WO2009151057A1 - Process for production of antioxidant active substance - Google Patents

Process for production of antioxidant active substance Download PDF

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Publication number
WO2009151057A1
WO2009151057A1 PCT/JP2009/060547 JP2009060547W WO2009151057A1 WO 2009151057 A1 WO2009151057 A1 WO 2009151057A1 JP 2009060547 W JP2009060547 W JP 2009060547W WO 2009151057 A1 WO2009151057 A1 WO 2009151057A1
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general formula
active substance
acid
group
antioxidant active
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PCT/JP2009/060547
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French (fr)
Japanese (ja)
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全宏 多田
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国立大学法人東京農工大学
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Priority to JP2010516861A priority Critical patent/JP5464444B2/en
Publication of WO2009151057A1 publication Critical patent/WO2009151057A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/347—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups
    • C07C51/367—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups by introduction of functional groups containing oxygen only in singly bound form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/22—Ortho- or ortho- and peri-condensed systems containing three rings containing only six-membered rings
    • C07C2603/26—Phenanthrenes; Hydrogenated phenanthrenes

Definitions

  • the present invention relates to a method for producing an antioxidant active substance. More specifically, the present invention relates to a method for producing carnosic acid and its derivatives from trees such as Sawara.
  • Lamiaceae plants such as rosemary, sage, perilla, oregano, basil, thyme, marjoram, peppermint are widely distributed throughout the world, and about 200 genera and 3500 species are known.
  • the antioxidant active substances contained in the Lamiaceae plants are roughly classified into four types: phenolic diterpenes, caffeic acid derivatives, flavonoids, and biphenyl derivatives.
  • rosemary has long been known as a spice for meat dishes and a folk medicine, and it is widely known that it contains a strong antioxidant active component typified by carnosic acid, carnosol, and rosmanol ( Non-patent document 1).
  • the above-mentioned antioxidant active substances contained in such rosemary have antibacterial activity, cranial nerve cell death prevention effect, cerebral blood treatment / prevention effect, Alzheimer's disease prevention effect, fat absorption prevention effect, anti-inflammatory effect, diabetes
  • Various activities such as blood glucose lowering effect and whitening effect of patients have been reported, and their use as food additives, supplements, medicines, etc. are being studied (Patent Document 1, Non-Patent Document 2 to Non-Patent Document 4). .
  • a neurite elongation agent containing at least one plant-derived extract selected from the group consisting of rosemary and sage as an active ingredient is disclosed (Patent Document 2). It is disclosed that the carnosic acid which is an example of the said extract is obtained by extracting predetermined parts, such as rosemary, with alcohol. However, carnosic acid separated from an extract of rosemary obtained by alcohol treatment or the like with respect to 5,000 grams of rosemary whole plant is only 1.5 grams, and there is a problem that the production efficiency is not sufficient.
  • rosemary leaves are treated with a lower alkyl alcohol aqueous solution in the presence of a water-soluble acid, and carnosic acid is optionally extracted.
  • a method including purification is disclosed (Patent Document 3 and Patent Document 4).
  • rosemary leaves are used as a starting material for carnosic acid.
  • the supply of rosemary leaves varies greatly depending on the production volume and price, and is not sufficient from the viewpoint of a production method that can supply carnosic acid in a large amount at a low cost.
  • Sawara is a special conifer of Japan, and its natural life is distributed a lot in Tochigi, Gunma, and Kamikochi, Nagano Prefecture.
  • the leaves and bark contain a large amount of related compounds such as piciferin acid, which is a raw material for carnosic acid (Non-patent Document 5).
  • Sawara contains a specific substance having activity similar to that of dibutylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) currently used as synthetic food additives.
  • BHT dibutylhydroxytoluene
  • BHA butylhydroxyanisole
  • the object of the present invention is to extract carnosic acid and derivatives thereof from evergreen broad-leaved trees such as Sawara, which are widely present in Japanese mountains, and extract an antioxidant active substance. May provide a method of manufacturing.
  • the inventors of the present invention obtained strong power by oxidizing the ortho position of phenol of a compound such as piciferin acid, which is an extract thereof, with a specific oxidizing agent. It has been found that carnosic acid and its derivatives, which are antioxidant active ingredients, can be produced, and the present invention has been completed.
  • R 1 is any one of COOR 3 , hydroxymethyl group and aldehyde group, and R 2 and R 3 each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.
  • R 2 and R 3 each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.
  • R 4 , R 5 and R 6 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, an allyl group, a phenyl group, a halogenated phenyl group, a halogen atom.
  • X represents a halogen atom, and n represents 1 or 2).
  • R 1 is any one of COOR 4 , hydroxymethyl group, and aldehyde group
  • R 2 , R 7, and R 4 are each independently a hydrogen atom or a C 1-6 carbon atom.
  • an antioxidant active substance typified by carnosic acid or the like can be produced easily and in large quantities from Sawara with an extremely high yield.
  • an antioxidant active substance such as carnosic acid can be produced by a simple process by using an inexpensive specific oxidation initiator.
  • the term “antioxidant active substance” refers to a compound having a strong antioxidant activity, such as carnosic acid, carnosol, rosmanol and the like.
  • the term “antioxidant active substance” is not limited to those extracted only from rosemary, but also uses Lamiaceae plants other than rosemary such as sage, perilla, oregano and basil as starting materials, or these starting materials. A composition produced using a combination of these as starting materials.
  • the raw material used in the method for producing an antioxidant active substance of the present invention only needs to contain, in its components, piciferic acid and its derivative, which are raw materials such as carnosic acid, which is the target substance of the production method of the present invention, although it does not restrict
  • the tree of the genus Sawara of the cypress family can be illustrated. Examples of the tree include Sawara, Shinobu hiba, Ogon Shinobu hiba, Himuro and Hiyoku hiba.
  • Sawara is preferable from the viewpoint of production and handling, and these raw materials can be mixed and used.
  • the general formulas for piciferic acid and its derivatives, which are starting materials for the antioxidant substance that is the target substance of the production method of the present invention, are shown below.
  • R 1 is any one of COOR 3 , a hydroxymethyl group, and an aldehyde group. That is, R 1 is a carboxyl group, a carboxylic ester group, a hydroxymethyl group, or an aldehyde group.
  • R 2 and R 3 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.
  • the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, an n-propyl group, and an i-propyl group. Examples thereof include a group, n-butyl group, sec-butyl group, tert-butyl group, n-pentyl group, n-hexyl group and the like.
  • piciferic acid is a diterpene most contained in Sawara, and is a chemically stable, colorless, and odorless crystal.
  • Sawara is an evergreen tree that can be obtained everywhere in Japan and can be obtained very easily. Furthermore, Sawara is a biomass that is readily available and present in large quantities. In the present invention, it is preferable to use the leaves and bark even though a lot of piciferin acid and its derivative compounds are included in the part of Sawara.
  • the method for extracting piciferin acid and the like from raw materials such as Sawara is not particularly limited, and for example, extraction can be performed by the following method. That is, it can be easily extracted by immersing a raw material such as sawara in a solvent or refluxing with a solvent.
  • the solvent that can be used include water, alcohol, alkane, carboxylic acid, ester, and ketone.
  • these solvents can be used alone, or two or more of these solvents can be appropriately mixed and used.
  • Examples of the alcohol include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl 1-propanol, t-butyl alcohol, 1-pentanol, 2-pentanol, and 3-pen.
  • Examples include butanol, 2-methyl 1-butanol, 3-methyl 1-butanol, 2 and 2 dimethyl 1-propanol.
  • Examples of the alkane include pentane, hexane, heptane, octane, nonane and decane.
  • ketones include acetone, methyl ethyl ketone (MEK), and diethyl ketone.
  • MEK methyl ethyl ketone
  • diethyl ketone two or more kinds of the above organic solvents can be adopted and used as a mixed solution.
  • the above solvents from the viewpoint of handling such as safety, it is preferable to use water, alcohol alone or a mixed solvent of water and alcohol. It is particularly preferable to use a mixed solvent of water and ethanol.
  • the extraction time and extraction temperature when performing the above solvent extraction can be appropriately set depending on the type of raw material.
  • the extraction temperature is preferably set to the reflux temperature, and the extraction reaction time can be 10 to 24 hours.
  • the method for producing an antioxidant active substance of the present invention employs a specific peroxide represented by the following general formula in the ortho-position oxidation reaction of phenol in the structural formulas of piciferic acid and derivatives thereof represented by the above chemical formula It has the characteristics. That is, in the present invention, an oxidant represented by the following general formula is used to oxidize the ortho position of phenol in piciferic acid and its derivatives.
  • R 4 , R 5 and R 6 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, an allyl group, a phenyl group, a halogenated group) (Represents either a phenyl group or a halogenated alkyl group.
  • X represents a halogen atom
  • n represents 1 or 2.
  • the methods adopted in the ortho-oxidation reaction of phenols include direct oxidation with selenium oxide, oxidation with benzoyl peroxide, air oxidation, and 2-Iodoxybenzoic acid (hereinafter referred to as “IBX”).
  • IBX 2-Iodoxybenzoic acid
  • examples of the oxidation method may include but are not limited thereto.
  • the use of various oxidizing agents can oxidize piciferin acid derivatives extracted from, for example, Sawara species.
  • benzoyl peroxide (mCBPO) having a halogenated phenyl in which R 4 and R 5 are phenyl halides, and a chloro having a halogenated phenyl and a halogenated alkyl group.
  • Acetylmetachlorobenzoyl peroxide (CAMCBPO) can be used, but is not limited to these oxidizing agents.
  • IBX and diacyl peroxide are used as the oxidizing agent, but the present invention is not limited to this.
  • a benzoic acid derivative represented by the above general formula can also be used as an oxidizing agent.
  • oxidation with IBX often further oxidizes the oxide, producing a complex mixture.
  • the oxide is further oxidized to produce ortho-quinone.
  • ortho-quinone is easily decomposed at anxiety points, so it is converted to catechol by rapid reduction with sodium hydride.
  • mCBPO benzoyl peroxide
  • AMCBPO benzoyl peroxide
  • the above IBX which is an oxidizing agent used in the present invention, can be easily produced from 2-iodobenzoic acid under predetermined conditions.
  • the reaction formula is shown below.
  • the reducing agent used for the reduction reaction of the generated orthoquinone after oxidation with IBX is not particularly limited as long as it is a catalyst having a mild reducing ability that does not reduce the carboxyl group.
  • Examples include lithium aluminum hydride [LiAlH (OR) 3 ], lithium borohydride (LiBH 4 ) sodium borohydride, lithium trialkylborohydride (LiR 3 BH), and dialkylaluminum hydride. These reducing agents may be used alone or in combination.
  • ester group after ortho-position oxidation can also be removed by hydrolysis.
  • a combination of a base such as sodium hydroxide, lithium hydroxide, potassium hydroxide, sodium carbonate or potassium carbonate and a solvent such as water-methanol or water-ethanol can be considered.
