JP7233085B2 - Method for separating selenoneine monomer - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act March 26, 2018 Presented in writing at the 2018 Spring Meeting of the Japanese Society of Fisheries Science. July 7, 2018 The 43rd▼ FEBS congress 2018, announced in writing.

The present invention relates to a method for separating selenoneine monomers from selenoneine-containing solutions.

Selenium is contained in fish tissues such as tuna and marlin at high concentrations, and is expected to be effective in preventing lifestyle-related diseases such as cancer and heart disease and aging. However, the low molecular weight selenium-containing compounds found in fish tissue have not been fully exploited. This is because the blood meat and internal organs of fish and shellfish may contain organic heavy metals such as methylmercury and cadmium, and are not suitable for use in pharmaceuticals, foods, and the like.
Therefore, the extraction of selenium-containing compounds from fish and shellfish tissue-derived concentrates to identify the substances, and the separation and purification of the selenium-containing compounds from the concentrates to produce high-purity products, Since it can be used in various applications, it has been strongly desired.

The inventors of the present application have elucidated the structure of a selenium-containing compound extracted from fish blood containing a large amount of organic selenium, and applied for a substance patent as a novel selenium compound (Patent Document 1). Later, this new selenium compound was named "selenoneine" because it is a compound in which the thioketone group of ergothioneine is replaced with a selenoketone group.

Selenoneine includes isomers of selenoneine monomers represented by structural formulas 1A and 1B below and selenoneine dimers (oxidized selenoneine) represented by compound 1C in which two compounds 1A are bonded.

It has been reported that selenoneine has various properties beneficial to the human body, including its strong antioxidant ability. ing.

In Patent Document 1 above, selenoneine was separated by high performance liquid chromatography (HPLC) using both a gel filtration column and an alkyl group-bonded column. However, although selenoneine can be purified from other selenium compounds by this method, selenoneine's sulfur analog, ergothioneine (see structural formula (2) below), was not sufficiently separated.

In addition, Non-Patent Document 1 describes separation and detection of ergothioneine and selenoneine using a C6 phenyl column in the HPLC method. However, the difference in retention time between ergothioneine and selenoneine when using this column is only about 24 seconds, and the selenoneine peak overlaps with the tail of the subsequent ergothioneine peak. is not suitable.

Non-Patent Document 2 describes a method for separating oxidized selenoneine (dimer) and ergothioneine (monomer) by the same HPLC method, and the resulting oxidized selenoneine has a purity of 98%. It is
However, in this method, since methanol is used to extract selenoneine, selenoneine is dimerized in ethanol, and although highly purified oxidized selenoneine is obtained, highly purified selenoneine monomer cannot be obtained. Since it is the selenoneine monomer that reacts in vivo, it is not appropriate to extract the selenoneine monomer using an organic solvent such as methanol. Moreover, in this method, since the selenoneine monomer in the sample is detected as selenoneine dimer, the content of the selenoneine monomer in the sample could not be analyzed.

Japanese Patent No. 5669056

Nina Kroepfl et al., Quantitative determination of the sulfur-containing antioxidant ergothioneine by HPLC/ICP-QQQ-MS, Journal of Analytical Atomic Spectometry, 2017, 32, 1571-1581

Nikolaus G. Turrini et al., Biosynthesis and isolation of selenoneine from genetically modified fission yeast, Metallomics, 2018.10.1532-1538, The Royal Society of Chemistry 2018

The present invention relates to a method for separating and purifying selenoneine as a monomer from a selenoneine-containing solution without dimerizing selenoneine.
In particular, ergothioneine, which is a sulfur analogue of selenoneine, has similar chemical properties to selenoneine, and when it coexists with selenoneine, it has been difficult to separate the two by conventional methods. As described above, selenoneine and ergothioneine are structurally different only in that the thioketone group is replaced with a selenoketone group. because there is no

However, it is said that selenoneine exhibits radical scavenging activity about 1000 times that of ergothioneine and about 500 times that of water-soluble vitamin E. In addition, selenoneine is rapidly taken up into cells from the medium of cultured cells via specific transporters, and enhances cell proliferation even under conditions of oxidative stress caused by hydrogen peroxide.
Therefore, the technical significance of separating and purifying selenoneine from ergothioneine is extremely significant.

