A method for establishing a clear heat and eyesight tea fingerprint by using high performance liquid chromatography and application thereof

Abstract

The application discloses a method for establishing a Qingre Mingmu tea fingerprint spectrum by using a high performance liquid chromatography method and application. By using the method, 17 fingerprint peaks can be confirmed, and three components of fried cassia seed, chrysanthemum and sweetleaf chrysanthemum in the Qingre Mingmu tea have corresponding attribution peaks. By comparison with reference substances and in combination with ultraviolet absorption spectrum characteristics, characteristic peaks of six chemical components, i.e., chlorogenic acid, rebaudioside A, stevioside, citranaxanthin, emodin and chrysophanol, are identified, wherein the rebaudioside A and the stevioside are characteristic peaks of the sweetleaf chrysanthemum, and the two characteristic peaks can distinguish the chrysanthemum and the sweetleaf chrysanthemum. The fingerprint spectrum detection method disclosed by the application can obtain a fingerprint spectrum with good separation degree and comprehensive chromatographic peaks, can more objectively and comprehensively evaluate the overall quality of the Qingre Mingmu tea, and lays a foundation for quality control of the Qingre Mingmu tea.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine analysis and detection technology, specifically to a method and application of establishing a fingerprint spectrum for a heat-clearing and vision-improving tea using high-performance liquid chromatography. Background Technology

[0002] Qingre Mingmu Tea is a traditional Chinese medicine preparation (WS3-B-1029-91) included in the "Pharmaceutical Standards of the Ministry of Health of the People's Republic of China" (Volume 5). It consists of three medicinal herbs: cassia seed (fried), chrysanthemum, and stevia. It has the effects of clearing heat and dispelling wind, calming the liver and improving eyesight, and can be used to treat hypertension, dizziness, headache, red and blurred vision, etc. Among them, cassia seed, the principal herb, has the effects of clearing heat and improving eyesight, and moistening the intestines and promoting bowel movements. Its main active ingredients are anthraquinones, naphthopyranones, amino acids, and inorganic elements. Representative anthraquinone components such as cassia seed extract, emodin, chrysophanol, emodin methyl ether, rhein, and aloe-emodin are characteristic components of cassia seed. Chrysanthemum refers to the dried capitulum of *Chrysanthemum morifolium* Ramat., a plant in the genus *Chrysanthemum* of the Asteraceae family. Modern pharmacological studies have shown that chrysanthemum possesses excellent biological activities, including anti-inflammatory, antioxidant, anti-tumor, neuroprotective, hyperuricemic prevention and treatment, and diabetes relief. These pharmacological effects are mainly related to its active ingredients, including phenolic acids, flavonoids, and polysaccharides. Stevia refers to the dried leaves of *Stevia rebaudiana* (Bertoni) Hemsl., a plant in the Asteraceae family. Stevia is mainly used to extract stevia glycosides as sweeteners or pharmaceutical excipients. However, the extraction of stevia glycosides generates a large amount of stevia waste. The active ingredients in this waste are mainly chlorogenic acid and neochlorogenic acid compounds, which possess excellent antioxidant, anti-inflammatory, and antibacterial functions.

[0003] Traditional Chinese medicine (TCM) fingerprinting is the analysis of TCM substances using modern techniques such as spectroscopy or chromatography to obtain results characterizing the overall features of TCM materials. It analyzes the chemical information of processed TCM to depict the peak distribution of specific chemical components, thereby achieving qualitative and quantitative evaluation of TCM quality. The construction and analysis of TCM fingerprinting can help ensure the consistency and quality of TCM and provide a scientific basis for the identification of medicinal materials and the optimization of production processes. The existing technology, "Study on HPLC Characteristic Fingerprint of Qingre Mingmu Tea and Determination of Multiple Component Content" (publication date: February 29, 2020), discloses the fingerprint spectrum of Qingre Mingmu Tea and identifies seven peaks: chlorogenic acid, cassia seed extract, aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether. However, among these 7 peaks, chlorogenic acid is a component shared by chrysanthemum and stevia, while the other 6 peaks are all attributed to roasted cassia seeds. Therefore, this fingerprint spectrum cannot effectively identify chrysanthemum and stevia in the heat-clearing and vision-improving tea, and thus cannot provide a good qualitative and quantitative evaluation of the quality of the heat-clearing and vision-improving tea.

[0004] Therefore, in order to more comprehensively monitor the quality of heat-clearing and vision-improving tea, improve the quality evaluation system of heat-clearing and vision-improving tea, and establish a fingerprint spectrum of heat-clearing and vision-improving tea with better separation and more comprehensive chromatographic peaks, it is of great significance. Summary of the Invention

[0005] To overcome the aforementioned defects and shortcomings in the existing technology, this invention provides a method and application for establishing a fingerprint spectrum of heat-clearing and vision-improving tea using high-performance liquid chromatography.

[0006] The first objective of this invention is to provide a high-performance liquid chromatography (HPLC) method for the pretreatment of heat-clearing and vision-improving tea.

[0007] The second objective of this invention is to provide a high-performance liquid chromatography method.

