Method for preparing high-purity hydroxytyrosol by using high-speed counter-current chromatography and high performance liquid chromatography in combined manner

A technology of high-speed countercurrent chromatography and high-performance liquid chromatography, which is applied in chemical instruments and methods, preparation of organic compounds, organic chemistry, etc., can solve the problem of high-purity hydroxytyrosol without batches, achieve fewer steps, increase yield, high cost effect

Active Publication Date: 2015-12-16
INST OF CHEM IND OF FOREST PROD CHINESE ACAD OF FORESTRY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the separation and preparation of hydroxytyrosol by high-speed counterc...

Method used

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  • Method for preparing high-purity hydroxytyrosol by using high-speed counter-current chromatography and high performance liquid chromatography in combined manner
  • Method for preparing high-purity hydroxytyrosol by using high-speed counter-current chromatography and high performance liquid chromatography in combined manner
  • Method for preparing high-purity hydroxytyrosol by using high-speed counter-current chromatography and high performance liquid chromatography in combined manner

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Separation of standard samples by high-speed countercurrent chromatography

[0033] 1. Experimental equipment:

[0034] FastChrome analytical high-speed countercurrent chromatograph (Jiangyin Countercurrent Technology Co., Ltd.); separation column volume: 25ml; value range: 0.56~0.91; OptiChromeA semi-preparative high-speed countercurrent chromatograph (Jiangyin Countercurrent Technology Co., Ltd.); spiral tube separation column (revolution Radius: 80cm; Value range: 0.50~0.80; Spiral tube inner diameter: 1.59mm), single column volume 180ml, double column volume 360ml.

[0035] 2. Experimental materials:

[0036] Analysis of pure isomers of phenolic samples: catechol, resorcinol, hydroquinone; n-hexane, ethyl acetate, methanol, distilled water

[0037] 3. Preparation of solvents and samples:

[0038] Prepare the methanol mixed solution of the phenolic sample, the concentration of the mixed sample: catechol 20mg / ml+ resorcinol 20mg / ml+ hydroquinone 10mg / ml; use 80% metha...

Embodiment 2

[0059] Example 2 High-speed countercurrent chromatography and high-performance liquid chromatography combined method

[0060] The first step, preparation of hydroxytyrosol ointment

[0061] The oleuropein extract of Olea europaea is enzymatically hydrolyzed, the solid-liquid ratio of the extract and the phosphate buffer solution of 4-7 is 1g:10-100mL, and the mass ratio of the extract and the biological enzyme is 1g:20-100mg, placed in 30 ℃~100℃ in a constant temperature water bath shaker. After enzymolysis for 1h-10h, filter, the filtrate is extracted with ethyl acetate, the ethyl acetate layer is concentrated and dried, the concentrate is hydroxytyrosol ointment, the yield of the ointment relative to the plant leaf powder is 1-20%, and the hydroxytyrosol content is 5~ 20%;

[0062] The second step, high-speed countercurrent chromatography separation

[0063] 1) Select the 3-6:4-8:3-6:4-8 solution of petroleum ether: ethyl acetate: methanol: water according to the volume ratio as t...

Embodiment 3

[0068] Example 3 Experiment of separating hydroxytyrosol ointment by high-speed countercurrent chromatography

[0069] 1. Solvent preparation: petroleum ether: ethyl acetate: methanol: water=2:3:2:3. Prepare 2000ml according to the volume ratio. The upper phase is the stationary phase and the lower phase is the mobile phase.

[0070] 2. Sample preparation: Take 50 mg of hydroxytyrosol ointment in Example 3 and dissolve it in 50 ml of mobile phase.

[0071] 3. System balance: pump full stationary phase at a speed of 50ml / min, pump in mobile phase at a speed of 5ml / min, start

[0072] 4.OptiChrome TM A-300 forward centrifugation, observe the UV3000 output to reach the system balance, calculate the stationary phase retention rate: 60%

[0073] 5. Sample loading: inject the sample into OptiChrome through the sample valve TM In A-300. Forward centrifugation: OptiChrome TM A-300 is set to forward centrifugal rotation, set. The sample gets a spectrum through the detector, as attached ...

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Abstract

The invention discloses a method for preparing high-purity hydroxytyrosol by using high-speed counter-current chromatography and high performance liquid chromatography in a combined manner. The method comprises the following steps of performing enzymolysis on an olive leaf extract; extracting with ethyl acetate and concentrating into hydroxytyrosol ointment of which hydroxytyrosol content is 5%-20%; mixing solvents such as petroleum ether, ethyl acetate, methanol and water proportionally; using the mixture as a high-speed counter-current solvent system under the condition that an upper phase is a fixed phase while a lower phase is a mobile phase; dissolving the hydroxytyrosol ointment in the lower phase to obtain a test article; fully filling multilayered coil separating columns of a high-speed counter-current chromatographic instrument in the fixed phase; injecting the mobile phase at revolving speed of 500-1800 r/min and at flowing speed of 1-5mL/min; and detecting by using an ultraviolet detector of which the wave length is 210-280 nanometers. Normal phase centrifugal rotation is implemented in a whole centrifugal process for 30-300 minutes. When the mobile phase begins to flow out of chromatographic columns, distillate is collected, and after the distillate is detected by HPLC (high performance liquid chromatography), the hydroxytyrosol of which the purity is 60-90% can be prepared. The method is easy to operate and low in pollution; and the purity of the separated hydroxytyrosol is high.

Description

1. Technical Field [0001] The invention relates to a method for separating hydroxytyrosol from plant leaves by enzymatic hydrolysis using high-speed countercurrent chromatography combined with high-performance liquid chromatography, and belongs to the field of natural medicine separation and medical and health care products. 2. Background technology [0002] At present, the separation technology of natural products in the world mostly adopts supercritical extraction technology, macroporous resin column chromatography technology, molecular distillation technology, membrane separation technology, etc. However, for natural products with low content and high biological activity, facing the society Advances and human requirements for high-tech refined products are extremely lacking in new technologies that can achieve separation and purification of high-purity substances and a considerable preparation capacity. If there is no such new technology, it will be difficult to realize the de...

Claims

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Application Information

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IPC IPC(8): C12P7/22C07C39/11C07C37/82
Inventor 王成章原姣姣叶建中陈虹霞周昊陶冉张宇思
Owner INST OF CHEM IND OF FOREST PROD CHINESE ACAD OF FORESTRY
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