MIXTURE OF INOSITOL DERIVATIVES

Abstract

A mixture of inositol derivatives in which a sugar is bonded to inositol includes an inositol derivative (A10) in which total sugars bonded to one inositol molecule is 10 or more in terms of monosaccharide units. In addition, a mixture of inositol derivatives in which a sugar is bonded to inositol includes 5% by mass or more of an inositol derivative (A7), in which total sugars bonded to one inositol molecule is 7 or more in terms of monosaccharide units, with respect to a total amount of inositol derivatives (100% by mass).

Claims

1. A mixture of inositol derivatives in which a sugar is bonded to inositol, the mixture of inositol derivatives comprising an inositol derivative (A10) in which total sugars bonded to one inositol molecule is 10 or more in terms of monosaccharide units.

2. The mixture of inositol derivatives according to claim 1, comprising 1% by mass or more of the inositol derivative (A10) with respect to a total amount of inositol derivatives (100% by mass).

3. The mixture of inositol derivatives according to claim 2, comprising 5% by mass or more of the inositol derivative (A10) with respect to a total amount of inositol derivatives (100% by mass).

4. The mixture of inositol derivatives according to claim 1, comprising an inositol derivative in which total sugars bonded to one inositol molecule is 11 or more in terms of monosaccharide units.

5. The mixture of inositol derivatives according to claim 4, comprising an inositol derivative in which total sugars bonded to one inositol molecule is 12 or more in terms of monosaccharide units.

6. The mixture of inositol derivatives according to claim 1, wherein the inositol derivative (A10) is at least one kind of inositol derivatives selected from the group consisting of the following (a) and (b): (a) an inositol derivative in which one or more glucoses, and one or more oligosaccharides containing glucose as a structural unit are respectively bonded to inositol; and (b) an inositol derivative in which one or more oligosaccharides containing glucose as a structural unit are bonded to inositol.

7. The mixture of inositol derivatives according to claim 1, further comprising: an inositol derivative (B10) in which total sugars bonded to one inositol molecule is less than 10 in terms of monosaccharide units, wherein the inositol derivative (B10) is at least one kind of inositol derivatives selected from the group consisting of the following (a) to (c): (a) an inositol derivative in which one or more glucoses, and one or more oligosaccharides containing glucose as a structural unit are respectively bonded to inositol; (b) an inositol derivative in which one or more oligosaccharides containing glucose as a structural unit are bonded to inositol; and (c) an inositol derivative in which one or more glucoses are bonded to inositol.

8. A mixture of inositol derivatives in which a sugar is bonded to inositol, the mixture of inositol derivatives comprising 5% by mass or more of an inositol derivative (A7), in which total sugars bonded to one inositol molecule is 7 or more in terms of monosaccharide units, with respect to a total amount of inositol derivatives (100% by mass).

9. The mixture of inositol derivatives according to claim 8, wherein the inositol derivative (A7) is at least one kind of inositol derivatives selected from the group consisting of the following (a) and (b): (a) an inositol derivative in which one or more glucoses, and one or more oligosaccharides containing glucose as a structural unit are respectively bonded to inositol; and (b) an inositol derivative in which one or more oligosaccharides containing glucose as a structural unit are bonded to inositol.

10. The mixture of inositol derivatives according to claim 8, further comprising: an inositol derivative (B7) in which total sugars bonded to one inositol molecule is less than 7 in terms of monosaccharide units, wherein the inositol derivative (B7) is at least one kind of inositol derivatives selected from the group consisting of the following (a) to (c): (a) an inositol derivative in which one or more glucoses, and one or more oligosaccharides containing glucose as a structural unit are respectively bonded to inositol; (b) an inositol derivative in which one or more oligosaccharides containing glucose as a structural unit are bonded to inositol; and (c) an inositol derivative in which one or more glucoses are bonded to inositol.

