A method for extracting and determining lilyside A and lilyside H in lily medicinal material

Abstract

The present invention provides a method for extracting robinoside A and robinoside H from lily medicinal materials, comprising: adding dilute methanol to dissolve a lily medicinal material sample and then ultrasonically extracting to provide a desired extract. The present invention further provides a method for determining robinoside A and robinoside H in lily medicinal materials. The present invention also provides a method for determining robinoside A and robinoside H in lily medicinal materials and its use in quality detection of lily medicinal materials. The method for extracting and determining robinoside A and robinoside H from lily medicinal materials provided by the present invention has good linear relationship, good repeatability, good precision, high accuracy and good stability, can effectively extract and accurately determine the content of robinoside A and robinoside H in lily medicinal materials, truly reflects the quality difference of robinoside A and robinoside H in lily medicinal materials, and comprehensively improves the quality control system of lily medicinal materials.

Description

Technical Field

[0001] The invention belongs to the technical field of detection of components of traditional Chinese medicines, and relates to a method for extracting and determining glycoside A and glycoside H in lily medicinal materials, and specifically relates to a method for extracting glycoside A and glycoside H in lily medicinal materials and a method for simultaneously determining the contents of glycoside A and glycoside H in lily medicinal materials by using high performance liquid chromatography. Background Art

[0002] Lily is the dried fleshy scales of Lilium lancifolium Thunb., Lilium brownii F.E.Brown var.viridulum Baker or Lilium pumilum DC. of the Liliaceae family. Lily scales mainly contain polysaccharides, phenolic acid glycerides, steroidal saponins, sterols, flavonoids, phenylpropanoids, alkaloids, phospholipids, inorganic salts and other ingredients. Phenolic acid glycerides are one of the major components of lily, including 1-O-feruloylglycerol, 1-Op-coumarylglycerol, lilyside A, lilyside B, lilyside C, lilyside D, lilyside E, lilyside H, etc., which have physiological functions such as antioxidant, anti-tumor, anti-proliferation, immunomodulatory and estrogen activity. Among them, lilyside A and lilyside H are isomers, and there is a mutual conversion process in lily. The results of pharmacological experiments show that it has significant antidepressant pharmacological activity.

[0003] Currently, the 2020 edition of the "Chinese Pharmacopoeia" only contains the content determination of lily polysaccharides, and lacks the content determination of characteristic indicator components.

[0004] The prior art document "Quality Rating and Analysis of Lily Medicinal Materials from Different Sources" (Liu Xiangdan, Chen Xun, Liu Changyu, Pei Gang, Zhou Ribao, Wang Zhi, Chen Naihong. Quality Evaluation and Analysis of Lily Medicinal Materials from Different Sources [J]. Journal of Hunan University of Traditional Chinese Medicine, 2019, 39(04): 480-484.) discloses a method for determining the content of lily glycoside A in lily medicinal materials, wherein the HPLC method is used to determine the content of lily glycoside A. The results show that the content of lily glycoside A in 54 batches of lily samples ranges from 0.019 to 0.599%. The chromatographic conditions of the method are: the chromatographic column is Supersil ODS-B (5μm, 4.6*250mm), the column temperature is 30℃, the injection volume is 10μl, the flow rate is 1.0ml / min, the mobile phase is 0.1% phosphoric acid aqueous solution: acetonitrile = 85:15 (V / V), and the detection wavelength is 310nm. This document does not report that lilyside A is unstable and has the problem of structural transformation, nor does it record a method for simultaneously determining the mutually convertible lilyside A and its isomer lilyside H. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a method for extracting and determining kingiglycoside A and kingiglycoside H in lily medicinal materials, which can effectively extract and accurately determine the content of kingiglycoside A and kingiglycoside H in lily medicinal materials.

[0006] To achieve the above-mentioned and other related purposes, the present invention provides a method for extracting lilyside A and lilyside H from lily medicinal materials in a first aspect, comprising: dissolving the lily medicinal material sample in dilute methanol and then ultrasonically extracting to provide the desired extract.

