WO2011065769A2 - Cell or culture extract of cladonia macilenta purple and/or biruloquinone as acetylcholinesterase inhibitors - Google Patents

Cell or culture extract of cladonia macilenta purple and/or biruloquinone as acetylcholinesterase inhibitors Download PDF

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WO2011065769A2
WO2011065769A2 PCT/KR2010/008416 KR2010008416W WO2011065769A2 WO 2011065769 A2 WO2011065769 A2 WO 2011065769A2 KR 2010008416 W KR2010008416 W KR 2010008416W WO 2011065769 A2 WO2011065769 A2 WO 2011065769A2
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biruloquinone
purple
cell
disease
extract
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WO2011065769A3 (en
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Jae-Seoun Hur
Young Jin Koh
Chang Tian Li
Heog Nuo
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Industry-Academy Cooperation Corps. Of Sunchon National University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
    • C07C49/753—Unsaturated compounds containing a keto groups being part of a ring containing ether groups, groups, groups, or groups
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
    • C07C49/753—Unsaturated compounds containing a keto groups being part of a ring containing ether groups, groups, groups, or groups
    • C07C49/755—Unsaturated compounds containing a keto groups being part of a ring containing ether groups, groups, groups, or groups a keto group being part of a condensed ring system with two or three rings, at least one ring being a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/40—Ortho- or ortho- and peri-condensed systems containing four condensed rings
    • C07C2603/42—Ortho- or ortho- and peri-condensed systems containing four condensed rings containing only six-membered rings
    • C07C2603/50—Pyrenes; Hydrogenated pyrenes

Definitions

  • the present invention relates to cell or culture extract of Cladonia macilenta purple, biruloquinone and pharmaceutically acceptable salts thereof useful for acetylcholinesterase (AChE) inhibitors, and pharmaceutical composition and functional food comprising the same.
  • AChE acetylcholinesterase
  • Acetylcholinesterase also known as AChE, is an enzyme that degrades (through its hydrolytic activity) the neurotransmitter acetylcholine (ACh) which mediates activity of parasympathetic neurons, producing choline and an acetate group. It is produced on the membrane of endoplasmic reticulum and moved to cellular membrane to perform its function. It is an important enzyme which is mainly found at cholinergic neurons and their surroundings, especially neuromuscular junctions, and also found in plasma, liver and other tissues.
  • ACh neurotransmitter acetylcholine
  • the acetylcholine is neurotransimitter which is secreted from the membrane of axon end to synaptic cleft and transfers action potential by way of binding the receptor of post-synaptic neuron. After transporting the excitation of the neuron, it is degraded by acetylcholinesterase into choline and acetate, then is transported, through reuptake, back into nerve terminals where it is used to synthesize new acetylcholine molecules.
  • AD Alzheimer’s disease
  • senile dementia is the most typical Acetylcholine-related disorders.
  • the main symptoms of the Alzheimer’s disease are a failure of memory, and a speech, a spatioperceptual, and a judgement disordors.
  • Various hypotheses have been announced for the pathogenesis and progress of the disease, but it is not known clearly.
  • the central nerve system of the patients suffering from AD is generally impaired, and particularly, cholinergic neuron is most severely impaired. Further, it was reported that such impairment of cholinergic neuron is deeply associated with the decreases of learning, memory, and cognitive skill of the patients suffering from AD. Efforts to improve the cholinergic neuron associated with AD have been dominated by the cholinergic hypothesis.
  • ACh precursor which is cholinergic neurotransmitter
  • a method to use cholinergic receptor agonists or a method to maintain a concentration of ACh in synapse to AchE inhibitor.
  • cholinergic receptor agonist has a disadvantage to be readily degraded in vivo , and thus, AChE inhibitor is generally used.
  • AChE inhibitors that have been approved by the United States Food and Drug Administration and used as therapeutic agents of AD are exemplified by tacrine (THA, Cognex ® ), donepezil (Acricept ® ), and rivastigmaine (Exelon ® ).
  • tacrine TAA, Cognex ®
  • donepezil Acricept ®
  • rivastigmaine Exelon ®
  • compounds derived from natural products are galanthamine ( Galanthus nivalis) , huperzine A ( Huperzia serrata) , and so on.
  • AChE inhibitors which keep the concentration of ACh by preventing degradation of ACh have the effect of improving cognitive function.
  • these AChE inhibitors are known to have such problems as hepatotoxicity, short half-life, low bioavailability, etc .
  • more studies have been performed to develop a novel AChE inhibitor.
  • neostigmine a type of AChE inhibitors, neostigmine, is sometimes used for the treatment of abdominal distension due to increased postoperative ileus or dysuria due to incompetence of the bladder detrusor.
  • myasthenia gravis is an autoimmune neuromuscular disease, occurred by destruction of acetylcholine receptors at the post-synaptic neuromuscular junction, thereby inhibiting the stimulative effect of the neurotransmitter acetylcholine. It is treated medically with acetylcholinesterase inhibitors such as mestinon or neostigmine. Mestinon is most frequently used for the treatment of myasthenia gravis, and can inhibit the hydrolytic activity of acetylcholinesterase, thereby increasing the acetylcholine concentration to remain the acetylcholine on the receptors in the muscle cells.
  • acetylcholinesterase inhibitors for the treatment of myasthenia gravis do not show selectivity to nicotinic or muscarinic acetylcholine receptor, and thus, the undesired effects for the stimulation of muscarinic receptors. Moreover, their absorption rates are very low, and their therapeutic ranges are narrow since they are partially effective.
  • Glaucoma is an ocular disease in which the optic nerve is damaged by increased intraocular pressure, leading to progressive, irreversible loss of vision.
  • acetylcholinesterase inhibitors facilitates reabsoprtion of aqueous fluid, thereby lowering the intraocular pressure.
  • special cautions are required since there exists risk occurring cataract when administering presently developed acetylcholinesterase inhibitors to treat glaucoma over 6 months.
  • acetylcholinesterase inhibitors have a lot of side effects such as hepatopathy, gastrointestinal disturbance (abdominal pain or diarrhea), myalgia, etc .
  • lichens are symbioants comprised of fungi, algae, and/or cyanobacteria, and classified into 14,000 species.
  • secondary metabolites of lichens as antimicrobial agents or antiherbivore agents.
  • some extracts of lichens have been found to be used as various medicines among people, and many metabolites of lichens have been found to have a lot of bioactivities, such as antitumour, antibacterial, antifungal, antiviral, antipyretic, antiinflammatory and also antioxidant activities (Oksanen, I. 2006. Ecological and biotechnological aspects of lichens. Applied Microbiology and Biotechnology. 73(4):723-734.). Therefore, much attention has been paid on the metabolites of lichens considerably as potential sources for pharmaceutical agents.
  • the present inventors have endeavored to find material which is able to treat various diseases/disorders associated with abnormal decrease in acetylcholine concentration; and therefore, have found that lichens forming fungi (LFF) is highly capable of inhibiting acetylcholinesterase.
  • LFF lichens forming fungi
  • It is still further object of the present invention to provide method for preventing or treating neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma comprising administering to a subject at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone, and pharmaceutically acceptable salt of biruloquinone.
  • It is still further object of the present invention to provide food for preventing or alleviating neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma comprising administering to a subject at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone, and pharmaceutically acceptable salt of biruloquinone.
  • Cell or culture extract of Cladonia macilenta purple, biruloquinone and pharmaceutically acceptable salt thereof are potent acetylcholinesterase inhibitors which can inhibits activity of acetylcholinesterase. Therefore, pharmaceutical composition or food comprising them can be useful for preventing or treating diseases/disorders associated with abnormal decrease in the acetylcholine concentration by the acetylcholinesterase activity such as neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma.
  • the cell or culture extract of Cladonia macilenta purple, biruloquinone and pharmaceutically acceptable salt thereof have safety on a living body since they are natural active ingredient derived from Cladonia macilenta purple. Accordingly, they can be administered without side effects to patients suffering from said diseases/disorders and subjects which wishes to prevent said diseases/disorders unlike conventional acetylcholinesterase inhibitors.
  • Fig. 1 a bar graph showing AChE inhibition activity of thallus extracts of 16 (sixteen) lichens which are collected from the Antarctic, Hungary and China;
  • Fig. 2 a bar graph showing AChE inhibition activity of lichens-forming fungus extract isolated from the thallus extracts of 16 (sixteen) lichens which are collected from the Antarctic, Hungary and China;
  • Fig. 3 a concentration-inhibition curve of biruloquinone on AChE
  • Fig. 4 results of analyzing cytotoxicity of biruloquinone on PC12 cell
  • Fig. 5 a bar graph showing ability of biruliquinone for prevention of oxidative stress
  • Fig. 6 Lineweaver-Burk plot showing inhibition behavior of biruloquinone on AChE.
