KR20180124432A - Composition for improving acne with antioxidant, antibacterial and anti-inflammatory properties - Google Patents

Abstract

The present invention relates to a composition for treating acne with antioxidant, antibacterial and anti-inflammatory properties, and more particularly, to a composition for treating acne with antioxidant, antibacterial and anti-inflammatory properties, which comprises a Pseudomonas aeruginosa culture solution extract containing Rhamnolipids and 7,10-dihydroxy-8(E)-octadecenoic acid as an active ingredient, and to a composition for treating acne, which further comprises a leaf extract of Pueraria montana to the composition.

Description

TECHNICAL FIELD The present invention relates to a composition for improving acne with antioxidant, antibacterial and anti-inflammatory properties,

The present invention relates to a composition for improving acne with antioxidative, antibacterial and anti-inflammatory properties, and more particularly, to a composition for improving acne which comprises Rhamnolipids and 7,10-dihydroxy-8 (E) -octadecenoic acid. A composition for improving acne with antioxidant, antibacterial and antiinflammatory activity, which comprises an extract of Pseudomonas aeruginosa culture fluid as an active ingredient, and a composition for improving acne, which further comprises a leaf extract thereof.

The exact cause of acne has not yet been elucidated in the field of skin care and is caused by the interaction of several factors, so there is no single effective treatment until now. Acne is a pilosebaceous disease characterized by lesions such as cotton papules, papules, pustules, and nodules that occur particularly during puberty, and the incidence of acne has increased in recent adults.

In Western civilized society, about 79-95% of teenagers have acne disease, and in the United States, about 4,000 to 50 million people are counted as acne patients. This is not limited to teenagers but is widely distributed among children and adults. In one study, 40% of adult men aged 25 or older and 54% of adult women reported experiencing acne. Acne became one of the major diseases that civilized society can not avoid.

As a pathogen of skin diseases such as acne, the production of free radicals due to environmental habits, eating habits, stress and physical strength, and the resulting oxidative stress of cells have been studied and understood. It is known that some of the oxygen used in human cells is partially reduced to active oxygen, and the resulting oxidative stress stimulates the cells to cause various diseases and skin diseases in the skin.

Nitric oxide (NO) is a kind of highly reactive biogenic radical that is formed by NOS (Nitric Oxide Synthase) composed of three types of neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (L-arginine) and oxygen (O ₂ ). In normal physiological state, it plays a physiologically important role such as nerve signal transmission and immunity through the death of bacteria. Excessive action may cause inflammation of the living body, Mutations, damage to tissues and nerves, and so on.

In addition, NO directly or indirectly regulates the activity of cyclooxygenase-2 (COX-2) and directly reacts with superoxide anions radicals to generate ONOO ^- (peroxynitrite anions) , Which increases the catalytic activity of COX-2 and indirectly triggers signaling cascade and regulates the expression of COX-2 in the transcription stage.

COX-2 is an inducible isoform of COX, which is one of the important factors controlling inflammatory response. It is involved in various intractable diseases and is known to be expressed acutely in pathological inflammatory reaction. In general, when the inflammatory reaction occurs in vivo, it is known that iNOS produces a large amount of NO and COX-2 expression is increased. Therefore, a substance that inhibits the production of iNOS and COX-2 is a potential modulator of inflammation including acne Is known to be high.

The pathogenesis of other pathways of acne includes increased sebum secretion by male hormones, increased hair follicle walls, inflammation induced by proliferation of Propionibacterium acnes (P. acnes), skin barrier abnormalities, Environmental factors, and reactivity of hair follicles. The most important cause of acne is the proliferation of acne-causing P. acnes. P. acnes is a lipophilic microorganism that grows in the follicle as an anaerobic mantle. Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) are aerobic skin strains normally distributed in the skin and are not the primary cause of inflammation. However, Deteriorate.

Inflammatory acne caused by bacterial infection among the causes of acne can be ameliorated by the administration of antibiotics. However, long-term use of tetracycline, clindamycin, and erythromycin, which are mainly used in the treatment of acne, , It may cause side effects or resistance, resulting in decreased therapeutic efficacy. In addition, the combination of retinoids, which have a strong solubilization function for inhibiting acne formation, and antibiotics for the inhibition of P. acnes proliferation may improve the therapeutic effect but also increase the side effects.

