WO2009020304A1

WO2009020304A1 - A composition comprising an extract of scrophularia radix for treating and preventing cognitive impairment involved disease

Info

Publication number: WO2009020304A1
Application number: PCT/KR2008/004466
Authority: WO
Inventors: Hi-Jae Cho; Yong-Wook Lee; Min-Ho Choi; Yong-Hoon Won; Chan-Sung Park; Yong-Hyuk Lee
Original assignee: Kwang Dong Pharmaceutical Co.
Priority date: 2007-08-09
Filing date: 2008-07-31
Publication date: 2009-02-12
Also published as: KR20090015704A; KR100890179B1

Abstract

The present invention relates to a composition comprising an extract of Scrophularia radix for treating and preventing cognitive impairment involved disease and the use thereof. The inventive extract of Scrophularia radix showed potent inhibiting effect on neuronal cell death and the loss of memory and improving effect on the loss of memory. Therefore, it can be used as the therapeutics or health food for treating and preventing cognitive impairment involved diseases caused by cognitive impairment.

Description

A COMPOSITION COMPRISING AN EXTRACT OF SCRO-

PHULARIA RADIX FOR TREATING AND PREVENTING

COGNITIVE IMPAIRMENT INVOLVED DISEASE

Technical Field

[1] The present invention relates to a composition comprising an extract of Scrophularia radix for treating and preventing cognitive impairment involved disease and the use thereof.

[2]

Background Art

[3] Cognitive impairment encompasses various syndromes, for example, physiological disorder, behavior disorder and endocrine disorder caused by brain disease, brain injury, intoxification etc, metabolic or nutritional abnormalities, and mental disease caused by drug etc and it also comprises amnesia belonged to temporal memory impairment disorder as well as dementia showing the common disorder in cognition and discretion according to the development stage of the disease.

[4] MCI (mild cognitive impairment), an initial stage of severed Alzheimer's disease, comprises various syndromes such as loss of the latest memory, the decline of learning ability, concentration power, thinking power and linguistic power, and the like. More than 80% of MCI patients frequently developed Alzheimer's disease within ten years after the syndrome, of which level is significantly higher considering the fact that only 1% of more than 65 year's old healthy peoples suffer with Alzheimer's disease (Peterson RC. et al., Mild Cognitive impairment: clinical characteristics and outcome, Arch. Neurol, 56, pp303-308, 1998; Collie A. & Maruff P., Neuropsychology of preclinical Alzheimer's disease and mild cognitive impairment, Neuroscience & Behavioral Review, 2Λ, pp 365-374, 2000). The disease is different from the age- associated memory impairment (AAMI) occurring by aging and closely related to Alzheimer's disease in consideration with their etiological origin and pathological progress, both of which show significant drop of memory ability and cognition ability. Alzheimer's disease oxurs within 70% of the patients suffering with dementia. The etiological origin of Alzheimer's disease has been not completely identified yet, however it is assumed to be caused by the degeneration of cholinergic neuron showing neurofibrillary tangles and the accumulation of neuritis plague (De Ia Torre JC, Alzheimer's disease; how does it start?, J. Alzheimer's Disease .4(6). pp497-512, 2002).

[5] Various factors such as excitotoxicity, oxidative stress, growth factor withdrawal, cytokines or toxin etc, may give rise to the injury of neuronal cell. Most of the neuronal cell injuries are attributed to the hyper-stimulation of ionotripic or metabotropic glutamate receptor (Vajda FJ., Neuroprotection and neurodegenerative disease, J. Clin. Neurosci., 9, pp4-8, 2002).

[6] There have been reported that mmole level of glutamate has been found in brain and it plays important role in the central excitatory neurotransmission by acting on N- methyl-D-aspartate (NMDA) receptor and non-NMDA receptor in brain. Those excitatory amino acid such as glutamate are involved in various pathways such as neuronal survival, synaptogenesis, neuronal plasticity, learning, memory process etc (Monaghan, D. T. et al., The excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system, Annual Review of Pharmacology and Toxicology, 29, pp365-402, 1989; Kruk, Z. L. et al., Neurotransmitters and Drugs, Chapman & Hall, London, ppl59-168, 1991).

[7] However, it has been reported that a glutamate causes the neuronal cell injury in

CNS through apparently discrete two pathways, i.e., an acute mechanism and chronic mechanism (Choi D. W., Glutamate neurotoxicity and diseases of the nervous system, Neuron, 1, pp623-634, 1988). For example, the abnormalities of glutamate neurotransmitter system may be involved in the diseases of the nervous system such as seizure (Zaczek, R and Coyle, J. T., Excitatory amino acid analogues: neurotoxicity and seizure, Neuropharmacology, 21, ppl5-26, 1982), Alzheimer's disease (Greenamyre, J. T. and Young, A. B., Excitatory amino acids and Alzheimer's disease, Neurobiology in Aging, JO, pp593-602, 1989), Parkinson's disease (Marino, M. J. et al., Glutamate receptor and Parkinson's disease: opportunities and intervention, Drugs Aging, 20(5). pp377-397, 2003), ischemia and spinal cord trauma (Albers, G. W. et al., N-methyl-D-aspartate antagonists: ready for clinical trial in brain ischemia, Annals of Neurology, 25, pp398-403, 1989) etc

[8]

[9] Scrophularia Radix, a dried radix of Scrophularia genus plants such as Scrophularia buergeriana, Scrophularia borealikoreana, Scrophularia takesimensis, Scrophularia kakudensis, Scrophularia koraiensis and the like, is distributed in Korea. The radix has been reported to treat intoxification, sore throat, rhinorrhagia etc and to contain alkaloid, sterol, amino acid, fatty acid such as oleic acid, linoleic acid, stearic acid etc (Chung B. S. and Shin M. K.; HyangyakDaesacheon, Youngrimsa., pp909-910, 1998). [10] Korea Patent Registration No. 10-0535266 disclosed anti-oxidant activity of the extract of Scrophularia Radix, which is confirmed by various experiments, such as the effect on the growth of human embryonic fibroblast, the expression of acidic beta- galactosidase, the expression of aging marker gene (p21), the DNA damage caused by oxidative stress and in vivo animal model test etc

[11] The present inventors already found that the phenylpropanoids such as p- methoxycinnamic acid; and phenylpropanoid esters such as p- methoxy-trans-dnnamoyl-4-alpha-L-rhamnose ester etc isolated from the extract soluble in the mixture solvent (CHCl₃: MeOH=I :1) of Scrophularia Radix, showed neuroprotective activity (Kim SR and Kim YC, Neuroprotective phenylpropanoid esters of rhamnose isolated from roots of Scrophularia buergeriana, Phytochemistry, 54, pp503-509, 2000; Korea Patent Publication No 10-2000-0008399 published on Feb. 07, 2000).

