A method for constructing a characteristic map and a reference map of persimmon calyx and its application

Abstract

The present invention discloses a method for constructing a characteristic spectrum and a reference spectrum of a persimmon calyx, and their application. The method comprises the following steps: taking persimmon calyx medicinal material powder, adding an extraction solvent, extracting after quantitative determination, cooling, replenishing the lost weight with the same solvent, shaking, filtering, and collecting a subsequent filtrate to obtain a test solution; and injecting the test solution and the reference solution into an ultra-high performance liquid chromatograph to obtain a characteristic spectrum. The characteristic spectrum, with betulinic acid, oleanolic acid, and ursolic acid as characteristic components, establishes seven characteristic peaks through an efficient detection method, and can relatively comprehensively reflect information on triterpenoid components in the persimmon calyx. The method is suitable for rapid quality control of persimmon calyx medicinal materials, decoction pieces, and intermediate and finished preparations containing persimmon calyx medicinal materials or decoction pieces. The method is efficient, easy to operate, and suitable for quality monitoring in continuous production.

Description

Technical Field

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

[0002] Persimmon calyx is a traditional Chinese medicine, documented as a medicinal herb as early as the Tang Dynasty. It has significant therapeutic effects in relieving nausea and vomiting, and is often used clinically with cloves. It is derived from the dried calyx of the plant Diospyros kaki Thunb. (Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China (Part I) [S]. Beijing: China Medical Science and Technology Press, 2020: 261-262.). The persimmon tree is cultivated in China and many other parts of the world. It contains a variety of chemical components, such as triterpenes, phenolic acids, and tannins, which have various pharmacological effects, including cardiovascular protection, antioxidant activity, and DNA damage protection (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 Sciences, 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 triterpenoid acids in persimmon calyx (Ren Lei, Feng Haiyan, Yang Xiaohui, et al. Determination of total triterpenoid acids in persimmon calyx by spectrophotometry [J]. Journal of Shijiazhuang University, 2014, 16(6): 28-30.). Characteristic fingerprints can better reflect the chemical composition information of traditional Chinese medicine and have 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, with only two papers. For example, Zhou Benhong et al. established the HPLC fingerprint of persimmon calyx (Zhou Benhong, Shen Heng, Wei Yan, et al. Study on the HPLC fingerprint of persimmon calyx [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, with common peaks of organic acids (such as gallic acid, protocatechuic acid) and flavonoids (such as hyperoside, isoquercetin, quercetin, kaempferol, luteolin, etc.). However, there is no report on the characteristic fingerprint of triterpenoid components. The content of this type of components is high and has 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, thereby strengthening the quality control of persimmon calyx and providing 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 the powder of persimmon calyx medicinal material, add the extraction solvent, extract after quantitative extraction, let 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, add solvent to prepare reference substance solution;

[0008] (3) The test solution and the reference solution were injected into 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 peak 1, peak 2, peak 3 and peak 7 were calculated. The relative retention times were within the range of ±10% of the specified values, and the specified values ​​of peak 1, peak 2, peak 3 and peak 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] 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 specifically 70%, 75%, 80%, 85%, 90%, 95%, and 100%; the ethanol volume fraction of the ethanol-water solution can be specifically 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%; considering that ethanol is non-toxic and has no pollution to the environment, the ethanol volume fraction can preferably be 80% to 95% ethanol.

[0013] The extraction method in step (1) includes ultrasonic treatment, heating under 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] The extraction time in step (1) is 15 to 60 minutes, specifically 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes or 60 minutes, which has little effect on the present invention. In some preferred embodiments, the extraction time is 30 minutes.

[0015] The mass-to-volume ratio of persimmon calyx medicinal material and decoction pieces powder to the extraction solvent is 1g:25mL. Too low a concentration of the test sample will hinder peak detection, while too high a concentration will overload the column and result in poor peak shape. For intermediates and finished products containing persimmon calyx medicinal material or decoction pieces, the mass-to-volume ratio of the test sample to the extraction solvent can be adjusted based on the proportion of persimmon calyx.

