Screening and determination method for antioxidant components in tea beverage

Abstract

The invention discloses a method for screening and determining antioxidant components in a tea beverage, and belongs to the technical field of tea beverage component analysis. The screening and determination method mainly comprises the following steps: diluting the tea beverage, separating out different components through a high performance liquid chromatograph, mixing with an Ag complex-luminol mixed solution, detecting a luminescence signal through a flow injection chemiluminiscence instrument, and comparing with a standard curve to obtain the content of the antioxidant component in the tea beverage. Different antioxidant components in the tea beverage are separated and screened out through high performance liquid chromatography, a luminous signal of a reducing substance is amplified through an Ag complex-luminol system, and then the content of the antioxidant components is measured through a chemiluminescence method, so that online and real-time detection of the types and the content of the antioxidant is truly realized; the time of incubation reaction with free radicals is saved, and the analysis efficiency is greatly improved. The method is suitable for detecting the variety and content of the antioxidant components in the tea beverage.

Description

Technical Field

[0001] The invention belongs to the technical field of tea beverage component analysis, and relates to an analysis of antioxidant components in tea beverages, in particular to a method for screening and determining antioxidant components in tea beverages. Background Art

[0002] Antioxidant effect is considered to be the most important part of the health benefits of tea beverages. Traditional antioxidant analysis is tedious and time-consuming, and screening and evaluation are carried out offline and step by step: first, the sample is extracted with extractants of different properties, and then the antioxidant properties of the extracts are determined by conventional methods (such as DPPH method, ABTS method). If it has a certain antioxidant property, it is then separated and purified to obtain the pure product of this antioxidant component step by step, and then quantitatively analyzed. Such an analysis process is not only time-consuming and labor-intensive, but also easy to cause the loss of active components in the tedious sample preparation process.

[0003] At present, in order to improve the analysis efficiency, an online screening method for detecting antioxidant active ingredients is adopted: each component to be tested in the mixture is separated by high performance liquid chromatography, and the separated components enter different activity detectors (such as DAD detector, electrochemical detector or fluorescence detector) online to obtain corresponding analysis and detection. The analysis of antioxidants is evaluated by measuring the capture ability of various free radicals. Therefore, the analysis of antioxidants requires two measurements. The samples are tested under two conditions, without adding free radicals and with adding free radicals. After mixing with free radicals, the incubation reaction is usually carried out for at least 30 minutes. Therefore, the screening and measurement time is long and the efficiency is low. Summary of the invention

[0004] The purpose of the present invention is to provide a method for screening and determining antioxidant components in tea beverages, so as to solve the current problems of long time and low efficiency in screening and determining antioxidant components.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for screening and determining antioxidant components in tea beverages comprises the following steps:

[0007] S1. Prepare Ag complex solution: Take AgNO 3 , KIO 4 , K 2 S 2 O 8 , KOH and water, heated for 30 min, and added NaOH solution to prepare Ag complex solution;

[0008] The chemical formula of the Ag complex is [Ag(HIO6 ) 2 ] 5– ;

[0009] S2. Prepare luminol solution: Take luminol reagent and NaOH solution to prepare luminol solution;

[0010] S3. Determination of standard curve of antioxidant components: Dissolve the antioxidant component standards in methanol-water solution, dilute with water to form standard solutions of different gradients, mix with Ag complex-luminol mixed solution, detect luminescent signals by flow injection chemiluminescence instrument, and draw a standard curve;

[0011] S4. Screening and determination of antioxidant components in tea beverages: dilute the tea beverage, separate different components by high performance liquid chromatography, mix with Ag complex-luminol mixed solution, detect the luminescent signal by flow injection chemiluminescence instrument, and compare with the standard curve to obtain the antioxidant components and content in the tea beverage;

[0012] The antioxidant ingredients include gallic acid, epicatechin gallate, epigallocatechin, catechin, epicatechin and epigallocatechin gallate.

[0013] As a limitation, the concentration of Ag ions in the Ag complex solution is 0.3×10 -4 mol / L, the concentration of NaOH is 0.12mol / L;

[0014] The concentration of luminol in luminol solution is 0.4×10 -8 mol / L, the concentration of NaOH is 0.01mol / L.

[0015] As another limitation, the separation conditions of the high performance liquid chromatograph are:

[0016] Chromatographic column: C18 reverse phase chromatographic column; flow rate: 1 mL / min; injection volume: 20 μL; mobile phase solution: acetonitrile for pump A and 0.1% acetic acid aqueous solution for pump B.

[0017] As a further limitation, the concentrations of gallic acid, epigallocatechin, epigallocatechin gallate and epicatechin gallate in the standard solutions of different gradients are 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 1.5 μg / mL and 2.0 μg / mL;

[0018] The concentrations of catechin and epicatechin were 0.02 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 1.0 μg / mL, and 2.0 μg / mL.

