JP5013886B2 - Novel longicyclene derivative and tyrosinase activity inhibitor containing the derivative - Google Patents

Description

  The present invention relates to a novel longicyclen derivative and a tyrosinase activity inhibitor containing the longicyclene derivative as an active ingredient.

  Longicyclen is a kind of terpenoid compound belonging to sesquiterpenes having a longifolene skeleton, and has antifungal, antiparasitic, and tonic effects. Naturally, longicyclen is known to be contained in essential oils of roots of Cryptomeria Japonica D. DON (cedar) and Chamaecyparis obtusa (Sieb. Et Zucc.) Endl. Shieh, Y. Iizuka and Y. Matsubara (1981), Agric. Biol. Chem., 6, 1493-1495, B. Shieh, YIizuka and Y. Matsubara (1981), Agric. Biol. Chem., 6, 1497 -1499).

  It is also known that terpenoid compounds can be converted to terpenoid derivatives by microbial conversion. Microbial conversion is a biological synthesis process that uses enzymes in the living body as biocatalysts to obtain the target compound, and is characterized by producing compounds in a site-specific manner under mild conditions. To do. Microbial conversion is therefore an advantageous method for the selective production of bioactive compounds.

  For example, Aspergillus niger which is a kind of fungus is (+)-Fencon (M. Miyazawa, K. Yamamoto, Y. Noma and H. Kameo, (1990), Chemistry Express, 5, 237-430), 1, 4 -Cineol (M.Miyazawa, Y.Noma, K.Yamamoto and H.Kameoka, (1992), Chemistry Express, 7, 305-308), (-)-α-bisabolol (M.Miyazawa, Y.Funatsu and H Kameoka, (1992), Chemistry Express, 7,573-576), (−)-cis-rose oxide (M. Miyazawa, K. Yokote and H. Kameoka (1995), Phytochemistry, 39, 85-89), (+) -Stereoselective oxidation of various natural terpenoids such as trans-rose oxide (M. Miyazawa, K. Yokote and H. Kameoka (1995), Phytochemistry, 39, 85-89), and conversion to terpenoid derivatives It has been known.

  An object of the present invention is to provide a novel longicyclen derivative, particularly a longicyclen derivative having an inhibitory action on tyrosinase activity. Another object of the present invention is to provide a tyrosinase activity inhibitor containing the longicyclene derivative as an active ingredient.

  For the purpose of creating a novel physiologically active substance, the present inventors have microbially converted various natural products with various microorganisms and searched for metabolites thereof. As a result, microorganisms belonging to the genus Aspergillus using Longgicyclen as a substrate. It was found that a substance having a strong tyrosinase activity inhibitory activity was produced in this culture. As a result of examining in detail the tyrosinase activity inhibitor produced in the culture of microorganisms belonging to the genus Aspergillus using Longgicyclen as a substrate, it was confirmed that it was a series of compounds having a Longgicyclen skeleton, and their isolation, Successfully purified.

〔一般名：
化合物〔ＩＩ〕：（−）−（１０Ｒ）−１０−ヒドロキシ−ロンギシクレン酸
化合物〔ＩＩＩ〕：（＋）−（１０Ｓ）−１０−ヒドロキシ−ロンギシクレン酸
化合物〔ＩＶ〕：（＋）−１０−オキソ−ロンギシクレン酸〕
により表される遊離形または塩形のロンギシクレン誘導体を提供する。 Thus, in a first aspect, the present invention provides a formula

〔common name:
Compound [II]: (−)-(10R) -10-hydroxy-longiciclenic acid compound [III]: (+)-(10S) -10-hydroxy-longiciclenic acid compound [IV]: (+)-10-oxo -Longiciclenic acid]
A longicyclene derivative represented by a free form or a salt form is provided.

  In another aspect, the present invention provides a tyrosinase activity inhibitor containing the longicyclene derivative as an active ingredient.

