CN108490095B - method for measuring contents of multiple components in gerbera piloselloides medicinal material - Google Patents
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Abstract
The invention relates to the technical field of traditional Chinese medicine detection, in particular to a method for determining the content of various components in gerbera pilosula medicinal material, which adopts a UPLC-PDA method and uses Waters BEH C18(2.1 × 150mm, 1.7 μm) as chromatographic column, gradient eluting with acetonitrile-0.1% formic acid water solution, and detectingThe length is 280nm and 328nm, the flow rate is 0.28mL/min, and the column temperature is 40 ℃. According to the method, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, luteolin and arbutin in the gerbera piloselloides medicinal material are well separated, the sample adding recovery rate of the method is 97.20-103.57%, and the RSD is less than or equal to 3.0%. The method is simple, convenient, accurate and good in repeatability, can be used for content determination of chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, luteolin and arbutin in the gerbera piloselloides medicinal material, and provides reference basis for quality control of the gerbera piloselloides.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine detection, in particular to a method for determining the content of various components in gerbera piloselloides medicinal material.
Background
The Gerberia pilosa is dry whole herb of Piloselloides hirsuta Hort (Forsk.) G.Jeffrey of Compositae, is also a drug for minority nationality in Guizhou province, and is named as 'added eight heavy Gerber (jab batnexjongxjud)' in Miao nationality medicine. The herba Gerberae Piloselloidis bitter and pungent in property and mild in taste, enters lung and stomach channels, has the effects of ventilating lung, relieving cough, inducing sweating, inducing diuresis, promoting qi circulation and activating blood circulation, and is mainly used for treating diseases such as cold cough, asthma, edema, dysuresia, infantile indigestion, amenorrhea, traumatic injury, carbuncle, furuncle and the like. According to records in Guiyang folk medicine, the large-flowered hedyotis herb is mainly used for treating diseases related to lung, such as wind cough, cough asthma, pulmonary abscess and the like. Modern pharmacological studies show that gerbera piloselloides has the effects of relieving cough, reducing sputum and relieving asthma.
At present, the gerbera piloselloides contains various phenolic acids and flavonoid components such as chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, luteolin and arbutin, and researches show that the components can be active components of the gerbera piloselloides. The gerbera piloselloides is collected in quality standards of 2003 edition of quality standards of traditional Chinese medicines and national medicines in Guizhou province, 2005 edition of quality standards of traditional Chinese medicine decoction pieces in Yunnan province, and the like, but the content of each beneficial component in the gerbera piloselloides is not determined, so that the quality standard of the gerbera piloselloides is not perfect at present, and the inherent quality of the gerbera piloselloides cannot be comprehensively controlled.
Therefore, it is urgent to find methods for simultaneously determining the content of various beneficial components in gerbera piloselloides medicinal material, such as chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, luteoloside and arbutin.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides a method for measuring the content of various components in gerbera piloselloides medicinal material, which comprises the following steps:
A method for measuring the contents of multiple components in Gerberaria pilosa (Roxb.) Craib for simultaneously measuring the contents of chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, luteoloside and arbutin in Gerberaria pilosa (Roxb) is characterized in that the UPLC-PDA method is used for measuring the contents of chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, luteoloside and arbutin in Gerberaria pilosa (18(2.1X 150mm, 1.7 μm) as a chromatographic column, and acetonitrile-0.1% formic acid aqueous solution was subjected to gradient elution while using detection wavelengths of 280nm and 328nm at a flow rate of 0.28mL/min at a column temperature of 40 ℃.
According to the UPLC-PDA method, the gerbera piloselloides medicinal material extracting solution is diluted to be used as a test solution.
The gerbera piloselloides medicinal material extracting solution is obtained by extracting a gerbera piloselloides medicinal material by using methanol as a solvent. The polarity of the methanol is higher than that of the ethanol, and the extraction rate is higher than that of the ethanol when the methanol is used for extracting effective components, so that the obtained result is more accurate.
