KR100558382B1 - A method for production of extract from ginkgo biloba l. by supercritical fluid extraction technique - Google Patents

Abstract

The present invention relates to a method for extracting and purifying ginkgo biloba extract containing a high amount of physiologically active ingredients such as bilobalide, gingkolide A, B, and C from ginkgo biloba L. with high purity. In addition, the present invention relates to a method for extracting and purifying the target ingredient in high yield and high purity by supercritical fluid extraction, which is economical, environmentally friendly, and non-toxic to humans, compared to conventional organic solvent extraction methods. According to this method, gingkolic acid components, which should not be included in the ginkgo biloba extract, can be completely removed at one time during the extraction process without separate removal process, and the ginkgo biloba extract extracted by this method is bilobalide. Because it contains a high purity of active physiologically active ingredients such as, gingkolide A, B, C, it can be usefully used as a thrombolytic agent, functional foods and cosmetic whitening agents and the like.

Description

A method for production of extract from ginkgo biloba l. by supercritical fluid extraction technique}

1 is a process diagram schematically showing the supercritical fluid extraction process of the present invention for obtaining high purity bilobalide, gingkolide A, B, C extracts,

Figure 2 is a schematic diagram of the supercritical fluid extraction apparatus used in the present invention,

Figure 3 is a mass spectrometry chromatogram showing the analysis results of bilobalide, gingkolide A, B, C in the ginkgo leaf extract prepared using the supercritical fluid extraction, purification according to the present invention,

Figure 4 is a mass spectrometry chromatogram showing the component analysis results of a conventional ginkgo biloba methanol extract.

<Brief description of symbols for the main parts of the drawings>

1 ... carbon dioxide supply 2 ... pressure regulator

3 ... gas booster 4 ... check valve

5 ... back pressure regulator 6 ... pressure transmitter

7 ... three-phase valve 8 ... extraction tank

9 ... Thermostat 10 ... Heating Jacket

11 ... flow regulator 12 ... primary collector

13 ... Secondary collector 14 ... Flow sensor

15 ... flow rate device 16 ... liquid pump

The present invention relates to a method for extracting, with high purity, a ginkgo leaf extract containing a high content of a bioactive active ingredient bilobalide, gingkolide A, B, C, etc. from Ginkgo biloba L. Specifically, according to the present invention, by performing supercritical extraction under optimum conditions using a supercritical solvent to which an optimum cosolvent is added to carbon dioxide, the above-mentioned target components can be extracted with high yield and high purity from the ginkgo biloba leaves. It is about how.

Ginkgo biloba L. has been used as a medicine for asthma and bronchitis for about 5,000 years, and it is still being evaluated. Recently, it has been widely used as an agent for treating senile peripheral circulation, cerebrovascular disorders and asthma in Asia and Europe (W. Schwabe and P. Kloss, Recovery of vasoactive drugs from leaves of Ginkgo biloba , German Patent, 1, 767, 098, May 31, 1972).

Ginkgo biloba extract was patented in Europe in 1972 and widely used as a therapeutic agent for the cardiovascular system (W. Schwabe and P. Kloss, Recovery of vasoactive drugs from leaves of Ginkgo biloba , German Patent, 1, 767, 098, May 31, 1972). The pharmacological action of ginkgo biloba leaves is mainly known to be based on flavonoid compounds and terpene compounds among ginkgo biloba leaves (R. Anton, Ginkgo et maladies vascularies, Plantes Medicinales et Phytotherapie, Tom XI, n ^o specials, 189-197). , 1977; H. Peter, J. Fisel and W. Weisser, Pharmacological study of the active substances of G. biloba , Arznem-Forsch, 16, 719-725, 1966). There have been many studies on the solvent-extraction of flavonoid compounds among the pharmacological components of the ginkgo biloba leaves.

However, all of the existing methods are only methods for extracting flavonoid compounds, and are not methods for efficiently extracting terpene compounds, which are components of ginkgo biloba, which have recently attracted more attention.

