Method for establishing fingerprint spectrum of heat-clearing and eyesight-improving tea by using high performance liquid chromatography method and application

Abstract

The invention discloses a method for establishing a heat-clearing and eyesight-improving tea fingerprint spectrum by using a high performance liquid chromatography method and an application of the heat-clearing and eyesight-improving tea fingerprint spectrum. By utilizing the method disclosed by the invention, 17 fingerprint peaks can be confirmed, and the three components including fried semen cassiae, chrysanthemum and stevia rebaudiana in the heat-clearing and eyesight-improving tea all have corresponding attribution peaks. According to the present invention, the characteristic peaks of the six chemical components such as chlorogenic acid, rebaudioside A, stevioside, aurantio-obtusin, emodin and chrysophanol are identified through the comparison of the reference substance and the combination of the ultraviolet absorption spectrum characteristics, the rebaudioside A and the stevioside are the characteristic peaks of stevia rebaudiana, and the chrysanthemum and the stevia rebaudiana can be separated by the two characteristic peaks; according to the fingerprint spectrum detection method disclosed by the invention, the fingerprint spectrum with good separation degree and comprehensive chromatographic peaks can be obtained, the overall quality of the heat-clearing and eyesight-improving tea can be evaluated more objectively and comprehensively, and a foundation is laid for quality control of the heat-clearing and eyesight-improving tea.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine analysis and detection. Specifically, it relates to a method for establishing a fingerprint of Qingre Mingmu Tea by high performance liquid chromatography and its application. Background Art

[0002] Qingre Mingmu Tea is a traditional Chinese medicine preparation (WS3-B-1029-91) included in the "Drug Standards of the Ministry of Health of the People's Republic of China - Traditional Chinese Medicine Formulas and Preparations (Volume 5)". It is composed of three Chinese medicinal materials: Semen Cassiae (fried), Flos Chrysanthemi, and Folium Steviae Rebaudianae, and has the effects of clearing heat and expelling wind, and soothing the liver and improving eyesight. It can be used for the treatment of hypertension, dizziness, headache, red and blurred eyes, etc. Among them, the monarch drug Semen Cassiae has the effects of clearing heat and improving eyesight, and laxative. Its main active ingredients include chemical components such as anthraquinones, naphthopyranones, amino acids, and inorganic elements. The representative components of anthraquinones, aurantio-obtusin, emodin, chrysophanol, physcion, rhein, and aloe-emodin are the characteristic components of Semen Cassiae. Flos Chrysanthemi is the dried capitulum of the plant Chrysanthemum morifolium Ramat. of the Compositae family. Modern pharmacological studies have shown that Flos Chrysanthemi has good bioactive functions such as anti-inflammatory, antioxidant, anti-tumor, neuroprotection, prevention and treatment of hyperuricemia, and alleviation of diabetes. These pharmacological effects are mainly related to its active ingredients, including phenolic acids, flavonoids, and polysaccharides. Folium Steviae Rebaudianae is the dried leaf of the plant Stevia rebaudiana (Bertoni) Hemsl. of the Compositae family. Folium Steviae Rebaudianae is mainly used to extract steviol glycosides as sweeteners or pharmaceutical excipients. However, a large amount of Folium Steviae Rebaudianae waste residue will be generated after extracting steviol glycosides. The main active ingredients in the Folium Steviae Rebaudianae waste residue are chlorogenic acid compounds such as chlorogenic acid and neochlorogenic acid. These active ingredients have good antioxidant, anti-inflammatory, antibacterial and other functions.

[0003] The fingerprint of traditional Chinese medicine is to analyze the substances of traditional Chinese medicine through modern technologies such as spectroscopy or chromatography, so as to obtain the results representing the overall characteristics of traditional Chinese medicinal materials. It analyzes the chemical information of the processed traditional Chinese medicine, depicts the peak distribution of specific chemical components, and thus realizes the qualitative and quantitative evaluation of the quality of traditional Chinese medicine. The construction and analysis of the fingerprint of traditional Chinese medicine can help ensure the consistency and quality of traditional Chinese medicine, and provide a scientific basis for the identification of the types of medicinal materials and the optimization of production processes. The prior art "Study on HPLC Characteristic Fingerprint of Qingre Mingmu Tea and Determination of Multi-component Contents" (Publication Date: February 29, 2020) discloses the fingerprint of Qingre Mingmu Tea and identifies 7 peaks of chlorogenic acid, aurantio-obtusin, aloe-emodin, rhein, emodin, chrysophanol and physcion. However, among these 7 peaks, chlorogenic acid is a component commonly contained in chrysanthemum and stevia rebaudiana, and the remaining 6 peaks all belong to stir-fried semen cassiae. Therefore, this fingerprint cannot effectively identify chrysanthemum and stevia rebaudiana in Qingre Mingmu Tea, and thus cannot well conduct qualitative and quantitative evaluation of the quality of Qingre Mingmu Tea.

[0004] Therefore, in order to more comprehensively monitor the quality of Qingre Mingmu Tea and improve the quality evaluation system of Qingre Mingmu Tea, it is of great significance to establish a fingerprint of Qingre Mingmu Tea with better resolution and more comprehensive chromatographic peaks. Summary of the Invention

[0005] To overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides a method and application for establishing a fingerprint of Qingre Mingmu Tea by using high performance liquid chromatography.

[0006] The first object of the present invention is to provide a pretreatment method for detecting Qingre Mingmu Tea by high performance liquid chromatography.

[0007] The second object of the present invention is to provide a high performance liquid chromatography method.

[0008] The third object of the present invention is to provide the application of the above pretreatment method and / or high performance liquid chromatography method in establishing the HPLC fingerprint of Qingre Mingmu Tea and / or the quality control of Qingre Mingmu Tea.

[0009] The fourth object of the present invention is to provide a method for constructing an HPLC fingerprint of Qingre Mingmu Tea.

[0010] The fifth object of the present invention is to provide a quality detection method for Qingre Mingmu Tea.

[0011] The sixth object of the present invention is to provide the application of the above high performance liquid chromatography method in detecting the contents of stir-fried semen cassiae, chrysanthemum and / or stevia rebaudiana in Qingre Mingmu Tea.

[0012] Therefore, the present invention claims to protect the following:

[0013] A pretreatment method for detecting Qingre Mingmu tea by high performance liquid chromatography. Take the Qingre Mingmu tea sample to be tested and ultrasonically extract it with a methanol aqueous solution with a volume fraction of 45-55% for 25-35 minutes.

[0014] Preferably, the mass-volume ratio of the Qingre Mingmu tea sample to be tested to methanol is 1 g:(45-55) mL.

[0015] More preferably, the mass-volume ratio of the Qingre Mingmu tea sample to be tested to methanol is 1 g:50 mL.

[0016] More preferably, the power of the ultrasonic wave is 280-320 W, and the frequency of the ultrasonic wave is 38-42 kHz.

[0017] Most preferably, the power of the ultrasonic wave is 300 W, and the frequency of the ultrasonic wave is 40 kHz.

[0018] More preferably, the time of ultrasonic extraction is 30 minutes.