11. The mixture of inositol derivatives according to claim 1, wherein the inositol is myo-inositol.

12. The mixture of inositol derivatives according to claim 1, which promotes cell activation.

13. A cell-activating agent comprising the mixture of inositol derivatives according to claim 1.

14. A composition for cell activation, comprising the mixture of inositol derivatives according to claim 1.

15. An external preparation for skin, comprising the mixture of inositol derivatives according to claim 1.

16. A cosmetic preparation comprising the mixture of inositol derivatives according to claim 1.

Description

EXAMPLES

[0200] Hereinafter, while the present invention will be described with reference to the following experimental examples, the present invention is not limited to the following experimental examples.

[Experimental Example 1] Production of Mixture of Inositol Derivatives

Example 1

[0201] Under the condition shown in Table 3, using a 5 L culture tank (model number MD-300, manufactured by Marubishi Bioengineering Co., Ltd.), myo-inositol (manufactured by Tsuno Rice Fine Chemicals Co., Ltd.) and -cyclodextrin (manufactured by ENSUIKO Sugar Refining Co., Ltd.) were reacted in the presence of CGTase (manufactured by Novozymes), and thereby inositol derivatives were generated. A composition of reaction solution in Table 3 indicates a final concentration.

TABLE-US-00003 TABLE 3 Composition of -cyclodextrin 459 g/L reaction solution Myo-inositol 116 g/L CGTase 0.5-SS/L Anhydrous citric acid 0.11 g/L Sodium citrate dihydrate 1.37 g/L Water pH 6.3 Temperature 50 C. Volume of reaction tank 5 L Amount of reaction solution 2.4 L Stirring rate 200 rpm Reaction time 48 hours

[0202] A diluted solution obtained by adding 3.0 L of water to 2.2 L of the reaction solution after the reaction was added to a stock solution tank of a membrane device in which a cross-flow ultrafiltration membrane (SIP-1013, Asahi Kasei Corporation) was placed. A circulation pump was operated to concentrate the stock solution to 1.1 L, and the filtrate was recovered. Thereafter, a step of adding 1 L of water to the stock solution tank and further recovering the filtrate was repeated 5 times. All the filtrates were mixed, and thereby 9.1 L of a recovered filtrate was obtained. Table 4 summarizes an amount of reaction solution, an amount of water added, and an amount of recovered filtrate in the present treatment. A treatment time indicates a time taken to obtain a recovered filtrate in which all the filtrates had been mixed.

TABLE-US-00004 TABLE 4 Amount of reaction solution 2.2 L Amount of water initially added 3 L Amount of water additionally added 1 L 5 Amount of recovered filtrate 9.1 L Treatment time 1.0 hour

[0203] The inositol derivatives were purified from the recovered filtrate, and thereby a mixture of the inositol derivatives was obtained. The obtained mixture of the inositol derivatives was subjected to HPLC analysis under the following conditions to examine a composition of the mixture of the inositol derivatives.

[0204] <Analysis Conditions>

[0205] Column: Shodex HILICpak VN-50 4D1

[0206] Eluent: CH.sub.3CN:water=60:40 (V:V)

[0207] Flow rate: 0.3 mL/min

[0208] Oven temperature: 40 C.

[0209] Detection: RI (Differential Refractive Index)

[0210] The results are shown in Table 8. In Table 8, a proportion of each inositol derivative is based on a total amount (100% by mass) of all inositol derivatives contained in the mixture.

Example 2

[0211] Under the condition shown in Table 5, myo-inositol was reacted with -cyclodextrin in the presence of CGTase, and thereby inositol derivatives were generated. A composition of reaction solution in Table 5 indicates a final concentration.