[0008] A third objective of this invention is to provide the application of the above-mentioned pretreatment method and / or high-performance liquid chromatography method in establishing the HPLC fingerprint of the heat-clearing and vision-improving tea and / or in the quality control of the heat-clearing and vision-improving tea.

[0009] The fourth objective of this invention is to provide a method for constructing an HPLC fingerprint of a heat-clearing and vision-improving tea.

[0010] The fifth objective of this invention is to provide a quality testing method for a heat-clearing and vision-improving tea.

[0011] The sixth objective of this invention is to provide the application of the above-described high-performance liquid chromatography method in the detection of the content of roasted cassia seed, chrysanthemum and / or stevia in a heat-clearing and vision-improving tea.

[0012] Therefore, this invention claims protection for the following:

[0013] A high-performance liquid chromatography (HPLC) method for the pretreatment of heat-clearing and vision-improving tea involves taking the tea sample to be tested and ultrasonically extracting it with a methanol aqueous solution of 45-55% (v / v) for 25-35 min.

[0014] Preferably, the mass-to-volume ratio of the heat-clearing and vision-improving tea sample to methanol is 1g:(45-55)mL.

[0015] More preferably, the mass-to-volume ratio of the heat-clearing and vision-improving tea sample to methanol is 1g:50mL.

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

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

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

[0019] A high-performance liquid chromatography (HPLC) method uses octadecylsilane-bonded silica gel as the packing material, acetonitrile as mobile phase A, and a 0.08–0.12% (w / w) phosphoric acid solution as mobile phase B. In a gradient elution program, the volume percentage change of mobile phase B in the mobile phase system is as follows:

[0020] From 0 to 12 minutes, the mobile phase B was 89%;

[0021] In 12–15 minutes, the mobile phase B decreased from 89% to 78%;

[0022] Within 15–40 minutes, the mobile phase decreased from 78% to 75%.

[0023] The mobile phase content decreased from 75% to 65% over 40–45 minutes.

[0024] 45–55 min, mobile phase 65%;

[0025] At 55–56 min, the mobile phase decreased from 65% to 60%;

[0026] 56–62 min, mobile phase 60%;

[0027] The mobile phase content decreased from 60% to 58% over 62–64 minutes.

[0028] The mobile phase content decreased from 58% to 48% over 64–79 minutes.

[0029] The mobile phase content decreased from 48% to 20% over 79–84 minutes.

[0030] 84–87 min, mobile phase 20%;

[0031] The mobile phase content increased from 20% to 89% in 87–89 minutes.

[0032] 89–95 min, mobile phase content 89%;

[0033] The detection wavelengths are gradient detection: 0–12 min, detection wavelength is 326 nm; 12–40 min, detection wavelength is 286 nm; 40–55 min, detection wavelength is 210 nm; 55–95 min, 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 a Waters XSelect HSS T3 column.

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

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

[0039] 0–12 min, flow rate 1.0 mL / min;

[0040] Over 12–15 minutes, the flow rate decreased from 1.0 mL / min to 0.8 mL / min;

[0041] 15–40 min, flow rate 0.8 mL / min;

[0042] Over 40–45 minutes, the flow rate was reduced from 0.8 mL / min to 0.5 mL / min.

[0043] 45–55 min, flow rate 0.5 mL / min;

[0044] 55–56 min, the flow rate was increased from 0.5 mL / min to 1.0 mL / min;

[0045] 56–95 min, flow rate 1.0 mL / min.

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

[0047] The application 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 in the quality control of Qingre Mingmu Tea.

[0048] A method for constructing an HPLC fingerprint of a heat-clearing and vision-improving tea involves processing a standard sample of the heat-clearing and vision-improving tea using any of the above-mentioned pretreatment methods to obtain a test solution.

[0049] Using chlorogenic acid solution as a reference solution, the standard sample of Qingre Mingmu Tea was detected by any of the above-mentioned high-performance liquid chromatography methods. The chromatogram was recorded, and the theoretical plate number calculated based on the chlorogenic acid peak was not less than 8000, thus obtaining the HPLC fingerprint of Qingre Mingmu Tea.

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

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

[0052] The HPLC fingerprint spectrum contains 17 fingerprint peaks. Peak 1 of the reference solution is designated as peak S. The relative retention times of the remaining peaks are within ±5% of a specified value. The specified values ​​are as follows:

[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] Peak 1 is a characteristic chromatographic peak of chlorogenic acid, peak 8 is a characteristic chromatographic peak of leboside A, peak 9 is a characteristic chromatographic peak of steviol glycoside, peak 10 is a characteristic chromatographic peak of cassia aurantium, peak 14 is a characteristic chromatographic peak of emodin, and peak 17 is a characteristic chromatographic peak of chrysophanol.

[0055] A method for quality testing of a heat-clearing and vision-improving tea involves processing the heat-clearing and vision-improving tea sample using any of the above-mentioned pretreatment methods to obtain a test solution of the sample.

[0056] The test solution of the sample is analyzed using any of the above-mentioned high-performance liquid chromatography (HPLC) methods, and the chromatogram is recorded to obtain the HPLC chromatogram of the sample. The HPLC chromatogram of the sample is compared with the reference fingerprint chromatogram. The sample is considered qualified only if it meets the following two conditions:

[0057] The HPLC fingerprint of the sample to be tested shows fingerprint peaks with retention times within ±5% of the specified value compared to the control fingerprint.

