WO2004084646A2

WO2004084646A2 - Cosmetic compositions comprising phytosterol esters

Info

Publication number: WO2004084646A2
Application number: PCT/IL2004/000264
Authority: WO
Inventors: Dorit Plat; Elzaphan Hotam; Orly Farkash
Original assignee: Enzymotec Ltd.
Priority date: 2003-03-27
Filing date: 2004-03-23
Publication date: 2004-10-07
Also published as: WO2004084646A3; IL155132A0

Abstract

The present invention generally relates to novel solid oil-based compositions for cosmetic applications and their production. In particular, the present invention provides novel non-aqueous lipid-based compositions for cosmetic applications containing a mixture of diglyceride(s), phytosterol ester(s) (PSE), and optionally monoglyceride(s), dissolved or dispersed in an oil, for example olive oil. The present invention also provides a process for preparing the novel composition compositions, and to various cosmetic uses of these novel compositions, for example ant-aging, anti-chapping, anti-irritancy, skin moisturizing/softening, line/wrinkle reduction and/or sunscreen protection.

Description

COSMETIC COMPOSITIONS COMPRISING PHYTOSTEROL ESTERS

FIELD OF THE INVENTION

The present invention generally relates to novel oil-based compositions for cosmetic applications and their production. In particular, the present invention provides novel non-aqueous lipid-based compositions for cosmetic applications containing a mixture of diglyceride(s), phytosterol ester(s) (PSE), and optionally monoglyceride(s), dissolved or dispersed in oil. The present invention also relates to various cosmetic uses of these novel compositions, for example anti-aging, anti-chapping, anti-irritancy, line/wrinkle reduction, skin moisturizing and softening, collagen recovery and/or sunscreen protection.

BACKGROUND OF THE INVENTION Skin requires a certain amount of moisture to maintain elasticity, a good feel and an aesthetically pleasing appearance. Water content of the skin is lost through evaporation and through contact with harsh chemicals such as soaps and detergents. Skin exposure to severe external environment together with aging, causes the roughness of the skin, which is also referred to as a dry skin. Age has a pronounced effect on the quality, appearance and nature of the skin. With age, the complexion of the skin, namely the color and appearance of the skin, deteriorates. This results in the wrinkled, sagging, reddish and leathery skin of the elderly. Exposure to sunlight and the destructive effect of free radical activity accelerate the deterioration of the complexion of the skin. Dryness in skin can be overcome by application of moisturizing agents through the vehicle of a cosmetic composition. In the past, consideration had been given to the need for anti-aging cosmetic compositions, but since the mechanism of aging had not been clear the advance in the development of anti-aging compositions was slow. In recent years, there have been advanced studies on aging. Drying conditions, oxidation and the effects of sunlight (UV rays) were mentioned as some of the direct factors relating to skin aging. Still, with conventional cosmetic compositions, the effort has been only to maintain moisture. Clearly, with moisture alone, it is impossible to slow down aging of the skin.

Compositions based on natural oil products are known in the art. For example, WO01/32035 discloses olive oil-based products, based on especially higher grades of olive oils (such as virgin olive oils), comprising plant stanol esters and/or plant sterol esters.

US Patent No. 5,843,499 discloses oil extractable from corn fiber that contains ferulate esters and use of the oil in a composition for reducing cholesterol level in the blood. US Patent No. 6,326,050 discloses a composition consisting of oil or fat, a diacylglycerol, a free phytosterol and tocopherol, dissolved or dispersed in the oil or fat.

None of the above mentioned publications describe a combination of phytosterol ester and diacylglycerol in oil and/or fat, for cosmetic applications.

A process for selective alcoholysis or esterification (interesterification) of free sterols (including phytosterols) in a fat-based product is disclosed in WO01/75083. The process may comprise use of an immobilized, optionally surfactant-coated, lipase. The reaction takes place in the presence of triglycerides contained in the fat-based product, optionally following addition of at least one carboxylic fatty acid or ester derivative thereof. As defined in this publication, "selective alcoholysis" means that the process causes alcoholysis or esterification of the free sterol, without causing a significant change in the identity or positional distribution of the fatty acyl groups on the glycerol backbone of the triglycerides present in the oil- or fat-based product, e.g. butterfat. This publication describes modified butterfat compositions and low-cholesterol food preparations obtained by the described process, and further describes fat-based products enriched with phytosterol esters that are obtained in situ. WO 01/75083 does not provide details of the composition and constituents of the products obtained by said selective esterification reaction or their physical properties.

There is a widely recognized need for, and it would be highly advantageous to produce oil compositions for cosmetic applications based on natural oil ingredients, which are safe to use, easy to prepare, and that can be applied in various cosmetic and/or therapeutic applications such as anti-aging, anti-chapping, anti-irritancy, line/wrinkle reduction, skin moisturizing and softening, collagen recovery and sunscreen protection.

SUMMARY OF THE PRESENT INVENTION The present invention generally relates to novel oil-based compositions for cosmetic applications and to their production. In particular, the present invention provides novel non-aqueous lipid-based compositions for cosmetic applications containing a mixture of diglyceride(s), phytosterol ester(s) (PSE), and optionally monoglyceride(s), and/or free fatty acids dissolved or dispersed in oil. The present invention also relates to various uses of these novel compositions, for example as anti- aging, anti-chapping, anti-irritancy, skin softening and moisturizing compositions, for line/wrinkle reduction and/or for protection from sun irradiation.

According to one aspect, the present invention provides a cosmetic composition comprising a first component comprising at least one diglyceride derivative and at least one phytosterol ester, which are dissolved or dispersed in an oil, and a second component comprising cosmetically acceptable diluent or carrier.

According to one embodiment, the first component is present in the cosmetic composition in an amount from about 1% to about 99% by weight based on the total weight of the cosmetic composition, preferably in an amount from about 1% to about 80% by weight based on the total weight of the cosmetic composition, more preferably in an amount from about 1% to 50% by weight based on the total weight of the cosmetic composition.

According to another embodiment, the oil is one or more of the oils selected from the group consisting of olive oil, avocado oil, canola oil, soybean oil, sunflower oil, peanut oil, almond oil, safflower oil, cottonseed oil, coconut oil, rice bran oil, mustardseed oil, camelina oil, chia oil, flaxseed oil, perilla oil, linseed oil, rapeseed oil, cocoa butter, palm oil, kernel oil, castor oil, sunflower oil, fish oil, corn oil, etheric oils and mixtures thereof. Preferably the oil is present in an amount of 50-98% by weight based on the total weight of the first component of the cosmetic composition. According to yet another embodiment, the phytosterol ester is phytosterol derived from β-sitosterol, campesterol, stigmasterol, brassicasterol, D5-avenasterol, β- sitostanol, campestanol and stanol. Preferably the phytosterol ester is an ester of a C₆- C₂₄, preferably C₁₆-C₁₈ saturated or unsaturated fatty acid. Particularly preferred phytosteryl esters are esters of fatty acid selected from the group consisting of oleic, linoleic, linolenic, palmitic and stearic acid, for example phytosterylpalmitate or phytosterylstearate.

According to yet another embodiment the phytosterol ester is present in an amount from about 1% to about 99% by weight based on the total weight of the first component of the cosmetic composition, preferably in an amount of 10-40% by weight based on the total weight of the first component of the cosmetic composition, more preferably at an amount of about 30% based on the total weight of the first component of the cosmetic composition.

The diglyceride derivative is preferably a 1,3-diacylglycerol, a 1,2- diacylglycerol or a combination thereof. Particularly preferred is a 1,3-diacylglycerol.

According to one embodiment, the diglyceride derivative is present in an amount from about 1% to about 99% based on the total weight of the first component of the cosmetic composition, preferably the diglyceride derivative is present in an amount of 10-50% by weight based on the total weight of the first component of the cosmetic composition, even more preferably at an amount of about 20% based on the total weight of the first component of the cosmetic composition. The optional component monoglyceride derivative is preferably a monoglyceride derivative of a saturated or unsaturated fatty acid such C₁₄-C₂ preferably C₁₆-C₁₈ saturated or unsaturated fatty acid. Particularly preferred monoglycerides are derivatives fatty acids selected from the group consisting of oleic, linoleic, linolenic, palmitic and stearic acid. Particularly preferred are derivatives of stearic acid and palmitic acid.

The cosmetic compositions of the present invention may be administered in any form commonly used for cosmetic application.

Thus, according to one embodiment, the second component of the cosmetic composition is selected from, but not limited to, an ointment, a cream, a lotion, an emulsion, a powder, oil, a gel, an aerosol, a water-oil emulsion, or any combination thereof. According to one preferred embodiment, the second component of the cosmetic composition of the present invention is a gel.