[0007] Preferably, the lily medicinal material sample is lily medicinal material powder obtained by crushing and sieving the lily medicinal material.

[0008] Preferably, the sieve is a No. 3 sieve.

[0009] Preferably, the dilute methanol is a methanol-water solution.

[0010] Preferably, the methanol aqueous solution is a methanol aqueous solution with a volume percentage of 30 to 70%, preferably 40 to 60%.

[0011] Preferably, the ratio of the mass of the added lily medicinal material sample to the volume of the added dilute methanol is 1:20-80, preferably 1:25-75, g / mL. For example, 1:25-50, 1:50-75, 1:25, 1:50, 1:75, g / mL.

[0012] Preferably, the ultrasonic extraction time is 40 to 80 minutes, preferably 45 to 75 minutes.

[0013] Preferably, the power of the ultrasonic extraction is 200-350W.

[0014] Preferably, the frequency of the ultrasonic extraction is 40-59 KHz.

[0015] Preferably, the ultrasonic extraction is followed by cooling, centrifugation and filtration.

[0016] Preferably, the step of cooling the mixture to room temperature is 20-30°C.

[0017] Preferably, the centrifugation time is 5-15 min.

[0018] Preferably, the centrifugal rotation speed is 8000-12000r / min.

[0019] Preferably, dilute methanol is added before centrifugation to make up for the weight loss, so as to quantify the sample.

[0020] Preferably, the filtration is to take the supernatant through a filtration membrane, discard the initial filtrate, and then take the subsequent filtrate.

[0021] More preferably, the filter membrane is a 0.22 μm filter membrane.

[0022] The second aspect of the present invention provides a method for determining the content of lilyside A and lilyside H in lily medicinal materials, comprising: using the extract obtained by the above-mentioned extraction method as a test solution, and adopting high performance liquid chromatography (HPLC) to detect and determine the content of lilyside A and lilyside H in the test solution.

[0023] Preferably, the CAS number of the oxalisin A is 114420-66-5, and the CAS number of the oxalisin H is 126239-77-8.

[0024] Preferably, high performance liquid chromatography (HPLC) is used for detection, comprising the following steps:

[0025] 1) Preparation of mixed reference solution: Add dilute methanol to dissolve the reference substances of lilioside A and lilioside H and make up to volume to prepare a mixed reference solution;

[0026] 2) Sample detection: Use high performance liquid chromatography (HPLC) to detect the test solution and the mixed reference solution in step 1), respectively, compare the retention time for qualitative analysis, and use the external standard method for quantitative analysis to determine the content of oxalisin A and oxalisin H in the test solution.

[0027] Preferably, in step 1), the mixed reference substance solution is prepared by first adding dilute methanol to prepare a mixed reference substance stock solution, and then diluting it step by step.

[0028] More preferably, in the mixed reference stock solution, the concentration of the oxalisin A is 20 μg / mL, and the concentration of the oxalisin H is 4 μg / mL.

[0029] Preferably, in step 1), the content of oxalisin A in the mixed reference solution ranges from 0.08624 to 0.8624 μg / mL, and the content of oxalisin H ranges from 0.01668 to 0.1668 μg / mL.

[0030] Preferably, in step 1), the concentration ratio of WBHGA to WBHGH in the same mixed reference solution is 5.0-5.5:1, so that WBHGA and WBHGH reach reaction equilibrium and no longer undergo mutual conversion.

[0031] Preferably, in step 1), the dilute methanol is a methanol-water solution.

[0032] More preferably, the methanol aqueous solution is a methanol aqueous solution with a volume percentage of 30 to 70%, preferably 40 to 60%.

[0033] Preferably, in step 2), in the high performance liquid chromatography (HPLC), the detector is an ultraviolet-visible spectroscopic detector (UV) or a diode array detector (DAD).

[0034] Preferably, in step 2), the chromatographic column in the high performance liquid chromatography is C 18 Chromatographic column.

[0035] More preferably, the chromatographic column in the high performance liquid chromatography is a Waters TM XBridge TM C 18 Chromatographic column (column length 150mm, inner diameter 4.6mm, filler particle size 3.5μm) or Waters TM XSelect TM HSS C 18 Chromatographic column (column length: 150 mm, inner diameter: 4.6 mm, filler particle size: 3.5 μm).