  • the target antioxidant active substance in the production method of the present invention can be produced by these reduction reactions or hydrolysis. As described above, in the present invention, it is possible to efficiently oxidize the ortho position of phenol of a compound such as piciferic acid using an available and inexpensive peroxide. As a result, an antioxidant active substance is produced with high efficiency. be able to.
  • the manufactured antioxidant active substance can be converted into carnosol and rosmanol by a known method.
  • carnosol can be produced when carnosic acid is oxidized by DDQ, silver oxide or air, and the quinone-quinone metide tautomer formed is lactonized.
  • Rosmanol can be produced by oxidation of carnosol with methylene chloride with pyridinium chlorochromate (PCC) or with air in the presence of sodium bicarbonate.
  • IBX As the oxidizing agent, IBX can be used, and the produced orthoquinone can be reduced to an ester group using a reducing agent such as sodium borohydride.
  • a reducing agent such as sodium borohydride.
  • the reaction formula is shown below.
  • carnosic acid can be produced by the following method for synthesizing carnosic acid using IBX.
  • Example 1 ⁇ Separation of piciferic acid> Sawara leaves (collected from the forest of Tokyo University of Agriculture and Technology, Harumi-cho, Fuchu-shi, Tokyo) were collected and used as starting materials. 160 g of the above Sawara leaves were weighed and refluxed in about 800 ml of methanol at about 65 ° C. for 24 hours. Next, the methanol extract obtained by the above refluxing operation was concentrated under reduced pressure, and then a liquid / liquid extraction operation was performed with ethyl acetate and water.
  • the ethyl acetate layer was concentrated, and the concentrate was subjected to column chromatography using silica gel and hexane / ethyl acetate (volume ratio 3: 1). After concentrating the fraction containing piciferin acid, perform column chromatography again in the same manner. The resulting fraction containing piciferin acid was crystallized using hexane / ethyl acetate (volume ratio 3: 1), and A certain 248 mg of piciferic acid was obtained.
  • the melting point and microstructure of the generated piciferin acid were measured by a melting point analyzer, 1 H-NMR, and 13 C-NMR, respectively.
  • the melting point measuring device used MEL-TEMP by Laboratory Device, and measured it on the conditions without correction
  • 1 H-NMR and 13 C-NMR measurements were performed using JEOL manufactured by JEOL Ltd. alpha-600 ( 1 H: 600 MHz, 13 C: 150.8 MHz) Using a spectrometer. Tetramethylsilane in deuterated chloroform was used as a standard.
  • FIG. 1 shows the analysis result of piciferic acid.
  • Piciferic acid 200 mg: 0.63 mmol was dissolved in methylene chloride (10 ml), and m-chlorobenzoyl peroxide (590 mg: 1.9 mmol) was added and dissolved. The solution was allowed to stand at room temperature under argon for 16 hours and then concentrated. The concentrated residue was dissolved in ethyl acetate, and hexane was added to separate the precipitated crystals. The mother liquor was further concentrated to obtain a mixture containing ortho-oxidized carnosic acid monoester.
  • the ester group was hydrolyzed under reductive conditions without further purification of the fraction containing carnosic acid monoester, and induced to a hydroxyl group.
  • a fraction (70 mg) containing carnosic acid monoester was dissolved in methanol (9 ml), 1% -NaOH (1 ml) and sodium borohydride (13.2 mg) were added, and the mixture was heated to reflux for 2 hours under argon.
  • the product was acidified with 1M hydrochloric acid and extracted with brine-ethyl acetate. The extract was washed with brine, dried over anhydrous magnesium sulfate and concentrated.
  • Example 2 ⁇ Synthesis of carnosic acid using chloroacetylmetachlorobenzoyl peroxide (CAMCBPO)> Chloroacetic acid (59.7 mg, 0.63 mmol) was dissolved in methylene chloride (15 ml), dicyclohexylcarbodiimide (DCC, 143.5 mg, 0.70 mmol) was added, and the mixture was stirred at 0 ° C. for 15 minutes under a stream of argon. mCPBA, 152.7 mg, 0.88 mmol) was added, and the mixture was stirred at 0 ° C.
  • DCC dicyclohexylcarbodiimide
  • Pisiferic acid was isolated and purified from Sawara in high yield, and this was ortho-oxidized to produce carnosic acid (Carnosic acid), the main antioxidant active substance of rosemary. It is understood that acid) can be produced efficiently.
  • the main antioxidant active substance of rosemary which has been attracting attention in recent years due to its preventive effect against Alzheimer's disease and various lifestyle-related diseases, is supplied from the leaves of Sawara planted in large quantities as wood resources. The technical significance is extremely large.
  • Example 4 ⁇ Measurement of antibacterial activity>
  • Carnosic acid (Example 4) which is the main antioxidant active substance of rosemary synthesized according to Example 1, and carsol (Example 5) synthesized from this carnosic acid were used for anti-methicillin-resistant Staphylococcus aureus activity (anti-MRSA). Activity) and anti-acne activity.
  • Acne (acne) is a chronic inflammatory disease that occurs in the skin from around puberty. Proliferation (infection) is thought to play an important role.
  • there are many types of antibacterial drugs for external use that have been approved for use as treatments for this disease, many of them are old, and few drugs can be applied to clinical treatment.
  • the measurement of anti-acne bacteria activity was performed as follows. That is, using the antioxidant substance obtained in Example 1 as a sample, diluting 10 times with Pluronic L44 (polyoxyethylene / polyoxypropylene), and diluting it 10% by weight in the medium for acne bacteria A sample was prepared at a concentration of 1.0% by weight. As a control, 10% by weight Pluronic L-44 was used. After acne bacteria were cultured in a GAM liquid medium for 24 hours, they were precipitated, and the GAM liquid medium was washed twice with an acne medium, and the sample was inoculated as an inoculum. Thereafter, the number of remaining bacteria was counted after 24 and 48 hours.
  • Pluronic L44 polyoxyethylene / polyoxypropylene
  • Counting was performed by serial dilution from 1.0 to 100,000 times using a medium for acne bacteria, and after anaerobic culture using a plate smearing method.
  • MRSA methicylene resistant Staphylococcus aureus
  • the main antioxidant active substance obtained by the method for producing the antioxidant active substance of the present invention has a remarkable antibacterial activity, and these results indicate that the antioxidant active substance of the present invention May be useful as a treatment for acne.
  • the method for producing an antioxidant active substance according to the present invention is a method for producing an antioxidant active substance using Sawara planted in large quantities as a wood resource, and can therefore contribute to the development of forestry and environmental technology fields. . Furthermore, the method for producing carnosic acid of the present invention can greatly contribute to technological innovation in the fields of medicine and medical technology.

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Abstract

Disclosed is a process for producing carnosic acid (the major antioxidant active substance contained in a rosemary plant) with high efficiency by carrying out the ortho-position oxidation of a phenol with an oxidizing agent (e.g., 2-iodoxybenzoic acid) and the subsequent ester removal reaction by using, as a starting material, pisiferic acid (the main component of Chamaecyparis pisifera which is a plant which has been planted in great numbers as wood resources). Specifically disclosed is a process for producing an antioxidant active substance contained in a rosemary plant and represented by general formula (5).  The process is characterized by comprising: a first step of oxidizing a pisiferic acid derivative represented by general formula (1) with an oxidizing agent represented by general formula (2) or (4); and a second step of reducing or hydrolyzing a pisiferic acid derivative intermediate produced in the first step.

Description

抗酸化活性物質の製造方法Method for producing antioxidant active substance
　本発明は、抗酸化活性物質の製造方法に関する。さらに詳しくは、サワラ等はじめとする樹木を原料とするカルノシン酸及びその誘導体の製造方法に関する。 The present invention relates to a method for producing an antioxidant active substance. More specifically, the present invention relates to a method for producing carnosic acid and its derivatives from trees such as Sawara.
　ローズマリー、セージ、シソ、オレガノ、バジル、タイム、マジョラム、ペパーミント等のシソ科植物は、全世界に広く分布し、約200属3500種が知られている。上記シソ科植物に含まれる抗酸化物活性物質を構造的な特徴から分類するとフェノール性ジテルペン、カフェ酸誘導体、フラボノイド、ビフェニル誘導体の4つの型に大別される。 Lamiaceae plants such as rosemary, sage, perilla, oregano, basil, thyme, marjoram, peppermint are widely distributed throughout the world, and about 200 genera and 3500 species are known. The antioxidant active substances contained in the Lamiaceae plants are roughly classified into four types: phenolic diterpenes, caffeic acid derivatives, flavonoids, and biphenyl derivatives.
　上記シソ科植物の中でも、ローズマリーは、古くから肉料理の香辛料や民間薬として知られ、カルノシン酸、カルノソール、ロスマノールに代表される強い抗酸化活性成分を含有することが広く知られている（非特許文献１）。近年、このようなローズマリーが含有する上記抗酸化活性物質は、抗菌活性、脳神経細胞死防止効果、脳血症の治療・予防効果、アルツハイマー病予防効果、脂肪吸収防止効果、抗炎症作用、糖尿病患者の血糖値低下効果、美白効果など様々な活性が報告されており、食品添加物、サプリメント、医薬等としての利用が検討されている（特許文献１及び非特許文献２ないし非特許文献４）。 Among the Lamiaceae plants, rosemary has long been known as a spice for meat dishes and a folk medicine, and it is widely known that it contains a strong antioxidant active component typified by carnosic acid, carnosol, and rosmanol ( Non-patent document 1). In recent years, the above-mentioned antioxidant active substances contained in such rosemary have antibacterial activity, cranial nerve cell death prevention effect, cerebral blood treatment / prevention effect, Alzheimer's disease prevention effect, fat absorption prevention effect, anti-inflammatory effect, diabetes Various activities such as blood glucose lowering effect and whitening effect of patients have been reported, and their use as food additives, supplements, medicines, etc. are being studied (Patent Document 1, Non-Patent Document 2 to Non-Patent Document 4). .
　例えば、ローズマリー及びセージからなる群より選択される少なくとも１種の植物由来の抽出物を有効成分として含有する、神経突起伸長剤が開示されている（特許文献２）。
　上記抽出物の一例であるカルノシン酸は、ローズマリー等の所定部位をアルコールにて抽出して得られることが開示されている。しかしながら、ローズマリーの全草5000グラムに対し、アルコール処理等を行い得られるローズマリーの抽出物から分離されたカルノシン酸は、わずか1.5グラムであり、製造効率としては十分でないという問題点がある。 For example, a neurite elongation agent containing at least one plant-derived extract selected from the group consisting of rosemary and sage as an active ingredient is disclosed (Patent Document 2).
It is disclosed that the carnosic acid which is an example of the said extract is obtained by extracting predetermined parts, such as rosemary, with alcohol. However, carnosic acid separated from an extract of rosemary obtained by alcohol treatment or the like with respect to 5,000 grams of rosemary whole plant is only 1.5 grams, and there is a problem that the production efficiency is not sufficient.
