A quality control method for mulberry seed formula granules and its application

Abstract

The present invention provides a quality control method for a formula granule of Corydalis chinensis and its application. The quality control method comprises the following steps: using high performance liquid chromatography to obtain a characteristic spectrum of the formula granule of Corydalis chinensis, taking the chromatographic peak of linoleic acid as the reference peak S in the characteristic spectrum, calculating the relative retention time of each characteristic peak and the reference peak S, wherein there are at least two characteristic peaks, and the relative retention time of the characteristic peaks is within ±10% of a specified value, the specified value is: 0.64 or 1.74, and the content of linoleic acid is not less than 4.9 μg / mL. The present invention constructs an HPLC characteristic spectrum of the formula granule of Corydalis chinensis by measuring the linoleic acid content in the formula granule of Corydalis chinensis, provides a method for constructing an HPLC characteristic spectrum of the formula granule of Corydalis chinensis and a quality control method thereof, strengthens specific identification and overall quality control, establishes a characteristic spectrum of the formula granule of Corydalis chinensis, and thus performs comprehensive quality control.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine detection and quality control, and particularly relates to a quality control method for Cuscuta australis formula granules and application thereof. Background Art

[0002] This product is the dried mature fruit of Broussonetia papyrifera (L.) Vent., a plant of the Moraceae family. This product is sweet and cold. It enters the liver and kidney meridians. Functions: nourishing the kidney and clearing the liver, improving eyesight, and promoting urination. It is used for liver and kidney deficiency, soreness of the waist and knees, asthenia and bone steaming, dizziness, eye film, edema and fullness. According to research, it was first recorded in the "Famous Doctors' Records" of the Han Dynasty. It is also known as Chu Shi, Gu Shi, Gu Zi, and Chu Tao. It is the dried mature fruit of Broussonetia papyrifera, a plant of the Moraceae family.

[0003] At present, the statutory standards do not fully control the medicinal materials of Cuscuta australis. The statutory standards only set standard limits on the properties, microscopy and inspection items of Cuscuta australis. In the prior art, Cuscuta australis medicinal materials have been widely studied, and traditional property identification and thin layer identification are commonly used identification methods. However, Cuscuta australis formula granules have lost the original appearance and properties of the medicinal materials. They are formula granules obtained by extracting, concentrating, drying, and preparing Cuscuta australis slices. There is a big difference between their material basis and the material basis of the medicinal materials.

[0004] In the prior art, for the determination of the total saponin content of the seeds of paper mulberry, 1 g of the seeds of paper mulberry powder (passed through a 20-mesh sieve) was accurately weighed and added with 50 mL of petroleum ether (60-90°C). After cold soaking for 12 hours, the residue was ultrasonically treated with 10 times the volume of petroleum ether for 30 minutes each time. The residue was ultrasonically treated with an 80% ethanol solution for 30 minutes each time, and the filtrate was combined and concentrated to 100 mL, and shaken well. 50 mL of the solution was accurately drawn and placed in an evaporating dish in a water bath and concentrated to dryness, and 10 mL of distilled water was added to dissolve. The aqueous solution was added to a ZS-101 macroporous resin column (1.0 cm × 10.0 cm), eluted with 50 mL of water, discarded the water, and then eluted with 80% ethanol solution. The 80% ethanol solution eluate was collected and placed in a 10 mL volumetric flask to the mark, and shaken well to obtain the test solution. Pipette 0.5 mL of the test solution into a 10 mL stoppered test tube, evaporate the solvent, accurately add 5 mL of perchloric acid, and color it in a 70°C water bath for 15 min. After cooling with ice water, scan at 200-700 nm. The results show that both have strong absorption peaks at 407 nm.

[0005] The defects of the prior art: the total saponin content is measured, the sample preparation process is very complicated, the effective ingredients are unclear, and the material basis is different from the formula granules for the medicinal materials. The prior art cannot accurately identify the formula granules of Cuscuta australis. Therefore, a quality control method for Cuscuta australis formula granules is provided to strengthen the specific identification and overall quality control, and comprehensive quality control of Cuscuta australis formula granules has important application value. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a quality control method for mulberry seed formula granules and its application. The present invention determines the linoleic acid in the mulberry seed formula granules, constructs the HPLC characteristic spectrum of the mulberry seed formula granules, provides a method for constructing the HPLC characteristic spectrum of the mulberry seed formula granules and a quality control method thereof, strengthens the specific identification and overall quality control, establishes the characteristic spectrum of the variety, and thus performs comprehensive quality control.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a quality control method for a granule of Codonopsis pilosula, the quality control method comprising the following steps:

[0009] High performance liquid chromatography is used to obtain a characteristic spectrum of the paper mulberry seed formula granules. In the characteristic spectrum, the chromatographic peak of linoleic acid is used as the reference peak S, and the relative retention time of each characteristic peak and the reference peak S is calculated. There are at least two characteristic peaks, and the relative retention time of the characteristic peaks is within ±10% of a specified value, and the specified value is: 0.64 or 1.74.

