KR100812922B1 - Skin whitening composition containing culture fluid or its extract of new microorganism - Google Patents

Abstract

A skin whitening composition comprising a culture solution of a novel Pseudomonas sp. microorganism or an extract thereof is provided to be useful for hyperpigmentation disorder such as freckles, liver spots, and senile keratosis, and skin beauty. A skin whitening composition comprises 0.0001-20 wt.% of a culture solution of Pseudomonas sp. microorganism(deposition no. KCTC 11119BP) or an extract of the culture solution as an effective ingredient, wherein the extract is a methylene chloride extract. The composition is formulated into face lotion, cream, lotion, pack, foundation or make-up base.

Description

Skin whitening composition containing culture fluid or its extract of new microorganism}

1 is a 16s rDNA sequencing analysis of the novel microorganism of the genus Pseudomonas according to the present invention.

Figure 2 is an experimental result evaluating the effect of Pseudomonas genus microorganism of the present invention on the pigment formation of Melan-a cells. Kojic acid was used as a positive control.

3a and 3b are the results of evaluating the whitening effect of the microbial culture extract of the present invention by measuring the melanin content in Melan-a cells or human melanocytes.

Figure 4 is an experimental result of evaluating the whitening effect of the novel microorganism Pseudomonas of the present invention using DOPA oxidase activity assay.

5 is an experimental result of evaluating the whitening effect of the novel microorganism of the present invention Pseudomonas through investigation of the expression level of MITF and tyrosinase.

6 is an experimental result evaluating the whitening effect of the novel microorganism of the present invention Pseudomonas using cultured skin. Fontana-masson staining was used, which is a result of 200 times magnification.

7 is an experimental result of evaluating the whitening effect of the novel Pseudomonas genus of the present invention using zebrafish.

In the figures, BE means bacterial extract according to the present invention, the result values being the mean value of five independent experiments, and * and ** are significant as p <0.01 and p <0.05, respectively. That means there is a difference.

The present invention relates to a composition for whitening skin containing a novel microbial culture of Pseudomonas or an extract of such a culture as an active ingredient.

Skin pigmentation results from the production and distribution of melanin in the epidermis. In mammalian melanocytes, melanin is synthesized in melanosomes containing the main pigment enzyme tyrosinase. Hyperpigmentation disorders, including surpluses, freckles, blemishes, and aging keratosis, are associated with abnormal accumulation of melanin pigments and studies are being actively conducted on skin whitening agents to improve such disorders.

Although some natural substances, such as kojic acid, arbutin and hydroquinone, are known to have tyrosinase inhibitory activity, they have the disadvantages of weak effects and side effects.

Recently, the sea has been recognized as a source of new biologically active substances, and active researches have been made to apply biologically active molecules separated from marine fauna and flora to the fields of pharmacy, food impact, cosmetics, agriculture, molecular biology, etc. It is becoming. In addition, marine invertebrates are reported to be involved in the production of various bioactive molecules, and these molecules have increased applicability in medical and industrial applications.

Therefore, the technical problem to be achieved by the present invention is to provide a skin whitening composition excellent in skin whitening effect and fewer side effects.

In order to achieve the above technical problem, the present invention provides a composition for skin whitening, comprising a culture solution or a culture solution extract of novel bacteria KCTC 11119BP of Pseudomonas.

The present invention is isolated from Pseudomonas species tidal KCTC 11119BP novel microorganism culture or the extract thereof is in a number of in vitro and in It is based on the fact that it exerts a surprising whitening effect in the evaluation of skin whitening effect in vivo .

Hereinafter, the composition for skin whitening of the present invention will be described in more detail.

The composition for skin whitening of the present invention includes a culture or culture extract of Pseudomonas genus novel microorganism KCTC 11119BP newly isolated, cultured and identified as an effective ingredient for skin whitening. The new microbial strain isolated and cultured in the present invention was identified as Pseudomonas species through molecular systemic analysis based on the 16s rDNA sequence, named " Pseudomonas sp. 54", dated April 17, 2007 It was deposited with KCTC 11119BP at the Korea Institute of Bioscience and Biotechnology.

Pseudomonas species extract of the novel microorganism culture or the culture of this invention is Melan-a cells, in using human melanocytes and the cultured skin engine in vitro evaluation and zebrafish in In vivo skin whitening evaluation experiments showed an amazing skin whitening effect. This melanin pigment inhibitory effect is thought to be exerted by inhibiting the expression and activity of microphthalmia-associated transcription factor (MITF) and tyrosinase, which are important for pigmentation.

The microorganism according to the present invention may be improved or improved to have equivalent activity or better activity by conventional physicochemical mutation methods and the like known in the art.

