PROCESS FOR PREPARING SPHINGOLIPIDS

Abstract

The invention provides a process for preparing sphingolipids, compositions comprising sphingolipids and further components, and for the use of the compositions.

Claims

1. A process for preparing sphingolipids of the general formula I ##STR00013## by reacting a first component, a lysosphingolipid of the general formula II ##STR00014## with a second component, an acyl group donor of the acyl group R.sup.1CO, wherein R.sup.1 represents a linear or branched alkyl chain having 2 to 55 carbon atoms that optionally contains one or more multiple bonds and/or aromatic or heteroaromatic rings, is optionally interrupted by oxygen atoms or ester or amide functionalities and is optionally substituted by at least one further group selected from alkyl, hydroxyl, keto or amine groups, R.sup.2 represents H, phosphocholine, serine, ethanolamine or a sugar, and X represents CH?CH, CH.sub.2CH.sub.2 or CH.sub.2HCOH, wherein the first and second components, based on the overall reaction mixture, account for a total of at least 70% by weight, and not more than 600 propyl laurate units of at least one carboxylic ester hydrolase from the E.C. 3.1.1 enzyme class per gram of first component are used in the entire reaction mixture, where one propyl laurate unit is defined as the amount of enzyme that synthesizes one ?mol of propyl laurate per minute from 1-propanol and lauric acid.

2. The process according to claim 1, wherein R.sup.2?H and X?CH.sub.2CH.sub.2.

3. The process according to claim 1, wherein the second component is selected from acyl group donors that provide an acyl group selected from the group of acyl groups of natural fatty acids.

4. The process according to claim 1, wherein the second component is selected from esters based on alkanols and polyols having up to 6 carbon atoms.

5. The process according to claim 1, wherein the first component used is sphinganine with R.sup.2?H and X?CH.sub.2CH.sub.2, and the second component used is an acyl group donor selected from the group consisting of coconut fat, palm kernel oil, olive oil, palm oil, argan oil, castor oil, linseed oil and babassu oil.

6. The process according to claim 1, wherein the reactants are present on commencement of the reaction in a molar ratio of first component to second component of 1:0.11 to 1:2000.

7. The process according to claim 1, wherein, based on the overall reaction mixture, the maximum total amount of any solvent present is less than 20% by weight.

8. The process according to claim 1, wherein it is conducted under anhydrous conditions.

9. The process according to claim 1, wherein the carboxylic ester hydrolase is selected from the group of the lipase from Thermomyces lanuginosus with accession number O59952, lipases A and B with accession number P41365 from Candida antarctica and the lipase from Mucor miehei with accession number P19515, the lipase from Humicola sp. with accession number O59952, the lipase from Rhizomucor javanicus with accession number S32492, the lipase from Rhizopus oryzae with accession number P61872, the lipases from Candida rugosa with accession number P20261, P32946, P32947, P3294 and P32949, the lipase from Rhizopus niveus accession number P61871, the lipase from Penicillium camemberti with accession number P25234, the lipases from Aspergillus niger with accession number ABG73613, ABG73614 and ABG37906 and the lipase from Penicillium cyclopium with accession number P61869, and their respective at least 60% homologues at the amino acid level.

10. A composition comprising A) at least one sphingolipid of the general formula I ##STR00015## B) at least one lysosphingolipid of the general formula II ##STR00016## C) at least one triglyceride of the general formula III ##STR00017## D) at least one diglyceride of the general formula IV ##STR00018## E) at least one monoglyceride of the general formula V ##STR00019## where R.sup.1 is the same or different and independently represents a linear or branched alkyl chain having 2 to 55 carbon atoms that optionally contains one or more multiple bonds and/or aromatic or heteroaromatic rings, is optionally interrupted by oxygen atoms or ester or amide functionalities and is optionally substituted by at least one further group selected from alkyl, hydroxyl, keto or amine groups, R.sup.2 is the same or different and independently represents H, phosphocholine, serine, ethanolamine or a sugar, and X represents CH?CH, CH.sub.2CH.sub.2 or CH.sub.2HCOH, wherein based on the total composition, the components are present as follows: A) 30% by weight to 98% by weight, B) 0.01% by weight to 60% by weight, C) 0.01% by weight to 60% by weight, D) 0.1% by weight to 30% by weight, E) 0.1% by weight to 30% by weight.