　また、上記抗酸化活性物質の一つであるカルノシン酸を抽出する方法として、ローズマリーの葉を水溶性酸の存在下で、低級アルキルアルコール水溶液で処理して、さらに任意にカルノシン酸を抽出して精製することを含む方法が開示されている（特許文献３及び特許文献４）。しかしながら、上記これらの方法においては、カルノシン酸の出発原料としてローズマリーの葉を使用している。ローズマリーの葉は、生産量と価格によりその供給量は大きく変わりものであり、安価にしかも大量にカルノシン酸を供給できる製造方法という観点からすれば十分ではない。 In addition, as a method of extracting carnosic acid, which is one of the above antioxidant active substances, rosemary leaves are treated with a lower alkyl alcohol aqueous solution in the presence of a water-soluble acid, and carnosic acid is optionally extracted. A method including purification is disclosed (Patent Document 3 and Patent Document 4). However, in these methods described above, rosemary leaves are used as a starting material for carnosic acid. The supply of rosemary leaves varies greatly depending on the production volume and price, and is not sufficient from the viewpoint of a production method that can supply carnosic acid in a large amount at a low cost.
　一方、サワラは、日本特産の針葉樹であり、天然生は栃木、群馬、及び長野県上高地などに多く分布している。その葉及び樹皮には、カルノシン酸の原料となる、ピシフェリン酸等の関連化合物を大量に含有する（非特許文献５）。さらにサワラには、現在、合成食品添加物として使用されているジブチルヒドロキシトルエン（BHT）やブチルヒドロキシアニソール（BHA）と同様活性を有する特定の物質が含まれていることが明らかになっている。上記合成食品添加物として使用されているジブチルヒドロキシトルエン（BHT）やブチルヒドロキシアニソール（BHA）等が発がん性など人体に影響を及ぼすことを鑑みれば、サワラを有効利用した活用法が期待されているところである。 On the other hand, Sawara is a special conifer of Japan, and its natural life is distributed a lot in Tochigi, Gunma, and Kamikochi, Nagano Prefecture. The leaves and bark contain a large amount of related compounds such as piciferin acid, which is a raw material for carnosic acid (Non-patent Document 5). Furthermore, it has been clarified that Sawara contains a specific substance having activity similar to that of dibutylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) currently used as synthetic food additives. Considering that dibutylhydroxytoluene (BHT) and butylhydroxyanisole (BHA), etc. used as the above synthetic food additives affect the human body such as carcinogenicity, the utilization method that uses sawara is expected. By the way.
　このようにサワラ等の樹木は、植物資源として有望であるが、それらの成分の際立った利用方法は開発されていない。一方、ローズマリー等の主要抗酸化活性物質であるカルノシン酸の効率的な製造方法は開示されていない。 As described above, trees such as Sawara are promising as plant resources, but no distinctive use of these components has been developed. On the other hand, an efficient method for producing carnosic acid, which is a main antioxidant active substance such as rosemary, is not disclosed.
　なお、本件特許出願人は、本件発明に関連する文献公知発明が記載された刊行物として、以下の技術文献を開示する。
特開２００１－１５８７４５号公報 特開２００７－２３０９４５号公報 特表２００１－５１８０７２号公報 特開２００３－５５６８６号公報 多田全宏、「シソ科香草に含まれる抗酸化活性物質の生理活性」、ＦＦＩジャーナル　オブ　ジャパン、２０００年、１８４号 S. C.Etter, 「Spices & Medicinal Plants」, Journal of Herbs,  11, 121-159 (2004) T. Satoh,K. Kosaka, K. Itoh, A. Kobayashi, M. Yamamoto, Y. Shimojo, C. Kitajima, J. Cui,J. Kamins, S. Okamoto, M. Izumi, T. Shirasawa, S. A. Lipton, 「J. Neurochem.」, 104, 1116-1131 (2008) K.Ninomiya, H. Matsuda, H. Shimoda, N. Nishida, N. Kasajima, T. Yoshino, T.Morikawa, M. Yoshikawa, 「Bioorg. Med. Chem. Lett.」, 14 (8), 1943-1946(2004) du Xiao,M. Kuroyanagi, T. Itani, H. Tatsuura, M. Udayama, M. Murakami, K. Umehara, N.Kawahara, 「Chem. Pharm. Bull.」, 49 (11), 1479-1481 (2001). In addition, this patent applicant discloses the following technical literature as a publication in which the literature well-known invention relevant to this invention was described.
JP 2001-158745 A JP 2007-230945 A JP-T-2001-518072 JP 2003-55686 A Zenta Tada, "Physiological activity of antioxidant active substances in Lamiaceae herbs", FFI Journal of Japan, 2000, No. 184 S. C. Etter, `` Spices & Medicinal Plants '', Journal of Herbs, 11, 121-159 (2004) T. Satoh, K. Kosaka, K. Itoh, A. Kobayashi, M. Yamamoto, Y. Shimojo, C. Kitajima, J. Cui, J. Kamins, S. Okamoto, M. Izumi, T. Shirasawa, SA Lipton , "J. Neurochem.", 104, 1116-1131 (2008) K. Ninomiya, H. Matsuda, H. Shimoda, N. Nishida, N. Kasajima, T. Yoshino, T. Morikawa, M. Yoshikawa, "Bioorg. Med. Chem. Lett.", 14 (8), 1943- 1946 (2004) du Xiao, M. Kuroyanagi, T. Itani, H. Tatsuura, M. Udayama, M. Murakami, K. Umehara, N. Kawahara, Chem. Pharm. Bull., 49 (11), 1479-1481 (2001) ).
　以上のような状況に鑑み、本発明の課題は、日本の山野に広く存在する樹木であるサワラ等の常緑広葉樹を出発原料とし、カルノシン酸及びその誘導体等を抽出して、抗酸化活性物質を製造する方法を提供することある。 In view of the situation as described above, the object of the present invention is to extract carnosic acid and derivatives thereof from evergreen broad-leaved trees such as Sawara, which are widely present in Japanese mountains, and extract an antioxidant active substance. May provide a method of manufacturing.
　本発明者等は、上記課題を解決すべく鋭意研究した結果、サワラ等を原料とし、その抽出物であるピシフェリン酸等の化合物のフェノールのオルト位を特定の酸化剤により酸化することにより、強抗酸化活性成分であるカルノシン酸及びその誘導体を製造することができることを見出し、本発明を完成するに至った。 As a result of diligent research to solve the above-mentioned problems, the inventors of the present invention obtained strong power by oxidizing the ortho position of phenol of a compound such as piciferin acid, which is an extract thereof, with a specific oxidizing agent. It has been found that carnosic acid and its derivatives, which are antioxidant active ingredients, can be produced, and the present invention has been completed.
　本発明は、以下の技術的事項から構成される。すなわち、
[1]　下記一般式（１）で表されるピシフェリン酸誘導体と、 The present invention is composed of the following technical matters. That is,
[1] A piciferin acid derivative represented by the following general formula (1):
Figure JPOXMLDOC01-appb-C000006
 （上記一般式（１）において、R1は、COOR3、ヒドロキシメチル基、アルデヒド基のいずれかであり、R2及びR3は、それぞれ独立に水素原子または炭素数１ないし６のアルキル基を表す。）
下記一般式（２）ないし一般式（４）で表されるいずれかの酸化剤、
Figure JPOXMLDOC01-appb-C000006
 (In the above general formula (1), R 1 is any one of COOR 3 , hydroxymethyl group and aldehyde group, and R 2 and R 3 each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. To express.)
Any of the oxidants represented by the following general formula (2) to general formula (4),
Figure JPOXMLDOC01-appb-C000007
Figure JPOXMLDOC01-appb-C000007
Figure JPOXMLDOC01-appb-C000008
Figure JPOXMLDOC01-appb-C000008
Figure JPOXMLDOC01-appb-C000009
 （上記一般式（２）及び（４）において、R４、R5及びR6は、それぞれ独立に水素原子または炭素数１ないし６のアルキル基、アリル基、フェニル基、ハロゲン化フェニル基、ハロゲン化アルキル基のいずれかを表す。上記一般式（３）において、Ｘは、ハロゲン原子、ｎは1又は２を表す。）
を、反応させることにより、酸化する第１の工程と、
前記第１の工程において生成するピシフェリン酸誘導体中間体を還元反応又は加水分解反応をさせる第２の工程を有することを特徴とする下記一般式（５）で表される
Figure JPOXMLDOC01-appb-C000009
 (In the above general formulas (2) and (4), R 4 , R 5 and R 6 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, an allyl group, a phenyl group, a halogenated phenyl group, a halogen atom. (In the general formula (3), X represents a halogen atom, and n represents 1 or 2).
A first step of oxidizing by reacting;
It has a 2nd process of carrying out a reductive reaction or a hydrolysis reaction of the piciferic acid derivative intermediate produced | generated in the said 1st process, It represents with following General formula (5) characterized by the above-mentioned.
Figure JPOXMLDOC01-appb-C000010
 抗酸化活性物質の製造方法。
（下記一般式（５）中、R1は、COOR4、ヒドロキシメチル基、アルデヒド基のいずれかであり、R2、R７及びR4は、それぞれ独立に水素原子または炭素数１ないし６のアルキル基、アリル基、フェニル基、ハロゲン化フェニル基のいずれかを表す。）
[２] 前記酸化剤が、一般式（３）において、Ｘ=Iであり、かつn＝１である２－ヨードキシ安息香酸（IBXと言う）であることを特徴とする[１]に記載の抗酸化活性物質の製造方法。
[３]前記酸化剤が、一般式（４）で表される２－ヨードキシ安息香酸トリアシルエステル誘導体であることを特徴とする[１]に記載のローズマリーの抗酸化活性物質の製造方法。
[４] [１]ないし[３]の何れかに記載の製造方法により、製造した抗酸化活性物質。
[５] [４]に記載の抗酸化活性物質を含有することを特徴とする糖尿病抑制剤。
[６] [４]に記載の抗酸化活性物質を含有することを特徴とするアルツハイマー病抑制剤。
[７] [４]に記載の抗酸化活性物質を含有することを特徴とするニキビ治療薬。
Figure JPOXMLDOC01-appb-C000010
 A method for producing an antioxidant active substance.
(In the following general formula (5), R 1 is any one of COOR 4 , hydroxymethyl group, and aldehyde group, and R 2 , R 7, and R 4 are each independently a hydrogen atom or a C 1-6 carbon atom. Represents any of an alkyl group, an allyl group, a phenyl group, and a halogenated phenyl group.)
[2] The oxidizing agent is 2-iodoxybenzoic acid (referred to as IBX) in which X = I and n = 1 in the general formula (3). A method for producing an antioxidant active substance.
[3] The method for producing a rosemary antioxidant active material according to [1], wherein the oxidizing agent is a 2-iodoxybenzoic acid triacyl ester derivative represented by the general formula (4).
[4] An antioxidant active substance produced by the production method according to any one of [1] to [3].
