WO2012169443A1

WO2012169443A1 - Method for manufacturing refined vegetable squalene and refined vegetable squalene

Info

Publication number: WO2012169443A1
Application number: PCT/JP2012/064286
Authority: WO
Inventors: 季之高橋; 秀忠永井; 光利東
Original assignee: Ｊｘ日鉱日石エネルギー株式会社
Priority date: 2011-06-06
Filing date: 2012-06-01
Publication date: 2012-12-13
Also published as: JPWO2012169443A1; KR20130143139A; TW201307278A

Abstract

Provided are a new method for manufacturing refined vegetable squalene that can easily provide vegetable squalene that has been refined to a high purity, and vegetable squalene that has been refined to a high purity. The method of manufacturing refined vegetable squalene includes at least a chromatography step of providing vegetable oils containing concentrated squalene to a silica column that uses hexane and/or heptane as a solvent and provides refined squalene as a flow-through fraction.

Description

Process for producing purified plant squalene and purified plant squalene

The present invention relates to a method for producing highly purified plant squalene and a highly purified plant squalene.

Squalene is a fat that belongs to terpenoids, is a colorless, odorless, tasteless, low-volatile oily liquid, has a relatively light oily feel, has excellent skin permeability and lubricity, and has very good elongation. In addition, since it has characteristics such as easy emulsification, it is widely used today as an oily base for various cosmetics and pharmaceuticals.

The natural squalene that has been used heretofore has been mainly animal squalene starting from deep-sea bream typified by sharks. However, due to factors such as environmental changes and overfishing, the catch of deep-sea bream has declined, making it difficult to supply natural squalene.

Therefore, plant squalene is attracting attention as a new natural squalene to replace animal squalene.

However, although plant squalene is mainly contained in plant oils and fats obtained from plant fruits and seeds, even if the content is high, it is about several thousand ppm. Therefore, in order to obtain the target plant squalene, a concentration treatment for increasing the content of squalene in the vegetable oil is required at a high magnification. In addition, the above-mentioned concentration treatment also concentrates other small / trace components, such as normal paraffin or various polar substances, that are similar in physical properties such as squalene and boiling point, contained in the vegetable oil and fat. In order to increase the purity of squalene, a purification process for separating and removing small / trace components other than these squalene from squalene is also necessary.

Conventionally, a method for producing plant squalene or plant squalane by concentrating and purifying squalene or squalene, which is a hydride thereof, from vegetable oil or hydride containing squalene is known (for example, Patent Document 1). See ~ 8). However, these methods still include problems that the process is complicated and that the purity of the obtained plant squalene or squalane is not sufficiently high, and it has been purified to high purity in this field. A new method capable of easily producing plant squalene and highly purified plant squalene produced by such a method have been anxious.

Japanese Patent Laid-Open No. 9-176057 JP-A-6-306387 JP-A-6-306388 Japanese Patent No. 3484227 JP-T-2004-502657 JP 2008-13477 A Japanese Patent No. 4642341 Japanese Patent No. 4424939

Therefore, the present invention aims to provide a new purified plant squalene production method capable of easily obtaining highly purified plant squalene, and a highly purified plant squalene. And

As a result of intensive studies to solve the above problems, the present inventors have purified the vegetable oil containing concentrated squalene by chromatography using a silica column, thereby purifying it with high purity. It has been found that plant squalene can be obtained, and the present invention has been completed.

The present invention includes the following.

[1] Purified vegetable squalene comprising at least a chromatography step of supplying vegetable oil containing concentrated squalene to a silica column as a hexane and / or heptane solution to obtain purified squalene as a flow-through fraction Manufacturing method.

[2] The production method of [1], wherein the purity of the purified plant squalene measured by a gas chromatography internal standard method is at least 93%.

[3] The method according to [1] or [2], wherein the vegetable oil containing concentrated squalene is roughly purified, and the normal paraffin content thereof is less than 1% by mass.

[4] Any of [1] to [3], wherein the vegetable oil containing concentrated squalene is roughly refined and has a purity of at least 90% as measured by gas chromatography internal standard method Manufacturing method.

[5] The method according to any one of [1] to [4], wherein the treatment temperature in the chromatography step is −10 to 15 ° C.

[6] Acetone is added to the flow-through fraction obtained from the chromatography step to obtain a homogeneous solution, then water is further added to the mixture, and the two layers are separated by allowing to stand, and the layer containing acetone and water is removed. The method according to any one of [1] to [5], further comprising a washing step of removing the water-soluble fraction contained in the flow-through fraction.

