Detection method of elaeagnus pungens leaf characteristic atlas and determination method of effective component content

Abstract

This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting characteristic chromatograms of Elaeagnus angustifolia leaves and a method for determining the content of active ingredients. The detection method includes the preparation of a test solution, followed by high-performance liquid chromatography (HPLC) determination under a specific gradient elution program to obtain characteristic chromatograms. This method establishes a comprehensive and rapid approach for detecting Elaeagnus angustifolia leaf pharmaceutical preparations, especially water-extracted preparations, which is of great significance for comprehensive quality testing and overall quality control.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting the characteristic spectrum of Elaeagnus angustifolia leaves and a method for determining the content of active ingredients. Background Technology

[0002] The raw material for Elaeagnus pungens leaf granules is the dried leaves of Elaeagnus pungens Thunb., a plant belonging to the Elaeagnaceae family. Elaeagnus pungens leaves are acidic and neutral in nature; they enter the lung meridian; they astringe the lungs, relieve asthma, and stop coughing; they are used for cough, asthma, hemoptysis, and external bleeding.

[0003] The main chemical components of Elaeagnus pungens leaves are flavonoids, etc. While Elaeagnus pungens leaves are not listed in the pharmacopoeia, and the literature describes their chemical components, on the one hand, determining or identifying the content of a single component in Elaeagnus pungens leaf granules cannot comprehensively detect and control their quality; on the other hand, combining the determination of a single component with the identification of other components in Elaeagnus pungens leaf granules is time-consuming and labor-intensive, making it difficult to widely apply in production practice. Furthermore, existing quality control technologies for Elaeagnus pungens leaves are all for processed leaf slices, not for water-extracted drug preparations. Therefore, the detection methods for processed Elaeagnus pungens leaf slices are not applicable to water-extracted drug preparations. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the existing technology, such as the inapplicability of the detection method of Elaeagnus pungens leaf slices to water-extracted drug preparations of Elaeagnus pungens leaves, and to provide a detection method of characteristic spectrum of Elaeagnus pungens leaves and a method for determining the content of effective components.

[0005] To this end, the present invention provides the following technical solution.

[0006] The first aspect of this invention provides a method for detecting the characteristic spectrum of Elaeagnus angustifolia leaves, comprising the following steps:

[0007] Preparation of test solution: Prepare a test solution from the test sample;

[0008] High performance liquid chromatography (HPLC): The determination was performed using HPLC. The chromatographic conditions included: acetonitrile as mobile phase A and formic acid solution or phosphoric acid solution as mobile phase B; gradient elution, with the gradient elution program including: 0-90 min, mobile phase A volume percentage 10% → 21%; mobile phase B volume percentage 90% → 79%.

[0009] The test sample was an aqueous extract of Elaeagnus pungens leaves.

[0010] The dosage form of the test sample is tablet, powder, ointment, capsule, pill, granule, honey-processed pill, sustained-release preparation, immediate-release preparation, controlled-release preparation, oral liquid preparation or injection preparation.

[0011] In this invention, Elaeagnus pungens leaf water extract refers to a pharmaceutical preparation obtained by extracting Elaeagnus pungens leaf medicinal material or decoction pieces with water.

[0012] In this invention, the preparation method of the Elaeagnus pungens leaf test sample is not specifically limited, and may include, but is not limited to, the following method: Take Elaeagnus pungens leaf slices, heat and reflux for extraction at least once, adding 8-14 times the weight of water each time, extracting for at least 0.5 hours, filtering, combining the filtrates, concentrating the filtrate to a relative density of 1.05-1.10 g / mL at 60°C, adding conventional excipients, and following conventional processes to prepare clinically acceptable tablets, capsules, pills, granules, honey-refined pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations, or injectable preparations. The pharmaceutically acceptable excipients include: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, and matrix, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitosan, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, croscarmellose sodium, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silica, etc.; suspending agents include: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch paste, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; flavoring agents include: sweeteners and various flavorings; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorethidium acetate, eucalyptus oil, etc.; matrix includes: PEG6000, PEG4000, insect wax, etc.

[0013] The chromatographic conditions also include: mobile phase B being 0.05-0.15% formic acid solution or 0.05-0.15% phosphoric acid solution; and / or, column temperature being 30-40℃; and / or, flow rate being 0.8-1.2 mL / min; and / or, detection wavelength being 220-380 nm; and / or, injection volume being 5-15 μL; and / or, chromatographic column being Waters XSelect HSS T3, with dimensions of 250 mm × 4.6 mm and 5 μm.

[0014] The chromatographic conditions were as follows: mobile phase B was 0.1% formic acid solution; column temperature was 35℃; flow rate was 1.0 mL / min; detection wavelength was 380 nm; and injection volume was 10 μL.

[0015] The preparation steps of the test sample solution include extracting the test sample with a solvent, filtering it, and taking the filtrate.

[0016] Preferably, the solvent used is a 50-80% methanol aqueous solution;

[0017] Preferably, the solvent used is a 50% methanol aqueous solution;

[0018] Preferably, the ratio of the mass of the test sample (in g) to the volume of the extraction solvent (in ml) is (0.2-0.4):(5-20).

[0019] The detection method further includes the preparation of a reference solution;

[0020] Preferably, the preparation of the reference solution includes the following steps: taking Elaeagnus pungens leaf reference material, extracting and filtering it, and collecting the filtrate;

[0021] Preferably, in the preparation of the reference solution, 50% to 80% methanol is used as the extraction solvent.