Although antimicrobial agents used in cosmetics are essential, synthetic materials used in the past can cause allergies to the skin. Therefore, research for searching natural products that are effective for acne skin, safe for skin and minimized side effects is required, and natural antimicrobial Interest in the market is also increasing rapidly.

Therefore, there is a need to develop a product that is safe for human body and has excellent improvement effect on acne skin.

Korean Patent Publication No. 10-2016-0025950 Korean Patent Publication No. 10-2016-0131755 Korean Patent Publication No. 10-2007-0027151 Korean Patent Publication No. 10-2001-0038285

It is an object of the present invention to provide a composition for improving acne with antioxidant, antibacterial and anti-inflammatory properties by solving the above-mentioned problems.

It is still another object of the present invention to provide a cosmetic composition for improving acne, a composition for external application for improving acne, and a composition for improving acne for treating, preventing or improving acne.

The present invention provides an antioxidant, antimicrobial and anti-inflammatory effect composition for improving acne comprising an extract of Pseudomonas aeruginosa culture solution containing rhamnose and hydroxy fatty acid as an active ingredient.

The present invention also provides a composition for improving acne characterized by further comprising a mulberry leaf extract in the composition for improving acne.

The composition for improving acne according to the present invention has antioxidative, antibacterial and anti-inflammatory properties derived from natural materials without using a chemical antibiotic, and has an acne-reducing effect without side effects on the human body.

The composition for improving acne according to the present invention is excellent in antimicrobial activity against acne causing bacteria and has excellent prevention and improvement effect against acne and can be used as a cosmetic, food or skin external composition composition.

Fig. 1 shows antimicrobial effects of seven kinds of dermatophytes of rumorifide and DOD at the same time. The photographs are shown together with the measured photographs and the measured inhibition rings.
2 shows the names of the strains used in the experiment.
Fig. 3 shows the result of MIC test for Staphylococcus epidermidis, which exhibited the effect of a substance containing both Ramanolipid and DOD in a growth inhibition ring assay.
Fig. 4 is a result of MIC test on Propionibacterium acnes showing the effect of a substance containing both Ramanoride and DOD in the growth inhibition assay.
FIG. 5 shows the DPPH radical scavenging activity of the extract of Liliaceae.
FIG. 6 shows the results of ABTS radical scavenging ability measurement of the 칡 leaf extract.
Fig. 7 shows the results of measurement of total polyphenolic contents of Leaf leaf extract.
FIG. 8 shows the results of measurement of cell viability of macrophages (Raw264.7) according to the concentration of Leaf leaf extract.
FIG. 9 shows the results of measurement of the inhibitory effect of Nitric oxide formation on macrophage (Raw 264.7) according to the concentration of Leaf leaf extract.
FIG. 10 shows the results of measuring the inhibitory effect of iNOS and COX-2 protein expression on macrophages (Raw 264.7) according to the concentration of 칡 leaf extract

Hereinafter, the present invention will be described in detail.

The present invention is not limited to the embodiments described below, but can be embodied in various other forms.

The definitions listed below are definitions of various terms used to describe the present invention. These definitions are used to describe only specific embodiments, unless otherwise limited, and are not intended to limit the invention.

The present invention relates to a composition for improving acne with antioxidative, antibacterial and anti-inflammatory properties derived from natural materials without using chemical antibiotics. The composition comprises a biosurfactant extracted from a strain of Pseudomonas aeruginosa, Leaf extract).

Biosurfactant is an industrially useful material that is comparable in chemical and physical properties to chemically synthesized surfactants but is non-toxic and easy to biodegrade as an environmentally friendly surfactant.

Sophorolipid is a glycolipid-based microbial surfactant that can be used as a detergent and an oil dispersant due to the physical and physiological properties of sophorolipid and has high antimicrobial activity against Gram-positive bacteria including acne bacteria. Otherwise, there is little side effect on the human body, and it does not cause resistance problem and is excellent in biodegradation.

In the case of the soporolipid, rhamnolipid is known to be produced by some Pseudomonas strains and has been reported to exhibit excellent dispersing ability and antimicrobial activity against Gram-positive microorganisms, and is being developed as a high-grade biosurfactant.

The worldwide surfactant market has been growing at a rapid rate of more than 50% a year since the early 1990s, and the demand is steadily increasing. In the case of biosurfactants, the demand for environmentally friendly products such as cosmetics, pharmaceuticals, foods, detergents, pulp and paper, and environmental purification varies depending on product characteristics.

