KR101149190B1 - NEUROPROTECTIVE CONSTITUENTS OF ERAGROSTIS FERRUGINEA AGAINST AβINDUCED TOXICITY AND PHARMACEUTICAL COMPOSITION COMPRISING THE SAME - Google Patents

Abstract

The present invention is an extract that has the activity of inhibiting brain cell damage by beta amyloid and the active ingredient tricin (tricin, compound 2), ageconyflavone (ageconyflavone A, compound 3), nectandrin B (nectandrin B) isolated from the extract , Compound 5) and 4-ketopinolesinol (4-ketopinoresinol, Compound 6) and a pharmaceutical composition for preventing or treating amyloid-related diseases containing the same. Heyeong extract and its active ingredient according to the present invention is a natural product extract relatively low side effects and toxic expression rate, bio-friendly and useful for the prevention or treatment of amyloid-related diseases such as Alzheimer's disease, Down syndrome, cognitive impairment, memory loss and amyloidosis Can be used.

Description

Extracts that inhibit beta amyloid toxicity and pharmaceutical compositions containing them {Neuroprotective constituents of Eragrostis ferruginea against Aβinduced toxicity and pharmaceutical composition comprising the same}

The present invention relates to an extract that inhibits beta amyloid toxicity and a pharmaceutical composition containing the same, and more particularly, the extract that has the activity of inhibiting brain cell damage caused by beta amyloid and tricin isolated from the extract It relates to an active ingredient such as and a pharmaceutical composition for preventing or treating amyloid-related diseases containing the same.

Dementia is a condition in which a person who normally maintains his or her daily life loses intellectual ability due to brain dysfunction. Dementia is common in the 8.2 ~ 10.8% of the aged population over 65 years old, and it is a representative old age disease that is becoming a serious social problem with the rapid aging of the population. Alzheimer's disease, Lewy body dementia, forehead dementia and vascular dementia are the most common causes of dementia.

In particular, Alzheimer's disease (AD) is the most common form of dementia, brain atrophy in the cortex or hippocampus, senile plaques, neurofibrillary tangles and nerve cells. It is characterized by histologic findings such as granulophobic degeneration (granulovacuolar degeneration) and hirano body, and beta amyloid (β-amyloid, Aβ) is a major component of senile plaques. It is known to induce neuronal cell death due to oxidative stress by generating reactive oxygen species (ROS).

On the other hand, memory loss, one of Alzheimer's symptoms, has been reported to be closely related to the cholinergic nervous system. It has been reported that beta amyloid protein inhibits the synthesis of acetylcholine in brain cells and increases the activity of acetylcholinesterase and thus is toxic to brain neurons (Small et al., Curr Alzheimer Res, 2004).

 Down's syndrome is a hereditary disorder caused by one more chromosome in the 21st chromosome. The accumulation of amyloid protein in the brain of patients with Down syndrome has been reported to be higher than that of the general population without Down syndrome, suggesting that amyloid is associated with the development of Down syndrome (Crystalet al., Neurobiology of Aging, Yasuhiro et al., Brain & Development, 1997).

Minimal cognitive impairment is a term coined by neuroscientists who study memory impairment, a new term that describes the intermediate state between normal physiological forgetfulness and memory impairment caused by Alzheimer's disease. The scientists classified patients with forgetfulness for their age but who did not have Alzheimer's dementia, and the term impaired by those patients is minimal cognitive impairment. It is still very subjective and inconclusive that this minimal cognitive impairment is an intermediate state between physiological memory impairment and dementia caused by dementia. The study of these people suggests that patients with these minimal cognitive impairments should be advised by a specialist for more accurate diagnosis and treatment, including those who are more likely to develop Alzheimer's disease. Recently, studies to investigate the effects of vitamin E and acetylcholinease inhibitors in patients with this minimal cognitive impairment. The study aimed to see if patients with minimal cognitive impairment could reduce the rate of development of Alzheimer's disease when taking vitamin E or acetylcholinease inhibitors.

Amyloidosis is also called amyloid degeneration. This is called because starch reacts like starch. It is translucent in wax shape, and even when dyed with purple pigment, it turns red and shows a dichroic reaction. This substance appears between the walls of blood vessels and surrounding fibers, or between cells of the spleen and liver or between the interstitial tissues of myocardium and heart muscle. Small amounts are called starch degeneration, but when they appear in large areas, they are called starchosis or amyloidosis.

Drugs for the treatment and improvement of Alzheimer's disease, which have been developed based on such etiology, include Tacrine and Aricept, which increase the amount of choline acting in the brain by reducing choline breakdown at synapses. And acetylcholinease inhibitors such as Exelon, etc. are used, but they have the effect of alleviating the symptoms of Alzheimer's only, are not effective and temporary, and have severe side effects such as hepatotoxicity. (Forchetti et al., Prim Care Companion J Clin Psychiatry, 2005).

