Phytoene-containing oleoresins and compositions

Abstract

The present invention provides a solvent-free non-GMO oleoresin obtained from Blakeslea trispora containing phytoene at a concentration of 10-40% (w / w). Further, the present invention relates to compositions containing said oleoresin, as well as topical and oral dosage forms containing said oleoresin. Use of said compositions and dosage forms in the treatment and prevention of various skin conditions and for improving skin quality is also disclosed and claimed.

Description

[Technical field]

[0001] The present invention relates to an oleoresin containing high concentrations of phytoene, and to a composition comprising said phytoene together with jojoba oil. The present invention also relates to the use of said phytoene-containing composition in the prevention and management of skin lesions caused by exposure to air pollution or UV radiation. [Background technology]

[0002] Phytoene is a largely colorless carotenoid found in many different species of fruit. In addition to acting as a key intermediate in the biosynthesis of other bioactive carotenoids, phytoene itself has several biological properties, including anti-inflammatory activity, antioxidant activity, and UV absorbing activity. These properties, combined with its lack of color, have led to the development of topical and cosmetic preparations containing phytoene for use in treating or preventing various skin conditions. One example of such a preparation that can be used to lighten or whiten the skin is described in US 8,398,958.

[0003] Phytoene can be obtained from several different sources, including various fruit species (e.g., tomato), algae (e.g., Dunaliella species), and molds, the most notable of which is Blakeslea trispora.

[0004] B. trispora is a tropical plant pathogen but is harmless to animals and humans. This species has been found to produce important carotenoids such as lycopene, β-carotene, phytoene and phytofluene and is therefore widely used to study the carotenoid synthesis pathway. Furthermore, by strain selection and growth conditions, it was found possible to produce high amounts of phytoene from B. trispora as described in Ukrainian patent UA85489. Furthermore, UA86556 discloses a method to obtain phytoene-containing biomass after incubation of the strain described in UA85489.

[0005] In recent years, the role of airborne pollution in causing disease has become of increasing concern. While the effects of air pollutants on cardiac and respiratory health have long been known, it is becoming clear that the skin is often targeted and adversely affected by such pollutants. Thus, a clear correlation has been found between the increase in airborne pollution and the number of new cases of acne, atopic dermatitis, urticaria, and other skin conditions in humans. Furthermore, airborne pollution is also thought to be an exacerbating factor for accelerated or premature skin aging. Finally, there is a growing body of knowledge linking air pollution to the incidence and / or severity of various skin cancers.

[0006] Airborne pollution can come from many different sources, but pollutants contained in automobile exhaust are of particular importance, given the daily exposure of a very large proportion of the world's population to such pollution. Of particular concern are pollutants contained in exhaust emitted by diesel engines.

[0007] Diesel exhaust contains a very large number of compounds, many of which are associated with significant health risks. In addition to the adverse effects caused by compounds dissolved in the gas phase, diesel exhaust also contains significant concentrations of diesel particulate matter (DPM). These particles, present in a variety of different size ranges, are of particular concern with regard to lung cancer and other severe lung diseases. However, recent studies have also shown a direct link between exposure of skin cells to DPM and various skin conditions (such as those mentioned above). Furthermore, several studies have been published that describe the effects of airborne particulate matter (including DPM) on skin cells. One such example is a study by Ryu et al. [Experimental & Molecular Medicine 51:108; 2019], in which the authors describe biochemical changes underlying keratinocyte senescence caused by exposure to airborne particulate matter. Other studies have also described increased levels of cytokines and interleukins, including IL-8, when cultured cells are exposed to DPM. Thus, for example, Reynolds et al. (2011) [Environmental Health Perspective 119(3):332-336] describe the biochemical pathways underlying the increased IL-8 secretion seen when epithelial cells are exposed to DPM.

[0008] There is a need for safe, naturally derived compositions that can be used to prevent, alleviate or treat skin conditions caused by or associated with exposure to air pollutants, particularly pollutants found in diesel exhaust, as well as skin conditions associated with sun exposure. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Pat. No. 8,398,958 [Patent Document 2] Ukrainian Patent No. 85489 [Patent Document 3] Ukrainian Patent No. 86556 [Non-patent literature]

[0010] [Non-Patent Document 1] Ryu et al.[Experimental & Molecular Medicine 51:108;2019] [Non-Patent Document 2] Reynolds et al. (2011) [Environmental Health Perspective 119(3):332-336] Summary of the Invention [Problem to be solved by the invention]

[0011] In one aspect of the present invention, the inventors have discovered that it is possible to produce a solvent-free, non-GMO oleoresin obtained from the fungus species Blakeslea trispora that contains phytoene in concentrations much higher (e.g., 20-30 times higher) than previously obtained. Furthermore, it has been unexpectedly found that there is selective enrichment of the phytosterol, ergosterol, in the oleoresin of the present disclosure when compared to the biomass from which the composition is derived. This phytoene-enriched oleoresin is used in certain embodiments as a starting material for the preparation of compositions and dosage forms as disclosed and described in more detail hereinafter.

[0012] In a further aspect, the inventors have found that compositions comprising Simmondsia Chinensis (jojoba) seed oil in combination with phytoene are particularly advantageous for use in protecting the skin against damage caused by air pollutants (such as diesel exhaust particulates (DPM)) and UV radiation, and for maintaining and improving skin quality.

[0013] Thus, the present invention primarily relates to a solvent-free non-GMO oleoresin obtained from Bracelea trispora containing phytoene in a concentration of 10-40% (w / w). [Means for solving the problem]

[0014] In a preferred embodiment, the concentration of phytoene in the oleoresin is in the range of 20-40% (w / w).

