Method for quantifying polyphenols

Abstract

To provide a highly sensitive method for quantifying polyphenols using ferric tartrate absorptiometry.SOLUTION: The present invention provides a method for quantifying polyphenols in a sample, in which a ferric tartrate reagent is added to a polyphenol-containing sample to develop color, and absorbance is measured after adjustment to a fixed volume. The method uses a 2-morpholinoethanesulfonic acid buffer solution to prepare and fix the volume of the ferric tartrate reagent, and adjusts the pH of a measurement solution to 8.0±0.5 before measuring the absorbance.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a highly sensitive method for quantifying polyphenols. [Background technology]

[0002] Polyphenols are a general term for natural organic compounds that have two or more hydroxyl groups in an aromatic ring among secondary metabolites of plants, and more than 5,000 types have been reported. Polyphenols are further classified into phenylpropanoids, flavonoids, and anthraquinones based on their structure. Polyphenols have long been known as the substances that cause astringency in plant foods, but in recent years, dietary polyphenols have been suggested to have preventive effects against various diseases, and their preventive effects against cardiovascular diseases, inflammation or allergies, tumors, osteoporosis, etc. have been reported. Such useful effects are thought to be due to the fact that polyphenols have antioxidant properties that detoxify active oxygen.

[0003] Polyphenols, which have preventive effects against various diseases, are not only contained in foods, but are also used as antioxidants, additives to foods and medicines, and as medicines. Therefore, although polyphenols, which are contained in foods and medicines and have useful physiological activities, are not specified in the nutritional labeling standards for foods, it is important to measure and manage their content in consideration of quality control and various expected physiological activities.

[0004] Testing methods for polyphenols include the Folin-Ciocalteu method, the Folin-Denis method, and the ferrous tartrate spectrophotometric method, and the ferrous tartrate spectrophotometric method is listed as an analytical method for green tea tannins in the Analysis Manual for the 2020 Edition (8th Revised Edition) of the Standard Tables of Food Composition in Japan. The ferrous tartrate method is a colorimetric method that utilizes the property that phenolic hydroxyl groups react quantitatively with iron ions to form a complex that turns blue.

[0005] However, polyphenols are known to be easily oxidized, and the phenolic hydroxyl groups are deprotonated during oxidation, which may reduce the efficiency of complex formation with iron ions. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Analysis Manual for the Standard Tables of Food Composition in Japan 2020 Edition (8th Revised Edition) (46-1. Ferrous Tartrate Absorption Spectrophotometric Method) Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to a highly sensitive method for quantifying polyphenols using ferric tartrate. [Means for solving the problem]

[0008] As a result of extensive research, the inventors of the present application have found that by using a 2-morpholinoethanesulfonic acid (hereinafter also referred to as MES) buffer as the buffer for ferric tartrate absorptiometry and adjusting the pH of the reaction solution within a predetermined range before measuring absorbance, more sensitive quantification is possible than when a conventional phosphate buffer is used.

[0009] That is, the present invention relates to the following 1) to 4). 1) A method for quantifying polyphenols in a sample by adding an iron tartrate reagent to a polyphenol-containing sample to develop color, and then measuring the absorbance after the volume has been determined. The iron tartrate reagent is prepared and the volume is determined using a 2-morpholinoethanesulfonic acid buffer solution, and the pH of the measurement solution is adjusted to 8.0±0.5 before the absorbance is measured. 2) The method according to 1), wherein the pH of the measurement solution is adjusted using trishydroxymethylaminomethane. 3) The method according to 1) or 2), wherein absorbance at 520 nm is measured. 4) The method according to any one of 1) to 3), wherein the polyphenol-containing sample is a plant containing polyphenols or an extract thereof. Effect of the Invention

[0010] According to the present invention, the polyphenol content can be accurately measured even in foods and medicines with low polyphenol content, enabling strict control of polyphenol intake, thereby making a great contribution to the food and medicine industries. [Brief description of the drawings]

[0011] [Figure 1] Measurement results of ethyl gallate using a conventional method (comparison example). [Diagram 2] Measurement results of ethyl gallate by the method of the present invention (Example). [Diagram 3] Effect of pH on absorbance. [Figure 4] Effect of adding sodium dodecyl sulfate on measurement results. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Preferred embodiments of the present invention will be described in detail below, however, the present invention is not limited to the following embodiments.

