阅读说明：本技术 大建中汤物质基准的质量控制方法 (Quality control method for soup material reference in large-scale construction ) 是由 张宁 王冰 邹纯朴 于 2020-06-12 设计创作，主要内容包括：本发明涉及一种大建中汤物质基准的质量控制方法,包括确立制备工艺和建立质量评价体系。首先在遵循古法的基础上对大建中汤及物质基准制备条件进行筛选比较,确定了其制备工艺。大建中汤中各味饮片经过煎煮、过滤、浓缩、干燥得到大建中汤物质基准。在此基础上,建立大建中汤物质基准的质量属性研究方法。大建中汤物质基准质量控制采用薄层色谱鉴别、含量测定、指纹图谱方法相结合。本发明建立了全面控制大建中汤物质基准的质量评价体系,为大建中汤复方制剂的合理开发应用提供了研究基础。(The invention relates to a quality control method of a soup material standard in the large-scale construction, which comprises the steps of establishing a preparation process and establishing a quality evaluation system. Firstly, screening and comparing the preparation conditions of the Dajianzhong soup and the material standard on the basis of following the ancient method, and determining the preparation process. The material standard of the Dajianzhong decoction is obtained by decocting, filtering, concentrating and drying various decoction pieces in the Dajianzhong decoction. On the basis, a quality attribute research method of the soup material standard in the large-scale construction is established. The quality control of the decoction in the large-scale building by the standard adopts the combination of thin-layer chromatography identification, content determination and fingerprint spectrum methods. The invention establishes a quality evaluation system for comprehensively controlling the material standard of the Dajianzhong decoction, and provides a research basis for reasonable development and application of the Dajianzhong decoction compound preparation.)

1. A quality control method for a soup material reference in the large-scale construction is characterized by comprising the following steps:

step S1, establishing a fingerprint of the standard of the soup material in the large building to be tested, wherein the chromatographic conditions are as follows: mobile phase 0.2% phosphoric acid (a) -acetonitrile (B), gradient elution, procedure:

detection wavelength: 210nm, column temperature: 30 ℃, flow rate: 1.0ml/min, sample size: 5 μ l, column: waters C18 chromatography columns;

step S2, determining whether the fingerprint of the standard soup material of the large building to be tested contains the following chromatographic peaks, and judging whether the quality of the soup of the large building to be tested is qualified:

2. the quality control method of the Jianzhong decoction substance reference according to claim 1, characterized by further determining whether the fingerprint of the Jianzhong decoction substance reference to be detected contains the following chromatographic peaks:

3. the quality control method of the large building middle soup material standard according to claim 1, characterized in that in the process of establishing the fingerprint of the large building middle soup material standard to be detected, the large building middle soup extract powder to be detected is prepared into a solution to be detected according to the following method: 2g of Dajianzhong decoction extract powder is taken, precisely weighed, added with 20ml of 70 percent methanol, ultrasonically treated (the frequency is 35kHz, the power is 250w) for 30min, filtered and measured.

4. The quality control method of the large building middle soup material reference according to claim 1, wherein in the process of establishing the fingerprint of the large building middle soup material reference to be detected, the preparation method of the mixed reference solution comprises the following steps: taking rutin, hyperoside, ginsenoside Rg1, and 6-gingerol, and dissolving with methanol.

5. The quality control method for the Dajianzhong soup material standard according to claim 1, further comprising the step of measuring the content of ginsenoside in the Dajianzhong soup extract powder to be measured, wherein the measured chromatographic conditions are as follows: mobile phase: water (a) -acetonitrile (B), gradient elution, specific gradient as follows:

the detection wavelength is 203nm, the column temperature is 35 ℃, the flow rate is 1.0ml/min, the sample injection amount is 10 mul, and the chromatographic column Waters symmetry shield C18(250mm × 4.6.6 mm, 5 mu m);

the preparation method of the solution to be detected comprises the following steps: precisely weighing 2g of extract powder of DAJIANZHONG decoction to be measured, adding 20ml of methanol, performing ultrasonic treatment (35kHz, 100w) for 1h, and filtering to obtain the final product; wherein the proportion of the added extract powder to the added methanol is as follows: 2g, 20 ml.

