CN111153842A - Method for preparing high-purity lycium barbarum zeaxanthin dipalmitate by using high-speed counter-current chromatography - Google Patents
Method for preparing high-purity lycium barbarum zeaxanthin dipalmitate by using high-speed counter-current chromatography Download PDFInfo
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- CN111153842A CN111153842A CN201811342521.XA CN201811342521A CN111153842A CN 111153842 A CN111153842 A CN 111153842A CN 201811342521 A CN201811342521 A CN 201811342521A CN 111153842 A CN111153842 A CN 111153842A
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- C07C403/06—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone having side-chains substituted by singly-bound oxygen atoms
- C07C403/12—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone having side-chains substituted by singly-bound oxygen atoms by esterified hydroxy groups
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Abstract
The invention discloses a method for preparing high-purity lycium ruthenicum zeaxanthin dipalmitate by using high-speed countercurrent chromatography (HSCCC), and particularly relates to the technical field of food processing. The method comprises pre-treating fructus Lycii powder by water washing and alcohol washing; extracting the lycium carotenoid by combining ultrasonic crushing with tetrahydrofuran; the mixed solution of normal hexane, dichloromethane and acetonitrile is used as an HSCCC solvent system. The method can prepare the zeaxanthin dipalmitate with the purity higher than 90% in a large scale by using the medlar as the raw material, has the advantages of high separation speed, large sample volume, low solvent consumption, high recovery rate, good repeatability and the like, can provide technical support for preparing the high-purity zeaxanthin dipalmitate in a large scale, and has important significance for the efficacy research and the product development of the zeaxanthin dipalmitate.
Description
Technical Field
The invention relates to a method for preparing high-purity zeaxanthin dipalmitate by using high-speed countercurrent chromatography (HSCCC), in particular to a method for preparing high-purity zeaxanthin dipalmitate by using medlar as a raw material.
Background
Lycium chinense is a deciduous shrub of Solanaceae, 1-3 m in height, and China is the main cultivation area. The fruits of Lycium barbarum, known as Lycium barbarum or Lycium barbarum, are mostly oblong ellipsoids, about 1-2cm long, and bright orange. As a traditional medicine and food dual-purpose resource, the medlar is rich in various bioactive substances and trace elements which are beneficial to human health, wherein carotenoid has the potential of promoting human health and strong characteristics of oxidation resistance, inflammation resistance and the like, and has attracted attention in recent years. Studies have shown that the daily intake of an appropriate amount of carotenoids reduces the risk of developing chronic and degenerative diseases such as type 2 diabetes, cardiovascular disease (CVD), cancer, age-related macular degeneration and cataracts. The carotenoids in the medlar mostly exist in the form of esters, and although the research on the purification of the carotenoids at home and abroad is relatively deep, the research objects are mostly free carotenoids with relatively simple compositions, and the purification report of the carotenoid esters is relatively few.
Chinese patent CN103655918A discloses a method for preparing medlar zeaxanthin dipalmitate, which only obtains medlar carotenoid crude extract, does not purify the zeaxanthin dipalmitate in the crude extract, and has low product purity; chinese patent CN108047110A discloses a method for purifying lycium ruthenicum zeaxanthin dipalmitate by using a preparative chromatography, which can obtain high-purity zeaxanthin dipalmitate, but has small sample amount and complex purification process, and is difficult to realize large-scale industrial production.
HSCCC is a chromatographic technique based on the principle of liquid-liquid partitioning, i.e. the separation of a target product is performed by using the difference in the partition coefficients of the components in the two phases. There is currently no report of large-scale preparation of high-purity zeaxanthin dipalmitate in HSCCC.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for preparing high-purity zeaxanthin dipalmitate by using HSCCC.
