JP2007300916A - Method for producing ergothioneine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for safely producing ergothioneine in high yield. <P>SOLUTION: The method for producing ergothioneine comprises the extraction of a mushroom containing ergothioneine in a solvent under crushing, the fractionation of the ergothioneine-containing extract by a fractionating means using adsorption chromatography, and the separation and purification of ergothioneine from an ergothioneine-containing fraction by high-performance liquid chromatographic instrument. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for producing ergothioneine, and more particularly to a method for producing ergothioneine that can produce ergothioneine at low cost.

In recent years, ergothioneine has attracted attention for its antioxidant properties, and has been attracting attention as a food, cosmetic or pharmaceutical product.
In order for humans to ingest such ergothioneine, it is usually possible to eat plants that have absorbed ergothioneine biosynthesized by fungi and mycobacteria from the roots.
However, since the amount of ergothioneine present in plants is extremely small, in order to consume a sufficient amount of ergothioneine, a very large amount of plants must be eaten.
For this reason, in order to produce ergothioneine in large quantities, chemical synthesis methods (see Patent Document 1 below) and fermentation methods (see Patent Document 2 below) have been proposed as methods for producing ergothioneine.
JP-T 8-501575 Japanese Patent Publication No. 43-20716

According to the chemical synthesis method or the fermentation method as the production method of ergothioneine, ergothioneine can be ingested without eating the plant.
However, according to the chemical synthesis method, since the process becomes complicated, the production cost of the obtained ergothioneine is extremely high. Moreover, since various chemicals are used in the chemical synthesis method, the obtained ergothioneine can be used for cosmetics that are not directly ingested into the body, but from the viewpoint of safety for food or medicine. There's a problem.
On the other hand, in the fermentation method, ergothioneine is produced using fungi, which is superior to the chemical synthesis method in terms of safety, but the yield is small, so the production cost of the obtained ergothioneine is still very high. is there.
Then, the subject of this invention is providing the manufacturing method of ergothioneine which can obtain ergothioneine with high yield and safety | security.

The inventors of the present invention have studied to solve the above-mentioned problem, knowing that mushrooms such as tamamogitake contain a large amount of ergothioneine, and using such a mushroom as a raw material, ergothioneine can be produced in large quantities and at low cost. As a result of repeated examination considering that it can be obtained, the present invention has been achieved.
That is, the present invention is to extract the ergothioneine-containing extract extracted from the ergothioneine-containing mushroom with a solvent by fractionation means using adsorption chromatography. In the method for producing ergothioneine, a fraction solution containing ergothioneine is separated and purified by a high performance liquid chromatography apparatus.
In this invention, the manufacturing cost of ergothioneine can be further reduced by using mushrooms that have been artificially cultivated.
For this mushroom, oyster mushrooms with a high content of ergothionein can be preferably used, and in particular, one or more mushrooms selected from Tamogitake, oyster mushroom, eringi and enokitake can be preferably used.

By pulverizing the mushroom and extracting it with a solvent, in particular, extracting it while pulverizing in a solvent, ergothioneine present in the cells of the mushroom can be effectively extracted into the solvent. As this solvent, water is preferably used from the viewpoint of safety.
Further, by extracting hot ergothioneine from mushrooms, pulverization of mushrooms and the like can be omitted, and the manufacturing process of ergothioneine can be simplified.
In the fractionation means of the present invention, an ion exchange resin having an adsorption ability to adsorb ergothioneine and adsorption chromatography can be used in combination. When an ion exchange resin is used, ionic compounds such as amino acids cannot be separated. For this reason, ergothioneine can be further concentrated by further fractionating the fractionated solution containing the fractionated ergothioneine using adsorption chromatography. As this ion exchange resin, a cation exchange resin can be suitably used.
Moreover, as an adsorption chromatography, the column chromatography with which the column was filled with the granular adsorbent can be used conveniently.
The “mushroom” referred to in the present invention includes not only a fruit body composed of a stem portion and an umbrella portion but also a sarcophagus portion.

The method for producing ergothioneine according to the present invention is to extract, concentrate and purify ergothioneine using mushrooms containing ergothioneine as a raw material. For this reason, the manufacturing cost of ergothioneine can be significantly reduced. Furthermore, when using mushrooms artificially cultivated, it is possible to further reduce the production cost of ergothioneine.
Moreover, when water or ethyl alcohol is used as a solvent used for extraction or the like, or when ergothioneine is extracted from mushrooms with hot water, the resulting ergothioneine can be used for food addition.