[0015] In another preferred embodiment, the concentration of phytoene in the oleoresin is in the range of 20-30% (w / w).

[0016] In many cases, the oleoresins disclosed above will contain one or more phytosterols. One of the major phytosterols present in oleoresins is ergosterol, which is generally present at a concentration in the range of about 0.5-1% (w / w). In a preferred embodiment, the ergosterol concentration is about 0.6% (w / w).

[0017] It should be noted that the term "about" is used throughout this disclosure (in relation to concentration percentage values) to indicate that concentrations that differ from the stated value by up to + / - 0.5% are included within the range defined by the stated concentration value or range.

[0018] In another aspect, the present invention provides a solvent-free non-GMO oleoresin obtained from Bracelea trispora containing phytoene at a concentration of 20-30% (w / w), a) culturing the fungal species Blakeslea trispora ( b) obtaining biomass from the cultured Blakeslea trispora; c) subjecting the biomass to supercritical carbon dioxide fluid extraction carried out at an operating pressure between 300-450 bar and an operating temperature between about 50°C and 65°C, thereby obtaining a phytoene-containing oleoresin; The present invention relates to a solvent free non-GMO oleoresin obtained or obtainable by a process comprising the steps of:

[0019] Preferably, the Blakeslea trispora cultivated in step (a) of the method disclosed above is a phyt 1+ / phyt 1- strain.

[0020] Typically, the oleoresin obtained by this process contains phytoene and one or more phytosterols at a concentration of 20-30% (w / w). In most embodiments, the one or more phytosterols comprises ergosterol as the major component. The concentration of ergosterol in the oleoresin generally ranges from 0.5-1% (w / w). In a preferred embodiment, the concentration of ergosterol in the oleoresin is about 0.6% (w / w).

[0021] In a further aspect, the present invention relates to a composition comprising a seed oil obtained from Simmondsia sinensis (jojoba) together with a phytoene-containing oleoresin as disclosed herein above. In a preferred embodiment, the concentration of phytoene in said composition is in the range of 0.01-10% (w / w). In particularly preferred embodiments, the concentration of phytoene is in the range of 1-2% (w / w), more preferably in the range of 1-1.5% (w / w). In some preferred embodiments, the concentration of phytoene in the composition is about 1% (w / w).

[0022] In yet a further aspect, the present invention relates to a dosage form comprising the phytoene-containing composition disclosed above together with one or more pharma- ceutically acceptable, nutraceutically acceptable, or cosmetically acceptable excipients.

[0023] In another aspect, the present invention relates to a dosage form comprising the phytoene-containing oleoresin disclosed above together with one or more pharma- ceutically acceptable, nutraceutically acceptable, or cosmetically acceptable excipients.

[0024] Further details regarding said dosage forms (both oral and topical) are provided herein below.

[0025] The present invention also encompasses a method for improving or maintaining skin quality and / or protecting against environmental damage in the form of skin (e.g., caused by air pollutants or UV radiation), said method comprising administering to a subject in need thereof the composition or dosage form disclosed above. In a preferred embodiment, the subject is a human subject. Further details regarding these methods are provided herein below. [Brief description of the drawings]

[0026] [Figure 1] Figure 1 is a flow chart summarizing the method for obtaining phytoene-rich biomass from Brackeslea trispora cultures. [Diagram 2] FIG. 2 is a flow chart summarizing the various process steps used to prepare the phytoene-containing oleoresin of the present invention. [Diagram 3] FIG. 3 is a graphical representation of results showing increased IL-8 production in cultured keratinocytes following treatment with diesel exhaust particulates, and inhibition of this effect when the cultured cells were treated with dilutions of the phytoene composition of the present invention. [Figure 4] FIG. 4 is a bar graph showing the inhibitory effect of phytoene-containing compositions of the present invention on reactive oxygen species (ROS) levels in cultured keratinocytes following exposure to diesel exhaust particulates. [Diagram 5] FIG. 5 is a bar graph showing the inhibitory effect of phytoene-containing compositions of the present invention on IL-8 levels in cultured keratinocytes following exposure to UVB radiation. [Figure 6] FIG. 6 is a graphical representation of results showing inhibition of IL-1α production by skin cells in an artificial skin model when the cells are treated with dilutions of a phytoene-containing composition of the present invention. [Figure 7] FIG. 7 is a graph showing the inhibitory effect of dilutions of a phytoene-containing composition on IL-6 production in an artificial skin model. [Figure 8] FIG. 8 presents data showing increased skin lightening caused by topical application of the composition of the present invention. [Figure 9] FIG. 9 presents data showing the reduction in skin redness caused by topical application of a composition of the present invention. [Figure 10] FIG. 10 presents data showing increased skin firmness caused by topical application of the composition of the present invention. [Figure 11] FIG. 11 presents data showing the increase in skin elasticity caused by topical application of a composition of the present invention. [Figure 12] FIG. 12 presents data showing the increase in skin smoothness caused by topical application of the composition of the present invention. [Figure 13] FIG. 13 presents data showing the improvement of various skin wrinkle parameters following topical application of the composition of the present invention. [Figure 14] FIG. 14 presents data showing the reduction in transepidermal water loss caused by topical application of a composition of the present invention. [Figure 15] FIG. 15 presents data showing reduced skin irritation following topical application of a composition of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] As explained herein above, in one aspect, the present invention relates to a solvent-free, non-GMO oleoresin obtained from the fungal species Blakeslea trispora, comprising phytoene at a concentration of 10-40% (w / w). In some preferred embodiments, the phytoene concentration of the oleoresin is in the range of 20-30% (w / w). In one preferred embodiment, the phytoene concentration in the oleoresin is about 20% (w / w).

