A mimensia contralateral extract, quality control method and application thereof

Abstract

The application discloses a mistletoe reference extract and a quality control method and application thereof, and relates to the field of medicine, in particular to a mistletoe reference extract and a quality control method and application thereof. The mistletoe reference extract is prepared by the following method: taking mistletoe medicinal materials, extracting by heating and refluxing with water, combining the extract, filtering, reducing pressure and concentrating the filtrate, centrifuging, filtering, adding equal amounts of ethyl acetate, stirring, standing, taking supernatant, recovering ethyl acetate under reduced pressure, dissolving with proper amount of methanol, centrifuging to take supernatant, adding proper amount of micro-powder silica gel, drying into thick paste under reduced pressure, freeze-drying, adding proper amount of micro-powder silica gel again, and crushing to obtain the mistletoe reference extract. In the quality control method, the detection conditions of thin layer chromatography and high performance liquid chromatography are optimized. The mistletoe reference extract provided by the application can be used as a reference material for detecting the quality of mistletoe medicinal materials and preparations, solves the problem of lack of reference materials, improves the quality control standard of mistletoe preparations, and is beneficial to improving the overall quality of the preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and in particular relates to a control extract of Caulis Spatholobi and a quality control method and application thereof. Background Art

[0002] Jinsha vine is the dried aerial part of Lygodinm japonicum (Thunb.) Sw., Lygodium microphyllum (Cav.) R.Br., and Lygodium flexuosum (L.) Sw., all members of the Lygodium family. It primarily contains volatile oils, flavonoids, phenolic acids, and phenylpropanoid compounds, and is listed in local medicinal material standards such as the "Guangxi Traditional Chinese Medicine Standards" and the "Guangxi Zhuang Medicine Quality Standards." Current quality control methods for Jinsha vine require the use of multiple single components as reference substances for identification and measurement. This is a cumbersome process and does not fully reflect the true quality of the medicinal material.

[0003] Sanjin Tablets is a Chinese patent medicine listed in the 2015 edition of the Chinese Pharmacopoeia. Jicosa chinensis is one of the five medicinal ingredients in the prescription. However, there is currently a lack of quality control methods for Jicosa chinensis under its standard thin-layer identification items, and there are no corresponding clear indicator components. There is also a lack of relevant reference substances, and basic research is relatively weak.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the existing technology and provide a control extract of Caulis Agrimoniae and its quality control method and application. The control extract of Caulis Agrimoniae provided by the present invention can be used as a reference substance to detect the quality of Caulis Agrimoniae medicinal materials and preparations, thus solving the problem of lack of reference substances and improving the quality control standards of Caulis Agrimoniae preparations, which is conducive to improving the overall quality of the preparations.

[0006] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:

[0007] The first object of the present invention is to provide a control extract of Caulis Agrimoniae, which is prepared by the following method:

[0008] Take the medicinal material of Jinsha Teng, extract it by heating and refluxing with water, combine the extracts, filter, concentrate the filtrate under reduced pressure, centrifuge, filter, add an equal amount of ethyl acetate, stir evenly, let it stand, take the supernatant, recover the ethyl acetate under reduced pressure, add an appropriate amount of methanol to dissolve it, centrifuge and take the supernatant, add an appropriate amount of micro-powder silica gel, dry it under reduced pressure to form a thick paste, freeze-dry it, add an appropriate amount of micro-powder silica gel, and crush it to obtain the product.

[0009] The present invention provides a control extract of the Herba Lycopodii, which can be used as a control substance to detect the quality of Herba Lycopodii medicinal materials and preparations, thereby solving the problem of lack of control substances. At the same time, it greatly simplifies the preparation steps of the control substances, is convenient for users, and is also conducive to improving detection efficiency.

[0010] In a further embodiment, the water content of the control extract of the Herba Lycopodii is no more than 5.0%.

[0011] In a further embodiment, the control extract of the Herba Lycopodii is a light brown or brown powder and is hygroscopic.

[0012] This invention tests and clarifies the shape and properties of a control extract of the Herba Caulis Sinensis, providing a theoretical basis for determining storage and packaging conditions. Based on the water content and hygroscopicity of the Herba Caulis Sinensis control extract, a fully enclosed brown ampoule can be used to prevent deliquescence and moisture absorption during storage and provide convenience for users. The extract should be stored at 4°C in the dark. Samples should be weighed quickly before use.

[0013] The second object of the present invention is to provide a quality control method for the control extract of the J. chinensis var. serrata as described above, comprising: detecting the moisture and hygroscopicity of the J. chinensis var. serrata extract; identifying the J. chinensis var. serrata extract by thin layer chromatography; and determining the content of the J. chinensis var. serrata extract by high performance liquid chromatography.

[0014] In a further embodiment, the method for identifying the extract of Rhizoma Cibotii using thin layer chromatography comprises:

[0015] (1) preparing a control extract solution of Caulis Spatholobifolia;

[0016] (2) preparing a test solution of the medicinal material of Caulis Spatholobi;

[0017] (3) According to the thin layer chromatography method, the test solution of the Jinsha Teng medicinal material and the control extract solution of the Jinsha Teng were spotted on the same thin layer plate, and a mixed solution of dichloromethane-methanol-formic acid was used as the developing solvent for thin layer identification.

