CN112444572A - Method for measuring luteolin content in pteris crassipes by HPLC-DAD - Google Patents

Method for measuring luteolin content in pteris crassipes by HPLC-DAD Download PDF

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CN112444572A
CN112444572A CN201910817951.0A CN201910817951A CN112444572A CN 112444572 A CN112444572 A CN 112444572A CN 201910817951 A CN201910817951 A CN 201910817951A CN 112444572 A CN112444572 A CN 112444572A
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luteolin
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pteris
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胡文忠
侯梦阳
修志龙
石玉生
郝可欣
孙兴盛
孙姣
王宇
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Dalian Minzu University
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Abstract

The invention belongs to the field of detection and quantitative analysis of active ingredients of traditional Chinese medicines, and discloses a method for determining the content of luteoloside in pteris latifolia by HPLC-DAD. The method specifically comprises the following steps: (1) collecting, drying and crushing rough pteris multifida; (2) preparing a luteoloside standard product and drawing a standard curve; (3) preparing a sample solution; (4) and (4) measuring the content of the luteoloside in the sample solution. The determination method is simple and convenient to operate, high in sensitivity and good in repeatability, can quickly and accurately determine the content of the luteoloside in the pteris latifolia, and provides scientific basis for accurately evaluating and effectively controlling the quality of the pteris latifolia.

Description

Method for measuring luteolin content in pteris crassipes by HPLC-DAD
Technical Field
The invention belongs to the field of detection and quantitative analysis of active ingredients of traditional Chinese medicines, and relates to a method for determining the content of luteoloside in pteris latifolia by HPLC-DAD.
Background
Pteris cretinica var. laeta (wall. ex Ettingsh.) is a variety of Pteris cretinica of Pteridaceae, and grows in acidic soil with elevation of 900-; further, they are distributed in Vietnam, Cambodia, Nipol, India, etc. The Pteris crassipes (Linn.) Kunth has high ornamental and medicinal values, is bitter in taste and cold in nature, is used as a medicine by whole herbs, and is used for diminishing inflammation, stopping dysentery, relaxing muscles and tendons and relieving pain in folk. However, the research on the chemical components of the rough pteris latiusculi is still in a relatively preliminary stage at present, and the qualitative and quantitative analysis of the effective components is not reported.
Luteolin (luteolin-7-O-glucoside) is the main effective component of Pteris crassipes, and modern pharmacological research shows that luteolin has various pharmacological effects of relieving asthma, protecting liver, resisting tumor, resisting fungi, resisting oxidation, treating diabetes, protecting heart, etc. However, the chemical components of the rough pteris fern are complex, the separation of the luteolin is difficult, the target components and impurities are separated during detection, and the optimization of chromatographic conditions is very difficult. Therefore, the development of a measuring method which is simple and convenient to operate, efficient, rapid, accurate, reliable and good in repeatability is used for analyzing the content of the luteolin in the pteris latifolia thumb, and the method has extremely important significance for quality control, development and utilization of the pteris latifolia thumb medicinal material.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the method for measuring the content of the luteoloside in the rough pteris latifolia by HPLC-DAD, the measuring method is simple and convenient to operate, high in sensitivity and good in repeatability, the content of the luteoloside in the rough pteris latifolia can be quickly and accurately measured, and scientific basis is provided for accurately evaluating and effectively controlling the quality of the rough pteris latifolia.
The above purpose of the invention is realized by the following technical scheme:
a method for measuring luteolin content in Pteris crassipes by HPLC-DAD comprises the following steps:
(1) preparation of a sample: collecting crude pteris latifolia medicinal materials, cleaning, drying, crushing and screening;
(2) preparation of a test solution: weighing rough pteris latifolia powder and extracting with ultrasound assistance to obtain a test solution;
(3) preparing a reference substance stock solution: accurately weighing luteolin reference substance, and preparing a series of reference substance solutions with different concentrations by using ethanol;
(4) drawing a standard curve: carrying out HPLC-DAD detection analysis on the reference substance solutions with different concentrations to obtain peak areas under the reference substance solutions with different concentrations, and carrying out linear regression analysis on the concentrations and the peak areas to obtain a standard curve regression equation;
(5) determining the content of luteoloside in the test solution: taking the test solution to carry out HPLC-DAD detection analysis to obtain the peak area of the luteolin in the test solution, and then calculating the content of the luteolin according to the regression equation of the standard curve of the luteolin.
