Characteristic identification method of Fritillaria cirrhosa in children's Qingfei granules

Abstract

This invention relates to a characteristic identification method for Fritillaria cirrhosa in Xiaor Qingfei granules. The Xiaor Qingfei granules are dissolved in water, then loaded onto the sample using a wet or dry method, and separated and enriched by normal-phase silica gel column chromatography. Gradient column chromatography is then performed sequentially using three mixtures of different polarities: dichloromethane:methanol (5:1), dichloromethane:methanol (2:1), and dichloromethane:methanol (1:1) as mobile phases. The fractions are collected in elution order and combined and concentrated to form fractions A and B. Thin-layer chromatography (TLC) analysis is performed on fractions A, B, and the Fritillaria cirrhosa extract for comparison. The developed TLC plate is then developed with potassium bismuth iodide as a colorimetric reagent; fractions A and B both show colorimetric spots corresponding to the Fritillaria cirrhosa extract. This method uses the identifying components in Fritillaria cirrhosa as a reference standard and combines column chromatography with a modified TLC method to characteristically identify Fritillaria cirrhosa in Xiaor Qingfei granules.

Description

Technical Field

[0001] The invention relates to an extraction and analysis method for identification components of Chinese patent medicines in the pharmaceutical industry, and in particular to a characteristic identification method for Fritillaria cirrhosa in Xiaoer Qingfei granules. Background Art

[0002] Chuanbei is the dried bulb of Fritilaria cirrhosa D.Don, Fritilariaunibracteata Hsiao et KCHsia, Fritilaria przewalskii Maxim., Fritilaria delavayi Franch., Fritilaria taipaiensis PY Li or Fritilaria unibracteata Hsiao et KC Hsiavar. wabuensis (SY Tanget SCYue) ZD Liu, S. Wang et SC Chen. It is commonly known as "pine shell", "green shell", "burnt shell" and "cultivated product" according to its characteristics. It is dug in summer and autumn or after the snow melts, and the fibrous roots, rough bark and mud and sand are removed, and it is dried in the sun or at low temperature. Fritillaria cirrhosa has been included in various editions of the "Pharmacopoeia of the People's Republic of China" from 1963 to the present. Its effects are clearing away heat and moistening the lungs, resolving phlegm and relieving cough, dispersing nodules and eliminating carbuncles. It is used for lung heat and dry cough, dry cough with little sputum, yin deficiency and fatigue cough, blood in sputum, scrofula, mastitis, and lung abscess. It is a dual-purpose medicinal material for food and medicine.

[0003] Xiaoer Qingfei Granule is a new drug exclusively produced by Hebei Julong Pharmaceutical Co., Ltd. in China. It is an empirical prescription created by hospital preparations after clinical practice. It is a children's medicine made with Fritillaria cirrhosa as the main drug, supplemented with Poria cocos, Pinellia ternata, and Stemona tuber. It has a definite therapeutic effect and low toxicity and side effects. It is mainly used clinically for symptoms such as cough, shortness of breath, and phlegm congestion caused by lower respiratory tract infections in children. In view of the fact that the qualitative identification method of Fritillaria cirrhosa in the original national drug standard (YBZ06062004-2007Z) has problems such as low sensitivity of detection components and high detection limit, in order to effectively control the quality of Xiaoer Qingfei Granule and ensure clinical effectiveness and safety, this technology is specially invented to perform characteristic identification of Fritillaria cirrhosa in Xiaoer Qingfei Granule. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a characteristic identification method of Fritillaria cirrhosa in Xiaoer Qingfei Granules which is suitable for drug quality control and has high sensitivity.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A characteristic identification method of Fritillaria cirrhosa in Xiaoer Qingfei granules.