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

[0059] The method for constructing the comparative fingerprint spectrum is as follows: the standard sample of Qingre Mingmu Tea is processed using any of the above-mentioned pretreatment methods to obtain the test solution;

[0060] Using chlorogenic acid solution as a reference solution, the test sample solution was detected using any of the above-mentioned high performance liquid chromatography methods, and the chromatogram was recorded. The theoretical plate number calculated based on the chlorogenic acid peak was not less than 8000, and the reference fingerprint chromatogram of the heat-clearing and vision-improving tea was obtained.

[0061] The reference fingerprint spectrum contains 17 fingerprint peaks, with peak number 1 of the reference solution designated as peak S. 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] Application of any of the above-mentioned high-performance liquid chromatography methods in the determination of the content of roasted cassia seed, chrysanthemum and / or stevia in heat-clearing and vision-improving tea.

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

[0065] This invention discloses a method and application for establishing a fingerprint spectrum of a heat-clearing and vision-improving tea using high-performance liquid chromatography (HPLC). Using this method, 17 fingerprint peaks can be identified, and the three components of the heat-clearing and vision-improving tea—fried cassia seed, chrysanthemum, and stevia—each have corresponding assigned peaks. Through comparison with reference standards and combined with ultraviolet absorption spectral characteristics, characteristic peaks of six chemical components—chlorogenic acid, leucine A, steviol, aurantium cassiaside, emodin, and chrysophanol—are identified. Among these, leucine A and steviol are characteristic peaks of stevia, and these two characteristic peaks can distinguish chrysanthemum from stevia. The fingerprint spectrum detection method disclosed in this invention can obtain fingerprint spectra with good separation and comprehensive chromatographic peaks, enabling a more objective and comprehensive evaluation of the overall quality of the heat-clearing and vision-improving tea, laying the foundation for quality control of this tea. Attached Figure Description

[0066] Figure 1 To determine the medicinal properties of the chromatographic peaks in the fingerprint spectrum.

[0067] Figure 2 To identify the chemical components of each medicinal ingredient by using fingerprint chromatographic peaks.

[0068] Figure 3 This is a comparison of the ultraviolet absorption spectra of the reference standard (top) and the test sample (bottom).

[0069] Figure 4 Fingerprint spectrum of a tea for clearing heat and improving eyesight.

[0070] Figure 5 The peak time of the fingerprint spectrum of the heat-clearing and vision-improving tea.

[0071] Figure 6 This is an enlarged image of peaks 8 and 9 in the fingerprint spectrum of the heat-clearing and vision-improving tea.

[0072] Figure 7 This is the chromatogram result for injection precision.

[0073] Figure 8 This is a repeatable chromatogram result.

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

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

[0076] Figure 11 Chromatographic results for robustness under different phosphoric acid concentrations.

[0077] Figure 12 The results are chromatograms showing the robustness under different column temperature conditions.

[0078] Figure 13 The chromatogram results are for Comparative Example 1.

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

[0080] Figure 15 Chromatograms of test solutions prepared with different extraction solvents.

[0081] Figure 16 The chromatogram results are for Comparative Example 4.

[0082] Figure 17 The chromatogram results are for Comparative Example 5. Detailed Implementation

[0083] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

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

[0085] The heat-clearing and vision-improving tea used in the examples and comparative examples is a traditional Chinese medicine heat-clearing and vision-improving tea approved by the State Food and Drug Administration (National Medicine Approval Number Z43020775).

[0086] Example 1: Peak assignment in the HPLC chromatogram of the heat-clearing and vision-improving tea

[0087] 1. Experimental Methods

[0088] (1) Preparation of the test solution

[0089] Take 0.5g of heat-clearing and vision-improving tea powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 50% methanol solution (v / v), weigh it, sonicate it (power 300W, frequency 40kHz) for 30min, cool it, weigh it again, replenish the lost weight with 50% methanol solution (v / v), shake it well, centrifuge it at 12000rpm for 5min, take the supernatant and filter it 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”, about 0.5g of fine powder of fried cassia seed, chrysanthemum and stevia were used to prepare three single herbal decoction sample solutions.

[0092] (3) HPLC detection

[0093] An octadecylsilane-bonded silica gel column (Waters XSelect HSS T3 column, 25 cm long, 4.6 mm inner diameter, 5 μm particle size) was used as the stationary phase. Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution (w / v) was used as mobile phase B. Gradient elution was performed according to the mobile phase gradient elution program shown in Table 1. The column temperature was 35 °C. 10 μL each of the test solution and the single-herb decoction piece solution were accurately injected into the liquid chromatograph, and the chromatograms were recorded.

[0094] Table 1 Elution conditions for liquid chromatography

[0095]

[0096] 2. Information on test samples and reference materials

[0097]

[0098] 3. Experimental Results

[0099] Chromatograms of the test sample and three single-herb decoction pieces are shown below. Figure 1 As shown. From Figure 1As can be seen from the results, peaks 1, 2, 3, and 4 are common to both chrysanthemum and stevia; peaks 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, and 17 are characteristic peaks specific to roasted cassia seed; and peaks 8 and 9 are characteristic peaks specific to stevia. The results show that all three herbs have corresponding classifications, and roasted cassia seed and stevia have specific characteristic peaks, as shown in Table 2.

