Extract of Arthrobacter agilis for use in particular in cosmetics

Abstract

Cosmetic, pharmaceutical or dietary composition comprising an extract of the bacteria Arthrobacter agilis, preferably rich in carotenoids.

Claims

1. A cosmetic, pharmaceutical, or dietary composition comprising: (a) an extract rich in carotenoids of the bacteria Arthrobacter agilis and (b) vitamin E or a derivative of vitamin E.

2. The composition according to claim 1, wherein the extract is obtained by culturing the bacterium in a medium consisting of 1 wt % of tryptone and 0.5 wt % of yeast extract.

3. The composition according to claim 1, wherein the extract is obtained from cells in the stationary phase.

4. The composition according to claim 1, wherein the extract corresponds to an apolar phase obtained from bacterial cells precipitated after being cultured in a culture medium.

5. The composition according to claim 4, wherein the apolar phase is obtained by adding an apolar solvent and a saturated salt solution to the precipitated bacterial cells.

6. The composition according to claim 5 wherein the bacterial cell precipitate is extracted using acetone and/or methanol.

7. The composition according to claim 5, wherein the apolar solvent is hexane.

8. The composition according to claim 5, wherein the saturated salt solution is a NaCl solution.

9. The composition according to claim 1, wherein the vitamin E derivative is trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), or tocopherol acetate.

10. The composition according to claim 1, wherein the extract represents from 0.0001 to 0.01% by dry weight of the composition.

11. The composition according to claim 1, wherein the composition is in the form of a lotion, cream, gel, spray, solutions, capsules or tablets.

12. The composition according to claim 1, further comprising an antioxidant.

13. The composition according to claim 12, wherein the antioxidant is selected from the group consisting of; N-acetyl-cysteine (NAC); butyl-hydroxy-anisole (BHA); vitamin C; glutathione; caffeic acid or (E) 3-(3,4-dihydroxyphenyl)prop-2-enoic acid; curcumin or diferuloylmethane; quercetin or quercetol; lycopene; coenzyme Q10 (Q10); and uric acid.

14. The composition of claim 1, comprising the extract rich in carotenoid of the bacteria Arthrobacter agilis and vitamin E.

Description

EMBODIMENTS

(1) The manner in which the invention can be produced and the advantages resulting from it will be better understood with the embodiments below, given as a rough guide and as part of a non-exhaustive list, with the help of the figures annexed.

(2) FIG. 1 corresponds to an image of inverse microscopy (magnification 100) of the strain SB5.

(3) FIG. 2 shows the HPLC spectrum of the SBE extract according to the invention and the absorbance spectrum for each of the six main peaks (graded 1 to 6).

(4) FIG. 3 shows the antioxidant power measured using the ABTS assay of the extract according to the invention (SBE) relative to other known antioxidants, expressed as Trolox equivalent.

(5) FIG. 4 shows the protective capacity of proteins, vis--vis the free radicals, of the extract according to the invention (SBE) compared to other antioxidants, expressed as Trolox equivalent (A), and compares this protective capacity to that of the fractions corresponding to the 6 peaks observed in HPLC (B).

(6) FIG. 5 shows the protective capacity of proteins, vis--vis the UVs, of the extract according to the invention (SBE) compared to other antioxidants, expressed as Trolox equivalent.

(7) FIG. 6 shows the monitoring of the activity of the alkaline phosphatase (AP) depending on the concentration of the extract according to the invention (SBE).

(8) FIG. 7 shows the protective capacity of proteins, vis--vis the free radicals, of the extract according to the invention (SBE) mixed with other antioxidants, expressed as Trolox equivalent.

(9) FIG. 8 shows the protective capacity of human cells (keratinocytes) and their DNA, vis--vis free radicals and UVs, of the extract according to the invention (SBE) relative to tocopheryl acetate using the comet assay.

