Method for detecting content of hyperoside in humifuse euphorbia herb sample and application thereof

Abstract

The invention discloses a method for detecting the content of hyperoside in a humifuse euphorbia herb sample. The detection method comprises the following steps: preparing a humifuse euphorbia herb sample test solution and a hyperoside reference substance solution; respectively detecting the reference substance solution and the test substance solution by using an ultra-performance liquid chromatograph, and comparing the maps of the reference substance solution and the test substance solution; a mobile phase used in the detection is a methanol-acetonitrile-0. 1% formic acid solution; and calculating the content of hyperoside in the humifuse euphorbia herb sample by utilizing a regression equation of the sample size and the peak area of the components. The quality detection method of the humifuse euphorbia herb sample provided by the invention is simple to operate, good in reproducibility and stability, high in accuracy, reliable in recovery rate and high in detection efficiency, and provides a basis for standardizing the market and reasonable development of humifuse euphorbia herb medicinal materials and preparations.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for detecting the content of hyperoside in Euphorbia humifusa sample, belonging to the technical field of pharmaceutical analysis. BACKGROUND

[0002] Euphorbia humifusa, the dry whole herb of Euphorbia pekinensis Rupr. or Euphorbia humifusa, has the effects of clearing heat, removing dampness, reducing jaundice and stopping bleeding, and can be used for treating dysentery, diarrhea, hematochezia, damp-heat jaundice and the like. Modern studies have shown that Euphorbia humifusa mainly contains flavonoids, triterpenes, coumarins, phenolic acids, alkaloids and the like, and has the pharmacological effects of antibacterial, anti-inflammatory, antioxidant, antiviral, antitumor and hypoglycemic.

[0003] As for the quality control of Euphorbia humifusa, the hydrochloric acid hydrolysis method is adopted to prepare the sample in the Chinese Pharmacopoeia, and quercetin is used as the content determination index to determine the content of quercetin in Euphorbia humifusa medicinal material (State Pharmacopoeia Commission. Chinese Pharmacopoeia (2020 Edition) Part I [S]. Beijing: China Medical Science and Technology Press, 2020: 131.). In the existing literature reports, the content determination research mainly focuses on organic acids and flavonoids, such as Zheng Xuanxing et al. who used ultra-high performance liquid chromatography-tandem mass spectrometry to determine the contents of gallic acid, rutin, isoquercitrin, ellagic acid, astragalin, luteolin, quercetin and kaempferol (Zheng Xuanxing, Zhang Yufan, She Yibin, et al. Simultaneous determination of the contents of eight components in Euphorbia humifusa by ultra-high performance liquid chromatography-tandem mass spectrometry [J]. Brand and Standardization, 2024, (03): 37.), Lin Zuwu established the HPLC content determination method of gallic acid, ellagic acid, quercetin, luteolin and apigenin in Euphorbia humifusa (Lin Zuwu. Simultaneous determination of the contents of five components in Euphorbia humifusa from different sources by HPLC [J]. Chinese Herbal Medicines, 2021, 44(07): 1701.), and Peng Jiming et al. used multi-wavelength UPLC method to determine the contents of gallic acid, ellagic acid, quercetin and kaempferol in Euphorbia humifusa (Peng Jiming, Lu Juan, Chen Zhe, et al. Multi-wavelength detection UPLC method for determining the contents of four index components in Euphorbia humifusa [J]. Journal of Pharmaceutical Analysis, 2017, 37(04): 589.), but there is no report on the content determination method of hyperoside.

[0004] Hyperoside is an important flavonoid glycoside compound, which has significant antioxidant, antitumor and anti-aging effects, and is consistent with the pharmacological effects of Euphorbia humifusa, so it has certain control significance to evaluate the quality of Euphorbia humifusa sample by using hyperoside.

[0005] In addition, the currently reported content determination methods are mainly established for Euphorbia humifusa medicinal materials, and there are few reports on the determination methods for Euphorbia humifusa preparations, therefore, the establishment of the determination method of hyperoside in Euphorbia humifusa preparations can provide a reference for the quality control of Chinese medicine preparations containing Euphorbia humifusa. SUMMARY

[0006] The present application aims to provide a method for detecting the content of hyperoside in Euphorbia humifusa sample and its application. In view of the fact that there are relatively few flavonoid component indicators that can be used for quality evaluation of Euphorbia humifusa preparation sample (such as standard decoction, formula granules, etc.) in the prior art, the present application proposes to apply hyperoside to the quality detection of Euphorbia humifusa sample (such as crude drug, decoction piece, standard decoction, formula granule, etc.), and establishes a content determination method using liquid chromatography as the analysis means, thereby providing a new analysis means for the internal quality control of Euphorbia humifusa sample.

