CN112083097A - A kind of thin-layer identification method for simultaneously identifying ferulic acid, verbisoflavone glucoside and hesperidin - Google Patents

Abstract

The invention discloses a thin-layer identification method for simultaneously identifying ferulic acid, mullein glucoside and hesperidin, belonging to the technical field of identification of traditional Chinese medicine compound preparations. In this technical scheme, through the preparation of the test solution, the preparation of the reference solution and the detection by thin-layer chromatography, as well as each specific control condition, the simultaneous qualitative identification of three traditional Chinese medicines, ferulic acid, verbisoflavone glucoside, and hesperidin is completed. Active ingredients, at the same time, involve a good degree of separation, effectively eliminate negative interference, and have a clear inspection, with good specificity and good reproducibility, which can effectively realize the quality control of compound preparations, realize the standardization of drug production, and then better In addition, it can effectively save solvent and labor costs, and shorten the identification time by about 70%.

Description

A kind of thin-layer identification for simultaneous identification of ferulic acid, verbisoflavone glucoside and hesperidin other way

technical field

The invention relates to a thin-layer identification method for traditional Chinese medicine compound preparations, in particular to a thin-layer identification method for simultaneously identifying ferulic acid, verbisoflavone glucoside and hesperidin, belonging to the technical field of traditional Chinese medicine compound preparation identification.

Background technique

Ferulic acid is a phenolic acid widely found in plants, mostly found in Chinese medicines such as Angelica and Chuanxiong. Its pharmacological activity is widely used, with anti-atherosclerosis, anti-platelet aggregation and thrombosis, antibacterial and anti-inflammatory, anti-tumor, anti-mutation , Increase immune function, enhance human sperm motility and motility, etc. Vervis isoflavone glucoside is an isoflavone compound, which is the active monomer component extracted from the dried roots of leguminous plants Astragalus mongolica and Astragalus capsulata. It is also one of the most active main components of the commonly used traditional Chinese medicine Astragalus. , antiviral, inhibition of melanin formation and immunosuppression. Hesperidin is widely found in leguminous, labiatae, disciaceae, rutaceae, citrus plants, and has anti-inflammatory, antioxidant, antibacterial, anticancer, immune regulation, radiation protection, and cardiovascular system protection and other pharmacological activities.

In the existing traditional Chinese medicine composition for treating qi and blood deficiency syndrome, it is composed of astragalus, angelica, jujube, wolfberry, dried tangerine peel or its extracts, and the combination of astragalus and angelica is used for yang to generate yin, and qi is prosperous. At the same time, supplemented with wolfberry, jujube and tangerine peel, the whole formula has the effect of nourishing liver and kidney, strengthening spleen and regulating qi, benefiting the body and strengthening the body, thereby enhancing the immune function of the body. People with weakened immune function caused by excessive fatigue, postpartum qi and blood deficiency, and cancer radiotherapy and chemotherapy. However, a set of standard and effective detection methods for ensuring product quality has not been established for the quality control of the traditional Chinese medicine composition. Therefore, in order to ensure the medication effect of the medicine, it is urgent to provide a set of simple and reliable quality standards.

In order to better control the quality of the combination, a method was established for the simultaneous determination of active components such as astragalus, tangerine peel, angelica, etc., while improving the quality control method, a more concise, rapid and accurate identification method was realized, and the cost was saved.

At present, due to the complex components of traditional Chinese medicine compound preparations, if the traditional thin-layer identification method is used, that is, if a thin-layer identification method is established for each medicinal material or each active ingredient, the operation process is cumbersome, complicated, time-consuming, and efficient. low, and the retrieval results are less accurate.

The paper "Study on the Content Determination of Various Components in Buzhong Yiqi Pills by One Test and Multiple Evaluation Method" mentioned that the one test and multiple evaluation method is to use the inherent relationship between drug compounds to realize the synchronization of one component to multiple components. The measurement and control better reflects the multi-component quality evaluation mode of traditional Chinese medicine, which is in line with the overall view of traditional Chinese medicine theory. In addition, it involves establishing the internal connection between various components in Buzhong Yiqi Pills and hesperidin, and realizing the simultaneous determination of multiple groups of Buzhong Yiqi Pills through the reference substance of hesperidin, which is the quality standard for Buzhong Yiqi Pills. The supplement and improvement of control methods provide reference.

The paper "Research on the Quality Standard of Fufang Yiqi Zhike Granules, Wang Jing et al." specifically disclosed: adding methanol to prepare the pretreatment of the mixed reference solution of glucosinolate and hesperidin, and its identification by thin layer chromatography, etc.

