KR960004022B1 - Method of extraction and purification of flavonoid compounds in ginko leaves - Google Patents

Abstract

The method for extracting and purifying a flavonoid cpd. from gingko leaves comprises (a) extracting and filtering the gingko leaves with an aqueous dioxane solution of pH 8-13 selected from dioxane/water, dioxane/lower alcohol/water and dioxane/lower ketone/water at 30˜55deg. C, (b) extracting the filtered aqueous solution layer with a water-insoluble organic solvent, and extracting and distributing the layer with an organic solvent i.e. butyl alcohol, amyl alcohol, methyl ethyl ketone or ethylacetate, (c) drying the organic solvent layer at 600mmHg or less, and (d) extracting it with an ethyl ether or a petroleum ether. The flavonoid cpd. is useful as a circulatory drug.

Description

Method for Extracting and Purifying Flavonoid Compounds from Ginkgo Leaves

1 is a schematic diagram of a method for extracting and purifying flavonoid compounds from ginkgo biloba leaves.

The present invention relates to a method for selectively extracting and purifying flavonoids, which is an agent for improving and treating circulatory diseases in ginkgo biloba, and in particular, an advanced method for extracting and purifying flavonoids with high concentrations, which is simple, convenient and economical, using a new selective reagent It is about.

Briefly, the characteristics of the present invention are characterized by obtaining a high purity flavonoid with high yield with ease and stability of the production process while controlling the concentration of chlorophyll using dioxane aqueous solution as an extraction solvent. In the present invention, 5-30% aqueous dioxane solution forms an azotropic compound to minimize chlorophyll extraction and increase the selective extraction content of flavonoids. In addition, by using a dioxane aqueous solution, the boiling point is lowered to lower the evaporation concentration temperature, the manufacturing cost required for extraction and disadvantages is reduced, and the process is easy and simple, so that a biologically active substance having high biological pharmacological effect can be easily obtained.

Ginkgo biloba is a tree that survives changes in climatic and geological conditions such as ice age and tectonic change for more than 200 million years. It is called "living fossil." It has been reported to have a substance resistant to (T. Major Randolph, Science, 1967, 157, 1270.). Although the therapeutic action of ginkgo biloba has been known for centuries, it is very common to find the modern pharmacological aspect, and there are three types of flavonoids in ginkgo biloba, namely dimeric flavonoids that form the apigenine skeleton. Quercetin Glycoside, Flavonoids, Campphenol Glycoside, Oligomeric Procyanidin and Polymeric Procyanidin. Pharmacological effect of ginkgo biloba The main ingredient of the extract quality of interest is diglyceride, which can be esterified with coumarin acid at the C ₆ position of Glucose. Quercetin glycolamno in the extract Side-coumaric esters (Quercetin-3-O-α- (6 "'-coumaroyl-glucosyl-β-1.4-Rhamnoside) are found only in ginkgo biloba, especially camphor phenol glycoramnoside coumaric esters (P. Braquet, C. Roumestand, B. Peryly, "Plant Flavonoids in Biology and Medicine" Strasburg, France, 1987) .In addition to polyphenols, Ginkgolide is an important ingredient that is unique to ginkgo biloba. ) And bilobalide-based diterpenes and sesquiterpenes, which are very promising for treatment in allergic diseases and hemodynamics today. They have long been studied and synthesized because they contained less than 0.01% in ginkgo biloba (Edwin B. Villbauer and Robert C. Anderson, J. Org. Chem., 1987, 52, 1186). This manufacturing method is nearly impossible to implement on a production scale, and at present it is the only way to obtain these from natural products for pharmacological and clinical trials.

Extraction of the vasoregulatory active ingredient from ginkgo biloba was realized after active research activities identified the main components of various classes and the fact that they were constant. That is, to obtain the active ingredient material from the ginkgo biloba, special techniques are required from the tillage method, harvest time, drying method and extraction process of the plant. Harvesting time to obtain high-density, high-quality vascular control active ingredients used in the treatment of ginkgo biloba is to have aromatic resin group, that is, after the ginkgo biloba is completely green, and it is well ventilated as soon as possible by the drying method. It is good to dry at low temperature. Once dried, ginkgo biloba leaves long-term studies unaffected, and in fact, plant components such as flavonoids, alkaloids, and terpenoids are said to have no component changes even after years of drying.

