Method for detecting content of vanillic acid-4-O-beta-D-glucoside in fructus broussonetiae sample and application of method

Abstract

The invention discloses a method for detecting the content of vanillic acid-4-O-beta-D-glucoside in a fructus broussonetiae sample and application of the method. A test solution and a reference solution are prepared respectively and injected into an ultra-high performance liquid chromatograph, and specific chromatograms of the reference solution and the test solution are compared; and calculating the content of the vanillic acid-4-O-beta-D-glucoside in the fructus broussonetiae sample by utilizing a regression equation of the sample size and the peak area of the vanillic acid-4-O-beta-D-glucoside. The method disclosed by the invention is good in specificity and durability, and a new analysis means can be provided for quality detection of the fructus broussonetiae sample; the specific component of vanillic acid-4-O-beta-D-glucoside is taken as an index, so that the method is more reasonable and accurate; the application range comprises detection of fructus broussonetiae medicinal materials, standard decoction, formula granules and other products containing fructus broussonetiae; the method is simple to operate, good in reproducibility and stability, reliable in recovery rate, low in detection cost, high in detection efficiency and small in environmental pollution.

Description

Technical Field

[0001] The invention relates to a method for detecting the content of vanillic acid-4-O-β-D-glucoside in a paper mulberry fruit sample and application thereof, belonging to the technical field of drug analysis. Background Art

[0002] Paper mulberry seeds are the dried mature fruits of Broussonetia papyrifera (L.) Vent., a plant of the Moraceae family. They are harvested in autumn when the fruits are ripe. They are cold in nature, sweet in taste, and enter the liver and kidney meridians. They have the effects of tonifying the kidney and clearing the liver, improving eyesight, and promoting diuresis. They are used for liver and kidney deficiency, soreness of the waist and knees, asthenia, dizziness, pannus, and edema. Modern research shows that paper mulberry seeds contain chemical components such as fatty oils, amino acids, pigments, and polysaccharides, and have antioxidant, lipid-lowering, immune-enhancing, anti-tumor, and liver-protecting effects (Jiang Qianqian, Liang Shaoyu, Li Zhongqiu, et al. Resource distribution, ancient and modern medicinal uses, chemical components, and pharmacological effects of paper mulberry fruits - Paper mulberry seeds [J]. Journal of Henan University of Science and Technology (Medical Edition), 2018, 36(03): 236-240.).

[0003] The main quality research on Cuscuta australis in the existing technology includes: The current "Chinese Pharmacopoeia" 2020 edition only includes properties, identification, inspection and extraction items under the Cuscuta australis medicinal material decoction pieces, and there are no content determination indicators. Liu Hongwei et al. determined the fat oil yield, water content, ash content, and extract content of Cuscuta australis (Liu Hongwei, Huang Yuanshuai, Xiong Jun, et al. Preliminary study on the quality standard of wild Cuscuta australis from different origins in Hunan [J]. World Composite Medicine, 2016, 2(02): 54-56.); Pang Suqiu et al. determined the total alkaloid content in Cuscuta australis by colorimetry (Pang Suqiu, Wang Guoquan, Huang Baokang, et al. Comparison of alkaloid content in Cuscuta australis from different origins [J]. Journal of Pharmaceutical Practice, 2010, 28(03): 223-224.); Xiong Shan et al. determined the total saponin content in Cuscuta australis by colorimetry (Xiong Shan, Chen Yuwu, Ye Zuguang. Determination of total saponin content in Cucurbita chinensis seeds [J]. Modern Chinese Medicine, 2009, 11(07): 26-27+44.); Chi Yuxin et al. used colorimetry to determine the total flavonoids content in Cucurbita chinensis seeds (Chi Yuxin, Dou Deqiang. Determination of total flavonoids content in Cucurbita chinensis seeds and Broussonetia scoparia leaves [J]. Modern Chinese Medicine, 2008, 10(11): 16-17.); Huang Baokang et al. analyzed the amino acids and fatty acids in Cucurbita chinensis seeds (Huang Baokang, Qin Luping, Zheng Hanchen, et al. Analysis of amino acid and fatty oil components in Cucurbita chinensis seeds [J]. Journal of Second Military Medical University, 2003(02): 213-217+227.). The patent (Zhang Zhiqiang, Shi Yuzhe, Meng Qinde, et al. A quality control method for paper mulberry seed formula granules and its application [P]. CN115561378A, 2023.01.03.) determines the linoleic acid content in paper mulberry seed formula granules. The linoleic acid content determination indicator is distributed in many plants and has low specificity.

