KR100554343B1 - Composition for inhibiting activity of acetylcholinesterase continiaing extract from chrysanthemum indicum - Google Patents

Abstract

 The present invention relates to a composition for inhibiting acetylcholinesterase comprising the extract of gakguk and a compound derived therefrom. In particular, the present invention not only can be effectively used for diseases caused by lack of acetylcholine by effectively inhibiting acetylcholinesterase activity, and in particular, it can be easily used for preventing dementia and enhancing memory and derived from it. One compound is provided.

Sensitization, dementia, memory enhancement, acetylcholinesterase

Description

COMPOSITION FOR INHIBITING ACTIVITY OF ACETYLCHOLINESTERASE CONTINIAING EXTRACT FROM CHRYSANTHEMUM INDICUM}

 Figure 1 shows briefly the mechanism of secretion and degradation of acetylcholine.

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a composition for inhibiting acetylcholinesterase comprising the extract of gakguk and a compound derived therefrom, and more particularly, to diseases caused by lack of acetylcholine by effectively inhibiting acetylcholinesterase activity. It relates to a gamguk extract which can be used and a compound derived therefrom.

[Private Technology]

Brain disease is the biggest cause of death in the past decade, but it is estimated that the damage caused by it is more difficult to diagnose and treat than other diseases.

In particular, dementia is a disease that depletes human life by leading to mental disorders such as memory impairment and loss of judgment. The causes of dementia are various, with about 50% being Alzheimer's dementia, 20-30% being vascular dementia, alcoholic dementia or Parkinson's dementia, and about 15-20% being caused by both Alzheimer's and vascular dementia. Alzheimer's disease (AD) dementia, also known as senile dementia, is known to be closely related to aging, although the cause of the disease has not been precisely identified.

Dementia has been reported to be caused by neuronal damage or decreased acetylcholine levels. Neuronal damage is caused by a number of factors, including free radicals, glutamate, calcium overload, nitrogen, amyloid beta protein, and cytokines, among which free radicals are produced during the metabolism of lipid peroxidation. Causing cellular damage (Beckman JS, et al., (1990) Proc. Natl. Acad. Sci ., 87: 1620; Radi R., et al., (1991) Arch. Biochem. Biophys ., 288, 481 ; Ljubuncic P., et al, ( 2000) GUT, 47 (5), 710;........ Maria-Liisa S., et al, (2001) Cell Biol lmmunol Pathol Intern Soc Nephrol, 59 ( 2): 481).

Acetylcholine (ACh) is biosynthesized in the cytoplasm of synaptic knobs of neurons and secreted from presynaptic and postsynaptic fibers of the sympathetic or parasympathetic nervous system, and the muscarinic receptors present in the postganglion of parasympathetic nerve fibers ( In response to a stimulus signal to the muscarin receptor, acetylcholine is degraded by acetylcholinesterase (FIG. 1). Decreased acetylcholine function has been reported to be a cause of dementia (Sefel, IH (1975) John Willey &Sono; New York, 18-99) and therefore acetylcholinesterase inhibitors are used to prevent dementia. Or it is thought that it can be treated. Drugs developed to date include lecithin as an acetylcholine precursor, RS-86 as a receptor agonist, nicotine as a acetylcholine precursor, and nicotine as a acetylcholinesterase inhibitor. There is Tacrine and Aricept recently approved in Korea. However, tacrine is very expensive, and causing hepatotoxicity is difficult to use long-term. In addition, acetylcholinesterase inhibitors such as donepezil (Donepezil), rivastigmine (galivatamine) has been developed, but due to toxicity and side effects are controversial.

Therefore, the development of natural resources effective for preventing and treating dementia while minimizing side effects is urgently required.

Accordingly, an object of the present invention is to provide an extract of gamguk that can inhibit acetylcholinesterase activity.

In addition, an object of the present invention is to provide a compound derived from a country that can inhibit acetylcholinesterase activity.