  • cell extract or culture extract of Cladonia macilenta purple which inhibits activity of AChE.
  • Cladonia maclienta is a widely contributed lichen-forming fungi (LFF) all over the world including Denmark, and there are 2 (two) morphologically and chemically different types of subcultures, i.e. , purple type and white type. Among these types, the purple type shows potent AChE inhibitory activity, while the white type showed low activity. In the present invention, said purple type refers to Cladonia macilenta purple or Cladonia macilenta.
  • LFF lichen-forming fungi
  • extract refers to material extract from Cladonia macilenta purple or cultural filtration thereof with conventional methods known in the related art. Further, the “extract” encompasses extracted fraction and dried powder thereof, and all formulated types using them.
  • solvent for extracting from Cladonia macilenta purple or its cultural filtration to obtain the inventive extract may be C 1-4 alcohol or its aqueous solution such as methanol, ethanol, propanol or butanol, hexane, ethylacetate, acetone, methylene chloride, or a mixture thereof, preferably C 1-4 alcohol such as methanol or ethanol.
  • Cladonia macilenta purple itself or its pulverized form may be used.
  • the amount of solvent to be used is in the range of 0.1 to 5L per 100g of dried Cladonia macilenta purple, and the non-limiting temperature for the extraction is in the range of 4 to 120°C.
  • the extraction may be conducted for 10 minutes to 30 days, and conventional extraction device, sonicator or fractionator. Then the solvent in the obtained extract may be removed by suction filtration and/or freeze-drying.
  • acetylcholinesterase also known as “AChE”
  • AChE is an enzyme that degrades (through its hydrolytic activity) the neurotransmitter acetylcholine, producing choline and an acetate group, and it has deep relevance to variation in concentration of acetylcholine.
  • acetylcholinesterase inhibitors have been used for the prevention and treatment of numerous diseases/disorders associated with abnormal decrease in the acetylcholine concentration, i.e ., neurodegenerative disease such as Alzheimer’s disease, Parkinson’s disease, ischemic brain disease, myasthenia gravis, defects of memory, alcohol addiction, etc ., myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma.
  • neurodegenerative disease such as Alzheimer’s disease, Parkinson’s disease, ischemic brain disease, myasthenia gravis, defects of memory, alcohol addiction, etc ., myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma.
  • the inventive cell or culture extract of Cladonia macilenta purple exhibited potent AChE inhibitory activity (about 60%), and shows superior AChE inhibitory activity to those of other thallus or lichen-forming fungi (LFF). Accordingly, the extract of Cladonia macilenta purple of the present invention are useful for the prevention and treatment of diseases/disorders associated with abnormal decrease in the acetylcholine concentration by the acetylcholinesterase activity, i.e., neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma.
  • the examples of the neurodegenerative disease are such as Alzheimer’s disease, Parkinson’s disease, ischemic brain disease, myasthenia gravis, defects of memory, alcohol addiction, etc.
  • biruloquinone of formula I derived from Cladonia macilenta purple, which have ability to inhibit AChE activity.
  • the biruloquinone of formula I is main compound isolated from Cladonia macilenta purple.
  • the pharmaceutically acceptable salts of the biruliquinone may include salts of acidic or basic group on the biruloquinone.
  • acidic or basic group are sodium, calcium and potassium salts of hydroxyl group, and hydrobromide salt, sulfate, hydrosulfate, phosphate, hydrogen phosphate, dihydrogen phosephate, acetate, succinate, citrate, tartrate, lactate, madelate, methane sulfonate (mesylate), and p-toluene sulfonate (tosylate) salts of amino group, which may be prepared through the methods/process known in the art.
  • acid-addition salt formed with free acid may also be useful for the pharmaceutically acceptable salts of the present invention.
  • the acid-addition salt may be obtained through conventional method, e.g., dissolving a compound with excess amount of acid solution, followed by precipitating of acid-addition salt with water miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Same molar amounts of a compound, and acid in water or alcohol (e.g ., glycol monomethyl ether) may be heated, and then the obtained mixture may be dried by evaporation or the precipitated salt may be filtered by suction.
  • water miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile.
  • Organic acid or inorganic acid may be used as said free acid.
  • the organic acid is hydrochloric acid, phosphoric acid, nitric acid, or tartaric acid
  • examples of the inorganic acid is methane sulfonic acid, p-toluene sulfonic acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, fumaric acid, mandelic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanilic acid, hydroiodic acid.
  • Alkaline metal salt or alkaline earth metal salt may be obtained through conventional method, e.g., dissolving a compound in excess amount of alkaline metal hydroxide or alkaline earth metal hydroxide solution, filtering non-dissolved salts of the compound, and evaporating and drying remained filtrate.
  • Sodium, potassium, or calcium salt may be preferable, and silver salt corresponding thereto, which is prepared by reacting the sodium, potassium, or calcium salt with suitable silver salt (e.g ., silver nitrate) may also be preferable.
  • suitable silver salt e.g ., silver nitrate
  • method for isolating the biruloquinone of formula I from cell extract or culture extract of Cladonia macilenta purple may comprise preparing extract from Cladonia macilenta purple with lower alcohol such as C 1-4 alcohol or ethyl acetate and eluting the extract on column with mixed solvent of toluene: ethyl acetate: formic acid (90: 20: 20, v/v).
  • inventive cell and culture extract of Cladonia macilenta purple, and biruloquinone of formula I isolated therefrom exhibit inhibitory activity on AChE with 27.1ug/ml of IC 50 , and they act as mixed type inhibitor. Further, they significantly reduce oxidative stress, and are found to have no toxicity though toxicity test.
  • inventive cell and culture extract of Cladonia macilenta purple, and biruloquinone are useful for the prevention and treatment of diseases/disorders associated with abnormal decrease in the acetylcholine concentration by the acetylcholinesterase activity, i.e. , neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma.
  • the examples of the neurodegenerative disease are such as Alzheimer’s disease, Parkinson’s disease, ischemic brain disease, myasthenia gravis, defects of memory, alcohol addiction, etc.
  • the present invention provides pharmaceutical composition for preventing or treating diseases/disorders associated with abnormal decrease in the acetylcholine concentration by the acetylcholinesterase activity, i.e. , neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma which comprises at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone, and pharmaceutically acceptable salt of biruloquinone.
  • neurodegenerative disease i.e. , neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma which comprises at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone, and pharmaceutically acceptable salt of biruloquinone.
  • neurodegenerative disease are as shown above.
  • the pharmaceutical composition of the present invention may be formulated so as to provide various formulations together with pharmaceutically acceptable carrier and/or excipient.
  • suitable carrier and excipient are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.
  • the inventive composition may further comprise filler, anticoagulant, humectant, lubricant, flavor, emulsifier, preservative, etc.
  • the pharmaceutical composition of the present invention may be administered via any of the common routes, such as oral, topical (including buccal, sublingual, dermal, and intraocular route), parenteral (subcutaneous, intradermal, intraperitoneal, intracerebrospinal, intramuscle, intravascular and intraacrticular route), or transdermal route, but the present invention is not limited to these exemplified modes of administration.
  • composition of the present invention may be used as the single therapy. But it may be simultaneously or consecutively administered together with other acetylcholinesterase inhibitor which is known in the art.
  • acetylcholinesterase inhibitor is Aricept (donepezil hydrochloride capsule commercialized by Eisai), Amirine (Nikken), SM-10888 (Sumitomo), MF-217 (Mediolanum Pharmaceutici-Angelini), Ro 46-5934, HP-290 (Hoesht-Russel), ENA 713 (Sandoz), Itameline (Hoesht-Russel), Metrifonate (Bayer-Wiles), TAK 147 (Takeda), CP 118.954 (Pfizer), Ganlanthamine (Naedheim), ONO 1603 (Ono), Zifrosilone (Marion Merrel Dow).
  • Such acetylcholinesterase inhibitors are disclosed Brufani et al.(Alzheimer Disease: From Molecular Biology to Therapy, eds. Becker et al., pp. 171-177 (1996)), Schmidt et al. (Alzheimer Disease: From Molecular Biology to Therapy, eds. Becker et al., pp. 217-221 (1996)), Vargas et al. (Alzheimer Disease: From Molecular Biology to Therapy, eds. Becker et al., pp. 251-255 (1996)), Greig et al. (Alzheimer Disease: From Molecular Biology to Therapy, eds. Becker et al., pp. 231-237 (1996)), and Giacobini (Alzheimer Disease: From Molecular Biology to Therapy, eds. Becker et al., pp. 187-204 (1996)).