[0028] It is noted that the term "oleoresin" is generally understood to refer to a semi-solid (or viscous liquid) extract comprising essential and / or fatty oils and other, mainly hydrophobic compounds. Historically, such oleoresins have been obtained exclusively by solvent extraction of biological material (e.g. plant material or biomass obtained after fermentation of microorganisms) and subsequent removal of said solvent. However, in the context of the present invention, which concerns a completely solvent-free composition, the oleoresin is obtained by supercritical fluid extraction, preferably supercritical carbon dioxide extraction. Furthermore, as herein above, the oleoresin is a "non-GMO" oleoresin, i.e. not obtained from genetically modified organisms.

[0029] In some embodiments of this aspect of the invention, the oleoresin further comprises additional biologically active compounds, particularly phytosterols. In some cases, the major phytosterol component of the oleoresin is ergosterol.

[0030] As described hereinabove, phytoene-rich biomass is obtained from Blakeslea trispora strains following the teachings of UA85489 and UA86556. The method is summarized in the flow chart provided in Figure 1.

[0031] The biomass (which typically contains 5-7% (w / w) phytoene and about 40%-50% oil) is then subjected to supercritical carbon dioxide fluid extraction carried out at an operating pressure between 300-450 bar. The extraction was carried out at a temperature of about 50°C to 65°C. After this extraction, an oleoresin (as hereinabove described) is obtained, which contains generally phytoene at a concentration of 20-30% (w / w) and a mixture of phytosterols (of which ergosterol is the main component) at a concentration of about 0.6% (w / w). The process is outlined in the flow diagram depicted in Figure 2.

[0032] As described herein above, the present invention also provides a composition comprising phytoene and phytosterol. In a preferred embodiment, the composition comprises 1% (w / w) phytoene and 0.02% (w / w) phytosterol. In another preferred embodiment, the composition comprises 0.1% (w / w) phytoene and 0.002% phytosterol.

[0033] In one embodiment of the method disclosed above, the phytoene present in the administered composition comprises 15-cis-phytoene. In some embodiments, greater than 50% (w / w) of the phytoene in the composition is 15-cis-phytoene. In other embodiments, greater than 90% (w / w) of the phytoene is 15-cis-phytoene.

[0034] As noted above, the present invention includes within its scope dosage forms comprising the phytoene-containing compositions disclosed above together with one or more pharma- ceutically acceptable, nutraceutical, or cosmetically acceptable excipients, which may be formulated for either topical or oral administration, as described in more detail hereinbelow.

[0035] In another aspect, the present invention provides a dosage form comprising the phytoene-containing oleoresin disclosed and described herein together with one or more pharma- ceutically acceptable, nutraceutical, or cosmetically acceptable excipients. Such a dosage form may be prepared to be suitable for topical administration. However, in a preferred embodiment, this type of dosage form is suitable for oral administration.

[0036] In a preferred embodiment, the dosage form is suitable for topical administration (i.e., for application to the skin or mucous membrane of a subject). For example, the composition may be formulated as a cream, lotion, ointment, gel, foam, salve, suspension, oil, or solution. Alternatively or additionally, the composition may be adsorbed onto a pad or other delivery device suitable for placement on or adherence to the skin or mucosal surface.

[0037] In some embodiments, topical dosage forms may be formulated as emulsions, such as oil-in-water or water-in-oil emulsions. In other cases, hydrophobic phytoene may be diluted with naturally occurring biocompatible diluents, such as vegetable oils (e.g., olive oil, sunflower oil, tomato oil, jojoba oil, etc.), liquid paraffin, or other conventional lipophilic diluents (e.g., squalene).

[0038] In some preferred embodiments, the concentration of phytoene in the topical dosage form ranges from 0.01 to 0.5% (w / w). In some preferred embodiments, the concentration of phytoene is 0.1%. In other preferred embodiments, the concentration of phytoene is 0.02% (w / w).

[0039] In another embodiment, the dosage form of the present invention is a dosage form suitable for oral administration as disclosed hereinabove. Preferably, such oral dosage form is selected from the group consisting of a tablet, a caplet, a capsule, a lozenge, a chewable tablet, a suspension, a syrup, an oil or a solution.

[0040] In some embodiments, the oral composition is in the form of a softgel capsule. In some embodiments, the oral composition is in the form of a drink, shot, gummy, or powder. In some embodiments, the oral composition is mixed or assimilated into foodstuffs such as chocolate, ice cream, etc.

[0041] Such unit dosage forms contain a safe and effective amount of the composition. Pharmaceutically acceptable carriers suitable for the preparation of unit dosage forms for oral administration are well known in the art. In some embodiments, tablets typically contain conventional pharma- ceutically compatible adjuvants such as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose; binders (hinders), such as starch, gelatin, and sucrose; disintegrants, such as starch, alginic acid, and croscarmellose; and lubricants, such as magnesium stearate, stearic acid, and talc. In one embodiment, glidants, such as silicon dioxide, can be used to improve the flow properties of the powder mixture. In one embodiment, coloring agents, such as FD&C dyes, can be added for appearance. Sweeteners and flavoring agents, such as aspartame, saccharin, menthol, peppermint, and fruit flavors, are useful adjuvants for chewable tablets. Capsules typically contain one or more solid diluents. In some embodiments, the selection of the carrier component will depend on secondary considerations such as taste, cost and storage stability, which are not critical for purposes of the present invention and can be readily made by one of ordinary skill in the art.