The composition for skin whitening of the present invention may contain a culture of the microorganism KCTC 11119BP or an extract thereof as an active ingredient, and may further include a suitable excipient or carrier. The culture is culture medium itself in which the microorganism according to the invention is cultured in a suitable liquid medium, filtrate (filtered or centrifuged supernatant) from which the strain is removed by filtration or centrifugation, the culture solution is sonicated or the Cell lysates obtained by treating lysozymes, but are not limited thereto.

The skin whitening composition of the present invention may be prepared in various formulations such as cosmetics, creams, lotions, packs, foundations, makeup bases, and the like, and specifically, applied in various properties such as liquid, cream, paste, solid, and the like. It is possible. In addition, a conventional cosmetic or skin ointment preparation method may be used to prepare a composition for skin whitening containing the novel microbial culture of the present invention or an extract thereof.

Meanwhile, in the composition for skin whitening of the present invention, the content of the culture of the present invention or the extract thereof is preferably 0.0001 to 20% by weight based on the total weight of the composition. If the content of the active ingredient is less than 0.0001% by weight, it is not preferable to achieve a sufficient whitening effect of the original desired skin, and if the content of the active ingredient exceeds 20% by weight, it may cause problems in the stability of the composition. This may cause problems in the appearance of the composition.

Extract of the novel microorganism KCTC 11119BP culture medium of the genus Pseudomonas of the present invention can be extracted through an extraction method well known to those skilled in the art. Extraction solvents include hexane, benzene, toluene, diethyl ether, chloroform, ethyl acetate, methylene chloride, 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethylformamide, dimethylsulfoxide, acetic acid and methanol, Alcohols having 1 to 4 carbon atoms, including ethanol, propylene glycol, propyl alcohol, butylene glycol, butyl alcohol, glycerin, and the like may be used, but are not limited thereto. In consideration of the skin whitening effect of the extract, Most preferably, chloride is used as the extraction solvent.

Hereinafter, examples and the like will be described in detail to help understand the present invention. However, embodiments according to the present invention can be modified in many different forms, the scope of the invention should not be construed as limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

For all the experiments, statistical significance analysis was performed using student t- test.

<Isolation, Culture and Identification of Microorganisms>

Microorganisms were separated from the tidal flats in Ganghwa-do, Korea by extraction using sterile seawater. The isolated microorganisms were mixed in a seawater complete agar culture medium (25% purified water containing 25 g bactotryptone in 1 liter, 3 g yeast extract, 3 ml glycerol and 15 g agar, 75% seawater, pH 7.0). In the culture of the liquid, a complete seawater culture medium (25% purified water including 25 g bactotryptone in 1 liter, 3 g yeast extract and 3 ml glycerol and 75% seawater, pH 7.0) was used. .

For identification of the isolated, cultured bacteria, 16s rDNA sequencing analysis method was used. PCR amplification of 16s rDNA was performed with primers 27F (5'-AGAGTTTGATCMGGCTCAG), 342R (5'-CACGGATCCCCACTGCTGCSYCCCGTAG), 66F (5'-GTGCTGCAGAACACATGCAAGTCGA), 1100R (5'-GGGTTGCGCTCGTC) GCCGCGCGG 5'-AAGGAGGTGWTCCARCC). The complete sequence was sorted using the GenBank BLAST program. 16s rDNA sequencing sequence of the isolated bacteria KCTC 11119BP is shown in Figure 1 below. From the results of FIG. 1, it was confirmed that the isolated microorganism was a novel microorganism belonging to Pseudomonas through molecular sequence analysis based on the nucleotide sequence.

<Production of Organic Extracts>

Microbial isolates were inoculated into one liter of seawater complete agar culture medium, and the culture medium was shaken at 30 ° C. for 5 days. After incubation, the microbial culture was divided into new containers of 250 ml each, to which the same volume of hexane, methylene chloride, acetone or methanol was added. Each mixture was shaken (170 rpm) at 30 ° C. for 4 hours and then the organic phase was recovered. The organic phase was dried using a rotary evaporator and the resulting dried material was used for the test described below. Stored at 4 ° C.

<Mushroom Tyrosinase Suppression Evaluation>

Mushroom tyrosinase inhibition experiment was used to evaluate the whitening effect of the microbial culture or extract of the present invention. Tyrosinase hydroxylates tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and is involved in the oxidation of DOPA and plays an important role in melanogenesis. Therefore, melanin production can be said to be mainly dependent on the expression and activation of tyrosinase.