[0036] Preferably, in step 2), the detection wavelength in the high performance liquid chromatography is 310-314 nm. More preferably, the detection wavelength is 312 nm.

[0037] Preferably, in step 2), the column temperature in the high performance liquid chromatography is 30-40°C.

[0038] Preferably, in step 2), the flow rate in the high performance liquid chromatography is 0.9 to 1.1 mL / min.

[0039] Preferably, in step 2), the injection volume in the high performance liquid chromatography method is 5 to 20 μL.

[0040] Preferably, in step 2), in the high performance liquid chromatography, the mobile phase is acetonitrile-0.09-0.11% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.09-0.11% phosphoric acid aqueous solution; the analysis time is 30 min; and gradient elution is used.

[0041] The above-mentioned 0.09-0.11% phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.09-0.11%.

[0042] More preferably, the specific procedure of the gradient elution is shown in Table 1, which is:

[0043] 0-15min, the volume ratio of phase A:phase B was 7-13:93-87-7-13:93-87;

[0044] 15-15.1min, the volume ratio of phase A:phase B is 7-13:93-87-20-40:80-60;

[0045] 15.1-22min, the volume ratio of phase A:phase B is 20-40:80-60-20-40:80-60;

[0046] 22-25min, the volume ratio of phase A:phase B is 20-40:80-60-7-13:93-87;

[0047] 25-30min, the volume ratio of phase A:phase B is 7-13:93-87-7-13:93-87.

[0048] Table 1

[0049]

[0050] Most preferably, the specific procedure of the gradient elution is:

[0051] 0-15min, volume ratio of phase A:phase B is 10:90-10:90;

[0052] 15-15.1min, the volume ratio of phase A:phase B is 10:90-30:70;

[0053] 15.1-22min, the volume ratio of phase A:phase B is 30:70-30:70;

[0054] 22-25min, the volume ratio of phase A:phase B is 30:70-10:90;

[0055] 25-30min, the volume ratio of phase A:phase B is 10:90-10:90.

[0056] Preferably, in step 2), the external standard method comprises the following steps:

[0057] A) preparing a series of mixed reference substance solutions of different concentrations according to step 1), and performing HPLC detection respectively, obtaining the linear relationship between the chromatographic peak areas of leucoside A and leucoside H and the corresponding contents of leucoside A and leucoside H, drawing the corresponding standard working curves, and calculating the regression equations of the standard working curves of leucoside A and leucoside H respectively;

[0058] B) The test solution is subjected to HPLC detection, and the chromatographic peak areas of the obtained glycoside A and glycoside H are substituted into the regression equation of the corresponding standard working curve of glycoside A and glycoside H in step A) to calculate the content of glycoside A and glycoside H in the test solution.

[0059] More preferably, in the standard working curve, the chromatographic peak areas of oxalisin A and oxalisin H are used as the ordinate (Y-axis), and the corresponding contents (ie, concentrations) of oxalisin A and oxalisin H are used as the abscissa (X-axis).

[0060] The third aspect of the present invention provides a method for extracting roquetiaside A and roquetiaside H from lily medicinal materials, and / or a method for determining roquetiaside A and roquetiaside H in lily medicinal materials, and the use of the method in quality inspection of lily medicinal materials.

[0061] As described above, the present invention provides a method for extracting and determining oxalisin A and oxalisin H in lily medicinal materials, which uses optimized pretreatment conditions and instrument detection methods to accurately detect oxalisin A and oxalisin H in lily medicinal materials quantitatively and qualitatively. In particular, for oxalisin A and oxalisin H, which are easy to convert into each other, the extraction and determination method can make the separation degree of oxalisin A and oxalisin H meet the requirements of the pharmacopoeia, and the WBHGA and WBHG reference substance solutions are stable and do not convert. While accurately detecting, it can avoid the influence of the mutual conversion of oxalisin A and oxalisin H on the detection results to a certain extent, improve the accuracy of the detection, and meet the detection requirements.

