KR20050067807A - Preparation method of Ultraviolet-A (UV-A) protecting terreusinol from the biotransformation of terreusinone by the Marine-derived Streptomyces sp. - Google Patents

Abstract

The present invention uses a bioconversion technology by marine actinomycetes The present invention relates to a method for preparing a novel compound tereusinol having excellent UV A blocking ability, and is isolated from Streptomyces sp. From marine plant Zostera marina and using this strain through bioconversion technology from teriusinone. There is an excellent effect of providing the novel compound tereusinol having excellent ultraviolet A blocking ability. Therefore, tereusinol, which is a novel compound of the present invention, has an effect of having an excellent UV-A blocking activity than oxybenzone, which is currently generally used as a sunscreen.

Description

Preparation method of Ultraviolet-A (UV-A) protecting terreusinol from the biotransformation of terreusinone by the Marine-derived Streptomyces sp.}

The present invention uses a bioconversion technology by marine actinomycetes The present invention relates to a method for preparing a novel compound tereusinol having excellent ultraviolet A blocking ability. More specifically, Streptomyces sp. Isolated from marine plant Zostera marina are isolated and identified, and a novel compound tereusinol having excellent UV A blocking ability from terreusinone by a bioconversion technique using this strain. The present invention relates to providing a method for obtaining tereusinol by preparing and preparing its molecular structure and physicochemical properties.

Ultraviolet light is an invisible light that appears outside the short wavelength of visible light and can cause skin cancer, erythema, and eye damage in the human body. The incidence of these diseases and other adverse effects on flora and fauna is expected to increase with increasing levels of ultraviolet light in sunlight. Long-term repeated exposure to solar ultraviolet radiation, particularly UV-B, is associated with the risk of non-melanoma skin cancers (NMSC) in sensitive whites, while strong and intermittent UV-B Exposure to cancer can be a very dangerous factor for malignant melanoma (MM). Several countries have reported increasing skin cancer in recent years. This trend can be thought to be primarily due to increased personal exposure.

Marine microorganisms have been demonstrated to be a rich source of structurally novel and biologically active natural materials for the development of fine chemicals (Faulkner, DJ Nat. Prod. Rep . 2002, 19, 1-47; Pietra, F. Nat. Prod. Rep . 1997, 14, 453-464).

The present inventors searched for UV-A blocking components in the material produced from the bacteria isolated from the marine host, and found that crude extracts of the seaweed engraftment Aspergillus sp. Have the appropriate UV-A blocking activity. And filed a patent.

Selectivity, on the other hand, is an essential requirement in organic synthetic chemistry. Even in complex or symmetric molecules without protecting groups, the regioselectivity of enzymes is a potent biocatalysis. As a result of the application of biocatalysts to natural marine materials, they are robust for the production of new, active and weakly toxic derivatives for the optimization of lead compounds and the establishment of SAR and structure-activity relationships (SAR). It became a tool.

In view of this, the present inventor intends to apply a biocatalyst to a natural material separated from marine plants, thereby separating actinomycetes from marine plants and inducing local selective oxidation of tereusinone using bioconversion technology from the strain. The inventors of the present invention have attempted to obtain a novel compound that is far superior to UV A blocking ability than the blocking agent.

Accordingly, an object of the present invention is to identify and identify marine actinomycetes in marine plant Malzalpi ( Zostera marina ), and UV-A blocking ability from tereusinone isolated from Aspergillus through bioconversion using the microbial catalytic ability of the strain. It is intended to provide novel compounds that are superior to others.

The object of the present invention is to isolate and identify marine actinomycetes from marine plant Malzalpi, and to separate and purified tereusinol through a two-step fermentation process by adding tereusinone to the strain culture medium and the molecular structure and physicochemical properties of the material. It was achieved by clarifying and comparatively examining the ultraviolet ray A blocking ability.

Hereinafter, the configuration and operation of the present invention.

The present invention is to identify and isolate marine actinomycetes from marine plant Malzalpi ( Zostera marina ); A two-step fermentation process step of adding tereusinone to the strain culture solution; Molecular structure and physicochemical characterization of tereusinol obtained from the above; And a comparative investigation of ultraviolet A blocking ability of the compounds.