Several organic solvent extracts of microbial cultures were mixed with 1 mM tyrosine, 34 units / ml mushroom tyrosinase and 0.1 M K-phosphate buffer (pH 6.8) and incubated at 37 ° C. for 20 minutes. After incubation, optical density was measured using an enzyme-linked immunosorbent assay reader at 470 nm.

Arbutin, a well known tyrosinase inhibitor, was evaluated as a positive control, and the effect of methylene chloride extract was superior to that of other organic solvent extracts. The methylene chloride extract showed about 90% tyrosinase inhibition rate compared to arbutin, which showed 97% tyrosinase inhibition efficiency.

In all subsequent evaluation experiments, methylene chloride extract of the novel microbial culture solution according to the present invention was used.

<Evaluation of whitening effect using Melan-a cells and melanocytes>

Melan-a cells and melanocytes used in the whitening evaluation experiment described below were cultured as follows.

Melan-a cells were cultured in RPMI 1640 medium supplemented with 4% fetal bovine serum, 100 uM β-mercaptoethanol, 1% antibiotic / antifungal solution and 200 nM TPA.

Human melanocytes were obtained from adult foreskin, 10% heat-inactivated fetal bovine serum, 1% antibiotic / antifungal solution, 24 ug / ml 3-isobutyl-1-methylxanthine, 80 nM 12- It was stored in F12 medium supplemented with O-tetradecanoyl phorbor-13-acetate, 1.2 ng / ml fibroblast growth factor and 0.1 ug / ml cholera toxin. In the experiments described below, melanocytes were used with 2 or 3 passages, 4% heat-inactivated fetal bovine serum, 0.6 ng / ml fibroblast growth factor, 5 ug / ml insulin, 1 ug / ml Vitamin E and 1 ug / ml transferrin were maintained in MCDB-153 medium supplemented.

Cell growth assay

Melan-a cells were placed at 2 × 10 ⁵ cells / cm ² in a culture dish. After incubation for 1 day, the medium was replaced with fresh medium containing various concentrations of microbial culture extract. Kojic acid was used as a positive control. After 3 days of culture, cells were counted using a Coulter counter. Cytotoxicity was assessed using trypan blue exclusion assay. No effect on viability was observed at the concentrations tested, which means that the bacterial extracts of the present invention have little or no cytotoxicity against Melan-a cells.

Experimental results using the methylene chloride extract of the bacterial culture medium according to the present invention is shown in FIG. As shown in Figure 2, the microbial extract of the present invention inhibited the pigment formation of Melan-a cells in a concentration-dependent manner, the pigment inhibitory effect was superior to that of kojic acid, which is widely known to have a whitening effect.

The effect of the bacterial extract of the present invention on human melanocyte viability was evaluated using trypan blue exclusion assay. No effect on viability was observed at the concentrations tested, meaning that the bacterial extracts of the present invention had little or no cytotoxicity against human melanocytes.

Melanin content measurement

Melan-a cells were treated with the microbial culture extract of the present invention as in the cell growth assay. Thereafter, pellets of 3 × 10 ⁵ Melan-a cells were lysed in 1 M aqueous NaOH solution, and the optical density was measured using an enzyme-linked immunosorbent assay reader at 490 nm. The melanin content was measured by comparing the absorbance to a standard curve obtained using synthetic melanin.

The results are shown in Figure 3a. As shown in Figure 3a, melanin content in Melan-a cells was reduced by treating the bacterial culture extract of the present invention.

Melanocytes were placed in culture dishes at 1.5 × 10 ⁵ cells / cm ² and evaluated similarly to Melan-a cells, except that they were treated with test samples for 5 days.

The results are shown in Figure 3b. As shown in Figure 3b, the bacterial culture extract of the present invention reduced the melanin content in human melanocytes concentration-dependently.

DOPA Oxidase  Activity analysis

In order to evaluate the inhibitory activity mechanism of the bacterial extract of the present invention on melanin production, the tyrosinase activity of Melan-a cells treated with methylene chloride extract was measured as in the previous experiment.

The degree of enzyme induction was assessed using spectrophotometric experiments using L-DOPA as the substrate. Melan-a cells treated with samples were lysed in 1% Triton-X and 10 mM L-DOPA. After 90 min incubation at 37 ° C., the absorbance was measured at 475 nm.

The results are shown in FIG. As shown in Figure 4, when the Pseudomonas genus microbial culture extract according to the present invention is treated at concentrations of 10, 50 and 100 ug / ml, the optical density is 90 ± 4, 66 ± 18 and It decreased to 58 ± 9% (n = 5, P <0.01). Decreased tyrosinase activity This level is consistent with the results of the melanin content experiment, which means that the bacterial extract of the present invention affects tyrosinase and consequently inhibits melanin synthesis of human melanocytes.

