WO2014109426A1 - Cosmetic composition comprising bacteriochlorophyll-containing microbial cell piece as active ingredient - Google Patents

Abstract

The present invention relates to a cosmetic composition for relieving skin irritation or moisturizing the skin, including, as an active ingredient, a bacteriochlorophyll-containing microbial cell extract acquired from Rhodobacter sphaeroides, and displays excellent skin irritation relieving effects and skin moisturizing effects.

Description

Cosmetic composition comprising bacteriochlorophyll-containing cell lysate as an active ingredient

The present invention relates to a cosmetic composition comprising a bacteriochlorophyll-containing cell lysate as an active ingredient, and more particularly, to alleviate skin irritation including a bacteriochlorophyll-containing cell lysate obtained from Rhodobacter sphaeroides as an active ingredient. It relates to a cosmetic composition for moisturizing or skin.

 Photosynthetic bacteria are independent nutrients and largely classified into green bacteria and red bacteria, and are widely distributed in rivers, lakes, and oceans. Photosynthetic bacteria play a major role in the circulation of carbon, nitrogen, and sulfur on the planet, and contribute greatly to the production of food and energy, to solving various environmental deterioration and pollution problems.

 In addition, photosynthetic bacteria are evaluated as excellent nutrients with high nutritional value, and are superior to yeast or chlorella as food for chlorella, daphnia and early flora and fauna. In particular, various bioactive components such as amino acids, vitamins, carotenoids, and bacteriochlorophyll in photosynthetic bacteria can be expected to nourish the skin and make the skin healthy.

 The cosmetics market in Korea is ranked 10th in the world and is regarded as an important market leading the trend of the global cosmetics market. Domestic cosmetics sales amount to W7trn and still show high growth.

 As the use of fermented microorganisms has increased in the cosmetic industry since the late 2000s, fermented cosmetics using microorganisms have grown to the largest cosmetic group after herbal cosmetics. SKII products, which have been selling high in Korea and overseas markets, have achieved great success by emphasizing yeast-derived “Pyterra”. In Korea, LG H & H's “Hu”, “Beautiful” and AMOREPACIFIC's “Hyosia” have fermentation technology as their main technology. The market size of fermented cosmetics is estimated to be close to about 1 trillion won, and most of them use lactic acid bacteria or yeast that are not harmful to humans, and thus, there are many limitations in expanding the variety of products and brands.

 Therefore, the exploration of skin physiological activity of cell components or culture medium of photosynthetic bacteria can be said to be an important research theme that can further expand the field of fermented cosmetics limited to lactic acid bacteria and yeast.

 Korean Patent Publication No. 10-2012-0098334 (published date 2012. 09. 05) includes duck oil, EM fermentation broth (fermentation solution of photosynthetic bacteria) and pozzo eggs to promote skin metabolism, moisturizing effect, and enhance cleansing power. A functional cosmetic soap composition is characterized by having an effect, an anti-aging effect of the skin by an antioxidant effect, an antibacterial effect and an environmental purification effect.

The present invention, as a photosynthetic bacterium, is intended to develop a cosmetic composition having a skin stimulating effect and skin moisturizing effect by using a cell lysate of Rhodobacter sphaeroides containing bacteriochlorophyll.

The present invention comprises the steps of culturing Rhodobacter sphaeroides containing bacteriochlorophyll (bacteriochlorophyll); after the culture, removing the culture medium and recovering the cells; Crushing the recovered cells; After crushing the cells, adding an extraction solvent and centrifuging; After centrifugation, recovering the supernatant; provides a cosmetic composition for alleviating skin irritation comprising a bacteriochlorophyll-containing extract prepared from the process as an active ingredient.

In another aspect, the present invention, the step of culturing Rhodobacter sphaeroides containing bacteriochlorophyll (bacteriochlorophyll); After the incubation, removing the culture solution and recovering the cells; Crushing the recovered cells; After crushing the cells, adding an extraction solvent and centrifuging; After centrifugation, recovering the supernatant; provides a moisturizing cosmetic composition comprising a bacteriochlorophyll-containing extract prepared from the process as an active ingredient.