An object of the present invention is to separate selenoneine monomers from a selenoneine-containing solution without dimerization, even if ergothioneine having similar chemical properties coexists.

〔２〕移動相媒体として水系溶液を用いる〔１〕に記載されるセレノネインモノマーの分離方法。
〔３〕分子排除クロマトグラフィーを行う工程をさらに含む、〔１〕または〔２〕に記載されるセレノネインモノマーの分離方法。
〔４〕セレノネインを熱水抽出する工程をさらに含む、〔１〕ないし〔３〕のいずれかに記載のセレノネインモノマーの分離方法。
〔５〕固定相としてアルキル基結合型シリカゲルを用いて（逆相）クロマトグラフィーを行う工程をさらに含む、〔１〕ないし〔４〕のいずれかに記載のセレノネインモノマーの分離方法。
〔６〕〔１〕ないし〔５〕のいずれかに記載のセレノネインモノマーの分離方法を含むセレノネインモノマーの製造方法。
〔７〕〔１〕ないし〔５〕のいずれかに記載のセレノネインモノマーの分離方法を含むセレノネインモノマーの分析方法。 In order to solve the above problems, the present disclosure employs a method having the following configuration.
[1] A method for separating a selenoneine monomer from a selenoneine-containing solution, comprising the step of performing (reverse phase) chromatography using pentabromobenzyl group-bonded silica gel (see structural formula (3)) as a stationary phase.

[2] The method for separating selenoneine monomers described in [1] using an aqueous solution as a mobile phase medium.
[3] The method for separating selenoneine monomers according to [1] or [2], further comprising the step of performing molecular exclusion chromatography.
[4] The method for separating selenoneine monomers according to any one of [1] to [3], further comprising a step of extracting selenoneine with hot water.
[5] The method for separating selenoneine monomers according to any one of [1] to [4], further comprising the step of performing (reverse phase) chromatography using alkyl-bonded silica gel as a stationary phase.
[6] A method for producing a selenoneine monomer, including the method for separating a selenoneine monomer according to any one of [1] to [5].
[7] A method for analyzing selenoneine monomer, including the method for separating selenoneine monomer according to any one of [1] to [5].

According to the selenoneine monomer separation method according to the present disclosure, it is possible to separate the selenoneine monomer from a selenoneine-containing solution containing ergothioneine, which has similar chemical properties and is difficult to separate.
In addition, since the selenoneine monomer of high purity was obtained, it became possible to develop reagents for chemical and biochemical experiments and standard products for analysis.
Furthermore, if a molecular exclusion column is combined, impurities with different molecular sizes contained in the selenoneine monomer-containing solution can be separated to increase the purity of the selenoneine monomer.

UV (260 nm) chromatogram with C30 column UV (260 nm) chromatogram when using a PBr column Ergothioneine standard ICP-MS chromatogram (SO-47.967) ICP-MS chromatogram of purified selenoneine (SO-47.967) ICP-MS chromatogram of purified selenoneine (SeO-95.9114) UV chromatogram of purified selenoneine (after concentration) (260nm, PBr column) UV chromatogram of purified selenoneine (260 nm, Ultrahydrogel 120 column) UV chromatogram of high-purity selenoneine (260 nm, Ultrahydrogel 120 column) UV (260 nm) chromatogram when using a C6 phenyl column (same conditions as in Example 1 except for the column) UV (260 nm) chromatogram when using a C6 phenyl column (same conditions as in Example 1 except for column and gradient conditions)

A method for separating selenoneine monomer from a selenoneine-containing solution of the present disclosure comprises performing (reverse phase) chromatography using pentabromobenzyl-bonded silica gel as the stationary phase.
In the present disclosure, the selenoneine monomer includes the isomers represented by the structural formulas 1A and 1B, but does not include the oxidized selenoneine (dimer) represented by the structural formula 1C.

