WO2021225363A1 - Anti-inflammatory or antidiabetic composition comprising metabolite of marine-drived fungus penicillium glabrum sf-7123 - Google Patents

Anti-inflammatory or antidiabetic composition comprising metabolite of marine-drived fungus penicillium glabrum sf-7123 Download PDF

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WO2021225363A1
WO2021225363A1 PCT/KR2021/005622 KR2021005622W WO2021225363A1 WO 2021225363 A1 WO2021225363 A1 WO 2021225363A1 KR 2021005622 W KR2021005622 W KR 2021005622W WO 2021225363 A1 WO2021225363 A1 WO 2021225363A1
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formula
inflammatory
present
pharmaceutical composition
compound
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Korean (ko)
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임정한
김일찬
한세종
오현철
손재학
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한국해양과학기술원
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention relates to an anti-inflammatory or anti-diabetic composition comprising a metabolite of the marine-derived fungus Penicillium glabrum SF-7123, and more particularly, to an anti-inflammatory or anti-diabetic composition comprising the metabolite It relates to a pharmaceutical composition for use, a food for improving inflammation or diabetes, and a method for isolating the metabolite.
  • Inflammation is an essential immune response of the body to injury or infection.
  • Macrophages and microglia resident macrophage-like cells of the central nervous system
  • They are activated in response to various stimuli and cause phagocytosis of damaged macrophages and nerve cells to protect tissues and prevent damage to the brain and body.
  • pro-inflammatory mediators including NO, prostaglandin E 2 (PGE 2 ), tumor necrosis factor (TNF)- ⁇ , interleukin (IL)-1 ⁇ and IL-6 ( Lappas, M., et al., Biol. Reprod . 2002 , 67 , 668-673.; Amor, S., et al., Immunology 2010 , 129 , 154-169.). Therefore, inhibiting the production of pro-inflammatory mediators can be considered as a major target for the prevention or treatment of inflammatory diseases.
  • iNOS inducible nitric oxide enzyme
  • COX- 2 cyclooxygenase-2
  • pro-inflammatory mediators including NO, prostaglandin E 2 (PGE 2 ), tumor necrosis factor (TNF)- ⁇ , interleukin (IL)-1 ⁇ and IL-6 ( Lappas, M., et al., Biol. Reprod . 2002 , 67 , 668-673.; Amor, S.
  • the RAW 264.7 cell line (Abelson murine leukemia virus-transformed macrophages derived from male BALB/c mice) has been widely used as an in vitro model for examining cells.
  • BV2 cells raf/myc-immortalized murine microglia
  • BV2 cells have been frequently used to model the response of microglia in vivo.
  • PTP1B Protein tyrosine phosphatase 1B
  • marine-derived fungi have been proposed as a unique source of biologically active secondary metabolites (Ebada, SS; Proksch, ed. Springer Berlin Heidelberg, Berlin, Heidelberg, Chapter 32; pp 759-788; 2015.).
  • biologically active compounds including polyketides (40%), alkaloids (20%), peptides (15%), terpenoids (15%), a variety of novel metabolites consisting of prenylated polyketides (7%) and shikimates (2%) and lipids (1%) were explored (Rateb, ME, Ebel, R. Nat. Prod. Rep . 2011 , 28 , 290-344).
  • studies on metabolites having an effect of inhibiting the production of pro-inflammatory mediators and PTP1B have not been reported.
  • the present inventors made diligent efforts to screen metabolites with excellent anti-inflammatory or antidiabetic activity.
  • metabolites of the marine-derived fungus Penicillium glabrum (SF-7123) were detected by LPS in RAW264.7 macrophages and BV2 microglia. It was confirmed that it has an anti-inflammatory effect and an anti-diabetic effect on the induced inflammatory response, and the present invention was completed.
  • An object of the present invention is to provide an anti-inflammatory or anti-diabetic pharmaceutical composition comprising a metabolite of Penicillium glabrum SF-7123 and a food for improving inflammation or diabetes, which has excellent anti-inflammatory or anti-diabetic activity have.
  • Another object of the present invention is to provide a method for isolating the metabolite.
  • Another object of the present invention is to provide a method for preventing or treating an inflammatory disease or diabetes comprising administering the metabolite or the pharmaceutical composition.
  • Another object of the present invention is to provide an anti-inflammatory or anti-diabetic use of the metabolite.
  • Another object of the present invention is to provide the use of the metabolite for the prevention or treatment of inflammatory diseases or diabetes.
  • Another object of the present invention is to provide the use of said metabolite in the manufacture of a medicament for the treatment of inflammatory diseases or diabetes.
  • the present invention provides an anti-inflammatory or anti-diabetic pharmaceutical composition
  • an extract of Penicillium glabrum SF-7123 comprising an extract of Penicillium glabrum SF-7123.
  • the present invention also provides a food for improving inflammation or diabetes containing the extract.
  • the present invention also comprises the steps of (a) culturing Penicillium glabrum (Penicillium glabrum) SF-7123; (b) extracting the strain culture obtained in step (a) with ethyl acetate (EtOAc); and (c) separating the ethyl acetate (EtOAc) extract obtained in step (b) by column chromatography.
  • R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 is aryl.
  • the present invention also provides an anti-inflammatory or anti-diabetic pharmaceutical composition
  • an anti-inflammatory or anti-diabetic pharmaceutical composition comprising one or more metabolites selected from the group consisting of the following Chemical Formulas 1 to 4:
  • R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 is aryl.
  • the present invention also provides a food for improving inflammation or diabetes comprising one or more metabolites selected from the group consisting of the following Chemical Formulas 1 to 4:
  • R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 is aryl.
  • the present invention also provides a method for preventing or treating an inflammatory disease or diabetes comprising administering the extract, the metabolite, or the pharmaceutical composition.
  • the present invention also provides the use of said extract or said metabolite for anti-inflammatory or anti-diabetic use.
  • the present invention also provides the use of said extract or said metabolite for the prevention or treatment of inflammatory diseases or diabetes.
  • the present invention also provides the use of said extract or said metabolite in the manufacture of a medicament for the treatment of an inflammatory disease or diabetes.
  • the present invention also provides the use of the extract or the metabolite in the manufacture of a functional food for the prevention or amelioration of inflammatory diseases or diabetes.
  • R 1 shows the effect of compound 1 in which R 1 is H on the mRNA expression of pro-inflammatory cytokines in LPS-induced BV2 cells.
  • Cells were pretreated with compound 1 with R 1 of H at the indicated concentration for 3 hours, followed by stimulation with LPS (1 ⁇ g/mL) for 24 hours.
  • Data represent mean values of three experiments ⁇ SD ( * p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001 compared to LPS treated group).
  • FIG. 2 shows the effect of compound 1, wherein R 1 is H, on protein expression of iNOS and COX-2 in LPS-induced BV2 cells (A) and RAW264.7 cells (B).
  • Cells were pretreated with Compound 1 (1.0, 2.0 and 4.0 ⁇ M) in which R 1 is H for 3 hours, followed by treatment with LPS (1 ⁇ g/mL) for 24 hours.
  • Western blot analysis was performed for iNOS and COX-2 expression and representative blots of three independent experiments are shown. Band intensities were quantified by densitometry and normalized to ⁇ -actin ( ** p ⁇ 0.01; *** p ⁇ 0.001 compared to LPS treated group).
  • 3A to 3H show the effect of Compound 1, wherein R 1 is H, on NF- ⁇ B activation in LPS-induced BV2 and RAW264.7 cells.
  • Cells were pretreated with compound 1 (1.0, 2.0 and 4.0 ⁇ M) with R 1 of H for 3 h, followed by stimulation with LPS for 1 h (* p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001). compared to the LPS-treated group).
  • 3A-3F show the results of Western blot analysis using specific anti-I ⁇ B- ⁇ , anti-p-I ⁇ B- ⁇ , anti-p65 and anti-p50 antibodies. Representative blots of three independent experiments are shown. Band intensities were quantified by densitometry and normalized to ⁇ -actin in the cytoplasmic fraction and PCNA in the nuclear fraction.
  • 3G and 3H show the results of measuring the NF- ⁇ B binding activity in the nuclear fraction using the NF- ⁇ B ELISA kit (Active Motif).
  • Figure 4 shows the effect of compound 1 in which R 1 is H on the activation of the MAPK pathway in LPS-induced BV2 and RAW264.7 cells.
  • Cells were pretreated with compound 1 (1.0, 2.0 and 4.0 ⁇ M) with R 1 of H for 3 h, followed by stimulation with LPS for 1 h.
  • the levels of phosphorylated-ERK (p-ERK), phosphorylated-JNK (p-JNK) and phosphorylated-p38 MAPK (p-p38 MAPK) were determined by Western blot analysis. Representative blots of three independent experiments are shown. Band intensities were quantified by densitometry and normalized to ⁇ -actin ( *** p ⁇ 0.001 compared to LPS treated group).
  • FIG. 5 shows Lineweaver-Burk plots for compound 3 (A) and compound 4 (B) in inhibition of PTP1B. Data represent mean ⁇ SD of three experiments. Concentrations ( ⁇ M) of compound 3 and compound 4 are indicated.
  • the metabolites of compounds 1 to 4 were isolated from the extract of the marine-derived fungus Penicillium glabrum SF-7123 (Accession No.: KCTC 14169BP), and the structures thereof were confirmed.
  • the metabolites (Compounds 1 to 4) were NO, prostaglandin E 2 (PGE 2 ), inducible nitric oxide enzyme (iNOS) and cyclooxygenase-2 (COX) in RAW264.7 macrophages and BV2 microglia. -2), and the like, by inhibiting the production of pro-inflammatory mediators and the expression of PTP1B, it was confirmed that it has a preventive or therapeutic effect on inflammatory diseases or diabetes.
  • the present invention in one aspect relates to a pharmaceutical composition for anti-inflammatory or antidiabetic comprising an extract of Penicillium glabrum SF-7123.
  • the extract relates to an anti-inflammatory or anti-diabetic pharmaceutical composition
  • an anti-inflammatory or anti-diabetic pharmaceutical composition comprising one or more metabolites selected from the group consisting of the following Chemical Formulas 1 to 4.
  • R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 aryl, preferably H, CH 3 or CH 2 CH 3 .
  • C 1-10 alkyl group means a linear or branched saturated hydrocarbon group having 1 to 10 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl , n-pentyl, n-hexyl, 1-ethylpropyl, 2,2-dimethylpropyl, and the like.
  • C 1-10 alkoxy group means a linear or branched alkyloxy group having 1*10 carbon atoms, for example, methoxy, ethoxy, propoxy, prop-2-oxy, butoxy, but-2- oxy, methylprop-2-oxy, isopropoxy, tert-butoxy, and the like.
  • C 3-10 cycloalkyl group means a saturated hydrocarbon ring having 3 * 10 carbon atoms, and in addition to monocycloalkyl groups exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like, polycycloalkyl groups, such as For example, a bicycloalkyl group, tricycloalkyl group, etc.
  • the bicycloalkyl group includes norbornyl, for example, exo-2-norbornyl, endo-2-norbornyl, 3-pinanyl, bi A cyclo[3.1.0]hexyl group, a bicyclo[2.2.1]heptyl group, a bicyclo[2.2.2]octo-2-yl group, etc., a tricycloalkyl group includes adamantyl, for example 1-adaman A tyl group, 2-adamantyl, etc. are mentioned.
  • a C 2-10 alkenyl group contains at least one double bond and is a straight or branched chain hydrocarbon radical having from 2 to 10 carbon atoms, for example, ethenyl, 2-propenyl, 3-butenyl, 2-part tenyl, 2-pentenyl, 3-pentenyl, 3-methyl-2-butenyl or 3-hexenyl and the like.
  • the C 6-10 aryl group means an aryl group having 6*10 carbon atoms, and examples thereof include a phenyl group, a naphthyl group, an anthryl group, and a phenanthryl group.
  • R 1 is CH 3 Metabolites of the formula (1) 9- O - is defined as methyl, New chroman menin (9- O -methylneuchromenin), the R 1 is H
  • the metabolite of Formula 1 is defined as neuchromenin, and the term "Compound 1" refers to a metabolite in which R 1 is CH 3 or H in Formula 1 above.
  • metabolites of R 2 to the formula II R 4 is CH 3, respectively, asterisks acid is defined as (asterric acid), the term "compound 2" the metabolic the R 2 to R 4 CH 3, respectively in the above formula (2) means body.
  • the metabolite of Formula 3 in which R 5 is CH 3 is defined as myxotrichin C (myxotrichin C), and the term “Compound 3” refers to a metabolite in which R 5 is CH 3 in Formula 3 above.
  • R 6 to R of 9 is CH 3
  • each metabolite of formula (4) is to be defined by the oxy Fu Nikon (deoxyfunicone)
  • the term "compound 4" is a metabolic
  • R 6 to R 9 is CH 3 respectively in the above formula (4) means body.
  • anti-inflammatory refers to the action of inhibiting or reducing inflammation.
  • the term "inflammation” is one of the defensive responses of biological tissues to certain stimuli, and is a biological defense mechanism that seeks to recover to its original state by removing injuries caused by various harmful stimuli.
  • Inflammation stimuli include infection or chemical and physical stimuli, and the inflammatory process can be divided into acute and chronic inflammation.
  • Acute inflammation is a short-term reaction within a few days, and plasma components or blood cells are involved in the removal of foreign substances by opening the microcirculation system.
  • Chronic inflammation has a long duration, and tissue proliferation is seen.
  • the anti-inflammatory or anti-diabetic pharmaceutical composition can be used for prevention, treatment or improvement of inflammatory diseases by having anti-inflammatory activity.
  • inflammatory disease is a generic term for diseases in which inflammation is the main lesion.
  • the term "extract” refers to a substance having anti-inflammatory activity isolated from the Penicillium glabrum SF-7123.
  • the extract is used in the sense of including not only the extract but also its dry powder or all forms formulated using the same.
  • the extract may be extracted using polar, non-polar, water, an organic solvent, or a mixed solvent thereof, and preferably may be extracted using an organic solvent.
  • the extracted liquid may be used in liquid form or may be used after concentration and/or drying.
  • the organic solvent is an anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, isopropanol, butanol, etc.), ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylform Amide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixture thereof can be used, and the extract is extracted by heating at room temperature or under conditions in which the active ingredient of the extract is not destroyed or minimized. can do.
  • methanol, ethanol, isopropanol, butanol, etc. ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylform Amide (DMF), dimethyl sulfoxide (DMSO
  • the degree of extraction and loss of the active ingredient of the extract may be different, so an appropriate organic solvent should be selected and used.
  • the extraction method is not particularly limited, and examples thereof include cold extraction, ultrasonic extraction, and reflux cooling extraction.
  • Filtration is a process of removing suspended solid particles from the extract, and may use cotton, nylon, etc. to filter out particles, ultrafiltration, cryofiltration, centrifugation, etc., but is not limited thereto.
  • a separation process by various chromatography chromatography according to size, charge, hydrophobicity or affinity may be further included.
  • Drying the filtrate includes, but is not limited to, freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, foam drying, high frequency drying, infrared drying, and the like. In some cases, a process of pulverizing the final dried extract may be added.
  • the anti-inflammatory or anti-diabetic pharmaceutical composition may be characterized in having one or more of the following characteristics.
  • NO nitric oxide
  • PGE 2 prostaglandin E 2
  • NF- ⁇ B nuclear factor kappa B
  • MAPK mitogen-activated protein kinase
  • the anti-inflammatory or anti-diabetic pharmaceutical composition is nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) It may be characterized in that it has a production inhibitory property.
  • NO is a small molecule that is an intracellular mediator produced by various immune cells and plays a pivotal role in the physiological and pathological conditions of inflammatory conditions.
  • PGE 2 can modulate immune and inflammatory responses.
  • nitric oxide is a substance whose amount is increased by nitric oxide synthase when inducing an inflammatory reaction in a cell, and is a molecule that is an indicator of the inflammatory response.
  • NOS nervous system nitric oxide synthase
  • the synthesized nitric oxide increases the production of cGMP in brain cells, thereby performing a function of storing information recognized from the outside for a long time.
  • NO is a free radical and is known to be involved in physiological and pathological processes.
  • NO is synthesized by L-Arginine oxidation by nitric oxide synthase (Atkan, et al., 75: 639-653, 2004).
  • PGE 2 prostaglandin E 2
  • COX-2 COX-2
  • PGE 2 is an inflammatory mediator produced at the site of inflammation by COX-2 called prostaglandin endoperoxide synthetase.
  • PGE 2 is associated with many cardiovascular diseases, arthritis, inflammatory bowel disease and chronic inflammatory diseases including chronic gastric ulcer (St-Onge, M. et al., Biochim. Biophys. Acta. 1771:1235-1245, 2007).
  • St-Onge M. et al., Biochim. Biophys. Acta. 1771:1235-1245, 2007.
  • Turini ME et al., Annu. Rev. Med. 53:35-57, 2002
  • Singh VP et al., Pharmacology 72:77-84, 2004).
  • the anti-inflammatory or anti-diabetic pharmaceutical composition may be characterized in that it inactivates NF- ⁇ B (nuclear factor kappa B) and MAPK (mitogen-activated protein kinase).
  • NF- ⁇ B nuclear factor kappa B
  • MAPK mitogen-activated protein kinase
  • nuclear factor-kappa B nuclear factor-kappa B
  • mitogen-activated protein kinase MAPK
  • NF- ⁇ B nuclear factor-kappa B
  • MAPK mitogen-activated protein kinase
  • I ⁇ B is phosphorylated and translocates NF- ⁇ B dimers (p50 and p65) to the nucleus, resulting in iNOS, COX-2, NO, PGE 2 , TNF- ⁇ , IL-1 ⁇ and IL It results in the transcription of inflammatory genes such as -6.
  • FIGS. 3G and 3H decreased the DNA binding activity of the p65 subunit in LPS-stimulated BV2 and RAW264.7 cells.
  • MAPK is a major signal transduction system that regulates cell growth and differentiation by transmitting this signal from the cell membrane to the nucleus in order to activate a receptor located on the cell membrane, such as a growth factor.
  • MAPKs play important roles in a variety of cellular conditions, such as apoptosis, cell growth, differentiation, proliferation and immune responses (Liu, Y., et al., Nat. Rev. Immunol . 2007 , 7 , 202-212.).
  • MAPK consists of three major signaling pathways, including extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2), c-Jun N-terminal kinase (JNK) and p38 MAPK.
  • p38 MAPK is one of the MAPKs regulating the inflammatory response, so it is considered a target for anti-inflammatory treatment.
  • Pre-treatment with compound 1 in which R 1 is H inhibited LPS-induced p38 MAPK phosphorylation in BV2 and RAW264.7 cells, but did not affect ERK and JNK MAPK ( FIG. 4 ).
  • the anti-inflammatory or anti-diabetic pharmaceutical composition is characterized in that it has an expression inhibitory property of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).
  • iNOS inducible nitric oxide synthase
  • COX-2 cyclooxygenase-2
  • compound 1 in which R 1 is H attenuated the protein expression of iNOS and COX-2 in a dose-dependent manner in both BV2 and RAW264.7 cells stimulated with LPS ( FIG. 2 ).
  • iNOS and COX-2 are pro-inflammatory mediators, iNOS is an inflammatory molecule that produces NO, and COX-2 produces PGE 2 . Excessive increased activity of iNOS and COX-2 may be the etiology of various inflammatory diseases.
  • COX-2 is an enzyme involved in the production of prostaglandin, a protein related to an inflammatory response, and an increase in intracellular COX-2 expression level can be an indicator indicating that an inflammatory response is in progress. have.
  • the anti-inflammatory or anti-diabetic pharmaceutical composition may be characterized in that it has the properties of inhibiting the expression of IL-1 ⁇ , IL-6 and TNF- ⁇ .
  • compound 1 wherein R 1 is H effectively inhibits the mRNA expression of IL-1 ⁇ , TNF- ⁇ and IL-6 when BV2 cells are activated with LPS (1 ⁇ g/mL) for 12 hours. decreased (Fig. 1).
  • the anti-inflammatory or anti-diabetic pharmaceutical composition may be characterized in that it has a property of inhibiting the expression of PTP1B.
  • compounds 3 and 4 were shown to inhibit the active PTP1B enzyme in a dose-dependent manner with IC 50 values of 19.2 ⁇ M and 24.3 ⁇ M, respectively (Table 3).
  • the pharmaceutical composition may be characterized in that it further comprises a pharmaceutically acceptable carrier, excipient or diluent.
  • Carriers, excipients and diluents that may be included in the composition include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil.
  • a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.
  • the pharmaceutical composition according to the present invention may be formulated and used in various forms according to conventional methods. Suitable dosage forms include tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, oral dosage forms such as aerosols, external preparations, suppositories, and sterile injection solutions.
  • Suitable dosage forms include tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, oral dosage forms such as aerosols, external preparations, suppositories, and sterile injection solutions.
  • the present invention is not limited thereto.
