CN115524410A - Method for measuring content of blood ginseng ethanol extract - Google Patents
Method for measuring content of blood ginseng ethanol extract Download PDFInfo
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- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 claims abstract description 62
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- 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
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- G01N30/02—Column chromatography
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- 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
- G01N30/14—Preparation by elimination of some components
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- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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Abstract
The invention discloses a method for measuring the content of a blood ginseng ethanol extract, which can effectively separate catechin from epicatechin in blood ginseng ethanol extract powder (ISs-EthE), wherein the linear range of the catechin of an ISs-EthE index component is 2.04 mu g.mL-1 to 32.74 mu g.mL-1, the linear range of the epicatechin of the ISs-EthE index component is 4.50 mu g.mL-1 to 71.20 mu g.mL-1, and the linearity of the catechin and the epicatechin in the linear range is good (R2 is more than 0.99). In addition, the results of precision, repeatability, stability, sample recovery rate and the like show that the analysis method is stable and feasible.
Description
Technical Field
The invention relates to a method for measuring the content of a blood ginseng ethanol extract, in particular to a method for measuring the content of catechin and epicatechin in the blood ginseng ethanol extract.
Background
Bloody ginseng (ISs) is a dry root of trichoderma aureocaudatum Lindl (indigo of Indigofera athyrioides, isatis) of the genus trichoderma, a leguminous plant, is a common medicine for Miao nationality, is collected by quality standards of Chinese medicinal materials and national medicinal materials in Guizhou province in 2003, has long application history, is sweet in taste, slightly bitter and warm in nature, and has the effects of nourishing yin, tonifying deficiency, regulating menstruation, absorbing blood, promoting blood circulation and relaxing muscles and tendons, is mainly used for treating metrorrhagia and metrostaxis, is mainly distributed in places such as Yunnan, guizhou, fujian, guangxi and the like, and particularly has wide distribution in Guizhou Yang, anshun, qianxian, qian southeast and the like.
At present, a great deal of research is carried out on components in the blood ginseng, but the content determination method of catechin and epicatechin in the ethanol extract of the blood ginseng is not reported yet, the invention establishes a high performance liquid analysis method of blood ginseng ethanol extract powder (ISs-EthE), and the analysis method is verified to be stable and feasible by methodology. .
Disclosure of Invention
The invention aims to provide a method for measuring the content of a blood ginseng ethanol extract. The invention adopts a high performance liquid analysis method to measure the contents of the chemical components of catechin and epicatechin of the blood ginseng ethanol extract powder (ISs-EthE).
The technical scheme of the invention is as follows:
a method for measuring the content of a blood ginseng ethanol extract comprises the following steps:
a. preparing blood ginseng ethanol extract powder: weighing appropriate amount of XUERENSHEN, cutting or chopping, reflux-extracting with 60% ethanol, filtering, reflux-extracting the residue with 60% ethanol, and filtering; mixing the two filtrates, recovering ethanol under reduced pressure, concentrating to a certain concentration, and vacuum drying to water content less than 5% to obtain blood Ginseng radix ethanol extract powder;
b. preparing a test sample: accurately weighing blood ginseng ethanol extract powder, adding methanol into a volumetric flask, performing ultrasonic treatment to completely dissolve the blood ginseng ethanol extract powder, and performing constant volume to obtain the ginseng extract powder;
c. preparation of mixed reference: accurately weighing catechin and epicatechin, respectively placing in a volumetric flask, adding methanol to completely dissolve them and fixing the volume to obtain stock solutions of catechin and epicatechin, accurately transferring the stock solutions of catechin and epicatechin, placing in the volumetric flask, fixing the volume with methanol to obtain a mixed reference stock solution of catechin and epicatechin, and storing at 4 ℃;
d. respectively carrying out UPLC determination on the test solution and the mixed reference solution under chromatographic conditions, effectively separating catechin and epicatechin in the blood ginseng ethanol extract powder, and determining the content of catechin and epicatechin;
e. the feasibility of the analysis method is evaluated by investigating specificity, linear range, precision, repeatability, stability and sample recovery rate.
