WO2006006751A1

WO2006006751A1 - A composition comprising purified extract of wild ginseng having neuronal cell protecting activity

Info

Publication number: WO2006006751A1
Application number: PCT/KR2004/001942
Authority: WO
Inventors: Yong Jin Park; In Sop Shim; Gyu Yong Song
Original assignee: Seocksanteo Medical Co., Ltd.
Priority date: 2004-07-15
Filing date: 2004-08-02
Publication date: 2006-01-19
Also published as: KR100452555B1

Abstract

The present invention relates to a use of a purified extract prepared by the method comprising the steps consisting of; subjecting the wild ginseng material to distillation extracting method with extracting solvent repeatedly; freezing the distilled extract to obtain frozen purified extract; thawing the extract and filtrating to obtain purified extract for the preparation of a pharmaceutical composition for the treatment and prevention of degenerative brain disease by protecting neuronal cell in human or mammal, and a composition comprising the same prepared by inventive method. The composition have potent neuronal cell protective activity, therefore, it can be used as the therapeutics for treating and preventing neuro-degenerative brain diseases caused by neuronal cell such as death stroke, apoplexy, dementia, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, Pick's disease and Creutzfeld-Jakob's disease and the like.

Description

A COMPOSITION COMPRISING PURIFIED EXTRACT OF WILD GINSENG HAVING NEURONAL CELL

PROTECTING ACTIVITY

Technical Field

[1] The present invention relates to a composition comprising purified extract of wild ginseng having neuronal cell protecting activity.

Background Art

[2] 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 chronic degenerative diseases have been more rapidly increased than acute infectious diseases having been main aetiology of death for 50 years.

[3] Among the chronic degenerative disease, a cerebrovascular disease to cause to death has become an important disease and ranked to the second frequent disease among lethal diseases to die due to single aetiology.

[4] Cerebrovascular disease can be classified into two types. One is a hemorrhagic brain disease mainly occurred by external impact sich as traffic accident resulting in cerebral hemorrhage and another is an ischemic brain disease mainly occurred by aging and other factors resulting in cerebrovascular occlusion.

[5] In case that temporary ischemia is occurred, the supply of oxygen and glucose are blocked to cause the decrease of ATP and edema and finally those serial phenomena give rise to extensive brain damage. The death of neuronal cells appears at con¬ siderable times after the ischemia, which is called as delayed neuronal death. Through transient forebrain ischemic model experiment using by Mongolian gerbil, it is reported that there occurred the death of neuronal cell at CAl region of hippocampus four days after the ischemia inducement (Kirino T. Sano K., Acta Neuropathol, 62 , pp201-208, 1984; Kirino T. Brain Research, 29 , pp57-69, 1982).

[6] There have been reported that the mechanism of neuronal cell death is classified into two types: one is an excitational neuronal cell death mechanism characterized that excess amount of glutamate is accumulated in outer cell after cerebral ishemia occurred and the glutamate is flowed into inner cell apoptosis to cause to neuronal cell death due to excess accumulation of calcium ion in inner cell (Kang T. C, et al., /. NeurocytoL, 30 , pp945-955, 2001); another is an oxidative neuronal cell death char- acterized that abrupt oxygen supply causes to the increase of in vivo radical resulting in damages of cytoplasm (Won M. H, et al., Brain Research, 836 , pp70-78, 1999; Sun A. Y., Chen Y. M., /. Biomed. ScL, 5 , pp401-414, 1998; Flowers F, Zimmerman J. J. New Horiz., 6 , ppl69-180.1998).

[7] There have been studied and developed to search effective substance effectively inhibiting neuronal cell death and the action mechanism of the substance till now, however, there has not yet reported on the substance to inhibit neuronal cell death ef¬ fectively. There have been several attempts to find effective agent till now. For example, t-PA (tissue Plasminogen activator), a sole FDA approved treating agent for ischemia, has thrombolytic activity which can dissolve blood thrombus to indice rapid supply of oxygen and glicose. Fbwever, it has several disadvantages such as necessity to instant use, the occurrence of hemorrhagic cerebrovascular disease caused by thinned blood vessel wall in case of excessive or frequent use of the agent. MK-801, a calcium channel blocker inhibit initial calcium influx effectively, however, the further development was postponed because of its adverse effect.

[8] Accordingly, there have been urgently needed to find effective substances providing with verified efficacy as well as low or at least toxicity from natural resource till now. Recently, alternative medicine, especially, Chinese medical therapy based on immune potentiating mechanism has been highlighted as an alternative method with conventional Western medicine to deviate the adverse effect of chemotherapy. Among the Chinese medical therapy, together with a use of plant extract extracted from Chinese drug showing immune potentiating activity and less toxicity, a use of acupuncture treatment with effective extract become highlighted in Korea, which comprises the steps consisting of: selecting plant or other natural resource having most effective activity on individual disease; extracting effective ingredient from the extract providing with maximized efficacy and minimized toxicity; inoculating or injecting the ingredient into the spots suitable for acupuncture or painful spots on the body and therefore it could endow with synergic effect due to the effect of acupuncture and the pharmacological effect of the ingredient. Fbwever, there have been needed to obtain more purified extract having less toxicity than crude form which could express its t oxicity in administrating into injection or acupuncture such as fever, pain, edema etc.

