Compositions and methods for inhibiting collagen loss - Patents.com

Abstract

The present invention is directed to compositions comprising phytoene, phytofluene, and zeta-carotene, and methods of using same, such as to reduce collagen loss.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 913,220, filed October 10, 2019, and entitled "COMPOSITIONS AND METHODS FOR INHIBITING COLLAGEN LOSS," the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates generally to the field of carotenoids and methods of using carotenoids, such as to treat skin-related conditions involving collagen loss. [Background technology]

[0003] The decline in the biological structure and function of skin with aging has received significant attention, primarily due to the increasing life expectancy of the population. Fibroblasts are the primary components of the dermis and are known to produce collagen and elastic fibers. A distinctive feature of photographically aged skin is photoelastosis, likely the end product of extracellular matrix (ECM) degradation. The ECM, which supports the cells of skin tissue, includes collagen, elastic fibers, and fibrillin.

[0004] Neutrophils have been reported to infiltrate skin after exposure to erythematous doses of ultraviolet B (UVB), which is solar simulating radiation (SSR) and a part of natural sunlight. Exposure of human skin to a certain threshold of UV, infrared radiation, or heat triggers neutrophil influx. These neutrophils are packed with potent proteolytic enzymes capable of degrading collagen and elastic fibers. Neutrophil-derived proteolytic enzymes are involved in the ECM damage observed in several non-dermatological conditions. Furthermore, it has been suggested that neutrophils, rather than keratinocytes and fibroblasts, may be key players in photoaging. Following exposure of white skin to erythematous doses of SSR, infiltrating neutrophils, rather than keratinocytes or fibroblasts, were reported to be the primary source of proteolytic enzymes, particularly matrix metalloproteinases (MMPs) and elastase. Oxygen-derived metabolites may induce the activation of proteolytic enzymes and / or prevent these enzymes from being inactivated by antiproteinases, thus inducing ECM damage and ultimately leading to solar elastosis.

[0005] There remains a great need for compositions and methods to prevent the release of neutrophil-derived proteolytic enzymes and thus protect against collagen damage. Summary of the Invention

[0006] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools, and methods that are meant to be exemplary and illustrative, not limiting in scope.

[0007] According to a first aspect, there is provided a composition comprising phytoene in an amount of 55-65% (w / w) of the total carotenoids in the composition, phytofluene in an amount of 10-20% (w / w) of the total carotenoids in the composition, zeta-carotene in an amount of 15-25% (w / w) of the total carotenoids in the composition, and an acceptable carrier.

[0008] According to another aspect, provided is a method for preventing or treating collagen loss in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition disclosed herein, thereby preventing or treating collagen loss in the subject.

[0009] According to another aspect, provided is a method for reducing the amount, activity, or both of any one of matrix metalloproteinases (MMPs), myeloperoxidase (MPO), superoxide (SO), elastase, nitric oxide (NO), and any combination thereof in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition disclosed herein.

[0010] In some embodiments, the weight ratio of the combined phytoene and phytofluene to zeta-carotene ranges from 15:1 (w / w) to 2:1 (w / w).

[0011] In some embodiments, the composition further comprises an additional carotenoid selected from the group consisting of lycopene, beta-carotene, gamma-carotene, and any combination thereof.

[0012] In some embodiments, the lycopene comprises lycopene in an amount less than 5% (w / w) of the total carotenoids in the composition, beta-carotene in an amount less than 5% (w / w) of the total carotenoids in the composition, gamma-carotene in an amount between 0.2 and 1.5% (w / w) of the total carotenoids in the composition, or a combination thereof.

[0013] In some embodiments, the composition comprises 10-15% (w / w) of the total carotenoids in the composition.

[0014] In some embodiments, the composition further comprises tocopherol.

[0015] In some embodiments, the tocopherol is in an amount of 10-30% (w / w) of the total carotenoids in the composition.

[0016] In some embodiments, the composition further comprises a phytosterol.

[0017] In some embodiments, the phytosterols are in an amount of 5-15% (w / w) of the total carotenoids in the composition.

[0018] In some embodiments, the composition is for use in reducing collagen loss, increasing collagen levels, or both.

[0019] In some embodiments, the prevention or treatment is a 50-90% reduction in collagen loss.

[0020] In some embodiments, the MMP is selected from MMP-9, MMP-8, MMP-1, or any combination thereof.

[0021] In some embodiments, the subject is suffering from a collagen loss-associated disease.

[0022] In some embodiments, the collagen loss-related disease is selected from the group consisting of an age-related disease, a skin disease, and an inflammatory disease.

[0023] In some embodiments, the skin disease comprises skin damage induced by any of radiation, oxidative stress, DNA damage, telomere shortening, inflammation, tobacco use, and any combination thereof.

[0024] In some embodiments, the radiation comprises UV radiation, visible light radiation, infrared radiation, or any combination thereof.

[0025] In some embodiments, the UV radiation is UVA, UVB, UVC, or any combination thereof.

[0026] In some embodiments, the subject has an increased amount, improved activity, or both, of any one of MMP, MPO, SO, elastase, NO, and any combination thereof in the skin, systemically, or both, compared to a control.

[0027] In some embodiments, administering includes administering orally, administering topically, or both.

[0028] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. Furthermore, the materials, methods, and examples are merely illustrative and are not intended to be necessarily limiting.

[0029] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0030] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the figures and by study of the following detailed descriptions. [Brief explanation of the drawings]