[7] The production method of [6], wherein the purity of the purified plant squalene obtained through the washing step is at least 96% as measured by a gas chromatography internal standard method.

[8] The production method according to any one of [1] to [7], wherein at least the chromatography step is performed in an inert gas atmosphere.

[9] The purified vegetable squalene comprises impurity 1, wherein the gas chromatogram of the purified vegetable squalene, the ratio RT of the retention time RT _S of the main peak at a retention time of RT ₁ and squalene peak of the impurity 1 ₁ / RT _s is in the range of 1.01 to 1.07, and the ratio of the peak area of the impurity 1 with the retention time RT ₁ to the total peak area is 0.0005 to 0.0020, [1] to [8] The production method according to any one of [8].

[10] Purified plant squalene having a purity of at least 96% as measured by a gas chromatography internal standard method.

[11] Purified plant squalene according to [10] comprising the following impurity 1: In gas chromatographic analysis of the purified plant squalene, the retention time RT ₁ of the impurity 1 peak and the main peak of squalene The ratio RT ₁ / RT _s to the retention time RT _S is in the range of 1.01 to 1.07, and the ratio of the peak area of the impurity 1 of the retention time RT _{1 to} the area of all the peaks is 0.0005 to 0 And in GC / MS analysis, the peak of impurity 1 with retention time RT ₁ in the gas chromatography analysis gives peaks with mass numbers 392, 253 and 199.

This specification includes the contents described in the specification of Japanese Patent Application No. 2011-126699, which is the basis of the priority of the present application.

According to the present invention, there are provided a method for producing purified plant squalene and a highly purified plant squalene, which can easily obtain plant squalene purified to a high purity. Thereby, highly purified vegetable squalene can be manufactured and supplied on an industrial scale.

FIG. 1 shows a gas chromatogram of the acetone-treated product (5) obtained in the example. FIG. 2-1 shows a gas chromatogram of olive squalene (before thin film centrifugal distillation treatment and urea treatment). FIG. 2-2 shows a gas chromatogram of olive squalene (before thin film centrifugal distillation treatment and urea treatment). FIG. 3-1 shows a gas chromatogram of purified squalene derived from a commercially available deep-sea shark. FIG. 3-2 shows a gas chromatogram of purified squalene derived from a commercially available deep-sea shark. FIG. 4-1 shows the results of GC analysis of the acetone-treated product (5) obtained in the example. The peak with a retention time of 43.567 minutes corresponds to “Impurity 1” contained in the purified plant squalene. FIG. 4-2 shows the result of GC / MS analysis of impurity 1. Peaks estimated as molecular ions, m / z 253, 199, 69, and other peaks can be seen at mass number (m / z) 392. FIG. 5-1 shows the result of GC analysis of purified squalene derived from commercially available deep-sea bream. FIG. 5-2 shows the results of GC / MS analysis of impurities (assigned to coprostan) that give a peak having a retention time of 42.181 minutes in the GC analysis of purified squalene derived from deep-sea sharks.

Hereinafter, the present invention will be described in detail. The following embodiments are exemplifications for explaining the present invention, and are not intended to limit the present invention only to these embodiments. The present invention can be implemented in various forms without departing from the gist thereof. can do.

The first aspect of the present invention includes at least a chromatography step of supplying vegetable oil containing concentrated squalene to a silica column as a hexane and / or heptane solution to obtain purified squalene as a flow-through fraction. The present invention relates to a method for producing purified plant squalene.

If the production method of the present invention is used, it is possible to easily obtain highly purified vegetable squalene from vegetable oil containing concentrated squalene.

Hereinafter, the method for producing the purified plant squalene of the present invention will be described in detail according to preferred embodiments.

The “vegetable fats and oils containing concentrated squalene” used in the method for producing purified vegetable squalene of the present embodiment can be obtained from vegetable fats and oils containing squalene as a starting material. The vegetable oil containing squalene is not particularly limited as long as it contains squalene, and examples thereof include olive oil, corn oil, soybean oil, safflower oil, rice bran oil, wheat germ oil, and palm oil. Even if the squalene content in these vegetable fats and oils is high, it is about several thousand ppm. The vegetable oil containing concentrated squalene used in the production method of the present embodiment is a vegetable oil with an increased squalene content obtained by concentrating the vegetable oil containing squalene.