[0004] The existing technical methods for quality control of Cuscuta australis have unclear components to be measured, cumbersome measurement methods with poor accuracy, and only control of medicinal materials. There is an urgent need for a quality control method with good specificity that can be applied to the entire process from medicinal materials to preparations. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for detecting the content of vanillic acid-4-O-β-D-glucoside in a paper mulberry seed sample with strong specificity and its application.

[0006] Technical solution: To solve the above technical problems, the present invention provides a method for detecting the content of vanillic acid-4-O-β-D-glucoside in a paper mulberry fruit sample, comprising the following steps:

[0007] (1) Take the powder of the seed of Corydalis chinensis sample, add 15-100 ml of the extraction solvent, seal it, weigh it, extract it, weigh it again, make up the lost weight with 10%-50% methanol, shake it well, filter it, and take the filtrate to obtain the test solution;

[0008] (2) Take an appropriate amount of vanillic acid-4-O-β-D-glucoside reference substance, accurately weigh it, and add 10% methanol to obtain the reference substance solution;

[0009] (3) Injecting the reference solution and the test solution into an ultra-high performance liquid chromatograph respectively, and comparing the characteristic spectra of the reference solution and the test solution;

[0010] (4) Using the regression equation of the injection volume and peak area of ​​vanillic acid-4-O-β-D-glucoside, Y=8434.6543X-5203.9643, R 2 =0.99990, calculate the content of vanillic acid-4-O-β-D-glucoside in the paper mulberry seed sample.

[0011] The Cuscuta australis sample is a Cuscuta australis medicinal material, a standard decoction, an extract, a formula granule, or a reference sample, a preparation intermediate or a finished product of a classic formula containing Cuscuta australis.

[0012] Wherein, the extraction method in step (1) includes ultrasonic treatment, heating reflux or shaking extraction.

[0013] Wherein, the extraction time in step (1) is 45-90 minutes.

[0014] Wherein, the extraction time in step (1) is 15-60min.

[0015] Wherein, the extraction solvent is alcohol or alcohol aqueous solution.

[0016] Preferably, the extraction solvent is methanol or methanol-water solution;

[0017] Preferably, the concentration of the methanol aqueous solution is less than or equal to 50% by volume of methanol.

[0018] Preferably, the extraction solvent is 10%-50% methanol.

[0019] Wherein, the chromatographic conditions in step (3) are: flow rate 0.20 ml / min to 0.30 ml / min, column temperature 25°C to 35°C, chromatographic column is BEH Sheild RP18, mobile phase A is acetonitrile or methanol, mobile phase B is phosphoric acid aqueous solution, acetic acid aqueous solution, formic acid aqueous solution or water, and elution mode is gradient elution or isocratic elution.

[0020] Preferably, the mobile phase A is methanol, and the mobile phase B is a phosphoric acid aqueous solution having a concentration of 0.1% by volume of phosphoric acid.

[0021] Preferably, the volume ratio of mobile phase A to mobile phase B is 4:96 to 50:50; the flow rate is 0.20-0.30 mL / min; the column temperature is 25-35°C; and an ultraviolet detector, evaporative light scattering detector, differential refractometer detector or charged aerosol detector is used.

[0022] Preferably, the detector is an ultraviolet detector, and the detection wavelength is 200nm to 300nm, preferably 254nm.

[0023] The mass volume ratio of the paper mulberry seed sample powder to 10%-50% methanol is 1g:(50-330mL).

[0024] The present invention also provides a method for recovering vanillic acid-4-O-β-D-glucoside from paper mulberry seeds, comprising the following steps:

[0025] (1) Take the powder of the seed of paper mulberry, add 15-100 ml of 10%-50% methanol, seal it, weigh it, extract it for 15-60 minutes, weigh it again, make up the lost weight with 10%-50% methanol, shake it well, filter it, and take the filtrate to obtain the test solution; the extraction method includes ultrasonic treatment, heating reflux or shaking extraction;

[0026] (2) The sample solution is injected into an ultra-high performance liquid chromatograph to recover vanillic acid-4-O-β-D-glucoside.

[0027] The Cuscuta australis sample is a Cuscuta australis medicinal material, a standard decoction, an extract, a formula granule, or a reference sample, a preparation intermediate or a finished product of a classic formula containing Cuscuta australis.

[0028] Wherein, the chromatographic conditions in step (2) are: flow rate 0.20 ml / min to 0.30 ml / min, column temperature 25°C to 35°C, chromatographic column BEH Sheild RP18, mobile phase A is acetonitrile or methanol, mobile phase B is phosphoric acid aqueous solution, acetic acid aqueous solution, formic acid aqueous solution or water, elution mode is gradient elution or isocratic elution, and detection wavelength is 254 nm.

