JP2779774B2 - Method for selective acylation of steryl glycosides - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a steryl acylated glycoside which can be used in the fields of health foods, pharmaceuticals, agricultural chemicals, cosmetics, etc., and more specifically, to select only a specific hydroxyl group of the sugar moiety of steryl glycoside. The present invention relates to a method for performing a specific acylation.

[0002]

2. Description of the Related Art It is known that steryl glycoside exists as a kind of glycoside in living bodies of animals, plants and microorganisms, and is considered to be involved in various physiological and biological phenomena. Further, the hydroxyl group of the sugar moiety is acylated, for example, steryl-6-O-acyl-β-D-glucoside or steryl-2.3.4.6-O-tetraacyl-
It is also well known that β-D-glucoside has an anti-inflammatory effect and is a useful compound as a pharmaceutical raw material (Japanese Patent Publication No. 53-53).
20986, JP-B-57-58357).

Hitherto, there have not been many proposals and reports on a method for producing such a steryl acylated glycoside, and the steryl glycoside and the acyl chloride are slightly dissolved in a pyridine solution as described in the above publication. The chemical reaction was carried out by stirring at 0 to 50 ° C. or room temperature to 70 ° C. for several hours to about 1 day, and the reaction product was purified by fractionating or recrystallizing on silica gel. Further, there is no example of acylating steryl glycoside using an enzyme.

In the production of steryl acylated glycosides, according to such a chemical synthesis method, good results can be obtained in terms of the reaction time and the conversion, but the primary hydroxyl group and the secondary hydroxyl group in the sugar moiety of the steryl glycoside can be obtained.
Since the graded hydroxyl group is non-selectively acylated, it has been substantially difficult to specify the type, position and number of the hydroxyl group into which the acyl group is to be introduced during the acylation reaction. Therefore, in the chemical synthesis method, the reactant is obtained as a mixture of various steryl acylated glycosides having different numbers and positions of acyl groups, and a great deal of effort is required to separate and purify the target steryl acylated glycoside having a specific structure. It had the disadvantage of requiring it.

[0005]

In view of the above situation, the present inventors have conducted intensive studies on a method for selectively acylating steryl glycoside, and as a result, a method using steryl glycoside and a fatty acid as raw materials, By using an enzyme as a catalyst under the reaction conditions, they have found that only a specific hydroxyl group of the saccharide moiety of steryl glycoside can be acylated, and have completed the present invention. That is, an object of the present invention is to develop a method for easily producing a specific steryl acylated glycoside that can selectively acylate a specific hydroxyl group of a sugar moiety of a steryl glycoside.

[0006]

The gist of the present invention to achieve the above object is to provide a steryl glycoside represented by the following general formula (I).

Embedded image HO—CH ₂ —Gly—O—St (I) [In the formula (I), Gly represents a glycoside residue that is each residue of glucose, galactose, fructose or mannose, and St represents sitosterol or campe. Each residue of sterol, stigmasterol, avenasterol, isofucosterol, brassicasterol or cholesterol is shown. And a linear or side-chain saturated or unsaturated fatty acid in the presence of lipase to form a solution of 0.1 to 0.1%.
The reaction is carried out in an aqueous solution or a chloroform solution containing 1 to 10% by weight of a lower alcohol, and a steryl acylated glycoside represented by the following general formula (II) is obtained.

Embedded image R-COO-CH ₂ -Gly-O-St (II) [In the formula (II), Gly and St are the same as those in the formula (I), and R represents a residue of the fatty acid. ] In a selective acylation method of steryl glycoside.

The steryl glycoside, which is one of the raw materials of the present invention, is represented by the general formula HO-GH ₂ -Gly-O-St, wherein Gly represents a glycoside residue. It is any of a glucose residue, a galactose residue, a fructose residue and a mannose residue. St represents a steryl residue, and specific examples include a sitosterol residue, a campesterol residue, a stigmasterol residue, an avenasterol residue, an isofucosterol residue, a brassicasterol residue, and a cholesterol residue. it can.

