KR20230151796A - Pharmaceutical composition comprising furanodienone derived from Curcuma phaeocaulis Valeton (Zingiberaceae) for prion disease caused by protein aggregation - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating prion disease, and health functional food and feed composition for preventing or alleviating prion disease, including an extract of Curcuma zedoaria. The extract according to the present invention inhibits the transformation of a prion protein and inhibits the increase of a scrapie prion protein.

Description

Pharmaceutical composition comprising furanodienone derived from Curcuma phaeocaulis Valeton (Zingiberaceae) for prion disease caused by protein aggregation}

The present invention relates to a pharmaceutical composition containing a furanodienone derived from Ahchul for the treatment of protein aggregation-based prion disease.

Prion diseases, also known as transmissible spongiform encephalopathies (TSE), are infectious diseases caused by non-nucleic acid protein-related pathogens called prions. This disease occurs in a variety of mammals, including humans. The most famous animal prion disease is bovine spongiform encephalopathy, which occurs in cattle. Human prion diseases include idiopathic, hereditary, and acquired Creutzfeldt-Jakob disease (CJD), fatal familial insomnia, and Gerstmann-Streusler-Scheinker syndrome.

Additionally, according to a recent study, prion disease was reported to cause Alzheimer's disease (Tomas Tousseyn, et al., Prion Disease Induces Alzheimer Disease-Like Neuropathologic Changes, Journal of Neuropathology & Experimental Neurology, Volume 74, Issue 9, September 2015 , Pages 873-888). In this study, when human brain aggregates were inoculated into brain homogenate suspensions derived from patients with sporadic Creutfeldt-Jakob disease, the brain aggregates produced large amounts of β-amyloid (Aβ) inclusions, whereas this was not the case in uninoculated controls. reported, and as a result of analyzing the hippocampus of prion disease patients, it was confirmed that APOE-4 and PrP ^Sc together cause neuron loss in prion disease. In addition, it was confirmed that the levels of hyperphosphorylated tau protein and hyperphosphorylated tau protein-positive neural network were higher in the prion disease and Alzheimer's disease groups compared to the control group.

The agent that causes the disease is called scrapie prion (PrP ^Sc ) and is an isoform of PrP that is misfolded through structural changes in cellular PrP (PrP ^C ). Because PrP ^Sc is hydrophobic, it forms various aggregates such as oligomers and amyloid. Neuropathologically, PrP ^Sc aggregates are often found as amyloid plaques in brain wounds. PrP ^Sc aggregates cause neuronal death, forming vacuoles, ultimately leading to spongiform degeneration, and these activate glial cells in the brain.

Prion diseases are known to be fatal without exception, but there is no cure. Developing treatments for prion diseases is still limited to the laboratory level due to the ineffectiveness or toxicity of drugs used in human clinical trials. For example, quinacrine effectively reduced PrP ^Sc levels in vitro but was ineffective in CJD patients, ultimately causing liver failure. Polypentosan sulfate used in human clinical trials was found to be effective only in limited cases and was therefore found to be unsuitable for treatment.

Meanwhile, Curcuma phaeocaulis Valeton (Zingiberaceae); CpV) has been used in East Asia as a traditional medicine to treat congestive syndromes such as mental disorders, menstrual pain, and arthralgia caused by blood stagnation. A recent investigation of chemicals in Achul showed that sesquiterpenoids, one of the main components of CpV, have anti-inflammatory, anti-tumor and anti-platelet aggregation activities. In addition, furanodienones are known to be compounds present in achul, but the efficacy of achul extract or furanodienones on the brain has not yet been revealed.

CN102091063B

Xiaoqin Wang et al., Antiproliferative activity of Curcuma phaeocaulis Valeton extract using ultrasonic assistance and response surface methodology, Prep Biochem Biotechnol. 2017 Jan 2; 47)1:19-31

The purpose of the present invention is to provide a pharmaceutical composition and feed composition for preventing and treating degenerative brain diseases.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating prion disease, a health functional food for preventing or alleviating prion disease, and a feed composition for preventing or treating prion disease, including furanodienone. . In one embodiment of the invention, furanodienones reduce the level of scrapie prion protein (PrP ^Sc ) in cells and/or inhibit aggregation of prion proteins in cells, and in another embodiment of the invention, the prion The disease includes bovine spongiform encephalopathy (BSE); advance disease of sheep and goats; Chronic wasting disease of deer, elk, moose, and caribou; Creutfeldt-Jakob disease, fatal familial insomnia, Gerstmann-Streussler-Scheinker syndrome, and Alzheimer's disease in humans.

By administering a composition containing furanodienones according to the present invention, bovine spongiform encephalopathy (BSE) in cattle, forward disease in sheep and goats, chronic wasting disease in deer, elk, moose, reindeer, etc., and Creutfeldt disease in humans. -Can treat, prevent, or alleviate Jakob disease, fatal familial insomnia, Gerstmann-Streussler-Scheinker syndrome, and Alzheimer's disease.