[9] There has been not reported or disclosed about a composition comprising purified extract of wild ginseng having neuronal cell protecting activity in any of above cited literatures, the disclosures of which are incorporated herein by reference.

[10] To investigate an effect of purified extract from wild ginseng prepared by the method of the present invention on the memory improvement and neuronal cell injury, the inventors of the present invention have intensively carried out several animal model experiments, and finally completed present invention by confirming that the purified extract inhibits the neuronal injury and shows neuronal cell protective activity

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

Disclosure

[12] The present invention provides a use of above extract for the preparation of phar¬ maceutical composition to treat and prevent degenerative brain disease by protecting neuronal cell in mammal or human.

[13] Accordingly, it is an object of the present invention to provide a use of purified extract prepared by the steps consisting of; subjecting the wild ginseng material to dis¬ tillation extracting method with extracting solvent repeatedly; freezing the distilled extract to obtain frozen purified extract; thawing the extract and filtrating to obtain purified extract for the preparation of a pharmaceutical composition for the treatment and prevention of degenerative brain disease by protecting neuronal cell in human or mammal.

[14] 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 or human comprising administering to said mammal or human an effective amount of a purified extract of wild ginseng prepared by above described method, together with a pharma¬ ceutically acceptable carrier thereof.

[15] The term disclosed herein 'extracting solvent' comprises water, lower alcohols such as methanol, ethanol, preferably water.

[16] The term disclosed herein 'wild ginseng material' comprises all the wild ginseng cultivated or naturally grown wild ginseng in the world, for example, Korea, Japan, Russia, China, Europe, America etc.

[17] The term disclosed herein 'degenerative brain disease' comprises stroke, apoplexy, dementia, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, Pick's disease and Creutzfeld-Jakob's disease and the like .

[18] 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.

[19] An inventive a purified extract of wild ginseng may be prepared in accordance with the following preferred embodiment. [20] Hereinafter, the present invention is described in detail.

[21] An inventive purified extract of wild ginseng can be prepared in detail by following procedures,

[22] The inventive purified extract of wild ginseng can be prepared by follows;

[23] Specifically, it is an object of the present invention to provide a method for preparing a purified extract from wild ginseng comprising the steps consisting of; subjecting the wild ginseng material to the 1 distillation extracting method with extracting solvent to obtain the I ^s distillate at the I^s step; subjecting the I^s distillate to the 2° distillation extracting method with extracting solvent to obtain the 2 ⁿ distillate at the 2ⁿ step; freezing the 2° distillate to obtain frozen purified extract at the 3 ^r step; thawing the extract and filtrating to obtain filtrated extract at the 4 step; heating the filtrate with double boiler and refreezing at the 5 step; thawing and sterilizing the extract to obtain the purified extract of the present invention at the 6 step.

[24] Specifically, At the 1 ^s step, it is preferable that the wild ginseng material is poured to polar solvent selected from water, lower alcohol sich as methanol, ethanol, propanol and the solvent mixture there of preferably, water, more preferably, water in the ratio ranging from 1.0 kg to 3.0 kg of material per liter of water and left alone at room temperature for the period ranging from 1 to 4 hours, preferably, 3 hours. Sub¬ sequently, it is heated with distillation apparatus at the temperature ranging from 60 to 120 ° C, preferably 80 to 100 ° C, more preferably, 100 ° C, for the period ranging from 6 to 24 hours, preferably, from 8 to 10 hours gradually to obtain the 1 distillate.

[25] At the 2° step, it is preferable that the I^s distillate is added to the boiling chip containing flask equipped with distillation apparatus and heated at the temperature ranging from 60 to 120 ° C, preferably 80 to 100 ° C, more preferably, 100 ° C, for the period ranging from 7 to 24 hours, preferably, from 8 to 12 hours gradually and the heating process is maintained to the extent that the volume of concentrate is reduced to be in the ranging 70 to 90(v/v)% to obtain the 2 ⁿ distillate.

[26] At the 3^r step, it is preferable that the 2° distillate is subjected to freezing process at the temperature ranging from -5 to -15 ° C, preferably from -8 to -12 ⁰ C and the process is maintained to the extent that the volume of un-frozen fraction is decreased to be less than 5% of the total volume. The un-frozen fraction is removed and the remaining distillate is subjecting to thawing process. Those freezing and removing un¬ frozen fraction processes can be repeated, preferably 1 to 6 times, to obtain frozen purified extract of the present invention. Through the 3^r step, toxic substance in wild ginseng material at the I^s step is almost or completely removed in purified extract prepared by this step.

[27] At the 4 step, it is preferable that the frozen purified extract at the 3 step is subjected to thawing process at room temperature and filtrating process is followed with filter paper having pore size ranging from 0.1 to 0.6 um to obtain filtrates.