[0029] The present invention also provides application of the method in quality control or quality detection of paper mulberry seed samples.

[0030] The invention establishes a quality control method for a paper mulberry seed sample, and specifically relates to a method for determining the content of vanillic acid 4-O-β-D-glucoside in the paper mulberry seed.

[0031] Vanillic acid-4-O-β-D-glucoside is a flavonoid glycoside component with a molecular formula of C 14 H 18 O 9 , and has good antioxidant (Yu H, Yang G, Sato M, et al. Antioxidant activities of aqueous extract from Stevia rebaudiana stem waste to inhibit fish oil oxidation and identificationof its phenolic compounds[J]. Food chemistry, 2017, 232: 379-386.)(Chemam Y, Benayache S, Marchioni E, et al. On-line screening, isolation and identificationof antioxidant compounds of Helianthemum ruficomum[J]. Molecules, 2017, 22(2): 239.), anti-inflammatory (Jung CJ, Park SM, Lee DG, et al. Adenophora Stricta Root ExtractAlleviates Airway Inflammation in Mice with Ovalbumin-Induced Allergic Asthma[J]. Antioxidants, 2023, 12(4): 922.) and other pharmacological activities, which are close to the pharmacological effects of Cucurbita chinensis. At the same time, there is no research and application of vanillic acid-4-O-β-D-glucoside as a content determination indicator in the quality control of Cuscuta australis seeds.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention establishes a method for determining the content of vanillic acid-4-O-β-D-glucoside in Cucurbita chinensis samples by ultra-high performance liquid chromatography and reasonably setting the chromatographic conditions. The method has good specificity and durability, and can provide a new analytical means for the quality detection of Cucurbita chinensis samples; 2. The method of the present invention uses the specific component of vanillic acid-4-O-β-D-glucoside as an indicator, rather than the sum of a certain type of components as a quality control indicator, which is more reasonable and accurate; 3. The scope of application of the method of the present invention is not only limited to Cucurbita chinensis medicinal materials, but also extends to the detection of standard decoctions, formula granules and other products containing Cucurbita chinensis; 4. The method of the present invention is simple to operate, has good reproducibility and stability, reliable recovery rate, low detection cost, high detection efficiency and low environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the ultraviolet absorption spectrum of the vanillic acid-4-O-β-D-glucoside reference substance solution;

[0034] Figure 2 The ultra-high performance liquid chromatograms of the formula granules of Cuscuta australis extracted with different solvents;

[0035] Figure 3 The ULTRA PERFORMANCE LIQUID CHROMATOGRAPHIC GRAIN EXTRACTED BY DIFFERENT WAYS;

[0036] Figure 4 The ULTRA PERFORMANCE LIQUID CHROMATOGRAPHIC GRAIN EXTRACTED WITH DIFFERENT VOLUME;

[0037] Figure 5 The ultra-high performance liquid chromatograms of the Cuscuta australis granules extracted at different times;

[0038] Figure 6 This is the linear relationship between the injection volume and the peak area value of the vanillic acid-4-O-β-D-glucoside reference substance in ultra-high performance liquid chromatography;

[0039] Figure 7 The ultra-high performance liquid chromatograms of the results of investigation at different flow rates;

[0040] Figure 8 The ultra-high performance liquid chromatograms of the investigation results at different temperatures;

[0041] Fig. 9 Ultra-high performance liquid chromatograms showing the results of investigations on different chromatographic columns;

[0042] Fig.10 This is the ultra-high performance liquid chromatogram of the specificity experimental results of the content determination of the formula granules of Cuscuta australis;

[0043] Fig.11This is the ultra-high performance liquid chromatogram of the specificity test results of the content determination of the standard decoction of Cuscuta australis;

[0044] Fig.12 This is the ultra-high performance liquid chromatogram of the specificity experimental results for the determination of the content of Cuscuta australis medicinal materials. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0046] Instruments, reagents and samples:

[0047] Waters Acquity UPLC ultra-high performance liquid chromatograph (Waters); Empower 3 workstation (Waters); Thermo Vanquish Flex ultra-high performance liquid chromatograph; Chromeleam 7.2SR4 Chameleon workstation; Agilent Technologies 1290 Infinity ultra-high performance liquid chromatograph; 1290 DAD diode array detector; 1290 MCT column oven; 1290 Vialsampler autosampler; 1290 Fiexible pump quaternary pump; Open LAB CDS2.3 chromatography workstation; electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); KQ-250B ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.); pure water system (Sartorius); AS165W centrifuge (Azov (Shanghai) Trading Co., Ltd.). Methanol (chromatographic grade, ThermoFisher); water is ultrapure water; phosphoric acid (chromatographic grade, Aladdin); other reagents are of analytical grade.

