CN109030681B - Method for identifying authenticity of subprostrate sophora - Google Patents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
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Abstract
The invention discloses a method for identifying the authenticity of subprostrate sophora, which is used for determining the content of phenylbenzofuran in a subprostrate sophora medicinal material sample to judge the authenticity of the subprostrate sophora medicinal material sample. The detection method provided by the invention is accurate and reliable, can effectively eliminate the interference of other impurities in the sample, can obtain a satisfactory separation detection effect, and is simple and convenient to operate, easy to implement, low in cost, strong in practicability and very suitable for detecting the product quality.
Description
Technical Field
The invention relates to the field of traditional Chinese medicine identification methods. More particularly, the invention relates to a method for identifying the authenticity of subprostrate sophora.
Background
The subprostrate sophora is the dry root and rhizome of leguminous plant sophora tonkinensis, has the effects of clearing away heat and toxic material, reducing swelling and relieving sore throat, contains matrine, oxymatrine, sophocarpine, dauricine, genistein, beta-sterol, flavone and the like, the existing Chinese pharmacopoeia has a simpler quality control method for the subprostrate sophora, only takes the content of the matrine and the oxymatrine as the index of the quality evaluation of medicinal materials, but the matrine and the oxymatrine commonly exist in leguminous plants sophora alopecuroides, sophora flavescens, sophora davidii, sophora moorcroftiana and the like instead of the specific components of the subprostrate sophora, and the content measurement of the matrine and the oxymatrine is difficult to integrally react with the quality of the medicinal materials.
The phenylbenzofuran is obtained by separating subprostrate sophora for the first time, has very obvious components for preventing and treating the rhinitis cancer, is one of the main active components of the subprostrate sophora for preventing and treating the cancer, establishes the quality control and the quality inspection standard of the subprostrate sophora by taking the phenylbenzofuran as an index component, and can better reflect the quality of the subprostrate sophora medicinal materials.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
The invention also aims to provide a method for identifying the authenticity of the subprostrate sophora root, which can better reflect the quality of the subprostrate sophora root medicinal material sample.
To achieve these objects and other advantages in accordance with the purpose of the present invention, a method for authenticating subprostrate sophora is provided, which determines the authenticity of a subprostrate sophora medicinal material sample by measuring the content of phenylbenzofuran in the subprostrate sophora medicinal material sample.
Preferably, when the mass fraction of the phenylbenzofuran in the subprostrate sophora medicinal material sample is not less than 0.04%, the subprostrate sophora medicinal material sample is genuine; when the mass fraction of the phenylbenzofuran in the subprostrate sophora medicinal material sample is lower than 0.04%, the subprostrate sophora medicinal material sample is a pseudo-inferior product.
Preferably, the phenylbenzofuran in the tonka-bean-root medicinal material sample is extracted and dissolved to obtain a phenylbenzofuran sample solution, and then the content of the phenylbenzofuran in the tonka-bean-root medicinal material sample is determined.
Preferably, the content of phenylbenzofuran in the subprostrate sophora medicinal material sample is obtained by measuring the phenylbenzofuran sample solution by adopting a liquid chromatography and an external standard method.
Preferably, the specific process for measuring the p-phenylbenzofuran sample solution by using the liquid chromatography and the external standard method comprises the following steps:
step one, preparing a phenylbenzofuran standard solution;
performing liquid chromatography detection on the phenylbenzofuran sample solution and the phenylbenzofuran standard solution to obtain a peak area of phenylbenzofuran in the phenylbenzofuran sample solution and a peak area of phenylbenzofuran in the phenylbenzofuran standard solution;
step three, calculating the mass fraction X% of phenylbenzofuran in the radix sophorae tonkinensis medicinal material sample according to the following formula:
wherein V is the volume of the phenylbenzofuran sample solution; a. thesIs the peak area of phenylbenzofuran in the phenylbenzofuran sample solution; a. thesdIs the peak area of phenylbenzofuran in a phenylbenzofuran standard solution; c is the concentration of the phenylbenzofuran standard solution; m is the mass of the subprostrate sophora medicinal material sample.
Preferably, the process for extracting and dissolving phenylbenzofuran in the subprostrate sophora medicinal material sample comprises the following steps: weighing tonka-bean root medicinal material sample powder, adding an extractant with the weight 5-20 times of the tonka-bean root medicinal material sample powder, performing reflux extraction for 1-2 times, 1-2 hours each time, performing reduced pressure rotary evaporation to recover the extractant until the extractant is evaporated to dryness to obtain a crude extract, adding water to dissolve the crude extract, passing through macroporous resin, eluting with 10-40% ethanol, then eluting with 60-95% ethanol, collecting 60-95% ethanol elution part, performing reduced pressure rotary evaporation to recover ethanol until the ethanol is evaporated to dryness to obtain a fine extract, and adding methanol to the fine extract to dissolve and fix the volume.