[0013] In the present invention, "polyphenol" refers to an organic compound that belongs to phenylpropanoids, flavonoids, and anthraquinones and has two or more hydroxyl groups on an aromatic ring. For example, phenylpropanoids include resveratrol, curcumin, cinnamic acid, caffeic acid, chlorogenic acid, etc., flavonoids include avigenin, luteonin, quercetin, rutin, kaempferol, epigallocatechin gallate, epicatechin gallate, pentagalloyl glucose, glucogallin, tannic acid, galloylbergenin, galloyl arbutin, etc., and anthraquinones include aloe-emodin, etc., but are not limited thereto. Also included are artificially synthesized polyphenols such as N2,N6-bis[N2,N6-bis(3,4,5-trihydroxybenzoyl)-lysyl]-N-(2-aminoethyl)-lysineamide (WO 2007 / 052641, hereinafter referred to as TGDK).

[0014] In the present invention, the "polyphenol-containing sample" includes plants or extracts thereof that contain such polyphenols (e.g., seaweed, vegetables, fruits such as citrus fruits and apples, grains or extracts thereof, etc.), foods and beverages (e.g., tea, wine, cacao, etc.), etc.

[0015] The method of the present invention is a method for quantifying the polyphenol content in a sample by adding an iron tartrate reagent to a polyphenol-containing sample to develop color, and then measuring the absorbance after the volume is determined. MES buffer is used to prepare and volume-determine the iron tartrate reagent, and the pH of the measurement solution is adjusted to 8.0±0.5 before measuring the absorbance. The method of quantifying the polyphenol content in a sample using ferric tartrate reagent is known as ferric tartrate spectrophotometry, and as mentioned above, is listed as an analytical method for tannins in green tea, for example, in the Analysis Manual for the 2020 Edition (8th Revised Edition) of the Standard Tables of Food Composition in Japan. In this method, the iron tartrate reagent used as a color developer is prepared from ferrous sulfate and sodium potassium tartrate, and is usually prepared by dissolving a predetermined amount of ferrous sulfate and sodium potassium tartrate in water. In the present invention, the iron tartrate reagent is prepared by dissolving ferrous sulfate and sodium potassium tartrate in MES buffer. For example, 100 mg of ferrous sulfate and 500 mg of sodium potassium tartrate can be dissolved in MES buffer and made up to 100 mL to prepare the ferric tartrate reagent.

[0016] In the ferric tartrate absorptiometry, the ferric tartrate reagent is added to the polyphenol-containing sample and reacted, and then the volume of the measurement sample is adjusted to a fixed amount. In the volume adjustment, water or a phosphate buffer solution (pH 7 to 8) is generally used, but in the present invention, an MES buffer solution is used.

[0017] In the present invention, the MES buffer used in the preparation of the iron tartrate reagent and the volumetric adjustment operation is prepared by dissolving 2-morpholinoethanesulfonic acid (MES) in purified water and adding NaOH dropwise to adjust the pH to a predetermined range.

[0018] The MES buffer may be at any concentration that has buffering capacity, but is preferably 10 mM or more, 50 mM or more, 100 mM or more, and preferably 500 mM or less, more preferably 250 mM or less, and even more preferably 100 mM.

[0019] The pH of the MES buffer may be within the range in which it has a buffering capacity, but is preferably 5.5 to 6.5, more preferably 5.5 to 6.0, and even more preferably 5.5.

[0020] The polyphenol-containing sample used for the measurement may be a sample extracted from a solid (e.g., a plant), or a liquid sample may be used as is. For extraction from a solid, for example, as described in the Analysis Manual 46-1 of the Standard Tables of Food Composition in Japan 2020 Edition (8th Edition), hot water may be poured into the solid and extracted at 80°C or higher, but is not limited thereto, and extraction may be performed with a liquid containing a surfactant, etc.