[0042] In one embodiment, the oral dosage form comprises a predetermined release profile. In one embodiment, the oral dosage form of the present invention comprises a sustained release tablet, capsule, lozenge or chewable tablet. In one embodiment, the oral dosage form of the present invention comprises a delayed release tablet, capsule, lozenge or chewable tablet. In one embodiment, the oral dosage form of the present invention comprises an immediate release tablet, capsule, lozenge or chewable tablet. In one embodiment, the oral dosage form is formulated according to a desired release profile of the pharmacoactive ingredient as known to those skilled in the art.

[0043] In addition to the phytoene and other optional active ingredients present in the compositions of the present invention, as described above, the dosage forms provided herein generally further comprise one or more excipients, which may include, for example, thickeners, humectants, stabilizers, colorants, diluents, bulking agents, controlled release polymers, and the like.

[0044] The various dosage forms types that can be used to formulate the compositions of the present invention are well known to those skilled in the art, and further details can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co, Easton, Pa, USA, 21 st These are available in standard reference works such as the 2006 edition.

[0045] As explained hereinabove, the present invention also encompasses a method of improving or maintaining skin quality and / or protecting skin morphology from environmental damage (e.g., caused by air pollutants or UV radiation), said method comprising administration of the composition or dosage form disclosed above to a subject in need thereof.

[0046] Thus, in one embodiment, the present invention relates to a method for protecting the skin of a subject exposed to air pollutants, comprising administering a composition comprising phytoene to a subject in need of such protection. Typically, in this embodiment, the composition of the present invention or a dosage form containing said composition can be administered topically to the skin of a subject as a preventative measure or to alleviate or treat existing lesions caused by exposure to diesel exhaust particulates (DPM) or other airborne pollutants. The composition can be administered for a short period of time (e.g., days or weeks) to prevent the development of new pollutant-related skin lesions, for example, if the subject knows that they are spending time in an environment where such pollutants are present. Alternatively, the composition can be administered for a longer period of time (e.g., weeks to months). When the composition is formulated as a cream, gel, ointment, or other relatively viscous dosage form, a small amount (e.g., about 0.5 to about 10 cm of cream) is rubbed into the target area of ​​the subject's skin surface. The frequency of such treatments may range from once per week to three or more times per day, depending on the exact concentration of phytoene and / or other active ingredients in the formulation, the age of the subject, and / or the reason for administering the formulation (e.g., to prevent new lesions, prevent premature skin aging, or treat existing lesions).

[0047] In another embodiment of this method, the composition of the method may be orally administered to a subject in need thereof.

[0048] It should be noted that the phrase "protecting the skin of a subject exposed to air pollutants" should be understood to refer to one or more of the prevention, mitigation, or treatment of diseases, disorders, and premature aging of the skin of a subject. These diseases and disorders can be any skin condition caused or aggravated by air pollutants. However, in some embodiments, the diseases and disorders are selected from the group consisting of acne, atopic dermatitis, urticaria, skin cancer, and premature skin aging.

[0049] The present invention also relates to a method for preventing, alleviating or treating a skin condition caused by or associated with exposure to air pollutants, comprising administering a composition or dosage form of the present invention to a human (or non-human mammalian) subject.

[0050] In one embodiment, the above-mentioned skin condition is an inflammatory condition mediated at least in part by the cytokines IL-1α, IL-8 and / or IL-6.

[0051] The term "air pollutants" as used in the context of the two methods disclosed above should be understood to include all types of harmful compounds, substances and agents that may be present in the air, either indoors or outdoors. Said harmful compounds, substances and agents may be dispersed or dissolved in the air, or may be present as liquid droplets, aerosols or as solid particulate matter. In the latter case, the particulate matter may have additional liquid or gaseous components adsorbed on its surface.

[0052] In one embodiment of the present invention, the method disclosed above is particularly directed to air pollutants emitted in the exhaust of vehicles, particularly those powered by diesel engines. In one embodiment, the method disclosed above is particularly directed to particulate matter (i.e., DPM) present in diesel exhaust. In yet other embodiments of the disclosed method, air pollutants are emitted from other forms of transportation (e.g., aircraft and diesel train engines) or industrial plants and factories.

[0053] While any suitable phytoene-containing composition may be used to practice the above disclosed methods of the present invention, in one preferred embodiment, the composition comprises naturally occurring, solvent-free, non-GMO phytoene.

[0054] In another embodiment, the present invention relates to a method for protecting the skin of a subject exposed to UV radiation, comprising administering a composition comprising phytoene to a subject in need of such protection. The term "UV radiation" refers to radiation present in sunlight to which the skin of a human subject is exposed, including all frequencies of UV radiation, particularly radiation in the UVA and UVB ranges.

[0055] In yet a further embodiment, the present invention relates to a method for improving one or more aspects of skin quality, comprising administering a composition comprising phytoene to a subject in need of such protection. Many different aspects of skin quality may be improved by this method. However, in a preferred embodiment, said aspect of skin quality is selected from the group consisting of evening and evening skin tone, reducing wrinkles, improving elasticity, protecting the natural skin barrier function, and improving the appearance of the skin.

[0056] In another aspect, the present invention provides a composition disclosed hereinabove for use as a medicament. In a preferred embodiment, the medicament is for use in protecting the skin of a subject exposed to air pollutants. In another embodiment, the medicament is for use in protecting the skin of a subject exposed to UV radiation. In yet a further embodiment, the medicament is for use in improving one or more aspects of skin quality, the improvement being preferably selected from the group consisting of evening and leveling skin tone, reducing wrinkles, improving elasticity, protecting natural skin barrier function, and improving skin appearance.