  • the pharmaceutical composition according to the present invention can be prepared in a suitable dosage form using a pharmaceutically inert organic or inorganic carrier. That is, when the formulation is a tablet, a coated tablet, a dragee, and a hard capsule, it may contain lactose, sucrose, starch or derivatives thereof, talc, calcium carbonate, gelatin, stearic acid or a pharmaceutically acceptable salt thereof. In addition, when the formulation is a soft capsule, it may contain vegetable oils, waxes, fats, semi-solid and liquid polyols. In addition, when the formulation is in the form of a solution or syrup, water, polyol, glycerol, and vegetable oil may be included.
  • the "pharmaceutically acceptable salt” refers to a formulation of a compound that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound.
  • the pharmaceutical salt is an acid that forms a non-toxic acid addition salt containing a pharmaceutically acceptable anion, for example, an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc., tartaric acid, formic acid, citric acid , organic carbonic acids such as acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, etc., sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.
  • acid addition salts formed with phonic acid and the like are included.
  • pharmaceutically acceptable salts of carboxylic acids include metal salts or alkaline earth metal salts formed with lithium, sodium, potassium, calcium, magnesium, etc., amino acid salts such as lysine, arginine, and guanidine, dicyclohexylamine, N organic salts such as -methyl-D-glucamine, tris(hydroxymethyl)methylamine, diethanolamine, choline and triethylamine, and the like.
  • Acids such as oxalic acid are not pharmaceutically acceptable, but can be used in the preparation of useful salts as intermediates for obtaining pharmaceutically acceptable salts.
  • the pharmaceutical composition according to the present invention may further include a preservative, a stabilizer, a wetting agent, an emulsifier, a solubilizing agent, a sweetener, a colorant, an osmotic pressure regulator, an antioxidant, and the like, in addition to the carrier described above.
  • the pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , sensitivity to drugs, administration time, administration route and excretion rate, duration of treatment, factors including concurrent drugs, and other factors well known in the medical field.
  • the pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • composition of the present invention can be used in combination with a pharmaceutical composition for treatment of inflammatory diseases, such as immunotherapy, chemotherapy, and radiation therapy, and pharmaceutical compositions for the treatment of other inflammatory diseases, which can be recognized by those skilled in the art as being effective in the prevention or treatment of inflammatory diseases. .
  • inflammatory diseases such as immunotherapy, chemotherapy, and radiation therapy
  • pharmaceutical compositions for the treatment of other inflammatory diseases which can be recognized by those skilled in the art as being effective in the prevention or treatment of inflammatory diseases.
  • the pharmaceutical composition of the present invention may be administered to an individual by various routes.
  • the mode of administration can be, for example, by subcutaneous, intravenous, intramuscular or intrauterine intrathecal or intracerebrovascular injection.
  • the pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient, along with several related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease.
  • the present invention relates to a food for improving inflammation or diabetes comprising an extract of Penicillium glabrum SF-7123.
  • the extract relates to a food for improving inflammation or diabetes containing one or more metabolites selected from the group consisting of the following Chemical Formulas 1 to 4.
  • R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 aryl, preferably H, CH 3 or CH 2 CH 3 .
  • the term "food” refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, There are vitamin complexes, health functional foods, and health foods, and includes all foods in the ordinary sense.
  • the functional food is the same term as food for special health use (FoSHU), and in addition to supplying nutrition, it is processed to efficiently exhibit bioregulatory functions and has high medical effects.
  • function (sex) means to obtain a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body.
  • the food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art.
  • the formulation of the food may be manufactured without limitation as long as it is a formulation recognized as a food, and the health functional food according to the present invention may be in the form of powder, granule, tablet, capsule or beverage.
  • the health food means a food having an active health maintenance or promotion effect compared to general food
  • the health supplement food means a food for the purpose of health supplementation.
  • the terms health functional food, health food, and dietary supplement are used interchangeably.
  • the food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used.
  • the composition may include additional ingredients that are commonly used in food to improve odor, taste, vision, and the like.
  • vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included.
  • it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr).
  • it may include amino acids such as lysine, tryptophan, cysteine, and valine.
  • the composition includes a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), a disinfectant (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), an antioxidant (butylhydroxyanisole (BHA), butylhydroxy Toluene (BHT), etc.), coloring agents (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (MSG, etc.), sweeteners (dulcin, cyclimate, saccharin, sodium, etc.) ), flavorings (vanillin, lactones, etc.), expanding agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (flavors), film agents, gum base agents, foam inhibitors, solvents, food additives such as improving agents ) may be included.
  • the additive may be selected according to the type of
  • It may further include a food pharmaceutically acceptable food supplement additive together with the Penicillium glabrum extract of the present invention, and may be used with other foods or food ingredients, and may be appropriately used according to a conventional method.
  • the mixed amount of the active ingredient may be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment).
  • the terms “improvement”, “prevention” and “treatment” should be interpreted in the broadest sense, and “improvement” means any action that temporarily/continuously relieves a disease or one or more clinical symptoms.
  • "Prevention” means preventing the progression of one or more of the clinical symptoms of a disease in a patient who may be exposed to or susceptible to the disease but has not yet experienced or exhibited symptoms of the disease.
  • Treatment means any action that arrests or reduces the development of a disease or one or more clinical symptoms thereof.
  • the fungal strain of SF-7123 was cultured on an agar Petri plate in a medium containing 3% NaCl at 25°C for 21 days. After extracting the culture medium with EtOAc, the filtered extract was concentrated in vacuo to obtain a crude extract, and compounds 1 to 4 were obtained from the crude extract using a chromatography method (Example) 3).
  • the present invention in another aspect, (a) Penicillium glabrum ( Penicillium glabrum ) culturing SF-7123; (b) extracting the strain culture obtained in step (a) with ethyl acetate (EtOAc); and (c) separating the ethyl acetate (EtOAc) extract obtained in step (b) by column chromatography.
  • EtOAc ethyl acetate
  • R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 aryl, preferably H, CH 3 or CH 2 CH 3 .
  • step (a) is a step of culturing Penicillium glabrum SF-7123, and the strain culture is preferably cultured in a medium containing nutrients that can be used by ordinary microorganisms.
  • a known nutrient source conventionally used for culturing mold can be used.
  • a carbon source glucose, starch syrup, dextrin, starch, molasses, animal oil, vegetable oil, etc.
  • a nitrogen source wheat bran, soybean meal, wheat, malt, cottonseed meal, fish meal, cornstarch, broth, yeast Extracts, ammonium sulfate, sodium nitrate, urea, etc.
  • shaking culture or stationary culture is preferable under aerobic conditions, but is not limited thereto.
  • the culture temperature is slightly different depending on the conditions when culturing under each of the above conditions, but it is usually preferred to culture at 20 to 37° C., more preferably at 25° C., but is not limited thereto.
  • the present invention relates to an anti-inflammatory or anti-diabetic pharmaceutical composition
  • an anti-inflammatory or anti-diabetic pharmaceutical composition comprising one or more metabolites selected from the group consisting of the following Chemical Formulas 1 to 4:
  • R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 is aryl.
  • R 1 to R 9 may be each independently H, CH 3 or CH 2 CH 3 , but is not limited thereto.
  • the present invention relates to a food for improving inflammation or diabetes comprising one or more metabolites selected from the group consisting of the following Chemical Formulas 1 to 4:
  • R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 is aryl.
  • R 1 to R 9 may be each independently H, CH 3 or CH 2 CH 3 , but is not limited thereto.
  • the present invention relates to a method for preventing or treating an inflammatory disease or diabetes comprising administering the extract, the metabolite, or the pharmaceutical composition of the Penicillium glabrum SF-7123. .
  • the present invention relates to the use of the extract of Penicillium glabrum SF-7123 or the metabolite for anti-inflammatory or anti-diabetes.
  • the present invention relates to the use of the extract or the metabolite of Penicillium glabrum SF-7123 for the prevention or treatment of inflammatory diseases or diabetes.
  • the present invention relates to the use of the extract or the metabolite of Penicillium glabrum SF-7123 in the manufacture of a medicament for the treatment of inflammatory diseases or diabetes.
  • the present invention relates to the use of the extract or the metabolite of the Penicillium glabrum SF-7123 in the manufacture of a functional food for the prevention or improvement of inflammatory diseases or diabetes.
  • Optical rotation was recorded using a Jasco P-2000 digital polarimeter.
  • UV spectra were recorded on a Beckman DU 800 UV-Visible Spectrophotometer.
  • IR spectra were obtained on an Agilent Cary 630 FT-IR spectrometer.
  • NMR spectra (1D and 2D) were recorded in CD 3 OD or CDCl 3 using a JEOL JNM ECP-400 spectrometer (400 MHz for 1 H and 100 MHz for 13 C) and the corresponding residual solvent signal (CD 3 OD: d H 3.30/ d C 49.0 and CDCl 3 : d H 7.26/ d C 77.0) referenced chemical shifts.
  • HRESIMS data were obtained using an ESI Q-TOF MS/MS system (AB SCIEX Triple). TLC was performed on Kieselgel 60 F 254 (1.05715; Merck) or RP-18 F 254s (Merck) plates. The spots were visualized by spraying 10% H 2 SO 4 aqueous solution followed by heating. Column chromatography was performed on YMC octadecyl-functionalized silica gel (C 18 ).
  • Example 2 Strain Materials and Fermentation.
  • Penicillium glabrum SF-7123 (Accession No.: KCTC 14169BP) was isolated from sediments collected on January 10, 2015 using a dredge in the Ross Sea (N 77 ⁇ 34.397′ W 166 ⁇ 10.8653′). 1 g of sample was mixed with sterile seawater (10 mL). A portion (0.1 mL) of the sample was treated using the spread plate method in potato dextrose agar (PDA) medium containing sterile seawater. Plates were incubated at 25° C. for 14 days. After subculture of the isolates several times, the final pure culture was selected and stored at -70°C.
  • PDA potato dextrose agar
  • Fungal strain SF-7123 was identified based on analysis of the 28S ribosomal RNA (rRNA) gene sequence. GenBank search using the 28S rRNA gene of SF-7123 (GenBank accession number KY563089) revealed 100%, 99.64% and 97.13% sequence identity to Penicillium glabrum (JN938946), P. spinulosum (HM469405) and P. multicolor (HM469407), respectively. shown as the closest match representing . Therefore, the marine-derived fungal strain SF-7123 was identified as Penicillium glabrum.
  • rRNA ribosomal RNA
  • Example 3 Extraction and isolation of compounds 1-4.
  • Fungal strain SF-7123 was cultured in 10 Fernbach flasks (300 mL) each containing 300 mL of PDA medium containing 3% NaCl (w/v). Flasks were individually inoculated with 2 mL of seed cultures of fungal strains and incubated at 25° C. for 14 days. The combined culture medium was extracted with EtOAc (4 L). The combined extraction solution was filtered through filter paper and then evaporated to dryness to obtain a crude extract SF7123 (1.0 g).
  • the SF7123-4 fraction was first separated with compound 3 (2.5 mg), followed by two SF7123-4-1 and SF7123 using Sephadex LH-20 as stationary phase and a 3/1 (v/v) mixture of MeOH in water as mobile phase.
  • the -4-2 sub-fraction was subjected to column chromatography (3 x 35 cm).
  • the SF7123-4-2 sub-fraction was then chromatographed on a RP C 18 column (1.2 x 30 cm) eluting with MeOH in water [2/3 (v/v)] to give 3 sub-fractions.
  • the SF7123-5 fraction was subjected to column chromatography packed with Sephadex LH-20 (2.0 x 30 cm). The column was then eluted with a mixture of MeOH in H 2 O [3/1 (v/v)] to separate into three subfractions SF7123-5-1 to SF7123-5-3.
  • Example 4 Determination of structures of compounds 1 to 4.
  • R 1 is CH 3
  • Table 1 Precise analysis of the COZY and HMBC spectra of Compound 1 in which R 1 is CH 3 (Table 1) is actually a derivative of nuchromenin in which a hydroxyl group is substituted with a methoxy group attached to C-9, in which R 1 is CH 3
  • R 1 is CH 3
  • Table 1 The planar structure of This assignment was confirmed by observation of the HMBC correlation from 9-OCH 3 to C-9.
  • R 1 is CH 3 structure of the compound 1 was determined as shown in formula 1 R 1 is CH 3, 9- O based on the above-described proof-methyl New chroman menin (9- O -methylneuchromenin) with Named.
  • the NMR data of Compound 1 in which R 1 is H was almost identical to the NMR data of Compound 1 in which R 1 is CH 3 and having a pyranchromene skeleton belonging to a citromycetin analog.
  • a scrutiny of 1 H and 13 C NMR data for compound 1 in which R 1 is H in the literature eventually led to the assignment of its structure as neuchromenin, which was found in Eupenicillium javanicum var. It was isolated from the culture medium of meloforme PF1181. It has been reported that neuchromenin can induce neurite outgrowth in PC12 rat pheochromocytoma cells (Tanada, Y.; Mori, K. European J. Org. Chem . 2001 , 2001 , 1963.).
  • the absolute configuration of naturally occurring neuchromenin was determined to be S by synthetic studies of the enantiomer of neuchromenin (Hayakawa, Y., et al., Tetrahedron Lett . 1996 , 37). , 6363-6364.). NMR data and specific rotation values of compound 1 in which R 1 is H and the literature (Tanada, Y.; Mori, K. European J. Org. Chem . 2001 , 2001 , 1963.) and the close similarity of specific rotation values suggested that the absolute configuration of compound 1 in which R 1 is H should be the same as that of (-)-neuchromenin. Therefore, it was suggested that the absolute configuration of compound 1 where R 1 is CH 3 is similar to the absolute configuration of compound 1 where R 1 is H, since they were isolated from chemical investigations on the same fungal strain.
  • diphenyl ether was isolated, which was identified as asteric acid ( compound 2 in which R 2 to R 4 is each independently CH 3 ) known as an endothelin binding inhibitor (Liao, WY, et al., J. Nat. Prod . 2012 , 75 , 630-635.; Ohashi, H., et al., Antibiot (Tokyo) . 1992 , 45 , 1684-1685.).
  • Example 5 Effect of secondary metabolites isolated from SF-7123 on the production of pro-inflammatory mediators.
  • LPS lipopolysaccharide
  • neurochromenin is formed by a methoxyl group (Compound 1 in which R 1 is CH 3) It was found that replacement of the hydroxyl group at the C-9 position of (neuchromenin, compound 1 in which R 1 is H) significantly reduced this effect.
  • the most active compound in this assay was identified as Compound 1 in which R 1 is H, and further studies were conducted to determine the additional anti-inflammatory effect of this compound and its underlying mechanism. As shown in FIG.
  • Example 6 PTP1B inhibitory effect of isolated compounds 1 to 4.
  • p-nitrophenyl phosphate was used as an enzyme substrate to evaluate the effect of the compound on PTP1B activity.
  • ursolic acid a known phosphatase inhibitor
  • ursolic acid a known phosphatase inhibitor
  • Example 7 Cell culture and viability assay.
  • RAW264.7 and BV2 cells were treated with Dulbecco's Fertilized Eagle supplemented with 10% fetal bovine serum (FBS), penicillin G (100 U/mL), streptomycin (100 mg/L) and L-glutamine (2 mM). It was maintained at 5 ⁇ 10 5 cells/mL in a medium (Dulbecco's modified eagle's medium, DMEM), and incubated at 37° C. in a humidified atmosphere containing 5% CO 2 . DMEM, FBS and other tissue culture reagents were purchased from Gibco BRL Co. Cell viability was determined using the previously described MTT assay (Cho, KH, et al., Neurochem. Int . 2016. 95. 55-62.).
  • Example 8 nitrite crystals.
  • Example 9 Preparation of cytoplasmic and nuclear fractions.
  • Cytoplasmic and nuclear fractions were extracted using an Affymetrix Nuclear Extraction kit (Affymetrix, Inc. Santa Clara, CA). Dissolution of each fraction was performed according to the manufacturer's instructions. Details of the assay are described in a previous report (Cho, KH, et al., Neurochem. Int . 2016. 95. 55-62.).
  • Example 10 Western blot analysis.
  • Example 11 DNA binding activity of NF- ⁇ B.
  • DNA-binding activity of NF- ⁇ B in nuclear extracts was measured using the TransAM® kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions. Analyzes were performed independently in triplicate.
  • the level of PGE 2 present in each sample was measured using a commercially available kit from R&D Systems (Minneapolis, Minn.). Three independent analyzes were performed according to the manufacturer's instructions.
  • Example 13 Quantitative real-time reverse transcriptase PCR (qRT-PCR).
  • PTP1B human, recombinant
  • Enzyme activity was measured in a reaction mixture containing 2 mM p-nitrophenyl phosphate (pNPP) in 50 mM citrate, pH 6.0 (composition: 0.1 M NaCl, 1 mM EDTA and 1 mM dithiothreitol [DTT]).
  • the reaction mixture was placed in an incubator maintained at 30° C. for 30 minutes, and 1N NaOH was added to terminate the reaction.
  • the amount of p-nitrophenol produced was estimated by measuring the increase in absorbance at 405 nm.
  • Non-enzymatic hydrolysis of 2 mM pNPP was corrected by measuring the increase in absorbance at 405 nm in the absence of PTP1B enzyme (Hamaguchi, T., et al., FEBS Lett . 1995 , 372 , 54-58.).
  • reaction mixtures consisted of different concentrations of pNPP as PTP1B substrate in the presence or absence of compounds 3 and 5, and the analysis was performed as described above.
  • Michaelis-Menten constant (Km) and maximum velocity (Vmax) of PTP1B were determined by Lineweaver-Burk plot using GraphPad Prism® 4 program (GraphPad Software Inc., USA).
  • a composition comprising a metabolite of the marine-derived fungus Penicillium glabrum SF-7123 is produced in LPS-stimulated RAW264.7 macrophages and BV2 microglia cells with NO, prostaglandin E 2 (PGE 2 ), the production of pro-inflammatory mediators such as inducible nitric oxide enzyme (iNOS) and cyclooxygenase-2 (COX-2) and inhibits the expression of PTP1B, thus preventing inflammatory diseases or diabetes or It can be usefully used for therapeutic purposes.
  • PGE 2 prostaglandin E 2
  • iNOS inducible nitric oxide enzyme
  • COX-2 cyclooxygenase-2

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Abstract

The present invention relates to: an anti-inflammatory or antidiabetic composition comprising a metabolite of marine-derived fungus Penicillium glabrum SF-7123; food for ameliorating inflammation or diabetes; and a method for separating the metabolite. The composition comprising a metabolite of marine-derived fungus Penicillium glabrum SF-7123, according to the present invention, inhibits the production of a pro-inflammatory mediator, such as NO, prostaglandin E2(PGE2), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and the expression of PTP1B, and thus can be useful in the prevention or treatment of inflammatory diseases or diabetes.

Description

해양 유래 진균 페니실리움 글라브룸 SF-7123의 대사체를 포함하는 항염증 또는 항당뇨용 조성물Composition for anti-inflammatory or anti-diabetes containing metabolite of marine-derived fungus Penicillium glabrum SF-7123
본 발명은 해양 유래 진균 페니실리움 글라브룸(Penicillium glabrum) SF-7123의 대사체를 포함하는 항염증 또는 항당뇨용 조성물에 관한 것으로, 더욱 상세하게는 상기 대사체를 포함하는 항염증 또는 항당뇨용 약학 조성물, 염증 또는 당뇨 개선용 식품 및 상기 대사체의 분리방법에 관한 것이다.The present invention relates to an anti-inflammatory or anti-diabetic composition comprising a metabolite of the marine-derived fungus Penicillium glabrum SF-7123, and more particularly, to an anti-inflammatory or anti-diabetic composition comprising the metabolite It relates to a pharmaceutical composition for use, a food for improving inflammation or diabetes, and a method for isolating the metabolite.
염증은 신체의 부상 또는 감염에 대한 필수적인 면역반응이다. 대식세포 및 미세아교세포(중추 신경계의 상주 대식세포-유사 세포)는 면역계에서 중요한 역할을 수행하는 것으로 반복적으로 보고되고 있다(Block, M.L., et al., Nat. Rev. Neurosci. 2007, 8, 57-69.; Sugama, S. Med. Hypotheses 2009, 73, 1031-1034.). 그들은 다양한 자극에 반응하여 활성화되며, 손상된 대식세포와 신경세포의 식세포작용을 일으켜 조직을 보호하고 뇌와 신체의 손상을 예방한다. 그러나, 지속적인 활성화는 세포 또는 조직에 유해하고 염증성 장 질환, 알츠하이머병, 파킨슨병 및 다발성 경화증을 포함하는 다양한 염증성 질환을 유발하는 유도성 산화질소 효소(iNOS), 시클로옥시게나아제-2(COX-2), NO, 프로스타글란딘 E2(PGE2), 종양 괴사 인자(TNF)-α, 인터루킨(IL)-1β 및 IL-6을 포함하는 염증 촉진성(pro-inflammatory) 매개체의 방출을 초래한다(Lappas, M., et al., Biol. Reprod. 2002, 67, 668-673.; Amor, S., et al., Immunology 2010, 129, 154-169.). 따라서, 염증 촉진성(pro-inflammatory) 매개체의 생성을 억제하는 것은 염증성 질환의 예방 또는 치료를 위한 주요 표적으로 간주될 수 있다. 수년 동안, RAW 264.7세포주(수컷 BALB/c 마우스로부터 유래된 아벨슨 뮤린 백혈병 바이러스-형질전환된 대식세포)는 세포를 조사하기 위한 시험관 내 모델에 널리 사용되어 왔다. 유사하게, BV2세포(raf/myc-불멸화된 뮤린 미세아교세포)는 생체 내에서 미세아교세포의 반응을 모델링하기 위해 자주 사용되어 왔다.Inflammation is an essential immune response of the body to injury or infection. Macrophages and microglia (resident macrophage-like cells of the central nervous system) have been repeatedly reported to play important roles in the immune system (Block, ML, et al., Nat. Rev. Neurosci . 2007 , 8 , 57-69.; Sugama, S. Med. Hypotheses 2009 , 73 , 1031-1034.). They are activated in response to various stimuli and cause phagocytosis of damaged macrophages and nerve cells to protect tissues and prevent damage to the brain and body. However, sustained activation is detrimental to cells or tissues and causes inducible nitric oxide enzyme (iNOS), cyclooxygenase-2 (COX- 2), resulting in the release of pro-inflammatory mediators including NO, prostaglandin E 2 (PGE 2 ), tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 ( Lappas, M., et al., Biol. Reprod . 2002 , 67 , 668-673.; Amor, S., et al., Immunology 2010 , 129 , 154-169.). Therefore, inhibiting the production of pro-inflammatory mediators can be considered as a major target for the prevention or treatment of inflammatory diseases. For many years, the RAW 264.7 cell line (Abelson murine leukemia virus-transformed macrophages derived from male BALB/c mice) has been widely used as an in vitro model for examining cells. Similarly, BV2 cells (raf/myc-immortalized murine microglia) have been frequently used to model the response of microglia in vivo.
