WO2007061243A1

WO2007061243A1 - Composition comprising the extract of cassiae semen for treating or preventing cognitive dysfunction

Info

Publication number: WO2007061243A1
Application number: PCT/KR2006/004961
Authority: WO
Inventors: Hyun Soo Kim; Jong Hoon Ryu; Ho Jae Park; Jae Sung Jung; Seung Joo Lee; Dong Hyun Kim; Ji Wook Jung; Byung Hoon Yoon
Original assignee: INNO PHARM CO Ltd
Current assignee: INNO PHARM CO Ltd
Priority date: 2005-11-24
Filing date: 2006-11-24
Publication date: 2007-05-31
Anticipated expiration: 2008-05-24
Also published as: KR20070054783A; KR100730302B1

Abstract

The present invention relates to a composition comprising the crude extract, polar-solvent soluble extract and non-polar solvent soluble extract of cassia semen showing potent inhibiting activity of acetyl choline esterase enzyme and promoting activity of memory learning function. Therefore, it can be used as the therapeutics or health food for treating and preventing cognitive function disorder without adverse action.

Description

COMPOSITION COMPRISING THE EXTRACT OF CASSIA SEMEN FOR TREATING OR PREVENTING COGNITIVE

DYSFUNCTION

Technical Field

[1] The present invention relates to a composition comprising the extract of Cassia semen showing preventive and treating activity of cognitive dysfunction disease and the use thereof.

[2]

Background Art

[3] CNS (Central Nervous System) consisting of brain and spinal cord which plays a main role in regulating life phenomenon is a essential organ governing all the human function through from sensory and (in)voluntary movement to thinking, memory, motion, language etc. Accordingly, a rapidly progressed apoptosis of neuronal cell caused by stroke, trauma etc as well as slowly progressed apoptosis such as degenerative disease occurring in CNS caused by senile dementia for example, Alzheimer's disease or Parkinson disease etc result in irreversible functional disorder of neuronal network, which give rise to immortal failure of human function in the end. Among them, the patients suffering from Alzheimer disease, a representative senile dementia have been increased in proportion to both of extended life-span and modernized welfare facility. According to the public survey of Korea Institute for Health and Social Affair, the ratio of older people among Korean people exceeds 7% in 2000, reaches to 8.3% (3,970,000) and shall approach to 14.4% in 2019. Especially, the ratio of more than 65 years old patient suffering with senile dementia is presumed to 8.2% in Korea. In Western countries, about 10% among more than 65 years old and about 40-50% among 80 years old patient suffers with senile dementia. Since more than five million patients suffer with the disease, the medical expense caused thereby is presumed to hundred billiard dollars in a year. There have been found that more than about two hundred thousand people are suffering from dementia in Korea. In America, it has been presumed the number of the patients be increased to two fold than the number of present patients in 2030 and fourteen million (more than 350%) in 2050.

[4] Dementia is classified into two types according to its etiology, i.e., one is Alzheimer type dementia and another is vascular type dementia. Alzheimer type dementia frequently occurring in Western countries is caused by the death of cholinergic neuron resulting from the accumulation of beta-amyloid at the specific region of brain whereas vascular type dementia occurring in Oriental countries is caused by the death of neuron in the region of hippocampus resulting from vascular ischemia occurred by vascular disorder caused by hypertension, stroke, hyperlipidemia etc. Although the etiological cause of each disease is different from each other, both of the diseases result in cognitive function disorder caused by reduced action of acetylcholine, a neurotransmitter associated with cognitive function. Actylcholine having quaternary amine structure is hydrolyzed into choline by the action of acetylcholine esterase enzyme. It has been reported that the inhibition of acetylcholine esterase increase the concentration of acetylcholine resulting in improvement of dementia. Several acetylcholine esterase inhibitors have been reported, for example, tacrine, donepezil, galantamine and rivastigmine.

