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WO2014098454A1 - Method for purifying d-chiro-inositol from solution comprising myo-inositol and d-chiro-inositol - Google Patents

Method for purifying d-chiro-inositol from solution comprising myo-inositol and d-chiro-inositol Download PDF

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Publication number
WO2014098454A1
WO2014098454A1 PCT/KR2013/011752 KR2013011752W WO2014098454A1 WO 2014098454 A1 WO2014098454 A1 WO 2014098454A1 KR 2013011752 W KR2013011752 W KR 2013011752W WO 2014098454 A1 WO2014098454 A1 WO 2014098454A1
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Prior art keywords
myo
inosi
chiro
supernatant
inositol
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French (fr)
Korean (ko)
Inventor
명현군
윤상활
이현서
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Solgent Co
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Solgent Co
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B63/00—Purification; Separation; Stabilisation; Use of additives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C29/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
    • C07C29/74—Separation; Purification; Use of additives, e.g. for stabilisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C29/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
    • C07C29/74—Separation; Purification; Use of additives, e.g. for stabilisation
    • C07C29/76—Separation; Purification; Use of additives, e.g. for stabilisation by physical treatment
    • C07C29/78—Separation; Purification; Use of additives, e.g. for stabilisation by physical treatment by condensation or crystallisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C35/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring
    • C07C35/02—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring monocyclic
    • C07C35/08—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring monocyclic containing a six-membered rings
    • C07C35/14—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring monocyclic containing a six-membered rings with more than one hydroxy group bound to the ring
    • C07C35/16—Inositols
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/14—The ring being saturated

Definitions

  • the present invention relates to a method for purifying and separating high concentrations of myo-inosino and D-chiro-inosino from a solution containing e.g.
  • D-chiro-inositol (cis— 1, 2, 4_t rans-3, 5,6-cyclohexol) is used for myo-inositol, cis ⁇ l, 2, 3,5—trans — 4,6 cyclo cyclohexanehexol) is a stereoisomer of the 3 hydroxyl group is epimerized (Fig. 1).
  • D-Cyrropyinosinos is reported to be an important mediator of insulin signaling as an important component of phosphoglycans (IPG) and is known to be effective in the treatment of type 2 diabetes.
  • D-Cyro-inosyl is primarily found in eukaryotes and is biosynthesized by epimerization of myo-inositol.
  • D-Cyro-inosyl is mainly produced by hydrochloric acid hydrolysis of D-pinitol or kasugamycin (US Pat No. 5827896, US Pat No. 5091596, US Pat No. 5463142). , US Pat No. 5714643).
  • raw materials such as pinir and kasugamycin are expensive, and organic synthesis method (US Pat No. 5406005, WO 96/25381) is known, it is not easy to separate by-products and thus it is economically low.
  • D-chiroybinosinos may be produced from myo-inosyl by the continuous action of inositol dehydrogenase and inosose isomerase enzymes. It can be produced by fermentation methods using recombinant cells transformed directly with Myo-Inosi, or transformed to express the enzyme. However, in the reaction product produced by the fermentation method or the enzyme reaction method, myo-inosino and D-chiro-inosino are mixed, and the mixed solution contains myo-inosino-D. The concentration of Cairo-inos is relatively low. Thus, with the Maio-Inoshi
  • -Inosino exists in this mixture.
  • the present inventors have tried to develop a method for purifying D-Cyro-Inosine with high purity from the stereoisomeric mixed solution of this inosine. As a result, the present inventors found that the solubility of myo-inos and D-chiro-inosio in the stereoisomer relationship was large in the aqueous solution.
  • the present invention has been completed by confirming that chiro-inosi can be efficiently separated and purified in high purity through a simple process.
  • the present invention provides a high purity of D-chiro-inositol from a solution containing myo-inosine and D-chiro-inositol.
  • a method for purifying there is provided a method comprising the following steps: (a) adding an organic solvent to a solution containing myo-inosine and D-chiro-inosine to form inosi in a precipitate; step; (b) separating the supernatant from the resulting inosi from the precipitate; And (c) drying the separated supernatant to obtain Inosi powder.
  • the present invention provides a method characterized by further comprising the following step after the step (c): (d) powdering the inosine obtained in the step (c) Dissolving in water to prepare an aqueous solution; (e) adding an organic solvent to the aqueous solution to generate an inosi precipitate; (f) separating the supernatant from the resulting inosi from the precipitate; And (g) drying the separated supernatant to obtain Inosi powder.
  • the invention provides a method according to claim 1, further comprising the steps of after said step (c): the INNO when obtained in (d) 'the step (C) Dissolving the powder in water to form an aqueous solution and producing inosi precipitates; (e) 'separating the supernatant from the precipitate created in the Inno when the aqueous solution; And (f) ' drying the supernatant to obtain Inosi powder.
  • the inositol mixed solution containing myo-inosino and D-chiro-inosino was obtained as a final reaction product by the method of producing D-chiro-inosino with myo-inosino as substrate. Can lose.
  • recombinant cells transformed with myo-inosio transporter, inosio dehydrogenase and inosose isomerase are used.
  • the reaction product cultured with the myo-inosine as a substrate was reacted with the reaction product D- PO 2014/098454
  • Ino City mixed with Cairo ⁇ Ino City is made of a solution.
  • the resting cells of the recombinant cells transformed to express the enzyme are inoculated into a minimal medium containing myo-inosi to convert myozynosi to chiro-inosi.
  • an inositol mixture is produced in which the reaction product, the remaining reaction product, myo-inosi and the reaction product, D-chirojan inosi, are mixed with the final culture product.
  • an enzyme reaction method for producing D-chiro-inosine from myo-inosine using an enzyme of myo-inosio, an inosio dehydrogenase and an inososomerase After the reaction was completed, the resulting reaction product was made, and the remaining reaction product was prepared by mixing the remaining reaction mixtures, such as Mio-Inoshi and Ekairo-Inoshi. Therefore, the method of the present invention is, for example, the myo-inosity produced as the final reaction solution of the fermentation method, the dormant cell conversion method and the enzyme reaction method for producing D-chiro-inosity from the above-mentioned myo-inoshi.
  • the mixed solution of myo-inosyl and D-chiro-inosyl to which the method of the present invention can be applied may be a nutrient medium (complete medium), for example, a fermentation broth using recombinant cells.
  • M9 may be the minimum medium, and in the case of reaction reaction by enzyme reaction method, it may be a complete enzyme reaction solution.
  • the final equilibrium concentration of D-chiropy Inosio in the final semi-aqueous solution of the process for preparing D-chiropyrosinos described above is about 14% 3 ⁇ 4, Myo-inos in the mixed solution of Inosyl.
  • D_Cairo-Inosio are present in ratio of about 86:14.
  • the method of the present invention is a mixture of myo-inosino and D-chiro-inosino by the difference in solubility of myo-inosino and D-chiro-inosino in the solution of D-chiro-inosino Purification is to increase the purity.
  • the difference in solubility of each inosin in an aqueous solution of Myo-inosyl and D-Cairo-inosyl in water is shown in FIG. 3.
  • an organic solvent is first added to a solution of inos containing the myo-inositol and D-chiropyonos. Precipitation of myo-inos and D-chiro-inos is induced by the addition of the organic solvent.
  • the precipitation rate of myo-inos and D-chiro-inos is increased by the addition of organic solvent, but compared to D-chiro-inos
  • the precipitation rate of myo-inos is much greater (see Figure 5). Therefore, by adding an organic solvent to the mixed solution of myo-inosino and D-chiro-inosino, D-chiro-inosio is precipitated in a higher amount than that of D-chiro-inosio. Can increase the concentration of D-Cairo-Inosino.
  • the organic solvent that can be used in the present invention is not particularly limited as long as it has a property of inducing precipitation of myo-inosine more than precipitation of D-chiro-inosine.
  • the organic solvent is alcohol, acetone, ethyl acetate, butyl acetate, 1, 3- butylene glycol or ether.
  • the organic solvent is an alcohol having 2 to 6 carbon atoms.
  • the organic solvent is ethane, isopropanol, or acetone.
  • the amount of the organic solvent added to the mixed solution of the myo-inosino and D-chiro-inosino is not particularly limited, and the inosin is suitable within the range of 0.1-9 times (v / v) compared to the mixed solution.
  • the amount can be selected and added.
  • the organic solvent is added to the inosin in an amount of 0.1-6 times (v / v) compared to the mixed solution.
  • the organic solvent is added to the inosin in an amount of 0.1-4 times (v / v) compared to the mixed solution.
  • the organic solvent is added to the inosin in an amount of 0.1 — 2 times (v / v) compared to the mixed solution. According to another embodiment of the present invention, the organic solvent is added to the inosin in the amount of 0.1 ⁇ 1.5 times (v / v) compared to the mixed solution.
  • Inositol precipitates induced by the addition of an organic solvent in the present invention include both myo-inosyl and D-chiro-inosyl, but the precipitation rate of myosin inosyl is higher. Oh-Innosi contains more. Step (b): separating the supernatant from the resulting inosi from the precipitate
  • the inos generated through step (a) is separated from the supernatant from the precipitate.
  • the inositol precipitate is a precipitate of myo-inos and D-chiro-inosit, but since the myosininosium exhibits a greater precipitation rate, the inositino is more likely to be the myo-inos than the D-chiro-inos. Poems are included in even greater amounts have. Due to the large precipitation of myo-inosyl, the concentration of myo-inosyl is lowered in the supernatant, and the concentration (purity) of D-chiro-inosylol is relatively increased. Separation of the supernatant from the precipitate by Inosi is carried out using a filtration or centrifugation method, preferably by using a filtration method. Step (c): drying the separated supernatant to obtain Inosi powder
  • the supernatant separated in step (b) is dried to obtain Inosi powder. Drying of the supernatant may be performed while heating the supernatant, or may be performed in a vacuum. Inosi obtained by drying the supernatant was composed of myo-inosino and D-chiro-inosino, but the myo-inosino and D-chiro-inosilo of step (a). The purity of D-Cairo-inosity is increased as compared to this containing solution.
  • the method of the present invention may further comprise the following steps (d) to (g) after step (C). Step (d): dissolving the powder of Inosi obtained in step (C) in water to prepare an aqueous solution .
  • An inosi-dissolved aqueous solution was prepared by dissolving the inosi powder obtained in step (C) in water.
  • the water is preferably distilled water.
  • the inosin powder is dissolved in water at a concentration of 20% (w / v) or less.
  • the inosin is dissolved in water at a concentration of 0.1% (w / v) or more and 20% (w / v) or less.
  • the inositol powder is dissolved at a concentration of 0.1% (w / v) or more and 18% (w / v) or less.
  • the inosin is dissolved in the powder at a concentration of 0.1% (w / v) or more and 15% (w / v) or less.
  • organic solvent is added to the aqueous solution in which the inositol obtained in step (d) is dissolved, thereby producing a precipitate of inositol.
  • the organic solvent is the same as described in step (a).
  • the organic solvent is not particularly limited as long as the organic solvent has a property of inducing precipitation of myo-inositol more than precipitation of D-chiro-inoshi.
  • the organic solvent is alcohol, acetone, ethyl acetate, butyl acetate, 1, 3- butylene glycol or ether.
  • the organic solvent is alcohol having 2 to 6 carbon atoms.
  • the organic solvent is ethanol, isopropanol, or acetone.
  • the amount of the organic solvent added to the aqueous solution may be added by selecting a suitable amount within the range of 0.1 to 10 times (v / v) relative to the aqueous solution.
  • the organic solvent is added in an amount of 1.5-9 times (v / v) relative to the aqueous solution.
  • the organic solvent is added in an amount of 9 times (v / v) relative to the aqueous solution.
  • the inos generated through step (e) is separated from the supernatant from the precipitate. Separation of the supernatant from the precipitate by the inosi is carried out using a filtration or centrifugal separation method, preferably using a filtration method. Since the inosi precipitate contains a large amount of myosininosylol, the supernatant has an increased purity of D-Cairo-inositol. Step (g): drying the separated supernatant to obtain inosyrol powder
  • the supernatant separated in step (f) is dried to obtain Inosi powder. Drying of the supernatant may be performed while heating the supernatant, or This can be done in a vacuum. Inosine obtained by drying the supernatant was composed of myo-inosino and D-chiro-inosino, but the purity of D-chiro-inosino was increased.
  • the method of the present invention further comprises the following steps after step (c)
  • Step (d) '- Step (f)' a may be further included.
  • Step (d) ' dissolution of the inositol powder obtained in step (c) in water to prepare an aqueous solution and inosi to produce a precipitate
  • An inosi-dissolved aqueous solution was prepared by dissolving the inosi powder obtained in step (c) in water.
  • the water is preferably distilled water.
  • the inosi powder is dissolved in water at a high concentration of more than 20% (w / v).
  • the inosi powder is dissolved in water at a concentration of greater than 2093 ⁇ 4 (w / v) up to 80% (w / v).
  • the inosi powder is dissolved in water at a concentration of 30% (w / v) or more and 70% (w / v) or less.
  • the solubility of myoininosi to water is up to about 15%, and the solubility of D-Cyro-inosino is up to about 55%, so that the inosi obtained in step (c) Maio the case of dissolving the "concentration of 70% of the powder in water - during the Inno will be dissolved at up to 15% density, and the other non-dissolved Maio - is produced when the Inno in the precipitate.
  • D-Cairo-Inosine is soluble in up to 55%, so all of the Cairo-Inosine is dissolved in this water.
  • Step (e) ' separating the supernatant from the inosolole precipitate produced in the aqueous solution
  • the supernatant is separated from the precipitate of Inosi produced in step (d) ' . Separation of the supernatant from the precipitate by the inosi is carried out using a filtration or centrifugation method, preferably by using a filtration method. Most of the inosi precipitate will contain myoininosi, and the supernatant has increased purity of D-Cairo-inossi.
  • Step (f) ' drying the supernatant to obtain Inosi powder.
  • the supernatant separated in Step (e) ' is dried to obtain Inosi powder. The supernatant may be dried while the supernatant is heated, or may be performed in a vacuum.
  • the purity of D-Cairo-Inosi in the dried powder when the inosi powder is dissolved in water at a concentration of about 70% (w / v) in step (d) ' Will be about 70% or more.
  • the present invention relates to a method for purifying D-Cyro-Inosyl with high purity from a mixed solution of Myo-Inosyl and D-Cyro-Inosyl. .
  • the method of the present invention is based on the principle of the difference in solubility between myoininosi and D—chiro-inosi.
  • the method of the present invention employs simple processes such as the addition of organic solvents, supernatant separation (filtration) and drying, and therefore, at low cost, yields a solution from the mixed solution of myoininosi and D-chiro-inosi. Inosi can be separated and purified in high purity.
  • the myo-inosine separated from the separated D-chiro-inos is reusable as a substrate.
  • the present invention utilizes the solubility difference between myo-inosino and D-chirofoinosinos to obtain high purity from the mixture of myo-inosino and D-chirofoinosinos. It relates to a method of purifying with.
  • the process can be carried out at low cost through simple processes such as adding an organic solvent to a mixed solution of Inosi, inducing Inosi to precipitate, separating the supernatant, and drying the supernatant.
  • the separated myo-inosity can be reused as a substrate in the method of producing D-chiro-inositol from the myo-inosino.
  • FIG. 2 shows a mixed solution of myo-inosine and D-chiropyonosinolol.
  • FIG. 3 shows the solubility of myopininosine and D-chiro-inosi in 25 ° C distilled water.
  • B solubility of myo-inosino
  • O solubility of D-chiro-inosino.
  • FIG. 4 shows inositol by adding ethanol to various types of mixed solutions (water, saline, enzyme reaction buffer, M9 medium, and TB medium) in which myo-inosino and D-chiro-inosothol are dissolved. The precipitates were precipitated and the concentrations of myo-inosine and D-chiro-inoshiro in the supernatant were measured.
  • B concentration of myo-inosyl
  • O concentration of D-chiro-inosyl.
  • FIG. 5 shows that ethane was added to various kinds of mixed solutions (water, saline, ⁇ small reaction buffer, M9 medium, and TB medium) in which myo-inosino and echairo-inosio were dissolved.
  • the precipitation rates of O-inosino and D-Cairo-inosinot are shown.
  • ⁇ The precipitation rate of Maio-Inoshiro
  • O The precipitation rate of D-Cairo-Inoshiro.
  • FIG. 6 shows inosin by adding ethane to water, a saline enzyme reaction mixture, M9 medium, and TB medium, which are various solvent mixture solutions in which myo-inositile and D-chiro-inosi are dissolved. After precipitation of the supernatant obtained by drying the inositol powder obtained by measuring the purity of the D-Cairo-Inoshile is shown.
  • Fig. 7 shows the case where the mixed powders of myo-inoshiro and D-chiro-inositol, which are dry powders of the supernatant obtained by adding primary ethane, are dissolved again in water. Show the dissolution properties of Inosi.
  • Panel A shows the kinetics of myo-inos and D-chiropyonos in the supernatant of aqueous solution.
  • Panel B shows the solubility of myofininos and D-chiro-inosino in the supernatant of aqueous solution.
  • Panel C shows the precipitation rates of myo-inoshiro and D-chiro-inosi in the supernatant of aqueous solution.
  • Panel D shows the purity of D-Cyro-Inosyl in solution increase in aqueous solution.
  • ⁇ Myo-Ino City
  • O D-Cairo Ino City.
  • Figure 8 is obtained by re-dissolving a mixed powder of myo-inosino and D-chiro-inosino, a dry powder of the supernatant obtained by adding primary ethanol, in water at various concentrations between 35% and 70%. This is the result of measuring the concentration of myosininosi and D-chiro-inosi in the supernatant after adding various amounts of ethanol to the aqueous solution of inosi.
  • Panel A is the concentration of myo-inosine in the supernatant.
  • Panel B is the concentration of D-Cyro-inosyl in the supernatant. ⁇ : 35%, O: 40%, ⁇ : 45%, V: 50%, ⁇ : 55%, : 60%, ⁇ : 65%, ⁇ : 70%.
  • FIG. 10 is prepared from an aqueous solution containing 70% of a dry powder of a supernatant obtained by adding primary ethanol (combined powders of myo-inos and D-chiropyinos) to remove the precipitate and to remove the supernatant. After re-dissolving, the Inosi, which has a weight ratio of D-Cairo-InoShitl added to 70% or more, is redissolved in a dry mixture with an aqueous solution of 10% to 50%, and then various amounts of secondary alcohol are added to each ino This is the result of measuring precipitation of poems. Panel A is the concentration of myo-inosi in the supernatant.
  • Panel B is the concentration of D-Cyro-inosyl in the supernatant.
  • Panel C is the precipitation rate of myo-inos.
  • Panel D is the precipitation rate of Ecuiro-Inosi. ⁇ : 10%, O: 20%, ⁇ : 30%, V: 40%, 50%.
  • 11 is an aqueous solution containing about 12% of dry powder (combined powders of myo-inos and D-chiro-inos) of the supernatant obtained by adding primary ethanol. This is the result of measurement of precipitation and purification characteristics when various amounts of ethanol were added.
  • Panel A is a result of measuring the concentration of myo-inosine and D-chiro-inosyl in the supernatant.
  • Panel B is the result of measuring the precipitation rates of myo-inosine and D-chiro-inosino.
  • Panel C is a result showing the purity of D-chirozyno inos in the supernatant.
  • substrates of recombinant cells transformed to express myo-inositol transporter, inosi dehydrogenase and inosose isomerase When culturing in a medium containing phosphorus myo-inosity, a mixed solution is prepared in which the ino-sidium mixed with the remaining myo-inosine and the reaction product D—Cairo-inosino are mixed.
  • an enzyme reaction for generating D-chiro-inosine from myo-inosity which is a substrate, by using a myo-inosity transporter, an inosine dehydrogenase, and an enzyme of inososomerase
  • the resultant reaction product was produced by mixing the remaining Maio-Inos and D-Cairo-Inos. Since the final equilibrium concentration of the product in the reaction product of the Cairo-Inosyl D-Cairo-Inosyl is about 14%, the final reaction of the Myo-Inosyl and D-Cairo-Inosyl In the mixture, myo-inos and D-chiro-inos are present at a ratio of about 86:14.
  • Myo-inosyl substrate which is a substrate in the reaction solution to generate E. coli-Inosio from myo-inosyl, is included at a concentration of about 150 g / L, considering the solubility in water.
  • the reaction product contains 129 g / L of myo-inosyl and 21 g / L of D-chiro-inosyl.
  • the mixture of Inosul is a fermentation method using a recombinant cell. It may be complete medium, for example TB (terrific broth), in the case of dormant cell conversion method may be M9 minimal medium or saline, in the case of enzyme reaction method may be a buffer of the enzyme reaction.
  • TB medium containing 0.5% (w / v) lactose, 12.9% (w / v) myo-inosity, and 2.1% (w / v) D-chirobinosinos for the fermentation method.
  • FIG. 4 shows the concentrations of myo-inosyl and D-chirojan inositol remaining in the supernatant obtained by adding ethane to the respective mixtures of ethane.
  • the concentration decreased rapidly according to the amount of ethanol added under all medium conditions, and the myo-inosine was approximately 99 from the initial 126 g / L concentration when about 1 multiple of ethane was added. g / L precipitated, leaving about 27 g / L of myo-inosyl.
  • the result shown in FIG. 4 is shown in FIG. 5 in terms of precipitation rate.
  • the precipitation rate of myo-inosi increased rapidly with the amount of ethanol, and when 1-fold ethanol was added, the precipitation rate of 70-80% was relatively similar in all reaction conditions. In most of the myo-inosity was confirmed to precipitate. In contrast, the precipitation rate of D-Cairo-inosyl was slightly different depending on the medium conditions. Until the addition of about 1-fold ethane, the sedimentation rate was about 15% or less in total.However, when 1.5-fold or more ethanol was added, it was found in M9 and TB media more than water, saline, and enzyme reaction complete solution. The precipitation rate increased rapidly. This may be due to the different amounts of salt in each solution, since the amount of salt in M9 and TB medium is higher than in water, saline and enzyme reaction buffer. Seems to be.
  • the purity of D-Cairo-Inosyl is calculated and shown in FIG. 6.
  • the purity of D-Cyro-Inosyl was found to be about 35-40% in all cultures when 1-fold ethanol was added. The loss ratio of the ino-seattle will increase. Taken together, about 1% of ethanol is added and about 80% of myo-inositol is precipitated under all kinds of culture conditions. It was found that the initial purity of D-Cairo-Inoshil, which was about 14%, could be increased to about 3-40%.
  • Example 3 Concentration drying and re-dissolution of the supernatant after treatment with primary ethane, the same amount (1: 1) of ethanol was mixed in the inositol mixture as described in Example 2, and then the precipitate was removed.
  • the supernatant obtained was heated at 8 (rc, concentrated and dried in a "vacuum-dried powder of the Inno when obtained after drying (Maio the eu Ino when a D- and Carr-heunhap the powder of the Inno-shi)
  • the dissolution characteristics of myo-inosyl and D-chiro-inosyl were observed at each concentration by dissolving in distilled water at various concentrations of 15% to 70%. Same as described in Example 1.
  • the results of analyzing the dissolution characteristics of myo-inosyl and D-chiro-inosyl are shown in FIG.
  • Panel A of FIG. 7 shows the supernatant concentration, that is, the solubility of the solution at each concentration, and it was confirmed that the solubility of the myo-inosi did not increase more than about 15% as shown in Example 2 above. Can.
  • solubility is about 55%, even when the dry powder was dissolved at a concentration of 70%, all of the D-Cyro-Inosy was dissolved.
  • the solubility and precipitation rate of the myo-inosino and D-chiro-inosino are shown in panel B and panel C of FIG.
  • Ethanol was added to each solution having a concentration of 35% or more, which was a concentration at which the saturated solution for myo-inosine, that is, the myo-inosine, began to sufficiently precipitate in the mixed solution for each concentration prepared in Example 3.
  • concentration of 35% or more was a concentration at which the saturated solution for myo-inosine, that is, the myo-inosine, began to sufficiently precipitate in the mixed solution for each concentration prepared in Example 3.
  • Panel A of FIG. 8 shows the concentration of myo-inosity in the supernatant, and the solubility of myo-inosine does not exceed 15%. Is showing. As the ethane was added, the concentration of myo-inosine decreased drastically, and it was confirmed that a large amount of myo-inosine was removed when 2 ethane was added. In the case of 65% and 70% solutions, the addition of 1-fold of ethane started to remove myoininosi. It was confirmed that the poem began to be removed.
  • Panel B of FIG. 8 shows the concentration in the supernatant of D-chiro-inosino, and even in the case of D-chiro-inosino, a small amount of ethane was added to precipitate rapidly.
  • D-Cyro-Inosi began to precipitate from 1-fold ethane for 65% and 70% -concentration solutions, and most precipitation occurred from 1.5-fold ethane.
  • concentration of the solution was low, that is, the degree of precipitation was weak at 35% or 40%, but it was confirmed that precipitation occurred at a level similar to that of the high concentration solution from the addition of about 2 times of ethanol.
  • FIG. 9 shows the concentration of inosine shown in FIG. 8 in terms of precipitation rate.
  • the precipitation rate increased rapidly with the addition of ethanol to the myo-inosino and D-chiro-inosino, and the precipitation rate of the myo-inosino and D-chiro-inosino The increase was similar.
  • the precipitate obtained after treating the primary ethane was removed, and the mixed dry matters of the dried myopia-inos and D-chiro-inosies were re-dissolved at a high concentration of 35% or more, and then the ethane was removed. It is not easy to separate myo-inos and d-chiro-inos through the method of addition Was confirmed.
  • Example 5 Purification Characteristics of D-Cyro-Inosine Treated with Secondary Ethane for Various Concentrated Solutions
  • Example 6 Purification characteristics when secondary ethanol was treated in a mixed dry solution of low concentration of myo-inosino and D-chiro-inosino Maio Inino City and Ekairo—Ino City's mixed building with low concentration
  • FIG. 11 shows the concentration of the supernatant after the addition of ethanol, and in the case of myo-inosity, there was no significant change until about one-fold ethanol was added, but when 1.5 times of ethane was added, More than myo-inos appeared to precipitate. In contrast, D-Cyro-Inosi was found to precipitate slightly in 1.5-fold ethanol, after which the precipitation did not increase significantly.
  • Panel B of FIG. 11 shows the precipitation rate, and in the case of myo-inosine, when about 9 times of ethanol was added, 90% or more of the precipitates were precipitated, and the precipitation rate of D-chiro-inosine was about 20%. appear.
  • FIG. Panel A of FIG. 12 shows the concentration of inosyl in the actual supernatant.
  • concentrations of ethane and isopropane in myo-inosity were not significantly different, but slightly lower in acetone.
  • concentration of isopropane was lower than that of ethane, whereas in the case of acetone, it was the same as the concentration without the organic solvent and did not precipitate at all.
  • isopropane was similar in precipitation rate to myo-inosine and higher in precipitation rate (loss rate) of D-Cairo-inosine, while acetone was higher in precipitation rate for myo-inosine. This is higher, and the loss rate of D-Cairo-Inosi seems to be less.
  • panel B of FIG. 12 which shows the relative value for the precipitation rate of ethane.
  • the precipitation rate of myo-inosyl is the same with respect to ethanol, and the precipitation rate of D-chiro-inosyl is as high as about 0.7.
  • Addition of an organic solvent in the same volume as the mixed solution containing inosi may induce precipitation of myoininosi in a relatively larger amount, and the supernatant contains myo-inosino and D-chiro. Inno City will exist at a ratio of about 60:40. Drying the supernatant gives a dry mixture of Inosi having a purity of 30% -40% D-Cyro-Inosi.
  • Inosi with a purity of 40%, is dissolved in water at low concentrations, such as about 15%, of the dry mixture, to which precipitation is induced again by adding 10 times the amount of the organic solvent of the solution volume. Drying the supernatant from which the precipitate was removed can obtain a dry mixture of Inosi having a purity of 90% or more of D-Cairo-Inosi.