단백질 티로신 포스파타제 1B(protein tyrosine phosphatase 1B, PTP1B)는 인슐린 및 렙틴 신호전달 경로의 주요 음성 조절제이다. 또한, 많은 연구에 따르면, 염증, 암 및 당뇨병과 같은 많은 병리생리학적 조건 하에서 PTP1B 발현이 증가하는 것으로 나타났으며, 이는 그러한 질환의 치료에서 PTP1B 억제제의 치료적 영향을 시사한다.Protein tyrosine phosphatase 1B (PTP1B) is a major negative regulator of the insulin and leptin signaling pathways. In addition, many studies have shown that PTP1B expression is increased under many pathophysiological conditions such as inflammation, cancer and diabetes, suggesting a therapeutic effect of PTP1B inhibitors in the treatment of such diseases.
한편, 해양 유래 진균은 생물학적으로 활성인 2차 대사체의 독특한 원천으로 제시되어 왔다(Ebada, S.S.; Proksch, ed. Springer Berlin Heidelberg, Berlin, Heidelberg, Chapter 32; pp 759-788; 2015.). 최근 몇 년 동안, 생물학적으로 활성인 화합물의 귀중한 자원으로서 해양 유래 진균에 대한 연구적 관심이 크게 증가하여, 폴리케타이드(40%), 알칼로이드(20%), 펩타이드(15%), 테르페노이드(15%), 프레닐화 폴리케타이드(7%) 및 시키메이트(2%) 및 지질(1%)로 구성된 다양한 새로운 대사체가 탐색되었다(Rateb, M.E., Ebel, R. Nat. Prod. Rep. 2011, 28, 290-344). 그러나, 염증 촉진성(pro-inflammatory) 매개체 및 PTP1B의 생성을 억제하는 효과를 갖는 대사체에 대한 연구는 보고된 바 없다. On the other hand, marine-derived fungi have been proposed as a unique source of biologically active secondary metabolites (Ebada, SS; Proksch, ed. Springer Berlin Heidelberg, Berlin, Heidelberg, Chapter 32; pp 759-788; 2015.). In recent years, there has been a significant increase in research interest in marine-derived fungi as valuable resources of biologically active compounds, including polyketides (40%), alkaloids (20%), peptides (15%), terpenoids (15%), a variety of novel metabolites consisting of prenylated polyketides (7%) and shikimates (2%) and lipids (1%) were explored (Rateb, ME, Ebel, R. Nat. Prod. Rep . 2011 , 28 , 290-344). However, studies on metabolites having an effect of inhibiting the production of pro-inflammatory mediators and PTP1B have not been reported.
이에, 본 발명자들은 항염증 또는 항당뇨 활성이 우수한 대사체를 스크리닝하고자 예의 노력한 결과, 해양 유래 진균 Penicillium glabrum(SF-7123)의 대사체들이 RAW264.7 대식세포 및 BV2 미세아교세포에서 LPS에 의해 유도되는 염증반응에 대한 항염증 효과 및 항당뇨 효과를 갖는 것을 확인하고, 본 발명을 완성하였다.Accordingly, the present inventors made diligent efforts to screen metabolites with excellent anti-inflammatory or antidiabetic activity. As a result, metabolites of the marine-derived fungus Penicillium glabrum (SF-7123) were detected by LPS in RAW264.7 macrophages and BV2 microglia. It was confirmed that it has an anti-inflammatory effect and an anti-diabetic effect on the induced inflammatory response, and the present invention was completed.
본 배경기술 부분에 기재된 상기 정보는 오직 본 발명의 배경에 대한 이해를 향상시키기 위한 것이며, 이에 본 발명이 속하는 기술분야에서 통상의 지식을 가지는 자에게 있어 이미 알려진 선행기술을 형성하는 정보를 포함하지 않을 수 있다.The information described in the background section is only for improving the understanding of the background of the present invention, and it does not include information forming prior art known to those of ordinary skill in the art to which the present invention pertains. may not be
발명의 요약Summary of the invention
본 발명의 목적은 항염증 또는 항당뇨 활성이 우수한 페니실리움 글라브룸(Penicillium glabrum) SF-7123의 대사체를 포함하는 항염증 또는 항당뇨용 약학 조성물 및 염증 또는 당뇨 개선용 식품을 제공하는 데 있다.An object of the present invention is to provide an anti-inflammatory or anti-diabetic pharmaceutical composition comprising a metabolite of Penicillium glabrum SF-7123 and a food for improving inflammation or diabetes, which has excellent anti-inflammatory or anti-diabetic activity have.
본 발명의 다른 목적은 상기 대사체의 분리방법을 제공하는 데 있다.Another object of the present invention is to provide a method for isolating the metabolite.
본 발명의 또 다른 목적은 상기 대사체 또는 상기 약학 조성물을 투여하는 단계를 포함하는 염증성 질환 또는 당뇨의 예방 또는 치료방법을 제공하는 데 있다.Another object of the present invention is to provide a method for preventing or treating an inflammatory disease or diabetes comprising administering the metabolite or the pharmaceutical composition.
본 발명의 또 다른 목적은 상기 대사체의 항염증 또는 항당뇨의 용도를 제공하는 데 있다.Another object of the present invention is to provide an anti-inflammatory or anti-diabetic use of the metabolite.
본 발명의 또 다른 목적은 염증성 질환 또는 당뇨의 예방 또는 치료를 위한 상기 대사체의 용도를 제공하는 데 있다.Another object of the present invention is to provide the use of the metabolite for the prevention or treatment of inflammatory diseases or diabetes.
본 발명의 또 다른 목적은 염증성 질환 또는 당뇨의 치료를 위한 약제의 제조에 있어 상기 대사체의 사용을 제공하는 데 있다.Another object of the present invention is to provide the use of said metabolite in the manufacture of a medicament for the treatment of inflammatory diseases or diabetes.
상기 목적을 달성하기 위하여, 본 발명은 페니실리움 글라브룸(Penicillium glabrum) SF-7123의 추출물을 포함하는 항염증 또는 항당뇨용 약학 조성물을 제공한다.In order to achieve the above object, the present invention provides an anti-inflammatory or anti-diabetic pharmaceutical composition comprising an extract of Penicillium glabrum SF-7123.
본 발명은 또한 상기 추출물을 포함하는 염증 또는 당뇨 개선용 식품을 제공한다.The present invention also provides a food for improving inflammation or diabetes containing the extract.
본 발명은 또한 (a) 페니실리움 글라브룸(Penicillium glabrum) SF-7123를 배양하는 단계; (b) 상기 (a) 단계에서 수득된 균주 배양물을 에틸아세테이트(EtOAc)로 추출하는 단계; 및 (c) 상기 (b) 단계에서 얻어진 에틸아세테이트(EtOAc) 추출물을 컬럼 크로마토그래피로 분리하는 단계를 포함하는 하기 화학식 1 내지 화학식 4의 대사체의 분리방법을 제공한다:The present invention also comprises the steps of (a) culturing Penicillium glabrum (Penicillium glabrum) SF-7123; (b) extracting the strain culture obtained in step (a) with ethyl acetate (EtOAc); and (c) separating the ethyl acetate (EtOAc) extract obtained in step (b) by column chromatography.
[화학식 1][Formula 1]
Figure PCTKR2021005622-appb-I000001
Figure PCTKR2021005622-appb-I000001
[화학식 2][Formula 2]
Figure PCTKR2021005622-appb-I000002
Figure PCTKR2021005622-appb-I000002
[화학식 3][Formula 3]
Figure PCTKR2021005622-appb-I000003
Figure PCTKR2021005622-appb-I000003
[화학식 4][Formula 4]
Figure PCTKR2021005622-appb-I000004
Figure PCTKR2021005622-appb-I000004
여기서, 상기 R1 내지 R9는 각각 독립적으로 H, 직쇄형 또는 분지쇄형의 C1-10 알킬, C1-10 알콕시, C3-10 사이클로알킬, C2-10 알케닐 또는 C6-10 아릴이다.Here, R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 is aryl.
본 발명은 또한 하기 화학식 1 내지 화학식 4로 구성된 군에서 선택되는 하나 이상의 대사체를 포함하는 항염증 또는 항당뇨용 약학 조성물을 제공한다:The present invention also provides an anti-inflammatory or anti-diabetic pharmaceutical composition comprising one or more metabolites selected from the group consisting of the following Chemical Formulas 1 to 4:
[화학식 1][Formula 1]
Figure PCTKR2021005622-appb-I000005
Figure PCTKR2021005622-appb-I000005
[화학식 2][Formula 2]
Figure PCTKR2021005622-appb-I000006
Figure PCTKR2021005622-appb-I000006
[화학식 3][Formula 3]
Figure PCTKR2021005622-appb-I000007
Figure PCTKR2021005622-appb-I000007
[화학식 4][Formula 4]
Figure PCTKR2021005622-appb-I000008
Figure PCTKR2021005622-appb-I000008
여기서, 상기 R1 내지 R9는 각각 독립적으로 H, 직쇄형 또는 분지쇄형의 C1-10 알킬, C1-10 알콕시, C3-10 사이클로알킬, C2-10 알케닐 또는 C6-10 아릴이다.Here, R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 is aryl.
본 발명은 또한 하기 화학식 1 내지 화학식 4로 구성된 군에서 선택되는 하나 이상의 대사체를 포함하는 염증 또는 당뇨 개선용 식품을 제공한다:The present invention also provides a food for improving inflammation or diabetes comprising one or more metabolites selected from the group consisting of the following Chemical Formulas 1 to 4:
[화학식 1][Formula 1]
Figure PCTKR2021005622-appb-I000009
Figure PCTKR2021005622-appb-I000009
[화학식 2][Formula 2]
Figure PCTKR2021005622-appb-I000010
Figure PCTKR2021005622-appb-I000010
[화학식 3][Formula 3]
Figure PCTKR2021005622-appb-I000011
Figure PCTKR2021005622-appb-I000011
[화학식 4][Formula 4]
Figure PCTKR2021005622-appb-I000012
Figure PCTKR2021005622-appb-I000012
여기서, 상기 R1 내지 R9는 각각 독립적으로 H, 직쇄형 또는 분지쇄형의 C1-10 알킬, C1-10 알콕시, C3-10 사이클로알킬, C2-10 알케닐 또는 C6-10 아릴이다.Here, R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 is aryl.
본 발명은 또한 상기 추출물, 상기 대사체 또는 상기 약학 조성물을 투여하는 단계를 포함하는 염증성 질환 또는 당뇨의 예방 또는 치료방법을 제공한다.The present invention also provides a method for preventing or treating an inflammatory disease or diabetes comprising administering the extract, the metabolite, or the pharmaceutical composition.
본 발명은 또한 상기 추출물 또는 상기 대사체의 항염증 또는 항당뇨의 용도를 제공한다.The present invention also provides the use of said extract or said metabolite for anti-inflammatory or anti-diabetic use.
본 발명은 또한 염증성 질환 또는 당뇨의 예방 또는 치료를 위한 상기 추출물 또는 상기 대사체의 용도를 제공한다.The present invention also provides the use of said extract or said metabolite for the prevention or treatment of inflammatory diseases or diabetes.
본 발명은 또한 염증성 질환 또는 당뇨의 치료를 위한 약제의 제조에 있어 상기 추출물 또는 상기 대사체의 사용을 제공한다.The present invention also provides the use of said extract or said metabolite in the manufacture of a medicament for the treatment of an inflammatory disease or diabetes.
본 발명은 또한 염증성 질환 또는 당뇨의 예방 또는 개선을 위한 기능성 식품의 제조에 있어 상기 추출물 또는 상기 대사체의 사용을 제공한다.The present invention also provides the use of the extract or the metabolite in the manufacture of a functional food for the prevention or amelioration of inflammatory diseases or diabetes.
도 1은 LPS-유도된 BV2세포에서 염증 촉진성 사이토카인의 mRNA 발현에 대한 R1이 H인 화합물 1의 효과를 나타낸다. 세포를 지정된 농도의 R1이 H인 화합물 1로 3시간 동안 전처리한 후, LPS(1㎍/mL)로 24시간 동안 자극시켰다. 데이터는 3개의 실험 ±SD의 평균값을 나타낸다(* p < 0.05; ** p < 0.01; *** p < 0.001는 LPS 처리된 군과 비교).1 shows the effect of compound 1 in which R 1 is H on the mRNA expression of pro-inflammatory cytokines in LPS-induced BV2 cells. Cells were pretreated with compound 1 with R 1 of H at the indicated concentration for 3 hours, followed by stimulation with LPS (1 μg/mL) for 24 hours. Data represent mean values of three experiments ±SD ( * p <0.05; ** p <0.01; *** p < 0.001 compared to LPS treated group).
도 2는 LPS-유도된 BV2세포(A) 및 RAW264.7세포(B)에서 iNOS 및 COX-2의 단백질 발현에 대한 R1이 H인 화합물 1의 효과를 나타낸다. 세포를 3시간 동안 R1이 H인 화합물 1 (1.0, 2.0 및 4.0μM)로 전처리한 후, LPS(1㎍/mL)로 24시간 동안 처리하였다. iNOS 및 COX-2 발현에 대한 웨스턴 블롯 분석을 수행하고 3개의 독립적인 실험의 대표적인 블롯을 나타내었다. 밴드 강도는 밀도측정법에 의해 정량화되고 β-액틴으로 정규화되었다(** p < 0.01; *** p < 0.001 는 LPS 처리된 군과 비교).2 shows the effect of compound 1, wherein R 1 is H, on protein expression of iNOS and COX-2 in LPS-induced BV2 cells (A) and RAW264.7 cells (B). Cells were pretreated with Compound 1 (1.0, 2.0 and 4.0 μM) in which R 1 is H for 3 hours, followed by treatment with LPS (1 μg/mL) for 24 hours. Western blot analysis was performed for iNOS and COX-2 expression and representative blots of three independent experiments are shown. Band intensities were quantified by densitometry and normalized to β-actin ( ** p <0.01; *** p < 0.001 compared to LPS treated group).
도 3a 내지 도 3h는 LPS-유도된 BV2 및 RAW264.7세포에서 NF-κB 활성화에 대한 R1이 H인 화합물 1의 효과를 나타낸다. 세포를 3시간 동안 R1이 H인 화합물 1(1.0, 2.0 및 4.0μM)로 전처리한 후, 1시간 동안 LPS로 자극하였다(* p < 0.05; ** p < 0.01; *** p < 0.001는 LPS 처리된 군과 비교).3A to 3H show the effect of Compound 1, wherein R 1 is H, on NF-κB activation in LPS-induced BV2 and RAW264.7 cells. Cells were pretreated with compound 1 (1.0, 2.0 and 4.0 μM) with R 1 of H for 3 h, followed by stimulation with LPS for 1 h (* p <0.05; ** p <0.01; *** p < 0.001). compared to the LPS-treated group).
도 3a 내지 도 3f는 특정한 항-IκB-α, 항-p-IκB-α, 항-p65 및 항-p50 항체를 사용하여 웨스턴 블롯 분석을 수행한 결과를 도시한다. 3개의 독립적인 실험의 대표적인 블롯을 나타내었다. 밴드 강도는 밀도측정법에 의해 정량화되고 세포질 분획에서 β-액틴 및 핵 분획에서 PCNA로 정규화되었다. 3A-3F show the results of Western blot analysis using specific anti-IκB-α, anti-p-IκB-α, anti-p65 and anti-p50 antibodies. Representative blots of three independent experiments are shown. Band intensities were quantified by densitometry and normalized to β-actin in the cytoplasmic fraction and PCNA in the nuclear fraction.
도 3g 및 도 3h는 핵 분획에서의 NF-κB 결합 활성을 NF-κB ELISA 키트(Active Motif)를 사용하여 측정한 결과를 도시한다. 3G and 3H show the results of measuring the NF-κB binding activity in the nuclear fraction using the NF-κB ELISA kit (Active Motif).
도 4는 LPS-유도된 BV2 및 RAW264.7세포에서 MAPK 경로의 활성화에 대한 R1이 H인 화합물 1의 효과를 나타낸다. 세포를 3시간 동안 R1이 H인 화합물 1(1.0, 2.0 및 4.0μM)로 전처리한 후, 1시간 동안 LPS로 자극하였다. 인산화된-ERK(p-ERK), 인산화된-JNK(p-JNK) 및 인산화된-p38 MAPK(p-p38 MAPK)의 수준은 웨스턴 블롯 분석에 의해 결정되었다. 3개의 독립적인 실험의 대표적인 블롯을 나타내었다. 밴드 강도는 밀도측정법에 의해 정량화되고 β-액틴으로 정규화되었다(*** p < 0.001는 LPS 처리된 군과 비교).Figure 4 shows the effect of compound 1 in which R 1 is H on the activation of the MAPK pathway in LPS-induced BV2 and RAW264.7 cells. Cells were pretreated with compound 1 (1.0, 2.0 and 4.0 μM) with R 1 of H for 3 h, followed by stimulation with LPS for 1 h. The levels of phosphorylated-ERK (p-ERK), phosphorylated-JNK (p-JNK) and phosphorylated-p38 MAPK (p-p38 MAPK) were determined by Western blot analysis. Representative blots of three independent experiments are shown. Band intensities were quantified by densitometry and normalized to β-actin ( *** p < 0.001 compared to LPS treated group).
도 5는 PTP1B의 억제에서 화합물 3(A) 및 화합물 4(B)에 대한 라인웨버-버크도면(Lineweaver-Burk plot)을 도시한다. 데이터는 3개의 실험의 평균값 ±SD를 나타낸다. 화합물 3 및 화합물 4의 농도(μM)가 표시된다.FIG. 5 shows Lineweaver-Burk plots for compound 3 (A) and compound 4 (B) in inhibition of PTP1B. Data represent mean ± SD of three experiments. Concentrations (μM) of compound 3 and compound 4 are indicated.
발명의 상세한 설명 및 바람직한 구현예DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.
본 발명의 일 실시예에서, 해양 유래 진균 페니실리움 글라브룸(Penicillium glabrum) SF-7123(기탁번호: KCTC 14169BP)의 추출물로부터 화합물 1 내지 4의 대사체를 분리하고 그 구조를 확인하였다. 또한, 상기 대사체(화합물 1 내지 4)가 RAW264.7 대식세포 및 BV2 미세아교세포에서 NO, 프로스타글란딘 E2(PGE2), 유도성 산화질소 효소(iNOS) 및 시클로옥시게나아제-2(COX-2) 등과 같은 염증 촉진성(pro-inflammatory) 매개체의 생성 및 PTP1B의 발현을 억제시킴으로써, 염증성 질환 또는 당뇨의 예방 또는 치료 효과를 갖는 것을 확인하였다. In one embodiment of the present invention, the metabolites of compounds 1 to 4 were isolated from the extract of the marine-derived fungus Penicillium glabrum SF-7123 (Accession No.: KCTC 14169BP), and the structures thereof were confirmed. In addition, the metabolites (Compounds 1 to 4) were NO, prostaglandin E 2 (PGE 2 ), inducible nitric oxide enzyme (iNOS) and cyclooxygenase-2 (COX) in RAW264.7 macrophages and BV2 microglia. -2), and the like, by inhibiting the production of pro-inflammatory mediators and the expression of PTP1B, it was confirmed that it has a preventive or therapeutic effect on inflammatory diseases or diabetes.
따라서, 본 발명은 일 관점에서 페니실리움 글라브룸(Penicillium glabrum) SF-7123의 추출물을 포함하는 항염증 또는 항당뇨용 약학 조성물에 관한 것이다.Accordingly, the present invention in one aspect relates to a pharmaceutical composition for anti-inflammatory or antidiabetic comprising an extract of Penicillium glabrum SF-7123.