(10) FIG. 9 shows the measurement of protein carbonylation evaluating the protective capacity of human cells (keratinocytes) and their proteins, vis--vis UV/visible light radiation, of the extract according to the invention (SBE) compared to tocopherol acetate.

1/ISOLATION AND CHARACTERISATION OF THE STRAIN SB5

(11) A bacterium (SB5) was isolated in the falling snow and selected for its resistance to UVC and its ability to grow rapidly. It has been characterised as belonging to arthrobacters, which are positive bacteria grams whose many species are extremophile or extremotolerant.

(12) Specifically, the isolated bacterium is a strain of Arthrobacter agilis. A. agilis is a non-sporulating and non-pathogenic gram-positive coccoid bacterium, of 0.8 to 1.2 mm in diameter and which may have from 0 to 3 flagella (Koch et al. (1995) Int J Syst Bacteriol. 45(4):837-9), isolated for the first time in 1889 (Ali-Cohen (1889) Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 6:33-36).

(13) FIG. 1 shows an inverse microscope image of the isolated strain, consistent with the description of the bacterium Arthrobacter agilis.

(14) In addition, the fact that the strain SB5 belongs to the species Arthrobacter agilis was confirmed by partial sequencing of its 16S RNA. The corresponding sequence (SEQ ID NO: 2) is as follows, the base N indicating that it could not be determined whether the nucleotide corresponded to A, C, G or T:

(15) TABLE-US-00002 ACATGCAAGTCGAACGATGAACCTCACTTGTGGGGGGATT AGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTT GACTCTGGGATAAGCCTGGGAAACCGGGTCTAATACTGGA TACGACCTTCTGGCGCATGCCATGTTGGTGGAAAGCTTTT GTGGTTTTGGATGGACTCGCGGCCTATCAGCTTGTTGGTG GGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTG AGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAG ACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGG GCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGAAGG CCTTCGGGTTGTAAACCTCTTTCAGTAGGGAAGAAGCCGG CCTTTTGGGTTGGTGACGGTACCTGCAGAAGAAGCGCCGG CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCA AGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGC GGTTTGTCGCGTCTGCCGTGAAAGTCCGGGGCTTAACTCC GGATCTGCGGNGGGTACGGGCAGACTAGAGTGCAGTAGGG GAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATA TCAGGAGGAACACCGATGGCGAAGGCAGGTNTCTGGGCTG TAACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGG ATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCAC TAGGTGTGGGGGACATTCCACGTTTTCCGCGCCGTAGCTA ACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGC TAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCG GAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTAC CAAGGCTTGACATGAACCGGAATGATGCAGAGATGTGTCA GCCACTTGTGGCCGGTTTACAGGTGGTGCATGGTTGTCGT CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA GCGCAACCCTCGTTCCATGTTGCCAGCGGGTTATGCCGGG GACTCATGGGAGACTGCCGGGGTCAACTCGGAGGAAGGTG GGGACGACGTCAAATCATCATGCCCCTTATGTCTTGGGCT TCACGCATGCTACAATGGCCGGTACAAAGGGTTGCGATAC TGTGAGGTGGAGCTAATCCCAAAAAGCCGGTCTCAGTTCG GATTGAGGTCTGCAACTCGACCTCATGAAGTTGGAGTCGC TAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCC CGGGCCTTGTACACACCGCCCGTCAAGTCACGAAAGTNGT AACACCCGAAGCCGGNGCCTAACCCCTTGNGGAGGGAGCC

(16) This sequence presents effectively more than 99.2% identity with the reference sequence SEQ ID NO: 1, confirming that this strain belongs to the species Arthrobacter agilis.

2/PREPARATION OF SBE EXTRACT

(17) 2-1. Culture of SB5 Strain:

(18) The strain is cultured in the LB medium without salt at a temperature of 25 C., aerobically and with vigorous stirring, until the stationary phase is reached, after about 3 days.

