COMPOSITION CONTAINING PSEUDOMONAS AERUGINOSA CULTURE SOLUTION EXTRACT HAVING ANTIBIOTIC AND ANTISEPTIC ACTIVITIES, AND USE THEREOF

Abstract

The present invention provides an antiseptic and antibiotic composition containing a Pseudomonas aeruginosa culture solution extract as an active ingredient. The present invention provides a composition for preventing or treating acne, containing a Pseudomonas aeruginosa culture solution extract as an active ingredient. The composition containing a Pseudomonas aeruginosa culture solution extract, of the present invention, exhibits a broad antibiotic spectrum and a high antioxidant effect against various bacteria, and the composition can be applied to a cosmetic composition such as a cosmetic product and can also be useful for antibiotic, sterilizing and antiseptic purposes in various fields such as food, medical supplies, agricultural chemicals and household items. The present invention identifies a specific active ingredient causing the antibiotic effect of a Pseudomonas aeruginosa culture solution extract, and provides the structure thereof.

Claims

1-17. (canceled)

18. A method for preserving or antibiotic treatment, comprising: contacting a composition comprising a Pseudomonas aeruginosa culture extract to a subject.

19. The method of claim 18, wherein the Pseudomonas aeruginosa culture extract is obtained by adding a hydrochloric acid solution to a Pseudomonas aeruginosa culture to form a precipitate through precipitation and then adding ethyl ether to the precipitate.

20. The method of claim 18, wherein the Pseudomonas aeruginosa culture extract contains rhamnolipid.

21. The method of claim 18, wherein the rhamnolipid is di-rhamnolipid represented by Chemical formula 1 or mono-rhamnolipid represented by Chemical formula 2: ##STR00002##

22. The method of claim 18, wherein the composition contains 0.005-1.0 parts by weight of the Pseudomonas aeruginosa culture extract on the basis of 100 parts by weight of the entire composition.

23. The method of claim 18, wherein the composition exhibits an antibacterial activity against at least one microorganism selected from the group consisting of Staphylococcus spp., Bacillus spp., Pseudomonas spp., Candida spp., Propionibacterium spp., Streptococcus spp., Proteus spp., Corynebacterium spp., Enterococcus spp., Klebsiella spp., and Escherichia coli.

24. A method for preventing or treating acne comprising: contacting composition comprising a Pseudomonas aeruginosa culture extract to a subject.

25. The method of claim 24, wherein the Pseudomonas aeruginosa culture extract is obtained by adding a hydrochloric acid solution to a Pseudomonas aeruginosa culture to form a precipitate through precipitation and then adding ethyl ether to the precipitate.

26. The method of claim 24, wherein the Pseudomonas aeruginosa culture extract contains rhamnolipid.

27. The method of claim 24, wherein the rhamnolipid is di-rhamnolipid represented by Chemical formula 1 or mono-rhamnolipid represented by Chemical formula 2: ##STR00003##

28. The method of claim 24, wherein the composition contains 0.005-1.0 parts by weight of the Pseudomonas aeruginosa culture extract on the basis of 100 parts by weight of the entire composition.

29. The method of claim 24, wherein the composition exhibits an antibacterial activity against at least one microorganism selected from the group consisting of Staphylococcus spp., Bacillus spp., Pseudomonas spp., Candida spp., Propionibacterium spp., Streptococcus spp., Proteus spp., Corynebacterium spp., Enterococcus spp., Klebsiella spp., and Escherichia coli.

30. A method of antioxidating, comprising: contacting composition containing a Pseudomonas aeruginosa culture extract to a subject.

31. The method of claim 30, wherein the Pseudomonas aeruginosa culture extract is obtained by adding a hydrochloric acid solution to a Pseudomonas aeruginosa culture to form a precipitate through precipitation and then adding ethyl ether to the precipitate.

32. The method of claim 30, wherein the Pseudomonas aeruginosa culture extract contains rhamnolipid.

33. The method of claim 30, wherein the rhamnolipid is di-rhamnolipid represented by Chemical formula 1 or mono-rhamnolipid represented by Chemical formula 2: ##STR00004##

34. The method of claim 30, wherein the composition contains 0.005-1.0 parts by weight of the Pseudomonas aeruginosa culture extract on the basis of 100 parts by weight of the entire composition.

35. The method of claim 30, wherein the composition exhibits an antibacterial activity against at least one microorganism selected from the group consisting of Staphylococcus spp., Bacillus spp., Pseudomonas spp., Candida spp., Propionibacterium spp., Streptococcus spp., Proteus spp., Corynebacterium spp., Enterococcus spp., Klebsiella spp., and Escherichia coli.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG. 1 illustrates results confirming antibacterial effects of Pseudomonas aeruginosa culture extract (R1) through paper disk diffusion test.

[0056] FIG. 2 illustrates graphs showing CFU as a result of preservative power test (challenge test) on toner dosage form of products containing Pseudomonas aeruginosa culture extract.

[0057] FIG. 3 illustrates images after plate smearing in preservative power test on toner dosage form of products containing Pseudomonas aeruginosa culture extract.

[0058] FIG. 4 illustrates results obtained by measuring reduction rate of acne-related trouble according to concentration of Pseudomonas aeruginosa culture extract (Red bars represent a balm dosage form, and purple bars represent a cream dosage form).

[0059] FIG. 5 illustrates results obtained by confirming, through photographing, the reduction of acne-related trouble after the composition containing Pseudomonas aeruginosa culture extract of the present invention is coated on skin of subjects.

[0060] FIG. 6 shows structures of rhamnose and rhamnolipids.

[0061] FIG. 7 illustrates analysis results of rhamnolipid through 1D 1 H-NMR.

[0062] FIG. 8 illustrates proton analysis results of rhamnose.

