A method for constructing a characteristic map of Rhizoma Lu

Abstract

The present invention belongs to the technical field of drug analysis, and in particular to a method for constructing a characteristic spectrum of Rhizoma Lucidum. The construction method includes the preparation of a test solution and a reference solution, and is determined according to high performance liquid chromatography; wherein, the chromatographic conditions of high performance liquid chromatography include: acetonitrile as mobile phase A, formic acid aqueous solution or phosphoric acid aqueous solution as mobile phase B, gradient elution; flow rate: 0-35min, flow rate is 0.1→X1ml / min; 35-45min, flow rate is any value in X1-X2ml / min. The spectrum obtained by the construction method has 8 common characteristic peaks, and these common characteristic peaks are well separated, the spectrum baseline is stable, the noise interference is small, the peak shape of the characteristic peak is good, and the separation degree is high, which provides a basis for the quality detection and control of Rhizoma Lucidum and its preparations, and realizes the characterization of the overall components of Rhizoma Lucidum and its preparations, and the spectrum obtained by the construction method has good reproducibility.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for constructing a characteristic spectrum of Rhizoma Lupuli. Background Art

[0002] Rhaponticum uniflorum (L.) DC. is a plant of Asteraceae. It is conical or flat, often twisted, of varying lengths, 1-2.5 cm in diameter, with dark brown, gray-brown or black-brown surface, rough, with longitudinal grooves and diamond-shaped reticular cracks; the outer layer is easy to peel off, the root head is swollen, with residual stems and scaly leaf bases, and gray-white villi on the top; the body is light, brittle, easy to break, the cross-section is uneven, gray-yellow, with cracks, some with star-shaped cracks in the center, gray-black or brown-black; it has a peculiar smell and a slightly bitter taste; it enters the stomach meridian.

[0003] The main components of the herb are phytoecdysone, terpenoids, and thiophenes, along with a small amount of acids and other types of compounds. Traditional medicine believes that the herb can clear away heat and detoxify, eliminate carbuncle, promote lactation, relax muscles and unblock meridians. It is used for breast carbuncle, carbuncle, back ulcer, scrofula, milk blockage, dampness and stiffness, and is included in the Pharmacopoeia of the People's Republic of China.

[0004] In recent years, the research of domestic and foreign medical workers on Qizhou Loulu, especially the quality standards of Qizhou Loulu preparations, is relatively simple, and cannot fully achieve accurate description and evaluation of the overall quality of Loulu products. In the prior art, the paper "HPLC method for simultaneous determination of the contents of six components in Loulu" published by Zhou Ting et al. provides a method for determining the contents of caffeic acid, glycyrrhizin, rosmarinic acid, oleanolic acid, β-sitosterol, and stigmasterol in Loulu medicinal materials. This detection method takes a long time, the baseline fluctuation of the spectrum is large, the proportion of the six index components in the chemical composition of Loulu is low, and the accuracy is low. For Loulu formula granules, this test method can only detect caffeic acid, and the detection information is not comprehensive, which is not suitable for the detection of Loulu formula granules. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the fingerprint spectrum of the radix sphaerocephala constructed by the prior art, such as incomplete detection information, few applicable preparation types, unstable spectrum baseline, large interference, and complex gradient elution procedure, thereby providing a method for constructing a characteristic spectrum of the radix sphaerocephala.

[0006] To this end, the present invention provides the following technical solutions.

[0007] The present invention provides a method for constructing a characteristic spectrum of a loquat, comprising the following steps:

[0008] Preparation of test sample solution: Prepare the test sample into test sample solution;

[0009] Preparation of reference solution: Prepare reference solution from control medicinal materials;

[0010] Determination: Determined by high performance liquid chromatography;

[0011] The chromatographic conditions of the high performance liquid chromatography method include: acetonitrile as mobile phase A, formic acid aqueous solution or phosphoric acid aqueous solution as mobile phase B, and gradient elution;

[0012] The gradient elution conditions were as follows: 0-35 min, 3→25% mobile phase A, 97→75% mobile phase B; 35-45 min, 25→80% mobile phase A, 75→20% mobile phase B;

[0013] The flow rate is: 0-35min, the flow rate is 0.1→X1ml / min; 35-45min, the flow rate is any value between X1-X2 ml / min; wherein, X1 is 0.1-0.2, and X2 is 0.1-0.2.

[0014] Among them, the flow rate of 0.1→X1ml / min means that the flow rate increases from 0.1ml / min to X1, and the flow rate of X1-X2ml / min means that the flow rate is any value between X1-X2.

[0015] The high performance liquid chromatography conditions also include: using an octadecylsilane bonded silica gel column as a chromatographic column, the chromatographic column specifications are: inner diameter 2.1 mm, column length 100 mm, particle size 1.7 μm; and / or,

[0016] The column temperature is 32-38°C; and / or,

[0017] The wavelength is 210-230nm; and / or,

[0018] The injection volume is 1-3 μL; and / or,

[0019] Use 0.01-0.03% phosphoric acid aqueous solution as mobile phase B.

[0020] The test sample is at least one of the following: Lophatherum gracile decoction pieces, Lophatherum formula granules, Lophatherum standard decoction freeze-dried powder, Lophatherum gracile extract powder and Lophatherum gracile medicinal materials.

[0021] The method for preparing the test solution comprises: taking the test sample, adding an extraction solvent, extracting, and filtering;

[0022] Preferably, when preparing the test solution, the extraction solvent is an organic solvent, preferably methanol.

[0023] The construction method, when the test sample is at least one of the formula granules of Rhizoma Lupuli, freeze-dried powder of Rhizoma Lupuli standard decoction and Rhizoma Lupuli extract powder, when preparing the test sample solution, the ratio of the mass of the test sample to the volume of the extraction solvent is (0.1-0.3) g: (5-20) ml.

