Physical and chemical identification method for Yanning granules

Abstract

The invention discloses a physical and chemical identification method for Yanning granules, which comprises the following steps: identifying dayflower, and taking Yanning granule powder; adding a mixed solution composed of trichloromethane, methanol and water, and mixing the mixed solution with the Yanning particle powder; performing treatment through ultrasonic waves; filtering, evaporating filtrate to dryness to obtain residues, and dissolving and diluting the residues with 28-32% methanol; filtering again, and taking a subsequent filtrate as a test solution; taking a common dayflower herb reference medicinal material, and preparing a reference medicinal material solution according to the steps S1b to S1d; according to a high performance liquid chromatography test, octadecylsilane chemically bonded silica is used as a filler; if a chromatographic peak consistent with the retention time of the chromatographic characteristic peak S of the reference medicinal material appears in the chromatogram of the test sample, the sample contains the dayflower. The method has the beneficial effects that the identification specificity is improved, the durability of the method is enhanced, the quality and consistency of medicines are guaranteed, clinical reasonable medication is assisted, and the treatment failure or adverse reaction caused by the quality problem of the medicines is reduced.

Description

Technical Field

[0001] The present invention relates to a method of identification, and more particularly to a method for physicochemical identification of Yanning Granules. Background Art

[0002] In the field of modern pharmaceutical R & D and production, the accurate control of drug quality is a key link to ensure the safety and efficacy of patients' medication. As a drug composed of three traditional Chinese medicine materials, namely Hedyotis diffusa, Commelina communis, and Monochasma sheareri, Yanning Granules have certain value in clinical applications. However, its original quality standard has obvious limitations;

[0003] From the perspective of the identification method, the physicochemical identification method in the original quality standard has poor specificity, which means that it is difficult to accurately distinguish the components of each medicinal material in Yanning Granules during the quality inspection of Yanning Granules, and it cannot provide strong guarantee for the quality of drugs. During the drug production process, factors such as the source of medicinal materials and the processing method may vary. If the identification method cannot accurately identify, it is difficult to ensure that each batch of Yanning Granules meets the quality requirements, which may in turn affect the clinical treatment effect;

[0004] In addition, the quality of drugs not only depends on the accurate identification of their components, but also is closely related to the stability and reliability of the identification method under different conditions. During the actual drug storage and experiment process, environmental factors such as temperature and humidity changes, as well as fine-tuning of the proportion of developing agent, different sources and qualities of thin-layer plates, and differences in sample application amounts during experimental operations may all affect the identification results. If the durability of the identification method is insufficient and it cannot maintain a stable identification effect when these factors change, it is difficult to obtain consistent and reliable test results under different laboratories and different operating conditions;

[0005] For example, temperature changes may affect the evaporation rate of the developing agent and its diffusion behavior on the thin-layer plate, thereby changing the position and clarity of the spots; humidity changes may cause changes in the activity of the thin-layer plate, affecting the separation effect of the sample on the plate, and thin-layer plates of different brands and qualities have differences in the properties and thickness of silica gel, which will also affect the identification results. Different sample application amounts may change the size and shape of the spots, thereby affecting the resolution and the accuracy of the identification results;

[0006] Therefore, in order to improve the quality control level of Yanning Granules, ensure the quality of drugs and clinical efficacy, it is urgent to establish a highly specific and durable identification method. This not only requires in-depth research on the characteristics of each medicinal material, but also needs to comprehensively consider various factors that may affect the identification results. By optimizing the experimental conditions and methods, it is ensured that the identification method can stably and accurately identify the components of each medicinal material in Yanning Granules under different environmental and operating conditions, providing more scientific and reliable technical support for the quality control of Yanning Granules. Summary of the Invention

[0007] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a method for physical and chemical identification of Yanning Granules that can effectively solve the above-mentioned technical problems.

[0008] To achieve the above object, the present invention provides a method for physical and chemical identification of Yanning Granules, including the identification of Commelina communis, specifically including:

[0009] S1: Take the powder of Yanning Granules;

[0010] S1a: Add a mixed solution composed of chloroform - methanol - water and mix it with the powder of Yanning Granules;

[0011] S1b: Treat it by ultrasonic wave;

[0012] S1c: Filter, evaporate the filtrate to dryness to obtain a residue, dissolve the residue with 28 - 32% methanol and dilute it to 10 ml;

[0013] S1d: Filter again, take the subsequent filtrate as the test solution;

[0014] S1e: Take another Commelina communis control medicinal material and prepare a control medicinal material solution according to the steps of S1b - S1d;

[0015] S2: Perform the test according to the high performance liquid chromatography method, using octadecylsilane chemically bonded silica gel as the filler;

[0016] Use acetonitrile as mobile phase A and 0.1 - 0.3% phosphoric acid solution as mobile phase B for gradient elution;

[0017] The number of theoretical plates calculated by the characteristic peak S is not less than 4000; Take the control medicinal material solution and the test solution respectively, inject them into the liquid chromatograph, measure, and record the chromatogram for 15 min;

[0018] S3: If a chromatographic peak with the same retention time as the characteristic peak S of the control medicinal material chromatogram appears in the test solution chromatogram, it indicates that the sample contains Commelina communis.

[0019] Furthermore, in S1, take 0.5 g of the powder of Yanning Granules;

[0020] In S1a, add 20 ml of a mixed solution composed of chloroform - methanol - water and mix it with the Yanning Granule powder;

[0021] In S1e, separately take 0.6 g of the Commelina communis control medicinal material and prepare a control medicinal material solution according to the steps of S1b - S1d.

[0022] Furthermore, in S1b, place the container filled with the S1a mixture in an ultrasonic instrument and perform treatment through ultrasonic waves; the treatment time is 20 minutes, the ultrasonic power is 480 kW, and the ultrasonic frequency is 40 Hz.

