WO2006009373A1

WO2006009373A1 - The purified leaf extract of ginkgo biloba having neuronal cell-protecting activity, the method for preparing them and the composition comprising the same

Info

Publication number: WO2006009373A1
Application number: PCT/KR2005/002288
Authority: WO
Inventors: Yong Nam Han; Jong Hoon Ryu
Original assignee: Seoul National University Industry Foundation
Priority date: 2004-07-22
Filing date: 2005-07-16
Publication date: 2006-01-26
Also published as: KR100623164B1; KR20060007861A

Abstract

The present invention relates to a novel purified leaf extract of Ginkgo biloba (GBB extract) having neuronal cell protecting activity prepared by inventive method for preparing them and the composition comprising the same having neuronal cell protecting activity for preventing and treating degenerative brain disease. The inventive method of the present invention can provide simpler and cheaper preparation than the conventional preparation method and the inventive extract prepared by the above described inventive method has more safe and effective treating efficacy than the ginkgo leaf extract prepared by the conventional extract by removing unfavorable ingredients which inhibits TF involved in blood coagulation system or shows side effect and increasing advantageous components such as terpene and biflavone showing potent neuro-protective activity. Ac¬ cordingly, it can be used as the therapeutics for treating and preventing neuro-degenerative brain diseases such as stroke, cerebral concussion, Huntington's disease, Creutzfeld- Jakob disease, AIz heimer's disease (AD), Parkinson's disease (PD), senile dementia and the like.

Description

THE PURIFIED LEAF EXTRACT OF GINKGO BILOBA

HAVING NEURONAL CELL-PROTECTING ACTIVITY, THE

METHOD FOR PREPARING THEM AND THE COMPOSITION

COMPRISING THE SAME Technical Field

[1] The present invention relates to a purified leaf extract of ginkgo biloba having neuronal cell protecting activity, the method for preparing them and the composition comprising the same having neuronal cell protecting activity for preventing and treating degenerative brain disease.

[2]

Background Art

[3] In the twentieth century, as the average life span of human has been increasing with the rapid development of life science and medicine, new social problems including increased population ratio of older people are coming to the front, especially, the de¬ generative neuronal diseases such as stroke, Alzheimer's disease (AD), Parkinson's disease (PD) etc., which are fatal functional disorder of neuronal system, have been increased.

[4] Cerebrovascular disease is classified into two types, hemorrhagic brain disease and ischemic brain disease: hemorrhagic brain disease such as cerebral hemorrhage occurs mainly by some traffic accidents and the ischemic brain disease frequently occurring in older people is caused by the occlusion of cerebral vessels.

[5] In case that temporary cerebral ischemia occurs, the supply of oxygen and glucose to brain is prevented and several syndrome such as ATP decrease and edema follows, which causes exclusive range of brain injury as the result. After the considerable time lapses, the apoptosis of neuronal cell occurs, which is called as delayed neuronal death. It has been reported that the neuronal cell death is caused by two mechanisms; one is excitation neuronal cell death mechanism; cerebral ischemia causes to excessive ac¬ cumulation of outer glutamate and those glutamate are influxed into inner cell which causes to neuronal cell death by excessive accumulation of intracellular calcium ion.

[6] On the base of those mechanism study, there have been endeavored to develop ef¬ fectively inhibiting substance of neuronal cell death or the mechanism thereof till now, however, the effective and satisfactory inhibitors of neuronal cell death have been nearly not yet found.

[7] Some of treating agent for cerebral ischemia, for example, NMDA (N-methyl-D-aspartate) glutamate receptor inhibitor, calcium ion channel blocker, protein kinase C inhibitor etc. have been developed, however those agents have been abandoned because of its adverse action in clinical trial.

[8] Recently, sodium channel blocker, NOS (Nitric Oxide Synthase) inhibitor and various kinds of neurotrophic factors have been reported to show neuronal cell protecting effect (Keyser J. D. et al, Trends NeuroscL, 22, pp535-540, 1999).

[9] The administration of treating agents showing various action mechanisms other than those showing simple mechanism or the co-administration of both treating agents having different mechanisms between each other might be more effective in treating brain stroke caused by various factors.

[10] There have been reported that conventionally available ginkgo products containing ginkgo extract have various pharmacological effects on lots of diseases, for example, depression, the injury of memory and concentration involved in brain disease, stoke, the brain injury caused by trauma, macular degeneration, dementia, anoxia, mental disease, and other mild CNS disease as well as anti-platelet activity, blood circulation action, free radical scavenging activity, however the potency of previous known ginkgo products has been reported to be too weak for applying in clinical use (Elovic E. P., et al., Journal of Head Trauma Rehabilitation, 16(6). pp603-607, 2001).

[H]

[12] The main ingredients of ginkgo leaves showing pharmacological activity are flavonoids, for example, flavonol glycosides classified into three categories, i.e., the glycosides having kaempferol, quercetin, and isorhamnetin aglycone and biflavonoids such as sciadopitysin, ginkgetin, isoginkgetin, bilobetin, amentoflavone etc; terpenes such as ginkgolide A, B, C, J and bilobalide; proanthocyanidin; and alkyl phenols known to cause allergic response.

[13] It has been reported that conventionally available ginkgo products containing ginkgo extract could be divided into two kinds of products, i.e., one standardized by containing 14% flavonol glycoside, 7% proanthocyanidin tannin, 6% terpene and mis¬ cellaneous biflavonoid and another standardized by containing 25% flavonol glycoside and 6% terpene (Maclennan K. M., et al., Progress in Neurobiology, 67, pp235-257, 2002).

[14]

[15] In the such aspect, there have been needed to re-evaluate pharmacologically active ingredient in ginkgo leaves since prior cited reference disclosed that the intravenous administration of conventionally available ginkgo products containing ginkgo extract in an amount of 200 mg/kg could not afford satisfactory reducing effect on the cerebral infarction lesion while high amount of bilobalide, ginkgolide A and B, i.e., each 10, 50 and 100mg/kg showed reducing effect on cerebral infarction in ischemia induced ex- perimental animal model (Krieglstein J., et al., European J. Pharmaceutical Sicences, 3, pp39-48, 1995). Accordingly, there have been still needed to develop more effective ginkgo leave product in treating or preventing cerebral infarction and ischemia.

[16] Tissue Factor (TF) such as a blood coagulation factor III (F III) existed in blood en¬ dothelial membrane is enters blood vessel when blood vessel is damaged or human body is stressed or suffered from various diseases such as cancer, diabetes, athe- riosclerosis etc. And it is bound to blood coagulation factor VII (FVII) to initiate the extrinsic pathway of blood coagulation system. Furthermore, it is bound to blood co¬ agulation factor IX (FIX) to initiate the intrinsic pathway of blood coagulation system, which causes blood coagulation involved in hemostatic reaction (Kelly, R. F., Tissue factor (thromboplastin), Wiley Encyclopedia of Molecular Medicine, 5, pp3181-3185, 2002).