The gerbera piloselloides medicinal material is extracted by taking methanol as a solvent, and the gerbera piloselloides medicinal material is subjected to reflux extraction by using 50% methanol, wherein the reflux extraction time is 2 hours. The extraction efficiency is higher at this moment, and the beneficial ingredients in the gerbera piloselloides medicinal material can not be damaged.
The dilution is to dilute the gerbera piloselloides medicinal material extracting solution by 5 times by using distilled water. The UPLC-PDA has better measuring effect after being diluted according to the proportion.
The gerbera piloselloides medicinal material is gerbera piloselloides medicinal material powder which is sieved by a third sieve. The gerbera piloselloides medicinal material powder which is sieved by a third sieve has better extraction effect.
Preferably, the UPLC-PDA method, wherein the test solution is prepared by: taking 0.5g of gerbera piloselloides medicinal material powder which passes through a third sieve, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 50% methanol, weighing, heating and refluxing for 2 hours, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, weighing 5mL of subsequent filtrate, transferring to a 25mL volumetric flask, diluting to a scale with distilled water, and shaking up to obtain a test solution. At the moment, the gerbera piloselloides medicinal material has higher extraction efficiency, the concentration of the test solution is moderate, and the measured result is more accurate.
The method comprises the following specific operation steps:
(1) preparing a reference substance solution and a test substance solution;
preparing reference solution, precisely weighing appropriate amounts of chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, luteolin and arbutin, respectively placing in 10mL measuring bottles, adding methanol to dissolve and fixing the volume to scale, respectively obtaining reference stock solutions with chlorogenic acid concentration of 1.80mg/mL, isochlorogenic acid A concentration of 0.987mg/mL, isochlorogenic acid B concentration of 0.963mg/mL, isochlorogenic acid C concentration of 0.963mg/mL, luteolin concentration of 0.991mg/mL and arbutin concentration of 1.92mg/mL, respectively precisely weighing the 6 reference stock solutions, diluting, placing in 10mL bottles, adding 50% methanol to dilute to scale, shaking to obtain mixed reference solution, wherein chlorogenic acid concentration is 9.00 μ g/mL, isochlorogenic acid A concentration is 24.7 μ g/mL, isochlorogenic acid B concentration is 9.63 g/mL, isochlorogenic acid C concentration is 9.63 g/mL, and arbutin concentration is 84.84 μ g/mL, and storing at 84. mu.00 μ g/mL.
Preparation of a test solution: taking about 0.5g of gerbera piloselloides medicinal material powder (screened by a third sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 50% methanol, weighing, heating and refluxing for 2 hours, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, weighing 5mL to 25mL of a volumetric flask of subsequent filtrate, diluting to a scale with distilled water, and shaking up to obtain a test solution.
(2) The determination process comprises the following steps: respectively taking mixed reference solution and sample solution, measuring with UPLC-PDA to obtain chromatogram, measuring arbutin at 280nm wavelength, measuring the rest 5 compounds at 328nm, and completely separating 6 components from other substance peaks. Wherein the chromatographic conditions are as follows: the chromatographic column is Waters BEH C18(2.1 × 150mm, 1.7 μm), gradient elution with acetonitrile-0.1% formic acid water solution as mobile phase, detection wavelength of 280nm and 328nm, flow rate of 0.28mL/min, column temperature of 40 deg.C, and sample injection amount of 10 μ L.
And step 3: and respectively carrying out linear relation investigation, precision experiment, repeatability experiment, stability experiment and sample-adding recovery rate experiment.
And (3) screening a sample preparation method:
in order to select a stable sample preparation method, the solvent, the extraction mode and the extraction time are used as variables, and the result of a screening test on the extraction method of the gerbera piloselloides is as follows:
from the above data, it can be seen that: when 50% methanol is used as a solvent, the extraction efficiency is higher and the impurities are less; compared with the water bath reflux extraction, the ultrasonic extraction has higher reflux extraction efficiency; the extraction effect of refluxing for 1h is poor, and the extraction effect of refluxing for 2h is not greatly different from that of refluxing for 3 h.
And (3) screening the measurement wavelength:
because the components to be detected in the gerbera piloselloides are all absorbed by ultraviolet, an ultraviolet detector is selected to detect the components to be detected.
