JP2013127027A - Ultraviolet absorber composition originated from plant inhabiting microbes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new ultraviolet absorber composition originated from a natural component.SOLUTION: The ultraviolet absorber composition contains a component originating from plant inhabiting microbes, and has an absorption peak of ultraviolet absorption spectrum in a wavelength region of 320-400 nm. From microbes inhabiting the phyllosphere exposed to various environmental stresses such as ultraviolet ray, an ultraviolet absorption component having an absorption effect, UV-A in particular, can be obtained. A cosmetic contains the composition with the ultraviolet absorption component. Also, there is provided a method for producing the ultraviolet absorber composition including the steps of culturing the microbes containing the component, extracting a solvent and separating the microbes from the solvent.

Description

  The present invention relates to an ultraviolet absorber composition derived from a microorganism that inhabits the surface of a plant such as an agricultural crop and a method for producing the same.

  Various types of UV inhibitors have been used in cosmetics, sunscreen products, and other industrial products in order to protect the skin and products from UV rays. For example, UV inhibitors used in cosmetics and sunscreen products mainly correspond to UV-A (320-400 nm long wavelength UV) and UV-B (280-320 nm medium wavelength UV) from various viewpoints. More specifically, reflective agents (such as titanium oxide and zinc oxide), absorbents (such as oxybenzone and avobenzone), and antioxidants (such as vitamins and carotenoids) are used.

  Among the above, there are few types of UV absorbers that are still in practical use, compared to reflective agents that are almost established as materials and antioxidants that already have various types. In addition, oxybenzone and avobenzone, which are existing UV-A UV absorbers used as cosmetic raw materials, are chemically synthesized products, but these substances may cause irritation and rough skin. In view of the above, it is desired in the market to be replaced by a substance derived from a natural component. Moreover, the recognition that UV-A is the main cause of photoaging of the skin is widespread, and consumers are highly interested in UV-A countermeasures.

  As a naturally occurring ultraviolet absorbing substance, mycosporin-like compounds, cytonemin, etc. produced by marine bacteria and cyanobacteria have been reported so far (Patent Documents 1, 2; Non-Patent Documents 1, 2, 3, 4). ). However, there have been no examples that have been discovered focusing on microorganisms that inhabit universally on the ground crops, and the UV-absorbing components they contain are not known. In addition, many of the known absorbents have properties with low polarity, and there are problems from the viewpoint of processing characteristics.

JP 2006-160662 A JP 2000-16976

Botanica Marina (Bot.Mar.) 16, 23 (1974) Marine Biology (Mar. Biol) 108, 157 (1990) Journal of Plankton Research (J. Planton Res.) 12, 909 (1990) Journal of Phycology (J. Physol.) 27, 395, (1991)

  An object of this invention is to provide the novel ultraviolet absorber composition which solves said subject.

  The inventors of the present application are able to obtain an ultraviolet ray absorbing component having an effective absorption effect of ultraviolet rays, particularly UV-A, from plant phytosphere microorganisms (plant-inhabiting microorganisms) exposed to various environmental stresses such as ultraviolet rays. As a result, the present invention has been completed.

That is, the present invention provides an ultraviolet absorber composition derived from plant-inhabiting microorganisms, which has an absorption peak of an ultraviolet absorption spectrum in a wavelength region of 320 to 400 nm.
Moreover, this invention provides the cosmetics containing the said ultraviolet absorber composition.
The present invention also includes a step of culturing a plant-inhabiting microorganism having an ultraviolet-absorbing component having an absorption peak of an ultraviolet absorption spectrum in a wavelength region of 320 to 400 nm, a step of extracting a plant-inhabiting microorganism culture with a solvent, and a solvent. The manufacturing method of the ultraviolet absorber composition containing the said ultraviolet absorption component including the process of isolate | separating an ultraviolet absorption component from the said is provided.

  According to the present invention, a novel ultraviolet absorber composition derived from natural components can be provided. Moreover, since the ultraviolet absorber composition of the present invention has a high polarity, it has an advantage in applicability to industrial products such as cosmetics and inks from the viewpoint of processing characteristics.

