KR960010622B1 - Method of extraction and purification for active substances from ginko herb - Google Patents

Abstract

The purification method of Ginko extracts (I) consists of (1) dissolving crude (I) in 50% ethanol solution, and (2) filtrating with ultrafiltration membranes in the range of 3000- 10000 dalton to discard protein, polysaccharide and proanthicyanins. (I) is safe for use.

Description

Extraction and Purification of Active Active Substances from Ginkgo Leaves

1, 2 and 3 respectively show absorption maximum curves as quantitative analysis results of proanthocyanidin compounds in the active substances extracted and purified in Examples 1, 2 and 8 according to the method of the present invention. It is a graph compared with the case of Comparative Example 2,

4 and 5 are chromatograms for quantitatively analyzing the amounts of active substances using high-performance liquid chromatography, respectively, for powders and standard products extracted in Example 2 according to the method of the present invention.

The present invention relates to a method for extracting and purifying an effective physiologically active substance from ginkgo biloba, and more particularly, to extract an effective physiologically active substance which can be used for the treatment of vascular and blood circulation disorders and senile diseases from ginkgo biloba without using harmful substances. The present invention also relates to a method for extracting and purifying high concentrations of active substances in a simple and economical way.

In general, the active gingo flavones contained in the ginkgo biloba has various effects, so drugs or health foods using ginkgo biloba extract have been developed.

Specifically, ginkgo biloba extract exhibits vasodilation and significantly increases peripheral blood flow and brain blood flow by blood flow promoting action and increases the concentration of ATP in human tissue. It is also known to promote energy metabolism in cells. In addition, it is found that there is an excellent prevention and treatment effect on cerebral ischemia, peripheral cerebral vasodilation and various hypoxia, and its use is further increased due to such efficacy.

There are several known methods for extracting the active ingredient from the ginkgo leaves.

For example, Japanese Patent Publication Nos. 46-28091 and 49-27323 disclose a method of extract-purifying an active ingredient using a halogenated hydrocarbon such as dichloromethane, chloroform and carbon tetrachloride as a method for preparing a ginkgo leaf extract. However, this method requires careful attention to the purification process or can cause environmental problems because the solvent is a very undesirable pest in pharmaceuticals, and it is also a concern for direct harm to the workers involved in extraction purification. Furthermore, Japanese Patent Publication No. 49-27323 points out that it is not preferable because toxic lead compounds are used in the extraction process.

In particular, Ginkgo biloba extract in Japanese Patent Publication No. 46-28091 is suitable as an oral administration agent, but it can be used as an injection only when an unknown substance that impairs the stability of the active ingredient is removed. It is stated that must be found.

In addition, Japanese Patent Publication No. 49-27323 found that an unknown substance that impairs the stability of the active substance mentioned in SO 46-28091 causes precipitation in solution, and the cause of precipitation is prodelpinidine. Although it is mentioned, the lead compound which is a harmful substance is used as an extraction method to reduce the residual amount in the extract by removing it.

In addition, there are methods of extracting ginkgo biloba extracts, such as Japanese Patent Publication Nos. 81-333, 90-5318, and 90-5319, and the Japanese Patent Publication No. 49-27323 of Patent Publication No. 81-333. As substantially the same method as above, there is a problem as described above using a halogenated hydrocarbon and lead acetate or lead hydroxide.

However, in the case of Korean Patent Publication Nos. 90-5318 and 90-5319, an improved method of the existing method is shown, but the former Korean Patent Publication No. 90-5318 uses a purified column as a purification process. There is a problem of using carbon tetrachloride, which is a halogenated aliphatic hydrocarbon, in the preparation of the column, and the latter Korean Patent Publication No. 90-5319 can evaluate some improvement points, but it is also necessary to pay attention because it uses bismuth compounds.

As described above, all known methods of preparing ginkgo biloba extract extracts have a lot of room for improvement due to problems of human hazards, environmental pollution, and problems that require careful attention in the process because they all use halogenated hydrocarbons or harmful heavy metal compounds. .

