WO2013047055A1 - Heat shock protein expression inducer - Google Patents
Heat shock protein expression inducer Download PDFInfo
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- WO2013047055A1 WO2013047055A1 PCT/JP2012/071660 JP2012071660W WO2013047055A1 WO 2013047055 A1 WO2013047055 A1 WO 2013047055A1 JP 2012071660 W JP2012071660 W JP 2012071660W WO 2013047055 A1 WO2013047055 A1 WO 2013047055A1
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- 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
- A61K31/365—Lactones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/58—One oxygen atom, e.g. butenolide
Definitions
- the present invention relates to an expression inducer of heat shock protein (heat shock protein) useful as a pharmaceutical product.
- HSP heat shock protein
- HSP has several families due to the difference in molecular weight. For example, HSP90 family (molecular weight: 90 kDa to 110 kDa), HSP70 family (molecular weight: 70 kDa to less than 80 kDa), HSP60 family (molecular weight: 60 kDa to 70 kDa). Less), and the low molecular weight HSP family (molecular weight: less than 60 kDa).
- HSP HSP70 and HSP60 families bind to denatured proteins and unwind them to natural folding (higher-order structure / folded structure), and are associated with third proteins and nucleic acids. It has been clarified that it has functions called so-called molecular chaperones, such as intracellular localization and involvement in membrane permeation (Non-patent Documents 1 and 2).
- the molecular chaperone refers to a series of proteins involved in the inhibition of folding following the synthesis of a polypeptide chain and irreversible thermal denaturation of the enzyme.
- One of the therapies using this induction (production) of HSP expression is cancer thermoimmunotherapy.
- heating the whole body induces the generation of HSP and activates the immune function of the whole body.
- it improves the discrimination ability between cancer cells and normal cells, has no side effects, and kills only cancer cells. It is a cure to let you.
- HSP expression (production) of this HSP is induced as a biological defense system not only at high temperatures but also when exposed to external stresses such as external injury, radiation, and ultraviolet rays. That is, stress on a living body, such as high temperature and ultraviolet rays, denatures cellular proteins, forms insoluble precipitates, and damages cells. Therefore, the expression of HSP is induced for the purpose of preventing damage to such cells.
- Non-patent Document 3 neuronal cell death that is a cause of brain diseases such as cerebral infarction and Alzheimer's disease is caused by damage of heat shock protein (HSP) that protects cells.
- HSP heat shock protein
- Non-patent Document 4 an HSP inducer is effective for ulcerative colitis and Alzheimer's disease.
- HSP heat shock protein
- a component having an effective HSP70 expression inducing action is an extremely potent pharmaceutical.
- the present inventors have further studied and searched for various highly safe components that induce HSP70 expression for various plant components (herbal medicines).
- Eupatorium lindleyanum above-ground dry crude drug (Yabatsui) extract was found to enhance the expression induction of HSP70.
- extremely strong HSP70 expression induction was observed in components (fractions) obtained by fractionating Yabatsui ethanol extract using various column chromatography or high performance liquid chromatography (HPLC).
- HPLC high performance liquid chromatography
- Non-Patent Document 5 Non-Patent Document 5
- an object of the present invention is to provide a heat shock protein expression inducer comprising Euparinolide A and B which are sesquiterpene lactones.
- the present invention for solving this problem has the following configuration.
- a heat shock protein (HSP) expression inducer comprising Euparinolide A and / or B (Eupalinolide A and / or B);
- the present invention provides a heat shock protein (HSP) expression inducer, particularly an expression inducer of HSP70, comprising Euparinolide A and / or B (Eupalinolide A and / or B), which is a sesquiterpene lactone compound.
- HSP expression inducer provided by the present invention is extracted and isolated from the above-ground dry herbal medicine (Yabatsui) of Eupatorium lindleyanum, and the HSP70 produced by the HSP expression inducing action is tyrosinase, and It controls microphthalmia-related transcription factor (MITF) and suppresses melanin production, and is useful as a therapeutic agent for brain diseases such as cerebral infarction and Alzheimer's disease, and ulcerative colitis.
- MTF microphthalmia-related transcription factor
- Example 6 it is the figure which showed the melanin content which shows the melanin production inhibitory effect by IBMX irritation
- Example 6 it is the figure which showed the tyrosinase activity which shows the melanin production inhibitory effect by IBMX irritation
- the present invention is a heat shock protein (HSP) expression inducer composed of Euparinolide A and / or B (Eupalinolide A and / or B). Regulates tyrosinase and microphthalmia-related transcription factor (MITF) and exerts the effect of suppressing melanin production, brain diseases such as cerebral infarction and Alzheimer's disease caused by HSP damage, ulcerative colitis It is useful as a therapeutic agent.
- HSP heat shock protein
- MITF microphthalmia-related transcription factor
- Euparinolide A and / or B which is an HSP expression inducer provided by the present invention is shown below. That is, it is a compound represented by the following chemical formula (I) for Euparinolide A and a compound represented by the following Chemical Formula (II) for Euparinolide B.
- the Euparinolides A and B are preferably obtained by extraction and isolation from the above-ground dry herbal medicine (Yabatsui) of Eupatorium lindleyanum.
- Euparinolide A And / or B is also a heat shock protein (HSP) inducer extracted from the above-ground dry herbal medicine (Yabatsui) of Eupatorium lindleyanum.
- HSP heat shock protein
- Supreme dolphins are perennials that belong to the Asteraceae genus Hydribana, and grow all over Japan and Southeast Asia.
- This above-ground dry herbal medicine is called Yabatsui (Nomaoi) and is a crude drug ingredient used as an antipyretic / antidote in China.
- the extraction and isolation of Euparinolide A and / or B, which is an HSP inducer provided by the present invention, from Yabatsui can be specifically performed as follows. That is, the above-ground dry herbal medicine of Eupatorium lindleyanum (hereinafter referred to as “Yabatsui” unless otherwise specified) is subjected to ethanol extraction to obtain an ethanol extract.
- Yabatsui is commercially available as a crude drug, and can be obtained from, for example, Maruzen Pharmaceutical Co., Ltd.
- the extraction time cannot be generally limited, it may be about 1 to 5 hours, preferably about 2 hours.
- the extraction temperature is preferably 70 ° C. to a temperature around the boiling point of ethanol.
- the cotton plug is filtered after cooling to obtain an ethanol extract.
- This extraction operation is repeated 2 to 5 times, preferably about 3 to 4 times, and the combined ethanol extraction solution is concentrated under reduced pressure to obtain a crude ethanol extract of Yabatsui as a residue.
- the target Euparinolide A and / or B is isolated. Specifically, it can be carried out as follows. . First, a crude ethanol extract of Yabatsui is degreased with hexane. Specifically, a crude ethanol extract is suspended in a 90% methanol solution, transferred to a separatory funnel, and extracted with a small amount of hexane to perform a degreasing treatment. The 90% methanol layer subjected to the degreasing treatment is dried under reduced pressure to obtain a 90% methanol solution fraction (fraction A).
- the 90% methanol solution fraction (fraction A) obtained above was subjected to polystyrene gel column chromatography, specifically, MCl ⁇ GEL CHP-20P (Wako Pure Chemical Industries) column chromatography, Elution is performed sequentially with water, 50% methanol, a stepwise gradient method of methanol solution, and an acetone solution to obtain each elution fraction. Induction of HSP expression was strongly observed in the methanol-eluted fraction (fraction B) obtained in this operation.
- Methanol elution fraction (fraction B), in which HSP expression induction was strongly observed, was further subjected to ODS column chromatography, and eluted in a stepwise gradient method using 50% methanol solution, 75% methanol solution, and methanol solution sequentially. Each elution fraction was obtained. Induction of HSP expression was strongly observed in the 50% methanol elution fraction (fraction C) obtained in this procedure.
- fraction C 50% methanol elution fraction
- HPLC high performance liquid chromatography
- fraction D1 to D12 Twelve elution fractions (fractions D1 to D12) were obtained.
- eluted fractions D1 to D12 it was found that particularly the fraction D3 and the fraction D7 have a very strong HSP expression inducing action.
- the identification of the HSP expression-inducing agent was examined.
- the fraction D3 contains Euparinolide A as the main component and the fraction D7 contains Euparinolide B as the main component.
- the present invention also provides extraction of Euparinolide A and / or B (Eupalinolide A and / or B) comprising extraction and fractionation treatment from above-ground dry herbal medicine (Yabatsui) of Eupatorium lindleyanum. It is also a method.
- the HSP expression inducer provided by the present invention exhibits an inhibitory action on melanin production by inducing the expression of HSP70, among other HSPs. That is, HSP70 controls tyrosinase and microphthalmia-related transcription factor (MITF: microphthalmia transcription factor) involved in melanin production, and as a result, the production of melanin is suppressed and the brain caused by the damage of HSP. It is useful as a therapeutic agent for brain diseases such as infarction and Alzheimer's disease, and ulcerative colitis.
- the HSP expression inducer provided by the present invention is suitable for use as, for example, a therapeutic agent for cerebral infarction or Alzheimer's disease such as cerebral infarction or Alzheimer's disease, ulcerative colitis, melanin production-suppressing cosmetic, particularly whitening cosmetic. ing.
- the HSP expression inducer provided by the present invention is a pharmaceutical preparation for oral administration such as tablets, capsules and powders, and parenteral administration such as injections and infusions.
- Skin cosmetics such as milky lotion, cream, cosmetic liquid, pack, foundation cosmetics such as makeup base lotion and makeup base cream, foundations for each dosage form such as emulsion, oily, solid, eye color, cheek color And makeup cosmetics such as hand cream, leg cream, neck cream, body lotion and the like.
- the HSP expression inducer provided by the present invention contains Euparinolide A and / or B
- the extract of Yabatsui obtained by the above extraction / isolation operation may be contained as it is. Moreover, it can use in a desired form with the other conventional component accept
- the content of Euparinolide A and / or B which is an HSP expression inducer provided by the present invention, cannot be generally limited, and the purpose of prevention / amelioration of the disease state, the sex of the user, the body weight However, it cannot be generally limited by various conditions such as age, dosage form, type and degree of disease state, use site, number of uses.
- it when it is administered as an oral preparation, it can be applied at a dose of 0.1 to 100 ⁇ g / kg / day once a day or several times a day, but the amount used is not necessarily limited to this range. It is not a thing.
- the extract of Yabatoi obtained by the above extraction / isolation operation is contained as it is, it is contained in an amount converted to the amount of Euparinolide A and / or B contained in the extracted fraction. be able to.
- the HSP expression inducer provided by the present invention is used for the purpose of medical effects and also used as a melanin production-suppressing cosmetic.
- Example 1 Extraction and Isolation of Euparinolide A and / or B 10.1 g of Yabatsui [Eupatorium lindleyanum above-ground dry herbal medicine] obtained from Maruzen Pharmaceutical Co., Ltd. was placed in a 500 mL eggplant-shaped flask. , 100 mL of ethanol was added, and the mixture was refluxed for 2 hours on a 70 ° C. water bath. After cooling, a cotton plug was filtered to obtain an ethanol extract. After this reflux extraction operation was repeated three times, the obtained ethanol extracts were combined and concentrated under reduced pressure to obtain an ethanol extract (499 mg).
- This ethanol extract was suspended in 100 mL of 90% methanol, transferred to a separatory funnel, treated with 30 mL of hexane three times, and degreased.
- the 90% methanol solution layer was dried under reduced pressure using an evaporator to obtain 310 mg of a 90% methanol solution fraction (fraction A).
- the 90% methanol solution fraction (fraction A) was The activity was strong.
- the methanol-eluted fraction (fraction B) having a high activity was subjected to Chromatorex ODS column chromatography ( ⁇ 15 mm ⁇ 150 mm), and stepwise from water to methanol (1: 0 ⁇ 0: 1, v / v). Elution was performed by a gradient method, whereby a 50% methanol-eluted fraction (fraction C) (49 mg) that could be eluted with a 50% methanol solution, a 75% methanol solution, and a methanol solution was obtained. Of the three fractions obtained, the 50% methanol elution fraction (Fraction C) was the most active.
- Example 2 Expression of HSP and cell viability B16 cells derived from mouse melanoma (RIKEN BioResource Center) were combined with various concentrations of Euparinolide A or B in DMEM medium (10% fetal bovine serum, 100 U / mL penicillin). , 100 U / mL streptomycin) at 37 ° C./5% CO 2 atmosphere for 24 hours.
- the expression level of HSP70 was measured by Western blotting using an anti-HSP70 antibody (Stressgen) after subjecting the cell culture solution to SDS-PAGE electrophoresis.
- Cell viability was measured by immunoblotting of whole cell extracts together with anti-HSP70 antibody or actin according to the MTT method.
- Example 3 Effect of HSP70 expression on AGS cells Human gastric mucosal epithelial cells (AGS cells) were combined with each concentration of Euparinolide A or B in RPMI medium (10% fetal bovine serum, 100 U / mL penicillin, 100 U / Incubation was carried out for 24 hours at 37 ° C./5% CO 2 atmosphere.
- the expression level of HSP70 was measured by Western blotting using an anti-HSP70 antibody (Stressgen) after subjecting the cell culture solution to SDS-PAGE electrophoresis. Cell viability was measured by immunoblotting of whole cell extracts together with anti-HSP70 antibody or actin according to the MTT method.
- Example 4 Effect of enhancing HSP70 expression with other stress AGS cells were combined with each concentration of Euparinolide A or B (0, 1 and 2 ⁇ g / mL) in RPMI medium (10% fetal bovine serum, 100 U / mL). Of penicillin, 100 U / mL streptomycin) at 37 ° C./5% CO 2 atmosphere for 3 hours. Next, a heat shock of 40 ° C./1 hour was applied, and then recovery from the heat shock was performed at 37 ° C. for 6 hours. On the other hand, AGS cells were similarly cultured with 3.5% ethanol as stress for 24 hours.
- HSP70 The expression level of HSP70 in AGS cells cultured without stress and each AGS cell cultured with heat shock stress or alcohol stress was subjected to SDS-PAGE electrophoresis in the same manner as in the above Examples. Thereafter, the expression level of HSP70 was measured by Western blotting using an anti-HSP70 antibody (Stressgen). Cell viability was determined by immunoblotting of whole cell extracts together with anti-HSP70 antibody or actin according to the MTT method.
- Example 5 Cytoprotective effect of Euparinolide A and B when exposed to stress AGS cells were pretreated with each Euparinolide A or B concentration (0, 2.5 and 5.0 ⁇ g / mL). Washed with fresh medium, and cultured in RPMI medium (10% fetal bovine serum, 100 U / mL penicillin, 100 U / mL streptomycin) at 37 ° C./5% CO 2 atmosphere for 18 hours. Finally, the cells were cultured with 3.5% ethanol or 15 ⁇ M menadione for 24 hours. The cell viability was determined by the MTT method as in the previous example. Apoptotic cells (cells in sub-G1) were counted by fluorescence activated cell sorting, and the values were expressed as mean ⁇ SD.