The UV absorption spectrum of the extract of Methylobacterium sp. W-213 strain is shown. A photograph of Methylobacterium sp. W-213 strain cultured in the medium is shown. A suspension of Methylobacterium sp. W-213 strains is shown. The basic compound component solution of Methylobacterium sp. W-213 strain is shown. The HPLC profile of the absorption component of Methylobacterium sp. W-213 strain is shown. The ultraviolet absorption spectrum of the suspension water of the dried microbial cells of Methylobacterium sp. W-213 strain, W-182 strain and Bacillus sp. No. 20 strain (control) is shown. The ultraviolet absorption spectrum of the methanol extract of Methylobacterium sp. W-213 stock | strain, commercial sunscreen cream A, and commercial sunscreen cream B is shown.

The present invention provides an ultraviolet absorber composition derived from plant-inhabiting microorganisms, which has an absorption peak of an ultraviolet absorption spectrum in a wavelength region of 320 to 400 nm.
The plant-inhabiting microorganism used in the present invention can be appropriately selected by those skilled in the art to have a desired ultraviolet absorption ability and to absorb ultraviolet rays in a desired wavelength region. For example, for a group of microorganisms isolated from a plant, plant extracts that are suitable for the present invention can be routinely selected by making extracts and measuring UV absorption spectra.

The type of plant-inhabiting microorganism is not particularly limited as long as it is selected by the above-described method and the like, and may be any of bacteria, yeast, mold, etc. Among them, bacteria are preferred, for example, Rhizobiales And those belonging to Methylobacteriaceae (Methylobacteriaceae) are preferred. Among them, bacteria belonging to the genus Methylobacterium are known to inhabit a lot of plant leaves and the like, and are particularly preferable for the purpose of the present invention.
In addition, examples of the plant serving as an isolation source of the plant-inhabiting microorganisms of the present invention include, but are not limited to, rice, wheat, barley, tomato, strawberry, leek, komatsuna, dung beetle, and soybean.

In the ultraviolet absorbent composition of the present invention, the mode in which plant-inhabiting microorganisms are used in the ultraviolet absorbent composition can be appropriately determined by those skilled in the art, and the culture can be obtained as it is or by drying the cells. You may use the obtained dried material for a ultraviolet absorber composition. Or you may use for the ultraviolet absorber composition the extract which extracted the plant inhabitant microorganisms cultured suitably with the appropriate solvent. Preferably, the ultraviolet absorbing component is separated from the solvent extracted from the cultured plant-inhabiting microorganisms by means such as ion exchange chromatography and blended into the ultraviolet absorbent composition.
The culture method is not particularly limited, and may be either liquid culture or solid culture, and can be appropriately determined according to the type of microorganism.

Moreover, when using an extract, the solvent can be suitably determined by those skilled in the art according to the type of microorganism, the type of final product in which the absorbent is used, and the like. For example, the solvent polarity parameter ET value (reference: Marcus, Y. 1998. The Property of Solvents, Wiley, ISBN: 978-0-471-98369-9) used as a general solvent polarity index is 45.0. It is possible to use a solvent of ˜65.0 (kcal / mol, 25 ° C.), more preferably a solvent having an ET value of 46.0 to 64.0, and still more preferably a solvent having an ET value of 48.0 to 63.5.
More specific solvents include water (ET value 63.1), methanol (55.5), n-butanol (49.7), 2-propanol (48.6), and ethanol (51.9). You may mix and use. For example, methanol and water can be mixed and used as 50-80% methanol.

  The ultraviolet absorbent composition of the present invention can be appropriately blended in cosmetics and the like. The type of cosmetic is not particularly limited, and examples thereof include sun cream, makeup base, solid or liquid foundation, UV prevention spray, milky lotion, hair conditioner, lipstick and the like.

  The ultraviolet absorbent composition of the present invention has UV-A absorption activity, and has an absorption peak in the wavelength region of 320 to 400 nm when the absorption spectrum is measured with a spectrophotometer. The ultraviolet absorbent composition of the present invention preferably has a maximum absorption peak in the ultraviolet wavelength region within the wavelength range of 320 to 400 nm, more preferably 340 to 380 nm, and even more preferably 355 to 365 nm.

Further, the ultraviolet absorbent composition of the present invention has a ratio of ultraviolet absorption spectrum [peak area of 340-380 nm] / [peak area of 320-400 nm] of 0.5 or more, preferably 0.55 or more, more preferably 0.60 or more. is there. The above-mentioned absorption spectrum area can be obtained by converting the ultraviolet absorption peak into an area value by an integration program of various spectrum analysis software such as half width method or SWIFT II-METHOD.
Examples of the present invention will be described below, but the present invention is not limited thereto.