[0057] In some preferred embodiments of the methods and uses disclosed above, the composition comprising phytoene is a composition as defined hereinabove and as claimed hereinbelow. In some embodiments of these methods and uses, the composition comprising phytoene is administered by topical application to the skin and / or mucous membranes of the subject. In other embodiments, the composition comprising phytoene is administered to the subject by oral route, preferably formulated as an oral dosage form, for example one of the oral dosage forms defined hereinabove.

[0058] As disclosed hereinabove, the composition used in the method disclosed above comprises phytoene obtained from the fungal species, Blakeslea trispora. In a particularly preferred embodiment, said Blakeslea trispora is a phyt 1+ / phyt 1- strain, such as the strain disclosed in UA85489, deposited in the depository of the Institute of Microbiology and Virology (named after DK Zabolotny), NAS, Ukraine, under the accession number F-100053.

[0059] Preferably, the phytoene contained in the composition used in the method disclosed above is obtained from biomass produced according to the teachings of UA86556. Further details of this method are provided herein below. The teachings of both UA85489 and UA86556 are incorporated herein in their entirety.

[0060] In a preferred embodiment, the composition administered in the method disclosed above contains phytoene in a concentration of 0.01% (w / w) to 10% (w / w). In a more preferred embodiment, the concentration of phytoene in the composition ranges from 0.1 to 2.5% (w / w). In an even more preferred embodiment, the concentration of phytoene in the composition is about 0.1% (w / w). In an even more preferred embodiment, the concentration of phytoene is about 1% (w / w).

[0061] In some embodiments of the methods of the invention, the composition administered further comprises one or more phytosterols. In one embodiment, the one or more phytosterols comprise ergosterol.

[0062] The present invention further encompasses the use of a non-GMO, solvent-free, phytoene-containing composition as defined herein in the manufacture of a pharmaceutical or cosmetic or dietary supplement.

[0063] In one embodiment, the present invention relates to the use of a non-GMO, solvent-free, phytoene-containing composition as defined herein in the manufacture of a pharmaceutical or cosmetic or dietary supplement for protecting the skin of a subject exposed to air pollutants.

[0064] In another embodiment, the present invention relates to the use of a non-GMO, solvent-free phytoene-containing composition as defined herein in the manufacture of a pharmaceutical or cosmetic or dietary supplement for preventing, alleviating or treating a skin condition caused by or associated with exposure to air pollutants.

[0065] In one embodiment, the present invention relates to the use of a non-GMO, solvent-free, phytoene-containing composition as defined herein in the manufacture of a pharmaceutical or cosmetic or dietary supplement for protecting the skin of a subject exposed to UV radiation.

[0066] In one embodiment, the invention relates to the use of a non-GMO, solvent-free, phytoene-containing composition as defined herein in the manufacture of a pharmaceutical or cosmetic or dietary supplement for improving one or more aspects of skin quality, in a preferred embodiment, said improvement being selected from the group consisting of evening and even skin tone, reducing wrinkles, improving elasticity, protecting the natural skin barrier function, and improving the appearance of the skin.

[0067] All of the various technical features explained in relation to the methods disclosed above apply equally to the different modes of use disclosed herein. EXAMPLES

[0068] Various features of the presently claimed invention will now be illustrated in the following non-limiting examples. [Example 1] Inhibition of DPM-induced IL-8 production in cultured cells by the compositions of the present invention Introduction: The aim of this study was to investigate whether phytoene can inhibit DPM-induced increase in the production of the pro-inflammatory cytokine Il-8 in cultured keratinocytes. This in vitro system serves as a model of the inflammatory response in skin cells observed in subjects that develop skin lesions and accelerate skin aging after exposure to air pollutants.

[0069] method: Spontaneously transformed adult human keratinocytes of the HaCaT strain were grown and maintained in filtered DMEM supplemented with 100 U / ml penicillin and 100 μg / ml streptomycin and 10% FBS. Aliquots of cells (approximately 2.5 × 10 5The cells (concentration: 100,000 cells / ml) were seeded into 96-well plates containing 170 μl / well of the above-mentioned medium. Each seeded well contained approximately 42,500 cells. The plates were then incubated at 37° C., 5% CO 2 After this time, the medium was aspirated and replaced with a dilution of a 1% (w / w) phytoene-containing composition of the present invention containing a final phytoene concentration of 56 ppm ("Test"), or the following three controls: - Vehicle control ("Control", FIG. 3) -DPM Stimulation Control ("DPM") Positive Control: Dexamethasone ("DEX")

[0070] The vehicle for preparing DPM, DEX and test solutions was PBS containing 100 μg / ml diesel exhaust particulates (DPM). All test articles and controls were plated in triplicate.

[0071] The plates were then incubated at 37°C and 5% CO 2 The cells were incubated at RT for an additional 24 hours at RT. At the end of this incubation period, the medium was aspirated and centrifuged at 250×g for 5 minutes to remove particulate matter. The clear supernatant was then frozen at −70° C. until IL-8 analysis could be performed.

[0072] Quantitative IL-8 analysis was performed using a commercially available ELISA kit (ELISA Max Deluxe set for human IL-8, supplied by Enco, catalog number B287549).

[0073] result: The results of this study are shown in Figure 3, which shows that treatment with DPM alone causes an approximately two-fold increase in IL-8 production compared to vehicle-only control (negative control). The dexamethasone positive control does not cause a significant change in the basal level of IL-8 production of unstimulated cells. However, it can be seen that the composition of the present invention ("Test") caused a significant and statistically significant (p<0.05) inhibition of DPM-stimulated IL-8 production when compared to the DPM-only group.

[0074] These results, obtained using a cultured human keratinocyte model, indicate that phytoene can reduce at least part of the inflammatory response in skin cells exposed to air pollutants such as DPM.