The technical scheme of the method is as follows:
(1) pulverizing dried fructus Lycii, placing fructus Lycii powder in 25 times volume of ultrapure water, performing ultrasonic treatment in ultrasonic cleaner (300W/40KHz) for 10-30min, and repeating the ultrasonic treatment for 1-3 times; adding a proper amount of 50-70% ethanol into the washed medlar, carrying out ultrasonic treatment for 10-30min, rinsing with ultrapure water for 1 time, and filtering to obtain a treated medlar sample;
(2) adding tetrahydrofuran with the volume 15-30 times that of the medlar sample obtained in the step (1), crushing for 10-40 minutes under the ultrasonic power of 300-650W, centrifuging the extracting solution, and performing rotary evaporation to dryness to obtain a medlar carotenoid crude extract;
(3) mixing 4-6 parts of n-hexane, 1-2 parts of dichloromethane and 2-4 parts of acetonitrile, and performing ultrasonic standing to obtain an HSCCC solvent system;
(4) dissolving the crude extract of the carotenoid of the medlar obtained in the step (2) into the upper phase and the lower phase with the same volume obtained in the step (3), and filtering the mixture through an organic filter membrane with the diameter of 0.45 mu m to obtain a sample for later use;
(5) setting the temperature of an HSCCC column to be 15-35 ℃, selecting the upper phase in the step (3) as a stationary phase, pumping the stationary phase into a main machine pipeline at the flow rate of 25mL/min, after the upper phase is filled with the column and flows out, pumping the lower phase at the flow rate of 3-11mL/min, setting the detection wavelength of 450nm, rotating the main machine in a forward rotation mode, adjusting the rotation speed to 500-900 rpm, after the mobile phase flows out from an outlet and the base line is stable, injecting the sample prepared in the step (4), and collecting target components according to the record map of a detector to obtain the high-purity zeaxanthin dipalmitate.
The invention has the following advantages:
the HSCCC is used for preparing the high-purity zeaxanthin dipalmitate for the first time.
The method adopts water washing and alcohol washing to remove impurities in the medlar, the pretreatment is simple and effective, more than 85 percent of soluble total sugar, protein, polyphenol, flavone and other substances in the raw materials can be removed, the carotenoid loss rate is less than 1 percent, and favorable conditions are provided for preparing the zeaxanthin dipalmitate by subsequent HSCCC purification in large scale.
The method can avoid the problems of irreversible adsorption of the solid support body to components, sample pollution, denaturation and the like, and has the advantages of high separation speed, large sample introduction amount, low solvent consumption, high recovery rate, good repeatability and the like. The purity of the zeaxanthin dipalmitate can reach more than 90 percent.
The method can provide technical support for large-scale production and preparation of high-purity zeaxanthin dipalmitate, and has important significance for efficacy research and product development of zeaxanthin dipalmitate.
Drawings
FIG. 1HSCCC separation of Lycium carotenoids profile;
FIG. 2 is a HPLC detection chart of a zeaxanthin dipalmitate standard;
FIG. 3 HPLC detection chart of zeaxanthin dipalmitate from HSCCC purification;
FIG. 4 TOF-MS graph of zeaxanthin dipalmitate obtained by HSCCC purification.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are further described below with reference to specific embodiments. The described embodiments are some, but not all embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The first embodiment is as follows:
(1) pulverizing dried fructus Lycii, placing fructus Lycii powder in 25 times volume of ultrapure water, ultrasonic cleaning in ultrasonic cleaner (300W/40KHz) for 10min, and repeating the treatment for 1 time; adding a proper amount of 50% ethanol into the washed medlar, carrying out ultrasonic treatment for 10min, rinsing with ultrapure water for 1 time, and filtering to obtain a treated medlar sample;
(2) adding tetrahydrofuran with the volume 15 times that of the medlar sample obtained in the step (1), crushing for 30 minutes under the ultrasonic power of 400W, centrifuging the extracting solution, and performing rotary evaporation to dryness to obtain a medlar carotenoid crude extract;
(3) mixing 10 parts of n-hexane, 3 parts of dichloromethane and 7 parts of acetonitrile, ultrasonically degassing, and standing for layering to obtain an upper phase and a lower phase;
(4) dissolving the crude extract of the carotenoid of the medlar obtained in the step (2) into the upper phase and the lower phase with the same volume obtained in the step (3), and filtering the mixture through an organic filter membrane with the diameter of 0.45 mu m for later use;
(5) setting the temperature of an HSCCC column to be 35 ℃, selecting the upper phase in the step (3) as a stationary phase, pumping the upper phase into a main machine pipeline at the flow rate of 25mL/min, after the upper phase is filled in the column and flows out, pumping the lower phase at the flow rate of 5mL/min, setting the detection wavelength to be 450nm, rotating the main machine in a forward rotation mode, adjusting the rotating speed to 700rpm, after the outlet flows out a mobile phase and the base line is stable, injecting the sample prepared in the step (4), and collecting target components according to the atlas to obtain the high-purity zeaxanthin dipalmitate.
