CN112326835A - Method for measuring content of chlorogenic acid in vanilla - Google Patents

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for measuring the content of chlorogenic acid in vanilla planifolia, which comprises the following steps: taking a proper amount of chlorogenic acid reference substance, adding 50% methanol to prepare a solution containing 4 mu g of chlorogenic acid per 1ml, taking 0.5g of dried and crushed herba valerianae latifoliae powder, placing the powder in a conical flask with a plug, adding 50% methanol into the conical flask, weighing the powder, carrying out ultrasonic treatment for 30 minutes at 53KHz, cooling the powder, weighing the powder again, supplementing the lost weight with 50% methanol, shaking the powder evenly, filtering the solution, precisely taking 5ml of subsequent filtrate, placing the subsequent filtrate in a 25ml brown measuring flask, adding 50% methanol to the scale, shaking the subsequent filtrate evenly, precisely sucking 10 mu l of the reference substance solution and 10 mu l of the test substance solution respectively, injecting the subsequent solutions into a liquid chromatograph, and measuring the subsequent solutions to obtain the chlorogenic acid; the method for measuring the content of chlorogenic acid in the vanilla planifolia has the advantages of simple and convenient operation, reliable method, accurate result and strong specificity, and can effectively improve the quality control level of the cymbidium planifolia.

Description

Method for measuring content of chlorogenic acid in vanilla

Technical Field

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for measuring the content of chlorogenic acid in vanilla planifolia.

Background

The afternoon vanilla is derived from dried whole herb of Chang, Anaphylis belleyana (J.F. Jeffr.) belonging to Compositae. Pungent, slightly bitter and warm in flavor. It enters lung and stomach meridians. Has effects of dispelling pathogenic wind and cold, relieving cough, eliminating phlegm, regulating stomach function and relieving diarrhea. Can be used for treating wind-cold type common cold, cough with excessive phlegm, and pharyngalgia; abdominal distension, abdominal pain, diarrhea, dysentery.

The active components or index components of the vanilla at noon are not detected in the current standard, and the report of analysis of the vanilla at noon is not found through retrieval, so that the pertinence and the accuracy of the quality control of the vanilla at noon are poor, and the quality of medicinal materials is difficult to control effectively. Through long-term research and detection of the applicant, the active ingredients in the cymbopogon flexuosus are mainly chlorogenic acid, so that corresponding research on the content determination is needed if the medicinal effect of the cymbopogon flexuosus is further researched, the content determination method of the chlorogenic acid has many methods, but many substances are similar to the molecular structure of the chlorogenic acid and exist in medicinal materials such as the cymbopogon flexuosus and the like, so the separation is often difficult in the content determination, the common determination methods comprise an ultraviolet spectrophotometry method, a high performance liquid chromatography method and the like, and the conventional methods either are difficult to completely separate the substances to be determined, are easy to interfere, or have long analysis time and low efficiency, so that the unique chlorogenic acid content determination method suitable for the cymbopogon flexuosus needs to be developed.

Disclosure of Invention

The invention aims to provide a unique chlorogenic acid content determination method suitable for the characteristics of cymbopogon flexuosus, which can not only separate substances to be determined more thoroughly, but also avoid interference and improve the determination efficiency.

In order to achieve the purpose, the invention adopts the technical scheme that: the method for measuring the content of chlorogenic acid in vanilla planifolia specifically comprises the following steps:

1) chromatographic conditions and system applicability experiments take octadecylsilane chemically bonded silica as a filler, acetonitrile-0.4% phosphoric acid (13:87) as a mobile phase, the detection wavelength is 328nm, and the number of theoretical plates is more than or equal to 3000 calculated according to the peak of chlorogenic acid;

2) preparation of control solutions: precisely weighing chlorogenic acid reference substance, placing into brown measuring flask, and adding 50% methanol to obtain solution containing 4 μ g per 1 ml;

3) preparation of a test solution: taking 0.5g of hyssop officinalis powder (screened by a No. three sieve), precisely weighing, placing in a conical flask with a plug, adding 50ml of 50% methanol, weighing, carrying out ultrasonic treatment for 30 minutes at 53KHz, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, precisely weighing 5ml of subsequent filtrate, placing in a brown measuring flask with 25ml, adding 50% methanol to scale, and shaking up to obtain the compound preparation;