11. A composition having a high oil content and comprising A) at least one sphingolipid of the general formula I ##STR00020## B) at least one lysosphingolipid of the general formula II ##STR00021## C) at least one triglyceride of the general formula III ##STR00022## D) at least one diglyceride of the general formula IV ##STR00023## E) at least one monoglyceride of the general formula V ##STR00024## where R.sup.1 is the same or different and independently represents a linear or branched alkyl chain having 2 to 55 carbon atoms that optionally contains one or more multiple bonds and/or aromatic or heteroaromatic rings, is optionally interrupted by oxygen atoms or ester or amide functionalities and is optionally substituted by at least one further group selected from alkyl, hydroxyl, keto or amine groups, R.sup.2 is the same or different and independently represents H, phosphocholine, serine, ethanolamine or a sugar, and X represents CH?CH, CH.sub.2CH.sub.2 or CH.sub.2HCOH, wherein the components are present in the composition as follows: A) 30 parts by weight to 98 parts by weight, B) 0.01 part by weight to 60 parts by weight, D) 0.1 part by weight to 30 parts by weight, E) 0.1 part by weight to 30 parts by weight, and, based on the overall composition, C) 60% by weight to 98% by weight.

12. The composition according to claim 10, wherein R.sup.2?H and X?CH.sub.2CH.sub.2 and R.sup.1 is selected from the group consisting of cocyol radicals, palmitoyl radicals, oleyl radicals, linoleyl radicals, linolenyl radicals, ricinoyl radicals, stearyl radicals, erucyl radicals and polyunsaturated fatty acyl radicals.

13. A cosmetic formulation comprising the composition according to claim 10.

14. A retaining skin moisture formulation, for avoiding skin dryness and/or for fortifying the skin barrier retaining, the skin moisture formulation comprising the composition according to claim 10.

15. A hair structure formulation, for repair of the hair structure after UV stress, for boosting the CMC complex in the hair and/or for protecting the hair, especially from protein degradation, the hair structure formulation comprising the according to claim 10.

16. The composition according to claim 10, wherein R.sup.2 is selected from the group consisting of sugar and H, and X is CH.sub.2CH.sub.2 wherein based on the total composition, the components are present as follows: A) 50% by weight to 90% by weight, B) 0.01% by weight to 45% by weight, C) 0.01% by weight to 45% by weight, D) 0.1% by weight to 20% by weight, E) 0.1% by weight to 20% by weight.

17. The composition according to claim 10, wherein R.sup.2 is H, and X is CH.sub.2CH.sub.2 wherein based on the total composition, the components are present as follows: A) 65% by weight to 85% by weight, B) 0.5% by weight to 10% by weight, C) 0.01% by weight to 10% by weight, D) 2% by weight to 15% by weight, and E) 2% by weight to 15% by weight.

18. The process according to claim 1, wherein the first and second components, based on the overall reaction mixture, account for a total of at least 95% by weight.

19. The process according to claim 1, wherein, based on the overall reaction mixture, the maximum total amount of any solvent present is less than 5% by weight.

20. The process according to claim 1, wherein the carboxylic ester hydrolase is selected from the group of the lipase from Thermomyces lanuginosus with accession number O59952, lipases A and B with accession number P41365 from Candida antarctica and the lipase from Mucor miehei with accession number P19515, the lipase from Humicola sp. with accession number O59952, the lipase from Rhizomucor javanicus with accession number 532492, the lipase from Rhizopus oryzae with accession number P61872, the lipases from Candida rugosa with accession number P20261, P32946, P32947, P3294 and P32949, the lipase from Rhizopus niveus accession number P61871, the lipase from Penicillium camemberti with accession number P25234, the lipases from Aspergillus niger with accession number ABG73613, ABG73614 and ABG37906 and the lipase from Penicillium cyclopium with accession number P61869, and their respective at least 95% homologues at the amino acid level.