When concentrating vegetable oils and oils containing squalene, not only squalene but also other minor / trace components contained in vegetable oils and oils whose physical properties (eg, boiling point) are similar to those of squalene are also concentrated. There is a case. When the vegetable oil containing concentrated squalene contains a large amount of these components, a crude refining treatment is performed to reduce the content of the component in the vegetable oil containing concentrated squalene. Is preferred. This rough purification treatment may be performed separately from the squalene concentration treatment, or the concentration treatment and the rough purification treatment may be performed in the same step.

The method for obtaining vegetable oil containing squalene concentrated by subjecting vegetable oil containing squalene to concentration treatment or rough purification treatment in addition to concentration treatment is not limited, and known methods can be used. These methods include, for example, simple distillation method, thin film centrifugal distillation method, thin film multistage centrifugal distillation method, short process distillation method, and conversion of fatty acid glycerides or free fatty acids contained in vegetable oils and fats to methyl ester or ethyl ester. The method of performing the distillation can be used. Moreover, since the distillate from the deoxidation and deodorization treatment generally performed when producing edible oil from vegetable oil containing squalene contains squalene in a concentrated form, this deacidification and deodorization treatment Can also be regarded as a concentration treatment of squalene or a concentration and crude purification treatment, and the deacidified and deodorized distillate is used as a vegetable oil containing the preferred concentrated squalene. In addition, since squalene is contained in the non-saponified fraction in the saponification treatment of vegetable oil containing squalene, this non-saponified product fraction is recovered and used as vegetable oil containing concentrated squalene. You can also

Further, when the vegetable oil containing concentrated squalene is further roughly purified, impurities derived from the vegetable oil contained in the concentrated vegetable oil containing squalene, particularly normal paraffin and various polarities. Remove material to reduce its content. As a crude purification method in this case, for example, urea or thiourea treatment can be used (Japanese Patent Laid-Open No. 9-176057).

That is, in the urea or thiourea treatment, vegetable oil containing vegetable squalene is mixed with an organic solvent containing urea or thiourea, heated to 40 to 120 ° C., and then 2 to 15 at 0 to 35 ° C. The cooling can be carried out slowly over time, and after cooling, the urea or thiourea crystals and the organic solvent can be removed.

Examples of the organic solvent used for the urea or thiourea treatment include lower alcohols having about 2 to 4 carbon atoms. Examples of such lower alcohol include isopropanol and ethanol (not particularly limited). In addition, when the intended use of the purified plant squalene is food, medicine, cosmetics, etc., it is not preferable to use a solvent such as methanol, which is a concern for safety due to mixing into the product. The amount of the organic solvent used in the urea or thiourea treatment is suitably 0.2 to 100 times the weight of the vegetable oil containing vegetable squalene, and particularly about 0.3 to 10 times. Is preferred.

As urea or thiourea, commercially available industrial urea and industrial thiourea conforming to JIS standards can be used. The amount used is preferably about 0.2 to 20 times, particularly about 0.3 to 5 times the weight of the raw material.

The squalene concentration treatment and rough purification treatment may be performed using any one of the above methods, including the case where the concentration treatment and the rough purification treatment are performed simultaneously, or may be used in combination of two or more.

The vegetable oil containing concentrated squalene contains impurities such as normal paraffin derived from vegetable oil and polar substances in addition to squalene, even when the above-described rough purification treatment is performed. It is preferable that the purity measured by the gas chromatography internal standard method in the vegetable oil containing concentrated squalene used in the present embodiment is at least 90%. The vegetable oil containing concentrated squalene is subjected to a rough purification treatment, and the content of normal paraffin thereof is preferably 2% by mass or less, more preferably less than 1% by mass, and Even more preferably, it is less than 9% by weight. In addition, when the vegetable oil containing concentrated squalene contains 2% by mass or more of normal paraffin, the fluidity deteriorates at room temperature, and when the content is further increased, it solidifies at room temperature and is difficult to use as a raw material. There is also a problem.

In the production method of this embodiment, vegetable oil containing concentrated squalene is dissolved in an oleophilic organic solvent, and the resulting solution is subjected to a chromatography step using a silica column. In chromatographic process using silica column, impurities (especially polar substances) other than squalene contained in vegetable oil containing concentrated squalene can be removed by adsorption to silica, and squalene can be purified efficiently. it can.

Here, as the “lipophilic organic solvent”, various lipophilic organic solvents can be used, but it is considered that the purified plant squalene as the target product is used for various cosmetics, foods, medicines, etc. Then, it is preferable to select from hexane and / or heptane. The amount of the oleophilic organic solvent used is suitably 0.25 to 3.00 times the mass of the vegetable oil containing concentrated squalene, and is particularly suitable for the vegetable property containing concentrated squalene. It is preferable to use the same amount as oil.