[0048] Vanillic acid-4-O-β-D-glucoside reference substance was purchased from Chengdu Pusi Biotechnology Co., Ltd. with batch number PS012936.

[0049] Cuscuta australis medicinal materials (YC1), Cuscuta australis standard decoction (DG1), Cuscuta australis formula granules (KL1, KL2, KL3) were all provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0050] Example 1: Construction of a method for determining the content of mulberry seed formula granules

[0051] 1. Preparation of reference solution

[0052] Take an appropriate amount of vanillic acid-4-O-β-D-glucoside reference substance, weigh it accurately, and add 10% methanol to make a solution containing 10 μg per 1 ml.

[0053] 2. Chromatographic conditions

[0054] (1) Determination of detection wavelength

[0055] The vanillic acid-4-O-β-D-glucoside reference solution was scanned at a wavelength of 190 to 400 nm and its UV absorption graph was recorded. The results are shown in Figure 1 The results showed that the vanillic acid-4-O-β-D-glucoside reference solution had strong absorption in the range of 200nm to 300nm and could be used for detection. Considering that there was an obvious absorption peak around 254nm, the detection wavelength could be preferably 254nm.

[0056] (2) Determination of chromatographic conditions

[0057] BEH Sheild RP18 (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.7 μm) was used as the chromatographic column; methanol-0.1% phosphoric acid solution (4:96) was used as the mobile phase; the detection wavelength was 254 nm; the flow rate was 0.25 ml / min; the column temperature was 30° C., and the elution was isocratic.

[0058] 3 Preparation of test solution

[0059] (1) Investigation of extraction solvent

[0060] Take an appropriate amount of Cuscuta chinensis formula granules (batch number: KL1), grind them into powder, take about 0.3g, and make 5 groups in total, with 2 parallel portions in each group, accurately weigh, accurately add 10% methanol, 30% methanol, 50% methanol, 70% methanol and 25ml of methanol respectively, seal, weigh, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and take the filtrate. Accurately take 2μl of each test solution, inject it into the ultra-high performance liquid chromatograph, and measure it according to the above chromatographic conditions, record the peak area value of vanillic acid-4-O-β-D-glucoside, calculate its content and RSD value, and the results are shown in Table 1. Figure 2 .

[0061] Table 1 Comparison of different extraction solvents

[0062]

[0063] The results show that the content is low and the peak shape is poor when 70% methanol and methanol are used as extraction solvents, and the extraction efficiency is close when 50% methanol, 30% methanol and 10% methanol are used. Considering the reduction of the use of organic solvents, the extraction solvent may preferably be 10% methanol.

[0064] (2) Investigation of different extraction methods

[0065] Take an appropriate amount of the formula granules of Cuscuta chinensis (batch number: KL1), grind them into powder, take about 0.3g, and make 3 groups in total, with 2 parallel portions in each group, accurately weigh them, put them in a stoppered conical bottle, accurately add 25ml of 10% methanol, seal them, weigh them, ultrasonically treat them (power 250W, frequency 40kHz), heat to reflux, and shake to extract for 30 minutes, let them cool, weigh them again, make up the lost weight with 10% methanol, shake them well, filter them, and take the filtrate. Accurately take 2μl of each test solution, inject them into the ultra-high performance liquid chromatograph, and determine them according to the above chromatographic conditions. Record the peak area value of vanillic acid-4-O-β-D-glucoside, calculate its content and RSD value, and the results are shown in Table 2. Figure 3 .

[0066] Table 2 Comparison of different extraction methods

[0067]

[0068]

[0069] The results show that the contents of vanillic acid-4-O-β-D-glucoside extracted by the three extraction methods are similar. Considering that the operation of ultrasonic treatment is relatively simple, ultrasonic treatment is the preferred extraction method.

[0070] (3) Investigation of different extraction volumes

[0071] Take an appropriate amount of Cuscuta chinensis formula granules (batch number: KL1), grind them into powder, take about 0.3g, and make 4 groups in total, with 2 parallel portions in each group, accurately weigh, accurately add 10% methanol 15ml, 25ml, 50ml, 100ml respectively, seal, weigh, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, let cool, weigh again, make up the lost weight with 10% methanol, shake well, filter, and take the filtrate. Accurately take 2μl of each test solution, inject it into the ultra-high performance liquid chromatograph, and measure it according to the above chromatographic conditions, record the peak area value of vanillic acid-4-O-β-D-glucoside, calculate its content and RSD value, and the results are shown in Table 3. Figure 4 .

