CN114920619A - Method for purifying plant-derived squalene - Google Patents
Method for purifying plant-derived squalene Download PDFInfo
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- CN114920619A CN114920619A CN202210567876.9A CN202210567876A CN114920619A CN 114920619 A CN114920619 A CN 114920619A CN 202210567876 A CN202210567876 A CN 202210567876A CN 114920619 A CN114920619 A CN 114920619A
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- squalene
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- derived squalene
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C7/00—Purification; Separation; Use of additives
- C07C7/005—Processes comprising at least two steps in series
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C11/00—Aliphatic unsaturated hydrocarbons
- C07C11/21—Alkatrienes; Alkatetraenes; Other alkapolyenes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C7/00—Purification; Separation; Use of additives
- C07C7/04—Purification; Separation; Use of additives by distillation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C7/00—Purification; Separation; Use of additives
- C07C7/12—Purification; Separation; Use of additives by adsorption, i.e. purification or separation of hydrocarbons with the aid of solids, e.g. with ion-exchangers
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses a method for purifying plant source squalene, which is characterized by comprising the following steps: s1, taking plant-derived squalene as a raw material, adding a sample into a molecular distillation preheating system for preheating, controlling a film scraping speed at a proper temperature and a proper vacuum degree, slowly feeding, and receiving a light component; s2, filling the obtained light component on a preparative chromatographic silica gel column, setting an automatic receiving mode, wherein the ultraviolet detection wavelength is 210nm, and eluting a sample by using a gradient mobile phase to obtain the purified squalene. The method prepares high-purity squalene from plant sources by combining molecular distillation and preparative chromatography, and the obtained squalene has high purity and is suitable for large-scale production.
Description
Technical Field
The invention belongs to the technical field of purification of natural products, and particularly relates to a method for purifying plant-derived squalene.
Background
Squalene is mainly obtained from liver oil of some deep-sea sharks (cartilaginous fish). The use of squalene from animal sources is limited by the presence of different Persistent Organic Pollutants (POPs) in the marine environment, such as PCB (polychlorinated biphenyls), PBDE (polybrominated diphenyl ethers), organochlorine pesticides, polycyclic aromatic hydrocarbons, dioxins and heavy metals, and by the concern for protecting marine life. The extraction of squalene from plants has therefore attracted considerable interest.
US patent No. US7161055B2 describes a process for separating and recovering trace components from vegetable oils, especially palm oil. Esterified palm oil is subjected to molecular distillation to produce a concentrate rich in minor constituents, such as tocopherols and squalene.
Direct distillation of vegetable oil is not a suitable method for obtaining squalene, since it is heat resistant due to its unsaturated linear chain. The literature in this field mentions some other methods for the separation and/or fractionation of the lipid fraction and the purification of squalene, which can also be applied industrially. They can be divided into two main types: one is organic solvent extraction, such as hexane extraction, followed by degumming and deacidification as necessary, and finally molecular distillation of the mixture; the other method is to extract oil from seeds by using supercritical fluid and fractionate the oil, but the requirements on equipment are high, the cost is high, and the purity of the obtained squalene cannot reach more than 95%.
The patent CN114252544A uses a chromatographic separation method to purify plant-derived squalene, the content can reach a level of 99%, but the sample amount is small, and the method is only suitable for analytical detection. The preparative chromatography is an automatic column-passing machine, can accurately calculate and quantify the mobile phase, and can greatly solve the problem of complicated operation of the column chromatography. And the current preparative chromatography technology is developed rapidly, and industrialization of a certain scale can be realized.
Therefore, the preparation of high-purity squalene of plant origin by means of a combination of molecular distillation and preparative chromatography is of great importance.
Disclosure of Invention
The invention mainly aims to overcome the defects in the prior art and provide a method for purifying plant-derived squalene. The invention adopts a mode of combining molecular distillation and preparative chromatography to prepare high-purity squalene from plant sources, and the obtained squalene has high content.
The purpose of the invention and the technical problem to be solved are realized by adopting the following technical scheme.
The invention provides a method for purifying plant-derived squalene, which comprises the following steps:
s1, taking plant source squalene as a raw material, adding a sample into a molecular distillation preheating system for preheating, controlling a film scraping speed under proper temperature and vacuum degree, slowly feeding, and receiving a light component;
s2, filling the obtained light component on a preparative chromatographic silica gel column, setting an automatic receiving mode, wherein the ultraviolet detection wavelength is 210nm, and eluting a sample by using a gradient mobile phase to obtain the purified squalene.
Preferably, the content of plant-derived squalene in step S1 is 80%.
Preferably, the plant-derived squalene in step S1 comprises olive oil, soybean oil.
Preferably, the preheating condition in step S1 is: the temperature is 60-70 ℃.
Preferably, the suitable temperature in step S1 is 180-.
Preferably, the weight of the light fraction obtained in step S1 is about 70 to 90% of the total raw material amount.
Preferably, the silica gel column in step S2 is C18 reverse phase silica gel, and the mobile phase is methanol: and (3) water.
Preferably, the elution rate in step S2 is 10 mL/min.