[0019] Due to the adoption of the above technical solution, the present invention has the following technical advances compared with the prior art:

[0020] ① The present invention provides a method for screening and determining antioxidant components in tea beverages, which separates and screens different antioxidant components in tea beverages by high performance liquid chromatography, amplifies the luminescent signal of reducing substances by an Ag complex-luminol system, and then determines the content of antioxidant components by chemiluminescence, thereby truly realizing online and real-time detection of the types and contents of antioxidants, eliminating the time for incubation reaction with free radicals, and greatly improving the analysis efficiency;

[0021] ② The present invention provides a method for screening and determining antioxidant components in tea beverages, which has high sensitivity and high accuracy of quantitative detection, and the method is simple, has few steps, and is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a schematic diagram of the HPLC-CL instrument device in Example 1;

[0023] Figure 2 The chemiluminescence chromatograms of the green tea and oolong tea samples diluted 100 times in Example 1 are shown. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below by specific examples. It should be understood that the described examples are only used to explain the present invention, and are not intended to limit the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

[0026] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0027] Example 1

[0028] This embodiment discloses a method for screening and determining antioxidant components in tea beverages, and detects antioxidant components in green tea and oolong tea, specifically comprising the following steps:

[0029] S1. Preparation of Ag complex solution

[0030] Take 1.36 g of AgNO 3 5.80g of KIO 4 , 3g K 2 S 2 O 8, 8g of KOH, add 200mL of water, boil and continue heating for 30min, concentrate to about 100mL, and obtain Ag complex stock solution, in which the concentration of Ag(Ш) ion is measured according to its molar absorption coefficient at 362nm (ε=1.26×10 4 L·mol -1 cm -1 );

[0031] The Ag complex refers to di(hydroperiodic acid)silver(Ш) complex, the chemical formula of which is [Ag(HIO 6 ) 2 ] 5– ;

[0032] Take a certain volume of the calibrated Ag complex stock solution and a certain volume of 1.0 mol / L NaOH solution in a 250 mL volumetric flask, add water to dilute to the scale line, and prepare a concentration of Ag (Ш) ions of 0.3×10 -4 mol / L, NaOH concentration of 0.12mol / L Ag complex solution, set aside;

[0033] S2. Preparation of luminol solution

[0034] Take a certain volume of 20mM luminol reagent and a certain volume of 1.0mol / L NaOH solution in a 250mL volumetric flask to obtain a luminol concentration of 0.4×10 -8 mol / L, NaOH with a concentration of 0.01mol / L luminol solution for standby use;

[0035] S3. Standard curve for determination of antioxidant components

[0036] Accurately weigh six standard substances: 5 mg each of gallic acid (GA), epigallocatechin (EGC), catechin (C), epicatechin (EC), epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), dissolve them in 50% methanol aqueous solution, transfer them to clean 10 mL volumetric flasks to make up the volume, obtain the standard stock solution of each substance (the concentration is 0.5 mg / mL), and store them in a refrigerator at 4°C;

[0037] A certain volume of the standard stock solutions of GA, EGC, EGCG and ECG were taken, and they were graded diluted with pure water to prepare standard solutions of different gradients, wherein the concentrations of the standard solutions were 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 1.5 μg / mL and 2.0 μg / mL, respectively; a certain volume of the standard stock solutions of C and EC were taken, and they were graded diluted with pure water to prepare standard solutions of different gradients, wherein the concentrations of the standard solutions were 0.02 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 1.0 μg / mL and 2.0 μg / mL, respectively;

[0038] The standard solutions of different gradients were mixed with the Ag complex-luminol mixed solution, and the luminescence signal was detected by flow injection chemiluminescence instrument to draw the standard curve. The results are shown in Table 1:

[0039] Table 1 Standard curve list

[0040]

[0041]

[0042] S4. Screening and determination of antioxidant components in tea beverages

[0043] Chemiluminescence (CL) is recognized as one of the most sensitive detection technologies. It is a highly sensitive trace analysis technology that quantifies target compounds by directly oxidizing target compounds to produce chemiluminescent substances, or by enhancing or inhibiting chemiluminescent substances. Combining chemiluminescence detection technology with high-performance liquid chromatography separation technology can achieve online screening and real-time detection of antioxidants, greatly improving the efficiency of screening and separation of antioxidants.

[0044] The goal of screening and determining antioxidants is to identify and screen reducing compounds. The Ag complex-luminol chemiluminescence system has strong oxidation efficiency and luminescence efficiency, and responds sensitively to reducing compounds. It is particularly suitable as a chemiluminescence system for screening and determining antioxidants. It can also avoid the common bubble problem in the luminol-hydrogen peroxide system and the self-absorption of chemiluminescence by unreacted potassium permanganate in the potassium permanganate system.

