Novel compounds and their use

文档序号：1821036 发布日期：2021-11-09 浏览：32次中文

阅读说明：本技术 新化合物及其应用 (Novel compounds and their use ) 是由张万皎小坂邦男松山惠介田中麻美曾田匡洋于 2020-02-07 设计创作，主要内容包括：本发明目的在于提供具有抗氧化作用的新化合物及其应用技术。提供由式(1)表示的化合物,解决上述课题。[式中,R～(1)～R～(3)中的任意一个为氢、乙酰基、2-丁烯酰基或2-甲基-2-戊烯酰基,其余2个为氢；且R～(4)～R～(6)中的任意一个为2-甲基-2-丁烯酰基,其余2个为氢]。(The invention aims to provide a novel compound with an antioxidation effect and an application technology thereof. Provided is a compound represented by the formula (1) and solving the above problems. [ in the formula, R 1 ～R 3 Any one of them is hydrogen, acetyl, 2-butenoyl or 2-methyl-2-pentenoyl, and the other 2 are hydrogen; and R is 4 ～R 6 Any one of them is 2-methyl-2-butenoyl, and the other 2 are hydrogen]。)

1. A compound represented by the following formula (1):

[ solution 1]

In the formula, R¹～R³Any one of them is hydrogen, acetyl, 2-butenoyl or 2-methyl-2-pentenoyl, and the other 2 are hydrogen; and R is⁴～R⁶Any one of them is 2-methyl-2-butenoyl, and the remaining 2 are hydrogen.

2. The compound according to claim 1, wherein in the formula (1), R is²Is hydrogen, acetyl, 2-butenoyl or 2-methyl-2-pentenoyl, R⁵Is 2-methyl-2-butenoyl, R¹、R³、R⁴And R⁶Is hydrogen.

3. A composition comprising a compound of claim 1 or 2.

4. The composition according to claim 3, further comprising at least one compound represented by the following formulae (6) to (8),

[ solution 2]

[ solution 3]

[ solution 4]

5. The composition according to claim 3 or 4, wherein the composition is a cosmetic composition or an antioxidant.

6. A method for producing the compound according to claim 1 or 2, which comprises:

(A) a step of culturing a microorganism having an ability to produce the compound of claim 1 or 2 in a medium; and

(B) a step of collecting the compound of claim 1 or 2 from the culture.

Technical Field

The invention relates to a novel compound and application thereof. More particularly, the present invention relates to a novel compound, a composition comprising the novel compound, a cosmetic composition or an antioxidant comprising the novel compound, and the like.

Background

Metabolites produced by microorganisms are mostly useful substances for human beings, and have been conventionally used as active ingredients for medicines, cosmetics, and the like.

For example, isolation of penicillin having antibacterial activity from penicillium is well known as a cause. Further, as described in patent documents 1 and 2, it has been reported that Trehangelin having membrane lipid disorder-inhibiting action, antibacterial activity, antioxidant action and the like is obtained from actinomycetes.

Thus, there are many possibilities that the examples of obtaining useful substances from microorganisms are not exhaustive and the search for new useful substances using microorganisms still remains.

[ Prior art documents ]

[ patent document ]

[ patent document 1] International publication No. 2014/034147

[ patent document 2] Japanese laid-open patent publication No. 2015-24985 "

Disclosure of Invention

Problems to be solved by the invention

Under such circumstances, an object of one embodiment of the present invention is to provide a novel compound having an antioxidant effect and a technique for using the same.

Means for solving the problems

The present inventors have conducted extensive studies to solve the above problems, and as a result, succeeded in identifying a novel compound having an antioxidant activity from a specific microorganism, specifically, Streptomyces lividans strain 1326, which is an actinomycete, and have completed the present invention. That is, one embodiment of the present invention is a compound represented by the following formula (1).

[ solution 1]

(in the formula, R¹～R³Any one of which is hydrogen, acetyl, 2-butenoyl or 2-methyl-2-pentenoyl, the remaining 2 are hydrogen, and R is⁴～R⁶Any one of which is 2-methyl-2-butenoyl and the remaining 2 are hydrogen).

Effects of the invention

According to one embodiment of the present invention, a novel compound having an antioxidant effect can be provided.

Drawings

FIG. 1 is a graph showing the results of evaluation of the antioxidant activity of a compound according to one embodiment of the present invention.

Detailed Description

One embodiment of the present invention is described in detail below.

In the present specification, "a to B" indicating a numerical range means "a to B inclusive", unless otherwise specified. In addition, when the structural formula in the present specification does not particularly indicate a stereostructure or the like, the compound represented by the structural formula in the present specification includes various stereoisomers such as tautomers, geometric isomers, optical isomers and the like, and a mixture thereof (including racemates).

[ 1. Compound ]

The present inventors have conducted detailed studies on various microorganisms in order to search for a novel compound having an antioxidant activity. Among them, a novel compound having an antioxidant activity was successfully identified from Streptomyces lividans strain 1326 of actinomycetes into which a gene related to a synthesis pathway of a specific compound has been introduced.

A compound according to an embodiment of the present invention (hereinafter, referred to as "the present compound") is a compound represented by the above formula (1). As shown in examples described later, the compound has an excellent antioxidant effect. Specific examples of the compound are described below. The compound of the present invention may be in the form of a solvate, for example, a hydrate, and such a solvate is also included in the scope of the present compound. In the present specification, "2-butenoyl" is used in the same sense as crotonyl and isocrotonyl, and "2-methyl-2-butenoyl" is used in the same sense as angeloyl.

In one embodiment of the present invention, the compound is preferably represented by the formula (1) wherein R is²Is hydrogen, acetyl, 2-butenoyl or 2-methyl-2-pentenoyl, R⁵Is 2-methyl-2-butenoyl, R¹、R³、R⁴And R⁶Is hydrogen.

In the preferred embodiment, R in the formula (1)²The compound referred to as "hydrogen (H)" is 3-O-angeloyltrehalose (3-O-angeloyltrehalase), and is represented by the following formula (2).

[ solution 2]

In the present specification, 3-O-angeloyltrehalose is a compound having the following physical properties.

