Method for detecting content of vanillic acid-4-o-beta-d-glucoside in fructus jujube samples and application thereof

Abstract

The application discloses a method for detecting the content of vanillic acid-4-O-beta-D-glucoside in Fructus Broussonetiae samples and application thereof, and respectively prepares a test sample solution and a control sample solution, injects the solutions into an ultra-high performance liquid chromatograph, compares the characteristic maps of the control sample solution and the test sample solution, calculates the content of vanillic acid-4-O-beta-D-glucoside in the Fructus Broussonetiae sample by using a regression equation of the injection amount and the peak area of vanillic acid-4-O-beta-D-glucoside. The method has good specificity and durability, can provide a new analysis means for quality detection of the Fructus Broussonetiae samples, is more reasonable and accurate by taking vanillic acid-4-O-beta-D-glucoside as an index, is suitable for detection of Fructus Broussonetiae medicinal materials, standard decoctions, formula granules and other products containing the Fructus Broussonetiae, has simple operation, good reproducibility and stability, reliable recovery rate, low detection cost, high detection efficiency and small environmental pollution.

Description

TECHNICAL FIELD

[0001] The application relates to a method for detecting the content of vanillic acid-4-O-beta-D-glucoside in a fructus broussonetiae sample and application thereof, and belongs to the technical field of pharmaceutical analysis. BACKGROUND

[0002] Fructus broussonetiae is the dried mature fruit of Broussonetia papyrifera (L.) Vent. of Moraceae. The fruit is harvested in autumn when it is mature. Fructus broussonetiae is cold in nature and sweet in flavor, and is attributed to the liver and kidney channels. Fructus broussonetiae has the effects of tonifying the kidney and clearing the liver, improving eyesight, and diuresis, and is used for liver and kidney deficiency, soreness of waist and knees, asthenia and bone steaming, dizziness and dim vision, and nebula in the eyes and edema. Modern studies have shown that fructus broussonetiae contains fatty oil, amino acids, pigments, and polysaccharides and other chemical components, and has the effects of antioxidation, blood lipid reduction, immune enhancement, anti-tumor, and liver function protection (Jiang Qianqian, Liang Shaoyu, Li Zhongqiu, et al. Resources distribution, ancient and modern medicine, chemical composition and pharmacological action of Broussonetia papyrifera fruit-fructus broussonetiae [J]. Journal of Henan University of Science and Technology (Medical Edition), 2018, 36(03): 236-240.).

[0003] The quality research on Fructus Broussonetiae in the prior art mainly includes: under the item of Fructus Broussonetiae medicinal materials and decoction pieces in the current Chinese Pharmacopoeia 2020 edition, only the appearance, identification, inspection and extract are recorded, and no content determination index is included. Liu Hongwei et al. determined the fat oil yield, moisture, ash content and extract of Fructus Broussonetiae medicinal materials (Liu Hongwei, Huang Yuanshuai, Xiong Jun, et al. Preliminary study on quality standards of wild Fructus Broussonetiae from different regions in Hunan province [J]. World Composite Medicine, 2016, 2(02): 54-56.); Pang Suqiu et al. determined the total alkaloid content in Fructus Broussonetiae medicinal materials by colorimetry (Pang Suqiu, Wang Guquan, Huang Baokang, et al. Comparison of alkaloid content in Fructus Broussonetiae from different regions [J]. Journal of Pharmacy Practice, 2010, 28(03): 223-224.); Xiong Shan et al. determined the total saponin content in Fructus Broussonetiae medicinal materials by colorimetry (Xiong Shan, Chen Yuwu, Ye Zuguang. Determination of total saponin content in Fructus Broussonetiae [J]. Chinese Modern Chinese Medicine, 2009, 11(07): 26-27+44.); Chi Yuxin et al. determined the total flavonoid content in Fructus Broussonetiae medicinal materials by colorimetry (Chi Yuxin, Dou Deqiang. Determination of total flavonoids in Fructus Broussonetiae and Broussonetia papyrifera leaves [J]. Chinese Modern Chinese Medicine, 2008, 10(11): 16-17.); Huang Baokang et al. analyzed the amino acids and fatty acids in Fructus Broussonetiae medicinal materials (Huang Baokang, Qin Luping, Zheng Hanchen, et al. Analysis of amino acid and fatty oil components in Fructus Broussonetiae [J]. Journal of the Second Military Medical University, 2003(02): 213-217+227.); a patent (Zhang Zhiqiang, Shi Yuzhe, Meng Qinde, et al. Quality control method of Fructus Broussonetiae formula granules and its application [P]. CN115561378A, 2023.01.03.) determines the linoleic acid content in Fructus Broussonetiae formula granules, and the linoleic acid content determination index is distributed in many plants, and the specificity is not strong.