Hydroxy fatty acid (HFA) is a form that has at least one hydroxyl group in the central chain of a common fatty acid. The hydroxyl group added to the fatty acid chain gives the fatty acid a unique property such as high viscosity and reactivity . Hydroxy fatty acids are classified into mono-, di-, and tri- (hydroxy) fatty acids depending on the number of hydroxyl groups to which they are attached and include a separate structure in addition to the hydroxyl group Or an epoxy-hydroxy fatty acid or an oxo-hydroxy fatty acid.

Hydroxy fatty acids are found in trace amounts in plants mainly in nature. However, hydroxy groups introduced into hydroxy fatty acids give fatty acids with specific properties such as high viscosity and reactivity, and thus they can be used in various industrial fields. As a result, it can be widely applied to various industries such as pesticides, medicines, high-functional resins and fiber materials, biodegradable plastic materials, lubricants, cosmetics and paints.

칡 is a perennial plant that has been eaten as a wild plant since long ago and has been used as a health food such as nourishing tonic. In the oriental medicine, it is said that the roots are used as a medicinal substance called 葛根, and it is said to have effects such as perspiration and fever. In recent years, there has been a tendency that the use of 칡 is limited, and the related research is also focused on the root, and domestic papers and patents about the skin pharmacological efficacy of 칡 leaf are very few.

The present invention relates to an antioxidant, antimicrobial and anti-inflammatory agent comprising, as an active ingredient, a Pseudomonas aeruginosa culture liquid extract containing Rhamnolipids and 7,10-dihydroxy-8 (E) -octadecenoic acid The composition for improving acne is provided.

In the present invention, by culturing Pseudomonas aeruginosa (KACC 10186), DOD (7,10-dihydroxy-8 (E) -octadecenoic acid) and Rhamnolipids, which have antimicrobial activity against acne, And the extract was used.

The present invention has found that DOD, which is effective for acne bacteria, is present in addition to the conventionally known substance rhamnolipid among the Pseudomonas aeruginosa culture extract.

This is the traditional acne remedy that causes side effects or resistance when used for a long time. It is complementary to the disadvantages of tetracycline, clindamycin, erythromycin and allergies known as side effects of other synthetic antimicrobials. And it is industrially useful material because it is easily biodegradable.

In one embodiment of the present invention, the composition for ameliorating acne may be a composition for improving acne, which further comprises a lupine extract.

In order to develop a better composition, the extract of P. aeruginosa culture solution of the present invention is enhanced by adding phytophthora extract obtained by pharmacological experiment, which has been proved to have antioxidant and anti-inflammatory effects.

칡 Leaf extract is a natural substance that has a strong antioxidant and anti-inflammatory action. It stimulates the cells inside the human body. It suppresses oxidative stress that causes various diseases and skin diseases in the skin. If it acts excessively, And damage the nervous system, and acts to control the inflammatory phenomenon that is harmful to human body.

The 칡 leaf extract according to the present invention is obtained by extracting 칡 leaf with an organic solvent.

By combining the extract of Pseudomonas aeruginosa with the extract of P. aeruginosa cultivated with the simultaneous production of rhamniripid and hydroxy fatty acid, the synergistic effect on the efficacy can be seen, and the antioxidant, antibacterial and antiinflammatory action can be uniformly used.

Mono-rhamnolipids of the culture solution of Pseudomonas aeruginosa (KACC 10186) according to the present invention are represented by the formula (1), and DOD (7,10-dihydroxy-8 (E) -octadecenoic acid) 2:

[Chemical Formula 1]

(2)

In one embodiment of the present invention, it may be a composition for improving acne characterized by exhibiting an antibacterial activity against Staphylococcus epidermidis or Propionibacterium acnes.

In one embodiment of the present invention, it may be a composition for improving acne, which comprises the rhamnolipid and the hydroxy fatty acid at a weight ratio of 1: 0.1 to 0.8.

In one embodiment of the present invention, the acne improvement composition comprising the extract of a culture solution of P. aeruginosa in which rhamnose and hydroxy fatty acid are simultaneously produced is contained in an amount of 0.001 to 20 parts by weight based on the total weight of the composition Or a composition for improving acne.

In one embodiment of the present invention, the use ratio of the extract of Pseudomonas aeruginosa culture and the extract of Liliaceae is not particularly limited and may be, for example, in a ratio of 1: 0.5 to 30. When the content ratio is within the above range, the antioxidant, antibacterial and anti-inflammatory effects can be maximized.