Therefore, the present inventors have studied the method of extracting and purifying the active ingredient from the ginkgo biloba without using any toxic organic solvents or heavy metal compounds used in the conventional method, and clearly identified the substance causing the precipitation in the ginkgo biloba extract. By using the ultrafiltration method, which has not been used before, based on this, it was found that not only harmful substances are used, but also the extract of purified ginkgo biloba extract can be economically extracted while greatly shortening the process, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a new method for producing high purity ginkgo biloba extract in high yield by using a harmless organic solvent and using ultrafiltration in extracting and purifying the active ingredient from the ginkgo biloba leaves.

Hereinafter, the present invention will be described in detail.

The present invention extracts ginkgo biloba leaves with a mixture of methanol or water and acetone or alcohol and adds an inorganic salt, followed by extraction with an organic solvent, and then removing impurities from the crude extract obtained to extract and purify the active substance. To obtain an extract from which the impurities were removed from the extract, the crude extract was concentrated under reduced pressure, and then ethanol aqueous solution was added to the residue, which was passed through an ultrafiltration membrane, and the resulting solution was treated with an aqueous ethanol solution and dried under reduced pressure to obtain a powder. It is characterized by.

Referring to the present invention in more detail as follows.

The present invention is characterized in that the crude extract is prepared by extracting from the ginkgo biloba with a mixed solvent or an organic solvent by a conventional method, followed by extraction and purification by ultrafiltration.

According to the present invention, the dried ginkgo biloba leaves are extracted with methanol or water and a mixed solution of alcohol such as acetone or methanol, ethanol or propanol at room temperature or boiling point temperature of the solvent. The extract thus obtained is separated from the residue and the organic solvent is removed under reduced pressure and concentrated to 10% or less of the total weight. At this time, water may be added to replace the organic component in the extract if necessary. Water was added to the concentrated extract concentrate as described above, and the dried extract was diluted to 10-20 wt% in the total aqueous solution. After cooling to 20 ° C. or lower, the fat-soluble component was precipitated and removed by filtration. Is added in a volume of 20-30% with stirring. Methyl ethyl ketone or a mixed organic solution of methyl ethyl ketone and acetone is added to this solution and left to stand. In this case, when the mixed organic solution is used, the ratio of methyl ethyl ketone: acetone is 9: 1 to 4: 6 in volume ratio. When the mixture is separated into two layers in this state, the active substance of ginkgo biloba leaves in the organic solvent layer It is obtained in the state containing a large quantity, and it concentrates so that solid content may be 50 weight% of the total solution by depressurizing this, and a crude extract is obtained.

According to the present invention, a 50v / v% ethanol aqueous solution is added to the concentrated crude extract to dilute the solid component to 5 to 20% by weight, and then a protein, a polysaccharide, and a polyphenol polymer contained in the crude extract of the ginkgo biloba extract. To effectively remove polymers such as anthocyanidin compounds, the diluted solution is passed through an ultrafiltration membrane with an exclusion limit of about 3,000 to 10,000 Daltons to extract only the active substance.

At this time, if the exclusion limit of the ultrafiltration membrane is lower than 3,000 Daltons, the extraction effect of the active active substances is lowered, and the yield is greatly lowered. If the upper limit is higher than 10,000 Daltons, the yield of the active active substances is increased, but the removal efficiency of the polymer is greatly decreased, and thus Since a large amount of the proanthocyanidin compound as an impurity remains, there is a problem of deterioration in purity.

The ultrafiltration membrane used in the present invention is a very thin and selective filtration membrane used to separate molecules of a substance dissolved in the solution according to the size by passing through the solution. Most of the ultrafiltration membranes include a solvent by filtering and purifying a polymer having a specific size or more. Molecular filtration membrane that quantitatively passes small molecules of.