[0007] 1) Dissolve the Xiaoer Qingfei Granules in water, then apply the sample by wet or dry method, and separate and enrich by normal phase silica gel column chromatography;

[0008] 2) Then, gradient column chromatography was performed using three mixed solutions of different polarities, namely, dichloromethane:methanol (5:1), dichloromethane:methanol (2:1), and dichloromethane:methanol (1:1), as mobile phases;

[0009] 3) Collect the fractions in the order of elution, and combine and concentrate the corresponding fractions into two parts, A and B;

[0010] 4) Perform thin layer chromatography analysis and comparison on Part A, Part B and Fritillaria cirrhosa extract, using dichloromethane:methanol:ammonia water (8:1:0.1) as the developing solvent;

[0011] 5) The developed thin layer chromatography plate was developed with potassium bismuth iodide as a color developer, and both part A and part B showed color spots corresponding to the extract of Fritillaria cirrhosa.

[0012] A further improvement of the technical solution of the present invention is that the required silica gel mesh number for normal phase silica gel column chromatography in step 1 is 300-400 meshes.

[0013] A further improvement of the technical solution of the present invention is: in step 2, after first using 2 to 3 times the column volume of the mobile phase of dichloromethane:methanol (5:1), use a mobile phase of dichloromethane:methanol (2:1); after using 3 to 4 times the column volume of the mobile phase of dichloromethane:methanol (2:1), use a mobile phase of dichloromethane:methanol (1:1) of 1 times the column volume.

[0014] A further improvement of the technical solution of the present invention is: part or all of the effluent equivalent to 2.5 to 4.0 times the column volume in step 3 is combined into part A; part or all of the effluent equivalent to 4.5 to 6.0 times the column volume is combined into part B.

[0015] A further improvement of the technical solution of the present invention is as follows: before thin layer chromatography analysis in step 4, part B is concentrated into a thick paste and then extracted by shaking with 12.5% ​​ammonia water and dichloromethane, and the concentrated liquid of the dichloromethane layer is taken for thin layer spotting.

[0016] A further improvement of the technical solution of the present invention is as follows: the preparation method of the Fritillaria cirrhosa medicinal material extract in step 4 is to extract the crushed Fritillaria cirrhosa medicinal material with 95% ethanol and 50% ethanol in turn by reflux, combine the extracts, concentrate and evaporate to dryness, knead the dry paste with anhydrous ethanol, and use the kneaded solution for thin layer spotting.

[0017] A further improvement of the technical solution of the present invention is: reflux extraction is performed twice, each time for 2 hours.

[0018] Due to the adoption of the above technical solution, the technical effects achieved by the present invention are as follows:

[0019] The characteristic identification method of Fritillaria cirrhosa in Xiaoer Qingfei granules of the present invention can obtain the characteristic components of two parts A and B in Xiaoer Qingfei granules through extraction and only one conventional column chromatography enrichment process, and at the same time, a proper amount of ammonia water is added to the thin layer chromatography developing agent to adjust the acidity and alkalinity to avoid tailing after the sample is developed. This identification method can avoid the use of a high performance liquid chromatography instrument with an evaporative light detector and greatly reduce the cost.

[0020] The identification method of the present invention is easy to operate, takes a short time, has significant technical advantages, is suitable for on-the-spot process detection and quality control of final products during product production, and effectively meets the needs of production and development of medicines, health products and foods using Fritillaria cirrhosa as main raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a flow chart of the method of the present invention;

[0022] Figure 2 is the thin layer chromatogram of Example 1;

[0023] Figure 3 It is the thin layer chromatogram of embodiment 2;

[0024] Figure 4 It is the thin layer chromatogram of Example 3 of the present invention. DETAILED DESCRIPTION

[0025] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0026] The present invention is a characteristic identification method of Fritillaria cirrhosa in Xiaoer Qingfei Granules, which is used for characteristic identification of Fritillaria cirrhosa in Xiaoer Qingfei Granules. Xiaoer Qingfei Granules is a new drug produced by Hebei Julong Pharmaceutical Co., Ltd.

