A method for determining the content of rosmarinic acid in a standard wind wheel vegetable soup and a method for constructing a characteristic spectrum

Abstract

The application relates to the technical field of pharmaceutical analysis, in particular to a method for determining the content of rosmarinic acid in a standard decoction of wind wheel herb and a method for constructing a characteristic spectrum. The high performance liquid chromatography method is used for determination, and the conditions of the high performance liquid chromatography method include that octadecylsilane bonded silica gel is used as a filler of a chromatographic column; methanol is used as a mobile phase A, and a 0.1% formic acid aqueous solution is used as a mobile phase B, and gradient elution is carried out. The method has strong specificity, good repeatability and durability, high stability and high accuracy; the constructed characteristic spectrum and detection method can quickly and comprehensively realize the purpose of combined control of multiple components in the standard decoction of wind wheel herb, realize quality control of the wind wheel herb from medicinal materials to preparations, and are favorable for guaranteeing the effectiveness of clinical medication.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical analysis, in particular to a method for determining the content of rosmarinic acid in a standard decoction of Clinopodium chinense (Benth.) O. Kuntze and a method for constructing a characteristic chromatogram. BACKGROUND

[0002] Clinopodium chinense (Benth.) O. Kuntze is the dry aboveground part of Clinopodium chinense (Benth.) O. Kuntze, also known as Daanxueliu, Dayexiangre, Yinfuneng, etc., and is distributed in East China, Southwest China, Hebei, Shaanxi, Gansu, Henan, Hubei, Hunan, and Guangxi, etc. Modern research shows that its main components include flavonoids, triterpene saponins, volatile oils, and other components such as alcohols, glycosides, and inorganic salts. Clinopodium chinense (Benth.) O. Kuntze has the effects of hemostasis, indirect promotion of blood clotting, contraction of blood vessels, contraction of the uterus, regulation of endocrine, and anti-inflammatory and antibacterial effects, etc. Clinopodium chinense (Benth.) O. Kuntze is mainly used for treating gynecological hemorrhagic diseases such as metrorrhagia, uterine fibroid hemorrhage, functional uterine bleeding, postpartum hemorrhage, and menorrhagia, etc. A small amount of literature reports that it can also be used for treating traumatic hemorrhage, gingival bleeding, upper gastrointestinal hemorrhage, epistaxis, hematuria, primary thrombocytopenic purpura, and simple purpura, etc.

[0003] The literature “Establishment of HPLC characteristic chromatogram of Clinopodium chinense (Benth.) O. Kuntze and determination of the contents of three components” discloses that a high-pressure liquid chromatography (HPLC) characteristic chromatogram of the chemical components of Clinopodium chinense (Benth.) O. Kuntze is established by using high-performance liquid chromatography, and the contents of three components are simultaneously determined. An Agilent 5TC-C18(2) chromatographic column (250 mm x 4.6 mm, 5 μm) is used, the mobile phase is methanol (A)-water (B), gradient elution is used, and the detection wavelengths are 290 nm (for jatropherol and isosyringidol) and 330 nm (for geniposide). The established HPLC characteristic chromatogram method and multi-component content determination method can provide a basis for evaluating the quality of Clinopodium chinense (Benth.) O. Kuntze.

[0004] The research objects of the above literatures are all radix salviae miltiorrhizae, but the application type research in the quality control field of radix salviae miltiorrhizae standard decoction and formula granules is not involved in the above literatures. Unlike the above literatures, the radix salviae miltiorrhizae standard decoction is a freeze-dried powder prepared by water extraction, concentration and drying of radix salviae miltiorrhizae pieces. Compared with the medicinal materials and pieces, the freeze-dried powder has lost its inherent form and the corresponding active ingredients have also changed. At present, the quality of radix salviae miltiorrhizae is controlled by microscopic and thin-layer identification, which cannot comprehensively reflect the internal quality. At present, there are very few literatures on the high performance liquid chromatography (HPLC) qualitative and quantitative detection technology of radix salviae miltiorrhizae. Therefore, it is urgent to establish a method for determining the content of rosmarinic acid in radix salviae miltiorrhizae standard decoction and constructing a characteristic spectrum by HPLC, so as to provide a basis for effectively controlling and more comprehensively evaluating the quality of radix salviae miltiorrhizae standard decoction. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a method for determining the content of rosmarinic acid in radix salviae miltiorrhizae standard decoction and a method for constructing a characteristic spectrum, which provides a guarantee for the quality and consistency evaluation of radix salviae miltiorrhizae standard decoction.

[0006] In a first aspect, the present application provides a method for determining the content of rosmarinic acid in radix salviae miltiorrhizae standard decoction, which adopts an external standard method and is determined by high performance liquid chromatography. The conditions of the high performance liquid chromatography include:

[0007] The chromatographic column uses octadecylsilane-bonded silica gel as the filler;

[0008] Methanol is used as the mobile phase A, and 0.05-0.3% formic acid aqueous solution is used as the mobile phase B for gradient elution;

[0009] The conditions of the gradient elution are as follows:

[0010]

[0011] The percentage in the table means volume percentage.

[0012] According to some embodiments of the present application, the mobile phase B is 0.05-0.3% formic acid aqueous solution in volume percentage.

[0013] Preferably, the mobile phase B is 0.1% formic acid aqueous solution in volume percentage.

[0014] According to some embodiments of the present application, the chromatographic column is InertSustain C18.

[0015] According to some embodiments of the present application, the specification of the chromatographic column is 4.6 mm x 250 mm, 5 μm.