In the present invention, ergothioneine-containing mushroom is used as a raw material. As for such mushrooms, by using artificially cultivated mushrooms, it is possible to stably obtain mushrooms as a raw material and to further reduce the production cost of ergothioneine finally obtained.
As a kind of mushroom, a large amount of ergothioneine is preferable for mushrooms in the family Amanita. In particular, as the content of ergothionein is large and it is artificially cultivated and easily available, one or more mushrooms selected from Tamogitake, Oystertake, Eringi and Enokitake can be used preferably.
Such “mushrooms” include not only a fruit body composed of a stem portion and an umbrella portion but also a sarcophagus portion.

In the present invention, ergothioneine is extracted with a solvent from ergothioneine-containing mushrooms. In this extraction, it is preferable to pulverize mushrooms and extract them with a solvent, in particular, to perform mushroom pulverization in a solvent and extract them while pulverizing mushrooms because ergothioneine can be sufficiently extracted from mushrooms in a short time. This pulverization can be performed with a commercially available pulverizer.
As such a solvent, a solvent capable of extracting ergothioneine in mushrooms, such as acetone, can be used, but a solvent that does not cause a problem even if ingested by the human body, for example, water or ethyl alcohol is preferable. In particular, when water is used as the solvent, it goes without saying that there is no problem even if the solvent is taken into the human body, but the used solvent can be easily discarded.
When mushrooms are pulverized and extracted with a solvent, an extract from which ergothioneine is extracted is separated from solids from a slurry composed of the pulverized mushroom and a solvent. In such a separation operation, the slurry is suction filtered using a filter medium such as filter paper, and then an alcohol solution such as ethanol is added to the filtrate and left to stand to precipitate a precipitate, and then the precipitate and the liquid are filtered. An extract is obtained by suction filtration using the filter medium. Alternatively, the filtrate may be freeze-dried after the slurry is suction filtered using a filter medium such as filter paper.
Thus, in order to make an ergothioneine-containing mushroom into a slurry, it is necessary to pulverize the mushroom into a slurry.
In this regard, by extracting ergothioneine from ergothioneine-containing mushrooms by hot water extraction, the mushrooms can be extracted without slurrying. According to such hot water extraction, ergothioneine can be extracted by manually holding mushrooms cut in hot water. For this reason, steps such as pulverization and slurrying of mushrooms can be omitted, and the ergothioneine-containing extract can be easily filtered.

The extract thus obtained is fractionated by a fractionation means using adsorption chromatography. As this adsorption chromatography, column chromatography in which a column is filled with a granular adsorbent can be suitably used.
Further, a plurality of fractions fractionated by such column chromatography are examined using thin layer chromatography in which a gel-like adsorbent is formed in a thin layer, and fractions containing ergothioneine are collected. Thus, the subsequent process can be efficiently performed by collecting the fraction solution containing ergothioneine.
In this column chromatography, an extract separated by a separation operation is attached to a particulate adsorbent such as alumina, and then the adsorbent is packed into a column and developed with ethanol-water, and the eluate is separated with a friction collector. Draw.
In thin-layer chromatography, a plurality of fractions are dropped into a thin layer made of a gel-like adsorbent such as silica gel and developed with methanol-water. Fractions containing a component with an Rf value of 0.27 divided by the solvent travel distance and a quenching UV (254 nm) are collected. Such a fraction contains ergothioneine.

By the way, since ergothioneine has an amphoteric ionic structure, ergothioneine can be separated from nonionic compounds such as saccharides by passing the ergothioneine-containing extract separated in the separation operation through an ion exchange resin. However, ionic compounds such as amino acids are easily adsorbed on the ion exchange resin, and ergothioneine cannot be separated from ionic compounds such as amino acids only with the ion exchange resin.
For this reason, as a fractionation means for fractionating an ergothioneine-containing extract, a fractionation means using an ion exchange resin and adsorption chromatography can be employed. As this ion exchange resin, a cation exchange resin can be suitably used.
In such fractionation means, the ergothioneine-containing extract separated by the separation operation is passed through ion exchange, and a plurality of fractions are collected. At this time, an ergothioneine-containing extract is attached to the ion exchange resin, and then a diluted ammonia solution is passed through to elute and separate the ergothioneine attached to the ion exchange resin. A plurality of fractionated liquids are inspected using thin layer chromatography in which a gel-like adsorbent is formed in a thin layer, and a fractional liquid containing ergothioneine is collected.