[0007] Technical scheme: The present application provides a method for detecting the content of hyperoside in Euphorbia humifusa sample, comprising the following steps:

[0008] (1) preparing a Euphorbia humifusa sample test solution and a hyperoside control solution;

[0009] (2) detecting the control solution and the test solution with an ultra-high performance liquid chromatograph, and comparing the chromatograms of the control solution and the test solution; the mobile phase used in the detection is methanol-acetonitrile-0.1% formic acid solution;

[0010] (3) calculating the content of hyperoside in the Euphorbia humifusa sample by using the regression equation of the injection amount and the peak area of hyperoside.

[0011] Further, the Euphorbia humifusa sample in step (1) includes crude drug, decoction piece, standard decoction, formula granule or preparation.

[0012] Further, the preparation method of the Euphorbia humifusa sample test solution in step (1) is as follows: taking Euphorbia humifusa sample powder, accurately weighing, adding extraction solvent, extracting, filtering, evaporating the filtrate to dryness, dissolving the residue with water, extracting with extraction reagent, evaporating the extract to dryness, dissolving the residue with extraction solvent, constant volume, filtering, and taking the filtrate to obtain the test solution.

[0013] Further, the extraction solvent includes water, 30% to 100% methanol, and the extraction reagent is ethyl acetate.

[0014] Further, the mass-volume ratio of the Euphorbia humifusa sample powder to the extraction solvent is 1g:30 to 100mL.

[0015] Further, the extraction method includes ultrasonic treatment, heating reflux or shaking.

[0016] Further, the extraction time is 15 to 60 minutes.

[0017] Further, the conditions of the ultra-high performance liquid chromatograph in step (2) are as follows: octadecylsilane-bonded silica gel as filler, column temperature 20-30 DEG C, isocratic elution, detection wavelength 255 nm or 355 nm, and the ratio of methanol-acetonitrile-0.1% formic acid solution is 28-30:1:69-71.

[0018] Further, the regression equation of the sample injection amount and peak area of hyperoside in step (3) is y=9242357.7074x-65247.1611, R=1.0000.

[0019] The application further provides a method for recovering hyperoside in a Euphorbia humifusa sample, comprising the following steps:

[0020] (1) taking Euphorbia humifusa sample powder, accurately weighing, adding an extraction solvent, extracting, filtering, evaporating the filtrate, dissolving the residue with water, extracting with an extraction reagent, evaporating the extract, dissolving the residue with an extraction solvent, constant volume, filtering, and taking the filtrate to obtain a test sample solution;

[0021] (2) injecting the test sample solution into an ultra-high performance liquid chromatograph, using methanol-acetonitrile-0.1% formic acid solution as the mobile phase, and recovering hyperoside.

[0022] The application further provides application of the above method in quality control or quality detection of the Euphorbia humifusa sample.

[0023] Advantages: compared with the prior art, the application has the following remarkable advantages: 1. the application controls the sample preparation method and the conditions of liquid chromatography reasonably, establishes a content determination method for determining hyperoside in the Euphorbia humifusa sample, and provides a new analysis means for quality detection of the Euphorbia humifusa sample; 2. the application is not only suitable for quality detection of medicinal materials, decoction pieces, standard decoctions and formula granules of Euphorbia humifusa, but also suitable for detection of other products containing Euphorbia humifusa; 3. in addition, the method of the application is simple in operation, good in reproducibility and stability, high in accuracy, reliable in recovery rate, and high in detection efficiency, and can provide a basis for standardizing the Euphorbia humifusa medicinal material and preparation market and reasonably developing the Euphorbia humifusa medicinal material. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a UV absorption spectrum of hyperoside;

[0025] Figure 2 is a liquid chromatogram of Euphorbia humifusa formula granules at different detection wavelengths: A: 255 nm wavelength; B: 355 nm wavelength;

[0026] Figure 3 is a liquid chromatogram of a control sample and Euphorbia humifusa formula granule sample with different mobile phases;

[0027] Figure 4 Liquid chromatogram of Euphorbiae Humifusae Formula Granules with different extraction solvents;

[0028] Figure 5 Liquid chromatogram of Euphorbiae Humifusae Formula Granules with different extraction methods;

[0029] Figure 6 Liquid chromatogram of Euphorbiae Humifusae Formula Granules with different extraction volumes;

[0030] Figure 7 Liquid chromatogram of Euphorbiae Humifusae Formula Granules with different extraction times;

