JP6400882B2 - Blood sugar level control agent containing specific cocoa polyphenol as active ingredient, and method for producing specific cocoa polyphenol - Google Patents

Description

The present invention, 4 and higher monomers of the cocoa polyphenols active ingredients relates catechin, a blood sugar control agent which does not substantially contain epicatechin and trimers following cocoa polyphenols. The present invention also relates to a method for producing a specific cocoa polyfer.

  It has been reported in the past that “procyanidins have an effect of suppressing an increase in blood glucose level” (for example, JP-A-9-291039). For this reason, procyanidins are utilized as active ingredients such as blood sugar level control agents. And procyanidins can be extracted from cacao, for example (for example, Japanese translations of PCT publication No. 2003-585111). Procyanidin is a form of polyphenol.

JP-A-9-291039 Special Table 2003-585111

  In recent years, there has been a demand for blood sugar level control agents and the like that can control blood sugar levels more efficiently than conventional blood sugar level control agents and the like.

  Then, the subject of this invention is providing the blood glucose level control agent etc. which use a specific polyphenol as an active ingredient which can control a blood glucose level more efficiently than the blood glucose level control agent etc. which used the procyanidin as an active ingredient conventionally. It is in.

The blood sugar level control agent according to the present invention contains a cocoa polyphenol mixture of tetramer or higher as an active ingredient and does not contain catechin, epicatechin and cocoa polyphenol of trimer or lower . However, this means that the blood glucose level control agent is “substantially” free of catechins, epicatechins and trimeric procyanidins. That is, this blood glucose level control agent has a ratio of “total mass of polyphenol mixture of tetramer or more” to “total mass of catechin, epicatechin and polyphenol of trimer or less” of 9 or more, preferably 10 or more. In other words, the polyphenol mixture is preferably 11 or more, 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 blood sugar level control agent according to the present invention is also an incretin production promoter in the body. Here, “ cocoa polyphenol” is a polyphenol derived from cacao . The “polyphenol” referred to here is preferably procyanidin. Furthermore, the cocoa polyphenol mixture of tetramer or higher is preferably orally administered at a daily dose of 1 mg / kg · Bw or higher on a dry matter basis. And this blood glucose level control agent can be added to food-drinks or a supplement.

In the blood sugar level control agent according to the present invention, the content of cocoa polyphenols having a trimer or less is 10% by mass or less. The content of the cocoa polyphenol of the 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.

  In addition, the above-mentioned active ingredient was added to an aqueous solution of acetonitrile and methanol (97 in a column having an inner diameter of 8.0 mm and a length of 100 mm using a particulate gel in which a glyceropropyl group is chemically bonded to a silica substrate as a filler). % By weight) of the aqueous methanol solution (97% by weight) is 0% by weight (0 minutes), 20% by weight (4-9 minutes), 40% by weight (13-18 minutes), 0% by weight (20 to 20%). 30 minutes) in a high-performance liquid chromatography apparatus that delivers liquid at a flow rate of 2.4 mL / min under a gradient condition of 30 min), after injecting 50 μL of the cacao extract raw material liquid (45 mg / mL) It is possible to exemplify that it is obtained by fractionating the fraction that flows out during the period up to, more preferably from 17 to 25 minutes. The solvent may be removed from this fraction, and the components in the fraction may be concentrated and / or dried.

  In addition, the above-mentioned active ingredients are “a guard column having an inner diameter of 5.0 mm and a length of 100 mm using a particulate gel in which a glyceropropyl group is chemically bonded to a silica substrate as a filler” and “glycero on a silica substrate. To this column having an inner diameter of 5.0 mm and a length of 300 mm using a particulate gel chemically bonded with a propyl group as a filler, acetonitrile and aqueous methanol solution (97% by mass) are mixed with aqueous methanol solution (97% by mass). Under a gradient condition in which the mass ratio is 0 mass% (0 minutes), 20 mass% (24 to 54 minutes), 40 mass% (78 to 108 minutes), and 0 mass% (120 to 180 minutes), 20. In a high-performance liquid chromatography apparatus that delivers liquid at a flow rate of 8 mL / min, the raw material liquid of cocoa extract (45 mg / mL) is injected from 1960 μL to 85 minutes to 150 minutes. , More preferably it is exemplified also be obtained by preparative fractions flowing out until 135 minutes to 90 minutes. The solvent may be removed from this fraction, and the components in the fraction may be concentrated and / or dried.