In the separation method of the present disclosure, selenoneine monomers are separated by liquid phase chromatography. In liquid phase chromatography, various columns are prepared according to the purpose of separation, and these columns are classified according to packing material and separation mode.
The alkyl group-bonded reversed-phase stationary phase column is a reversed-phase column that uses a reversed-phase mode, and the column is packed with silica gel to which functional groups are bonded. In a reversed-phase column, when a sample is injected into the column, the components in the sample are separated by being eluted in order from the one with the highest polarity.

The column used in the present disclosure is a pentabromobenzyl group-bonded column in which fine silica gel is modified with pentabromobenzyl groups and has the function of separating hydrophilic compounds in reversed-phase mode (hereinafter "PBr column" called).

The present disclosure is based on the new finding that the use of a PBr column for liquid phase chromatography can more efficiently separate selenoneine and ergothioneine than the C6 phenyl column used in the conventional method. It is a thing.

In general, the pentabromobenzyl group and the C6 phenyl group (see structural formula (4)) both have aromatic rings and have similar chemical structures. is predicted to be impossible. That is, it was difficult to predict that the separation of selenoneine monomers and ergothioneine monomers (Resolution, hereinafter sometimes referred to as R) can be increased by using a PBr column compared to a C6 phenyl column. .
Here, the degree of separation indicates how much a certain peak is separated from adjacent peaks. The degree of separation is represented by Equation (1).

formula 1

In the separation method of the present disclosure, it is preferable to use an aqueous solution as the mobile phase in the step of performing (reverse phase) chromatography using a PBr column as the stationary phase. In the present invention, the aqueous solvent is not particularly limited as long as it is a solvent containing water as a main component, but it may be (acetic acid water), (ammonium acetate water), (formic acid water), ammonium formate water and water. is preferred, and water is more preferred.

In the separation method of the present disclosure, in the step of performing (reverse phase) chromatography using a PBr column as the stationary phase, the chromatography is not particularly limited as long as selenoneine monomers and ergothioneine monomers can be separated well, and open columns, Flash columns, HPLC, etc. can be used, but HPLC is preferred. The flow rate of the mobile phase may be appropriately adjusted depending on the size of the column used.

A selenoneine-containing solution to be treated in the present disclosure can be any solution that contains selenoneine. Selenoneine is contained in fish tissues such as tuna and marlin and in whale meat at high concentrations. It is processed material. In addition, a wide range of tissues other than fish and cetacean tissues can be processed, and examples thereof include chicken liver and pig kidney.

In addition to natural products, selenoneine-containing culture solutions and cell extracts obtained by culturing genetically modified fission yeast strains are also targeted.

In addition to ergothioneine, selenoneine-containing mixtures contain various contaminants. Therefore, in selenoneine separation, the following extraction methods and various columns in chromatography are used in combination to remove various contaminants according to the characteristics of each, so that the selenoneine monomer can be purified more efficiently. can do well.

Preferably, the separation method of the present disclosure further includes the step of removing contaminants using an alkyl group-bonded reversed-phase stationary phase column as the stationary phase. Although there is no particular limitation on the alkyl group-bonded reversed-phase stationary phase column, a column packed with silica gel to which a C6 to C30 alkyl group is bonded can be used, and C8, C18, and C30 columns are particularly preferred. A C30 column is more preferred. By using this, contaminants (mainly hydrophobic compounds) having a polarity different from that of selenoneine can be removed.

Also, the separation method of the present disclosure preferably further comprises the step of performing molecular exclusion chromatography. Separation using a molecular exclusion column packed with porous spherical silica gel can separate the components of the sample based on the difference in molecular size. This utilizes the phenomenon that silica gel has fine pores, and small molecules enter the pores and are eluted later than large molecules.

