JPH09322794A - Production of phenolic acid saccharide ester - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject compound useful as a tumor necrosis factor production inhibitor having an antiinflammatory action, etc., by reacting a phenolic acid alcohol ester with a saccharide in the presence of an enzyme capable of cleavable the ester bond in a solvent. SOLUTION: A phenolic acid alcohol ester in which the phenolic acid- corresponding site of the phenolic acid alcohol ester is ferulic acid residue, p-coumaric acid residue, caffeic acid residue, sinapinic acid residue or cinnamic acid residue (e.g. trichloroethyl ferulate) is reacted with a saccharide such as an acidic saccharide, a neutral saccharide, an aminosaccharide or an oligo saccharide or polysaccharide containing these monosaccharides as constituting saccharides in the presence of an enzyme capable of cleaving the ester bond (e.g. lipase) in a solvent (e.g. pyridine) to obtain the objective phenolic acid saccharide ester useful as a tumor necrosis factor production inhibitor having an antiinflammatory action, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a phenolic acid sugar ester using an enzyme, and production of tumor necrosis factor (TNF) containing ferulic acid which is one of phenolic acids or a ferulic acid sugar ester as an active ingredient. Regarding inhibitors.

[0002]

BACKGROUND OF THE INVENTION Phenolic acid esters have various physiological activities. For example, it is known that ferulic acid ester, which is a typical phenolic acid ester, has a function of regulating the function of the diencephalon (Japanese Patent Publication No. 43-1972). Also, ferulic acid itself has an antioxidant effect (Toda S., Ki
mura M. and Ohnishi M .: Planta Medical 57,8-10 (199
0)), which is known to have anti-inflammatory activity (Chawla,
A, S. et al. Indian J. Exp. Biol).

Known methods for producing a ferulic acid sugar ester include a method of extracting from a natural product such as bamboo shoots and rice bran, and a method of synthesizing ferulic acid and sugar by dehydration condensation. However, in the method of extracting from a natural product, since a small amount of ferulic acid sugar ester is contained in the natural product, a large amount of natural product raw material is necessary to obtain a sufficient amount of ferulic acid sugar ester. Further, the method of chemically synthesizing has a poor yield and is not suitable for use as a food material.

As a method for synthesizing a sugar ester, there is known a so-called transesterification method in which an alcohol ester and a sugar are synthesized by allowing a lipase or the like to act under a condition with a small amount of water (JP-A-62-195292). Sho 60-70094, Japanese Patent Laid-Open No.
5-168489, JP 60-227687). However, due to the low negative charge voltage of the carbonyl carbon of ferulic acid, there are still no known examples of successful synthesis of phenolic acid sugar ester including ferulic acid sugar ester by the ester substitution method.

By the way, the inflammatory reaction is a vital defense reaction necessary for the living body in a normal state, but its overshoot leads to a pathological state. TNF was originally discovered as a substance that damages tumors, but recently it was found that it plays an important role in the inflammatory response (Yamazaki Masatoshi, Inflammation 10,163). Therefore, it is possible to reduce a pathological inflammatory condition by suppressing the production of TNF. Therefore, various substances have been investigated for TNF production inhibitory activity, but there are few substances having such activity, and they are only found in perilla extract and ginger extract (Japanese Patent Laid-Open No. 4-312058). -79852 publication, Masatoshi Yamazaki, Bioregulatory function of foods, pp. 423-430, published by Academic Publishing Center).

[0006]

DISCLOSURE OF THE INVENTION The present invention provides an efficient method for producing a phenolic acid sugar ester having various physiological activities and a new use of ferulic acid sugar ester, which is one of the compounds. The purpose is to do.

[0007]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventors have decided to produce a phenolic acid sugar ester by transesterification by devising various reaction conditions. We have succeeded, and have found that a ferulic acid sugar ester, which is a kind of this phenolic acid sugar ester, strongly suppresses the production of TNF, and completed the present invention.