[0024] When the test sample is at least one of the decoction pieces of Rhizoma Cyrthospermi and the medicinal materials of Rhizoma Cyrthospermi, the preparation method of the test sample solution comprises: taking the test sample, adding 15-20 times of water, decocting, filtering, evaporating the filtrate to dryness, and obtaining a water extract; adding an extraction solvent to the water extract for ultrasonic or reflux extraction, and obtaining a subsequent filtrate. The ratio of the water extract mass to the extraction solvent volume is (0.1-0.3) g: (5-20) ml.

[0025] When the test sample is at least one of the formula granules of Rhizoma Lumifuga, the freeze-dried powder of the standard decoction of Rhizoma Lumifuga and the extract powder of Rhizoma Lumifuga, the preparation method of the test sample solution comprises: taking the test sample, adding an extraction solvent, ultrasonic or reflux extraction, filtering, and taking a filtrate. The ratio of the mass of the test sample to the volume of the extraction solvent is (0.1-0.3) g: (5-20) ml.

[0026] The preparation method of the reference solution comprises: taking a reference medicinal material, adding water to extract, filtering and then evaporating to dryness to obtain a water extract; adding an extraction solvent to the water extract, extracting, and filtering;

[0027] Preferably, when preparing the reference solution, the extraction solvent is an organic solvent, preferably methanol.

[0028] Specifically, the preparation method of the reference solution includes: taking a reference medicinal material, adding 15-20 times water, decocting, filtering, evaporating the filtrate to dryness, and obtaining a water extract; adding an extraction solvent to the water extract for ultrasonic or reflux extraction, and obtaining a subsequent filtrate.

[0029] Furthermore, when preparing the reference solution, the ratio of the mass of the water extract to the volume of the extraction solvent is (1.0-2.0) g: (20-40) ml.

[0030] The characteristic spectrum obtained by the construction method includes 8 characteristic peaks;

[0031] Taking peak 6 as the reference peak, the specified values ​​of relative retention times of peaks 1, 2, 3, 4, 7 and 8 are 0.25, 0.41, 0.66, 0.70, 1.07 and 1.27 respectively;

[0032] Among them, the relative retention time of the characteristic peak is within ±10% of the specified value.

[0033] The construction method also includes the preparation of a turosterone reference solution and a β-ecdysterone reference solution;

[0034] Preferably, the steps of preparing the turxosterone reference substance solution include: taking a turxosterone reference substance and adding a solvent;

[0035] Preferably, the preparation step of the β-ecdysterone reference solution comprises: taking a β-ecdysterone reference solution and adding a solvent.

[0036] Further, each 1 ml of the turosterone reference solution contains 50-100 μg of the turosterone reference;

[0037] Each 1 ml of β-ecdysterone reference solution contains 0.15-0.2 mg of turosterone reference.

[0038] The technical solution of the present invention has the following advantages:

[0039] 1. A method for constructing a characteristic spectrum of Rhizoma Lumbricata provided by the present invention, the method comprising preparing a test solution and a reference solution, and determining the solution by high performance liquid chromatography; wherein the chromatographic conditions of the high performance liquid chromatography comprise: using acetonitrile as mobile phase A, using formic acid aqueous solution or phosphoric acid aqueous solution as mobile phase B, and gradient elution; the gradient elution conditions are as follows: 0-35 min, 3→25% mobile phase A, 97→75% mobile phase B; 35-45 min, 25→80% mobile phase A, 75→20% mobile phase B; the flow rate is: 0-35 min, the flow rate is 0.1→X1 ml / min; 35-45 min, the flow rate is any value between X1-X2 ml / min; wherein X1 is 0.1-0.2, and X2 is 0.1-0.2. The spectrum obtained by the construction method has 8 common characteristic peaks, and these common characteristic peaks are well separated. The spectrum baseline is stable, the noise interference is small, the peak shape of the characteristic peaks is good, and the separation degree is high. It provides a basis for the quality detection and control of the radix spatholobi and its preparations, realizes the characterization of the overall components of the radix spatholobi and its preparations, and the spectrum obtained by the construction method has good reproducibility.

[0040] This construction method can obtain 8 common characteristic peaks with only 2 elution levels, and the separation between the characteristic peaks is high, the elution level is simple, and the baseline of the obtained spectrum is stable.

[0041] The construction method provided by the present invention can better characterize the overall material basis components in the radix sutchuenensis preparation by controlling the chromatographic conditions, can control the relative retention time of the characteristic peaks of each component, and realize the quality control of the radix sutchuenensis preparation.

[0042] 2. The method for constructing the characteristic spectrum of the Rhizoma Lucidum provided by the present invention has good peak shape and separation degree of the characteristic peak obtained by the method, good repeatability, and better separation efficiency, peak capacity and sensitivity. The method for constructing is simple, time-consuming, and efficient, and has the advantages of being simple, stable, high in precision, and good in repeatability. The method provided by the present invention is scientific and reliable, can be applied to the construction of the characteristic spectrum of the Rhizoma Lucidum of different preparation types, can control the consistency of the quality of the Rhizoma Lucidum preparations, and quickly realize the quality control of the Rhizoma Lucidum of different types of preparations.

[0043] In the spectrum obtained by the construction method of the present invention, peak No. 5 is turosterone, peak No. 6 is β-ecdysterone, and the β-ecdysterone peak has good separation and moderate area. It can be used as a reference to achieve the accuracy of quality control of lonicera japonica in a qualitative manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0045] Figure 1 It is the characteristic spectrum of 15 batches of lyophilized powder of standard decoction of Rhizoma Lupuli and 3 batches of Rhizoma Lupuli formula granules in Example 1 of the present invention;

[0046] Figure 2 It is a comparison characteristic spectrum of the reference substance solution and the Rhizoma Lupuli in Example 1 of the present invention;

[0047] Figure 3 It is the characteristic spectrum obtained by investigating the extraction solvent of the test solution in Experimental Example 1 of the present invention;

[0048] Figure 4 This is the characteristic spectrum obtained by investigating the wavelength in Experimental Example 5 of the present invention;

[0049] Figure 5 It is the characteristic spectrum obtained by investigating the construction method in Experimental Example 6 of the present invention;