Terpene compounds include bilovalide, gingcholides A, B, C, D, M, and J. Gingcholides are platelets induced by antagonists such as Adenosine diphosphate (ADP), arachidonic acid, collagen, and thrombin. It has been reported to be a potent antagonist that does not inhibit aggregation but selectively inhibits platelet aggregation induced by platelet activators (CA Demopoulos, RN Pinckard and DJ Hanahan, Platelet-activating factor: Evidence for 1-). O -alkyl-2-acetyl-sn-glycero-3-phosphbochloine as the active component (a new class of lipid mediators), J. Biol. Chem. 254, 9355-9358, 1979; P. Braquet and JJ Godfriod, PAF -acether specific binding sites, Trend Pharmacol.Sci., 7, 397-403, 1986). Gingcolide is a competitive reaction with platelet activators and is useful for asthma, bronchitis, senile dementia, allergies, heart disease, shock symptoms, rheumatic diseases and other circulatory diseases. It is very difficult to extract and separate them effectively, and research on this is underway.

To date, a method of selectively extracting and purifying terpene-based compounds, ie, bilobalide, gingkolide A, B, C, and the like from ginkgo biloba leaves may include extracting the extract after primary extraction using methanol, acetone, and the like. It uses complex separation and purification of the steps. The usual method is as follows.

First, the dried ginkgo biloba extract is extracted with a water-acetone (1: 1) solution and the filtrate is concentrated. After evaporation of acetone from the extract, the remaining aqueous layer is extracted with normal hexane to remove nonpolar material. The remaining aqueous layer is acidified to pH 2.0 with 1 normal hydrochloric acid, and the aqueous layer is extracted with ethyl acetate. The ethyl acetate extract is dried again, and then the concentrate is suspended in water, extracted with diethyl ether, and then water is removed with sodium sulfate to prepare the desired bilovalide, gingkolide A, B, and C concentrates.

Such conventional extraction and purification methods have a large amount of extract of the concentrate, but other unnecessary components are extracted together in the resulting extract, and there is a high possibility that a residual solvent remains in the desired extract due to the use of various organic solvents harmful to the human body. In addition, due to the excessive amount of organic solvent used, there are many problems in terms of cost and environmental pollution. Furthermore, according to the above method, since ginkgolic acid, which is known as a toxic component of ginkgo biloba leaves, cannot be removed, as shown in FIG. 4, a separate step of removing gingkolic acid must be performed. There is a problem.

Accordingly, the present invention overcomes the limitations of the conventional ginkgo biloba extract method, and the supercritical fluid technology having various advantages such as no residual solvent in the extract and no pollution is generated from bilobalide and gingkolide A. The purpose of this study was to extract, purify, and extract high-purity, high-yield components.

Supercritical Fluid Extraction Technology is a technology that uses fluids above the critical temperature and pressure to replace existing processes in the extraction and refining fields of pharmaceuticals, food processing and petrochemical refining. Received attention as an environmentally friendly clean technology (CD Bevan and PS Marshall.The use of supercritical fluids in the isolation of natural products, Nat. Prod. Analytical supercritical fluid extraction of natural products, Phytochem.Anal. 7, 1-15, 1996). In particular, in advanced countries, gas and liquids have been used in traditional processes for the past 30 years because of the rise of energy resource prices, the environmental hazards of traditional separation processes, and the expansion of demand for special purpose new materials that cannot be manufactured by gas or liquid processes. We have been devoting ourselves to the development of new process fluid technology that uses supercritical fluid technology as a process fluid. As a result, technology that uses supercritical fluid as a process fluid is rapidly spreading to various industries such as fine chemicals, energy, environment, and new materials, and is replacing various traditional separation technologies with new technologies using supercritical fluid processes.