The present invention is a terresinone represented by the following structural formula (I) isolated from the seaweed larvae Aspergillus terreus strain through a biotransformation technique using Streptomyces sp. MFAac18 KACC 91085 strain It provides a novel Terreusinol (2), which is excellent in UV A blocking ability prepared in (Terreusinone, 1).

... (I)

Terreusinone (1) isolated from Aspergillus terreus MFA460 KACC 93010 strain used in the present invention is a dimer having a chiral symmetric structure, and is a microbial catalyst as dipyrroloquinone having ultraviolet A blocking ability. It is used as a substrate for the preparation of tereusinol, a novel compound having excellent UV A blocking ability through oxidation according to a two-stage fermentation process.

The novel method for preparing terecinin with excellent UV A blocking ability is characterized by comprising the following steps:

Streptomyces sp. MFAac18 KACC 91085 strain isolated from marine plant Zostera marina was subjected to stationary culture in SWS medium (consisting of 0.1 parts by weight, 1.0 part by weight of soluble starch and 100 parts by weight of seawater),

The culture was added to the culture solution by adding tereusinone,

Ethyl acetate extracted from the culture solution obtained above to obtain an organic layer, which was dried, filtered and concentrated under reduced pressure to obtain a viscous brown extract,

The extract was subjected to silica gel column chromatography using a solvent of n-hexane: ethyl acetate (100: 0 to 0: 100) to obtain reversed phase HPLC (YMC ODS-A) using methanol as a solvent. Carried out to obtain red teriusinol.

The present invention measured and corrected the melting point on an Electrothermal model IA 9100 micro-melting point instrument.

Concentration was measured on a Perkin Elmer model 341 polarimeter.

IR spectra were measured with a Bruker FT-IR model IFS-88 spectrophotometer.

Hereinafter, the configuration of the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to the following Examples.

EXAMPLE

Example 1 Marine Plant Malzalpi Zostera marina Isolation and Identification of Marine Actinomycetes

Through bioconversion technology using microbial catalytic capacity, microorganisms isolated from marine plants Malzalpi were identified to obtain new substances with UV A blocking ability.

For this purpose, YPG agar consisting of 0.5% yeast extract, 0.5% peptone, 1% glucose, 1.6% agar, 40% deionized water and 60% seawater from Zostera marina , a marine plant collected from Bijindo, Tongyeong, South Gyeongsang Province Actinomycetes MFAac18 were isolated from the medium in which penicillin and streptomycin were added at 250 μg / mL, respectively.

The culture was identified by fatty acid composition analysis as an off-white vegetable mycelium, showing typical growth characteristics of actinomycetes belonging to the genus Streptomyces. Therefore, the present inventor named the strain Streptomyces sp. MFAac18, and deposited on November 18, 2003 to the Center for Agricultural Microorganisms (KACC) was given the number of KACC 91085.

Example 2 Structure and Physicochemical Characterization of Novel Metabolites Obtained by Bioconversion of Tereusinone of the Invention

A two-step fermentation protocol from tereusinone using the strain isolated in Example 1 (Orabi, KY et al, J. Nat. Prod. , 63, 396-398 (2000); Smith RV et al., J. According to Pharm. Sci. , 64, 1737-1759 (1975), bioconversion secondary metabolites of tereusinone were prepared.

First, SWS medium containing 0.1 parts by weight of exhaustion, 1.0 parts by weight of soluble starch and 100 parts by weight of seawater was prepared, and then autoclave at 121 ° C. for 15 minutes. Streptomyces strain obtained in Example 1 was pre-cultured for 1 week in a culture flask for 3 L containing 1 L of sterile medium at 29 ℃ (political culture). Two-step culture was performed using a one-step culture medium incubated for one week. The culture was incubated for 24 hours prior to the addition of tereusinone, followed by incubation at 29 ° C. for 5 weeks by adding 0.75 mL of dimethyl formamide (DMF) in which 20 mg of tereusinone was dissolved (political culture).

Tereusinone used in the above Aspergillus terreus ( Aspergillus terreus ) MFA 460 KACC 93010 strain incubated for 30 days at 29 ℃ using SWS medium, to obtain the ethyl acetate extract in the final culture silica gel flash chromatography (The solution mixed with ethyl acetate and n-hexane was used as a solvent.) Tereusinone purified by HPLC (using a solvent mixed with methanol: water = 5: 1) was used.