[0031] [Figures 1A-1C]Column graph showing the kinetics of matrix metalloproteinase 9 (MMP-9) secretion from human neutrophils stimulated by tumor necrosis factor alpha (TNFα, 1A), interleukin 8 (IL8, 1B), and formyl-methionine-leucine-phenylalanine (fMLP, 1C) agonists. Values ​​represent the mean ± standard error of the mean (SEM) of five independent experiments. [Figures 2A-2C] Column graphs showing the kinetics of myeloperoxidase (MPO) secretion from human neutrophils stimulated with TNFα (2A), IL8 (2B), and fMLP (2C) agonists. Values ​​represent the mean ± SEM of five independent experiments. [Figure 3A-3C] 1 is a column graph showing the inhibitory effect of Lumenato (Golden Tomato Supercritical Extract or GTE) on MMP-9 secretion. Human neutrophils were stimulated with TNFα (3A), IL8 (3B), and fMLP (3C) agonists, and a dose-dependent inhibition of MMP-9 secretion was observed in the presence of GTE. Values ​​represent the mean ± SEM of three independent experiments. [Figures 4A-4C] 1 is a column graph showing the inhibitory effect of GTE on MPO secretion. Human neutrophils were stimulated with TNFα (4A), IL8 (4B), and fMLP (4C) agonists, and a dose-dependent inhibition of MPO secretion was observed in the presence of GTE. Values ​​represent the mean ± SEM of three independent experiments. [Figures 5A-5C] Figure 1 shows a column graph depicting the inhibitory effect of GTE on superoxide secretion. Human neutrophils were stimulated with TNFα (5A), IL8 (5B), and fMLP (5C) agonists, and a dose-dependent inhibition of superoxide secretion was observed in the presence of GTE. Values ​​represent the mean ± SEM of three independent experiments. [Figures 6A-6S]Immunofluorescence micrographs and column graphs show that the addition of GTE dose-dependently prevented neutrophil-induced collagen-3 damage in normal human dermal fibroblasts (NHDFs). Neutrophils were stimulated with 100 ng / ml TNFα and incubated in the presence of increasing concentrations of GTE: 6.5 μg / ml (6C, 6J), 13 μg / ml (6D, 6K), 26 μg / ml (6E, 6L), 52 μg / ml (6F, 6M), and 104 μg / ml (6G, 6N). Controls: negative (no TNFα; 6A and 6H), positive (no GTE; 6B and 6I). (6A-6G) are representative immunofluorescent staining of collagen-3 at 40x magnification. (6H-6N) are representative immunofluorescent staining of collagen-3 at 200x magnification. (6A-6N) Ten fields of each treatment were scanned in each experiment. (6O) Column graph showing the prevention of collagen damage by GTE, analyzed by densitometry of immunofluorescent staining for collagen-3. Values ​​represent the mean of five separate experiments. (6P) and (6Q) Column graphs showing the effect of increasing concentrations of LycoDerm and LycoMato, respectively, on preventing collagen loss. High concentrations of either LycoDerm or LycoMato were toxic to cells. In contrast, no cytotoxic effect was observed with GTE at 104 μg / ml (the highest concentration used). (6R) Graph showing densitometry of immunofluorescent staining for collagen-3 (6A-6N). Shown are the means ± SEM of three separate experiments. (6S) Graph showing the amount of procollagen-3 secreted into the supernatant of cocultures. Shown are the means ± SEM of three separate experiments, each performed in triplicate. The horizontal dashed line indicates the effect of Lumenato without the effect of neutrophils. Lum - Lumenato (μg / ml), Fib - fibroblasts, Neu - neutrophils. [Figure 7A-7C]7A and 7B are column graphs showing that GTE induced inhibition of MMP-9 release from neutrophils cocultured with NHDFs (described in Figure 6). MMP-9 was quantified in the coculture supernatant (7A). Values ​​represent the average of five separate experiments. (7B) and (7C) are column graphs showing the effect of increasing concentrations of LycoDerm and LycoMato, respectively, on MMP-9 levels. High concentrations of either LycoDerm or LycoMato were toxic to the cells. In contrast, no cytotoxic effect was observed at 104 μg / ml of GTE (the highest concentration used). [Figures 8A-8C] 8A is a vertical graph showing that GTE induced inhibition of MPO release from neutrophils cocultured with NHDFs (described in Figure 6). Quantification of MPO was determined based on its activity in the coculture supernatant (8A). Values ​​represent the average of five separate experiments. (8B) and (8C) are vertical bar graphs showing the effect of increasing concentrations of LycoDerm and LycoMato, respectively, on MPO activity. High concentrations of either LycoDerm or LycoMato were toxic to the cells. In contrast, no cytotoxic effect was observed at 104 μg / ml of GTE (the highest concentration used). [Figure 9] 1 is a column graph showing that GTE induced inhibition of superoxide release from neutrophils co-cultured with NHDFs (described in FIG. 6). Superoxide levels were detected using Amplex red. Values ​​represent the average of five different experiments. [Figure 10] 1 is a column graph showing that GTE induced inhibition of MMP-8 (collagenase) release from neutrophils co-cultured with NHDFs (as described in FIG. 6). MMP-8 was quantified in the supernatant of the co-culture. Values ​​represent the average of five different experiments. [Figure 11] 1 is a column graph showing that GTE induced inhibition of elastase release from neutrophils co-cultured with NHDFs (as described in FIG. 6). Quantification of elastase was determined based on its activity in the supernatant of the co-culture. Values ​​represent the average of five different experiments. [Figures 12A-12B]12A and 12B are column graphs showing the effect of pulp or GTE on nitric oxide (NO) release from macrophages stimulated with lipopolysaccharide (LPS). Pulp was dissolved in medium and filtered. GTE was dissolved in DMSO. At all concentrations tested, there was no effect on cell viability, as determined by MTT reduction. (12A) shows the effect of filtered pulp, and (12B) shows the effect of GTE on NO production. Results represent the average of two experiments performed. [Figures 13A-13C] These are column graphs showing that the addition of tocopherols and phytosterols to stimulated neutrophils at the ratios present in GTE resulted in significant synergistic inhibition of MPO release and activity from TNFα-activated neutrophils. Addition of tocopherols (13A) or phytosterols (13B) alone had no effect on MPO release or activity from TNFα-activated neutrophils. In contrast, 225 μg / ml tocopherol and 62.5 μg / ml phytosterol synergized to approximately 20% inhibition of MPO activity, whereas 10-fold higher or lower ratios did not (13C). [Figure 14] Figure 1 is a column graph showing that the addition of zeta-carotene together with either phytoene, tocopherol, or phytosterol to stimulated neutrophils at the ratios present in GTE resulted in a significant synergistic inhibition of MPO activity. Addition of zeta-carotene, tocopherol, phytoene, or phytosterol alone had a maximum of 5% inhibition of MPO activity or in most cases had no effect. Addition of zeta-carotene with either tocopherol, phytoene, or phytosterol at the ratios present in GTE induced a synergistic inhibition of MPO activity or TNFα release from activated neutrophils. [Figures 15A-15B]15A and 15B are graphs showing that GTE (e.g., Lumenato) capsules increased the bioavailability of carotenoids. Phytoene, phytofluene, and zeta-carotene, the primary carotenoids in golden tomato extract, have been shown to be well absorbed. Zeta-carotene was very well absorbed, as reflected by the rapid increase in plasma concentrations. (15A) and (15B) are graphs showing the bioavailability of phytoene and phytofluene alone. [Figures 16A-16B] 16A and 16B are graphs showing that H2O2 induces cell death and increases MMP1 secretion in a dose-dependent manner. Addition of Lumenato at the indicated concentrations before H2O2 increased cell viability (16A) and decreased MMP1 secretion (16B). [Figure 17] This graph shows that GTE (e.g., Lumenato) dose-dependently enhanced activation of ARE / Nrf2 transcriptional activity. Lycomato is shown as a reference. The concentrations shown are those of lycopene (for Lycomato) and phytoene (for Lumenato). [Figure 18] Includes a column graph showing the levels of procollagen-3 in the co-culture supernatant. Shown is the mean ± SEM of four different experiments, each in triplicate. Lumenato, ranging from 6.5 to 104 μg / ml, was added to seeded fibroblasts at two different concentrations. [Figure 19] 1 includes a graph showing that Lumenato corrects H2O2-induced decrease in collagen 1a1 secretion. DETAILED DESCRIPTION OF THE INVENTION

[0032] In some embodiments, the present invention is directed to compositions comprising multiple carotenoids. The present invention is based, in part, on the surprising discovery that tomato extracts containing high amounts of phytoene, phytofluene, and zeta-carotene prevent collagen loss, inhibit matrix metalloproteinase 9 (MMP-9) and myeloperoxidase (MPO) activity at higher levels than other tomato extracts, and have reduced cytotoxicity compared to the same tomato extracts.

[0033] In some embodiments, the composition comprises phytoene, phytofluene, and zeta-carotene. In one embodiment, the composition comprises phytoene, phytofluene, zeta-carotene, and an additional carotenoid. In one embodiment, the composition comprises phytoene, phytofluene, zeta-carotene, lycopene, beta-carotene, gamma-carotene, tocopherol, and phytosterol.

[0034] In some embodiments, the carotenoid is a natural carotenoid extracted, isolated, or purified from a fruit, vegetable, or plant (including plant parts). In another embodiment, the carotenoid is a carotenoid extracted from a tomato plant. In another embodiment, the carotenoid is a carotenoid extracted from a tomato fruit. In another embodiment, the tomato carotenoid is a carotenoid-enriched tomato extract. In another embodiment, the tomato carotenoid is an all-natural carotenoid-rich tomato extract. In another embodiment, the tomato carotenoid is a tomato carotenoid complex. In another embodiment, the tomato carotenoid complex comprises a phytonutrient complex including multiple carotenoids (such as phytoene, phytofluene, zeta-carotene, beta-carotene), tocopherol, and phytosterol. In some embodiments, the carotenoid is a synthetic carotenoid.

[0035] In some embodiments, the present invention provides a tomato extract obtained by an innovative extraction protocol. This particular extract, containing phytoene, phytofluene, and zeta-carotene (in amounts specified below), exhibits reduced cytotoxicity. In some embodiments, the reduced cytotoxicity is compared to other tomato extracts. In some embodiments, the reduced toxicity allows for the compositions of the present invention to be provided to a subject in need thereof in larger doses without reducing the subject's survival, well-being, or both. In some embodiments, administering the compositions of the present invention to a subject in need thereof can enhance the efficacy of treatment by providing active ingredients such as phytoene, phytofluene, and zeta-carotene in higher amounts that enhance the therapeutic effect, without reducing the subject's survival rate, health, or both due to high cytotoxicity.

