Construction method and identification method of UPLC (unified Power level liquid chromatography) characteristic spectrum of kadsura longepedunculata medicinal material
阅读说明：本技术 一种南五味子药材uplc特征图谱的构建方法及其鉴别方法 (Construction method and identification method of UPLC (unified Power level liquid chromatography) characteristic spectrum of kadsura longepedunculata medicinal material ) 是由 陈芳 梁月仪 刘晓霞 李乐 孙冬梅 王利伟 陈向东 魏梅 程学仁 于 2019-11-14 设计创作，主要内容包括：本发明涉及一种南五味子药材UPLC特征图谱构建方法及其鉴别方法,所述的南五味子药材UPLC特征图谱的构建方法包含如下步骤：(1)精密称取南五味子药材,制备得到南五味子药材供试品溶液；(2)将南五味子药材供试品溶液采用超高效液相色谱仪分析,得到南五味子药材UPLC特征图谱。本发明采用UPLC法能从根本上快速、准确的鉴别和区分南五味子,具有一定的特征性；本发明所述的方法稳定且专属性强,基线平稳,为南五味子药材质量控制提供科学的新方法；本发明使南五味子药材的质量控制从原来的某几个成分含量测定,上升为对整个药品的品质检测,避免了单一的化学成分检验的缺陷。(The invention relates to a construction method and an identification method of UPLC (ultra performance liquid chromatography) characteristic spectrum of kadsura longepedunculata medicinal material, wherein the construction method of the UPLC characteristic spectrum of the kadsura longepedunculata medicinal material comprises the following steps: (1) precisely weighing the kadsura longepedunculata medicinal material, and preparing a kadsura longepedunculata medicinal material test solution; (2) analyzing the sample solution of the Kadsura longepedunculata medicinal material by using an ultra-high performance liquid chromatograph to obtain a UPLC characteristic spectrum of the Kadsura longepedunculata medicinal material. The method adopts a UPLC method, can fundamentally, quickly and accurately identify and distinguish the kadsura longepedunculata, and has certain characteristics; the method is stable, has strong specificity and stable baseline, and provides a scientific new method for controlling the quality of the kadsura longepedunculata medicinal material; the invention improves the quality control of the kadsura longepedunculata medicinal material from the original content measurement of certain components to the quality detection of the whole medicine, and avoids the defect of single chemical component detection.)
1. A construction method of UPLC characteristic spectrum of Kadsura longepedunculata medicinal material is characterized by comprising the following steps:
(1) precisely weighing the kadsura longepedunculata medicinal material, and preparing a kadsura longepedunculata medicinal material test solution;
(2) analyzing the sample solution of the Kadsura longepedunculata medicinal material by using an ultra-high performance liquid chromatograph to obtain a UPLC characteristic spectrum of the Kadsura longepedunculata medicinal material.
2. The method for constructing the UPLC feature map of the kadsura longepedunculata medicinal material according to claim 1, wherein the chromatographic conditions of the ultra-high performance liquid chromatograph analysis are as follows: using octadecylsilane chemically bonded silica as a filling agent, acetonitrile as a mobile phase A, and 0.05-0.15% phosphoric acid aqueous solution as a mobile phase B, and performing gradient elution at a flow rate of 0.25-0.35 mL/min, a column temperature of 25-35 ℃, a detection wavelength of 210-250 nm, and a sample introduction amount of 0.6-1.8 μ l.
3. The method for constructing the UPLC feature map of the Kadsura longepedunculata medicinal material according to claim 2, wherein gradient elution conditions are as follows: the volume fraction of the mobile phase A is changed to 2-10% and the volume fraction of the mobile phase B is changed to 98-90% in 0-10 min; 10-11 min, the volume fraction of the mobile phase A is changed to 10-51%, and the volume fraction of the mobile phase B is changed to 90-49%; the volume fraction of the mobile phase A is changed to 51-54% and the volume fraction of the mobile phase B is changed to 49-46% in 11-26 min; 26-40 min, the volume fraction of the mobile phase A is changed to 54-95%, and the volume fraction of the mobile phase B is changed to 46-5%; the volume fraction of the mobile phase A is changed to 95-2% and the volume fraction of the mobile phase B is changed to 5-98% in 40-40.01 min; and (3) 40.01-45 min, wherein the volume fraction of the mobile phase A is 2%, and the volume fraction of the mobile phase B is 98%.
