Identification and use of active ingredients

Abstract

Inventive aspects disclosed herein includes an oily barley extract derived from barley grass exposed to light, the extract having a molecular oxygen partial pressure of at least about 180 mbar and a chlorophyll concentration of about 180 mg / kg.

Description

[0001] IDENTIFICATION AND USE OF ACTIVE INGREDIENTS

[0002] Claim of Priority

[0003] This patent application claims the benefit of priority to NL Application Serial No. N2035682, filed August 25, 2023, which is incorporated by reference herein in its entirety.

[0004] Field

[0005] The disclosure is generally directed to identification and use of active ingredients capable of multiple modes of action simultaneously, including but not limited to increasing ceramide synthesis, collagen synthesis, ATP synthesis, and presence of oxygen in cells.

[0006] Background

[0007] Barley is one of the oldest cultured plants. It may be the first cereal domesticated, cultivated in the Middle East, around 7000 BC. Traces of barley are found in the Neolithic period. Barley was considered sacred by ancient Greeks. The ancient Egyptians believed that barley was cultivated by the goddess, Isis.

[0008] Brief Description of the Figures

[0009] FIG. 1 is an illustration of the presence of ceramides (visible via immunostaining) in human keratinocyte samples in skin explants that are treated with a formulation containing 0.5% barley extract versus explant skin samples that are treated with an identical formulation save for the presence of the barley extract.

[0010] FIG. 2 is a graph showing the expression of genes responsible for governing skin barrier strength.

[0011] FIG. 3 is another graph showing analysis of expression of genes responsible for governing skin barrier strength using the same protocol as discussed with respect to FIG. 2.

[0012] FIG. 4 is a graph showing expression of genes responsible for governing skin hydration

[0013] FIG. 5 is a graph showing barley extract’s impact in increasing ATP synthesis.

[0014] FIG. 6 is a graph showing barley extract's impact in increasing collagen synthesis is shown in fibroblasts as assessed in vitro. FIG. 7 is a graph showing gene expression where Normal Human Fibroblasts were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing FBS (fetal bovine serum) at 2%.

[0015] FIG. 8 is a graph showing results where Normal Human Fibroblasts were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing FBS (fetal bovine serum) at 2%.

[0016] FIG. 9 are graphs showing results of Normal human skin explants were treated with the barley at 0.5% for 24 hours and incubated at 37°C, 5% CO2.

[0017] Summary

[0018] Various aspects of the instant disclosure relate to a method of improving the aesthetic appearance of skin of a human affected by aging resulting from both decreased ATP synthesis and decreased skin barrier reinforcement within comprising topically applying thereto a therapeutically effective amount of an active agent wherein the active agent is selected as fulfilling the following criteria: the active agent both upregulates one or more of the genes listed in FIGS. 1-5 and upregulates ATP synthesis when applied to fibroblasts at a sufficient concentration (w / w) in a cosmetically acceptable vehicle for a time sufficient to achieve an improvement in the aesthetic appearance of said skin of said human.

[0019] Various aspects further relate to a method for screening for putative active agents capable of efficacious improvement of aesthetic appearance of human skin, comprising culturing a fibroblast cell culture in the presence or absence of the putative active agent; measuring the relative amount of gene expression of two or more of the genes listed in FIGS. 1-5 in the treated and untreated cells; and selecting the putative active agent for use if the putative active agent upregulates at least two of the genes over baseline.

[0020] Various aspects further relate to a method for providing a personalized method for improvement of aesthetic appearance of skin, comprising obtaining a sample of skin cells from an individual; assessing the level of gene expression of at least 2 of the genes listed in FIGS. 1-5 in a cultured sample of the obtained skin cells; and suggesting an aesthetic appearance improvement regimen to the individual comprising use of a cosmetic product including an active agent increases gene synthesis of at least two of the genes listed in FIGS. 1-5 when applied to fibroblasts at a concentration of 0.50%(w / w) if the cultured sample shows expression of any two of the genes listed in FIGS. 1-5 elevated relative to baseline.