As the glycoside residue and the steryl residue, one or two or more of the above-mentioned specific examples can be used as a raw material. Among them, sitosteryl-β-D-glucoside, campesteryl-β- D
-Glucoside, stigmasteryl-β-D-glucoside, brassicasteryl-β-D-glucoside, cholesteryl-β-D-glucoside, sitosteryl-β-D-galactoside, campesteryl-β-D-galactoside,
Stigmasteryl-β-D-galactoside, brassicasteryl-β-D-galactoside, cholesteryl-β-D
-Galactoside is more preferred, furthermore sitosteryl-, campesteryl- and / or stigma-β-D
-Glucoside, cholesteryl-β-D-glucoside is most preferred.

The steryl glycoside may be derived from a natural product obtained by fractionating lecithin from soybean, rapeseed, sesame, egg yolk or the like by silica gel chromatography, or may be any of the various steryl glycosides described above. A compound obtained by etherifying a sterol halide (eg, chloride, bromide, iodide, etc.) with glucose, galactose, fructose or mannose may be used.

Another source of the present invention is a fatty acid. Fatty acids that can be used in the present invention are straight-chain or side-chain saturated or unsaturated fatty acids. In the case of unsaturated fatty acids, they can be used regardless of their cis- and trans-forms. The number of carbon atoms is not particularly limited, but fatty acids that can be easily obtained as practical raw materials are those having 2 to 50 carbon atoms. These fatty acids are exemplified below, of which linear, saturated or unsaturated fatty acids having 2 to 22 carbon atoms are preferred.

The fatty acids which can be used in the present invention are acetic acid, lactic acid, butyric acid, valeric acid, caproic acid, caprylic acid, 3,5,5
5-trimethylpentanoic acid, nonanoic acid, isononanoic acid (e.g., Equasid 9 manufactured by Idemitsu Petrochemical Co., Ltd.), capric acid, lauric acid, tridecanoic acid, isotridecanoic acid (e.g., EQUACID 1 manufactured by Idemitsu Petrochemical Co., Ltd.)
3), myristic acid, pentadecanoic acid, palmitic acid,
Palmitooleic acid, isopalmitic acid, heptadecanoic acid, stearic acid, isostearic acid (methyl branched isostearic acid manufactured by Emery), oleic acid, elaidic acid, petroselic acid, linoleic acid, α- and γ-linolenic acid, dihomoγ -Linolenic acid, eleostearic acid, 9- or 12-ketostearic acid, 9,10-dihydroxystearic acid, 12-hydroxystearic acid, ricinoleic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, behenic acid, docosahexaenoic acid Lignoceric acid (carbon number: 24, hereinafter referred to as C ₂₄ ), cellotic acid (C ₂₆ ), montanic acid (C ₂₈ ), melicic acid (C
₃₀ ), laxeronic acid (C ₃₂ ), gedic acid (C ₃₄ ) and the like.

Palm oil-decomposed fatty acids which are mixed fatty acids;
Soybean oil-decomposed fatty acid, rapeseed oil-decomposed fatty acid, safflower oil-decomposed fatty acid, linseed oil-decomposed fatty acid, fish oil or hydrogenated fish oil-decomposed fatty acid, Unicid 425, a linear saturated fatty acid manufactured by Petrolite USA (acid value 94, average molecular weight 59)
6), Unicid 550 (acid value 72, average molecular weight 7)
78), Unicid 700 (acid value 63, average molecular weight 888) and the like can also be used.

A major feature of the present invention resides in that the above-mentioned steryl glycoside is reacted with a fatty acid using lipase under specific reaction conditions. By such an acylation reaction, only the primary hydroxyl group bonded to the 6-position carbon of the sugar moiety of the steryl glycoside (however, in the case of fructoside, the 1-position carbon) is selectively acylated. Therefore, the step of purifying the reactants can be greatly simplified as compared with the conventional method.

The type of lipase used in the present invention includes
The origin of which is Candida, Penicillium, Pseudomonas, Mucor, Chromobacterium, Geotricum, Aspergillus, Rhizopus, etc., among which, preferably, Candida, Penicillium, Pseudomonas, Lipases from the genus Mucor and Chromobacterium, most preferably lipases from the genus Candida. Preferably, as an example of the origin of the lipase, Candida syrup L.
dia cylindracea), penicillium
Camemberti (Penicillium camem)
bertii), Pseudomonas sp. (Pseu)
domonas sp. ), Mucor javanicus, Mucor miehei, Chromobacterium v
iscosum), Geotricum Candy Dam (G
eotrichum candidum). These lipases are commercially available and can be easily obtained.