The present invention also provides a method for preparing the innovative compositions of the present invention. The diglyceride derivatives and phytosterol esters may be obtained by any conventional enzymatic or non-enzymatic procedure. For example, the first component of the cosmetic compositions of the present invention may be prepared by conventional methods, by mixing (or blending) the desired amounts of diacylglycerol(s) and phytostanol ester(s) with the oil and/or fat, further mixing (or blending) the first component and the diluent or carrier. Preferably, the first component of the cosmetic compositions of the present invention is obtained by inter-esterification reaction between phytosterol(s) and triglyceride(s) present in the oil and/or fat. In accordance with this preferred embodiment, the first component of the cosmetic compositions of the present invention is produced in situ from the oil which is used as a base in the oil composition. An enzymatic reaction is performed in order to form the monglyceride, diglyceride and/or phytosterol esters in situ from the oil, by adding a lipase immobilized on a resin in a microaqueous medium, in accordance with the methods described in PCT International Patent Publications WOOO/56869, WO01/62906 and WO01/75083, assigned to the Assignees of the present invention, the contents of which are incorporated by reference herein.

Thus, the present invention provides a process for preparing the first component of the cosmetic compositions of the present invention, which comprises the following steps:

(a) mixing at least one phytosterol and an oil; (b) optionally adding at least one carboxylic fatty acid or ester derivative thereof;

(c) adding to the mixture of (a) or (b) an immobilized lipase complex;

(d) incubating the mixture of (c) for a period of time sufficient to give a product comprising a phytosterol ester and a diglyceride in oil, wherein said product comprises two distinct fractions, an oil fraction and a sediment fraction;

(e) optionally removing the immobilized lipase complex; and (f) separating the oil fraction from the sediment fraction.

According to one embodiment, the immobilized lipase complex is surfactant- coated.

A process for obtaining the combinations used by the present invention is described in detail in co-owned, co-pending Israel Patent Application No. 147942 and International Publication No. WO03/06444, fully incorporated herein by reference. These patent applications describe a composition of matter comprising diacyl glycerol(s), mainly 1,3-diacyl glycerols (DAG) and phytosterol esters, dispersed in an edible oil and/or edible fat. According to another embodiment, the oil fraction resulting from the enzymatic reaction comprises diglycerides, triglycerides, phytosterol esters and, optionally, monoglycerides. According to yet another embodiment, the sediment fraction which results from the enzymatic reaction comprises diglycerides, triglycerides, phytosterol esters and optionally monoglycerides. The lipase complex, which is preferably immobilized onto an insoluble matrix, possesses a high level of sterol-specific alcoholytic and/or esterification activity and minimal acidolytic and interesterification activities. In preferred embodiments, the lipase is selected from the group consisting of Aspergillus niger, Candida antarctica, Candida cylindracea, Candida rugosa, Mucor miehei, Pseudomonas cepacia, Rhizopus niveus, Rhizopus arrhizus, Hog pancreas, Aspergillus oryzae, Candida lipolytica, Mucor javanicus, Pencillium roqueforti, Pseudomonas fluorescens, Rhizomucor miehei, wheat germ, Chromobact. viscosum, lipoprotein from Pseudomonas species, lipoprotein from Pseudomonas species B, and Thermomyces lanuginosus.

Furthermore, the present invention encompasses cosmetic and therapeutic uses of the novel compositions provided herein. For example, the oil-based compositions are useful as anti-aging compositions, anti-irritant compositions, as skin moisturizing and/or softening in the reduction or prevention of skin lines and/or wrinkles and in the recovery of collagen. Furthermore, the novel compositions are useful as sunscreens.

According to one embodiment, the present invention discloses the use in cosmetic products of a composition comprising an oil, at least one diglyceride derivative and at least one phytosterol ester. The cosmetic products are exemplified by, but not restricted to a lipstick, a daily cream, a daily cream for dry skin, and eye cream, a neck cream, a night cream, a nourishing musk, a hair nourishing musk, an hand cream, a body cream, a foot cream, an all purpose cream, a bath oil, a baby oil, a baby cream and a baby paste. According to one embodiment, the composition of the present invention is applied to the skin, the nails, the hair, or the eyelashes.

According to another embodiment, the composition is applied as a care or makeup composition.

According to yet another embodiment, the composition is applied for delaying or lowering the effects of the aging on the skin by any one of the methods selected from a group consisting of: a method for counteracting the harmful effects of free radicals on the skin, a method for treating sensitive skins and making up sensitive skins.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be understood and appreciated more fully from the following detailed description taken in conjunction with the appended drawings which depict:

FIG. 1 is a graph showing the percent increase in skin moisturization following skin treatment with the compositions of the present invention vs. control (no treatment).

FIG. 2 is a graph showing the percent increase in skin softening following skin treatment with the compositions of the present invention.

FIG. 3 is a graph showing efficacy of the compositions of the present invention (16% PS-E top, 1; 35% PS-E bottom, 2; solid mixture, 3) in skin moisturization and softening compared with the efficacy of natural formulations. FIG. 4 is a graph showing collagen recovery in vitro by the compositions of the present invention (FGS7, black; FGS8S, white).

FIG. 5 is a graph showing the anti-inflammatory activity of test material FGS7 (A) and FGS8S (B) at different concentrations (10%, full circle; 20%, cross; 30%, empty circle) vs. undamaged skin (diamond) and untreated skin (square).

FIG. 6 is a graph showing the reduction in facial lines and wrinkles as a result of treatment with the compositions of the present invention (FGS7, black; FGS8S, white).

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention generally relates to novel oil-based compositions for cosmetic applications and to their production. In particular, the present invention provides novel non-aqueous lipid-based compositions for cosmetic applications containing a mixture of diglyceride(s), phytosterol ester(s) (PSE), and optionally monoglyceride(s), and/or free fatty acids dissolved or dispersed in an oil. The present invention also relates to various uses of these novel compositions, for example as anti- aging, anti-chapping, anti-irritancy, skin softening and moisturizing compositions, line/wrinkle reduction, collagen recovery and/or sunscreen protection. The term "cosmetic application" as used herein refers to use of the compositions of the present application for any dermatological condition for the purpose of treatment, prophylaxis and/or aesthetics.

Thus, in one embodiment, the present invention provides a cosmetic composition comprising a first component comprising at least one diglyceride derivative and at least one phytosterol ester, which are dissolved or dispersed in an oil, and a second composition comprising a cosmetically acceptable diluent or carrier.

I. Composition content A. Oil

The oils used in the compositions of the present invention are preferably oils of natural origin. The term "natural origin" as used herein refers to all plant, fish and vegetable oils. However, the use of synthetic oils is also within the scope of the present invention, so long as the synthetic oils are suitable for use in cosmetic applications. The natural oil is preferably one or more of the oils from the group of olive oil, avocado oil, canola oil, soybean oil, sunflower oil, peanut oil, almond oil, safflower oil, cottonseed oil, coconut oil, rice bran oil, mustardseed oil camelina oil, chia oil, flaxseed oil, perilla oil, linseed oil, rapeseed oil, cocoa butter, palm oil, kernel oil, castor oil, sunflower oil, fish oil, corn oil and etheric oils. Particularly preferred is olive oil. The natural oil is preferably one or more of the oils from the group of olive oil, avocado oil, canola oil, soybean oil, sunflower oil, peanut oil, almond oil, safflower oil, cottonseed oil, coconut oil, rice bran oil, mustardseed oil camelina oil, chia oil, flaxseed oil, perilla oil, linseed oil, rapeseed oil, cocoa butter, palm oil, kernel oil, castor oil, sunflower oil, fish oil, corn oil and etheric oils. Particularly preferred is olive oil. Oils of synthetic origin which may be used in the composition of the present invention include hydrocarbon oils and liquid paraffins such as ozokerite, squalane, pristane, paraffin, ceresine, squalene, vaseline, microcrystalline wax, and liquid isoparaffm; and silicone oils such as dimethylpolysiloxane. Also suitable are synthetic esters such as synthetic ester oils, isopropyl myristate, cetyl octanate, octyldodecyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, decyl oleate, hexyldecyl dimethyloctanate, cetyl lactate, myristyl lactate, lanolin acetate, isocetyl stearate, isocetyl isostearate, cholesteryl 12-hydroxystearate, ethylene glycol di- 2-ethylhexylate, dipentaerythritol fatty acid ester, N-alkylglycol monoisostearate, neopentylglycol dicaproate, diisostearyl malate, glycerin di-2-heptyl undecanoate, trimethylopropane tri-2-ethylhexylate, trimethylopropane trusostearate, pentaerythritol tetra-2-ethylhexylate, glycerin tri-2-ethylhexylate, trimethylopropane trusostearate, cetyl-2-ethylhexanoate, 2-ethylhexyl palmitate, glycerin trimyristate, glyceride tri-2- heptylundecanoate, castor oil fatty acid methyl ester, oil oleate, cetostearyl alcohol, acetoglyceride, 2-heptylundecyl palmitate, diisobutyl adipate, N-lauroyl-L-glutamate-2- octyl dodecyl ester, di-2-heptylundecyl adipate, ethyl laurate, di-2-ethylhexyl sebatate, 2-hexyldecyl myristate, 2-hexyldecyl palmitate, 2-hexyldecyl adipate, diisopropyl sebatate, 2-ethylhexyl succinate, ethyl acetate, butyl acetate, amyl acetate, triethyl citrate, etc.