[0075] [Example 2] Inhibition of DPM-induced reactive oxygen species (ROS) production in cultured cells by the compositions of the present invention Introduction: Airborne particulate matter from fossil fuel combustion (e.g., DPM) can induce oxidative stress via the production of reactive oxygen species (ROS), which are strongly correlated with airway inflammation and asthma. ROS entities include superoxide radicals (O 2 ·- ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH · ) and singlet oxygen ( 1 O 2 This study examined the effect of the compositions of the present invention on the production of NOS in response to exposure of cultured keratinocytes to DPM.

[0076] method: Spontaneously transformed adult human keratinocytes of the HaCaT strain were grown, maintained, plated, and exposed to control and test substances as described above in Example 1. The control substances used in this study were as follows: - Vehicle control ("Control", FIG. 4) -DPM -N-acetylcysteine ​​(NAC; positive control)

[0077] The test material used was a dilution of the composition of the present invention (ie, containing non-GMO, solvent-free phytoene and jojoba oil) resulting in a final phytoene concentration of 56 ppm.

[0078] Quantitative analysis of ROS content in treated and control cell supernatants was performed using a commercially available kit that uses a fluorogenic dye (DCFDA) to measure hydroxyl, peroxyl and other intracellular reactive oxygen species ROS activity.

[0079] result: FIG. 4 shows that DPM treatment increased ROS levels approximately 40-fold over the unstimulated control, but the composition of the present invention ("Test") inhibited this increase in ROS concentration to a highly significant extent, and was slightly less effective than the NAC positive control.

[0080] [Example 3] Inhibition of UVB-induced IL-8 production in cultured cells by compositions of the present invention Introduction: In this study, the effect of the compositions of the present invention on the production of IL-8 in UVB-exposed cultured keratinocytes was examined.

[0081] method: Spontaneously transformed adult human keratinocytes of the HaCaT strain were grown, maintained, plated, and treated with control and test substances as described above in Example 1. To induce an inflammatory response in the cultured cells, the cultured cells were irradiated with a UVB lamp. The control substances used in this study were as follows: - Vehicle control ("Control", FIG. 5) -UVB exposure only ("untreated") -4-aminobenzoic acid ("PABA"; positive control)

[0082] The test material used was a dilution of the composition of the present invention (ie, containing non-GMO, solvent-free phytoene and jojoba oil) resulting in a final phytoene concentration of 56 ppm.

[0083] IL-8 concentrations in cultured cell supernatants were measured as described in Example 1 above.

[0084] result: It can be seen from Figure 5 that UVB exposure caused a significant increase in IL-8 production compared to the vehicle control. It is further noted that the test compositions of the present invention caused a significant decrease in this UVB-stimulated IL-8 production to a greater extent than the inhibition caused by the positive control (the commercial sunscreen ingredient PABA).

[0085] [Example 4] Inhibition of DPM-induced inflammatory responses in a 3D reconstructed skin model Introduction: To further explore the in vitro results shown in Example 1 (herein above), a reconstructed human epidermis (RHE) model was used as a test platform. The reconstructed skin used in the experiments described in this Example hereinafter, commercially available under the trade name "EpiDerm", is a highly differentiated 3D tissue model consisting of normal human-derived epidermal keratinocytes cultured on cell culture inserts. These inserts allow the culture of the reconstructed tissue at an air-liquid interface, thereby readily allowing the evaluation of topically administered agents on the skin.

[0086] method: EpiDerm reconstructed skin system was obtained from the supplier MatTek (catalog number EPI-200) and the tissue was handled according to the manufacturer's instructions. Briefly, upon delivery, the tissue was removed from its agar medium, washed, and then incubated at 4°C for 24 hours at 5% CO. 2 The samples were then left overnight at 37°C in the environment and then harvested.

[0087] The tissues were then treated with topically applied DPM (100 μg / ml in PBS, 10 μl / sample) in the presence or absence of phytoene oleoresin diluted in jojoba wax at concentrations of 0.1, 1 and 2.5% in a final volume of 30 μl. The following control groups were also set up: naive tissue (untreated control), vehicle (jojoba wax), DPM stimulated tissue and N-acetylcysteine ​​(NAC; positive control). After addition of the test substances and controls, the reconstructed skin cultures were incubated at 4°C for 24 hours at 5% CO 2 The mixture was incubated at 37C in the environment for a further 48 hours.

[0088] At the end of the treatment period, media was collected from tissue control implants and centrifuged at 250×g for 5 min to remove particulate matter, and the clear supernatant was then frozen at −70° C. prior to cytokine analysis.

[0089] IL-1α and IL-6 levels in the thawed medium were determined using commercially available ELISA kits (ELISA Max Deluxe Set human IL-1α Cat. No. B265424; ELISA Max Deluxe Set human IL-6 Cat. No. B2852558; both supplied by Enco) according to the manufacturer's instructions.

[0090] result: The results of this study are shown in Figures 6 and 7. The bar graph shown in Figure 6 indicates that some, but not all, dilutions of phytoene-containing oleoresin caused a decrease in IL-1α production by skin cells in the EpiDerm artificial skin model. It should be noted that a 1% concentration of this test material caused a quantitatively similar decrease in IL-1α production as that caused by the anti-inflammatory positive control (N-acetylcysteine; NAC). Furthermore, the decrease in IL-1α levels caused by this concentration of test material was statistically significant (p<0.05) when compared to both DPM-only and vehicle-only samples.

[0091] The results of IL-6 production are shown in Figure 7. From this graph it can be seen that all concentrations of phytoene containing oleoresin dilutions tested caused a significant decrease (p<0.05) in IL-6 production when compared to the DPM only sample.