[0063] FIG. 9 illustrates 2D-NMR analysis results.

[0064] FIG. 10 illustrates 2D NMR ROESY test results.

[0065] FIG. 11 illustrates results obtained by measuring relative density of rhamnose in sample through 1D 1H-NMR spectra preliminary analysis.

[0066] FIG. 12 illustrates antioxidative effect test results of Pseudomonas aeruginosa culture extract R1.

[0067] FIG. 13 illustrates test results of antibiotic activity of R1 and fractions isolated from R1 against S. aureus.

[0068] FIG. 14 illustrates LC analysis results of R1 and fractions isolated from R1.

[0069] FIG. 15 illustrates structure analysis results of active ingredient peak 1 and peak 2 utilizing LC-MS and LC-MS/MS.

[0070] FIG. 16 illustrates LC analysis results of R1.

MODE FOR CARRYING OUT THE INVENTION

[0071] Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

EXAMPLES

Materials and Method

[0072] 1. Production of Biosurfactant

[0073] Prior to test, strain (CJM01) was selected. The results of identification showed Pseudomonas aeruginosa. The strain was inoculated in M9 medium (CaCl.sub.2 0.015 g/L, Na.sub.2HPO.sub.4 6 g/L, KH.sub.2PO.sub.4 3 g/L, NaCl 0.5 g/L, NH.sub.4Cl 1 g/L, MgSO.sub.4 0.0625 g/L, glucose 20 g/L), and cultured at 37 C. for 72 hours. After the culture, the culture was centrifuged to obtain cell-completely-removed culture. The cell-removed culture was lowered to pH 2 using HCl, and then precipitated at 4 C. for one day. After the precipitation, centrifugation was performed to remove the supernatant and obtain only precipitate. The precipitate was extracted by ethyl ether, and then only ethyl ether layer was separated, followed by concentration using a rotary evaporator concentrator to remove all ethyl ether, thereby obtaining an extract (biosurfactants crude), which was named R1.

[0074] 2. Antibacterial Power Test of Extracted Biosurfactant (Paper Disk Diffusion Test)

[0075] The mixture state of biosurfactant, extracted from microorganism (P. aeruginosa) culture, was named R1. Paper disk diffusion test was conducted on gram positive pathogens (Staphylococcus aureus and Bacillus cereus) and gram negative pathogens (Pseudomonas aeruginosa and Escherichia coli).

[0076] S. aureus, B. cereus, P. aeruginosa, and E.coli each were inoculated in Luria-Bertani (LB) medium (tryptone 10 g/L, NaCl 10 g/L, yeast extract 5 g/L), and liquid-cultured at 37 C. in aerobic conditions for 18 hours. After the culture, 200 l of each bacteria culture was plated on LB agar plate. R1 extracted from microorganism (P. aeruginosa) culture was dissolved at a concentration of 2 mg/ml in methanol. Methyl paraben, phenoxy ethanol, and Naturotics, as controls, were dissolved at a concentration of 10 mg/ml in methanol. 20 l of the dissolved R1, methyl paraben, phenoxy ethanol, and Naturotics solutions each were put on paper disks (diameter: 6 mm), and then the paper disks were sufficiently dried. After that, the paper disks were placed on the agar plates plated with S. aureus, B. cereus, P. aeruginosa, and E.coli each. Each agar plate was cultured at 37 C. in aerobic conditions for 18 hours. After 18 hours, the clear zones generated around the paper disks were observed.

[0077] 3. Preservative Power Test of Extracted Biosurfactant (Challenge Test)

[0078] In order to investigate the possibility of R1 as a preservative compared with methyl paraben, the preservative power test was conducted by measuring the sterilizing power of a toner dosage form and a lotion dosage form of R1 against gram positive pathogens (Staphylococcus aureus and Bacillus cereus) and gram negative pathogens (Pseudomonas aeruginosa and Escherichia coli). The preservative power test was conducted on the basis of the U.S. Cosmetics, Toiletry, and Fragrance Association (CTFA), and the basis is that a particular material has a possibility as a preservative if the particular material in dosage forms of a toner or lotion kills 99.9% of inoculated bacteria within 7 days. Prior to the test, toner and lotion were manufactured, and the compositions thereof were shown in tables 1 and 2.

TABLE-US-00001 TABLE 1 Toner composition R1- R1- R1- R1 or Methyl R1 or Methyl R1 or Methyl 0% 0.01% 0.05% paraben-0.1% paraben-0.5% paraben-1% MeoH Hyarluonic acid 2 g 2 g 2 g 2 g 2 g 2 g 2 g Glycerin 3 g 3 g 3 g 3 g 3 g 3 g 3 g Aloewater 40 g 40 g 40 g 40 g 40 g 40 g 40 g Calendula extract 2.8 g 2.8 g 2.8 g 2.8 g 2.8 g 2.8 g 2.8 g Distilled water 52.2 g 52.18 g 52.1 g 52 g 51.2 g 50.2 g 50.2 g R1 or Methyl 0 g 20 l 100 l 200 l 1 ml 2 ml 0 paraben (500 mg/ml MeOH) MeOH 0 0 0 0 0 0 2 ml Total 100 g 100 g 100 g 100 g 100 g 100 g 100 g

TABLE-US-00002 TABLE 2 Lotion composition R1 or Methyl R1 or Methyl paraben-0% paraben-0.5% MeOH Aloewater 82.5 g 81.5 g 81.5 g Glycerin 5 g 5 g 5 g Sorbitan olivate 1.5 g 1.5 g 1.5 g Olive oil 7 g 7 g 7 g Hyarluronic acid 2 g 2 g 2 g Calendula extract 2 g 2 g 2 g R1 or Methyl paraben 0 1 ml 0 (500 mg/ml MeOH) MeOH 0 0 1 ml Total 100 g 100 g 100 g