[0016] Wherein, the solvent in step (2) is alcohol or alcohol-water solution, wherein the methanol volume fraction of the methanol-water solution is not less than 70%, specifically 70%, 75%, 80%, 85%, 90%, 95%, or 100%; the ethanol volume fraction of the ethanol-water solution is not less than 50%, specifically 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%; 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 chromatography in step (3) includes ZORBAX SB-C18 RRHD, Poroshell HPH-C18 or Poroshell 120EC-C18.

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

[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] Extract 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 ultimately establishes a characteristic spectrum determination method for triterpenoid components such as oleanolic acid, ursolic acid, and betulinic acid in persimmon calyx samples, providing a new analytical method for quality testing 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 rational 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 profiles of the elution gradients in Tables 2 to 5 in Example 1 are: 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-5 mmol / L ammonium formate aqueous solution as the mobile phase, and D: methanol-5 mmol / L ammonium acetate aqueous solution as the mobile phase;

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

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

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

[0036] Figure 6This is an overlay of characteristic spectra of multiple batches of persimmon calyx samples in Example 3;

[0037] Figure 7 This 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] Figure 9 This is the liquid phase spectrum of the extraction method in Example 2;

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

[0041] Figure 11 This is the liquid phase spectrum observed at different chromatographic column temperatures in Example 1;

[0042] Figure 12 This is the liquid phase spectrum of different flow rates in Example 1;

[0043] Figure 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 will be further described below with reference to 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 and methanol (chromatographic grade, Thermo Fisher); formic acid, ammonium formate, and ammonium acetate (chromatographic grade, Aladdin); methanol and ethanol (analytical grade, Sinopharm Chemical Reagent Co., Ltd.).

[0047] Betulinic acid (batch number 15286, purity ≥97.2%) was purchased from Shanghai Shidande 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 the 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 kaki Thunb., a plant of the Ebenaceae family.

[0049] Example 1: Detection of the Characteristic Spectrum of Persimmon Calyx

[0050] 1. Preparation of test solution

[0051] Take about 1 g of persimmon calyx powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 95% ethanol, stopper it, weigh it, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, 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 amounts of betulinic acid reference substance, oleanolic acid reference substance and ursolic acid reference substance, add 95% ethanol to prepare 1 ml of a solution containing 190 μg betulinic acid, 70 μg oleanolic acid and 235 μg ursolic acid, which is used as the reference substance solution.

[0054] 3. Establishment of Chromatographic Analysis Conditions

[0055] 3.1 An Infinity Lab Poroshell 120Aq-C18 column (2.1 × 150 mm, 2.7 μm) was used with acetonitrile as mobile phase A and 0.1% formic acid in water as mobile phase B, following the gradient specified in Table 1. The flow rate was 0.30 mL / min and the column temperature was 35°C. Detection was performed using an electrospray ionization detector.

[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 column for this type of chromatographic peak. Therefore, a ZORBAX SB-C18RRHD (Agilent, 2.1×150 mm, 1.8 μm) column was replaced, and 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 column (Agilent, 2.1 × 150 mm, 1.8 μm) was used with a flow rate of 0.30 mL / min and a column temperature of 25°C. Detection was performed using an electrospray detector. Four mobile phase systems, methanol-0.1% formic acid aqueous solution, methanol:acetonitrile (9:1)-0.1% formic acid aqueous solution, methanol-5 mmol / L ammonium formate aqueous solution, and methanol-5 mmol / 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 showed that the separation effect of each chromatographic peak using methanol-5mmol / L ammonium acetate aqueous solution system was relatively good. Figure 2 However, the elution time was long and the chromatographic peak distribution was dispersed, so the elution gradient and column temperature were further optimized.

[0070] 3.3 After several optimizations, the chromatographic time was shortened appropriately, 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 column (Agilent, 2.1×150 mm, 1.8 μm) 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 shown in Table 6. Detection was performed using an electrospray detector.