[0018] Furthermore, in the developing solvent, the volume ratio of dichloromethane:methanol:formic acid is (8-12):2:0.2.

[0019] When the above-mentioned developing agent is used and the proportions of the components in the developing agent are as above, the developing effect is good, the difference in development on different thin layer plates is small, it is more stable, and the developing agent is non-toxic and easy to use.

[0020] As a preferred embodiment, the volume ratio of dichloromethane:methanol:formic acid is 8:2:0.2.

[0021] A further solution is to use high performance liquid chromatography to determine the content of the extract of Rhizoma Cibotii.

[0022] (1) Prepare reference solutions of caffeic acid, isoquercetin, kaempferol-3-O-rutinoside and astragaloside reference substances;

[0023] (2) preparing a control extract solution of Caulis Spatholobi;

[0024] (3) The content of each component in the reference substance and the reference extract of Rhizoma Coptidis was detected by high performance liquid chromatography. The chromatographic conditions included: octadecylsilane bonded silica gel as the filler of the chromatographic column; acetonitrile as the mobile phase A, 0.4% phosphoric acid as the mobile phase B, gradient elution; column temperature of 30°C, detection wavelength of 354 nm; flow rate of 1 mL / min; the theoretical plate number calculated based on the caffeic acid peak should be no less than 10,000.

[0025] Furthermore, the conditions of the gradient elution include:

[0026]

[0027] The present invention explores the optimal chromatographic conditions and elution conditions through a large number of condition experiments. Under the above method, the obtained chromatogram has good separation, less interference, good peak shape, high stability, good methodological verification effect, and is conducive to accurately measuring the content of each component.

[0028] The third object of the present invention is to provide a method for detecting the quality of a Caulis Agrimoniae preparation using the Caulis Agrimoniae control extract as described above, characterized in that it comprises:

[0029] (1) preparing a control extract solution of Caulis Spatholobifolia;

[0030] (2) preparing a test solution of the genus Rhizoma Coptidis preparation;

[0031] (3) According to the thin layer chromatography method, the test solution of the Jinsha Teng preparation and the control extract solution of the Jinsha Teng were spotted on the same thin layer plate, and cyclohexane-ethyl acetate-formic acid-acetone was used as the developing solvent for thin layer chromatography identification;

[0032] Preferably, the Rhizoma Corydalis preparation includes Sanjin preparation.

[0033] The present invention applies the control extract of J. chinensis to the quality inspection of J. chinensis preparations, such as Sanjin tablets, which fills the detection gap of indicators in J. chinensis, further improves the quality control standards of J. chinensis preparations, and is beneficial to improving the overall quality of the preparations.

[0034] Preferably, the thin layer plate is a high-efficiency silica gel G thin layer plate.

[0035] Furthermore, the volume ratio of cyclohexane:ethyl acetate:formic acid:acetone in the developing solvent is 15:15:0.5:1.

[0036] The effect of development is good, and the difference of development on different thin layer plates is small, and it is more stable.

[0037] Compared with the prior art, the present application has the following beneficial effects:

[0038] 1、The gold-thread clematis reference extract provided by the present application can be used as a reference material for detecting the quality of gold-thread clematis medicinal materials and preparations, solving the problem of lack of reference materials; meanwhile, the preparation steps of the reference materials are greatly simplified, which is convenient for users and is also conducive to improving the detection efficiency.

[0039] 2、The present application proposes and optimizes the quality control conditions for the gold-thread clematis reference extract, which is conducive to the production and use of the gold-thread clematis reference extract. In addition, a draft quality standard is proposed, which is conducive to product quality control.

[0040] 3、The present application applies the gold-thread clematis reference extract to the quality detection of gold-thread clematis preparations, such as Sanjin tablets, fills the blank of index detection of gold-thread clematis, further perfects the quality control standard of gold-thread clematis preparations, and is conducive to improving the overall quality of the preparations.

[0041] The specific embodiments of the present application will be described in further detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0042] The accompanying drawings are part of the present application and serve to provide a further understanding of the present application, the illustrative embodiments of the present application and their descriptions serve to explain the present application, but do not constitute an improper limitation on the present application. Obviously, the accompanying drawings described below are only some embodiments, and other drawings can be obtained by those skilled in the art without creative labor. In the drawings:

[0043] Figure 1 is the development effect diagram under the condition of using silica gel G-Merck plate and developing agent dichloromethane-methanol-formic acid (8:2:0.2) in embodiment 3 of the present application;

[0044] Figure 2 is the development effect diagram under the condition of using silica gel G-Merck plate and developing agent dichloromethane-methanol-formic acid (10:2:0.2) in embodiment 3 of the present application;