[5]

[6] There have been reported that cassia semen, semen of Cassia torra L and Cassia obtusifolia L beloned to Leguminosae contains several anthraquinones such as chrysophanol, emodin, rhein etc (J. A. Duke, Handbook of phytochemical constituents of GRAS herbs and other economic plants, Herbal Reference Library, CRC press, Boca Raton, Florida ppl43-144, 1992; Q. Zhang et al., Chinese Herb, 21, pp79-81, 1996) and showed various activities such as antagonistic activity of aflatoxin B 1 in Ames test (J. S. Choi et al., Planta Medica, 63, ppll-14, 1997); anti-mutagenic activity (N. J. Hao et al., Mutation Research, 328 ppl83-191, 1995); anti-hepatotoxic activity (H. S. et al., Journal of the Korean Society of Food Science and Nutrition, 30f 6^*). ppl 177-1183, 2002); and reducing activity of hyperlipidemia (U. K. Patil et al., Journal of Ethnopharmacology, 90f2-3^*>. pp249-252, 2004).

[7] However, there has been not reported or disclosed about therapeutic effect on cognitive function disorder of cassia semen in any of above cited literatures, the disclosures of which are incorporated herein by reference.

[8]

[9] To investigate an inhibiting effect ofcassia semen on the cognitive function disorder through already well-known screening tests, the inventors of the present invention have performed various in vitro and in vivo tests such as passive avoidance test and acetylcholine esterase inhibition test, and finally completed present invention by confirming that the crude extract, polar solvent and non-polar solvent soluble extract of cassia semen recover cognition function disorder and inhibit the activity of acetylcholine esterase enzyme.

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

[H]

Disclosure of Invention Technical Problem

[12] The present invention provides a pharmaceutical composition comprising a crude extract of cassia semen as an active ingredient in an effective amount to treat and prevent cognitive dysfunction disease by inhibiting acetyl choline esterase enzyme.

[13] The present invention also provides a use of an extract of cassia semen for the preparation of pharmaceutical composition to treat and prevent cognitive dysfunction disease by inhibiting acetyl choline esterase enzyme in mammal or human.

[14] The present invention also provides a health food comprising an extract of cassia semen for the prevention or alleviation of cognitive dysfunction disease by inhibiting acetyl choline esterase enzyme.

[15]

Technical Solution

[16] Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising the crude extract, polar solvent soluble or non-polar solvent soluble extract of cassia semen as an active ingredient for the treatment and prevention of cognitive dysfunction disease by inhibiting acetyl choline esterase enzyme.

[17]

[18] Above described crude extract comprises the extract prepared by extracting plant material with water, lower alcohols such as methanol, ethanol and the like, or the mixtures thereof, preferably the mixture of water and ethanol, more preferably, the mixture of water and ethanol with mixed ratio ranging from 60 to 90 %(v/v), most preferably, about 85% (v/v) ethanol solution.

[19] Above described polar solvent soluble extract can be prepared by extracting above crude extract with polar solvent, for example, water, lower alcohol such as methanol, ethanol, butanol and the like, or the mixtures thereof, preferably, butanol.

[20] Above described non-polar solvent soluble extract can be prepared by extracting above crude extract with non-polar solvent, for example, hexane, ethyl acetate or dichloromethane, preferably chloroform.

[21]

[22] It is an object of the present invention to provide a use of a crude extract, polar solvent soluble or non-polar solvent soluble extract of cassia semen for the preparation of therapeutic agent for the treatment and prevention of cognitive dysfunction disease by inhibiting acetyl choline esterase enzyme in human or mammal.

[23]

[24] It is an object of the present invention to provide a method of treating or preventing cognitive dysfunction disease by inhibiting acetyl choline esterase enzyme in a mammal comprising administering to said mammal an effective amount of crude extract, polar solvent soluble or non-polar solvent soluble extract of cassia semen, together with a pharmaceutically acceptable carrier thereof.

[25]

[26] It is another object of the present invention to provide a health food or food additives comprising crude extract, polar solvent soluble or non-polar solvent soluble extract of cassia semen, together with a sitologically acceptable additive for the prevention and improvement of cognitive dysfunction disease by inhibiting acetyl choline esterase enzyme.

[27]

[28] The term 'cognitive function disorder' disclosed herein includes Alzheimer type dementia, cerebrovascular type dementia, pick's disease, Creutzfeldt-jakob's disease, dementia caused by cephalic damage, Parkinson's disease, and so on, preferably, Alzheimer's disease or Parkinson disease.

[29]

[30] The term "cassia semen" disclosed herein includes a semen of Cassia torra L or

Cassia obtusifolia L.