[0016] According to some embodiments of the present application, the conditions of the high performance liquid chromatography further include that the flow rate of the mobile phase is 0.5-1.0 mL / min.

[0017] Preferably, the conditions of the high performance liquid chromatography further include that the flow rate of the mobile phase is 0.8 mL / min.

[0018] According to some embodiments of the present application, the conditions of the high performance liquid chromatography further include that the column temperature of the chromatographic column is 30-35℃.

[0019] Preferably, the conditions of the high performance liquid chromatography further include that the column temperature of the chromatographic column is 30℃.

[0020] According to some embodiments of the present application, the conditions of the high performance liquid chromatography further include that the injection volume is 5-10 μL.

[0021] Preferably, the conditions of the high performance liquid chromatography further include that the injection volume is 10 μL.

[0022] According to some embodiments of the present application, the conditions of the high performance liquid chromatography further include that the detection wavelength is 330 nm.

[0023] According to some embodiments of the present application, the detector of the high performance liquid chromatography is an ultraviolet detector.

[0024] According to some embodiments of the present application, the theoretical plate number of the high performance liquid chromatography calculated according to the rosmarinic acid peak is not less than 2000.

[0025] According to some embodiments of the present application, the content determination method further includes a preparation of a test sample solution; the preparation step includes mixing a standard decoction of wind wheel herb with a solvent, and ultrasonicating, to obtain the test sample solution.

[0026] According to some embodiments of the present application, the mass concentration of the test sample solution is 1-5 mg / mL.

[0027] According to some embodiments of the present application, the solvent of the test sample solution is water or an alcohol-water mixed solution.

[0028] According to some embodiments of the present application, the solvent is a methanol aqueous solution with a volume percentage of 50-70%, an ethanol aqueous solution with a volume percentage of 50-70%, or water.

[0029] Preferably, the solvent is a methanol aqueous solution with a volume percentage of 50-60%.

[0030] More preferably, the solvent is a methanol aqueous solution with a volume percentage of 50%.

[0031] According to some embodiments of the present application, the ultrasonic conditions comprise at least one of i-iii:

[0032] i. power: 400-600 W;

[0033] ii. frequency: 30-50 kHz;

[0034] iii. time: 20-40 min.

[0035] According to some embodiments of the present application, the content determination method further comprises a preparation of a reference solution, wherein the preparation step comprises mixing a reference with a solvent, and obtaining the same.

[0036] According to some embodiments of the present application, the reference is rosmarinic acid.

[0037] According to some embodiments of the present application, the reference solution has a mass concentration of 20-100 μg / mL.

[0038] According to some embodiments of the present application, the solvent of the reference solution is water or an alcohol-water mixed solution.

[0039] According to some embodiments of the present application, the solvent is a methanol aqueous solution with a volume percentage of 50-70%, an ethanol aqueous solution with a volume percentage of 50-70%, or water.

[0040] Preferably, the solvent is a methanol aqueous solution with a volume percentage of 50-60%.

[0041] More preferably, the solvent is a methanol solution.

[0042] In a second aspect of the present application, a method for constructing a characteristic chromatogram of a standard decoction of windwheel spirose is provided, wherein a reference solution, a control medicinal material solution, and a sample solution are determined by high performance liquid chromatography, and common peaks are calibrated, and the characteristic chromatogram is obtained; the high performance liquid chromatography adopts the above conditions, wherein the wavelength of the characteristic chromatogram is 283 nm, and the other conditions remain unchanged.

[0043] According to some embodiments of the present application, the characteristic chromatogram comprises seven characteristic peaks, wherein peak 5 rosmarinic acid is used as a reference peak, and the relative retention time of each of the other characteristic peaks to the reference peak is calculated, and the relative retention time should be within ±10% of a specified value, and the specified value is: peak 1: 0.66, peak 2: 0.80, peak 3: 0.91, peak 4: 0.96, peak 6: 1.50, and peak 7: 1.65.

[0044] According to some embodiments of the present application, the preparation step of the control medicinal material solution comprises mixing a control medicinal material with a solvent, and ultrasonic treatment, and obtaining the same.

[0045] According to some embodiments of the present application, the quality concentration of the control medicinal material solution is 1-5 mg / mL.

[0046] According to some embodiments of the present application, the solvent is water or an alcohol-water mixed solution.

[0047] According to some preferred embodiments of the present application, the solvent is a methanol-water solution with a volume percentage of 50-70%, an ethanol-water solution with a volume percentage of 50-70%, or water.

[0048] Preferably, the solvent is a methanol-water solution with a volume percentage of 50-60%.

[0049] More preferably, the solvent is a methanol-water solution with a volume percentage of 50%.

[0050] According to some embodiments of the present application, the ultrasonic conditions include at least one of the following i-iii:

[0051] i. Power: 400-600 W;

[0052] ii. Frequency: 30-50 kHz;

[0053] iii. Time: 20-40 min.

[0054] In a third aspect, the present application provides the use of the above-mentioned content determination method or construction method in the detection of the quality of the standard decoction of Herba Leonuri.

[0055] The present application has at least the following advantages:

[0056] The present application provides a characteristic chromatogram and a content determination method for the standard decoction of Herba Leonuri, determines 7 common peaks including rosmarinic acid, and realizes good separation of the chromatographic peaks in the constructed HPLC characteristic chromatogram, so that the characteristic chromatogram information is rich and the chromatographic peak shape is good, which can comprehensively reflect the main chemical component information of the standard decoction of Herba Leonuri, and the detection method has good stability and repeatability, and is convenient for popularization and application.