[0010] Cuscuta australis has the effects of lowering blood lipids, resisting atherosclerosis, resisting platelet aggregation and thrombosis. Its pharmacological effects are mainly due to the linoleic acid in the fatty acids in its oil, but the existing methods cannot detect the linoleic acid in its oil. The existing technology has the technical defects of being unable to identify Cuscuta australis formula granules and being unable to determine the linoleic acid content in its oil. Based on this, the present invention provides a method for constructing an HPLC characteristic spectrum of Cuscuta australis formula granules and a quality control method thereof, strengthens specific identification and overall quality control, establishes a characteristic spectrum of the variety, and thus performs comprehensive quality control. Cuscuta australis formula granules all present 3 characteristic peaks in the HPLC characteristic spectrum, of which peak 2 should be consistent with the retention time of the linoleic acid reference substance, and peak 2 is taken as the S peak, and the relative retention time of characteristic peaks 1 and 3 is calculated, and the relative retention time should be within ±10% of the specified value.

[0011] Preferably, at least three characteristic peaks are presented in the characteristic spectrum of the Cuscuta australis formula granules, namely peak 1, peak 2 and peak 3, the relative retention time of peak 1 is 0.64±10%, peak 2 is the chromatographic peak of linoleic acid, and the relative retention time of peak 3 is 1.74±10%.

[0012] Preferably, the step of obtaining the characteristic spectrum of the mulberry seed formula granules by high performance liquid chromatography comprises:

[0013] (1) Mixing the mulberry seed granules and methanol, and ultrasonically treating them to prepare a test solution; preparing the mulberry seed reference medicinal material into a reference medicinal material reference solution; and mixing the linoleic acid reference substance with methanol to prepare a reference substance solution;

[0014] (2) The test solution, the control medicinal material reference solution and the reference solution were tested by high performance liquid chromatography to obtain the content of linoleic acid in the Cuscuta australis granules and the characteristic spectrum of the Cuscuta australis granules.

[0015] Preferably, in step (1), the mass ratio of the paper mulberry seeds formula particles to methanol is 1:(20-100), for example, it can be 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or 1:100, etc.

[0016] Preferably, the power of the ultrasonic treatment is 300-500 W, for example, 300 W, 350 W, 400 W, 450 W or 500 W, etc., the frequency of the ultrasonic treatment is 30-50 kHz, for example, 30 kHz, 35 kHz, 40 kHz, 45 kHz or 50 kHz, etc., and the time of the ultrasonic treatment is 10-40 min, for example, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min or 40 min, etc.

[0017] Preferably, the mass concentration of linoleic acid in the reference solution is 110-150 μg / mL, for example, it can be 110 μg / mL, 115 μg / mL, 120 μg / mL, 125 μg / mL, 130 μg / mL, 135 μg / mL, 140 μg / mL, 145 μg / mL or 150 μg / mL, etc.

[0018] Preferably, in step (1), the control medicinal material reference solution is prepared by a method comprising the following steps: mixing the Cucurbita chinensis reference medicinal material with methanol, ultrasonically treating, filtering, and taking the filtrate to obtain the control medicinal material reference solution.

[0019] Preferably, the mass ratio of the Cuscuta australis reference medicinal material to methanol is 1:(10-30), for example, it can be 1:10, 1:15, 1:20, 1:25 or 1:30, etc.

[0020] Preferably, the power of the ultrasonic treatment is 300-500 W, for example, 300 W, 350 W, 400 W, 450 W or 500 W, etc., the frequency of the ultrasonic treatment is 30-50 kHz, for example, 30 kHz, 35 kHz, 40 kHz, 45 kHz or 50 kHz, etc., and the time of the ultrasonic treatment is 10-40 min, for example, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min or 40 min, etc.

[0021] Preferably, in step (2), the chromatographic conditions of the HPLC are as follows: the chromatographic column of the HPLC is filled with octadecylsilane bonded silica gel; 0.08-0.12% phosphoric acid (for example, 0.08%, 0.09%, 0.1%, 0.11% or 0.12%, etc.) is used as mobile phase A, acetonitrile is used as mobile phase B, and the volume ratio of mobile phase A to mobile phase B is (70-80):(20-30) for isocratic elution, and the volume ratio can be, for example, 70:30, 72:28, 74:26, 75:25, 76:24, 78:22 or 80:20, etc.

[0022] Preferably, the chromatographic column comprises ZORBAX Eclipse XDB-C18, 4.6×250 mm, 5 μm, Agilent 5TC-C18, 4.6×250 mm, 5 μm or XTERRA MS C18, 4.6×250 mm, 5 μm.

[0023] Preferably, the flow rate of the mobile phase in the high performance liquid chromatography is 0.8-1.2 mL per minute, for example, it can be 0.8 mL, 0.9 mL, 1.0 mL, 1.1 mL or 1.2 mL, etc., the column temperature is 28-32°C, for example, it can be 28°C, 29°C, 30°C, 31°C or 32°C, etc., the detection wavelength is 203-210 nm, for example, it can be 203 nm, 205 nm or 210 nm, etc., and the number of theoretical plates calculated based on the linoleic acid peak should be not less than 5000.