のいずれかで表されるロンギシクレン誘導体の製造方法であって、
（１）ロンギシクレンを基質として、当該ロンギシクレン誘導体を生産する能力を有するアスペルギルス属に属する微生物によりロンギシクレンを処理する工程、そして
（２）当該誘導体を採取する工程
を含むことを特徴とする方法を提供する。 In yet another aspect, the present invention provides a compound of formula

A method for producing a longicyclene derivative represented by any of the following:
(1) A method comprising treating longicyclen with a microorganism belonging to the genus Aspergillus having the ability to produce the longicyclen derivative using longicyclen as a substrate; and (2) collecting the derivative. .

Effects of the present invention

  Surprisingly, the longicyclene derivative obtained in the present invention exhibits a remarkable activity inhibitory action on tyrosinase. Therefore, the longicyclene derivative of the present invention is extremely useful as a therapeutic agent for pigment diseases such as vitiligo and melanosis, a raw material for whitening cosmetics, a browning inhibitor for foods, and a pest control agent.

  Tyrosinase is an enzyme widely distributed in animals, plants, and microorganisms, and is an enzyme that catalyzes the synthesis of melanin pigment that is a causative substance of spots and buckwheat. Therefore, tyrosinase inhibitors are extremely useful as therapeutic agents for pigment diseases such as vitiligo and melanosis, raw materials for whitening cosmetics, browning inhibitors for foods, pest control agents and the like. Conventionally, hydroquinones, ascorbic acids, glutathione, kojic acid, arbutin and the like are known as compounds having tyrosinase inhibitory activity. However, these compounds have drawbacks such as low stability over time, low black prevention, strong cytotoxicity, and decreased activity due to oxidation in the presence of moisture (for example, JP-A-60-56912 and JP-A-2001). No. 316268), a compound having an inhibitory action on tyrosinase activity without such inconvenience has been demanded.

Definitions of Terms Terms used herein have the meanings commonly understood by those of ordinary skill in the art unless otherwise indicated.

Method for Producing Longicyclen Derivative To produce a longicyclen derivative which is the target substance of the present invention, longicyclen is used as a substrate, and longicyclen is treated with a longicyclen derivative-producing bacterium belonging to the genus Aspergillus, which is a fungus, or its in vivo enzyme, A longicyclen derivative may be collected. The term “treatment” as used herein is meant to include conventional microbial conversion means such as contact between longicyclen and bacterial cells, culture in which longicyclen is contained in the culture medium of the bacterial cells, and fermentation. "Means a process involving conventional separation, extraction and purification means.

  An example of an Aspergillus genus microorganism used for the production of a longicyclen derivative is Aspergillus niger, which has been deposited as NBRC 4414 with the National Institute of Technology and Evaluation.

  In vivo enzymes mean any enzyme that is identified as an enzyme that the bacterium uses in the reaction process, and the bacterium itself is used by placing it under appropriate conditions, for example, within the cells. The reaction can proceed as in the case.

  As a medium used for culturing a longicyclen derivative-producing bacterium, a conventional medium suitable for growing the bacterium to be used or desired microbial conversion can be appropriately selected and includes a nutrient source that can be used by the bacterium. It may be liquid or solid, but it is preferable to use a liquid medium in order to obtain a large amount of longicyclene derivatives.

  In this medium, substances necessary for culturing the bacterium, for example, a carbon source that can be assimilated by the bacterium, a nitrogen source that can be digested, an inorganic substance, and the like are appropriately blended. As the carbon source, glucose, sucrose, maltose, lactose, starch and the like are used, and as the nitrogen source, yeast extract, meat extract, peptone, polypeptone, malto extract, sodium nitrate and the like are used. The medium may contain salts such as sodium, potassium, calcium, magnesium and the like.

  The pH and temperature conditions of the medium can be used under any conditions that are suitable for the growth of the bacteria to be used. Such conditions are known, or those skilled in the art can appropriately use the conventional means. Can be set. For example, when the bacterium is Aspergillus niger, the initial pH is about 3 to 4, preferably 3.5, and the culture temperature is about 15 to about 35 ° C., preferably 25 to 30 ° C. .