4. The method for constructing the UPLC characteristic spectrum of the Kadsura heteroclita medicinal material according to claim 1, wherein the test solution is prepared by the following steps: precisely weighing 0.3-0.7 g of kadsura longepedunculata powder, placing the powder in a container, precisely adding 40-60 ml of 60-80% ethanol, weighing, carrying out ultrasonic treatment for 20-40 minutes, cooling, weighing again, supplementing the lost weight with 60-80% ethanol, shaking up, filtering, and taking the subsequent filtrate to obtain the kadsura longepedunculata powder.
5. The method for constructing the UPLC characteristic spectrum of the Kadsura heteroclita medicinal material according to claim 1, wherein the test solution is prepared by the following steps: weighing 0.5g of fructus Schisandrae Sphenantherae powder, precisely weighing, placing in a container, precisely adding 50ml of 70% ethanol, weighing, ultrasonically treating for 30 min, cooling, weighing again, supplementing the lost weight with 70% ethanol, shaking, filtering, and collecting the filtrate.
6. The method for identifying the kadsura longepedunculata medicinal material is characterized by comprising the following steps of:
(1) precisely weighing the kadsura longepedunculata medicinal material to be identified, and preparing a kadsura longepedunculata medicinal material sample solution to be identified;
(2) precisely absorbing the kadsura longepedunculata medicinal material sample solution to be identified, injecting the solution into an ultra-high performance liquid chromatograph, and measuring to obtain a UPLC characteristic spectrum of the kadsura longepedunculata medicinal material to be identified;
(3) and comparing the measured UPLC characteristic spectrum with the UPLC characteristic spectrum of the Kadsura longepedunculata medicinal material constructed by the method of any one of claims 1 to 5, and if the UPLC characteristic spectrum is consistent with the UPLC characteristic spectrum of the Kadsura longepedunculata medicinal material, determining the Kadsura longepedunculata medicinal material with qualified quality.
7. The method for identifying Kadsura longepedunculata medicinal material according to claim 6, wherein the chromatographic conditions of the ultra high performance liquid chromatograph analysis are as follows: using octadecylsilane chemically bonded silica as a filling agent, acetonitrile as a mobile phase A, and 0.05-0.15% phosphoric acid aqueous solution as a mobile phase B, and performing gradient elution at a flow rate of 0.25-0.35 mL/min, a column temperature of 25-35 ℃, a detection wavelength of 210-250 nm, and a sample introduction amount of 0.6-1.8 μ l.
8. The method for identifying Kadsura longepedunculata medicinal material according to claim 6, wherein the gradient elution condition is as follows: the volume fraction of the mobile phase A is changed to 2-10% and the volume fraction of the mobile phase B is changed to 98-90% in 0-10 min; 10-11 min, the volume fraction of the mobile phase A is changed to 10-51%, and the volume fraction of the mobile phase B is changed to 90-49%; the volume fraction of the mobile phase A is changed to 51-54% and the volume fraction of the mobile phase B is changed to 49-46% in 11-26 min; 26-40 min, the volume fraction of the mobile phase A is changed to 54-95%, and the volume fraction of the mobile phase B is changed to 46-5%; the volume fraction of the mobile phase A is changed to 95-2% and the volume fraction of the mobile phase B is changed to 5-98% in 40-40.01 min; and (3) 40.01-45 min, wherein the volume fraction of the mobile phase A is 2%, and the volume fraction of the mobile phase B is 98%.
9. The method for identifying Kadsura heteroclita medicinal material according to claim 6, wherein the sample solution of Kadsura heteroclita medicinal material to be identified is prepared by the following steps: precisely weighing 0.3-0.7 g of kadsura longepedunculata powder, placing the powder in a container, precisely adding 40-60 ml of 60-80% ethanol, weighing, carrying out ultrasonic treatment for 20-40 minutes, cooling, weighing again, supplementing the lost weight with 60-80% ethanol, shaking up, filtering, and taking the subsequent filtrate to obtain the kadsura longepedunculata powder.
