JP2011157293A - Antioxidant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel nature-derive compound employable for an antioxidant, and an antioxidant using the same. <P>SOLUTION: There is provided a triprenylphenol compound represented by formula (I). The antioxidant comprises the triprenylphenol compound. X is -CHY-C(CH<SB>3</SB>)<SB>2</SB>Z; Y and Z are each -H or -OH, or linked together to form a single bond; and R<SP>1</SP>is an aryl, or a heterocyle or the like. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a novel triprenyl phenol compound and an antioxidant using the same.

  Active oxygen is a highly reactive substance that oxidizes others in a short time and disappears itself. It is known that if it is overproduced in vivo or the rate of disappearance is reduced, it will have a harmful effect due to oxidation. . Oxidative stress due to excessive active oxygen is considered to cause various diseases to the living body. Such diseases include, for example, brain disorders such as schizophrenia and manic depression, adult respiratory distress syndrome, arteriosclerosis, hypertension, circulatory disorders such as thrombosis, renal disorders such as nephritis and renal failure, alcohol Hepatic disorders such as hepatitis and cirrhosis, diabetic complications such as cataracts and peripheral neuropathy, gastrointestinal disorders such as gastric ulcer and duodenal ulcer, rheumatoid arthritis, cancer, accelerated aging, and UV damage. For this reason, various antioxidants that effectively remove active oxygen or suppress generation thereof have been developed (for example, Patent Documents 1 to 3).

  On the other hand, SMTP (Stachybotrys microspora triprenyl phenol) compounds are a group of compounds having a triprenyl phenol skeleton produced by filamentous fungi, and are known to have a thrombolysis promoting action and an angiogenesis inhibiting action (for example, patents). References 4-6). Regarding the thrombolysis-promoting action, the SMTP compound leads to a change in plasminogen conformation. As a result, the sensitivity of plasminogen to t-PA and the binding of plasminogen to thrombus are increased. It has been suggested that the mechanism of action promotes (see, for example, Non-Patent Document 1).

JP 2001-316262 A JP 2006-298807 A JP 2007-320859 A JP 2004-224737 A Japanese Patent No. 4313049 WO2007 / 111203 pamphlet

FEBS Letter 1997; 418: 58-62

However, the pathogenesis of disease that appears to be due to excess active oxygen is not fully elucidated, and existing antioxidants may not always be effective in all situations. Some naturally derived antioxidants do not have sufficient antioxidant activity.
Accordingly, an object of the present invention is to provide a novel naturally-derived compound that can be used as an antioxidant and an antioxidant using the same.

  The present invention provides a triprenyl phenol compound represented by the following formula (I) and an antioxidant containing the triprenyl phenol compound.

In the above general formula (I), X is —CHY—C (CH ₃ ) ₂ Z, and Y and Z are —H or —OH, respectively, or together form a single bond. R ¹ is the following substituent, and * represents a bonding position with a nitrogen atom.

  ADVANTAGE OF THE INVENTION According to this invention, the novel naturally derived compound which can be used for an antioxidant, and an antioxidant using the same are provided.

Hereinafter, the present invention will be described in detail.
The triprenyl phenol compound of the present invention is represented by the above general formula (I).
The triprenyl phenol compound is a compound in which a predetermined substituent is directly bonded to the nitrogen atom at the 1-position in addition to the triprenyl phenol skeleton. As a compound having a similar triprenyl phenol skeleton, orniprabin showing a high thrombolysis promoting action is known, but the triprenyl phenol compound represented by the above general formula (I) is similar to the triprenyl phenol. Despite having a skeleton, the effect of promoting thrombolysis is weak. For this reason, this triprenyl phenol compound can be used as an antioxidant even in a situation where the thrombolysis promoting action is not preferred.
The compound represented by the general formula (I) is preferably a triprenyl phenol compound from the viewpoint of antioxidant activity.