[0022] The characteristic spectrum of Elaeagnus pungens leaves obtained by the detection method includes at least 7 characteristic peaks;

[0023] Using peak 4 as the reference peak, the specified values ​​for the relative retention times of each characteristic peak are as follows: peak 1 is 0.30, peak 2 is 0.88, peak 3 is 0.91, peak 5 is 1.05, peak 6 is 1.31, and peak 7 is 2.17.

[0024] The relative retention time of each characteristic peak is within ±10% of the specified value;

[0025] Preferably, peak 4 is rutin, peak 5 is kaempferol-3-O-neohesperidin, peak 6 is astragaloside, and peak 7 is cypermethrin.

[0026] The detection method further includes the preparation of a reference solution;

[0027] Preferably, the reference standard includes at least one of rutin and cypermethrin;

[0028] Preferably, the mass of each reference standard in each 1 ml of reference solution is 10–40 μg.

[0029] A second aspect of the present invention provides a method for determining the content of active ingredients in Elaeagnus pungens leaves, using the above-mentioned detection method;

[0030] Preferably, the active ingredient is cypermethrin.

[0031] The technical solution of this invention has the following advantages:

[0032] 1. The present invention provides a method for detecting the characteristic chromatogram of Elaeagnus pungens leaves. This method includes the preparation of a test solution and determination by high-performance liquid chromatography (HPLC) under a specific gradient elution program to obtain the characteristic chromatogram. This method establishes a comprehensive and rapid method for detecting Elaeagnus pungens leaf pharmaceutical preparations, especially water-extracted preparations, which is of great significance for comprehensive quality testing and overall quality control. The characteristic chromatogram obtained by this method has 7 common characteristic peaks, and identifies the specific components corresponding to 4 characteristic peaks. The known peaks account for a large proportion, exhibiting strong specific representativeness. Using rutin as a reference peak, the relative retention times of each characteristic peak are calculated, which is beneficial for comprehensive quality testing and overall quality control of Elaeagnus pungens leaf test samples, thereby contributing to the safety and uniformity of Elaeagnus pungens leaf pharmaceuticals.

[0033] The method of this invention produces a feature spectrum with good separation of each feature peak. Methodological verification shows that the method of this invention has high precision, good stability and repeatability. Attached Figure Description

[0034] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 These are chromatograms obtained from different gradient elution programs in Section 1.1 of Experimental Example 1 of this invention; a is the chromatogram obtained from gradient elution program 1, b is the chromatogram obtained from gradient elution program 2, c is the chromatogram obtained from gradient elution program 3, and d is the chromatogram obtained from gradient elution program 4. Figure 2 The chromatograms obtained at different wavelengths in Section 1.2 of Experimental Example 1 of this invention are shown, where a to i correspond to wavelengths of 220nm, 240nm, 260nm, 280nm, 300nm, 320nm, 340nm, 360nm, and 380nm, respectively. Figure 3 These are chromatograms obtained from different mobile phase systems in Experimental Example 1, Section 1.3 of this invention; a is a chromatogram of water as the mobile phase, b is a chromatogram of acetonitrile-0.1% formic acid system as the mobile phase, and c is a chromatogram of acetonitrile-0.1% phosphoric acid system as the mobile phase. Figure 4 These are chromatograms obtained at different flow rates in Section 1.4 of Experimental Example 1 of this invention; a is the chromatogram at a flow rate of 0.8 ml / min, b is the chromatogram at a flow rate of 1.0 ml / min, and c is the chromatogram at a flow rate of 1.2 ml / min. Figure 5These are chromatograms obtained at different column temperatures in Section 1.5 of Experimental Example 1 of this invention; a is a chromatogram at a column temperature of 30°C, b is a chromatogram at a column temperature of 35°C, and c is a chromatogram at a column temperature of 40°C. Figure 6 These are chromatograms obtained by different instruments in Experimental Example 1, Section 1.6 of this invention; a is the chromatogram obtained by Agilent, and b is the chromatogram obtained by Waters. Figure 7 These are chromatograms obtained from the chromatographic conditions and system adaptability of Experimental Example 1 of the present invention; a is the chromatogram of the reference standard; b is the chromatogram of the test sample; Figure 8 These are chromatograms obtained from different extraction solvent concentrations in Section 2.1 of Experimental Example 2 of this invention; a is a chromatogram of 20% methanol, b is a chromatogram of 50% methanol, and c is a chromatogram of 80% methanol. Figure 9 These are chromatograms obtained at different extraction times in Section 2.2 of Experimental Example 2 of this invention; a is the chromatogram with an extraction time of 30 min, b is the chromatogram with an extraction time of 45 min, and c is the chromatogram with an extraction time of 60 min. Figure 10 These are chromatograms obtained with different extraction powers in Section 2.3 of Experimental Example 2 of this invention; a is the chromatogram with an extraction power of 150W, b is the chromatogram with an extraction power of 250W, and c is the chromatogram with an extraction power of 350W. Figure 11 These are chromatograms obtained from different sample amounts in Section 2.4 of Experimental Example 2 of this invention; a is the chromatogram with a sample amount of 0.2g, b is the chromatogram with a sample amount of 0.3g, and c is the chromatogram with a sample amount of 0.4g. Figure 12 These are chromatograms obtained from different extraction solvent volumes in Section 2.5 of Experimental Example 2 of this invention; a is a chromatogram with an extraction solvent volume of 5 ml, b is a chromatogram with an extraction solvent volume of 10 ml, and c is a chromatogram with an extraction solvent volume of 15 ml. Figure 13 These are the characteristic chromatograms of 15 batches of Elaeagnus pungens leaf formulation granules in Experimental Example 3 of this invention; Figure 14 This is a control feature map generated from the feature maps of 15 batches of Elaeagnus pungens leaf formulation granules in Experimental Example 3 of this invention; Figure 15 This is a chromatogram of the Elaeagnus pungens leaf control material in Experiment Example 3 of this invention; Figure 16 This is the spectrum of the negative sample in the specificity test in section 4.2 of Experimental Example 4 of this invention. Detailed Implementation