[5] An antidiabetic agent comprising the antioxidant active substance according to [4].
[6] An Alzheimer's disease inhibitor comprising the antioxidant active substance according to [4].
[7] An acne therapeutic agent comprising the antioxidant active substance according to [4].
　本発明によれば、サワラから極めて高い収率にて、カルノシン酸等に代表される抗酸化活性物質を簡易かつ大量に製造することができる。また、本発明によれば、安価な特定の酸化開始剤を使用することにより、簡易な工程によりカルノシン酸等の抗酸化活性物質を製造することができる。 According to the present invention, an antioxidant active substance typified by carnosic acid or the like can be produced easily and in large quantities from Sawara with an extremely high yield. In addition, according to the present invention, an antioxidant active substance such as carnosic acid can be produced by a simple process by using an inexpensive specific oxidation initiator.
　以下、本発明の実施の形態を詳細に説明する。 Hereinafter, embodiments of the present invention will be described in detail.
（サワラ等の樹木ついて）
　本発明の抗酸化活性物質の製造方法は、まず、サワラ等樹木から、ピシフェリン酸及びその誘導体を抽出し、特定の過酸化物により、ピシフェリン酸等のフェノールのオルト位を酸化し、その後還元反応または加水分解する工程を有することを特徴とするものである。 (For trees such as Sawara)
In the method for producing an antioxidant active substance of the present invention, first, piciferin acid and its derivative are extracted from a tree such as sawara, and the ortho-position of phenol such as piciferin acid is oxidized with a specific peroxide, and then a reduction reaction is performed. Or it has the process of hydrolyzing.
　本発明において、抗酸化活性物質とは、例えばカルノシン酸、カルノソール、ロスマノール等に代表される強い抗酸化活性を有する化合物をいう。本発明において「抗酸化活性物質」とは、ローズマリーのみから抽出したものした以外にも、セージ、シソ、オレガノ、バジル等のローズマリー以外のシソ科植物を出発原料とし、又はこれらの出発原料を組み合わせたものを出発原料として製造される組成物を含むものである。本発明の抗酸化活性物質の製造方法に使用される原料は、本発明の製造方法の目的物質であるカルノシン酸等の原料となる、ピシフェリン酸及びその誘導体をその成分に含んでいればよく、特に制限されるものではないが、例えば、ヒノキ科のサワラ属の樹木を例示することができる。上記樹木としては、例えばサワラ、シノブヒバ、オウゴンシノブヒバ、ヒムロおよびヒヨクヒバを例示することができる。上記例示した針葉樹の中でも、その生産量及び取り扱いの観点から、サワラが好ましく、これらの原料を混合して使用することもできる。以下、本発明の製造方法の目的物質である抗酸化活性物質の出発原料となるピシフェリン酸及びその誘導体の一般式を示す。 In the present invention, the term “antioxidant active substance” refers to a compound having a strong antioxidant activity, such as carnosic acid, carnosol, rosmanol and the like. In the present invention, the term “antioxidant active substance” is not limited to those extracted only from rosemary, but also uses Lamiaceae plants other than rosemary such as sage, perilla, oregano and basil as starting materials, or these starting materials. A composition produced using a combination of these as starting materials. The raw material used in the method for producing an antioxidant active substance of the present invention only needs to contain, in its components, piciferic acid and its derivative, which are raw materials such as carnosic acid, which is the target substance of the production method of the present invention, Although it does not restrict | limit in particular, For example, the tree of the genus Sawara of the cypress family can be illustrated. Examples of the tree include Sawara, Shinobu hiba, Ogon Shinobu hiba, Himuro and Hiyoku hiba. Among the above-mentioned examples of conifers, Sawara is preferable from the viewpoint of production and handling, and these raw materials can be mixed and used. The general formulas for piciferic acid and its derivatives, which are starting materials for the antioxidant substance that is the target substance of the production method of the present invention, are shown below.
Figure JPOXMLDOC01-appb-C000011
Figure JPOXMLDOC01-appb-C000011
　上記一般式において、R1は、COOR3、ヒドロキシメチル基、アルデヒド基のいずれかである。すなわち、R1は、カルボキシル基、カルボン酸エステル基、ヒドロキシメチル基、アルデヒド基である。 In the above general formula, R 1 is any one of COOR 3 , a hydroxymethyl group, and an aldehyde group. That is, R 1 is a carboxyl group, a carboxylic ester group, a hydroxymethyl group, or an aldehyde group.
　R2、R３は、それぞれ独立に、水素原子又は炭素数1ないし６のアルキル基であり、炭素数１ないし６のアルキル基としては、メチル基、エチル基、n-プロピル基、i-プロピル基、n-ブチル基、sec-ブチル基、tert-ブチル基、n-ペンチル基、n-ヘキシル基等を例示することができる。上記一般式において、R1がカルボキシル基（R1=COOH）かつR2が水素原子(R2=H)である場合には、下記化学式で示されるピシフェリン酸となる。なお、ピシフェリン酸は、サワラに最も多く含まれているジテルペンであり、かつ化学的に安定かつ、無色、無臭の結晶である。また、R1がメトキシカルボニル基（R1=COOCH3）かつR2が水素原子(R2=H)である場合には、ピシフェリン酸メチル、R1がカルボキシル基（R1=COOH）かつR2が水素原子(R2= CH3)である場合には、o-メチルピシフェリン酸メチルとなる。 R 2 and R 3 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. Examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, an n-propyl group, and an i-propyl group. Examples thereof include a group, n-butyl group, sec-butyl group, tert-butyl group, n-pentyl group, n-hexyl group and the like. In the above general formula, when R 1 is a carboxyl group (R 1 = COOH) and R 2 is a hydrogen atom (R 2 = H), it becomes piciferic acid represented by the following chemical formula. In addition, piciferic acid is a diterpene most contained in Sawara, and is a chemically stable, colorless, and odorless crystal. When R 1 is a methoxycarbonyl group (R 1 = COOCH 3 ) and R 2 is a hydrogen atom (R 2 = H), methyl piciferate, R 1 is a carboxyl group (R 1 = COOH) and R When 2 is a hydrogen atom (R 2 = CH 3 ), it becomes methyl o-methylpiciferate.
Figure JPOXMLDOC01-appb-C000012
Figure JPOXMLDOC01-appb-C000012
　サワラは、日本国の至るところで入手することができる常緑高木であり、きわめて容易に入手することができる。さらに、サワラは、入手容易でありかつ、大量に存在するバイオマスである。なお、本発明においては、サワラの部位の中で、ピシフェリン酸とその誘導体化合物を多く含むのでもその葉と樹皮を使用するのが好ましい。 Sawara is an evergreen tree that can be obtained everywhere in Japan and can be obtained very easily. Furthermore, Sawara is a biomass that is readily available and present in large quantities. In the present invention, it is preferable to use the leaves and bark even though a lot of piciferin acid and its derivative compounds are included in the part of Sawara.
　サワラ等の原料から、ピシフェリン酸等を抽出する方法としては、特に制限されるものではないが、例えば、以下の方法により抽出することができる。すなわち、サワラ等の原料を溶媒に浸漬又は溶媒によって還流することにより容易に抽出することができる。使用できる溶媒としては、水、アルコール、アルカン、カルボン酸、エステル、ケトンを例示することができる。また、これら溶媒は、単独で使用することもでき、これら２種類以上を適宜混合して使用することもできる。 The method for extracting piciferin acid and the like from raw materials such as Sawara is not particularly limited, and for example, extraction can be performed by the following method. That is, it can be easily extracted by immersing a raw material such as sawara in a solvent or refluxing with a solvent. Examples of the solvent that can be used include water, alcohol, alkane, carboxylic acid, ester, and ketone. In addition, these solvents can be used alone, or two or more of these solvents can be appropriately mixed and used.
　上記アルコールとしては、メタノール、エタノール、１－プロパノール、２－プロパノール、１－ブタノール、２－ブタノール、２－メチル１－プロパノール、ｔ－ブチルアルコール、１－ペンタノール、２－ペンタノール、３－ペンタノール、２－メチル１－ブタノール、３－メチル１－ブタノール、２、２ジメチル１－プロパノールなどが挙げられる。また上記アルカンとしては、ペンタン、へキサン、ヘプタン、オクタン、ノナン、デカンなどが挙げられる。 Examples of the alcohol include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl 1-propanol, t-butyl alcohol, 1-pentanol, 2-pentanol, and 3-pen. Examples include butanol, 2-methyl 1-butanol, 3-methyl 1-butanol, 2 and 2 dimethyl 1-propanol. Examples of the alkane include pentane, hexane, heptane, octane, nonane and decane.
　ケトンとしては、アセトン、メチルエチルケトン（ＭＥＫ）、ジエチルケトンが挙げられる。また、必要に応じて、上記の有機溶媒から二種類以上を採択して混合溶液として使用することもできる。上記溶媒の中でも、安全性等の取り扱いの観点から、水、アルコールの単独又は水とアルコールとの混合溶媒を使用することが好ましい。特に好ましくは水とエタノールの混合溶媒とするのが好ましい。 Examples of ketones include acetone, methyl ethyl ketone (MEK), and diethyl ketone. In addition, if necessary, two or more kinds of the above organic solvents can be adopted and used as a mixed solution. Among the above solvents, from the viewpoint of handling such as safety, it is preferable to use water, alcohol alone or a mixed solvent of water and alcohol. It is particularly preferable to use a mixed solvent of water and ethanol.
　上記溶媒抽出を行う場合の抽出時間及び抽出温度は、原料の種類により適宜設定することができる。例えば、原料をサワラとし、抽出する溶媒としてメタノールを採択して抽出する場合には、その抽出温度を還流温度にする事が望ましく、抽出反応時を１０時間ないし２４時間とすることができる。上記抽出方法により、生成した下記一般式で示されるピシフェリン酸及びその誘導体を含有する溶液を抽出後、濃縮し、カラムクロマトグラフィーにて分離後、再結晶によって精製することができる。 The extraction time and extraction temperature when performing the above solvent extraction can be appropriately set depending on the type of raw material. For example, when the raw material is sawara and methanol is extracted as the solvent to be extracted, the extraction temperature is preferably set to the reflux temperature, and the extraction reaction time can be 10 to 24 hours. By the above extraction method, a solution containing the generated piciferin acid represented by the following general formula and its derivative can be extracted, concentrated, separated by column chromatography, and then purified by recrystallization.
（ピシフェリン酸のオルト位酸化）
　本発明の抗酸化活性物質の製造方法は、上記化学式で示されるピシフェリン酸及びその誘導体の構造式中、フェノールのオルト位酸化反応に際して、下記一般式で示される特定の過酸化物を採択した点に特徴を有する。すなわち、本発明においては、下記一般式で表される酸化剤を使用し、ピシフェリン酸及びその誘導体のフェノールのオルト位を酸化するものである。 (Ortho oxidation of piciferic acid)
The method for producing an antioxidant active substance of the present invention employs a specific peroxide represented by the following general formula in the ortho-position oxidation reaction of phenol in the structural formulas of piciferic acid and derivatives thereof represented by the above chemical formula It has the characteristics. That is, in the present invention, an oxidant represented by the following general formula is used to oxidize the ortho position of phenol in piciferic acid and its derivatives.
