JP2010236905A - Method for analysis of anthocyanin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of determining anthocyanin by using an organic solvent in place of acetonitrile as a moving phase, in analysis of the anthocyanin by HPLC. <P>SOLUTION: In the method wherein the anthocyanin included in an analysis sample is determined by being separated by high-performance liquid chromatography (HPLC), the moving phase is ethanol and/or water. In the method, for example, a material having a filler (ODS) formed by chemically bonding an octadecyl group to a silica gel carrier is used a column for use in HPLC, and a detection wavelength of the anthocyanin is set at 520 nm, and the anthocyanin included in a sample such as delphinidin-3-O-glucoside, delphinidin-3-O-rutinoside, cyanidin-3-O-glucoside, or cyanidin-3-O-rutinoside can be quantitatively analyzed simply. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a method for analyzing anthocyanins using HPLC.

Anthocyanin is a glycoside component in which anthocyanidin is bound to sugar as an aglycon. As anthocyanidins, for example, delphinidin, cyanidin, malvidin, pelargonidin, peonidin, petunidin having a chemical structure represented by the following formula (I): )and so on. Anthocyanins are called anthocyanidin glucoside when glucose is bound to the anthocyanidins as a glycoside. In addition to glucose, which is a monosaccharide, sugars found in anthocyanins include galactose, arabinose, disaccharides, rutinose, and soforose. Among them, anthocyanins derived from cassis (English name Black currant) are called cassis anthocyanins. Cassis anthocyanins are known to have useful effects such as a visual improvement function, a blood fluidity improvement function, and a blood pressure lowering function (Patent Document 1).

As a conventionally known method for analyzing anthocyanin, a method using high performance liquid chromatography (HPLC) is known (Non-patent Documents 1 and 2). In the methods of Non-Patent Documents 1 and 2, reverse phase HPLC using an octadecylsilyl silica (ODS) packed column is used, and a solvent of acetonitrile / water is used as a mobile phase.

WO2001 / 01798

Arapitsas et al., Talanta 2008, 74, 1218-1223 Matsumoto et al., Journal of Agricultual and Food Chemistry, March 2001, Vol. 49, No. 3, 1541-1545

However, due to the recession in the automobile industry in recent years, supply of acetonitrile, which is a byproduct of acrylonitrile, is insufficient, and an alternative is demanded. An object of the present invention is to provide a method that enables anthocyanins to be quantified even when an organic solvent instead of acetonitrile is used as a mobile phase in the analysis of anthocyanins by HPLC.

As a result of intensive studies to achieve the above object, the present inventors have found that the problem can be solved by providing the analysis method shown below. That is,
(1) A method for separating and quantifying anthocyanins contained in an analytical sample by high performance liquid chromatography (HPLC), wherein the mobile phase is ethanol and / or water.
(2) The method according to (1), wherein phosphoric acid is added to water of the mobile phase.
(3) The method according to (1) or (2), wherein the column used for HPLC is a column having a packing material (ODS) in which an octadecyl group is chemically bonded to a silica gel carrier.
(4) The method according to any one of (1) to (3), wherein the detection wavelength of anthocyanin is 520 nm.
(5) Anthocyanins are a group of delphinidin-3-O-glucoside (D3G), delphinidin-3-O-rutinoside (D3R), cyanidin-3-O-glucoside (C3G), cyanidin-3-O-lutinoside (C3R) The method according to any one of (1) to (4), wherein the method is one or more substances selected from the group consisting of:
(6) The method according to any one of (1) to (5), wherein the analysis sample is a beverage containing cassis anthocyanin.

The method of the present invention using ethanol as an alternative to acetonitrile as the mobile phase of HPLC enables quantitative analysis of anthocyanins contained in an anthocyanin-containing composition without being affected by insufficient supply of acetonitrile.

