WO2008044848A1 - A composition comprising an extract of rhei rhizoma or physcion compound isolated therefrom for treating or preventing cognitive dysfunction and the use thereof - Google Patents

A composition comprising an extract of rhei rhizoma or physcion compound isolated therefrom for treating or preventing cognitive dysfunction and the use thereof Download PDF

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Publication number
WO2008044848A1
WO2008044848A1 PCT/KR2007/004905 KR2007004905W WO2008044848A1 WO 2008044848 A1 WO2008044848 A1 WO 2008044848A1 KR 2007004905 W KR2007004905 W KR 2007004905W WO 2008044848 A1 WO2008044848 A1 WO 2008044848A1
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Prior art keywords
extract
rhei
physdon
isolated therefrom
compound isolated
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PCT/KR2007/004905
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French (fr)
Inventor
Hoon Yi Jung
Chan Mi Joung
Eun Joo Nam
Hee Jin Ha
Jinha Lee
Hyunsik Kim
Hee Kim
Minsik Hwang
Young Ho Kim
Eunhui Lee
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Digital Biotech Co., Ltd
Ilsung Pharmaceuticals Co., Ltd
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Publication of WO2008044848A1 publication Critical patent/WO2008044848A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
    • A61K36/708—Rheum (rhubarb)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/03—Organic compounds
    • A23L29/035—Organic compounds containing oxygen as heteroatom
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/322—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the nervous system or on mental function
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material