In pharmacological and clinical pathology, pharmacological effects of ginkgo biloba leaf have been reported for arterial circulation, capillary circulation, venous circulation and vigorous metabolic activation of cells, and lower risk of microcirculating thrombus (H. Peter, J.). Fisel, W. Wiesser, Arznei Forsch, 1966, 16, 719 and G. Lagrue; J. Baill et., A. Behar, Sem. Hop. Paris, 1978, 54, 214. In addition, in the clinical and pharmacological studies conducted to study the therapeutic effect on post-thrombotic disease, blood pressure lowering and capillary peripheral vascular relaxation were stabilized (H. Trounier, Arzneim. Forsch, 1968, 18, 551.) The clinical pharmacological effects are effective for the treatment of cerebral vascular disease according to the condition such as senile dementia and Alzheimerqud, cerebrovascular or traumatic injury, psychiatric behavioral disease caused by senescence, peripheral vascular disease, ear disease suspected of ischemic predisposition, Assessments were made (J. Taillandier, A. Ammar; JP Rabourdin; J. Pichon; S. Niddan; H. Pierart, Presse Med., 1986, 15, 1583.).

As such, the biological pharmacological effects of ginkgo biloba leaves are very large (L. Karcher, P. Zagemann, J. Krieglstein, Naunyn-Schmiederber's Arch. Pharmcol, 1980, 327, 31.). One of the existing methods for extracting and purifying flavonoids from ginkgo leaves has been disclosed in Korean Patent No. 81-333. In this method, after extraction for 2-6 hours at 40-100 ° C. using 20-50% low aliphatic ketone or low aliphatic alcohol aqueous solution, the fat is removed with an organic solvent (CHCl ₃ , CCl ₄ ) which is not mixed with water. The layer was evaporated and concentrated through an adsorbent (Celite, Al ₂ O ₃ , CaCO ₃ , Sucrose), dissolved in ethanol, precipitated by the addition of lead compound, the organic solvent layer was separated and concentrated under reduced pressure, dissolved in ethanol, and left for 1 day before filtration. The filtrate was concentrated under reduced pressure to obtain flavonoids, but this method has a disadvantage in that the process is very complicated. In addition, Korean Patent No. 86-651 discloses extracting ginkgo biloba leaves using an ultrasonic extractor (frequency 20-50KHz), and then removes fat using an organic solvent (chloroform, ethyl acetate, ether, benzene, toluene, etc.) After transferring to butyl alcohol and sedimentation using activated carbon and inorganic salts to obtain an active ingredient, but the yield is 0.6-3.5% has a disadvantage of low efficiency. In addition, Korean Patent Nos. 90-5318 and 90-5319 extract ginkgo biloba leaves in aqueous alkali solution (NH ₄ OH, Ca (OH) _2, etc.) at 20-50 ° C. for 3-5 hours and acid treatment of the filtrate to pH 2 After adjusting to -5, the aqueous layer was extracted with lower alcohol, ethyl acetate, lower ketones, dissolved in alcohols and treated with alkali to adjust pH to 6-8, and then effective at yields of 12% and 2.5-9%, respectively. The active material is obtained but the process is complicated. Recently known Korean Patent No. 91-2391 discloses an enzyme (maserosin, cellulase C and cellulase NC, a mixture of maserosine and cellulase) in an aqueous solution of ginkgo biloba, followed by stirring and extraction at a predetermined temperature, followed by Semen was obtained and the solution was passed through a resin column (Amberlite resin XAD-2) and then molten with methanol to obtain pure flavonoids. However, the process is complicated and the yield is low at 1.0-2.0%, making it difficult to produce.

The inventors of the present invention have researched to solve the problems of the prior art, and have thus invented a method for extracting and purifying flavonoids having a high concentration in a simple, convenient and economical manner by using an optional new extraction reagent.