4) the determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

The beneficial technical effects of the invention are as follows:

the Wuxiangcao is a commonly used medicinal material in Yi nationality, and is collected in Yi nationality's medicine of Yunnan province medicinal material Standard 2005 edition, and the content determination item is not specified in the Standard, and at the same time, the Wuxiangcao is also the current oneNo correlation with measurement of the content of vanilla at noon was found. Chlorogenic acid has antibacterial, antiviral, immunity enhancing, peptic ulcer resisting, liver protecting, and antiinflammatory effects^]The like, has the effects of expelling wind and removing cold with the hyssop officinalis, has the effects of relieving cough and reducing sputum, harmonizing stomach and stopping diarrhea, and is used for treating wind-cold type common cold, cough with excessive phlegm and pharyngalgia; the therapeutic effects of abdominal distension and pain, diarrhea and dysentery are positively correlated and are main active ingredients, and through research, the method can be used as a main quality index of the cymbopogon flexuosus and can be used for quality control of cymbopogon flexuosus medicinal materials and quality evaluation of the cymbopogon flexuosus medicinal materials, decoction pieces and related products.

Detailed Description

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 only a part of the embodiments of the present invention, and not all 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.

The embodiments of the invention relate to samples and reagents and devices as follows:

1. sample source: the samples are respectively collected from Chuxiong Zhou Dayao county and Dalizhou Xiangyun county in Yunnan province, are identified as the cymbopogon flexuosus by professional technicians of companies, are extracted and identified to be qualified, are sent to Kunming plant research institute of Chinese academy of sciences, are identified as the compositae plant, namely the cymbopogon flexuosus, and are consistent with the expression of the cymbopogon flexuosus basic source.

2. And (3) reagent sources: methanol (analytically pure, Tianjin, Fengshanji chemical reagent science and technology limited), water (purified water, manufactured by company), ethanol (analytically pure, Tianjin, Fengshanji chemical reagent science and technology limited), phosphoric acid (analytically pure, Tianjin, Fengshanji chemical reagent science and technology limited), glacial acetic acid (analytically pure, Tianjin, Funing Fine chemical industry limited), methanol (chromatographically pure, Saimer Feishehiji science and technology (China) limited), and acetonitrile (chromatographically pure, Sigma Aldrich (Shanghai) trade limited).

3. Description of main instruments: the HPLC is Agilent 1100 (Agilent USA), the electronic balance is Mettler AB 204-N and MS105 (Mettler-Torilduo instruments, Inc.), the dual-beam UV-visible photometer is UV-1800APC (Shanghai Meiji instruments, Inc.), and the instruments are qualified and in the valid period.

Example (b):

the method for measuring the content of chlorogenic acid in vanilla planifolia specifically comprises the following steps:

1) chromatographic conditions and system applicability experiments take octadecylsilane chemically bonded silica as a filler, acetonitrile-0.4% phosphoric acid (13:87) as a mobile phase, the detection wavelength is 328nm, and the number of theoretical plates is more than or equal to 3000 calculated according to the peak of chlorogenic acid; wherein the content of the first and second substances,

selection of detection wavelength: respectively preparing chlorogenic acid solution by using acetonitrile-0.4% phosphoric acid solution (13:87), methanol-0.4% phosphoric acid solution and acetonitrile-0.5% glacial acetic acid solution, scanning by using an ultraviolet spectrophotometer within the range of 200 plus 400nm, and selecting 328nm as the detection wavelength;

selection of mobile phase: preparing a sample, and respectively using the three mobile phases for determination, wherein the acetonitrile-0.4% phosphoric acid solution (13:87) has less impurity peaks, good resolution and high determination content, so the acetonitrile-0.4% phosphoric acid solution (13:87) is selected as the mobile phase of the method;

2) preparation of control solutions: precisely weighing chlorogenic acid reference substance, placing into brown measuring flask, and adding 50% methanol to obtain solution containing 4 μ g per 1 ml;

3) preparation of a test solution: taking 0.5g of hyssop officinalis powder (a third sieve), precisely weighing, placing in a conical flask with a plug, adding 50ml of 50% methanol, weighing, carrying out ultrasonic treatment for 30 minutes at 53KHz, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, precisely weighing 5ml of subsequent filtrate, placing in a brown measuring flask with 25ml, adding 50% methanol to scale, and shaking up to obtain the compound preparation;

4) the determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

Selection of sample preparation methods in the examples: the medicinal part of the product is whole herb, so the whole plant sample is selected, crushed and screened by a third sieve, water, methanol and ethanol are respectively used as solvents, the cold soaking method, the reflux method and the ultrasonic extraction method are used for carrying out experiments, and finally, the ultrasonic treatment by 50 percent methanol is carried out for 40 minutes to obtain the product with high content, good separation degree and few impurity peaks.