(19) The cells are then collected by centrifugation, typically at a speed of 5000 rpm for about 15 minutes. The pellet is then dried by storage in the dark at 4 C.

(20) 2-2. Isolation of the SBE Extract:

(21) The pellet is taken with 6 ml acetone per gram of pellet, optionally in the presence of methanol, for example in a methanol/acetone mixture (5/1). This extraction step takes place for several hours, typically 18 hours, at 4 C. in the dark.

(22) The suspension in acetone, or in an acetone/methanol mixture, is completed by addition of hexane, preferably at half the volume of acetone. A saturated solution of NaCl (sodium chloride) is then added until the separation of the biphasic mixture, with an aqueous phase on one hand and an apolar phase on the other. Preferably, the aqueous phase is again extracted using hexane and the hexane phases are combined.

(23) The apolar or hexane phase is evaporated, preferably under vacuum at 25 C.

(24) The SBE extract can be preserved as is at 20 C. or may be taken as a solution, for example in the DMSO (dimethylsulfoxide) or THF (tetrahydrofuran) or propanediol to be tested on live cells, and is highly soluble in dichloromethane and acetone.

(25) The carotenoid concentration is determined by measuring the absorbance at 512 nm in DMSO or 502 nm in propanediol.

(26) The HPLC spectrum of the extract and the absorbance spectrum for each of the six main peaks are shown in FIG. 2. Comparison of these spectra with the literature data (Fong et al, Appl Microbiol Biotechnol (2001) 56: 750-756) showed that the extract contains significant quantity of carotenoids, with a characteristic absorption spectrum of 3 fingers to 490 nm. As described in Fong et al, (Appl Microbiol Biotechnol (2001) 56: 750-756), the absorption spectrum also revealed the presence of glycosylated forms and different isomers of these carotenoids.

3/ACTIVITIES OF THE SBE EXTRACT

(27) 3-1. ABTS Assay:

(28) As mentioned above, this assay is conventionally used in the cosmetics and agri-food industry helps establish, in a purely chemical manner, the total antioxidant capacity of an extract. Although this assay is incomplete because it measures the antioxidant activity of the extract only when dealing with a particular kind of radical, it has the advantage, by way of its standardisation with Trolox, of generating results that are comparable with thousands of other molecules tested in this manner.

(29) This assay was carried out as described in Re et al., Free Radic. Biol. Med. (1999) 26(9-10): 1231-7.

(30) FIG. 3 shows that the extract according to the invention (SBE) shows antioxidant capacity over 4 times higher than that of the reference antioxidant, Trolox.

(31) In the context of the invention, a Trolox equivalent corresponding to the number of moles of the product tested helps obtain an effect equivalent to that of 1 mol Trolox. The extract according to the invention was quantified with the use of carotenoids as a marker of the extract. Thus, 1 mol of the SBE extract corresponds to 1 mole of carotenoids as measured by absorbance at a wavelength corresponding to the maximum absorption and deducted from the molar extinction coefficient of the carotenoids.

(32) 3-2. Protection of Proteins Against Free Radicals:

(33) This test consists of measuring at 405 nm the activity of alkaline phosphatase (AP) in the presence of free radicals, especially hydroxyl (OH.), and a compound or extract, in order to assess the protective capacity vis--vis the proteins of the said compound or extract.

(34) Materials and Methods:

(35) Reagents:

(36) Buffer: Tris Buffered Saline powder (Sigma; Product reference: T6664-10PAK);

(37) Enzyme: Alkaline phosphatase 10 KU at 125 U/4 in buffered glycerol solution (Sigma; Product reference: P0114);

(38) Oxidizing Agent:

(39) 1/Hydrogen Peroxide at 30% or 9M (Sigma; Product reference: H1009-5ML): solution B1;

(40) 2/Iron II sulphate heptahydrate (FeSO4, 7H.sub.2O) 0.1M (Fluka; Product reference: 44970): solution B2;

(41) Substrate: solution of 4-nitrophenyl phosphate (Alkaline Phosphatase yellow substrate; Sigma; Product reference: P7998-100ML).

