KR20110057831A - Acetylcholinesterase inhibitors comprising the extracts of cladonia macilenta purple or culture borth and/or biruloquinone - Google Patents

Abstract

PURPOSE: A Cladonia macilenta purple, culture extract, biruloquinone compound isolated from the same are provided to treat and prevent anaplasia central nervous system diseases and glaucoma. CONSTITUTION: A Cladonia macilenta purple or extract isolated from culture of the fungi has an ability of suppressing acetylcholine esterase activity. Biruloguinone derived from Cladonia macilenta purple is denoted by chemical formula 1. A pharmaceutical composition for preventing and treating anaplasia central nervous system diseases and glaucoma contains Cladonia macilenta purple extract, culture extract, biruloquinone of chemical formula 1, and pharmaceutically acceptable salt thereof. The anaplasia central nervous system diseases are Alzheimer's diseases, Parkinson's disease, ischemia, myasthenia gravis, or memory loss.

Description

The present invention relates to an acetylcholinesterase inhibitor comprising Cladonia maxilentera purple cells or a culture extract thereof and / or a derivative thereof from a plant, and / or an acetylcholinesterase inhibitor comprising the same as a derivative of Cladonia macilenta purple or culture birch and / or biruloquinone,

The present invention relates to Cladonia < RTI ID = 0.0 > maculenta purple cells or a culture extract thereof, acetylcholinesterase (hereinafter also referred to as " AChE ") inhibitor comprising at least one member selected from the group consisting of Biruloquinone and a pharmaceutically acceptable salt thereof, ≪ / RTI > and pharmaceutical compositions and functional foods containing the same.

Acetylcholinesterase (AChE) is an enzyme that hydrolyzes acetylcholine (ACh), one of the neurotransmitters that mediate the activity of parasympathetic nerves in the body, into choline and acetate, and is formed in the membrane of the endoplasmic reticulum It moves to the cell membrane and performs its function. It is the most widely distributed enzyme in the plasma, liver and other tissues.

In general, the acetylcholine is a substance that is released into a gap called a synaptic cleft in the axonal terminal membrane of neuron and transmits action potential by binding to a receptor of post-synaptic neuron cell membrane. After the excitement is transmitted, it is known that it is decomposed into choline and acetate by the acetylcholinesterase (AChE) enzyme and then absorbed into the cell for reuse.

A vigorous study has shown that many physical and mental illnesses are highly correlated with reduced levels of abnormal acetylcholine levels associated with the mechanism of acetylcholine synthesis and degradation.

BACKGROUND ART Alzheimer's disease, an aging dementia typical of acetylcholine-related diseases, is a representative degenerative brain nervous system disease accompanied by symptoms such as memory decline, language, spatial perception and judgmental disorders. Various hypotheses have been published on the cause and progress of the disease, but it is not clear yet. However, in the brain of patients with Alzheimer 's disease, various nervous systems involving various neurotransmitters are damaged, and damage of the cholinergic nerve is known to be the most serious. In addition, such cholinergic nerve damage has been reported to be closely related to learning, memory and cognitive decline in Alzheimer's disease patients. The pathogenesis of Alzheimer's disease is called 'Cholinergic Hypothesis in Alzheimer's disease' and based on this, the development of therapeutic agents has been actively developed in order to improve the damaged cholinergic nerve. Methods for ameliorating impaired cholinergic neurons include administering a synthetic precursor of acetylcholine (ACh), a cholinergic neurotransmitter, a receptor agonist of cholinergic receptors, or degrading ACh And the ACh inhibitor is administered to maintain the concentration of ACh at the synapse. However, the agonists of cholinergic receptors have disadvantages in that they are easily hydrolyzed in vivo, and practically, a method using an AChE inhibitor is predominant. In fact, all of the drugs developed and used by the FDA for the treatment of Alzheimer's disease are AChE inhibitors such as tacrine, donepezil, and rivastigmine. Among them, compounds derived from natural products include physostigmine (Physostigma venesosum ), galanthamine ( Galanthus nivalis ), huperzine A ( Huperzia serrata ), and the like.

