KR101195228B1 - A method for extracting terpene oil from natural product - Google Patents

Abstract

The present invention relates to a method for extracting fragrance components having a headache treatment effect from natural products. More particularly, supercritical extraction of pine leaves using at least one solvent selected from the group consisting of methanol, ethanol, acetonitrile, petroleum ether and hexane It relates to a method for extracting the terpene component by.

Description

A method for extracting terpene oil from natural product

The present invention relates to a method for extracting fragrance ingredients having a headache treatment effect from natural products.

Let's take a look at the essential oil extraction method using the portable steam distillation small essential oil extractor. First, the branches or leaves cut to about 1cm in length are put in a distillation tank in a separately manufactured stainless steel net. Put water in the distillation tank so that the sample will not be submerged, connect the cooling water to the cooler, and ignite. Subsequently, the refined oil flowing out while heating for 2 to 3 hours after the internal temperature of the distillation tank reaches 100 degreeC is collected in a rectifier collector. At this time, terpene essential oil is lighter than water, so it floats on the water. If it is collected in a separatory filter and left for 20 minutes, it is divided into an essential oil layer and a water layer. Then, you only have to discard the water and collect only the essential oils. The essential oil collected by repeating this process several times is filtered to remove impurities contained in the essential oil. Finally, about 1% of anhydrous sodium sulfate (drying at 105 DEG C for at least 3 hours and cooling in a desiccator) of the essential oil weight is added thereto, and purified terpene essential oil is obtained by dehydrating for 24 hours in a cold dark place.

In the case of extracting the essential oil in the above manner, the oil recovery percentage is not only a small amount but also requires a lot of working time in the construction method, there is an economic problem that increases the cost accordingly. In addition, when the material is put in a small distillation tank and boiled by the so-called distillation method, since the active ingredient inherent in essential oils is extracted from the water vapor after being dissolved in a large amount of water, there is a problem in yield, that is, mass production is difficult. In addition, because it contains chemicals such as anhydrous sodium sulfate, it can not be used directly in the extracted state because of the possibility of remaining toxicity. In addition, there is a problem in economy because high energy is used through several distillation processes.

The common method of extracting active ingredients from copper and plants is to use organic solvents such as water, methanol, ethanol, hexane, etc. The extraction method using organic solvents is easy to apply, while selecting an appropriate solvent, remaining of organic solvent, Difficulties include removal of solvents, environmental pollution, low extraction yield, and usually require a long extraction time of about 24 to 48 hours. However, for commercial use, it is necessary to shorten the stability and extraction time of the solvent, to acquire the environmental pollution and the ingredients to be extracted and to have the activity, but there is no clear method for this. And terpene-like fragrance can relieve headaches by refreshing the effect of forest bathing, but due to the unique unique fragrance can also give a sense of rejection.

In the present invention, to overcome the limitations of the conventional phytoncide extraction method, there is no residual chemical component in the extract, does not cause pollution, and introduces a supercritical fluid technology having various advantages of high efficiency to extract the phytoncide component from the pine leaves It was intended to apply.

It is therefore an object of the present invention to provide a new method for extracting phytoncide components from pine leaf.

In order to achieve the above object, the present invention provides a method for extracting a terpene component by supercritical extraction of a pine leaf using at least one solvent selected from the group consisting of methanol, ethanol, acetonitrile, petroleum ether and hexane.

In one embodiment of the present invention, the supercritical extraction conditions are preferably performed in the range of 30 ℃ to 50 ℃ but is not limited thereto.

In another embodiment of the present invention, the terpene component is gamma-cardinene, delta-cadinene, gamma-murorene, low macrone-Germacrene-D, polyaltic acid ( polyalthic acid, bornyl acetate, beta-caryophyllene, cis-3-hexenyl cinnamate, menthol, liguloxidol, phytol and neoyi Somethol (Neoisomenthol) is preferably one compound selected from the group consisting of, but is not limited thereto.

According to a preferred embodiment of the present invention, the solvent is preferably added to the solvent in order to supplement the non-polarity of carbon dioxide.