Collecting tissue sample materials of the rough pteris multifida plants in the step (1), cleaning the tissue sample materials by using deionized water, drying the tissue sample materials to constant weight, crushing the tissue sample materials and sieving the crushed tissue sample materials with a 100-mesh sieve.
Weighing 2.0g of sample powder obtained in the step (1) in the step (2), adding 100mL of 70% ethanol solution, performing ultrasonic extraction (200W, 40KHz) at 60 ℃ for 45min, cooling to room temperature, supplementing lost extraction solution with 70% ethanol solution, filtering, and filtering 1mL of filtrate with 0.45-micrometer organic microporous filter membrane to obtain a test solution.
Taking 14.2mg of a luteolin reference substance in the step (3), putting the luteolin reference substance in a 100mL volumetric flask, adding ethanol to dissolve the luteolin reference substance and fixing the volume to a scale, and completely dissolving and shaking up to obtain a reference substance stock solution A (0.142 mg/mL); then, 1, 2, 3, 4, 5 and 6mL of the reference stock solution A are respectively measured and placed in a 25mL volumetric flask, ethanol is added to dissolve the reference stock solution A and the volume is determined to the scale, and a series of standard stock solutions (5.68, 11.36, 17.04, 22.72, 28.4 and 34.08 mu g/mL) are obtained after shaking up.
The chromatographic conditions adopted in the steps (4) and (5) are chromatographic columns: YMC-Pack ODS-A (250 mm. times.4.6 mm,5 μm); a detector: SPD-20A; mobile phase: phase A is 0.5 ‰ (V: V) trifluoroacetic acid-water solution, and phase B is methanol; gradient elution procedure: 0-30min, 30-60% B, 30-35min, 60-100% B, 35-40min, 100% B; sample introduction volume: 20 mu L of the solution; column temperature: room temperature; flow rate: 0.5 mL/min; detection wavelength: 350 nm.
Compared with the prior art, the invention has the beneficial effects that:
the HPLC-DAD method for determining the content of the luteoloside in the rough pteris multifida provided by the invention is simple, convenient, feasible, high in separation degree, efficient, time-saving, good in precision, stability and repeatability, accurate and reliable in result, and has important significance for controlling the quality of the rough pteris multifida.
Drawings
FIG. 1 is a chemical structural diagram of luteolin;
FIG. 2 is a standard curve of the luteolin control of example 1;
FIG. 3 is a diagram showing the ultraviolet absorption spectrum of luteolin;
FIG. 4 is an HPLC-DAD chromatogram of the test solution in example 1 at a detection wavelength of 350 nm.
FIG. 5 is an HPLC-DAD chromatogram of the test solution in comparative example 1 at a detection wavelength of 350 nm.
FIG. 6 is an HPLC-DAD chromatogram of the test solution in comparative example 2 at a detection wavelength of 350 nm.
FIG. 7 is an HPLC-DAD chromatogram of the test solution in comparative example 3 at a detection wavelength of 350 nm.
FIG. 8 is an HPLC-DAD chromatogram of the test solution in comparative example 4 at a detection wavelength of 350 nm.
FIG. 9 is an HPLC-DAD chromatogram of the test solution in comparative example 5 at a detection wavelength of 350 nm.
FIG. 10 is an HPLC-DAD chromatogram of the test solution in comparative example 6 at a detection wavelength of 350 nm.
FIG. 11 is an HPLC-DAD chromatogram of the test solution in comparative example 7 at a detection wavelength of 350 nm.
Detailed Description
The invention is described in more detail below with reference to specific examples, without limiting the scope of the invention. Unless otherwise specified, the experimental methods adopted by the invention are all conventional methods, and experimental equipment, materials, reagents and the like used in the experimental method can be obtained from commercial sources.
Instruments and reagents
1 apparatus
LC-20AD type high performance liquid chromatograph (equipped with SPD-M20A type DAD detector, Shimadzu corporation, Japan); KQ-5200DE type numerically controlled ultrasonic cleaner (Kunshan Shumei ultrasonic Instrument Co., Ltd.); quinix 35-1CN type electronic balance (Beijing Western Jie balance instruments Co., Ltd.).
2 materials and reagents
The samples were collected from Kunming city, Yunnan province and identified as Pteris cretica var.