That is, the present invention is a method for producing a phenolic acid sugar ester, which comprises reacting a phenolic acid alcohol ester with a sugar in a solvent using an enzyme capable of cleaving the ester bond. Further, the present invention is a TNF production inhibitor characterized by containing ferulic acid sugar ester or ferulic acid as an active ingredient. Hereinafter, the present invention will be described in detail.

1. Method for Producing Phenolic Acid Sugar Ester In the present invention, a phenolic acid sugar ester is produced by reacting a phenolic acid alcohol ester with a sugar in a solvent using an enzyme.

The phenolic acid alcohol ester used herein may be any one, but the phenolic acid equivalent site of the ester is a ferulic acid residue, a p-coumaric acid residue, a caffeic acid residue, a sinapinic acid residue or A cinnamic acid residue is preferred, and a ferulic acid residue is particularly preferred. The alcohol-corresponding portion of the ester is preferably a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and particularly 1 to 4 carbon atoms.
The halogen-substituted or unsubstituted alkyl group of is preferable. The halogen-substituted alkyl group may be any as long as it can form an ester bond with phenolic acid,
A halogen-substituted methyl group and a halogen-substituted ethyl group are preferable, and a halogen-substituted ethyl group is particularly preferable. CCl as the halogen-substituted methyl group and the halogen-substituted ethyl group
_{3 -, CHCl 2 -, CH} 2 Cl-, CF 3 -, CHF
_{2 -, CH 2 F-, CBr} 3 -, CHBr 2 -, CH 2 Br
_{-, CCl 3 CH 2 -,} CHCl 2 CH 2 -, CH 2 ClC
_{H 2 -, CF 3 CH 2} -, CHF 2 CH 2 -, CH 2 FCH 2
_{-, CBr 3 CH 2 -,} CHBr 2 CH 2 -, CH 2 BrC
H ₂ − and the like can be exemplified, and among these, C is particularly preferable.
Cl ₃ CH ₂ - is preferred. As the unsubstituted alkyl group,
Examples include CH ₃ −, C ₂ H ₅ −, C ₃ H ₇ −, C ₄ H ₉ −, and among these, C ₄ H ₉ − is particularly preferable.
Phenolic alcohol can be synthesized by dehydration condensation of phenolic acid and alcohol in the presence of sulfuric acid.

The saccharides in the present invention refer to aldehydes, ketones and acids of polyalcohols, as well as polyalcohols themselves, their derivatives and condensates, and include any of monosaccharides, oligosaccharides and polysaccharides. Specifically, arabinose,
Xylose, glucose, fructose, mannose,
Fucose, neutral monosaccharides such as galactose, dextrin, cyclodextrin, maltose, maltooligosaccharides, fructooligosaccharides, lactose sucrose, isomaltooligosaccharides, cellooligosaccharides, neutral oligosaccharides such as cellobiose, amylose, amylopectin, cellulose,
Neutral polysaccharides such as mannan, ascorbic acid, acidic monosaccharides such as N-acetylneuraminic acid, N-glycolneuraminic acid, polysialic acid, pectin, alginic acid, acidic polysaccharides such as carrageenan, N-acetylglucosamine, Examples thereof include amino sugars such as N-acetylgalactosamine, oligosaccharides of amino sugars such as chitooligosaccharides and chitosan oligosaccharides, and polysaccharides of amino sugars such as chitin and chitosan.

