JP2007197346A - Stabilization agent for s-adenosylmethionine and stabilization method for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a stabilization agent containing a substance having an effect for stabilizing S-adenosylmethionine (SAM), and to provide a stabilization method using the substance. <P>SOLUTION: The stabilization agent for the S-adenosylmethionine contains a phosphate compound as an active ingredient. The stabilization method for the S-adenosylmethionine comprises adding a phosphate compound to the S-adenosylmethionine. Thereby, the stabilization agent and the stabilization method which each can more effectively stabilize SAM over a long period than conventional agents and conventional methods are obtained. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a stabilizer and a stabilization method for stabilizing S-adenosylmethionine.

  In recent years, S-adenosylmethionine (SAM) has attracted attention because of its remarkable curative and preventive effects on alcoholic hepatitis and other liver diseases. Furthermore, a healing effect for depression, a function recovery effect in osteoarthritis, an improvement effect on senile dementia, and the like have been reported (Patent Document 1).

  This SAM is known to have the ability to produce high yields of yeasts, especially brewing yeasts such as sake yeast. However, since SAM is a chemically unstable substance and rapidly decomposes even at room temperature, it has been a major obstacle when used as a pharmaceutical or a supplement, although the utility of SAM is attracting attention. In the US, etc., SAM tosylate, paratoluene sulfonate, and sulfosartylate are stabilized and partially used, but these are not recognized as food additives. A stabilization method for enabling SAM to be used as a safer supplement or part of food is more desirable. As such a SAM stabilization method, it has been reported that trehalose has an effect of stabilizing SAM (Biochemica et Biophysica Acta, 1573 (2002) pp.105-108). There are also many SAMs that are stabilized and sold in the form of hydrochloride. However, these stabilizing effects are not sufficient, and development of a method with higher stability is necessary.

JP 2005-261361 Biochemica et Biophysica Acta, 1573 (2002) pp.105-108

  Accordingly, an object of the present invention is to provide a stabilizer containing a substance having an effect of stabilizing SAM and a stabilization method using the substance.

  As a result of intensive studies, the present inventors have found that a phosphate compound has an effect of stabilizing SAM over a long period of time, and have completed the present invention.

  That is, the present invention provides a stabilizer for S-adenosylmethionine containing a phosphate compound as an active ingredient. The present invention also provides a method for stabilizing S-adenosylmethionine, which comprises adding a phosphate compound to S-adenosylmethionine.

  According to the present invention, a stabilizer and a stabilization method capable of stabilizing SAM more effectively over a longer period than conventional methods are provided. Since the phosphate compound used in the stabilizer and stabilization method of the present invention is also recognized as a food additive, according to the present invention, SAM can be used more safely as a supplement or part of food. . The present invention pioneers the use of SAM which has been difficult to use in practice, although various effects such as liver disease, depression, osteoarthritis, and senile dementia are obvious.

The phosphoric acid compound used in the stabilizer and stabilization method of the present invention is a compound that has H ₂ PO _4- in the structural formula or a compound that dissolves in water to give a compound having H ₂ PO _4- or a salt thereof. Say. Specifically, metaphosphoric acid _(HPO 3), phosphoric acid (orthophosphoric _acid, H 3 _PO 4), pyrophosphoric acid _{(H 2 PO 4 -H 2 PO} 3), polyphosphoric acid _{(H 2 PO 4 - (HPO} 3) _n-2- H ₂ PO ₃ (n ≧ 3)) and salts thereof. Of these, phosphoric acid, pyrophosphoric acid, and polyphosphoric acid are preferable, and the above-mentioned salt is preferably an alkali metal salt, but is not limited thereto. A compound having a structure in which two or more molecules of phosphoric acid are dehydrated and condensed can be collectively referred to as polyphosphoric acid. In this specification, a compound having a structure in which two molecules of phosphoric acid are dehydrated and condensed is referred to as pyrophosphoric acid, three molecules. A structure in which the above phosphoric acid is dehydrated and condensed is expressed as polyphosphoric acid. In addition, the said phosphoric acid compound can be used individually or can use 2 or more types together.

  The stabilizer of the present invention contains the above phosphoric acid compound as an active ingredient. The stabilizer can be prepared in the form of a solid such as a powder or granules or a solution such as an aqueous solution. In the case of a solid, as described later, for example, it can be added to a solution containing SAM and dissolved. When the stabilizer is used, it is used so that the weight of the phosphate compound contained in the stabilizer is preferably 1 to 100 times, more preferably 5 to 20 times the SAM weight. For example, when the stabilizer of the present invention is used in a 10-fold amount, if the SAM concentration in the solution containing SAM to be stabilized is 0.2 mg / ml, the amount of phosphate compound is 2 mg per 1 ml of the SAM solution. Use stabilizers.