[28] At the 5 step, the filtrate prepared in above step is heated with double boiler at the temperature ranging from 60 to 12O ⁰ C, preferably 80 to 100 ⁰ C, in the period ranging from 20 to 40 mins, preferably, 30 mins, and is refrozen completely at the temperature ranging from -30 to -1O ⁰ C, preferably, at -15 ⁰ C tO obtain frozen purified extract.

[29] At the 6 step, the frozen filtrate is thawed and sterilized to obtain final purified extract of the present invention.

[30] Alternatively, additional step i.e., maturing wild ginseng material in maturing apparatus can be subjected before the 1 step. Specifically, it is preferable that the wild ginseng material is poured to maturing apparatus being constricted with equivalent ratio of various material, for example, equivalent ratio of Kaolinite (china clay), yellow earth, tourmaline, mineral stone, jade stone, vein stone (ui-wang seok), germanium stone, seriate and selenium stone, and designed by the present inventors as shown in Hg. 1, and the entrance is closed with elvan stone. The maturing process is subject to heat treatment to mature at the temperature ranging from 50 to 100 ° C, preferably from 60 to 80 ° C, for the period ranging from 6 to 24 hours, preferably, from 7 to 9 hours gradually and drying the matured material for the period ranging 5 to 5 hours, preferably, 5 hour is followed to obtain matured wild ginseng.

[31] In a preferred embodiment of present invention, above described maturing apparatus can be constricted as follows:

[32] Maturing apparatus can be constricted with the structure characterizing in: having outer wall made from cement material; basement as a heating part; stainless steel made entrance where wild ginseng material is poured located in the upper central part: kaoline layer located in uttermost downward part; yellow earth layer located in upper side of kaoline layer; tourmaline layer located in upper side of yellow earth layer; mineral stone layer located in right-handed side of entrance; jade stone layer located in upper side of mineral stone layer; vein stone (ui-wang seok) layer located in upper side of jade stone layer; germanium stone layer located in left handed side of entrance; seriate layer located in upper side of germanium stone layer; selenium stone layer located in upper side of seriate layer and elvan stone layer using as a covering lid. st

[33] In a preferred embodiment of present invention, in above described the 1 step, the concentration of the 1 ^s distillate can be controlled by changing the ratio of material pursuant to the purpose of the present invention.

[34] In above described purification process, the extract can be separated into two parts, i.e., frozen part and unfrozen part according to the difference with specific ingredients. Since the ingredients having stronger toxicity being contained in the extract become frozen slower than those having weaker toxicity because of their low freezing point, the unfrozen part containing most of toxic ingredient can be removed by repeating the above freezing processes.

[35] Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising a purified extract of wild ginseng prepared by above described method as an active ingredient in an effective amount to treat and prevent degenerative brain disease by protecting neuronal cell.

[36] It is an object of the present invention to provide a use of a purified extract of wild ginseng prepared by above described method for the preparation of therapeutic agent for the treatment and prevention of degenerative brain disease by protecting neuronal cell in human or mammal.

[37] 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 or human comprising administering to said mammal or human an effective amount of a purified extract of wild ginseng prepared by above described method, together with a pharma¬ ceutically acceptable carrier thereof.

[38] Through the animal model experiments to confirm the effect of the purified extract of the present invention on the neuronal cell injury caused by forebrain ischemia, present inventors confirm that the purified extract shows potent protecting effect on neuronal cell injury and improving effect on learning memory disorder.

[39] Therefore, the purified extract of the present invention can be useful in treating and preventing degenerative brain disease caused by neuronal cell death.

[40] In addition to the efficacy, the purified extract of the present invention can be used safely in long-term administration since it has been used as a commercial crude drug since long years ago.

[41] The inventive composition for treating and preventing degenerative brain disease by protecting neuronal cell may comprises above extracts as 0.01 ~ 50 % by weight based on the total weight of the composition.

[42] 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 ).

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

[44] 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.

[45] Ibr 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. Ibr topical administration, the extract of the present invention can be formulated in the form of ointments and creams.

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

[47] 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.

[48] 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 ad¬ ministration, 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 10 g/kg, preferably, 1 to 3 g/kg 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.

[49] 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 intracerebro ventricular injection or acupuncture injection onto the spots suitable for acupuncture.

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

[51] 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.

Description Of Drawings

[52] The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

[53] Hg. 1 shows the result determining the correct choice number of main path in radial arm maze experiment after occurring of forebrain apparatus ischemia;

[54] Hg. 2 shows the result determining the error rate number of main path in radial arm maze experiment after occurring of forebrain apparatus ischemia;

[55] Hg. 3 represents the photograph observing the degree of neuronal cell injury at hippocampus region in brain tissue using by Cresyl violet staining method;

[56] Hg. 4 represents the graph showing the degree of neuronal cell injury at hippocampus region in brain tissue using by Cresyl violet staining method;

[57] Hg. 5 presents the AchE expression degree at CAl region of hippocampus using by AchE staining method;

[58] Hg. 6 presents the AchE expression degree at CA3 region of hippocampus using by AchE staining method;

[59] Hg. 7 depicts the ChAT expression degree at CAl region of hippocampus using by

ChAT staining method;

[60] Hg. 8 depicts the ChAT expression degree at CA3 region of hippocampus using by ChAT staining method.

