WO2014092175A1

WO2014092175A1 - Specific polyphenol used in food and drink products, supplements, pharmaceuticals, and the like and method for manufacturing said specific polyphenol

Info

Publication number: WO2014092175A1
Application number: PCT/JP2013/083429
Authority: WO
Inventors: みどり夏目; 公一郎角
Original assignee: 株式会社明治
Priority date: 2012-12-14
Filing date: 2013-12-13
Publication date: 2014-06-19
Also published as: CN104837824A; CN104837824B; JP6297501B2; HK1207646A1; JPWO2014092175A1

Abstract

The present invention addresses the problem of providing a method for manufacturing a comparatively high-purity ≥4-mer polyphenol by precisely isolating said ≥4-mer polyphenol from a polyphenol mixture. This method for manufacturing a specific polyphenol uses a non-acid-containing eluent to isolate a ≥4-mer polyphenol from a polyphenol mixture via gel filtration fractionation, e.g. liquid chromatography or gel filtration chromatography.

Description

Specified polyphenols used in foods, drinks, supplements and pharmaceuticals, etc.

The present invention relates to a specific polyphenol useful for foods and drinks, supplements, pharmaceuticals, and the like, and a method for producing the same.

Polyphenols are said to "suppress the action of active oxygen, which is said to cause various diseases such as cancer and arteriosclerosis", and "have an allergy suppressing effect and a stress suppressing effect". For this reason, plants containing polyphenols, processed products and extracts of the plants, and the like are widely used as raw materials and additives in the fields of foods and drinks, supplements and pharmaceuticals. In particular, cacao is richer in polyphenols than other plants. For this reason, cocoa powder and cocoa extract are attracting particular attention as such raw materials and additives.

Examples of the cocoa-derived polyphenol (hereinafter also referred to as “cocoa polyphenol”) include, for example, a monomer catechin, a dimer procyanidin B2, a trimer procyanidin C1, and a tetramer cinnamtannin A2. Etc.

By the way, conventionally, what gives various actions and effects as described above to a living body is a cocoa polyphenol having a degree of polymerization of 3 or less (that is, monomer, dimer and trimer) that can be absorbed from the intestinal tract. Has been considered to be a polyphenol component. Under such circumstances, “a method for separating (±) -catechin, (±) -epicatechin and their procyanidins (including dimers and trimers) from cacao polyphenols” has been proposed in the past ( For example, Japanese translations of PCT publication No. 2009-501161), “Methods for extracting and separating“ catechin, epicatechin and their procyanidins ”or“ higher procyanidins ”from cacao polyphenols” have been proposed (for example, Japanese translation of PCT publication 2003 -535111).

Special table 2009-501161 Special table 2003-535111 gazette

However, in recent years, it has been found that polyphenols of tetramer or higher also have beneficial actions, effects, or uses.

Therefore, an object of the present invention is to provide a method for producing a polyphenol having a relatively high purity and a tetramer having a relatively high purity by accurately separating the polyphenol having a tetramer or more from the polyphenol mixture.

In the method for producing a specific polyphenol according to the first aspect of the present invention, an eluent containing no acid (hereinafter also referred to as “acid-free eluent”) is used to convert a polyphenol of tetramer or higher from a polyphenol mixture into a gel filtration fraction. Drawn. Here, “polyphenol” is a general term for plant components having a plurality of phenolic hydroxyl groups. The “polyphenol mixture” referred to here is, for example, a cacao extract. Further, the tetramer or higher polyphenol (hereinafter also referred to as “specific polyphenol”) is preferably a tetramer or higher procyanidin. “Procyanidin” is an oligomer of catechins such as catechin and epicatechin. The upper limit of the degree of polymerization of polyphenol or procyanidin is preferably 20. Further, for the gel filtration fraction, for example, liquid chromatography (including high performance liquid chromatography) or gel filtration chromatography can be used. At this time, it is preferable to use silica particles chemically bonded with glyceropropyl groups as the gel filtration material.

In this specific polyphenol production method, it is preferable to change the composition of the acid-free eluent as the eluent in the gel filtration fractionation. The composition of the acid-free eluent is preferably changed in at least four stages. It is because it can suppress effectively that this specific polyphenol mixes the polyphenol below a trimer.