[31]

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

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

[33]

[34] An inventive extracts isolated from cassia semen may be prepared in accordance with the following preferred embodiment.

[35]

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

[37]

[38] An inventive extract of cassia semen can be prepared in detail by following procedures,

[39]

[40] The inventive crude extract of cassia semen can be prepared by follows; a semen of

Cassia torra L or Cassia obtusifolia L is dried, cut, crushed and mixed with 5 to 25-fold, preferably, approximately 10 fold volume of distilled water, lower alcohols such as methanol, ethanol, butanol and the like, or the mixtures thereof, preferably the mixture of water and ethanol; the solution is treated with hot water at the temperature ranging from 20 to 100°C, preferably from 60 to 100°C, for the period ranging from 1 to 24 hours with extraction method by the extraction with hot water, cold water, reflux extraction, or ultra-sonication extraction, preferably, ultra-sonication extraction, with 1 to 5 times, preferably 2 to 3 times, consecutively; the residue is filtered to obtain the supernatant to be concentrated with rotary evaporator, at the temperature ranging from 20 to 100°C, preferably from 50 to 70°C and then dried by vacuum freeze-drying, hot air-drying or spray drying to obtain dried crude extract powder of cassia semen which can be soluble in water, lower alcohols, or the mixtures thereof.

[41]

[42] Additionally, polar solvent soluble and non-polar solvent soluble extract of present invention can be prepared by following procedure; the crude extract prepared by the above-described step, is suspended in water, and then is mixed with 1 to 100-fold, preferably, 1 to 5-fold volume of non polar solvent such as ethyl acetate, chloroform, hexane and the like; the non-polar solvent soluble layer is collected to obtain non-polar solvent soluble extract of the present invention and remaining polar solvent soluble layer is collected to obtain polar solvent soluble extract of the present invention which is soluble in water, lower alcohols, or the mixtures thereof. 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^r Ed. pp6-7, 1998).

[43]

[44] Through several in vitro and in vivo experiments including acetylcholine esterase inhibition test and passive avoidance test, the crude extract, polar-solvent soluble extract and non-polar solvent soluble extract showed potent inhibiting activity of acetyl choline esterase enzyme and promoting activity of memory learning function confirmed by passive avoidance test.

[45]

[46] In accordance with another aspect of the present invention, there is provided provide a pharmaceutical composition comprising the crude extract, polar solvent soluble or non-polar solvent soluble extract of cassia semen prepared by the above described preparation method as an active ingredient for the treatment and prevention of cognitive dysfunction disease by inhibiting acetyl choline esterase enzyme.

[47]

[48] It is an object of the present invention to provide a method of treating or preventing cognitive function disorder by inhibiting acetyl choline esterase enzyme in a mammal comprising administering to said mammal an effective amount of the crude extract, polar solvent soluble or non-polar solvent soluble extract of cassia semen prepared by the above described preparation method, together with a pharmaceutically acceptable carrier thereof.

[49]

[50] It is an object of the present invention to provide a use of the crude extract, polar solvent soluble or non-polar solvent soluble extract of cassia semen prepared by the above described preparation method for the manufacture of therapeutic agent for the treatment and prevention of cognitive function disorder by inhibiting acetyl choline esterase enzyme.

[51]

[52] 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.

[53]

[54] The inventive composition for treating and preventing cognitive function disorder may comprises above extracts as 0.01 ~ 50 % by weight based on the total weight of the composition.

[55]

[56] 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).

[57]

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

[59]

[60] 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.

[61]

[62] 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.

[63]

[64] 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), or injectable preparation (solution, suspension, emulsion).

[65]

[66] 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.

[67]

[68] 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.

[69]

[70] 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.

[71]

[72] It is another object of the present invention to provide a health food or food additives comprising the crude extract, polar solvent soluble or non-polar solvent soluble extract of cassia semen, together with a sitologically acceptable additive for the prevention and improvement of cognitive function disorder. The health food of the present invention comprises the above described extract as 0.01 to 80 %, preferably 1 to 50 % by weight based on the total weight of the composition.

[73]

[74] Above health food can be contained in health food, health beverage etc, and may be used as powder, granule, tablet, chewing tablet, capsule, beverage etc. [75]

[76] The health food of the present invention comprises above extracts as 0.01 to 80 %, preferably 1 to 50 % by weight based on the total weight of the composition.