Figure 1 shows the anti-prion activity of Achul extract in ScN2a cells, according to an embodiment of the present invention: (A) Western blot and densitometric analysis in four repeated Western blots, showing the anti-prion activity of Achul extract in ScN2a cells. showed a decrease in PK-resistant PrP ^Sc levels; (B) Extract concentration-dependent reduction of PK-resistant PrP ^Sc in ScN2a cells; (C) HPLC chromatogram of Achul extract.
Figure 2 shows the in vivo prion disease inhibitory effect of an extract of Ahchul according to an embodiment of the present invention: (A) Administration of 0, 100, or 200 mg/kg of Ahchul extract twice a week for 4 weeks. Disease incidence curve for each group of prion-infected mice (n=9/group); (b) Western blot for PK-resistant PrP ^Sc in mouse brains collected from different groups at the end point of each mouse.
Figure 3 shows the solvent fraction of the Ahchul extract according to an embodiment of the present invention: (A) a schematic diagram of the fraction of the Ahchul extract; (B) HPLC profile of the extract (HX, EA, ED, and DW).
Figure 4 shows the results confirming the effectiveness of reducing the infectivity of prions by the extract of Achul according to an embodiment of the present invention: standard scrapie cell analysis at passages 4 and 10 after prion infection; NBH: Brain homogenate suspension from normal mouse without prion infection; SBH: Brain homogenate suspension after euthanizing mice that developed disease due to prion infection.
Figure 5 shows the results of Western blot and densitometry analysis for PK-resistant PrP ^Sc in cells cultured with the crude extract and fractions by solvent according to an embodiment of the present invention.
Figure 6 is a preparative HPLC chromatogram of an n-hexane extract according to an embodiment of the present invention.
Figure 7 is an HPLC profile of the HX1 to HX12 subfractions of the hexane fraction (HX) obtained through preparative HPLC according to an embodiment of the present invention.
Figure 8 shows the results of Western blot and densitometry analysis of PK-resistant PrP ^Sc in ScN2a cells cultured with Achul crude extract, HX-fraction, and small fractions of HX1 to HX12 according to an embodiment of the present invention.
Figure 9 shows the chemical structures of curzerenone (1) and furanodienone (2).
Figure 10 is a ¹ H NMR spectrum of curzerenone.
Figure 11 is a ¹³ C NMR spectrum of curzerenone.
Figure 12 is an ESI-MS spectrum of curzerenone.
Figure 13 is a ¹ H NMR spectrum of furanodienone.
Figure 14 is a ¹³ C NMR spectrum of furanodienone.
Figure 15 is an ESI-MS spectrum of furanodienone.
Figure 16 confirms that the level of PrP ^Sc in ScN2a cells decreases in a concentration-dependent manner of furanodienone: PK-resistant PrP ^Sc was measured by densitometry after four Western blots.
Figure 17 shows the concentration-dependent inhibitory effect of furanodienone on PrP modification in RT-QuIC.

Hereinafter, the present invention will be described in detail through embodiments of the present invention with reference to the attached drawings. However, the following embodiments are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.

In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, preferred methods and samples are described in this specification, but similar or equivalent methods are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are hereby incorporated by reference.

“Achul” is Curcuma phaeocaulis Valeton, and is also called Bongahul, Gwangchul, and Bongchul. It is known to be effective in breaking blood, removing stagnant blood, and eliminating pain. Traditionally, it is effective in treating meningitis, ovarian ovaries, emphysema, diaphragm, urinary tract, small intestine, dropsy, heart pain, food fluoride, dehydration, spleen, celiac disease, bruising, tonic acid, etc. It was used to treat lower blood pressure and hysteresis.

The term “extract” in the present invention may refer to an extract obtained by extracting a herbal medicine using an appropriate solvent, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, and a crude product or purified product thereof. The Achul extract can be prepared using general extraction, separation and purification methods known in the art. The extraction method may include, but is not limited to, boiling water extraction, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction.

As used herein, the term "about" or "approximately" means within an acceptable margin of error for a particular value as determined by a person skilled in the art, depending on how that value is measured or determined, i.e., the limits of the measurement system. It will depend in part on For example, “about” can mean within 3 or more than 3 standard deviations, depending on the practice in the art. Alternatively, “about” can mean a range of less than or equal to 10%, more preferably less than or equal to 5%, and even more preferably less than or equal to 1% of a given value.

The present invention relates to a pharmaceutical composition for preventing or treating prion disease containing furanodienone.

The term prion disease in the present invention is also called infectious spongiform encephalopathy. Prion diseases are caused by infectious proteins called prions, which are produced by misfolding of endogenous prion proteins. Prion diseases include bovine spongiform encephalopathy (BSE) in cattle, BSE in sheep and goats, chronic wasting disease in deer, elk, moose, and reindeer, Creutfeldt-Jakob disease, fatal familial insomnia, and Gerstmann-Jakob disease in humans. Includes Stureussler-Scheinker syndrome, and Alzheimer's disease.

A further aspect of the invention relates to pharmaceutical compositions comprising the furanodienones described herein. In one embodiment, the pharmaceutical composition further comprises one or more carriers, diluents and/or excipients.

The furanodienones described herein can be administered as pharmaceutical compositions or medicaments for therapeutic or prophylactic treatment, and may contain a pharmaceutically acceptable carrier, optionally containing one or more adjuvants, stabilizers, etc. It may be administered in the form of any suitable pharmaceutical composition that may contain. In one embodiment, the pharmaceutical composition is used for therapeutic or prophylactic treatment, e.g., for treating prion diseases such as those described herein, e.g., bovine spongiform encephalopathy (BSE) in cattle, BSE in sheep and goats, deer, For use in treating, alleviating or preventing chronic wasting disease in elk, moose, reindeer, etc., Creutfeldt-Jakob disease, fatal familial insomnia, Gerstmann-Streussler-Scheinker syndrome, and Alzheimer's disease in humans. It is for.

