CN111351887A

CN111351887A - Simultaneous determination of ginsenoside Rb1、Rc、Ra1、Ra2、Ra3Method for measuring the content of

Info

Publication number: CN111351887A
Application number: CN201911288387.4A
Authority: CN
Inventors: 金永日; 李绪文; 王紫琪; 张佳音; 马双成
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2019-12-13
Filing date: 2019-12-13
Publication date: 2020-06-30
Anticipated expiration: 2039-12-13
Also published as: CN111351887B

Abstract

The invention discloses a method for simultaneously measuring ginsenoside Ra by utilizing high performance liquid chromatography₁、Ra₂、Ra₃、Rb₁The specific content is that an amide chromatographic column is selected as a chromatographic column; the mobile phase is acetonitrile (B) and water (A) for gradient elution, and the specific conditions are 0-30 min and 10-18% of A; 30-60 min, 18-15% A; the column temperature is 30 ℃; the detection wavelength is 203 nm; the flow rate was 1.0 mL/min. The invention establishes the simultaneous determination of ginsenoside Ra for the first time₁、Ra₂、Ra₃The content method simultaneously overcomes the defect that the prior art can not use the ginsenoside Rb₁And ginsenoside Ra₃Ginsenoside Rc and ginsenoside Ra₁The separation results in a higher measurement result, canCan quickly and accurately measure ginsenoside Ra simultaneously₁、Ra₂、Ra₃、Rb₁And the Rc content.

Description

Simultaneous determination of ginsenoside Rb1、Rc、Ra1、Ra2、Ra3Method for measuring the content of

Technical Field

The invention relates to a method for simultaneously measuring ginseng in panax plants and related products thereof by adopting an HPLC methodSaponin Ra₁、Ra₂、Ra₃、Rb₁The invention relates to a method for Rc content, belonging to the invention creation in the field of traditional Chinese medicine.

Background

Ginsenoside Rg₁、Re、Rb₁、Rb₂、Rb₃Rc and Rd are ginsenoside compounds which are discovered at the earliest time and occupy very important positions in the research of the chemical components and the biological activity of the panax plants. The ginsenosides measured under the content determination items of ginseng, American ginseng, red ginseng, ginseng stem and leaf total saponins, ginseng total saponins, pseudo-ginseng triol saponins and pseudo-ginseng total saponins in the first part of the pharmacopoeia of 2015 edition of China relate to at least two of the ginsenosides. In addition, in 2 months in 2003, ginsenoside Rg is determined in "high performance liquid chromatography determination of ginsenoside in health food" in "health food inspection and evaluation technical Specification" issued by Ministry of health of the people's republic of China at that time₁、Re、Rb₁、Rb₂And Rc and Rd are 6 kinds of ginsenoside.

Except the content determination method in the statutory, the method relates to ginsenoside Rg in the panax plants and different medicinal parts thereof₁、Re、Rb₁、Rb₂、Rb₃The content determination of Rc and Rd can be up to hundreds of documents, wherein the vast majority of methods for determining the content of ginsenoside are high performance liquid chromatography, the stationary phase is octadecylsilane chemically bonded silica (C-18 or ODS), and the mobile phase is acetonitrile-water, methanol-water or acetonitrile-0.1% phosphoric acid water solution for gradient elution.

Ginsenoside Ra₁、Ra₂、Ra₃It is found that ginsenoside Rg₁、Re、Rb₁、Rb₂、Rb₃Ginsenosides found shortly after Rc, Rd are more polar than the above ginsenosides. Ginsenoside Ra has been described so far₁、Ra₂、Ra₃The research on the aspects of biological activity, content measurement and the like is hardly reported, which is probably related to the great difficulty in separating and purifying the saponins. Especially in the content measurement, the inventor has recently excluded [ Zhang Jia YinStudy of high-grade ginsenoside component in Ginseng [ D]Gillen university, 2017, published by Unitry-C18 (4.6mm × 250mm,5 μm,100A) chromatography column using acetonitrile-phosphoric acid aqueous solution as mobile phase, at 203nm wavelength for determination of ginsenoside Ra in various Panax species₁、Ra₂、Ra₃No other reports are found for the determination of the content of (A). The inventors have next conducted on the ginsenoside Ra₁、Ra₂、Ra₃Further studies on the content determination of (A) have shown that it cannot be used for ginsenoside Ra₁、Ra₂、Ra₃Accurate quantitative analysis. As for the simultaneous determination of ginsenoside Ra by high performance liquid chromatography₁、Ra₂、Ra₃、Rb₁The method of Rc content is not yet reported.