Description

[0102] The following figures are part of the examples:

[0103] FIG. 1: Probability of survival of the individual hairs

[0104] FIG. 2: Probability of survival of alkali-straightened hair after treatment with ceramides

[0105] FIG. 3: Probability of survival of ethnic hair

EXAMPLES

Example 1: Determination of the Specific Activity of the Enzyme Used in PLU

[0106] To determine the enzymatic activity in PLU (propyl laurate units), 1-propanol and lauric acid are mixed homogeneously in an equimolar ratio at 60? C. The reaction is started with addition of enzyme and the reaction is timed. Samples are taken from the reaction mixture at intervals, and the content of lauric acid converted is determined by means of titration with potassium hydroxide solution. Enzyme activity in PLU is found from the rate at which 1 g of the enzyme in question synthesizes 1 ?mol of propyl laurate per minute at 60? C.; cf. also US20070087418, especially

[0107] The manufacturer Novozymes, for example, states 10000 PLU/g in its Novozym 435 product data sheet.

Example 2: Reaction of Sphinganine with Olive Oil, Olive Oil-Based Ceramide NG

[0108] 12 g of olive oil together with 8 g of sphinganine are dissolved in 2.2 g of 2-methyl-2-butanol. The mixture is heated to 100? C., blanketed with nitrogen to 0.05 bar and stirred. On attainment of the reaction temperature, 120 PLU of Novozym 435 are added and stirring is continued. After 24 h, Novozym 435 was filtered off. The reaction mixture thus obtained, based on sphinganine, reached a conversion of >98.5%. After the reaction, the solvent is removed by distillation and the product mixture obtained is used in application tests.

Example 3: Composition Having a High Oil Content

[0109] 10 g of a composition from Example 2 are dissolved in 90 g of a 1:1 (by weight) mixture of olive oil and castor oil. This gives a composition having a high oil content, containing 10% of a mixture of olive oil-based ceramide NG, glycerol, glycerides and sphinganine with 45% olive oil and 45% castor oil.

Example 4: Reaction of Sphinganine with Castor Oil

[0110] 25 g of castor oil are mixed with 16 g of sphinganine. The mixture is heated to 100? C., blanketed with nitrogen to 0.05 bar and stirred. On attainment of the reaction temperature, 80 PLU of Novozym 435 (based on sphinganine) are added and stirring is continued. After 24 h, Novozym 435 was filtered off. The reaction mixture thus obtained was blended with 60 ml of castor oil. Based on sphinganine, the reaction reached a conversion of >98.5%.

Example 5: Effect of the Amount of Solvent

[0111] 12 g of olive oil are mixed with 8 g of sphinganine, and the amount of 2-methyl-2-butanol stated below in the table is added. The mixture is heated to 80? C., blanketed with nitrogen to 0.05 bar and stirred. On attainment of the reaction temperature, the amount of Novozym 435 specified is added and stirring is continued. After 2 h, a sample was taken in each case and analysed by means of GC for the residual sphinganine content. The results show that better conversions can be achieved with decreasing amount of solvent when less enzyme is used. The negative control conducted without addition of solvent and enzyme gave a non-zero conversion.

TABLE-US-00001 Amount of enzyme in 2-Methyl-2-butanol Conversion PLU/g of sphinganine solvent in % in % 120 10 26 25 10 24 120 1 27 25 0 28

Example 6: Further Synthesis Examples

[0112] The general procedure was as described in Example 2 without the use of solvents.

TABLE-US-00002 Conversion Amount Amount of after 24 h of acyl Amount of enzyme in % based donor in sphinganine in PLU/g of Temperature on Name Acyl donor g in g sphinganine ? C. sphinganine TG1 Olive oil 448 41 200 95 >98 TG2 Olive oil 29 41 200 100 >98 Castor oil 31 TG3 Castor oil 24 16 120 85 >98 TG4a Rapeseed oil 73 25 100 90 >95 TG4b Rapeseed oil 88 10 100 90 >95 TG5 Sesame oil 10 7 150 90 >95 TG6 Palm kernel oil 56 25 100 90 >95 TG7a Palm oil 70 25 100 90 >95 TG7b Palm oil 84 10 100 90 >95 TG8 Almond oil 10 7 100 90 >90 TG9 Sorbitan 60 50 100 90 >70 monooleate TG10 Sorbitan 58 50 100 90 >70 monolaurate TG11 Sorbitan trioleate 83 50 100 90 >70 TG12 Soya oil 87 10 100 90 >90 TG13 Glycol distearate 50 25 100 90 >70 TG14 Cetyltriglyceride 83 50 100 90 >90