2 is a chromatogram of anthocyanins separated by the HPLC analysis method according to Example 1. 2 is a chromatogram of anthocyanins separated by the HPLC analysis method according to Comparative Example 1. The graph which shows that the analysis method by the comparative example 1 and the analysis method by Example 1 have a correlation.

Hereinafter, embodiments of the present invention will be described in detail.
The substance quantified by the method of the present invention is anthocyanin, and as anthocyanin (AC), delphinidin, cyanidin, malvidin, pelargonidin, peonidin, petunidin (Petunidin) ) And other anthocyanidins such as glucose, rutinose, galactose, arabinose, and soforose are glycosides, preferably delphinidin-3-O-glucoside (D3G). , Delphinidin-3-O-rutinoside (D3R), cyanidin-3-O-glucoside (C3G), cyanidin-3-O-rutinoside (C3R) and the like.

Analytical samples targeted by the method of the present invention include natural products (such as walnuts, cranberries, cassis, grape skins, purple potato skins) and extracts thereof, foods and drinks, pharmaceuticals, cosmetics, and other anthocyanins. Any sample that is expected to contain. When the sample is a liquid, it may be measured by diluting with 0.1N hydrochloric acid so that the total of all components of anthocyanin is 10 to 100 mg / 100 g. In the case of a solid, the sample is extracted with an appropriate solvent to obtain an analysis sample. A preferred analytical sample targeted by the method of the present invention is a beverage containing cassis anthocyanin.

In the method of the present invention, anthocyanins are separated and quantified by high performance liquid chromatography (HPLC). The column used for the analysis is preferably a reverse phase column, and the reverse phase column is a silica gel carrier in which octadecyl group is chemically bonded (ODS), butyl group is chemically bonded (C4), and octyl group is chemically bonded. Filler (CS), filler with chemically bonded phenyl group (Ph), filler with chemically bonded alkyl chain of C30 (C30), octadecyl group bonded to synthetic polymer (ODP) or reverse phase synthetic polymer carrier An amide-bonded reversed phase carrier can be used.

As a mobile phase for HPLC analysis, water and a polar organic solvent soluble in water can be used, and ethanol, methanol and the like can be used, and ethanol is particularly preferable. The mixing ratio with water can be 0 to 100%. It is preferable to make the mobile phase acidic for stability of components to be separated and analyzed. 0.001% to 5% of trifluoroacetic acid (TFA), phosphoric acid, formic acid, acetic acid, trichloroacetic acid (TCA), perchloric acid, etc. You may mix. Preferably, 0.01% to 1.0% phosphoric acid is used.

Specifically, using an ODS column (Zorbax SB-C18: 4.6 mm x 150 mm, Agilent), 0.5% phosphoric acid aqueous solution was used for the A solution, ethanol was used for the B solution, the flow rate was 0.8 ml / min, % → B solution 13% (20 minutes), B solution 13% → B solution 13% (3 minutes), B solution 13% → B solution 80% (0.01 minutes), then B solution 80% → B solution 80% ( 4.99 minutes), elution is performed at a column temperature of 40 ° C., and anthocyanins are detected by measuring absorbance at 520 nm. Anthocyanins can be quantified from the peak area values of each substance. Delphinidin-3-O-glucoside (D3G) is 8.9 minutes, delphinidin-3-O-rutinoside (D3R) is 10.2 minutes, cyanidin-3-O-glucoside (C3G) is 12.6 minutes, cyanidin-3-O-lutinoside ( C3R) eluted at 14.9 minutes. However, it is not necessarily limited to this analysis condition, and all conditions that can be quantified can be used.

  Hereinafter, although an example is shown and explained in detail, the technical scope of the present invention is not limited to these examples.

Example 1
Beverages containing cassis anthocyanins (samples 1 to 5) were diluted with 0.1N hydrochloric acid so that the total of all components of anthocyanins was 10 to 100 mg / 100 g, and analyzed by HPLC under the following conditions.