[0036] In some embodiments, the compositions of the present invention provide high amounts of carotenoids with reduced toxicity compared to extracts from other plants, fruits, or vegetables, such as tomatoes. In some embodiments, the compositions of the present invention provide improved therapeutic efficacy with reduced toxicity compared to extracts from other plants, fruits, or vegetables, such as tomatoes.

[0037] In some embodiments, the compositions of the present invention comprise natural carotenoids, synthetic carotenoids, or any combination thereof.

[0038] In some embodiments, the composition comprises phytoene in an amount of 10-40% (w / w), 15-35% (w / w), 20-45% (w / w), 25-35% (w / w), 20-30% (w / w), or 30-50% (w / w) of the total carotenoids of the composition, with each possibility representing a separate embodiment of the present invention.

[0039] In some embodiments, the composition comprises phytofluene in an amount of 1-10% (w / w), 3-12% (w / w), 4-14% (w / w), 5-10% (w / w), 8-15% (w / w), or 2-9% (w / w) of the total carotenoids of the composition, with each possibility representing a separate embodiment of the present invention.

[0040] In some embodiments, the composition comprises zeta-carotene in an amount of 4-20% (w / w), 6-18% (w / w), 5-15% (w / w), 6-12% (w / w), 9-17% (w / w), or 10-17% (w / w) of the total carotenoids in the composition, with each possibility representing a separate embodiment of the present invention.

[0041] In some embodiments, the weight ratio of the combined phytoene and phytofluene to zeta-carotene ranges from 20:1 (w / w) to 3:1 (w / w), 15:1 (w / w) to 3:1 (w / w), 20:1 (w / w) to 6:1 (w / w), 15:1 (w / w) to 2:1 (w / w), 17:1 (w / w) to 4:1 (w / w), 16:1 (w / w) to 7:1 (w / w), 13:1 (w / w) to 8:1 (w / w), 10:1 (w / w) to 3:1 (w / w), or 15:1 (w / w) to 10:1 (w / w). Each possibility represents a separate embodiment of the present invention.

[0042] In some embodiments, the composition further comprises an additional carotenoid. As used herein, "additional carotenoid" refers to any carotenoid or metabolite thereof other than or different from phytoene, phytofluene, and zeta-carotene.

[0043] In some embodiments, the additional carotenoid is selected from lycopene, beta-carotene, gamma-carotene, and any combination thereof.

[0044] In some embodiments, the composition comprises lycopene in an amount of less than 10% (w / w), less than 7% (w / w), less than 5% (w / w), 3% (w / w), less than 2% (w / w), or less than 1% (w / w) of the total carotenoids in the composition, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, the composition comprises lycopene in an amount of 1-3% (w / w), 1-5% (w / w), 2-6% (w / w), 0.5-4.5% (w / w), 0.1-3% (w / w), 0.6-4.8% (w / w), or 2.5-4% (w / w) of the total carotenoids in the composition. Each possibility represents a separate embodiment of the present invention.

[0045] In some embodiments, the composition comprises beta-carotene in an amount of less than 10% (w / w), less than 7% (w / w), less than 5% (w / w), 3% (w / w), less than 2% (w / w), or less than 1% (w / w) of the total carotenoids in the composition, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, the composition comprises beta-carotene in an amount of 1-3% (w / w), 1-5% (w / w), 2-6% (w / w), 0.5-4.5% (w / w), 0.1-3% (w / w), 0.6-4.8% (w / w), or 2.5-4% (w / w) of the total carotenoids in the composition. Each possibility represents a separate embodiment of the present invention.

[0046] In some embodiments, the composition comprises gamma macarotene in an amount of at least 0.15% (w / w), at least 0.18% (w / w), at least 0.2% (w / w), at least 0.25% (w / w), at least 0.35% (w / w), at least 0.5% (w / w), at least 0.75% (w / w), at least 0.9% (w / w), at least 1% (w / w), at least 1.2% (w / w), at least 1.35% (w / w), or at least 1.7% (w / w) of the total carotenoids in the composition, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention. In some embodiments, the composition comprises gamma carotene in an amount of 0.15-3% (w / w), 0.2-2% (w / w), 0.2-1.5% (w / w), 0.5-3% (w / w), 0.7-1.6% (w / w), 0.4-2.8% (w / w), or 1.2-3.2% (w / w) of the total carotenoids in the composition, with each possibility representing a separate embodiment of the present invention.

[0047] In some embodiments, the composition comprises lycopene in an amount less than 5% (w / w) of the total carotenoids in the composition, beta-carotene in an amount less than 5% (w / w) of the total carotenoids in the composition, gamma-carotene in an amount between 0.2 and 1.5% (w / w) of the total carotenoids in the composition, or a combination thereof.

[0048] In some embodiments, the composition includes additional carotenoid in an amount of 0.1-3% (w / w), 0.2-3.5% (w / w), 0.5-2.5% (w / w), 0.15-1.75% (w / w), 0.35-2.75% (w / w), 0.8-4% (w / w), 1-5% (w / w), or 1.5-4.75% (w / w). Each possibility represents a separate embodiment of the present invention.

[0049] In some embodiments, lycopene in an amount less than 5% (w / w) of the total carotenoids in the composition.

[0050] In some embodiments, the composition comprises 5-25% (w / w), 10-15% (w / w), 12-35% (w / w), 3-17% (w / w), 2-20% (w / w), or 1-30% (w / w) of total carotenoids of the composition, with each possibility representing a separate embodiment of the present invention.

[0051] In some embodiments, the composition further comprises a tocopherol (e.g., vitamin E). In some embodiments, the composition comprises tocopherol in an amount of 1-30% (w / w), 3-35% (w / w), 5-25% (w / w), 2-20% (w / w), 4-41% (w / w), 8-32% (w / w), or 13-39% (w / w) of the composition. Each possibility represents a separate embodiment of the present invention.

[0052] In some embodiments, the weight ratio of combined phytoene and phytofluene to tocopherol ranges from 20:1 (w / w) to 3:1 (w / w), 15:1 (w / w) to 3:1 (w / w), 20:1 (w / w) to 6:1 (w / w), 17:1 (w / w) to 4:1 (w / w), 16:1 (w / w) to 7:1 (w / w), 13:1 (w / w) to 8:1 (w / w), 10:1 (w / w) to 3:1 (w / w), or 15:1 (w / w) to 10:1 (w / w), with each possibility representing a separate embodiment of the present invention.

[0053] In some embodiments, the weight ratio of zeta-carotene to tocopherol ranges from 3:1 (w / w) to 1:3 (w / w), 3:1 (w / w) to 1:2 (w / w), 3:1 (w / w) to 1:1 (w / w), 2:1 (w / w) to 1:1 (w / w), 2:1 (w / w) to 1:2 (w / w), 2:1 (w / w) to 1:3 (w / w), 1:1 (w / w) to 1:2 (w / w), or 1:1 (w / w) to 1:3 (w / w), with each possibility representing a separate embodiment of the present invention.

[0054] In some embodiments, the composition further comprises a phytosterol.

[0055] In some embodiments, the phytosterol is selected from cholesterol brassicasterol, campesterol, stigmasterol, β-sitosterol, Δ5-avenasterol, Δ7-avenasterol, Δ7-stigmasterol, and any combination thereof.

[0056] In some embodiments, the composition comprises phytosterols in an amount of 1-20% (w / w), 2-19% (w / w), 10-25% (w / w), 5-25% (w / w), 8-16% (w / w), 6-18% (w / w), 3-20% (w / w), 4-17% (w / w), or 5-15% (w / w) of the composition. Each possibility represents a separate embodiment of the present invention. Each possibility represents a separate embodiment of the present invention.

