KR102286776B1 - A composition comprising quercetin 3-O-a-L-arabinofuranoside or 2-oxopomolic acid for preventing or treating neurodegenerative disease - Google Patents

Abstract

The present invention relates to a composition for preventing or treating degenerative brain disease comprising quercetin 3-OaL-arabinofuranoside or 2-oxofomolic acid isolated from apple ( Malus domestica ). The compound isolated from the apple has excellent sEH inhibitory activity and excellent acetylcholinesterase and butyrylcholinesterase inhibitory activity, so it can be usefully used as a composition for preventing or treating degenerative brain disease.

Description

A composition for preventing or treating degenerative brain disease, comprising quercetin 3-O-a-L-arabinofuranoside or 2-oxopomolic acid for preventing or treating neurodegenerative disease

The present invention relates to a composition for preventing or treating degenerative brain disease comprising quercetin 3-OaL-arabinofuranoside or 2-oxofomolic acid isolated from apple ( Malus domestica ).

Neurodegenerative disease is a disease that occurs in the brain among degenerative diseases that occur with aging. It is known to be caused by the death of cranial nerve cells, which are the most important for information transmission in the cranial nervous system, problems with the formation or function of synapses that transmit information between cranial nerve cells and cranial nerve cells, and ideal symptoms or reduction of the electrical activity of cranial nerves. . Representative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (Eunsu Son et al., Journal of the Korea Academia-Industrial Cooperation Society) , 12(10), 4411-4417, 2011).

Neurodegenerative diseases such as Alzheimer's disease cause a decrease in the production of neurotransmitters such as acetylcholine, which is involved in learning and memory, in the brain by killing nerve cells, and it is further exacerbated by enzymes that hydrolyze acetylcholine. can be

Acetylcholine is a neurotransmitter related to memory and thinking, and its concentration is decreased in certain regions of the brain of Alzheimer's disease patients (Tricco, A. C. et al., Syst Rev., 1, 31, 2012). Therefore, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity inhibitors are used for treatment (Alzheimer's Association, Alzheimers Dement., 8(2), 131-168, 2012).

Most Alzheimer's drugs currently used in countries are acetylcholinesterase (AChE) inhibitors, such as tacrine (trade name: cognex), donepezil (trade name: aricept), and Rivasti. Rivastigmine (trade name: exelon) and galanthamine (trade name: reminyl) were launched, and cognitive function of Alzheimer's patients was improved to some extent. However, due to the characteristics of degenerative brain disease, long-term administration of the drug is required, and the above drugs have problems such as hepatotoxicity, vomiting, and various side effects including loss of appetite. Therefore, the development of a new therapeutic agent that can block the progression of degenerative brain disease is an urgent task.

Soluble epoxide hydrolase (sEH) is arachidonic acid (Spector, AA, J Lipid Res., 50, 52-56, 2009), linoleic acid (Moghaddam, MF et al., Nat Med., 3 ( 5), 562-566, 1997) and other endogenous chemical mediators such as lipid epoxides (Carroll, MA et al., Thorax, 55, 13-16, 2000). Epoxyeicosatrienoic acid (EET) is rapidly metabolized to dihydroxyeicosatrienoic acids (DHETs) by sEH and inactivated (Capdevila, JH et al. , J Lipid Res, 41(2), 163-181, 2000).

sEH inhibitors have been reported to be useful in the treatment of inflammatory diseases, cardiovascular diseases, metabolic syndrome, stroke, neurological diseases, and degenerative brain diseases (US Patent No. 08815951; International Patent Publication No. 2003002555; US Patent Publication No. 20080221105) US Patent Publication No. 20060148744; Shen, HC, Expert Opin Ther Pat., 20(7), 941-956, 2010; Fang, X. et al., Drugs of the Future, 34(7), 579- 585, 2009; Morisseau, C. et al., Annu Rev Pharmacol Toxicol., 53, 37-58, 2013; Hashimoto, K., Front Pharmacol., 10(36), 2019; Wagner, KM et al., Pharmacol Ther., 180, 62-76, 2017).

On the other hand, as a prior literature related to a composition for preventing or treating degenerative brain disease containing quercetin 3-OaL-arabinofuranoside or 2-oxofomolic acid, a prior paper [Hyun-Kyung Kim, Learning ability performance and memory of fruit and vegetable peel extracts Effects on enhancement, The Journal of the Convergence on Culture Technology (JCCT), 4(3), 261-267, 2018], the mixed extract of apple peel and pear peel compared to the extracts of apple peel and pear peel, respectively, acetylcholine It has been disclosed that it is an anti-dementia substance that effectively acts on memory and learning promotion by stimulating the cholinergic nervous system by inhibiting esterase, promoting short-term memory and long-term memory activity. , 25(1), 99-108, 2014] have disclosed the NHE inhibitory activity of apple extract, but quercetin 3-OaL-arabinofuranoside or 2-oxofomolic acid of the present invention is used for the treatment of degenerative brain diseases There are no previous reports confirming that it is effective.