[0057] The present application constructs an HPLC characteristic chromatogram for the standard decoction of Herba Leonuri based on a high-performance liquid chromatograph, detects the characteristic chromatogram, can control the characteristic components in the standard decoction of Herba Leonuri as a whole, can effectively ensure the stability of the overall quality of the standard decoction of Herba Leonuri, and makes the quality control technology of the standard decoction of Herba Leonuri more perfect and scientific; and the method has the advantages of high precision, good stability, good repeatability, high accuracy, and the like, and provides an effective basis for the quality identification of traditional Chinese medicinal materials.

[0058] The chromatographic condition in the application can be used for detecting the rosmarinic acid content and characteristic spectrum of the standard decoction of Sanguisorba officinalis simultaneously, reduces the solvent consumption and the analysis cost.

[0059] Definitions:

[0060] The Sanguisorba officinalis in the application refers to the dried leaves of Sanguisorba officinalis.

[0061] Other features and advantages of the application will be set forth in the following description, and in part will become apparent from the description, or can be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0062] The above and / or additional aspects and advantages of the application will become apparent and be readily understood by considering the following detailed description, including the accompanying drawings, in which:

[0063] Figure 1 The chromatogram of the organic phase investigation result in Example 1;

[0064] Figure 2 The chromatogram of the optimal absorption wavelength investigation in Example 1 (210nm);

[0065] Figure 3 The chromatogram of the optimal absorption wavelength investigation in Example 1 (230nm);

[0066] Figure 4 The chromatogram of the optimal absorption wavelength investigation in Example 1 (254nm);

[0067] Figure 5 The chromatogram of the optimal absorption wavelength investigation in Example 1 (283nm);

[0068] Figure 6 The chromatogram of the optimal absorption wavelength investigation in Example 1 (320nm)

[0069] Figure 7 The chromatogram of the chromatographic column selection result in Example 1;

[0070] Figure 8 The chromatogram of the gradient selection result in Example 1;

[0071] Figure 9 The superimposed chromatogram of the characteristic spectrum of 15 batches of Sanguisorba officinalis standard decoction in Example 1;

[0072] Figure 10 The control characteristic spectrum of Sanguisorba officinalis standard decoction in Example 1;

[0073] Figure 11 The characteristic spectrum of Sanguisorba officinalis control medicinal material in Example 1;

[0074] Figure 12 Reference standard chromatogram for Example 1;

[0075] Figure 13 Chromatogram for Example 2 at the wavelength of maximum absorption investigated (330 nm);

[0076] Figure 14 Chromatogram for Example 2 for standard broth specificity results;

[0077] Figure 15 Reference standard curve for rosmarinic acid reference standard for Example 2;

[0078] Figure 16 Chromatogram for Satureja montana for acetonitrile-0.1% phosphoric acid mobile phase for Comparative Example 1;

[0079] Figure 17 Chromatogram for Satureja montana for acetonitrile-0.1% formic acid mobile phase for Comparative Example 1;

[0080] Figure 18 Chromatogram for Satureja montana for methanol-0.1% phosphoric acid mobile phase for Comparative Example 1;

[0081] Figure 19 Chromatogram for Satureja montana for methanol-0.1% formic acid mobile phase for Comparative Example 1;

[0082] Figure 20 Chromatogram for Comparative Example 2 for Gradient Method 1 results;

[0083] Figure 21 Chromatogram for Comparative Example 2 for Gradient Method 2 results;

[0084] Figure 22 Chromatogram for Comparative Example 2 for Gradient Method 3 results;

[0085] Figure 23 Chromatogram for Comparative Example 3 for Column A results;

[0086] Figure 24 Chromatogram for Comparative Example 3 for Column B results;

[0087] Figure 25 Chromatogram for Comparative Example 3 for Column C results;

[0088] Figure 26 Chromatogram for Comparative Example 4 for regular method results;

[0089] Figure 27 Chromatogram for Comparative Example 4 for Example 1 method of the present application results;

[0090] Figure 28 Chromatogram for Comparative Example 5 for different concentrations of formic acid. Detailed Implementation

[0091] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0092] The freeze-dried powder of the blood-stopping (Gynostemma pentaphyllum) standard decoction of the present invention is prepared by the following method:

[0093] Take the raw herb *Gnaphalium affine* (also known as *Gnaphalium affine*), remove impurities, moisten slightly with water, cut into sections, dry, and sift to remove ash, obtaining *Gnaphalium affine* slices. Take 100g of *Gnaphalium affine* slices and place them in an electric ceramic kettle. Add water and decoct twice: For the first decoction, add 15 times the amount of water (by weight) and soak for 30 minutes. Bring to a boil over high heat, then simmer over low heat for 30 minutes. Filter the decoction through a 200-mesh sieve while hot and set aside. For the second decoction, add 12 times the amount of water (by weight), heat to a boil over high heat, then simmer over low heat for 20 minutes. Filter the decoction through a 200-mesh sieve while hot. Combine the two decoctions and concentrate under reduced pressure at 65℃ to approximately 120g of extract. Dispense into 6mL–14mL containers, each with a volume of 2mL–3mL. After dispensing, freeze-dry, remove, and cap with an aluminum cap to obtain the final product. Properties of the freeze-dried powder of the standard decoction of *Gnaphalium affine* (also known as *Gnaphalium affine*): Brown to reddish-brown powder; slight odor, slightly bitter taste. The preparation process complies with the relevant provisions under the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules".