The triprenyl phenol compound of the present invention can be obtained by either a chemical production method by chemical synthesis or a biological production method using living organisms.
When the triprenyl phenol compound of the present invention is produced by chemical synthesis, it is preferable to use an intermediate having no side chain on the chromanlactam nitrogen atom in the triprenyl phenol skeleton from the viewpoint of production efficiency. Examples of such intermediates include those disclosed in WO 2007/111203.
In order to produce the triprenylphenol compound of the present invention by a biological production method, a filamentous fungus can be used as a living organism, and from the viewpoint of production efficiency, a filamentous fungus such as Stachybotrys or Memnoniella is preferred. Is selected. Particularly preferred production bacteria are Stachybotrys microspora and the like, and more preferred is S. microspora IFO30018 strain, but the present invention is not limited to this bacteria.

In the biological production method, it can be more easily obtained from the viewpoint of production efficiency that the method comprises culturing in a medium in which a specific nitrogen compound is added to the filamentous fungus. Examples of such a production method include those disclosed in the aforementioned WO 2007/111203.
Briefly describing this production method, a culture step of culturing filamentous fungi in a culture solution containing a specific added amine compound described later, and a separation step of separating the triprenyl phenol compound from the culture after the culture step Is included.
The added amine compound only needs to be present during the culturing process of the filamentous fungus and may be present from the initial stage of the culture, but is preferably added at the middle stage of the culture from the viewpoint of production efficiency.

When an added amine compound is added in the middle of the culture, the filamentous fungus culturing step includes the first culturing step using a restricted medium having an amine compound content of 0.5% by mass or less, and the added amine compound. And a second culturing step with the production medium.
When a limited medium in which the content of amine compound is limited to 0.5% by mass is used as the medium used in the first culturing step, after the middle stage of culturing to move to the second culturing step, a larger amount than before Intermediate compounds can be obtained. Moreover, after producing a large amount of the intermediate compound in this manner, the target triprenylphenol compound is obtained efficiently and with high selectivity by performing the second culturing step with the production medium containing the added amine compound. be able to. In the present specification, the “amine compound” includes an added amine compound unless otherwise specified.

The amine compound that can be contained in the restriction medium acts as a nitrogen source for growth of filamentous fungi in the restriction medium, a growth promoting factor, or a production promoting factor for the triprenyl phenol compound precursor. As a form of addition, it can be used as a natural mixture such as yeast extract, bouillon, peptone, tryptone, soy bean meal, pharma media, corn steep liquor, fish meat extract, etc., or as a purified compound. Since natural mixtures contain a wide variety of amine compounds, it is preferable to limit the amount in a limiting medium.
In this case, the amine compound is preferably 0.5% by mass or less with respect to the total volume of the restricted medium, preferably from 0.01 to 0.5% by mass from the viewpoint of fungal growth, production amount and production selectivity. Preferably it can be 0.1 mass%-0.3 mass%. When it exceeds 0.5 mass%, things other than the target compound are produced at the same time, resulting in inferior selectivity and reduced production efficiency. On the other hand, if it is less than 0.01% by mass, the growth of filamentous fungi may be inferior, which is not preferable. Moreover, when adding a refinement | purification compound as an amine compound, the quantity and kind of the range in which the growth of the filamentous fungus used for production and the production of a triprenyl phenol compound precursor occur favorably are used.

  In the second culture step after the middle stage of culture, a production medium containing an added amine compound is used. Here, the “intermediate culture period” can be a predetermined period from the start of the culture for reliably continuing the first culture step, preferably the second day after the start of the culture, more preferably the fourth day or later. . If this period is too short, for example, if the culture in the production medium is started immediately after the start of the culture, the amount of intermediate compound necessary to obtain the target triprenyl phenol compound may be insufficient, and the The prenylphenol compound may not be produced.

  The production medium used in the second culture step can be composed of the same composition as that of the restriction medium except that it contains an added amine compound for obtaining the target triprenylphenol compound. For this reason, the culturing in the second culturing step may be performed by adding an added amine compound to the restriction medium used in the first culturing step, or the newly prepared added amine compound-containing medium is added as it is. May be.

The additive amine compound may be any compound corresponding to the side chain of the lactam ring nitrogen of the target triprenyl phenol compound. Specifically, naphthylamine, glucosamine, galactosamine, aminoisobutyric acid, aminoantipyrine, dinitrotyrosine, amino -N-butyric acid can be mentioned, and these added amine compounds may be used alone or in combination.
Among these, from the viewpoint of antioxidant activity, 1-naphthylamine, D-(+)-glucosamine, D-(+)-galactosamine, DL-3-aminoisobutyric acid, 3-aminoantipyrine, 4-amino-n- Butyric acid and L-3,5-dinitrotyrosine are preferred.