[0031] Figure 8 Liquid chromatogram of Euphorbiae Humifusae Formula Granules with different extraction times

[0032] Figure 9 Standard curve of Hyperoside;

[0033] Figure 10 Liquid chromatogram of Euphorbiae Humifusae Formula Granules for content determination specificity experiment results;

[0034] Figure 11 Liquid chromatogram of Euphorbiae Humifusae Formula Granules with different flow rates;

[0035] Figure 12 Liquid chromatogram of Euphorbiae Humifusae Formula Granules determined by using different column temperatures;

[0036] Figure 13 Liquid chromatogram of Euphorbiae Humifusae Formula Granules determined by using different columns;

[0037] Figure 14 Determination chromatogram of Euphorbiae Humifusae medicinal materials; Figure 15 Chromatogram for determination of the content of Hyperoside in Euphorbiae Humifusae standard decoction. DETAILED DESCRIPTION

[0038] The technical solutions of the present application will be further described below in combination with the drawings.

[0039] The instruments, reagents and samples used in the following examples are as follows:

[0040] Waters ACQUITY UPLC H-Class ultra-high performance liquid chromatograph system; Waters Quaternary Solvent Manager four-element pump; Sample Manager-FTN automatic sampler; Waters UPLC PDA detector; Empower 3 chromatographic workstation; Thermo vanquish ultra-high performance liquid chromatograph (Thermo Corporation); KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); METTLER TOLEDO XP6 millionth balance (Metller-Toledo (Shanghai) Instrument Co., Ltd.); ME204E / 02 electronic analytical balance (Metller-Toledo Instrument (Shanghai) Co., Ltd.); GKC114 temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); HY-4 oscillator (Jintan City Ke Xing Instrument Factory); Milli-Q IQ water purification system (Millipore Corporation); AS165W centrifuge (Acesse (Shanghai) Trading Co., Ltd.); TGL-16C centrifuge (Shanghai Anting Scientific Instrument Factory); methanol (chromatographically pure, Thermo Fisher Corporation); acetonitrile (chromatographically pure, Thermo Fisher Corporation); water is ultrapure water; formic acid (chromatographically pure, aladdin Corporation); other reagents are analytically pure.

[0041] Hyperoside reference substance (batch number 111521-201809, purity ≥ 94.9%) was purchased from China Institute for Drug Control.

[0042] Euphorbiae humifusae formula granules were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd. (batch numbers: 23050019, 23050029, 23050039).

[0043] Example 1: Method for determining the content of hyperoside in Euphorbiae humifusae formula granules

[0044] 1. Preparation of reference solution and determination of detection wavelength

[0045] An appropriate amount of hyperoside reference substance was accurately weighed and dissolved in 70% methanol to prepare a reference solution with a mass concentration of 50 μg / mL. The ultraviolet absorption spectrum of the reference solution was collected, and the results are shown in Figure 1 .

[0046] From the analysis Figure 1 , it can be seen that hyperoside has strong ultraviolet absorption between 245 nm and 265 nm or between 345 nm and 365 nm. Considering that the ultraviolet absorption near 255 nm or 355 nm is relatively high, the detection wavelength can be preferably 255 nm or 355 nm.

[0047] 2. Preparation of test solution

[0048] Take the Euphorbia humifusa formula granules (batch number: 23050029) appropriate amount, grind finely, take about 0.5g, accurately weigh, place in a conical bottle with a stopper, accurately add 70% methanol 25ml, tightly seal, weigh, ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the weight loss with 70% methanol, shake well, filter, take all the filtrate, evaporate to dryness, add water 25ml to dissolve the residue, extract with ethyl acetate for 3 times, 25ml each time, combine the ethyl acetate liquid, evaporate to dryness, add 70% methanol to dissolve and transfer to a 5ml volumetric flask, add 70% methanol to the mark, shake well, filter, take the filtrate, and get it. Record the chromatogram at 255nm and 355nm respectively, see Figure 2 .

[0049] From Figure 2 It can be seen that the Euphorbia humifusa sample can be well detected at 255nm or 355nm. The subsequent examples use the detection wavelength of 255nm.