[0023] Furthermore, in S2, use acetonitrile as mobile phase A and 0.2% phosphoric acid solution as mobile phase B.

[0024] Furthermore, in S2, the flow rate is 0.9 ml per minute; the column temperature is 25 °C; the detection wavelength is 250 nm; the number of theoretical plates calculated based on the characteristic peak S is not less than 4000; separately take 15 μl of the control medicinal material solution and 5 μl of the test solution, inject them into the liquid chromatograph, determine, and record the chromatogram for 15 min.

[0025] Furthermore, it includes the identification of Monochasma sheareri, specifically including:

[0026] Preparation of the test solution: Take the Yanning Granule powder; add water to dissolve it, extract it 3 times with ethyl acetate, combine the ethyl acetate solutions and evaporate to dryness, dissolve the residue in methanol to obtain the test solution;

[0027] Preparation of the negative control solution: Take the Monochasma sheareri negative sample powder, add water to dissolve it, extract it 3 times with ethyl acetate, combine the ethyl acetate solutions and evaporate to dryness, dissolve the residue in methanol to obtain the negative control solution;

[0028] Preparation of the reference solution: Take luteolin reference substance, add methanol to make a solution containing 0.1 mg per 1 ml to obtain the reference solution;

[0029] Determination: Take the control solution, the test solution, and the negative control solution, respectively spot them on the same silica gel thin layer plate, use chloroform - methanol - formic acid - water as the developing agent, develop, take out, dry, spray with aluminum trichloride test solution, heat to make the spots develop color, and view under an ultraviolet lamp;

[0030] Results and analysis: In the chromatogram of the test solution, at the position corresponding to the reference solution, there are fluorescent spots showing the same color, with no interference from the negative, and the Rf value is 0.52, indicating that this identification method has specificity.

[0031] Furthermore, it also includes the durability verification, specifically including:

[0032] SS1: According to the preparation method of the test solution, the reference solution, the test solution and the negative control solution were respectively investigated under different conditions of the proportion change of chloroform in the developing agent by ±1, temperature change and relative humidity change on different thin-layer plates, as well as the sample application amount, to verify the durability of the identification method of Monochasma sheareri Maxim.;

[0033] Developing agent: When the proportion of chloroform changes by ±1 based on the set value, the spots on the thin-layer chromatography are clear, the resolution is good, and there is no tailing phenomenon;

[0034] Thin-layer plate: including self-made thin-layer plates and commercially available thin-layer plates. For self-made thin-layer plates, the separation degree of characteristic spots is poor and the tailing is serious; for commercially available thin-layer plates, the spots are clear, the resolution is good, and there is no tailing;

[0035] Temperature: Under different temperature conditions of 4°C, 25°C and 40°C, the spots are clear, the resolution is good, and there is no tailing;

[0036] Humidity: When the relative humidity is 32%, the separation degree of characteristic spots is relatively poor; when the relative humidity is 44% and 75%, the spots are clear, the resolution is good, and there is no tailing;

[0037] Sample application amount: Under different sample application amount conditions, the spots are clear, the resolution is good, and there is no tailing;

[0038] SS2: According to the above investigation results, for the specific identification of Monochasma sheareri Maxim., commercially available thin-layer plates are selected, and the relative humidity is preferably not less than. The spots with different sample application amounts are clear and the resolution is good. For the convenience of sample application, 1 μl is selected as the sample application amount.

[0039] Furthermore, in the preparation of the test solution as described above, the temperature changes include 4°C, 25°C and 40°C; the relative humidity changes are 32%, 44% and 75%.

[0040] Furthermore, the preparation of the test solution and the preparation of the negative control solution specifically include

[0041] Preparation of the test solution: Take 2 g of Yanning Granule powder, add 20 ml of water to dissolve it, extract it with ethyl acetate 3 times, 25 ml each time, combine the ethyl acetate extracts and evaporate to dryness, and dissolve the residue in 2 ml of methanol to obtain the test solution;

[0042] Preparation of the negative control solution: Take 2 g of Monochasma sheareri Maxim. negative sample powder, add 20 ml of water to dissolve it, extract it with ethyl acetate 3 times, 25 ml each time, combine the ethyl acetate extracts and evaporate to dryness, and dissolve the residue in 2 ml of methanol to obtain the negative control solution.

[0043] Furthermore, the determination specifically includes:

[0044] Take 3 μl of the control solution, 5 μl each of the test solution and the negative control solution, and spot them on the same silica gel thin-layer plate respectively. Use chloroform-methanol-formic acid-water as the developing solvent, develop, take out, air dry, spray with aluminum chloride test solution, heat at 105 °C until the spots develop color, and examine under an ultraviolet lamp at 365 nm.

[0045] The beneficial effects of the present invention are as follows:

[0046] 1) Improve the specificity of identification. The established identification methods for Commelina communis and Monochasma sheareri are highly specific. For the identification of Commelina communis, high-performance liquid chromatography is used. Through specific sample treatment, mobile phase gradient elution, and comparison with the chromatographic peaks of the reference medicinal material, the presence of Commelina communis in the sample can be accurately judged. For the identification of Monochasma sheareri, luteolin is used as the reference substance, developed and visualized on a silica gel thin-layer plate. The test sample chromatogram shows the same fluorescent spots as the corresponding position of the reference substance solution, and there is no interference from the negative sample, effectively avoiding the problem that the original identification method cannot accurately distinguish the medicinal material components and improving the accuracy of the quality detection of Yanning Granules.