In one embodiment of the present invention, the composition for improving acne, which further comprises a fungus leaf extract in the extract of the Pseudomonas aeruginosa culture solution in which the rhamnolipid and the hydroxy fatty acid are co-produced, comprises 0.001 to 20 parts by weight The composition for improving acne is preferably 1 to 10 parts by weight, more preferably 5 to 10 parts by weight. When the content is less than 0.0001 part by weight, the effect of treating, preventing and improving acne is insufficient. When the content is more than 20 parts by weight, the effect of increasing the content is not increased and the stability of the shape is not secured.

In one embodiment of the present invention, the composition for improving acne may be a cosmetic composition for improving acne, a composition for improving acne, or a composition for external application for improving acne.

The composition according to the present invention can be prepared into any formulation conventionally produced in the art. For example, they may be formulated as solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays, But is not limited thereto.

Hereinafter, specific embodiments of the present invention will be described. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

< Preparation Example  And Experimental Example >

[ Preparation Example  One] Randomized  Preparation of a substance containing DOD at the same time

1-1. Culture of Pseudomonas aeruginosa KACC 10186 strain

Pseudomonas aeruginosa KACC 10186 strain was produced under the following culture conditions for the co-production of monoramnolipid and dihydroxy fatty acid with vegetable oil as a substrate.

Pseudomonas air Rouge labor KACC 10186 to M-NB medium (0.7% glycerol, 0.1% meat extract (beef extract), 0.2% yeast extract, 0.5% peptone, 0.5% sodium chloride (NaCl), 0.02% magnesium sulfate strain (MgSO ₄₎ Lt; RTI ID = 0.0 &gt; 30 C. &lt; / RTI &gt;

1% olive oil was added as a substrate to the culture medium of KACC 10186, which was cultured in M-NB medium for 24 hours, and further cultured and terminated. After the completion of the incubation, the same amount of ethyl acetate was added to extract the supernatant, and the extract was concentrated using a rotary evaporator.

The crude extract obtained through the above procedure contains 7,10-dihydroxy-8 (E) -octadecenoic acid (DOD), which is an antibacterial active material, and mono Randomized feeds are included.

1-2. Partial purification of crude extract (acidification method)

The concentrated crude extract was mixed with distilled water at a ratio of 1: 5, and then the pH of the solution was adjusted to 2-4 by adding 5N hydrochloric acid (HCl), followed by storage at 4 ° C for 24 hours. After the precipitate and the supernatant were separated, hexane was mixed with the precipitate to extract the rhamnolipid and then concentrated using a rotary evaporator. After that, the sample was pulverized by freeze-drying to conduct the experiment.

[ Preparation Example  2] Production of 칡 leaf extract

The extracts were prepared by filtration through a filter paper, concentrated under reduced pressure, and then concentrated under reduced pressure. The extracts were washed three times with distilled water and dried at room temperature for 24 hours using 70% And lyophilized to be powdered. Storage was kept in a 4 ° C refrigerator room and used as a test sample.

[ Experimental Example 1 ] Randomized  Antibacterial effect of substances containing DOD at the same time

1-1. Culture of bacteria

In order to investigate the antimicrobial effect of substances containing both ranorifide and DOD, seven strains were grown and used in the experiment.

Candida albicans were cultured in a 5% CO 2 incubator at 25 ° C. Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Propionibacterium acnes, Escherichia coli, Enterobacter cloacae, Escherichia coli, The bacteria were cultured at 37 ℃ in a 5% CO2 incubator.

1-2. Inhibition of growth  Measure

The antimicrobial activity was measured by a paper disc agar diffusion method. Each of the strains cultured on the solid plate culture medium was inoculated into 10 mL of liquid culture medium by inoculating 1 mL of the culture medium for 18-24 h and then inoculated with 10 mL of the culture broth in 0.1 mL of the culture broth for 3-6 h, The number of bacteria on the plate medium is about 1? 10 ⁷ &lt; / RTI &gt;

After the sterilized paper disc was placed on a solid plate medium, 0.05 mL of the diluted sample solution was absorbed per disc and incubated for 24 to 72 hours in an incubator under conditions suitable for each microorganism. The diameter of the clear zone (mm) of the sample was measured.