[0050] Figure 6 This is the characteristic spectrum obtained by investigating the elution gradient in Experimental Example 7 of the present invention;

[0051] Figure 7 This is the characteristic spectrum obtained by investigating the flow velocity in Experimental Example 8 of the present invention;

[0052] Figure 8 This is the characteristic spectrum obtained by investigating the mobile phase in Experimental Example 9 of the present invention;

[0053] Fig. 9This is the characteristic spectrum obtained from the delay experiment of Experimental Example 10 of the present invention;

[0054] Fig.10 This is the characteristic spectrum obtained by investigating the preparation type in Experimental Example 11 of the present invention. DETAILED DESCRIPTION

[0055] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0056] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0057] Reagents and instruments used in the following examples

[0058] Reagents:

[0059] The reference medicinal material of Lophatherum gracile was batch number 121036-201603, China Food and Drug Inspection Institute;

[0060] Tuxedosterone reference substance batch number: AF21101506, Chengdu Aifa Biotechnology Co., Ltd., purity greater than 98%;

[0061] β-ecdysterone reference substance batch number: 111638-201706, China Food and Drug Inspection Institute, purity 98.4%;

[0062] The batch numbers of Lophatherum gracile medicinal materials, Lophatherum gracile standard decoction freeze-dried powder, and Lophatherum gracile formula granules are as follows:

[0063]

[0064] Acetonitrile: Merck, chromatographic grade;

[0065] Formic acid: Fisher Chemical, chromatographic grade;

[0066] Phosphoric acid: Fisher Chemical, chromatographic grade;

[0067] Methanol and ethanol were of analytical grade; water was distilled water from Watsons.

[0068] instrument:

[0069] Waters ACQUITY H-Class high performance liquid chromatograph, PDA Detector;

[0070] TUV Detector UV detector, Empower 3 chromatography workstation;

[0071] ME104E electronic balance, Mettler Toledo;

[0072] JY20002 electronic balance, Mettler Toledo;

[0073] DZKW-4 electronic constant temperature water bath, Beijing Zhongxing Weiye Instrument Co., Ltd.;

[0074] KQ-300DB CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.

[0075] Example 1

[0076] This embodiment provides a method for constructing a characteristic spectrum of a loquat, comprising the following steps:

[0077] When the test sample is a formula granule, freeze-dried powder or extract powder, the preparation method of the test solution includes: taking an appropriate amount of the test sample, about 0.2g, accurately weighing, accurately adding 15ml of 70% methanol, ultrasonically treating for 40min, taking out and cooling, filtering, taking the filtrate, and obtaining the test solution. When the test sample is a decoction piece or medicinal material, the preparation method of the test solution includes: taking the test sample, placing it in a stoppered conical flask, adding 20 times water, decocting and extracting for 40min, filtering, evaporating the filtrate, accurately adding 70% methanol ultrasonically treating for 40min, taking the filtrate, and obtaining the test solution.

[0078] Preparation of reference solution: Take the reference medicinal material of Rhizoma Corydalis, place it in a stoppered conical flask, add 20 times water, decoct and extract for 40 minutes, filter, evaporate the filtrate, add 70% methanol ultrasonic extraction for 40 minutes, take the filtrate to obtain the reference solution.

[0079] Preparation of turosterone reference solution: Take turosterone reference, add 70% methanol to dissolve it, and prepare a turosterone reference solution containing 100 μg per 1 ml.

[0080] Preparation of β-ecdysterone reference solution: Take β-ecdysterone reference, add 70% methanol to dissolve, and prepare a β-ecdysterone reference solution containing 0.2 mg per 1 ml.

[0081] Preparation of caffeic acid reference solution: Take caffeic acid reference, add 70% methanol, dissolve, and prepare a caffeic acid reference solution containing 70 μg per 1 ml.

[0082] Determination method: 1 μL of reference solution, reference solution and test solution were injected into the liquid chromatograph for determination to obtain characteristic spectra. The high performance liquid chromatography conditions are as follows: chromatographic column with octadecylsilane bonded silica gel as filler, ACQUITY UPLC BEH Shield C18, specification: 2.1mm×100mm 1.7μm; acetonitrile as mobile phase A, 0.02% phosphoric acid aqueous solution as mobile phase B, gradient elution according to the provisions of Table 1, column temperature of 35°C, wavelength of 225nm, flow rate as shown in Table 1, theoretical plate number calculated according to β-ecdysterone peak should be not less than 8000.

[0083] Table 1 Gradient elution program and flow rate

[0084] Time (min) Flow rate ml / min Mobile phase A (%) Mobile phase B (%) 0-35 0.1→0.2 3→25 97→75 35-45 0.2 25→80 75→20

[0085] Confirmation of characteristic peaks

[0086] Take 15 batches of lyophilized powder of standard decoction of Rhizoma Lumifuga and 3 batches of Rhizoma Lumifuga formula granules, prepare the test solution of lyophilized powder of standard decoction of Rhizoma Lumifuga and the test solution of Rhizoma Lumifuga formula granules according to the above steps, and measure according to the above chromatographic conditions to obtain the characteristic spectrum of each test solution. The results are shown in Table 2 and Figure 1 .in, Figure 1 S1-S15 correspond to the characteristic spectra of 15 batches of freeze-dried powder of standard decoction of Rhizoma Lupuli, and S16-S18 correspond to the characteristic spectra of 3 batches of Rhizoma Lupuli formula granules.

[0087] Eight peaks with good repeatability were selected as common characteristic peaks, namely Peak 1, Peak 2, Peak 3, Peak 4, Peak 5, Peak 6, Peak 7 and Peak 8; the relative retention times of these eight common characteristic peaks were slightly different, and the average value of the relative retention time was selected as the measured value. Peak 6 was used as the reference peak, and the specified values ​​of the relative retention times of Peak 1, Peak 2, Peak 3, Peak 4, Peak 7 and Peak 8 were 0.25, 0.41, 0.66, 0.70, 1.07 and 1.27, respectively, with an allowable error of ±10%.