Preferably the oil is present in an amount of 50-98% by weight based on the total weight of the first component of the cosmetic composition. Certain non-limiting embodiments depicting the amount of the oil are: 80-98%, 90-98% 90-95%, 95-98%, 92-97%, 90%, 92%, 95%, 97% and the like.

B. Phytosterols The term "phytosterols" covers plant sterols and plant stanols. Plant sterols are naturally occurring substances present in the diet as minor components of vegetable oils. Plant sterols have a role in plants similar to that of cholesterol in mammals, e.g. forming cell membrane structures. Phytosterols are chemically similar to cholesterol. Cholesterol, however, only occurs in animals and is not found in plants. The cylclopentanoper- hydrophenanthrene ring structure of the sterol molecule is common to all sterols; the differences are primarily in the structure of the side chains. For example, β-sitosterol differs from cholesterol by the presence of an ethyl group at the 24^th carbon position of the side chain.

In the case of campesterol, this position is occupied by a methyl group. Chemically, the phytosterols are classified as 4-desmethylsterols of the cholestane series.

Phytosterols and phytosterol esters are currently being used in three main industrial categories, namely pharmaceuticals, cosmetics and alimentary products. Since phytosterols are not fat-soluble, esterification processes were developed for producing fat-soluble phytosterol esters. The phytosterol esters suitable for use in the present invention are preferably phytosterols derived from beta- sitosterol, campesterol, stigmasterol, brassicasterol and stanol. Preferably the phytosterol ester is a phytosterol ester, such as an ester of a C₆- C₂₄, preferably C₁₆-C₁₈ saturated or unsaturated fatty acid. Particularly preferred phytosteryl esters are esters of fatty acid selected from the group consisting of oleic, linoleic, linolenic, palmitic and stearic acid, for example phytosterylpalmitate or phytosterylstearate. The phytosterol ester may be used in any suitable amount, ranging from 1-99% by weight based on the total weight of the first component of the cosmetic composition. Preferably the phytosterol ester is present in an amount of 10-40% by weight based on the total weight of the first component of the cosmetic composition, even more preferably at an amount of about 30% based on the total weight of the first component of the cosmetic composition.

C. Diglycerides (DAG) Diglycerides contain two fatty acids per molecule of fat. The major part of the two fatty acids are located mainly on each end of the fat molecule (generally referred as 1,3 positions), or on the end and the middle end of the fat molecule (generally referred to as Im positions).

The diglyceride derivative is preferably a l,3-diacylglycerol(s), a 1,2- diacylglycerol(s) or a combination thereof. Particularly preferred is a 1,3- diacylglycerol(s). The structure of the diacyl glycerol(s) depends on the particular oil and/or fat used for dissolving or dispersing the phytosterol ester(s). For example, when olive oil is used, the diacyl glycerol mainly consists of 1,3-dioleyl glycerol. Generally speaking, fatty acid moieties of the DAG include oleic, palmitic, palmitoleic, stearic, linoleic, linolenic, and eicosanoic fatty acids (DAGs being 1,2 dioleyl, 1,3 dioleyl, 1,2- oleyl palmitoyl and 1,3 oleyl palmitoyl glycerols).

The amount of diglyceride contained in the oil and or fat may range from lwt% to about 99wt%. Preferably the diglyceride derivative is present in an amount of 10-

50% by weight based on the total weight of the first component of the cosmetic composition, even more preferably at an amount of about 20% based on the total weight of the first component of the cosmetic composition.

The molar ratio of phytosterol and/or phytosterol ester(s) to diglycerides in the oil is from about 5:1 to about 1:5, for example 5:1, 2:1, 1:1, 1:2, or 1:5.

D. Monoglycerides

The optional component monoglyceride derivative is preferably a monoglyceride derivative of a saturated or unsaturated fatty acid such C₁₄-C₂₄, preferably C₁₆-C₁₈ saturated or unsaturated fatty acid. Particularly preferred monoglycerides are derivatives fatty acids selected from the group consisting of oleic, linoleic, linolenic, palmitic and stearic acid. Particularly preferred are derivatives of stearic acid and palmitic acid.

E. DermatogieaUy acceptable cosmetic carriers

The topical skin composition of the present invention may appropriately contain, in addition to the aforementioned ingredients, a variety, of ingredients or carriers which are usually employed in cosmetics. Examples of such ingredients or carriers include humectants, powders, gelation agents, thickeners, surfactants, emulsifiers, anti- inflammatory agents, antioxidants, pH regulating agents, chelating agents, preservatives, dyes, perfumes, UV absorbing-agents, UV protecting agents, existing skin-aging preventive or retarding agents such as collagen, and existing hair-growth suppressing agents.

For example, the compositions may comprise surfactants. Suitable surfactants are anionic surfactants, cationic surfactants, zwitterionic surfactants and nonionic (lipophilic and hydrophillic surfactants). Preferably the surfactant is present in an amount of 0.01-5% by weight based on the total weight of the composition. Certain non- limiting embodiments depicting the amount of the surfactant are 0.01-1%, 0.01-0.1%, 0.05-0.1%, 1-2%, 1-2.5%, 0.5-2%, 0.5-5% and the like. Suitable anionic surfactants are, for example, soap ingredients, sodium laurate, sodium palmitate, and other fatty acid soaps, sodium laurosulfate, potassium laurosulfate, and other higher alkyl sulfate ester salts, POE laurosulfate triethanol amine, sodium POE laurosulfate, and other alkyl ester sulfate ester salts, sodium lauroylsarcosine and other N-acylsarcosine acids, sodium N-myristyl-N-methyltaurine, sodium coconut oil fatty acid methyl tauride, sodium laurylmethyl tauride, and other higher fatty acid amide sulfonates, sodium POE oleyl ether phosphate, POE stearyl ether phosphate, and other phosphate ester salts, sodium di-2-ethylhexylsulfosuccinate, sodium monolauroylmonoethanol amide polyoxyethylene sulfosuccinate, sodium laurylpolypropylene glycol sulfosuccinate, and other sulfosuccinates, linear sodium dedecylbenzensulfonate, linear dodecylbenzensulfonate triethanol amine, linear dodecyl benzensulfate, and other alkylbenzensulfonates, sodium N-lauroylglutamate, disodium N-stearoylglutamate, monosodium N-myristoyl-L-glutamate, and other N- acylglutamates, sodium hardened castor oil fatty acid glycine sulfate and other higher fatty acid ester sulfate ester salts, Turkey red oil and other sulfated oils, POE alkyl ether carboxylic acid, POE alkylaryl ether carboxylate, -olefinsulfates, higher fatty acid ester sulfonates, secondary alcohol sulfate ester salts, higher fatty acid alkylolamide sulfate ester salts, sodium lauroyl monoethanolamide succinate, N-palmitoyl asparaginate ditriethanol amine, sodium caseine, etc. Suitable cationic surfactants are, for example, stearyl trimethyl ammonium chloride, lauryl trimethyl ammonium chloride, and other alkyl trimethyl ammonium salts, distearyldimethyl ammonium chloride, dialkyldimethyl ammonium chloride salts, poly(N,N'-dimethyl-3,5-methylenepiperidinium)chloride, cetylpyridinium chloride and other alkyl pyridinium salts, alkyl quaternary ammonium salts, alkyl dimefhylbenzyl ammonium salts, alkyl isoquinolinium salts, dialkyl morphonium salts, POE alkyl amines, alkyl amine salts, polyamine fatty acid derivatives, amyl alcohol fatty acid derivatives, benzalkonium chloride, benzethonium chloride, etc.