Definitions

  • the present invention relates to a composition
  • a composition comprising an extract of Rhei rhizama or physdon compound isolated therefrom showing preventive and treating activity of cognitive dysfunction disease and the use thereof.
  • CNS Central Nervous System
  • 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.
  • 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' 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.
  • 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.
  • 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.
  • the ratio of more than 65 years old patient suffering with senile dementia is presumed to 8.2% in Korea.
  • about 10% among more than 65 years old and about 40-50% among 80 years old patient suffers with senile dementia.
  • the medical expense caused thereby is presumed to hundred billion dollars in a year.
  • more than about two hundred thousand people are suffering from dementia in Korea.
  • 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.
  • Alzheimer' disease initiated with cognitive function disorder is one of long- term degenerative diseases resulting in the breakdown of human nature
  • acetylcholinesterase inhibitor such as Aricept ⁇ (Pfizer Co.), Exelon ®(Novartis Co.), Reiminyl ®(fanssen Co.) or NMDA receptor antagonist such as Ebixa ®(Lundbeck Co.).
  • the acetylcholine esterase inhibitor could just alleviate reduced cognitive ability and could not satisfactorily treat etiological cause of the disease.
  • the drug shows temporarily alleviated effect on only some of patients (about 40-30%), it could not maintain its potency for a long time moreover it shows various side effects such as hepato-toxidty, vomiting, and anorexia in case of long-term treatment. Accordingly, there has been urgently needed to develop new therapeutic agent to prevent and treat the disease nowadays.
  • Many multi-national pharmaceutical companies have been invested on the development in a large scale and in particular, focused in the development for beta- or gamma secretase inhibitor reducing the reproduced amount of beta-amyloid consisting of about 40 amino acids which has been presumed to be an etiological factor of Alzheimer disease.
  • beta amyloid vaccine could alleviate cognitive function in animal model test and improve the activity of brain cell as well as damaged brain neuronal cells, resulting in alleviating Alzheimer syndrome. (Janus et al., Nature 408, pp979-982, 2000; Morgan et al., Nature 408, pp982-985, 2000)
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an extract of Rhei rhia)ma or physdon compound isolated therefrom as an active ingredient in an effective amount to treat and prevent cognitive function disorder.
  • the present invention also provides a use of an extract of Rhei rhiaama or physdon compound isolated therefrom for the manufacture of pharmaceutical composition to treat and prevent cognitive function disorder.
  • the present invention also provides a health care food comprising an extract of Rhei rhiaama or physdon compound isolated therefrom for the prevention or improvement of cognitive function disorder.
  • the present invention to provide a pharmaceutical composition
  • a pharmaceutical composition comprising an extract of Rhei rhizama or physdon compound isolated therefrom as an active ingredient and pharmaceutically acceptable carrier, diluents or adjuvants to treat and prevent cognitive function disorder.
  • cognitive function disorder includes Alzheimer type dementia, cerebrovascular type dementia, pick' disease, Creutzfeldt-jakob' disease, dementia caused by cephalic damage, Parkinson' disease, and so on, preferably, Parkinson' disease.
  • an extract includes a crude extract which can be extracted with polar solvent such as water, C1-C4 lower alcohol such as methanol, ethanol, butanol etc or the mixture thereof, preferably, methanol and an organic solvent soluble extract which can be extracted with organic solvent such as acetone, di- ethylether, ethylacetate, chloroform, dichloromethane, or the mixture thereof, preferably, diethylether.
  • polar solvent such as water, C1-C4 lower alcohol such as methanol, ethanol, butanol etc or the mixture thereof, preferably, methanol and an organic solvent soluble extract which can be extracted with organic solvent such as acetone, di- ethylether, ethylacetate, chloroform, dichloromethane, or the mixture thereof, preferably, diethylether.
  • Rheum officinale BAILL Rheum coreanum NAKAI or Rheum palmatum L. belonged to Polygonaceae.
  • the inventive extract of Rhei rhizama or physdon compound isolated therefrom of the present invention can be prepared by the procedure comprising the steps consisting of; adding 0.05 to 0.3-fold, preferably, 0.1 to 0.2-fold volume of polar solvent such as water, C1-C4 lower alcohol such as methanol, ethanol, butanol etc or the mixture thereof, preferably, methanol to dried rhizama of Rheum undulatum L, Rheum officinale BAILL, Rheum coreanum NAKAI or Rheum palmatum L; extracting with extraction method by the reflux extraction, or ultra- sonication extraction, preferably, reflux extarction for the period ranging from 0.5 to 10 hours, preferably, 2 to 5 hours, at the temperature ranging from 50 to 120 0 C, preferably, 90 to 100 0 C; subjecting the solution with filtering to obtain the supernatant to be concentrated with rotary evaporator, at the temperature ranging from 35 to 45 0 C
  • a phar- maceutical composition comprisingan extract of Rhei rhiaama or physdon compound isolated therefrom prepared by the above-described preparation method as an active ingredient in an effective amount to treat and prevent cognitive function disorder.
  • composition of the present invention can contain about 0.01 ⁇ 30
  • the inventive composition for treating and preventing cognitive function disorder may comprises the above compound as 0.01 ⁇ 30 % by weight based on the total weight of the composition.
  • 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 'Pharmaceutical Sdence (Mack Publishing co, Easton PA).
  • 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 benaaate, propylhydroxy benaaate, talc, magnesium stearate and mineral oil.
  • pharmaceutically acceptable carriers e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose,
  • 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.
  • compositions of the present invention can be dissolved in oils, propylene gljcol or other solvents that are commonly used to produce an injection.
  • suitable examples of the carriers include physiological saline, polyethylene glyx>l, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them.
  • the extract of the present invention can be formulated in the form of ointments and creams.
  • compositions 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).
  • 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.
  • the desirable dose of the inventive extract or compound of the present 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 from 0.0001 to 100 mg/kg, preferably, 0.001 to 10 mg/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 or compound should be present between 0.01 to 30% by weight, preferably 0.5 to 40% by weight based on the total weight of the composition.
  • 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.
  • Theinventive extract or compoundof the present invention also can be used as a main component or additive and aiding agent in the preparation of various functional health food and health care food.
  • functional health food defined herein the functional food having enhanced functionality such as physical functionality or physiological functionality by adding the compound of the present invention to conventional food to prevent or improve cognitive function disorder in human or mammal.
  • a substance is added to a food for a specific purpose in that food, it is referred to as a direct additive and indirect food additives are those that become part of the food in trace amounts due to its packaging, storage or other handling.
  • Health foods can be contained in food, health beverage, dietary therapy etc, and may be used as a form of powder, granule, tablet, chewing tablet, capsule, beverage etc for preventing or improvingcognitive function disorder.
  • above described extract or compound can be added to food or beverage for prevention and improvement of cognitive function disorder.
  • the amount of above described compound in food or beverage as a functional health food or health care food may generally range from about 0.01 to 100 w/w % of total weight of food for functional health food composition.
  • the preferable amount of the compound of the present invention in the functional health food, health care food or special nutrient food may be varied in accordance to the intended purpose of each food, it is preferably used in general to use as a additive in the amount of the extract or compound of the present invention ranging from about 0.01 to 5% in food such as noodles and the like, from 40 to 100% in health care food on the ratio of 100% of the food composition.