10. The method for identifying Kadsura heteroclita medicinal material according to claim 6, wherein the sample solution of Kadsura heteroclita medicinal material to be identified is prepared by the following steps: weighing 0.5g of fructus Schisandrae Sphenantherae powder, precisely weighing, placing in a container, precisely adding 50ml of 70% ethanol, weighing, ultrasonically treating for 30 min, cooling, weighing again, supplementing the lost weight with 70% ethanol, shaking, filtering, and collecting the filtrate.
Technical Field
The invention belongs to the field of traditional Chinese medicine identification, and particularly relates to a construction method of a UPLC (ultra performance liquid chromatography) characteristic spectrum of a kadsura longepedunculata medicinal material and an identification method thereof.
Background
Dried mature fruits of Kadsura longipedunculata, a magnolia plant, Schisandra sphenanthera, are collected from the first edition of Chinese pharmacopoeia 2015. Picking in autumn when the fruits are ripe, drying in the sun, and removing fruit stalks and impurities. The book of Ben Jing records: the Kadsura longepedunculata has the effects of tonifying qi, coughing and ascending qi, hurting and emaciating, tonifying deficiency, strengthening yin and benefiting male essence. The kadsura longepedunculata traditional Chinese medicine has the effects of nourishing and strengthening, has extremely high medicinal value, has the effects of tonifying qi and promoting the production of body fluid, astringing lung and nourishing kidney, stopping diarrhea, arresting seminal emission, soothing nerves and the like, and is used for treating symptoms of chronic cough, deficient asthma, nocturnal emission, spermatorrhea, enuresis, frequent micturition, chronic diarrhea, spontaneous perspiration, night sweat, body fluid consumption, thirst, shortness of breath, pulse deficiency, internal heat, thirst, palpitation, insomnia and the like. Fructus Schisandrae Sphenantherae mainly contains lignanoids such as deoxyschizandrin, schisantherin A, schisantherin B, D, and E, and volatile oil components. Modern pharmacological research shows that the kadsura longepedunculata has obvious effects of resisting oxidation, cancer and virus, protecting liver and the like, and can be used for treating diseases such as diabetes, infantile diarrhea, insomnia, intractable urticaria and the like.
In recent years, due to the diversity and complexity of effective components of traditional Chinese medicines, the traditional single component content measurement is not enough to explain the quality of the traditional Chinese medicines, and the specificity is also lacking. The traditional Chinese medicine fingerprint spectrum can reflect the commonness among traditional Chinese medicine individuals comprehensively and can also reflect the uniqueness among traditional Chinese medicine populations. The traditional Chinese medicine quality is controlled by using the traditional Chinese medicine fingerprint, the information is more comprehensive and abundant than the information provided by the prior method, and a comprehensive quality control means can be provided for the quality control of the kadsura longepedunculata.
Currently, identification methods include traditional methods and chromatographic methods combined with modern technology. At present, the traditional simple methods such as eye-viewing, mouth-tasting, nose-smelling, hand-touching and the like are very limited in Chinese medicinal material identification due to strong subjectivity, and chromatographic methods such as UV, IR, NIR and TLC have a large number of false positive and false negative problems, which easily cause the deviation of conclusion. At present, the research of the literature is not much about the research of the fingerprint spectrum of the kadsura longepedunculata, so that the invention adopts an ultra-performance liquid chromatography (UPLC method) to establish the characteristic spectrum of the kadsura longepedunculata and can quickly and accurately identify the medicinal materials of the kadsura longepedunculata.
Disclosure of Invention
The invention aims to provide a construction method of a UPLC characteristic spectrum of a Kadsura japonica medicinal material and an identification method thereof, the method is rapid, stable and strong in specificity, can more comprehensively reflect the characteristics of the Kadsura japonica medicinal material, and provides a scientific and new method for quality control of the Kadsura japonica medicinal material.
