Method for detecting content of tea polyphenol

Abstract

The invention discloses a method for detecting the content of tea polyphenol. The detection method can be used for simultaneously detecting the content of 12 tea polyphenols in the tea product, the mobile phase is simple to prepare, and the use of controlled chemical reagents is avoided; universal instruments and equipment are used, and popularization is convenient; the detection efficiency is high; the precision and the accuracy are high; and one or more tea polyphenols can be monitored at the same time. The detection method is suitable for detecting various samples including raw materials, finished products and the like, and has high practicability.

Description

Technical Field

[0001] The invention relates to a method for detecting the content of tea polyphenols. Background Art

[0002] Tea polyphenols is the general name for polyphenols in tea leaves. It is a white amorphous powder that is easily soluble in water, soluble in ethanol, methanol, acetone, and ethyl acetate, but insoluble in chloroform. The content of tea polyphenols in green tea is relatively high, accounting for 15% to 30% of its mass. The main components of tea polyphenols are: flavanones, anthocyanidins, flavonols and anthocyanidins, and phenolic acids and phenolic acids. Among them, flavanones (mainly catechin compounds) are the most important, accounting for 60% to 80% of the total tea polyphenols, followed by flavonoids, and the content of other phenolic substances is relatively small.

[0003] Tea polyphenols are widely used in medicine, food industry, cosmetics and daily necessities due to their excellent natural antioxidant properties.

[0004] At present, the commonly used methods for detecting the content of tea polyphenols include spectral analysis, electrochemical analysis, capillary electrophoresis, etc. Among them, spectral analysis can only measure the content of total polyphenols, and cannot quantitatively analyze different types of tea polyphenol compounds at the same time. In addition, it is easily interfered by other phenolic compounds during the test, resulting in high measurement results. Electrochemical analysis can only quantitatively test the total polyphenol content, and cannot measure multiple compounds at the same time. In addition, it is necessary to select a suitable working electrode to obtain reliable results. Capillary electrophoresis has relatively poor repeatability, low analysis accuracy, large errors, and the instrumentation is not popular. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defect of the prior art that there are few detection technologies for the content of multiple tea polyphenols in tea products, and provide a quantitative detection method for tea polyphenols in tea products. The detection method of the present invention mainly detects tea polyphenols in tea raw materials, and its mobile phase is simple to prepare, avoiding the use of controlled chemical reagents; using general instruments and equipment, it is easy to promote; the detection efficiency is high; the detection can be completed in 25 minutes, with high precision and accuracy; and the content of 12 kinds of tea polyphenols can be detected simultaneously. The detection method of the present invention is suitable for detecting various tea products and has strong practicality.

[0006] The present invention provides a method for detecting the content of tea polyphenols, which comprises the following steps: subjecting a tea product to gradient elution in a high performance liquid chromatography; The stationary phase of the high performance liquid chromatography is C18; The gradient elution is a multi-stage gradient elution; the mobile phase of the high performance liquid chromatography is methanol and 0.01% phosphoric acid aqueous solution, wherein the percentage of phosphoric acid aqueous solution refers to the percentage of phosphoric acid volume to water volume.

[0007] The multi-stage gradient elution comprises the following steps, wherein: In the first gradient, the volume ratio of methanol is 15-25%, and the volume ratio of the phosphoric acid aqueous solution is 75-85%, preferably, the volume ratio of methanol is 20%, and the volume ratio of the phosphoric acid aqueous solution is 80%; the elution time is 13-17 minutes, preferably, the elution time is 15 minutes; In the second gradient, the volume ratio of methanol is 25-35%, and the volume ratio of the phosphoric acid aqueous solution is 65-75%, preferably, the volume ratio of methanol is 30%, and the volume ratio of the phosphoric acid aqueous solution is 70%; the elution time is 3-7 minutes, preferably, the elution time is 5 minutes; In the third gradient, the volume ratio of methanol is 35-45%, and the volume ratio of the phosphoric acid aqueous solution is 55-65%, preferably, the volume ratio of methanol is 40%, and the volume ratio of the phosphoric acid aqueous solution is 60%; the elution time is 3-7 minutes, preferably, the elution time is 5 minutes; In the present invention, the type of tea can be selected from green tea, black tea, white tea, snow tea, oolong tea, yellow tea, white tea, black tea, scented tea, jasmine, Tieguanyin, vegetarian tea, Pu'er tea, Kuding tea, and one or more of Dahongpao, Xiongdan tea, ginseng tea, and Pu'er tea. The type of tea product can be one or more of tea leaves, tea freeze-dried powder, tea soup, and tea extracts. The tea extracts include but are not limited to active ingredients of tea polyphenols extracted by water extraction, alcohol extraction, or other methods. Preferably, the type of tea is green tea, and the tea product is one or more of tea leaves, tea freeze-dried powder, and tea extracts; further, the present invention can also be used to detect various beverages and skin care products containing tea polyphenols.