Any enzyme may be used as long as it can cleave the ester bond of a phenolic acid alcohol ester, for example, lipase, esterase,
Serine protease or the like can be used. As lipase, lipase derived from Wheat germ, Porcine panc
lipase from reas, lipase from Candida cylindracea, lipase from Pseudomonas species (above, Si
gma), lipase from Aspergillus niger, Muco
r javanicus-derived lipase, Candida rugosa-derived lipase, Rhizopus species-derived lipase (all manufactured by Amano Pharmaceutical Co., Ltd.), Candida cylindracea-derived lipase (produced by Meito Sangyo Co., Ltd.), Rhizopus japonicus-derived lipase (produced by Nagase & Co., Ltd.) Etc. can be used. Esterase derived from Porcine liver (Sigma
Streptomyces rochei-derived carboxylesterase (Wako Pure Chemical Industries), Candida cylindracea-derived cholesterol esterase (Seikagaku Corporation), Bo
Cholesterol esterase from vine pancreas, Po
Pseu, a cholesterol esterase derived from rcine liver
Cholesterol esterase derived from domonas species (above manufactured by Sigma) can be used. As the serine protease, thermolysin derived from Bacillus thermoproteolyticus, chymotrypsin derived from Bovine pancreas, subtilisin derived from Bacillus licheniformis (above manufactured by Sigma), protease derived from Bacillus subtilis (manufactured by Amano Pharmaceutical Co., Ltd.) and the like can be used. The enzyme can be immobilized on a suitable carrier for the purpose of improving pH and temperature, improving stability of organic solvent, maintaining activity, and reusing. As the immobilization carrier, cellulose, dextran, polystyrene, polyacrylamide, polyvinylalcohol, ion exchange resin, magnetic material, activated carbon,
Alumina, photo-crosslinked resin, alginate, celite and the like can be used.

Any solvent may be used, but when water is used as the solvent, the phenolic acid alcohol ester is hydrolyzed and the yield of the phenolic acid sugar ester is deteriorated. Further, in a nonpolar solvent such as hexane, neither the phenolic acid alcohol ester nor the phenolic acid sugar ester is easily dissolved, which is not preferable. Therefore,
In the present invention, it is preferable to use a solvent having polarity. Specifically, methanol, ethanol, isopropanol, isobutanol, acetone, dioxane,
Tetrahydrofuran, acetonitrile, dimethylformamide, pyridine, dimethylsulfoxide, 2-pyrrolidone, tert-amyl alcohol, ethyl ether, isopropyl ether and the like can be mentioned.

The mixing ratio of the phenolic acid alcohol ester and the sugar is not particularly limited, but for example, when pyridine is used as the solvent, it is preferably 10: 1 to 1:10 and 3: 1 to 1: 3. Is more preferable. The amount of enzyme used in the reaction is preferably 100 to 1,000,000 U, and more preferably 1000 to 100,000 U per 1 g of phenolic acid alcohol ester. The reaction temperature may be set so as to be the optimum temperature for the enzyme to be used. For example, as an enzyme, Pseudomonas spec
If ies-derived lipase or Aspergillus niger-derived lipase is used, 20 to 50 ° C. is preferable, and 4
0 to 45 ° C is more preferable. The reaction time varies depending on the amount of enzyme, the reaction temperature, etc., and may be determined according to the progress of the reaction. Usually, a sufficient amount of phenolic sugar ester is produced in about 48 to 72 hours.

In order to isolate and purify the phenolic acid sugar ester from the obtained reaction solution, first, the reaction solution is centrifuged (for example, 15000 rpm, 5 min), and the supernatant is subjected to a pore size of 0.22.
Remove insoluble matters such as enzymes with a filter of about μm.
Next, the obtained solution is concentrated to dryness, distilled water is added, and the water-soluble fraction is separated. From this aqueous solution fraction, for example, Iner
tsil ODS column was used (eluent 30% methanol)
The phenolic sugar ester fraction can be separated by HPLC.

2. TNF production inhibitor containing ferulic acid or ferulic acid sugar ester as an active ingredient The TNF production inhibitor of the present invention contains ferulic acid or ferulic acid sugar ester as an active ingredient.

Since ferulic acid is commercially available as a reagent, it can be used. The ferulic acid sugar ester can be produced from the ferulic acid sugar ester and sugar using an enzyme as described above, or can be extracted from a natural product containing the ferulic acid sugar ester such as rice bran. The method for extracting ferulic acid sugar ester from rice bran is, for example,
Ishii and Hiroi's method (Ishii T. and Hiroi T.:Carb
ohydr., 206,297,1990). In this method, the homogenized rice bran is defatted, treated with dricerase, and the ferulic acid sugar ester is isolated and purified by column chromatography.

Ferulic acid and ferulic acid sugar ester are
It has an action of suppressing the production of TNF. As mentioned above, TNF
Is closely related to the inflammatory reaction, and therefore, it is possible to suppress pathological inflammation by formulating ferulic acid or a ferulic acid sugar ester and administering it to the human body.