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Abstract

The present invention relates to a method for isolating and highly purifying D-chiro-inositol from a mixed solution of myo-inositol and D-chiro-inositol. The method according to the present invention adds an organic solvent into a solution comprising myo-inositol and D-chiro-inositol, and induces myo-inositol to precipitate, thereby increasing the purity of D-chiro-inositol in the mixed solution. Accordingly, the method according to the present invention can separate, with high purity, D-chiro-inositol from a mixed solution of myo-inositol and D-chiro-inositol simply and economically.

Description

【명세서】  【Specification】

【발명의 명칭】  [Name of invention]

마이오-이노시를 및 D-카이로 -이노시를이 포함된 용액으로부터 D- 카이로ᅳ이노시를을 정제하는 방법  Method for purifying D-chiropyonosino from a solution containing myo-inosine and D-chiro-inosino

【기술 분야】 [Technical field]

본 발명은 마이오-이노시를 및 D—카이로 -이노시를이 포함된 용액으로부터 으카이로—이노시를을 고순도로 분리하여 정제하는 방법에 관한 것이다.  The present invention relates to a method for purifying and separating high concentrations of myo-inosino and D-chiro-inosino from a solution containing e.g.

【배경 기술】 [Background technology]

D-카이로-이노시틀 (D-chi ro-inositol, c i s— 1， 2 , 4_t rans-3 ,5,6- cyclohexol)은 마이오一이노시를 (myo— inositol , cis一 l,2,3,5—trans— 4,6一 cyclohexanehexol)의 스테레오이소머로서 3 번 하이드록실 그룹이 에피머화 된 형태이다 (도 1). D-카이로ᅳ이노시를은 이노시를 포스포글리칸 (IPG)의 주요 구성성분으로 인슐린 신호전달의 증요한 매개체로 보고되어 있으며， 제 2 형 당뇨의 치료에 효과가 있다고 알려져 있다. D-카이로 -이노시를은 주로 진핵생물에서 발견되며， 마이오 -이노시틀의 에피머화에 의해 생합성된다. D-카이로 -이노시를은 주로 피니를 (D— pinitol)이나 카스가마이신 (kasugamycin)의 염산 가수분해에 의해 생산하고 있다 (U.S. Pat No. 5827896, U.S. Pat No. 5091596, U.S. Pat No. 5463142, U.S. Pat No.5714643). 그러나, 원료인 피니를이나 카스가마이신이 고가이며， 유기합성법 (U.S. Pat No. 5406005, W0 96/25381)도 알려져 있으나， 부산물의 분리가 용이하지 않아 경제성이 낮다. D-카이로ᅳ이노시를은 마이오-이노시를로부터 이노시를 디하이드로게나아제 (inositol dehydrogenase) 및 이노소스 이소머라아제 ( inosose isomerase) 효소의 연속작용에 의해 생성될 수 있으며， 상기 효소를 마이오 -이노시를에 직접 반웅시키거나, 상기 효소를 발현하도록 형질전환된 재조합 세포를 이용한 발효 방법에 의해 생산될 수 있다. 그러나, 상기 발효방법 또는 효소반응법을 통해 생산된 반웅생성물에는 마이오—이노시를과 D-카이로- 이노시를이 흔합되어 있고, 흔합용액중에는 마이오ᅳ이노시를에 비해 D- 카이로 -이노시를의 농도가 상대적으로 낮다. 따라서， 마이오 -이노시를과D-chiro-inositol (cis— 1, 2, 4_t rans-3, 5,6-cyclohexol) is used for myo-inositol, cis 一 l, 2, 3,5—trans — 4,6 cyclo cyclohexanehexol) is a stereoisomer of the 3 hydroxyl group is epimerized (Fig. 1). D-Cyrropyinosinos is reported to be an important mediator of insulin signaling as an important component of phosphoglycans (IPG) and is known to be effective in the treatment of type 2 diabetes. D-Cyro-inosyl is primarily found in eukaryotes and is biosynthesized by epimerization of myo-inositol. D-Cyro-inosyl is mainly produced by hydrochloric acid hydrolysis of D-pinitol or kasugamycin (US Pat No. 5827896, US Pat No. 5091596, US Pat No. 5463142). , US Pat No. 5714643). However, although raw materials such as pinir and kasugamycin are expensive, and organic synthesis method (US Pat No. 5406005, WO 96/25381) is known, it is not easy to separate by-products and thus it is economically low. D-chiroybinosinos may be produced from myo-inosyl by the continuous action of inositol dehydrogenase and inosose isomerase enzymes. It can be produced by fermentation methods using recombinant cells transformed directly with Myo-Inosi, or transformed to express the enzyme. However, in the reaction product produced by the fermentation method or the enzyme reaction method, myo-inosino and D-chiro-inosino are mixed, and the mixed solution contains myo-inosino-D. The concentration of Cairo-inos is relatively low. Thus, with the Maio-Inoshi