Mode for Invention

[61] 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.

[62] The present invention is more specifically explained by the following examples, tbwever, it should be understood that the present invention is not limited to these examples in any manner.

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

[64] Reference Example 1. Preparation of experimental animal

[65] Aprague-dawley male rats weighing from 260 to 300g were purchased from Sahm- yook animal lab. center located in Seoul . The rats were acclimated to the experimental condition (Temperature 22 + 3⁰C, humidity: 50 + 1O⁰C, light: 12hours per day) and freely access to water and feed (Samyang oil feed Co. Ltd, Seoul , Korea ) to use in following experiments.

[66] Example 1. Preparation of the purified extract of naturally grown wild ginseng

[67] 2.5 kg of Sanyang wild ginseng originated from naturally grown wild ginseng was washed with salt water and sliced into piece at the width of 3mm, poured into flask containing 1 liter of water and left alone for three hours at 2O⁰C.

[68] The flask was equipped with distillation apparatus and heated at 90 ⁰C for eight

St St hours to obtain the 1 distillate. 5g of boiling chip and the 1 distillate were poured flask equipped with distillation apparatus and the flask was further heated at 93 ⁰C for ten hours to obtain the 2 distillate. The 2 distillate was frozen to the extent that the volume of unfrozen part become less than 5% of total volume at -10 ⁰C and the unfrozen part was discarded. Remaining frozen part was thawed at room temperature. Above freezing and thawing steps were further repeated two times. After the frozen part was thawed, it was filtered with filter paper having a pore size of 0.4 um and the filtrate was heated in double boiler at 90 ⁰C for 30 mins. The filtrate was cooled and frozen completely at -15 ⁰C. The frozen purified extract was thawed at room temperature, sterilized and stored in sterilized bottle maintaining the temperature at below 4 ⁰C to use as a sample in following experiments.

[69] Example 2. Preparation of the purified extract of cultivated wild ginseng [70] 2 kg of cultivated wild ginseng (or 400 g of dried wild ginseng) supplied with

Kyunghee University (Seoul, Korea) was washed with salt water and sliced into piece at the width of 3mm, poured into flask containing 1 liter of water and left alone for three hours at 2O⁰C.

[71] Further steps were performed with the methods similar to those in Example 1 to obtain purified extract of cultivated wild ginseng and it was used as a sample in following experiments.

[72] Comparative Example 1. Preparation of not purified extract of naturally grown wild ginseng

[73] 2.5 kg of Sanyang wild ginseng originated from naturally grown wild ginseng was washed with salt water and sliced into piece at the width of 3 mm, poured into flask containing 1 liter of water and heated at 100 ⁰C for eight. The distillate was filtered with filter paper having a pore size ranging from 0.1 to 0.6 um. The filtrate was cooled at room temperature, sterilized and stored in sterilized bottle maintaining the temperature at below 4 ⁰C to use as a sample in following experiments.

[74] Comparative Example 2. Preparation of not purified extract of cultivated wild ginseng

[75] 2 kg of cultivated wild ginseng originated from naturally grown wild ginseng was washed with salt water and sliced into piece at the width of 3 mm, poured into flask containing 1 liter of water and heated at 100 ⁰C for eight. The distillate was filtered with filter paper having a pore size ranging from 0.1 to 0.6 um. The filtrate was cooled at room temperature, sterilized and stored in sterilized bottle maintaining the temperature at below 4 ⁰C to use as a sample in following experiments.

[76] Experimental Example 1. Toxicity test of the purified extract of wild ginseng

[77] To compare the toxicity of extracts in Examples 1, 2 and Comparative Examples 2, the toxicity test using by experimental animals obtained in Reference Example 1 was performed as follows:

[78] The change of body temperature and formation of spot on the back of rats were determined as the indications of toxicity of extracts. Experimental animals were grouped into three, i.e., the groups treated with the extract prepared in Example 1, 2 and Comparative Example 2 of which group is consisted of five rats respectively.

[79] To check whether the change of body temperature and formation of spot on the back of rats administrated with each samples occurred or not, each 20 ml of extracts prepared in Example 1, 2 and Comparative Example 2 was orally administrated into rats and the body temperature of the rats and the spot formation of rats were determined and observed at 0.5, 1 and 2 hours after the administration.

[80] At the result, while the rats treated with extracts in Examples 1, 2 did not show any change in body temperature, the rats treated with extract in Comparative Example 2 showed increased body temperature. Furthermore, while the rats treated with extracts in Examples 1, 2 did not show any spot formation on the back, and the rats treated with extract in Comparative Example 2 showed several numbers of spot on the back. Those results showed that the purified extracts prepared in Examples 1, 2 have no toxicity while the unpurified extract prepared in Comparative Examples 2 has several toxicity such as the increase of body temperature and spot formation ( See Table 1).

[81] [Table 1]

[82] Experimental Example 2. Determination of Effect on the memory injury using Morris's water maze apparatus [83] To determine the effect of the extract of the present invention on the memory injury, Morris's water maze experiment was performed by modifying the procedure disclosed in the literature (Morris R, Neurosci. Method, 11: 47-60, 1984).

