Construction method and application of persimmon calyx characteristic spectrum and control spectrum

Abstract

The invention discloses a construction method and application of a persimmon calyx characteristic chromatogram and a control chromatogram, and the construction method comprises the following steps: taking persimmon calyx medicinal material powder, adding an extraction solvent, quantifying, extracting, cooling, complementing the lost weight by using the same solvent, shaking uniformly, filtering, and taking a subsequent filtrate to obtain a test solution; and respectively injecting the test solution and the reference solution into an ultra-high performance liquid chromatograph to obtain the characteristic chromatogram. The specific chromatogram takes betulinic acid, oleanolic acid and ursolic acid as characteristic components, seven characteristic peaks are established through an efficient detection method, information of triterpenoid components in persimmon calyx can be comprehensively reflected, and the method is suitable for rapid quality control of persimmon calyx medicinal materials, decoction pieces and preparation intermediate products and finished products containing the persimmon calyx medicinal materials or the decoction pieces. The method is efficient, convenient to operate and suitable for quality monitoring of continuous production.

Description

Technical Field

[0001] The invention relates to a construction method of a persimmon calyx characteristic spectrum and a reference spectrum and application thereof, and belongs to the field of persimmon calyx analysis. Background Art

[0002] Persimmon calyx is a traditional Chinese medicine. It has been used as medicine since the Tang Dynasty. It has a significant therapeutic effect on relieving adverse reactions and stopping vomiting. It is often used in combination with cloves in clinical practice. It comes from the dried calyx of Diospyros kaki Thunb. of the Ebenaceae family (Chinese Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China (Part I) [S]. Beijing: China Medical Science and Technology Press, 2020: 261-262.). Persimmon trees are cultivated in China and many parts of the world. They contain a variety of chemical components, such as triterpenes, phenolic acids, tannins, etc., and have multiple pharmacological effects such as cardiovascular protection, antioxidant, and anti-DNA damage (Butta SM, Sultanb MT, Aziza M., et al. Persimmon (Diospyros kaki) fruit: hidden phytochemicals and health claims [J]. EXCLI Journal, 2015, 14: 542-561.).

[0003] Regarding the quality control methods of persimmon calyx, the most reported methods are content determination. For example, Zhu Zhiyong et al. used HPLC to determine the content of ursolic acid and oleanolic acid in persimmon calyx (Zhu Zhiyong, Zheng Feng, Chen Wu. Determination of the content of ursolic acid and oleanolic acid in persimmon calyx by HPLC [J]. Anhui Agricultural Science, 2007, 35(31): 9840-9841.), Huang Wenping et al. used HPLC to determine the content of gallic acid in persimmon calyx (Huang Wenping et al. Ping, Huang Luqiang, Song Yonggui, et al. Comparison of gallic acid content in persimmon calyx from different origins [J]. Journal of Jiangxi University of Traditional Chinese Medicine, 2014, 26 (3): 58-59, 62.), Ren Lei et al. used spectrophotometry to determine the content of total triterpenic acid in persimmon calyx (Ren Lei, Feng Haiyan, Yang Xiaohui, et al. Determination of total triterpenic acid in persimmon calyx by spectrophotometry [J]. Journal of Shijiazhuang College, 2014, 16 (6): 28-30.). The characteristic fingerprint spectrum can reflect more information on the chemical composition of traditional Chinese medicine and has been widely used in the quality evaluation of traditional Chinese medicine and its products. At present, there are few studies on the characteristic fingerprint of persimmon calyx medicinal materials, with only two papers. For example, Zhou Benhong et al. established the HPLC fingerprint of persimmon calyx medicinal materials (Zhou Benhong, Shen Heng, Wei Yan, et al. Study on the HPLC fingerprint of persimmon calyx medicinal materials [J]. Journal of Guangdong Pharmaceutical College, 2011, 27(2): 147-150.). Patent CN118090985A established a characteristic spectrum for the water-soluble components in persimmon calyx. The common peaks are organic acids (such as gallic acid and protocatechuic acid) and flavonoids (such as hyperoside, isoquercetin, quercetin, kaempferol, luteolin, etc.). However, there is no report on the characteristic fingerprint of triterpenoid components. This type of component has a high content and significant pharmacological activity, which is of great significance for the quality control of persimmon calyx. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for constructing a characteristic map and a reference map of persimmon calyx and its application, so as to strengthen the quality control of persimmon calyx and provide a reference for the quality analysis of persimmon calyx samples.