  The added amine compound that can be contained in the production medium in the second culture step may be present in the medium in an amount necessary to obtain the target triprenylphenol compound. From the viewpoint of production amount, it is preferably 0.01% by mass to 1% by mass, and more preferably 0.1% by mass to 0.5% by mass.

  In addition to the above components, the restriction medium and the production medium contain, in addition to the above components, an additive component of a synthetic medium usually used for culturing the above microorganisms for the purpose of promoting the production of compounds by the microorganisms. Examples of additional components that can be added to the restriction medium include nutrient sources such as glucose, sucrose, dextrin, animal oil, vegetable oil, vitamins such as chlorine, nitric acid, sulfuric acid, phosphoric acid, sodium, potassium, calcium, magnesium, cobalt , And other inorganic salts capable of generating ions.

Among inorganic salts, in particular, inorganic salts capable of generating metal ions, and from the viewpoint of increasing the production amount of the product and production efficiency, it can be preferably added to a restricted medium. Examples of such metal ions include magnesium ions, cobalt ions, iron ions, calcium ions, potassium ions, and sodium ions.
These inorganic salts and metal ions may be used alone or in combination of two or more.

The first culture step using the restriction medium is continued until the middle stage of culture in which a sufficient amount of the intermediate compound is obtained to efficiently obtain the target triprenylphenol compound. From the viewpoint of efficient production of the triprenylphenol compound, the second culturing step using the production medium is preferably performed after 2 days from the start of cultivation of the filamentous fungus, more preferably from 4 days after the start of the culture.
The second culture step is terminated by stopping the culture when the amount of triprenylphenol compound produced is maximum. The period of the second culture step varies depending on the state of the microorganism and the size of the culture system, but is generally 1 to 5 days, and preferably 1 to 3 days from the viewpoint of production.

The first and second culturing steps in this production method are usually performed by static culture or shaking culture using the above-mentioned medium. When shake culture is applied, it may be carried out at a rate normally applied in fungal culture.
The culture temperature in the first and second culture steps can be appropriately set according to the growth conditions of fungi at various temperatures, but is generally 4 to 50 ° C, preferably 15 to 37 ° C, more preferably 20 -30 ° C, most preferably room temperature (25 ° C). Outside this range, a triprenyl phenol compound cannot be produced efficiently. Moreover, generally the pH of the culture medium used can be 3-9, Preferably it can be set to 5-6.

  In addition, you may provide a preculture process in order to stabilize the production ability by microorganisms before the 1st and 2nd culture process. The medium used in the preculture step may be a normal growth medium used for maintaining microorganisms.

The obtained triprenyl phenol compound can be obtained by collecting and purifying from the culture. The recovery / purification method may be any means as long as it can recover and purify the triprenylphenol compound released into the medium, and examples thereof include liquid chromatography, solvent extraction, and crystallization. The product is preferably recovered and purified in two or more stages from the viewpoint of recovery efficiency.
In these recovery / purification methods, it is preferable to select a solvent or the like by utilizing the fact that the triprenyl phenol compound is lipophilic.
When collecting and purifying the triprenylphenol compound from the culture, it is preferable to remove the cells from the culture in advance. In that case, a solvent such as methanol is added to the culture to extract the triprenyl phenol compound, and filtration or the like may be used for the subsequent removal of the cells.

The antioxidant of the present invention contains the above triprenyl phenol compound as an active ingredient.
As described above, since the triprenyl phenol compound of the present invention is an antioxidant having a weak thrombolysis promoting activity, various symptoms caused by active oxygen can be alleviated without causing thrombolysis.