(1) Selection of plant-inhabiting microorganisms A total of 239 strains of bacteria isolated from rice leaf sheath, petals of various plants, wheat ears, strawberry leaves, and chingensai leaves were tested and potato peptone glucose (PPG: potato, 200 g; peptone, 5 g; glucose, 5 g; disodium phosphate, 3 g; monopotassium phosphate, 0.5 g; sodium chloride, 3 g; distilled water, 1000 ml) were smeared and cultured at 25 ° C. for 5 days. After culturing, the cells on the plate were scraped off, and about 10 to 45 mg of dried cells of each strain was obtained by freeze-drying or the like. 4 ml of a 50% methanol solution was added to the dried microbial cell, and extracted by shaking (160 rpm) for 20 minutes after sonication for 5 minutes. After shaking, the extract was collected by centrifugation (10000 rpm, 10 minutes), and this extraction process was repeated twice to finally obtain about 12 ml of extract. Put 100 ul of the obtained extract in a 96-well microplate, and use the multi-spectrophotometer (Viento, manufactured by Dainippon Pharmaceutical Co., Ltd.) and analysis software KcJunior (produced by Dainippon Pharmaceutical Co., Ltd.) Absorption spectrum was measured. The measurement was performed by a spectrum scan method in which a wavelength range of 200 to 500 nm was continuously measured at intervals of 10 nm. As a result, it was found that the extract of 17 strains including the W-213 strain showed a characteristic spectrum having a maximum absorption peak around 360 nm (FIG. 1 shows the spectrum of the W213 strain). This spectrum was equivalent to that of avobenzone, an existing chemically synthesized absorbent.

  As a result of identifying these 17 strains based on the base sequence of the rRNA gene, all of them were identified as Methylobacterium bacteria. A photograph of Methylobacterium sp. W-213 showing a characteristic UV absorption spectrum is shown in FIG.

(2) Extraction solvent Using the dried cells of Methylobacterium sp. W-213 obtained by the same method as in (1) above as a starting material, extraction with various extraction solvents is performed in the same procedure as in (1). The concentration of the extract was adjusted to 1 ml per 1 mg of dry cell weight. Absorbance at a wavelength of 360 nm for 100 μl of each solvent-extracted solution was measured using a spectrophotometer (Ultrospec 2000, Pharmacia) and analysis software SWIFT II-METHOD (Pharmacia; Wavescan program, 1.0 nm step, scan speed 6200 nm / min) It was measured by. The absorbance (360 nm) of the extract obtained with each extraction solvent and the ET value (kcal / mol, 25 ° C.) of the solvent are shown below.

From the above, it can be seen that absorption at 360 nm is confirmed when plant inhabiting microorganisms are extracted with a solvent having a solvent polarity parameter ET value of 48.6 to 63.1.

(3) Extraction of UV-absorbing components from plant-inhabiting microorganisms
Using Methylobacterium sp. W-213 strain, cells were obtained by shaking culture on PPG liquid medium and smear culture on PPG agar plate (FIG. 3). An active extract (1.13 g) was obtained by extracting 8.169 g of the dried product obtained by drying the cells with about 3 liters of 80% methanol. The extracted product was stepwise eluted with methanol and then concentrated ammonia: methanol = 5: 95 using a solid phase cartridge column (PoraPak, Waters) according to the usage attached to the column, and concentrated ammonia: methanol = 5: 95. A liquid fraction, 81.4 mg of a yellowish brown basic absorption component, was obtained as the active fraction (FIG. 4).

(4) HPLC analysis of extracted components In order to separate the absorbing components, analysis by HPLC (Waters, 600 Controller, 2414 Detecter) was performed. The column is Atlantis T3 (3μm, 4.6 x 150mm, manufactured by Waters). Elution conditions are 0 to 8 minutes, H ₂ O is eluted with a linear gradient of 0.5 ml / min → 0.7 ml / min, 8 → 12 Elution was performed with a linear gradient of H ₂ O → 100% methanol at a flow rate of 0.7 ml / min until min, and elution with 100% methanol at a flow rate of 0.7 ml / min until 12 → 30 min. As a result, five large peaks were detected, and all the peaks showed the maximum absorption peak with respect to the wavelength of absorbance of 360 nm. Therefore, it was found that the present absorption component is composed of at least five kinds of absorbing substances ( FIG. 5).

