HPLC (High Performance Liquid Chromatography) method for utilizing chiral column to separate, identify and prepare monomer matter from cucurbitacin mixture

A technology of cucurbitacins and mixtures, which is applied in the field of medicine and can solve problems such as ineffective separation of compounds

Inactive Publication Date: 2014-09-10
SHENYANG PHARMA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the relevant experiments of the present invention, common chromatographic columns such as C18 columns, C8 columns, amino columns, and pure silica gel columns have been used, but the above compounds cannot be effectively separated.

Method used

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  • HPLC (High Performance Liquid Chromatography) method for utilizing chiral column to separate, identify and prepare monomer matter from cucurbitacin mixture
  • HPLC (High Performance Liquid Chromatography) method for utilizing chiral column to separate, identify and prepare monomer matter from cucurbitacin mixture
  • HPLC (High Performance Liquid Chromatography) method for utilizing chiral column to separate, identify and prepare monomer matter from cucurbitacin mixture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 uses Preparation of five kinds of cucurbitacins by AD-H chiral column

[0079] 1 Experimental equipment and chromatographic conditions

[0080] P230 high-pressure constant-flow pump (preparative type), UV228 ultraviolet-visible detector (Dalian Yilite Scientific Instrument Co., Ltd.); Chiralpak AD-H chiral column (preparative type, 10 μm Daicel Chemical Industry Co., Ltd.)

[0081] Mobile phase: n-hexane: isopropanol: ethanol: trifluoroacetic acid (75:15:10:0.1) (v / v)

[0082] Flow rate: 3.0mL / min

[0083] Column temperature: room temperature

[0084] Detection wavelength: 220nm

[0085] Injection volume: 200μL

[0086] 2 Preparation of samples

[0087] Take 200 mg of cucurbitacin mixture (containing 15% cucurbitacin B), add 5 mL of ethanol to dissolve it, filter through a 0.45 μm microporous membrane, and inject the sample (separation effect is as follows: figure 2 As shown), the effluent was collected in sections to obtain a solution containing fi...

Embodiment 2

[0112] Example 2 Analytical type under different mobile phases Separation of Cucurbitacin Raw Material by AD-H Chiral Column

[0113] Other operations are the same as embodiment 1, the difference is:

[0114] With volume ratio n-hexane: ethanol: isopropanol: glacial acetic acid=70: 20: 10: 0.1 as the mobile phase cucurbitacin mixture for separation and identification, the separation effect is as follows Figure 4-A mentioned.

[0115] Or volume ratio n-hexane: ethanol: trifluoroacetic acid=70: 30: 0.1 is that mobile phase cucurbitacin mixture is separated and identified, and its separation effect is as follows Figure 4-B mentioned.

[0116] Or with volume ratio normal hexane: isopropanol=80:20 is that mobile relative raw material is separated, and its separation effect is as follows Figure 4-C shown.

Embodiment 3

[0117] Various cucurbitacin monomer content determination methodological investigation in the cucurbitacin mixture of embodiment 3

[0118] Standard curve Accurately weigh the appropriate amount of cucurbitacin E, isocucurbitacin B, 3-epi-isocucurbitacin B, dihydrocucurbitacin B and cucurbitacin B reference substances, dissolve and dilute to a concentration of about 40 μg mL with mobile phase -1 (Cucurbitacin E, Isocucurbitacin B, 3-Epi-Isocucurbitacin B), 0.1mg·mL -1 (dihydrocucurbitacin B) and 0.7mg·mL -1 (cucurbitacin B) mixed solution as a control stock solution. Precisely measure 1.0, 3.0, 5.0, 7.0, and 9.0 mL of the reference substance stock solution and put it in a 10 mL measuring bottle, dilute to the mark with mobile phase, shake well, and prepare a series of reference substance solutions. Precisely draw 20 μL each of the series of control solutions, inject them into the liquid chromatograph, and record the peak areas of the chromatographic peaks of the five compone...

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Abstract

The invention discloses an HPLC (High Performance Liquid Chromatography) method for utilizing a chiral column to separate, identify and prepare a monomer matter from a cucurbitacin mixture. The process conditions of the HPLC method are as follows: the chiral column using an amylose derivative as a solid phase is taken as a chromatographic column; more than two mixtures of isopropanol, alcohol, methyl alcohol, acetonitrile, normal hexane, isohexane, n-pentane, trifluoroacetic acid and glacial acetic acid are taken as mobile phases; and a detection wavelength is 220nm-270nm. The HPLC method provided by the invention is characterized by simple operation, high accuracy and strong specificity. The HPLC method can be used for excellently separating matters, such as cucurbitacin B, iso-cucurbitacin B, dihydro cucurbitacin B, cucurbitacin E, and the like, from the cucurbitacin mixture, can be used for preparing various cucurbitacin monomers from the cucurbitacin mixture and also can be used for accurately measuring contents of cucurbitacin raw materials and various cucurbitacins in a prepared product.

Description

technical field [0001] The invention belongs to the technical field of medicine, specifically relates to the separation, preparation and identification of a cucurbitacin mixture, and more specifically relates to the separation, identification and preparation of a cucurbitacin mixture by using a chiral column. Background technique [0002] Cucurbitacins are a class of highly oxidized triterpenes, widely distributed in plants, with more than 200 species. Pharmacological experiments show that cucurbitacin has various biological activities such as anti-tumor, anti-chemical carcinogenic, liver protection, and improving immunity. The cucurbitacin preparation extracted from muskmelon pedicles in my country has been used clinically, and its tablets are recorded in the Ministry of Health. Standard "Prescriptions of Traditional Chinese Medicine" Volume 19. Clinical trials have proved that it has a significant effect on the treatment of primary liver cancer and hepatitis. Cucurbitacin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/36G01N30/08
Inventor 邓意辉李晓峰季秀峰
Owner SHENYANG PHARMA UNIVERSITY
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