[12] However, there has been not reported or disclosed on the treating and preventing activity of an extract of Scrophularia radix for cognitive impairment involved disease, in any of above cited literatures, the disclosures of which are incorporated herein by reference.

[13] To investigate an protecting or improving effect of Scrophularia radix on neuronal cell death and the loss of memory through several experiments, for example, a protecting activity test from a neuronal cell viability using by Sprague-Dawley rat to confirm the protecting effect on neuronal cell death, and the various animal model test such as passive avoidance test, Morris water maze test, and anti-dementia test to confirm the improving effect on the loss of memory, and finally completed the present invention by confirming that the extract inhibits the neuronal cell death, as well as improves the loss of memory.

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

[15]

Disclosure of Invention Technical Problem

[16] The present invention provides a pharmaceutical composition comprising an extract of Scrophularia radix as an active ingredient in an effective amount to treat and prevent a cognitive impairment involved disease caused by cognitive impairment.

[17] The present invention also provides a use of the above extract for the preparation of pharmaceutical composition to treat and prevent cognitive impairment involved disease caused by cognitive impairment in mammal or human.

[18] The present invention also provides a health care food or food additives comprising the above extract for the prevention or alleviation of cognitive impairment involved disease caused by cognitive impairment.

[19]

[20]

Technical Solution

[21] Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising an extract of Scrophularia radix as an active ingredient for the treatment and prevention of cognitive impairment involved disease caused by cognitive impairment.

[22]

[23] The present invention also provide a pharmaceutical composition comprising an extract of Scrophularia radix as an active ingredient, and the pharmaceutically acceptable carriers, adjuvants, or diluents,for the treatment and prevention of cognitive impairment involved disease by caused by cognitive impairment.

[24] The term "extract" disclosed herein comprises the extract prepared by extracting plant material with water, lower alcohols such as methanol, ethanol, etc or the mixtures thereof, preferably, the mixture solvent of water and ethanol, more preferably, the mixture solvent of water and ethanol with the mixed ratio ranging from 30 to 90% (v/vΨc), more preferably, from 50 to 90% (v/vΨc), the most preferably, 65 to 85% (v/v%) in the present invention , which comprise the extract of Scrophularia buergeriana, Scrophularia borealikoreana, Scrophularia takesimensis, Scrophularia kakudensis, Scrophularia koraiensis and the like, preferably, Scrophularia buergeriana.

[25]

[26] It is an object of the present invention to provide a use of an extract of Scrophularia radix for the preparation of therapeutic agent for the treatment and prevention of cognitive impairment involved disease caused by cognitive impairment in human or mammal.

[27]

[28] It is an object of the present invention to provide a method of treating and preventing cognitive impairment involved disease caused by cognitive impairment in a mammal comprising administering to said mammal an effective amount of an extract of Scro- phularia radix, together with a pharmaceutically acceptable carrier thereof.

[29]

[30] It is another object of the present invention to provide a health care food or food additives comprising an extract of Scrophularia radix, together with a sitologically acceptable additive for the prevention and improvement of cognitive impairment involved disease caused by cognitive impairment.

[31]

[32] The term "cognitive impairment" disclosed herein comprises physiological disorder, behavior disorder and endocrine disorder caused by brain disease, brain injury, intoxi- fication etc, metabolic or nutritional abnormalities, and mental disease caused by drug etc, a memory decline, and memory impairment, preferably, a memory decline, and memory impairment.

[33] The term "cognitive impairment" disclosed herein comprises for example, Alzheimer type dementia, cerebrovascular type dementia, Pick's disease, Creutzfeldt-jakob's disease, dementia caused by cephalic damage, Parkinson's disease, and the like, preferably, Alzheimer type dementia or cerebrovascular type dementia,.

[34]

[35] 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.

[36]

[37] The health food of the present invention comprises the above extracts as 0.01 to 80

%, preferably 1 to 50 % by weight based on the total weight of the composition.

[38]

[39] The above described health care food can be contained in health care food, health beverage etc, and may be used as powder, granule, tablet, chewing tablet, capsule, beverage etc

[40]

[41] An inventive extract isolated from Scrophularia radix, may be prepared in accordance with the following preferred embodiment.

[42]

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

[44]

[45] An inventive extract of Scrophularia radix, can be prepared in detail by following procedures,

[46] [47] The inventive extract of Scrophularia radix, can be prepared by the procedure comprising the steps; of mixing the Scrophularia radix with 1 to 30-fold, preferably, approximately 5 to 15-fold volume of distilled water, lower alcohols such as methanol, ethanol, butanol and the like, or the mixtures thereof, preferably a mixture of ethanol and water based on the volume of the plant; subject to the extraction with the extraction method by the extraction with hot water, cold water, reflux extraction, or ultra- sonication extraction, preferably, the extraction with hot water, at the temperature ranging from 10 to 200⁰C, preferably from 50 to 100⁰C, for the period ranging from 30 mins to 20 hours, preferably, 1 to 5 hours; consecutively, filtering the residue to obtain the supernatant, concentrating and drying the supernatant by vacuum freeze-drying, hot air-drying or spray drying, preferably, spray drying to obtain inventive extract powder of Scrophularia radix, of the present invention.

[48]

[49] Also, the above described procedures may be modified or subjected to further step to fractionate or isolate more potent fractions or compounds by conventional procedure well- known in the art, for example, the procedure disclosed in the literature (Harborne J. B., Phytochemical methods: A guide to modern techniques of plant analysis, 3^rd Ed. pp6-7, 1998).

[50]

[51] Present inventors have investigated the effect of the inventive extract of Scrophularia radix on neuronal cell death and the loss of memory through several experiments, for example, a protecting activity test from a neuronal cell viability using by Sprague- Dawley rat to confirm the protecting effect on neuronal cell death, and the various animal model test such as a passive avoidance test, Morris water maze test, and anti- dementia test to confirm the improving effect on the loss of memory, and finally found that the inventive extract prepared by the above-described procedure significantly inhibited the neuronal cell death, as well as improved the loss of memory.

[52]

[53] In accordance with another aspect of the present invention, there is provided a pharmaceutical composition comprising an extract of Scrophularia radix prepared by the above preparation method as an active ingredient for the treatment and prevention of cognitive impairment involved disease caused by cognitive impairment.