[0004] The prior art method for quality control of Fructus Broussonetiae is not clear enough in the tested components, the determination method is complicated and the accuracy is poor, and only the medicinal materials are controlled, so a quality control method with good specificity and applicable to the whole process from medicinal materials to preparations is urgently needed. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a method for detecting the content of vanillic acid-4-O-β-D-glucoside in Fructus Broussonetiae samples and its application.

[0006] Technical scheme: In order to solve the above technical problems, the present application provides a method for detecting the content of vanillic acid-4-O-β-D-glucoside in Fructus Broussonetiae samples, which comprises the following steps:

[0007] (1) take the sample powder of Fructus Broussonetiae, add extraction solvent 15-100 ml, seal tightly, weigh, extract, weigh again, make up the weight loss with 10%-50% methanol, shake well, filter, take the filtrate, and obtain the test solution;

[0008] (2) take vanillic acid-4-O-β-D-glucoside control substance, weigh accurately, add 10% methanol, and obtain the control solution;

[0009] (3) inject the control solution and the test solution into the ultra-high performance liquid chromatograph, and compare the characteristic chromatograms of the control solution and the test solution;

[0010] (4) use the regression equation Y=8434.6543X-5203.9643, R 2 =0.99990 of the injection amount and the peak area of vanillic acid-4-O-β-D-glucoside to calculate the content of vanillic acid-4-O-β-D-glucoside in the Fructus Broussonetiae sample.

[0011] Among them, the Fructus Broussonetiae sample is a medicinal material, a standard decoction, an extract, a formula granule, or a benchmark sample of a classic famous prescription containing Fructus Broussonetiae, an intermediate product or a finished product of a preparation.

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

[0013] Among them, the extraction time in step (1) is 45-90 min.

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

[0015] Among them, the extraction solvent is an alcohol or an alcohol aqueous solution.

[0016] Preferably, the extraction solvent is methanol or a methanol aqueous 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] Among them, the chromatographic conditions in step (3) are: flow rate 0.20 ml / min-0.30 ml / min, column temperature 25℃-35℃, chromatographic column BEH Sheild RP18, mobile phase A acetonitrile or methanol, mobile phase B phosphoric acid aqueous solution, acetic acid aqueous solution, formic acid aqueous solution or water, and elution mode gradient elution or isocratic elution.

[0020] Preferably, the mobile phase A is methanol, and the mobile phase B is an aqueous phosphoric acid solution with a concentration of 0.1% in terms of volume percentage of phosphoric acid.

[0021] Preferably, the volume ratio of the mobile phase A to the 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℃; and an ultraviolet detector, an evaporative light scattering detector, a differential refractometer detector or an electrospray detector is used.

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

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

[0024] The application also provides a method for recovering Vanillic acid 4-O-β-D-glucoside from Chinese walnut, comprising the following steps:

[0025] (1) A sample powder of Chinese walnut is taken, 10%-50% methanol 15-100ml is added, the bottle is tightly capped, the weight is determined, extraction is carried out for 15-60min, the weight is determined again, 10%-50% methanol is added to make up for the weight loss, the mixture is shaken and filtered, and the filtrate is taken as the test solution; the extraction mode includes ultrasonic treatment, heating reflux or shaking extraction;

[0026] (2) The test solution is injected into an ultra-high performance liquid chromatograph, and Vanillic acid 4-O-β-D-glucoside can be recovered.

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

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

[0029] The application also provides application of the method in quality control or quality detection of a sample of Chinese walnut.

[0030] The application establishes a quality control method for a sample of Chinese walnut, and particularly relates to a content determination method for Vanillic acid 4-O-β-D-glucoside in Chinese walnut.