[0045] Figure 3 is the development effect diagram under the condition of using silica gel G-Merck plate and developing agent dichloromethane-methanol-formic acid (12:2:0.2) in embodiment 3 of the present application;

[0046] Figure 4This is a diagram showing the development effect of Example 3 of the present invention using Qingdao Ocean Silica G plate and a developing solvent of dichloromethane-methanol-formic acid (8:2:0.2);

[0047] Figure 5 This is a diagram showing the unfolding effect of a polyamide film in Example 3 of the present invention;

[0048] Figure 6 This is a diagram showing the development effect of using methanol-glacial acetic acid-water (4:1:5) as the developing agent in Example 3 of the present invention;

[0049] Figure 7 The chromatograms of the reference solution and the test solution in Example 4, wherein the reference extract of Caulis Agrimoniae (A), Caulis Agrimoniae medicinal material (B) and the reference solution (C); 1: caffeic acid, 2: isoquercetin, 3: kaempferol-3-O-rutinoside, 4: astragaloside;

[0050] Figure 8 is a chromatogram using a Waters column (A) and an Agilent column (B) in Example 4;

[0051] Figure 9 The durability of the method in Example 4 is achieved by using Waters symmetry shield TM Chromatogram of RP18 column;

[0052] Figure 10 This is a chromatogram obtained by using a SHISEIDO CAPCELL PAKC18 column in the method for the robustness test of Example 4;

[0053] Figure 11 This is a chromatogram obtained by using an ECLIPSE XDB-C18 column in the robustness test of the method in Example 4;

[0054] Figure 12 This is the result diagram of method 1 in Example 5;

[0055] Figure 13 This is the result diagram of method 2 in Example 5.

[0056] It should be noted that these drawings and textual descriptions are not intended to limit the conceptual scope of the present invention in any way, but rather to illustrate the concept of the present invention for those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0058] Instruments and reagents: Waters 2695 high performance liquid chromatograph. Chromatographic column: Agilent ZORBAX SB-C18 (250×4.6 mm, 5 μm) and Waters symmetry shield TM RP18 (250 × 4.6 mm, 5 μm). Ultrasonic cleaner (model KQ-500E, Kunshan Ultrasonic Instrument Co., Ltd.). Electronic balance (model XS105DU, model FX-200). Silica gel G thin layer plates (Merck and Qingdao Ocean Company) and polyamide film (Xijia Biochemical Plastic Factory, Luqiao, Taizhou, Zhejiang Province). Methanol, acetonitrile, formic acid (chromatographic grade, Fisher, USA), ethanol, aluminum chloride, dichloromethane, n-butanol, glacial acetic acid, and ethyl acetate (analytical grade, Sinopharm Group Corporation). Water was prepared using a Milli-Q system (Millipore, USA).

[0059] Control extracts of Rhizoma Cibotii (Batch Nos. 191103, 191104, and 190904), Rhizoma Cibotii medicinal materials (Batch Nos. 191103, 191104, and 190904), and Sanjin Tablets (Batch Nos. 190904, 190930, and 190935) were provided by Guilin Sanjin Pharmaceutical Co., Ltd. Reference substances: caffeic acid (110885-201703, 99.7%), isoquercetin (111809-201804, 97.2%), and kaempferol-3-O-rutinoside (112007-201602, 90.8%) were provided by the China Food and Drug Administration. Astragaloside (98.61%) was provided by Guilin Sanjin Pharmaceutical Co., Ltd.

[0060] Example 1 Preparation of the control extract of Rhizoma Cibotii

[0061] Take the medicinal material of Jinsha Teng, heat and reflux extract with water twice, each time for 1 hour, combine the extracts, filter, and concentrate the filtrate under reduced pressure to a clear paste of about 1.5-2 times the weight of the medicinal material. Centrifuge, filter, add an equal amount of ethyl acetate, stir evenly, let it stand, take the supernatant, recover the ethyl acetate under reduced pressure, add an appropriate amount of methanol to dissolve, centrifuge and take the supernatant, add an appropriate amount of micro-powdered silica gel, dry under reduced pressure to a thick paste, freeze-dry, add an appropriate amount of micro-powdered silica gel, and crush to obtain the product. The Jinsha Teng control extract is a light brown or brown powder.

[0062] Referring to the method of this example, three batches of Rhizoma Cibotii control extracts (batch numbers: 191103, 191104, and 190904) were prepared.

[0063] Example 2 Detection of Moisture and Hygroscopicity of the Control Extract of Caulis Agrimoniae

[0064] 1. Moisture detection

[0065] method:

[0066] Take a culture dish with a diameter of about 12 cm, add an appropriate amount of phosphorus pentoxide desiccant to a thickness of 0.5 to 1 cm, and place it in a reduced pressure desiccant with a diameter of 30 cm.

