JPH0586396A - Method for purifying hinokitiol - Google Patents

Abstract

(57) [Summary] The present invention provides a method for purifying hinokitiol from a tree essential oil containing hinokitiol, which comprises a step of purifying an acidic oil obtained from the tree essential oil by centrifugal liquid-partition chromatography. Provide a way. The method of the present invention is industrially advantageous because high-purity hinokitiol can be easily purified in high yield.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a method for purifying hinokitiol. More specifically, it relates to a method for purifying hinokitiol, which is used as a component of antibacterial agents, hair nourishing agents, etc., from tree essential oils such as hiba oil, by centrifugal liquid-partition chromatography.

[0002]

2. Description of the Related Art Conventionally, as a method for separating and purifying hinokitiol from tree essential oils such as red hinoki oil, Heinez, Nezuko essential oil, Taiwan cypress oil, and Hiba oil, steam-distilled or solvent-extracted Hiba wood chips and sawdust. A method of sequentially refining the obtained Hiba oil by a chemical treatment is performed.

That is, as a method for separating hinokitiol from hiba oil, (1) the acidic oil separated from the essential oil by alkali extraction is washed with sodium bicarbonate to remove the carboxylic acid, and treated with a strong alkali to make the hinokitiol water-soluble. After separation from essential oil components, treatment with calcium chloride to separate as calcium salt and free with hydrochloric acid, or method for obtaining hinokitiol via sodium salt (Japanese Patent Publication Sho 3)
(3-8518), (2) Benzene is added to a hinokitiol-containing vegetable oil, and a caustic soda solution is further added to the oil to shake the mixture to improve the separation of the oil and the alkaline aqueous layer, and then the alkaline aqueous layer is cooled to remove hinokitiol Na salt. Obtained (JP-A-51-23244), (3) Hiva oil after alkaline treatment, the ether solution of acidic oil obtained by neutralizing the aqueous layer is adsorbed on a silica gel or alumina packed column to give petroleum ether, alcohol. (4) In addition, a method of eluting with a method of purification (Japanese Patent Laid-Open No. 61-278596),
Phosphoric acid is added to acidic oil to make hinokitiol phosphate,
A method of hydrolyzing this to obtain hinokitiol is also known ("Mystery of Aomori Hiba" Okabe, Saito, Otomo,
Kudo, published by Aomori Hiba Kenkyukai).

[0004]

However, when these methods are examined, the above (1), (2),
The methods (3) and (4) are complicated in operation, and it is not easy to separate and purify hinokitiol from several other tropolone compounds or phenols in acidic oil, and further by recrystallization purification or column chromatography. It has the drawback of requiring repeated purification. In addition, the methods (1) and (2) have a large number of steps, and have a problem that the hinokitiol recovery rate is reduced accordingly. In addition, the method (3) is expensive because an expensive filler is used, and it is complicated to replace the filler.

The hinokitiol content of red hinoki oil, Heinez, Nezuko essential oil, Taiwan cypress oil, and Hiba oil is 0.1.
It is a mixture of several 10 kinds of components, with a trace amount of several%. The acidic oil (1 to 20% of the essential oil) obtained after neutralization by transferring to an aqueous layer by alkali treatment contains about 10 kinds of components such as hinokitiol and other tropolones, phenols and carboxylic acids. There is. In the conventional method, as described above, carboxylic acids are removed by heavy water treatment or calcium salts are reacted with calcium chloride to remove phenols, and the fact that phenol salts are slightly more soluble in warm water than tropolone salt is used. Methods such as washing with warm water to remove the phenol salt or adding phosphoric acid to form the phosphate salt and separating the tropolone salt from the phenols have been carried out. Furthermore, hinokitiol is purified from tropolones by distillation, column chromatography, recrystallization, etc., but as mentioned above, the complexity and loss of the operation during this period are industrially important problems.

Hinokitiol is now widely used as a safe antibacterial agent as a food preservative, an antibacterial agent in packaging materials for transporting fresh foods, a hair nourishing agent utilizing the skin contraction effect of hinokitiol, and an agricultural antibacterial agent. However, natural hinokitiol is particularly useful as a food preservative and an antibacterial agent, and technical solutions to the above problems are urgently needed.
Although no satisfactory method has been found at present, the development of a more effective hinokitiol purification method is expected, and the object of the present invention is exactly at this point.