[84] 2-1. Preparation of animal model having memory injury [85] 1 ug of acetylcholine like 192 Saporin (ATS, San Diego, CA. USA) damaging only neuronal cell was injected into the specific region of cerebrum medial septum (AP:-0.2, L: + 0.3, H: -6.2) isolated from the r ats anesthetized with 50 mg/kg (i.p.) of sodium pentobarbital using by stereotaxic technique. The injection was performed by using perfusion pump (Pump 22, Harvard Apparatus, South Natick, MA) connected to gas -tight 1 mlof glass syringe (Hamilton, Reno, NV, USA) with polyethylene tube and the syringe was removed 5 mins after the injection at the speed of 0.2 ml/min.

[86] 2-2. Determination of the effect on memory injury [87] The experimental apparatus consisted of a circular water tank (diameter 180 cm, height 50 cm), containing water maintaining the temperature at 22 + 2 ⁰C in a depth of 30 cm. The apparatuses necessary to the experiment such as video cam, water temperature control apparatus etc were set up neighbor the water tank. Hidden platform was prepared by attaching a support to circular transparent acryl having a diameter of 12 cm and positioned at 1.5cm below the water surface. Water maze was divided into four sections i.e., northeast, northwest, southeast, southwest section. The hidden platform was positioned at the center of northeast section and one of other sections was starting point.

[88] The administration of samples were performed the day after finishing day of preparation of animal model and artificial cerebrospinal fluid containing 145mM NaCl, 2.7 mM KCl, 1.2 mM CaCl and 1.0 mM MgCl was injected as Sham. Each 0.05 CE of the control group containing only saline and sample treatment groups with the extract prepared in Example 2 and Comparative Example 1 was injected to both sides of specific spots called as JokSamri spot positioned at about 9 cm upward from malleolus by U-100 insulin gauage needle (l/2cc, Becton Dicken and company, USA) like as a acupuncture. The experimental animals were trained for six days four times a day and the swimming examination allowing the rats to free swimming without hidden platform was performed at 7 day. All the behaviors of rats were recorded by video camera and the duration time (sec) to reach hidden platform from starting platform in the experiment.

[89] At the result, while control group showed significant learning disorder syndrome, the acupuncture treatment group with the extract in Example 2 and Comparative Example 1 showed potent improving activity of learning memory. In particular, the acupuncture treatment group with the extract in Example 2 showed more potent activity than that of the treatment group with the extract in Comparative Example 1 ( See Table 2).

[90] [Table 2]

[91] Experimental Example 3. Determination of Effect on the memory injury using radial arm maze apparatus [92] To determine the effect of the extract in the present invention on the memory injury, a radial arm maze experiment was performed by modifying the procedure disclosed in the literature (Zea L. E., Stroke, 20: 84-91, 1989).

[93] 3-1. Preparation of animal model having memory injury [94] Temporary forebrain animal model occluding middle brain artery was prepared by using intraaluminal suture method as follows:

[95] The rats prepared in Reference Example 1 was orally anesthetized with 5% isoflurane gas (mixed gas with the gas mixture consisting of 70% N O and 30% O gas and 5% isoflurane) and maintained with 1% isoflurane gas during experiment.

[96] The determination probe of body temperature was inserted to the rectum of the rat and the body temperature of the rats was maintained to be 38 ⁰C using by heating lamp and heating mattress. To occlude the middle brain artery, th e skin at the center of mouse neck was excised to expose the CCA (common carotid artery) and intraluminal filament ( ?02.8 mm, rounded tip) was inserted into ICA (internal carotid artery) in order that the terminal of the tip can be reached at the proximal region of cerebral artery. CCA and ECA were ligated with ligature and ICA (Internal Carotid Artery) and pterygopalatine artery were also isolated and two hours after the occlusion of blood flow, the filament was removed right-handed CCA was ligated to allow the blood flow to reperfusion through collateral circulation.

[97] The experimental animals were divided into five groups, i.e., a Sham group as a control group (n=6), the forebrain ischemia occurring group (n=7), the acupuncture treatment group with physiological saline after occurring forebrain ischemia (n=6), and the acupuncture treatment group with the extract prepared in Example 1 after occurring forebrain ischemia (n=4) and the extract prepared in Example 2 after occurring forebrain ischemia (n=5) as sample treatment groups. The acupuncture treatment with 100mg/kg of control and sample was injected into both sides of specific spots called as JokSamri spot once within in one hour before occurring ischemia at every 10 o'clock for two weeks.