[0045] Green tea and oolong tea beverages were filtered through a 0.45 μm water filter membrane, diluted 100 times, and added to the HPLC-CL instrument. The schematic diagram of the HPLC-CL instrument is shown in the figure. Figure 1 As shown;

[0046] Depend on Figure 1It can be seen that, driven by the peristaltic pump, a certain amount of Ag complex solution and luminol solution are first mixed to react, and then mixed with different components separated by high performance liquid chromatography (HPLC) in a glass coil. When there is no reducing compound flowing out, the Ag complex-luminol mixed solution will produce a baseline signal. When there is a reducing compound flowing out, the chemiluminescence signal is sharply amplified, and the degree of amplification is proportional to its content. Therefore, the luminescence signal is detected by a flow injection chemiluminescence instrument and compared with the standard curve, the content of the compound in the sample can be accurately determined. The chemiluminescence chromatograms of green tea and oolong tea are shown in the following figure. Figure 2 As shown;

[0047] Depend on Figure 2 It can be seen that under the above experimental conditions, gallic acid (GA), epigallocatechin (EGC), catechin (C), epicatechin (EC), epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) can be detected in both green tea and oolong tea beverages. Substituting the peak areas of the above six antioxidants at the retention times in the sample spectra into the standard curve equation respectively, it can be obtained that the antioxidant components in green tea include GA, EGC, EGCG, ECG, C and EC, and their contents are 10.80μg / mL, 24.87μg / mL, 5.66μg / mL, 8.54μg / mL, 68.28μg / mL and 13.14μg / mL; the antioxidant components in oolong tea include GA, EGC, C, EC, EGCG and ECG, and their contents are 2.8μg / mL, 46.84μg / mL, 2.58μg / mL, 9.16μg / mL, 75.72μg / mL and 9.96μg / mL, respectively.

[0048] The separation conditions of the high performance liquid chromatography are:

[0049] Chromatographic column: C18 reverse phase column; flow rate: 1 mL / min; injection volume: 20 μL; mobile phase solution: acetonitrile for pump A and 0.1% acetic acid aqueous solution for pump B; gradient elution program is shown in Table 2:

[0050] Table 2 HPLC gradient elution program

[0051] Time(min) <![CDATA[V A %]]> <![CDATA[V B %]]> Flow rate (mL / min) Time(min) 0 0 100 1.0 0 6 7 93 1.0 6 10 17 83 1.0 10 20 17 83 1.0 20 21 28 72 1.0 21 30 7 93 1.0 30 35 7 93 1.0 35

Claims

1. A method for screening and determining antioxidant components in tea beverages, characterized in that: The following steps are involved: S1. Preparation of Ag complex solution: Take AgNO3, KIO4, K2S2O8, KOH and water, heat for 30 min, add NaOH solution to prepare Ag complex solution; Among them, the chemical formula of the Ag complex is [Ag(HIO6)2] 5– ; S2. Prepare luminol solution: Take luminol reagent and NaOH solution to prepare luminol solution; S3. Determination of standard curve of antioxidant components: Dissolve the antioxidant component standards in methanol-water solution, dilute with water to form standard solutions of different gradients, mix with Ag complex-luminol mixed solution, detect luminescent signals by flow injection chemiluminescence instrument, and draw a standard curve; S4. Screening and determination of antioxidant components in tea beverages: dilute the tea beverage, separate different components by high performance liquid chromatography, mix with Ag complex-luminol mixed solution, detect the luminescent signal by flow injection chemiluminescence instrument, and compare with the standard curve to obtain the antioxidant components and content in the tea beverage; The antioxidant ingredients include gallic acid, epicatechin gallate, epigallocatechin, catechin, epicatechin and epigallocatechin gallate.

2. The method for screening and determining antioxidant components in tea beverages according to claim 1, characterized in that: In the Ag complex solution, the concentration of Ag ions is 0.3×10 -4 mol / L, the concentration of NaOH is 0.12mol / L; The concentration of luminol in luminol solution is 0.4×10 -8 mol / L, the concentration of NaOH is 0.01mol / L.

3. The method for screening and determining antioxidant components in a tea beverage according to claim 1 or 2, characterized in that: The separation conditions of the high performance liquid chromatograph are: Chromatographic column: C18 reverse phase column; flow rate: 1 mL / min; injection volume: 20 μL; mobile phase solution: acetonitrile for pump A and 0.1% acetic acid aqueous solution for pump B.

4. The method for screening and determining antioxidant components in tea beverages according to claim 3, characterized in that: In the standard solutions of different gradients, the concentrations of gallic acid, epigallocatechin, epigallocatechin gallate and epicatechin gallate are 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 1.5 μg / mL and 2.0 μg / mL; The concentrations of catechin and epicatechin were 0.02 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 1.0 μg / mL, and 2.0 μg / mL.

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