(42) Kit:

(43) 96-well plate; Buffer: 20 ml of a TBS solution0.05 M, pH 8; Solution A (enzyme): 10 l of the stock solution diluted at 1/1000 (or 1.25 U i.e. 0.125 U/L) in the buffer; 100 l of solution B1 and 100 l of solution B2 or solution B corresponding to the mixture of solutions B1 and B2 at a suitable dilution in the buffer (for example 3 ml of solution B at 90 mM, obtained from 30 l of solution B1+30 L of solution B2 i.e. a 1/100 dilution); Solution C (substrate): 5 ml of the commercial solution.
Protocol: Dilute the solution A in the buffer at a suitable dilution; Insert, in each well, 10 l of the diluted solution A so as to have an enzyme amount between 0.005 to 0.05 U; Add 10 l of the product or the extract to be tested, optionally diluted in the buffer in each well. The individual wells can be used to test a range of dilution; Add 30 l of solution B to each well. The concentration of the solution B is adjusted to obtain 90% inactivation of the amount of enzyme present. The final concentration of the oxidising agent equal to 30% of the concentration of the solution B; Incubate for 15 minutes at 37 C. without stirring; Add 50 l of solution C. The reaction volume is thus equal to 100 l; Place the plate at 37 C. with stirring; Read the absorbance at 405 nm for 20 minutes (kinetic) or after 5 minutes of reaction (selective).
Results

(44) FIG. 4A shows that the extract according to the invention (SBE) has a protective capacity of proteins, against free radicals, about 100 times higher than that of the Trolox, and more than 6 times higher than all the antioxidants tested. The difference between this result and the antioxidant potential measured by ABTS demonstrates that the protection of proteins is not linked only to the antioxidant potential of SBE.

(45) Furthermore, the same technique was used to compare the effect of 6 fractions (graded SB1 to SB6) corresponding to the 6 major peaks identified by HPLC analysis to that of the total extract SBE.

(46) It is clear from FIG. 4B that the SBE extract has a protective capacity against free protein radicals which is 2 to 4 times higher than that of each of the peaks that it comprises and demonstrates a synergistic effect between the various fractions of the extract.

(47) 3-3. Protection of Proteins Against UV:

(48) This test is similar to that described in the previous section. It consists of measuring at 405 nm the activity of alkaline phosphatase (AP), irradiated using UV-C (254 nm), in the presence of a compound or an extract, in order to assess the protective capacity vis--vis the proteins of the said compound or extract.

(49) Materials and Methods:

(50) The alkaline phosphatase was subjected to UV radiation at 254 nm corresponding to the UV-C. Under constant stirring, the UV doses resulting in 90% inactivation of the enzyme were applied to a reaction medium and under conditions similar to those mentioned above. Note that the volume occupied by the oxidising agent is replaced with the buffer. In practice, a lamp with a power equal to 0,0365 J/cm.sup.2/min a lamp, with an exposure time of 1 or 2 hours.

(51) Results

(52) FIG. 5 shows that the extract according to the invention (SBE) has a protein protection capacity against UV, which is more than 300 times higher than that of the Trolox and almost 40 times greater than all the antioxidants tested.

(53) This very strong protective capacity of the extract according to the invention may be explained by several combined effects of the extract in the presence: an effect of neutralising oxygen singlets (.sup.1O.sub.2) generated by UV; a screen, filter or shield effect by direct UV absorption; a classic antioxidant effect, i.e. the neutralisation of free radicals generated by oxygen singlet (.sup.1O.sub.2).

(54) 3-4. Protection of Proteins:

(55) The activity of alkaline phosphatase was measured in the absence of oxidative stress but in the presence of increasing concentrations of the extract according to the invention (SBE).