(1) The characteristics are as follows: white powder

(2) Molecular weight: 424

(3) The molecular formula is as follows: c₁₇H₂₈O₁₂

(4) Melting point: 117-120 deg.C

(5) High resolution mass spectrometry [ M + NH₄]⁺Theoretical value (m/z): 442.1925, found value (m/z): 442.1901

(6) Uv absorption maximum (in methanol): 220nm

(7)¹H-NMRδppm：1.86(3H,dd,J₁＝1.5Hz,J₂＝1.5Hz),1.93(3H,dd,J₁＝7.2Hz,J₂＝1.5Hz),3.15(1H,ddd,J₁＝9.5Hz,J₂＝9.1Hz,J₃＝5.7Hz),3.2～3.7(9H,m),3.78(1H,ddd,J₁＝10.0Hz,J₂＝4.0Hz,J₃＝2.3Hz),4.37(1H,t,J＝5.9Hz),4.48(1H,t,J＝5.9Hz),4.72(1H,d,J＝6.4Hz),4.77(1H,d,J＝5.6Hz),4.84(1H,d,J＝5.7Hz),4.84(1H,d,J＝6.7Hz),4.91(1H,d,J＝3.6Hz),4.95(1H,d,J＝3.6Hz),5.02(1H,d,J＝7.0Hz),5.21(1H,t,J＝9.5Hz),6.04(1H,dq,J₁＝7.2Hz,J₂＝1.5Hz)

(8) Solubility in solvent: is difficult to dissolve in methanol

In the preferred embodiment, R in the formula (1)²Is "acetyl ((CO) CH₃) The compound of (1) is 3-O-acetyl-3 '-O-angeloyltrehalose (3-O-acetyl-3' -O-angeloyltrehalose), and is represented by the following formula (3).

[ solution 3]

In the present specification, 3-O-acetyl-3' -O-angeloyltrehalose is a compound having the following physical properties.

(1) The characteristics are as follows: white powder

(2) Molecular weight: 466

(3) The molecular formula is as follows: c₁₉H₃₀O₁₃

(4) Melting point: 160-161 ℃ C

(5) High resolution mass spectrometry [ M + NH₄]⁺Theoretical value (m/z): 484.2030, found value (m/z): 484.2001

(6) Uv absorption maximum (in methanol): 219nm

(7)¹H-NMRδppm：1.87(3H,dd,J₁＝1.4Hz,J₂＝1.4Hz),1.93(3H,dd,J₁＝7.1Hz,J₂＝1.4Hz),2.05(3H,s),3.2～3.5(2H,m),3.4～3.7(6H,m),3.7～3.8(2H,m),4.45(1H,t,J＝5.6Hz),4.48(1H,t,J＝5.6Hz),4.94(1H,d,J＝7.3Hz),4.95(1H,d,J＝6.7Hz),4.97(2H,d,J＝3.7Hz),5.02(1H,d,J＝6.2Hz),5.03(1H,d,J＝7.0Hz),5.14(1H,t,J＝9.5Hz),5.24(1H,t,J＝9.5Hz),6.05(1H,dq,J₁＝7.1Hz,J₂＝1.4Hz)

(8) Solubility in solvent: is difficult to dissolve in methanol

In the preferred embodiment, R in the formula (1)²Is "isocrotonyl ((CO) HC ═ CHCH₃) The compound is 3-O-angeloyl-3 '-O-isocrotonyl trehalose (3-O-angeloyl-3' -O-isocrotonyl trehalose) and is represented by the following formula (4).

[ solution 4]

In the present specification, 3-O-angeloyl-3' -O-isocrotonyl trehalose is a compound having the following physical properties.

(1) The characteristics are as follows: white powder

(2) Molecular weight: 492

(3) The molecular formula is as follows: c₂₁H₃₂O₁₃

(4) Melting point: 155-158 DEG C

(5) High resolution mass spectrometry [ M + NH₄]⁺Theoretical value (m/z): 510.2187, found value (m/z): 510.2225

(6) Uv absorption maximum (in methanol): 227nm

(7)¹H-NMRδppm：1.87(3H,dd,J₁＝1.4Hz,J₂＝1.4Hz),1.94(3H,dd,J₁＝7.1Hz,J₂＝1.4Hz),2.10(3H,dd,J₁＝7.2Hz,J₂＝1.7Hz),3.2～3.5(2H,m),3.4～3.7(6H,m),3.7～3.8(2H,m),4.46(1H,t,J＝5.9Hz),4.48(1H,t,J＝5.9Hz),4.92(1H,d,J＝7.2Hz),4.93(1H,d,J＝7.2Hz),4.97(1H,d,J＝3.1Hz),4.98(1H,d,J＝3.1Hz),5.01(1H,d,J＝7.3Hz),5.02(1H,d,J＝7.4Hz),5.23(1H,t,J＝9.5Hz),5.26(1H,t,J＝9.5Hz),5.85(1H,dd,J₁＝11.5Hz,J₂＝1.7Hz),6.05(1H,dq,J₁＝7.1Hz,J₂＝1.4Hz),6.39(1H,dq,J₁＝11.5Hz,J₂＝7.2Hz)

(8) Solubility in solvent: is difficult to dissolve in methanol

In the preferred embodiment, R in the formula (1)²Is "2-methyl-2-pentenoyl ((CO) C (CH)₃)＝CHCH₂CH₃) "is 3-O- (2-methyl-2-butenoyl) -3 '-O- (2-methyl-2-pentenoyl) trehalose (3-O- (2-methyl-2-butenoyl) -3' -O- (2-methyl-2-pentenoyl) trehalose), and is represented by the following formula (5).

[ solution 5]

In the present specification, 3-O- (2-methyl-2-butenoyl) -3' -O- (2-methyl-2-pentenoyl) trehalose has a structure represented by the above formula (5) and has the following physical properties.

(1) The characteristics are as follows: white powder

(2) Molecular weight: 520

(3) The molecular formula is as follows: c₂₃H₃₆O₁₃

(4) Melting point: 176 to 180 DEG C

(5) High resolution mass spectrometry [ M + NH₄]⁺Theoretical value (m/z): 538.2500, found value (m/z): 538.2495

(6) Uv absorption maximum (in methanol): 227nm

(7)¹H-NMRδppm：0.98(3H,t,J＝7.5Hz),1.8～1.9(6H,m),1.94(3H,dd,J₁＝7.2Hz,J₂＝1.5Hz),2.41(2H,ddq,J₁＝7.5Hz,J₂＝7.5Hz,J₃＝1.1Hz),3.2～3.5(2H,m),3.4～3.7(6H,m),3.7～3.8(2H,m),4.48(2H,t,J＝5.6Hz),4.917(1H,d,J＝7.3Hz),4.925(1H,d,J＝7.3Hz),4.98(2H,d,J＝3.7Hz),5.01(2H,d,J＝7.4Hz),5.26(1H,t,J＝9.6Hz),5.27(1H,t,J＝9.5Hz),5.93(1H,dt,J₁＝7.5Hz,J₂＝1.4Hz),6.05(1H,dq,J₁＝7.2Hz,J₂＝1.5Hz)

(8) Solubility in solvent: is difficult to dissolve in methanol

[ 2. composition ]

A composition according to an embodiment of the present invention (hereinafter referred to as "the present composition") contains at least one of the present compounds represented by the above formulas (1) to (5).

In another embodiment, the present composition preferably further contains at least one compound represented by the following formulae (6) to (8).