Analytically pure ethanol, chromatographically pure methanol, chromatographically pure trifluoroacetic acid (Shanghai Aladdin Biotechnology Ltd.);
luteolin control (not less than 98%, Shanghai-sourced leaf Biotech limited); ultrapure water.
Example 1
A method for measuring luteolin content in Pteris crassipes by HPLC-DAD comprises the following steps:
(1) preparation of a sample: collecting tissue sample material of rough pteris latifolia plant, cleaning with deionized water, drying to constant weight, pulverizing, and sieving with 100 mesh sieve.
(2) Preparation of a test solution: taking rough pteris latifolia powder, and performing ultrasonic-assisted extraction to obtain a test solution;
weighing 2.0g of the sample powder obtained in the step (1), adding 100mL of 70% ethanol solution, carrying out ultrasonic extraction at 60 ℃ for 45min (200W, 40KHz), cooling to room temperature, supplementing lost extraction solution with 70% ethanol solution, filtering, and filtering 1mL of filtrate through a 0.45-micrometer organic microporous filter membrane to obtain a sample solution.
(3) Preparing a reference substance stock solution: accurately weighing 14.2mg of luteolin control, placing in a 100mL volumetric flask, adding ethanol for dissolving, fixing the volume to scale, dissolving completely, and shaking to obtain control stock solution A (0.142 mg/mL); then, 1, 2, 3, 4, 5 and 6mL of the reference stock solution A are respectively measured and placed in a 25mL volumetric flask, ethanol is added to dissolve the reference stock solution A and the volume is determined to the scale, and a series of standard stock solutions (5.68, 11.36, 17.04, 22.72, 28.4 and 34.08 mu g/mL) are obtained after shaking up.
(4) Drawing a standard curve: taking the reference substance solutions with different concentrations in the step (3) to perform HPLC-DAD detection analysis to obtain peak areas under the reference substance solutions with different concentrations, performing linear regression analysis by taking the mass concentration of the luteolin as a horizontal coordinate (x) and the peak area (y), and solving a regression equation of a standard curve of the luteolin as follows: y is 71752.8199x +117499.2933, and has a good linear relation (R is in a range of the mass concentration of the luteolin between 5.68 and 34.08 mu g/mL)2＝0.9998)。
The chromatographic conditions used for the determination were as follows:
a chromatographic column: YMC-Pack ODS-A (250 mm. times.4.6 mm,5 μm); a detector: SPD-20A; mobile phase: phase A is 0.5 ‰ (V: V) trifluoroacetic acid-water solution, and phase B is methanol; gradient elution procedure: 0-30min, 30-60% B, 30-35min, 60-100% B, 35-40min, 100% B; sample introduction volume: 20 mu L of the solution; column temperature: room temperature; flow rate: 0.5 mL/min; detection wavelength: 350 nm.
(5) Determining the content of luteoloside in the test solution: and (4) carrying out HPLC-DAD detection analysis on the test solution, testing the conditions in the same step (4), obtaining the peak area of the luteolin in the test solution, and then calculating the content of the luteolin according to a standard curve regression equation of the luteolin.
(6) And (3) precision test: taking the reference substance stock solution A, continuously measuring for 6 times in one day by using the chromatographic conditions in the step (3), recording the peak area of the luteolin, and calculating to obtain the Relative Standard Deviation (RSD) of 0.94%, which indicates that the method has good precision in one day; the RSD of the method is measured to be 1.17 percent after the RSD is measured for 3 days and 1 time every day, which shows that the precision degree of the method is better in the daytime.
(7) And (3) repeatability test: and (3) taking the sample solution in the step (2), continuously measuring for 6 times by using the chromatographic condition in the step (3), recording the peak area of the luteolin, and measuring to obtain the RSD of 0.83%, which shows that the method has good repeatability.
(8) And (3) stability test: and (3) taking the test solution in the step (2), measuring for 0 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours by using the chromatographic conditions in the step (3), recording the peak area of the luteolin, and measuring to obtain the RSD of 1.07 percent, which indicates that the test solution has better stability within 24 hours.
(9) Sample recovery rate test: precisely adding 6 parts of test solution with known content into the reference solution A, continuously measuring by using the chromatographic conditions in the step (3), recording the peak area of the luteolin, and measuring the average sample recovery rate of the luteolin to be 98.62% and the RSD to be 1.05%.