As a result of intensive studies by the inventors of the present application, when this specific polyphenol production method is used, a tetraphenol or higher polyphenol is accurately separated from a polyphenol mixture to produce a relatively high purity tetramer or higher polyphenol. It became clear that it could be done. At this time, it is preferable that this specific polyphenol does not contain catechin, epicatechin and procyanidins of trimer or less. However, this means “substantially” free of catechins, epicatechins and trimeric procyanidins. That is, this specific polyphenol has a ratio of “total mass of polyphenols of tetramer or more” to “total mass of catechin, epicatechin and polyphenols of trimer or less” of 9 or more, preferably 10 or more, more preferably 11 In other words, it can be paraphrased as a polyphenol mixture, more preferably 12 or more. The upper limit of this ratio is infinity (∞), and the higher the ratio, the better. For example, the upper limit of this ratio is 100.

The specific polyphenol according to the second aspect of the present invention has a polyphenol content of trimer or less of 10% by mass or less. The content of polyphenols of trimer or less is preferably 9% by mass or less, more preferably 8% by mass or less, further preferably 7% by mass or less, and 6% by mass or less. Particularly preferred is 5% by mass or less.

At this time, for example, when cacao polyphenol is used as the polyphenol, from the viewpoint of safety, flavor, physical properties, etc., the specific polyphenol is a raw material for food and drink (material, composition), a raw material for supplement (material, composition). And as a raw material for pharmaceuticals (raw material, composition) and the like, more preferably as a raw material for food and drink and a raw material for supplement, and further more preferable as a raw material for food and drink.

3 is a chart showing the results of gel filtration fractionation according to Example 1. It is a chart which shows the result of the gel filtration fractionation concerning Example 2. 2 is a chart showing analysis results of the content of high polymerization procyanidins contained in each fraction after gel filtration fractionation according to Example 2 (a) cocoa extract (CLPr), b) low polymerization fraction (CLPr− L), c) High polymerization fraction (CLPr-H)). It is a chart which shows the result of the gel filtration fraction which concerns on the comparative example 1.

The tetrameric or higher cocoa procyanidins (hereinafter also referred to as “highly polymerized procyanidins”) according to an embodiment of the present invention can be obtained by subjecting a cocoa extract to gel filtration fractionation. In this gel filtration fractionation, an “eluent containing no acid” is used as the eluent. In addition, a cocoa extract may purchase a commercial item etc., and may extract it from a cocoa bean and cacao powder by a well-known method. The “cocoa beans” that can be used in the embodiment of the present invention are not limited by the production area, the growth situation, the presence or absence of roasting, and the like.

For gel filtration fractionation, known means such as high performance liquid chromatography (HPLC) and gel filtration chromatography (GFC) can be used. From the viewpoint of ease of operation and reproducibility, it is preferable to use HPLC at the laboratory scale, and it is possible to scale up to the pilot plant scale or the actual equipment scale as long as fractionation efficiency equivalent to the laboratory scale can be obtained. It is. In such a case, as one embodiment of the gel filtration material, “silica particles chemically bonded with glyceropropyl groups” for normal phase separation are used to elute from molecules with low polarity (in the present invention, polyphenols having a low degree of polymerization). It is preferable to carry out HPLC, GFC, etc. using the principle of elution in order.

The “eluent containing no acid” as used herein means an eluent substantially free of acetic acid, phosphoric acid, formic acid, trifluoroacetic acid and the like that is generally added to the eluent. In the embodiment of the present invention, as such an eluent, for example, a polar organic solvent such as acetonitrile / methanol, water, or a mixed solution thereof is preferably used. “No acid” is judged based on the fact that it does not substantially affect the gel filtration fraction of cocoa polyphenols of trimer or less and cocoa polyphenols (cacaoprocyanidins) of tetramer or more. be able to.

Hereinafter, the present invention will be described in more detail with reference to examples. In addition, this invention is not limited to the Example shown below. In the following, the term “low polymerization” means that the degree of polymerization is 1 or more and 3 or less, that is, a monomer to trimer, and the term “high polymerization” means that the degree of polymerization is 4 That is, it means a tetramer or more.

<Example 1>
(1) Preparation of raw material solution After CLPr was dissolved in an aqueous methanol solution (50% by mass) so that the concentration of the cacao extract (hereinafter also referred to as “CLPR”) native to Ecuador was 30 mg / mL, this CLPr solution was The raw material liquid was prepared by filtering through a regenerated cellulose membrane having a pore diameter of 0.45 μm.