D-카이로 -이노시를의 흔합물로부터 D-카이로 -이노시를을 농도를 높여 고순도로 정제하는 방법의 개발이 요구된다. 본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다. 【발명의 상세한 설명】 Development of a method for purifying D-Cyro-Inosyl with high purity from a mixture of D-Cyro-Inosyl is required. Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained. [Detailed Description of the Invention]

【기술적 과제】  [Technical problem]

형질전환세포를 이용한 발효방법 또는 효소반응 방법에 의해 마이오- 이노시를을 기질로 하여 D-카이로 -이노시를을 생산하는 공정의 최종 반응 생성물내에서 마이오 -이노시를과 D-카이로 -이노시를이 흔합되어 존재한다. 본 발명자들은 이러한 이노시를의 입체이성질체 흔합용액으로부터 D- 카이로—이노시를만을 고순도로 정제하는 방법을 개발하고자 연구 노력하였다. 그 결과 본 발명자들은 입체이성질체 관계에 있는 마이오- 이노시를과 D—카이로—이노시를이 수용액 상에서 큰 용해도 차이를 보임을 확인하였고, 이러한 용해도 차이를 이용하여 마이오-이노시를로부터 D- 카이로 -이노시를을 간단한 공정을 통해 효율적으로 분리하여 고순도로 정제할 수 있음을 확인함으로써 본 발명을 완성하였다.  Myo-inosine and D-chiro in the final reaction product of the process for producing D-chiro-inosine as a substrate by fermentation or enzymatic reaction using transformed cells. -Inosino exists in this mixture. The present inventors have tried to develop a method for purifying D-Cyro-Inosine with high purity from the stereoisomeric mixed solution of this inosine. As a result, the present inventors found that the solubility of myo-inos and D-chiro-inosio in the stereoisomer relationship was large in the aqueous solution. The present invention has been completed by confirming that chiro-inosi can be efficiently separated and purified in high purity through a simple process.

따라서 , 본 발명의 목적은 마이오-이노시를 및 D—카이로 -이노시를이 함유된 용액으로부터 D-카이로 -이노시를을 고순도로 정제하는 방법을 제공하는 데에 있다.  Accordingly, it is an object of the present invention to provide a method for purifying D-chiro-inosi with high purity from a solution containing myo-inosi and D-chiro-inosi.

, ,

본 발명의 목적 및 장점은 하기의 발명의 상세한 설명， 청구의 범위 및 도면에 의해 보다 명확하게 된다.  The objects and advantages of the invention will become apparent from the following detailed description, claims and drawings.

【기술적 해결방법】 Technical Solution

본 발명의 일 양태에 따르면, 본 발명은 마이오-이노시를 및 D- 카이로—이노시를이 함유된 용액으로부터 D-카이로 -이노시톨을 고순도로 정제하는 방법으로서， 다음의 단계를 포함하는 방법을 제공한다: (a) 마이오-이노시를 및 D-카이로 -이노시를이 함유된 용액에 유기용매를 첨가하여 이노시를 침전물을 생성시키는 단계; (b) 상기 생성된 이노시를 침전물로부터 상층액을 분리하는 단계; 및 (c) 상기 분리한 상층액을 건조시켜 이노시를 분말을 얻는 단계 . According to one aspect of the present invention, the present invention provides a high purity of D-chiro-inositol from a solution containing myo-inosine and D-chiro-inositol. As a method for purifying, there is provided a method comprising the following steps: (a) adding an organic solvent to a solution containing myo-inosine and D-chiro-inosine to form inosi in a precipitate; step; (b) separating the supernatant from the resulting inosi from the precipitate; And (c) drying the separated supernatant to obtain Inosi powder.

본 발명의 바람직한 구현예에 의하면, 본 발명은 상기 단계 (c) 이후에 다음의 단계를 더욱 포함하는 것을 특징으로 하는 방법을 제공한다: (d) 상기 단계 (c)에서 얻은 이노시를 분말을 물에 용해시켜 수용액을 제조하는 단계; (e) 상기 수용액에 유기용매를 첨가하여 이노시를 침전물을 생성시키는 단계; (f) 상기 생성된 이노시를 침전물로부터 상층액을 분리하는 단계; 및 (g) 상기 분리한 상층액을 건조시켜 이노시를 분말을 얻는 단계.  According to a preferred embodiment of the present invention, the present invention provides a method characterized by further comprising the following step after the step (c): (d) powdering the inosine obtained in the step (c) Dissolving in water to prepare an aqueous solution; (e) adding an organic solvent to the aqueous solution to generate an inosi precipitate; (f) separating the supernatant from the resulting inosi from the precipitate; And (g) drying the separated supernatant to obtain Inosi powder.

본 발명의 다른 바람직한 구현예에 의하면， 본 발명은 상기 단계 (c) 이후에 다음의 단계를 더 포함하는 것을 특징으로 하는 방법을 제공한다: (d)' 상기 단계 (C)에서 얻은 이노시를 분말을 물에 용해시켜 수용액을 제조하고 이노시를 침전물을 생성시키는 단계; (e)' 상기 수용액에서 생성된 이노시를 침전물로부터 상층액을 분리하는 단계; 및 (f)' 상기 상층액을 건조시켜 이노시를 분말을 얻는 단계. 이하에서 본 발명의 방법을 각 단계에 따라 보다 상세히 설명한다. 단계 (a): 마이오—이노시를 및 D-카이로 -이노시를이 함유된 용액에 유기용매를 첨가하여 이노시를 침전물을 생성시키는 단계 According to another preferred embodiment of the invention, the invention provides a method according to claim 1, further comprising the steps of after said step (c): the INNO when obtained in (d) 'the step (C) Dissolving the powder in water to form an aqueous solution and producing inosi precipitates; (e) 'separating the supernatant from the precipitate created in the Inno when the aqueous solution; And (f) ' drying the supernatant to obtain Inosi powder. Hereinafter, the method of the present invention will be described in more detail with each step. Step (a): adding an organic solvent to a solution containing myo-inosine and D-chiro-inosino to form inosi precipitate

마이오 -이노시를과 D-카이로 -이노시를이 함유된 이노시틀 흔합용액은 마이오 -이노시를을 기질로 하여 D-카이로 -이노시를을 생산하는 방법에서 최종 반웅 산물로 얻어 질 수 있다. 예를 들어, 마이오-이노시를 트랜스포터 (myo— inositol transporter) , 이노시를 디하이드로게나아제 (inositol dehydrogenase) 및 이노소스 이소머라아제 (inosose isomerase)를 발현하도록 형질전환된 재조합 세포를 이용한 발효 방법에서 마이오 -이노시를을 기질로 하여 배양한 배양 산물에는 반응하고 남은 반웅기질 마이오 -이노시를과 반응 생성물인 D- P O 2014/098454 The inositol mixed solution containing myo-inosino and D-chiro-inosino was obtained as a final reaction product by the method of producing D-chiro-inosino with myo-inosino as substrate. Can lose. For example, recombinant cells transformed with myo-inosio transporter, inosio dehydrogenase and inosose isomerase are used. In the fermentation method, the reaction product cultured with the myo-inosine as a substrate was reacted with the reaction product D- PO 2014/098454

카이로ᅳ이노시를이 흔합된 이노시를 흔합용액이 만들어진다 . 또한 , 상기 효소를 발현하도록 형질전환된 재조합 세포의 휴면세포 (resting cell)를 마이오 -이노시를을 포함하는 최소배지에 접종하여 마이오ᅳ이노시를을 으 카이로 -이노시를로 전환하는 방법의 경우에도 배양한 최종 배양 산물에 반웅하고 남은 반웅 기질 마이오 -이노시를과 반웅 생성물인 D-카이로ᅳ 이노시를이 흔합된 이노시틀 흔합물이 만들어진다. 또한， 마이오-이노시를 트랜스포터， 이노시를 디하이드로게나아제 및 이노소스 이소머라아제의 효소를 사용하여 마이오-이노시를로부터 D-카이로 -이노시를을 생성시키는 효소 반웅법에서 반웅을 완료한 최종 반웅 산물에 반웅하고 남은 반웅 기질 마이오 -이노시를과 으카이로 -이노시를이 흔합된 이노시톨 흔합 용액이 만들어진다. 따라서， 본 발명의 방법은 예컨대 상술한 마이오- 이노시를로부터 D-카이로 -이노시를을 생산하는 발효방법， 휴면세포 전환방법 및 효소 반응법의 최종 반응용액으로 만들어지는 마이오- 이노시를과 으카이로 -이노시를의 흔합용액으로부터 D-카이로 -이노시를을 고순도로 정제하는 방법에 적용될 수 있다. 본 발명의 방법이 적용될 수 있는 마이오-이노시를 및 D-카이로 -이노시를의 흔합용액은 예컨대 재조합 세포를 사용한 발효액일 경우 영양배지 (완전배지)일 수 있으며， 휴면세포 전환법에 의한 반웅액일 경우 M9 최소배지일 수 있으며 , 효소반웅법에 의한 반응액일 경우 효소 반응 완층액이 될 수 있다. 상기 설명된 D-카이로ᅳ 이노시를을 제조하는 방법의 최종 반웅용액내에서 D-카이로ᅳ이노시를의 최종 평형 농도는 약 14%이므 ¾， 이노시를의 흔합 용액내의 마이오- 이노시를과 D_카이로 -이노시를은 약 86 :14의 비율로 존재한다. Ino City mixed with Cairo ᅳ Ino City is made of a solution. In addition, the resting cells of the recombinant cells transformed to express the enzyme are inoculated into a minimal medium containing myo-inosi to convert myozynosi to chiro-inosi. In the case of the method, an inositol mixture is produced in which the reaction product, the remaining reaction product, myo-inosi and the reaction product, D-chirojan inosi, are mixed with the final culture product. In addition, an enzyme reaction method for producing D-chiro-inosine from myo-inosine using an enzyme of myo-inosio, an inosio dehydrogenase and an inososomerase After the reaction was completed, the resulting reaction product was made, and the remaining reaction product was prepared by mixing the remaining reaction mixtures, such as Mio-Inoshi and Ekairo-Inoshi. Therefore, the method of the present invention is, for example, the myo-inosity produced as the final reaction solution of the fermentation method, the dormant cell conversion method and the enzyme reaction method for producing D-chiro-inosity from the above-mentioned myo-inoshi. It can be applied to a method of purifying D-Cairo-inosity with high purity from a mixed solution of E. co. The mixed solution of myo-inosyl and D-chiro-inosyl to which the method of the present invention can be applied may be a nutrient medium (complete medium), for example, a fermentation broth using recombinant cells. In the case of reaction solution, M9 may be the minimum medium, and in the case of reaction reaction by enzyme reaction method, it may be a complete enzyme reaction solution. The final equilibrium concentration of D-chiropy Inosio in the final semi-aqueous solution of the process for preparing D-chiropyrosinos described above is about 14% ¾, Myo-inos in the mixed solution of Inosyl. And D_Cairo-Inosio are present in ratio of about 86:14.

본 발명의 방법은 마이오 -이노시를과 D-카이로 -이노시를의 용해도 차이에 의해 마이오ᅳ이노시를과 D-카이로ᅳ이노시를 흔합 용액내에서 D- 카이로 -이노시를의 순도를 높여 정제하는 방법이다. 마이오 -이노시를과 D- 카이로 -이노시를이 물에 용해된 수용액내에서 각각의 이노시를의 용해도 차이는 도 3에 나타나 있다. 본 발명의 방법에서， 먼저 상기 마이오- 이노시틀과 D-카이로ᅳ이노시를을 함유한 이노시를 용액에 유기용매를 첨가한다. 상기 유기용매의 첨가에 의해 마이오 -이노시를과 D-카이로- 이노시를의 침전이 유도된다. 유기용매 첨가에 의해 마이오 -이노시를과 D- 카이로 -이노시를의 침전율이 모두 증가되지만, D-카이로 -이노시를에 비해 마이오 -이노시를의 침전율이 훨씬 크게 증가한다 (도 5 참조). 따라서， 마이오 -이노시를과 D-카이로 -이노시를이 흔합된 용액에 유기용매를 첨가하여 D-카이로-이노시를 보다 마이오 -이노시틀을 더 많은 양으로 침전시켜 용액중의 D-카이로—이노시를의 농도를 증대시킬 수 있다. The method of the present invention is a mixture of myo-inosino and D-chiro-inosino by the difference in solubility of myo-inosino and D-chiro-inosino in the solution of D-chiro-inosino Purification is to increase the purity. The difference in solubility of each inosin in an aqueous solution of Myo-inosyl and D-Cairo-inosyl in water is shown in FIG. 3. In the method of the present invention, an organic solvent is first added to a solution of inos containing the myo-inositol and D-chiropyonos. Precipitation of myo-inos and D-chiro-inos is induced by the addition of the organic solvent. The precipitation rate of myo-inos and D-chiro-inos is increased by the addition of organic solvent, but compared to D-chiro-inos The precipitation rate of myo-inos is much greater (see Figure 5). Therefore, by adding an organic solvent to the mixed solution of myo-inosino and D-chiro-inosino, D-chiro-inosio is precipitated in a higher amount than that of D-chiro-inosio. Can increase the concentration of D-Cairo-Inosino.

본 발명에서 사용할 수 있는 유기용매는 D-카이로 -이노시를의 침전 보다 마이오 -이노시를의 침전을 더욱 크게 유도할 수 있는 특성을 갖는 것이면 특별하게 한정되지 않는다. 본 발명의 일 구현예에 의하면, 상기 유기용매는 알코올， 아세톤， 에틸아세테이트， 부틸아세테이트, 1， 3- 부틸렌글리콜 또는 에테르이다. 본 발명의 다른 구현예에 의하면， 상기 유기용매는 탄소수 2— 6개의 알코올이다. 본 발명의 또 다른 구현예에 의하면, 상기 유기용매는 에탄을, 이소프로판올, 또는 아세톤이다.  The organic solvent that can be used in the present invention is not particularly limited as long as it has a property of inducing precipitation of myo-inosine more than precipitation of D-chiro-inosine. According to one embodiment of the invention, the organic solvent is alcohol, acetone, ethyl acetate, butyl acetate, 1, 3- butylene glycol or ether. According to another embodiment of the present invention, the organic solvent is an alcohol having 2 to 6 carbon atoms. According to another embodiment of the present invention, the organic solvent is ethane, isopropanol, or acetone.

상기 마이오 -이노시를과 D-카이로 -이노시틀의 흔합용액에 첨가되는 유기용매의 양은 특별히 한정되지 않고, 이노시를 흔합용액 대비 0.1 - 9배 (v/v)의 범위 내에서 적합한 양을 선택하여 첨가할 수 있다. 본 발명의 일 구현예에 의하면 상기 유기용매는 이노시를 흔합용액 대비 0.1 - 6배 (v/v)의 양으로 첨가된다. 본 발명의 다른 구현예에 의하면 상기 유기용매는 이노시를 흔합용액 대비 0.1 - 4배 (v/v)의 양으로 첨가한다. 본 발명의 다른 구현예에 의하면 상기 유기용매는 이노시를 흔합용액 대비 0.1 — 2배 (v/v)양으로 첨가한다. 본 발명의 또 다른 구현예에 의하면 상기 유기용매는 이노시를 흔합용액 대비 0.1 ᅳ 1.5배 (v/v)양으로 첨가한다.  The amount of the organic solvent added to the mixed solution of the myo-inosino and D-chiro-inosino is not particularly limited, and the inosin is suitable within the range of 0.1-9 times (v / v) compared to the mixed solution. The amount can be selected and added. According to one embodiment of the present invention, the organic solvent is added to the inosin in an amount of 0.1-6 times (v / v) compared to the mixed solution. According to another embodiment of the present invention, the organic solvent is added to the inosin in an amount of 0.1-4 times (v / v) compared to the mixed solution. According to another embodiment of the present invention, the organic solvent is added to the inosin in an amount of 0.1 — 2 times (v / v) compared to the mixed solution. According to another embodiment of the present invention, the organic solvent is added to the inosin in the amount of 0.1 ᅳ 1.5 times (v / v) compared to the mixed solution.