[0050] 3. Determination of analysis method

[0051] 3.1 Chromatographic conditions

[0052] HSS T3 (Waters, 2.1x100mm, 1.8μm) chromatographic column was used, and methanol-0.1% formic acid (28:72), methanol-acetonitrile-0.1% phosphoric acid (28:2:70), methanol-acetonitrile-0.1% phosphoric acid (29:1:70), methanol-acetonitrile-0.1% phosphoric acid (30:1:69), methanol-acetonitrile-0.1% formic acid solution (28:1:71) were used as mobile phase, column temperature was 30℃, flow rate was 0.30mL / min. UV detector was used for detection, and the detection wavelength was 255nm. The theoretical plate number should not be less than 5000 calculated by hyperoside. Accurately take 1μL of the control solution and the test solution, respectively, inject into the ultra-high performance liquid chromatograph, and determine. Calculate by external standard point method, and get it.

[0053] The results are as follows Figure 3The peak shape of the hyperoside was poor and easy to tail when methanol-0.1% formic acid (28:72) was used as the mobile phase; the peak of the hyperoside was out quickly and was sandwiched in other chromatographic peaks in the sample, which was not conducive to the determination when methanol-acetonitrile-0.1% phosphoric acid (28:2:70) was used as the mobile phase; the peak time of the hyperoside was increased and the separation from other chromatographic peaks was improved when the proportion of acetonitrile was reduced and methanol-acetonitrile-0.1% phosphoric acid (29:1:70) was used as the mobile phase; the peak time of the hyperoside was advanced when the proportion of methanol was increased and the proportion of 0.1% phosphoric acid was reduced, and methanol-acetonitrile-0.1% phosphoric acid (30:1:69) was used as the mobile phase, but the peak shape of the hyperoside in the sample was slightly poor; the peak time of the hyperoside was increased and the peak shape of the hyperoside in the sample was improved when the proportion of methanol was reduced and the proportion of 0.1% phosphoric acid was increased, and methanol-acetonitrile-0.1% phosphoric acid (28:1:71) was used as the mobile phase, and the separation from other chromatographic peaks was good, and the detection could be completed within 24 minutes, and the retention time of the hyperoside was about 19.2 minutes, so methanol-acetonitrile-0.1% phosphoric acid (28:1:71) was finally selected as the mobile phase.

[0054] 3.2 Investigation of preparation of test solution

[0055] (1) Investigation of extraction solvent

[0056] Investigation method: take the E.J. formula granules (batch number: 23050029) in an appropriate amount, grind finely, take about 0.5 g, a total of 5 groups, 2 parallel each time, accurately weigh, place in a conical flask with a plug, accurately add water, 30% methanol, 50% methanol, 70% methanol, and methanol 25 ml respectively, weigh, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the weight loss with the corresponding solvent, shake well, filter, take all the filtrate, evaporate to dryness, add water 25 ml to redissolve, extract with ethyl acetate for 3 times, 25 ml each time, combine the ethyl acetate liquid, evaporate to dryness, add 70% methanol to dissolve and transfer to a 5 ml volumetric flask, add 70% methanol to the mark, shake well, filter, take the filtrate, and it is obtained. Accurately take 1 μL of each test solution, inject into the liquid chromatograph, sample according to the chromatographic conditions in item 3.1, calculate the content of hyperoside, and the results are shown in Table 1, Figure 4 .

[0057] Table 1 Comparison of different extraction solvents

[0058]

[0059] Conclusion: The content of hyperoside is relatively close when the above solvents are used to prepare the sample solution, and all of them can be used to prepare the sample of Herba Humuli. Considering that the content of hyperoside is relatively high when 70% methanol is used, 70% methanol is preferred.

[0060] (2) Investigation of extraction method

[0061] Investigation method: Take Herba Humuli Dispensing Granules (batch number: 23050029) and grind them finely. Take about 0.5 g and divide them into 3 groups, each with 2 parallel samples. Precisely weigh them and place them in conical bottles with stoppers. Precisely add 25 ml of 70% methanol, weigh, and extract by ultrasonic (power 250 W, frequency 40 kHz) for 30 minutes, shake extraction for 30 minutes, and heat reflux for 30 minutes. Cool, re-weigh, make up the weight loss with 70% methanol, shake well, filter, take all the filtrate, evaporate to dryness, dissolve the residue in 25 ml of water, extract with ethyl acetate for 3 times, each time with 25 ml, combine the ethyl acetate liquid, evaporate to dryness, dissolve the residue in 70% methanol, transfer to a 5 ml volumetric flask, add 70% methanol to the calibration mark, shake well, filter, take the filtrate, and you get the sample solution. Precisely take 1 μL of each sample solution and inject it into the ultra-high performance liquid chromatograph. Sample according to the chromatographic conditions in item "3.1" and calculate the content of hyperoside. The results are shown in Table 2, Figure 5 .

