Quality detection or quality control method of polygonatum odoratum formula granules

Abstract

The invention discloses a quality detection or quality control method of polygonatum odoratum formula granules, which comprises the following steps: preparation of a test solution, preparation of a reference solution, liquid chromatography detection and data analysis, and the data analysis comprises the step of calculating the content of (3R)-5, 7-Dihydroxy-6-methyl-3-(4 '-hydroxybenzyl) chroman-4-on in a polygonatum odoratum formula granule sample. The detection method is rapid and accurate in analysis, free of interference of negative samples, high in specificity, good in reproducibility and stability, low in detection cost and high in detection efficiency, and a basis can be provided for internal quality monitoring, control and quality evaluation of the polygonatum odoratum formula granules.

Description

Technical Field

[0001] The invention relates to a quality detection or quality control method for polygonatum odoratum formula granules, belonging to the technical field of drug detection. Background Art

[0002] Polygonatum odoratum is the dried rhizome of Polygonatum odoratum (Mill.) Druce of the Liliaceae family. It is a commonly used Chinese medicine with a slightly cold nature and sweet taste. It has the effects of nourishing yin and moistening dryness, promoting body fluid and quenching thirst. It is used to treat lung and stomach yin damage, dryness and cough, dry throat and thirst, internal heat and thirst. Modern pharmacological studies have shown that Polygonatum odoratum has the effects of strengthening the heart, lowering blood pressure, lowering blood sugar, lowering blood lipids, enhancing immune function, anti-aging, anti-fatigue, and anti-oxidation. It is often used in combination with other Chinese medicines in clinical practice to treat diseases such as yin damage, cough and thirst, asthenia and fever, easy hunger after digestion, frequent urination, etc., and has a strong clinical application value. In addition to medicinal use, Polygonatum odoratum is used as a health product and food, and there is a broad market demand and export.

[0003] Polygonatum odoratum contains a variety of chemical components, which mainly include steroidal saponins, homoisoflavonoids, volatile oils and polysaccharides. Flavonoids in Polygonatum odoratum are one of the main components of its pharmacological effects. Flavonoids are a natural antioxidant that has anti-aging and immune-enhancing effects. It has been reported in the literature (Zhou X, Zhang Y, Zhao H, et al. Antioxidant homoisoflavonoids from Polygonatum odoratum [J]. Food Chemistry, 2015, 186: 63-68.) that three new homoisoflavonoids and eight homoisoflavonoids isolated from Polygonatum odoratum all showed strong antioxidant activity, and compounds with dihydroxylated B rings showed stronger antioxidant activity than VC.

[0004] In recent years, the quality inspection methods for Polygonatum odoratum have mainly focused on sugar components, including the 2020 edition of the Chinese Pharmacopoeia, which uses colorimetry to determine the polysaccharide content in Polygonatum odoratum as a quality control method. However, this method is cumbersome and has poor accuracy. In addition, the addition of excipients during the production process of Polygonatum odoratum formula granules will interfere with the colorimetric determination of polysaccharides. Therefore, polysaccharide indicators are not suitable for quality control of Polygonatum odoratum formula granules. There are also reports on the determination of monosaccharides such as fructose and glucose as a quality control method for Polygonatum odoratum, but the monosaccharide specificity is poor and exists in many traditional Chinese medicines. For flavonoid components, efficient and green extraction and separation technologies and detection technologies need to be further developed and improved, so as to provide a solid foundation for the further development and utilization of Polygonatum odoratum and its quality control. 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 quickly and accurately detecting or controlling the quality of Polygonatum odoratum formula granules.

[0006] Technical solution: To solve the above technical problems, the present invention provides a method for detecting the content of (3R)-5,7-Di hydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one in Polygonatum odoratum formula granules, comprising the following steps:

[0007] (1) Take the powder of Polygonatum odoratum formula granules, dissolve it with the extraction solvent, weigh it, extract it, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, accurately take the filtrate, evaporate it to dryness, add 50% methanol to dissolve the residue, and obtain the test solution;

[0008] (2) Take (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one and add solvent to prepare a reference solution;

[0009] (3) Take the test solution and the reference solution, inject them into the ultra-high performance liquid chromatograph respectively, compare the detection spectrum of the test solution with the detection spectrum of the reference solution, and use the peak area of ​​the chromatographic peak to calculate the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one in the test sample according to the peak area normalization method.

