Separation and detection method of ginsenoside Rh2 chiral isomer
Through high-performance liquid chromatography, the C-20 chiral isomer of ginseng saponin Rh2 was successfully isolated and detected by using a binary mobile phase of n-hexane and ethanol bonded chiral chromatography column, which solved the problem of lack of effective separation methods in the prior art and achieved efficient and accurate separation detection effect.
Patent Information
- Application Number
- CN202411726528.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-28
- Publication Date
- 2025-03-07
AI Technical Summary
The prior art lacks effective methods for isolating and detecting the C-20 chiral isomer of ginsenoside Rh2, resulting in slow progress in its pharmacological activity.
High performance liquid chromatography (HPLC) method was used to separate and detect the chiral isomer of ginseng saponin Rh2 using a binary mobile phase of n-hexane and ethanol, combined with a polysaccharide derivative-bonded chiral chromatography column.
This method can effectively separate two C-20 chiral isomers in the Rh2 mixed ginseng saponin, which are simple to operate and have high accuracy. It is suitable for optical purity determination and quality control of Rh2 single isomers of ginseng saponin.
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Figure CN119574733A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of analytical chemistry, in particular to a method for separating and detecting ginsenoside Rh2 chiral isomers. Background Art
[0002] Chirality is one of the most important properties in nature. According to statistics, about 60% of the existing drugs on the market have chiral centers, and the proportion of drugs from natural sources is even higher. The structural differences between stereoisomers of chiral drugs often cause them to have different pharmacological activities and pharmacokinetic processes in vivo, and the use of enantiomeric mixtures can sometimes lead to serious toxic side effects. FDA, EMEA and NMPA have successively issued corresponding policies and regulations to set requirements for the composition and pharmacological and toxicological activities of stereoisomers in the process of new drug research and development. This has made the research on chiral drugs a hot spot and frontier in international new drug research. Due to the lack of effective and widely applicable chiral separation analysis methods, chiral drug separation has become a hot spot and difficulty throughout the research and development of chiral drugs.
[0003] Ginsenoside Rh2 is extracted from ginseng and has anti-tumor, reversal of multidrug resistance, and anti-fatigue activities. The chiral carbon at the C-20 position of ginsenoside Rh2 gives it two isomers, namely (20 R )-ginsenoside Rh2 and (20 S )-ginsenoside Rh2. At present, there are literature reports that the C-20 isomers of ginsenoside compounds have different pharmacological activities and pharmacokinetic properties. For example, (20 R )-Rg3 is (20 S )-Rg3 exhibited better antioxidant, antitumor and neuroprotective activities, but (20 S )-Rg3 ratio (20 R )-Rg3 has better inhibitory activity on 5-HT3. However, there are no patents or literature reports on the method of separating chiral isomers of ginsenoside Rh2, which has led to slow progress in the study of the effect of C-20 chirality in ginsenoside Rh2 on pharmacological activity.
[0004] Therefore, it is urgent to establish a separation and detection method for ginsenoside Rh2 chiral isomers to provide a guarantee for subsequent pharmacology and quality control research on single isomers of ginsenoside Rh2. Summary of the invention
[0005] The purpose of the present invention is to provide a method for separating and detecting ginsenoside Rh2 chiral isomers, which can effectively separate two C-20 chiral isomers in the ginsenoside Rh2 racemic isomer and is easy to operate and has high precision.
[0006] To achieve the purpose of the present invention, the technical solution is as follows: A method for separating and detecting ginsenoside Rh2 chiral isomers, wherein: the method uses high performance liquid chromatography for separation and detection, wherein: the mobile phase is a binary mobile phase composed of n-hexane and an alcohol organic solvent, and the volume ratio is 60:40-95:5; the preparation of the test solution includes: taking a sample to be tested, dissolving and diluting it with the mobile phase.
[0007] Furthermore, in the above method, the alcohol organic solvent in the mobile phase is selected from isopropanol, ethanol or a combination of the two.
[0008] Furthermore, the concentration of the test solution is 300 μg mL -1 .
[0009] Furthermore, the chromatographic column in the high performance liquid chromatography is a polysaccharide derivative bonded chiral chromatographic column; wherein the high performance liquid chromatography condition is that the flow rate of the mobile phase is 0.5 to 2.0 mL min -1 ; Detection wavelength is 190-250 nm; Column temperature is 20-30 o C.
[0010] Furthermore, in the method, the injection volume is 20 μL.
[0011] Further, the mobile phase is a combination of normal hexane and ethanol. Further, the mobile phase is normal hexane and ethanol, and the volume ratio is 70:30.
[0012] Furthermore, in the above technical solution, the flow rate of the mobile phase is 1.0 mL min -1 , column temperature is 25 o C; the detection wavelength is 210 nm.
[0013] Furthermore, the polysaccharide derivative bonded chiral chromatographic column is a cellulose bio-bonded chiral chromatographic column. Chromatographic column; the specifications of the chromatographic column are 250 mm × 4.6 mm, 5 μm.