[ solution 6]

[ solution 7]

[ solution 8]

The compounds represented by the above formulae (6) to (8) are referred to as "Trehangelin a", "Trehangelin B", and "Trehangelin C", respectively. In the present specification, the compounds represented by the above formulas (6) to (8) are collectively referred to as "trehangeolin".

The present compound contained in the present composition has an antioxidant effect, and therefore, can be used in various fields requiring antioxidant substances, for example, in the fields of cosmetics, medicines, foods, drinks, perfumes, pigments, synthetic resins, adhesives, fuels, and the like. Further, since the compounds represented by the above formulae (6) to (8) have membrane lipid disorder-inhibiting action, antibacterial activity, antioxidant action and other functions, the present composition containing these compounds can be expected to have a wider range of effects, synergistic effects with antioxidant action and the like.

The amount of the present compound contained in the present composition is preferably about 0.00001 to 5% by weight, but may be appropriately adjusted depending on various conditions such as the form of the preparation to be used and the object to be used. The amount of the present compound contained in the present composition is preferably 0.00001 to 0.5 wt%, and more preferably 0.0001 to 0.05 wt%.

The amount of the compound represented by the above formulae (6) to (8) contained in the present composition is preferably about 0.0001 to 10% by weight, but may be appropriately adjusted depending on various conditions such as the form of the preparation to be used and the object to be used. The amount of the compound contained in the present composition is preferably 0.0001 to 1% by weight, more preferably 0.001 to 0.1% by weight.

In the present composition, the mixing ratio of the compound represented by formula (1) of the present invention to trehangeolin may be, for example, 0.01: 1-10: 1, preferably 0.4: 1.

in the present composition, the mixing ratio of the compound represented by the formula (2) of the present invention to trehangeolin may be, for example, 0.01: 1-10: 1, preferably 0.2: 1.

in the present composition, the mixing ratio of the compound represented by the formula (3) of the present invention to trehangeolin may be, for example, 0.01: 1-10: 1, preferably 0.04: 1.

in the present composition, the mixing ratio of the compound represented by the formula (4) of the present invention to trehangeolin may be, for example, 0.01; 1-10: 1, preferably 0.05: 1.

in the present composition, the mixing ratio of the compound represented by formula (5) of the present invention to trehangeolin may be, for example, 0.01: 1-10: 1, preferably 0.05: 1.

in the present composition, the mixing ratio of the compounds represented by the formulae (2) to (5) of the present invention to Trehangelin may be, for example, 0.01: 1-10: 1, preferably 0.34: 1.

the composition may also contain substances other than Trehangelin.

Hereinafter, a "cosmetic composition" and an "antioxidant" will be described as examples of the present composition.

[ 3. cosmetic compositions ]

In one embodiment of the present invention, the present composition is preferably a cosmetic composition (hereinafter, referred to as "the present cosmetic composition").

The cosmetic composition preferably contains at least one compound represented by the above formulas (1) to (5), and more preferably further contains at least one compound represented by the above formulas (6) to (8).

In addition to the above compounds, the cosmetic composition may contain oils and fats such as vegetable oils, higher fatty acids, higher alcohols, silicones (silicones), anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, preservatives, saccharides, metal ion blocking agents, polymers such as water-soluble polymers, thickeners, powder components, ultraviolet absorbers, ultraviolet screening agents, moisturizers such as hyaluronic acid, perfumes, pH adjusting agents, drying agents, and the like. The cosmetic composition may further contain other medicinal components such as vitamins, skin activators, blood circulation promoters, resident bacteria control agents, active oxygen scavengers, anti-inflammatory agents, anticancer agents, whitening agents, bactericides, and the like, physiologically active components, and the like.

Examples of the oils and fats include liquid oils and fats such as camellia oil, evening primrose oil, macadamia nut oil, olive oil, rapeseed oil, corn oil, sesame oil, jojoba oil, germ oil, wheat germ oil, triglyceride, and tricaprylin; solid fats and oils such as cacao butter, coconut oil, hydrogenated coconut oil, palm kernel oil, wood wax kernel oil, hydrogenated oil, and hydrogenated castor oil; waxes such as beeswax, candelilla wax, cotton wax, bran wax, lanolin acetate, liquid lanolin, and sugar cane wax; liquid paraffin; squalene; squalane; microcrystalline waxes, and the like.

Examples of the higher fatty acid include lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and the like.

Examples of the higher alcohols include linear alcohols such as lauryl alcohol, stearyl alcohol, cetyl alcohol, and cetearyl alcohol; and branched alcohols such as monostearyl glyceryl ether, lanolin alcohol, cholesterol, phytosterol, and octyldodecanol.

Examples of the silicone include dimethylpolysiloxane and methylphenylpolysiloxane as chain polysiloxanes; decamethylpolysiloxane of cyclic polysiloxane, and the like.

Examples of the anionic surfactant include fatty acid salts such as sodium laurate, higher alkyl sulfate salts such as sodium lauryl sulfate, alkyl ether sulfate salts such as POE lauryl sulfate triethanolamine, N-acylsarcosine, sulfosuccinate, and N-acylamino acid salts.

Examples of the cationic surfactant include alkyltrimethylammonium salts such as stearyltrimethylammonium chloride, benzalkonium chloride, and benzethonium chloride.

Examples of the amphoteric surfactant include betaines such as alkyl betaine and amido betaine, which are surfactants.

Examples of the nonionic surfactant include sorbitan fatty acid esters such as sorbitan monooleate, and hydrogenated castor oil derivatives.

Examples of the preservative include methyl paraben and ethyl paraben.

Examples of the metal ion-blocking agent include ethylenediaminetetraacetic acid and ethylenediaminetetraacetic acid sodium salt.

Examples of the polymer include vinyl polymers such as gum arabic, tragacanth gum, polygalactose, guar gum, carageenan, pectin, agar gum, quince seed, polydextrose, carboxymethyl starch, collagen, casein, gelatin, methyl cellulose, methylhydroxypropyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose (CMC), sodium alginate, and carboxyvinyl polymers (CARBOPOL, etc.), and bentonite.

Examples of the thickener include carrageenan, tragacanth gum, quince seed, casein, dextrin, gelatin, CMC, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxyvinyl polymer, guar gum, xanthan gum, and the like.

Examples of the powder component include pearl pigments such as talc, kaolin, mica, silica, zeolite, polyethylene powder, polystyrene powder, cellulose powder, inorganic white pigment, inorganic red pigment, titanium oxide-coated mica, titanium oxide-coated talc, and colored titanium oxide-coated mica; and organic pigments such as red 201 and red 202.

Examples of the ultraviolet absorber include p-aminobenzoic acid, phenyl salicylate, isopropyl p-methoxycinnamate, octyl p-methoxycinnamate, and 2, 4-dihydroxydiphenyl ketone.

Examples of the ultraviolet shielding agent include titanium oxide, talc, carmine (carmine), bentonite, kaolin, and zinc oxide.