[0062] The method is simple to operate and easy to control. The prepared extract has good stability. The standard curves of the two components determined have good linear relationships within their respective ranges. The method has good repeatability, good precision, high accuracy, and good stability. The accuracy meets the pharmacopoeia standards and the method has good durability. The method can effectively extract and accurately determine the contents of lilyside A and lilyside H in lily medicinal materials, truly reflect the quality difference between lilyside A and lilyside H in lily medicinal materials, comprehensively improve the quality control system of lily medicinal materials, and provide a basis for the quality control of lily medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 It shows the HPLC chromatograms of blank solvent, lilyoside A reference solution, lilyoside H reference solution, lilyoside A and lilyoside H mixed reference solution and lily medicinal material test solution under the specified wavelength conditions in the present invention. DETAILED DESCRIPTION

[0064] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.

[0065] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0066] The reagents and instruments used in the following examples are as follows:

[0067] 1. Reagents

[0068] Lily medicinal material (origin: Longhui, Hunan, origin: Lilium brownii FEBrownvar. viridulum Baker). Wangbaiheglycoside A reference substance (Shanghai Shidande Standard Technology Service Co., Ltd., batch number 10874, purity ≥ 98.0%), Wangbaiheglycoside H reference substance (Shanghai Shidande Standard Technology Service Co., Ltd., batch number 9560, purity ≥ 98.0%).

[0069] Acetonitrile (chromatographic grade, Thermo Fisher Scientific Inc.); phosphoric acid (chromatographic grade, Shanghai Anpu Laboratory Technology Co., Ltd.); anhydrous methanol (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); ultrapure water was prepared by a Milli-Q ultrapure water treatment system.

[0070] 2. Instruments

[0071] Waters e2695-2498 high performance liquid chromatograph and Empower chromatography workstation (Waters, USA); Waters Acquity Arc high performance liquid chromatograph and Empower chromatography workstation (Waters, USA); Agilent 1260 InfinityⅡ high performance liquid chromatograph and OpenLab CDS workstation (Agilent, USA); XSR205 DU / A electronic balance and ME204E / 02 electronic balance (Mettler Toledo (Shanghai) Co., Ltd.); SK7200H ultrasonic cleaning machine (Shanghai Kedao Ultrasonic Instrument Co., Ltd.); Milli-Q Direct A10 water purifier (Merck Chemical Technology (Shanghai) Co., Ltd.); Waters TM XBridge TM C18 (3.5μm, 4.6×150mm) chromatographic column, Waters TM XSelect TM HSS C18 (3.5 μm, 4.6×150 mm) chromatographic column (Agilent, USA).

[0072] Example 1

[0073] After the lily medicinal material sample is crushed, it is passed through a No. 3 sieve. The obtained lily medicinal material powder is accurately weighed to 1g, placed in a stoppered conical bottle, and 50mL of 50% methanol aqueous solution is accurately added, the bottle is sealed, and the weight is weighed. Ultrasonic extraction is performed at a power of 350W and a frequency of 53kHz for 60 minutes, cooled, and the weight is weighed again, and the lost weight is supplemented with 50% methanol aqueous solution, shaken, and centrifuged at a speed of 10000r / min for 10 minutes. The supernatant is filtered through a membrane, and after discarding the initial filtrate, the subsequent filtrate is taken to obtain the required extract sample 1#.

[0074] Example 2

[0075] After the lily medicinal material sample is crushed, it is passed through a No. 3 sieve. The obtained lily medicinal material powder is accurately weighed to 1g, placed in a stoppered conical bottle, and 60mL of 40% methanol aqueous solution is accurately added, the bottle is sealed, and the weight is weighed. Ultrasonic extraction is performed at a power of 250W and a frequency of 50kHz for 50 minutes, cooled, and the weight is weighed again. The lost weight is supplemented with 40% methanol aqueous solution, shaken, and centrifuged at a speed of 11000r / min for 12 minutes. The supernatant is filtered through a membrane, and after discarding the initial filtrate, the subsequent filtrate is taken to obtain the required extract sample 2#.