The application of early supercritical fluid technology mainly to natural product extraction and purification was 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 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 different types of supercritical fluid candidates, with the added benefit of using carbon dioxide. Carbon dioxide is not only present in nature indefinitely, but also a large amount of substances generated in the steel industry and petrochemical industry. In addition, carbon dioxide is a colorless, odorless, harmless to the human body and chemically very stable material. In addition, carbon dioxide has a lower critical temperature (31.1 ° C) and critical pressure (7.4 MPa) than any other fluid, and can be easily adjusted to supercritical conditions, which has great advantages in terms of environmentally friendly properties and efficient energy use. Furthermore, when this technology is applied in the field of separation and purification of natural physiologically active substances, problems occurring in the existing processes, such as human toxicity due to organic solvents remaining in the final product, high cost, environmental pollution due to waste solvents, Denaturation and low extraction selectivity can be addressed or complemented in many ways.

The supercritical fluid technology using carbon dioxide has not yet been applied to extract and purify the bioactive components of ginkgo biloba, particularly terpene-based components. This is because when the supercritical fluid extraction using carbon dioxide is simply applied to the extract of ginkgo biloba, the extraction of the supercritical fluid is difficult to extract due to the polarity of the components such as bilovalide, gingkolide A, B, and C. The content in the interior is too small. Thus, the components could not be obtained in the desired yield and purity in practical applications.

In the present invention, in extracting and purifying bilobalide, gingkolide A, B, and C among the active ingredients of ginkgo biloba, the conventional organic solvent extraction and yield are similar, but also harmful to humans, environmental toxicity of the organic solvent extraction method In order to solve high cost, low selectivity, and effectively remove gingic acid, which is a toxic component of ginkgo biloba in one extraction process, supercritical fluid technology, which is currently being spotlighted as a new extraction and purification technology, is used to extract active ingredient of ginkgo biloba. It is intended to apply.

In order to achieve the above object, the present invention is extracted and purified by an extraction method comprising a supercritical fluid extraction step using a mixed fluid containing 70 to 97% by volume of carbon dioxide and 30 to 3% by volume of a co-solvent in a supercritical state, Provided ginkgo biloba extract comprising high purity of bilovalide, gingkolide A, B, C, and less than 5ppm gingkolic acid.

According to a preferred embodiment of the present invention, the cosolvent preferably comprises at least one selected from the group consisting of ethanol, methanol, water, normal hexane, and ethyl acetate.

According to a preferred embodiment of the present invention, the mixed fluid preferably comprises 70 to 97 parts by weight of carbon dioxide, 28 to 1 parts by weight of lower alcohol and 2 to 10 parts by weight of water.

In order to achieve the above object, the present invention is characterized in that it comprises a supercritical extraction using a mixed fluid comprising 70 to 97 parts by volume of carbon dioxide in a supercritical state and 30 to 3 parts by volume of a cosolvent Provided is a method for preparing ginkgo biloba extract comprising bilobalide, gingkolide A, B, and C.

Furthermore, in order to achieve the above object, the present invention, the normal hexane extraction step of primary removal of lipids, chlorophyll, gingkolic acid, etc. by cold ginkgo biloba leaves in normal hexane; A first supercritical extraction step of secondarily removing residual normal hexane, trace lipids, and gingic acid by performing supercritical extraction using pure supercritical carbon dioxide on the residue after the normal hexane extraction; And performing a second supercritical extraction with respect to the residue remaining after the first supercritical extraction with a fluid of a supercritical carbon dioxide mixed with a co-solvent containing low alcohol and water. Provided is a method for preparing ginkgo biloba extract comprising Gingcholides A, B, and C.

According to one embodiment of the invention, the preparation method may further comprise the step of purifying with an ion exchange resin for the extract obtained in the second supercritical extraction step.

Preferably, the cosolvent of the second supercritical extraction step may be a lower alcohol, and more preferably the supercritical mixed fluid of the second supercritical extraction step may further include water.

According to one embodiment of the present invention, the supercritical mixed fluid of the second supercritical extraction step may include 70 to 97 parts by weight of carbon dioxide and 3 to 30 parts by volume of cosolvent, or 70 to 97 parts by weight of carbon dioxide, 1 to 28 parts by weight of lower alcohol and 2 to 10 parts by weight of water.