- FIG. 5 shows the result of culturing with 3.5% ethanol, and it is found that Euparinolide A or B increases the cell viability in a concentration-dependent manner.
- FIG. 6 shows the results of culturing with 15 ⁇ M menadione, and it is found that Euparinolide A or B increases the cell viability in a concentration-dependent manner as in the case of 3.5% ethanol.
- Example 6 Effect of inhibiting melanin production by IBMX stimulation with Euparinolide A and B B16 cells were cultured together with Euparinolide A or B concentrations (5.0 and 10.0 ⁇ g / mL). The cells were further cultured for 72 hours or 48 hours with or without the addition of 100 ⁇ M IBMX (3-isobutyl-1-methylxanthine: Sigma). The protein amount of each sample was determined by Bio-Rad protein assay kit, and after aligning the protein amount with the same amount, the absorbance at 490 nm was measured with a plate reader (Fluostar Galaxy). The amount of melanin during cell extraction was measured and displayed in comparison with the control. In addition, tyrosinase activity was displayed in comparison with the control.
- IBMX 3-isobutyl-1-methylxanthine
- FIGS. FIG. 7 shows the melanin content
- FIG. 8 shows the tyrosinase activity.
- Euparinolide A or B suppresses melanin production and reduces tyrosinase activity.
- the suppression of melanin production and the decrease in tyrosinase activity by addition of IBMX are strong.
- Example 7 Confirmation of HSP70 expression
- a human fibroblast cell line (NBIRGB: RIKEN, BioResource Center) was seeded so as to obtain a cell suspension of 1 ⁇ 10 2 cells / mL. Culturing was performed at 37 ° C. for 24 hours in a 5% carbon dioxide atmosphere. The immunoblot method described above with Euparinolide A or B extracted and isolated in Example 1 above, and the Yabatsui solvent extract (fractions A to C) obtained in the separation process of Example 1 in this culture solution Then, induction of HSP70 expression was examined. Evaluation was expressed as the minimum effective concentration (MEC 2.0 ( ⁇ g / ml)) that doubled the expression of HSP70.
- MEC 2.0 ⁇ g / ml
- HSP expression by Euparinolide A and / or B which is an HSP expression inducer
- HSP70 expression induction is caused by brain diseases such as cerebral infarction and Alzheimer's disease, ulcerative colitis, etc. It is useful as a pharmaceutical agent as a therapeutic agent, and further becomes a melanin production-suppressing cosmetic.
- Application Example 2 Injection Euparinolide A or B was dissolved in sterile purified water for injection together with sodium chloride and polysorbate 20 as a solubilizing agent to obtain an injection containing 1%.
- Application Example 3 1% (w / w) of euparinolide A or B, 10% (w / w) sucrose, 0.05% (w / w) benzalkonium chloride in 5% aqueous xylitol solution And then freeze-dried.
- An intravenous injection preparation was obtained by adding 0.5% carmellose or water for injection separately filled into the resulting freeze-dried preparation.
- the present invention provides a heat shock protein (HSP) expression inducer comprising Euparinolide A and / or B.
- HSP heat shock protein
- Euparinolide A and / or B provided by the present invention induces the expression of HSP, particularly HSP70, and is useful as a therapeutic agent for brain diseases such as cerebral infarction and Alzheimer's disease caused by HSP damage, ulcerative colitis, etc.
- HSP70 whose expression is induced controls melanin production, and as a result, suppresses the production of melanin. As a result, it is possible to provide a cosmetic having an effect of preventing and improving stains. It is very specific.
Abstract
Provided is a heat shock protein expression inducer based on a heat shock protein (HSP) expression-inducing action. The present invention is a heat shock protein (HSP) expression inducer comprising eupalinolide A and/or B, the heat shock protein expression inducer being obtained by extracting eupalinolide A and/or B from the dried aerial part of the herb Eupatorium lindleyanum, and is also a method for extracting eupalinolide A and/or B, the method comprising fractionating an ethanol extract obtained by the ethanol extraction of Eupatorium lindleyanum, the fractionation being performed by a variety of column chromatography and HPLC techniques.
Description
本発明は、医薬品として有用な熱ショックタンパク質（ヒートショックプロテイン）の発現誘導剤に関する。
The present invention relates to an expression inducer of heat shock protein (heat shock protein) useful as a pharmaceutical product.
細胞、組織あるいは固体においては、一般的な生理的温度より３℃以上高い温度に晒されたときに、生体の防御システムの一つとして、特異的タンパク質の発現（産生）が誘導されることが知られている。
このタンパク質は、ＳＤＳ－ＰＡＧＥ（ドデシル硫酸ナトリウム－ポリアクリルアミドゲル電気泳動法）によって測定した場合、分子量範囲１０～１１０ＫＤａを有する一群のタンパク質として存在しており、熱ショックタンパク質（ヒートショックプロテイン：heat shock proteins、以下、「ＨＳＰ」と称する場合もある）と呼ばれている。 In cells, tissues, or solids, expression (production) of specific proteins is induced as one of the defense systems of living bodies when exposed to a temperature of 3 ° C. or higher than a general physiological temperature. Are known.
This protein exists as a group of proteins having a molecular weight range of 10 to 110 KDa as measured by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and is a heat shock protein (heat shock protein: heat shock protein). proteins, hereinafter referred to as “HSP”).
このタンパク質は、ＳＤＳ－ＰＡＧＥ（ドデシル硫酸ナトリウム－ポリアクリルアミドゲル電気泳動法）によって測定した場合、分子量範囲１０～１１０ＫＤａを有する一群のタンパク質として存在しており、熱ショックタンパク質（ヒートショックプロテイン：heat shock proteins、以下、「ＨＳＰ」と称する場合もある）と呼ばれている。 In cells, tissues, or solids, expression (production) of specific proteins is induced as one of the defense systems of living bodies when exposed to a temperature of 3 ° C. or higher than a general physiological temperature. Are known.
This protein exists as a group of proteins having a molecular weight range of 10 to 110 KDa as measured by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and is a heat shock protein (heat shock protein: heat shock protein). proteins, hereinafter referred to as “HSP”).
ＨＳＰは、その分子量の相違により幾つかのファミリーが形成されており、例えば、ＨＳＰ９０ファミリー（分子量：９０ｋＤａ以上１１０ｋＤａ以下）、ＨＳＰ７０ファミリー（分子量：７０ｋＤａ以上８０ｋＤａ未満）、ＨＳＰ６０ファミリー（分子量：６０ｋＤａ以上７０ｋＤａ未満）、及び低分子量ＨＳＰファミリー（分子量：６０ｋＤａ未満）のように分類されている。
HSP has several families due to the difference in molecular weight. For example, HSP90 family (molecular weight: 90 kDa to 110 kDa), HSP70 family (molecular weight: 70 kDa to less than 80 kDa), HSP60 family (molecular weight: 60 kDa to 70 kDa). Less), and the low molecular weight HSP family (molecular weight: less than 60 kDa).
ＨＳＰの機能は多岐にわたっており、例えば、ＨＳＰ７０及びＨＳＰ６０ファミリーは、変性タンパク質に結合して、天然のフォールディング（高次構造・折り畳み構造）に巻き戻す作用や、第三のタンパク質や核酸との会合、細胞内での局在化や膜透過への関与など、いわゆる分子シャペロンと呼ばれる機能を担っていることが明らかにされている（非特許文献１及び２）。
なお、分子シャペロンとは、ポリペプチド鎖の合成に引き続くフォールディングや酵素の不可逆的な熱変性の抑制に関与している一連の蛋白質をさす。 The functions of HSP are diverse. For example, the HSP70 and HSP60 families bind to denatured proteins and unwind them to natural folding (higher-order structure / folded structure), and are associated with third proteins and nucleic acids. It has been clarified that it has functions called so-called molecular chaperones, such as intracellular localization and involvement in membrane permeation (Non-patentDocuments 1 and 2).
The molecular chaperone refers to a series of proteins involved in the inhibition of folding following the synthesis of a polypeptide chain and irreversible thermal denaturation of the enzyme.
なお、分子シャペロンとは、ポリペプチド鎖の合成に引き続くフォールディングや酵素の不可逆的な熱変性の抑制に関与している一連の蛋白質をさす。 The functions of HSP are diverse. For example, the HSP70 and HSP60 families bind to denatured proteins and unwind them to natural folding (higher-order structure / folded structure), and are associated with third proteins and nucleic acids. It has been clarified that it has functions called so-called molecular chaperones, such as intracellular localization and involvement in membrane permeation (Non-patent
The molecular chaperone refers to a series of proteins involved in the inhibition of folding following the synthesis of a polypeptide chain and irreversible thermal denaturation of the enzyme.
このＨＳＰ発現の誘導（産生）を利用した療法の一つとして、癌温熱免疫療法がある。すなわち、全身を加温することによりＨＳＰの生成を誘導し、全身の免疫機能を活発にするのと同時に、がん細胞と正常細胞の識別能力を向上させ、副作用が無く、癌細胞のみを死滅させる治療法である。
One of the therapies using this induction (production) of HSP expression is cancer thermoimmunotherapy. In other words, heating the whole body induces the generation of HSP and activates the immune function of the whole body. At the same time, it improves the discrimination ability between cancer cells and normal cells, has no side effects, and kills only cancer cells. It is a cure to let you.
ところで、このＨＳＰは、高温ばかりではなく、外的傷害、放射線、紫外線などの外界からのストレスに晒された場合にも、生体防御システムとして、その発現（産生）が誘導される。
すなわち、高温、紫外線などの生体に対するストレスは、細胞のタンパク質を変性させ、不溶性沈殿を形成して細胞にダメージを与える。したがって、かかる細胞へのダメージを防御する目的で、ＨＳＰの発現が誘導されることとなる。 By the way, expression (production) of this HSP is induced as a biological defense system not only at high temperatures but also when exposed to external stresses such as external injury, radiation, and ultraviolet rays.
That is, stress on a living body, such as high temperature and ultraviolet rays, denatures cellular proteins, forms insoluble precipitates, and damages cells. Therefore, the expression of HSP is induced for the purpose of preventing damage to such cells.
すなわち、高温、紫外線などの生体に対するストレスは、細胞のタンパク質を変性させ、不溶性沈殿を形成して細胞にダメージを与える。したがって、かかる細胞へのダメージを防御する目的で、ＨＳＰの発現が誘導されることとなる。 By the way, expression (production) of this HSP is induced as a biological defense system not only at high temperatures but also when exposed to external stresses such as external injury, radiation, and ultraviolet rays.
That is, stress on a living body, such as high temperature and ultraviolet rays, denatures cellular proteins, forms insoluble precipitates, and damages cells. Therefore, the expression of HSP is induced for the purpose of preventing damage to such cells.
かかる観点から、水生プランクトンであるアルテミアの孵化直前の耐久卵から水抽出した活性エキス（アルテミアエキス）成分が、ヒト皮膚細胞においてＨＳＰ７０の産生を誘発することが見出され、かかる作用を利用した抗皮膚ストレス（ストレス防御）用化粧料が提案されている（特許文献１）。
From this point of view, it has been found that an active extract (artemia extract) component extracted from a durable egg immediately before hatching of artemia, an aquatic plankton, induces the production of HSP70 in human skin cells. Cosmetics for skin stress (stress protection) have been proposed (Patent Document 1).
更に最近、脳梗塞やアルツハイマー病など脳疾患の原因である神経細胞死が、細胞を保護する熱ショックタンパク質（ＨＳＰ）の損傷によって生じることが明らかにされた（非特許文献３）。
また、ＨＳＰ誘導物質が潰瘍性大腸炎やアルツハイマー病に有効であることが示されている（非特許文献４）。 More recently, it has been clarified that neuronal cell death that is a cause of brain diseases such as cerebral infarction and Alzheimer's disease is caused by damage of heat shock protein (HSP) that protects cells (Non-patent Document 3).
Moreover, it has been shown that an HSP inducer is effective for ulcerative colitis and Alzheimer's disease (Non-patent Document 4).
また、ＨＳＰ誘導物質が潰瘍性大腸炎やアルツハイマー病に有効であることが示されている（非特許文献４）。 More recently, it has been clarified that neuronal cell death that is a cause of brain diseases such as cerebral infarction and Alzheimer's disease is caused by damage of heat shock protein (HSP) that protects cells (Non-patent Document 3).
Moreover, it has been shown that an HSP inducer is effective for ulcerative colitis and Alzheimer's disease (Non-patent Document 4).
したがって、ヒートショックプロテイン（ＨＳＰ）の発現誘導は、種々の病態の治療に有効であり、また、メラニン産生を抑制するものであり、効果的なＨＳＰ７０発現誘導作用を有する成分は、極めて有力な医薬品、或いは化粧品となる。
かかる観点より本発明者は、更に検討を進め、各種植物成分（生薬）について、ＨＳＰ７０発現を誘導する安全性の高い成分の検索を行ってきていたが、その検討の中で、植物成分としてサワヒヨドリ（Eupatorium lindleyanum）の地上部の乾燥生薬（ヤバツイ）の抽出物が、ＨＳＰ７０の発現誘導を強化させることを見出した。
特に、ヤバツイのエタノール抽出エキスを、各種カラムクロマトグラフィー、或いは高速液体クロマトグラフィー（ＨＰＬＣ）等で分画して得た成分（画分）には、極めて強いＨＳＰ７０の発現誘導が認められることを確認し、その画分の主成分を検討したところ、セスキテルペンラクトン化合物であるユーパリノライドＡ（Eupalinolide Ａ）、及びユーパリノライドＢ（Eupalinolide Ｂ）であることが判明した。 Therefore, induction of heat shock protein (HSP) expression is effective in the treatment of various pathological conditions and suppresses melanin production, and a component having an effective HSP70 expression inducing action is an extremely potent pharmaceutical. Or cosmetics.
From this point of view, the present inventors have further studied and searched for various highly safe components that induce HSP70 expression for various plant components (herbal medicines). (Eupatorium lindleyanum) above-ground dry crude drug (Yabatsui) extract was found to enhance the expression induction of HSP70.
In particular, it was confirmed that extremely strong HSP70 expression induction was observed in components (fractions) obtained by fractionating Yabatsui ethanol extract using various column chromatography or high performance liquid chromatography (HPLC). When the main component of the fraction was examined, it was found to be Euparinolide A (Eupalinolide A) and Euparinolide B (Eupalinolide B), which are sesquiterpene lactone compounds.