[17] Accordingly, present inventors have studied to develop new simple method for de¬ termining the activity of tissue factor to screen and search new anti- agglutinating entity from various natural resources (Han Y. N., et al., Arch. Pharm. Res., 21, pp549-554, 1998) and found various TF inhibitors from Chinese quince (Chaenomes sinensis (THUNB) Koehne) (Lee M. H., et al., Arch. Pharm. Res., 25, pp842-850, 2002; Lee M. H., et al., Planta Medica, 69, pp327-331, 2003).

[18]

[19] However, there has not beenreported or disclosed on a purified leaf extract of ginkgo biloba having neuronal cell protecting activity, the new method for preparing the same in any of above cited literatures, the disclosures of which are incorporated herein by reference.

[20] To investigate an effect of a purified leaf extract of Ginkgo biloba prepared by inventive method of the present invention on neuronal cell death through several biochemical experiments, the inventors of the present invention have intensively carried out several experiments such as in vitro test determining inhibiting effect on tissue factor activity and in vivo animal test using MCAO rat model and finally completed present invention by confirming that the purified leaf extract of Ginkgo biloba prepared by inventive method did not inhibit tissue factor activity and shows potent neuronal cell protective activity.

[21] These and other objects of the present invention will become apparent from the detailed disclosure of the present invention provided hereinafter.

[22]

Disclosure of Invention Technical Problem

[23] The present invention provides novel purified leaf extract of Ginkgo biloba having neuronal cell protecting activity prepared by inventive method for preparing them and the composition comprising the same having neuronal cell protecting activity for preventing and treating degenerative brain disease.

[24] The present invention also provides a use of novel purified leaf extract of ginkgo biloba prepared by inventive method for the manufacture of the medicament to treat and prevent degenerative brain disease by protecting neuronal cell in mammal or human in need thereof.

[25]

Technical Solution

[26] Accordingly, it is an object of the present invention to provide novel purified leaf extract of ginkgo biloba prepared by the steps consisting of: extracting dried powder type ginkgo leave with sole organic solvent selected from methanol, ethanol and acetone or the solvent mixture mixed with water, preferably, extracting with reflux dis¬ tillation with mixed solvent with a mixed ratio ranging from 30 to 60 % (v/v) at the temperature ranging from 90 to 100⁰C for the period ranging from 1 to 12 hours or extracting with percolation method for the period ranging from 1 to 7 days to obtain crude extract; adding hexane thereto to obtain their residue removed hexane- soluble layer and adding ethylacetate to remaining residue to obtain ethylacetate soluble layer with collecting ethylacetate soluble layer by washing said layer with distilled water to concentrate remaining ethylacetate soluble layer; adding ethanol thereto to be left alone at room temperature for the period ranging from 3 to 24 hours, filtering to obtain their filtrate; concentrating said filtrate and drying to obtain purposed purified leaf extract of ginkgo biloba showing potent neuronal cell protective activity without un¬ favorable ingredients (Hereinafter which is called as "GBB" herein).

[27]

[28] The term "GBB extract" disclosed herein is characterized in comprising increased pharmacologically favorable components showing potent neuronal cell protective activity without unfavorable ingredients which has no neuronal cell protective activity, specifically, exemplary favorable components showing potent neuronal cell protective activity are about 15% terpene compound consisting of about 11% ginkgolides such as ginkgolide A, B, C and J and about 4% bilobalide; about 4.5% biflavonoid such as sci- adopitisin, ginkgetin, isoginkgetin, bilobetin, amentoflavone etc.; exemplary un¬ favorable components which has no neuronal cell protective activity are less than 8% flavonol glycoside as an ingredient having no neuronal cell protective activity and less than 5ppm of unfavorable component.

[29] Additionally, the term "GBB extract" disclosed herein is characterized in that the extract substantially removed unfavorable components such as proanthocyanidine having prolonging bleeding time caused by inhibiting TF involved in blood co¬ agulation system, alkyl phthalic acid which prolongs bleeding time as well as gives rise to diarrhea, and flavonol glycoside having little neuronal cell-protecting activity in brain.

[30]

[31]

[32] An inventive extracts isolated from ginkgo leaf may be prepared in accordance with the following preferred embodiment.

[33]

[34] Hereinafter, the present invention is described in detail.

[35]

[36] The inventive ginkgo leaf extract can be prepared as follows;

[37] At the 1^st step, dried ginkgo leaf is sliced, mixed and extracted with 1 to 20-fold, preferably, approximately 2 to 10 fold volume of distilled water, lower alcohols such as methanol, ethanol, butanol, acetone and the like, or the mixtures thereof, preferably mixed solvent with water and other solvent such as methanol, ethanol or acetone with the mixed ratio ranging from 1:0.1 to 1:10, more preferably, from 1:0.2 to 1:3 in the temperature ranging from 20 to 120⁰C, preferably from 90 to 100⁰C for the period ranging from 1 hour to four days, preferably from 2 hours to three days with con¬ ventional extraction method by stirring extraction with water, for example, hot-water extraction, percolation extraction, reflux extraction, or ultra- sonication extraction, preferably reflux extraction or percolating method, with 1 to 5 times, preferably 2 to 4 times to obtain the crude extract of ginkgo leaf;

[38] At the 2ⁿ step, adding equivalent volume of hexane or heptane solvent to said crude extract prepared in step 1, extracting and fractionating repeatedly said extract to obtain polar lower layer containing the 1^st purified extract removed upper layer containing abundant unfavorable component such as alkyl phthalic acid (Hereinafter which is called as "Fr. H" herein);

[39] At the 3^r step, adding about 1 to 100, preferably 10 to 20 fold volume of ethylacetate solvent to said 2ⁿ purified extract prepared in step 2, extracting and frac¬ tionating repeatedly said extract to collect ethylacetate- soluble layer containing the 2nd purified extract;

[40] At the 4^th step, adding about 1 to 1/100, preferably 1/10 to 1/5 volume of distilled water to said 2ⁿ purified extract prepared in step 3, extracting and fractionating repeatedly said extract to obtain upper layer containing the 3^r purified extract removed lower layer mainly containing water-soluble fraction (Hereinafter which is called as "Fr. W" herein);

[41] At the 5 step, adding about 1 to 100, preferably 10 to 20 fold volume of ethanol solvent to said 3^r purified extract prepared in step 4, subjecting the solution to being left alone at R. T. for the period ranging from 3 to 14 hrs, preferably, 6 to 12 hrs, filtering said solution to remove residual fraction containing abundant unnecessary component for example, the proanthocyanidin-polysaccharide complex (Hereinafter which is called as "Fr. P" herein), flavonol glycoside (Hereinafter which is called as "Fr. C" herein), and other unfavorable component, subjecting to lyophilzation to obtain dried purified ginkgo leaf extract (Hereinafter which is called as "GBB extract" herein), which contains abundant amount of neuro-protective terpene (Hereinafter which is called as "Fr. A" herein), and biflavonoid components (Hereinafter which is called as "Fr. B" herein) showing potent neuronal cell protective activity.