[0062] Table 2 Comparison of extraction methods

[0063]

[0064] Conclusion: When ultrasonic treatment, heat reflux, and shake extraction are used, the content of hyperoside has little difference. Considering that ultrasonic treatment is simple to operate, ultrasonic treatment is preferred.

[0065] (3) Investigation of the volume of extraction solvent

[0066] Investigation method: Take Herba Humuli Dispensing Granules (batch number: 23050029) and grind them finely. Take about 0.5 g and divide them into 3 groups, each with 2 parallel samples. Precisely weigh them and place them in conical bottles with stoppers. Precisely add 15 ml, 25 ml, and 50 ml of 70% methanol, respectively, weigh, and extract by ultrasonic (power 250 W, frequency 40 kHz) for 30 minutes. Cool, re-weigh, make up the weight loss with 70% methanol, shake well, filter, take all the filtrate, evaporate to dryness, dissolve the residue in 15 ml, 25 ml, and 50 ml of water, respectively, extract with ethyl acetate for 3 times, each time with the corresponding volume, combine the ethyl acetate liquid, evaporate to dryness, dissolve the residue in 70% methanol, transfer to a 5 ml volumetric flask, add 70% methanol to the calibration mark, shake well, filter, take the filtrate, and you get the sample solution. Precisely take 1 μL of each sample solution and inject it into the ultra-high performance liquid chromatograph. Sample according to the chromatographic conditions in item "3.1" and calculate the content of hyperoside. The results are shown in Table 3,Figure 6 .

[0067] Comparison of extraction volume

[0068]

[0069]

[0070] Conclusion: When the extraction volume is 15 mL, 25 mL, 50 mL, the content of hyperoside obtained is not much different, indicating that the extraction is sufficient when the extraction solvent is 15 mL. Considering the differences between different batches of samples, in order to ensure sufficient extraction, the amount of solvent added is selected as 25 mL.

[0071] (4) Investigation of extraction time

[0072] Investigation method: Take Euphorbia Formula Granules (batch number: 23050029) of appropriate amount, grind finely, take about 0.5 g, a total of 4 groups, 2 replicates in each group, accurately weigh, place in a conical flask with a plug, accurately add 25 ml of 70% methanol, weigh, ultrasonic extraction (power 250 W, frequency 40 kHz) for 15 minutes, 30 minutes, 45 minutes, 60 minutes respectively, cool, weigh again, make up the weight loss with 70% methanol, shake well, filter, take all the filtrate, evaporate to dryness, add 25 ml of water to the residue, dissolve and transfer to a 5 ml volumetric flask, add 70% methanol to the mark, shake well, filter, take the filtrate, and obtain. Accurately take 1 μL of each test solution, inject into the ultra-high performance liquid chromatograph, and calculate the content of hyperoside according to the chromatographic conditions in item "3.1". The results are shown in Table 4, Figure 7 .

[0073] Comparison of extraction time

[0074]

[0075] Conclusion: Different ultrasonic treatment time, the content of hyperoside obtained is not much different, indicating that the extraction is sufficient when the extraction time is 15 minutes. Considering the differences between different batches of samples, in order to ensure sufficient extraction, the extraction time is selected as 30 minutes.

[0076] (5) Investigation of different extraction times

[0077] Method: Take a suitable amount of Euphorbia Formula Granules (batch number: 23050029), grind finely, take about 0.5g, 3 groups, each group in parallel 2 copies, accurately weigh, place in a conical bottle with a plug, accurately add 25ml of 70% methanol, weigh, ultrasonic extraction (power 250W, frequency 40kHz) for 30 minutes, cool, reweigh, make up the weight loss with 70% methanol, shake well, filter, take all the filtrate, evaporate to dryness, add 25ml of water to the residue, redissolve, extract with ethyl acetate for 2, 3 and 4 times, each time with 25ml, combine the ethyl acetate liquid, evaporate to dryness, add 70% methanol to the residue to dissolve and transfer to a 5ml volumetric flask, add 70% methanol to the mark, shake well, filter, take the filtrate, and obtain. Accurately pipette 1μL of each test solution into the ultra-high performance liquid chromatograph, inject the sample according to the chromatographic conditions in item "3.1", and calculate the content of hyperoside. The results are shown in Table 5, Figure 8 .

[0078] Table 5 Investigation of different extraction times

[0079]

[0080] Conclusion: When the extraction times are 2, 3 and 4 times, the content of hyperoside basically does not increase with the increase of extraction times. From the perspective of extraction sufficiency, the extraction times are determined to be 3 times.