[0036] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0037] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0038] Instruments and reagents

[0039] instrument

[0040] Chromatograph 1: Agilent LC 1260 High Performance Liquid Chromatograph: 1260 Quant Pump VL quaternary pump, 1260 Visalasamper injector, ICC column oven, 1260 DAD WR detector, chromatography workstation; Chromatograph 2: Waters 2695 High Performance Liquid Chromatograph, including a quaternary gradient pump (Alliance 2695 model), a 120-position high-performance autosampler, an original imported chromatography column oven, a Waters 2998 diode array UV detector, and an Empower chromatography workstation.

[0041] Electronic analytical balances: Sartorius SQP SECURA225 D-1CN (1 / 100,000) (Sartorius Scientific Instruments (Beijing) Co., Ltd.), Shimadzu AY (1 / 200,000) (Shimadzu Corporation, Japan). Ultrasonic instrument: KQ-400KDB high-power CNC ultrasonic instrument (Kunshan Ultrasonic Instrument Co., Ltd.). Water bath: HH-S6 digital display constant temperature water bath (Jiangsu Jinyi Instrument Technology Co., Ltd.).

[0042] Chromatographic columns: Waters XSelect HSS T3 (column length 250 mm, inner diameter 5 mm, particle size 4.6 μm); SHIMADZUShim-pack GISS (column length 250 mm, inner diameter 5 mm, particle size 4.6 μm); Agilent A2000250X046 (column length 250 mm, inner diameter 5 mm, particle size 4.6 μm).

[0043] reagents

[0044] Acetonitrile was of chromatographic grade, and water was Watson's distilled water; all other reagents were of analytical grade.

[0045] Drug testing

[0046] Silver calcinate reference standard (purchased from Chengdu Manster Biotechnology Co., Ltd., batch number MUST-22031012, purity ≥99.74%). Elaeagnus pungens leaf reference material (purchased from Lemeitian Pharmaceutical / Dest Biotechnology, batch number DSTYH012301).

[0047] In a specific embodiment, the preparation method of Elaeagnus pungens leaf formula granules is as follows: take Elaeagnus pungens leaf slices, heat and reflux extract twice. For the first extraction, add 12 times the weight of water and soak for 30 minutes, heat and reflux extract for 0.5 hours, filter, add 10 times the weight of water for the second extraction and extract for 0.5 hours, filter, combine the filtrates, concentrate the filtrate to a relative density of 1.05 g / mL at 60℃, spray dry, add maltodextrin as an excipient to the dry powder, mix evenly, and then perform dry granulation to produce Elaeagnus pungens leaf formula granules. Fifteen batches of Elaeagnus pungens leaf slices were taken and made into fifteen batches of Elaeagnus pungens leaf formula granules. The fifteen batches of Elaeagnus pungens leaf formula granules S1 to S15 are 1902002W, 1904002S, 1901001S, 1903001W, 1905001W, 1910002S, 1912001W, 2004001S, 2011002W, 2105001S, 2108002S, 2112002S, 2203001W, 2209003S, and 2212001S.

[0048] Preparation method of Elaeagnus pungens leaf pills: Take 500g of Elaeagnus pungens leaf slices, add 6000mL of water and extract for 1 hour, filter, add 5000mL of water to the residue and decoct for 1 hour, filter, and combine the filtrates; concentrate under vacuum to a flow paste with a relative density of 1.05-1.15g / mL, spray dry to prepare intermediate extract, add appropriate excipients to make soft material, pass through a sieve and knead into pills.

[0049] Example 1

[0050] This embodiment provides a method for detecting the characteristic spectrum of Elaeagnus angustifolia leaf formulation granules, including:

[0051] Preparation of the test solution: Take the Elaeagnus pungens leaf formula granules, grind them finely, take about 0.3g, accurately weigh them, place them in a stoppered conical flask, add 5mL of 50% methanol, sonicate (power 350W, frequency 40kHz) for 1h, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0052] Preparation of reference solution: Take 2g of Elaeagnus pungens leaf reference material, place it in a stoppered conical flask, add 40ml of water, heat under reflux for 1h, filter, evaporate the filtrate to dryness, add 5mL of 50% methanol to the residue and sonicate (power 350W, frequency 40kHz) for 30min, take it out, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0053] Preparation of reference solution: Take appropriate amounts of rutin, cypermethrin, kaempferol 3-O neohesperidin and astragaloside reference standards, add 80% methanol to prepare a mixed solution containing 20 μg of each per 1 mL, as the reference solution.

[0054] 10 μL of each of the test solution, reference solution, and standard solution were injected into the high-performance liquid chromatograph for determination. The chromatographic conditions were as follows: octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B, with gradient elution. The gradient elution program included: 0-90 min, mobile phase A volume percentage 10% → 21%; mobile phase B volume percentage 90% → 79%; detection wavelength 380 nm, column temperature 35℃, flow rate 1.0 mL / min; the theoretical plate number calculated based on the arginine peak should be no less than 3000.