  Since longicyclen derivative-producing bacteria have the property of growing by attaching to a stationary object, stationary culture is desirable. Also, the growth of an object that adheres to the microorganism, such as aluminum foil, is promoted by placing it in a liquid medium. Is done. Although the culture period is not constant, it is desirable to culture until the concentration of the longicyclen derivative to be produced is maximized. The number of days required for this is usually about 10 days in the case of stationary culture using a liquid medium.

  The obtained culture solution is separated into cells and culture solution by means such as centrifugation or filtration. Both of these have an inhibitory effect on tyrosinase activity, but a longicyclene derivative is contained in a large amount in the culture solution. In order to extract longicyclene derivatives from the collected culture broth, after making a saturated solution such as sodium chloride, an organic solvent miscible with water, for example, lower alcohols such as methanol and ethanol, acetone, methyl ethyl ketone and the like may be used. . Further, an organic solvent immiscible with water, such as chloroform, methylene chloride, ethyl acetate, etc. may be used.

  If the solvent is distilled off from the extract thus obtained under reduced pressure, a crude extract containing a longicyclene derivative can be obtained.

  In order to isolate and purify the longicyclene derivative from this crude extract, the usual means for isolating and purifying fat-soluble low-molecular substances can be applied. Specifically, gel filtration chromatography using Sephadex LH-20 (manufactured by Pharmacia, registered trademark), adsorption chromatography using an adsorbent such as silica gel, high performance liquid chromatography using a normal phase carrier such as silica gel, etc. Or they may be implemented in combination.

  When Sephadex LH-20 is used, it is generally eluted with a combination of a polar organic solvent and a nonpolar organic solvent, for example, a mixed solvent such as methanol and chloroform or methylene chloride.

  When adsorption chromatography using silica gel is used, it is suitable to use a mixed solvent such as hexane and chloroform, ethyl acetate or methylene chloride as an elution solvent.

  In the case of high performance liquid chromatography using silica gel as a carrier, a mixed solvent of methylene chloride and benzene or methanol or methylene chloride alone is used as an elution solvent. By applying such purification means, one or more of the longicyclene derivatives shown above are isolated.

Tyrosinase activity inhibitor The longicyclen derivative obtained by the present invention, especially the above-mentioned compounds [II] to [IV], show a remarkable activity suppressing action on tyrosinase. The activity can be easily confirmed by an appropriate in vitro or in vivo test, for example, the test described in Example 2. The longicyclene derivative of the present invention preferably has a tyrosinase activity of 10% or more, preferably 20%, when 0.5 mM of the compound is added to a tyrosinase (Sigma) solution (528 units / mL) and incubated at 37 ° C. for 60 minutes. As mentioned above, it can inhibit 30% or more especially preferably.

  Therefore, the longicyclene derivative of the present invention can be used as a tyrosinase activity inhibitor. Preferably, the longicyclene derivative of the present invention appropriately uses conventional auxiliaries such as excipients, pH adjusters, fragrances, surfactants, chelating agents, thickeners, preservatives and the like by conventional formulation means. It can be prepared and used in any dosage form such as liquid, emulsion, powder, granule, tablet, lotion, ointment, injection solution, cream and the like. For example, when the longicyclen derivative of the present invention is formulated as a cosmetic, a dosage form such as lotion, emulsion, cream or the like is preferable.

  In addition, the tyrosinase activity inhibitor containing the longicyclene derivative of the present invention can be administered in any form, for example, oral, enteral, parenteral, topical or transdermal, when intended for administration to animals. When it is intended to be blended in food, it can be blended in food by means of conventional mixing or the like.