Suitable Zwitterionic surfactants are for example, sodium 2-undecyl-N,N,N-

(hydroxyethylcarboxymethyl)-2-imidazoline, 2-cocoyl-2-imidazoliniumhydroxide- 1 - carboxyethyloxy-2-sodium salt, and other imidazoline family bipolar surfactants, 2- heptadecyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, lauryldimethyl- aminoacetate betaine, alkyl betaine, amide betaine, sulfo betaine, and other betaine family surfactants etc.

Suitable lipophilic nonionic surfactants are, for example, sorbitan monooleate, sorbitan monoisostearate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate, diglycerol sorbitan penta-2- ethylhexylate, diglycerol sorbitan tetra-2-ethylhexylate, and other sorbitan fatty acid esters, glycerol cotton seed oil fatty acid mono ester, glycerin monoerucate, glycerin sesquioleate, glycerin monostearate, glycerin alpha-oleate pyroglutamate, monostearate glycerin malic acid and other glycerin polyglycerin fatty acids, propylene glycol monostearate and other propylene glycol fatty acid esters, hardened castor oil derivatives, glycerin alkyl ethers, polyoxyethylene methylpolysiloxane copolymers, etc.

Suitable hydrophilic nonionic surfactants are, for example, POE sorbitan monooleate, POE-sorbitan monostearate, POE-sorbitan monooleate, POE-sorbitan tetraoleate, and other POE sorbitan fatty acid esters, POE-sorbite monolaurate, POE- sorbite monooleate, POE-sorbite pentaoleate, POE-sorbite monostearate, and other POE sorbite fatty acid esters, POE-glycerin monostearate, POE-glycerin monoisostearate, POE-glycerin trusostearate, and other POE glycerin fatty acid esters, POE monooleate, POE distearate, POE monodioleate, distearate ethylene glycol, and other POE fatty acid esters, POE lauryl ethers, POE oleyl ethers, POE stearyl ethers, POE phenyl ethers, POE2-octyldodecyl ethers, POE cholestanol ethers, and other POE alkyl ethers, POE octyl phenyl ethers, POE nonyl phenyl ethers, POE dinonyl phenyl ethers, and other POE alkyl phenyl ethers, pluronic and other pluaronics, POE-POP cetyl ethers, POE.POP-2-decyltetradecyl ethers, POE-POP monobutyl ethers, POE-POP hydrated lanolin, POE.POP glycerin ethers, and other POE-POP alkyl ethers, tetronic and other tetra-POE-tetra-POP ethylene diamine condensation products, POE castor oil, POE hardened castor oil, POE hardened castor oil monoisostearate, POE hardened castor oil trusostearate, POE hardened castor oil monopyroglutamate monoisostearate diester, POE hardened castor oil maleic acid and other POE castor oil hardened castor oil derivatives, POE sorbite beeswax and other POE beeswax lanolin derivatives, coconut oil fatty acid diethanol amide, laurate monoethanol amide, fatty acid isopropanol amide, and other alkanol amides, POE propylene glycol fatty acid esters, POE alkylamines, POE fatty acid amides, sucrose fatty acid esters, POE nonylphenyl formaldehyde condensation products, alkylethoxydimethylamineoxide, trioleylphosphoric acid etc.

Silicones may also be used in the present invention. Suitable silicones are, for example, dimethyl polysiloxane, methylphenyl polysiloxane, methylhydrogen polysiloxane, and other chain like polysiloxanes, decamethyl polysiloxane, dodecamethyl polysiloxane, tetramethyltetrahydrogen polysiloxane, and other cyclic polysiloxanes, silicone resins forming 3D net structures, silicone rubber, etc.

As preservatives, methylparaben, ethylparaben, butylparaben, etc. may be used.

Optionally, the compositions described herein may contain a known sunscreen. Preferably, the sunscreens contain at least one UVA filter and at least one UVB filter. Oil-soluble UVB products include 3-benzylidenecamphor derivatives, 4-aminobenzoic acid derivatives, esters of cinnamic acid, derivatives of benzophenone, esters of benzylidenemalonic acid. Water-soluble UVA filters include salts of 2- phenylbenzimidazole-5-sulphonic acid, sulphonic acid derivatives of benzophenones and sulphonic acid derivatives of 3-benzylidenecamphor. Effective amounts of sunscreens will be known to those of skill in the art. Preferably, sunscreens will comprise between about 0.1% and 30% by weight of the total preparation.

Suitable thickeners are, for example, carragheenin, karaya gum, tragacanth gum, carob gum, quince seed (Marumero), caseine, dextrin, gelatin, sodium pectinate, sodium alginate, methyl cellulose, ethyl cellulose, CMC, hydroxyethyl cellulose, hydroxypropyl cellulose, PVA, PVM, PVP, sodium polyacrylate, carboxyvinyl polymer, loqust bean gum, guar gum, tamarind gum, dialkyldimethyl ammonium sulfate cellulose, xanthane gum, aluminum magnesium silicate, bentonite, hectonite, etc.

Suitable powder components are, for example, talc, kaolin, mica, sericite, dolomite, phlogopite, synthetic mica, lepidolite, biotite, lithia mica, vermiculite, magnesium carbonate, calcium carbonate, aluminum silicate, barium silicate, calcium silicate, magnesium silicate, strontium silicate, metal salts of tungstenic acid, magnesium, silica, zeolite, barium sulfate, sintered calcium sulfate (sintered gypsum), calcium phosphate, fluorapatite, hydroxyapatite, ceramic powder, metal soap (zinc myristate, calcium palmitate, ammonium stearate), boronitride, and other inorganic powders, polyamide resin powder (nylon powder), polyethylene powder, methyl polymefhacrylate powder, polytetrafluoroethylene powder, cellulose powder, and other organic powders.

Pigments may also be used in the compositions of the present invention, such as titanium dioxide, zinc oxide, and other inorganic white pigments, iron oxide (bengara), iron titanate, and other inorganic red pigments, gamma-iron oxide and other inorganic brown pigments, yellow iron oxide, yellow earth, and other inorganic yellow pigments, black iron oxide, carbon black, lower titanium oxide, and other inorganic black pigments, mango violet, cobalt violet, and other inorganic violet pigments, chromium oxide, chromium hydroxide, cobalt titanate, and other inorganic green pigments, prussian blue, ultramarine, and other inorganic blue pigments, titanium oxide coated talc, colored titanium oxide coated mica, bismuth oxichloride, fish scales, and other pearl pigments, aluminum powder, copper powder, and other metal powder pigments, Red No. 201, Red No. 202, Red No. 204, Red No. 205, Red No. 220, Red No. 226, Red No. 228, Red No. 405, Orange No. 203, Orange No. 204, Yellow No. 204, Yellow No. 401, Blue No. 404, and other organic pigments, Red No. 3, Red No. 104, Red No. 106, Red No. 227, Red No. 230, Red No. 401, Red No. 505, Orange No. 205, Yellow No. 4, Yellow No. 5, Yellow No. 202, Yellow No. 203, Green No. 3, Blue No. 1, and other zirconium, barium, or aluminum lakes and other organic pigments, chlorophyll, beta- carotene, and other natural colors, perfumes, water, alcohol, titanium yellow, Safflower Yellow, Safflower Red, and other coloring agents, etc. may also be suitably blended into the cosmetic composition of the present invention if necessary.

F. Other Additives

Other optional ingredients which can be present in the cosmetic compositions of the present invention include the flavor oils which were described above, fat soluble vitamins such as vitamin A and E, esters of vitamin A (e.g., acetate, propionate or palmitate) and of vitamin E (e.g., acetate or sorbate), sunscreens such as octyl methoxycinnamate and butyl methoxydibenzoylmethane, sunblocks such as titanium dioxide and zinc oxide, germicides such as triclosan, anti-inflammatory agents such as hydrocortisone, lipid materials such as ceramides and liposomes and skin care actives.

The cosmetic compositions may comprise ingredients conventionally employed in cosmetic compositions such as mascara, foundation or lipcare products. The compositions of the invention may also contain various pharmacologically active substances usable in the treatment of diseases of the integuments and of the nails.

Skin care active ingredients can be added to the cosmetic compositions of the present invention. These include, but are not limited to vitamin C and its derivatives (e.g., ascorbyl palmitate, ascorbyl phosphate and its salts such as magnesium or sodium), vitamin D, pathenol, retinoic acid, zinc oxide, beta-glycyerhetic acid; chamomile oil; ginko biloba extract; pyroglutamic acid, salts or esters; sodium hyaluronate; 2-hydroxyoctanoic acid; sulfur; salicylic acid; carboxmethyl cysteine, and mixtures thereof. These additives, both fat soluble and water soluble, will normally be present in amounts of less than about 10% by weight, and generally in the range of about 0.01% to about 5%, preferably from about 0.01% to about 3%, most preferably from about 0.1% to about 1%, by weight.