  • the composition of the present invention may be administered at an early stage of onset of neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder or glaucoma, or it may also be administered after progression of the diseases.
  • the composition may be administered after 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours of onset of neurodegenerative disease, but it is not limited thereto.
  • the composition of the present invention may be administered for the prevention of neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder or glaucoma.
  • the inventive composition may be repeatedly administered as the need arises. For example, in case of clinical need, it may be administered daily, or at suitable intervals, e.g., 1 to 12 weeks, preferably 3 to 8 week, but not limited thereto.
  • composition of the present invention may be formulated so as to provide immediate or sustained release of active ingredient.
  • Such formulation may be tablet, powder, pill, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft or hard capsule, sterilized injectable solution, sterilized powder, etc.
  • the composition of the present invention may be preferably provided in the form of single dose.
  • composition of the present invention may be administered at “therapeutically effective amount.”
  • therapeutically effective amount refers to amount enough to treat diseases/disorders.
  • the therapeutically effective amount of the composition ought to be determined in light of various relevant factors including the condition to be treated, the age, weight, health, reaction susceptibility and sex of the individual patient, food, administration time, administration route, excretion rate, period of treatment, the severity of the patient's symptom and, medication combined or simultaneously administered with the inventive composition, and the factors known in the medical field; and, therefore, the above dose should not be intended to limit the scope of the invention in any way.
  • single dose of the inventive composition for animal except human may be administered in amount of 200 to 10,000 IU/kg, preferably 200 to 5,000 IU/kg, more preferably 200 to 2,000 IU/kg, most preferably 500 to 1,000 IU.
  • single dose the inventive composition for human may be administered in amount of 25,000 to 45,000 IU, preferably 25,000 to 40,000 IU, more preferably 30,000 to 40,000 IU, regardless of body weight.
  • the present invention relates to method for preventing or treating diseases/disorders associated with abnormal decrease in the acetylcholine concentration by the acetylcholinesterase activity such as neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma comprising administering to a subject at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone, and pharmaceutically acceptable salt of biruloquinone.
  • a subject in need of treatment in the present invention may be one which wishes to prevent neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma as well as one suffered therefrom.
  • subject means human, primates, and certain mammals such as dog, horse, cat, sheep, pig, cattle, etc. , in need of the treatment.
  • the present invention relates to health food for preventing or alleviating diseases/disorders associated with abnormal decrease in the acetylcholine concentration by the acetylcholinesterase activity such as neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder or glaucoma which comprises at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone, and pharmaceutically acceptable salt of biruloquinone.
  • diseases/disorders associated with abnormal decrease in the acetylcholine concentration by the acetylcholinesterase activity such as neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder or glaucoma which comprises at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone, and pharmaceutically acceptable salt of biruloquinone.
  • the health food of the present invention may comprise at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone, and pharmaceutically acceptable salt of biruloquinone alone or together with other food or other food component.
  • inventive health food may be used according to conventional method. The ratio of components contained in the food ought to be determined in light of purpose for using (prevention, health or therapeutic treatment).
  • the extract of Cladonia macilenta purple, biruloquinone or pharmaceutically aceeptable salt thereof may be added in amount of 10% by weight or less, preferably 5% by weight or less for preparing food or beverage.
  • food for long-term ingestion for health maintenance may comprise less amount of said active ingredients than such amount.
  • more amount of active ingredients than such amount may be used for the food of the present invention since the active ingredients have no problem in safety.
  • the health food which can comprise the extract of Cladonia macilenta purple, biruloquinone or pharmaceutically aceeptable salt thereof are meat, sausage, bread, chocolate, candies, snacks, cookies, pizza, ramen or other noodles, gums, dairy products including icecream, various soups, beverage, tea, drink, alcoholic beverage and Vitamin composition.
  • the food of the present invention encompasses all kinds of conventional health food.
  • the health beverage may contain various flavoring agent, natural hydrocarbon, etc. , as an additional component like conventional beverages.
  • the natural hydrocarbons may be monosaccharide such as glucose or fructose, disaccharide such as maltose or sucrose, natural sweetener such as dextrin or cyclodextrin or synthetic sweetener such as saccharin or aspartame.
  • the ratio of the natural hydrocarbon is generally about 0.01 to 0.4g, preferably 0.02 to 0.03g per 100ml of the health beverage.
  • the food of the present invention may further contain various nutritional supplements, vitamin, electrolyte, savoring agent, coloring agent, pectic acid and salt thereof, alginic acid and salt thereof, organic acid, protective colloid viscosity agent, pH controlling agent, stabilizer, preservative, glycerin, alcohol, and carbonatization agent used for a carbonated drink.
  • the food of the present invention may contain pulp (fruit flesh) for natural fruit juice, fruit juice beverage and vegetable beverage. The ratios of such additives are not critical, and are generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the inventive food.
  • Example 1 Prepeartions of lichen thallus extract and linchen forming fungi extract
  • lichen forming fungi were isolated from lichen thallus by Yamamoto’s method (Yamamoto, Y., Mizuguchi, R. and Yamada, Y. 1985. Tissue cultures of Usnea rubescens and Ramalina yasudae and production of usnic acid in their cultures. Agricultural and Biological Chemistry. 49(11):3347-3348.) and cultured on MY medium at 15°C for 60 days. Thereafter, they were dried and collected 50g lichen thallus and LFF, respectively. They were extracted with 1L acetone or methanol for 3 hours, and this process repeated 3 times. Acetone or methanol extracts were then evaporated in vacuum at 40°C, and dissolved in 10% of DMSO, then diluted to 1 mg/ml.
  • Yamamoto Yamamoto, Y., Mizuguchi, R. and Yamada, Y. 1985. Tissue cultures of Usnea rubescens and Ramalina yasudae and production
  • Example 2 AChE inhibitory activities of lichen thallus extract and LFF extract
  • AChE inhibitory activities of lichen thallus extract and LFF extract gained from example 1 were studied by the colorimetric method described at Ellman et al.(Ellman, G.E., Courtnery, D., Andres, V., Featherstone, R.M., A new and rapid colorimetric determination of acetylcholinesterase activity., Biochem. Pharmacol. 1961, 7, 88-95) and using acetylthiocholine iodide as a substrate.
  • PC12 cell cultures were homogenized in a Glass-Col ® homogenizer (Terre-Haute, Ind., USA) with 5 vol of homogenation buffer (10 mM Tris-HCl(pH7.2), containing 1 M NaCl, 50 nM MgCl 2 and 1% TritonX-100), and then centrifuged at 10,000g for 30 min. The resulting supernatant was used as an enzyme source. All extraction steps were carried out at 4°C. The protein concentration was determined using the bicinchoninic acid kit (Sigma Co., St. Louis, Mo., USA) with bovine serum albumin as protein standard.
  • the rates of hydrolysis by AChE were monitored spectrophotomatically using a 96-well microtiter plate format. Freeze-dried extracts were initially dissolved in 5% dimethyl sulfoxide, and the solution was diluted to 1,000ug/ml. Each extract was mixed with enzyme solution and incubated at 37°C for 15min. Absorbance at 405 nm was read immediately after adding an Ellman’s reaction mixture(0.5 mM acetylthiocholine, 1 mM 5,5-dithiobis(2-nitrobenzoic acid)(DTNB)) in 50 mM sodium phosphate buffer (pH 8.0) to the above reaction mixture. Reading was repeated for 10min at 2 min intervals to verify that the reaction occurred linearly.
  • AChE inhibitory activity of lichen thallus extract and LFF extract were caculated as an inhibition percentage of the maximum activity(negative control wells), and determined IC 50 . These experiments were performed at 3 times, and the average was used to determine IC 50 .
  • AChE inhibitory activity of lichen thallus extract was summarized in figure 1
  • AChE inhibitory activity of LFF extract was summarized in figure 2.
  • Figures 1 and 2 showed that the AChE inhibitory activity of lichen thallus extract was about 15-35% and the AChE inhibitory activity of LFF extract was below 30% except Cladonia macilenta purple.
  • the AChE inhibitory activity of Cladonia macilenta purple was about 60% when treated with 1 mg/ml Cladonia macilenta purple.
  • the high AChE inhibitory activity in low LFF purification fold means the usable possibility of the fucntional material.
  • Example 3 Isolation and identification of biruloquinone derived from Cladonia macilenta purple extracts
  • the method described in example 2 was used to determine the AChE inhibition activity of biruloquinone. As such result, the inhibition is in a dose-dependent manner (Fig. 3). The inhibition percentage increased rapidly with the increase of biruloquinone concentration from 0 to 100ug/ml. The concentration that was required for the 50% enzyme inhibition IC 50 was 27.1ug/ml(83.1uM). This result suggested that biruloquinone derived from Cladonia macilenta purple extracts had AChE inhibitory activity and increased acetylcholine.