Preferably, the extractant is a methanol solution with a mass fraction of 45-100%, an ethanol solution with a mass fraction of 45-99.8% (when the ethanol concentration is 99.8%, the ethanol solution is analytically pure absolute ethanol), or an acetone solution with a mass fraction of 45-100%.
Preferably, the extractant is an 80% ethanol solution by mass fraction.
Preferably, the phenylbenzofuran is 2- (2', 4' -dihydroxyphenyl) -5, 6-dioxymethylenebenzofuran with the structural formula
Preferably, the process for extracting and dissolving phenylbenzofuran in the subprostrate sophora medicinal material sample comprises the following steps: weighing the powder of a subprostrate sophora medicinal material sample, adding an extractant with the weight 5-20 times, performing reflux extraction for 1-2 times, performing 1-2 h each time, performing reduced pressure rotary evaporation to recover the extractant until the extractant is evaporated to dryness to obtain a crude extract, adding water to dissolve the crude extract, passing through macroporous resin, eluting with 39% ethanol, then eluting with 95% ethanol, collecting an ethanol elution part with 95%, performing reduced pressure rotary evaporation to recover ethanol until the ethanol is evaporated to dryness to obtain a fine extract, and adding methanol to the fine extract to dissolve and fix the volume.
Preferably, octadecylsilane chemically bonded silica is used as a filler for a chromatographic column used for liquid chromatography detection, and the column temperature is 25-30 ℃; the detector used for liquid chromatography detection is an ultraviolet detector, and the use wavelength is 205-280 nm; the sample injection amount during liquid chromatography detection is 10-20 mul, and the flow rate is 1.0 ml/min; gradient elution is adopted in liquid chromatography detection, wherein acetonitrile is an A phase, water is a B phase, and the proportion of the acetonitrile is changed as follows: in 0-5 min, the acetonitrile accounts for 25%; in 5-55 min, the proportion of acetonitrile is gradually increased from 25% to 50%; and in 55-80 min, the proportion of acetonitrile is gradually increased from 50% to 95%.
The invention at least comprises the following beneficial effects: the method can accurately identify the authenticity of the subprostrate sophora medicinal material by extracting the main active ingredient phenylbenzofuran in the subprostrate sophora and taking the content of phenylbenzofuran as the index of the subprostrate sophora quality standard evaluation, has the advantages of higher detection sensitivity and accuracy, high repeatability, simplicity, convenience and feasibility, realizes the stable control of the quality of the subprostrate sophora medicinal material and the preparation thereof, and provides theoretical and method support for the application and popularization of the subprostrate sophora in Guangxi and the development of the national medicine. The method is verified through a linear relation test, a precision test, a stability test, a repeatability test and a sample adding recovery rate test, and the result shows that the detection method provided by the invention is accurate and reliable, can effectively eliminate the interference of other magazines in the sample and can obtain a satisfactory detection effect; the method is simple and convenient to operate, easy to implement, low in cost, high in practicability and very suitable for detecting the product quality.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a liquid chromatogram of a phenylbenzofuran sample solution according to one embodiment of the present invention;
FIG. 2 is a liquid chromatogram of a phenylbenzofuran standard solution according to one embodiment of the present invention;
FIG. 3 is a graph of a phenylbenzofuran standard according to the linear relationship test of the present invention.
Detailed Description
The present invention is further described in detail below with reference to the drawings and examples so that those skilled in the art can practice the invention with reference to the description.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.
< example >
And (3) producing areas of the subprostrate sophora medicinal material samples: guangxi medicinal plant garden.