The apparatus used in the chromatography step can be performed using an apparatus for performing general column chromatography, and the equilibration and flow rate of the column are determined by the vegetable oil and fat containing the column size and concentrated squalene. It can be set as appropriate depending on the amount of processing.

The silica to be packed in the silica column is not particularly limited as long as polar impurities can be removed by adsorption, and crushed silica or spherical silica can be used. Specifically, silica gel PSQ-100B (trade name) manufactured by Fuji Silysia Chemical Co., Ltd., Daiso Gel (trade name) (trade name) manufactured by Daiso Corporation, Wakogel (trade name) manufactured by Wako Pure Chemical Industries, Ltd. (50- 200 μm spherical shape).

In the chromatography step, impurities other than squalene, particularly polar substances, in the vegetable oil containing concentrated squalene are adsorbed to the silica column. Therefore, the vegetable squalene refine | purified with high purity can be obtained by collect | recovering the passage fraction (passage liquid) of a silica column. That is, a plant having a gas chromatography relative purity of 95% or more, preferably 96% or more, and a purity measured by gas chromatography internal standard method of 93% or more, preferably 94% or more. Squalene can be obtained.

The purity of squalene in purified vegetable squalene or vegetable oil containing concentrated squalene is determined by gas chromatography using a capillary column (hereinafter also referred to as “GC”). it can. In this specification, “gas chromatography relative purity” of squalene (hereinafter sometimes simply referred to as “relative purity”) refers to all of the samples derived from the sample detected by GC of the sample containing the target squalene. The percentage of the peak area attributed to squalene relative to the peak area. Here, the peak attributed to squalene refers to a peak attributed to all squalene isomers. Further, in this specification, “purity measured by gas chromatography internal standard method” of squalene (hereinafter sometimes simply referred to as “internal standard method purity”) refers to the purity obtained by the following method. . First, as a standard squalene, a reagent dodecane as an internal standard substance is added to a reagent squalene (indicated purity: 99.5% or more) at a predetermined mass ratio, and this is further diluted to a predetermined concentration with normal hexane. For GC. The resulting calculates a ratio A _s between the relative purity RP _s and squalene area of the peak of the peak area and from dodecane attributed to (including all isomers) standard squalene from the chromatogram. On the other hand, the sample containing squalene to be measured is added with dodecane at the same mass ratio and diluted with normal hexane, and then GC analysis is performed. From the obtained chromatogram, the ratio A between the area of the peak attributed to squalene and the area of the peak derived from dodecane is calculated. Then, the internal standard method purity of squalene in the sample is calculated by the following formula.

Internal standard method purity = RP _s × A / A _s (%)

A more specific GC analysis method is as follows.

Take 20 mg of dodecane and 50 mg of standard squalene (display purity 99.5% or more, manufactured by Wako Pure Chemical Industries, Ltd.) accurately weighed in a 100 mL volumetric flask and prepare a standard squalene solution by measuring up with HPLC grade normal hexane. To do. On the other hand, 20 mg of dodecane and 50 mg of sample precisely weighed in a 100 mL volumetric flask are taken, and a sample solution is prepared by measuring up with HPLC grade normal hexane.

The gas chromatograph is GC-2010 (OCI specification) manufactured by Shimadzu Corporation, the detector is FID, the analytical column is J & W Scientific DB-5 manufactured by Agilent Technologies, the column equilibration time is 0 minutes, the sample injection amount is 1 μL, Injection mode is full volume injection, carrier gas is He, control mode is flow rate 15.0 mL / min, purge dose is 1.5 mL / min, total analysis time is 35 minutes, detector temperature is 280 ° C., makeup flow rate is 20 mL / min Min, the hydrogen flow rate is 40 mL / min, the air flow rate is 400 mL / min, the temperature program for the vaporization chamber and the temperature program for the column oven are as shown in Tables 1 and 2, respectively. Peak separation in the resulting chromatogram is performed by splitting from the baseline.