[0094] Example 1: Establishment of UPLC characteristic spectrum method for standard decoction of *Gnaphalium affine* (a type of herb).

[0095] 1. Instruments and reagents

[0096] 1.1 Instruments: LC-40D XR high-performance liquid chromatograph (Shimadzu Corporation, Japan); Agilent 1290 high-performance liquid chromatograph (Agilent Technologies, USA); KQ-500DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); MS / 05DU / A electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); ME104E / 02 electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); MS / 303 / 02 electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); MWO-ABS20-DW ultrapure water system (Mingwo Technology);

[0097] Chromatographic column: InertSustain C18 chromatographic column (5 μm, 4.6 mm x 250 mm).

[0098] 1.2 Reagents: methanol, acetonitrile were chromatographically pure (TEDIA); water was ultrapure water (laboratory self-made); other reagents were analytical pure.

[0099] 1.3 Reagents: naringin (purity 98.3%, Chengdu Keluoma Biotechnology Co., Ltd., CHB201208); rosmarinic acid (purity 90.5%, China Institute for Food and Drug Control, 111871-201706); control drug material of wind wheel (self-made).

[0100] 2. Investigation of chromatographic conditions

[0101] 2.1 Investigation of mobile phase: two different organic phases of methanol and acetonitrile were investigated, and the peak shape of methanol was better, so methanol was finally selected as the organic phase, and the results are shown in Figure 1 .

[0102] 2.2 Investigation of wavelength: investigate the chromatogram of blood flow (wind wheel) at different wavelengths of 210 nm, 230 nm, 254 nm, 283 nm and 320 nm, and finally select 283 nm as the characteristic spectrum detection wavelength, the chromatographic peak is more, and the impurity interference is less, and the results are shown in Figures 2 to 6 .

[0103] 2.3 Selection of chromatographic column: investigate the chromatogram of blood flow (wind wheel) under three different chromatographic columns of InertSustain C18, Agilent ZORBAX SB-C18 and Agilent Eclipse Plus C18, and select InertSustain C18 when the resolution is better, finally select InertSustain C18 chromatographic column, and the results are shown in Figure 7 . Investigate the chromatogram of blood flow (wind wheel) under three different gradients, and the gradient elution is shown in the table below, select gradient elution 3 when the resolution is better, finally select gradient elution 3, and the results are shown in Tables 1-3 Figure 8 .

[0104] Table 1 Gradient elution 1

[0105]

[0106] Table 2 Gradient elution 2

[0107]

[0108]

[0109] Table 3 Gradient elution 3

[0110]

[0111] 3 Determination of chromatographic conditions

[0112] 3.1 Chromatographic conditions and system suitability test

[0113] Take octadecylsilane-bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); take methanol as mobile phase A and take 0.1% formic acid aqueous solution as mobile phase B, and perform gradient elution under the following specified gradient elution conditions; flow rate 0.8 mL / min; column temperature 30 °C; detection wavelength 330 nm; and the theoretical plate number calculated according to the rosmarinic acid peak should not be less than 2000. The specified gradient elution program is as follows: (in the table, the percentage means volume percentage.)

[0114]

[0115] 3.2 Preparation of solutions

[0116] (1) Preparation of the reference solution of the control: take naringin and rosmarinic acid control substances in appropriate amounts, accurately weigh, add methanol to prepare a mixed solution containing 40 μg of naringin and 40 μg of rosmarinic acid per 1 mL, as the reference solution of the control.

[0117] (2) Preparation of the solution of the control medicinal material: take 0.1 g of the control medicinal material of bleeding stope, add 25 mL of 50% methanol, ultrasonically treat (power 500 W, frequency 40 kHz) for 20 minutes, cool, shake well, and filter, as the solution of the control medicinal material.

[0118] (3) Preparation of the test solution: take about 0.1 g of the freeze-dried powder of the product, accurately weigh, place in a conical flask with a stopper, accurately add 25 mL of 50% methanol, weigh, ultrasonically treat (power 500 W, frequency 40 kHz) for 20 minutes, cool, weigh again, make up the weight loss with 50% methanol, shake well, and filter, to obtain the test solution.

[0119] 3.3 Determination method: take 10 μL of the obtained reference solution of the control, the solution of the control medicinal material, and the test solution respectively, inject into the high performance liquid chromatograph, and determine, to obtain the test solution.

[0120] 4 Identification and selection of characteristic peaks

[0121] Establishment of sample characteristic chromatogram: prepare 15 batches of standard decoction test solution of bleeding stope (wind wheel vegetable), inject sample respectively, and record the characteristic chromatogram of 15 batches of standard decoction of bleeding stope (wind wheel vegetable).

[0122] The test solution chromatogram should show 7 characteristic peaks, which should correspond to the retention times of the 7 characteristic peaks in the chromatogram of the reference substance of the control medicinal material. The retention time of one peak should correspond to the retention time of the corresponding reference peak of the control substance. The peak corresponding to the rosmarinic acid reference peak is the S peak. The relative retention times of the other characteristic peaks to the S peak should be within ±10% of the specified values, which are 0.66 (peak 1), 0.80 (peak 2), 0.91 (peak 3), 0.96 (peak 4), 1.50 (peak 6), and 1.65 (peak 7). The characteristic chromatogram of the standard decoction of Sanguisorba officinalis (L.) Moech (Sanguisorba officinalis) was obtained, as well as the standard decoction control characteristic chromatogram, the reference substance solution of the control medicinal material, and the chromatogram of the reference substance of the control substance, as shown in the following table. Figures 9 to 12

[0123] 5. Methodology verification of characteristic chromatogram

[0124] 5.1 Reproducibility test

[0125] Sanguisorba officinalis (L.) Moech standard decoction lyophilized powder was weighed at about 0.1 g (batch number DGZS-PF-YL-208-210801), and 6 samples were prepared in parallel according to the test sample method. The chromatograms were recorded and integrated, with peak 5 as the reference peak, and the relative retention times and relative peak areas of each characteristic peak were calculated. The results are shown in the following tables. The relative retention times of each characteristic peak were less than 1%, and the relative peak area RSD was less than 12%. The test showed that the method had good reproducibility, and the results are shown in Tables 4-5.