(10) And (3) sample determination: taking 6 batches of rough pteridium aquilinum samples, and carrying out sample injection detection according to the method. As a result, the content of luteolin in the 6 batches of samples was 3.738, 3.762, 3.767, 3.769, 3.784, 3.803 and 3.816mg/g, respectively.
Comparative example 1
(1) Preparation of a sample: collecting tissue sample material of rough pteris latifolia plant, cleaning with deionized water, drying to constant weight, pulverizing, and sieving with 100 mesh sieve.
(2) Preparation of a test solution: taking rough pteris latifolia powder, and performing ultrasonic-assisted extraction to obtain a test solution;
weighing 2.0g of the sample powder obtained in the step (1), adding 100mL of 80% ethanol solution, performing ultrasonic extraction at 55 ℃ for 1h, cooling to room temperature, supplementing lost extraction solution with 70% ethanol solution, filtering, and filtering 1mL of filtrate through a 0.45-micrometer organic microporous filter membrane to obtain a test sample solution.
(3) Preparing a reference substance stock solution: accurately weighing 14.2mg of luteolin control, placing in a 100mL volumetric flask, adding ethanol for dissolving, fixing the volume to scale, dissolving completely, and shaking to obtain control stock solution A (0.142 mg/mL); then, 1, 2, 3, 4, 5 and 6mL of the reference stock solution A are respectively measured and placed in a 25mL volumetric flask, ethanol is added to dissolve the reference stock solution A and the volume is determined to the scale, and a series of standard stock solutions (5.68, 11.36, 17.04, 22.72, 28.4 and 34.08 mu g/mL) are obtained after shaking up.
(4) Drawing a standard curve: and (4) carrying out HPLC-DAD detection analysis on the reference substance solutions with different concentrations in the step (3) to obtain peak areas of the reference substance solutions with different concentrations, and carrying out linear regression analysis on the peak areas (y) and the abscissa (x) by taking the mass concentration of the luteolin to obtain a regression equation of the standard curve of the luteolin.
The chromatographic conditions used for the determination were as follows:
a chromatographic column: YMC-Pack ODS-A (250 mm. times.4.6 mm,5 μm); a detector: SPD-20A; mobile phase: phase A is 0.1 ‰ (V: V) trifluoroacetic acid-water solution, and phase B is methanol; gradient elution procedure: 0-30min, 30-60% B, 30-35min, 60-100% B, 35-40min, 100% B; sample introduction volume: 20 mu L of the solution; column temperature: room temperature; flow rate: 0.5 mL/min; detection wavelength: 350 nm.
(5) Determining the content of luteoloside in the test solution: and (4) carrying out HPLC-DAD detection analysis on the test solution, testing the conditions in the same step (4), obtaining the peak area of the luteolin in the test solution, and then calculating the content of the luteolin according to a standard curve regression equation of the luteolin.
Comparative example 2
(1) Preparation of a sample: collecting tissue sample material of rough pteris latifolia plant, cleaning with deionized water, drying to constant weight, pulverizing, and sieving with 100 mesh sieve.
(2) Preparation of a test solution: taking rough pteris latifolia powder, and performing ultrasonic-assisted extraction to obtain a test solution;
weighing 2.0g of the sample powder obtained in the step (1), adding 100mL of 70% ethanol solution, carrying out ultrasonic extraction at 60 ℃ for 45min (200W, 40KHz), cooling to room temperature, supplementing lost extraction solution with 70% ethanol solution, filtering, and filtering 1mL of filtrate through a 0.45-micrometer organic microporous filter membrane to obtain a sample solution.
(3) Preparing a reference substance stock solution: accurately weighing 14.2mg of luteolin control, placing in a 100mL volumetric flask, adding ethanol for dissolving, fixing the volume to scale, dissolving completely, and shaking to obtain control stock solution A (0.142 mg/mL); then, 1, 2, 3, 4, 5 and 6mL of the reference stock solution A are respectively measured and placed in a 25mL volumetric flask, ethanol is added to dissolve the reference stock solution A and the volume is determined to the scale, and a series of standard stock solutions (5.68, 11.36, 17.04, 22.72, 28.4 and 34.08 mu g/mL) are obtained after shaking up.
(4) Drawing a standard curve: and (4) carrying out HPLC-DAD detection analysis on the reference substance solutions with different concentrations in the step (3) to obtain peak areas of the reference substance solutions with different concentrations, and carrying out linear regression analysis on the peak areas (y) and the abscissa (x) by taking the mass concentration of the luteolin to obtain a regression equation of the standard curve of the luteolin.