[0019] A high performance liquid chromatography method. Using octadecylsilane bonded silica as the filler of the chromatographic column, acetonitrile as mobile phase A, and a phosphoric acid solution with a mass fraction of 0.08-0.12% as mobile phase B. In the gradient elution program, the volume percentage change of mobile phase B in the mobile phase system is as follows:

[0020] 0-12 min, mobile phase B is 89%;

[0021] 12-15 min, mobile phase B drops from 89% to 78%;

[0022] 15-40 min, the mobile phase drops from 78% to 75%;

[0023] 40-45 min, the mobile phase drops from 75% to 65%;

[0024] 45-55 min, the mobile phase is 65%;

[0025] 55-56 min, the mobile phase drops from 65% to 60%;

[0026] 56-62 min, the mobile phase is 60%;

[0027] 62-64 min, the mobile phase drops from 60% to 58%;

[0028] 64-79 min, the mobile phase drops from 58% to 48%;

[0029] 79-84 min, the mobile phase drops from 48% to 20%;

[0030] 84-87 min, the mobile phase is 20%;

[0031] 87 - 89 min, the mobile phase increases from 20% to 89%;

[0032] 89 - 95 min, the mobile phase is 89%;

[0033] The detection wavelength is gradient detection: 0 - 12 min, the detection wavelength is 326 nm; 12 - 40 min, the detection wavelength is 286 nm; 40 - 55 min, the detection wavelength is 210 nm; 55 - 95 min, the detection wavelength is 286 nm.

[0034] Preferably, the column temperature of the chromatographic column is 34 - 36 °C.

[0035] More preferably, the column temperature of the chromatographic column is 35 °C.

[0036] More preferably, the chromatographic column is Waters XSelect HSS T3 chromatographic column.

[0037] More preferably, the Waters XSelect HSS T3 chromatographic column has a column length of 25 cm, an inner column diameter of 4.6 mm, and a particle size of 5 μm.

[0038] Preferably, the flow rate of the gradient elution program is:

[0039] 0 - 12 min, the flow rate is 1.0 mL / min;

[0040] 12 - 15 min, the flow rate decreases from 1.0 mL / min to 0.8 mL / min;

[0041] 15 - 40 min, the flow rate is 0.8 mL / min;

[0042] 40 - 45 min, the flow rate decreases from 0.8 mL / min to 0.5 mL / min;

[0043] 45 - 55 min, the flow rate is 0.5 mL / min;

[0044] 55 - 56 min, the flow rate increases from 0.5 mL / min to 1.0 mL / min;

[0045] 56 - 95 min, the flow rate is 1.0 mL / min.

[0046] Preferably, the mass fraction of the phosphoric acid solution is 0.1%.

[0047] Use of any of the above - mentioned pretreatment methods and / or high - performance liquid chromatography methods in establishing the HPLC fingerprint of Qingre Mingmu tea and / or quality control of Qingre Mingmu tea.

[0048] A method for constructing an HPLC fingerprint of a heat-clearing and eyesight-improving tea, comprising treating a heat-clearing and eyesight-improving tea standard sample using any of the above-mentioned pretreatment methods to obtain a test solution to be tested;

[0049] Using chlorogenic acid solution as a reference solution, use any of the above-mentioned high performance liquid chromatography methods to detect the standard sample of the heat-clearing and eyesight-improving tea, record the chromatogram, and the theoretical plate number calculated based on the chlorogenic acid peak is not less than 8000 to obtain the HPLC fingerprint of the heat-clearing and eyesight-improving tea.

[0050] Preferably, the reference solution is prepared by fully mixing chlorogenic acid and methanol aqueous solution, wherein the mass volume ratio of chlorogenic acid to methanol is (58-62) μg:1 mL.

[0051] More preferably, the mass volume ratio of the chlorogenic acid to methanol is 60 μg:1 mL.

[0052] The HPLC fingerprint spectrum contains 17 fingerprint peaks, with the No. 1 peak corresponding to the reference solution as the S peak, and the relative retention times of the remaining peaks are within ±5% of the specified values, and the specified values are:

[0053] Peak 1 (S peak) 1.000, peak 2 2.434, peak 3 2.568, peak 4 2.780, peak 5 2.997, peak 6 3.239, peak 7 5.183, peak 8 5.445, peak 9 5.480, peak 10 6.411, peak 11 7.231, peak 12 7.645, peak 13 7.735, peak 14 8.181, peak 15 8.520, peak 16 8.658, peak 17 8.837;

[0054] The peak No. 1 is the characteristic chromatographic peak of chlorogenic acid, the peak No. 8 is the characteristic chromatographic peak of rebaudioside A, the peak No. 9 is the characteristic chromatographic peak of stevioside, the peak No. 10 is the characteristic chromatographic peak of aurantium dulcisin, the peak No. 14 is the characteristic chromatographic peak of rhamnosin, and the peak No. 17 is the characteristic chromatographic peak of chrysophanol.

[0055] A method for testing the quality of heat-clearing and eyesight-improving tea, comprising treating a sample of the heat-clearing and eyesight-improving tea using any of the above-mentioned pretreatment methods to obtain a test sample solution of the sample;

[0056] Use any of the above-mentioned high performance liquid chromatography methods to test the test sample solution, record the chromatogram, obtain the HPLC spectrum of the test sample, compare the HPLC spectrum of the test sample with the reference fingerprint spectrum, and judge it as qualified only if it meets the following two conditions:

[0057] The HPLC fingerprint of the sample to be tested exhibits a fingerprint peak whose corresponding retention time in the control fingerprint is within ±5% of the specified value;

[0058] Calculate the fingerprint peaks according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint. The similarity between the HPLC fingerprint of the test sample and the control fingerprint is not less than 0.90;

[0059] The construction method of the control fingerprint is as follows: use any of the above-mentioned pretreatment methods to treat the standard sample of Qingre Mingmu tea to obtain the test sample solution for testing;

[0060] Use the chlorogenic acid solution as the reference solution, and use any of the above-mentioned high performance liquid chromatography methods to detect the test sample solution for testing, record the chromatogram, and the theoretical plate number is not less than 8000 calculated according to the chlorogenic acid peak to obtain the control fingerprint of Qingre Mingmu tea;

[0061] The control fingerprint contains 17 fingerprint peaks. Taking the corresponding peak 1 of the reference solution as the S peak, the specified values are as follows:

[0062] Peak 1 (S peak) 1.000, Peak 2 2.434, Peak 3 2.568, Peak 4 2.780, Peak 5 2.997, Peak 6 3.239, Peak 7 5.183, Peak 8 5.445, Peak 9 5.480, Peak 10 6.411, Peak 11 7.231, Peak 12 7.645, Peak 13 7.735, Peak 14 8.181, Peak 15 8.520, Peak 16 8.658, Peak 17 8.837.

[0063] Use of any of the above-mentioned high performance liquid chromatography methods in the determination of the contents of Cassia obtusifolia, Chrysanthemum morifolium and / or Stevia rebaudiana in Qingre Mingmu tea.

