KR100573632B1 - A novel compound inhibiting acetylcholine esterase produced by Aspergillus terreus Fb501 - Google Patents

Abstract

The present invention has an acetylcholine esterase inhibitory activity, and is produced from a new meroterpenoid compound (terreulactone), terreulactone A, B, which is produced from Aspergillus terreus FB501 strain. C, D and a method for producing the same. Terreulactone A, B, C, D has an excellent activity of selectively inhibiting acetylcholine esterase, so it can be used for the prevention and treatment of dementia, which is a very useful invention in the pharmaceutical industry.

Aspergillus terius Fb501, acetylcholinesterase, teriulactone, dementia

Description

Novel compound inhibiting acetylcholine esterase produced by Aspergillus terreus Fb501

1 shows hydrogen nuclear magnetic resonance ( ¹ H-NMR) and carbon nuclear magnetic resonance ( ¹³ C-NMR) spectra of teriulactone A materials.

Figure 2 shows the hydrogen nuclear magnetic resonance ( ¹ H-NMR) and carbon nuclear magnetic resonance ( ¹³ C-NMR) spectra of the teriulactone B material.

3 shows hydrogen nuclear magnetic resonance ( ¹ H-NMR) and carbon nuclear magnetic resonance ( ¹³ C-NMR) spectra of teriulactone C materials.

4 shows the hydrogen nuclear magnetic resonance ( ¹ H-NMR) and carbon nuclear magnetic resonance ( ¹³ C-NMR) spectra of the teriulactone D material.

The present invention is produced from the Aspergillus terreus Fb501 strain, a novel substance exhibiting inhibitory activity against acetylcholinesterase, terreulactone A, B, C, D and a method for producing the same It is about.

Due to the rapid increase of the elderly population due to an aging society, senile dementia has become a serious social problem, so research for prevention and treatment of it is very important. Dementia is divided into Alzheimer's dementia, vascular dementia, and AIDS-related dementia. Alzheimer's disease is more than half of the dementia patients in Korea and cerebral vascularity is about 30-40%. Alzheimer's disease is a progressive neurodegenerative disorder that causes damage to the central nervous system and damages memory, thinking and behavior. At present, studies on the causes of Alzheimer's disease and treatments are being actively conducted, but there are no reports on the causes or treatments of the breakthrough. What is observed in the brain of patients with Alzheimer's dementia is a histopathologic feature of a decrease in acetylcholine at the end of the cholinergic nervous system in the brain where beta-amyloid accumulates. The concentration of acetylcholine in the brain of dementia patients is about 50% less than normal. Accordingly, a method for increasing the concentration of acetylcholine at the synapse has been studied as a treatment for dementia.

Acetylcholine is a method of increasing the concentration of acetylcholine at the synapse of patients with Alzheimer's disease, which inhibits acetylcholinesterase, an acetylcholinesterase, thereby preventing the removal of acetylcholine from the synaptic cleft and increasing the concentration of acetylcholine. The study of esterase inhibitors has proceeded. Recently, tacrine (tetrahydroaminoacridine) has been licensed and marketed by the US FDA as a treatment for Alzheimer's disease. Tacrine is a potent acetylcholinesterase inhibitor, but also has a strong inhibitory activity against butyrylcholinesterase and has problems of liver toxicity. Accordingly, to develop a therapeutic agent without the side effect of liver toxicity caused by butyrylcholinesterase inhibitory activity, butyrylcholinesterase is a selective acetylcholinesterase that strongly inhibits acetylcholinesterase without inhibiting it. The development of inhibitors is called for.

Despite the limitation that acetylcholinesterase inhibitors are not fundamental dementia treatments and only relieve symptoms, the market for acetylcholinesterase inhibitors has grown rapidly every year in the dementia market, where no clear dementia treatment has been developed. have. Very recently, Aricept, which has low butyrylcholinesterase inhibitory activity and low side effects, has been developed and marketed by Ezai-Pfizer Co., Ltd., and has been well received.

Accordingly, it is an object of the present invention to provide a novel substance for preventing and treating dementia that selectively inhibits acetylcholinesterase.

It is also an object of the present invention to provide a method for preparing a new substance that selectively inhibits acetylcholinesterase.