[0100] Table 2. Results of chromatographic peak attribution to medicinal flavor.

[0101]

[0102] Example 2: Peak identification in the HPLC chromatogram of the heat-clearing and vision-improving tea

[0103] 1. Experimental Methods

[0104] Peak localization studies were conducted on the main components of each herb: chlorogenic acid, leboside A, stevioside, cassia seed extract, emodin, and chrysophanol. Chlorogenic acid was assigned to chrysanthemum and stevia, leboside A to stevia, stevioside to stevia, cassia seed extract to roasted cassia seed, emodin to roasted cassia seed, and chrysophanol to roasted cassia seed.

[0105] A DAD detector was used to scan the control solution and the test solution across the entire wavelength range of 190–400 nm to obtain the UV absorption spectra of the absorption peaks of each compound and the test solution. By comparing the retention times and UV absorption spectra of the peaks of the compounds and the test solution, the main characteristic peaks of the characteristic spectra were identified.

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

[0107] (2) Preparation of reference solution

[0108] Accurately weigh appropriate amounts of chlorogenic acid reference standard, levodocin A reference standard, steviol reference standard, cassia seed reference standard, rhein reference standard and chrysophanol reference standard, respectively, and add 50% methanol solution (v / v) to prepare a solution containing 30 μg per 1 mL.

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

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

[0111]

[0112]

[0113] 3. Experimental Results

[0114] The control solution and the test solution were scanned across the entire wavelength range of 190–400 nm using a DAD detector. The UV absorption spectra of the absorption peaks of each reference standard and the test solution are shown below. Figure 2 and Figure 3 As shown in Table 3, the mass spectrometry analysis results of the heat-clearing and vision-improving tea are as follows.

[0115] Table 3. Chromatographic peak identification results of the heat-clearing and vision-improving tea.

[0116] Peak Compound Name Retention time (min) Belonging to the medicinal flavor 1 chlorogenic acid 9.87 Chrysanthemum, Stevia 8 Leptoside A 53.75 Stevia 9 Stevioside 54.10 Stevia 10 Orange-yellow cassia seed extract 63.30 Fried Cassia Seeds 14 emodin 80.77 Fried Cassia Seeds 17 rhein 87.24 Fried Cassia Seeds

[0117] Based on Table 3, combined with the retention times and UV absorption spectra of the compounds and test solutions (see Table 3), Figure 2 and Figure 3 The results showed that the retention times of compounds chlorogenic acid, lebodiin A, steviol, cassia aurantium, emodin, and chrysophanol were consistent with those of peaks 1 (9.87 min), 8 (53.75 min), 9 (54.10 min), 10 (63.30 min), 14 (80.77 min), and 17 (87.24 min) in the chromatogram of the test sample. Therefore, peaks 1, 8, 9, 10, 14, and 17 were identified as compounds chlorogenic acid, lebodiin A, steviol, cassia aurantium, emodin, and chrysophanol, respectively.

[0118] Given that the chlorogenic acid chromatographic peak (peak 1) has a high response value and good resolution, chlorogenic acid was selected as the reference peak and labeled as peak S. At the same time, the chlorogenic acid standard was used as the reference.

[0119] Example 3: A method for constructing the HPLC characteristic chromatogram of a heat-clearing and vision-improving tea.

[0120] 1. Experimental Methods

[0121] (1) Preparation of the test sample: Same as in Example 1;

[0122] (2) Preparation of reference solution

[0123] Take an appropriate amount of chlorogenic acid reference standard, accurately weigh it, and add 50% methanol solution (v / v) to prepare a solution containing 30 μg per 1 mL, which is the reference solution.

[0124] (3) HPLC detection

[0125] An octadecylsilane-bonded silica gel column (Waters XSelect HSS T3 column, 25 cm long, 4.6 mm inner diameter, 5 μm particle size) was used as the stationary phase. Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution (w / v) was used as mobile phase B. Gradient elution was performed according to the mobile phase gradient elution program shown in Table 1 of Example 1. The column temperature was 35 °C. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 8000. 10 μL of each of the reference solution and the test solution were accurately pipetted into the liquid chromatograph and the chromatograms were recorded.

[0126] 2. Information on test samples and reference materials

[0127] Serial Number Product Name source purity% batch number 1 Tea for clearing heat and improving eyesight Zhuzhou Qianjin Pharmaceutical Co., Ltd. / S11230001 2 chlorogenic acid National Institutes for Food and Drug Control (NIFDC) 96.3 110753-202119

[0128] 3. Experimental Results

[0129] Characteristic chromatograms of the heat-clearing and vision-improving tea sample are as follows: Figure 4 and Figure 5 As shown, from Figure 4 It can be seen that there are 17 characteristic peaks in the characteristic spectrum of the heat-clearing and vision-improving tea sample. Among them, peak 1 (S peak) is the characteristic peak of chlorogenic acid, peak 8 is the characteristic peak of leucodilin A, peak 9 is the characteristic peak of steviol glycoside, peak 10 is the characteristic peak of cassia oleracea, peak 14 is the characteristic peak of emodin, and peak 17 is the characteristic peak of chrysophanol.