[42] Accordingly, the present invention also provide above described purifying method for preparing inventive GBB extract of the present invention showing potent neuronal cell protective activity.

[43] It is an object of the present invention also provides novel method for preparing novel purified leaf extract of ginkgo biloba prepared by the procedure consisting of the steps: extracting dried powder type ginkgo leave with sole organic solvent selected from methanol, ethanol and acetone or the solvent mixture mixed with water, preferably, extracting with reflux distillation with mixed solvent with a mixed ratio ranging from 30 to 60 % (v/v) at the temperature ranging from 90 to 100⁰C for the period ranging from 1 to 12 hours or extracting with percolation method for the period ranging from 1 to 7 days to obtain crude extract; adding hexane thereto to obtain their residue removed hexane-soluble layer and adding ethylacetate to remaining residue to obtain ethylacetate soluble layer with collecting ethylacetate soluble layer by washing said layer with distilled water to concentrate remaining ethylacetate soluble layer; adding ethanol thereto to be left alone at room temperature for the period ranging from 3 to 24 hours, filtering to obtain their filtrate; concentrating said filtrate and drying to obtain purposed purified leaf extract of ginkgo biloba showing potent neuronal cell protective activity without unfavorable ingredients.

[44] The method for purifying ginkgo extract disclosed herein can provide various advantages over the conventional method: it provides simpler and cheaper preparation than the conventional preparation method and the inventive extract prepared by above described inventive method has more safe and effective treating efficacy than the ginkgo leaf extract prepared by the conventional extract by removing unfavorable in¬ gredients which inhibits TF involved in blood coagulation system or shows side effect and increasing advantageous components such as terpene and biflavone showing potent neuro-protective activity.

[45] The present invention provides a GBB extract prepared by above -described step showing potent neuronal cell protective activity. [46]

[47]

[48] It is an object of the present invention to provide a pharmaceutical composition comprising GBB extract as an active ingredient for the treatment and prevention of brain degenerative disease caused by neuronal apoptosis.

[49]

[50] It is an object of the present invention to provide a use of GBB extract for the manufacture of the medicament to treat and prevent degenerative brain disease by protecting neuronal cell in mammal or human in need thereof.

[51]

[52] It is an object of the present invention to provide a method of treating or preventing degenerative brain disease by protecting neuronal cell in a mammal comprising ad¬ ministering to said mammal an effective amount of GBB extract, together with a phar¬ maceutically acceptable carrier thereof.

[53]

[54]

[55] Above described degenerative brain disease comprises stroke, cerebral concussion,

Huntington's disease, Creutzf eld- Jakob disease, Alzheimer's disease (AD), Parkinson's disease (PD), senile dementia and the like.

[56]

[57] The pharmaceutical composition of the present invention can contain about 0.01 ~

50 % by weight of the above extract based on the total weight of the composition.

[58]

[59]

[60] Present inventors have performed several experiments such as in vitro test de termining inhibiting effect on tissue factor (TF) involved in blood coagulation and in vivo animal test using middle cerebral artery occlusion (MCAO) and finally confirmed that the GBB extract inhibit the neuronal cell death and show potent preventing activity of the neuronal cell injury caused by brain ischemia.

[61]

[62] In accordance with another aspect of the present invention, there is provided a phar¬ maceutical composition comprising GBB extract prepared by above preparation method for the treatment and prevention of degenerative brain disease by protecting neuronal cell as active ingredients.

[63]

[64] It is another of the present invention to provide a treating method and preventing method comprising administering a pharmaceutical composition comprising GBB extract prepared by above preparation method to degenerative brain of mammals including human.

[65]

[66] The inventive composition for treating and preventing degenerative brain disease by protecting neuronal cell may comprise above extracts as 0.001 ~ 50 % by weight based on the total weight of the composition. If the amount of the extract is less than 0.001 % (w/w), over dosing administration may be required to obtain effective efficacy and if the amount of the extract is more than 50 % (w/w), it is not economical since the efficacy of abundant extract may be equal to that of lesser amount of extract. However, it is preferable that the amount of extract is controlled according to the using method and the using purpose of the composition.

[67]

[68] The inventive composition may additionally comprise conventional carrier, adjuvants or diluents in accordance with a using method well known in the art. It is preferable that said carrier is used as appropriate substance according to the usage and application method, but it is not limited. Appropriate diluents are listed in the written text of Remington's Pharmaceutical Science (Mack Publishing co, Easton PA).

[69]

[70] Hereinafter, the following formulation methods and excipients are merely exemplary and in no way limit the invention.

[71]

[72] The composition according to the present invention can be provided as a phar¬ maceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.

[73]

[74] For example, the compositions of the present invention can be dissolved in oils, propylene glycol or other solvents that are commonly used to produce an injection. Suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them. For topical administration, the extract of the present invention can be formulated in the form of ointments and creams. [75]

[76] Pharmaceutical formulations containing present composition may be prepared in any form, such as oral dosage form (granule, powder, tablet, capsule, suspension, emulsion, syrup, aerosol spray, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol, plasters and the like), or injectable preparation (solution, suspension, emulsion).

[77]

[78] The composition of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.

[79]

[80] The desirable dose of the inventive extract or composition varies depending on the condition and the weight of the subject, severity, drug form, route and period of admin¬ istration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging from O.Olmg /kg to 500mg /kg per day, by weight/day of the inventive extract or compounds of the present invention. The dose may be administered in single or divided into several times per day. In terms of composition, the amount of inventive extract should be present between 0.01 to 50% by weight, preferably 0.5 to 40% by weight based on the total weight of the composition.

[81]

[82] The pharmaceutical composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intra¬ cutaneous, intrathecal, epidural or intracerebroventricular injection.

[83]

[84] Inventive extract of the present invention have no toxicity and adverse effect therefore; they can be used with safety.

[85]

[86]

Advantageous Effects

[87] The inventive method of the present invention can provide simpler and cheaper preparation than the conventional preparation method and the inventive extract prepared by the above described inventive method has more safe and effective treating efficacy than the ginkgo leaf extract prepared by the conventional extract by removing unfavorable ingredients which inhibits TF involved in blood coagulation system or shows side effect and increasing advantageous components such as terpene and biflavone showing potent neuro-protective activity. Accordingly, it can be used as the therapeutics for treating and preventing neuro-degenerative brain diseases such as stroke, cerebral concussion, Huntington's disease, Creutzfeld-Jakob disease, Alzheimer's disease (AD), Parkinson's disease (PD), senile dementia and the like.

[88]

Best Mode for Carrying Out the Invention

[89] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[90] The present invention is more specifically explained by the following examples.