Figure JPOXMLDOC01-appb-C000013
Figure JPOXMLDOC01-appb-C000013
Figure JPOXMLDOC01-appb-C000014
Figure JPOXMLDOC01-appb-C000014
Figure JPOXMLDOC01-appb-C000015
 （上記一般式（２）及び一般式（４）において、また、R４、R５及びR６は、それぞれ独立に水素原子または炭素数１ないし６のアルキル基、アリル基、フェニル基、ハロゲン化フェニル基、ハロゲン化アルキル基のいずれかを表す。上記一般式（３）において、Ｘは、ハロゲン原子、ｎは1又は２を表す。）
Figure JPOXMLDOC01-appb-C000015
 (In the above general formula (2) and general formula (4), R 4 , R 5 and R 6 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, an allyl group, a phenyl group, a halogenated group) (Represents either a phenyl group or a halogenated alkyl group. In the general formula (3), X represents a halogen atom, and n represents 1 or 2.)
　一般に、フェノール類のオルト位酸化反応において採択される方法としては、セレン酸化物による直接酸化方法、過酸化ベンゾイルによる酸化方法、空気酸化、2-Iodoxybenzoic acid（以下、「ＩＢＸ」と言います。）による酸化等方法を例示することができるが、これに限定されるものではない。本発明においては、種々の酸化剤を使用することによって、例えばサワラ属樹種等から抽出される、ピシフェリン酸誘導体を酸化することができる。例えば、ジアシル類を表す上記一般式（２）において、R４及びR５をハロゲン化フェニルとしたハロゲン化フェニルとしたメタクロロ過酸化ベンゾイル(mCBPO)と、ハロゲン化フェニルとハロゲン化アルキル基としたクロロアセチルメタクロロ過酸化ベンゾイル（CAMCBPO）を使用することができるが、これらの酸化剤に何ら限定されない。また、上記ＩＢＸの中でも酸化効率及び安全性の観点から、上記一般式（３）の中でも、Ｘをヨウ素とし、ｎ＝１としたＩＢＸが特に好ましい。この点、後述する実施例においては、酸化剤としてＩＢＸや過酸化ジアシルを用いたがこれに限定されない。本発明においては、酸化剤として、上記一般式で表される安息香酸誘導体を使用することもできる。 In general, the methods adopted in the ortho-oxidation reaction of phenols include direct oxidation with selenium oxide, oxidation with benzoyl peroxide, air oxidation, and 2-Iodoxybenzoic acid (hereinafter referred to as “IBX”). Examples of the oxidation method may include but are not limited thereto. In the present invention, the use of various oxidizing agents can oxidize piciferin acid derivatives extracted from, for example, Sawara species. For example, in the above general formula (2) representing a diacyl, benzoyl peroxide (mCBPO) having a halogenated phenyl in which R 4 and R 5 are phenyl halides, and a chloro having a halogenated phenyl and a halogenated alkyl group. Acetylmetachlorobenzoyl peroxide (CAMCBPO) can be used, but is not limited to these oxidizing agents. Among the above IBXs, from the viewpoint of oxidation efficiency and safety, among the above general formula (3), IBX is particularly preferable in which X is iodine and n = 1. In this regard, in Examples described later, IBX and diacyl peroxide are used as the oxidizing agent, but the present invention is not limited to this. In the present invention, a benzoic acid derivative represented by the above general formula can also be used as an oxidizing agent.
　上記酸化剤の中でも、ＩＢＸによる酸化では、酸化物が更に酸化され、複雑な混合物を生成することが多い。ＩＢＸによるオルト位酸化では、酸化物が更に酸化され、オルト－キノンを生成するが、一般的にオルト－キノンは不安点で分解しやすいので水素化ナトリウムよって速やかに還元することによってカテコールに変換した。また、安全かつ取り扱いが簡便な酸化剤としてメタクロロ過酸化ベンゾイル（ｍCBPO）とクロロアセチルメタクロロ過酸化ベンゾイル（CAMCBPO）を開発した。 Of the above oxidizing agents, oxidation with IBX often further oxidizes the oxide, producing a complex mixture. In the ortho-position oxidation by IBX, the oxide is further oxidized to produce ortho-quinone. Generally, however, ortho-quinone is easily decomposed at anxiety points, so it is converted to catechol by rapid reduction with sodium hydride. . We also developed benzoyl peroxide (mCBPO) and benzoyl peroxide (CAMCBPO) as oxidants that are safe and easy to handle.
　なお、本発明で使用する酸化剤である上記ＩＢＸは、２－ヨード安息香酸から所定の条件にて、簡易に製造することができる。以下に反応式を示す。 The above IBX, which is an oxidizing agent used in the present invention, can be easily produced from 2-iodobenzoic acid under predetermined conditions. The reaction formula is shown below.
Figure JPOXMLDOC01-appb-C000016
Figure JPOXMLDOC01-appb-C000016
（オルト位酸化後のエステル基の還元的除去）
　ＩＢＸによる酸化後の生成したオルトキノンの還元反応に使用される還元剤としては、カルボキシル基を還元しない程度の緩やかな還元性を有する触媒であれば特に制限されるものではないが、たとえば、トリアルコキシ水素化アルミニウムリチウム[LiAlH(OR)3]、水素化ホウ素リチウム（LiBH4）水素化ホウ素ナトリウム、トリアルキル水素化ホウ素リチウム(LiR3BH)及びジアルキル水素化アルミニウムを例示することができる。なお、これらの還元剤は単独で使用してもよいし、組み合わせて使用してもよい。 (Reductive removal of ester group after ortho-position oxidation)
The reducing agent used for the reduction reaction of the generated orthoquinone after oxidation with IBX is not particularly limited as long as it is a catalyst having a mild reducing ability that does not reduce the carboxyl group. Examples include lithium aluminum hydride [LiAlH (OR) 3 ], lithium borohydride (LiBH 4 ) sodium borohydride, lithium trialkylborohydride (LiR 3 BH), and dialkylaluminum hydride. These reducing agents may be used alone or in combination.
（オルト位酸化後のエステル基の加水分解）
　また、オルト位酸化後のエステル基は加水分解によっても除去することができる。加水分解反応の触媒としては、水酸化ナトリウム、水酸化リチウム、水酸化カリウム、炭酸ナトリウム、炭酸カリウムなどの塩基類と水―メタノール、水―エタノールなどの溶媒の組み合わせが考えられる。 (Hydrolysis of ester group after ortho-position oxidation)
Moreover, the ester group after ortho-position oxidation can also be removed by hydrolysis. As a catalyst for the hydrolysis reaction, a combination of a base such as sodium hydroxide, lithium hydroxide, potassium hydroxide, sodium carbonate or potassium carbonate and a solvent such as water-methanol or water-ethanol can be considered.
　これらの還元反応または加水分解により、本発明の製造方法において目的とする抗酸化活性物質を製造することができる。このように本発明においては、入手可能で安価な過酸化物を使用してピシフェリン酸等の化合物のフェノールのオルト位を効率よく酸化することができる結果、抗酸化活性物質を高い効率で製造することができる。 The target antioxidant active substance in the production method of the present invention can be produced by these reduction reactions or hydrolysis. As described above, in the present invention, it is possible to efficiently oxidize the ortho position of phenol of a compound such as piciferic acid using an available and inexpensive peroxide. As a result, an antioxidant active substance is produced with high efficiency. be able to.
　さらに、本発明においては、製造された抗酸化活性物質を既知の方法によりカルノソールおよびロスマノールに変換することができる。例えば、カルノソールは、カルノシン酸をDDQ、酸化銀または空気などによって酸化すると、生成するキノンーキノンメチド互変異性体がラクトン化し、製造することがきる。ロスマノールは、カルノソールを塩化メチレン中、ピリジニウムクロロクロメート（PCC）による酸化、または、炭酸水素ナトリウム存在下、空気による酸化によって製造することができる。 Furthermore, in the present invention, the manufactured antioxidant active substance can be converted into carnosol and rosmanol by a known method. For example, carnosol can be produced when carnosic acid is oxidized by DDQ, silver oxide or air, and the quinone-quinone metide tautomer formed is lactonized. Rosmanol can be produced by oxidation of carnosol with methylene chloride with pyridinium chlorochromate (PCC) or with air in the presence of sodium bicarbonate.
　酸化剤として、ＩＢＸを使用し、さらに生成したオルトキノンを水素化ホウ素ナトリウム等の還元剤を使用してエステル基を還元することができる。以下に反応式を示す。さらに、加水分解をすることにより、以下のＩＢＸを用いたカルノシン酸の合成方法により、カルノシン酸を製造することができる。 As the oxidizing agent, IBX can be used, and the produced orthoquinone can be reduced to an ester group using a reducing agent such as sodium borohydride. The reaction formula is shown below. Furthermore, by carrying out hydrolysis, carnosic acid can be produced by the following method for synthesizing carnosic acid using IBX.
Figure JPOXMLDOC01-appb-C000017
Figure JPOXMLDOC01-appb-C000017
　このように本発明の製造方法によればカルノシン酸及びその誘導体の抗酸化活性物質を簡易かつ容易にしかも大量に製造することができる。更にこれらの抗酸化活性物質を原料としてカルノソール、ロスマノール等の有用な化合物を大量に製造することができる。 Thus, according to the production method of the present invention, it is possible to easily and easily produce a large amount of carnosic acid and its antioxidant active substance. Furthermore, a large amount of useful compounds such as carnosol and rosmanol can be produced from these antioxidant active substances.
　以下、本発明について実施例を用いて説明するが、本発明は、何らこれらに限定されるものではない。 Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto.
（実施例１）
＜ピシフェリン酸の分離＞
　サワラの葉（東京都府中市晴見町東京農工大学農学部森林から採取）を採取し、そのまま出発原料とした。上記サワラの葉160ｇを秤量し、メタノール約800ml中で約65℃にて、24時間還流した。次に、上記還流操作により得られたメタノール抽出液を減圧濃縮した後、酢酸エチルと水により液／液による抽出操作を行った。さらに、上記抽出操作後、酢酸エチル層を濃縮し、その濃縮物をシリカゲル、ヘキサン／酢酸エチル（体積比3：1）を用いて、カラムクロマトグラフィーを行った。ピシフェリン酸を含む画分を濃縮後、再度同様にカラムクロマトグラフィーを行い、得られたピシフェリン酸を含む画分をヘキサン／酢酸エチル（体積比3：1）を用いて結晶化させ、目的化合物であるピシフェリン酸248 mgを得た。 Example 1
<Separation of piciferic acid>
Sawara leaves (collected from the forest of Tokyo University of Agriculture and Technology, Harumi-cho, Fuchu-shi, Tokyo) were collected and used as starting materials. 160 g of the above Sawara leaves were weighed and refluxed in about 800 ml of methanol at about 65 ° C. for 24 hours. Next, the methanol extract obtained by the above refluxing operation was concentrated under reduced pressure, and then a liquid / liquid extraction operation was performed with ethyl acetate and water. Further, after the above extraction operation, the ethyl acetate layer was concentrated, and the concentrate was subjected to column chromatography using silica gel and hexane / ethyl acetate (volume ratio 3: 1). After concentrating the fraction containing piciferin acid, perform column chromatography again in the same manner. The resulting fraction containing piciferin acid was crystallized using hexane / ethyl acetate (volume ratio 3: 1), and A certain 248 mg of piciferic acid was obtained.