Disclosure of Invention

The present inventors have continued research on methods for extracting and separating ginsenoside, identifying structure, and measuring content, and have recently completed the present invention, i.e., a new method for simultaneously measuring ginsenoside Ra by high performance liquid chromatography is first established₁、Ra₂、Ra₃、Rb₁And Rc content.

The present inventors have found for the first time the problem of the prior art that when octadecylsilane chemically bonded silica (C-18 or ODS) column is selected and high performance liquid chromatography is used to measure the content of common ginsenoside, the Ra of ginsenoside can not be measured₁With Rc, ginsenoside Ra₃And Rb₁Completely separated, thereby failing to correctly determine ginsenoside Rb₁、Rc、Ra₁、Ra₃The specific procedures and the research results of (1) are as follows.

According to the high performance liquid chromatography conditions for determining the ginsenoside content in 2015 edition Chinese pharmacopoeia, a commonly used ODS (Agilent ZORBAX SB-C18(4.6 × 250mm,5 mu m) chromatographic column) is selected, a mobile phase is subjected to gradient elution by acetonitrile (B) and water (A), wherein the gradient elution is performed for 0-35 min, 19% of B, 35-55 min, 19-29% of B, 55-70 min, 29% of B, 70-100 min and 29-40% of B, the flow rate is 1.0mL/min, the column temperature is 30 ℃, the detection wavelength is 203nm, the sample injection volume is 1.0mL/min, the detection temperature is high: 20 μ L, 8 ginsenosides Ra were investigated₁、Ra₂、Ra₃、Rb₁、Rb₂、Rb₃Rc and Rd separating effect. The experimental results are shown in figure 1.

Only 6 chromatographic peaks appear in the chromatogram, indicating that the above-mentioned 8 ginsenosides cannot be completely separated by the above-mentioned HPLC conditions. By adding Ra to the mixed standard solution₁、Ra₂、Ra₃、Rb₁、Rb₂、Rb₃And single standard solutions of Rc and Rd, and chromatographic peaks corresponding to the components are confirmed. As a result, it was found that ginsenoside Ra₁Rc, ginsenoside Ra₃、Rb₁The peak appears at the same position, namely the ginsenoside Ra under the condition₁Rc overlap, ginsenoside Ra₃、Rb₁And (4) overlapping. The ginsenosides corresponding to the chromatographic peaks are as follows: chromatographic peak 1-ginsenoside Ra₂(ii) a Chromatographic peak 2-ginsenoside Rb₁+ ginsenoside Ra₃(ii) a Chromatographic peak 3-ginsenoside Rc + ginsenoside Ra₁(ii) a Chromatographic peak 4-ginsenoside Rb₂(ii) a Chromatographic peak 5-ginsenoside Rb₃(ii) a Chromatographic peak 6-ginsenoside Rd. In order to eliminate the factors of low column efficiency of the chromatographic column or difference in separating capacity of different chromatographic columns, C-18 chromatographic columns of other four different manufacturers with different models are selected, and the 8 ginsenosides Ra are continuously investigated according to the liquid phase conditions₁、Ra₂、Ra₃、Rb₁、Rb₂、Rb₃Tnature C18(4.6 × 250mm,5 μm), Unitry C18(4.6 × 250mm,5 μm,100A), COSMOSIL C18-AR-II (4.6 × 250mm,5 μm) and Agilent Eclipse XDB-C18(4.6 × 250mm,5 μm), respectively, the experimental results are shown in FIGS. 2-5.