[0014] Furthermore, the method may also include the determination of the content of ginsenoside Rh2 isomer in the test sample, specifically: taking (20 R )-ginsenoside Rh2 reference substance and / or (20 S )-ginsenoside Rh2 reference and test samples were dissolved in a solution of n-hexane and ethanol (volume ratio of 70:30) to a concentration of 300 μg mL -1 For the sample solution to be tested, take 20 μL of the test solution and the reference solution respectively, inject them into the liquid chromatograph, use the above separation and detection parameters, record the chromatogram, and calculate the peak area according to the external standard method.
[0015] Compared with the prior art, the present invention has the following significant advantages: the present invention provides a method for separating and detecting ginsenoside Rh2 chiral isomers by normal phase high performance liquid chromatography, which can effectively separate the two C-20 chiral isomers in the ginsenoside Rh2 racemic body, and has the advantages of simple solution preparation, convenient operation, fast separation speed and high precision. The present invention can also be used for the determination of the optical purity of a single isomer of ginsenoside Rh2, and is suitable for the quality control of such compounds. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 It is the HPLC chromatogram of the separation of ginsenoside Rh2 racemic isomer in Example 1 of the present invention. DETAILED DESCRIPTION
[0017] The embodiments of the present invention are described in detail below with reference to the accompanying drawings. Example
[0018] A method for separating and detecting ginsenoside Rh2 chiral isomers comprises the following steps: Sample preparation: Weigh 3 mg of the purchased ginsenoside Rh2 racemic body, place it in a 10 mL brown volumetric flask, filter, dissolve it with n-hexane and ethanol (volume ratio of 70:30) and dilute to the mark, shake well, and obtain a sample concentration of 300 μg mL -1 The test solution was o C Save; The determination was carried out under the following chromatographic conditions: Chromatographic instrument: Agilent 1200 high performance liquid chromatograph; Chromatographic column: Chromatographic column (250 mm × 4.6 mm, 5 μm); Mobile phase: n-hexane-ethanol (v: v = 70: 30); Flow rate: 1.0 mL min -1 ; Column temperature: 25°C; Detection wavelength: 210 nm; Injection volume: 20 μL; The obtained chromatogram is as follows Figure 1 The chromatographic parameters are shown in Table 1.
[0019] Table 1
[0020] As shown in Table 1, the normal phase HPLC separation of ginsenoside Rh2 in this example has a separation degree greater than 1.5, and the separation effect is good, which can provide support for subsequent quantitative and qualitative studies of ginsenoside Rh2.
Claims
1. A method for separating and detecting ginsenoside Rh2 chiral isomers, characterized in that: The method uses high performance liquid chromatography for separation, wherein: the mobile phase is a binary mobile phase consisting of n-hexane and an alcohol organic solvent, with a volume ratio of 60:40 to 95:5; the preparation of the test solution includes: taking a sample to be tested, dissolving and diluting it with the mobile phase.
2. The method for separating and detecting ginsenoside Rh2 chiral isomers according to claim 1, characterized in that: The alcohol organic solvent in the mobile phase is selected from isopropanol, ethanol or a combination of the two.
3. The method for separating and detecting ginsenoside Rh2 chiral isomers according to claim 1, characterized in that: The concentration of the test solution is 300 μg mL -1 .
4. The method for separating and detecting ginsenoside Rh2 chiral isomers according to claim 1, characterized in that: The chromatographic column in the high performance liquid chromatography is a polysaccharide derivative bonded chiral chromatographic column; wherein the high performance liquid chromatography condition is that the flow rate of the mobile phase is 0.5 to 2.0 mL min -1 ; Detection wavelength is 190~250 nm; Column temperature is 20~30 ℃.
5. The method for separating and detecting ginsenoside Rh2 chiral isomers according to claim 4, characterized in that: In the described method, the injection volume was 20 μL.
6. The method for separating and detecting ginsenoside Rh2 chiral isomers according to claim 2, characterized in that: The mobile phase is a combination of n-hexane and ethanol.
7. The method for separating and detecting ginsenoside Rh2 chiral isomers according to claim 6, characterized in that: The mobile phase was hexane and ethanol in a volume ratio of 70:
30.
8. The method for separating and detecting ginsenoside Rh2 chiral isomers according to claim 4, characterized in that: The flow rate of the mobile phase was 1.0 mL min -1 , column temperature was 25 ℃; detection wavelength was 210 nm.
9. The method for separating and detecting ginsenoside Rh2 chiral isomers according to claim 4, characterized in that: The polysaccharide derivative bonded chiral chromatographic column is Chromatographic column; the specifications of the chromatographic column are 250 mm × 4.6 mm, 5 μm.
10. The method for separating and detecting ginsenoside Rh2 chiral isomers according to any one of the above claims, characterized in that: The method also includes the quantitative determination of ginsenoside Rh2 isomers in the test sample: taking (20 R )-ginsenoside Rh2 reference substance and / or (20 S )-ginsenoside Rh2 reference substance and test substance were dissolved in n-hexane and alcohol organic solvent solution to prepare corresponding test solutions, injected into liquid chromatograph, and the chromatogram was recorded. The peak area was calculated according to the external standard method.