[0007]

Means for Solving the Problems The present inventors have made various studies to develop an effective purification method for hinokitiol which can solve the above problems. As a result, the acidic oil obtained by neutralizing the water layer separated from the neutral oil by alkaline water treatment of tree essential oil such as hinoki thiol-containing red hinoki oil, Heinez, Nezuko essential oil, Taiwan cypress oil, and Hiba oil. As a result of performing a separation operation using a centrifugal liquid-partition chromatography device (CPC), it was discovered that hinokitiol can be separated with high purity and high yield, and the present invention has been completed.

That is, the gist of the present invention is a method for purifying hinokitiol from a tree essential oil containing hinokitiol, which is characterized by comprising a purification step of centrifugal oil-liquid partition chromatography of an acidic oil obtained from the tree essential oil. The present invention relates to a method for purifying hinokitiol.

Examples of the tree essential oils used in the present invention include Bamboo oil, red hinoki oil, Heinez, Nezuko essential oil, hiba oil, taiwan hinoki cypress oil, and the like. It is not limited. The acidic oil can be easily obtained from such a tree essential oil by a known method of treating the tree essential oil with an alkali and then neutralizing it as described above. Also, the centrifugal liquid distribution chromatography device used here is that of Miki Engineering Co., Ltd., but other companies can be used as well.

Centrifugal liquid-partition chromatography uses a polar solvent or a non-polar solvent as a stationary phase and the other as a mobile phase as a method for separating organic compounds, and a cell having many chambers is rotatably shaken by centrifugal force. However, the device elutes the sample from the stationary phase to the mobile phase due to the difference in solubility. The advantage of using this device for purification of hinokitiol is that column chromatography or high performance liquid chromatography for large-scale separation can be used to separate a large number of high-boiling components, but these are packed with expensive silica gel. Although a large amount of the agent is used and the replacement of the filler is complicated, the cost is low by the amount of the filler because the filler is not used in this device, and the post-treatment is not complicated because it is only washed with the solvent. That is the point. A small machine (LLN type manufactured by Miki Engineering Co., Ltd.) was used for this CPC device this time, but there is also a large machine, which can be industrialized by processing a large amount of samples.

Centrifugal liquid distribution chromatography (CP
C) When purifying hinokitiol from acidic oil obtained from tree essential oil using an apparatus, since hinokitiol is easily dissolved in a polar solvent and hardly dissolved in a nonpolar solvent, a polar solvent is usually used as a stationary phase and a mobile phase is used. Is used as a non-polar solvent (forward elution). That is, as the stationary phase, acetonitrile, methanol, water, acetone and other polar solvents are used alone or in combination. As the mobile phase, hexane, pentane, petroleum ether, benzene,
n-Butanol and other organic solvents are used. If the elution of hinokitiol is slow, it is possible to switch to a method that uses a non-polar solvent such as hexane for the stationary phase and a polar solvent such as acetonitrile or methanol for the mobile phase (reversal elution) to elute hinokitiol early. ..

The sample to be purified usually has a sample volume of 1
It is dissolved in a stationary phase or stationary phase-mobile phase solvent in an amount of -100 to 100 times, preferably 3 to 100 times, and then injected. The centrifuge operates at 5 to 30 ° C and 300 to 1300 rpm,
First, the stationary phase is filled with the solvent, and then the solvent of the mobile phase is pumped into the centrifuge together with the sample. The amount of liquid pumped depends on the capacity of the cell in the centrifuge. To confirm the elution of hinokitiol, use the UV-Visible Absorption Monitor attached, and at the same time, activate the fraction collector to fractionate the eluted components, analyze them by gas chromatography, and reconfirm the components to confirm the fractionation. The target hinokitiol can be obtained by collecting and concentrating with an evaporator.