  • Example 4 Toxicity assay of biruloquinone on injured PC12 cells
  • PC12 cells were purchased from Korean Cell Line Bank (KCLB). They were cultured in growth medium RPMI-1640 with l-glutamine, 5% fetal bovine serum(FBS), 10% horse serum, and antibiotics. The cells were maintained in 5% CO2 incubator at 37°C.
  • KCLB Korean Cell Line Bank
  • Viable PC 12 cells (10 4 /well) were plated into 96-well collagen-coated plate 24 hours before the experiments. On the experiment day, the cells were cultured with gradient concentration of biruloquinone (6.25, 12.5, 25, 50 and 100 ⁇ g/ml) for 3 hours.
  • the cell viability was determined by conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) reduction assay(Shi, D. H., Wu, J. H., Ge, H. M. and Tan, R. X. 2009. Protective effect of hopeahainol A, a novel acetylcholinesterase inhibitor, on hydrogen peroxide-induced injury in PC12 cells.
  • Viable cells(10 4 /well) were plated into 96-well collagen-coated plated 24 hours before the experiments to test oxidative stress of biruloquione.
  • the cells were cultured with 100 ⁇ M DCFH-DA for 30min. After DCFH-DA was removed, the cells were washed and incubated with RPMI medium.
  • PC12 cells were then pretreated with various concentrations of biruloquione(25, 12.5, 6.25 and 1 ⁇ g/ml) for 30min. The cells were then treated with or without 200 ⁇ M H 2 O 2 for 30 min and the fluorescence was finally quantified using fluorescent microplate reader(SpectraMax, Molecular Devices, California, US).
  • the in vitro oxidative stress prevention of biruloquinone were summarized in figure 5.
  • Example 6 Inhibition kinetic assay of biruloquinone on AChE
  • Example 8 The preparation of table including cell or culture extract of Cladonia macilenta purple, or biruloquinone
  • the above components were added to the suggested content and uniformly mixed. Thereafter, it stirred and granulated.
  • the intended refinement in which the active ingredient of cell or culture extract of Cladonia macilenta purple, or biruloquinone per 1 refinement was included was manufactured.
  • Example 9 The manufacture of the capsule including cell or culture extract of Cladonia macilenta purple, or biruloquinone
  • the above components were added to the suggested content and uniformly mixed. Thereafter, it filled in the gelatin capsule of the proper size so that 200mg of cell or culture extract of Cladonia macilenta purple, or 1mg biruloquinone per 1 capsule was included and the intended capsule was manufactured.
  • Example 10 The manufacture of chewing tablet including cell or culture extract of Cladonia macilenta purple, or biruloquinone
  • the above components were mixed with the suitable amount of purified water. Thereafter, it dissolved while heating, cooled, put into molder, and the chewing tablet of the intended shape was manufactured so that 200mg of cell or culture extract of Cladonia macilenta purple, or 1mg biruloquinone per 1 tablet was included.
  • Example 11 The manufacture of the candy including cell or culture extract of Cladonia macilenta purple, or biruloquinone
  • the water and granulated sugar syrup of the rest which was molten cell or culture extract of Cladonia macilenta purple, or biruloquinone were added to of the rest melting. Temperature was then raised to 145°C. The fire was drawn. Spice and tartaric acid were added and it mixed. After it cooled to 75 ⁇ 80°C, it molded to the star roller and candy containing cell or culture extract of Cladonia macilenta purple, or biruloquinone was manufactured.
  • Example 12 the manufacture of the drink including cell or culture extract of Cladonia macilenta purple, or biruloquinone
  • the above components were mixed to the suggested content with the suitable amount of purified water. After it heated and dissolved, the drink cooled, charged in container. Thereby the drink incluing 200mg of cell or culture extract of Cladonia macilenta purple, or 1mg biruloquinone per 1 bottle of 200ml capacity was manufactured.

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Abstract

The present invention relates to cell or culture extract of Cladonia macilenta purple, biruloquinone and pharmaceutically acceptable salts thereof which have acetylcholinesterase (AChE) inhibitory activity, and pharmaceutical composition and functional food for preventing, treating, or alleviating neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma, comprising the same.

Description

CELL OR CULTURE EXTRACT OF CLADONIA MACILENTA PURPLE AND/OR BIRULOQUINONE AS ACETYLCHOLINESTERASE INHIBITORS
The present invention relates to cell or culture extract of Cladonia macilenta purple, biruloquinone and pharmaceutically acceptable salts thereof useful for acetylcholinesterase (AChE) inhibitors, and pharmaceutical composition and functional food comprising the same.
Acetylcholinesterase, also known as AChE, is an enzyme that degrades (through its hydrolytic activity) the neurotransmitter acetylcholine (ACh) which mediates activity of parasympathetic neurons, producing choline and an acetate group. It is produced on the membrane of endoplasmic reticulum and moved to cellular membrane to perform its function. It is an important enzyme which is mainly found at cholinergic neurons and their surroundings, especially neuromuscular junctions, and also found in plasma, liver and other tissues.
Generally, the acetylcholine is neurotransimitter which is secreted from the membrane of axon end to synaptic cleft and transfers action potential by way of binding the receptor of post-synaptic neuron. After transporting the excitation of the neuron, it is degraded by acetylcholinesterase into choline and acetate, then is transported, through reuptake, back into nerve terminals where it is used to synthesize new acetylcholine molecules.
After active research of acetylcholine, numerous physical and mental diseases/disorders are significantly related with abnormal decrease in acetylcholine concentration concerning the synthesis and degradation mechanisms for acetylcholine.
Alzheimer’s disease (AD), which is known as senile dementia, is the most typical Acetylcholine-related disorders. The main symptoms of the Alzheimer’s disease are a failure of memory, and a speech, a spatioperceptual, and a judgement disordors. Various hypotheses have been announced for the pathogenesis and progress of the disease, but it is not known clearly. However, the central nerve system of the patients suffering from AD is generally impaired, and particularly, cholinergic neuron is most severely impaired. Further, it was reported that such impairment of cholinergic neuron is deeply associated with the decreases of learning, memory, and cognitive skill of the patients suffering from AD. Efforts to improve the cholinergic neuron associated with AD have been dominated by the cholinergic hypothesis. Based on this hypothesis, the development of therapeutics has been actively made in direction of improving the damaged cholinergic neurons. To improve the impaired cholinergic neuron, there is a method to use ACh precursor which is cholinergic neurotransmitter, a method to use cholinergic receptor agonists, or a method to maintain a concentration of ACh in synapse to AchE inhibitor. However, cholinergic receptor agonist has a disadvantage to be readily degraded in vivo, and thus, AChE inhibitor is generally used.
Actually, AChE inhibitors that have been approved by the United States Food and Drug Administration and used as therapeutic agents of AD are exemplified by tacrine (THA, Cognex®), donepezil (Acricept®), and rivastigmaine (Exelon®). Among them, compounds derived from natural products are galanthamine (Galanthus nivalis), huperzine A (Huperzia serrata), and so on.
These AChE inhibitors which keep the concentration of ACh by preventing degradation of ACh have the effect of improving cognitive function. However, these AChE inhibitors are known to have such problems as hepatotoxicity, short half-life, low bioavailability, etc. Thus, more studies have been performed to develop a novel AChE inhibitor.
Meanwhile, the other pathological studies which are related with choline nervous system have been reported that gastrointestinal movement is controlled by nerve stimulation and acetylcholine improves stomach exercise. Therefore, a type of AChE inhibitors, neostigmine, is sometimes used for the treatment of abdominal distension due to increased postoperative ileus or dysuria due to incompetence of the bladder detrusor.
Further, myasthenia gravis is an autoimmune neuromuscular disease, occurred by destruction of acetylcholine receptors at the post-synaptic neuromuscular junction, thereby inhibiting the stimulative effect of the neurotransmitter acetylcholine. It is treated medically with acetylcholinesterase inhibitors such as mestinon or neostigmine. Mestinon is most frequently used for the treatment of myasthenia gravis, and can inhibit the hydrolytic activity of acetylcholinesterase, thereby increasing the acetylcholine concentration to remain the acetylcholine on the receptors in the muscle cells. Although it has advantage to show therapeutic effects in a short period of time, there often exist some side effects of abdominal pain or diarrhea according to its dose. Further, presently developed acetylcholinesterase inhibitors for the treatment of myasthenia gravis do not show selectivity to nicotinic or muscarinic acetylcholine receptor, and thus, the undesired effects for the stimulation of muscarinic receptors. Moreover, their absorption rates are very low, and their therapeutic ranges are narrow since they are partially effective.