Therefore, beta amyloid (Aβ) is the most important substance that makes neurofibromas that cause amyloid-related diseases such as Alzheimer's disease, and many research institutes at home and abroad have developed and inhibited Aβ production in order to develop the fundamental therapeutic agents for amyloid-related diseases such as Alzheimer's disease. The elimination of Aβ or inhibition of Aβ aggregation is a major target in the treatment and control of Alzheimer's disease and the like, and the search for a drug that finds a substance having inhibitory effect on beta amyloid formation or suppresses apoptosis of neurons. Has been working hard.

In Alzheimer's research, plants have not been of much interest. Only EGb 761 of Ginkgo biloba and Hurperzine A of Chinese lychee have been studied to show effects on Alzheimer's disease. In recent years, however, more research has been published on plant materials and extracts that are effective in Alzheimer's disease. Calebin-A and curcumun from turmeric and extracts from plants such as green tea and wolfberry have been shown to protect neurons against Aβ-induced cytotoxicity.

On the other hand, Eragrostis ferruginea (Thunb.) P. Beauv. Is a perennial herb that is common in Korea on roadsides, grasses, and glades. Stems are flat and several clusters make a big abandonment. Leaves are lined, pointed, very tough, 20-40cm long, 2-6mm butterfly. There is hair at the bottom of the surface and at the top of the leaf. In August-September, reddish brown flowers bloom in cones. Isaac is long oval, 20-40cm long. Each branch has one node, a few hairs in the armpit, and there is a ring-shaped yellow gland on the upper part of the spikes split from the inflorescence. Fine grain is 5 ~ 10mm long, 5 ~ 10 small flowers hang, and anther is 0.8 ~ 1.2mm long. Poyoung has 1 vein with basso, and Hoyoung has a narrow egg shape, falling earlier than Nayoung and slightly longer. Fruits are oval, slightly flat oval. It is mainly used for grasses and all directions, and it is used for farm animals as feed, and leaves are used as baby substitutes. It is distributed in Korea, China, and the Himalayas.

His roots have been used in the treatment of cancer and diabetes in oriental medicine (Kang, 2008; Nishiya et al. , 1991). However, there is no known neuroprotective effect on brain cells. In addition, diterpenoids isolated from the roots of the herb have been reported (Nishiya et al., 1991), but no chemical composition or physiological activity of the other parts of the herb has been reported.

Haass, C., and Selkoe, D. J. Cellular Processing of Beta-Amyloid Precursor Protein and the Genesis of Amyloid Beta-Peptide. Cell, 75, 1039-1042 (1993). Kang, B. H. Ecological flora of Korean medicinal plants. Geobook, Seoul, Korea, (2008). Kelly, B. L., and Ferreira, A. beta-amyloid-induced dynamin 1 degradation is mediated by N-methyl-D-aspartate receptors in hippocampal neurons. Journal of Biological Chemistry, 281, 28079-28089 (2006). Kwon, Y. E. Inhibitory petency of acetylcholinesterase and amyloid beta (1-42) peptide aggregation to the extracts of enthusiasm reducing herbal. Kor. J. Pharmacogn., 38, 308-311 (2007). Kwon, Y. S., and Kim, C. M. Antioxidant constituents from the stem of Sorghum bicolor. Arch. Pharmacal Res., 26, 535-539 (2003). Nishiya, K., Kimura, T., Takeya, K., Itokawa, H., and Lee, S. R. Diterpenoids from Eragrostis ferruginea. Phytochemistry, 30, 2410-11 (1991). Nkengfack, A. E., Kouam, J., Vouffo, T. W., Meyer, M., Tempesta, M. S., and Fomum, Z. T. An isoflavanone and a coumestan from Erythrina sigmoidea. Phytochemistry, 35, 521-6 (1994). Otsuka, H., Takeuchi, M., Inoshiri, S., Sato, T., and Yamasaki, K. Phenolic compounds from Coix lachryma-jobi var. ma-yuen. Phytochemistry, 28, 883-6 (1989). Park, S. Y., and Kim, D. S. Discovery of natural products from Curcuma longa that protect cells from beta-amyloid insult: a drug discovery effort against Alzheimer's disease. J Nat Prod, 65, 1227-31 (2002). Pietrzik, C., and Behl, C. Concepts for the treatment of Alzheimer's disease: molecular mechanisms and clinical application. Int. J. Exp. Pathol., 86, 173-185 (2005). Shimomura, H., Sashida, Y., and Oohara, M. Lignans from Machilus thunbergii. Phytochemistry, 27, 634-6 (1988). Tomazela, DM, Pupo, MT, Passador, EAP, Da Silva, MFDGF, Vieira, PC, Fernandes, JB, Rodrigues Fo, E., Oliva, G., and Pirani, JR Pyrano chalcones and a flavone from Neoraputia magnifica and their Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase-inhibitory activities. Phytochemistry, 55, 643-651 (2000). Vyas, A. V., and Mulchandani, N. B. Polyoxygenated flavones from Ageratum conyzoides. Phytochemistry, 25, 2625-7 (1986). Wang, D., Li, F., and Jiang, Z. Osteoblastic proliferation-stimulating activity of Psoralea corylifolia extracts and two of its flavonoids. Planta Med., 67, 748-749 (2001).