On July 13, 2016, a patent document with a publication number of CN105758985A and the title of "a quality detection method for Tongkang tablets" was disclosed, wherein, it was disclosed that the wavelength was 365nm under the ultraviolet lamp, and sprayed with 10% Aluminum trichloride ethanol solution and heating at 105 ° C until the spots are clear, to identify the isoflavone glucoside and hesperidin; on September 23, 2015, a publication number CN104931640A was published, and the name was "an improved animal The patent document of the thin-layer chromatography detection method of the traditional Chinese medicine composition of immunity ", wherein, disclose: 1mg/mL Verisiflavone glucoside reference substance solution is detected by thin-layer chromatography, and the inspection wavelength under 254nm is the silica gel GF254 thin-layer plate. Silica gel plate, etc.; on March 28, 2012, a patent document with a publication number of CN102393440A, entitled "Method for detecting hesperidin in Huoxiangzhengqi tablets" by thin-layer chromatography, was disclosed, wherein: hesperidin control The product solution was detected by thin layer chromatography, sprayed with aluminum trichloride test solution, and inspected under ultraviolet light with a wavelength of 365 nm.

Although the prior art discloses the theoretical basis for the simultaneous determination of multiple components in it by liquid chromatography, and the detection of the active ingredients in traditional Chinese medicines (verasco isoflavone glucoside or hesperidin), but for different detections Subjects and traditional Chinese medicine compositions with more complex ingredients, which were not effective in simultaneously identifying ferulic acid, vervescoflavonoid glucoside and hesperidin.

SUMMARY OF THE INVENTION

The present invention aims to solve the problems of the prior art, and proposes a thin-layer identification method for simultaneously identifying ferulic acid, velocidone glucoside and hesperidin. In this technical scheme, through the preparation of the test solution, the preparation of the reference solution, and the detection by thin-layer chromatography, as well as each specific control condition, it is effective to simultaneously qualitatively identify three traditional Chinese medicines: ferulic acid, verbisoflavone glucoside, and hesperidin. and the identification method is simple and quick to operate, and provides an effective basis for the identification of compound preparations.

In order to achieve the above technical purpose, the following technical solutions are proposed:

A thin-layer identification method for simultaneously identifying ferulic acid, verbisoflavone glucoside and hesperidin, comprising the following steps:

A. Preparation of the test solution: take 3g of the traditional Chinese medicine compound preparation, add 20mL of ethyl acetate, heat under reflux for 1h, filter, and evaporate the filtrate to dryness; add 1mL of methanol to the filter residue and dissolve to obtain the test solution;

Wherein, evaporating to dryness specifically refers to making the solvent in the filtrate zero;

Among them, the heating and refluxing adopts a conventional electronic constant temperature water bath (for example: the model is DK-98-11, Beijing Zhongxing Weiye Instrument Co., Ltd.);

Wherein, in parts by weight, the traditional Chinese medicine compound preparation includes the following components: 25-35 parts of Astragalus or its extract, 5-7 parts of Angelica or its extract, 8-12 parts of jujube or its extract, Lycium barbarum 5 to 10 parts of its extract, 5 to 8 parts of tangerine peel or its extract, and the traditional Chinese medicine composition at least includes the following active ingredients: mullein isoflavone glucoside ≥ 0.10 mg/g, astragaloside ≥ 0.45 mg/g, astragalus Acid ≥0.05mg/g, Lycium barbarum polysaccharide ≥8.0mg/g, Hesperidin ≥5.0mg/g;

B. Preparation of reference solution: respectively take ferulic acid reference substance, verbisoflavone glucoside reference substance and hesperidin reference substance, add methanol, and prepare 1mg/L ferulic acid reference substance solution and 1mg/L mullein reference substance respectively. Flavonoid glucoside solution and 1mg/L hesperidin reference solution, for later use;

C. Preparation of reference medicinal material solution: respectively take 0.5 g of Angelica reference medicinal material, 1 g of Astragalus reference medicinal material and 1 g of tangerine peel reference medicinal material, add 28-32 mL of water, boil for 30 minutes, place at room temperature, filter, and evaporate the filtrate to dryness until the moisture is zero; Add 1 mL of methanol and dissolve to obtain three reference medicinal material solutions;

D. Detection: Using thin-layer chromatography, take 5-20 μL of the test solution, 2-15 μL of the ferulic acid reference solution, 2-15 μL of the isoflavone glucoside reference solution, and 5-15 μL of the hesperidin reference solution. and 10 μL of each of the three reference medicinal material solutions, respectively, were placed on the same GF254 thin-layer plate, unfolded with a developing agent, and taken out, dried in the air until the developing agent evaporated, and the fluorescent spots were checked at 254 nm, and then sprayed with a chromogenic agent 10% Aluminum trichloride ethanol solution, dry the color developer, heat it at 105 °C until the spots on the plate develop clear color, and inspect it under a UV lamp (365 nm);

D. Analysis of results: In the chromatogram of the test product at 254nm, the same spots are displayed in the corresponding position of the ferulic acid reference solution and the reference solution of Verisiflavone glucoside; in the chromatogram of the test solution at 365nm, the The hesperidin reference solution showed fluorescent spots of the same color on the corresponding position of the chromatogram.