[0047] 2) Enhance the durability of the method. A comprehensive durability verification was carried out on the identification method for Monochasma sheareri, including the investigation of conditions such as different thin-layer plates, developing solvent ratios, temperature, humidity, and sample spotting amounts. The results show that under various condition changes, the method can still maintain good identification effects. For example, the spots on the commercially available thin-layer plates are clear and the resolution is good under different conditions, providing a stable and reliable technical means for the quality detection of Yanning Granules and ensuring consistent test results under different laboratory and operating conditions.

[0048] 3) Ensure the quality and consistency of the drug: An accurate and reliable identification method helps to ensure that the quality of each batch of Yanning Granules meets the standards. By accurately identifying the medicinal material components, the impact of differences in the source of medicinal materials and processing methods on the drug quality during the production process can be effectively monitored, ensuring the stability and consistency of the drug quality, providing an effective quality control tool for drug manufacturers, and improving the production management level of enterprises.

[0049] 4) Facilitate rational clinical medication. The reliable identification method provides a guarantee for the safety of clinical medication. It has the beneficial effects of ensuring the stable quality of Yanning Granules, helping to improve the clinical treatment effect, and reducing treatment failures or adverse reactions caused by drug quality problems. Description of the Drawings

[0050] Figure 1 It is the specificity diagram for the identification of Commelina communis;

[0051] Figure 2 It is the specific component spectrogram for the identification of Commelina communis;

[0052] Figure 3 It is the reference chromatogram;

[0053] Figure 4 It is the specific TLC diagram of Monochasma sheareri. Specific implementation manners

[0054] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0055] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the accompanying drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus cannot be construed as a limitation of the present invention. In addition, the terms "first", "second", and "third" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0056] In the description of the present invention, it should be noted that unless otherwise clearly specified and defined, the terms "installation", "setting", and "connection" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0057] As Figures 1 to 4 shown

[0058] For identification, this preparation is composed of three herbs: Hedyotis diffusa, Commelina communis, and Monochasma sheareri. The specific property investigation of the physicochemical identification in the original quality standard was carried out. The results showed that the specific property was not strong, so it was not included in the standard. Referring to the qualitative identification methods in Part I of the Chinese Pharmacopoeia 2020 Edition and relevant literature, the qualitative research on Hedyotis diffusa, Commelina communis, and Monochasma sheareri in the prescription was carried out. The results showed that the identification methods for the three herbs of Hedyotis diffusa, Commelina communis, and Monochasma sheareri all had specificity, so they were included in the main text of the quality standard. The specific test work is described in detail as follows:

[0059] 1) Physicochemical identification

[0060] Yanning Granule sample: YZ20220501, a self-made pilot-scale sample.

[0061] Commelina communis negative control, Y20220101, prepared by taking the prescription herbs without Commelina communis according to the process.

[0062] Commelina communis positive control: DY20220101, prepared by taking Commelina communis herbs according to the process.

[0063] Negative control of Monochasma sheareri (S. Moore) Maxim.: L20220101. Take the prescription herbs without Monochasma sheareri (S. Moore) Maxim. and prepare them according to the process by oneself.

[0064] Positive control of Monochasma sheareri (S. Moore) Maxim.: DL20220101. Take the Monochasma sheareri (S. Moore) Maxim. herbs and prepare them according to the process by oneself.

[0065] Negative control of Hedyotis diffusa Willd., B20220101. Take the prescription herbs without Commelina communis L. and prepare them according to the process by oneself.

[0066] Positive control of Hedyotis diffusa Willd.: DB20220101. Take the Commelina communis L. herbs and prepare them according to the process by oneself.

[0067] According to the identification item in the quality standard of Yanning Granules (with sugar), carry out physical and chemical identification experiments on Yanning Granules (sugar-free).

[0068] Under the original standard identification item:

[0069] (1) Take 5 g of this product, soak it in 20 ml of ethanol for 10 minutes, filter, take 2 ml of the filtrate, add 2 drops of ferric trichloride test solution, and it will show dark green immediately.

[0070] (2) Take 5 ml of the remaining filtrate in step (1), evaporate to dryness, dissolve it in 5 ml of chloroform, take the chloroform solution, add 2 drops of sulfuric acid, it will show light yellow after standing, and heat it in a water bath, it will turn pink.

[0071] Solution preparation:

[0072] Step ①: Take 5 g of this product, soak it in 20 ml of ethanol for 10 minutes, filter, take 2 ml of the filtrate, and add 2 drops of ferric trichloride test solution.

[0073] Step ②: Take 5 ml of the remaining filtrate in step ①, evaporate to dryness, dissolve it in 5 ml of chloroform, take the chloroform solution, add 2 drops of sulfuric acid, it will show light yellow after standing, and heat it in a water bath.

[0074] Take the positive and negative controls of Hedyotis diffusa Willd., Commelina communis L., and Monochasma sheareri (S. Moore) Maxim. respectively and conduct tests according to steps ① and ②.

[0075] Results:

[0076] The results of step ① show that the test solution shows the same dark green as each positive and negative control solution, and the method is not specific.

[0077] The results of step ② show that the test solution shows the same pink as each positive and negative control solution, and the method is not specific.

[0078] Conclusion:

[0079] Both physical and chemical identification methods are not specific, so they are not included in the standard text.

[0080] ②Qualitative identification of Hedyotis diffusa Willd

[0081] Hedyotis diffusa Willd negative control: B20220201, prepared according to the process from the prescription without Hedyotis diffusa Willd medicinal materials.

[0082] Hedyotis diffusa Willd positive control: DB20220201, prepared according to the process from Hedyotis diffusa Willd medicinal materials.

[0083] Hedyotis diffusa Willd medicinal materials: P-210801, provided by Zhejiang Ainuo Biopharmaceutical Co., Ltd. after passing the inspection. Hedyotis diffusa Willd reference medicinal materials: (121183-201605), purchased from the National Institutes for Food and Drug Control.