Experimental results show that the separation effects of four different types of C-18 chromatographic columns are similar, and ginsenoside Ra can not be separated₃、Rb₁Separately, ginsenoside Ra cannot be extracted₁Rc were completely separated. Ginsenoside Ra₁Rc still peaked at the same position, ginsenoside Ra₃、Rb₁Still peaked at the same position, i.e. ginsenoside Ra₁Overlap with Rc, ginsenoside Ra₃And Rb₁And (4) overlapping. The ginsenosides corresponding to the chromatographic peaks are as follows: chromatographic peak 1-ginsenoside Ra₂(ii) a Chromatographic peak 2-ginsenoside Rb₁+ ginsenoside Ra₃(ii) a Chromatographic peak 3-ginsenoside Rc + ginsenoside Ra₁(ii) a Chromatographic peak 4-ginsenoside Rb₂(ii) a Chromatographic peak 5-ginsenoside Rb₃(ii) a Chromatographic peak 6-ginsenoside Rd. Similarly, ginsenoside Ra can not be detected by the method adopted in ginsenoside high performance liquid chromatography assay in health food released in health food inspection and evaluation technical Specification published in 2 months 2003₃、Rb₁Separately, ginsenoside Ra cannot be extracted₁Rc is completely separated; ginsenoside Ra₁Rc still peaked at the same position, ginsenoside Ra₃、Rb₁Still peaked at the same position, i.e. ginsenoside Ra₁Overlap with Rc, ginsenoside Ra₃And Rb₁The problem of overlap. The chromatogram is shown in FIGS. 6-10. The first peak in the figure is ginsenoside Ra₃And Rb_1，The second peak is ginsenoside Ra₁And Rc.

The above research results show that Rb cannot be accurately measured by using ODS chromatographic column₁、Rc、Ra₁、Ra₂、Ra₃The content of (a).

In order to solve the above-mentioned significant defects in the prior art, the invention screens chromatographic columns with various different stationary phases and mobile-phase-relative ginsenoside Rb₁、Rc、Ra₁、Ra₂、Ra₃The separation effect of (2) finally finds that the amide chromatographic column is directed to ginsenoside Rb₁、Rc、Ra₁、Ra₂、Ra₃Has good separation effect.

The stationary phase of the amide chromatographic column is derivatives of silicon dioxide and amino or amido, etc., and the mobile phase is an organic solvent miscible with water. When the mobile phase flows through the stationary phase, a water-rich layer is established on the surface of the stationary phase, so that the analyte is distributed between the mobile phase and the hydrophilic layer, and the method is particularly suitable for separating hydrophilic substances and compounds with larger polarity. Therefore, the experiment researches the amide chromatographic column on 8 hydrophilic ginsenoside Rb₁、Rb₂、Rb₃、Rc、Rd、Ra₁、Ra₂、Ra₃The separation effect of (1).

The chromatographic column is an Acchrom XAmide (5 mu m,100A,4.6 × 250mm) amide column, the mobile phase is acetonitrile (B) and water (A), the gradient elution conditions are 0-30 min, 10-18% of A, 30-60 min, 18-15% of A, the flow rate is 1.0mL/min, the column temperature is 30 ℃, the detection wavelength is 203nm, and the HPLC chromatogram of 8 hydrophilic ginsenosides separated by the amide chromatographic column is shown in figure 11.

The experimental result shows that only 7 chromatographic peaks appear in the chromatogram, which indicates that the 8 hydrophilic ginsenosides can not be completely separated under the mobile phase condition. By adding Ra to the mixed standard solution₁、Ra₂、Ra₃、Rb₁、Rb₂、Rb₃And single standard solutions of Rc and Rd, and chromatographic peaks corresponding to the components are confirmed. As a result, it was found that ginsenoside Ra could be purified by using an amide column chromatography₁With Rc, ginsenoside Ra₃And Rb₁Completely separating, but not separating ginsenoside Rb₂And Rb₃Completely separating, i.e. under the above conditions, ginsenoside Rb₂And Rb₃And (4) overlapping.