The said triprenyl phenol compound contained in the antioxidant of this invention may be used independently, and may be used in combination of multiple. In the antioxidant, the triprenyl phenol compound may be in a free form, a pharmaceutically acceptable salt or ester form, or a solvate form.
Inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, citric acid, formic acid, fumaric acid, malic acid, acetic acid, succinic acid, tartaric acid, methanesulfonic acid, p-toluenesulfonic acid, Suitable for the formation of pharmaceutically acceptable salts of the compounds of the present invention. In addition, compounds containing alkali metals or alkaline earth metals such as sodium, potassium, calcium, magnesium, basic amines, or basic amino acids are also suitable for the formation of pharmaceutically acceptable salts of the compounds of the present invention. is there. Also, alcohols or carboxylic acids having 1 to 10 carbon atoms, preferably methyl alcohol, ethyl alcohol, acetic acid, or propionic acid are suitable for forming a pharmaceutically acceptable ester of the compound of the present invention. . Water and the like are also suitable for forming a pharmaceutically acceptable solvate of the compounds of the present invention.

Further, the dosage form of the present thrombolytic agent can be appropriately changed according to various administration forms. Examples of oral dosage forms include tablets, capsules, powders, fine granules, granules, solutions or syrups, and parenteral dosage forms include injections, drops, suppositories, inhalants, Examples thereof include patches, ointments, cream preparations and the like.
In order to maintain these forms, additives such as well-known solvents and excipients that can be used in these applications can be included.

The antioxidant of the present invention is preferably administered in an amount of 1 to 1000 mg per adult as an effective dose per adult. There is no restriction | limiting in particular in the frequency | count of administration, It may be used by single administration, may be used by repeated administration, and may be used by continuous administration. The administration interval and administration period can be selected by those skilled in the art according to clinical findings, imaging findings, blood findings, comorbid diseases, past medical history, and the like.
The antioxidant of the present invention may be used without being limited to human use. As another application object, you may use for domestic animals, such as a cow, a horse, and a sheep, pets, such as a dog, a cat, and a monkey.

As described above, the antioxidant of the present invention has a weak thrombolysis-promoting activity. Therefore, bleeding conditions such as gastrointestinal bleeding, urinary tract bleeding, and hemoptysis; high blood pressure, high risk of bleeding such as after major surgery; It can be effectively used even in a state where a coagulant or heparin or the like is used; liver disorder, acute pancreatitis; or a history of hypersensitivity to a thrombolytic agent.
Moreover, since the antioxidant of this invention has weak thrombolysis promotion activity, it is not limited to a pharmaceutical use, You may use it as an antioxidant in cosmetics and foodstuffs.

  Examples of the present invention will be described below, but the present invention is not limited thereto. Unless otherwise specified, “%” is based on mass.

[Example 1]
(1) Synthesis of compound SMTP-16
The spore of Stachybotrys microspora IFO30018 strain (Fermentation Laboratory) was inoculated into a 500 ml Erlenmeyer flask containing 100 ml of the seed culture medium, and seed culture was performed at 180 rpm and 25 ° C. for 4 days using a rotary shaker. As the seed culture medium, glucose (4%), soybean meal (0.5%), dry bouillon (0.3%), and powdered yeast extract (0.3%) are dissolved in water, and the pH is adjusted to 5. The antifoaming agent CB442 (0.01%) (0.1 g / ml acetone solution added at 1 ml / L) (Nippon Yushi Chemical Co., Ltd.) was added, and 100 ml each was dispensed to the incubator, and then the autoclave ( 121 ° C., 15 min) was used.

5 ml of this culture solution was inoculated into a 500 ml 1 Erlenmeyer flask containing 100 ml of the main culture medium, and main culture was carried out at 180 rpm and 25 ° C. for 5 days using a rotary shaker.
The main culture medium (restriction medium) is sucrose (5%), powdered yeast extract (0.1%), NaNO ₃ (0.3%), K ₂ HPO ₄ (0.1%), MgSO ₄ · 7H ₂ O (0.05%), KC1 (0.05%), CoCl ₂ .6H ₂ O (0.00025%), FeSO ₄ .7H ₂ O (0.0015%), CaCl ₂ .2H ₂ O ( 0.00065%) is dissolved in water, adjusted to pH 5.8 using HCl, and defoamer CB442 (0.01%) (0.1 g / ml acetone solution is added at 1 ml / L) (Nippon Oils Chemicals, Japan) was added, and 100 ml each was dispensed to the incubator, and then autoclaved (121 ° C., 15 min).