Flavor oils such as peppermint oil, orange oil, citrus oil, wintergreen oil can be used along with an alcohol or glycerin. Flavor oils are usually mixed in a solvent such as ethanol to dilute the flavor. The flavor oils useful herein can be derived from natural sources or be synthetically prepared. Generally flavor oils are mixtures of ketones, alcohols, fatty acids, esters and terpenes. The term "flavor oil" is generally recognized in the art to be a liquid which is derived from botanical sources, i.e. leaves, bark, or skin of fruits or vegetables, and which are usually insoluble in water. The level of flavor oil used can range from 0% to about 5%, preferable from 0% to about 1%.

Moisturizers may also be included into the present compositions. Preferred moisturizers include pyrrolidone carboxylic acid, sodium lactate or lactic acid, urea, guanidine, glyceric acid and its salts (e.g., calcium salt), petrolatum, collagen, .alpha. - hydroxy propylglyceryl ether, .alpha. -hydroxy acids (e.g., ethylglycolic acid, leucic acid, mandelic acid, glycolic acid), glucosamines and elastin fibers, D-panthenol, allantoin and hyaluronic acid and chondroitin sulfate. Examples of suitable moisturizers can be found in Cosmetic Bench Reference, p. 1.30-1.32 (1996), herein incorporated by reference. Another optional component is silica. Silica is generally preferred for use at levels of from about 1% and about 5%. Hypoallergenic compositions can be made from the liquid crystal, wax, oil and colors herein. These compositions should not contain fragrances, flavor oils, lanolin, sunscreens, particularly PABA, or other sensitizers or potential sensitizers and irritants.

The compositions of the present invention can also be made into long lasting or non-transferable cosmetic compositions. Detailed discussions of such lipsticks are found in Japanese Patent Publication Hei No. 6-199630 and European Patent Application 748622, both of which are herein incorporated by reference in their entirety.

Additional optional materials that can be incorporated in the compositions of the present invention can be found in PCT application WO 97/39733, to Oblong et al.

II. Methods of Preparation

The present invention also provides a method for preparing the innovative compositions of the present invention. The diglyceride derivatives and phytosterol esters may be obtained by any conventional enzymatic or non-enzymatic procedure. For example, the first component of the cosmetic compositions of the present invention may be prepared by conventional methods, by mixing (or blending) the desired amounts of diacylglycerol(s) and phytostanol ester(s) with the oil and/or fat, further mixing (or blending) the first component and the diluent or carrier.

Preferably, the first component of the cosmetic compositions of the present invention is obtained by inter-esterification reaction between phytosterol(s) and triglyceride(s) present in the oil and/or fat. In accordance with this preferred embodiment, the first component of the cosmetic compositions of the present invention is produced in situ from the oil which is used as a base in the oil composition. An enzymatic reaction is performed in order to form the monglyceride, diglyceride and/or phytosterol esters in situ from the oil, by adding a lipase immobilized on a resin in a microaqueous medium, in accordance with the methods described in PCT International Patent Publications WO00/56869, WO01/62906 and WO01/75083, assigned to the Assignees of the present invention, the contents of which are incorporated by reference herein. Thus, the present invention provides a process for preparing the first component of the cosmetic compositions of the present invention, which comprises the following steps:

(c) adding to the mixture of (a) or (b) an immobilized lipase complex which may optionally be surfactant-coated;

(e) optionally removing the immobilized lipase complex, and

(f) separating the oil fraction from the sediment fraction.

A process for obtaining the combinations used by the present invention as the first component of the cosmetic composition is described in detail in co-owned, co- pending Israel Patent Application No. 147942, and International PCT Application No. PCT/IL03/0081, fully incorporated herein by reference. These patents describe a composition of matter comprising diacyl glycerol(s), mainly 1,3 -diacyl glycerols (DAG) and phytosterol esters, dispersed in edible oil and/or edible fat.

In one embodiment, the oil fraction which results from the enzymatic reaction comprises diglycerides, triglycerides, phytosterol esters and optionally monoglycerides. In another embodiment, the sediment fraction which results from the enzymatic reaction comprises diglycerides, triglycerides, phytosterol esters and optionally monoglycerides. The lipase complex, which is preferably immobilized onto an insoluble matrix, possesses a high level of sterol-specific alcoholytic and/or esterification activity and minimal acidolytic and interesterification activities. In preferred embodiments, the lipase is selected from the group consisting of Aspergillus niger, Candida antarctica, Candida cylindracea, Candida rugosa, Mucor miehei, Pseudomonas cepacia, Rhizopus niveus, Rhizopus arrhizus, Hog pancreas, Aspergillus oryzae, Candida lipolytica, Mucor javanicus, Pencillium roqueforti, Pseudomonas fluorescens, Rhizomucor miehei, wheat germ, Chromobact. viscosum, lipoprotein from Pseudomonas species, lipoprotein from Pseudomonas species B, and Thermomyces lanuginosus.

The term "interesterification" as used herein, refers to the reaction of a first ester with a second ester leading to a mix up between the acyl and the alcohol moieties.

III. Cosmetic and therapeutic uses

The present invention encompasses various cosmetic and/or therapeutic uses of the novel compositions provided herein. For example, the oil-based compositions are useful as anti-aging compositions, anti-irritant compositions, in the reduction or prevention of skin lines and/or wrinkles and in the recovery of collagen. Furthermore, the novel compositions are useful as sunscreens.

Thus, in one embodiment, the cosmetic composition may be used as anti-aging compositions to prevent and or reduce the effects of aging on the skin of a subject. In another embodiment, the cosmetic compositions have applications as anti-irritant dermatological compositions. In a further embodiment, the cosmetic compositions can be used in moisturizing lotions/creams as skin moisturizers and/or softeners. In another embodiment, the cosmetic compositions can be used in reducing, preventing or treating skin lines and/or wrinkles which result from aging, from long exposure to sunlight, from exposure to chemicals and the like. In yet further embodiment, the cosmetic compositions may be used for the recovery of collagen.

In yet another embodiment, the cosmetic compositions may be used as sunscreen protectors.

In yet further embodiment, the present invention discloses the use in cosmetic products of a composition comprising an oil, at least one diglyceride derivative and at least one phytosterol ester. The cosmetic products are exemplified by, but not restricted to a lipstick, a daily cream, a daily cream for dry skin, and eye cream, a neck cream, a night cream, a nourishing musk, a hair nourishing musk, an hand cream, a body cream, a foot cream, an all purpose cream, a bath oil, a baby oil, a baby cream and a baby paste. The cosmetic compositions of the present invention can be applied in a wide range of application forms used for the external skin. It may take forms such as creams, lotions, emulsions, powders, oils, gels, ointments, aerosols, water-oil two-phase systems, water- oil-powder three-phase systems, aqueous solutions, dissolvable systems, etc. That is, the cosmetic compositions may be applied in the above various forms for facial cleansers, toilet water, emulsions, creams, gels, essences (beauty lotions), packs, masks, and other types of cosmetics. Further, in a makeup cosmetic composition, they may be used for a wide range of types of cosmetics composition such as foundations while in toiletry products they may be used for body soap, facial soap, etc.

The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

Example 1: Efficacy of the Oil-Based Compositiong as Skin Moisturization And

Softening

Objectives The objective of these studies was to determine the efficacy of the oil-based compositions according to the present invention in improving the appearance and another qualitative aspects of skin, during an eight hours usage period.

Materials and Methods The compositions tested for their ability to moisturize and soften skin are described in table 1 below.

Table 1: The Oil-Based Component Of The Cosmetic Compositions

Application of test compositions to subject was as follows:

1. No Treatment.

2. Sample 2: Carbopol Vehicle only 3. Sample 3 : SEG at 20% in Carbopol Gel

4. Sample 4: S IL at 20% in Carbopol Gel

5. Sample 5: S7S at 20% in Carbopol Gel

6. Sample 6: OOPE Used neat

Five (5) female subjects (age 35+), with dry skin and exhibiting moderate to severe dry skin in the lower leg area participated in this 8 -Hours Product Efficacy In- Use- Study. Subjects applied each sample composition to each of the defined sites located in the calf region of their lower legs. Skin was prewetted with water prior to application. All test samples were randomized among the six test sites on both the left and right legs of subjects. At 0, 4 and 8 hours post-application skin impedance and skin softening measurement were taken.

Protocol

A. Week -1 to 0: Subject dry out To ensure that all subjects are at a baseline value and to factor out differences in effects of current skin care regimens, a one-week dry out period preceded the study. On day -7 subjects received a bar of soap (Neutrogena) to wash their entire calf/lower legs as often as they choose. No moisturizer, sunscreen, nor body products on their calf/lower legs was allowed during this phase of the study, as well as excessive UV exposure, and tanning salons.

