阅读说明：本技术 一种小叶金花草的鉴别方法及其应用 (Method for identifying small-leaf golden camellia and application thereof ) 是由 陈锋 李嘉 张颖 杨海船 何春欢 张赟赟 巫凯 于 2020-05-25 设计创作，主要内容包括：本发明公开了一种小叶金花草的鉴别方法及其应用,属于药物分析技术领域。本发明鉴别方法包括供试品溶液的制备方法和高效液相色谱检测条件；用中药指纹图谱相似度软件进行处理,得小叶金花草的指纹图谱；将相关色谱数据导入SPSS软件进行聚类分析和主成分分析,并得到相关分析结果。本发明建立了小叶金花草HPLC指纹图谱的共有模式,指认了11个共有峰,其中4个共有峰为已知成分,涵盖了药材指标性成分的大部分信息,该方法简便、快速、重现性好,填补了小叶金花草质量控制方法的空白,具有较高的应用价值。(The invention discloses an identification method of small-leaf golden camellia and application thereof, belonging to the technical field of drug analysis. The identification method comprises a preparation method of a test solution and high performance liquid chromatography detection conditions; processing with traditional Chinese medicine fingerprint similarity software to obtain fingerprint of herba Gerberae Piloselloidis; and (4) importing the related chromatographic data into SPSS software for cluster analysis and principal component analysis, and obtaining a related analysis result. The method establishes a common mode of the HPLC fingerprint spectrum of the California leaflet, identifies 11 common peaks, wherein 4 common peaks are known components and cover most information of index components of medicinal materials, and has the advantages of simplicity, rapidness, good reproducibility, filling of the blank of the quality control method of the California leaflet, and high application value.)

1. The method for identifying the small leaf golden camellia is characterized by comprising the following specific steps of:

(1) preparation of test solution

Beating a small leaf golden camellia sample to be tested into powder, and screening the powder by a third sieve; taking 1.0g of small-leaf golden camellia powder, precisely weighing, placing in a round-bottom flask, precisely adding 30mL of methanol-acetic acid-water (45:5:50) solution, weighing, heating in a water bath and refluxing for 1.5h, cooling to room temperature, weighing again and complementing the mass, shaking up, filtering, and taking a subsequent filtrate as a sample solution;

(2) preparation of control solutions

Respectively taking appropriate amount of protocatechuic acid, vanillic acid, caffeic acid and chicoric acid as reference substances, precisely weighing, adding methanol for dissolving to obtain mixed reference substance solution containing the 4 components with mass concentrations of 5.07, 8.26, 6.53 and 32.46 mu g/mL respectively, filtering, and taking the subsequent filtrate as the reference substance solution;

(3) high performance liquid chromatography detection and fingerprint generation

Sucking the test solution obtained in the step (1), injecting the test solution into a high performance liquid chromatograph for high performance liquid chromatography detection, and recording a fingerprint within 105 minutes;

the conditions of the high performance liquid chromatography are as follows: using an Agilent XDB-C18(250 mm. times.4.6 mm. times.5 μm) column, mobile phase: acetonitrile (A) -0.4% phosphoric acid solution (B), and gradient elution (0-10 min, 10% A; 10-60 min, 10% A → 30% A; 60-105 min, 30% A); flow rate: 1.0 mL/min; detection wavelength: 260 nm; column temperature: 25 ℃; sample introduction amount: 10 μ L, assay time: 105 min;

(4) similarity analysis

Introducing the chromatogram into software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system for analysis, selecting a reference spectrum, performing chromatogram peak matching by adopting a multipoint correction method, and selecting a median method to generate a reference spectrum; performing similarity evaluation by adopting software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system and taking the HPLC control spectrum of the medicinal material sample as a reference;

(5) identification and correlation analysis of common peaks

Determining 11 common peaks of the medicinal material sample, comparing with the mixed reference solution, and performing sample injection detection according to the chromatographic conditions in the step (3); setting a reference peak, and calculating the relative retention time and the relative peak area of other common peaks;

(6) cluster analysis

By utilizing a data management function of a Waters empower workstation, importing the relative peak areas of 11 common peaks as original data, introducing SPSS software, and performing cluster analysis on the small-leaf golden clover by using a group mean number coupling method and combining a squared Euclidean distance as a measurement standard;

(7) principal component analysis

And carrying out standardization treatment on the peak area of each common peak by using SPSS software, and carrying out principal component analysis on 11 common peaks obtained by the fingerprint of the small leaf golden camellia medicinal material.

2. The method of claim 1, wherein the common peaks at 1, 4, 5 and 8 are identified as protocatechuic acid, vanillic acid, caffeic acid and chicoric acid in step (5).

3. The method for identifying the California microphylla as claimed in claim 1, wherein the fingerprint is applied to quality control of the California microphylla.

