JP2008174488A - Method for producing phenyl glucoside derivative from raw sugar - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an industrial method for refining and producing a fraction comprising phenyl glucoside derivative contained in raw sugar using water alone without using an organic solvent. <P>SOLUTION: The method for isolating a fraction comprising a phenyl glucoside derivative is characterized by subjecting an aqueous solution of raw sugar to column chromatography wherein a strong acid type ion exchange resin is used as a support and water is used as an eluent to isolate a fraction having a maximum absorption at an ultraviolet wavelength of 272 nm. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to a method for separating a phenylglucoside derivative contained in crude sugar.
More specifically, the present invention relates to an industrial separation method using a strongly acidic ion exchange resin and water of a fraction containing a phenylglucoside derivative which is contained in crude sugar and exhibits maximum absorption with respect to ultraviolet rays having a wavelength of 272 nm.

  It has already been clarified that the black substance (hereinafter also referred to as a pigment component) contained in the non-sucrose fraction in brown sugar has an inhibitory action on the increase of neutral fat and insulin in serum (for example, non-patent literature) 1). It has also been reported that the active ingredient is considered to be 3,4-dimethoxyphenyl-β-D-glucoside or 2,4-dimethoxyphenyl-β-D-glucoside (for example, Non-Patent Document 2). ).

  Furthermore, it has been clarified that the black substance has an antiallergic action, can prevent and improve arteriosclerosis (for example, Patent Document 1), and has effects such as skin activation / whitening action (for example, patents). Reference 2).

  And in any case, the manufacture of the sucrose-free pigment component extract having the above-mentioned effects in the above-mentioned literature is activated carbon (Non-patent Literature 2) or amber resin which is a polystyrene resin in which the crude sugar is dissolved in water. Adsorbed to a non-polar adsorbent such as XAD-2 (Patent Documents 1 and 2), eluted with water until no sweetness is found, then eluted with 20-30% alcohol to elute the dye component and elute When the color of the liquid almost disappeared, it was eluted with 95-99% high-concentration alcohol to completely elute the dye components, and these alcohol-containing eluates were combined and evaporated to dryness to give about 0. A column chromatography method using a non-polar adsorbent has been used that yields a pigment component purified extract powder that does not contain sucrose in a yield of 5%.

As the alcohol used in the above method, ethanol is used because the purified extract powder is used for pharmaceuticals and cosmetics. For example, to obtain 375 g of pigment component purified extract powder from 75 kg of crude sugar, about 200 L of 20% ethanol, Approximately 100 L of 95% ethanol was required.
And, the above-mentioned ethanol distillation recovery requires labor and expense, and further involves the risk of ignition.

Kimura et al., Pharmaceutical Journal, 102 (7), 1982, 666-669. Kimura et al., Japanese and Chinese Journal of Medicine, Vol1, No. 1, 1984, 178-179 JP-A-57-88107 Patent 59-48809

  Therefore, industrial use for purifying a fraction containing 3,4-dimethoxyphenyl-β-D-glucoside, which is a phenylglucoside derivative contained in crude sugar, using only water without using an organic solvent such as ethanol as described above. Development of a simple purification method and manufacturing method has been awaited.

  As a result of extensive research, the present inventors have used a strongly acidic ion exchange resin as a support instead of the non-polar adsorbent described above, so that only the water is used as an eluent. , 4-dimethoxyphenyl-β-D-glucoside-containing fractions were found industrially purified, and this study was completed.

  Thus, according to the present invention, a crude sugar aqueous solution is subjected to column chromatography using a strongly acidic ion exchange resin as a carrier and water as an eluent, and a fraction exhibiting maximum absorption with respect to ultraviolet rays having a wavelength of 272 nm is fractionated. A method for separating a fraction containing a phenylglucoside derivative is provided.

  According to the present invention, an inexpensive and safe industrial purification of a fraction containing a phenylglucoside derivative, particularly a fraction containing 3,4-dimethoxyphenyl-β-D-glucoside, with only a strongly acidic ion exchange resin and water. A manufacturing method is provided.

The term “crude sugar” used in the present invention means any of crude sucrose, crude sucrose extract or dried product thereof obtained from sugarcane or sugar radish.
In addition, column chromatography using a strongly acidic ion exchange resin as a carrier and water as an eluent used in the present invention is not only a chromatography using a normal glass column, but also a larger, dispersed in water. It also means a chromatography method using a metal purification tower packed with the above ion exchange resin.