According to a preferred embodiment of the present invention, the mixed fluid is preferably maintained at 30 ° C. or more and 7.4 MPa or more.

Hereinafter, the present invention will be described.

Supercritical Fluid Extraction Technology is a technology that uses fluids above the critical temperature and pressure to replace existing processes in the extraction and purification fields of pharmaceuticals, food processing and petrochemicals. It is attracting attention as an environmentally friendly clean technology. (CD Bevan and PS Marshall.The use of supercritical fluid in the isolation of natural products, Nat.Prod.Rep. 11, 451-576, 1994) New process fluids that use supercritical fluid technology as process fluids in advanced countries for over 30 years, avoiding the concept of using gas or liquid in traditional processes due to the expansion of demand for special purpose new materials that cannot be manufactured by gas or liquid processes. Care has been taken in the development of the technology. As a result, the supercritical fluid process is rapidly spreading to various industries such as fine chemicals, energy, environment, and new materials, and the movement to replace various traditional separation technologies with the supercritical fluid process is accelerating.

The areas where early supercritical fluid technology is mainly applied to extracts from natural products are limited to non-polar substances such as spices, cosmetics, fats, low-cost foods and flavoring ingredients, but recently, due to the development of several phenomenological characteristics and additional technologies related to this technology, It has been applied to the extraction, purification of polar, small amount and expensive natural medicines. There are many types of supercritical fluid candidates. Among them, carbon dioxide is not only present in nature in abundantly, but also abundantly generated in steel and petrochemical industry, and is colorless, odorless, harmless to human body, and chemically stable. Best of all, it has a lower critical temperature (31.1 ° C) and critical pressure (73.8 bar) than any other fluid, making it easy to adjust to supercritical conditions, resulting in high energy efficiency. In addition, it can solve the problems occurring in the existing process, that is, human toxicity by the organic solvent remaining in the final product.

Hereinafter, the present invention will be described in detail. The present invention

1) a pretreatment step of crushing pine leaves containing phytoncide;

2) filling the pine tree leaves crushed in step 1 into the supercritical fluid extraction vessel;

3) extracting the extract by inputting a supercritical fluid through a heat exchanger to the extractor filled with the raw material powder in step 2;

4) separating the supercritical fluid and the extraction mixture by depressurizing the pressure reducing valve through a pressure reducing valve in a pressure reducing separator; And

5) The supercritical fluid separated in step 4 is recycled by depressurization by a pump with the supercritical fluid supplemented from the outside after the circulation and the supercritical fluid extraction method comprising the steps of sequentially collecting the separated extract It provides a method for producing phytoncide-containing extract using.

Extraction from pine leaves containing various components by the conventional organic solvent extraction method, selection of solvents, residual organic solvents, difficulty in removing solvents, environmental pollution, low extraction yield, and usually 24-48 hours It will have various problems such as long time extraction time. On the other hand, extraction using the supercritical fluid extraction method can eliminate the problems caused by the organic solvent extraction method, it is possible to obtain a high purity phytoncide component.

In step 1 of the above method, crushed to a size of about 1 mm in diameter, and ethanol and water may be further added to the extractor of step 2 as a co-solvent. The supercritical fluid of step 3 is preferably carbon dioxide, the temperature of the supercritical fluid is 30 ℃, the pressure is preferably 250 bar.

As a result of using the supercritical fluid extraction method, the inventors of the present invention have obtained useful extracts as described above, which have no loss, high extraction efficiency, and higher extracts than conventional extraction methods. Since the extract is known to be effective in alleviating the headache treatment and forest effects, respiratory diseases, atopic dermatitis, the composition containing the supercritical fluid extract may also be used for alleviating or treating diseases related to the efficacy. .

The present inventors further performed extraction using ethanol, methanol, petroleum ether, acetonitrile, nucleic acid, and the like as cosolvents. Accordingly, it was confirmed that selective extraction was possible depending on the cosolvent. Therefore, according to the present invention, if the co-solvent is different in the process of extracting supercritical fluid according to the component to be obtained, there is an advantage of obtaining various extracts.