[0084] Methyl deacetylasperulate reference substance: Methyl deacetylasperulate reference substance (content 94.3%, batch number: 111786-201602, for content determination), purchased from the National Institutes for Food and Drug Control.

[0085] Refer to the content under the item of "Identification" in Part I of the Chinese Pharmacopoeia (2020 Edition) [1] "Yanning Syrup" and the literature [2-3] to conduct a preliminary exploration experiment on the identification of Hedyotis diffusa Willd. The test was carried out with Hedyotis diffusa Willd medicinal materials as the control.

[0086] In the exploration experiment, first dissolve the test solution in water, extract with ethyl acetate, evaporate to dryness, and dissolve the residue in methanol to prepare the test solution. Three kinds of thin-layer plates, namely silica gel G, GF254, and polyamide film, and three kinds of developing agents, namely chloroform-methanol-water, chloroform-ether, and acetic acid, were investigated. The results showed that the spots had no specificity. Then, using ethanol as the extraction solvent, the test solution was prepared by soaking and ultrasonic extraction respectively, and three kinds of developing agents, namely chloroform-methanol-water, the lower layer solution of chloroform-methanol-water, and ethyl acetate-methanol-water, were investigated;

[0087] For the three kinds of developing agents, the results showed that the spots had specificity, but the resolution was poor and the tailing was serious, not meeting the inspection requirements; therefore, the thin-layer identification of Hedyotis diffusa Willd was not included in the standard text.

[0088] ③Qualitative identification of Commelina communis L

[0089] Commelina communis L reference medicinal materials: (121663-201602), purchased from the National Institutes for Food and Drug Control.

[0090] A. Exploration of Commelina communis L identification I (thin-layer identification)

[0091] Commelina communis L contains a variety of flavonoid compounds, and flavonoid compounds generally have significant pharmacological and physiological activities. Therefore, in the research, the extraction of flavonoid components was mainly selected for identification, and the test was carried out with Commelina communis L medicinal materials as the control.

[0092] In the exploratory experiment, ethanol was used as the extraction solvent, and the samples were extracted by soaking and refluxing methods respectively to prepare the test solution. After thin-layer development, no specific spots were detected. Then, the test solution was evaporated to dryness, the residue was dissolved in water, extracted with ethyl acetate, evaporated to dryness, and the residue was dissolved in ethyl acetate to prepare the test solution. The test solution was developed with chloroform-methanol-water, the lower layer solution of chloroform-methanol-water, and chloroform-ether respectively. As a result, no specific flavonoid components of Commelina communis L. were detected in the samples.

[0093] Then, Yanning Granules were ultrasonically extracted with 70% ethanol. The extract was loaded onto a silica gel column and eluted successively with ethyl acetate-methanol-water, chloroform-methanol-water, chloroform-methanol-water, and chloroform-methanol-water. The eluates were evaporated to dryness respectively, and the residues were dissolved in methanol to prepare their respective test solutions. The test solutions were developed with methanol-acetic acid-water, chloroform-acetic acid-water, and toluene-ethyl acetate as the developing agents, developed, and examined, but no specific spots of Commelina communis L. were detected either.

[0094] Therefore, it was considered to use HPLC method for the specific identification of Commelina communis L.

[0095] B. Exploration of the Identification of Commelina communis L. II (HPLC method)

[0096] In view of the fact that the specific properties of Commelina communis L. could not be characterized in the thin-layer identification exploration experiment, referring to the literature "Study on Flavonoid Components of Commelina communis L.", it was considered to establish a characteristic chromatogram by liquid chromatography method and use the relative retention time method (using methyl deacetylasperulosidate reference substance to locate the characteristic peaks of Commelina communis L.) to identify Commelina communis L. in the prescription.

[0097] First, the gradient elution sample preparation method in the literature was used to prepare samples of the eluates in three stages: chloroform-methanol-water, chloroform-methanol-water, and chloroform-methanol-water. In the eluate sample in the chloroform-methanol-water stage, there were two chromatographic peaks corresponding to the positive control and the test solution, and there was no interference from the negative control. Then, the sample preparation method was simplified, and a chloroform-methanol-water mixed solution was directly used for ultrasonic extraction. The results showed that the method had specificity, but in the durability investigation experiment, it was found that the relative retention time had a large deviation and was not suitable for inclusion in the standard.

[0098] C. Exploration of the Identification of Commelina communis L. III (HPLC method)

[0099] In "Identification Exploration of Commelina communis II", the relative retention time method (using methyl deacetylasperate reference substance to locate the characteristic peak of Commelina communis) had a large error, so the reference medicinal material of Commelina communis was used for location. Among them, characteristic peak 2 was greatly affected by the phosphoric acid concentration, which affected the judgment of specificity, while characteristic peak 1 was less affected by the phosphoric acid concentration. Therefore, only characteristic peak 1 was included in the standard, and the methodological verification was carried out according to the verification indicators (specificity and durability) under the identification item in the Guidelines for Method Verification of the Fourth Part of the Chinese Pharmacopoeia (2020 Edition). The results showed that this method had specificity and good durability, so it could be used for the qualitative identification of Commelina communis and was included in the main text of the standard. The specific work is described in detail as follows.

[0100] (1) Experimental materials

[0101] High performance liquid chromatograph: Waters alliance e265#L185M4690A

[0102] Detector: Waters 2998PDA Detector#18998720A

[0103] Chromatographic column: GL Sciences InertSustain AQ-C18

[0104] (2) Experimental content

[0105] a. Investigation of the sample preparation method for test samples

[0106] Investigation of the extraction solvent. The test sample solutions were prepared by extraction with chloroform-methanol-water, anhydrous methanol, and 50% methanol respectively. The results showed that when chloroform-methanol-water was used as the solvent, the target peak area was larger and the impurity peaks were fewer. Therefore, chloroform-methanol-water was used as the extraction solvent.