In the chromatography step, impurities that are polar substances are mainly removed by adsorption onto silica, but impurities that are nonpolar substances typified by normal paraffin are difficult to remove by adsorption onto silica. When the content of normal paraffin in the flow-through fraction of the chromatography step using the silica column is larger than the predetermined value, the solution containing the flow-through fraction (flowing solution in the chromatography step) is cooled to make normal paraffin as a solid. The normal paraffin content in the refined vegetable squalene can be further reduced by precipitating and subjecting it to a step of removing solid content by filtration (cooling / separation step). In particular, in the GC analysis of the pass-through fraction, when the ratio of the total area of the peaks having a shorter retention time than the peak attributed to squalene with respect to the total peak area exceeds 0.005, the peak area is increased by the cooling / separation step. The ratio can be 0.002 or less. The temperature in the cooling / separation step is preferably −10 to 15 ° C.

Further, the chromatography step and the cooling / separation step can be performed in one step. That is, by cooling a solution in which vegetable oil containing concentrated squalene is dissolved in a lipophilic organic solvent, and / or subjecting the solution to a chromatography step using a silica column under cooling, At the same time, the normal impurities that are precipitated as a solid in the solution can be removed by filtration using a silica column as a filter medium. In this case, it is not necessary to provide the cooling / separation process as a separate process, and the process can be simplified. In this case, the cooling temperature of the solution and / or the treatment temperature in the chromatography step is preferably −10 to 15 ° C.

Next, the purified plant obtained by subjecting the flow-through obtained by the chromatography step (including the cooling / separation step in some cases) to a washing step of washing with a hydrophilic organic solvent and water. It is possible to further improve the purity of the crystalline squalene. Here, the “hydrophilic organic solvent” includes various hydrophilic organic solvents. In consideration of using the obtained purified plant squalene for various cosmetics, foods, medical uses, etc., acetone is preferable. .

The washing step can be performed by the following procedure. A hydrophilic organic solvent is added to and mixed with a passing solution comprising a purified vegetable squalene and a lipophilic solvent obtained by the chromatography step (including the cooling / separation step in some cases) A good solution. Furthermore, a small amount of water is added to the solution, mixed and allowed to stand to separate into a layer of a mixture containing a hydrophilic organic solvent and water and a layer of a mixture containing squalene and a lipophilic organic solvent. Let me. And the refined vegetable squalene can be obtained by fractionating the layer of the mixture containing a squalene and a lipophilic organic solvent, and collect | recovering the fractions contained in this layer. In this washing step, water-soluble impurities contained in the flow-through liquid are transferred to the layer of the mixture containing the hydrophilic organic solvent and water and removed from the mixture containing squalene and the lipophilic organic solvent. Furthermore, plant squalene purified to a high purity can be obtained. That is, a purified plant squalene having a relative purity of 97% or more, preferably 98% or more, and an internal standard method purity of 95% or more, preferably 96% or more can be obtained.

The amount of the hydrophilic organic solvent used in the washing step is 0.1 to 3 times, preferably 0.1 to 2 times, more preferably the amount of the passing liquid obtained by the chromatography step. Is 0.5 to 1 times the amount. The amount of a small amount of water to be added later varies depending on the amount of the hydrophilic organic solvent used, but is 0.01 to 0.5 times the amount of the hydrophilic organic solvent, preferably 0.1. Double amount to 0.3 times amount.

A purified vegetable squalene can be obtained by removing the lipophilic organic solvent from the flow-through obtained from the chromatography step (optionally including the cooling / separation step and / or the washing step). . The method for removing the solvent is not particularly limited, and a known method such as steam distillation or spraying under reduced pressure can be used.

Note that squalene has a property of being easily oxidized, and when it is oxidized, it generates a specific odor. Therefore, the squalene contains the concentrated squalene as a raw material, the chromatography step, the cooling / separation step, the washing step. Concentration treatment, crude refining treatment for obtaining vegetable oil and fat, and possibility of contact of squalene with air, including storage and transfer of vegetable oil and fat containing purified squalene or refined vegetable squalene It is preferable to perform a certain process, process, etc. in inert gas atmosphere, such as nitrogen.

The purified plant squalene obtained through the chromatography step or the washing step contains at least a specific impurity (hereinafter referred to as “impurity 1”). Impurity 1, wherein the chromatography step or in the gas chromatogram of vegetable squalene purified obtained through the washing step, the ratio of the retention times RT _S of the main peak of a retention time impurities 1 RT ₁ and squalene RT 1 _/ RT _s is in the range of 1.01 to 1.07, and the peak area of impurity 1, it is preferable the ratio of the area of all peaks is 0.0005 to 0.0020. Although the chemical structure of impurity 1 to which the peak of retention time RT ₁ in the gas chromatogram is assigned has not been specified, terpene-like was found from the results of GC / MS analysis and NMR analysis after fractionation by liquid chromatography. It is estimated that the substance has the following structure.