  The day of inoculation was defined as day 0 of culture, and on day 4 of culture (after 96 hours), 100 mg of 1-naphthylamine powder was added to the medium as a production medium, and the culture was continued. About 24 hours later, 200 ml of methanol was added to terminate the culture. Then, it extracted by shaking for about 3 hours at 180 rpm and 25 degreeC using the rotary shaker.

  From 300 ml of the obtained culture extract, cells were removed using a Buchner funnel to obtain a culture supernatant. Concentration was performed using a rotary evaporator under reduced pressure by a water pump. Water was added to make 100 ml, and the pH was adjusted to around 7. Extraction was performed twice with an equal amount of ethyl acetate and once with a half amount. The organic phase was dehydrated with sodium sulfate, filtered and then evaporated to dryness (450 mg). MeOH was added to 100 mg / ml, and the supernatant was collected by centrifugation. Reverse phase HPLC is a column; Inertsil PREP-ODS (diameter: 30 × 250 mm) (GL Science Co., Tokyo, Japan), temperature: 40 ° C., flow rate: 25 ml / min, detection wavelength: 260 nm, developing solvent: 100% methanol The peak with a retention time of 12.0 minutes was collected. Methanol was concentrated and dried by a rotary evaporator to obtain a purified product of compound SMTP-16 at a yield of 2304 mg per liter of culture solution.

The properties of the compound SMTP-16 were confirmed as follows.
MALDI-TOF-MS was measured using Voyager-DE STR (Applied Biosystem) and α-cyano-4-hydroxycinnamic acid as a matrix in positive ion mode.
UV was measured in methanol using a 320 spectrophotometer (Hitachi).
As the FT-IR, JIR-WINSPEC50 (JEOL) was used. A sample dissolved in acetone was applied to rock salt and measured. NMR was measured at ¹ H 600 MHz and ¹³ C 150 MHz using Alpha 600 (JEOL). The sample was DMSO (dimethyl sulfoxide) -d ₆ solution.
The physicochemical properties of the compound SMTP-16 are shown below.

(2) Synthesis of SMTP-33 Pre-culture was performed in the same manner as (1) above.
The main culture was performed in the same manner as in the above (1) except that D-(+)-glucosamine was used as the organic amine compound added on the fourth day of the main culture.
From 300 ml of the obtained culture extract, cells were removed using a Buchner funnel to obtain a culture supernatant. From 300 ml of the obtained culture extract obtained by concentration using a rotary evaporator under reduced pressure by a water pump, the cells were removed using a Buchner funnel to obtain a culture supernatant. Concentration was performed using a rotary evaporator under reduced pressure by a water pump. MeOH was added thereto, and the dissolved fraction was pretreated with Lichrolut RP-18 (100 mg). Reverse phase HPLC was performed with a column; Inertsil PREP-ODS (diameter: 30 × 250 mm), temperature: 40 ° C., flow rate: 25 ml / min, detection wavelength: 260 nm, developing solvent: 75% methanol containing 50 mM ammonium acetate, retention time: 9. A 4-minute peak was collected. Methanol was distilled off using a rotary evaporator, followed by extraction with ethyl acetate. The extract was dried over anhydrous sodium sulfate, then concentrated and dried to obtain 324 mg of a purified product SMTP-33 per liter of culture solution. Yield.
The properties of the compound SMTP-33 were confirmed in the same manner as (1) above. However, the NMR using acetone -d ₆ as a solvent.
The physicochemical properties of the compound SMTP-33 are shown below.