Dioxane aqueous solution in the present invention is a mixed organic solvent containing dioxane + water, dioxane + alcohol (methyl alcohol, ethyl alcohol, isopropyl alcohol, etc.) or ketones (acetone, methyl ethyl ketone) + water. And ketones collectively refer to mixed organic solvents containing less than 20% of the total.

The present invention is described in detail as follows. In the present invention, in extracting and purifying the vascular control active ingredient from the ginkgo biloba, the ginkgo biloba is dried and pulverized in a well-ventilated place at 10-20 ° C (10-30mesh) to use an aqueous solution of 5-30% dioxane at 20-50 ° C. The mixture was extracted with reflux cooling, and adjusted to pH 8-13 using sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium phosphate, sodium carbonate and the like to increase solubility. Flavonoid glycosides are relatively well soluble in water, but glassy forms are poorly soluble, and flavonoid glycosides have a good extraction of 3 hydroxyl groups in strong alkalis and 7 hydroxyl positions in weak alkalis. Good extraction can increase yield and purity, respectively. After extracting by this method for 1-5 hours, the filtrate was concentrated under reduced pressure (60 mmHg or less), extracted with an organic solvent (ethyl ether, petroleum ether, etc.) insoluble in water to remove chlorophyll and fatty acids, and the aqueous layer was Then, the mixture is extracted and partitioned with an organic solvent (butyl alcohol, amyl alcohol, methyl ethyl ketone, ethyl acetate or a mixture thereof). The organic solvent layer is dried under reduced pressure (60 mmHg or less; hereafter referred to as 600 mmHg or less) to obtain a high concentration of flavonoid compound. In order to confirm the residual amount of dioxane used during extraction, it was confirmed that there was no residual amount of dioxane using gas chromatography, and the dried product was dissolved in ethyl alcohol, filtered, and dried under reduced pressure (500 mmHg, 40 ° C) to obtain a pure flavonoid compound. .

The vasoregulatory active substance obtained in the above method is a tan pigment, which contains quercetin glycoside, camphor phenol glycoside, and isorhamnetin glycoside in flavonoid glycosides, and the thin layer chromatography of glycosides listed in Table 1 As a result of qualitative analysis under the conditions of the graph, the quercetin glycoside, isoramnetine glycoside, and camphor phenol glycoside Rf values were 0.39, 0.53, and 0.58, respectively. Acid hydrolysis to quantitatively analyze each of them, followed by high performance liquid chromatography according to the conditions listed in Table 2, showed that the RT (Retention Time, minutes) of quercetin, camphor phenol and isoramnetine differed slightly depending on the column efficiency. However, they were 15.44, 25.34 and 28.54, respectively.

TABLE 1

Analysis conditions of thin layer chromatography

TABLE 2

Analysis Conditions of High Performance Liquid Chromatography

Example 1

1 g of 30% dioxane aqueous solution was added to 100 g of dry powder of ginkgo biloba and extracted for 5 hours while refluxing at 35 ± 5 ° C., and the filtrate was concentrated under reduced pressure (400 ± 50 mmHg). 500 ml of 1% aqueous ammonium solution was added to the concentrated solution, followed by stirring and filtration. The filtrate was extracted with amyl alcohol, and the organic solvent layer was dried under reduced pressure (500 ± 50 mmHg, 30 ± 5 ° C.) to obtain a dried product. The dried product was again dissolved in ethanol, and the insoluble part was filtered, and the filtrate was dried under reduced pressure (300 ± 5 mmHg, 40 ± 5 ° C.) to obtain 11.5 g of powder. Qualitative analysis of flavonoids, an active ingredient thereof, was tested according to the conditions of Table 1, and quantitative analysis was more than 24% as a total flavonoid as a result of analyzing the purity according to the conditions of Table 2.

Example 2

To 100 g of ginkgo biloba dry powder was added 500 ml of dioxane: methanol: water = 1: 1: 1 mixed solution, extracted at 45 ± 5 ° C. for 3 hours, and then filtered twice. The filtrate was collected, concentrated to 30% of the filtrate, and 100 ml of saturated sodium chloride aqueous solution was added to the concentrated solution, followed by stirring and filtering. The filtrate was extracted with butyl alcohol: amyl alcohol (4: 1), and the organic solvent layer was dried under reduced pressure (400 ± 50 mmHg, 45 ± 5 ° C.) to obtain 10.7 g of an active powder. According to the analytical method of Table 1 and Table 2, the purity was more than 24% as a total flavonoid.

