CN109030681B

CN109030681B - How to identify the authenticity of Shandougen

Info

Publication number: CN109030681B
Application number: CN201811102881.2A
Authority: CN
Inventors: 缪剑华; 宋志军; 姚彩云; 闫炳雄; 李秋萍; 欧春丽
Original assignee: Guangxi Botanical Garden of Medicinal Plants
Current assignee: Guangxi Botanical Garden of Medicinal Plants
Priority date: 2018-09-20
Filing date: 2018-09-20
Publication date: 2021-10-01
Anticipated expiration: 2038-09-20
Also published as: CN109030681A

Abstract

The invention discloses a method for identifying the authenticity of the mountain bean root. The authenticity of the mountain bean root medicinal material sample is judged by measuring the content of phenylbenzofuran in the mountain bean root 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, and can obtain a satisfactory separation and detection effect. The method is simple to operate, easy to implement, low in cost and strong in practicability, and is very suitable for the detection of product quality.

Description

Method for identifying authenticity of subprostrate sophora

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. the_sIs the peak area of phenylbenzofuran in the phenylbenzofuran sample solution; a. the_sdIs 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. the_sIs the peak area of phenylbenzofuran in the phenylbenzofuran sample solution; a. the_sdIs 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

1. A method for identifying the authenticity of Lentinus chinensis, characterized in that, by determining the content of phenylbenzofuran in the sample of Radix Radix et Rhizoma When the mass fraction of phenylbenzofuran is not less than 0.04%, the samples of Radix japonica are genuine; when the mass fraction of phenylbenzofuran in the samples is less than 0.04%, the samples of Radix Radix are fake and inferior;

Firstly, extract and dissolve phenylbenzofuran in the samples of Radix japonicas to obtain a sample solution of phenylbenzofuran, and then determine the content of phenylbenzofurans in the samples of Radix japonicas;

The phenylbenzofuran sample solution was determined by liquid chromatography and external standard method, and the content of phenylbenzofuran in the rhizoma rhizome sample was obtained;

The process of extracting and dissolving the phenylbenzofuran in the Shandou root medicinal material sample is as follows: weighing the Shandou root medicinal material sample powder, adding 5 to 20 times the weight of the extractant, and refluxing for 1 to 2 times, 1 to 2 times each time. For 2h, the extractant was recovered by rotary evaporation under reduced pressure until evaporated to dryness to obtain the crude extract, which was dissolved in water, and the macroporous resin was eluted with 39% ethanol and then eluted with 95% ethanol. The ethanol is recovered by rotary evaporation to evaporate to dryness to obtain the refined extract, and the refined extract is dissolved in methanol to constant volume, and the extractant is an ethanol solution with a mass fraction of 80%;

The phenylbenzofuran is 2-(2',4'-dihydroxyphenyl)-5,6-dioxymethylenebenzofuran;

The chromatographic column used in the liquid chromatography detection is made of octadecylsilane-bonded silica gel as the filler, and the column temperature is 25~30 °C; the detector used in the liquid chromatography detection is an ultraviolet detector with a wavelength of 280 nm; The sample injection volume during detection is 10~20µl, and the flow rate is 1.0ml/min; the liquid chromatography detection adopts gradient elution, in which acetonitrile is phase A, water is phase B, and the ratio of acetonitrile changes as follows: in 0~5min, The proportion of acetonitrile was 25%; in 5~55min, the proportion of acetonitrile gradually increased from 25% to 50%; in 55~80min, the proportion of acetonitrile gradually increased from 50% to 95%.

2. the method for identifying the authenticity of Radix japonica as claimed in claim 1, is characterized in that, the concrete process that adopts liquid chromatography and external standard method to measure phenylbenzofuran sample solution comprises the following steps:

Step 1, prepare phenylbenzofuran standard solution;

Step 2. Perform liquid chromatography detection on the phenylbenzofuran sample solution and the phenylbenzofuran standard solution, and measure the peak area of the phenylbenzofuran and the phenylbenzofuran in the phenylbenzofuran sample solution. Peak area of phenylbenzofuran in furan standard solution;

Step 3, according to the following formula, calculate the mass fraction X% of phenylbenzofuran in the Rhododendron chinensis medicinal material sample:

Wherein, V is the volume of the phenylbenzofuran sample solution; As is the peak area of the _{phenylbenzofuran in the phenylbenzofuran sample solution; Asd} _is the phenylbenzofuran in the phenylbenzofuran standard solution The peak area of ; c is the concentration of the standard solution of phenylbenzofuran; m is the mass of the mountain bean root medicinal material sample.