[0079] 4. Test on Toner Dosage Form Against Gram Positive Pathogens (Staphylococcus Aureus and Bacillus Cereus)

[0080] R1 dissolved in methanol was added to the prepared toner dosage form, so that the final concentration of R1 had 0%, 0.01%, 0.05%, and 0.1%, and the S. aureus and B. cereus cultures each were inoculated in each container. In addition, methyl paraben dissolved in methanol was added to the toner dosage form, so that the final concentration of the methyl paraben had 0%, 0.1%, 0.5%, and 1%, and the S. aureus and B. cereus cultures each were inoculated in each container. The number of inoculated bacterial cells was 10.sup.6-10.sup.7 colony forming unit (CFU)/ml. While the bacteria-inoculated toners were kept at room temperature, each toner was diluted immediately, one day, three days, five days, and seven days after the inoculation of bacteria, and 200 l of the diluted toner was plated on LB agar plate. The death or not of bacteria was checked by calculating the number of cells and then comparing the calculated number with the number of initially inoculated bacteria. As control, only methanol was added to the toner dosage form, and the bacteria culture was inoculated, followed by repeated tests.

[0081] 5. Test on Toner Dosage Form Against Gram Negative Pathogen (Pseudomonas Aeruginosa ) and Fungus (Candida Albicans)

[0082] R1 and methyl paraben, which were dissolved in methanol, each were added to the toner dosage form, so that the final concentrations of R1 and the methyl paraben had 0%, 0.1%, 0.5%, and 1%, and the P. aeruginosa, C. albicans cultures each were inoculated in each container. The number of inoculated bacterial cells was 106-107 colony forming unit (CFU)/ml. While the bacteria-inoculated toners were kept at room temperature, each toner was diluted immediately, one day, three days, five days, and seven days after the inoculation of bacteria, and 200 l of the toner inoculated with P. aeruginosa was plated on LB agar plate, and 200 l of the toner inoculated with C. albicans was plated on YPD agar plate. The death or not of bacteria was checked by calculating the number of cells and then comparing the calculated number with the number of initially inoculated bacteria. As control, only methanol was added to the toner dosage form, and the bacteria culture was inoculated, followed by repeated tests.

[0083] *YPD agar (peptone 5 g/L, yeast extract 5 g/L, dextrose 5 g/L, agar 15 g/L)

[0084] 6. Test on Lotion Dosage Form Against Gram Positive Pathogens (Staphylococcus Aureus and Bacillus Cereus), Gram Negative Pathogen (Pseudomonas Aeruginosa), and Fungus (Candida Albicans)

[0085] R1 and methyl paraben, which were dissolved in methanol, each were added to the toner dosage form, so that the final concentration of each of R1 and the methyl paraben had 0% and 0.5%, and the S. aureus, B. cereus, P. aeruginosa, and C. albicans cultures each were inoculated in each container. The number of inoculated bacterial cells was 106-107 colony forming unit (CFU)/ml. While the bacteria-inoculated lotions were kept at room temperature, each lotion was diluted immediately, one day, three days, five days, and seven days after the inoculation of bacteria, and 200 l of the lotions inoculated with S. aureus, B. cereus, and P. aeruginosa each were as plated on LB agar plate, and 200 l of the lotion inoculated with C. albicans was plated on YPD agar plate. The death or not of bacteria was checked by calculating the number of cells and then comparing the calculated number with the number of initially inoculated bacteria. As control, only methanol was added to the lotion dosage form, and the bacteria culture was inoculated, followed by repeated tests.

[0086] 7. Acne-Related Trouble Reduction Effect of Products Containing Extracted Biosurfactant

[0087] 7-1. Kind, Dose, and Dosage Form of Products

[0088] Natural cosmetic products containing biosurfactant R1 extracted from microorganism (P. aeruginosa) culture were manufactured. The products were applied (5 g/one person/two weeks) to subjects having acne-related trouble symptoms (pustule acne, acne miliaris, acne scar, pimples, etc.), and then the acne-related trouble reduction effect was observed.

TABLE-US-00003 TABLE 3 Kind of product Kind 1 Cream containing microorganism culture extract-0% 2 Cream containing microorganism culture extract-0.02% 3 Cream containing microorganism culture extract-0.1% 4 Balm containing microorganism culture extract-0% 5 Balm containing microorganism culture extract-0.02% 6 Balm containing microorganism culture extract-0.1% 7 Balm containing microorganism culture extract-0.5%

TABLE-US-00004 TABLE 4 Composition of balm dosage form product containing 0.5% R1 mixture Ingrednet g % Bees wax 7 23.33333 Tocopheryl acetate 0.3 1 Calendula extract 0.1 0.333333 Tea tree E.O 0.3 1 Sunflower seed oil 0.15 0.5 Rosemary leaf extract Tocopherol 0.15 0.5 Grape E.O 0.2 0.666667 Oenothera biennis oil 5 16.66667 Simmondsia chinensis oil 3 10 Organic tamanu oil 13.65 45.5 R1 (Mixture) 0.15 0.5 Total 30 100

TABLE-US-00005 TABLE 5 Composition of balm dosage form product cotnaing 0.1% R1 mixture Ingredient g % Bees wax 7 23.33333 Tocopheryl acetate 0.3 1 Calendula extract 0.1 0.333333 Tea tree E.O 0.3 1 Sunflower seed oil 0.15 0.5 Rosemary leaf extract Tocopherol 0.15 0.5 Grape E.O 0.2 0.666667 Oenothera biennis oil 5 16.66667 Simmondsia chinensis oil 3 10 Organic tamanu oil 13.77 45.9 R1 (Mixture) 0.03 0.1 Total 30 100