[0008] The types of tea polyphenols detectable by the present invention are gallic acid (hereinafter referred to as GA), (-)-gallocatechin / epicatechin (hereinafter referred to as GC), (-)-epigallocatechin (hereinafter referred to as EGC), (-)-catechin (hereinafter referred to as C), epicatechin (hereinafter referred to as EC), (-)-epigallocatechin gallate (hereinafter referred to as EGCG), (-)-gallocatechin gallate (hereinafter referred to as GCG), (-)-epicatechin gallate (hereinafter referred to as ECG), (-)-gallic acid catechin ester (hereinafter referred to as CG), theophylline (hereinafter referred to as TP), theobromine (hereinafter referred to as TB), and caffeine (hereinafter referred to as CAF). Preferably, the method of the present invention can simultaneously and accurately detect the contents of the above 12 types of tea polyphenols.

[0009] Preferably, the chromatographic column of the HPLC is Kromasil 100-5-C18, 4.6*250mm*5μm.

[0010] Preferably, the column temperature of the HPLC can be a conventional column temperature in the art, preferably 28-35°C, more preferably 30°C.

[0011] Preferably, the detection wavelength of the HPLC is a conventional detection wavelength in the art, preferably 270 nm to 280 nm, more preferably 278 nm.

[0012] Preferably, the flow rate of the HPLC can be a conventional flow rate in the art, preferably 0.8 mL / min to 1.2 mL / min, more preferably 1.0 mL / min.

[0013] Preferably, the injection volume of the HPLC is 5 to 15 μL, preferably 10 μL.

[0014] Based on the common sense in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain the preferred embodiments of the present invention.

[0015] The reagents and raw materials used in the present invention are commercially available.

[0016] The positive and progressive effects of the present invention are: the mobile phase preparation of the tea polyphenol content detection method described in the present invention is simple, avoiding the use of controlled chemical reagents; using general instruments and equipment, it is easy to promote; the detection efficiency is high; the precision and accuracy are high; and the content of 12 kinds of tea polyphenols can be monitored simultaneously. In a preferred embodiment of the present invention, a single determination only takes 25 minutes, and 12 kinds of tea polyphenols can be monitored simultaneously; the peak area and mass concentration of the 12 kinds of tea polyphenols show a good linear relationship, the method quantitative limit is 2.5~25mg / L, the recovery rate is 94%~103%, and RSD≤2.8%. The detection method of the present invention is suitable for detecting various tea products and has strong practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Chromatogram of mixed standard solution of 12 compounds Figure 2 Luna Phenyl-Hexyl Chromatogram Figure 3 YMC Triart PFP Chromatogram Figure 4 Detection results of different elution modes Figure 5 Detection results of different mobile phases Figure 6 Chromatogram of West Lake Longjing Tea Freeze-dried Powder Figure 7 Chromatogram of Anji White Tea Freeze-dried Powder Figure 8 Chromatogram of Biluochun Green Tea Freeze-dried Powder Fig. 9 Chromatogram of Hengjiuyuan Green Tea Freeze-dried Powder Fig.10 Chromatogram of Chunlun Green Tea Freeze-dried Powder Fig.11 Chromatogram of West Lake Longjing extract Implementation

[0018] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0019] Example 1 Sample preparation Weigh 250 mg of ascorbic acid, add 500 mL of pure water and 500 mL of methanol to dissolve, and prepare a stable solution with a VC concentration of 250 mg / L.

[0020] Mixed standard solution Accurately weigh about 50 mg of EGC, EGCG and about 40 mg of EC into a 50 mL volumetric flask, dissolve and dilute to the mark with stabilizing solution and shake well as mother solution 1; Accurately weigh about 20 mg each of GA, GC, GCG, ECG, C and about 10 mg of CG into a 50 mL volumetric flask, dissolve and dilute to the mark with stabilizing solution and shake well as mother solution 2; Accurately weigh about 10 mg each of TB and TP and about 40 mg of CAF into a 50 mL volumetric flask, dissolve and dilute to the mark with stabilizing solution and shake well as mother solution 3.

[0021] Take 0.25, 0.5, 1, 2, 5 mL of mother solution 1, and take 0.125, 0.25, 0.5, 1, 2.5 of mother solution 2 and 3 respectively, and place them in a 10 mL volumetric flask, dilute to the scale with stabilizing solution and shake well. The concentrations (mg / L) of the series of standard solutions are as follows: Table 1 Standard solution concentration (mg / L)

[0022] Example 2 Optimization of Chromatographic Conditions In order to determine the content of tea polyphenols, an Agilent HPLC 1200 high performance liquid chromatograph was used, coupled with a diode array detector, and a chromatographic column Kromasil 100-5-C18 4.6*250mm*5um purchased from Nouryon; the chromatographic conditions are shown in Table 2, the standard solution prepared in Example 1 was used for loading, methanol and 0.01% phosphoric acid aqueous solution were used as mobile phases, and the gradient elution method shown in Table 3 was used for elution. The feed volume was 10uL, the column temperature was 30°C, and the detection wavelength was 278nm.