Preferably, 10 μL of the test solution, 5 μL of the ferulic acid reference solution, 5 μL of the isoflavone glucoside reference solution, and 10 μL of the hesperidin reference solution were taken, respectively, and placed on the same GF254 thin-layer plate.

Preferably, the developing agent is chloroform-methanol-acetone=15:3.5:1.5.

By adopting this technical solution, the beneficial technical effects brought are as follows:

1) The present invention is based on thin-layer chromatography, which can simultaneously identify three active ingredients of traditional Chinese medicines: ferulic acid, verisoflavone glucoside, and hesperidin. The identification method is simple and quick to operate, and provides an effective basis for the identification of compound preparations. In addition, the degree of separation involved in this technical solution is good, negative interference is effectively eliminated, and the inspection is clear, with good specificity and good reproducibility, which can effectively realize the quality control of compound preparations, realize the standardization of drug production, and further improve the Good to meet drug safety, production process stability and controllability;

2) In the step A of the present invention, ethyl acetate is used as the solvent, and according to the principle of similar compatibility, the effective components in the traditional Chinese medicine compound preparation with complex components can be extracted to a large extent, and a better separation effect can be obtained, which is convenient for later detection and analysis. Accuracy and precision of judgment. Methanol is added to the filter residue, the polarity of methanol is greater than that of ethyl acetate, and the use of methanol can effectively ensure that the proposed ethyl acetate-extracted components are dissolved to a greater extent;

3) In step D of the present invention, GF254 thin-layer plate is selected, and a specific fluorescent agent is added to the GF254 thin-layer plate. The fluorescent agent emits green fluorescence at a wavelength of 254 nm, and most of the compounds with aromatic rings or conjugated systems are in Under the wavelength of 254nm, there will be ultraviolet absorption, which will cover up the green fluorescence and form dark spots; while at the wavelength of 254nm, the isoflavone glucoside and ferulic acid can form more obvious dark spots. In addition, the GF254 thin-layer plate can also check the fluorescence at 366nm, so as to better distinguish hesperidin; in addition, drying the color developer can effectively prevent the color developer from being dried during heating. The problem of poor color rendering effect.

Hesperidin belongs to the dihydroflavonoid glycosides. Spraying 10% aluminum trichloride ethanol solution as a color developer will produce a chemical reaction, which can be excited by the ultraviolet light of 365nm wavelength. After the aluminum ion is complexed with the color developer, It can display obvious fluorescence at 365nm, that is, the identification method is based on rational principles and rigorous design, and can effectively improve the accuracy of simultaneous identification of three traditional Chinese medicine components: ferulic acid, vervescoflavonoid glucoside and hesperidin;

4) In the present invention, through a GF254 thin-layer plate and two inspection conditions, 3 kinds of medicinal flavors are identified, and the three kinds of traditional Chinese medicine components of ferulic acid, verisin isoflavone glucoside and hesperidin are simultaneously identified for the first time, effectively saving Solvent and labor costs, and the identification time is shortened by about 70%. Only a single sample preparation method can be used for the identification of other components containing these three components. At the same time, it also overcomes the interference of other complex components in traditional Chinese medicine, and ensures the identification of accuracy.

Description of drawings

Figure 1 is a thin-layer chromatography development diagram (1), wherein No. 1: Verisiflavone glucoside; No. 2: Hesperidin; No. 3: Ferulic acid; Nos. 4-6: Samples;

Figure 2 is a thin-layer chromatographic development diagram (2), wherein No. 1: Verisiflavone glucoside; No. 2: Hesperidin; No. 3: Ferulic acid; Nos. 4-6: Samples;

Figure 3 is a thin-layer chromatography development diagram (3), wherein No. 1: Verisiflavone glucoside; No. 2: Hesperidin; No. 3: Ferulic acid; Nos. 4-6: Samples;

Figure 4 is a thin-layer chromatography development diagram (4), wherein No. 1: Verisiflavone glucoside; No. 2: Hesperidin; No. 3: Ferulic acid; Nos. 4-6: Samples;

Figure 5 is a thin-layer chromatographic development diagram (5), wherein No. 1: Verbasidone glucoside; No. 2: Hesperidin; No. 3: Ferulic acid; Nos. 4-6: Samples;

Figure 6 is a thin-layer chromatographic development diagram (6), wherein No. 1: Verisiflavone glucoside; No. 2: Hesperidin; No. 3: Ferulic acid; Nos. 4-6: Samples;

Figure 7 is a thin-layer chromatographic development diagram (VII), wherein No. 1: Verisiflavone glucoside; No. 2: Hesperidin; No. 3: Ferulic acid; Nos. 4-6: Samples;

Figure 8 is a thin-layer chromatography development diagram (8), wherein No. 1: Verisiflavone glucoside; No. 2: Hesperidin; No. 3: Ferulic acid; Nos. 4-6: Samples;