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating amyloid-related diseases, containing as an active ingredient an effective extract of Alzheimer's disease, Down's syndrome, cognitive impairment, memory loss and amyloidosis, which are safe and have little side effects to the human body.

It is another object of the present invention to provide a food composition for preventing or improving amyloid-related diseases, including as an active ingredient extracts that are effective and effective in Alzheimer's disease, Down's syndrome, cognitive disorders, memory loss and amyloidosis, while being safe with almost no side effects in the human body.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

The inventors performed repeated column chromatography purification and preparative HPLC of the above-mentioned EtOAc-dissolving extracts of the above-mentioned grounds to prepare a new flavonoid 7-dimethylageconiflavone A (compound 1) and five known compounds 2- 6 (see Figure 1) was separated. Using the compounds 1-6 (7-demethylageconyflavone A, tricin, ageconyflavone A, corylin, nectandrin-B, 4-ketopinoresinol) found in the above-mentioned test, the neuronal protective effect against Aβ-induced cytotoxicity, Trisine (Tricin, Compound 2) showed potent neuronal protective effect in PC12 cells (ED ₅₀ , 20.3 uM), Ageconyflavone A (Compound 3), Nectandrin-B (Compound 5) , 4-ketopinolesinol (4-ketopinoresinol, Compound 6) was also confirmed to exhibit a neuroprotective effect (ED ₅₀ , 58.7, 44.1, 54.8 uM) to complete the present invention.

According to an aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating amyloid-related diseases, which contains the extract of Eragrostis ferruginea as an active ingredient.

In the composition of the present invention, the aged extract may be extracted using water, an organic solvent or a mixed solvent thereof, which is a well-known extraction solvent for conventional extraction of natural products, preferably the methanol or ethanol extract of the do. Here, methanol or ethanol may be used by diluting with water in 50% to 100%, but preferably extracted with 100% methanol or ethanol to elute the active ingredient. The methanol or ethanol extract may be further fractionated using an organic solvent having a different polarity, and may be further purified by various chromatography.

In the present invention, the extract may include any of the extracts, fractions and purified products obtained in each step of extraction, fractionation and purification, any dilution or concentrate thereof, or dried products thereof.

Such extracts, which are the raw materials of the present invention, may be formulated in the form of granules, tablets, capsules or drinks by conventional methods known in the art. Moreover, in order to make storage and handling easy, you may add the carrier used for normal formulation, such as dextrin and cyclodextrin, and other arbitrary preparations.

In addition, it is appropriate to administer the soybean extract of the present invention so that the daily intake of adult 1 ~ 3000mg. In addition, the dosage can be appropriately increased or decreased depending on age, symptoms, and the like.

In the present invention, the amyloid-related diseases include all diseases caused by amyloid accumulation, degeneration or toxicity thereof, but preferably include Alzheimer's disease, Down syndrome, cognitive impairment, memory loss and amyloisosis. It is characterized in that it is any one selected from the group and more preferably Alzheimer disease (Alzheimer disease).

According to another aspect of the present invention, the present invention is tricin (Tricin) represented by the formula (2) or a pharmaceutically acceptable salt thereof, ageconyflavone A (Ageconyflavone A) represented by the formula (3) or a pharmaceutically acceptable Possible salts, nectandrin-B represented by formula 5 or a pharmaceutically acceptable salt thereof, and 4-ketopinoresinol represented by formula 6 or a pharmaceutically acceptable salt thereof It provides a pharmaceutical composition for preventing or treating amyloid-related diseases containing at least one compound selected from the group consisting of possible salts as an active ingredient.

Pharmaceutically acceptable salts of the above formulas, unless otherwise indicated, include salts of hydroxy groups which may be present in compounds of formula, and salts of hydroxy groups include alkali metal salts such as lithium, sodium, potassium, magnesium, Alkaline earth metal salts, such as calcium, are mentioned. Preferably, salts with physiologically acceptable sodium, potassium, calcium, and the like may be cited, and these salts may be prepared through a method or a process for preparing a salt known in the art.

Compounds 2, 3, 5 or 6, such as tricin, used in the present invention may be purchased or synthesized from commercially purified compounds, but are preferably used in the extracts of plants, especially those containing the compounds ( Eragrostis ferruginea ). The separated one can be used, and any separation method from the extract can be used as long as it can separate tricin from the same. For example, the polar or non-polar solvent soluble extract may be purified by fractionation and washing several times with distilled water or the like, followed by further conventional fractionation processes (Harborne J.B. et al., Phytochemical methods: A guide to modern techniques of plant analysis., 3rd Ed., pp 6-7, 1998).

In addition, compounds 2, 3, 5 or 6, such as tricin of the present invention, may be prepared with pharmaceutically acceptable non-toxic salts and solvates according to methods conventional in the art. As the pharmaceutically acceptable salt, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and acid or alcohol (eg, glycol monomethylether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered.

Pharmaceutically acceptable salts of compounds 2, 3, 5 or 6 of the present invention include salts of acidic or basic groups which may be present in compounds 2, 3, 5 or 6 of the present invention unless otherwise indicated. . For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, including, but not limited to, salts known in the art. It may be manufactured through a manufacturing method or a manufacturing process.