[77]

[78] Above health food can be contained in health food, health beverage etc, and may be used as powder, granule, tablet, chewing tablet, capsule, beverage etc.

[79]

[80] Also, the present invention provide a composition of the health food beverage for the prevention and improvement of cognitive function disorder adding 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 of proper amount.

[81]

[82] To develop for health 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.

[83]

[84] Also, the extract of the present invention will be able to prevent and improve cognitive function disorder by way of adding to child and infant food, such as modified milk powder, modified milk powder for growth period, modified food for growth period.

[85]

[86] 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 IOOD of the health beverage composition.

[87]

[88] Providing that the health beverage composition of present invention contains 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, cy- clodextrin; 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 D of present beverage composition.

[89]

[90] 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 acid and the salt thereof, alginic acid and the salt thereof, organic acid, 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.

[91]

[92] 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.

[93]

[94] The extract of the present invention may be 20 to 90 % high concentrated liquid, power, or granule type.

[95]

[96] Similarly, the extract of present invention can comprise additionally one or more than one of lactose, casein, dextrose, glucose, sucrose and sorbitol.

[97]

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

[99]

[100] 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.

[101] Advantageous Effects

[102] As described in the present invention, the inventive extract of cassia semen showing potent inhibiting activity of acetyl choline esterase enzyme and promoting activity of memory learning function. Therefore, it can be used as the therapeutics or health food for treating and preventing cognitive function disorder without adverse action. [103]

Brief Description of the Drawings [104] 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; [105]

[106] Fig. 1 shows the TLC result of the extract of cassia semen;

[107] Fig. 2 shows the latency time in passive avoidance test when the crude extract of cassia semen was administrated; [108] Fig. 3 shows the latency time in passive avoidance test when the crude extract of cassia semen was administrated in a dose dependent manner; [109] Fig. 4 represents the latency time in passive avoidance test when the polar solvent soluble extract and non-polar solvent soluble extract of cassia semen was administrated in a dose dependent manner; [110] Fig. 5 represents the latency time in passive avoidance test when the butanol soluble extract of cassia semen was administrated. [Ill]

Best Mode for Carrying Out the Invention [112] 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. [113] [114] 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. [115]

Mode for the Invention [116] The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope. [117]

[118] Example 1. Preparation of the crude extract of cassia semen

[119] [120] 1-1. Extraction by ultra-sonication method

[121] lkg of dried Cassia tora Lpurchased from Kyung-dong Market located in Seoul was cut into small pieces, mixed with 10 fold volume of 50%, 85% and 100% ethanol and the mixture was subjected to sonication extraction (Branson Co. U.S.A.) for 1 hour twice and the extract was filtered with filter paper to remove the debris.

[122] The filtrates were pooled and concentrated by rotary evaporator (N- 1000, Eyela Co.

Japan) at 55 ~ 65 under reduced pressure and dried with freezing dryer (Speed Spec 3000, Bio-Rad Co. U.S.A.) to obtain various crude extract of cassia semen as follows:

[123] 50% ethanol soluble extract (yield: 20.4%) designated as 'Et 50' hereinafter;

[124] 85% ethanol soluble extract (yield: 8.5%) designated as 'Et 85' hereinafter;

[125] 100% ethanol soluble extract (yield: 10%) designated as 'Et 100' hereinafter.

[126]

[127] 1-2. Extraction by reflux extraction method

[128] Additionally, lkg of dried Cassia tora Lpurchased from Kyung-dong Market located in Seoul was cut into small pieces, mixed with 10 fold volume of 85% ethanol and the mixture was subjected to reflux extraction for 2 hour at 70 °C and the extract was filtered with filter paper to remove the debris.

[129] The filtrates were pooled and concentrated by rotary evaporator (N- 1000, Eyela Co.

Japan) at 55 ~ 65 °C under reduced pressure and dried with freezing dryer (Speed Spec 3000, Bio-Rad Co. U.S.A.) to obtain crude extract of cassia semen (yield: 10.25%) designated as 'Heat 85' hereinafter.

[130]

[131] Example 2. Preparation of polar solvent and non-polar solvent soluble extract

[132] The dried crude extract prepared in Example 1 was subject to fractionation by following procedure.