Full wavelength scanning was performed by PDA detector, and the maximum absorption wavelength of each component was as follows:
| composition (I) | Wavelength of maximum absorption (nm) |
| Arbutin | 194.9、221.1、280.0 |
| Chlorogenic acid | 326.1 |
| Oleuropein | 347.9 |
| Isochlorogenic acid B | 328.0 |
| Isochlorogenic acid A | 327.4 |
| Isochlorogenic acid C | 328.6 |
From the data, the maximum absorption wavelength of arbutin is greatly different from the maximum absorption wavelength of other components, the detection sensitivity can be obviously influenced when the detection is carried out at the same wavelength, the content of 6 components is determined by selecting double wavelengths (280nm and 328nm) in a comprehensive consideration manner, arbutin is determined at 280nm, and the rest 5 components are determined under the condition of 328nm, so that the aim of simultaneously determining various chemical components of different types under the same chromatographic condition is fulfilled.
In order to select proper chromatographic conditions, different chromatographic columns, different mobile phase systems, different mobile phase pH and different mobile phase elution gradients are investigated, repeated tests are carried out for many times, and finally, a 0.1% formic acid aqueous solution and acetonitrile are selected as mobile phases to carry out gradient elution, so that 6 components such as arbutin, chlorogenic acid, galuteolin, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C and the like are well separated.
The experiment adopts an HPLC-PAD method to carry out content measurement on 6 chemical components of arbutin, chlorogenic acid, galuteolin, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C in the gerbera piloselloides, the method is simple and convenient to process, the 6 components are well separated under the same chromatographic condition, the linearity is good through methodology verification, the repeatability, the precision, the stability and the recovery rate are good, and the method can be used for controlling the quality of the gerbera piloselloides.
Compared with the prior art, the invention has the technical effects that:
the invention adopts a UPLC-PDA method and uses Waters BEH C18(2.1 × 150mm, 1.7 μm) as chromatographic column, gradient eluting with acetonitrile-0.1% formic acid water solution, detecting wavelength of 280nm and 328nm, flow rate of 0.28mL/min, and column temperature of 40 deg.C. According to the method, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, luteolin and arbutin in the gerbera piloselloides medicinal material are well separated, the sample adding recovery rate of the method is 97.20-103.57%, and the RSD is less than or equal to 3.0%. The method is simple, convenient, accurate and good in repeatability, can be used for content determination of chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, luteolin and arbutin in the gerbera piloselloides medicinal material, and provides reference basis for quality control of the gerbera piloselloides.
Drawings
Fig. 1 is a diagram of a system suitability experiment UPLC.
Wherein, the corresponding relation of each absorption peak is as follows: 1-arbutin; 2-chlorogenic acid; 3-luteolin; 4-isochlorogenic acid B; 5-isochlorogenic acid A; 6-isochlorogenic acid C.
Detailed Description
The technical solution of the present invention is further defined in step with reference to the specific embodiments, but the scope of the claims is not limited to the description.
Example (b):
instruments and materials:
the acquisition UPLC-PDA system (Waters, including a binary ultra-high pressure gradient pump, an autosampler, a chromatographic column incubator, an array diode detector, and an Empower workstation), an EL204 ten-thousandth electronic balance (Metler-Torlduo instruments Shanghai Co., Ltd.), an ultrapure water machine (Sichuan Walter science and technology development Co., Ltd.), and a DK-98-II type electric heating thermostat water bath (Tester instruments Co., Tianjin).
The arbutin reference (lot: 111951-201301), chlorogenic acid (lot: 110753) and luteolin (lot: 111720-201609) were purchased from the China institute for testing food and drug; isochlorogenic acid A (batch: MUST-16031611), isochlorogenic acid B (batch: MUST-16031612) and isochlorogenic acid C (batch: MUST-16031613) were purchased from Chengdumant Biotech GmbH/institute of academic sciences, China.
Methanol (analytically pure), ethanol (analytically pure) and acetonitrile (chromatographically pure) are purchased from chemical reagents of national drug group, ltd; formic acid (chromatographically pure) was purchased from TEDIA ltd. The experimental gerbera Piloselloides medicinal material is identified as dry whole herb of pilocarpus Piloselloides hirsuta (Forsk.) G.Jeffrey by crude drug of college of pharmacy of university of medical science of Guizhou and professor Zhanxu auxiliary professor of medicinal botanicals.