The collected fraction is neutralized with an acid such as hydrochloric acid, if necessary, and then separated into multiple fractions using gel filtration column chromatography to remove salts and impurities such as ammonium chloride. You may draw. In this case, the gel-like adsorbent is examined using a thin layer chromatography in which a thin layer is formed, and an ergothioneine-containing fraction is collected.
The collected fraction containing ergothioneine is fractionated into a plurality of fractions using adsorption chromatography. As this adsorption chromatography, column chromatography in which a column is filled with a granular adsorbent can be suitably used.
Further, a plurality of fractions fractionated by such column chromatography are also examined using thin layer chromatography in which a gel-like adsorbent is formed in a thin layer, and fractions containing ergothioneine are collected. .

Thus, the concentrated residue obtained by concentrating the collected fractions containing lugothionein under reduced pressure is dissolved in water, and then the solution obtained by removing insoluble components is placed in a high-performance liquid chromatography apparatus. Supply and separate and purify ergothioneine.
In this high performance liquid chromatography apparatus, a stationary phase having an alkyl chain mainly composed of a triacontyl group (C30) is used as the stationary phase packed in the column.
With respect to the effluent flowing out from the column, a UV detector provided as a detector detects a peak that absorbs ultraviolet light having a wavelength of 258 nm, and a predetermined peak that appears at a predetermined time is collected.
The obtained fraction is lyophilized to a powder. This powder, ^{1 H} spectrum measured by NMR is a ^{1 H} spectrum identical spectrum of ergothioneine, the obtained powder was ergothioneine.

As described above, in the method for producing ergothioneine according to the present invention described above, ergothioneine contained in the mushroom used as a raw material can be easily extracted, concentrated and purified. For this reason, the manufacturing cost of ergothioneine can be significantly reduced compared with the synthesis method and the fermentation method.
Further, water or water-ethanol can be used for the extract or the developing solution, which is safe for the human body and easy to process the treated solution.
Furthermore, artificially cultivated mushrooms can be used as a raw material, and mushrooms can be cultivated stably even in summer when the consumption of mushrooms is reduced and production is adjusted. For this reason, mushroom artificial cultivation can be performed stably throughout the year.

Tamogitake fruit body (fresh weight 500 g) is cut into small pieces and placed in a blender, and then distilled water (1.5 liters) is added and blended for 1 to 2 minutes to form a bowl-like slurry.
The obtained slurry is subjected to suction filtration using filter paper using Hyflo Supercell as a filter aid. The filtrate obtained is added with 2.3 times the amount of ethanol and allowed to stand overnight, and then filtered with suction using filter paper with Hyflo Supercell as a filter aid.
The obtained filtrate (5.3 liters) is passed through an alumina column. This alumina column is a column (with an inner diameter of 50 mm and a height of 210 mm) packed with 500 g of alumina [Alminium oxide 90 active neutral (Merck)].

A concentrate (4 g) obtained by concentrating the solution passing through the alumina column under reduced pressure is fractionated by column chromatography. The column used for this column chromatography is a column (with an inner diameter of 24 mm and a height of 200 mm) packed with 80 g of alumina [Alminium oxide 90 active neutral (Merck)]. The resulting concentrate was adhered to the alumina, eluted with ethanol-water (volume ratio 7: 3), and fractionated with a fraction collector.
The obtained fractions are examined by thin layer chromatography on silica gel [TLC; aluminium sheet, silica gel 60 F254 (Merck)]. In this thin layer chromatography, the reaction was developed with methanol-water (6: 1), the Rf value (spot movement distance divided by solvent movement distance) was 0.27, and quenching was performed with UV (254 nm). Collect the fractions containing the indicated ingredients. The collected fractions were concentrated under reduced pressure to obtain 1.4 g of concentrated residue.