[0088] Table 2 Relative retention time of 15 batches of freeze-dried powder of standard decoction of Rhizoma Lu

[0089]

[0090] The characteristic spectra of 15 batches of freeze-dried powder of standard decoction of Rhizoma Lupuli were imported into the "Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine" (2012.1 version), and the similarities between the characteristic spectra of 15 batches of freeze-dried powder of standard decoction of Rhizoma Lupuli and the common pattern control characteristic spectra were 0.912, 0.965, 0.990, 0.970, 0.986, 0.923, 0.957, 0.949, 0.969, 0.983, 0.971, 0.978, 0.986, 0.981, and 992, respectively, and the similarities were all above 90%, indicating that the 15 batches of freeze-dried powder of standard decoction of Rhizoma Lupuli had a high degree of fit with the common pattern control spectra, and the 8 characteristic peaks were relatively stable. The detection method was used to well control the quality of Rhizoma Lupuli.

[0091] Characteristic peak identification:

[0092] By comparing the reference solution and the characteristic spectrum of the radix sphaerocarpon, we can see Figure 2 The characteristic spectrum constructed by the present invention contains caffeic acid, but the content is low and it is not used as a common characteristic peak. Peak 5 is turcesterone and peak 6 is β-ecdysterone. Figure 2 2a is the comparison spectrum of turosterone reference substance, caffeic acid reference substance and Rhizoma Lupulinae; 2b is the comparison spectrum of β-ecdysterone and Rhizoma Lupulinae.

[0093] Experimental Example 1 Investigation of extraction solvent

[0094] This experimental example provides an investigation of the extraction solvent of the test solution, specifically including: taking 4 portions of the same batch of Lophatherum formula granules (batch number KL190418-072350-01), preparing 4 portions of test solution according to the "Test solution" item of Example 1, and obtaining the characteristic spectra of the test solution obtained with different extraction solvents according to the "Assay method" item of Example 1, see Figure 3 ; The extraction solvents of the four test sample solutions were: methanol, 30% methanol, 50% methanol, and 70% methanol, and the amount of extraction solvent added was 20 ml. Figure 3 The characteristic spectra in the figure are 30% methanol, 50% methanol, 70% methanol and methanol from top to bottom.

[0095] It can be seen from the figure that when different proportions of methanol are extracted, the content of each characteristic peak increases with the increase of methanol proportion, and 70% methanol and methanol are slightly larger, but the difference between the two is not large. Considering the economy, 70% methanol is preferred as the extraction solvent for the characteristic spectrum of the loofah.

[0096] Experimental Example 2 Investigation of the concentration of the test solution

[0097] This experimental example investigates the concentration of the test solution, specifically including: taking 4 portions of the same batch of Lophatherum formula granules (batch number KL190418-072350-01), preparing 4 test solutions according to the "Test Solution" item of Example 1, obtaining the characteristic peak areas of the characteristic spectra of test solutions of different concentrations according to the "Determination Method" item described in Example 1, and comparing the peak areas under the same conditions converted according to the sampling amount and sample concentration. The results are shown in Tables 3 and 4; the ratios of the mass of the test sample to the volume of the extraction solvent (70% methanol) in the 4 test solutions are: 0.3g:15ml, 0.2g:15ml, 0.2g:20ml, 0.1g:15ml, respectively.

[0098] Table 3 Characteristic peak areas of characteristic spectra obtained from test solution at different concentrations

[0099] Peak concentration Sampling volume g 1 2 3 4 5 6 7 8 0.3-15 0.3012 261157 1398148 192946 183258 310245 1131245 166845 216789 0.2-15 0.2006 175347 940825 129016 122687 210058 756214 113796 146983 0.2-20 0.2018 131257 698456 96279 92015 157234 560895 83049 109015 0.1-15 0.1035 88158 476513 65578 62678 103894 379875 55489 73192

[0100] Table 4 Mean peak areas of characteristic peaks converted to different concentrations of test solution under the same conditions

[0101] concentration 1 2 3 4 5 6 7 8 0.3-15 13005.8 69628.9 9608.9 9126.4 15450.4 56336.9 8309.0 10796.3 0.2-15 13111.7 70350.8 9647.3 9174.0 15707.2 56546.4 8509.2 10990.8 0.2-20 13008.6 69222.6 9542.0 9119.4 15583.2 55589.2 8230.8 10804.3 0.1-15 12776.5 69059.9 9504.1 9083.8 15057.1 55054.3 8041.9 10607.5

[0102] It can be seen from the table that the characteristic peak graphs of the test solutions with different concentrations are not much different. Taking into account the differences between batches, in order to ensure complete extraction, 0.2g:15ml is used as the concentration of the test solution.

[0103] Experimental Example 3 Investigation of the preparation method of the test solution

[0104] This experimental example investigates the preparation method of the test solution, specifically including: taking 2 portions of the same batch of Lophatherum formula granules (batch number KL190418-072350-01), according to the "determination method" item described in Example 1, obtaining the peak areas of the characteristic peaks of the characteristic spectrum of the test solution with different concentrations, and comparing the peak areas under the same conditions converted according to the sampling amount and sample concentration, the results are shown in Tables 5 and 6; the preparation method of the test solution adopts ultrasonic extraction and reflux extraction respectively, specifically: taking an appropriate amount of the test sample, grinding it, taking about 0.2g, accurately weighing, accurately adding 30ml of 30% methanol, sealing, weighing the weight, reflux extraction or ultrasonic extraction for 40min, cooling, weighing again, supplementing the lost weight with 30% methanol, shaking, filtering, accurately taking 1μL of the filtrate, and injecting it into a high performance liquid chromatograph; wherein, the power of ultrasonic extraction is 250W, the frequency is 40kHz; the reflux temperature is 90℃.