In addition, sterile medium was used as a substrate control, and microorganisms were removed and cultured in sterile medium under the same conditions as described above. In addition, the culture control subtracted the substrate during fermentation to grow microorganisms under the same conditions. After 5 weeks of culture, each control was harvested and analyzed by TLC. The culture was filtered through a cotton cloth and the filtrate was extracted with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfide (anhydrous Na ₂ SO ₄ ), filtered through a silicide glass, and concentrated under reduced pressure to give a viscous brown extract (75 mg).

The brown extract (75 mg) was passed through silica gel flash column chromatography, and the elution of the n-hexane-ethyl acetate (100: 0 to 0: 100) solvent was performed serially to dissolve the substrate (tereusinone) and the metabolite (tere). Fractions A and B, respectively, comprising (usinol). Each fraction was separated and purified by reverse phase YMC ODS-A gel flash column chromatography using methanol, and then purified by HPLC (ODS-A, methanol) to obtain 10 mg of substrate and 5 mg of metabolite, respectively.

¹ H (400 MHz) and ¹³ C NMR (100 MHz) spectra were obtained on a JEOL JNM-ECP 400 NMR spectrophotometer using TMS or solvent peaks as standard. MS spectra were obtained on a JEOL JMS-700 NMR spectrophotometer. UV / Visible spectra were measured on a Hitachi U-2001 UV / Vis spectrophotometer. CD spectra were obtained on a JASCO J-715 spectropolarimeter.

Tereusinol is a red solid and the NMR spectral results are shown in Table 1:

[α] +33.8 ^o ( c 0.6, MeOH); IR (KBr) ν _max 3403, 3200, 1626, 1467, 1162, 1056, 990, 837, 744 cm ⁻¹ ; UV (MeOH) λ _max nm (log ε) 356 (3.87), 285 (4.10), 247 (4.53); LREIMS m / z 346 [M] ⁺ (2), 328 [MH ₂ 0] ⁺ (9); 310 [M-2H ₂ O] ⁺ (7); 288 [M- (CH ₃ ) ₂ CO] ⁺ (45); 270 [288-H ₂ O] ⁺ (100); 59 (94); HREIMS m / z 346.1529 (C ₁₈ H ₂₂ N ₂ O ₅ , 346.1518)

NMR Spectrum Results of Tereusinol location δ H (mult., J) δc (mult) HMBC (H to C) 1233a44a5677a88a1'1'-OH2'3'4'1 "1" -OH 2 "2" -OH3 "4" 12.21 (s) 6.28 (s) 11.91 (s) 6.31 (s) 4.23 (dd, 6.0, 5.0) 5.18 (d, 5.0) 1.90 (qqd, 6.8, 6.6, 6.0) 0.86 (d, 6.6) ^c 0.76 (d , 6.8) ^c 4.31 (d, 5.3) 5.34 (d, 5.3) 4.38 (s) 1.06 (s) 1.04 (s) 143.4 (s) 104.4 (d) 125.9 (s) 173.9 (s) 130.9 (s) 141.4 (s) 105.5 (d) 125.7 (s) 174.0 (s) 131.3 (s) 71.5 (d) 33.8 (d) 18.1 ( q) ^d 18.7 (q) ^d 73.6 (d) 71.7 (s) 26.2 (q) 25.3 (q) 2, 3, 3a, 8, 8a 1 '2, 3a, 4, 8a 4, 4a, 6, 7, 7a 1 ", 4a, 6, 7a, 8 2, 2 '3, 3' 4'1 ', 2'1', 2, 3 ', 4'1', 2 ', 4'1', 2 ', 3'2 ", 3", 4 ", 6, 71 ", 2" 1 ", 2", 3 ", 4" 1 ", 2", 4 "1", 2 ", 3"

a: measured under the conditions of DMSO-d ₆ . ¹ H NMR measured at 400 MHz and ¹³ C NMR at 100 MHz.

b: Including results by DEPT, HM QC and HMBC.

c, d: interchangeable within each column.

... (I)

Tereusinol ( 2 ) obtained in Example 2 was isolated as a reddish solid, and the molecular formula established according to HREIMS and ¹³ C NMR method was C ₁₈ H ₂₂ N ₂ O ₅ with 9 degrees of unsaturation. As shown in Table 1, the general characteristics of the ultraviolet, infrared, and NMR spectra of terreusinol are due to the ¹ H and ¹³ C NMR signals due to the presence of two slightly different side chains due to local selective oxidation in one direction of the two side chains. It was very similar to terreusinone except for the double.