  The strong acid type ion exchange resin may be either a sodium type or a potassium type ion exchange resin, and any gel type or porous type ion exchange resin can be used. However, the elution rate and the ease of regeneration of the used resin can be improved. A gel type is preferable from the viewpoint.

  Examples of the gel-type sodium-type or potassium-type strongly acidic ion exchange resin that can be used in the present invention include SK1B, SK104, SK110, SK112, SK116, PK208, PK212, PK216 as commercially available Diaion (trademark) series. , PK220, PK228, HPK25, UBK530, UBK535, UBK550 and UBK555 (above, manufactured by Mitsubishi Chemical Corporation), IR120B, IR120BN, IR124, XT1006CP, IR118, IIR120B, IIR120BN, Amberlist (trademark) 31, Amberlite CG120 for chromatograph, CG6000 (above, manufactured by Organo Corporation), Dowex (trademark) series, 50WX8 200, 50WX4-400, 50WX2-100, 50WX4-200, 50WX2-200, 50WX2-400, 50WX8-400, 50WX8-100, HCR-W2, HCR-S (above, manufactured by Dow Chemical Japan Co., Ltd.) Can be mentioned.

  Among the above, Diaion (trademark) UBK series for chromatographic separation is preferable, and UBK530 is more preferable from the viewpoint of availability, price, and resolution.

  The term “water” used in the present invention means deionized water, distilled water or deionized distilled water, but deionized water is sufficient for the use of the present invention.

  In addition, one of the features of the present invention is that the sodium-type or potassium-type strongly acidic ion exchange resin in the present invention can be used as it is simply by purchasing the above-mentioned commercial product and washing it with deionized water. .

  In addition, the amount of the ion exchange resin used in the present invention is preferably 10 to 20 times (volume ratio), more preferably 15 to 18 times (volume ratio) with respect to 1 kg of the crude sugar powder.

  In the industrial production method of a phenylglucoside derivative from a crude sugar according to the present invention, a crude sugar aqueous solution is subjected to column chromatography using a strongly acidic ion exchange resin as a carrier and water as an eluent, and has a maximum absorption with respect to ultraviolet rays having a wavelength of 272 nm. This is done by fractionating the fractions indicating.

  More specifically, for example, a crude sugar aqueous solution is prepared by dissolving crude sugar in 2.5 times (volume ratio) of water and filtering off insoluble matter. Then, according to a conventional method, the crude sugar aqueous solution is poured from the top of the column packed with a strongly acidic cation exchange resin onto the top of the packing with water as an eluent, and the eluent is dropped from the column to adsorb the crude sugar onto the packing. Let

  Next, the sucrose is eluted by elution from the column at a flow rate of 6 to 8 L / hour using 70 to 90 times the volume (volume ratio) of the crude sugar at room temperature. When the eluate is fractionated from 3 to 5 L from the start of coloration, it has no sweetness, shows a value of 0 by the sugar content Brix of the saccharimeter, and shows a light absorbance of 1.0 or more in the ultraviolet absorption spectrum at a wavelength of 272 nm. A yellow elution fraction can be obtained. The elution fractions thus obtained are combined and concentrated under reduced pressure at a temperature of 60 ° C. or lower to obtain a phenylglucoside derivative-containing fraction, and further concentrated to dryness under reduced pressure to obtain a concentrated dry powder.

Therefore, the term “phenylglucoside derivative-containing fraction” used in the present invention means any of a phenylglucoside derivative-containing eluate, a vacuum concentrate of the eluate, and a concentrated dry powder.
The strongly acidic ion exchange resin used in the present invention can be regenerated and used repeatedly after use.

The properties of the phenylglucoside derivative-containing purified concentrated dry powder obtained by the production method according to the present invention are exactly the same as those obtained by the conventional adsorbent method described in Non-Patent Document 2, and show the following properties.
(1) The concentrated dry powder is a tan brown slightly hygroscopic powder, soluble in water and alcohol, and insoluble in hexane petroleum ether and ether.
(2) A 1% aqueous solution of the concentrated dry powder has a pH of about 7.5.