The acylation reaction of the present invention can be carried out in an aqueous system or a substantially non-aqueous system. 0.1-10 for aqueous reaction
% By weight, preferably 1 to 10% by weight, more preferably 5% by weight.
An aqueous solution containing a lower alcohol of 7 to 7% by weight is used as a solvent, and 0.1 to 10% by weight, preferably 0.1 to 5% by weight, more preferably 0.5 to 1% by weight in a substantially non-aqueous reaction. A chloroform solution containing a lower alcohol is used as a solvent. In the aqueous reaction, the reaction can be performed even in an aqueous solution containing no lower alcohol. Here, the lower alcohol is an aliphatic alcohol having 1 to 4 carbon atoms,
Specifically, methyl alcohol, ethyl alcohol, n-
One or more alcohols selected from the group consisting of propyl alcohol, isopropyl alcohol, n-butyl alcohol, sec-butyl alcohol (2-butyl alcohol), and tert-butyl alcohol (2-methyl-2-propyl alcohol) It is. Of these, ethyl alcohol and tert-butyl alcohol are desirable. If the alcohol has more than 4 carbon atoms, the acylation reaction tends to be inhibited. In the substantial non-aqueous reaction using the chloroform solution as a solvent, the acylation reaction proceeds more rapidly than the aqueous reaction in which the reaction substrate (raw material) may be in a dispersed state.

In order to carry out the present invention by an aqueous reaction, the required amounts of steryl glycoside and fatty acid are combined with a small amount (0.1 to 10% by weight) of a lower alcohol (preferably tert-butyl alcohol) as described above. ) Is dissolved or dispersed in an aqueous solution, preferably a buffer solution having a pH of 5 to 9, and most preferably a pH of 6 to 8, and lipase of 0.1 to 10% by weight based on the raw material is added thereto. Gently shake, stir or stand at a temperature that does not activate for 50 to 200 hours. When the pH is out of the above range, the acylation reaction hardly proceeds.

To carry out the reaction in a substantially non-aqueous reaction, a small amount (0.1 to 5% by weight) of the same raw materials as in the case of the aqueous reaction is used.
May be dissolved in a chloroform solution containing a lower alcohol (preferably ethyl alcohol), lipase may be added in the same manner as in the aqueous reaction, and the mixture may be gently shaken, stirred, or allowed to stand at 20 to 70 ° C. for 50 to 200 hours. In this substantially non-aqueous reaction, the acylation reaction is inhibited in any case where the lower alcohol concentration is out of the above range.

The acylation reaction rate at the time of reaction equilibrium varies depending on the charge ratio of the raw materials.
When β-D-glycoside: fatty acid = 1: 5 (molar ratio), it is about 70% based on the primary hydroxyl group to be acylated of steryl-β-D-glycoside.

Thus, any of the reactants obtained by the above-mentioned aqueous reaction and substantially non-aqueous reaction is a primary compound bonded to the 6-position carbon of the sugar moiety of the starting material steryl glycoside (however, in the case of fructoside, the 1-position carbon). Only the hydroxyl groups are specifically acylated, and those in which the other hydroxyl groups (secondary hydroxyl groups) of the sugar moiety are acylated and other by-products are scarcely produced. For this reason, the desired steryl acylated glycoside can be isolated by a simple treatment such as cooling precipitation utilizing the difference in the solubility of each component in the reaction product and solvent separation. During the acylation reaction of the present invention, when unsaturated fatty acids, especially linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, and highly unsaturated fatty acids such as fish oil-decomposed fatty acids are used as raw materials, inert gas such as nitrogen is contained in the reaction vessel. Oxidation degradation can be prevented by introducing a gas and keeping the atmosphere of an inert gas.