6. The quality control method of the Jianzhong decoction substance reference according to claim 1, which is characterized by further comprising the step of measuring the content of 6-gingerol in the Jianzhong decoction extract powder to be measured, wherein the measured chromatographic conditions comprise that a mobile phase is water (A) -acetonitrile (B), gradient elution is performed for 0-35 min, 38-39% B, 35-40 min and 39-90% B, the column temperature is 30 ℃, the flow rate is 1.0ml/min, the detection wavelength is 280nm, the sample injection amount is 20 μ l, and a chromatographic column Kromasil100-5C18(250mm × 4.6.6 mm, 5 μm);

the preparation method of the solution to be detected comprises the following steps: precisely weighing 1g of extract powder of DAJIANZHONG decoction to be measured, adding 75% methanol 20ml, weighing, performing ultrasonic treatment (frequency 35kHz and power 250w) for 45min, cooling, weighing, supplementing the weight loss with 75% methanol, and filtering; wherein the proportion of the added extract powder to the added methanol is as follows: 20ml for 1 g.

7. The quality control method of the Dajianzhong soup material reference according to claim 1, further comprising the step of measuring the content of rutin and hyperoside in the Dajianzhong soup extract powder to be measured, wherein the chromatographic conditions for measurement are that a Kromasil100-5C18(4.6mm × 250mm, 5 μm) chromatographic column is adopted, the mobile phase is 0.3% formic acid (A) -acetonitrile (B), gradient elution is performed for 0-40 min, 14-16% B, 40-41 min, 16-90% B, 41-50 min and 90% B, the flow rate is 1.0ml/min, the column temperature is 35 ℃, the detection wavelength is 256nm, and the sample injection amount is 5 μ l;

the preparation method of the solution to be detected comprises the following steps: precisely weighing 1g of extract powder of DAJIANZHONG decoction to be measured, adding 75% methanol 30ml, weighing, performing ultrasonic treatment (frequency 35kHz and power 250w) for 30min, standing at room temperature, weighing, supplementing the weight loss with 75% methanol, and filtering with 0.22 μm filter membrane; wherein the proportion of the added extract powder to the added methanol is as follows: 1g, 30 ml.

8. The quality control method for the material reference of the Dajianzhong decoction according to claim 1, further comprising the step of ginseng thin layer identification, which is specifically as follows:

preparing a test solution: taking 1g of DAJIANZHONGTANG extract powder, adding 10ml of water-saturated n-butanol, performing ultrasonic treatment (frequency 35kHz and power 250w) for 30min, filtering, adding 3 times of ammonia solution into the filtrate, shaking for extraction, standing, collecting the upper layer solution, evaporating to dry, and dissolving the residue with 0.5ml of methanol to obtain the final product; preparing Ginseng radix blank solution and Ginseng radix control solution by the same method;

sucking the three solutions, dropping on the same silica gel G thin layer plate, spreading with subnatant of chloroform-ethyl acetate-methanol-water (15:40:22:10) at-20 deg.C, taking out, air drying, spraying 10% ethanol sulfate solution, heating at 105 deg.C for color development, observing under fluorescent lamp and ultraviolet lamp (365nm), wherein the chromatogram of the sample shows the same color spot or fluorescent spot at the corresponding position of the chromatogram of the medicinal material, and the blank solution of Ginseng radix has no interference, and the quality is qualified.

9. The quality control method of the material standard of the Dajianzhong decoction according to claim 1, which is characterized by further comprising the step of identifying rhizoma zingiberis and pericarpium zanthoxyli by thin-layer chromatography, and specifically comprises the following steps:

preparing a test solution: taking 1g of Dajianzhong decoction extract powder to be detected, adding 20ml of ethyl acetate, carrying out ultrasonic treatment (frequency 35kHz and power 250w) for 30min, filtering, evaporating filtrate to dryness, and dissolving residues in 0.5ml of methanol to obtain the extract powder; preparing blank solutions of rhizoma Zingiberis and fructus Zanthoxyli by the same method; preparing medicinal solution from 0.5g of rhizoma Zingiberis and fructus Zanthoxyli powder by the same method;

sucking 5 solutions, dropping on the same silica gel G thin layer plate, developing with petroleum ether (60-90 deg.C) -chloroform-ethyl acetate (2:1:1), taking out, air drying, spraying 5% vanillin sulfuric acid test solution, heating at 105 deg.C until color development is clear, and observing to obtain qualified product with the same color spot in the corresponding position of medicinal material chromatogram and no interference.

Technical Field

The invention relates to the field of medicine quality control, in particular to a quality control method of a soup material standard in the large-scale construction.

