JPH09143197A - Production of ecdysteroid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a compound useful for the control of harmful life and the control of the growth of useful life, etc., in high purity and yield by extracting a vegetable containing an ecdysteroid with a solvent, adjusting the extracted liquid to an alkaline state, treating with an adsorbent and eluting the adsorbed component. SOLUTION: The objective ecdysteroid expressed by the formula (Me is methyl) is produced in high purity and yield by extracting an ecdysteroid- containing vegetable (e.g. Pfaffia stenophylla) with a water-containing alcohol containing a 1-3C alcohol, adjusting the extracted liquid to an alkaline state of pH8-12, treating the pH-adjusted extracted liquid with an adsorbent such as a hydrophobic adsorbent, washing the adsorbent with an alkaline washing liquid of pH8-12 and an acidic washing liquid of pH1-5, eluting the component adsorbed to the adsorbent using an elution solvent having a 1-3C alcohol content higher than the content in the extraction liquid used in the adsorption treatment, treating the eluted liquid with an anion exchange resin and crystallizing the ecdysteroid from the eluted liquid subjected to the ion exchange treatment.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing ecdysteroids [(poly) hydroxysteroids such as 20-hydroxyecdysone] which are insect molting hormones useful for the protection of pests and the growth control of useful organisms. .

[0002]

2. Description of the Related Art Ecdysteroids are contained in the animal body of the phylum Arthropoda, Nematoda, Flatworm, Annelida, and Ciliophora, and induce morphology of insects and arthropods. It is a hormone that plays an important role in growth, differentiation and reproduction. In addition, administration of ecdysteroids from outside the animal body affects the normal molting of many insects, induces developmental abnormalities, exhibits juvenile hormone-like activity, promotes egg and sperm formation, or Inhibit. These various physiological actions indicate that ecdysteroids are useful for controlling pests and controlling the growth of useful organisms. However, the ecdysteroid content in the animal body is usually very small.

[0003] In the plant kingdom, there are known plants containing a relatively large amount of ecdysteroids and steroids having a similar structure. For example, Pafia, Inokozuchi, Kiransou, Inumasaki, Vitex, and the like are attracting attention as useful resources containing ecdysteroids. As a method for isolating and purifying ecdysteroids from these plants, for example, Japanese Patent Publication No.
-28038 discloses a method for extracting ecdysteroids from Lamiaceae plants, and JP-B-46-31230 and JP-B-46-31554 disclose a method for extracting ecdysteroids from dogwood. There is. Japanese Patent Application Laid-Open No. 59-10600 discloses a method for obtaining β-ecdysteroid from Pfaffia paniculata , and Japanese Patent Application Laid-Open No. 63-2928 discloses Pfaffia iresinoides.
oides ) to obtain 20-hydroxyecdysteroids. Japanese Laid-Open Patent Publication No. 1-135794 discloses Vitex stricker.
Method for obtaining ecdysteroid from i ), JP-A 2-24
Japanese Patent Publication No. 0096 discloses that Pfaffi
a method of obtaining 20-hydroxyecdysteroids from A. stenophylla ) has been proposed. Also, Japanese Patent Application Laid-Open No. 1-31
7397 discloses a method of infecting an ecdysteroid-containing plant with a bacterium of the genus Agrobacterium, culturing the tissue, and extracting the ecdysteroid from the culture.

Further, in JP-A-4-279528, an ecdysteroid-containing plant is extracted with water, a hydrophilic solvent or a mixed solvent thereof, and an alkali-adjusted extract is treated with a reverse phase adsorbent. Then, the reverse phase adsorbent is washed with an alkaline cleaning solution containing 20% by volume or less of the lower alcohol, and the adsorbed component of the reverse phase system adsorbent is eluted with an elution solvent containing 20 to 60% by volume of the lower alcohol. Discloses a method for crystallization and isolation of ecdysteroid.

However, even if the plant species are the same, the ecdysteroid-containing plant as a raw material is
The ecdysteroid content varies depending on the cultivation conditions. Particularly in a plant having a low ecdysteroid content, the content of impurities is relatively large, and therefore it is necessary to repeat the separation operation, and the purification process is complicated. Moreover,
Since the operation of crystallizing ecdysteroid from the eluate is repeated, the loss of ecdysteroid occurs and the yield decreases. Therefore, it is difficult to industrially and efficiently produce ecdysteroids from plants efficiently.

[0006]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a method for producing ecdysteroids, which enables high-purity ecdysteroids to be obtained in high yield even from plants having a low ecdysteroid content. To provide.
Another object of the present invention is to provide a method for producing ecdysteroid, which enables highly pure ecdysteroid to be obtained in a high yield by a relatively simple operation.