1-3. MIC  Test

Staphylococcus epidermidis (Staphylococcus epidermidis), which has antibacterial activity among seven strains, Propionibacterium acnes (Propionibacterium acnes) The experiment was conducted to determine the minimum inhibitory concentration (RAM) concentration of the test compound. Bacteria were obtained by inoculating 1 ml of each strain 1 cultivated in a solid plate culture medium into 10 ml of a liquid medium, incubating them for 18 to 24 hours, activating them, and subsequently inoculating 0.1 ml of the bacterial solution into 10 ml of the liquid medium for 3 to 6 hours . The number of each bacteria used in the experiment was adjusted to about 1 × 10 ^{6, and} then diluted 10 ^-1 in the liquid medium. The ranorifide used in the experiments was diluted to concentrations of 0.01%, 0.02%, 0.04%, 0.08%, 0.16%, 0.31%, 0.63%, 1.25% and 2.5%. 100 μL of each culture medium was dispensed into 96-well plates in a 96-well plate. 100 μL of each culture medium was added to each well of the 96-well plate. 100 μL of rhamnoside was added to the control, and 600 nm The absorbance was measured.

2-1. Inhibition of growth  Measurement result

Antimicrobial activities of co - products of Randomized and ROD were measured using various strains (Escherichia coli, Enterobacter cloace, Staphylococcus epidermidis, Pseudomonas auruginosa, Stapylococcus aureus, Propionibacterium acnes, Candida albicans).

As a result, the clear zone for Staphylococcus epidermidis was measured at 20 mm at 0.5 mg / disc condition, and Propionibacterium acnes, a causative agent of acne, The clear zone for the bacteria was measured to be 21 mm, confirming the excellent antibacterial effect against these fungi.

2-2. MIC  Test results

The MIC test was conducted to confirm the minimal inhibitory concentration of the co-products of Randomized and DOD. Staphylococcus epidermidis, Staphylococcus epidermidis, Staphylococcus epidermidis, Experiments on Propionibacterium acnes showed that the minimum inhibitory concentration of Staphylococcus epidermidis was 0.16% and the minimum inhibitory concentration of Propionibacterium acnes was 0.02 %.

[ Experimental Example 2 ] Antioxidative Effect of Leaf Extracts

1-1. DPPH  Radical Scatters

The antioxidative effect of DPPH radical scavenging was measured by Blois method. To 120 μL of each sample solution, 60 μL of 0.45 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH) was added and reacted for 15 minutes. The absorbance at 517 nm was measured and the result was calculated.

DPPH radical scavenging ability (%) = (1-absorbance group in the sample addition group / absorbance in the no-addition group) × 100

1-2. ABTS  Radical Scatters

The antioxidative effect of ABTS ⁺ radical scavenging was measured by Van den Berg method. A mixture of 7 mM 2,2 azinobis (3-ethylbenzothiazoline-6-sulfonic acid) and 2.4 mM potassium persulfate was mixed with 24 mM allowed to stand for a time and was used after dilution with 99% ethanol after forming the ABTS ⁺ radical. was placed a 100 μL ABTS ⁺ solution was diluted to 100 μL of each sample solution was reacted for 7 minutes in a dark room by measuring the absorbance at 734 nm results Respectively.

ABTS ⁺ radical scavenging ability (%) = (1-absorbance group in the sample addition group / absorbance in the no-addition group) × 100

1-3. Total polyphenol content (total polyphenolic  contents)

Quantification of polyphenol compounds was determined by the Folin-danis method. Add 50 μL of the diluted sample solution to 50 μL of phenol reagent (Folin-Ciocalteu phenol reagent), react at room temperature for 3 min, add 50 μL of 0.7 M sodium carbonate (Na ₂ CO ₃ ) solution, And the absorbance at 730 nm was measured. The polyphenol content was calculated in comparison with a previously prepared absorbance graph of a reference substance tannic acid.

2-1. DPPH  Radical Scatters  Measurement result

The DPPH radical is a stable free radical which is very stable to organic solvents such as alcohol and reacts with antioxidants. It is characterized by decolorization by a proton-radical scavenger in yellow, The difference of the absorbance by 517 nm becomes maximum at around 517 nm. As a result of this experiment, the antioxidative effect of the substance can be confirmed and it is widely used to search for antioxidant activity by using a relatively simple method. Leaf extract showed a concentration - dependent DPPH elimination ability and the effect was constantly maintained. The effect was more than 76% at 1,000 ㎍ / mL concentration.