본 발명에 있어서, 상기 추출물은 하기 화학식 1 내지 화학식 4로 구성된 군에서 선택되는 하나 이상의 대사체를 포함하는 항염증 또는 항당뇨용 약학 조성물에 관한 것이다.In the present invention, the extract relates to an anti-inflammatory or anti-diabetic pharmaceutical composition comprising one or more metabolites selected from the group consisting of the following Chemical Formulas 1 to 4.
[화학식 1][Formula 1]
Figure PCTKR2021005622-appb-I000013
Figure PCTKR2021005622-appb-I000013
[화학식 2][Formula 2]
Figure PCTKR2021005622-appb-I000014
Figure PCTKR2021005622-appb-I000014
[화학식 3][Formula 3]
Figure PCTKR2021005622-appb-I000015
Figure PCTKR2021005622-appb-I000015
[화학식 4][Formula 4]
Figure PCTKR2021005622-appb-I000016
Figure PCTKR2021005622-appb-I000016
여기서, 상기 R1 내지 R9는 각각 독립적으로 H, 직쇄형 또는 분지쇄형의 C1-10 알킬, C1-10 알콕시, C3-10 사이클로알킬, C2-10 알케닐 또는 C6-10 아릴이며, 바람직하게는 H, CH3 또는 CH2CH3이다.Here, R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 aryl, preferably H, CH 3 or CH 2 CH 3 .
C1-10 알킬기는 탄소수 1~10의 직쇄형 또는 분지쇄형의 포화 탄화수소기를 의미하고, 예를 들어, 메틸, 에틸, 프로필, 이소프로필, n-부틸, sec-부틸, tert-부틸, 이소부틸, n-펜틸, n-헥실, 1-에틸프로필, 2,2-디메틸프로필 등을 들 수 있다.C 1-10 alkyl group means a linear or branched saturated hydrocarbon group having 1 to 10 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl , n-pentyl, n-hexyl, 1-ethylpropyl, 2,2-dimethylpropyl, and the like.
C1-10 알콕시기는 탄소수 1*10의 직쇄형 또는 분지쇄형의 알킬옥시기를 의미하고, 예를 들어, 메톡시, 에톡시, 프로폭시, 프로프-2-옥시, 부톡시, 부트-2-옥시, 메틸프로프-2-옥시, 이소프로폭시, tert-부톡시 등을 들 수 있다.C 1-10 alkoxy group means a linear or branched alkyloxy group having 1*10 carbon atoms, for example, methoxy, ethoxy, propoxy, prop-2-oxy, butoxy, but-2- oxy, methylprop-2-oxy, isopropoxy, tert-butoxy, and the like.
C3-10 사이클로알킬기는 탄소수 3*10의 포화 탄화수소 고리를 의미하고, 사이클로프로필, 사이클로부틸, 사이클로펜틸, 사이클로헥실, 사이클로헵틸, 사이클로옥틸 등으로 예시되는 모노사이클로알킬기 외에, 폴리사이클로알킬기, 예를 들어, 비사이클로알킬기, 트리사이클로알킬기 등도 포함되며, 비사이클로알킬기로는, 노르보르닐, 예를 들어, exo-2-노르보르닐, endo-2-노르보르닐, 3-피나닐, 비사이클로[3.1.0]헥실기, 비사이클로[2.2.1]헵틸기, 비사이클로[2.2.2]옥토-2-일기 등, 트리사이클로알킬기로는 아다만틸, 예를 들어, 1-아다만틸기, 2-아다만틸 등을 들 수 있다.C 3-10 cycloalkyl group means a saturated hydrocarbon ring having 3 * 10 carbon atoms, and in addition to monocycloalkyl groups exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like, polycycloalkyl groups, such as For example, a bicycloalkyl group, tricycloalkyl group, etc. are also included, and the bicycloalkyl group includes norbornyl, for example, exo-2-norbornyl, endo-2-norbornyl, 3-pinanyl, bi A cyclo[3.1.0]hexyl group, a bicyclo[2.2.1]heptyl group, a bicyclo[2.2.2]octo-2-yl group, etc., a tricycloalkyl group includes adamantyl, for example 1-adaman A tyl group, 2-adamantyl, etc. are mentioned.
C2-10 알케닐기는 하나 이상의 이중 결합을 함유하고 2 내지 10개의 탄소 원자를 갖는 직쇄형 또는 분지쇄형 탄화수소 라디칼, 예를 들면, 에테닐, 2-프로페닐, 3-부테닐, 2-부테닐, 2-펜테닐, 3-펜테닐, 3-메틸-2-부테닐 또는 3-헥세닐 등을 의미한다.A C 2-10 alkenyl group contains at least one double bond and is a straight or branched chain hydrocarbon radical having from 2 to 10 carbon atoms, for example, ethenyl, 2-propenyl, 3-butenyl, 2-part tenyl, 2-pentenyl, 3-pentenyl, 3-methyl-2-butenyl or 3-hexenyl and the like.
C6-10 아릴기는 탄소수 6*10의 아릴기를 의미하고, 예를 들어 페닐기, 나프틸기, 안트릴기, 페난트릴기를 들 수 있다.The C 6-10 aryl group means an aryl group having 6*10 carbon atoms, and examples thereof include a phenyl group, a naphthyl group, an anthryl group, and a phenanthryl group.
본 발명에 있어서, R1이 CH3인 화학식 1의 대사체는 9-O-메틸뉴크로메닌(9-O-methylneuchromenin)으로 정의되며, R1이 H인 화학식 1의 대사체는 뉴크로메닌(neuchromenin)으로 정의되고, 용어 "화합물 1"은 상기 화학식 1에서 R1이 CH3 또는 H인 대사체를 의미한다.In the present invention, R 1 is CH 3 Metabolites of the formula (1) 9- O - is defined as methyl, New chroman menin (9- O -methylneuchromenin), the R 1 is H The metabolite of Formula 1 is defined as neuchromenin, and the term "Compound 1" refers to a metabolite in which R 1 is CH 3 or H in Formula 1 above.
또한, R2 내지 R4가 각각 CH3인 화학식 2의 대사체는 아스테릭산(asterric acid)으로 정의되며, 용어 "화합물 2"는 상기 화학식 2에서 R2 내지 R4가 각각 CH3인 대사체를 의미한다.In addition, metabolites of R 2 to the formula II R 4 is CH 3, respectively, asterisks acid is defined as (asterric acid), the term "compound 2" the metabolic the R 2 to R 4 CH 3, respectively in the above formula (2) means body.
또한, R5가 CH3인 화학식 3의 대사체는 믹소트리친 C(myxotrichin C)로 정의되며, 용어 "화합물 3"은 상기 화학식 3에서 R5가 CH3인 대사체를 의미한다.In addition, the metabolite of Formula 3 in which R 5 is CH 3 is defined as myxotrichin C (myxotrichin C), and the term “Compound 3” refers to a metabolite in which R 5 is CH 3 in Formula 3 above.
또한, R6 내지 R9가 각각 CH3인 화학식 4의 대사체는 데옥시푸니콘(deoxyfunicone)으로 정의되며, 용어 "화합물 4"는 상기 화학식 4에서 R6 내지 R9가 각각 CH3인 대사체를 의미한다.Also, R 6 to R of 9 is CH 3, each metabolite of formula (4) is to be defined by the oxy Fu Nikon (deoxyfunicone), the term "compound 4" is a metabolic R 6 to R 9 is CH 3, respectively in the above formula (4) means body.
본 발명에서 용어 "항염증"은 염증을 억제하거나 감소시키는 작용을 의미한다.In the present invention, the term "anti-inflammatory" refers to the action of inhibiting or reducing inflammation.
본 발명에서 용어 "염증"이란, 어떤 자극에 대한 생체조직의 방어반응의 하나로, 각종 유해한 자극에 의한 상해를 제거하여 원래의 상태로 회복하려는 생체방어 기전이다. 염증의 자극에는, 감염 혹은 화학적, 물리적 자극이 있으며, 염증의 과정은 급성과 만성 염증의 2가지로 나눌 수 있다. 급성염증은 수일 이내의 단기적인 반응이며, 혈장성분이나 혈구 등이 미소순환계를 게재하여 이물 제거에 관련한다. 만성염증은 지속시간이 길며, 조직의 증식 등이 보여진다.In the present invention, the term "inflammation" is one of the defensive responses of biological tissues to certain stimuli, and is a biological defense mechanism that seeks to recover to its original state by removing injuries caused by various harmful stimuli. Inflammation stimuli include infection or chemical and physical stimuli, and the inflammatory process can be divided into acute and chronic inflammation. Acute inflammation is a short-term reaction within a few days, and plasma components or blood cells are involved in the removal of foreign substances by opening the microcirculation system. Chronic inflammation has a long duration, and tissue proliferation is seen.
본 발명에 있어서, 항염증 또는 항당뇨용 약학 조성물은 항염증 활성을 가짐으로써 염증성 질환의 예방, 치료 또는 개선에 이용될 수 있다.In the present invention, the anti-inflammatory or anti-diabetic pharmaceutical composition can be used for prevention, treatment or improvement of inflammatory diseases by having anti-inflammatory activity.
본 발명에 있어서 "염증성 질환"은 염증을 주 병변으로 하는 질병을 총칭하는 것이다.In the present invention, "inflammatory disease" is a generic term for diseases in which inflammation is the main lesion.
예를 들어, 각종 암, 부종, 알레르기, 천식, 결막염, 치주염, 중이염, 인후염, 편도염, 폐렴, 위궤양, 위염, 크론병, 대장염, 치질, 통풍, 강직성 척추염, 류마티스성 열, 루푸스, 섬유근통 (fibromyalgia), 건선 관절염, 골관절염, 류마티스성 관절염, 견관절주위염, 건염, 건초염, 근육염, 간염, 방광염, 신장염, 쇼그렌 증후군(sjogren's syndrome) 중증근무력증, 다발성 경화증 피부염, 알러지, 비염, 간직성 척추염, 건주위염, 제1형 당뇨병, 피부경화증(scleroderma), 퇴행성 신경질환, 제2형 당뇨병, 규폐증, 죽상동맥경화증, 백반증, 결막염 및 자가면역 질환으로 구성된 군으로부터 선택되는 어느 하나에 해당할 수 있으나, 이에 제한되는 것은 아니다.For example, various cancers, edema, allergy, asthma, conjunctivitis, periodontitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia ), psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, tendinitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, myasthenia gravis, multiple sclerosis dermatitis, allergies, rhinitis, salivary spondylitis, peritenitis, It may correspond to any one selected from the group consisting of type 1 diabetes, scleroderma, neurodegenerative disease, type 2 diabetes, silicosis, atherosclerosis, vitiligo, conjunctivitis and autoimmune diseases, but is limited thereto it is not
본 발명에서 용어 "추출물"은 상기 페니실리움 글라브룸(Penicillium glabrum) SF-7123로부터 분리된 항염증 활성을 가지는 물질을 말한다. 또한 본 발명에서 추출물은 추출액뿐만 아니라 이의 건조 분말 또는 이를 이용하여 제형화된 모든 형태를 포함하는 의미로 사용된다. 본 발명에서 추출물은 극성, 비극성, 물, 유기 용매 또는 이의 혼합 용매를 사용하여 추출할 수 있으며, 바람직하게는 유기 용매를 사용하여 추출할 수 있다. 추출한 액은 액체 형태로 사용하거나 또는 농축 및/또는 건조하여 사용할 수 있다. 상기 유기용매는 탄소수 1 내지 4의 무수 또는 함수 저급알코올(메탄올, 에탄올, 이소프로판올, 부탄올 등), 에틸렌, 아세톤, 헥산, 에테르, 클로로포름, 에틸아세테이트, 부틸아세테이트, 디클로로메탄, N, N-디메틸 포름아미드(DMF), 디메틸설폭사이드(DMSO), 1,3-부틸렌글리콜, 프로필렌글리콜 또는 이들의 혼합용매 등을 사용할 수 있으며, 추출물의 유효 성분이 파괴되지 않거나 최소화된 조건에서 실온 또는 가온하여 추출할 수 있다. 추출하는 유기용매에 따라 추출물의 유효성분의 추출정도와 손실정도가 차이가 날 수 있으므로, 알맞은 유기용매를 선택하여 사용하도록 한다. 추출 방법은 특별히 제한되지 않고, 예를 들어 냉침 추출, 초음파 추출, 환류 냉각 추출 등이 있다. 여과는 추출액으로부터 부유하는 고체 입자를 제거하는 과정으로, 면, 나일론 등을 이용하여 입자를 걸러 내거나 한외여과, 냉동여과법, 원심분리법 등을 사용할 수 있으나, 이에 제한되지 않는다. 또한, 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 크로마토그래피)에 의한 분리 과정을 추가로 포함할 수 있다. 여액을 건조하는 단계는 동결건조, 진공건조, 열풍건조, 분무건조, 감압건조, 포말건조, 고주파건조, 적외선건조 등을 포함하나 이에 제한되지 않는다. 경우에 따라, 최종 건조된 추출물을 분쇄하는 공정을 추가할 수 있다.In the present invention, the term "extract" refers to a substance having anti-inflammatory activity isolated from the Penicillium glabrum SF-7123. In addition, in the present invention, the extract is used in the sense of including not only the extract but also its dry powder or all forms formulated using the same. In the present invention, the extract may be extracted using polar, non-polar, water, an organic solvent, or a mixed solvent thereof, and preferably may be extracted using an organic solvent. The extracted liquid may be used in liquid form or may be used after concentration and/or drying. The organic solvent is an anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, isopropanol, butanol, etc.), ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylform Amide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixture thereof can be used, and the extract is extracted by heating at room temperature or under conditions in which the active ingredient of the extract is not destroyed or minimized. can do. Depending on the organic solvent to be extracted, the degree of extraction and loss of the active ingredient of the extract may be different, so an appropriate organic solvent should be selected and used. The extraction method is not particularly limited, and examples thereof include cold extraction, ultrasonic extraction, and reflux cooling extraction. Filtration is a process of removing suspended solid particles from the extract, and may use cotton, nylon, etc. to filter out particles, ultrafiltration, cryofiltration, centrifugation, etc., but is not limited thereto. In addition, a separation process by various chromatography (chromatography according to size, charge, hydrophobicity or affinity) may be further included. Drying the filtrate includes, but is not limited to, freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, foam drying, high frequency drying, infrared drying, and the like. In some cases, a process of pulverizing the final dried extract may be added.
본 발명에 있어서, 상기 항염증 또는 항당뇨용 약학 조성물은 하나 이상의 하기 특징을 가지는 것을 특징으로 할 수 있다.In the present invention, the anti-inflammatory or anti-diabetic pharmaceutical composition may be characterized in having one or more of the following characteristics.
1) 산화질소(NO) 및 프로스타글란딘 E2(PGE2)의 생성 억제;1) inhibition of the production of nitric oxide (NO) and prostaglandin E 2 (PGE 2 );
2) NF-κB(nuclear factor kappa B) 및 MAPK(mitogen-activated protein kinase)의 불활성화;2) inactivation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK);
3) 유도성 산화질소 효소(inducible nitric oxide synthase; iNOS) 및 시클로옥시게나아제-2(COX-2)의 발현 억제;3) inhibition of expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2);
4) IL-1β, IL-6 및 TNF-α의 발현 억제; 및4) inhibition of expression of IL-1β, IL-6 and TNF-α; and
5) PTP1B(protein tyrosine phosphate 1B)의 발현 억제.5) Inhibition of expression of PTP1B (protein tyrosine phosphate 1B).
본 발명에 있어서, 상기 항염증 또는 항당뇨용 약학 조성물은 산화질소(NO) 및 프로스타글란딘 E2(PGE2)의 생성 억제 특성을 갖는 것을 특징으로 할 수 있다.In the present invention, the anti-inflammatory or anti-diabetic pharmaceutical composition is nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) It may be characterized in that it has a production inhibitory property.
본 발명의 일 실시예에서, 화학식 1 내지 4의 화합물이 NO 및 PGE2의 과잉 생성을 억제하는 것을 확인하였다(표 2). NO는 다양한 면역 세포에서 생성되는 세포 내 매개체인 작은 분자이며, 염증 증상의 생리학적 및 병리학적 상태에 중추적인 역할을 한다. 또한, PGE2는 면역 반응과 염증 반응을 조절할 수 있다.In one embodiment of the present invention, it was confirmed that the compounds of Formulas 1 to 4 inhibit the excessive production of NO and PGE 2 (Table 2). NO is a small molecule that is an intracellular mediator produced by various immune cells and plays a pivotal role in the physiological and pathological conditions of inflammatory conditions. In addition, PGE 2 can modulate immune and inflammatory responses.
본 발명에서 용어 "산화질소(NO)"는 세포 내 염증반응 유발시 산화질소 합성효소에 의해 생성량이 증가하는 물질로, 염증반응의 지표가 되는 분자이다. 신경계에서 산화질소는 신경세포에 존재하는 신경계 산화질소 합성효소(NOS)에 의하여 합성된다. 상기 합성된 산화질소는 뇌세포에서 cGMP의 생성을 증가시키고, 이로 인하여 외부로부터 인지한 정보를 오랫동안 저장하는 기능을 수행한다. NO는 자유 라디칼로 생리학적, 병리학적 과정에 관련된 것으로 알려져 있다. NO는 산화질소 합성효소에 의해 엘-아르기닌(L-Arginine) 산화에 의해 합성된다(Atkan, et al., 75: 639-653, 2004).In the present invention, the term “nitric oxide (NO)” is a substance whose amount is increased by nitric oxide synthase when inducing an inflammatory reaction in a cell, and is a molecule that is an indicator of the inflammatory response. In the nervous system, nitric oxide is synthesized by the nervous system nitric oxide synthase (NOS) present in nerve cells. The synthesized nitric oxide increases the production of cGMP in brain cells, thereby performing a function of storing information recognized from the outside for a long time. NO is a free radical and is known to be involved in physiological and pathological processes. NO is synthesized by L-Arginine oxidation by nitric oxide synthase (Atkan, et al., 75: 639-653, 2004).
본 발명에서 용어 "프로스타글란딘 E2(PGE2)"는 프로스타글란딘 엔도페록시드 합성효소라고 불리는 COX-2에 의해 염증 부위에서 생성되는 염증 매개체이다. PGE2는 많은 심장혈관 질환, 관절염, 염증성 장 질환 및 만성 위궤양을 포함하는 만성 염증성 질환과 관련되어 있다(St-Onge, M. et al., Biochim.Biophys.Acta. 1771:1235-1245, 2007; Turini, M.E. et al., Annu.Rev.Med. 53:35-57, 2002; Rocca, B. et al., Int.Immunopharmacol. 2: 603-630, 2002; Singh, V.P. et al., Pharmacology 72:77-84, 2004).In the present invention, the term "prostaglandin E 2 (PGE 2 )" is an inflammatory mediator produced at the site of inflammation by COX-2 called prostaglandin endoperoxide synthetase. PGE 2 is associated with many cardiovascular diseases, arthritis, inflammatory bowel disease and chronic inflammatory diseases including chronic gastric ulcer (St-Onge, M. et al., Biochim. Biophys. Acta. 1771:1235-1245, 2007). Turini, ME et al., Annu. Rev. Med. 53:35-57, 2002; Rocca, B. et al., Int. Immunopharmacol. 2: 603-630, 2002; Singh, VP et al., Pharmacology 72:77-84, 2004).
본 발명에 있어서, 상기 항염증 또는 항당뇨용 약학 조성물은 NF-κB(nuclear factor kappa B) 및 MAPK(mitogen-activated protein kinase)를 불활성화하는 것을 특징으로 할 수 있다.In the present invention, the anti-inflammatory or anti-diabetic pharmaceutical composition may be characterized in that it inactivates NF-κB (nuclear factor kappa B) and MAPK (mitogen-activated protein kinase).
본 발명의 일 실시예에서, LPS로 유도된 세포에서 R1이 H인 화합물 1이 NF-κB의 활성화를 억제하고(도 3) p38 MAPK 인산화를 억제함(도 4)을 확인하였다.In an embodiment of the present invention, it was confirmed that Compound 1 in which R 1 is H in LPS-induced cells inhibited the activation of NF-κB ( FIG. 3 ) and inhibited p38 MAPK phosphorylation ( FIG. 4 ).