(2) Gel filtration fraction The environmental conditions of the high performance liquid chromatography (hereinafter also referred to as “HPLC”) system were set as shown below. Then, 50 μL of the above raw material solution is injected into the HPLC system, and the elution fraction from 8 to 16 minutes after the injection of the raw material solution is defined as a low polymerization fraction (hereinafter also referred to as “CLPr-L”). The subsequent elution fractions from 17 minutes to 25 minutes were collected as high polymerization fractions (hereinafter also referred to as “CLP-H”) (see FIG. 1). As shown in FIG. 1, in this example, the peaks of the trimer and the tetramer are completely separated. Therefore, CLPr-L mainly contains catechin, epicatechin and dimers and trimers thereof, and CLPr-H mainly contains procyanidins of tetramer or higher. That is, the content of highly polymerized procyanidins contained in each fraction and the yield of each fraction were the same as the results of Example 2 described later. As the fraction point, a point in time where CLPr-H contained no catechin, epicatechin and dimer or trimer procyanidin as much as possible was set.

(Environmental conditions of HPLC system)
Apparatus: Semi-preparative liquid chromatography system (manufactured by Shimadzu, system configuration: LC-6AD, 2 units, CBM-20A, CTO-20A, SIL-10AF, SPD-20A, FRC-10A, LCsolution software)
Column: Deverosil 100 Diol-5 8.0 × 300mm
・ Flow rate: 2.4 ml / min ・ Detection wavelength: UV 280 nm
Eluent: acetonitrile and methanol aqueous solution (97% by mass)
Gradient: (mass% of aqueous methanol solution (97 mass%)) 0 (0 min), 20 (4-9 min), 40 (13-18 min), 0 (20-30 min)

<Example 2>
(1) Preparation of raw material liquid CLPr was prepared in the same manner as in Example 1.
(2) Gel filtration fraction The environmental conditions of the HPLC system were changed as shown below, and the above-mentioned raw material liquid was injected into the HPLC system in 1960 μL. Then, the elution fraction from 45 to 85 minutes after injection of the raw material liquid was CLPr-L, and the elution fraction from 90 to 135 minutes after injection of the raw material liquid was CLPr-H. Each was fractionated (see FIG. 2).

(Environmental conditions of HPLC system)
-Equipment: Liquid chromatography system for large volume fractionation (manufactured by Shimadzu, system configuration: LC-8A, 2 units, FCV-130AL, SIL-10AP, SPD-20AV, FRC-10A, LCsolution software)
Guard column: Deverosil 100 Diol-10 50 × 100mm
-This column: Deverosil 100 Diol-10 50x300mm
・ Flow rate: 20.8 ml / min ・ Detection wavelength: UV 280 nm
Eluent: acetonitrile and methanol aqueous solution (97% by mass)
Gradient: (mass% of aqueous methanol solution (97 mass%)) 0 (0 minutes), 20 (24 to 54 minutes), 40 (78 to 108 minutes), 0 (120 to 180 minutes)

(3) Analysis of the content of highly polymerized procyanidins contained in each fraction The elution fractions of the above-mentioned CLPr-L and CLPr-H were collected at 2 to 4 mL, respectively, and each was dried to dryness using a centrifugal evaporator. . Next, each dried eluate fraction was redissolved with 100 μL of an aqueous methanol solution (50% by mass) to prepare a sample.

Using the procyanidins normal phase HPLC measurement method (Kelm, MA et al. J. Agric Food Chem 2006, 54 (5); p.1571-1576) according to the method of Kelm et al. When the analysis was performed with the equivalent amount of epicatechin, the analysis chart shown in FIG. 3 and the results shown in Table 1 were obtained.

CLPr-H contained tetramer or higher procyanidin at 90% by mass or more. On the other hand, procyanidins of tetramer or higher were not detected in CLPr-L. These procyanidins were quantified by preparing a calibration curve with epicatechin according to the method of reverse phase HPLC analysis and calculating the procyanidin concentration of each degree of polymerization as epicatechin equivalent.

(4) Yield of each fraction Each of the above-mentioned elution fractions of CLPr-L and CLPr-H was concentrated under reduced pressure by removing an organic solvent. The concentrated elution fraction was transferred to a metal container while washing with ultrapure water, and freeze-dried at −80 ° C. Then, the weight of each elution fraction was weighed to determine the yields of CLPr-L and CLPr-H. The yield of CLPr-L was 33%, and the yield of CLPr-H was 40%. Met.