[0024] As a preferred technical solution of the present invention, the quality control method comprises the following steps:

[0025] (1) Mix the mulberry seed granules and methanol in a mass ratio of 1:(20-100), and perform ultrasonic treatment at 300-500W and 30-50kHz for 10-40min to prepare a test solution;

[0026] Mix the Cuscuta australis reference medicinal material with methanol at a mass ratio of 1:(10-30), perform ultrasonic treatment at 300-500W and 30-50kHz for 10-40min, filter, and take the filtrate to obtain a reference medicinal material solution;

[0027] The linoleic acid reference substance is mixed with methanol to prepare a reference substance solution, wherein the mass concentration of linoleic acid in the reference substance solution is 110-150 μg / mL.

[0028] (2) The test solution, the reference medicinal material reference solution and the reference substance solution are tested by high performance liquid chromatography; the chromatographic column of the high performance liquid chromatography is filled with octadecylsilane bonded silica gel; 0.08-0.12% phosphoric acid is used as mobile phase A, acetonitrile is used as mobile phase B, and the volume ratio of mobile phase A to mobile phase B is (70-80):(20-30) for isocratic elution; the chromatographic column includes ZORBAX Eclipse XDB-C18, 4.6×250mm, 5μm, Agilent 5TC-C18, 4.6×250mm, 5μm or XTERRA MS C18, 4.6×250mm, 5μm; the flow rate of the mobile phase in the high performance liquid chromatography is 0.8-1.2mL per minute, the column temperature is 28-32°C, the detection wavelength is 203-210nm, and the number of theoretical plates calculated based on the linoleic acid peak should be not less than 5000;

[0029] The content of linoleic acid in the paper mulberry seed formula granules and the characteristic spectrum of the paper mulberry seed formula granules are obtained, wherein the chromatographic peak of linoleic acid in the characteristic spectrum is used as the reference peak S, and the relative retention time of each characteristic peak and the reference peak S is calculated, wherein there are at least two characteristic peaks, and the relative retention time of the characteristic peaks is within ±10% of a specified value, and the specified value is: 0.64 or 1.74.

[0030] In the second aspect, the present invention provides application of the quality control method of the mulberry seed formula granules described in the first aspect in quality inspection of the mulberry seed formula granules.

[0031] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The present invention constructs the UPLC characteristic spectrum of the granules of the seed of Cuscuta australis, which fully demonstrates the chemical composition characteristics of the granules of the seed of Cuscuta australis;

[0034] (2) The characteristic spectrum constructed by the present invention can fully reflect the characteristic peak information of the sample, and the method is stable, with high precision and good reproducibility;

[0035] (3) The HPLC characteristic spectrum of the Cuscuta australis granules established in the present invention can be used to control the quality of the Cuscuta australis granules, providing a rapid and reliable detection method for the identification of the Cuscuta australis granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the control characteristic spectrum in Example 1;

[0037] Figure 2 It is the HPLC characteristic spectrum of 5 batches of Cuscuta australis granules in Example 1;

[0038] Figure 3 This is the precision investigation result of the characteristic spectrum of the granules of the mulberry seed formula in Example 3;

[0039] Figure 4 This is the result of the repeatability investigation of the characteristic spectrum of the granules of the mulberry seed formula in Example 3;

[0040] Figure 5 This is the intermediate precision investigation result of the characteristic spectrum of the granules of the seed of paper mulberry in Example 3;

[0041] Figure 6 This is the result of stability study of the characteristic spectrum of the granules of the mulberry seed formula in Example 4;

[0042] Figure 7 is a chromatogram of different flow rates of the granules of the seed of Cuscuta australis in Example 4;

[0043] Figure 8 It is the chromatogram of different column temperatures of the granules of the seed of Cuscuta australis in Example 4;

[0044] Fig. 9 It is the characteristic spectrum of different chromatographic columns of the formula granules of the seed of paper mulberry in Example 4;

[0045] Fig.10 is the chromatogram corresponding to the gradient elution procedure 1 in Example 8;

[0046] Fig.11 This is the chromatogram corresponding to the gradient elution program 2 in Example 8. DETAILED DESCRIPTION

[0047] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0048] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0049] The sources of materials and instruments in the following specific embodiments are as follows:

[0050] 1. Reagents and test drugs

[0051] Comparative medicinal materials:

[0052] Linoleic acid reference substance: batch number: 111622-201704, purity 99.8%, China Food and Drug Inspection Institute;

[0053] Cuscuta australis formula granules: Beijing Kangrentang Pharmaceutical Co., Ltd.; batch numbers include: 202109091, 202109092, 202109093, 202109094, and 202109095 granules.

[0054] Reagents:

[0055] Methanol: Sinopharm Chemical Reagent Co., Ltd.;

[0056] Acetonitrile: Fisher Chemical chromatographic grade;

[0057] Phosphoric acid: Fisher Chemical chromatographic grade.