Fig. 9 is a TLC development diagram (9), wherein No. 1: sample; No. 2: Angelica reference medicinal material; No. 3: Astragalus reference medicinal material; No. 4: Lycium barbarum reference medicinal material; No. 5: Jujube reference medicinal material; No. 1: tangerine peel control medicinal material;

Figure 10 is a TLC development diagram (ten), wherein No. 1: sample; No. 2: Angelica reference medicinal material; No. 3: Astragalus reference medicinal material; No. 4: Lycium barbarum reference medicinal material; No. 5: Jujube reference medicinal material; No. 1: tangerine peel control medicinal material;

Figure 11 shows the expanded TLC (254nm) of the sample size of the reference substance, in which the sample sizes of No. 1-15 are: 2 μL, 5 μL, 8 μL, 10 μL, 15 μL, 2 μL, 5 μL, 8 μL, 10 μL, 15 μL, respectively. , 2 μL, 5 μL, 8 μL, 10 μL, 15 μL; No. 1-5: Ferulic acid; No. 6-10: Verisiflavone glucoside; No. 11-15: Hesperidin;

Figure 12 is the expanded TLC (365nm) view of the spotting amount of the reference substance, wherein the spotting amounts of No. 1-15 are: 2 μL, 5 μL, 8 μL, 10 μL, 15 μL, 2 μL, 5 μL, 8 μL, 10 μL, 15 μL, respectively. , 2 μL, 5 μL, 8 μL, 10 μL, 15 μL; No. 1-5: Ferulic acid; No. 6-10: Verisiflavone glucoside; No. 11-15: Hesperidin;

Figure 13 is a thin-layer chromatogram (254nm) developed for the inspection of the amount of the sample to be tested, wherein the amount of the samples numbered 1-4 are: 5 μL, 10 μL, 15 μL, and 20 μL;

Figure 14 is a thin-layer chromatogram (365nm) developed for the inspection of the amount of the sample to be tested, wherein the amount of the samples numbered 1-4 are: 5 μL, 10 μL, 15 μL, and 20 μL;

Figure 15 shows the TLC development diagram of different manufacturers of TLC (Silida, 254nm), wherein No. 1: Ferulic acid; No. 2: Angelica reference material; No. 3: Sample; No. 4: Sample; No. 5: Verisiflavone glucoside; No. 6: Astragalus reference medicinal material; No. 7: Sample; No. 8: Sample; No. 9: Hesperidin; No. 10: Reference medicinal material of tangerine peel; No. 11: Sample; No. 12: Sample;

Figure 16 is the TLC development diagram (Silida, 365nm) of different manufacturers of TLC, wherein No. 1: Ferulic acid; No. 2: Angelica reference medicinal material; No. 3: Sample; No. 4: Sample; No. 5: Verisiflavone glucoside; No. 6: Astragalus reference medicinal material; No. 7: Sample; No. 8: Sample; No. 9: Hesperidin; No. 10: Reference medicinal material of tangerine peel; No. 11: Sample; No. 12: Sample;

Figure 17 is the TLC development diagram of different manufacturers of TLC (Qingdao Ocean, 254nm), wherein No. 1: Ferulic acid; No. 2: Angelica reference medicinal material; No. 3: Sample; No. 4: Sample; No. 5 No. 6: Astragalus reference medicinal material; No. 7: Sample; No. 8: Sample; No. 9: Hesperidin; No. 10: Reference medicinal material of tangerine peel; No. 11: Sample; No. 12: Sample;

Figure 18 is the TLC development diagram of different manufacturers' TLC (Qingdao Ocean, 365nm), wherein No. 1: Ferulic acid; No. 2: Angelica reference medicinal material; No. 3: Sample; No. 4: Sample; No. 5 No. 6: Astragalus reference medicinal material; No. 7: Sample; No. 8: Sample; No. 9: Hesperidin; No. 10: Reference medicinal material of tangerine peel; No. 11: Sample; No. 12: Sample;

Figure 19 is the TLC development diagram of different manufacturers' TLC (Qingdao Yumin, 254nm), wherein No. 1: Ferulic acid; No. 2: Angelica reference medicinal material; No. 3: Sample; No. 4: Sample; No. 5: Verisiflavone glucoside; No. 6: Astragalus reference medicinal material; No. 7: Sample; No. 8: Sample; No. 9: Hesperidin; No. 10: Reference medicinal material of tangerine peel; No. 11: Sample; No. 12: Sample;

Figure 20 is the TLC development diagram of different manufacturers' TLC (Qingdao Yumin, 365nm), wherein No. 1: Ferulic acid; No. 2: Angelica reference medicinal material; No. 3: Sample; No. 4: Sample; No. 5: Verisiflavone glucoside; No. 6: Astragalus reference medicinal material; No. 7: Sample; No. 8: Sample; No. 9: Hesperidin; No. 10: Reference medicinal material of tangerine peel; No. 11: Sample; No. 12: Sample;