[0057] In some embodiments, the weight ratio of combined phytoene and phytofluene to phytosterols ranges from 20:1 (w / w) to 3:1 (w / w), 15:1 (w / w) to 3:1 (w / w), 20:1 (w / w) to 6:1 (w / w), 17:1 (w / w) to 4:1 (w / w), 16:1 (w / w) to 7:1 (w / w), 13:1 (w / w) to 8:1 (w / w), 10:1 (w / w) to 3:1 (w / w), or 15:1 (w / w) to 10:1 (w / w), with each possibility representing a separate embodiment of the present invention.

[0058] In some embodiments, the weight ratio of zeta-carotene to phytosterols ranges from 6:1 (w / w) to 2:1 (w / w), 5:1 (w / w) to 2:1 (w / w), 4:1 (w / w) to 2:1 (w / w), 3:1 (w / w) to 2:1 (w / w), 6:1 (w / w) to 3:1 (w / w), 5:1 (w / w) to 3:1 (w / w), 4:1 (w / w) to 3:2 (w / w), or 6:1 (w / w) to 3:1 (w / w), with each possibility representing a separate embodiment of the present invention.

[0059] Methods for determining the amount of phytonutrients such as carotenoids are common and will be apparent to those skilled in the art. Non-limiting examples of such methods include, but are not limited to, gas chromatography, liquid chromatography, and mass spectrometry.

[0060] In some embodiments, the composition is an oral composition or a topical composition. In some embodiments, the composition is a pharmaceutical or nutraceutical composition. In some embodiments, the composition comprises a pharmaceutical or nutraceutical acceptable carrier or excipient. In some embodiments, the composition further comprises a cosmeceutical composition. In some embodiments, the composition comprises a functional cosmetically acceptable excipient. In some embodiments, the composition as described herein is an ingestible skin composition.

[0061] 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 a food item such as chocolate, ice cream, or other.

[0062] In some embodiments, the compositions of the present invention are for use in reducing collagen loss, increasing collagen levels, or both.

[0063] The method for measuring collagen level is common and will be clear to those skilled in the art.Non-limiting examples of the method for determining or measuring collagen level include immunofluorescence microscopy, electron microscopy, Western blot and qRT-PCR, some of which are exemplified below.For example, by comparing the collagen level measured under the presence and absence of the composition of the present invention, collagen loss, increased collagen level, or both can be determined.

[0064] In some embodiments, the compositions of the present invention have an activity that increases collagen levels. In some embodiments, the activity that increases collagen levels includes any activity selected from increasing collagen synthesis, increasing collagen weight per tissue weight, increasing the number, length, or both of collagen fibrils, increasing the amount of native or properly folded collagen, decreasing the amount of structurally damaged collagen, and any combination thereof.

[0065] In some embodiments, the collagen comprises procollagen 3, collagen 3, collagen 1a1, or any combination thereof.

[0066] In some embodiments, the collagen is procollagen 3. In some embodiments, the collagen is collagen 3. In some embodiments, the collagen is collagen 1a1. In some embodiments, the collagen is a combination of collagen 3, procollagen 3, and collagen 1a1.

[0067] In one embodiment, the compositions of the present invention may be provided to an individual per se, hi one embodiment, the compositions of the present invention may be provided to an individual as part of a pharmaceutical composition or a dietary supplement composition that includes a pharmaceutically acceptable carrier.

[0068] In one embodiment, a "pharmaceutical composition," "cosmeceutical composition," or "nutraceutical composition" refers to a preparation of a composition described herein that includes other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical, cosmeceutical, or nutraceutical composition is to facilitate administration of the composition to an organism.

[0069] In some embodiments, a process is provided for producing a composition comprising phytoene in an amount of 20-30% (w / w), phytofluene in an amount of 5-10% (w / w), zeta-carotene in an amount of 5-15% (w / w), and an acceptable carrier. In some embodiments, the process comprises extracting golden tomatoes as disclosed herein. In some embodiments, the compositions of the present invention comprise a golden tomato extract produced by the process disclosed herein.

[0070] In one embodiment, a "combined preparation" specifically defines a "kit of parts" in the sense that the previously defined combining components can be administered independently or by using different fixed combinations with different amounts of the combining components, i.e., simultaneously, concurrently, individually, or sequentially. In some embodiments, the components of the kit of parts can then be administered, for example, simultaneously or chronologically staggered, i.e., at different time points, with equal or different time intervals for any part of the kit of parts. In some embodiments, the ratio of the total amounts of the combination partners can be administered in the combined preparation. In one embodiment, the combined preparation can be modified, for example, to address the needs of a patient subpopulation being treated or the needs of a single patient, the different needs of which may be due to the specific disease, disease severity, age, sex, or weight, which can be easily generated by a person skilled in the art.

[0071] In one embodiment, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier," used interchangeably, refer to a carrier or diluent that does not cause significant irritation to a mammal and does not interfere with the biological activity and properties of the administered composition. These phrases include adjuvants.

[0072] In one embodiment, "excipient" refers to an inert substance added to a composition to further facilitate administration of an active ingredient. In one embodiment, excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0073] Techniques for drug formulation and administration are found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference in its entirety.

[0074] In one embodiment, suitable routes of administration include, for example, oral, rectal, transmucosal, nasal, parenteral, or parenteral delivery (including intramuscular, subcutaneous, and intramedullary injection, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection).

[0075] According to some embodiments, there is provided a method for treating or preventing collagen loss in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition of the present invention.

[0076] In some embodiments, the subject is suffering from a collagen loss-associated disease.

[0077] In some embodiments, the subject is a healthy human. In some embodiments, the subject suffers from collagen loss due to aging, exposure to environmental factors such as UV, pollution, and smoke, lack of sleep, stress, or any combination thereof.

[0078] In some embodiments, a subject suffering from a collagen loss-associated disease is characterized by having reduced skin elasticity, reduced epidermal thickness, increased skin vulnerability to injury (such as mechanical injury), increased skin wrinkling, sagging, or both, and any combination thereof.

[0079] Methods for determining either skin elasticity or epidermal thickness are common and apparent to those skilled in the art, and non-limiting examples of such methods include, but are not limited to, optical measurements and histological analysis of skin biopsies by suction or indentation of the skin and subsequent detection of skin displacement.

[0080] According to some embodiments, there is provided a method for inhibiting or reducing the amount, activity, or both of any one of MMP, MPO, superoxide (SO), elastase, nitric oxide (NO), and any combination thereof in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition of the invention.

[0081] In one embodiment, the MMP is MMP-9, MMP-8, MMP-1, or any combination thereof.

[0082] Methods for determining the amount, activity, or both of any one of MMP, MPO, SO, elastase, and NO are common and will be apparent to those skilled in the art. Non-limiting examples of these methods include, but are not limited to, ELISA, immunohistochemistry, oxidation assays, and enzyme assays such as those exemplified below, and / or others.

[0083] In some embodiments, inhibition or reduction is by 50-60%, 50-75%, 50-90%, 50-99%, 65-95%, 70-90%, or 75-99% relative to a control, with each possibility representing a separate embodiment of the present invention.

[0084] As used herein, a control includes the skin of a healthy subject. In some embodiments, the control is a healthy skin sample obtained, isolated, or obtained from the same subject (e.g., a subject suffering from a collagen loss-related disease).

[0085] In some embodiments, administering comprises orally administering, in some embodiments, administering comprises topically administering, in some embodiments, administering comprises a combination of orally administering and topically administering.

[0086] In some embodiments, the collagen loss-related disease is selected from an age-related disease, a skin disease, and an inflammatory disease.

[0087] As used herein, an "age-related disease" refers to a disease or related condition that has an incidence that increases rapidly with age. As used herein, the term "rapidly" means exponentially.

[0088] Non-limiting examples of age-related conditions, which may be collectively referred to as "lesser diseases of aging" (LAA), include, but are not limited to, general muscle weakness, cold intolerance, age-related cognitive decline including mild memory loss, skin wrinkling, delayed healing of skin bruises, wasting (total weight loss), muscle loss, and decreased bone mineral density. Non-limiting examples of age-related diseases include cardiovascular disease, cancer, arthritis, dementia, cataracts, osteoporosis, metabolic diseases including diabetes, increased cholesterol and lipid profiles, high blood pressure, and neurodegenerative diseases including, but not limited to, Alzheimer's disease.