These AChE inhibitors have the effect of reducing the degradation of cognitive function by inhibiting the degradation of ACh by maintaining the concentration of ACh. However, as problems such as hepatotoxicity, short half-life or low bioavailability are raised, Of AChE inhibitors, which have a low side effect.

And, according to another pathological study of the cholinergic system, gastrointestinal motility is regulated by nerve stimulation, and acetylcholine has been reported to enhance this gastrointestinal motility. Therefore, neostigmine, an AChE inhibitor, may be used for the treatment of abdominal distension due to postoperative ileus or dysuria due to incompetence of bladder detrusor muscle.

In addition, myasthenia gravis is a neuromuscular disease caused by an acetylcholine receptor in the muscle membrane that is destroyed by an autoimmune disease, and acetylcholine secreted from the nerve end portion is not sufficiently transferred to the muscle membrane. As part of the treatment, acetylcholinesterase inhibitors such as mestinone and neostigmine are used. Mastinon, which is most commonly used in the treatment of severe myasthenia gravis, also inhibits the action of acetylcholinesterase, which degrades acetylcholine, a neurotransmitter, to increase the concentration of acetylcholine in the body so that acetylcholine may remain in the muscle receptor for a long time . However, there are some cases in which abdominal pain or diarrhea is caused depending on the dose of the drug. In addition, mastinon and other AChE inhibitors for myasthenia gravis developed to date have insufficient selective discrimination against nicotine-type and muscarinic acetylcholine receptors, resulting in adverse effects due to the excitement of muscarinic receptors in patients during drug administration , The rate of absorption is very low, and the treatment effect is partially observed, thus the treatment range is narrow.

In addition, it has been reported that AChE inhibitors effectively reduce the intraocular pressure by accelerating the reabsorption of waterproofing even in the case of glaucoma, which is an eye disease that causes visual impairment due to optic nerve damage due to the elevation of the intraocular pressure. However, currently developed AChE inhibitors require caution because they may cause cataracts when used for more than 6 months for glaucoma treatment.

Although alternatives to acetylcholine synthesis have been suggested once by administering acetylcholine precursors such as choline to effectively treat and prevent various diseases caused by decreased levels of the neurotransmitter acetylcholine, Due to the drawback that it can not pass through the cerebral blood vessels and the effect is terminated in a short time, a drug treatment method for suppressing the hydrolyzing action of acetylcholine using an acetylcholine esterase inhibitor such as tacrine . However, acetylcholinesterase inhibitors currently on the market have been reported to have side effects such as hepatic damage due to toxicity, gastrointestinal disorders such as abdominal pain and diarrhea, and muscle aches as described above.

Lichens, on the other hand, are symbiotic complexes of fungi and algae or / and cyanobacteria, and are classified into about 14,000 species. It has once been proposed to use secondary metabolites of lichens as antimicrobial agents or antiherbivore agents. In addition, several licorice extracts have been used in various fields in the private sector, and the metabolites of lichens have been used as antibiotic agents, antimycobacterial agents, antiviral agents, analgesic agents, antipyretic properties, etc. Have a variety of biological activities. Thus, lichens metabolites are of considerable interest as sources of potential pharmacological agents.

Accordingly, the present inventors have sought to find a substance capable of improving various diseases due to the reduction of the abnormal concentration of acetylcholine in natural products by inhibiting the decomposition of acetylcholine. As a result, It was completed.

It is an object of the present invention to provide a Cladonia macilenta purple cell extract or a culture extract thereof having an acetylcholinesterase enzyme activity inhibiting ability.

Another object of the present invention is to provide a biruloquinone compound derived from Cladonia macilenta purple represented by the following Chemical Formula 1 having an acetylcholinesterase enzyme activity inhibitory activity, Salt.

It is another object of the present invention to provide a method for the production of Cladonia macilenta purple cell extract, a culture extract thereof, a compound of Biruloquinone represented by the following formula (1) and a pharmaceutically acceptable salt thereof, The present invention provides a pharmaceutical composition for the prevention and treatment of any one or more diseases selected from the group consisting of degenerative central nervous system diseases including one or more diseases, asthenic and bladder smooth muscle weakness, and glaucoma diseases.