Further, Glaucoma is an ocular disease in which the optic nerve is damaged by increased intraocular pressure, leading to progressive, irreversible loss of vision. In this regard, it is reported that acetylcholinesterase inhibitors facilitates reabsoprtion of aqueous fluid, thereby lowering the intraocular pressure. However, special cautions are required since there exists risk occurring cataract when administering presently developed acetylcholinesterase inhibitors to treat glaucoma over 6 months.
To effectively prevent and treat various diseases/disorders associated with decrease in acetylcholine concentration, the alternative method of administering precursors of acetylcholine to facilitate the synthesis of acetylcholine was once suggested. However, due to their short-term effects without entering blood brain barrier (BBB), the drug treatment with acetylcholinesterase such as tacrine to hydrolytic activity of acetylcholine has been mainly used.
As mentioned above, it is reported that presently developed and commercialized acetylcholinesterase inhibitors, however, have a lot of side effects such as hepatopathy, gastrointestinal disturbance (abdominal pain or diarrhea), myalgia, etc.
Meanwhile, lichens are symbioants comprised of fungi, algae, and/or cyanobacteria, and classified into 14,000 species. Once, it was suggested to use secondary metabolites of lichens as antimicrobial agents or antiherbivore agents. Further, some extracts of lichens have been found to be used as various medicines among people, and many metabolites of lichens have been found to have a lot of bioactivities, such as antitumour, antibacterial, antifungal, antiviral, antipyretic, antiinflammatory and also antioxidant activities (Oksanen, I. 2006. Ecological and biotechnological aspects of lichens. Applied Microbiology and Biotechnology. 73(4):723-734.). Therefore, much attention has been paid on the metabolites of lichens considerably as potential sources for pharmaceutical agents.
In this regard, the present inventors have endeavored to find material which is able to treat various diseases/disorders associated with abnormal decrease in acetylcholine concentration; and therefore, have found that lichens forming fungi (LFF) is highly capable of inhibiting acetylcholinesterase.
Accordingly, it is an object of the present invention to provide cell extract or culture extract of Cladonia macilenta purple which inhibits activity of AChE.
It is another object of the present invention to provide biruloquinone of formula I derived from Chladonia macilenta purple, or pharmaceutically acceptable salt thereof which have ability to inhibit AChE activity.
It is further object of the present invention to provide use of cell or culture extract of Cladonia macilenta purple, biruloquinone, or pharmaceutically acceptable salt of biruloquinone as AChE inhibitor.
It is still further object of the present invention to provide pharmaceutical composition for preventing or treating neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma which comprises at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone, and pharmaceutically acceptable salt of biruloquinone.
It is still further object of the present invention to provide use of cell or culture extract of Cladonia macilenta purple, biruloquinone, or pharmaceutically acceptable salt of biruloquinone for preventing or treating neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder or glaucoma.
It is still further object of the present invention to provide method for preventing or treating neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma comprising administering to a subject at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone, and pharmaceutically acceptable salt of biruloquinone.
It is still further object of the present invention to provide food for preventing or alleviating neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma comprising administering to a subject at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone, and pharmaceutically acceptable salt of biruloquinone.
Cell or culture extract of Cladonia macilenta purple, biruloquinone and pharmaceutically acceptable salt thereof are potent acetylcholinesterase inhibitors which can inhibits activity of acetylcholinesterase. Therefore, pharmaceutical composition or food comprising them can be useful for preventing or treating diseases/disorders associated with abnormal decrease in the acetylcholine concentration by the acetylcholinesterase activity such as neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma.
In particular, the cell or culture extract of Cladonia macilenta purple, biruloquinone and pharmaceutically acceptable salt thereof have safety on a living body since they are natural active ingredient derived from Cladonia macilenta purple. Accordingly, they can be administered without side effects to patients suffering from said diseases/disorders and subjects which wishes to prevent said diseases/disorders unlike conventional acetylcholinesterase inhibitors.
The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, which respectively show:
Fig. 1: a bar graph showing AChE inhibition activity of thallus extracts of 16 (sixteen) lichens which are collected from the Antarctic, Hungary and China;
Fig. 2: a bar graph showing AChE inhibition activity of lichens-forming fungus extract isolated from the thallus extracts of 16 (sixteen) lichens which are collected from the Antarctic, Hungary and China;
Fig. 3: a concentration-inhibition curve of biruloquinone on AChE;
Fig. 4: results of analyzing cytotoxicity of biruloquinone on PC12 cell;
Fig. 5: a bar graph showing ability of biruliquinone for prevention of oxidative stress; and
Fig. 6: Lineweaver-Burk plot showing inhibition behavior of biruloquinone on AChE.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Further, all documents mentioned herein are incorporated by reference in their entireties.
In accordance with one aspect of the present invention, there is provided cell extract or culture extract of Cladonia macilenta purple which inhibits activity of AChE.
Cladonia maclienta is a widely contributed lichen-forming fungi (LFF) all over the world including Denmark, and there are 2 (two) morphologically and chemically different types of subcultures, i.e., purple type and white type. Among these types, the purple type shows potent AChE inhibitory activity, while the white type showed low activity. In the present invention, said purple type refers to Cladonia macilenta purple or Cladonia macilenta.
The term “extract” as used herein refers to material extract from Cladonia macilenta purple or cultural filtration thereof with conventional methods known in the related art. Further, the “extract” encompasses extracted fraction and dried powder thereof, and all formulated types using them.
In the present invention, solvent for extracting from Cladonia macilenta purple or its cultural filtration to obtain the inventive extract may be C1-4 alcohol or its aqueous solution such as methanol, ethanol, propanol or butanol, hexane, ethylacetate, acetone, methylene chloride, or a mixture thereof, preferably C1-4 alcohol such as methanol or ethanol. For the extraction, Cladonia macilenta purple itself or its pulverized form may be used. The amount of solvent to be used is in the range of 0.1 to 5L per 100g of dried Cladonia macilenta purple, and the non-limiting temperature for the extraction is in the range of 4 to 120℃. Further, the extraction may be conducted for 10 minutes to 30 days, and conventional extraction device, sonicator or fractionator. Then the solvent in the obtained extract may be removed by suction filtration and/or freeze-drying.
In the present invention, “acetylcholinesterase”, also known as “AChE”, is an enzyme that degrades (through its hydrolytic activity) the neurotransmitter acetylcholine, producing choline and an acetate group, and it has deep relevance to variation in concentration of acetylcholine. Accordingly, acetylcholinesterase inhibitors have been used for the prevention and treatment of numerous diseases/disorders associated with abnormal decrease in the acetylcholine concentration, i.e., neurodegenerative disease such as Alzheimer’s disease, Parkinson’s disease, ischemic brain disease, myasthenia gravis, defects of memory, alcohol addiction, etc., myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma.
The inventive cell or culture extract of Cladonia macilenta purple exhibited potent AChE inhibitory activity (about 60%), and shows superior AChE inhibitory activity to those of other thallus or lichen-forming fungi (LFF). Accordingly, the extract of Cladonia macilenta purple of the present invention are useful for the prevention and treatment of diseases/disorders associated with abnormal decrease in the acetylcholine concentration by the acetylcholinesterase activity, i.e., neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma. The examples of the neurodegenerative disease are such as Alzheimer’s disease, Parkinson’s disease, ischemic brain disease, myasthenia gravis, defects of memory, alcohol addiction, etc.
In accordance with another aspect of the present invention, there is provided biruloquinone of formula I derived from Cladonia macilenta purple, which have ability to inhibit AChE activity.
<Formula I>
Figure PCTKR2010008416-appb-I000001
The biruloquinone of formula I is main compound isolated from Cladonia macilenta purple. In the present invention, the “biruloquinone,” in case, encompasses its pharmaceutically acceptable salts.
Unless described otherwise, the pharmaceutically acceptable salts of the biruliquinone may include salts of acidic or basic group on the biruloquinone. Examples of such acidic or basic group are sodium, calcium and potassium salts of hydroxyl group, and hydrobromide salt, sulfate, hydrosulfate, phosphate, hydrogen phosphate, dihydrogen phosephate, acetate, succinate, citrate, tartrate, lactate, madelate, methane sulfonate (mesylate), and p-toluene sulfonate (tosylate) salts of amino group, which may be prepared through the methods/process known in the art.
In addition, acid-addition salt formed with free acid may also be useful for the pharmaceutically acceptable salts of the present invention. The acid-addition salt may be obtained through conventional method, e.g., dissolving a compound with excess amount of acid solution, followed by precipitating of acid-addition salt with water miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Same molar amounts of a compound, and acid in water or alcohol (e.g., glycol monomethyl ether) may be heated, and then the obtained mixture may be dried by evaporation or the precipitated salt may be filtered by suction.