4. The method for identifying the golden camellia lobus as claimed in claim 1, wherein the fingerprint is applied to the detection of the golden camellia lobus extract and the preparation.

Technical Field

The invention relates to the technical field of pharmaceutical analysis, and particularly relates to a method for identifying California lobus, which is used for analyzing main components in medicinal materials and evaluating the quality of the medicinal materials, and an application of the California lobus.

Background

The small leaf golden flower herb is derived from Onychium japonicum (Thunb.) Kze, which is a common traditional Chinese medicine in Guangxi Yao nationality and Zhuang nationality of China fern family, namely, golden flower fern, small pheasant tail, Japanese Stenoloma chusana, and the like. It is bitter in taste, and has effects of clearing away heat and toxic materials, promoting diuresis, and stopping bleeding. It is mainly used for treating wind-heat type common cold, cough due to lung heat, acute gastroenteritis, jaundice, hematochezia, etc. At present, the research on the small leaf golden camellia is mainly in the aspects of crude drug discrimination, gametophyte development, separation of chemical components, pharmacological action and the like, and the research on the aspect of quality evaluation is not seen.

The traditional Chinese medicine has complex components and various drug effects, and HPLC is the mainstream analysis means of the traditional Chinese medicine, and has important significance for the quality evaluation of the medicinal materials. The demand of traditional Chinese medicines and traditional Chinese medicine preparations is rapidly increased all over the world, so the evaluation and control of the quality of medicinal materials are particularly critical, and because the traditional Chinese medicines are difficult to completely characterize all compounds, but the compounds usually have synergistic effect in the aspect of treatment, and a proper analysis method is necessary to find. The research on the traditional Chinese medicine fingerprint is not only an identification method of the traditional Chinese medicine, but also one of the important means for evaluating the quality standard, is widely accepted by scholars at home and abroad, gradually becomes a trend, and can more comprehensively reflect the overall chemical characteristics of the traditional Chinese medicine. The chemical components of the traditional Chinese medicine are complex, the content of the traditional Chinese medicine can be influenced by various factors, and the richer material basic research method has high application value.

Therefore, the identification method of the California leaflet and the application thereof are problems to be solved urgently by the technical personnel in the field.

Disclosure of Invention

In view of the above, the invention provides the identification method and the application of the small leaf golden camellia, the method is simple, convenient, quick and good in reproducibility, fills the blank of the quality control method of the small leaf golden camellia, and has higher application value.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for identifying small leaf golden camellia comprises the following specific steps:

(1) preparation of test solution

Beating a small leaf golden camellia sample to be tested into powder, and screening the powder by a third sieve; taking 1.0g of small-leaf golden camellia powder, precisely weighing, placing in a round-bottom flask, precisely adding 30mL of methanol-acetic acid-water (45:5:50) solution, weighing, heating in a water bath and refluxing for 1.5h, cooling to room temperature, weighing again and complementing the mass, shaking up, filtering, and taking a subsequent filtrate as a sample solution;

(2) preparation of control solutions

Respectively taking appropriate amount of protocatechuic acid, vanillic acid, caffeic acid and chicoric acid as reference substances, precisely weighing, adding methanol for dissolving to obtain mixed reference substance solution containing the 4 components with mass concentrations of 5.07, 8.26, 6.53 and 32.46 mu g/mL respectively, filtering, and taking the subsequent filtrate as the reference substance solution;

(3) high performance liquid chromatography detection and fingerprint generation

Sucking the test solution obtained in the step (1), injecting the test solution into a high performance liquid chromatograph for high performance liquid chromatography detection, and recording a fingerprint within 105 minutes;

the conditions of the high performance liquid chromatography are as follows: using an Agilent XDB-C18(250 mm. times.4.6 mm. times.5 μm) column, mobile phase: acetonitrile (A) -0.4% phosphoric acid solution (B), and gradient elution (0-10 min, 10% A; 10-60 min, 10% A → 30% A; 60-105 min, 30% A); flow rate: 1.0 mL/min; detection wavelength: 260 nm; column temperature: 25 ℃; sample introduction amount: 10 μ L, assay time: 105 min;

(4) similarity analysis

Introducing the chromatogram into software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system for analysis, selecting a reference spectrum, performing chromatogram peak matching by adopting a multipoint correction method, and selecting a median method to generate a reference spectrum; performing similarity evaluation by adopting software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system and taking the HPLC control spectrum of the medicinal material sample as a reference;

(5) identification and correlation analysis of common peaks

Determining 11 common peaks of the medicinal material sample, comparing with the mixed reference solution, and performing sample injection detection according to the chromatographic conditions in the step (3); the reference peak was set and the relative retention time and relative peak area of the other common peaks were calculated.