[0072] Table 3 Comparison of different extraction volumes

[0073]

[0074]

[0075] The results show that the contents of vanillic acid-4-O-β-D-glucoside extracted with different amounts of solvent are similar. Considering the comprehensive extraction and solvent saving, the preferred amount of solvent is 25 ml.

[0076] (4) Investigation of different extraction times

[0077] Take an appropriate amount of Cuscuta chinensis formula granules (batch number: KL1), grind them into powder, take about 0.3g, and make 4 groups in total, with 2 parallel portions in each group, accurately weigh them, put them in a stoppered conical bottle, accurately add 25ml of 10% methanol, seal them tightly, weigh them, and ultrasonically treat them (power 250W, frequency 40kHz) for 15 minutes, 30 minutes, 45 minutes, and 60 minutes respectively, take them out, let them cool, weigh them again, make up the lost weight with 10% methanol, shake them well, filter them, and take the filtrate to obtain them. Accurately take 2μl of each test solution, inject them into the ultra-high performance liquid chromatograph, and determine them according to the above chromatographic conditions, record the peak area value of vanillic acid-4-O-β-D-glucoside, calculate its content and RSD value, and the results are shown in Table 4. Figure 5 .

[0078] Table 4 Comparison of different extraction times

[0079]

[0080] The results show that the extraction is relatively sufficient after 45 minutes of ultrasonic treatment. In order to ensure complete extraction and save time, the extraction time is preferably 45 minutes.

[0081] According to the above research results, the optimal method for preparing the test solution is:

[0082] Take an appropriate amount of paper mulberry seed granules, grind them into powder, take about 0.3g, weigh it accurately, put it in a stoppered conical flask, accurately add 25ml of 10% methanol, stopper it, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 45 minutes, cool it, weigh it again, make up the lost weight with 10% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0083] (5) Testing of different batches of mulberry seed granules

[0084] Take appropriate amount of samples of 3 batches of Cuscuta australis formula granules, with 2 parallel samples from each batch, prepare the test solution according to the above test solution preparation method, inject into ultra performance liquid chromatography, determine the peak area value of vanillic acid-4-O-β-D-glucoside according to the above chromatographic conditions, and calculate the content of vanillic acid-4-O-β-D-glucoside in Cuscuta australis formula granules using the correction factor. The results are shown in Table 5.

[0085] Table 5 Determination of the content of vanillic acid-4-O-β-D-glucoside in the formula granules of Cuscuta australis (correction factor method)

[0086]

[0087] The results showed that the content of vanillic acid-4-O-β-D-glucoside in different batches of Cucurbitacin mulberry seed granules was relatively consistent, and the RSD of the determination results of parallel samples from the same batch was less than 2%, indicating that the selection of vanillic acid-4-O-β-D-glucoside as an indicator in this method is feasible and has good repeatability.

[0088] Example 2: Methodological verification of the content determination method

[0089] 1. Linear relationship

[0090] 0.5 μl, 1.0 μl, 1.5 μl, 2.0 μl, 2.5 μl, 3.0 μl, 3.5 μl and 4.0 μl of vanillic acid-4-O-β-D-glucoside (0.0100548 mg / ml) reference substance were accurately drawn respectively and injected into ultra performance liquid chromatography. The standard curve was plotted with peak area (AU) as ordinate and injection volume (μg) as abscissa to obtain the regression equation: Y=8434.6543X-5203.9643, R 2 =0.99990, the results are shown in Table 6, Figure 6 .

[0091] Table 6 Relationship between peak area and injection volume of vanillic acid-4-O-β-D-glucoside reference substance

[0092]

[0093]

[0094] The results showed that the injection amount of vanillic acid-4-O-β-D-glucoside was in the range of 5.03μg to 40.22μg, and there was a good linear relationship between the injection amount and the peak area value.

[0095] 2. Precision test

[0096] Accurately pipette 2 μl of the Cuscuta australis test solution (batch number of Cuscuta australis formula granules: KL1), inject into ultra-high performance liquid chromatography, measure according to the above chromatographic conditions, inject 6 times continuously, record the peak area value of vanillic acid-4-O-β-D-glucoside, calculate the relative standard deviation, and the results are shown in Table 7.

[0097] Table 7 Instrument precision test

[0098]

[0099] The results showed that the RSD value of the peak area of ​​vanillic acid-4-O-β-D-glucoside was 0.16%, and the instrument precision was good.

[0100] 3. Stability test

[0101] Take 1 portion of Cuscuta chinensis sample to prepare the test solution (batch number of Cuscuta chinensis formula granules: KL1), inject 2 μl every 4 hours, record the peak area value of vanillic acid-4-O-β-D-glucoside, calculate its RSD, and measure for a total of 24 hours. The results are shown in Table 8.