The obtained concentrated residue (1.4 g) was dissolved in water (3 ml), and this solution was supplied to high performance liquid chromatography (HPLC). In such high performance liquid chromatography (HPLC), water as an eluent is supplied to a column [Develosil C30-UG-5 (20 × 250 mm)] with a guard column (20 × 50 mm), and the flow rate of the effluent from the column Was adjusted to 5 ml / min. For the effluent from the column, a peak that absorbs ultraviolet light having a wavelength of 258 nm was detected by a UV detector. A peak that absorbs ultraviolet rays having a wavelength of 258 nm is shown in FIG. In FIG. 1, the horizontal axis represents time (retention time) from the start of outflow of the effluent from the column.
In this example, components of absorption peaks appearing at a retention time of 22 to 24 minutes were collected. The fraction obtained by high performance liquid chromatography (HPLC) was lyophilized to obtain 100 mg of white powder.
About this white powder, ¹ H spectrum was measured by NMR. The result of the measurement is shown in FIG. The ¹ H spectrum shown in FIG. 2 is the same as the ¹ H spectrum shown in FIG. 3 measured by NMR for ergothioneine.
Therefore, the white powder obtained is ergothioneine.

  Among the fractions fractionated by column chromatography in Example 1, the fractions adjacent to the fraction subjected to high performance liquid chromatography (HPLC) in Example 1 were prepared in the same manner as in Example 1. After the treatment, components of the absorption peak appearing at a retention time of 22 to 24 minutes were collected using high performance liquid chromatography (HPLC). The fraction obtained by high performance liquid chromatography (HPLC) was lyophilized to obtain 50 mg of white powder. FIG. 4 shows a peak that absorbs ultraviolet light having a wavelength of 258 nm with respect to the effluent from the column of high performance liquid chromatography (HPLC).

Enokitake fruit body (fresh weight 110 g) is put in a blender, distilled water (330 ml) is added and blended for 1 to 2 minutes to form a cocoon-shaped slurry.
The obtained slurry is subjected to suction filtration using filter paper using Hyflo Supercell as a filter aid. The obtained filtrate was freeze-dried to obtain a water extract (7.4 g).
The resulting water extract is fractionated by column chromatography. The column used for this column chromatography is a column (100 mm inner diameter, 200 mm height) packed with 100 g of alumina [Alminium oxide 90 active neutral (Merck)]. The obtained water extract was adhered to the alumina, eluted with ethanol-water (volume ratio 7: 3), and fractionated with a fraction collector.
The obtained fractions are examined by thin layer chromatography on silica gel [TLC; aluminium sheet, silica gel 60 F254 (Merck)]. In this thin layer chromatography, the reaction was developed with methanol-water (6: 1), the Rf value (spot movement distance divided by solvent movement distance) was 0.27, and quenching was performed with UV (254 nm). Collect the fractions containing the indicated ingredients. The collected fractions were concentrated under reduced pressure to obtain 1.7 g of concentrated residue.

The obtained concentrated residue (1.7 g) is dissolved in water (3 ml), and then filtered using a syringe with a filter of cellulose acetate (Cellulose Acetate 0.45 mm: Advantec) to remove insoluble components.
Subsequently, the obtained filtrate was supplied to high performance liquid chromatography (HPLC). In such high performance liquid chromatography (HPLC), water as an eluent is supplied to a column [Develosil C30-UG-5 (20 × 250 mm)] with a guard column (20 × 50 mm), and the flow rate of the effluent from the column Was adjusted to 5 ml / min. For the effluent from the column, a peak that absorbs ultraviolet light having a wavelength of 258 nm was detected by a UV detector. A peak that absorbs ultraviolet rays having a wavelength of 258 nm is shown in FIG. In FIG. 5, the horizontal axis represents time (retention time) from the start of outflow of the effluent from the column.
In this example, components of absorption peaks appearing at a retention time of 22 to 24 minutes were collected. The fraction obtained by high performance liquid chromatography (HPLC) was lyophilized to obtain 12 mg of white powder.
About this white powder, ¹ H spectrum was measured by NMR. The measurement results are shown in FIG. The ¹ H spectrum shown in FIG. 6 is the same as the ¹ H spectrum shown in FIG. 3 measured by NMR for ergothioneine.
Therefore, the white powder obtained is ergothioneine.