Preferred conditions for the supercritical fluid extraction are not particularly limited as long as the temperature and pressure to create a supercritical fluid state, particularly preferably 30 ℃ temperature and pressure is 250 bar pressure conditions. The extraction is performed for 2 hours at a flow rate of 1 ml / min under these temperature and pressure conditions. In Example 1 below, it is shown that selective extraction of a substance having an effect of alleviating headache treatment according to a cosolvent is possible.

In the present invention, a supercritical fluid is used to extract the fragrance component having a headache treatment effect from pine nuts. First, the pine needles are cut into various sizes, solvent extracted, and the appropriate size is selected. Next, in order to extract various terpene-based substances, called phytoncide, among the fragrances contained in the pine tree, the optimum conditions are established by changing variables such as time, temperature, pressure, type and content of modifier I would like to. The gas to be tested in the supercritical fluid state is carbon dioxide, which is less toxic than other liquefied gases, and has an advantage of easily reaching a critical point. Fractions extracted with supercritical fluids are analyzed by Gas Chromatography-Mass Spectrometry Detector (GC-MSD) to determine the type and composition of extracts in the form of mixed components.

In the present invention, in obtaining a phytoncide, a component of the terpene family through pine tree extraction, by using a supercritical fluid extraction method with the addition of a cosolvent, various terpene family components can be extracted in a yield exceeding the conventional organic solvent extraction method. It can be said to be an environmentally friendly method that solves the problem of residual solvent by using less than the amount of organic solvent used in the prior art. The present invention including such contents can be used without any side effects or toxicity in vivo by modern people exposed to various adult diseases such as not only headache relief but also sterilization, antibacterial, stress relief, bronchial, asthma, rhinitis, allergy, insomnia, etc. Can contribute to.

1 is a picture of pine leaves,
Figure 2 is a photograph of the supercritical fluid extraction apparatus of the present invention,
3 is a photograph of the vessel used for extraction,
4 shows GC-MSD analysis conditions of the present invention,
5 is a mass spectrum for delta-Cadinene (MW 204.20),
6 is the mass spectrum for gamma-Cadinene (MW 204.20),
7 is the mass spectrum for Polyalthic acid (MW 316.00),
8 is a mass spectrum for trans bornyl acetate (MW 196.28),
9 is the mass spectrum for gamma-Muurolene (MW 204.35),
10 is the mass spectrum for cis 3-hexenyl cinnamate (MW 230.00),
11 is the mass spectrum for Liguloxidol (MW 238.00),
12 is the mass spectrum for beta-caryophyllene (MW 204.35),
13 is the mass spectrum for Germacrene-D (MW 204.35),
14 is the mass spectrum for Menthol (MW 156.26),
15 is the mass spectrum for Neoisomenthol (MW 156.26), and
16 shows the mass spectrum for Phytol (MW 296.53).

 The present invention will now be described in more detail by way of non-limiting examples. However, the following examples are set forth to explain the present invention and the scope of the present invention is not to be construed as being limited by the following examples.

Example

First, select the size of the pine leaf to be used for extraction. The average length of the pine leaves is 5 ~ 6 cm (Fig. 1) when the extract without extracting the pine leaves as it is, the surface area that the sample can interact with the supercritical fluid is reduced. Therefore, it is necessary to control the size of the pine leaf because the supercritical fluid is not suitable for extracting phytoncide components into the sample.

To this end, 3 g of pine are 0.5 cm. Three sizes of 1.0 cm and 1.5 cm were cut and immersed in methanol. After soaking for 3 days, the solvent was removed using a rotary evaporator and the scent was checked. The reason for selecting a suitable size from the extracted fragrance is that the terpene-based components extracted from pine leaves are well known as fragrance components that naturally volatilized in coniferous forests. The temperature of the water bath was 30 ° C. because the fragrance components could evaporate during removal of the solvent. Supercritical fluid extraction was performed with the size of pine needles selected in this way.