[54]

[55] It is another aspect of the present invention to provide a method for treating and preventing cognitive impairment involved disease caused by cognitive impairment of mammals including human comprising administering to said mammal an effective amount of an extract of Scrophularia radix prepared by the above preparation method, together with a pharmaceutically acceptable carrier thereof.

[56]

[57] It is an object of the present invention to provide a use of an extract of Scrophularia radix prepared by the above preparation method for the preparation of therapeutic agent for the treatment and prevention of cognitive impairment involved disease caused by cognitive impairment in human or mammal.

[58]

[59] The inventive composition for treating and preventing said disease may comprises the above extracts as 0.01 ~ 50 % by weight based on the total weight of the composition.

[60]

[61] 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).

[62]

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

[64]

[65] The composition according to the present invention can be provided as a pharmaceutical 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.

[66]

[67] 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.

[68] Specifically, the composition according to the present invention can further comprise other components such as surfactant and co- solvent besides the inventive extract in the present invention to improve the bio-availability of the composition with the mixed ratio ranging from 1:0.1-50: 0.1-50, preferably, 1:0.1-20: 0.1-20, based on the weight of each component.

[69] The surfactant and co-solvent used herein may increase the solubility of the composition, maintaining the physical and chemical stability of the composition by way of forming self-micelle drug delivery system in case that the inventive extract can not soluble thoroughly in solution or in body fluid.

[70] Preferable said surfactant can comprise nonionic surfactants, anionic surfactants, cationic surfactants, ambivalent surfactants and the like in the present invention.

[71] Specifically, preferable non-ionic surfactants can comprise self-emulsified monostearic add glycerin, propylene glycol fatty add ester, glycerin fatty add ester, polyglycerin fatty add ester, sorbitan fatty add ester, polyoxyethylene (POE) sorbitan fatty add ester, POE sorbitan fatty add ester, POE glycerin fatty add ester, POE alkyl ether, POE fatty add ester, POE solid pimaja oil, POE pimaja oil, POE-POP copolymer, POE-POP alkyl ether, polyether modified silicone, lauric add alkanol amide, alkyl amine oxide, hydrogen addition soybean phospholipid and the like.

[72] Preferable anionic surfactants can comprise fatty add soap, a-acyl sulfonic add salt, alkyl sulfonic add salt, alkyl ally sulfonic add, alkyl naphthalene sulfonic add salt, alkyl sulfonic add salt, POE alkylether sulfate salt, alkyl amide sulfate salt, alkyl phosphate salt, POE alkyl phosphate salt, alkylamide phosphate salt, alkyloylalkyl taurine salt, N-acyl-amino add salt, POE alkyl ether carboxylic add salt, alkyl sulfo suαinic aid salt, alkyl sulfo-acetic add salt, acylated hydrolysable collagen peptide salt, perfluoro alkyl phosphate ester and the like.

[73] Preferable cationic surfactant can comprise alkyl trimethyl ammonium chloride, stearyl trimethyl ammonium chloride, stearyl trimethyl ammonium bromide, setoste- aryltrimethyl ammonium chloride, distearyl dimethyl ammonium chloride, stearyl dimethyl benzyl ammonium chloride, vehenyltrimethyl ammonium bromide, ben- zalkonium chloride, diethylamino ethyl amide stearic add, dimethylaminopropyl amide stearic acid, lanolin derivatives quaternary ammonium and the like.

[74] Preferable ambivalent surfactants can comprise carboxy betaine type, amide betaine type, hydroxy sulfo betaine type, phosphobetaine type, aminocarboxylic acid, imidazoline derivatives type, amide amine type and the like.

[75] Preferable said co-solvent can comprise at least one selected from the group consisting of propylene carbonate, propylene glycol, ethanol, diethylene glycol monoethyl ether, glycofurol, polyethylene glycol, dimethyl isosorbide and methyl pyrrolidone, preferably, diethylene glycol monoethyl ether, propylene glycol or polyethylene glycol in the present invention.

[76] The pharmaceutical composition of the present invention may combine with other ingredients conventionally used in the art, if necessary, together with above described ingredients, for example, the fatty add or fatty add alcohol to increase the bioavailability such as dtric add, olied add, stearyl alcohol, myristic add, linoleic add, lauric add, capric add, caprylic add, caproic add and the like; sugar component such as white sugar, wheat-gluten, refined sugar, gelatin, sugar, and starch syrup and the like; lubricants such as magnesium stearic add, talc and the like; adjuvants such microcrystalline cellulose, caldum hydrogen phosphate, starch, mannitol and the like; anti-oxidants such as butyrated hydroxyl toluene, sodium bisulfate alpha-tocopherol, vitamin, beta-carotene, acetic add, fumaric add, nalic add, butyrated hydroxyl anisole, propyl gallate, sodium ascorbate and the like; flavoring agent such as mixed fruit flavor, apple flavor, strawberry flavor, cherry flavor, peppermint flavor, yoghurt flavor, or drink flavor and the like; a preservative such as benzoic add, sodium benzoic add, ethyl paraben, methyl paraben, propyl paraben and the like; aromatics such as menthol, menthol oil, orange oil, clove oil, dnnamon oil, strawberry essence, and the commerdally available fruit flavor or plant essence; sweetener such as refined sugar, grape sugar, fruit sugar, aspartame, stevioside, sorbitol, mannitol, oligosaccharide, starch syrup, and the like; food coloring agent such as Green 3, Red 2, Red 3, Blue 1, Blue 2, Yellow 4, Yellow 4, Yellow 5, soluble mannitol, caramel, titanium oxide, or ferrous oxide and the like; pH controller such as sodium carbonate, sodium hydroxide, caldum hydroxide, Methanol amine, monoethanol amine, dtric add, sodium dtrate, malic add, sodium malate, fumaric add, sodium fumaric add, suαinic add, sodium succinic add, sodium hydroxide, sodium hydrogen phosphate and the like; and viscosity modifier such as hydroxypropyl cellulose, hydroxy propyl methyl cellulose, hydroxyl ethyl cellulose, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, acada, bentonite, alginic add, propyleneglycol alginate, polyvinylpyrrolidone, polyvinyl alcohol, carbopol, polycarbopyl and the like, however, which are enumerated for exemplary explanation but not intended to limited thereto in the present invention.