[0107] Investigation of the extraction time. The target peak areas in the samples were compared after ultrasonic extraction for 20 min, 30 min, and 40 min. The results showed little difference among the three, indicating that the target substance had been completely extracted after 20 min of ultrasonic extraction. Therefore, 20 min was used as the extraction time.

[0108] b. Investigation of the preparation method for the solution of reference medicinal material

[0109] Chloroform-methanol-water, 95% ethanol extraction, and anhydrous ethanol extraction were successively used, and the target peak could be extracted in all cases. Therefore, the same method as that for the preparation of the test sample solution was adopted, using chloroform-methanol-water as the extraction solvent, and the concentrations of the solution of reference medicinal material and the test sample solution were adjusted to be basically the same.

[0110] c. Investigation of the mobile phase gradient

[0111] After exploring the mobile phase gradient, the exclusive characteristic peaks of Commelina communis were identified, and the mobile phase gradient was adjusted and optimized. It was found that the exclusive target peaks of Commelina communis could be separated under the gradient conditions close to the pure water phase, and the peak purity showed that there were no co-eluting substances in the target peaks. Given that the retention time of the characteristic peaks was within 15 minutes, combined with the peak elution situation in the chromatogram, the elution time of the pure water phase and the chromatogram acquisition time were set to 15 minutes. After 15 minutes, an organic phase elution gradient was set for a certain period of time to elute the residual components of a single injection in the chromatographic column and avoid affecting the next injection.

[0112] By comparing the overall separation of the chromatogram and the results of the separation degree, purity, retention time, etc. of the characteristic peaks, an HPLC method for the exclusive identification of Commelina communis was obtained.

[0113] d. Investigation of phosphoric acid concentration

[0114] After investigation, under the same chromatographic conditions, there were many non-target peaks in the chromatogram with pure water as the mobile phase, which affected the identification of the target peaks. In the chromatograms with 0.1%, 0.2%, and 0.3% phosphoric acid aqueous solutions as the mobile phase, the retention time of the second characteristic peak was significantly affected by the change in phosphoric acid concentration, which affected the judgment of exclusivity, so it was not included in the standard. The retention time of the first characteristic peak was less affected by the phosphoric acid concentration and was basically the same. Therefore, for the first characteristic peak, this method was durable between 0.1% and 0.3% phosphoric acid concentration. Considering the configuration error of the mobile phase in actual operation, 0.2% phosphoric acid aqueous solution was selected as the aqueous phase of the mobile phase, and the first characteristic peak was included in the standard.

[0115] e. Preliminary formulation of the method

[0116] Take 0.5 g of the powder of this product, add 20 ml of chloroform-methanol-water, sonicate for 20 minutes (480 kW, 40 Hz), filter, evaporate the filtrate to dryness, dissolve the residue in 30% methanol and dilute to 10 ml, filter, and take the subsequent filtrate as the test solution. Separately take 0.6 g of the control crude drug of Commelina communis and prepare the control crude drug solution in the same way. Test according to the high performance liquid chromatography method (General Principles 0512), using octadecylsilane chemically bonded silica as the filler; using acetonitrile as mobile phase A and 0.2% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in Table 2; respectively take 15 μl of the control crude drug solution and 5 μl of the test solution, inject them into the liquid chromatograph, determine, and record the chromatogram for 15 minutes. The chromatogram in the test solution should show chromatographic peaks with the same retention time as the characteristic peak S in the chromatogram of the control crude drug.

[0117] f. Methodological verification

[0118] Exclusivity

[0119] Precisely pipette 5 μl each of the blank solvent (30% methanol), the test solution, and the negative control prepared according to the method of "Preparation of the Test Solution" (prepared by self-making according to the prescription without Commelina communis L. according to the process), and 15 μl of the reference medicinal material solution into the liquid chromatograph, and record the chromatogram within 15 minutes. As can be seen from the figure, the reference medicinal material and the sample have the same peak 1, and the spectra Figure 1 are consistent, and there is no interference from the negative control, indicating that this identification method has specificity and meets the requirements.

[0120] Instrument precision

[0121] Precisely pipette 5 μl of the test solution into the liquid chromatograph and inject samples continuously for 5 times. The results are shown in Table 1. Table 1 Results of the injection precision test of the reference substance solution

[0122] Sample number Peak area Average value RSD(%) 1 60261 2 60234 3 60211 60288.4 0.20 4 60279 5 60457

[0123] Table 1

[0124] Analysis: For 5 consecutive injections, the RSD% of the target peak area is 0.20%, meeting the requirements.

[0125] Solution stability

[0126] Inject the reference medicinal material solution and the test solution on the first day, the second day, and the third day respectively. The results are shown in Table 2, indicating that both solutions are stable.

[0127]

[0128]

[0129] Table 2 Investigation of solution stability

[0130] Analysis: For both solutions within 3 days, the RSD% of the target peak is less than 1.0%, indicating that the solutions are stable within three days.

[0131] Robustness

[0132] During the investigation of phosphoric acid concentration, concentrations of 0.1%, 0.2%, and 0.3% phosphoric acid have been investigated. The results show that under the three conditions, the resolution and the number of theoretical plates of the target peak meet the requirements.

[0133] The investigation results for different column temperatures, different flow rates, different instruments, and different chromatographic columns are shown in Table 3 below:

[0134]

[0135]

[0136] Table 3 Analysis of the investigation results of column temperature: The resolution of the target peak is greatly affected by temperature, so the column temperature is specified as 25 °C in the standard.