Another object of the present invention is to provide a method for preventing and treating any one or more diseases from the group consisting of degenerative central nervous system diseases, insomnia of the gastrointestinal tract and bladder smooth muscle, and glaucoma disease by administering the pharmaceutical composition to an individual will be.

It is another object of the present invention to provide a method for the production of Cladonia macilenta purple cell extract, a culture extract thereof, a compound of Biruloquinone represented by the following formula (1) and a pharmaceutically acceptable salt thereof, The present invention provides a health food for preventing or ameliorating one or more diseases selected from the group consisting of degenerative central nervous system diseases including one or more diseases, asthenia and bladder smooth muscle weakness, and glaucoma diseases.

In one embodiment, the present invention relates to a Cladonia macilenta purple cell extract or a culture extract thereof having an acetylcholinesterase enzyme activity inhibiting ability.

Cladonia macilent is a lichen forming fungus that lives in Denmark and other countries. It is a fungus that exists morphologically and chemically in two different types, purple type and pink type. The present inventors have found that Clodoniamascilenta of purple type among the above types has acetylcholinesterase inhibitory activity as compared with pink type Clodnia maxilenta. Therefore, in the present invention, a purple type of clodonia maxilante is conveniently classified into Cladonia < RTI ID = 0.0 > macilenta purple) or clodonia maicilente .

In the present invention, the term "extract" means Cladonia macilenta purple or a substance extracted by a conventional method in a culture filtrate thereof. In the present invention, the term "extract" is used to include not only the above-mentioned extracted fraction but also its dry powder or any form formulated using the same.

In the present invention, Cladonia macilenta purple or a solvent for extracting the extract from the culture filtrate thereof may be a C ₁ -C ₄ alcohol such as methanol, ethanol, propanol, butanol or an aqueous solution thereof, hexane, ethyl Acetate, acetone, methylene chloride, and the like, and it is preferable to use a C ₁ -C ₄ alcohol such as methanol or ethanol. In this case, Cladonia macilenta purple may be used as it is or may be used after grinding. The amount of solvent used may be 0.1 to 5 L per 100 g of dried Cladonia macilenta purple . The temperature for the extraction may be 4 to 120 캜, but is not limited thereto. The extraction time is not particularly limited, but may be 10 minutes to 30 days, and conventional extraction equipment, ultrasonic pulverization extractor, or fractionator may be used. The produced extract may then be subjected to vacuum filtration and / or lyophilization to remove the solvent.

In the present invention, "acetylcholinesterase" is an enzyme that hydrolyzes acetycholine (hereinafter also referred to as ACh), which is one of neurotransmitters that mediate the activity of parasympathetic nerves in the body, with choline and acetate , Which is closely related to the increase and decrease of the concentration of cheneacetylcholine. Thus, the present invention provides a method for treating or preventing various diseases, such as Alzheimer's disease, Parkinson's disease, Ischemia, myasthenia gravis, A substance that inhibits the action of acetylcholinesterase in the treatment and prevention of disorders of the gastrointestinal tract and bladder smooth muscle, such as disorders and alcohol poisoning diseases, and glaucoma diseases, has been used as a therapeutic agent.

The Cladonia macilenta purple cells or the culture extract thereof of the present invention inhibits the activity of acetylcholinesterase by about 60% and is remarkably superior even when compared with other body extracts of the genus or other lichen-forming mold extracts It has an inhibitory effect. Accordingly, the Cladonia macilenta purple extract of the present invention is useful for the treatment of diseases associated with a decrease in the level of abnormal acetylcholine caused by the activity of acetylcholinesterase, such as degenerative central nervous system diseases, gastrointestinal and bladder smooth muscle Asthenia, and glaucoma disease. Examples of the degenerative central nervous system diseases include Alzheimer ' s disease, Parkinson ' s disease, Ischemia, myasthenia gravis, memory disorders and alcohol poisoning diseases.