[0058] 2. Instruments

[0059] High performance liquid chromatograph: Shimadzu LC-2010AHT high performance liquid chromatograph (TUV detector, LCsolution workstation);

[0060] Ultrasonic cleaning instrument: KQ-500DA ultrasonic cleaning instrument (power 300W, frequency 40kHz, Kunshan Ultrasonic Instrument Co., Ltd.);

[0061] Chromatographic column:

[0062] ZORBAX Eclipse XDB-C18 (4.6×250mm, 5μm);

[0063] Agilent 5TC-C18 (4.6×250mm, 5μm);

[0064] XTERRA MS C18 (4.6×250mm, 5μm).

[0065] Example 1 Characteristic Spectrum Construction

[0066] Preparation of reference solution: Take about 2.0 g of Cuscuta australis reference medicinal material, accurately weigh it, put it in a stoppered conical flask, accurately add 25 mL of methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate to obtain the reference solution.

[0067] Take an appropriate amount of linoleic acid reference substance, weigh it accurately, and add methanol to make a solution containing 130 μg per 1 mL as the reference substance solution.

[0068] Preparation of test solution: Grind the granules of Cuscuta australis, take about 0.5 g, weigh accurately, put it in a stoppered conical flask, add 25 mL of methanol accurately, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 min, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0069] Determination method: Accurately pipette 5 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0070] The chromatographic conditions are as follows: using octadecylsilane bonded silica gel as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); isocratic elution with acetonitrile-0.1% phosphoric acid (75:25); flow rate 1.0 mL per minute; column temperature 30°C; detection wavelength 203 nm, detection time 60 min. The theoretical plate number calculated based on the linoleic acid peak should not be less than 5000.

[0071] The characteristic spectrum of the control is as follows Figure 1 As shown, the HPLC characteristic spectra of 5 batches of Cuscuta australis granules are as follows Figure 2 As shown in the figure, R(3): control characteristic spectrum, S5(3): 202109091 particles, S4(3): 202109092 particles, S3(3): 202109093 particles, S2(3): 202109094 particles, S1(3): 202109095 particles.

[0072] The HPLC characteristic spectrum of the formula granules of Cuscuta australis showed three characteristic peaks, of which peak 2 should be consistent with the retention time of the linoleic acid reference substance. Peak 2 was taken as the S peak, and the relative retention time of characteristic peaks 1 and 3 was calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.64 (peak 1) and 1.74 (peak 3).

[0073] The Chinese medicine chromatographic fingerprint similarity evaluation software is used for evaluation. If the sample to be identified is consistent with the characteristic spectrum of Cuscuta australis, it is the Cuscuta australis formula granule.

[0074] Example 2 Content Determination

[0075] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0076] Preparation of reference solution: Take about 2.0 g of Cuscuta australis reference medicinal material, accurately weigh it, put it in a stoppered conical flask, accurately add 25 mL of methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate to obtain the reference solution.

[0077] Take an appropriate amount of linoleic acid reference substance, weigh it accurately, and add methanol to make a solution containing 130 μg per 1 mL as the reference substance solution.

[0078] Preparation of test solution: Grind the granules of Cuscuta australis, take about 0.5 g, weigh accurately, put it in a stoppered conical flask, add 25 mL of methanol accurately, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 min, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0079] Determination method: Accurately pipette 5 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0080] The chromatographic conditions are as follows: using octadecylsilane bonded silica gel as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); isocratic elution with acetonitrile-0.1% phosphoric acid (75:25); flow rate 1.0 mL per minute; column temperature 30°C; detection wavelength 203 nm, detection time 60 min. The theoretical plate number calculated based on the linoleic acid peak should not be less than 5000.

[0081] The HPLC characteristic spectrum of the formula granules of Cuscuta australis showed three characteristic peaks, of which peak 2 should be consistent with the retention time of the linoleic acid reference substance. Peak 2 was taken as the S peak, and the relative retention time of characteristic peaks 1 and 3 was calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.64 (peak 1) and 1.74 (peak 3).

[0082] The samples of the formula granules of Cuscuta australis and the calculation results of their similarity are shown in Table 1:

[0083] Table 1

[0084] serial number batch number Similarity with the control map R Control feature map 1.000 S1 202109091 1.000 S2 202109092 1.000 S3 202109093 1.000 S4 202109094 1.000 S5 202109095 1.000

[0085] As can be seen from Table 1, the similarities between the characteristic spectra of the five batches of Cuscuta australis formula granules and the control characteristic spectra are all 1.000, indicating that the differences in the spectra are small.

[0086] Example 3 Methodology Verification

[0087] 1. Precision

[0088] Take an appropriate amount of the sample of the formula granules of Cuscuta australis (202109093), grind it into powder, take 0.5g, prepare the test solution according to the preparation method of the test solution, and measure it according to the chromatographic conditions of Examples 1 and 2. Inject 6 injections continuously to measure the instrument precision. Take the second peak as the reference peak, calculate its relative peak area and relative retention time. And calculate RSD.