In the gas chromatogram of purified plant squalene, the ratio of the peak area of the impurity 1 to the total peak area is 0.0020 or less, so that it can be used for bases and foods of pharmaceuticals and cosmetics, especially for pharmaceuticals. Can also be used. On the other hand, the ratio of the area of the peak of impurity 1 to the area of all peaks is less than 0.0005, for example, it is necessary to extremely increase the contact time with the silica column in the production method of the present embodiment. Thus, it becomes difficult to produce the purified plant squalene with economic rationality.

According to the production method of the present embodiment, plant squalene is obtained in a high purity of 98% or more as relative purity and 96% or more as internal standard method purity from vegetable oil containing concentrated squalene. Plant squalene that can be applied to various uses as a base for pharmaceuticals and cosmetics and foods can be produced.

In addition, the refined plant squalene can be obtained by hydrogenating the refined plant squalene obtained by the production method of the present embodiment according to a conventional method. The purified plant squalene thus obtained has a high purity as with the purified plant squalene obtained by the production method of the present embodiment.

The second aspect of the present invention relates to a purified plant squalene having a purity of at least 96% as measured by a gas chromatography internal standard method.

Hereinafter, the purified plant squalene of the present invention will be described according to a preferred embodiment.

The purified plant squalene of the present embodiment can be produced by the above-described method for producing purified plant squalene of the present invention.

High-purity squalene is manufactured and supplied for animal squalene derived from deep sea bream. However, for vegetable squalene, the squalene content in the vegetable oil containing squalene, which is the starting material, is extremely low, and the fats and oils contain many impurities that approximate physical properties to squalene. It was difficult to obtain a highly pure product by concentration and purification. Therefore, the refined vegetable squalene of this embodiment is a highly pure vegetable squalene which is not conventionally known.

In addition, when the refined plant squalene of this embodiment is compared with the conventionally known refined animal squalene, not only the starting material is different but also the composition itself is different. That is, first, purified plant squalene and purified animal squalene are different from each other in the composition of the squalene isomers constituting each. Although many isomers exist in squalene, the composition of isomers constituting squalene differs depending on whether the starting material is plant or animal. Moreover, the refined plant squalene and the refined animal squalene have different impurity compositions. Purified plant squalene and purified animal squalene inevitably contain trace amounts of impurities, but the composition of these impurities depends on whether the starting material is plant or animal. is there. Thus, the refined plant squalene is different from those of the refined animal squalene in the chemical composition of the main component and the chemical composition as a composition including impurities.

The purified plant squalene of this embodiment contains at least an impurity 1. Impurity 1, in a gas chromatography analysis of the purified vegetable squalene in this embodiment, the ratio _RT 1 / RT _s and the retention time RT _S of the main peak of a retention time impurities 1 RT ₁ and squalene 1.01 Is characterized by a ratio of the area of the peak of impurity 1 to the area of all peaks being 0.0005 to 0.0020. Further, in the GC / MS analysis, the peak of the impurity 1 having the retention time RT ₁ in the gas chromatography analysis gives peaks of mass numbers 392, 253, and 199.

The impurity 1 specified by GC analysis and GC / MS analysis contained in the purified plant squalene of the present embodiment is a terpene-like compound whose structure is not specified as described above. And it was confirmed that this impurity 1 is not contained in the refined animal squalene. The purified animal squalene contains a plurality of impurities that give a peak having a retention time ratio close to that of the impurity 1 in the GC analysis. From the results of GC / MS analysis, all of the impurities are the impurities. It has been confirmed that the substance is different from 1. That is, the impurity 1 gives a large peak to mass numbers (m / z) 392, 253, and 199 in the GC / MS analysis, but the plurality of impurities in the purified animal squalene are all GC / In the MS analysis, the three mass number peaks are not given together.

From the above, when the purified squalene contains the impurity 1, it is determined that the purified squalene is vegetable.

When the ratio of the peak area of impurity 1 and the area of all peaks in the GC analysis of the purified plant squalene of this embodiment exceeds 0.0020, it is particularly difficult to use it for pharmaceutical purposes. On the other hand, in order to perform purification so that the ratio of the peak areas is less than 0.0005, for example, in chromatography using a silica column, it is necessary to extremely increase the contact time with the raw material silica. It becomes difficult to produce plant squalene purified with reasonableness.

The refined plant squalene of the present embodiment has the above-mentioned properties, and can be used for pharmaceuticals and cosmetic bases and foods, which are difficult with conventional plant squalene, especially for pharmaceuticals.

