ° C. to 30 ° C., for 2 days to 4 days. A crude seed extract 50 is obtained by subjecting it to the extraction process 10 carried out in . Next, the crude seed extract 50 is subjected to a hydration/concentration treatment 11 in which 0.5 times the volume of water is used with respect to the crude seed extract and then concentrated, and the ethanol is volatilized and the volume is reduced to 0.2 times. to obtain an emulsified crude seed extract 51.

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 amount of inorganic salt is added to the crude seed extract, followed by heating for several seconds to several minutes. After carrying out the acid/salting/standing treatment 12 of standing for several hours, centrifugation is performed to obtain a first precipitate 52 and a first supernatant 53 in a wet state.

As shown in FIG. 1, in the method of the first aspect of the present invention, a first precipitate 52 is dried to remove water from the precipitate, and then ethanol is added to the resulting dry matter. It is subjected to a drying/washing process 20 in which lightly shaking and centrifuging are repeated multiple times for washing. A second precipitate 60 is obtained after washing and a second supernatant 61 is obtained by collecting the supernatant after each wash. The second precipitate 60 is preferably a triterpenoid-containing seed extract with a high limonin content relative to the nomirin content.

The second supernatant 61 is subjected to a standing treatment 21 of standing for several minutes to several hours to several days. A third precipitate 62 and a third supernatant 63 are obtained by centrifuging after the standing 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 are repeated multiple times while still wet. A fourth precipitate 70 is obtained after washing and a fourth supernatant 71 is obtained by collecting the supernatant after each wash. The fourth precipitate 70 is preferably a triterpenoid-containing seed extract with a high nomirin content relative to the limonin content.

The fourth supernatant 71 is dried to remove moisture, then a small amount of triterpenoid-insoluble solvent (poor solvent) is added to the resulting dried product and mixed, and then for several minutes to several hours to several days. It is subjected to a drying/solvent addition/standing treatment 31 of standing still. A fifth precipitate 72 and a fifth supernatant 73 are obtained by centrifuging the solution after the treatment 31 . The fifth precipitate 72 is subjected to a washing process 32 by repeating washing with ethanol and centrifugation multiple 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 high nomirin content relative to the limonin content, and is preferably a triterpenoid-containing seed extract substantially free of limonin. more preferred.

A 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 derived from citrus seeds and contains triterpenoids at a higher concentration than in the prior art. Alternatively, from the viewpoint of the cost in manufacturing products by applying it as a raw material of a composition for external use, the content of triterpenoids per dry mass of the extract is, for example, as a lower limit, 1 wt% or more, preferably 40 wt%. The upper limit is 100 wt% or less, preferably 90 wt% or less. The triterpenoid-containing seed extract of one aspect of the invention is obtained by the method of one aspect of the invention.

The use of the triterpenoid-containing seed extract of the present invention is not particularly limited. Expecting activity, it can be used as a raw material for each composition such as oral compositions such as food and drink and pharmaceuticals, and external compositions such as cosmetics, or as the composition itself. The triterpenoid content in the triterpenoid-containing seed extract of the present invention can be measured by the method described in Examples below.

The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these Examples, and the present invention can take various aspects as long as the problems of the present invention can be solved. .

[Example 1. Method for measuring nomirin and limonin]
The contents (mg) of nomilin and limonin were 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 at a flow rate of 1 ml/min and a measurement wavelength of 215. Set at 0 nm, limonin was determined by a calibration curve method as a peak with a retention time of approximately 16 minutes and nomirin approximately 17 minutes.

[Example 2. Method for producing shikuwasa seed extract containing high nomilin and limonin (1)]
10.0 g of dried shikuwasa seeds were roughly chopped with scissors. The chopped seeds were added to 50 mL of 80 vol% ethanol and subjected to extraction treatment at room temperature for 3 days to obtain a Shikuwasa seed crude extract. By drying and concentrating the Shikuwasa seed crude extract, 583.8 mg of a concentrate of Shikuwasa seed crude extract was obtained. The nomilin and limonin contents of the concentrate were determined to be 34.9 mg (6.0 wt %) and 34.5 mg (5.9 wt %) respectively.

20 ml of water was added to 50 ml of the Shikuwasa seed crude extract, and then subjected to a concentration treatment. When the ethanol was volatilized, the concentration treatment was terminated to obtain a seed crude extract. The resulting crude seed extract was an emulsion and did not undergo layer separation by centrifugation.