[0005] Technical solution: To solve the above technical problems, the present invention provides a method for constructing a characteristic map of persimmon calyx, comprising the following steps:

[0006] (1) Take persimmon calyx powder, add extraction solvent, extract after quantitative extraction, cool, make up the lost weight with the same solvent, shake well, filter, and take the filtrate to obtain the test solution;

[0007] (2) Take betulinic acid reference substance, oleanolic acid reference substance and ursolic acid reference substance, and add solvent to prepare reference substance solution;

[0008] (3) The test solution and the reference solution were injected into an ultra-high performance liquid chromatography, respectively, to obtain a characteristic spectrum; the characteristic spectrum had 7 common peaks, betulinic acid was taken as the reference, and peak 4 was the chromatographic peak of betulinic acid; oleanolic acid was taken as the reference, and peak 5 was the chromatographic peak of oleanolic acid; ursolic acid was taken as the reference, and peak 6 was the chromatographic peak of ursolic acid; the peak corresponding to the oleanolic acid reference was the S peak, and the relative retention times of peaks 1, 2, 3 and 7 were calculated, and the relative retention times were within the range of ±10% of the specified values, and the specified values ​​of peaks 1, 2, 3 and 7 were 0.27, 0.41, 0.44 and 1.21, respectively.

[0009] Wherein, the extraction solvent in step (1) is alcohol or alcohol-water solution.

[0010] Wherein, the methanol volume fraction of the methanol-water solution is not less than 70%, and the ethanol volume fraction of the ethanol-water solution is not less than 50%.

[0011] Wherein, the extraction solvent in step (1) includes 70% to 100% methanol or 50% to 95% ethanol.

[0012] Among them, the methanol volume fraction of the methanol-water solution can be 70%, 75%, 80%, 85%, 90%, 95%, and 100%; the ethanol volume fraction of the ethanol-water solution can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%; considering that ethanol is non-toxic and does not pollute the environment, the ethanol volume fraction is preferably 80% to 95% ethanol.

[0013] Wherein, the extraction method in step (1) includes ultrasonic treatment, heating reflux or shaking extraction. In order to simplify the extraction operation, ultrasonic treatment is preferably used. The power and frequency of the ultrasound have little effect on the present invention. In some preferred embodiments, the power of the ultrasonic treatment is 600W and the frequency is 40kHz.

[0014] Wherein, the extraction time in step (1) is 15 to 60 minutes, specifically 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min, which has little effect on the present invention. In some preferred embodiments, the extraction time is 30 min.

[0015] Among them, the mass volume ratio of persimmon calyx medicinal materials and decoction pieces powder to the extraction solvent is 1g:25mL. If the concentration of the test sample is too low, it is not conducive to the detection of the chromatographic peak. If the concentration is too high, the chromatographic column will be overloaded, resulting in poor chromatographic peak shape. The preparation intermediates and finished products containing persimmon calyx medicinal materials or decoction pieces can adjust the mass volume ratio of the sample to be tested and the extraction solvent according to the proportion of persimmon calyx.

[0016] Wherein, in step (2), the solvent is alcohol or alcohol-water solution, wherein the methanol volume fraction of the methanol-water solution is not less than 70%, and can specifically be 70%, 75%, 80%, 85%, 90%, 95%, or 100%; the ethanol volume fraction of the ethanol-water solution is not less than 50%, and can specifically be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%; and considering that ethanol is non-toxic and has no pollution to the environment, the ethanol volume fraction is preferably 80% to 95% ethanol.

[0017] Specifically, the concentration of the betulic acid reference substance is 190 μg / mL, the concentration of the oleanolic acid reference substance is 70 μg / mL, and the concentration of the ursolic acid reference substance is 235 μg / mL.

[0018] Wherein, the chromatographic column of the ultra-high performance liquid chromatograph in step (3) comprises ZORBAX SB-C18 RRHD, Poroshell HPH-C18 or Poroshell 120EC-C18.

[0019] Among them, the chromatographic column length is 150mm, the inner diameter is 2.1mm, and the filler particle size is 1.8-2.7μm.

[0020] Wherein, the column temperature of the ultra-high performance liquid chromatograph in step (3) is 25°C to 35°C.

[0021] Wherein, the mobile phase of the ultra-high performance liquid chromatography in step (3) is methanol-5mmol / L ammonium acetate aqueous solution.