(3) Infrared absorption spectrum ν _max (Nujol; cm ⁻¹ ): 3300, 1590, 1020 and 720.
(4) UV absorption spectrum λ _max (nm) (water): 272, 320.
(5) When 2 to 3 drops of 5% aqueous solution of the above-concentrated dry powder is added to 5 mL boiling boiling reagent solution, a red precipitate is formed. Moreover, it is negative even if ferric chloride test solution is added to 5% aqueous solution of this product. Furthermore, precipitation does not occur even when a gelatin test solution is added to a 5% aqueous solution.
(6) Thin layer chromatography

When 10 mg of the above concentrated dry powder is dissolved in 1 mL of water and tested by the Japanese Pharmacopoeia General Test Method Thin-layer Chromatography under the conditions described below, a single Rf between about 0.6 and 0.85 is used. A marked red spot.
Carrier: Silica gel 60F ₂₅₄ (Merck, thickness 0.25mm)
Sample attachment amount: 10 μL
Developing solvent: Chloroform: Methanol: Water (65:35:10) lower layer Developing distance: 10 cm
Detection: Heated at 105 ° C for 5 minutes after spraying with p-anisaldehyde reagent solution

(7) Crystallization of 3,4-dimethoxyphenyl-β-D-glucoside from concentrated dry powder When the above concentrated dry powder is recrystallized from isopropyl alcohol, a white crystalline powder having a melting point of 154-156 ° C is obtained. can get.

  When the crystalline powder was subjected to thin layer chromatography under the conditions described in (6) above, a remarkable red spot appeared in Rf0.8.

Elemental analysis, mass spectrometry and ¹ H-NMR spectrum (400 MHz, CD ₃ OD) of the above crystalline powder were measured and found to be completely consistent with the data of 3,4-dimethoxyphenyl-β-D-glucoside. Powder was confirmed to be 3,4-dimethoxyphenyl-β-D-glucoside.

  In addition, HPLC analysis of the above crystalline powder (column: Phenomenex Synergi 4u Fusion-RP 80A (250 mm × 4.6 mm), mobile phase: 0.1% phosphoric acid aqueous solution; acetonitrile = 95%; 5%, detection wavelength: 282 nm) As a result, it was found that the purity was 96% or more.

  Therefore, the term “phenylglucoside derivative-containing fraction” used in the present invention means a fraction containing 3,4-dimethoxyphenyl-β-D-glucoside as a main component.

Comparative Example 1
Conventional adsorbent method 5 kg of Okinawa brown sugar is dissolved in 25 L of water, and 500 g of Amberlite XAD-2, which is a polystyrene-based adsorbent resin, is dispersed in 1.5 L of water and injected into a column with an inner diameter of 8 cm as an eluent. The water was allowed to flow down at a rate of 20 mL / min to adsorb the dye, then the column was eluted with water until the sweetness disappeared, and the sugar was completely eluted.

The eluent was then changed to 20% ethanol and eluted at a rate of 10 mL / min to elute the black material in brown sugar from the adsorbent. Elution was continued with the same eluent until the eluate disappeared.
After the eluate became transparent, the eluent was changed to 95% ethanol, elution was continued, and the same operation was performed until the eluate disappeared. The fraction obtained with 95% ethanol was concentrated to dryness under reduced pressure at 60 ° C. or lower to obtain 6 g of a black brown residue. This was dissolved in 2 L of ethanol by heating, filtered after cooling, concentrated again under reduced pressure at a temperature of 60 ° C. or lower, and further concentrated to dryness under reduced pressure to obtain 5.2 g of brown powder having no sweetness.

Example 1
Production by Ion Exchange Resin Method 1 kg of crude sugar powder was dissolved in 2.5 L of deionized water, and an aqueous solution of crude sugar obtained by filtering insolubles was obtained. This aqueous solution was injected into the upper part of a column in which 17 L of a sodium cation exchange resin (Mitsubishi Chemical UBK530) was dispersed and packed in deionized water according to a conventional method, and adsorbed on the resin.
Next, at room temperature, 85 L of ion exchange water was used to elute sucrose by elution from the above column at a flow rate of 8 L / hour.