After putting eringi fruit body (fresh weight 105 g) in a blender, distilled water (300 ml) is added and blended for 1 to 2 minutes to form a bowl-like slurry.
The obtained slurry is subjected to suction filtration using filter paper using Hyflo Supercell as a filter aid. The obtained filtrate was freeze-dried to obtain a water extract (5.6 g).
The resulting water extract is fractionated by column chromatography. The column used for this column chromatography is a column (100 mm inner diameter, 240 mm height) packed with 100 g of alumina [Alminium oxide 90 active neutral (Merck)]. The obtained water extract was adhered to the alumina, eluted with ethanol-water (volume ratio 7: 3), and fractionated with a fraction collector.
The obtained fractions are examined by thin layer chromatography on silica gel [TLC; aluminium sheet, silica gel 60 F254 (Merck)]. In this thin layer chromatography, the reaction was developed with methanol-water (6: 1), the Rf value (spot movement distance divided by solvent movement distance) was 0.27, and quenching was performed with UV (254 nm). Collect the fractions containing the indicated ingredients. The collected fractions were concentrated under reduced pressure to obtain 0.2 g of concentrated residue.

The obtained concentrated residue (0.2 g) is dissolved in water (2 ml), and then filtered using a syringe with a filter of cellulose acetate (Cellulose Acetate 0.45 mm: Advantec) to remove insoluble components.
Subsequently, the obtained filtrate was supplied to high performance liquid chromatography (HPLC). In such high performance liquid chromatography (HPLC), water as an eluent is supplied to a column [Develosil C30-UG-5 (20 × 250 mm)] with a guard column (20 × 50 mm), and the flow rate of the effluent from the column Was adjusted to 5 ml / min. For the effluent from the column, a peak that absorbs ultraviolet light having a wavelength of 258 nm was detected by a UV detector. A peak that absorbs ultraviolet rays having a wavelength of 258 nm is shown in FIG. In FIG. 7, the horizontal axis represents time (retention time) from the start of outflow of the effluent from the column.
In this example, components of absorption peaks appearing at a retention time of 22 to 24 minutes were collected. The fraction obtained by high performance liquid chromatography (HPLC) was lyophilized to obtain 9 mg of white powder.
About this white powder, ¹ H spectrum was measured by NMR. The measurement results are shown in FIG. The ¹ H spectrum shown in FIG. 8 is the same as the ¹ H spectrum shown in FIG. 3 measured by NMR for ergothioneine.
Therefore, the white powder obtained is ergothioneine.

After adding oyster mushroom fruit bodies (fresh weight 89 g) to a blender, distilled water (270 ml) is added and blended for 1 to 2 minutes to form a bowl-like slurry.
The obtained slurry is subjected to suction filtration using filter paper using Hyflo Supercell as a filter aid. The obtained filtrate was freeze-dried to obtain a water extract (6 g).
The resulting water extract is fractionated by column chromatography. The column used for this column chromatography is a column (100 mm inner diameter, 240 mm height) packed with 100 g of alumina [Alminium oxide 90 active neutral (Merck)]. The obtained water extract was adhered to the alumina, eluted with ethanol-water (volume ratio 7: 3), and fractionated with a fraction collector.
The obtained fractions are examined by thin layer chromatography on silica gel [TLC; aluminium sheet, silica gel 60 F254 (Merck)]. In this thin layer chromatography, the reaction was developed with methanol-water (6: 1), the Rf value (spot movement distance divided by solvent movement distance) was 0.27, and quenching was performed with UV (254 nm). Collect the fractions containing the indicated ingredients. The collected fractions were concentrated under reduced pressure to obtain 0.3 g of concentrated residue.