[0130] Using the characteristic peak of chlorogenic acid as a reference peak (S peak), the relative retention times of characteristic peaks 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, and 17 were calculated. These relative retention times were within ±5% of the specified values. The specified values ​​were 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), and 8.837 (peak 17). Among these, [the remaining values ​​are missing from the original text]. Figure 6 As shown, the fingerprinting method established in this invention can identify the characteristic peaks of stevia, namely leucodilin A (peak 8) and steviol glycoside (peak 9), indicating that the established fingerprinting method can effectively separate and detect the chemical components in the finished product of the heat-clearing and vision-improving tea, and can be applied to the detection and analysis of the finished product of the heat-clearing and vision-improving tea.

[0131] Example 4: A quality testing method for a heat-clearing and vision-improving tea

[0132] 1. HPLC chromatogram of the heat-clearing and vision-improving tea sample.

[0133] According to “(1) Preparation of test solution” in Example 3, the test sample of Qingre Mingmu Tea was prepared into a test solution of the test sample.

[0134] According to “(3) HPLC detection” in Example 3, 10 μL of the test solution of the sample to be tested was precisely pipetted into the liquid chromatograph, and the chromatogram was recorded to obtain the HPLC chromatogram of the sample to be tested, which is the heat-clearing and vision-improving tea.

[0135] 2. Compare with fingerprint patterns

[0136] Using a standard sample of Qingre Mingmu tea, a control fingerprint chromatogram was constructed according to the method in Example 3. This control fingerprint chromatogram contained 17 fingerprint peaks, with peak 1 corresponding to the reference solution designated as peak S. The specified values ​​were as follows:

[0137] 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.

[0138] 3. Judgment Criteria

[0139] The following two conditions must be met for the test sample of the heat-clearing and vision-improving tea to be deemed qualified:

[0140] The HPLC chromatogram of the heat-clearing and vision-improving tea sample showed fingerprint peaks with retention times within ±5% of the specified values ​​in the control fingerprint chromatogram.

[0141] The fingerprint peaks were calculated using the HPLC fingerprint similarity evaluation system for traditional Chinese medicine. The similarity between the HPLC fingerprint of the test solution and the control fingerprint was not less than 0.90.

[0142] Experimental Example 1: Sample Injection Precision Test

[0143] 1. Experimental Methods

[0144] Samples of Qingre Mingmu Tea (S2-6) with batch number S20240415 were taken and tested according to the method in Example 3. The samples were injected six times consecutively using the HPLC detection method in Example 3. The sample numbers were recorded as Precision 1 (S2), Precision 2 (S3), Precision 3 (S4), Precision 4 (S5), and Precision 5 (S6), and the chromatograms were recorded for each sample. Using the retention time and peak area of ​​peak 1 as a reference, the relative retention time and relative peak area of ​​each fingerprint peak were calculated.

[0145] The method of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" was used to automatically match the chromatographic peaks of repeatable HPLC chromatograms to form a common pattern diagram. The chromatogram of the heat-clearing and vision-improving tea sample S1 with batch number S11240004 before sterilization was used as the reference chromatogram to calculate the similarity.

[0146] 2. Experimental Results

[0147] The results are shown in Tables 4 and 5. The maximum RSD of the relative retention time of the 17 fingerprint peaks was 0.1% (n=5), which is less than 5%, and the maximum RSD of the relative peak area was 4.5% (n=5). Figure 7 As shown in Table 6, the similarity of the fingerprint spectra collected in the five trials was greater than 0.95, indicating that the instrument and experimental method had good precision.

[0148] Table 4. Injection Precision (RSD of Relative Retention Times of Each Peak)

[0149] Peak 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)

[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] Experiment Example 2 Repeatability Test

[0156] 1. Experimental Methods

[0157] Six samples of the heat-clearing and vision-improving tea with batch number S20240415 were taken. Test solutions were prepared according to the method in Example 3, and the samples were analyzed using the HPLC detection method in Example 3. The six test solutions were numbered as Repeatability 1 (S2), Repeatability 2 (S3), Repeatability 3 (S4), Repeatability 4 (S5), Repeatability 5 (S6), and Repeatability 6 (S7), respectively, and the chromatograms were recorded. Using the retention time and peak area of ​​peak 1 as a reference, the relative retention time and relative peak area of ​​each fingerprint peak were calculated.

[0158] The method of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" was used to automatically match the chromatographic peaks of repeatable HPLC chromatograms to form a common pattern diagram. The chromatogram of the heat-clearing and vision-improving tea sample S1 with batch number S11240004 before sterilization was used as the reference chromatogram to calculate the similarity.