Example 7: Application Data for Skin: Effect on Skin Moisture, Dryness Measured by Flakiness, Skin Barrier Measured by General Condition of Skin In Vivo

[0113] To determine the skincare properties of the composition having a high oil content from Example 3, an in vivo study was conducted. The sequence of the study was as follows: The subjects each received 2 test formulations that had to be applied to the inside of the underarm twice daily for a duration of 2 weeks. Before commencement of use and after 1 and 2 weeks, a special camera (Visioscan VC 98, Courage & Khazaka, Cologne) was used to take a black and white image of the skin. Using these images, the camera software calculated skin flakiness and skin roughness. The values are calculated from the grey level distribution of the images; therefore, the results have no unit.

[0114] The subjects also received a questionnaire for assessment of the care properties of the test formulations. A total of 25 subjects took part in the study.

TABLE-US-00003 Formulation with active Ingredient Vehicle ingredient Glyceryl Stearate Citrate 2.00% 2.00% Cetearyl Alcohol 1.00% 1.00% Caprylic/Capric Triglyceride 4.80% 4.80% C12-15 Alkyl Benzoate 4.00% 4.00% Example 3 (% based on sphingolipid) 1.00% Carbomer 0.20% 0.20% Ethylhexylglycerin, Phenoxyethanol 1.00% 1.00% Sodium hydroxide (10% in water) 0.70% 0.70% Water to 100% to 100%

TABLE-US-00004 Decrease in skin Decrease in skin roughness flakiness Vehicle Example 3 Vehicle Example 3 After 1 week ?1.58 ?11.08 ?0.0271 ?0.1292 After 2 weeks ?5.61 ?15.04 ?0.0761 ?0.1313

TABLE-US-00005 The product reduces The product supplies The product reduces the flakiness of the the skin with sufficient My skin condition has the flakiness of the dry skin moisture improved dry skin Vehicle Example 3 Vehicle Example 3 Vehicle Example 3 Agree completely 5.3 13.6 22.7 30.4 4.5 4.5 Agree 31.6 40.9 45.5 52.2 22.7 54.5 Neither agree nor 57.9 40.9 31.8 17.4 72.7 40.9 disagree Disagree if anything 5.3 4.5 0.0 0.0 0.0 0.0 Disagree completely 0.0 0.0 0.0 0.0 0.0 0.0

[0115] Results of the Subject Questionnaire.

[0116] Both skin flakiness and skin roughness were more greatly reduced after application of the formulation comprising the composition having a high oil content from Example 3 than after application of the vehicle formulation. These results were also confirmed by the subjects.

[0117] Distinctly higher agreement is seen here with the statements The formulation reduces the flakiness of the dry skin, The product supplies the skin with sufficient moisture and My skin condition has improved with the test formulation containing the product from Example 3 than in the case of the vehicle formulation.

Example 8: Application Data for Hair: Repair of UV-Damaged Hair

[0118] In the example which follows, the repair effect of a test conditioner containing the product from Example 2 is to be compared to the repair effect of ceramide NP.

[0119] Caucasian, undamaged hair was irradiated with UV for 24 h. For this purpose, a UV chamber was used (Dr. H?nle, Sol 2). The hair tresses were irradiated with a power of 910 W/m.sup.2. Subsequently, the hair was treated as follows:

[0120] 1. Washing with a test shampoo consisting of sodium lauryl ether sulfate and cocamidopropylbetaine.

[0121] 2. Application of the test conditioner; the contact time was 5 min. Subsequently, the hair tresses were rinsed under flowing tap water at a temperature of 37? C. for 1 min.

[0122] 3. Drying of the hair tresses with a hairdryer for 3 minutes.

[0123] These three treatment steps were conducted a total of 5?.