본 발명에서 유기용매의 첨가에 의해 유도되는 이노시틀 침전물에는 마이오 -이노시를과 D-카이로 -이노시를이 모두 포함되어 있으나, 마이오ᅳ 이노시를의 침전율이 더 높으므로, 마이오 -이노시를이 더 많이 포함되어 있다. 단계 (b): 상기 생성된 이노시를 침전물로부터 상층액을 분리하는 단계  Inositol precipitates induced by the addition of an organic solvent in the present invention include both myo-inosyl and D-chiro-inosyl, but the precipitation rate of myosin inosyl is higher. Oh-Innosi contains more. Step (b): separating the supernatant from the resulting inosi from the precipitate

상기 단계 (a)를 통해 생성된 이노시를 침전물로부터 상층액을 분리한다. 상기 이노시틀 침전물은 마이오-이노시를 및 D-카이로- 이노시틀의 침전물이지만， 마이오ᅳ이노시를이 더 큰 침전율을 나타내므로， D-카이로-이노시를 보다는 마이오 -이노시를이 더욱 많은 양으로 포함되어 있다. 마이오—이노시를의 다량 침전으로 인해 상층액 중에는 마이오- 이노시를의 농도가 낮아지게 되며， 반대로 D-카이로ᅳ이노시를의 농도 (순도)는 상대적으로 증가하게 된다. 이노시를 침전물로부터 상층액의 분리는 여과 또는 원심분리 방법을 사용하며， 바람직하게는 여과 방법을 사용하여 행한다. 단계 (c): 상기 분리한 상층액을 건조시켜 이노시를 분말을 얻는 단계 The inos generated through step (a) is separated from the supernatant from the precipitate. The inositol precipitate is a precipitate of myo-inos and D-chiro-inosit, but since the myosininosium exhibits a greater precipitation rate, the inositino is more likely to be the myo-inos than the D-chiro-inos. Poems are included in even greater amounts have. Due to the large precipitation of myo-inosyl, the concentration of myo-inosyl is lowered in the supernatant, and the concentration (purity) of D-chiro-inosylol is relatively increased. Separation of the supernatant from the precipitate by Inosi is carried out using a filtration or centrifugation method, preferably by using a filtration method. Step (c): drying the separated supernatant to obtain Inosi powder

상기 단계 (b)에서 분리한 상층액을 건조시켜 이노시를 분말을 얻는다. 상기 상층액의 건조는 상층액을 가열하면서 행할 수 있으며， 또는 진공상태에서 행할 수 있디-. 상층액을 건조시켜 얻은 이노시를 건조물에는 마이오 -이노시를과 D-카이로—이노시를이 흔합되어 있으나， 상기 단계 (a)의 마이오-이노시를 및 D-카이로 -이노시롤이 함유된 용액에서와 비교하여 D- 카이로 -이노시를의 순도가 증가되어 있다. 본 발명의 방법은 상기 단계 (C) 이후에 추가적으로 하기 단계 (d) ― 단계 (g)를 더욱 포함할 수 있다. 단계 (d) : 상기 단계 (C)에서 얻은 이노시를 분말을 물에 용해시켜 수용액을 제조하는 단계 . The supernatant separated in step (b) is dried to obtain Inosi powder. Drying of the supernatant may be performed while heating the supernatant, or may be performed in a vacuum. Inosi obtained by drying the supernatant was composed of myo-inosino and D-chiro-inosino, but the myo-inosino and D-chiro-inosilo of step (a). The purity of D-Cairo-inosity is increased as compared to this containing solution. The method of the present invention may further comprise the following steps (d) to (g) after step (C). Step (d): dissolving the powder of Inosi obtained in step (C) in water to prepare an aqueous solution .

상기 단계 (C)에서 얻은 이노시를의 분말을 물에 용해시켜 이노시를이 용해된 수용액을 제조한다 . 상기 물은 바람직하게는 증류수이다. 본 발명의 일 구현예에 의하면 상기 이노시를 분말은 물에 20%(w/v) 이하의 농도로 용해시킨다. 본 발명의 다른 구현예에 의하면， 상기 이노시를 분말은 물에 0.1%(w/v) 이상 20%(w/v) 이하의 농도로 용해시킨다. 본 발명의 다른 구현예에 의하면 상기 이노시틀 분말은 0.1% (w/v) 이상 18%(w/v) 이하의 농도로 용해시킨다. 본 발명의 또 다른 구현예에 의하면 상기 이노시를 분말은 0.1% (w/v) 이상 15% (w/v) 이하의 농도로 용해시킨다. 단계 (e): 상기 수용액에 유기용매를 첨가하여 이노시를 침전물을 생성시키는 단계 An inosi-dissolved aqueous solution was prepared by dissolving the inosi powder obtained in step (C) in water. The water is preferably distilled water. According to one embodiment of the present invention, the inosin powder is dissolved in water at a concentration of 20% (w / v) or less. According to another embodiment of the present invention, the inosin is dissolved in water at a concentration of 0.1% (w / v) or more and 20% (w / v) or less. According to another embodiment of the present invention, the inositol powder is dissolved at a concentration of 0.1% (w / v) or more and 18% (w / v) or less. According to another embodiment of the present invention, the inosin is dissolved in the powder at a concentration of 0.1% (w / v) or more and 15% (w / v) or less. Step (e): adding an organic solvent to the aqueous solution to precipitate inosi Generating step

상기 단계 (d)에서 얻은 이노시롤이 용해된 수용액에 유기용매를 첨가하여 이노시틀의 침전물을 생성시킨다. 상기 유기용매는 상기 단계 (a)에서 설명된 내용과 동일하다. 유기용매는 D-카이로 -이노시를의 침전 보다 마이오 -이노시틀의 침전을 더욱 크게 유도할 수 있는 특성을 갖는 것이면 특별하게 한정되지 않는다. 본 발명의 일 구현예에 의하면 상기 유기용매는 알코올， 아세톤, 에틸아세테이트， 부틸아세테이트， 1, 3- 부틸렌글리콜 또는 에테르이다. 본 발명의 다른 구현예에 의하면 상기 유기용매는 탄소수 2-6개의 알코을이다. 본 발명의 또 다른 구현예에 의하면, 상기 유기용매는 에탄올， 이소프로판올, 또는 아세톤이다. 본 발명의 일 구현예에 의하면， 상기 수용액에 첨가되는 유기용매의 양은 수용액 대비 0.1 - 10배 (v/v)의 범위 내에서 적합한 양을 선택하여 첨가할 수 있다. 본 발명의 다른 구현예에 의하면， 유기용매는 수용액 대비 1.5 - 9배 (v/v)의 양으로 첨가한다. 본 발명의 또 다른 구현예에 의하면, 유기용매는 수용액 대비 9배 (v/v)의 양으로 첨가한다. 상기 이노시를 침전물은 마이오-이노시를 및 D-카이로 -이노시를의 침전물이지만, 마이오- 이노시를이 더 큰 침전율을 나타내므로， D-카이로-이노시를 보다는 마이오- 이노시를이 더욱 많은 양으로 포함되어 있다. 단계 (f): 상기 생성된 이노시를 침전물로부터 상층액을 분리하는 단계  An organic solvent is added to the aqueous solution in which the inositol obtained in step (d) is dissolved, thereby producing a precipitate of inositol. The organic solvent is the same as described in step (a). The organic solvent is not particularly limited as long as the organic solvent has a property of inducing precipitation of myo-inositol more than precipitation of D-chiro-inoshi. According to one embodiment of the invention the organic solvent is alcohol, acetone, ethyl acetate, butyl acetate, 1, 3- butylene glycol or ether. According to another embodiment of the present invention, the organic solvent is alcohol having 2 to 6 carbon atoms. According to another embodiment of the present invention, the organic solvent is ethanol, isopropanol, or acetone. According to one embodiment of the present invention, the amount of the organic solvent added to the aqueous solution may be added by selecting a suitable amount within the range of 0.1 to 10 times (v / v) relative to the aqueous solution. According to another embodiment of the present invention, the organic solvent is added in an amount of 1.5-9 times (v / v) relative to the aqueous solution. According to another embodiment of the present invention, the organic solvent is added in an amount of 9 times (v / v) relative to the aqueous solution. The inosi precipitates are the precipitates of myo-inosio and D-chiro-inosino, but since the myo-inosio exhibits a greater precipitation rate, the inosio-inos than the Dio-inosino Poems are included in even greater amounts. Step (f): separating the supernatant from the resulting inosi from the precipitate

상기 단계 (e)를 통해 생성된 이노시를 침전물로부터 상층액을 분리한다. 이노시를 침전물로부터 상층액의 분리는 여과 또는 원심분리 방법을 사용하며， 바람직하게는 여과방법을 사용하여 행한다. 이노시를 침전물에는 마이오ᅳ이노시롤이 다량 포함되어 있으므로 상층액에는 D- 카이로 -이노시틀의 순도가 증가되어 있다. 단계 (g): 상기 분리한 상층액을 건조시켜 이노시롤 분말을 얻는 단계  The inos generated through step (e) is separated from the supernatant from the precipitate. Separation of the supernatant from the precipitate by the inosi is carried out using a filtration or centrifugal separation method, preferably using a filtration method. Since the inosi precipitate contains a large amount of myosininosylol, the supernatant has an increased purity of D-Cairo-inositol. Step (g): drying the separated supernatant to obtain inosyrol powder

상기 단계 (f)에서 분리한 상층액을 건조시켜 이노시를 분말을 얻는다. 상기 상층액의 건조는 상층액을 가열하면서 행할 수 있으며， 또는 진공상태에서 행할 수 있다. 상층액을 건조시켜 얻은 이노시를 건조물에는 마이오ᅳ이노시를과 D-카이로 -이노시를이 흔합되어 있으나， D-카이로- 이노시를의 순도가 증가되어 있다. 본 발명의 방법은 상기 단계 (c) 이후에 추가적으로 하기 단계The supernatant separated in step (f) is dried to obtain Inosi powder. Drying of the supernatant may be performed while heating the supernatant, or This can be done in a vacuum. Inosine obtained by drying the supernatant was composed of myo-inosino and D-chiro-inosino, but the purity of D-chiro-inosino was increased. The method of the present invention further comprises the following steps after step (c)

(d)' - 단계 (f)' 를 더욱 포함할 수 있다. 단계 (d)' ： 상기 단계 (c)에서 얻은 이노시틀 분말을 물에 용해시켜 수용액을 제조하고 이노시를 침전물을 생성시키는 단계 (d) '- Step (f)' a may be further included. Step (d) ' : dissolution of the inositol powder obtained in step (c) in water to prepare an aqueous solution and inosi to produce a precipitate

상기 단계 (c)에서 얻은 이노시를의 분말을 물에 용해시켜 이노시를이 용해된 수용액을 제조한다. 상기 물은 바람직하게는 증류수이다. 본 발명의 일 구현예에 의하면 상기 이노시를 분말은 물에 20%(w/v) 초과의 고농도로 용해시킨다. 본 발명의 다른 구현예에 의하면 상기 이노시를 분말은 물에 209¾(w/v) 초과 80%(w/v) 이하의 농도로 용해시킨다. 본 발명의 또 다른 구현예에 의하면 상기 이노시를 분말은 물에 30%(w/v) 이상 70%(w/v) 이하의 농도로 용해시킨다. 하기 구체적인 실시예에 의하면, 물에 대한 마이오ᅳ이노시를의 용해도는 최대 약 15% 이며, D-카이로 -이노시를의 용해도는 최대 약 55%이므로， 상기 단계 (c)에서 얻은 이노시를' 분말을 물에 70%의 농도로 용해시키는 경우 마이오 -이노시를은 최대 15% 농도로 용해될 것이며, 나머지 용해되지 않은 마이오 -이노시를은 침전물로 생성된다. D-카이로ᅳ이노시를은 최대 55%의 용해도이므로 모든 으카이로 -이노시를이 물에 용해된다. 단계 (e)' ： 상기 수용액에서 생성된 이노시롤 침전물로부터 상층액을 분리하는 단계 An inosi-dissolved aqueous solution was prepared by dissolving the inosi powder obtained in step (c) in water. The water is preferably distilled water. According to one embodiment of the present invention, the inosi powder is dissolved in water at a high concentration of more than 20% (w / v). According to another embodiment of the invention the inosi powder is dissolved in water at a concentration of greater than 209¾ (w / v) up to 80% (w / v). According to another embodiment of the present invention, the inosi powder is dissolved in water at a concentration of 30% (w / v) or more and 70% (w / v) or less. According to the following specific examples, the solubility of myoininosi to water is up to about 15%, and the solubility of D-Cyro-inosino is up to about 55%, so that the inosi obtained in step (c) Maio the case of dissolving the "concentration of 70% of the powder in water - during the Inno will be dissolved at up to 15% density, and the other non-dissolved Maio - is produced when the Inno in the precipitate. D-Cairo-Inosine is soluble in up to 55%, so all of the Cairo-Inosine is dissolved in this water. Step (e) ' : separating the supernatant from the inosolole precipitate produced in the aqueous solution

상기 단계 (d)' 에서 생성된 이노시를의 침전물로부터 상층액올 분리한다. 이노시를 침전물로부터 상층액의 분리는 여과 또는 원심분리 방법을 사용하며, 바람직하게는 여과방법을 사용하여 행한다. 이노시를 침전물에는 대부분이 마이오ᅳ이노시를이 포함되어 있을 것이며, 상충액에는 D-카이로 -이노시를의 순도가 증가되어 있다. 단계 (f)' ： 상기 상층액을 건조시켜 이노시를 분말을 얻는 단계 상기 단계 (e)' 에서 분리한 상충액을 건조시켜 이노시를 분말을 얻는다. 상기 상층액의 건조는 상층액올 가열하면서 행할 수 있으며， 또는 진공상태에서 행할 수 있다. 상층액을 건조시켜 얻은 이노시를 건조물에는 마이오 -이노시를과 D-카이로 -이노시를이 흔합되어 있으나, D-카이로ᅳ 이노시를의 순도가 증가되어 있다. 하기 구체적인 일 실시예에 의하면, 상기 단계 (d)' 에서 이노시를 분말을 물에 약 70%(w/v)의 농도로 용해시킨 경우 건조된 분말에서의 D-카이로 -이노시를의 순도는 약 70% 이상이 될 것이다. 본 발명의 이점 및 특징올 요약하면 다음과 같다. The supernatant is separated from the precipitate of Inosi produced in step (d) ' . Separation of the supernatant from the precipitate by the inosi is carried out using a filtration or centrifugation method, preferably by using a filtration method. Most of the inosi precipitate will contain myoininosi, and the supernatant has increased purity of D-Cairo-inossi. Step (f) ' : drying the supernatant to obtain Inosi powder. The supernatant separated in Step (e) ' is dried to obtain Inosi powder. The supernatant may be dried while the supernatant is heated, or may be performed in a vacuum. Inosi obtained by drying the supernatant was composed of Myo-inos and D-Cairo-inos, but the purity of D-Cairo Inos was increased. According to one specific embodiment below, the purity of D-Cairo-Inosi in the dried powder when the inosi powder is dissolved in water at a concentration of about 70% (w / v) in step (d) ' Will be about 70% or more. The advantages and features of the present invention are summarized as follows.

(i) 본 발명은 마이오—이노시를 및 D-카이로 -이노시를의 흔합용액으로부터 D-카이로 -이노시를을 고순도로 정제하는 방법에 관한 것이다. .  (i) The present invention relates to a method for purifying D-Cyro-Inosyl with high purity from a mixed solution of Myo-Inosyl and D-Cyro-Inosyl. .