[0067] Determination method: Take 2-4 g of the control extract of the Herba Caulis Sinensis (Coptissinus) and mix thoroughly. Aliquots of approximately 0.5-1 g are placed in a weighing bottle that has been dried and weighed under the same conditions as the test sample. The amount is accurately weighed. The bottle is capped and placed in the above-described vacuum desiccator. The pressure is reduced to below 2.67 kPa (20 mmHg) for half an hour. The sample is then allowed to stand at room temperature for 24 hours. Connect an anhydrous calcium chloride drying tube to the outlet of the vacuum desiccator, open the stopcock, wait until the internal and external pressures are equal, close the stopcock, open the desiccator, close the bottle cap, remove the weighing bottle, and quickly and accurately weigh the sample. Calculate the water content (%) of the sample.

[0068] result

[0069] The moisture contents of the three batches of control extracts of Rhizoma Cibotii were 2.3% (batch number 191103), 1.4% (batch number 191104), and 1.8% (batch number 190904), respectively.

[0070] 2. Hygroscopicity

[0071] method:

[0072] The hygroscopicity of a drug refers to the ability or degree of moisture absorption of the substance under certain temperature and humidity conditions. The specific test method is as follows:

[0073] 1) Take a dry, stoppered glass weighing bottle (outer diameter 50 mm, height 15 mm) and place it in a suitable constant temperature desiccator at 25°C ± 1°C (with ammonium chloride or ammonium sulfate saturated solution placed at the bottom) or an artificial climate chamber (set temperature 25°C ± 1°C, relative humidity 80% ± 2%) the day before and accurately weigh it (m1).

[0074] 2) Take an appropriate amount of the control extract of the Herba Cynanchifoliae as the test sample, place it in the above-mentioned weighing bottle and spread it evenly in the weighing bottle. The thickness of the test sample is generally about 1 mm, and weigh it accurately (m2).

[0075] 3) Open the weighing bottle and place it with the bottle cap under the above constant temperature and humidity conditions for 24 hours.

[0076] 4) Close the weighing bottle and weigh accurately (m3). Percentage weight gain = (m3 - m2) / (m2 - m1) × 100%.

[0077] 5) Description of hygroscopic characteristics and definition of hygroscopic weight gain. Deliquescence: Absorbs sufficient water to form a liquid. Extremely hygroscopic: Hygroscopic weight gain of not less than 15%. Some hygroscopic: Hygroscopic weight gain of less than 15% but not less than 2%. Slightly hygroscopic: Hygroscopic weight gain of less than 2% but not less than 0.2%. No or almost no hygroscopicity: Hygroscopic weight gain of less than 0.2%.

[0078] Results: The hygroscopic weight gains of the three batches of Rhizoma Cibotii control extracts were 7.8% (batch number 191103), 6.5% (batch number 191104), and 8.3% (batch number 190904), respectively. The hygroscopic characteristics were all described as hygroscopic.

[0079] Example 3 Thin-layer identification of the control extract of Caulis Agrimoniae

[0080] The chemical composition of Herba Caulis Agrimoniae is primarily composed of flavonoids and phenolic acid compounds. These components have low specificity for thin-layer chromatography (TLC) identification. Therefore, a TLC identification method was established by comparing Herba Caulis Agrimoniae with a control extract. The traditional chloroform-methanol-formic acid system is often used to develop flavonoids and phenolic acid components, considering the strong toxicity of chloroform. This experiment replaced chloroform with dichloromethane and used different ratios of dichloromethane-methanol-formic acid to explore more favorable TLC development conditions. The development effects of silica gel G plates and polyamide films were also examined as follows:

[0081] 1. Sample extraction method: Weigh 1 g of Radix Glehniae chinensis, add 25 mL of water, extract under reflux in a water bath for 1 h, let cool and filter, extract the filtrate with ethyl acetate twice, 20 mL each time, combine the ethyl acetate, evaporate to dryness, and dissolve it with 1 mL of methanol to obtain the test solution.

[0082] Take 50 mg of the control extract of Caulis Agrimoniae, dissolve it in methanol and then dilute the volume to 10 mL to obtain the control extract solution of Caulis Agrimoniaee.

[0083] Development method: According to the thin layer chromatography method (General Method 0502), take 1 μl of each of the above two solutions and spot them on the same silica gel G thin layer plate or polyamide film. Use dichloromethane-methanol-formic acid as the developing solvent. Develop, remove, dry, spray with 3% aluminum trichloride ethylphenol solution, heat at 105°C until the spots are clearly colored, and examine under ultraviolet light (365nm).

[0084] Result judgment: In the control extract chromatogram, fluorescent spots of the same color should appear at the corresponding positions in the chromatogram of the J. chinensis medicinal material.

[0085] 2. Thin layer identification results

[0086] The samples were developed using silica gel G thin layer plates (Merck and Qingdao Ocean), polyamide films and developing agents in different proportions. The sample volume was 1 μl. Spots E1, E2, and E3 were control extracts of Caulis Agrimoniae (corresponding batch numbers were 191103, 191104, and 190904), and spots L1, L2, and L3 were Caulis Agrimoniae medicinal materials (corresponding batch numbers were 191103, 191104, and 190904).