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] The present invention discloses a method for establishing a fingerprint of Qingre Mingmu tea by high performance liquid chromatography and its application. By using the method of the present invention, 17 fingerprint peaks can be confirmed, and there are corresponding attribution peaks for the three components of Cassia obtusifolia, Chrysanthemum morifolium and Stevia rebaudiana in Qingre Mingmu tea. Through comparison with reference substances and combined with the characteristics of ultraviolet absorption spectra, the characteristic peaks of 6 chemical components, namely chlorogenic acid, rebaudioside A, stevioside, aurantio-obtusin, emodin and chrysophanol, are identified. Among them, rebaudioside A and stevioside are the characteristic peaks of Stevia rebaudiana, and these two characteristic peaks can distinguish Chrysanthemum morifolium from Stevia rebaudiana. The fingerprint detection method disclosed by the present invention can obtain a fingerprint with good resolution and comprehensive chromatographic peaks, and can more objectively and comprehensively evaluate the overall quality of Qingre Mingmu tea, laying a foundation for the quality control of Qingre Mingmu tea. Description of the Drawings

[0066] Figure 1 It is the attribution of the chromatographic peaks of the fingerprint.

[0067] Figure 2 To identify the chemical components of each medicinal flavor based on the chromatographic peaks of the fingerprint.

[0068] Figure 3 It is a comparison chart of the ultraviolet spectrum absorption of the reference substance (upper) and the test sample (lower).

[0069] Figure 4 It is the fingerprint of Qingre Mingmu Tea.

[0070] Figure 5 It is the peak emergence time of the fingerprint of Qingre Mingmu Tea.

[0071] Figure 6 It is the enlarged chromatogram of peak No. 8 and peak No. 9 in the fingerprint of Qingre Mingmu Tea.

[0072] Figure 7 It is the result of the chromatogram of injection precision.

[0073] Figure 8 It is the result of the repeatability chromatogram.

[0074] Figure 9 It is the result of the intermediate precision chromatogram.

[0075] Figure 10 It is the result of the stability chromatogram.

[0076] Figure 11 It is the result of the durability chromatogram under different phosphoric acid concentration conditions.

[0077] Figure 12 It is the result of the durability chromatogram under different column temperature conditions.

[0078] Figure 13 It is the result of the chromatogram of Comparative Example 1.

[0079] Figure 14 It is the chromatogram obtained according to the gradient conditions shown in Table 24 in Comparative Example 2.

[0080] Figure 15 It is the chromatogram of the test sample solution prepared with different extraction solvents.

[0081] Figure 16 It is the result of the chromatogram of Comparative Example 4.

[0082] Figure 17 It is the result of the chromatogram of Comparative Example 5. Specific implementation manners

[0083] The present invention will be further described below in conjunction with specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0084] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0085] The Qingre Mingmu tea used in the examples and comparative examples is the traditional Chinese medicine Qingre Mingmu tea (Approval No. Z43020775) approved by the State Food and Drug Administration.

[0086] Attribution of chromatographic peaks in the HPLC chromatogram of Qingre Mingmu tea in Example 1

[0087] 1. Experimental method

[0088] (1) Preparation of test solution

[0089] Take 0.5 g of the powder of Qingre Mingmu tea, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 50% methanol solution (v / v), weigh it, ultrasonically treat it (power 300 W, frequency 40 kHz) for 30 min, let it cool, weigh it again, make up the lost weight with 50% methanol solution (v / v), shake well, centrifuge at 12000 rpm for 5 min, and take the supernatant for filtration to obtain the test solution.

[0090] (2) Preparation of single-herb decoction pieces solution

[0091] According to the preparation method of “(1) Preparation of test solution”, take about 0.5 g of the fine powder of stir-fried semen cassiae, flos chrysanthemi, and stevia rebaudiana respectively to prepare 3 single-herb decoction pieces sample solutions.

[0092] (3) HPLC detection

[0093] Use octadecylsilane chemically bonded silica gel as the filler (Waters XSelect HSS T3 chromatographic column, column length 25 cm, column inner diameter 4.6 mm, particle size 5 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution (w / v) as mobile phase B, and perform gradient elution according to the mobile phase gradient elution program shown in Table 1; the column temperature is 35 °C. Accurately pipette 10 μL of the test solution and the single-herb decoction pieces solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0094] Table 1 Liquid chromatographic elution conditions

[0095]

[0096] 2. Information of test samples and reference substances

[0097]

[0098] 3. Experimental results

[0099] The chromatograms of the test sample and the 3 single-herb decoction pieces samples are as Figure 1 shown. From Figure 1It can be seen that peaks 1, 2, 3, and 4 are the common peaks of chrysanthemum and stevia; peaks 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, and 17 are the exclusive characteristic peaks of stir-fried semen cassiae; peaks 8 and 9 are the exclusive characteristic peaks of stevia. The results show that each of the three herbs has corresponding attribution, and stir-fried semen cassiae and stevia have exclusive characteristic peaks, as shown in Table 2.

[0100] Table 2 Results of the attribution of chromatographic peaks to herbs

[0101]

[0102] Identification of chromatographic peaks in the HPLC chromatogram of Qingre Mingmu Tea in Example 2

[0103] 1. Experimental method

[0104] Select the reference substances of the main components of each herb: chlorogenic acid, rebaudioside A, stevioside, aurantio-obtusin, emodin, and chrysophanol for peak localization studies. Among them, chlorogenic acid belongs to chrysanthemum and stevia, rebaudioside A belongs to stevia, stevioside belongs to stevia, aurantio-obtusin belongs to stir-fried semen cassiae, emodin belongs to stir-fried semen cassiae, and chrysophanol belongs to stir-fried semen cassiae.

[0105] Use a DAD detector to perform full-wavelength scanning on the reference solution and the test solution at 190 - 400 nm to obtain the ultraviolet absorption diagrams of each compound and the absorption peaks of the test solution. By comparing the retention times and ultraviolet absorption diagrams of the peaks of the compounds and the test solution, the main characteristic peaks of the characteristic chromatogram are identified.

[0106] (1) Preparation of the test solution: The same as in Example 1;

[0107] (2) Preparation of the reference solution

[0108] Weigh accurately appropriate amounts of chlorogenic acid reference substance, rebaudioside A reference substance, stevioside reference substance, aurantio-obtusin reference substance, emodin reference substance, and chrysophanol reference substance respectively, and make a solution containing 30 μg per 1 mL with 50% methanol solution (v / v) to obtain the reference solution.

[0109] (3) Detection method: Take the test solution and 6 reference solutions and perform detection according to the chromatographic conditions in Table 1 of Example 1.

[0110] 2. Information on test samples and reference substances

[0111]

[0112]

[0113] 3. Experimental results

[0114] The control solution and the test solution were scanned at full wavelength from 190 to 400 nm using a DAD detector. The UV absorption spectra of the absorption peaks of each reference substance and the test substance are as shown in Figure 2 and Figure 3 . The results of the mass spectrometry analysis of Qingre Mingmu Tea are shown in Table 3.