(5) UV absorber composition using a culture of plant-inhabiting microorganisms Methylobacterium sp. W-213 and W-182 strains isolated from wheat ears and strawberry leaves isolated from wheat ears, and as controls Using Bacillus sp. No. 20 strain isolated from tomato leaves, each cell was obtained by smear culture on a potato, peptone, glucose (PPG) agar plate. The dried product obtained by drying the cells is suspended in sterile water at a concentration of 0.1 mg / ml, and the UV absorption spectrum of the suspension (100 ul) and the peak area in the wavelength range of 320-400 nm are measured with a spectrophotometer (Ultrospec 2000, manufactured by Pharmacia) and analysis software SWIFT II-METHOD (Pharmacia; Wavescan program, 1.0 nm step, scan speed 6200 nm / min). As a result, absorption peaks in the wavelength range of 320-400 nm were not confirmed in the cell suspension of Bacillus sp. No. 20, whereas it was confirmed in Methylobacterium sp. W-213 and W-182. The peak areas were 205.9 and 204.4, respectively (FIG. 6).

(6) Difference in absorption spectrum between commercially available cosmetics and ultraviolet absorbent composition of the present invention Dry cells of Methylobacterium sp. W-213 obtained by the same method as in (1) above and commercially available sunscreen creams A and B Extracted with methanol in the same procedure as in (1), and adjusted the concentration of each extract to 1 ml per 1 mg of dry weight of cells or cream. . 100 μl of each concentration-adjusted extract was tested, and the ultraviolet absorption spectrum and peak areas in the wavelength range of 320-400 nm and 340-380 nm were measured and compared using the same spectrophotometer and analysis software as in (5) (FIG. 7). ).
Here, for each sample, the peak area of 320-400 nm, the peak area of 340-380 nm, and the ratio of [peak area of 340-380 nm] / [peak area of 320-400 nm] are shown below.

  As described above, since the component of the present invention has a specific absorption ability in the UV-A region, for example, by adding it to an inexpensive commercial sunscreen product having no absorption ability in the UV-A region, An excellent effect can be exhibited as a derived component.

Claims (10)

  An ultraviolet absorber composition derived from a plant-inhabiting microorganism, having an absorption peak of an ultraviolet absorption spectrum in a wavelength region of 320 to 400 nm.   The ultraviolet absorbent composition according to claim 1, wherein the ratio of [peak area at 340 to 380 nm] / [peak area at 320 to 400 nm] in the ultraviolet absorption spectrum is 0.5 or more.   The ultraviolet absorber composition according to claim 1 or 2, wherein the plant-inhabiting microorganism is a microorganism that inhabits rice, wheat, barley, tomato, strawberry, leek, komatsuna, chingensai, or soybean.   The ultraviolet absorber composition according to any one of claims 1 to 3, comprising a plant-inhabiting microorganism extract obtained by extracting plant-inhabiting microorganisms with a solvent having a solvent polarity parameter ET value of 45.0 to 65.0.   The ultraviolet absorber composition according to any one of claims 1 to 4, wherein the solvent is a mixture of methanol and water.   The ultraviolet absorber composition according to any one of claims 1 to 5, wherein the plant-inhabiting microorganism is a bacterium of the genus Methylobacterium.   Cosmetics containing the ultraviolet absorber composition according to any one of claims 1 to 6.   The step of culturing plant-inhabiting microorganisms having an ultraviolet-absorbing component having an absorption peak of the ultraviolet absorption spectrum in the wavelength region of 320 to 400 nm, the step of extracting the plant-inhabiting microorganism culture with a solvent, and separating the ultraviolet-absorbing component from the solvent The manufacturing method of the ultraviolet absorber composition containing the said ultraviolet absorption component including the process to do.   The production method according to claim 8, wherein the plant-inhabiting microorganisms are extracted with a solvent having a solvent polarity parameter ET value of 45.0 to 65.0.   The production method according to claim 8 or 9, wherein the plant-inhabiting microorganism is a bacterium of the genus Methylobacterium.