[133]

[134] 2-1. Preparation of non-polar solvent soluble fraction

[135] 2g of the extract of "Et 85" was dissolved in 1000ml of distilled water and 1 ImI of hexane was added thereto in seperatory funnel to isolate hexane soluble extract (yield: 10.25%; designated as 'Fr 4' hereinafter) to use as a sample in the following experiments.

[136] 1 ImI of chloroform was added to remaining residue to isolate chloroform soluble extract (yield: 18.4%; designated as 'Fr 3' hereinafter) to use as a sample in the following experiments.

[137]

[138] 2-2. Preparation of polar solvent soluble fraction

[139] 1 lmlof butanol was added to remaining residue produced in 2-1 to isolate butanol soluble extract (yield: 17.4%; designated as 'Fr 2' hereinafter) to use as a sample in the following experiments.

[140] The remaining residue solved in water layer(yield: 41.8%, designated as^vFrl^v hereinafter) is to use as a sample in the following experiments.

[141] After the fractionation of each hexane, chloroform, butanol and water soluble extract, the isolation was confirmed by TLC analysis and the result was shown in Fig. 1.

[142]

[143] Reference Example 1. Drug and Reagent

[144] Scopolamine and tacrine were purchased from Sigma- Aldrich Chemistry Co. and other reagents used in following experiment were purchased from conventional companies.

[145]

[146] Reference Example 2. Preparation of experiment animal

[147] 6- weeks old ICR mouse weighing about 26 to 28g was procured from Orient Co.

Ltd. (Seoul), was acclimated to clean cage in the Kyung Hee University for 5 days providing with free access to water. The breeding condition was maintained to the temperature at 23+2°C, the relative humidity of 55+10%, and to be automatically controlled to dark/light cycle at the interval of 12 hours.

[148]

[149] Reference Example 3. Statistics

[150] All the experimental result was calculated by ANOVA (one way analysis of variance) statistics program and the statistics significance was tested by Student- Newman-Keuls test at the level (p<0.05).

[151]

[152] Experimental Example 1. Passive Avoidance Test

[153]

[154] 1-1. Treatment of test group

[155] For passive avoidance test, the crude extract, hexane-soluble extract, chloroform soluble extract and butanol soluble water extract were dissolved in 10% Tween 80 (Polyoxyethylene sorbitan monooleate; Sigma Co., USA) and the various concentrations of each extract was orally administered to the mice as a test group. 1.0mg/kg of tacrine was used as a positive control group and 10% Tween solution was used as a negative control group. 10 mice per each test group were used in the experiment.

[156]

[157] 1-2. Acquisition Test

[158] An automated system with a shuttle box was used to evaluate the effects of the extracts on learning and memory associated with neuronal cell growth. [159] Avoidance shuttle box (40x20x20cm, Gemini Co., U.S.A.) was divided into two chambers of equal size and had the 3mm thickness of grid in interval of 0.5D on the floor of the box. A light chamber is equipped with an illuminator. 30 minutes after the treatment of each drug, lmg/kg dose of scopolamine was intraperitoneally administrated into the mice. 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 (0.5mA, for 3 sec) to the mouse through the grid floor when the mouse preferring darkness went out from light chamber and entered the dark chamber.

[160]

[161] 1-3. Memory Test

[162] 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 (300 sec), it was assigned a value of 300 sec as its latency.

[163]

[164] 1-4. Test Result

[165] As shown in Fig. 2, the latency time of the test group treated with Et 85 and Et 50 was significantly increased to 152.75+13.69 (p<0.001) and 93.25+10.30 (p<0.005) respectively while the latency time was the shortest in scopolamine-induced amnesia mouse (46.63+9.27 sec). In the dose-dependent test for Et 85, the latency time of the test group treated with 25mg/kg, 50mg/kg and 100mg/kg of Et 85 was significantly increased to 146.50+14.19 (p<0.005), 183.88+17.13 (p<0.001) and 138.38+13.65 (p< 0.005) respectively while the latency time was the shortest in scopolamine-induced amnesia mouse (46.63+9.27 sec) as shown in Fig. 3. In the comparison test between the extract showing different solubility, the latency time of the test group treated with butanol soluble extract (fr. 2) was significantly increased to 110.750+11.523 (p<0.005) while the latency time was the shortest in scopolamine-induced amnesia mouse (20.250+4.57 sec) as shown in Fig. 4. In the dose-dependent test for Fr. 2, the latency time of the test group treated with 5mg/kg of Fr. 2 was most increased among the test groups with different dosing as shown in Fig. 5.