  The SAM stabilized by the present invention is not particularly limited, but may be extracted from a microorganism having SAM production ability such as yeast. The extract may be in the form of a solution such as an aqueous solution, or in the form of a solid obtained by freeze-drying or drying the extract. When the extract is in a solution form, the SAM in the extract can be stabilized by adding the stabilizer of the present invention in a solution form to the extract and mixing. The SAM in the extract can also be stabilized by adding the solid form of the stabilizer of the present invention to the extract in the form of a solution and dissolving it. When the extract is in solid form, the extract is added and dissolved in the stabilizer of the present invention in solution form, or the solid stabilizer of the present invention and the extract are mixed in advance. Alternatively, the SAM in the extract can be stabilized by dissolving in a solvent such as water. When the stabilizer of the present invention is in the form of a solution, the solvent is not particularly limited as long as it is a solvent that can dissolve a phosphate compound and is harmless to the human body, but water is used from the viewpoint of safety and cost. preferable.

  The SAM stabilized according to the present invention can be well stored for a long time even in a liquid state. Moreover, even when the stabilized SAM in a liquid state is freeze-dried or dried to obtain a solid state, it can be stored well.

  Hereinafter, the present invention will be specifically described based on examples. However, the present invention is not limited to the following examples.

1. Production and extraction of SAM First, 5 ml L-shaped tube using yeast Saccharomyces cerevisiae K-9 (Association No. 9 sake yeast) and YPD medium (yeast extract 1%, peptone 2%, glucose 2%) as the culture medium. And pre-cultured by shaking at 30 ° C. for 24 hours. Thereafter, a medium comprising sucrose 5%, yeast extract 0.3%, KH ₂ PO ₄ 0.4%, K ₂ HPO ₄ 0.2%, MgSO ₄ 7H ₂ 0.05%, L-methionine 0.15% (hereinafter referred to as D medium) In 78 ml, the pre-cultured yeast cells were inoculated so that OD ₆₆₀ = 0.1, and cultured in a 200 ml baffled Erlenmeyer flask with shaking at 30 ° C. for 2 days. The yeast cells that produced and accumulated a large amount of SAM in this manner were cultured and then collected by centrifugation, and the cells were washed twice with sterile water.

  The washed cells were suspended in 10 ml of sterilized water, frozen overnight at −30 ° C., and then dissolved the next day to extract SAM from the cells. Subsequently, the supernatant was collected by centrifugation and filtered through a 0.2 μm membrane filter to obtain an extract of SAM produced by yeast cells.

2. SAM analysis SAM analysis was measured by capillary electrophoresis in the following manner. The analysis conditions are as follows.
Analyzer Capillary ion analyzer (Waters)
Analysis conditions Capillary: AccuSep, 75μm × 60 cm
Buffer: 40 mM sodium phosphate buffer (pH 2.5)
Voltage: positive, 20 kV
Sampling: 30 seconds hydrostatic method Temperature: 25 ℃
Detection: UV 254nm

3. SAM stability test (part 1) (freeze-dried storage)
To the SAM extract (0.2 mg / ml), a substance to be subjected to the stabilization test was added in an amount 10 times the SAM weight, and each was subdivided into a predetermined amount, and then lyophilized. The lyophilized product composed of the SAM and the additive substance was stored in the dark at 25 ° C. and 37 ° C., and the remaining amount of SAM was measured over time. Thereby, the stable preservation | save state of SAM in the dry state of various additive substances was investigated. Each independent test was performed three times.

  (i) The SAM extracted from yeast was lyophilized as it was (indicated as “stock solution” in FIG. 1), and (ii) lyophilized after addition of dilute hydrochloric acid to 0.1N HCl (“0.1N HCl”) Display), and (iii) 10 times the SAM weight of phosphoric acid, that is, commercially available phosphoric acid is 85% pure, so 2.42 mg per ml was added to the SAM extract (0.2 mg / ml) and lyophilized. The remaining amount of SAM was examined for the three samples (labeled “phosphoric acid”) immediately after the start of storage on day 0 and then in the dark at 37 ° C. on day 3, week 1 and month 1. The results are shown in FIG.

  Many SAMs are sold in the form of hydrochloride as a reagent. In the form of hydrochloride, it was certainly shown that the extract was more stable than the one that was cryopreserved as it was, but in the state where phosphoric acid was added to form phosphate, it was stored more stably than the hydrochloride. It was shown that.