Examples of the humectant include polyethylene glycol, propylene glycol, dipropylene glycol, 1, 3-butylene glycol, glycerin, diglycerin, polyglycerin, xylitol, maltitol, maltose, sorbitol, glucose, fructose, sodium chondroitin sulfate, sodium hyaluronate, sodium lactate, pyrrolidone carboxylic acid, and cyclodextrin.

Examples of the pharmaceutically effective component include the following vitamins: vitamin A oils, retinol, and other vitamins A; vitamin B2 such as riboflavin; vitamin B6 such as pyridoxine hydrochloride; vitamin C such as L-ascorbic acid, L-ascorbic acid phosphate, L-ascorbic acid monopalmitate, L-ascorbic acid dipalmitate, and L-ascorbic acid-2-glucoside; pantothenic acids such as calcium pantothenate; vitamin D compounds such as vitamin D2 and cholecalciferol; vitamin E compounds such as alpha-tocopherol, tocopherol acetate, nicotinic acid, DL-alpha-tocopherol, etc.

Substances which can be contained in the cosmetic composition, and further skin whitening agents such as placentin, glutathione, and saxifrage extract; skin activators such as royal jelly and beech extract; blood circulation promoter such as capsaicin, zingerone, cantharides tincture, ichthyol, caffeine, tannin, and gamma-rice furfuryl alcohol; anti-inflammatory agents such as glycyrrhizic acid derivatives, glycyrrhetic acid derivatives, azulene and the like; amino acids such as arginine, serine, leucine, and tryptophan; resident bacteria control agents such as maltose sucrose condensates; lysozyme chloride and the like.

Further, substances that may be contained in the cosmetic composition, there are exemplified various extracts such as chamomile extract, parsley extract, beech extract, wine yeast, grapefruit extract, honeysuckle extract, rice extract, grape extract, hop extract, rice bran extract, loquat extract, phellodendron bark extract, coix seed extract, swertia herb extract, sweet clover extract, birch extract, licorice extract, peony extract, soapwort extract, luffa extract, capsicum extract, lemon extract, gentian extract, perilla extract, aloe extract, rosemary extract, sage extract, thyme extract, tea extract, seaweed extract, cucumber extract, clove extract, carrot extract, aesculus hippocastanum extract, witch hazel extract, mulberry extract, and the like.

The cosmetic composition can be applied in the following forms, for example: liquid agents such as aqueous solution, oil, lotion (deposition), and suspension; semi-solid agents such as gels and creams; solid agents such as powder, granule, capsule, microcapsule, solid, etc. The preparation can be made into various dosage forms such as emulsion, gel, cream, ointment, plaster, cataplasm, spray, suppository, injection, powder, granule, lozenge, pill, syrup, and larynx lozenge by conventional known method. It can be applied by coating, attaching, spraying, drinking, etc. onto the body. Of these, lotions, emulsions, creams, ointments, plasters, poultices (poultices), sprays and the like are particularly suitable for external preparations for skin.

The cosmetic composition can be skin cosmetics such as astringent, lotion, cream, and mud film; cosmetics such as foundation cream, makeup cream, foundation in the form of emulsion or cream or ointment, lipstick, eye color, cheek color, etc.; body cosmetics such as hand cream, leg cream, body lotion, etc.; bathing agent, oral cavity cosmetic, hair cosmetic, etc.

[ 4. antioxidants ]

In one embodiment of the present invention, the present composition is preferably an antioxidant (hereinafter referred to as "present antioxidant").

The antioxidant can be formulated by a conventional method using a generally pharmaceutically acceptable carrier.

When the composition is formulated into a solid preparation for oral administration, an excipient may be added to the main drug, and further, after a binder, a disintegrating agent, a lubricant, etc. are added as necessary, the composition is prepared into a solvent, granules, powder, capsules, etc. by a conventional method.

When the injection is prepared, a pH regulator, a buffer, a stabilizer, a cosolvent and the like can be added into the main medicine as required, and the injection for subcutaneous or intravenous injection is prepared by a conventional method.

When formulated as an external preparation for skin, it can be formulated according to the present cosmetic composition described above. For example, the ointment may be an ointment comprising a hydrophobic or anhydrous solvent selected from one or a mixture of two or more selected from fatty acid esters, higher alcohols and propylene carbonate in which the present compounds represented by the above formulae (1) to (5) are dissolved, and a lipophilic base selected from one or a mixture of two or more selected from white petrolatum, yellow petrolatum, liquid paraffin and liquid paraffin.

In addition, as a cream preparation, a cream preparation comprising the compound, an oil phase component, a water phase component, and 2 or more surfactants in an amount of 2.5 to 7.5 parts by weight can be formed, wherein the oil phase component comprises a solid oil component comprising 5 to 20 parts by weight of white vaseline and 5 to 15 parts by weight of higher alcohols, and a liquid oil component comprising 3 to 10 parts by weight of squalane. The oil phase component of the cream can be added with other solid oil component and liquid oil component besides the white vaseline, higher alcohols and squalane.

The main drug may be added with pH regulator, buffer, stabilizer, cosolvent, etc. as required, and made into semi-solid preparation such as ointment or cream, liquid preparation such as lotion, etc., or external preparation such as patch by conventional method.

The antioxidant may be administered systemically or locally.

In the case of systemic administration, the composition can be administered into blood vessels, tissues, gastrointestinal tracts, mucous membranes, etc. in the form of aqueous injection, oily injection, troche, granule, liquid, capsule, soft capsule, nasal liquid, nasal powder, etc. The amount to be administered varies depending on the degree of symptoms, age, type of disease, etc., but generally 50 to 500mg per day is administered to an adult 1 day in 1 to several times.

In the case of topical application, the topical agent may be applied directly to the affected part of the skin in the form of a semisolid preparation such as ointment or cream, a liquid preparation such as lotion, or a patch. The amount to be applied varies depending on the degree of symptoms, age, type of disease, etc., but may be applied according to the method of application of the general anti-inflammatory skin external preparation. For example, an appropriate amount of the external preparation may be applied 1 time to several times a day in accordance with the symptoms, or may be applied several times in accordance with the symptoms.

[ 5. Process for producing the Compound ]

A method for producing the present compound according to an embodiment of the present invention (hereinafter, referred to as "present production method") includes: (A) a step of culturing a microorganism having the ability to produce the present compound represented by the above formulae (1) to (5) in a medium; and (B) a step of collecting the present compound represented by the above formulae (1) to (5) from the above culture.

The compound can be produced by culturing a microorganism having the ability to produce the compound in a medium, accumulating the compound in the culture, and collecting the compound from the culture (separation, extraction, purification).

In the present production method, the "microorganism having the ability to produce the present compound" is not particularly limited as long as it is a microorganism having the ability to produce the present compound. In addition, all strains having the ability to produce the present compound, such as gene recombinant strains and unidentified wild strains, are included in strains that can be used in the present production method, including mutant strains thereof.