(ii) 본 발명의 방법은 마이오ᅳ이노시를와 D—카이로 -이노시를의 용해도 차이 원리에 기초한 것이다.  (ii) The method of the present invention is based on the principle of the difference in solubility between myoininosi and D—chiro-inosi.

(iii) 본 발명의 방법에서 유기용매를 첨가하여 D-카이로 -이노시를의 용해도 감소 보다 마이오 -이노시를의 용해도를 더 크게 감소시켜 결과적으로 D-카이로-이노시를 보다 마이오-이노시틀의 침전을 더 크게 유도한다.  (iii) the addition of an organic solvent in the process of the present invention reduces the solubility of myo-inosine more significantly than the decrease of the solubility of D-chiro-inosyl, resulting in more D-chiro-inosio Induces greater precipitation of inositol.

(iv) 본 발명의 방법은 유기용매의 첨가， 상층액 분리 (여과) 및 건조와 같은 간단한 공정을 사용하므로 저비용으로 마이오ᅳ이노시를과 D- 카이로 -이노시를의 흔합용액으로부터 으카이로 -이노시를을 고순도로 분리 정제할 수 있다.  (iv) The method of the present invention employs simple processes such as the addition of organic solvents, supernatant separation (filtration) and drying, and therefore, at low cost, yields a solution from the mixed solution of myoininosi and D-chiro-inosi. Inosi can be separated and purified in high purity.

(V) 본 발명의 방법에 의하면 분리된 D-카이로-이노시를로부터 분리된 마이오 -이노시를은 기질로서 재사용이 가능하다.  (V) According to the method of the present invention, the myo-inosine separated from the separated D-chiro-inos is reusable as a substrate.

【유리한 효과】 Advantageous Effects

본 발명은 마이오 -이노시를과 D-카이로ᅳ이노시를의 용해도 차이를 '이용하여 마이오-이노시를 및 D-카이로ᅳ이노시를의 흔합용액으로부터 으 카이로ᅳ이노시를을 고순도로 정제하는 방법에 관한 것이다. 본 발명의 방법은 이노시를의 흔합용액에 유기용매 첨가， 이노시를 침전의 유도, 상층액의 분리, 및 상층액의 건조와 같은 간단한 공정을 통해 저비용으로The present invention utilizes the solubility difference between myo-inosino and D-chirofoinosinos to obtain high purity from the mixture of myo-inosino and D-chirofoinosinos. It relates to a method of purifying with. Of the present invention The process can be carried out at low cost through simple processes such as adding an organic solvent to a mixed solution of Inosi, inducing Inosi to precipitate, separating the supernatant, and drying the supernatant.

D-카이로 -이노시를의 순도를 크게 높일 수 있는 이점이 있다. 또한， 본 발명의 방법에 의하면 분리된 마이오 -이노시를은 마이오-이노시를로부터 D- 카이로 -이노시틀을 생산하는 방법에서 기질로서 재사용이 가능하다. There is an advantage that can greatly increase the purity of D-Cairo-Inoshi. In addition, according to the method of the present invention, the separated myo-inosity can be reused as a substrate in the method of producing D-chiro-inositol from the myo-inosino.

【도면의 간단한 설명】 [Brief Description of Drawings]

도 1은 입체이성질체인 마이오-이노시를 및 D-카이로 -이노시를의 구조를 보여준다.  1 shows the structures of the stereoisomers myo-inos and D-chiro-inos.

도 2는 마이오-이노시를 및 D-카이로ᅳ이노시롤의 흔합용액으로부터 FIG. 2 shows a mixed solution of myo-inosine and D-chiropyonosinolol.

D-카이로 -이노시를을 고순도로 정제하는 본 발명의 정제 공정을 보여준다. 도 3은 25 °C 증류수에서 마이오ᅳ이노시를과 D-카이로 -이노시를의 용해도를 보여준다. B: 마이오 -이노시를의 용해도， O: D-카이로- 이노시를의 용해도. A purification process of the present invention for purifying D-Cairo-inosity with high purity is shown. FIG. 3 shows the solubility of myopininosine and D-chiro-inosi in 25 ° C distilled water. B: solubility of myo-inosino, O: solubility of D-chiro-inosino.

도 4는 마이오-이노시를 및 D-카이로—이노시틀이 용해되어 있는 다양한 종류의 흔합 용액 (물， 식염수, 효소반웅완충액， M9 배지, 및 TB 배지)에 에탄올을 첨가하여 이노시틀을 침전시킨 후 상층액내 마이오- 이노시를 및 D-카이로 -이노시롤의 농도를 측정한 결과이다. B: 마이오- 이노시를의 농도， O: D-카이로 -이노시를의 농도.  FIG. 4 shows inositol by adding ethanol to various types of mixed solutions (water, saline, enzyme reaction buffer, M9 medium, and TB medium) in which myo-inosino and D-chiro-inosothol are dissolved. The precipitates were precipitated and the concentrations of myo-inosine and D-chiro-inoshiro in the supernatant were measured. B: concentration of myo-inosyl, O: concentration of D-chiro-inosyl.

도 5는 마이오-이노시를 및 으카이로 -이노시를이 용해되어 있는 다양한 종류의 흔합 용액 (물， 식염수， ≤소반응완충액， M9 배지， 및 TB 배지)에 에탄을을 첨가한 경우 마이오-이노시를 및 D-카이로 -이노시틀의 침전율을 나타낸다. 園: 마이오 -이노시를의 침전율， O: D-카이로- 이노시를의 침전율.  FIG. 5 shows that ethane was added to various kinds of mixed solutions (water, saline, ≦ small reaction buffer, M9 medium, and TB medium) in which myo-inosino and echairo-inosio were dissolved. The precipitation rates of O-inosino and D-Cairo-inosinot are shown.園: The precipitation rate of Maio-Inoshiro, O: The precipitation rate of D-Cairo-Inoshiro.

도 6은 마이오-이노시틀 및 D-카이로 -이노시를이 용해되어 있는 다양한 종류의 용매 흔합 용액인 물， 식염수 효소반웅완층액， M9 배지， 및 TB 배지에 에탄을을 첨가하여 이노시를을 침전시킨 후 얻은 상층액을 건조하여 얻은 이노시틀 분말 중의 D-카이로 -이노시를의 순도를 측정한 결과를 보여준다.  FIG. 6 shows inosin by adding ethane to water, a saline enzyme reaction mixture, M9 medium, and TB medium, which are various solvent mixture solutions in which myo-inositile and D-chiro-inosi are dissolved. After precipitation of the supernatant obtained by drying the inositol powder obtained by measuring the purity of the D-Cairo-Inoshile is shown.

도 7은 1차 에탄을을 첨가하여 얻은 상층액의 건조 분말인 마이오- 이노시를과 D-카이로 -이노시틀의 흔합 분말을 물에 다시 용해한 경우의 각 이노시를의 용해 특성을 보여준다. 패널 A는 수용액의 상층액중의 마이오- 이노시를 및 D-카이로ᅳ이노시를의 동도를 보여준다. 패널 B는 수용액의 상층액중의 마이오ᅳ이노시를 및 D-카이로 -이노시를의 용해도를 보여준다. 패널 C는 수용액의 상층액중의 마이오-이노시롤 및 D-카이로 -이노시를의 침전율을 보여준다. 패널 D는 수용액의 상충액 증의 D-카이로 -이노시를의 순도를 보여준다. ■： 마이오 -이노시틀， O: D-카이로ᅳ이노시를. Fig. 7 shows the case where the mixed powders of myo-inoshiro and D-chiro-inositol, which are dry powders of the supernatant obtained by adding primary ethane, are dissolved again in water. Show the dissolution properties of Inosi. Panel A shows the kinetics of myo-inos and D-chiropyonos in the supernatant of aqueous solution. Panel B shows the solubility of myofininos and D-chiro-inosino in the supernatant of aqueous solution. Panel C shows the precipitation rates of myo-inoshiro and D-chiro-inosi in the supernatant of aqueous solution. Panel D shows the purity of D-Cyro-Inosyl in solution increase in aqueous solution. ■ ： Myo-Ino City, O: D-Cairo Ino City.

도 8은 1차 에탄올을 첨가하여 얻은 상충액의 건조 분말인 마이오- 이노시를과 D-카이로—이노시를의 흔합 분말을 35% 내지 70% 사이의 다양한 농도로 물에 다시 용해시켜 얻은 이노시를 수용액에 다양한 양의 에탄을올 첨가한 후 상층액의 마이오ᅳ이노시를과 D-카이로 -이노시를의 농도를 측정한 결과이다. 패널 A는 상층액의 마이오 -이노시를의 농도이다. 패널 B는 상층액의 D-카이로 -이노시를의 농도이다. 議: 35%， O: 40%, ▲： 45%, V： 50%, ♦: 55%, ： 60%, ► : 65%, ^： 70%.  Figure 8 is obtained by re-dissolving a mixed powder of myo-inosino and D-chiro-inosino, a dry powder of the supernatant obtained by adding primary ethanol, in water at various concentrations between 35% and 70%. This is the result of measuring the concentration of myosininosi and D-chiro-inosi in the supernatant after adding various amounts of ethanol to the aqueous solution of inosi. Panel A is the concentration of myo-inosine in the supernatant. Panel B is the concentration of D-Cyro-inosyl in the supernatant.議: 35%, O: 40%, ▲: 45%, V: 50%, ♦: 55%, ： 60%, ►: 65%, ^: 70%.

도 9는 1차 에탄올을 첨가하여 얻은 상층액의 건조 분말인 마이오- 이노시틀과 D-카이로 -이노시를의 흔합 분말을 35% 내지 70% 사이의 다양한 농도로 물에 다시 용해시켜 얻은 이노시를 수용액에 다양한 양의 에탄을을 첨가한 후 마이오ᅳ이노시를 및 D-카이로 -이노시를의 침전율을 측정한 결과이다. 패널 A는 마이오 -이노시톨의 침전율이다. 패널 B는 으카이로- 이노시를의 침전율이다. ■: 35%, O: 40%, A: 45%, V： 50%, ♦: 55%, <]： 60% , ►： 65%, 70%.  9 is obtained by re-dissolving a mixed powder of myo-inositol and D-chiro-inosino, the dry powder of the supernatant obtained by adding primary ethanol, in water at various concentrations between 35% and 70%. Inosi was added to the aqueous solution of various amounts of ethane, and then the precipitation rates of myoininosi and D-chiro-inosi were measured. Panel A is the precipitation rate of myo-inositol. Panel B is the precipitation rate of Ecuiro-Inosi. ■: 35%, O: 40%, A: 45%, V: 50%, ♦: 55%, <]: 60%, ►: 65%, 70%.

도 10은 1차 에탄올을 첨가하여 얻은 상층액의 건조 분말 (마이오- 이노시를과 D-카이로ᅳ이노시를의 흔합 분말)을 70% 함유하는 수용액으로 제조하여 침전물을 제거하고 상층액을 재건조하여 D-카이로 -이노시틀의 비율이 70% 이상으로 중가된 이노시를 건조 흔합물을 10% 내지 50% 농도의 수용액으로 재용해한 후 다양한 양의 2차 알코을을 첨가하여 각 이노시를의 침전양상을 측정한 결과이다. 패널 Α는 상층액 중의 마이오- 이노시를의 농도이다. 패널 B는 상층액 중의 D-카이로 -이노시를의 농도이다. 패널 C는 마이오—이노시를의 침전율이다. 패널 D는 으카이로 -이노시를의 침전율이다. ■: 10%, O: 20%, ▲： 30%, V： 40%, 50%.  FIG. 10 is prepared from an aqueous solution containing 70% of a dry powder of a supernatant obtained by adding primary ethanol (combined powders of myo-inos and D-chiropyinos) to remove the precipitate and to remove the supernatant. After re-dissolving, the Inosi, which has a weight ratio of D-Cairo-InoShitl added to 70% or more, is redissolved in a dry mixture with an aqueous solution of 10% to 50%, and then various amounts of secondary alcohol are added to each ino This is the result of measuring precipitation of poems. Panel A is the concentration of myo-inosi in the supernatant. Panel B is the concentration of D-Cyro-inosyl in the supernatant. Panel C is the precipitation rate of myo-inos. Panel D is the precipitation rate of Ecuiro-Inosi. ■: 10%, O: 20%, ▲: 30%, V: 40%, 50%.

도 11 은 1 차 에탄올을 첨가하여 얻은 상층액의 건조 분말 (마이오- 이노시를과 D-카이로—이노시를의 흔합 분말)을 약 12% 함유하는 수용액에 다양한 양의 에탄올을 첨가한 경우의 침전 및 정제 특.성을 측정한 결과이다. 패널 A 는 상층액 중의 마이오-이노시를 및 D-카이로 -이노시를의 농도를 측정한 결과이다. 패널 B 는 마이오-이노시를 및 D-카이로 -이노시를의 침전율을 측정한 결과이다. 패널 C 는 상층액내의 D-카이로ᅳ이노시를의 순도를 나타낸 결과이다. 빼: 마이오 -이노시를, O: D—카이로 -이노시를. 도 12 는 마이오-이노시를 및 D-카이로-이노시를이 익 ^ 13:2 의:¾ 중량비율로 용해된 M9 최소배지에 다양한 유기용매를 첨가한 경우에 마이오-이노시를 및 D-카이로 -이노시를의 침전 특성을 측정한 결과이다. 패널 A 는 에탄올, 이소프로판을 및 아세톤을 첨가한 경우의 침전특성이다. 여기에서 흰색 막대는 마이오 -이노시를의 농도를， 진회색 막대는 D-카이로- 이노시를의 농도를 나타낸다. 패널 B 는 에탄을에 대한 이소프로판을 및 아세톤의 상대적인 침전 특성을 보여준다. 여기에서 연회색 막대는 D- 카이로-이노시를이고， 진회색 막대는 마이오 -이노시를을 나타낸다. 【발명의 실시를 위한 형태】 11 is an aqueous solution containing about 12% of dry powder (combined powders of myo-inos and D-chiro-inos) of the supernatant obtained by adding primary ethanol. This is the result of measurement of precipitation and purification characteristics when various amounts of ethanol were added. Panel A is a result of measuring the concentration of myo-inosine and D-chiro-inosyl in the supernatant. Panel B is the result of measuring the precipitation rates of myo-inosine and D-chiro-inosino. Panel C is a result showing the purity of D-chirozyno inos in the supernatant. Sub: Maio-Inoshi, O: D-Cairo-Inoshi. 12 shows myo-inosine and myo-inosine when various organic solvents were added to the M9 minimum medium dissolved at a ratio of : ¾ weight ratio of myo -inosino and D-chiro-inosino. It is the result of measuring precipitation characteristics of D-Cyro-Inosi. Panel A shows precipitation characteristics when ethanol, isopropane and acetone are added. Where the white bar represents the concentration of myo-inosyl and the dark gray bar represents the concentration of D-chiro-inosyl. Panel B shows the relative precipitation characteristics of isopropane and acetone for ethane. Here the light gray bar represents D-Cairo-Inos, and the dark gray bar represents Myo-Inos. [Form for implementation of invention]