Figure 21 is a thin layer chromatogram (254nm) developed in the environment at 25°C, wherein No. 1: Ferulic acid; No. 2: Angelica reference material; No. 3: Sample; No. 4: Sample; No. 5: Verisiflavone glucose No. 6: Astragalus reference medicinal material; No. 7: Sample; No. 8: Sample; No. 9: Hesperidin; No. 10: Reference medicinal material of tangerine peel; No. 11: Sample; No. 12: Sample;

Figure 22 is a thin layer chromatogram (365nm) developed in the environment at 25°C, wherein No. 1: Ferulic acid; No. 2: Angelica reference material; No. 3: Sample; No. 4: Sample; No. 5: Verisiflavone glucose No. 6: Astragalus reference medicinal material; No. 7: Sample; No. 8: Sample; No. 9: Hesperidin; No. 10: Reference medicinal material of tangerine peel; No. 11: Sample; No. 12: Sample;

Figure 23 is a thin layer chromatogram (254nm) developed in the environment at 8°C, wherein No. 1: Ferulic acid; No. 2: Angelica reference material; No. 3: Sample; No. 4: Sample; No. 5: Verisiflavone glucose No. 6: Astragalus reference medicinal material; No. 7: Sample; No. 8: Sample; No. 9: Hesperidin; No. 10: Reference medicinal material of tangerine peel; No. 11: Sample; No. 12: Sample;

Figure 24 is a thin layer chromatogram (365nm) developed in the environment at 8°C, wherein No. 1: Ferulic acid; No. 2: Angelica reference material; No. 3: Sample; No. 4: Sample; No. 5: Verisiflavone glucose No. 6: Astragalus reference medicinal material; No. 7: Sample; No. 8: Sample; No. 9: Hesperidin; No. 10: Reference medicinal material of tangerine peel; No. 11: Sample; No. 12: Sample;

Figure 25 is the thin layer chromatogram (254nm) developed in the environment under the humidity of 32%, wherein, No. 1: Ferulic acid; No. 2: Angelica reference material; No. 3: Sample; No. 4: Sample; No. 5: Mullein isoflavones Glucoside; No. 6: Astragalus reference material; No. 7: Sample; No. 8: Sample; No. 9: Hesperidin; No. 10: Chenpi control material; No. 11: Sample; No. 12: Sample;

Figure 26 is the thin layer chromatogram (365nm) developed in the environment under the humidity of 32%, wherein, No. 1: ferulic acid; No. 2: Angelica reference medicinal material; No. 3: sample; No. 4: sample; No. 5: mullein isoflavones Glucoside; No. 6: Astragalus reference material; No. 7: Sample; No. 8: Sample; No. 9: Hesperidin; No. 10: Chenpi control material; No. 11: Sample; No. 12: Sample;

Figure 27 is a thin layer chromatogram (254nm) developed in an environment with a humidity of 75%, wherein No. 1: Ferulic acid; No. 2: Angelica reference material; No. 3: Sample; No. 4: Sample; No. 5: Verisin isoflavones Glucoside; No. 6: Astragalus reference material; No. 7: Sample; No. 8: Sample; No. 9: Hesperidin; No. 10: Chenpi control material; No. 11: Sample; No. 12: Sample;

Figure 28 is a thin layer chromatogram (365nm) developed in an environment with a humidity of 75%, wherein No. 1: ferulic acid; No. 2: Angelica reference medicinal material; No. 3: sample; No. 4: sample; No. 5: mullein isoflavones Glucoside; No. 6: Astragalus reference material; No. 7: Sample; No. 8: Sample; No. 9: Hesperidin; No. 10: Chenpi control material; No. 11: Sample; No. 12: Sample;

Figure 29 is a verification thin-layer chromatogram (254nm), wherein No. 1: Ferulic acid; No. 2: Angelica reference medicinal material; No. 3: Sample; No. 4: Sample; No. 5: Verisiflavone glucoside; No. 6: Astragalus Reference medicinal material; No. 7: sample; No. 8: sample; No. 9: hesperidin; No. 10: reference medicinal material of tangerine peel; No. 11: sample; No. 12: sample;

Figure 30 is a verification thin layer chromatogram (365nm), wherein No. 1: Ferulic acid; No. 2: Angelica reference medicinal material; No. 3: Sample; No. 4: Sample; No. 5: Verisiflavone glucoside; No. 6: Astragalus Reference medicinal material; No. 7: sample; No. 8: sample; No. 9: hesperidin; No. 10: reference medicinal material of tangerine peel; No. 11: sample; No. 12: sample.