Preferably, the first and second supercritical extraction step may be performed at a flow rate of 0.5 to 2 L / min at a temperature of 40 ~ 100 ℃ and a pressure of 3000 ~ 7000 psi.

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

The structural features of the extraction method of the present invention will be described with reference to FIGS. 1 and 2.

In order to remove the gingic acid which is a toxic component in ginkgo biloba among the problems of the prior art, supercritical extraction is performed using pure supercritical carbon dioxide. Since gingkolic acid is a relatively low polar material, it can be confirmed that the pure supercritical carbon dioxide extraction is very effective.

In addition, the second supercritical extraction is continuously performed on the remaining residues after the first supercritical extraction as described above. Among the problems of the prior art in the second supercritical extraction, the nonpolar material in the supercritical extraction method using carbon dioxide is In order to solve the problem that the highly polar substances (Bilovalide, Gingcollide A, B, C, etc.) are hardly extracted, the supercritical extraction is performed by adding a co-solvent to the supercritical carbon dioxide. Do it. Through this method, highly polar bilovalide, gingkolide A, B, C and the like could be extracted in high yield.

By performing supercritical extraction in two stages as described above, the problem of excessive use of solvents and the presence of residual solvents in the extract, which is a problem of the conventional organic solvent extraction method, is solved together with bilivalide and gingolide. Ginkgo leaf extract containing A, B, and C in high purity and high yield can be extracted and purified.

According to a preferred embodiment of the present invention, before the first supercritical extraction, organic solvent extraction is performed to more effectively remove the toxic components in the ginkgo biloba leaves. Although it will not restrict | limit especially if it is an organic solvent used for normal ginkgo leaf extraction, Preferably, extraction is performed using normal hexane. Extraction conditions use the usual conditions, but preferably, when using Korean ginkgo leaves collected around May, 1 to 2 parts by weight of dry ginkgo leaves and 1 to 2 parts by weight of normal hexane, the temperature of 40 to 80 ℃ Extraction is carried out by cooling for 1 to 3 hours at.

When the organic solvent extraction is carried out in the same manner as described above, various toxic components are extracted, so the filter is discarded, the residue is taken out, dried, and then used for primary supercritical extraction, which is a feature of the present invention.

As described above, the purpose of the first supercritical extraction is to completely remove toxic components such as gingkolic acid and residual solvent, which are relatively nonpolar and leave highly active ingredients, so that no pure solvent is added. Preference is given to using supercritical carbon dioxide.

The extraction process is performed by extracting normal hexane into the extraction tank 8 and filling the remaining ginkgo leaf residue up to 80% of the volume of the extraction tank 8, and then extracting the carbon dioxide heated by the gas booster 3 to a desired pressure. To extract).

At this time, the temperature and pressure of the carbon dioxide is not particularly limited as long as it is a temperature and pressure capable of creating a supercritical state, but a temperature of 40 to 100 ° C. and a pressure of 3000 to 7000 psi are preferable, and particularly preferably, 80 Condition of temperature and pressure of 3000 psi. Under such temperature and pressure conditions, extraction is performed at a flow rate of 0.5 to 2 L / min (at room temperature and normal pressure) for 1 to 2 hours. The temperature is extracted by the temperature controller 9 at the desired temperature and the flow rate is the desired flow rate by the flow rate controller 11.

After performing the first supercritical extraction under the same conditions as above, the extract was filtered, the extract containing gingkolic acid was discarded, and only the residue was taken and used for the second supercritical extraction.

Since the purpose of the second supercritical extraction is to extract the components of bilovalide, gingcholides A, B, and C in high yield, pure supercritical carbon dioxide alone does not achieve the desired purpose. Only by performing supercritical extraction can the components be extracted with high purity. As the cosolvent, lower alcohols such as ethanol and methanol or normal hexane, ethyl acetate and the like can be used. Preferably, lower alcohols, in particular ethanol, are most preferably used. The volume ratio of the preferred cosolvent is 3 to 30 parts by volume of the cosolvent relative to 70 to 97 parts by weight of carbon dioxide, as described above.