In the step a, a proper amount of the panax sanguinea medicinal material is weighed, cut or chopped, 5-7 times of 60% ethanol is added for reflux extraction for 1-3 hours, the mixture is filtered, 3-5 times of 60% ethanol is added for reflux extraction for 1-2 hours, the two filtrates are combined, the ethanol is recovered under reduced pressure and concentrated to a certain concentration, and the ethanol extract powder of the panax sanguinea is obtained after vacuum drying until the water content is less than 5%.
In the step a, weighing a proper amount of a panax sanguinea medicinal material, cutting or chopping the panax sanguinea medicinal material, adding 6 times of 60% ethanol for reflux extraction for 2 hours, filtering, adding 4 times of 60% ethanol for reflux extraction for 1.5 hours to filter residues, filtering, combining two filtrates, recovering ethanol under reduced pressure, concentrating to a certain concentration, and vacuum drying until the water content is less than 5%, thus obtaining the panax sanguinea ethanol extract powder.
In the method for measuring the content of the blood ginseng ethanol extract, in the step b, the preparation of the test sample comprises the following steps: accurately weighing 0.050g of blood ginseng ethanol extract powder, adding methanol into a 10mL volumetric flask, performing ultrasonic treatment to completely dissolve the powder, and fixing the volume to 10mL to obtain the ginseng ethanol extract powder.
Specifically, in the method for measuring the content of the blood ginseng ethanol extract, in the step b, the ultrasonic frequency is 53kHz.
In the aforementioned method for measuring the content of the blood ginseng ethanol extract, in step c, the mixed reference substance is prepared by: accurately weighing 0.01635g of catechin and 0.01424g of epicatechin in 10mL volumetric flasks respectively, adding methanol to completely dissolve the catechin and the epicatechin, fixing the volume to 10mL to obtain a stock solution of the catechin and the epicatechin, accurately transferring 2mL of the catechin stock solution and 5mL of the epicatechin stock solution, placing the stock solution in a 100mL volumetric flask, and fixing the volume to 100mL with methanol to obtain a solution containing 32.70 mu g/mL -1 Catechin and 71.20. Mu.g/mL -1 The mixed reference substance stock solution of epicatechin is stored at 4 ℃;
in the foregoing method for measuring the content of the blood ginseng ethanol extract, the chromatographic conditions in step d are: chromatographic column ComatexTMC 18 :250 mm. Times.4.6 mm,5 μm, acetonitrile as mobile phase A,0.2% phosphoric acid water as mobile phase B, 1.0 mL. Min sulfuric acid -1 Detecting wavelength is 280nm, column temperature is 30 ℃, sample is added by 10 mu L for gradient elution, and the gradient elution sequence is as follows: 0-40min,8-14% A.
Compared with the prior art, the invention has the following beneficial effects:
the invention establishes a high performance liquid analysis method of blood ginseng ethanol extract powder (ISs-EthE), which can effectively separate catechin and epicatechin in the ISs-EthE, wherein the linear range of the ISs-EthE index component catechin is 2.04 mu g.mL < -1 > -32.74 mu g.mL < -1 >, the linear range of the ISs-EthE index component epicatechin is 4.50 mu g.mL < -1 > -71.20 mu g.mL < -1 >, and the linearity of the ISs-EthE index component epicatechin is good within the linear range (R2 > 0.99). In addition, the results of precision, repeatability, stability, sample recovery rate and the like show that the analysis method is stable and feasible.
Description of the drawings:
FIG. 1. Control and ISs-EthE chromatograms (a: blank methanol, b: mixed control, c: ISs-EthE; 1 for catechin, 2 for epicatechin);
FIG. 2is a graph showing the linear relationship between catechins in ISs-EthE;
FIG. 3is a line graph of epicatechin in ISs-EthE;
FIG. 4 Linear Range investigation chromatogram (a: 2.04. Mu.g. ML) -1 Catechin and 4.45. Mu.g/mL -1 Epicatechin, b: 4.09. Mu.g/mL -1 Catechin and 8.90. Mu.g/mL -1 Epicatechin, c: 8.18. Mu.g/mL -1 Catechin and 17.80. Mu.g/mL -1 Epicatechin, d: 16.35. Mu.g/mL -1 Catechin and 35.60. Mu.g/mL -1 Epicatechin, e: 32.70. Mu.g/mL -1 Catechins and 71.20. Mu.g.mL -1 Epicatechin);
FIG. 5 precision assay chromatogram;
FIG. 6 reproducibility test chromatogram;
FIG. 7 stability test chromatogram;
FIG. 8 sample recovery test chromatogram.