The obtained concentrated residue (0.3 g) is dissolved in water (2 ml) and then filtered using a syringe with a cellulose acetate (Cellulose Acetate 0.45 mm: Advantec) filter to remove insoluble components.
Subsequently, the obtained filtrate was supplied to high performance liquid chromatography (HPLC). In such high performance liquid chromatography (HPLC), water as an eluent is supplied to a column [Develosil C30-UG-5 (20 × 250 mm)] with a guard column (20 × 50 mm), and the flow rate of the effluent from the column Was adjusted to 5 ml / min. For the effluent from the column, a peak that absorbs ultraviolet light having a wavelength of 258 nm was detected by a UV detector. A peak that absorbs ultraviolet light having a wavelength of 258 nm is shown in FIG. In FIG. 9, the horizontal axis represents time (retention time) from the start of outflow of the effluent from the column.
In this example, components of absorption peaks appearing at a retention time of 22 to 24 minutes were collected. The fraction obtained by high performance liquid chromatography (HPLC) was lyophilized to obtain 30 mg of white powder.
About this white powder, ¹ H spectrum was measured by NMR. The measurement results are shown in FIG. The ¹ H spectrum shown in FIG. 10 is the same as the ¹ H spectrum shown in FIG. 3 measured by NMR for ergothioneine.
Therefore, the white powder obtained is ergothioneine.

After extracting Tamogitake fruit bodies (fresh weight 5 kg) by immersing them in acetone (15 liters) for 1 week, the extract from which Tamogitake fruit bodies were removed was concentrated to about 3 liters. Ethyl acetate was added to this concentrated solution for partition extraction, and the aqueous layer (2.2 liters) was separated and concentrated to concentrated (1.7 liters). 1.4 liters of the concentrated solution was lyophilized to obtain a solid (65.79 g).
Further, a part of the obtained solid (37.53 g) is fractionated by column chromatography. The column used for this column chromatography was Sephadex G-10 (inner diameter 4.0 mm, height 50 mm), eluted with ethanol-water (volume ratio 7: 3), and fractionated with a fraction collector. did.
Next, among the obtained fractions, the fraction having the highest UV absorption at a wavelength of 254 nm was concentrated under reduced pressure to obtain a concentrated liquid (72.5 mg), and the concentrated liquid was supplied to high performance liquid chromatography (HPLC). . In such high performance liquid chromatography (HPLC), water as an eluent is supplied to a column [Develosil C30-UG-5 (20 × 250 mm)] with a guard column (20 × 50 mm), and the flow rate of the effluent from the column Was adjusted to 5 ml / min. For the effluent from the column, a peak that absorbs ultraviolet light having a wavelength of 258 nm was detected by a UV detector.
In this example, components of absorption peaks that appear at a retention time of 18 to 21 minutes were collected. The fraction obtained by high performance liquid chromatography (HPLC) was lyophilized to obtain 44.2 mg of white powder.
When this white powder was measured for ¹ H spectrum by NMR, it was the same as the ¹ H spectrum shown in FIG. 3 which was measured by NMR for ergothioneine.
Therefore, the white powder obtained is ergothioneine.

Tamogitake fruiting body (fresh weight 1 kg) was divided into 5 times and subjected to blender treatment. In one blender treatment, 200 ml of acetone and 200 ml of distilled water were added to 200 g of the fruit body of Tamogitake in a blender to obtain a bowl-like slurry.
The slurry that had been subjected to the blender treatment 5 times was collected, and Hyflo Supercell was used as a filter aid, and suction filtration was performed using filter paper to obtain a 2-liter filtrate. The obtained filtrate was concentrated under reduced pressure and lyophilized to obtain a lyophilized extract (40 g).
This lyophilized extract was redissolved in 100 ml of distilled water and passed through a column (diameter 45 mm × height 410 mm) packed with a cation exchange resin (Amberlite IR120B H ⁺ form). Cations were adsorbed. Further, this column was washed with 1 liter of distilled water.
Next, the ion exchange resin packed in the column was washed with ammonia water diluted to 1/5 (1.5 liters), and the cationic compound adsorbed on the ion exchange resin was eluted. 300 ml each of the eluate flowing out from the lower end side of the column was collected (a total of 3 fractions were collected).

The obtained fraction is examined by thin layer chromatography on silica gel [TLC; aluminum sheet, silica gel 60 F254 (Merck)]. This thin layer chromatography was developed with methanol-water (6: 1). In the inspection using such thin layer chromatography, an Rf value (a value obtained by dividing the moving distance of the spot by the moving distance of the solvent) is 0.27, and a preparative solution containing a component that exhibits quenching in UV (254 nm) is obtained. And collected as a fraction containing ergothioneine.
After the collected fraction was neutralized with 6N hydrochloric acid, gel filtration column chromatography (diameter 65 mm × height 380 mm) using Sephadex G-10 was used to remove ammonium chloride and other impurities. In this gel filtration column chromatography, water was used as an eluent. As for the fractions fractionated by gel filtration column chromatography, the fractions containing a component having an Rf value of 0.27 and quenching in UV (254 nm) were collected by inspection using thin layer chromatography. It was. The collected fractions were lyophilized to obtain a lyophilized product (9.5 g).