The inventors further found that when lower alcohols are used as cosolvents, the addition of a certain percentage of water leads to higher extraction yields of highly polar bilovalides, gingkolides A, B, and C. . Therefore, according to one aspect of the present invention, in the second supercritical extraction, it is preferable to perform supercritical extraction with a fluid in which water is further mixed with a mixture of supercritical carbon dioxide and a cosolvent. At this time, the volume ratio is preferably 70 to 97 parts by weight of carbon dioxide, 1 to 28 parts by weight of lower alcohol and 2 to 10 parts by weight of water.

 Preferred conditions for supercritical extraction are not particularly limited as long as they are temperatures and pressures capable of producing a supercritical state, but temperatures of 40 to 100 ° C. and pressures of 3000 to 7000 psi are preferred, particularly preferably 80 ° C. Temperature and pressure conditions of 7000 psi. Under such temperature and pressure conditions, extraction is performed at a flow rate of 0.5 to 2 L / min (at room temperature and normal pressure) for 1 to 2 hours.

After performing the second supercritical extraction, Filtered Discard the residue, take only the extract, and continue to separate and purify the desired components.

The extract obtained by the second supercritical extraction will contain a large amount of terpene components such as bilobalide, gingkolide A, B, and C. The extract was concentrated under reduced pressure, dissolved in ethanol, and then the extracted extract was separated and purified from an ion exchange resin (HP-20).

The ginkgo biloba extract of the present invention thus separated and purified includes high purity bilobalide, gingkolides A, B, and C, and has a gingkolic acid content of 5 ppm or less.

In Examples 1 to 4 below, in order to find the optimum temperature and pressure of the supercritical carbon dioxide of the present inventors, by measuring the extraction amount of bilobalide, gingkolide A, B, C from ginkgo biloba according to temperature and pressure change After selecting the optimum temperature and pressure conditions, various types of organic solvents are added to the supercritical carbon dioxide under the selected optimal conditions to select the cosolvent by changing the extraction efficiency, and then changing the volume ratio of the selected cosolvent The optimum cosolvent volume ratio was determined by looking at the changes in the extraction amounts of,,,, and.

Finally, in Example 5, after performing supercritical fluid extraction on the ginkgo biloba leaves under the optimum conditions determined in Examples 1 to 4, separation and purification, high purity including bilivalide, gingkolide A, B, and C The extract was concentrated to complete the present invention.

Through the following examples will be described in more detail the configuration of the present invention. However, the scope of the present invention is not limited only to the following examples.

Example  One

Choosing the optimal temperature and pressure for supercritical carbon dioxide

In order to determine the optimum temperature and pressure conditions for supercritical carbon dioxide extraction for extracting bilobalide, gingkolide A, B, and C from ginkgo biloba with high purity, the supercritical extractor shown in FIG. The degree of extraction of bilovalide, gingkolide A, B, and C in ginkgo biloba was measured with a conventional supercritical carbon dioxide extractor without cosolvents, while changing in the range of ˜100 ° C. and changing the pressure in the range of 3000 to 7000 psi.

As a result, as shown in Table 1, the optimum yield was shown at 80 ℃, 5000psi. However, although it was the highest at 80 ℃, 7000psi supercritical carbon dioxide conditions, the extraction rate was not satisfactory compared to the organic solvent extraction, it was considered to add a co-solvent to increase the polarity of the supercritical carbon dioxide.