[0087] The results of developing with silica gel G-Merck plate in different developing agents are as follows Figure 1-3 As shown in the development results, it can be seen that the dichloromethane-methanol-formic acid (8:2:0.2) system has a better development effect. Compared with Merck and Qingdao Ocean Silica G plate ( Figure 1 and Figure 4 ), the expansion difference is smaller.

[0088] The results of polyamide film development are as follows Figure 5 As shown, the dichloromethane-methanol-formic acid (10:2:0.2) developing system is better.

[0089] Try using methanol-glacial acetic acid-water (4:1:5) as the developing solvent, the development results are as follows Figure 6 As shown, the effect is poor.

[0090] Therefore, based on the above results, dichloromethane-methanol-formic acid was used as the developing solvent, wherein the volume ratio of dichloromethane:methanol:formic acid was 8:2:0.2 or 10:2:0.2, preferably 8:2:0.2.

[0091] Example 4 High Performance Liquid Chromatography Determination of the Control Extract of Caulis Agriophyllae

[0092] 1. Chromatographic conditions: Determined according to the high performance liquid chromatography method (General Chapter 0512) of the 2015 edition of the Chinese Pharmacopoeia.

[0093] The chromatographic column is Waters symmetry shield TM RP18 (250×4.6mm, 5um), acetonitrile as mobile phase A, 0.4% phosphoric acid as mobile phase B, gradient elution, elution program as shown in Table 1 below; column temperature 30°C, detection wavelength 354nm, flow rate 1mL / min; accurately aspirate 20μl of each reference solution and test solution, inject into liquid chromatograph, and measure.

[0094] Table 1 Elution program

[0095] Time (min) Mobile phase B (%) 0-40 90→75 40-45 75→60 45-50 60→10

[0096] 2. Solution Preparation

[0097] Preparation of reference solution: Take appropriate amount of caffeic acid, isoquercetin, kaempferol-3-O-rutinoside and astragalin reference substances, accurately weigh them, and add methanol to make a solution containing 0.01 mg of caffeic acid, 0.04 mg of isoquercetin, 0.06 mg of kaempferol-3-O-rutinoside and 0.02 mg of astragalin per 1 mL.

[0098] To prepare the test solution, take about 50 mg of the control extract of Jinsha Teng, accurately weigh it, place it in a 25 mL volumetric flask with a stopper, add 10% methanol to the scale, shake well, and ultrasonically extract for 30 minutes. Take the supernatant to obtain the test solution.

[0099] 3. Results: Accurately pipette 20μl of reference solution and test solution respectively, inject into liquid chromatograph and measure. The chromatogram of the test solution shows a chromatographic peak with the same retention time as the reference solution. The chromatogram is as follows: Figure 7 As shown, among them, the control extract of Jinsha Teng (A), the medicinal material of Jinsha Teng (B) and the reference substance (C); 1: caffeic acid, 2: isoquercetin, 3: kaempferol-3-O-rutinoside, 4: astragaloside.

[0100] By comparing the spectra of caffeic acid, isoquercetin, kaempferol-3-O-rutinoside and astragaloside reference substances, the corresponding peaks in the control extracts were detected with the spectra of the reference substances. Figure 1 Therefore, the indicator components in the control extract were determined.

[0101] 4. Exploration of chromatographic conditions

[0102] (1) In this experiment, 0.4% phosphoric acid and acetonitrile were used as mobile phases, and the chromatographic columns used were Agilent ZORBAXSB-C18 (250×4.6mm, 5um) and Waters symmetry shield TM RP18 (250×4.6mm, 5um) was used to optimize the chromatographic conditions. By optimizing the gradient elution program, the chromatogram was as follows: Figure 8 As shown in the figure, the separation effects of the two chromatographic columns can meet the determination requirements. After further comparison of separation and peak shape, Waters symmetry shield TM The RP18 column was used for analysis, and the optimized elution procedure is shown in Table 1 above.

[0103] (2) Detection wavelength selection

[0104] Because the composition of the control extract of Rhizoma Cibotii is relatively complex at different wavelengths, a comparison of the chromatograms at different wavelengths revealed that a detection wavelength of approximately 354 nm resulted in less interference with the target components (caffeic acid, isoquercetin, kaempferol-3-O-rutinoside, and astragalin), better peak shape, and more consistent absorbance values. Therefore, a detection wavelength of 354 nm was selected for this experiment.

[0105] 5. Methodological Investigation

[0106] Preparation of reference solution: Take appropriate amount of caffeic acid, isoquercetin, kaempferol-3-O-rutinoside and astragalin reference substances, accurately weigh them, and add methanol to make a solution containing 0.01 mg of caffeic acid, 0.04 mg of isoquercetin, 0.06 mg of kaempferol-3-O-rutinoside and 0.02 mg of astragalin per 1 mL.

[0107] Preparation of test solution: Take about 50 mg of the product, accurately weigh it, place it in a 20 mL volumetric flask with a stopper, add methanol to dissolve it to the scale, shake it well, filter it, and take the filtrate to obtain the test solution.