[0023] Table 2 Chromatographic conditions:

[0024] Table 3 Elution gradient

[0025] Test results see Figure 1 The results showed that the chromatogram showed 12 characteristic peaks, which were symmetrical and sharp, from left to right, namely GA, GC, TB, EGC, TP, C, CAF, EGCG, EC, GCG, ECG, and CG. The detection cycle can be completed within 25 minutes.

[0026] Example 3 Test results (1) Linear range and limit of quantification The series of mixed standard solutions in Example 1 were subjected to chromatographic detection (detection conditions are shown in Tables 2 and 3), and the peak area A was used for linear regression of mass concentration ρ (mg / L). The 12 tea polyphenols had a good linear relationship within their respective linear ranges. The results are shown in Table 4.

[0027] Table 4 Linear range, correlation coefficient, quantitation limit and detection limit are shown in the table below

[0028] Recovery and precision Accurately weigh 12 portions of tea freeze-dried powder sample matrix 50 mg (accurate to 0.1 mg) into a 25 mL volumetric flask, and prepare the test solution according to the preparation method of the test solution described in Example 1, of which 6 bottles are added with mixed standard solution according to STD1 concentration as the matrix low concentration precision test solution; the other 6 bottles are added with mixed standard solution according to STD4 concentration as the matrix high concentration precision test solution. After constant volume, 10 μL is taken for injection and detection (the detection conditions are shown in Tables 2 and 3), and the standard curve is used for quantitative analysis. The recovery rate and precision of the two high and low spiked concentrations are investigated. The experimental results are shown in Table 5. The recovery rates of the 12 tea polyphenols are 94%~103%, and RSD≤2.8%.

[0029] Table 5 Method spike recovery and precision

[0030] (3) Actual sample testing Preparation of test samples: Accurately weigh 50 mg (accurate to 0.1 mg) of different tea freeze-dried powder samples into a 25 mL volumetric flask, dissolve with stabilizing solution, dilute to scale, and shake well; filter an appropriate amount with a 0.45 um organic phase PTFE filter membrane, and the specific results of the sample injection test are shown in Table 6, and the test spectrum is shown in Figure 6-10 .

[0031] Weigh 0.5g of West Lake Longjing tea leaves (accurate to 0.1mg) into a 150mL beaker, add solvent (water / methanol = 1 / 1), and stir and extract at 50℃ for 30 minutes. To obtain West Lake Longjing tea extract, weigh 50mg of tea extract (accurate to 0.1mg) into a 25mL volumetric flask, dissolve with stabilizing solution, dilute to scale, and shake well; the specific results of the sample injection test are shown in Table 6, and the test spectrum is shown in Fig.11 .

[0032] In order to verify the credibility of this detection method, the total amount of tea polyphenols in each tea freeze-dried powder and tea extract was tested using the fourth part of GBT8313-2018 Folin phenol method, and the data are shown in Table 6. It has been verified that the test method of the present invention can accurately detect the content of 12 kinds of tea polyphenols, and the detection cycle is short and the data credibility is high.

[0033] Table 6 Test results

[0034] ND in the table means not detected This method uses C18 chromatographic column as filler, and uses methanol-0.01% phosphoric acid aqueous solution mobile phase combined with gradient elution. It can well separate and simultaneously determine 12 kinds of tea polyphenols. Within a certain mass concentration range, the peak area and mass concentration of the 12 kinds of tea polyphenols show a good linear relationship. The method quantitative limit is 2.5~25mg / L, the recovery rate is 94%~103%, and RSD≤2.8%. This method has many advantages: simple preparation of mobile phase, avoiding the use of controlled chemical reagents; using general instruments and equipment, easy to promote; high detection efficiency; high precision and accuracy; can simultaneously monitor the content of 12 kinds of tea polyphenols. The quantitative limit is as low as 2.5~25mg / L; 12 kinds of tea polyphenols are monitored simultaneously; it is suitable for all kinds of samples including raw materials and finished products, etc. Therefore, this method has strong practicality and is of great help to the government in strengthening market monitoring and improving the quality control of raw materials and products of enterprises.