Preferably, the separation method of the present disclosure further comprises the step of hot water extraction of the selenoneine. By hot water extraction, selenoneine can be efficiently extracted, and contaminants insoluble in hot water can be efficiently removed.

In addition, before treatment by the method of the present disclosure or in the middle of the treatment, the selenoneine-containing mixture is subjected to a treatment such as concentration while heating with a rotary evaporator to increase the concentration of selenoneine. Good for raising.
By using the separation method of the present invention, it becomes possible to produce a higher purity selenoneine monomer and to more accurately quantify the selenoneine monomer content.

<Step 1> Preparation of Selenoneine-Containing Sample A genetically modified fission yeast strain (P3nmt1-egt1+) was cultured to synthesize selenoneine. The fission yeast strain used was obtained from the Yeast Genetic Resource Center, Graduate School of Science, Osaka City University. EMM-2 medium, which is generally used for culturing fission yeast, was used for culturing the strain. P3nmt1-egt1+ was cultured on agarose-containing YES medium, a single colony was dissolved in 100 mL of EMM-2 medium, and cultured with shaking at 28°C for 48 hours using a baffled shaking Erlenmeyer flask to obtain a yeast culture solution. After adding 100 mL of EMM2 medium to a new shaking Erlenmeyer flask with baffles, 1 mL of yeast culture medium and 10 μL of 100 mM selenate aqueous solution (final concentration 10 μM) were added. Ten cells with the same composition were prepared and cultured with shaking at 28°C for 48 hours.

<Step 2> Extraction of selenoneine-containing extract
About 1000 mL of the culture solution cultured with 10 μM selenate was dispensed into 250 mL aliquots into centrifuge bottles and centrifuged at 5000 rpm and 4° C. for 15 minutes. The medium was removed by decantation, and 100 mL of ultrapure water was added to the resulting bacterial mass to dissolve the bacterial mass. The centrifuge bottle containing the lysate was heated in boiling water for 15 minutes. After cooling at room temperature, it was centrifuged at 5000 rpm and 4°C for 15 minutes. The supernatant was filtered and sterilized through a 0.22 μm membrane filter (AGC Techno Glass Co., Ltd.) to obtain a selenoneine-containing extract. The amount of selenoneine in this selenoneine-containing extract was 4.59 μmol.

<Step 3> Crude purification of selenoneine The selenoneine-containing extract obtained in step 2 was concentrated with a rotary evaporator while being heated at 50°C. The concentrate was dispensed into brown vials and subjected to HPLC-UV-vis (manufactured by Agilent) at 50 µL. A C30 column (Develosil C30-UG-5 column, 4.6×50 mm, 5 μm, Nomura Chemical Co., Ltd.) was used after being heated to 40° C. in a column oven. Elution was performed in a binary gradient mode in which two types of mobile phases were gradually mixed, and the mobile phases used were 0.1% acetic acid in water (A) and 0.1% acetic acid in acetonitrile (B). Gradient conditions and flow rates are listed in Table 1. 2.6 to 6.0 minutes after the start of the analysis, the liquid eluted only with 0.1% acetic acid water was collected in a glass vial (Agilent Technologies, Inc.) using a fraction collector. The entire amount of the selenoneine-containing extract was subjected to HPLC, and the obtained fractions were mixed to obtain a selenoneine crudely purified liquid. FIG. 1 shows a UV (260 nm) chromatogram obtained when the concentrate was subjected to a C30 column under the conditions described above. Fractions containing selenoneine are shaded.