SAM stability test (2) (freeze-dried storage)
(i) The SAM extracted from yeast was lyophilized as it was (designated as “stock solution” in FIG. 2), and (ii) 10 times the SAM weight of trehalose reported to improve the storage stability of SAM when added ( SAM extract (0.2 mg / ml) added and freeze-dried (2.0 mg), and (iii) polyphosphoric acid mixture (containing 85.8% pyrophosphate) 10 times the SAM weight (SAM extract (0.2 mg FIG. 2 shows the results of a SAM storage test conducted at 37 ° C. for one week, one month, and three months for three of those added and lyophilized at 2.0 mg / ml).

  As a result of the above test, trehalose did not have an effect of increasing the storage stability. On the other hand, in the case of the polyphosphoric acid mixture, almost 100% of SAM remained even after 3 months, and a very good SAM storage effect was observed.

  As described above, as shown in FIGS. 1 and 2, high storage stability was confirmed in a SAM dry state in a polyphosphoric acid mixture mainly containing a phosphoric acid compound, particularly pyrophosphoric acid.

SAM stability test (Part 3) (Aqueous solution storage)
To the SAM extract, 10 times the SAM weight of the substance to be used for the stabilization test was added, and each aliquot was aliquoted, then stored in the dark at 37 ° C. in the form of an aqueous solution. The remaining amount was measured. Thereby, the stable preservation | save state of SAM in the aqueous solution state of various additive substances was investigated. Each independent test was performed three times.

  (i) SAM-containing aqueous solution extracted from yeast (SAM amount 2 mg / ml) (indicated as “stock solution” in FIG. 3), (ii) sodium polyphosphate 10 times the amount of SAM (ie 20 mg / ml) 1 week, 1 month, and 3 months when stored in a dark place at 37 ° C, with the addition of (III) sodium pyrophosphate 10 times the amount of SAM (20 mg / ml) FIG. 3 shows the SAM residual ratio.

  In the SAM itself extracted from yeast, the SAM was almost completely decomposed and disappeared in about one month. On the other hand, when sodium polyphosphate was added, 80% or more of SAM remained after 3 months, and almost 100% of SAM remained in sodium pyrophosphate. It was confirmed that stable SAM storage stability was exhibited even in an aqueous solution state.

It is a figure which shows the residual rate (%) of SAM after preserve | saving the SAM solution which added 0.1N hydrochloric acid or phosphoric acid as a stabilizer, and the SAM solution which does not add anything in a lyophilized state for various periods. It is a figure which shows the residual ratio (%) of SAM after preserve | saving the SAM solution which added trehalose or polyphosphoric acid as a stabilizer, and the SAM solution which does not add anything in a lyophilized | freeze-dried state for various periods. It is a figure which shows the residual rate (%) of SAM after preserve | saving the SAM solution which added sodium polyphosphate or sodium pyrophosphate as a stabilizer, and the SAM solution which does not add anything in a solution state for various periods.

Claims (11)

  A stabilizer for S-adenosylmethionine containing a phosphate compound as an active ingredient.   The stabilizer according to claim 1, wherein the phosphoric acid compound is at least one selected from the group consisting of metaphosphoric acid, phosphoric acid, pyrophosphoric acid, polyphosphoric acid, and salts thereof.   The stabilizer according to claim 2, wherein the phosphoric acid compound is at least one selected from the group consisting of phosphoric acid, pyrophosphoric acid, polyphosphoric acid, and alkali metal salts thereof.   The stabilizer according to any one of claims 1 to 3, wherein the S-adenosylmethionine is extracted from a microorganism having an ability to produce S-adenosylmethionine.   The stabilizer according to claim 4, wherein the extract from the microorganism is in the form of a solution, a lyophilized product or a dried product.   A method for stabilizing S-adenosylmethionine, comprising adding a phosphate compound to S-adenosylmethionine.   The method according to claim 6, wherein the phosphoric acid compound is at least one selected from the group consisting of metaphosphoric acid, phosphoric acid, pyrophosphoric acid, polyphosphoric acid, and salts thereof.   The method according to claim 7, wherein the phosphoric acid compound is at least one selected from the group consisting of phosphoric acid, pyrophosphoric acid, polyphosphoric acid, and alkali metal salts thereof.   The method according to any one of claims 6 to 8, wherein the phosphate compound is used in a weight of 5 to 20 times the weight of the S-adenosylmethionine.   The method according to any one of claims 6 to 9, wherein the S-adenosylmethionine is extracted from a microorganism having an ability to produce S-adenosylmethionine. The extract from the microorganism is in the form of a solution, a lyophilized product or a dried product, and the phosphate compound is added to the extract and maintained in a solution state or the phosphate compound is added to the extract. The method according to claim 10, wherein the added solution is lyophilized or dried to maintain the solid state.

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