In the ¹ H and ¹³ C NMR spectra of terreusinol, two new methyl groups [δH 1.04, 1.06 (3H, s, H3-3 ″, 4 ″, respectively) on the low-field side; Addition of one new quaternary carbon with δc 26.2, 25.3 (C-3 ″, 4 ″)] and hydroxyl group [δH 4.38 (1H, s, 2 ″ -OH); δc 71.7 (C-2 ″)] Signals were observed, showing that the hydroxyl group is bound to the 2 "-carbon.

¹ H and ¹³ C NMR spectral analyzes, including DEPT, HMQC and HMBC experiments of tereusinol, showed characteristic signals with two halves of isobutyl-pyrroloketone in each halves. One of the materials was identical to tereusinone in the NMR data. The other was 1,2-dihydroxyisobutyl pyrroloketone in MR data.

Based on the evidence presented above, the structure of tereusinol is 2-[(1R) -1-hydroxyisobutyl] -6-[(1R) -1,2-dihydroxyisobutyl] -1H, 5H-pyrrolo [2,3 -b] indole-4,8-dione.

In addition, the orientation of pyrrolo [2,3- b ] indole-4,8-dione of dipyrroloquinone in tereusinol was determined by H-3 [δH 6.28 (1H, s)] and C-4 (δc 173.9) and H Supported by the HMBC correlation of -7 [δH 6.31 (1H, s)] and C-8 (δc174.0).

Example 3: Comparison of the UV Blocking Capacity of the Invention Tereusinol of the Invention

The UV-A blocking ability of tereusinone and the novel compound tereusinol of the present invention and its conventional sunscreen, oxybenzone, was compared.

To this end, 1000 μg / mL, 100 μg / mL, 10 μg / mL of tereusinone and tereusinol (1 mg) were weighed and dissolved in MeOH (1 mL) in steps (1 mg / mL). Samples at mL and 1 μg / mL concentrations were prepared. A solution of oxybenzone dissolved in MeOH (0.1 mg / mL) was diluted 10 times step by step and used as an internal standard. Each sample and standard (200 μl) prepared above was taken in a 96-well microtiter tray, and then the absorbance was measured at 340 nm using a microplate reader to calculate the ED ₅₀ .

As a result, there was observed a strong UV-A blocking action of tereusinol (ED ₅₀ , 150 μM) than tereusinone (ED ₅₀ , 163 μM) and oxybenzone (ED ₅₀ , 350 μM).

As described through the above embodiment, the present invention uses a bioconversion technology by marine actinomycetes The present invention relates to a method for preparing a novel compound tereusinol having excellent UV A blocking ability, and is isolated from Streptomyces sp. From marine plant Zostera marina and using this strain through bioconversion technology from teriusinone. There is an excellent effect of providing the novel compound tereusinol having excellent ultraviolet A blocking ability. Therefore, tereusinol, a novel compound of the present invention, has an effect of having excellent UV-A blocking activity than oxybenzone, which is currently generally used as a sunscreen.

Figure 1 shows the molecular structure of the novel compound tereusinol excellent in UV A blocking ability prepared using the bioconversion technology by the strain Streptomyces genus of the present invention.

Claims (3)

Tereusinol represented by the following structural formula (I) characterized by obtaining Streptomyces sp. MFAac18 KACC 91085 strain from Zostera marina through the bioconversion of tereusinone using a microbial catalyst: ... (I) A process for preparing a novel terecinol having excellent UV A blocking ability, comprising the following steps: Streptomyces sp . MFAac18 KACC 91085 strain isolated from marine plant Zostera marina was subjected to stationary culture in SWS medium (consisting of 0.1 parts by weight, 1.0 part by weight of soluble starch and 100 parts by weight of seawater), The culture was added to the culture solution by adding tereusinone, Ethyl acetate extracted from the culture solution obtained above to obtain an organic layer, which was dried, filtered and concentrated under reduced pressure to obtain a viscous brown extract, The extract was subjected to silica gel column chromatography using a solvent of n-hexane: ethyl acetate (100: 0 to 0: 100) to obtain reversed phase HPLC (YMC ODS-A) using methanol as a solvent. Carried out to obtain red teriusinol. A composition for sunscreen A according to claim 1, comprising tereusinol as an active ingredient.