Detailed Description
The present invention is further illustrated by the following examples, which are not to be construed as limiting the invention.
Example 1:
preparing blood ginseng ethanol extract powder: weighing a proper amount of panax sanguinea medicinal material, cutting or chopping, adding 6 times of 60% ethanol for reflux extraction for 2 hours, filtering, adding 4 times of 60% ethanol for reflux extraction for 1.5 hours into filter residues, filtering, combining the two filtrates, recovering ethanol under reduced pressure, concentrating to a certain concentration, and vacuum drying until the water content is less than 5% to obtain panax sanguinea ethanol extract powder.
Preparing a test sample: accurately weighing 0.050g of blood ginseng ethanol extract powder, adding methanol into a 10mL volumetric flask, and performing ultrasonic treatment at ultrasonic frequency of 53kHz to obtain a solution with constant volume of 10mL.
Preparation of mixed reference: accurately weighing 0.01635g of catechin and 0.01424g of epicatechin in 10mL volumetric flasks respectively, adding methanol to completely dissolve the catechin and the epicatechin, and fixing the volume to 10mL to obtain a stock solution of the catechin and the epicatechin, accurately transferring 2mL of the catechin stock solution and 5mL of the epicatechin stock solution, placing the stock solution in a 100mL volumetric flask, and fixing the volume to 100mL by using methanol to obtain a solution containing 32.70 mu g/mL -1 Catechin and 71.20. Mu.g/mL -1 The mixed reference substance stock solution of epicatechin is stored at 4 ℃;
chromatographic conditions are as follows: chromatographic column ComatexTMC 18 :250 mm. Times.4.6 mm,5 μm, acetonitrile as mobile phase A,0.2% phosphoric acid water as mobile phase B, sulfuric acid 1.0 mL. Min -1 Detecting wavelength is 280nm, column temperature is 30 ℃, and gradient elution is carried out by injecting 10 mu L of sample, and the gradient elution sequence is as follows: 0-40min,8-14% A.
And (3) detection: respectively carrying out UPLC measurement on the test solution and the mixed reference solution under chromatographic conditions, and measuring the content of catechin and epicatechin.
Experimental example: study of detection method
1 materials of the experiment
1.1 reagent
| Name (R) | Batch number | Manufacturer of the product |
| Acetonitrile | 21040484 | Tedia Inc. of USA |
| Phosphoric acid | 10015406 | SINOPHARM CHEMICAL REAGENT Co.,Ltd. |
| Catechin | 170209 | PureChemS TANDARD |
| Epimetechin | 110878-201703 | China Institute for food and drug control |
| Xuezhen medicine | 20210322 | GUIYANG DECHANGXIANG PHARMACEUTICAL Co.,Ltd. |
1.2 instruments
| Name (R) | Manufacturer of the product |
| LC-2030C3D high performance liquid chromatograph | Japan Shimadzu institute of technology |
| AUW120D type electronic analytical balance (0.01 mg) | Shimadzu Corp. |
Preparation of 2ISs-EthE
Weighing a proper amount of the panax ginseng medicinal material, cutting or chopping, adding 6 times of ethanol (60%) for reflux extraction for 2 hours, filtering, adding 4 times of ethanol (60%) for reflux extraction for filter residue for 1.5 hours, and filtering. Mixing the two filtrates, recovering ethanol under reduced pressure, concentrating to certain concentration, and vacuum drying to water content less than 5% to obtain blood Ginseng radix ethanol extract (ISs-EthE) extract powder with an extraction rate of about 18%.
Establishment of 3ISs-EthE in vitro analysis method
3.1 chromatographic conditions
Chromatographic column cometex tmc 18 (250 mm. Times.4.6 mm,5 μm), acetonitrile as mobile phase A,0.2% phosphoric acid water as mobile phase B, 1.0 mL-min -1 The flow rate of (2) was measured at a detection wavelength of 280nm and a column temperature of 30 ℃ and a sample of 10. Mu.L was injected to carry out gradient elution, the elution sequence being shown in Table 1.