Background

The DAJIANZHONG decoction is from Zhang Zhongjing, JINKUIAOLAO, and is prepared from fructus Zanthoxyli (for removing sweat), Zingiberis rhizoma, and Ginseng radix. It is mainly used for treating deficiency of middle-jiao and yang, and yin cold and internal excess, and has effects of warming middle-jiao, tonifying deficiency, dispelling cold, and lowering adverse qi. The Dajianzhong decoction has wide clinical application value, is always considered by doctors as a typical prescription for treating deficiency-cold and abdominal pain of spleen and stomach, and mainly treats abdominal pain caused by yang deficiency of spleen and stomach and internal excess of yin cold. Dajianzhong decoction is good at treating deficiency-cold syndrome of middle energizer and also can be used for treating yang deficiency cold and insect stirring. The Dajianzhong decoction is listed in the first published classical name prescription 100, and shows that the Dajianzhong decoction has high development and application values.

The Dajianzhong decoction is one of three prescriptions of a prescription system established by the philosophy of 'jianzhong method'. Zhang Zhongjing can constantly care the spleen and stomach when using drugs to treat diseases, and is combined with porridge except for the combination of the drugs. Modern people have fast and busy life rhythm and increasingly more patients with diseases related to the spleen and the stomach in clinic, and the Jianzhong series of prescriptions are widely applied to conditioning the spleen and the stomach in the middle jiao, so that a relatively independent treatment method is formed.

The Dajianzhong decoction has a decoction and taking method in the 'golden lack essentials', but due to the great difference between the modern and the Chinese-generation measuring units, the modern weighing parameters and decoction parameters are not clear, and the 'Chinese pharmacopoeia' only has a detection method of a single medicine, so that a compound (decoction and substance basis) of the Dajianzhong decoction does not originate from a traditional preparation method and accords with a preparation method of a modern preparation technology, and an objective compound (decoction and substance basis) quality evaluation system is lacked.

Patent document CN103237899A, published japanese patent No. 2013.08.07, discloses a bioassay method for dajianzhong soup and a quality control method using the same. The biological assay method of the Dajianzhong decoction comprises the steps of adding a test sample containing the Dajianzhong decoction into cultured cells which produce serotonin, and then measuring the content of the serotonin in culture supernatant. The quality management method of the Dajianzhong decoction preparation is characterized in that the biological determination method is used for evaluating the pharmacological activity of a reference preparation and a tested preparation which have approved clinical pharmacological effects and are used as the Dajianzhong decoction under the same condition, and evaluating the equivalence of the reference preparation and the tested preparation.

Journal literature (Li Xiang, Sun Yangzhi, Gaobaong, etc. the research on the quality standard of Dajianzhong decoction granules [ J ] the Chinese medicine academic newspaper, 2013,28(12):1867 and 1869.) discloses the quality standard of the Dajianzhong decoction granules. The method comprises the following steps: qualitatively identifying Ginseng radix in DAJIANZHONG decoction granule by thin layer chromatography, and measuring contents of ginsenoside Rg1, Re, and Rb1 in Ginseng radix by high performance liquid chromatography.

However, the quality control method of the standard of the soup material in the large-scale construction is not available at present.

Disclosure of Invention

The invention aims to provide a quality control method of the material reference of the Dajianzhong decoction aiming at the defects in the prior art, and provides a reliable research method and an evaluation system of the quality attribute for the development of a classical famous Dajianzhong decoction compound preparation.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a quality control method for a material reference of a soup in the large-scale building comprises the following steps:

step S1, establishing a fingerprint of the standard of the soup material in the large building to be tested, wherein the chromatographic conditions are as follows: mobile phase 0.2% phosphorus (a) -acetonitrile (B), gradient elution, procedure:

detection wavelength: 210nm, column temperature: 30 ℃, flow rate: 1.0ml/min, sample size: 5 μ l, column: waters C18 chromatography columns;

step S2, determining whether the fingerprint of the standard soup material of the large building to be tested contains the following chromatographic peaks, and judging whether the quality of the soup of the large building to be tested is qualified:

note: the following average retention time/retention time units are all min

As a preferred example, whether the fingerprint of the soup material reference in the capital construction to be tested contains the following chromatographic peaks is further determined:

as another preferred example, in the process of establishing the fingerprint of the standard of the material of the junior middle-jiao decoction to be detected, the junior middle-jiao decoction extract powder to be detected is prepared into the solution to be detected according to the following method: 2g of Dajianzhong decoction extract powder is taken, precisely weighed, added with 20ml of 70 percent ethanol, ultrasonically treated (the frequency is 35kHz, the power is 250w) for 30min, filtered and measured.