Preparation of control solution:
precisely weighing appropriate amounts of chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, luteolin and arbutin, respectively placing in 10mL measuring bottles, adding methanol to dissolve and fixing the volume to scale, respectively obtaining reference stock solutions with chlorogenic acid concentration of 1.80mg/mL, isochlorogenic acid A concentration of 0.987mg/mL, isochlorogenic acid B concentration of 0.963mg/mL, isochlorogenic acid C concentration of 0.963mg/mL, luteolin concentration of 0.991mg/mL and arbutin concentration of 1.92mg/mL, respectively diluting the 6 reference stock solutions, placing in 10mL measuring bottles, adding 50% methanol to dilute to scale, shaking to obtain a mixed reference solution, weighing chlorogenic acid concentration of 9.00 μ g/mL, isochlorogenic acid A concentration of 24.7 μ g/mL, isochlorogenic acid B concentration of 9.63 μ g/mL, isochlorogenic C concentration of 9.63 g/mL, and arbutin concentration of 84.84 μ g/mL, and storing at 84. deg.3 μ g/mL.
Preparing a test solution:
taking about 0.5g of crude gerbera piloselloides medicinal material powder passing through a third sieve, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 50% methanol, weighing, heating and refluxing for 2 hours, cooling, weighing again, supplementing the loss weight with 50% methanol, shaking up, filtering, measuring the subsequent filtrate in a volumetric flask with 5mL to 25mL, diluting to scale with distilled water, shaking up, filtering with a 0.22 mu m microporous membrane, taking the subsequent filtrate as a sample solution, and performing sample injection analysis.
Chromatographic conditions are as follows:
the chromatographic column is Waters BEH C18(2.1X 150mm, 1.7 μm) and acetonitrile (A) -0.1% formic acid aqueous solution (B) as a mobile phase, and the elution was carried out by gradient elution at detection wavelengths of 280nm and 328nm at a flow rate of 0.28mL/min at a column temperature of 40 ℃ and a sample volume of 10. mu.L, and the elution procedure is shown in Table 1.
TABLE 1 mobile phase gradient elution Table
And (3) system adaptability examination:
taking 5 μ L of the mixed reference solution and the sample solution respectively, measuring with UPLC-PDA under the above chromatographic conditions to obtain chromatogram, and completely separating 6 compounds from other substance peaks, as shown in figure 1.
2.4.2 Linear relationship investigation
The mixed control solutions 1, 2, 4, 6, 8 and 10. mu.L were precisely pipetted and measured under the above chromatographic conditions, and the sample volumes were plotted as abscissa and peak areas as ordinate to obtain working curves, as shown in Table 2.
TABLE 2 regression equation and Linear Range
And (3) precision experiment:
taking the same sample solution, carrying out continuous sample injection for 6 times, calculating the RSD of the peak areas of arbutin, chlorogenic acid, galuteolin, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C to be 0.82%, 1.9%, 1.7%, 1.4%, 1.5% and 1.3% respectively, and indicating that the precision of the instrument is good by result.
And (3) repeatability experiment:
taking 6 parts of the same batch medicinal materials, precisely weighing, preparing a test solution according to the preparation method of the test solution, respectively measuring the average content of arbutin, chlorogenic acid, luteolin, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C in the 6 parts of the test solution to be 0.728%, 0.0671%, 0.0197%, 0.0278%, 0.142% and 0.0390%, respectively obtaining the content results RSD of 1.7%, 2.3%, 1.7%, 1.0% and 1.8%, and indicating good repeatability.
Stability test:
respectively preparing the test solution according to the preparation method of the test solution, respectively injecting samples for 0, 2, 4, 8, 12 and 24 hours, and measuring the peak areas of arbutin, chlorogenic acid, galuteolin, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C as index components. The peak areas RSD were calculated to be 1.8%, 1.6%, 1.4%, 1.2%, 1.9%, 1.4%, respectively, indicating that the sample solutions were stable for 0 to 24 hours.