The technical problem to be solved by the invention is realized by the following technical scheme:
a construction method of a UPLC characteristic spectrum of a kadsura longepedunculata medicinal material comprises the following steps:
(1) precisely weighing the kadsura longepedunculata medicinal material, and preparing a kadsura longepedunculata medicinal material test solution;
(2) analyzing the sample solution of the Kadsura longepedunculata medicinal material by using an ultra-high performance liquid chromatograph to obtain a UPLC characteristic spectrum of the Kadsura longepedunculata medicinal material.
Preferably, the chromatographic conditions for the ultra high performance liquid chromatograph analysis are as follows: using octadecylsilane chemically bonded silica as a filling agent, acetonitrile as a mobile phase A, and 0.05-0.15% phosphoric acid aqueous solution as a mobile phase B, and performing gradient elution at a flow rate of 0.25-0.35 mL/min, a column temperature of 25-35 ℃, a detection wavelength of 210-250 nm, and a sample introduction amount of 0.6-1.8 μ l.
As a most preferred scheme, the chromatographic conditions for the hplc analysis are: using octadecylsilane chemically bonded silica as filler, acetonitrile as mobile phase A, and 0.1% phosphoric acid aqueous solution as mobile phase B, and performing gradient elution at flow rate of 0.3mL/min, column temperature of 30 deg.C, detection wavelength of 230nm, and sample amount of 1 μ l.
As a preferred embodiment, the gradient elution conditions are: the volume fraction of the mobile phase A is changed to 2-10% and the volume fraction of the mobile phase B is changed to 98-90% in 0-10 min; 10-11 min, the volume fraction of the mobile phase A is changed to 10-51%, and the volume fraction of the mobile phase B is changed to 90-49%; the volume fraction of the mobile phase A is changed to 51-54% and the volume fraction of the mobile phase B is changed to 49-46% in 11-26 min; 26-40 min, the volume fraction of the mobile phase A is changed to 54-95%, and the volume fraction of the mobile phase B is changed to 46-5%; the volume fraction of the mobile phase A is changed to 95-2% and the volume fraction of the mobile phase B is changed to 5-98% in 40-40.01 min; and (3) 40.01-45 min, wherein the volume fraction of the mobile phase A is 2%, and the volume fraction of the mobile phase B is 98%.
As a preferable scheme, the test solution is prepared by the following steps: precisely weighing 0.3-0.7 g of kadsura longepedunculata powder, placing the powder in a container, precisely adding 40-60 ml of 60-80% ethanol, weighing, carrying out ultrasonic treatment for 20-40 minutes, cooling, weighing again, supplementing the lost weight with 60-80% ethanol, shaking up, filtering, and taking the subsequent filtrate to obtain the kadsura longepedunculata powder.
As a most preferred embodiment, the test solution is prepared by the following steps: weighing 0.5g of fructus Schisandrae Sphenantherae powder, precisely weighing, placing in a container, precisely adding 50ml of 70% ethanol, weighing, ultrasonically treating for 30 min, cooling, weighing again, supplementing the lost weight with 70% ethanol, shaking, filtering, and collecting the filtrate.
The invention also provides an identification method of the schisandra sphenanthera medicinal material, which comprises the following steps:
(1) precisely weighing the kadsura longepedunculata medicinal material to be identified, and preparing a kadsura longepedunculata medicinal material sample solution to be identified;
(2) precisely absorbing the kadsura longepedunculata medicinal material sample solution to be identified, injecting the solution into an ultra-high performance liquid chromatograph, and measuring to obtain a UPLC characteristic spectrum of the kadsura longepedunculata medicinal material to be identified;
(3) and comparing the UPLC characteristic spectrum with the constructed UPLC characteristic spectrum of the Kadsura longepedunculata medicinal material, and if the UPLC characteristic spectrum is consistent with the UPLC characteristic spectrum of the Kadsura longepedunculata medicinal material, determining the Kadsura longepedunculata medicinal material with qualified quality.