  • the health beverage composition of present invention contains above described extract or compound as an essential component in the indicated ratio
  • the other component can be various deodorant or natural carbohydrate etc such as conventional beverage.
  • natural carbohydrate are monosaccharide such as gluoose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol, and erythritol etc.
  • natural deodorant such as taumatin, stevia extract such as levaudioside A, gljcyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al.
  • 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 m# of present beverage composition.
  • 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, chooolate 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, gljcerin, 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.
  • addable food comprising aforementioned extract therein are various food, beverage, gum, vitamin complex, health improving food and the like.
  • Inventive extract or compound of the present invention has no toxicity and side effect therefore; they can be used with safe.
  • Fig. 1 shows the isolation scheme for inventive extract and compound from the extract of Rhei rhiaama
  • Fig. 2 shows the inhibitory effect of the organic solvent soluble extract of Rhei rhia)ma on the aggregation and cytotoxicity of beta amyloid
  • Fig. 3 shows the inhibitory effect of Rh-7 on the aggregation and cytotoxity of beta amyloid
  • Fig. 1 shows the isolation scheme for inventive extract and compound from the extract of Rhei rhiaama
  • Fig. 2 shows the inhibitory effect of the organic solvent soluble extract of Rhei rhia)ma on the aggregation and cytotoxicity of beta amyloid
  • Fig. 3 shows the inhibitory effect of Rh-7 on the aggregation and cytotoxity of beta amyloid
  • Fig. 4 represents the inhibitory effect of physdon on the aggregation and cytotoxity of beta amyloid
  • Fig. 5 represents the inhibitory effect of the organic solvent soluble extract of Rhei rhia)ma on the cytotoxicity of beta amyloid
  • FIG. 6 presents the result of memory learning study (Y maze test) using physdon;
  • FIG. 7 depicts the result of memory learning study (PA test) using physdon
  • FIG. 8 depicts comparison of mouse brain staining between control group(a,c,e) and test group(b,d,f) treated with physdon.
  • Example 1 Preparation of an extract of Rhei rhizoma and physcion compound isolated therefrom (See Fig. 1)
  • the structure of physcion compound was determined by comparing the physicochemical property of physcion compound with the data disclosed in already-known literature (Chu-Hyun Lee, Arch Pharm Res., Vol26( 5), pp367-374, 2003).
  • ThT(Thioflavin T) was diluted with 5OmM glydne buffer solution and the diluted solution was added to each well by 150 ⁇ l/well.
  • the absorbance was determined by microplate reader (SAFIRE, TECAN) at 450 nm excitation wavelength/ 480 nm emission wavelength and the inhibition activity of the test sample on beta amyloid aggregation was transformed into IC .
  • Physdon compound also showed potent inhibitory effect on the aggregation of beta amyloid (See Fig. 4).
  • HT22 mouse neuronal cell line was incubated in DMEM (Dulbe ⁇ x)' Modified Eagle'Medium, Gibco-BRL) medium supplemented with 10% FBS (Fetal Bovine Serum, Hjclone) and 1% penicillin/strep tomydn (Sigma Co.) Prior to test, HT22 cell was incubated on 96 well plates with a density of 5x10 cell/well and further incubated in serum free DMEM medium for 1 hour before the treatment of test sample. Various concentration of diethylether extract prepared in Example 1 used as a test sample was added thereto and incubated for 1 hour.
  • Aggregated beta amyloid (US peptide) was treated thereto to the concentration of 25 ⁇ M and incubated for 18 hours to induce cell necrosis.
  • 5mg/ml of MTT (3-(4,5-dimethyl-2-thia»lyl)-2,5-diphenyl-2H-tetraz3lium bromide) solution was added each well with 15 ⁇ l/well and the well was incubated for 4 hours.
  • Dissolving buffer solution (10% SDS, 50% dimethyl formamide, pH 4.7) was added to each well with 100 ⁇ l/well and reacted for overnight. 18 hours after the reaction, the absorbance of solution was determined by microplate reader (SUNRISE, TECAN) at 570 run/ 630 nm wavelength (Gillardon, F. et al., Brain Research, 706(D ppl69-172, 1996).
  • HT22 cell was incubated in accordance with similar method disclosed in 1-1-2 and various concentration of the test sample prepared in Example 1 was added to the cell to incubate for 18 hours. MTT solution and dissolving buffer solution was added to cell serially and the absorbance 63 was determined by microplate reader (SUNRISE, TECAN) at 570 nm.
  • Synthetic beta amyloid 1-42 was dissolved in DMSO in order to be 250 ⁇ M solution and diluted with PBS to 1OnM and aggregated at 37 0 C for four days (Passive Avoidance test) or six days (Y maze test).
  • Example 1-3 was administrated into the mice at the interval of once a day in case of Y maze test and the test samples were continuously administrated for three days in case of passive avoidance test dividing into three test groups, i.e., 10 mg/kg treatment group, 100 mg/kg treatment group and 150mg/kg treatment group., Each test was performed at the next day of the administration.
  • Y-maze test was performed two days after the administration of beta amyloid.
  • Black acrylic Y maze box consists of three arms (length: 40cm, Height: 10cm, Width: 5cm) having identical angle between each other.
  • the mice were positioned at the center of maze and let to move freely for eight minutes with the maze.
  • the entering order of mice in the pathway was observed and the entering latency time was determined when four limbs was entered within the pathway.
  • the determined spontaneous alteration behavior was calculated by following empirical formula 1 and the actual alteration was assigned to one time at the time that mice was entered three pathways continuously.
  • mice weighing 25g were initially placed in the light chamber and allowed for habituation. The door was then opened and as soon as mice preferring darkness went out from light chamber and entered the dark chamber the door was closed immediately, and an electric shock (0.25 mA, 3s, once) was delivered to the mouse through the grid floor for 3 sec.
  • the change of latency time means the decline or recovery of memory and the lengthened latency time means the increased memory.
  • the latency time treated with physdon compound significantly increased compared with the sham control group (p ⁇ 0.05).
  • the mouse brain was delivered, kept in 10% formalin solution for 24 hours and transferred to 30% sucrose solution. After fixing the brain, the brain was performed to coronal section with a width of 40 ⁇ m using by cryostat. The sliced brain was performed to Cresyl violet staining to confirm the injury of brain neuronal cell, to ChAT staining to confirm the injury of cholinergic neuron and to GFAP staining to confirm the activation of astrocytes.
  • tissue was placed on gelatin-coated slide to stain with Cresyl violet, the tissue was performed to dehydration using ethanol. The tissue was incubated for about 3 minutes and dipped into 0.5% Cresyl violet solution for 30 minutes. After the solution was performed to re-hydration with ethanol, the slice was dipped into xylene for 3 minutes. The dried tissue was fixed with Canada balsam mounting medium.
  • test group treated with physdon compound showed more enriched neuronal cells than the control group treated with only beta amyloid.
  • PBST was used to washing tissues.
  • the tissue was pretreated with 0.5% H O and then treated with 5% FBS at room temperature for 1 hour to remove non-spedfic binding.
  • the tissue was incubated at 4 0 C for overnight using by mouse anti-GFAP (1:200) monoclonal antibody and goat-anti-ChAT (1:200) polyclonal antibody.
  • mouse anti-GFAP (1:200) monoclonal antibody
  • goat-anti-ChAT (1:200) polyclonal antibody.
  • a horse radish peroxidase-conjugated anti-mouse IgG and anti- goat IgG secondary antibody (1:600) was incubated at room temperature for 1 hour and detected by DAB kit after the incubation.
  • the inoculation of beta-amyloid into the cerebral ventricle induced the activation of astrocyte confirmed by GFAP staining.
  • Tablet preparation was prepared by mixing above components and entabletting. [201]
  • 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.
  • Liquid preparation was prepared by dissolving active component, filling all the components and sterilizing by conventional liquid preparation method. [224]
  • Vitamin mixture optimum amount
  • Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85 0 C for 1 hour, filtered and then filling all the components in 1000ml ample and sterilizing by conventional health beverage preparation method.
  • an extract of Rhei rhizoma or physdon compound isolated therefrom inhibits beta amyloid aggregation as well as the toxicity and cell apoptosis caused by beta amyloid resulting in stimulating the proliferation of neuronal cells, Therefore, it can be used as the therapeutics or health care food for treating and preventing cognitive function disorder without adverse action.