The chromatographic conditions used for the determination were as follows:
a chromatographic column: YMC-Pack ODS-A (250 mm. times.4.6 mm,5 μm); a detector: SPD-20A; mobile phase: phase A is trifluoroacetic acid-water solution with 0.1 per mill (V: V), phase B is acetonitrile; gradient elution procedure: 0-30min, 30-60% B, 30-35min, 60-100% B, 35-40min, 100% B; sample introduction volume: 20 mu L of the solution; column temperature: room temperature; flow rate: 0.5 mL/min; detection wavelength: 350 nm.
(5) Determining the content of luteoloside in the test solution: and (4) carrying out HPLC-DAD detection analysis on the test solution, testing the conditions in the same step (4), obtaining the peak area of the luteolin in the test solution, and then calculating the content of the luteolin according to a standard curve regression equation of the luteolin.
Comparative example 3
(1) Preparation of a sample: collecting tissue sample material of rough pteris latifolia plant, cleaning with deionized water, drying to constant weight, pulverizing, and sieving with 100 mesh sieve.
(2) Preparation of a test solution: taking rough pteris latifolia powder, and performing ultrasonic-assisted extraction to obtain a test solution;
weighing 2.0g of the sample powder obtained in the step (1), adding 100mL of 70% ethanol solution, carrying out ultrasonic extraction at 60 ℃ for 45min (200W, 40KHz), cooling to room temperature, supplementing lost extraction solution with 70% ethanol solution, filtering, and filtering 1mL of filtrate through a 0.45-micrometer organic microporous filter membrane to obtain a sample solution.
(3) Preparing a reference substance stock solution: accurately weighing 14.2mg of luteolin control, placing in a 100mL volumetric flask, adding ethanol for dissolving, fixing the volume to scale, dissolving completely, and shaking to obtain control stock solution A (0.142 mg/mL); then, 1, 2, 3, 4, 5 and 6mL of the reference stock solution A are respectively measured and placed in a 25mL volumetric flask, ethanol is added to dissolve the reference stock solution A and the volume is determined to the scale, and a series of standard stock solutions (5.68, 11.36, 17.04, 22.72, 28.4 and 34.08 mu g/mL) are obtained after shaking up.
(4) Drawing a standard curve: and (4) carrying out HPLC-DAD detection analysis on the reference substance solutions with different concentrations in the step (3) to obtain peak areas of the reference substance solutions with different concentrations, and carrying out linear regression analysis on the peak areas (y) and the abscissa (x) by taking the mass concentration of the luteolin to obtain a regression equation of the standard curve of the luteolin.
The chromatographic conditions used for the determination were as follows:
a chromatographic column: YMC-Pack ODS-A (250 mm. times.4.6 mm,5 μm); a detector: SPD-20A; mobile phase: phase A is trifluoroacetic acid-water solution with 0.5 per mill (V: V), phase B is acetonitrile; gradient elution procedure: 0-30min, 30-60% B, 30-35min, 60-100% B, 35-40min, 100% B; sample introduction volume: 20 mu L of the solution; column temperature: room temperature; flow rate: 0.5 mL/min; detection wavelength: 350 nm.
(5) Determining the content of luteoloside in the test solution: and (4) carrying out HPLC-DAD detection analysis on the test solution, testing the conditions in the same step (4), obtaining the peak area of the luteolin in the test solution, and then calculating the content of the luteolin according to a standard curve regression equation of the luteolin.
Comparative example 4
(1) Preparation of a sample: collecting tissue sample material of rough pteris latifolia plant, cleaning with deionized water, drying to constant weight, pulverizing, and sieving with 100 mesh sieve.
(2) Preparation of a test solution: taking rough pteris latifolia powder, and performing ultrasonic-assisted extraction to obtain a test solution;
weighing 2.0g of the sample powder obtained in the step (1), adding 100mL of 70% ethanol solution, carrying out ultrasonic extraction at 60 ℃ for 45min (200W, 40KHz), cooling to room temperature, supplementing lost extraction solution with 70% ethanol solution, filtering, and filtering 1mL of filtrate through a 0.45-micrometer organic microporous filter membrane to obtain a sample solution.