Hereinafter, the first aspect and the second aspect of the present invention will be described in detail for each step.

<Cultivation Step: Culture of Rhodobacter Spoidoids Containing Bacteriochlorophyll

This step is a step of culturing Rhodobacter sphaeroides containing bacteriochlorophyll (bacteriochlorophyll).

In the present invention, Rhodobacter sphaeroides belonging to the non-sulfur bacterium among photosynthetic bacteria are used, because it was confirmed that the bacteriochlorophyll was contained as a result of the experiment of the present invention. Rhodobacter spheroides are rod-shaped or spherical with a width of 0.5-1.2 μm and a length of 2-2.5 μm. In the absence of oxygen, carbon assimilation can be done using light like plants, and in the absence of oxygen, oxygen and organic matter can be used to grow.

On the other hand, bacteriochlorophyll (bacteriochlorophyll) is an assimilation pigment of the photosynthetic bacteria, chlorophyll contained in the red bacteria such as red staphylococcus bacterium, red staphylococcus bacterium is called bacteriobiridine. There are several pigments such as a and b in the bacteriochlorophyll, and the clear main pigment of the structure is sometimes called the bacteriochlorophyll a. The structure is a have one of the four blood rolhaek common green chlorophyll-a of the plant _{(C 55 H 72 MgN 4 O} 5) reduced form _{(C 55 H 74 MgN 4 O} 5). In alcoholic solutions, there are major absorption peaks at 357.5 nm and 770 nm, which are bound to proteins in cells and show absorption peaks at 380 nm and 890, 870, 850, and 800 nm. Of these, a light portion of 890 nm is used for photosynthesis, and fluoresces there.

In this step, Rhodobacter sphaeroides containing bacteriochlorophyll having the same characteristics as described above are used. The culture method may use a culture method well known in the art for photosynthetic bacteria. Since the present invention uses the extract obtained from the lysate of the cells, the culturing is better if the method can maximize the yield of the cells.

Cell recovery step; Remove the culture solution and collect the cells>

This step is to remove the culture medium and recover the cells after the culture. The culture solution can be removed from the cells by a variety of methods, for example, can be removed after separation from the cells by centrifugation.

<Cutting of Cells: Crushing of Recovered Cells>

This step is to crush the cells recovered above. Since the present invention uses an extract derived from the lysate of the cells, the cells should be crushed. Crushing the cells is a variety of physical and chemical methods, preferably by applying an ultrasonic wave to the cells.

<Centrifugation Step: Add Extraction Solvent and Centrifuge>

This step is a step of centrifugation after the cell disruption, the extraction solvent is added. In this step, the extraction solvent is added to the cell lysate and centrifuged. Through this step, the useful substance is dissolved in the extraction solvent and extracted. At this time, the kind and amount of useful components to be extracted are changed according to the type of the extraction solvent, the extraction solvent is preferably used any one selected from water, ethanol aqueous solution, aqueous methanol solution, ethyl acetate, chloroform and hexane or a mixture thereof It is good.

Supernatant recovery step: recovering the supernatant

 This step is to recover the supernatant after the centrifugation. This step is a process of separating the supernatant containing the useful component extracted through the extraction solvent. The supernatant of this step contains bacteriochlorophyll as a useful component.

 In the present invention, a cosmetic composition for skin irritation relief or skin moisturizing using the bacteriochlorophyll-containing extract prepared through the above steps as an active ingredient is prepared.

 The cosmetic composition according to the present invention may contain, in addition to the above-mentioned extracts, other ingredients that can give a synergistic effect to the main effect, preferably within the range of not impairing the main effect of the present invention. It may also contain auxiliaries commonly used in the cosmetic field such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, odorants and dyes. The amounts of these various adjuvants are amounts commonly used in the art, such as 0.0001 to 30% by weight relative to the total weight of the composition.

 On the other hand, the formulation of the cosmetic composition of the present invention is not limited to a specific kind, for example, made up of a lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) type and water-in-oil (W / O) type Basic cosmetic formulations; It may be any one selected from oil-in-water and water-in-oil makeup base, foundation formulation.