[0089] As used herein, the phrase "skin disease" refers to any pathological condition of the skin or a symptom thereof. In one embodiment, collagen loss is a pathogenic factor leading to the development of a skin disease. In one embodiment, collagen loss is a pathophysiological factor responsible for the development, initiation, progression, or any combination thereof, of a skin disease. In one embodiment, collagen loss is a condition characteristic of a developing or ongoing skin disease.

[0090] In some embodiments, the skin disease is an immune-related skin disease. In one embodiment, the immune-related skin disease is an inflammatory skin disease, an autoinflammatory skin disease, or an autoimmune skin disease.

[0091] As used herein, the phrase "inflammatory disease" refers to any disease involving a multicomponent response of an organism (e.g., cells of the immune system, molecular signal mediators such as cytokines, etc.) to a harmful exogenous entity, such as a bacterium, fungus, virus, protozoan, allergen, etc.

[0092] In some embodiments, the skin disease is characterized or determined based on skin damage. In some embodiments, the skin damage is caused by any of the following: radiation, oxidative stress, DNA damage, telomere shortening, inflammation, tobacco use, and any combination thereof.

[0093] In some embodiments, the radiation includes any wavelength of radiation within the optical spectrum. As used herein, the term "optical spectrum" refers to any wavelength within the optical spectrum. -9 m~10 -3 In some embodiments, the radiation wavelengths within the light spectrum include UV radiation, visible light radiation, infrared radiation, or a combination thereof. In some embodiments, exposure to radiation includes exposure to sunlight.

[0094] As used herein, the term "ultraviolet (UV) radiation" encompasses any wavelength in the UV range. In some embodiments, UV is UV radiation. In some embodiments, UV radiation is UVA radiation, UVB radiation, UVC radiation, or any combination thereof.

[0095] As used herein, the term "infrared" encompasses wavelengths ranging from 700 nm to 1000 nm (with frequencies from 430 THz to 300 GHz).

[0096] In some embodiments, the subject has an increased amount, improved activity, or both, of any one of MMP, MPO, SO, elastase, NO, and any combination thereof in the skin, systemically, or both, compared to a control.

[0097] In some embodiments, the increase is 1-10%, 5-30%, 15-50%, 25-75%, 70-150%, 100-350%, 250-550%, 500-750%, or 700-1,000% compared to the control, with each possibility representing a separate embodiment of the present invention.

[0098] As used herein, the term "treatment" or "treating" of a disease, disorder, or condition includes alleviating at least one symptom thereof, reducing its severity, or inhibiting its progression. Treatment does not necessarily mean that the disease, disorder, or condition is completely cured. To be an effective treatment, a composition useful herein need only reduce the severity of the disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide an improvement in the quality of life of the patient or subject.

[0099] As used herein, the term "prevention" of a disease, disorder, or condition encompasses delaying, preventing, suppressing, or inhibiting the onset of the disease, disorder, or condition. As used in accordance with the presently described subject matter, the term "prevention" refers to a prophylactic process in which a subject is exposed to a presently described composition or formulation prior to the induction or onset of the disease / disorder process. This can be done when an individual has a genetic lineage that predisposes to the development of the disease / disorder to be prevented. For example, this may apply to an individual whose ancestors predispose to a particular type of inflammatory disease. The term "suppression" is used to describe a state in which the disease / disorder process has already begun but overt symptoms of the condition have not yet materialized. Thus, an individual's cells may have the disease / disorder, but the outward signs of the disease / disorder have not yet been clinically recognized. In either case, the term prophylaxis can be applied to encompass both prevention and suppression. Conversely, the term "treatment" refers to the clinical application of an active agent to combat an existing condition in which clinical symptoms have already been realized in the patient.

[0100] In some embodiments, prevention includes reducing disease severity, delaying disease onset, reducing the cumulative incidence of disease, or any combination thereof.

[0101] As used herein, the term "subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject for whom treatment is desired, particularly mammalian subjects, such as humans.

[0102] In the discussion, unless otherwise specified, adjectives such as "substantially" and "about" modifying conditions or relationship characteristics of feature(s) of embodiments of the invention are understood to mean that such conditions or characteristics are defined within acceptable tolerances for the practice of the embodiments for the intended application. Unless otherwise indicated, the word "or" in the specification and claims is not considered to be an exclusive "or," but rather an inclusive "or," indicating at least one, or any combination, of the items with which it is connected.

[0103] The terms "a" and "an," as used above and elsewhere in this specification, should be understood to refer to "one or more" of the listed components. It will be clear to those skilled in the art that the use of the singular includes the plural unless otherwise specified. Thus, in this application, the terms "a," "an," and "at least one" are used interchangeably.

[0104] For purposes of better understanding the present teachings, and without in any way limiting the scope of the teachings, unless otherwise expressly stated, all numbers expressing quantities, percentages, or ratios, as well as other numerical values ​​used in the specification and claims, should be understood in all instances to be modified by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0105] In the description and claims of this application, the verbs "comprise," "include," and "have," and each of their conjugations, are used to indicate that the object(s) of the verb are not necessarily a complete list of the components, elements, or parts of the subject of the verb.

[0106] Other terms used herein are meant to be defined by their well-known meanings in the art.

[0107] As used herein, unless specifically stated otherwise or clear from context, the term "or" is understood to be inclusive.

[0108] Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising" indicate the inclusion of a recited integer or group of integers, but not the exclusion of other integers or groups of integers.

[0109] As used herein, the term "consists essentially of," or variations such as "consist essentially of" or "consisting essentially of," as used throughout the specification and claims, refers to the optional inclusion of the recited integer or group of integers, and any stated integer or group of integers that do not materially alter the basic or novel characteristics of the specified method, structure, or composition.

[0110] As used herein, terms such as "comprises," "comprising," "containing," "having," and the like can mean "comprise," "comprises," etc., and "consisting essentially of" or "consists essentially" likewise have the meaning under U.S. patent law, where the terms are open-ended, allowing for the presence of more than what is recited, but excluding prior art embodiments, so long as the basic or novel characteristics of what is recited are not changed by the presence of more than what is recited. In one embodiment, the terms "comprises," "comprising," and "having" are interchangeable with "consisting."

[0111] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as described hereinabove and as claimed in the claims section below finds experimental support in the following examples. [Example]

[0112] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include chemical, molecular, biochemical, and cell biological techniques. Such techniques are fully explained in the literature, for example, "Molecular Cloning: A Laboratory Manual" by Sambrook et al. (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); "Cell Biology: A Laboratory Handbook" Volumes I-III Cellis, JE, ed. (1994); The Organic Chemistry of Biological Pathways by John McMurry and Tadhg Begley (Roberts and Company, 2005); Organic Chemistry of Enzyme-Catalyzed Reactions by Richard Silverman (Academic Press, 2002); Organic Chemistry (6 th Edition) by Leroy “Skip” G Wade, Organic Chemistry by TW Graham Solomons and, Craig Fryhle.

[0113] material and method GTE preparation Dried and crushed golden tomato (Lycopersicon esculentum Mill. (Fam. Solanaceae) or Solanum lycopersicum) pulp was extracted using supercritical CO2 extraction at a pressure of 360 bar and a temperature of 60°C. The amount of crude extract from the raw material was 2.7% (w / w).

[0114] Non-limiting examples of compounds identified in supercritically extracted golden tomatoes, including their relative amounts, are set forth below (Table 1). The total weight of the extract was 7.55 grams. [Table 1]

[0115] LycoDerm, LycoMato content Standardized to 10% of the total carotenoid GTE extract, it is called Lumenato. A comparison of the active ingredients in Lumenato, Lycoderm, and Lyc-O-Mato 6% is shown in Table 2. [Table 2]

[0116] Preparation of human neutrophils Blood was collected from healthy volunteers. Neutrophils were obtained within 1 hour of collection with a purity of 95% by Ficoll-Hypaque centrifugation, dextran sedimentation, and hypotonic lysis of red blood cells. Cells were counted, and their viability was determined by trypan blue exclusion.