[0007]

Means for Solving the Problems As a result of intensive studies to achieve the above-mentioned object, the present inventors have found that, in the method described in the above-mentioned prior art document, (1) after the extract is adsorbed with an adsorbent, it is alkaline. Even if a plant with a low ecdysteroid content is treated with a high-purity ecdysteroid, washing with a combination of a cleaning solution and an acidic cleaning solution or (2) treating the eluate of the components adsorbed on the adsorbent with an anion exchange resin The inventors have found that a disteroid can be efficiently obtained in a high yield, and completed the present invention.

That is, in the present invention, a step of extracting an ecdysteroid-containing plant with a solvent, a step of treating an alkaline-adjusted extract with an adsorbent, and a step of treating the adsorbent with an alkaline cleaning solution and an acidic cleaning solution. And a step of eluting the component adsorbed on the adsorbent and isolating the ecdysteroid from the eluate to produce ecdysteroid. In this method, the eluate may be treated with an anion exchange resin, and the ecdysteroid may be crystallized from the ion-exchanged eluate for isolation. Furthermore, in the method of the present invention, a step of extracting the ecdysteroid-containing plant with a hydrous alcohol containing a C _1-3 alcohol, a step of adjusting the pH of the extract to 8 to 12, and
a step of treating the pH-adjusted extract with a hydrophobic adsorbent,
The adsorbent is treated with an alkaline cleaning solution having a pH of 8 to 12 and p
A step of washing with an acidic washing solution of H1 to 5; a step of eluting the components adsorbed on the adsorbent using an elution solvent having a higher content of C _1-3 alcohol than the extract used for the adsorption treatment; Also included is a method for producing ecdysteroids, which comprises a step of treating a liquid with an anion exchange resin and a step of crystallizing ecdysteroids from the ion-exchange treated eluate. In the present specification, the term “ecdysteroid-containing plant” is used to include an induced root produced by tissue culture of a plant, callus, or a plant regenerated from these.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION According to the method of the present invention, ecdysteroids can be efficiently obtained even if the content of ecdysteroids is small. Therefore, the type of plant is not particularly limited as long as it contains ecdysteroids. . More than 250 species of ecdysteroid-containing plants are distributed in 33 families [Journal of Scientific and Industrial Research (Journal of Scientific and Industr
ial Research), 38 , 632-647 (1979)], for example, JP-A-4-279528. Examples of ecdysteroid-containing plants include the following plants. The mainspring (Osmda japonica) and other mainspring (Osm
unda) genus; Shishigashira (Blechnum niponicum) and Osashida
(B. amabile) and other genus (Blechnum); Cheilanthes argentea and C. bran
dtii) and other genus Cheilanthes;
Lepidoptera (Lemmaphyllum) such as astrea microphyllus)
Genus: L. thelypteris
a) Genus; dogfish (Podocarpus macrophyllum), eel (P.
nagi) and brown pine (P. elatus)
genus rpus); Polypodium genus such as Polypodium veitchii and Polypodoum vulgare; Pteridi such as Pteridium aquilinum and P. japonicum.
um) genus; yew (Taxus cuspidata), yew vaceta (T.
baceata), Taxus genus such as Taxus cuspidata (T. cuspidata); Serratula coronata
Such as Serratula genus; Dcrydium intermedium and other Dcrydium genus; Abuta velutina and other Abuta genus; Cesbium porche la castrum Sesbium
vium); Pfaffia iresinoi
des), P. paniculata,
P. stenophylla, P. tuberosa, P. pulverulenta, P. glomerata
genus of Pfaffia such as omerata);
genus Achyranthes, such as yranthes japonica) and chickweed (A. fauriei);
genus Cyathula such as athula proatrata; Bosae such as Pozea and Elbamora
ea) genus; Ajuga reptans var. atropurpurea, Ajuga japonica, A. decumbens,
A. pygmaea, A. nipp
onensis), holly (A. incisa), etc .; Ajuga; Vitex sereti (Vitex sereti); Vitex remanni (V. rehmanni);
Madiensis (V. madiensis), Vitex cilsiflora (V. thyrsiflora), Vitex glabrata
(V. glabrata), Vitex Micranta (V. micrant
ha), Vitex Doniana (V. doniana), Vitex Strickeri (V. strickeri), Vitex Mombasae (V. mombasae), Vitex Buchanani (V.
buchananii), Vitex Ferguinea (V. ferrugi)
nea), V. megapotamic
a), Vitex genus such as V. celebica.

Among these plants, plants having a high ecdysteroid content, for example, plants of the genus Paphia such as Paphia paniculata, Paphia iresinoides, Paphia stenophylla; Use of a plant of the genus Vitex such as or vitex micranta is advantageous for producing a large amount of ecdysteroid by an industrial process. The Paphia plant is a medicinal plant mainly growing in South America, and the Vitex plant is a woody plant growing naturally in Africa.

