CN111505172A

CN111505172A - Method for measuring content of 10-DAB III in branches and leaves of taxus chinensis

Info

Publication number: CN111505172A
Application number: CN202010515399.2A
Authority: CN
Inventors: 蒋元烨; 曹勤; 韩吉; 林辉; 田继云; 李美莺; 冯明丽; 耿赟; 公培强
Original assignee: SHANGHAI PUTUO DISTRICT CENTRAL HOSPITAL
Current assignee: SHANGHAI PUTUO DISTRICT CENTRAL HOSPITAL
Priority date: 2020-06-08
Filing date: 2020-06-08
Publication date: 2020-08-07

Abstract

The invention relates to a method for measuring the content of 10-DAB III in branches and leaves of taxus chinensis, which adopts a water-acetone (50:50) mixed solvent for ultrasonic extraction, adopts a 2.1 × mm,1.7 mu m phenyl column, and adopts the flow rate of 0.3-0.6m L/min.

Description

Method for measuring content of 10-DAB III in branches and leaves of taxus chinensis

Technical Field

The invention relates to the technical field of taxus chinensis branch and leaf content determination, in particular to a method for determining the content of 10-DAB III in taxus chinensis branch and leaf.

Background

Taxus chinensis is an ancient natural rare anticancer plant, belongs to Taxus genus (Taxus genus) of Taxaceae (Taxaceae), and has a history of 250 ten thousand years on earth, Taxus chinensis is a unique variety in China, is a Taxus plant with fastest growth and most extensive distribution, Taxol (Taxol) found in Taxus chinensis var mairei at present is one of the effective anticancer substances recognized in the world, is distributed in roots, barks, stems and leaves of Taxus chinensis var mairei, but has limited content, 10-deacetylbaccatin III (10-DAB) is an index component of Taxus chinensis var mairei, is rich in content, can be used for synthesizing Taxol or docetaxel with stronger activity, and solves the problem of limited Taxol resources to a certain extent.

Chinese patent document CN104892550A discloses a method for extracting and separating 10-deacetylbaccatin III (10-DAB III) from taxus chinensis. Chinese patent document CN1473821A discloses a method for extracting and purifying 10-deacetylbaccatin III (10-DAB III). However, the method for measuring the content of 10-DAB III in the branches and leaves of the taxus chinensis has no report at present about short analysis time, simple sample pretreatment method and good separation effect.

Disclosure of Invention

The first purpose of the invention is to provide a method for measuring the content of 10-DAB III in taxus chinensis branches and leaves aiming at the defects in the prior art.

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

a method for measuring the content of 10-DAB III in branches and leaves of taxus chinensis adopts a water-acetone (50:50) mixed solvent for ultrasonic extraction, a 2.1 × 100mm and 1.7 mu m phenyl column is adopted in the method, and the flow rate is 0.3-0.6m L/min.

The flow rate was 0.6m L/min.

The ultrasonic treatment conditions are 50W of power, 53kHz of frequency and 30 minutes of time.

The regression equation in linear relation examination is that y is 5357700x-10710, and r is 0.9999, which shows that 10-DAB III has good linearity in 0.0191-0.609 mg/m L.

The method has the advantages that the water-acetone (50:50) mixed solvent is adopted for ultrasonic extraction in the pretreatment method for measuring the content of 10-DAB III in the branches and leaves of the taxus chinensis, so that the extraction of effective components can be guaranteed, meanwhile, the impurities are few, the baseline is stable, and the separation degree and the peak shape are remarkably improved.

Drawings

FIG. 1 UP L C chromatogram (A) of a sample of Taxus chinensis;

FIG. 2 UP L C chromatogram (B) blank;

FIG. 3 UP L C chromatogram (C) standard.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

Example 1

1 Material

The instrument comprises Waters ACQIUTY UP L C H C L ASS, an ultrasonic cleaner, an electronic balance;

materials: 10-Dab iii control, taxus mairei leaf powder (18 mesh sieve); acetonitrile, formic acid (chromatographically pure, Thremo); ultrapure water, other analytical reagents were purchased from national sources.

2 methods and results are shown in fig. 1, fig. 2 and fig. 3.

2.1 preparation of control solutions

Accurately weighing 12.18mg of 10-Dab III reference substance into a volumetric flask with the volume of 20m L, adding methanol to a constant volume to scale, uniformly mixing to prepare a standard substance solution with the volume of 0.609mg/m L, and diluting to 0.3045,0.1523,0.762,0.381 and 0.191mg/m L.

2.2 chromatographic Condition and System suitability test

Chromatographic column, Acquity BEH Phenyl (2.1 × 100mm,1.7 μm) mobile phase acetonitrile (A) and water (B), gradient elution conditions of 0-0.315% A, 0.3-5.022% A, 5.0-6.095% A, 6.0-9.095% A, flow rate of 0.6m L min^-1The column temperature is 55 ℃, the ultraviolet detection wavelength is 227nm, the injection volume is 2 mu L, and the running time is 9 min.

2.3 selection of sample extraction solvent

(1) Weighing 3.33g of taxus chinensis var mairei leaf powder, placing the taxus chinensis var mairei leaf powder into a conical flask with a plug, respectively adding 100.0m L (50% acetone solution) (A), methanol (B), ethanol (C) and acetone (D), sealing the mouth of the conical flask, performing ultrasonic extraction for 30mins (with the power of 53khz) at room temperature, filtering the solution through a 0.22-mum filter membrane to obtain a sample, and performing sample injection for 2-mum L analysis.