TABLE-US-00006 TABLE 6 Composition of balm dosage form product cotnaing 0.02% R1 mixture Ingredient g % Bees wax 7 23.33333 Tocopheryl acetate 0.3 1 Calendula extract 0.1 0.333333 Tea tree E.O 0.3 1 Sunflower seed oil 0.15 0.5 Rosemary leaf extract Tocopherol 0.15 0.5 Grape E.O 0.2 0.666667 Oenothera biennis oil 5 16.66667 Simmondsia chinensis oil 3 10 Organic tamanu oil 13.77 45.9 R1 (Mixture) 0.03 0.1 Total 30 100

TABLE-US-00007 TABLE 7 Composition of balm dosage form product cotnaing 0% R1 mixture Ingredient g % Bees wax 7 23.33333 Tocopheryl acetate 0.3 1 Calendula extract 0.1 0.333333 Tea tree E.O 0.3 1 Sunflower seed oil 0.15 0.5 Rosemary leaf extract Tocopherol 0.15 0.5 Grape E.O 0.2 0.666667 Oenothera biennis oil 5 16.66667 Simmondsia chinensis oil 3 10 Organic tamanu oil 13.8 46 R1 (Mixture) 0 0 Total 30 100

TABLE-US-00008 TABLE 8 Composition of cream dosage form product cotnaing 0% R1 mixture Material g % Witch hazel water 18.5 37 Golden Simmondsia chinensis oil 3 6 Oenothera odorata oil 2.5 5 Olive-emulsified wax 2.5 5 Aloe vera leaf extract 18 36 Glycerine Glycerylacrylate/acrylic acid copolymer Propylene glycol Centella asiatica extract 3 6 Tocopheryl acteate 1 2 Tee tree essencial oil 0.5 1 Grafefruit essential oil 0.5 1 Natural medicinal herb preservative 0.5 1 R1 (mixture) 0 0 Total 50 100

TABLE-US-00009 TABLE 9 Composition of cream dosage form product cotnaing 0.02% R1 mixture Material g % Witch hazel water 18.49 36.98 Golden Simmondsia chinensis oil 3 6 Oenothera odorata oil 2.5 5 Olive-emulsified wax 2.5 5 Aloe vera leaf extract 18 36 Glycerine Glycerylacrylate/acrylic acid copolymer Propylene glycol Centella asiatica extract 3 6 Tocopheryl acteate 1 2 Tee tree essencial oil 0.5 1 Grafefruit essential oil 0.5 1 Natural medicinal herb preservative 0.5 1 R1 (mixture) 0.01 0.02 Total 50 100

TABLE-US-00010 TABLE 10 Composition of cream dosage form product cotnaing 0.1% R1 mixture Matrial g % Witch hazel water 18.45 36.9 Golden mondsia chinensis 3 6 Oenothera odorata 2.5 5 Olive-emulsified wax 2.5 5 Aloe vera f extract 18 36 Glycerine Glycerylacrylate/acrylic acid copolymer Propylene glycol Centella asiatica ract 3 6 Tocopheryl acteate 1 2 Tee tree essencial oil 0.5 1 Grafefruit essential oil 0.5 1 Natural medicinal herb preservative 0.5 1 R1 (mixture) 0.05 0.1 Total 50 100

[0089] 7-2. Subjects

[0090] The present study was directed to observational study clinical tests of control and test groups by a single center, and the purpose of the present study was to evaluate the effectiveness and safety of products, such as reducing acne-related trouble symptoms, when the products were applied to subjects having acne-related troubles.

[0091] Primary endpoint is the reduction rate of acne-related trouble symptoms two weeks after the use of the products, and it was evaluated whether there is a difference in the reduction rate of acne-related trouble symptoms between before and after testing. The total number of subjects was set to 30 in consideration of a drop-out rate of about 10%, on the basis of clinical tests carried out in the past. Three subjects who did not sign a consent form for personal reasons did not participate in the present clinical test, and the total number of subjects participating in the present clinical test was 27. The drop-out rate was 0%, and thus, the total number of subjects who received final evaluations was 27.

[0092] 7-3. Methods

[0093] 5 g of test product was used for two weeks, and a cream or balm type product was applied to the skin lesion site designated by a tester twice or more (morning, evening, frequently) every day for two weeks.

[0094] Participants of the clinical test for evaluating acne-related trouble cosmetic evaluations were collected from students of Inha University and Incheon University, and the clinical test was conducted on voluntary applicants who were agreeable to the standard of application and were not agreeable to the standard of exception. All subjects were selected through screening. A total of 27 subjects agreed to participate in the test, and 16 were men and 11 were women. The ages of the subjects were distributed between 20 and 28. The number of drop-out subjects was zero, and the number of final subjects who completed evaluations was 27.

[0095] For the evaluation of acne-related trouble reduction effect, at every visit, visual evaluation, and image photographing were conducted, and a survey containing subjective opinions of the subject, including directly checking the extent of change (extent of feeling change and absence or presence of abnormal responses) every day, was conducted. For the visual evaluation, at day 0 of the clinical test, the number of ongoing acnes on the lesion site, which was designated by the tester before the product was applied, was set as an initial point, and the reduction degree by naked eyes at such a lesion site two weeks after testing was digitalized. The lesion site of each subject participating in the clinical test was image-photographed before testing and at every visit for clinical test measurement. In addition, the survey about subjective opinions of the subject was conducted

[0096] 7-4. Effect Evaluation Standards, Evaluation Methods, and Analysis Methods

[0097] Subjects suitable for the standard of selection and subjects who used supplied products for 2 weeks were selected as effectiveness evaluation subjects. On weeks 0, 1, and 2, a visual evaluation with respect to acne-related trouble reduction, such as checking the number of ongoing acnes and photographing images, was conducted, and a survey containing subjective opinions of the subjects was conducted. Summary and analysis of test results were conducted by statistical estimation scheme.