[0102] Table 8 Stability test results

[0103]

[0104]

[0105] The results showed that the RSD value of the peak area of ​​vanillic acid-4-O-β-D-glucoside was 0.48%, and the test solution had good stability within 24 hours.

[0106] 4. Repeatability test

[0107] Take an appropriate amount of Cuscuta chinensis sample (batch number of Cuscuta chinensis formula granules: KL1), grind it into powder, take about 0.3 g, weigh it accurately, and make 6 parallel portions. Prepare sample test solutions according to the test solution preparation method, inject 2 μl of each portion, record the peak area value of vanillic acid-4-O-β-D-glucoside, calculate the content and RSD value of vanillic acid-4-O-β-D-glucoside, and the results are shown in Table 9.

[0108] Table 9 Repeatability test

[0109]

[0110] The results showed that the RSD value of the peak area of ​​vanillic acid-4-O-β-D-glucoside was 0.32%, and the repeatability test was good.

[0111] 5. Accuracy test

[0112] Take 0.15g of Cuscuta australis sample with known content (batch number of Cuscuta australis formula granules: KL1), accurately weigh, add 5ml, 10ml and 15ml of vanillic acid-4-O-β-D-glucoside (0.00907mg / ml) reference solution three times, prepare the sample recovery test solution according to the test solution preparation method, chromatographic conditions: BEH Sheild RP18 (column length 100mm, inner diameter 2.1mm, particle size 1.7μm) as the chromatographic column; methanol-0.1% phosphoric acid solution (4:96) as the mobile phase; detection wavelength 254nm; flow rate 0.25ml / min; column temperature 30℃, isocratic elution, inject 2μl respectively, calculate the recovery rate and RSD value by the following formula, the results are shown in Table 10.

[0113]

[0114] Table 10 Vanillic acid-4-O-β-D-glucoside accuracy test

[0115]

[0116] Results: The recovery rate of vanillic acid-4-O-β-D-glucoside in Cucurbitacin was 99.38%-103.87%, and the RSD value was 1.23%. The accuracy test was good.

[0117] 6. Intermediate precision test

[0118] A sample of Cuscuta australis (batch number of Cuscuta australis formula granules: KL1) was taken and prepared into sample test solutions by different experimenters according to the test solution preparation method. 2 μl of the sample was injected into instruments of different models at different times and tested according to the above-mentioned chromatographic conditions. The peak area value of vanillic acid-4-O-β-D-glucoside was recorded, and the content and RSD value of vanillic acid-4-O-β-D-glucoside were calculated. The results are shown in Table 11.

[0119] Table 11 Intermediate precision test

[0120]

[0121]

[0122] The results showed that the RSD value of vanillic acid-4-O-β-D-glucoside was 1.67% and the intermediate precision was good.

[0123] 7. Durability test

[0124] (1) Investigation of different flow rates

[0125] Take 1 sample (batch number of Cuscuta australis granules: KL1), prepare the test solution according to the preparation method of the test solution, and investigate the influence of three flow rates of 0.20ml / min, 0.25ml / min, and 0.30ml / min on the content determination. The results are shown in Table 12. Figure 7 . Among them, BEH Sheild RP18 (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.7 μm) was used as the chromatographic column; methanol-0.1% phosphoric acid solution (4:96) was used as the mobile phase; the detection wavelength was 254 nm; the column temperature was 30° C., and the elution was isocratic.

[0126] Table 12 Investigation of different flow rates

[0127]

[0128] The results showed that when the flow rate was within the range of 0.20 ml / min to 0.30 ml / min, the chromatographic peak separation effect of vanillic acid-4-O-β-D-glucoside met the requirements and the measured contents were basically consistent, indicating that a slight change in the flow rate had no significant effect on the determination of vanillic acid-4-O-β-D-glucoside content and had good durability.

[0129] (2) Investigation of different column temperatures

[0130] Take 1 sample (batch number of Cuscuta australis granules: KL1), prepare the test solution according to the preparation method of the test solution, and investigate the influence of three temperatures of 25℃, 30℃ and 35℃ on the content determination. The results are shown in Table 13. Figure 8 . Among them, BEHSheild RP18 (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.7 μm) was used as the chromatographic column; methanol-0.1% phosphoric acid solution (4:96) was used as the mobile phase; the detection wavelength was 254 nm; the flow rate was 0.25 ml / min, and the elution was isocratic.