TABLE 1 gradient elution sequence
| Time(min) | A(％) | B(％) |
| 0 | 8 | 92 |
| 40 | 14 | 86 |
3.2 preparation of the solution
Preparation of mixed reference: catechin (0.01635 g) and epicatechin (0.01424 g) were weighed precisely in 10mL volumetric flasks, and methanol was added thereto to completely dissolve the catechins and epicatechin, respectively, to obtain a 10mL volume of stock solutions of catechins and epicatechins. Precisely transferring 2mL catechin stock solution and 5mL epicatechin stock solution into a 100mL volumetric flask, and diluting to 100mL with methanol to obtain a solution containing 32.70. Mu.g.mL -1 Catechin and 71.20. Mu.g/mL -1 A stock solution of a mixed control of epicatechin was stored at 4 ℃.
Preparing a test sample: accurately weighing ISs-EthE extract powder (0.050 g) in a 10mL volumetric flask, adding methanol, performing ultrasonic treatment at 53kHz to completely dissolve the ISs-EthE extract powder, and performing constant volume to 10mL.
3.3 methodological inspection
3.3.1 specificity
And (4) analyzing the reference substance solution and the test substance solution under the item of '3.1', and comparing the obtained chromatogram of the test substance with the chromatogram of the reference substance for analysis.
3.3.2 Linear Range inspection
Precisely sucking mixed reference substance stock solutions under the items of 0.625, 1.25, 2.5, 5.0 and 10.0mL for 3.2', respectively placing in 10mL volumetric flasks, adding methanol to constant volume to obtain 2.04, 4.09, 8.18, 16.35 and 32.70. Mu.g.mL -1 The catechin concentration is 4.45, 8.90, 17.80, 35.60, 71.20 μ g/mL -1 The mixed control solutions of epicatechin series concentration were analyzed under the term "3.1" to measure the peak areas thereof. And (5) performing linear regression by taking the concentration (x) of the reference substance as a horizontal coordinate and the peak area (y) of the component to be detected as a vertical coordinate to obtain a regression equation.
3.3.3 precision
Precisely sucking 2.5mL of mixed reference substance stock solution under the item 3.2', fixing the volume to 10mL by using methanol, carrying out sample injection analysis under the item 3.1, repeating for 6 times, calculating the actually measured concentration of catechin and epicatechin according to a follow standard curve, and calculating the RSD of the catechin and epicatechin.
3.3.4 reproducibility
Precisely weighing the same batch of ISs-EthE samples (50.0 mg), paralleling 5 parts, preparing treatment samples according to the test solution preparation under the item of '3.2 solution preparation', analyzing according to the item of '3.1', calculating the actually measured content of catechin and epicatechin according to a following standard curve, and calculating the repeatability of the RSD investigation method.
3.3.5 stability
Precisely weighing the same batch of ISs-EthE samples (50.0 mg), paralleling 5 parts, preparing a sample solution under the item of 3.2, respectively injecting samples for analysis at 0h, 2h, 4h, 6h, 8h, 10h and 12h by a chromatographic method under the item of 3.1, calculating the actually measured content of catechin and epicatechin according to a following standard curve, and calculating the RSD of the actually measured content to investigate the stability of the samples.
3.3.6 sample recovery
Weighing an ISs-EthE sample (about 25.0 mg) with a known content, paralleling 5 parts, preparing a treatment sample according to a test sample solution under the item of '3.2 solution preparation', analyzing according to the item of '3.1', calculating the actually measured content of catechin and epicatechin according to a following standard curve, respectively adding a standard substance which is 1.0 time of the content of the obtained two components, and calculating the recovery rate, wherein the formula is shown in formula 1.1.
3.3.7 assay
Precisely weighing the same batch of ISs-EthE samples (50.0 mg), preparing and processing the samples according to the item of '3.2 solution preparation', analyzing according to the item of '3.1', calculating the actual measurement content of the catechins and the epicatechins in the samples according to a following standard curve, calculating the content of two components in the ISs-EthE, paralleling 5 parts, and solving for RSD.
4 results of the experiment
4.1 specificity
Under the chromatographic conditions, the chromatographic peaks of the ISs-EthE test sample and the control (catechin and epicatechin) have the same retention time and can be completely separated (FIG. 1).