[0098] 8. NMR Analysis

[0099] NMR analysis was performed on biosurfactant R1 extracted from microorganism (P. aeruginosa) culture, biosurfactants R2 and R3 obtained by changing culturing and extraction manners with respect to the same P. aeruginosa, and biosurfactant R90 containing 90% of rhamnolipid (one kind of biosurfactant), purchased from SIGMA-ALDRICH.

[0100] The structure of rhamnolipid contains one or two rhamnose (sugar) moieties (left panel of FIG. 6) and lipid moiety containing two aliphatic chains (right panel of FIG. 6). The diversity of the rhamnolipid structure is due to the number of rhamnose rings, one or two, and the structures (length and saturation degree) of two aliphatic chains. Four different biosurfactant extracts (samples 0, 1, 2, and 3) and commercially accessible rhamnolipid (sample 90) each were dissolved in chloroform-methanol mixture (2:1), followed by NMR analysis.

[0101] 9. Antioxidative Effect of Pseudomonas Aeruginosa Culture Extract

[0102] For the application of Pseudomonas aeruginosa culture extract as a preservative, the test on an antioxidative effect thereof was conducted.

[0103] DPPH radical shows purple, and reacts with an antioxidant to show yellow. The color change of the DPPH radical is distinctive as it reacts with a reagent having excellent antioxidative ability. R1, ascorbic acid (AA), and methyl paraben (MTP) each were dissolved at different concentrations in methanol. The thus obtained samples (100 l) each were dissolved at a concentration of 0.006% in methanol, and mixed with 100 l of the DPPH radical solution to prepare a total volume of 200 l, followed by reaction in a light-blocked space for 30 minutes. After completion of the reaction, the absorbance of each mixture was measured at 517 nm to determine antioxidative ability of each sample.

[0104] 10. Search of Active Ingredients Causing Antibiotic Effect of Pseudomonas Aeruginosa Culture Extract

[0105] The Pseudomonas aeruginosa culture extract is in a mixture state of several ingredients rather than a single ingredient. Tests for searching an active ingredient causing an antibiotic effect, among various ingredients contained in the Pseudomonas aeruginosa culture extract, and discovering the structure thereof were conducted.

[0106] Separation Of Mixture State of R1 Extracted from Microorganism (P. Aeruginosa) Culture

[0107] 1 mg of a mixture state of R1 extracted from a microorganism (P. aeruginosa) culture was dissolved in methanol, and then injected in the sep-Pak vac 1cc tc18 cartridge column. After that, the column was washed with solvent A, and solvent B as a development solvent was injected thereinto. Solvent B with different concentrations was injected into the column, and the fractions passing through the column were collected, and named fraction 1, fraction 2, fraction 3, fraction 4, fraction 5, and fraction 6. The different concentrations of solvent B injected to the column were 50%, 55%, 60%, 65%, 70%, and 75%. The compositions of solvents A and B are shown in table 11.

TABLE-US-00011 TABLE 11 Compositions of solvents A and B Solvent A 0.1% Formic acid in water Solvent B 0.1% Formic acid in ACN

[0108] Analysis of Antibiotic Activity of Fractions Separated from R1

[0109] Each of the obtained fractions was dried, and then dissolved at a concentration of 10 mg/ml in methanol. 20 l of each of the fractions dissolved in methanol was dropped on an agar plate plated with the S. aureus culture, and cultured at 37 C. for 18 hours. After 18 hours, the clear zone generated on the agar plate was observed.

[0110] Analysis of Structures of Fractions Separated from R1 Through LC/MS

[0111] LC-MS for structural analysis of the separated fractions was conducted using 1.8 m 2.1*100 mm SB-Aq RRHD (analysis column) and 2.1*5 mm 1.8 m Zorbax SB-C8 (guard column) in a negative mode of ion polarity. The separated fractions were dissolved in methanol to prepare samples. 20 l of each sample was injected into HPLC equipped with the columns, and then was analyzed, together with a mass spectrometer connected thereto. In HPLC, a mixture of solvent A and solvent B was used as a development solvent, and the initial mixing percent of solvent A and solvent B was 50%. The sequential order was as follows. 100% solvent B (19 min), 100% solvent B (20.5 min), 50% solvent B (21 min), 50% solvent B (25 min), HPLC flow rate: 0.15 ml/min

[0112] The scanned mass range was 751000 m/z. MS/MS was conducted on a particular ingredient causing an antibiotic effect.

Results

[0113] 1. Test on Antibiotic Power of Extracted Biosurfactant

[0114] The mixture state of biosurfactant, extracted from microorganism (P. aeruginosa) culture, was named R1. The antibiotic power test was conducted on gram positive pathogens (Staphylococcus aureus and Bacillus cereus) and gram negative pathogens (Pseudomonas aeruginosa and Escherichia coli). As a result of the paper-disk diffusion test, R1 was a significantly large clear zone at a concentration, which was five-fold smaller than those of methyl paraben, phenoxy ethanol, and Naturotics (FIG. 1).

[0115] 2. Test on Preservative Power of Extracted Biosurfactant

[0116] As a result of the preservative power test, in a toner dosage form, R1 killed 99.9% of the inoculated bacteria within 7 days against all the inoculated bacteria, and especially, R1 killed 99.9% of the inoculated bacteria at a concentration of 0.01%, which was 10-fold smaller than that of methyl paraben, in only one day against gram positive pathogens Staphylococcus aureus and Bacillus cereus. R1 killed 99.9% of the inoculated bacteria at a concentration of 0.5%, which was the same concentration as methyl paraben, within five days against gram negative pathogen (Pseudomonas aeruginosa) and fungus (Candida albicans) (FIGS. 2 and 3). In addition, in a lotion dosage form, R1 killed 99.9% of the inoculated bacteria at a concentration of 0.5%, which was the same concentration as methyl paraben, within seven days against all the inoculated bacteria (table 12). These results show that R1 has a possibility as a preservative.