(3) Synthesis of SMTP-34 Pre-culture was performed in the same manner as (1) above.
The main culture was performed in the same manner as in the above (1) except that the organic amine compound added on the fourth day of the main culture was D-(+)-galactosamine.
From 300 ml of the obtained culture extract, cells were removed using a Buchner funnel to obtain a culture supernatant. From 300 ml of the obtained culture extract obtained by concentration using a rotary evaporator under reduced pressure by a water pump, the cells were removed using a Buchner funnel to obtain a culture supernatant. Concentration was performed using a rotary evaporator under reduced pressure by a water pump. MeOH was added thereto, and the dissolved fraction was pretreated with Lichrolut RP-18 (100 mg). Reverse phase HPLC was performed with a column; Inertsil PREP-ODS (diameter: 30 × 250 mm), temperature: 40 ° C., flow rate: 25 ml / min, detection wavelength: 260 nm, developing solvent: 75% methanol containing 50 mM ammonium acetate, retention time: 9. A 7 minute peak was collected. Methanol was distilled off using a rotary evaporator, and the mixture was extracted with ethyl acetate. The extract was dried over anhydrous sodium sulfate, concentrated and dried to obtain 283 mg of purified compound SMTP-34 per liter of culture solution. Yield.
The properties of the compound SMTP-34 were confirmed in the same manner as (1) above. However, the NMR using acetone -d ₆ as a solvent.
The physicochemical properties of the compound SMTP-34 are shown below.

(4) Synthesis of SMTP-35 Pre-culture was performed in the same manner as (1) above.
Main culture was carried out in the same manner as in the above (1) except that the organic amine compound added on the fourth day of main culture was DL-3-aminoisobutyric acid.
From 300 ml of the obtained culture extract, cells were removed using a Buchner funnel to obtain a culture supernatant. From 300 ml of the obtained culture extract obtained by concentration using a rotary evaporator under reduced pressure by a water pump, the cells were removed using a Buchner funnel to obtain a culture supernatant. Concentration was performed using a rotary evaporator under reduced pressure by a water pump. MeOH was added thereto, and the dissolved fraction was pretreated with Lichrolut RP-18 (100 mg). Reverse phase HPLC was performed with a column; Inertsil PREP-ODS (diameter: 30 × 250 mm), temperature: 40 ° C., flow rate: 25 ml / min, detection wavelength: 260 nm, developing solvent: 75% methanol containing 50 mM ammonium acetate, retention time: 9. A 6 minute peak was collected. Methanol was distilled off using a rotary evaporator, followed by extraction with ethyl acetate. The extract was dried over anhydrous sodium sulfate, and then concentrated and dried to obtain 35 mg of purified compound SMTP-35 per liter of culture solution. Yield.
The properties of the compound SMTP-35 were confirmed in the same manner as (1) above. However, the NMR using acetone -d ₆ as a solvent.
The physicochemical properties of the compound SMTP-35 are shown below.

(5) Synthesis of SMTP-38 Pre-culture was performed in the same manner as (1) above.
Main culture was performed in the same manner as in the above (1) except that 4-aminoantipyrine was used as the organic amine compound added on the fourth day of the main culture.
From 300 ml of the obtained culture extract, cells were removed using a Buchner funnel to obtain a culture supernatant. From 300 ml of the obtained culture extract obtained by concentration using a rotary evaporator under reduced pressure by a water pump, the cells were removed using a Buchner funnel to obtain a culture supernatant. Concentration was performed using a rotary evaporator under reduced pressure by a water pump. MeOH was added thereto, and the dissolved fraction was pretreated with Lichrolut RP-18 (100 mg). Reverse phase HPLC was carried out with a column; Inertsil PREP-ODS (diameter 30 × 250 mm), temperature 40 ° C., flow rate: 25 ml / min, detection wavelength: 260 nm, developing solvent: 75% methanol containing 50 mM ammonium acetate, retention time 17. A 6 minute peak was collected. Methanol was distilled off using a rotary evaporator, followed by extraction with ethyl acetate. The extract was dried over anhydrous sodium sulfate, and then concentrated and dried to obtain 458 mg of a purified compound SMTP-38 per liter of culture solution. Yield.
The properties of the compound SMTP-38 were confirmed in the same manner as (1) above. However, the NMR using acetone -d ₆ as a solvent.
The physicochemical properties of the compound SMTP-38 are shown below.