[77]

[78] Furthermore, the other ingredients addable to the above described component and the amount thereof are not limited within the scope of the purpose and the effect of the present invention, however, it is preferable that the amount of the other ingredients ranges from 0.01 to 5%, more preferably, 0.01 to 3% in that of total composition.

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

[80] Specifically, the present invention provide oral preparation comprising an extract of

Scrophularia radix as an active ingredient for the treatment and prevention of cognitive impairment involved disease caused by cognitive impairment. The oral preparation of the present invention can be prepared in the form of powder, tablet, hard capsule, soft capsule, caramel type or jelly type chewing tablet, liquid syrup, preferably, soft capsule, which may be prepared by mixing the inventive extract with said surfactant and co-solvent together and heating the mixture at appropriate temperature, preferably, about 8O⁰C, to formulate inventive oral preparation aαjording to the well-known procedure in the art.

[81] 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.

[82]

[83] 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 administration, 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.

[84]

[85] 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, intracutaneous, intrathecal, epidural or intracerebroventricular injection.

[86]

[87] It is another object of the present invention to provide a health care food or food additives comprising an extract of Scrophularia radix, together with a sitologically acceptable additive for the prevention and improvement of cognitive impairment involved disease caused by cognitive impairment.

[88] Also, the present invention provide a composition of the health food beverage for the prevention and improvement of said disease adding the above described extract 0.01 to 80 % by weight, amino acids 0.001 to 5 % by weight, vitamins 0.001 to 2 % by weight, sugars 0.001 to 20 % by weight, organic acids 0.001 to 10 % by weight, sweetener and flavors with proper amount.

[89]

[90] The above described inventive extract can be added to food and beverage for the prevention and improvement of said disease with a sitologically acceptable additive well-known in the art, for example, vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in, pectic add and the salt thereof, alginic add and the salt thereof, organic add, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al.

[91]

[92]

[93] To develop for health care food, examples of addable food comprising the above- described extract of the present invention are various food, beverage, gum, vitamin complex, health improving food and the like, and can be used as power, granule, tablet, chewing tablet, capsule or beverage etc

[94]

[95] Also, the extract of the present invention will be able to prevent, and improve said disease by comprising to child and infant food, such as modified milk powder, modified milk powder for growth period, modified food for growth period.

[96]

[97] The above described composition therein can be added to food, additive or beverage, wherein, the amount of above described extract in food or beverage may generally range from about 0.1 to 80w/w%, preferably 1 to 50 w/w% of total weight of food for the health food composition and 1 to 30 g, preferably 3 to 10 g on the ratio of 10Om^ of the health beverage composition.

[98]

[99] Providing that the health beverage composition of present invention may contain the above described extract as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol, and erythritol etc As the other deodorant than aforementioned ones, natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al., may be useful favorably. The amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100 mi of present beverage composition.

[100]

[101] The other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese chocolate et al., pectic add and the salt thereof, alginic add and the salt thereof, organic add, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al. The other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination. The ratio of the components is not so important but is generally range from about 0 to 20 w/w % per 100 w/w % present composition. Examples of addable food comprising aforementioned extract therein are various food, beverage, gum, vitamin complex, health improving food and the like.

[102]

[103] The inventive composition may additionally comprise one or more than one of organic acid, such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid; phosphate, such as phosphate, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate; natural anti-oxidants, such as polyphenol, catechin, α- tocopherol, rosemary extract, vitamin C, green tea extract, licorice root extract, chitosan, tannic acid, phytic acid etc

[104]

[105] The inventive extract may be 20 to 90% high concentrated liquid, power, or granule type and may comprise additionally one or more than one of lactose, casein, dextrose, glucose, sucrose and sorbitol.

[106]

[107] The inventive extract has no toxicity and adverse effect therefore; they can be used with safe.

[108]

[109] 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.

[HO]

Advantageous Effects

[111] As described in the present invention, the inventive extract of Scrophularia radix, showed potent inhibiting effect on neuronal cell death and the loss of memory and improving effect on the loss of memory being confirmed by various in vitro tests, therefore, it can be used as the therapeutics or health food for treating and preventing cognitive impairment involved diseases caused by cognitive impairment.

[112]

Brief Description of the Drawings

[113] 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 aαjompanying drawings, in which;

[114]

[115] Fig. 1 shows the result of passive avoidance test treated with inventive extract;

[116] Fig. 2 shows the effect of inventive extract on passive avoidance test treated at once;

[117] Fig. 3 shows the effect of inventive extract on passive avoidance test treated for 15 days once per day;

[118] Fig. 4 represents the effect of inventive extract on water maze test treated at once; [119] Fig. 5 represents the effect of inventive extract on water maze test treated for 15 days once per day; [120] Fig. 6 presents the relative composition ratio (MCA; p-methoxycinnamic acid) of the inventive extract (SB70E) against the CM extract (SBCM); [121] Fig 7 depicts the compared effect of the inventive extract (SB70E) and CM extract

(SBCM) on passive avoidance test. [122]

Best Mode for Carrying Out the Invention [123] 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. [124] [125]

Mode for the Invention [126] 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. [127] [128] The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope. [129] [130] Comparison Example 1. Preparation of the extract of Scrophulaήa buergeriana

(CM) [131] 1 kg of dried radix of Scrophulαriα buergeriana purchased from Kyung-dong Market located in Seoul was cut into small pieces, mixed with 10 L of the mixture solvent

(CHCl₃-MeOH=I :1, designated as "CM" hereinafter) and the mixture was subjected to extraction at 8O⁰C for 3 hours. [132] The extract was filtered with filter paper to remove the debris and the filtrates were pooled, concentrated by rotary evaporator (N- 1000, Eyela Co. Japan) at 55 ~ 65⁰C under reduced pressure and dried with spray dryer to obtain dried CM soluble extract of Scrophularia buergeriana. (Designated as "SBCM", hereinafter) The extract was used to compare with the inventive extract in Experimental Examples. [133] [134] Example 1. Preparation of the extract of Scrophularia buergeriana . [135]

[136] 1-1. 30% Ethanol Extract

[137] 1 kg of dried radix of Scrophularia buergeriana purchased from Kyung-dong Market located in Seoul was cut into small pieces, mixed with 10 L of 30% ethanol and the mixture was subjected to extraction at 8O⁰C for 3 hours.