In another aspect, the present invention relates to a biruloquinone compound derived from Cladonia macilenta purple having an acetylcholinesterase enzyme activity inhibitory activity represented by the following formula (1).

The biruloquinone compound represented by Formula 1 is the main compound isolated from Cladonia macilenta purple. In addition, in the present invention, the above-mentioned birruquinone compound is used as a case that includes a pharmaceutically acceptable salt of the birruquinone compound.

The pharmaceutically acceptable salts of the present invention include salts of acidic or basic groups that may be present in the compounds of the present invention, unless otherwise indicated. For example, erythropoietic salts include the sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include iodobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen phosphate (Acetate), succinate, citrate, tartrate, lactate, mannate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts, ≪ / RTI >

Pharmaceutically acceptable salts are also useful as acid addition salts formed by free acids. The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration.

As the organic acid, an organic acid and an inorganic acid can be used. As the inorganic acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acid include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, Glucuronic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid and the like can be used.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt in particular, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

In one specific embodiment, the method of separating the biruloquinone compound represented by the above formula (1) from Cladonia macilenta purple cells and culture medium is a method of separating Cladonia macilenta purple with a carbon number Preparing an extract using 1 to 4 lower alcohols, methanol or ethyl acetate, and extracting the extract on a column with toluene: ethyl acetate: formic acid (90: 20: 2 v / v) . ≪ / RTI >

The Cladonia < RTI ID = 0.0 > maculenta purple) and the culture solution thereof exhibited an IC ₅₀ (amount indicating 50% enzyme inhibition) of 0.584 mg / ml in the acetylcholinesterase inhibitory activity of the biruloquinone compound represented by the above formula (1) . Thus, it is possible to prevent and treat diseases associated with a decrease in the concentration of abnormal acetylcholine caused by the activity of acetylcholinesterase, for example, degenerative central nervous system diseases, gastrointestinal and bladder weakness, and glaucoma diseases. Examples of the degenerative central nervous system diseases include Alzheimer ' s disease, Parkinson ' s disease, Ischemia, myasthenia gravis, memory disorders and alcohol poisoning diseases.

Accordingly, the present invention provides an acetylcholinesterase inhibitor comprising any one or more of the group consisting of Cladonia macilenta purple extract, Biruloquinone and pharmaceutically acceptable salts thereof, The present invention provides a pharmaceutical composition for the prevention and treatment of various diseases caused by a decrease in the abnormal concentration of the compound of the present invention, i.e., degenerative central nervous system diseases, gastrointestinal tract and bladder smooth muscle, and glaucoma disease. The degenerative central nervous system diseases include Alzheimer ' s disease, Parkinson ' s disease, Ischemia, myasthenia gravis, memory disorders and alcoholism diseases.

The pharmaceutical composition of the present invention can also be mixed with a pharmaceutically acceptable carrier or excipient according to a conventional method or diluted with a diluent. Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. If necessary, the composition may further contain a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

The pharmaceutical composition of the present invention may be administered orally, topically (including buccal, sublingual, skin and intra-ocular), parenteral (subcutaneous, intradermal, intracerebroventricular, intraperitoneal, intramuscular, Intraperitoneal, intramuscular, intravenous, intravenous, intramuscular, intravenous, intraperitoneal, intraperitoneal, intrathecal, intraperitoneal, intraperitoneal, intramuscular, intravenous have. In the case of parenteral administration, for example, intravenous administration can be by needle or a compatible means, and may contain a pharmaceutically acceptable carrier. The compositions of the present invention are preferably administered via intravenous route, at a suitable dosage, and in liquid form. In the case of oral administration, for example, the composition of the present invention may be administered in a liquid or solid form together with a suitable carrier.