[0022] Wherein, the flow rate of the ultra-high performance liquid chromatography in step (3) is 0.28 ml / min to 0.32 ml / min.

[0023] Wherein, the elution gradient of the ultra-high performance liquid chromatograph in step (3) is 0-2 min, the volume fraction of mobile phase A is 70%, and the volume fraction of mobile phase B is 30%; 2-15 min, the volume change fraction of mobile phase A is 70→77%, and the volume change fraction of mobile phase B is 30→23%; 15-18 min, the volume change fraction of mobile phase A is 77→84%, and the volume change fraction of mobile phase B is 23→16%; 18-35 min, the volume fraction of mobile phase A is 84%, and the volume fraction of mobile phase B is 16%.

[0024] Wherein, the ultra-high performance liquid chromatograph in step (3) adopts a charged aerosol detector for detection.

[0025] The present invention also provides application of the construction method in identifying persimmon calyx or controlling the quality of persimmon calyx.

[0026] Among them, the above-mentioned persimmon calyx is one or more of persimmon calyx medicinal materials, decoction pieces, and preparation intermediates or finished products containing persimmon calyx medicinal materials or decoction pieces.

[0027] The characteristic spectrum of the present invention can be used for the detection of persimmon calyx medicinal materials, decoction pieces, and preparation intermediates or finished products containing persimmon calyx medicinal materials or decoction pieces. The detection method comprises the following steps:

[0028] Extracting the sample to be tested with an extraction solvent to prepare a solution of the sample to be tested;

[0029] The test solution is tested by liquid chromatography, and the test spectrum obtained is compared with the characteristic spectrum constructed by the method of the claim; if the characteristic spectra of the two are consistent, it is a qualified product; if the characteristic spectra of the two are inconsistent, it is an unqualified product.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the present invention increases the number of detectable chromatographic peaks by optimizing the detector, improves the separation effect of the chromatographic peaks by optimizing the liquid chromatography conditions, and finally establishes a characteristic spectrum determination method including triterpenoid components such as oleanolic acid, ursolic acid and betulinic acid in persimmon calyx samples, providing a new analytical means for the quality detection of persimmon calyx samples. In addition, the method of the present invention is simple to operate, has good reproducibility and stability, and has high detection efficiency, which can provide a basis for standardizing the market and reasonable development of persimmon calyx medicinal materials and preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the liquid phase spectrum of the elution gradient of Table 1 in Example 1;

[0032] Figure 2 The liquid phase spectrum of the elution gradient of Table 2 to Table 5 in Example 1: A: methanol-0.1% formic acid aqueous solution as the mobile phase, B: methanol: acetonitrile 9:1-0.1% formic acid aqueous solution as the mobile phase, C: methanol-5mmol / L ammonium formate aqueous solution as the mobile phase, D: methanol-5mmol / L ammonium acetate aqueous solution as the mobile phase;

[0033] Figure 3 is the liquid phase spectrum of the elution gradient of Table 6 in Example 1;

[0034] Figure 4 These are the detection spectra investigated by different detectors in Example 1: A: chromatogram of the persimmon calyx sample obtained by using an ELSD detector, B: chromatogram of the persimmon calyx sample obtained by using a CAD detector;

[0035] Figure 5 is the liquid phase spectrum of the reference substance and the sample in Example 3;

[0036] Figure 6It is a superposition of characteristic spectra of multiple batches of persimmon calyx samples in Example 3;

[0037] Figure 7 is the reference characteristic spectrum of the persimmon calyx medicinal material in Example 3;

[0038] Figure 8 This is the liquid phase spectrum of the extraction solvent investigation in Example 2;

[0039] Fig. 9 This is the liquid phase spectrum investigated by the extraction method in Example 2;

[0040] Fig.10 This is the liquid phase spectrum of the extraction time investigation in Example 2;

[0041] Fig.11 is the liquid phase spectrum investigated at different chromatographic column temperatures in Example 1;

[0042] Fig.12 is the liquid phase spectrum investigated at different flow rates in Example 1;

[0043] Fig.13 It is the liquid phase spectrum investigated by different chromatographic columns in Example 1. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0045] Instruments, reagents and test drugs

[0046] Thermo Vanquish ultra-high performance liquid chromatograph (Thermo Fisher); Thermo Vanquish charged aerosol detector (CAD) (Thermo Fisher); Chromeleam 7.2SR4 Chameleon workstation; 1 / 10,000 electronic analytical balance (Shanghai Mettler-Toledo Instrument Co., Ltd.); PL-J100 mechanical ultrasonic cleaning machine (Dongguan Kangshijie Ultrasonic Technology Co., Ltd.); TGL-16C high-speed desktop centrifuge (Shanghai Anting Scientific Instrument Factory); IQ 7003 pure water system (Millipore); acetonitrile, methanol (chromatographic grade, Thermo Fisher); formic acid, ammonium formate, ammonium acetate (chromatographic grade, Aladdin); methanol, ethanol (analytical grade, Sinopharm Chemical Reagent Co., Ltd.).