  The analysis conditions were Zorbax SB-C18 (4.6 mm x 150 mm, Agilent) as the column, 0.5% phosphoric acid aqueous solution for solution A, ethanol for solution B, 10 ml of solution B at a flow rate of 0.8 ml / min. B solution 13% (20 minutes), B solution 13% → B solution 13% (3 minutes), B solution 13% → B solution 80% (0.01 minutes), then B solution 80% → B solution 80% (4.99 minutes) ) Gradient elution. The column temperature was 40 ° C. and anthocyanins were detected by measuring the absorbance at 520 nm, and the anthocyanins were quantified from the peak area values of the respective substances. For HPLC, an HPLC system (Tosoh Corporation) using a multi-station LC-8020 Model II was used.

  Under these conditions, delphinidin-3-O-glucoside (D3G) is 8.9 minutes, delphinidin-3-O-lutinoside (D3R) is 10.2 minutes, cyanidin-3-O-glucoside (C3G) is 12.6 minutes, cyanidin-3- O-Rutinoside (C3R) eluted at 14.9 minutes and was well separated (FIG. 1).

  In addition, D3G, D3R, C3G, and C3R were obtained by purifying from cassis-concentrated juice according to the method of Matsumoto et al. (Matsumoto et al. J. Agricultual and Food Chemistry, March 2001, Vol. Using the sample as a standard, a calibration curve was prepared to quantify the four components of anthocyanins in each sample.

Comparative Example 1
The samples 1 to 5 described in Example 1 were quantitatively analyzed for the four components of anthocyanins D3G, D3R, C3G, and C3R under the same conditions as in Example 1 except that the B solution of the eluate was changed to acetonitrile.
Under these conditions, D3G, D3R, C3G, and C3R were eluted and separated at 7.9 minutes, 9.2 minutes, 11.7 minutes, and 13.8 minutes, respectively (FIG. 2).

アントシアニン4成分の各成分の量および4成分の総量について、横軸を比較例1による方法で得られた量とそれに対応する実施例1による方法によって得られた量を縦軸になるようにプロットしたグラフが図3である。そのグラフから相関係数を求めると1.00となった。
従って、HPLCでのカシスアントシアニン分析方法において移動相としてアセトニトリルの代わりにエタノールを使用しても定量分析は可能であることが判明した。 Test example 1
For the samples 1 to 5 used in Example 1 and Comparative Example 1, the content of the four components of D3G, D3R, C3G, and C3R and the total amount of the four components were quantified by the method of Comparative Example 1 or Example 1. The results are shown in Table 1.

For the amount of each component of the four anthocyanins and the total amount of the four components, the horizontal axis plots the amount obtained by the method according to Comparative Example 1 and the corresponding amount obtained by the method according to Example 1 on the vertical axis. The resulting graph is shown in FIG. The correlation coefficient obtained from the graph was 1.00.
Therefore, it was found that quantitative analysis is possible even when ethanol is used instead of acetonitrile as the mobile phase in the method for analyzing Cassis anthocyanin by HPLC.

The present invention allows ethanol to be used as an alternative to acetonitrile in the analysis of anthocyanins using acetonitrile as the mobile phase for HPLC analysis.

Claims (6)

A method for separating and quantifying anthocyanins contained in an analytical sample by high performance liquid chromatography (HPLC), wherein the mobile phase is ethanol and / or water. The method according to claim 1, wherein phosphoric acid is added to water of the mobile phase. 3. The method according to claim 1, wherein the column used for HPLC is a column having a packing material (ODS) in which an octadecyl group is chemically bonded to a silica gel support. The method according to any one of claims 1 to 3, wherein the detection wavelength of anthocyanin is 520 nm. The anthocyanins are selected from the group of delphinidin-3-O-glucoside (D3G), delphinidin-3-O-lutinoside (D3R), cyanidin-3-O-glucoside (C3G), cyanidin-3-O-lutinoside (C3R) The method according to claim 1, wherein the method is one or more substances. 6. The method according to claim 1, wherein the analysis sample is a beverage containing cassis anthocyanin.

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