In order to industrially produce ecdysteroids, plants containing a small amount of ecdysteroid green compounds, particularly plants of the genus Vitex and Paphia, are useful. The method of the present invention is also effective for plants having a low ecdysteroid content due to individual differences, collection times, cultivation conditions, etc., and is particularly effective for obtaining ecdysteroids from plants containing a large amount of acidic impurities.

Ecdysteroids are the leaves of these plants,
Since it is present in almost all organs such as stems, bark, roots and fruits, the organs of plants used for the extraction process are not particularly limited. Preferable sites include roots, which have a high ecdysteroid content and are low in contaminants. The plant may be a fresh material or a dry material. It is useful to use the plant in powder form in order to enhance the extraction efficiency. The ecdysteroid contained in the plant has 20-
Hydroxyecdysone, 22-hydroxyecdysone,
20-hydroxyecdysone, which is a typical ecdysteroid, includes ponasterone and inocosterone.
It is represented by the following equation.

[0013]

Embedded image The method of the present invention comprises an extraction step of extracting an ecdysteroid-containing plant with a solvent, an adsorption treatment step of treating an alkaline-adjusted extract with an adsorbent, and a washing step of treating the adsorbent with an alkaline or acidic washing solution. And an elution step of eluting the components adsorbed on the adsorbent, and an isolation step of isolating ecdysteroid from the eluate.

[Extraction Step] The plant is subjected to an extraction step with water, an organic solvent (particularly a hydrophilic organic solvent) or a mixed solvent thereof. As the organic solvent, a hydrophilic organic solvent, for example, methanol, ethanol, propanol,
Isopropanol, butanol, isobutanol, t-
Monohydric lower alcohols such as butanol, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol,
1,3-butanediol, 1,4-butanediol, polyhydric alcohol such as glycerol, methyl cellosolve,
Examples thereof include cellosolves such as ethyl cellosolve, ethers such as dioxane and tetrahydrofuran, and ketones such as acetone and methyl ethyl ketone. These organic solvents may be mixed and used.

Of these hydrophilic organic solvents, water-soluble organic solvents such as monovalent C _1-4 alcohols (especially water-soluble C _1-3 alcohols such as methanol and ethanol) and acetone are preferable. In particular, methanol and / or ethanol are preferable as the water-soluble alcohol, and methanol and the like are useful for effectively extracting ecdysteroid. It should be noted that extraction with water alone may cause foaming accompanying the extraction operation depending on the plant material, which may reduce workability efficiency.Extraction with the organic solvent alone may result in a large amount of components having a smaller polarity than ecdysteroids. It is easy to extract. Therefore, a more preferable extraction solvent is water, a mixed solvent of water / hydrophilic organic solvent (hydrous organic solvent), and particularly hydrous alcohol (for example, an aqueous solution of methanol or ethanol).

In a water-containing organic solvent such as a water-containing alcohol, the content of the organic solvent is within a range that does not impair the extraction efficiency of the ecdysteroid, depending on the type of the organic solvent and the plant.
For example, the organic solvent is 2.5 to 70% by volume, preferably 5 to
It is about 50% by volume and 5 to 30% by volume (for example, 5 to
It is often selected from the range of about 20% by volume. If the content of the organic solvent is too large, not only the amount of the lipophilic impurities such as chlorophyll and fat and oil components to be extracted increases, but also a step of distilling off the organic solvent in a subsequent step is required.

The extraction can be carried out by a conventional method such as cold soaking, warm soaking, refluxing, ultrasonic treatment, or the like, while standing or stirring. In order to enhance the extraction efficiency, hot extraction is preferable, and the extraction temperature is, for example, 30 ° C or higher (30 to
120 ° C.), particularly 50 ° C. to reflux temperature. The extraction time can be selected according to the extraction temperature, for example, in the case of cold extraction, for example, 1 to 15 days, preferably about 1 week, and in the case of hot extraction, for example, 30 minutes to 24 hours, preferably Is about 2 to 8 hours. The extraction operation may be performed under normal pressure or under pressure.

If necessary, the pH of the extraction solvent may be adjusted to be alkaline in advance. In this case, the pH adjusting step for adjusting the extract to be alkaline as described later is not always necessary. In order to remove impurities, the extract is usually subjected to an adsorption treatment step after being subjected to a filtration step and the like.