Example 3

100 g of dried ginkgo biloba powder was extracted with 500 ml of 25% aqueous dioxane solution at 40 ± 5 ° C. for 3 hours, and then filtered twice. Collect the filtrate and concentrate under reduced pressure (400 ± 50mmHg), extract the concentrated solution with ethyl ether, and extract the supernatant twice with 200ml of butyl alcohol, and dry the organic solvent layer under reduced pressure (250 ± 50mmHg, 45 ± 3 ℃). 7.9 g of an active substance flavonoid was obtained. The qualitative and quantitative analysis of Table 1 and Table 2 showed that the purity was over 30% as total flavonoids.

Example 4

Add 100 ml of 10% dioxane aqueous solution to 100 g of dried powder of Ginkgo biloba, adjust the pH to 12 ± 0.5 with 1N sodium hydroxide to increase the solubility, extract 2 times at 40 ± 5 ℃ for 3 hours, and collect the filtrate and concentrate under reduced pressure (350 ± 50mmHg). I was. The concentrated solution was extracted with butyl alcohol: amyl alcohol (3: 1), and the organic solvent layer was concentrated under reduced pressure (350 ± 50 mmHg, 40 ± 3 ° C) to obtain 10.4 g of an active substance. According to the analysis conditions of Table 1 and Table 2, the qualitative and quantitative analysis showed that the purity was more than 24% as total flavonoids.

Example 5

100 g of dried ginkgo biloba powder was extracted and filtered twice at 40 ± 5 ° C. for 2 hours with dioxane: acetone: distilled water = 1: 1: 1 mixture solution. The filtrate was removed fat with petroleum ether and the aqueous layer was extracted again with butyl alcohol, and the organic solvent layer was dried under reduced pressure (350 ± 50 mmHg, 40 ± 3 ° C.) to obtain 7.3 g of a blood vessel regulating active substance. Qualitative and quantitative analysis of Table 1 and Table 2 showed that the total flavonoid purity was more than 30%.

Example 6

Add 100 ml of 20% dioxane aqueous solution to 100 g of dry powder of ginkgo biloba, adjust pH 11 ± 0.5 with 1N potassium hydroxide, extract twice at 50 ± 5 ℃ for 4 hours, collect filtrate and concentrate under reduced pressure to 50% (300 ± 50mmHg). It was. Butyl alcohol: It was partitioned into methyl ethyl ketone (1: 4), and the organic solvent layer was dried under reduced pressure (250 ± 50 mmHg, 35 ± 5 ° C) to obtain 10.5 g of a flavonoid compound. The compound was analyzed according to Table 1 and Table 2 and the purity of the total flavonoids was 24% or more.

Example 7

500 g of 10% dioxane aqueous solution was added to 100 g of dried powder of ginkgo biloba, adjusted to pH 11 ± 0.5 with 1N sodium hydroxide, extracted twice with 4 hours while refluxing at 45 ± 5 ℃, filtered and concentrated under reduced pressure (250). ± 50 mmHg). Fat was extracted with petroleum ether, and the aqueous layer was extracted and distributed with butyl alcohol: ethyl acetate (1: 4), and the organic solvent layer was dried under reduced pressure (250 ± 50 mmHg, 35 ± 5 ° C.) to obtain 7.1 g of an active substance. This material was analyzed according to the analysis conditions of Table 1 and Table 2, and the total flavonoid purity was 30% or more.

Example 8

100 g of ginkgo biloba dry powder was extracted and filtered twice with 40 ml of a mixed solution of dioxane: ethyl acetate: distilled water (1: 1: 1) at 40 ± 5 ° C. Collect the filtrate and concentrate under reduced pressure (250 ± 50mmHg), add 100ml of 1% aqueous ammonium chloride solution, stir, leave, and filter. The filtrate is extracted with butyl alcohol: methyl ethyl ketone (1: 5) and partitioned into an organic solvent layer. The organic solvent layer was dried under reduced pressure (250 ± 50 mmHg, 35 ± 5 ° C.) to obtain 10.1 g of an active substance. According to the analytical conditions of Table 1 and Table 2, this active substance was found to have a total flavonoid purity of 24% or more.