[0137] Investigation of Flow Rate in Table 4

[0138]

[0139]

[0140] Table 4

[0141] Analysis: At a flow rate of 1.0 ml / min, in the control crude drug solution, the resolution between the target peak and the previous peak did not reach complete separation. However, from the chromatogram, it could achieve the purpose of identifying Commelina communis, that is, the retention times of the target peaks in the control crude drug solution and the test solution were consistent.

[0142] Investigation of Different Instruments and Chromatographic Columns in Table 5

[0143]

[0144]

[0145] Table 5

[0146] Analysis: Experiments were carried out using different chromatographic columns and different instruments. The results showed that in the test solution chromatogram, 1 characteristic peak corresponding to the control crude drug chromatogram appeared within 15 min, and there was no interference from the negative sample. The resolution of both peaks reached more than 1.5, meeting the requirements. However, the separation effect of the CSTCHROM Daiso C18 - BP chromatographic column was not as good as that of the GL Sciences InertSustain AQ - C18 chromatographic column. Therefore, it is recommended to use the GL Sciences InertSustain AQ - C18 chromatographic column.

[0147] Conclusion: The HPLC identification method for Commelina communis has strong specificity and good durability.

[0148] Combined with the above content, the final method is as follows:

[0149] A method for physical and chemical identification of Yanning Granules, including the identification of Commelina communis, specifically including:

[0150] S1: Take the powder of Yanning Granules;

[0151] In this embodiment, in S1, take 0.5 g of the powder of Yanning Granules;

[0152] S1a: Add a mixed solution composed of chloroform - methanol - water and mix it with the powder of Yanning Granules; specifically, in S1a, add 20 ml of the mixed solution composed of chloroform - methanol - water and mix it with the powder of Yanning Granules;

[0153] S1b: Perform treatment by ultrasonic waves; preferably, specifically, in S1b, place the container filled with the S1a mixture in an ultrasonic instrument and perform treatment by ultrasonic waves; the treatment time is 20 minutes, the ultrasonic power is 480 kW, and the ultrasonic frequency is 40 Hz.

[0154] S1c: Filter, evaporate the filtrate to dryness to obtain a residue, dissolve the residue with 28 - 32% methanol and dilute to 10 ml;

[0155] S1d: Filter again, take the subsequent filtrate as the test solution;

[0156] S1e: Separately take the Commelina communis control medicinal material and prepare a control medicinal material solution according to the steps of S1b - S1d; specifically, in S1e, separately take 0.6 g of the Commelina communis control medicinal material and prepare a control medicinal material solution according to the steps of S1b - S1d.

[0157] S2: Perform the test according to the high - performance liquid chromatography method (General Principles 0512), using octadecylsilane chemically bonded silica gel as the filler;

[0158] Use acetonitrile as mobile phase A and 0.1 - 0.3% phosphoric acid solution as mobile phase B for gradient elution; specifically including:

[0159] In S2, use acetonitrile as mobile phase A and 0.2% phosphoric acid solution as mobile phase B. Respectively take 15 μl of the control medicinal material solution and 5 μl of the test solution, inject them into the liquid chromatograph, determine, and record the chromatogram for 15 min.

[0160] The number of theoretical plates calculated by the characteristic peak S should be not less than 4000; respectively take the control medicinal material solution and the test solution, inject them into the liquid chromatograph, determine, and record the chromatogram for 15 min;

[0161] If a chromatographic peak with a retention time consistent with that of the characteristic peak S in the control medicinal material chromatogram appears in the test solution chromatogram, it indicates that the sample contains Commelina communis.

[0162] In the present invention, it also includes the identification of Monochasma sheareri (specific method)

[0163] Specifically: Preparation of the test solution: Take the powder of Yanning Granules; specifically: Take 2 g of the pilot - scale sample (YZ20220501) powder of Yanning Granules, add water to dissolve it, extract with ethyl acetate for 3 times, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue with methanol to obtain the test solution;

[0164] Preparation of the negative control solution: Take the powder of the Monochasma sheareri negative sample (L20220101), add water to dissolve it, extract with ethyl acetate for 3 times, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue with methanol to obtain the negative control solution;

[0165] Preparation of reference solution: Take luteolin reference substance, add methanol to make a solution containing 0.1 mg per 1 ml to obtain the reference solution;

[0166] Among them, further, the preparation of the test solution and the preparation of the negative control solution specifically include

[0167] Preparation of test solution: Take 2 g of Yanning Granule powder, add 20 ml of water to dissolve it, extract with ethyl acetate 3 times, 25 ml each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 2 ml of methanol to obtain the test solution;

[0168] Preparation of negative control solution: Take 2 g of Monochasma sheareri var. wulingense negative sample powder, add 20 ml of water to dissolve it, extract with ethyl acetate 3 times, 25 ml each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 2 ml of methanol to obtain the negative control solution.

[0169] Determination: Take the reference solution, test solution, and negative control solution, and spot them on the same silica gel thin layer plate respectively. Use chloroform - methanol - formic acid - water as the developing agent, develop, take out, dry, spray with aluminum trichloride test solution, heat to make the spots develop color, and examine under an ultraviolet lamp;

[0170] Further, the determination specifically includes:

[0171] Take 3 μl of the reference solution, 5 μl each of the test solution and the negative control solution, and spot them on the same silica gel thin layer plate respectively. Use chloroform - methanol - formic acid - water as the developing agent, develop, take out, dry, spray with aluminum trichloride test solution, heat at 105 °C to make the spots develop color, and examine under an ultraviolet lamp at 365 nm.

[0172] Results and analysis: In the chromatogram of the test sample, at the position corresponding to the reference solution, fluorescent spots of the same color appear, there is no interference from the negative, and the Rf value is 0.52 (see Figure 4 ), indicating that this identification method has specificity.