[Adsorption treatment step] If the extract is left for a long period of time, it may gradually become acidic and a precipitate may be formed. The formed precipitate may be used when a column packed with an adsorbent is used.
It also causes column clogging. Suppresses the formation of precipitates,
In order to increase the yield and purity of ecdysteroid, it is useful to adjust the pH of the extract solution to alkaline and subject it to the adsorption treatment step. As described above, when the alkaline cleaning liquid is used in the extraction step, the pH adjusting step is not always necessary, but even in this case, it is preferable to use the extraction liquid whose pH is adjusted to alkaline.

The pH of the pH-adjusted extract may be in the range that does not impair the adsorption efficiency and operability of ecdysteroids, and for example, pH7.
5 to 14, preferably about 8 to 12 (for example, 9 to 11)
Degree). The pH of the extract is an inorganic base (for example, alkali metal hydroxide such as sodium hydroxide and potassium hydroxide, alkali metal carbonate such as sodium carbonate and potassium carbonate, alkali metal carbonate such as sodium hydrogen carbonate and potassium hydrogen carbonate). Bicarbonate, ammonia, etc.),
It can be adjusted using an organic base (eg, trimethylamine, triethylamine, etc.). As a pH adjuster, an inorganic base is usually used in many cases. If necessary, the pH-adjusted extract may be concentrated and subjected to adsorption treatment. Further, the organic solvent content (for example, the water-soluble C _1-3 alcohol content) of the extract used for the adsorption treatment may be adjusted to a range that does not impair the adsorption efficiency, for example, about 1 to 15% by volume.

Adsorption treatment In the adsorption treatment step, ecdysteroid is separated from the extract or its concentrate using an adsorbent. As an adsorbent,
Activated carbon (powdered, fibrous, honeycomb, etc.),
Conventional adsorbents such as alumina can be used. Preferred adsorbents are hydrophobic adsorbents (reverse-phase adsorbents) having a large adsorption efficiency and adsorption amount for ecdysteroids, for example, chemically bonded silica gel such as octadecylsilane, hydrophobic adsorbents such as activated carbon (reverse-phase adsorbent). System adsorbents), especially hydrophobic (reverse phase) polymer adsorbents, and the like. Hydrophobic (reverse phase system)
The polymer adsorbent is a copolymer having a huge network structure and has a large adsorption amount of ecdysteroid. The resin matrix of the polymer adsorbent is usually a copolymer of styrene-divinylbenzene, acrylic acid ester, methacrylic acid ester and the like. Hydrophobic (reverse phase) polymer adsorbents include, for example, "XAD resin" from Organo Co., Ltd. and "HAD" from Mitsubishi Chemical Co., Ltd.
P resin ”and the like.

The adsorbent may be pretreated prior to the adsorption treatment. This pretreatment can be performed, for example, by washing the adsorbent with a solvent such as methanol to remove impurities, and then further washing with water or the like to remove the solvent. The adsorption treatment with the adsorbent may be either a batch method or a column method, and a column chromatographic method capable of efficiently treating with a relatively small amount of the adsorbent may be used. The adsorption treatment should be performed at least once,
The ratio of the adsorbent to the extract can be selected according to the saturated adsorption amount of the adsorbent and the like.

[Washing Step] The first feature of the present invention is that after the adsorption treatment, the adsorbent is washed with both the alkaline washing solution and the acidic washing solution in the washing step. As long as the cleaning treatment is carried out by alkali cleaning and acid cleaning, either alkali cleaning or acid cleaning may precede. In addition,
After the alkali cleaning, the cleaning treatment is often performed with an acidic cleaning solution. Further, the alkali cleaning and the acid cleaning may be performed plural times. After washing with an alkaline washing solution or an acidic washing solution, the adsorbent may be directly subjected to acidic washing or alkaline washing, but after each washing treatment (particularly after the preceding washing treatment), the pH of the washing liquid is moderate with water etc. In many cases, washing is performed until it reaches the nature range (for example, pH of about 6 to 8).

The washing with the alkaline washing solution and the acidic washing solution can be carried out by using the degree of coloring of the washing solution as an index, and in many cases, the washing is usually performed until the color of the washing solution does not become lighter. The washing may be performed by changing the concentrations of the basic component, the acid component and the hydrophilic organic solvent of the washing liquid stepwise or continuously. Further, the cleaning with the alkaline cleaning solution and the acidic cleaning solution is carried out alternately in combination with an alcohol aqueous solution containing a lower alcohol such as methanol at a concentration of 20% by volume or less (for example, using an alkaline cleaning solution or an acidic cleaning solution and an alcohol aqueous solution). You may do it.

When the adsorbent is washed by combining the alkaline washing liquid and the acidic washing liquid, not only the pigment components such as dark brown but also neutral, acidic and basic impurities can be efficiently removed and the yield is lowered. Without it, the purity of ecdysteroid can be effectively increased.