Experimental technique NMR was measured using 500 MHz ( ¹ H) and 125 MHz ( ¹³ C) with TMS as an internal standard and CDCl ₃ .
GC is a HP 5890A gas chromatograph (manufactured by Hewlett-Packard) equipped with a hydrogen flame ionization detector, a capillary column (DB-5, manufactured by Shimadzu Corporation: length 30 m × inner diameter 0.25 mm), and a 20: 1 split injection unit. It was used. As the mobile phase, helium gas was used at a flow rate of 1 mL / min. The oven temperature was programmed from 90 ° C. to 230 ° C. with a heating rate of 4 ° C./min. The inlet temperature was set to 270 ° C., and the detector temperature was set to 280 ° C. The peak area was calculated using an HP 3396 Series 2 detector (manufactured by Hewlett-Packard).

  GC / MS is a gas chromatograph (HP 5890A, manufactured by Hewlett Packard) equipped with a split injection unit, capillary column (HP-5MS, manufactured by Hewlett Packard: length 30 m × inner diameter 0.25 mm), and mass spectrometer (HP 5972A). , Made by Hewlett-Packard). The temperature raising program is the same as GC. As the mobile phase, helium gas was used at a flow rate of 1 mL / min. The ion source temperature was 280 ° C., and the electron energy was 70 electron volts (eV). As the ionization method, an electron impact method (EI) was used.

IR spectra were obtained with a Perkin-Elmer 1760X spectrometer.
TLC used was a TLC plate (Merck: layer thickness 0.25 mm) coated with silica gel 60 F254 using CHCl ₃ as a developing solvent.
The developing solvent for silica gel column chromatography was a hexane-ethyl acetate system.

Aspergillus niger pre-culture Aspergillus niger (NBRC 4414) spores stored at low temperature were sterilized in a culture medium (1.5% sucrose, 1.5% glucose, 0.5% polypeptone, magnesium sulfate (septahydrate). ) Shake flask containing 0.05%, potassium chloride 0.05%, dipotassium phosphate 0.1%, ferrous sulfate (septahydrate) 0.001%, and distilled water, pH 7.2) Planted in and cultured at 27 ° C. for 1 day.

Aspergillus niger cultured before aging was transplanted to a culture medium (20 mL in a 50 mL Petri dish) and cultured for 1 day (until mycelia accounted for 60-80% of the surface area of the culture medium). After Aspergillus niger grew, 60 mg of (+)-longicycrene [I] (manufactured by Fluka) was added to the medium, and the culture was continued at 27 ° C. for 10 days. As a result, it was converted into compounds [II] to [IV]. Then, it was saturated with sodium chloride and extracted with ethyl acetate. The extract was analyzed by thin layer chromatograph (TLC), gas chromatography (GC) and gas chromatography mass spectrometry (GC / MS). Compounds [II] to [IV] were not detected by TLC, GC and GC-MS analysis of Aspergillus niger culture medium to which no substrate was added. The time course of metabolites is shown in FIG. The ratio between [I] and compounds [II], [III], [IV] was determined based on the peak areas of GC and GC / MS (FIG. 1). After 10 days, about 80% of (+)-longisiclen was metabolized. After 10 days of conversion, Compound [II] was 27%, Compound [III] was 23%, and Compound [IV] was 30%.

In order to clarify the metabolic pathway of microbial conversion by Aspergillus niger, small amounts of [II], [III] and [IV] were cultured with Aspergillus niger at 27 ° C. for 10 days. [II] and [III] were microbially converted to [IV] by Aspergillus niger as detected by TLC, GC and GC / MS methods. And [IV] was not metabolized (FIGS. 3 and 4). In this way, the metabolic pathway of [I] microbial conversion by Aspergillus niger was derived from the time course (FIG. 2) and the structure of the metabolite (FIG. 1).
In FIG. 2, □: compound [I], ▲: compound [II], ●: compound [III], ▼: compound [IV].

Aspergillus niger precultured in (+)-longisiclen for 10 days in microorganism conversion Example 1 was transplanted to 1800 mL of the same medium as in Example 1, and stirred and cultured at 27 ° C. under aeration conditions for 1 day. After Aspergillus niger grew, 600 mg of (+)-longisiclen was added to the medium, and the culture was continued for 10 days.