B. Hour 0 to Hour 8 - Use of Test Products On Day 0 baseline measurements of the six defined sites were obtained for all subjects. The skin of the defined sites was then re- wetted with water; the excess water removed by padding with tissue paper and test products applied to the test sites in normal use fashion. Skin moisturization and softening effects were measured again at 4 hours and 8 hours after the first application of the test compositions: a. Skin Moisturization via Impedance Measurement b. Skin Softening via Gas Bearing Electrodynamometer

Techniques and instrumental methods:

1. Skin Hydration -Impedance Measurements The impedance meter is a well documented and widely used device to measure changes in skin moisture content. Each test day the unit was calibrated according to instrument instructions. A notation of temperature and relative humidity was taken.

Skin moisturization was evaluated as follows: On each test site, two separate readings were taken, recorded and averaged.

2. Skin Softening via Gas Bearing Electro-dynamometer

Research in many different laboratories has proven that the viscoelastic properties of skin correlate well with skin moisturization. In this study, skin suppleness/elasticity were assessed by a Gas Bearing Electro-dynamometer. Measurements were taken from the 3 different predetermined sites on the subjects' calf/lower legs area by attaching the probe to the skin surface with a double-stick tape. A force of approximately 3.5 gm was applied parallel to the skin surface and the skin displacement was accurately measured. Skin suppleness was then calculated and expressed as DSR (Dynamic Spring Rate in gm/mm). Results

1. Impedance Measurement of Skin Moisture and Hydration

Skin moisture content was assessed via impedance measurements on the defined sites at the calf/lower legs area of subjects. Skin was pre wetted with water prior to application of test products. Measurements were taken in duplicate and were averaged. Results are presented in Table 2 and in Figure 1 as percent increase in skin moisture. During the treatment period, all four test compositions showed skin moisture efficacy to a varying degree at 4 hours post-treatment. At eight hours post-treatment, the effects of skin moisture dimimshed considerably albeit the order of activity for the four compositions remained the same. Comparatively, the order of efficacy was found to be Sample 5 (S7S) > Sample 6 (OOPE) ~ Sample 4 (S1L) > Sample 3 (SEG).

Table 2: Skin Moisturizing by the Test Compositions

4 HOURS 8 HOURS

1. No Treatment 2.2% -0.6%

2. Carbopol Vehicle 7.2% 1.4%

3. SEG 28.1% 9.2%

4. S1L 34.2% 16.3%

5. ^ o 39.4% 23.6%

6. OOPE 34.7% 18.2%

2. Skin Suppleness via Gas Bearing Electrodynamometer Skin softness/suppleness was measured using the Gas Bearing Electrodynamometer. Measurements were obtained on the defined sites on the subject's lower legs area and are expressed as gm mm (Dynamic Spring Rate). Results are presented in Table 3 and in Figure 2 as percent increase in skin softening. As the results indicate, single treatment with Sample 3 (SEG), Sample 4 (S1L), Sample 5 (S7S) and Sample 6 (OOPE) resulted in an increase in skin softening to a varying degree at four hours post- treatment. At eight hours post-treatment, the skin softening effects diminished considerably. After 4 h, Sample 5 (S7S) ~ Sample 4 (S1L) > Sample 6 (OOPE) > Sample 3 (SEG). These results compliment those found in the skin moisture testing.

Table 3; Skin Softening by the Test Compositions

4 HOURS 8 HOURS

1. No Treatment 4.0% -0.7%

2. Carbopol Vehicle 4.2% -1.5%

3. SEG 17.6% 6.1%

4. S1L 22.4% 13.8%

5. S7S 23.3% 16.2%

6. OOPE 20.7% 15.6%

3. Efficacy versus SEG (OLIVE OIL) Results are depicted in Table 4 and in Figure 3 as percent improvement:

Table 4: Skin Moisturizing and Softening by the Te§t Compounds Compared to Pure Olive Oil

Moisturization Softening

4h 8h 4h 8h

S1L 22% 77% 27% 126%

S7S 40% 156% 32% 166%

OOPE 23% 98% 18% 156%

Conclusions

This study evaluated the clinical efficacy of four test compositions, SAMPLE 3

(SEG), SAMPLE 4 (S1L), SAMPLE 5 (S7S) and SAMPLE 6 (OOPE, Olive Oil

Enriched W/Phytosterol Esters), over eight hours usage period. All four test compositions tested were all effective in improving skin moisturization and skin softening to a varying degree. When used as per protocol instructions, good improvements in all clinical parameters tested were observed at 4 hours post-treatment and the improvements diminished considerably at 8 hours post-treatment.

A. SKIN MOISTURIZATION - Sample S7S demonstrated the highest efficacy, it was superior at all time periods.

B. SKIN SOFTENING -

Sample 5 (S7S) demonstrated the highest efficacy, it was superior at 4 hours and also slightly better than Sample 6 (OOPE) at 8 hours.

All test samples were superior to Olive Oil (SEG) with test sample 5 (S7S) demonstrating the highest efficacy. OOPE samples have longer lasting efficacy (8 hours) when compared to Olive Oil (SEG).

Example 2: Cosmetic Compositions According To The Present Invention

A. The novel cosmetic compositions according to the present invention may be used in various applications as described herein above. Table 5 summarizes examples of products in which the oil-based first component of the present invention were used, replacing the commonly used castor oil and waxes, further containing carbomer (2%) and water.

Table 5: Products containing oil-based compositions of the present invention

B. Specific example for the beneficial use of the cosmetic composition according to the present invention is in lipsticks, where moisturizing solid oil phase is required. Table 6 describes an example of the cosmetic composition according to the present invention - a lipstick composition. The oil enriched with phytosterol esters and diglycerides contain 25% phytosterol esters, 15% diglycerides, 11% monoglycerides, 6% free fatty acid and 43% olive oil triglycerides.

Table 6: lipstick composition comprising the oil-based first component "Multi Oil"

Example 3: In vitro Cell Culture Testing to Assess Collagen Synthesis

Objective

To determine the "in vitro" activity of two test materials, FG S7 & FG S8S, in stimulating Collagen synthesis.

Test Materials

1. FG S7 comprising 25% Phytosterol esters, 15% diglycerides, 3% monoglycerides, 6% free fatty acids and 49% olive oil triglycerides.

2. FG S8S comprising 28% Phytosterol esters, 15% diglycerides, 3% monoglycerides, 6% free fatty acids and 48% triglycerides.

Tests were performed with each of the above defined test materials at 3 different concentrations: 10%, 20% and 30% in a carbopol vehicle.

Procedure

Neonatal fibroblasts (ATCC or Clonetics) were plated in MEM (Modified Eagles Media, GIBCO or equivalent) in flat-bottomed 96-well microtitre tissue culture plates at a concentration of about 500,000 cells/ml. Cells were incubated for 48 hours at 37°C and 5% CO₂ to establish exponential growth. Fresh media was added along with tritiated-Proline (specific activity 52 mCi/mmol, at a concentration of 10 μCi/ml) and cells were further incubated at 37°C, 5% CO₂ for an additional 24 hours.

Test materials were diluted to the appropriate concentration in PBS and added as 10 μl aliquots to the appropriate wells.

After an additional 24 hours of growth, cell growth was terminated. Excess PBS was added to each well along with 0.001% of unlabelled collagen Type 3, (University of North Texas) to act as a carrier. Monoclonal antibodies of Type 1 and 3 (University of North Texas) were added in excess. Samples were allowed to react for 24 hours.

Antibody-antigen complexes were isolated and purified via repeated buffer washings and low speed centrifugation.

Samples were mixed with 10ml 10% TCA and incubated in boiling water for 30 minutes. Precipitates were collected by centrifugation, washed and filtered using a 0.22 μm Millipore filter.

The filters were dried and immersed in 15ml of scintillation fluid such as Amersham PCS or Picofluor 30S.

Radioactivity (indicative of collagen specific protein incorporation) in the individual samples was counted with a Packard Tricarb Liquid Scintillation Counter or equivalent, using external standards to compensate quenching.

Results obtained from the experimental wells were compared to controls.

Results

Prior to radioactive labeling, cultures were mixed with the two test materials at varying concentration in a carbopol gel vehicle. After the incubation period, total protein was precipitated and filtered. The precipitated protein recovered was then counted for trituim activity via scintillation counting. The amount of radioactivity (tritium) incorporation in the various test cultures was determined and presented in the following table 7 and in Figure 4. Table 7: Normalized DPM Proline Specific Protein (Collagen Recovery

* Results are average of two trials.