Therefore, the invention discusses the determination of ginsenoside Ra by using an amide chromatographic column₁、Ra₂、Ra₃、Rb₁And 5 HPLC analysis methods of the content of Rc hydrophilic ginsenoside.

1. Chromatographic conditions

The chromatographic column is an Acchrom XAmide (5 mu m,100A,4.6 × 250mm) amide column, the mobile phase is acetonitrile (B) and water (A), the gradient elution conditions are that the flow rate is 1.0mL/min, the column temperature is 30 ℃, and the detection wavelength is 203nm, wherein the gradient elution conditions are that the concentration of A is 10-18% in 0-30 min, the concentration of A is 30-60 min, and the concentration of A is 18-15%.

2. Preparation of Standard solutions

Accurate weighing of ginsenoside Ra₁、Ra₃2.00mg each of ginsenoside Ra₂1.00mg of ginsenoside Rb₁And Rc of 7.00mg respectively, dissolving with appropriate amount of methanol, metering to volume in 10ml volumetric flask, shaking, and making into mixed standard solution containing ginsenoside Ra₁、Ra₂、Ra₃、Rb₁The concentrations of Rc were 0.197mg/mL, 0.099mg/mL, 0.196mg/mL, 0.694mg/mL, and 0.696mg/mL, respectively.

3. Preparation of test solution

Respectively taking 2g of ginseng rootlets, American ginseng and pseudo-ginseng medicine powder (sieved by a 40-mesh sieve), carrying out reflux extraction for 3 times, wherein an extraction solvent is methanol, the extraction time is 1.5h, 1h and 0.5h sequentially, the using amount of the methanol is 20mL, combining 3 times of extracting solutions, filtering, carrying out reduced pressure concentration, diluting with water, carrying out adsorption by a D101 macroporous adsorption resin column, desorbing by absolute ethyl alcohol, collecting eluent, carrying out reduced pressure concentration to dryness, dissolving the methanol, fixing the volume to a 10mL volumetric flask, and shaking up to obtain a sample solution for later use.

Drawings

FIG. 1 HPLC chart of Agilent ZORBAX SB-C18 column

FIG. 2 HPLC chart of a Tnature C18(4.6 × 250mm,5 μm) chromatographic column

FIG. 3 HPLC plot of a column for the Unit C18(4.6 × 250mm,5 μm,100A)

FIG. 4 HPLC chart of COSMOSIL C18-AR-II (4.6 × 250mm,5 μm) column

FIG. 5 HPLC chart of Agilent Eclipse XDB-C18(4.6 × 250mm,5 μm) column

FIG. 6 HPLC chart of Agilent ZORBAX SB-C18 column

FIG. 7 HPLC chart of a Tnature C18(4.6 × 250mm,5 μm) column

FIG. 8 HPLC plot of a column for the Unit C18(4.6 × 250mm,5 μm,100A)

FIG. 9 HPLC chart of COSMOSIL C18-AR-II (4.6 × 250mm,5 μm) column

FIG. 10 HPLC chart of Agilent Eclipse XDB-C18(4.6 × 250mm,5 μm) column

Fig. 11 HPLC diagram of amide column separation of 8 hydrophilic ginsenosides, peak position composition of 1: rd; 2: rc; 3: rb₂+Rb₃；4：Rb₁；5：Ra₂；6：Ra₁；7：Ra₃

FIG. 12 ginsenoside Ra₁、Ra₂、Ra₃、Rb₁And an HPLC chart of the Rc standard, wherein the peak position composition is 1: rc; 2: rb₁；3：Ra₂；4：Ra₁；5：Ra₃

FIG. 13 HPLC chart of the ginseng extract, each peak position component is 1: rc; 2: rb₁；3：Ra₂；4：Ra₁；5：Ra₃

Example 1

1. Chromatographic conditions

The chromatographic column is an Acchrom XAmide (5 mu m,100A,4.6 × 250mm) amide column, the mobile phase is acetonitrile (B) and water (A), the gradient elution condition is that the flow rate is 1.0mL/min, the column temperature is 30 ℃, the detection wavelength is 203nm, and the standard product and the sample HPLC chromatogram map are shown in the attached figures 12-13 under the condition that the gradient elution condition is 0-30 min, 10-18% of A, 30-60 min and 18-15% of A.

