GLYCOSYLATED BACTERIORUBERINS AND INDUSTRIAL APPLICATIONS THEREOF

Abstract

The invention concerns glycosylated bacterioruberins isolated from monoglycosylated bacterioruberins, diglycosylated bacterioruberins, triglycosylated bacterioruberins, tetraglycosylated bacterioruberins, pentaglycosylated bacterioruberins, hexaglycosylated bacterioruberins, heptaglycosylated bacterioruberins, octaglycosylated bacterioruberins, nonaglycosylated bacterioruberins, decaglycosylated bacterioruberins, undecaglycosylated bacterioruberins, and dodecaglycosylated bacterioruberins. In particular, the present invention concerns the purification or synthesis of glycosylated forms of the carotenoid bacterioruberin, as well as its applications, in particular in the fields of pharmaceuticals, dermocosmetics and nutraceuticals and biotechnology.

Claims

1-15. (canceled)

16. An isolated glycosylated bacterioruberin, wherein the glycosylated bacterioruberin is a monoglycosylated bacterioruberin, diglycosylated bacterioruberin, triglycosylated bacterioruberin, tetraglycosylated bacterioruberin, pentaglycosylated bacterioruberin, hexaglycosylated bacterioruberin, heptaglycosylated bacterioruberin, octaglycosylated bacterioruberin, nonaglycosylated bacterioruberin, decaglycosylated bacterioruberin, undecaglycosylated bacterioruberin, or dodecaglycosylated bacterioruberin, and wherein the glycosylated bacterioruberin has a purity of at least 70% by weight.

17. The isolated glycosylated bacterioruberin of claim 16, wherein the glycosylated bacterioruberin has a purity of at least 80%, 90%, 95%, or 99% by weight.

18. The isolated glycosylated bacterioruberin of claim 16, wherein the glycosylated bacterioruberin is a monoglycosylated bacterioruberin, diglycosylated bacterioruberin, triglycosylated bacterioruberin, or tetraglycosylated bacterioruberin.

19. The isolated glycosylated bacterioruberin of claim 16, wherein the glycosylated bacterioruberin is separated and purified from an extract of an extremophilic bacterium.

20. The isolated glycosylated bacterioruberin of claim 19, wherein the extremophilic bacterium is of the Micrococcaceae family.

21. A composition comprising at least one glycosylated bacterioruberin, wherein the composition does not comprise a non-glycosylated form of bacterioruberin, and wherein the composition comprises one or more pharmaceutically acceptable excipients.

22. The composition of claim 21, wherein the composition comprises at least one isolated glycosylated bacterioruberin, wherein the glycosylated bacterioruberin is a monoglycosylated bacterioruberin, diglycosylated bacterioruberin, triglycosylated bacterioruberin, tetraglycosylated bacterioruberin, pentaglycosylated bacterioruberin, hexaglycosylated bacterioruberin, heptaglycosylated bacterioruberin, octaglycosylated bacterioruberin, nonaglycosylated bacterioruberin, decaglycosylated bacterioruberin, undecaglycosylated bacterioruberin, or dodecaglycosylated bacterioruberin, and wherein the glycosylated bacterioruberin has a purity of at least 70% by weight.

23. The composition of claim 21, wherein the composition comprises a mixture of monoglycosylated bacterioruberins, diglycosylated bacterioruberins, triglycosylated bacterioruberins, and tetraglycosylated bacterioruberins.

24. The composition of claim 21, wherein the composition comprises a mixture of glycosylated bacterioruberins comprising monoglycosylated bacterioruberins and diglycosylated bacterioruberins, and wherein the mixture comprises 20 to 80% by weight of monoglycosylated bacterioruberins and 20 to 80% by weight of diglycosylated bacterioruberins with respect to the total weight of the mixture of glycosylated bacterioruberins.

25. The composition of claim 21, wherein the composition is formulated as a therapeutic formulation.

26. The composition of claim 21, wherein the composition is in the form of a food supplement.

27. The composition of claim 21, wherein the composition is formulated as a cosmetic composition suitable for topical application.