[166]

[167] Experimental Example 2. Inhibition test of acety choline esterase activity

[168]

[169] 2-1. Treatmenr of test group

[170] For inhibition test of acetylcholine esterase activity, all the reagents, i.e., acetylcholine esterase, acetylcholine iodide, 5, 5-dithio-bis-(2-nitrobenzoic acid), neostigmine bromide etc were purchased from Sigma- Aldrich Chemistry (USA).

[171] [172] 2-2. Inhibition test [173] The inhibition test of acetylcholine esterase activity was determined by the procedure disclosed in the procedure (Ellman et al., Biochem. Pharmacol., July, 7 pp88-95, 1961).

[174] Delivered mouse brain was added to 10-fold volume of PBS 1 solution (12.5M sodium phosphate buffer, pH 7.0, 40OmM NaCl), macerated with Teflon glass tube and centrifuged at the speed of 100Ox g for 10 minutes. PBSl and Triton X-100 were added to the supernatant with mixing for 30 minutes and centrifuged at the speed of 100Ox g for 10 minutes to obtain its supernatant to use as an enzyme in the experiment.

[175] 1.5 ml of test solution containing the test sample or reagents, 2.6ml of buffer solution, 20 μl of 75mM acetylcholine iodide solution and 0.1ml of Ellman's reagent were mixed together and pre-incubated at 25°C for 30 minutes. 0.4ml of enzyme solution was added thereto and the absorbance was determined at 410 nm.

[176] The inhibition of acetylcholine esterase activity was calculated by following Empirical formulae 1.

[177] [178] MathFigure 1

Inhibition (%) = (0. D. of test groiφ-O.D. of blank group)/(ϋ.D. of control group-O.D. of blank group)x 100

[179] [180] At the result, the extract of 'Et 85' showed potent inhibitory activity of acetylcholine esterase activity (IC = 81.5 μg/ml) and the inhibitory activity of each test sample at the dose of lOOμg/ml was shown in Table 1. The butanol soluble extract of cassia semen (Fr.2) showed most potent inhibitory activity among the test groups.

[181] [182] Table 1

[183] Experimental Example 9. Toxicity test

[184]

[185] Methods (I)

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

Dawley rats (235+1Og, Jung-Ang Lab Animal Inc.) were performed using the extract of the present invention. Four group consisting of 10 mice or rats was administrated orally intraperitoneally with 250mg/kg, 500mg/kg, 1000mg/kg and 5000mg/kg of test sample or solvents (0.2 D, Lp) respectively and observed for 2 weeks.

[187]

[188] Methods (2)

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

[190]

[191] Results

[192] 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.

[193]

[194] 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.

[195]

[196] Preparation of powder

[ 197] Dried powder of Example 1-1 50mg

[198] Lactose lOOmg

[199] Talc lOmg

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

[201]

[202] Preparation of tablet

[203] Dried powder of Example 1 -2 50mg

[204] Corn Starch lOOmg

[205] Lactose lOOmg

[206] Magnesium Stearate 2mg

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

[208] [209] Preparation of capsule

[210] Dried powder of Example 1 -2 50mg

[211] Corn starch 1 OOmg

[212] Lactose lOOmg

[213] Magnesium Stearate 2mg

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

[215]

[216] Preparation of injection

[217] Dried powder of Example 1 -2 50mg

[218] Distilled water for injection optimum amount

[219] PH controller optimum amount

[220] 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.

