Noreugenin glycoside derivatives

Abstract

A use of noreugenin glycoside derivatives of formulas I, II and III as a self-tanning substance or to increase the synthesis of melanin, improve melanin transport and/or improve the distribution of melanin in suprabasal layers, preparations containing noreugenin glycoside derivatives of formulas I, II and III, and noreugenin glycoside derivatives of formulas I, II and III.

Claims

1. A method for tanning, comprising applying to skin a self-tanning substance comprising at least one compound of the formula I or II ##STR00008## wherein R stands for rhamnosyl, and wherein the radical R may be in the form of a D- or L-enantiomer or an - or -anomer.

2. The method according to claim 1, wherein said self-tanning substance is applied to skin until to increase melanin synthesis, improve melanin transport and/or improve the distribution of melanin in suprabasal strata.

3. The method according to claim 1, wherein the compound of formula I is 7-O--L-rhamnosylnoreugenin (7-[(6-deoxy--L-mannopyranosyl)oxy]-5-hydroxy-2-methyl-4H-1-benzopyran-4-one) (Ib) and the compound of formula II is 5-O--L-rhamnosylnoreugenin (5-[(6-deoxy--L-mannopyranosyl)oxy]7-hydroxy-2-methyl-4H-1-benzopyran-4-one)(IIa).

4. A method for producing a preparation according to claim 3, wherein the at least one compound of formulas I or II is mixed with a vehicle suitable for topical application.

5. A preparation containing at least one compound of formulas I or II ##STR00009## wherein R stands for the radical rhamnosyl, and wherein R may be present as a D- or L-enantiomer or an - or -anomer, as well as a cosmetically suitable vehicle.

6. The preparation according to claim 5, wherein the at least one compound of formulas I or II is present in an amount of 0.01 to 10 wt %.

7. The preparation according to claim 5, wherein the preparation contains at least one additional self-tanning substance.

8. A compound of formulas I or II ##STR00010## wherein R stands for rhamnosyl, wherein R may be present in the form of a D- or L-enantiomer or an - or -anomer.

9. The compound according to claim 8, wherein the compound of formula I is 7-O--L-rhamnosylnoreugenin (Ib) and the compound of formula II is 5-O--L-rhamnosylnoreugenin (IIa).

Description

FIGURES

(1) FIG. 1: Biotransformation after 6 hours to produce noreugenin rhamnosides. The annotated peaks from left to right are noreugenin 5-rhamnoside, noreugenin 7-rhamnoside, noreugenin with corresponding retention times of 46 min, 55 min and 60 min.

(2) FIG. 2: RP18 chromatogram of noreugenin 5-rhamnoside.

(3) FIG. 3: RP18 chromatogram of noreugenin 7-rhamnoside.

(4) FIG. 4: Ion scan of noreugenin rhamnosides.

(5) The examples should illustrate the present invention without limiting its scope in any way.

EXAMPLES

Example 1: Experimental Procedure: Evaluating the Tanning Properties of the Test Preparations Using Reconstituted Human Epidermis with Melanocytes of Phototype II

(6) The epidermis with melanocytes of phototype II is reconstituted in an air/liquid interface system (directly at the air-liquid interface) in a culture medium from day 0 to day 4 for a complete differentiation. The culture is then continued from day 4 to day 14 in this same medium but without bovine pituitary extract (BPE). The test preparations were administered in the cooling medium from day 4 to day 14. The medium and the test preparations are each added on day 4, day 7 and day 11. DMSO and THF are tested in parallel and IBMX (100 M) is used as the positive reference.

Example 2: Experimental Procedure: Semiquantification of the Melanin Content by Fontana-Masson Staining and Image Analysis

(7) After embedding in paraffin, a Fontana-Masson staining of the tissue sections is performed. The microscope slides are covered with a special medium and examined using a light microscope of the Leica DM2000 type with a digital camera. After staining, images are recorded and a quantification is performed by image analysis (on the basis of 12 images). For quantification of the melanin content in each image, the Leica QWin3 software is used. Two measurements are carried out. The first measurement gives the total intensity of the coloration, i.e., a lighter or darker melanin is reflected. The second measurement relates to the marked image area, i.e., the area of the epidermis containing the melanin. In these measurements all cell layers except the stratum corneum are included.

Example 3: Tanning Properties of the Substances (Ia), (Ib) and (IIa)

(8) The procedure used was the same as that carried out in example 1 and 2. After determining the cytotoxic concentrations, substance (Ia) is tested at 8 M, substance (Ib) at 40 M (solvent: 0.1% THF) and substance (IIa) at 20 M (solvent: 0.1% DMSO).

(9) TABLE-US-00001 Quantification after Preparation Fontana-Masson staining Quantification relative to the untreated controls IBMX (100 M) 152% (p < 0.05) Substance (Ia, 8 M) 125% Substance (Ib, 40 M) 174% (p < 0.05) Substance (IIa, 20 M) 143% (p < 0.05)

(10) For IBMX a significant increase in the melanin content relative to the untreated controls is found. The same thing is true for substance (Ib) relative to THF and substance (IIa) relative to DMSO. For substance (Ia) an unambiguous tendency to increase pigmentation was detected.

(11) Tanning properties of the substances (Ib) and (IIa), of a mixture of the two substances and rhamnose by itself

(12) In a second experiment the substances (Ib) and (IIa), a mixture of the two substances and rhamnose alone for comparison of the sugar radical.

(13) The procedure was the same as that described in Examples 1 and 2.