The term “pharmaceutical composition” relates to a formulation comprising therapeutically effective substances, preferably together with pharmaceutically acceptable carriers, diluents and/or excipients. The pharmaceutical composition is useful for reducing the severity of, preventing, or treating a disease or disorder by administering the pharmaceutical composition to an individual. Pharmaceutical compositions are also known in the art as pharmaceutical formulations.

The pharmaceutical composition herein may contain one or more adjuvants or may be administered together with one or more adjuvants. The term “adjuvant” refers to a compound that prolongs, enhances, or accelerates an immune response. Adjuvants include a heterogeneous group of compounds such as oil emulsions (eg Freund's adjuvant), mineral compounds (eg alum), bacterial products (eg pertussis toxin) or immune-stimulating complexes.

The pharmaceutical composition according to the present specification is generally applied as a “pharmaceutically effective amount” and as a “pharmaceutically acceptable formulation.”

The term “pharmaceutically acceptable” refers to the non-toxicity of a substance that does not interact with the action of the active ingredients of the pharmaceutical composition.

The term “pharmaceutically effective amount” or “therapeutically effective amount” means the amount that, alone or in combination with additional administration, achieves the desired response or desired effect. When treating a particular disease, the desired response preferably means inhibition of progression of the disease. This includes slowing down the progression of the disease, and in particular stopping or reversing the progression of the disease. The desired response in the treatment of a disease may also be delaying the onset or preventing the onset of the disease or condition. The effective amount of the composition described herein will depend on the condition being treated, the severity of the disease, the individual characteristics of the patient, including age, physical condition, height and weight, the duration of treatment, the type of concomitant therapy (if any), the specific route of administration and the like. It will be decided depending on factors. Accordingly, the administered dosage of the compositions described herein may be determined according to these various properties. If the response in the patient is not sufficient with the first dose, higher doses (or effectively higher doses achieved by other, more localized routes of administration) may be used.

Pharmaceutical compositions herein may include salts, buffers, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions herein include one or more pharmaceutically acceptable carriers, diluents and/or excipients.

Preservatives suitable for use in the pharmaceutical compositions herein include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

As used herein, the term “excipient” refers to a substance that may be present in the pharmaceutical compositions herein but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

The term “diluent” means a substance that dilutes and/or thins. Additionally, the term “diluent” includes any one or more of fluids, liquids or solid suspensions and/or mixed media. Examples of suitable diluents include ethanol, glycerol, and water.

The term “carrier” refers to a substance, which may be natural, synthetic, organic, or inorganic, that is combined with the active ingredient to facilitate, enhance, or facilitate administration of the pharmaceutical composition. As used herein, a carrier can be one or more compatible solid or liquid fillers, diluents, or encapsulating materials suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's, Ringer's lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and, especially, biocompatible lactide polymers, lactide/glycols. ride copolymers or polyoxyethylene/polyoxy-propylene copolymers. In one embodiment, the pharmaceutical composition herein comprises isotonic saline solution.

Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

Pharmaceutical carriers, excipients or diluents may be selected depending on the intended route of administration and standard pharmaceutical practice.

In one embodiment, the pharmaceutical compositions described herein can be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for topical or systemic administration. Systemic administration may include enteral administration or parenteral administration, involving absorption through the gastrointestinal tract. As used herein, “parenteral administration” means administration by any means other than via the gastrointestinal tract, such as by intravenous injection. In one embodiment, the pharmaceutical composition is formulated for oral administration. In another embodiment, it is formulated for intravenous administration.

As used herein, the term “co-administration” means administering several compounds or compositions to the same patient. The various compounds or compositions may be administered simultaneously, essentially simultaneously, or sequentially.

The materials, compositions and methods described herein can be used to treat individuals with diseases, such as prion diseases. Particularly preferred diseases are Creutfeldt-Jakob disease or Alzheimer's disease.

The term “disease” refers to an abnormal condition affecting an individual's body. A disease is often interpreted as a medical condition associated with specific symptoms and signs. The disease may be caused by factors originating from an external source, such as an infectious disease, or may be caused by an internal dysfunction, such as an autoimmune disease. In humans, “disease” is used in a broader sense to refer to any condition that, upon contact with an individual, causes pain, dysfunction, suffering, social problems, death, or similar problems in the afflicted individual. In a broader sense, it sometimes includes injuries, disabilities, disabilities, syndromes, infections, isolated symptoms, aberrant acts and structural and functional atypical deformities, which in other contexts and for other purposes may be considered distinct categories. You can. Diseases generally affect individuals not only physically but also emotionally, as living with various diseases can change an individual's perspective on life and personality.

In this context, the terms “treatment”, “treating” or “therapeutic intervention” mean the management and care of an individual for the purpose of combating a condition such as a disease or disorder. The term refers to alleviating symptoms or complications, delaying the progression of a disease, disorder or condition, ameliorating or alleviating symptoms and complications, and/or curing or eliminating a disease, disorder or condition, as well as treating a disease, disorder or condition. It is intended to include the full spectrum of treatment for a given condition suffering from an individual, such as the administration of a therapeutically effective pharmaceutical composition, wherein prophylaxis refers to the treatment of a disease, condition or disorder in an individual for the purpose of eradicating the disease, condition or disorder. It will be understood as the management and care of and includes the administration of active ingredients to prevent the onset of symptoms or complications.