[0055] Example 2

[0056] This embodiment provides a method for detecting the characteristic spectrum of Elaeagnus angustifolia leaf formulation granules, including:

[0057] Preparation of the test solution: Weigh approximately 0.3g of Elaeagnus pungens leaf pills accurately, place them in a stoppered conical flask, add 5mL of 50% methanol, sonicate (350W power, 40kHz frequency) for 1h, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0058] Preparation of reference solution: Take 2g of Elaeagnus pungens leaf reference material, place it in a stoppered conical flask, add 40ml of water, heat under reflux for 1h, filter, evaporate the filtrate to dryness, add 5mL of 50% methanol to the residue and sonicate (power 350W, frequency 40kHz) for 30min, take it out, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0059] Preparation of reference solution: Take appropriate amounts of rutin, cypermethrin, kaempferol 3-O neohesperidin and astragaloside reference standards, add 80% methanol to prepare a mixed solution containing 20 μg of each per 1 mL, as the reference solution.

[0060] 10 μL of each of the test solution, reference solution, and standard solution were injected into the high-performance liquid chromatograph for determination. The chromatographic conditions were as follows: octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B, with gradient elution. The gradient elution program included: 0-90 min, mobile phase A volume percentage 10% → 21%; mobile phase B volume percentage 90% → 79%; detection wavelength 380 nm, column temperature 35℃, flow rate 1.0 mL / min; the theoretical plate number calculated based on the arginine peak should be no less than 3000.

[0061] Example 1: Determination of Chromatographic Conditions

[0062] 1.1 Gradient elution procedure

[0063] Preparation of the test solution: Take the Elaeagnus pungens leaf formula granules, grind them finely, take about 0.3g, accurately weigh them, place them in a stoppered conical flask, add 5mL of 50% methanol, sonicate (power 350W, frequency 40kHz) for 1h, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0064] Pipette 10 mL of the test solution into the high-performance liquid chromatograph (HPLC) and determine its composition. The gradient elution program is used as the variable, and the gradient elution programs are shown in the table below. The chromatographic conditions are as follows: octadecylsilane-bonded silica gel is used as the stationary phase (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile is used as mobile phase A, and 0.1% formic acid solution is used as mobile phase B, with gradient elution; the detection wavelength is 360 nm, the column temperature is 35 ℃, and the flow rate is 1.0 mL / min.

[0065] Table 1 Gradient elution program 1

[0066] Time (min) Mobile phase A (%) Mobile phase B (%) 0～40 5～90 95～10 40～45 90 10 45～47 90～5 10～95 45～60 5 95

[0067] Table 2 Gradient elution program 2

[0068] Time (min) Mobile phase A (%) Mobile phase B (%) 0～25 15 90 25～30 15～17 85～83 30～45 17～22 83～78 45～70 22～34 78～66 70～75 34～35 66～65 75～76 35～90 65～10 76～80 90 10 80～81 90～15 10～85 81～90 15 85

[0069] Table 3 Gradient elution program 3

[0070] Time (min) Mobile phase A (%) Mobile phase B (%) 0～5 10 90 5～55 10～20 90～80 55～70 20～35 80～65 70～71 35～90 65～10 71～80 90 10 80～81 90～10 10～90 81～90 10 90

[0071] Table 4 Gradient elution program 4

[0072] Time (min) Mobile phase A (%) Mobile phase B (%) 0～90 10→29 90→71

[0073] Table 5. Chromatographic peak system suitability parameters for the chromatograms obtained from gradient elution programs 1-4.

[0074]

[0075]

[0076] The chromatograms obtained by comparing different gradient elution programs are shown below. Figure 1 The preferred gradient elution program is one that provides rich chromatographic information, good separation of major chromatographic peaks, a stable baseline, and a reasonable analysis time. The results indicate that the separation of chromatographic peaks improves with increasing elution time. Compared to the previous gradients, gradient elution program 4 provides better separation, richer chromatographic information, better resolution, and a more stable baseline. Therefore, gradient elution program 4 is the preferred option in this invention.

[0077] 1.2 Detection Wavelength

[0078] Prepare the test solution according to Section 1.1 of this experiment. Using the detection wavelength as a variable, inject the test solution into the liquid chromatograph under the chromatographic conditions determined in Section 1.1 and perform a full-wavelength scan. See [link to experiment description]. Figure 2 Based on the amount of information contained in the chromatograms, the chromatograms at nine different absorption wavelengths (220 nm, 240 nm, 260 nm, 280 nm, 300 nm, 320 nm, 340 nm, 360 nm, and 380 nm) were compared. The number of detected chromatographic peaks, response value, retention time, and system suitability were used as evaluation criteria to select the appropriate detection wavelengths.

[0079] The results showed that the chromatographic peak baseline was stable at a wavelength of 380 nm, and the separation effect of each characteristic peak was good. Therefore, the absorption wavelength was determined to be 380 nm.

[0080] 1.3 Mobile Phase System

[0081] The test solution was prepared according to Section 1.1 of this experiment. The mobile phase type was used as a variable, and the chromatographic conditions determined in Section 1.1 and the wavelength of 380 nm determined in Section 1.2 were used for detection. The mobile phase systems were water, acetonitrile-0.1% formic acid, and acetonitrile-0.1% phosphoric acid. The results are shown in [Figure 1]. Figure 3 and the table below.

[0082] Table 6. Parameters for evaluating system applicability under different mobile phase systems.