4.2 Linear Range inspection
The linear regression equation for catechin in ISs-EthE was y =6659x-404.29 (R) 2 = 0.9999), linear range of 2.04 μ g · mL -1 ～32.74μg·mL -1 (FIG. 2). The linear regression equation for epicatechin in ISs-EthE was y =7148.2x-659.54 (R) 2 = 1), linear range of 4.50 μ g · mL -1 ～71.20μg·mL -1 (FIG. 3). From this, it was found that the two components of ISs-EthE were well linear (R) in the linear range 2 >0.999)。
(chromatogram is shown in FIG. 4)
4.3 precision
The precision results (table 2) show that RSD of catechin concentration in ISs-EthE was 0.69%, RSD of epicatechin concentration was 1.07%, and RSD values met requirements, indicating that instrument precision was good. (chromatogram is shown in FIG. 5)
TABLE 2 precision test results (n = 6)
| |
1 | 2 | 3 | 4 | 5 | 6 | RSD(％) |
| Catechin (mug. ML) -1 ) | 8.39 | 8.46 | 8.34 | 8.41 | 8.46 | 8.33 | 0.69 |
| Epicatechin (μ g. ML) -1 ) | 18.55 | 18.57 | 18.42 | 18.35 | 18.43 | 18.03 | 1.07 |
4.4 repeatability
The reproducibility results (table 3) show that RSD values for catechin and epicatechin content in ISs-EthE are 2.11% and 1.11%, respectively, indicating that the method has good reproducibility. (chromatogram is shown in FIG. 6)
TABLE 3 repeatability test results (n = 6)
| |
1 | 2 | 3 | 4 | 5 | 6 | RSD(％) |
| Catechin (mug) | 109.48 | 109.76 | 110.28 | 104.89 | 111.73 | 109.13 | 2.11 |
| Epicatechin (μ g) | 191.44 | 189.55 | 192.20 | 186.12 | 189.91 | 190.25 | 1.11 |
4.5 stability
After content analysis of ISs-EthE at different time points, RSD values of catechin and epicatechin contents in the ISs-EthE extract powder were 2.12% and 2.37%, respectively (Table 4), indicating that the ISs-EthE sample has good stability. (chromatogram is shown in FIG. 7)
TABLE 4 stability test results (n = 7)
| Time (h) | 0 | 2 | 4 | 6 | 8 | 10 | 12 | RSD(％) |
| Catechin (mug) | 103.31 | 104.39 | 104.28 | 107.44 | 108.10 | 108.21 | 108.71 | 2.12 |
| Epicatechin (μ g) | 206.20 | 208.62 | 205.13 | 199.27 | 196.58 | 196.63 | 202.80 | 2.37 |
4.6 sample recovery
The experimental results of sample recovery (table 5) show that the sample recovery rates of catechin and epicatechin in ISs-EthE were (103.87 ± 2.97)%, and (100.53 ± 2.87)%, respectively, and the RSD values of both were 2.86%, which were less than 3%, and met the methodology requirements. (chromatogram is shown in FIG. 8)
TABLE 5 sample application recovery test results (n = 6)
4.7 assay
The content detection experiment result shows that the contents of catechin and epicatechin in the ISs-EthE are 2183.74 mu g.g respectively -1 And 3797.33. Mu.g.g -1 The RSD of the two is respectively 2.09 percent and 1.06 percent, and the composite requirement is met.
5 conclusion and discussion
The chapter establishes a high performance liquid phase analysis method of ISs-EthE, which can effectively separate the catechins and epicatechins in the ISs-EthE, wherein the linear range of the index component catechin of the ISs-EthE is 2.04 mu g.mL -1 ～32.74μg·mL -1 . The linear range of epicatechin serving as an ISs-EthE index component is 4.50 mu g/mL -1 ～71.20μg·mL -1 Both of which are good in linearity in the linear range (R) 2 >0.99). In addition, the results of precision, repeatability, stability, sample loading recovery rate and the like show that the analysis method is stable and feasible.