Example 9

100 g of dried ginkgo biloba powder was extracted with dioxane: ethyl acetate: distilled water (1: 1: 4) mixed solution at 500m with reflux cooling at 35 ± 5 ° C for 5 hours using a Soxhlet apparatus, and the extract was concentrated under reduced pressure (300 ± 50mmHg). I was. After removing the fat with ethyl ether, the aqueous solution was partitioned and transferred to butyl alcohol: ethyl acetate (1: 5) mixed solvent, and the organic solvent layer was dried under reduced pressure (250 ± 50 mmHg, 35 ± 5 ° C.) to give 7.6 g of a flavonoid compound. Got. According to the analysis conditions of Table 1 and Table 2, the purity was more than 30% as the total flavonoids.

Example 10

100 g of dried ginkgo biloba powder was added to 500 ml of 20% aqueous dioxane solution, and the pH was adjusted to 11 ± 0.5 with 1N potassium hydroxide. The extract was filtered twice at 40 ± 5 ° C for 2 hours, filtered and concentrated under reduced pressure (300 ± 50mmHg). Then, the mixture was extracted and distributed with a mixed solution of butyl alcohol: methyl ethyl ketone (1: 4). The mixture was dried under reduced pressure (350 ± 50mmHg, 40 ± 5 ℃) and filtered and extracted again with petroleum ether to obtain 5.4g of active active material. The material was analyzed under the analysis conditions in Table 1 and Table 2 as a total flavonoid. Got more than%.

Example 11

100 g of dried ginkgo biloba powder was added to 500 ml of 25% dioxane aqueous solution, the pH was adjusted to 12 ± 0.5 with 1N sodium hydroxide, and extracted and filtered twice at 40 ± 5 ° C. for 2 hours, and the filtrates were concentrated under reduced pressure (400 ± 50 mmHg). Then extracted with butyl alcohol: ethyl acetate (1: 3). The powder was dried under reduced pressure (250 ± 50mmHg, 35 ± 5 ℃) and filtered and filtered again with ethyl ether to obtain 5.2g of the active substance, which was 40% of the total flavonoids according to the analysis conditions of Table 1 and Table 2. It was above.

Example 12

100 g of dry powder of ginkgo biloba was added to 500 ml of a mixed solution of dioxane: ethyl acetate: distilled water (1: 1: 1), and extracted and filtered twice at 40 ± 5 ° C for 2 hours. The filtrates were combined and concentrated under reduced pressure (300 ± 50 mmHg), and the concentrate was extracted and distributed with butyl alcohol: ethyl acetate (1: 4). The solution was dried under reduced pressure (300 ± 50mmHg, 35 ± 5 ℃), extracted with ethyl ether, filtered and filtered to obtain 5.1g of the active substance. The active substance was purified as total flavonoids according to Table 1 and Table 2. It was over%.

Claims (3)

The ginkgo biloba leaves were extracted and filtered at 30-55 ° C. with dioxane aqueous solution having a pH range of 8-13 selected from the group consisting of dioxane + water, dioxane + lower alcohol + water and dioxane + lower ketone + water. The aqueous layer was extracted with an organic solvent insoluble in water, and the aqueous layer obtained after extraction was extracted and distributed with an organic solvent or a mixture thereof selected from the group consisting of butyl alcohol, amyl alcohol, methyl ethyl ketone and ethyl acetate, and then an organic solvent layer. Method for extracting and purifying flavonoid compounds from ginkgo leaves, characterized in that the dried under reduced pressure to 600mmHg or less. The method of claim 1, wherein the lower alcohol and lower ketone in the dioxane aqueous solution is less than 20% of the total. The method of claim 1, further comprising drying the organic solvent layer to 600 mmHg or less, followed by extraction with ethyl ether or petroleum ether.