The method for identifying the authenticity of the subprostrate sophora comprises the following steps:
weighing 2g of sample powder of the radix sophorae tonkinensis, adding an extracting agent with the weight being 20 times of that of the sample powder of the radix sophorae tonkinensis, performing reflux extraction for 1 time, wherein the extraction time is 2 hours, performing reduced pressure rotary evaporation to recover the extracting agent until the extracting agent is evaporated to dryness to obtain a crude extract, adding 20ml of water for dissolving, passing through macroporous resin, eluting with 39% ethanol, then eluting with 95% ethanol, collecting an elution part of the 95% ethanol, performing reduced pressure rotary evaporation to recover the ethanol until the ethanol is evaporated to dryness to obtain a fine extract, adding the methanol for dissolving the fine extract to a volumetric flask with the volume being 10ml, and obtaining a phenylbenzofuran sample solution, wherein the extracting agent is an ethanol solution with the mass fraction of 80%;
weighing a phenylbenzofuran monomer, adding methanol to dissolve the phenylbenzofuran monomer to prepare a phenylbenzofuran standard solution with the concentration of 1.41 mg/ml;
step three, performing liquid chromatography detection on the phenyl benzofuran sample solution and the phenyl benzofuran standard solution, wherein the conditions of the liquid chromatography detection are as follows:
a chromatographic column: waters Sunfire-C18(4.6 mm. times.250 mm, 5 μm);
column temperature: 30 ℃;
a detector: an ultraviolet detector with a wavelength of 280 nm;
sample introduction amount: 20 mu l of the mixture;
flow rate: 1.0 ml/min;
mobile phase: acetonitrile as mobile phase a and water as mobile phase B, gradient elution was performed as per table 1:
TABLE 1
The liquid chromatogram of the phenylbenzofuran sample solution is shown in FIG. 1, and the liquid chromatogram of the phenylbenzofuran standard solution is shown in FIG. 2.
Step four, calculating the mass fraction X% of phenylbenzofuran in the radix sophorae tonkinensis medicinal material sample according to the following formula:
wherein V is the volume of the phenylbenzofuran sample solution and the unit is ml; a. thesIs the peak area of phenylbenzofuran in the phenylbenzofuran sample solution; a. thesdIs the peak area of phenylbenzofuran in a phenylbenzofuran standard solution; c is the concentration of the phenylbenzofuran standard solution, and the unit is mg/ml; m is the mass of the subprostrate sophora medicinal material sample, and the unit is g. The mass fraction of phenylbenzofuran in the subprostrate sophora medicinal material sample of this example is 0.082%.
Fifthly, when the mass fraction of the phenylbenzofuran in the subprostrate sophora medicinal material sample is not lower than 0.04%, the subprostrate sophora medicinal material sample is genuine; when the mass fraction of the phenylbenzofuran in the subprostrate sophora medicinal material sample is lower than 0.04%, the subprostrate sophora medicinal material sample is a pseudo-inferior product. According to the above criteria, the sample of the tonka-bean-root medicinal material of this embodiment is genuine.
Wherein, the phenylbenzofuran described in this example is 2- (2', 4' -dihydroxyphenyl) -5, 6-dioxymethylenebenzofuran.
< Linear relationship test >
Weighing phenylbenzofuran monomersAdding methanol to prepare standard solutions with the solubility of 0.24mg/ml, 0.28mg/ml, 0.72mg/ml, 0.96mg/ml, 1.2mg/ml and 1.41mg/ml respectively, and performing liquid chromatography detection on the standard solutions with different concentrations according to the liquid chromatography detection conditions of the above examples to obtain a standard curve chart shown in figure 3, wherein the ordinate in the standard curve chart is the chromatographic peak area, the abscissa is the sample injection amount, the unit is mug, and the linear equation of the standard curve is that y is 4 × 106x-130105，R20.9999. The result shows that when the sample amount of the phenylbenzofuran standard solution is between 0.24 and 1.44 mu g, the sample amount and the peak area have good linear relation.
< precision test >
And (3) absorbing the phenylbenzofuran standard solution with the same concentration, repeatedly injecting for 6 times under the same liquid chromatogram detection condition as the embodiment, recording the liquid chromatogram obtained each time, analyzing and comparing the peak areas of the liquid chromatograms, and calculating that the relative standard deviation of the peak areas of the liquid chromatograms is 1.19%, which indicates that the method provided by the invention has good precision.
< stability test >
Absorbing 6 parts of phenyl benzofuran standard solution with the same concentration, respectively placing 1ml of each part of phenyl benzofuran standard solution and 6 parts of phenyl benzofuran standard solution for 0, 1, 2, 4, 8 and 12 hours, respectively recording liquid chromatogram of 6 parts of phenyl benzofuran standard solution by adopting the same liquid chromatogram detection conditions as the examples, analyzing and comparing peak areas of the chromatogram, obtaining a relative standard deviation of 1.86%, and indicating that the phenyl benzofuran standard solution is stable within 12 hours.
< reproducibility test >
Weighing 6 parts of the powder of the same batch of the tonka-bean-root medicinal material sample, wherein each part is about 1g, detecting the content of the phenylbenzofuran in each part of the sample by the same method as the embodiment, and analyzing and comparing to obtain a relative deviation value of 1.28%, which indicates that the method provided by the invention has good repeatability.