[0047] Betulinic acid (batch number 15286, purity ≥ 97.2%) was purchased from Shanghai Stand Biotechnology Co., Ltd. Oleanolic acid (batch number 110709-201808, purity ≥ 91.1%) and ursolic acid (batch number 110742-201622, purity ≥ 94.7%) were purchased from China Food and Drug Administration.

[0048] The persimmon calyx medicinal material samples were provided by Tianjiang Pharmaceutical Co., Ltd. and were numbered S1 to S20. They were identified as dried persistent calyx of Diospyros kakiThunb., a plant of the Ebenaceae family.

[0049] Example 1: Detection of the characteristic spectrum of persimmon calyx medicinal material

[0050] 1. Preparation of test solution

[0051] Take about 1 g of persimmon calyx powder, accurately weigh it, put it in a stoppered conical flask, accurately add 25 mL of 95% ethanol, stopper it, weigh it, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up for the weight loss, shake it well, filter it, and take the filtrate to obtain it.

[0052] 2. Preparation of reference substance solution

[0053] Take appropriate amount of betulinic acid reference substance, oleanolic acid reference substance and ursolic acid reference substance, add 95% ethanol to prepare 1 ml solution containing 190 μg betulinic acid, 70 μg oleanolic acid and 235 μg ursolic acid as reference substance solution.

[0054] 3. Establishment of Chromatographic Analysis Conditions

[0055] 3.1 An InfinityLab Poroshell 120Aq-C18 (2.1×150 mm, 2.7 μm) column was used, with acetonitrile as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B, and eluted according to the gradient specified in Table 1; the flow rate was 0.30 mL per minute; the column temperature was 35° C. An electrospray detector was used for detection.

[0056] Table 1 Elution gradient program

[0057]

[0058] Under this condition, the 18-min chromatographic peaks are stacked together and a good analysis cannot be obtained. Figure 1 This was due to the strong elution capacity of acetonitrile and the poor selectivity of the chromatographic column for this type of chromatographic peak. Therefore, the ZORBAX SB-C18RRHD (Agilent, 2.1×150mm, 1.8μm) chromatographic column was replaced, and the mobile phase A was changed from acetonitrile to a solution containing methanol, and the elution gradient was further optimized.

[0059] 3.2 A ZORBAX SB-C18 RRHD (Agilent, 2.1×150mm, 1.8μm) column was used with a flow rate of 0.30mL per minute; the column temperature was 25°C; and an electrospray detector was used for detection. Four different mobile phase systems, methanol-0.1% formic acid aqueous solution, methanol:acetonitrile (9:1)-0.1% formic acid aqueous solution, methanol-5mmol / L ammonium formate aqueous solution, and methanol-5mmol / L ammonium acetate aqueous solution, were used for elution according to the gradients specified in the table below.

[0060] Table 2 Methanol-0.1% formic acid gradient elution program

[0061]

[0062] Table 3 Methanol: acetonitrile (9:1)-0.1% formic acid gradient elution program

[0063]

[0064] Table 4 Methanol-5mmol / L ammonium formate gradient elution program

[0065]

[0066] Table 5 Methanol-5mmol / L ammonium acetate gradient elution program

[0067]

[0068]

[0069] The results show that the separation effect of each chromatographic peak using methanol-5mmol / L ammonium acetate aqueous solution system is relatively good. Figure 2 However, the elution time was long and the chromatographic peaks were dispersed, so the elution gradient and column temperature were further optimized.

[0070] 3.3 After multiple optimizations, the chromatogram time was appropriately shortened, and each chromatographic peak was well separated and evenly distributed. Figure 3 , the final chromatographic conditions were determined as follows:

[0071] A ZORBAX SB-C18 RRHD (Agilent, 2.1×150 mm, 1.8 μm) column was used, methanol was used as mobile phase A, 5 mmol / L ammonium acetate aqueous solution was used as mobile phase B, and elution was performed according to the gradient in Table 6; an electrospray detector was used for detection.