　上記生成したピシフェリン酸の融点及びその微細構造をそれぞれ、融点測定器及び1H－NMR、13C－NMRにより測定した。なお、融点測定器は、 Laboratory Device社製MEL-TEMPを使用し、補正なし条件下で測定した。1H－NMRおよび13C－NMRの測定は、日本電子社製JEOL
alpha-600 (1H: 600 MHz, 13C: 150.8 MHz) スペクトロメーターを使用し。重クロロホルム中テトラメチルシランを標準として測定した。 The melting point and microstructure of the generated piciferin acid were measured by a melting point analyzer, 1 H-NMR, and 13 C-NMR, respectively. In addition, the melting point measuring device used MEL-TEMP by Laboratory Device, and measured it on the conditions without correction | amendment. 1 H-NMR and 13 C-NMR measurements were performed using JEOL manufactured by JEOL Ltd.
alpha-600 ( 1 H: 600 MHz, 13 C: 150.8 MHz) Using a spectrometer. Tetramethylsilane in deuterated chloroform was used as a standard.
　図１にピシフェリン酸の分析結果を示す。
　ピシフェリン酸:
m.p. 174-180 ℃ ; 1H-NMR
(600 MHz, CDCl3) δ: 6.89 (1H, s), 6.67 (1H, s), 3.10 (1H, sept., J = 6.6 Hz), 2.89
(1H, dd, J = 16.2, 6.0 Hz), 2.82-2.76 (2H, m), 2.46 (1H, dddd, J = 13.1, 12.0,
11.0, 5.9 Hz), 1.94 (1H, m), 1.87 (1H, ddd, J = 11.0, 4.4, 2.8 Hz), 1.60 (1H,
ddd, J = 13.7, 3.4, 3.1 Hz), 1.49 (1H, dd, J = 13.2, 1.8 Hz), 1.45 (1H, d, J =
12.6 Hz), 1.24 (2H, m), 1.22 (3H, d, J = 6.6 Hz), 1.21 (3H, d, J = 6.6 Hz),
0.96 (3H, s), 0.82 (3H, s) FIG. 1 shows the analysis result of piciferic acid.
Piciferic acid:
mp 174-180 ℃; 1 H-NMR
(600 MHz, CDCl 3 ) δ: 6.89 (1H, s), 6.67 (1H, s), 3.10 (1H, sept., J = 6.6 Hz), 2.89
(1H, dd, J = 16.2, 6.0 Hz), 2.82-2.76 (2H, m), 2.46 (1H, dddd, J = 13.1, 12.0,
11.0, 5.9 Hz), 1.94 (1H, m), 1.87 (1H, ddd, J = 11.0, 4.4, 2.8 Hz), 1.60 (1H,
ddd, J = 13.7, 3.4, 3.1 Hz), 1.49 (1H, dd, J = 13.2, 1.8 Hz), 1.45 (1H, d, J =
12.6 Hz), 1.24 (2H, m), 1.22 (3H, d, J = 6.6 Hz), 1.21 (3H, d, J = 6.6 Hz),
0.96 (3H, s), 0.82 (3H, s)
13C-NMR (150 MHz,
CDCl3) δ: 181.2, 150.6, 138.2, 133.5, 129.2,
127.4, 112.3, 52.2, 47.5, 41.7, 36.7, 34.0, 32.1, 29.3, 26.8, 22.6, 22.3, 20.3,
20.1, 18.6. 13 C-NMR (150 MHz,
CDCl 3 ) δ: 181.2, 150.6, 138.2, 133.5, 129.2,
127.4, 112.3, 52.2, 47.5, 41.7, 36.7, 34.0, 32.1, 29.3, 26.8, 22.6, 22.3, 20.3,
20.1, 18.6.
＜ピシフェリン酸のオルト位酸化＞
　ピシフェリン酸（200mg：0.63mmol）を塩化メチレン（10ml）に溶解させた後、m‐クロロ過酸化ベンゾイル（590mg:1.9mmol）を加え溶解させた。この溶液を室温、アルゴン下で16時間放置した後、濃縮した。濃縮残渣は酢酸エチルに溶かし、ヘキサンを加えて析出した結晶をロ別した。母液を更に濃縮しオルト位酸化されたカルノシン酸モノエステル含む混合物が得られた。この混合物をカラムクロマトグラフィー（酢酸エチルーヘキサン）を行いカルノシン酸モノエステルの粗生成物（130 mg）を得た。
　カルノシン酸モノエステル : light yellow powder ; 1H-NMR
(600 MHz, CDCl3) δ： 8.20 (1H, s), 8.10 (1H, dd, J = 7.5, 1.8 Hz), 7.60 (1H, dd, J =
8.4, 1.8 Hz), 7.45 (1H, t, J = 8.4 Hz), 6.69 (1H, s), 3.35-3.32 (1H,m),
2.97-2.86 (2H, m), 2.40-2.33 (1H, m), 1.91-1.87 (2H, m), 1.61-1.59 (2H, m),
1.52-1.48 (1H, m), 1.34-1.25 (2H, m), 1.20 (3H, d, J = 6.6 Hz), 1.17 (3H, d, J
= 6.6 Hz), 1.01 (3H, s), 0.89 (3H, s)  <Orthic oxidation of piciferic acid>
Piciferic acid (200 mg: 0.63 mmol) was dissolved in methylene chloride (10 ml), and m-chlorobenzoyl peroxide (590 mg: 1.9 mmol) was added and dissolved. The solution was allowed to stand at room temperature under argon for 16 hours and then concentrated. The concentrated residue was dissolved in ethyl acetate, and hexane was added to separate the precipitated crystals. The mother liquor was further concentrated to obtain a mixture containing ortho-oxidized carnosic acid monoester. This mixture was subjected to column chromatography (ethyl acetate-hexane) to obtain a crude product (130 mg) of carnosic acid monoester.
Carnosic acid monoester: light yellow powder; 1 H-NMR
(600 MHz, CDCl 3 ) δ: 8.20 (1H, s), 8.10 (1H, dd, J = 7.5, 1.8 Hz), 7.60 (1H, dd, J =
8.4, 1.8 Hz), 7.45 (1H, t, J = 8.4 Hz), 6.69 (1H, s), 3.35-3.32 (1H, m),
2.97-2.86 (2H, m), 2.40-2.33 (1H, m), 1.91-1.87 (2H, m), 1.61-1.59 (2H, m),
1.52-1.48 (1H, m), 1.34-1.25 (2H, m), 1.20 (3H, d, J = 6.6 Hz), 1.17 (3H, d, J
= 6.6 Hz), 1.01 (3H, s), 0.89 (3H, s)
13C-NMR (150 MHz,
CDCl3) δ： 180.17, 163.70, 140.06, 136.69,
135.71, 134.81, 133.62, 130.97, 130.32, 129.94, 128.43, 124.77, 119.2, 53.82,
48.36, 41.59, 34.32, 34.29, 32.59, 31.99, 27.64, 22.94, 22.75, 21.16, 20.05,
18.54 13 C-NMR (150 MHz,
CDCl 3 ) δ: 180.17, 163.70, 140.06, 136.69,
135.71, 134.81, 133.62, 130.97, 130.32, 129.94, 128.43, 124.77, 119.2, 53.82,
48.36, 41.59, 34.32, 34.29, 32.59, 31.99, 27.64, 22.94, 22.75, 21.16, 20.05,
18.54
　以上の結果より、カルノシン酸モノエステルを含む分画をこれ以上精製することなく、還元的条件下でエステル基を加水分解し、水酸基への誘導を行なった。カルノシン酸モノエステル
を含む分画(70 mg)をメタノール（9ｍｌ）に溶かし、1％-NaOH（1ｍｌ）と水素化ホウ素ナトリウム（13.2 ｍｇ）を加え、アルゴン下2時間加熱還流した。生成物は1M塩酸で酸性にし、食塩水―酢酸エチルで抽出した。抽出液は食塩水で洗浄、無水硫酸マグネシウムで乾燥、濃縮した。濃縮残渣はヘキサン抽出し、抽出液を濃縮し、濃縮残渣をシリカゲルカラムクロマトグラフィー（ヘキサン－酢酸エチル　5：1後に3：1）で精製し、カルノシン酸 (23 mg)を得た。 From the above results, the ester group was hydrolyzed under reductive conditions without further purification of the fraction containing carnosic acid monoester, and induced to a hydroxyl group. A fraction (70 mg) containing carnosic acid monoester was dissolved in methanol (9 ml), 1% -NaOH (1 ml) and sodium borohydride (13.2 mg) were added, and the mixture was heated to reflux for 2 hours under argon. The product was acidified with 1M hydrochloric acid and extracted with brine-ethyl acetate. The extract was washed with brine, dried over anhydrous magnesium sulfate and concentrated. The concentrated residue was extracted with hexane, the extract was concentrated, and the concentrated residue was purified by silica gel column chromatography (hexane-ethyl acetate 5: 1 followed by 3: 1) to obtain carnosic acid (23 mg).
　カルノシン酸: 1H-NMR (600 MHz, CDCl3) δ: 6.58
(1H, s), 3.35-3.31 (1H,m), 3.20 (1H, sept, J = 7.2 Hz), 2.85-2.79 (2H, m),
2.42-2.37 (1H, m), 1.92-1.78 (2H, m), 1.65-1.49 (3H, m), 1.36-1.22 (2H, m),
1.22 (3H, d, J = 3.0 Hz), 1.20 (3H, d, J = 3.0 Hz), 1.01 (3H, s), 0.93 (3H, s);
13C-NMR (150 MHz, CDCl3) δ: 182.63,
142.06, 141.53, 133.70, 128.92, 122.04, 119.44, 53.96, 48.76, 41.88, 34.44,
34.41, 32.60, 31.48, 27.16, 22.68, 22.47, 21.85, 20.38, 18.88.