[0027] like Figure 1 As shown in the figure, the characteristic identification method of Fritillaria cirrhosa in Xiaoer Qingfei Granules, the specific steps are as follows:

[0028] 1) Dissolve the Xiaoer Qingfei granules in a small amount of water, then apply the sample by wet or dry method, and separate and enrich by normal phase silica gel column chromatography. Preferably, the required silica gel mesh number for normal phase silica gel column chromatography is 300-400 mesh.

[0029] 2) Then, gradient column chromatography is performed using three different polarity mixtures of dichloromethane:methanol (5:1), dichloromethane:methanol (2:1), and dichloromethane:methanol (1:1) as mobile phases. Specifically, preferably, 2 to 3 column volumes of mobile phase dichloromethane:methanol (5:1) are used first, and then 3 to 4 column volumes of mobile phase dichloromethane:methanol (2:1) are used; after 3 to 4 column volumes of mobile phase dichloromethane:methanol (2:1) are used, 1 column volume of mobile phase dichloromethane:methanol (1:1) is used.

[0030] 3) Collect the fractions in the order of effluent, and combine and concentrate the corresponding fractions into two parts, A and B. Specifically, part or all of the effluent equivalent to 2.5 to 4.0 times the column volume is combined into part A; part or all of the effluent equivalent to 4.5 to 6.0 times the column volume is combined into part B.

[0031] 4) Perform thin layer chromatography analysis and comparison on Part A, Part B, and the extract of Fritillaria cirrhosa, with the developing solvent being dichloromethane:methanol:ammonia water (8:1:0.1). Preferably, before performing thin layer chromatography analysis, concentrate Part B into a thick paste and then extract it by shaking with 12.5% ​​ammonia water and dichloromethane, and take the concentrated solution of the dichloromethane layer for thin layer spotting.

[0032] The preparation method of the extract of Fritillaria cirrhosa is to extract the crushed Fritillaria cirrhosa with 95% ethanol and 50% ethanol in turn, usually twice with 95% ethanol and 50% ethanol, each time for 2 hours. Then, the extracts are combined, concentrated and evaporated to dryness, and the dry paste is kneaded with anhydrous ethanol, and the kneaded solution is used for thin layer spotting.

[0033] 5) The developed thin layer chromatography plate was developed with potassium bismuth iodide as a color developer, and both part A and part B showed color spots corresponding to the extract of Fritillaria cirrhosa.

[0034] When this method is used, an appropriate amount of ammonia water is added to the thin layer chromatography developer to adjust the acidity and alkalinity to avoid tailing of the sample after development.

[0035] This method uses the identified components in Fritillaria cirrhosa as the reference standard, and can intuitively and quickly know the identification results. This method uses a combination of column chromatography and improved thin layer chromatography (TLC) to enrich the content of the corresponding components in Xiaoer Qingfei Granules to perform characteristic identification of Fritillaria cirrhosa in Xiaoer Qingfei Granules; it is convenient and fast, and the results are accurate.

[0036] The following are specific embodiments

[0037] Example 1

[0038] Take 100g of Xiaoer Qingfei Granules, grind it into powder, add a small amount of water to dissolve it, and mix it with 60-100 mesh coarse silica gel. Then load 300-400 mesh column chromatography silica gel into a 7cm diameter chromatographic column, vacuum it and tighten it to a height of 26cm. The column volume is about 1000.09 cm 3 , then add sample-mixing silica gel and prepare for elution.

[0039] Specific elution steps: 100 mL is a cup of fractions. First, dichloromethane: methanol (5:1) is used as the mobile phase for elution and separation. After the 21st cup of fractions, dichloromethane: methanol (2:1) is used for elution. After the 54th cup of fractions, dichloromethane: methanol (1:1) is used for elution, and this is up to the 64th cup of fractions. Combine the 26th to 40th cups of fractions and concentrate them to a small volume for thin layer spotting, which is part A. Combine the 46th to 60th cups of fractions and concentrate them to a thick paste. Then shake and extract the thick paste with 12.5% ​​ammonia water and dichloromethane. Take the concentrated liquid of the dichloromethane layer for thin layer spotting, which is part B.