The method of administering the present TNF production inhibitor to the human body is not particularly limited, and it may be administered orally, parenterally, or by inhalation. Oral administration is generally preferred. In addition, it can be used as an external medicine which is formulated into a cream or the like and applied to a skin inflammation site.

In the present TNF production inhibitor, ferulic acid or a ferulic acid sugar ester can be compounded with a pharmacologically acceptable solid or liquid pharmaceutical carrier. Formulated T
NF production inhibitors include injections, tablets, capsules, powders,
It may be in the form of fine granules, liquid preparations, syrups, suspensions, emulsions and the like. Examples of the pharmaceutical carrier include excipients, binders, lubricants, coating agents, solubilizers, emulsifiers, suspending agents, stabilizers, solvents and the like which are usually used in such a form. The concentration of ferulic acid or ferulic acid sugar ester in the preparation may be determined depending on the form of the preparation. The dose of the present TNF production inhibitor in the human body varies depending on the form of preparation, the condition to be treated and the like, but is usually 5 to 20 mg / kg (body weight) per day for an adult.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION

[0022]

【Example】

[Example 1] Production of ferulic acid sugar ester <Synthesis of trichloroethyl ferulate> Ferulic acid (manufactured by Tsukino Food Industry Co., Ltd.) 3.88 g was suspended in benzene 150 ml, trichloroethanol 8.96 g, sulfuric acid 0.5 m.
Then, 5 g of molecular sieve 4A (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was refluxed for 1 hour. After cooling, the reaction solution was purified water 10
After washing twice with 0 ml each and drying over anhydrous sodium sulfate,
The solvent was distilled off. The residue was applied to silica gel (10 g) column chromatography and eluted with chloroform to obtain 3.2 ml of trichloroethyl ferulate.

<Synthesis of Butyl Ferulate> Ferulic Acid 1
0 g was dissolved in 200 ml of n-butanol, 6 ml of concentrated sulfuric acid was added to this mixed solution, a small amount of zeolite was added, and the mixture was heated under reflux for 1 hour in an eggplant flask with a cooling tube set. The reaction solution was neutralized with sodium carbonate, the generated butyl ferulate was extracted with ethyl acetate, and concentrated to dryness. The yield of butyl ferulate obtained in this way was 11.9 g.

<Synthesis of ferulic acid sugar ester> 80 mg of sugar was dissolved in 1 ml of pyridine heated in advance to 80 ° C. or higher, and 200 mg of ferulic acid alcohol ester obtained above was dissolved, and then Pseudomonas specie
s-derived lipase (25U / mg, Sigma) 24mg or
Aspergillus niger-derived lipase (60 U / mg, manufactured by Amano Pharmaceutical Co., Ltd.) 200 mg was added, and the mixture was stirred at 40 ° C. for 1 to 7 days,
The reaction was performed. The combination of sugar, ferulic acid alcohol ester and lipase used in the reaction is as follows.

Reaction liquid A: trichloroethyl ferulate +
D-Glucose + Aspergillus niger-derived lipase reaction solution B: Trichloroethyl ferulate + D-glucose + Pseudomonas species-derived lipase reaction solution C: Trichloroethyl ferulate + L-arabinose + Aspergillus niger-derived lipase reaction solution D: Trichloroethyl ferulate + D- Xylose + Lipase derived from Aspergillus niger Reaction solution E: Butyl ferulate + D-glucose + Asperg
illus niger-derived lipase reaction solution F: butyl ferulate + D-glucose + Pseudo
Lipase derived from monas species

The reaction solution obtained above was spotted on one end of a thin layer plate, and production of ferulic acid sugar ester was confirmed by thin layer chromatography. As the thin layer plate, a RP-18F _254s (0.25 mm, glass) plate manufactured by Merck was used, and development was performed using 30% methanol as a mobile phase.
Spots were confirmed at detection wavelengths of 254 nm and 365 nm, and sugars were detected by spraying a mixture of aniline, diphenylamine, phosphoric acid and acetone (4 ml: 4 g: 30 ml: 200 ml) and heating on a hot plate to develop color. Let

The results are shown in FIGS. 1, 2 and 3. FIG.
Shows the case where the sugar was colored, FIG. 2 shows the case where the detection wavelength was 365 nm, and FIG. 3 shows the case where the detection wavelength was 254 nm.
The numbers in the figure indicate the following.