[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, and detect according to the chromatographic conditions under "3.3" of this example. Accurately draw 3 μL of the test solution, respectively use evaporative light scattering detector (ELSD) and electrospray detector (CAD) for sample injection 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 one 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 at 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 effect of the seven characteristic peaks in the range of 25°C to 35°C is good, and the column temperature changes within this range can meet the system suitability requirements. Figure 11 .

[0078] 3.6 Flow rate investigation

[0079] Take one 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 effect of the seven characteristic peaks is 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. Figure 12 .

[0080] 3.7 Chromatographic column inspection

[0081] Take one 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 results of three 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, and this method has good durability for different chromatographic columns. See Figure 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) 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 shown in Table 6. The flow rate was 0.30 mL / min, the column temperature was 30°C, and detection was performed using an electrospray ionization detector.

[0084] Example 2: Investigation of the Preparation Method of the Test Solution of the Persimmon Calyx Medicinal Material Characteristic Spectrum

[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 25 mL, respectively, plug, weigh, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let cool, weigh again, make up for the weight loss, shake well, filter, take the filtrate, accurately draw 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 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, which had a low extraction efficiency, the chromatograms of the test sample solutions prepared with the other five extraction solvents all showed seven characteristic peaks, and all of them could be used as extraction solvents for the characteristic spectrum of persimmon calyx.

[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 and extract for 30 minutes, let cool, weigh again, make up for the weight loss, shake well, filter, take the filtrate, accurately draw 3 μL of each test solution, and measure according to the method under "3.3" of Example 1 to calculate the extraction efficiency. The results are shown in Table 8 and Figure 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 seven characteristic peaks, and there was no significant difference in extraction efficiency. All three methods can be used to prepare test solutions with characteristic chromatograms of persimmon calyx. Considering the ease of operation, ultrasonic treatment was the preferred 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, and ultrasonically treat (power 600 W, frequency 40 kHz) for 15 minutes, 30 minutes, 45 minutes, and 60 minutes, respectively, let cool, weigh again, make up for the weight loss, shake well, filter, take the filtrate, accurately draw 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 Figure 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 the test sample with the characteristic spectrum 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, place it in a stoppered conical flask, accurately add 25 mL of 95% ethanol, seal it, weigh it, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, 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

[0104] 1. Preparation of test solution:

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

[0106] 2. Preparation of reference substance solution:

[0107] Take appropriate amounts of betulinic acid reference substance, oleanolic acid reference substance and ursolic acid reference substance, add 95% ethanol to prepare 1 mL of a solution containing 190 μg betulinic acid, 70 μg oleanolic acid and 235 μg ursolic acid, which is used as the 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 chromatography instrument, 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 maps

[0112] A ZORBAX SB-C18 RRHD column (Agilent, 2.1×150 mm, 1.8 μm) was used with methanol as mobile phase A and 5 mmol / L aqueous ammonium acetate as mobile phase B, following the gradient shown in Table 6. The flow rate was 0.30 mL / min, the column temperature was 30°C, and detection was performed using an electrospray ionization detector. Chromatograms of 20 batches of persimmon calyx medicinal materials were recorded. The 20 batches of persimmon calyx medicinal materials chromatograms were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" and peak matching was performed using the median method with a time window of 0.1 s. Multi-point calibration was performed, and a total of 7 relatively obvious common peaks were identified, as shown in Figure 6. 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 peaks 1, 2, 3, and 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 one sample of persimmon calyx medicinal material (number S2) and prepare the test solution according to the test solution confirmed in Example 2. Accurately pipette 3 μL and inject 6 times continuously. Measure under the optimal chromatographic conditions and record the chromatogram. Peaks 4, 5, and 6 should correspond to the retention times of the betulinic acid, oleanolic acid, and ursolic acid reference peaks, respectively; the peak corresponding to the oleanolic acid reference peak is the S peak. Calculate the relative retention time and relative peak area of ​​the remaining characteristic peaks.