In order to obtain higher purity hinokitiol, the high-purity hinokitiol collected in the first time is further added to CPC.
By subjecting it to centrifugal separation-liquid partition chromatography a plurality of times such as by applying it to an apparatus, hinokitiol of higher purity can be obtained. However, since the hinokitiol thus obtained contains the isomer β-drabulin in an amount of 5 to 50%, it can be hydrogenated by a conventional method at room temperature and atmospheric pressure using a catalyst such as Pd or Pt. Then, β-drabulin is quantitatively converted to hinokitiol. The obtained product is recrystallized using a solvent such as petroleum ether, hexane and ligroin to obtain natural hinokitiol crystals (mp 48 to 52 ° C.).

Thus, the method of separating and refining the acidic oil obtained by subjecting the hinokitiol-containing tree essential oil to alkali treatment and neutralization as it is by centrifugal liquid-partition chromatography can reduce the steps so far and reduce the loss of hinokitiol purification. In addition, the hinokitiol having a purity of usually 80% or more is obtained when it is applied to the CPC device once, and the hinokitiol having a purity of 99% or more (including β-drabulin) is obtained in the second time. Thus, according to the method of the present invention, by having a purification step by centrifugal liquid-partition chromatography, it is possible to obtain high-purity hinokitiol in a high yield. You may perform in combination. For example, the obtained acidic oil may be subjected to CPC after being subjected to distillation purification, or may be subjected to column chromatography in advance and then subjected to CPC.

[0015]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these preparation examples and examples. Preparation Example 1 Preparation of Hiba Oil (Solvent Extraction) 3 L-glass Kolben equipped with a Teflon stirring blade and a motor, 250 g of Hiba sawdust and 8 parts of toluene.
00 g was added, and the mixture was heated and extracted at 90 ° C. for 12 hours. The obtained extract is cooled and then filtered to give 800 g of sawdust.
After washing with water and collecting and concentrating the toluene solution, the Hiba oil 8.
74 g (3.5% based on sawdust) were obtained. When this was quantified by gas chromatography, 3.28% by weight of hinokitiol (including the isomer β-drabulin) was contained. The extraction efficiency of hinokitiol is better than that of the steam distillation method of Preparation Example 2.

Preparation Example 2 Preparation of Hiba Oil (Steam Distillation) 1000 g of water was added to 250 g of Hiba sawdust and steam distillation was carried out. When water was successively added and continued, 2.5 g of Hiba oil (1% based on sawdust) was added 12 hours later. ) Was obtained as distilled water. When this was quantified by gas chromatography, hinokitiol (including β-drabulin) was contained in an amount of 2% by weight.

Preparation Example 3 Preparation of Hiba Oil (Solvent Extraction of Steam Distillation Residue) Using the same apparatus as in Preparation Example 1, 316 g (air-dried product) of oak scraps after steam distillation were charged, and toluene 159 was prepared.
Extraction was performed with 4 g for 12 hours while heating at 90 ° C. The obtained extract was concentrated to obtain 4.45 g of hiba oil. It was 1.41% with respect to the remaining shavings. As a result of quantification by gas chromatography, it contained 6.68% by weight of hinokitiol (including β-drabulin).

Preparation Example 4 Preparation of Hiba oil (solvent extraction of steam distilled water) Distilled water 4467 obtained by steam distillation of Preparation Example 2
1000 ml of ethyl acetate was added to g and the mixture was shaken with a separating funnel to extract the dissolved oil. Furthermore, the extract obtained by similarly performing ethyl acetate extraction twice was concentrated to 0.60.
g oil was obtained. It was 0.08% with respect to the used sawdust. When quantified by gas chromatography, it contained 24% by weight of hinokitiol (including β-drabulin).

Preparation Example 5 Preparation of Acid Oil 500 g of steam distilled Hiba oil (10.0 g of hinokitiol)
3) by adding 500 g of 10% caustic soda aqueous solution
After stirring at room temperature for 0 minutes, suspend the oil and water in hexane.
00 ml was added and stirred. After separating the hexane layer and an aqueous layer in the separatory funnel, and the 6N-H ₂ SO ₄ was added to the aqueous layer pH2
It was set to 4. Hexane, toluene, etc. were added to the aqueous layer, and the mixture was extracted with a solvent and then concentrated to obtain 45.0 g of acidic oil. When hinokitiol in the acidic oil was quantified by gas chromatography, it was found to contain 20.0% by weight of hinokitiol.