본 발명에서, 핵 인자-카파 B(nuclear factor-kappa B, NF-κB) 및 미토겐-활성화 단백질 키나아제(mitogen-activated protein kinase, MAPK)는 염증 촉진성(pro-inflammatory) 사이토카인 및 매개체의 발현 조절에 관여하는 필수적인 전사인자이며, 따라서 염증반응에서 중요한 역할을 한다. NF-κB는 일반적으로 억제제 카파 B-α(inhibitor kappa B-α, IκB-α)에 의해 세포질에서 정지된다. 일단 LPS와 같은 염증 자극제에 의해 자극되면, IκB는 인산화되고 NF-κB 이량체(p50 및 p65)를 핵으로 전좌시켜 iNOS, COX-2, NO, PGE2, TNF-α, IL-1β 및 IL-6와 같은 염증성 유전자의 전사를 초래한다. R1이 H인 화합물 1은 IκB-α의 인산화 및 분해를 역전시키고(도 3A 및 3B), NF-κB 이량체(p50 및 p65)의 핵 전좌를 차단하였으며(도 3C, 3D, 3E 및 3F), LPS-자극된 BV2 및 RAW264.7세포에서 p65 서브유닛의 DNA 결합 활성을 감소시켰다(도 3G 및 3H).In the present invention, nuclear factor-kappa B (nuclear factor-kappa B, NF-κB) and mitogen-activated protein kinase (MAPK) are pro-inflammatory cytokines and mediators It is an essential transcription factor involved in expression regulation and thus plays an important role in the inflammatory response. NF-κB is normally arrested in the cytoplasm by the inhibitor kappa B-α (IκB-α). Once stimulated by an inflammatory stimulant such as LPS, IκB is phosphorylated and translocates NF-κB dimers (p50 and p65) to the nucleus, resulting in iNOS, COX-2, NO, PGE 2 , TNF-α, IL-1β and IL It results in the transcription of inflammatory genes such as -6. Compound 1, in which R 1 is H, reversed phosphorylation and degradation of IκB-α ( FIGS. 3A and 3B ) and blocked nuclear translocation of NF-κB dimers (p50 and p65) ( FIGS. 3C, 3D, 3E and 3F). ), decreased the DNA binding activity of the p65 subunit in LPS-stimulated BV2 and RAW264.7 cells ( FIGS. 3G and 3H ).
본 발명에서 용어 "MAPK"는 성장인자 등이 세포막에 위치한 수용체를 활성화하려면 이 신호를 세포막으로부터 핵으로 전달함으로써 세포의 성장과 분화를 조절하는 주요 신호전달계이다. MAPK는 세포 사멸, 세포 성장, 분화, 증식 및 면역반응과 같은 다양한 세포 조건에서 중요한 역할을 한다(Liu, Y., et al., Nat. Rev. Immunol. 2007, 7, 202-212.). MAPK는 세포 외 신호 조절 키나아제 1 및 2(ERK1/ERK2), c-Jun N-말단 키나아제(JNK) 및 p38 MAPK를 포함하는 세 가지의 주요 신호전달 경로로 구성된다. 특히, p38 MAPK는 염증반응을 조절하는 MAPK 중 하나이므로, 항염증 치료의 표적으로 간주된다. R1이 H인 화합물 1을 사용한 전처리(pre-treatment)는 BV2 및 RAW264.7세포에서 LPS-유도된 p38 MAPK 인산화를 억제했지만, ERK 및 JNK MAPK에는 영향을 미치지 않았다(도 4). In the present invention, the term "MAPK" is a major signal transduction system that regulates cell growth and differentiation by transmitting this signal from the cell membrane to the nucleus in order to activate a receptor located on the cell membrane, such as a growth factor. MAPKs play important roles in a variety of cellular conditions, such as apoptosis, cell growth, differentiation, proliferation and immune responses (Liu, Y., et al., Nat. Rev. Immunol . 2007 , 7 , 202-212.). MAPK consists of three major signaling pathways, including extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2), c-Jun N-terminal kinase (JNK) and p38 MAPK. In particular, p38 MAPK is one of the MAPKs regulating the inflammatory response, so it is considered a target for anti-inflammatory treatment. Pre-treatment with compound 1 in which R 1 is H inhibited LPS-induced p38 MAPK phosphorylation in BV2 and RAW264.7 cells, but did not affect ERK and JNK MAPK ( FIG. 4 ).
본 발명에 있어서, 상기 항염증 또는 항당뇨용 약학 조성물은 유도성 산화질소 효소(inducible nitric oxide synthase; iNOS) 및 시클로옥시게나아제-2(COX-2)의 발현 억제 특성을 가지는 것을 특징으로 할 수 있다.In the present invention, the anti-inflammatory or anti-diabetic pharmaceutical composition is characterized in that it has an expression inhibitory property of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). can
본 발명의 일 실시예에서, R1이 H인 화합물 1은 LPS로 자극된 BV2 및 RAW264.7세포 둘 다에서 용량-의존적 방식으로 iNOS 및 COX-2의 단백질 발현을 약화시켰다(도 2). iNOS 및 COX-2는 염증 촉진성 매개체로서, iNOS는 NO를 생성하는 염증성 분자이며, COX-2는 PGE2를 생성한다. iNOS 및 COX-2의 과도한 활성 증가는 다양한 염증질환의 병인일 수 있다.In one embodiment of the present invention , compound 1 in which R 1 is H attenuated the protein expression of iNOS and COX-2 in a dose-dependent manner in both BV2 and RAW264.7 cells stimulated with LPS ( FIG. 2 ). iNOS and COX-2 are pro-inflammatory mediators, iNOS is an inflammatory molecule that produces NO, and COX-2 produces PGE 2 . Excessive increased activity of iNOS and COX-2 may be the etiology of various inflammatory diseases.
본 발명에서 용어 "COX-2"는 염증반응과 관련된 단백질인 프로스타글라딘을 생성하는데 관여하는 효소로, 세포내 COX-2 발현 수준의 증가는 염증반응이 진행되고 있음을 나타내는 지표가 될 수 있다.In the present invention, the term "COX-2" is an enzyme involved in the production of prostaglandin, a protein related to an inflammatory response, and an increase in intracellular COX-2 expression level can be an indicator indicating that an inflammatory response is in progress. have.
본 발명에 있어서, 상기 항염증 또는 항당뇨용 약학 조성물은 IL-1β, IL-6 및 TNF-α의 발현 억제 특성을 가지는 것을 특징으로 할 수 있다.In the present invention, the anti-inflammatory or anti-diabetic pharmaceutical composition may be characterized in that it has the properties of inhibiting the expression of IL-1β, IL-6 and TNF-α.
본 발명의 일 실시예에서, R1이 H인 화합물 1은 BV2세포가 12시간 동안 LPS(1㎍/mL)로 활성화될 때, IL-1β, TNF-α 및 IL-6의 mRNA 발현을 효과적으로 감소시켰다(도 1). 이러한 결과는 R1이 H인 화합물 1이 전사 수준(transcriptional level)에서 전 염증성 사이토카인의 유전자 발현을 약화시킨다는 것을 의미한다. TNF-α, IL 등과 같은 전 염증성 사이토카인의 과잉 생성은 염증질환의 발병에 기여한다.In one embodiment of the present invention , compound 1 wherein R 1 is H effectively inhibits the mRNA expression of IL-1β, TNF-α and IL-6 when BV2 cells are activated with LPS (1 μg/mL) for 12 hours. decreased (Fig. 1). These results suggest that compound 1, in which R 1 is H, attenuates gene expression of pro-inflammatory cytokines at the transcriptional level. Excessive production of pro-inflammatory cytokines such as TNF-α and IL contributes to the pathogenesis of inflammatory diseases.
본 발명에 있어서, 상기 항염증 또는 항당뇨용 약학 조성물은 PTP1B의 발현 억제 특성을 가지는 것을 특징으로 할 수 있다.In the present invention, the anti-inflammatory or anti-diabetic pharmaceutical composition may be characterized in that it has a property of inhibiting the expression of PTP1B.
본 발명의 일 실시예에서, 화합물 3 및 4는 각각 IC50값이 19.2μM 및 24.3μM인 용량-의존적 방식으로 활성 PTP1B 효소를 억제하는 것으로 나타났다(표 3).In one embodiment of the present invention, compounds 3 and 4 were shown to inhibit the active PTP1B enzyme in a dose-dependent manner with IC 50 values of 19.2 μM and 24.3 μM, respectively (Table 3).
본 발명에 있어서, 상기 약학 조성물은 약학적으로 허용되는 담체, 부형제 또는 희석제를 추가로 포함하는 것을 특징으로 할 수 있다.In the present invention, the pharmaceutical composition may be characterized in that it further comprises a pharmaceutically acceptable carrier, excipient or diluent.
상기 조성물에 포함될 수 있는 담체, 부형제 및 희석제는 비제한적으로 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시 벤조에이트, 프로필히드록시 벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등이 있다. 상기 조성물을 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다.Carriers, excipients and diluents that may be included in the composition include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil. When formulating the composition, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.
본 발명에 따른 약학 조성물은 통상의 방법에 따라 다양한 형태로 제형화하여 사용될 수 있다. 적합한 제형으로는 정제, 환제, 산제, 과립제, 당의정, 경질 또는 연질의 캡슐제, 용액제, 현탁제 또는 유화액제, 주사제, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액 등이 있으나, 이에 한정되는 것은 아니다.The pharmaceutical composition according to the present invention may be formulated and used in various forms according to conventional methods. Suitable dosage forms include tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, oral dosage forms such as aerosols, external preparations, suppositories, and sterile injection solutions. The present invention is not limited thereto.
본 발명에 따른 약학 조성물은 약학적으로 불활성인 유기 또는 무기 담체를 이용하여 적합한 제형으로 제조할 수 있다. 즉, 제형이 정제, 코팅된 정제, 당의정 및 경질 캡슐제인 경우 락토스, 수크로스, 전분 또는 그 유도체, 탈크, 칼슘 카보네이트, 젤라틴, 스테아르산 또는 그 약학적으로 허용가능한 염을 포함할 수 있다. 또한, 제형이 연질 캡슐제인 경우에는 식물성 오일, 왁스, 지방, 반고체 및 액체의 폴리올을 포함할 수 있다. 또한, 제형이 용액 또는 시럽 형태인 경우, 물, 폴리올, 글리세롤, 및 식물성 오일 등을 포함할 수 있다.The pharmaceutical composition according to the present invention can be prepared in a suitable dosage form using a pharmaceutically inert organic or inorganic carrier. That is, when the formulation is a tablet, a coated tablet, a dragee, and a hard capsule, it may contain lactose, sucrose, starch or derivatives thereof, talc, calcium carbonate, gelatin, stearic acid or a pharmaceutically acceptable salt thereof. In addition, when the formulation is a soft capsule, it may contain vegetable oils, waxes, fats, semi-solid and liquid polyols. In addition, when the formulation is in the form of a solution or syrup, water, polyol, glycerol, and vegetable oil may be included.
상기 "약학적으로 허용가능한 염"이란, 화합물이 투여되는 유기체에 심각한 자극을 유발하지 않고 화합물의 생물학적 활성과 물성들을 손상시키지 않는 화합물의 제형을 의미한다. 상기 약학적 염은, 약학적으로 허용되는 음이온을 함유하는 무독성 산부가염을 형성하는 산, 예를 들어, 염산, 황산, 질산, 인산, 브롬화수소산, 요오드화수소산 등과 같은 무기산, 타타르산, 포름산, 시트르산, 아세트산, 트리클로로아세트산, 트리플로로아세트산, 글루콘산, 벤조산, 락트산, 푸마르산, 말레인산, 살리신산 등과 같은 유기 카본산, 메탄설폰산, 에탄술폰산, 벤젠설폰산, p-톨루엔설폰산 등과 같은 설폰산 등에 의해 형성된 산부가염이 포함된다. 예를 들어, 약학적으로 허용되는 카르복실산 염에는, 리튬, 나트륨, 칼륨, 칼슘, 마그네슘 등에 의해 형성된 금속염 또는 알칼리 토금속 염, 라이신, 아르지닌, 구아니딘 등의 아미노산 염, 디시클로헥실아민, N-메틸-D-글루카민, 트리스(히드록시메틸) 메틸아민, 디에탄올아민, 콜린 및 트리에틸아민 등과 같은 유기염 등이 포함된다. 옥살산 (oxalic)과 같은 산은 약학적으로 허용되는 것은 아니지만 약학적으로 허용되는 염을 얻기 위한 중간체로서, 유용한 염의 제조에 사용될 수 있다. The "pharmaceutically acceptable salt" refers to a formulation of a compound that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound. The pharmaceutical salt is an acid that forms a non-toxic acid addition salt containing a pharmaceutically acceptable anion, for example, an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc., tartaric acid, formic acid, citric acid , organic carbonic acids such as acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, etc., sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. acid addition salts formed with phonic acid and the like are included. For example, pharmaceutically acceptable salts of carboxylic acids include metal salts or alkaline earth metal salts formed with lithium, sodium, potassium, calcium, magnesium, etc., amino acid salts such as lysine, arginine, and guanidine, dicyclohexylamine, N organic salts such as -methyl-D-glucamine, tris(hydroxymethyl)methylamine, diethanolamine, choline and triethylamine, and the like. Acids such as oxalic acid are not pharmaceutically acceptable, but can be used in the preparation of useful salts as intermediates for obtaining pharmaceutically acceptable salts.
본 발명에 따른 약학 조성물은 상기의 담체 외에도 보존제, 안정화제, 습윤제, 유화제, 용해제, 감미제, 착색제, 삼투압 조절제, 산화방지제 등을 더 포함할 수 있다.The pharmaceutical composition according to the present invention may further include a preservative, a stabilizer, a wetting agent, an emulsifier, a solubilizing agent, a sweetener, a colorant, an osmotic pressure regulator, an antioxidant, and the like, in addition to the carrier described above.
본 발명에 따른 약학 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명에 따른 약학 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , sensitivity to drugs, administration time, administration route and excretion rate, duration of treatment, factors including concurrent drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.
본 발명의 조성물은 염증성 질환의 예방 또는 치료에 효과가 있는 것으로 당업자에게 인식될 수 있는 면역요법, 화학요법 및 방사선요법 등과 같은 염증성 질환의 치료법 및 다른 염증성 질환의 치료용 약학 조성물 등과 함께 사용될 수 있다.The composition of the present invention can be used in combination with a pharmaceutical composition for treatment of inflammatory diseases, such as immunotherapy, chemotherapy, and radiation therapy, and pharmaceutical compositions for the treatment of other inflammatory diseases, which can be recognized by those skilled in the art as being effective in the prevention or treatment of inflammatory diseases. .
본 발명의 약학 조성물은 개체에 다양한 경로로 투여될 수 있다. 투여의 방식은, 예를 들면, 피하, 정맥, 근육 또는 자궁 내 경막 또는 뇌혈관 내 주사에 의해 투여될 수 있다. 본 발명의 약학 조성물은 치료할 질환, 투여 경로, 환자의 연령, 성별 및 체중 및 질환의 중등도 등의 여러 관련 인자와 함께, 활성성분인 약물의 종류에 따라 결정된다.The pharmaceutical composition of the present invention may be administered to an individual by various routes. The mode of administration can be, for example, by subcutaneous, intravenous, intramuscular or intrauterine intrathecal or intracerebrovascular injection. The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient, along with several related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease.
본 발명은 다른 관점에서, 페니실리움 글라브룸(Penicillium glabrum) SF-7123의 추출물을 포함하는 염증 또는 당뇨 개선용 식품에 관한 것이다.In another aspect, the present invention relates to a food for improving inflammation or diabetes comprising an extract of Penicillium glabrum SF-7123.
본 발명에 있어서, 상기 추출물은 하기 화학식 1 내지 화학식 4로 구성된 군에서 선택되는 하나 이상의 대사체를 포함하는 염증 또는 당뇨 개선용 식품에 관한 것이다.In the present invention, the extract relates to a food for improving inflammation or diabetes containing one or more metabolites selected from the group consisting of the following Chemical Formulas 1 to 4.
[화학식 1][Formula 1]
Figure PCTKR2021005622-appb-I000017
Figure PCTKR2021005622-appb-I000017
[화학식 2][Formula 2]
Figure PCTKR2021005622-appb-I000018
Figure PCTKR2021005622-appb-I000018
[화학식 3][Formula 3]
Figure PCTKR2021005622-appb-I000019
Figure PCTKR2021005622-appb-I000019
[화학식 4][Formula 4]
Figure PCTKR2021005622-appb-I000020
Figure PCTKR2021005622-appb-I000020
여기서, 상기 R1 내지 R9는 각각 독립적으로 H, 직쇄형 또는 분지쇄형의 C1-10 알킬, C1-10 알콕시, C3-10 사이클로알킬, C2-10 알케닐 또는 C6-10 아릴이며, 바람직하게는 H, CH3 또는 CH2CH3이다.Here, R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 aryl, preferably H, CH 3 or CH 2 CH 3 .
본 발명에서 용어 "식품"은 육류, 소시지, 빵, 초콜릿, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올음료, 비타민 복합제, 건강기능식품 및 건강식품 등이 있으며, 통상적인 의미에서의 식품을 모두 포함한다.In the present invention, the term "food" refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, There are vitamin complexes, health functional foods, and health foods, and includes all foods in the ordinary sense.
상기 건강기능(성) 식품(functional food)이란, 특정보건용 식품(food for special health use, FoSHU)과 동일한 용어로, 영양 공급 외에도 생체조절기능이 효율적으로 나타나도록 가공된 의학, 의료효과가 높은 식품을 의미한다. 여기서 "기능(성)"이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 식품은 당 업계에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조시에는 당 업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한 상기 식품의 제형 또한 식품으로 인정되는 제형이면 제한 없이 제조될 수 있으며, 본 발명에 따른 건강기능식품은 분말, 과립, 정제, 캡슐 또는 음료의 형태일 수 있다.The functional food (functional food) is the same term as food for special health use (FoSHU), and in addition to supplying nutrition, it is processed to efficiently exhibit bioregulatory functions and has high medical effects. means food. Here, "function (sex)" means to obtain a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body. The food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, the formulation of the food may be manufactured without limitation as long as it is a formulation recognized as a food, and the health functional food according to the present invention may be in the form of powder, granule, tablet, capsule or beverage.
상기 건강식품(health food)은 일반식품에 비해 적극적인 건강유지나 증진 효과를 가지는 식품을 의미하고, 건강보조식품(health supplement food)은 건강보조 목적의 식품을 의미한다. 경우에 따라, 건강 기능 식품, 건강 식품, 건강보조식품의 용어는 혼용된다.The health food means a food having an active health maintenance or promotion effect compared to general food, and the health supplement food means a food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and dietary supplement are used interchangeably.
상기 식품조성물은 생리학적으로 허용 가능한 담체를 추가로 포함할 수 있는데, 담체의 종류는 특별히 제한되지 않으며 당해 기술 분야에서 통상적으로 사용되는 담체라면 어느 것이든 사용할 수 있다.The food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used.
또한, 상기 조성물은 식품에 통상 사용되어 냄새, 맛, 시각 등을 향상시킬 수 있는 추가 성분을 포함할 수 있다. 예들 들어, 비타민 A, C, D, E, B1, B2, B6, B12, 니아신(niacin), 비오틴(biotin), 폴레이트(folate), 판토텐산(panthotenic acid) 등을 포함할 수 있다. 또한, 아연(Zn), 철(Fe), 칼슘(Ca), 크롬(Cr), 마그네슘(Mg), 망간(Mn), 구리(Cu), 크륨(Cr) 등의 미네랄을 포함할 수 있다. 또한, 라이신, 트립토판, 시스테인, 발린 등의 아미노산을 포함할 수 있다.In addition, the composition may include additional ingredients that are commonly used in food to improve odor, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included. Also, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr). In addition, it may include amino acids such as lysine, tryptophan, cysteine, and valine.
또한, 상기 조성물은 방부제(소르빈산 칼륨, 벤조산나트륨, 살리실산, 데히드로초산나트륨 등), 살균제(표백분과 고도 표백분, 차아염소산나트륨 등), 산화방지제(부틸히드록시아니졸(BHA), 부틸히드록시톨류엔(BHT) 등), 착색제(타르색소 등), 발색제(아질산 나트륨, 아초산 나트륨 등), 표백제(아황산나트륨), 조미료(MSG 등), 감미료(둘신, 사이클레메이트, 사카린, 나트륨 등), 향료(바닐린, 락톤류 등), 팽창제(명반, D-주석산수소칼륨 등), 강화제, 유화제, 증점제(호료), 피막제, 검기초제, 거품억제제, 용제, 개량제 등의 식품 첨가물(foodadditives)을 포함할 수 있다. 상기 첨가물은 식품의 종류에 따라 선별되고 적절한 양으로 사용될 수 있다.In addition, the composition includes a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), a disinfectant (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), an antioxidant (butylhydroxyanisole (BHA), butylhydroxy Toluene (BHT), etc.), coloring agents (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (MSG, etc.), sweeteners (dulcin, cyclimate, saccharin, sodium, etc.) ), flavorings (vanillin, lactones, etc.), expanding agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (flavors), film agents, gum base agents, foam inhibitors, solvents, food additives such as improving agents ) may be included. The additive may be selected according to the type of food and used in an appropriate amount.
본 발명의 페니실리움 글라브룸 추출물과 함께 식품학적으로 허용가능한 식품 보조 첨가제를 더 포함할 수 있으며, 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 그의 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다.It may further include a food pharmaceutically acceptable food supplement additive together with the Penicillium glabrum extract of the present invention, and may be used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment).
본 발명에서 용어 "개선", "예방" 및 "치료"는 최광의의 개념으로 해석되어야 하며, "개선"이란 질환 또는 하나 이상의 임상적 증상을 일시적/지속적으로 완화시키는 모든 행위를 의미한다. "예방"이란, 질환에 노출되거나 질환에 걸리기 쉬울 수 있으나 질환의 증상을 아직 경험하거나 드러내지 아니한 환자에게서 질환의 임상적 증상 중 하나 이상이 진행되지 아니하도록 하는 것을 의미한다. "치료"란, 질환 또는 이의 하나 이상의 임상적 증상의 발달을 저지 또는 감소시키는 모든 행위를 의미한다.In the present invention, the terms "improvement", "prevention" and "treatment" should be interpreted in the broadest sense, and "improvement" means any action that temporarily/continuously relieves a disease or one or more clinical symptoms. "Prevention" means preventing the progression of one or more of the clinical symptoms of a disease in a patient who may be exposed to or susceptible to the disease but has not yet experienced or exhibited symptoms of the disease. "Treatment" means any action that arrests or reduces the development of a disease or one or more clinical symptoms thereof.