Preferably, the chromatographic conditions for the ultra high performance liquid chromatograph analysis are as follows: using octadecylsilane chemically bonded silica as a filling agent, acetonitrile as a mobile phase A, and 0.05-0.15% phosphoric acid aqueous solution as a mobile phase B, and performing gradient elution at a flow rate of 0.25-0.35 mL/min, a column temperature of 25-35 ℃, a detection wavelength of 210-250 nm, and a sample introduction amount of 0.6-1.8 μ l.
As a most preferred scheme, the chromatographic conditions for the hplc analysis are: using octadecylsilane chemically bonded silica as filler, acetonitrile as mobile phase A, and 0.1% phosphoric acid aqueous solution as mobile phase B, and performing gradient elution at flow rate of 0.3mL/min, column temperature of 30 deg.C, detection wavelength of 230nm, and sample amount of 1 μ l.
As a preferred embodiment, the gradient elution conditions are: the volume fraction of the mobile phase A is changed to 2-10% and the volume fraction of the mobile phase B is changed to 98-90% in 0-10 min; 10-11 min, the volume fraction of the mobile phase A is changed to 10-51%, and the volume fraction of the mobile phase B is changed to 90-49%; the volume fraction of the mobile phase A is changed to 51-54% and the volume fraction of the mobile phase B is changed to 49-46% in 11-26 min; 26-40 min, the volume fraction of the mobile phase A is changed to 54-95%, and the volume fraction of the mobile phase B is changed to 46-5%; the volume fraction of the mobile phase A is changed to 95-2% and the volume fraction of the mobile phase B is changed to 5-98% in 40-40.01 min; and (3) 40.01-45 min, wherein the volume fraction of the mobile phase A is 2%, and the volume fraction of the mobile phase B is 98%.
As a preferable scheme, the test solution is prepared by the following steps: precisely weighing 0.3-0.7 g of kadsura longepedunculata powder, placing the powder in a container, precisely adding 40-60 ml of 60-80% ethanol, weighing, carrying out ultrasonic treatment for 20-40 minutes, cooling, weighing again, supplementing the lost weight with 60-80% ethanol, shaking up, filtering, and taking the subsequent filtrate to obtain the kadsura longepedunculata powder.
As a most preferred embodiment, the test solution is prepared by the following steps: weighing 0.5g of fructus Schisandrae Sphenantherae powder, precisely weighing, placing in a container, precisely adding 50ml of 70% ethanol, weighing, ultrasonically treating for 30 min, cooling, weighing again, supplementing the lost weight with 70% ethanol, shaking, filtering, and collecting the filtrate.
Has the advantages that: (1) the method adopts a UPLC method, can fundamentally, quickly and accurately identify and distinguish the kadsura longepedunculata, and has certain characteristics; (2) the method is stable, has strong specificity and stable baseline, and provides a scientific new method for controlling the quality of the kadsura longepedunculata medicinal material; (3) the invention determines 7 characteristic peaks in total by measuring 13 batches of kadsura longepedunculata medicinal materials in different producing areas, identifies chemical components of 7 common peaks, takes the
Drawings
FIG. 1 is a UPLC characteristic spectrum overlay of 13 batches of Kadsura longepedunculata medicinal materials;
FIG. 2 is a UPLC comparison characteristic spectrum of 13 batches of Kadsura longepedunculata medicinal material;
FIG. 3 is an identification chart of UPLC chromatographic peak reference sample of Kadsura longepedunculata medicinal material;
FIG. 4 is a chemical structure diagram of schisantherin A;
FIG. 5 is a chemical structural diagram of schisantherin B;
FIG. 6 is a chemical structure diagram of schisantherin C;
FIG. 7 is a chemical structure diagram of schisantherin D;
FIG. 8 is a chemical structure diagram of deoxyschizandrin;
FIG. 9 is a chemical structure diagram of protocatechuic acid;
FIG. 10 is a chemical structure diagram of anwurtzitaxenic acid;
FIG. 11 is UPLC characteristic spectrum of Kadsura longepedunculata medicinal material to be identified.
The labels in the figure are: the
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but 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.
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