2-2. ABTS  Radical Scatters  Measurement result

The ABTS cation radical is a type of cyanide radical that reacts with antioxidants and is characterized by discoloration by light green. Hydrogen is known to be able to measure both hydrogen-donating antioxidant and chain-breaking antioxidant, and it is one of the commonly used methods for measuring antioxidant capacity, as it is relatively easy to use as DPPH . The antioxidative effect of 칡 leaf extracts was demonstrated by the concentration dependent ABTS scavenging ability and its effect was over 94% at 1,000 ㎍ / mL concentration.

2-3. Total polyphenol content (total polyphenolic  contents) measurement result

The content of total polyphenol compounds was calculated using a standard curve using tannic acid. Total polyphenol content of 칡 leaf extract was 58.56 mg TAE / g.

The content of polyphenol compounds was found to have an effect on DPPH and ABTS radical scavenging ability.

[ Experimental Example 3 ] Anti-inflammatory effect of 칡 leaf extract

1-1. Cell culture

To determine the anti-inflammatory effect, RAW 264.7 murine macrophage cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin (100 units / mL) And incubated at 37 ° C in a 5% CO ₂ incubator (CO ₂ incubator).

1-2. Cells according to the concentration of 칡 leaf extract Survival rate  Measure

Cell viability was measured according to Carmichael's method. Cell line RAW 264.7 was seeded in 96 well plates at 1 x 10 ⁴ cells / well and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. Subsequently, each sample solution was treated at different concentrations and incubated at 37 ° C in a 5% CO 2 incubator for 24 hours. In the control group, the same amount of DMEM medium as the sample was added and cultured under the same conditions. Then, 0.02 mL of MTT solution prepared at 5 mg / mL concentration was added and incubated for 4 h. Then, the culture solution was removed, 0.1 mL of dimethyl sulfoxide (DMSO) was added to each well, And the absorbance at 540 nm was measured.

Cell viability (%) = (absorbance of sample group / absorbance of group without addition) x 100

1-3. Measuring the inhibitory effect of nitric oxide formation

The amount of nitric oxide (NO) produced was measured by using a griess reagent system. The cell line RAW 264.7 was seeded in a 96-well plate at 5 x 10 ⁴ cells / well and cultured in a 5% CO 2 incubator at 37 ° C for 24 hours. Lipopolysaccharide (LPS) was treated at 1 ppm / well to induce the inflammatory response of the cells. After treatment with each sample solution, the cells were incubated at 37 ° C in a 5% CO 2 incubator for 24 hours Lt; / RTI &gt; In the control group, the same amount of DMEM medium as the sample was added and cultured under the same conditions. Then, 100 μL of the cell culture was transferred to a new 96-well plate, and 100 μL of a griess reagent was added. After reacting at room temperature for 5 minutes, absorbance was measured at 540 nm and the result was calculated.

NO production amount (%) = (absorbance of sample addition group / absorbance of no addition group) x 100

1-4. iNOS , The inhibitory effect of COX-2 protein expression

The cell line RAW 264 was seeded in a 96-well plate at 2 × 10 ⁵ cells / well and cultured in a 5% CO 2 incubator at 37 ° C. for 24 hours. Lipopolysaccharide (LPS) was treated at a concentration of 1 ppm / well to induce the inflammatory response of the cells after the exchange of the serum with the serum-free medium. Each sample solution was treated at different concentrations and then incubated at 37 ° C in a 5% CO 2- Lt; / RTI &gt; In the control group, the same amount of DMEM medium as the sample was added and cultured under the same conditions. After removing the medium and washing once with PBS, the cells were lysed with 60 μL / well of RIPA buffer containing protease inhibitor (0.1%) and centrifuged to remove 4 And centrifuged at 15,000 rpm for 15 minutes.

The supernatant obtained by centrifugation was quantified by Bradford assay and 20 μL of the protein was separated by 10% SDS-PAGE and transferred to a PVDF membrane using a power supply electrophoresis, The cells were blocked with 5% skin milk, primary antibody, washing, secondary antibody and washing procedure using a digital reciprocating shaker, followed by reaction with HRP substrate reagent for 2 minutes, The band was identified using a western imaging system instrument and the results were calculated.

2-1. Cells according to the concentration of 칡 leaf extract Survival rate  Measurement result

The cell viability of macrophage cells (RAW264.7) was determined by MTT assay. Leaf extract showed stable cell viability up to 100 μg / mL and cell viability tended to decrease sharply. Therefore, the highest concentration of 칡 leaf extract available for cell experiments using macrophage cells was set at 100 μg / mL.