본 발명의 일 실시예에서, SF-7123의 진균 균주를 25℃에서 21일 동안 3%의 NaCl을 함유하는 배지에서 한천 페트리 플레이트 상에서 배양하였다. 배양 배지를 EtOAc로 추출한 후, 여과된 추출물을 진공에서 농축시켜 조추출액(crude extract)을 수득하고, 크로마토그래피 방법을 사용하여 조추출액(crude extract)으로부터 화합물 1 내지 화합물 4를 수득하였다(실시예 3).In one embodiment of the present invention, the fungal strain of SF-7123 was cultured on an agar Petri plate in a medium containing 3% NaCl at 25°C for 21 days. After extracting the culture medium with EtOAc, the filtered extract was concentrated in vacuo to obtain a crude extract, and compounds 1 to 4 were obtained from the crude extract using a chromatography method (Example) 3).
따라서, 본 발명은 또 다른 관점에서, (a) 페니실리움 글라브룸(Penicillium glabrum) SF-7123를 배양하는 단계; (b) 상기 (a) 단계에서 수득된 균주 배양물을 에틸아세테이트(EtOAc)로 추출하는 단계; 및 (c) 상기 (b) 단계에서 얻어진 에틸아세테이트(EtOAc) 추출물을 컬럼 크로마토그래피로 분리하는 단계를 포함하는 하기 화학식 1 내지 화학식 4의 대사체의 분리방법에 관한 것이다:Accordingly, the present invention in another aspect, (a) Penicillium glabrum ( Penicillium glabrum ) culturing SF-7123; (b) extracting the strain culture obtained in step (a) with ethyl acetate (EtOAc); and (c) separating the ethyl acetate (EtOAc) extract obtained in step (b) by column chromatography.
[화학식 1][Formula 1]
Figure PCTKR2021005622-appb-I000021
Figure PCTKR2021005622-appb-I000021
[화학식 2][Formula 2]
Figure PCTKR2021005622-appb-I000022
Figure PCTKR2021005622-appb-I000022
[화학식 3][Formula 3]
Figure PCTKR2021005622-appb-I000023
Figure PCTKR2021005622-appb-I000023
[화학식 4][Formula 4]
Figure PCTKR2021005622-appb-I000024
Figure PCTKR2021005622-appb-I000024
여기서, 상기 R1 내지 R9는 각각 독립적으로 H, 직쇄형 또는 분지쇄형의 C1-10 알킬, C1-10 알콕시, C3-10 사이클로알킬, C2-10 알케닐 또는 C6-10 아릴이며, 바람직하게는 H, CH3 또는 CH2CH3이다.Here, R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 aryl, preferably H, CH 3 or CH 2 CH 3 .
본 발명에 있어서, (a) 단계는 페니실리움 글라브룸(Penicillium glabrum) SF-7123를 배양하는 단계로서, 균주 배양은 통상의 미생물이 사용할 수 있는 영양원을 함유하는 배지에서 배양하는 것이 바람직하다. 영양원으로는 종래 곰팡이의 배양에 이용되고 있는 공지의 영양원을 사용할 수 있다. 예를 들어, 탄소원으로는 글루코오스, 물엿, 덱스트린, 전분, 당밀, 동물유, 식물유 등을 사용할 수 있으며, 질소원으로는 밀기울, 대두박, 소맥, 맥아, 면실박, 어박, 콘스팁리커, 육즙, 효모 추출물, 황산암모늄, 질산소다, 요소 등을 사용할 수 있고, 필요에 따라, 식염, 칼륨, 마그네슘, 코발트, 염소, 인산, 황산 및 기타 이온생성을 촉진하는 무기염류를 첨가하면 매우 효과적이다. 배양방법으로는 호기적 조건에서는 진탕 배양 또는 정치배양이 바람직하나 이에 한정되지 않는다.In the present invention, step (a) is a step of culturing Penicillium glabrum SF-7123, and the strain culture is preferably cultured in a medium containing nutrients that can be used by ordinary microorganisms. As the nutrient source, a known nutrient source conventionally used for culturing mold can be used. For example, as a carbon source, glucose, starch syrup, dextrin, starch, molasses, animal oil, vegetable oil, etc. can be used, and as a nitrogen source, wheat bran, soybean meal, wheat, malt, cottonseed meal, fish meal, cornstarch, broth, yeast Extracts, ammonium sulfate, sodium nitrate, urea, etc. can be used, and if necessary, adding salt, potassium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other inorganic salts that promote ion formation is very effective. As a culture method, shaking culture or stationary culture is preferable under aerobic conditions, but is not limited thereto.
배양온도는 상기의 각 조건들에서 배양할 경우 조건에 따라 약간씩 상이하나, 보통 20 내지 37℃에서 배양하는 것이 바람직하고, 25℃에서 배양하는 것이 더욱 바람직하지만, 이에 한정되지 않는다.The culture temperature is slightly different depending on the conditions when culturing under each of the above conditions, but it is usually preferred to culture at 20 to 37° C., more preferably at 25° C., but is not limited thereto.
본 발명은 또 다른 관점에서, 하기 화학식 1 내지 화학식 4로 구성된 군에서 선택되는 하나 이상의 대사체를 포함하는 항염증 또는 항당뇨용 약학 조성물에 관한 것이다:In another aspect, the present invention relates to an anti-inflammatory or anti-diabetic pharmaceutical composition comprising one or more metabolites selected from the group consisting of the following Chemical Formulas 1 to 4:
[화학식 1][Formula 1]
Figure PCTKR2021005622-appb-I000025
Figure PCTKR2021005622-appb-I000025
[화학식 2][Formula 2]
Figure PCTKR2021005622-appb-I000026
Figure PCTKR2021005622-appb-I000026
[화학식 3][Formula 3]
Figure PCTKR2021005622-appb-I000027
Figure PCTKR2021005622-appb-I000027
[화학식 4][Formula 4]
Figure PCTKR2021005622-appb-I000028
Figure PCTKR2021005622-appb-I000028
여기서, 상기 R1 내지 R9는 각각 독립적으로 H, 직쇄형 또는 분지쇄형의 C1-10 알킬, C1-10 알콕시, C3-10 사이클로알킬, C2-10 알케닐 또는 C6-10 아릴이다.Here, R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 is aryl.
본 발명에 있어서, 상기 R1 내지 R9는 각각 독립적으로 H, CH3 또는 CH2CH3일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, R 1 to R 9 may be each independently H, CH 3 or CH 2 CH 3 , but is not limited thereto.
본 발명은 또 다른 관점에서, 하기 화학식 1 내지 화학식 4로 구성된 군에서 선택되는 하나 이상의 대사체를 포함하는 염증 또는 당뇨 개선용 식품에 관한 것이다:In another aspect, the present invention relates to a food for improving inflammation or diabetes comprising one or more metabolites selected from the group consisting of the following Chemical Formulas 1 to 4:
[화학식 1][Formula 1]
Figure PCTKR2021005622-appb-I000029
Figure PCTKR2021005622-appb-I000029
[화학식 2][Formula 2]
Figure PCTKR2021005622-appb-I000030
Figure PCTKR2021005622-appb-I000030
[화학식 3][Formula 3]
Figure PCTKR2021005622-appb-I000031
Figure PCTKR2021005622-appb-I000031
[화학식 4][Formula 4]
Figure PCTKR2021005622-appb-I000032
Figure PCTKR2021005622-appb-I000032
여기서, 상기 R1 내지 R9는 각각 독립적으로 H, 직쇄형 또는 분지쇄형의 C1-10 알킬, C1-10 알콕시, C3-10 사이클로알킬, C2-10 알케닐 또는 C6-10 아릴이다.Here, R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 is aryl.
본 발명에 있어서, 상기 R1 내지 R9는 각각 독립적으로 H, CH3 또는 CH2CH3일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, R 1 to R 9 may be each independently H, CH 3 or CH 2 CH 3 , but is not limited thereto.
본 발명은 또 다른 관점에서, 상기 페니실리움 글라브룸(Penicillium glabrum) SF-7123의 추출물, 상기 대사체 또는 상기 약학 조성물을 투여하는 단계를 포함하는 염증성 질환 또는 당뇨의 예방 또는 치료방법에 관한 것이다.In another aspect, the present invention relates to a method for preventing or treating an inflammatory disease or diabetes comprising administering the extract, the metabolite, or the pharmaceutical composition of the Penicillium glabrum SF-7123. .
본 발명은 또 다른 관점에서, 상기 페니실리움 글라브룸(Penicillium glabrum) SF-7123의 추출물 또는 상기 대사체의 항염증 또는 항당뇨의 용도에 관한 것이다.In another aspect, the present invention relates to the use of the extract of Penicillium glabrum SF-7123 or the metabolite for anti-inflammatory or anti-diabetes.
본 발명은 또 다른 관점에서, 염증성 질환 또는 당뇨의 예방 또는 치료를 위한 상기 페니실리움 글라브룸(Penicillium glabrum) SF-7123의 추출물 또는 상기 대사체의 용도에 관한 것이다.In another aspect, the present invention relates to the use of the extract or the metabolite of Penicillium glabrum SF-7123 for the prevention or treatment of inflammatory diseases or diabetes.
본 발명은 또 다른 관점에서, 염증성 질환 또는 당뇨의 치료를 위한 약제의 제조에 있어 상기 페니실리움 글라브룸(Penicillium glabrum) SF-7123의 추출물 또는 상기 대사체의 사용에 관한 것이다.In another aspect, the present invention relates to the use of the extract or the metabolite of Penicillium glabrum SF-7123 in the manufacture of a medicament for the treatment of inflammatory diseases or diabetes.
본 발명은 또 다른 관점에서, 염증성 질환 또는 당뇨의 예방 또는 개선을 위한 기능성 식품의 제조에 있어 상기 페니실리움 글라브룸(Penicillium glabrum) SF-7123의 추출물 또는 상기 대사체의 사용에 관한 것이다.In another aspect, the present invention relates to the use of the extract or the metabolite of the Penicillium glabrum SF-7123 in the manufacture of a functional food for the prevention or improvement of inflammatory diseases or diabetes.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예1: 일반적인 실험 절차.Example 1: General experimental procedure.
광학 회전은 Jasco P-2000 디지털 편광계를 사용하여 기록되었다. UV 스펙트럼은 Beckman DU 800 UV-가시광선 분광 광도계에서 기록되었다. IR 스펙트럼은 Agilent Cary 630 FT-IR 분광계에서 수득하였다. NMR 스펙트럼(1D 및 2D)은 JEOL JNM ECP-400 분광계(1H의 경우 400MHz 및 13C의 경우 100MHz)를 사용하여 CD3OD 또는 CDCl3에 기록하고 상응하는 잔류 용매 신호(CD3OD: d H 3.30/d C 49.0 및 CDCl3: d H 7.26/d C 77.0)와 관련하여 화학적 이동이 참조되었다. HMQC 및 HMBC 실험은 각각 1 J CH = 140 Hz 및 n J CH = 8 Hz에서 최적화되었다. HRESIMS 데이터는 ESI Q-TOF MS/MS 시스템(AB SCIEX Triple)을 사용하여 수득하였다. TLC는 Kieselgel 60 F254(1.05715; Merck) 또는 RP-18 F254s(Merck) 플레이트에서 수행되었다. 스팟은 10%의 H2SO4 수용액을 분무한 다음 가열함으로써 시각화 하였다. 컬럼 크로마토그래피는 YMC 옥타데실-작용화된 실리카 겔(C18)상에서 수행하였다. Optical rotation was recorded using a Jasco P-2000 digital polarimeter. UV spectra were recorded on a Beckman DU 800 UV-Visible Spectrophotometer. IR spectra were obtained on an Agilent Cary 630 FT-IR spectrometer. NMR spectra (1D and 2D) were recorded in CD 3 OD or CDCl 3 using a JEOL JNM ECP-400 spectrometer (400 MHz for 1 H and 100 MHz for 13 C) and the corresponding residual solvent signal (CD 3 OD: d H 3.30/ d C 49.0 and CDCl 3 : d H 7.26/ d C 77.0) referenced chemical shifts. HMQC and HMBC experiments were optimized at 1 J CH = 140 Hz and n J CH = 8 Hz, respectively. HRESIMS data were obtained using an ESI Q-TOF MS/MS system (AB SCIEX Triple). TLC was performed on Kieselgel 60 F 254 (1.05715; Merck) or RP-18 F 254s (Merck) plates. The spots were visualized by spraying 10% H 2 SO 4 aqueous solution followed by heating. Column chromatography was performed on YMC octadecyl-functionalized silica gel (C 18 ).
실시예 2: 균주 물질 및 발효.Example 2: Strain Materials and Fermentation.
Penicillium glabrum SF-7123(기탁번호: KCTC 14169BP)은 2015년 1월 10일에 Ross Sea(N 77ο34.397′ W 166ο10.8653′)에서 준설선(dredge)을 사용하여 수집된 퇴적물에서 분리되었다. 샘플 1g을 멸균된 해수(10mL)와 혼합하였다. 멸균된 해수를 함유하는 감자 덱스트로스 한천(PDA) 배지에서 스프레드 플레이트 방법을 사용하여 샘플의 일부(0.1mL)를 처리하였다. 플레이트를 25℃에서 14일 동안 배양하였다. 분리물을 여러 번 계대배양한 후, 최종 순수 배양물을 선택하고 -70℃에서 보존하였다. 진균 균주 SF-7123은 28S 리보솜 RNA(rRNA) 유전자 서열의 분석에 기초하여 확인되었다. SF-7123(GenBank 수탁 번호 KY563089)의 28S rRNA 유전자를 사용한 GenBank 검색은 Penicillium glabrum (JN938946), P. spinulosum (HM469405) 및 P. multicolor (HM469407)를 각각 100%, 99.64% 및 97.13%의 서열 동일성을 나타내는 가장 근접한 매칭으로 나타냈다. 따라서, 해양-유래 진균 균주 SF-7123은 페니실리움 글라브룸(Penicillium glabrum)으로 동정되었다.Penicillium glabrum SF-7123 (Accession No.: KCTC 14169BP) was isolated from sediments collected on January 10, 2015 using a dredge in the Ross Sea (N 77ο34.397′ W 166ο10.8653′). 1 g of sample was mixed with sterile seawater (10 mL). A portion (0.1 mL) of the sample was treated using the spread plate method in potato dextrose agar (PDA) medium containing sterile seawater. Plates were incubated at 25° C. for 14 days. After subculture of the isolates several times, the final pure culture was selected and stored at -70°C. Fungal strain SF-7123 was identified based on analysis of the 28S ribosomal RNA (rRNA) gene sequence. GenBank search using the 28S rRNA gene of SF-7123 (GenBank accession number KY563089) revealed 100%, 99.64% and 97.13% sequence identity to Penicillium glabrum (JN938946), P. spinulosum (HM469405) and P. multicolor (HM469407), respectively. shown as the closest match representing . Therefore, the marine-derived fungal strain SF-7123 was identified as Penicillium glabrum.
실시예 3: 화합물 1 내지 4의 추출 및 분리.Example 3: Extraction and isolation of compounds 1-4.
진균 균주 SF-7123을 3%의 NaCl(w/v)을 함유하는 300mL의 PDA 배지를 각각 함유하는 10개의 Fernbach 플라스크(300mL)에서 배양하였다. 플라스크에 2mL의 진균 균주의 종자 배양물을 개별적으로 접종하고, 14일 동안 25℃에서 배양하였다. 합쳐진 배양 배지를 EtOAc(4L)로 추출하였다. 합쳐진 추출 용액을 여과지를 통해 여과한 다음 증발건조시켜 조추출액(crude extract) SF7123(1.0g)을 수득하였다. 조추출액(crude extract)을 H2O에서 20, 30, 40, 50, 60, 80 및 100%(v/v) MeOH(각각 400mL)의 단계적 구배로 용리시키면서 역상(RP) C18 플래시 컬럼 크로마토그래피(4.5 x 30cm)에서 분별한 후, SF7123-1 내지 SF7123-8의 7개의 분획을 연속적으로 수득하였다. SF7123-4분획을 먼저 화합물 3(2.5mg)로 분리하고, 고정상으로서 Sephadex LH-20및 이동상으로서 물 중의 MeOH의 3/1(v/v) 혼합물을 사용하여 2개의 SF7123-4-1 및 SF7123-4-2서브분획을 컬럼 크로마토그래피(3 x 35cm)에 적용하였다. 이어서, SF7123-4-2 서브분획을 물 중 MeOH[2/3(v/v)]로 용리시키면서 RP C18 컬럼(1.2 x 30cm)상에서 크로마토그래피하여 3개의 서브분획을 수득하였다. 이들 중에서, SF7123-4-2-1서브분획을 C18 prep HPLC[16분에 걸쳐 H2O 중 27 내지 43%의 CH3CN(0.1% HCOOH)]에서 추가로 분리한 후, R1이 H인 화합물 1(6.5mg, tR = 15분)을 수득하였다. 이와 유사하게, SF7123-4-2-2서브분획을 C18 prep HPLC[16분에 걸쳐 H2O 중 35 내지50%의 CH3CN(0.1% HCOOH)]를 사용하여 정제한 후, R1이 CH3인 화합물 1(4.3mg, tR = 15.5분)을 수득하였다. SF7123-5 분획을 Sephadex LH-20 (2.0 x 30cm)으로 패킹된 컬럼 크로마토그래피에 적용하였다. 이어서, 컬럼을 H2O[3/1(v/v)] 중의 MeOH의 혼합물로 용리시켜 SF7123-5-1 내지 SF7123-5-3의 3개의 서브분획으로 분리하였다. 제1서브분획인 SF7123-5-1에 C18 prep HPLC[18분에 걸쳐 물 중 48 내지 70%의 CH3CN(0.1% HCOOH)]를 적용하여 화합물 4(6mg, tR = 17분)를 수득하였다. 다른 화합물 2(2mg, tR = 23분)은 RP C18 prep HPLC[25분에 걸쳐 H2O 중 35 내지 65%의 CH3CN(0.1% HCOOH)]에 의해 제2서브분획인 SF7123-5-2로부터 분리하였다.Fungal strain SF-7123 was cultured in 10 Fernbach flasks (300 mL) each containing 300 mL of PDA medium containing 3% NaCl (w/v). Flasks were individually inoculated with 2 mL of seed cultures of fungal strains and incubated at 25° C. for 14 days. The combined culture medium was extracted with EtOAc (4 L). The combined extraction solution was filtered through filter paper and then evaporated to dryness to obtain a crude extract SF7123 (1.0 g). Reversed phase (RP) C 18 flash column chromatography eluting the crude extract with a step gradient of 20, 30, 40, 50, 60, 80 and 100% (v/v) MeOH (400 mL each ) in H 2 O After fractionation on a graph (4.5 x 30 cm), 7 fractions of SF7123-1 to SF7123-8 were successively obtained. The SF7123-4 fraction was first separated with compound 3 (2.5 mg), followed by two SF7123-4-1 and SF7123 using Sephadex LH-20 as stationary phase and a 3/1 (v/v) mixture of MeOH in water as mobile phase. The -4-2 sub-fraction was subjected to column chromatography (3 x 35 cm). The SF7123-4-2 sub-fraction was then chromatographed on a RP C 18 column (1.2 x 30 cm) eluting with MeOH in water [2/3 (v/v)] to give 3 sub-fractions. Of these, the SF7123-4-2-1 subfraction was further separated on C 18 prep HPLC [27-43% CH 3 CN in H 2 O over 16 min (0.1% HCOOH)], after which R 1 was H, compound 1 (6.5 mg, t R = 15 min) was obtained. Similarly, the SF7123-4-2-2 subfraction was purified using C 18 prep HPLC [35-50% CH 3 CN in H 2 O (0.1% HCOOH) over 16 min], followed by R 1 This CH 3 compound 1 (4.3 mg, t R = 15.5 min) was obtained. The SF7123-5 fraction was subjected to column chromatography packed with Sephadex LH-20 (2.0 x 30 cm). The column was then eluted with a mixture of MeOH in H 2 O [3/1 (v/v)] to separate into three subfractions SF7123-5-1 to SF7123-5-3. Compound 4 (6 mg, t R = 17 min) was applied to the first subfraction, SF7123-5-1, by C 18 prep HPLC [48-70% CH 3 CN (0.1% HCOOH) in water over 18 min]. was obtained. Another compound 2 (2 mg, t R = 23 min) was prepared by RP C 18 prep HPLC [35-65% CH 3 CN in H 2 O (0.1% HCOOH) over 25 min] as the second subfraction, SF7123- 5-2.