 The cosmetic composition of the present invention exhibits an excellent skin irritation alleviating effect and a skin moisturizing effect.

 1 is a result of analysis of bacteriochlorophyll for photosynthetic microbial strains. At this time, the culture was all carried out under aerobic conditions.

 2 is Rhodobacter sproidoids (Rhodobacter sphaeroides) According to the fractional concentration of DS-EBN-BL6, it shows the toxicity and proliferative effect of skin keratinocyte line (HaCaT; Keratinocyte).

 3 is Rhodobacter sproidoids (Rhodobacter sphaeroides) According to the fractional concentration of DS-EBN-BL6, it shows the toxic and proliferative effect of skin dermal cell line (HS68; Fibroblast).

 4 is Rhodobacter spheroid death (Rhodobacter sphaeroides) According to the fractional concentration of DS-EBN-BL6, it shows the toxicity and proliferative effect of melanin forming cell line (B16F10; Melanocyte).

5 shows the expression level of TNF-α according to the concentration of Rhodobacter sphaeroides DS-EBN-BL6 water extraction fraction.

Figure 6 shows the ability to inhibit the growth of keratinocytes (HaCaT; Keratinocyte) according to the concentration of bacteriochlorophyll (Bacteriochlorophyll), Astaxanthin (Astaxathin), Xanthophyll, Aminolevulinic acid (ALA).

Figure 7 shows the amount of TNF-a expression according to the concentration of bacteriochlorophyll (Bacteriochlorophyll), astaxanthin (Astaxathin), Xanthophyll, aminodebulic acid (ALA).

Figure 8 shows the results of artificial skin irritation of Rhodobacter sphaeroides DS-EBN-BL6 water extract fractions.

9 shows liposome formulation stability results of photosynthetic microbial water extract fractions.

10-14 show the results of clinical trials of photosynthetic microbial containing and non-containing products.

Hereinafter, the content of the present invention will be described in more detail through the following examples. However, the scope of the present invention is not limited only to the following examples, but includes modifications of equivalent technical ideas.

Example 1 Analysis of the Content of Bacteriochlorophyll in Various Photosynthetic Bacteria

In this example, the bacteriochlorophyll content in cells of various photosynthetic bacteria was analyzed. The strains used for bacteriochlorophyll analysis were Rhodobacter capsulatus, Rhodobacter sphaeroides and Rhodobacter azotoformans , photosynthetic microbial strains isolated from our soil, river and wastewater sludge. As a control, Rhodopseudomonas palustris ATCC 17001 (Koo Ja Yong, Soo Kyung Lee, Myung Kyu Lee (2003), Phototropic Bacteria Producing technic development, Using agriculture and lomestic animals' waste.) Was used.

0.5 g of K ₂ HPO _4, 0.5 g of KH ₂ PO ₄ , 2.7 g of DL-Malic acid, 0.8 g of Ammonium phosphate, 3.76 g of MSG, 1 g of Tryptone, 2 g of Yeast extract, and TE 2.1 ml The photosynthetic microorganisms were inoculated in the medium, and these were incubated for 48 hours at 150 rpm at a light source of 30 lux or more and a temperature of 30 ° C. using a shake incubator.

 Bacteriochlorophyll analysis was performed according to the method described in Clayton, R. K. 1966. Spectroscopic analysis of bacteriochlorophylls in vitro and in vivo.Photochem.Photobiol.

 ① Each photosynthetic microbial culture was centrifuged at 6,000 rpm for 10 minutes (Supra 25K, KOREA) and the supernatant was removed.

 ② Collect the photosynthetic microbial pellet from which the supernatant was removed, and use Sonicator (VCX-750, USA) for 20 minutes at 20 kHz frequency and 25% amplitude for 2 minutes and then stop for 10 minutes using 25 mm solid probe. Cells were disrupted.

 ③ The crushed cells were reacted for 30 minutes while blocking the light using a solvent prepared in methyl alcohol: acetone = 7: 2.

 ④ After the reaction was completed, the photosynthetic microorganism lysate was centrifuged at 12,000 rpm for 10 minutes (Supra 25K, KOREA), and the supernatant was collected.

 ⑤ Optical Density Value (770 nm) of the collected supernatant was measured.