The present inventors, while recognizing the problems of the prior art as described above and searching for acetylcholinesterase inhibitory active substances from natural products such as microorganisms and plants, are still reported from Aspergillus terreus Fb501 strains. Unidentified new terpene-based material and terurilactone

It was named (terreulactone), and the present invention was completed by identifying the physicochemical properties of the terurilactone, examining and confirming the biological activity, and establishing the preparation method thereof.

The present invention has a acetylcholine esterase inhibitory activity, a new meroterpenoid type material, terurilactone, produced from the Aspergillus terreus FB501 strain

(terreulactone) relates to A, B, C, D.

The present invention also relates to a method for producing terululactone A, B, C, D from the Aspergillus terreus FB501 strain.

The present invention comprises the steps of obtaining Aspergillus terius Fb501 separated and identified from soil; Culturing the Aspergillus terius Fb501 in a nutrient medium; Extracting the cultured cells with an organic solvent and performing column chromatography to obtain active fractions teriulactone A, B, C, D; And assaying the acetylcholinesterase inhibitory activity of the teriulactones A, B, C, and D.

The terurilactone production strain used in the present invention is a strain newly isolated from the soil.

When the teriulactone producing strain of the present invention was incubated for 10 days in MEA, CYA and CzA medium, its culture characteristics are shown in Table 1 below. The growth of the strain was very good and the diameter of the colony was different according to the culture temperature and the type of medium. The surface color was yellowish brown and the back side was grayish brown ~ brown.

Type of badge MEA CYA CzA Diameter of colonies at 25 ° C incubation (cm) 2.8 ~ 3.0 3.4 ~ 3.9 5.0 ~ 5.4 Diameter of colonies at 37 ° C incubation (cm) 8.0-9.0 - 9.0 ~ 9.4 Conidia Brownish yellow Yellowish brown Brownish orange Mycelium White, soft velvet-shaped fur White, fleecy floccose White, soft velvet-shaped fur Reverse Grayish yellow Wood brown Brown Exudate none yellow none Pigment yellow Amber color Amber color

Morphological characteristics of the teriulactone producing strain of the present invention was observed by using an optical microscope after incubation for 9 days at 25 ℃ in CzA medium. The conidiophore originated from the basal hyphae, and the conidial heads were densely columnar and 120-200 μm at maturity. Tipes are sometimes found to be black in color and the conidia were globose to subglobose with a diameter of about 2.5 μm.

As a result of identification of the above morphological and culture characteristics, the strain was found to be a new strain belonging to Aspergillus terius. The strain was named Aspergillus terreus Fb501, and was deposited on November 30, 2003 by the strain depositing institution of Korea Research Institute of Bioscience and Biotechnology, and was given accession number 0000-00000.

The general properties of the fungus are not constant, but are easily changed naturally or artificially, and this Aspergillus terius Fb501 strain is also prone to change. Therefore, aspergillus terius Fb501 of the present invention, as well as mutant strains (natural or mutagenic strains) derived from this strain, strains newly produced by a conjugate or genetic engineering method to produce teriulactone medulla It will be apparent to those skilled in the art that everything is included in the scope of the present invention.

Cultivation of the Aspergillus terius Fb501 strain for producing teriulactone in the present invention is carried out in a medium containing a nutrient source that can be used by conventional microorganisms. As a nutrient source, the well-known nutrient source currently used for the cultivation of a mold is used. For example, as a carbon source, glucose, starch syrup, dextrin, starch, molasses, animal oil, vegetable oil, etc. may be used, and as nitrogen sources, bran, soybean meal, wheat, malt, cottonseed gourd, fishmeal, corn stew ricker, gravy, yeast extract, sulfuric acid Ammonium, sodium nitrate, urea and the like can be used. If desired, it is more effective to add salts, potassium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, and other inorganic salts that promote ion generation. In addition, the solid medium containing bran as a main component has the effect of promoting the growth of bacteria and the production of quinolactacin material. As a culture method, shaking culture or political culture is possible under aerobic conditions.

     The culture temperature is slightly different depending on the conditions when incubated in each of the above conditions, it is usually appropriate to incubate at 20-37 ℃, in most cases it is incubated at 26-30 ℃. In addition, in the culture period of the shaking culture, politics culture, the production of quinolactacin material reached the highest when cultured for 4 to 7 days.