[0040] Preparation of Fritillaria cirrhosa extract: The crushed Fritillaria cirrhosa extract was extracted with 95% ethanol and 50% ethanol in turn, twice for 2 hours each time. All the extracts were combined, concentrated and evaporated to dryness. The dry paste was squeezed with anhydrous ethanol and the squeezed solution was used for thin layer spotting.

[0041] Silica gel thin layer chromatography identification: Part A, Part B, and Sichuan Fritillaria extract were subjected to thin layer chromatography analysis and comparison, and the developing solvent was dichloromethane: methanol: ammonia water (8:1:0.1); the developed thin layer chromatography plate was developed with potassium bismuth iodide as a color developer. In the chromatograms of Part A and Part B, spots of the same color were shown at the corresponding positions of the chromatograms of Sichuan Fritillaria, as shown in the attached figure. Figure 2 shown.

[0042] The chromatogram shows that the sample contains Fritillaria cirrhosa that meets the requirements.

[0043] Example 2

[0044] Take 100g of Xiaoer Qingfei Granules, grind it into powder, add a small amount of water to dissolve it, and mix it with 60-100 mesh coarse silica gel. Then load 300-400 mesh column chromatography silica gel into a 7cm diameter chromatographic column, vacuum it and tighten it to a height of 26cm. The column volume is about 1000.09 cm 3 , then add sample-mixing silica gel and prepare for elution.

[0045] Specific elution steps: 100 mL is a cup of fractions. First, dichloromethane: methanol (5:1) is used as the mobile phase for elution and separation. After the 22nd cup of fractions, dichloromethane: methanol (2:1) is used for elution. After the 53rd cup of fractions, dichloromethane: methanol (1:1) is used for elution, and the 63rd cup of fractions is used. Combine the 30th to 32nd cup of fractions and concentrate them to a small volume for thin layer spotting, which is part A. Combine the 48th to 52nd cup of fractions and concentrate them to a thick paste. Then shake and extract the thick paste with 12.5% ​​ammonia water and dichloromethane. Take the concentrated liquid of the dichloromethane layer for thin layer spotting, which is part B.

[0046] Preparation of Fritillaria cirrhosa extract: The crushed Fritillaria cirrhosa extract was extracted with 95% ethanol and 50% ethanol in turn, twice for 2 hours each time. All the extracts were combined, concentrated and evaporated to dryness. The dry paste was squeezed with anhydrous ethanol and the squeezed solution was used for thin layer spotting.

[0047] Silica gel thin layer chromatography identification: Part A, Part B, and Sichuan Fritillaria extract were subjected to thin layer chromatography analysis and comparison, and the developing solvent was dichloromethane: methanol: ammonia water (8:1:0.1); the developed thin layer chromatography plate was developed with potassium bismuth iodide as a color developer. In the chromatograms of Part A and Part B, spots of the same color were shown at the corresponding positions of the chromatograms of Sichuan Fritillaria, as shown in the attached figure. Figure 3 shown.

[0048] The chromatogram shows that the sample contains Fritillaria cirrhosa that meets the requirements.

[0049] Example 3

[0050] Take 300g of Xiaoer Qingfei Granules, grind them into powder, add a small amount of water to dissolve, and mix with 60-100 mesh coarse silica gel. Then put 300-400 mesh column chromatography silica gel into a 3cm diameter chromatographic column, vacuum tighten it to a height of 35cm, and the column volume is about 247.275 cm 3 , then add sample-mixing silica gel and prepare for elution.