[138] The extract was filtered with filter paper to remove the debris and the filtrates were pooled, concentrated by rotary evaporator (N- 1000, Eyela Co. Japan) at 55 ~ 65⁰C under reduced pressure and dried with spray dryer to obtain 210 g of dried 30% ethanol soluble extract of Scrophularia buergeriana. (Designated as "SB30E" hereinafter)

[139]

[140] 1-2. 40% Ethanol Extract

[141] All the procedure was similarly performed to that in Example 1-1 excepting that the extraction solvent was 40% ethanol instead of 30% ethanol and 173g of dried 40% ethanol soluble extract of Scrophularia buergerian was obtained. (Designated as "SB40E" hereinafter)

[142]

[143] 1-3. 50% Ethanol Extract

[144] All the procedure was similarly performed to that in Example 1-1 excepting that the extraction solvent was 50% ethanol instead of 30% ethanol and 149 g of dried 50% ethanol soluble extract of Scrophularia buergeriana was obtained. (Designated as "SB50E" hereinafter)

[145]

[146] 1-4. 60% Ethanol Extract

[147] All the procedure was similarly performed to that in Example 1-1 excepting that the extraction solvent was 60% ethanol instead of 30% ethanol and 121 g of dried 60% ethanol soluble extract of Scrophularia buergeriana was obtained. (Designated as "SB60E" hereinafter)

[148]

[149] 1-5. 70% Ethanol Extract

[150] All the procedure was similarly performed to that in Example 1-1 excepting that the extraction solvent was 70% ethanol instead of 30% ethanol and 114 g of dried 70% ethanol soluble extract of Scrophularia buergeriana was obtained. (Designated as "SB70E" hereinafter) [152] 1-6. 80% Ethanol Extract

[153] All the procedure was similarly performed to that in Example 1-1 excepting that the extraction solvent was 80% ethanol instead of 30% ethanol and 103 g of dried 80% ethanol soluble extract of Scrophularia buergeriana was obtained. (Designated as "SB80E" hereinafter)

[154]

[155] 1-7. 90% Ethanol Extract

[156] All the procedure was similarly performed to that in Example 1-1 excepting that the extraction solvent was 90% ethanol instead of 30% ethanol and 89 g of dried 90% ethanol soluble extract of Scrophularia buergeriana was obtained. (Designated as "SB90E" hereinafter)

[157]

[158] 1-8. 99% Ethanol Extract

[159] All the procedure was similarly performed to that in Example 1-1 excepting that the extraction solvent was 99% ethanol instead of 30% ethanol and 83 g of dried 99% ethanol soluble extract of Scrophularia buergeriana was obtained. (Designated as "SB99Ξ" hereinafter)

[160]

[161] Reference Example 1. Component analysis

[162] The component, especially, dnnamate derivatives such as cinnamic acid (a), p- methoxycinnamic acid (b), harpagoside (c), and 8-O-( E-methoxycinnamoyl)harpagide (d) contained in the respective extract prepared in Example 1, was determined to identify the active ingredient of the extract and the analysis data was shown in Table 1.

[163]

[164] Table 1

[Table 1] [Table ]

[165] As shown in Table 1, the amount of dnnmate derivatives in more than 90% ethanol soluble extract was the higher level than those in less than 90% ethanol soluble extract and the amount of the compounds in less than 80% ethanol soluble extract was sharply decreased.

[166] [167] Experimental Example 1. Effect on Neuronal cell viability [168] The primary cultures of the cortex of cerebrum isolated from the fetus (17-19 days pregnancy) of Sprague-Dawley rat (female, 15 weeks, 287.1-303.2 g, Seoul National University), were prepared according to the procedure disclosed in the literature (Kim Y.C. et al., Ginsenosides RbI and Rg3 protect cultured rat cortical cells from glutamate-induced neurodegeneration, Journal of Neuroscience Research, 53_, pp426-432, 1998). The culture cell was incubated in DMEM (Gibco BRL Co., Ltd., USA), supplemented with penollin (100 IU/ml, Sigma Co.), Streptomydn (10 mg/ml, Sigma Co.) and 10% heat-inactivated fetal calf serum for 3 days at 37⁰C in 5% CO ₂ and 95% air condition in a humidified incubator and 50 micromole 5-fhx>rodeoxyuridine was added thereto to stop the division of non-neuronal cells. [169] The respective extracts prepared in Example were dissolved in DMSO to the extent the final concentration reached to 0.1%) to uses as a test sample and it has been confirmed that DMSO did not affect the activity of cell survival through preliminary test. The cell was washed with DMEM and incubated with the test sample for 1 hour. The culture was treated with 50 mM glutamate and, washed with DMEM medium 30 mins after the treatment.

[170] The neuronal cell viability of each group was determined by using MTT assay method (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) together with the analysis of LDH activity to asses the protecting effect of the inventive extract on the neuronal cell viability against glutamate toxicity.

[171] The result was shown in following Table 2. [172] [173] Table 2 [Table 2] [Table ]

[174] [175] [176] As shown in Table 2, it has been confirmed that the 70% ethanol and 80% ethanol soluble extract of Scrophularia buergeriana showed the higher cell viability than those of other extracts. The above-described result is a rather opposite to the already disclosed result that phenylpropanoid derivatives having alpha, beta-unsaturated ester moiety and para-methoxy group such as p-methoxy cinnamic acid, played a important role in treating and preventing cognitive impairment disorder since the group treated with the extracts of Scrophularia buergeriana soluble in 70% and 80% ethanol (SB70E & SB 80E) known to contain less amount of p-methoxy dnnamic add, showed the higher cell viability than the group treated with the extracts of Scrophularia buergeriana soluble in more than 90% ethanol (SB90E & SB99E) known to contain more abundant amount of p-methoxy dnnamic add. ( See , Kim SR and Kim YC, Neuroprotective phenylpropanoid esters of rhamnose isolated from roots of Scrophularia buergeriana, Phytochemistry, 54, pp5O3-5O9, 2000; and Kim SR et al., Four new neuroprotective iridoid glycosides from Scrophularia buergeriana,root, J. Nat. Prod., 65, pp 1696- 1699, 2002)

[177]

[178] Experimental Example 2. Passive Avoidance Test

[179] To investigate the improving effect of the inventive extract prepared in Example 1 on the cognitive impairment disorder (namely, memory improvement), passive avoidance test was performed by the procedure disclosed in the literature (Kim SR et al., Anti- amnestic adtivity of E-p-methoxydnnamic add from Scrophularia buergerianajoot, Cognitive Brain Research, 17, pp454-461, 2003).

[180]

[181] An automated system with a shuttle box was used to evaluate the effects of the extract on the learning and memory assodated with neuronal cell growth.