[0031] Vanillic acid-4-O-beta-D-glucoside is a flavonoid glycoside component, and its molecular formula is C 14 H 18 O9, 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 identification of its phenolic compounds [J]. Food chemistry, 2017, 232: 379-386.)(Chemam Y, Benayache S, Marchioni E, et al. On-line screening, isolation and identification of antioxidant compounds of Helianthemum ruficomum [J]. Molecules, 2017, 22(2): 239.), anti-inflammatory (Jung C J, Park S M, Lee D G, et al. Adenophora Stricta Root Extract Alleviates Airway Inflammation in Mice with Ovalbumin-Induced Allergic Asthma [J]. Antioxidants, 2023, 12(4): 922.) and other pharmacological activities, which are similar to the pharmacological effects of Fructus Trema. At the same time, there is no research and application of vanillic acid-4-O-beta-D-glucoside as a content determination index in the quality control of Fructus Trema.

[0032] Beneficial effects: compared with the prior art, the present application has the following obvious advantages: 1. the present application establishes a content determination method of vanillic acid-4-O-beta-D-glucoside in Fructus Trema sample by ultra-high performance liquid chromatography, and reasonably sets the chromatographic conditions, which has good specificity and durability, and can provide a new analysis means for the quality detection of Fructus Trema sample; 2. the method of the present application takes vanillic acid-4-O-beta-D-glucoside as an index, instead of taking the total of a certain component as a quality control index, which is more reasonable and accurate; 3. the method of the present application is not only limited to Fructus Trema medicinal materials, but also expands to the detection of standard decoction, formula granules and other products containing Fructus Trema; 4. the method of the present application is simple in operation, good in reproducibility and stability, reliable in recovery rate, low in detection cost, high in detection efficiency and small in environmental pollution. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 UV absorption spectrum of vanillic acid-4-O-β-D-glucoside reference solution;

[0034] Figure 2 UPLC chromatogram of Fructus Malli extractive granules extracted with different solvents;

[0035] Figure 3 UPLC chromatogram of Fructus Malli extractive granules extracted with different methods;

[0036] Figure 4 UPLC chromatogram of Fructus Malli extractive granules extracted with different volumes;

[0037] Figure 5 UPLC chromatogram of Fructus Malli extractive granules extracted with different times;

[0038] Figure 6 Linear relationship diagram of injection volume and peak area value of vanillic acid-4-O-β-D-glucoside reference in UPLC;

[0039] Figure 7 UPLC chromatogram of different flow rate investigation results;

[0040] Figure 8 UPLC chromatogram of different temperature investigation results;

[0041] Figure 9 UPLC chromatogram of different column investigation results;

[0042] Figure 10 UPLC chromatogram of content determination specificity experiment results of Fructus Malli extractive granules;

[0043] Figure 11 UPLC chromatogram of content determination specificity experiment results of Fructus Malli standard decoction;

[0044] Figure 12 UPLC chromatogram of content determination specificity experiment results of Fructus Malli medicinal materials. DETAILED DESCRIPTION

[0045] The technical solutions of the present application will be further described below in combination with the drawings.

[0046] Instruments, reagents and samples:

[0047] Waters Acquity UPLC ultra-high performance liquid chromatograph (Waters Corporation); Empower 3 workstation (Waters Corporation); 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 automatic sampler; 1290 Fiexible pump four-component pump; Open LAB CDS2.3 chromatography workstation; electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); pure water system (Sartorius Corporation); AS165W centrifuge (Avestin (Shanghai) Trading Co., Ltd.). Methanol (chromatographically pure, Thermo Fisher Scientific); water is ultrapure water; phosphoric acid (chromatographically pure, aladdin); other reagents are analytical pure.

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

[0049] The bark of Catalpa ovata (YC1), Catalpa ovata standard decoction (DG1), and Catalpa ovata formula granules (KL1, KL2, and KL3) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0050] Example 1, Construction of Catalpa ovata Formula Granule Content Determination Method

[0051] 1. Preparation of reference substance solution

[0052] An appropriate amount of vanillic acid-4-O-β-D-glucoside reference substance was accurately weighed and dissolved in 10% methanol to prepare 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 substance solution was scanned at 190-400 nm, and the ultraviolet absorption spectrum was recorded, as shown in Figure 1 . The results showed that the vanillic acid-4-O-β-D-glucoside reference substance solution had strong absorption in the range of 200-300 nm, which could be used for detection. Considering that there is a significant absorption peak around 254 nm, the detection wavelength can be preferably 254 nm.