TABLE-US-00005 TABLE 5 Composition of -cyclodextrin 246 g/L reaction solution Myo-inositol 98 g/L CGTase 1.0-SS/L Anhydrous citric acid 0.11 g/L Sodium citrate dihydrate 1.37 g/L Water pH 6.3 Temperature 50 C. Volume of reaction tank 1 L Amount of reaction solution 0.51 L Stirring rate 200 rpm Reaction time 22.5 hours

[0212] The reaction solution after the reaction was ultrafiltered using a cross-flow ultrafiltration membrane (SIP-0013, Asahi Kasei Corporation), and the filtrate was recovered. The inositol derivatives were purified from the recovered filtrate, and further fractionated using activated carbon. 600 mL of the filtrate 10-fold diluted with ion exchange water was allowed to pass through an activated carbon column (a column in which a glass chromatograph tube with 40 mm diameter1000 mm was filled with activated carbon (manufactured by NACALAI TESQUE, INC., for column chromatography)), and thereafter, 400 mL of ion exchange water, 700 mL of 5% ethanol, 1000 mL of 10% ethanol, 1000 mL of 15% ethanol, 1400 mL of 20% ethanol, and 500 mL of 30% ethanol were allowed to pass through the activated carbon column sequentially. Thereafter, 50% ethanol was allowed to pass through the activated carbon column, the eluted fraction was recovered, and thereby a mixture of the inositol derivatives was obtained.

[0213] The obtained inositol derivatives were subjected to HPLC analysis in the same manner as in Example 1 to examine a composition of the mixture of the inositol derivatives. The results are shown in Table 8.

Example 3

[0214] Under the condition shown in Table 6, myo-inositol was reacted with -cyclodextrin in the presence of CGTase, and thereby inositol derivatives were generated. A composition of reaction solution in Table 6 indicates a final concentration.

TABLE-US-00006 TABLE 6 Composition of -cyclodextrin 258 g/L reaction solution Myo-inositol 110 g/L CGTase 1.0-SS/L Anhydrous citric acid 0.11 g/L Sodium citrate dihydrate 1.37 g/L Water pH 6.3 Temperature 50 C. Volume of reaction tank 5 L Amount of reaction solution 3.2 L Stirring rate 250 rpm Reaction time 24 hours

[0215] The reaction solution after the reaction was ultrafiltered using a cross-flow ultrafiltration membrane (SIP-0013, Asahi Kasei Corporation), and the filtrate was recovered. The inositol derivatives were purified from the recovered filtrate, and further fractionated using activated carbon. 1460 mL of the filtrate was allowed to pass through an activated carbon column (a column in which a glass chromatograph tube with 40 mm diameter1000 mm was filled with activated carbon (manufactured by NACALAI TESQUE, INC., for column chromatography)), and thereafter, 2070 mL of ion exchange water and 4140 mL of 10% ethanol were allowed to pass through the activated carbon column sequentially. The eluted fraction was recovered, and thereby a mixture of the inositol derivatives was obtained.

[0216] The obtained inositol derivatives were subjected to HPLC analysis in the same manner as in Example 1 to examine a composition of the mixture of the inositol derivatives. The results are shown in Table 8.

Example 4

[0217] In the same manner as in Example 3, and under the condition shown in Table 6, myo-inositol was reacted with -cyclodextrin in the presence of CGTase, and thereby inositol derivatives were generated.

[0218] The reaction solution after the reaction was ultrafiltered using a cross-flow ultrafiltration membrane (SIP-0013, Asahi Kasei Corporation), and the filtrate was recovered. The inositol derivatives were purified from the recovered filtrate, and further fractionated using activated carbon. 1460 mL of the filtrate was allowed to pass through an activated carbon column (a column in which a glass chromatograph tube with 40 mm diameter1000 mm was filled with activated carbon (manufactured by NACALAI TESQUE, INC., for column chromatography)), and thereafter, 2070 mL of ion exchange water was allowed to pass through the activated carbon column. The eluted fraction was recovered, and thereby a mixture of the inositol derivatives was obtained.

[0219] The obtained inositol derivatives were subjected to HPLC analysis in the same manner as in Example 1 to examine a composition of the mixture of the inositol derivatives. The results are shown in Table 8.

Comparative Example 1

[0220] Under the condition shown in Table 7, myo-inositol was reacted with -cyclodextrin in the presence of CGTase, and thereby inositol derivatives were generated. A composition of reaction solution in Table 7 indicates a final concentration.