[182]

[183] Avoidance shuttle box (40x20x20 cm, Gemini Co., U.S.A.) was divided into two chambers of equal size and had the 3 mm thickness of grid in interval of 0.5 cm on the floor of the box. A light chamber is equipped with an illuminator. 60 minutes after the treatment of inventive extract, 1 mg/kg dose of scopolamine was subcutaneously administrated into male ICR mice (8 weeks, 25.6-30.2 g, Seoul National University) to induce memory decline. 30 minutes after the treatment, the mouse was initially placed in the light chamber to perform acquisition training was carried out, which delivering the electrical foot shock (0.1 rriA/10 g body weight, for 2 sec) by stainless steel road to the mouse through the grid floor when the mouse preferring darkness went out from light chamber and entered the dark chamber, the inventive extract was dissolved in 5% Tween 80 (Vehicle; Polyoxyethylene sorbitan monooleate; Sigma Co., USA) and the various concentrations of each extract was orally administered to the mice as a test group 90 mins before the acquisition trial. 5% Tween solution was used as a negative control group. 10 mice per each test group were used in the experiment. 24 hours after the acquisition trial, the identical experiment was performed again with mouse to measure the latency time staying at the light chamber. The data was regarded as the index which meant the memory on previous training by electronic shock. Latency to enter the dark compartment was measured for 300 sec If it did not enter the dark chamber within the cut-off time (180 sec), it was assigned a value of 180 sec as its latency.

[184]

[185] As shown in Fig. 1, the latency time of the test group treated with the extracts of Scrophularia buergeriana was significantly more increased by more than 60% than that of control group, especially, the group treated with 70% ethanol and 80% ethanol soluble extract of Scrophularia buergeriana showed the higher improving effect on memory ability than those treated with more than 90% ethanol soluble extract or less than 60% ethanol soluble extract.

[186] Various concentrations of 70% ethanol soluble extract showed the most potent activity, were administrated to the mice in a dose dependent manner with the similar method to the above-described procedure to perform a passive avoidance test and the result was shown in Fig. 2 and Fig. 3.

[187] In the dose-dependent test for SB70E, the latency time of the test group treated with 50 mg/kg, and 100 mg/kg of SB70E was significantly increased by 51% and 61% respectively comparing with the control group, which confirmed that the improved memory activity ( See Fig. 2). After performing a passive avoidance test using by various concentrations of 70% ethanol soluble extract, i.e., 3 mg/kg, 10 mg/kg, and 30 mg/kg once a day for 15 days, the extract (SB70E) increased the memory activity in a dose dependent manner ( See Fig. 3).

[188]

[189] Experimental Example 3. Morris water maze test

[190] To investigate the improving effect of the inventive extract prepared in Example 1 on the cognitive impairment disorder (namely, memory improvement), Morris water maze test was performed by the procedure disclosed in the literature (R. G. Morris, Development of a water maze procedure for studying spatial learning in the rat, J. Neurosci. Meth., U, pp47-60, 1984).

[191] Various concentrations of 70% ethanol soluble extract dissolved in 5% (wΛO

Tween80 solution (vehicle), i.e., 10 mg/kg, 30 mg/kg, and 100mg/kg, were orally administrated to the rats used passive avoidance test once a day for 4 days excluding the first day for one shot-treatment test. Additionally, various concentrations of 70% ethanol soluble extract dissolved in 5% (wΛO Tween80 solution (vehicle), i.e., 3 mg/ kg, 10 mg/kg, and 30 mg/kg, were orally administrated to the rats once a day for 15 days and then the above-described one shot-treatment test followed. 60 mins after the treatment of test sample and Tween80 solution used as a negative control group, 1 mg/kg of scopolamine was subcutaneously administrated into the rats to induce memory deficit. All the mice were used to perform water-maze test every day 30 mins after the treatment of the scopolamine or 5% Tween80.

[192] Water-maze test was assessed in a water tank which consisted of circular tub (inner dimensions: diameter 90 cm, depth: 45 cm) divided into four sections (quadrants) and filled with clear tap water at a temperature of approximately 22-23⁰C. A glass escape platform (diameter 6 cm, height: 25 cm) submerged 1 cm below the surface of the water was positioned at the intermediates between the center and edge of the pool. The mice was accustomed to the experimental environment for 30 sec by entering into the pool without submerged platform and then water maze test was performed for 4 days at the interval of 30 mins, twice a day to record the swimming behavior. The movement of the rat were recored automatically by a video-tracking system (Etho Vision, Noldus Information Technology, Wageningen, The Netherlands). The test consisted of the 1 ^st trial and 2^nd trial using the same mice positioned at the same platform and the trails were performed at the interval of several hours.

[193] At the test, "Escape latency" is the time(s) taken to find and escape onto the submerged platform and stayed over 10 sees. "Mean escape latency" is the average time(s) per rat within three sessions per day, and was taken as 120 sec in a case that the mean escape latency exceeds over 120 sec 15 sessions per rat was performed for the four day's period. The reduction of escape latency in the 1 ^st trial aαjording to the time indicated long-term memory ability whereas the reduction of the interval between the 1 st trial and the 2^nd trial indicated a short-term memory ability.

[194] As can be seen the result in case of treatment of SB70E for once ( See Fig.

4) and for 15 days once a day ( See Fig. 5), the group treated with only Tween80 solution showed significant difference from the Escape latency of the 1 ^st trial and the second trail performed at the 1 ^st day and 2^nd day, and reached to certain escape latency at the 2^nd day whereas the group treated with scopolamine showed no difference from the escape latency of the 1 ^st trial and 2^nd trial for four day's test. However, there showed a significantly decrease in Escape latency of the 1 ^st trial and 2^nd trail for test group treated with inventive extract, and the group had reached to certain escape latency at the 2^nd day. Additionally, there also showed a significantly decrease in Escape latency of the 1^st trial and 2^nd trial for test groups treated with inventive extract and scopolamine, which means that the inventive extract is effective in improving short-term memory in consideration with result that the group treated with only scopolamine showed unchanged Escape latency in the 2 ^nd trial. Through the result performing long- term memory test for five days, it has been confirmed that the inventive extract also showed potent improving effect on long-term memory considering the result that there also showed a significant decrease in Escape latency.