The term “therapeutic treatment” refers to any treatment that improves the health status of an individual and/or prolongs (increases) the lifespan of an individual. The treatment may eliminate the disease in the individual, stop or slow the progression of the disease in the individual, inhibit or slow the progression of the disease in the individual, reduce the frequency or severity of symptoms in the individual, and/or treat the individual currently suffering from or previously suffering from the disease. There may be a reduction in recurrence.

The term “prophylactic treatment” or “prophylactic treatment” refers to any treatment intended to prevent a disease from developing in an individual. The terms “prophylactic treatment” or “prophylactic treatment” are used interchangeably herein.

The terms “individual” and “entity” are used interchangeably herein. These terms refer to humans or other mammals (e.g., mice, rats, rabbits, dogs, cats, cattle, pigs, sheep, horse, deer, or primate), but may or may not have the disease or disorder. In many embodiments, the subject is a human or domestic animal. Unless otherwise specified, the terms “individual” and “subject” do not refer to a specific age and therefore encompass adults, older adults, children, and newborns. In embodiments herein, “subject” or “individual” is “patient.”

The term “patient” refers to an individual or entity in need of treatment, specifically an individual or entity suffering from a disease.

The present invention provides a health functional food containing furanodienone for preventing or alleviating prion disease.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or alleviating prion disease.

The term “health functional food” in the present invention refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Health Functional Food, and provides nutrients for the structure and function of the human body. It means ingestion for the purpose of obtaining useful effects for health purposes such as regulation or physiological action.

In addition to the above health functional food composition, various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid. It may further contain carbonating agents used in beverages. The ratio of the ingredients added is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food of the present invention.

The health functional food of the present invention may contain common food additives, and its suitability as a food additive is determined in accordance with the general provisions and general test methods of the food additive code approved by the Food and Drug Administration, unless otherwise specified. Judgment is made according to specifications and standards.

Examples of items listed in the Food Additives Code include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; It includes, but is not limited to, mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations.

For example, health functional foods in the form of tablets are made by granulating a mixture of the furanodienone with excipients, binders, disintegrants, and other additives in a conventional manner, then adding a lubricant, etc., and compression molding, The furanodienone powder can be directly compression molded. In addition, the health functional food in the form of tablets may contain flavoring agents, etc., if necessary.

Among capsule-type health functional foods, hard capsules can be manufactured by filling a regular hard capsule with a mixture of the furanodienone and additives such as excipients, while soft capsules can be prepared by mixing the furanodienone with excipients, etc. It can be manufactured by filling a mixture mixed with additives into a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary.

The health functional food in the form of a pill can be prepared by molding a mixture of the furanodienone and excipients, binders, disintegrants, etc. using a known method. If necessary, it can be coated with white sugar or other coating agents. , or the surface can be coated with substances such as starch or talc.

Health functional foods in the form of granules can be manufactured into granules using a mixture of the furanodienon and excipients, binders, disintegrants, etc., using a known method, and may contain flavoring agents, flavoring agents, etc. as needed. You can.

The health functional foods include beverages, meat, chocolate, foods, and confectionery. These may include pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin supplements, and health supplements.

One embodiment of the present invention provides a feed composition for preventing or alleviating prion disease containing furanodienone.

The term “feed composition” of the present invention includes any feed, diet, feed supplement, or substance that may contain protein, carbohydrate and/or crude fat. Feed may also contain supplementary substances or additives such as minerals, vitamins and seasonings. These feed compositions may or may not be nutritionally complete. In certain embodiments, animal feed compositions according to the present invention are nutritionally complete feed compositions.

As used herein, “nutritionally complete” means that the composition provides complete and balanced nutritional requirements for the animal. In certain embodiments, such animals are livestock. Therefore, a nutritionally adequate feed is a feed that can be fed to the animal, for example the livestock, as its only ration and is capable of sustaining life without additional feed (except water). The feed composition may contain carriers, diluents or excipients. Depending on the intended use, carriers, diluents or excipients may be selected to be suitable for animal use. In certain embodiments, the animal is a domestic animal, and may include, but is not limited to, a cow, sheep, goat, deer, elk, moose, or reindeer. Typically, a nutritionally complete composition includes one or more sources of protein, such as protein extracts, sources of vitamins, minerals, trace elements, and fat.

As used herein, “feed supplements” means nutrients such as, but not limited to, vitamins and minerals, substances for nutritional or physiological purposes, or plants and plant preparations to supplement deficiencies in an animal’s regular diet. It means a concentrated source of. In certain embodiments, the feed supplement is sold in the form of capsules, lozenges, tablets, pills, powder packets, or liquid form (ampoules, vials with droppers).

Illustratively, the feed composition described herein includes protein, crude fat, ash, crude fiber, starch, calcium, phosphorus, sodium, chloride, potassium, magnesium, iron, water, copper, manganese, zinc, selenium, and vitamin A. , vitamin D3, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin B7, vitamin B9, choline chloride, arachidonic acid, W3 fatty acid, or W6 fatty acid.

As used herein, the term “animal” or “animals” refers to ruminants, poultry, pigs, mammals, horses, mice, rats, rabbits, guinea pigs, hamsters, cattle, cats or canines. The term animal may refer more specifically to non-human animals. It can refer to domestic or wild animals. In certain embodiments, the non-human animal can be livestock.

Reference to literature and studies referenced herein is not intended as an admission that any foregoing content is relevant to prior art. All references to the content of these documents are based on information available to the applicant and are not considered as any admission as to the accuracy of the content of these documents.