[0089] The precision test results of the characteristic spectrum of the formula granules of Cuscuta australis are as follows Figure 3 As shown in the figure, S6(3): 202109093 granule-6, S5(3): 202109093 granule-5, S4(3): 202109093 granule-4, S3(3): 202109093 granule-3, S2(3): 202109093 granule-2, S1(3): 202109093 granule-1. The retention time and relative retention time table of the precision investigation of the granules of the formula of Cuscuta australis are shown in Table 2, and the peak area and relative peak area table of the precision investigation of the granules of the formula of Cuscuta australis are shown in Table 3.

[0090] Table 2

[0091]

[0092]

[0093] Table 3

[0094]

[0095] The precision test results show that the relative retention time RSD of the three identification peaks of the six-needle determination results is in the range of 0-0.10%, indicating that the precision of the instrument is good.

[0096] 2. Repeatability

[0097] Using Shimadzu LC-2010AHT high performance liquid chromatograph, take an appropriate amount of the sample of the formula granules of Cuscuta australis (202109093 granules), grind it into powder, take 0.5 g, take 6 portions, prepare the test solution according to the preparation method of the test solution, and measure it according to the chromatographic conditions of Examples 1 and 2. Take the characteristic peak of linoleic acid (the second peak) as the reference peak, calculate its relative peak area and relative retention time, and calculate RSD.

[0098] The repeatability test results of the characteristic spectrum of the granules of the formula of Cuscuta australis are as follows Figure 4As shown in the figure, S6(3): 202109093 granule-6, S5(3): 202109093 granule-5, S4(3): 202109093 granule-4, S3(3): 202109093 granule-3, S2(3): 202109093 granule-2, S1(3): 202109093 granule-1. The retention time and relative retention time table of the repeatability investigation of the granules of the formula of Cuscuta australis are shown in Table 4, and the peak area and relative peak area table of the repeatability investigation of the granules of the formula of Cuscuta australis are shown in Table 5.

[0099] Table 4

[0100]

[0101] Table 5

[0102]

[0103] The results of the repeatability experiment showed that the relative retention time RSD of the three identification peaks of the six repeatability experimental samples was in the range of 0.0-0.07%, and the relative peak area RSD was in the range of 0.36-3.62%, indicating that the repeatability of the characteristic spectrum was good.

[0104] 3. Intermediate precision

[0105] Using a Waters 2695 high performance liquid chromatograph, take an appropriate amount of the sample of the formula granules of Cuscuta australis (batch number: 202109093), grind it into powder, take 0.5 g, take 6 portions, prepare the test solution according to the preparation method of the test solution, and measure it according to the chromatographic conditions of Examples 1 and 2. Take the characteristic peak of linoleic acid (the second peak) as the reference peak, calculate its relative peak area and relative retention time, and calculate RSD.

[0106] The results of the intermediate precision investigation of the characteristic spectrum of the formula granules of Cuscuta australis are as follows Figure 5 As shown in the figure, S6: 202109093 granule-6, S5: 202109093 granule-5, S4: 202109093 granule-4, S3: 202109093 granule-3, S2: 202109093 granule-2, S1: 202109093 granule-1. The retention time and relative retention time table of the intermediate precision inspection of the formula granules of Cuscuta australis are shown in Table 6, and the peak area and relative peak area table of the intermediate precision inspection of the formula granules of Cuscuta australis are shown in Table 7.

[0107] Table 6

[0108]

[0109] Table 7

[0110]

[0111] The results of the intermediate precision experiment showed that the relative retention time RSD of the three identification peaks of the six samples was in the range of 0-0.10%, indicating that the intermediate precision of the characteristic spectrum was good and the characteristic spectrum method met the analysis requirements between different instruments.

[0112] Example 4 Durability

[0113] 1. Stability

[0114] An appropriate amount of Cuscuta australis formula granule sample (202109093) was ground into powder. 0.5 g was taken and the test solution was prepared according to the preparation method of the test solution. The test was carried out at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h, respectively. The retention time and peak area of ​​each common peak were recorded, and the RSD was calculated.

[0115] The results of the stability test of the characteristic spectrum of the granules of the seed of mulberry are as follows Figure 6 As shown, S1 to S8 in the figure are 0h to 24h of the above sample list. The retention time and relative retention time table of the stability study of the granules of the formula of Cuscuta chinensis are shown in Table 8, and the peak area and relative peak area table of the stability study of the granules of the formula of Cuscuta chinensis are shown in Table 9.

[0116] Table 8

[0117]

[0118]

[0119] Table 9

[0120]

[0121] The results of the stability experiment showed that after examining the stability of the solution for 24 hours, the relative retention time RSD of the three identification peaks was in the range of 0 to 0.33%. The characteristic spectrum analysis method of the paper mulberry formula particles used in this experiment was stable, reliable and reproducible.

[0122] 2. Investigation of different flow rates

[0123] Take an appropriate amount of the Cuscuta chinensis formula granule sample (2021090903), grind it into powder, take 0.5 g, prepare the test solution according to the preparation method of the test solution, and measure it according to the chromatographic conditions of Examples 1 and 2. This experiment examined the flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min to examine the durability of the experimental method for different flow rates.