[0072] Table 6 Methanol-0.1% formic acid gradient elution program

[0073]

[0074] 3.4 Comparison of different detectors

[0075] Take 1 sample of persimmon calyx medicinal material (No. S2), prepare the test solution according to the method under "1. Preparation of test solution" of this example, detect according to the chromatographic conditions under "3.3" of this example, accurately draw 3 μL of the test solution, use evaporative light scattering detector (ELSD) and electrospray detector (CAD) for sample injection and measure, and record the chromatogram. The results show that ( Figure 4 ), the electrospray ionization detector can detect more chromatographic peaks than the evaporative light scattering detector, and is more suitable for the detection of the characteristic spectrum of persimmon calyx.

[0076] 3.5 Column temperature investigation

[0077] Take 1 sample of persimmon calyx medicinal material (No. S2), prepare the test solution according to the method under "1. Preparation of test solution" in Example 1, accurately draw 3 μL, and follow the gradient elution conditions under "3.3" in Example 1 with a flow rate of 0.30 ml / min. Compare the sample separation effects when the column temperature is 25°C, 30°C, and 35°C. The results show that the separation effects of the 7 characteristic peaks in the range of 25°C to 35°C are all good, and the column temperature changes within this range can meet the system suitability requirements. See Fig.11 .

[0078] 3.6 Flow rate investigation

[0079] Take 1 sample of persimmon calyx medicinal material (No. S2), prepare the test solution according to the method under "1. Preparation of test solution" in Example 1, accurately draw 3 μL, and follow the gradient elution conditions under "3.3" in Example 1 to compare the sample separation effects at flow rates of 0.28 ml / min, 0.30 ml / min, and 0.32 ml / min. The results show that the separation effects of the 7 characteristic peaks are good when the flow rate is in the range of 0.28 ml / min to 0.32 ml / min, and the column temperature changes within this range can meet the system suitability requirements. See Fig.12 .

[0080] 3.7 Chromatographic column investigation

[0081] Take 1 sample of persimmon calyx medicinal material (No. S2), prepare the test solution according to the method under "1. Preparation of test solution" in Example 1, accurately draw 3 μL, and follow the gradient elution conditions under "3.3" in Example 1 to compare the analytical effects of 3 different types of chromatographic columns, namely ZORBAX SB-C18 RRHD (Agilent, 2.1×150mm, 1.8μm), Poroshell HPH-C18 (Agilent, 2.1×150mm, 1.9μm), and Poroshell 120EC-C18 (Agilent, 2.1×150mm, 2.7μm). All three chromatographic columns can present 7 characteristic peaks. This method has good durability for different chromatographic columns. Fig.13 .

[0082] The optimal chromatographic conditions were finally determined to be:

[0083] A ZORBAX SB-C18 RRHD (Agilent, 2.1×150 mm, 1.8 μm) chromatographic column was used with methanol as mobile phase A and 5 mmol / L ammonium acetate aqueous solution as mobile phase B, and elution was performed according to the gradient in Table 6; the flow rate was 0.30 mL per minute; the column temperature was 30°C; and detection was performed using an electrospray detector.

[0084] Example 2: Investigation of the method for preparing the test solution of the characteristic spectrum of the persimmon calyx medicinal material

[0085] 1. Investigation of extraction solvent

[0086] Take 1 portion of the persimmon calyx medicinal material (No. S2), take about 1 g, accurately weigh, place in a stoppered conical flask, parallel 6 portions, accurately add 50% methanol, 70% methanol, methanol, 50% ethanol, 80% ethanol, 95% ethanol 25mL, seal, weigh, ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, let cool, weigh again, make up for the weight loss, shake well, filter, take the filtrate, accurately absorb 3μL of each test solution, and determine according to the method under "3.3" of Example 1 to calculate the extraction efficiency. The results are shown in Table 7 and Figure 8 .

[0087] Table 7 Comparison of extraction efficiency of different extraction solvents (peak area / sample weight)

[0088]

[0089]

[0090] The results showed that, except for 50% methanol with low extraction efficiency, the chromatograms of the test sample solutions prepared with the other five extraction solvents all showed 7 characteristic peaks, and all of them could be used as extraction solvents for the characteristic spectrum of persimmon calyx medicinal materials.