The composition of the present invention may also be administered to an individual simultaneously or sequentially with other acetylcholinesterase inhibitors known in the art. The known acetylcholinesterase inhibitor may be of various types, such as Aricept, a capsule containing donepezil hydrochloride, which is typically sold by Eisai, Amici of Nikken Pharmaceuticals, Amirine, SM-10888 from Sumitomo, MF-217 from Mediolanum Pharmaceutici-Angelini, Ro 46-5934 from Hoesht-Russel, HP -290, ENA 713 of Sandoz, Itameline of Bast-Wiles, Metrifonate of Bayer-Wiles, TAK 147 of Takeda, Pfizer's CP 118.954, Naedheim Pharmaceuticals' Galanthamine, ONO ONO 1603, and Marion Merrel Dow's Zifrosilone. Such acetylcholinesterase inhibitors are described in Brufani et al., Alzheimer Disease: From Molecular Biology to Therapy, eds. Becker et al., Pp. 171-177 (1996); Schmidt et al., Alzheimer Disease Alzheimer Disease: From Molecular Biology to Therapy, eds., Becker et al., Pp. 217-221 (1996); 1996); and Giacobini, Alzheimer Disease: From Molecular Biology to Therapy, eds. Becker et al., (1996); and Greig et al., Alzheimer Disease: , pp. 187-204 (1996)).

The composition of the present invention may be administered at the onset of degenerative central nervous system disease, gastrointestinal tract and bladder smooth muscle, and early onset of glaucoma disease, or even after the disease has progressed considerably. For example, it may be administered at about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours after the onset of symptoms of degenerative central nervous system disease, but not limited thereto, It can also be administered when the disease progresses significantly and the relevant symptoms appear overall. The composition of the present invention can also be administered for the prevention of degenerative diseases of the central nervous system, inactivity of the gastrointestinal tract and bladder smooth muscle, and glaucoma disease. Such administration may be repeated as necessary. For example, it may be administered repeatedly daily if clinically necessary, or may be administered, but not limited thereto, after a suitable time interval, for example every 1 to 12 weeks, preferably every 3 to 8 weeks Do not.

The compositions of the present invention may be formulated using methods well known in the art so as to provide for rapid or delayed release of the active ingredient after administration to a subject. Formulations may be in the form of tablets, powders, pills, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like. The formulations will conveniently be in a disposable dosage form.

The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease, and the effective dose level is the severity of the disease; Age, weight, health, sex of the patient; The sensitivity of the patient to the drug; Administration time, route of administration and rate of release; Treatment period; Factors including, but not limited to, the drugs used in combination with or co-used with the compositions of the present invention used, and other factors well known in the medical arts. For example, the single dose of the composition of the present invention may be administered in an amount of 200 to 10,000 units (IU) / body weight (kg), preferably about 200 to 5000 units / body weight (kg) ), More preferably 200 to 2000 units / body weight (kg), most preferably 500 to 1000 units / body weight (kg). In the case of humans, the dose may be 20,000 to 45,000 units, preferably 25,000 to 40,000 units, and more preferably 30,000 to 40,000 units per administration regardless of body weight in consideration of safety.

In another aspect, the present invention provides a pharmaceutical composition comprising at least one member selected from the group consisting of a Cladonia macilenta purple cell extract, a culture extract thereof, Biruloquinone and a pharmaceutically acceptable salt thereof, As the acetylcholinesterase inhibitor, a pharmaceutical composition for the prevention and treatment of various diseases caused by a decrease in the abnormal concentration of acetylcholine, i.e., degenerative central nervous system diseases, gastrointestinal tract and bladder smooth muscle weakness, and glaucoma disease, ≪ / RTI >

As used herein, the term "treatment" refers to therapeutic treatment and prevention or prevention measures. Thus, those in need of treatment will already include those with degenerative central nervous system disease, asthenia and bladder weakness, asthenia, and those with glaucoma disease as well as those who wish to prevent such diseases. As used herein, the term "individual" refers to any mammal in need of treatment, including humans, primates and livestock, breeding, pet or sports animals such as dogs, horses, cats, sheep, pigs, cows and the like.

In another aspect, the present invention provides a pharmaceutical composition comprising at least one member selected from the group consisting of a Cladonia macilenta purple cell extract, a culture extract thereof, Biruloquinone and a pharmaceutically acceptable salt thereof, An acetylcholinesterase inhibitor, and a health food for preventing various diseases caused by a decrease in an abnormal concentration of acetylcholine, that is, a degenerative central nervous system disease, asthenia and bladder weakness, and glaucoma disease.