Isolation of metabolites After 10 days of microbial conversion, the media and mycelium were separated by filtration. The medium was saturated with sodium chloride and extracted with ethyl acetate. Furthermore, the mycelium was extracted with ethyl acetate. The ethyl acetate extract was mixed and dehydrated with sodium sulfate, and then the solvent was distilled off under reduced pressure to obtain 752 mg of a crude extract. The extract was subjected to column chromatography using a hexane-ethyl acetate mixture and 300 mesh silica gel. 150 mg of unreacted (+)-longicyclene was recovered. Compound [II] 36 mg, [III] 28 mg, and [IV] 83 mg were isolated.

Compound [II]
The structure of compound [II], (−)-(10R) -10-hydroxy-longiciclenic acid was determined from the following MS, IR and NMR data. In [II] high resolution fast atom bombardment mass spectrometry (HR-FABMS (POS.)), The m / z value was 251.1646, and the molecular formula was C ₁₅ H ₂₃ O ₃ . In the IR and ¹³ C-NMR spectrum of [II], hydroxyl groups (IR: 3432 cm ⁻¹ , δ _C 79.8 ppm (CH)) and carboxyl groups (IR: 1683 cm ⁻¹ , δ _C 178.5 ppm (C)) Existence was shown. ¹ H- and ¹³ C-NMR signals were similar to the substrate except for the presence of a new methine group and aprotic carbon. Further, the signals of methylene group and methyl group disappeared. Regarding ¹ H-NMR, the presence of three methyl groups was indicated. This was confirmed by assignment of two-dimensional NMR (COSY (correlation spectroscopy), HMQC (heteronuclear multiple quantum correlation), and HMBC (heteronuclear multiple bond correlation)). The characteristic HMBC spectral correlation has two methyl protons (δ _H 1.01, 1.08 ppm; C-14, C-15) and a new methine carbon (δ _C 79.8 ppm; C-10). Two methine protons (δ _H 1.78, 2.11 ppm; C-) having a methine proton (δ _H 1.43; C-1), a novel aprotic carbon (δ _C 178.5 ppm; C-13). 4, C-2) was observed. From this, it is considered that [II] was produced by hydroxylating the 10th carbon of [I] and carboxylating the 12th carbon. Furthermore, the absolute configuration of [II] was determined by the new Mosher method (I. Ohtani, T. Kusumi (1991), J. Am. Chem. Soc., 113, 4092-4096). That is, [II] was converted to (+)-(−)-α-methoxy-α- (trifluoromethyl) phenylacetic acid (MTPA-Cl) in dichloromethane and N, N-dimethyl-4-aminopyridine (DMAP). To give (+)-MTPA ester and (−)-MTPA ester, respectively. The Δδ value [δ (−) − δ (+)] indicated that the absolute configuration of carbon at the 10-position was R-form. Specific rotation showed (-)-body. From these data, the structure of [II] was determined to be a novel compound (-)-(10R) -10-hydroxy-longiciclenic acid.