 Analysis of the bacteriochlorophyll for each photosynthetic microorganism strain was as shown in FIG. 1.Rhodobacter sphaeroidesIn the case of DS-EBN-BL6, the components of the bacteriochlorophyll were not the highest compared to other strains, but when converted based on the number of viable cells, it was found to contain the most content of all components.

 Based on this, in the present invention, Rhodobacter sphaeroides having excellent production capacity of bacteriochlorophyll (Rhodobacter sphaeroides) DS-EBN-BL6 was selected as the experimental strain and the skin cells and the improvement effect were verified below. Rhodobacter SpiroidesRhodobacter sphaeroides) DS-EBN-BL6 was deposited on August 9, 2010 with the Korea Research Institute of Bioscience and Biotechnology. KCTC11738BP was given.

Example 2: Confirming the skin improvement effect of the selected photosynthetic microorganisms ( In vitro )]

 Prior to the skin improvement test, samples were prepared using various solvents to obtain a fraction of photosynthetic bacterial cells, and the method was as follows.

 ① Water extraction, ② EtOH extraction, ③ 70% EtOH extraction, ④ MeOH extraction, ⑤ 70% MeOH extraction, ⑥ Hexane extraction, ⑦ Hexane: EtOH = 3: 1 extraction, ⑧ Chloroform: MeOH = 2: 1 extraction.

 Extraction proceeded slowly at 12O &lt; 0 &gt; C for 12 hours. After extraction for 12 hours in each solvent, the extract was filtered and dried in vacuo to obtain an extract. Thereafter, the photosynthetic microbial extract was dissolved in DMSO (dimethyl sulfoxide), and the following efficacy evaluation experiment was conducted.

MTT assay was performed to investigate the effects of photosynthetic microorganisms on skin cell growth. Fibroblast, HaCaT, and B16F10 cells at a concentration of 7 × 10 ³ were inoculated in 96 well plates and incubated for 24 hours, and then photosynthetic microbial lysates were treated in medium to further culture for 24 hours. At this time, the group treated with the photosynthetic microbial culture medium and the culture supernatant from which the cells were removed were used as a control group and compared with the results of the experimental group samples.

As a result, as shown in Figures 2 to 4 concentration-dependent toxicity was expressed in the fraction using hexane, chloroform and MeOH. However, due to the characteristics of each cell line, it is good to have the compatibility of growth inhibition (HaCaT, B16F10) and growth proliferation (Fibroblast), and based on the safety of the solvent used, it is considered that the fraction using water is most suitable. In other words, the fractions using water were more toxic to keratinocytes and melanin-forming cell lines than 'medium used for culturing R. sphaeroides DS-EBN-BL6' and 'supernatant without cells used for fractionation'. It is excellent in terms of effectiveness because it has a growth proliferation effect on the dermal cell line.

 Meanwhile, the anti-inflammatory activity was measured. The anti-inflammatory activity was evaluated by measuring the reduction effect of TNF-α, which is a representative inflammatory cytokine, and dexamethasone was used as a control. TNF-α is a 'tumor necrosis factor' that signals tumor cells to kill themselves, inhibits intracellular replication of viruses, stimulates macrophages, and promotes inflammatory responses. are signaling molecules that are widely involved in the innate immune response.

For experiments, 1 × 10 ⁵ concentrations of HaCaT cells were inoculated into 12 well plates and incubated for 24 hours, and then the samples were treated in different concentrations in the medium with UV irradiation for the purpose of inducing TNF-α and further cultured for 24 hours. It was. When not treated with UV, the group treated with dexamethasone treated with 1 μM / ml after UV was used as a control and compared with the results of the samples.

 In the case of the TNF-α assay, HaCaT cells were used to irradiate UV at 312 nm at 12.5 mJ, and then treated each fraction, incubated for 24 hours, and measured TNF-α secreted into the medium. The recovered medium was measured for absorbance at 450 nm using a microplate reader (Perkin Elmer, USA) using the TNF-α kit (invitrogen, # KHC3012).