[0124] Test Formulations:

[0125] Shampoo

TABLE-US-00006 Ingredient Concentration Sodium Laureth Sulfate (28%) 32.10% Water 60.50% Cocomidopropyl Betaine (47.5%) 6.40% Phenoxyethanol, Ethylhexylglycerin 1.00%

TABLE-US-00007 Ingredient Vehicle Example 2 Ceramide NP Ceteareth-25 0.50% 0.50% 0.50% Cetearyl Alcohol 4.00% 4.00% 4.00% Cetrimonium Chloride 1.50% 1.50% 1.50% Ceramide mixture from 0.10% Example 2 (% based on sphingolipid) Ceramide NP 0.1% Water to 100% to 100% to 100% Phenoxyethanol, Ethylhexylglycerin 1.00% 1.00% 1.00% Lactic acid (10%) pH 4.0-4.5 pH 4.0-4.5 pH 4.0-4.5

[0126] Test Conditioner for the Repair of UV-Damaged Hair.

[0127] For quantification of the repair effect, a hair breakage measurement was conducted (Cyclic Tester, Diastron Limited, UK). In this measurement, individual hairs are extended at a constant force until they break. The number of extension cycles survived is recorded and serves to calculate the probability of survival. About 50 individual hairs were used for each measurement.

[0128] FIG. 1 shows that the probability of survival of UV-irradiated hair in the hair breakage test is much smaller than in the case of unirradiated hair (virgin caucasian). Treatment with the ceramide-containing test formulations increased the probability of survival again. It can be seen that the formulation containing the product from Example 2 leads to a higher probability of survival than ceramide NP tested for comparison.

Example 9: Application Data for Hair: Protection from Protein Degradation

[0129] Ethnic hair was chemically straightened. For this purpose, a standard formulation based on guanidinium carbonate and calcium hydroxide having a pH of about 12 was used. Subsequently, the hair was treated as follows: [0130] 1. Washing with a shampoo consisting of sodium lauryl ether sulfate and cocamidopropylbetaine. [0131] 2. Application of the test conditioner; the contact time was 5 min. Subsequently, the hair tresses were rinsed under flowing tap water at a temperature of 37? C. for 1 min. [0132] 3. Drying of the hair tresses with a hairdryer for 3 minutes.

[0133] These three treatment steps were conducted a total of 5?.

[0134] Test Formulation:

TABLE-US-00008 Ingredient Concentration Sodium Laureth Sulfate (28%) 32.10% Water 60.50% Cocomidopropyl Betaine (47.5%) 6.40% Phenoxyethanol, Ethylhexylglycerin 1.00%

[0135] Shampoo

TABLE-US-00009 Formulation with Ingredient Vehicle Example 2 ceramide II Ceteareth-25 0.50% 0.50% 0.50% Cetearyl Alcohol 4.00% 4.00% 4.00% Cetrimonium Chloride 1.50% 1.50% 1.50% Ceramide mixture from 0.10% Example 2 (% based on sphingolipid) Ceramide II (Sederma) 0.10% Water to 100% to 100% to 100% Phenoxyethanol, 1.00% 1.00% 1.00% Ethylhexylglycerin Lactic acid (10%) pH 4.0-4.5 pH 4.0-4.5 pH 4.0-4.5

[0136] Test Conditioner

[0137] For quantification of the repair effect, a hair breakage measurement was conducted (Cyclic Tester, Diastron Limited, UK). In this measurement, individual hairs are extended at a constant force until they break. The number of extension cycles survived is recorded and serves to calculate the probability of survival. About 50 individual hairs were used for each measurement.

[0138] FIG. 2 shows that the probability of survival of alkali-straightened hair in the hair breakage test is much smaller than in the case of unstraightened hair (virgin). Treatment with the ceramide-containing test formulations increased the probability of survival again. It has been found that, surprisingly, the inventive compositions (Example 2) lead to a higher probability of survival than ceramide II (pure sphinganine C18), which is customary for commercial hair care products.

Example 10: Application Data for Hair: Repair of the Hair after Acidic Straightening

[0139] Ethnic hair was acid-straightened and aftertreated with a conditioner that contained one of the claimed compositions with elevated triglyceride content, consisting of: 10% olive oil-based ceramide (Example 2). For comparison, the process was conducted with a corresponding amount of pure ceramide NG or pure olive oil.