Hydrochloric acid was added to 15 ml of the obtained crude seed extract to adjust the pH to 1 to 3, and then 50 to 100 mg of NaCl was added and lightly shaken to obtain a crude extract mixture. The resulting crude extract mixture was allowed to stand for 5 minutes and then centrifuged to obtain a first supernatant and a first precipitate. When the contents of nomirin and limonin were measured for the first supernatant, both substances were below the detection limit.

The first precipitate was subjected to a freeze-drying process to obtain a dry freeze-dried product. 165 mg of the resulting lyophilisate was washed twice with 1 ml of ethanol. That is, after ethanol was added to the freeze-dried product, the mixture was gently shaken and centrifuged to obtain a precipitate and a supernatant. The same operation was repeated once for this precipitate to obtain a second precipitate of 36.6 mg, and the second supernatant was obtained by collecting the supernatant. The nomilin and limonin contents of the second precipitate were determined to be 11.1 mg (30.2 wt%) and 16.0 mg (43.8 wt%) respectively. That is, it was possible to obtain a Shikuwasa seed extract containing 74% or more of triterpenoids such as nomilin and limonin.

The second supernatant was allowed to stand for one day and then centrifuged to obtain a third precipitate of 22.3 mg and a third supernatant of 106.0 mg. The content of nomirin and limonin in the third precipitate was determined to be 8.6 mg (38.7 wt%) and 5.8 mg (26.1 wt%) respectively. In addition, the content of nomilin and limonin in the third supernatant was measured and found to be 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 nomirin and limonin were obtained as the second and third precipitates, respectively. Since the contents of nomilin and limonin in the Shikuwasa seed crude extract were 33.1 mg and 32.5 mg, respectively, based on this, the amounts of nomilin and limonin in the second and third precipitates were Recoveries were 60% and 67%, respectively.

In addition, considering that the content of limonin in the Shikuwasa seed crude extract was less, it was a surprising result that the content of limonin in the second precipitate was higher than that in nomirin. Therefore, this method was found to be an excellent method for obtaining large amounts of limonin.

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

300 ml of water was added to 1 L of the Shikuwasa seed crude extract, and then subjected to a concentration treatment. When the ethanol was volatilized, the concentration treatment was terminated to obtain 150 ml of a seed crude extract. The resulting crude seed extract was an emulsion and did not undergo layer separation by centrifugation.

Hydrochloric acid was added to 50 ml of the obtained crude seed extract 3 times (150 ml in total) to adjust the pH to 1 to 3, and then 150 mg of NaCl was added in 3 portions, and shaken lightly to obtain a coarse extract. An extract mixture was obtained. The resulting crude extract mixture was allowed to stand for 5 minutes and then centrifuged to obtain a first supernatant and a first precipitate. When the content of nomirin and limonin in the supernatant was measured, both substances were below the lower limit of detection.

3.3 g of the first precipitate was washed three times with 1 ml of ethanol. That is, after ethanol was added to the first precipitate, the mixture was gently shaken and centrifuged to obtain a precipitate and a supernatant. For this precipitate, the same operation was repeated twice to obtain 901.4 mg of the fourth precipitate, and the supernatant was collected to obtain the fourth supernatant. At this time, in order to dissolve nomilin and precipitate limonin, the first precipitate was used in a water-containing state without being dried. The content of nomirin and limonin in the fourth precipitate was determined to be 394.5 mg (43.8 wt%) and 336.5 mg (37.3 wt%) respectively. That is, it was possible to obtain a Shikuwasa seed extract containing 81% or more of triterpenoids such as nomilin and limonin.

The fourth supernatant was lyophilized to remove water. A mixed solution of 2.4 g of the obtained freeze-dried 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. By repeating washing of the fifth precipitate with ethanol and centrifugation three times, 339.8 mg of the sixth precipitate and 2,100 mg of the sixth supernatant were obtained. The content of nomirin and limonin in the sixth precipitate was determined to be 257.0 mg (75.6 wt%) and 0 mg (0 wt%) respectively. Also, the contents of nomilin and limonin in the sixth supernatant were measured 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 nomirin and limonin were obtained as the fourth and sixth precipitates, respectively. Since the contents of nomilin and limonin in the Shikuwasa seed crude extract were 1,200 mg and 1,000 mg, respectively, based on this, the amounts of nomilin and limonin in the fourth and sixth precipitates were Recoveries were 54% and 34%, respectively.