2 is a supercritical fluid extraction device used in the present invention. The device consists of a fluid delivery section, an extraction section and a collection section. The fluid delivery portion consists of a carbon dioxide pump required to move and press carbon dioxide out of the cylinder and a modifier pump that moves a small amount of polar solvent to compensate for the nonpolarity of the carbon dioxide. The extraction part is a space for extracting fragrance components using a supercritical fluid under a constant temperature and pressure, and is composed of an extraction vessel (vessel, FIG. 3) and an oven for maintaining in a supercritical state. Finally, the collecting part consists of a back pressure device and a collecting device. In the collecting portion, the extracted fragrance components are depressurized by the reverse pressure regulating device. As a result, the supercritical fluid is discharged by converting carbon dioxide into gas and the extract is collected in the collector.

Experiment using varying the extraction time, temperature, pressure to extract the fragrance components in high yield using the supercritical fluid extraction device. Extraction conditions are as follows. Extraction time is based on 2 hours, the temperature is changed by 5 ~ 10 ℃ based on 30 ℃, experiment by changing the pressure based on the critical pressure of carbon dioxide. In addition, a small amount of polar modifier is used to prevent the polar fragrance component from being extracted due to the nonpolarity of supercritical carbon dioxide. Cosolvents were ethanol, methanol, hexane, acetonitrile, petroleum ether organic solvent.

Extracts are analyzed by GC-MSD after removing residual solvent using rotary evaporator. Analysis conditions of the essential oil component are shown in FIG. 4. The obtained mass spectrum is obtained by searching Wiley 138 data base by HP 59970C Chemstation data system and mass spectral data in various literatures.

Pine leaves collected from Hwacheon-gun were used to extract the fragrances contained in the pine leaves. 3 g of pine leaves were cut into sizes of 0.5 cm, 1.0 cm, and 1.5 cm to be suitable for extraction, soaked in methanol (methanol) and left for 3 days. The reason for cutting the raw material into such a small size is to allow the supercritical fluid to penetrate the surface of the raw material well. The extract was removed solvent by rotary evaporator. At this time, the temperature of the water bath was 30 ° C to prevent evaporation of the fragrance component while removing the solvent. The degree of aroma of the extract from which the solvent was removed was compared. As a result, when the pine tree was cut to 1.0 cm, the darkest aroma was extracted. This is because the terpene-based compound is a fragrance component, so if the sample is cut too small, the fragrance component contained in the pine leaves may be volatilized during processing. Supercritical fluid extraction was performed with the sample of this size.

Modifier Compounds Terpenes
Methanol Delta-cadinene,
Gamma-Murourolene
Germacrene-d
Polyalthic acid
Sesquiterpene Diterpene acid
Acetonitrile Delta-cadinene,
Gamma-cadinene,
Gamma-Murourolene,
Germacrene-d
Sesquiterpene

Petroleum ether trans bornyl acetate
Gamma-Murourolene,
Germacrene-D,
Beta-caryophyllene,
cis-3-hexenyl cinnamate Bicyclic monoterpene
Sesquiterpene
-
Ethanol Menthol,
trans bornyl acetate
Beta-caryophyllene Monoterpene alcohol Bicyclic monoterpene Sesquiterpene
Hexane trans bornyl acetate
Gamma-Murourolene
Liguloxidol Bicyclic monoterpene Sesquiterpene

Table 1 is a table showing the extract composition according to the cosolvent.

Supercritical fluid extraction attempted to find the optimal extraction conditions by varying the temperature, pressure, time and cosolvent parameters. First, 1.5 g of pine needles were cut into 1.0 cm in the extraction container (Fig. 2), and the extraction time was fixed for 2 hours, the temperature was 37 ° C., and the pressure was 250 bar. Cosolvents were injected into methanol, ethanol, acetonitrile, petroleum Ether, and hexane at 0.2 mL (0.2 mL / min) per minute. The solvent of the obtained extract was removed and then dissolved in 1 mL ethanol and analyzed by GC / MSD.