[0010] Wherein, the extraction solvent in step (1) comprises 30%-100% methanol.

[0011] Preferably, the extraction solvent in step (1) comprises 50%-100% methanol.

[0012] Wherein, the volume of the extraction solvent in step (1) is 20-100 ml.

[0013] Preferably, the volume of the extraction solvent in step (1) is 25-100 ml.

[0014] Preferably, the volume percentage of alcohol in the alcohol aqueous solution is 100%.

[0015] Wherein, the dosage ratio of the Polygonatum odoratum formula granule powder to the extraction solvent is 1g:20-100mL, preferably 1g:25mL.

[0016] Wherein, the extraction method in step (1) includes ultrasonic extraction or heating reflux; the extraction time is 15-60 minutes.

[0017] Preferably, the extraction time in step (1) is 30-60 minutes.

[0018] The preferred extraction method is ultrasonic extraction.

[0019] The preferred extraction time is 15-30 min, more preferably 30 min.

[0020] Wherein, the chromatographic conditions of the ultra-high performance liquid chromatography described in step (3) are as follows: the mobile phase is acetonitrile: 0.1% phosphoric acid = 45:55, acetonitrile: 0.1% formic acid = 45:55 or acetonitrile: water = 40-60:40-60; the chromatographic column is ZORBAX SB-Aq, YMC-Triart-C18 or Shim-pack GIST C18-AQ; the elution mode is isocratic elution: the flow rate is 0.8-1.2 mL / min; the column temperature is 20-30° C.; and the detection wavelength is 260 nm to 330 nm.

[0021] The preferred mobile phase volume ratio is 45:55 acetonitrile-water binary system, the preferred detection wavelength is 292 nm, the preferred flow rate is 1 mL / min, and the preferred column temperature is 25°C.

[0022] The linear relationship between the peak area of ​​the chromatographic peak in step (3) and the injection amount of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one is: Y=-0.0157+58.186X, R=0.99992, wherein X is the injection amount of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one, and Y is the peak area of ​​the chromatographic peak; the injection amount of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one is 0.0100 μg to 0.0501 μg.

[0023] The present invention also provides a method for recovering (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one from the granules of Polygonatum odoratum formula, comprising the following steps:

[0024] (1) Take the powder of Polygonatum odoratum formula granules, dissolve it with the extraction solvent, weigh it, extract it, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, accurately take the filtrate, evaporate it to dryness, add 50% methanol to dissolve the residue, and obtain the test solution;

[0025] (2) The sample solution is injected into a high performance liquid chromatograph to recover (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one.

[0026] The present invention also provides application of the method in quality detection and / or quality control of Polygonatum odoratum formula granules.

[0027] The method of the invention uses (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one as an index and adopts high performance liquid chromatography to detect the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one to detect and monitor the intrinsic quality of the polygonatum odoratum formula granules.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The detection method of the yuzhu formula granules provided by the present invention has fast and accurate analysis, no interference from negative samples, strong specificity, good reproducibility and stability, the obtained chromatogram has a stable baseline, good separation, and a good chromatographic peak shape, low detection cost and high detection efficiency, and can accurately detect the content of isoflavone components in the yuzhu sample; 2. It provides a set of fast, efficient and feasible detection methods for the quality control of the yuzhu formula granules; 3. It can provide a basis for the intrinsic quality monitoring, control and quality evaluation of the yuzhu formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The UV spectrum of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one reference substance at a wavelength of 190nm to 400nm;

[0030] Figure 2 UPLC spectrum of the test product of Polygonatum odoratum formula granules under the conditions of mobile phase system and ratio;

[0031] Figure 3 UPLC spectra of the test samples of Polygonatum odoratum formula granules under different extraction solvent conditions;

[0032] Figure 4 is the linear relationship diagram of the reference substance;

[0033] Figure 5 This is a specificity test for the test product of Polygonatum odoratum formula granules;