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Abstract

The present invention relates to a composition comprising an extract of Rhei rhizoma or physdon compound isolated therefrom showing potent inhibiting effect on the aggregation and toxicity of beta-amyloid and recovering activity of memory learning disorder confirmed by Y maze study and Passive Avoidance study. Therefore it can be used as the therapeutics or health care food for treating and preventingcognitive function disorder with safe.

Description

Description
A COMPOSITION COMPRISING AN EXTRACT OF
RHEI RHIZOMA OR PHYSCION COMPOUND
ISOLATED THEREFROM FOR TREATING OR
PREVENTING COGNITIVE DYSFUNCTION AND THE
USE THEREOF Technical Field
[1] The present invention relates to a composition comprising an extract of Rhei rhizama or physdon compound isolated therefrom showing preventive and treating activity of cognitive dysfunction disease and the use thereof. Background Art
[2] 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' 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 billion 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.
[3] Since Alzheimer' disease initiated with cognitive function disorder is one of long- term degenerative diseases resulting in the breakdown of human nature, there have been tried to develop effective and preventive drugs till now, for example, acetylcholinesterase inhibitor such as Aricept ©(Pfizer Co.), Exelon ®(Novartis Co.), Reiminyl ®(fanssen Co.) or NMDA receptor antagonist such as Ebixa ®(Lundbeck Co.). However, the acetylcholine esterase inhibitor could just alleviate reduced cognitive ability and could not satisfactorily treat etiological cause of the disease. Although the drug shows temporarily alleviated effect on only some of patients (about 40-30%), it could not maintain its potency for a long time moreover it shows various side effects such as hepato-toxidty, vomiting, and anorexia in case of long-term treatment. Accordingly, there has been urgently needed to develop new therapeutic agent to prevent and treat the disease nowadays. Many multi-national pharmaceutical companies have been invested on the development in a large scale and in particular, focused in the development for beta- or gamma secretase inhibitor reducing the reproduced amount of beta-amyloid consisting of about 40 amino acids which has been presumed to be an etiological factor of Alzheimer disease. The basic study on the Alzheimer disease has been actively attempted in Korea however the development of Alzheimer treating agent has been merely progressed till now. Since there have been found in animal model test as well as clinical trial that the development of gamma secretase inhibitor is associated with considerable toxicity, it has been proved to be not recommendable whereas the development of beta secretase inhibitor is recommendable as proven by gene deficiency transformed animal model test. It is also regarded as a safe tool to focus on targeting the factors involved in beta amyloid aggregation. There has been reported that "phenserine"developed by Axonyx Co. in USA has been progressed in Clinical trial 2 phase and it shows dual activities of inhibiting cholinesterase as well as beta amyloid aggregation. (Greig et al., J. Med. Chem. 44 pp4062-4071, 2001; www.medicalnewstoday.com; www.alzforum.org/drg/drc)
[4] The development of vaccine using beta amyloid has been known as another possible method. There has been reported that the serial study on the vaccine progressed by Elan Co. failed because of its un-predictable side effects such as encephalitis during clinical trial. However, it has been reported that beta amyloid vaccine could alleviate cognitive function in animal model test and improve the activity of brain cell as well as damaged brain neuronal cells, resulting in alleviating Alzheimer syndrome. (Janus et al., Nature 408, pp979-982, 2000; Morgan et al., Nature 408, pp982-985, 2000)
[5]
[6] There have been reported that the extract of Rhei rhizoma, a rhizama of Rheum undulatum L, Rheum officinale BAILL, Rheum coreanum NAKAI or Rheum palmatum L belonged to Polygonaceae, contains several dianthrone glyx>sides such as sennoside A-F, and several anthraquinones such as chrysophanol, emodin, aloe- emodin, rhein, physdon, dtreorosein etc (Il-Moo Chang et al, Oriental Medicine and Medical Science Encyclopedia, Seoul National University, 2003).
[7] There have been also reported on the pharmacological activity of Rhei rhiaama, for example, anti-hyperlipidemia activity (Korean Patent No. 2004-0027100), antibacterial activity (Mi-kyung Lim, Korean J. Soc. Food Cookery ScL, 19 p4, 2003) etc. However, there has been not reported or disclosed about therapeutic effect on cognitive function disorder of an extract of Rhei rhiaama or physdon compound isolated therefrom in any of above dted literatures, the disclosures of which are incorporated herein by reference.
[8]
[9] To investigate an inhibiting effect of an extract of Rhei rhizoma or physdon compound isolated therefrom on cognitive function disorder through already well- known screening tests, the inventors of the present invention have intensively screened various plants showing potent inhibiting activity of beta-amyloid aggregation and memory learning recovery study using passive avoidance test etc, and finally completed present invention by confirming that an extract of Rhei rhiaama or physdon compound isolated therefrom inhibits beta-amyloid aggregation and cell cytotoxidty resulting in stimulating the proliferation of neuronal cells as well as recovers memory learning injury caused by neuronal cell injury
[10]
[11] These and other objects of the present invention will become apparent from the detailed disclosure of the present invention provided hereinafter.
[12]
Disclosure of Invention Technical Problem
[13] The present invention provides a pharmaceutical composition comprising an extract of Rhei rhia)ma or physdon compound isolated therefrom as an active ingredient in an effective amount to treat and prevent cognitive function disorder.
[14] The present invention also provides a use of an extract of Rhei rhiaama or physdon compound isolated therefrom for the manufacture of pharmaceutical composition to treat and prevent cognitive function disorder. [15] The present invention also provides a health care food comprising an extract of Rhei rhiaama or physdon compound isolated therefrom for the prevention or improvement of cognitive function disorder.
[16]
Technical Solution
[17] Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising an extract of Rhei rhizama or physdon compound isolated therefrom, as an active ingredient in an effective amount to treat and prevent cognitive function disorder.
[18]
[19] Spedfically, the present invention to provide a pharmaceutical composition comprising an extract of Rhei rhizama or physdon compound isolated therefrom as an active ingredient and pharmaceutically acceptable carrier, diluents or adjuvants to treat and prevent cognitive function disorder.
[20] It is an object of the present invention to provide a method of treating or preventing cognitive function disorder in a mammal comprising administering to said mammal an effective amount of an extract of Rhei rhizama or physdon compound isolated therefrom, together with a pharmaceutically acceptable carrier thereof.
[21] It is an object of the present invention to provide a use of an extract of Rhei rhizama or physdon compound isolated therefrom for the manufacture of therapeutic agent for the treatment and prevention of cognitive function disorder.
[22] The term "cognitive function disorder" disclosed herein includes Alzheimer type dementia, cerebrovascular type dementia, pick' disease, Creutzfeldt-jakob' disease, dementia caused by cephalic damage, Parkinson' disease, and so on, preferably, Parkinson' disease.
[23] The term "an extract "disclosed herein includes a crude extract which can be extracted with polar solvent such as water, C1-C4 lower alcohol such as methanol, ethanol, butanol etc or the mixture thereof, preferably, methanol and an organic solvent soluble extract which can be extracted with organic solvent such as acetone, di- ethylether, ethylacetate, chloroform, dichloromethane, or the mixture thereof, preferably, diethylether.
[24] The term "Rhei rhiz3ma"disclosed herein includes a rhizama of Rheum undulatum L,
Rheum officinale BAILL, Rheum coreanum NAKAI or Rheum palmatum L. belonged to Polygonaceae.
[25] [26] An inventive extract of Rhei rhizama or physdon compound isolated therefrom may be prepared in accordance with the following preferred embodiment.
[27]
[28] Hereinafter, the present invention is described in detail.
[29]
[30] An inventive extract of Rhei rhizama or physdon compound isolated therefrom can be prepared in detail by following procedures,