These ultrafiltration membranes are used in the manufacturing process of pharmaceuticals such as vaccines beyond the research field in biotechnology. For example, high concentrations of cells cultured during vaccine and interferon production are separated from the medium, and the concentration and washing of biopolymers such as proteins It is used for destingting, separating hemoglobin and enzymes from erythrocyte lysates and removing pyrogen such as colloidal microorganisms.

In the present invention, by using a new ultrafiltration membrane that can be fractionated according to the molecular weight as described above for the extraction of natural products that have never been considered at all, it is surprising to know that it is possible to obtain a very high purity of the active substance more simply than the existing known method It became.

That is, in the present invention, by using an ultrafiltration method that has not been used for the extraction of active ingredients from natural products in the past, there is no fear of chemical property change due to a phase change as compared with the conventional method. And high concentration of flavone glycoside, an active ingredient of ginkgo biloba leaves, and very high purity extract with little polymer impurities such as proteins, sugars and proanthocyanidins was obtained.

Therefore, when passing through the ultrafiltration membrane can be obtained by simultaneously separating the portion of the concentrated polymer material and the portion containing little such polymer material is good extraction and purification effect, in particular in the present invention molecular weight of about 3,000 ~ 10,000 Daltons The use of those having an exclusion limit prevents the passage of polymers such as proteins, polysaccharides and polyphenol polymers, so that only the active substances required by the present invention can be selectively extracted. After use, the ultrafiltration membrane is washed with water and can be reused repeatedly. In case of ultrafiltration, no pretreatment process is required, and at room temperature without special conditions.

Thus, the extract extracted with the ultrafiltration membrane according to the present invention is concentrated under reduced pressure, and once again dissolved in 85 ~ 95v / v% ethanol aqueous solution, and then allowed to stand overnight at room temperature to precipitate, which is filtered to obtain a clear filtrate. , This process is to remove dissolved ammonium sulfate remaining in the extract until the extraction due to the water mixed when the extraction using the mixed organic solution before the ultrafiltration membrane extraction, the filtrate obtained through this process is dried under reduced pressure The target product is obtained with a powder having a residual moisture of 5% by weight or less.

The ginkgo biloba active active material obtained through the above process is an orange-yellow amorphous hygroscopic powder containing a large amount of flavone glycosides such as quercetin glycosides, camphorol glycosides, and isoramnetine glycosides. High purity extract with high content of anthocyanidin compound is greatly reduced, and no precipitation is produced even when used as an oral solution or injectable solution according to a conventional method. This is very simple because it removes all impurities such as proanthocyanidin, a polyphenolic polymer that affects stability due to the formation of precipitates when used as an oral solution or an injectable solution at once, by ultrafiltration. By increasing the purity of the process can be used as a liquid or injection.

As a result, in the present invention, as a result of constant research to clarify the fact that the precipitate forming material that inhibits the stability of the ginkgo biloba extract is prodelpinidine, all of the proanthocyanidin compounds such as prodelinidine and procyanidin are found in the precipitate forming material. To identify new facts that precipitate stability hinders stability and to prepare ginkgo biloba extracts that do not contain these precipitate forming substances, ultrafiltration has not been used for extraction of active substances from ginkgo biloba as well as conventional natural product extraction. It is possible to achieve the purpose by simply extract purification of the ginkgo biloba extract, which almost all of the high-polymerization proanthocyanidin compound of the precipitate-forming material is removed.

As described above, by extracting by ultrafiltration according to the present invention, the ginkgo biloba leaf extract can be obtained with a high purity and high yield through a simple and improved process. As follows.

First, it does not use harmful substances that have been conventionally used in almost all extraction methods.

Second, the complex impurity removal process was greatly shortened after the mixed solvent extraction.

Third, it was found to be a proanthocyanidin compound of the precipitate-forming material in the preparation of the injection and greatly improved the purity by removing almost all of it.

Fourth, by providing a high-purity ginkgo leaf extract from which the precipitate-forming substance is removed during the preparation of the injection, it is possible to provide a raw material that can be used as a liquid or injection without side effects.