The fingerprint is a spectrum or chromatogram obtained by measuring various chemical components of the traditional Chinese medicine, and is an effective method for evaluating the quality of the traditional Chinese medicine, identifying the truth and the falseness, distinguishing species and ensuring the consistency and the stability by researching gene segments of the traditional Chinese medicine in combination with HPLC. Taking 13 batches of California microphylla as an example, 11 common peaks in a high performance liquid fingerprint are identified, and 4 common peaks are determined to be protocatechuic acid, vanillic acid, caffeic acid and chicoric acid respectively through comparison of a reference substance.

(6) Cluster analysis

By utilizing a data management function of a Waters empower workstation, taking the relative peak areas of 11 common peaks as original data, importing SPSS25.0 software, and carrying out cluster analysis on the California microphylla by taking a mean number coupling method and combining a squared Euclidean distance as a measurement standard.

China is wide in territory, has complex natural geographic environment, and has different environments such as sunshine, temperature, soil texture and the like, necessary favorable conditions are provided for the growth of medicinal plants and animals, meanwhile, the production and the quality of the Chinese medicinal materials have certain regionality, so that the content of each compound in different batches of medicinal materials has certain difference, the objective of cluster analysis is to collect data for classification on the basis of similarity, and related data obtained from a fingerprint map is subjected to cluster analysis by using SPSS software, so that the cluster analysis can be used as preliminary exploratory analysis, and certain conclusions can be visually and concisely obtained. The method is used for scientifically classifying the California minor medicinal materials of different batches, so that rules are summarized and reference is provided for quality evaluation.

(7) Principal component analysis

And carrying out standardization treatment on the peak area of each common peak by using SPSS software, and carrying out principal component analysis on 11 common peaks obtained by the fingerprint of the small leaf golden camellia medicinal material.

The principal component analysis is a bilinear model method, a maximum variance principle is utilized, a plurality of independent variables contained in original data are subjected to linear fitting, new low-dimensional variables are used for replacing original high-dimensional variables, namely principal components, all the principal components are not related to each other, so that the principal components can reflect most of information of the original variables, the contained information is not overlapped with each other, and then the dimensionality reduction of the data is realized. The method is provided for exploring the correlation of the common components in the California microphylla of different batches, the SPSS software is used for carrying out principal component analysis on the related data obtained from the fingerprint, the scores of the principal components are converted from the variance and weight results, and the method has important reference value for evaluating the quality of medicinal materials.

Further, step (5) confirmed that common peaks No. 1, 4, 5, 8 were protocatechuic acid, vanillic acid, caffeic acid, and chicoric acid, respectively.

Further, the application of the fingerprint spectrum in quality control of the California microphylla.

Further, the application of the fingerprint in the detection of the California microphylla extract and a preparation is provided.

The invention combines a plurality of research means and analysis results, so that the quality control method of the California microphylla is expanded and improved compared with the prior art, and the modernization pace of the traditional Chinese medicine is followed, which is beneficial to forming the quality standard of the California microphylla, and the medicinal material can better serve for human.

According to the technical scheme, compared with the prior art, the invention discloses an identification method of the small leaf golden camellia and application thereof, in particular to a construction method of a small leaf golden camellia fingerprint and application of the small leaf golden camellia fingerprint; the method comprises a preparation method of a test solution and high performance liquid chromatography detection conditions; processing with traditional Chinese medicine fingerprint similarity software to obtain fingerprint of herba Gerberae Piloselloidis; and (4) importing the related chromatographic data into SPSS software for cluster analysis and principal component analysis, and obtaining a related analysis result. The method establishes a common mode of the HPLC (high performance liquid chromatography) fingerprint of the California leaflet, identifies 11 common peaks, wherein 4 common peaks are known components and cover most information of index components of medicinal materials.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a drawing showing an HPLC control fingerprint spectrum R of a sample of a medicinal material of the present invention;

FIG. 2 is a schematic diagram of HPLC overlay fingerprints of 13 batches of the medicinal material samples according to the present invention;

FIG. 3 is a chromatogram of a mixed control according to the invention;

FIG. 4 is a diagram showing the result of cluster analysis of 13 batches of herbs in this invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Waters e2695 series quaternary gradient pump high performance liquid chromatograph (wawter, usa); an XS205 electronic balance (mettler-toledo, switzerland); KS-500DE ultrasonic cleaner (Kunshan Jielimei ultrasonic instruments Co., Ltd.); HH-S4 model digital display constant temperature water bath (Jintani medical instrument factory).

Protocatechuic acid (China institute for testing and testing food and drug; batch No. 110809-; vanillic acid (China institute for food and drug assay, batch No. 110776-201503, purity 99.8%); caffeic acid (Kyorman Biotech, Inc., lot number: MUST-18032003, 99.48% pure); chicoric acid (Doudnerite Biotech, Inc., batch No.: MUST-18031720, purity 99.33%); acetonitrile (Fisher corporation, USA, chromatographic grade), phosphoric acid as analytical grade, water as ultra pure water.