28. The composition of claim 27, wherein the at least one glycosylated bacterioruberin is present in an amount ranging from 0.0001 to 0.5% by weight with respect to the total weight of the cosmetic composition.

29. A method for preventing oxidative stress in a subject, the method comprising: administering to the subject a composition comprising at least one glycosylated bacterioruberin, wherein the composition does not comprise a non-glycosylated form of bacterioruberin, and wherein the composition comprises one or more pharmaceutically acceptable excipients.

30. The method of claim 29, wherein the composition: combats cellular aging of the skin; scavenges radicals; protects protein degradation; protects proteome degradation; protects cellular detoxification mechanisms; or protects DNA repair systems.

31. The method of claim 30, wherein the cellular aging of the skin is due to solar radiation, UV radiation, or visible light.

32. The method of claim 29, wherein administering the composition comprises applying the composition to the skin of the subject.

Description

[0199] The manner in which the invention may be implemented and the concomitant advantages will become more apparent from the following exemplary embodiments, which are given by way of non-limited indication and with the support of the accompanying figures.

[0200] FIG. 1 shows the composition of an extract of carotenoids from the isolate SB5 of the species A. agilis.: BR=-bacterioruberin, BR-MonoG: monoglycosylated form, BR-DiG: diglycosylated form, BR-DiG2: Another form of BR-DiG, BR-TetraG: tetraglycosylated form.

[0201] FIG. 2 shows the protection provided by several carotenoids against UV-B-induced protein carbonylation in Normal Human Keratinocytes (NHK). DMSO=dimethyl sulphoxide (solvent control); BR=-bacterioruberin; BR-DiG=diglycosylated bacterioruberins;: BR-MonoG+BR-DiG=mixture of mono- and di-glycosylated bacterioruberins.

EXEMPLARY EMBODIMENTS

Example I

Purification of the Glycosylated Bacterioruberins of an Extract of Carotenoids of the Bacterium A. agilis

I-1 Aim of the Study

[0202] The aim of this study was to isolate and quantify the molecules contained in an extract of total carotenoids from the bacterium A. agilis.

I-2 Materials and Methods

I-2.1 Extract

[0203] The total extract of carotenoids from the bacterium A. agilis contained in the GREENTECH starting material known as Miroruberine and corresponding to the INCI name Micrococcus lysate was used in this study; it was derived from the strain SB5. This so-called SBE extract could be obtained by using the method described in the publication of patent application WO 2014/167247.

I-2.2 Column and Thin-Layer Chromatography

[0204] The SBE extract was taken up in tetrahydrofuran (THF) until it had completely dissolved. A step for separating the glycosylated Bacterioruberins by silica gel chromatography was carried out in a glass column after diluting the SBE in THF.

[0205] 1. Suspension of the silica gel in a DCM/methanol mixture (10/1) before being poured into the column

[0206] 2. After sedimentation of the silica gel, 1 cm of sand is added before carrying out 3 washes with the DCM/methanol mixture.

[0207] 3. 0.5 mL of SBE diluted in THF was deposited on the sand and allowed to stand for 5 minutes

[0208] 4. 50 mL of DCM/methanol mixture (10/1) was added slowly, allowing fractions 1, 2, 3 and 4 to be collected

[0209] 5. 40 mL of DCM/methanol mixture (8/2) was added slowly, allowing the collection of fractions 5 and 6

[0210] 6. 40 mL of DCM/methanol mixture (5/5) was added slowly, allowing the collection of fraction 7

[0211] 7. 40 mL of DCM/methanol mixture (3/7) was added slowly to allow the collection of fraction 8

[0212] 8. All of the fractions were then compared by TLC (DCM/methanol (10/1)) with SBE and quantified by absorption (using the absorption maximum for each fraction).