[0056] (2) Determination of chromatographic conditions

[0057] The BEH Sheild RP18 column (100 mm in length, 2.1 mm in inner diameter, and 1.7 μm in particle size) 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 isocratic elution was performed.

[0058] 3 Preparation of test solution

[0059] (1) Investigation of extraction solvent

[0060] A certain amount of Fructus Toreniae Dispensing Granules (batch number: KL1) was finely ground, about 0.3 g was taken, and the total was 5 groups, each group in parallel 2 times. The accurately weighed sample was precisely added with 10% methanol, 30% methanol, 50% methanol, 70% methanol, and methanol 25 ml respectively, tightly sealed, weighed, ultrasonically treated (power 250 W, frequency 40 kHz) for 30 minutes, cooled, weighed again, the lost weight was made up with the corresponding solvent, shaken well, filtered, and the filtrate was taken, namely the test solution. 2 μl of each test solution was precisely taken and injected into the ultra-high performance liquid chromatograph, and the peak area value of vanillic acid-4-O-β-D-glucoside was recorded by the above chromatographic conditions. The content and RSD value were calculated, and the results were shown in Table 1. Figure 2 .

[0061] Table 1 Comparison of different extraction solvents

[0062]

[0063] From the results, it can be seen that the content is lower and the peak shape is poorer when 70% methanol and methanol are used as the extraction solvent. When 50% methanol, 30% methanol, and 10% methanol are used, the extraction efficiency is close. Considering the reduction of the use of organic solvents, the extraction solvent can be preferably 10% methanol.

[0064] (2) Investigation of different extraction methods

[0065] A certain amount of Fructus Toreniae Dispensing Granules (batch number: KL1) was finely ground, about 0.3 g was taken, and the total was 3 groups, each group in parallel 2 times. The accurately weighed sample was precisely added with 10% methanol 25 ml in a conical flask with a plug, tightly sealed, weighed, ultrasonically treated (power 250 W, frequency 40 kHz), heated to reflux, and shaken for 30 minutes. The cooled sample was weighed again, the lost weight was made up with 10% methanol, shaken well, filtered, and the filtrate was taken, namely the test solution. 2 μl of each test solution was precisely taken and injected into the ultra-high performance liquid chromatograph, and the peak area value of vanillic acid-4-O-β-D-glucoside was recorded by the above chromatographic conditions. The content and RSD value were calculated, and the results were shown in Table 2. Figure 3 .

[0066] Comparison of different extraction methods

[0067]

[0068]

[0069] From the results, the content of vanillic acid-4-O-β-D-glucoside obtained by the three extraction methods is similar. Considering the operation of ultrasonic treatment is relatively simple, the extraction method is preferably ultrasonic treatment.

[0070] (3) Investigation of different extraction volumes

[0071] Take a suitable amount of formula granules of Fructus Broussonetiae (batch number: KL1), grind finely, take about 0.3 g, a total of 4 groups, 2 replicates in each group, accurately weigh and place in a stoppered conical flask, accurately add 10% methanol 15 ml, 25 ml, 50 ml, and 100 ml, respectively, seal tightly, weigh, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with 10% methanol, shake well, filter, and take the filtrate, i.e. the sample solution. Accurately pipette 2 μl of each sample solution, inject into the ultra-high performance liquid chromatograph, determine according to the above chromatographic conditions, record the peak area value of vanillic acid-4-O-β-D-glucoside, calculate the content and RSD value, the results are shown in Table 3, Figure 4 .

[0072] Table 3 Comparison of different extraction volumes

[0073]

[0074]

[0075] From the results, the content of vanillic acid-4-O-β-D-glucoside obtained by different solvent amounts is similar. From the comprehensive consideration of sufficient extraction and solvent saving, the solvent amount is preferably 25 ml.