[0021] Detailed Description

[0022] The following detailed description includes references to the accompanying drawings, which form a part of the detailed description. The drawings show, by way of illustration, specific aspects in which the disclosure may be practiced. These aspects, which are also referred to herein as “examples,” are described in enough detail to enable those skilled in the art to practice the disclosure. The aspects may be combined, other aspects may be utilized, or structural, and logical changes may be made without departing from the scope of the present disclosure. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present disclosure is defined by the appended claims and their equivalents.

[0023] In this document, the terms “a” or “an” are used to include one or more than one and the term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation.

[0024] A “therapeutically effective amount” (or “effective amount”) of a compound with respect to use in treatment, refers to an amount of the compound in a preparation which, when administered as part of a desired dosage regimen (to a mammal, such as a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment.

[0025] Furthermore, all publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0026] Collagen synthesis and degradation play a role in common skin concerns. Reduction in collagen I, the collagen type in skin, is associated with loss of firmness and elasticity of skin and leads to wrinkling associated with aging. Collagen is the body's major structural protein and gives skin strength, durability, and a smooth, plump appearance. It is created by fibroblasts, specialized skin cells located in the dermis, in a process that involves conversion of preprocollagen I to procollagen I and eventually to tropocollagen, the form that forms collagen fibers. Collagen I is degraded in the skin by matrix metalloproteinases (MMPs). Gross J, et al. Biochem Biophys Res Commun 1974; 61 :605-12. MMPs are a family of related zinc-dependent proteases, including the metalloproteinases (MMP-1, -8, and -13) and gelatinases (MMP-2 and 9).

[0027] One inventive cosmetic formulation aspect includes the use of a barley extract formulation for increasing skin barrier strength and increasing collagen synthesis as identified by a particular panel assay, wherein the barley extract formula has a molecular oxygen content of about 180 mbar partial pressure, as measured by an oximeter and a chlorophyll content of about 180 mg / kg. Barley extract aspects disclosed herein are obtained from barley grass as a leaf extract. Barley grass naturally contains a large amount of chlorophyll. The barley extract aspects also include this large amount of chlorophyll obtained from increased photosynthesis due to special lighting. Barley grass for the barley extract aspects claimed herein is harvested one week after seeding, or when young leaves have a nutritional peak with respect to chlorophyll, before plants begin to produce flowers and seeds. The oily barley extract aspects include chlorophyll and oxygen. This molecular oxygen acts to generate energy in skin cells by increasing generation of adenosine triphosphate, ATP needed to boost production of essential cellular proteins.

[0028] The barley employed in the barley extract aspects was exposed to a 75 Watt, 2400 Lux light source. Testing has also been performed on separate samples using exposure to 60 Watt, 600 Lux and 100 Watt, 20000 Lux light sources, respectively. The data shows a partial oxygen pressure of about 180 mbars for barley exposed to 60 Watts / 600 Lux; 183 mbars for barley exposed to 75 Watts / 2400 Lux and 178 mbars for barley exposed to 100 Watts / 20000 Lux. The data shows that the most intense light did not produce barley having the highest oxygen partial pressure. The barley exposed to 75 Watts / 2400 Lux produced extract with the highest oxygen partial pressure.

[0029] Barley extract aspects disclosed herein are obtained from barley cultivated under a specific type of lighting. Lighting used to make the barley extract is 2400 Lux, as measured with a luximeter. A Lux is equal to one lumen per square meter.

[0030] It has been found that an oily barley extract having barley exposed to the higher light intensity is identified as having selective skin barrier strengthening and simultaneous energy generating properties. This oily barley extract demonstrates an increase in oxygen compared to formulas without the barley extract.

[0031] Molecular oxygen from ambient air is absorbed within the skin following an oxygen gradient.