Organic acid or inorganic acid may be used as said free acid. Examples of the organic acid is hydrochloric acid, phosphoric acid, nitric acid, or tartaric acid, and examples of the inorganic acid is methane sulfonic acid, p-toluene sulfonic acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, fumaric acid, mandelic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanilic acid, hydroiodic acid.
Further, pharmaceutically acceptable metal salt formed with base may also be useful for the present invention. Alkaline metal salt or alkaline earth metal salt may be obtained through conventional method, e.g., dissolving a compound in excess amount of alkaline metal hydroxide or alkaline earth metal hydroxide solution, filtering non-dissolved salts of the compound, and evaporating and drying remained filtrate.
Sodium, potassium, or calcium salt may be preferable, and silver salt corresponding thereto, which is prepared by reacting the sodium, potassium, or calcium salt with suitable silver salt (e.g., silver nitrate) may also be preferable.
In one specific example of the present invention, method for isolating the biruloquinone of formula I from cell extract or culture extract of Cladonia macilenta purple may comprise preparing extract from Cladonia macilenta purple with lower alcohol such as C1-4 alcohol or ethyl acetate and eluting the extract on column with mixed solvent of toluene: ethyl acetate: formic acid (90: 20: 20, v/v).
The inventive cell and culture extract of Cladonia macilenta purple, and biruloquinone of formula I isolated therefrom exhibit inhibitory activity on AChE with 27.1ug/ml of IC50, and they act as mixed type inhibitor. Further, they significantly reduce oxidative stress, and are found to have no toxicity though toxicity test. Thus, inventive cell and culture extract of Cladonia macilenta purple, and biruloquinone are useful for the prevention and treatment of diseases/disorders associated with abnormal decrease in the acetylcholine concentration by the acetylcholinesterase activity, i.e., neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma. The examples of the neurodegenerative disease are such as Alzheimer’s disease, Parkinson’s disease, ischemic brain disease, myasthenia gravis, defects of memory, alcohol addiction, etc.
Accordingly, the present invention provides pharmaceutical composition for preventing or treating diseases/disorders associated with abnormal decrease in the acetylcholine concentration by the acetylcholinesterase activity, i.e., neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma which comprises at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone, and pharmaceutically acceptable salt of biruloquinone. Examples of the neurodegenerative disease are as shown above.
The pharmaceutical composition of the present invention may be formulated so as to provide various formulations together with pharmaceutically acceptable carrier and/or excipient. Example of suitable carrier and excipient are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Optionally, the inventive composition may further comprise filler, anticoagulant, humectant, lubricant, flavor, emulsifier, preservative, etc.
The pharmaceutical composition of the present invention may be administered via any of the common routes, such as oral, topical (including buccal, sublingual, dermal, and intraocular route), parenteral (subcutaneous, intradermal, intraperitoneal, intracerebrospinal, intramuscle, intravascular and intraacrticular route), or transdermal route, but the present invention is not limited to these exemplified modes of administration.
Further, the composition of the present invention may be used as the single therapy. But it may be simultaneously or consecutively administered together with other acetylcholinesterase inhibitor which is known in the art. Examples of such acetylcholinesterase inhibitor is Aricept (donepezil hydrochloride capsule commercialized by Eisai), Amirine (Nikken), SM-10888 (Sumitomo), MF-217 (Mediolanum Pharmaceutici-Angelini), Ro 46-5934, HP-290 (Hoesht-Russel), ENA 713 (Sandoz), Itameline (Hoesht-Russel), Metrifonate (Bayer-Wiles), TAK 147 (Takeda), CP 118.954 (Pfizer), Ganlanthamine (Naedheim), ONO 1603 (Ono), Zifrosilone (Marion Merrel Dow). Such acetylcholinesterase inhibitors are disclosed Brufani et al.(Alzheimer Disease: From Molecular Biology to Therapy, eds. Becker et al., pp. 171-177 (1996)), Schmidt et al. (Alzheimer Disease: From Molecular Biology to Therapy, eds. Becker et al., pp. 217-221 (1996)), Vargas et al. (Alzheimer Disease: From Molecular Biology to Therapy, eds. Becker et al., pp. 251-255 (1996)), Greig et al. (Alzheimer Disease: From Molecular Biology to Therapy, eds. Becker et al., pp. 231-237 (1996)), and Giacobini (Alzheimer Disease: From Molecular Biology to Therapy, eds. Becker et al., pp. 187-204 (1996)).
The composition of the present invention may be administered at an early stage of onset of neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder or glaucoma, or it may also be administered after progression of the diseases. For example, the composition may be administered after 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours of onset of neurodegenerative disease, but it is not limited thereto. Also, the composition of the present invention may be administered for the prevention of neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder or glaucoma. The inventive composition may be repeatedly administered as the need arises. For example, in case of clinical need, it may be administered daily, or at suitable intervals, e.g., 1 to 12 weeks, preferably 3 to 8 week, but not limited thereto.
The composition of the present invention may be formulated so as to provide immediate or sustained release of active ingredient. Such formulation may be tablet, powder, pill, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft or hard capsule, sterilized injectable solution, sterilized powder, etc. The composition of the present invention may be preferably provided in the form of single dose.
Further, the composition of the present invention may be administered at “therapeutically effective amount.” The “therapeutically effective amount,” as used herein, refers to amount enough to treat diseases/disorders.
It should be understood that the therapeutically effective amount of the composition ought to be determined in light of various relevant factors including the condition to be treated, the age, weight, health, reaction susceptibility and sex of the individual patient, food, administration time, administration route, excretion rate, period of treatment, the severity of the patient's symptom and, medication combined or simultaneously administered with the inventive composition, and the factors known in the medical field; and, therefore, the above dose should not be intended to limit the scope of the invention in any way. For example, single dose of the inventive composition for animal except human may be administered in amount of 200 to 10,000 IU/kg, preferably 200 to 5,000 IU/kg, more preferably 200 to 2,000 IU/kg, most preferably 500 to 1,000 IU. Further, single dose the inventive composition for human may be administered in amount of 25,000 to 45,000 IU, preferably 25,000 to 40,000 IU, more preferably 30,000 to 40,000 IU, regardless of body weight.
In another embodiment, the present invention relates to method for preventing or treating diseases/disorders associated with abnormal decrease in the acetylcholine concentration by the acetylcholinesterase activity such as neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma comprising administering to a subject at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone, and pharmaceutically acceptable salt of biruloquinone.
The terms “treating” and “treatment,” as used herein, refer to therapeutic measure and preventing or protecting method. Thus, a subject in need of treatment in the present invention may be one which wishes to prevent neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma as well as one suffered therefrom. The term “subject,” as used herein, means human, primates, and certain mammals such as dog, horse, cat, sheep, pig, cattle, etc., in need of the treatment.
In still another embodiment, the present invention relates to health food for preventing or alleviating diseases/disorders associated with abnormal decrease in the acetylcholine concentration by the acetylcholinesterase activity such as neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder or glaucoma which comprises at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone, and pharmaceutically acceptable salt of biruloquinone.
The health food of the present invention may comprise at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone, and pharmaceutically acceptable salt of biruloquinone alone or together with other food or other food component. Further, the inventive health food may be used according to conventional method. The ratio of components contained in the food ought to be determined in light of purpose for using (prevention, health or therapeutic treatment). Generally, the extract of Cladonia macilenta purple, biruloquinone or pharmaceutically aceeptable salt thereof may be added in amount of 10% by weight or less, preferably 5% by weight or less for preparing food or beverage. However, food for long-term ingestion for health maintenance may comprise less amount of said active ingredients than such amount. Also, more amount of active ingredients than such amount may be used for the food of the present invention since the active ingredients have no problem in safety.
There is no limit of kinds of the health food. Examples of the health food which can comprise the extract of Cladonia macilenta purple, biruloquinone or pharmaceutically aceeptable salt thereof are meat, sausage, bread, chocolate, candies, snacks, cookies, pizza, ramen or other noodles, gums, dairy products including icecream, various soups, beverage, tea, drink, alcoholic beverage and Vitamin composition. The food of the present invention encompasses all kinds of conventional health food.
If the food of the present invention is health beverage, the health beverage may contain various flavoring agent, natural hydrocarbon, etc., as an additional component like conventional beverages. The natural hydrocarbons may be monosaccharide such as glucose or fructose, disaccharide such as maltose or sucrose, natural sweetener such as dextrin or cyclodextrin or synthetic sweetener such as saccharin or aspartame. The ratio of the natural hydrocarbon is generally about 0.01 to 0.4g, preferably 0.02 to 0.03g per 100ml of the health beverage.