[0076] (4) Investigation of different extraction times

[0077] Take a suitable amount of formula granules of Fructus Broussonetiae (batch number: KL1), grind finely, take about 0.3 g, a total of 4 groups, 2 replicates in each group, accurately weigh and place in a stoppered conical flask, accurately add 10% methanol 25 ml, seal tightly, weigh, ultrasonic treatment (power 250 W, frequency 40 kHz) for 15 minutes, 30 minutes, 45 minutes, and 60 minutes, respectively, take out, cool, re-weigh, make up the weight loss with 10% methanol, shake well, filter, and take the filtrate, i.e. the sample solution. Accurately pipette 2 μl of each sample solution, inject into the ultra-high performance liquid chromatograph, determine according to the above chromatographic conditions, record the peak area value of vanillic acid-4-O-β-D-glucoside, calculate the content and RSD value, the results are shown in Table 4,Figure 5 .

[0078] Table 4 Comparison of different extraction times

[0079]

[0080] From the results, it can be seen 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 preferred test solution preparation method is as follows:

[0082] Take an appropriate amount of Cortex Fraxini Granules, grind finely, take about 0.3 g, accurately weigh, place in a conical flask with a stopper, accurately add 10% methanol 25 ml, tightly seal, weigh, ultrasonic treatment (power 250 W, frequency 40 kHz) for 45 minutes, cool, weigh again, make up the weight loss with 10% methanol, shake well, filter, take the filtrate, and obtain.

[0083] (5) Detection of different batches of Cortex Fraxini Granules

[0084] Take an appropriate amount of 3 batches of Cortex Fraxini Granules sample, 2 parallel samples for each batch, prepare the test solution according to the above test solution preparation method, inject into the ultra-high performance liquid chromatograph, measure 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 Cortex Fraxini Granules using the correction factor. The results are shown in Table 5.

[0085] Table 5 Content determination of vanillic acid-4-O-β-D-glucoside in Cortex Fraxini Granules (correction factor method)

[0086]

[0087] The results show that the content of vanillic acid-4-O-β-D-glucoside in different batches of Cortex Fraxini Granules is consistent, and the determination results of parallel samples of the same batch have an RSD less than 2%, indicating that the use of vanillic acid-4-O-β-D-glucoside as an index is feasible and has good repeatability.

[0088] Example 2, Methodology verification of content determination construction method

[0089] 1. Linear relationship

[0090] Take 0.5 μl, 1.0 μl, 1.5 μl, 2.0 μl, 2.5 μl, 3.0 μl, 3.5 μl, 4.0 μl of vanillic acid-4-O-β-D-glucoside (0.0100548 mg / ml) control 0.5 μl, 1.0 μl, 1.5 μl, 2.0 μl, 2.5 μl, 3.0 μl, 3.5 μl, 4.0 μl, inject into the ultra-high performance liquid chromatograph, and determine according to the above chromatographic conditions, take the peak area (AU) as the vertical coordinate and the injection amount (μg) as the horizontal coordinate to draw the standard curve, and 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 amount of vanillic acid-4-O-β-D-glucoside control

[0092]

[0093]

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

[0095] 2. Precision test

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

[0097] Table 7 Instrument precision test

[0098]

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

[0100] 3. Stability test

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

[0102] Table 8 Determination results of the stability test

[0103]

[0104] ​

[0105] The results show that the RSD value of the peak area of vanillic acid-4-O-β-D-glucoside is 0.48%, and the test solution is stable within 24 hours.

[0106] 4. Reproducibility test

[0107] Take a certain amount of Fructus Spondiis Mitratae sample (batch number of Fructus Spondiis Mitratae formula granules: KL1), grind it finely, take about 0.3 g, accurately weigh, parallel 6 times, respectively according to the preparation method of the test solution to prepare the sample test solution, respectively inject 2 μl, record the peak area value of vanillic acid-4-O-β-D-glucoside, calculate the content of vanillic acid-4-O-β-D-glucoside and the RSD value, and the results are shown in Table 9.

[0108] Table 9 Reproducibility test

[0109]

[0110] The results show that the RSD value of the peak area of vanillic acid-4-O-β-D-glucoside is 0.32%, and the reproducibility test is good.