[0159] 2. Experimental Results

[0160] The results are shown in Tables 7 and 8. The maximum RSD of the relative retention time of the 17 fingerprint peaks was 0.2% (n=6), which is less than 5%, and the maximum RSD of the relative peak area was 7.8% (n=6). See Tables 9 and 8 for further details. Figure 8 As shown, the similarity of the fingerprint spectra collected in the six studies was greater than 0.95, indicating that the method has 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)

[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 Methods

[0171] Different personnel collected samples of Qingre Mingmu Tea (S2-6) with batch number S20240415 at different times. Test solutions were prepared according to the method in Example 3. Using different instruments, samples were injected six times consecutively according to the HPLC detection method in 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. Chromatograms were recorded for each sample. Using the retention time and peak area of ​​peak 1 as a reference, the relative retention time and relative peak area of ​​each fingerprint peak were calculated.

[0172] Take one sample of the test solution and perform six consecutive injections. Record the chromatograms 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), respectively. Calculate the similarity between these chromatograms and the chromatograms of the test solution in the "II. Repeatability" section. The chromatograms of the test solution in the "II. Repeatability" section are recorded as repeatability 1 (S2), repeatability 2 (S3), repeatability 3 (S4), repeatability 4 (S5), repeatability 5 (S6), and repeatability 6 (S7), respectively.

[0173] The method of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" was used to automatically match the chromatographic peaks of repeatable HPLC chromatograms to form a common pattern diagram. The chromatogram of the heat-clearing and vision-improving tea sample S1 with batch number S11240004 before sterilization was used as the reference chromatogram to calculate the similarity.

[0174] 2. Experimental Results

[0175] The results are shown in Tables 10 and 11. The maximum RSD of the relative retention time of the 17 fingerprint peaks was 0.8% (n=12), which is less than 5%, and the maximum RSD of the relative peak area was 14.6% (n=12). See Tables 12 and 11 for further details. Figure 9 As shown, the similarity of the fingerprint spectra collected in the 12 collections was greater than 0.95, indicating that the instrument and experimental method had good precision.

[0176] Table 10 Intermediate Precision (RSD of Relative Retention Times of Each Peak)

[0177]

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

[0179]

[0180]

[0181] Table 12 Intermediate Precision (Similarity)

[0182]

[0183]

[0184] Experiment Example 4 Stability Test

[0185] 1. Experimental Methods

[0186] A sample of heat-clearing and vision-improving tea (S1) with batch number S20240415 was taken. The test solution was prepared according to the method in Example 3. The samples were left at room temperature for 0, 4, 8, 13, 18, 35, 52, 69, and 86 hours, respectively. The samples were then analyzed using the HPLC detection method in Example 3. The sample numbers were recorded as 0h (S1), 4h (S2), 8h (S3), 13h (S4), 18h (S5), 35h (S6), 52h (S7), 69h (S8), and 86h (S9), respectively. Chromatograms were recorded for each sample. Using the retention time and peak area of ​​peak 1 as a reference, the relative retention time and relative peak area of ​​each fingerprint peak were calculated.

[0187] The method of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" was adopted to automatically match the chromatographic peaks of repeatable HPLC chromatograms to form a common pattern diagram. The chromatogram of the sample injected at 0h was used as the reference chromatogram to calculate the similarity.

[0188] 2. Experimental Results

[0189] The results are shown in Tables 13 and 14. The maximum RSD of the relative retention time for the 17 fingerprint peaks was 0.2% (n=9), and the maximum RSD of the relative peak area was 8.7% (n=9). See Tables 15 and 14 for further details. Figure 10 As shown, the similarity of the fingerprint spectra collected in the nine tests was greater than 0.95, indicating that the test solution was stable after being placed at room temperature for 86 hours.

[0190] Table 13 Solution stability (RSD of relative retention times of each peak)

[0191]

[0192]

[0193] Table 14 Solution stability (RSD of relative peak areas)

[0194] Peak 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 Methods

[0200] Take the heat-clearing and vision-improving tea sample (S2-4) with batch number S20240415, and prepare the test solution and reference solution according to the method of Example 3. According to the detection conditions shown in Table 16, accurately pipette the blank solvent (50% methanol solution (v / v)), reference solution and test solution, and inject them 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] The method of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" was used to automatically match the chromatographic peaks of repeatable HPLC chromatograms to form a common pattern diagram. The chromatogram of the heat-clearing and vision-improving tea sample S1 with batch number S11240004 before sterilization was used as the reference chromatogram to calculate the similarity.

[0202] Table 16 Durability Testing Conditions

[0203]

[0204] 2. Experimental Results

[0205] (1) Different phosphoric acid concentrations

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

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

[0208]

[0209] Table 18 Robustness (RSD of relative peak area) - 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 Tables 20 and 21. Under different column temperatures, the maximum RSD of the relative retention time of the 17 fingerprint peaks was 3.7% (n=3), which is less than 5%, and the maximum RSD of the relative peak area was 26.6% (n=3). See Tables 22 and 21 for further details. Figure 12 As shown, the chromatogram at column temperature of 38℃ is denoted as S2, the chromatogram at column temperature of 40℃ is denoted as S3, and the chromatogram at column temperature of 42℃ is denoted as S4. The similarity of the fingerprint chromatograms collected in the three tests was greater than 0.95, indicating that the test solution has good robustness under different column temperature conditions.