 In the composition of the present invention, the compound 2, 3, 5 or 6 may preferably be included in 0.0001 to 90% by weight relative to the total weight of the composition. In the composition of the present invention, preferably the composition may have a powder, granule, tablet, capsule, injection, cream, gel, patch, spray or ointment formulation formulation. Pharmaceutical compositions containing compounds 2, 3, 5 or 6, such as tricin of the present invention as an active ingredient, may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical compositions containing the compound 2, 3, 5 or 6 as an active ingredient, respectively, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, or the like in the form of sterile injectable solutions Can be formulated and used.

In addition, carriers, excipients and diluents that may be included in the composition containing the compound 2, 3, 5 or 6 as an active ingredient, lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol Starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate And mineral oils. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose, or the like in the extract. Or lactose, gelatin, or the like is mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.0001 to l00 mg / kg, preferably at 0.001 to 10 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The compounds of the present invention can be administered by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

According to another aspect of the present invention, the present invention provides a food composition for preventing or ameliorating amyloid-related diseases containing the extract as an active ingredient.

The extract is an active ingredient Trisin (Tricin) represented by the following formula (2) or a pharmaceutically acceptable salt thereof, Ageconyflavone A (Ageconyflavone A) represented by the formula (3) or a pharmaceutically acceptable salt thereof, Nectandrin-B represented by 5 or a pharmaceutically acceptable salt thereof, and 4-ketopinoresinol represented by the formula (6) or a pharmaceutically acceptable salt thereof It is characterized by containing at least one compound selected from the group as an active ingredient.

 In the present specification, the term "food" means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through some processing process. Includes food, food additives, nutraceuticals and beverages.

 In the food composition for preventing or improving amyloid-related diseases of the present invention, preferably the compound 2, 3, 5 or 6 may be included in 0.0001 to 90% by weight relative to the total weight of the composition. Foods for ameliorating or preventing amyloid-related diseases of the present invention may further include food acceptable food additives, and may be formulated in the form of pills, powders, granules, acupuncture, tablets, capsules or beverages.

 In addition, the compound 2, 3, 5 or 6 of the present invention can be used as a food supplement of health supplements for the prevention of amyloid-related diseases such as Alzheimer's. Foods to which the compounds 2, 3, 5 or 6 of the present invention can be added include various foods, for example, beverages, gums, teas, vitamin complexes and health supplements. , Tablets, capsules or beverages.

At this time, the amount of the compound 2, 3, 5 or 6 in the food or beverage can generally be added in 0.0001 to 90% by weight of the total food weight in the case of the food composition of the present invention, in the case of a health beverage composition based on 100ml To 0.0001 to 20% by weight.

Food additives other than Compounds 2, 3, 5 or 6 as defined in the present invention include food additives customary in the art, such as flavors, flavors, colorants, fillers, stabilizers and the like, and are exemplified below. .

In the case of the beverage composition, there are no particular limitations on the liquid components which contain other than the compounds 2, 3, 5 or 6 as essential ingredients in the indicated ratios, and various flavors or natural carbohydrates as additional ingredients are used as in general beverages. It may contain. Examples of the natural carbohydrates include, for example, monosaccharides such as glucose and fructose, for example, disaccharides such as maltose and sucrose, for example, conventional sugars such as polysaccharides such as dextrin and cyclodextrin, and xylitol. Sugar alcohols such as sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0.001 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

As described above, tricin (Tricin, Compound 2), ageconyflavone (ageconyflavone A, Compound 3), nectanrin B (nectandrin B, Compound 5) and 4 isolated from the extract and the extract according to the present invention -Ketopinolesinol (4-ketopinoresinol, Compound 6) is a natural product extract with relatively low side effects and toxicity, and is bio-friendly and prevents amyloid-related diseases such as Alzheimer's disease, Down's syndrome, cognitive impairment, memory loss and amyloidosis. It is effective in treatment.

Therefore, compound 2, 3, 5, and 6 isolated from the extract and the extract according to the present invention, which can be easily obtained in Korea, etc., can be used inexpensively and usefully as a material of drugs and functional foods for amyloid-related diseases.

1 is a diagram showing the structure of compound 1-6.
FIG. 2 is a diagram showing key HMBC correlations of Compound 1. FIG.
3A and 3B are diagrams showing nuclear magnetic resonance (NMR) spectra of compound 1. FIG.
4A and 4B are diagrams showing nuclear magnetic resonance (NMR) spectra of compound 2. FIG.
5A and 5B are diagrams showing nuclear magnetic resonance (NMR) spectra of compound 3.
6A and 6B are diagrams showing nuclear magnetic resonance (NMR) spectra of compound 4. FIG.
7A and 7B are diagrams showing nuclear magnetic resonance (NMR) spectra of compound 5. FIG.
8A and 8B are diagrams showing nuclear magnetic resonance (NMR) spectra of compound 6.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

MATERIALS AND METHODS

Melting points were measured without correction with a Fisher-Johns melting point apparatus. NMR spectra were obtained with TMS as an internal standard using a Varian 500 MHz NMR spectrometer and chemical shifts were expressed as δ values. ESI-MS was measured using a Waters Q-TOF micro mass spectrometer. UV and IR spectra were obtained using Mecasys Optizen 2120 UV and Varian 640-IR, respectively. Chromatography was performed on silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck), Sephadex LH-20 (18-111 μm, GE Healthcare), and ODS-A (12 nm, S-75 μm, YMC) Was performed using. TLC was performed using a precoated silica gel 60 F ₂₅₄ plate (0.25 mm, Merck). Preparative HPLC was performed using a Varian Prostar 210 system.

MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl-tetrazolium bromide) and SDS (sodium dodecyl sulfate) were purchased from Sigma (St Louis, Mo, USA). Aβ (25-35) is Bachem California Inc. (Torrance, CA, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and horse serum were purchased from Invitrogen (Carlsbad, CA, USA).

Plant materials

The aerial parts of Eragrostis ferruginea were collected in Deokso, Namyangju, Gyeonggi-do, South Korea in September 2006, and confirmed by Kang Byung-hwa, one of the inventors. Samples were stored at Korea University in Seoul, Korea (voucher # EF20060900).

Example 1. Extraction and isolation of extracts

The ground part was then dried in the wind and then powdered to a uniform size with a grinder. A powder sample (2.2 kg) was extracted with methanol (MeOH) (x3) and methanol extract (200.4 g) was dissolved in water and fractionated with n- hexane and H ₂ O. The aqueous layer was extracted again with EtOAc.

EtOAc-soluble extract (13.5 g) was subjected to silica gel column chromatography using CHCl ₃ -MeOH gradient as the mobile phase to obtain 13 fractions (F01-F13). Fraction F05 (3.9 g) was chromatographed again on silica gel with stepwise gradient elution with n -hexane-EtOAc solvent to separate into 12 fractions (F14-F24).

Fraction F24 (1.3 g) was passed through a column containing Sephadex LH-20 gel using CHCl ₃ -MeOH (1: 1 v / v) for elution to separate compound 2 (59.2 mg) and 10 fractions ( F25-F34) was prepared by TLC analysis. F28 (179.1 mg) was passed through a C ₁₈ reverse phase silica gel column (C ₁₈ reversed-phase silica gel column) using a H ₂ O-MeOH gradient, there is provided a seven fractions (F35-F41), the compound 3 (50.7 mg ) Was obtained. Compound 1 (5.1 mg) was isolated at F39 by preparative HPLC (column: YMC J'sphere ODS-H80, 4 μm, 250 × 20 mm id, 50-55% MeOH, flow rate 10.0 mL / min). F29 (89.1 mg) was obtained was passed through a C ₁₈ reverse phase silica gel column (C ₁₈ reversed-phase silica gel column) using a H ₂ O-MeOH gradient compound 4 (10.0 mg). Fraction F21-22 (224.0 mg) was purified by column chromatography on Sephadex LH-20 gel in CHCl ₃ -MeOH (1: 1 v / v) and then 8 fractions (F42-49) by TLC analysis. Divided by. Compound 5 (46.4 mg) and Compound 6 (6.0 mg) were separated from fractions F44 and F45 by Sephadex LH-20 column chromatography, respectively.

Physicochemical properties and instrumental analysis of Compound 1 (7-Demethylageconyflavone A) are as follows (see FIGS. 3A and 3B).

Yellow resin;

m.p. 159-161 ° C .;

UV (MeOH) λ _max (log ε): 210 (4.49), 330 (4.26) nm;

IR (ATR) ν _max : 1680, 1633, 1467, 1450, 1335, 1259, 1202, 1110, 1027, 932, 841, 801 cm ⁻¹ ;

ESI-MS m / z 343.3 [M + H] ⁺ , 341.3 [M-H] ^- ;

HRESI-MS m / z 341.0670 [M-H] ^- (calcd. For C ₁₈ H ₁₄ 0 ₇ , 341.0661);

_{^{1 H-NMR (DMSO- d 6}} , 500 MHz) δ: 10.79 (brs, 7-OH), 7.59 (1H, brs, H-2 '), 7.58 (1H, brd, J = 8.5 Hz, H-6 '), 7.07 (1H, d, J = 8.5 Hz, H-5'), 6.89 (1H, s, H-8), 6.65 (1H, s, H-3), 6.13 (2H, s, -OCH ₂ O-), 3.79 (3H, s, 5-OCH ₃ ), 3.77 (3H, s, 6-OCH ₃ );

¹³ C-NMR (DMSO- d ₆ , 125 MHz) δ: 175.6 (C-4), 159.7 (C-2), 156.0 (C-7), 153.6 (C-9), 152.0 (C-5), 149.9 (C-4 '), 148.1 (C-3'), 139.2 (C-6), 124.8 (C-1 '), 121.0 (C-6'), 111.1 (C-10), 108.6 (C- 5 '), 106.3 (C-3), 106.0 (C-2'), 101.8 (-OCH ₂ O-), 99.8 (C-8), 61.7 (5-OCH ₃ ), 60.8 (6-OCH ₃ ) .