[0111] 5. Accuracy test

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

[0113]

[0114] Table 10 Accuracy test of vanillic acid-4-O-β-D-glucoside

[0115]

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

[0117] 6. Intermediate precision test

[0118] Take the sample of Fructus Terminaliae (batch number of Fructus Terminaliae formula granules: KL1), and prepare sample test solution by different experimenters according to the preparation method of test solution, respectively, 2 μl is injected at different times on different types of instruments, and the content of vanillic acid-4-O-β-D-glucoside and RSD value are calculated according to the above chromatographic conditions, and the results are shown in Table 11.

[0119] Table 11 Intermediate precision test

[0120]

[0121]

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

[0123] 7. Durability test

[0124] (1) Different flow rate investigation

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

[0126] Table 12 Different flow rate investigation

[0127]

[0128] The results show that the flow rate is within 0.20 ml / min-0.30 ml / min, the separation effect of vanillic acid-4-O-β-D-glucoside chromatographic peak meets the requirements, the measured content is basically consistent, and it is indicated that the slight change of flow rate has no significant influence on the determination of vanillic acid-4-O-β-D-glucoside content, and the durability is good.

[0129] (2) Different column temperature investigation

[0130] Take 1 part of sample (batch number of Fructus Terminaliae formula granules: KL1), and prepare test solution according to the preparation method of test solution, and investigate the influence on content determination under 25°C, 30°C and 35°C temperatures, and the results are shown in Table 13, Figure 8The BEH Shield RP18 column (100 mm in length, 2.1 mm in inner diameter, and 1.7 μm in particle size) 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 isocratic elution was performed.

[0131] Table 13: Investigation of different column temperatures

[0132]

[0133] The results show that when the column temperature is in the range of 25-35°C, the separation effect of the vanillic acid-4-O-β-D-glucoside chromatographic peak meets the requirements, the measured content is basically consistent, and it is indicated that the slight change of the column temperature has no significant influence on the determination of the vanillic acid-4-O-β-D-glucoside content, and the durability is good.

[0134] (3) Investigation of different chromatographic columns

[0135] A sample (batch number of the Fructus Chebulae dispensing granules: KL1) was taken, the test sample solution was prepared according to the preparation method of the test sample solution, and three kinds of chromatographic columns, BEH C18 (2.1x100 mm, 1.7 μm), BEH Shield RP18 (2.1x100 mm, 1.7 μm), and Poroshell 120EC-C18 (2.1x100 mm, 1.9 μm), were used to analyze the Fructus Chebulae granule sample. The results are shown in Table 14, Figure 9 The 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 isocratic elution was performed.

[0136] Table 14: Investigation of different chromatographic columns

[0137]

[0138] The results show that the separation degree of the vanillic acid-4-O-β-D-glucoside chromatographic peak meets the requirements in the three kinds of chromatographic columns, and there is no significant difference in the measured results, which indicates that the different chromatographic columns have little influence on the determination results of the sample content, and the durability is good.

[0139] 8. Specificity test

[0140] Take the above Fuling prescription granules test solution (batch number: KL1), vanillic acid-4-O-β-D-glucoside control solution and negative sample solution each 2 μl, inject into liquid chromatograph, according to the above chromatographic conditions (with BEH Sheild RP18 (column length is 100 mm, inner diameter is 2.1 mm, particle size is 1.7 μm) as chromatographic column; with methanol-0.1% phosphoric acid solution (4:96) as mobile phase; detection wavelength is 254 nm; flow rate is 0.25 ml / min; column temperature is 30 ℃, isocratic elution) determination, record chromatogram, results are shown in Figure 10 . Among them, the negative sample is auxiliary material and solvent.

[0141] The results show that: the auxiliary material and solvent do not interfere with the determination of vanillic acid-4-O-β-D-glucoside in Fuling prescription granules, the method has strong specificity, which is suitable for the content determination of Fuling prescription granules.

[0142] Example three, the specificity experiment of vanillic acid-4-O-β-D-glucoside content determination in Fuling standard decoction

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

[0144] Results: the solvent does not interfere with the determination of vanillic acid-4-O-β-D-glucoside in Fuling standard decoction, the method has strong specificity, which is suitable for the content determination of Fuling standard decoction.