[0091] 2. Investigation of extraction methods

[0092] Take 1 portion of the persimmon calyx medicinal material (No. S2), take about 1 g, accurately weigh, place in a stoppered conical flask, parallel 3 portions, accurately add 25 mL of 95% ethanol, seal, weigh, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, heat reflux extraction for 30 minutes, shake extraction for 30 minutes, let cool, weigh again, make up for the weight loss, shake well, filter, take the filtrate, accurately absorb 3 μL of each test solution, and determine according to the method under "3.3" of Example 1 to calculate the extraction efficiency. The results are shown in Table 8 and Fig. 9 .

[0093] Table 8 Comparison of extraction efficiency of different extraction methods (peak area / sample weight)

[0094]

[0095] The results showed that the chromatograms of the test solutions prepared by the three different extraction methods all showed 7 characteristic peaks, and there was no significant difference in extraction efficiency, and all of them could be used to prepare the test solution of the characteristic spectrum of Diospyros kaki. Considering the simplicity of operation, ultrasonic treatment was preferred as the extraction method.

[0096] 3. Investigation of extraction time

[0097] Take 1 portion of the persimmon calyx medicinal material (No. S2), take about 1 g, accurately weigh, place in a stoppered conical flask, parallel 4 portions, accurately add 25 mL of 95% ethanol, seal, weigh, ultrasonically treat (power 600 W, frequency 40 kHz) for 15 minutes, 30 minutes, 45 minutes, and 60 minutes, cool, weigh again, make up for the weight loss, shake well, filter, take the filtrate, accurately absorb 3 μL of each test solution, and determine according to the method under "3.3" of Example 1 to calculate the extraction efficiency. The results are shown in Table 9 and Fig.10 .

[0098] Table 9 Comparison of extraction efficiency at different extraction times (peak area / sample weight)

[0099]

[0100]

[0101] The results showed that the chromatograms of the test sample solutions prepared at different extraction times all showed 7 characteristic peaks, and the extraction efficiency was not much different, and they could all be used to prepare test samples with characteristic spectra of persimmon calyx.

[0102] In summary, the preparation method of the test solution of the present invention is confirmed: take about 1 g of persimmon calyx medicinal material powder, accurately weigh it, put it in a stoppered conical flask, accurately add 25 mL of 95% ethanol, seal it, weigh it, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up for the weight loss, shake it well, filter it, and take the filtrate to obtain it.

[0103] Example 3: Establishment of the characteristic spectrum of persimmon calyx medicinal material

[0104] 1. Preparation of test solution:

[0105] Take about 1 g of 20 batches of persimmon calyx medicinal powder (numbered S1 to S20), accurately weigh it, put it in a stoppered conical flask, accurately add 25 mL of 95% ethanol, stopper it, weigh it, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up for the weight loss, shake it well, filter it, and take the filtrate to obtain it.

[0106] 2. Preparation of reference substance solution:

[0107] Take appropriate amount of betulinic acid reference substance, oleanolic acid reference substance and ursolic acid reference substance, add 95% ethanol to prepare 1 mL of solution containing 190 μg betulinic acid, 70 μg oleanolic acid and 235 μg ursolic acid as reference substance solution.

[0108] 3. Accurately pipette 3 μL of the reference solution and the test solution respectively, inject them into the ultra-high performance liquid chromatograph, record the chromatogram, and obtain the characteristic spectrum of the persimmon calyx medicinal material.

[0109] 4. Identification of chromatographic peaks

[0110] The reference solutions of betulinic acid, oleanolic acid and ursolic acid were used for positioning. The results showed that ( Figure 5 ): Peak 4 is betulinic acid, Peak 5 is oleanolic acid, and Peak 6 is ursolic acid.

[0111] 5. Selection of common peaks and establishment of reference characteristic profiles

[0112] A ZORBAX SB-C18 RRHD (Agilent, 2.1×150mm, 1.8μm) column was used, methanol was used as mobile phase A, 5mmol / L ammonium acetate aqueous solution was used as mobile phase B, and elution was performed according to the gradient in Table 6; the flow rate was 0.30mL per minute; the column temperature was 30°C; an electrospray detector was used for detection, and chromatograms of 20 batches of persimmon calyx medicinal materials were recorded. The chromatograms of 20 batches of persimmon calyx medicinal materials were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition", and the median method was used with a time window of 0.1s. Through multi-point calibration, peak matching was performed, and a total of 7 relatively obvious common peaks were determined, see Figure 6The generated control feature map is shown in Figure 7 . Figure 7 There were 7 characteristic peaks in the chromatogram of the test sample, among which peak 4, peak 5 and peak 6 corresponded to the retention times of the reference peaks of betulinic acid, oleanolic acid and ursolic acid, respectively.