[0081] After the above investigation, the preparation method of the test solution is determined as follows:

[0082] Take a suitable amount of Euphorbia Formula Granules, grind finely, take about 0.5g, accurately weigh, place in a conical bottle with a plug, accurately add 25ml of 70% methanol, tightly plug, weigh, ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes, cool, reweigh, make up the weight loss with 70% methanol, shake well, filter, take all the filtrate, evaporate to dryness, add 25ml of water to the residue, redissolve, extract with ethyl acetate for 3 times, each time with 25ml, combine the ethyl acetate liquid, evaporate to dryness, add 70% methanol to the residue to dissolve and transfer to a 5ml volumetric flask, add 70% methanol to the mark, shake well, filter, take the filtrate, and obtain.

[0083] (6) Detection of different batches

[0084] Take 3 batches of different batches of samples, prepare the samples according to the preparation method of the test solution determined in item "3.2", analyze according to the chromatographic conditions in item "3.1", and the specific results are shown in Table 6:

[0085] Table 6 Content of hyperoside in Euphorbia Formula Granules of different batches

[0086]

[0087] From the results, the hyperoside content in different batches of Euphorbia humifusa samples was between 0.431-0.489 mg / g.

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

[0089] 1. Linear relationship

[0090] Respectively, 0.1 μl, 0.2 μl, 0.4 μl, 0.8 μl, 1.0 μl, 1.4 μl, 1.8 μl, 2.0 μl, 2.5 μl of hyperoside control solution (0.0994552 mg / ml) was precisely taken and injected into the liquid chromatograph, the sample was injected under the chromatographic conditions in item "3.1" of Example 1, the standard curve was plotted with the peak area as the vertical coordinate and the injection amount as the horizontal coordinate, the regression equation was obtained, the results are shown in Table 7, and the graph is shown in Figure 9 .

[0091] Table 7 Linear regression equation of hyperoside

[0092]

[0093] The results showed that the injection amount of hyperoside was in the range of 0.0099455 μg-0.2486380 μg, and the injection amount and the peak area showed a good linear relationship.

[0094] 2. Precision test

[0095] Investigation method: the same batch of samples (batch number: 23050029) was taken, the test sample solution was prepared according to the test sample preparation method determined in item "3.2" of Example 1, 1 μL of the test sample solution was precisely taken and injected into the ultra-high performance liquid chromatograph, the sample was injected under the chromatographic conditions in item "3.1" of Example 1, and the relative standard deviation was calculated by continuously injecting 6 times, the results are shown in Table 8.

[0096] Table 8 Instrument precision test (peak area)

[0097]

[0098] Results: the peak area RSD was less than 1%, and the instrument precision test was good.

[0099] 3. Reproducibility test

[0100] Investigation method: about 0.5 g of the same batch of Euphorbia humifusa samples (batch number: 23050029) was precisely weighed in parallel for 6 times, the samples were prepared according to the preparation method of the test sample solution in item "3.2" of Example 1, and were analyzed by injecting the sample under the chromatographic conditions in item "3.1" of Example 1, the content and RSD value were calculated, the results are shown in Table 9.

[0101] Table 9 Reproducibility test of Euphorbia humifusa sample content

[0102]

[0103] Results: The RSD of hyperoside content is less than 3%, and the repeatability test is good.

[0104] 4. Intermediate precision test

[0105] Investigation method: Take Euphorbia humifusa sample (batch number: 23050029), and prepare test solution according to the preparation method of test sample solution in item “3.2” of Example 1 by two experimenters respectively. 1 μl is injected at different times, on different instruments respectively, the peak area value is measured, the content of hyperoside is calculated, and the RSD is calculated. The results are shown in Table 10.

[0106] Table 10 Intermediate precision test

[0107]

[0108] Results: The RSD of hyperoside content on different instruments is 1.41%, and the intermediate precision test is good.

[0109] 5. Accuracy test

[0110] Investigation method: Take 0.25 g of sample with known content (batch number: 23050029, content: 0.473 mg / g), and accurately weigh, in parallel for 6 times, add 5 ml, 10 ml and 15 ml of hyperoside (0.012498 mg / ml) control solution respectively, each for 3 times, prepare test solution according to the preparation method of test sample solution in item “3.2” of Example 1, and inject 1 μl according to the chromatographic conditions in item “4.1” of Example 1. The recovery rate and RSD value are calculated according to the following formula, and the results are shown in Table 11.

[0111]

[0112] Table 11 Accuracy test

[0113]

[0114]

[0115] Results: The recovery rate of hyperoside is between 96.79% and 103.86%, and the accuracy test is good.