Claims (7)
1. A method for measuring the content of a blood ginseng ethanol extract is characterized by comprising the following steps: the method comprises the following steps:
preparing blood ginseng ethanol extract powder: weighing appropriate amount of XUERENSHEN, cutting or chopping, reflux-extracting with 60% ethanol, filtering, reflux-extracting the residue with 60% ethanol, and filtering; mixing the two filtrates, recovering ethanol under reduced pressure, concentrating to a certain concentration, and vacuum drying to water content less than 5% to obtain blood Ginseng radix ethanol extract powder;
preparing a test sample: accurately weighing blood ginseng ethanol extract powder, adding methanol into a volumetric flask, performing ultrasonic treatment to completely dissolve the blood ginseng ethanol extract powder, and fixing the volume to obtain the blood ginseng ethanol extract powder;
preparation of mixed reference: accurately weighing catechin and epicatechin, respectively placing in a volumetric flask, adding methanol to completely dissolve them and fixing the volume to obtain stock solutions of catechin and epicatechin, accurately transferring the stock solutions of catechin and epicatechin, placing in the volumetric flask, fixing the volume with methanol to obtain a mixed reference stock solution of catechin and epicatechin, and storing at 4 ℃;
respectively carrying out UPLC determination on the test solution and the mixed reference solution under chromatographic conditions, effectively separating catechin and epicatechin in the blood ginseng ethanol extract powder, and determining the content of catechin and epicatechin;
the feasibility of the analysis method is evaluated by investigating specificity, linear range, precision, repeatability, stability and sample recovery rate.
2. The method for measuring the content of the ethanol extract of blood ginseng according to claim 1, wherein the method comprises the following steps: in the step a, weighing a proper amount of the panax ginseng medicinal material, cutting or chopping, adding 5-7 times of 60% ethanol for reflux extraction for 1-3h, filtering, adding 3-5 times of 60% ethanol for reflux extraction for 1-2h to filter residues, filtering, combining the two filtrates, recovering ethanol under reduced pressure, concentrating to a certain concentration, and vacuum drying until the water content is less than 5% to obtain the panax ginseng ethanol extract powder.
3. The method for measuring the content of the blood ginseng ethanol extract according to claim 1 or 2, wherein the method comprises the following steps: in the step a, a proper amount of the panax sanguinea medicinal material is weighed, cut or cut, added with 6 times of 60% ethanol for reflux extraction for 2 hours, filtered, added with 4 times of 60% ethanol for reflux extraction for 1.5 hours, filtered, combined with two filtrates, subjected to reduced pressure recovery of ethanol and concentration to a certain concentration, and vacuum-dried until the water content is less than 5%, so that the panax sanguinea ethanol extract powder is obtained.
4. The method for measuring the content of the ethanol extract of blood ginseng according to claim 1, wherein the method comprises the following steps: in the step b, preparing a test article: accurately weighing 0.050g of blood ginseng ethanol extract powder, adding methanol into a 10mL volumetric flask, performing ultrasonic treatment to completely dissolve the powder, and fixing the volume to 10mL to obtain the blood ginseng ethanol extract powder.
5. The method for measuring the content of the ethanol extract of blood ginseng according to claim 1 or 4, wherein the method comprises the following steps: in the step b, the ultrasonic frequency is 53kHz.
6. The method of claim 1, wherein the ethanol extract of Panax ginseng C.A. MeyerThe content determination method of the substance is characterized in that: in the step c, preparing a mixed reference substance: accurately weighing 0.01635g of catechin and 0.01424g of epicatechin in 10mL volumetric flasks respectively, adding methanol to completely dissolve the catechin and the epicatechin, and fixing the volume to 10mL to obtain a stock solution of the catechin and the epicatechin, accurately transferring 2mL of the catechin stock solution and 5mL of the epicatechin stock solution, placing the stock solution in a 100mL volumetric flask, and fixing the volume to 100mL by using methanol to obtain a solution containing 32.70 mu g/mL -1 Catechin and 71.20. Mu.g/mL -1 The mixed control stock solution of epicatechin was stored at 4 ℃.
7. The method for measuring the content of the ethanol extract of blood ginseng according to claim 1, wherein the method comprises the following steps: the chromatographic conditions in the step d are as follows:
chromatographic column cometex tmc 18 :250 mm.times.4.6 mm,5 μm, acetonitrile as mobile phase A,0.2% phosphoric acid water as mobile phase B, sulfuric acid 1.0 mL. Min -1 Detecting wavelength is 280nm, column temperature is 30 ℃, and gradient elution is carried out by injecting 10 mu L of sample, and the gradient elution sequence is as follows: 0-40min,8-14% A.
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