[195]

[196] Experimental Example 4. Comparison of Component analysis

[197] To verify the superior utility of inventive extract over the already known extract disclosed in the prior art (Korea Patent Publication No. 10-2000-0008399), the component analysis and yield analysis on the 70% ethanol soluble extract prepared in Example 1 (SB70E) and CM-soluble extract (SBCM) prepared in Comparison Example, were performed to compare with each other in respect to industrial utility such as economic aspect etc as follows:

[198]

[199] 4-1. Preparation of standard solution

[200] lό.Omg, 15.5mg, lO.lmg and 3.1mg of each dried extract prepared in Example 1 and Comparison Example, were dissolved in 90% methanol to the extent to the volume of standard solution had reached to 1000ml.

[201]

[202] 4-2. Analysis condition

[203] The amount of HS (Harpagoside), HG (Harpagide), MCA (p-methoxydnnamic acid) and CA (Cinnamic acid) contained in each extract was determined by HPLC-UV detector. The column was reverse-phase Cl 8 column (18.5 micrometer filler, diameter: 4.6 mm, length: 150 mm) running with the mixture solvent of 1% acetic acid and acet- onitrile (77:33) with the speed of 1.0 ml/min, and the temperature of column had maintained to 3O⁰C. The detecting wavelength of the detector was 280 nm and the result was shown in following Table 3 and Fig. 6.

[204]

[205] Table 3 [Table 3] [Table ]

[206] [207] At the result, the 70% ethanol soluble extract of Scrophularia buergeriana showed 14-fold higher yield compared with CM extract of Scrophularia buergeriana disclosed in the prior art, which means that the requiring cost for preparing the inventive extract is the very cheaper than that for preparing CM extract.

[208] Additionally, it has been confirmed that the inventive extract contains about 7-fold, 5.6-fold, and about 4.2-fold more abundant active ingredients in respect to HS, HG, MCA and CA comparing with that of CM extract.

[209] [210] Experimental Example 5. Comparison of the anti-dementia activity [211] To compare the anti-dementia activity of the inventive extract and the already known extract disclosed in the prior art (Korea Patent Publication No. 10-2000-0008399), passive avoidance test was performed by the procedure disclosed in the literature (Kim SR et al., Anti-amnestic adtivity of E-p-methoxydnnamic add from Scrophularia buergeriana root, Cognitive Brain Research, J/7, pp454-461, 2003).

[212] [213] Male Sprague-Dawley mouse (10 weeks, 305.8-333.4g, Chung-ang Experimental Animal, Korea) was acclimated for 7 days and the mice were divided into three groups with 12 mice/each group, i.e., normal group, negative control group and test sample group. Physiological saline was orally administrated into each normal group and negative control group and the test samples, i.e., SB70E and SBCM were orally administrated into the test sample group in a dose of 100mg/kg and 17mg/kg respectively. 1.5 hours after the treatment, physiological saline solution was subcu- taneously administrated into the normal group and scopolamine was subcutaneously administrated into the negative control group and test sample group in a dose of 2mg/kg induce memory decline.

[214] Avoidance shuttle box (40x20x20 cm, Gemini Co., U.S.A.) was divided into two chambers of equal size and had the 3mm thickness of grid in interval of 0.5 cm on the floor of the box. A light chamber is equipped with an illuminator. 30 minutes after the treatment with scopolamine, the mouse was initially placed in the light chamber to perform acquisition training by delivering the electrical foot shock (0.1mA/ 1Og body weight, for 2 sec) with a stainless steel road through the grid floor when the mouse preferring darkness went out from light chamber and entered the dark chamber, the inventive extract was dissolved in 5% Tween80 (Vehicle; Polyoxyethylene sorbitan monooleate; Sigma Co., USA) and the various concentrations of each extract was orally administered to the mice as a test group 90 mins before the acquisition trial. 5% Tween solution was used as a negative control group. 10 mice per each test group were used in the experiment. 24 hours after the acquisition trial, the identical experiment was performed again with mouse to measure the latency time staying at the light chamber. The data was regarded as the index which meant the memory on previous training by electronic shock. Latency to enter the dark compartment was measured for 300 sec If it did not enter the dark chamber within the cut-off time (180 sec), it was assigned a value of 180 sec as its latency and the result was shown in Table 4 and Fig. 7.

[215] [216] Table 4 [Table 4] [Table ]

[217] [218] As shown in Table 4 and Fig. 7, the test group treated with the 70% ethanol soluble extract of Scrophularia buergeriana showed more than about 6.7-fold higher improving effect on memory ability compared with that treated with the CM soluble extract of Scrophularia buergeriana (17mg/kg).

[219]

[220] Accordingly, the inventive extract of the present invention shows the superior technical effect over the already known extract disclosed in the prior art (Korea Patent Publication No. 10-2000-0008399), in respect to various factors such that the inventive extract contains more abundant amount of active ingredients and more potent anti- dementia activity regardless of the amount of dnnamate derivatives compared with already known extract disclosed in the prior art.

[221]

[222] 6-1. Methods (D

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

Dawley rats (235+10 g, Jung-Ang Lab Animal Inc.) were performed using the extract of the Example l(SB70E). Four group consisting of 10 mice or rats was administrated orally intraperitoneally with 250 mg/kg, 500 mg/kg, 1000 mg/kg and 5000 mg/kg of test sample or solvents (0.2 m-6, i.p.) respectively and observed for 2 weeks.

[224]

[225] 6-2. Methods (2)

[226] The acute toxicity tests on ICR mice and Sprague-Dawley rats were performed using the extract of the Example 1. Four group consisting of 10 mice or rats was administrated intraperitoneally with 25 mg/kg, 250 mg/kg, 500 mg/kg and 725 mg/kg of test sample or solvents (0.2 m-6, i.p.), respectively and observed for 24 hours.

[227]

[228] 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.

[229]

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

[231]

[232] Preparation of powder

[233] Dried powder of SB70E 50mg [234] Lactose lOOmg

[235] Talc lOmg

[236] Powder preparation was prepared by mixing above components and filling sealed package. [237]

[238] Preparation of tablet (I)

[239] Dried powder of SB80E lOOmg

[240] Microcrystalline cellulose 750mg

[241] Silicone dioxide 2mg

[242] Sodium starch glycolate 20mg

[243] Magnesium Stearate 3mg

[244] Hydroxypropylmethylcellulose optimum amount

[245] Ethyl cellulose optimum amount

[246] Polyvinyl alcohol optimum amount

[247] Talc optimum amount

[248] Xanthane gum optimum amount

[249] Qiinoline yellow ws aluminium lake optimum amount

[250] Tablet preparation was prepared by mixing the above components and entabletting. [251]

[252] Preparation of tablet (2)

[253] Dried powder of SB70E lOOmg

[254] Anhydrous calcium hydrogen phosphate 750mg

[255] Silicone dioxide 2mg

[256] Sodium starch glycolate 20mg

[257] Magnesium Stearate 3mg

[258] Titan oxide optimum amount

[259] Dethylpthalate optimum amount

[260] Alluraed ac aluminium lake optimum amount

[261] lecithin optimum amount

[262] Indigo carmine aluminium lake optimum amount

[263] Tablet preparation was prepared by mixing the above components and entabletting. [264]

[265] Preparation of capsule

[266] Dried powder of SB60E lOmg

[267] Crystalline cellulose 3mg [268] Lactose 14.8mg

[269] Magnesium Stearate 0.2mg

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

[271]

[272] Preparation of injection

[273] Dried powder of SB50E lOmg

[274] mannitol 180mg

[275] Distilled water for injection 2974mg

[276] Na₂HPO₄12H₂O 26mg

[277] 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 conventional injection preparation method.