In addition to foregoing, the food of the present invention may further contain various nutritional supplements, vitamin, electrolyte, savoring agent, coloring agent, pectic acid and salt thereof, alginic acid and salt thereof, organic acid, protective colloid viscosity agent, pH controlling agent, stabilizer, preservative, glycerin, alcohol, and carbonatization agent used for a carbonated drink. Further, the food of the present invention may contain pulp (fruit flesh) for natural fruit juice, fruit juice beverage and vegetable beverage. The ratios of such additives are not critical, and are generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the inventive food.
The present invention will be described in further detail with reference to the following Examples. However, it should be understood that the present invention is not restricted by the specific Examples.
Example 1: Prepeartions of lichen thallus extract and linchen forming fungi extract
16 lichen thalli were collected from Antarctica, Hungary and China from 2007 to 2008. Further, lichen forming fungi (LFF) were isolated from lichen thallus by Yamamoto’s method (Yamamoto, Y., Mizuguchi, R. and Yamada, Y. 1985. Tissue cultures of Usnea rubescens and Ramalina yasudae and production of usnic acid in their cultures. Agricultural and Biological Chemistry. 49(11):3347-3348.) and cultured on MY medium at 15℃ for 60 days. Thereafter, they were dried and collected 50g lichen thallus and LFF, respectively. They were extracted with 1L acetone or methanol for 3 hours, and this process repeated 3 times. Acetone or methanol extracts were then evaporated in vacuum at 40℃, and dissolved in 10% of DMSO, then diluted to 1 mg/ml.
Example 2: AChE inhibitory activities of lichen thallus extract and LFF extract
The AChE inhibitory activities of lichen thallus extract and LFF extract gained from example 1 were studied by the colorimetric method described at Ellman et al.(Ellman, G.E., Courtnery, D., Andres, V., Featherstone, R.M., A new and rapid colorimetric determination of acetylcholinesterase activity., Biochem. Pharmacol. 1961, 7, 88-95) and using acetylthiocholine iodide as a substrate. Firstly, PC12 cell cultures were homogenized in a Glass-Col® homogenizer (Terre-Haute, Ind., USA) with 5 vol of homogenation buffer (10 mM Tris-HCl(pH7.2), containing 1 M NaCl, 50 nM MgCl2 and 1% TritonX-100), and then centrifuged at 10,000g for 30 min. The resulting supernatant was used as an enzyme source. All extraction steps were carried out at 4℃. The protein concentration was determined using the bicinchoninic acid kit (Sigma Co., St. Louis, Mo., USA) with bovine serum albumin as protein standard. The rates of hydrolysis by AChE were monitored spectrophotomatically using a 96-well microtiter plate format. Freeze-dried extracts were initially dissolved in 5% dimethyl sulfoxide, and the solution was diluted to 1,000ug/ml. Each extract was mixed with enzyme solution and incubated at 37℃ for 15min. Absorbance at 405 nm was read immediately after adding an Ellman’s reaction mixture(0.5 mM acetylthiocholine, 1 mM 5,5-dithiobis(2-nitrobenzoic acid)(DTNB)) in 50 mM sodium phosphate buffer (pH 8.0) to the above reaction mixture. Reading was repeated for 10min at 2 min intervals to verify that the reaction occurred linearly. The percentage of the AChE inhibitory activity of lichen thallus extract and LFF extract were caculated as an inhibition percentage of the maximum activity(negative control wells), and determined IC50. These experiments were performed at 3 times, and the average was used to determine IC50. AChE inhibitory activity of lichen thallus extract was summarized in figure 1, and AChE inhibitory activity of LFF extract was summarized in figure 2.
Figures 1 and 2 showed that the AChE inhibitory activity of lichen thallus extract was about 15-35% and the AChE inhibitory activity of LFF extract was below 30% except Cladonia macilenta purple. The AChE inhibitory activity of Cladonia macilenta purple was about 60% when treated with 1 mg/ml Cladonia macilenta purple. The high AChE inhibitory activity in low LFF purification fold means the usable possibility of the fucntional material.
Example 3: Isolation and identification of biruloquinone derived from Cladonia macilenta purple extracts
1L methanol extracts of Cladonia macilenta purple or its fermentation broth of example 1 were extracted with ethyl acetate and concentrated in vacuum for 3 hours. The concentrated extracts was then performed open column chromatography in room temperature, and gaind 10 fractions. The purple crystals of fractions were then analyzed by MS and NMR, and identified as biruloquinone(chemical structure 1).
The method described in example 2 was used to determine the AChE inhibition activity of biruloquinone. As such result, the inhibition is in a dose-dependent manner (Fig. 3). The inhibition percentage increased rapidly with the increase of biruloquinone concentration from 0 to 100ug/ml. The concentration that was required for the 50% enzyme inhibition IC50 was 27.1ug/ml(83.1uM). This result suggested that biruloquinone derived from Cladonia macilenta purple extracts had AChE inhibitory activity and increased acetylcholine.
Example 4: Toxicity assay of biruloquinone on injured PC12 cells
4-1. Purchase and cultures of cell
PC12 cells were purchased from Korean Cell Line Bank (KCLB). They were cultured in growth medium RPMI-1640 with l-glutamine, 5% fetal bovine serum(FBS), 10% horse serum, and antibiotics. The cells were maintained in 5% CO2 incubator at 37℃.
4-2. Cell toxicity assay
Viable PC 12 cells (104/well) were plated into 96-well collagen-coated plate 24 hours before the experiments. On the experiment day, the cells were cultured with gradient concentration of biruloquinone (6.25, 12.5, 25, 50 and 100μg/ml) for 3 hours. The cell viability was determined by conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) reduction assay(Shi, D. H., Wu, J. H., Ge, H. M. and Tan, R. X. 2009. Protective effect of hopeahainol A, a novel acetylcholinesterase inhibitor, on hydrogen peroxide-induced injury in PC12 cells. Environmental Toxicology and Pharmacology. 28(1):30-36.). 0.5mg/ml MTT solution was added to the medium and incubation for 4 hours. Cells were lysed in DMSO and the amount of MTT formazan was quantified by determining the absorbance at 570 nm using a microplate reader (VERSAmax, Molecular Devices, California, US). The in vitro toxicity of biruloquinone on PC12 cells was summarized in figure 4.
The results showed that the cell viability of PC12 cells which was co-cultured with biruloquinone(6.25, 12.5, 25 and 50μg/ml) and 0.5% of DMSO was not significantly differ with that of cells without any treatments, indicating the low in vitro toxicity of biruloquinone on PC12 cells. But the cell viability of PC12 cells which was co-cultured with 100ug/ml biruloquinone was significantly differ with that of cells without any treatments, indicating the in vitro toxicity of biruloquinone on PC12 cells.
Example 5: Cellular oxdative stress assay of biruloquinone
Viable cells(104/well) were plated into 96-well collagen-coated plated 24 hours before the experiments to test oxidative stress of biruloquione. On the experiment day, the cells were cultured with 100μM DCFH-DA for 30min. After DCFH-DA was removed, the cells were washed and incubated with RPMI medium. PC12 cells were then pretreated with various concentrations of biruloquione(25, 12.5, 6.25 and 1μg/ml) for 30min. The cells were then treated with or without 200μM H2O2 for 30 min and the fluorescence was finally quantified using fluorescent microplate reader(SpectraMax, Molecular Devices, California, US). The in vitro oxidative stress prevention of biruloquinone were summarized in figure 5.
The results showed that cells pretreated with 12.5 and 25ug/ml biruloquinone markedly reduced the H2O2-induced oxidative stress.
Example 6: Inhibition kinetic assay of biruloquinone on AChE
Kinetic studies were performed using AChE from electric eel. Enzyme activities were determined using four concentration of substrate(250, 500, 1000 and 2000μM) and two concentration of biruloquinone(61 and 122 μM). The inhibition type of biruloquinone on the enzyme was assayed by a Lineweaver-Burk plot. Plots of the slopes or intercepts versus the inhibitor concentrations gave estimates of KI(the dissociation constant for inhibitor binding to enzyme)and KIS(the dissociation constant for inhibitor binding to enzyme-substrate complex). And the results were summarized in figure 6.
The results showed that the inhibition behavior of biruloquinone gave increasing slopes and growing y-axis intercepts with higher concentration of biruloquinone, indicating that biruloquinone is a mixed-Ⅱ inhibitor. And the KI(204μM) is larger than that of KIS(12.5μM), indicating that the affinity of inhibitor for enzyme-substrate complex is stronger than that for free enzyme.