2. Method for preparing sample solution

In order to reduce the influence of impurities on the separation effect, a macroporous adsorption resin purification method is adopted. Taking 2g of ginseng medicinal material powder (screened by a 40-mesh sieve), carrying out reflux extraction for 3 times, wherein an extraction solvent is methanol, the extraction time is 1.5h, 1h and 0.5h in sequence, the using amount of the methanol is 20mL, combining the extracting solutions obtained in the 3 times, filtering, concentrating under reduced pressure, diluting with water, adsorbing by a D101 macroporous adsorption resin column, desorbing by ethanol, collecting eluent, concentrating under reduced pressure to be dry, dissolving the methanol, fixing the volume to a 10mL volumetric flask, and shaking up to obtain a sample solution for later use.

3. Methodology investigation

3.1 Linear relationship investigation

Accurate weighing of ginsenoside Ra₁、Ra₃10.00mg each of ginsenoside Ra₂5.00mg of ginsenoside Rb₁And Rc 35.00mg each, dissolved in an appropriate amount of methanol and fixed to a volume in a 10mL volumetric flask, and shaken well to prepare a mixed standard solution I. Precisely transferring 5.00mL, 2.00mL, 1.00mL, 0.50mL, 0.20mL and 0.06mL to 10mL volumetric flasks of the mixed standard solution respectively, adding a proper amount of methanol for dissolving, fixing the volume to the scribed line, and shaking up to obtain a series of mixed standard solutions II, III, IV, V, VI and VII for later use. Mixing the standard solutions I-VII with ginsenoside Ra₁The concentrations of (A) are respectively 0.987mg/mL, 0.493mg/mL, 0.197mg/mL, 0.099mg/mL, 0.049mg/mL, 0.020mg/mL and 0.006 mg/mL; ginsenoside Ra₂The concentration of (b) is 0.494mg/mL and 0.247m respectivelyg/mL, 0.099mg/mL, 0.049mg/mL, 0.025mg/mL, 0.010mg/mL, 0.003 mg/mL; ginsenoside Ra₃The concentrations of (A) are 0.982mg/mL, 0.491mg/mL, 0.196mg/mL, 0.098mg/mL, 0.049mg/mL, 0.020mg/mL, 0.006mg/mL, respectively; ginsenoside Rb₁The concentrations of (a) are 3.472mg/mL, 1.736mg/mL, 0.694mg/mL, 0.347mg/mL, 0.174mg/mL, 0.069mg/mL, 0.021mg/mL, respectively; the concentrations of ginsenoside Rc were 3.479mg/mL, 1.740mg/mL, 0.696mg/mL, 0.348mg/mL, 0.174mg/mL, 0.070mg/mL, and 0.021mg/mL, respectively. Filtering the series of mixed standard solutions with 0.22 μm filter membrane, injecting 20 μ L into high performance liquid chromatograph, performing parallel sample injection for 3 times, and calculating the relation between average peak area and sample injection amount to obtain ginsenoside Ra₁、Ra₂、Ra₃、Rb₁The linear relationship of Rc, experimental data and results are shown in tables 1-6.