■ 이하， 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로， 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다. 실시예 실시예 1: 마이오 -이노시를과 D-카이로 -이노시를의 용해도 측정 마이오-이노시를 (myo-inositol， Ml)과 D-카이로-이노시를 (D-chiro- inositol, DCI)의 용해도를 측정하였다. 먼저 마이오 -이노시를과 D- 카이로 -이노시를올 상온 (약 25°C)에서 1차 증류수에 각각 59 (w/v) 내지 80%(w/v)의 농도가 되도록 용해시켰다. 각 용액에서 용해되지 않고 침전된 부분을 원심분리를 통하여 분리하고 이노시를이 완전히 용해된 상층액 부분을 취하여 각각의 농도를 HPLC로 분석하였다. 분석조건은 HPLC(Shimadzu LClOAvp)를 이용하여 , Kromasil 5NH2 칼럼 (4.6 麵 X 250瞧), 이동상 75% 아세토니트릴， 칼럼 온도 40°C, RI 검출기를 이용하였다. 그 결과는 도 3에 나타내었다. 마이오-이노시를 (Ml)의 용해도는 25°C에서 약 15%(w/v)로 나타났으며, D—카이로—이노시를 (DCI)의 경우 약 55%(w/v) 전후로 나타나， 마이오 -이노시를과 D-카이로 -이노시를의 용해도 차이가 매우 큰 것으로 확인하였다. 실시예 2： D-카이로-이노시를의 고순도 정제를 위한 1차 에탄올 처리 마이오 -이노시를과 D-카이로 -이노시틀의 흔합액은 마이오 -이노시를을 기질로하여 D-카이로 -이노시를을 생산하는 다양한 방법을 통해 얻어진다. 예를 들어， 마이오-이노시를 트랜스포터 (myo-inositol transporter), 이노시를 디하이드로게나아제 (inositol dehydrogenase) 및 이노소스 이소머라아제 (inosose isomerase)를 발현하도록 형질전환된 재조합 세포를 기질인 마이오 -이노시를을 포함하는 배지에서 배양하는 경우 배양한 배지에는 반웅하고 남은 마이오 -이노시를과 반웅 생성물인 D—카이로- 이노시를이 흔합된 이노시를 흔합용액이 만들어진다. 또한， 상기 효소를 발현하도록 형질전환된 재조합 세포의 휴면세포를 마이오 -이노시를을 포함하는 최소배지에서 접종하여 마이오 -이노시를을 으카아로 -이노시를로 전환하는 방법의 경우에도 배양한 최종 배양 산물에 반웅하고 남은 마이오- 이노시를과 반웅 생성물인 D-카이로 -이노시틀의 이노시를 흔합물이 만들어진다. 또한， 마이오-이노시를 트랜스포터， 이노시를 디하이드로게나아제 및 이노소스 이소머라아제의 효소를 사용하여 기질인 마이오-이노시를로부터 D-카이로 -이노시를을 생성시키는 효소 반응방법의 반웅을 완료한 최종 반웅산물에 반웅하고 남은 마이오—이노시를과 D- 카이로 -이노시를이 흔합된 흔합 용액이 만들어진다. 상기 D—카이로- 이노시를의 제조방법에서 반웅 생성물내의 생성물인 으카이로 -이노시를의 최종 평형 농도는 약 14%이므로， 마이오—이노시를과 D-카이로—이노시를의 최종 반응 흔합물에서 마이오 -이노시를과 D-카이로 -이노시를은 약 86 :14의 비율로 존재한다. 마이오-이노시를로부터 으카이로 -이노시를을 생성하는 반응전의 반웅액 내에서 기질인 마이오 -이노시를은 물에 대한 용해도를 고려할 때 약 150 g/L의 농도로 포함되며， 반응 완료후 반응 생성물에는 129g/L의 마이오 -이노시를과 21g/L의 D-카이로 -이노시를이 포함되어 있다. 이노시를의 흔합액은 재조합 세포를 사용한 발효방법인 경우 발효가 완료된 완전배지, 예를 들어 TB (terrific broth)일 수 있으며， 휴면세포를 이용한 전환법인 경우 M9 최소배지 또는 식염수일 수 있으며 , 효소반웅법인 경우 효소반응의 완충액이 될 수 있다. 이와 같은 다양한 반웅액의 조성에서 에탄올을 첨가하여 마이오-이노시를 (Ml)과 D-카이로-이노시를 (DCI)의 분리를 시험하였다. 발효방법의 경우를 실험하기 위해서 0.5%(w/v) 락토오스， 12.9%(w/v) 마이오 -이노시를, 2.1%(w/v) D-카이로ᅳ이노시를을 첨가한 TB 배지 [terrific broth, 1.2% (w/v) 트립톤， 2.4% 효모추출물, 0.4% 글리세롤, 0.Γ7Μ K2HP04, 0.72M H2P04]를 사용하였다. 휴면세포 전환법의 경우를 실험하기 위해서 0.5% (w/v) 락토오스， 12.9% (w/v) 마이오 -이노시를, 2.1% (w/v) D-카이로 -이노시를을 첨가한 M9 최소배지 [6.4%(w/v) Na2HP04.7H20, 1.5% (w/v) KH2P04 , 0.25% (w/v) NaCl , 0.5% (w/v) NH4C1 , 2 mM MgS04, 0.1 mM CaCl2]와， 0.5% (w/v) 락토오스， 12.9% (w/v) 마이오 -이노시를, 2.1% (w/v) D-카이로—이노시를을 첨가한 식염수 (0.85% NaCl)를 사용하였다. 효소반웅법의 경우를 실험하기 위해서 50 mM Tris-HCl (pH8.0), 1 mM MgS04, 0.1 mM MnS04, 0.5 mM NAD의 조성의 반웅 완충액에 12.9% (w/v) 마이오 -이노시를, 2.1% (w/v) D-카이로- 이노시를을 첨가하여 사용하였다. 대조구로서는 12.9%(w/v) 마이오- 이노시를， 2.1%(w/v) D-카이로 -이노시를이 포함된 증류수를 사용하였다. 상기 제조한 이노시를 흔합액에 에탄올을 가하여， 이노시를 흔합액：에탄올의 비율이 각각 10:0, 9:1(0.11배)， 8:2(0.25배), 7:3(0.43배)， 6:4(0.67배)， 5:5(1배), 4:6(1.5배)， 3:7(2.33배)， 2:8(4배)， 1:9(9배)로 되도록 하여 완전히 흔합한 후 실온 (약 25°C)에서 1시간 동안 진탕하였다. 이를 3，500 rpm 에서 30분간 원심분리한 후 상층액을 취하여 HPLC로 마이오 -이노시를과 으카이로 -이노시를의 농도를 분석하였다. HPLC분석 방법은 실시예 1에서 설명된 방법과 같다. 분석한 결과는 도 4， 도 5 및 도 6에 나타내었다. ■ Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. . EXAMPLES Example 1 Determination of Solubility of Myo-inosyl and D-Cyro-inosyl Myo-inositol (Mo) and D-chiro-inositol (D-chiro-inositol) , Solubility of DCI) was measured. First, myo-inosino and D-chiro-inosiol were dissolved in primary distilled water at room temperature (about 25 ° C.) to a concentration of 59 (w / v) to 80% (w / v), respectively. The undissolved portions in each solution were separated by centrifugation and each concentration was analyzed by HPLC, taking the supernatant portion in which the inos were completely dissolved. Analytical conditions were performed using HPLC (Shimadzu LClOAvp), Kromasil 5NH2 column (4.6 x 250 cc), mobile phase 75% acetonitrile, column temperature 40 ° C, RI detector. That The results are shown in FIG. The solubility of myo-inosyl (Ml) was found to be about 15% (w / v) at 25 ° C, and D-chiro-inosyl was about 55% (w / v) for (DCI). It was confirmed that the difference in solubility between myo-inos and D-chiro-inosio was very large. Example 2 Primary Ethanol Treatment for High Purity Purification of D-Cyro-Inosyl A mixture of Myo-Inosyl and D-Cairo-Inosyl was prepared using D-Cyro-Inosio as a substrate. It is obtained through various methods of producing Cairo-Inosi. For example, substrates of recombinant cells transformed to express myo-inositol transporter, inosi dehydrogenase and inosose isomerase When culturing in a medium containing phosphorus myo-inosity, a mixed solution is prepared in which the ino-sidium mixed with the remaining myo-inosine and the reaction product D—Cairo-inosino are mixed. In addition, in the case of the method of inoculating the dormant cells of the recombinant cells transformed to express the enzyme in a minimal medium containing myo-inosi to convert myo-inosi to ekaa-inosi A mixture is formed between the remaining myo-inos and the inos of D-Cairo-inositite, the reaction product of the final culture product. In addition, an enzyme reaction for generating D-chiro-inosine from myo-inosity, which is a substrate, by using a myo-inosity transporter, an inosine dehydrogenase, and an enzyme of inososomerase After the reaction of the method was completed, the resultant reaction product was produced by mixing the remaining Maio-Inos and D-Cairo-Inos. Since the final equilibrium concentration of the product in the reaction product of the Cairo-Inosyl D-Cairo-Inosyl is about 14%, the final reaction of the Myo-Inosyl and D-Cairo-Inosyl In the mixture, myo-inos and D-chiro-inos are present at a ratio of about 86:14. Myo-inosyl substrate, which is a substrate in the reaction solution to generate E. coli-Inosio from myo-inosyl, is included at a concentration of about 150 g / L, considering the solubility in water. Upon completion the reaction product contains 129 g / L of myo-inosyl and 21 g / L of D-chiro-inosyl. The mixture of Inosul is a fermentation method using a recombinant cell. It may be complete medium, for example TB (terrific broth), in the case of dormant cell conversion method may be M9 minimal medium or saline, in the case of enzyme reaction method may be a buffer of the enzyme reaction. The separation of myo-inosyl (Ml) and D-chiro-inosy (DCI) was tested by adding ethanol in the composition of these various reaction solutions. TB medium containing 0.5% (w / v) lactose, 12.9% (w / v) myo-inosity, and 2.1% (w / v) D-chirobinosinos for the fermentation method. [terrific broth, 1.2% (w / v) tryptone, 2.4% yeast extract, 0.4% glycerol, 0.Γ 7Μ K 2 HP0 4 , 0.72MH 2 P0 4 ] was used. To test the case of dormant cell conversion, 0.5% (w / v) lactose, 12.9% (w / v) myo-inosity and 2.1% (w / v) D-chiro-inosity were added. M9 minimal medium [6.4% (w / v) Na 2 HP0 4 .7H 2 0, 1.5% (w / v) KH 2 P0 4, 0.25% (w / v) NaCl, 0.5% (w / v) NH 4 C1, 2 mM MgS0 4 , 0.1 mM CaCl 2 ], 0.5% (w / v) lactose, 12.9% (w / v) myo-inos, 2.1% (w / v) D-Cairo-inos Saline (0.85% NaCl) to which was added was used. To test the case of enzyme reaction, 12.9% (w / v) myo-ino in reaction mixture of 50 mM Tris-HCl (pH8.0), 1 mM MgSO 4 , 0.1 mM MnSO 4 , 0.5 mM NAD The city was used with the addition of 2.1% (w / v) D-Cyro-inosi. Distilled water containing 12.9% (w / v) myo-inosi and 2.1% (w / v) D-chiro-inosi was used as a control. Ethanol was added to the prepared inosin mixture, and the inosin mixture was 10: 0, 9: 1 (0.11 times), 8: 2 (0.25 times), 7: 3 (0.43 times), respectively. ), 6: 4 (0.67x), 5: 5 (1x), 4: 6 (1.5x), 3: 7 (2.33x), 2: 8 (4x), 1: 9 (9x) After complete mixing it was shaken for 1 hour at room temperature (about 25 ° C). After centrifugation at 3,500 rpm for 30 minutes, the supernatant was collected, and the concentrations of myo-inosyl and eucairo-inosyl were analyzed by HPLC. HPLC analysis method is the same as described in Example 1. The analysis results are shown in FIGS. 4, 5, and 6.

도 4는 각 이노시를 흔합액에 에탄을 첨가한 후 생성된 침전물을 제거하여 얻은 상층액내에 남아있는 마이오 -이노시를과 D-카이로ᅳ 이노시틀의 농도를 나타낸 것이다. 마이오 -이노시를의 경우， 모든 배지 조건에서 에탄올 첨가량에 따라 농도가 빠르게 감소하여， 약 1 배수의 에탄을을 첨가한 경우 마이오 -이노시를은 초기 126 g/L 농도로부터 약 99 g/L이 침전되어 약 27 g/L의 마이오 -이노시를이 남아 있는 것으로 나타났다. 이에 비해, D-카이로 -이노시를의 감소량은 매우 낮아서 약 1 배수의 에탄을을 첨가하면 초기 17 g/L의 농도에서 15 g/L으로 감소되어 약 2 g/L만이 침전되는 것으로 나타났다. FIG. 4 shows the concentrations of myo-inosyl and D-chirojan inositol remaining in the supernatant obtained by adding ethane to the respective mixtures of ethane. In the case of myo-inosyl, the concentration decreased rapidly according to the amount of ethanol added under all medium conditions, and the myo-inosine was approximately 99 from the initial 126 g / L concentration when about 1 multiple of ethane was added. g / L precipitated, leaving about 27 g / L of myo-inosyl. In comparison, the decrease in D-Cyro-Inosi was very low, and the addition of about 1 multiple of ethane reduced to 15 g / L at the initial concentration of 17 g / L, leaving only about 2 g / L of precipitation.

상기 도 4에 나타난 결과를 침전율로 환산하여 도 5에 나타내었다. 마이오 -이노시를의 침전율은 에탄올 첨가량에 따라 급격히 증가하였으며 , 1 배수의 에탄올을 첨가하였을 때 모든 반응액 조건에서 비교적 유사하게 70- 80%의 침전율올 보였고， 약 2.5 배 이상의 에탄을 첨가 조건에서는 대부분의 마이오 -이노시를이 침전되는 것을 확인하였다. 이에 반하여 D- 카이로 -이노시를의 침전율은 배지조건에 따라서 약간 다르게 나타났다. 약 1 배의 에탄을을 첨가할 때까지는 비교적 유사하게 전체적으로 약 15% 이하의 침전율을 보였으나， 1.5 배 이상의 에탄올을 첨가한 경우 물， 식염수， 효소반웅 완층액의 경우보다 M9 배지와 TB 배지에서 침전율이 급격하게 증가하는 양상을 보였다. 이러한 양상은 각 용액에 포함된 염 (salt)의 양이 다르기 때문인 것으로 판단되는데 , 물， 식염수， 효소반웅 완충액에 함유되어있는 염의 양에 비해 M9 배지와 TB 배지에 함유된 염의 양이 더 많기 때문인 것으로 보인다.  The result shown in FIG. 4 is shown in FIG. 5 in terms of precipitation rate. The precipitation rate of myo-inosi increased rapidly with the amount of ethanol, and when 1-fold ethanol was added, the precipitation rate of 70-80% was relatively similar in all reaction conditions. In most of the myo-inosity was confirmed to precipitate. In contrast, the precipitation rate of D-Cairo-inosyl was slightly different depending on the medium conditions. Until the addition of about 1-fold ethane, the sedimentation rate was about 15% or less in total.However, when 1.5-fold or more ethanol was added, it was found in M9 and TB media more than water, saline, and enzyme reaction complete solution. The precipitation rate increased rapidly. This may be due to the different amounts of salt in each solution, since the amount of salt in M9 and TB medium is higher than in water, saline and enzyme reaction buffer. Seems to be.