Physical and chemical properties and results of the instrumental analysis of Compound 2 (Tricin) are as follows (see FIGS. 4A and 4B).

Yellow powder;

ESI-MS, m / z : 331.3 [M + H] ⁺ ;

ESI-MS, m / z : 329.3 [M ⁻ H] ⁻ ;

¹ H- and ¹³ C-NMR data (DMSO- d ₆ ) are consistent with published values (Kwon and Kim, 2003).

Physical and chemical properties and results of instrumental analysis of Compound 3 (Ageconyflavone A) are as follows (see FIGS. 5A and 5B).

Yellow resin;

ESI-MS, m / z : 357.3 [M + H] ⁺ ;

¹ H-NMR (DMSO- d ₆ , 500 MHz) δ: 7.64 (1H, s, H-2 '), 7.63 (1H, s, H-6'), 7.09 (1H, d, J = 8.5 Hz, H -5 '), 6.72 (1H, s, H-3), 6.14 (2H, s, OCH ₂ O), 3.93 (3H, s, OCH ₃ ) and 3.78 (6H, d, J = 17.5 Hz, 2OCH ₃ );

¹³ C-NMR (DMSO- d ₆ , 125 MHz) δ: 175.7 (C-4), 159.8 (C-2), 157.4 (C-7), 153.9 (C-9), 151.5 (C-5), 150.1 (C-4 '), 148.2 (C-3'), 139.7 (C-6), 124.7 (C-1 '), 121.1 (C-6'), 112.0 (C-10), 108.6 (C-5 '), 106.5 (C-3), 106.0 (C-2'), 102.0 (OCH ₂ O), 97.4 (C-8), 61.8 (OCH ₃ ), 61.0 (OCH ₃ ), 56.4 (OCH ₃ ).

Physical and chemical properties and results of instrumental analysis of Compound 4 (Corylin) are as follows (see FIGS. 6A and 6B).

Light yellow resin;

ESI-MS, m / z : 319.3 [M ⁻ H] ⁻ ;

¹ H-NMR (Acetone, 500 MHz) δ: 8.18 (1H, s, H-2), 8.06 (1H, d, J = 8.5 Hz, H-5), 7.37 (1H, dd, J = 2.5 Hz and 8.5 Hz, H-2 '), 7.32 (1H, d, J = 2.0 Hz, H-6'), 7.00 (1H, dd, J = 2.5 Hz and 9.0 Hz, H-6), 6.91 (1H, d, J = 2.5 Hz, H-8), 6.77 (1H, d, J = 8.5 Hz, H-3 '), 6.44 (1H, d, J = 9.5 Hz, H-4 "), 5.75 (1H, d, J = 10.0 Hz, H-3 ″), 1.43 (6H, s, 2 ″ -2CH ₃ );

¹³ C-NMR (Acetone, 125 MHz) δ: 175.7 (C-4), 163.4 (C-7), 158.9 (C-9), 153.8 (C-4 '), 153.5 (C-2), 131.9 (C -3 "), 130.6 (C-2 '), 128.6 (C-5), 128.0 (C-6'), 125.9 (C-1 '), 125.0 (C-3), 123.0 (C-4") , 122.0 (C-5 '), 118.6 (C-10), 116.7 (C-3'), 115.8 (C-6), 103.3 (C-8), 77.2 (C-2 "), 28.4 (2CH ₃ ).

Physical and chemical properties and the results of the instrumental analysis of Compound 5 (Nectandrin-B) are as follows (see FIGS. 7A and 7B).

Yellow resin;

+11.25°(Acetone; c 0.48); [α]

+ 11.25 ° (Acetone; c 0.48);

ESI-MS, m / z : 345.4 [M + H] ⁺ ; ESI-MS, m / z : 343.4 [M ⁻ H] ⁻ ;

¹ H-NMR (Acotone, 500 MHz) δ: 7.08 (2H, d, J = 2.0 Hz, H-2 ', 2 "), 6.92 (2H, dd, J = 2.0 Hz and 8.0 Hz, H-6', 6 "), 6.81 (2H, d, J = 8.0 Hz, H-5 ', 5"), 4.42 (2H, d, J = 6.5 Hz, H-2,5), 3.85 (6H, s, 2OCH ₃ ), 2.25-2.28 (2H, m, H-3,4), 1.01 (6H, d, J = 7.0 Hz, 3,4-CH ₃ );

¹³ C-NMR (Acetone, 125 MHz) δ: 147.4 (C-3 ', 3 "), 145.9 (C-4', 4"), 134.2 (C-1 ', 1 "), 119.0 (C-6' , 6 "), 114.6 (C-5 ', 5"), 109.8 (C-2', 2 "), 87.2 (C-2,5), 55.3 (2OCH ₃ ), 44.6 (C-3,4) , 12.2 (2CH ₃ ).

Physicochemical properties and results of instrumental analysis of Compound 6 (4-Ketopinoresinol) are as follows (see FIGS. 8A and 8B).