TABLE 1 ginsenoside Ra₁Linear relationship data of

TABLE 2 ginsenoside Ra₂Linear relationship data of

TABLE 3 ginsenoside Ra₃Linear relationship data of

TABLE 4 ginsenoside Rb₁Linear relationship data of

TABLE 5 Linear relationship data for ginsenoside Rc

TABLE 6 ginsenoside Ra₁、Ra₂、Ra₃、Rb₁Results of investigation of the Linear relationship of Rc

3.2 detection and quantitation limits

Precisely transferring the mixed standard solution VI in the 3.1, gradually diluting with chromatographic methanol, recording the concentration of each compound in the diluted mixed standard solution, filtering with a 0.22 mu m filter membrane, and injecting 20 mu L into a high performance liquid chromatograph. The sampling amount of S/N-3 is the detection limit; the sample size of S/N10 is the limit of quantitation. The results are shown in Table 7.

TABLE 7 detection limit and quantitation limit test results

3.3 precision

Mixing 3.1 with standard solution II, filtering with 0.22 μm filter membrane, injecting 20 μ L into high performance liquid chromatograph, injecting sample for 6 times per day, and recording Ra₁、Ra₂、Ra₃、Rb₁The peak area of Rc and calculating its RSD value. Injecting sample once a day for 6 days, and recording Ra₁、Ra₂、Ra₃、Rb₁Rc and calculating its RSD value, ginsenoside Ra₁、Ra₂、Ra₃、Rb₁RSD values of Rc peak areas are all less than 2%, indicating that the precision of the instrument is good. The results are shown in tables 8-9.

TABLE 8 results of precision examination within day

TABLE 9 results of precision between days

3.4 repeatability

Preparing 6 parts of sample solution by 3.2 method, filtering with 0.22 μm filter membrane, sampling for three times, sampling for 20 μ L each time, and measuring ginsenoside Ra₁、Ra₂、Ra₃、Rb₁And the peak area of Rc, the content of each compound was calculated by an external standard method, and the RSD value was calculated. Ginsenoside Ra₁、Ra₂、Ra₃、Rb₁And the RSD value of the Rc content is less than 2 percent, which shows that the method has good repeatability. The results are shown in tables 10-11.

TABLE 10 ginsenoside Ra₁、Ra₂、Ra₃Result of repetitive investigation of

TABLE 11 ginsenoside Rb₁Results of reproducibility evaluation of Rc

3.5 stability

A sample solution was prepared as described in 3.2, and passed through a 0.22 μm filter and injected every 2h for 12h in succession, 20 μ L each time. Recording ginsenoside Ra₁、Ra₂、Ra₃、Rb₁Retention time and peak area of Rc, and calculating RSD value. The results show that the ginsenoside Ra₁、Ra₂、Ra₃、Rb₁Retention time of Rc, and RSD value of Peak areaAll are less than 2 percent, which shows that the stability of the test solution is good within 12 hours. The results are shown in tables 12-13.

TABLE 12 ginsenoside Ra₁、Ra₂、Ra₃Stability test result of

TABLE 13 ginsenoside Rb₁Results of stability test of Rc

3.6 sample recovery

Collecting 6 parts of Ginseng radix powder (sieved with 60 mesh sieve), and dividing 6 parts into three groups (each group is composed of ginsenoside Ra in the materials) with 2g₁、Ra₂、Ra₃、Rb₁The standard, 3.2, was added to 80%, 100%, 120% of the known content of Rc to prepare 6 sample solutions. Filtering with 0.22 μm filter membrane, sampling for three times, sampling for 20 μ L each time, and measuring ginsenoside Ra₁、Ra₂、Ra₃、Rb₁Peak area of Rc, calculated content, recovery and RSD value. The results show that the ginsenoside Ra₁、Ra₂、Ra₃、Rb₁The Rc recovery rate is between 96.67% and 102.86%, and the RSD values of the recovery rates are all less than 2%, which shows that the recovery rate of the method is good. The results are shown in tables 14-18.