[0032] Assay combinations assessing the ability of an active ingredient to both increase ATP synthesis and increase gene expression associated with upregulation of lipid transporters (ABCA12), ceramide synthesis (PNPLA1 and GBA); fatty free acid synthesis (ELOVL1, ELOVL4, FASN,SCD), cholesterol synthesis (HMGCR, HMGSC1), and / or PPARa synthesis) have also been newly shown to be useful in selecting active ingredients capable of improving the aesthetic appearance of skin.

[0033] In addition, assay combinations assessing the ability of an active to increase expression of genes associated with cornified envelope formation (IVL, SPRR1 A, TGM1); stratum corneum cohesion (CDSN, CLDN4, OCLN); desquamation regulation (KLK5 and KLK7), have been newly shown to be predictive in assessing the ability of an active ingredient to improve aesthetic appearance.

[0034] In addition, assay combinations assessing the ability of an active to upregulate genes associated with promotion of skin hydration (AQP3, AQP9, HAS2) have been newly shown to be predictive in assessing the ability of an active ingredient to improve aesthetic appearance.

[0035] All living organisms that depend upon oxygen obtain their energy from a respiratory system where molecular oxygen is reduced to water and energy in the form of ATP. Any living organism requires energy in enough quantity to stay metabolically active, repair and regenerate for cell division. In particular, human cells from skin, directly exposed to sun, are highly dependent upon energy to fight damage and to fight cell deregulation. Cellular energy levels decrease with age and these deficiencies are highly correlated with alteration of human skin structure. This mechanism produces signs of aging.

[0036] Oxygen is an essential factor in collagen production. Appropriate molecular oxygen concentration is essential in allowing normal hydroxylation of collagen and accumulation of neo synthesized collagen is increased in tissue with elevated PO(2). When oxygen is brought to fibroblasts, it increases their synthetic capacity.

[0037] Oxygen supply by microcirculation and skin oxygen content in the skin depend upon factors like age, sex, and microangiopathic risk. Thus, smokers have a reduced amount of oxygen in their skin. Male subjects in general have lower PO(2) levels than women. In women, PO(2) levels decrease rapidly from ages 30 to 45 and then start increasing again after 50 years. This increase may be linked to changes of skin thickness in relation to hormonal changes and hormonal replacement therapy. Aging is accompanied by a decrease of oxygen content in the skin .

[0038] The aspects are described in sufficient detail to enable those skilled in the art to practice the disclosure. Other aspects may be utilized and formulation and method of using changes may be made without departing from the scope of the disclosure. The detailed description is not to be taken in a limiting sense, and the scope of the disclosure is defined only by the appended claims, along with the full scope of equivalents to which such claims are entitled. It will be appreciated by those skilled in the art that changes could be made to the aspects described above without departing from the broad inventive concept thereof. It is understood, therefore, that this disclosure is not limited to the particular aspects disclosed, but it is intended to cover modifications within the spirit and scope of the present disclosure as defined by the present description.

[0039] Examples

[0040] With reference to figure 1 an illustration of the presence of ceramides (visible via immunostaining) in human keratinocyte samples in skin explants that are treated with a formulation containing 0.5% barley extract versus explant skin samples that are treated with an identical formulation save for the presence of the barley extract is presented. Normal human skin explants were treated with the barley at 0.5% for 24 hours and incubated at 37°C, 5% CO2. Then ceramides immunostaining was performed on formol-fixed-paraffin embedded skin sections with a monoclonal anti-ceramides antibody, using an amplifier system avidin / biotin, and revealed by VIP, a substrate of peroxidase giving a violet staining once oxidized. Results were expressed by the surface of the immunostaining. As can be seen in the depth of staining, application of the formulation containing 0.5 % barley extract resulted in an increase in ceramide production in explant skin cells of 28% after 24 hours.