The pH of the alkaline cleaning alkaline cleaning solution, as in the pH adjustment step, PH7.5～14, preferably about 8 to 12 (e.g., about 9-11). The alkaline cleaning liquid is preferably an alkaline aqueous solution or an organic solvent-containing alkaline aqueous solution, and the base of the alkaline cleaning liquid has the above-mentioned pH.
The same base as described in the adjustment step is exemplified. Further, the organic solvent in the organic solvent-containing alkaline aqueous solution includes the same hydrophilic organic solvent as exemplified in the extraction step. Of the above organic solvents, lower alcohols, preferably C _1-4 alcohols, and particularly water-soluble C _1-3 alcohols (among others, methanol and ethanol) are preferable. The content of the hydrophilic organic solvent in the alkaline cleaning liquid is lower than the content of the organic solvent in the extraction solvent and can be selected within a range capable of suppressing the elution of ecdysteroids, and, for example, 2.5 to
It can be selected from the range of about 20% by volume, preferably about 5 to 20% by volume, more preferably about 5 to 15% by volume.

The pH of the acidic cleaning acidic cleaning solution, for example, PH1～6, preferably 1 to 5 (e.g., 1-4), more preferably about 2 to 3. The acidic cleaning liquid is preferably an acidic aqueous solution or an organic solvent-containing acidic aqueous solution. The pH of the acidic cleaning solution is
Conventional acid components such as inorganic acids (hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, etc.), organic acids (formic acid, acetic acid, propionic acid, trichloroacetic acid, trifluoroacetic acid, oxalic acid, malonic acid,
Adjusted using carboxylic acids such as succinic acid and glutaric acid, oxycarboxylic acids such as glycolic acid, lactic acid, tartaric acid and citric acid) and buffers (eg sodium citrate-hydrochloric acid buffer, tartaric acid-sodium tartrate etc.) it can. These acid components may be used alone or in combination of two or more. An inorganic acid such as hydrochloric acid is often used as the acid component. In the acidic aqueous solution containing the organic solvent, the organic solvent includes the same hydrophilic organic solvent as exemplified in the extraction step. Of the above organic solvents, lower alcohols, preferably C _1-4 alcohols, especially water-soluble C _1-3
Alcohols (among others, methanol and ethanol) are preferred. The content of the hydrophilic organic solvent in the acidic cleaning solution is lower than the content of the organic solvent in the extraction solvent, and can be selected within a range capable of suppressing the elution of ecdysteroids.
-20% by volume, preferably 5-20% by volume (eg 1
0 to 20% by volume), more preferably 5 to 15% by volume.

[Elution Step] After the washing step, the components adsorbed on the adsorbent are eluted with an elution solvent. As the elution solvent, a solvent capable of eluting ecdysteroid, for example, the same hydrophilic organic solvent as described above (for example, monovalent lower alcohol such as methanol, ethanol, propanol, isopropanol, butanol, isobutanol, t-butanol). , Cellulosolves such as methyl cellosolve and ethyl cellosolve, ethers such as dioxane and tetrahydrofuran, ketones such as acetone and methyl ethyl ketone), and water-containing solvents containing these hydrophilic solvents. Of the hydrophilic organic solvents, lower alcohols, preferably C _1-4 alcohols, especially water-soluble C _1-3 alcohols (among others, methanol and ethanol) are preferable, and methanol having high elution efficiency is often used.

When the organic solvent such as methanol alone is eluted, impurities may be easily eluted and it may be difficult to isolate the ecdysteroid by crystallization. Therefore, a particularly preferable elution solvent is a water-containing organic solvent. The content of the organic solvent (hydrophilic organic solvent such as C _1-3 alcohol) in the water-containing organic solvent (elution solvent) is in a range larger than the content of the organic solvent in the extract or the washing solution subjected to the adsorption treatment, For example, 20 to 95% by volume, preferably 25
To 70% by volume (for example, 25 to 60% by volume), and more preferably about 30 to 50% by volume.
The elution may be performed by changing the concentration of the hydrophilic organic solvent stepwise or continuously. The eluate eluted with the solvent may be subjected to a concentration step of concentrating or drying under normal pressure or reduced pressure. The eluate is often concentrated in order to improve the work efficiency for isolating ecdysteroids.

[Isolation step] High-purity ecdysteroid can be obtained by isolation from the eluate. Isolation of ecdysteroids can be carried out by various methods, such as concentration, crystallization,
It can be carried out using an isolation means such as recrystallization, extraction, partitioning, chromatography and the like. A preferred method includes a step of treating the eluate with an anion exchange resin and a step of isolating ecdysteroid from the eluate treated with the anion exchange resin, in which the ecdysteroid is isolated by crystallization. May be isolated. By treating the eluate with the anion exchange resin, to remove the acidic impurities remaining in the eluate, while suppressing the loss of ecdysteroid,
It is possible to suppress coloring of ecdysteroids and obtain highly pure ecdysteroids.