[0105] Table 5 Peak areas of characteristic peaks in the spectra obtained by different test solution preparation methods

[0106] Extraction method Sample weight g 1 2 3 4 5 6 7 8 Ultrasound 0.2011 175536 941177 129522 123916 216547 753648 115134 147535 Reflow 0.2018 180448 944875 130306 124567 216895 754806 116417 147006

[0107] Table 6 Peak areas of characteristic peaks in the spectra obtained by different test solution preparation methods under the same conditions

[0108] Extraction method 1 2 3 4 5 6 7 8 Ultrasound 872.88 4680.14 644.07 616.19 1076.81 3747.63 572.52 733.64 Reflow 894.19 4682.23 645.72 617.28 1074.80 3740.37 576.89 728.47

[0109] As can be seen from the table, there is little overall difference between ultrasound and reflux. Considering the ease of operation, ultrasonic extraction is preferred as the extraction method for the test solution.

[0110] Experimental Example 4 Investigation of extraction time

[0111] This experimental example investigates the extraction time, specifically including: taking 3 portions of the same batch of Lophatherum formula granules (batch number KL190418-072350-01), and obtaining the peak area of ​​the characteristic peak of the characteristic spectrum of the test solution according to the "Determination Method" item described in Example 1, and comparing them under the same conditions according to the sampling amount and sample concentration. The results are shown in Tables 7 and 8; the difference in the preparation of these 3 test solutions is that the ultrasonic extraction time is different, and the ultrasonic extraction time is 20min, 40min and 60min respectively.

[0112] Table 7 Peak areas of characteristic peaks obtained at different extraction times

[0113] Extraction time Sampling volume g 1 2 3 4 5 6 7 8 Ultrasound 20min 0.2025 178437 945740 122027 146764 212804 758315 117361 146275 Ultrasound 40min 0.2011 175536 941177 129522 123916 216547 753648 115134 147535 Ultrasound 60min 0.2033 176243 944372 124568 134607 217083 758006 115661 144674

[0114] Table 8 Mean peak areas of characteristic peaks obtained at different extraction times under the same conditions

[0115] Peak 1 2 3 4 5 6 7 8 Ultrasound 20min 881.17 4670.32 602.60 724.76 1050.88 3744.77 579.56 722.35 Ultrasound 40min 872.88 4680.14 644.07 616.19 1076.81 3747.63 572.52 733.64 Ultrasound 60min 866.91 4645.21 612.73 662.11 1067.80 3728.51 568.92 711.63

[0116] It can be seen from the table that the peak areas of the characteristic peaks obtained at different ultrasonic extraction times are not much different. Considering the differences between batches, 40 min was used as the ultrasonic extraction time.

[0117] Experimental Example 5 Investigation of Detection Wavelength

[0118] This experimental example investigates the detection wavelength, specifically including: the formula granules of Lou Lu (batch number KL190418-072350-01) are used according to the "determination method" item described in Example 1 to obtain a test solution, and then the test solution is subjected to a full wavelength scan of 210-400nm to obtain a 3D chromatogram of the full wavelength scan, as shown in FIG. Figure 4 As shown in a in . Figure 4 The bf in the figure corresponds to the spectra with wavelengths of 210nm, 215nm, 220nm, 225nm and 230nm. It can be seen from the figure that the characteristic peak information of 210-230nm is basically consistent. Since the peak at 225nm is more abundant and the peak shape is better than other wavelengths, combined with the overall height uniformity of the characteristic peaks, using 225nm as the detection wavelength is the most preferred option.

[0119] Experimental Example 6 Investigation of Construction Method

[0120] This experimental example investigates the method for constructing a characteristic spectrum, specifically comprising: taking 6 portions of the same batch of Lophatherum formula granules (batch number KL190418-072350-01), obtaining a test solution according to the "Assay Method" item described in Example 1, and obtaining a characteristic spectrum of the test solution according to the "Assay Method" item described in Example 1, the difference being that the chromatographic column, flow rate and gradient elution procedure are different, see Figure 5 , as follows:

[0121] (1) Condition 1: Agilent SB (2.1×100 mm, 1.8 μm) was used as the chromatographic column, the flow rate was 0.3 ml / min, the gradient elution was shown in Table 9, and the spectrum was shown in Figure 5 a in;

[0122] Table 9

[0123]

[0124]

[0125] (2) Condition 2: CORTECS UPLC T3 (2.1×100 mm 1.6 μm) was used as the chromatographic column. The flow rate and gradient elution are shown in Table 10. The spectrum is shown in Figure 5 b in;

[0126] Table 10

[0127] Time (min) Flow rate (ml / min) Acetonitrile (%) 0.02% Phosphoric acid (%) 0-7 0.1→0.2 5 95 7-10 0.2 5→12 95→88 10-25 0.2 12→22 88→78 25-30 0.2 22→60 78→40 30-40 0.2 60→100 40→0

[0128] (3) Condition 3: Shimadzu GL Sciences Inertsil ODS column (2.1×150 mm 1.6 μm) was used as the chromatographic column, the flow rate was 0.25 ml / min, the gradient elution program was shown in Table 11, and the spectrum was shown in Figure 5 c in;

[0129] Table 11

[0130] Time (min) Acetonitrile (%) 0.02% Phosphoric acid (%) 0-20 2→17 98→83 20-30 17→30 83→70 30-35 30→100 70→0 35-40 100→2 0→98

[0131] (4) Condition 4: BEH Shield column (2.1×100 mm 1.7 μm) was used as the chromatographic column. The flow rate and gradient elution program are shown in Table 12. The spectrum is shown in Figure 5 d in;

[0132] Table 12

[0133] Time (min) Flow rate (ml / min) Acetonitrile (%) 0.02% Phosphoric acid (%) 0-20 0.1 5→15 95→85 20-30 0.1→0.2 15→21 85→79 30-45 0.2 21→40 79→60 45-55 0.2 40→100 60→0 55-60 0.2→0.1 100→5 0→95

[0134] (5) Condition 5: CORTECS UPLC C18 column (2.1×100 mm 1.6 μm) was used as the chromatographic column. The gradient elution program and flow rate are shown in Table 13. The spectrum is shown in Figure 5 e in;