かかる観点より本発明者は、更に検討を進め、各種植物成分（生薬）について、ＨＳＰ７０発現を誘導する安全性の高い成分の検索を行ってきていたが、その検討の中で、植物成分としてサワヒヨドリ（Eupatorium lindleyanum）の地上部の乾燥生薬（ヤバツイ）の抽出物が、ＨＳＰ７０の発現誘導を強化させることを見出した。
特に、ヤバツイのエタノール抽出エキスを、各種カラムクロマトグラフィー、或いは高速液体クロマトグラフィー（ＨＰＬＣ）等で分画して得た成分（画分）には、極めて強いＨＳＰ７０の発現誘導が認められることを確認し、その画分の主成分を検討したところ、セスキテルペンラクトン化合物であるユーパリノライドＡ（Eupalinolide Ａ）、及びユーパリノライドＢ（Eupalinolide Ｂ）であることが判明した。 Therefore, induction of heat shock protein (HSP) expression is effective in the treatment of various pathological conditions and suppresses melanin production, and a component having an effective HSP70 expression inducing action is an extremely potent pharmaceutical. Or cosmetics.
From this point of view, the present inventors have further studied and searched for various highly safe components that induce HSP70 expression for various plant components (herbal medicines). (Eupatorium lindleyanum) above-ground dry crude drug (Yabatsui) extract was found to enhance the expression induction of HSP70.
In particular, it was confirmed that extremely strong HSP70 expression induction was observed in components (fractions) obtained by fractionating Yabatsui ethanol extract using various column chromatography or high performance liquid chromatography (HPLC). When the main component of the fraction was examined, it was found to be Euparinolide A (Eupalinolide A) and Euparinolide B (Eupalinolide B), which are sesquiterpene lactone compounds.
これらセスキテルペンラクトンであるユーパリノライドＡ及びＢは、すでに公知の化合物であるが、これまでの研究では、その構造決定と、細胞毒性に関する報告（非特許文献５）のみであって、このものにＨＳＰの発現を誘導する作用があることについては、一切記載されていない。
These esquiterpene lactones, Euparinolides A and B, are already known compounds. However, in the research so far, only the structure determination and cytotoxicity report (Non-Patent Document 5) have been reported. No mention is made of that it has an effect of inducing the expression of HSP.
したがって、本発明は、セスキテルペンラクトンであるユーパリノライドＡ及びＢからなるヒートショックプロテイン発現誘導剤を提供することを課題とする。
Therefore, an object of the present invention is to provide a heat shock protein expression inducer comprising Euparinolide A and B which are sesquiterpene lactones.
かかる課題を解決するための本発明は、その態様は、以下の構成からなる。
（１）ユーパリノライドＡ及び／又はＢ（Eupalinolide Ａ及び／又はＢ）からなるヒートショックプロテイン（ＨＳＰ）発現誘導剤；
（２）ユーパリノライドＡ及び／又はＢ（Eupalinolide Ａ及び／又はＢ）が、サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（ヤバツイ）から抽出したものである上記（１）に記載のヒートショックプロテイン（ＨＳＰ）発現誘導剤；
（３）サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（ヤバツイ）より、以下の抽出・分画処理することからなるユーパリノライドＡ及び／又はＢ（Eupalinolide Ａ及び／又はＢ）の抽出方法であって、
（ａ）サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（ヤバツイ）をエタノール抽出して得た抽出物を、ヘキサンにより脱脂処理を行った後、９０％メタノール溶液に溶解して得た９０％メタノール溶液画分（画分Ａ）を得、
（ｂ）次いで、上記で得た９０％メタノール溶液画分（画分Ａ）を、ＭＣｌ・ＧＥＬ ＣＨＰ－２０Ｐカラムクロマトグラフィーに付し、順次、水溶出－５０％メタノール溶出－メタノール溶出－アセトン溶出を行い、メタノール溶出画分（画分Ｂ）を得、
（ｃ）上記で得たメタノール溶出画分（画分Ｂ）を、更にＯＤＳカラムクロマトグラフィーに付し、順次５０％メタノール溶液、７５％メタノール溶液、及びメタノール溶液で溶出を行い、５０％メタノール溶出画分（画分Ｃ）を得、
（ｄ）最後に、上記で得た５０％メタノール溶出画分（画分Ｃ）をＨＰＬＣ（カラム：ＡＲ－ＩＩ ＯＤＳ）に付し、４０％メタノールにて溶出すること、
からなる抽出方法；
である。 The present invention for solving this problem has the following configuration.
(1) a heat shock protein (HSP) expression inducer comprising Euparinolide A and / or B (Eupalinolide A and / or B);
(2) The heat shock protein according to (1) above, wherein Euparinolide A and / or B (Eupalinolide A and / or B) is extracted from the above-ground dry herbal medicine (Yabatsui) of Eupatorium lindleyanum (HSP) expression inducer;
(3) It is an extraction method of Euparinolide A and / or B (Eupalinolide A and / or B) comprising the following extraction and fractionation treatment from the above-ground dry herbal medicine (Yabatsui) of Eupatorium lindleyanum And
(A) 90% methanol solution obtained by degreasing the extract obtained by ethanol extraction of dry crude herb (Yabatsui) from Eupatorium lindleyanum with hexane and then dissolving in 90% methanol solution To obtain a fraction (fraction A)
(B) Next, the 90% methanol solution fraction (fraction A) obtained above was subjected to MCl · GEL CHP-20P column chromatography, followed by water elution, 50% methanol elution, methanol elution, and acetone elution. To obtain a methanol elution fraction (fraction B),
(C) The methanol elution fraction (Fraction B) obtained above was further subjected to ODS column chromatography, and the elution was carried out sequentially with 50% methanol solution, 75% methanol solution and methanol solution, and 50% methanol elution. Fraction (Fraction C)
(D) Finally, the 50% methanol elution fraction obtained above (fraction C) is subjected to HPLC (column: AR-II ODS) and eluted with 40% methanol.
An extraction method comprising:
It is.
（１）ユーパリノライドＡ及び／又はＢ（Eupalinolide Ａ及び／又はＢ）からなるヒートショックプロテイン（ＨＳＰ）発現誘導剤；
（２）ユーパリノライドＡ及び／又はＢ（Eupalinolide Ａ及び／又はＢ）が、サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（ヤバツイ）から抽出したものである上記（１）に記載のヒートショックプロテイン（ＨＳＰ）発現誘導剤；
（３）サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（ヤバツイ）より、以下の抽出・分画処理することからなるユーパリノライドＡ及び／又はＢ（Eupalinolide Ａ及び／又はＢ）の抽出方法であって、
（ａ）サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（ヤバツイ）をエタノール抽出して得た抽出物を、ヘキサンにより脱脂処理を行った後、９０％メタノール溶液に溶解して得た９０％メタノール溶液画分（画分Ａ）を得、
（ｂ）次いで、上記で得た９０％メタノール溶液画分（画分Ａ）を、ＭＣｌ・ＧＥＬ ＣＨＰ－２０Ｐカラムクロマトグラフィーに付し、順次、水溶出－５０％メタノール溶出－メタノール溶出－アセトン溶出を行い、メタノール溶出画分（画分Ｂ）を得、
（ｃ）上記で得たメタノール溶出画分（画分Ｂ）を、更にＯＤＳカラムクロマトグラフィーに付し、順次５０％メタノール溶液、７５％メタノール溶液、及びメタノール溶液で溶出を行い、５０％メタノール溶出画分（画分Ｃ）を得、
（ｄ）最後に、上記で得た５０％メタノール溶出画分（画分Ｃ）をＨＰＬＣ（カラム：ＡＲ－ＩＩ ＯＤＳ）に付し、４０％メタノールにて溶出すること、
からなる抽出方法；
である。 The present invention for solving this problem has the following configuration.
(1) a heat shock protein (HSP) expression inducer comprising Euparinolide A and / or B (Eupalinolide A and / or B);
(2) The heat shock protein according to (1) above, wherein Euparinolide A and / or B (Eupalinolide A and / or B) is extracted from the above-ground dry herbal medicine (Yabatsui) of Eupatorium lindleyanum (HSP) expression inducer;
(3) It is an extraction method of Euparinolide A and / or B (Eupalinolide A and / or B) comprising the following extraction and fractionation treatment from the above-ground dry herbal medicine (Yabatsui) of Eupatorium lindleyanum And
(A) 90% methanol solution obtained by degreasing the extract obtained by ethanol extraction of dry crude herb (Yabatsui) from Eupatorium lindleyanum with hexane and then dissolving in 90% methanol solution To obtain a fraction (fraction A)
(B) Next, the 90% methanol solution fraction (fraction A) obtained above was subjected to MCl · GEL CHP-20P column chromatography, followed by water elution, 50% methanol elution, methanol elution, and acetone elution. To obtain a methanol elution fraction (fraction B),
(C) The methanol elution fraction (Fraction B) obtained above was further subjected to ODS column chromatography, and the elution was carried out sequentially with 50% methanol solution, 75% methanol solution and methanol solution, and 50% methanol elution. Fraction (Fraction C)
(D) Finally, the 50% methanol elution fraction obtained above (fraction C) is subjected to HPLC (column: AR-II ODS) and eluted with 40% methanol.
An extraction method comprising:
It is.
本発明により、セスキテルペンラクトン化合物であるユーパリノライドＡ及び／又はＢ（Eupalinolide Ａ及び／又はＢ）からなるヒートショックプロテイン（ＨＳＰ）発現誘導剤、特にＨＳＰ７０の発現誘導剤が提供される。
本発明が提供するＨＳＰ発現誘導剤は、サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（ヤバツイ）から抽出・単離されたものであり、ＨＳＰの発現誘導作用により、特に生成したＨＳＰ７０がチロシナーゼ、及び小眼球症関連転写因子（ＭＩＴＦ：microphthalmia転写因子）を制御させ、メラニン産生を抑制するとともに、脳梗塞やアルツハイマー病など脳疾患、潰瘍性大腸炎などの治療剤として有用である。 The present invention provides a heat shock protein (HSP) expression inducer, particularly an expression inducer of HSP70, comprising Euparinolide A and / or B (Eupalinolide A and / or B), which is a sesquiterpene lactone compound.
The HSP expression inducer provided by the present invention is extracted and isolated from the above-ground dry herbal medicine (Yabatsui) of Eupatorium lindleyanum, and the HSP70 produced by the HSP expression inducing action is tyrosinase, and It controls microphthalmia-related transcription factor (MITF) and suppresses melanin production, and is useful as a therapeutic agent for brain diseases such as cerebral infarction and Alzheimer's disease, and ulcerative colitis.
本発明が提供するＨＳＰ発現誘導剤は、サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（ヤバツイ）から抽出・単離されたものであり、ＨＳＰの発現誘導作用により、特に生成したＨＳＰ７０がチロシナーゼ、及び小眼球症関連転写因子（ＭＩＴＦ：microphthalmia転写因子）を制御させ、メラニン産生を抑制するとともに、脳梗塞やアルツハイマー病など脳疾患、潰瘍性大腸炎などの治療剤として有用である。 The present invention provides a heat shock protein (HSP) expression inducer, particularly an expression inducer of HSP70, comprising Euparinolide A and / or B (Eupalinolide A and / or B), which is a sesquiterpene lactone compound.
The HSP expression inducer provided by the present invention is extracted and isolated from the above-ground dry herbal medicine (Yabatsui) of Eupatorium lindleyanum, and the HSP70 produced by the HSP expression inducing action is tyrosinase, and It controls microphthalmia-related transcription factor (MITF) and suppresses melanin production, and is useful as a therapeutic agent for brain diseases such as cerebral infarction and Alzheimer's disease, and ulcerative colitis.
本発明は、上記したように、その基本は、ユーパリノライドＡ及び／又はＢ（Eupalinolide Ａ及び／又はＢ）からなるヒートショックプロテイン（ＨＳＰ）発現誘導剤であって、特に、ＨＳＰ７０の生成により、チロシナーゼ及び小眼球症関連転写因子（ＭＩＴＦ：microphthalmia転写因子）を制御させ、メラニン産生を抑制する効果を発揮するとともに、ＨＳＰの損傷によって生じる脳梗塞やアルツハイマー病などの脳疾患、潰瘍性大腸炎などの治療剤として有用である。
As described above, the present invention is a heat shock protein (HSP) expression inducer composed of Euparinolide A and / or B (Eupalinolide A and / or B). Regulates tyrosinase and microphthalmia-related transcription factor (MITF) and exerts the effect of suppressing melanin production, brain diseases such as cerebral infarction and Alzheimer's disease caused by HSP damage, ulcerative colitis It is useful as a therapeutic agent.
本発明が提供するＨＳＰ発現誘導剤であるユーパリノライドＡ及び／又はＢ（Eupalinolide Ａ及び／又はＢ）の化学式を以下に示した。
すなわち、ユーパリノライドＡにあっては下記化学式（Ｉ）、ユーパリノライドＢにあっては下記化学式（ＩＩ）で示される化合物である。 The chemical formula of Euparinolide A and / or B (Eupalinolide A and / or B) which is an HSP expression inducer provided by the present invention is shown below.
That is, it is a compound represented by the following chemical formula (I) for Euparinolide A and a compound represented by the following Chemical Formula (II) for Euparinolide B.
すなわち、ユーパリノライドＡにあっては下記化学式（Ｉ）、ユーパリノライドＢにあっては下記化学式（ＩＩ）で示される化合物である。 The chemical formula of Euparinolide A and / or B (Eupalinolide A and / or B) which is an HSP expression inducer provided by the present invention is shown below.
That is, it is a compound represented by the following chemical formula (I) for Euparinolide A and a compound represented by the following Chemical Formula (II) for Euparinolide B.
両者は同一の分子式（Ｃ２４Ｈ３０Ｏ９）を有し、その化学構造式も極めて類似するものであるが、立体的構造として、その４位において、ユーパリノライドＡがＺ型を保持しているのに対して、ユーパリノライドＢがＥ型を保持している点で相違するのみである。
Both have the same molecular formula (C 24 H 30 O 9 ), and their chemical structural formulas are also very similar, but as a steric structure, Euparinolide A retains the Z form at the 4-position. However, the only difference is that Euparinolide B retains the E type.
このユーパリノライドＡ及びＢは、好ましくは、サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（ヤバツイ）から抽出・単離することにより得ることができ、本発明の一態様としては、ユーパリノライドＡ及び／又はＢが、サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（ヤバツイ）から抽出したものであるヒートショックプロテイン（ＨＳＰ）誘導剤でもある。
The Euparinolides A and B are preferably obtained by extraction and isolation from the above-ground dry herbal medicine (Yabatsui) of Eupatorium lindleyanum. As an aspect of the present invention, Euparinolide A And / or B is also a heat shock protein (HSP) inducer extracted from the above-ground dry herbal medicine (Yabatsui) of Eupatorium lindleyanum.
サワヒヨドリ（Eupatorium lindleyanum）は、キク科ヒヨドリバナ属植物に属し、日本全国、東南アジアに生育する多年草である。この地上部乾燥生薬は、ヤバツイ（野馬追）と称され、中国においては解熱・解毒薬として使用されている生薬成分である。
Supreme dolphins (Eupatorium lindleyanum) are perennials that belong to the Asteraceae genus Hydribana, and grow all over Japan and Southeast Asia. This above-ground dry herbal medicine is called Yabatsui (Nomaoi) and is a crude drug ingredient used as an antipyretic / antidote in China.