1: Fr.5 described later, 2: Fr.6 described later,
3: ferulic acid, 4: reaction liquid A, 5: reaction liquid B, 6: reaction liquid C, 7: reaction liquid D, 8: reaction liquid E, 9: reaction liquid F

As shown in FIG. 1, in lanes 4 to 9, spots detected by coloring of sugar were confirmed at positions having higher polarity than ferulic acid. This material could also be detected with light at wavelengths of 254 nm and 365 nm as shown in FIGS. The Rf value of each lane is 0.25 for lane 1, 0.14 for lane 2, 0.10 for lane 3,
Lane 4 is 0.19, Lane 5 is 0.19, Lane 6 is 0.12, Lane 7 is 0.12, Lane 8 is 0.16,
Lane 9 was 0.16. From the above results, it was confirmed that glucose ferulate was produced in the reaction solutions A, B, E, and F, arabinose ferulate was produced in the reaction solution C, and xylose ferulate was produced in the reaction solution D.

[Example 2] <Separation and purification of ferulic acid sugar ester> Commercially available rice bran,
Six times the amount of distilled water was added, homogenized, and filtered on a stainless steel analytical sieve (40-60 mesh). After further washing with 24 volumes of distilled water, it was washed with 6 volumes of ethanol. Six times the amount of benzene: ethanol (2: 1) was added to the residue, and the lipid was extracted at room temperature for 17 to 20 hours. After removing the lipid extract using an analytical sieve, the residue was washed with 3 volumes of benzene: ethanol (2: 1) and then 6 times.
A defatted rice bran was prepared by washing with twice the amount of acetone.

The defatted rice bran thus obtained was suspended in 50 times the amount of distilled water, and then purified dolicerase was added at a ratio of 180 mg / l, and the pH was adjusted to 5 with acetic acid. After incubation at 30 ° C for 15 hours, the reaction was stopped by heating at 100 ° C for 15 minutes. The filtrate was separated using a brush cloth and concentrated by an evaporator. Five times the amount of ethanol was added to the concentrated solution, which was left overnight and the insoluble material was removed with paper. In addition, the purified dolicerase was obtained from Kyowa Fermentation using Fr.
It was prepared by purification by the method of y (Fry SC: Biochem. J. 203, 493, 1982). At this time, the cellulase activity and xylanase activity of dolicerase were determined by measuring the amount of purified reducing sugar using Avicel and xylan as substrates (So.
mogyi M.:J.Biol. Chem., 195,19,1952, Nelson N.:J.Bi
ol. Chem., 153, 375, 1944).

The obtained ethanol-soluble substance was treated with Sephadex.
Fractionation was performed with LH-20 (φ2.6 × 90 cm). Distilled water was used as the eluent. The peak was detected by UV absorption at 320 nm to detect phenolic acid, and the phenol-sulfuric acid method (Dubois M.et
al.:Anal.Chem.,28,350,1956) after the reaction 49
Sugar detection was performed at 0 nm. By this fractionation operation, the ferulic acid sugar ester fraction fraction 5 (Fr.5) and fraction 6 (Fr.6) were separated.

<Tumor Necrosis Factor Production Inhibition Test> 0.4 ml of each of the following test solutions and muramyl dipeptide 2 were added to a 7-week-old inbred C3H / He female mouse (purchased from Japan Biomaterials Co., Ltd.).
7 mg / kg was orally administered at the same time.

(1) 1% ferulic acid, (2) 5% ferulic acid, (3) perilla extract (manufactured by Amino Up Chemical),
(4) 1% Fr.5, (5) 1% Fr.6, (6) 5% Fr.6,
(7) Distilled water

(Control) After 3 hours, 25 μg of lipopolysaccharide was intravenously administered. Then, 2 hours later, blood was collected from the heart to separate serum. Three mice were used for each test solution to collect serum.