<Comparative Example 1>
(1) Preparation of raw material liquid CLPr was prepared in the same manner as in Example 1.
(2) Gel filtration fraction The environmental conditions of the HPLC system were changed as shown below, and 50 μL of the above raw material solution was injected into the HPLC system. Example 1 except that the elution fraction from 8 minutes to 18.5 minutes after injection of the raw material liquid was CLPr-L, and the elution fraction from 19 minutes to 30 minutes after injection of the raw material liquid was CLPr-H. In the same manner as above, each fraction was collected (see FIG. 4). As shown in FIG. 4, in this comparative example, the peaks of the trimer and the tetramer are close to each other. For this reason, CLPr-H contains a relatively large amount of catechin, epicatechin and their dimers and trimers procyanidins, and it was difficult to obtain high-purity CLPr-H. .

(Environmental conditions of HPLC system)
Apparatus: Semi-preparative liquid chromatography system (manufactured by Shimadzu, system configuration: LC-6AD, 2 units, CBM-20A, CTO-20A, SIL-10AF, SPD-20A, FRC-10A, LCsolution software)
Column: Deverosil 100 Diol-5 8.0 × 300mm
・ Flow rate: 2.4 ml / min ・ Detection wavelength: UV 280 nm
Eluent: Acetonitrile / acetonitrile solution (2% by mass) and acetic acid / methanol aqueous solution (acetic acid: 2% by mass, methanol 95% by mass)
Gradient: (mass% of acetic acid / methanol aqueous solution) 0 (0 min), 40 (18-23 min), 0 (25-35 min)

<Consideration from results of Examples and Comparative Examples>
Comparing “FIG. 1 and FIG. 2 showing the results of gel filtration fractionation according to Example 1 and Example 2” and “FIG. 4 showing the results of gel filtration fractionation according to Comparative Example 1”, 3 in the former It can be seen that the time interval between the detection peak of the monomer and the detection peak of the tetramer is longer than the time interval in the latter. For this reason, in Example 1 and Example 2, it is thought that highly polymerized procyanidins (that is, procyanidins of tetramer or higher) having higher purity than before could be fractionated. Usually, in order to fractionate polyphenols from a polyphenol crude product with high accuracy, it is common to add an acid to the eluent of the gel filtration fraction. On the other hand, in the gel filtration fractionation method according to this example, the time interval between the detection peak of the trimer and the detection peak of the tetramer could be widened by not intentionally adding an acid. As a result, high-purity highly polymerized procyanidins could be obtained.

In the method for producing the specific polyphenol according to the present invention, highly-purified highly polymerized cacaoprocyanidins (that is, cacaoprocyanidins having a tetramer or higher) can be obtained. The method for producing a specific polyphenol according to the present invention is not limited to extraction of highly polymerized procyanidins from cocoa powder and cocoa extract by gel filtration fractionation, but other plants and foods and drinks containing polyphenols such as tea and fruits. It can also be applied when polyphenols are extracted by gel filtration fractionation (eg grapes, apples, blueberries, etc.), cereals (sesame seeds, soybeans, buckwheat etc.) or powders thereof, or extracts thereof.

Moreover, the highly polymerized procyanidins produced by the method for producing the specific polyphenol according to the present invention can be used not only as an active ingredient of pharmaceuticals such as blood glucose level control agents, but also added to foods and drinks, supplements, etc. It can also be used. In addition, these pharmaceuticals etc. may be administered orally, may be administered by tube, and may be administered enterally.

Claims

A method for producing a specific polyphenol, which comprises subjecting a polyphenol mixture to gel filtration fractionation of a tetramer or higher polyphenol from a polyphenol mixture using an eluent containing no acid.
The method for producing a specific polyphenol according to claim 1, wherein gel filtration fractionation is performed while changing the composition of the eluent.
The method for producing a specific polyphenol according to claim 1 or 2, wherein silica particles chemically bonded with glyceropropyl groups are used as the gel filtration material.
A specific polyphenol containing a polyphenol having a tetramer or higher produced by the method for producing a specific polyphenol according to any one of claims 1 to 3.
The specific polyphenol whose content rate of the polyphenol below a trimer is 10 mass% or less.