Fifth, while maintaining the content of flavone glycoside, an active active substance contained in the final extract at a high level, the extraction yield was greatly improved to obtain the target compound in high yield.

Sixth, the ultrafiltration membrane used for extraction is economically advantageous because it can be washed and reused.

Seventh, it is possible to extract by a simple process without temperature conditions or special pretreatment during extraction by ultrafiltration.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited by the Examples.

Example 1

100 g of dried ginkgo biloba leaves are filled in a cone container and 930 ml of 60v / v% acetone aqueous solution is added to leach the leaf powder. After 12 hours, the resultant was filtered under pressure and the filtrate was concentrated under reduced pressure to a total amount of 150 ml. Water was added to the concentrated solution to make 200 ml, the mixture was stirred vigorously, cooled to 15 ° C. for 12 hours, and the supernatant was separated and filtered.

After stirring the filtrate, 50 g of ammonium sulfate was added, and after 15 minutes, 37 ml of methyl ethyl ketone and 25 ml of acetone were added thereto, and the mixture was left to stir after mixing. The organic solvent layer was separated, and the mixture was repeatedly extracted three times with the same amount of methyl ethyl ketone and acetone. Separate and add. The methyl ethyl ketone and acetone layers are concentrated under reduced pressure so that the total amount is 10 g.

68 ml of 50v / v% ethanol aqueous solution was added to the obtained concentrate, followed by dissolution, followed by filtration with Amicon's Diaflo Ultrafiltration Membranes YM3 (exclusion limit: 3,000 Daltons) at a pressure of 4 atm. The filtrate was concentrated under reduced pressure to 5 g, dissolved in 25 ml of 90v / v% ethanol aqueous solution, and allowed to stand at room temperature overnight. The filtrate was filtered clearly and dried under reduced pressure to a maximum of 60 ° C. 2.16 g of active active material (orange yellow powder) are obtained.

Example 2

The target product was obtained in the same manner as in Example 1, but using an ultrafiltration membrane having an exclusion limit of 5,000 Daltons. The results obtained by repeating this process seven times were as shown in Table 1 below, and as a result, 2.2 g of an active substance (orange powder) was obtained.

* The above unit is the unit of content of the active substance contained in the final target product.

Example 3

100 g of dried ginkgo biloba powder was filled into a cone container, and the same procedure as in Example 2 was carried out except that 930 ml of methanol was used instead of an aqueous 60v / v% acetone solution to obtain 2.17 g of an active substance (orange powder).

Example 4

2.54 g of active active material (orange powder) was prepared in the same manner as in Example 2 except that 100 g of dried ginkgo biloba powder was filled in a cone container and 930 ml of 60v / v% methanol aqueous solution was used instead of 60v / v% acetone aqueous solution. Got.

Example 5

An active material of 2.2 g (orange powder) was prepared in the same manner as in Example 2 except that 100 g of dried ginkgo biloba powder was filled into a cone container and 930 ml of an aqueous 60v / v% ethanol solution was used instead of an aqueous 60v / v% acetone solution. Got.

Example 6

1.66 g of an active substance (orange powder) was prepared in the same manner as in Example 2 except that 100 g of dried ginkgo biloba powder was filled into a cone container and 930 ml of an aqueous 60v / v% isopropanol solution was used instead of an aqueous 60v / v% acetone solution. Got.

Example 7

The same procedure as in Example 2 was carried out, but after passing through the ultrafiltration membrane, the filtrated aqueous solution of ethanol was concentrated under reduced pressure to half the amount, and 50 g of ammonium sulfate was added. After 15 minutes, 37 ml of methyl ethyl ketone and 25 ml of acetone were added thereto, followed by mixing and stirring. After separating the organic solvent layer, the mixture was extracted and separated three times by the same amount of methyl ethyl ketone and acetone mixed solution.

The methyl ethyl ketone and acetone layer was reduced in pressure to a total amount of 5 g, dissolved in 25 ml of 90v / v% ethanol aqueous solution, and allowed to stand at room temperature overnight, then filtered clearly and dried under reduced pressure at a maximum of 60 ° C. 1.95 g of an active substance (orange powder) was obtained.