As another preferred example, in the process of establishing the fingerprint of the soup material standard in the large building to be tested, the preparation method of the mixed reference solution comprises the following steps: taking rutin, hyperoside, ginsenoside Rg1, and 6-gingerol, and dissolving with methanol.

As another preferred example, the method further comprises the step of measuring the content of ginsenoside in the extract powder of the decoction of rhubarb and rhubarb to be measured, wherein the chromatographic conditions are as follows: mobile phase: water (a) -acetonitrile (B), gradient elution, specific gradient as follows:

the detection wavelength is 203nm, the column temperature is 35 ℃, the flow rate is 1.0ml/min, the sample injection amount is 10 mul, and the chromatographic column Waters symmetry shield C18(250mm × 4.6.6 mm, 5 mu m);

the preparation method of the solution to be detected comprises the following steps: 2g of Dajianzhong decoction extract powder to be measured is precisely weighed, 20ml of methanol is added, ultrasonic treatment (35kHz, 100w) is carried out for 1h, and filtration is carried out, thus obtaining the extract powder; wherein the proportion of the added extract powder to the added methanol is as follows: 2g, 20 ml.

As another preferred example, the method also comprises the step of measuring the content of 6-gingerol in the extract powder of the Jianzhong decoction to be measured, wherein the chromatographic conditions for measurement comprise that the mobile phase comprises water (A) -acetonitrile (B), the gradient elution is carried out for 0-35 min, 38-39% of B, 35-40 min, 39-90% of B, the column temperature is 30 ℃, the flow rate is 1.0ml/min, the detection wavelength is 280nm, the sample injection amount is 20 mu l, and the chromatographic column Kromasil100-5C18(250mm × 4.6.6 mm, 5 mu m);

the preparation method of the solution to be detected comprises the following steps: precisely weighing 1g of Dajianzhong decoction extract powder to be measured, adding 20ml of 75% methanol, weighing, performing ultrasonic treatment (frequency 35kHz and power 250w) for 45min, cooling, weighing, supplementing the weight loss with 75% methanol, and filtering to obtain the final product; wherein the proportion of the added extract powder to the added methanol is as follows: 20ml for 1 g.

As another preferred example, the method also comprises the step of measuring the content of rutin and hyperin in the Dajianzhong decoction extract powder to be measured, wherein the chromatographic conditions for measurement are that a Kromasil100-5C18(4.6mm × 250mm, 5 mu m) chromatographic column is adopted, the mobile phase is 0.3 percent formic acid (A) -acetonitrile (B), gradient elution is carried out for 0-40 min, 14-16 percent B, 40-41 min, 16-90 percent B, 41-50 min and 90 percent B, the flow rate is 1.0ml/min, the column temperature is 35 ℃, the detection wavelength is 256nm, and the sample introduction amount is 5 mu l;

the preparation method of the solution to be detected comprises the following steps: precisely weighing 1g of extract powder of DAJIANZHONG decoction to be measured, adding 75% methanol 30ml, weighing, performing ultrasonic treatment (frequency 35kHz and power 250w) for 30min, standing at room temperature, weighing, supplementing the weight loss with 75% methanol, and filtering with 0.22 μm filter membrane; wherein the proportion of the added extract powder to the added methanol is as follows: 1g, 30 ml.

As another preferred example, the method further comprises the step of ginseng thin-layer chromatography identification, and specifically comprises the following steps:

preparing a test solution: taking 1g of extract powder, adding 10ml of water-saturated n-butanol, performing ultrasonic treatment (frequency of 35kHz and power of 250w) for 30min, filtering, adding 3 times of ammonia test solution into filtrate, shaking for extraction, standing, taking upper layer solution, evaporating to dryness, and dissolving residue with 0.5ml of methanol to obtain the final product; preparing Ginseng radix blank solution and Ginseng radix control solution by the same method;

sucking the three solutions, dropping on the same silica gel G thin layer plate, spreading with subnatant of chloroform-ethyl acetate-methanol-water (15:40:22:10) at-20 deg.C, taking out, air drying, spraying 10% ethanol sulfate solution, heating at 105 deg.C for color development, observing under fluorescent lamp and ultraviolet lamp (365nm), wherein the chromatogram of the sample shows the same color spot or fluorescent spot at the corresponding position of the chromatogram of the medicinal material, and the blank solution of Ginseng radix has no interference, and the quality is qualified.