Tyrosinase  And MITF of Weston Blot  analysis

Microphthalmia-associated transcription factor (MITF) is involved in pigmentation, proliferation and survival of melanocytes and strongly regulates the expression of tyrosinase, which means that MITF is an important transcriptional regulator in the melanogenesis process. . Therefore, it was evaluated whether the bacterial extract of the present invention regulates MITF expression.

For Western blot analysis, melan-a cells were treated with 50 ug / ml microbial extract for 3 days and then lysed. 10 ug of protein per lane was electrophoresed using sodium dodecyl sulphate-polyacrylamide gel and blotted onto nitrocellulose paper. Blots were incubated with rabbit polyclonal antibodies against tyrosinase, diluted 1: 2000 and then treated with horseradish peroxidase-binding secondary antibody. The monoclonal antibody of MITF was diluted 1: 200. Bound antibodies were analyzed using the enhanced chemiluminescence plus kit (Amersham Int., UK). To quantify the level of expression, the intensity of the bands was analyzed with a densitometer using Image-Pro Plus version 4.5 and expressed as relative intensity to actin.

The results are shown in FIG. As shown in Figure 5, the microbial extract of the present invention reduced the expression of MITF in Melan-a cells, and also reduced the expression of tyrosinase protein. These results indicate that the novel microbial culture of Pseudomonas genus of the present invention reduces melanin production through MITF inhibition, and MITF inhibition reduces the expression and activity of tyrosinase.

<Evaluation of Whitening Effect Using Cultured Skin>

Cultured skin was used to evaluate the skin whitening effect of the bacterial culture extract of the present invention.

Skin samples of human breast skin were incubated. Petri dishes were incubated in DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Skin specimens were placed on a stainless steel grid, incubated at 37 ° C. and 5% CO ₂ , and the medium changed every three days.

Cultured skin organs were treated with 100 ug / ml microbial culture extract according to the present invention. After 3 days incubation, the samples were fixed in 4% buffered formaldehyde and embedded into paraffin sections. Thereafter, it was stained with hematocrine and eosin for microscopic observation. Melanin pigments were observed using Fontana-Masson staining. Image analysis was performed using Image Pro Plus Version 4.5 and the pigmented area / epidermal area (PA / EA) was measured.

6 shows the results of the comparison with the negative control which was not treated with the microbial extract according to the present invention. As shown in Figure 6, the bacterial extract of the present invention inhibited pigment formation in the skin organs. Image analysis showed that the PA / EA in the skin treated with the extract of the present invention was 88 ± 47.5, whereas the PA / EA in the control skin was 227 ± 36.6.

<Evaluation of Whitening Effect Using Zebrafish>

After the mature zebrafish was purchased, 10-15 zebrafish were stored in a 5 liter acrylic tank at 28.5 ° C., 14/10 hour day / night circulation. The fetus was obtained by a natural scattering method and used in the experiments described later.

Synchronized embryos were collected and 3-4 fetuses per well were arranged using a pipette in a 96-well containing 200 ul of fetal medium. As a test material, the microbial culture extract was dissolved in 0.1% DMSO and added to the fetal medium for 63 hours from 9 hpf to 72 hpf. The effect of the cultures of the present invention on pigment formation in zebrafish was observed using a stereomicroscope. Stirring was often done as well as during the medium change to spread the test material evenly. In all experiments, 0.2 mM PTU was used to generate transparent Zebrafish without affecting the developmental process and used as a positive control.

Body pigmentation was evaluated based on the phenotype at 35 hpf and 55 hpf for melanogenesis inhibitors, and body pigment formation was evaluated based on the phenotype at 60 hpf for melanogenesis stimulants (α-MSH). For observation, the fetus was removed with forceps and anesthetized with tricaine methanesulfonate. After mounting with 3% methyl cellulose on the depression slides, pictures were read using stereomicroscope MZ16 (Leica Microsystems, Germany).

The experimental results are shown in FIG. 7. As shown in Figure 7, the bacterial culture extract of the present invention significantly inhibited the pigmentation of zebrafish. These results indicate that the novel microorganism of Pseudomonas of the present invention can be used as a whitening agent in skin hyperpigmentation disorder.

The present invention provides a composition for skin whitening useful for hyperpigmentation disorders, skin care, etc. including surplus, freckles, blemishes, senile keratosis.

Claims (2)

Pseudomonas (Pseudomonas) microorganisms of the skin whitening composition comprising a culture or extract of the culture medium of KCTC 11119BP as an active ingredient. The composition for skin whitening according to claim 1, wherein the extract is a methylene chloride extract.

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