< sample recovery test >
Weighing 6 parts of subprostrate sophora medicinal material samples, respectively adding 0.5ml of phenylbenzofuran standard solution with the concentration of 1.44mg/ml, and measuring by adopting the same method as the embodiment, calculating the recovery rate to obtain the average recovery rate of 99.41 percent and the relative standard deviation of 1.92 percent, which indicates that the method provided by the invention has better sample-adding recovery rate.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (2)
1. The method for identifying the authenticity of the subprostrate sophora root is characterized in that the authenticity of the subprostrate sophora root medicinal material sample is judged by measuring the content of phenyl benzofuran in the subprostrate sophora root medicinal material sample, and when the mass fraction of the phenyl benzofuran in the subprostrate sophora root medicinal material sample is not less than 0.04%, the subprostrate sophora root medicinal material sample is genuine; when the mass fraction of the phenylbenzofuran in the subprostrate sophora medicinal material sample is lower than 0.04%, the subprostrate sophora medicinal material sample is a pseudo-inferior product;
firstly, extracting and dissolving phenylbenzofuran in a radix sophorae tonkinensis medicinal material sample to obtain a phenylbenzofuran sample solution, and then measuring the content of phenylbenzofuran in the radix sophorae tonkinensis medicinal material sample;
measuring the phenyl benzofuran sample solution by liquid chromatography and external standard method to obtain content of phenyl benzofuran in radix Sophorae Tonkinensis sample;
the process for extracting and dissolving phenylbenzofuran in the subprostrate sophora medicinal material sample comprises the following steps: weighing subprostrate sophora medicinal material sample powder, adding an extractant with the weight 5-20 times, performing reflux extraction for 1-2 times, each time for 1-2 h, performing reduced pressure rotary evaporation to recover the extractant until the extractant is evaporated to dryness to obtain a crude extract, adding water for dissolution, passing through macroporous resin, eluting with 39% ethanol, then eluting with 95% ethanol, collecting 95% ethanol elution part, performing reduced pressure rotary evaporation to recover ethanol until the ethanol is evaporated to dryness to obtain a fine extract, adding methanol to the fine extract for dissolution and volume determination, wherein the extractant is an ethanol solution with the mass fraction of 80%;
the phenylbenzofuran is 2- (2', 4' -dihydroxyphenyl) -5, 6-dioxymethylenebenzofuran;
a chromatographic column used for liquid chromatography detection takes octadecylsilane chemically bonded silica as a filler, and the column temperature is 25-30 ℃; the detector used for liquid chromatography detection is an ultraviolet detector, and the wavelength of the detector is 280 nm; the sample injection amount during liquid chromatography detection is 10-20 mu l, and the flow rate is 1.0 ml/min; gradient elution is adopted in liquid chromatography detection, wherein acetonitrile is an A phase, water is a B phase, and the proportion of the acetonitrile is changed as follows: in 0-5 min, the acetonitrile accounts for 25%; in 5-55 min, the proportion of acetonitrile is gradually increased from 25% to 50%; and in 55-80 min, the proportion of acetonitrile is gradually increased from 50% to 95%.
2. The method for authenticating the authenticity of subprostrate sophora as claimed in claim 1, wherein the specific process of determining the p-phenylbenzofuran sample solution by using liquid chromatography and external standard method comprises the following steps:
step one, preparing a phenylbenzofuran standard solution;
performing liquid chromatography detection on the phenylbenzofuran sample solution and the phenylbenzofuran standard solution to obtain a peak area of phenylbenzofuran in the phenylbenzofuran sample solution and a peak area of phenylbenzofuran in the phenylbenzofuran standard solution;
step three, calculating the mass fraction X% of phenylbenzofuran in the radix sophorae tonkinensis medicinal material sample according to the following formula:
wherein V is the volume of the phenylbenzofuran sample solution; a. thesIs the peak area of phenylbenzofuran in the phenylbenzofuran sample solution; a. thesdIs the peak area of phenylbenzofuran in a phenylbenzofuran standard solution; c is the concentration of the phenylbenzofuran standard solution; m is the mass of the subprostrate sophora medicinal material sample.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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Application publication date: 20181218 Assignee: Pubei County Hongwei Nut Planting Co.,Ltd. Assignor: GUANGXI BOTANICAL GARDEN OF MEDICINAL PLANTS Contract record no.: X2023980046052 Denomination of invention: Method for identifying the authenticity of Shandou root Granted publication date: 20211001 License type: Common License Record date: 20231108 |
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