本願発明が提供するＨＳＰ誘導剤であるユーパリノライドＡ及び／又はＢのヤバツイからの抽出、単離は、具体的には以下のようにして行うことができる。
すなわち、サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（以下、特に断らない限り、「ヤバツイ」と称する。）をエタノール抽出してエタノール抽出物を得る。
なお、ヤバツイは、生薬として市販されており、例えば、丸善製薬（株）から入手することができる。
抽出にあたっては、ヤバツイ１００ｇに対して、エタノールを０．５～２Ｌ程度加え、加熱還流条件下で抽出を行うのがよい。抽出時間は一概に限定できないが、１～５時間程度、好ましくは２時間程度でよい。また、抽出温度は、７０℃～エタノールの沸点付近の温度で行うのがよい。抽出操作を完了した段階で、冷却後綿栓濾過を行い、エタノール抽出液を得る。
この抽出操作を２～５回、好ましくは３～４回程度繰り返し、併せたエタノール抽出溶液を減圧下に濃縮し、残渣として、ヤバツイの粗エタノール抽出物を得る。 The extraction and isolation of Euparinolide A and / or B, which is an HSP inducer provided by the present invention, from Yabatsui can be specifically performed as follows.
That is, the above-ground dry herbal medicine of Eupatorium lindleyanum (hereinafter referred to as “Yabatsui” unless otherwise specified) is subjected to ethanol extraction to obtain an ethanol extract.
Yabatsui is commercially available as a crude drug, and can be obtained from, for example, Maruzen Pharmaceutical Co., Ltd.
In the extraction, it is preferable to add about 0.5 to 2 L of ethanol to 100 g of Yabatsui and perform the extraction under heating and refluxing conditions. Although the extraction time cannot be generally limited, it may be about 1 to 5 hours, preferably about 2 hours. The extraction temperature is preferably 70 ° C. to a temperature around the boiling point of ethanol. When the extraction operation is completed, the cotton plug is filtered after cooling to obtain an ethanol extract.
This extraction operation is repeated 2 to 5 times, preferably about 3 to 4 times, and the combined ethanol extraction solution is concentrated under reduced pressure to obtain a crude ethanol extract of Yabatsui as a residue.
すなわち、サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（以下、特に断らない限り、「ヤバツイ」と称する。）をエタノール抽出してエタノール抽出物を得る。
なお、ヤバツイは、生薬として市販されており、例えば、丸善製薬（株）から入手することができる。
抽出にあたっては、ヤバツイ１００ｇに対して、エタノールを０．５～２Ｌ程度加え、加熱還流条件下で抽出を行うのがよい。抽出時間は一概に限定できないが、１～５時間程度、好ましくは２時間程度でよい。また、抽出温度は、７０℃～エタノールの沸点付近の温度で行うのがよい。抽出操作を完了した段階で、冷却後綿栓濾過を行い、エタノール抽出液を得る。
この抽出操作を２～５回、好ましくは３～４回程度繰り返し、併せたエタノール抽出溶液を減圧下に濃縮し、残渣として、ヤバツイの粗エタノール抽出物を得る。 The extraction and isolation of Euparinolide A and / or B, which is an HSP inducer provided by the present invention, from Yabatsui can be specifically performed as follows.
That is, the above-ground dry herbal medicine of Eupatorium lindleyanum (hereinafter referred to as “Yabatsui” unless otherwise specified) is subjected to ethanol extraction to obtain an ethanol extract.
Yabatsui is commercially available as a crude drug, and can be obtained from, for example, Maruzen Pharmaceutical Co., Ltd.
In the extraction, it is preferable to add about 0.5 to 2 L of ethanol to 100 g of Yabatsui and perform the extraction under heating and refluxing conditions. Although the extraction time cannot be generally limited, it may be about 1 to 5 hours, preferably about 2 hours. The extraction temperature is preferably 70 ° C. to a temperature around the boiling point of ethanol. When the extraction operation is completed, the cotton plug is filtered after cooling to obtain an ethanol extract.
This extraction operation is repeated 2 to 5 times, preferably about 3 to 4 times, and the combined ethanol extraction solution is concentrated under reduced pressure to obtain a crude ethanol extract of Yabatsui as a residue.
上記で得たヤバツイの粗エタノール抽出物を分画操作することにより、目的とするユーパリノライドＡ及び／又はＢを単離するのであるが、具体的には以下のようにして行うことができる。
最初に、ヤバツイの粗エタノール抽出物を、ヘキサンにより脱脂処理を行う。具体的には、粗エタノール抽出物を９０％メタノール溶液に懸濁させ、分液漏斗に移して少量のヘキサンにより抽出することにより脱脂処理を行う。
脱脂処理した９０％メタノール層を減圧乾固し、９０％メタノール溶液画分（画分Ａ）が得られる。 By subjecting the crude ethanol extract of Yabatsui obtained above to fractionation, the target Euparinolide A and / or B is isolated. Specifically, it can be carried out as follows. .
First, a crude ethanol extract of Yabatsui is degreased with hexane. Specifically, a crude ethanol extract is suspended in a 90% methanol solution, transferred to a separatory funnel, and extracted with a small amount of hexane to perform a degreasing treatment.
The 90% methanol layer subjected to the degreasing treatment is dried under reduced pressure to obtain a 90% methanol solution fraction (fraction A).
最初に、ヤバツイの粗エタノール抽出物を、ヘキサンにより脱脂処理を行う。具体的には、粗エタノール抽出物を９０％メタノール溶液に懸濁させ、分液漏斗に移して少量のヘキサンにより抽出することにより脱脂処理を行う。
脱脂処理した９０％メタノール層を減圧乾固し、９０％メタノール溶液画分（画分Ａ）が得られる。 By subjecting the crude ethanol extract of Yabatsui obtained above to fractionation, the target Euparinolide A and / or B is isolated. Specifically, it can be carried out as follows. .
First, a crude ethanol extract of Yabatsui is degreased with hexane. Specifically, a crude ethanol extract is suspended in a 90% methanol solution, transferred to a separatory funnel, and extracted with a small amount of hexane to perform a degreasing treatment.
The 90% methanol layer subjected to the degreasing treatment is dried under reduced pressure to obtain a 90% methanol solution fraction (fraction A).
次いで、上記で得られた９０％メタノール溶液画分（画分Ａ）を、ポリスチレンゲルカラムクロマトグラフィー、具体的には、ＭＣｌ・ＧＥＬ ＣＨＰ－２０Ｐ（和光純薬工業）カラムクロマトグラフィーに付し、順次、水、５０％メタノール、メタノール溶液の段階的グラジエント法、及びアセトン溶液にて溶出を行い、各溶出画分を得る。
かかる操作において得られたメタノール溶出画分（画分Ｂ）に、ＨＳＰ発現誘導が強く認められた。 Next, the 90% methanol solution fraction (fraction A) obtained above was subjected to polystyrene gel column chromatography, specifically, MCl · GEL CHP-20P (Wako Pure Chemical Industries) column chromatography, Elution is performed sequentially with water, 50% methanol, a stepwise gradient method of methanol solution, and an acetone solution to obtain each elution fraction.
Induction of HSP expression was strongly observed in the methanol-eluted fraction (fraction B) obtained in this operation.
かかる操作において得られたメタノール溶出画分（画分Ｂ）に、ＨＳＰ発現誘導が強く認められた。 Next, the 90% methanol solution fraction (fraction A) obtained above was subjected to polystyrene gel column chromatography, specifically, MCl · GEL CHP-20P (Wako Pure Chemical Industries) column chromatography, Elution is performed sequentially with water, 50% methanol, a stepwise gradient method of methanol solution, and an acetone solution to obtain each elution fraction.
Induction of HSP expression was strongly observed in the methanol-eluted fraction (fraction B) obtained in this operation.
ＨＳＰ発現誘導が強く観察されたメタノール溶出画分（画分Ｂ）を、更にＯＤＳカラムクロマトグラフィーに付し、順次５０％メタノール溶液、７５％メタノール溶液、及びメタノール溶液による段階的グラジエント法で溶出を行い、それぞれの溶出画分を得た。
かかる操作において得られた５０％メタノール溶出画分（画分Ｃ）に、ＨＳＰ発現誘導が強く認められた。 Methanol elution fraction (fraction B), in which HSP expression induction was strongly observed, was further subjected to ODS column chromatography, and eluted in a stepwise gradient method using 50% methanol solution, 75% methanol solution, and methanol solution sequentially. Each elution fraction was obtained.
Induction of HSP expression was strongly observed in the 50% methanol elution fraction (fraction C) obtained in this procedure.
かかる操作において得られた５０％メタノール溶出画分（画分Ｃ）に、ＨＳＰ発現誘導が強く認められた。 Methanol elution fraction (fraction B), in which HSP expression induction was strongly observed, was further subjected to ODS column chromatography, and eluted in a stepwise gradient method using 50% methanol solution, 75% methanol solution, and methanol solution sequentially. Each elution fraction was obtained.
Induction of HSP expression was strongly observed in the 50% methanol elution fraction (fraction C) obtained in this procedure.
次いで、ＨＳＰ発現誘導が強く観察された５０％メタノール溶出画分（画分Ｃ）を、高速液体クロマトグラフィー（ＨＰＬＣ、カラム：ＡＲ－ＩＩ ＯＤＳ）に付し、４０％メタノールにて溶出して全１２個の溶出画分（画分Ｄ１～Ｄ１２）を得た。
かくして得られた溶出画分Ｄ１～Ｄ１２のうち、特に、画分Ｄ３並びに画分Ｄ７に、極めて強いＨＳＰ発現誘導作用があることが判明した。 Next, the 50% methanol elution fraction (fraction C), in which HSP expression induction was strongly observed, was subjected to high performance liquid chromatography (HPLC, column: AR-II ODS) and eluted with 40% methanol. Twelve elution fractions (fractions D1 to D12) were obtained.
Of the eluted fractions D1 to D12 thus obtained, it was found that particularly the fraction D3 and the fraction D7 have a very strong HSP expression inducing action.
かくして得られた溶出画分Ｄ１～Ｄ１２のうち、特に、画分Ｄ３並びに画分Ｄ７に、極めて強いＨＳＰ発現誘導作用があることが判明した。 Next, the 50% methanol elution fraction (fraction C), in which HSP expression induction was strongly observed, was subjected to high performance liquid chromatography (HPLC, column: AR-II ODS) and eluted with 40% methanol. Twelve elution fractions (fractions D1 to D12) were obtained.
Of the eluted fractions D1 to D12 thus obtained, it was found that particularly the fraction D3 and the fraction D7 have a very strong HSP expression inducing action.
この画分Ｄ３及びＤ７について、ＨＳＰ発現誘導作用物質の特定を検討したところ、画分Ｄ３には、ユーパリノライドＡが、画分Ｄ７にはユーパリノライドＢが主成分として含有されていることが判明した。
かかる画分Ｄ３及びＤ７について、一般的に汎用されている単離・結晶化等の操作を行い、目的とするユーパリノライドＡ及び／又はＢを得ることができる。
したがって、本発明は、また、サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（ヤバツイ）より、抽出・分画処理することからなるユーパリノライドＡ及び／又はＢ（Eupalinolide Ａ及び／又はＢ）の抽出方法でもある。 Regarding the fractions D3 and D7, the identification of the HSP expression-inducing agent was examined. As a result, the fraction D3 contains Euparinolide A as the main component and the fraction D7 contains Euparinolide B as the main component. There was found.
With respect to the fractions D3 and D7, generally used operations such as isolation and crystallization can be performed to obtain the desired Euparinolide A and / or B.
Therefore, the present invention also provides extraction of Euparinolide A and / or B (Eupalinolide A and / or B) comprising extraction and fractionation treatment from above-ground dry herbal medicine (Yabatsui) of Eupatorium lindleyanum. It is also a method.
かかる画分Ｄ３及びＤ７について、一般的に汎用されている単離・結晶化等の操作を行い、目的とするユーパリノライドＡ及び／又はＢを得ることができる。
したがって、本発明は、また、サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（ヤバツイ）より、抽出・分画処理することからなるユーパリノライドＡ及び／又はＢ（Eupalinolide Ａ及び／又はＢ）の抽出方法でもある。 Regarding the fractions D3 and D7, the identification of the HSP expression-inducing agent was examined. As a result, the fraction D3 contains Euparinolide A as the main component and the fraction D7 contains Euparinolide B as the main component. There was found.
With respect to the fractions D3 and D7, generally used operations such as isolation and crystallization can be performed to obtain the desired Euparinolide A and / or B.
Therefore, the present invention also provides extraction of Euparinolide A and / or B (Eupalinolide A and / or B) comprising extraction and fractionation treatment from above-ground dry herbal medicine (Yabatsui) of Eupatorium lindleyanum. It is also a method.
本発明が提供するＨＳＰ発現誘導剤は、ＨＳＰのなかでも、特に、ＨＳＰ７０の発現誘導によるメラニン産生抑制作用を発揮する。
すなわち、ＨＳＰ７０は、メラニン産生に関与するチロシナーゼ及び小眼球症関連転写因子（ＭＩＴＦ：microphthalmia転写因子）を制御させるものであり、その結果、メラニンの産生が抑制されるとともに、ＨＳＰの損傷によって生じる脳梗塞やアルツハイマー病などの脳疾患、潰瘍性大腸炎などの治療剤として有用である。 The HSP expression inducer provided by the present invention exhibits an inhibitory action on melanin production by inducing the expression of HSP70, among other HSPs.
That is, HSP70 controls tyrosinase and microphthalmia-related transcription factor (MITF: microphthalmia transcription factor) involved in melanin production, and as a result, the production of melanin is suppressed and the brain caused by the damage of HSP. It is useful as a therapeutic agent for brain diseases such as infarction and Alzheimer's disease, and ulcerative colitis.
すなわち、ＨＳＰ７０は、メラニン産生に関与するチロシナーゼ及び小眼球症関連転写因子（ＭＩＴＦ：microphthalmia転写因子）を制御させるものであり、その結果、メラニンの産生が抑制されるとともに、ＨＳＰの損傷によって生じる脳梗塞やアルツハイマー病などの脳疾患、潰瘍性大腸炎などの治療剤として有用である。 The HSP expression inducer provided by the present invention exhibits an inhibitory action on melanin production by inducing the expression of HSP70, among other HSPs.
That is, HSP70 controls tyrosinase and microphthalmia-related transcription factor (MITF: microphthalmia transcription factor) involved in melanin production, and as a result, the production of melanin is suppressed and the brain caused by the damage of HSP. It is useful as a therapeutic agent for brain diseases such as infarction and Alzheimer's disease, and ulcerative colitis.