[0034] Figure 6This is a delayed test of the test product of Polygonatum odoratum formula granules;

[0035] Figure 7 This is the UPLC spectrum of the test product of Yuzhu Formula Granule under different flow rate conditions;

[0036] Figure 8 This is the UPLC spectrum of the test product of Yuzhu Formula Granule under different column temperature conditions;

[0037] Fig. 9 This is the UPLC spectrum of the test sample of Polygonatum odoratum formula granules under different chromatographic column inspection conditions. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0039] 1. Instruments, reagents and samples

[0040] (1) Instruments: Thermo Vanquish high performance liquid chromatograph; Chameleon 7.2 workstation (Thermo Corporation), Agilent 1260 high performance liquid chromatograph, 1260 TCC detector, 1260 ALS autosampler, 1260 Quat pump VL quaternary pump, Chrome Leon chromatography workstation, KQ-250E ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.); ME204E / 02 electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); METTLER TOLEDO XP6 one-millionth balance, GKC114 temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); HY-4 oscillator (Jintan Kexing Instrument Factory); TGL-16C centrifuge (Shanghai Anting Scientific Instrument Factory); Milli-Q pure water system (Millipore Corporation).

[0041] (2) Reagents: acetonitrile (chromatographic grade, Thermo Fisher); water was ultrapure water; other reagents were of analytical grade.

[0042] (3) Samples:

[0043] Reference substance: (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one (batch number: 114008) was purchased from Shanghai Shidande Biotechnology Co., Ltd. for content determination. The content was 98% and no treatment was required before use.

[0044] Preparation of reference solution: Take an appropriate amount of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one, accurately weigh it, and add 50% methanol to make a solution with a concentration of 1 μg / mL to obtain the reference solution.

[0045] Test sample: Polygonatum odoratum formula granules (batch numbers 19060139, 19060149, and 19060159) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0046] Example 1 Determination of chromatographic conditions

[0047] 1. Determination of detection wavelength

[0048] Collect the UV absorption spectrum of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one reference substance at 210-400nm. The results are as follows Figure 1 As shown, in the ultraviolet absorption spectrum, the (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one reference substance has a high absorption between 260nm and 330nm, which can be used as the detection wavelength. Considering that there is a maximum absorption around 292nm, the detection wavelength is preferably 292nm.

[0049] 2. Selection of mobile phase

[0050] Take the Yuzhu Formula Granules (batch number: 19060139) of this product, grind it into fine powder, take about 1g, a total of 3 groups, 2 parallel portions in each group, accurately weigh, place in a stoppered conical bottle, accurately add 25ml of methanol, seal, weigh, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, let cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately draw 20ml of the filtrate, evaporate to dryness, add 50% methanol to dissolve the residue, transfer to a 5ml volumetric bottle, and obtain the test solution. Accurately draw 20μl of each test solution and inject it into the high performance liquid chromatograph. The chromatographic conditions of this embodiment are: YMC-Triart-C18 chromatographic column (column length is 250mm, inner diameter is 4.6mm, particle size is 5μm); elution mode adopts isocratic elution; flow rate is 1mL / min; column temperature is 30℃; detection wavelength is 292nm. The number of theoretical plates calculated based on the (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one peak should be no less than 3000. The mobile phase ratio was optimized according to the existing experimental conditions (condition S1: acetonitrile: 0.1% phosphoric acid = 45:55; condition S2: acetonitrile: 0.1% formic acid = 45:55; condition S3: acetonitrile: water = 40:60; condition S4: acetonitrile: water = 45:55; condition S5: acetonitrile: water = 50:50; condition S6: acetonitrile: water = 55:45; condition S7: acetonitrile: water = 60:40), see for details. Figure 2 .Depend on Figure 2 It can be seen that under the above conditions, the target components can obtain acceptable separation. There is little difference between formic acid, phosphoric acid and pure water for the aqueous phase. From the perspective of economy and environmental protection, an acetonitrile-water mobile phase system is used. The acetonitrile ratio of the S3 condition is low, and the detection time is long. Therefore, from the perspective of time cost, the acetonitrile-water ratio is preferably S4 to S7, which can save about 10 minutes compared to the S3 condition. The following experiment uses acetonitrile-water with a volume ratio of 45:55 as the mobile phase.