Detailed ways

By clearly and completely describing the technical solutions in the embodiments of the present invention below, it is obvious that the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

In the following embodiments, the instruments involved include:

Semi-automatic thin layer spotter (CAMAG Lionmat-5), thin layer imaging system (CAMAG TLC Visμalizer), ultrapure water machine (cell type 1810A, Shanghai Moller Scientific Instrument Co., Ltd.), ultrasonic cleaner (KQ5200DB type, 600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.), silicone G thin-layer board (Qingdao Ocean Chemical Factory Branch, Tianjin Silida Technology Co., Ltd., Qingdao Yumin Chemical Factory;

In the following examples, the reagents involved include: methanol, ethyl acetate, chloroform, acetone, etc., all of which are of analytical grade;

In the following examples, the reference substances involved include: ferulic acid (China Institute for Food and Drug Control, batch number: 110773-201614); hesperidin (China Institute for Food and Drug Control, batch number: 110721-201818); Flavonoid glucoside (China Institute for Food and Drug Control, batch number: 111920-201608);

In the following examples, the reference medicinal materials involved include: Angelica sinensis reference medicinal material (China Institute for Food and Drug Control, batch number: 120927-201617); Astragalus (Mongolia) reference medicinal material (China Food and Drug Control Institute, batch number: 120974-201612 ); tangerine peel reference medicinal material (China Institute for Food and Drug Control, batch number: 120969-201510); the traditional Chinese medicine compound preparation involved is Guiqi Qizhi ointment, which contains angelica, astragalus, jujube, tangerine peel and wolfberry.

Example 1

A thin-layer identification method for simultaneously identifying ferulic acid, verbisoflavone glucoside and hesperidin, comprising the following steps:

A. Preparation of the test solution: take 3g of the traditional Chinese medicine compound preparation, add 20mL of ethyl acetate, heat under reflux for 1h, filter, and evaporate the filtrate to dryness; add 1mL of methanol to the filter residue and dissolve to obtain the test solution;

Wherein, evaporating to dryness specifically refers to making the solvent in the filtrate zero;

Among them, the heating and refluxing adopts a conventional electronic constant temperature water bath (for example: the model is DK-98-11, Beijing Zhongxing Weiye Instrument Co., Ltd.);

Wherein, in parts by weight, the traditional Chinese medicine compound preparation includes the following components: 25-35 parts of Astragalus or its extract, 5-7 parts of Angelica or its extract, 8-12 parts of jujube or its extract, Lycium barbarum or its extract 5 to 10 parts of the extract, 5 to 8 parts of dried tangerine peel or its extract, and the traditional Chinese medicine composition at least includes the following active ingredients: mullein isoflavone glucoside ≥ 0.10 mg/g, astragaloside ≥ 0.45 mg/g, ferulic acid ≥ 0.05mg/g, Lycium barbarum polysaccharide ≥8.0mg/g, Hesperidin ≥5.0mg/g;

B. Preparation of reference solution: respectively take ferulic acid reference substance, verbisoflavone glucoside reference substance and hesperidin reference substance, add methanol, and prepare 1mg/L ferulic acid reference substance solution and 1mg/L mullein reference substance respectively. Flavonoid glucoside solution and 1mg/L hesperidin reference solution, for later use;

C. Preparation of reference medicinal material solution: respectively take 0.5 g of Angelica reference medicinal material, 1 g of Astragalus reference medicinal material and 1 g of tangerine peel reference medicinal material, add 28-32 mL of water, boil for 30 minutes, place at room temperature, filter, and evaporate the filtrate to dryness until the moisture is zero; Add 1 mL of methanol and dissolve to obtain three reference medicinal material solutions;

D. Detection: Using thin-layer chromatography, take 10 μL of the test solution, 5 μL of the ferulic acid reference solution, 5 μL of the isoflavone glucoside reference solution, 10 μL of the hesperidin reference solution, and 10 μL of each of the three reference medicinal material solutions. , respectively point on the same GF254 thin layer plate, develop with the developing agent chloroform-methanol-acetone = 15:3.5:1.5, take out, dry until the developing agent volatilizes, check the fluorescent spots at 254nm, and then spray to develop color Add 10% aluminum trichloride ethanol solution, dry the color developer, heat it at 105 °C until the spots on the plate are clearly colored, and inspect it under a UV lamp (365 nm);

D. Analysis of results: In the chromatogram of the test product at 254nm, the same spots are displayed in the corresponding position of the ferulic acid reference solution and the reference solution of Verisiflavone glucoside; in the chromatogram of the test solution at 365nm, the The hesperidin reference solution showed fluorescent spots of the same color on the corresponding position of the chromatogram.

Example 2

In this embodiment, the effects of different deployment systems on the results of the technical solution are studied to further illustrate the technical solution.

Take 3 g of Guiqi Qilu ointment, prepare the test solution according to the proposed method, and prepare the reference solution according to the proposed method;

Expand system one, chloroform: methanol: acetone = 15: 3.5: 1.5;

Expand system two, dichloromethane: methanol: acetone = 15: 3.5: 1.5;

Take 5 μL of each of the test solution and reference solution prepared according to the proposed method, point them on the same GF254 plate, and deploy them in the expansion system 1 and 2 respectively, take them out to dry, and inspect according to the proposed inspection method. Better results.

Example 3

In this embodiment, the influence of specificity on the results of the technical solution is studied to further illustrate the technical solution.