したがって、本発明が提供するＨＳＰ発現誘導剤は、例えば、脳梗塞やアルツハイマー病などの脳疾患、潰瘍性大腸炎などの治療剤、メラニン産生抑制化粧料、特に美白化粧料として使用するのに適している。
本発明が提供するＨＳＰ発現誘導剤は、医薬品として錠剤、カプセル、粉末剤などの経口投与剤、注射剤、点滴剤などの非経口投与剤とし、化粧料としては、具体的には、化粧水、乳液、クリーム、美容液、パック等の皮膚化粧料、メイクアップベースローション、メイクアップベースクリーム等の下地化粧料、乳液剤、油性、固形状等の各剤型のファンデーション、アイカラー、チークカラー等のメイクアップ化粧料、ハンドクリーム、レッグクリーム、ネッククリーム、ボディローション等の身体用化粧料等を挙げることができる。 Therefore, the HSP expression inducer provided by the present invention is suitable for use as, for example, a therapeutic agent for cerebral infarction or Alzheimer's disease such as cerebral infarction or Alzheimer's disease, ulcerative colitis, melanin production-suppressing cosmetic, particularly whitening cosmetic. ing.
The HSP expression inducer provided by the present invention is a pharmaceutical preparation for oral administration such as tablets, capsules and powders, and parenteral administration such as injections and infusions. Skin cosmetics such as milky lotion, cream, cosmetic liquid, pack, foundation cosmetics such as makeup base lotion and makeup base cream, foundations for each dosage form such as emulsion, oily, solid, eye color, cheek color And makeup cosmetics such as hand cream, leg cream, neck cream, body lotion and the like.
本発明が提供するＨＳＰ発現誘導剤は、医薬品として錠剤、カプセル、粉末剤などの経口投与剤、注射剤、点滴剤などの非経口投与剤とし、化粧料としては、具体的には、化粧水、乳液、クリーム、美容液、パック等の皮膚化粧料、メイクアップベースローション、メイクアップベースクリーム等の下地化粧料、乳液剤、油性、固形状等の各剤型のファンデーション、アイカラー、チークカラー等のメイクアップ化粧料、ハンドクリーム、レッグクリーム、ネッククリーム、ボディローション等の身体用化粧料等を挙げることができる。 Therefore, the HSP expression inducer provided by the present invention is suitable for use as, for example, a therapeutic agent for cerebral infarction or Alzheimer's disease such as cerebral infarction or Alzheimer's disease, ulcerative colitis, melanin production-suppressing cosmetic, particularly whitening cosmetic. ing.
The HSP expression inducer provided by the present invention is a pharmaceutical preparation for oral administration such as tablets, capsules and powders, and parenteral administration such as injections and infusions. Skin cosmetics such as milky lotion, cream, cosmetic liquid, pack, foundation cosmetics such as makeup base lotion and makeup base cream, foundations for each dosage form such as emulsion, oily, solid, eye color, cheek color And makeup cosmetics such as hand cream, leg cream, neck cream, body lotion and the like.
本発明が提供するＨＳＰ発現誘導剤は、ユーパリノライドＡ及び／又はＢを含有するものであるが、上記の抽出・単離操作により得たヤバツイの抽出物を、そのまま含有してもよい。
また、各種製剤として使用されている薬理学的に許容されている他の慣用成分と共に、所望の形態で使用することができる。 Although the HSP expression inducer provided by the present invention contains Euparinolide A and / or B, the extract of Yabatsui obtained by the above extraction / isolation operation may be contained as it is.
Moreover, it can use in a desired form with the other conventional component accept | permitted pharmacologically used as various preparations.
また、各種製剤として使用されている薬理学的に許容されている他の慣用成分と共に、所望の形態で使用することができる。 Although the HSP expression inducer provided by the present invention contains Euparinolide A and / or B, the extract of Yabatsui obtained by the above extraction / isolation operation may be contained as it is.
Moreover, it can use in a desired form with the other conventional component accept | permitted pharmacologically used as various preparations.
この場合において、本発明が提供するＨＳＰ発現誘導剤であるユーパリノライドＡ及び／又はＢの含有量は、一概に限定できず、また、病態の予防・改善の目的、用いる人の性別、体重、年齢、剤型、病態の種類や程度、使用部位、使用回数などの種々の条件により一概に限定できない。
例えば、経口剤として投与する場合には、０．１～１００μｇ／ｋｇ／日で、一日１回から数回に分けて適用することができるが、使用量は、必ずしもこの範囲に限定されるものではない。
また、上記の抽出・単離操作により得たヤバツイの抽出物を、そのまま含有する場合には、その抽出画分に含有されるユーパリノライドＡ及び／又はＢの量に換算した量で含有させることができる。 In this case, the content of Euparinolide A and / or B, which is an HSP expression inducer provided by the present invention, cannot be generally limited, and the purpose of prevention / amelioration of the disease state, the sex of the user, the body weight However, it cannot be generally limited by various conditions such as age, dosage form, type and degree of disease state, use site, number of uses.
For example, when it is administered as an oral preparation, it can be applied at a dose of 0.1 to 100 μg / kg / day once a day or several times a day, but the amount used is not necessarily limited to this range. It is not a thing.
In addition, when the extract of Yabatoi obtained by the above extraction / isolation operation is contained as it is, it is contained in an amount converted to the amount of Euparinolide A and / or B contained in the extracted fraction. be able to.
例えば、経口剤として投与する場合には、０．１～１００μｇ／ｋｇ／日で、一日１回から数回に分けて適用することができるが、使用量は、必ずしもこの範囲に限定されるものではない。
また、上記の抽出・単離操作により得たヤバツイの抽出物を、そのまま含有する場合には、その抽出画分に含有されるユーパリノライドＡ及び／又はＢの量に換算した量で含有させることができる。 In this case, the content of Euparinolide A and / or B, which is an HSP expression inducer provided by the present invention, cannot be generally limited, and the purpose of prevention / amelioration of the disease state, the sex of the user, the body weight However, it cannot be generally limited by various conditions such as age, dosage form, type and degree of disease state, use site, number of uses.
For example, when it is administered as an oral preparation, it can be applied at a dose of 0.1 to 100 μg / kg / day once a day or several times a day, but the amount used is not necessarily limited to this range. It is not a thing.
In addition, when the extract of Yabatoi obtained by the above extraction / isolation operation is contained as it is, it is contained in an amount converted to the amount of Euparinolide A and / or B contained in the extracted fraction. be able to.
本発明が提供するＨＳＰ発現誘導剤は、上記したように、医療効果を目的として使用されるとともに、メラニン産生抑制化粧料として使用される。
As described above, the HSP expression inducer provided by the present invention is used for the purpose of medical effects and also used as a melanin production-suppressing cosmetic.
以下に本発明を、ヤバツイからのユーパリノライドＡ及び／又はＢの抽出・単離の実際、並びに、ＨＳＰ７０の発現の誘導の試験を説明しながら、より詳細に説明していく。
Hereinafter, the present invention will be described in more detail while explaining the actual extraction and isolation of Euparinolide A and / or B from Yabatsui, and the test of induction of HSP70 expression.
なお、以下の実施例に示す材料、使用量、割合、処理内容、処理手順等は、本発明の趣旨を逸脱しない限り、適宜、変更することができる。したがって、本発明の範囲は以下に示す具体例に限定されるものではない。
It should be noted that the materials, amounts used, ratios, processing details, processing procedures, and the like shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention is not limited to the specific examples shown below.
実施例１：ユーパリノライドＡ及び／又はＢの抽出・単離
丸善製薬（株）から入手したヤバツイ［サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬］の１０．１ｇを５００ｍＬのナス型フラスコに入れ、１００ｍＬのエタノールを加え、７０℃の水浴上で２時間還流抽出した。冷却後、綿栓濾過してエタノール抽出液を得た。
この還流抽出操作を３回繰り返した後、得られたエタノール抽出液を合わせて減圧濃縮し、エタノール抽出物（４９９ｍｇ）を得た。
このエタノール抽出物を１００ｍＬの９０％メタノールに懸濁させ、分液漏斗に移して３０ｍＬのヘキサンにて３回処理を行い、脱脂した。９０％メタノール溶液層をエバポレーターで減圧乾固し、３１０ｍｇの９０％メタノール溶液画分（画分Ａ）を得た。
この段階で得られた、ヘキサン溶液溶解画分と、９０％メタノール溶液画分（画分Ａ）について、免疫ブロット法により活性を検討したところ、９０％メタノール溶液画分（画分Ａ）が、活性が強いものであった。 Example 1: Extraction and Isolation of Euparinolide A and / or B 10.1 g of Yabatsui [Eupatorium lindleyanum above-ground dry herbal medicine] obtained from Maruzen Pharmaceutical Co., Ltd. was placed in a 500 mL eggplant-shaped flask. , 100 mL of ethanol was added, and the mixture was refluxed for 2 hours on a 70 ° C. water bath. After cooling, a cotton plug was filtered to obtain an ethanol extract.
After this reflux extraction operation was repeated three times, the obtained ethanol extracts were combined and concentrated under reduced pressure to obtain an ethanol extract (499 mg).
This ethanol extract was suspended in 100 mL of 90% methanol, transferred to a separatory funnel, treated with 30 mL of hexane three times, and degreased. The 90% methanol solution layer was dried under reduced pressure using an evaporator to obtain 310 mg of a 90% methanol solution fraction (fraction A).
When the activity was examined by immunoblotting for the hexane solution-dissolved fraction and 90% methanol solution fraction (fraction A) obtained at this stage, the 90% methanol solution fraction (fraction A) was The activity was strong.
丸善製薬（株）から入手したヤバツイ［サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬］の１０．１ｇを５００ｍＬのナス型フラスコに入れ、１００ｍＬのエタノールを加え、７０℃の水浴上で２時間還流抽出した。冷却後、綿栓濾過してエタノール抽出液を得た。
この還流抽出操作を３回繰り返した後、得られたエタノール抽出液を合わせて減圧濃縮し、エタノール抽出物（４９９ｍｇ）を得た。
このエタノール抽出物を１００ｍＬの９０％メタノールに懸濁させ、分液漏斗に移して３０ｍＬのヘキサンにて３回処理を行い、脱脂した。９０％メタノール溶液層をエバポレーターで減圧乾固し、３１０ｍｇの９０％メタノール溶液画分（画分Ａ）を得た。
この段階で得られた、ヘキサン溶液溶解画分と、９０％メタノール溶液画分（画分Ａ）について、免疫ブロット法により活性を検討したところ、９０％メタノール溶液画分（画分Ａ）が、活性が強いものであった。 Example 1: Extraction and Isolation of Euparinolide A and / or B 10.1 g of Yabatsui [Eupatorium lindleyanum above-ground dry herbal medicine] obtained from Maruzen Pharmaceutical Co., Ltd. was placed in a 500 mL eggplant-shaped flask. , 100 mL of ethanol was added, and the mixture was refluxed for 2 hours on a 70 ° C. water bath. After cooling, a cotton plug was filtered to obtain an ethanol extract.
After this reflux extraction operation was repeated three times, the obtained ethanol extracts were combined and concentrated under reduced pressure to obtain an ethanol extract (499 mg).
This ethanol extract was suspended in 100 mL of 90% methanol, transferred to a separatory funnel, treated with 30 mL of hexane three times, and degreased. The 90% methanol solution layer was dried under reduced pressure using an evaporator to obtain 310 mg of a 90% methanol solution fraction (fraction A).
When the activity was examined by immunoblotting for the hexane solution-dissolved fraction and 90% methanol solution fraction (fraction A) obtained at this stage, the 90% methanol solution fraction (fraction A) was The activity was strong.
この活性の強かった９０％メタノール溶出画分を、ＭＣｌ・ＧＥＬ ＣＨＰ－２０Ｐカラムクロマトグラフィー（φ１７ｍｍ×１００ｍｍ：和光純薬工業）の段階的グラジエント法に付し、順次、水（８０ｍＬ）、５０％メタノール（１００ｍＬ）、メタノール（１００ｍＬ）、及びアセトン（１００ｍＬ）溶液にて溶出を行い、各溶出画分を得た。
その結果、水溶出画分（１１９ｍｇ）、５０％メタノール溶出画分（４９ｍｇ）、メタノール溶出画分（画分Ｂ）（１２５ｍｇ）及びアセトン溶出画分（２０ｍｇ）を得た。
得られた四つの画分のうち、メタノール溶出画分（画分Ｂ）（１２５ｍｇ）が最も活性が強いものであった。 This 90% methanol-eluted fraction with strong activity was subjected to a stepwise gradient method of MCl · GEL CHP-20P column chromatography (φ17 mm × 100 mm: Wako Pure Chemical Industries), followed by water (80 mL), 50% Elution was performed with methanol (100 mL), methanol (100 mL), and acetone (100 mL) solution to obtain each elution fraction.
As a result, a water elution fraction (119 mg), a 50% methanol elution fraction (49 mg), a methanol elution fraction (fraction B) (125 mg), and an acetone elution fraction (20 mg) were obtained.
Of the four fractions obtained, the methanol-eluted fraction (Fraction B) (125 mg) was the most active.
その結果、水溶出画分（１１９ｍｇ）、５０％メタノール溶出画分（４９ｍｇ）、メタノール溶出画分（画分Ｂ）（１２５ｍｇ）及びアセトン溶出画分（２０ｍｇ）を得た。
得られた四つの画分のうち、メタノール溶出画分（画分Ｂ）（１２５ｍｇ）が最も活性が強いものであった。 This 90% methanol-eluted fraction with strong activity was subjected to a stepwise gradient method of MCl · GEL CHP-20P column chromatography (φ17 mm × 100 mm: Wako Pure Chemical Industries), followed by water (80 mL), 50% Elution was performed with methanol (100 mL), methanol (100 mL), and acetone (100 mL) solution to obtain each elution fraction.
As a result, a water elution fraction (119 mg), a 50% methanol elution fraction (49 mg), a methanol elution fraction (fraction B) (125 mg), and an acetone elution fraction (20 mg) were obtained.
Of the four fractions obtained, the methanol-eluted fraction (Fraction B) (125 mg) was the most active.
次いで、活性の強かったメタノール溶出画分（画分Ｂ）を、Chromatorex ＯＤＳカラムロマトグラフィー（φ１５ｍｍ×１５０ｍｍ）に付し、水－メタノール（１：０→０：１、ｖ／ｖ）の段階的グラジエント法により溶出し、これにより、順次５０％メタノール溶液、７５％メタノール溶液、及びメタノール溶液で溶出を行い得た５０％メタノール溶出画分（画分Ｃ）（４９ｍｇ）を得た。
得られた三つの画分のうち、５０％メタノール溶出画分（画分Ｃ）が最も活性が強いものであった。 Subsequently, the methanol-eluted fraction (fraction B) having a high activity was subjected to Chromatorex ODS column chromatography (φ15 mm × 150 mm), and stepwise from water to methanol (1: 0 → 0: 1, v / v). Elution was performed by a gradient method, whereby a 50% methanol-eluted fraction (fraction C) (49 mg) that could be eluted with a 50% methanol solution, a 75% methanol solution, and a methanol solution was obtained.