[31]
[32] The inventive extract of Rhei rhizama or physdon compound isolated therefrom of the present invention can be prepared by the procedure comprising the steps consisting of; adding 0.05 to 0.3-fold, preferably, 0.1 to 0.2-fold volume of polar solvent such as water, C1-C4 lower alcohol such as methanol, ethanol, butanol etc or the mixture thereof, preferably, methanol to dried rhizama of Rheum undulatum L, Rheum officinale BAILL, Rheum coreanum NAKAI or Rheum palmatum L; extracting with extraction method by the reflux extraction, or ultra- sonication extraction, preferably, reflux extarction for the period ranging from 0.5 to 10 hours, preferably, 2 to 5 hours, at the temperature ranging from 50 to 120 0C, preferably, 90 to 100 0C; subjecting the solution with filtering to obtain the supernatant to be concentrated with rotary evaporator, at the temperature ranging from 35 to 45 0C; drying to obtain dried inventive crude extract powder of Rhei rhizamaat 1 s step; suspending said crude extract in distilled water and adding organic-solvent soluble extract which can be extracted with organic solvent such as acetone, diethylether, ethylacetate, chloroform, dichloromethane, or the mixture thereof, preferably, diethylether thereto; mixing and subjecting to fractionation with 3 to 6 times to obtain inventive organic solvent soluble extract of Rhei rhizama at 2 n step; subjecting the organic solvent soluble extract to repeating silica gel column chromatography (Silica gel 60, 70-230 mesh) eluting with solvent mixture (Hexane: ethylacetate) increasing the polarity of the eluting solvent to obtain inventive physdon compound of the present invention.
[33] 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 Ei. pp6-7, 1998).
[34]
[35] In accordance with another aspect of the present invention, there is provided a phar- maceutical composition comprisingan extract of Rhei rhiaama or physdon compound isolated therefrom prepared by the above-described preparation method as an active ingredient in an effective amount to treat and prevent cognitive function disorder.
[36] It is an object of the present invention to provide a method of treating or preventing cognitive function disorder in a mammal comprising administering to said mammal an effective amount ofan extract of Rheirhizamaor physdon compoundisolated therefrom prepared by the above-described preparation method, together with a pharmaceutically acceptable carrier thereof.
[37] It is an object of the present invention to provide a use ofan extract of Rhei rhizama or physdon compound isolated therefrom prepared by above described preparation method for the manufacture of therapeutic agent for the treatment and prevention of cognitive function disorder.
[38]
[39] The pharmaceutical composition of the present invention can contain about 0.01 ~ 30
% by weight of the above extract based on the total weight of the composition.
[40]
[41] Through various screening test to determine inhibiting effect ofan extract of Rhei rhizama or physdon compound isolated therefrom inhibit beta-amyloid aggregation as well as the toxidty and cell apoptosis caused by beta amyloid resulting in stimulating the proliferation of neuronal cells and moreover recover memory learning injury caused by neuronal cell injury.
[42]
[43] The inventive composition for treating and preventing cognitive function disorder may comprises the above compound as 0.01 ~ 30 % by weight based on the total weight of the composition.
[44]
[45] 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 'Pharmaceutical Sdence (Mack Publishing co, Easton PA).
[46]
[47] Hereinafter, the following formulation methods and exripients are merely exemplary and in no way limit the invention.
[48] [49] 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 benaaate, propylhydroxy benaaate, 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.
[30]
[51] For example, the compositions of the present invention can be dissolved in oils, propylene gljcol or other solvents that are commonly used to produce an injection. Suitable examples of the carriers include physiological saline, polyethylene glyx>l, 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.
[52]
[53] 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).
[54]
[55] 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.
[56]
[57] The desirable dose of the inventive extract or compound of the present 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 from 0.0001 to 100 mg/kg, preferably, 0.001 to 10 mg/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 or compound should be present between 0.01 to 30% by weight, preferably 0.5 to 40% by weight based on the total weight of the composition.
[58]
[59] 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.
[60]
[61] Theinventive extract or compoundof the present invention also can be used as a main component or additive and aiding agent in the preparation of various functional health food and health care food.
[62]
[63] Accordingly, it is the other object of the present invention to provide a functional health food comprisingan extract of Rhei rhizama or physdon compound isolated therefrom for aiding cognitive function disorder.
[64]
[65] The term "functional health food"defined herein the functional food having enhanced functionality such as physical functionality or physiological functionality by adding the compound of the present invention to conventional food to prevent or improve cognitive function disorder in human or mammal.
[66]
[67]
[68] It is the other object of the present invention to provide a health care food comprising an extract of Rhei rhizama or physdon compound isolated therefrom,together with a sitologically acceptable additive for the prevention and improvement of cognitive function disorder.
[69]
[70] The term "health care food"defined herein the food containing the compound of the present invention showing no spedfic intended effect but general intended effect in a small amount of quantity as a form of additive or in a whole amount of quantity as a form of capsule, pill, tablet etc. [71]
[72] The term "sitologically acceptable additive"defined herein any substance the intended use which results or may reasonably be expected to result-directly or indirectly-in its becoming a component or otherwise affecting the characteristics of any food for example, thickening agent, maturing agent, bleaching agent, se- questerants, humectant, anti-caking agent, clarifying agents, curing agent, emulsifier, stabilizer, thickner, bases and acid, foaming agents, nutrients, coloring agent, flavoring agent, sweetner, preservative agent, antioxidant, etc, which shall be explained in detail as follows.
[73] If a substance is added to a food for a specific purpose in that food, it is referred to as a direct additive and indirect food additives are those that become part of the food in trace amounts due to its packaging, storage or other handling.
[74]
[75] Above described health foods can be contained in food, health beverage, dietary therapy etc, and may be used as a form of powder, granule, tablet, chewing tablet, capsule, beverage etc for preventing or improvingcognitive function disorder.
[76]
[77] Also, above described extract or compound can be added to food or beverage for prevention and improvement of cognitive function disorder. The amount of above described compound in food or beverage as a functional health food or health care food may generally range from about 0.01 to 100 w/w % of total weight of food for functional health food composition. In particular, although the preferable amount of the compound of the present invention in the functional health food, health care food or special nutrient food may be varied in accordance to the intended purpose of each food, it is preferably used in general to use as a additive in the amount of the extract or compound of the present invention ranging from about 0.01 to 5% in food such as noodles and the like, from 40 to 100% in health care food on the ratio of 100% of the food composition.
[78]
[79] Providing that the health beverage composition of present invention contains above described extract or compound 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 gluoose, 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, gljcyrrhizin 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 m# of present beverage composition.
[80]
[81] 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, chooolate 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, gljcerin, 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.
[82]
[83] Inventive extract or compound of the present invention has no toxicity and side effect therefore; they can be used with safe.
[84]