Compound [III]
The second metabolite, compound [III], (+)-(10S) -10-hydroxy-longiciclenic acid, has a molecular formula of C ₁₅ H ₂₂ O ₃ , m / by high resolution electron impact mass spectrometry (HR-EIMS). The z value was 250.1580. In the IR and ¹³ C-NMR spectrum of [III], the hydroxyl group (IR: 3430 cm ⁻¹ , δ _C 76.7 ppm (CH)) and the carboxyl group (IR: 1682 cm ⁻¹ , δ _C 178.7 ppm (C)) Existence was shown. ¹ H- and ¹³ C-NMR signals were similar to the substrate except for the presence of a new methine group and aprotic carbon. Further, the signals of methylene group and methyl group disappeared. Regarding ¹ H-NMR, the presence of three methyl groups was indicated. This was confirmed by assignment of two-dimensional NMR (COSY, HMQC, and HMBC). The characteristic HMBC spectral correlation has two methyl protons (δ _H 0.95, 1.08 ppm; C-14, C-15) and a new methine carbon (δ _C 79.7 ppm; C-10). Two methine protons (δ _H 2.05, 1.) having a methine proton (δ _H 1.41-1.44; C-1), a novel aprotic carbon (δ _C 178.7 ppm; C-13). 74-1.77 ppm; C-4, C-2) was observed. From this, it is considered that [III] was produced by hydroxylating the 10th carbon of [I] and carboxylating the 12th carbon. Furthermore, it was estimated by NOE analysis that the absolute configuration of [III] is S-form. When the resonance of the α-methyl group at position 15 in [II] was saturated by irradiation with a decoupling pulse, an increase in the signal intensity of hydrogen at position 10 was observed. However, NOE analysis between the β-methyl group at the 14-position and the hydrogen at the 10-position showed that the hydroxyl group at the 10-position carbon is in the α-position. Specific rotation showed (+)-body. From these data, the structure of [III] was determined to be a novel compound (+)-(10S) -10-hydroxy-longiciclenic acid.

Compound [IV]
The third metabolite, compound [IV], (+)-10-oxo-longicyrene, had a molecular formula of C ₁₅ H ₂₀ O ₃ and an m / z value of 248.1416 according to HR-EIMS. In the IR and ¹³ C-NMR spectrum of [IV], a carbonyl group (IR: 1674 cm ⁻¹ , δ _C 215.9 ppm (C)) and a carboxyl group (IR: 1698 cm ⁻¹ , δ _C 179.2 ppm (C)) The presence of was shown. ¹ H- and ¹³ C-NMR signals were similar to the substrate except for the presence of a new methine group and aprotic carbon. Further, the signals of methylene group and methyl group disappeared. Regarding ¹ H-NMR, the presence of three methyl groups was indicated. This was confirmed by assignment of two-dimensional NMR (COSY, HMQC, and HMBC). The characteristic HMBC spectral correlation shows two methyl protons (δ _H 1.15, 1.19 ppm; C-14, C-15) and a new aprotic carbon (δ _C 215.9 ppm; C-10). Two methine protons (δ _H 1.99, 2 having a methine proton (δ _H 1.57 -1.61; C-1), a novel aprotic carbon (δ _C 179.2 ppm; C-13). 20-2.21 ppm; C-4, C-2) was observed. From this, it is considered that [IV] was produced by oxidation of the 10th carbon of [I] and carboxylation of the 12th carbon. Specific rotation showed (+)-body. From these data, the structure of [IV] was determined to be a novel compound (+)-10-oxo-longiciclenic acid.

Compound [II]: colorless crystals, [α] _D ^21.5 -1.7 (CHCl ₃ ; c = 0.4), HR-FABMS (POS.): M / z 251.1646 [M + H] ⁺ , molecular formula C ₁₅ H ₂₃ O ₃ , EIMS m / z: [M] ⁺ 250 (5), 232 (15), 204 (12), 162 (48), 108 (59), 91 (72), 69 (100), 41 (86), IR (KBr method) ν _max cm ⁻¹ : 3407, 2962, 2930, 1677, 1434, ¹ H and ¹³ C-NMR are shown in Tables 1 and 2.

Compound [III]: colorless crystals, [α] _D ^21.5 +1.6 (CHCl ₃ ; c = 0.4), HR-EIMS: m / z 250.1580 [M + H] ⁺ , molecular formula C ₁₅ H ₂₂ O ₃ , EIMS m / z: [M] ⁺ 250 (4), 232 (15), 204 (14), 143 (55), 105 (68), 91 (100), 69 (94), 41 (99), IR (KBr method ) ν _max cm ⁻¹ : 3420, 2960, 2923, 1682, 1456, ¹ H and ¹³ C-NMR are shown in Tables 1 and 2.