[0108] (1) Sensitivity and linearity

[0109] 1, 5, 10, 20, and 30 μl of the prepared reference solution were accurately injected, and regression calculation was performed using the peak area Y and the injection volume X. The results showed that the linear relationship was good within the range (R>0.999). Using the stepwise dilution method, a signal-to-noise ratio (S / N) greater than 3 was defined as the detection limit, and a signal-to-noise ratio (S / N) greater than 10 was defined as the quantification limit. The results are shown in Table 2.

[0110] Table 2 Linear range, detection limit and quantification limit of the method

[0111]

[0112] (2) Precision

[0113] 20 μl of the reference solution was injected six times continuously under the above chromatographic conditions, and the peak areas were recorded. The RSDs (n=6) for the peak areas of caffeic acid, isoquercetin, kaempferol-3-O-rutinoside, and astragalin were 0.3%, 0.2%, 0.3%, and 0.2%, respectively, indicating good instrument precision.

[0114] Table 3 Precision test results

[0115]

[0116]

[0117] (3) Repeatability

[0118] Take the control extract (191103) 6 parts, accurately weigh, according to the preparation method of test solution, sample solution is prepared, according to the above chromatographic conditions, 20 μl is injected, respectively, the content of caffeic acid, isoquercitrin, kaempferol-3-O-rutinoside and astragalin is calculated, the average content (n=6) of the above four components is: 0.51%, 0.16%, 0.24% and 0.75%, the RSD is 0.4%, 0.5%, 0.4% and 0.5% respectively, indicating that the method repeatability is good.

[0119] Table 4 repeatability test results

[0120] serial number Caffeic acid% Isoquercetin% Kaempferol-3-o-rutinoside% Astragaloside% 1 0.503 0.162 0.237 0.744 2 0.503 0.161 0.237 0.743 3 0.506 0.162 0.238 0.745 4 0.506 0.162 0.238 0.75 5 0.508 0.163 0.239 0.752 6 0.506 0.163 0.238 0.747 mean 0.51 0.16 0.24 0.75 RSD% 0.4 0.5 0.4 0.5

[0121] (4) Stability

[0122] Take the same test solution, place at room temperature, respectively, at 0, 2, 5, 12, 24, 36, 48 h, sample determination, record peak area. The results of caffeic acid, isoquercitrin, kaempferol-3-O-rutinoside and astragalin peak area RSD (n=7) are 0.3%, 0.6%, 0.6% and 0.2% respectively, indicating that the method has good stability.

[0123] Table 5 stability test results

[0124]

[0125] (5) Recovery

[0126] Take the known content of 6 parts of control extract (191103) about 25 mg, accurately weigh, respectively, add the control solution 10 mL prepared, according to the preparation method of test solution, sample solution is prepared, according to the above chromatographic conditions, 20 μl is injected, respectively, the recovery of caffeic acid, isoquercitrin, kaempferol-3-O-rutinoside and astragalin is calculated, the average recovery (n=6) of the above four components is: 100.1%, 97.7%, 92.5% and 101.0%, the RSD is 0.3%, 0.5%, 0.3% and 1.0% respectively, indicating that the method has good accuracy.

[0127] Table 6 recovery test results

[0128]

[0129] (5) Method durability

[0130] This example is to select different chromatographic column to investigate the durability of the method, respectively, ECLIPSE XDB-C18 (250 x 4.6 mm, 5 um), Waters symmetry shicld TMRP18 (250 x 4.6 mm, 5 um), SHISEIDO CAPCELL PAK C18 (250 x 4.6 mm, 5 um) chromatographic column, 10% methanol extract (A), Dendrolium canariense Wall. (B), control extract (C) and control solution (D) were analyzed by using the established chromatographic conditions, respectively, and the results are shown in Table 2. Figures 9-11 The results show that the separation degree of the target components is good, and the method has good durability.

[0131] (6) Content determination

[0132] Three batches of control extracts were determined by the method established in this example, and the determination results are shown in Table 7.

[0133] Table 7 Determination results of three batches of control extracts

[0134] extract Batch 101103 Batch 101104 Batch 190904 Caffeic acid 0.50％ 0.64％ 0.56％ Isoquercetin 0.16％ 0.14％ 0.21％ Kaempferol-3-O-rutinoside 0.24％ 0.11％ 0.36％ Astragaloside 0.74％ 0.66％ 0.80％

[0135] Example 5 Dendrolium canariense Wall. control extract for thin layer identification study of Sanjin tablets

[0136] Sanjin tablets are prepared by decocting Dendrolium canariense Wall., Chrysanthemum root, Smilax, Capra hircus and Centella asiatica, and involve complex chemical components. In this experiment, different extraction solvents, extraction methods, and different developing systems were explored, and the suitable thin layer identification method was optimized by using polyamide film to develop, according to the effect of developing spots and comparison with negative samples. The specific research contents are as follows:

[0137] 1. Method

[0138] Method 1: Take 10 Sanjin tablets, grind them finely, add 50 mL of water, and reflux extract in a water bath for 1 h. After cooling, filter the solution. Extract the filtrate with ethyl acetate twice, 20 mL each time. Combine the ethyl acetate and evaporate to dryness. Dissolve the residue in 1 mL of methanol to obtain the test solution. Prepare a negative test solution by the same method without Dendrolium canariense Wall. Prepare a control extract solution by dissolving 50 mg of Dendrolium canariense Wall. control extract in methanol and diluting to 10 mL. Test the solutions prepared by the above methods according to the thin layer chromatography (General Rule 0502). Apply 1 μl of each solution to the same polyamide film. Use methanol-glacial acetic acid-water (4:1:5) and dichloromethane-methanol-formic acid (10:2:0.2) as developing agents, respectively. Develop, remove, dry, and spray with 3% aluminum chloride ethanol solution. Heat at 105°C until the spots develop clear color, and observe under ultraviolet light (365 nm).

[0139] Method 2: Grind 10 Sanjin tablets, add 20 mL of methanol, and ultrasonically extract for 1 hour. Cool and filter to obtain the test solution. Similarly, prepare a negative test solution by taking Sanjin tablets without the Jinsha Teng medicinal material. Separately, dissolve 50 mg of the Jinsha Teng control extract in methanol and dilute to 10 mL to obtain the control extract solution. Perform the thin-layer chromatography (TLC) test according to General Method 0502. Apply 5 μl of each solution prepared above to the same silica gel G thin-layer plate using cyclohexane-ethyl acetate-formic acid-acetone (15:15:0.5:1) as the developing solvent. Develop the plate, remove the plate, air-dry, and examine under ultraviolet light (365 nm).

[0140] Result judgment: In the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position in the chromatogram of the control extract of the Herba Lycopodii.

[0141] 2. Experimental results

[0142] (1) Results of Method 1

[0143] BK is the negative Sanjin tablet, E is the control extract, S1, S2, and S3 are Sanjin tablets (corresponding batch numbers are 190904, 190930, and 190935), and different development systems are as follows. Figure 12 As shown in the figure, the methanol-glacial acetic acid-water (4:1:5) system has a slightly better development effect and less interference from negative samples.

[0144] (2) Results of Method 2

[0145] E1, E2, and E3 are control extracts (corresponding to batch numbers 191103, 191104, and 190904), and S1, S2, and S3 are Sanjin tablets (corresponding to batch numbers 190904, 190930, and 190935). The results of different thin layer plate developments are shown in Figure 2. Figure 13 As shown, the high-efficiency thin layer plate has a better unfolding effect, while the domestic Qingdao marine thin layer plate has an average unfolding effect.

[0146] From a comprehensive comparison, the development effect of method 2 is relatively stable, so method 2 was used as the sample treatment method, and cyclohexane-ethyl acetate-formic acid-acetone (15:15:0.5:1) was used as the developing solvent.

[0147] Example 6 Draft Quality Standard for the Control Extract of Caulis Agrimoniae

[0148] Rhizoma Cibotii control extract

[0149] This product is a control extract made by extracting the dried aerial parts of Lygodium japonicum (Thunb.) Sw., Lygodium microphyllum (Cav.) R.Br., and Lygodium flexuosum (L.) Sw. of the Lygodium family.

[0150]

Properties

[0151]

Identification

[0152] (2) Take approximately 50 mg of this product, accurately weigh it, and place it in a 25 mL volumetric flask with a stopper. Add 10% methanol solution to the mark, shake well, and extract by ultrasonication for 30 minutes. Collect the supernatant to obtain the test solution. Except for the test solution, the rest of the test should be conducted according to the method under [Assay]. The chromatogram of the test sample should show a chromatographic peak with the same retention time as the chromatographic peaks of caffeic acid, isoquercetin, kaempferol-3-O-rutinoside, and astragalin.

[0153] [Inspection] Moisture content: not more than 5.0% (General Rule 0832, Method 3).

[0154] Hygroscopic, hygroscopic (General Rule 9103).

[0155] [Content Determination] Determine according to high performance liquid chromatography (General Rule 0512).

[0156] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as the filler, acetonitrile as mobile phase A, and 0.4% phosphoric acid as mobile phase B, with gradient elution as specified in the table below; column temperature 30°C, detection wavelength 354 nm; flow rate 1 mL / min; the number of theoretical plates calculated based on the caffeic acid peak should be no less than 10,000.

[0157] Time (min) Mobile phase B (%) 0-40 90→75 40-45 75→60 45-50 60→10

[0158] Preparation of reference solution: Take appropriate amount of caffeic acid, isoquercetin, kaempferol-3-O-rutinoside and astragalin reference substances, weigh accurately, and add methanol to make a solution containing 0.01 mg of caffeic acid, 0.04 mg of isoquercetin, 0.06 mg of kaempferol-3-O-rutinoside and 0.02 mg of astragalin per 1 mL.

[0159] Preparation of test solution: Take about 50 mg of the product, accurately weigh it, place it in a 20 mL volumetric flask with a stopper, add methanol to dissolve it to the scale, shake it well, filter it, and take the filtrate to obtain the test solution.