[0115] Table 3 Identification Results of Chromatographic Peaks of Qingre Mingmu Tea

[0116] Peak Number Compound Name Retention Time (min) Attributed Flavor 1 Chlorogenic Acid 9.87 Chrysanthemum, Stevia Rebaudiana 8 Rebaudioside A 53.75 Stevia Rebaudiana 9 Stevioside 54.10 Stevia Rebaudiana 10 Aurantio-obtusin 63.30 Fried Semen Cassiae 14 Emodin 80.77 Fried Semen Cassiae 17 Chrysophanol 87.24 Fried Semen Cassiae

[0117] According to Table 3, combining the retention times and UV absorption spectra of the compounds and the test solution (see Figure 2 and Figure 3 ), it is shown that the retention times of the compounds chlorogenic acid, rebaudioside A, stevioside, aurantio-obtusin, emodin, and chrysophanol are consistent with those of peak 1 (9.87 min), peak 8 (53.75 min), peak 9 (54.10 min), peak 10 (63.30 min), peak 14 (80.77 min), and peak 17 (87.24 min) in the test substance chromatogram. Therefore, peak 1, peak 8, peak 9, peak 10, peak 14, and peak 17 are respectively identified as the compounds chlorogenic acid, rebaudioside A, stevioside, aurantio-obtusin, emodin, and chrysophanol.

[0118] In view of the relatively high response value and good resolution of the chlorogenic acid chromatographic peak (peak 1), chlorogenic acid was selected as the reference peak for the reference substance and marked as peak S. At the same time, the chlorogenic acid reference substance was used as the reference object.

[0119] Example 3 A Method for Constructing the HPLC Characteristic Chromatogram of Qingre Mingmu Tea

[0120] 1. Experimental Method

[0121] (1) Preparation of the test substance: The same as in Example 1;

[0122] (2) Preparation of the reference substance solution

[0123] An appropriate amount of chlorogenic acid reference substance was accurately weighed and dissolved in 50% methanol solution (v / v) to prepare a solution containing 30 μg per 1 mL, which is the reference substance solution.

[0124] (3) HPLC Detection

[0125] Using octadecylsilyl silica gel as the filler (Waters XSelect HSS T3 chromatographic column, column length 25 cm, column inner diameter 4.6 mm, particle size 5 μm); using acetonitrile as mobile phase A and 0.1% phosphoric acid solution (w / v) as mobile phase B, gradient elution was carried out according to the mobile phase gradient elution program shown in Table 1 of Example 1; the column temperature was 35°C. The number of theoretical plates calculated based on the chlorogenic acid peak should be not less than 8000. Accurately pipette 10 μL each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0126] 2. Information of the test sample and reference substance

[0127] Serial Number Product Name Source Purity % Batch Number 1 Heat-Clearing and Eyesight-Improving Tea Zhuzhou Qianjin Pharmaceutical Co., Ltd. / S11230001 2 Chlorogenic Acid National Institutes for Food and Drug Control 96.3 110753-202119

[0128] 3. Experimental results

[0129] The characteristic chromatogram of the test sample of Qingre Mingmu Tea is as shown in Figure 4 and Figure 5 . It can be seen from Figure 4 that there are 17 characteristic peaks in the characteristic chromatogram of the test sample of Qingre Mingmu Tea. Among them, peak 1 (S peak) is the characteristic peak of chlorogenic acid, peak 8 is the characteristic peak of rebaudioside A, peak 9 is the characteristic peak of stevioside, peak 10 is the characteristic peak of aurantio-obtusin, peak 14 is the characteristic peak of emodin, and peak 17 is the characteristic peak of chrysophanol.

[0130] Taking the chlorogenic acid characteristic peak as the reference peak (S peak), calculate the relative retention times of characteristic peaks 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17. The relative retention times are within ±5% of the specified values; the specified values are 1.000 (peak 1, S peak), 2.434 (peak 2), 2.568 (peak 3), 2.780 (peak 4), 2.997 (peak 5), 3.239 (peak 6), 5.183 (peak 7), 5.445 (peak 8), 5.480 (peak 9), 6.411 (peak 10), 7.231 (peak 11), 7.645 (peak 12), 7.735 (peak 13), 8.181 (peak 14), 8.520 (peak 15), 8.658 (peak 16), 8.837 (peak 17). Among them, as shown in Figure 6 the fingerprint method established in the present invention can identify the characteristic peaks of rebaudioside A (peak 8) and stevioside (peak 9) of Stevia rebaudiana, indicating that the established fingerprint method can effectively separate and detect the chemical components in the finished product of Qingre Mingmu Tea and can be applied to the detection and analysis of the finished product of Qingre Mingmu Tea.

[0131] Example 4 A quality detection method for Qingre Mingmu Tea

[0132] 1. HPLC chromatogram of the test sample of Qingre Mingmu Tea

[0133] According to "(1) Preparation of the test solution" in Example 3, prepare the test sample solution of the Qingre Mingmu tea to be tested from the sample to be tested.

[0134] According to "(3) HPLC detection" in Example 3, precisely pipette 10 μL of the test sample solution of the Qingre Mingmu tea to be tested, inject it into the liquid chromatograph, record the chromatogram, and obtain the HPLC chromatogram of the Qingre Mingmu tea sample to be tested.

[0135] 2. Control fingerprint chromatogram

[0136] Use the standard sample of Qingre Mingmu tea to construct a control fingerprint chromatogram according to the method of Example 3. This control fingerprint chromatogram contains 17 fingerprint peaks. Taking the 1st peak corresponding to the reference substance solution as the S peak, the specified values are as follows:

[0137] The 1st peak (S peak): 1.000, the 2nd peak: 2.434, the 3rd peak: 2.568, the 4th peak: 2.780, the 5th peak: 2.997, the 6th peak: 3.239, the 7th peak: 5.183, the 8th peak: 5.445, the 9th peak: 5.480, the 10th peak: 6.411, the 11th peak: 7.231, the 12th peak: 7.645, the 13th peak: 7.735, the 14th peak: 8.181, the 15th peak: 8.520, the 16th peak: 8.658, the 17th peak: 8.837.

[0138] 3. Judgment criteria

[0139] The test sample of Qingre Mingmu tea can be judged as qualified only when the following two conditions are met:

[0140] The HPLC chromatogram of the test sample of Qingre Mingmu tea shows fingerprint peaks with corresponding retention times within ±5% of the specified values in the control fingerprint chromatogram;

[0141] Calculate the fingerprint peaks according to the similarity evaluation system for traditional Chinese medicine chromatographic fingerprint chromatograms. The similarity between the HPLC fingerprint chromatogram of the test solution and the control fingerprint chromatogram is not less than 0.90.

[0142] Experimental example 1 Injection precision test

[0143] 1. Experimental method

[0144] Take the Qingre Mingmu tea sample (S2 - 6) with batch number S20240415, prepare the test sample solution according to the method of Example 3, and inject samples continuously 6 times according to the HPLC detection method of Example 3. The sample numbers are respectively recorded as precision 1 (S2), precision 2 (S3), precision 3 (S4), precision 4 (S5), and precision 5 (S6), and record the chromatograms respectively. Taking the retention time and peak area of the 1st peak as references, calculate the relative retention time and relative peak area of each fingerprint peak.