(6) Synthesis of SMTP-39 Pre-culture was performed in the same manner as (1) above.
The main culture was performed in the same manner as in the above (1) except that the organic amine compound added on the fourth day of the main culture was changed to 3,5-dinitrotyrosine.
From 300 ml of the obtained culture extract, cells were removed using a Buchner funnel to obtain a culture supernatant. From 300 ml of the obtained culture extract obtained by concentration using a rotary evaporator under reduced pressure by a water pump, the cells were removed using a Buchner funnel to obtain a culture supernatant. Concentration was performed using a rotary evaporator under reduced pressure by a water pump. MeOH was added thereto, and the dissolved fraction was pretreated with Lichrolut RP-18 (100 mg). Reversed phase HPLC was performed with a column; Inertsil PREP-ODS (diameter 30 × 250 mm), temperature 40 ° C., flow rate: 25 ml / min, detection wavelength: 260 nm, developing solvent: 75% methanol containing 50 mM ammonium acetate, retention time 24. A peak of 8 minutes was collected. Methanol was distilled off using a rotary evaporator, followed by extraction with ethyl acetate. The extract was dried over anhydrous sodium sulfate and then concentrated and dried to obtain 53 mg of purified compound SMTP-39 per liter of culture solution. Yield.
The properties of the compound SMTP-39 were confirmed in the same manner as (1) above. However, the NMR using acetone -d ₆ as a solvent.
The physicochemical properties of the compound SMTP-39 are shown below.

(6) Synthesis of SMTP-40 Pre-culture was performed in the same manner as (1) above.
The main culture was performed in the same manner as in the above (1) except that 4-amino-n-butyric acid was used as the organic amine compound added on the fourth day of the main culture.
From 300 ml of the obtained culture extract, cells were removed using a Buchner funnel to obtain a culture supernatant. From 300 ml of the obtained culture extract obtained by concentration using a rotary evaporator under reduced pressure by a water pump, the cells were removed using a Buchner funnel to obtain a culture supernatant. Concentration was performed using a rotary evaporator under reduced pressure by a water pump. MeOH was added thereto, and the dissolved fraction was pretreated with Lichrolut RP-18 (100 mg). Reverse phase HPLC was performed with a column; Inertsil PREP-ODS (diameter: 30 × 250 mm), temperature: 40 ° C., flow rate: 25 ml / min, detection wavelength: 260 nm, developing solvent: 75% methanol containing 50 mM ammonium acetate, retention time: 9. A 5 minute peak was collected. Methanol was distilled off using a rotary evaporator, followed by extraction with ethyl acetate. The extract was dried over anhydrous sodium sulfate, and then concentrated and dried to obtain 130 mg of purified compound SMTP-40 per liter of culture solution. Yield.
The properties of the compound SMTP-40 were confirmed in the same manner as (1) above. However, the NMR using acetone -d ₆ as a solvent.
The physicochemical properties of the compound SMTP-40 are shown below.

[Example 2]
<Antioxidant activity of SMTP compound>
The following ORAC (Oxygen Radical Absorbance Capacity) tests were conducted in order to examine the antioxidant activity of various triprenyl phenol compounds (hereinafter sometimes collectively referred to as “SMTP compounds”).
SMTP-16 was evaluated by modified H-ORAC (mH-ORAC), and SMTP-33, SMTP-34, SMTP-35, SMTP-38, SMTP-39 and SMTP-40 were evaluated by H-ORAC. . Orniprabin (SMTP-7) was used as a control SMTP compound. Each SMTP compound evaluated by H-ORAC was an aqueous sodium salt solution, and each compound evaluated by mH-ORAC was used as an acetone solution.

The test compound was appropriately diluted with a buffer (75 mM phosphate buffer pH 7.4) in H-ORAC and used for measurement. In mH-ORAC, an acetone solution was diluted with water to obtain a 50% (v / v) acetone solution, and then appropriately diluted with a 50% (v / v) acetone solution to prepare a solution 40 times the final concentration. This solution was diluted 10-fold with a buffer solution and used for measurement (acetone final concentration 1.25%). For the standard substance Trolox, 500 μM / buffer was appropriately diluted with buffer.
Add 50 μL of the test compound solution or Trolox solution to a 96-well microplate (clear, flat bottom, black wall), and then add 100 μL of fluorescent substance fluorescein 140 nM / buffer (final concentration 70 nM) and incubate at 37 ° C. for 10 minutes. did.
The free radical generator 2,2′-azobisamidinopropane dihydrochloride 48 mM / buffer (final concentration 12 mM) 50 μL was added, and the fluorescence intensity was measured every 2 minutes at an excitation wavelength of 485 nm and a fluorescence wavelength of 535 nm for 90 minutes. A calibration curve was prepared at a final Trolox concentration of 5-15 μM. The measurement was carried out at a concentration of n = 3, and the time was plotted on the horizontal axis, the fluorescence intensity was plotted on the vertical axis, and evaluation was performed using a value obtained by subtracting the area under the blank curve from the area under the curve of the sample. The results are shown as equivalent Trolox. The results are shown in Table 8.