[0041] With reference to figure 2 an analysis of expression of genes responsible for governing skin barrier strength, is presented. As can be seen in Figure 2, use of 0.5% of barley extract results in increased expression of certain genes associated with skin barrier reinforcement. The protocol for gene expression involved culturing normal human keratinocytes were in KSFM (Keratinocytes serum-free medium). The barley was diluted in the culture medium at 0.5% and applied on the cells for 48 hours, cells were incubated at 37°C, 5% CO2. Non-treated cells were incubated under the same conditions. Total RNA were extracted with the Kingfisher Flex (Thermo Fischer Scientific) by using NucleoMag RNA kit (Macherey -Nagel) and quantified with a spectrophotometer at 260nm. First strand cDNA synthesis was performed with the High cDNA Reverse Transcription kit. Real-Time PCR was carried out with the QuantStudio 7 Flex Real-Time PCR System (Thermo Fischer Scientific) by using the TaqMan primers and probes specific to the studied genes. Relative changes in gene expression were calculated according to the 2-AACT method with the 7300 System software™.

[0042] The results shown in figure 2 are summarized below in Table 1.

[0043] Table 1

[0044] With reference to figure 3 an additional analysis of expression of genes responsible for governing skin barrier strength using the same protocol as discussed with respect to figure 2. As can be seen in Figure 3, use of 0.5% of barley extract results in increased of certain genes associated with skin barrier reinforcement.

[0045] The results shown in figure 3 are summarized below in Table 2.

[0046] Table 2

[0047] With reference to figure 4 an analysis of expression of genes responsible for governing skin hydration, is presented. Using the same protocol as discussed with respect to figure 2. As can be seen in figure 4, use of 0.5 % of barley extract results in increased expression of genes associated with skin hydration. The results shown in figure 4 are summarized below in Table 3.

[0048] Table 3

[0049] With reference to figure 5 barley extract’s impact in increasing ATP synthesis. Normal human keratinocytes were cultured in serum-free KGM-2 medium is presented. Normal human fibroblasts were cultured in DMEM (Dulbecco’s Modified Eagle Medium) containing FBS (fetal bovine serum) at 2%. The barley was diluted in the culture medium at 0.1% and applied on the cells for 72 hours, cells were incubated at 37°C, 5% CO2. Non-treated cells were incubated under the same conditions. Cell ATP content was assessed by using a bioluminescence microplate assay based. The ATP level was normalized by the total cellular protein content. As shown in figure 5, ATP synthesis in keratinocytes increased by 51% relative to a composition that does not include the barley extract and was increased by 14% in fibroblasts relative to a composition that does not include the barley extract.

[0050] With reference to figure 6 barley extract’ s impact in increasing collagen synthesis is shown in fibroblasts as assessed in vitro. Results are shown that evaluate the potential of barley extract on collagen-I neosynthesis. To accomplish this an in vitro test on cells culture of normal human dermal fibroblasts was used. Barley extract was tested at different doses 0.1%, 0.25% and 0.5% and was put in contact with cells for 72 hours while some cells were left untreated as control. It is possible to increase the doses beyond 0.5%. In parallel, a positive control was tested made of sodium ascorbate 50 pg / ml.

[0051] Normal Human Fibroblasts were cultured in DMEM (Dulbecco’s Modified Eagle Medium) containing FBS (fetal bovine serum) at 2%. The barley was diluted in the culture medium at 0.5% and applied on the cells for 24 hours, cells were incubated at 37°C, 5% CO2. Non-treated cells were incubated under the same conditions. The cell proliferation was assessed by quantifying the total DNA content by using the CyQuant Cell Proliferation Assay kit.

[0052] The test included measuring collagen-I level in cells culture using ELISA assay on untreated cells, cells treated with positive control and cells treated with Barley extract at different doses. Additionally a 61% increase in collagen synthesis was shown when the barley was included, relative to a solution that did not include barley.