Since Guiqi Qizhi ointment is a compound preparation, its composition is relatively complex, and several medicinal materials may coexist with the same component, so the specificity investigation interferes greatly, and the negative control is not considered. Astragalus, jujube, dried tangerine peel, wolfberry), add 30 mL of water, heat and cook for 30 minutes, let it cool and filter, evaporate the filtrate to dryness, and add 20 mL of ethyl acetate to prepare a reference medicinal material solution in the same way.

Take the reference medicinal material solution, the reference substance solution and the test solution solution, and point them on the same thin-layer plate in the same way to compare the source of the ingredients. The results showed that the test solution chromatogram and the reference medicinal material chromatogram showed the same color fluorescent main spots at the corresponding positions, and it was concluded that the method could identify three active ingredients (as shown in Figure 9-10).

Example 4

In this example, the influence of the spotting amount of ferulic acid reference substance solution, hesperidin reference substance solution, verbisoflavone glucoside reference substance solution and test solution solution on the results of this technical solution is studied to further illustrate this technical solution .

Take 2 μL, 5 μL, 8 μL, 10 μL and 15 μL of each of the 3 reference solutions and place them on the same GF254 plate respectively, and inspect according to the proposed inspection method. The results are shown in Figure 11-12; , 15μL and 20μL, respectively, were placed on the same GF254 plate and inspected according to the proposed inspection method. The results are shown in Figure 13-14;

The results showed that: the ferulic acid reference solution and the Verisiflavone glucoside reference solution had clear fluorescent spots in 2-15 μL, the hesperidin reference solution had clear fluorescent spots in 5-15 μL, and the spotting amount of the test solution was 5-20 μL. The fluorescent spots are clear; therefore, based on the sample cost, experimental conditions and the clarity of the fluorescent spots, etc., it is preferable to use 5 μL of the ferulic acid reference solution, 5 μL of the mullein glucoside reference solution, and 5 μL of hesperidin. The sample volume of the reference solution is 10μL; the sample volume of the test sample is 10μL.

Example 5

In this embodiment, the influence of the thin-layer boards of different manufacturers on the results of the technical solution is studied to further illustrate the technical solution.

Silica gel GF ₂₅₄ thin-layer boards from three different manufacturers (Tianjin Silida Technology Co., Ltd., Qingdao Ocean Chemical Factory Branch and Qingdao Yumin Chemical Factory Branch respectively) were selected, and the amount of ferulic acid reference solution was sampled respectively. 5 μL, 5 μL of Verisiflavone glucoside reference solution, 10 μL of hesperidin reference solution, 10 μL of test solution, spot on the same silica gel GF ₂₅₄ thin-layer plate, and Expand according to the selected test method, take out, dry, take out, dry, inspect at 254 nm, then spray 10% aluminum trichloride ethanol solution, heat at 105 ° C until the spot color is clear, place sunlight and ultraviolet light (365nm). To test the effect of unfolding different thin-layer boards, the unfolding result of the cuttable board of Tianjin Silida Technology Co., Ltd. is used, as shown in Figure 15-16; As shown in Figure 19-20, the glass plate expansion result of Qingdao Yumin Chemical Plant Branch is used.

The results show that by analyzing and comparing the thin-layer chromatograms developed by different thin-layer plates, under the selected thin-layer chromatographic conditions, different thin-layer plates can achieve the purpose of identification.

Example 6

In this embodiment, the effects of different temperature and humidity on the results of the technical solution are studied to further illustrate the technical solution. Combined with the requirements of conventional standards, choose to place the thin-layer board at 8°C and 25°C under the condition of relative humidity of 60% for comparison; and choose to place the thin-layer board at 25°C The relative humidity of 32% and 75% were developed and compared.

Under the condition of relative humidity of 60%, the temperature of the thin-layer development environment was changed respectively, and the sampled thin-layer plate was taken and placed in the environment of 8 °C and 25 °C for development; then taken out, dried, and inspected at 254 nm. Spray 10% aluminum trichloride ethanol solution, heat at 105 °C until the spot color is clear, and check under sunlight and ultraviolet light (365 nm). The results of the thin-layer chromatogram developed at a temperature of 25°C are shown in Figures 21-22; the results of the thin-layer chromatogram developed at a temperature of 8°C are shown in Figures 23-24.

Under the condition of temperature of 25°C, change the relative humidity of the thin-layer development environment respectively, take the thin-layer plate that has been spotted and place it in the environment of relative humidity of 32% and 75%, take it out, dry it, and inspect it at 254nm. Spray with 10% aluminum trichloride ethanol solution, heat at 105°C until the spot color is clear, and check under sunlight and ultraviolet light (365nm). Figure 25-26 shows the expansion results under a relative humidity of 32%, and Figure 27-28 shows the expansion results under a relative humidity of 75%.

The results show that under different temperature and humidity and selected TLC conditions, different temperature and humidity can achieve the purpose of identification.

Example 7

In this example, a sample of the compound preparation of traditional Chinese medicine-Guiqi Qizhi ointment is verified to further illustrate the technical solution.