Extraction Bilovalide (μg / g) Gingcolide A (μg / g) Gingcolide ratio (µg / g) Gingcolide (μg / g) 40 ℃, 3000 psi supercritical carbon dioxide 5.72 (± 1.93) 13.27 (± 3.30) 4.10 (± 1.25) - 40 ℃, 7000 psi supercritical carbon dioxide - 25.00 (± 6.83) 14.47 (± 2.27) - 60 ℃, 3000 psi supercritical carbon dioxide 3.80 (± 1.51) 14.97 (± 3.77) 1.30 (± 0.20) - 60 ℃, 7000 psi supercritical carbon dioxide 3.18 (± 1.19) 28.07 (± 3.14) 15.03 (± 2.82) - 80 ℃, 3000 psi supercritical carbon dioxide 1.52 (± 0.49) 12.38 (± 2.57) 1.13 (± 0.48) - 80 ℃, 7000 psi supercritical carbon dioxide 5.34 (± 0.65) 34.07 (± 2.37) 29.61 (± 2.36) - 100 ℃, 3000 psi supercritical carbon dioxide 1.31 (± 0.51) 10.14 (± 1.87) 1.17 (± 0.56) - 100 ℃, 7000 psi supercritical carbon dioxide 4..56 (± 0.98) 33.78 (± 1.87) 28.31 (± 1.22) -

Example  2

Cosolvent Selection for Supercritical Fluid Extraction

Bilovalide and Gingcola when 5% by volume of various cosolvents such as normal hexane, ethyl acetate, acetonitrile and methanol are added to supercritical carbon dioxide at 80 ℃ and 7000 psi selected as the optimum temperature and pressure for supercritical carbon dioxide extraction Yield A, B, C extraction yield was highest when methanol was added. Therefore, when methanol was selected as a cosolvent, the extraction yield was measured by varying the amount of addition. As shown in Table 2, the highest extraction yield was obtained when 20 vol% was added.

Extraction Bilovalide (μg / g) Gingcolide A (μg / g) Gingcolide ratio (µg / g) Gingcolide (μg / g) Supercritical Carbon Dioxide with 5% Methanol 36.16 (± 11.91) 12.85 (± 1.66) 9.70 (± 1.83) 5.15 (± 2.29) Supercritical Carbon Dioxide with 10% Methanol 38.15 (± 10.59) 14.59 (± 1.22) 12.56 (± 1.87) 7.86 (± 2.31) Supercritical Carbon Dioxide with 20% Methanol 48.39 (± 3.77) 17.42 (± 1.50) 13.72 (± 1.64) 10.86 (± 1.82)

Example  3

Effect of Ethanol on Supercritical Fluid Extraction

Supercritical fluid extraction was performed by converting 20% methanol selected in Example 1 to 20% ethanol at 80 ° C., 7000 psi, selected as the optimal temperature and pressure for supercritical carbon dioxide extraction. As a result, as shown in Table 3, the extraction yield of ethanol was higher than that of methanol. This suggests that the extraction can be performed only with non-toxic carbon dioxide-ethanol in the extraction of the extract containing high purity bilobalide, gingkolide A, B, C from the ginkgo leaf for the purpose of the present invention.

Extraction Bilovalide (μg / g) Gingcolide A (μg / g) Gingcolide ratio (µg / g) Gingcolide (μg / g) Methanol extraction 835.92 (± 38.30) 252.18 (± 5.53) 201.37 (± 8.89) 214.97 (± 9.76) Supercritical Carbon Dioxide with 20% Methanol 48.39 (± 3.77) 17.42 (± 1.50) 13.72 (± 1.64) 10.86 (± 1.82)

However, as can be seen from Table 3, the extraction efficiency when 20% ethanol is added as supersolvent to supercritical carbon dioxide is less than 10% extraction efficiency compared to conventional organic solvent extraction (methanol extraction). Since the efficiency was too low, the addition of another cosolvent was necessary.

Therefore, in Example 4 described below, water was added to the supercritical carbon dioxide and ethanol mixture was measured to increase the extraction yield by increasing the desorption rate of the target material from the plant substrate.

Example  4

Effect of Water as Cosolvent on Supercritical Fluid Extraction

In order to increase the supercritical fluid extraction efficiency to extract the desired bilobalide, gingkolide A, B, and C components from the ginkgo biloba, the extraction efficiency was measured by adding ethanol and water to the supercritical carbon dioxide. . As shown in Table 4, the addition of water resulted in an increase in the extraction efficiency of bilovalide, gingkolide A, B, and C, which was not expected in other cosolvents, which exceeded the conventional organic solvent extraction process. Indicated.