TABLE-US-00012 TABLE 12 Preservative power test of R1 in dosage form (%-survival rate of inoculated bacteria) Day 0 Day 1 Day 3 Day 5 Day 7 Staphylococcus 0% 100% 6.74% 0.06% 30% 78% aureus MeOH 100% 15.70% 4.27% 0.04% 0.04% R10.5% 100% 0% 0% 0% 0% MTP0.5% 100% 0% 0% 0% 0% Bacillus 0% 100% 0.09% 0.23% 35% 54% cereus MeOH 100% 0.17% 0.02% 0.02% 0.02% R10.5% 100% 0.01% 0% 0% 0% MTP0.5% 100% 0.12% 0.06% 0.02% 0.01% Pseudomonas 0% 100% 0.30% 0.60% 0.02% 0.02% aeruginosa MeOH 100% 7.30% 0.80% 0.03% 0.02% R10.5% 100% 7.30% 0.60% 0.006% 0% MTP0.5% 100% 0% 0% 0% 0% Candida 0% 100% 37.50% 1% 0.5%.sup. 0.5%.sup. albicans MeOH 100% 27.50% 1.80% 0.4%.sup. 0.3%.sup. R10.5% 100% 16.30% 0.10% 0% 0% MTP0.5% 100% 0% 0% 0% 0%

[0117] 3. Acne-Related Trouble Reduction Effect of Products Containing Extracted Biosurfactant

[0118] Natural cosmetic products containing biosurfactant R1 extracted from the microorganism (P. aeruginosa) culture were manufactured, and then applied to subjects having acne-related trouble symptoms. As a result, the acne-related trouble reduction rate was significantly increased in the subjects using products containing R1 rather than the subjects not containing R1, and abnormal skin responses (itching, flushing, red marks, swelling, furuncles, prick, etc.) were not observed in all the subjects.

TABLE-US-00013 TABLE 13 Acne-related trouble reduction rate Subject Extract content and product No. Gender dosage form Reduction rate 1 Female 0.5%/Balm 57% 2 Male 0.1%/Balm 57% 3 Male 0.02%/Cream 75% 4 Male 0.02%/Balm 75% 5 Male 0%/Cream 60% 6 Female 0%/Balm 47% 7 Female 0.1%/Balm 43% 8 Female 0.5%/Balm 50% 9 Female 0%/Cream 20% 10 Female 0.1%/Balm 100% 11 Female 0.02%/Balm 50% 12 Female 0%/Cream 38% 13 Male 0.1%/Cream 54% 14 Male 0.02%/Balm 100% 15 Male 0.1%/Cream 80% 16 Female 0.02%/Cream 88% 17 Female 0%/Cream 31% 18 Female 0.02%/Balm 50% 19 Male 0.1%/Balm 64% 20 Male 0.1%/Balm 60% 21 Male 0.02%/Cream 63% 22 Male 0.02%/Balm 57% 23 Male 0.1%/Balm 60% 24 Male 0.5%/Balm 60% 25 Male 0.5%/Balm 65% 26 Male 0.5%/Balm 100% 27 Male 0.02%/Cream 100%

[0119] At day 0 of the clinical test, the number of ongoing acnes on the lesion site, which was designated by the tester before the product was applied, was set as an initial point, and the reduction degree by naked eyes at such a lesion site two weeks after testing was digitalized. The results verified that the subjects using the products containing the microorganism culture extract showed higher reduction rates when compared with the products not containing the microorganism culture extract. It was verified that the highest reduction rate was showed in the subjects using, especially, a cream type product containing a 0.02% microorganism culture extract, and next, a high reduction rate was showed in the subjects using a cream type product containing a 0.1% microorganism culture extract (FIG. 4). Based on these results, it can be considered that the natural cosmetic products containing the microorganism culture extract prepared by the present inventors are helpful in the reduction of acne. The image photographing results of the subjects are shown in FIGS. 5a to 5i.

TABLE-US-00014 TABLE 14 Overall effectiveness evaluation Subject No. Evaluatioin Subject No. Evaluation 1 4 15 4 2 3 16 4 3 4 17 3 4 5 18 4 5 4 19 4 6 3 20 4 7 3 21 3 8 3 22 3 9 3 23 4 10 5 24 4 11 3 25 4 12 3 26 5 13 4 27 4 14 4 Average 3.7

[0120] 5: Symptoms appeared, 4: Symptoms generally distinctively improved, 3: Symptoms generally slightly improved, 2: Symptoms not changed compared with before test, 1: Symptoms worse than before test

TABLE-US-00015 TABLE 15 Product satisfaction evaluation results after use Subject No. Evaluatioin Subject No. Evaluation 1 3 15 5 2 4 16 3 3 4 17 4 4 5 18 5 5 4 19 4 6 4 20 4 7 4 21 4 8 3 22 4 9 3 23 4 10 5 24 4 11 2 25 3 12 4.5 26 5 13 4 27 4 14 4 Average 3.9

[0121] 1: Uncertain, 2: Unsatisfactory, 3: Mediocre, 4: Satisfactory, 5: Very satisfactory

[0122] Two weeks after the clinical test, the general satisfaction of the subjects in the acne-related trouble reduction effect was evaluated as being high. The most striking changes shown in the survey evaluation and visual evaluation were skin troubles, such as pustule acne and temporary pimples. On the other hand, acne miliaris and acne scars were relatively less changed. In addition, it was verified that the reduction effect and the satisfaction were higher in the subject using cream dosage form of products rather than balm dosage form of products.