The determination of whether or not the microorganism is a "microorganism having the ability to produce the present compound" can be carried out by culturing the subject microorganism under conditions (culture temperature, pH, medium components, etc.) in which the microorganism can appropriately proliferate, and examining the presence or absence of the present compound in the culture. If the present compound is present in the culture, the microorganism is identified as a microorganism having the ability to produce the present compound. The conditions under which the microorganism of the subject can be appropriately proliferated may be appropriately set according to the microorganism to be cultured.

In the present specification, the term "mutant strain" refers to a strain which has a different bacteriological property or gene from a microorganism having the ability to produce the present compound by inducing stimulation by mutation artificially or in nature, and such a mutant strain includes not only a strain derived from a microorganism having the ability to produce the present compound but also a strain from which a microorganism having the ability to produce the present compound is derived. In the present specification, regardless of whether the mutant strain has an actual derivation trace, for example, a strain having a gene highly similar (for example, 80% or more, 85% or more, 90% or more, 95% or more, etc.) to a gene of a microorganism having the ability to produce the present compound (for example, 16S rRNA gene) is also included in the mutant strain. Such mutant strains can be produced artificially or collected naturally as long as the ability to produce the compound is maintained.

In the present specification, the "recombinant strain" means a strain in which a gene is artificially introduced from the outside into a wild strain of a microorganism. The recombinant gene is not particularly limited as long as it has the ability to produce the compound. The gene recombinant strain is also referred to as a gene recombinant.

The host of the recombinant gene is not particularly limited, and for example, a microorganism having no synthetic gene of the compound on its genome can be used. In this case, the compound can be produced by introducing a synthetic gene of the compound into the microorganism.

In another embodiment, a microorganism having a synthetic gene of the present compound in its genome can be used as a host. Such a microorganism has the ability to produce the present compound without introducing a gene, but when a synthetic gene of the present compound is introduced into such a microorganism, there is an advantage that the amount of the present compound produced is increased. Examples of such microorganisms include Rhodosporidium sp (Polymorphospora rubera) K07-0510 strain (Japanese accession No. NITE BP-01411). Further, Rhodosporidium sp (Polymorphospora rubra) K07-0510 strain is included in the "microorganism having the ability to produce the present compound" even when no gene is introduced, since the present compound can be produced from the monomer as described above.

Examples of the host of the recombinant strain include those belonging to the genus Escherichia (Escherichia), Corynebacterium (Corynebacterium), Brevibacterium (Brevibacterium), Bacillus (Bacillus), Microbacterium (Microbacterium), Serratia (Serratia), Pseudomonas (PSEudomonas), Agrobacterium (Agrobacterium), Alicyclobacillus (Alicyclobacillus), Anabaena (Anabaena), Synechocystis (Analysis), Arthrobacter (Arthrobacter), Azotobacter (Azobactrobacter), Chromobacterium (Chromobacterium), Erwinia (Erwinia), Methylobacterium (Methylobacterium), Schimidium (Phormidium), Rhodobacterium (Rhodobacter), Rhodopseudomonas (Rhodomonas), Rhodospirillum (Rhodococcus), Rhodospirillum (Streptomyces), Streptomyces (Streptomyces), and Streptomyces (Streptomyces). Preferably, the microorganism is a microorganism belonging to the genus Escherichia (Escherichia), Corynebacterium (Corynebacterium), Brevibacterium (Brevibacterium), Bacillus (Bacillus), Pseudomonas (PSEudomonas), Agrobacterium (Agrobacterium), Alicyclobacillus (Alicyclobacillus), Anabaena (Anabaena), Arthrobacter (Anabaena), Azotobacter (Azobacter), Chromobacterium (Anabaena), Methylobacterium (Methylobacterium), Phormidium (Phormidium), Rhodobacterium (Rhodobacterium), Rhodopseudomonas (Rhodopseudomonas), Rhodospirillum (Rhodospirillum), Scenedesmus (Scenedesmus), Streptomyces (Synechococcus), Microbacterium (Zymomonas, etc. Examples of the actinomycetes include Streptomyces albus (Streptomyces albus), Streptomyces lividans (Streptomyces lividans), Streptomyces chromofuscus (Streptomyces chromofuscus), Streptomyces exfoliatus (Streptomyces exfoliatus), and Streptomyces argentatus (Streptomyces argenteus).

The host of the recombinant gene strain is not particularly limited as long as it has the ability to produce the compound when a desired gene is artificially introduced from outside, but preferably an actinomycete, more preferably an actinomycete belonging to the genus Streptomyces, and particularly preferably Streptomyces lividans.

The recombinant strain can be prepared, for example, by the method described in Japanese patent laid-open publication No. 2017-158546. Briefly, genes encoding enzymes related to Trehangelin synthesis (enoyl-CoA hydratase, 3-ketoacyl-CoA synthase, acyltransferase, and 3-ketoacyl-CoA reductase) are obtained based on the disclosure of japanese patent laid-open No. 2017-158546, and a recombinant strain (recombinant gene) contained in the "microorganism having the ability to produce the present compound" is obtained by introducing the obtained genes into a vector and introducing the vector into a host based on the disclosure of the document.

That is, in another embodiment of the present invention, a method for producing the present compound comprises: (C) a step of culturing a genetic recombinant (for example, actinomycetes) into which a gene encoding an enzyme involved in the synthesis of Trehangelin has been introduced in a medium; and (D) a step of collecting the present compound represented by the above formulae (1) to (5) from the above culture.

In this embodiment, the compound can be produced by culturing a recombinant gene (e.g., actinomycetes) into which a group of enzymes involved in trehangeolin synthesis has been incorporated in a medium, accumulating the compound in the culture, and collecting (isolating, extracting, purifying) the compound from the culture.

The medium used for culturing the microorganism having the ability to produce the present compound may be any medium that can be used as a nutrient source for the microorganism. For example, commercially available nitrogen sources such as peptone, meat extract, corn steep liquor, cottonseed meal, peanut meal, soybean meal, yeast extract, NZ amine (NZ-amine), casein hydrate, sodium nitrate, ammonium sulfate and the like; carbohydrates such as glycerol, starch, glucose, galactose, and mannose; or a carbon source such as fat; and inorganic salts such as sodium chloride, phosphate, calcium carbonate, magnesium sulfate, and the like. In addition, a trace amount of metal salt, animal oil, vegetable oil, mineral oil, or the like as an antifoaming agent may be added as necessary. Any culture medium of known microorganisms can be used as long as it is useful for producing the compound using a production bacterium.

The culture of the microorganism having the ability to produce the present compound can be carried out by shaking culture for several days to 2 weeks at a temperature within a range (for example, 10 to 40 ℃ C., preferably 25 to 30 ℃ C.) in which the producing microorganism develops and produces the present compound. The culture conditions can be appropriately selected according to the properties of the producing strain of the present compound to be used, with reference to the description of the present specification.