(3) Preparing a reference substance stock solution: accurately weighing 14.2mg of luteolin control, placing in a 100mL volumetric flask, adding ethanol for dissolving, fixing the volume to scale, dissolving completely, and shaking to obtain control stock solution A (0.142 mg/mL); then, 1, 2, 3, 4, 5 and 6mL of the reference stock solution A are respectively measured and placed in a 25mL volumetric flask, ethanol is added to dissolve the reference stock solution A and the volume is determined to the scale, and a series of standard stock solutions (5.68, 11.36, 17.04, 22.72, 28.4 and 34.08 mu g/mL) are obtained after shaking up.
(4) Drawing a standard curve: and (4) carrying out HPLC-DAD detection analysis on the reference substance solutions with different concentrations in the step (3) to obtain peak areas of the reference substance solutions with different concentrations, and carrying out linear regression analysis on the peak areas (y) and the abscissa (x) by taking the mass concentration of the luteolin to obtain a regression equation of the standard curve of the luteolin.
The chromatographic conditions used for the determination were as follows:
a chromatographic column: YMC-Pack ODS-A (250 mm. times.4.6 mm,5 μm); a detector: SPD-20A; mobile phase: phase A is 0.5 ‰ (V: V) acetic acid-water solution, and phase B is methanol; gradient elution procedure: 0-30min, 30-60% B, 30-35min, 60-100% B, 35-40min, 100% B; sample introduction volume: 20 mu L of the solution; column temperature: room temperature; flow rate: 0.5 mL/min; detection wavelength: 350 nm.
(5) Determining the content of luteoloside in the test solution: and (4) carrying out HPLC-DAD detection analysis on the test solution, testing the conditions in the same step (4), obtaining the peak area of the luteolin in the test solution, and then calculating the content of the luteolin according to a standard curve regression equation of the luteolin.
Comparative example 5
(1) Preparation of a sample: collecting tissue sample material of rough pteris latifolia plant, cleaning with deionized water, drying to constant weight, pulverizing, and sieving with 100 mesh sieve.
(2) Preparation of a test solution: taking rough pteris latifolia powder, and performing ultrasonic-assisted extraction to obtain a test solution; weighing 2.0g of the sample powder obtained in the step (1), adding 100mL of 70% ethanol solution, carrying out ultrasonic extraction at 60 ℃ for 45min (200W, 40KHz), cooling to room temperature, supplementing lost extraction solution with 70% ethanol solution, filtering, and filtering 1mL of filtrate through a 0.45-micrometer organic microporous filter membrane to obtain a sample solution.
(3) Preparing a reference substance stock solution: accurately weighing 14.2mg of luteolin control, placing in a 100mL volumetric flask, adding ethanol for dissolving, fixing the volume to scale, dissolving completely, and shaking to obtain control stock solution A (0.142 mg/mL); then, 1, 2, 3, 4, 5 and 6mL of the reference stock solution A are respectively measured and placed in a 25mL volumetric flask, ethanol is added to dissolve the reference stock solution A and the volume is determined to the scale, and a series of standard stock solutions (5.68, 11.36, 17.04, 22.72, 28.4 and 34.08 mu g/mL) are obtained after shaking up.
(4) Determining the content of luteoloside in the test solution: taking the test solution to carry out HPLC-DAD detection analysis to obtain the peak area of the luteolin in the test solution, and then calculating the content of the luteolin according to the regression equation of the standard curve of the luteolin.
The chromatographic conditions used for the determination were as follows:
a chromatographic column: YMC-Pack ODS-A (250 mm. times.4.6 mm,5 μm); a detector: SPD-20A; mobile phase: phase A is 0.5 ‰ (V: V) trifluoroacetic acid-water solution, and phase B is methanol; gradient elution procedure: 0-60min, 10-100% B; sample introduction volume: 20 mu L of the solution; column temperature: room temperature; flow rate: 0.5 mL/min; detection wavelength: 350 nm.
(5) Determining the content of luteoloside in the test solution: and (4) carrying out HPLC-DAD detection analysis on the test solution, testing the conditions in the same step (4), obtaining the peak area of the luteolin in the test solution, and then calculating the content of the luteolin according to a standard curve regression equation of the luteolin.
Comparative example 6
(1) Preparation of a sample: collecting tissue sample material of rough pteris latifolia plant, cleaning with deionized water, drying to constant weight, pulverizing, and sieving with 100 mesh sieve.