 Experimental results show that photosynthetic microbial water fractions are toxic to keratinocytes and melanin forming cells and have a growth-proliferating effect on dermal cells. Was excellent (FIG. 5).

Example 3 Indicator Material Utilization Test for Efficacy Expression In vitro )]

 Since the amount of the bacteriochlorophyll contained in the photosynthetic microorganism is not high, it was intended to find out how much is effective. Bacteriochlorophyll, carotenoids (Astaxathin, Xanthophyll), aminolevulinic acid (ALA), which are known to be useful substances in photosynthetic microorganisms, were tested. Attempts to determine whether the data is reproduced and whether other substances are involved.

 As shown in FIG. 6, bacteriochlorophyll was most excellent in virulence expression of HaCaT cells, and astaxanthin corresponding to xanthophylls among carotenoid pigments was excellent. In addition, as a result of experimenting with their TNF-a inhibitory ability, astaxanthin and bacteriochlorophyll were inhibited at the lowest concentration, as shown in FIG.

Example 4 Skin Efficacy Verification Using Artificial Skin

 For skin irritation, skin sensitization test is performed using artificial skin EPI-300 tissue. To evaluate. The test results of each sample were as shown in FIG. 8. When a group with cell viability of 50% or less compared to the control group was considered to have a stimulus, there was no significant stimulus in most samples (see FIG. 8).

Example 5: Stability Test in Liposomal Formulations of Photosynthetic Microbial Water Extract Fractions

 In this example, liposomes including photosynthetic bacterial fractions were prepared to check their stability. In preparing liposomes, hydrogenated purified soy lecithin was used to eliminate unsaturation. Phospholipid general composition of the purified soy lecithin was shown in Table 1 below.

Table 1

ingredient	weight%
Phosphatidylcholine	70-95
Phosphatidylethanolamine	0-15
Phosphatidyl Inositol	0-2
Lysophosphatidylcholine	0-5

Dissolved in. The solvent was evaporated from the obtained solution to form a thin film. Liposomes of a certain size were then prepared by hydration and sonication at 60 ° C. above the phase transition temperature of the lipid.

 As a result of measuring formulation safety at 40 ° C. for 6 months, it was found that the size of liposomes was well maintained without significant change (see FIG. 9).

Example 6: Clinical Trial (Formulation Development)

 Cosmetic formulations were developed using photosynthetic bacterial fractions. To the cream base of the following Table 4, the photosynthetic microbial culture lysate of the present invention was added to 10% to complete the formulation and proceed with the clinical experiment.

TABLE 2

division	Sysmedic cream	content(%)
Award	DI-Water	70.708
	EDTA-2NA	0.02
	Glycerin	4
	1.3-BG	2
	Keltrol-F	0.05
Thickener	Carbopol # 940	0.1
antiseptic	DM	0.2
	DP	0.1
Oil part Ⅰ	Stearic acid	One
	Kalcol 6870	One
	Kalcol 8688	0.8
	GMS 105	One
	Ari 165	0.3
	Mango butter	0.3
	Myrj 52s	1.2
	PhytoSqualane	2
	CIO	3
	Jojoba oil	3
	LP 70	2
	DC 200 / 100cs	0.5
	Phytoshpningosine	0.15
	Cholestrol	0.5
	PC 95	0.05
Oil part Ⅱ	Borage seed oil	0.3
	DC345	2
extract	Gemall 115	0.2
	Vitamin E Acetate	0.5
	Allantoin	0.1
	DL-Panthenol	0.2
	Chamomile Ext.	One
	Licolice BG 100	0.2
	SC-Glucan	One
	Niacinamide	0.001
	Guava Ext.	0.001
	KXDUW0028 (incense)	0.18
pH regulator	TEA	0.14
Thickener	Rheocare ATH	0.2
Remarks	HM: Homo Mixer / PM: Paddle Mixer
-Working process- 1. Put the water part in order and heat it to 80 ~ 85 ℃ transparently. Then add a thickener. 2. Put the preservative, oil part I, extract, and cream base sequentially in the oil part and heat it to 85 ~ 90 ℃. 3. After confirming that oil part I is completely dissolved, add oil part Ⅱ at about 80 ℃. * Borage oil is added just before emulsification because there is a risk of deterioration. Mix until 4. After confirming that the oil content is completely dissolved, it is added to the water phase at 75 to 80 ° C. to give the first emulsification, and homogenize at 4,000 to 4,500 rpm (HM) for 3 minutes. Add extract and flavor at 55 ° C and homogenize for 5 min at 3,500-4,000 rpm (HM). After adding the pH adjuster at 45 ~ 50 ℃ homogenization for 3 minutes at 3,500 ~ 4,000rpm (HM).