[0021] In addition, when using a highly turbid sample, the turbidity of the measurement solution can be reduced by adding a surfactant to the measurement system (iron tartrate reagent or measurement solution) within a range that does not interfere with the formation of a complex between polyphenols and iron ions.As such a surfactant, any of cationic surfactants, anionic surfactants, amphoteric surfactants and nonionic surfactants may be used. For example, cationic surfactants include hexadecyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium chloride (CTAC) and cetylpyridinium chloride (HPC). Anionic surfactants include sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LiDS), sodium deoxycholate (SDC) and sodium Examples of amphoteric surfactants include 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), 3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonate (CHAPSO), 3-[Dimethyl(tetradecyl)ammonio]propane-1-sulfonate (Zwittergent3-14) and n-Dodecyl-N,N-Dimethyl-3-Ammonio-1-Propanesulfonate (Anzergent3-12). Examples of nonionic surfactants include Polyethyleneglycol-[4-(1,1,3,3-tetramethylbutyl)phenyl]-ether (TritonX-100), Polyethylene glycol mono(4-nonylphenyl) ether (NP-40), Polyethylene glycol lauryl ether (Briji35), Polyoxyethylene sorbitan Examples of such antibacterial agents include, but are not limited to, polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monooleate (Tween 80) and octyl β-D-glucopyranoside.

[0022] In addition, in the method for quantifying polyphenols of the present invention, polyphenols may be separated in advance from a polyphenol-containing sample by combining with a separation technique such as high performance liquid chromatography, or polyphenols complexed with iron ions after reaction with an iron tartrate reagent may be separated and subjected to measurement.

[0023] In the method for quantifying polyphenols of the present invention, the standard solution for preparing a calibration curve may be ethyl gallate as described in the Analysis Manual 46-1. Ferrous Tartrate Absorption Spectrophotometric Method, 2020 Edition (8th Edition) of the Standard Tables of Food Composition in Japan, but known concentrations of resveratrol, curcumin, cinnamic acid, caffeic acid, chlorogenic acid, avigenin, luteonin, quercetin, rutin, kaempferol, gallic acid, epigallocatechin gallate, epicatechin gallate, pentagalloylglucose, glucogallin, tannic acid, galloylbergenin, galloylarbutin, aloe-emodin, etc. may also be used.

[0024] In the method for quantifying polyphenols of the present invention, the absorbance measurement is carried out after previously adjusting the pH of the measurement solution to 8.0±0.5. This improves the efficiency of complex formation between the iron tartrate reagent and polyphenols, making it possible to detect polyphenols with high sensitivity. The pH (8.0±0.5) can be adjusted preferably using 2 M trishydroxymethylaminomethane, but a buffer solution having buffering capacity in the basic range, such as piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) (POPSO), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS), N-[Tris(hydroxymethyl)methyl]glycine (Tricine), N,N-Bis(2-hydroxyethyl)glycine (Bicine), and N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), or potassium hydroxide or sodium hydroxide may also be used.

[0025] In the method for quantifying polyphenols of the present invention, absorbance is measured at 520 to 540 nm. The 2020 Edition (8th Edition) of the Standard Tables of Food Composition in Japan, Analysis Manual 46-1. In the conventional method described in the ferric tartrate spectrophotometric method, the absorbance is measured at 540 nm, but in the present invention, the maximum absorption wavelength shifts to 520 nm because the pH is adjusted to 8.0, so it is preferable to measure the absorbance at 520 nm for sensitive analysis. Thus, according to the method for quantifying polyphenols of the present invention, it is possible to accurately measure the polyphenol content in a polyphenol-containing sample having a low polyphenol content, as shown in the Examples described below. EXAMPLES

[0026] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0027] Comparative Example 1: Quantitative determination of polyphenols by ferric tartrate spectrophotometry Standard Tables of Food Composition in Japan 2020 Edition (8th Edition) Analysis Manual 46-1. Polyphenols were quantified according to the ferric tartrate absorptiometry method as follows. (1) Preparation of polyphenol-containing samples Ethyl gallate was dissolved in water to prepare a 1000 μM ethyl gallate solution, which was then serially diluted to prepare 500, 250, 125, 62.5, 31.3 and 15.6 μM ethyl gallate solutions.