In extracting aroma of pine, sesquiterpene compounds were commonly extracted when cosolvent was extracted using methanol and acetonitrile. In other words, in the case of Methanol -cadinene delta, gamma -Muurolene, Germacrene-D, In the case of acetonitrile, the delta -cadinene, -cadinene gamma, gamma -Muurolene, Germacrene-D have been extracted were all these compounds (C ₅ H ₈ ) Is a sesquiterpene in the form of ₃ . Diterpene acid-based polyalthic acid was also extracted.

When petroleum ether was used as the cosolvent, trans-bornyl acetate, gamma-Muurolene, Germacrene-D, beta-caryophyllene, and cis-3-hexenyl cinnamate were extracted to obtain the highest number of components. In particular, the monoterpene ((C ₅ H ₈ ) ₂ ) -based compound (trans bornyl acetate), which is known to have the highest aroma among terpene-based compounds, was extracted. Indeed, trans-bornyl acetate is already used industrially as a pine needle fragrance. It is also characterized by the extraction of cis-3-hexenyl cinnamate, which does not belong to the terpene family but is known as a fragance material. When petroleum ether was used as a cosolvent, the largest number of components were extracted from petroleum ether mixed with pentane (C ₅ H ₁₂ ) and hexane (C ₆ H ₁₄ ), and terpene-based compounds were hydrocarbon compounds. It is thought to be.

In the case of ethanol, it is noteworthy that different terpene family compounds were extracted. Unlike other cosolvents, monoterpene alcohol menthol, bicyclic monoeterpene trans bornyl acetate and sesquiterpene beta-caryophyllene were extracted under the same conditions. In particular, menthol is a substance known for its peppermint scent and relieves pain and itching. When hexane was used as a cosolvent, transgulantyl acetate, gamma-Muurolene, and Liguloxidol, which are well known as fragrances from the odor, were obtained. All mass spectrums listed in the specification of the present invention were found to be components after comparison by searching the Wiley 138 data base.

Next, in order to confirm the difference according to the extraction temperature, the components extracted under 35 ° C. and 50 ° C. (250 bar, ethanol modifier, 2 hr) were analyzed by GC / MSD. As a result, almost the same components were extracted, Menthol at 35 ℃, neoisomenthol (FIG. 15) was extracted at 50 ℃. Menthol and neoisomenthol are both monoterpene series, but the experimental formula is the same, but the structural isomers have different structural formulas.

EtOH (35 ℃) EtOH (50 ℃) Menthol,
Trans bornyl acetate,
Beta-caryophyllene Neoisomenthol,
Trans bornyl acetate,
Beta-caryophyllene

Table 2 is a table showing the difference between the extracts according to the temperature.

Finally, 85% methanol (15% H ₂ O + 85% methanol) and 100% methanol were extracted at a temperature of 35 ° C. for 2 hours under a pressure of 250 bar to confirm the difference according to the cosolvent. Analyzed. The results are shown in Table 3. Almost the same components were extracted. However, when 85% methanol was used as a cosolvent, diterpene alcohol-based phytol was extracted. Phytol is the main ingredient in the sessile odor of sesame leaves and has recently been known to kill cancer cells.

85% MeOH 100% MeOH Delta-cadinene,
Gamma-cadinene,
Phytol Delta-cadinene,
Gamma-Murourolene
Germacrene-d

Table 3 is a table comparing the extract according to the cosolvent.

Claims (3)

Using supercritical extraction method, pine leaves were used as supercritical fluid solvent, carbon dioxide and co-solvent using acetonitrile. Delta-cadinene, gamma-cadinene, gamma-murorene ), And the Germacrene-D compound, or
Using petroleum ether as cosolvent, transbornyl acetate, gamma-murorene, and Germacrene-D, beta-caryophyllene, cis-3-hexenyl cinnamate ( hexenyl cinnamate), or
A method of extracting transbornyl acetate, gamma-murorene, and liguloxidol using hexane as a cosolvent. The method of claim 1, wherein the pine leaf has a size of 1.0 cm.
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