As the results indicate, a control with no additives resulted in an average of 11434 dpm of tritiated proline incorporation. In heat shocked culture where the cells were so severely damaged, very little tritiated proline incorporation was detected. On the other hand, in culture supplemented with highly active collagen synthesis stimulator "Epidermal Growth Factor, EGF" a very high tritiated proline incorporation was found (+213%).

For the two test materials FG S7 and FG S8S; both showed good activity in stimulating collagen synthesis at 10% concentration. As the concentration increases, the collagen synthesis activity increases accordingly. Comparatively, test material FG S8S was shown to be more active than FG S7 in stimulating collagen synthesis at the three concentration level tested (10%, 20% and Conclusions

Both test materials FG S7 and FG S8S were found to be highly active in promoting/stimulating collagen synthesis. While good activity was evident at 10% concentration, optimum activity was found at 30% concentration. Comparatively, FG S8S was shown to be more effective than FG S7.

Example 4: Anti Inflammatory Effect of the Oil Based Compositions

Objective To determine the anti-inflammatory activity of the above defined test materials,

FG S7 and FG S8S, via the measurements of the release of Interleukin in inflamed fibroblasts.

Test Materials 1. FG S7 comprising 25% Phytosterol esters, 15% diglycerides, 3% monoglycerides, 6% free fatty acids and 49% olive oil triglycerides.

Procedure

Anti-Inflammatory Activity-Inhibition of Interleukin la

The skin normally contains a reservoir of Interleukin la (ILla), which is mobilized and released in the lower layers of the skin during inflammatory reactions. A test was developed wherein the levels of ILla in the outer layers of the skin are quantified via antibody-ELISA assay. During an inflammatory response, ILla levels are decreased in the skin surface layers, as ILla participates in the inflammatory response. A defined area on the skin is extracted with phosphate buffer at a neutral pH. The extract is mixed with ILla specific antibody and the amount of ILla is quantified via the antibody-antigen reaction, measured with an ELISA reader.

Results

The following validation table (table 8) demonstrates that normal skin, not exposed to an inflammatory assault, contains high levels of ILla that is present in the surface skin layers. Test validation was performed by exposing the skin to defined irritating conditions such as UV or SLS treatment. After exposure of the skin to UV or a chemical irritant the surface levels of ILla dropped dramatically (almost 50%). After 24 hours, the level has started to return to normal. Pre-treatment with sphingosine, a known anti-inflammatory agent significantly reduced the effects of UV on ILla level, as expected, through membrane stabilization.

Table 8: Control & Validation Table of IL-la levels

* Units are arbitrary units of the ELISA reader

The anti inflammatory activity of the test materials FG S7 and FG S8S was evaluated by applying the test materials on a skin area prior to exposure to the chemical irritant SLS. Application of test material was at three concentrations of 10%, 20%, and 30% in carbopol. Table 9 below and Figure 5 show that both test materials possess anti- inflammatory activity. Both test materials were effective at all concentrations tested; however, test material FG S8S was shown to have better anti-inflammatory activity than FG S7. These results show that the oil-based compositions according to the present invention may be used as anti-inflammatory agents.

Table 9: Anti-Inflammatory effect of the test materials

* Units are arbitrary units of the ELISA reader

Conclusions

The study evaluated the anti-inflammatory activity of two test materials FG S8S and FG S7. The results suggested that both test materials were active in providing anti- inflammatory activity throughout a concentration range between 10% to 30%. The activity is concentration dependent with optimum activity seen at 30% concentration. Comparatively, test material FG S8S was shown to be more effective than FG S7.

Example 5; Efficacy of the Oil-Based Compositions in Improving the Appearance of Skin

Objective The objective of this study was to determine the efficacy of the above defined test materials, FG S7 and FG S8S in improving the appearance and qualitative aspects of skin during a one month usage period.

Test Materials

Procedure Subjects

Five (5) subjects (age 35+), with normal to dry and dry skin and exhibiting some of the visible signs of aging (wrinkles, lack of firmness etc.) participated and completed this one-month product efficacy in-use-study. Subjects applied the test material FS S7 to half of their face and then applied test material FG S8S to the other side of their face after facial cleansing, twice a day (am & pm). All of the subjects visited the test center at Day 0 (baseline) and after 1 month, for evaluation of skin texture and lines and wrinkles reduction by trained technicians.

Exclusion criteria for participating in the study were as follows: 1. Subjects currently using or having used within the last six months Retin-A, or analogs, Corticosteroids, Benzoyl Peroxide, and Topical Anti-biotics.

2. Subjects having had facial peels or Dermabrasion within the last year.

3. Subjects with psoriasis, eczema, or atopic dermatitis. 4. Subjects currently on test in any other clinical study for topical or systemic medications or products.

5. Subjects with known communicable disease.

6. Subjects on medication will be reviewed on a case-by-case basis. 7. Subjects who are pregnant or intend to become pregnant within the next 60 days.

8. Subjects who are nursing/lactating.

9. Subjects who, in the opinion of the Principal investigator, may express a negative reaction to a test sample based on medical or other pre-test condition.

Procedure

Week -1 to 0 - Subject dry out

To ensure that all subjects are at a baseline value and to factor out differences in effects of current skin care regimens, a one week dry out period preceded the study. On day -7 subjects visited the test center and received a bar of soap (Neutrogena) to wash their entire facial area as often as they choose. During this phase of study, subjects were not allowed to use any moisturizer, sunscreen, and liquid make-up. Subjects were also instructed to avoid excessive UV exposure, and they were not allowed to visit tanning salons. Subjects were allowed to use eye, face (blush only) and lip products during this and subsequent phases of the study.

Day 0 to Month 1 - Use of Test Products

On Day 0 all subjects visited the test center during the morning with no product applied to their faces. Instructions on how to use the test product, and a daily diary to record their use of the products and their observations were given. The instructions were:

1. To clean the face with the Neutrogena soap provided during the dry out period. 2. To apply test material A (FS S7) to half of their face and then apply material B (FG S8S) to the other side of their face, twice a day (am & pm) throughout the treatment period. Assignment of materials A and B were randomized at left and right sides of the subjects' faces.

Thereafter, baseline measurements were taken using the technique of Line & Wrinkle Reduction and Skin Smoothness via Replicas and Image Analysis:

Silicon (Siflo™ ) replicas of the skin texture of the canthus area were taken by placing adhesive rings on the canthus area at a defined orientation and pouring dental silicon replicating material inside the ring. As soon as the silicon dries (2-3 minutes) it was removed, and the replicas were labeled with the subject number and visit number. At the end of the study, the replicas were analyzed via digital image analysis for lines and wrinkles and overall skin texture. After 1 month, final measurements were taken by the same procedure.

Results

Computer Image Analysis of Replicas

Changes in skin texture were quantified by taking Siflo™ Replicas of the canthus area and analyzing the replicas with a computer image analysis system. After the replicas were photographed and the black and white photos were scanned into the image processing computer program, images were converted into black and white pixels where black pixels highlighting only the lines and wrinkles. These images were quantified as the total number of white (non wrinkles) and black (wrinkles and lines) pixels and compared to a predetermined threshold value. Results are presented in Table 10 and Figure 6. As the results indicate, significant reductions in facial lines and wrinkles of 30.1% and 37.3% were observed after 1 month of treatment with test products FG S7 and FG S8S respectively. Comparatively, product FG S8S induced slightly better wrinkle reduction than product FG S7. Table 10: Wrinkle Reduction by the Oil-Based Compositions

* Paired T-Test at 95% confidence value; results were compared to baseline values

Conclusions This study evaluated the clinical efficacy of the oil-based compositions FG S7 and FG S8S, over a one month usage period. The results of the study show that both products tested were highly effective in reducing superficial facial lines and wrinkles. Comparatively, product FG S8S was found to be more effective than product FG S7.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed chemical structures and functions may take a variety of alternative forms without departing from the scope of the invention, which is defined in the claims which follow.

Claims

1. A cosmetic composition comprising: a first component comprising an oil, at least one diglyceride derivative and at least one phytosterol ester; and a second component comprising a cosmetically acceptable diluent or carrier.

2. The cosmetic composition of claim 1 wherein the first component is present in an amount of from about 1 to about 99% by weight based on the total weight of the cosmetic composition.

3. The cosmetic composition of claim 2 wherein the first component is present in an amount of from about 1 to about 50% by weight based on the total weight of the cosmetic composition.

4. The cosmetic composition of claim 1, wherein said diglyceride derivative and phytosterol ester are dissolved or dispersed in said oil.