[85] 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.
[86]
[87]
Advantageous Effects
[88] An extract of Rhei rhizama or phy scion compound isolated therefrominhibits beta amyloid aggregation as well as the toxicity and cell apoptosis caused by beta amyloid resulting in stimulating the proliferation of neuronal cells, Therefore, it can be used as the therapeutics or health care food for treating and preventing cognitive function disorder without adverse action.
[89] [90]
Brief Description of the Drawings
[91] 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; [92] [93] Fig. 1 shows the isolation scheme for inventive extract and compound from the extract of Rhei rhiaama; [94] Fig. 2 shows the inhibitory effect of the organic solvent soluble extract of Rhei rhia)ma on the aggregation and cytotoxicity of beta amyloid; [95] Fig. 3 shows the inhibitory effect of Rh-7 on the aggregation and cytotoxity of beta amyloid; [96] Fig. 4 represents the inhibitory effect of physdon on the aggregation and cytotoxity of beta amyloid; [97] Fig. 5 represents the inhibitory effect of the organic solvent soluble extract of Rhei rhia)ma on the cytotoxicity of beta amyloid;
[98] Fig. 6 presents the result of memory learning study (Y maze test) using physdon;
[99] Fig. 7 depicts the result of memory learning study (PA test) using physdon;
[100] Fig. 8 depicts comparison of mouse brain staining between control group(a,c,e) and test group(b,d,f) treated with physdon. [101]
Best Mode for Carrying Out the Invention [102] 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. [103] [104] The present invention is more spedfically explained by the following examples.
However, it should be understood that the present invention is not limited to these examples in any manner. [105]
Mode for the Invention [106] The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope. [107] [108] Example 1. Preparation of an extract of Rhei rhizoma and physcion compound isolated therefrom (See Fig. 1)
[109]
[110] 1-1. Preparation of methanol soluble extract
[111] 6kg of dried of Rhei rhiaama purchased from Kyung-dong Market located in Seoul was cut into small pieces, mixed with 1.5 L of methanol and subjected to reflux- extraction for 3 hours at three times. The residue is filtered to obtain the supernatant and the filtrate was dried with vaαum evaporator at 4O0C to obtain 2.3kg of methanol soluble extract of Rhei rhiaama.
[112]
[113] 1-2. Preparation of diethylether soluble extract
[114] 2.3 kg of methanol soluble extract was suspended in distilled water and diethylether solvent was added thereto. The suspension was subject to fractionation at 3 to 4 times to obtain water soluble extract and diethylether soluble extract. The diethylether soluble extract was concentrated and dried to obtain 268g of diethylether soluble extract of Rhei rhiaama.
[115]
[116] 1-3. Preparation of phvsdon compound
[117] As shown in Fig. 1, 268g of diethylether soluble extract of Rhei rhioma was loaded to Silica gel column chromatography (Silica gel 60, 70-230 mesh) eluting with solvent mixture (hexane:ethylacetate= 100:1)3 to obtain 18 purified fractions. To purify further, the 6 , 7 and 8 fractions among said fractions (designated as "Rh-6, Rh-7 and Rh-8"respectively, hereinafter) was loaded to Silica gel column chromatography (Silica gel 60, 70-230 mesh) eluting with solvent mixture (hexane:dichloromethane= 3:1) to obtain 1.5 g of physcion compound. The structure of physcion compound was determined by comparing the physicochemical property of physcion compound with the data disclosed in already-known literature (Chu-Hyun Lee, Arch Pharm Res., Vol26( 5), pp367-374, 2003).
[118]
[119] [Chemical Formula 1]
[120]
Figure imgf000014_0001
[123] Phvsdon [124] [125] [126] Experimental Example 1. In vitro activity test [127] 1-1. Preparation of experiment [128] 1-1-1. Inhibition test of beta amyloid aggregation [129] [130] Synthetic beta amyloid 1-42 (BACHEM) was dissolved in DMSO in order to 250 μm solution and diluted with PBS into 1/10 on fluorescent black plate to induce aggregation. By comparing with inhibition activity of the extract and compound prepared in Example 1 on beta amyloid aggregation, the test sample showing more than 50% inhibition activity at 10 μg/ml was chosen to use and added to react for 1 hour at room temperature. ThT(Thioflavin T) was diluted with 5OmM glydne buffer solution and the diluted solution was added to each well by 150μl/well. The absorbance was determined by microplate reader (SAFIRE, TECAN) at 450 nm excitation wavelength/ 480 nm emission wavelength and the inhibition activity of the test sample on beta amyloid aggregation was transformed into IC .
50
[131] [132] As can be shown in Figs. 2-4, the diethytether soluble extract prepared in Example 1-2 showed potent inhibitory activity (IC value= 1.1 μg/ml; See Fig. 2). The purified
50 extracts, i.e., Rh-6, Rh-7 and Rh-8, also showed more than 50% inhibitory activity, especially, Rh-7 showed most potent inhibitory activity among them (IC value= 1.25 μg/ml) of beta- amyloid aggregation (See Fig. 3). Physdon compound also showed potent inhibitory effect on the aggregation of beta amyloid (See Fig. 4).
[133] [134] 1-1-2. Inhibition test of beta amyloid toxicity [135] HT22 mouse neuronal cell line was incubated in DMEM (Dulbeαx)' Modified Eagle'Medium, Gibco-BRL) medium supplemented with 10% FBS (Fetal Bovine Serum, Hjclone) and 1% penicillin/strep tomydn (Sigma Co.) Prior to test, HT22 cell was incubated on 96 well plates with a density of 5x10 cell/well and further incubated in serum free DMEM medium for 1 hour before the treatment of test sample. Various concentration of diethylether extract prepared in Example 1 used as a test sample was added thereto and incubated for 1 hour. Aggregated beta amyloid (US peptide) was treated thereto to the concentration of 25 μM and incubated for 18 hours to induce cell necrosis. 5mg/ml of MTT (3-(4,5-dimethyl-2-thia»lyl)-2,5-diphenyl-2H-tetraz3lium bromide) solution was added each well with 15 μl/well and the well was incubated for 4 hours. Dissolving buffer solution (10% SDS, 50% dimethyl formamide, pH 4.7) was added to each well with 100 μl/well and reacted for overnight. 18 hours after the reaction, the absorbance of solution was determined by microplate reader (SUNRISE, TECAN) at 570 run/ 630 nm wavelength (Gillardon, F. et al., Brain Research, 706(D ppl69-172, 1996).
[136]
[137] As can be shown in Fig. 5, 10 μg/ml of the diethytether soluble extract prepared in Example 1-2 completely inhibits the cell necrosis caused by beta- amyloid.
[138]
[139] 1-1-3. Determination of cytotoxicity
[140] To determine the toxicity of test sample, HT22 cell was incubated in accordance with similar method disclosed in 1-1-2 and various concentration of the test sample prepared in Example 1 was added to the cell to incubate for 18 hours. MTT solution and dissolving buffer solution was added to cell serially and the absorbance 63 was determined by microplate reader (SUNRISE, TECAN) at 570 nm.
[141]
[142] At the result, it has been confirmed that the diethytether soluble extract prepared in Example 1-2 showed no cytotoxicity.
[143]
[144]
[145] Experimental Example 2. In vivo activity test
[146]
[147] 2-1. Experimental Design
[148] For passive avoidance test, male ICR mouse weighing 25g purchased from Samtaco Co. was bred with five mice per cage and the cage was kept with following condition maintaining the temperature of 22+20C and the relative humidity of 50+50C under the regularly controlled light/dark condition with an interval of 12 hours.
[149] Synthetic beta amyloid 1-42 (BACFJEM) was dissolved in DMSO in order to be 250μM solution and diluted with PBS to 1OnM and aggregated at 370C for four days (Passive Avoidance test) or six days (Y maze test).
[130] Aggregated beta amyloid 1-42 was administrated into the mice according to the procedure disclosed in the literature (Lausen & Belknap, J. Pharmacol. Methods, 16 pp355-357, 1986).
[151] Five microlmliter of aggregated beta amyloid 1-42 was administrated into the 2.4mm depth of bregma region with 50μl of Hamilton micro-syringe equipped with 26-gauge needle. The behavior tests were divided into Y maze test and PA (passive avoidance) test after the beta amyloid administration. Y maze test was performed 2 days after the administration and PA test was 3 days after the administration. Each test was done with more than 10 mice.
[152] At the end of the experiment, the brain of animals was delivered and kept in 10% formalin solution to staining.
[153]
[154] 2-2. Drug Treatment
[155] After the administration of beta amyloid, the physdon compound prepared in