2-2. Nitric oxide production inhibitory effect measurement result

To determine the inhibitory effect of LPS- induced macrophage cells (RAW264.7) on the production of NO by nitric oxide (NO) treatment, Were measured. The effect of 칡 leaf extract on the cell viability of the highest concentration was 54%, indicating that it effectively inhibited the formation of nitrogen oxides .

2- 3. iNOS , The inhibitory effect of COX-2 protein expression

Western blot analysis was performed to examine the anti - inflammatory effect obtained from the nitric oxide inhibitory activity experiment and the anti - inflammatory mechanism in the intracellular protein unit. As a result, it was confirmed that the 칡 leaf extract inhibits the production of intracellular inflammation-related proteins iNOS and COX-2 with excellent effects. The effect at the highest available concentration of 100 μg / mL inhibited iNOS protein production by about 99% and COX-2 protein by about 98%.

< Example  And Comparative Example >

An emulsion for acne was prepared by a method known in the art in the composition of the following [Table 1]. The units in Table 1 below are% by weight.

Ingredients Content (% by weight) Example 1 Example 2 Comparative Example 1 Comparative Example 2 Randomized and DOD Simultaneous Products 1.0 1.0 - - 칡 leaf extract - 1.0 1.0 - glycerin 5.0 5.0 5.0 5.0 Stearic acid 8.0 8.0 8.0 8.0 Squalane 5.0 5.0 5.0 5.0 Self emulsifying glycerin monostearate 2.5 2.5 2.5 2.5 Polyoxyethylene sorbitan monostearate 1.5 1.5 1.5 1.5 Propylene glycol 4.0 4.0 4.0 4.0 Stearyl glycyrrhetinate 0.2 0.2 0.2 0.2 vaseline 2.0 2.0 2.0 2.0 Antioxidant Suitable amount Suitable amount Suitable amount Suitable amount Spices Suitable amount Suitable amount Suitable amount Suitable amount antiseptic Suitable amount Suitable amount Suitable amount Suitable amount Purified water Balance Balance Balance Balance

[Table 2] briefly shows the effect of the formulation of the preparation method according to [Table 1].

Example 1 Example 2 Comparative Example 1 Comparative Example 2 Anti-inflammatory effect △ ◎ ◎ x Antioxidative effect ○ ○ ○ x Antimicrobial effect ◎ ◎ x x

⊚: very good / ∘: good / △: fair / x: no effect

The antioxidant effect of [Table 3] was measured as a DPPH radical scavenging activity measurement method. The formulations of the Examples and Comparative Examples were used as they were.

The antimicrobial effect of [Table 3] was measured as a paper disc agar diffusion method, and the growth inhibition rings (mm) were shown using the formulations of Examples and Comparative Examples at 0.5 mg / disc. A is B is the result for Staphylococcus epidermidis and B is for Propionibacterium acnes.

The anti-inflammatory effect of [Table 3] was measured as a measurement of nitric oxide production in RAW264.7 macrophages. The formulations of Examples and Comparative Examples were diluted 1000 times and the amount of nitric oxide produced in comparison with the untreated group was expressed as%.

Item Example 1 Example 2 Comparative Example 1 Comparative Example 2 DPPH radical scavenging ability
(Antioxidative effect) 58.34% 90.16% 88.56% 5.22% Growth inhibition ring size (mm)
(Antibacterial effect) A 20 22 0 0 B 21 23 0 0 Nitric oxide production
(Anti-inflammatory effect) 100% 80% 85% 100%

[Table 3] shows antioxidant, antibacterial and anti-inflammatory effects of Examples 1 and 2 in which the co-products of Ramanolipid and DOD in the present invention and the leaf extract thereof were added.

Hereinafter, [Table 4] to [Table 9] are given as formulation examples to specifically explain the use of the substance according to the present invention based on the results of the above experimental examples. It should be understood, however, by those skilled in the art that these formulation examples can be modified in any other proportions and forms, and that the scope of the present invention is not construed as being limited to the formulation examples described below It is.