[0117] cell culture Normal human dermal fibroblasts (NHFD) from adult donors (PROMOCELL, Heidelberg, Germany) were cultured in fibroblast growth medium-2 (PROMOCELL) supplemented with fibroblast growth medium-2 (final supplement concentrations in the medium: 0.002% (v / v) fetal bovine serum, 1 mg / ml basic fibroblast recombinant human growth factor, and 5 μg / ml recombinant human insulin), 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin (Beit-Haemek, Israel). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. NHFDs were seeded into 24-well plates when they reached >80% confluency.

[0118] Immunofluorescence analysis For immunofluorescence detection, NHFDs were fixed with methanol for 3 minutes at 20°C and then washed with phosphate-buffered saline (PBS). Fixed NHFDs were incubated with anti-collagen-3 antibody (Santa Cruz) 1:50 in 5% (w / v) BSA / PBS for 90 minutes at room temperature. Cells were washed three times with PBS and incubated with Cy3 anti-mouse (1:100 in 5% (w / v) BSA / PBS; Jackson ImmunoResearch Laboratories, Inc., PA, USA) for 60 minutes at room temperature. Cells were washed three times with PBS, and nuclei were stained with DAPI. Next, a final wash was performed, and cells were analyzed using a fluorescence microscope (Olympus, BX60, Hamburg, Germany). Collagen-3 fluorescence intensity was determined using the CellProfiler program. The percentage of fluorescent area was determined using the CellProfiler program.

[0119] Superoxide production Cytochrome C reduction - The generation of superoxide anion by neutrophils was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome c by the microtiter plate technique. Cells (2.5 × 10 5 Cells (cells / well) were suspended in 100 μl of Hank's Balanced Salt Solution (HBSS) containing 150 mM ferricytochrome c. Following reduction of acetylferricytochrome c, absorbance at 550 nm was measured at 2-minute intervals in a Thermomax microplate reader (Molecular Devices, Mellon Park, CA). The maximum rate of superoxide generation was determined by the extinction coefficient E 550 =21mM -1 ·cm -1 Determined using nmol O 2- / 10 6 Expressed as cells / min.

[0120] Amplex red - A highly sensitive fluorescent biosensor, Amplex red, is utilized for its horseradish peroxidase (HRP)-dependent oxidation. The oxidation of Amplex red occurs outside the cell via HRP. HRP is the first product of NADPH oxidase, O2. - Resting and activated neutrophils (2 × 10 4 Microglia (cells / well) were suspended in KRPG buffer (phosphate buffer, 145 mM NaCl, 4.86 mM KCl, 1.22 mM MgSO, 5.5 mM D-glucose, 0.54 mM CaCl, pH 7.35) containing HRP (0.1 units / ml) and Amplex red (50 μM). Fluorescence was recorded on a microplate reader at an excitation wavelength of 535 nm and an emission wavelength of 595 nm. Background fluorescence was measured in the absence of microglial cells.

[0121] Myeloperoxidase (MPO) activity 100 μl of 37°C O-dianisidine hydrochloride solution (1 mg O-dianisidine, 10 ml phosphate buffer pH 6.0 + 0.0015% H2O2) was added to 100 μl of supernatant in a 96-well plate just before optical density, followed by absorbance changes at 450 nm at 2-min intervals in a Thermomax microplate reader (Molecular Devices, Melno Park, CA).

[0122] Twenty minutes before the addition of 100 ng / ml TNFα, tocopherols and phytosterols were added at 5 × 10 5 The mixture was added to human neutrophils and maintained overnight at 37°C in a humidified atmosphere containing 5% CO. MPO activity was measured in the supernatant of the cultured neutrophils.

[0123] Zeta-carotene, phytoene, tocopherol, phytosterol, or a combination thereof was added to 5 × 10 5 The mixture was added to human neutrophils and maintained overnight at 37°C in a humidified atmosphere containing 5% CO. MPO activity was measured as described above.

[0124] MMP-9 Human MMP-9 concentrations in cell culture supernatants were quantified by ELISA kit (R&D systems Minneapolis, MN, USA).

[0125] MMP-8 (collagenase) Human MMP-8 concentrations in cell culture supernatants were quantified by an ELISA kit (OriGene Technologies, Inc. Rockville, MD, USA).

[0126] Elastase activity N-Methoxysuccinyl-ala-ala-pro-Val-P-nitroaniline was added to 100 μl of the supernatant to a final concentration of 0.5 mM, followed by overnight incubation at 37° C. The absorbance at 405 nm was measured with a Thermomax microplate reader (Molecular Devices, Melno Park, CA).

[0127] Macrophage isolation and culture Peritoneal macrophages were collected from the peritoneal cavity of 6-8 week-old male ICR mice (Harlan, Israel) after intraperitoneal injection of 1.5 ml of thioglycollate broth (4%) 4 days before collection. Peritoneal macrophages were washed three times with PBS, followed by hypotonic lysis of red blood cells, and a highly enriched (90-95%) macrophage cell population was collected. Macrophages were identified by FACS analysis using FITC-conjugated rat anti-mouse F4 / 80 (MCA497F) (Serotec, Oxford, England) by flow microfluorometry on a FACS (Becton Dickinson, Mountain View, CA). For each sample, 10,000 light-scatter gated live cells were analyzed. Peritoneal macrophages (1 x 10 6Cells (number of cells / well) were cultured in 96-well plates at 37°C in a 5% CO atmosphere in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin (Beit-Haemek, Israel). Cells were stimulated with 100 ng / ml LPS from Salmonella enterica serovar Typhimurium in the absence or presence of components. LPS was purchased from Sigma, Israel.

[0128] All compounds were dissolved in DMSO, and the amount of DMSO did not exceed 0.1–0.2% in the test plates. An appropriate amount of DMSO (0.1–0.2%) was added to the control plates. The % inhibition in each tube test was calculated relative to its control.

[0129] Nitric oxide (NO) production assay NO levels in cell culture supernatants were determined by assaying nitrite concentrations using Griess reagent and sodium nitrite as a standard.

[0130] Cell survival Cell viability was assessed by cell count using trypan blue exclusion or by colorimetric MTT ([3-4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium) metabolic activity assay. For MTT measurements, cells were cultured in 96-well plates. MTT was dissolved in culture medium (5 mg / ml) and added to each sample in an amount equivalent to 10% of the culture medium volume. After 4 hours of incubation, formazan crystals were dissolved in 100 mM HCl, 10% Triton X-100, and then dissolved in an equal volume of isopropanol to the culture medium. The culture medium served only as background. Absorbance intensity was measured at 570 nm with a reference wavelength of 690 nm using a Thermomax microplate reader (Molecular Devices, Mellon Park, CA, USA).

[0131] Procollagen-3 The human procollagen-3 concentration in the cell culture supernatant was quantified using a Human PIIINP (N-terminal procollagen III propeptide) ELISA kit (Elabscience Biotechnology Inc. Houston, Texas, USA). Because the concentration of procollagen-3 in the fibroblast supernatant was very low, the supernatant was concentrated by evaporation.

[0132] Collagen-3 Human collagen-3 concentrations in cell culture lysates and supernatants were quantified by a human collagen, type III, alpha 1 (COL3A1) ELISA kit (Cusabio Technology Inc., Houston, Texas, USA).

[0133] Collagen 1a1 After incubating the cells with Lumenato for 24 hours, hydrogen peroxide (H2O2) was added and the cells were incubated for another 24 hours to induce oxidative stress. The incubation medium was collected and the level of collagen1a1 protein secretion into the medium was analyzed using a specific ELISA detection kit.

[0134] statistical analysis Significant differences between evaluated parameters were determined by ANOVA using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) followed by Bonferroni post-hoc correction for multiple comparisons. P values ​​less than 0.05 were considered statistically significant. Significance: * - p < 0.05, ** - p < 0.001, *** - p < 0.0001.