Example 1-3 was administrated into the mice at the interval of once a day in case of Y maze test and the test samples were continuously administrated for three days in case of passive avoidance test dividing into three test groups, i.e., 10 mg/kg treatment group, 100 mg/kg treatment group and 150mg/kg treatment group., Each test was performed at the next day of the administration.
[156]
[157]
[158] 2-3 Behavior procedure
[159]
[160] 2-3-1. AD acute model experiment- Y maze test
[161] Y-maze test was performed two days after the administration of beta amyloid. Black acrylic Y maze box consists of three arms (length: 40cm, Height: 10cm, Width: 5cm) having identical angle between each other. The mice were positioned at the center of maze and let to move freely for eight minutes with the maze. The entering order of mice in the pathway was observed and the entering latency time was determined when four limbs was entered within the pathway. To determine the spatial memory, the determined spontaneous alteration behavior was calculated by following empirical formula 1 and the actual alteration was assigned to one time at the time that mice was entered three pathways continuously. [162] [Empirical Formula 1]
[163]
[164] Spontaneous alteration (%) = [actual alteration/ total arm entries -2] x 100
[165]
[166] 10mg/kg, 100mg/kg and 150mg/kg of the compound was orally directly administrated into the mice treated with beta amyloid through intracerebro- ventricular pathway once a day for two days and the recovering effect of the compound on brain damage was found through Y maze test. The treatment groups of 10 mg/kg, 100 mg/kg and 150mg/kg of physdon showed potent recovering activity of memory learning disorder caused by beta amyloid administration (See Fig. 6).
[167]
[168] 2-3-2. AD acute model experiment- Passive Avoidance test
[169] Passive Avoidance test was performed three days after the administration of beta amyloid. Black avoidance shuttle box was divided into two chambers of equal size (18cm L x 9.5cm W x 17cm H) partitioned by compartment door (4cm L x 3.5cm W) allowing electricity to run on the floor of the dark compartment. A light chamber is equipped with a 20-W lamp on the hinged plexiglass lid and the mice were allowed to enter dark chamber through compartment door.
[170] The experiments consisted of training and test sessions.
[171] In the training session, male mice weighing 25g were initially placed in the light chamber and allowed for habituation. The door was then opened and as soon as mice preferring darkness went out from light chamber and entered the dark chamber the door was closed immediately, and an electric shock (0.25 mA, 3s, once) was delivered to the mouse through the grid floor for 3 sec.
[172]
[173] At 24 hours after the training session, 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 0.25mA of electronic shock for 3 second. Latency to enter the dark compartment from the light compartment was measured as a step through latency. 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. Passive avoidance test was performed using by the mice orally administrated with test sample prepared in Example in the dose of amount of 10 mg/kg and 100 mg/ kg once a day for three days.
[174] As shown in the Fig. 7, the change of latency time means the decline or recovery of memory and the lengthened latency time means the increased memory. In the sham operated control group, there was no change in latency time and in the vehicle control group administered with solvent, the latency time treated with physdon compound significantly increased compared with the sham control group (p<0.05).
[175]
[176] 2-3-3. Immunochemistrv brain staining of AD acute model experiment
[177] At the end of behavior test, the mouse brain was delivered, kept in 10% formalin solution for 24 hours and transferred to 30% sucrose solution. After fixing the brain, the brain was performed to coronal section with a width of 40μm using by cryostat. The sliced brain was performed to Cresyl violet staining to confirm the injury of brain neuronal cell, to ChAT staining to confirm the injury of cholinergic neuron and to GFAP staining to confirm the activation of astrocytes.
[178]
[179] (a) Cresyl Violet Staining
[180] After the tissue was placed on gelatin-coated slide to stain with Cresyl violet, the tissue was performed to dehydration using ethanol. The tissue was incubated for about 3 minutes and dipped into 0.5% Cresyl violet solution for 30 minutes. After the solution was performed to re-hydration with ethanol, the slice was dipped into xylene for 3 minutes. The dried tissue was fixed with Canada balsam mounting medium.
[181] As can be shown in Figs. 8a and 8b, the test group treated with physdon compound showed more enriched neuronal cells than the control group treated with only beta amyloid.
[182]
[183] (b) Immunohistochemistrv
[184] In the washing process between all the antibody incubation, PBST was used to washing tissues. To reduce the activity of endogenous peroxidase activity, the tissue was pretreated with 0.5% H O and then treated with 5% FBS at room temperature for 1 hour to remove non-spedfic binding. The tissue was incubated at 40C for overnight using by mouse anti-GFAP (1:200) monoclonal antibody and goat-anti-ChAT (1:200) polyclonal antibody. A horse radish peroxidase-conjugated anti-mouse IgG and anti- goat IgG secondary antibody (1:600) was incubated at room temperature for 1 hour and detected by DAB kit after the incubation. The inoculation of beta-amyloid into the cerebral ventricle induced the activation of astrocyte confirmed by GFAP staining.
[185] As can be shown in Fig. 8c and 8d, it has been confirmed that the treatment with physdon did not inhibit the activation of astrocyte and the number and intensity of cholinergic neuron in the region of caudate putamen were increased by the treatement of physdon compound being confirmed by staining with ChAT, a cholinergic neuron marker. (See Figs. 8e and 8f). [186] [187] Hereinafter, the formulating methods and kinds of esipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows. [188]
[189] Preparation of powder [190] Physdon 30mg [191] Lactose lOOmg [192] TalclOmg [193] Powder preparation was prepared by mixing above components and filling sealed package. [194]
[195] Preparation of tablet [196] Physdon 50mg [197] Corn Starch lOOmg [198] Lactose lOOmg [199] Magnesium Stearate 2mg
[200] Tablet preparation was prepared by mixing above components and entabletting. [201]
[202] Preparation of capsule [203] Physdon 50mg [204] Corn starch lOOmg [205] Lactose lOOmg [206] Magnesium Stearate 2mg [207] Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method. [208]
[209] Preparation of injection [210] Physdon 50mg
[211] Distilled water for injection optimum amount [212] PH controller optimum amount [213] 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.
[214]
[215] Preparation of liquid
[216] Physάon 0.1~80g
[217] Sugar 5~10g
[218] Citric acid 0.05-0.3%
[219] Caramel 0.005-0.02%
[220] Vitamin C 0.1-1%
[221 ] Distilled water 79-94%
[222] CO gas 0.5-0.82%
2
[223] Liquid preparation was prepared by dissolving active component, filling all the components and sterilizing by conventional liquid preparation method. [224]
[225] Preparation of health food
[226] Physdon lOOOmg
[227] Vitamin mixture optimum amount
[228] Vitamin A acetate 70μg
[229] Vitamin E l.Omg
[230] Vitamin B 0.13mg
[231] Vitamin B 2 0.15mg
[232] Vitamin B6 0.5mg
[233] Vitamin B 12 0.2mg
[234] Vitamin C lOmg
[235] Biotin lOμg
[236] Amide nicotinic add 1.7mg
[237] Folic aid 50μg
[238] Caldum pantothenic add 0.5mg
[239] Mineral mixture optimum amount
[240] Ferrous sulfate 1.75mg
[241] Zinc oxide 0.82mg
[242] Magnesium carbonate 25.3mg
[243] Monopotassium phosphate 15mg
[244] Dicaldum phosphate 55mg
[245] Potassium dtrate 90mg [246] Calcium carbonate lOOmg
[247] Magnesium chloride 24.8mg
[248] The above-mentioned vitamin and mineral mixture 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.
[249]
[230] Preparation of health beverage
[251] Phy scion lOOOmg
[252] Citric acidlOOOmg
[253] Oligosaccharide 10Og
[254] Apricot concentration2g
[255] Taurine Ig
[256] Distilled water900ml
[257]
[258] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 850C for 1 hour, filtered and then filling all the components in 1000ml ample and sterilizing by conventional health beverage preparation method.
[259]
[260]
[261] 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.
[262]
[263]
Industrial Applicability
[264] As described in the present invention, an extract of Rhei rhizoma or physdon compound isolated therefrominhibits beta amyloid aggregation as well as the toxicity and cell apoptosis caused by beta amyloid resulting in stimulating the proliferation of neuronal cells, Therefore, it can be used as the therapeutics or health care food for treating and preventing cognitive function disorder without adverse action.
[265]