I-2.3 Separation of Fractions by HPLC

[0213] Equipment: Nexera XR, binary pump (Shimadzu)

[0214] Column: C18; Intersustainable Swift 5 m 4.6150 mm, manufacturer: GL Sciences

[0215] Mobile phases:

[0216] A: 20% H.sub.2O in MeOH

[0217] B 20% EtOAc in MeOH

[0218] Flow: 1.5 mL/min

[0219] Injection volume: 50 L

TABLE-US-00001 TABLE 1 A B 1 min 100 0 20 min 0 100

I-3 Results and Discussion

[0220] In this purification, the first step for separation by column chromatography made it possible to collect the various fractions the purity of which was confirmed by TLC and by HPLC-DAD, by comparing it with the absorbance spectrum for the native extract; the quantification of the various forms was carried out by UV absorption at 500 nm.

[0221] Each of the molecules of each of the fractions collected by chromatography was identified by Maldi-TOF-TOF spectroscopy using an AUTOFLEX instrument (Brucker: method: CHCA and DHB Matrix without TFA in reflector acquisition). The distribution between the different forms was calculated by combining the results obtained with the quantifications carried out on these fractions by HPLC-DAD (FIG. 1). Fraction 1 corresponds to beta-carotene, a by-product of the synthesis of bacterioruberin, but this molecule represented only 0.79% of the extract. Fraction 4 had the same extract profile for Halobacter salinarium (Halorubin), the majority molecule of which is bacterioruberin (BR). This molecule represents approximately half of the dry extract (FIG. 1).

[0222] Two diglycosylated forms (BR-DiG1 and BR-DiG2) which migrated separately were identified. The diglycosylated forms represented >22% of the extract.

[0223] The monoglycosylated form BR-MonoG represented >26% of the extract. The tetraglycosylated form (BR-TetraG) represented only 0.01% of the extract (FIG. 1).

Example II

Protection Conferred by Several Carotenoids against UV-B-Induced Carbonylation in Human Keratinocytes

II-1 Aim of the Study

[0224] The aim of this study was to compare the effect of protecting the keratinocyte proteins from UV stress for the various molecules derived from a carotenoid extract of the SB5 strain of the actinobacterium A. agilis according to Example I. In particular, the following molecules were the subject of the study: [0225] -bacterioruberin (BR) [0226] the monoglycosylated form of bacterioruberin (MonoG-BR) [0227] the two diglycosylated forms of bacterioruberin, (BR-DiG1 and BR-DiG2); these forms were combined and studied together (BR-DiG)

[0228] A known carotene, a xanthophyll and a glycosylated carotenoid were also tested: [0229] lycopene (carotene) [0230] astaxanthin (xanthophyll) [0231] crocin (glycosylated carotenoid).

[0232] The total carbonylation of the proteins was measured in the presence or absence of UV-B stress.

II-2 Materials and Methods

II-2.1 Experimental Design

[0233] 1Test different stress doses (UV) on 12 wp (well plate) (aggregation): [0234] UV-B (80; 40; 20; 15; 10 mJ/cm2)

[0235] 2Based on 1-, test 2 different stress doses (UV) on a small flask for carbonylation;

[0236] 3Test the molecules against UV stress on 54 flasks for carbonylation: 8 molecules+control, in triplicate, with and without UV;

[0237] 4Measure the carbonylation of the 108 conditions (in duplicate).

II-2.2 Cell Treatment and Irradiation

[0238] UV-B stress: Lamp at 313 nm, dose of 80; 40; 20; 10mJ/cm.sup.2 for dose selection and 15 mJ/cm.sup.2 for molecule testing

II-2.3 Test Molecules and Concentrations

[0239] BR, 100 nM [0240] BR-DiG, 100 nM [0241] BR-MonoG +BR-DiG 100 nM (50+50 nM) [0242] lycopene 100 nM [0243] astaxanthin 100 nM [0244] crocin 100 nM. [0245] the solvent (DMSO) represented the control.