In particular, it was confirmed that the yield of the most physiologically active gingkolide ratio among the various ginkgo biloba leaves increased by 40% compared to methanol extraction.

Extraction Bilovalide (μg / g) Gingcolide A (μg / g) Gingcolide ratio (µg / g) Gingcolide (μg / g) Supercritical Carbon Dioxide with 20% Ethanol 64.10 (± 4.84) 19.95 (± 1.73) 17.84 (± 3.46) 16.09 (± 2.27) Supercritical Carbon Dioxide with 16% Ethanol and 4% Water 764.71 (± 18.17) 201.71 (± 26.30) 278.70 (± 16.72) 169.73 (± 12.28)

Example  5

Extraction and Purification of Bilovalide, Gingcholide A, Rain, Sea from Ginkgo Biloba

First, a sample of 1 kg of ginkgo biloba leaves was cooled at room temperature and atmospheric pressure for 3 hours using 3 liters of normal hexane, and then lipids, chlorophyll, and gingic acid were removed.

After extracting normal hexane and filtering with filter paper, The residue was dried in an oven for 1 hour, placed in an extraction tank, filled up to 80% of the volume of the extraction tank, and passed through the extraction tank with carbon dioxide raised to the desired pressure by a gas booster.

Temperature and flow rate are controlled by thermostat and flow rate regulator, and the residual normal hexane, lipid, chlorophyll and gingic acid with pure supercritical carbon dioxide of 0.5 L / min (at room temperature, normal pressure) at 80 ℃, 3000 psi pressure. Etc. were removed.

After the extraction, the residue and the extract were automatically separated by a filtration device at the end of the extraction tank. The remaining residue was added to 80 parts of 80 ° C., 7000 psi supercritical carbon dioxide, and 16 parts of ethanol and 4 parts of water were extracted at a flow rate of 400 L / min (at room temperature and atmospheric pressure) for 3 hours.

The extract was concentrated under reduced pressure and dissolved in 50% ethanol. The dissolved extract was further purified and separated from 100 g of ion exchange resin (HP-20).

As a solvent for separation and purification, 2 L of 50% ethanol was removed to remove impurities, and then 4 L of 70% ethanol was further developed to obtain a desired extract. The yield of the extract and purified product was 5g per kg of ginkgo biloba.

Experimental Example  One

Analysis of Active Ingredients in Extracts

The analysis of the extract obtained from Example 5 and the bilovalide, gingkolide A, B, and C in the purified product were carried out in the following manner.

In the present invention, the mass spectrometer was mounted on the high pressure liquid chromatography for analysis of the components. The quantitative analysis of bilobalide, gingkolide A, B, and C in ginkgo biloba is carried out by conventional ultraviolet spectroscopy and refractive index measuring instruments. Although there were difficulties due to the difficulty of separation analysis due to the similarity of structures, the present invention was reproducibly analyzed using a mass spectrometer to solve this problem.

The mass spectrometry method used in the present invention is a method for selectively quantitatively distinguishing each molecular weight. The high-pressure liquid chromatography used was Agilent's 1100 series, including autoinjectors, full-wave ultraviolet absorbers, column ovens, and high-speed pumps. The mass spectrometer was Agilent's 1100 LC / MSD ion trap mass spectrometer. A pressure of 50 psi, a flow rate of 10 L / min nitrogen was used as the spray gas, the ionization temperature was 350 ° C., and the capillary bolt was -4 kV. In addition, all the mass spectrometry was measured within the range of molecular weight m / z 100-1500 by anionization. The high pressure liquid chromatography column used was a Curosil-PFP column (150 mm (4.6 mm, 5 μm) from Phenomenex. The mobile phase was analyzed by varying the composition of water and acetonitrile (initial 90:10; after 10 minutes , 70:30; after 30 minutes, 30:70; after 60 minutes, 15:85) The flow rate was 0.3 ml / min per minute.