[0092] These data demonstrate that exposure of in vivo skin model to DPM causes increased production of inflammatory mediators, and treatment with the phytoene-containing composition of the present invention can inhibit this aspect of inflammatory response.Therefore, these results strongly suggest that the composition of the present invention may be valuable in preventing, alleviating or treating skin damage caused by skin exposure to environmental pollutants and irritants, such as DPM.

[0093] [Example 5] Clinical Trials of the Effect of the Compositions of the Present Invention on Various Aspects of Skin Quality Introduction: The following skin quality characteristics were examined in a group of healthy volunteers before and after topical treatment with the compositions of the present application: -Skin tones -Skin redness - Skin firmness -Skin elasticity -Smoothness of the skin -Wrinkle condition -Skin barrier function -Sedative effect

[0094] Methods and Results: A group of 33 female volunteers (average age 50 years) were randomly selected for this study. The facial skin of the volunteers was treated with twice daily applications of the composition of the present invention (containing 1.5% (w / w) phytoene in jojoba oil) for up to 28 days.

[0095] Skin tone was assessed using a Mexameter® MX 18 device (supplied by Courage+Khazaka electronic GmbH, Germany) to assess the melanin levels of the subjects. As can be seen from the results shown in Figure 8, application of the composition of the present invention for 14 days reduced the melanin index by an average of 6%, while 28 days of treatment reduced it by an average of 10%, indicating a significant increase in skin brightness as a result of this treatment.

[0096] Skin redness was assessed using a Mexameter® MX 18 device. Figure 9 shows a significant reduction in erythema at both 14 and 28 days after treatment when compared to the initial measurements before treatment.

[0097] Skin firmness and skin elasticity were evaluated using a suction-based device, Cutometer® MPA 580 (sold by Courage+Khazaka electronic GmbH, Germany). Figure 10 shows an increase in skin firmness (i.e., a decrease in skin deformability depth) at both 14 and 28 days. Similarly, as shown in Figure 11, there was a significant increase in skin elasticity at these two time points.

[0098] Skin smoothness was assessed using the Primos 3D Lite system (distributed by Canfield Scientific, New Jersey, USA). As shown in Figure 12, a significant increase in smoothness was seen at both the 14 and 28 day time points.

[0099] Wrinkle analysis was performed using the Primos 3D Lite system. Figure 13 shows that all of the various parameters measured (i.e., wrinkle length, depth, number, volume and area) improved after 28 days of treatment, with the most significant change seen in wrinkle number (a 20% reduction).

[0100] Skin barrier function Skin hydration was used as a measure of skin barrier function and was assessed using a Tewameter® transepidermal water loss (TEWL) device. As shown in Figure 14, treatment with the composition of the present invention significantly reduced water loss at both 14 and 28 days.

[0101] Skin Soothing The ability of the composition of the present invention to soothe irritated skin was evaluated using the score made by subjects after applying 10% lactic acid solution to the skin surface.As can be seen in Figure 15, treatment with the composition of the present invention for 14 days reduced irritation by more than 20%.This skin soothing effect was even more pronounced after 28 days of treatment, with a 45% reduction in reported irritation.

[0102] [Example 6] Face cream formulations comprising the compositions of the present invention

[0103] [Table 1]

[0104] The phytoene-containing oleoresin of the present invention, prepared as hereinbefore described and summarized in Figure 1, is mixed with a face cream formulation prepared from the ingredients summarized in the table above. The final phytoene concentration of the formulation is 0.1% (w / w).

[0105] [Example 7] Hair cream formulation comprising the composition of the present invention

[0106] [Table 2]

[0107] The phytoene-containing oleoresin of the present invention, prepared as hereinbefore described and summarized in Figure 1, is mixed with a hair cream formulation prepared from the ingredients summarized in the table above. The final phytoene concentration of the formulation is 0.1% (w / w).

[0108] [Example 8] Orally administered softgel capsules containing the phytoene composition of the present invention. Phytoene oleoresin softgel capsules are prepared according to standard procedures known to those skilled in the art, each capsule containing oleoresin prepared according to the present invention such that the final concentration of phytoene in said capsule is in the range of 0.01-10% (w / w).

[0109] [Example 9] Efficacy of phytoene oleoresin compositions in protecting healthy skin from UV-induced photooxidative damage method A double-blind, placebo-controlled, parallel-group clinical biostudy. After a 5-week run-in period during which intake of phytoene-rich foods and antioxidant supplements is restricted, subjects are randomized to receive either a softgel capsule of Formulation 1 or an identical-looking softgel capsule as placebo daily for 12 weeks.

[0110] Number of subjects (planning and analysis) 60 subjects to complete the study and be analyzed.

[0111] Diagnosis and main inclusion criteria Fitzpatrick skin type I~IV, BMI ≤30kg / m 2 and healthy men and women aged 20-65 with healthy eating habits.

[0112] Test products, doses and modes of administration One softgel capsule (as described in Example 8 above) was administered orally daily.

[0113] Reference therapy, dosage and mode of administration A placebo was administered orally daily.

[0114] Treatment period 12 weeks.

[0115] Protection against erythema measured by Chromometry Colorimetry is a method of measuring skin color using a colorimeter. Skin color is measured using the L*a*b* scale, where L* reflects the brightness of the color on a grayscale, a* reflects the contrast between red and green, and b* reflects the contrast between blue and yellow.

[0116] Subjects are tested for protection from UV radiation-induced erythema: an area of ​​each subject's skin is exposed to UV radiation and the occurrence of erythema is measured 24 hours after exposure.