Sample recovery rate experiment:
weighing 9 parts of the sample amount which is 50 percent of the sample amount in the preparation method of the test solution, respectively measuring the content of each component in the gerbera piloselloides according to the repeatability to be the background value, respectively adding 50 percent, 100 percent and 150 percent of reference solution, respectively preparing 3 parts of the test solution according to the preparation method of the test solution at each level, measuring according to the chromatographic conditions and calculating the recovery rate of 6 components, and the result is shown in table 3. The average recovery rate of arbutin is 99.69%, the average recovery rate of RSD (%) is 1.7%, the average recovery rate of chlorogenic acid is 100.9%, the average recovery rate of RSD (%) is 1.9%, the average recovery rate of luteolin is 99.92%, the average recovery rate of RSD (%) is 1.8%, the average recovery rate of isochlorogenic acid A is 100.7%, the average recovery rate of RSD (%) is 1.2%, the average recovery rate of isochlorogenic acid B is 101.1%, the average recovery rate of RSD (%) is 1.9%, the average recovery rate of isochlorogenic acid C is 99.80%, and the average recovery rate of RSD (%) is 2.0%, which indicates that the.
TABLE 3 sample recovery test results
Sample assay
Gerbera piloselloides of different producing areas of Guizhou is collected, and the content of arbutin, chlorogenic acid, luteolin, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C is determined by the established method, and the result is shown in Table 4.
Table 4 content of 6 ingredients in different batches of gerbera piloselloides (%, n ═ 2)
From the above, the method of the invention can better separate chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, luteolin and arbutin in the gerbera piloselloides medicinal material, has the advantages of simplicity, accuracy and good repeatability, can be used for content determination of chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, luteolin and arbutin in the gerbera piloselloides medicinal material, and provides reference basis for quality control of the gerbera piloselloides.
Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above-described embodiments, and many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (7)
1, A method for measuring the content of multiple components in Gerberae piloselloides, which is used for simultaneously measuring the content of chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, luteoloside and arbutin in Gerberae piloselloides, and is characterized in that the UPLC-PDA method is adopted for measurement, and Waters BEH C is used for measurement182.1 × 150mm 1.7 μm is chromatographic column, acetonitrile-0.1% formic acid water solution is used for gradient elution, detection wavelengths of 280nm and 328nm are simultaneously used, flow rate is 0.28mL/min, column temperature is 40 ℃, and the specific elution procedure is as follows:
。
2. The method for measuring the contents of various components in the gerbera piloselloides medicinal material according to claim 1, wherein the UPLC-PDA method is used for diluting the gerbera piloselloides medicinal material extracting solution to be used as a test solution.
3. The method for measuring the contents of various components in the gerbera piloselloides medicinal material according to claim 2, wherein the gerbera piloselloides medicinal material extracting solution is obtained by extracting the gerbera piloselloides medicinal material by using methanol as a solvent.
4. The method for measuring the contents of various components in the gerbera piloselloides oliv medicinal material according to claim 3, wherein the step of extracting the gerbera piloselloides with methanol as a solvent is to perform reflux extraction on the gerbera piloselloides with 50% methanol, and the reflux extraction time is 2 hours.
5. The method for measuring the contents of various components in the gerbera piloselloides oliv medicinal material according to claim 2, wherein the dilution is performed by diluting the gerbera piloselloides oliv medicinal material extract with distilled water by 5 times.
6. The method for measuring the contents of various components in the gerbera piloselloides medicinal material according to claim 2, wherein the gerbera piloselloides medicinal material is gerbera piloselloides medicinal material powder which is sieved by a third sieve.
7. The method for measuring the contents of various components in the gerbera piloselloides medicinal material according to claim 1, wherein the UPLC-PDA method is characterized in that a test solution is prepared by the following method: taking 0.5g of gerbera piloselloides medicinal material powder which passes through a third sieve, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 50% methanol, weighing, heating and refluxing for 2 hours, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, weighing 5mL of subsequent filtrate, transferring to a 25mL volumetric flask, diluting to a scale with distilled water, and shaking up to obtain a test solution.
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