[0135] Example 1 GTE reduces the secretion of degradative enzymes from stimulated neutrophils. To study the effect of GTE on the release of enzymes that can damage collagen, we first determined the kinetics of release of such enzymes from activated neutrophils. Neutrophils were activated by 100 ng / ml TNFα or 100 ng / ml IL8, which are released and activated by skin cells during UV exposure. Additionally, neutrophils were cultured at 5 × 10 -7 Human neutrophils were activated with fMLP. As shown in Figure 1, the addition of TNFα to human neutrophils induced a significant (p<0.001) release of MMP-9 at 2 hours of activation (16.52 ± 0.5 ng / ml), which persisted at the same level at 4 hours. The addition of IL8 induced a dose-dependent release of MMP-9 during 4 hours of activation. A significant (p<0.001) release of MMP-9 was detected at 2 hours of activation with IL8 (9.12 ± 0.2 ng / ml), which further increased significantly (p<0.0001) to 17.86 ± 0.4 ng / ml at 4 hours. fMLP induced a rapid and significant (p<0.001) release of MMP-9 at 30 minutes of activation (10.41 ± 0.4 ng / ml), which gradually increased to 13.45 ± 0.3 ng / ml during 4 hours of activation. As shown in Figure 2, the addition of either TNFα or IL8 induced a significant (p<0.0001) release of MPO at 2 h of activation, which further increased at 4 h to 0.28 ± 0.001 ng / ml and 0.4 ± 0.08 ng / ml, respectively. fMLP induced a rapid and significant (p<0.0001) release of MPO to 0.54 ± 0.004 ng / ml at 30 min, which was sustained at this level at 4 h. Activation of neutrophils for 24 h did not increase MMP-9 or MPO release compared to 4 h (data not shown).

[0136] To study the effect of GTE on MMP-9 or MPO release, neutrophils were activated with TNFα or IL8 for 4 hours and fMLP for 30 minutes. Prior to activation, GTE was added to the neutrophils for 10 minutes at 37°C. As shown in Figure 3, the addition of GTE at concentrations ranging from 26 to 210 μg / ml dose-dependently inhibited MMP-9 release from neutrophils stimulated with either TNFα, IL8, or fMLP. The maximum inhibition of MMP-9 secretion was 95.4 ± 4.6%, 83.19 ± 12.0%, or 76.6 ± 12.0% from neutrophils stimulated with either TNFα, IL8, or fMLP, respectively. Similarly, GTE induced a dose-dependent inhibition of MPO from neutrophils stimulated with TNFα, IL8, or fMLP, with a maximum inhibition of 46.0 ± 6.7%, 33.86 ± 0.7%, or 38.5 ± 4.5%, respectively (Fig. 4 ).

[0137] Next, the effect of GTE on superoxide release from neutrophils was analyzed by cytochrome C reduction. Because superoxide is unstable, analysis was performed immediately after the addition of each of the aforementioned agonists (e.g., TNFα, IL8, etc.). As shown in Figure 5, the addition of GTE for 10 min prior to stimulation induced a dose-dependent inhibition of superoxide production, reaching a maximum inhibition of 63.27 ± 13%, 67.55 ± 3.4%, and 56.81 ± 2.4% when neutrophils were stimulated with TNFα, IL8, and fMLP, respectively.

[0138] Example 2 GTE reduces collagen loss in fibroblast-neutrophil co-cultures. To study the effect of GTE on collagen-3 damage induced by activated neutrophils, we used optimal conditions for culturing fibroblasts and neutrophils. To obtain confluent cultures, we used 1 × 10 5 2 × 10 fibroblasts were seeded for 24 h. 5The addition of activated neutrophils induced significant collagen-3 damage of approximately 30%, as shown by collagen-3 immunofluorescence staining, but did not destroy the cultures (Figures 6A-6N). Cell viability was measured for each treatment and was found to be unaffected by the addition of neuropils stimulated in the absence or presence of GTE. The concentration of neutrophils used in the coculture experiments was lower than that used in the neutrophil studies (Figures 1-5). Neutrophils were incubated with GTE for 10 min at 37 °C before being added to the cultures for 24 h. As shown by collagen-3 immunofluorescence staining (Figures 6A-6N), the addition of neutrophils preincubated with GTE at concentrations ranging from 6.5 to 104 μg / ml prior to addition to the fibroblast cultures resulted in a dose-dependent inhibition of collagen-3 damage. Maximum prevention of collagen damage was achieved at 52 μg / ml GTE (approximately 80% protection). Quantification of collagen-3 levels in the cultures, determined by densitometry and shown in Figure 6O, was greater than that of other tomato-derived compositions (Figures 6P-6Q). The presence of Lumenato in the coculture caused a dose-dependent increase in secreted procollagen-3 to 68.21 ± 7.1 pg / ml in the presence of 104 μg / ml Lumenato (Figure 6S). Subtracting the level of procollagen-3 induced by neutrophils (shown by the horizontal dashed line) showed an increase of approximately 10 pg / ml in the presence of 104 μg / ml Lumenato. Furthermore, overnight addition of Lumenato to fibroblasts did not affect fibroblast counts measured by MTT or DAPI staining (data not shown). Nevertheless, the addition of Lumenato induced a significant increase in procollagen-3 levels in the supernatant in a dose-dependent manner (Figure 6E).

[0139] Treatment of NHDF cells with 50 μM HO resulted in a roughly 10-fold decrease in collagen 1a1 secretion (Figure 19). The lowest concentration of Lumenato used completely abolished the decrease in collagen secretion. Given Lumenato's ability to reduce MMP1 secretion, these results suggest that Lumenato is a promising agent for increasing skin collagen levels and reducing skin aging in vivo.

[0140] The amounts / levels of degradative enzymes released under the aforementioned co-culture system (as shown in Figure 6) were measured in the supernatant after 24 h of culture. Because a lower concentration of neutrophils was used in the co-culture experiments (2.5 × 10 5 5 x 10 per ml 6 The concentrations of GTE used in the coculture experiments (cells / ml) were lower than those used to study their effects on neutrophils (Figures 1-5). As shown in Figure 7A, dose-dependent inhibition of MMP-9 was observed with GTE but not with other tomato-derived compositions (Figures 7B-7C). GTE suppression was approximately 12.5 ± 3.9% inhibition. MPO release, determined based on activity in the supernatant, was significantly inhibited at GTE concentrations as low as 13 μg / ml and gradually increased, reaching 45.8 ± 5.6% inhibition at the maximum concentration tested (Figure 8A). The GTE MPO inhibitory effect was approximately twofold compared to other tomato-derived compositions (Figures 8B-8C). The effect of GTE on superoxide generation was studied by Amplex Red, performed immediately after neutrophil stimulation (superoxide is unstable). As shown in Figure 9, GTE induced a significant dose-dependent inhibition reaching 78.5 ± 0.9% inhibition. The beneficial effect of GTE in coculture was further illustrated by the inhibition of MMP-8 (collagenase), as shown in Figure 10. GTE induced a gradual inhibition of MMP-8 release, with a maximum inhibition of 43.0 ± 7.1% achieved at 26 μg / ml of GTE and sustained at higher GTE concentrations. Elastase activity was not significantly elevated in the presence of GTE in cocultures of stimulated neutrophils and fibroblasts (Figure 11).

[0141] Example 3 GTE reduces nitric oxide production in stimulated macrophages. Macrophages were stimulated with LPS and then incubated with increasing concentrations of either filtered pulp or GTE. As shown in Figure 12, GTE was twice as effective at inhibiting nitric oxide (NO) production compared to filtered pulp. GTE was found to inhibit NO production in a dose-dependent manner, starting at a concentration of only 5 μg / ml, demonstrating approximately 20% inhibition. In the presence of a GTE concentration of 100 μg / ml, the level of NO production inhibition continued to increase to approximately 55%.