Preparation Example 6 Preparation of acidic oil 10 g of calcium hydroxide was added to 50 g of solvent extracted Hiba oil (containing 1.75 g of hinokitiol), and the mixture was stirred at 25 ° C. for 5 hours. 100 ml of hexane was added to the mixture, and after suction filtration, the yellowish white solid on the filter paper was further washed with hexane,
Vacuum drying was performed in a desiccator. Yellowish white solid 11
g in a 300 ml Erlenmeyer flask and 50 ml of toluene
2N while stirring with a magnetic stirrer
Hydrochloric acid was added until pH 2-3. The mixed solution was transferred to a separatory funnel, the toluene layer was dried over Na ₂ SO ₄ , and then concentrated by an evaporator to obtain 4.1 g of an acidic oil (containing 1.6 g of hinokitiol).

Example 1 10.0 g of the acidic oil obtained in Preparation Example 5 (containing 2.0 g of hinokitiol) was dissolved in a mixed solvent of 20 ml of hexane and 20 ml of acetonitrile, and a centrifugal liquid-liquid partition chromatography device (Miki Engineering ( Ltd., LL
Type) and separated and purified. The stationary phase was acetonitrile and the mobile phase was hexane.
00 rpm, mobile phase hexane flow rate is 15 ml / mi
I drove with n. The elution of hinokitiol was confirmed by an ultraviolet-visible absorption monitor (wavelength 254 nm), and at the same time that a hinokitiol peak appeared, fractionation was performed using a fraction collector. After 4 hours, 3600 ml of hexane was fed, the operation was terminated, the fractionated fractions were concentrated, and the weight was measured and gas chromatographic analysis was performed. As a result, 1.56 g of a fraction of hinokitiol having a purity of 81.7% (hinokitiol 1.
27 g) and a fraction having a purity of 62.7% of 0.89 g (hinokitiol 0.56 g) and 1.06 g (hinokitiol 0.17 g) were obtained.

Again, 1.56 g of a fraction of hinokitiol having a purity of 80% or more (containing 1.27 g of hinokitiol) was added.
When subjected to a CPC apparatus under the same conditions as above, the GC purity is 99.5.
% Of hinokitiol was obtained, and the average GC purity of 99.
1.38 g of 0% hinokitiol was obtained. The isomer β-drabulin was also contained in these hinokitiol in an amount about half that of hinokitiol.

0.18 g of 5% palladium carbon and 15 g of ethanol were added to 1.38 g of hinokitiol having a GC purity of 99.0%, hydrogen reduction was carried out at 25 ° C. and normal pressure for 30 minutes, and the reaction solution was filtered and concentrated. , Hexane to the residue 2
When ~ 3 g was added and recrystallized, colorless crystals were precipitated, and the crystals were dried at 30 ° C with a vacuum pump to have a GC purity of 99.4%.
1.1 g of colorless crystals of hinokitiol (mp.
C) was obtained. Hinokitiol in the recrystallization mother liquor can be used for the next CPC purification. The yield (recovery rate) from the acidic oil to the product of hinokitiol is 95%. Therefore, the yield from Hiba oil to acid oil is 90%.
Therefore, the yield from Hiba oil to hinokitiol product is 85.5%.

The solvent used for the operation of the CPC device is recovered by simple distillation and reused. Gas chromatography used for the analysis is Shimadzu GC-14A, data processor Shimadzu C
-R6A, column ULBON HR-1701 Capillary column 25m, FID, He50cc / min,
1.0 kg / cm ² , split ratio 1: 100, 60 ° C
To 250 ° C, temperature rise 8 ° C / min, chart Spe
ed 0.5 cm / min.

Example 2 Fraction with a GC purity of 62.7% obtained in Example 1 0.89
g (hinokitiol 0.56 g) as in Example 1
It was subjected to PC separation and purification. Mobile phase hexane flow rate 20m
When the centrifuge was operated at 1000 rpm at 25 ° C. at 1 / min, a hinokitiol fraction separated in 3 hours was obtained. The obtained fractions were analyzed by GC and concentrated together to obtain 0.59 g (hinokitiol 0.52 g) and 0.15 g (hinokitiol 0.04 g) of a fraction having a GC purity of 87.5%. It was When the fraction having a GC purity of 87.5% was separated and purified again by CPC, 0.52 g of colorless crystals of hinokitiol having a GC purity of 99.5% (mp.
C) was obtained.