[0124] Chromatograms of different flow rates of Cuscuta australis granules Figure 7As shown in the figure, S1: flow rate 0.8mL / min, S2: flow rate 1.0mL / min, S3: flow rate 1.2mL / min. The retention time and relative retention time table corresponding to different flow rates are shown in Table 10, and the peak area and relative peak area table corresponding to different flow rates are shown in Table 11.

[0125] Table 10

[0126]

[0127] Table 11

[0128]

[0129] Summary: From the above data, it can be seen that the RSD of the relative retention time of the three peaks is between 0 and 0.86%, and the separation of the chromatographic peaks is good at different flow rates. This method has good durability to flow rates.

[0130] 3. Investigation of different column temperatures

[0131] Take an appropriate amount of the sample of the formula granules of Cuscuta australis (202109093), grind it into powder, take 0.5 g, prepare the test solution according to the preparation method of the test solution, and measure it according to the chromatographic conditions of Examples 1 and 2. This experiment examined the column temperatures of 28°C, 30°C and 32°C to examine the durability of the experimental method for different column temperatures.

[0132] Chromatograms of different column temperatures of Cuscuta australis granules Figure 8 As shown in the figure, S1: column temperature 28°C, S2: column temperature 30°C, S3: column temperature 32°C. The relative retention time table of different column temperatures is shown in Table 12, and the relative peak area table of different column temperatures is shown in Table 13. In the table, 1, 2, and 3 correspond to column temperatures of 28°C, 30°C, and 32°C, respectively.

[0133] Table 12

[0134]

[0135] Table 13

[0136]

[0137] Summary: From the above data, it can be seen that the RSDs of the relative retention times of the three peaks are all in the range of 0-0.12%, and the RSDs of the relative peak areas are 0.59% and 2.70%, respectively, indicating that this method has good durability to column temperature.

[0138] 4. Investigation of different chromatographic columns

[0139] Take an appropriate amount of Cuscuta australis formula granule sample (202109093), grind it into powder, take 0.5 g, prepare the test solution according to the preparation method of the test solution, and use different types of chromatographic columns for determination to examine the durability of the experimental method for different chromatographic columns.

[0140] The stationary phase is as follows:

[0141] ZORBAX Eclipse XDB-C18 (4.6×250mm, 5μm);

[0142] Agilent 5TC-C18 (4.6×250mm, 5μm);

[0143] XTERRA MS C18 (4.6×250mm, 5μm).

[0144] Characteristic spectra of different chromatographic columns of Cuscuta australis granules Fig. 9 As shown in the figure, S1: ZORBAX Eclipse XDB-C18; S2: Agilent 5TC-C18; S3: XTERRA MS C18. The relative retention time schedules of different chromatographic columns are shown in Table 14.

[0145] Table 14

[0146] Chromatographic columns Peak 1 Peak 2 Peak 3 ZORBAX Eclipse XDB-C18 0.632 1.000 1.748 Agilent 5TC-C18 0.689 1.000 1.593 XTERRA MS C18 0.678 1.000 1.617 RSD(%) 4.530 0.000 5.055

[0147] Summary: As can be seen from the figure above, this method is not very specific in the selection of stationary phase. From the above results, it can be seen that ZORBAX Eclipse XDB-C18, Agilent 5TC-C18, and XTERRA MS C18 can all obtain good characteristic spectra. Although the Agilent 5TC-C18 and XTERRA MS C18 chromatographic columns have good separation effects, the peaks have shifted to varying degrees relative to the retention time, but they are all within the acceptable range, and the peak shape of XTERRA MS C18 is also good. Comprehensively comparing the peak shape, separation degree and other aspects of ZORBAXEclipse XDB-C18 and XTERRA MS C18, they are relatively excellent. Therefore, the method has good durability in the selection of stationary phase. It is recommended to use ZORBAX Eclipse XDB-C18 and XTERRA MS C18 for this method.

[0148] Example 5 Investigating the effects of different column temperatures on content and characteristic spectra

[0149] Test method: Take an appropriate amount of the sample of the formula granules of Cuscuta australis (202109093), grind it into powder, take 0.5g, prepare the test solution according to the preparation method of the test solution, and measure it according to the chromatographic conditions of Examples 1 and 2. This experiment examined the column temperatures of 22°C, 30°C and 38°C to examine the effects of different column temperatures on the content and characteristic spectra.

[0150] The relative retention time table of different column temperatures is shown in Table 15, and the relative peak area table of different column temperatures is shown in Table 16. In the table, 1, 2, and 3 correspond to column temperatures of 22°C, 30°C, and 38°C, respectively.

[0151] Table 15

[0152]

[0153] Table 16

[0154]

[0155] As can be seen from the above table, the RSDs of the relative retention times of the three peaks are all in the range of 0 to 0.12%, and the RSDs of the relative peak areas are 0.59% and 2.70%, respectively, indicating that this method has good durability to column temperature.