9-O-메틸뉴크로메닌(9-O-methylneuchromenin, R1이 CH3인 화합물 1)은 황색 분말(Yellow powder)이며, [a]D -344 (c = 0.72, MeOH)이고, 1H 및 13C NMR 데이터는 표 1을 참조하며, HRESIMS는 m/z 263.0911 [M + H]+ (C14H15O5에 대한 계산치, 263.0919)이다.9- O -methylneuchromenin (9- O- methylneuchromenin, compound 1 in which R 1 is CH 3 ) is a yellow powder, [ a ] D -344 ( c = 0.72, MeOH), and 1 H and 13 C NMR data see Table 1, HRESIMS is m/z 263.0911 [M + H] + ( calculated for C 14 H 15 O 5 , 263.0919).
실시예 4: 화합물 1 내지 4의 구조 결정.Example 4: Determination of structures of compounds 1 to 4.
R1이 CH3인 화합물 1을 비정질 분말로서 분리하고 -344.63(c 0.48, CH3OH)의 [α]20 D값으로 광학 활성을 가졌다. R1이 CH3인 화합물 1의 HRESIMS는 m/z 263.0911 [M + H]+(C14H15O5에 대한 계산치, 263.0919)에서 이온 피크를 보이므로, C14H14O5의 분자식으로 결정하였다. R1이 CH3인 화합물 1의 1H NMR 스펙트럼(표 1)은 δH 6.34(1H, s, H-7) 및 7.06(1H, s, H-10)에서 2개의 방향족 양성자에 대한 전형적인 신호를 나타내고, 메톡시 양성자에 대한 δH 3.83(3H, H-12)에서 단일 피크 및 δH 1.55(3H, d, J = 6.4, H-11)에서 메틸 신호를 나타냈다. 또한, δH 4.71(1H, m, H-2)에서 옥시메틴 양성자, 4.73(1H, d, J = 12.0, H-5a) 및 5.03(1H, d, J = 12.0, H-5b)에서 옥시메틸렌 양성자에 상응하는 3개의 다운필드-시프트된 신호가 존재하였다. δH 2.53(2H, m, H-3)에서의 나머지 신호는 메틸렌 양성자에 할당되었다. R1이 CH3인 화합물 1의 13C NMR 스펙트럼은 δC 191.0(C-4)에서 카르보닐기, δC 104.8(C-7) 및 δC 108.9(C-10)에서 2개의 방향족 메틴 탄소에 할당된 9개의 sp2 신호를 포함하는 14개의 탄소를 나타냈다. 또한 δC 102.4(C-4a), 156.3(C-6a), 154.5(C-8), 144.8(C-9), 107.9(C-10a), 및 165.7(C-10b)에서 6개의 sp2 4차 탄소 신호가 스펙트럼에 존재하였다. δC 77.3 및 δC 64.1에서 2개의 산소화된 탄소는 각각 메틴(C-2) 및 메틸렌(C-5)으로 확인되었다. 또한, 화합물 1의 13C NMR 스펙트럼은 δC 57.0(C-12)에서 메톡시기, δC 43.5(C-3)에서 메틸렌 탄소 및 δC 20.6(C-11)에서 메틸기에 대한 신호를 나타냈다. 광범위한 문헌 스크리닝 과정에서, R1이 CH3인 화합물 1의 1H 및 13C NMR 데이터와 뉴크로메닌(neuchromenin)에 대한 보고된 값의 데이터의 비교(Tanada, Y.; Mori, K. European J. Org. Chem. 2001, 2001, 1963.; Hayakawa, Y., et al., Tetrahedron Lett. 1996, 37, 6363-6364.)한 결과, R1이 CH3인 화합물 1에서의 메톡시기의 존재를 제외하고는 구조적으로 매우 유사함을 나타냈다. R1이 CH3인 화합물 1의 COZY 및 HMBC 스펙트럼의 정밀한 분석(표 1)은 실제로 하이드록시기를 C-9에 부착된 메톡시기로 치환한 뉴크로메닌의 유도체로서 R1이 CH3인 화합물 1의 평면구조를 확인하였다. 이 할당은 9-OCH3에서 C-9로의 HMBC 상관관계의 관찰에 의해 확인되었다. 결과적으로, 전술한 증거에 기초하여, R1이 CH3인 화합물 1의 구조는 R1이 CH3인 화학식 1과 같이 결정되었고, 9-O-메틸뉴크로메닌(9-O-methylneuchromenin)으로 명명되었다. Compound 1 in which R 1 is CH 3 was isolated as an amorphous powder and had optical activity with a [α] 20 D value of -344.63 (c 0.48, CH 3 OH). HRESIMS of Compound 1 in which R 1 is CH 3 shows an ion peak at m/z 263.0911 [M + H] + ( calculated for C 14 H 15 O 5 , 263.0919), so the molecular formula of C 14 H 14 O 5 decided. 1 H NMR spectrum (Table 1) of compound 1 in which R 1 is CH 3 shows typical signals for two aromatic protons at δ H 6.34 (1H, s, H-7) and 7.06 (1H, s, H-10). , and a single peak at δ H 3.83 (3H, H-12) and a methyl signal at δ H 1.55 (3H, d, J = 6.4, H-11) for the methoxy proton. In addition, oxymethine protons at δ H 4.71 (1H, m, H-2), oxymethine protons at 4.73 (1H, d, J = 12.0, H-5a) and 5.03 (1H, d, J = 12.0, H-5b) There were three downfield-shifted signals corresponding to methylene protons. The remaining signal at δ H 2.53 (2H, m, H-3) was assigned to the methylene proton. 13 C NMR spectrum of compound 1 in which R 1 is CH 3 is assigned to a carbonyl group at δ C 191.0 (C-4) and two aromatic methine carbons at δ C 104.8 (C-7) and δ C 108.9 (C-10) It showed 14 carbons containing 9 sp 2 signals. 6 sp 2 at δ C 102.4 (C-4a), 156.3 (C-6a), 154.5 (C-8), 144.8 (C-9), 107.9 (C-10a), and 165.7 (C-10b) A quaternary carbon signal was present in the spectrum. The two oxygenated carbons at δ C 77.3 and δ C 64.1 were identified as methine (C-2) and methylene (C-5), respectively. In addition, the 13 C NMR spectrum of Compound 1 showed a signal for a methoxy group at δ C 57.0 (C-12), a methylene carbon at δ C 43.5 (C-3), and a methyl group at δ C 20.6 (C-11). In the course of extensive literature screening, comparison of 1 H and 13 C NMR data of compound 1 in which R 1 is CH 3 with data of reported values for neuchromenin (Tanada, Y.; Mori, K. European J) Org. Chem . 2001 , 2001 , 1963.; Hayakawa, Y., et al., Tetrahedron Lett . 1996 , 37 , 6363-6364.) As a result, R 1 is CH 3 The presence of a methoxy group in Compound 1 , except that they are structurally very similar. Precise analysis of the COZY and HMBC spectra of Compound 1 in which R 1 is CH 3 (Table 1) is actually a derivative of nuchromenin in which a hydroxyl group is substituted with a methoxy group attached to C-9, in which R 1 is CH 3 Compound 1 The planar structure of This assignment was confirmed by observation of the HMBC correlation from 9-OCH 3 to C-9. As a result, by, R 1 is CH 3 structure of the compound 1 was determined as shown in formula 1 R 1 is CH 3, 9- O based on the above-described proof-methyl New chroman menin (9- O -methylneuchromenin) with Named.
Figure PCTKR2021005622-appb-T000001
Figure PCTKR2021005622-appb-T000001
R1이 H인 화합물 1에 대한 NMR 데이터는 시트로마이세틴(citromycetin) 유사체에 속하는 피란크로멘(pyranchromene) 골격을 갖는 R1이 CH3인 화합물 1의 NMR 데이터와 거의 동일하였다. 문헌에서 R1이 H인 화합물 1에 대한 1H 및 13C NMR 데이터의 면밀한 조사는 결국 뉴크로메닌(neuchromenin)으로서 이의 구조의 할당으로 이어졌으며, 이는 Eupenicillium javanicum var. meloforme PF1181의 배양액으로부터 분리되었다. 뉴크로메닌(neuchromenin)은 PC12 래트 크롬친화성세포종(pheochromocytoma) 세포의 신경돌기 성장을 유도할 수 있는 것으로 보고되었다(Tanada, Y.; Mori, K. European J. Org. Chem. 2001, 2001, 1963.). 천연적으로 생성된 뉴크로메닌(neuchromenin)의 절대 배열은 뉴크로메닌(neuchromenin)의 거울상 이성질체의 합성 연구에 의해 S인 것으로 결정되었다(Hayakawa, Y., et al., Tetrahedron Lett. 1996, 37, 6363-6364.). R1이 H인 화합물 1의 NMR 데이터 및 특정 회전값과 문헌(Tanada, Y.; Mori, K. European J. Org. Chem. 2001, 2001, 1963.)의 NMR 데이터 및 특정 회전값의 밀접한 유사성은 R1이 H인 화합물 1의 절대 배열이 (-)-뉴크로메닌(neuchromenin)의 절대 배열과 같아야 한다는 것을 암시했다. 따라서, 그들은 동일한 진균 균주에 대한 화학적 조사로부터 분리되었기 때문에, R1이 CH3인 화합물 1의 절대 배열은 R1이 H인 화합물 1의 절대 배열과 유사하다는 것이 제안되었다.The NMR data of Compound 1 in which R 1 is H was almost identical to the NMR data of Compound 1 in which R 1 is CH 3 and having a pyranchromene skeleton belonging to a citromycetin analog. A scrutiny of 1 H and 13 C NMR data for compound 1 in which R 1 is H in the literature eventually led to the assignment of its structure as neuchromenin, which was found in Eupenicillium javanicum var. It was isolated from the culture medium of meloforme PF1181. It has been reported that neuchromenin can induce neurite outgrowth in PC12 rat pheochromocytoma cells (Tanada, Y.; Mori, K. European J. Org. Chem . 2001 , 2001 , 1963.). The absolute configuration of naturally occurring neuchromenin was determined to be S by synthetic studies of the enantiomer of neuchromenin (Hayakawa, Y., et al., Tetrahedron Lett . 1996 , 37). , 6363-6364.). NMR data and specific rotation values of compound 1 in which R 1 is H and the literature (Tanada, Y.; Mori, K. European J. Org. Chem . 2001 , 2001 , 1963.) and the close similarity of specific rotation values suggested that the absolute configuration of compound 1 in which R 1 is H should be the same as that of (-)-neuchromenin. Therefore, it was suggested that the absolute configuration of compound 1 where R 1 is CH 3 is similar to the absolute configuration of compound 1 where R 1 is H, since they were isolated from chemical investigations on the same fungal strain.
나머지 화합물의 구조는 문헌에서 이전에 공개된 데이터의 구조와 비교하여 그들의 NMR 및 MS 데이터의 분석에 기초하여 결정되었다. 이 연구에서 디페닐 에테르가 분리되었으며, 이는 엔도텔린 결합 억제제로 알려진 아스테릭산(R2 내지 R4가 각각 독립적으로 CH3인 화합물 2)으로 확인되었다(Liao, W.Y., et al., J. Nat. Prod. 2012, 75, 630-635.; Ohashi, H., et al., Antibiot (Tokyo). 1992, 45, 1684-1685.). R5가 CH3인 화합물 3은 시트로마이세틴(citromycetin) 유도체인 믹소트리친 C(myxotrichin C)로 확인되었다(Yuan, C., et al., Myxotrichum sp.. Phytochemistry Letters 2013, 6, 662-666.). 이 대사체의 생물학적 효과에 대한 선행 보고는 없다. R6 내지 R9가 각각 독립적으로 CH3인 화합물 4의 구조는 페놀 폴리케타이드(phenolic polyketide)로 분류될 수 있고 푸니콘(funicone) 유사체에 속하는 데옥시푸니콘(deoxyfunicone)으로 밝혀졌으며(Sassa, T., et al., Agric. Biol. Chem. 1991, 55, 2415-2416.), 이는 HIV-1-통합 억제제로서 설명되었다(Singh, S.B., et al., J. Ind. Microbiol. Biotechnol. 2003, 30, 721-731.).The structures of the remaining compounds were determined based on analysis of their NMR and MS data compared to the structures of previously published data in the literature. In this study, diphenyl ether was isolated, which was identified as asteric acid ( compound 2 in which R 2 to R 4 is each independently CH 3 ) known as an endothelin binding inhibitor (Liao, WY, et al., J. Nat. Prod . 2012 , 75 , 630-635.; Ohashi, H., et al., Antibiot (Tokyo) . 1992 , 45 , 1684-1685.). Compound 3 in which R 5 is CH 3 was identified as myxotrichin C, a citromycetin derivative (Yuan, C., et al., Myxotrichum sp. Phytochemistry Letters 2013 , 6 , 662) -666.). There are no prior reports on the biological effects of this metabolite. The structure of compound 4 in which R 6 to R 9 is each independently CH 3 can be classified as a phenolic polyketide and was found to be deoxyfunicone belonging to a funicone analog (Sassa). , T., et al., Agric. Biol. Chem . 1991 , 55 , 2415-2416.), which has been described as an HIV-1-integration inhibitor (Singh, SB, et al., J. Ind. Microbiol. Biotechnol). ( 2003 , 30 , 721-731.).
실시예 5: 염증 촉진성(pro-inflammatory) 매개체 생성에 미치는 SF-7123에서 분리된 2차 대사체의 영향.Example 5: Effect of secondary metabolites isolated from SF-7123 on the production of pro-inflammatory mediators.
그람 음성 박테리아의 주요 세포벽 성분인 지질다당류(lipopolysaccharide, LPS)는 대식세포 및 미세아교세포에 대한 강력한 활성화제이므로, LPS로 이들 세포를 치료하는 것은 염증 현상과 관련된 연구에서 자주 사용된다(Zhang, H., et al., Int. Immunopharmacol. 2017, 52, 93-100.,20). 본 명세서에서, 분리된 화합물 1 내지 화합물 4 모두에 대해 LPS-유도된 BV2 미세아교세포 및 RAW264.7 대식세포에서 NO 및 PGE2의 과잉생성에 미치는 효과를 조사함으로써 그들의 항신경염증 및 항염증 효과를 평가하였다. 또한, LPS-유도된 BV2 미세아교세포 및 RAW264.7 대식세포에서 NO의 과잉생성에 미치는 효과를 조사하는 데 항염증 및 항암효과를 갖는 물질인 뷰테인(butein)을 양성 대조군으로 사용하였다. LPS 자극은 두 세포 모두에서 NO 및 PGE2의 생성을 증가시켰다. R1이 H인 화합물 1, 화합물 3 및 화합물 4를 사용한 전처리(pre-treatment)는 표 2에 나타낸 바와 같이 상이한 IC50값으로 이들 반응을 약화시켰다. R1이 CH3인 화합물 1 및 2는 80.0mM에서 NO 생성의 변경에 크게 영향을 미치지 않았다. 이 연구에서, R1이 CH3인 화합물 1 및 R1이 H인 화합물 1의 IC50값 및 이들 각각의 구조에 기초하여, 메톡실기(R1이 CH3인 화합물 1)에 의해 뉴로크로메닌(neuchromenin, R1이 H인 화합물 1)의 C-9위치에서 하이드록실기의 대체가 이 효과를 현저하게 감소시킨다는 것을 알게 되었다. 이 분석에서 가장 활성인 화합물은 R1이 H인 화합물 1인 것으로 확인되었고, 이 화합물의 추가의 항염증 효과 및 이의 기저 메커니즘을 알아내기 위해 추가적인 연구를 진행하였다. 도 1에 도시된 바와 같이, R1이 H인 화합물 1은 BV2세포가 12시간 동안 LPS(1㎍/mL)로 활성화될 때, IL-1β, TNF-α 및 IL-6의 mRNA 발현을 효과적으로 감소시켰다. 또한, R1이 H인 화합물 1은 LPS로 자극된 BV2 및 RAW264.7세포 둘 다에서 용량-의존적 방식으로 iNOS 및 COX-2의 단백질 발현을 약화시켰다(도 2). 관찰된 효과가 그들의 세포독성으로부터 발생할 가능성을 배제하기 위해, MTT 분석을 수행한 결과, 화합물 1 내지 4는 최대 80.0mM의 용량 범위 내에서 이들 세포에 세포독성이 없는 것으로 나타났다(데이터는 표시되지 않음).Since lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria, is a potent activator for macrophages and microglia, treatment of these cells with LPS is frequently used in studies related to inflammatory phenomena (Zhang, H. ., et al., Int. Immunopharmacol . 2017 , 52 , 93-100., 20). In the present specification, by examining the effects on the overproduction of NO and PGE 2 in LPS-induced BV2 microglia and RAW264.7 macrophages for all of the isolated compounds 1 to 4, their anti-neuroinflammatory and anti-inflammatory effects was evaluated. In addition, to investigate the effect on the overproduction of NO in LPS-induced BV2 microglia and RAW264.7 macrophages, butein, a substance having anti-inflammatory and anticancer effects, was used as a positive control. LPS stimulation increased the production of NO and PGE 2 in both cells. Pre-treatment with compound 1, compound 3 and compound 4 in which R 1 is H attenuated these responses with different IC 50 values as shown in Table 2. Compounds 1 and 2 in which R 1 is CH 3 did not significantly affect the alteration of NO production at 80.0 mM. In this study, based on the IC 50 values of Compound 1 in which R 1 is CH 3 and Compound 1 in which R 1 is H and their respective structures, neurochromenin is formed by a methoxyl group (Compound 1 in which R 1 is CH 3) It was found that replacement of the hydroxyl group at the C-9 position of (neuchromenin, compound 1 in which R 1 is H) significantly reduced this effect. The most active compound in this assay was identified as Compound 1 in which R 1 is H, and further studies were conducted to determine the additional anti-inflammatory effect of this compound and its underlying mechanism. As shown in FIG. 1 , Compound 1 in which R 1 is H effectively inhibited the mRNA expression of IL-1β, TNF-α and IL-6 when BV2 cells were activated with LPS (1 μg/mL) for 12 hours. decreased. In addition, compound 1, in which R 1 is H, attenuated protein expression of iNOS and COX-2 in a dose-dependent manner in both BV2 and RAW264.7 cells stimulated with LPS ( FIG. 2 ). To rule out the possibility that the observed effects arise from their cytotoxicity, MTT assays were performed and showed that compounds 1-4 were not cytotoxic to these cells within a dose range of up to 80.0 mM (data not shown). ).
Figure PCTKR2021005622-appb-T000002
Figure PCTKR2021005622-appb-T000002
실시예 6: 분리된 화합물 1 내지 4의 PTP1B 억제 효과.Example 6: PTP1B inhibitory effect of isolated compounds 1 to 4.
분리된 화합물의 항염증 및 항신경염증 효과의 평가에 더하여, 본 실험에서는 p-니트로페닐 포스페이트(pNPP)를 효소 기질로 사용하여 PTP1B 활성에 대한 화합물의 효과를 평가하였다. 본 실험에서, 공지된 포스파타제 억제제인 우르솔산(ursolic acid)을 양성 대조군으로 사용하였다(Na, M., et al., Planta Med 2006, 72, 261-263.; Zhang, W., Biochem Biophy Acta 2006, 1760, 1505-1512.). 시험된 화합물 중에서, 화합물 3 및 4는 각각 IC50값이 19.2μM 및 24.3μM인 용량-의존적 방식으로 활성 PTP1B 효소를 억제하는 것으로 나타났다(표 3). In addition to evaluating the anti-inflammatory and anti-neuroinflammatory effects of the isolated compound, in this experiment, p-nitrophenyl phosphate (pNPP) was used as an enzyme substrate to evaluate the effect of the compound on PTP1B activity. In this experiment, ursolic acid, a known phosphatase inhibitor, was used as a positive control (Na, M., et al., Planta Med 2006 , 72 , 261-263.; Zhang, W., Biochem Biophy Acta) 2006 , 1760 , 1505-1512.). Among the compounds tested, compounds 3 and 4 were shown to inhibit the active PTP1B enzyme in a dose-dependent manner with IC 50 values of 19.2 μM and 24.3 μM, respectively (Table 3).
Figure PCTKR2021005622-appb-T000003
Figure PCTKR2021005622-appb-T000003
또한, PTP1B 촉매화된 pNPP 가수분해의 동역학적 프로파일에 대한 화합물 3 및 5의 효과를 방법에 기재된 바와 같이 분석하였다. 상이한 농도에서 화합물 3의 존재 및 부재 하에 pNPP 기질에 대해 효소 분석을 수행한 경우, 도 5A에 나타낸 바와 같이 억제제에 대한 라인웨버-버크도면(Lineweaver-Burk plot)에서 Km은 억제제 농도가 증가함에 따라 변하지 않았지만, Vmax는 감소하였다. 따라서, 화합물 3는 비경쟁적 억제제로서 결정되었으며, 이는 화합물이 PTP1B 내의 알로스테릭 부위 또는 효소 기질에 결합할 수 있음을 시사한다. 한편, 동역학 분석을 통해, 라인웨버-버크도면(Lineweaver-Burk plot)이 Vmax값의 변화없이 Km의 증가를 보였기 때문에, 화합물 4의 억제 모드가 경쟁적 모드임을 나타냈다(도 5B). 이 결과는 화합물 4가 PTP1B 내의 활성 부위에 결합할 수 있음을 나타낸다.In addition, the effect of compounds 3 and 5 on the kinetic profile of PTP1B catalyzed pNPP hydrolysis was analyzed as described in Methods. When enzymatic assays were performed on pNPP substrates in the presence and absence of compound 3 at different concentrations, Km in the Lineweaver-Burk plot for inhibitors as shown in FIG. 5A was determined as the inhibitor concentration increased. Although unchanged, Vmax decreased. Therefore, compound 3 was determined as a non-competitive inhibitor, suggesting that compound may bind to an allosteric site or enzyme substrate in PTP1B. On the other hand, through kinetic analysis, the Lineweaver-Burk plot showed an increase in Km without a change in Vmax, indicating that the inhibitory mode of compound 4 was a competitive mode (FIG. 5B). This result indicates that compound 4 can bind to the active site in PTP1B.