[0020]

【Example】

Example 1 Oil (precipitate) of sesame crude oil obtained in the sesame oil production process
Was extracted with aqueous ethanol (β-sitosteryl-β-D-glucoside: campesteryl-β-D-glucoside: stigma-β-D-glucoside: β-sitosteryl-β-D-galactoside).
5.0 g of a 63: 20: 15: 2 mixture (weight ratio) and 100 g of palmitic acid were placed in a glass reaction vessel,
Chloroform 500 containing 0.5% by weight of ethanol
Add and dissolve the lipase, and then lipase OF (manufactured by Meito Sangyo Co., Ltd., lipase derived from Candida cylindrace)
After adding 5.0 g, the head space was replaced by blowing nitrogen gas, and shaken at 60 ° C. for 160 hours (1
20 rpm) to carry out an acylation reaction. The acylation reaction rate (based on the primary hydroxyl group to be acylated of the raw material steryl-β-D-glycoside, the same applies hereinafter) was 68%.

Next, the reaction product was filtered through a filter paper to remove lipase, the solvent in the filtrate was distilled off under reduced pressure, 300 ml of n-hexane was added to the residue, and the residue was filtered through a filter paper at room temperature (25 ° C.). Was removed together with n-hexane to collect insolubles. This insoluble matter was dispersed in 50 ml of ethyl acetate, and the insoluble matter (unreacted steryl-β-D-glycoside) was dissolved.
Was filtered through filter paper at the same temperature, and ethyl acetate was distilled off from the filtrate under reduced pressure to obtain 5.1 g of white crystals.

As a result of analyzing the obtained crystals by high performance liquid chromatography and ¹³ C-NMR, this component was bonded to carbon 6 of the sugar moiety of the steryl-β-D-glycoside (mixture) used as the starting material. Steryl-6-O-palmitoyl-β-D-glycoside in which only the hydroxyl group is selectively acylated (composition ratio of steryl-
β-D-glycoside). Steryl-B-
¹³ C-NM before and after acylation of D-glycoside
From the comparison of the R spectra (FIGS. 1 and 2), a signal 1 (173.9 ppm) derived from carbon of the carbonyl group was newly generated by the acylation treatment (FIG. 2), and 6 The signal 2 derived from the carbon at position 2 was shifted (62.4 ppm in FIG. 1 was 64.4 ppm in FIG. 2), and it was confirmed that the hydroxyl group of this carbon was acylated.

Example 2 In Example 1, soybean lecithin was replaced by steryl-β-D-glycoside (5.0 g) derived from sesame, and steryl-β separated by silica gel column treatment with hydrous ethanol.
-D-glycoside (a mixture of β-sitosteryl-β-D-glucoside: candosteryl-β-D-glucoside: stigmasteryl-β-D-glucoside = 59: 21: 20 (weight ratio)) (5.0 g) And palmitic acid (1
00g) with oleic acid (100 g), and lipase OF (5.0 g) with lipase GC (Amano Pharmaceutical Co., Ltd.)
Lipase from Geotricum candy dam
The acylation reaction and the purification treatment were carried out under the same conditions except for (3.0 g) to obtain 5.3 g of white crystals. This was analyzed in the same manner as in Example 1. As a result, it was found that the component was steryl-β-D-glycoside (mixture) in which only the primary hydroxyl group bonded to the 6-position carbon of the sugar moiety of the starting material was selectively acylated. 6-O-oleoyl-β-D-glycoside (composition ratio was the same as that of steryl-β-D-glycoside as a raw material).

Example 3 Steril-β-D-glycoside 10 used in Example 2
g and 30 g of oleic acid in a glass reaction vessel,
0.1 containing 5% by weight of tert-butyl alcohol
M phosphate buffer (pH 7.0) (1000 ml) was added and dispersed, and lipase OF (used in Example 1) was further added.
After adding 5 g, the acylation reaction was performed by gentle stirring (120 rpm) at 60 ° C. for 120 hours. The acylation reaction rate was 72%. The reaction product was treated as in Example 1 to obtain 10.2 g of white crystals. As a result of analysis by the method described in Example 1, the component of the crystal was found to be steryl-
1 of the 6-position carbon of the sugar moiety of β-D-glycoside (mixture)
Steryl-6 in which only the hydroxyl group is selectively acylated
O-oleoyl-β-D-glycoside (composition ratio was the same as that of steryl-β-D-glycoside as a raw material).