[0113] The peak corresponding to the oleanolic acid reference substance peak is the S peak. Calculate the relative retention times of Peak 1, Peak 2, Peak 3 and Peak 7. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.27 (Peak 1), 0.41 (Peak 2), 0.44 (Peak 3), and 1.21 (Peak 7).

[0114] Example 4: Methodological Investigation of the Characteristic Spectrum of Persimmon Calyx

[0115] 1. Precision test

[0116] Take 1 sample of persimmon calyx medicinal material (No. S2), prepare the test solution according to the test solution confirmed in Example 2, accurately draw 3 μL, inject 6 times continuously, measure according to the optimal chromatographic conditions, and record the chromatogram. Among them, peak 4, peak 5 and peak 6 should correspond to the retention time of the reference peak of betulinic acid, oleanolic acid and ursolic acid reference substance respectively; the peak corresponding to the reference peak of oleanolic acid reference substance is S peak, and the relative retention time and relative peak area of ​​the remaining characteristic peaks are calculated.

[0117] Table 10 Precision test results (relative retention time)

[0118]

[0119] Table 11 Precision test results (relative peak area)

[0120]

[0121]

[0122] The results of the precision test showed that the relative retention time RSD values ​​of each characteristic peak of Persimmon Calyx were less than 1%, and the relative peak area RSD values ​​were less than 3%, indicating that the instrument had good precision.

[0123] 2. Repeatability test

[0124] Take 6 samples of persimmon calyx medicinal material (number S2), prepare the test solution according to the method confirmed in Example 2, accurately draw 3 μL respectively, measure according to the optimal chromatographic conditions, and record the chromatogram. Among them, peak 4, peak 5 and peak 6 should correspond to the retention time of the reference peak of betulinic acid, oleanolic acid and ursolic acid reference substance respectively; the peak corresponding to the reference peak of oleanolic acid reference substance is S peak, and calculate the relative retention time and relative peak area of ​​the remaining characteristic peaks.

[0125] Table 12 Repeatability test results (relative retention time)

[0126]

[0127] Table 13 Repeatability test results (relative peak area)

[0128]

[0129]

[0130] The results of the repeatability test showed that the relative retention time RSD values ​​of each characteristic peak of the persimmon calyx medicinal material were all less than 1%, and the relative peak area RSD values ​​were all less than 3%, indicating that the method had good repeatability.

[0131] 3. Stability test

[0132] Take 1 sample of persimmon calyx medicinal material (No. S2), prepare the test solution according to the method confirmed in Example 2, and measure according to the optimal chromatographic conditions at 0h, 2h, 4h, 8h, 12h, 18h, and 24h, accurately draw 3μL injection measurement, and record the chromatogram. Among them, peak 4, peak 5 and peak 6 should correspond to the retention time of the reference peak of betulinic acid, oleanolic acid, and ursolic acid reference substance respectively; the peak corresponding to the reference peak of oleanolic acid reference substance is S peak, and the relative retention time and relative peak area of ​​the remaining characteristic peaks are calculated.

[0133] Table 14 Stability test results (relative retention time)

[0134]

[0135] Table 15 Stability test results (relative peak area)

[0136]

[0137] The results of the stability test showed that the relative retention time RSD values ​​of each characteristic peak of Persimmon Calyx were less than 1%, and the relative peak area RSD values ​​were less than 3%, indicating that the method had good stability.

[0138] Example 5: Detection of Persimmon Calyx Medicinal Material

[0139] 1. Preparation of sample solution

[0140] Take about 1 g of persimmon calyx powder, accurately weigh it, put it in a stoppered conical flask, accurately add 25 mL of 95% ethanol, stopper it, weigh it, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up for the weight loss, shake it well, filter it, and take the filtrate to obtain it.

[0141] 2. Detection

[0142] Accurately pipette 3 μL of the sample solution to be tested, inject it into the ultra-high performance liquid chromatograph, and perform the measurement under the optimal chromatographic conditions.