[0083]

[0084] The results showed that the chromatograms eluted with 0.1% formic acid solution and 0.1% phosphoric acid solution had good peak shapes and resolution. Considering that the acid strength of 0.1% phosphoric acid solution is greater than that of 0.1% formic acid solution, in order to prolong the service life of the chromatographic column, 0.1% formic acid solution was selected as mobile phase B.

[0085] 1.4 flow rate

[0086] The test solution was prepared according to Section 1.1 of this experiment. Using flow rate as a variable, the test solution was injected into the liquid chromatograph under the chromatographic conditions determined in Section 1.1 and the wavelength of 380 nm determined in Section 1.2. The flow rates were 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min, respectively. The separation effect of different flow rates on the granule chromatogram of the Elaeagnus pungens leaf formulation was investigated. The results are shown in [Figure 1]. Figure 4 and the table below.

[0087] Table 7. Parameters for assessing system suitability at a flow rate of 0.8 ml / min

[0088] Peak Retention time Peak area Peak Resolution Asymmetry Theoretical number of plates 1 13.222 175.883 15.024 3.287 1.128 29445 2 35.735 238.594 13.856 1.167 1.082 99690 3 36.876 340.222 20.290 2.537 1.141 109311 4 40.428 78.773 4.157 7.486 1.101 103280 5 42.111 348.542 18.570 3.392 1.152 117602 6 49.457 39.824 1.954 14.737 1.331 152987 7 83.684 579.602 24.436 54.345 1.145 296514

[0089] Table 8. Parameters for assessing system suitability at a flow rate of 1.0 ml / min

[0090] Peak Retention time Peak area Peak Resolution Asymmetry Theoretical number of plates 1 11.012 141.297 11.060 2.870 1.144 16866 2 31.987 194.668 11.128 1.640 1.084 81967 3 33.163 282.563 16.987 2.653 1.176 91949 4 36.259 56.974 3.149 6.701 1.251 89118 5 37.895 272.688 14.891 3.407 1.091 101066 6 45.185 33.551 1.669 7.927 0.888 133932 7 78.725 469.462 20.626 55.390 1.147 278965

[0091] Table 9. Parameters for assessing system suitability at a flow rate of 1.2 ml / min

[0092] Peak Retention time Peak area Peak Resolution Asymmetry Theoretical number of plates 1 9.092 118.647 11.156 2.744 1.116 16761 2 29.000 166.729 9.729 1.805 1.115 66392 3 30.213 243.300 14.158 2.714 1.188 74146 4 33.014 58.147 2.698 5.622 1.568 57175 5 34.669 211.640 12.180 3.237 1.181 87305 6 41.864 30.216 1.343 6.731 0.886 85286 7 74.839 379.799 16.870 55.523 1.124 252496

[0093] The results showed that, based on the chromatographic peak separation effect, the flow rate was determined to be 1.0 ml / min.

[0094] 1.5 column temperature

[0095] The test solution was prepared according to Section 1.1 of this experiment. Using column temperature as a variable, the test solution was injected into the liquid chromatograph under the chromatographic conditions determined in Section 1.1 and the wavelength of 380 nm determined in Section 1.2. The column temperatures were 30℃, 35℃, and 40℃. The results are shown in [Figure 1]. Figure 5 and the table below.

[0096] Table 10 Parameters for System Suitability Testing at 30℃ Column Temperature

[0097]

[0098]

[0099] Table 11 Parameters for System Suitability Testing at 35℃ Column Temperature

[0100] Peak Retention time Peak area Peak Resolution Asymmetry Theoretical number of plates 1 10.702 137.173 11.345 2.977 1.052 17384 2 31.493 205.899 11.614 1.547 0.994 77348 3 32.693 287.586 17.442 2.689 1.118 88513 4 35.741 59.672 3.281 6.546 1.345 83995 5 37.388 273.801 15.184 3.392 1.028 98041 6 47.121 287.116 14.219 4.444 1.130 125531 7 78.276 494.224 21.458 55.387 1.081 278863

[0101] Table 12 Parameters for System Suitability Testing at 40℃ Column Temperature

[0102] Peak Retention time Peak area Peak Resolution Asymmetry Theoretical number of plates 1 10.511 140.288 11.534 2.684 1.142 16721 2 30.344 189.916 11.553 2.048 1.085 77017 3 31.559 290.996 17.634 2.810 1.115 85856 4 34.298 68.347 3.510 6.046 1.000 83978 5 35.947 291.739 15.666 3.484 1.376 91322 6 43.486 41.239 1.558 0.626 2.009 50873 7 76.172 464.332 20.750 55.692 1.080 265631

[0103] The results showed that the elution time was relatively prolonged when the column temperature was 30℃; the elution time was shortened when the column temperature was 40℃; and the system adaptability parameters of each chromatographic peak were relatively good and the separation effect was the best when the column temperature was 35℃. Considering all factors, the column temperature was determined to be 35℃.

[0104] 1.6 Instrument Selection

[0105] Two test solutions were prepared according to Example 1, and the chromatograms were used as the variable for determination according to Example 1. The results are shown in [Figure 1]. Figure 6 The results are shown in the table below. Chromatographs 1 and 2 are Agilent and Waters, respectively. The results indicate that different chromatographs have little impact on the information content of chromatographic peaks and system adaptability parameters; therefore, a stationary liquid chromatograph is not required at this time, and all chromatographs are applicable.