However, it should be understood that the present invention is not limited to these examples in any manner.

[91]

Mode for the Invention

[92] The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

[93] Example 1. Preparation of 1 ^st purified extract of ginkgo leaf

[94] 1-1. the 1 ^~purified extract from ethanol soluble extract

[95] 3kg of dried and powdered ginkgo leaf (Ginkgo biloba L.) harvested from on

September to November 2001 at South Korea was mixed with 30 L of 45% (v/v) ethanol solution and subjected to reflux extraction in the temperature ranging from 90 to 100 ⁰C in the period ranging from 2 to 5 hours. Resulting extract was cooled at R. T. and subjected to filtration using by filter paper to collect their filtrate. Above procedure was repeated again. Residual ginkgo leaf was washed with 15 L of 45% (v/v) ethanol solution and the washed fraction was added to the filtrate. 6OL of hexane was added thereto and subjected to extraction three times. The lower layer of 45% EtOH layer was concentrated in vacuum at below 55 ⁰C to the extent that the weight of concentrate was about 3kg, wherein concentration of EtOH in the concentration was less than 5% (v/v).

[96]

[97] 1-2. the 1 ^~purified extract from acetone soluble extract

[98] 3kg of dried and powdered ginkgo leave (Ginkgo biloba L.) prepared in Example

1-1 was subjected to percolation extraction, i.e., 12L of acetone was added thereto and the solution was left alone at R. T. for three days. Three days after, the extract was eluted and 8L of acetone was added to remaining dried leave. The elution was performed to the extent that the volume of elution was 8L and percolation extraction was performed for 2 days. Two days after, the extract was eluted and 8L of acetone was added to remaining dried leave again. The elution was performed to the extent that the volume of elution was to be 8L and percolation extraction was performed for 2 days twice. The collected elution was filtrated and the filtrate was concentrated in vacuuo with removing acetone solvent to obtain 654g of acetone soluble extract. The extract was dissolved in 3 L of ethanol and 6L of distilled water was added dropwisely and diluted to the extent that the alcohol concentration was reached to 33%. The diluted solution was left alone at the temperature ranging from 10 to 15°C for 24 hours and the solution was filtrated to obtain their filtrate. 9L of hexane was added thereto and the solution was extracted with 9L of hexane two times. The remaining 33% ethanol layer was concentrated with vacuum at below 55°C to the extent the weight of concentrate was to about 2kg, wherein the concentration of ethanol in concentration was to be below 5% (v/v).

[99]

[100] Example 2. Preparation of GBB purified extract of ginkgo leaf

[101]

[102] To obtain ethylacetate soluble extract from the above concentrate in Example 1, the concentrate was cooled at R. T. and extracted with 3L of ethylacetate three times. The ethyl acetate layer was collected and washed with 0.9-1.8 L of distilled water. The isolated ethylacetate soluble layer was concentrated to collect. About 1 L of ethanol was added thereto and the solution was left alone at R. T. for over 12 hours to be pre¬ cipitation. The solution was filtrated to obtain their filtrate. The filtrate was con¬ centrated and dried to obtain about 54 to 66g of ethylacetate soluble purified ginkgo extract, which is called as GBB extract hereinafter.

[103]

[104] Comparative Example 1. Conventional preparation of ginkgo leaf extract (1)

[105]

[106] Respective 2L of 67%(v/v) acetone in water, 100% methanol, 100% acetone and

45(v/v%) ethanol was added to lOOg of dried ginkgo leaf prepared in Example 1 and the solution was subjected to percolation extraction for three days. Each extracted solution was filtrate with filter paper and each filtered was concentrated in vacuum at 50⁰C to obtain 36.7g of 67%(v/v) acetone-soluble extract, 23.3g of 100% methanol- soluble extract, 21.8g of 100% acetone- soluble extract and 26.2g of 45(v/v%) ethanol- soluble extract respectively.

[107]

[108] Comparative Example 2. Conventional preparation of ginkgo leaf extract (2) [109]

[110] Step 1. 67%fv/v) acetone soluble extract

[111] 6OL of 67%(v/v) acetone in water was added to 6.0kg of dried ginkgo leaf prepared in Example 1 and the solution was subjected to percolation extraction at R. T. for three days. The extracted solution was filtrated with filter paper and each filtrate was con¬ centrated in vacuum at below 50⁰C.

[112]

[113] Step 2. Preparation of Fr. H and Hl

[114] The concentrate prepared in step 1 was cooled at R. T. and extracted with 6 L of chloroform three times. The residue (Fr. Hl) was used in following step 4 and the chloroform layer was collected, concentrated to obtain 354g of chloroform extract. The chloroform extract was dissolved in 3 L of 90% (v/v) methanol solvent and extracted with 3 L of hexane three times. The collected hexane layer was concentrated in vacuum at below 50⁰C to obtain 209g of hexane soluble layer (Fr. H).

[115]

[116] Step 3. Preparation of Fr. A and Al

[117] The remaining 90%(v/v) methanol soluble layer prepared in step 2 was diluted with

6 L of distilled water to be 30%(v/v) methanol solution. The solution was left alone at R. T. for overnight and subjected to centrifugation to isolate their precipitate fraction (Fr. Al). The supernatant was extracted with 9 L of ethylacetate at three times and the ethylacetate soluble layer was collected, concentrated in vacuum to obtain 150g of ethylacetate soluble fraction (Fr. A).

[118]

[119] Step 4. Preparation of Fr. B and Bl

[120] The Fr. Hl and Fr. Al prepared in Step 2 and 3 respectively were mixed and suspended with water. The suspension was extracted with 6 L of ethylacetate at three times and the ethylacetate layer was collected, concentrated in vacuum at below 50⁰C to obtain 82g of ethylacetate soluble fraction (Fr. B) and the residue fraction (Fr. Bl) was used in following step 5.

[121]

[122] Step 5. Preparation of Fr. C and Cl

[123] The Fr. B 1 prepared in Step 4 was extracted with 6 L of butanol three times and the upper butanol layer was collected, concentrated in vacuum at below 60⁰C to obtain 71Og of butanol soluble fraction (Fr. C). The remaining water fraction (Fr. Cl) was used in following step 6.

[124]

[125] Step 6. Preparation of Fr. P and Pl

[126] The middle layer between butanol layer and water layer prepared in Step 5 was collected, concentrated, and subjected to lyophilization to obtain 57g of dark reddish- brown powder fraction (Fr. P). The remaining lower water layer fraction (Fr. Pl) was used in following step 7.

[127]

[128] Step 7. Preparation of Fr. W

[129] The water layer prepared in Step 6 was collected and concentrated in vacuum at below 60⁰C to obtain 62Og of remaining fraction (Fr. W).

[130]

[131] Experimental Example 1. Identification of active ingredients in GBB extract

[132]

[133] To identify the active ingredients in GBB extract prepared in Example 2, following analytic procedures were performed:

[134]

[135] 1-1. Determination of alkylphtalic acid content.