[0160] Determination method: Separately and closely aspirate 20 μl of the reference solution and the test solution, inject them into the liquid chromatograph, determine, and calculate the contents of caffeic acid, isoquercetin, kaempferol-3-O-rutinoside and astragaloside.

[0161] This product, calculated on a dry basis, contains no less than 0.30% caffeic acid (C9H8O4) and no less than 0.5% isoquercetin (C 21 H 20 O 12 ) shall not be less than 0.10%, containing kaempferol-3-O-rutinoside (C 27 H 30 O 15 ) shall not be less than 0.10%, containing astragalin (C 21 H 20 O 11 ) shall not be less than 0.50%.

[0162]

Storage

[0163] Example 7 Draft Quality Standard for Sanjin Tablets

[0164] Three gold pieces

[0165] [Identification] Take 15 tablets (small tablets) or 10 tablets (large tablets) of this product, grind them into powder, add 20 mL of methanol, perform ultrasonic extraction for 1 hour, let cool and filter to obtain the test solution. Separately, take 50 mg of the control extract of Jinsha Teng, dissolve it in methanol and dilute to 10 mL to obtain the control extract solution. According to the thin layer chromatography method (General Rule 0502), take 5 μl of each of the above two solutions and spot them on the same high-performance silica gel G thin layer plate. Use cyclohexane-ethyl acetate-formic acid-acetone (15:15:0.5:1) as the developing solvent, develop, remove, dry, and examine under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the control extract.

[0166] The above merely describes the preferred embodiments of the present application, and is not intended to limit the present application in any form. Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make some changes or modifications to the above-mentioned technical content with the prompt as equivalent embodiments of equivalent changes without departing from the technical solution of the present application. Any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application, which does not depart from the technical solution of the present application, still belongs to the scope of the present application.

Claims

1. A quality control method for a control extract of Caulis Spatholobi, characterized in that: include: The control extract of Rhizoma Cibotii was prepared by the following method: Take the medicinal material of Jinsha Teng, extract it by heating under reflux with water, combine the extracts, filter, concentrate the filtrate under reduced pressure, centrifuge, filter, add an equal amount of ethyl acetate, stir evenly, let it stand, take the supernatant, recover the ethyl acetate under reduced pressure, add an appropriate amount of methanol to dissolve it, centrifuge and take the supernatant, add an appropriate amount of micro-powdered silica gel, dry it under reduced pressure to form a thick paste, freeze-dry it, add an appropriate amount of micro-powdered silica gel, and crush it to obtain the product; Detect the moisture and hygroscopicity of the extract of Rhizoma Cibotii; identify the extract of Rhizoma Cibotii by thin layer chromatography; and determine the content of the extract of Rhizoma Cibotii by high performance liquid chromatography. The method for determining the content of the extract of Rhizoma Cibotii using high performance liquid chromatography includes: (1) Prepare reference solutions of caffeic acid, isoquercetin, kaempferol-3-O-rutinoside and astragaloside reference substances; (2) preparing a control extract solution of Rhizoma Cibotii; (3) The content of each component in the reference substance and the reference extract solution of the Herba Caulis Sinensis was determined by high performance liquid chromatography. The chromatographic conditions included: the chromatographic column was filled with octadecylsilane bonded silica gel; acetonitrile was used as mobile phase A, 0.4% phosphoric acid was used as mobile phase B, and gradient elution was performed; the detection wavelength was 354 nm, and the gradient elution conditions included: 。 2. The quality control method according to claim 1, characterized in that Methods for identifying the extract of Rhizoma Cibotii using thin layer chromatography include: (1) Preparing a control extract solution of Rhizoma Cibotii; (2) Prepare the test solution of the medicinal material of Rhizoma Coptidis; (3) According to the thin layer chromatography method, the test solution of the Jinsha Teng medicinal material and the control extract solution of the Jinsha Teng were spotted on the same thin layer plate, and a mixed solution of dichloromethane-methanol-formic acid was used as the developing solvent for thin layer identification.

3. The quality control method according to claim 2, characterized in that: In the developing solvent, the volume ratio of dichloromethane:methanol:formic acid is (8-12):2:0.

2.

4. The quality control method according to claim 3, characterized in that: In the developing solvent, the volume ratio of dichloromethane:methanol:formic acid is 8:2:0.

2.

5. The quality control method according to claim 1, characterized in that: In the method for determining the content of the extract of Rhizoma Cibotii using high performance liquid chromatography: The column temperature was 30°C; the flow rate was 1 mL / min; the theoretical plate number calculated based on the caffeic acid peak should be no less than 10,000.

6. The quality control method according to any one of claims 1 to 5, characterized in that: The water content in the control extract of the Herba Lycopodii is no more than 5.0%.

7. The quality control method according to any one of claims 1 to 5, characterized in that: The control extract of the Herba Lycopodii is a light brown or brown powder and has hygroscopic properties.

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