Compound [IV]: colorless crystals, [α] _D ^21.5 +22.3 (CHCl ₃ ; c = 0.5), HR-EIMS: m / z 248.1416 [M + H] ⁺ , molecular formula C ₁₅ H ₂₀ O ₃ , EIMS m / z: [M] ⁺ 248 (51), 230 (34), 202 (16), 123 (69), 107 (77), 93 (100), 69 (94), 41 (90), IR (KBr method ) ν _max cm ⁻¹ : 2965, 2926, 1698, 1674, ¹ H and ¹³ C-NMR are shown in Tables 1 and 2.

Tyrosinase inhibitory activity Tyrosinase activity using L-tyrosine and L-dopa as substrates was measured spectrophotometrically. 680 μL of 0.1 M phosphate buffer (pH 6.8), 80 μL of 20% polyoxyethylene-nonyl-phenyl ether, 100 mL of 0.03% L-tyrosine or L-dopa, and 40 μL of compound [II] (DMSO solution) In addition to the test tube, it was incubated at 37 ° C. for 10 minutes. Thereafter, tyrosinase (Sigma) solution (528 units / mL, dissolved in 0.1 M phosphate buffer) was added and incubated at 37 ° C. for 60 minutes. The enzyme activity was determined by measuring the change in absorbance (475 nm) of melanin synthesized by tyrosinase. Arbutin was used as a comparative control substance.

Compounds [III] and [IV] were similarly tested. Tyrosinase activity is expressed by the following formula tyrosinase activity (%) = [(A−B) / (Cp−Cn)] × 100
[In this formula, A is the absorbance of the test substance (0.1 M phosphate buffer, polyoxyethylene-nonyl-phenyl ether, L-tyrosine or L-dopa, compound solution, and tyrosinase), B is blank (0. Absorbance of 1M phosphate buffer, polyoxyethylene-nonyl-phenyl ether, distilled water, compound solution, and tyrosinase), Cp is positive control (0.1M phosphate buffer, polyoxyethylene-nonyl-phenyl ether, L Absorbance of tyrosine or L-dopa, DMSO, and tyrosinase), Cn indicates the absorbance of negative controls (0.1 M phosphate buffer, polyoxyethylene-nonyl-phenyl ether, distilled water, and tyrosinase). ]
Is obtained. The results are shown in Table 3 and FIG.

図５中、◆：ロンギシクレン、■：化合物［ＩＩ］、▲：化合物［ＩＩＩ］、×：化合物［ＩＶ］、◇：アルブチンである。

In FIG. 5, ♦: longicyclene, ■: compound [II], ▲: compound [III], x: compound [IV], ◇: arbutin.

  Therefore, the compounds [II] to [IV] have significantly more tyrosinase inhibitory activity than longicyclen and arbutin.

(+)-Longicyclen [I] shows the change in structure when microbially converted by Aspergillus niger. The time course of the relative abundance of (+)-longisiclene [I] and each of compounds [II] to [IV] when (+)-longicyclen [I] is microbially converted by Aspergillus niger is shown. The microbial conversion of compound [II] into compound [IV] by Aspergillus niger and its change with time are shown. The microbial conversion of compound [III] to compound [IV] by Aspergillus niger and its change with time are shown. (+)-Longycyclene [I], compounds [II] to [IV], and arbutin (control) show tyrosinase inhibitory activity.

Claims (5)

formula

A longicyclene derivative in free or salt form. formula

A longicyclene derivative in free or salt form. formula

A longicyclene derivative in free or salt form.   A tyrosinase activity inhibitor containing the longicyclene derivative according to any one of claims 1 to 3 as an active ingredient.

A method for producing a longicyclene derivative represented by any of the following:
(1) A method comprising treating Longicyclen with a microorganism belonging to the genus Aspergillus having the ability to produce the Longicyclen derivative using Longicyclen as a substrate, and (2) collecting the derivative.

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