[0145] Using the method of "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition)", the chromatographic peaks of the reproducible HPLC chromatograms were automatically matched to form a common pattern diagram. Taking the chromatogram of the Qingre Mingmu tea sample S1 with the pre-sterilization batch number S11240004 as the reference chromatogram, the similarity was calculated.

[0146] 2. Experimental results

[0147] The results are shown in Tables 4 and 5. The maximum RSD of the relative retention times of the 17 fingerprint peaks was 0.1% (n = 5), less than 5%, and the maximum RSD of the relative peak areas was 4.5% (n = 5). As Figure 7 shown in Tables 5 and 6, the similarities of the fingerprint chromatograms collected 5 times were all greater than 0.95, indicating good precision of the instrument and experimental method.

[0148] Table 4 Injection precision (RSD of relative retention times of each peak)

[0149] Peak Number Precision 1 Precision 2 Precision 3 Precision 4 Precision 5 RSD 1(S) 1.000 1.000 1.000 1.000 1.000 0.0％ 2 2.434 2.437 2.438 2.436 2.438 0.1％ 3 2.568 2.573 2.574 2.571 2.573 0.1％ 4 2.780 2.785 2.785 2.782 2.784 0.1％ 5 2.997 3.002 3.003 2.999 3.001 0.1％ 6 3.239 3.245 3.247 3.241 3.243 0.1％ 7 5.183 5.190 5.196 5.188 5.190 0.1％ 8 5.445 5.451 5.459 5.451 5.451 0.1％ 9 5.480 5.487 5.495 5.486 5.487 0.1％ 10 6.411 6.421 6.430 6.421 6.421 0.1％ 11 7.231 7.242 7.251 7.241 7.241 0.1％ 12 7.645 7.656 7.666 7.656 7.656 0.1％ 13 7.735 7.746 7.757 7.746 7.746 0.1％ 14 8.181 8.192 8.203 8.192 8.191 0.1％ 15 8.520 8.532 8.544 8.533 8.534 0.1％ 16 8.658 8.669 8.681 8.670 8.672 0.1％ 17 8.837 8.848 8.861 8.849 8.852 0.1％

[0150] Table 5 Injection precision (RSD of relative peak areas of each peak)

[0151]

[0152]

[0153] Table 6 Injection precision (similarity)

[0154] S1 S2 S3 S4 S5 S6 S1 1.000 0.983 0.983 0.983 0.983 0.983 S2 0.983 1.000 1.000 1.000 1.000 1.000 S3 0.983 1.000 1.000 1.000 1.000 1.000 S4 0.983 1.000 1.000 1.000 1.000 1.000 S5 0.983 1.000 1.000 1.000 1.000 1.000 S6 0.983 1.000 1.000 1.000 1.000 1.000

[0155] Experimental Example 2 Reproducibility test

[0156] 1. Experimental method

[0157] Six portions of the Qingre Mingmu tea sample with the batch number S20240415 were taken. The test solution was prepared according to the method of Example 3 and injected for determination according to the HPLC detection method of Example 3. The sample numbers of the six test solutions were respectively recorded as Reproducibility 1 (S2), Reproducibility 2 (S3), Reproducibility 3 (S4), Reproducibility 4 (S5), Reproducibility 5 (S6), and Reproducibility 6 (S7), and the chromatograms were recorded. Taking the retention time and peak area of Peak 1 as the reference, the relative retention times and relative peak areas of each fingerprint peak were calculated.

[0158] Using the method of "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition)", the chromatographic peaks of the reproducible HPLC chromatograms were automatically matched to form a common pattern diagram. Taking the chromatogram of the Qingre Mingmu tea sample S1 with the pre-sterilization batch number S11240004 as the reference chromatogram, the similarity was calculated.

[0159] 2. Experimental results

[0160] The results are shown in Tables 7 and 8. The maximum RSD of the relative retention times of 17 fingerprint peaks was 0.2% (n = 6), which was less than 5%. The maximum RSD of the relative peak areas was 7.8% (n = 6). As shown in Tables 9 and Figure 8 as follows, the similarities of the fingerprint chromatograms collected 6 times were all greater than 0.95, indicating that the method had good repeatability.

[0161] Table 7 Repeatability (RSD of relative retention times of each peak)

[0162]

[0163]

[0164] Table 8 Repeatability (RSD of relative peak areas of each peak)

[0165]

[0166]

[0167] Table 9 Repeatability (similarity)

[0168] S1 S2 S3 S4 S5 S6 S7 S1 1.000 0.983 0.982 0.982 0.981 0.983 0.983 S2 0.983 1.000 0.999 0.999 0.998 0.998 0.999 S3 0.982 0.999 1.000 1.000 1.000 1.000 1.000 S4 0.982 0.999 1.000 1.000 0.999 0.999 1.000 S5 0.981 0.998 1.000 0.999 1.000 1.000 1.000 S6 0.983 0.998 1.000 0.999 1.000 1.000 1.000 S7 0.983 0.999 1.000 1.000 1.000 1.000 1.000

[0169] Experimental Example 3 Intermediate Precision Test

[0170] 1. Experimental Method

[0171] Samples of Qingre Mingmu Tea (S2-6) with batch number S20240415 were taken by different personnel at different times. The test solution was prepared according to the method of Example 3. Using different instruments, the test solution was injected continuously 6 times according to the HPLC detection method of Example 3. The sample numbers were recorded as Precision 1-1, Precision 1-2, Precision 1-3, Precision 1-4, Precision 1-5, Precision 1-6, Precision 2-1, Precision 2-2, Precision 2-3, Precision 2-4, Precision 2-5, and Precision 2-6 respectively, and the chromatograms were recorded. Using the retention time and peak area of Peak 1 as the reference, the relative retention times and relative peak areas of each fingerprint peak were calculated.

[0172] Take the chromatograms of one of the test samples injected continuously for 6 times, numbered as intermediate precision 1 (S8), intermediate precision 2 (S9), intermediate precision 3 (S10), intermediate precision 4 (S11), intermediate precision 5 (S12), and intermediate precision 6 (S13), and calculate the similarity jointly with the chromatograms of the test sample solutions in "II. Repeatability". Among them, the chromatograms of the test sample solutions in "II. Repeatability" are numbered as repeatability 1 (S2), repeatability 2 (S3), repeatability 3 (S4), repeatability 4 (S5), repeatability 5 (S6), and repeatability 6 (S7).

[0173] Adopt the method of "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)", automatically match the chromatographic peaks of the repeatability HPLC chromatograms to form a common pattern diagram, and use the chromatogram of sample S1 of Qingre Mingmu Tea with the pre-sterilization batch number of S11240004 as the reference chromatogram to calculate the similarity.