In the present invention, the degenerative central nervous system diseases include Alzheimer ' s disease, Parkinson ' s disease, Ischemia, myasthenia gravis, memory disorders and alcohol poisoning diseases.

The health food of the present invention may be prepared by adding one or more components from the group consisting of Cladonia macilenta purple cell extract, culture extract thereof, Biruloquinone and pharmaceutically acceptable salts thereof, Can be used together with food or food ingredients, and can be suitably used according to conventional methods. The mixing amount of the above components may be appropriately determined according to the intended use (prevention, health or therapeutic treatment). Generally, in the production of foods or beverages, the components of the present invention are added in an amount of 10% by weight or less, preferably 5% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

When the health food of the present invention is a health drink, various flavoring agents, natural carbohydrates and the like may be added as additional components such as ordinary beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame, and the like . The ratio of the natural carbohydrate is generally about 0.01 to 0.4 g, preferably about 0.02 to 0.03 g per 100 ml of the health drink of the present invention.

In addition to the above, the health food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health food of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention.

The Cladonia macilenta purple cell extract, its culture extract, Biruloquinone and its pharmaceutically acceptable salts according to the present invention are effective acetylcholinesterase inhibiting activity of acetylcholinesterase As a cholinesterase inhibitor, a pharmaceutical composition or a health food containing the same as an active ingredient can be used as a cholinesterase inhibitor, which is produced by decreasing the concentration of acetylcholine in the body such as degenerative central nervous system diseases, asthenia and bladder smooth muscle weakness, And can be effectively used for treating and preventing various diseases.

Particularly, a Cladonia macilenta purple cell extract, a culture extract thereof, Biruloquinone and a pharmaceutically acceptable salt thereof are natural active substances extracted from Cladonia macilenta purple , It has an advantage that it can be safely administered to a patient suffering from a related disease and a patient for preventing related diseases without side effects due to toxicity compared with conventional acetylcholinesterase inhibitors.

The present invention will be explained in more detail through the following examples. These embodiments are merely illustrative of the present invention in order that the present invention may be understood more easily, and thus the scope of the present invention should not be limited to these embodiments.

Example 1: Preparation of fungal extracts of lichen and lichen forming

From the thallus of 16 lichen species collected from Antarctica, Hungary and China from 2007 to 2008, Yamamoto's method (Amamoto, Y., Kinoshita, Y and Yoshimura, 2002. Culture of thallus fragments to form lichens culturing, biochemistry, ecophysiology and use in biomonitoring, ed by I. Kranner, RP Beckett and AK Varma, pp 34-46 Springer-Verlag, NewYork): and redifferentiation of lichens in:. Protocols in lichenology.... The lichen forming fungi (LFF) was isolated and cultured for 60 days in MY medium at 15 ° C. This was dried, and 50 g of the fungus of the lichen tissue and the lichen forming fungi were respectively collected and repeatedly extracted 3 times for 3 hours with 1 L of acetone or methanol. The extract was concentrated under reduced pressure at 40 ° C and redissolved in 10% DMSO to be diluted to 1 mg / ml.

Example 2: Inhibitory activity of AChE on fungal extracts of lichen and lichen forming