The following description is provided to enable those skilled in the art to make and use various implementations. Descriptions of specific devices, techniques and uses are provided by way of example only. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and uses without departing from the spirit and scope of the various embodiments. . Accordingly, the various embodiments are not intended to be limited to the examples described and shown herein, but are intended to be accorded the scope consistent with the claims.

Example

Example 1. Materials and Methods

1.1 Preparation of Achul extract

The rhizome of Achul was purchased (Gyeongdong Market), and 600 g of the sample was refluxed and extracted three times with methanol for 2 hours. The filtrate was evaporated using a rotary evaporator under reduced pressure conditions to obtain 31.1 g of crude extract. The crude extract was stored at -80°C and dissolved in DMSO for analysis.

1.2 Cell-based PrP ^Sc analyze

The anti-prion activity of the extract, fractions of the extract, and extract-derived compounds (furanodienones and curzerenone) was measured by measuring the levels of proteinase K (PK)-resistant PrP ^Sc ( Waqas M, Trinh HT, Lee S, Kim DH, Lee SY, Choe KK, et al. Decrease of protease-resistant PrP ^Sc level in ScN2a cells by polyornithine and polyhistidine . J Microbiol Biotechnol 2018;28:2141-4).

ScN2a cells were cultured and inoculated with achul extract. Cells were disrupted in lysis buffer (20 mM Tris (pH 8.0), 150 mM NaCl, 0.5% Nonidet P-40, and 0.5% sodium deoxycholate), and cell lysates were analyzed by Western blot. Quantitative protein analysis was performed with the Pierce BCA protein assay kit (ThermoFisher Scientific, USA), and equal volumes (0.2 mg) of cell lysates were incubated with 20 μg/ml PK (Roche, Switzerland) for 1 h at 37°C. . After 2 hours, the reaction was terminated using 2 mM phenylmethylsulfonyl fluoride (PMSF, Merck Millipore, Germany) and centrifuged at 16,000 xg at 4°C for 1 hour. The pellet was incubated in 0.25 M Tris (pH 6.8, Sigma, USA) solvent with 8% SDS (sodium dodecyl sulfate, Sigma, USA), 30% glycerol (Sigma, USA), 0.02% bromophenol blue (Sigma, USA), and After being dissolved in 4X sample loading buffer containing 8% β-mercaptoethanol (Sigma, USA), it was heated at 95°C for 5 minutes and SDS-PAGE was performed. Samples separated from the gel were transferred to Immobilon-P polyvinylidene difluoride membrane (Merck Millipore, Germany) and incubated at room temperature with 5% Difco skim milk (BD, USA) in TBS buffer supplemented with 0.1% Tween 20 (Sigma, USA). Blocked for 1 hour. After overnight incubation with mouse monoclonal anti-PrP antibody 6D11 (1:30,000-fold dilution, Biolegend, USA), incubated at room temperature using HRP-fused goat anti-mouse IgG (1:10,000-fold dilution, Invitrogen, USA) antibody. The membrane was incubated for 1 hour. PK-resistant PrP ^Sc bands were detected with Amersham ECL Prime Western Blotting Detection Reagent (Cytiva, USA) and visualized with Gbox Chemi XR5 image analysis system (Syngene, UK). Separately, immunodetection of β-actin levels was performed on the same amount (20 μg) of cell lysate without PK digestion. The experimental method was the same as above, and mouse monoclonal anti-β-actin (Santa Cruz Biotechnology, USA) was diluted 1:5000 and used as the primary antigen. Signal intensity was quantified densitometrically using GeneTools software (Syngene, UK).

1.3 Breeding period analysis

To evaluate the efficacy of Achul extract on the course of the disease, the disease onset period was measured in a prion disease mouse model. Animal experiments were conducted with the approval of Hanyang University's Animal Experiment Ethics Committee.

After 1 week of adaptation, 5-week-old female CD-1 mice (Samtaco, Korea) were treated with mouse-adapted scrapie RML prions (hereinafter referred to as “SBH”), 1% (w) prepared from mice in which prion disease was finally induced. /v) 50 μl of brain homogenate suspension was injected intraperitoneally. 100 mg/kg to 200 mg/kg of Achul extract was provided three times a week for 4 weeks starting the day after prion inoculation. They were reared under 12:12 day:night conditions until they showed obvious prion-related neuropathological signs such as weight loss, hunched posture, reduced locomotion, and tail stiffness. If the mouse showed two or more clinical signs, it was humanely euthanized using carbon dioxide, the breeding period was measured for each mouse, and the average breeding period and standard mean error for each group were calculated and compared.

The group of mice without these signs that received the extract of Achul was observed until 452 days post-inoculation (dpi) and euthanized. Prion-infected mice administered only the carrier (10% DMSO in PBS) were used as controls.

1.4 PrP using brain tissue ^Sc analyze

Ryou C, Titlow WB, Mays CE, Bae Y, Kim S. The suppression of prion propagation using poly-L ^- lysine by targeting plasminogen that stimulates prion protein conversion. It was measured according to the method described in Biomaterials 2011;32:3141-9.

Brain homogenate suspension (10%, w/v) in PBS was prepared using OMNI Bead Ruptor 24 (PerkinElmer, USA) and silicon beads. After centrifugation at 1000 x g for 5 minutes at room temperature, the separated supernatant was quantitatively analyzed, and 0.2 mg of brain homogenate suspension was mixed with 2% Sarkosyl PBS. PK digestion and Western blot were performed in the same manner as above.