Brown resin;

+12.44°(Acetone; c 0.50); [α]

+ 12.44 ° (Acetone; c 0.50);

ESI-MS, m / z : 371.1 [M−H] ⁻¹ ;

H-NMR (Acotone, 500 MHz) δ: 7.04 (2H, sx 2, 2OH), 6.82-6.91 (6H, m, Ar-H), 5.42 (1H, d, J = 3.5 Hz, H-7), 5.22 (1H, d, J = 3.5 Hz, H-7 '), 4.34 (1H, d, J = 2.5 Hz, H-9'), 4.05 (1H, dd, J = 4.5 and 9.5 Hz, H-9 ' ), 3.87 (6H, d, J = 4.5 Hz, 20CH ₃ ), 3.69 (1H, dd, J = 4.0 and 9.0 Hz, H-8), 3.39-3.44 (1H, m, H-8 ');

¹³ C-NMR (Acetone, 125 MHz) δ: 176.9 (C-9), 147.7, 147.5, 146.8, 146.2 (C-3, C-3 ', C-4, C-4'), 132.3, 131.6 (C -1, C-1 '), 118.8, 113.4 (C-6, C-6'), 114.9, 114.7 (C-5, C-6 '), 109.6, 109.5 (C-2, C-2') , 84.9 (C-7 '), 83.6 (C-7), 72.6 (C-9'), 55.5, 55.4 (20CH ₃ ), 52.8 (C-8 '), 49.5 (C-8).

Example 2. Aβ _25-35 Standard solution (Aβ _25-35 stock solution manufacturing

Aβ is a potential cause of AD (Haass and Selkoe, 1993) and Aβ oligomers are known to be more toxic to neurons than monomers (soluble forms) of fibrils (Kelly and Ferreira, 2006). Thus, Aβ 25-35 was used in the present invention pre-aggregated before use. Briefly, Aβ (25-35) (1 mg) was dissolved in 1 ml DMEM medium and incubated in a 37 ° C. water bath for 3 days to induce aggregation. Aggregated Aβ (25-35) was then diluted to 100 μg / ml and stored at −70 ° C. until use.

Example 3.Assay for the ability to protect PC12 cells against Aβ insult

In order to determine the activity of compounds for inhibiting or treating neurotoxicity induced by Aβ, we conducted bioassays according to previously known methods (Park and Kim, 2002). Since the conversion of MTT into MTT formazan in cells directly reflects cell survival, the experimental compound is evaluated to protect PC12 cells from damage by Aβ (25-35) by enhancing the conversion of these cells. It became.

For analysis, we dispensed 90 μL of exponential PC12 cells (4 × 10 ⁴ cells per well) in 96-well tissue culture plates for at least 2 hours. Cells were then pretreated with varying concentrations of experimental methanol extracts, rosmarinic acid (positive control) or DMSO (negative control). After 1 hour, Aβ (25-35) aggregates (10 μM) were added to the pretreated cells and incubated for 24 hours. For analysis, cells were treated with MTT solution (20 μL per well, 1 mg / mL stock solution) at 37 ° C. for 3 hours and then lysed overnight at 37 ° C. with 100 μL lysis buffer. . We measured the optical density of the resulting solutions at 590 nm using a microplate reader. The inventors then created a concentration-dependent curve for each extract and expressed the results in an ED ₅₀ value μM. The ED ₅₀ value of the samples was defined as the concentration (μM) for cell 50% survival under damage by Aβ.

Conclusion and consideration

The inventors performed repeated column chromatography purification and preparative HPLC of the above-mentioned EtOAc-dissolving extracts of the above-mentioned grounds to obtain a new flavonoid 7-dimethylageconylflavone A (compound 1) and five known compounds 2- 6 (see Figure 1) was separated.

Compound 1 separated as yellow resin and had a melting point of 158-161 ° C. M / z 341.0670 through HRESIMS (negative ion mode) (calcd for C 18 H 13 O 7, 341.0661.) In the [M ^- H] - to check the molecular ion peak, it was found the molecular formula C ₁₈ H ₁₄ O ₇ The 18 carbon atoms (two methoxyls, one methylenedioxy, five methines, and ten quaternary carbons) were identified by ¹³ C-NMR and HSQC spectra.

Δ _H 7.59 (1H, brs, H-2 ′), 7.58 (1H, brd, J = 8.5 Hz, H-6 ′) and 7.07 (1H, d, J = 8.5 Hz from the ¹ H-NMR spectrum of Compound 1 , AB-5-type aromatic proton signals in H-5 ') and two singlet aromatic protons in 6.89 (1H, s, H-8) and 6.65 (1H, s, H-3) Two singlet aromatic proton signals were identified. In addition, a characteristic signal of two methoxyl units at δ _H 3.79 (3H, s, C-5-OCH ₃ ) and 3.77 (3H, s, C-6-OCH ₃ ), δ _H 6.13 (2H , s, OCH ₂ O) showed a methylenedioxy signal (methylenedioxy signal) and exchangeable proton signal (exchangeable proton signal) in δ _H 10.79 (brs, 7-OH).