Accordingly, in the process of studying quercetin 3-OaL-arabinofuranoside or 2-oxofomolic acid, the present inventors confirmed that the compounds have excellent sEH inhibitory activity as well as acetylcholinesterase and butyrylcholinesterase inhibitory activity. invention could be completed.

US Patent No. 08815951, Inhibitors of epoxide hydrolases for the treatment of inflammation, August 26, 2014, registered. US Patent Publication No. 20080221105, SOLUBLE EPOXIDE HYDROLASE INHIBITORS FOR TREATMENT OF METABOLIC SYNDROME AND RELATED DISORDERS, published September 11, 2008. US Patent Publication No. 20060148744, Use of cis-epoxyeicosantrienoic acids and inhibitors of soluble epoxide hydrolase to reduce damage from stroke, July 06, 2006, published. International Patent Publication No. 2003002555, METHODS OF USING SOLUBLE EPOXIDE HYDROLASE INHIBITORS, January 09, 2003, registered.

Kim Hyun-kyung, Effects of fruit and vegetable peel extracts on learning ability performance and memory enhancement, The Journal of the Convergence on Culture Technology (JCCT), 4(3), 261-267, 2018. Eunsoo Son et al., R&D and patent trend analysis of therapeutics for degenerative brain disease, Journal of the Korea Academia-Industrial Cooperation Society, 12(10), 4411-4417, 2011. Alzheimer's Association, 2012 Alzheimer's disease facts and figures, Alzheimers Dement., 8(2), 131-168, 2012. Capdevila, J. H. et al., Cytochrome P450 and arachidonic acid bioactivation: Molecular and functional properties on the arachidonate monooxygenase, J Lipid Res, 41(2), 163-181, 2000. Carroll, M. A. et al., A new class of lipid mediators: Cytochrome P450 arachidonate metabolites, Thorax, 55, 13-16, 2000. Fang, X. et al., Soluble epoxide hydrolase as a therapeutic target for cardiovascular diseases, Drugs of the Future, 34(7), 579-585, 2009. Hashimoto, K., Role of Soluble Epoxide Hydrolase in Metabolism of PUFAs in Psychiatric and Neurological Disorders, Front Pharmacol., 10(36), 2019. Moghaddam, M. F. et al., Bioactivation of leukotoxins to their toxic diols by epoxide hydrolase, Nat Med., 3(5), 562-566, 1997. Morisseau, C. et al., Impact of soluble epoxide hydrolase and epoxyeicosanoids on human health, Annu Rev Pharmacol Toxicol., 53, 37-58, 2013. Shen, H. C., Soluble epoxide hydrolase inhibitors: a patent review, Expert Opin Ther Pat., 20(7), 941-956, 2010. Spector, A. A., Arachidonic acid cytochrome P450 epoxygenase pathway, J Lipid Res., 50, 52-56, 2009. Tricco, A. C. et al., Efficacy of cognitive enhancers for Alzheimer's disease: protocol for a systematic review and network meta-analysis, Syst Rev., 1, 31, 2012. Verma, V. et al., Sodium-hydrogen exchanger inhibitory potential of Malus domestica, Musa × paradisiaca, Daucus carota, and Symphytum officinale, J Basic Clin Physiol Pharmacol., 25(1), 99-108, 2014. Wagner, K. M. et al., Soluble epoxide hydrolase as a therapeutic target for pain, inflammatory and neurodegenerative diseases, Pharmacol Ther., 180, 62-76, 2017.

An object of the present invention is to provide a composition for preventing or treating degenerative brain disease comprising quercetin 3-OaL-arabinofuranoside or 2-oxofomolic acid isolated from apple ( Malus domestica ).

The present invention relates to a composition for preventing or treating degenerative brain disease comprising quercetin 3-O-a-L-arabinofuranoside or 2-oxofomolic acid.

The compound may be synthesized according to a conventional method in the art and may be prepared as a pharmaceutically acceptable salt, but is preferably obtained by fractionation by chromatography from an apple ( Malus domestica ) extract.

The apple extract is an apple extracted with water, C1 to C4 alcohol or a mixed solvent thereof, and the C1 to C4 alcohol may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol and isobutanol. More preferably, an apple is extracted with alcohol or an aqueous alcohol solution, concentrated and suspended in water, and then fractionated with at least one solvent selected from the group consisting of ethyl acetate, n-butanol, and n-hexane is used.