It is another object of the present invention to provide a composition which is harmless to the human body and has a dementia prevention and memory enhancing effect.

In order to achieve the above object, the present invention provides a composition for inhibiting acetylcholinesterase comprising the extract as an active ingredient.

In another aspect, the present invention provides a composition for inhibiting acetylcholinesterase comprising a compound represented by Formula 1 or Formula 2 as an active ingredient:

(Formula 1)

(Formula 2)

Hereinafter, the present invention will be described in detail.

The present invention relates to a gamguk extract or a compound derived from gamguk that can be used as an acetylcholinesterase inhibitor to prevent and treat dementia and memory loss.

Chrysanthemum indicum is a plant that is harmless to the human body, which is used as medicine or tea in everyday life.

The gamguk extract is provided by extracting or fractionating the extraction part of gamgug with water or organic solvent. The extract may be a part of the whole country, or part of constituting the country such as persimmon flower, persimmon stem, persimmon root and persimmon leaves, the organic solvent is alcohol having 1 to 5 carbon atoms, hexane, chloroform, methylene chloride and ethyl It may be selected from the group consisting of acetates. The alcohol having 1 to 5 carbon atoms may be specifically ethanol, methanol, propanol, isopropanol or butanol, wherein the alcohol may be used as a 100% stock solution but may be an alcoholic water having an alcohol content of 95 to 50%. However, the organic solvent is not limited to the examples described above, and all known solvents may be used.

Specific method for producing the extract of gamguk, immersed in the root, stem, leaves, flowers or dried mixture of gamguk by adding alcohol or water of 1 to 5 carbon atoms in a ratio of 1: 1 to 20 by weight, then filtration and concentrated under reduced pressure or lyophilization It is. In addition, one or more organic solvents may be sequentially added to the gamguk alcohol extract prepared by the above method according to polarity to extract fractions for each organic solvent.

In one embodiment of the present invention, ethanol was added to gamguk to prepare gamguk ethanol extract, and hexane, methylene chloride, ethyl acetate and butanol were sequentially added to the ethanol ethanol extract and fractionated to remove gamguk hexane extract, gamuk methylene chloride extract, and gamguk. Ethyl acetate extract, gamgutan butanol extract and gamgug water extract were obtained. The extracts of the Korean soups showed antioxidant and acetylcholinesterase inhibitory effects. In particular, the acetylcholinesterase inhibitory effect was very excellent in the gamguk ethyl acetate extract and the gamguk butanol extract, and the active compounds were separated therefrom.

In addition, the present invention relates to a compound derived from the country having antioxidant activity and acetylcholinesterase inhibitory effect, specifically 5,7-dihydroxy-4'-methoxyflavone-7-O represented by the following formula (1) -Beta-D-galactosyranoside and 5,7-dihydroxy-4'-methoxyflavone-7-O-lutinoside represented by the formula (2).

(Formula 1)

(Formula 2)

Accordingly, the present invention provides a composition for inhibiting acetylcholinesterase, comprising the persimmon extract or a compound derived therefrom as an active ingredient. The active ingredient may be used alone, and may further include other pharmacologically acceptable groups or excipients. In this case, the active ingredient in the composition for inhibiting acetylcholinesterase may be 0.1 to 99% by weight, but the preferred content is appropriately adjusted according to the method of use and purpose of use of the composition.

Carriers or excipients that can be used in the composition for inhibiting acetylcholinesterase may use a suitable material depending on the dosage form, and when formulated, conventional fillers, extenders, wetting agents, disintegrating agents or surfactants may be used. Representative diluents or excipients include water, dextrins, calcium carbonates, lactose, propylene glycol, liquid paratins, talc, isosaccharides, sodium metabisulfite. Methylparaben, propylparaben, magnesium stearate, lactose, physiological saline, perfumes and pigments.