Example 3 50.0 g of Taiwan cypress wood oil (4.1 Hinokitiol)
When 50 g of a 10% aqueous sodium hydroxide solution is allowed to act on g) and treated in the same manner as in Preparation Example 1, 8.0 g (1
6% by weight) was obtained. When hinokitiol in acidic oil was quantified by gas chromatography, hinokitiol was 5
The content was 1.3% by weight. 8.0 g of the obtained acidic oil was separated and purified using a CPC device under the same conditions as in Example 1, and as a result, 3.2 g (hinokitiol 2.7 g) of a fraction of hinokitiol having a GC purity of 85.6%, GC purity 6
1.9 g of 1.7% fraction (1.2 g of hinokitiol)
And other fractions of 2.3 g (0.2 g of hinokitiol) were obtained. When 3.2 g of a fraction having a purity of 85.6% was again subjected to CPC separation and purification under the same conditions, the GC purity was 99.2%.
2.7 g (mp 52) of colorless crystals of hinokitiol
C) was obtained.

Example 4 4.1 g of the acidic oil obtained in Preparation Example 6 (containing 1.6 g of hinokitiol) was added to 20 g of hexane in the same manner as in Example 1.
When dissolved in a mixed solvent of 20 ml of ml-acetonitrile and purified and separated once by a CPC device under the same conditions, GC purity is 8
1.2% of 3.2% fraction (1.0 g of hinokitiol)
Then, 0.8 g (hinokitiol 0.5 g) of a fraction having a purity of 63.2% and 0.4 g (hinokitiol 0.1 g) of the other fractions were obtained.

Example 5 4.40 g of hiba oil (containing 0.29 g of hinokitiol) obtained in Preparation Example 3 was treated by the method of Preparation Example 5 to obtain 1.04 g of acidic oil (0.26 g of hinokitiol).
was gotten. When this was subjected to CPC separation and purification once under the same conditions as in Example 2, the fraction with a GC purity of 86.0% was 0.1%.
9 g (0.16 g of hinokitiol) and a purity of 62.1
% Fraction 0.10 g (hinokitiol 0.06 g) and other fractions 0.10 g (hinokitiol 0.04 g) were obtained.

Example 6 0.60 g of Hiba oil (containing 0.14 g of hinokitiol) obtained in Preparation Example 4 was treated by the method of Preparation Example 5 to obtain 0.33 g of acidic oil (0.13 g of hinokitiol). When this was subjected to CPC separation and purification once under the same conditions as in Example 2, 0.12 g of a fraction having a GC purity of 84.9% (hinokitiol 0.08 g) and a fraction having a purity of 64.2% of 0.
06 g (hinokitiol 0.04 g) and other fractions 0.30 g (hinokitiol 0.01 g) were obtained.

Example 7 10.0 g of the acidic oil (containing 2.00 g of hinokitiol) obtained in Preparation Example 5 was dissolved in a mixed solvent of 20 ml of hexane-18 ml of acetonitrile-2 ml of water, and the stationary phase was acetonitrile-water (9: 1). ), The mobile phase was hexane, and the CPC separation / purification was performed with a centrifuge at 25 ° C., 1100 rpm and a mobile phase hexane feed rate of 15 ml / min. After 5 hours, 4500 ml of hexane was fed, and the process was completed. As a result, the fraction having a GC purity of 88.2% was 1.47 g (hinokitiol 1.30 g) and the fraction having a purity of 64.7% was 0.
86 g (hinokitiol 0.56 g) and other fractions 0.49 g (hinokitiol 0.13 g) were obtained.

[0031]

Industrial Applicability The method of the present invention is industrially advantageous because high-purity hinokitiol can be easily purified in high yield.

Claims (1)

[Claims] 1. A method for purifying hinokitiol from a tree essential oil containing hinokitiol, which comprises a step of purifying an acidic oil obtained from the tree essential oil by centrifugal liquid-partition chromatography.