<Step 4> Purification of Selenoneine The selenoneine crudely purified liquid obtained in Step 3 was heated at 50°C and concentrated using a rotary evaporator. The concentrate was dispensed into a vial, and 15 μL was applied to HPLC-UV1200 series (Agilent Technologies, Inc.). As the column, a PBr column (Cosmosil PBr packed column, 4.6×250 mn, 5 μm, Nacalai Tesque, Inc.) was heated to 40° C. and used. Elution was performed in a binary gradient mode in which two types of mobile phases were gradually mixed, and water (C) and acetonitrile (D) were used as mobile phases. Gradient conditions and flow rates are listed in Table 2. A liquid eluted with only water at 16.1 to 18.0 minutes after the start of analysis was collected in a glass vial using a fraction collector. When the resolution, which is a value that indicates how far a peak is separated from adjacent peaks, is calculated for ergothioneine and selenoneine peaks, it is 4.16. was much higher. The entire crude selenoneine solution was subjected to HPLC, and the resulting fractions were combined and concentrated using a rotary evaporator. This was used as a purified selenoneine solution, which was adjusted to 200 μL with ultrapure water. FIG. 2 shows a UV (260 nm) chromatogram obtained when the selenoneine crude solution obtained in step 3 was subjected to a PBr column under the conditions described above. The shaded portion indicates the fraction containing selenoneine.

<Step 5> Analysis of Selenoneine The purified selenoneine solution obtained in Step 4 was analyzed by LC-ICP-MS and LC-PDA-MS. ICP-MS analysis conditions were referred to Patent Document 1 and Non-Patent Document 1. That is, HPLC pump (Pu712, GL Science Co., Ltd.), sample injector (9725i, Rheodyne Co., Ltd.), column oven (C0631A, GL Science Co., Ltd.), gel filtration column (Ultrahydrogel 120, inner diameter 7.8 mm × column length 300 mm, Waters Co., Ltd.). .) and ICP-MS (ELAN DRCII, Perkin-Elmer) were connected, and a 0.1 M ammonium acetate aqueous solution was flowed at a flow rate of 1.0 mL. The column was equilibrated with a 0.1 M ammonium acetate aqueous solution and heated to 40°C in a column oven before use. Oxygen was introduced as a reaction gas into the dynamic reaction cell (DRC) of the ICP-MS, and sulfur-32 oxide SO (molecular weight 47.967) and selenium-80 oxide SeO (molecular weight 95.9114) were monitored. .

For LC-PDA-MS analysis, the Ultimate 3000 (Thermo Fisher Scientific Co., Ltd.) system was used as the LC-PDA system. Ltd.) analyzed selenoneine under isocratic conditions in which a 0.1% acetic acid aqueous solution was flowed at a flow rate of 0.3 mL/min. Selenoneine was detected using a PDA detector in the Ultimate 3000 system and a time-of-flight mass spectrometer micrOTOFQII (Bruker Japan Co., Ltd.) as an MS system. Detection by PDA detected absorption at wavelengths 195-800 nm and monitored absorption at 260 nm derived from the imidazole ring of selenoneine. Detection by MS extracted m/z 278.0402, which is [M+H] ⁺ of selenoneine's compositional formula C ₉ H ₁₅ O ₂ N ₃ Se, from the measurement data.

Simultaneous analysis of sulfur and selenium by ICP-MS confirmed that selenoneine was isolated and purified from ergothioneine. That is, in FIG. 4, which shows the chromatogram obtained by the SO-monitored analysis, none of the standard ergothioneine peaks shown in FIG. 3 can be seen, and in FIG. , a distinct peak was observed.
As a result of LC-MS analysis, it was confirmed that the purified selenoneine solution before concentration contained monomeric selenoneine. In addition, according to the UV chromatogram (260 nm, PBr column: Fig. 6) obtained by analyzing the purified selenoneine solution after concentration, in addition to monomeric selenoneine, a trace amount of dimeric selenoneine is contained. was confirmed.