The compound can be collected by extraction from the culture medium using a water-immiscible organic solvent such as ethyl acetate. In addition to the present extraction method, it is also possible to purify to purity by appropriately combining or repeating known methods for collecting fat-soluble substances such as absorption chromatography, partition chromatography, gel filtration chromatography, extraction by thin layer chromatography, centrifugal countercurrent partition chromatography, high-speed liquid chromatography and the like.

The present compound can be collected, for example, by the methods described in examples.

One embodiment of the present invention includes the following configurations.

<1> a compound represented by the following formula (1).

[ solution 9]

<2>A kind of<1>The compound of (1), wherein R is²Is hydrogen, acetyl, 2-butenoyl or 2-methyl-2-pentenoyl, R⁵Is 2-methyl-2-butenoyl, R¹、R³、R⁴And R⁶Is hydrogen.

<3> a composition comprising the compound of <1> or <2 >.

<4> the composition of <3>, which further comprises at least one compound represented by the following formulae (6) to (8).

[ solution 10]

[ solution 11]

[ solution 12]

<5> the composition according to <3> or <4>, wherein the composition is a cosmetic composition or an antioxidant.

<6> A method for producing the compound according to <1> or <2>, the method comprising:

(A) culturing a microorganism having the ability to produce the compound of <1> or <2> in a medium; and

(B) collecting the compound of <1> or <2> from the culture.

The present invention is not limited to the above embodiments, and various modifications can be made within the scope shown in the claims, and embodiments obtained by appropriately combining technical means described in different embodiments are also included in the technical scope of the present invention.

[ examples ] A method for producing a compound

The present invention will be described in further detail with reference to examples, but the present invention is not limited to these examples.

[ 1. preparation of production Strain ]

Polymorphospora rubra K07-0510 strain of actinomycetes was cultured at an appropriate temperature (for example, 27 ℃) for several days in YD medium containing 1% yeast extract and 1% glucose. After the culture, the cells were collected from the obtained culture solution by centrifugation. Then, chromosomal DNA was isolated and purified from the obtained bacterial cells according to a conventional method (Molecular Cloning second edition).

For convenience, the sequence numbers: 1 (orf), wherein the nucleotide sequences are designated as orf A, orf B, orf C, orf D, and orf A to D in the order named from the beginning with the smaller nucleotide sequence number: the positions and functions in 1 are as follows.

orfA (SEQ ID NO: 2, base numbers 1 to 828 in SEQ ID NO: 1, encoding enoyl-CoA hydratase);

orfB (SEQ ID NO: 3, base No. 875-1900 at SEQ ID NO: 1, encoding 3-ketoacyl-coenzyme A synthetase);

orfC (SEQ ID NO: 4, nucleotide numbers 1905-2684 of SEQ ID NO: 1, encoding acyltransferase);

orfD (SEQ ID NO: 5, base No. 2681-3475 in SEQ ID NO: 1, encoding 3-ketoacyl-coenzyme A reductase).

A recombinant plasmid capable of sufficiently expressing the above 4 genes was constructed by the following method using the PCR method [ Science, 230, 1350(1985) ].

Chromosomal DNA of Polymorphospora rubra K07-0510 strain of actinomycetes was used as a template by using the sequence No: a sense strand primer having a PstI restriction enzyme site and a ribosome binding sequence at the 5' end represented by 6, SEQ ID NO: an antisense strand primer having a StuI restriction site at the 5' -end, denoted by 7, and Taq DNA polymerase (manufactured by Roche biosciences) were used to amplify DNA expressing orfA, orfB, orfC, and orfD (hereinafter referred to as "orfABCD") by PCR using a DNA thermal cycler (manufactured by applied biosystems). The PCR was carried out in 1 cycle of reaction steps of 95 ℃ for 30 seconds, 68 ℃ for 30 seconds and 72 ℃ for 4 minutes, 30 cycles of the reaction were carried out, and then the reaction was carried out at 72 ℃ for 10 minutes. The amplified DNA fragment was purified by agarose gel electrophoresis, and cleaved with restriction enzymes PstI and StuI, thereby obtaining a DNA fragment containing the DNA expressing orfABCD treated with PstI and StuI (hereinafter, referred to as "DNA fragment containing orfABCD").

pOSV556t (nat. chem., 3, 338, 2011) was cleaved with restriction enzymes PstI and StuI to obtain PstI and StuI-treated pOSV556t fragment. The thus obtained PstI and StuI-treated orfbcd-containing DNA fragment and PstI and StuI-treated plosv 556t fragment were mixed and subjected to ligation reaction, thereby obtaining a recombinant DNA.

Using the recombinant DNA, E.coli Top10 strain was transformed according to a conventional method, and the genetic recombinant was plated on LB agar medium containing 100. mu.g/ml ampicillin and cultured overnight at 37 ℃. Plasmids containing the recombinant DNA were isolated from the genetic recombinants according to the prescribed method. The recombinant DNA was determined to be orfABCD by sequencing the DNA sequence, and this plasmid was named pOSV 556-orfABCD.

pOSV556-orfABCD was introduced into E.coli ET12567/pUZ8002 strain (Practical Streptomyces Genetics, 2000) by a conventional method, and E.coli ET12567/pUZ8002/pOSV556-orfABCD strain exhibiting resistance to 50. mu.g/ml of kanomycin, 25. mu.g/ml of chloramphenicol and 100. mu.g/ml of ampicillin was obtained. Further, pOSV556-orfABCD was ligated from E.coli ET12567/pUZ8002 strain to Streptomyces lividans 1326 strain (department of Biogenetic resources (NBRC) of the national department of Biotechnology, independently administrative department of human products evaluation technology, NBRC No. 15675) which was transferred to actinomycetes, by a conventional method, to thereby obtain Streptomyces lividans/pOSV 556-orfABCD which exhibited resistance to hygromycin of 50. mu.g/ml.

[ 2. isolation and characterization of novel Compounds ]

Streptomyces lividans/pOSV 556-orfABCD obtained as described above was cultured in 500ml of a liquid medium containing 1% of yeast extract and 1% of glucose at 27 ℃ for 1 day with shaking. Subsequently, 50ml of a 20% trehalose aqueous solution was added, followed by further shake culture at 27 ℃ for 4 days. To the resulting culture solution was added 500ml of ethanol and stirred for 1 hour. Then, ethanol in the extract was distilled off under reduced pressure, and 250ml of ethyl acetate was added to the obtained aqueous solution and sufficiently stirred, followed by recovery of an ethyl acetate layer. Thereafter, the mixture was concentrated and dried to dissolve in 100ml of 0.1% formic acid.

Purification of the novel compound was performed using a concentrated dried sample derived from 500mL of the culture broth obtained above. For purification, the following column and elution solvent were used.