1.5 Serum chemistry test

Whole mouse blood from the group administered the carrier and extract extract was collected, serum was separated and analyzed using an automated chemical analyzer Fuji DRI-CHEM 4000i (Fujifilm, Japan) and the proposed colorimetric slide (Fujifilm, Japan).

1.6 Tissue structure and immunohistochemistry

The extracted mouse brain was fixed with 4% paraformaldehyde in PBS (Sigma, USA) overnight at 4°C, and paraffin blocks were prepared with a tissue processor Citadel 2000 (ThermoFisher Scientific, UK). Tissue fixed in paraffin was cut into 6 μm thick pieces and H&E staining was performed. Mouse brain tissue was observed under a light microscope, and histological images were obtained with an Axioscan Z1 slide scanner (Zeiss, Germany). The degree of vacuole formation was measured as the number of vacuoles per unit area using Zen2 software (Zeiss, Germany). Immunohistochemistry of glial fibrillary acidic protein (GFAP) was performed by M.O.M. It was performed with an immunodetection kit (Vector Laboratories, USA) according to the manufacturer's instructions.

Specifically, brain tissue from which paraffin was removed was boiled in 10 mM sodium citrate buffer (pH 6.0) for 10 minutes and incubated in quenching solution (0.3% H ₂ O ₂ ) for 5 minutes. After blocking with MOM blocking reagent for 1 hour, mouse monoclonal anti-GFAP antibody (diluted 1:500 times with MOM diluent, Sigma, USA) and biotinylated anti-mouse IgG (diluted 1:250 times, Vector Laboratories, USA) were used sequentially for 1 hour each to culture the brain tissue. Washed with PBS at each step. Biotinylated molecules were detected using VECTASTAIN®Elite ABC-HRP Kit, Peroxidase (Vector Laboratories, USA). Detection of specific staining was performed using Vector DAB Substrate (Vector Laboratories, USA). Fear formation and GFAP signaling were analyzed independently three times in the same regions of cortex, hippocampus, hypothalamus, striatum, and thalamus.

1.7 In vitro Prion infection assay

To evaluate the efficacy of Achul extract against prion infection, the standard scrapie cell assay (SSCA) was performed with minor modifications. N2a cells (2.4 × 10 ⁴ cells) were cultured in 24-well dishes for 4 days in the presence of RML-SBH at a final concentration of 0.9% with or without the addition of 50 μg/ml of Achul extract. N2a cells not infected with prions were cultured as a control. Cells were maintained for several passages. At passages 4 and 10, 2.5 × 10 cells were seeded and immobilized in opaque 96-well plates activated with 0.45 μm hydrophobic ^, highly protein-binding Immobilon-P membrane (Merck Millipore, Germany). Membranes were incubated with 5 μg/ml PK in RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1% sodium deoxycholate, 1% Nonidet P-40, and 0.1% SDS) for 90 min and with 2mM PMSF in PBS for 20 min. . Afterwards, it was exposed to 3 M guanidine thiocyanate for 10 minutes and washed thoroughly with PBS. After blocking with Superblock (ThermoFisher Scientific, USA) solution, PrP ^Sc was specifically labeled with mouse monoclonal anti-PrP antibody 6D11, and the same concentration of alkaline dephosphorylase-fused mouse IgG (Abcam, UK) was used for Western analysis. It was used for blot analysis. After color development using NBT/BCIP (nitro-blue tetrazolium chloride/5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt) substrate, the number of infected cells was measured using a S6 universal M2 ELISPOT instrument (ImmunoSpot, USA). and analyzed using ImmunoSpot software (ImmunoSpot, USA).

1.8 Activity tracking identification of active ingredients in Achul extract

The crude extract was dissolved in water and successively fractionated with n -hexane and ethyl acetate. Among the extracts, the active n -hexane extract (HX) was further separated by preparative HPLC. The anti-prion activity of each fraction was confirmed through the cell-based PrP ^Sc assay described above. Finally, the compounds were purified from the active fraction and their chemical structures were confirmed by ESI-MS and NMR spectra.

1.9 Real-time causal pathogen amplification detection method (RT-QuIC) analysis

RT-QuIC analysis was performed based on previous studies (Kang HE, Mo Y, Abd Rahim R, Lee HM, Ryou C. Prion Diagnosis: Application of real-time quaking-induced conversion. BioMed Res Int'l 2017;2017 :5413936.; Caughey B, Baron GS, Chesebro B, Jeffrey M. Getting a grip on prions: oligomers, amyloids, and pathological membrane interactions. Annu Rev Biochem 2009;78:177-204.; Wilham JM, Orru CD, Bessen RA, Atarashi R, Sano K, Race B, et al. Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays. PLoS Pathog 2010;6:e1001217.; Schmitz M, Cramm M, Llorens F, Muller-Cramm D, Collins S, Atarashi R, et al. The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases. Nat Protoc 2016;11:2233-42.). The reaction mixture was prepared in a 96-well black plate with a transparent, flat bottom (Corning, USA). Final concentration of 100 μg/ml recombinant mouse PrP (23-231), 5 μl of 1 × ¹⁰ diluted RML-SBH, and RT-QuIC assay buffer (200 mM NaCl, 10 mM thioflavin T (ThT), 0.002% SDS) , 10 mM EDTA, and 10 mM phosphate buffer (pH 7.4)) were mixed in a 100 μl reaction volume. To test the anti-prion converting activity of furanodienones and curzerenones, RT-QuIC assays were performed in the presence or absence of the compounds at given concentrations. Reaction plates were incubated at 42°C for 90 to 120 hours with selective mixing in a FLUOstar Omega (BMG Labtech, Germany) instrument. PrP aggregation was measured by measuring ThT fluorescence intensity every 15 min with excitation at 450 nm and emission at 480 nm. AUC values were calculated by subtracting the background fluorescence intensity. The length of the lag phase was confirmed using time course data of ThT fluorescence.