The experiments related to the embodiments of the present invention are illustrated as follows:

1. investigation of Linear relationships

Precisely sucking 5, 10, 15, 20, 25, 30, 35, 40 and 45 mul of chlorogenic acid reference solution (4.232 mug/ml), respectively injecting into a liquid chromatograph, measuring according to preset chromatographic conditions, performing linear regression by using peak area and sample volume, drawing a standard curve by using the sample volume (mug) as an abscissa and the integrated value of the peak area of the reference as an ordinate, wherein the regression equation is that Y is 2541.1x +17.68(r is 0.9990), and the sample volume range is: the linearity is good between 0.02116-0.19044 ug.

Control concentration (4.232. mu.g/ml)

2. Precision test

The control solution (10. mu.l) was precisely aspirated, the sample injection was repeated 5 times in succession under predetermined chromatographic conditions, the peak area was measured, and RSD (%) was calculated to be 0.61%.

Control concentration (4.232. mu.g/ml)

3. Stability test

Weighing herba Cymbopogonis Citrari 0.5g, precisely weighing, preparing solution according to the preparation method of test solution, measuring once every 2 hr for 7 times, and calculating RSD (%) according to the measured peak area. RSD ═ 0.014%.

4. Repeatability test

Weighing 0.5g of vanilla planifolia, processing 5 parts of vanilla planifolia in parallel, precisely weighing, preparing solution according to the preparation method of the test solution, measuring according to the preset chromatographic condition, and calculating the RSD (%) of the content according to the peak area. RSD is 0.08%.

5. Sample application recovery test

Weighing 0.25g of vanilla planifolia, parallelly processing 9 parts, precisely weighing, adding an appropriate amount of chlorogenic acid reference solution, preparing a solution according to a method for preparing a test solution, and measuring according to a preset chromatographic condition, wherein the recovery rate is 101.3-106.8%, the average recovery rate is 104.5%, and the RSD is 1.74%, and the result meets the requirement.

Sample preparation: fragrant hair, producing area: yao county lot number 20191108; the content is measured as follows: 0.504%.

Chlorogenic acid control solution: 0.1326mg/ml

6. Reproducibility test

Three batches of the vanilla samples were tested under the above test conditions in the laboratory of Yunan Jinbi pharmaceutical center (Column model: design OOG-4252-EO Luna 5. mu. m C18(2)100A LC Column 250X 4.6mm Yue Xue), Chuxiong State food and drug laboratory, Chuxiong medical research institute (Shimadzu LC-2030 high performance liquid chromatograph. Column model: Shim-pack VP-ODS analytical Column, C185. mu.l, 4.6mm X25 cm), and the content was calculated and RSD (%) was calculated. RSD is respectively 1.93%, 4.43% and 3.57%, and meets the requirement.

Measuring 15 batches of samples in different production areas according to the measuring method, and according to the measuring result, the content limit of chlorogenic acid in the product is defined as that the product contains chlorogenic acid (C) calculated according to the dry product₁₆H₁₈O₉) Not less than 0.18%.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (1)

1. The method for measuring the content of chlorogenic acid in vanilla planifolia is characterized by comprising the following steps:

1) chromatographic conditions and system applicability experiments take octadecylsilane chemically bonded silica as a filler, acetonitrile-0.4% phosphoric acid (13:87) as a mobile phase, the detection wavelength is 328nm, and the number of theoretical plates is more than or equal to 3000 calculated according to the peak of chlorogenic acid;

2) preparation of control solutions: precisely weighing chlorogenic acid reference substance, placing into brown measuring flask, and adding 50% methanol to obtain solution containing 4 μ g per 1 ml;

3) preparation of a test solution: taking 0.5g of hyssop officinalis powder (screened by a No. three sieve), precisely weighing, placing in a conical flask with a plug, adding 50ml of 50% methanol, weighing, carrying out ultrasonic treatment for 30 minutes at 53KHz, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, precisely weighing 5ml of subsequent filtrate, placing in a brown measuring flask with 25ml, adding 50% methanol to scale, and shaking up to obtain the compound preparation;

4) the determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.