II-2.4 Culture and Treatment of Keratinocytes

[0246] Immortalized NHEK/SVTERT 3-5 keratinocytes were seeded with approximately one million cells/plate in 108 plates (diam: 100 mm, 56 cm.sup.2), corresponding to 36 conditions in the biological triplicatesthe cells were cultured to a confluence of 90 to 95% in the keratinocyte medium (Keratinocyte-SFM from Gibco, 17005075) at 37 C. and 5% CO.sub.2.

[0247] The cells were incubated with the test molecules for 2 hours.

[0248] After 2 h of pretreatment, the medium was removed and replaced with PBS. Half of the plates were irradiated with 15 mJ/cm.sup.2 UV-B.

[0249] Following the irradiation in each plate, Keratinocyte-SFM medium was added and the cells were incubated for 24 h at 37 C., 5% CO.sub.2.

[0250] After removing the medium from all the plates, the cells were washed with PBS and then trypsinized with trypsin-EDTA (0.25%) in HBSS (1) in the presence of phenol red (Capricorn Scientific, TRY-3B). After collecting the plates, the cells were counted, transferred into 1.5 mL tubes and the pellets were stored at 80 C.

II-2.5 Measurement of Protein Carbonylation

[0251] The cells were lysed by adding 100 L of UTC lysis buffer (UTC: urea, thiourea & CHAPS) to each tube containing the cell pellet. The lysis of the cells was carried out on a microtube shaker at a temperature of 4 C. for 45 min; 600 rpm.

[0252] After that, the samples were centrifuged and the supernatant was collected; the granules were discarded.

[0253] The supernatant was transferred into a 3 kDa Amicon filter and rinsed with PBS (approximately 100 L of protein for 400 L of PBS). The samples were centrifuged for 40 min at +4 C. at 14000 rpm. The procedure was repeated 3 times for each sample. After the third centrifugation, the Amicon filters were placed in a new tube and centrifuged for 3 minutes at 2000 rpm; the eluates were used in the remainder of the analysis.

[0254] The protein concentration was measured by means of a Bradford protein quantification assay.

[0255] Each 15 g sample of protein was stained with 0.15 L of 5 mM CF647 dye. The staining was carried out overnight at +4 C., 600 rpm.

[0256] The protein samples were loaded onto 12.5% acrylamide gels. The electrophoresis was carried out at 80V until the samples were removed from the stacking gel, after which the voltage was increased to 120V until the samples reached the end of the gel.

[0257] The gels were then collected and rinsed several times in ultrapure water. Next, the gels were stained with 1 purple dye (SERVA Purple 43386.01) from a 250 concentrated form in order to examine the expression of the proteins. This step was carried out at 50 rpm on a shaker for 10 min at ambient temperature.

[0258] The images were digitized with the Amersham Typhoon Biomolecular Imager for both dyes. Fluorescence scanning was at 635 nm for CF647 and at 532 nm for SERVA violet.

[0259] Subsequently, the analysis of the scan was carried out with Life Science Research Bio-Rad's Image Lab software by selecting each channel and normalizing for background noise.

[0260] The total carbonylation of each culture was finally measured using a Western blot method (Oxy-blot).

II-3 Results and Discussions

[0261] The overall results of the experiments are shown in FIG. 2. In this figure, the levels of carbonylation before and after UV-B stress are compared. Comparison with the DMSO solvent, which should not offer significant protection, makes it possible to determine whether a compound or mixtures of compounds protects the cellular proteins from UV-B-induced carbonylation. Surprisingly, diglycosylated bacterioruberins (BR-DiG) and the mixture of mono- and diglycosylated bacterioruberins (BR-MonoG+BR-DiG) protect more effectively than all the other carotenes and xanthophylls which were tested, including crocin, which is a glycosylated carotenoid. The protection conferred in each case is greater than that observed with -bacterioruberin (BR).

Example III

O/W SPF50+Emulsion

[0262] The percentages indicated are given by weight of product with respect to the total weight of the composition in the tables below.