[0142] Example 4 The multiple phytonutrient compositions synergistically inhibit MPO activity. We investigated the effects of multiple phytonutrients on MPO activity in stimulated neutrophils. Addition of tocopherol or phytosterol alone did not affect MPO release or activity from TNFα-activated neutrophils (Figures 13A-13B). However, supplementation with 225 μg / ml tocopherol and 62.5 μg / ml phytosterol produced a synergistic effect, inhibiting MPO activity by approximately 20% compared to the control (Figure 13C).

[0143] We also investigated the effect of adding zeta-carotene together with phytoene, tocopherol, or phytosterol on MPO activity. Addition of zeta-carotene, tocopherol, phytoene, or phytosterol alone had little inhibitory effect on 5% inhibition of MPO activity (Figure 14). However, addition of zeta-carotene with either tocopherol, phytoene, or phytosterol at the ratio present in GTE induced synergistic inhibition of MPO activity or release from TNFα-activated neutrophils (Figure 14).

[0144] Example 5 Lumenato capsules with improved carotenoid bioavailability We investigated the bioavailability of carotenoids delivered in Lumenato capsules. The following carotenoids were quantified in plasma at 1, 2, 3, and 4 weeks after administration: The main carotenoids in Lumenato (golden tomato extract), phytoene, phytofluene, and zeta-carotene, were well absorbed (Figure 15). Zeta-carotene was found to be highly absorbed, as reflected by a rapid rise in plasma concentrations (Figure 15A).

[0145] Example 6 Balances oxidative stress-induced damage to skin cells Next, we investigated whether Lumenato, a golden tomato extract, enhances skin elasticity and prevents oxidative stress-induced skin damage in human keratinocytes. Reactive oxygen species (ROS), such as H2O2, are known to induce inflammatory processes in the skin, increase matrix metalloproteinase (MMP) production in skin cells, and cause collagen degradation.

[0146] We induced oxidative stress in KERTr human keratinocytes using hydrogen peroxide (H2O2) and analyzed the effects on cell number (cell death) and MMP1 secretion. Cell viability was measured using an XTT cell proliferation kit, and MMP1 levels were measured by ELISA. Treatment of cells with H2O2 resulted in dose-dependent cell death (Figure 16A) and increased MMP1 secretion (Figure 16B). Addition of Lumenato at the indicated concentrations before H2O2 increased cell viability and decreased MMP1 secretion.

[0147] One possible explanation for the protective effect described above is activation of the antioxidant response element transcription system (ARE / Nrf2). Therefore, we measured the activation of this transcription system using a reporter gene assay. A dose-dependent activation of ARE / Nrf2 transcriptional activity by Lumenato was observed (Figure 17).

[0148] Example 7 Skin condition and appearance We developed a baseline skin questionnaire that included 14 questions asking participants about the condition and appearance of their skin before and after taking the study product, Lumenato (e.g., at baseline and after 12 weeks). The results show that satisfaction levels improved significantly for 11 of the 14 questions (excluding questions 3, 10, and 11) (Table 3). [Table 3]

[0149] Additionally, we developed a skin update questionnaire containing 14 questions asking participants about the condition and appearance of their skin while taking the study product, Lumenato (Weeks 4, 8, and 12). The results (Table 4) show that satisfaction levels improved significantly for 10 of the 14 questions (excluding questions 3, 8, 11, and 12). [Table 4] JPEG0007732981000005.jpg17170

[0150] While the present invention has been specifically described, those skilled in the art will appreciate that many variations and modifications are possible. Accordingly, the present invention should not be construed as limited to the specifically described embodiments, and the scope and spirit of the present invention will be more readily understood by reference to the following claims.

Claims

1. A pharmaceutical composition for use in reducing collagen loss, increasing collagen levels, or both, comprising phytoene in an amount of 55-65% (w / w) of the total carotenoids in the composition, phytofluene in an amount of 10-20% (w / w) of the total carotenoids in the composition, zeta-carotene in an amount of 15-25% (w / w) of the total carotenoids in the composition, tocopherol in an amount of 10-30% (w / w) of the total carotenoids in the composition, and an acceptable carrier, wherein the amounts of zeta-carotene and either the tocopherol or phytoene in the pharmaceutical composition synergistically inhibit myeloperoxidase (MPO) activity, and the pharmaceutical composition is characterized by reduced toxicity to fibroblasts compared to a tomato extract control.

2. 2. The pharmaceutical composition for use according to claim 1, wherein the weight ratio of the combined phytoene and phytofluene to the zeta-carotene ranges from 15:1 (w / w) to 2:1 (w / w).

3. 3. The pharmaceutical composition for use according to claim 1 or 2, further comprising an additional carotenoid selected from the group consisting of lycopene, beta-carotene, gamma-carotene, and any combination thereof.

4. 4. The pharmaceutical composition for use according to claim 3, comprising the lycopene in an amount of less than 5% (w / w) of the total carotenoids in the composition, the beta-carotene in an amount of less than 5% (w / w) of the total carotenoids in the composition, the gamma-carotene in an amount of 0.2-1.5% (w / w) of the total carotenoids in the composition, or a combination thereof.

5. 2. The pharmaceutical composition for use according to claim 1, comprising an amount of 10-15% (w / w) of the total carotenoids in said composition.

6. A pharmaceutical composition for use according to claim 1, wherein the pharmaceutical composition is a tomato extract composition.

7. 10. The pharmaceutical composition for use according to claim 1, further comprising a phytosterol.

8. 8. The pharmaceutical composition for use according to claim 7, comprising said phytosterol in an amount of 5-15% (w / w) of the total carotenoids in said composition.

9. 1. A pharmaceutical composition for use in preventing or treating collagen loss in a subject in need thereof, comprising phytoene in an amount of 55-65% (w / w) of the total carotenoids in the composition, phytofluene in an amount of 10-20% (w / w) of the total carotenoids in the composition, zeta-carotene in an amount of 15-25% (w / w) of the total carotenoids in the composition, tocopherol in an amount of 10-30% (w / w) of the total carotenoids in the composition, and an acceptable carrier, wherein the amounts of zeta-carotene and any one of the tocopherol or phytoene in the pharmaceutical composition synergistically inhibit myeloperoxidase (MPO) activity, and the pharmaceutical composition is characterized by reduced toxicity to fibroblasts compared to a tomato extract control.

10. 10. The pharmaceutical composition for use according to claim 9, wherein said prevention or treatment is to reduce collagen loss by 50 to 90%.

11. The pharmaceutical composition for use according to claim 9, wherein the prevention or treatment of collagen loss comprises reducing the amount, activity, or both of any one of matrix metalloproteinases (MMPs), myeloperoxidase (MPO), superoxide (SO), elastase, nitric oxide (NO), and any combination thereof in the subject in need thereof.

12. 12. The pharmaceutical composition for use according to claim 11, wherein the MMP is selected from MMP-9, MMP-8, MMP-1, or any combination thereof.

13. 10. The pharmaceutical composition for use according to claim 9, wherein the subject is suffering from a collagen loss-related disease.

14. 14. The pharmaceutical composition for use according to claim 13, wherein the collagen loss-related disease is selected from the group consisting of age-related diseases, skin diseases, and inflammatory diseases.

15. 15. The pharmaceutical composition for use according to claim 14, wherein the skin disease comprises skin damage induced by any of the following: radiation, oxidative stress, DNA damage, telomere shortening, inflammation, tobacco use, and any combination thereof.

16. 16. The pharmaceutical composition for use according to claim 15, wherein the radiation comprises UV radiation, visible light radiation, infrared radiation, or any combination thereof.

17. 17. The pharmaceutical composition for use according to claim 16, wherein the UV radiation is UVA, UVB, UVC, or any combination thereof.

18. 10. The pharmaceutical composition for use according to claim 9, wherein the subject has an increased amount, increased activity, or both, of any one of MMP, MPO, SO, elastase, NO, and any combination thereof in the skin, systemically, or both, compared to a control.

19. The pharmaceutical composition for use according to claim 9, wherein the pharmaceutical composition is administered orally, topically, or both.

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