Example 4 36 g of cholesterol was dissolved in 120 ml of dichloromethane, and tin chloride was added. The solution was added dropwise to a solution of glucose pentaacetate in dichloromethane (37.5 g / 120 ml of dichloromethane) and left at room temperature for 6 hours. Then, 1 liter of saturated saline was added, and the deposited precipitate was removed by filtration with filter paper, and the filtrate was washed with water until it became neutral.
The solvent was removed to obtain crude tetraacetylcholesteryl-β-D-glucoside. Then add this to ethyl alcohol 400
and 2 g of sodium methoxide was added thereto.
After 6 hours, the ethyl alcohol was distilled off, 1 liter of water was added, and the resulting precipitate was collected by centrifugation. The precipitate was washed with 800 ml of acetone, 50 ml of hexane and then 50 ml of warm acetone to obtain cholesteryl-β-D-glucoside.

Cholesteryl-β-D-glucoside 10
g, 30 g of 12-hydroxystearic acid and 5 g of lipase TOYO (manufactured by Asahi Kasei Corporation, lipase derived from Chromobacterium biscosam), and 1000 ml of chloroform containing 1% by weight of n-propyl alcohol was used as a reaction solvent. Was subjected to an acylation reaction at 55 ° C. for 95 hours (acylation rate of reaction product: 70%). Then, lipase was removed from the reaction product and the reaction product was subjected to solvent fractionation in the same manner as in Example 1 to obtain 7.4 g of white crystals. As a result of analysis by the method described in Example 1, this crystal component was found to be cholesteryl-6-O in which only the primary hydroxyl group at the 6-position carbon of the glucose residue of cholesteryl-β-D-glucoside as a raw material was specifically acylated. -12-hydroxystearoyl-
β-D-glucoside.

Comparative Examples 1-3 The acylation reaction was carried out under the same conditions as in Example 1 except that chloroform was replaced with n-hexane, benzene or carbon tetrachloride. They were 1%, 0.5% and 0.1%, respectively.

Comparative Example 4 The acylation reaction was carried out under the same conditions as in Example 1 except that the ethanol content was increased from 0.5% by weight to 12% by weight, but the ethyl esterification reaction of the fatty acid proceeded remarkably. However, the acylation reaction rate of the reaction product was as low as 5%.

[0029]

According to the present invention, only the specific primary hydroxyl group of the saccharide moiety of the steryl glycoside can be selectively acylated, and the purification step of the reaction product can be simplified, so that the steryl acylated glycoside having the specific structure can be obtained. Can be easily manufactured.

[Brief description of the drawings]

FIG. 1 is a view showing a ¹³ C-NMR spectrum of steryl-β-D-glycoside (before acylation treatment) described in Example 1.

FIG. 2 is a view showing a ¹³ C-NMR spectrum of a product obtained by acylating the steryl-β-D-glycoside with palmitic acid in Example 1 and removing and purifying unreacted substances.

Claims (3)

(57) [Claims] 1. A steryl glycoside represented by the following general formula (I): HO-CH ₂ -Gly-O-St (I) [In the formula (I), Gly represents glucose, galactose, fructose or mannose. And St represents each residue of sitosterol, campesterol, stigmasterol, avenasterol, isofucosterol, brassicasterol or cholesterol. And a linear or side-chain saturated or unsaturated fatty acid in the presence of lipase to form a solution of 0.1 to 0.1%.
Reaction is carried out in an aqueous solution or a chloroform solution containing 1 to 10% by weight of a lower alcohol, and a steryl acylated glycoside represented by the following general formula (II): R-COO-CH ₂ -Gly-O-St (II) [In the formula (II), Gly and St are the same as in the formula (I), and R represents a residue of the fatty acid. ], And a method for selective acylation of steryl glycosides. 2. The lipase is derived from the genus Candida, Penicillium, Pseudomonas, Mucor, Geotricum or Chromobacterium.
The method described in. 3. The method according to claim 1, wherein the lower alcohol is one or more selected from aliphatic alcohols having 1 to 4 carbon atoms.