[0174] 2. Experimental results

[0175] The results are shown in Table 10 and Table 11. The maximum RSD of the relative retention times of 17 fingerprint peaks is 0.8% (n = 12), less than 5%, and the maximum RSD of the relative peak areas is 14.6% (n = 12). As shown in Table 12 and Figure 9 as shown, the similarities of the fingerprint chromatograms collected 12 times are all greater than 0.95, indicating that the precision of the instrument and the experimental method is good.

[0176] Table 10 Intermediate precision (RSD of relative retention times of each peak)

[0177]

[0178] Table 11 Intermediate precision (RSD of relative peak areas of each peak)

[0179]

[0180]

[0181] Table 12 Intermediate precision (similarity)

[0182]

[0183]

[0184] Experimental example 4 Stability test

[0185] 1. Experimental method

[0186] Take the Qingre Mingmu tea sample (S1) with the batch number S20240415, prepare the test solution according to the method of Example 3, place it at room temperature for 0, 4, 8, 13, 18, 35, 52, 69, 86 h respectively, inject and determine according to the HPLC detection method of Example 3, and record the chromatograms with the sample numbers recorded as 0 h (S1), 4 h (S2), 8 h (S3), 13 h (S4), 18 h (S5), 35 h (S6), 52 h (S7), 69 h (S8) and 86 h (S9) respectively. Take the retention time and peak area of Peak 1 as the reference, and calculate the relative retention time and relative peak area of each fingerprint peak.

[0187] Adopt the method of "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition)", automatically match the chromatographic peaks of the repetitive HPLC chromatograms to form a common pattern diagram, take the chromatogram of the injection at 0 h as the reference chromatogram, and calculate the similarity.

[0188] 2. Experimental Results

[0189] The results are shown in Table 13 and Table 14. The maximum RSD of the relative retention time of 17 fingerprint peaks is 0.2% (n = 9), and the maximum RSD of the relative peak area is 8.7% (n = 9). As shown in Table 15 and Figure 10 As shown, the similarity of the fingerprint chromatograms collected 9 times is greater than 0.95, indicating that the test solution is stable when placed at room temperature for 86 h.

[0190] Table 13 Solution Stability (RSD of Relative Retention Time of Each Peak)

[0191]

[0192]

[0193] Table 14 Solution Stability (RSD of Relative Peak Area of Each Peak)

[0194] Peak Number 0h 4h 8h 13h 18h 35h 52h 69h 86h RSD 1(S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.0％ 2 0.218 0.205 0.209 0.219 0.206 0.219 0.220 0.224 0.222 3.2％ 3 3.202 3.195 3.185 3.206 3.175 3.173 3.152 3.122 3.083 0.4％ 4 1.739 1.739 1.729 1.750 1.723 1.734 1.730 1.718 1.704 0.5％ 5 2.163 2.174 2.165 2.198 2.203 2.232 2.260 2.293 2.316 1.2％ 6 3.501 3.504 3.490 3.522 3.502 3.523 3.539 3.538 3.553 0.4％ 7 1.205 1.227 1.292 1.236 1.195 1.183 1.191 1.260 1.212 3.2％ 8 1.556 1.553 1.630 1.566 1.572 1.576 1.584 1.576 1.587 1.8％ 9 0.510 0.480 0.416 0.455 0.483 0.535 0.448 0.462 0.511 8.7％ 10 3.694 3.695 3.669 3.713 3.692 3.699 3.699 3.425 3.803 0.4％ 11 1.806 1.805 1.802 1.813 1.813 1.807 1.817 1.809 1.806 0.2％ 12 0.839 0.821 0.835 0.829 0.855 0.821 0.824 0.835 0.828 1.5％ 13 0.945 0.939 0.934 0.950 0.951 0.942 0.937 0.945 0.941 0.7％ 14 0.350 0.357 0.385 0.361 0.392 0.368 0.371 0.379 0.407 4.5％ 15 3.117 3.121 3.043 3.047 3.013 2.892 2.843 2.686 2.594 2.8％ 16 1.805 1.780 1.709 1.685 1.625 1.454 1.319 1.150 1.014 7.6％ 17 1.082 1.100 1.116 1.129 1.142 1.216 1.260 1.281 1.285 4.1％

[0195] Table 15 Solution Stability (Similarity)

[0196]

[0197]

[0198] Experimental Example 5 Durability

[0199] 1. Experimental Method

[0200] Take the Qingre Mingmu tea sample (S2-4) with the batch number S20240415, prepare the test solution and the reference solution according to the method of Example 3. Respectively, according to the detection conditions shown in Table 16, precisely pipette the blank solvent (50% methanol solution (v / v)), the reference solution and the test solution, and inject and measure according to the HPLC detection method of Example 3. Using the retention time and peak area of Peak 1 as a reference, calculate the relative retention time and relative peak area of each fingerprint peak.

[0201] Adopt the method of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)", automatically match the chromatographic peaks of the reproducible HPLC chromatogram to form a common pattern diagram. Take the chromatogram of the Qingre Mingmu tea sample S1 with the batch number S11240004 before sterilization as the reference chromatogram and calculate the similarity.

[0202] Table 16 Durability detection conditions

[0203]

[0204] 2. Experimental results

[0205] (1) Different phosphoric acid concentrations

[0206] The results are shown in Table 17 and Table 18. Under different phosphoric acid concentration conditions, the maximum RSD of the relative retention time of 17 fingerprint peaks is 2.9% (n = 3), which is less than 5%, and the maximum RSD of the relative peak area is 26.5% (n = 3). As shown in Table 19 and Figure 11 The chromatogram at a phosphoric acid concentration of 0.09% is denoted as S2, the chromatogram at a phosphoric acid concentration of 0.10% is denoted as S3, and the chromatogram at a phosphoric acid concentration of 0.11% is denoted as S4. The similarity of the fingerprint chromatograms collected 3 times is greater than 0.95, indicating that the test solution has good durability under different phosphoric acid concentration conditions.

[0207] Table 17 Durability (RSD of relative retention time of each peak) - Different phosphoric acid concentrations

[0208]

[0209] Table 18 Durability (RSD of relative peak area of each peak) - Different phosphoric acid concentrations

[0210]

[0211]

[0212] Table 19 Durability (similarity) - Different phosphoric acid concentrations

[0213] S1 S2 S3 S4 S1 1.000 0.975 0.983 0.973 S2 0.975 1.000 0.996 0.999 S3 0.983 0.996 1.000 0.994 S4 0.973 0.999 0.994 1.000

[0214] (2) Different column temperatures

[0215] The results are shown in Table 20 and Table 21. The maximum RSD of the relative retention times of 17 fingerprint peaks under different column temperature conditions is 3.7% (n = 3), which is less than 5%. The maximum RSD of the relative peak areas is 26.6% (n = 3). As shown in Table 22 and Figure 12 , the chromatogram with a column temperature of 38 °C is denoted as S2, the chromatogram with a column temperature of 40 °C is denoted as S3, and the chromatogram with a column temperature of 42 °C is denoted as S4. The similarities of the fingerprint spectra collected 3 times are all greater than 0.95, indicating that the test solution has good durability under different column temperature conditions.