[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) and prepare the test solution according to the method confirmed in Example 2. Accurately pipette 3 μL of each solution, measure under the optimal chromatographic conditions, and record the chromatogram. Peaks 4, 5, and 6 should correspond to the retention times of the betulinic acid, oleanolic acid, and ursolic acid reference substance peaks, respectively; the peak corresponding to the oleanolic acid reference substance peak is the S peak. Calculate the relative retention times and relative peak areas 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 were less than 1%, and the relative peak area RSD values ​​were less than 3%, indicating that the method had good repeatability.

[0131] 3. Stability test

[0132] Take one sample of persimmon calyx medicinal material (number S2), prepare a 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 of sample for measurement, and record the chromatogram. Peaks 4, 5, and 6 should correspond to the retention times of the betulinic acid, oleanolic acid, and ursolic acid reference substance peaks, respectively; the peak corresponding to the oleanolic acid reference substance peak is the 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

[0139] 1. Preparation of the sample solution to be tested

[0140] Take about 1 g of persimmon calyx powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 95% ethanol, stopper it, weigh it, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, 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 chromatography instrument, and measure it according to the optimal chromatographic conditions.

[0143] By comparing the sample to be tested with Figure 6 The reference characteristic spectrum of the medicinal material persimmon calyx shows 7 characteristic peaks. Among them, peak 4, peak 5, and peak 6 should correspond to the retention times 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. The calculated relative retention times of peaks 1, 2, 3, and 7 are 0.27, 0.41, 0.45, and 1.22, respectively, all of which are within the ±10% range 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). It can be considered that the quality of the sample to be tested is stable, 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 the powder of the medicinal material of persimmon calyx, add the extraction solvent, extract after quantitative extraction, let it cool, make up the lost weight with the same solvent, shake it well, filter it, and take the filtrate to obtain the test solution; the extraction solvent is 70%~100% methanol or 50%~95% ethanol; (2) Take betulinic acid reference substance, oleanolic acid reference substance and ursolic acid reference substance, add solvent to prepare reference substance solution; (3) Take the test solution and the reference solution and inject them into the ultra-high performance liquid chromatography instrument respectively to obtain the characteristic spectrum; The characteristic spectrum has 7 common peaks. Taking betulic acid as a reference substance, peak 4 is the chromatographic peak of betulic acid; taking oleanolic acid as a reference substance, peak 5 is the chromatographic peak of oleanolic acid; taking ursolic acid as a reference substance, 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. 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; the chromatographic column of the ultra-high performance liquid chromatograph comprises ZORBAX SB-C18 RRHD, Poroshell HPH-C18 or Poroshell 120 EC-C18; the mobile phase of the ultra-high performance liquid chromatograph is methanol-5mmol / L ammonium acetate aqueous solution; the flow rate is 0.28ml / min~0.32ml / min; the ultra-high performance liquid chromatograph adopts a charged aerosol detector for detection; the elution gradient of the ultra-high performance liquid chromatograph is 0~2min, the volume fraction of mobile phase A is 70%, and the volume fraction of mobile phase B is 30%; 2~15min, the volume change fraction of mobile phase A is 70→77%, and the volume change fraction of mobile phase B is 30→23%; 15~18min, the volume change fraction of mobile phase A is 77→84%, and the volume change fraction of mobile phase B is 23→16%; 18~35min, the volume fraction of mobile phase A is 84%, and the volume fraction of mobile phase B is 16%.

2. 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.

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

4. A method for constructing a persimmon calyx reference map, characterized in that: The following steps are involved: (1) Prepare multiple batches of persimmon calyx test sample solutions; (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 3 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, making up the lost weight with the same solvent, shaking, filtering, and taking a filtrate to obtain the test solution; (3) The obtained characteristic spectrum was imported into the Chinese medicine chromatographic fingerprint similarity evaluation system to establish a reference spectrum of persimmon calyx.

5. Use of the construction method according to any one of claims 1 to 4 in identifying persimmon calyx or controlling its quality.

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