[136] Ig of GBB extract prepared in Example 2 was dissolved in 5ml of EtOH and heptane solvent was added thereto to use as a test solution adjusted to 25ml. Ap¬ propriate amount of alkyl phthalic acid was dissolve in heptane to be various con¬ centrations of standard solution, i.e., 3, 4, 5, 6, 7 microgram/25ml respectively.

[137]

[138] Each 5 microliter of standard solution and test solution was loaded onto silica gel plate and subjected to chromatography using a developing solvent (Hexane: Ethylacetate: Acetic acid= 40:10:1). The spot of alkyl phthalic acid showed pink flu¬ orescence (R =0.38) in UV light (254nm). The size and color concentration of resulting spots were analyzed and compared.

[139]

[140] 1-2. Quantitative analysis of gingkolide A. B. C and J content.

[141] The qualitative analysis for identifying the amount of ginkgolide was performed by the procedure disclosed in the procedure (Tang C, et al, /. Pharm. Biomed., Anal., 33, pp811-817, 2003).

[142]

[143] 1-3. Quantitative analysis of biflavonoids content.

[144] The qualitative analysis for identifying the amount of biflavonoids was performed by the procedure disclosed in the procedure (Chang S. K. et al, Kor. J. Pharmacogn., 24m. pp54-57, 1993).

[145]

[146] 1-4 Quantitative analysis of flavonol glycoside content.

[147] The qualitative analysis for identifying the amount of flavonol glycosides was performed by the procedure disclosed in the procedure (Kang G. S. et al, Kor. J. Pharmacogn., 2A(I). pp47-53, 1993).

[148]

[149] 1-5 Quantitative analysis of amino acid content.

[150] The qualitative analysis for identifying the amount of amino acid was performed using by commercial amino acid analyzer (Hewlett Packard 1100 Series, column: Waters Symmetry C18 (4.6x250mm, 5micrometer)).

[151]

[152] 1-6 Quantitative analysis of proanthocvanidin content.

[153] The qualitative analysis for identifying the amount of proanthocyanidin was performed by the procedure disclosed in the procedure (Broadhust R. B., et al, /. ScL Fd. Agric, 29, pp788-794, 1978).

[154]

[155] At the result, it has been confirmed that the "GBB extract" prepared in Example 2 contains less than 5ppm of alkylphthalic acid showing adverse reaction, 11.0+2.0% gingkolides such as ginkgolide A, B, C and J, 4.0+2.0% bilobalide, 15.0+3.0% terpene, 4.5+1.5% biflavonoids such as sciadopitisin, ginkgetin, isoginkgetin, bilobetin, and amentoflavone, less than 8% flavonol glycoside having coumaroyl group and sub¬ stantially does not contain proanthocyanidin.

[156] On the other hand, the 67% acetone soluble ginkgo leaf extract (Fr. A+Fr.C) prepared by Conventional preparation method (Comparative Example 2) showed quite different ingredient spectra from the GBB extract prepared by the inventive preparation method, for example, the former extract and the latter extract contain 20-30% and less than 8% flavonol glycoside; about 10% and little proanthocyanidin; about 6% and 15% terpene; little and 4.5% biflavone respectively. Additionally, whereas the former extract contains significant amount of amino acid, especially arginine, proline or alanine, the latter contains little amino acid. Furthermore, the former extract is prepared by the procedure consisting of 15 steps however the latter extract is prepared by the procedure consisting of only 4 5 steps (Maclennan K. M., Progress in Neurobiology, 67, pp235-257, 2002). Accordingly, it is confirmed that the latter method is more efficient and economic than the former method.

[157]

[158] Experimental Example 2. Identification of tissue factor inhibiting ingredients

[159]

[160] To identify the tissue factor inhibiting ingredients in the extract prepared in

Comparative Example 2, various phytochemical analysis were performed:

[161] Through the experiment to identify active ingredients to inhibit tissue factor, it is confirmed that the alkyl phthalic acid of following Chemical Formulae 1 and methyl alkyl phthalate of following Chemical Formulae 2 showed potent inhibiting activity of tissue factor. [162] [163] Chemistry Figure 1

[164] [165] Pale yellow powder, [166] m.p: about 40 ⁰C, [167] TLC Rf= 0.38 in UV light (254nm) (Hexane: Ethylacetate: Acetic acid= 40:10:1), [168] UVλmax(ε,hexane):247.7(18,070),319.6(10,030), [169] IRV max(KBr,cm^"1): 3009,3000, 1647, 1609, 1449, 1302, 1246, 1211, 822,707, [170] ¹H-NMR (500MHz, CDCl ): 0.82 (3H, t, /=7.1Hz, H' 15), 1.51 (2H, quintet, / =7.5Hz, H' 14), 2.90 (2H, t, /=7.5Hz, H' 1), 5.27 (2H, m, H' 8 and H' 9), 6.68 (IH, dd, /= 7.4, 1.2 Hz, H-6), 6.78 (IH, dd, /= 8.2, 1.2 Hz, H-4), 7.25 (IH, dd, /= 7.4, 8.2 Hz, H-5), l l.O (br.s),

[171] 13

C-NMR (75MHz, CDCl ) δ: See Table 1, [172] EI-MS m/z (%): 374 (20, M⁺), 356(22, M⁺-H O), 346(5, M⁺-CO), 338 (13, 356- H O), 328 (6, 356-CO), 152 (100).

[173] [174] ChemistryFigure 2

[175] [176] UVλmax(ε,hexane):245.7(12,010),315.4(7,220), [177] IRV max(KBr,cm¹): 3059, 1736, 1667, 1609, 1449, 1315, 1250, 1211, 816,709, [178] ¹H-NMR (300MHz, CDCl ): 0.89 (3H, t, /= 6.9Hz, H' 15), 1.53 (2H, quintet, / =7.5Hz, H' 14), 2.87 (2H, t, /=7.5Hz, H' 1), 3.96 (3H, s, OCH ), 5.35 (2H, m, H' 8 and H' 9), 6.72 (IH, dd, /= 7.5, 1.2 Hz, H-6), 6.84 (IH, dd, /= 8.4, 1.2 Hz, H-4), 7.28 (IH, dd, /= 7.5, 8.4 Hz, H-5), 11.1 (IH, s. COOH),

[179] ¹³C-NMR (75MHz, CDCl ) δ: See Table 1, [180] EI-MS m/z (%): 388 (49, M⁺), 360(31, M⁺-H₂O), 334(69), 328(21), 166 (100). [181] [182] Table 1

[183] [184] Experimental Example 3. Determination of tissue factor inhibiting activity [185] [186] To identify the tissue factor inhibiting ingredients in the extract prepared in Comparative Example 2, following experiment was performed by determining prothrombin time disclosed in the literature (Han Y. N., et al., Arch. Pharm, Res., 2K51 pp549-554, 1998).