The obtained lyophilizate is fractionated by column chromatography. The column used for this column chromatography is a column (inner diameter 40 mm, height 175 mm) filled with 200 g of alumina [Alminium oxide 90 active neutral (Merck)]. The obtained freeze-dried product is adhered to such alumina by dissolving it in a small amount of water.
In such column chromatography, ethanol-water (volume ratio 7: 3) was poured into the column as an eluent and fractionated. As for the obtained fraction solution, the fraction solution containing a component having an Rf value of 0.27 and quenching by UV (254 nm) was collected by inspection using thin layer chromatography. The collected fractions were lyophilized to obtain a lyophilized product (0.5 g).

The obtained lyophilized product was supplied to high performance liquid chromatography (HPLC). In such high performance liquid chromatography (HPLC), water as an eluent is supplied to a column [Develosil C30-UG-5 (20 × 250 mm)] with a guard column (20 × 50 mm), and the flow rate of the effluent from the column Was adjusted to 5 ml / min. For the effluent from the column, a peak that absorbs ultraviolet light having a wavelength of 258 nm was detected by a UV detector. FIG. 11 shows the change in ultraviolet absorption over time.
In this example, absorption peak components appearing at 22-24 minutes over time (retention time) were collected. When the fractionated fraction was freeze-dried, 98 mg of white powder was obtained.
When the ¹ H spectrum of the obtained white powder was measured by NMR, it was the same as the ¹ H spectrum measured by NMR for ergothioneine. Therefore, the white powder obtained is ergothioneine.

Cut Tamogi bamboo fruit body (fresh weight 1.9kg) by hand into a suitable size, put it in a container, add 8 liters of water to the container, drop the lid, boil for 10 minutes and perform the first extraction It was. After the completion of the first extraction, the container was allowed to cool and filtered using a cloth bag.
The fruit body of Tamogitake filtered in a cloth bag was put into a container again, and 3 liters of water was further added to the container, dropped and covered, and boiled for 10 minutes to perform a second extraction. After the completion of the second extraction, the container was allowed to cool, and the second filtration was performed using the cloth bag used for the first filtration.
The content of ergothioneine in the filtrate obtained by the first filtration and the second filtration was measured using high performance liquid chromatography (HPLC).

In this measurement, first, 1 ml collected from each filtrate was passed through a Sep-Pak ™ Plus C18 cartridge (Waters) in order to remove the fat-soluble components in the filtrate.
Subsequently, the content of ergothioneine in the solution from which the fat-soluble component was removed was subjected to high performance liquid chromatography (HPLC). In this high performance liquid chromatography (HPLC), water as an eluent is supplied to a column [Develosil C30-UG-5 (4.6 × 250 mm)] with a guard column (4.6 × 10 mm), and the effluent from the column. Was adjusted to 0.8 ml / min. For the effluent from the column, a peak that absorbs ultraviolet light having a wavelength of 258 nm was detected by a UV detector. FIG. 12 shows the change in ultraviolet absorption over time. The change with time shown in FIG. 12 is for the first filtrate, and the absorption peak E that appears at 6.9 to 7.0 minutes with time (retention time) is the absorption peak of ergothioneine. When the component of this absorption peak E was fractionated, 0.98 g of ergothioneine could be obtained.
Similarly, when the first filtrate was subjected to high performance liquid chromatography (HPLC), an absorption peak E appeared at the same position as in FIG. 12, and the components of this absorption peak E were collected. However, 0.17 g of ergothioneine could be obtained.
The component of absorption peak E is the absorption peak of ergothioneine, which means that a solution in which only ergothioneine is dissolved in advance is subjected to high performance liquid chromatography (HPLC), and an absorption peak of ergothioneine appears at the position of absorption peak E. I have confirmed.