[0140] The repair effect and the boosting of the CMC complex by means of Hair fatigue alpha were measured.

[0141] Ethnic hair was chemically straightened. For this purpose, an acidic standard formulation having a pH of about 1-2 was used in combination with the use of a straightening iron. Subsequently, the hair was treated as follows: [0142] 1. Washing with a shampoo consisting of sodium lauryl ether sulfate and cocamidopropylbetaine. [0143] 2. Application of the test conditioner; the contact time was 5 min. Subsequently, the hair tresses were rinsed under flowing tap water at a temperature of 37? C. for 1 min. [0144] 3. Drying of the hair tresses with a hairdryer for 3 minutes.

[0145] These three treatment steps were conducted a total of 5?.

[0146] The test conditioner contained either no active ingredient (vehicle, 1% olive oil, 0.1% ceramide NG) or the claimed compositions having elevated triglyceride content.

TABLE-US-00010 Ingredient Concentration Sodium Laureth Sulfate (28%) 32.10% Water 60.50% Cocomidopropyl Betaine (47.5%) 6.40% Phenoxyethanol, Ethylhexylglycerin 1.00%

[0147] Shampoo

TABLE-US-00011 Formulation Formulation with with Olive oil ceramide Ingredient Vehicle Example 3 formulation NG Ceteareth-25 0.50% 0.50% 0.50% 0.50% Cetearyl Alcohol 4.00% 4.00% 4.00% 4.00% Cetrimonium 1.50% 1.50% 1.50% 1.50% Chloride Ceramide mixture 1.00% from Example 3 (% based on sphingolipid) Olive oil 1.00% Ceramide NG 0.10% Water to 100% to 100% to 100% to 100% Phenoxyethanol, 1.00% 1.00% 1.00% 1.00% Ethylhexylglycerin Lactic acid (10%) pH 4.0-4.5 pH 4.0-4.5 pH 4.0-4.5 pH 4.0-4.5

[0148] Test Conditioner

[0149] For quantification of the repair effect, a hair breakage measurement was conducted (Cyclic Tester, Diastron Limited, UK). In this measurement, individual hairs are extended at a constant force until they break. The number of extension cycles survived is recorded and serves to calculate the probability of survival. About 50 individual hairs were used for each measurement.

[0150] Surprisinglyas shown in FIG. 3it was found that only the inventive composition could achieve an appropriate repair effect, whereas ceramide NG and olive oil were not able to achieve any repair effect.

Example 11: Application Data on Formulability: Crystal-Free Incorporation into Cosmetic Oils

[0151] The low solubility of sphingolipids and their high tendency to recrystallize have always made it difficult to stably incorporate them into cosmetic formulations.

[0152] It has been found that, surprisingly, the use of the inventive compositions having a high oil content and an elevated triglyceride content leads to distinct simplification of incorporability of the sphingolipids present into cosmetic oils than in the case of prior art sphingolipids.

[0153] For this purpose, the solubility temperatures of the sphingolipids were ascertained: A final concentration of 0.1% ceramide in cosmetic oil was chosen. The ceramide compositions were heated and stirred gradually in the cosmetic oils in a beaker on a hotplate. The solubility temperature was attained as soon as the solution was clear. Subsequently, the mixture was stirred at the solubility temperature ascertained for 1 hour. After it had cooled down to room temperature, the formulation was examined.

TABLE-US-00012 Example 3 Pure ceramide NP Solubility Appear- Solubility Appear- temper- ance temper- ance Emollient Polarity ature at RT ature at RT Octyl- polar 40? C. clear 70? C. crystals dodecanol TEGOSOFT polar 35? C. clear 65? C. crystals APM TEGOSOFT polar - 35? C. clear 95? C. crystals CT medium polar TEGOSOFT medium 45? C. clear 90? C. crystals M polar TEGOSOFT non- 50? C. clear 100? C. crystals OS polar Mineral Oil non- 60? C. cloudy 110? C. crystals polar

[0154] The data show a distinct improvement in incorporability of the inventive compositions having a high oil content into cosmetic oils compared to pure ceramides.