< purification of the 1 st time >

Pipe column: ULTRA PACK ODS-SM-50B(manufactured by Shanshan Co., Ltd.)

Dissolution solvent: water/acetonitrile/formic acid (850/150/1)

< purification of 2 nd time >

Pipe column: 10g of Silica PSQ-100B was used in an open column (open column)

Dissolution solvent: ethyl acetate

The purity of the separated liquid during purification was confirmed by HPLC. Recovering LC purity above 95%, freeze drying, and purifying. HPLC and LC/MS were performed under the following analytical conditions.

< conditions for HPLC analysis >

The device comprises the following steps: promience UFLC system (Shimadzu corporation)

Pipe column: YMC-Pack ODS-AQ (manufactured by YMC corporation)

Mobile phase: water/acetonitrile/formic acid (850/150/1)

Flow rate: 1 ml/min

Detection: 220nm

< LC/MS analysis conditions >

The device comprises the following steps: agilent Technologies 6224TOF LC/MS (manufactured by Agilent Technologies Co., Ltd.)

Pipe column: YMC-Pack ODS-AQ (manufactured by YMC corporation)

Mobile phase: water/acetonitrile/formic acid (850/150/1)

Flow rate: 0.5 ml/min

Thus, novel compound A (25mg), novel compound B (12mg), novel compound C (15mg) and novel compound D (20mg) were obtained. These compounds were analyzed by the following melting point and NMR.

(melting Point and NMR analysis result)

The melting point and NMR were measured for the above novel compounds A, B, C and D under the following analytical conditions.

< conditions for measuring melting Point >

The device comprises the following steps: BUCHI Melting Point B-545 (manufactured by BUCHI Co., Ltd.)

< NMR analysis conditions >

The device comprises the following steps: BRUKER AMX 400 (manufactured by Bruker Co., Ltd.)

Solvent: DMSO-d6

Internal standard: TMS

As a result, the new compounds A, B, C and D were identified as 3-O-angeloyltrehalose, 3-O-acetyl-3 ' -O-angeloyltrehalose, 3-O-angeloyl-3 ' -O-isocrotonyl trehalose, and 3-O- (2-methyl-2-butenoyl) -3 ' -O- (2-methyl-2-pentenoyl) trehalose, respectively.

[ 3. evaluation of physiological Functions ]

The physiological function of the compound obtained above was evaluated. Evaluation of physiological function was carried out using the tools described in Carbonic acid, a hydrocarbon-type electrochemical compound, protection nerves in vitro and in vivo tissue activation of the Keap1/Nrf2 pathway via S-activation of targeted tissues on Keap1.Takumi Satoh et al, Journal of neurochemistry. Vol.104, pp.1116-1131, (2008).

Briefly, ARPE-19 cells were seeded on a 24-well plate using 10% FBS-DMEM/F12 medium (manufactured by GIBCO Co., Ltd.) to achieve 50% concentration. After 24 hours, a mixed solution (carrier solution) of 0.5. mu.g of pGL3-GSTYAARE-Luciferase vector, 0.5. mu.g of pSV-betaGAL vector and 50. mu.L of Opti-MEM I (manufactured by GIBCO Co., Ltd.) was prepared for each well; and (2) a mixed solution of 2. mu.L of lipofectamine 2000 (manufactured by Saimer Feishell science) and 50. mu.L of Opti-MEM I (manufactured by GIBCO Co., Ltd.) (lipofectamine 2000 solution), and allowed to stand at room temperature for 5 minutes. The solution of the carrier was mixed with the solution of lipofectamine 2000 and allowed to stand at room temperature for 20 minutes. The mixture (100. mu.L per 1 well) was added to cultured cells (ARPE-19 cells) in CO₂The culture was carried out in an incubator for 6 hours. Next, the medium was exchanged with new 10% FBS-DMEM/F12 medium (0.5 ml/well) and cultured for 18 hours. Thereafter, the medium was exchanged for new 10% FBS-DMEM/F12 medium (0.3[ mL/well) or 10% FBS-DMEM/F12 medium (0.3 mL/well) containing 20mg/mL of various Trehangeolin derivatives and cultured for 24 hours. After removing the medium, 150. mu.L of Passive Lysis Buffer (Passive Lysis Buffer, manufactured by Promega corporation) was added to prepare a cell lysate.

To a 96-well plate, 100. mu.L of a cell lysate and 100. mu.L of a β GAL assay kit solution (manufactured by Promega corporation) were added, and the cells were cultured at 37 ℃ for 3 hours. Thereafter, the β GAL activity was confirmed by measuring the absorbance at 405 nm.

To a 96-well plate different from the above, 20. mu.L of a cell lysis solution was added one well, and 100. mu.L of a luciferase assay kit solution (manufactured by Promega corporation) was added one well. Thereafter, luciferase activity was measured by measuring fluorescence. The obtained luciferase activity was corrected by dividing the result by β GAL activity, and the antioxidant responsive element (antioxidant responsive element) transcriptional activity was calculated. The results are shown in FIG. 1.

(results)

According to FIG. 1, 3-O-angeloyltrehalose, 3-O-acetyl-3 ' -O-angeloyltrehalose, 3-O-angeloyl-3 ' -O-isocrotonoyl trehalose and 3-O- (2-methyl-2-butenoyl) -3 ' -O- (2-methyl-2-pentenoyl) trehalose all showed 1.24 to 2.09 times of luciferase activity as compared to the control. Accordingly, it is known that 3-O-angeloyltrehalose, 3-O-acetyl-3 ' -O-angeloyltrehalose, 3-O-angeloyl-3 ' -O-isocrotonoyl trehalose and 3-O- (2-methyl-2-butenoyl) -3 ' -O- (2-methyl-2-pentenoyl) trehalose have an antioxidant effect.

In addition, it is found that 3-O-angeloyl trehalose has the same degree of antioxidant effect as Trehangelin a, and 3-O-angeloyl-3 '-O-isocrotonyl trehalose and 3-O- (2-methyl-2-butenoyl) -3' -O- (2-methyl-2-pentenoyl) trehalose have higher antioxidant effect than Trehangelin a, as compared with Trehangelin a, which is a known substance.

[ 4. prescription example ]

(toning lotion)

As a cosmetic containing the compound of one embodiment of the present invention, a cosmetic lotion was produced from the following composition. The following components (1) to (10) were added to the following component (11) at room temperature and stirred. Thereafter, component (12) was added and uniformly dissolved to obtain a lotion (unit is wt%).

(1) Glycerol 9.5

(2)1, 3-butanediol 4.5

(3) Glucose 1.5

(4) Ethanol 5.0

(5) Carboxyvinyl Polymer 0.02

(6) Dipotassium glycyrrhizinate 0.1

(7) Sodium hyaluronate 0.1

(8) Compound 0.1 of one embodiment of the present invention

(9) Citric acid 0.05

(10) Sodium citrate 0.1

(11) Ion exchange water balance

(12) 0.01 part of potassium hydroxide

[ industrial applicability ]

The invention relates to a novel compound with antioxidation, which can be applied to the fields of cosmetics, medicines and the like.