<Step 6> Quantification of Selenoneine and Calculation of Recovery Rate The total selenium concentration of the purified selenoneine solution obtained by the method of step 4 was measured by the fluorescence method described in Patent Document 1 for novel selenium-containing compounds. As a result, the total selenium concentration was 13.72 mM. From the results of the ICP-MS chromatogram in FIG. 5, the total selenium concentration and selenoneine concentration are equal, so the amount of selenoneine in the purified selenoneine solution was calculated to be 2.744 μmol.
In addition, the amount of selenoneine in the selenoneine-containing extract in step 2 was measured using the diluted solution of this sample as a standard for quantitative determination. As a result, the amount of selenoneine in the selenoneine-containing extract was 4.59 μmo1. When the recovery rate of selenoneine by this purification method was calculated from these values, the recovery rate was 59.78%.
In addition, the purity of the purified selenoneine solution was measured by absorption at 260 nm using HPLC in step 3 of Example 1 and 0.1% ammonium acetate water, Ultrahydrogel 120 column, and LC conditions in step 5, and the purity was 74.4%. rice field. FIG. 7 shows a UV chromatogram obtained by analyzing this purified selenoneine solution.

<High Purification of Purified Selenoneine Solution>
The purified selenoneine solution was purified by Ultrahydrogel 120 column. Using the HPLC used in Step 3 of Example 1 and the 0.1% aqueous ammonium acetate, Ultrahydrogel 120 column, and LC conditions of Example 2, the eluate with a retention time of 9.8 to 11.0 minutes was collected in a glass vial with a fraction collector. 79 μL of the purified selenoneine solution was subjected to HPLC to obtain an eluate. The eluate was concentrated while being heated to 50° C. using a rotary evaporator to obtain 35 μL of a highly purified selenoneine solution. When the purity of the highly purified selenoneine solution was measured by the method of Example 1, the purity was 93.3%. A UV chromatogram obtained by analyzing this highly purified selenoneine solution is shown in FIG. It can be seen that the peaks that appear as impurities in the chromatogram of FIG. 7 have disappeared. Moreover, the recovery rate at this time was 57.5%.

[Comparative Example 1]
Example 1 was carried out under the same conditions except that a C6 phenyl column was used instead of the PBr column, and the results of analysis of the obtained samples are shown in FIG.
As is clear from these results, the retention times of ergothioneine and selenoneine were 4.822 minutes and 5.214 minutes, respectively, but their base portions overlapped and they could not be separated. Moreover, the difference was 0.4 minutes and the degree of resolution was 0.99, and it was not recognized that the peaks were completely separated.

[Comparative Example 2]
In Example 1, a C6 column was used instead of the PBr column, and the elution was performed under the following gradient conditions (see Table 3) described in Non-Patent Document 1. The results of analyzing the obtained samples are shown in FIG. shown in
As is clear from this result, the retention times of ergothioneine and selenoneine are 3.741 minutes and 4.079 minutes, respectively, and separation is possible, but the difference is only 0.3 minutes, and the degree of resolution is only 1.09. However, it is clear that separation becomes more difficult as the amount of injection increases.

Claims (6)

from a solution containing selenoneine, comprising the step of performing (reverse phase) chromatography using aqueous acetic acid, aqueous ammonium acetate, aqueous formic acid, aqueous ammonium formate and water as the mobile phase and using pentabromobenzyl group-bonded silica gel as the stationary phase. method for the separation of selenoneine monomers. 2. A method for separating selenoneine monomers according to claim 1, further comprising the step of performing molecular exclusion chromatography. 3. The method for separating selenoneine monomers according to claim 1 or 2 , further comprising a step of extracting selenoneine with hot water. 4. The method for separating selenoneine monomers according to any one of claims 1 to 3 , further comprising a step of performing (reverse phase) chromatography using alkyl-bonded silica gel as a stationary phase. A method for producing selenoneine monomer, comprising the method for separating selenoneine monomer according to any one of claims 1 to 4 . A method for analyzing selenoneine monomer, comprising the method for separating selenoneine monomer according to any one of claims 1 to 4 .

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