Example 8

In the same manner as in Example 2, 3.17 g of an active substance (yellowish brown powder) was obtained using an ultrafiltration membrane having a bezel limit of 10,000 Daltons.

Reference Example

In the same manner as in Example 1, 0.67 g of an active substance (orange powder) was obtained using an ultrafiltration membrane having an exclusion limit of 1,000 Daltons.

Comparative Example 1

100 g of dried ginkgo biloba leaves are filled in a cone container and 930 ml of 60v / v% acetone aqueous solution is added to leach the leaf powder. After 12 hours, the resultant was filtered under pressure, and the filtrate was concentrated to 150 ml under reduced pressure. Water was added to the concentrated solution to make 200 ml, the mixture was stirred vigorously, cooled to 15 ° C. for 12 hours, and the supernatant was separated and filtered. After stirring the filtrate, 50 g of ammonium sulfate was added, and after 15 minutes, 37 ml of methyl ethyl ketone and 25 ml of acetone were added thereto, and the mixture was left to stir after mixing. The organic solvent layer was separated, and the mixture was repeatedly extracted three times with the same amount of methyl ethyl ketone and acetone. Separate and add. The methyl ethyl ketone and acetone layer are concentrated under reduced pressure. The residue obtained is dissolved in 25 ml of 90v / v% ethanol aqueous solution, and is allowed to stand at room temperature overnight. The resulting residue is filtered clearly and dried under reduced pressure at a maximum of 60 ° C. As a result, 3.7 g effective active material (brown powder) was obtained.

Comparative Example 2

100 g of dried ginkgo biloba leaves are filled in a cone container and 930 ml of 60v / v% acetone aqueous solution is added to leach the leaf powder. After 12 hours of filtration, the filtrate was extracted three times with 100 ml of carbon tetrachloride to separate the aqueous acetone layer. The aqueous acetone layer is concentrated under reduced pressure to 150 ml. Water was added to this solution to make 200 ml, the mixture was stirred vigorously, left to cool at 15 ° C. for 12 hours, and the supernatant was separated and filtered. 50 g of ammonium sulfate was added while stirring the filtrate, and after 15 minutes, the mixture was extracted and separated three times with methyl ethyl ketone and acetone mixed solution. The methyl ethyl ketone and acetone layer are concentrated under reduced pressure. The residue obtained is dissolved in 25 ml of 90v / v% ethanol aqueous solution, and is allowed to stand at room temperature overnight. The resulting residue is filtered clearly and dried under reduced pressure at a maximum of 60 ° C. As a result, 3.13 g of an active substance (brown powder) was obtained.

Comparative Example 3

100 g of dried ginkgo biloba leaves are filled in a cone container and 930 ml of 60v / v% acetone aqueous solution is added to leach the leaf powder. The aqueous acetone layer is concentrated under reduced pressure to a total amount of 150 ml. Water was added to the concentrated solution to make 200 ml, the mixture was stirred vigorously, cooled to 15 ° C. for 12 hours, and the supernatant was separated and filtered. While stirring the filtrate, 50 g of ammonium sulfate was added, and after 15 minutes, the mixture was repeatedly separated and separated three times with a mixture of methyl ethyl ketone and acetone. The methyl ethyl ketone and acetone layers were concentrated under reduced pressure to a total amount of 10 g.

68 ml of a 50v / v% ethanol aqueous solution was added to the obtained concentrate, followed by dissolution. 30 ml of a 10% aqueous solution of lead acetate [Pb (COOCH) .3HO] was added thereto, stirred, and left to stand. The precipitate formed was filtered off. Concentrate the aqueous solution of ethanol under reduced pressure in half, add 50 g of ammonium sulfate, and after 15 minutes, add 37 ml of methyl ethyl ketone and 25 ml of acetone, and after stirring, separate the organic solvent layer, and then use an equal amount of methyl ethyl ketone and acetone. Extract three times and combine. The methyl ethyl ketone and acetone layers are concentrated under reduced pressure so that the total amount is 10 g. The obtained concentrated solution was dissolved in 25 ml of 90v / v% ethanol aqueous solution, left at room temperature for one night, then filtered clearly and dried under reduced pressure up to 60 ° C. As a result, 1.22 g effective active material (orange yellow powder) was obtained.