[0051] Example 2 Preparation of test solution

[0052] 1. Investigation of extraction solvent

[0053] Take an appropriate amount of Polygonatum odoratum formula granules (batch number: 19060139), grind it into powder, take about 1g, and make 5 groups in total, with 2 parallel portions in each group. Weigh accurately, place in a stoppered conical flask, and accurately add 25ml of water, 30% methanol, 50% methanol, 70% methanol, and methanol, respectively. Seal the flask, weigh the weight, and ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes. Let it cool, weigh the weight again, make up the lost weight with the corresponding solvent, shake well, filter, accurately draw 20ml of the filtrate, evaporate to dryness, add 50% methanol to dissolve the residue, and transfer to a 5ml volumetric flask to obtain. Accurately draw 20μl of each test solution, inject into a high performance liquid chromatograph, and determine according to the chromatographic conditions determined in Example 1 to calculate the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one. The results are shown in Table 1. Figure 3 .

[0054] Table 1 Comparison of different extraction solvents

[0055]

[0056] Conclusion: From the results, it can be seen that methanol has a higher extraction rate and a smaller relative standard deviation, so methanol is selected as the extraction solvent.

[0057] 2. Investigation of extraction methods

[0058] Take the Yuzhu Formula Granules (batch number: 19060139) of this product, grind it into fine powder, take about 1g, and make 3 groups in total, with 2 parts in each group, accurately weigh it, put it in a stoppered conical bottle, accurately add 25ml of methanol, seal it, weigh it, ultrasonically treat it (power 600W, frequency 40kHz), heat it under reflux, and shake it for 30 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, accurately take 20ml of the filtrate, evaporate it to dryness, add 50% methanol to dissolve the residue, and transfer it to a 5ml volumetric bottle to obtain it. Accurately take 20μl of each test solution, inject it into a high performance liquid chromatograph, and measure it according to the chromatographic conditions determined in Example 1 to calculate the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one. The results are shown in Table 2.

[0059] Table 2 Comparison of different extraction methods

[0060]

[0061]

[0062] Conclusion: The results show that the extraction efficiencies of ultrasonic extraction and heating reflux are similar. Considering the convenience of ultrasonic treatment, ultrasonic treatment was selected as the extraction method.

[0063] 3. Extraction time investigation

[0064] Take the formula granules of Polygonatum odoratum (batch number: 19060139), grind them into powder, take about 1g, and make 4 groups in total, with 2 parts in parallel for each group, accurately weigh them, put them in a stoppered conical bottle, accurately add 25ml of methanol, seal them, weigh them, and treat them with ultrasound (power 600W, frequency 40kHz) for 15 minutes, 45 minutes, 30 minutes, and 60 minutes respectively, let them cool, weigh them again, make up the lost weight with methanol, shake them well, filter them, accurately take 20ml of the filtrate, evaporate them to dryness, add 50% methanol to dissolve the residue, and transfer them to a 5ml volumetric bottle to obtain them. Accurately take 20μl of each test solution, inject them into a high performance liquid chromatograph, and measure them according to the chromatographic conditions determined in Example 1 to calculate the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one. The results are shown in Table 3.

[0065] Table 3 Comparison of different extraction times

[0066]

[0067]

[0068] Conclusion: The results show that the contents of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one measured at different extraction times are similar. To ensure sufficient extraction, the extraction time was determined to be 30 minutes.

[0069] 4. Investigation of extraction solvent volume

[0070] Take an appropriate amount of Yuzhu Formula Granules (batch number: 19060139), grind it into powder, take about 1g, and make 4 groups in total, with 2 parts in parallel for each group, accurately weigh it, put it in a stoppered conical bottle, accurately add 20ml, 25ml, 50ml, and 100ml of methanol respectively, plug it tightly, weigh it, and perform ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, accurately take 20ml of the filtrate, evaporate it to dryness, add 50% methanol to dissolve the residue, and transfer it to a 5ml volumetric bottle to obtain it. Accurately take 20μl of each test solution, inject it into a high performance liquid chromatograph, and measure it according to the chromatographic conditions determined in Example 1 to calculate the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one. The results are shown in Table 4.