The composition for inhibiting acetylcholinesterase of the present invention can be used as a medicament, a food additive or a food, and when used as a medicament, the administration method can be oral or parenteral. The formulation of the composition may vary depending on the method of use, but may include PLASTERS, GRANULES, LOTIONS, POWDERS, Syrups, LIQUIDS AND SOLUTIONS, and Aerosols. AEROSOLS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS, SUSPENSIONS, INFUSIONS, TABLETS, INJECTIONS, CAPSULES AND PILLS And the like.

The dosage of the composition for inhibiting acetylcholinesterase should be determined in consideration of the age, sex, condition, absorbency of the active ingredient in the body, inactivation rate, and the drug used in combination. From 1 mg / kg body weight to 500 mg / kg body weight.

The composition for inhibiting acetylcholinesterase according to the present invention effectively inhibits the degradation of acetylcholinesterase and thus is applicable to the prevention and treatment of diseases caused by the lack of acetylcholine. Representative diseases include dementia, memory loss, vascular brain disease and concentration loss (Giacobini E. Int J Geriatr Psychiatry. 2003 Sep; 18 (Suppl 1): S1-5, Grantham C, Geerts H. Neurol Sci. 2002 Nov 15; 203-204: 131-6)

 Since the composition for inhibiting acetylcholinesterase of the present invention also has an antioxidant effect, it can be used very effectively for senile dementia, which regards free radical as the etiology.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example 1-2 Preparation of Gammon Ethanol and Water Extract

2 kg of flower powder of persimmon soup was immersed in 8 L of ethanol or water, and then refluxed for 4 hours and extracted three times. All the extracts were collected and concentrated under reduced pressure to obtain 520 g and 600 g of ethanol extract of Example 1 (Example 1) and extract of Example 2 (Korean Example 2).

Example 3-7 Preparation of Fractions of Ethanol Extracts

N-hexane: methanol: water was added 10: 1: 9 to 300 g of the ethanol extract of Gam-Gook and fractionated to obtain 55 g (Example 3).

Methylene chloride, ethyl acetate and butanol were then added sequentially to the residue, followed by 43 g of methane chloride extract (Example 4), 25 g of ethyl acetate acetate (Example 5), 55 g of glutinous butanol extract (Example 6), and 110 g (Example 7) of Persimmon water extract was obtained.

Example 8: Separation of Compounds Derived from Persimmon Extract

The active compounds were isolated and identified from each of the extracts of Gamyeo ethyl acetate of Example 5.

20 g of Gam-guk ethyl acetate fraction was adsorbed on a silica gel-filled column (70-230 mesh, Art, 7734 Merck), and then eluted by flowing it over a column with a gradient of 0 to 100% methanol in a mixed solvent of diethyl ether and methanol. Each was isolated and identified using spectral data (NMR, IR, UV) to identify 5,7-dihydroxy-4'-methoxyflavone-7-O-beta-D-galactosyranoside (Example 7). ) 0.5 g was obtained.

5,7-Dihydroxy-4'-methoxyflavone-7-O-beta-D-galactosyranoside:

Molecular formula C ₂₂ H ₂₂ O ₁₀ , Molecular weight: 446.41 Melting point: 259-261 ° C., Specific light intensity: [α] 25 D = -36.6 ° (C 0.55, DMF), UV (λ max, nm) (MeOH): 269 (4.28) ), 325 (4.32); (+ AlCl 3) 277 (4.24), 300 (4.18), 343.5 (4.32)

IRν (cm-1, KBr): 3360 (br.,-OH), 1650 (flavone> CO), 1600, 1485 (aromatic), 1160 (ether linkage), 820 (p-substituted phenyl ring)

¹ H-NMR (DMSO-d6, 400 MHz) δ: 7.96 (H2 ', 6'), 7.03 (8.0, H3 ', 5'), 6.75 (2, H6), 6.85 (2, H8), 6.38 ( s, H3), 3.79 (OMe4), 5.30 (Gal-H-1), 3.0-3.70 (other galactosyl protone)

MS: M + = 446, m / z = "284 (100), 241, 152, 132"

Example 9 Separation of Compounds Derived from Persimmon Extract

40 g of Gam-gukbutanol extract was adsorbed on a silica gel-filled column (70-230 mesh, Art, 7734 Merck), and then methylene chloride and methanol were flowed through the column with a gradient of 0 to 100% to separate the eluted compounds. Identification using data (NMR, IR, UV) yielded 1.69 g of 5,7-dihydroxy-4'-methoxyflavone-7-O-lutinoside (Example 8).