Water, C1 to C4 alcohol, or a mixed solution thereof used for extraction may be used in an amount of 1 to 100 times the weight of the apple used, and the above process may be repeated 1 to 5 times.

The extraction time of the extract is not particularly limited, but it is preferable to extract within 10 minutes to 1 day, and the extraction method includes all commonly used extraction methods, for example, pressure extraction, immersion (cold-chilling, warm-chilling), ultrasonic extraction, A reflux extraction method and the like can be used, and as an extraction device, a conventional extraction device, an ultrasonic crushing extractor, or a fractionator can be used.

In addition, the extract is formed using the extract itself and the extract, such as the extract obtained from the extraction process, the diluted or concentrated liquid of the extract, the dried product obtained by drying the extract, the prepared or purified product of the extract, or a mixture thereof. All possible formulations of extracts are included.

The chromatography is silica gel column chromatography (silica gel column chromatography), flash column chromatography (flash column chromatography), Sephadex LH-20 column chromatography (sephadex LH-20 column chromatography), RP-18 column chromatography (RP) -18 column chromatography), thin layer chromatography (TLC), medium pressure liquid chromatography, vacuum liquid column chromatography, and high performance liquid chromatography, HPLC) and the like may be used, but is not particularly limited thereto.

The degenerative brain disease (neurodegenerative disease) may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick's disease, amyotrophic axonal sclerosis, and Creutzfeldt-Jakob disease, but is not particularly limited thereto.

The pharmaceutical composition according to the present invention may be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier. “Pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans. In addition, the composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively.

Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl paraoxybenzoate, propyl paraoxybenzoate, talc, magnesium stearate, and mineral oil, but is not limited thereto. In the case of formulation, it is prepared using diluents or excipients, such as commonly used fillers, stabilizers, binders, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient, for example, starch, microcrystalline cellulose, sucrose or lactose, to the compound of the present invention; It is prepared by mixing low-substituted hydroxypropyl cellulose, hypromellose, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, etc. may be used. In order to formulate the formulation for parenteral administration, the compound or a pharmaceutically acceptable salt thereof is sterilized and/or adjuvants such as preservatives, stabilizers, wetting agents or emulsification accelerators, salts and/or buffers for regulating osmotic pressure, and other treatments It can be prepared as a solution or suspension by mixing it with water with useful substances, and it can be prepared in ampoules or vial unit dosage form.

The pharmaceutical composition comprising the compound disclosed in the present invention as an active ingredient may be administered to mammals such as mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection. The dosage may vary depending on the age, sex, weight, specific disease or pathology to be treated, the severity of the disease or pathology, administration time, administration route, absorption, distribution and excretion rate of the drug, the type of other drugs used, and the prescriber's It will depend on judgment, etc. Dosage determination based on these factors is within the level of the skilled artisan, and dosages generally range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

In another aspect, the present invention relates to a health functional food for preventing or improving degenerative brain disease comprising quercetin 3-O-aL-arabinofuranoside or 2-oxofomolic acid as an active ingredient.

The health functional food refers to food manufactured or processed using raw materials or ingredients having useful functionality, and includes, for example, health supplements, functional foods, nutritional supplements, supplements, and the like.

The quercetin 3- O- aL-arabinofuranoside or 2-oxofomolic acid is preferably 0.001% to 50% by weight, more preferably 0.001% to 30% by weight based on the total weight of the total health functional food. , most preferably 0.001 wt% to 10 wt% may be added.

The health functional food of the present invention includes the form of tablets, capsules, pills, and liquids, and the present invention quercetin 3- O- aL-arabinofuranoside or 2-oxofomolic acid can be added to the food. , for example, various foods, beverages, gum, tea, vitamin complexes, and the like.

The present invention relates to a composition for preventing or treating degenerative brain disease comprising quercetin 3-OaL-arabinofuranoside or 2-oxofomolic acid isolated from apple ( Malus domestica ). The compound isolated from the apple has excellent sEH inhibitory activity and excellent acetylcholinesterase and butyrylcholinesterase inhibitory activity, so it can be usefully used as a composition for preventing or treating degenerative brain disease.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided so that this disclosure will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

<Example 1. Isolation of quercetin 3-O-a-L-arabinofuranoside or 2-oxofomolic acid from apple>

Apple ( M. domestica ) peel and flesh sliced and dried at room temperature for 1 week. The dried pulp (1.6 kg) and peel (260.0 g) were ultrasonically extracted three times with methanol at 35 ± 3 °C, and then the solvent was removed under reduced pressure to obtain a methanol extract (725.0 g of pulp, 36 g of peel), and the methanol pulp extract Alternatively, each of the bark extracts was suspended in distilled water and fractionated with n-hexane, ethyl acetate and n-butanol to obtain each fraction.