[0076] Example 3

[0077] 1. Preparation of test solution

[0078] The extract sample 1# prepared in Example 1 was used as the test solution 1#.

[0079] 2. Preparation of mixed reference solution

[0080] Take the reference substances of lilioside A and lilioside H, weigh them accurately, and add 50% methanol aqueous solution to prepare a mixed reference substance stock solution. In the mixed reference substance stock solution, the concentration of lilioside A is 20 μg / mL, and the concentration of lilioside H is 4 μg / mL.

[0081] The mixed reference substance stock solution was then diluted stepwise with dilute methanol and fixed to volume to prepare a series of mixed reference substance solutions of different concentrations. In a series of mixed reference substance solutions of different concentrations, the content of lilyside A ranged from 0.08624 to 0.8624 μg / mL, and the content of lilyside H ranged from 0.01668 to 0.1668 μg / mL. The concentration ratio of lilyside A to lilyside H in the same mixed reference substance solution was 5.0 to 5.5:1.

[0082] 3. Determination

[0083] High performance liquid chromatography (HPLC) was used to detect the test solution 1# and a series of mixed reference solutions of different concentrations, and the retention time was compared for qualitative analysis, and the external standard method was used for quantitative analysis. That is, a series of mixed reference solutions of different concentrations were respectively subjected to HPLC detection, and the linear relationship between the chromatographic peak area of ​​lilyoside A and lilyoside H and the corresponding concentration of lilyoside A and lilyoside H was obtained, and the corresponding standard working curves were drawn, and the regression equations of the standard working curves of lilyoside A and lilyoside H were respectively calculated. Then, the test solution 1# was subjected to HPLC detection, and the chromatographic peak areas of lilyoside A and lilyoside H obtained were substituted into the regression equations of the corresponding standard working curves of lilyoside A and lilyoside H to calculate the concentrations of lilyoside A and lilyoside H in the test solution 1#.

[0084] Among them, the high performance liquid chromatography method includes the following detection conditions:

[0085] The detector is UV-visible spectrophotometer (UV); the chromatographic column is Waters TM XBridge TM C 18 Chromatographic column (column length: 150 mm, inner diameter: 4.6 mm, filler particle size: 3.5 μm); detection wavelength: 312 nm; column temperature: 35° C.; flow rate: 1.0 mL / min; injection volume: 10 μL; mobile phase: acetonitrile-0.10% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.10% phosphoric acid aqueous solution; analysis time: 30 min; gradient elution.

[0086] The specific procedure of gradient elution is:

[0087] 0-15min, volume ratio of phase A:phase B is 10:90-10:90;

[0088] 15-15.1min, the volume ratio of phase A:phase B is 10:90-30:70;

[0089] 15.1-22min, the volume ratio of phase A:phase B is 30:70-30:70;

[0090] 22-25min, the volume ratio of phase A:phase B is 30:70-10:90;

[0091] 25-30min, the volume ratio of phase A:phase B is 10:90-10:90.

[0092] Example 4

[0093] Take 50 mL of 50% methanol aqueous solution and perform extraction according to Example 1 to obtain a blank solvent sample.

[0094] Take the reference substance of lilyoside A and prepare it according to step 2 of Example 3 to obtain a reference substance solution of lilyoside A. Similarly, take the reference substance of lilyoside H and prepare it according to step 2 of Example 3 to obtain a reference substance solution of lilyoside H.

[0095] The blank solvent sample, Wang Baihe glycoside A reference solution, and Wang Baihe glycoside H reference solution were measured according to step 3 of Example 3, and the measurement results were compared with the results of test solution 1# and mixed reference solution. Specific data are shown in Figure 1 .Depend on Figure 1 It can be seen that the method can effectively separate oxalisin A and oxalisin H, keep the contents of oxalisin A and oxalisin H stable, avoid the mutual conversion of oxalisin A and oxalisin H, and improve the accuracy of detection.

[0096] Example 5

[0097] 1. Preparation of test solution

[0098] The extract sample 1# prepared in Example 1 was used as the test solution 2#.