[0116] 6. Specificity test

[0117] Investigation method: take hyperoside reference substance solution, then take the Dichondra repens formula granules sample (batch number: 23050029) and the formula granules excipient without Dichondra repens as negative samples, respectively prepare solutions according to the preparation method of the test sample solution determined in item “3.2” under Example 1, and sample by the chromatographic conditions under item “3.1” in Example 1. The results are shown in Table 1. Figure 10 .

[0118] Conclusion: according to the results, there is no chromatographic peak at the retention time corresponding to the reference substance in the negative chromatogram, indicating that the determination of hyperoside in Dichondra repens by this method has no interference, and the method has strong specificity.

[0119] 7. Durability test

[0120] 7.1 Investigation of different flow rates

[0121] Investigation method: take the Dichondra repens formula granules sample (batch number: 23050029) 1 part, prepare the test sample solution according to the preparation method of the test sample solution determined in item “3.2” under Example 1, and sample by the chromatographic conditions under item “3.1” in Example 1, to investigate the influence of the separation of chromatographic peaks under 0.25 mL / min, 0.30 mL / min, and 0.35 mL / min flow rates. The results are shown in Table 12. Figure 11 , Table 12.

[0122] Table 12 Investigation of different flow rates

[0123]

[0124] Results: the separation effect of chromatographic peaks is good between 0.25 mL / min and 0.35 mL / min flow rates, and the determination of hyperoside content is relatively close, with good durability.

[0125] 7.2 Investigation of column temperature

[0126] Investigation method: take the Dichondra repens formula granules sample (batch number: 23050029) 1 part, prepare the test sample solution according to the preparation method of the test sample solution determined in item “3.2” under Example 1, and sample by the chromatographic conditions under item “3.1” in Example 1, to investigate the influence of the separation of chromatographic peaks under 20 ℃, 25 ℃, and 30 ℃ temperatures. The results are shown in Table 13. Figure 12 , Table 13.

[0127] Table 13 Investigation of different column temperatures

[0128]

[0129] Results: the separation effect of chromatographic peaks is good between 20 ℃ and 30 ℃ column temperatures, and the determination of hyperoside content is relatively close, with good durability.

[0130] 7.3 Investigation of chromatographic column

[0131] Method: Take Euphorbia humifusa formula granules sample (batch number: 23050029) 1 part, prepare the test sample solution according to the preparation method of the test sample solution determined in item “3.2” of Example 1, sample according to the chromatographic conditions in item “3.1” of Example 1, and analyze using three kinds of chromatographic columns, Eclipse Plus C18 (Agilent, 2.1 x 100 mm, 1.8 μm), HSS T3 (Waters, 2.1 x 100 mm, 1.8 μm), and SB C18 RRHD (Agilent, 2.1 x 100 mm, 1.8 μm) respectively. The results are shown in Table 14. Figure 13

[0132] Table 14 Comparison of different chromatographic columns

[0133]

[0134] Results: Hyperoside has good separation effect on the three different types of octadecylsilane-bonded silica gel chromatographic columns, and the measured content results are close, indicating that the chromatographic column has little effect on the determination results of the sample.

[0135] Example 3: Determination of the content of hyperoside in Euphorbia humifusa medicinal materials

[0136] Take three batches of Euphorbia humifusa medicinal material samples (provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.), powder (pass through a No. 3 sieve), accurately weigh about 1.0 g, place in a conical flask with a plug, accurately add 25 ml of 70% methanol, tightly plug, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the weight loss with 70% methanol, shake well, filter, take all the filtrate, evaporate to dryness, add 25 ml of water to the residue, redissolve, extract with ethyl acetate for 3 times, 25 ml each time, combine the ethyl acetate liquid, evaporate to dryness, add 70% methanol to dissolve and transfer to a 5 ml volumetric flask, add 70% methanol to the mark, shake well, filter, take the filtrate as the Euphorbia humifusa medicinal material test sample solution.

[0137] Sample according to the chromatographic conditions in item “3.1” of Example 1 for analysis, the results are shown in Table 15, Figure 14 .

[0138] Table 15 Content of hyperoside in Euphorbia humifusa medicinal materials

[0139]

[0140] The results show that hyperoside can be detected in Euphorbia humifusa medicinal materials, and this method can be used for the content determination of Euphorbia humifusa medicinal materials.