[0216] Table 20 Robustness (RSD of relative retention time of each peak) - at different column temperatures

[0217]

[0218]

[0219] Table 21 Robustness (RSD of relative peak area) - at different column temperatures

[0220] Peak 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 Methods

[0225] The experiment was conducted according to the sample preparation method and chromatographic conditions described in Zhou Jie et al.'s "Study on HPLC Characteristic Fingerprint and Determination of Multiple Component Content of Qingre Mingmu Tea" (publication date: February 29, 2020).

[0226] 1. Preparation of the test sample

[0227] Take about 1.0g of the heat-clearing and vision-improving tea powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of methanol-ethyl acetate (volume ratio 2:1), weigh it, sonicate it (power 300W, frequency 40kHz) for 45min, cool it, weigh it again, replenish the lost amount with methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0228] 2. Determination method

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

[0230] 3. Chromatographic conditions

[0231] Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A and 0.5% phosphoric acid solution (w / v) was used as mobile phase B, and elution was performed according to the conditions in Table 23; the flow rate was 1.0 mL per minute; the detection wavelength was 210 nm, and full wavelength scanning was performed; the column temperature was 30 °C.

[0232] Table 23 Elution conditions for liquid chromatography

[0233] Time (minutes) 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 follows Figure 13 As shown, under this gradient condition, the main chromatographic information is concentrated in the first 60 minutes. The peaks are more concentrated and information-rich from 18 to 30 minutes, but the resolution is poor. The peaks are better separated from 38 to 55 minutes. However, there is a very large peak at the 8-minute position in the chromatogram. Comparison revealed that this peak was introduced by the blank solvent. Ethyl acetate has two main absorption peaks in the UV region, one of which is located in the 210-230 nm range. It is speculated that this peak belongs to ethyl acetate. Therefore, this method suffers from excessive interference from the blank solvent.

[0236] Comparative Example 2

[0237] 1. Preparation of the test solution

[0238] Take about 0.5g of the heat-clearing and vision-improving tea powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of 70% methanol solution (v / v), weigh it, sonicate it (power 300W, frequency 40kHz) for 45min, cool it, weigh it again, replenish the lost amount with 70% methanol solution (v / v), shake it well, filter it, and take the filtrate to obtain the test solution.

[0239] 2. HPLC detection

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

[0241] Table 24 Elution conditions for liquid chromatography

[0242]

[0243] 3. Experimental Results

[0244] like Figure 14As shown in Table 24, under the gradient conditions, there is a lot of chromatographic information in the first 30 minutes, but the resolution is poor. The resolution of each chromatographic peak is better from 38 to 55 minutes. Therefore, the gradient needs to be reduced and the flow rate slowed down in the first 30 minutes.

[0245] Comparative Example 3

[0246] 1. Preparation of the test solution

[0247] Take four portions of the heat-clearing and vision-improving tea powder (S11230001), each approximately 0.5g, and accurately weigh them. Place them in stoppered conical flasks and accurately add 25mL each of 25% methanol solution (v / v), 50% methanol solution (v / v), 75% methanol solution (v / v), and 50% ethanol solution (v / v). Weigh the flasks and sonicate them (300W power, 40kHz frequency) for 30 minutes. Let them cool and weigh them again. Make up for the lost weight with 25% methanol solution (v / v), 50% methanol solution (v / v), 75% methanol solution (v / v), and 50% ethanol solution (v / v). Shake well and centrifuge at 12000rpm for 5 minutes. Filter the supernatant to obtain the test solution.

[0248] 2. HPLC detection

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

[0250] Table 25 Elution conditions for liquid chromatography

[0251]

[0252] 3. Experimental Results

[0253] The results are as follows Figure 15 As shown, the extraction rates of the compounds with retention times of 82–88 min varied among the different extraction solvents. The 50% methanol solution (v / v) had the highest extraction rate. Therefore, the 50% methanol solution (v / v) was selected as the extraction solvent, which is the extraction solvent of Example 3.

[0254] Comparative Example 4

[0255] 1. Preparation of the test solution

[0256] 1) Test Solution 1: Prepared according to the method described in "Test Solution" in Zhou Jie et al.'s "Study on Characteristic Fingerprint of Heat-Clearing and Vision-Improving Tea by HPLC and Determination of Multiple Component Content" (Publication Date: February 29, 2020):

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

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

[0259] 2. HPLC detection

[0260] An octadecylsilane-bonded silica gel column (Waters XSelect HSS T3 column, 25 cm long, 4.6 mm inner diameter, 5 μm particle size) was used as the stationary phase. Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution (w / v) was used as mobile phase B. Gradient elution was performed according to the mobile phase gradient elution program shown in Table 1. The column temperature was 35 °C. 10 μL of each of the test solutions, 1 and 2, was accurately injected into the HPLC system, and the chromatograms were recorded.

[0261] 3. Experimental Results

[0262] like Figure 16 As shown, the sample peak response obtained by the prior art test preparation method is significantly lower than that of the test preparation method in Example 3 of the present invention. Furthermore, the extraction solvent in the prior art test preparation method 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 the test solution

[0265] The test solution was prepared according to the method described in Example 3, "Preparation of Test Solution".

[0266] 2. HPLC detection

[0267] The test solutions were tested according to the "HPLC detection method" of Example 3 and Comparative Example 1, respectively.