[0106] Table 13 Results of the investigation using different instruments

[0107]

[0108] The final chromatographic conditions were determined as follows: Octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B, with gradient elution. The gradient elution program included: 0-90 min, mobile phase A volume percentage 10% → 21%; mobile phase B volume percentage 90% → 79%; detection wavelength 380 nm, column temperature 35℃, and flow rate 1.0 mL / min. The theoretical plate number, calculated based on the arginine peak, should be no less than 3000.

[0109] Chromatographic conditions and system suitability: System suitability parameters for the reference solution and the Elaeagnus pungens leaf test solution are shown in [reference needed]. Figure 7 .

[0110] Example 2: Preparation of the test solution

[0111] 2.1 Extraction solvent concentration

[0112] Preparation of the test solution: Take the granulated formula of Elaeagnus angustifolia leaves, grind them finely, accurately weigh about 0.3 g, place them in a stoppered conical flask, add 5 mL of extraction solvent, sonicate (power 350 W, frequency 40 kHz) for 1 h, cool, shake well, filter, and collect the filtrate to obtain the test solution. The extraction solvents used were 20% methanol, 50% methanol, and 80% methanol.

[0113] The chromatographic conditions determined in Experimental Example 1 were followed, and the results are shown in [Figure Number]. Figure 8 and the table below.

[0114] Table 14 Adaptability parameters of chromatographic peak systems for different extraction solvents

[0115]

[0116] Based on the chromatographic peak parameters, the optimal extraction solvent is 50% methanol, considering the total peak area and peak shape.

[0117] 2.2 Extraction time

[0118] The preparation method of the test solution, with extraction time as the variable, included: taking granulated Elaeagnus pungens leaves, grinding them finely, accurately weighing approximately 0.3 g, placing it in a stoppered conical flask, adding 5 mL of 50% methanol, sonicating (power 250 W, frequency 40 kHz), cooling, shaking well, filtering, and collecting the filtrate. The sonication times were 30 min, 45 min, and 60 min, respectively. The chromatographic conditions determined according to Experimental Example 1 were used for determination, and the results are shown in [Figure 1]. Figure 9 and the table below.

[0119] Table 15 Extraction time parameters for chromatographic peak system adaptability.

[0120]

[0121] The results showed that different extraction times had little effect on the retention time of each peak. The peak areas at 30 min and 45 min did not change much, while the peak area at 60 min was generally improved. The separation and tailing factor were generally better. Therefore, the optimal extraction time was 60 min.

[0122] 2.3 Extraction Power

[0123] The test solution was prepared using the ultrasonic treatment power as a variable: Elaeagnus angustifolia leaf granules were finely ground, and approximately 0.3 g was accurately weighed and placed in a stoppered conical flask. 5 mL of 50% methanol was added, and the solution was ultrasonically treated (frequency 40 kHz) for 60 min. After cooling, the solution was shaken well, filtered, and the filtrate was collected. The ultrasonic treatment powers were 150 W, 250 W, and 350 W. The chromatographic conditions determined according to Experimental Example 1 were used, and the results are shown below. Figure 10 and the table below.

[0124] Table 16 Parameters for evaluating the system adaptability of chromatographic peaks at different ultrasonic powers

[0125]

[0126]

[0127] Based on the extraction process, the resolution of each chromatographic peak, asymmetry, theoretical plate number, etc., the optimal ultrasonic power is 350W.

[0128] 2.4 Sample Size

[0129] The sample size was used as the variable. Preparation of the test solution: Elaeagnus angustifolia leaf granules were finely ground. Different masses of the test sample were accurately weighed and placed in stoppered conical flasks. 5 mL of 50% methanol was added, and the solution was sonicated (350 W, 40 kHz) for 60 min. After cooling, the solution was shaken well, filtered, and the filtrate was collected. The sample sizes were 0.2 g, 0.3 g, and 0.4 g, respectively. The chromatographic conditions determined in Example 1 were followed, and the results are shown in the figure. Figure 11 and the table below.

[0130] Table 17 Parameters for Adaptability of Chromatographic Peak System to Different Sample Sizes

[0131]

[0132] The results showed that the sample amounts of 0.2g, 0.3g, and 0.4g had no effect on the retention time of each chromatographic peak, nor on the resolution, symmetry factor, or theoretical plate number. When the sample amount was 0.3g, the peak areas of each chromatographic peak were relatively moderate. Therefore, 0.3g is the preferred amount.

[0133] 2.5 Amount of extraction solvent

[0134] With the amount of extraction solvent as the variable; preparation of the test solution: Take approximately 0.3 g of the Elaeagnus angustifolia leaf granules, grind them finely, and accurately weigh each granule. Place them in a stoppered conical flask, add 50% methanol, sonicate (350 W, 40 kHz) for 60 min, cool, shake well, filter, and collect the filtrate. The amounts of 50% methanol added were 5 ml, 10 ml, and 15 ml, respectively. The chromatographic conditions determined according to Experimental Example 1 were used, and the results are shown below. Figure 12 and the table below.

[0135] Table 18 Parameters for Adaptability of Chromatographic Peak System under Different Extraction Solvent Amounts

[0136]

[0137] The results showed that the amount of extraction solvent had no effect on the retention time of each chromatographic peak, and had little effect on the resolution, symmetry factor, and theoretical plate number; when the volume of 50% methanol was 5 ml, the peak area of ​​the characteristic peaks in the chromatogram was relatively moderate. Therefore, the optimal amount of extraction solvent is 5 ml.