Treatment Process with Anion Exchange Resin As the anion exchange resin, various resins having anion exchange ability, for example, strongly basic ion exchange resins containing quaternary ammonium, primary amines, secondary amines, etc. Weakly basic anion exchange resins containing primary amines, tertiary amines, etc. can be used. The anion exchange resin may be a gel type (crosslinking type) of a polymer phase or a porous type in which pores are developed. The resin matrix of the anion exchange resin can be usually composed of a copolymer of styrene-divinylbenzene, acrylic acid ester, methacrylic acid ester and the like. Preferred anion exchange resins include weakly basic resins that have a low possibility of degrading ecdysteroids, particularly acrylic resins that have a low adsorption capacity for ecdysteroids. The anion exchange resin is available, for example, as "IRA resin" from Organo Corporation, "DIAION resin" from Mitsubishi Chemical Corporation, and the like.

The anion exchange resin is preferably pretreated prior to the treatment. This pretreatment is, for example, after treating the adsorbent with an acidic aqueous solution containing an acid component,
It can be carried out by treating with a basic aqueous solution containing a basic component and further washing with water. When hydrochloric acid is used as the acid component and sodium hydroxide is used as the base component, the resin is made chloride ion type, then the resin is made free with an aqueous sodium hydroxide solution, and excess base component is removed by washing with water. The anion exchange resin can be pretreated.

The treatment with the anion exchange resin may be carried out by either a batch method or a column method. The treatment conditions such as concentration, temperature and time can be used to enhance the efficiency of the ion exchange reaction and the ecdysteroid is not decomposed. You can choose from the conditions.
The amount of the anion exchange resin used depends on the amount of acidic impurities in the eluate, but 100 ml or less of the granular anion exchange resin (for example, 5 to 7) per 10 L of the eluate from the adsorbent.
0 ml), preferably about 10 to 50 ml. The treatment temperature is, for example, about room temperature to 50 ° C., and the treatment time can be selected within the range of the time when the ion exchange reaction is completed, but the treatment time is advantageously short.

Isolation of Ecdysteroid As described above, the ecdysteroid can be isolated from the eluate treated with the anion exchange resin by a conventional isolation means. In order to further increase the purity of the ecdysteroid, it is preferable to isolate the ecdysteroid by crystallization. Crystallization of ecdysteroids may be carried out using one or more organic solvents, but water, particularly a mixed solvent of water and a lower alcohol (first mixed solvent), an ester and a lower alcohol. It is preferable to use a mixed solvent of (2nd mixed solvent). When the eluate treated with an anion exchange resin is crystallized with water or the mixed solvent, the purity is 9
0% or more of high-purity ecdysteroid is obtained as a crystalline substance. In particular, it is preferable to alternately crystallize the ecdysteroid from the eluate using the first mixed solvent or two mixed solvents of water and the second mixed solvent. When two mixed solvents are used, the first mixed solvent or water can remove impurities having a higher polarity than ecdysteroids, and the second mixed solvent can remove impurities having a lower polarity than ecdysteroids. Thus, high-purity ecdysteroid having a purity of 95% or more can be obtained.

As the lower alcohol, a lower alcohol having 1 to 4 carbon atoms, preferably a water-soluble C _1-3 alcohol, more preferably methanol or ethanol,
In particular, methanol is mentioned. The ratio of water to lower alcohol may be in the range where ecdysteroids can be crystallized, and for example, water / lower alcohol = 50 to 99/50 to
It is about 1 (capacity part), preferably about 80 to 99/20 to 1 (capacity part). Examples of the esters include methyl acetate, ethyl acetate, butyl acetate, isobutyl acetate and the like. Preferred esters include ethyl acetate. The ratio of the ester and the lower alcohol may be within the range in which the ecdysteroid can be crystallized, and for example, esters / lower alcohol = 50 to 95/50 to 5 (volume part), preferably 70 to 95 / It is about 30 to 5 (capacity part). Crystallization of ecdysteroid with a solvent can be repeated if necessary in combination with a concentration operation.