Under the condition, the purity of the zeaxanthin dipalmitate can reach 93.1 percent.
Example two:
(1) pulverizing dried fructus Lycii, placing fructus Lycii powder in 2 times volume of ultrapure water, performing ultrasonic treatment in ultrasonic cleaner (300W/40KHz) for 10min, and repeating the ultrasonic treatment for 1 time; adding a proper amount of 70% ethanol into the washed medlar, carrying out ultrasonic treatment for 10min, rinsing with ultrapure water for 1 time, and filtering to obtain a treated medlar sample;
(2) adding tetrahydrofuran with the volume of 25 times of that of the medlar sample obtained in the step (1), crushing for 40 minutes under 500W ultrasonic power, centrifuging the extracting solution, and performing rotary evaporation to dryness to obtain a medlar carotenoid crude extract;
(3) mixing 3 parts of n-hexane, 1 part of dichloromethane and 2 parts of acetonitrile, ultrasonically degassing, and standing for layering to obtain an upper phase and a lower phase;
(4) dissolving the crude extract of the carotenoid of the medlar obtained in the step (2) into the upper phase and the lower phase with the same volume obtained in the step (3), and filtering the mixture through an organic filter membrane with the diameter of 0.45 mu m to obtain a sample for later use;
(5) setting the temperature of an HSCCC column to be 25 ℃, selecting the upper phase in the step (3) as a stationary phase, pumping the upper phase into a main machine pipeline at the flow rate of 25mL/min, after the upper phase is filled in the column and flows out, pumping the lower phase at the flow rate of 5mL/min, setting the detection wavelength to be 450nm, rotating the main machine in a forward rotation mode, adjusting the rotating speed to 800rpm, after the outlet flows out a mobile phase and the base line is stable, injecting the sample prepared in the step (4), and collecting target components according to the atlas to obtain the high-purity zeaxanthin dipalmitate.
Under the condition, the purity of the zeaxanthin dipalmitate can reach 93.3 percent.
Claims (5)
1. A method for preparing high-purity lycium ruthenicum zeaxanthin dipalmitate by using high-speed countercurrent chromatography (HSCCC) is characterized by comprising the following operation steps: washing and alcohol washing to process the medlar sample; ultrasonic crushing to assist tetrahydrofuran extraction to obtain a carotenoid crude product; preparing a mixed solution of normal hexane, dichloromethane and acetonitrile as an HSCCC solvent system, carrying out ultrasonic degassing, and standing for layering to obtain an upper phase and a lower phase; the high-purity zeaxanthin dipalmitate is prepared by HSCCC.
2. The pretreatment method of claim 1 for treating a powder of Lycium barbarum fruit, comprising: pulverizing dried fructus Lycii, placing fructus Lycii powder in 25 times volume of ultrapure water, ultrasonic treating in ultrasonic cleaner (300W/40KHz) for 10-30min, and repeating for 1-3 times; and adding a proper amount of 50-70% ethanol into the washed medlar, carrying out ultrasonic treatment for 10-30min, rinsing with ultrapure water for 1 time, and filtering to obtain a treated medlar sample.