[0145] Example four, the specificity experiment of vanillic acid-4-O-β-D-glucoside content determination in Fuling medicinal materials

[0146] Take about 2g of Vaccinium Fructus (batch number: YC1), accurately weigh, and place in a Soxhlet extractor. Add petroleum ether (30-60°C) in proper amount, heat and reflux for 2 hours. Discard the petroleum ether liquid, and dry the solvent in the residue along with the filter paper cylinder. Move into a conical flask with a plug, accurately add 10% methanol 50ml, weigh, heat and reflux for 90 minutes, weigh again, make up the weight loss with 10% methanol, shake well, filter, and take the filtrate, which is Vaccinium Fructus sample solution.

[0147] Take 2ul of Vaccinium Fructus sample solution, vanillic acid-4-O-β-D-glucoside control solution and blank solvent, inject into the ultra-high performance liquid chromatograph, and determine according to the above chromatographic conditions, record the chromatogram, and the results are shown in Figure 12 .

[0148] Results: The solvent has no interference with the determination of vanillic acid-4-O-β-D-glucoside in Vaccinium Fructus. The method has strong specificity and is suitable for the content determination of Vaccinium Fructus.

[0149] Example Five

[0150] Take 3 batches of Vaccinium Fructus formula granules, take 2 samples in parallel for each batch, and prepare the sample solution according to the sample solution preparation method in Example One. 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. Calculate the content of vanillic acid-4-O-β-D-glucoside in Vaccinium Fructus sample using the regression equation of the injection amount and peak area of vanillic acid-4-O-β-D-glucoside in Example Two. The results are shown in Table 15.

[0151] Table 15 Determination of vanillic acid-4-O-β-D-glucoside in Vaccinium Fructus formula granules (standard curve method)

[0152]

[0153]

[0154] The results show that the content of vanillic acid-4-O-β-D-glucoside in different batches of Vaccinium Fructus formula granules calculated by the standard curve method has little difference from the correction factor method. The determination results of parallel samples of the same batch have RSD less than 2%, indicating that the method for determining vanillic acid-4-O-β-D-glucoside in Vaccinium Fructus sample is stable, feasible, reproducible, and reliable.

Claims

1. A method for detecting vanillic acid-4- in paper mulberry fruit samples. O - β The method for determining the content of -D-glucosides is characterized by... Includes the following steps: (1) Take the powdered paper mulberry fruit sample, add 15-100 ml of 10%-50% methanol, seal tightly, weigh, extract, weigh again, replenish the lost weight with 10%-50% methanol, shake well, filter, and take the filtrate to obtain the test solution; the paper mulberry fruit sample is the medicinal material, standard decoction or formula granules of paper mulberry fruit; (2) Take vanillic acid-4- O-β Weigh an appropriate amount of 3-D-glucoside reference standard accurately, add 10% methanol to obtain the reference standard solution; (3) Inject the reference solution and the test solution into the ultra-high performance liquid chromatograph and compare the characteristic chromatograms of the reference solution and the test solution; the ultra-high performance liquid chromatograph uses a BEH Sheild RP18 column with a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.7 μm; the mobile phase is methanol-0.1% phosphoric acid solution with a volume ratio of methanol to 0.1% phosphoric acid solution of 4:96; the elution method is isocratic elution; and the detection wavelength is 254 nm. (4) Using vanillic acid-4- O-β The regression equation for the injection volume and peak area of ​​-D-glucoside is Y = 8434.6543X - 5203.9643, R. 2 =0.99990, calculate vanillic acid-4- in the Broussonetia papyrifera fruit sample. O-β The content of -D-glucoside.

2. The method according to claim 1, characterized in that, The extraction methods described in step (1) include ultrasonic treatment, heating and reflux, or shaking extraction.

3. The method according to claim 1, characterized in that, The extraction time described in step (1) is 15-60 min.

4. The method according to claim 1, characterized in that, The chromatographic conditions in step (3) are: flow rate 0.20 ml / min ~ 0.30 ml / min, column temperature 25℃ ~ 35℃.

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

6. The application of the method according to any one of claims 1-5 in the quality control of paper mulberry fruit samples.

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