2.4 Linear relationship investigation

According to the chromatographic conditions under item 2.2, respectively adding 10-Dab III standard substance solution 2 mu L with the concentrations of 0.609, 0.3045,0.1523, 0.0762, 0.0381 and 0.0191mg/m L, drawing a standard curve by taking the concentrations as abscissa and peak areas as ordinate, and finally obtaining a regression equation of y-5357700 x-10710 and r-5357700 x-107100.9999, indicating that 10-DAB III is at 0.0191_～0.609mg/m L was linear well.

2.5 precision test

Continuously feeding high, medium and low concentration standard solutions for 5 times according to the above chromatographic conditions, wherein RSD is 0.3, 0.2, 0.2%; the samples were injected continuously for 5 days, with RSD of 0.7, 0.9, 1.1%, indicating good precision within and during the day.

2.6 stability test

The sample solution of sample No. 1 is sampled and analyzed for 0, 1, 2, 4, 6, 8, 12 and 24 hours respectively, the RSD is 1.21%, and the result shows that the stability of the sample is good when the method is used for detection.

TABLE 1 stability test results

2.7 repeatability test

6 parts of the same batch of samples are taken in parallel, prepared according to a method of 2.3 and then subjected to sample injection analysis, and the result shows that the RSD is 1.13%. The method is applicable to extraction and analysis of 10-DAB III in the sample.

TABLE 2 results of the repeatability experiments

2.8 sample recovery test

Accurately weighing 6 parts of branches and leaves of the taxus mairei No. 1 sample with the determined content, wherein each part is about 1.67g, accurately adding 10-DAB III reference substance solution respectively to prepare a test solution, and calculating that the average recovery rate of 10-DAB III is 96.07 percent and the RSD is 1.42 percent.

TABLE 3 sample recovery test

2.9 measurement of 10-DAB III content in branches and leaves of southern Taxus chinensis

Precisely weighing 3.3g of 13 batches of taxus chinensis branch and leaf powder (screened by a second sieve) with different producing areas and categories, placing the powder into a conical flask with a stopper, precisely adding 100m L of 50% acetone solution, closing the stopper, weighing, carrying out ultrasonic treatment (power of 50W and frequency of 53kHz) for 30 minutes, cooling, weighing, complementing the weight with an extraction solution, shaking uniformly, centrifuging, taking supernatant, and carrying out sample injection analysis, wherein the result is shown in Table 4. the content difference of 10-DAB III in taxus chinensis leaf samples of different types is large, the content of the 5 # sample is the highest and is 1.483%, the content of the 8 # sample is only 0.178%, the content of 10-DAB III in taxus chinensis leaves of other batches is respectively about 1.1% and 0.5%, the content difference of the 10-DAB III in taxus chinensis leaves of different categories is different, chromatograms are different, and the established by the method can meet the content analysis of all taxus chinensis leaf samples of a new method.

TABLE 4 results of content measurement

Discussion:

1. at present, the pretreatment method for measuring the content of 10-DAB III in branches and leaves of taxus chinensis is complex. In the existing reports, methanol and ethanol are adopted for ultrasonic extraction, dichloromethane or normal hexane is used for extraction, then the mixture is evaporated to dryness to fix the volume, the steps are complex, and the chromatogram resolution is still poor. The invention adopts water-acetone (50:50) mixed solvent for ultrasonic extraction, which can ensure extraction of effective components, and has less impurities, stable baseline and significantly improved separation degree and peak shape.

2. The existing analysis method for determining 10-DAB III in taxus chinensis leaves mostly adopts HP L C, the analysis time is long, one sample can be analyzed within 25 minutes generally, the built ultra-high performance liquid chromatography UHP L C has the characteristics of high efficiency, rapidness and simplicity and convenience in operation, one sample can be analyzed within 9 minutes, the time is greatly saved, and the analysis efficiency is improved by nearly 3 times.

3. Flow rate we further improved the instrument parameters in the experiment, typically UHP L C flow rate 0.3m L/min, flow rate 0.3m L/min, and other conditions of analysis were not changed, 10-DAB iii peak time was about 9 minutes, sample analysis time was 20 minutes, based on this, we further pursued efficient sample analysis efficiency, increasing flow rate to 0.6m L/min, saving sample analysis time, and impurities did not interfere with sample detection (2) column temperature, because of the higher flow rate used in this method, column pressure could be reduced using high column temperature, increasing column lifetime, when flow rate 0.6m L/min, mobile phase was the initial proportion, column temperature was 30 ℃, column pressure was as high as 7400bar, under this condition sample analysis was more damaging to the instrument and column for a long time, when column temperature was increased to about 55 ℃, pressure was 5700bar, sample could be analyzed normally.

4. Selection of chromatographic column the chromatographic column used in this experiment was an Acquity BEH Phenyl (2.1 × 100mm,1.7 μm) Phenyl column, furthermore, we used a conventional C column₁₈And C₈When the chromatographic column is used for sample analysis, 10-DAB III is found in C₁₈The peak pattern was severely smeared during on-column analysis at C₈The peak is fast on the chromatographic column, and the impurity greatly interferes the detection.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims

1. A method for measuring the content of 10-DAB III in branches and leaves of taxus chinensis is characterized in that a water-acetone (50:50) mixed solvent is adopted for ultrasonic extraction, a 2.1 × 100mm 1.7 mu m phenyl column is adopted for the method, and the flow rate is 0.3-0.6m L/min.

2. The method of claim 1, wherein the flow rate is 0.6m L/min.

3. The method of claim 1, wherein: the ultrasonic treatment conditions are 50W of power, 53kHz of frequency and 30 minutes of time.

4. The method of claim 1, wherein: regression equation in linear relationship investigation: y-5357700 x-10710 and r-0.9999, indicating that 10-DAB III is at 0.0191_～0.609mg/m L was linear well.