[0126] Table 4 Reproducibility data of Sanguisorba officinalis (L.) Moech standard decoction (relative retention time)

[0127]

[0128] Table 5 Reproducibility data of Sanguisorba officinalis (L.) Moech standard decoction (relative peak area)

[0129]

[0130]

[0131] 5.2 Precision test

[0132] Sanguisorba officinalis (L.) Moech standard decoction lyophilized powder was weighed at about 0.1 g (batch number DGZS-PF-YL-208-210801), and prepared according to the test sample method. The chromatograms were recorded and integrated, with peak 5 as the reference peak, and the relative retention times and relative peak areas of each characteristic peak were calculated. The results are shown in the following tables. The relative retention times of each characteristic peak were less than 1%, and the relative peak area RSD was less than 9%. The test showed that the method had good precision, and the results are shown in Tables 6-7.

[0133] ​Table 6 Precision data of standard decoction of Sanguisorba officinalis (Radix Mume) (relative retention time)

[0134]

[0135] Table 7 Precision data of standard decoction of Sanguisorba officinalis (Radix Mume) (relative peak area)

[0136]

[0137]

[0138] 5.3 Stability test

[0139] About 0.1 g of lyophilized powder of standard decoction of Sanguisorba officinalis (Radix Mume) (batch number DGZS-PF-YL-208-210801) was weighed, prepared according to the test sample method, and injected at different time points within 12 h, the chromatogram was recorded and integrated, and the relative retention time and relative peak area of each characteristic peak were calculated with peak 5 as the reference peak. The results are shown in the following tables. The relative retention time of each characteristic peak was less than 1%, and the relative peak area RSD was less than 9%. The test showed that the test sample solution was stable within 12 h, and the results are shown in Tables 8-9.

[0140] Table 8 Stability data of standard decoction of Sanguisorba officinalis (Radix Mume) (relative retention time)

[0141]

[0142] Table 9 Stability data of standard decoction of Sanguisorba officinalis (Radix Mume) (relative peak area)

[0143]

[0144] 6 Determination of relative retention time of characteristic peaks

[0145] Through the above series of tests and optimization, under the optimal chromatographic conditions of the characteristic spectrum, 7 characteristic peaks should appear in the chromatogram of the test sample solution; the peaks were identified by reference to the control substance. Peak 2 is naringin, peak 5 is rosmarinic acid, and peak 5 has a high peak area and a moderate retention time, so peak 5 (rosmarinic acid) is used as the S peak. Analyze the fingerprints of 15 batches of standard decoction of Sanguisorba officinalis (Radix Mume), calculate the relative retention time of the 7 common peaks relative to peak 5, and the results are shown in Table 10.

[0146] Table 10 Relative retention time results of standard decoction of Sanguisorba officinalis (Radix Mume)

[0147]

[0148] Conclusion: The test solution chromatogram should show 7 characteristic peaks, which should correspond to the retention times of the 7 characteristic peaks in the reference chromatogram of the control medicinal material. The retention time of one peak should correspond to the retention time of the corresponding reference peak. The peak corresponding to the rosmarinic acid reference peak is the S peak. Calculate the relative retention time of each characteristic peak to the S peak. The relative retention time should be within ±10% of the specified value, which is 0.66 (peak 1), 0.80 (peak 2), 0.91 (peak 3), 0.96 (peak 4), 1.50 (peak 6), and 1.65 (peak 7).

[0149] Example 2: Establishment of UPLC content determination method for standard decoction of Sanguisorba officinalis

[0150] Based on the construction method of the UPLC characteristic spectrum of the standard decoction of Sanguisorba officinalis (S. officinalis) in Example 1, Example 2 also conducted the following methodological investigation, established the UPLC content determination method for the standard decoction of S. officinalis, which specifically includes:

[0151] 1.1 Instruments and equipment

[0152] LC-20AT high performance liquid chromatograph (Shimadzu Corporation); KM-500DB ultrasonic cleaner (Kunshan Meimei Ultrasonic Instrument Co., Ltd.); T-214 electronic balance (Beijing Sartorius Instrument System Co., Ltd.); XPE105 electronic balance (Mettler-Toledo Instrument Co., Ltd.).

[0153] 1.2 Column information

[0154] InertSustain C18 column (5 μm, 4.6*250 mm)

[0155] 1.3 Reagents

[0156] Methanol for analysis (Hunan Huihong Reagent Co., Ltd., batch number: 202110233); methanol for chromatography (American Tian Di Co., Ltd., batch number: 21095151); formic acid for chromatography (Tianjin Kemio Chemical Reagent Co., Ltd., batch number: 20201110); water for Yibao water (Hualun Yibao Beverage Co., Ltd., batch number: 20211105).