실시예 7: 세포 배양 및 생존능력 분석.Example 7: Cell culture and viability assay.
RAW264.7 및 BV2세포는 10%의 소태아혈청(fetal bovine serum, FBS), 페니실린 G(100U/mL), 스트렙토마이신(100mg/L) 및 L-글루타민(2mM)으로 보충된 둘베코수정이글배지(Dulbecco's modified eagle's medium, DMEM)에서 5 Х 105 cells/mL로 유지되었고, 5%의 CO2를 함유하는 가습 대기에서 37℃에서 배양하였다. DMEM, FBS 및 기타 조직 배양 시약은 Gibco BRL Co에서 구입하였다. 세포 생존능력은 이전에 기술된 MTT 분석법을 사용하여 측정하였다(Cho, K.H., et al., Neurochem. Int. 2016. 95. 55-62.).RAW264.7 and BV2 cells were treated with Dulbecco's Fertilized Eagle supplemented with 10% fetal bovine serum (FBS), penicillin G (100 U/mL), streptomycin (100 mg/L) and L-glutamine (2 mM). It was maintained at 5 Х 10 5 cells/mL in a medium (Dulbecco's modified eagle's medium, DMEM), and incubated at 37° C. in a humidified atmosphere containing 5% CO 2 . DMEM, FBS and other tissue culture reagents were purchased from Gibco BRL Co. Cell viability was determined using the previously described MTT assay (Cho, KH, et al., Neurochem. Int . 2016. 95. 55-62.).
실시예 8: 아질산염 결정.Example 8: nitrite crystals.
NO 생성의 지표인 배지 중의 아질산염 농도는 Griess 반응에 의해 측정하였다. 자세한 절차는 이전의 보고서(Cho, K.H., et al., Neurochem. Int. 2016. 95. 55-62.)에 기재되어 있다.The concentration of nitrite in the medium, which is an indicator of NO production, was measured by the Griess reaction. The detailed procedure is described in a previous report (Cho, KH, et al., Neurochem. Int . 2016. 95. 55-62.).
실시예 9: 세포질 및 핵 분획의 제조.Example 9: Preparation of cytoplasmic and nuclear fractions.
세포질 및 핵 분획은 Affymetrix Nuclear Extraction kit(Affymetrix, Inc. Santa Clara, CA)를 사용하여 추출하였다. 각 분획의 용해는 제조자의 지시에 따라 수행하였다. 분석에 대한 세부적인 사항은 이전의 보고서(Cho, K.H., et al., Neurochem. Int. 2016. 95. 55-62.)에 기재되어 있다.Cytoplasmic and nuclear fractions were extracted using an Affymetrix Nuclear Extraction kit (Affymetrix, Inc. Santa Clara, CA). Dissolution of each fraction was performed according to the manufacturer's instructions. Details of the assay are described in a previous report (Cho, KH, et al., Neurochem. Int . 2016. 95. 55-62.).
실시예 10: 웨스턴 블롯 분석.Example 10: Western blot analysis.
세포를 200g에서 3분 동안 원심분리하여 수득하였다. 이어서, 세포를 PBS로 세척하고 25mM Tris-HCl 완충제(pH 7.6), 150mM NaCl, 1% NP-40, 1% sodium deoxycholate 및 0.1% SDS를 함유하는 RIPA 용해 완충제를 사용하여 용해시켰다. 1차 항체는 Santa Cruz Biotechnology(COX-2: sc-1745; iNOS: sc-650; IкB-α: sc-371; p-IкB-α: sc-8404; p50: sc-7178; 및 p65: sc-8008)에서 구입하였다. sc-8008). 2차 항체는 Millipore(마우스: ap124p; 염소: ap106p 및 토끼: ap132p)에서 구입하였다. 웨스턴 블롯 분석에 대한 세부적인 사항은 이전의 보고서(Cho, K.H., et al., Neurochem. Int. 2016. 95. 55-62.)에 기재되어 있다.Cells were harvested by centrifugation at 200 g for 3 minutes. Cells were then washed with PBS and lysed using RIPA lysis buffer containing 25 mM Tris-HCl buffer (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS. Primary antibodies were prepared from Santa Cruz Biotechnology (COX-2: sc-1745; iNOS: sc-650; IKB-α: sc-371; p-IKB-α: sc-8404; p50: sc-7178; and p65: sc -8008). sc-8008). Secondary antibodies were purchased from Millipore (mouse: ap124p; goat: ap106p and rabbit: ap132p). Details of Western blot analysis are described in a previous report (Cho, KH, et al., Neurochem. Int . 2016. 95. 55-62.).
실시예 11: NF-кB의 DNA 결합 활성.Example 11: DNA binding activity of NF-κB.
핵 추출물에서 NF-кB의 DNA-결합 활성은 제조사의 지시에 따라 TransAM® kit(Active Motif, Carlsbad, CA)를 사용하여 측정하였다. 분석은 독립적으로 3회 수행하였다.The DNA-binding activity of NF-κB in nuclear extracts was measured using the TransAM® kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions. Analyzes were performed independently in triplicate.
실시예 12: PGE2 분석.Example 12: PGE 2 analysis.
각 샘플에 존재하는 PGE2의 수준은 R&D Systems (Minneapolis, MN)로부터 상업적으로 이용 가능한 키트를 사용하여 측정하였다. 제조자의 지시에 따라 3회의 독립적인 분석을 수행하였다. The level of PGE 2 present in each sample was measured using a commercially available kit from R&D Systems (Minneapolis, Minn.). Three independent analyzes were performed according to the manufacturer's instructions.
실시예 13: 정량적 실시간 역전사효소 PCR (qRT-PCR).Example 13: Quantitative real-time reverse transcriptase PCR (qRT-PCR).
정량적 역전사 중합효소 연쇄 반응 분석의 세부적인 사항은 이전의 보고서(Cho, K.H., et al., Neurochem. Int. 2016. 95. 55-62.)에 기재되어 있다. cDNA의 PCR 증폭을 위한 최적의 조건은 제조자의 지시에 따라 설정되었다. 사용된 프라이머 서열은 하기 표 4와 같다. Details of quantitative reverse transcription polymerase chain reaction analysis have been described in a previous report (Cho, KH, et al., Neurochem. Int . 2016. 95. 55-62.). Optimal conditions for PCR amplification of cDNA were established according to the manufacturer's instructions. The primer sequences used are shown in Table 4 below.
Figure PCTKR2021005622-appb-T000004
Figure PCTKR2021005622-appb-T000004
실시예 14: PTP1B 분석.Example 14: PTP1B analysis.
PTP1B(인간, 재조합)는 BIOMOL Research Laboratories, Inc.에서 구입하였다. 효소 활성은 50mM 시트레이트, pH 6.0(구성: 0.1M NaCl, 1mM EDTA 및 1mM dithiothreitol [DTT])에 2mM p-니트로페닐 포스페이트(pNPP)를 포함하는 반응 혼합물에서 측정하였다. 반응 혼합물을 30℃에서 30분 동안 유지된 인큐베이터에 넣고, 1N NaOH를 첨가하여 반응을 종결시켰다. 생성된 p-니트로페놀의 양은 405nm에서 흡광도의 증가를 측정함으로써 추정하였다. 2mM pNPP의 비효소 가수분해는 PTP1B 효소가 없는 상태에서 405nm에서 흡광도의 증가를 측정함으로써 정정하였다(Hamaguchi, T., et al., FEBS Lett. 1995, 372, 54-58.). 동역학적 분석을 위해, 반응 혼합물은 화합물 3 및 5의 존재 또는 부재 하에 PTP1B 기질로서 상이한 농도의 pNPP로 구성되었으며, 분석은 상기 기술한 바와 같이 수행하였다. PTP1B의 Michaelis-Menten 상수(Km) 및 최대 속도(Vmax)는 GraphPad Prism® 4 프로그램(GraphPad Software Inc., USA)을 사용하여 라인웨버-버크도면(Lineweaver-Burk plot)에 의해 결정하였다.PTP1B (human, recombinant) was purchased from BIOMOL Research Laboratories, Inc. Enzyme activity was measured in a reaction mixture containing 2 mM p-nitrophenyl phosphate (pNPP) in 50 mM citrate, pH 6.0 (composition: 0.1 M NaCl, 1 mM EDTA and 1 mM dithiothreitol [DTT]). The reaction mixture was placed in an incubator maintained at 30° C. for 30 minutes, and 1N NaOH was added to terminate the reaction. The amount of p-nitrophenol produced was estimated by measuring the increase in absorbance at 405 nm. Non-enzymatic hydrolysis of 2 mM pNPP was corrected by measuring the increase in absorbance at 405 nm in the absence of PTP1B enzyme (Hamaguchi, T., et al., FEBS Lett . 1995 , 372 , 54-58.). For kinetic analysis, reaction mixtures consisted of different concentrations of pNPP as PTP1B substrate in the presence or absence of compounds 3 and 5, and the analysis was performed as described above. Michaelis-Menten constant (Km) and maximum velocity (Vmax) of PTP1B were determined by Lineweaver-Burk plot using GraphPad Prism® 4 program (GraphPad Software Inc., USA).
실시예 15: 통계적 분석.Example 15: Statistical Analysis.
데이터는 3번 이상의 독립적인 실험 중 평균 ±S.D로 표시된다. 3개 이상의 그룹을 비교하기 위해, 일원 분산 분석(one-way analysis of variance, ANOVA) 후 Tukey의 다중 비교 테스트를 수행하였다. 통계 분석은 GraphPad Prism software, version 3.03 (GraphPad Software Inc.)을 사용하여 수행하였다.Data are presented as mean ± S.D of at least 3 independent experiments. To compare three or more groups, one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test was performed. Statistical analysis was performed using GraphPad Prism software, version 3.03 (GraphPad Software Inc.).
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. will be. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.
본 발명에 따르면 해양 유래 진균 페니실리움 글라브룸(Penicillium glabrum) SF-7123의 대사체를 포함하는 조성물은 LPS-자극된 RAW264.7 대식세포 및 BV2 미세아교세포에서 NO, 프로스타글란딘 E2(PGE2), 유도성 산화질소 효소(iNOS) 및 시클로옥시게나아제-2(COX-2) 등과 같은 염증 촉진성(pro-inflammatory) 매개체의 생성 및 PTP1B의 발현을 억제하므로, 염증성 질환 또는 당뇨의 예방 또는 치료 용도로 유용하게 사용될 수 있다.According to the present invention, a composition comprising a metabolite of the marine-derived fungus Penicillium glabrum SF-7123 is produced in LPS-stimulated RAW264.7 macrophages and BV2 microglia cells with NO, prostaglandin E 2 (PGE 2 ), the production of pro-inflammatory mediators such as inducible nitric oxide enzyme (iNOS) and cyclooxygenase-2 (COX-2) and inhibits the expression of PTP1B, thus preventing inflammatory diseases or diabetes or It can be usefully used for therapeutic purposes.
전자파일 첨부하였음.An electronic file is attached.
Figure PCTKR2021005622-appb-I000033
Figure PCTKR2021005622-appb-I000033

Claims (11)

  1. 페니실리움 글라브룸(Penicillium glabrum) SF-7123의 추출물을 포함하는 항염증 또는 항당뇨용 약학 조성물. Penicillium glabrum ( Penicillium glabrum ) An anti-inflammatory or anti-diabetic pharmaceutical composition comprising an extract of SF-7123.
  2. 제1항에 있어서, 상기 추출물은 하기 화학식 1 내지 화학식 4로 구성된 군에서 선택되는 하나 이상의 대사체를 포함하는 항염증 또는 항당뇨용 약학 조성물:According to claim 1, wherein the extract is an anti-inflammatory or anti-diabetic pharmaceutical composition comprising one or more metabolites selected from the group consisting of the following Chemical Formulas 1 to 4:
    [화학식 1][Formula 1]
    Figure PCTKR2021005622-appb-I000034
    Figure PCTKR2021005622-appb-I000034
    [화학식 2][Formula 2]
    Figure PCTKR2021005622-appb-I000035
    Figure PCTKR2021005622-appb-I000035
    [화학식 3][Formula 3]
    Figure PCTKR2021005622-appb-I000036
    Figure PCTKR2021005622-appb-I000036
    [화학식 4][Formula 4]
    Figure PCTKR2021005622-appb-I000037
    Figure PCTKR2021005622-appb-I000037
    여기서, 상기 R1 내지 R9는 각각 독립적으로 H, 직쇄형 또는 분지쇄형의 C1-10 알킬, C1-10 알콕시, C3-10 사이클로알킬, C2-10 알케닐 또는 C6-10 아릴임.Here, R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 Aryl.
  3. 제1항에 있어서, 하나 이상의 하기 특징을 가지는 것을 특징으로 하는 약학 조성물:The pharmaceutical composition according to claim 1, characterized in that it has one or more of the following characteristics:
    1) 산화질소(NO) 및 프로스타글란딘 E2(PGE2)의 생성 억제;1) inhibition of the production of nitric oxide (NO) and prostaglandin E 2 (PGE 2 );
    2) NF-кB(nuclear factor kappa B) 및 MAPK(mitogen-activated protein kinase)의 불활성화;2) inactivation of nuclear factor kappa B (NF-KB) and mitogen-activated protein kinase (MAPK);
    3) 유도성 산화질소 효소(inducible nitric oxide synthase; iNOS) 및 시클로옥시게나아제-2(COX-2)의 발현 억제;3) inhibition of expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2);
    4) IL-1β, IL-6 및 TNF-α의 발현 억제; 및4) inhibition of expression of IL-1β, IL-6 and TNF-α; and
    5) PTP1B(protein tyrosine phosphate 1B)의 발현 억제.5) Inhibition of expression of PTP1B (protein tyrosine phosphate 1B).
  4. 제1항에 있어서, 약학적으로 허용되는 담체, 부형제 또는 희석제를 추가로 포함하는 것을 특징으로 하는 약학 조성물.The pharmaceutical composition according to claim 1, further comprising a pharmaceutically acceptable carrier, excipient or diluent.
  5. 페니실리움 글라브룸(Penicillium glabrum) SF-7123의 추출물을 포함하는 염증 또는 당뇨 개선용 식품.A food for improving inflammation or diabetes, comprising an extract of Penicillium glabrum SF-7123.
  6. 제5항에 있어서, 상기 추출물은 하기 화학식 1 내지 화학식 4로 구성된 군에서 선택되는 하나 이상의 대사체를 포함하는 염증 또는 당뇨 개선용 식품:The food for improving inflammation or diabetes according to claim 5, wherein the extract contains one or more metabolites selected from the group consisting of the following Chemical Formulas 1 to 4:
    [화학식 1][Formula 1]
    Figure PCTKR2021005622-appb-I000038
    Figure PCTKR2021005622-appb-I000038
    [화학식 2][Formula 2]
    Figure PCTKR2021005622-appb-I000039
    Figure PCTKR2021005622-appb-I000039
    [화학식 3][Formula 3]
    Figure PCTKR2021005622-appb-I000040
    Figure PCTKR2021005622-appb-I000040
    [화학식 4][Formula 4]
    Figure PCTKR2021005622-appb-I000041
    Figure PCTKR2021005622-appb-I000041
    여기서, 상기 R1 내지 R9는 각각 독립적으로 H, 직쇄형 또는 분지쇄형의 C1-10 알킬, C1-10 알콕시, C3-10 사이클로알킬, C2-10 알케닐 또는 C6-10 아릴임.Here, R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 Aryl.
  7. (a) 페니실리움 글라브룸(Penicillium glabrum) SF-7123를 배양하는 단계;(A) Penicillium glabrum ( Penicillium glabrum ) culturing SF-7123;
    (b) 상기 (a) 단계에서 수득된 균주 배양물을 에틸아세테이트(EtOAc)로 추출하는 단계; 및(b) extracting the strain culture obtained in step (a) with ethyl acetate (EtOAc); and
    (c) 상기 (b) 단계에서 얻어진 에틸아세테이트(EtOAc) 추출물을 컬럼 크로마토그래피로 분리하는 단계를 포함하는 하기 화학식 1 내지 화학식 4의 대사체의 분리방법:(c) a method for separating metabolites of the following Chemical Formulas 1 to 4, comprising separating the ethyl acetate (EtOAc) extract obtained in step (b) by column chromatography:
    [화학식 1][Formula 1]
    Figure PCTKR2021005622-appb-I000042
    Figure PCTKR2021005622-appb-I000042
    [화학식 2][Formula 2]
    Figure PCTKR2021005622-appb-I000043
    Figure PCTKR2021005622-appb-I000043
    [화학식 3][Formula 3]
    Figure PCTKR2021005622-appb-I000044
    Figure PCTKR2021005622-appb-I000044
    [화학식 4][Formula 4]
    Figure PCTKR2021005622-appb-I000045
    Figure PCTKR2021005622-appb-I000045
    여기서, 상기 R1 내지 R9는 각각 독립적으로 H, 직쇄형 또는 분지쇄형의 C1-10 알킬, C1-10 알콕시, C3-10 사이클로알킬, C2-10 알케닐 또는 C6-10 아릴임.Here, R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 Aryl.
  8. 하기 화학식 1 내지 화학식 4로 구성된 군에서 선택되는 하나 이상의 대사체를 포함하는 항염증 또는 항당뇨용 약학 조성물:A pharmaceutical composition for anti-inflammatory or antidiabetic comprising at least one metabolite selected from the group consisting of the following Chemical Formulas 1 to 4:
    [화학식 1][Formula 1]
    Figure PCTKR2021005622-appb-I000046
    Figure PCTKR2021005622-appb-I000046
    [화학식 2][Formula 2]
    Figure PCTKR2021005622-appb-I000047
    Figure PCTKR2021005622-appb-I000047
    [화학식 3][Formula 3]
    Figure PCTKR2021005622-appb-I000048
    Figure PCTKR2021005622-appb-I000048
    [화학식 4][Formula 4]
    Figure PCTKR2021005622-appb-I000049
    Figure PCTKR2021005622-appb-I000049
    여기서, 상기 R1 내지 R9는 각각 독립적으로 H, 직쇄형 또는 분지쇄형의 C1-10 알킬, C1-10 알콕시, C3-10 사이클로알킬, C2-10 알케닐 또는 C6-10 아릴임.Here, R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 Aryl.
  9. 제8항에 있어서, 상기 R1 내지 R9는 각각 독립적으로 H, CH3 또는 CH2CH3인 것을 특징으로 하는 항염증 또는 항당뇨용 약학 조성물.The pharmaceutical composition for anti-inflammatory or antidiabetic according to claim 8, wherein R 1 to R 9 are each independently H, CH 3 or CH 2 CH 3 .
  10. 하기 화학식 1 내지 화학식 4로 구성된 군에서 선택되는 하나 이상의 대사체를 포함하는 염증 또는 당뇨 개선용 식품:A food for improving inflammation or diabetes comprising one or more metabolites selected from the group consisting of the following Chemical Formulas 1 to 4:
    [화학식 1][Formula 1]
    Figure PCTKR2021005622-appb-I000050
    Figure PCTKR2021005622-appb-I000050
    [화학식 2][Formula 2]
    Figure PCTKR2021005622-appb-I000051
    Figure PCTKR2021005622-appb-I000051
    [화학식 3][Formula 3]
    Figure PCTKR2021005622-appb-I000052
    Figure PCTKR2021005622-appb-I000052
    [화학식 4][Formula 4]
    Figure PCTKR2021005622-appb-I000053
    Figure PCTKR2021005622-appb-I000053
    여기서, 상기 R1 내지 R9는 각각 독립적으로 H, 직쇄형 또는 분지쇄형의 C1-10 알킬, C1-10 알콕시, C3-10 사이클로알킬, C2-10 알케닐 또는 C6-10 아릴임.Here, R 1 to R 9 are each independently H, linear or branched C 1-10 alkyl, C 1-10 alkoxy, C 3-10 cycloalkyl, C 2-10 alkenyl or C 6-10 Aryl.
  11. 제10항에 있어서, 상기 R1 내지 R9는 각각 독립적으로 H, CH3 또는 CH2CH3인 것을 특징으로 하는 염증 또는 당뇨 개선용 식품.The food for improving inflammation or diabetes according to claim 10, wherein R 1 to R 9 are each independently H, CH 3 or CH 2 CH 3 .
PCT/KR2021/005622 2020-05-07 2021-05-04 Anti-inflammatory or antidiabetic composition comprising metabolite of marine-drived fungus penicillium glabrum sf-7123 WO2021225363A1 (en)

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