상기 도 4 및 도 5에 나타난 실험 결과를 토대로 D-카이로- 이노시를의 순도를 계산하여 도 6에 나타내었다. D-카이로 -이노시를의 순도는 1 배수의 에탄올을 첨가하였을 경우 모든 종류의 배양액에서 35- 40% 정도로 나타났으며， 그 이상의 양의 에탄을을 첨가하면 순도가 증가하지만， D-카이로 -이노시틀의 손실율이 커지게 된다. 이상의 결과를 종합하면 약 1 배수의 에탄올을 첨가할 경우 모든 종류의 배양액 조건에서 약 80%의 마이오 -이노시톨이 침전되고， 이때 D-카이로 -이노시를의 침전율， 즉 손실율은 15% 이하로 나타났으몌 초기 14% 정도이었던 D-카이로- 이노시를의 순도를 약 3으40%로 증가시킬 수 있었다. 에탄을 첨가량이 1 배수를 초과할 경우 D-카이로 -이노시를의 침전율 (손실율)이 급격히 증가므로， 약 1 배수의 에탄올을 첨가하는 경우 D—카이로 -이노시를의 손실율을 최소로 하면서 마이오 -이노시를을 침전시켜 분리 및 제거할 수 있을 것으로 기대된다. 상기한 실험에서 이노시를이 흔합된 배양액에 대하여 약 1 배수의 에탄올을 첨가하여 침전시켰을 경우 마이오 -이노시를과 D-카이로 -이노시틀의 비율은 약 63:37 정도인 것으로 나타났다. 실시예 3: 1차 에탄을 처리 후 상층액의 농축 건조 및 재용해 상기 실시예 2에서 설명된 방법과 같이， 이노시틀 흔합액에 동량 (1:1)의 에탄올을 흔합한 후 침전물을 제거하여 얻은 상층액을 8(rc로 가열하고' 진공 상태에서 농축 및 건조하였다. 건조후 얻은 이노시를의 건조 분말 (마이오ᅳ이노시를과 D-카아로 -이노시를의 흔합 분말)을 15% - 70%의 다양한 농도로 증류수에 용해시켜 각 농도에서의 마이오 -이노시를과 D-카이로 -이노시를의 용해 특성을 관찰하였다. HPLC를 아용한 이노시를의 분석 방법은 실시예 1에서 설명된 내용과 동일하다. 마이오 -이노시를과 D- 카이로 -이노시를의 용해특성을 분석한 결과는 도 7에 나타내었다. Based on the experimental results shown in FIGS. 4 and 5, the purity of D-Cairo-Inosyl is calculated and shown in FIG. 6. The purity of D-Cyro-Inosyl was found to be about 35-40% in all cultures when 1-fold ethanol was added. The loss ratio of the ino-seattle will increase. Taken together, about 1% of ethanol is added and about 80% of myo-inositol is precipitated under all kinds of culture conditions. It was found that the initial purity of D-Cairo-Inoshil, which was about 14%, could be increased to about 3-40%. If the amount of ethane added exceeds 1, the precipitation rate (loss rate) of D-Cyro-Inosyl is rapidly increased, and when about 1 multiple of ethanol is added, the loss of D-Cyro-Inosyl is minimized. It is expected that OH-Inosi can be separated and removed by precipitation. In the above experiments, when Inosi was precipitated by adding about 1 multiple of ethanol to the mixed culture solution, The ratio of D-Cairo-inositole was found to be about 63:37. Example 3: Concentration drying and re-dissolution of the supernatant after treatment with primary ethane, the same amount (1: 1) of ethanol was mixed in the inositol mixture as described in Example 2, and then the precipitate was removed. the supernatant obtained was heated at 8 (rc, concentrated and dried in a "vacuum-dried powder of the Inno when obtained after drying (Maio the eu Ino when a D- and Carr-heunhap the powder of the Inno-shi) The dissolution characteristics of myo-inosyl and D-chiro-inosyl were observed at each concentration by dissolving in distilled water at various concentrations of 15% to 70%. Same as described in Example 1. The results of analyzing the dissolution characteristics of myo-inosyl and D-chiro-inosyl are shown in FIG.

도 7의 패널 A는 각 농도별 용액의 상층액 농도， 즉 용해도를 나타낸 것으로 마이오 -이노시를의 용해도의 경우 상기 실시예 2에서 나타난 바와 같이 약 15% 이상으로는 더 증가하지 않는 것을 확인할 수 있었다. 한편， D-카이로 -이노시를의 경우 용해도가 약 55%이므로 건조분말을 70% 농도로 용해시킨 경우에서도 모든 D-카이로—이노시를이 용해된 것을 확인할 수 있었다. 마이오 -이노시를과 D-카이로 -이노시를의 용해특성을 용해도와 침전율로 표시한 결과는 각각 도 7의 패널 B와 패널 C에 나타내었다. 마이오 -이노시를은 약 20% 용액부터 용해도가 감소하기 시작하여 용액의 농도가 증가할수록 계속적으로 감소하였고， 침전율은 용액의 농도가 증가함에 따라 증가하였다ᅳ D-카이로 -이노시를의 경우 용해도는 용액의 농도가 70%인 경우에 이르기까지 100%을 유지하였으며, 이에 따라 침전율도 0%로서 전혀 침전되지 않는 것으로 나타났다. 결론적으로 최대 70% 용액올 제조하는 경우 마이오 -이노시를은 약 13% 만이 용해되었고, D-카이로 -이노시를은 모두 용해되었다. 이때 D—카이로 -이노시를의 경우 순도는 약 73% 정도인 것으로 측정되었다 (도 7의 패널 D). 실시예 4: 2차 에탄올 처리에 따른 정제 특성  Panel A of FIG. 7 shows the supernatant concentration, that is, the solubility of the solution at each concentration, and it was confirmed that the solubility of the myo-inosi did not increase more than about 15% as shown in Example 2 above. Could. On the other hand, in the case of D-Cyro-Inosyl, solubility is about 55%, even when the dry powder was dissolved at a concentration of 70%, all of the D-Cyro-Inosy was dissolved. The solubility and precipitation rate of the myo-inosino and D-chiro-inosino are shown in panel B and panel C of FIG. The solubility of myo-inosine began to decrease from about 20% of the solution, and then decreased as the concentration of the solution increased, and the precipitation rate increased with increasing concentration of the solution. The solubility was maintained at 100% until the concentration of the solution was 70%, so that the precipitation rate was 0%, indicating no precipitation at all. In conclusion, only about 13% of myo-inosity was dissolved and all of D-Cyro-inosity was dissolved when up to 70% solution was prepared. In this case, the purity of the D—Cairo-Inoshiro was measured to be about 73% (Panel D of FIG. 7). Example 4: Purification Characteristics by Secondary Ethanol Treatment

실시예 3에서 제조한 각 농도별 흔합용액에서 마이오 -이노시를에 대한 포화용액, 즉, 마이오 -이노시를이 충분히 침전되기 시작하는 농도인 35% 이상이 되는 농도의 각 용액에 에탄올을 첨가량을 달리하여 첨가하였다. 에탄을 첨가 후 침전물은 원심분리를 통하여 제거하고 상층액에 존재하는 이노시를을 HPLC를 이용하여 분석하였다. HPLC를 이용한 분석 방법은 실시예 1의 방법과 동일하였다. 이노시를 농도를 분석한 결과는 도 8에 나타내었다. Ethanol was added to each solution having a concentration of 35% or more, which was a concentration at which the saturated solution for myo-inosine, that is, the myo-inosine, began to sufficiently precipitate in the mixed solution for each concentration prepared in Example 3. By varying the amount Added. After addition of ethane, the precipitate was removed by centrifugation and the inos present in the supernatant were analyzed by HPLC. The analysis method using HPLC was the same as that of Example 1. The results of analyzing the concentration of inos are shown in FIG. 8.

도 8의 패널 A는 상층액 중의 마이오-이노시를 농도를 나타낸 것이며， 마이오 -이노시를의 용해도가 15%를 넘지 않으므로 에탄올을 첨가함에 따라 마이오 -이노시를이 침전되는 양상을 보여주고 있다. 에탄을을 첨가함에 따라 마이오 -이노시를의 농도가 급격히 감소하며， 2 배수의 에탄을 첨가시 많은 양의 마이오 -이노시를이 제거된 것을 확인할 수 있었다. 65% 및 70% 농도 용액의 경우 1 배수의 에탄을을 첨가한 경우 마이오ᅳ이노시를이 제거되기 시작하였고， 이보다 낮은 농도의 용액의 경우에도 1.5 배의 에탄올을 첨가함에 따라 마이오 -이노시를이 제거되기 시작함을 확인하였다. 도 8의 패널 B은 D-카이로ᅳ이노시를의 상층액내의 농도를 나타낸 것이며， D-카이로—이노시를의 경우에도 적은 양의 에탄을이 첨가되면서 급격히 침전되는 것으로 나타났다. 마이오ᅳ이노시를에서와 마찬가지로 65% 및 70% 농도 용액의 경우 1 배수의 에탄을부터 D-카이로 -이노시를이 침전되기 시작하였고, 1.5 배수의 에탄을에서 대부분의 침전이 일어났다. 용액의 농도가 낮을 경우， 즉 35% 또는 40%에서는 침전의 정도가 미약하였으나， 약 2 배의 에탄올을 첨가하면서부터는 고농도의 용액에서와 비슷한 수준으로 침전이 일어남을 확인하였다.  Panel A of FIG. 8 shows the concentration of myo-inosity in the supernatant, and the solubility of myo-inosine does not exceed 15%. Is showing. As the ethane was added, the concentration of myo-inosine decreased drastically, and it was confirmed that a large amount of myo-inosine was removed when 2 ethane was added. In the case of 65% and 70% solutions, the addition of 1-fold of ethane started to remove myoininosi. It was confirmed that the poem began to be removed. Panel B of FIG. 8 shows the concentration in the supernatant of D-chiro-inosino, and even in the case of D-chiro-inosino, a small amount of ethane was added to precipitate rapidly. As with myoininosity, D-Cyro-Inosi began to precipitate from 1-fold ethane for 65% and 70% -concentration solutions, and most precipitation occurred from 1.5-fold ethane. When the concentration of the solution was low, that is, the degree of precipitation was weak at 35% or 40%, but it was confirmed that precipitation occurred at a level similar to that of the high concentration solution from the addition of about 2 times of ethanol.

상기 실험결과로부터 마이오 -이노시를과 D-카이로—이노시를이 함께 고농도로 존재하는 경우에는 에탄올을 첨가하여 마이오-이노시를로부터 D- 카이로 -이노시를을 분리하는 것이 용이하지 않다는 것을 알 수 있었다.  From the above experimental results, it is not easy to separate D-chiro-inosine from myo-inosino by adding ethanol when the concentration of myo-inosine and D-chiro-inosino is present together. I could not see.

도 9는 도 8에 나타낸 이노시를의 농도를 침전율로 환산하여 나타낸 것이다. 마이오 -이노시를과 D—카이로-이노시를 모두 에탄올이 첨가됨에 따라 급격히 침전율이 증가하는 것으로 나타났으며， 침전율에 대하여 마이오 -이노시를과 D-카이로-이노시를에서 침전율의 증가양상은 유사하게 나타났다. 도 9로 나타낸 결과에 의해서도 1차 에탄을을 처리후 얻은 침전물을 제거하고 건조한 마이오ᅳ이노시를과 D-카이로 -이노시를의 흔합 건조물을 35% 이상의 고농도로 재용해시킨 후 에탄을을 첨가하는 방법을 통해서는 마이오 -이노시를과 D-카이로 -이노시를을 분리하는 것이 용이하지 않은 것으로 확인되었다. 실시예 5: D-카이로-이노시를 비율이 높아진 다양한 농도의 용액에 대한 2차 에탄을 처리에 따른 정제 특성 FIG. 9 shows the concentration of inosine shown in FIG. 8 in terms of precipitation rate. The precipitation rate increased rapidly with the addition of ethanol to the myo-inosino and D-chiro-inosino, and the precipitation rate of the myo-inosino and D-chiro-inosino The increase was similar. According to the results shown in FIG. 9, the precipitate obtained after treating the primary ethane was removed, and the mixed dry matters of the dried myopia-inos and D-chiro-inosies were re-dissolved at a high concentration of 35% or more, and then the ethane was removed. It is not easy to separate myo-inos and d-chiro-inos through the method of addition Was confirmed. Example 5 Purification Characteristics of D-Cyro-Inosine Treated with Secondary Ethane for Various Concentrated Solutions

실시예 3에서 1차 에탄올 처리 후 침전물을 제거하여 얻은 상층액을 건조한 이노시를 분말을 증류수에 용해시켜 약 70%농도의 아노시를 용액을 만들었을 경우 마이오ᅳ이노시를의 용해도의 한계 때문에 D-카이로- 이노시를의 순도가 약 73%까지 상승한다는 것을 확인하였다 (도 7의 패널 D 참조)ᅳ 이어서, 1차 에탄올 처리 후 얻은 건조한 이노시를 분말을 약 70% 농도로 용해시킨 용액의 상층액을 다시 건조하여, D-카이로ᅳ이노시를의 비율 (순도)이 70% 이상으로 높아진 이노시를의 건조 흔합물을 얻고， 이를 10% 내지 50% 농도로 증류수에 다시 용해하였다. 이 수용액에 2차 에탄을을 다양한 첨가량으로 첨가하여 정제 특성을 확인하였다. 이 실험은 D-카이로 -이노시를의 순도가 높을 경우 에탄올 처리에 의하여 침전되는 양상을 확인하기 위한 것이다. 실험결과는 도 10에 나타내었다. 도 10의 패널 A와 패널 B에는 D-카이로 -이노시를의 비율이 70% 이상으로 높을 경우 에탄올의 첨가량을 0.5배 - 4배 까지 첨가한 경우에서 상층액의 마이오- 이노시를 농도와 으카이로ᅳ이노시롤의 농도를 측정한 결과가 나타나 있다. 이노시를 흔합물의 농도가 높을수록 적은 양의 에탄올에서 침전이 이루어졌으며, 에탄올 첨가량을 달리하면서 나타난 침전양상은 마이오- 이노시를과 D-카이로-이노시롤에서 차이가 없었다. 각 이노시를 화합물에 대한 침전율을 환산하여 도 10의 패널 C와 패널 D에 나타내었다. 약 3배 이상의 에탄올 첨가량에서 마이오ᅳ이노시를이 J 약간 침전되는 양상을 나타냈으나， 그 비율이 20% 미만으로서 실제 D—카이로 -이노시를의 순도에 미치는 영향은 미미한 것으로 나타났다. 상기 실험결과에 의해 D-카이로— 이노시를의 농도가 높은 흔합물 , 또는 이노시를 흔합물의 농도가 높은 용액에서 에탄올 첨가에 의한 용해도 차이로 D—카이로ᅳ이노시롤과 마이오- 이노시를을 분리하는 것은 용이하지 않은 것으로 나타났다. 실시예 6: 저농도의 마이오 -이노시를과 D-카이로 -이노시를의 혼합 건조물 용액에 2차 에탄올을 처리한 경우의 정제 특성 마이오ᅳ이노시를 및 으카이로—이노시를의 흔합 건조물을 저농도인The limit of solubility of myosininosine in Example 3 when the supernatant obtained by removing the precipitate after the first ethanol treatment was dissolved in distilled water by inosin powder, and the solution of the inosin of about 70% concentration. Therefore, it was confirmed that the purity of D-Cyro-inosity was increased to about 73% (see panel D of FIG. 7). Next, the dry inos obtained after primary ethanol treatment was dissolved in the powder at about 70% concentration. The supernatant of the solution was dried again to obtain a dry mixture of inos with an increase in the ratio (purity) of D-chiropy Inosine to 70% or more, which was dissolved again in distilled water at a concentration of 10% to 50%. . Secondary ethane was added to this aqueous solution in various amounts to confirm the purification characteristics. This experiment is to confirm the precipitation of ethanol treatment when the purity of D-Cyro-Inosyl is high. The experimental results are shown in FIG. 10. Panels A and B of FIG. 10 show the concentration of myo-inosine in the supernatant when the amount of D-Cyro-inosine is higher than 70% and the amount of ethanol added is 0.5--4 times. The result of having measured the concentration of acro uroinosinolol is shown. The higher the concentration of the inosi, the more precipitated the ethanol was, and the difference in the amount of ethanol added was not different between myo-inosyl and D-chiro-inosirole. Each inos is shown in Panel C and Panel D of FIG. 10 in terms of precipitation rates for the compounds. But it did receive the Maio aspect J is a little precipitate during eu Ino at least about 3-fold amount of ethanol, as the ratio is less than 20% of the actual D- Cairo-effect on the purity of the Inno when appeared to be insignificant. According to the results of the experiment, D-chiro-inosinol and myo-inosino were mixed with a high solubility of D-Cyro-Inosine or a solution of high concentration of Inosio-C. It was found that it was not easy to separate them. Example 6: Purification characteristics when secondary ethanol was treated in a mixed dry solution of low concentration of myo-inosino and D-chiro-inosino Maio Inino City and Ekairo—Ino City's mixed building with low concentration