[187] [188] 3-1. Isolation of tissue factor [189] 20ml of physiological saline solution was added to 5g of brain and lung tissue isolated from Sprague-dawley male mice. The tissue was macerated for 1 min, centrifuged at 60⁰C for 20 mins (2000 rpm) and the supernatant was further subjected to ultra-centrifugation at 4°C for 1 hour (105,000 x g). The precipitate was suspended with 5ml of physiological saline solution, which was kept at -15°C before use as a tissue factor crude solution.

[190]

[191] 3-2. Determination of tissue factor activity

[192] 100 micro-liter of tissue factor crude solution and 100 micro-liter of 0.313% blood plasma sodium citrate were added to plastic test tube dipped in water bath maintaining 4°C and mixed together to determine the plasma coagulation time (prothrombin time). The determination was repeated again to calculate the mean value of prothrombin times. The blood clotting stimulating activity (%) according to various concentrations of tissue factor was calculated in the basis that the tissue factor activity at the time at 18 sec. for lung tissue and 30 sec. for brain tissue. The concentration of tissue factor showing 50% blood clotting stimulating activity (%) was set to unit 1 and the con¬ centration of test sample inhibiting tissue factor by 50% was determined.

[193]

[194] 3-3. Determination of inhibiting activity for tissue factor

[195] 100 micro-liter of test sample mixture mixed diluted tissue factor crude solution and extract solution dissolved in 70% (v/v) acetone solution (0.25mM Tris buffer solution 30 % and acetone 70%) prepared by the method in the procedure (Lee M. H., Planta Medica, 69, pp327-331, 2003) with a ratio of 70:30 was used to determine the prothrombin time according to the procedure disclosed in Experimental Example 3-2 and the inhibiting percentage (%) was calculated by following Empirical Formula 1.

[196] [Empirical Formula 1]

[197] Inhibition Percentage (%)= (Au-Bu/Au) x 100

[198] Au: Tissue factor activity where only tissue factor was added without inhibitor.

[199] Bu: Tissue factor activity where both of tissue factor and inhibitor were added.

[200]

[201] At the result, 67%(v/v) acetone- soluble extract (IC = 0.51 microgram) showed most potent inhibiting activity of tissue factor among four kinds of extracts prepared in Comparative Example 1 whereas 100% acetone-soluble extract and 45(v/v%) ethanol- soluble extract showed about 1/20 activity of 67%(v/v) acetone- soluble extract. The fractions prepared in Comparative Example 2 showed inhibiting activity in following order i.e., Fr. P>Fr.H>Fr.W>Fr.C however Fr. A and Fr. B did not show any inhibiting activity of tissue factor ( See Table 2)

[202]

[203] Table 2

Tissue Factor Inhibition

[204] [205] Experimental Example 4. Determination of tissue factor inhibiting activity of isolated ingredients

[206] To identify and determine the proanthocyandin, a tissue factor inhibiting ingredient, isolated from the extract prepared in Comparative Example 2, acidic vanillin col- orimetric method disclosed in the literature (Broadhust R. B. et al., /. ScL Fd. Agric, 29, pp788-794, 1978) was used.

[207] At the result, quercetin a flavonol glycoside isolated from Fr.C containing mainly flavonol glycoside and proanthocyanidin showed mere inhibiting activity of tissue factor. The ginkgolidessuch as ginkgolide A, B, C, J, bilobalide, isolated from Fr. A containing abundant terpene and the biflavonoids such as sciadopitisin, ginkgetin, isoginkgetin, and bilobetin isolated from Fr. B did not show any inhibition effect on tissue factor ( See. Table 3)

[208] [209] Table 3 Tissue Factor Inhibition (IC )

[210] Experimental Example 5. Study on the brain tissue factor activity and bleeding time of alkyl phtalic acid, GBB extract and Fr.C

[211] [212] Through the above-described experiments, it has been found that the main inhibiting ingredient of tissue factor in ginkgo leaf is pro-anthocyanidin. Since the ginkgo leaf extract prepared by the conventional preparation method (Fr. C) contains abundant alkyl phthalic acid and proanthocyanidin, the present inventors have studied to confirm whether the Fr. C inhibits tissue factor in animal model test or not and the GBB extract of the present invention inhibits or not through following procedure:

[213] [214] Male Sprague-Dawley rats weighing 200 23Og were divided into eight groups and each group consists of six rats. At the 1st day, the experimental animals were starved providing with water only from a.m. 1:00 and the suspended feed with 1% CMC solution containing various test samples, i.e., 0.2, 0.6, 2.0mg/kg of alkyl phthalic acid treatment group, 200mg/kg of GBB extract treatment group and 0.5, 3.0, 18.0 mg/kg of Fr.C treatment group, were orally administrated into the animals twice from p.m. 5:00 to the next day a.m. 10:00 twice. Only 1% CMC instead of test sample was orally ad¬ ministrated as a control group. At that time, the half of above dosage were orally ad¬ ministrated twice at a interval of 12 hours and 2 hours after the second administration, sodium barbital was administrated with peritoneal injection in a dosage of 40mg/kg to anesthetize the rats. The tail of anesthetized rats was cut at 0.3 cm distant from tail end and then the tail was dipped in physiological saline solution at 37.5°C to determine their hemostatic time. After the determination, the rats were sacrificed and the brain of rats was delivered to determine their tissue factor activities according to the procedure disclosed in Experimental Example 3. The mean value of the determined values obtained from each test groups was expressed by ±standard deviation (*p<0.05, **p< 0.01). After the feed was administrated at second time, the animal behavior especially the number of diarrhea was observed and the number of rats showing diarrhea was counted.

[215] At the result, the GBB extract treatment group did not show any diarrhea and did not inhibit tissue factor nor prolong bleeding time whereas the Fr. C treatment group prepared by the conventional preparation method showed adverse reaction, i.e., diarrhea and inhibited tissue factor resulting in prolonging bleeding time caused by abundant alkylphthalic acid and proanthocyanidin ( See Table 4).

[216] [217] Table 4

[218] [219] Experimental Example 6. Determination of neuro-protective activity in orally administered MCAO animal model with test samples

[220] [221] To determine the effect of test samples on the brain neuronal cell, following animal experiment was performed in this experiment.

[222] [223] 6-1. Experimental animal and administration method

[224] Seven weeks old male Sprague-Dawley rats weighing 260 to 27Og were used as ex¬ perimental animals.

[225] The animals providing with free access to water and feed were acclimated with following housing condition maintaining the temperature of 23+2 ⁰C and the relative humidity of 55+10⁰C under the regularly controlled light/dark condition, i.e., light from am 7:00 to pm 7:00.

[226]

[227] 6-2. Experimental Procedure

[228] MCAO (middle cerebral artery occlusion) model test disclosed in the literature

(Nagasawa H. et al., Stroke, 20, ppl037-1043, 1989) was performed as follows.