[98] 3-2. Determination of the effect on memory injury

[99] The radial maze experimental apparatus consisted of wooden box which has octagonal central platform and eight paths reached out from the central platform in every 45 ° degree, i.e., a radial shape. The central platform was 50 cm wide and 25 cm high and main path was 70 cm long and connected with the paths having a size of 10x25 cm each path not allowing the rats to escape. 7x5x3 cm size of a vessel (food dish) to provide rats with water and food was positioned at the end of main path. To record the behavior of rats, video camera was set up and the experiment was performed for 1 week after two weeks of treatment. The visit number and visit error number to main path were counted by recording the behavior of rats using by vise camera. The rats were fasted until 36 hours before the test, transferred to behavior ob¬ servation room and acclimated to environment for 30 mins. The rats were put in the maze and acclimated to the environment for 1 min. each exit was open to run freely and the rat visiting main path was allowed to eat the food as a compensation, tbwever, the rats visiting through same main path were not allowed and it was checked as an error. The test was stopped when the rats could not visit eight main paths for 5 mins and it is regarded as a failure. Af er the learning test prosecuted for 5 days, learning ability examination was performed to the rats passed the test. The treatment before memory test and the procedure before the entry of the 4 main path were performed similarly. The main path was closed simultaneously with the entry with the 4 path and the test was postponed for 30 sec. The exit of main path was open again to allow the rat to pass remaining four paths, to eat food and finally to be free. The number of errors till the end of test was regarded as an index of memory.

[100] All the determination values were expressed as mean + SE and the statistical analysis between respective test groups was calculated by using SPSS for window. The comparison between the behavior determination value of respective groups was performed by using repeated ANOVA test and the differences were considered significant for P<0.05.

[101] At the result obtained by the experiment calculating the visiting number of main path within 300 sec for 5 days, the group treated with the extract prepared by Example 2 showed significant increase comparing with those of control group treated nothing and control group treated with only physiological saline (P<0.01) at the 4 day. At the 6 day, the group treated with the extract prepared by Example 2 showed significant increased the correct choice number of main path in radial arm maze experiment after occurring of forebrain apparatus ischemia comparing with that of control group treated with only physiological saline (P<0.05) and the determined values between the groups showed significant difference (F(4,27)=5.248, P<0.05) as can be seen in Table 3 and

Rg. l.

[102] [Table 3]

[103] At the result obtained by the experiment calculating the error rate number of visiting main path again within 300 sec for 5 days, the group treated with the extract prepared by Example 2 (F(4,23)=7.363, P<0.01) showed significant increase comparing with those of negative control group treated nothing and control group treated with only physiological saline (P<0.01) at the 4 day. At the 6 day, the groups treated with the extract prepared by Example 1 (P<0.01) and Example 2 (P<0.05) showed significant decrease comparing with that of control group treated nothing and the group treated with the extract prepared by Example 2 showed significant decrease comparing with that of control group treated with only physiological saline (P<0.05) at the 5 day. At the 6 day, the group treated with the extract prepared by Example 2 showed significant decrease comparing with control group treated with nothing (P< 0.05) and the determined values between the groups showed significant difference (F(4,27)=0.435, P<0.01) as can be seen in Table 4 and Hg. 2.

[104] [Table 4]

[105] Experimental Example 4. Protection effect on the brain neuronal cell using by Histochemical analysis

[106] 4-1. Preparation of animal tissue [107] The rats prosecuted the Experimental Example 3 was anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and 200 ml of 0.9% saline solution and 800 ml of 4% formalin fixative dissolved in phosphate buffer were perfused through heart. At this time, 200 ml of the fixative solution was perfused for 2 mins with a high speed and remaining fixative solution for 35 mins with a slow aped. The fixed brain of rats were delivered and fixed again with same fixative for 2 hours. PBS solution containing 20% sicrose was added thereto and stored at 4 ⁰C for a night. On next day, the brain was quick- frozen and the hippocampus organ deliver from brain was sliced to the piece at the width of 30 um .

[108] 4-2. Observation of brain tissue using by cresyl violet staining method [109] The brain tissue prepared in Experimental Example 4-1 was washed with PBS solution several times, defatted and dehydrated by subsequently soaking in xylene for 5 mins, in 100% alcohol for 2 mins, in 95% alcohol for 1 min, in 70% alcohol for 1 min, and in distilled water for 2 mins. The tissue was stained with cresyl violet buffer for 5 mins and the stained tissue was observed in optical spectroscopy with high resolution (x200). The position of probe was detected by consulting the coordinate of brain tissue atlas authored by Paxinos and Watson and the number of neuronal cell was determined. The determined values were performed by one-way ANOVA and the post- hoc survey was applied to Turkey test. T he differences were considered significant for P<0.05.

[HO] At the result, the effect of each groups on the injuries of neuronal cell in hippocampus were shown in Hg. 3 and Hg. 4. [111] The staining degree of neuronal cell in hippocampus region was shown in Table 5, and the groups showed significant difference between each other (F(4,83)=5.310, P< 0.05). At the result of post-hoc survey, the group treated with the extract prepared by Example 2 showed significantly increased neuronal cell comparing with that of control group (P<0.05), which means that the acupuncture treatment group with purified extract of wild ginseng of the present invention has potent preventing activity of neuronal cell injury at hippocampus region caused by forebrain ischemia.