[0157] 1.4 Reagents

[0158] Naringin (purity 98.3%, Chengdu Keluoma Biological Technology Co., Ltd., CHB201208); rosmarinic acid (purity 90.5%, China Institute for Drug Control, 111871-201706).

[0159] 2 Selection of chromatographic conditions

[0160] 2.1 Selection of the measured component

[0161] Rosmarinic acid was selected as the evaluation index to verify the chromatographic conditions and system suitability for the determination of the content of the standard decoction of the Xueshuailu (Sedum lineare Thunb.) medicinal material, and the sample pretreatment method was investigated to determine the test sample preparation method.

[0162] 2.2 Selection of wavelength

[0163] Referring to the “2.2 Investigation of wavelength” in “Case 1”, in the chromatogram of Xueshuailu (Sedum lineare Thunb.), the maximum absorption of rosmarinic acid was at a wavelength of 330 nm; finally, the maximum absorption wavelength 330 nm of rosmarinic acid was selected as the detection wavelength for the content determination, as shown in the following figure. Figure 13

[0164] According to the wavelength scanning graph from 190 nm to 800 nm, the rosmarinic acid reference substance with a retention time of 41.956 min had a large peak at 329 nm, and the scaling factor was 1.00, so 330 nm was selected as the detection wavelength for the content determination.

[0165] 3 Investigation of test sample preparation method

[0166] 3.1 Selection of extraction time

[0167] The effects of different ultrasonic times on the chromatogram of the standard decoction were investigated. 0.1 g of sample was added to 25 ml of 50% methanol and ultrasonicated for 20 minutes, 30 minutes, and 40 minutes, respectively. The results showed that the contents of rosmarinic acid were 7.40 mg / g, 4.98 mg / g, and 7.41 mg / g, respectively. The contents of rosmarinic acid were higher and had little difference when ultrasonicated for 20 minutes and 40 minutes. Finally, ultrasonication for 20 minutes was selected, and the results are shown in the following Table 11.

[0168] Table 11 Contents of rosmarinic acid in Xueshuailu (Sedum lineare Thunb.) standard decoction under different ultrasonic times

[0169]

[0170] 3.2 Selection of solvent amount

[0171] The effects of different solvent amounts on the content of rosmarinic acid in the standard decoction were investigated. 0.1 g of sample was added to 10 ml, 25 ml, and 50 ml of 50% methanol, respectively, and ultrasonicated for 20 minutes. The results showed that the contents of rosmarinic acid were 4.55 mg / g, 7.40 mg / g, and 7.24 mg / g, respectively. The content was the highest when 25 ml was used. Finally, about 0.1 g of sample was selected, and 25 ml of 50% methanol was added. The results are shown in the following Table 12.

[0172] Table 12 Contents of rosmarinic acid in Xueshuailu (Sedum lineare Thunb.) standard decoction under different solvent amounts ​

[0173]

[0174] 3.3 Determination of chromatographic conditions

[0175] The chromatographic column was an InertSustain C18 column (5 μm, 4.6*250 mm, SN: 20F0125895); methanol was used as mobile phase A, and 0.1% formic acid aqueous solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the column temperature was 30℃; the flow rate was 0.8 mL / min, and the wavelength was 330 nm (for content determination). The injection volume was 10 μl, and the theoretical plate number calculated based on the rosmarinic acid peak should not be less than 2000.

[0176]

[0177] Preparation of the test solution: Accurately weigh approximately 0.1 g of the lyophilized powder of this product, place it in a stoppered conical flask, accurately add 25 ml of 50% methanol, weigh, sonicate (500 W, 40 kHz) for 20 minutes, cool, weigh, make up the weight loss with 50% methanol, shake well, and filter to obtain the test solution. Preparation of the reference solution: Accurately weigh an appropriate amount of rosmarinic acid reference standard, add methanol to prepare a solution containing 40 μg per ml. Assay: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph, and determine the result.

[0178] 4. Validation of content determination method

[0179] 4.1 Specificity

[0180] Prepare a test solution using the standard decoction of *Rhizoma Cynanchi Amaranthus* (batch number DGZS-PF-YL-208-210801). Determine the chromatographic properties under the conditions described above, and plot the chromatograms of rosmarinic acid reference standard, the blank of the *Rhizoma Cynanchi Amaranthus* standard decoction (50% methanol), and its excipient (dextrin). The results are shown in the figure below. In the chromatogram of the test solution, a peak with the same retention time as the reference standard is observed. No interference is found from the blank and the excipient (dextrin). The experiment demonstrates that this method has good specificity. Figure 14 As shown.

[0181] 4.2 Linear Relationship

[0182] Accurately weigh an appropriate amount of rosmarinic acid reference standard, add methanol to prepare a solution containing 239.80 μg / ml, which serves as the reference standard stock solution. Dilute and measure the solution sequentially, record the chromatograms and integrate them. Using rosmarinic acid as the reference peak, calculate the peak area. The results are shown in the table below. Plot a standard curve with concentration on the x-axis and peak area on the y-axis. The results are shown in the figure below. y = 44233x - 67581, r = 0.9997. The results indicate that rosmarinic acid exhibits good linearity in the range of 4.80 μg / ml to 239.80 μg / ml.Figure 15 Results are shown in Table 13 below.

[0183] Table 13 Rosmarinic acid concentration and peak area data

[0184]

[0185] 4.3 Reproducibility test

[0186] About 0.1 g of standard decoction freeze-dried powder of Salvia miltiorrhiza (batch number DGZS-PF-YL-208-210801) was weighed, and 6 samples were prepared in parallel according to the test sample method. The chromatogram was recorded and integrated according to the law, the content of rosmarinic acid was calculated, the results are shown in the table below, and the content RSD value is less than 1%. The test showed that the method was reproducible well, and the results are shown in Table 14.