[0099] 2. Preparation of mixed reference solution

[0100] Take the reference substances of lilioside A and lilioside H, and prepare a series of mixed reference substance solutions of different concentrations according to step 2 in Example 3.

[0101] 3. Determination

[0102] High performance liquid chromatography (HPLC) was used to detect the test solution 2# and a series of mixed reference solutions of different concentrations, and the retention time was compared for qualitative analysis, and the external standard method was used for quantitative analysis. The concentrations of oxalisin A and oxalisin H in the test solution 2# were obtained. The specific quantitative process was the same as step 3 in Example 3.

[0103] Among them, the high performance liquid chromatography method includes the following detection conditions:

[0104] The detector is a diode array detector (DAD); the chromatographic column is a Waters TM XSelect TM HSS C 18 Chromatographic column (column length: 150 mm, inner diameter: 4.6 mm, filler particle size: 3.5 μm); detection wavelength: 312 nm; column temperature: 34° C.; flow rate: 1.5 mL / min; injection volume: 15 μL; mobile phase: acetonitrile-0.10% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.10% phosphoric acid aqueous solution; analysis time: 30 min; gradient elution.

[0105] The specific procedure of gradient elution is:

[0106] 0-15min, the volume ratio of phase A:phase B is 11:89-11:89;

[0107] 15-15.1min, the volume ratio of phase A:phase B is 11:89-35:65;

[0108] 15.1-22min, the volume ratio of phase A:phase B is 35:65-35:65;

[0109] 22-25min, the volume ratio of phase A:phase B is 35:65-11:89;

[0110] 25-30min, the volume ratio of phase A:phase B is 11:89-11:89.

[0111] Example 6

[0112] The method for detecting lilyside A and lilyside H in the lily medicinal material sample of the present invention was methodologically verified, and the performance index results are as follows.

[0113] 1. Linear relationship

[0114] Accurately weigh appropriate amounts of reference substances of lilioside A and lilioside H, and add 50% methanol aqueous solution according to step 2 in Example 3 to prepare a series of 6 mixed reference substance solutions of different concentrations. In the 6 mixed reference substance solutions, 1 ml of lilioside A contains 86.24, 64.68, 43.12, 21.56, 17.248, and 8.624 μg, respectively, and 1 ml of lilioside H contains 16.68, 12.51, 8.34, 4.17, 3.336, and 1.668 μg, respectively. Then, according to the chromatographic conditions of step 3 in Example 3, a series of 1 μL mixed reference solution were accurately pipetted and injected into the high performance liquid chromatograph, and the injection mass (μg) of glycoside A and glycoside H was used as the abscissa (X-axis), and the corresponding peak areas of glycoside A and glycoside H were used as the ordinate (Y-axis) for regression, and a standard curve was drawn. The standard regression equation, correlation coefficient, and linear range of glycoside A and glycoside H were calculated. The specific results are shown in Table 2.

[0115] According to Table 2, the linear relationship between oxalisin A and oxalisin H is good within the respective injection mass concentration ranges, indicating that the method of the present invention has a wide linear range and high accuracy.

[0116] Table 2

[0117]

[0118] 2. Stability

[0119] Take the lily medicinal material sample of batch number A2021092801, prepare the test solution according to step 1 in Example 3, and analyze the samples at 0h, 1h, 2h, 4h, 8h, 12h, 16h, 20h, and 24h according to the chromatographic conditions of step 3 in Example 3, and record the chromatographic peak area data of Wang Bailioside A and Wang Bailioside H. The specific results are shown in Table 3. The results show that the RSD of the chromatographic peak area of ​​Wang Bailioside A and Wang Bailioside H within 24h is less than 2%, indicating that the test solution has no effect on the results when detected within 24h, and has good stability.

[0120] Table 3

[0121]

[0122] 3. Instrument precision

[0123] Lily medicinal material sample with batch number A2021092801 was taken, and the test solution was prepared according to step 1 in Example 3. The sample was continuously injected and tested 6 times according to the chromatographic conditions of step 3 in Example 3, and the chromatographic peak area data was recorded. The results are shown in Table 4. The results show that the instrument precision RSD of Wang Bailioside A and Wang Bailioside H are both less than 1%, indicating that the instrument precision is good.