3. The extraction method for extracting the lycium carotenoid according to the claim 1 is characterized in that: adding 15-30 times of tetrahydrofuran into the fructus Lycii sample, crushing for 10-40min under 300W-650W ultrasonic power, centrifuging the extractive solution, and rotary evaporating to dryness to obtain fructus Lycii carotenoid crude extract.
4. The HSCCC solvent system of claim 1 configured with a mobile phase and a stationary phase, wherein: mixing 4-6 parts of n-hexane, 1-2 parts of dichloromethane and 2-4 parts of acetonitrile, carrying out ultrasonic treatment, and standing to obtain the HSCCC solvent system. Setting the temperature of an HSCCC column to be 15-35 ℃, selecting an upper phase as a stationary phase, pumping into a main machine pipeline at a flow rate of 25mL/min, pumping into a lower phase at a flow rate of 3-11mL/min after the upper phase is filled with the column and flows out, setting a detection wavelength of 450nm, rotating the main machine in a forward rotation mode, adjusting the rotation speed to 500-900 rpm, feeding samples after a mobile phase flows out from an outlet and a base line is stable, and collecting target components according to a detection map to obtain the high-purity zeaxanthin dipalmitate.
5. The method for preparing high-purity lycium ruthenicum zeaxanthin dipalmitate by using HSCCC according to claim 1, wherein: the purity of the zeaxanthin dipalmitate can reach more than 90 percent.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111675640A (en) * | 2020-07-27 | 2020-09-18 | 中国科学院兰州化学物理研究所 | Method for separating and preparing high-purity zeaxanthin |
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| WO2009063100A1 (en) * | 2007-11-13 | 2009-05-22 | Universidad De Almería | Extraction of carotenoids using a single-phase ternary blend of ethanol:hexane:water. |
| CN104447470A (en) * | 2014-12-19 | 2015-03-25 | 宁夏农林科学院 | Method for preparing lycium barbarum lutein by HSCCC (high-speed countercurrent chromatography) separation |
| CN106543769A (en) * | 2016-10-31 | 2017-03-29 | 中国科学院兰州化学物理研究所 | The method that the carotenoidses monomer pigment based on cryptoxanthin is extracted in Fructus Lycii |
| CN107151203A (en) * | 2016-03-03 | 2017-09-12 | 复旦大学 | Separate the method for preparing natural naphthoquinone compound |
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- 2018-11-08 CN CN201811342521.XA patent/CN111153842A/en active Pending
Patent Citations (4)
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| WO2009063100A1 (en) * | 2007-11-13 | 2009-05-22 | Universidad De Almería | Extraction of carotenoids using a single-phase ternary blend of ethanol:hexane:water. |
| CN104447470A (en) * | 2014-12-19 | 2015-03-25 | 宁夏农林科学院 | Method for preparing lycium barbarum lutein by HSCCC (high-speed countercurrent chromatography) separation |
| CN107151203A (en) * | 2016-03-03 | 2017-09-12 | 复旦大学 | Separate the method for preparing natural naphthoquinone compound |
| CN106543769A (en) * | 2016-10-31 | 2017-03-29 | 中国科学院兰州化学物理研究所 | The method that the carotenoidses monomer pigment based on cryptoxanthin is extracted in Fructus Lycii |
Non-Patent Citations (9)
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111675640A (en) * | 2020-07-27 | 2020-09-18 | 中国科学院兰州化学物理研究所 | Method for separating and preparing high-purity zeaxanthin |
| CN111675640B (en) * | 2020-07-27 | 2021-06-22 | 中国科学院兰州化学物理研究所 | Method for separating and preparing high-purity zeaxanthin |
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