Thus, the ergothioneine in the filtrate obtained by each filtration of the 1st time and the 2nd time is contained.
Therefore, for these filtrates as well, fractionation treatment by fractionation means employing an alumina column as adsorption chromatography used in Example 1, or cation exchange resin as ion exchange resin used in Example 7 And ergothioneine can be further separated and purified by high performance liquid chromatography (HPLC) after fractionation by a fractionation means using both of them and an alumina column as adsorption chromatography.

It is a chart which shows the UV absorption condition of the effluent which flows out from a column, when the fraction liquid obtained using the alumina column as adsorption chromatography for the extract liquid extracted from a Tamogi-Dake fruit body is supplied to a high performance liquid chromatography. . The separated aliquot by high performance liquid chromatography shown in Figure 1 is the ^{1 H} spectrum measured by NMR of the white powder obtained by freeze-drying. The ¹ H spectrum was measured by NMR for ergothioneine. A chart showing the UV absorption status of the effluent that flows out from the column when the fraction obtained by using an alumina column as an adsorption chromatography for another extract extracted from the fruit body of Tamogi-Dake is supplied to high-performance liquid chromatography. It is. A chart showing the UV absorption status of the effluent that flows out from the column when the fraction obtained by using an alumina column as an adsorption chromatography for another extract extracted from the fruit body of Enokitake is supplied to high performance liquid chromatography It is. The separated liquid was fractionated by high performance liquid chromatography shown in Figure 5 is the ^{1 H} spectrum measured by NMR of the white powder obtained by freeze-drying. A chart showing the UV absorption status of the effluent flowing out from a column when a fraction obtained by using an alumina column as an adsorption chromatography for another extract extracted from the eringi fruiting body is supplied to high performance liquid chromatography. It is. The separated liquid was fractionated by high performance liquid chromatography shown in Figure 7 is the ^{1 H} spectrum measured by NMR of the white powder obtained by freeze-drying. A chart showing the UV absorption status of the effluent that flows out from the column when a fraction obtained by using an alumina column as an adsorption chromatography for another extract extracted from the oyster mushroom fruit body is supplied to high-performance liquid chromatography. It is. FIG. 10 is a ¹ H spectrum measured by NMR of a white powder obtained by freeze-drying a fraction collected by high performance liquid chromatography shown in FIG. 9. UV of the effluent that flows out from the column when the fraction obtained by using a fractionation means that uses a cation exchange resin as an ion exchange resin and an alumina column as an adsorption chromatography is supplied to high performance liquid chromatography. It is a chart which shows an absorption condition. It is a chart which shows the UV absorption condition of the effluent which flows out from a column, when the extract obtained by performing hot water extraction to a Tamogi-Dake fruit body is supplied to a high performance liquid chromatography.

Claims (11)

The extract containing ergothioneine extracted from the ergothioneine-containing mushroom with a solvent was fractionated by fractionation means using adsorption chromatography,
Among the obtained fractions, the ergothioneine-containing fraction is separated and purified by a high performance liquid chromatography apparatus using a ergothioneine-containing fraction.   The method for producing ergothioneine according to claim 1, wherein mushrooms that are artificially cultivated are used.   The method for producing ergothioneine according to claim 1 or 2, wherein mushrooms from the family Amanita are used.   The method for producing ergothioneine according to claim 1 or 2, wherein one or more mushrooms selected from mushrooms, oyster mushrooms, oyster mushrooms, eringgi and enokitake are used.   The method for producing ergothionein according to claim 1, wherein mushrooms are crushed and extracted with a solvent.   The method for producing ergothioneine according to any one of claims 1 to 5, wherein the ergothioneine-containing mushroom is extracted while being pulverized in a solvent.   The method for producing ergothioneine according to any one of claims 1 to 6, wherein water is used as a solvent for extracting ergothioneine from mushrooms.   The method for producing ergothioneine according to claim 1, wherein ergothioneine is extracted from mushrooms with hot water.   As fractionation means, an ion exchange resin having adsorption ability to adsorb ergothioneine and adsorption chromatography are used, and a fractionated solution containing ergothioneine fractionated using the ion exchange resin is further separated using the adsorption chromatography. The method for producing ergothioneine according to any one of claims 1 to 8.   The method for producing ergothioneine according to claim 9, wherein a cation exchange resin is used as the ion exchange resin.   The method for producing ergothionein according to any one of claims 1 to 10, wherein column chromatography in which a column is filled with a granular adsorbent is used as the adsorption chromatography.

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