[0131] Table 13 Investigation of different column temperatures

[0132]

[0133] The results showed that when the column temperature was in the range of 25℃~35℃, the separation effect of the chromatographic peak of vanillic acid-4-O-β-D-glucoside met the requirements and the measured contents were basically consistent, indicating that slight changes in column temperature had no significant effect on the determination of vanillic acid-4-O-β-D-glucoside content and had good durability.

[0134] (3) Investigation of different chromatographic columns

[0135] Take one sample (batch number of the mulberry seed granules: KL1), prepare the test solution according to the preparation method of the test solution, and use three chromatographic columns: BEH C18 (2.1×100mm, 1.7μm), BEH Sheild RP18 (2.1×100mm, 1.7μm), and Poroshell 120EC-C18 (2.1×100mm, 1.9μm) to analyze the mulberry seed granules. The results are shown in Table 14. Fig. 9 The mobile phase was methanol-0.1% phosphoric acid solution (4:96); the detection wavelength was 254 nm; the flow rate was 0.25 ml / min; the column temperature was 30°C, and the elution was isocratic.

[0136] Table 14 Investigation of different chromatographic columns

[0137]

[0138] The results showed that the separation of the chromatographic peak of vanillic acid-4-O-β-D-glucoside in the three chromatographic columns met the requirements, and there was no significant difference in the measured results, indicating that different chromatographic columns had little effect on the determination results of sample content and had better durability.

[0139] 8. Specificity test

[0140] Take 2 μl of the test solution of the above-mentioned Cuscuta australis formula granules (batch number: KL1), vanillic acid-4-O-β-D-glucoside reference solution and negative sample solution, inject them into the liquid chromatograph, and measure according to the above-mentioned chromatographic conditions (BEH Sheild RP18 (column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm) as the chromatographic column; methanol-0.1% phosphoric acid solution (4:96) as the mobile phase; detection wavelength 254 nm; flow rate 0.25 ml / min; column temperature 30°C, isocratic elution), record the chromatogram, and the results are shown in the table. Fig.10 Among them, negative samples are excipients and solvents.

[0141] The results showed that the excipients and solvents had no interference in the determination of vanillic acid-4-O-β-D-glucoside in the formula granules of Cuscuta australis. The method had strong specificity and was suitable for the content determination of Cuscuta australis formula granules.

[0142] Example 3. Specificity experiment for determining the content of vanillic acid-4-O-β-D-glucoside in standard decoction of Cucurbitacin Take about 0.3 g of standard decoction of Cucurbitacin (batch number: DG1), accurately weigh it, put it in a Soxhlet extractor, add an appropriate amount of petroleum ether (30-60° C.), heat and reflux for 2 hours, discard the petroleum ether liquid, evaporate the solvent from the residue, and move it into a stoppered conical flask together with the filter paper tube, accurately add 50 ml of 10% methanol, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 45 minutes, weigh it again, make up the lost weight with 10% methanol, shake it well, filter it, and take the filtrate to obtain the test solution of standard decoction of Cucurbitacin.

[0143] Take 2 μl of each of the test solution of the standard decoction of Cuscuta australis, the reference solution of vanillic acid-4-O-β-D-glucoside and the blank solvent, inject them into the ultra-high performance liquid chromatograph, measure according to the above chromatographic conditions, record the chromatogram, and the results are shown in the table. Fig.11 .

[0144] Results: The solvent had no interference on the determination of vanillic acid-4-O-β-D-glucoside in the standard decoction of Cuscuta australis. The method had strong specificity and was suitable for the content determination of the standard decoction of Cuscuta australis.

[0145] Example 4: Specificity experiment for determination of vanillic acid-4-O-β-D-glucoside content in Cuscuta australis

[0146] Take about 2g of Cuscuta australis medicinal material (batch number: YC1), accurately weigh it, put it in a Soxhlet extractor, add an appropriate amount of petroleum ether (30-60℃), heat and reflux for 2 hours, discard the petroleum ether liquid, evaporate the solvent from the residue, and transfer it together with the filter paper tube into a stoppered conical flask, accurately add 50ml of 10% methanol, weigh the weight, heat and reflux for 90 minutes, weigh it again, make up the lost weight with 10% methanol, shake well, filter, and take the filtrate to obtain the Cuscuta australis medicinal material test solution.

[0147] Take 2 μl of each of the test solution of Cuscuta australis, the reference solution of vanillic acid-4-O-β-D-glucoside and the blank solvent, inject them into the ultra-high performance liquid chromatograph, measure according to the above chromatographic conditions, record the chromatogram, and the results are shown in Fig.12 .

[0148] Results: The solvent had no interference on the determination of vanillic acid-4-O-β-D-glucoside in Cucurbitacin. The method had strong specificity and was suitable for the content determination of Cucurbitacin.