[0124] Table 4

[0125]

[0126] 4. Repeatability

[0127] Take 6 portions of lily medicinal material sample with batch number A2021092801, accurately weigh them, prepare the test solution according to step 1 in Example 3, measure according to the chromatographic conditions of step 3 in Example 3, calculate the content, and the specific results are shown in Table 5. The results show that the RSD of the content of lilyside A and lilyside H are both less than 1%, indicating that the method has good repeatability.

[0128] Table 5

[0129]

[0130] 5. Spike recovery rate

[0131] Weigh 9 portions of lily medicinal material powder samples of known concentration (known contents of lilyside A and lilyside H are 1.0004 mg / g and 0.1173 mg / g, respectively), 0.5 g per portion, accurately weighed. Accurately add lilyside A reference substance and lilyside H reference substance, respectively, the amount of reference substance added is 50%, 100% and 150% of the content in the test sample (three replicates for each concentration), prepare the test solution according to step 1 in Example 3, and according to the chromatographic conditions of step 3 in Example 3, respectively, inject and analyze, record the peak area, calculate the sample recovery rate and RSD of each component according to the measured amount and the added amount, the results are shown in Table 6. It can be seen from Table 6 that the sample recovery rates of lilyoside A are between 90% and 108%, and the sample recovery rates of lilyoside H are between 85% and 110%, and the RSDs are less than 1%, which meet the relevant requirements under the "9101 Guidelines for Validation of Analytical Methods" of the 2020 edition of the "Chinese Pharmacopoeia" (Part 4).

[0132] Table 6

[0133]

[0134] 6. Intermediate precision

[0135] Different experimenters from the technical center of Shanghai Hutchison Pharmaceuticals Co., Ltd. took samples of lily medicinal materials from the same batch (batch number A2021092801), prepared the test solution according to step 1 in Example 3, injected the sample and tested it according to the chromatographic conditions of step 3 in Example 3, and calculated the content of lily glycoside A and lily glycoside H. The results are shown in Table 7. The results show that the relative deviation of the detection amount of lily glycoside A and lily glycoside H determined by different experimenters is less than 0.5%. At the same time, the difference between the two determinations is 0.07% (i.e., the relative deviation of the total detection amount of WBHGA+WBHGH), and the intermediate precision is good.

[0136] Table 7

[0137]

[0138] 7. Durability

[0139] The test solution was prepared according to step 1 in Example 3, and the test was performed according to the chromatographic conditions of step 3 in Example 3. According to the content of "Durability" under "9101 Validation Guidelines for Analytical Methods" of the 2020 edition of the Chinese Pharmacopoeia (Part IV), the changes in the detection values ​​of Wang Bailioside A and Wang Bailioside H after slight changes in pH, column temperature, chromatographic column, and liquid chromatography instrument were investigated. The specific results are shown in Table 8. The results show that after slight changes in pH, column temperature, chromatographic column, and liquid chromatography instrument, the detection values ​​of Wang Bailioside A and Wang Bailioside H changed little, and the method had good durability.

[0140] Table 8

[0141]

[0142] Example 7

[0143] 22 batches of lily medicinal material samples were collected, and the test solution was prepared according to step 1 in Example 3, and the chromatographic conditions of step 3 in Example 3 were used for determination. The specific results are shown in Table 9. The results showed that the content of lilyside A in the 22 batches of lily medicinal materials ranged from 863.38 to 1583.57 μg / g, and the content of lilyside H ranged from 107.33 to 256.32 μg / g. This method can be used for the quality control of lilyside A and lilyside H in lily medicinal materials.