TABLE-US-00002 TABLE 2 INCI name % Aqua/water/eau 49.816 Octocrylene 9.00 Dicaprylyl carbonate 8.4973 Methylene bis-benzotriazolyl 6.00 tetramethylbutylphenol [nano] Glycerin 5.00 Butyl methoxydibenzoylmethane 4.50 Dimethicone 3.00 Tribehenin PEG-20 esters 3.00 Bis-ethylhexyloxyphenol methoxyphenyl triazine 2.50 C10-18 triglycerides 2.00 Methylpropanediol 1.40 Sucrose stearate 1.00 Decyl glucoside 0.90 Hydroxyethyl acrylate/sodium acryloyldimethyl 0.88 taurate copolymer Pentylene glycol 0.50 Tocopheryl acetate 0.50 Sodium citrate 0.20 1,2-hexanediol 0.125 Caprylyl glycol 0.125 Mannitol 0.10 Xylitol 0.10 Citric acid 0.09 Polysorbate 60 0.06 Sorbitan isostearate 0.06 Bacterioruberin monoglycoside (in accordance 0.50 with the invention) Bacterioruberin diglycoside (in accordance 0.50 with the invention) Rhamnose 0.05 Dipropylene glycol 0.048 Xanthan gum 0.024 Ectoin 0.01 Glycyrrhiza glabra (licorice) root extract 0.01 Tocopherol 0.0027 Fructooligosaccharides 0.001 Caprylic/capric triglyceride 0.00095 Laminaria ochroleuca extract 0.00005

Example IV

Day CreamO/W Emulsion

[0263]

TABLE-US-00003 TABLE 3 INCI name % Aqua/water/eau 55.788 Dicaprylyl carbonate 25.0873 Glycerin 5.00 Dimethicone 3.00 Tribehenin PEG-20 esters 3.00 C10-18 triglycerides 2.00 Methylpropanediol 1.40 Sucrose stearate 1.00 Hydroxyethyl acrylate/sodium acryloyldimethyl 0.88 taurate copolymer Pentylene glycol 0.50 Tocopheryl acetate 0.50 Sodium citrate 0.20 1,2-hexanediol 0.125 Caprylyl glycol 0.125 Mannitol 0.10 Xylitol 0.10 Citric acid 0.09 Polysorbate 60 0.06 Sorbitan isostearate 0.06 Rhamnose 0.05 Bacterioruberin monoglycoside (in accordance 0.04 with the invention) Bacterioruberin diglycoside (in accordance 0.03 with the invention) Bacterioruberin tetraglycoside (in accordance 0.03 with the invention) Ectoin 0.01 Glycyrrhiza glabra (licorice) root extract 0.01 Tocopherol 0.0027 Fructooligosaccharides 0.001 Caprylic/capric triglyceride 0.00095 Laminaria ochroleuca extract 0.00005

Example V

Day CreamSerum

[0264]

TABLE-US-00004 TABLE 4 INCI name % Aqua/water/eau 70.044252 Isostearyl isostearate 6.00 Limnanthes alba (meadowfoam) seed oil 5.9982 Glycerin 5.00 Propanediol 3.00 Boron nitride 2.00 Lauroyl lysine 2.00 C10-18 triglycerides 1.50 C12-16 alcohols 1.20 Pentylene glycol 1.00 Hydrogenated lecithin 0.40 Palmitic acid 0.40 Caprylic/capric triglyceride 0.29928 1,2-hexanediol 0.25 Caprylyl glycol 0.25 Sodium polyacrylate 0.25 Fragrance (parfum) 0.20 Xanthan gum 0.18 Sclerotium gum 0.08 Lecithin 0.076 Bacterioruberin tetraglycoside (in 0.06 accordance with the invention) Pullulan 0.06 Silica 0.004

BIBLIOGRAPHY

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[0271] Mohamed H E, van de Meene A M L, Roberson R W, Vermaas W F J. Myxoxanthophyll is required for normal cell wall structure and thylakoid organization in the cyanobacterium, Synechocystis sp strain PCC 6803 (2005) J Bacteriol. 187:6883-6892.

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