5. The cosmetic composition of claim 1, wherein said oil is selected from the group consisting of olive oil, avocado oil, canola oil, soybean oil, sunflower oil, peanut oil, almond oil, safflower oil, cottonseed oil, coconut oil, rice bran oil, mustardseed oil camelina oil, chia oil, flaxseed oil, perilla oil, linseed oil, rapeseed oil, cocoa butter, palm oil, kernel oil, castor oil, sunflower oil, fish oil, corn oil, etheric oils, and mixtures thereof.

6. The cosmetic composition of claim 5, wherein said oil is olive oil.

7. The cosmetic composition of any one of claims 1-6, wherein said diglyceride derivative is a 1,3-diacylglycerol, a 1,2-diacylglycerol or a combination thereof.

8. The cosmetic composition of any one of claims 1-7 wherein said phytosterol is beta- sitosterol, campesterol, stigmasterol, brassicasterol, D5-avenasterol, β- sitostanol, campestanol and stanol.

9. The cosmetic composition of any one of claims 1-8, wherein said phytosterol ester is an ester of aC₆-C₂₄ saturated or unsaturated fatty acid.

10. The cosmetic composition of claim 9, wherein said phytosterol ester is an ester of C₁₆-C₁₈ saturated or unsaturated fatty acid.

11. The cosmetic composition of any one of claims 9-10 wherein said fatty acid is selected from the group consisting of oleic, linoleic, linolenic, palmitic and stearic acid.

12. The cosmetic composition according to any one of claim 1-11, further comprising a monoglyceride derivative.

13. The cosmetic composition of claim 12, wherein said monoglyceride is a derivative of a C₁₄-C₂₄ saturated or unsaturated fatty acid.

14. The cosmetic composition of claim 13, wherein said monoglyceride is a derivative of a C₁₆-C₁₈ saturated or unsaturated fatty acid.

15. The cosmetic composition of any one of claims 13-14, wherein said fatty acid is selected from the group consisting of oleic, linoleic, linolenic, palmitic and stearic acid.

16. The cosmetic composition of any one of claims 1-11, further comprising free fatty acids.

17. The cosmetic composition of any one of claims 1-11, wherein said phytosterol ester is present in an amount of from about 1% to about 99% by weight based on the total weight of the first component.

18. The cosmetic composition of claim 17, wherein said phytosterol ester is present in an amount of from about 10 to about 40% by weight based on the total weight of the first component.

19. The cosmetic composition of claim 18, wherein said phytosterol ester is present in an amount of about 30% by weight based on the total weight of the first component.

20. The cosmetic composition of any one of claims 1-11, wherein said diglyceride derivative is present in an amount of from about 1% to about 99% by weight based on the total weight of the first component.

21. The cosmetic composition of claim 20, wherein said diglyceride derivative is present in an amount of from about 10 to about 50% by weight based on the total weight of the first component.

22. The cosmetic composition of claim 21, wherein said diglyceride derivative is present in an amount of about 20% by weight.

23. The cosmetic composition of claim 1, wherein said second component is an ointment, a cream, a lotion, an emulsion, a powder, an oil, a gel, an aerosol, a water-oil emulsion, or any combination thereof.

24. The composition of claim 1 wherein said cosmetic composition is a gel.

25. The cosmetic composition of claim 1, for use as an anti-aging composition.

26. The cosmetic composition of claim 1, for use as an anti-irritant composition.

27. The cosmetic composition of claim 1 , for use as a skin moisturizer or softener.

28. The cosmetic composition of claim 1, for use in reduction, prevention or treatment of skin chapping.

29. The cosmetic composition of claim 1, for use in reduction, prevention or treatment of skin lines or wrinkles.

30. The cosmetic composition of claim 1, for use as a sunscreen.

31. The cosmetic composition of claim 1 , for use for collagen recovery.

32. A process for preparing the cosmetic composition of claim 1, said process comprising the steps of preparing the first component and mixing said first component with said diluent and carrier, thereby preparing said cosmetic composition.

33. The process of claim 32, wherein the process for preparing the first component comprises the steps of mixing said oil, said diglyceride derivative and said phytosterol ester, thereby preparing said first component.

34. The process of claim 32, wherein the process for preparing the first component comprises the steps of: a. mixing at least one phytosterol and an oil; b. optionally adding at least one carboxylic fatty acid or ester derivative thereof; c. adding to the mixture of (a) or (b) an immobilized lipase complex; d. incubating the mixture of (c) for a period of time sufficient to give a product comprising a phytosterol ester and a diglyceride in oil, wherein said product comprises two distinct fractions, an oil fraction and a sediment fraction; e. optionally removing said immobilized lipase complex, and f. separating said oil fraction from said sediment fraction.

35. The process of claim 34, wherein the lipase is surfactant-coated.

36. The process of claim 34, wherein said oil fraction comprises diglycerides, triglycerides and phytosterol esters.

37. The process of claim 36, said oil fraction further comprising monoglycerides.

38. The process of claim 34, wherein said sediment fraction comprises diglycerides, triglycerides and phytosterol esters.

39. The process of claim 38, wherein said sediment fraction further comprises monoglycerides .

40. The process of claim 34, wherein said lipase complex possesses a high level of sterol-specific alcoholytic and/or esterification activity and minimal acidolytic and interesterification activities.

41. The process of claim 40, wherein said lipase is selected from the group consisting of Aspergillus niger, Candida antarctica, Candida cylindracea, Candida rugosa, Mucor miehei, Pseudomonas cepacia, Rhizopus niveus, Rhizopus arrhizus, Hog pancreas, Aspergillus oryzae, Candida lipolytica, Mucor javanicus, Pencillium roqueforti, Pseudomonas fluorescens,

Rhizomucor miehei, wheat germ, Chromobact. viscosum, lipoprotein from Pseudomonas species, lipoprotein from Pseudomonas species B and Thermomyces lanuginosus.

42. The process according to claims 34, 40 and 41, wherein the lipase is immobilized onto an insoluble matrix.

43. The process of claims 34 and 37-39, wherein said lipase is surfactant coated.

44. The process of claim 32, wherein the oil in said first component is selected from the group consisting of olive oil, avocado oil, canola oil, soybean oil, sunflower oil, peanut oil, almond oil, safflower oil, cottonseed oil, coconut oil, rice bran oil, mustardseed oil, camelina oil, chia oil, flaxseed oil, perilla oil, linseed oil, rapeseed oil, cocoa butter, palm oil, kernel oil, castor oil, sunflower oil, fish oil, corn oil etheric oils and mixtures thereof.

45. The process of claim 44, wherein said oil is olive oil.

46. The process of claim 32, wherein the diglyceride in said first component is a 1,3-diacylglycerol, a 1,2-diacylglycerol or a combination thereof.

47. The process of claim 32, wherein the phytosterol in said first component is beta-sitosterol, campesterol, stigmasterol, brassicasterol and stanol.

48. The process of claim 47, wherein said phytosterol ester is an ester of a C₆-C₂₄ saturated or unsaturated fatty acid.

49. The process of claim 48, wherein said phytosterol ester is an ester of a C₁₆-C₁₈ saturated or unsaturated fatty acid.

50. The process of any one of claims 48-49 wherein said fatty acid is selected from the group consisting of oleic, linoleic, linolenic, palmitic and stearic acid.

51. The process according to claim 32, wherein said product further comprises a monoglyceride derivative.

52. The process of claim 51, wherein said monoglyceride is a derivative of a C₁₄- C₂₄ saturated or unsaturated fatty acid.

53. The process of claim 49, wherein said monoglyceride is a derivative of a C₁₆- C₁₈ saturated or unsaturated fatty acid.

54. The process of any one of claims 52-53 wherein said fatty acid is selected from the group consisting of oleic, linoleic, linolenic, palmitic and stearic acid.

55. The process of claim 32, wherein said product further comprises free fatty acids.

56. Use in cosmetic products of a composition comprising an oil, at least one diglyceride derivative and at least one phytosterol ester.

57. The use according to claim 56 wherein the cosmetic product is selected from the group consisting of a lipstick, a daily cream, a daily cream for dry skin, and eye cream, a neck cream, a night cream, a nourishing musk, a hair nourishing musk, an hand cream, a body cream, a foot cream, an all purpose cream, a bath oil, a baby oil, a baby cream and a baby paste.

58. The use according to claim 56 wherein the cosmetic product is applied to the skin, the nails, the hair, or the eyelashes.

59. The use according to claim 56 wherein the cosmetic product is applied for delaying or lowering the effects of the aging on the skin by any one of the methods selected from a group consisting of: a method for counteracting the harmful effects of free radicals on the skin, a method for treating sensitive skins and making up sensitive skins.