(Ellman, GE, Courtnery, D., Andres, V., Featherstone, RM, A new and rapid colorimetric method for investigating the AChE inhibitory activity of the extracts of mung bean- Determination of acetylcholinesterase activity., Biochem. Pharmacol. 1961, 7, 88-95) and acetylthiocholine iodide as an enzyme reaction substrate. First, the PC12 cell culture broth was homogenized with a homogenizer (Terre-Coefficient) with 5 vol of homogenization buffer (containing 10 mM Tris-HCl (pH 7.2), 1 M NaCl, 50 mM MgCl2 and 1% Triton X- Haute, Ind., USA) and centrifuged at 10,000 xg for 30 min. The centrifugation supernatant was used as the source of the enzyme. All extraction steps were carried out at 4 ° C. Protein concentration was measured by bicinchoninic acid (BCA) kit (Sigma Co., St. Louis, Mo., USA) using bovine serum albumin as a standard protein. The hydrolysis rate by AChE was monitored by spectrophotometric change using a 96 well microtiter plate format. The lyophilized extract was initially dissolved in 5% dimethylsulfoxide and then diluted to 1,000 ug / ml. Each extract was mixed with the enzyme solution and incubated for 15 minutes at 37 ° C. Next, the absorbance change at 405 nm was measured after addition of 50 mM phosphate buffer (pH 8.0), Elman reaction reagent (0.5 mM acetylthiocolchine, 1 mM 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) Respectively. The recordings were made for 10 minutes at 2 minute intervals to confirm that the reaction occurred linearly and the% inhibition due to the presence of the extract of the present invention was calculated. The% inhibition was measured as the concentration of extract (IC ₅₀ ) that produced AChE inhibition. The rate of the reaction was calculated using at least three measurements, and the% inhibition due to the presence of the test compound was calculated for the control without the extract. As a result, the AChE inhibitory activity of the extract of the root of the lichen was shown in Fig. 1, and the AChE inhibitory activity of the extract of the lichen-forming fungus was shown in Fig.

As can be seen from Figs. 1 and 2, the AChE inhibition rate was about 15 to 35% in the case of extracts of lichen, and in the case of the lichen-forming fungus extract, the extract of Cladonia macilenta purple was about 30 % AChE inhibitory activity. In particular, the AChE inhibitory activity of Cladonia maxilenta purple showed about 60% higher activity at a concentration of 1.0 mg / ml, indicating that it could be used as a functional material having a high inhibitory activity even at a low purification level.

Example 3: Isolation and activity of Biruloquinone compounds inhibiting AChE from Cladonia maxilenta purple extracts

1 L of the methanol extract of Cladonia maxilenta purple of Example 1 or the culture filtrate thereof was extracted with 3 L of ethyl acetate for 1 hour and then concentrated under reduced pressure. The concentrate was subjected to column chromatography under normal temperature conditions to obtain 10 The fractions were obtained. As a result of confirming the purple color of the fraction by MS and NMR, it was confirmed to be Biruloquinone which is the compound of the above formula (1).

The AChE inhibitory activity of the separated viruloquinone compound was measured by the method described in Example 2, and the IC ₅₀ (amount indicating 50% enzyme inhibition) was 0.584 mg / ml. From these results, it was suggested that the viruloquinone purified from Cladonia masilenta purple inhibits AChE contained in neurons and has an excellent effect of increasing the amount of acetylcholine.

Example 4: Preparation of Purified Extracts containing Cladonia maxilenta purple extract, its culture extract or a compound containing a birchuoquinone

Ingredient content

200 mg of Cladonians maxilentea extract or its culture extract, or 1 mg of a birrudoquinone compound

Corn starch 68 mg

Lactose 90 mg

40 mg of microcrystalline cellulose

Magnesium stearate 2 mg

According to the conventional preparation method of tablets, the above ingredients were added in the prescribed amounts, uniformly mixed, stirred, and then granulated. After drying, the tablets were used to produce the desired tablets containing the active ingredient Cladonia maxilenta purple extract, the culture extract thereof or the birudoquinone compound per one tablet.

Example 5: Preparation of capsid preparation containing Clodoniamascilenta purple extract, its culture extract or a birudoquinone compound

Ingredient content

200 mg of Cladonians maxilentea extract or its culture extract, or 1 mg of a birrudoquinone compound

Corn starch 68 mg

Lactose 90 mg

40 mg of microcrystalline cellulose

Magnesium stearate 2 mg

According to a conventional method for preparing a capsule, the above ingredients are added in the prescribed amounts and uniformly mixed. Then, 200 mg of Cladonia massilenta extract and / or a culture extract thereof or 1 mg of a biruloquinone compound per capsule are contained The gelatin capsule was appropriately sized to fill the desired capsule.