[0216] Table 20 Durability (RSD of relative retention time of each peak) - Different column temperatures

[0217]

[0218]

[0219] Table 21 Durability (RSD of relative peak area of each peak) - Different column temperatures

[0220] Peak Number 38℃ 40℃ 42℃ RSD % 1(S) 1.000 1.000 1.000 0.0％ 2 0.197 0.218 0.217 5.6％ 3 3.592 3.202 2.937 10.2％ 4 1.598 1.739 1.612 4.7％ 5 2.255 2.163 2.231 2.2％ 6 3.587 3.501 3.506 1.4％ 7 1.234 1.205 1.342 5.7％ 8 1.415 1.556 1.574 5.7％ 9 0.302 0.510 0.494 26.6％ 10 3.581 3.694 3.862 3.8％ 11 1.813 1.806 1.788 0.7％ 12 0.822 0.839 0.806 2.0％ 13 0.957 0.945 0.939 1.0％ 14 0.382 0.350 0.366 4.4％ 15 2.926 3.117 2.849 4.7％ 16 1.551 1.805 1.355 14.4％ 17 1.165 1.082 1.264 7.8％

[0221] Table 22 Durability (Similarity) - Different column temperatures

[0222] S1 S2 S3 S4 S1 1.000 0.985 0.983 0.973 S2 0.985 1.000 0.997 0.995 S3 0.983 0.997 1.000 0.997 S4 0.973 0.995 0.997 1.000

[0223] Comparative Example 1

[0224] I. Experimental method

[0225] The experiment was carried out according to the test solution preparation method and chromatographic conditions in "Study on HPLC Characteristic Fingerprint Spectrum and Determination of Multi-component Contents of Qingre Mingmu Tea" by Zhou Jie et al. (Publication date: February 29, 2020).

[0226] 1. Preparation of test solution

[0227] Take about 1.0 g of Qingre Mingmu Tea powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of methanol-ethyl acetate (volume ratio 2:1), weigh it, ultrasonically treat it (power 300 W, frequency 40 kHz) for 45 min, let it cool, weigh it again, make up the lost amount with methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0228] 2. Determination method

[0229] Accurately pipette 10 μL of the test solution, inject it into the liquid chromatograph, determine it, and record the chromatogram.

[0230] 3. Chromatographic conditions

[0231] Using octadecylsilane-bonded silica gel as the filler; using acetonitrile as mobile phase A and 0.5% phosphoric acid solution (w / v) as mobile phase B, eluting according to the conditions in Table 23; the flow rate is 1.0 mL per minute; the detection wavelength is 210 nm, with full wavelength scanning; the column temperature is 30 °C.

[0232] Table 23 Liquid Chromatography Elution Conditions

[0233] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0～12 15 85 12～20 15→28 85→72 20～36 28→40 72→60 36～50 40→50 60→50 50～60 50→55 50→45 60～68 55→62 45→38 68～74 62→85 38→15 74～90 85→15 15→85

[0234] II. Experimental Results

[0235] The results are as Figure 13 shown. Under this gradient condition, the main chromatographic information is concentrated in the first 60 min. The chromatographic peaks are relatively concentrated from 18 to 30 min, with a large amount of information but poor resolution. The peaks are well separated from 38 to 55 min. However, there is a large chromatographic peak at the 8-min position in the chromatogram. After comparison, this chromatographic peak is introduced by the blank solvent. Ethyl acetate has 2 main absorption peaks in the ultraviolet region of the absorption spectrum, one of which is located at 210 - 230 nm. It is speculated that this chromatographic peak is ethyl acetate. Therefore, this method has the problem of excessive interference from the blank solvent.

[0236] Comparative Example 2

[0237] 1. Preparation of Test Solution

[0238] Take about 0.5 g of Qingre Mingmu Tea powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 70% methanol solution (v / v), weigh it, ultrasonically treat it (power 300 W, frequency 40 kHz) for 45 min, let it cool, weigh it again, make up the lost amount with 70% methanol solution (v / v), shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0239] 2. HPLC Detection

[0240] Using octadecylsilane-bonded silica gel as the filler, using acetonitrile as mobile phase A and 0.1% phosphoric acid solution (w / v) as mobile phase B, eluting according to the conditions in Table 24; the flow rate is 1.0 mL per minute; the column temperature is 30 °C. Accurately pipette 10 μL of the test solution prepared in "1. Preparation of Test Solution" and inject it into the liquid chromatograph for determination, and record the chromatogram.

[0241] Table 24 Liquid Chromatography Elution Conditions

[0242]

[0243] 3. Experimental Results

[0244] As Figure 14As shown, under the gradient conditions shown in Table 24, there is more chromatographic information in the first 30 minutes, but the separation is poor. The separation of the chromatographic peaks from 38 to 55 minutes is better, so the gradient in the first 30 minutes needs to be reduced and the flow rate needs to be slowed down.

[0245] Comparative Example 3

[0246] 1. Preparation of test solution

[0247] Take 4 portions of heat-clearing and eye-brightening tea powder (S11230001), each portion is about 0.5g, accurately weigh, put in a stoppered conical flask, accurately add 25% methanol solution (v / v), 50% methanol solution (v / v), 75% methanol solution (v / v), 50% ethanol solution (v / v) 25mL each, weigh the weight, ultrasonically treat (power 300W, frequency 40kHz) for 30min, cool, weigh again, make up the lost weight with 25% methanol solution (v / v), 50% methanol solution (v / v), 75% methanol solution (v / v), 50% ethanol solution (v / v), shake well, centrifuge at 12000rpm for 5min, take the supernatant and filter to obtain the test solution.

[0248] 2.HPLC detection

[0249] Use octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, 0.1% phosphoric acid solution (w / v) as mobile phase B, and perform elution according to the conditions in Table 25; flow rate is 1.0 mL per minute; column temperature is 30°C. Accurately pipette 10 μL of the test solution prepared in "1. Preparation of test solution", inject into liquid chromatograph, measure, and record the chromatogram.

[0250] Table 25 Liquid chromatography elution conditions

[0251]

[0252] 3. Experimental results

[0253] The results are as follows Figure 15 As shown, the extraction rates of the compounds with retention time of 82 to 88 min by various extraction solvents are different to a certain extent, among which 50% methanol solution (v / v) has the highest extraction rate. Therefore, the extraction solvent selected is 50% methanol solution (v / v), i.e., the extraction solvent of Example 3.

[0254] Comparative Example 4

[0255] 1. Preparation of test solution

[0256] 1) Test solution 1: Prepared according to the method of "Test solution" in "Study on HPLC characteristic fingerprint of heat-clearing and eye-brightening tea and determination of multi-component content" by Zhou Jie et al. (publication date: February 29, 2020):

[0257] Take about 1.0 g of the Qingre Mingmu tea powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of methanol-ethyl acetate (2:1), weigh it, ultrasonically treat it (power 300 W, frequency 40 kHz) for 45 minutes, let it cool, weigh it again and make up the lost amount with methanol, shake well, filter through a 0.45 μm microporous membrane to obtain the test solution.

[0258] 2) Test solution 2: Prepare the test solution according to the method of "Preparation of test solution" in Example 3.