[0149] Embodiment 5

[0150] Take an appropriate amount of 3 batches of mulberry fruit formula granule samples, 2 copies of each batch, prepare the test solution according to the test solution preparation method in Example 1, inject into the ultra-high performance liquid chromatograph, and determine the content of vanillic acid-4-O-β-D-glucoside according to the above chromatographic conditions. The content of vanillic acid-4-O-β-D-glucoside in the mulberry fruit sample was calculated using the regression equation of the injection amount and peak area of ​​vanillic acid-4-O-β-D-glucoside in Example 2. The results are shown in Table 15.

[0151] Table 15 Determination of the content of vanillic acid-4-O-β-D-glucoside in the formula granules of Cuscuta australis (standard curve method)

[0152]

[0153]

[0154] The results showed that the results calculated by the standard curve method for the content of vanillic acid-4-O-β-D-glucoside in different batches of Cucurbitacin formula granules were not much different from those by the correction factor method, and the RSD of the determination results of parallel samples from the same batch was less than 2%, indicating that this method for determining the vanillic acid-4-O-β-D-glucoside index in Cucurbitacin samples was stable and feasible, with good repeatability and reliable results.

Claims

1. A method for detecting the content of vanillic acid-4-O-β-D-glucoside in a sample of paper mulberry fruit, characterized in that: The following steps are involved: (1) Take the powder of the seed of Corydalis chinensis sample, add 15-100 ml of 10%-50% methanol, seal it, weigh it, extract it, weigh it again, make up the lost weight with 10%-50% methanol, shake it well, filter it, and take the filtrate to obtain the test solution; (2) Take an appropriate amount of vanillic acid-4-O-β-D-glucoside reference substance, accurately weigh it, and add 10% methanol to obtain the reference substance solution; (3) Injecting the reference solution and the test solution into an ultra-high performance liquid chromatograph respectively, and comparing the characteristic spectra of the reference solution and the test solution; (4) Using the regression equation of the injection volume and peak area of ​​vanillic acid-4-O-β-D-glucoside, Y=8434.6543X-5203.9643, R 2 =0.99990, calculate the content of vanillic acid-4-O-β-D-glucoside in the paper mulberry seed sample.

2. The method according to claim 1, characterized in that: The Cuscuta australis sample is a Cuscuta australis medicinal material, a standard decoction, an extract, a formula granule, or a reference sample, a preparation intermediate product, or a finished product of a classic formula containing Cuscuta australis.

3. The method according to claim 1, characterized in that: The extraction method in step (1) includes ultrasonic treatment, heating reflux or shaking extraction.

4. The method according to claim 1, characterized in that: The extraction time in step (1) is 15-60 minutes.

5. The method according to claim 1, characterized in that: The chromatographic conditions in step (3) are: The flow rate is 0.20ml / min~0.30ml / min, the column temperature is 25℃~35℃, the chromatographic column is BEH Sheild RP18, the mobile phase is methanol-0.1% phosphoric acid solution, the elution mode is gradient elution or isocratic elution, and the detection wavelength is 254nm.

6. The method according to claim 1, characterized in that: The mass volume ratio of the paper mulberry seed sample powder to 10%-50% methanol is 1g:(50-330mL).

7. A method for recovering vanillic acid-4-O-β-D-glucoside from paper mulberry seeds, characterized in that: The following steps are involved: (1) Take the powder of the seed of paper mulberry, add 15-100 ml of 10%-50% methanol, seal it, weigh it, extract it for 15-60 minutes, weigh it again, make up the lost weight with 10%-50% methanol, shake it well, filter it, and take the filtrate to obtain the test solution; the extraction method includes ultrasonic treatment, heating reflux or shaking extraction; (2) The sample solution is injected into an ultra-high performance liquid chromatograph to recover vanillic acid-4-O-β-D-glucoside.

8. The method according to claim 7, characterized in that: The Cuscuta australis sample is a Cuscuta australis medicinal material, a standard decoction, an extract, a formula granule, or a reference sample, a preparation intermediate product, or a finished product of a classic formula containing Cuscuta australis.

9. The method according to claim 1, characterized in that: The chromatographic conditions in step (2) are: flow rate 0.20 ml / min-0.30 ml / min, column temperature 25° C.-35° C., chromatographic column BEH Sheild RP18, mobile phase methanol-0.1% phosphoric acid solution, elution mode gradient elution or isocratic elution, and detection wavelength 254 nm.

10. Use of the method according to any one of claims 1 to 6 in quality control or quality detection of paper mulberry seed samples.

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