　以上より、ピシフェリン酸からカルノシン酸までの2段階の総収率は25-55％であった。 Carnosic acid: 1 H-NMR (600 MHz, CDCl 3 ) δ: 6.58
(1H, s), 3.35-3.31 (1H, m), 3.20 (1H, sept, J = 7.2 Hz), 2.85-2.79 (2H, m),
2.42-2.37 (1H, m), 1.92-1.78 (2H, m), 1.65-1.49 (3H, m), 1.36-1.22 (2H, m),
1.22 (3H, d, J = 3.0 Hz), 1.20 (3H, d, J = 3.0 Hz), 1.01 (3H, s), 0.93 (3H, s);
13 C-NMR (150 MHz, CDCl 3 ) δ: 182.63,
142.06, 141.53, 133.70, 128.92, 122.04, 119.44, 53.96, 48.76, 41.88, 34.44,
34.41, 32.60, 31.48, 27.16, 22.68, 22.47, 21.85, 20.38, 18.88.
From the above, the total yield of the two steps from piciferic acid to carnosic acid was 25-55%.
（実施例２）
＜クロロアセチルメタクロロ過酸化ベンゾイル（CAMCBPO）を用いたカルノシン酸の合成＞
　クロロ酢酸（59.7
mg, 0.63 mmol）を塩化メチレン（15 ml）に溶解し、ジシクロへキシルカルボジイミド（DCC,　143.5 mg, 0.70 mmol）を加え、アルゴン気流下0 ℃で15 分撹拌した後、メタクロロか過安息香酸（mCPBA,　152.7 mg, 0.88 mmol)を加え、アルゴン気流下0 ℃で30 分撹拌し,クロロアセチルメタクロロ過酸化ベンゾイル（CAMCBPO）を生成させた。この反応液に、ピシフェリン酸(100 mg, 0.32
mmol)を加え、アルゴン気流下0 ℃→室温で66 時間撹拌した。セライトにより、dicyclohexylureaを濾過し、母液を濃縮後、酢酸エチルとブラインによって液‐液抽出した後、MgSO4で乾燥し、濃縮した。残渣をシリカゲルカラムクロマトグラフィー(hexane : EtOAc = 5 : 1)に供し、カルノシン酸モノエステルを含む租生成画分（71.1 mg）を得た。この画分をこれ以上精製することなく、MeOH(9 ml)に溶解し、水素化ホウ素ナトリウム(35.9 mg, 0.95 mmol)を加え、さらに水酸化ナトリウム水溶液（1%水溶液, 1 ml）を加えアルゴン気流下2時間加熱還流した。1 N塩酸によって反応を停止し、ヘキサンで液‐液抽出し、有機層を飽和NaHCO3及びブラインで洗浄した後MgSO4で乾燥し、濃縮した。残渣をシリカゲルカラムクロマトグラフィー(hexane : EtOAc = 10 : 1)に供し、租生成であるカルノシン酸（23.3
mg, 0.07 mmol, 22.4%）を得た（化１３を参照）。）
Figure JPOXMLDOC01-appb-C000018
 （実施例３）
　フェノール類のオルト位酸化反応において使用する、酸化剤としてＩＢＸを使用し、生成物を還元する事によって、カルノシン酸を製造した。
＜2-iodxybenzoic
acid （ＩＢＸ）の製造＞
　過硫酸カリウム塩（デュポン社商品名：Oxone）(14.9 g, 26.0 mmol)を水(90 ml)に溶解し、2-iodobenzoic acid （2.0 g, 8.00 mmol）を加え、70℃で３時間攪拌した。反応液を1時間氷冷し、生成した結晶を吸引濾過し、水(6×40 ml)とacetone(2×40
ml)で洗浄した。結晶個体を室温で乾燥させＩＢＸ（0.57 g, 2.03 mmol）を収率25%で得た。 (Example 2)
<Synthesis of carnosic acid using chloroacetylmetachlorobenzoyl peroxide (CAMCBPO)>
Chloroacetic acid (59.7
mg, 0.63 mmol) was dissolved in methylene chloride (15 ml), dicyclohexylcarbodiimide (DCC, 143.5 mg, 0.70 mmol) was added, and the mixture was stirred at 0 ° C. for 15 minutes under a stream of argon. mCPBA, 152.7 mg, 0.88 mmol) was added, and the mixture was stirred at 0 ° C. for 30 minutes under a stream of argon to produce chloroacetylmetachlorobenzoyl peroxide (CAMCBPO). To this reaction mixture, picipheric acid (100 mg, 0.32
mmol) was added, and the mixture was stirred at 0 ° C. → room temperature for 66 hours under an argon stream. Dicyclohexylurea was filtered through Celite, and the mother liquor was concentrated, liquid-liquid extracted with ethyl acetate and brine, dried over MgSO 4 and concentrated. The residue was subjected to silica gel column chromatography (hexane: EtOAc = 5: 1) to obtain a crude fraction (71.1 mg) containing carnosic acid monoester. This fraction was dissolved in MeOH (9 ml) without further purification, sodium borohydride (35.9 mg, 0.95 mmol) was added, and sodium hydroxide aqueous solution (1% aqueous solution, 1 ml) was added. The mixture was heated to reflux for 2 hours under an air stream. The reaction was quenched with 1 N hydrochloric acid, liquid-liquid extracted with hexane, and the organic layer was washed with saturated NaHCO 3 and brine, then dried over MgSO 4 and concentrated. The residue was subjected to silica gel column chromatography (hexane: EtOAc = 10: 1) to produce carnosic acid (23.3
mg, 0.07 mmol, 22.4%) was obtained (see chemical formula 13). )
Figure JPOXMLDOC01-appb-C000018
 (Example 3)
Carnosic acid was produced by reducing the product using IBX as the oxidizing agent used in the ortho-position oxidation reaction of phenols.
<2-iodxybenzoic
Production of acid (IBX)>
Potassium persulfate (DuPont brand name: Oxone) (14.9 g, 26.0 mmol) was dissolved in water (90 ml), 2-iodobenzoic acid (2.0 g, 8.00 mmol) was added, and the mixture was stirred at 70 ° C. for 3 hours. . The reaction solution was ice-cooled for 1 hour, and the produced crystals were filtered by suction, and water (6 × 40 ml) and acetone (2 × 40
ml). The crystalline solid was dried at room temperature to obtain IBX (0.57 g, 2.03 mmol) in a yield of 25%.
　ＩＢＸ ; white
powder ; m.p. ; 232-233 ℃ ; 1H-NMR (600 MHz, d6-DMSO) δ ;
8.15(1H, d, J = 7.8 Hz), 8.04(1H, d, J = 7.2 Hz), 8.00(1H, t, J = 7.9 Hz),
7.85(1H, t, J = 7.8 Hz) ; 13C-NMR
(150 MHz, d6-DMSO) δ ; 167.49, 146.55, 133.38, 132.95, 131.43, 130.08, 124.99 IBX; white
powder; mp; 232-233 ° C; 1 H-NMR (600 MHz, d 6 -DMSO) δ;
8.15 (1H, d, J = 7.8 Hz), 8.04 (1H, d, J = 7.2 Hz), 8.00 (1H, t, J = 7.9 Hz),
7.85 (1H, t, J = 7.8 Hz); 13 C-NMR
(150 MHz, d 6 -DMSO) δ; 167.49, 146.55, 133.38, 132.95, 131.43, 130.08, 124.99
＜ＩＢＸを用いたカルノシン酸の合成＞
　ピシフェリン酸（316.4
mg, 1.00 mmol）を無水DMF（5 ml）に溶解し、IBX（336.0 mg, 1.20 mmol）を加え、アルゴン気流下室温で1時間攪拌した。TLCにて酸化反応進行を確認後、反応液にNaBH4(378.3 mg,
10 mmol)を加えアルゴン気流下室温で6時間攪拌した。その後1 M HClを加え反応を停止させ、Hexaneで液－液抽出した後、有機層をbrineで洗浄した。その後MgSO4で乾燥し、濃縮した後にシリカゲルカラムクロマトグラフィー(hexane : EtOAc = 10 : 1)で精製し、カルノシン酸（114.3
mg, 0.34 mmol）を34%の収率で得た。 <Synthesis of carnosic acid using IBX>
Piciferic acid (316.4
mg, 1.00 mmol) was dissolved in anhydrous DMF (5 ml), IBX (336.0 mg, 1.20 mmol) was added, and the mixture was stirred at room temperature for 1 hour under an argon stream. After confirming the progress of the oxidation reaction by TLC, NaBH 4 ( 378.3 mg,
10 mmol) was added, and the mixture was stirred at room temperature for 6 hours under an argon stream. Thereafter, 1 M HCl was added to stop the reaction, liquid-liquid extraction with Hexane was performed, and then the organic layer was washed with brine. Thereafter, it was dried over MgSO 4 , concentrated and then purified by silica gel column chromatography (hexane: EtOAc = 10: 1) to obtain carnosic acid (114.3
mg, 0.34 mmol) was obtained in a yield of 34%.
　カルノシン酸;
yellow powder ; 1H-NMR
(600 MHz, CDCl3) δ ;
6.57 (1H, s), 3.30 (1H,d, J = 12 Hz ), 3.17 (1H, sept, J = 7.2 Hz), 2.86-2.78
(2H, m), 2.42-2.35 (1H, m), 1.88-1.85 (1H, m), 1.75 (1H, t, J = 12 Hz),
1.62-1.56 (2H, m), 1,48 (1H, d, J = 12 Hz), 1.34-1.23 (2H, m), 1.21 (3H, d, J =
3.0 Hz), 1.20 (3H, d, J = 3.0 Hz), 1.01 (3H, s), 0.90 (3H, s); 13C-NMR (150 MHz, CDCl3) δ ; 183.39, 142.10,
141.32, 133.79, 129.02, 122.08, 119.38, 53.95, 48.67, 41.81, 34.46, 34.33,
32.59, 31.44, 27.13, 22.49, 22.12, 21.69, 20.31, 18.83 Carnosic acid;
yellow powder; 1 H-NMR
(600 MHz, CDCl 3 ) δ;
6.57 (1H, s), 3.30 (1H, d, J = 12 Hz), 3.17 (1H, sept, J = 7.2 Hz), 2.86-2.78
(2H, m), 2.42-2.35 (1H, m), 1.88-1.85 (1H, m), 1.75 (1H, t, J = 12 Hz),
1.62-1.56 (2H, m), 1,48 (1H, d, J = 12 Hz), 1.34-1.23 (2H, m), 1.21 (3H, d, J =
3.0 Hz), 1.20 (3H, d, J = 3.0 Hz), 1.01 (3H, s), 0.90 (3H, s); 13 C-NMR (150 MHz, CDCl 3 ) δ; 183.39, 142.10,
141.32, 133.79, 129.02, 122.08, 119.38, 53.95, 48.67, 41.81, 34.46, 34.33,
32.59, 31.44, 27.13, 22.49, 22.12, 21.69, 20.31, 18.83
　以上の実施例から、サワラから高い収量でピリフェリン酸（Pisiferic acid）を分離精製し、これをオルト位酸化することにより、ローズマリーの主要抗酸化活性物質である
カルノシン酸（Carnosic
acid）を効率的に製造する事ができることが理解される。 From the above examples, Pisiferic acid was isolated and purified from Sawara in high yield, and this was ortho-oxidized to produce carnosic acid (Carnosic acid), the main antioxidant active substance of rosemary.