Comparative Example 4

100 g of dried ginkgo biloba leaves are filled in a cone container and 930 ml of 60v / v% acetone aqueous solution is added to leach the leaf powder. The aqueous acetone layer is concentrated under reduced pressure to a total amount of 150 ml. Water was added to the concentrated solution to make 200 ml, the mixture was stirred vigorously, cooled to 15 ° C. for 12 hours, and the supernatant was separated and filtered. 50 g of ammonium sulfate was added while stirring the filtrate, and after 15 minutes, the mixture was extracted and separated three times with methyl ethyl ketone and acetone mixed solution. The methyl ethyl ketone and acetone layers were concentrated under reduced pressure to a total amount of 10 g.

68 ml of a 50v / v% ethanol aqueous solution was added to the obtained concentrate, followed by dissolution. 30 ml of a 10% aqueous solution of lead acetate [Pb (COOCH) .3HO] was added thereto, stirred, and left to stand. The precipitate formed was filtered off. About 5 g of an ethanol aqueous solution was concentrated under reduced pressure, and the obtained concentrated solution was dissolved in 25 ml of 90v / v% ethanol aqueous solution, and allowed to stand at room temperature for one night, then filtered clearly and dried under reduced pressure to a maximum of 60 ° C. As a result, 1.11 g effective active material (dark yellow powder) was obtained.

Experimental Example 1

The powders obtained in Examples 1 to 8 and Reference Examples, together with the powders obtained in Comparative Examples 1 to 4, are polyphenol polymers that produce precipitates in the preparation of liquids or injectables in effective active materials using an ultraviolet / visible absorption spectrophotometer. Quantitative analysis was performed to determine the content of residual proanthocyanidin compounds.

Among them, Examples 1, 2, and 8 using the ultrafiltration method instead of using the harmful substance lead acetate and Comparative Example 2, which also did not use the lead acetate which is also a hazardous substance, were prepared in the same conditions. Comparison was made with 1 degree, 2 degrees and 3 degrees.

From these results, it can be seen that in the case of Examples 1, 2, 8, significantly lower absorbance than each of Comparative Example 2. Therefore, in the case of the present invention, it can be seen that the content (residual amount) of the proanthocyanidin compound is much lower as compared with the conventional one having the conditions.

[Test Methods]

20 mg of the active substance extracted from Ginkgo biloba are precisely weighed and dissolved in a mixed solution of isopropanol: 3 normal hydrochloric acid (5: 1) to make 25 ml. Take 5 ml of the filtrate and transfer to a 10 ml flask. After cooling for 10 minutes, add isopropanol: 3 normal hydrochloric acid (5: 1) mixed solution to fill the mark and measure the absorbance at 556 nm.

0.5 mg of the indicator substance proanthocyanidins had an absorbance of 0.477 at 10 ml according to the above conditions.

Experimental Example 2

The powders obtained in Examples 1 to 8 were treated with Comparative Example 1, Comparative Example 2, Comparative Example 3, and lead acetate aqueous solution, and then the powders obtained in Comparative Example 4, which were not subjected to an extraction process using an organic solvent, Quantitative analysis of the amount of the active flavone glycoside as a quantitative analysis using high performance liquid chromatography (HPLC) as shown in Table 2 below.

In addition, confirmation of homogeneity of the components of the active substance obtained according to the method of the present invention was confirmed through FIG. 4, which is a chromatogram of the powder obtained in Example 2, and FIG. 5, which is a chromatogram of a standard product.