[0123] It could be seen through clinical test results, visual evaluation, survey evaluation, and image photographing for two weeks, that the natural cosmetic products containing the microorganism culture extract prepared by the present inventors are helpful in the reduction of the acne-related troubles.

[0124] 4. NMR Analysis

[0125] General Observation

[0126] All the samples contained a rhamnolipid set composed of four main ingredients (two types of di-rhamnolipids and two types of mono-rhamnolipids). The content percents of di-rhamnolipids of fraction A were different in all samples (up to 30% in sample 90, and 65% or more in samples 2 and 4) (table 17). In addition, samples 0, 1, 2, and 3 contained aromatic ingredients that could be anticipated to have an effect on antibacterial activity.

[0127] 1D-NMR Analysis

[0128] In the 1D 1H-NMR spectrum of sample 1, moieties corresponding to rhamnolipids are shown in FIG. 7. Signal intensities (integrals) are shown below the spectrum. All signal intensities were standardized according to the metal peak at 0.82 ppm, and the peaks correspond to six protons (2 methyl groups). Therefore, a single proton peak seems to have a signal intensity of approximately 100 units. For example, two groups of peaks shown at 1.4-1.5 ppm and 2.4-2.6 ppm exhibit signal intensities of 400, which are consistent with four proton signals shown at C.sub.2H.sub.2/C.sub.5H.sub.2 and C.sub.7H.sub.2/C.sub.10H.sub.2 groups, respectively. The mass ratio of mono- and di-rhamnolipids may be determined by comparing the intensities of signals obtained from lipid and rhamnose groups.

[0129] If a sample contains only mono-rhamnolipid, only a single proton from the rhamnose is consistent with one proton present in the lipid moiety. Alternatively, if a sample contains only di-rhamnolipid, the signals may be consistent with 2-to-1. As validated in FIG. 8, H1 and H4 lipid protons (dark orange-color boxes) show the same type of signals, and the sum of the intensities thereof is approximately 100, which means that the number of this type of proton in each rhamnolipid molecule in the mixture is exactly one.

[0130] Resultantly, the sum of signal intensities obtained the H1/1, H2/2, H3/3, H5/5 rhamnose protons (blue boxes in FIG. 8) was higher than the value expected in the mono-rhamnolipid mixture. A single H1 proton per molecule in the mono-rhamnolipid mixture must show a signal having a value of 100. Three, H2 H3, and H5 protons must show signals having a value of 300. However, these signal intensities in sample 1 were measured to be approximately 150-480. It may be estimated from these signal intensity values that approximately 60% of the di-rhamnolipid was present in sample 1. By similar analysis, 1D 1H-NMAR spectra were performed on samples 0, 2, 3, and 90.

[0131] 2D-NMR Analysis

[0132] The presence of di-rhamnolipid in the samples was confirmed by 2D 1H-13C-HSQC test. The formation of -L-Rhap (1.fwdarw.2)--L-Rhap bond caused a strong NMR signal shift of C2 and C1 atoms of di-rhamnose connected via oxygen atom (structure in FIG. 6). C.sup.1/1H and C.sup.2/2H sections in HSQC spectrum are shown in FIG. 9. The cross-peaks shown at 102 ppm (13C scale, red box) are consistent with the correlation coefficient of H1/C1 in the di-rhamnolipid. On the other hand, the cross-peaks shown at 95-95 ppm are consistent with the correlation coefficient of H1/C1 in the mono-rhamnolipid (structure in FIG. 6). Two cross-peaks shown at 79 ppm (blue boxes in FIG. 9) are consistent with two C2 atoms of the di-rhamnolipid.

[0133] Spin System Allocation

[0134] In order to measure relative densities and numbers of different rhamnolipids in the samples, the spin systems were separated using 2D NMR spectra (HSQC, TOCSY, COSY-DQF), and these were consistent with rhamnose ring structure and fatty acid tail structure of the rhamnolipid molecule. In addition, six unknown systems (systems 1-6) of the rhamnose moiety and eight systems (systems 7-14) of the fatty acid tail could be proved. The systems were previously allotted to the rhamnose moiety (first () or second () single ring structure in di-rhamnose), and the location of the hydroxyl fatty acid (directly or indirectly connected to rhamnose, FIG. 6) is based on the known chemical shift (part 3, 2D-NMR analysis, shown in FIG. 9). Systems 1 and 2 were allotted to the second rhamnose ring structure of the di-rhamnose, and systems 3 and 4 were allotted to the first rhamnose ring structure of the di-rhamnose. Systems 5 and 6 are consistent with the rhamnose ring structure of the mono-rhamnolipid. Systems 7 and 8 as well as minimum ingredients 9 and 10 thereof were allotted to the second fatty acid of the lipid (not directly connected to the rhamnose moiety). Resultantly, systems 11-14 were allotted to the first fatty acid chain, and directly connected to the rhamnose moiety.

[0135] Spin System Connection

[0136] After the allotment of the spin systems for structural moieties of the rhamnolipid molecule, several possible methods for connecting the spin systems into the rhamnolipid were conducted. In order to sense the dipole-dipole interaction through the surfaces of the protons present on the rhamnose ring structure and the protons of the fatty acid, 2D NMR ROESY test was conducted. As anticipated, the cross-peaks of H1 proton of rhamnose systems 3-6 and H1 protons of fatty acid systems 11-14 were sensed (FIG. 10). This test allows the connection between the spin system allotted to the first ring structure or a single structure of rhamnose and the spin system allotted to the fatty acid.