[0138] The final method for preparing the test solution was determined as follows: Take an appropriate amount of this product, grind it into a fine powder, weigh approximately 0.3 g accurately, place it in a stoppered conical flask, add 5 mL of 50% methanol, sonicate (power 350 W, frequency 40 kHz) for 1 hour, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0139] Identification and designation of characteristic peaks in Experiment Example 3

[0140] Fifteen batches of Elaeagnus pungens leaf granules were taken, and test solutions of Elaeagnus pungens leaf granules were prepared according to Example 1. Reference solutions of Elaeagnus pungens leaf and a mixed reference solution of rutin and cynomolgus glycoside were also prepared according to Example 1. Characteristic chromatograms of the 15 batches of Elaeagnus pungens leaf granules were determined, and the results are shown in the table below. Figure 13 The 2012 version of the fingerprint chromatogram similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" compiled by the Pharmacopoeia Commission was used to generate a reference characteristic chromatogram. (See attached image.) Figure 14 , Figure 15For reference, the HPLC characteristic chromatogram of the Elaeagnus pungens leaf formula granules showed seven chromatographic peaks. Peak 4 was used as the reference peak; peak 1 was 0.306, peak 2 was 0.883, peak 3 was 0.916, peak 5 was 1.045, peak 6 was 1.315, and peak 7 was 2.174. The relative retention times of each characteristic peak were within ±10% of the specified values.

[0141] Through reference standard localization and identification, peak 4 was identified as rutin, peak 5 as kaempferol 3-O neohesperidin, peak 6 as astragaloside, and peak 7 as cynomol glycoside.

[0142] Similarity results of characteristic maps of 15 batches of Elaeagnus pungens leaf formula granules: 0.988, 0.994, 0.987, 0.989, 0.995, 0.977, 0.972, 0.994, 0.988, 0.982, 0.979, 0.989, 0.985, 0.981, 0.969.

[0143] Table 19. Results of relative retention times of 15 batches of Elaeagnus pungens leaf granules.

[0144]

[0145] Table 20. Comparison charts of Elaeagnus pungens leaf granule formulations and their relative retention times.

[0146] name Peak 1 Peak 2 Peak 3 Peak 4 (S) Peak 5 Peak 6 Peak 7 Retention time 11.094 31.999 33.173 36.234 37.874 47.636 78.769 Relative retention time 0.306 0.883 0.916 1.000 1.045 1.315 2.174

[0147] Table 21. Comparison of relative peak areas in Elaeagnus pungens leaf granule formulations.

[0148] name Peak 1 Peak 2 Peak 3 Peak 4(s) Peak 5 Peak 6 Peak 7 Peak area 7444 10794 16626 3843 15729 14104 24064 relative peak area 1.937 2.809 4.327 1.000 4.093 3.670 6.262

[0149] Table 22 Relative retention times of characteristic chromatograms of Elaeagnus pungens leaves compared to other medicinal materials

[0150] name Peak 1 Peak 2 Peak 3 Peak 4(s) Peak 5 Peak 6 Peak 7 Retention time 11.12 32.033 33.206 36.307 37.954 47.676 78.796 Relative retention time 0.306 0.882 0.915 1.000 1.045 1.313 2.170

[0151] Table 23 Relative peak areas of characteristic spectra of Elaeagnus pungens leaves as a control medicinal material

[0152] name Peak 1 Peak 2 Peak 3 Peak 4(s) Peak 5 Peak 6 Peak 7 Peak area 2923 8953 13833 3313 15096 14441 25553 relative peak area 0.882 2.703 4.176 1.000 4.557 4.359 7.714

[0153] Experiment Example 4: Methodological Validation

[0154] 4.1 System Applicability

[0155] 4.1.1 Instrument precision test

[0156] The same sample solution of Elaeagnus pungens leaf granules was injected six times under the chromatographic conditions of Example 1. Chromatograms were recorded, and the relative retention times and relative peak areas of seven characteristic peaks were determined and analyzed. The results are shown in the table below. The results indicate that the instrument precision is good. In the table, t / ts represents the relative retention time, and A / As represents the relative peak area.

[0157] Table 24 Relative Retention Time of Instrument Precision

[0158]

[0159] Table 25 Instrument Precision Relative Peak Area

[0160]

[0161] 4.1.2 Method repeatability test

[0162] Six parallel test solutions were prepared using Elaeagnus angustifolia leaf granules from the same batch. The solutions were analyzed under the chromatographic conditions described in Example 1. The results are shown in the table below. The results indicate that the method has good repeatability.

[0163] Table 26 Relative Retention Time for Method Repeatability (n=6)

[0164] Table 27 Relative peak areas for method repeatability (n=6)

[0165]

[0166] 4.1.3 Intermediate Precision

[0167] The same batch of Elaeagnus angustifolia leaf granules was used, and three inspectors prepared test solutions according to Example 1 and measured the results, which are shown in the table below. The results indicate that the intermediate precision (by different personnel) of this method is good.

[0168] Table 28 Intermediate Precision Relative Retention Time (Different Operators)

[0169]

[0170]

[0171] Table 29 Relative Peak Areas for Intermediate Precision (Different Operators)

[0172]

[0173] 4.2 Specificity Test

[0174] The test solution and negative blank solution were prepared according to Example 1. The negative sample was maltodextrin, an excipient used in the production of Elaeagnus pungens leaf granules. The results were measured, the chromatograms were recorded, and the influence of the negative sample was investigated. The results are shown in [Figure 1]. Figure 16 Experimental results show that negative samples do not cause interference.