Sequence listing

<110> changlie trades; school courthouse north research institute

<120> novel compound and use thereof

<130> CPWNHK0004

<150> JP 2019-068164

<151> 2019-03-29

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 3475

<212> DNA

<213> Rhodosporidium rubrum (Polymorphospora rubra)

<400> 1

gtgacccgac cggacgcccc gggcccaccg gtcgccgccc cggacccggc cgacgccgtc 60

gtgacggccg tcgagccgta cgtggtccgg gcgacgatca accgaccggc ccgccgcaac 120

gccatcgacc tcgccgtgat cgagggcctc gaacgcgcca tcgacctcgc cgaggcgacc 180

ggcgcccggg tcctggtgct gcgcggcgcc ggcggcacct tctgctccgg cgccgacctg 240

cgcgtcctgg aggagatgag cgtcgacccg caccgggtgg agacgttcat ggtgcggctc 300

gccctcgtgc tgcgccggct ggagaccgcc cggttcgtct cggtcgcggt cgtcgagggg 360

cacgccgtcg cgggcggctg cgagatcctg ctcgcctgcg acgtctcggt cgccgccacc 420

gacgcccgca tcggcgaccg ccatctcgag tacgggctgg tccccgccgc cggcggctcg 480

gtacggctgg cccgcaccct gcccaaggcc cggggcaact acctgctgct cgccgccgac 540

ctgctcaccg gggagcaggc ggcgcagtgg ggactggtca gcgtcgcggt gccgccaacc 600

gacctggaac cccgggtgga cgccctggtc gggcggctcg tcggccacag cgccgacgcg 660

ctcgccgtgg tcaagaagat ggtctggacc gccgaccacg aaccccggcc ggacgccatg 720

tcctgggaac gccggctctt cctgcgccac ctcggctccg aagacgtgtc cgaggggctg 780

cgcgccttcc gcgagcggcg ccggccggcg ttccgcgccg atgactgact ccgtacggcc 840

gcccgaccgg acggcccgac cgcaaggaga ccacatgtcg acctcgacgg tgatcggaac 900

aggttcgtac ctaccccgcc gcgtcctcag cagcggcgaa ctggcccgcc gggtcggtgt 960

ggcggagaac tggatcgtgg agaagaccgg gatccgggaa cgccgggtgg ccgccgacga 1020

ggaggccacc tccgacctgg ccacccgggc cgcgcgccgg gcgctgcgga ccgcccggct 1080

ggacccggcc gacgtcgacc tgatcgtgct ggcgacctcc acaccggacc ggccgatgcc 1140

ggccaccgcc agcaccgtgc aggccaacct cggcgcccgc caggcggtcg cgttcgacgt 1200

cgacgcggtg tgcagcggct tcgtgtacgc cctcgtcgtc gcccactcga tgctgaacag 1260

cgagggctgg gcccgtaccg cgctggtgat cggcgccgac acgtactcgc gggtcctgga 1320

ctacaccgac cggcgtaccg cggtgctctt cggcgacggt gccggcgcgg tcgtcctcgg 1380

ccgggagacc ggcggcggga gcggaatccg ggccaccacc ctgggcaccg acggcaccct 1440

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cgaggccggc gaccactact tcgcgatgcg cggcggcgac gtccgcaggc tggccaacca 1560

ggtcttcccg gcactggtcg ggcaactgct caaggcggcc tcgctcgacc tggaccaggt 1620

cgacctgatc gccgcccacc aggccaacgg cacgatgctc accgactggt cgcgggacct 1680

cggcctgcgc ccgggggtgc tgcaccgcac cgtcgagcgg tacggcaaca ccggtgccgc 1740

ctcggtcccg gtcaccctcg acgacgccgt acgcaccggg cggctcggtg ccgccgccac 1800

cctgctgatg gtcgccttcg gcggcgggat gacctggggc ggcgtcgccc tggactggtc 1860

cgccgacccg tcggttcccc gctccaactg cgtgaggtga agtgatgacc acgtccgcac 1920

tcggcgccga accgggcgcc accgatccgc cggccgcccc gccggtctcc tacgccccgt 1980

ccggccggat gccccggacg atcaaccgct ggccgcgccc gttcgcgccg cgcaacatcc 2040

tcggcgtgct gcgggcccgg ctgcgcggcg ccacgatcag cctcaaccag gtcaagttcg 2100

acgagtccac cgcggagcgc aacgcgctgg tgatggaaca cgcgctgctg cggcacagcc 2160

acgtcgcccg gtacgcgatc gtcggcccgt tcacctcgct gttcaaggtc cgggtcggcc 2220

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cgaccagtca cgtcttcccg gtcaacgccg agttcggctt ctgcgcgggg gagtggccgg 2340

aggtgcccgg caccgaggtc ggcgccgacg cgtggatcgg cgccggcgcg gtggtccggg 2400

ccggcgtccg gatcgggcac ggcgcgatcg tcgcggccgg cgcggtcgtc acccgcgacg 2460

tcgccgacta cgagatcgtg gccggggtgc cggcccgccg gctgcggtcc cggttccccg 2520

acgacctggc cgaacggctg gtcgcgctgg cctggtggga ctggccgccg gggttcatca 2580

aggccaacat cgacctgttc cagcggccgc tgaccgccga caccctggcc gcgctggagg 2640

agcgggcccg cgccctgccc acacccccgg gtggtgccgc gtgaccgccg gtacgggcca 2700

gccggtcgtc gccgcgcccg gcagggcgct cgccgaccgg gtggtggtgg tgaccggcgc 2760

cagccgcggt gtcggccgcg acctcgcccg ggtgttcgcc gaccacggcg cccgcctcgg 2820

cctgctggcc cgcagccggg acgcgctcga cgacctcggc ggcacgctca ccgccgccgg 2880

tgccgacgtc ctcgccgtgc cgtgcgacgt cggcgaaccg gactcgctgg ccggcgcggt 2940

cgacgccgtc gccgggcact tcggagggat cgactcggtc gtcgtcaacg ccggcatctc 3000

gcccgtcgcc cgtcgggcgc accacctgcc gatcgacgcc tggcacgacg tgctggcgac 3060

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gcgtggccgg ctggtgttca ccacctcggt catggccgcc accccgcggc gcgggctgag 3180

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ccggttcggc cgcgccgacg agttggccgc cgcgttcgtc ttcctggccg gtgacgcctg 3420

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<210> 2

<211> 828

<212> DNA

<213> Rhodosporidium rubrum (Polymorphospora rubra)

<400> 2