[112] [Table 5]

[113] 4-3. Observation of brain neuronal cell using by AchE staining method [114] The brain tissue prepared in Experimental Example 4-1 was washed with PBS solution at three times. The tissue was soaked in weak green colored attaining solution prepared by mixing the solution containing 250 mg of acetylcholine iodide dissolved in 325 ml of 0.1 M sodium hydrogen phosphate with mixture solution mixed with 25 ml of 0.1 M sodium citrate, 50 ml of 3OmM copper sulfate, 50 ml of 5mM potassium ferricyanide and 50 ml of distilled water, and incubated at room temperature for 1 to 2 hours. Resulting brain tissue was observed by optical spectroscopy conditioned with multiplied ratio(xlOO) with a rectangle grid (200x200 um). The density of AchE neuronal cell was determined by using Scion image program (Scion Corp. MD. USA ).

[115] At the result, the effect of each groups on the expression degree of AchE at hippocampus were shown in Hg. 5 and Hg. 6. [116] The AchE expression degree at CAl region of hippocampus was shown in Table 6 and the groups showed significant difference between each other (F(4,80)=6.747, P< 0.001). At the result of post-hoc survey, the group treated with the extract prepared by Example 2 showed significantly increased neuronal cell comparing with those of control group (P<0.05) and the group treated with saline solution (P<0.05). The AchE expression at CA3 region was shown in Table 7 and the groups did not show significant difference between each other (F(4,80)=0.395, P<0.811), which means that the acupuncture treatment group with purified extract of wild ginseng of the present invention has potent preventing activity of neuronal cell injury at hippocampus region caused by forebrain ischemia.

[117] [Table 6]]

[118]

[119] 4-4. Observation of brain neuronal cell using by ChAT Immuno- histocemistrv

[120] The brain tissue prepared in Experimental Example 4-1 was washed with PBS solution at three times. Primary sheep polyclonal ChAT antibody (Cambridge Research Biochemicals, Wilmington , DE , USA ) conventionally used in the study of the ChAT gene expression, was used in this experiment. Primary antibody was prepaed by gene expression. The primary antibody was prepared by diluting with (x2000) 2% rabbit serum and 0.1% sodium azide (Sigma, St, MO, USA) in PBST solution prepared by adding 0.3% Triton X-100 to PBS. The brain tissue was incubated at 4 ⁰C for 72 hours stirring continuously and washed with PBST at over three times. The tissue was reacted with (x200) diluted biotinylated anti-sheep serum (Vector Lab. Burlingame, CA, USA) with PBST containing 2% rabbit serum at room temperature for 2 hours and washed with PBS solution three times. The tissue was soaked in avidine- biotin-peroxidase complex (Vectastain-Elite ABC kit, Vector Lab. Inc., USA) at room temperature for 2 hours and washed with PBS at several times. The tissue was reinforced by nickel chloride and expressed by staining agent (3, 3'-diamino benzidine tetrahydrochloride, DAB). Resulting brain tissue was fixed at gelatine-coated slide, covered by cover slide removing air, and observed by optical spectroscopy. Resulting brain tissue was observed by optical spectroscopy conditioned with multiplied ratio (xlOO) with a rectangle grid (200x200 um ). The density of ChAT neuronal cell was determined by using Scion image program (Scion Corp. MD. USA ).

[121] At the result, the effect of each groups on the expression degree of ChAT at hippocampus were shown in Hg. 7 and Hg. 8.

[122] The ChAT expression degree at CAl region of hippocampus was expressed in Table 8 and the groups showed significant difference between each other (F(4,86)=6.252, P<0.001). The ChAT expression degree at CA3 region of hippocampus was expressed in Table 9 and the groups showed significant difference between each other (F(4,86)=5.262, P<0.01). At the result of post-hoc survey, the group treated with the extract prepared by Example 2 showed significantly increased expression of ChAT comparing with those of control group (P<0.05) statistically, which means that the acupuncture treatment group with purified extract of wild ginseng of the present invention has potent preventing activity of neuronal cell injury at hippocampus region caused by forebrain ischemia.

[123] [Table 8]

[124]

[125] Experimental Example 5. Toxicity test [126] Methods (1) [127] 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 extract of the Example 1. lour group consisting of 10 mice or rats was administrated orally in- traperitoneally with 250mg/kg, 500mg/kg, 1000mg/kg and 5000mg/kg of test sample or solvents (0.2 ml , i.p.) respectively and observed for 2 weeks. [128] Methods (2)

[129] The acute toxicity tests on ICR mice and Sprague-Dawley rats were performed using the extract of the Example 2. lour group consisting of 10 mice or rats was ad¬ ministrated intraperitoneally with 25mg/kg, 250mg/kg, 500mg/kg and 725mg/kg of test sample or solvents (0.2 ml, Lp.), respectively and observed for 24 hours.

[130] Results

[131] 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 were potent and safe.

[132] 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.

[133] Preparation of powder

[134] Dried powder of Example 1 300mg

[135] Lactose lOOmg

[136] Talc lOmg

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

[ 138] Preparation of tablet

[139] Dried powder of Example 1 50mg

[140] Corn starch lOOmg

[141] Lactose lOOmg

[142] Magnesium Stearate 2mg

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

[144] Preparation of capsule

[145] Dried powder of Example 1 50mg

[146] Corn starch lOOmg

[147] Lactose lOOmg

[148] Magnesium Stearate 2mg

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

[150] Preparation of injection

[151] Dried powder of Example2 50mg

[152] Distilled water for injection optimum amount

[153] PH controller optimum amount [154] 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.