In this method, it was possible to obtain a fourth precipitate and a sixth precipitate with a high nomirin content. In particular, it was a very surprising result that the sixth precipitate contained nomirin and little limonin. Therefore, this method was found to be an excellent method for obtaining a large amount of nomirin.

INDUSTRIAL APPLICABILITY The present invention is useful in the fields of food and beverages, pharmaceuticals, quasi-drugs, cosmetics, etc., and particularly antitumor compositions, compositions for activating detoxification metabolic enzymes, compositions for shortening sleep time during anesthesia, and analgesics. It is useful in that it can be used to manufacture a composition for use in food, a composition for improving serum lipid metabolism, or a raw material for these compositions.

1 citrus seeds 2 limonin-rich seed extract 3 nomirin-rich seed extract 10 extraction treatment 11 hydration treatment and concentration treatment 12 acid treatment, salting treatment and standing treatment 20 drying treatment and washing treatment 21 standing treatment 30 washing treatment 31 drying treatment, solvent addition treatment and standing treatment 32 washing treatment 50 crude seed extract 51 crude seed extract 52 first precipitate 53 first supernatant 60 second precipitate 61 second supernatant 62 3 precipitate 63 third supernatant 70 fourth precipitate 71 fourth supernatant 72 fifth precipitate 73 fifth supernatant 74 sixth precipitate 75 sixth supernatant

Claims (7)

A method for producing a triterpenoid-containing seed extract, comprising the following steps (1) to ( 5 ) , wherein the triterpenoids are nomilin and limonin .
(1) A step of subjecting citrus seeds to an extraction treatment using 60 vol% to 80 vol% ethanol to obtain a seed crude extract (2) A step of subjecting the seed crude extract to a hydration treatment and a concentration treatment a step of obtaining a crude seed extract (3) a step of subjecting the crude seed extract to acid treatment and salting treatment to obtain a triterpenoid-containing seed extract as a first precipitate;
(4) subjecting the first precipitate to drying treatment and washing treatment to obtain a triterpenoid-containing seed extract and a second supernatant as a second precipitate;
(5) a step of obtaining a triterpenoid-containing seed extract as a third precipitate by subjecting the second supernatant to a standing treatment; A method for producing a triterpenoid-containing seed extract, comprising the following steps (1)-(3) and (6)-(7), wherein the triterpenoids are nomirin and limonin.
(1) A step of obtaining a seed crude extract by subjecting citrus seeds to extraction treatment using 60 vol% to 80 vol% ethanol
(2) a step of obtaining a crude seed extract by subjecting the crude seed extract to a hydration treatment and a concentration treatment;
(3) subjecting the crude seed extract to acid treatment and salting treatment to obtain a triterpenoid-containing seed extract as a first precipitate; (6) subjecting the first precipitate to a washing treatment; obtaining a triterpenoid-containing seed extract and a fourth supernatant as a fourth precipitate (7) subjecting the fourth supernatant to drying treatment, solvent addition treatment using ethanol, and standing treatment; obtaining a triterpenoid-containing seed extract as a fifth precipitate by 3. The method of claim 2 , further comprising step (8) of:
(8) a step of obtaining a triterpenoid-containing seed extract as a sixth precipitate by subjecting the fifth precipitate to a washing treatment; The citrus fruits include Citrus depressa, C. unshiu, C. tachibana, C. leiocarpa, C. tardiva, C. succosa, and C. citrus fruit. kinokuni), C. erythrosa, C. sunki, C. deliciosa, C. nobilis, C. retuculata, Dancy tangerine (C. tangerina), Hanayu ( C. hanayu), mandarin orange (C. reticulata), sunki (C. sunki), korai tachibana (C. nippokoreana), shiranuhi and at least one citrus selected from the group consisting of Kiyomi, claims 1 to 3 A method according to any one of 2. The method of claim 1, wherein the second precipitate is a triterpenoid-containing seed extract having a high limonin content relative to the nomilin content. 3. The method of claim 2 , wherein said fourth precipitate or said fifth precipitate is a triterpenoid-containing seed extract having a high content of nomirin relative to the content of limonin. 4. The method of claim 3, wherein the sixth precipitate is a triterpenoid-containing seed extract having a high nomirin content relative to the limonin content.

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