These data assume that Compound 1 has a flavone skeleton comprising one hydroxyl group, two methoxyl units, and one methylenedioxy substituent, and this inference is confirmed by 2D NMR techniques. It became. The complete structure of compound 1, including the position of the functional group, was determined by HMBC NMR technique (FIG. 2). Thus, the methylenedioxy proton signal at δ _H 6.13 showed an HMBC correlation with δ _C 148.1 (C-3 ′) and 149.9 (C-4 ′), which was C-3 ′ and C-4 'Confirmed the location. In addition, the positions of the two methoxyl groups are long-range HMBC relationship between δ _H 3.79 and δ _C 152.0 (C-5), and δ _H 3.77 and δ _C 139.2 (C-6), respectively. Decided by. The spectroscope data of these compounds 1 are almost identical to the data of the aggeniconiflavone A (compound 3) isolated and identified in the present invention except that there is no single methoxyl signal. Based on these observations, the structure of compound 1 was found to be 7-dimethylagegoniflavone A (7-hydroxy-3 ', 4'-methylenedioxy-5,6-dimethoxyflavone).

In addition, the structure of the known compound 2-6, NMR data analysis and physical data and spectral data are compared with the values in the literature, respectively, tricin (compound 2; Kwon and Kim, 2003), aggiconiflavone ( ageconyflavone A, Compound 3 ; Tomazela et al. , 2000; Vyas and Mulchandani, 1986), Corylin (Compound 4; Nkengfack et al. , 1994; Wang et al. , 2001), Nectandrin B, Compound 5; Shimomura et al. , 1988) and 4-ketopinoresinol (Compound 6; Otsuka et al. , 1989).

All compounds were tested for neuroprotective effects on Aβ-induced toxicity in PC12 cells. As a result, as shown in Table 1, tricin (Compound 2) exhibited a strong neuroprotective effect of the ED ₅₀ value (positive control, rosmarinic acid, ED ₅₀ value, 23.6 μM) of 20.3 μM. Ageconiflavone A (compound 3), nectandrin-B (compound 5), and 4-ketopinolesinol (compound 6) had moderate neuroprotective effects with ED ₅₀ values of 58.7, 44.1 and 54.8 μM, respectively. Indicated. In contrast, 7-dimethylageconiflavones A (compound 1) and coryline (compound 4) had no neuroprotective effect on Aβ-induced toxicity (ED ₅₀ values> 100 μg / mL). While Ageconiflavone A (Compound 3) exhibits a moderate (ED ₅₀ 54.8 μM) neuroprotective effect on Aβ-induced toxicity, its 7- O- dimethylated compound, 7-dimethylageconiflavone A (Compound 1) was found to have no PC12 cytoprotective effect against Aβ-induced toxicity. Table 1 below shows the neuronal protective effects of compounds 1-6 against Aβ induced toxicity.

Compounds ED ₅₀ (μM) ¹ 7-Demethylageconyflavone A ( 1 ) > 100 Tricin ( 2 ) 20.3 Ageconyflavone A ( 3 ) 58.7 Corylin ( 4 ) > 100 Nectandrin-B ( 5 ) 44.1 4-Ketopinoresinol ( 6 ) 54.8 Rosmarinic acid ² 23.6

¹ Concentration achieving 50% cell viability under Aβinsult;

² Positive control.

Preparation Example 1 Preparation of Health Food

Heraldic Extract Powder 1000mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods. Furthermore, instead of the extract, 1000 mg of compound 2, 3, 5 or 6 may be mixed.

Although specific portions of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto. It is therefore intended that the scope of the present invention be defined by the appended claims and their equivalents.

Claims (8)

Containing Eragrostis ferruginea extract as an active ingredient,
A pharmaceutical composition for preventing or treating amyloid-related diseases selected from the group consisting of Alzheimer's disease, Down syndrome, cognitive impairment, memory loss and amyloidosis. The composition of claim 1, wherein the extract is a methanol or ethanol extract. delete Tricin represented by the following formula (2) or a pharmaceutically acceptable salt thereof, ageconyflavone A (Ageconyflavone A) or a pharmaceutically acceptable salt thereof represented by the formula (3), and 4- represented by the formula (6) A group consisting of Alzheimer's disease, Down syndrome, cognitive impairment, memory loss and amyloidosis, containing as an active ingredient at least one compound selected from the group consisting of 4-ketopinoresinol or a pharmaceutically acceptable salt thereof Pharmaceutical composition for preventing or treating amyloid-related diseases selected from.
(2)

(Formula 3)

(Formula 6)

delete Containing the extract as an active ingredient,
Food composition for preventing or ameliorating amyloid-related diseases selected from the group consisting of Alzheimer's disease, Down syndrome, cognitive impairment, memory loss and amyloidosis. The method according to claim 6, wherein the extract is the active ingredient Tricin (Tricin) represented by the formula (2) or a pharmaceutically acceptable salt thereof, Ageconyflavone A (Ageconyflavone A) represented by the formula (3) Acceptable salts, nectandrin-B represented by Formula 5 or a pharmaceutically acceptable salt thereof, and 4-ketopinoresinol represented by Formula 6, or a pharmaceutical thereof A composition comprising one or more compounds selected from the group consisting of acceptable salts as an active ingredient.
(2)

(Formula 3)

(Formula 5)

(Formula 6)

delete

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