[0135] Table 13

[0136] Time (min) Flow rate (ml / min) Acetonitrile (%) 0.02% Phosphoric acid (%) 0-35 0.1→0.2 3→25 97→75 35-45 0.2 25→80 75→20 45-50 0.2→0.1 80→3 20→97

[0137] (6) Condition 6: Hypersil GOLD aQ column (2.1×100 mm 1.9 μm) was used as the chromatographic column. The flow rate and gradient elution program are shown in Table 14. The spectrum is shown in Figure 5 f in;

[0138] Table 14

[0139]

[0140]

[0141] from Figure 5 It can be seen that compared with these six methods, the construction method provided by the present invention is simpler, and the separation degree of each characteristic peak in the obtained spectrum is better. From the characteristic spectra obtained by the above six methods, the basic information of the characteristic spectra of Rhizoma Lucidum obtained by these six methods is basically the same. From the analysis of the separation of characteristic peaks, Shimadzu GL Sciences InertsilODS column, BEH Shield column and CORTECS UPLC C18 column are preferably used as the chromatographic column of the present invention; from the universality of the chromatographic column, BEH Shield column is considered as the optimal chromatographic column.

[0142] Experimental Example 7 Investigation of Gradient Elution Procedure

[0143] This experimental example investigated the gradient elution procedure, specifically including: taking 6 portions of the same batch of Lophatherum formula granules (batch number KL190418-072350-01), preparing 6 portions of test solution according to the "Preparation of test solution" described in Example 1, and then obtaining characteristic spectra using different elution procedures according to the "Assay method" item described in Example 1; wherein, gradient elution procedures AF are shown in Tables 15-20, and the results are shown in Figure 6 6a-6f in .

[0144] Table 15 Gradient elution program A

[0145] Time (min) Flow rate (ml / min) Acetonitrile (%) 0.02% (phosphoric acid%) 0-7 0.1→0.2 5 95 7-10 0.2 5→13 95→87 10-25 0.2 13→21 87→79 25-30 0.2 21→60 79→40 30-40 0.2 60→100 40→0 40-45 0.2→0.1 100→5 0→95

[0146] Table 16 Gradient elution program B

[0147] Time (min) Flow rate (ml / min) Acetonitrile (%) 0.02% (phosphoric acid%) 0-7 0.1 5 95 7-10 0.1→0.2 5→13 95→87 10-16 0.2 13 87 16-25 0.2 13→22 87→78 25-40 0.2 22→60 78→40 40-45 0.2→0.1 60→5 40→95

[0148] Table 17 Gradient elution program C

[0149] Time (min) Flow rate (ml / min) Acetonitrile (%) 0.02% (phosphoric acid%) 0-20 0.1 5→15 95→85 20-30 0.1→0.2 15→21 85→79 30-45 0.2 21→40 79→60 45-55 0.2 40→100 60→0 55-60 0.2→0.1 100→5 0→95

[0150] Table 18 Gradient elution program D

[0151] Time (min) Flow rate (ml / min) Acetonitrile (%) 0.02% (phosphoric acid%) 0-20 0.1→0.2 2→15 98→85 20-30 0.2 15→21 85→79 30-38 0.2→0.3 21→42 79→58 38-45 0.3 42→90 58→10

[0152] Table 19 Gradient elution program E

[0153] Time (min) Flow rate (ml / min) Acetonitrile (%) 0.02% (phosphoric acid%) 0-35 0.1→0.2 3→25 97→75 35-45 0.2 25→80 75→20 45-50 0.2→0.1 80→3 20→97

[0154] Table 20 Gradient elution program F

[0155] Time (min) Flow rate (ml / min) Acetonitrile (%) 0.02% (phosphoric acid%) 0～14 0.3 10→20 90→80 14～23 0.3 20→55 80→45 23～25 0.3 55→100 45→0

[0156] from Figure 6 It can be seen that before 5min, the large polarity peak of gradient elution program A was not separated, and the small peak at 14min was not completely separated. Before 5min, the large polarity peak of gradient elution program B had a separation trend, and the small peak at 14min was basically separated, with a large overall capacity. The gradient elution degree C reduced the separation of characteristic peaks at 22min. There was a package peak at 12min in the gradient elution degree D. The characteristic peaks of gradient elution degree E were basically separated. Peak No. 1, Peak No. 2 and Peak No. 3 could not be effectively identified in the gradient elution program F. The polarity of these three characteristic peaks was large, and the polarity of the mobile phase needed to be increased to elute them. Therefore, the gradient elution program of 0-45min in the gradient elution program E was used as the gradient elution program of the present invention.

[0157] Experimental Example 8 Investigation of flow rate

[0158] This experimental example investigated the flow rate, specifically including: taking 5 portions of the same batch of Lophatherum formula granules (batch number KL190418-072350-01), preparing 5 portions of test solution according to the "Preparation of test solution" described in Example 1, and then obtaining characteristic spectra at different flow rates according to the "Assay method" described in Example 1, see Figure 7 ; where the flow rate is as follows:

[0159] Flow rate 1: 0.2 ml / min;

[0160] Flow rate 2: 0.1 ml / min;

[0161] Flow rate 3: 0-35min: 0.1→0.15ml / min, 35-45min: 0.15ml / min, 45-50min: 0.15→0.10ml / min;

[0162] Flow rate 4: 0.1→0.25 ml / min for 0-35 min, 0.25 ml / min for 35-45 min, 0.25→0.10 ml / min for 45-50 min;

[0163] Flow rate 5: 0.1→0.3 ml / min for 0-35 min, 0.30 ml / min for 35-45 min, 0.30→0.10 ml / min for 45-50 min.