(56) FIG. 6 shows that the AP activity increases in the presence of the extract according to the invention (SBE) at very low doses. This increase may be due to the protection of the enzyme during incubation and agitation. It is also conceivable that some of the oxidation of the enzyme stored at 20 C. is reduced by the SBE extract, which could increase its activity by rejuvenation.

(57) On the basis of FIG. 6, a suitable concentration of the extract according to the invention (SBE) is between 0.001 and 100 m, preferably between 0.1 and 1 M. However, these are concentrations to be achieved in the target cells of the skin. Thus, the concentration in the cosmetic compositions must be much higher to account for the losses related to skin penetration.

(58) An interaction between the enzyme and the extract could be the cause of this boosting action. By extension, the extract according to the invention is likely to protect the proteins in the body, especially those directly involved in protecting the body against oxidative stress or in the repair of oxidative damage.

(59) 3-5. Synergy with Other Antioxidants:

(60) To identify antioxidants that may increase the effectiveness of the protein protection by the extract according to the invention (SBE), the test described in the section 3-2 was carried out by mixing the extract according to the invention (SBE) and a dozen known antioxidants, taken individually. It is necessary to identify molecules with which the extract according to the invention (SBE) combines the antioxidant effects or to highlight synergies beyond additive effects. Another advantage of such mixtures is the possible stabilisation of the extract according to the invention (SBE).

(61) FIG. 7 shows that in the presence of all the antioxidants tested, and especially in the presence of Trolox, the extract according to the invention (SBE) has a strong synergy, which significantly increases (by a factor of 5 with Trolox) its protein protection capacity against free radicals.

(62) 3-6. Protection of Human Cells:

(63) A/Comet Assay:

(64) To confirm the protection by the extract according to the invention (SBE) of human cells, measures to protect primary cultures of keratinocytes and their DNA facing oxidative stress induced by UV/visible light were implemented through the comet assay, as described by Ostling, O., and K. J. Johanson. (Biochemical and biophysical research communications 123.1 (1984): 291-298) and Singh, Narendra P., et al. (Experimental cell research 175.1 (1988): 184-191). It is essential to show the protection activity of the extract according to the invention (SBE) in a cell model and compare it to a reference antioxidant, tocopheryl acetate.

(65) Materials and Methods:

(66) The antioxidant properties of the SBE and tocopherol acetate (Ac-Toc) against UVB/UVA/VISIBLE radiation (Irradiated: 290 nm-800 nm) were assessed by the comet assay (alkaline version) on primary cultures of normal human keratinocytes.

(67) The antioxidant properties were evaluated at concentrations of 500 nM for SBE and 50 M for Ac-Toc. Both products were dissolved in tetrahydrofuran at a final concentration of 1%. The antioxidant properties have been defined as the ability to reduce the number of single-stranded breaks of the DNA of cells which occur after UVB/UVA/visible radiation (290-800 nm) and these properties were measured after contact with the products for 120 minutes at 37 C. The radiation was delivered by a solar simulator Suntest CPS+ (Atlas Material Testing Technology BV, Moussy le Boeuf, France). The total radiation dose was 12.0 J/cm2 for a period of 2.7 minutes (rated power of the lamp750 W/m2). Negative controls included non-irradiated keratinocytes treated by THF (1%) and by Ac-Toc (50 M) and SBE (500 nM) in 1% THF.

(68) Results

(69) FIG. 8 shows that the extract according to the invention (SBE) has a high cell protective capacity against oxidative stress and that it helps to protect cell DNA better than Tocopherol acetate, while being 100 times less concentrated. Its in vitro protective capacity is thus over 100 times greater than that of tocopherol acetate.

(70) B/Carbonylation Test:

(71) By following the same UV/visible irradiation protocol as the one used above for the comet assay, the rate of carbonylation of keratinocytes in the presence of SBE and tocopherol acetate was measured.