Skin toner Ingredients Content (% by weight) Randomized and DOD Simultaneous Products 0.5 칡 leaf extract 0.5 glycerin 5.0 1,3-butylene glycol 3.0 ethanol 5.0 Polyoxyethylene nonylphenyl ether 0.5 Spices Suitable amount antiseptic Suitable amount Purified water Balance

Convergent lotion ( astrigent ) Ingredients Content (% by weight) Randomized and DOD Simultaneous Products 0.5 칡 leaf extract 0.5 glycerin 3.0 Citric acid 0.1 ethanol 10.0 Polyoxyethylene oleyl ether 1.0 Sorbitol 2.0 Spices Suitable amount antiseptic Suitable amount Purified water Balance

Lotion( emulsion )
Ingredients Content (% by weight) Randomized and DOD Simultaneous Products 0.5 칡 leaf extract 0.5 glycerin 5.0 1,3-butylene glycol 8.0 Squalane 10.0 Polyoxyethylene sorbitan monooleate 2.0 Triethanolamine 1.5 Glyceryl stearate 0.5 Stearyl glycyrrhetinate 0.2 Carboxyvinyl polymer 0.1 Arginine 0.1 Spices Suitable amount antiseptic Suitable amount Purified water Balance

Essence Ingredients Content (% by weight) Randomized and DOD Simultaneous Products 0.5 칡 leaf extract 0.5 Sorbitol 8.0 Polyoxyethylene glycol 1500 6.0 ethanol 5.0 glycerin 3.0 1,3-butylene glycol 3.0 Polyoxyethylene oleyl alcohol ether 1.0 olive oil 0.3 Hyaluronic acid 0.2 Antioxidant Suitable amount Spices Suitable amount antiseptic Suitable amount Purified water Balance

Gel (gel) Ingredients Content (% by weight) Randomized and DOD Simultaneous Products 0.5 칡 leaf extract 0.5 ethanol 10.0 glycerin 4.0 Propylene glycol 4.0 Polyoxyethylene hardened castor oil 0.1 Carboxy polymer 0.3 Triethanolamine 0.3 Antioxidant Suitable amount Spices Suitable amount antiseptic Suitable amount Purified water Balance

Ointment Ingredients Content (% by weight) Randomized and DOD Simultaneous Products 3.0 칡 leaf extract 3.0 Cetostearyl alcohol 2.0 Self emulsifying monostearic acid 2.0 Stearic acid 1.0 Wax 4.0 Squalane 7.0 Monostearin glycerin 3.0 Sorbitan monostearate 1.0 Polysorbate 80 3.0 glycerin 5.0 Propylene glycol 4.0 Spices Suitable amount Pigment Suitable amount Vaseline Balance

Claims (12)

Antioxidant, antimicrobial, and anti-inflammatory agents containing Pseudomonas aeruginosa culture liquid extract containing Rhamnolipids and 7,10-dihydroxy-8 (E) -octadecenoic acid as active ingredients, A composition for improving acne.
The method according to claim 1,
A composition for improving acne, which further comprises a leaf extract.
3. The method according to claim 1 or 2,
The extract of Pseudomonas aeruginosa culture solution containing the above-mentioned rhamnoglyphides and hydroxy fatty acids is prepared by mixing mono-rhamnolipids represented by the formula (1) and DOD (7,10-dihydroxy-8 (E) -octadecenoic acid The composition for ameliorating acne characterized by comprising:
[Chemical Formula 1]

(2)

3. The method according to claim 1 or 2,
Wherein the composition for improving acne has an antibacterial activity against Staphylococcus epidermidis or Propionibacterium acnes.
The method according to claim 1,
A composition for improving acne, which comprises the rhamnolipid and the hydroxy fatty acid at a weight ratio of 1: 0.1 to 0.8.
6. The method of claim 5,
Wherein the composition for improving acne contains 0.001 to 20 parts by weight based on the total weight of the composition.
3. The method of claim 2,
Wherein the use ratio of the Pseudomonas aeruginosa culture solution extract to the Leaf extract is in a weight ratio of 1: 0.5 to 30.
8. The method of claim 7,
Wherein the composition for improving acne is contained in an amount of 0.001 to 20 parts by weight based on the total weight of the composition.
3. The method according to claim 1 or 2,
Wherein the composition for improving acne is a composition for improving acne.
3. The method according to claim 1 or 2,
Wherein the composition for improving acne is a composition for external application for skin for improving acne.
3. The method according to claim 1 or 2,
Wherein the composition for improving acne is a composition for improving acne.
The method of claim 9, wherein
The composition for improving acne has a formulation selected from the group consisting of solution, suspension, emulsion, paste, gel, sol, cream, lotion, powder, soap, cleansing oil, powder foundation, emulsion foundation, wax foundation and spray , A composition for improving acne.

Images