[0071] Table 4 Comparison of different extraction solvent volumes

[0072]

[0073] Conclusion: The results show that the extraction rates of different extraction volumes are similar, and the measured contents of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one are similar, indicating that the extraction is basically complete when the extraction solvent volume is 20 mL. In order to ensure sufficient extraction, the extraction solvent volume is selected to be 25 mL.

[0074] Based on the above results, the chromatographic conditions used in the following Example 3 of the present invention are: YMC-Triart-C18 chromatographic column (column length 250mm, inner diameter 4.6mm, particle size 5μm); acetonitrile-water (45:55) as mobile phase; isocratic elution; flow rate 1mL / min; column temperature 30℃; detection wavelength 292nm. The theoretical plate number calculated based on the (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one peak should not be less than 5000.

[0075] The preparation method of the test solution is as follows: take an appropriate amount of the product, grind it into powder, take about 1g, accurately weigh it, put it in a stoppered conical flask, accurately add 25ml of methanol, weigh it, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, accurately absorb 20ml of the filtrate, evaporate it to dryness, dissolve the residue in 50% methanol and transfer it to a 5ml volumetric flask, add 50% methanol to the scale, shake it well, filter it, and take the filtrate to obtain it.

[0076] Example 3: Methodological verification of the method for detecting the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one in Polygonatum odoratum formula granules

[0077] 1. Linear

[0078] 5.0 μl, 10.0 μl, 15.0 μl, 20.0 μl and 25.0 μl of the reference solution ((3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one concentration of 2.00312 μg / mL) were accurately drawn and injected into the high performance liquid chromatograph. The standard curve was plotted with the peak area as the ordinate and the injection volume as the abscissa according to the above chromatographic conditions, and the regression equation was obtained: Y = -0.0157 + 58.186X, R = 0.99992. The results are shown in Table 5 and Figure 4 .

[0079] Table 5 Relationship between peak area integral value of reference substance and injection volume

[0080]

[0081] The results showed that when the injection amount of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one was in the range of 0.0100μg to 0.0501μg, there was a good linear relationship between the injection amount and the peak area value.

[0082] 2. Precision

[0083] 2.1 Instrument precision

[0084] Take Polygonatum odoratum formula granules (batch number: 19060139) and prepare the test solution according to the test solution preparation method determined in Example 2. Accurately pipette 20 μL and inject it into the high performance liquid chromatograph. Determine according to the chromatographic conditions determined in Example 1. Inject 6 times continuously, record the peak area measurement value, and calculate the relative standard deviation. The results are shown in Table 6.

[0085] Table 6 Instrument precision test

[0086]

[0087] The results show that the precision test of the instrument is good.

[0088] 2.2 Intermediate precision

[0089] Take Yuzhu Formula Granules (Batch No.: 19060139), and two experimenters prepared 3 test solutions according to the test solution preparation method determined in Example 2. 20 μL was injected on different instruments at different times, and the peak area value of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one was determined according to the chromatographic conditions determined in Example 1, and its content and RSD were calculated. The results are shown in Table 7.

[0090] Table 7 Intermediate precision test

[0091]

[0092] Results: The RSD of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one content measured by intermediate precision test was 0.92%, and the result was good.

[0093] 2.3 Repeatability

[0094] Take an appropriate amount of Polygonatum odoratum formula granules (batch number: 19060139), grind it into powder, take about 1 g, accurately weigh it, and make 6 parallel portions. Prepare the test solution according to the test solution preparation method determined in Example 2, inject 20 μL, and determine the peak area value of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one according to the chromatographic conditions determined in Example 1, calculate its content and RSD, and the results are shown in Table 8.

[0095] Table 8 Repeatability test results of samples

[0096]

[0097] Results: The RSD of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one content measured by repeatability test was 0.87%, and the result was good.