TABLE-US-00007 TABLE 7 Composition of -cyclodextrin 224 g/L reaction solution Myo-inositol 73 g/L CGTase 0.5-SS/L Anhydrous citric acid 0.11 g/L Sodium citrate dihydrate 1.37 g/L Water pH 6.3 Temperature 50 C. Volume of reaction tank 1 L Amount of reaction solution 0.56 L Stirring rate 200 rpm Reaction time 22.5 hours

[0221] The reaction solution after the reaction was ultrafiltered using a cross-flow ultrafiltration membrane (SIP-0013, Asahi Kasei Corporation), and the filtrate was recovered. The inositol derivatives were purified from the recovered filtrate, and further fractionated using activated carbon. 611 mL of the filtrate 10-fold diluted with ion exchange water was allowed to pass through an activated carbon column (a column in which a glass chromatograph tube with 40 mm diameter1000 mm was filled with activated carbon (manufactured by NACALAI TESQUE, INC., for column chromatography)), and thereafter, 400 mL of ion exchange water, 780 mL of 10% ethanol, and 780 mL of 20% ethanol were allowed to pass through the activated carbon column sequentially. Thereafter, 780mL of 30% ethanol was allowed to pass through the activated carbon column, the eluted fraction was recovered, and thereby a mixture of the inositol derivatives was obtained.

[0222] The obtained inositol derivatives were subjected to HPLC analysis in the same manner as in Example 1 to examine a composition of the mixture of the inositol derivatives. The results are shown in Table 8.

TABLE-US-00008 TABLE 8 Number of Comparative monosaccharide Example Example Example Example Example units 1 2 3 4 1 1 9 0 8 3 1 2 12 5 20 13 20 3 12 5 20 16 30 4 12 14 17 16 24 5 11 22 13 13 14 6 9 21 9 11 7 7 8 15 6 8 3 8 6 9 4 7 1 9 5 4 2 9 0 10 4 2 0 0 0 11 4 1 0 2 0 12 or more 8 2 1 2 0 (Unit: % by mass)

[0223] As shown in Table 8, the mixtures of inositol derivatives of Examples 1 to 4 contained more inositol derivatives having a larger number of monosaccharide units than the mixture of inositol derivatives of Comparative Example 1. Regarding inositol derivatives having 7 or more monosaccharide units, 35% by mass thereof was contained in Example 1, 33% by mass thereof was contained in Example 2, 13% by mass thereof was contained in Example 3, and 28% by mass thereof was contained in Example 4, whereas only 4% by mass of the inositol derivatives having 7 or more monosaccharide units was contained in Comparative Example 1. In addition, in Example 1, a proportion of inositol derivatives having 9 or more monosaccharide units was 21% by mass, and a proportion of an inositol derivative having 10 or more monosaccharide units was 16% by mass, whereas in Comparative Example 1, the inositol derivatives having 9 or more monosaccharide units were 0% by mass. In Example 1, 12% by mass of inositol derivatives having 11 or more monosaccharide units and 8% by mass of inositol derivatives having 12 or more monosaccharide units were contained. In Examples 2 to 4, the inositol derivatives having 10 or more monosaccharide units were also contained. In Example 2, the inositol derivatives having 10 or more monosaccharide units, the inositol derivatives having 11 or more monosaccharide units, and the inositol derivatives having 12 or more monosaccharide units were contained. Among Examples 1 to 4, Example 1 had the highest content of the inositol derivatives having 10 or more monosaccharide units.

[0224] Based on the above results, it was confirmed that a mixture of inositol derivatives containing the inositol derivatives having 10 or more monosaccharide units can be obtained by reacting inositol and dextrin by the methods shown in Examples 1 to 4. In addition, it was also confirmed that a mixture of inositol derivatives which has a high content of inositol derivatives having a large number of monosaccharide units (monosaccharide units of 7 or more) can be obtained.

[Experimental Example 2] Cell Activation Test Using Normal Human Fibroblasts

[0225] As cells, cells obtained by treating normal human fibroblasts (RIKEN BioResource Research Center) with hydrogen peroxide were used. As a medium, Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum was used.