A preferred method of the invention is a combination of the preferred embodiments described above for the isolation of ecdysteroids.
That is, a preferred method is to use (1) an ecdysteroid-containing plant in water, preferably a hydrous alcohol containing a water-soluble alcohol (for example, a water-soluble alcohol content of 0 to 20).
Extraction step of extraction with (% by volume), (2) pH adjustment step of adjusting the pH of the extraction solution to alkaline, (3) Adsorption treatment step of treating the pH-adjusted extraction solution with a hydrophobic (reverse phase) adsorbent (4) A washing step of washing the adsorbent with an alkaline washing solution and an acidic washing solution (which may contain a water-soluble alcohol in a concentration of 0 to 20% by volume), (5) p
An extraction solvent that has been adjusted to H and has a higher water-soluble alcohol content than the extraction liquid or washing liquid that has been subjected to the adsorption treatment step (for example, an elution solvent containing water-soluble alcohol at a concentration of 20% by volume or more)
An elution step of eluting the components adsorbed on the adsorbent using
(6) An isolation step of isolating ecdysteroid from the eluate is included. In the (6) isolation step, (6a) a treatment step of treating the eluate with a weakly basic anion exchange resin, and (6b) a crystallization step of isolating ecdysteroid from the elution treatment solution by crystallization. It is preferable that By such a method, ecdysteroid having a purity of 90% or more can be obtained in a high yield in a single process. Further, even if inexpensive and safe water and lower alcohol are used as the extract, washing solution, and eluate, highly pure ecdysteroid can be obtained in a high yield, which is industrially advantageous.

The crystals thus obtained were subjected to thin layer chromatography (adsorbent: Merck, Kieselgel 60).
F254, developing solvent: chloroform / methanol / water =
Lower layer of 7/3/1 (capacity ratio)), main wavelength 254 nm
When irradiated with UV light, it gave a single spot, vanillin: sulfuric acid: ethanol (3g: 1.5ml: 100m
l) After spraying the solution, heating at 100 ° C. for about 5 minutes gives a green spot, confirming that it is an ecdysteroid. Furthermore, high performance liquid chromatography (HPLC) (eluent: acetonitrile / water = 17 /
83 or dichloromethane / isopropyl alcohol / water = 100/40/3) shows a single peak.

The ecdysteroid obtained by the method of the present invention usually has a purity of 90% or more and can be used as a standard reagent for various physiological activity tests and analytical tests. The purity of ecdysteroid can be measured by a conventional assay method such as NMR, HPLC, and ultraviolet absorption spectrum.

[0039]

According to the method of the present invention, since the adsorbent is washed with the alkaline washing solution and the acidic washing solution, it is possible to obtain a high-purity ecdysteroid with a high yield even in a plant having a low ecdysteroid content. . Further, a highly pure ecdysteroid can be obtained in a high yield by a relatively simple operation.

[0040]

EXAMPLES The present invention will be described below in more detail with reference to Examples, but the present invention is not limited to these Examples. Comparative Example 1 2.4 L of a 10 vol% methanol aqueous solution was added to 600 g of Puffia stenophylla powder, and the mixture was heated under reflux at 80 ° C. for 5 hours. The obtained extract was filtered through Celite, the residue was washed with water, the wash and the filtrate were combined, and the ecdysteroid content in the solution was quantified by HPLC to give 20-hydroxyecdysteroid. (Hereinafter, referred to as ecdysone unless otherwise specified).

Hydrophobic (reverse phase) polymer adsorbent [Amberlite XAD-2000, manufactured by Organo Corporation] 0.1 L
Was packed in a column with 0.5 L of methanol, and water of 0.
Washed with 5 L. The pH of the ecdysone-containing solution was adjusted to about 10 using sodium hydroxide, and 1 L of 3 L of the pH adjusting solution was passed through a column for adsorption treatment. The column was washed successively with 1 L of water adjusted to pH 10 with a 1N sodium hydroxide aqueous solution and 1 L of water, and then the adsorbed components were eluted with 2 L of a 40 vol% methanol aqueous solution.
The solid content in the eluate was 3.1 g, the ecdysone content in the solid content of the eluate was 0.83 g (purity 27%), and the ecdysone fractionation rate was 89% with respect to the amount of ecdysone loaded on the column. It was

1 L of the 2 L of the eluate was concentrated under reduced pressure, and the crystals produced in the concentration process were collected by filtration and dried to obtain 0.26 g of crystals. The ecdysone content in the crystal was measured by HPLC to find that it was 0.24 g (purity 9
2%), and the ecdysone fractionation rate in the crystallization operation was 58%, the ecdysone fractionation rate was 51% with respect to the amount of ecdysone in the extract, and the yield based on the raw material was 0.26%.