Of the three fractions obtained, the 50% methanol elution fraction (Fraction C) was the most active.
得られた三つの画分のうち、５０％メタノール溶出画分（画分Ｃ）が最も活性が強いものであった。 Subsequently, the methanol-eluted fraction (fraction B) having a high activity was subjected to Chromatorex ODS column chromatography (φ15 mm × 150 mm), and stepwise from water to methanol (1: 0 → 0: 1, v / v). Elution was performed by a gradient method, whereby a 50% methanol-eluted fraction (fraction C) (49 mg) that could be eluted with a 50% methanol solution, a 75% methanol solution, and a methanol solution was obtained.
Of the three fractions obtained, the 50% methanol elution fraction (Fraction C) was the most active.
最後に、得られた５０％メタノール溶出画分（画分Ｃ）を高速液体クロマトグラフィー（ＨＰＬＣ，Cosmosil ＡＲ－ＩＩ ＯＤＳカラム：φ２０ｍｍ×３５０ｍｍ）に付し、４０％メタノールにて分取し、１２個の溶出画分（画分Ｄ１～Ｄ１２）を得た。
これらの溶出画分Ｄ１～Ｄ１２について活性を検討したところ、画分Ｄ３及び画分Ｄ７が最も活性が強いものであり、画分Ｄ３より式（Ｉ）で示されるユーパリノライドＡを４．６ｍｇ（収率：０．０５％）、画分Ｄ７より式（ＩＩ）で示されるユーパリノライドＢを４．５ｍｇ（収率：０．０４％）得ることができた。
これらの化学構造は、赤外線吸収スペクトル、Ｈ１或いはＣ１３磁気共鳴スペクトル等の標品との比較による物理化学的手段により決定することができた。 Finally, the obtained 50% methanol elution fraction (fraction C) was subjected to high performance liquid chromatography (HPLC, Cosmosil AR-II ODS column: φ20 mm × 350 mm), and fractionated with 40% methanol. Elution fractions (fractions D1 to D12) were obtained.
When the activities of these eluted fractions D1 to D12 were examined, fraction D3 and fraction D7 had the strongest activity, and 4.6 mg of Euparinolide A represented by the formula (I) was obtained from fraction D3. (Yield: 0.05%) From the fraction D7, 4.5 mg (yield: 0.04%) of Euparinolide B represented by the formula (II) could be obtained.
These chemical structures could be determined by physicochemical means by comparison with standard products such as infrared absorption spectrum, H 1 or C 13 magnetic resonance spectrum.
これらの溶出画分Ｄ１～Ｄ１２について活性を検討したところ、画分Ｄ３及び画分Ｄ７が最も活性が強いものであり、画分Ｄ３より式（Ｉ）で示されるユーパリノライドＡを４．６ｍｇ（収率：０．０５％）、画分Ｄ７より式（ＩＩ）で示されるユーパリノライドＢを４．５ｍｇ（収率：０．０４％）得ることができた。
これらの化学構造は、赤外線吸収スペクトル、Ｈ１或いはＣ１３磁気共鳴スペクトル等の標品との比較による物理化学的手段により決定することができた。 Finally, the obtained 50% methanol elution fraction (fraction C) was subjected to high performance liquid chromatography (HPLC, Cosmosil AR-II ODS column: φ20 mm × 350 mm), and fractionated with 40% methanol. Elution fractions (fractions D1 to D12) were obtained.
When the activities of these eluted fractions D1 to D12 were examined, fraction D3 and fraction D7 had the strongest activity, and 4.6 mg of Euparinolide A represented by the formula (I) was obtained from fraction D3. (Yield: 0.05%) From the fraction D7, 4.5 mg (yield: 0.04%) of Euparinolide B represented by the formula (II) could be obtained.
These chemical structures could be determined by physicochemical means by comparison with standard products such as infrared absorption spectrum, H 1 or C 13 magnetic resonance spectrum.
以上の分画処理の流れ（フロー）を、図１に示した。
The flow of the above fractionation process is shown in FIG.
実施例２：ＨＳＰの発現と細胞生存率
マウスメラノーマ由来のＢ１６細胞（理研バイオリソースセンター）を、各濃度のユーパリノライドＡ又はＢと共に、ＤＭＥＭ培地（１０％の牛胎児血清、１００Ｕ／ｍＬのペニシリン、１００Ｕ／ｍＬのストレプトマイシン）で、３７℃／５％ＣＯ２雰囲気下で２４時間培養した。
ＨＳＰ７０の発現量は、細胞培養液をＳＤＳ－ＰＡＧＥ電気泳動にかけた後、抗ＨＳＰ７０抗体（Stressgen社）を用いたウエスタン・ブロット法によりＨＳＰ７０の発現量を測定した。
細胞生存率は、ＭＴＴ法に従い、全細胞の抽出物を、抗ＨＳＰ７０抗体又はアクチンと共にイムノブロット法により測定した。 Example 2: Expression of HSP and cell viability B16 cells derived from mouse melanoma (RIKEN BioResource Center) were combined with various concentrations of Euparinolide A or B in DMEM medium (10% fetal bovine serum, 100 U / mL penicillin). , 100 U / mL streptomycin) at 37 ° C./5% CO 2 atmosphere for 24 hours.
The expression level of HSP70 was measured by Western blotting using an anti-HSP70 antibody (Stressgen) after subjecting the cell culture solution to SDS-PAGE electrophoresis.
Cell viability was measured by immunoblotting of whole cell extracts together with anti-HSP70 antibody or actin according to the MTT method.
マウスメラノーマ由来のＢ１６細胞（理研バイオリソースセンター）を、各濃度のユーパリノライドＡ又はＢと共に、ＤＭＥＭ培地（１０％の牛胎児血清、１００Ｕ／ｍＬのペニシリン、１００Ｕ／ｍＬのストレプトマイシン）で、３７℃／５％ＣＯ２雰囲気下で２４時間培養した。
ＨＳＰ７０の発現量は、細胞培養液をＳＤＳ－ＰＡＧＥ電気泳動にかけた後、抗ＨＳＰ７０抗体（Stressgen社）を用いたウエスタン・ブロット法によりＨＳＰ７０の発現量を測定した。
細胞生存率は、ＭＴＴ法に従い、全細胞の抽出物を、抗ＨＳＰ７０抗体又はアクチンと共にイムノブロット法により測定した。 Example 2: Expression of HSP and cell viability B16 cells derived from mouse melanoma (RIKEN BioResource Center) were combined with various concentrations of Euparinolide A or B in DMEM medium (10% fetal bovine serum, 100 U / mL penicillin). , 100 U / mL streptomycin) at 37 ° C./5% CO 2 atmosphere for 24 hours.
The expression level of HSP70 was measured by Western blotting using an anti-HSP70 antibody (Stressgen) after subjecting the cell culture solution to SDS-PAGE electrophoresis.
Cell viability was measured by immunoblotting of whole cell extracts together with anti-HSP70 antibody or actin according to the MTT method.
その結果を図２に示した。
図２中、上段にユーパリノライドＡの結果を、下段にユーパリノライドＢの結果を示した。
図中に示した結果からも判明するように、ユーパリノライドＡ及びＢは、濃度依存的にＨＳＰを発現しており、その時の細胞生存率に変化がなく、安全なものであることが確認された。 The results are shown in FIG.
In FIG. 2, the result of Euparinolide A is shown in the upper part, and the result of Euparinoride B is shown in the lower part.
As can be seen from the results shown in the figure, Euparinolide A and B express HSP in a concentration-dependent manner, and it is confirmed that the cell viability at that time is unchanged and safe. It was done.
図２中、上段にユーパリノライドＡの結果を、下段にユーパリノライドＢの結果を示した。
図中に示した結果からも判明するように、ユーパリノライドＡ及びＢは、濃度依存的にＨＳＰを発現しており、その時の細胞生存率に変化がなく、安全なものであることが確認された。 The results are shown in FIG.
In FIG. 2, the result of Euparinolide A is shown in the upper part, and the result of Euparinoride B is shown in the lower part.
As can be seen from the results shown in the figure, Euparinolide A and B express HSP in a concentration-dependent manner, and it is confirmed that the cell viability at that time is unchanged and safe. It was done.
実施例３：ＡＧＳ細胞におけるＨＳＰ７０発現効果
ヒト胃粘膜上皮細胞（ＡＧＳ細胞）を、ユーパリノライドＡ又はＢの各濃度と共に、ＲＰＭＩ培地（１０％の牛胎児血清、１００Ｕ／ｍＬのペニシリン、１００Ｕ／ｍＬのストレプトマイシン）で、３７℃／５％ＣＯ２雰囲気下で２４時間培養した。
ＨＳＰ７０の発現量は、細胞培養液をＳＤＳ－ＰＡＧＥ電気泳動にかけた後、抗ＨＳＰ７０抗体（Stressgen社）を用いたウエスタン・ブロット法により測定した。
細胞生存率は、ＭＴＴ法に従い、全細胞の抽出物を、抗ＨＳＰ７０抗体又はアクチンと共にイムノブロット法により測定した。 Example 3: Effect of HSP70 expression on AGS cells Human gastric mucosal epithelial cells (AGS cells) were combined with each concentration of Euparinolide A or B in RPMI medium (10% fetal bovine serum, 100 U / mL penicillin, 100 U / Incubation was carried out for 24 hours at 37 ° C./5% CO 2 atmosphere.
The expression level of HSP70 was measured by Western blotting using an anti-HSP70 antibody (Stressgen) after subjecting the cell culture solution to SDS-PAGE electrophoresis.
Cell viability was measured by immunoblotting of whole cell extracts together with anti-HSP70 antibody or actin according to the MTT method.
ヒト胃粘膜上皮細胞（ＡＧＳ細胞）を、ユーパリノライドＡ又はＢの各濃度と共に、ＲＰＭＩ培地（１０％の牛胎児血清、１００Ｕ／ｍＬのペニシリン、１００Ｕ／ｍＬのストレプトマイシン）で、３７℃／５％ＣＯ２雰囲気下で２４時間培養した。
ＨＳＰ７０の発現量は、細胞培養液をＳＤＳ－ＰＡＧＥ電気泳動にかけた後、抗ＨＳＰ７０抗体（Stressgen社）を用いたウエスタン・ブロット法により測定した。
細胞生存率は、ＭＴＴ法に従い、全細胞の抽出物を、抗ＨＳＰ７０抗体又はアクチンと共にイムノブロット法により測定した。 Example 3: Effect of HSP70 expression on AGS cells Human gastric mucosal epithelial cells (AGS cells) were combined with each concentration of Euparinolide A or B in RPMI medium (10% fetal bovine serum, 100 U / mL penicillin, 100 U / Incubation was carried out for 24 hours at 37 ° C./5% CO 2 atmosphere.
The expression level of HSP70 was measured by Western blotting using an anti-HSP70 antibody (Stressgen) after subjecting the cell culture solution to SDS-PAGE electrophoresis.
Cell viability was measured by immunoblotting of whole cell extracts together with anti-HSP70 antibody or actin according to the MTT method.
その結果を、図３に示した。
図３中、上段にユーパリノライドＡの結果を、下段にユーパリノライドＢの結果を示した。
図中に示した結果からも判明するように、ＡＧＳ細胞においても、実施例２と同様にユーパリノライドＡ及びＢは、濃度依存的にＨＳＰ７０を発現しており、その時の細胞生存率に変化がなく、安全なものであることが確認された。 The results are shown in FIG.
In FIG. 3, the result of Euparinolide A is shown in the upper part, and the result of Euparinoride B is shown in the lower part.
As can be seen from the results shown in the figure, also in AGS cells, Euparinolide A and B expressed HSP70 in a concentration-dependent manner as in Example 2, and the cell viability at that time changed. It was confirmed that it was safe.
図３中、上段にユーパリノライドＡの結果を、下段にユーパリノライドＢの結果を示した。
図中に示した結果からも判明するように、ＡＧＳ細胞においても、実施例２と同様にユーパリノライドＡ及びＢは、濃度依存的にＨＳＰ７０を発現しており、その時の細胞生存率に変化がなく、安全なものであることが確認された。 The results are shown in FIG.
In FIG. 3, the result of Euparinolide A is shown in the upper part, and the result of Euparinoride B is shown in the lower part.
As can be seen from the results shown in the figure, also in AGS cells, Euparinolide A and B expressed HSP70 in a concentration-dependent manner as in Example 2, and the cell viability at that time changed. It was confirmed that it was safe.
実施例４：他のストレスとのＨＳＰ７０発現増強効果
ＡＧＳ細胞を、ユーパリノライドＡ又はＢの各濃度（０、１及び２μｇ／ｍＬ）と共に、ＲＰＭＩ培地（１０％の牛胎児血清、１００Ｕ／ｍＬのペニシリン、１００Ｕ／ｍＬのストレプトマイシン）で、３７℃／５％ＣＯ２雰囲気下で３時間培養した。次いで、４０℃／１時間のヒートショックを与え、続いて３７℃にて６時間ヒートショックからの回復を行った。
一方、ＡＧＳ細胞を同様に、ストレスとして３．５％エタノールと共に２４時間培養した。
ストレスを与えないで培養したＡＧＳ細胞、並びにヒートショックストレス或いはアルコールストレスを与えて培養した各ＡＧＳ細胞におけるＨＳＰ７０の発現量を、上記実施例と同様に、細胞培養液をＳＤＳ－ＰＡＧＥ電気泳動にかけた後、抗ＨＳＰ７０抗体（Stressgen社）を用いたウエスタン・ブロット法によりＨＳＰ７０の発現量を測定した。
また、細胞生存率は、ＭＴＴ法に従い、全細胞の抽出物を、抗ＨＳＰ７０抗体又はアクチンと共にイムノブロット法により測定した。 Example 4: Effect of enhancing HSP70 expression with other stress AGS cells were combined with each concentration of Euparinolide A or B (0, 1 and 2 μg / mL) in RPMI medium (10% fetal bovine serum, 100 U / mL). Of penicillin, 100 U / mL streptomycin) at 37 ° C./5% CO 2 atmosphere for 3 hours. Next, a heat shock of 40 ° C./1 hour was applied, and then recovery from the heat shock was performed at 37 ° C. for 6 hours.
On the other hand, AGS cells were similarly cultured with 3.5% ethanol as stress for 24 hours.
The expression level of HSP70 in AGS cells cultured without stress and each AGS cell cultured with heat shock stress or alcohol stress was subjected to SDS-PAGE electrophoresis in the same manner as in the above Examples. Thereafter, the expression level of HSP70 was measured by Western blotting using an anti-HSP70 antibody (Stressgen).