[0098] 3. Accuracy (sample recovery test)

[0099] Take 0.5 g of a sample with a known content (Yuzhu formula granules, (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenz yl)chroman-4-one content: 0.00831 mg / g), a total of 9 portions, accurately weighed, and added three kinds of reference solution of high, medium and low concentrations (the amount added in Table 9), respectively, and prepare the sample recovery test solution according to the test solution preparation method determined in Example 2. According to the chromatographic conditions of Example 1, 20 μL were injected respectively, and the recovery rate and RSD were calculated according to the following formula. The results are shown in Table 9.

[0100]

[0101] Table 9 Recovery test results

[0102]

[0103]

[0104] Results: The recovery rate of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one was between 93.26% and 101.66%, and the accuracy test was good.

[0105] 4. Exclusivity

[0106] The auxiliary materials added to the granules of Polygonatum odoratum formula are maltodextrin, silicon dioxide and magnesium stearate. This experiment investigates the effect of negative samples lacking Polygonatum odoratum on the content determination of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one in the granules of Polygonatum odoratum formula. The negative sample lacking Polygonatum odoratum (auxiliary materials) and the granules of Polygonatum odoratum formula were taken to prepare negative sample solutions and test sample solutions according to the test sample preparation method determined in Example 2, and the samples were tested according to the chromatographic conditions determined in Example 1. The results are shown in Table 1. Figure 5 .

[0107] Results: There was no chromatographic peak in the negative chromatogram at the retention time corresponding to the reference substance, indicating that the excipients and solvents had no interference with the determination of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one. This method was specific for determining the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one in Polygonatum odoratum granules.

[0108] 5. Latency

[0109] Take Yuzhubiao Formula Granules (Batch No.: 19060139), prepare the test solution according to the preparation method of the test solution determined in Example 2, and analyze the sample according to the chromatographic conditions obtained in Example 1. Under the same chromatographic conditions, keep the elution gradient when the acetonitrile ratio is the highest, double the elution time, record the chromatogram, and the results are shown in Figure 6 .

[0110] Results: No obvious chromatographic peaks were eluted after the original gradient elution, indicating that the chromatographic conditions basically met the principle of maximum information content.

[0111] 6. Durability

[0112] 6.1 Stability test

[0113] Take Yuzhubiao formula granules (batch number: 19060139) to prepare a test solution according to the preparation method of the test solution in Example 2, and measure according to the chromatographic conditions of Example 1 at 0h, 4h, 8h, 12h, 16h, and 24h, respectively, and record the change of the peak area of ​​(3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one, and calculate its RSD. The results are shown in Table 10.

[0114] Table 10 Stability test results

[0115]

[0116] Results: From the above data, it can be seen that the peak area RSD value of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one peak within 24 hours was 1.08%, which met the requirements of the system suitability test.

[0117] 6.2 Investigation of different flow rates

[0118] The Yuzhubiao formula granules (batch number: 19060139) were used to prepare the test solution according to the preparation method of the test solution in Example 2 and the chromatographic conditions of Example 1 were used to investigate the chromatographic peak separation effect at different flow rates (0.8 mL / min, 1.0 mL / min, 1.2 mL / min). The experimental results are shown in Tables 11 and Figure 7 .

[0119] Table 11 Investigation results of different flow rates

[0120]

[0121] Results: At different flow rates, the measured (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one contents were similar and the peak separation was good, indicating that the durability was good when the flow rate fluctuated in the range of 0.8mL / min to 1.2mL / min.

[0122] 6.3 Investigation of different column temperatures

[0123] The test solution was prepared by the method for preparing the test solution in Example 2 using Yuzhubiao Formula Granules (Batch No.: 19060139), and the chromatographic peak separation effect at different column temperatures (20°C, 25°C, 30°C) was investigated. Other chromatographic conditions were the same as in Example 2. The experimental results are shown in Tables 12 and Figure 8 .

[0124] Table 12 Investigation results of different column temperatures

[0125]

[0126] Results: Under different column temperatures, the chromatographic peak separation effect was good, and the measured (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one content was similar, indicating that the column had good durability when the temperature fluctuated within the range of 20℃~30℃.