[0164] Figure 7 7a-7e in the figure correspond to the characteristic spectra obtained at flow rates 1-5, respectively. As can be seen from the figure, the spectrum obtained with an initial flow rate of 0.2ml / min has a faster peak elution before 10min, and the separation is relatively small. The spectrum obtained when the initial flow rate is 0.1ml / min has a moderate separation degree; when the flow rate is 0.25ml / min and 0.30ml / min between 35-45min, the small peaks show a peak-wrapping state, and when the flow rate is reduced to 0.1ml / min, 0.15ml / min, and 0.20ml / min, the separation degree of each peak is better. Therefore, the initial flow rate is determined to be 0.1 ml / min, and the flow rate at 35-45 min is any value between 0.1 and 0.2 ml / min. The flow rate of the present invention is determined as follows: 0-35 min, flow rate 0.1→X1 ml / min; 35-45 min, flow rate is any value between X1 and X2 ml / min; wherein X1 is 0.1-0.2, and X2 is 0.1-0.2.

[0165] Experimental Example 9 Investigation of mobile phase

[0166] This experimental example investigated the mobile phase, specifically including: taking 2 portions of the same batch of Lou Lu formula granules (batch number KL190418-072350-01), preparing 2 portions of test solution according to the "Preparation of test solution" described in Example 1, and then obtaining the characteristic spectrum of the test solution according to the "Assay" item described in Example 1 using different mobile phases, see Figure 8 ; Wherein, 0.02% formic acid aqueous solution and water were used to replace the mobile phase B 0.02% phosphoric acid solution, respectively. Figure 8 a in Figure 8 b in.

[0167] from Figure 8 As can be seen from b in Figure 1, the characteristic information in the spectrum of Lu Lu obtained by using water as the elution solvent is relatively small; Figure 8 As can be seen from a in FIG. 1 , when formic acid and phosphoric acid are used as mobile phase B, there is basically no difference in the characteristic spectrum information of Lu Lu. Considering the peak shape of the characteristic peak, the mobile phase B is preferably a phosphoric acid solution.

[0168] Experimental Example 10 Delay Test

[0169] This experimental example is a delayed experiment. The test solution of the Lophatherum formula granules was prepared according to the "Preparation of the test solution" described in Example 1, and then the chromatogram of 2 times the retention time of the mobile phase was recorded according to the "Determination method" described in Example 1. The results are shown in Fig. 9 .

[0170] It can be seen from the figure that no obvious hysteresis peak appears after 45 minutes, so the construction method of the present invention meets the analysis requirements.

[0171] Experimental Example 11 Investigation of Preparation Types

[0172] This experimental example investigated different preparations of Rhizoma Cyperi. Rhizoma Cyperi decoction pieces, standard decoction freeze-dried powder and formula granules were used to prepare the test solution according to the "Preparation of Test Solution" described in Example 1, and then the characteristic spectra of Rhizoma Cyperi of different preparations were obtained according to the "Assay Method" described in Example 1. The results are shown in Fig.10 .

[0173] As can be seen from the figure, the construction method provided by the present invention is applicable to different preparation types of Rhizoma Lupuli, such as decoction pieces, standard decoction freeze-dried powder and formula granules.

[0174] Experimental Example 12 Methodological Investigation

[0175] (1) Repeatability study

[0176] Take 6 portions of the same batch of Lophatherum formula granules (batch number: 190418-072350-01), use Waters UPLCH-Class, TUV detector, prepare the test solution according to the "Preparation of test solution" in Example 1, and then obtain the characteristic spectrum of the test solution according to the "Chromatographic conditions" and "Determination" in Example 1, calculate its relative peak area and relative retention time, and calculate RSD. The results are shown in Tables 21 and 22.

[0177] Table 21 Repeatability of characteristic spectrum of Loulu formula granules - relative retention time

[0178]

[0179] Table 22 Repeatability of characteristic spectrum of Loulu formula granules - relative peak area

[0180]

[0181] As can be seen from the table, the relative retention time RSD of each characteristic peak is in the range of 0.02%-0.07%, and the relative peak area RSD is in the range of 1.14%-1.96%, indicating that the repeatability of the characteristic spectrum is good.

[0182] (2) Intermediate precision study

[0183] Take 6 portions of the same batch of Lophatherum formula granules (batch number: 190418-072350-01), and prepare the test solution according to the item "Preparation of test solution" in Example 1. Then, according to the "Chromatographic conditions" in Example 1, use Shimadzu LC-30AD and TUV detector to obtain the characteristic spectrum of the test solution, calculate its relative peak area and relative retention time, and calculate RSD. The results are shown in Tables 23 and 24.

[0184] Table 23 Intermediate precision study of characteristic spectrum of Loulu formula granules - relative retention time

[0185]

[0186] Table 24 Intermediate precision study of characteristic spectrum of Loulu formula granules - relative peak area

[0187]

[0188] As can be seen from the table, the relative retention time RSD of the eight identified peaks in the characteristic peak spectrum obtained by Shimadzu LC-30AD is in the range of 0.04%-0.76%; the relative retention time RSD between different instruments is in the range of 0.27%-5.48%; the relative peak area RSD range is 0.58%-1.67%, indicating that the intermediate precision of the characteristic spectrum meets the analysis requirements.

[0189] (3) Investigation of stability

[0190] Take 8 portions of the same batch of Lophatherum formula granules (batch number: 190418-072350-01), prepare the test solution according to the "Preparation of test solution" in Example 1, and place it at room temperature for 0h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h, respectively. Then, obtain the characteristic spectrum of the test solution according to the "Chromatographic conditions" and "Determination" in Example 1, calculate the relative peak area and relative retention time, and calculate the RSD. The results are shown in Tables 25 and 26.

[0191] Table 25 Stability study of characteristic spectrum of Loulu formula granules - relative retention time

[0192]

[0193] Table 26 Stability study of characteristic spectrum of granules of Loulu formula - relative peak area

[0194]

[0195]

[0196] As can be seen from the table, after examining the solution stability for 24 hours, the relative retention time RSD of each characteristic peak is in the range of 0.38%-1.13%; the relative peak area RSD is in the range of 0.43%-3.53%, indicating that the characteristic spectrum analysis method of the loofah formula granules adopted in the present invention is stable, reliable and has good reproducibility.