[229] After the artery occlusion and reperfusion, ischemia has been maintained for 22 hours and the body temperature of the rats was maintained to 37+0.5⁰C using by heating pad and heat lamp to prevent the decrease of body temperature during the operation. Various concentrations of inventive GBB extract, i.e., 10, 20, 40, 80 mg/kg dissolved in physiological saline solution were orally administrated into experimental animals at 2 hours after the inducement of ischemia and reperfusion as a test group and the MCI- 186, well-known agent for acute brain stroke, was administrated in a dose of 10mg/kg as a positive control (Tanaka M., Folia Pharmacol. Japan, 119. pp301-308, 2002). Equivalent amount of physiological saline solution to above test samples was orally administrated as negative control group.

[230]

[231] 6-3. Histological Examination

[232] To evaluate the histological study of brain tissue injury, 24 hours after the MCAO, the animal brain was removed and stained with 2,3,5-triphenyltetrazolium chloride reagent. The stained brain tissue slices were dipped into 10% formalin neutral buffer solution to fix and the resulting data was stored in computer program

[233] The infarct area (mm ) was determined by computerized digital analyzer and the infarct volume (mm ) was calculated by multiplying the sum of infarct area in each slice with the width of each slices. The determined value was expressed by mean+SEM (*p<0.05, **p<0.01).

[234] As can be seen in Table 5, the infarct volume of negative control showed

350.6+20.3mm (Mean+SEM) and that of positive control showed significantly reduced to 180.3+19.0mm (Mean+SEM). Respective inventive GBB extract treatment groups with 10mg/kg, 20mg/kg, 40mg/kg and 80mg/kg also significantly reduced the infarct volume by 31.3%, 54.2%, 55.3% and 60.0% (p<0.01), respectively whereas the ginkgo leaf extract prepared by the conventional method did not show any reducing effect on the brain tissue injury at the dosage of 200 mg/kg treatment group (Krieglstein J. et al., Eur. J. Pharm. Sci., 3, pp39-48, 1995). Accordingly, it is confirmed that the inventive GBB extract has more potent reducing effects on the brain tissue injury and potent protecting activity than positive control treated with MIC- 186.

[235] [236] Table 5

[237] [238] Experimental Example 7. Determination of neuro-protective activity in in¬ travenously administered MCAO animal model with test samples

[239] [240] To determine the effect of test samples on the brain neuronal cell, the similar procedure to those disclosed in Experimental Example 6 excepting that the testing samples were administrated intravenously to experimental animals was performed to compare with orally administration test and the resulting determined values were expressed by Mean+SEM(**p<0.01).

[241] [242] As can be seen in Table 6, the inventive GBB extract treatment groups (GBB 1) with 20mg/kg intravenously showed significant neuro-protecting activity (47.9%) and the inventive GBB extract treatment groups (GBB 2) with 20mg/kg orally showed also significant neuro-protecting activity (51.6%).

[243] [244] Table 6

[245] [246] Experimental Example 8. Determination of neuro-protective activity in orally administered MCAO animal model with the ginkgo fractions, GBB extract and the combination thereof

[247] [248] To determine the neuro-protective effect of test samples on the brain neuronal cell, the similar procedure to those disclosed in Experimental Example 6 excepting that the testing samples include the ginkgo fractions prepared in Comparative Example 2, i.e., Fr. A, Fr. B. Fr. C, Fr. H and Fr. P and the combination with GBB extract ad¬ ministrated orally to experimental animals was performed and the resulting determined values were expressed by Mean+SEM (**p<0.01).

[249] As can be seen in Table 7, the test groups treated with the ginkgo fractions prepared in Comparative Example 2, i.e., Fr. A, Fr. B. Fr. C, Fr. H and Fr. P with 20mg/kg orally showed relatively lower neuro-protecting activity however the combined test group treated with 10mg/kg of the inventive GBB extract and 10mg/kg of Fr. A comprising terpene such as ginkgolide and bilobalide, or Fr. B comprising biflavonoids prepared in Comparative Example 2 showed significant preventing effect on brain tissue injury. Accordingly, it is confirmed that the combination of the inventive GBB extract and Fr. A or Fr. B showed synergistic neuro-protective activity. On the contrary, the combination of the GBB extract and the other ginkgo fractions prepared in Comparative Example 2, i.e., Fr.C, Fr.H or Fr. P comprising abundant tissue factor inhibitors showed reduced or disappeared neuro-protective activity.

[250] Table 7

[251] [252] Experimental Example 9 Determination of neuro-protective activity in MCAO animal model according to the administration time of test samples

[253] [254] To determine the therapeutic time of the inventive GBB extract on the brain tissue injury, the similar procedure to those disclosed in Experimental Example 6 was performed i.e., the 40mg/kg of the GBB extract was orally administrated at the interval of 1 and 3 hours (Gl), 2 and 4 hours (G2), 3 and 5 hours (G3) after the inducement of ischemia. 10mg/kg of MCI- 186, a well-known agent for acute brain stroke, was ad¬ ministrated as a positive control and the equivalent amount of physiological saline solution was orally administrated as a negative control group. The resulting determined values were expressed by Mean+SEM (*p<0.05, **p<0.01).

[255] As can be seen in Table 8, the GBB extract treatment groups, i.e., Gl, G2 and G3 test groups showed 59.4%, 56.1% and 49.0% neuro-protective activity respectively. Accordingly, it is confirmed that more prompt treatment of GBB extract enhances neuro-protective activity and the GBB extract treatment within 5 hours after the inducement of ischemia obtain the most effective treating activity.

[256] Table 8

[257]

[258] Experimental Example 10. Toxicity test

[259] Methods

[260] The acute toxicity tests on ICR mice (mean body weight 25+5g) and Sprague-

Dawley rats (235+1Og, Jung-Ang Lab Animal Inc.) were performed using the inventive GBB extract. Four group consisting of 10 mice or rats was administrated orally with 100, 500, 1000, and 2000mg/kg of test sample or solvents (0.2 D, i.p.) re¬ spectively and observed for 2 weeks.

[261] Result

[262] There were no treatment-related effects on mortality, clinical signs, body weight changes and gross findings in any group or either gender. These results suggested that the extract prepared in the present invention was potent and safe.

[263]

[264] Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

[265]

[266] Preparation of powder

[267] Dried powder of the GBB extract 50mg

[268] Lactose lOOmg

[269] Talc lOmg

[270] Powder preparation was prepared by mixing above components and filling sealed package.

[271]

[272] Preparation of tablet

[273] Dried powder of the GBB extract 50mg

[274] Corn Starch lOOmg

[275] Lactose lOOmg

[276] Magnesium Stearate 2mg

[277] Tablet preparation was prepared by mixing above components and entabletting.