[0187] Table 14 Reproducibility content data of standard decoction of Salvia miltiorrhiza (Bupleurum)

[0188]

[0189] 4.4 Precision test

[0190] About 0.1 g of standard decoction freeze-dried powder of Salvia miltiorrhiza (batch number DGZS-PF-YL-208-210801) was weighed, and the test sample solution was prepared. The sample was injected 6 times in succession, and the chromatogram was recorded and integrated according to the law. The peak area was calculated with rosmarinic acid as the reference peak. The results are shown in the table below, and the reference peak area RSD is less than 3%. The test showed that the method was precise, and the results are shown in Table 15.

[0191] Table 15 Precision peak area data of standard decoction of Salvia miltiorrhiza (Bupleurum)

[0192]

[0193]

[0194] 4.5 Stability test

[0195] About 0.1 g of standard decoction freeze-dried powder of Salvia miltiorrhiza (batch number DGZS-PF-YL-208-210801) was weighed, and the test sample solution was prepared. The sample was injected at different time points within 12 hours, and the chromatogram was recorded and integrated according to the law. The peak area of rosmarinic acid was calculated. The results are shown in the table below, and the reference peak area RSD is less than 3%. The test showed that the peak area of the test sample solution was basically stable within 12 hours, and the results are shown in Table 16.

[0196] Table 16 Stability peak area data of standard decoction of Salvia miltiorrhiza (Bupleurum)

[0197]

[0198] 4.6 Intermediate precision

[0199] Take the blood flow (wind wheel menu) standard decoction freeze-dried powder about 0.1 g (batch DGZS-PF-YL-208-210801), prepared in parallel 6, according to the law of determination, record chromatogram and integral, calculate the peak area and content of rosmarinic acid, the results are shown in the table below, with the repeatability of the results of the content of RSD in 2%, the test showed that: the intermediate precision of this method is good, the results are shown in Table 17.

[0200] Table 17 blood flow (wind wheel menu) standard decoction intermediate precision content data

[0201]

[0202] 4.7 Recovery test

[0203] Take the blood flow (wind wheel menu) standard decoction freeze-dried powder 6 (batch DGZS-PF-YL-208-210801) measured content, about 0.05 g each, accurately weighed, respectively, parallel to add rosmarinic acid, according to the preparation method of test solution, according to the law of determination, record chromatogram and integral, the results are shown in the table, the recovery rate is between 97.72% ~ 101.51% of the specified range, RSD less than 2%, the test showed that: the method of standard recovery is good, the results are shown in Table 18.

[0204] Table 18 blood flow (wind wheel menu) standard decoction accuracy data

[0205]

[0206] 4.8 durability

[0207] Take the blood flow (wind wheel menu) standard decoction freeze-dried powder about 0.1 g (batch DGZS-PF-YL-208-210801), preparation of test solution. According to the law of determination, record chromatogram and integral, with rosmarinic acid as reference peak, calculate the content, the results are shown in the table. Under different chromatographic conditions, the RSD value of rosmarinic acid content is less than 5%, the test showed that: the method of durability is good, the results are shown in Table 19.

[0208] Table 19 blood flow (wind wheel menu) standard decoction durability data

[0209]

[0210] 5 standard decoction sample determination

[0211] The content of rosmarinic acid in 15 batches of standard decoction was determined according to the law. The measured content of rosmarinic acid in 15 batches of standard decoction was in the range of 7.4-12.9 mg / g, the average content was 10.8 mg / g, the content range of mean ± 30% was 7.6-14.0 mg / g, the SD value was 1.7, and the content range of mean ± 3SD was 5.6-16.0 mg / g. The above data showed that the content distribution of 15 batches of standard decoction was relatively discrete. The measured transfer rate of rosmarinic acid was in the range of 20.2%-57.0%, the average transfer rate was 35.5%, the transfer rate range of mean ± 30% was 24.9%-46.2%, the SD value was 11.2, and the transfer rate range of mean ± 3SD was 1.9%-69.1%. According to the data distribution of 15 batches of standard decoction, the content limit range was determined as mean + 3SD, and the content of rosmarinic acid in standard decoction was finally determined as 5.6-16.0 mg / g. The upper limit of content transfer rate was mean + 3SD, and the lower limit was the minimum measured value. The content transfer rate range of rosmarinic acid in standard decoction was finally determined as 20.2%-69.0%.

[0212] Table 20 Content determination results of 15 batches of standard decoction of Sanguisorba officinalis (Sanguisorba officinalis)

[0213]

[0214]

[0215] Selection and investigation of mobile phase types in Comparative Example 1

[0216] The gradient elution was performed according to the preset gradient in Table 3 at a flow rate of 0.8 ml / min and a column temperature of 30 ℃, and the changes of chromatographic peaks were investigated by using four different mobile phases of A: acetonitrile-0.1% phosphoric acid; B: acetonitrile-0.1% formic acid; C: methanol-0.1% phosphoric acid; and D: methanol-0.1% formic acid, so as to determine the chromatographic conditions of the characteristic spectrum of Sanguisorba officinalis.