After elution of sucrose, when the eluate from the column started to color, it was fractionated by 4 L to obtain fractions 1-20. Each fraction was measured with a sugar content meter (MASTE, manufactured by Atago Co., Ltd.). Fractions 8 to 16 having a sugar content Prix of 0, no sweetness, and a maximum ultraviolet absorption wavelength of 272 nm and an absorbance of 1.0 or more were combined. The eluate was concentrated under reduced pressure at 60 ° C., and the residue was further concentrated to dryness under vacuum at 60 ° C. and dried to contain a phenylglucoside derivative containing 3,4-dimethoxyphenyl-β-D-glucoside as a main component. 5 g of dried powder was obtained in 0.5% yield.
In addition, the absorption spectrum of the ultraviolet part and visible part of the fraction containing a phenyl glucoside derivative containing 3,4-dimethoxyphenyl-β-D-glucoside as a main component is shown in FIG.

Example 2
Production of 3,4-dimethoxyphenyl-β-D-glucoside 1 kg of the dry fraction of the phenylglucoside derivative-containing fraction obtained by the method described in Example 1 was dissolved in 60 L of deionized water, and the insoluble matter was filtered off. A separate aqueous solution of crude sugar was obtained. This aqueous solution was injected into the upper part of a resin tower (column) filled with 50 L of a sodium cation exchange resin (UBK 530 manufactured by Mitsubishi Chemical) suspended in deionized water and adsorbed on the resin.

Next, at room temperature, 250 L of deionized water was used and eluted from the column at a rate of 25 L / hour to elute sucrose.
After elution of sucrose, when the eluate from the column started to color, it was fractionated by 12.5 L to obtain fractions 1-20. In the thin layer chromatography, a red spot was confirmed at an Rf of about 0.8, and fractions 7 to 13 having a maximum ultraviolet absorption wavelength of 272 nm were combined, concentrated under reduced pressure at 60 ° C., and further concentrated to dryness under vacuum to remain. 65 g of product was obtained.

The residue was dissolved by heating in 3.5 L of isopropyl alcohol and recrystallized to obtain 8.4 g of a crystalline powder of crude 3,4-dimethoxyphenyl-β-D-glucoside.
This crude crystal powder was dissolved in 8 L of deionized water, and again injected from the upper part of the resin tower packed with 6.5 L of sodium-type strongly acidic ion exchange resin (UBK 530, manufactured by Mitsubishi Chemical Corporation) and adsorbed on the resin.

Using 30 L of deionized water, the column was eluted from the column at a flow rate of 4 L / hour. After the flow-down solution was slightly colored, the solution was fractionated by 1.5 L and fractions 1 to 20 were collected.
In fractions 1 to 20, the fractions 9 to 14 in which a single red spot can be confirmed by Rf0.8 by the thin layer chromatography described above were combined, concentrated under reduced pressure at 60 ° C., and further concentrated to dryness under vacuum. 4.2 g of residue was obtained.

  This residue was dissolved in 200 mL of hot isopropyl alcohol, recrystallized and dried to obtain 0.8 g of 3,4-dimethoxyphenyl-β-D-glucoside, a white crystalline powder having a melting point of 155 to 156 ° C. Obtained.

The resulting white crystalline powder was completely consistent with the 3,4-dimethoxyphenyl-β-D-glucoside preparation in mass spectrometry, HPLC analysis and ¹ H-NMR spectrum.

  According to the present invention, without using an organic solvent, a fraction containing a phenylglucoside derivative, particularly a fraction containing 3,4-dimethoxyphenyl-β-D-glucoside, which contains only a strongly acidic ion exchange resin and water, is inexpensive. And a safe industrial production method is provided.

It is an absorption spectrum in the ultraviolet and visible part of a fraction containing a phenylglucoside derivative mainly composed of 3,4-dimethoxyphenyl-β-D-glucoside.

Claims (4)

  Phenylglucoside characterized by subjecting a crude sugar aqueous solution to column chromatography using a strongly acidic ion exchange resin as a carrier and water as an eluent, and fractionating a fraction exhibiting maximum absorption with respect to ultraviolet rays having a wavelength of 272 nm A method for separating a derivative-containing fraction.   The separation method according to claim 1, wherein the phenylglucoside derivative is 3,4-dimethoxyphenyl-β-D-glucoside.   The separation method according to claim 1, wherein the strongly acidic ion exchange resin is a sodium or potassium ion exchange resin.   The separation method according to claim 1, wherein the crude sugar is crude sucrose obtained from sugarcane or sugar radish.

Images