15% 이하 농도의 용액으로 제조하여 여기에 에탄올을 첨가하는 실험을 수행하였다. 1차 에탄을 처리 후 상층액의 건조분말이 완전히 녹는 양의 물을 첨가하여 용해시켰으며， 이때 총 이노시를 함량으로 약 11.5%가 되었고, 각각의 농도는 마이오-이노시를 75 g/L 및 D-카이로-이노시를 40 g/L 이었다. 이 흔합용액에 에탄올을 최대 19배 까지 첨가하면서 침전 및 분리 양상을 확인하였다. 그 결과는 도 11에 나타내었다. 도 11의 패널 A는 에탄올 첨가 후 상층액의 농도를 나타낸 것이며， 마이오 -이노시를의 경우 약 1 배수의 에탄올올 첨가할 때까지 큰 변화가 없었으나， 1.5 배의 에탄을이 첨가되면 절반 이상의 마이오 -이노시를이 침전되는 것으로 나타났다. 이에 반하여 D-카이로 -이노시를은 1.5 배수의 에탄올에서 약간 침전되는 것으로 나타났으며， 이후에도 침전량이 크게 증가되지는 않았다. 도 11의 패널 B는 침전율을 나타낸 것이며， 마이오 -이노시를의 경우 약 9배의 에탄올을 첨가할 경우 90% 이상 침전되었으며， 이때 D-카이로- 이노시를의 침전율은 약 20%인 것으로 나타났다. 따라서 약 9 배 양의 에탄을을 첨가하면 대부분의 마이오ᅳ이노시를을 ' 제거하면서 D-카이로- 이노시를 손실율을 20% 이하로 줄일 수 있고， D-카이로ᅳ이노시틀의 순도를 90% 이상으로 정제 가능하였다 (도 11의 패널 C 참조). 실시예 7: 에탄올 이외의 유기용매에 의한 마이오ᅳ이노시를과 D- 카이로 -이노시를의 선택적 침전 An experiment was conducted in which a solution of 15% concentration or less was added to ethanol. After treatment with primary ethane, the dry powder of supernatant was dissolved in the amount of completely dissolved water. At this time, the total amount of inos was about 11.5%, and each concentration was 75 g / ml of myo-inos. L and D-Cairo-inosity was 40 g / L. Precipitation and separation were confirmed by adding up to 19-fold ethanol to the mixed solution. The results are shown in FIG. Panel A of FIG. 11 shows the concentration of the supernatant after the addition of ethanol, and in the case of myo-inosity, there was no significant change until about one-fold ethanol was added, but when 1.5 times of ethane was added, More than myo-inos appeared to precipitate. In contrast, D-Cyro-Inosi was found to precipitate slightly in 1.5-fold ethanol, after which the precipitation did not increase significantly. Panel B of FIG. 11 shows the precipitation rate, and in the case of myo-inosine, when about 9 times of ethanol was added, 90% or more of the precipitates were precipitated, and the precipitation rate of D-chiro-inosine was about 20%. appear. Thus D- Cairo while adding ethanol to approximately 92 times the amount by removing most of the Maio when Ino eu '- it is possible to reduce a loss rate when the Inno below 20%, the purity of the D- Cairo eu Ino during frame Purification was possible above 90% (see panel C in FIG. 11). Example 7 Selective Precipitation of Myoininosi and D-Cyro-Inosi by Organic Solvents Other Than Ethanol

상기 실시예 1 내지 6의 실험결과에서 마이오—이노시를과 D-카이로- 이노시를의 큰 용해도 차이에 의해 에탄올을 이용한 분리가 가능함을 확인하였다. 2개의 탄소를 갖는 에탄올 이외의 다른 유기용매에 의해서도 이러한 분리가 가능한지 확인하기 위해 탄소수 3개의 알코을인 이소프로판올과 케톤인 아세톤을 이용하여 분리를 시도하였다. 마이오ᅳ 이노시틀과 D_카이로 -이노시를의 흔합용액 제조 시， 마이오 -이노시를 및 으 카이로 -이노시를의 비율은 일반적인 반응평형 도달 농도인 약 13 :2로 하였으며ᅳ M9 최소배지 조건으로 전체 농도가 약 15%가 되도록 제조하였다. 제조한 이노시를의 흔합용액에 동일부피의 에탄올, 이소프로판올， 아세톤을 첨가하여 실은에서 1시간 동안 진탕한 후 원심분리하고, 그 상층액을 HPLC로 분석하였다. 결과는 도 12에 나타내었다. 도 12의 패널 A는 실제 상층액에 존재하는 이노시를의 농도를 나타낸 것이다. 마이오 -이노시를의 경우 에탄을과 이소프로판을은 그 농도가 크게 차이나지 않았으나， 아세톤에서는 약간 낮게 나타났다. 으카이로—이노시를의 경우， 이소프로판을에서 에탄을에 비해 농도가 낮았던 반면， 아세톤의 경우에는 유기용매를 처리하지 않았던 농도와 같게 나타나 전혀 침전되지 않은 것으로 나타났다 . 즉， 이소프로판을은 에탄을과 비교할 때 마이오- 이노시를 침전율은 비슷하고， D-카이로 -이노시를의 침전율 (손실율)이 더 높았던 반면， 아세톤은 마이오 -이노시를에 대한 침전율이 더 높고， D- 카이로 -이노시를의 손실율이 적은 것으로 보인다. 이는 에탄을의 침전율에 대한 상대값을 나타낸 도 12의 패널 B에서 쉽게 볼 수 있다. 이소프로판을은 에탄올에 대하여 마이오 -이노시를의 침전율은 동일하고, D- 카이로—이노시를의 침전율은 약 0.7 수준으로 높다. 반면 아세톤의 경우는 마이오 -이노시를의 침전율이 약 0.1배 높으며， D-카이로 -이노시틀에 대해서는 약 1배 수준이 낮은 것으로 나타났다. 따라서 에탄을 이외의 유기용매가 에탄을과 같이 마이오ᅳ이노시롤과 D-카이로 -이노시를의 분리에 사용할 수 있는 것을 확인하였으며, 아세톤의 경우 에탄을에 비해 그 분리 특성이 유리할 수도 있다는 사실을 확인하였다. 본 발명의 실험 결과를 요약하면 다음과 같다. In the experimental results of Examples 1 to 6 it was confirmed that the separation using ethanol due to the large difference in solubility between myo-inosino and D-chiro-inosino. In order to confirm that such separation is possible by an organic solvent other than ethanol having two carbons, separation was attempted using isopropanol having 3 carbon atoms and acetone having ketone. In the preparation of the mixed solution of Myo Ino-Shittle and D_Cairo-Ino City, the ratio of Myo-Ino City and U-Cairo-Ino City was about 13: 2, which is a typical reaction equilibrium concentration. It was prepared such that the total concentration was about 15% under the minimum medium conditions. The same volume of ethanol, isopropanol, and acetone was added to the prepared solution of Inosi, and the mixture was shaken at room temperature for 1 hour, followed by centrifugation. Analyzed by HPLC. The results are shown in FIG. Panel A of FIG. 12 shows the concentration of inosyl in the actual supernatant. The concentrations of ethane and isopropane in myo-inosity were not significantly different, but slightly lower in acetone. In the case of Eucairo-Inosyl, the concentration of isopropane was lower than that of ethane, whereas in the case of acetone, it was the same as the concentration without the organic solvent and did not precipitate at all. That is, isopropane was similar in precipitation rate to myo-inosine and higher in precipitation rate (loss rate) of D-Cairo-inosine, while acetone was higher in precipitation rate for myo-inosine. This is higher, and the loss rate of D-Cairo-Inosi seems to be less. This can be easily seen in panel B of FIG. 12 which shows the relative value for the precipitation rate of ethane. For isopropane, the precipitation rate of myo-inosyl is the same with respect to ethanol, and the precipitation rate of D-chiro-inosyl is as high as about 0.7. On the other hand, in the case of acetone, the precipitation rate of myo-inosyl is about 0.1 times higher, and that of D-Cairo-inositol is about 1 times lower. Therefore, it was confirmed that organic solvents other than ethane can be used to separate myopium inosylol and D-chiro-inosi as ethane, and that acetone may have advantageous separation properties compared to ethane. I confirmed the fact. The experimental results of the present invention are summarized as follows.

( i ) 유기용매 1차 처리 방법에 의하면 D-카이로 -이노시를이 약 30% 내지 40%의 순도를 갖는 이노시를 흔합물을 얻을 수 있다. 재조합 세포 발효 방법, 휴면세포 전환 방법， 및 효소반웅방법을 통해 얻은 최종 반웅액에는 마이오—이노시를과 D_카이로 _이노시틀 흔합물이 약 15%의 농도로 용해되어 있고ᅳ 마이오ᅳ이노시를과 D-카이로-이노시를 비율은 약 13:2으로 용해되어 있다. 여기에 이노시를이 포함된 흔합용액과 동일한 부피의 유기용매를 첨가하면 마이오ᅳ이노시를을 상대적으로 더 많은 양으로 침전이 유도되고, 상충액에는 마이오 -이노시를과 D-카이로ᅳ이노시를이 약 60： 40 비율로 존재하게 된다. 상층액을 건조하면 30% - 40%의 D-카이로- 이노시를 순도를 갖는 이노시를 건조흔합물을 얻을 수 있다. (i) According to the organic solvent primary treatment method, it is possible to obtain a mixture of inosyl having a purity of about 30% to 40% of D-chiro-inosine. Recombinant cell fermentation, dormant cell transformation method, and the final banung solution obtained by the enzymatic banung methods Maio - the Inno when the D _ Cairo _ Ino when the frame common compound is dissolved at a concentration of about 15%, and eu Maio The ratio of kinosinos and D-chiro-inosio is dissolved at about 13: 2. Addition of an organic solvent in the same volume as the mixed solution containing inosi may induce precipitation of myoininosi in a relatively larger amount, and the supernatant contains myo-inosino and D-chiro. Inno City will exist at a ratio of about 60:40. Drying the supernatant gives a dry mixture of Inosi having a purity of 30% -40% D-Cyro-Inosi.

(ii) 유기용매 1차 처리 방법에 의해 얻은 D-카이로-이노시를 30% - 40%의 순도의 이노시를 건조 흔합물을 고농도， 예컨대 약 70%의 농도로 물에 용해시켜 생성된 침전물을 제거하고 상층액을 건조시키면 70% - 80%의 으카이로-이노시를 순도를 갖는 아노시를 건조 흔합물을 얻을 수 있다. (ii) 30% of D-Cyro-inosity obtained by the organic solvent primary treatment method. Drying the inosi of 40% purity in water at high concentration, such as about 70%, to remove the precipitate formed, and drying the supernatant, 70%-80% of the Ekairo-Inoshi The dry mixture having the anosine can be obtained.

(iii) 유기용매 1차 처리 방법에 의해 얻은 D-카이로ᅳ이노시를 30% - (iii) 30% of D-chiropyinosine obtained by the organic solvent primary treatment method.

40%의 순도의 이노시를 건조 흔합물을 저농도， 예컨대 약 15%의 농도로 물에 용해시키고, 여기에 용액 부피의 그 10배의 유기용매을 첨가하여 다시 침전을 유도한다. 침전물을 제거한 상층액을 건조시키면 90% 이상의 D- 카이로-이노시를 순도를 갖는 이노시를 건조 흔합물을 얻을 수 있다. 이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바， 당업계의 통상의 지식올 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며， 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다. Inosi, with a purity of 40%, is dissolved in water at low concentrations, such as about 15%, of the dry mixture, to which precipitation is induced again by adding 10 times the amount of the organic solvent of the solution volume. Drying the supernatant from which the precipitate was removed can obtain a dry mixture of Inosi having a purity of 90% or more of D-Cairo-Inosi. As described above in detail a specific part of the present invention, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

【청구의 범위】 [Range of request] 【청구항 1】  [Claim 1] 마이오ᅳ이노시를 및 으카이로 -이노시를이 함유된 용액으로부터 으 카이로 -이노시를을 고순도로 정제하는 방법으로서， 다음의 단계를 포함하는 방법：  A method of high purity purification of Cairo-Inosio from a solution containing Myoinino-shi and Eucairo-inosino, comprising the following steps: (a) 마이오-이노시를 및 D-카이로 -이노시를이 함유된 용액에 유기용매를 첨가하여 이노시를 침전물을 생성시키는 단계;  (a) adding an organic solvent to a solution containing myo-inosine and D-chiro-inosio to produce inosi precipitate; (b) 상기 생성된 이노시를 침전물로부터 상층액을 분리하는 단계; 및 (b) separating the supernatant from the resulting inosi from the precipitate; And (c) 상기 분리한 상층액을 건조시켜 이노시를 분말을 얻는 단계. (c) drying the separated supernatant to obtain Inosi powder. 【청구항 2】 [Claim 2] 제 1 항에 있어서, 상기 단계 (c) 이후에 다음의 단계를 더욱 포함하는 것을 특징으로 하는 방법:  The method of claim 1, further comprising the following steps after step (c): (d) 상기 단계 (c)에서 얻은 이노시를 분말을 물에 용해시켜 수용액을 제조하는 단계;  (d) dissolving the inosine obtained in step (c) in water to prepare an aqueous solution; (e) 상기 수용액에 유기용매를 첨가하여 이노시를 침전물을 생성시키는 단계;  (e) adding an organic solvent to the aqueous solution to generate an inosi precipitate; (f) 상기 생성된 이노시를 침전물로부터 상층액을 분리하는 단계; 및 (f) separating the supernatant from the resulting inosi from the precipitate; And (g) 상기 분리한 상충액을 건조시켜 이노시를 분말을 얻는 단계. (g) drying the separated supernatant to obtain Inosi powder. 【청구항 3] [Claim 3] 제 2 항에 있어서， 상기 단계 (d)에서， 이노시를 분말을 20%(w/v) 이하의 농도로 물에 용해시키는 것을 특징으로 하는 방법.  The method of claim 2, wherein in step (d), Inosi is dissolved in water at a concentration of 20% (w / v) or less. 【청구항 4】 [Claim 4] 제 1 항에 있어서， 상기 단계 (c) 이후에 다음의 단계를 더 포함하는 것을 특징으로 하는 방법 :  The method of claim 1, further comprising the following steps after step (c): (d)' 상기 단계 (c)에서 얻은 이노시를 분말을 물에 용해시켜 수용액을 제조하고 이노시를 침전물을 생성시키는 단계; (d) 'a step of the Inno when powder obtained in the above step (c) was dissolved in water to prepare an aqueous solution, and the precipitate generated when the Eno; (e)' 상기 수용액에서 생성된 이노시를 침전물로부터 상층액을 분리하는 단계 ; 및 (f)' 상기 상층액을 건조시켜 이노시를 분말을 얻는 단계. (e) 'separating the supernatant from the precipitate created in the Inno when the aqueous solution; And (f) ' drying the supernatant to obtain Inosi powder. 【청구항 5】 [Claim 5] 제 4 항에 있어서， 상기 단계 (d)' 에서， 이노시를을 20%(w/v) 초과 - 70%(w/v) 이하의 농도로 물에 용해시키는 것을 특징으로 하는 방법 . 5. The process of claim 4, wherein in step (d) ' , inosi is dissolved in water at a concentration of greater than 20% (w / v)-up to 70% (w / v). 【청구항 6】 [Claim 6] 제 1 항 또는 제 2 항에 있어서, 상기 단계 (a) 또는 (e)에서의 유기용매는 알코을， 아세톤, 에틸아세테이트， 부틸아세테이트， 1,3- 부틸렌글리콜 또는 에테르인 것을 특징으로 하는 방법.  The method according to claim 1 or 2, wherein the organic solvent in step (a) or (e) is alcohol, acetone, ethyl acetate, butyl acetate, 1,3-butylene glycol or ether. 【청구항 7】 [Claim 7] 제 2 항 또는 제 4 항에 있어서， 상기 단계 (d) 또는 (d)' 에서의 물은 증류수인 것을 특징으로 하는 방법. The method according to claim 2 or 4, wherein the water in step (d) or (d) ' is distilled water. 【청구항 8】 [Claim 8] 제 1 항에 있어서, 상기 단계 (a)에서 유기용매의 첨가량은 마이오- 이노시를 및 D-카이로 -이노시를이 함유된 용액 대비 0.1 - 9배 (v/v)인 것을 특징으로 하는 방법 .  The method of claim 1, wherein the amount of the organic solvent added in step (a) is 0.1-9 times (v / v) compared to a solution containing myo-inosine and D-chiro-inosino. Way . 【청구항 9】 [Claim 9] 제 2 항에 있어서, 상기 단계 (e)에서 유기용매의 첨가량은 수용액 대비 1 - 10배 (v/v)인 것을 특징으로 하는 방법.  The method of claim 2, wherein the amount of the organic solvent added in step (e) is 1 to 10 times (v / v) of the aqueous solution.
PCT/KR2013/011752 2012-12-17 2013-12-17 Method for purifying d-chiro-inositol from solution comprising myo-inositol and d-chiro-inositol Ceased WO2014098454A1 (en)

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