Hereinafter, the present invention will be described in more detail by way of examples. However, the technical scope of the present invention is not limited to the following examples.

Purity of olive squalene (hereinafter referred to as “0LSQE”) (source: SOSRACCORPORACION ALIMENTARIA, SA Madrid, Spain, internal standard method purity of 85%) was obtained by thin film centrifugal distillation treatment and urea treatment, internal standard method purity of 90.9% And purified as a raw material. The normal paraffin content of the raw material was 0.85% by mass.

The raw material (57.7 g) was dissolved in an equal amount (57.7 g) of hexane (HPLC grade: the same applies to hexane described below), and then a silica column Sep-Pak VAX PS-2 (registered trademark) (Waters Impurity adsorption treatment was carried out. The adsorption treatment was carried out at a flow rate of 2 mL / min using a Shimadzu HPLC pump AV-10. The column treatment was performed at 15 ° C.

¡All parts where squalene may come into contact with air were replaced with nitrogen gas. In addition, the measurement of the squalene purity in the raw material and the purified squalene was performed using the above-described method by GC.

After the feed of the raw material was completed, the recovered material (squalene amount: 32.5 g) until the raw material / hexane solution did not flow out from the lower part of the column was designated as “silica filtered product (1)”.

A part of the silica filter product (1) is taken out, treated with an evaporator at 40 ° C. for 2 hours, and then the solvent is distilled off by vacuum drying (40 ° C.) for 3 hours. The amount of purified plant squalene recovered And the purity was confirmed.

Subsequently, the same amount (57.7 g) of hexane as the raw material in which 0 LSQE remaining in the column was allowed to flow (squalene amount: 24.0 g) was designated as “hexane-cleaned product (2)”.

Furthermore, after the hexane-cleaned product (2) (squalene amount: 15.6 g) was dissolved in hexane (57.7 g) in an amount equivalent to that of the raw material, an impurity adsorption treatment was performed on the silica column in the same manner as described above. A part of the hexane-cleaned product (2) was taken out and the recovered amount and purity were confirmed in the same manner as in the above method.

After completion of feeding the hexane-cleaned product (2), the hexane-cleaned product (2) / the product collected until the hexane solution no longer comes out from the bottom of the column (squalene amount: 6.3 g) was designated as “silica filtered product (3)”. A part of the silica filtered product (3) was taken out, and the recovered amount and purity were confirmed in the same manner as in the above method.

Subsequently, 0LSQE remaining in the column with the same amount (57.7 g) of hexane as the raw material was washed and recovered (amount of squalene: 7.8 g) was designated as “hexane-cleaned product (4)”. A part of the hexane-cleaned product (4) was taken out and the recovered amount and purity were confirmed in the same manner as in the above method.

"Silica filtered product (1)", "Silica filtered product (3)" and "Hexane-cleaned product (4)" were mixed (200g) and transferred to a separatory funnel, 0.2g of the total amount of 40g Of acetone was added, and after replacing with nitrogen gas, the mixture was shaken 100 times. After shaking, 8 g of water was added and shaken again 100 times, and the mixture was allowed to stand for 20 minutes. The content liquid separated into two layers, a hexane layer and an acetone / water layer. After removing the lower acetone / water layer, the recovered upper hexane fraction (squalene amount: 45.7 g) was designated as “acetone treated product (5)”. A part of the acetone-treated product (5) was taken out, and the recovered amount and purity were confirmed in the same manner as in the above method.

Table 3 shows the purity (relative purity and internal standard method purity) and step recovery in each step.

In the GC analysis of the acetone-treated product (5), the peak area of the impurity 1 having a ratio (RT ₁ / RT _s ) of the retention time RT ₁ of the impurity 1 to the retention time RT _s of the main peak of squalene is 1.014 The ratio of the total area of all the peaks of the purified plant squalene was 0.00065. FIG. 1 shows a GC chromatogram of a typical acetone-treated product (5).

Figures 2-1 and 2-2 show gas chromatograms of olive squalene used (before thin film centrifugal distillation and urea treatment) for reference.

Furthermore, Figures 3-1 and 3-2 show gas chromatograms of purified squalene derived from commercially available deep-sea bream as a reference.

Further, GC / MS analysis of the acetone-treated product (5) was performed. The result is shown in FIG. The column temperature program for GC analysis in this GC / MS analysis is different from that for the GC analysis.