[0035] Comparative Example 1: Different chromatographic columns were selected for detection, such as the chromatographic column Luna Phenyl-Hexyl, 4.6*250mm*5um, purchased from the United States Fenome Corporation, and the chromatographic column YMC Triart PFP4.6*250mm*5um purchased from Japan Vimexi Co., Ltd. Two different chromatographic columns were investigated, with the same elution gradient and flow rate. The detection results are shown in Example 2. Figure 2 , Figure 3 , we can know that: In the chromatogram obtained by Luna Phenyl-Hexyl detection, ECG and CG were not baseline-separated, and the contents of ECG and CG could not be accurately detected. In the chromatogram obtained by YMC Triart PFP column detection, the detection time was more than 35 minutes, and the detection efficiency was low.

[0036] Comparative Example 2: Different mobile phases and elution gradients were used for detection, wherein the mobile phase was methanol and 0.2% phosphoric acid water; the gradient elution mode was selected as follows for elution: 0-7 min: the volume ratio of methanol to 0.2% phosphoric acid water was 28:72, 7-15 min: the volume ratio of methanol to 0.2% phosphoric acid water was 20:80, 15-35 min: the volume ratio of methanol to 0.2% phosphoric acid water was 28:72. The remaining steps were the same as in Example 2, and the results were as follows: Figure 4 From the chromatogram, it can be seen that the GA and GC peaks interfere with each other; the TB and EGC peaks interfere with each other; the TP and EGCG peaks interfere with each other; the CAF and GCG peaks overlap; the content of the above 8 tea polyphenols cannot be accurately measured.

[0037] Comparative Example 3: Different mobile phases were used for detection. The mobile phase was acetonitrile: 0.01% phosphoric acid water for elution. The other test conditions were the same as those in Example 2. The test results are shown in Figure 5 ,As can be seen from the figure, the C and CAF peaks cannot be separated, and the C and CAF contents cannot be ,accurately analyzed.

Claims

1. A method for detecting the content of tea polyphenols, comprising the following steps: subjecting a tea product to gradient elution in a high performance liquid chromatography; The stationary phase of the high performance liquid chromatography is C18; The mobile phase of the high performance liquid chromatography is methanol and 0.01% phosphoric acid aqueous solution, wherein: The percentage of phosphoric acid aqueous solution refers to the percentage of phosphoric acid volume to water volume; The gradient elution is a multi-stage gradient elution.

2. The detection method according to claim 1, wherein The multi-stage gradient elution comprises the following steps: The first gradient has a volume ratio of 15-25% methanol and a volume ratio of 75-85% phosphoric acid aqueous solution, preferably, a volume ratio of 20% methanol and a volume ratio of 80% phosphoric acid aqueous solution; the elution time is 13-17 minutes, preferably, the elution time is 15 minutes; The second gradient, the volume ratio of methanol is 25-35%, the volume ratio of phosphoric acid aqueous solution is 65-75%, preferably, the volume ratio of methanol is 30%, and the volume ratio of phosphoric acid aqueous solution is 70%; the elution time is 3-7 minutes, preferably, the elution time is 5 minutes; The third gradient, the volume proportion of methanol is 35-45%, the volume proportion of phosphoric acid aqueous solution is 55-65%, preferably, the volume proportion of methanol is 40%, and the volume proportion of phosphoric acid aqueous solution is 60%; the elution time is 3-7 minutes, preferably, the elution time is 5 minutes.

3. The detection method according to claim 1, wherein the tea polyphenols can be selected from the group consisting of gallic acid (hereinafter referred to as GA), (-)-gallocatechin / epicatechin (hereinafter referred to as GC), (-)-epigallocatechin (hereinafter referred to as EGC), (-)-catechin (hereinafter referred to as C), epicatechin (hereinafter referred to as EC), (-)-epigallocatechin gallate (hereinafter referred to as EGCG), (-)-gallocatechin gallate (hereinafter referred to as GCG), (-)-epicatechin gallate (hereinafter referred to as ECG), (-)-gallic acid catechin ester (hereinafter referred to as CG), theophylline (hereinafter referred to as TP), theobromine (hereinafter referred to as TB), and caffeine (hereinafter referred to as CAF). Preferably, the contents of the above 12 tea polyphenols are tested simultaneously.

4. The detection method according to claim 1, wherein the chromatographic column of the high performance liquid chromatography is Kromasil 100-5-C18 4.6*250mm*5μm.

5. The detection method according to claim 1, wherein the column temperature of the HPLC is 28-35°C, preferably 30°C.

6. The detection method according to claim 1, wherein the detection wavelength of the HPLC is conventionally 270 nm to 280 nm in the art, preferably 278 nm.

7. The detection method according to claim 1, wherein the flow rate of the HPLC is 0.8 mL / min to 1.2 mL / min, preferably 1.0 mL / min.

8. The detection method according to claim 1, wherein the injection volume of the HPLC is 5 to 15 μL, preferably 10 μL.

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