96 L of L929 cells (manufactured by Dainippon Pharmaceutical Co., Ltd.)
ll Flat bottom plate, 8.0 × 10 ⁴ pieces / 100 μl / wel
The sample serum obtained above was added with 50 μl of a 10 to 10 ⁸ -fold diluted solution of the sample serum obtained above. L929 cells are Eagle containing 10% fetal bovine serum (Nichirei Co., Ltd.)
An e's minimal essential medium (EMEM), which was subcultured every 3 to 4 days, was used. Also, in each well,
To enhance the sensitivity, actinomycin D (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the culture solution so that the final concentration was 1 μg / ml, and the mixture was cultured at 37 ° C. under 5% CO _{2 for} 18 hours.
After removing the medium, the cells were washed once with a phosphate buffer solution (PBS), added with 25 μl / well of 0.5% crystal violet-20% methanol, and stained for 20 minutes. And each
The well was washed with PBS and dried, then 0.5% SDS was added at 100 μl / well, shaken with a micromixer, and the absorbance at 540 nm was measured with a microplate radar (Bio-Rad). The amount (U) of tumor necrosis factor (TNF) in serum was calculated assuming that the concentration of cytocidal activity at 50% was 1U. FIG. 4 shows the results.

As shown in FIG. 4, not only perilla extract, which was conventionally known to have a TNF production inhibitory effect, but also ferulic acid, Fr.5 and Fr.6 inhibited TNF production. Particularly, in the case of Fr.6, when it was administered at a concentration of 5%, the production amount of TNF was suppressed by 80%. On the other hand, in the case of ferulic acid, even if the concentration was increased to 5%, a large suppressing effect like Fr.6 was not recognized.

Reference Example 1 Preparation of Polyethylene Glycol Modifying Enzyme Preparation of polyethylene glycol (PEG) modifying enzyme was carried out using In
ada et al. (InadaY. et al: Biochemical and Biophys
ical Reseach Communications., 122, 845, 1984). First, 0.4M borate buffer (pH 1
0) 18 mg of Candida cylindracea-derived lipase (Meito Sangyo Co., Ltd.) was dissolved in 3.2 ml, and 0.8 g of methoxypolyethylene glycol (activated PEG, Sigma) activated by cyanuric chloride was added to this solution. 37 ℃,
Stir for 1 hour. 0.1 M borate buffer (pH 8.
0) After adding 100 ml to stop the reaction, unreacted PE
G was removed by ultrafiltration (Amicon PM30 membrane). Next, it was dialyzed in distilled water and freeze-dried to obtain a white dried product.

Example 3 <Synthesis of ferulic acid glucose ester> Ferulic acid 1
Dissolve 00 g in 500 g of trichloroethanol, add 0.5 ml of sulfuric acid and molecular sieve (MS) 4A, and add 7
The reaction was carried out at 0 ° C for 1 hour. After cooling, trichloroethanol was distilled off, and the residue was converted to silica gel (Wakogel C-200, 75
0 g) Load on a column chromatograph, chloroform:
Elute the reaction product with methanol (50: 1) and
0 g of trichloroethyl ferulate were obtained. The purity of the product was analyzed by HPLC using an Inertsil Sil column (GL Science). The molecular weight of the product was analyzed by a gas mass spectrometer (Shimadzu QP-5000).

Pyridine 5 dehydrated with anhydrous sodium sulfate
40 g of D-glucose was dissolved in 00 ml, and 40 g of the above trichloroethyl ferulate was dissolved in this solution. Next, lipase A6 (manufactured by Amano Pharmaceutical Co., Ltd.) was added to this solution and reacted at 40 ° C. for 3 days.