[0268] 3. Experimental Results

[0269] like Figure 17 As shown, the test solution was detected under two chromatographic conditions. The chromatographic conditions of the present invention showed a significantly better separation effect than those of Comparative Example 1 within 16 to 34 minutes.

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

Claims

1. A method for constructing an HPLC fingerprint of a heat-clearing and vision-improving tea, characterized in that, Includes the following steps: Pretreatment method: Take the heat-clearing and vision-improving tea sample to be tested and extract it with a methanol aqueous solution with a volume fraction of 45-55% by ultrasonic extraction for 25-35 min; Preparation of reference solutions: Chlorogenic acid reference standard, levofloxacin A reference standard, steviol reference standard, cassia aurantium reference standard, rhein reference standard and chrysophanol reference standard were respectively prepared by dissolving chlorogenic acid reference standard, levofloxacin A reference standard solution, steviol reference standard solution, cassia aurantium reference standard solution, rhein reference standard solution and chrysophanol reference standard solution in 50% methanol aqueous solution; High-performance liquid chromatography (HPLC) method: Octadecylsilane-bonded silica gel was used as the column packing material. Acetonitrile was used as mobile phase A, and 0.08–0.12% (w / w) phosphoric acid solution was used as mobile phase B. In the gradient elution program, the volume percentage change of mobile phase B in the mobile phase system was as follows: From 0 to 12 min, the mobile phase B was 89%; In 12–15 minutes, the mobile phase B decreased from 89% to 78%. Within 15–40 minutes, the mobile phase B decreased from 78% to 75%. Over 40–45 minutes, the mobile phase B decreased from 75% to 65%. 45–55 min, mobile phase B is 65%; At 55–56 min, the mobile phase B decreased from 65% to 60%; 56–62 min, mobile phase B is 60%; At 62–64 min, the mobile phase B decreased from 60% to 58%; Between 64 and 79 minutes, the mobile phase B decreased from 58% to 48%. Between 79 and 84 minutes, the mobile phase B decreased from 48% to 20%. 84–87 min, mobile phase B is 20%; At 87–89 min, the mobile phase B increased from 20% to 89%; 89–95 min, mobile phase B is 89%; The detection wavelengths were gradient detection: 326 nm for 0–12 min; 286 nm for 12–40 min; 210 nm for 40–55 min; and 286 nm for 55–95 min.

2. The construction method according to claim 1, characterized in that, In the pretreatment method, the mass-to-volume ratio of the heat-clearing and vision-improving tea sample to the methanol aqueous solution is 1 g: (45-55) mL.

3. The construction method according to claim 1, characterized in that, The column temperature of the chromatographic column is 34–36 °C.

4. The construction method according to claim 1, characterized in that, The flow rate of the gradient elution process is: 0–12 min, flow rate 1.0 mL / min; The flow rate was reduced from 1.0 mL / min to 0.8 mL / min over 12–15 min. 15–40 min, flow rate 0.8 mL / min; The flow rate was reduced from 0.8 mL / min to 0.5 mL / min over 40–45 min. 45–55 min, flow rate 0.5 mL / min; 55–56 min, the flow rate was increased from 0.5 mL / min to 1.0 mL / min; 56–95 min, flow rate 1.0 mL / min.

5. The application of the construction method according to any one of claims 1 to 4 in the quality control of the heat-clearing and vision-improving tea.

6. A method for quality testing of a heat-clearing and vision-improving tea, characterized in that, The heat-clearing and vision-improving tea sample was processed using the pretreatment method described in claim 1 to obtain the test solution of the sample. The test solution of the sample to be tested is detected using the high-performance liquid chromatography method described in claim 1, and the chromatogram is recorded to obtain the HPLC chromatogram of the sample to be tested. The HPLC chromatogram of the sample to be tested is compared with the reference fingerprint chromatogram. The sample is deemed qualified only if it meets the following two conditions: The HPLC fingerprint of the sample to be tested shows fingerprint peaks with retention times within ±5% of the specified value compared to the control fingerprint. The fingerprint peaks were calculated according to the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine. The similarity between the HPLC fingerprint of the test sample and the control fingerprint was not less than 0.

90. The method for constructing the comparative fingerprint spectrum is as follows: the standard sample of the heat-clearing and vision-improving tea is processed using the pretreatment method described in claim 1 to obtain the test sample solution; Using chlorogenic acid standard solution as a reference solution, the test sample solution was detected by the high performance liquid chromatography method described in claim 1, and the chromatogram was recorded. The theoretical plate number calculated based on the chlorogenic acid peak was not less than 8000, and a reference fingerprint chromatogram of the heat-clearing and vision-improving tea was obtained. The reference fingerprint spectrum contains 17 fingerprint peaks, with peak number 1 of the reference solution designated as peak S. The specified values ​​are as follows: 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.

7. The quality inspection method according to claim 6, characterized in that, In the chlorogenic acid reference solution, the mass-to-volume ratio of chlorogenic acid to methanol aqueous solution is (28-32) μg: 1 mL.

8. The application of the construction method according to any one of claims 1 to 4 in detecting the content of roasted cassia seed, chrysanthemum and / or stevia in a heat-clearing and vision-improving tea.

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