[0226] The mixtures of inositol derivatives of Examples 1 to 4 and Comparative Example 1 were dissolved in purified water and added to the medium so that a final concentration became 10 mg/L, and thereby a medium was prepared (Examples 1 to 4 and Comparative Example 1). As a control, a medium in which purified water was added instead of the inositol derivatives was prepared (control). The cells were cultured in these media for 24 hours at 37 C. in a 5% CO.sub.2 atmosphere.

[0227] After 24 hours from the start of culture, the cells were recovered and washed with phosphate buffered saline. Thereafter, a cell proliferative activity was measured. The measurement of the cell proliferative activity was performed by a method using WST-8 (a living cell count measuring reagent SF, NACALAI TESQUE, INC.), which is a tetrazolium salt that generates water-soluble formazan. Thereafter, the number of cells was measured by a neutral red method, and a value representing the cell proliferative activity was divided by a value representing the number of cells to calculate the cell activation activity.

[0228] The results are shown in Table 9. In Table 9, the cell activation activity was expressed as a relative value when defining the cell activation activity of the control in which purified water was added as 1.00. In Examples 1 to 4, it was confirmed that the cell activation activity was improved as compared with that of Comparative Example 1. Among Examples 1 to 4, the cell activation activity in Example 1 was the highest, and the cell activation activity in Example 3 was the lowest. Based on these results, it was confirmed that the cell activation activity improves as a content of the inositol derivatives having 7 or more monosaccharide units or the inositol derivatives having 10 or more monosaccharide units increases.

TABLE-US-00009 TABLE 9 Comparative Control Example 1 Example 2 Example 3 Example 4 Example 1 Addition Purified 10 10 10 10 10 concentration water (mg/L) Cell activation 1.00 1.25 1.21 1.12 1.15 0.98 activity

[Experimental Example 3] Cell Activation Test Using Normal Human Epidermal Keratinocytes

[0229] As cells, cells obtained by subjecting normal human epidermal keratinocytes (NHEK cells, KURABO) to ultraviolet light irradiation were used. The ultraviolet light irradiation was carried out by recovering cells cultured in a medium, washing them with phosphate buffered saline, and then irradiating them with 60 mJ/cm.sup.2 of ultraviolet B wave in the presence of phosphate buffered saline. As a medium, Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum was used.

[0230] The mixtures of inositol derivatives of Examples 1 to 4 and Comparative Example 1 were dissolved in purified water and added to the medium so that a final concentration became 10 mg/L, and thereby a medium was prepared (Examples 1 to 4 and Comparative Example 1). As a control, a medium in which purified water was added instead of the inositol derivatives was prepared (control). The cells were cultured in these media for 24 hours at 37 C. in a 5% CO.sub.2 atmosphere.

[0231] After 24 hours from the start of culture, the cell proliferative activity was measured in the same manner as in Experimental Example 2, and the cell activation activity was calculated.

[0232] The results are shown in Table 10. In Table 10, the cell activation activity was expressed as a relative value when defining the cell activation activity of the control in which purified water was added as 1.00. In Examples 1 to 4, it was confirmed that the cell activation activity was improved as compared with that of Comparative Example 1. Among Examples 1 to 4, the cell activation activity in Example 1 was the highest, and the cell activation activity in Example 3 was the lowest. Based on these results, it was recognized that, even in the case of normal human epidermal keratinocytes, the cell activation activity tends to improve as a content of the inositol derivatives having 7 or more monosaccharide units or the inositol derivatives having 10 or more monosaccharide units increases.

TABLE-US-00010 TABLE 10 Comparative Control Example 1 Example 2 Example 3 Example 4 Example 1 Addition Purified 10 10 10 10 10 concentration water (mg/L) Cell activation 1.00 1.27 1.17 1.15 1.23 1.01 activity

INDUSTRIAL APPLICABILITY

[0233] According to the present invention, a mixture of inositol derivatives which has an improved cell-activating effect, and use applications thereof are provided.