Preferably, the elution mode in step S2 is gradient elution performed by an automatic column chromatography, specifically: the initial mobile phase was methanol: water (V: V) = 50-70%: 50-30%, linear transition to methanol: water (V: V) =100%: 0.
The purpose of the invention and the technical problem to be solved are also realized by adopting the following technical scheme.
The invention also provides squalene obtained by the method for purifying plant-derived squalene, wherein the purity of the obtained squalene is more than or equal to 95%.
By the technical scheme, the invention at least has the following advantages:
1. the raw material used by the invention is plant-derived squalene, and compared with the prior art, the source of the squalene is rich and easy to obtain.
2. The invention adopts molecular distillation and preparative chromatography to purify squalene, heavy components are cut off through the molecular distillation, light components are reserved, the light components are filled on a silica gel column, the purification of the squalene is realized by adjusting the type, proportion and gradient of a mobile phase, and the purity of the obtained squalene is high and is more than or equal to 95%.
3. The invention adopts molecular distillation and preparative chromatography to purify squalene, can carry out large-scale purification of raw materials, and is suitable for industrial production.
In summary, the purification method of the present invention has the advantages and practical values described above, and similar designs are not published or used in similar products, but rather are innovative, and it has great improvements in product composition or function, great progress in technology, and good and practical effects, and has enhanced multiple functions compared with the existing methods, thus being more practical, having industrial wide-ranging value, and being a novel, advanced and practical new design.
The foregoing is a summary of the present invention, and in order to provide a clear understanding of the technical means of the present invention and to be implemented in accordance with the present specification, the following is a detailed description of the preferred embodiments of the present invention.
Specific embodiments of the present invention are given in detail by the following examples.
Detailed Description
To further illustrate the technical means and effects of the present invention for achieving the predetermined objects, the following detailed description of the embodiments, methods and effects of the adjuvant and the herbal water-aqua according to the present invention will be given with reference to the preferred embodiments.
Example 1
Adding squalene with 80% olive oil content into a molecular distillation preheating system, preheating to 60 deg.C, controlling the film scraping speed at 200rpm under 190 deg.C and vacuum degree of less than or equal to 5Pa, slowly feeding, and receiving 80% light component. Loading the obtained light component onto C18 reverse phase silica gel column, loading 1g/100g, setting ultraviolet detection wavelength at 210nm, eluting the sample with gradient mobile phase, and eluting under the following conditions: methanol: water (50%: 50%) linearly transitioned to methanol over 120 minutes: water (100%: 0%) and an elution rate of 10 ml/min. The squalene content obtained was 95.6% by gas chromatography.
Example 2
Adding squalene with soybean oil content of 80% into a molecular distillation preheating system, preheating to 70 deg.C, controlling film scraping speed at 180rpm under 200 deg.C and vacuum degree of less than or equal to 8Pa, slowly feeding, and receiving 80% of light components. Loading the light components onto a C18 reverse phase silica gel column, wherein the loading amount is 1g/100g, the ultraviolet detection wavelength is set to be 210nm, and eluting the sample by using a gradient mobile phase, wherein the elution conditions are as follows: methanol: water (70%: 30%) linearly transitioned to methanol over 120 minutes: water (100%: 0%) and an elution rate of 10 ml/min. The squalene content obtained was 96.1% by gas chromatography.
Example 3
Adding squalene with soybean oil content of 80% into a molecular distillation preheating system, preheating to 60 deg.C, controlling film scraping speed at 220rpm under 180 deg.C and vacuum degree of less than or equal to 5Pa, slowly feeding, and receiving 80% light component. Loading the light components onto a C18 reverse phase silica gel column, wherein the loading amount is 1g/100g, the ultraviolet detection wavelength is set to be 210nm, and eluting the sample by using a gradient mobile phase, wherein the elution conditions are as follows: methanol: water (60%: 40%) linearly transitioned to methanol within 120 minutes: water (100%: 0%) and elution rate 10 ml/min. The squalene content obtained was 95.0% by gas chromatography.
Comparative example 1
In the embodiment, the squalene in the soybean oil deodorized distillate is extracted by a method combining saponification pretreatment and secondary column chromatography, and the specific implementation steps are as follows:
separating unsaponifiable matters: taking 108g of soybean oil deodorized distillate (the squalene content is 1.96 percent), adding a small amount of TBHQ as a protective agent, adding 320ml of 1.5mol/L NaOH-methanol solution under the condition of stirring, refluxing and saponifying at 80 ℃ for 1h, adding 300ml of distilled water after the reaction is finished, stirring for 2 minutes, taking out and cooling to room temperature. Extracting with petroleum ether for three times, each time 500mL, combining the petroleum ether layers, and washing the petroleum ether layers with 5% saline water to neutrality. And removing the solvent by rotary evaporation at 60 ℃ after the petroleum ether layer is dehydrated to obtain the unsaponifiable matter, wherein the content of squalene in the unsaponifiable matter is 12.48 percent.