[0173] Further, it also includes (specificity method)

[0174] SS1: According to the preparation of the test solution, the reference solution, test solution, and negative control solution are respectively investigated under different conditions of ±1 change in the proportion of chloroform in the developing agent, temperature change, and relative humidity change on different thin layer plates, as well as the sample application amount investigation. Specifically, in the preparation of the test solution, the temperature changes include 4 °C, 25 °C, and 40 °C; the relative humidity changes are 32%, 44%, and 75%.

[0175] Verify the durability of the Monochasma sheareri var. wulingense identification method:

[0176] Developing solvent: When the proportion of chloroform varies by ±1 based on the set value, the spots on the thin-layer chromatography are clear, the resolution is good, and there is no tailing phenomenon.

[0177] Thin-layer plate: It includes self-made thin-layer plates and commercially available thin-layer plates. The characteristic spots of the self-made thin-layer plates have poor resolution and serious tailing; for the two commercially available brand thin-layer plates, the spots are clear, the resolution is good, and there is no tailing.

[0178] Temperature: Under different temperature conditions of 4°C, 25°C, and 40°C, the spots are clear, the resolution is good, and there is no tailing.

[0179] Humidity: When the relative humidity is 32%, the resolution of the characteristic spots is relatively poor; when the relative humidity is 44% and 75%, the spots are clear, the resolution is good, and there is no tailing.

[0180] Sample application volume: Under different sample application volume conditions, the spots are clear, the resolution is good, and there is no tailing.

[0181] For the specific results of the robustness investigation, please refer to Table 6.

[0182]

[0183]

[0184] Table 6

[0185] SS2: Conclusion: According to the above investigation results, for the specific identification of Monochasma sheareri, it is advisable to choose a commercially available thin-layer plate with a relative humidity not less than 40%. The spots with different sample application volumes are clear and the resolution is good. For the convenience of sample application, 1 μl is selected as the sample application volume.

[0186] Detection of three batches of samples

[0187] Three batches of commercially produced finished products were determined according to the proposed method. Chromatographic peaks consistent with the retention time of the characteristic peak S of the reference medicinal material chromatogram appeared in the chromatograms of the three batches of test samples.

[0188] ③ Detection of three batches of samples

[0189] Three batches of sugar-free granules of Yanning Granules YZ20221001, YZ2022110, and YZ20221102 were detected according to the above method.

[0190] ④ Qualitative identification of Monochasma sheareri

[0191] After consulting relevant literature, it was found that Monochasma sheareri contains a large amount of flavonoid components, mainly including caffeic acid, luteolin and other components. Identification tests were carried out on these two components. The results showed that caffeic acid does not have specificity, but luteolin has strong specificity and good method durability. Therefore, it was included in the main text of the standard. The specific work is described in detail as follows.

[0192] A. Exploration of the Identification of Monochasma sheareri Maxim

[0193] a. Caffeic acid

[0194] Referring to the content under the item of "Identification" in Part I of the Chinese Pharmacopoeia (2020 Edition) and querying relevant literature, caffeic acid is one of the active components of Monochasma sheareri Maxim. Therefore, caffeic acid was first selected as the control for the exploratory experiment in the research.

[0195] In the exploratory experiment, after dissolving the test sample in water first, it was extracted with ethyl acetate, the solvent was evaporated to dryness, the residue was dissolved in methanol to prepare the test solution. Using a polyamide film as the thin-layer plate and 36% acetic acid as the developing agent for development, the results showed that there was interference in the negative test, indicating that caffeic acid in Monochasma sheareri Maxim was not specific.

[0196] b. Luteolin

[0197] By querying relevant literature, it was confirmed that luteolin was one of the effective components of Monochasma sheareri Maxim, and the specific identification method of Monochasma sheareri Maxim was explored using luteolin as the reference substance.

[0198] In the exploratory experiment, after dissolving the sample in water, it was extracted with ethyl acetate, evaporated to dryness, and the residue was dissolved in methanol to obtain the test solution. Using a silica gel G thin-layer plate, chloroform-methanol-formic acid as the developing agent and aluminum trichloride test solution as the color-developing agent for the experiment, the results showed that luteolin in Monochasma sheareri Maxim was specific.

[0199] c. Optimization of the Identification Method of Monochasma sheareri Maxim

[0200] By querying and referring to relevant literature, the thin-layer identification method of Monochasma sheareri Maxim was optimized.

[0201] The methods for preparing the test solution by extraction with ethyl acetate, extraction with diethyl ether, extraction with water-saturated n-butanol, soaking with ethanol, and ultrasonic treatment with ethanol were explored, and chloroform-methanol-formic acid, toluene-ethyl acetate-formic acid, and chloroform-methanol-formic acid-water were used as the developing agents for development respectively. The results showed that when the sample was prepared by extraction with ethyl acetate and developed with chloroform-methanol-formic acid-water as the developing agent, the spots were clearly colored, the resolution was good, and it was easier to observe.

[0202] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations according to the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field of the present invention based on the concept of the present invention through logical analysis, reasoning, or limited experiments on the basis of the prior art should fall within the protection scope determined by the claims.

Claims

1. A method for physical and chemical identification of Yanning granules, characterized in that: Including identification of Commelina, specifically including: S1: Take Yanning granule powder; S1a: adding a mixed solution consisting of chloroform-methanol-water and mixing with the Yanning granule powder; S1b: treatment by ultrasound; S1c: Filter, evaporate the filtrate to dryness to obtain a residue, dissolve the residue in 28-32% methanol and dilute; S1d: Filter again and take the filtrate as the test solution; S1e: Take another Commelina communis control medicinal material, and prepare a control medicinal material solution according to the steps of S1b-S1d; S2: According to the HPLC test, octadecylsilane bonded silica gel is used as the filler; Acetonitrile was used as mobile phase A and 0.1-0.3% phosphoric acid solution was used as mobile phase B for gradient elution; The number of theoretical plates calculated based on the characteristic peak S is not less than 4000; respectively take the control medicinal material solution and the test sample solution, inject them into the liquid chromatograph, measure, and record the chromatogram for 15 minutes; S3: If the chromatogram of the test sample shows a chromatographic peak with a retention time consistent with the chromatographic characteristic peak S of the reference medicinal material, it indicates that the sample contains Commelina.