Example 6: Preparation of chewing gum containing Clodonia maicilenta purple extract or its culture extract or a birchuoquinone compound

Ingredient content

200 mg of Cladonians maxilentea extract or its culture extract, or 1 mg of a birrudoquinone compound

Granule sugar syrup 240 mg

Spices (peppermint or lemon flavor) 2 mg

Corn starch 60 mg

100 mg mannitol

20 mg of magnesium stearate

Purified Water

According to the conventional chewing definition manufacturing method, the above ingredients are mixed in an appropriate amount of water, dissolved with heating, cooled, and placed in a molding machine so that the desired shape of the chewing gum is mixed with 200 mg of Cladonia maxilenta extract or its Cultured extract, or 1 mg of a viruloquinone compound.

Example 7: Preparation of Candida including Clodonia maicilenta purple extract, its culture extract, or a birudoquinone compound

Ingredient content

200 mg of Cladonians maxilentea extract or its culture extract, or 1 mg of a birrudoquinone compound

640㎎ granulated sugar syrup

Spices (peppermint or lemon flavor) 2 mg

230 mg of starch syrup

50% tartaric acid 20 mg

Purified Water

The granulated sugar syrup was heated to 110 DEG C while completely dissolving the granulated sugar syrup in a small amount of water, and then the remaining water and starch syrup obtained by dissolving the Cladonia massilenta extract, its culture extract or the birroquinone compound were added, and the temperature was raised to 145 deg. The flame was turned off, and the flavor and tartaric acid were added and mixed. The mixture was cooled to 75 to 80 캜 and molded into a shaping roller to prepare a candy containing Cladonia maxilenta extract, its culture extract or a birchuoquinone compound.

Example 8: Preparation of beverage containing Clodoniamascilenta purple extract, its cultured extract, or a birudoquinone compound

Ingredient content

200 mg of Cladonians maxilenta extract or its culture extract or 1 mg of a birrudoquinone compound

Concentrated juice 2g

12 grams of sucrose

100 mg of sodium citrate

70 mg flavoring

Water appropriate amount

According to the usual method of producing the beverage, the above ingredients are mixed in a suitable amount of water in the prescribed amount, heated and dissolved, and then cooled and filled into a container to obtain 200 mg of Clodnia maxilentea extract or A drink containing the cultured extract thereof, or 1 mg of a biruoquinone compound was prepared.

FIG. 1 shows acetylcholinesterase inhibitory activity of thallus extract of 16 lichen species collected from Antarctica, Hungary and China.

FIG. 2 is a graph showing the acetylcholinesterase inhibitory activity of the extract of the fungus forming fungus isolated from the thallus of 16 lichen species collected from Antarctica, Hungary and China.

Claims (6)

An extract derived from Cladonia macilenta purple cells or culture thereof having an acetylcholinesterase enzyme activity inhibiting ability. A biruloquinone compound derived from Cladonia macilenta purple having an acetylcholinesterase enzyme activity inhibitory activity represented by the following formula (1). Formula 1

A degenerative central nervous system comprising at least one member selected from the group consisting of a Cladonia macilenta purple cell extract, a culture extract thereof, Biruloquinone represented by the above formula (1) and a pharmaceutically acceptable salt thereof, Diseases, gastrointestinal tract and bladder weakness, and a pharmaceutical composition for the prevention and treatment of glaucoma disease. 4. The method of claim 3 wherein the degenerative central nervous system disease is selected from the group consisting of Alzheimer ' s disease, Parkinson ' s disease, Ischemia, myasthenia gravis, memory disorders and alcoholism disorders A pharmaceutical composition which is at least one disease. A degenerative central nervous system comprising at least one member selected from the group consisting of a Cladonia macilenta purple cell extract, a culture extract thereof, Biruloquinone represented by the above formula (1) and a pharmaceutically acceptable salt thereof, Diseases, incompetence of gastrointestinal tract and bladder smooth muscle, and health food for the prevention and improvement of glaucoma disease. 6. The method of claim 5, wherein the degenerative central nervous system disease is selected from the group consisting of Alzheimer ' s disease, Parkinson ' s disease, Ischemia, myasthenia gravis, memory disorders, Health food that is one or more diseases.

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