Example 2. Results and Analysis

2.1 Extracts from ScN2a cells show PrP ^Sc reduce the level

A cell-based PrP ^Sc assay was performed to confirm the anti-prion efficacy of the sprout extract in ScN2a cells, a persistent prion-infected, immortalized neuroblastoma cell line. In the PrP ^Sc assay, the level of PrP ^Sc in ScN2a cells cultured for 4 days with 50 μg/ml of Achul extract was significantly reduced to less than about 20% compared to the control group cultured with carrier (Figure 1A), and this efficacy appeared in a concentration-dependent manner (Figure 1B). This explains that Achul extract inhibits prions.

2.2 Achul extract alters the course of prion disease

The anti-prion efficacy of the extract was evaluated in mice infected with the mouse-adapted RML scrapie prion. When 100 mg/kg of Achul extract was administered, the rearing period was extended by 1 week compared to the control group that received only the carrier (Table 1).

army breeding period
Mean ± SEM (days) n/n ₀ carrier 202.4 ± 4.44 9/9 Achul extract 100 mg/kg 209.0 ± 6.3 9/9 Achul extract 200 mg/kg 212.8 ± 4.71 6/9

On the other hand, in the group that received 200 mg/kg of Achul extract, one-third of the mice remained healthy until 452 dpi, the end point of the experiment, showing that it had a preventive effect against prion disease. The remaining two-thirds of the mice showed signs of prion disease, but their housing period extended beyond 10 days compared to the control group.

As a result of comparing each group using the log-rank method, the average rearing period of the group administered 200 mg/kg of Achul extract showed a statistically significant difference compared to the group administered only the carrier (Figure 2A). These results mean that the course of prion disease changed in the group administered 200 mg/kg of achul extract.

Accumulation of PrP ^Sc in the mouse brain was confirmed through Western blot. The level of PrP ^Sc in the brains of mice administered 100 mg/kg of Achul extract was similar to that in the brains of control mice (Figure 2B). However, it was confirmed that the level of PrP ^Sc in the brains of mice that developed prion disease and received 200 mg/kg of Achul extract was reduced compared to the level of PrP ^Sc in the brains of control mice. In addition, it was confirmed that PrP ^Sc in the brain of mice that received 200 mg/kg of Achul extract and survived until the end of the experiment was rapidly reduced (Figure 2B). These results show that prion-related biochemical events in the course of the disease are completely altered by administering achul extract to prion-infected mice. These results were not found in most existing in vivo activity tests (Ryou C, Titlow WB, Mays CE, Bae Y, Kim S. The suppression of prion propagation using poly-L-lysine by targeting plasminogen that stimulates prion protein conversion Transmission of Creutzfeldt-Jakob disease from humans to transgenic mice expressing chimeric human-mouse prion protein Proc Natl Acad Sci USA 1994;91:9936-40.; Vickery CM, Lockey R, Holder TM, Thorne L, Beck KE, Wilson C, et al. Assessing the susceptibility of transgenic mice overexpressing deer prion protein to bovine spongiform encephalopathy . .J Virol 2014;88:1830-3).

2.3 Extract of achrysalis prevents neuropathological deterioration

To confirm the efficacy of Ahchul extract on neurodegeneration that occurs during the course of prion disease, vacuole formation and gliosis were examined through brain sections of prion-infected mice that were or were not administered 100 to 200 mg/kg of Ahchul extract. was confirmed. In particular, we sought to investigate altered pathological characteristics in brain sections of mice that received 200 mg/kg of Achul extract and did not develop prion disease.

Through H&E staining, it was confirmed that fear, which was found throughout various brain regions of mice in the control group, was reduced in a dose-dependent manner in the group of mice administered Achul extract (Figure 3A). When fear was counted in both the 100 mg/kg and 200 mg/kg groups, the results showed that Achul extract reduced cavernous degeneration at a statistically significant level in the cerebral cortex, hippocampus, and striatum, and in the hypothalamus and thalamus. It was confirmed that this was not the case. Specifically, fear formation was reduced dose-dependently by 40% to 55% in the cerebral cortex, 43% to 49% in the hippocampus, and 54% to 68% in the striatum (Figure 3A).

Astrocytes were identified in mouse brain through GFAP immunohistochemistry. Astrocyte activation was significantly less advanced in all brain regions of the three mice receiving 200 mg/kg of Achul extract, thus reducing the incidence of disease compared to the group receiving 100 mg/kg of Achul extract and the group receiving only the vehicle. It shows that no signs appear (Figure 3B). Through quantification of these GFAP-positive astrocytes, it was confirmed that active astrocytes were reduced by 62% to 91% depending on the mouse brain region. It shows that administration of Achul extract can effectively prevent brain damage caused by prion disease through reduction of vacuole formation and active glial cells.