2. The method for physical and chemical identification of Yanning granules as claimed in claim 1, characterized in that: In S1, 0.5 g of Yanning granule powder is taken; In the S1a, 20 ml of a mixed solution consisting of chloroform-methanol-water is added and mixed with the Yanning granule powder; In the above S1e, 0.6 g of Commelina communis reference medicinal material is taken and a reference medicinal material solution is prepared according to the steps of S1b-S1d.

3. The method for physical and chemical identification of Yanning granules as claimed in claim 2, characterized in that: In the S1b, the container containing the S1a mixture is placed in an ultrasonic instrument and treated with ultrasound; the treatment time is 20 minutes, the ultrasound power is 480 kW, and the ultrasound frequency is 40 Hz.

4. The method for physical and chemical identification of Yanning granules as claimed in claim 3, characterized in that: In S2, acetonitrile is used as mobile phase A and phosphoric acid solution is used as mobile phase B.

5. The method for physical and chemical identification of Yanning granules as claimed in claim 2, characterized in that: In the S2, the flow rate is 0.9 ml per minute; the column temperature is 25° C.; the detection wavelength is 250 nm; the number of theoretical plates calculated based on the characteristic peak S is not less than 4000; 15 μl of the control medicinal material solution and 5 μl of the test sample solution are respectively taken, injected into the liquid chromatograph, measured, and the chromatogram is recorded for 15 minutes.

6. The method for physical and chemical identification of Yanning granules according to claim 1, characterized in that: It also includes identification of velvet antler grass, specifically: Preparation of the test solution: Take Yanning granule powder; add water to dissolve it, extract it with ethyl acetate three times, combine the ethyl acetate solution and evaporate it to dryness, add methanol to dissolve the residue to obtain the test solution; Preparation of negative control solution: Take the negative sample powder of Pteroantler Herb, add water to dissolve it, extract it with ethyl acetate for 3 times, combine the ethyl acetate solution and evaporate it to dryness, add methanol to dissolve the residue to obtain a negative control solution; Preparation of reference solution: Take luteolin reference substance and add methanol to make a 1 ml solution containing 0.1 mg to obtain reference solution; Determination: Take the control solution, test solution and negative control solution, spot them on the same silica gel thin layer plate, use chloroform-methanol-formic acid-water as the developing agent, develop, take out, dry, spray with aluminum chloride test solution, heat to color the spots, and inspect under ultraviolet light; Results and analysis: In the chromatogram of the test sample, a fluorescent spot of the same color appeared at the corresponding position of the reference solution, with no interference in the negative test and an Rf value of 0.52, indicating that this identification method is specific.

7. The method for physical and chemical identification of Yanning granules as claimed in claim 6, characterized in that: It also includes durability verification, specifically: SS1: According to the preparation of the test solution, the reference solution, test solution and negative control solution were placed on different thin layer plates, and different conditions such as the change of chloroform ratio in the developing agent ±1, temperature change and relative humidity change, as well as the spotting amount were investigated to verify the durability of the identification method of velvet antler grass; Developing agent: When the ratio of chloroform changes by ±1 based on the set value, the spots of thin layer chromatography are clear, the separation is good, and there is no tailing phenomenon; Thin layer plates: including homemade thin layer plates and commercial thin layer plates. The homemade thin layer plates have poor spot separation and severe tailing; the commercial thin layer plates have clear spots, good separation and no tailing. Temperature: Under different temperature conditions of 4℃, 25℃ and 40℃, the spots are clear, well separated and without tailing; Humidity: When the relative humidity is 32%, the separation of characteristic spots is poor; when the relative humidity is 44% and 75%, the spots are clear, the separation is good, and there is no tailing; Spotting amount: Under different spotting amount conditions, the spots are clear, the separation is good, and there is no tailing; SS2: According to the above investigation results, commercially available thin layer plates were selected for the specific identification of deer antler grass. The relative humidity should not be lower than , and the spots of different spotting amounts should be clear with good separation. In order to facilitate spotting, 1μl was selected as the spotting amount.

8. The method for physical and chemical identification of Yanning granules as claimed in claim 7, characterized in that: In the preparation of the test solution, the temperature changes included 4°C, 25°C and 40°C; the relative humidity changes were 32%, 44% and 75%.

9. The method for physical and chemical identification of Yanning granules as claimed in claim 8, characterized in that: The preparation of the test solution and the preparation of the negative control solution specifically include: Preparation of test solution: Take 2g of Yanning granule powder, add 20ml of water to dissolve, extract with ethyl acetate 3 times, 25ml each time, combine the ethyl acetate solution and evaporate to dryness, add 2ml of methanol to dissolve the residue to obtain the test solution; Preparation of negative control solution: Take 2g of negative sample powder of Pilosa antler grass, add 20ml of water to dissolve it, extract it with ethyl acetate for 3 times, 25ml each time, combine the ethyl acetate liquid and evaporate to dryness, add 2ml of methanol to dissolve the residue to obtain a negative control solution.

10. The method for physical and chemical identification of Yanning granules according to claim 9, characterized in that: The determination specifically includes Take 3μl of control solution, 5μl of test solution and negative control solution, spot them on the same silica gel thin layer plate, use chloroform-methanol-formic acid-water as the developing agent, develop, take out, dry, spray with aluminum chloride test solution, heat at 105℃ to color the spots, and examine under ultraviolet light at 365nm.

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