[0053] With reference to figure 7, results are shown where Normal Human Fibroblasts were cultured in DMEM (Dulbecco’s Modified Eagle Medium) containing FBS (fetal bovine serum) at 2%. The barley was diluted in the culture medium at 0.5% and applied on the cells for 24 hours, cells were incubated at 37°C, 5% CO2. Nontreated cells were incubated under the same conditions. The cell proliferation was assessed by quantifying the total DNA content by using the CyQuant Cell Proliferation Assay kit.

[0054] With reference to figure 8 Normal Human Fibroblasts were cultured in DMEM (Dulbecco’s Modified Eagle Medium) containing FBS (fetal bovine serum) at 2%. The barley was diluted in the culture medium at 0.5% and applied on the cells for 48 hours, cells were incubated at 37°C, 5% CO2. Non-treated cells were incubated under the same conditions. Total RNA were extracted with the Kingfisher Flex (Thermo Fischer Scientific) by using NucleoMag RNA kit (Macherey -Nagel) and quantified with a spectrophotometer at 260nm. First strand cDNA synthesis was performed with the High cDNA Reverse Transcription kit. Real-Time PCR was carried out with the QuantStudio 7 Flex Real-Time PCR System (Thermo Fischer Scientific) by using the TaqMan primers and probes specific to the studied genes. Relative changes in gene expression were calculated according to the 2-AACT method with the 7300 System software™.

[0055] With reference to figure 9 Normal human skin explants were treated with the barley at 0.5% for 24 hours and incubated at 37°C, 5% CO2. Then AQP3 immunostaining was performed on frozen skin sections with a polyclonal anti- AQP3 antibody and revealed by fluorescent second antibody. Results were expressed by the surface of the immunostaining.

Claims

What is claimed is:

1. A method of improving the aesthetic appearance of skin of a human affected by aging resulting from both decreased ATP synthesis and decreased skin barrier reinforcement within comprising topically applying thereto a therapeutically effective amount of an active agent wherein the active agent is selected as fulfilling the following criteria: the active agent both upregulates one or more of the genes listed in FIGS. 1- 5 and upregulates ATP synthesis when applied to fibroblasts at a sufficient concentration (w / w) in a cosmetically acceptable vehicle for a time sufficient to achieve an improvement in the aesthetic appearance of said skin of said human.

2. The method of claim 1, wherein the criteria of increasing collagen synthesis in fibroblasts by at least 50% is also achieved.

3. The method of claim 1, wherein the active agent comprises at least one extract of barley.

4. The method of claim 1, wherein the extract of barley is present in a concentration in range of from about 0.02 wt% to about 2 wt%.

5. A method for screening for putative active agents capable of efficacious improvement of aesthetic appearance of human skin, comprising culturing a fibroblast cell culture in the presence or absence of the putative active agent; measuring the relative amount of gene expression of one or more of the genes listed in FIGS. 1-5 and of ATP synthesis in the treated and untreated cells; and selecting the putative active agent for use if the putative active agent upregulates ATP synthesis at least one of the genes over baseline.

6. A method for providing a personalized method for improvement of aesthetic appearance of skin, comprising obtaining a sample of skin cells from an individual; assessing the level of gene expression of at least one of the genes listed in FIGS. 1-5 and of ATP synthesis in a cultured sampleof the obtained skin cells; and suggesting an aesthetic appearance improvement regimen to the individual comprising use of a cosmetic product including an active agent which increases ATP synthesis and expression of at least one of the genes listed in FIGS. 1-5 when applied to fibroblasts at a concentration of 0.50%(w / w) if the cultured sample shows increased ATP synthesis and expression of at least one of the genes listed in FIGS. 1-5 elevated relative to baseline.

7. The method of any of claims 1 through 6 wherein the assay used for selection is any of those described in FIGs. 1-5.

8. The method of any of claims 1-7 wherein the gene expression is increased by at least 10% over baseline.

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