[0141] Example 4: Determination of the content of hyperoside in Euphorbia humifusa standard decoction ​

[0142] Preparation of Euphorbia humifusa standard decoction: Take 100 g of Euphorbia humifusa decoction pieces, weigh and place in a sand pot, soak in 1400 ml of water for 30 min, use YMW mechanical split decoction pot, first boil with strong fire, then simmer for 20 min with gentle fire, select 200 mesh standard sieve and filter while hot, cool the filtrate in a cold water bath; add 1200 ml of water for the second decoction, boil with strong fire, then simmer for 15 min with gentle fire, filter while hot with a 200 mesh standard sieve, cool the filtrate in a cold water bath; combine the two filtrates and concentrate under reduced pressure at 65°C; concentrate to a relative density of 1.03-1.07 (65°C), then divide the concentrated liquid into Westlin bottles, and place in a freeze dryer, freeze-dry, to obtain Euphorbia humifusa standard decoction.

[0143] Take 3 batches of Euphorbia humifusa standard decoction samples, grind finely, take about 0.3 g, weigh accurately, place in a conical flask with a stopper, add 25 ml of 70% methanol accurately, seal tightly, weigh again, ultrasonic treat (power 250 W, frequency 40 kHz) for 30 min, cool, weigh again, make up the weight loss with 70% methanol, shake well, filter, take the filtrate, extract with ethyl acetate 3 times, 25 ml each time, combine the ethyl acetate liquid, evaporate to dryness, add 70% methanol to dissolve and transfer to a 5 ml volumetric flask, add 70% methanol to the mark, shake well, filter, take the filtrate as the Euphorbia humifusa standard decoction test sample solution.

[0144] According to the chromatographic conditions in item "3.1" of Example 1, the results are shown in Table 16, Figure 15 .

[0145] Table 16 Content of hyperoside in Euphorbia humifusa standard decoction

[0146]

[0147] The results show that hyperoside can be detected in Euphorbia humifusa standard decoction, and this method can be used for the content determination of Euphorbia humifusa standard decoction.

Claims

1. A method for detecting the content of hyperoside in a Euphorbia humifusa sample, characterized in that, It comprises the following steps: (1) preparing a sample solution of Euphorbia humifusa and a control solution of hyperoside; (2) detecting the control solution and the sample solution respectively by using an ultra-high performance liquid chromatograph, and comparing the chromatograms of the control solution and the sample solution; the mobile phase used in the detection is methanol-acetonitrile-0.1% formic acid solution; (3) calculating the content of hyperoside in the sample of Euphorbia humifusa by using the regression equation of the injection amount and the peak area of hyperoside.

2. The method of claim 1, wherein, The sample of Euphorbia humifusa in step (1) includes crude drugs, decoction pieces, standard decoction, formula granules or preparations.

3. The method of claim 1, wherein, The preparation method of the sample solution of Euphorbia humifusa in step (1) is as follows: taking the powder of Euphorbia humifusa, accurately weighing, adding an extraction solvent, extracting, filtering, evaporating the filtrate, dissolving the residue with water, extracting with an extraction reagent, evaporating the extract, dissolving the residue with the extraction solvent, constant volume, filtering, and taking the filtrate to obtain the sample solution.

4. The method of claim 3, wherein, The extraction solvent includes water, 30% to 100% methanol, and the extraction reagent is ethyl acetate.

5. The method of claim 3, wherein, The mass-volume ratio of the powder of Euphorbia humifusa to the extraction solvent is 1g:30 to 100mL.

6. The method of claim 3, wherein, The extraction method includes ultrasonic treatment, heating reflux or shaking.

7. The method of claim 3, wherein, The extraction time is 15 to 60 minutes.

8. The method of claim 1, wherein, The conditions of the ultra-high performance liquid chromatograph in step (2) are as follows: octadecylsilane-bonded silica gel as the filler, column temperature of 20 to 30℃, isocratic elution, detection wavelength of 255nm or 355nm, and the ratio of methanol-acetonitrile-0.1% formic acid solution is 28 to 30:1:69 to 71.

9. A method of recovering hyperoside from a sample of Euphorbia humifusa, characterized by, It comprises the following steps: (1) taking the powder of Euphorbia humifusa, accurately weighing, adding an extraction solvent, extracting, filtering, evaporating the filtrate, dissolving the residue with water, extracting with an extraction reagent, evaporating the extract, dissolving the residue with the extraction solvent, constant volume, filtering, and taking the filtrate to obtain the sample solution; (2) injecting the sample solution into an ultra-high performance liquid chromatograph, and using methanol-acetonitrile-0.1% formic acid solution as the mobile phase to recover hyperoside.

10. The use of the method of any one of claims 1 to 8 in the quality control or quality detection of Euphorbia humifusa.

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