After removing the insoluble matter of the obtained enzyme synthesis reaction solution with filter paper (Advantec 5A), the solvent was distilled off and the residue was dissolved in methanol. The methanol-soluble fraction was redissolved in distilled water and centrifuged, and the supernatant was C-18 (GL Science).
Purified on column. After eluting with distilled water, 30% ethanol and methanol were successively eluted. After concentrating the 30% methanol elution fraction, reverse phase HPLC (Inertsil ODS-80A:
GL Science), 4 peaks a to d
Two peaks were recognized (Fig. 5). Of these components, only the component of peak a was separated again by reverse phase HPLC, and it was separated into two components, peak a and peak c (FIG. 6). Thus, the two peaks were considered to be due to the difference in the glucose anomeric structure, and the following structural analysis was carried out with the components of peak a and peak c as a mixture.

<Infrared absorption spectrum (IR) measurement> IR
The measurement was carried out by the KBr tablet method using FTIR-8200PC (manufactured by Shimadzu Corporation). As a result of the measurement, -OH around 3400 cm ^-1
(Stretching), -CH (stretching) and -CH near 2940 cm ^-1
₂ and CH ₃ (reverse symmetrical expansion and contraction), 1692 cm ^-1 at -C = O
(Stretch), aromatic -C = C- at 1632 cm ^-1 (stretch), aromatic -C = C at 1599 cm ^-1 , 1518 cm ^-1
-(In-plane skeletal vibration), an absorber derived from sugar type absorption was observed in the fingerprint area at 900 to 800 cm ^-1 . Since the absorption at 1667 cm ^-1 observed with ferulic acid was shifted to the high wave number side to 1692 cm ^-1 , the existence of an ester bond was considered.

<FAB-MS measurement> FAB-MS measurement was carried out with a ZAB-HF mass spectrometer (VG). Glycerol is used for the matrix and the ion acceleration voltage is 8.0 kV
Was measured. Measurement results, positive FAB-MS
To m / z 357 (M + H) ⁺ (Fig. 7), negative FAB-MS
At m / z 355 (MH) ^- (Fig. 8), a strong molecular ion peak was observed. From this result, the molecular weight of the compound was 356.
It was found that it was equal to the molecular weight of ferulic acid and glucose which were ester-bonded.

<Measurement of Nuclear Magnetic Resonance Spectrum> A UNITY INOVA 600 type was used for NMR measurement. ^In the case of ¹ H-NMR, D ₂ O containing 5% CD ₃ OD is used as a solvent,
HDO was measured as an internal standard. ¹³ C-NMR used CD ₃ OD as an internal standard. Also, DQF-C
OSY measurement and HMQC ( ¹ H observation CH-COSY) measurement were also performed. The measurement results of ¹ H-NMR are shown in Table 1, and ¹³ C-N
Table 2 shows the measurement results of MR.

[0045]

[Table 1]

[0046]

[Table 2]

From ¹ H-NMR, a chemical shift of the anomeric proton of glucose was confirmed, and it was considered that peaks a and c were due to the difference in the anomeric structure. Furthermore, the fact that the binding constant J _7.8 was 15.9 Hz indicates that ferulic acid exists as a trans form. Also,
^{From 13} C-NMR, the bonding position of ferulic acid was 62 ppm signal at C-6 position of unsubstituted glucose, and it was shifted to 64.5 ppm in a low magnetic field. It was shown to be attached at the -6 position. That is, it was revealed that peak a was 6-O-feruloyl-β-glucopyranoside and peak c was 6-O-feruloyl-α-glucopyranoside (FIG. 9).

[0048]

INDUSTRIAL APPLICABILITY The present invention provides a novel method for producing a phenolic acid sugar ester utilizing an ester substitution reaction. The present invention also provides a novel TNF production inhibitor containing ferulic acid or a ferulic acid sugar ester as an active ingredient. T
Since NF is a cytokine associated with inflammation in the human body, the present inhibitor that suppresses this production can be used as an anti-inflammatory agent.

[Brief description of drawings]

FIG. 1 is a photograph showing a thin layer chromatogram of an enzyme synthesis product.

FIG. 2 is a photograph showing a thin layer chromatogram of an enzyme synthesis product.

FIG. 3 is a photograph showing a thin layer chromatogram of an enzyme synthesis product.