(2) Preparation of a test solution: taking rough pteris latifolia powder, and performing ultrasonic-assisted extraction to obtain a test solution; weighing 2.0g of the sample powder obtained in the step (1), adding 100mL of 70% ethanol solution, carrying out ultrasonic extraction at 60 ℃ for 45min (200W, 40KHz), cooling to room temperature, supplementing lost extraction solution with 70% ethanol solution, filtering, and filtering 1mL of filtrate through a 0.45-micrometer organic microporous filter membrane to obtain a sample solution.
(3) Preparing a reference substance stock solution: accurately weighing 14.2mg of luteolin control, placing in a 100mL volumetric flask, adding ethanol for dissolving, fixing the volume to scale, dissolving completely, and shaking to obtain control stock solution A (0.142 mg/mL); then, 1, 2, 3, 4, 5 and 6mL of the reference stock solution A are respectively measured and placed in a 25mL volumetric flask, ethanol is added to dissolve the reference stock solution A and the volume is determined to the scale, and a series of standard stock solutions (5.68, 11.36, 17.04, 22.72, 28.4 and 34.08 mu g/mL) are obtained after shaking up.
(4) Determining the content of luteoloside in the test solution: taking the test solution to carry out HPLC-DAD detection analysis to obtain the peak area of the luteolin in the test solution, and then calculating the content of the luteolin according to the regression equation of the standard curve of the luteolin.
The chromatographic conditions used for the determination were as follows:
a chromatographic column: YMC-Pack ODS-A (250 mm. times.4.6 mm,5 μm); a detector: SPD-20A; mobile phase: phase A is 0.5 ‰ (V: V) acetic acid-water solution, and phase B is methanol; gradient elution procedure: 0-15min, 20-30% B, 15-40min, 30-45% B, 40-50min, 45-70% B, 50-60min, 70-95% B, 60-70min, 95% B; sample introduction volume: 20 mu L of the solution; column temperature: room temperature; flow rate: 0.5 mL/min; detection wavelength: 350 nm.
(5) Determining the content of luteoloside in the test solution: and (4) carrying out HPLC-DAD detection analysis on the test solution, testing the conditions in the same step (4), obtaining the peak area of the luteolin in the test solution, and then calculating the content of the luteolin according to a standard curve regression equation of the luteolin.
Comparative example 7
(1) Preparation of a sample: collecting tissue sample material of rough pteris latifolia plant, cleaning with deionized water, drying to constant weight, pulverizing, and sieving with 100 mesh sieve.
(2) Preparation of a test solution: taking rough pteris latifolia powder, and performing ultrasonic-assisted extraction to obtain a test solution; weighing 2.0g of the sample powder obtained in the step (1), adding 100mL of 70% ethanol solution, carrying out ultrasonic extraction at 60 ℃ for 45min (200W, 40KHz), cooling to room temperature, supplementing lost extraction solution with 70% ethanol solution, filtering, and filtering 1mL of filtrate through a 0.45-micrometer organic microporous filter membrane to obtain a sample solution.
(3) Preparing a reference substance stock solution: accurately weighing 14.2mg of luteolin control, placing in a 100mL volumetric flask, adding ethanol for dissolving, fixing the volume to scale, dissolving completely, and shaking to obtain control stock solution A (0.142 mg/mL); then, 1, 2, 3, 4, 5 and 6mL of the reference stock solution A are respectively measured and placed in a 25mL volumetric flask, ethanol is added to dissolve the reference stock solution A and the volume is determined to the scale, and a series of standard stock solutions (5.68, 11.36, 17.04, 22.72, 28.4 and 34.08 mu g/mL) are obtained after shaking up.
(4) Determining the content of luteoloside in the test solution: taking the test solution to carry out HPLC-DAD detection analysis to obtain the peak area of the luteolin in the test solution, and then calculating the content of the luteolin according to the regression equation of the standard curve of the luteolin.
The chromatographic conditions used for the determination were as follows:
a chromatographic column: YMC-Pack ODS-A (250 mm. times.4.6 mm,5 μm); a detector: SPD-20A; mobile phase: phase A is 0.5 ‰ (V: V) acetic acid-water solution, and phase B is methanol; gradient elution procedure: 0-25min, 25% -45% B, 25-40min, 45-65% B, 40-60min, 65-90% B; sample introduction volume: 20 mu L of the solution; column temperature: room temperature; flow rate: 0.5 mL/min; detection wavelength: 350 nm.