(14) After determining the cytotoxic concentrations of these substances, substance (Ib) is tested at 40 M in DMSO (0.1%), substance (IIa) is tested at 20 M in DMSO (0.1%), the mixture of (Ib)/(IIa) is tested at 40/20 M in DMSO (0.1% and rhamnose is tested at 40 M in THF (0.2%).

(15) The results obtained have confirmed the findings of the first study.

(16) TABLE-US-00002 Quantification after Preparation Fontana-Masson staining Quantification relative to the untreated controls IBMX (100 M) 150% (p < 0.01) Rhamnose (40 M) 109% Substance (Ib, 40 M) 167% (p < 0.01) Substance (IIa, 20 M) 147% (p < 0.05) Mixture (Ib/IIa, 40 M/20 M) 140%

(17) For IBMX a significant increase in the melanin content relative to the untreated controls was found. The same thing is also true of substance (Ib) relative to DMSO and substance (IIa) relative to DMSO. For the mixture (Ib/IIa) a definite tendency to increase pigmentation was detected. With rhamnose the tendency to increase melanin synthesis is only faintly pronounced.

Example 5: Production of Noreugenin 5-Rhamnoside (IIa) and Noreugenin 7-Rhamnoside (Ib)

(18) The biotransformation is carried in a fermentative process in 3 liter agitated Erlenmeyer flasks with a fermentation volume of 500 mL. To this were added the transgenic strains E. coli BL21 (DE3) pET19::mgtB for the glucosylation or E. coli Rosetta gami 2 (DE3) pET19::gtfC were tested in Luria-Bertani (LB) complex medium at 28 C. and found to have an optical density (OD.sub.600) of 0.8. By adding 50 M isopropyl--D-thiogalactopyranoside (IPTG), the glycosyl transferase is expressed overnight at 17 C. Then the cells are centrifuged for 20 minutes at 17 C. and 7500 g and then resuspended in 0.1M phosphate buffer with 0.15M sodium chloride. To this culture are added 1 (w/v) % glucose and 0.2 mM noreugenin for the reaction and incubated further at 28 C. After 24 hours, the reaction is terminated by centrifuging the cells at 4 C. and 10,000 g for 20 minutes. The glycosides are in the supernatant after the reaction and are isolated by means of chromatographic purification. The first separation step takes place on an RT 250-10 Pharm prep column 100 RP-18e (10 m) from Merck. The entire culture supernatant is enriched on the column by means of the pump system at a flow rate of 10 mL/min on the preparative HPLC system 1260 Infinity from Agilent and then eluted from the column again with a linear gradient of ascending acetonitrile concentration and thereby fractionated.

(19) The preparative RP18-HPLC method as well as the analytical RP18 methods are based on a step gradient with the eluents A (H.sub.2O+0.01% (v:v) trifluoroacetic acid) and eluent B (acetonitrile+0.01% (v:v) trifluoroacetic acid) having the following steps:

(20) 0-10 min 5% B,

(21) 10-20 min 10% B,

(22) 20-30 min 15% B,

(23) 30-40 min 20% B,

(24) 40-50 min 30% B,

(25) 50-60 min 100% B,

(26) 60-70 min 5% B.

(27) Even in the first chromatography step, the individual compounds can be separated well from one another (see FIG. 1). Acetonitrile is removed using an evaporator from Bchi Labortechnik GmbH and the aqueous solution of the individual noreugenin glycosides is lyophilized. The dried powder is dissolved in 30% acetonitrile (v:v). The second purification step for separation of the remaining impurities is carried out using a Hypersil Gold PFP (pentafluorophenyl) phase 2504.6 mm from Thermo Scientific on a VWR Hitachi LaChrom Elite system. An isocratic run with 30% acetonitrile and a flow of 1 mL/min yields the target substances in a purity of more than 95%. The pure substances are dried, thus yielding a lyophilized powder.

(28) For the analysis and process control, analytical HPLC with a diode array detector (VWR Hitachi see above) and mass spectrometry are performed. Mass spectrometric data is recorded using the microOTOF-Q with electron spray ionization (ESI) from Bruker Dalton. The samples are measured in the negative ion mode and injected using a syringe pump at a flow rate of 200 L/min.

(29) FIG. 1 shows the supernatant of a fermentation process after a reaction time of 6 hours. The non-glycosylated starting material noreugenin elutes at a retention time of 60 minutes. The noreugenin 7-rhamnoside has a retention time of 55 minutes, and the most hydrophilic molecule is noreugenin 5-rhamnoside with a retention time of 46 minutes.

(30) The purified products are illustrated in FIGS. 2 and 3 and show a purity of >95% deteiniined by HPLC.

(31) For unambiguous identification, the mass of the aforementioned fractions is determined. In the negative ion mode, the rhamnosides are presented as shown in FIG. 4. The molecule has a single charge and a molecular weight of m/z 337. Two molecules stored together and showing a charge appear as a mass of m/z 675 in the mass spectrum.

(32) NMR data on noreugenin 5-rhamnoside (IL):

(33) .sup.1H-NMR (DMSO-, 400 MHz): =6.62 (d, 1H, J=2.2 Hz), 6.58 (d, 1H, J=2.2 Hz), 5.99 (d, 1H), 5.38 (d, 1H), 4.00 (s, 1H), 3.89-3.92 (dd, 1H, J.sub.1=3.10 Hz, J.sub.2=9.2 Hz), 3.49-3.54 (m, 7H), 3.36 (m, 1H), 2.30 (s, 3H).