2.4 Acul Extract in vitro Prevents prion infection in

To confirm the prion disease prevention effect of Achul extract, cell-based prion infection was performed in the presence of Achul extract. N2a cells were cultured with RML scrapie prions for 24 hours, and passaged 4 times in prion-free culture medium, confirming the formation of PrP ^Sc -positive cells (Figure 4). However, when prions were infected in the presence of Achul extract, the number of PrP ^Sc positive cells was substantially reduced. This efficacy was more evident when prion-infected N2a cells were cultured for 4 passages followed by an additional 6 passages in prion-free culture, and most prion infections and propagation were completely inhibited.

Through the results of in vitro prion infection experiments, it was confirmed that Achul extract reduces prion infectivity. Additionally, these results demonstrate that asymptomatic mice that do not accumulate PrP ^Sc and show neuropathological characteristics in the brain are not caused by an experimental error during the prion infection stage in animal experiments.

2.5 Cell-based PrP after activity tracking identification of Achul extract ^Sc Assay-based identification of anti-prion active ingredients: curzerenone and furanodienone

Activity tracking identification was performed to confirm the active ingredient of the Achul crude extract with prion inhibitory activity. After solvent fractionation, ScN2a cells were cultured using the crude extract and its extracted fractions according to the weight ratio of each fraction (Figure 3 and Table 2).

fraction mass (g) Mass ratio (%) Concentration (μg/ml) CpV Ext 4.26 100 50 HX 1.85 43.4 21.7 EA 1.72 40.4 20.2 ED 0.13 3.1 1.5 D.W. 0.56 13.1 6.6

CpV Ext: Achul crude extract HX: n-hexane fraction

EA: ethyl acetate fraction

ED: unmixed fraction between ethyl acetate and water fractions

DW: water fraction

The levels of PrP ^Sc in ScN2a cells cultured in the HX fraction were similar to those observed in the total effusion extract (Figure 5). Additional activity tracking identification with purified sub-fractions of HX was performed in the same manner. HX was separated into 12 subfractions by preparative HPLC (Figures 6 and 7). Each subfraction was used for culturing ScN2a cells, and the subfractions with reduced levels of PrP ^Sc compared to the level of PrP ^Sc in the control group using the carrier were HX7, HX8, and HX9 (FIG. 8). Two compounds were isolated from the HX7, HX8, and HX9 subfractions, and their chemical structures were confirmed to be curzerenone and furanodienone (FIGS. 9 to 15).

2.6 Furanodienones bind to PrP in ScN2a cells ^Sc reduce the level

To confirm the anti-prion activity of the identified compounds (curzerenone and furanodienone) contained in the active subfraction of HX, PrP ^Sc levels were measured in ScN2a cells by culturing with these compounds. Furanodienones reduced the level of PrP ^Sc in ScN2a cells in a concentration-dependent manner (FIG. 16). However, curzerenone increased the level of PrP ^Sc and appeared to be unaffected by the concentration of the compound. These results indicate that the anti-prion activity of Achul extract is due to furanodienone.

2.7 Furanodienones inhibit PrP changes

To confirm the biochemical method of furanodienone activity, RT-QuIC analysis was performed simulating the change of PrP ^C to a misfolded isoform through prion seeding. When furanodienone was added in the RT-QuIC reaction, PrP aggregation was reduced due to the generation of misfolded isoforms due to the transformation process (FIG. 17). In the RT-QuIC reaction, furanodienone concentration was inversely correlated with the calculated AUC and showed a proportional correlation with the length of the lag phase. These results show that furanodienones inhibit PrP changes.

For example, for claim construction purposes, the claims set forth below should not be construed in any way narrower than their literal language, and thus exemplary embodiments from the specification should not be read as claims. Accordingly, it should be understood that the present invention has been described by way of example and not as a limitation on the scope of the claims. Accordingly, the invention is limited only by the following claims. All publications, issued patents, patent applications, books and journal articles cited in this application are each hereby incorporated by reference in their entirety.

Claims (12)

A pharmaceutical composition for preventing or treating prion disease, comprising furanodienone. The pharmaceutical composition of claim 1, wherein the furanodienone reduces the level of scrapie prion protein (PrP ^Sc ) in cells. The pharmaceutical composition according to claim 1, wherein furanodienone inhibits aggregation of prion protein in cells. The method of claim 1, wherein the prion disease is bovine spongiform encephalopathy (BSE); advance disease of sheep and goats; Chronic wasting disease of deer, elk, moose, and caribou; Creutfeldt-Jakob disease, fatal familial insomnia, Gerstmann-Streussler-Scheinker syndrome, and Alzheimer's disease in humans, pharmaceutical compositions. Health functional food for preventing or alleviating prion disease, containing furanodienone. The health functional food according to claim 5, wherein furanodienon reduces the level of scrapie prion protein (PrP ^Sc ) in cells. The health functional food according to claim 5, wherein furanodienon inhibits the aggregation of prion protein in cells. The method of claim 5, wherein the prion disease is bovine spongiform encephalopathy (BSE); advance disease of sheep and goats; Chronic wasting disease of deer, elk, moose, and caribou; Creutfeldt-Jakob disease, fatal familial insomnia, Gerstmann-Streussler-Scheinker syndrome, and Alzheimer's disease in humans, health functional food. A feed composition for preventing or treating prion disease, comprising furanodienone. 10. The feed composition according to claim 9, wherein the furanodienones reduce the level of intracellular scrapie prion protein (PrP ^Sc ). The feed composition according to claim 9, wherein furanodienon inhibits aggregation of prion protein in cells. The method of claim 9, wherein the prion disease is bovine spongiform encephalopathy (BSE); advance disease of sheep and goats; A feed composition for chronic wasting disease of deer, elk, moose, and caribou.