[0028] (2) Preparation of iron tartrate reagent Weigh out 500 mg of iron(II) sulfate heptahydrate and 100 mg of sodium potassium iron tartrate tetrahydrate, dissolve them in water to make 100 mL, and use these as the iron tartrate reagent.

[0029] (3) Quantification of polyphenols 90 μL of the tartaric acid reagent prepared in (2), 90 μL of each concentration of ethyl gallate solution, and 270 μL of 1 / 15 M phosphate buffer were mixed, and 12 μL of 1 / 15 M phosphate buffer was added to the mixture, and the absorbance at a wavelength of 540 nm was measured. Common logarithms were calculated for each concentration of ethyl gallate and the measured values, and an approximate straight line and multiple correlation coefficient (r 2 ) was calculated. The 1 / 15M phosphate buffer solution was prepared by mixing 1 / 15M disodium hydrogen phosphate solution (11.867g / L) and 1 / 15M potassium dihydrogen phosphate solution (9.073g / L) in a ratio of 84:16, and adjusting the pH to 7.5 using a pH meter.

[0030] The results are shown in Figure 1. Concentration-dependent changes in absorbance were observed at the five higher concentrations (62.5-1000μM), but the measured values ​​at 15.6 and 31.3μM were similar, indicating that quantitative analysis is difficult in these concentration ranges. In addition, no concentration-dependent changes in absorbance were observed at two concentrations, so the multiple correlation coefficient was 0.9836.

[0031] Example 1: Quantification of polyphenols using improved ferric tartrate spectrophotometric method (1) Preparation of polyphenol-containing samples Ethyl gallate was dissolved in water to prepare a 1000 μM ethyl gallate solution, which was then serially diluted to prepare 500, 250, 125, 62.5, 31.3 and 15.6 μM ethyl gallate solutions.

[0032] (2) Preparation of iron tartrate reagent 500 mg of iron (II) sulfate heptahydrate and 100 mg of sodium potassium iron tartrate tetrahydrate were weighed out and dissolved in 0.1 M MES buffer to make 100 mL, and these were used as iron tartrate reagent. The 0.1 M MES buffer solution was prepared by dissolving MES in water to a final concentration of 0.1 M and adjusting the pH to 5.5 with a sodium hydroxide solution.

[0033] (3) Quantification of polyphenols 90 μL of the tartaric acid reagent prepared in (2), 90 μL of each concentration of ethyl gallate solution, and 270 μL of 0.1 M MES buffer were mixed. 12 μL of 2 M trishydroxymethylaminomethane solution was added to adjust the pH to 8.0, and the absorbance at a wavelength of 520 nm was measured. Common logarithms were calculated for each concentration of ethyl gallate and the measured values, and an approximate straight line and multiple correlation coefficient (r 2 ) was calculated.

[0034] The results are shown in Figure 2. A concentration-dependent change in absorbance was confirmed at all measured concentration levels from 15.6 to 1000 μM, and the fit with the approximate line was good, with a multiple correlation coefficient of 0.9961. Therefore, it was confirmed that in the ferric tartrate absorptiometry method, it is possible to increase the sensitivity of the quantification limit to one-quarter the concentration by using an MES buffer solution and adjusting the pH to 8.0 before measuring absorbance (Figure 2).