[0117] For this analysis, Δa* was defined as the difference between the level of erythema development 24 hours after UV irradiation before feeding and the level of erythema development 24 hours after UV irradiation after feeding. A statistically significant change in erythema formation between the Formulation 1 group and the placebo group is expected.

[0118] Cytokine levels Mean IL-1α, IL-6, IL-10 and TNF-α levels are assessed by polymerase chain reaction (PCR). Subjects participating in the clinical trial are assayed for IL-1α, IL-6, IL-10 and TNF-α levels. Samples are obtained after UVB irradiation prior to randomization and after UVB irradiation following 12 weeks of treatment with Formulation 1 or placebo.

[0119] Expected Results: Analysis of cytokine mRNA from skin biopsies will be statistically significantly different between treatment groups.

[0120] [Example 10] The compositions of the present invention reduce the secretion of degradative enzymes from stimulated neutrophils. To study the effect of the composition of the present invention on the release of enzymes that can cause damage to collagen, we first determine the kinetics of release of such enzymes from activated neutrophils. Neutrophils are activated by 100 ng / ml TNFα or 100 ng / ml IL8, which are released and activated by skin cells during exposure to UV light. Furthermore, for comparison, neutrophils are treated with 5×10 7 Activated by fMLP.

[0121] To study the effect of the phytoene-containing compositions of the present invention on the release of MMP-9 or MPO, neutrophils should be activated with TNFα or IL8 for 4 hours and with fMLP for 30 minutes. The compositions are added to the neutrophils for 10 minutes at 37° C. prior to activation. Inhibition of cytokine release from stimulated neutrophils is measured and a dose-dependent inhibition is expected.

[0122] [Example 11] Phytoene-Containing Compositions of the Invention Reduce Collagen Loss in Fibroblast-Neutrophil Co-Cultures To study the effect of the composition of the present invention on collagen-3 damage induced by activated neutrophils, we use optimal conditions for cultured fibroblasts and neutrophils. 5 Fibroblasts are plated for 24 hours to obtain confluent cultures, and cell viability is measured for each treatment.

[0123] Neutrophils are incubated with the compositions of the invention for 10 minutes at 37° C. before being added to the cultures for 24 hours. Quantitation of collagen-3 levels in the cultures should be determined by densitometry.

[0124] The presence of the composition of the invention in the co-culture is expected to have a dose-dependent increase in secreted pro-collagen-3.

[0125] Treatment of NHDF cells with 50 mM H2O2 reduces collagen lal secretion by approximately 10-fold. The compositions of the present invention are expected to completely reverse the reduction in collagen secretion, suggesting a role for the compositions in increasing skin collagen levels and reducing skin aging in vivo.

Claims

1. A solvent-free, non-GMO oleoresin obtained from Blakeslea trispora containing phytoene at a concentration of 10-40% (w / w).

2. An oleoresin as described in claim 1, having a phytoene concentration of 20 to 40% (w / w).

3. An oleoresin as described in claim 1, having a phytoene concentration of 20 to 30% (w / w).

4. The oleoresin of claim 1 further comprising one or more phytosterols.

5. The oleoresin of claim 4, wherein one or more phytosterols include ergosterol.

6. The oleoresin described in claim 5, wherein the concentration of ergosterol is in the range of 0.5 to 1% (w / w).

7. The oleoresin of claim 6, wherein the concentration of ergosterol is about 0.6% (w / w).

8. A composition comprising the oleoresin of claim 1 and Simmondsia sinensis (jojoba) seed oil.

9. The composition of claim 8, wherein the concentration of phytoene is in the range of 0.01% to 10% (w / w).

10. The composition of claim 9, wherein the concentration of phytoene is in the range of 1 to 2% (w / w).

11. A dosage form comprising the composition of claim 8 and one or more pharmaceutically acceptable, nutraceutical acceptable, or cosmetically acceptable excipients.

12. The dosage form of claim 11, suitable for topical administration.

13. The dosage form of claim 12, selected from the group consisting of a cream, lotion, ointment, gel, foam, salve, suspension, oil or solution.

14. The dosage form of claim 12, wherein the concentration of phytoene in the dosage form is in the range of 0.01 to 0.5% (w / w).

15. The dosage form of claim 11, suitable for oral administration.

16. The dosage form of claim 15, selected from the group consisting of a tablet, caplet, capsule, syrup, oil, soft gel capsule, troche, chewable tablet, suspension or solution.

17. A composition comprising phytoene for use in protecting the skin of a subject exposed to air pollutants.

18. A composition comprising phytoene for use in protecting the skin of a subject exposed to UV radiation.

19. A composition comprising phytoene for use in improving one or more aspects of skin quality, the composition being the composition described in claim 6.

20. The composition described in claim 19, wherein the improvement in one or more aspects of skin quality is selected from the group consisting of evening and averaging skin tone, reducing wrinkles, improving elasticity, protecting the natural skin barrier function, and improving the appearance of the skin.

21. A solvent-free, non-GMO oleoresin obtained from Blakeslea trispora containing phytoene at a concentration of 20-30% (w / w), comprising: a) culturing a phyt 1+ / phyt 1- strain of the fungal species Blakeslea trispora; b) obtaining biomass from the cultured Blakeslea trispora; c) subjecting the biomass to supercritical carbon dioxide fluid extraction carried out at an operating pressure of between 300 and 450 bar and an operating temperature of about 50°C to 65°C, thereby obtaining an oleoresin comprising phytoene at a concentration of 20-30% (w / w) and ergosterol; 1. A solvent-free non-GMO oleoresin obtained by a process comprising:

22. An oleoresin obtained according to claim 21, wherein the concentration of ergosterol is about 0.6% (w / w).

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