[0127] 6.4 Investigation of different chromatographic columns

[0128] The test solution was prepared from Yuzhubiao Formula Granules (batch number: 19060139) according to the preparation method of the test solution in Example 2, and the separation effects of different chromatographic columns ZORBAX SB-Aq (Agilent, 4.6×250mm, 5.0μm), YMC-Triart-C18 (YMC, 4.6×250mm, 5.0μm), and Shim-pack GIST C18-AQ (Agilent, 4.6×250mm, 5.0μm) on the Yuzhu Formula Granules were investigated respectively. The other chromatographic conditions were the same as in Example 2. The experimental results are shown in Tables 13 and 14. Fig. 9 .

[0129] Table 13 Investigation results of different chromatographic columns

[0130]

[0131] Results: The separation of the three chromatographic columns was good and the retention time was moderate, which showed that the chromatographic columns had little influence on the determination results of the samples and this method had universal adaptability.

[0132] Example 4: Determination of the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one in Polygonatum odoratum formula granules

[0133] Take 3 batches of Polygonatum odoratum formula granules, prepare samples according to the preparation method of the test solution determined in Example 2, prepare reference solutions according to Example 2, and perform sample injection analysis according to the chromatographic conditions determined in Example 2, inject 20 μL, record the peak area, and calculate the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one. The specific results are shown in Table 14.

[0134] Table 14 Determination of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one content in different batches of Polygonatum odoratum formula granules

[0135]

[0136] The results show that the present invention can be used to determine the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl) in the yuzhu formula granules, providing technical support for the quality control of the yuzhu formula granules.

Claims

1. A method for detecting the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one in Polygonatum odoratum formula granules, characterized in that: The following steps are involved: (1) Take the powder of Polygonatum odoratum formula granules, dissolve it with the extraction solvent, weigh it, extract it, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, accurately take the filtrate, evaporate it to dryness, add 50% methanol to dissolve the residue, and obtain the test solution; (2) Take (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one and add solvent to prepare a reference solution; (3) Injecting the test solution and the reference solution into a high performance liquid chromatograph respectively, comparing the detection spectrum of the test solution with the detection spectrum of the reference solution, and calculating the content of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one in the test sample by using the peak area of ​​the chromatographic peak according to the peak area normalization method.

2. The method according to claim 1, characterized in that: The extraction solvent in step (1) comprises 30%-100% methanol.

3. The method according to claim 1, characterized in that: The volume of the extraction solvent in step (1) is 20-100 ml.

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

5. The method according to claim 1, characterized in that: The mobile phase of the ultra-high performance liquid chromatograph in step (3) is acetonitrile:0.1% phosphoric acid=45:55, acetonitrile:0.1% formic acid=45:55 or acetonitrile:water=40-60:40-60.

6. The method according to claim 1, characterized in that: The chromatographic column of the ultra-high performance liquid chromatograph in step (3) is ZORBAX SB-Aq, YMC-Triart-C18 or Shim-pack GIST C18-AQ.

7. The method according to claim 1, characterized in that: The elution mode of the ultra-high performance liquid chromatograph in step (3) is isocratic elution; the flow rate is 0.8-1.2 mL / min; the column temperature is 20-30° C.; and the detection wavelength is 260 nm to 330 nm.

8. The method according to claim 1, characterized in that: The linear relationship between the peak area of ​​the chromatographic peak and the injection amount of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one in step (3) is: Y=-0.0157+58.186X, R=0.99992, wherein X is the injection amount of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one, and Y is the peak area of ​​the chromatographic peak; the injection amount of (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one is 0.0100 μg to 0.0501 μg.

9. A method for recovering (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one from Polygonatum odoratum formula granules, characterized in that: The following steps are involved: (1) Take the powder of Polygonatum odoratum formula granules, dissolve it with the extraction solvent, weigh it, extract it, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, accurately take the filtrate, evaporate it to dryness, add 50% methanol to dissolve the residue, and obtain the test solution; (2) The sample solution is injected into a high performance liquid chromatograph to recover (3R)-5,7-Dihydroxy-6-methyl-3-(4'-hydroxybenzyl)chroman-4-one.

10. Application of the method according to any one of claims 1 to 9 in quality detection or quality control of Polygonatum odoratum formula granules.

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