(72) Materials and Methods:

(73) 1Culture of Keratinocytes and Irradiation

(74) Normal human keratinocytes (NHK) (T6)-10.sup.6 cells/condition Conditions: 1% THF solvent control SBE 200 nM Tocopherol acetate 20 M Each condition is carried out in triplicate. Contact with KHNs in different experimental conditions for 2 hours. UVB/UVA/Visible irradiation at 120 kJ/m.sup.2 of the cells placed in PBS by a Suntest CPS+ solar simulator (Atlas Material Testing Technology) at 4 C. Negative controls are maintained at 4 C. for the same duration as the irradiation. Trypsination of the cells post irradiation. Rinsing of the iced PBS. Elimination of the PBS and immediate freezing of the cell pellets at 80 C.
2Measurement of the Carbonylation The proteins are extracted after lysis of the cells and their concentration is measured using the Bradford test. The proteins are derived by 2,4-dinitrophenylhydrazine (DNPH), which binds to the carbonyl groups. The carbonylation rate is then measured on ELISA plates (OxyELISA Oxidized Protein Quantitation Kit, Millipore) at 450 nm.
Results

(75) FIG. 9 shows that the rate of carbonylation of proteins of the keratinocytes significantly increases after UV/visible irradiation. This increase decreases significantly (P-value <0.01) by the addition of the SBE extract or tocopherol acetate which protects the protein against oxidation. This protection is similar (P-value>0.01) for SBE and tocopherol acetate although the SBE extract is 100 times less concentrated.

(76) Although slightly less strong, the proteome protection capacity of the SBE is the same as that observed through the comet assay and 100 times that of tocopherol acetate, which corresponds to the results obtained below based on the measurement of the activity of alkaline phosphatase (AP).

(77) The protection of cells observed using the comet assay seems mainly driven by the ability of SBE to protect the proteome.

4/COSMETIC COMPOSITION OF THE SBE EXTRACT

(78) The cosmetic compositions typically comprise from 0.0001 to 0.01 wt % of the extract (dry) according to the invention.

(79) A cream can have the following composition (% by weight), the agent comprising the extract according to the invention being preferably integrated into isononyl isononanoate.

(80) TABLE-US-00003 % by INCI Name weight Aqua 78.290000 Xanthan Gum 0.100000 Ammonium Acryloyldimethyltaurate/ 0.800000 VP Copolymer & Aqua Glycerin 5.000000 Butylene Glycol 2.000000 Phenoxyethanol 0.350000 Disodium EDTA 0.200000 Chlorphenesin 0.260000 Steareth-21 1.160000 Steareth-2 1.840000 Dimethicone 2.000000 Glyceryl Dibehenate & Tribehenin & 0.500000 Glyceryl Behenate Isononyl Isononanoate 5.000000 Acacia Decurrens/Jojoba/Sunflower Seed 1.000000 Wax/Polyglyceryl-3 Esters Cetyl Alcohol 1.500000

(81) Typically, these compositions may have a colour ranging from light pink to red, because of the colour of the extract according to the invention.

(82) These embodiments show that the particular choice of the bacterium Arthrobacter agilis as a source of carotenoids (found in many species, including algae plants, cyanobacteria, fungi, etc.) has unexpected benefits within the context of the applications: Relatively easy culturing of the bacterium and preparation of the extract (unlike many extremophile organisms including halobacter); Synergy between the different fractions of the extract; Contribution of different isoforms in the overall activity of the extract (unlike for example the only deinoxanthin of Deinococcus radiodurans); Very strong anti-radical and anti-UV activity relative to known antioxidants; Synergy with known antioxidants; Protective effect never revealed vis--vis the proteome, which goes beyond the antioxidant capacity: The antioxidant capacity of SBE extract is 4 times higher than that of Trolox, while its capacity to protect the proteins is 100 times stronger than that of Trolox, in the context of both biochemical tests as well as cell cultures.