  In addition, although it is a reference value, the ORAC value of (alpha) -tocopherol measured by the ORAC test similar to the above was 0.50 +/- 0.02 with respect to Trolox 1.00 (Huang et al.,). J. Agric. Food Chem., 50, 1815-1821 (2002)).

As is clear from Table 8, all of the SMTP compounds of the present invention exhibit a free radical scavenging ability equivalent to or higher than Trolox, and in particular, SMTP-35 has a higher free radical scavenging ability than SMTP-7. You can see that
Therefore, the SMTP compound of the present invention can be used as an antioxidant having high antioxidant activity.

[Example 3]
<Thrombolytic activity evaluation>
As for the various triprenyl phenol compounds obtained as described above, the thrombolytic activity was performed as follows as the activity to promote plasminogen activation by the urokinase (u-PA) catalyst, and the performance was evaluated.
As a comparative example, orniprabin (SMTP-7) was used. Each compound is as follows.

Utilizing that plasmin cleaves the peptide bond of the synthetic chromogenic substrate VLK-pNA (Val-Leu-Lys-p-nitroanilide) to produce p-nitroarinin (pNA), the yellow color absorbed at 405 nm of pNA By measuring the color development, the plasminogen activation promoting activity of the sample is measured. An MTP-500 type microplate reader (Corona Electric Co., Ltd.) was used as a measuring device, and measurement was performed with a 96-well round bottom microplate.
The measurement conditions were a kinetic measurement at 37 ° C. and a dual wavelength of 405 nm (activity) -595 nm (background), and measurement was performed 60 times per minute.
The purified sample was DMSO solution or an aqueous solution of sodium salt. It was diluted with TBS / T (50 mM Tris-HCl, 100 mM NaCl and 0.01% Tween 80, pH 7.4) to obtain a measurement sample. To 15 μl of sample, 35 μl of each reaction solution (prepared to have final concentrations of 0.1 mM VLK-pNA, 50 nM Glu-plasminogen, 50 U / ml u-PA by TBS / T) is added, and 50 μl is added. / Well, each concentration was measured in triplicate.
Moreover, it reacted using the reaction liquid which does not contain u-PA as a blank, and the value was subtracted from the value obtained by said reaction. Absorbance against the square of time was plotted, the slope was taken as the initial reaction rate, and comparison was made with no triprenylphenol compound added as a control, and the degree of activity of each compound was determined.

  The results are shown in Table 9. “10-fold accelerating activity concentration” represents a concentration at which 10-fold accelerating activity is obtained when the value when a reaction solution (control) containing no SMTP compound is used is 1. “Maximum promoting activity” represents the magnification at which the promotion of plasminogen activation by the SMTP compound is maximized, and the SMTP concentration at that time. In the table, NA indicates that the maximum acceleration does not reach 10 times and cannot be measured. Further, “−” in the table indicates that no promotion of plasminogen activation is observed, and thus the concentration of SMTP that gives the maximum promotion activity cannot be expressed.

  As shown in Table 9, it can be seen that none of the SMTP compounds of the present invention exhibits thrombolytic activity. From this, the triprenyl phenol compound according to the present invention can be used without thrombolysis.

  Therefore, according to the present invention, it is possible to provide a novel naturally-occurring triprenylphenol compound exhibiting antioxidant activity and an antioxidant containing the same.

Claims (2)

Triprenyl phenol compound represented by the following formula (I).

[Wherein X is —CHY—C (CH ₃ ) ₂ Z, and Y and Z are each —H or —OH, or together form a single bond. R ¹ is the following substituent, and * represents a bonding position with a nitrogen atom. ]
  The antioxidant containing the triprenyl phenol compound of Claim 1.