(5) Determining the content of luteoloside in the test solution: and (4) carrying out HPLC-DAD detection analysis on the test solution, testing the conditions in the same step (4), obtaining the peak area of the luteolin in the test solution, and then calculating the content of the luteolin according to a standard curve regression equation of the luteolin.
As can be seen from the comparative example experimental result data chart, the quantitative and qualitative analysis of luteolin in the pteris Rohdea can be accurately separated only under the specific limited conditions of the extraction conditions and the chromatographic detection in the technical scheme of example 1 of the application; the method solves the problem that the heteropak substances in the pteris rough, which are close to the luteolin and are difficult to distinguish, has high separation degree and smooth peak shape, so that the luteolin in the pteris rough can be accurately quantitatively and qualitatively analyzed, and the existing problems that the separation degree is poor and the separation cannot be accurately carried out are overcome; the peak shape is not smooth and the accurate quantification can not be realized.
The embodiments described above are merely preferred embodiments of the invention, rather than all possible embodiments of the invention. Any obvious modifications to the above would be obvious to those of ordinary skill in the art, but would not bring the invention so modified beyond the spirit and scope of the present invention.

Claims (5)

1. A method for measuring the content of luteolin in Pteris crassipes by HPLC-DAD is characterized by comprising the following steps:
(1) preparation of a sample: collecting crude pteris latifolia medicinal materials, cleaning, drying, crushing and screening;
(2) preparation of a test solution: weighing rough pteris latifolia powder and extracting with ultrasound assistance to obtain a test solution;
(3) preparing a reference substance stock solution: accurately weighing luteolin reference substance, and preparing a series of reference substance solutions with different concentrations by using ethanol;
(4) drawing a standard curve: carrying out HPLC-DAD detection analysis on the reference substance solutions with different concentrations to obtain peak areas under the reference substance solutions with different concentrations, and carrying out linear regression analysis on the concentrations and the peak areas to obtain a standard curve regression equation;
(5) determining the content of luteoloside in the test solution: taking the test solution to carry out HPLC-DAD detection analysis to obtain the peak area of the luteolin in the test solution, and then calculating the content of the luteolin according to the regression equation of the standard curve of the luteolin.
2. The method for determining the content of luteolin in Pteridium crassimum by HPLC-DAD as claimed in claim 1, wherein the tissue sample material of Pteridium crassimum plant in step (1) is collected, washed with deionized water, dried to constant weight, pulverized and sieved with 100 mesh sieve.
3. The method for determining the content of luteolin in the pteris crassipes by HPLC-DAD as claimed in claim 1, wherein in the step (2), 2.0g of the sample powder obtained in the step (1) is weighed, 100mL of 70% ethanol solution is added, and ultrasonic: 200W, 40 KHz; extracting for 45min, cooling to room temperature, supplementing lost extraction solution with 70% ethanol solution, filtering, and filtering 1mL filtrate with 0.45 μm organic microporous membrane to obtain test solution.
4. The method for measuring the content of the luteolin in the pteris crassipes by HPLC-DAD as claimed in claim 1, wherein in the step (3), 14.2mg of the luteolin control is taken and placed in a 100mL volumetric flask, ethanol is added for dissolving, the volume is fixed to the scale, and after complete dissolution and shaking up, the reference stock solution A is obtained; then, 1mL, 2 mL, 3 mL, 4 mL, 5mL, 6mL of the reference stock solution a are measured respectively, placed in a 25mL volumetric flask, dissolved by adding ethanol and fixed to the volume to be calibrated, and shaken up to obtain a series of standard stock solutions.
5. The method for determining the content of luteolin in pteris crassipes by HPLC-DAD as claimed in claim 1, wherein the chromatographic conditions adopted in the steps (4) and (5) are that the chromatographic column: YMC-Pack ODS-A (250 mm. times.4.6 mm,5 μm); a detector: SPD-20A; mobile phase: phase A is 0.5 ‰ (V: V) trifluoroacetic acid-water solution, and phase B is methanol; gradient elution procedure: 0-30min, 30-60% B, 30-35min, 60-100% B, 35-40min, 100% B; sample introduction volume: 20 mu L of the solution; column temperature: room temperature; flow rate: 0.5 mL/min; detection wavelength: 350 nm.
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