Cell viability was determined by immunoblotting of whole cell extracts together with anti-HSP70 antibody or actin according to the MTT method.
ＡＧＳ細胞を、ユーパリノライドＡ又はＢの各濃度（０、１及び２μｇ／ｍＬ）と共に、ＲＰＭＩ培地（１０％の牛胎児血清、１００Ｕ／ｍＬのペニシリン、１００Ｕ／ｍＬのストレプトマイシン）で、３７℃／５％ＣＯ２雰囲気下で３時間培養した。次いで、４０℃／１時間のヒートショックを与え、続いて３７℃にて６時間ヒートショックからの回復を行った。
一方、ＡＧＳ細胞を同様に、ストレスとして３．５％エタノールと共に２４時間培養した。
ストレスを与えないで培養したＡＧＳ細胞、並びにヒートショックストレス或いはアルコールストレスを与えて培養した各ＡＧＳ細胞におけるＨＳＰ７０の発現量を、上記実施例と同様に、細胞培養液をＳＤＳ－ＰＡＧＥ電気泳動にかけた後、抗ＨＳＰ７０抗体（Stressgen社）を用いたウエスタン・ブロット法によりＨＳＰ７０の発現量を測定した。
また、細胞生存率は、ＭＴＴ法に従い、全細胞の抽出物を、抗ＨＳＰ７０抗体又はアクチンと共にイムノブロット法により測定した。 Example 4: Effect of enhancing HSP70 expression with other stress AGS cells were combined with each concentration of Euparinolide A or B (0, 1 and 2 μg / mL) in RPMI medium (10% fetal bovine serum, 100 U / mL). Of penicillin, 100 U / mL streptomycin) at 37 ° C./5% CO 2 atmosphere for 3 hours. Next, a heat shock of 40 ° C./1 hour was applied, and then recovery from the heat shock was performed at 37 ° C. for 6 hours.
On the other hand, AGS cells were similarly cultured with 3.5% ethanol as stress for 24 hours.
The expression level of HSP70 in AGS cells cultured without stress and each AGS cell cultured with heat shock stress or alcohol stress was subjected to SDS-PAGE electrophoresis in the same manner as in the above Examples. Thereafter, the expression level of HSP70 was measured by Western blotting using an anti-HSP70 antibody (Stressgen).
Cell viability was determined by immunoblotting of whole cell extracts together with anti-HSP70 antibody or actin according to the MTT method.
その結果を、図４に示した。
図４中、上段にヒートショックを与えた場合の結果を、下段にアルコールストレスを与えた場合の結果を示した。
図中に示した結果からも判明するように、ヒートショックを与えない場合であってもＨＳＰ７０の発現は誘導されているが、ヒートショックを与えた場合においては、ＨＳＰ７０がより多く誘導されることが判明した。
アルコールストレスを与えた場合であっても同様であった。
以上の結果から、ユーパリノライドＡ又はＢは、他のストレスによりＨＳＰ７０発現の増強効果があることが理解される。 The results are shown in FIG.
In FIG. 4, the results when heat shock is given to the upper stage and the results when alcohol stress is given to the lower stage are shown.
As can be seen from the results shown in the figure, expression of HSP70 is induced even when heat shock is not applied, but more HSP70 is induced when heat shock is applied. There was found.
The same was true even when alcohol stress was applied.
From the above results, it is understood that Euparinolide A or B has an effect of enhancing HSP70 expression by other stress.
図４中、上段にヒートショックを与えた場合の結果を、下段にアルコールストレスを与えた場合の結果を示した。
図中に示した結果からも判明するように、ヒートショックを与えない場合であってもＨＳＰ７０の発現は誘導されているが、ヒートショックを与えた場合においては、ＨＳＰ７０がより多く誘導されることが判明した。
アルコールストレスを与えた場合であっても同様であった。
以上の結果から、ユーパリノライドＡ又はＢは、他のストレスによりＨＳＰ７０発現の増強効果があることが理解される。 The results are shown in FIG.
In FIG. 4, the results when heat shock is given to the upper stage and the results when alcohol stress is given to the lower stage are shown.
As can be seen from the results shown in the figure, expression of HSP70 is induced even when heat shock is not applied, but more HSP70 is induced when heat shock is applied. There was found.
The same was true even when alcohol stress was applied.
From the above results, it is understood that Euparinolide A or B has an effect of enhancing HSP70 expression by other stress.
実施例５：ストレスに晒した場合のユーパリノライドＡ及びＢの細胞保護作用
ＡＧＳ細胞を、ユーパリノライドＡ又はＢの各濃度（０、２．５及び５．０μｇ／ｍＬ）と共に前処理し、新鮮な培地で洗浄し、ＲＰＭＩ培地（１０％の牛胎児血清、１００Ｕ／ｍＬのペニシリン、１００Ｕ／ｍＬのストレプトマイシン）で、３７℃／５％ＣＯ２雰囲気下で１８時間培養した。最後に３．５％エタノール、又は１５μＭのmenadioneと共に２４時間培養した。
細胞の生存率は、前実施例と同様にＭＴＴ法により行った。アポトーシスを起こした細胞（sub-G1中の細胞）は、fluorescence activated cell sortingによりカウントした、値は、平均値±S.D.で表示した。 Example 5: Cytoprotective effect of Euparinolide A and B when exposed to stress AGS cells were pretreated with each Euparinolide A or B concentration (0, 2.5 and 5.0 μg / mL). Washed with fresh medium, and cultured in RPMI medium (10% fetal bovine serum, 100 U / mL penicillin, 100 U / mL streptomycin) at 37 ° C./5% CO 2 atmosphere for 18 hours. Finally, the cells were cultured with 3.5% ethanol or 15 μM menadione for 24 hours.
The cell viability was determined by the MTT method as in the previous example. Apoptotic cells (cells in sub-G1) were counted by fluorescence activated cell sorting, and the values were expressed as mean ± SD.
ＡＧＳ細胞を、ユーパリノライドＡ又はＢの各濃度（０、２．５及び５．０μｇ／ｍＬ）と共に前処理し、新鮮な培地で洗浄し、ＲＰＭＩ培地（１０％の牛胎児血清、１００Ｕ／ｍＬのペニシリン、１００Ｕ／ｍＬのストレプトマイシン）で、３７℃／５％ＣＯ２雰囲気下で１８時間培養した。最後に３．５％エタノール、又は１５μＭのmenadioneと共に２４時間培養した。
細胞の生存率は、前実施例と同様にＭＴＴ法により行った。アポトーシスを起こした細胞（sub-G1中の細胞）は、fluorescence activated cell sortingによりカウントした、値は、平均値±S.D.で表示した。 Example 5: Cytoprotective effect of Euparinolide A and B when exposed to stress AGS cells were pretreated with each Euparinolide A or B concentration (0, 2.5 and 5.0 μg / mL). Washed with fresh medium, and cultured in RPMI medium (10% fetal bovine serum, 100 U / mL penicillin, 100 U / mL streptomycin) at 37 ° C./5% CO 2 atmosphere for 18 hours. Finally, the cells were cultured with 3.5% ethanol or 15 μM menadione for 24 hours.
The cell viability was determined by the MTT method as in the previous example. Apoptotic cells (cells in sub-G1) were counted by fluorescence activated cell sorting, and the values were expressed as mean ± SD.
その結果を、図５及び図６に示した。
図５は、３．５％エタノールと共に培養した結果であるが、ユーパリノライドＡ又はＢは、濃度依存的に細胞生存率を高めていることが判明する。
また、図６は１５μＭのmenadioneと共に培養した結果であるが、３．５％エタノールの場合と同様、ユーパリノライドＡ又はＢは、濃度依存的に細胞生存率を高めていることが判明する。 The results are shown in FIG. 5 and FIG.
FIG. 5 shows the result of culturing with 3.5% ethanol, and it is found that Euparinolide A or B increases the cell viability in a concentration-dependent manner.
FIG. 6 shows the results of culturing with 15 μM menadione, and it is found that Euparinolide A or B increases the cell viability in a concentration-dependent manner as in the case of 3.5% ethanol.
図５は、３．５％エタノールと共に培養した結果であるが、ユーパリノライドＡ又はＢは、濃度依存的に細胞生存率を高めていることが判明する。
また、図６は１５μＭのmenadioneと共に培養した結果であるが、３．５％エタノールの場合と同様、ユーパリノライドＡ又はＢは、濃度依存的に細胞生存率を高めていることが判明する。 The results are shown in FIG. 5 and FIG.
FIG. 5 shows the result of culturing with 3.5% ethanol, and it is found that Euparinolide A or B increases the cell viability in a concentration-dependent manner.
FIG. 6 shows the results of culturing with 15 μM menadione, and it is found that Euparinolide A or B increases the cell viability in a concentration-dependent manner as in the case of 3.5% ethanol.
実施例６：ユーパリノライドＡ及びＢによるＩＢＭＸ刺激によるメラニン生成抑制効果
Ｂ１６細胞を、ユーパリノライドＡ又はＢの各濃度（５．０及び１０．０μｇ／ｍＬ）と共に培養した。細胞を、さらに１００μＭのＩＢＭＸ（3-isobutyl-1-methylxanthine：Sigma社）の添加、或いは無添加にて７２時間、或いは４８時間培養した。
各サンプルの蛋白質量を、Bio-Rad protein assay kitにより求め、同量の蛋白質量に揃えた後、４９０ｎｍの吸光度を、プレートリーダー（Fluostar Galaxy社）により測定した。
細胞抽出中のメラニンの量を測定し、コントロールとの比較で表示した。
また、チロシナーゼ活性をコントロールとの比較で表示した。 Example 6: Effect of inhibiting melanin production by IBMX stimulation with Euparinolide A and B B16 cells were cultured together with Euparinolide A or B concentrations (5.0 and 10.0 μg / mL). The cells were further cultured for 72 hours or 48 hours with or without the addition of 100 μM IBMX (3-isobutyl-1-methylxanthine: Sigma).
The protein amount of each sample was determined by Bio-Rad protein assay kit, and after aligning the protein amount with the same amount, the absorbance at 490 nm was measured with a plate reader (Fluostar Galaxy).
The amount of melanin during cell extraction was measured and displayed in comparison with the control.
In addition, tyrosinase activity was displayed in comparison with the control.
Ｂ１６細胞を、ユーパリノライドＡ又はＢの各濃度（５．０及び１０．０μｇ／ｍＬ）と共に培養した。細胞を、さらに１００μＭのＩＢＭＸ（3-isobutyl-1-methylxanthine：Sigma社）の添加、或いは無添加にて７２時間、或いは４８時間培養した。
各サンプルの蛋白質量を、Bio-Rad protein assay kitにより求め、同量の蛋白質量に揃えた後、４９０ｎｍの吸光度を、プレートリーダー（Fluostar Galaxy社）により測定した。
細胞抽出中のメラニンの量を測定し、コントロールとの比較で表示した。
また、チロシナーゼ活性をコントロールとの比較で表示した。 Example 6: Effect of inhibiting melanin production by IBMX stimulation with Euparinolide A and B B16 cells were cultured together with Euparinolide A or B concentrations (5.0 and 10.0 μg / mL). The cells were further cultured for 72 hours or 48 hours with or without the addition of 100 μM IBMX (3-isobutyl-1-methylxanthine: Sigma).
The protein amount of each sample was determined by Bio-Rad protein assay kit, and after aligning the protein amount with the same amount, the absorbance at 490 nm was measured with a plate reader (Fluostar Galaxy).
The amount of melanin during cell extraction was measured and displayed in comparison with the control.
In addition, tyrosinase activity was displayed in comparison with the control.
その結果を、図７及び図８に示した。
図７に、メラニン含有量を示し、図８にチロシナーゼ活性を示した。
いずれの場合において、ユーパリノライドＡ又はＢは、メラニン生成を抑制し、チロシナーゼ活性も低下させていることが理解される。
特に、ＩＢＭＸの添加におけるメラニン生成の抑制、並びにチロシナーゼ活性の低下は強いものであることが判明した。 The results are shown in FIGS.
FIG. 7 shows the melanin content, and FIG. 8 shows the tyrosinase activity.
In any case, it is understood that Euparinolide A or B suppresses melanin production and reduces tyrosinase activity.
In particular, it has been found that the suppression of melanin production and the decrease in tyrosinase activity by addition of IBMX are strong.
図７に、メラニン含有量を示し、図８にチロシナーゼ活性を示した。
いずれの場合において、ユーパリノライドＡ又はＢは、メラニン生成を抑制し、チロシナーゼ活性も低下させていることが理解される。
特に、ＩＢＭＸの添加におけるメラニン生成の抑制、並びにチロシナーゼ活性の低下は強いものであることが判明した。 The results are shown in FIGS.
FIG. 7 shows the melanin content, and FIG. 8 shows the tyrosinase activity.
In any case, it is understood that Euparinolide A or B suppresses melanin production and reduces tyrosinase activity.
In particular, it has been found that the suppression of melanin production and the decrease in tyrosinase activity by addition of IBMX are strong.
実施例７：ＨＳＰ７０発現の確認
培養液を満たした培養皿に、ヒト線維芽細胞株（NBIRGB：理化学研究所、バイオリソースセンター）を１×１０２個／ｍＬの細胞懸濁となるように播種し、５％炭酸ガス雰囲気下に３７℃にて２４時間培養を行った。
この培養液に、上記実施例１で抽出・単離したユーパリノライドＡ又はＢ、並びに実施例１の分離過程で得たヤバツイの溶媒抽出物（画分Ａ～Ｃ）を上記したイムノブロット法に従ってＨＳＰ７０の発現誘導を検討した。
評価は、ＨＳＰ７０の発現を２倍にする最小有効濃度（ＭＥＣ２．０（μｇ／ｍｌ））として表示した。
あわせて、その時の細胞生存率（Safe Induction（SI）Index）（％）も検討した。
その結果を下記表１に示した。
なお、表中には、実施例１で調製したヤバツイの溶媒抽出物として画分Ａ～Ｃの結果も示した。 Example 7: Confirmation of HSP70 expression In a culture dish filled with a culture solution, a human fibroblast cell line (NBIRGB: RIKEN, BioResource Center) was seeded so as to obtain a cell suspension of 1 × 10 2 cells / mL. Culturing was performed at 37 ° C. for 24 hours in a 5% carbon dioxide atmosphere.
The immunoblot method described above with Euparinolide A or B extracted and isolated in Example 1 above, and the Yabatsui solvent extract (fractions A to C) obtained in the separation process of Example 1 in this culture solution Then, induction of HSP70 expression was examined.
Evaluation was expressed as the minimum effective concentration (MEC 2.0 (μg / ml)) that doubled the expression of HSP70.
In addition, the cell viability (Safe Induction (SI) Index) (%) at that time was also examined.
The results are shown in Table 1 below.