TABLE 14 ginsenoside Ra₁Investigation result of sample recovery rate

TABLE 15 ginsenoside Ra₂Investigation result of sample recovery rate

TABLE 16 ginsenoside Ra₃Is added withSample recovery rate investigation result

TABLE 17 ginsenoside Rb₁Investigation result of sample recovery rate

TABLE 18 examination of sample recovery rates of ginsenoside Rc

Example 2 ginsenoside Ra in three Panax plants₁、Ra₂、Ra₃、Rb₁Measurement of Rc content 1 chromatographic conditions

2. Preparation of Standard solutions

3. Preparation of test solution

4. Determination of content

Take Ra₁、Ra₂、Ra₃、Rb₁Mixing the Rc mixed external standard solution and three test solutions, filtering with 0.22 μm filter membrane, respectively injecting into high performance liquid chromatograph, injecting 20 μ L each time, recording peak area, and calculating ginsenoside Ra by external standard method₁、Ra₂、Ra₃、Rb₁And the content of Rc. The results are shown in Table 19.

TABLE 19 ginsenoside Ra in Panax plants₁、Ra₂、Ra₃、Rb₁Content of Rc

The experimental result shows that the ginsenoside Ra in the ginseng₁、Ra₂、Ra₃、Rb₁And the content of Rc is respectively as follows: 0.107%, 0.068%, 0.048%, 0.428% and 0.422%; ginsenoside Ra in Notoginseng radix₁、Ra₂、Ra₃、Rb₁And the content of Rc is respectively as follows: 0.008%, 0.004%, 0.027%, 0.875% and 0.010%; ginsenoside Rb in American ginseng₁And the content of Rc is respectively as follows: 0.630% and 0.066%, and ginsenoside Ra was not detected₁、Ra₂、Ra₃。

Example 3 ginsenoside Rb₁Comparative analysis of results of Rc content measurement

The method adopts C-18 chromatographic column to treat ginsenoside Rb in three Panax plants₁Rc was determined and the results were analyzed in comparison to the amide column assay established herein.

1. Chromatographic conditions

The chromatographic column is Tnature C18(4.6 × 250mm,5 mu m), the mobile phase is acetonitrile (B) and water (A), the gradient elution condition is 0-35 min, 19% of B, 35-55 min, 19-29% of B, 55-70 min, 29% of B, 70-100 min, 29-40% of B, the flow rate is 1.0mL/min, the column temperature is 30 ℃, and the detection wavelength is 203 nm.

2. Standard substance solution

Accurate weighing of ginsenoside Rb₁And Rc of 4.00mg respectively, dissolving with appropriate amount of methanol, fixing the volume in a 5mL volumetric flask, shaking up, and preparing ginsenoside Rb in the mixed standard solution₁The concentrations of Rc were 0.794mg/mL and 0.795mg/mL, respectively.

3. Test solution

Prepared by the method of 3.4.2.

4. Determination of content

Collecting ginsenoside Rb according to 3.4.1 chromatography condition₁Mixing Rc with external standard solution and three test solutions, filtering with 0.22 μm filter membrane, respectively injecting 20 μ L into high performance liquid chromatograph, sampling for 3 times, recording peak area, and calculating ginsenoside Rb by external standard method₁And the content of Rc. The results are shown in tables 20 to 21.

TABLE 20C-18 chromatographic column determination of ginsenoside Rb in Panax plants₁In an amount of

TABLE 21C-18 column determination of ginsenoside Rc content in Panax plants

5. Analysis of results

Ginsenoside Rb in three Panax plants measured by two chromatographic columns₁Comparative results of the contents of Rc and Rc are shown in Table 3.23.

TABLE 3.23 amide column and C18 column to determine ginsenoside Rb₁Comparison of Rc content

As can be seen from the table, ginsenoside Rb in Ginseng radix and Notoginseng radix measured by C-18 chromatographic column₁The content of Rc was higher than that measured by an amide column. This is in contrast to ginsenoside Rb when measuring the content with C-18 column₁With ginsenoside Ra₃Overlapping ginsenoside Rc and ginsenoside Ra₁Overlap, resulting in ginsenoside Rb₁The peak area of Rc becomes larger. The two methods for determining American ginseng are basically consistent because American ginseng does not contain ginsenoside Ra₁、Ra₂、Ra₃。