[0156] Example 6 Investigating the effects of different flow rates on content and characteristic spectra

[0157] Test method: Take an appropriate amount of the sample of the formula granules of Cuscuta australis (202109093), grind it into powder, take 0.5g, prepare the test solution according to the preparation method of the test solution, and measure it according to the chromatographic conditions of Examples 1 and 2. This experiment examined the flow rates of 0.5mL / min, 1.0mL / min, and 1.5mL / min to examine the effects of different flow rates on the content and characteristic spectra.

[0158] The relative retention time table of different flow rates is shown in Table 17, and the relative peak area table of different flow rates is shown in Table 18. In the table, 1, 2, and 3 correspond to a flow rate of 0.5 mL / min, a flow rate of 1.0 mL / min, and a flow rate of 1.5 mL / min, respectively.

[0159] Table 17

[0160]

[0161] Table 18

[0162]

[0163] As can be seen from the above table, the RSD of the relative retention time of the three peaks is between 0.04% and 0.86%, and the separation of the chromatographic peaks is good at different flow rates. This method has good durability to the flow rate.

[0164] Example 7 Investigating the effects of different elution procedures on content and characteristic spectra

[0165] Test method: Take an appropriate amount of linoleic acid reference substance, accurately weigh it, and add methanol to make a solution containing 0.16 mg per 1 mL. Take 6 portions of mulberry seeds granules with a known content (2021009091, mulberry seeds content is 6.5 mg / g), each about 0.25 g, accurately weigh it, accurately add the corresponding linoleic acid reference substance solution, and make up to 25 mL with methanol. The rest of the operation is the same as in Example 1. The recovery rate is calculated according to the following formula. The results are as follows.

[0166]

[0167] Elution procedure 1: using 0.1% phosphoric acid as mobile phase A and acetonitrile as mobile phase B, the volume ratio of mobile phase A to mobile phase B is 75:25 for isocratic elution.

[0168] Elution procedure 2: using 0.1% phosphoric acid as mobile phase A and acetonitrile as mobile phase B, with the volume ratio of mobile phase A to mobile phase B being 78:22 for isocratic elution.

[0169] Elution procedure 3: using 0.1% phosphoric acid as mobile phase A and acetonitrile as mobile phase B, with the volume ratio of mobile phase A to mobile phase B being 72:28 for isocratic elution.

[0170] Elution procedure 4: using 0.08% phosphoric acid as mobile phase A and acetonitrile as mobile phase B, with the volume ratio of mobile phase A to mobile phase B being 75:25 for isocratic elution.

[0171] Elution procedure 5: using 0.12% phosphoric acid as mobile phase A and acetonitrile as mobile phase B, with the volume ratio of mobile phase A to mobile phase B being 75:25 for isocratic elution.

[0172] Elution procedure 6: using 0.1% phosphoric acid as mobile phase A and acetonitrile as mobile phase B, with the volume ratio of mobile phase A to mobile phase B being 65:35 for isocratic elution.

[0173] Elution procedure 7: using 0.1% phosphoric acid as mobile phase A and acetonitrile as mobile phase B, with the volume ratio of mobile phase A to mobile phase B being 85:15 for isocratic elution.

[0174] The results of the accuracy experiment are shown in Table 19:

[0175] Table 19

[0176] Elution procedure Sample volume Measured amount Recovery rate Elution procedure 1 1.631mg 3.044mg 87％ Elution procedure 2 1.631mg 3.158mg 94％ Elution procedure 3 1.631mg 3.011mg 85％ Elution Procedure 4 1.631mg 3.028mg 86％ Elution procedure 5 1.631mg 3028mg 86％ Elution Procedure 6 1.631mg 3.060mg 88％ Elution Procedure 7 1.631mg 3.109mg 91％

[0177] From the results in Table 19, it can be seen that the mobile phase is 0.1% phosphoric acid as mobile phase A and acetonitrile as mobile phase B. The closer the volume ratio of mobile phase A to mobile phase B is to 80:20, the higher the isocratic elution recovery rate and the more accurate the content determination result.

[0178] Example 8 Investigating the elution effect of the gradient elution program

[0179] Test method:

[0180] Preparation method of test solution: Take the formula granules of Cuscuta chinensis, grind them into powder, take about 0.5g, weigh accurately, put them in a stoppered conical bottle, add 25mL of methanol accurately, ultrasonically treat (power 300W, frequency 40kHz) for 30min, let cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain. Determine according to the following chromatographic conditions.

[0181] Gradient elution program 1: 0.1% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution. The gradient is shown in Table 20.

[0182] Table 20

[0183] Flow rate mL / min Flow rate mL / min Mobile phase A Mobile phase A 0 1.0 75 25 15 1.0 75 25 17 1.0 82 18 30 1.0 82 18 32 1.0 80 20 42 1.0 80 20

[0184] The results of gradient elution program 1 are as follows Fig.10 As shown, from Fig.10 It can be seen that the peak shapes and separation effects of each characteristic peak are already good, but they can still be adjusted appropriately.

[0185] Gradient elution program 2: 0.1% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution. The gradient is shown in Table 21.