FIG. 4 is a graph showing the effect of a ferulic acid sugar ester fraction on the suppression of TNF production in mice.

FIG. 5 is a diagram showing the results of analysis of a 30% methanol elution fraction by reverse phase HPLC.

FIG. 6 is a diagram showing the results of analysis of components of peak a by reverse phase HPLC.

FIG. 7 is a diagram showing a FAB-MS spectrum (positive ions).

FIG. 8 is a diagram showing a FAB-MS spectrum (negative ions).

FIG. 9 shows the structure of glucose ferulic acid ester.

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 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Keitaro Kikuta 1102-2 Mikura, Omiya City, Saitama Prefecture (72) Inventor Hitoshi Aoki 4-2-21 Yayoi-cho, Nakano-ku, Tokyo

Claims (13)

[Claims] 1. A process for producing a phenolic acid sugar ester, which comprises reacting a phenolic acid alcohol ester with a sugar in a solvent using an enzyme capable of cleaving the ester bond. 2. A phenolic acid equivalent site of a phenolic acid alcohol ester is a ferulic acid residue, a p-coumaric acid residue, a caffeic acid residue, a sinapinic acid residue or a cinnamic acid residue. Item 1. A method for producing a phenolic acid sugar ester according to Item 1. 3. The method for producing a phenolic acid sugar ester according to claim 1, wherein the alcohol equivalent site of the phenolic acid alcohol ester is a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms. . 4. The method for producing a phenolic acid sugar ester according to claim 3, wherein the substituted or unsubstituted alkyl group having 1 to 4 carbon atoms is a halogen-substituted or unsubstituted alkyl group. 5. The method for producing a phenolic acid sugar ester according to claim 4, wherein the halogen-substituted or unsubstituted alkyl group is a butyl group or a halogen-substituted ethyl group. 6. The halogen-substituted ethyl group is CCl ₃ CH _{2
-, CHCl 2 CH 2 -,} CH 2 ClCH 2 -, CF 3 CH 2
_{-, CHF 2 CH 2 -,} CH 2 FCH 2 -, CBr 3 CH 2
-, CHBr ₂ CH ₂ -, or CH ₂ BrCH ₂ - preparation of phenolic acid sugar esters according to claim 5, characterized in that the. 7. The phenol according to any one of claims 1 to 6, wherein the sugar is an acidic sugar, a neutral sugar, an amino sugar, or an oligosaccharide or a polysaccharide having them as a constituent sugar. Method for producing acid sugar ester. 8. An acidic sugar, a neutral sugar, an amino sugar, or an oligosaccharide or a polysaccharide having them as constituent sugars is L-arabinose, D-xylose, D-glucose, D-fructose, D-galactose, L-. Fucose, D-mannose, ascorbic acid, N-acetylneuraminic acid, N-
Glycolneuraminic acid, polysialic acid, N-acetylglucosamine, N-acetylgalactosamine, dextrin, cyclodextrin, amylose, amylopectin, pectin, cellulose, serobios, cellooligosaccharide, mannan, alginic acid, carrageenan, maltose, maltooligosaccharide, fructooligosaccharide, The method for producing a phenolic acid sugar ester according to claim 7, which is lactose sucrose or isomaltooligosaccharide. 9. The method for producing a phenolic acid sugar ester according to claim 1, wherein the enzyme capable of cleaving the ester bond is esterase, lipase, or serine protease. 10. The method for producing a phenolic acid sugar ester according to claim 1, wherein the solvent is a polar solvent. 11. The polar solvent is methanol, ethanol, isopropanol, isobutanol, acetone, dioxane, tetrahydrofuran, acetonitrile, dimethylformamide, pyridine, dimethyl sulfoxide, 2-pyrrolidone, tert-amyl alcohol, ethyl ether, or isopropyl. It is ether, The manufacturing method of the phenolic acid sugar ester of Claim 10 characterized by the above-mentioned. 12. A tumor necrosis factor production inhibitor comprising a ferulic acid sugar ester as an active ingredient. 13. A tumor necrosis factor production inhibitor, which comprises ferulic acid as an active ingredient.