As a result of intensive studies by the inventors of the present application, it has been clarified that the blood sugar level control agent according to the present invention can control the blood sugar level more efficiently than the conventional one. That is, the tetraphenol or higher polyphenol mixture according to the present invention can be used as an active ingredient of a blood glucose level control agent . However, this blood glucose level control agent is substantially free of catechin, epicatechin and cocoa polyphenols of trimer or less.

3 is a chart showing the results of gel filtration fractionation according to Example 1. It is a chart which shows the analysis result of the content rate of the high polymerization degree procyanidin contained in each fraction after the gel filtration fractionation concerning Example 1, (a) Cocoa extract (CLPr), b) Low polymerization fraction (CLPr- L), c) High polymerization fraction (CLP-H)). It is a bar graph which compares and compares the blood insulin concentration of each mouse | mouth when the cocoa extract which concerns on Example 1, a high polymerization procyanidin, and a low polymerization procyanidin are each administered to a mouse | mouth. In the figure, “Water” indicates water, “High-DP” indicates CLPr-H, and “Low-DP” indicates CLPr-L. FIG. 5 is a bar graph showing comparison of blood insulin concentrations of mice when procyanidin B2, procyanidin C1 and cinnamtannin A2 are respectively administered to mice. In the figure, “Water” represents water, “EC” represents epicatechin, “PcB2” represents procyanidin B2, “PcC1” represents procyanidin C1, and CTA2 represents cinnamtannin A2. It is a chart which shows the result of the gel filtration fractionation concerning Example 2.

  The tetramer 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.

As a result of the analysis by the inventors of the present application, it was found that the highly polymerized procyanidin according to the present invention specifically promotes incretin production as compared with catechin, epicatechin and procyanidins of trimer or less. Incretin is known as a substance that affects the production of insulin, which is one factor that controls blood glucose levels. Therefore, the highly polymerized procyanidin according to the present invention can be used as an active ingredient of a blood sugar level control agent and an incretin production promoter in the body. In such a case, the blood sugar level control agent according to the present invention preferably does not contain catechin, epicatechin and procyanidins of trimer or less. However, this means “substantially” free of catechins, epicatechins and trimeric procyanidins. That is, the blood glucose level control agent according to the present invention has a ratio of “total mass of polyphenols of tetramer or higher” to “total mass of polyphenols of catechin, epicatechin and trimers” of 9 or higher, preferably 10 or higher. In other words, it can be paraphrased as a polyphenol mixture, more preferably 11 or more, and still 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.

Incidentally, about the blood glucose level controlling agent according to the embodiment of the present invention, in order to effectively express the blood glucose control effect, highly polymerized procyanidin (dry matter) of 1 mg / kg · Bw or more per day, preferably It is preferable to orally administer to the living body at 2 mg / kg · Bw or more per day, more preferably 5 mg / kg · Bw or more per day, and even more preferably 10 mg / kg · Bw or more per day. The upper limit of this value is infinity (∞), and the higher the value, the better. However, for example, the upper limit of this value is 1000 mg / kg · Bw or less per day. For example, it is preferable to contain highly polymerized procyanidin (dried product) in an amount of 1 g or more per packaging form.

  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, 1960 μL of the above raw material solution is injected into the HPLC system, and the elution fraction from 45 minutes to 85 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 90 to 135 minute elution fractions were collected as high polymerization fractions (hereinafter also referred to as “CLPr-H”) (see FIG. 1). As shown in FIG. 1, in this example, the peaks of the trimer and the tetramer are completely separated. For this reason, CLPr-L mainly contains catechin, epicatechin and their dimers and trimers, and CLPr-H mainly contains procyanidins of tetramer or higher. 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: Liquid chromatography system for large volume fractionation (manufactured by Shimadzu Corporation, system configuration: LC-8A, 2 units, FCV-130AL, SIL-10AP, SPD-20AV, FRC-10A, LCsolution software)
Guard column: Deverosil 100 Diol-10 50 × 100 mm
-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 in 2 to 4 mL, respectively, and each was dried to dryness with 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. 2 and the results shown in Table 1 were obtained.

  CLPr-H contained tetramer or higher procyanidin at 90% by mass or higher. 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 The above-mentioned elution fractions of CLPr-L and CLPr-H were depressurized with an evaporator to remove the organic solvent and concentrated. 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.