The content of the bilovalide, gingkolide A, B, C in the purified product analyzed in the same manner as shown in Figure 3 and Table 5 below. As shown in FIG. 3 and Table 5, the purified product extracted and purified in the present invention had a higher content of bilovalide, gingkolide A, B, and C, up to 240%, than the ginkgo biloba extract.

Extraction Bilovalide (μg / g) Gingcolide A (μg / g) Gingcolide ratio (µg / g) Gingcolide (μg / g) Ginkgo leaf refined 18.40 (± 0.71) 5.67 (± 0.23) 4.82 (± 0.15) 2.11 (± 0.11) Existing product 15.01 (± 0.86) 4.57 (± 0.16) 2.03 (± 0.23) 4.12 (± 0.10)

Experimental Example  2

Analysis of Gingkolic Acid Content in Extracts

Gingkolic acid-based substances that exhibit the toxicity of ginkgo biloba leaf include the following three representative substances, the content of the following substances in the product produced by the extraction of conventional organic solvent extraction method and supercritical extraction method It measured by the same method as 1, and compared.

As a result of the quantitative analysis, the ginkgo biloba extract product produced by the supercritical extraction method of the present invention contained all of the toxic substances gingic acid 1, 2, and 3 in an amount of 0.001 mg / g or less. However, in the organic solvent extract, the gingic acid was found to be 4.5 mg / g, 123 mg / g and 44.0 mg / g, respectively, as can be expected from FIG. 4.

In the present invention, in the preparation of ginkgo biloba extract, by using a supercritical fluid extraction method to add a co-solvent, bilobalide, gingkolide A, B, C is extracted in a yield that exceeds the conventional organic solvent extraction method, At the same time, it can be said to be an environmentally friendly method that solves the residual solvent problem by reducing the amount of the organic solvent used in the conventional organic solvent extraction method by more than 80%.

In addition, the present invention has solved the complicated and time-consuming problem of the conventional organic solvent extraction method has developed a process for the extraction and purification of bilivalide, gingkolide A, B, C, which can simplify the process and reduce the extraction time. Further, the present invention overcomes the problems of low purity of the bilivalide, gingkolide A, B, C extracted by the conventional organic solvent extraction method, high-purity bilobalide, gingkolide A, B, C By manufacturing, it can be usefully used for high-end applications such as pharmaceuticals, food, cosmetics.

Claims (11)

delete delete delete delete (1) a normal hexane extraction step of coldly ginkgo biloba leaves in normal hexane to remove lipids, chlorophyll and gingic acid; (2) supercritical extraction is carried out by passing pure supercritical carbon dioxide to the residue after the extraction of the normal hexane at a flow rate of 0.5 to 2 L / min at a temperature of 40 to 100 ° C. and a pressure of 3000 to 7000 psi to obtain residual normal hexane, A first supercritical extraction step of secondary removal of trace lipids, gingic acid and the like; (3) Residue after the first supercritical extraction with a mixed fluid comprising 70 to 97 parts by volume of supercritical carbon dioxide and 3 to 30 parts by volume of methanol or methanol or a mixture thereof, the same as the first supercritical extraction. Performing a second supercritical extraction under conditions to obtain an extract; And (4) separating and purifying the extract obtained by the second supercritical extraction using an ion exchange resin; Comprising a, containing a bilobalide, gingkolide A, B, C, and a method for producing a ginkgo leaf extract containing less than 5ppm gingkolic acid. delete delete 6. The method according to claim 5, wherein the supercritical mixed fluid of step (3) further comprises water, including bilovalide, gingkolides A, B, and C, and containing 5 ppm or less of gingkolic acid. Method for producing a ginkgo leaf extract comprising. delete The method of claim 8, wherein the supercritical mixed fluid of step (3) comprises 70 to 97 parts by weight of carbon dioxide, 1 to 28 parts by weight of ethanol or methanol or a mixture thereof and 2 to 10 parts by weight of water. A method for preparing ginkgo biloba extract, comprising bilobalide, gingkolide A, B, and C, and less than 5 ppm gingkolic acid. delete

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