[221]

[222] Preparation of liquid

[223] Dried powder of Example 1-2 0.1 ~80g

[224] Sugar 5~10g

[225] Citric acid 0.05-0.3%

[226] Caramel 0.005-0.02%

[227] Vitamin C 0.1-1%

[228] Distilled water 79-94%

[229] CO₂ gas 0.5-0.82%

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

[231]

[232] Preparation of health food

[233] Extract of Example 1-1 lOOOmg

[234] Vitamin mixture optimum amount

[235] Vitamin A acetate 70mg

[236] Vitamin E l.Omg

[237] Vitamin B₁ 0.13mg

[238] Vitamin B 0.15mg

[239] Vitamin B6 0.5mg

[240] Vitamin B 12 0.2mg

[241] Vitamin C lOmg

[242] Biotin lOmg [243] Amide nicotinic acid 1.7mg

[244] Folic acid 50mg

[245] Calcium pantothenic acid 0.5mg

[246] Mineral mixture optimum amount

[247] Ferrous sulfate 1.75mg

[248] Zinc oxide 0.82mg

[249] Magnesium carbonate 25.3mg

[250] Monopotassium phosphate 15mg

[251 ] Dicalcium phosphate 55mg

[252] Potassium citrate 90mg

[253] Calcium carbonate lOOmg

[254] Magnesium chloride 24.8mg

[255] 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.

[256]

[257] Preparation of health beverage

[258] Extract of Example 1-2 lOOOmg

[259] Citric acid lOOOmg

[260] Oligosaccharide lOOg

[261] Apricot concentration 2g

[262] Taurine Ig

[263] Distilled water 900D

[264]

[265] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85°C for 1 hour, filtered and then filling all the components in IOOOD ample and sterilizing by conventional health beverage preparation method.

[266]

[267] 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.

[268]

Industrial Applicability

[269] As described in the present invention, through several in vitro and in vivo experiments including acetylcholine esterase inhibition test and passive avoidance test, the crude extract, polar-solvent soluble extract and non-polar solvent soluble extract of cassia semen showed potent inhibiting activity of acetyl choline esterase enzyme and promoting activity of memory learning function confirmed by passive avoidance test. Therefore, it can be used as the therapeutics or health food for treating and preventing cognitive function disorder without adverse action.

Claims

[I] A pharmaceutical composition comprising the crude extract of cassia semen as an active ingredient for the treatment and prevention of cognitive dysfunction disease by inhibiting acetyl choline esterase enzyme.

[2] The pharmaceutical composition according to claim 1 wherein said crude extract is extracted with the solvent selected from the group consisting of water, lower alcohol and the mixture thereof.

[3] A pharmaceutical composition comprising the polar solvent soluble extract of cassia semen as an active ingredient for the treatment and prevention of cognitive dysfunction disease by inhibiting acetyl choline esterase enzyme.

[4] The pharmaceutical composition according to claim 3 wherein said polar solvent soluble extract is extracted with the solvent selected from the group consisting of water, lower alcohol and the mixture thereof.

[5] A pharmaceutical composition comprising the non-polar solvent soluble extract of cassia semen as an active ingredient for the treatment and prevention of cognitive dysfunction disease by inhibiting acetyl choline esterase enzyme.

[6] The pharmaceutical composition according to claim 5 wherein said non-polar solvent soluble extract is extracted with the solvent selected from the group consisting of hexane, ethylacetate, dichloromethane and chloroform.

[7] The pharmaceutical composition according to any of claim 1, 3 or 5 wherein said extract is extracted from the plant consisting of Cassia torra L or Cassia ob- tusifolia L.

[8] The pharmaceutical composition according to any of claim 1, 3 or 5 wherein said cognitive dysfunction disease comprises Alzheimer type dementia, cerebrovascular type dementia, pick's disease, Creutzfeldt-jakob's disease, dementia caused by cephalic damage and Parkinson's disease.

[9] A use of a crude extract, polar solvent soluble or non-polar solvent soluble extract of cassia semen for the preparation of therapeutic agent for the treatment and prevention of cognitive dysfunction disease by inhibiting acetyl choline esterase enzyme in human or mammal.

[10] A method of treating or preventing cognitive dysfunction disease by inhibiting acetyl choline esterase enzyme in a mammal comprising administering to said mammal an effective amount of crude extract, polar solvent soluble or non-polar solvent soluble extract of cassia semen, together with a pharmaceutically acceptable carrier thereof.

[II] A health food or food additives comprising crude extract, polar solvent soluble or non-polar solvent soluble extract of cassia semen, together with a sitologically acceptable additive for the prevention and improvement of cognitive dysfunction disease by inhibiting acetyl choline esterase enzyme.

[12] The health food according to claim 11 wherein said health food is provided as powder, granule, tablet, chewing tablet, capsule or beverage type.