<Confirmation of blood glucose level control action for each eluted fraction>
(1) Measurement of blood insulin concentration of each fraction with respect to living body After 4-week-old male ICR-mouse was fasted overnight (18 hours), the mouse was divided into Sample A, Sample B and Sample C. Then, the above-mentioned CLPr was orally administered to specimen A, CLPr-L to specimen B, and CLPr-H to specimen C at 10 mg / kg · Bw each. Then, 60 minutes after administration, each specimen was sacrificed, and blood was collected from the heart of each specimen. And blood of each specimen was coagulated and plasma was collected.

  Next, the insulin concentration in plasma obtained from each sample was measured using an insulin measurement kit (manufactured by Shiba Goat Co., Ltd., Levis (registered trademark) insulin mouse kit). The measurement results are shown in FIG. In the breeding of mice, normal feed was given and water was not restricted.

  As is clear from FIG. 3, the blood insulin concentration of the sample C administered with CLPr-H was higher than the blood insulin concentrations of the sample A and the sample B. That is, it has been clarified that CLPr-H is superior to CLPr and CLPr-L in the function of increasing the blood insulin concentration in the living body.

(2) Reference Example After a 4-week-old male ICR-mouse was fasted overnight (18 hours), the mouse was divided into Sample D, Sample E, and Sample F. Then, procyanidin B2 (dimer procyanidin) is sample D, procyanidin C1 (trimer procyanidin) is specimen E, and cinnamtannin A2 (tetramer procyanidin) is 10 mg / kg for specimen F. -Orally administered in increments of Bw. Next, 60 minutes after the administration, each specimen was sacrificed, and blood was collected from the heart of each specimen. Then, blood of each specimen was coagulated and plasma was collected.

  Next, the insulin concentration in plasma obtained from each sample was measured using an insulin measurement kit (Shibayagi Co., Ltd., Levis (registered trademark) insulin mouse kit). The measurement results are shown in FIG. In the breeding of mice, normal feed was given and water was not restricted.

  As is clear from FIG. 4, the blood insulin concentration of the sample F administered with cinnamtannin A2 was significantly higher than the blood insulin concentrations of the sample D and the sample E. That is, it has been clarified that cinnamtannin A2 is superior to procyanidin B2 and procyanidin C1 in the function of increasing the blood insulin concentration in the living body.

<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 50 μL of the raw material solution was injected into the HPLC system. Then, the elution fraction from 8 minutes to 16 minutes after injection of the raw material liquid was CLPr-L, and the elution fraction from 17 minutes to 25 minutes after injection of the raw material liquid was CLPr-H. Each was fractionated (see FIG. 5).

(Environmental conditions of HPLC system)
Apparatus: Semi-preparative liquid chromatography system (manufactured by Shimadzu Corporation, 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 x 300 mm
・ 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 minutes), 20 (4-9 minutes), 40 (13-18 minutes), 0 (20-30 minutes)

  The content of highly polymerized procyanidins contained in each fraction and the yield of each fraction were the same as those in Example 1. Moreover, the blood glucose level control action of each fraction was the same as the result of Example 1.

The tetramer or higher polyphenol (highly polymerized procyanidin) according to the present invention can be effectively utilized as an active ingredient of a blood glucose level control agent for patients requiring control of blood glucose level such as diabetic patients. Moreover, by adding the blood sugar level control agent according to the present invention to foods and drinks, supplements and the like, it is possible to provide foods and drinks, supplements and the like effective for the prevention and improvement of diabetes.

Claims (2)

Contains cacao- derived procyanidins including tetramer, pentamer, hexamer , heptamer and octamer, and has a cacao- derived procyanidin content of 10% by mass or less. A blood glucose level control agent for orally administering the procyanidin mixture in an amount in the range of 1 mg / kg · Bw to 1000 mg / kg · Bw per day on the dry matter basis. Contains cacao- derived procyanidins including tetramer, pentamer, hexamer , heptamer and octamer, and has a cacao- derived procyanidin content of 10% by mass or less. Blood glucose level control method in which the procyanidin mixture is orally administered to a living body in an amount ranging from 1 mg / kg · Bw to 1000 mg / kg · Bw on a dry matter basis per day (excluding the act of treating a person).

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