[0137] No cross-peaks were observed between H1 protons of systems 1 and 2 allotted to the protons of the second ring structure in the di-rhamnose and the fatty acid. Ultimately, the spin systems were summarized as four rhamnolipid molecules I-IV (table 16).

TABLE-US-00016 TABLE 16 Rhamnose moieties/spin systems Lipid moieties/spin systems Molecule -L-Rhap-1 Linker -L-Rhap-1 Linker Fatty acid 1 Linker Fatty acid 2 I 1 (2) 1-O-2 3 1-O-1 11 2-C.sup.o--O-4 8 II 1 (2) 4 13 7 III 5 14 7 (8) IV 6 12 7 (8)

[0138] Relative Densities of Molecules I-IV in NMR Sample

[0139] Preliminary analysis of 1D 1H-NMR spectra clearly shows relative densities of rhamnose in the samples (FIG. 11). The same results were observed in the fatty acid systems. The relative densities of molecules I-IV were estimated using signal intensities, and the signal intensities were measured by 1D 1H NMR spectra in order to obtain well separated signals allotted to the H1/H1 and H2/H2 rhamnose protons and H1 and H4 fatty acid protons. The analysis was summarized in table 16.

[0140] As a result of NMR analysis of biosurfactant R1 extracted from the microorganism (P. aeruginosa) culture, biosurfactants R2 and R3 obtained by changing culturing and extraction manners with respect to the same P. aeruginosa, and biosurfactant R90 containing 90% of rhamnolipids, purchased from SIGMA-ALDRICH, it was found that all the respective extracts and purchased R90 contained rhamnolipids. These results validated that R1 extracted from the microorganism (P. aeruginosa) culture contains rhamnolipid-based biosurfactant (FIG. 11 and table 16). In addition, it could be seen through NMR analysis that five samples contained rhamnolipids and the content percentages thereof were different. In addition, the rhamnolipids may probably be associated with other biological studies.

TABLE-US-00017 TABLE 17 Population in sample Molecule dl-Rhamnolipid mono-Rhamnolipid 0 1 2 3 90 I {1/2}-3-O-11-8 24 33 15 19 35 II {1/2}-4-O-13-7 25 33 52 50 III 5-O-14-{7/8} 31 16 25 22 47 IV 6-O-12-{7/8} 20 18 8 9 18

[0141] 5. Antioxidative Effect of Pseudomonas Aeruginosa Culture Extract

[0142] In order to investigate the antioxidative effect of Pseudomonas aeruginosa culture extract R1, DPPH scavenging activity test was conducted. As a result, R1 showed the same level of antioxidative effect as ascorbic acid (AA), which is a representative ingredient that has an antioxidative effect at a concentration of 0.5% or more. On the other hand, the methyl paraben (MTP) exhibited a significantly low antioxidative effect at the same concentration when compared with R1. It is considered on the basis of these results that Pseudomonas aeruginosa culture extract R1 having an antioxidative effect can be applied as a preservative (FIG. 12).

[0143] 6. Search of Active Ingredients Causing Antibiotic Effect of Pseudomonas Aeruginosa Culture Extract

[0144] A mixture state of R1 extracted from the microorganism (P. aeruginosa) culture was separated to obtain fraction 1, fraction 2, fraction 3, fraction 4, fraction 5, and fraction 6. As a result of testing antibacterial activity of each of the obtained samples against S. aureus, fraction 1, fraction 2, and fraction 3 exhibited antibacterial activity corresponding to R1; fraction 4 and fraction 5 exhibited a relatively weaker antibacterial activity than R1; and fraction 6 did not exhibit antibacterial activity (FIG. 13). All the samples were subjected to LC analysis, and as a result, two common peaks, which are high in fraction 1, fraction 2, and fraction 3 and are gradually reduced in fraction 4 and fraction 5, were found, and the two peaks were named peak 1 and peak 2 (FIG. 14). In connection with the antibiotic activity test, the presence or absence of peak 1 and peak 2 were considered to determine antibacterial activity. As a result of MS and MS/MS tests of peak 1 and peak 2, peak 1 was analyzed as a di-rhamnolipid having a molecular weight of 650 and a structure of Rha-Rha-C10-C10, and peak 2 was analyzed as a mono-rhamnolipid having a molecular weight of 504 and a structure of Rha-C10-C10 (FIGS. 15a-15d). It was verified that R1 has various structures of rhamnolipids (FIG. 16 and table 18), and of them, the rhamnolipids having structures of Rha-Rha-C10-C10 and Rha-C10-C10 are active ingredients having antibacterial activity. In addition, the antibacterial activities of rhamnolipids (R90, R95) extracted from the culture of P. aeruginosa purchased from SIGMA-ALDRICH KOREA against S. aureus were significantly low compared with R1 (FIG. 17).

TABLE-US-00018 TABLE 18 MS of main peaks (R1) Number Name Symbol Formula (DB) Avg Mass Base Peak RT Vol % 1 Di-rhamnolipid Rha-Rha-C10-C8 C30H54O13 622.715 621.3529 4.059 5.45 2 Di-rhamnolipid Rha-Rha-C10-C10 C32H58O13 650.7773 649.3865 5.289 14.22 3 Di-rhamnolipid Rha-Rha-C10-C12:1 C34H60O13 676.7992 675.4063 6.266 6.33 4 Mono-rhamnolipid Rha-C10-C10 C26H48O9 504.6391 503.3306 6.772 16.66 5 Mono-rhamnolipid Rha-C10-C12:1 C28H59O9 530.6705 529.342 7.916 6.29 6 Mono-rhamnolipid Rha-C10-C12 C28H52O9 532.6893 531.3579 8.451 7.74 7 2-Nor-1,3-seco-1,25-dihydroxyvitamin D3 C26 H44 O3 404.5802 403.3085 12.059 1.36

[0145] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.