As another preferred example, the method further comprises the step of identifying the dried ginger and the pepper by thin-layer chromatography, and specifically comprises the following steps:

preparing a test solution: taking 1g of Dajianzhong decoction extract powder to be detected, adding 20ml of ethyl acetate, carrying out ultrasonic treatment (frequency 35kHz and power 250w) for 30min, filtering, evaporating filtrate to dryness, and dissolving residues in 0.5ml of methanol to obtain the extract powder; preparing blank solutions of rhizoma Zingiberis and fructus Zanthoxyli by the same method; preparing medicinal solution from 0.5g of rhizoma Zingiberis and fructus Zanthoxyli powder by the same method;

sucking 5 solutions, dropping on the same silica gel G thin layer plate, developing with petroleum ether (60-90 deg.C) -chloroform-ethyl acetate (2:1:1), taking out, air drying, spraying 5% vanillin sulfuric acid test solution, heating at 105 deg.C until color development is clear, and observing to obtain qualified product with the same color spot in the corresponding position of medicinal material chromatogram and no interference.

The invention has the advantages that:

1. according to the method for measuring the content of the relevant components in the pepper in 2015 edition of Chinese pharmacopoeia, through reference of documents, the pepper contains flavonoid components and mainly contains rutin, hyperin and the like, so that the method for simultaneously measuring the HP L C of the rutin and the hyperin in the pepper fills the gap of the lack of measurement of the content of the main components of the pepper in the current edition of pharmacopoeia.

2. Corresponding characteristic spots exist in the identification of the medicinal material → the medicinal piece → the decoction in the great Jian Zhong → the material reference thin-layer chromatography, a basis is provided for tracing the characteristic components in the material reference to the medicinal material, and the content measurement proves that a certain correlation exists between the medicinal material → the medicinal piece → the decoction in the great Jian Zhong → the material reference.

3. Establishing a characteristic map of the standard of the soup material in the large-scale building, making up the defect that a single component cannot reflect the whole compound, and integrally evaluating the variety and the content of the internal chemical substances of the standard of the soup material in the large-scale building.

4. The quality evaluation method established by the invention has good precision, stability and repeatability.

5. The preparation and quality evaluation system of the Dajianzhong decoction material standard is researched by taking the Dajianzhong decoction as a research object and establishing a preparation and quality evaluation system of the Dajianzhong decoction material standard according to the technical requirements of documents such as simplified registration approval management regulations of ancient classical famous prescription Chinese medicinal compound preparations, and reporting data requirements (request comments) of the ancient classical famous prescription Chinese medicinal compound preparations material standard, and the like, thereby providing a research work basis for reasonable development and application of the compound preparation.

Drawings

FIG. 1 is a thin layer chromatogram for identification of Ginseng radix in the standard of the decoction in the middle of Mass and Mass.

FIG. 2 is a thin-layer chromatogram for identification of Zingiberis rhizoma and Zanthoxyli fructus in the standard of the material of DAJIANZHONG decoction.

FIG. 3 is a finger print of DAJIANZHONG decoction detected at different wavelengths.

FIG. 4 is a finger print of DAJIANZHONG decoction detected at different column temperatures.

FIG. 5 shows the fingerprint of Dajianzhong decoction at different flow rates.

FIG. 6 is a chromatogram of different extraction methods of the decoction material standard in the great Jian.

FIG. 7 is a chromatogram of different ultrasonic extraction times of the decoction material standard in the great Jian.

FIG. 8 is a chromatogram obtained by ultrasonic extraction of different concentrations of methanol based on the decoction in the middle of the great-aged building.

FIG. 9. control chromatogram of mixed standards.

FIG. 10 is the fingerprint of DAJIANZHONG decoction and each negative sample.

FIG. 11 is a fingerprint of DAJIANZHONG decoction.

FIG. 12 is a comparative fingerprint of the soup material standard in the great construction.

FIG. 13.15 overlay of fingerprint spectrum of Dajianzhong decoction of batch.

FIG. 14.15 overlay of fingerprint spectra of soup material standard in batch of large-scale construction.

Detailed Description

The following detailed description of the present invention will be made with reference to the accompanying drawings. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. Wherein, the reagents or instruments used are not indicated by manufacturers, and are all conventional products which can be obtained commercially.