[0035] Example 2: Consideration of measurement conditions 1) As in Example 1, ethyl gallate was dissolved in water to prepare a 1000 μM ethyl gallate solution, which was then serially diluted to prepare a 100 μM ethyl gallate solution. Also, an iron tartrate reagent was prepared using 0.1 M MES buffer as in Example 1, and 90 μL of the tartaric acid reagent, 90 μL of the 100 μM ethyl gallate solution, and 270 μL of the 0.1 M MES buffer were mixed together. Then, absorbance measurements were performed for the following cases: no 2M trishydroxymethylaminomethane solution was added, pH was adjusted to 7.5 by adding 9 μL of 2M trishydroxymethylaminomethane solution, and pH was adjusted to 8.0 by adding 12 μL of 2M trishydroxymethylaminomethane solution. The absorbance measurements were performed at wavelengths of 400 to 800 nm at 10 nm intervals. 2) The results are shown in Figure 3. When 2M trishydroxymethylaminomethane solution was not added, the maximum absorption wavelength was around 540 nm, but the peak shape was low and broad. On the other hand, when 2M trishydroxymethylaminomethane solution was added and the pH was adjusted to 7.5 or 8.0, it was confirmed that the peak became high and sharp. It was also confirmed that the maximum absorption wavelength shifted to 510 nm when the pH was adjusted to 7.5, and to around 520 nm when the pH was adjusted to 8.0. Therefore, it is necessary to adjust the pH with 2M trishydroxymethylaminomethane solution in order to perform the analysis with high sensitivity, and it is also necessary to change the wavelength for measuring absorbance to the lower wavelength side.

[0036] Reference Example 1: Examination of the amount of tris(hydroxymethylaminomethane) added In order to adjust the pH of the reaction solution for ferric tartrate spectrophotometric assay using MES buffer to 8.0, the necessary amount of addition was determined using 2 M trishydroxymethylaminomethane solution.

[0037] An iron tartrate reagent was prepared using 0.1 M MES buffer in the same manner as in Example 1, and 90 μL of the tartaric acid reagent and 360 μL of the 0.1 M MES buffer were mixed together. 2, 8, 9, 10, or 12 μL of a 2 M trishydroxymethylaminomethane solution was added thereto, and the pH of each solution was measured.

[0038] [Table 1]

[0039] The measurement results are shown in Table 1. The pH increased depending on the amount of 2M tris(hydroxymethylaminomethane) solution added, and the pH reached 8.0 when 12 μL was added. Although the pH does not necessarily have to be adjusted with 2M tris(hydroxymethylaminomethane) solution, by checking the relationship between the amount added and the pH of the reaction solution in advance, the amount added can be determined, which improves operability during analysis.

[0040] Reference Example 2: Effect of adding surfactant on measured values In order to suppress the turbidity of the sample to be measured, the effect on absorbance when a surfactant was added to the measurement system was evaluated.

[0041] 500 mg of iron (II) sulfate heptahydrate and 100 mg of sodium potassium iron tartrate tetrahydrate were weighed out, and SDS was added to the iron tartrate reagent so that the final concentrations were 0.4, 0.8, 1.6, 3.2, and 6.4%, and the mixture was dissolved in 0.1M MES buffer to make 100 mL. These were used as iron tartrate reagents, and 90 μL of 1000 μM ethyl gallate solution prepared in the same manner as in Example 1 was added to 90 μL of iron tartrate reagent containing SDS at each concentration, and 270 μL of 0.1M MES buffer was further added and mixed. 12 μL of 2M trishydroxymethylaminomethane solution was added thereto, and the absorbance at a wavelength of 520 nm was measured.

[0042] As shown in the measurement results in Figure 4, no significant change in absorbance was observed between 0 and 3.2% SDS concentration, but the measurement result for 6.4% showed a decrease in absorbance of about 10%. Therefore, when adding SDS to the ferric tartrate reagent, it is considered appropriate to set the SDS concentration at 3.2% or less.

Claims

1. A method for quantifying polyphenols in a sample by adding an iron tartrate reagent to a polyphenol-containing sample to develop color, measuring the absorbance after volumetric determination, in which 2-morpholinoethanesulfonic acid buffer is used to prepare and volumetrically determine the iron tartrate reagent, and the pH of the measurement solution is adjusted to 8.0±0.5 before measuring the absorbance.

2. 2. The method according to claim 1, wherein the pH of the measurement solution is adjusted using trishydroxymethylaminomethane.

3. 10. The method of claim 1, wherein absorbance at 520 nm is measured.

4. The method according to any one of claims 1 to 3, wherein the polyphenol-containing sample is a polyphenol-containing plant or an extract thereof.

Images