Claims

Claims
[1] A pharmaceutical composition comprising an extract of Rhei rhizama or physdon compound isolated therefrom as an active ingredient and pharmaceutically acceptable carrier, diluents or adjuvants to treat and prevent cognitive function disorder.
[2] The pharmaceutical composition according to claim 1, said extract is a crude extract or an organic solvent soluble extract of Rhei rhiaama.
[3] The pharmaceutical composition according to claim 2, said crude extract can be extracted with a polar solvent selected from water, C1-C4 lower alcohol or the mixture thereof.
[4] The pharmaceutical composition according to claim 2, said organic solvent soluble extract can be extracted with acetone, diethylether, ethylacetate, chloroform, dichloromethane, or the mixture thereof.
[5] The pharmaceutical composition according to claim 1, wherein said cognitive function disorder is selected from Alzheimer type dementia, cerebrovascular type dementia, pick' disease, Creutzfeldt-jakob' disease, dementia caused by cephalic damage or Parkinson' disease.
[6] The pharmaceutical composition according to claim 1, wherein said Rhei rhia)ma is selected from a rhiaama of Rheum undulatum L, Rheum officinale BAILL, Rheum coreanum NAKAI or Rheum palmatum L. belonged to Polygonaceae.
[7] A method of treating or preventing cognitive function disorder in a mammal comprising administering to said mammal an effective amount of an extract of Rhei rhia)ma or physdon compound isolated therefrom, together with a pharmaceutically acceptable carrier thereof.
[8] A use of an extract of Rhei rhiaama or physdon compound isolated therefrom for the manufacture of therapeutic agent for the treatment and prevention of cognitive function disorder.
[9] A health care food comprising an extract of Rhei rhiaama or physdon compound isolated therefrom, together with a sitologically acceptable additive for the prevention and improvement of cognitive function disorder.
[10] The health care food according to claim 9 wherein said health food is provided as powder, granule, tablet, chewing tablet, capsule or beverage type.
PCT/KR2007/004905 2006-10-10 2007-10-09 A composition comprising an extract of rhei rhizoma or physcion compound isolated therefrom for treating or preventing cognitive dysfunction and the use thereof WO2008044848A1 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009145557A2 (en) * 2008-05-27 2009-12-03 Korea Research Institute Of Chemical Technology Use of physcion-8-o-beta-d-glucopyranoside or physcion for promoting osteoblast activity
CN101961326A (en) * 2010-10-29 2011-02-02 西南交通大学 Application of physcion in preparing medicines for treating depression
CN104288242A (en) * 2014-09-28 2015-01-21 江西百神药业股份有限公司 Total anthraquinone and compositions thereof treating Parkinson's disease

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101877860B1 (en) * 2017-05-16 2018-07-13 한국과학기술연구원 Compositions for preventing or treating cognitive impairment-related disease comprising extracts of Euonymus hamiltonianus Wall.

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020087723A (en) * 2001-05-16 2002-11-23 김호철 Extract of Rheum palmatum L. having neuroprotective effects and pharmaceutical composition containing the same

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020087723A (en) * 2001-05-16 2002-11-23 김호철 Extract of Rheum palmatum L. having neuroprotective effects and pharmaceutical composition containing the same

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
GU J. ET AL.: "Rhubarb extracts in treating complications of severe cerebral injury", CHIN. MED. J. (ENG), vol. 113, no. 6, 2000, pages 529 - 531 *
LEE H.C. ET AL.: "Screening of the acetylcholinesterase inhibitors from water extracts of the medicinal plants", KOREAN J. ORIENTAL PHYSIOLOGY & PATHOLOGY, vol. 16, no. 2, 2002, pages 215 - 219, XP053003917 *
PARK C.H. ET AL.: "The effects of Rheum palmatum (RHP) extract on the Alzheimer's disease model", J. ORIENTAL NEUROPSYCHIATRY, vol. 16, no. 1, 2005, pages 67 - 80, XP053008982 *
TIAN J. ET AL.: "A clinical study on compound da huang (radix et Rhizoma rhei) preparations for improvement of senile persons' memory ability", J. TRADIT. CHEM., vol. 17, no. 3, 1997, pages 168 - 173 *
ZHANG Q. ET AL.: "Effect of Rheum palmatum decoction on increasing intelligence", ZHONG YAO CAI, vol. 24, no. 10, 2001, pages 728 - 730 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009145557A2 (en) * 2008-05-27 2009-12-03 Korea Research Institute Of Chemical Technology Use of physcion-8-o-beta-d-glucopyranoside or physcion for promoting osteoblast activity
WO2009145557A3 (en) * 2008-05-27 2010-03-04 Korea Research Institute Of Chemical Technology Use of physcion-8-o-beta-d-glucopyranoside or physcion for promoting osteoblast activity
CN101961326A (en) * 2010-10-29 2011-02-02 西南交通大学 Application of physcion in preparing medicines for treating depression
CN104288242A (en) * 2014-09-28 2015-01-21 江西百神药业股份有限公司 Total anthraquinone and compositions thereof treating Parkinson's disease

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