Primary column chromatography: and (2) taking the unsaponifiable matters obtained in the step (1) as raw materials, taking methanol as a mobile phase, diluting the unsaponifiable matters by a small amount of methanol, adding the unsaponifiable matters into a chromatographic column with JK206 anion exchange resin as a filler, eluting the mobile phase at a certain flow rate, collecting the mobile phase, stopping collecting when the color is colorless, and combining the collected mobile phases to obtain the squalene preparation liquid A.
Recovering sterol by collecting squalene preparation liquid A obtained by first-stage column chromatography, evaporating to remove solvent, and dissolving with methanol at a ratio of V Concentrate ∶V Methanol And the ratio is 1: 5. Placing in a refrigerator at-4 deg.C for 12 hr to obtain white crystal, and vacuum filtering to obtain phytosterol. Recrystallizing for many times according to the steps to obtain the high-purity phytosterol, wherein the purity can reach more than 98 percent. The obtained filtrate is squalene preparation liquid B.
Secondary column chromatography: and (3) concentrating the squalene preparation liquid B at 60 ℃ in vacuum to obtain a second-stage column chromatography raw material. Placing on the top of a silica gel column (100-200 meshes), separating by silica gel column chromatography, eluting with petroleum ether, collecting colorless part, and concentrating to obtain plant squalene, wherein the content of squalene is 72.85% and the total extraction rate is 85.44%.
As can be seen from comparison of examples 1 to 3 with comparative example 1, the method for purifying squalene derived from plants of the present invention, which uses a combination of molecular distillation and preparative chromatography, has a higher purity of squalene product, a higher separation efficiency, a higher degree of automation, a fewer number of steps of the whole process, and a simpler process, compared to the conventional method.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A method for purifying plant-derived squalene, comprising the steps of:
s1, taking plant-derived squalene as a raw material, adding a sample into a molecular distillation preheating system for preheating, controlling a film scraping speed at a proper temperature and a proper vacuum degree, slowly feeding, and receiving a light component;
s2, filling the obtained light component on a preparative chromatographic silica gel column, setting an automatic receiving mode, wherein the ultraviolet detection wavelength is 210nm, and eluting a sample by using a gradient mobile phase to obtain the purified squalene.
2. The method for purifying plant-derived squalene according to claim 1, wherein the content of the plant-derived squalene in step S1 is 80%.
3. The method for purifying plant-derived squalene according to claim 1, wherein the plant-derived squalene in step S1 comprises olive oil and soybean oil.
4. The method for purifying plant-derived squalene according to claim 1, wherein the preheating conditions in step S1 are: the temperature is 60-70 ℃.
5. The method as claimed in claim 1, wherein the suitable temperature in step S1 is 180 ℃ and 200 ℃, the vacuum degree is less than or equal to 10Pa, and the scraping speed is 200 ± 20 rmp.
6. The method for purifying plant-derived squalene according to claim 1, wherein the weight of the light fraction obtained in step S1 is about 70-90% of the total raw material.
7. The method for purifying plant-derived squalene according to claim 1, wherein the silica gel column in step S2 is C18 reverse phase silica gel, and the mobile phase is methanol: and (3) water.
8. The method for purifying plant-derived squalene according to claim 1, wherein the elution rate in step S2 is 10 mL/min.
9. The method for purifying plant-derived squalene according to claim 1, wherein the elution manner in step S2 is gradient elution by an automatic column chromatography, specifically: the initial mobile phase was methanol: water (V: V) = 50-70%: 50-30%, linear transition to methanol: water (V: V) =100%: 0.
10. Squalene obtained by the method for purifying plant-derived squalene according to any one of claims 1-9, wherein the squalene obtained has a purity of 95% or more.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210567876.9A CN114920619A (en) | 2022-05-24 | 2022-05-24 | Method for purifying plant-derived squalene |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210567876.9A CN114920619A (en) | 2022-05-24 | 2022-05-24 | Method for purifying plant-derived squalene |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101597204A (en) * | 2009-07-09 | 2009-12-09 | 天津大学 | With the sweet oil is the method that raw material extracts high-purity squalene |
| CN101830770A (en) * | 2010-06-02 | 2010-09-15 | 天津大学 | Method for extracting squalene from vegetable oil deodorized distillate |
| CN114252544A (en) * | 2021-11-16 | 2022-03-29 | 湖南醇健制药科技有限公司 | Chromatographic separation, purification, detection and analysis method of squalene and pure squalene product |
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2022
- 2022-05-24 CN CN202210567876.9A patent/CN114920619A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101597204A (en) * | 2009-07-09 | 2009-12-09 | 天津大学 | With the sweet oil is the method that raw material extracts high-purity squalene |
| CN101830770A (en) * | 2010-06-02 | 2010-09-15 | 天津大学 | Method for extracting squalene from vegetable oil deodorized distillate |
| CN114252544A (en) * | 2021-11-16 | 2022-03-29 | 湖南醇健制药科技有限公司 | Chromatographic separation, purification, detection and analysis method of squalene and pure squalene product |
Non-Patent Citations (1)
| Title |
|---|
| 戴宇航: "橄榄油中角鲨烯的分离纯化工艺研究", 中国优秀博硕士学位论文全文数据库(硕士)工程科技Ⅰ辑, pages 018 - 47 * |
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