[0259] 2. HPLC detection

[0260] Use octadecylsilane-bonded silica gel as the filler (Waters XSelect HSS T3 chromatographic column, column length 25 cm, column inner diameter 4.6 mm, particle size 5 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution (w / v) as mobile phase B, and perform gradient elution according to the mobile phase gradient elution program shown in Table 1; the column temperature is 35°C. Accurately pipette 10 μL of test solution 1 and test solution 2 respectively, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0261] 3. Experimental results

[0262] As Figure 16 shown, the peak response of the sample obtained by the test solution preparation method of the prior art is significantly lower than that of the test solution preparation method of Example 3 of the present invention, and the extraction solvent in the test solution preparation method of the prior art is less safe than that of the present invention, the ultrasonic time is longer than that of the present invention, and the extraction rate is lower than that of the present invention.

[0263] Comparative Example 5

[0264] 1. Preparation of test solution

[0265] Prepare according to the method of "Preparation of test solution" in Example 3 to obtain the test solution.

[0266] 2. HPLC detection

[0267] Detect the test solution respectively according to the "HPLC detection method" of Example 3 and Comparative Example 1.

[0268] 3. Experimental results

[0269] As Figure 17 shown, when the test solution is detected under two chromatographic conditions, the separation effect of the chromatographic conditions of the present invention is significantly better than that of Comparative Example 1 in the range of 16 - 34 minutes.

[0270] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A pretreatment method for detecting Qingre Mingmu tea by high performance liquid chromatography, characterized in that, Take the sample of the heat-clearing and eye-soothing tea to be tested, and ultrasonically extract it with a methanol aqueous solution with a volume fraction of 45-55% for 25-35 min.

2. The pre-treatment method according to claim 1, characterized in that, The mass-volume ratio of the sample of the heat-clearing and eye-soothing tea to be tested to methanol is 1 g:(45-55) mL.

3. An HPLC method, characterized in that, Using octadecylsilane-bonded silica gel as the filler of the chromatographic column, acetonitrile as mobile phase A, and a phosphoric acid solution with a mass fraction of 0.08-0.12% as mobile phase B. In the gradient elution program, the volume percentage change of mobile phase B in the mobile phase system is as follows: 0-12 min, mobile phase B is 89%; 12-15 min, mobile phase B decreases from 89% to 78%; 15-40 min, the mobile phase decreases from 78% to 75%; 40-45 min, the mobile phase decreases from 75% to 65%; 45-55 min, the mobile phase is 65%; 55-56 min, the mobile phase decreases from 65% to 60%; 56-62 min, the mobile phase is 60%; 62-64 min, the mobile phase decreases from 60% to 58%; 64-79 min, the mobile phase decreases from 58% to 48%; 79-84 min, the mobile phase decreases from 48% to 20%; 84-87 min, the mobile phase is 20%; 87-89 min, the mobile phase increases from 20% to 89%; 89-95 min, the mobile phase is 89%; The detection wavelength is gradient detection: 0-12 min, the detection wavelength is 326 nm; 12-40 min, the detection wavelength is 286 nm; 40-55 min, the detection wavelength is 210 nm; 55-95 min, the detection wavelength is 286 nm.

4. The high performance liquid chromatography method according to claim 3, characterized in that, The column temperature of the chromatographic column is 34-36 °C.

5. The high performance liquid chromatography method according to claim 3, characterized in that, The flow rate of the gradient elution program is as follows: 0-12 min, the flow rate is 1.0 mL / min; 12-15 min, the flow rate decreases from 1.0 mL / min to 0.8 mL / min; 15-40 min, the flow rate is 0.8 mL / min; 40-45 min, the flow rate decreases from 0.8 mL / min to 0.5 mL / min; 45-55 min, the flow rate is 0.5 mL / min; 55-56 min, the flow rate increases from 0.5 mL / min to 1.0 mL / min; 56-95 min, the flow rate is 1.0 mL / min.

6. The application of the pretreatment method according to any one of claims 1-2 and / or the high performance liquid chromatography method according to any one of claims 3-5 in establishing the HPLC fingerprint of the heat-clearing and eye-soothing tea and / or the quality control of the heat-clearing and eye-soothing tea.

7. A method for constructing an HPLC fingerprint of a heat-clearing and eye-clearing tea, characterized in that, Use the pretreatment method according to any one of claims 1-2 to process the standard sample of the heat-clearing and eye-soothing tea to obtain the test sample solution for testing. Using the chlorogenic acid solution as the reference solution, use the high performance liquid chromatography method according to any one of claims 3-5 to detect the standard sample of the heat-clearing and eye-soothing tea, record the chromatogram, and the theoretical plate number is not less than 8000 calculated by the chlorogenic acid peak to obtain the HPLC fingerprint of the heat-clearing and eye-soothing tea.

8. The construction method according to claim 7, characterized in that, The preparation method of the reference solution is: fully mix chlorogenic acid with a methanol aqueous solution, and the mass-volume ratio of chlorogenic acid to methanol is (58-62) μg:1 mL.

9. A quality inspection method for a heat-clearing and eyesight-improving tea, characterized in that, The test sample of Qingre Mingmu tea is processed using the pretreatment method according to any one of claims 1 to 2 to obtain the test solution of the test sample; The test solution of the test sample is detected using the high performance liquid chromatography method according to any one of claims 3 to 5, the chromatogram is recorded, the HPLC fingerprint of the test sample is obtained, and the HPLC fingerprint of the test sample is compared with the control fingerprint. It can be determined to be qualified only when the following two conditions are met: The HPLC fingerprint of the test sample shows fingerprint peaks corresponding to the retention times in the control fingerprint within ±5% of the specified values; Calculating the fingerprint peaks according to the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, the similarity between the HPLC fingerprint of the test sample and the control fingerprint is not less than 0.90; The method for constructing the control fingerprint is: the standard sample of Qingre Mingmu tea is processed using the pretreatment method according to any one of claims 1 to 2 to obtain the test solution for testing; Using the chlorogenic acid solution as the reference solution, the test solution for testing is detected using the high performance liquid chromatography method according to any one of claims 3 to 5, the chromatogram is recorded, the theoretical plate number calculated by the chlorogenic acid peak is not less than 8000, and the control fingerprint of Qingre Mingmu tea is obtained; The control fingerprint contains 17 fingerprint peaks, with the corresponding 1st peak of the reference solution as the S peak, and the specified values are respectively: 1st peak (S peak) 1.000, 2nd peak 2.434, 3rd peak 2.568, 4th peak 2.780, 5th peak 2.997, 6th peak 3.239, 7th peak 5.183, 8th peak 5.445, 9th peak 5.480, 10th peak 6.411, 11th peak 7.231, 12th peak 7.645, 13th peak 7.735, 14th peak 8.181, 15th peak 8.520, 16th peak 8.658, 17th peak 8.

837.

10. Use of the high performance liquid chromatography method according to any one of claims 3 to 5 in detecting the contents of Cassia obtusifolia, Chrysanthemum morifolium and / or Stevia rebaudiana in Qingre Mingmu tea.

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