[0144] Table 9

[0145]

[0146] In summary, the present invention provides a method for extracting and determining oxalisin A and oxalisin H in lily medicinal materials, which has good linear relationship, good repeatability, good precision, high accuracy and good stability, and can effectively extract and accurately determine the content of oxalisin A and oxalisin H in lily medicinal materials, truly reflect the quality difference of oxalisin A and oxalisin H in lily medicinal materials, and comprehensively improve the quality control system of lily medicinal materials. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

[0147] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for determining lilyside A and lilyside H in lily medicinal materials, comprising: The extract obtained by the extraction method of lily glycoside A and lily glycoside H in lily medicinal materials is used as the test solution, and the content of lily glycoside A and lily glycoside H in the test solution is determined by high performance liquid chromatography; The detection is carried out by high performance liquid chromatography, including the following steps: 1) Preparation of mixed reference solution: dissolve the reference substances of lilioside A and lilioside H in methanol-water solution and make up to volume to prepare mixed reference solution; 2) Sample detection: Use high performance liquid chromatography to detect the test solution and the mixed reference solution in step 1) respectively, compare the retention time for qualitative analysis, and use the external standard method for quantitative analysis to determine the content of oxalisin A and oxalisin H in the test solution; In step 2), the detection conditions of the high performance liquid chromatography include: the detector is a UV-visible spectrophotometer or a diode array detector; the chromatographic column is C 18 Chromatographic column; detection wavelength 310~314nm; mobile phase acetonitrile-0.09~0.11% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.09~0.11% phosphoric acid aqueous solution; analysis time 30min; gradient elution; The specific procedure of the gradient elution is: 0-15min, volume ratio of phase A:phase B was 10:90-10:90; 15-15.1min, the volume ratio of phase A:phase B is 10:90-30:70; 15.1-22min, the volume ratio of phase A:phase B is 30:70-30:70; 22-25min, the volume ratio of phase A:phase B is 30:70-10:90; 25-30min, volume ratio of phase A:phase B is 10:90-10:90; The method for extracting lilyside A and lilyside H from lily medicinal materials comprises: adding a lily medicinal material sample to a methanol aqueous solution and then ultrasonically extracting the sample to provide a desired extract.

2. The method for determining the content of lilyside A and lilyside H in lily medicinal materials according to claim 1, characterized in that: Step 1) includes any one or more of the following conditions: C1) The content of oxalisin A in the mixed reference solution is in the range of 0.08624-0.8624 μg / mL, and the content of oxalisin H is in the range of 0.01668-0.1668 μg / mL; C2) the concentration ratio of oxalisin A to oxalisin H in the same mixed reference solution is 5.0-5.5:1; C3) The methanol aqueous solution is a methanol aqueous solution with a volume percentage of 30-70%.

3. The method for determining the content of lilyside A and lilyside H in lily medicinal materials according to claim 1, characterized in that: In step 2), the detection conditions of the high performance liquid chromatography method further include any one or more of the following conditions: D1) Column temperature is 30~40℃; D2) Flow rate is 0.9~1.1mL / min; D3) The injection volume is 5~20μL.

4. The method for determining the content of lilyside A and lilyside H in lily medicinal materials according to claim 1, characterized in that: These include the following conditions: A1) the lily medicinal material sample is lily medicinal material powder obtained by crushing and sieving the lily medicinal material; A2) the methanol aqueous solution is a methanol aqueous solution with a volume percentage of 30-70%; A3) the ratio of the mass of the lily medicinal material sample added to the volume of the methanol aqueous solution added is 1:20-80; A4) The ultrasonic extraction time is 40 to 80 minutes; A5) The power of the ultrasonic extraction is 200-350W; the frequency of the ultrasonic extraction is 40-59KHz; A6) After the ultrasonic extraction, the mixture is cooled, centrifuged and filtered.

5. The method for determining the content of lilyside A and lilyside H in lily medicinal materials according to claim 4, characterized in that: The A6) item includes any one or more of the following conditions: B1) cooling to room temperature; B2) The centrifugation time is 5-15 minutes; B3) The centrifugal speed is 8000-12000r / min; B4) The filtration is to take the supernatant through the filter membrane, discard the initial filtrate, and take the subsequent filtrate.

6. Use of the method for determining the content of lilyside A and lilyside H in lily medicinal materials according to any one of claims 1 to 5 in quality inspection of lily medicinal materials.

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