[0186] Preparation method of test solution:

[0187] Take the formula granules of Cuscuta australis, grind them into powder, take about 0.5g, weigh it accurately, put it in a stoppered conical flask, add 25mL of 70% methanol accurately, treat it ultrasonically (power 300W, frequency 40kHz) for 30min, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0188] Table 21

[0189] Flow rate mL / min Flow rate mL / min Mobile phase A Mobile phase A 0 1.0 75 25 15 1.0 75 25 17 1.0 82 18 30 1.0 82 18 32 1.0 80 20 42 1.0 80 20

[0190] The results of gradient elution program 2 are as follows Fig.11 As shown, from Fig.11 It can be seen that the peak shapes of the characteristic peaks are poor and the separation effect is also poor, so the chromatographic conditions need to be improved.

[0191] In summary, the present invention determines the linoleic acid content in the Cuscuta australis formula granules and constructs the HPLC characteristic spectrum of the Cuscuta australis formula granules, provides a method for constructing the HPLC characteristic spectrum of the Cuscuta australis formula granules and a quality control method thereof, strengthens specific identification and overall quality control, establishes the characteristic spectrum of the Cuscuta australis formula granules, and thus performs comprehensive quality control.

[0192] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A quality control method for mulberry seed granules, characterized in that: The quality control method comprises the following steps: A high performance liquid chromatography method is used to obtain a characteristic spectrum of the mulberry seed formula granules, wherein the chromatographic peak of linoleic acid in the characteristic spectrum is used as a reference peak S, and the relative retention time of each characteristic peak and the reference peak S is calculated, wherein there are at least two characteristic peaks, and the relative retention time of the characteristic peaks is within ±10% of a specified value, and the specified value is: 0.64 or 1.74; In the characteristic spectrum of the Cuscuta chinensis formula granules, at least three characteristic peaks are presented, namely peak 1, peak 2 and peak 3, the relative retention time of peak 1 is 0.64±10%, peak 2 is the chromatographic peak of linoleic acid, and the relative retention time of peak 3 is 1.74±10%; The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column of the high performance liquid chromatography uses octadecylsilane bonded silica gel as a filler; 0.08-0.12% phosphoric acid is used as mobile phase A, acetonitrile is used as mobile phase B, and the volume ratio of the mobile phase A to the mobile phase B is (70-80):(20-30) for isocratic elution; The chromatographic columns include ZORBAX Eclipse XDB-C18, 4.6×250 mm, 5 μm, Agilent 5TC-C18, 4.6×250 mm, 5 μm or XTERRA MS C18, 4.6×250 mm, 5 μm.

2. The quality control method of the mulberry seed granules according to claim 1, characterized in that: The steps of obtaining the characteristic spectrum of the mulberry seed formula granules by high performance liquid chromatography include: (1) Mixing the mulberry seed granules and methanol, and ultrasonically treating them to prepare a test solution; preparing the mulberry seed reference medicinal material into a reference medicinal material reference solution; and mixing the linoleic acid reference substance with methanol to prepare a reference substance solution; (2) The test solution, the control medicinal material reference solution and the reference solution were tested by high performance liquid chromatography to obtain the content of linoleic acid in the Cuscuta australis granules and the characteristic spectrum of the Cuscuta australis granules.

3. The quality control method of the mulberry seed formula granules according to claim 2, characterized in that: In step (1), the mass ratio of the paper mulberry seeds formula particles to methanol is 1:(20-100).

4. The quality control method of the mulberry seed granules according to claim 2, characterized in that: The power of the ultrasonic treatment is 300-500W, the frequency of the ultrasonic treatment is 30-50kHz, and the time of the ultrasonic treatment is 10-40min.

5. The quality control method of the paper mulberry seed granules according to claim 2, characterized in that: In step (1), the mass concentration of linoleic acid in the reference solution is 110-150 μg / mL.

6. The quality control method of the paper mulberry seed granules according to claim 2, characterized in that: In step (1), the control medicinal material reference solution is prepared by a method comprising the following steps: mixing the Cuscuta australis reference medicinal material with methanol, ultrasonically treating, filtering, and taking the filtrate to obtain the control medicinal material reference solution.

7. The quality control method of the mulberry seed granules according to claim 6, characterized in that: The mass ratio of the Cuscuta australis reference medicinal material to methanol is 1:(10-30).

8. The quality control method of the mulberry seed granules according to claim 6, characterized in that: The power of the ultrasonic treatment is 300-500W, the frequency of the ultrasonic treatment is 30-50kHz, and the time of the ultrasonic treatment is 10-40min.

9. The quality control method of the paper mulberry seed granules according to claim 2, characterized in that: The flow rate of the mobile phase in the high performance liquid chromatography method is 0.8-1.2 mL per minute, the column temperature is 28-32° C., the detection wavelength is 203-210 nm, and the theoretical plate number calculated based on the linoleic acid peak should be no less than 5000.

10. Use of the quality control method of the Cuscuta australis formula granules according to any one of claims 1 to 9 in the quality inspection of the Cuscuta australis formula granules.

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