5,7-Dihydroxy-4'-methoxyflavone-7-O-lutinoside:

Molecular Formula: C ₂₈ H ₃₂ O ₁₄ , Molecular Weight: 592.55, Melting Point 272-276 ° C, Specific Radiance: [α] 24 D = -90.3 ° (C Py)

UV (λ max, nm) (MeOH): 270,324; (+ NaOMe) 280, 368; (+ AlCl3) 276, 301, 343, 384; (+ AlCl + HCl) 277,300,339,384 (MeOH + NaOAc) 271,320; (+ NaOAc + H3BO3) 269,328

IRν (cm-1, KBr): 3420 (OH), 1640 (a, b-unsaturated ketone), 1480, 1600 (aromatic C = C), 1070, 1030 (glucosodic C-O)

1 H-NMR (DMSO-d 5, 400 MHz) δ: 1.09 (3H, d, 6.1, CH ₃ of rhamnose), 3.87 (3H, s, -OMe), 4.53 (1H, s, anomeric H of glucose), 6.45 (1H, d, 2.0, H6), 6.79 (1H, d, 2.0, H8), 6.94 (1H, s, H3), 7.15 (2H, d, 9.0, H3 'and H5'), 8.05 (2H, d , 9.0, H2 'and H6'),

¹³ C-NMR (DMSO-d5, 75.5 MHz) δ: 163.9 (2), 103.8 (3), 181.9 (4), 161.1 (5), 99.9 (6), 162.4 (7), 94.7 (8), 156.9 (9), 105.4 (10), 122.7 (1 '), 128.4 (2'), 114.7 (3 '), 162.9 (4'), 114.7 (5 '), 128.4 (6'), 55.5 (OCH ₃ ) , 99.9 (1 ''), 73.0 (2 ''), 76.2 (3 ''), 69.6 (4 ''), 75.6 (5 ''), 66.1 (6 ''), 100.5 (1 ''')

HRMS (FAB +) [M + l] Exact mass: 593.1870, Found: 593.1875.

Experimental Example 1: Antioxidant Effect

DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals are purple (517 nm absorbance) when dissolved in a solution such as methanol, forming a stable form of radicals. In the presence of a compound that eliminates DPPH radicals, purple light is lost, and the radical scavenging ability of a particular compound can be measured.

Samples of Examples 1 to 8 and 0.1 ml of the samples were mixed with 2.9 ml of DPPH 10 ^-4 M solution, respectively, and reacted at 30 ° C for 30 minutes. The absorbance was then measured at 517 nm and the relative radical scavenging ability of the sample was converted to 0% when no sample was added (Brand-Williams, W., Cuvelier, ME, Berset, C. (1995) Lebensm.-Wiss u.Technol. , 28 (1), 25-30). The results are shown in Table 1 below.

sample Antioxidant Effect DPPH (IC ₅₀ ) Example 1 250.8 μg / ml Example 3 240.5 μg / ml Example 4 220.3 μg / ml Example 5 160.5 μg / ml Example 6  90.4 μg / ml Example 7 120.6 μg / ml Example 8 51.5 μg / ml Example 9 37.4 μg / ml BHT (Synthetic Antioxidant) 15.5 μg / ml L-ascorbate  10.6 μg / ml

In Table 1, all of the samples of Examples 1 to 9 of the present invention showed antioxidant activity, and in particular, the compounds from Examples 8 and 9 were somewhat lower than the synthetic antioxidants BHT and L-ascorbate. It showed excellent antioxidant activity.