Among the fractions, an apple peel ethyl acetate fraction (16.2 g) was subjected to silica gel column chromatography (CH ₂ Cl ₂ -MeOH (30:1→1:1)) to obtain eight small fractions (PE1-PE8). PE8 in the small fraction was subjected to RP-18 silica gel column chromatography (MeOH-H ₂ O (2:1→1:1)) to obtain quercetin 3-OaL-arabinofuranoside (20.0 mg) having the following chemical structure .

Next, the apple pulp ethyl acetate fraction (18.1 g) of the fractions was subjected to vacuum liquid column chromatography (CH ₂ Cl ₂ -MeOH (50:1→1:1)) to 6 small fractions (FE1- FE6) was obtained. FE2 in the small fraction was subjected to RP-18 silica gel column chromatography (MeOH-H ₂ O (3:1)) to obtain 2-oxofomolic acid (16.3 mg) having the following chemical structure.

<Example 2. Confirmation of sEH inhibitory activity>

To measure the sEH inhibitory effect, first, 130 μl of sEH (~16 ng/mL) dissolved in 25 mM bis-Tris-HCl buffer (pH 7.0) containing 0.1% BSA (pH 7.0) and 20 μl of the compound dissolved in methanol were added to a 96-well plate. , 50 μl of a buffer containing 5 μM PHOME was added. After reacting at 37° C. for 40 minutes, IC ₅₀ values were calculated and shown in Table 1.

compound IC ₅₀ Quercetin 3-OaL-
Arabinofuranoside 39.3±3.4 μM 2-oxoformolic acid 11.4±2.7 μM Positive control (AUDA) 1.5±1.2 nM

<Example 3. Confirmation of acetylcholinesterase and butyrylcholinesterase inhibitory activity>

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activity was measured using the method of Ellman et al. The yellow color generated by reacting AChE and BuChE with thiocholine produced by the reaction of acetylthiocholine iodide and butyrylthiocholine iodide with the color developer DTNB was measured using absorbance at 405 nm. .

In a 96-well microplate, 130 μl each of 0.0325 U/ml of AChE or BuChE, 20 μl of each of the compounds of the present invention, quercetin 3-OaL-arabinofuranoside or 2-oxofomolic acid, 0.125 mM acetylthiocholine iodide, or 25 μl of butyrylthiocholine iodide, 25 μl of 0.625 mM DTNB was added, and absorbance was measured at 30 second intervals at 405 nm and 37° C., and IC ₅₀ values were calculated and shown in Table 2.

compound IC ₅₀ (μM) AChE BuChE Quercetin 3-OaL-
Arabinofuranoside 86.4±3.9 125.3±1.4 2-oxoformolic acid 71.5±4.3 106.7±1.3

Referring to Table 2 above, the compound of the present invention was confirmed to have inhibitory activity of acetylcholinesterase or butyrylcholinesterase, so that quercetin 3-OaL-arabinofuranoside or 2-oxofomolic acid is degenerative brain including Alzheimer's disease It can be seen that it can be usefully used as a preventive and therapeutic agent for diseases.

<Formulation Example 1. Preparation of tablets>

20 g of the present invention quercetin 3- O- aL-arabinofuranoside or 2-oxofomolic acid were mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid, respectively. After adding a 10% gelatin solution to this mixture, it was ground and passed through a 14 mesh sieve. This was dried, and 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate were added thereto, and the resulting mixture was made into tablets.

<Formulation Example 2. Preparation of capsules>

After mixing 100 mg of quercetin 3- O- aL-arabinofuranoside or 2-oxofomolic acid of the present invention, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate of the present invention, The ingredients were mixed and filled into gelatin capsules to prepare capsules.

<Formulation Example 3. Preparation of injection>

1 g of the present invention quercetin 3- O- aL-arabinofuranoside or 2-oxofomolic acid, 0.6 g of sodium chloride, and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20° C. for 30 minutes.

Claims (4)

A pharmaceutical composition for preventing or treating Alzheimer's disease, comprising quercetin 3- O- aL-arabinofuranoside or 2-oxofomolic acid as an active ingredient. delete A health functional food for preventing or improving Alzheimer's disease, comprising quercetin 3- O-aL-arabinofuranoside or 2-oxofomolic acid as an active ingredient. delete