[Test Methods]

10 mg of the active substance extracted from Ginkgo biloba is precisely weighed, dissolved in 20 ml of methanol, 10 ml of hydrochloric acid is added, and hydrolyzed by heating in a boiling water bath for 1 hour. After cooling, it is made into a sample solution with exactly 50 ml of methanol. Standard solutions were prepared using indicators such as netine and analyzed by high performance liquid chromatography.

[Analysis Condition]

Instrument: Waters 6000A Pump

Waters U6K Injector

Schimadzu auto integrator

A. Column: 3.9 × 150mm Nova-Pak C18, Waters

B. Mobile Solvent: Water: Methanol: Acetic Acid (60: 40: 3)

C. Flow Rate: 1.0ml / min

D. Detector: UV 365nm

E. Injection volume: 10μl

F. Sensitivity: 0.1Aufs.

G. Record Sensitivity: 0.1cm / min

In the case of the embodiment (ultrafiltration method) according to the extraction and purification method of the present invention from the analysis results, Comparative Example 1 in which the ultrafiltration method was not performed or Comparative Example 2 without using a lead compound (Japanese Patent Publication No. 46-28091) It can be seen that the content of proanthocyanidins is significantly reduced compared to).

In addition, when comparing the Example (Ultrafiltration method) of the present invention with Comparative Example 3 (Japanese Patent Publication No. 49-27323) using a lead compound as a harmful component, it was carried out in comparison with Comparative Example 3 in that it used lead acetate as a hazardous substance. Although the example is excellent, in Example, the method of the present invention uses 100 ml of 95v / v% ethanol, 30ml of 10v / v% lead acetate in Comparative Example 3, instead of using only one simple ultrafiltration, filtration, concentration, 60v / Example in terms of yield or proanthocyanidin content (remaining rate), even using unfavorable methods such as v% ethanol use, ammonium sulfate use, stirring, filtration, three times extraction with methyl ethyl ketone and acetone mixed solvent. Since it is only maintained at about the same level as compared to this, it can be seen that the method of the present invention has an advanced effect of shortening the process and using no harmful substances.

In addition, in the case of Comparative Example 4, which is carried out in the same manner as in Comparative Example 3 using a lead compound but extracted with lead acetate and ethanol instead of the ultrafiltration method in Example, and no other processes were carried out, Examples of the present invention It can be seen that the content of the active active material is significantly lower and the yield is significantly lower than that of the present invention. The low content of proanthocyanidin is low in the active active material and the yield is low, and therefore, the proanthocyanidin content is also low. It is possible to confirm that the effect is greatly reduced compared to the present invention in any aspect.

From the above results, it can be seen that the present invention can significantly reduce the process and obtain a high purity, high yield of ginkgo biloba extract without using an active substance compared to the conventional method, in particular, the desired total active substance yield Comparing to the method of the present invention can be seen that greatly improved not only in the purity but also the yield.

On the other hand, in the case of the reference example compared to Examples 1 to 8, the ultrafiltration method was used, but the exclusion limit was 1,000 Daltons, and the yield was lower than that of the Example, so the ultrafiltration membrane exclusion limit was 3,000 Daltons or more. In the case of Example 8, when the exclusion limit was used at 10,000 Daltons, the content of proanthocyanidin was slightly increased compared to the other examples, so that the exclusion limit was higher than 10,000 Daltons. It can be seen that the use of an ultrafiltration membrane is undesirable.

Claims (1)

The ginkgo biloba leaves are initially extracted with a mixed solution of methanol, water, acetone or alcohol, added with an inorganic salt, and then extracted again with an organic solvent. The crude extract is then extracted and purified by removing impurities from the obtained crude extract. A 50v / v% aqueous ethanol solution was added to the residue obtained by concentration under reduced pressure, so that the solid component was 5-20% by weight, and the protein was passed through an ultrafiltration membrane having a limit of 3,000-10,000 Daltons at 4 atm. And extracting and purifying the active substance from ginkgo biloba leaves, characterized in that it removes polysaccharide and proanthocyanidin components.