[0175] 4.3 Durability

[0176] 4.3.1 Stability

[0177] The Elaeagnus angustifolia leaf granule test solution was prepared according to Example 1, and the relative retention time and relative peak area of ​​the seven common peaks were measured at 0, 2, 4, 8, 12, 18, and 24 hours after preparation. The stability of the test solution was determined by analysis, and the results are shown in the table below. The results indicate that the method is stable within 24 hours.

[0178] Table 30 Relative retention time of stability

[0179]

[0180] Table 31 Relative Peak Area for Stability Assessment

[0181]

[0182]

[0183] 4.3.2 Different chromatographic columns

[0184] Using the chromatographic column as a variable, the test solution was prepared and measured according to Example 1. The chromatographic columns were: (1) watersXSelect HSS T3 (column length 250 mm, inner diameter 5 mm, particle size 4.6 μm); (2) SHIMADZU Shim-pack GISS (column length 250 mm, inner diameter 5 mm, particle size 4.6 μm); (3) Agilent A2000250X046 (column length 250 mm, inner diameter 5 mm, particle size 4.6 μm). Chromatograms were recorded, and the results are shown in the table below. The results show that different brands of chromatographic columns have a significant impact on the relative retention time and relative peak area of ​​the characteristic chromatograms. It is recommended to choose the watersXSelect HSS T3 (column length 250 mm, inner diameter 5 mm, particle size 4.6 μm) column.

[0185] Table 32 Relative peak areas were investigated using different chromatographic columns.

[0186]

[0187] Table 33 Relative peak areas were investigated using different chromatographic columns.

[0188]

[0189] Example 3

[0190] This embodiment provides a method for determining the content of cypermethrin in 15 batches of Elaeagnus angustifolia leaf granules. The content was determined using the detection method provided in Example 1. Each batch was measured twice, and the average value was taken. The results are as follows:

[0191] Table 34. Content of cypermethrin in different batches of Elaeagnus pungens leaf granules

[0192]

[0193] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for detecting the characteristic map of Elaeagnus angustifolia leaves, characterized in that, Includes the following steps: Preparation of the test solution: The test sample is prepared into a test solution; the test sample is an aqueous extract of Elaeagnus pungens leaves; a 50-80% methanol aqueous solution is used when preparing the test solution; Rutin, cypermethrin, kaempferol 3-O neohesperidin, and astragalin were used as reference standards. High-performance liquid chromatography (HPLC): The determination was performed using HPLC. The chromatographic conditions included: a WatersXSelect HSS T3 column, acetonitrile as mobile phase A, and 0.05-0.15% formic acid solution or 0.05-0.15% phosphoric acid solution as mobile phase B; gradient elution, with the following elution program: 0-90 min, mobile phase A volume percentage 10%→21%; mobile phase B volume percentage 90%→79%; detection wavelength 380 nm.

2. The detection method according to claim 1, characterized in that, The dosage form of the test sample is tablet, powder, ointment, capsule, pill, granule, sustained-release preparation, immediate-release preparation, controlled-release preparation, oral liquid preparation or injection preparation.

3. The detection method according to claim 1 or 2, characterized in that, The chromatographic conditions also include: a column temperature of 30-40℃; and / or a flow rate of 0.8-1.2 mL / min; and / or an injection volume of 5-15 µL; and / or a column size of 250 mm × 4.6 mm and 5 μm.

4. The detection method according to claim 1 or 2, characterized in that, The chromatographic conditions were as follows: mobile phase B was 0.1% formic acid solution; column temperature was 35℃; flow rate was 1.0 mL / min; and injection volume was 10 µL.

5. The detection method according to claim 1 or 2, characterized in that, The preparation steps of the test sample solution include extracting the test sample with a solvent, filtering it, and taking the filtrate.

6. The detection method according to claim 5, characterized in that, The solvent used is a 50% methanol aqueous solution.

7. The detection method according to claim 5, characterized in that, The ratio of the mass of the test sample to the volume of the extraction solvent is (0.2-0.4):(5-20), where the mass of the test sample is in g and the volume of the extraction solvent is in ml.

8. The detection method according to claim 1 or 2, characterized in that, It also includes the preparation of reference solutions.

9. The detection method according to claim 8, characterized in that, The preparation of the reference solution includes the following steps: taking Elaeagnus pungens leaves as a reference herb, extracting and filtering them, and collecting the filtrate.

10. The detection method according to claim 8, characterized in that, In the preparation of the reference solution, 50%–80% methanol was used as the extraction solvent.

11. The detection method according to claim 1 or 2, characterized in that, The characteristic spectrum of Elaeagnus pungens leaves obtained by the detection method includes at least 7 characteristic peaks; Using peak 4 as the reference peak, the specified values ​​for the relative retention times of each characteristic peak are as follows: peak 1 is 0.30, peak 2 is 0.88, peak 3 is 0.91, peak 5 is 1.05, peak 6 is 1.31, and peak 7 is 2.

17. The relative retention time of each characteristic peak is within ±10% of the specified value.

12. The detection method according to claim 11, characterized in that, Peak 4 is rutin, peak 5 is kaempferol-3-O-neohesperidin, peak 6 is astragaloside, and peak 7 is cypermethrin.

13. The detection method according to claim 1 or 2, characterized in that, It also includes the preparation of reference solutions.

14. The detection method according to claim 1 or 2, characterized in that, The mass of rutin in each 1 ml of the reference solution is 10-40 μg; The mass of ginsenoside in each 1 ml of reference solution is 10-40 μg.

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