[0217] The peak shape was better, the separation degree was better, and the baseline was more stable by using methanol-0.1% formic acid as the mobile phase. Therefore, methanol-0.1% formic acid was selected as the mobile phase for the characteristic spectrum, as shown in the chromatogram results. Figures 16 to 19

[0218] Comparison of different gradient methods in Comparative Example 2

[0219] The elution was performed by using different gradient methods at a column temperature of 30 ℃ and according to the preset chromatographic column and mobile phase (chromatographic column: InertSustain C18; methanol as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B), the changes of three different gradient methods were investigated, and the chromatographic conditions of the characteristic spectrum of Sanguisorba officinalis were determined.

[0220] Table 21 Gradient method 1

[0221] ​

[0222] Table 22 Gradient method 2

[0223]

[0224] Table 23 Gradient method 3

[0225]

[0226]

[0227] Gradient method is 3 (gradient elution procedure specified in Example 1), peak shape is better, separation is better, and baseline is more stable. Therefore, the characteristic map gradient method is 3, as shown in the chromatogram results Figures 20 to 22 .

[0228] Comparative Example 3 Comparison of different chromatographic columns

[0229] Gradient elution was carried out according to the gradient elution procedure specified in Example 1, three different chromatographic columns A: InertSustain C18; B: ZORBAX Eclipse Plus C18; C: Ultimate HPLC column MB-C18 were investigated to investigate the changes of chromatographic peaks, and the determination of the chromatographic conditions of bleeding.

[0230] Among the three different chromatographic columns, the chromatographic column A has better peak shape and separation degree, and is representative, so the bleeding method adopts the chromatographic column A, as shown in the chromatogram results Figures 23 to 25 .

[0231] Comparative Example 4

[0232] The conventional chromatographic conditions are as follows: the chromatographic column is Ultimate LP-C18 (4.6 mm x 250 mm, 5 μm) ; the mobile phase is acetonitrile-0.2% phosphoric acid solution (20:80, V / V) ; the flow rate is 1.0 mL / min; the column temperature is 30°C; and the detection wavelength is 330 nm. The conventional chromatographic conditions are compared with the bleeding method in Example 1.

[0233] The conventional method is not as good as Example 1 in separation effect and peak shape, and the conventional method is poor, mainly reflected in that the separation effect cannot be achieved, there are included impurity peaks, tailing, etc., and the characteristic map of bleeding can be studied by only changing the detection wavelength, so the bleeding method of Example 1 is better than the conventional method, and the results are shown as follows Figures 26 to 27 .

[0234] Comparative Example 5 Comparison of different concentrations of formic acid

[0235] The gradient elution was carried out at a flow rate of 1.0 ml / min, column temperature of 30℃, and specified gradient elution program, and the changes of chromatographic peaks were investigated with A: methanol-0.1% formic acid; B: methanol-0.3% formic acid; two different mobile phases, so as to determine the chromatographic conditions for the determination of the bloodletting method.

[0236] At different concentrations, more peaks appeared at low concentrations, and the separation effect of 0.1% formic acid was better than that of 0.3% formic acid; and high-difficulty acid caused great damage to the chromatographic column, and the chromatographic column also had requirements for acid and alkali, therefore, 0.1% formic acid was adopted, and the results are shown in Table 1. Figure 28

[0237] The embodiments of the present application are described in detail above in combination with the drawings, but the present application is not limited to the above-described embodiments, and various changes can be made within the knowledge range possessed by those skilled in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.​

Claims

1. A method for determining the content of rosmarinic acid in a standard decoction of Artemisia scoparia, characterized by, The determination by high performance liquid chromatography also includes preparation of the test solution and the control solution; The solvent of the test solution and the control solution is 50-70% methanol aqueous solution by volume percentage; The conditions of the high performance liquid chromatography include: The chromatographic column is packed with octadecylsilane-bonded silica gel; The detection wavelength is 330 nm; Methanol is used as the mobile phase A, and 0.1-0.2% formic acid aqueous solution by volume percentage is used as the mobile phase B for gradient elution; The conditions of the gradient elution are as follows:

2. The assay method of claim 1, wherein, The conditions of the high performance liquid chromatography also include at least one of i-iii: i. flow rate: 0.5-1.0 mL / min; ii. column temperature: 30-35℃; iii. injection volume: 5-20 μL.

3. The assay of claim 1, wherein, The mass concentration of the test solution is 1-5 mg / mL; The mass concentration of the control solution is 20-100 μg / mL.

4. The assay of claim 1, wherein, The theoretical plate number is calculated based on the rosmarinic acid peak, and is not less than 2000.

5. A method for constructing a characteristic map of a standard decoction of windwheel clover, characterized in that, The control solution, the control medicinal material solution and the test solution are determined by high performance liquid chromatography to calibrate the common peak, i.e. the characteristic chromatogram is obtained, wherein the conditions of the high performance liquid chromatography are as described in any one of claims 1 to 4, and the wavelength for the characteristic chromatogram is 283 nm, and the other conditions remain unchanged.

6. The construction method of claim 5, wherein, The characteristic chromatogram includes seven characteristic peaks, wherein, taking peak 5 rosmarinic acid as the reference peak, the relative retention time of each of the other characteristic peaks to the reference peak is calculated, and the relative retention time should be within ±10% of the specified value, and the specified value is: peak 1: 0.66, peak 2: 0.80, peak 3: 0.91, peak 4: 0.96, peak 6: 1.50, peak 7: 1.

65.

7. The construction method of claim 5, wherein, The mass concentration of the control medicinal material solution is 1-5 mg / mL.

8. Application of the content determination method of any one of claims 1 to 4 or the construction method of any one of claims 5 to 7 in detecting the quality of the standard decoction of Anemone vitifolia.

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