Experimental Example 2: Inhibitory Effect of Acetylcholinesterase

The activity of acetylcholinesterase was confirmed by experiments according to known methods (Ellman, GL, courtney, KD, Andres, Jr. V., Featherstone, RM (1961) Biochem. Pharmacol. 7, 88-95). It reacts with thiochoine produced by hydrolysis of acetylcholine, an enzyme substrate, with 5,5'-dithio-bis- (2-nitrobenzoic acid) (DTNB) to produce new dithio compounds and 2- After the thio anion of nitrobenzoic acid is made, the anion is detected by UV. Acetylcholinesterase activity was measured by measuring the rate of production of anions, and the results are shown in Table 2 below.

sample Inhibitory effect of acetylcholinesterase (IC ₅₀ ) Example 1 12.2 μg / ml Example 3 10.0 μg / ml Example 4 9.7 μg / ml Example 5 7.4 μg / ml Example 6 5.8 μg / ml Example 7 20.6 μg / ml Example 8 6.8 μM Example 9  3.2 μM Galantamine 2.3 μM

In Table 2, the inhibitory activity against acetylcholinesterase was found to inhibit acetylcholinesterase activity by about 50% at 12.2 μg / ml of the ethanol extract of Example 1 in the case of gamguk extract, Example 5 Ethyl acetate extract and butanol extract of Example 6 showed a strong inhibitory activity of 7.4 ㎍ / ㎖ and 5.8 ㎍ / ㎖, respectively.

In addition, the compound of Example 9 was the strongest IC ₅₀ was 3.2 μM, the compound of Example 8 was also 6.8 μM showed an excellent acetylcholinesterase inhibitory effect. This is similar to the activity of galantamine used as a standard comparator, and it is rare that the inhibitory activity against acetylcholinesterase in phenolic substance is comparable to that of amine galantamine.

In addition, the extracts of compounds 1 and 2 were also confirmed in the extract of Gam-guk-su in Example 2, and similar antioxidant effects and acetylcholinesterase inhibitory activity were confirmed.

Example 9: Preparation of acetylcholinesterase inhibitory composition

9-1. Injection manufacturing

100 mg of the compound of Example 8, 3.0 mg of sodium metabisulfite, 0.8 mg of methylparaben, and 0.1 mg of propylparaben were mixed in sterile distilled water for injection to prepare a final volume of 2 ml of injection, followed by ampoule sterilization.

9-2. Tablet manufacturing

200 mg of the compound of Example 8, 100 mg of lactose, 100 mg of starch, and 600 mg of magnesium stearate were mixed and compressed into tablets.

9-3. Capsule Manufacturing

100 mg of the compound of Example 8, 50 mg of lactose, 50 mg of starch, 2 mg of talc and 600 mg of magnesium stearate were mixed and filled into gelatin capsules to prepare a capsule.

9-4. Liquid manufacturing

1000 mg of the compound of Example 8, 20 g of sugar, 20 g of isomerized sugar and an appropriate amount of lemon flavor were added to purified water to prepare a total of 100 ml, and filled into a brown bottle and sterilized to prepare a liquid.

As described above, the gamgukchi extract of the present invention and the compounds derived therefrom can be effectively used for diseases caused by lack of acetylcholine by effectively inhibiting acetylcholinesterase activity, in particular, preventing dementia And it can be easily used for memory enhancement.

Claims (6)

A composition for preventing or treating dementia comprising ethanol extract of Gamkook as an active ingredient. delete The composition of claim 1, wherein the ethanol ethanol extract is a fraction obtained by sequentially adding hexane, methylene chloride, ethyl acetate and butanol to the ethanol ethanol extract. A composition for preventing or treating dementia comprising the compound represented by Formula 1 or Formula 2 as an active ingredient: (Formula 1)

(Formula 2)

delete 5. The composition of claim 1, wherein the composition is a food, food additive.

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