In the table, the results of fractions A to C as the solvent extract of Yabatsui prepared in Example 1 are also shown.
培養液を満たした培養皿に、ヒト線維芽細胞株（NBIRGB：理化学研究所、バイオリソースセンター）を１×１０２個／ｍＬの細胞懸濁となるように播種し、５％炭酸ガス雰囲気下に３７℃にて２４時間培養を行った。
この培養液に、上記実施例１で抽出・単離したユーパリノライドＡ又はＢ、並びに実施例１の分離過程で得たヤバツイの溶媒抽出物（画分Ａ～Ｃ）を上記したイムノブロット法に従ってＨＳＰ７０の発現誘導を検討した。
評価は、ＨＳＰ７０の発現を２倍にする最小有効濃度（ＭＥＣ２．０（μｇ／ｍｌ））として表示した。
あわせて、その時の細胞生存率（Safe Induction（SI）Index）（％）も検討した。
その結果を下記表１に示した。
なお、表中には、実施例１で調製したヤバツイの溶媒抽出物として画分Ａ～Ｃの結果も示した。 Example 7: Confirmation of HSP70 expression In a culture dish filled with a culture solution, a human fibroblast cell line (NBIRGB: RIKEN, BioResource Center) was seeded so as to obtain a cell suspension of 1 × 10 2 cells / mL. Culturing was performed at 37 ° C. for 24 hours in a 5% carbon dioxide atmosphere.
The immunoblot method described above with Euparinolide A or B extracted and isolated in Example 1 above, and the Yabatsui solvent extract (fractions A to C) obtained in the separation process of Example 1 in this culture solution Then, induction of HSP70 expression was examined.
Evaluation was expressed as the minimum effective concentration (MEC 2.0 (μg / ml)) that doubled the expression of HSP70.
In addition, the cell viability (Safe Induction (SI) Index) (%) at that time was also examined.
The results are shown in Table 1 below.
In the table, the results of fractions A to C as the solvent extract of Yabatsui prepared in Example 1 are also shown.
表中の結果より明らかなように、ユーパリノライドＡ及びＢには、極めて強いＨＳＰ７０の発現誘導作用があることが判明する。
また、画分Ａ～Ｃにあっても、ＨＳＰ７０発現誘導作用があることが判明する。
ヤバツイ自体はすでに中国において解熱・解毒薬として使用されている生薬であることから、ヤバツイから抽出・単離したユーパリノライドＡ及びＢは、細胞毒性がなく、安全なものであることが判明した。 As is clear from the results in the table, it is found that Euparinolide A and B have a very strong HSP70 expression-inducing action.
It is also found that even in fractions A to C, there is an HSP70 expression inducing action.
Yabatsui itself is a herbal medicine already used in China as an antipyretic / antidote, so it was found that Euparinolide A and B extracted and isolated from Yabatsui are safe and non-cytotoxic. .
また、画分Ａ～Ｃにあっても、ＨＳＰ７０発現誘導作用があることが判明する。
ヤバツイ自体はすでに中国において解熱・解毒薬として使用されている生薬であることから、ヤバツイから抽出・単離したユーパリノライドＡ及びＢは、細胞毒性がなく、安全なものであることが判明した。 As is clear from the results in the table, it is found that Euparinolide A and B have a very strong HSP70 expression-inducing action.
It is also found that even in fractions A to C, there is an HSP70 expression inducing action.
Yabatsui itself is a herbal medicine already used in China as an antipyretic / antidote, so it was found that Euparinolide A and B extracted and isolated from Yabatsui are safe and non-cytotoxic. .
既に上記で説明したように、ＨＳＰ発現誘導剤であるユーパリノライドＡ及び／又はＢによるＨＳＰの発現、特にＨＳＰ７０の発現誘導は、脳梗塞やアルツハイマー病などの脳疾患、潰瘍性大腸炎などの治療剤としての医薬品として有用であり、さらにメラニン産生抑制化粧料となる。
As already explained above, HSP expression by Euparinolide A and / or B, which is an HSP expression inducer, in particular, HSP70 expression induction is caused by brain diseases such as cerebral infarction and Alzheimer's disease, ulcerative colitis, etc. It is useful as a pharmaceutical agent as a therapeutic agent, and further becomes a melanin production-suppressing cosmetic.
以下に、本発明が提供するＨＳＰ発現誘導剤であるユーパリノライドＡ及びＢの製剤応用例を示した。
応用例１：皮膚外用剤
以下の処方により、皮膚外用剤（クリーム剤）を得た。
スクワラン ２０重量％
ミツロウ ５
精製ホホバ油 ５
グリセリンモノステアレート ２
ソルビタンモノステアレート ２
ポリオキシエチレン（２０）ソルビタンモノステアレート ２
グリセリン ５
ユーパリノライドＡ １
ユーパリノライドＢ １
精製水 １００とする残部 Examples of pharmaceutical applications of Euparinolide A and B, which are HSP expression inducers provided by the present invention, are shown below.
Application Example 1: External preparation for skin An external preparation for skin (cream) was obtained according to the following formulation.
Squalane 20% by weight
Beeswax 5
Refined jojoba oil 5
Glycerol monostearate 2
Sorbitan monostearate 2
Polyoxyethylene (20)sorbitan monostearate 2
Glycerin 5
Upalino Ride A 1
Upalino Ride B 1
The balance of 100 purified water
応用例１：皮膚外用剤
以下の処方により、皮膚外用剤（クリーム剤）を得た。
スクワラン ２０重量％
ミツロウ ５
精製ホホバ油 ５
グリセリンモノステアレート ２
ソルビタンモノステアレート ２
ポリオキシエチレン（２０）ソルビタンモノステアレート ２
グリセリン ５
ユーパリノライドＡ １
ユーパリノライドＢ １
精製水 １００とする残部 Examples of pharmaceutical applications of Euparinolide A and B, which are HSP expression inducers provided by the present invention, are shown below.
Application Example 1: External preparation for skin An external preparation for skin (cream) was obtained according to the following formulation.
Polyoxyethylene (20)
The balance of 100 purified water
応用例２：注射剤
ユーパリノライドＡ又はＢを、塩化ナトリウム、及び溶解補助剤としてポリソルベート２０ともに注射用滅菌精製水に溶解し、１％含有注射剤を得た。 Application Example 2: Injection Euparinolide A or B was dissolved in sterile purified water for injection together with sodium chloride andpolysorbate 20 as a solubilizing agent to obtain an injection containing 1%.
ユーパリノライドＡ又はＢを、塩化ナトリウム、及び溶解補助剤としてポリソルベート２０ともに注射用滅菌精製水に溶解し、１％含有注射剤を得た。 Application Example 2: Injection Euparinolide A or B was dissolved in sterile purified water for injection together with sodium chloride and
応用例３：静脈注射剤
ユーパリノライドＡ又はＢの１％（ｗ／ｗ）、シュークロース１０％（ｗ／ｗ）、塩化ベンザルコニウム０．０５％（ｗ／ｗ）を５％キシリトール水溶液に溶解した後、凍結乾燥した。得られた凍結乾燥剤に、別にバイアル充填した０．５％カルメロースあるいは注射用水を加えることにより静脈注射用剤を得た。 Application Example 3: 1% (w / w) of euparinolide A or B, 10% (w / w) sucrose, 0.05% (w / w) benzalkonium chloride in 5% aqueous xylitol solution And then freeze-dried. An intravenous injection preparation was obtained by adding 0.5% carmellose or water for injection separately filled into the resulting freeze-dried preparation.
ユーパリノライドＡ又はＢの１％（ｗ／ｗ）、シュークロース１０％（ｗ／ｗ）、塩化ベンザルコニウム０．０５％（ｗ／ｗ）を５％キシリトール水溶液に溶解した後、凍結乾燥した。得られた凍結乾燥剤に、別にバイアル充填した０．５％カルメロースあるいは注射用水を加えることにより静脈注射用剤を得た。 Application Example 3: 1% (w / w) of euparinolide A or B, 10% (w / w) sucrose, 0.05% (w / w) benzalkonium chloride in 5% aqueous xylitol solution And then freeze-dried. An intravenous injection preparation was obtained by adding 0.5% carmellose or water for injection separately filled into the resulting freeze-dried preparation.
以上記載のように、本発明により、ユーパリノライドＡ及び／又はＢからなるヒートショックプロテイン（ＨＳＰ）発現誘導剤が提供される。
本発明が提供するユーパリノライドＡ及び／又はＢは、ＨＳＰ、特にＨＳＰ７０の発現を誘導し、ＨＳＰの損傷によって生じる脳梗塞やアルツハイマー病などの脳疾患、潰瘍性大腸炎などの治療剤として有用であるとともに、発現が誘導されたＨＳＰ７０がメラニン産生を制御させ、その結果、メラニンの産生を抑制するものであり、その結果、シミの予防・改善作用のある化粧料を提供することができる、極めて特異的なものである。 As described above, the present invention provides a heat shock protein (HSP) expression inducer comprising Euparinolide A and / or B.
Euparinolide A and / or B provided by the present invention induces the expression of HSP, particularly HSP70, and is useful as a therapeutic agent for brain diseases such as cerebral infarction and Alzheimer's disease caused by HSP damage, ulcerative colitis, etc. In addition, HSP70 whose expression is induced controls melanin production, and as a result, suppresses the production of melanin. As a result, it is possible to provide a cosmetic having an effect of preventing and improving stains. It is very specific.
本発明が提供するユーパリノライドＡ及び／又はＢは、ＨＳＰ、特にＨＳＰ７０の発現を誘導し、ＨＳＰの損傷によって生じる脳梗塞やアルツハイマー病などの脳疾患、潰瘍性大腸炎などの治療剤として有用であるとともに、発現が誘導されたＨＳＰ７０がメラニン産生を制御させ、その結果、メラニンの産生を抑制するものであり、その結果、シミの予防・改善作用のある化粧料を提供することができる、極めて特異的なものである。 As described above, the present invention provides a heat shock protein (HSP) expression inducer comprising Euparinolide A and / or B.
Euparinolide A and / or B provided by the present invention induces the expression of HSP, particularly HSP70, and is useful as a therapeutic agent for brain diseases such as cerebral infarction and Alzheimer's disease caused by HSP damage, ulcerative colitis, etc. In addition, HSP70 whose expression is induced controls melanin production, and as a result, suppresses the production of melanin. As a result, it is possible to provide a cosmetic having an effect of preventing and improving stains. It is very specific.
Claims (3)
- ユーパリノライドＡ及び／又はＢ（Eupalinolide Ａ及び／又はＢ）からなるヒートショックプロテイン（ＨＳＰ）発現誘導剤。 A heat shock protein (HSP) expression inducer comprising Euparinolide A and / or B (Eupalinolide A and / or B).
- ユーパリノライドＡ及び／又はＢ（Eupalinolide Ａ及び／又はＢ）が、サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（ヤバツイ）から抽出したものである請求項１に記載のヒートショックプロテイン（ＨＳＰ）発現誘導剤。 The heat shock protein (HSP) expression according to claim 1, wherein Euparinolide A and / or B (Eupalinolide A and / or B) is extracted from the above-ground dry herbal medicine (Yabatsui) of Eupatorium lindleyanum. Inducer.
- サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（ヤバツイ）より、以下の抽出・分画処理することからなるユーパリノライドＡ及び／又はＢ（Eupalinolide Ａ及び／又はＢ）の抽出方法であって、
（ａ）サワヒヨドリ（Eupatorium lindleyanum）の地上部乾燥生薬（ヤバツイ）をエタノール抽出して得た抽出物を、ヘキサンにより脱脂処理を行った後、９０％メタノール溶液に溶解して得た９０％メタノール溶液画分（画分Ａ）を得、
（ｂ）次いで、上記で得た９０％メタノール溶液画分（画分Ａ）を、ＭＣｌ・ＧＥＬ ＣＨＰ－２０Ｐカラムクロマトグラフィーに付し、順次、水溶出－５０％メタノール溶出－メタノール溶出－アセトン溶出を行い、メタノール溶出画分（画分Ｂ）を得、
（ｃ）上記で得たメタノール溶出画分（画分Ｂ）を、更にＯＤＳカラムクロマトグラフィーに付し、順次５０％メタノール溶液、７５％メタノール溶液、及びメタノール溶液で溶出を行い、５０％メタノール溶出画分（画分Ｃ）を得、
（ｄ）最後に、上記で得た５０％メタノール溶出画分（画分Ｃ）をＨＰＬＣ（カラム：ＡＲ－ＩＩ ＯＤＳ）に付し、４０％メタノールにて溶出すること、
からなる抽出方法。 A method of extracting Euparinolide A and / or B (Eupalinolide A and / or B) comprising the following extraction and fractionation treatment from the above-ground dry herbal medicine (Yabatsui) of Eupatorium lindleyanum,
(A) 90% methanol solution obtained by degreasing the extract obtained by ethanol extraction of dry crude herb (Yabatsui) of Eupatorium lindleyanum with hexane, and then dissolving in 90% methanol solution To obtain a fraction (fraction A)
(B) Next, the 90% methanol solution fraction (fraction A) obtained above was subjected to MCl · GEL CHP-20P column chromatography, followed by water elution, 50% methanol elution, methanol elution, and acetone elution. To obtain a methanol elution fraction (fraction B),
(C) The methanol elution fraction (Fraction B) obtained above was further subjected to ODS column chromatography, and the elution was carried out sequentially with 50% methanol solution, 75% methanol solution and methanol solution, and 50% methanol elution. Fraction (Fraction C)
(D) Finally, the 50% methanol elution fraction obtained above (fraction C) is subjected to HPLC (column: AR-II ODS) and eluted with 40% methanol.
Extraction method consisting of.
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| CN117137905A (en) * | 2023-10-30 | 2023-12-01 | 中国中医科学院中药研究所 | Application of eupatorium sesquiterpene lactone compound in preparation of medicine or health-care product for preventing/treating psoriasis |
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| CN105461782A (en) * | 2016-01-14 | 2016-04-06 | 郑平珍 | Novel triterpene compound and preparation and medical application thereof |
| WO2017204321A1 (en) * | 2016-05-26 | 2017-11-30 | 株式会社アミノアップ化学 | Sleep improving agent |
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| CN114432298A (en) * | 2022-01-24 | 2022-05-06 | 苏州大学 | Application of eupatorium lindleyanum sesquiterpene lactone in preparation of medicine for preventing or treating pulmonary fibrosis |
| CN117137905A (en) * | 2023-10-30 | 2023-12-01 | 中国中医科学院中药研究所 | Application of eupatorium sesquiterpene lactone compound in preparation of medicine or health-care product for preventing/treating psoriasis |
| CN117137905B (en) * | 2023-10-30 | 2024-03-01 | 中国中医科学院中药研究所 | Application of eupatorium sesquiterpene lactone compounds in preparation of medicines for preventing/treating psoriasis |
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