Example 7: Molecular docking of biruloquinone into Elecyrophorus electricus AChE
Molecular docking was performed by Accelrys Discovery Studio V2.5 softerware. The high-resolution X-ray crystal structure of Elecyrophorus electricus AChE (PDB code: 1C2B) were downloaded from protein data bank. A 3-D structure of the biruloquinone molecular was drawn by Chem Office Ultra 2008 V1, Cambridge Software. Then these two molecular were defined as receptor and ligand respectively. The binding sphere of the receptor was selected from the active site of the protein. Then hydrogens were added and CHARMm force fields were employed. LigandFit (LibDock) module was used to dock the ligand into the binding sphere. All other parameters were set as default. The binding interactional energy, including whole energy and electrostatic energy, was calculated and optimized. The docked conformations were then used to analyze the binding interactions.
The results showed that the main biding sites of biruloquinone with AChE were realized through the direct hydrogen bond between the C-12 O atom and the OH group on Ser203 amino acid residue located in the active site of AChE. A hydrogen bond was also formed between the C-8 OH group and O atom with the OH group on Tyr124 amino acid residue (data was not shown).
Example 8: The preparation of table including cell or culture extract of Cladonia macilenta purple, or biruloquinone
Component contents
200mg of cell or culture extract of Cladonia macilenta purple, or 1mg biruloquinone
Corn starch 68mg
Lactose 90mg
Fine crystalloid cellulose 40mg
Magnesium stearate 2mg
According to the manufacturing method of the conventional refinement, the above components were added to the suggested content and uniformly mixed. Thereafter, it stirred and granulated. By using the tablet machine after drying, the intended refinement in which the active ingredient of cell or culture extract of Cladonia macilenta purple, or biruloquinone per 1 refinement was included was manufactured.
Example 9: The manufacture of the capsule including cell or culture extract of Cladonia macilenta purple, or biruloquinone
Component content
200mg of cell or culture extract of Cladonia macilenta purple, or 1mg biruloquinone
Corn starch 68mg
Lactose 90mg
Fine crystalloid cellulose 40mg
Magnesium stearate 2mg
According to the manufacturing method of the conventional capsule, the above components were added to the suggested content and uniformly mixed. Thereafter, it filled in the gelatin capsule of the proper size so that 200mg of cell or culture extract of Cladonia macilenta purple, or 1mg biruloquinone per 1 capsule was included and the intended capsule was manufactured.
Example 10: The manufacture of chewing tablet including cell or culture extract of Cladonia macilenta purple, or biruloquinone
Component content
200mg of cell or culture extract of Cladonia macilenta purple, or 1mg biruloquinone
Granulated sugar syrup 240mg
Spice (peppermint or lemon flavor) 2mg
Corn starch 60mg
Mannitol 100mg
Magnesium stearate 20mg
A suitable amount of purified water
According to the manufacturing method of the conventional chewing tablet, the above components were mixed with the suitable amount of purified water. Thereafter, it dissolved while heating, cooled, put into molder, and the chewing tablet of the intended shape was manufactured so that 200mg of cell or culture extract of Cladonia macilenta purple, or 1mg biruloquinone per 1 tablet was included.
Example 11: The manufacture of the candy including cell or culture extract of Cladonia macilenta purple, or biruloquinone
Component content
200mg of cell or culture extract of Cladonia macilenta purple, or 1mg biruloquinone
Granulated sugar syrup 240mg
Spice (peppermint or lemon flavor) 2mg
Starch syrup 230mg
50% tartaric acid 20mg
A suitable amount of purified water
After it heated to 110℃ while completely melting the granulated sugar syrup at a small amount of water, the water and granulated sugar syrup of the rest which was molten cell or culture extract of Cladonia macilenta purple, or biruloquinone were added to of the rest melting. Temperature was then raised to 145℃. The fire was drawn. Spice and tartaric acid were added and it mixed. After it cooled to 75~80℃, it molded to the star roller and candy containing cell or culture extract of Cladonia macilenta purple, or biruloquinone was manufactured.
Example 12: the manufacture of the drink including cell or culture extract of Cladonia macilenta purple, or biruloquinone
Component content
200mg of cell or culture extract of Cladonia macilenta purple, or 1mg biruloquinone
Concentrated fruit juice 2g
Sucrose 12g
Sodium citrate 100mg
Spice 70mg
A suitable amount of purified water
According to the manufacturing method of the conventional drink, the above components were mixed to the suggested content with the suitable amount of purified water. After it heated and dissolved, the drink cooled, charged in container. Thereby the drink incluing 200mg of cell or culture extract of Cladonia macilenta purple, or 1mg biruloquinone per 1 bottle of 200ml capacity was manufactured.
While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.

Claims (7)

  1. Extract derived from Cladonia macilenta purple cell or culture thereof which has acetylcholinesterase inhibitory activity.
  2. Biruloquinone of formula I derived from Cladonia macilenta purple, or pharmaceutically acceptable salt thereof which has acetylcholinesterase inhibitory activity.
    <Formula I>
    Figure PCTKR2010008416-appb-I000002
  3. Pharmaceutical composition for preventing or treating neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract, or urinary bladder or glaucoma which comprises at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone of formula I, and pharmaceutically acceptable salt of biruloquinone.
    <Formula I>
    Figure PCTKR2010008416-appb-I000003
  4. The composition of claim 3, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, ischemic brain disease, myasthenia gravis, defects of memory, and alcohol addiction.
  5. Method for preventing or treating neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma comprising administering the pharmaceutical composition of claim 3 or 4 to a subject.
  6. Health food for preventing or alleviating neurodegenerative disease, myasthenia of smooth muscle of gastrointestinal tract or urinary bladder, or glaucoma which comprises at least one selected from the group consisting of cell or culture extract of Cladonia macilenta purple, biruloquinone of formula I, and pharmaceutically acceptable salt of biruloquinone.
    <Formula I>
    Figure PCTKR2010008416-appb-I000004
  7. The food of claim 6, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, ischemic brain disease, myasthenia gravis, defects of memory, and alcohol addiction.
PCT/KR2010/008416 2009-11-25 2010-11-25 Cell or culture extract of cladonia macilenta purple and/or biruloquinone as acetylcholinesterase inhibitors WO2011065769A2 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2467063C1 (en) * 2011-05-05 2012-11-20 Федеральное государственное автономное образовательное учреждение высшего профессионального образования "Северо-Восточный федеральный университет имени М.К. Аммосова" Method of producing highly active solid-phase antibiotic biopreparation of reindeer moss from lichen fronds
CN113373098A (en) * 2021-07-28 2021-09-10 西安医学院 Stenotrophomonas H2 strain for treating Alzheimer disease and application thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100509440B1 (en) 2003-01-14 2005-08-22 임강현 Pharamceutical preparation comprising extract of Yak-kong (Rhynchosia nolubilis) for Alzheimer's disease, dementia, amyosthenia of smooth muscle, and glaucoma
KR100550358B1 (en) 2005-05-20 2006-02-08 임강현 Food product and feed product comprising extract of Yak-kong (Rhynchosia nolubilis or Rhynchosia volubilis) for Alzheimer's disease, dementia, amyosthenia of smooth muscle, and glaucoma
CN101525631A (en) * 2008-03-03 2009-09-09 迈德金生物技术（北京）有限公司 Thallus extract and preparation method thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HERZ, W. ET AL.: 'Progress in the chemistry of organic natural products.' PROGRESS IN THE CHEMISTRY OF ORGANIC NATURAL PRODUCTS. vol. 81, 2001, WIEN NEW YORK, page 195 *
JEE, J-H. ET AL.: 'Effects of phenanthrene exposure on the acetyl- cholinesterase activity of olive flounder(Paralichthys olivaceus).' J. FISH. SCI. TECH. vol. 6, no. 4, 2003, pages 225 - 227 *
LUO, 0. ET AL.: 'Acetylcholinesterase inhibitor from secondary metabolites of lichen forming fungi Cladonia macilenta.' THE 8TH ANNUAL MEETING OF THE JAPANESE SOCIETY FOR LICHENOLOGY 12 July 2009, *
PODTEROB, A. P.: 'Chemical composition of lichens and their medical applications.' PHARMACEUTIAL CHEMISTRY JOURNAL vol. 42, no. 10, 2008, pages 582 - 588 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2467063C1 (en) * 2011-05-05 2012-11-20 Федеральное государственное автономное образовательное учреждение высшего профессионального образования "Северо-Восточный федеральный университет имени М.К. Аммосова" Method of producing highly active solid-phase antibiotic biopreparation of reindeer moss from lichen fronds
CN113373098A (en) * 2021-07-28 2021-09-10 西安医学院 Stenotrophomonas H2 strain for treating Alzheimer disease and application thereof
CN113373098B (en) * 2021-07-28 2022-07-01 西安医学院 Stenotrophomonas H2 strain for treating Alzheimer disease and application thereof

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