[278] Preparation of capsule

[279] Dried powder of the GBB extract 50mg

[280] Corn starch lOOmg

[281] Lactose lOOmg

[282] Magnesium Stearate 2mg

[283] Capsule preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

[284]

[285] Preparation of injection

[286] Dried powder of the GBB extract 50mg

[287] Distilled water for injection optimum amount

[288] PH controller optimum amount

[289] Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2ml ample and sterilizing by con¬ ventional injection preparation method.

[290]

[291] Preparation of liquid

[292] Dried powder of the GBB extract 0.1 ~80g

[293] Sugar 5~ 1Og

[294] Citric acid 0.05-0.3%

[295] Caramel 0.005-0.02%

[296] Vitamin C 0.1-1%

[297] Distilled water 79-94%

[298] CO₂ gas 0.5-0.82%

[299] Liquid preparation was prepared by dissolving active component, filling all the components and sterilizing by conventional liquid preparation method.

[300]

[301] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

[302]

Industrial Applicability

[303] As described in the present invention, the inventive method of the present invention can provide simpler and cheaper preparation than the conventional preparation method and the inventive extract prepared by the above described inventive method has more safe and effective treating efficacy than the ginkgo leaf extract prepared by the con¬ ventional extract by removing unfavorable ingredients which inhibits TF involved in blood agglutination system or shows side effect and increasing advantageous components such as terpene and biflavone showing potent neuro-protective activity. Accordingly, it can be used as the therapeutics for treating and preventing neuro¬ degenerative brain diseases such as stroke, cerebral concussion, Huntington's disease, Creutzfeld- Jakob disease, Alzheimer's disease (AD), Parkinson's disease (PD), senile dementia and the like. [304] [305] [306]

Claims

[1] A novel purified leaf extract of ginkgo biloba prepared by the steps consisting of: extracting dried powder type ginkgo leave with sole organic solvent selected from methanol, ethanol and acetone or the solvent mixture mixed with water, preferably, extracting with reflux distillation with mixed solvent with a mixed ratio ranging from 30 to 60 % (v/v) at the temperature ranging from 90 to 100⁰C for the period ranging from 1 to 12 hours or extracting with percolation method for the period ranging from 1 to 7 days to obtain crude extract; adding hexane thereto to obtain their residue removed hexane-soluble layer and adding ethylacetate to remaining residue to obtain ethylacetate soluble layer with collecting ethylacetate soluble layer by washing said layer with distilled water to concentrate remaining ethylacetate soluble layer; adding ethanol thereto to be left alone at room temperature for the period ranging from 3 to 24 hours, filtering to obtain their filtrate; concentrating said filtrate and drying to obtain purposed purified leaf extract of ginkgo biloba showing potent neuronal cell protective activity without unfavorable ingredients.

[2] The extract according to claim 1, wherein said extract comprise about 15% terpene compound consisting of about 11% ginkgolides such as ginkgolide A, B, C and J and about 4% bilobalide, about 4.5% biflavonoid such as sciadopitisin, ginkgetin, isoginkgetin, bilobetin, amentoflavone etc showing potent neuronal cell protective activity; less than 8% flavonol glycoside having no neuronal cell protective activity and less than 5ppm of unfavorable component.

[3] The extract according to claim 1, wherein said extract substantially removes un¬ favorable component such as proanthocyanidine having prolonging bleeding time caused by inhibiting TF involved in blood coagulation system, alkyl phthalic acid which prolongs bleeding time as well as gives rise to diarrhea, and flavonol glycoside having little neuronal cell-protecting activity in brain.

[4] A method for preparing novel purified leaf extract of ginkgo biloba as set forth in

Claim 1 prepared by the procedure consisting of the steps: extracting dried powder type ginkgo leaf with solvent mixture mixed with water and methanol, ethanol or acetone at the temperature ranging from 90 to 100⁰C for the period ranging from 1 to 12 hours or extracting with percolation method for the period ranging from 1 to 7 days to obtain crude extract; adding hexane thereto to obtain their residue removed hexane-soluble layer and adding ethylacetate to remaining residue to obtain ethylacetate soluble layer with collecting ethylacetate soluble layer by washing said layer with distilled water to concentrate remaining ethylacetate soluble layer; adding ethanol thereto to be left alone at room temperature for the period ranging from 3 to 24 hours, filtering to obtain their filtrate; concentrating said filtrate and drying to obtain purposed purified leaf extract of ginkgo biloba showing potent neuronal cell protective activity without unfavorable ingredients.

[5] The method according to claim 4, wherein said procedure consists of the steps: at the 1^st step, dried ginkgo leaf is sliced, mixed and extracted with 2 to 10 fold volume of mixed solvent with water and other solvent such as methanol, ethanol or acetone with the mixed ratio ranging from 1:0.2 to 1:3 in the temperature ranging from 90 to 100⁰C for the period ranging from 2 hours to three days with reflux extraction or percolating method with 2 to 4 times to obtain the crude extract of ginkgo leaf; at the 2ⁿ step, adding equivalent volume of hexane or heptane solvent to said crude extract prepared in step 1, extracting and frac¬ tionating repeatedly said extract to obtain polar lower layer containing the 1^st purified extract removed upper layer containing abundant unfavorable component such as alkyl phthalic acid; at the 3^r step, adding about 10 to 20 fold volume of ethylacetate solvent to said 1^st purified extract prepared in step 2, extracting and fractionating repeatedly said extract to collect ethylacetate- soluble layer containing the 2nd purified extract; at the 4 step, adding about 1/10 to 1/5 volume of distilled water to said 2ⁿ purified extract prepared in step 3, extracting and fractionating repeatedly said extract to obtain upper layer containing the 3^r purified extract removed lower layer mainly containing water-soluble fraction; at 5 step, adding 10 to 20 fold volume of ethanol solvent to said 3^r purified extract prepared in step 4, subjecting the solution to being left alone at R. T. for the period ranging from 6 to 12 hrs, filtering said solution to remove residual fraction containing the proanthocyanidin-polysaccharide complex, flavonol glycoside, and other unfavorable component, subjecting to lyophilzation to obtain dried purified ginkgo leaf extract, which contains abundant amount of neuro-protective terpene, and biflavonoid showing potent neuronal cell protective activity.

[6] A pharmaceutical composition comprising the purified ginkgo leaf extract prepared by the method as set forth in claim 4 as an active ingredient, for the treatment and prevention of brain degenerative disease caused by neuronal apoptosis.

[7] The pharmaceutical composition according to claim 6 wherein said composition is form of granule, powder, tablet, capsule, suspension, emulsion, syrup, or aerosol spray.

[8] The pharmaceutical composition according to claim 6 wherein said composition is form of topical preparation, suppository or injection. [9] The pharmaceutical composition according to claim 6 wherein said degenerative brain disease comprises stroke, cerebral concussion, Huntington's disease, Creutzfeld- Jakob disease, Alzheimer's disease (AD), Parkinson's disease (PD) and senile dementia.