[0143] By comparing the sample to be tested with Figure 6 The reference characteristic spectrum of the persimmon calyx medicinal material shows that the sample to be tested can detect 7 characteristic peaks, among which peak 4, peak 5, and peak 6 should correspond to the retention time of the betulinic acid reference substance, oleanolic acid reference substance, and ursolic acid reference substance reference substance peaks, respectively. The peak corresponding to the oleanolic acid reference substance peak is the S peak, and the relative retention times of peak 1, peak 2, peak 3, and peak 7 are calculated to be 0.27, 0.41, 0.45, and 1.22, respectively, which are in line with the ±10% range of the specified value. The specified values ​​are: 0.27 (peak 1), 0.41 (peak 2), 0.44 (peak 3), and 1.21 (peak 7). It can be considered that the sample to be tested has stable quality, meets the quality requirements, and is a qualified medicinal material.

Claims

1. A method for constructing a characteristic spectrum of persimmon calyx, characterized in that: The following steps are involved: (1) Take persimmon calyx powder, add extraction solvent, extract after quantitative extraction, cool, make up the lost weight with the same solvent, shake well, filter, and take the filtrate to obtain the test solution; (2) Take betulinic acid reference substance, oleanolic acid reference substance and ursolic acid reference substance, and add solvent to prepare reference substance solution; (3) Inject the test solution and the reference solution into an ultra-high performance liquid chromatograph to obtain characteristic spectra; The characteristic spectrum has 7 peaks in total, betulic acid is taken as the reference substance, and peak 4 is the chromatographic peak of betulic acid; oleanolic acid is taken as the reference substance, and peak 5 is the chromatographic peak of oleanolic acid; ursolic acid is taken as the reference substance, and peak 6 is the chromatographic peak of ursolic acid; the peak corresponding to the oleanolic acid reference substance is the S peak, and the relative retention times of peaks 1, 2, 3 and 7 are calculated, and the relative retention times are within the range of ±10% of the specified values, and the specified values ​​of peaks 1, 2, 3 and 7 are 0.27, 0.41, 0.44 and 1.21, respectively.

2. The construction method according to claim 1, characterized in that: The extraction solvent in step (1) includes 70% to 100% methanol or 50% to 95% ethanol.

3. The construction method according to claim 1, characterized in that: The extraction method in step (1) includes ultrasonic treatment, heating reflux or shaking extraction; the extraction time is 15 to 60 minutes.

4. The construction method according to claim 1, characterized in that: The chromatographic column of the ultra-high performance liquid chromatography in step (3) includes ZORBAX SB-C18 RRHD, Poroshell HPH-C18 or Poroshell 120EC-C18.

5. The construction method according to claim 1, characterized in that: The column temperature of the ultra-high performance liquid chromatograph in step (3) is 25°C to 35°C.

6. The construction method according to claim 1, characterized in that: The mobile phase of the ultra-high performance liquid chromatograph in step (3) is methanol-5mmol / L ammonium acetate aqueous solution; the flow rate is 0.28ml / min to 0.32ml / min.

7. The construction method according to claim 1, characterized in that: The ultra-high performance liquid chromatograph in step (3) uses a charged aerosol detector for detection.

8. The construction method according to claim 1, characterized in that: The elution gradient of the ultra-high performance liquid chromatograph in step (3) is 0-2 min, the volume fraction of mobile phase A is 70%, and the volume fraction of mobile phase B is 30%; 2-15 min, the volume change fraction of mobile phase A is 70→77%, and the volume change fraction of mobile phase B is 30→23%; 15-18 min, the volume change fraction of mobile phase A is 77→84%, and the volume change fraction of mobile phase B is 23→16%; 18-35 min, the volume fraction of mobile phase A is 84%, and the volume fraction of mobile phase B is 16%.

9. A method for establishing a comparison map of persimmon calyx, characterized in that: The following steps are involved: (1) preparing multiple batches of persimmon calyx test sample solutions respectively; (2) The test solution is tested according to the method for constructing the characteristic spectrum of the persimmon calyx according to any one of claims 1 to 8 to obtain the characteristic spectrum of the persimmon calyx; the preparation method of the test solution comprises the following steps: taking persimmon calyx medicinal material powder, adding an extraction solvent, extracting after quantitative determination, cooling, supplementing the lost weight with the same solvent, shaking well, filtering, and taking a filtrate to obtain the test solution; (3) The obtained characteristic spectrum was imported into the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint to establish the reference spectrum of persimmon calyx.

10. Use of the construction method according to any one of claims 1 to 9 in identifying persimmon calyx or controlling its quality.

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