It is understood that acid) can be produced efficiently.
　上記実施例は、近年、アルツハイマー病や様々な生活習慣病の予防効果があることで注目されているローズマリーの主用抗酸化活性物質を、木材資源として大量植林されているサワラの葉から供給する事を可能にしたものであり、その技術的意義は極めて大きい。 In the above examples, the main antioxidant active substance of rosemary, which has been attracting attention in recent years due to its preventive effect against Alzheimer's disease and various lifestyle-related diseases, is supplied from the leaves of Sawara planted in large quantities as wood resources. The technical significance is extremely large.
（実施例４及び実施例５）
＜抗菌活性の測定＞
　実施例１によって合成したローズマリーの主要抗酸化活性物質であるカルノシン酸（実施例４）およびこのカルノシン酸から合成したカルソール(実施例５)を用いて、抗メチシリン耐性黄色ブドウ球菌活性（抗ＭＲＳＡ活性）および抗アクネ菌活性を測定した。すなわち、ニキビ（座瘡）は思春期頃から皮膚に生ずる慢性炎症性疾患で、ニキビの発症と悪化は食生活やストレスなどの要因と毛穴に寄生したアクネ菌や黄色ブドウ球菌、皮膚ブドウ球菌の増殖（感染症）が重要な役割を果たすと考えられている。現在までに本疾患の治療薬として使用が認められている医療用外用抗菌薬は、種類が少なく古いものが多く、臨床治療に応用できる薬剤が少ない。このため、日本のみならず海外でもニキビ（座瘡）の治療効果を持つ新規の有効な治療薬が求められている。このような観点から、合成したローズマリーの主要抗酸化活性物質を用いて、抗メチシリン耐性黄色ブドウ球菌活性および抗アクネ菌活性を測定した。また、比較例として、実施例４及び実施例５と同様な条件下におけるバンコマイシン（比較例１）及びアンピシリン（比較例２）の場合の抗メチシリン耐性黄色ブドウ球菌活性（抗ＭＲＳＡ活性）および抗アクネ菌活性を測定した。測定結果を表１に示す。 (Example 4 and Example 5)
<Measurement of antibacterial activity>
Carnosic acid (Example 4), which is the main antioxidant active substance of rosemary synthesized according to Example 1, and carsol (Example 5) synthesized from this carnosic acid were used for anti-methicillin-resistant Staphylococcus aureus activity (anti-MRSA). Activity) and anti-acne activity. Acne (acne) is a chronic inflammatory disease that occurs in the skin from around puberty. Proliferation (infection) is thought to play an important role. To date, there are many types of antibacterial drugs for external use that have been approved for use as treatments for this disease, many of them are old, and few drugs can be applied to clinical treatment. For this reason, there is a need for a new effective therapeutic agent having a therapeutic effect for acne (acne) not only in Japan but also overseas. From such a viewpoint, the anti-methicillin-resistant Staphylococcus aureus activity and the anti-acne activity were measured using the main antioxidant active substance of rosemary synthesized. Further, as comparative examples, anti-methicillin-resistant Staphylococcus aureus activity (anti-MRSA activity) and anti-acne in the case of vancomycin (Comparative Example 1) and ampicillin (Comparative Example 2) under the same conditions as in Examples 4 and 5 The fungal activity was measured. The measurement results are shown in Table 1.
　なお、抗アクネ菌活性の測定は、以下のように行った。すなわち、実施例１で得られた抗酸化活性物質を試料として、プルロニックL４４（ポリオキシエチレン・ポリオキシプロピレン）にて１０倍にて希釈し、これをアクネ菌用培地に１０重量％希釈することで試料を１．０重量％濃度に調製した。コントロールとしては１０重量％プルロニックL－４４を用いた。GAM液体培地にてアクネ菌を２４時間培養した後、沈殿させ、アクネ菌用培地にて上記GAM液体培地を２回洗浄し、これを接種菌液として試料に接種した。その後、２４時間、４８時間後に残存菌数をカウントした。カウントは、アクネ菌用培地を用いて１．０～１００,０００倍まで段階希釈し、平板塗抹法を用い嫌気的に培養後カウントした。なお、上記抗アクネ菌活性測定と同様の条件にてメチシレン耐性黄色ブドウ球菌（ＭＲＳＡ）に対する抗菌活性の測定を行った。 In addition, the measurement of anti-acne bacteria activity was performed as follows. That is, using the antioxidant substance obtained in Example 1 as a sample, diluting 10 times with Pluronic L44 (polyoxyethylene / polyoxypropylene), and diluting it 10% by weight in the medium for acne bacteria A sample was prepared at a concentration of 1.0% by weight. As a control, 10% by weight Pluronic L-44 was used. After acne bacteria were cultured in a GAM liquid medium for 24 hours, they were precipitated, and the GAM liquid medium was washed twice with an acne medium, and the sample was inoculated as an inoculum. Thereafter, the number of remaining bacteria was counted after 24 and 48 hours. Counting was performed by serial dilution from 1.0 to 100,000 times using a medium for acne bacteria, and after anaerobic culture using a plate smearing method. In addition, the antibacterial activity with respect to methicylene resistant Staphylococcus aureus (MRSA) was measured on the same conditions as the said anti-acne bacteria activity measurement.
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-T000019
　表１によれば、本発明の抗酸化活性物質の製造方法により得られた主要な抗酸化活性物質は、顕著な抗菌活性を有しており、これらの結果は、本発明の抗酸化活性物質がニキビ治療薬として有用である可能性を示している。 According to Table 1, the main antioxidant active substance obtained by the method for producing the antioxidant active substance of the present invention has a remarkable antibacterial activity, and these results indicate that the antioxidant active substance of the present invention May be useful as a treatment for acne.
　本発明の抗酸化活性物質の製造方法は、木材資源として大量に植林されているサワラを原料とした抗酸化活性物質の製造方法であるので、林業及び環境技術分野の発展に貢献することができる。さらに、本発明のカルノシン酸の製造方法は、医薬及び医療技術分野の技術革新に大きく貢献することができる。 The method for producing an antioxidant active substance according to the present invention is a method for producing an antioxidant active substance using Sawara planted in large quantities as a wood resource, and can therefore contribute to the development of forestry and environmental technology fields. . Furthermore, the method for producing carnosic acid of the present invention can greatly contribute to technological innovation in the fields of medicine and medical technology.

Claims (7)

  1. 下記一般式（１）で表されるピシフェリン酸誘導体と、
    Figure JPOXMLDOC01-appb-C000001
     （上記一般式（１）において、R1は、COO R3、ヒドロキシメチル基、アルデヒド基のいずれかであり、R2及びR3は、それぞれ独立に水素原子または炭素数１ないし６のアルキル基を表す。）
    下記一般式（２）ないし一般式（４）で表されるいずれかの酸化剤、
    Figure JPOXMLDOC01-appb-C000002
    Figure JPOXMLDOC01-appb-C000003
    Figure JPOXMLDOC01-appb-C000004
      
    （上記一般式（２）及び（４）において、R４、R5及びR6は、それぞれ独立に水素原子または炭素数１ないし６のアルキル基、アリル基、フェニル基、ハロゲン化フェニル基、ハロゲン化アルキル基のいずれかを表す。上記一般式（３）において、Ｘは、ハロゲン原子、ｎは1又は２を表す。）
    を、反応させることにより、酸化する第１の工程と、
    前記第１の工程において生成するピシフェリン酸誘導体中間体を還元反応又は加水分解反応をさせる第２の工程を有することを特徴とする下記一般式（５）で表される
    Figure JPOXMLDOC01-appb-C000005
     抗酸化活性物質の製造方法。
    （下記一般式（５）中、R1は、COOR4、ヒドロキシメチル基、アルデヒド基のいずれかであり、R2、R７及びR4は、それぞれ独立に水素原子または炭素数１ないし６のアルキル基、アリル基、フェニル基、ハロゲン化フェニル基のいずれかを表す。）     A piciferin acid derivative represented by the following general formula (1);
    Figure JPOXMLDOC01-appb-C000001
     (In the above general formula (1), R 1 is any one of COO R 3 , a hydroxymethyl group, and an aldehyde group, and R 2 and R 3 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. Represents.)
    Any of the oxidants represented by the following general formula (2) to general formula (4),
    Figure JPOXMLDOC01-appb-C000002
    Figure JPOXMLDOC01-appb-C000003
    Figure JPOXMLDOC01-appb-C000004
    (In the above general formulas (2) and (4), R 4 , R 5 and R 6 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, an allyl group, a phenyl group, a halogenated phenyl group, a halogen atom. (In the general formula (3), X represents a halogen atom, and n represents 1 or 2).
    A first step of oxidizing by reacting;
    It has a 2nd process of carrying out a reductive reaction or a hydrolysis reaction of the piciferic acid derivative intermediate produced | generated in the said 1st process, It represents with following General formula (5) characterized by the above-mentioned.
    Figure JPOXMLDOC01-appb-C000005
     A method for producing an antioxidant active substance.
    (In the following general formula (5), R 1 is any one of COOR 4 , hydroxymethyl group, and aldehyde group, and R 2 , R 7, and R 4 are each independently a hydrogen atom or a C 1-6 carbon atom. Represents any of an alkyl group, an allyl group, a phenyl group, and a halogenated phenyl group.
  2. 　前記酸化剤が、一般式（３）において、Ｘ=Iであり、かつn＝１である２－ヨードキシ安息香酸（IBXと言う）であることを特徴とする請求項１に記載の抗酸化活性物質の製造方法。 2. The antioxidant activity according to claim 1, wherein the oxidizing agent is 2-iodoxybenzoic acid (referred to as IBX) wherein X = I and n = 1 in the general formula (3). A method for producing a substance.
  3. 　前記酸化剤が、一般式（４）で表される２－ヨードキシ安息香酸トリアシルエステル誘導体であることを特徴とする請求項１に記載のローズマリーの抗酸化活性物質の製造方法。 The method for producing a rosemary antioxidant active substance according to claim 1, wherein the oxidant is a 2-iodoxybenzoic acid triacyl ester derivative represented by the general formula (4).
  4. 　請求項１ないし請求項３の何れか1項に記載の製造方法により、製造した抗酸化活性物質。 An antioxidant active substance produced by the production method according to any one of claims 1 to 3.
  5. 　請求項４に記載の抗酸化活性物質を含有することを特徴とする糖尿病抑制剤。 A diabetes inhibitor comprising the antioxidant active substance according to claim 4.
  6. 　請求項４に記載の抗酸化活性物質を含有することを特徴とするアルツハイマー病抑制剤。 An Alzheimer's disease inhibitor comprising the antioxidant active substance according to claim 4.
  7. 　請求項４に記載の抗酸化活性物質を含有することを特徴とするニキビ治療薬。
          A therapeutic agent for acne comprising the antioxidant active substance according to claim 4.
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