Example 4 ginsenoside Ra in three Panax plants₁、Ra₂、Ra₃、Rb₁Measurement of Rc content

1. Chromatographic conditions

The chromatographic column is a TOSOH TSKgel Amide-80(4.6mm I.D × 25cm,5 mu m) Amide column, the mobile phase is acetonitrile (B) and water (A), the gradient elution condition is 0-30 min, 10-18% A, 30-60 min, 18-15% A, the flow rate is 1.0mL/min, the column temperature is 30 ℃, and the detection wavelength is 203 nm.

2. Preparation of Standard solutions

3. Preparation of test solution

4. Determination of content

Take Ra₁、Ra₂、Ra₃、Rb₁Mixing the Rc mixed external standard solution and three test solutions, filtering with 0.22 μm filter membrane, respectively injecting into high performance liquid chromatograph, injecting 20 μ L each time, recording peak area, and calculating ginsenoside Ra by external standard method₁、Ra₂、Ra₃、Rb₁And the content of Rc. The results are shown in Table 24.

TABLE 24 ginsenoside Ra in Panax plants₁、Ra₂、Ra₃、Rb₁Content of Rc

In conclusion, the new ginsenoside Ra established by the invention₁、Ra₂、Ra₃、Rb₁The HPLC content determination method of Rc overcomes the defect that the existing content determination method can not determine the ginsenoside Ra₃And ginsenoside Rb₁Ginsenoside Ra₁Has a significant defect separated from ginsenoside Rc, and eliminates ginsenoside Rb₁And ginsenoside Ra₃Ginsenoside Ra₁And the mutual interference of the content measurement of ginsenoside Rc, the ginsenoside Ra is realized₁、Ra₂、Ra₃、Rb₁And Rc are simultaneously measured, and the accuracy of the measurement result is ensured.

The invention discloses brand-new ginsenoside Ra₁、Ra₂、Ra₃、Rb₁And method for measuring Rc content by HPLC. It should be noted that, for those skilled in the art, it is possible to make improvements and modifications to the technical solutions of the present invention and the aforementioned embodiments, or to substitute part of the technical features thereof, with reference to the research procedures and embodiments disclosed in the present invention. The invention therefore claims that these are based on octadecylsilane chemically bonded silica (C: (A))C18, ODS) can not extract ginsenoside Ra₃And ginsenoside Rb₁Ginsenoside Ra₁Any further screening, modification, equivalent substitution, improvement and the like which are made separately from the important finding of ginsenoside Rc in the present invention shall be included in the scope of protection of the present invention.

Claims

1. Simultaneous determination of ginsenoside Rb₁、Rc、Ra₁、Ra₂、Ra₃The method for measuring the content of (1) is characterized in that the chromatographic column is an amide chromatographic column.

2. The content measurement method according to claim 1, wherein the mobile phase used is acetonitrile (B) and water (a), and the gradient elution condition is: 0-30 min, 10-18% A; 30-60 min, 18-15% A. The flow rate was 1.0 mL/min.

3. The method according to claim 1, wherein the column temperature is 30 ℃.

4. The assay of claim 1, wherein the detector is a solid-liquid separator.

5. The method according to claim 1, wherein the detection wavelength is 203 nm.

6. The assay of claim 1, wherein the flow rate is 1.0 mL/min.

7. The method for measuring the content of claim 1, wherein the preparation method of the test solution comprises the steps of taking 2g of ginseng medicinal material powder, carrying out reflux extraction with methanol for 3 times, wherein the extraction time is 1.5h, 1h and 0.5h in sequence, and the using amount of the methanol is 20mL, combining the 3 times of extracting solutions, filtering, concentrating under reduced pressure, diluting with water, adsorbing by a D101 macroporous adsorption resin column, desorbing with ethanol, collecting eluent, concentrating under reduced pressure to dryness, dissolving the methanol, fixing the volume to a 10mL volumetric flask, and shaking up.