[0051] Specific elution steps: 50 mL is one cup of fraction, first use dichloromethane: methanol (5:1) as the mobile phase for elution and separation, after the 14th cup of fraction, use dichloromethane: methanol (2:1) for elution, after the 32nd cup of fraction, use dichloromethane: methanol (1:1) for elution, and so on until the 37th cup of fraction. Combine the 14th to 19th cups of fractions and concentrate them to a small volume for thin layer spotting, which is part A. Combine the 26th to 29th cups of fractions and concentrate them to a thick paste, then shake and extract the thick paste with 12.5% ​​ammonia water and dichloromethane, and take the concentrated liquid of the dichloromethane layer for thin layer spotting, which is part B.

[0052] Preparation of Fritillaria cirrhosa extract: The crushed Fritillaria cirrhosa extract was extracted with 95% ethanol and 50% ethanol in turn, twice for 2 hours each time. All the extracts were combined, concentrated and evaporated to dryness. The dry paste was squeezed with anhydrous ethanol and the squeezed solution was used for thin layer spotting.

[0053] Silica gel thin layer chromatography identification: Part A, Part B, and Sichuan Fritillaria extract were subjected to thin layer chromatography analysis and comparison, and the developing solvent was dichloromethane: methanol: ammonia water (8:1:0.1); the developed thin layer chromatography plate was developed with potassium bismuth iodide as a color developer. In the chromatograms of Part A and Part B, spots of the same color were shown at the corresponding positions of the chromatograms of Sichuan Fritillaria, as shown in the attached figure. Figure 4 shown.

[0054] The chromatogram shows that the sample contains Fritillaria cirrhosa that meets the requirements.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A characteristic identification method of Fritillaria cirrhosa in Xiaoer Qingfei granules, comprising the following steps: 1) Dissolve the Xiaoer Qingfei Granules in water, then apply the sample by wet or dry method, and separate and enrich by normal phase silica gel column chromatography; 2) Then, gradient column chromatography was performed using three mixed solutions of different polarities as mobile phases, with a dichloromethane:methanol ratio of 5:1, a dichloromethane:methanol ratio of 2:1, and a dichloromethane:methanol ratio of 1:1; 3) Collect the fractions in the order of elution, and combine and concentrate the corresponding fractions into two parts, A and B; 4) Perform thin layer chromatography analysis and comparison on Part A, Part B and Fritillaria cirrhosa extract, using a developing solvent of dichloromethane:methanol:ammonia water in a ratio of 8:1:0.1; 5) The developed thin layer chromatography plate is developed with bismuth potassium iodide as a color developer, and both part A and part B show color points corresponding to the extract of Fritillaria cirrhosa. In step 2, first use 2 to 3 column volumes of mobile phase with a ratio of dichloromethane to methanol of 5:1, then use a mobile phase with a ratio of dichloromethane to methanol of 2:1; use 3 to 4 column volumes of mobile phase with a ratio of dichloromethane to methanol of 2:1, then use 1 column volume of mobile phase with a ratio of dichloromethane to methanol of 1:1; Part or all of the effluent in step 3 equivalent to 2.5 to 4.0 times the column volume is combined into part A; part or all of the effluent equivalent to 4.5 to 6.0 times the column volume is combined into part B; In step 4, before thin layer chromatography analysis, part B is concentrated into a thick paste and then extracted with 12.5% ​​ammonia water and dichloromethane by shaking, and the concentrated dichloromethane layer is taken for thin layer spotting; The preparation method of the extract of Fritillaria cirrhosa in step 4 is to extract the crushed Fritillaria cirrhosa with 95% ethanol and 50% ethanol in turn, combine the extracts, concentrate and evaporate to dryness, dissolve the dry paste with anhydrous ethanol, and use the squeezed solution for thin layer spotting.

2. The characteristic identification method of Fritillaria cirrhosa in Xiaoer Qingfei Granule according to claim 1, characterized in that: The required silica gel mesh number for normal phase silica gel column chromatography in step 1 is 300-400 mesh.

3. The characteristic identification method of Fritillaria cirrhosa in Xiaoer Qingfei Granule according to claim 1, characterized in that: The extraction was performed under reflux twice, each time for 2 hours.

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