Example 1 0.2 L of the adsorbent [Amberlite XAD-2000 manufactured by Organo Co., Ltd.] used in Comparative Example 1 was packed in a column using 1 L of methanol and washed with 1 L of water. 2 L of the ecdysone-containing solution adjusted to pH 10 in Comparative Example 1 was passed through the column for adsorption treatment. The column was sequentially washed with 2 L of water adjusted to pH 10 with 1N aqueous sodium hydroxide solution and 2 L of water, and then washed with 1N aqueous hydrochloric acid solution to adjust the pH.
After sequentially washing with 2 L of water adjusted to 2 and 2 L of water, 4
The adsorbed component was eluted with 4 L of 0% by volume aqueous methanol solution. The solid content in the eluate was 3.45 g, the ecdysone content in the eluate was 1.75 g (purity 51%), and the ecdysone fractionation rate was 94% with respect to the amount of ecdysone loaded on the column. .

1 L of 4 L of the eluate was concentrated under reduced pressure, and the crystals produced in the concentration process were collected by filtration and dried to obtain 0.36 g of crystals. The ecdysone content in the crystals was measured by HPLC to find that it was 0.33 g (purity 9
2%), the ecdysone fractionation rate in the crystallization operation was 75%, the ecdysone fractionation rate was 71% with respect to the amount of ecdysone in the extract, and the yield based on the raw material was 0.36%.

Examples 2 to 4 Powdery and granular anion exchange resins shown in the table [Organo Corporation]
Manufactured by Amberlite IRA series] was washed with 25 ml of 1N aqueous hydrochloric acid solution, then activated with 25 ml of 1N aqueous sodium hydroxide solution, and washed with water until the washing solution became neutral. The anion exchange resin used in each example is as follows. Example 2: Organo Co., Amberlite IRA-
35 (weakly basic acrylic anion exchange resin) Example 3: Amberlite IRA- manufactured by Organo Corporation
94s (weakly basic styrene-based anion exchange resin) Example 4: Organo Co., Amberlite IRA-
401 (strongly basic styrene-based anion exchange resin) 3 L of the eluate prepared in Example 1 was 75 m under reduced pressure.
The solution was concentrated to 1 and the concentrated solution was divided into 25 ml. To each of the divided concentrated liquids (25 ml) was added 16.7 ml of methanol to prepare a 40% by volume aqueous methanol solution, and 70 rp
The anion exchange resin was added under stirring of m and the stirring was continued until the pH of the mixed solution became constant.

The anion exchange resin was removed from the mixed solution by filtration, the anion exchange resin was washed twice with 5 ml of a 40% by volume aqueous methanol solution, and the filtrate and the washing solution were combined to prepare an ion exchange treatment solution. The solvent of the ion exchange treatment liquid was distilled off under reduced pressure, and the crystals produced in the distillation process (concentration process) were collected by filtration and dried to obtain crystals. When the ecdysone content in the crystals was measured by HPLC, the results shown in the table were obtained. In the table, "EC" indicates ecdysone.

[0047]

[Table 1] As is clear from the table, by combining the alkaline cleaning and acidic cleaning of the adsorbent with the ion exchange treatment of the eluate,
High-purity ecdysone can be obtained with a high fractionation rate.

Claims (7)

[Claims] 1. A step of extracting an ecdysteroid-containing plant with a solvent, a step of treating an alkaline-adjusted extract with an adsorbent, a step of treating the adsorbent with an alkaline cleaning solution and an acidic cleaning solution, and the adsorption. A process for producing ecdysteroid, which comprises a step of eluting a component adsorbed to the agent and isolating the ecdysteroid from the eluate. 2. The alkaline cleaning solution and the acidic cleaning solution are 0.
The method for producing an ecdysteroid according to claim 1, which comprises ˜20% by volume of a lower alcohol. 3. An alkaline cleaning liquid having a pH of 8 to 12, and p
The method for producing an ecdysteroid according to claim 1 or 2, which uses an acidic washing solution of H1 to H5. 4. The method for producing an ecdysteroid according to claim 1, wherein the eluate is treated with an anion exchange resin, and the ecdysteroid is crystallized and isolated from the ion-exchanged eluate. 5. The method for producing an ecdysteroid according to claim 4, wherein the anion exchange resin is a weakly basic anion exchange resin. 6. The method for producing an ecdysteroid according to claim 4, wherein the anion exchange resin is an acrylic anion exchange resin. 7. A step of extracting an ecdysteroid-containing plant with a hydrous alcohol containing a C _1-3 alcohol,
A step of adjusting the pH of the extract to 8 to 12, a step of treating the pH adjusted extract with a hydrophobic adsorbent, and a step of washing the adsorbent with an alkaline cleaning solution of pH 8 to 12 and an acidic cleaning solution of pH 1 to 5. A step of eluting components adsorbed on the adsorbent using an elution solvent having a higher content of C _1-3 alcohol than the extract used for the adsorption treatment, and a step of treating the eluate with an anion exchange resin And a step of crystallizing ecdysteroid from the ion-exchange treated eluate.