In the GC chromatogram of FIG. 4-1, the peak with a retention time of 43.567 minutes corresponds to “Impurity 1” contained in the purified plant squalene. The mass spectrum of impurity 1 is shown in FIG. In the mass spectrum, a peak estimated as a molecular ion at a mass number (m / z) 392, m / z 253, 199, 69, and other peaks are seen.

For further comparison, a GC analysis of purified squalene derived from commercially available deep-sea bream was performed. The result is shown in FIG.

In the GC chromatogram of the purified plant squalene shown in FIG. 4-1, a shoulder peak of the squalene isomer is observed on the short retention time side (retention time 41.50 to 41.70 minutes) of the main peak. . On the other hand, in the GC chromatogram of purified animal squalene shown in FIG. 5-1, a peak attributed to the isomer of squalene is hardly observed. This indicates that there is a difference in the isomer composition of squalene, which is the main component, between purified plant squalene and purified animal squalene.

In the GC chromatogram of purified animal squalene shown in FIG. 5-1, the peak of impurities having a retention time ratio approximate to the peak attributed to impurity 1 contained in the purified plant squalene is the main. Five things are observed. Each mass spectrum of these peaks is different from the mass spectrum of impurity 1 contained in the purified plant squalene, and does not show both m / z 392, 253, and 199 peaks. It belongs to a substance different from the impurity 1. In addition, the peak of each impurity in the GC chromatogram of the purified animal squalene is attributed as shown in Table 4 from the comparison between the mass spectrum and the library. Among these, a mass spectrum of an impurity (assigned to coprostan) that gives a peak at a retention time of 42.181 minutes in the GC chromatogram is shown as an example in FIG.

As is clear from the above results, it was possible to obtain purified plant squalene having a high purity, which could not be obtained conventionally, by the above method. The purified plant squalene obtained has an isomer composition and an impurity composition of squalene different from the purified animal squalene.

According to the present invention, there are provided a method for producing a purified plant squalene that can easily obtain a highly purified plant squalene, and a highly purified plant squalene. This is expected to contribute greatly to the production of various cosmetics, pharmaceuticals, foods and the like.

All publications, patents and patent applications cited in this specification shall be incorporated into the present specification as they are.

Claims

A method for producing purified vegetable squalene, comprising at least a chromatography step of supplying vegetable oil containing concentrated squalene to a silica column as a hexane and / or heptane solution to obtain purified squalene as a flow-through fraction .
The production method according to claim 1, wherein the purity of the purified plant squalene measured by gas chromatography internal standard method is at least 93%.
3. The production method according to claim 1 or 2, wherein the vegetable oil containing concentrated squalene is roughly refined and has a normal paraffin content of less than 1% by mass.
The vegetable oil containing concentrated squalene is crudely purified and has a purity measured by gas chromatography internal standard method of at least 90%. Manufacturing method.
The production method according to claims 1 to 4, wherein a treatment temperature in the chromatography step is -10 to 15 ° C.
Acetone is added to the flow-through fraction obtained from the chromatography step to obtain a homogeneous solution, and then water is further added to the mixture, mixed and allowed to stand to separate into two layers, and the layer containing acetone and water is removed. The production method according to any one of claims 1 to 5, further comprising a washing step of removing a water-soluble fraction contained in the flow-through fraction.
The production method according to claim 6, wherein the purity of the purified plant squalene obtained through the washing step is at least 96% as measured by a gas chromatography internal standard method.
The production method according to any one of claims 1 to 7, wherein at least the chromatography step is performed in an inert gas atmosphere.
Purified vegetable squalene comprises impurity 1, wherein the gas chromatogram of the purified vegetable squalene ratio RT 1 / RT and retention time RT S of the main peak at a retention time of RT 1 and squalene peak of the impurity 1 s is in the range of 1.01 to 1.07, the ratio of the area and the total peak area of the peak of the impurity 1 retention times RT 1 is 0.0005 to 0.0020, more of claims 1 to 8 The production method according to claim 1.
Purified plant squalene having a purity measured by gas chromatography internal standard method of at least 96%.
The purified plant squalene of claim 10 comprising the following impurity 1: In the gas chromatographic analysis of the purified plant squalene, the retention time RT 1 of the impurity 1 peak and the main peak of squalene time RT ratio RT 1 / RT s and S is in the range of 1.01 to 1.07, and the peak area of the impurity 1 retention times RT 1, the ratio 0.0005 to zero area of all peaks. And in GC / MS analysis, the peak of impurity 1 with retention time RT 1 in the gas chromatography analysis gives peaks of mass numbers 392, 253 and 199.