According to the proposed method, the sample was verified, and the results showed that: under the condition of 254nm, in the chromatogram of the test substance, the same spot was displayed on the chromatogram corresponding to the ferulic acid reference substance and the Vervis isoflavone glucoside reference substance; at 365nm Under the conditions, in the chromatogram of the test product, the fluorescent spots of the same color are displayed on the corresponding position of the chromatogram of the reference substance of hesperidin.

Claims (9)

1. A thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin is characterized by comprising the following steps:

A. preparing a test solution: adding ethyl acetate into the Chinese medicinal compound preparation, heating and refluxing, filtering, and evaporating filtrate; adding methanol into the filter residue, and dissolving to obtain a test solution;

B. preparation of control solutions: respectively taking ferulic acid reference substance, calycosin glucoside reference substance and hesperidin reference substance, adding methanol, and respectively preparing ferulic acid reference substance solution, calycosin glucoside reference substance solution and hesperidin reference substance solution for use;

C. preparing a reference medicinal material solution: respectively taking radix Angelicae sinensis control medicinal material, radix astragali control medicinal material and pericarpium Citri Tangerinae control medicinal material, boiling in water, standing to normal temperature, filtering, and evaporating to dryness; adding methanol into the residue, and dissolving to obtain three reference medicinal solutions;

D. and (3) detection: respectively taking 5-20 muL of a test solution, 2-15 muL of a ferulic acid reference solution, 2-15 muL of a calycosin glucoside reference solution, 5-15 muL of an hesperidin reference solution and 10 muL of each of three reference medicinal material solutions by adopting a thin layer chromatography, respectively dropping the solutions on the same GF254 thin layer plate, spreading the solutions by using a developing agent, taking out the solutions, airing the solutions until the developing agent is volatilized, inspecting fluorescent spots at 254nm, spraying a 10% aluminum trichloride ethanol solution of a color developing agent, airing the color developing agent, heating the solutions at 105 ℃ until the spots on the plate are clearly developed, and inspecting the fluorescent spots at 365 nm;

D. and (4) analyzing results: in the chromatogram of the test sample under 254nm, the same spots are displayed on the corresponding positions of the ferulic acid reference solution and the calycosin glucoside reference solution in the chromatogram; and in the chromatogram of the test solution under 365nm, fluorescent spots with the same color are displayed at the corresponding positions of the chromatogram with the hesperidin reference solution.

2. The thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin as claimed in claim 1, wherein said Chinese medicinal compound preparation comprises radix astragali, radix Angelicae sinensis, fructus Jujubae, fructus Lycii, pericarpium Citri Tangerinae or their extracts.

3. The thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin according to claim 1, wherein the compound traditional Chinese medicine preparation comprises the following components in parts by weight: 25-35 parts of astragalus membranaceus or extract thereof, 5-7 parts of angelica sinensis or extract thereof, 8-12 parts of Chinese date or extract thereof, 5-10 parts of medlar or extract thereof, and 5-8 parts of pericarpium citri reticulatae or extract thereof.

4. The thin-layer chromatography identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin as claimed in claim 1, 2 or 3, wherein said Chinese medicinal composition comprises at least the following effective ingredients: calycosin glucoside is greater than or equal to 0.10 mg/g, astragaloside is greater than or equal to 0.45mg/g, ferulic acid is greater than or equal to 0.05mg/g, lycium barbarum polysaccharide is greater than or equal to 8.0mg/g, and hesperidin is greater than or equal to 5.0 mg/g.

5. The thin-layer identification method for identifying ferulic acid, calycosin glucoside and hesperidin simultaneously as claimed in claim 1, wherein in step A, 3g of the compound Chinese medicinal preparation is taken, 20mL of ethyl acetate is added, heating and refluxing are carried out for 1h, filtering is carried out, and the filtrate is evaporated to dryness; and adding 1mL of methanol into the filter residue, and dissolving to obtain a test solution.

6. The thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin as claimed in claim 1, wherein the concentrations of ferulic acid control solution, calycosin glucoside control solution and hesperidin control solution are all 1 mg/L.

7. The thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin as claimed in claim 1, wherein in step C, 0.5g of radix Angelicae sinensis control drug, 1g of radix astragali control drug and 1g of pericarpium Citri Tangerinae control drug are respectively taken, added with 28-32mL of water, boiled for 30min, placed to normal temperature, filtered and evaporated to dryness; adding 1mL of methanol into the filter residue, and dissolving to obtain three reference medicinal material solutions.

8. The thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin as claimed in claim 1, wherein in step D, 10 μ L of test solution, 5 μ L of ferulic acid control solution, 5 μ L of calycosin glucoside control solution and 10 μ L of hesperidin control solution are respectively spotted on the same GF254 thin-layer plate.

9. The thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin as claimed in claim 1, wherein said developing solvent is chloroform-methanol-acetone = 15: 3.5: 1.5.

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