[278]

[279] Preparation of liquid

[280] Dried powder of SB90E 20mg

[281] Isomerized Sugar 1Og

[282] mannitol 5g

[283] microcrystalline cellulose optimum amount

[284] calcium hydrogen phosphate optimum amount

[285] Dstilled water optimum amount

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

[287]

[288] Preparation of health food

[289] Dried powder of SB40E lOOOmg

[290] Vitamin mixture optimum amount

[291 ] Vitamin A acetate 70mg

[292] Vitamin E l.Omg

[293] Vitamin B ₁ 0.13mg

[294] Vitamin B₂ 0.15mg

[295] Vitamin B6 0.5mg

[296] Vitamin B 12 0.2mg

[297] Vitamin C lOmg

[298] Biotin lOmg [299] Amide nicotinic add 1.7mg

[300] Folic add 50mg

[301] Caldum pantothenic add 0.5mg

[302] Mineral mixture optimum amount

[303] Ferrous sulfate 1.75mg

[304] Zinc oxide 0.82mg

[305] Magnesium carbonate 25.3mg

[306] Monopotassium phosphate 15mg

[307] Dcaldum phosphate 55mg

[308] Potassium dtrate 90mg

[309] Caldum carbonate lOOmg

[310] Magnesium chloride 24.8mg

[311] The above-mentioned vitamin and mineral mixture may be varied in may ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention.

[312]

[313] Preparation of health beverage

[314] Dried powder of SB70E lOOOmg

[315] Citric add lOOOmg

[316] Oligosaccharide lOOg

[317] Apricot concentration 2g

[318] Taurine Ig

[319] Distilled water 900m£

[320]

[321] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85⁰C for 1 hour, filtered and then filling all the components in 2000m# ample and sterilizing by conventional health beverage preparation method.

[322]

[323] 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.

[324]

[325] Industrial Applicability

[326] As described in the present invention, the inventive extract of Scrophularia radix, showed potent inhibiting effect on neuronal cell death and the loss of memory and improving effect on the loss of memory being confirmed by various in vitro tests such as a protecting activity test from a neuronal cell viability as well as animal model test such as passive avoidance test, and Morris water maze test, therefore, it can be used as the therapeutics or health food for treating and preventing cognitive impairment involved diseases caused by cognitive impairment.

[327]

Claims

[1] A pharmaceutical composition comprising an extract of Scrophularia radix as an active ingredient, and the pharmaceutically acceptable carriers, adjuvants, or diluents, for the treatment and prevention of cognitive impairment involved disease by caused by cognitive impairment.

[2] The pharmaceutical composition according to claim 1 wherein said extract is extracted with the mixture solvent of water and ethanol.

[3] The pharmaceutical composition according to claim 2 wherein said extract is extracted with the mixture solvent of water and ethanol with the mixed ratio ranging from 30 to 90% (v/V%).

[4] The pharmaceutical composition according to claim 3 wherein said extract is extracted with the mixture solvent of water and ethanol with the mixed ratio ranging from 50 to 90% (v/V%).

[5] The pharmaceutical composition according to claim 4 wherein said extract is extracted with the mixture solvent of water and ethanol with the mixed ratio ranging from 65 to 85% (v/v%).

[6] The pharmaceutical composition according to claim 1 wherein said cognitive impairment is selected from Alzheimer type dementia, cerebrovascular type dementia, Pick's disease, Creutzfeldt-jakob's disease, dementia caused by cephalic damage, or Parkinson's disease .

[7] The pharmaceutical composition according to claim 1 wherein said composition is in the form of oral dosage form such as a powder, tablet, capsule, suspension, emulsion, aerosol, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, or granule; topical preparation such as a cream, ointment, lotion, gel, balm, patch, paste, suppository preparation, spray solution, or aerosol and the like; or injectable preparation such as injection, solution, suspension, or emulsion.

[8] A method for treating and preventing cognitive impairment involved disease caused by cognitive impairment of mammals including human comprising administering to said mammal an effective amount of an extract of Scrophularia radix, together with a pharmaceutically acceptable carrier thereof.

[9] A use of an extract of Scrophularia radix prepared by the above preparation method for the preparation of therapeutic agent for the treatment and prevention of cognitive impairment involved disease caused by cognitive impairment in human or mammal.

[10] A health care food comprising an extract of Scrophularia radix, together with a sitologically acceptable additive for the prevention and improvement of cognitive impairment involved disease caused by cognitive impairment.

[11] The health care food according to claim 10 wherein said extract is extracted with the mixture solvent of water and ethanol.

[12] The health care food according to claim 11 wherein said extract is extracted with the mixture solvent of water and ethanol with the mixed ratio ranging from 30 to

90% (v/v%).

[13] The health care food according to claim 12 wherein said extract is extracted with the mixture solvent of water and ethanol with the mixed ratio ranging from 50 to

90% (v/v%).

[14] The health care food according to claim 13 wherein said extract is extracted with the mixture solvent of water and ethanol with the mixed ratio ranging from 65 to

85% (v/v%).

[15] The health care food according to claim 10 wherein said "cognitive impairment" is selected from Alzheimer type dementia, cerebrovascular type dementia, Pick's disease, Creutzfeldt-jakob's disease, dementia caused by cephalic damage, or

Parkinson's disease.

[16] The health care food according to claim 15 wherein said health food is provided as powder, granule, tablet, chewing tablet, capsule or beverage type.

[17] A food additive comprising an extract of Scrophularia radix, together with a sitologically acceptable additive for the prevention and improvement of cognitive impairment involved disease caused by cognitive impairment.