[0197] (4) Durability assessment

[0198] Take 3 portions of the same batch of Lophatherum formula granules (batch number: 190418-072350-01), and prepare the test solution according to the "Preparation of test solution" in Example 1. Then, obtain the characteristic spectrum of the test solution at different column temperatures according to the "Chromatographic conditions" and "Determination" in Example 1, calculate the relative retention time, and calculate the RSD. The results are shown in Table 27.

[0199] Table 27 Durability study of characteristic spectrum of loulu formula granules - relative retention time

[0200]

[0201] The relative retention time RSDs of the eight characteristic peaks in the characteristic spectra under different column temperature conditions were in the range of 0.03%-5.19%, indicating that the method had good durability for column temperatures between 32-38°C.

[0202] (5) Investigation of different wavelengths

[0203] Take 3 portions of the same batch of Lophatherum formula granules (batch number: 190418-072350-01), and prepare the test solution according to the "Preparation of test solution" in Example 1. Then, obtain the characteristic spectrum of the test solution at different wavelengths according to the "Chromatographic conditions" and "Determination" in Example 1, calculate the relative retention time, and calculate the RSD. The results are shown in Table 28.

[0204] Table 28 Relative retention time of characteristic spectra of Loulu formula granules at different wavelengths

[0205]

[0206] It can be seen from the table that the relative retention time RSD of the eight characteristic peaks in the characteristic spectrum under different wavelength conditions is in the range of 0.00%-0.02%, indicating that the method has good durability at wavelengths between 222-228nm.

[0207] (6) Investigation of the concentration of mobile phase B phosphoric acid solution

[0208] Take 3 portions of the same batch of Lophatherum formula granules (batch number: 190418-072350-01), and prepare the test solution according to the "Preparation of test solution" in Example 1. Then, obtain the characteristic spectrum of the test solution at different mobile phase B concentrations according to the "Chromatographic conditions" and "Determination" in Example 1, calculate the relative retention time, and calculate the RSD. The results are shown in Table 29.

[0209] Table 29 Characteristic spectrum of Loulu formula granules Mobile phase B concentration investigation-relative retention time

[0210]

[0211] It can be seen from the table that under different flow rate conditions, the relative retention time RSD of the eight characteristic peaks in the characteristic spectrum is in the range of 0.14%-2.72%; the method has good durability for the phosphoric acid concentration at 0.01%-0.03% characteristic peaks.

[0212] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the scope of protection of the invention.

Claims

1. A method for constructing a characteristic spectrum of Rhizoma Lupuli, characterized in that: The following steps are involved: Preparation of the test solution: taking the test sample, adding methanol, extracting, filtering, and preparing the test sample into the test solution; the test sample is at least one of the following: the decoction pieces of Rhizoma Lupuli, the formula granules of Rhizoma Lupuli, the freeze-dried powder of the standard decoction of Rhizoma Lupuli, the extract powder of Rhizoma Lupuli and the medicinal materials of Rhizoma Lupuli; Preparation of reference solution: Prepare reference solution from control medicinal materials; Preparation of reference solutions: Prepare turosterone reference solution and β-ecdysterone reference solution; Determination: Determined by ultra-high performance liquid chromatography; The chromatographic conditions of the ultra-high performance liquid chromatography method include: using an octadecylsilane bonded silica gel column as the chromatographic column, the chromatographic column specifications are: inner diameter 2.1 mm, column length 100 mm, particle size 1.7 μm; column temperature 32-38°C; wavelength 210-230 nm; injection volume 1-3 μL; acetonitrile as mobile phase A, 0.02% formic acid aqueous solution or 0.01-0.03% phosphoric acid aqueous solution as mobile phase B, gradient elution; The gradient elution conditions were as follows: 0-35 min, 3→25% mobile phase A, 97→75% mobile phase B; 35-45 min, 25→80% mobile phase A, 75→20% mobile phase B; The flow rate is: 0-35min, the flow rate is 0.1→X1ml / min; 35-45min, the flow rate is any value between 0.1-0.2 ml / min; wherein X1 is 0.1-0.

2.

2. The construction method according to claim 1, characterized in that: When the test sample is at least one of the formula granules of Rhizoma Lophatherum, freeze-dried powder of Rhizoma Lophatherum standard decoction and Rhizoma Lophatherum extract powder, when preparing the test sample solution, the ratio of the mass of the test sample to the volume of the extraction solvent is (0.1-0.3) g:(5-20) ml.

3. The construction method according to claim 1, characterized in that: The preparation method of the reference solution comprises: taking a reference medicinal material, adding water to extract, filtering and then evaporating to dryness to obtain a water extract; adding an extraction solvent to the water extract, extracting, and filtering.

4. The construction method according to claim 3, characterized in that: When preparing the reference solution, the extraction solvent is an organic solvent.

5. The construction method according to claim 4, characterized in that: The extraction solvent was methanol.

6. The construction method according to any one of claims 3 to 5, characterized in that: When preparing the reference solution, the ratio of the mass of the water extract to the volume of the extraction solvent is (1.0-2.0) g: (20-40) ml.

7. The construction method according to any one of claims 1 to 3, characterized in that: The characteristic spectrum obtained by the construction method includes 8 characteristic peaks; Taking peak 6 as the reference peak, the specified values ​​of relative retention times of peaks 1, 2, 3, 4, 7 and 8 are 0.25, 0.41, 0.66, 0.70, 1.07 and 1.27 respectively; Among them, the relative retention time of the characteristic peak is within ±10% of the specified value.

8. The construction method according to claim 1, characterized in that: The preparation steps of the turesterone reference substance solution include: taking a turesterone reference substance and adding a solvent.

9. The construction method according to claim 1, characterized in that: The preparation steps of the β-ecdysterone reference substance solution include: taking the β-ecdysterone reference substance and adding a solvent.

10. The construction method according to claim 8 or 9, characterized in that: Each 1 ml of turosterone reference solution contains 50-100 μg of turosterone reference; Each 1 ml of β-ecdysterone reference solution contains 0.15-0.2 mg of turosterone reference.

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