When cultivated under the conditions described above, a teriulactone-based material can be obtained. Since the present material is present not only in the culture medium but also in the cell part, it can be efficiently extracted and purified by the following method. That is, an organic solvent such as acetone is added to the culture solution and the mycelium, followed by stirring to extract the active ingredient from the cell, and then the acetone is evaporated and the solvent is extracted with ethyl acetate and chloroform. The chloroform solvent layer containing the active ingredient thus obtained is concentrated under reduced pressure to remove chloroform, followed by silica gel column chromatography using a solvent having a chloroform: methanol of 50: 1 to 10: 1. The active fraction thus obtained was purified by Sephadex LH-20 column chromatography using methanol as a solvent, and then subjected to HPLC again to obtain pure teriulactone of the present invention.

The scope of the present invention includes teriulactone as well as all derivatives thereof including inorganic or organic salts, esters, hydrates and solvates and isomers thereof.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited only to these Examples, and the substance of the present invention can be obtained even if the media type, culture conditions, extraction and purification methods are drastically changed. This is within the scope of the present invention.

Example 1 Culture of Aspergillus terius Fb 501 Strain

In order to cultivate Aspergillus terreus Fb501 strains, which were distributed by the Korea Research Institute of Bioscience and Biotechnology, a genetic deposit center, strains were cultured with yeast extract 0.3%, malt extract 0.3%, Medium containing 0.5% tryptone and 1% glucose was used after adjusting the pH to 5.5 before sterilization.

A 100 ml Erlenmeyer flask containing 20 ml of the seed medium was sterilized at 121 ° C. for 20 minutes, inoculated with Aspergillus terius Fb501 strain, shaken at 28 ° C. for 3 days, and used as a primary seed culture solution. Then, the seed culture was inoculated into a 500 ml Erlenmeyer flask containing sterilized bran and incubated at 28 ° C. for 7 days.

Example 2 Isolation and Purification of Terriuractone

Acetone was added to the bran culture cultured in Example 1 and stirred to extract the active ingredient from the cells, and then the acetone was evaporated and the solvent was extracted three times with ethyl acetate. The ethyl acetate solvent layer containing the active ingredient obtained in this manner was concentrated under reduced pressure to remove ethyl acetate, and silica gel column chromatography was then performed with a solvent having a chloroform: methanol of 100: 1 to 10: 1. The active fractions obtained in this manner were concentrated under reduced pressure to obtain an oily crude active ingredient, followed by Sephadex LH-20 column chromatography using methanol as a solvent. The active fractions were subjected to HPLC under acetonitrile: 55: 45 solvent conditions to obtain four compounds consisting of active fractions teriulactones A, B, C, and D.

The physicochemical properties and chemical structure of the teriulactone A, B, C, D of the present invention purified as described above are as follows.

(1) terreulactone A

Appearance of substance: White Powder

Molecular Weight: 510

Molecular Formula: C ₂₆ H ₃₀ O ₉

UV absorption spectrum [UVγ _max ^MeOH nm (logε)]: 214 (85,459), 253 (10969), 331 (11275)

Infrared Absorption Spectrum [IR (KBr) υcm ^-1 ]: 3437, 2924, 1800, 1754, 1704, 1572, 1259, 1178

Nuclear Magnetic Resonance (NMR) Absorption Spectrum: Hydrogen magnetic resonance ( ¹ H-NMR) and carbon nuclear magnetic resonance ( ¹³ C-) measured using chloroform (CDCl ₃ ) as a solvent and tetramethylsilane (TMS) as standard. NMR) spectrum is shown in FIG.

(2) terreulactone B

Appearance of substance: White Powder

Molecular Weight: 466

Molecular Formula: C ₂₇ H ₃₀ O ₇

UV absorption spectrum [UVγ _max ^MeOH nm (logε)]: 208 (41400), 247 (21200), 331 (22900)

Infrared Absorption Spectrum [IR (KBr) υcm ^-1 ]: 3432, 2926, 1671, 1569, 1514, 1260, 1180

Nuclear magnetic resonance (NMR) absorption spectra: Hydrogen magnetic resonance ( ¹ H-NMR) and carbon nuclear magnetic resonance ( ¹³ C-) measured using chloroform (CDCl ₃ ) as a solvent and tetramethylsilane (TMS) as a standard. NMR) spectra are shown in FIG. 2.