[155] Preparation of liquid

[156] Dried powder of Example 2 0.1~80g

[157] Sugar 5~10g

[158] Citric aid 0.05-0.3%

[159] Caramel 0.005-0.02%

[160] Vitamin C 0.1-1%

[161] Distilled water 79-94%

[162] CO gas 0.5-0.82%

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

[164] The invention being thus described, it will be obvious that the same may be varied in many ways. Sich 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.

Industrial Applicability

[165] As described in the present invention, the purified extracts of wild ginseng prepared by inventive method have potent neuronal cell protective activity, therefore, it can be used as the therapeutics for treating and preventing neuro-degenerative brain diseases caused by neuronal cell sich as death stroke, apoplexy, dementia, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, Pick's disease and Creutzfeld-Jakob's disease and the like .

Claims

[1] L A use of a purified extract prepared by the method comprising the steps consisting of; subjecting an wild ginseng material to distillation extracting method with extracting solvent repeatedly; freezing the distilled extract to obtain frozen purified extract; thawing the extract and filtrating to obtain purified extract for the preparation of a pharmaceutical composition for the treatment and prevention of degenerative brain disease by protecting neuronal cell in human or mammal.

[2] 2. The use according to claim 1, said extracting solvent is selected from water, C -C lower alcohol or the mixture thereof.

1 4

[3] 3. The use according to claim 2, said extracting solvent is water.

[4] 4. The use according to claim 1, said wild ginseng comprises cultivated ginseng and naturally grown wild ginseng.

[5] 5. A use of a purified extract of wild ginseng prepared by the method comprising the steps consisting of; subjecting an wild ginseng material to the 1 distillation extracting method with extracting solvent to obtain the 1 distillate at the I^s step; subjecting the I^s distillate to the 2° distillation extracting method with extracting solvent to obtain the 2 distillate at the 2 step; freezing the 2° distillate to obtain frozen purified extract at the 3^r step; thawing the extract and filtrating to obtain filtrated extract at the 4 step; heating the filtrate with double boiler and refreezing at the 5 step; thawing and sterilizing the extract to obtain the purified extract of the present invention at the 6 step to obtain purified extract for the preparation of a pharmaceutical composition for the treatment and prevention of degenerative brain disease by protecting neuronal cell in human or mammal.

[6] 6. The use according to claim 5, said the 1 ^s step is consisting of: pouring water to said material in the ratio ranging from 1.0 kg to 3.0 kg of material per liter of water; being left alone at room temperature for the period ranging from 1 to 4 hours; subsequently, heating with distillation apparatus at below 100 ⁰ C for the period ranging from 6 to 24 hours to obtain the I^s distillate.

[7] 7. The use according to claim 5, said the 2 step is consisting of: adding boiling chip containing flask equipped with distillation apparatus to the 1 ^s distillate; heating at 100 ° C, for the period ranging from 7 to 24 hours gradually to the extent that the volume of concentrate is rediced to be in the ranging 70 to 90(v/v)% to obtain the 2 ⁿ distillate.

[8] 8. The use according to claim 5, said the 3 ^r step is consisting of: freezing the 2° distillate at the temperature ranging from -5 to -15 ⁰ C to the extent that the volume of un-frozen fraction is decreased to be less than 5% of the total volume; removing the un-frozen fraction; thawing remaining distillate; repeating those freezing and removing un-frozen fraction processes at 1 to 6 times to obtain frozen purified extract of the present invention.

[9] 9. The use according to claim 5, said the 4 step is consisting of: thawing the frozen purified extract at the 3 step at room temperature; and filtrating with filter paper having pore size ranging from 0.1 to 0.6 um to obtain filtrates.

[10] 10. The use according to claim 5, said the 5 step is consisting of: heating with double boiler at 100 ° C in the period ranging from 20 to 40 mins; and refreezing to obtain frozen purified extract.

[11] 11. The use according to claim 1 wherein said pharmaceutical composition comprises oral dosage form sich as powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule or aerosol.

[12] 12. The use according to claim 1 wherein said pharmaceutical composition comprises topical preparation sich as cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol, and injectable preparation sich as acupuncture injectable preparation.

[13] 13. The use according to claim 1 wherein said pharmaceutical composition is administered via spots orally, rectally or by intravenous, intramuscular, subcutaneous, intracutaneous, intrathecal, epidural or intracere- broventriαilar injection or acupuncture injection onto the spots suitable for acupuncture.

[14] 14. The use according to claim 13 wherein said pharmaceutical composition is administered via acupuncture injection onto the spots suitable for acupuncture.

[15] 15. A method of treating or preventing degenerative brain disease by protecting neuronal cell in a mammal or human comprising administering to said mammal or human an effective amount of a purified extract of wild ginseng prepared by the method as set forth in claim 1, together with a pharmaceutically acceptable carrier thereof.