 The test group of the photosynthetic microbial fraction product prepared above and the control group other than the control group were commissioned to the PNK (P & K) Skin Clinical Research Center, and the "human efficacy evaluation for alleviation of itching caused by dry skin" was conducted according to the standard test method (SOP). It was.

 In clinical trials, five items were measured: ① skin moisture content, ② epidermal moisture evaporation, ③ skin temperature measurement, ④ skin pH measurement, and ⑤ SCORAD index change rate.

TABLE 3

Age (years)	Number of people	%
1 to 9	22	47.8
10 to 19	9	19.6
20 to 29	10	21.7
30 to 39	5	10.9

 Compliance with clinical product use was calculated as a percentage of the number of uses used over the course of the trial. The total compliance of the 43 who completed the test was 95.7%, with a maximum compliance of 100% and a minimum compliance of 81.5%. Since no subjects showed less than 80% compliance in this trial, all 43 subject data were used for outcome analysis. Three of the 46 patients who participated in this trial were eliminated due to follow-up failure, and a total of 43 completed the trial. The dropout information was as follows (Table 4).

Table 4

Dropout	12N14-A1-14	12N14-A1-18	12N14-A1-46
Reason for dropping out	Tracking failure	Tracking failure	Tracking failure
Dropout date	D28	D14	D14
gender	female	male	male
age	22	20	7

 The clinical trial results of the cream base product containing 10% photosynthetic microbial fraction and the cream base product to which the photosynthetic microbial fraction was not added were as shown in FIGS. 10 to 14. The skin moisture content was increased by 15.25% after 2 weeks and 28.26% after 4 weeks of use compared to the change rate of the control product. As for the epidermal moisture evaporation rate, it was found to increase 9.42% after 2 weeks and 16.59% after 4 weeks of use, compared to the improvement rate of the control product. The change in skin temperature was found to increase 0.14% after two weeks of use and 0.57% after four weeks of use, compared to the rate of improvement of the control product. The SCORAD index increased by 1.39% after 2 weeks of use and 0.07% after 4 weeks of use, compared to the rate of change of the control product.

  As a result, it was found that the cream base product containing photosynthetic microorganisms improved skin dryness as compared to the product containing no photosynthetic microorganisms. At this time, the difference with the control product is not a large phenomenon due to the fact that the cream base formulation has some effect on skin improvement.

[Correction under Rule 91 14.03.2013]

Claims (4)

1. Culturing Rhodobacter sphaeroides containing bacteriochlorophyll;

    After the incubation, removing the culture solution and recovering the cells;

    Crushing the recovered cells;

    After crushing the cells, adding an extraction solvent and centrifuging;

    After the centrifugation, recovering the supernatant; a cosmetic composition for alleviating skin irritation, comprising a bacteriochlorophyll-containing extract prepared from the process comprising as an active ingredient.
2. Culturing Rhodobacter sphaeroides containing bacteriochlorophyll;

    After the incubation, removing the culture solution and recovering the cells;

    Crushing the recovered cells;

    After crushing the cells, adding an extraction solvent and centrifuging;

    After the centrifugation, recovering the supernatant; moisturizing cosmetic composition comprising a bacteriochlorophyll-containing extract prepared from the process comprising as an active ingredient.
3.  The method according to claim 1 or 2,

    Crushing the cells,

    Cosmetic composition, characterized in that performed by applying ultrasonic waves to the cells.
4.  The method according to claim 1 or 2,

    The solvent,

    Cosmetic composition, characterized in that any one selected from water, aqueous ethanol, aqueous methanol solution, ethyl acetate, chloroform and hexane or a mixture thereof.

Images