(3) terreulactone C

Appearance of substance: White Powder

Molecular Weight: 468

Molecular Formula: C ₂₇ H ₃₂ O ₇

UV absorption spectrum [UVγ _max ^MeOH nm (logε)]: 210 (29700), 252 (14400), 330 (18800)

Infrared Absorption Spectrum [IR (KBr) υcm ^-1 ]: 3403, 2938, 1702, 1673, 1569, 1514, 1260, 1182

Nuclear magnetic resonance (NMR) absorption spectra: Hydrogen magnetic resonance ( ¹ H-NMR) and carbon nuclear magnetic resonance ( ¹³ C-) measured using chloroform (CDCl ₃ ) as a solvent and tetramethylsilane (TMS) as a standard. NMR) spectrum is shown in FIG. 3.

(4) terreulactone D

Appearance of substance: White Powder

Molecular Weight: 556

Molecular Formula: C ₃₀ H ₃₆ O ₁₀

UV absorption spectrum [UVγ _max ^MeOH nm (logε)]: 213 (39300), 252 (17000),

331 (15200)

Infrared Absorption Spectrum [IR (KBr) υcm ^-1 ]: 3430, 2944, 1688, 1610, 1583, 1505, 1199, 1126

Nuclear Magnetic Resonance (NMR) Absorption Spectrum: Hydrogen magnetic resonance ( ¹ H-NMR) and carbon nuclear magnetic resonance ( ¹³ C-) measured using chloroform (CDCl ₃ ) as a solvent and tetramethylsilane (TMS) as a standard. NMR) spectra are shown in FIG. 4.

Example 3: Acetylcholinesterase inhibitory activity of quinolactacin

The acetylcholinesterase inhibitory activity of the quinolactacin substance, teriulactone A, B, C, and D obtained in Example 2 was measured using an Elllman's coupled enzyme assay method. As enzyme, acetylcholinesterase purified from electric eel or brain homogenates of male SD rats 8-10 weeks old was used, and acetylthiocholine was used as substrate. (acetylthiocholine), 5,5'-dithio-bis (2-nitrobenzoic acid) as a coupling reagent of the hydrolyzate thiocholine ) Was reacted under the condition of sodium phosphate buffer (pH 7.3) to measure the initial rate for 60 seconds at 412 nm. Enzyme inhibitory activity was defined as the initial rate ratio of the inhibitory reaction to the control reaction. Butylyl choline esterase inhibitory activity was carried out in the same manner as described above using butylyl choline esterase as an enzyme and S-butyrylthiocholine iodide as a substrate. The results are shown in Table 2 below.

matter IC ₅₀ (μM) Selectivity (butylyl choline esterase / acetyl choline esterase) Acetylcholinesterase Butylyl choline esterase Terriuractone A 0.23 > 200 > 869 Terriuractone B 0.09 > 200 > 2222 Terriuractone C 0.06 > 200 > 3333 Terriuractone D 0.42 > 200 > 476 Taclean 0.09 0.01 0.05

As can be seen in Table 2, the effective concentrations (IC ₅₀ ) showing the inhibitory activity of teriulactone A, B, C and D on acetylcholinesterase were 0.23, 0.09, 0.06 and 0.42 μM, respectively. On the other hand, the effective concentration (IC ₅₀ ) which shows the inhibitory activity of the teriulactone A, B, C, and D with respect to butylyl choline esterase was 200 micrometers or more. Thus, teriulactones A, B, C, and D showed 869, 2222, 3333, 476 times more potent selective activity to acetylcholinesterase than butylylcholinesterase. On the other hand, the well-known dementia treatment tacrine showed very low selectivity because of the inhibitory activity of acetylcholinesterase and butylylcholinesterase at 0.09 and 0.01 μM, respectively. Therefore, the teriulactone compound showed more than 5009 times of selective acetylcholinesterase inhibitory activity compared to tacrine.

As described above, the present invention Aspergillus terius FB501                     

Meroterpene- based material produced from Aspergillus terreus Fb501 strain

(meroterpenoid), terreulactone A, B, C, and D have excellent activity to selectively inhibit acetylcholine esterase, which can be used for the prevention and treatment of dementia. It is a useful invention.

Claims (3)

Meloterpenoid terreulactone B, C, D and pharmaceutically acceptable salts thereof represented by the following formulas (I) to (III). (I) Terriuractone B

(II) Terriuractone C

(III) Terriuractone D

A composition for inhibiting acetylcholinesterase containing teriulactone B, C and D of claim 1. Culturing Aspergillus terreus Fb501 strain in a nutrient medium; And extracting the cultured cells with an organic solvent and performing column chromatography to obtain active fractions of terryuractone B, C and D of claim 1.

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