Identification and use of active ingredients

Abstract

The presently disclosed inventive aspects include an oily barley extract extracted from barley grass exposed to light, the extract having a molecular oxygen partial pressure of at least about 180 millibar and a chlorophyll concentration of about 180 mg / kg.

Description

Priority Statement

[0001] This patent application claims priority to Dutch application No. N2035682, filed on August 25, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure generally relates to the identification and use of active ingredients capable of performing multiple modes of action simultaneously, including but not limited to increasing ceramide synthesis, collagen synthesis, ATP synthesis, and the presence of oxygen in cells. Background Technology

[0003] Barley is one of the earliest cultivated plants. It was likely the first grain domesticated and cultivated in the Middle East around 7000 BC. Traces of barley have been found in the Neolithic period. Barley was considered sacred by the ancient Greeks. The ancient Egyptians believed that barley was cultivated by the goddess Isis. Summary of the Invention

[0004] Various aspects of this disclosure relate to a method for improving the aesthetic appearance of aging-affected human skin, said aging being caused by both decreased ATP synthesis and weakened skin barrier function. The method comprises topically applying a therapeutically effective amount of an active agent, wherein the active agent is selected to meet the following criteria: when applied to fibroblasts at a sufficient concentration (w / w) in a cosmetically acceptable medium and sustained for a sufficient duration to achieve an improvement in the aesthetic appearance of said human skin, said active agent is upregulated. Figures 1 to 5 It includes one or more genes listed in the table and upregulates ATP synthesis.

[0005] Various aspects further relate to a method for screening putative active agents capable of effectively improving the aesthetic appearance of human skin, the method comprising culturing fibroblast cultures in the presence or absence of said putative active agent; measuring the effects of treated and untreated cells. Figures 1 to 5 The relative levels of gene expression of two or more genes listed in the table; and if the putative activator upregulates at least two of the genes relative to baseline, then the putative activator is selected for use.

[0006] Various aspects further relate to a method for providing a personalized approach to improve the aesthetic appearance of skin, the method comprising obtaining a skin cell sample from an individual; evaluating the cultured sample of the obtained skin cells in... Figures 1 to 5 Gene expression levels of at least two genes listed; and if the cultured sample shows relative to baseline. Figures 1 to 5If the expression of any two genes listed above is elevated, then an aesthetic appearance improvement program is recommended to the individual. This program includes the use of cosmetics containing an active agent that, when applied to fibroblasts at a concentration of 0.50% (w / w), increases... Figures 1 to 5 Gene synthesis of at least two of the genes listed in the document. Attached Figure Description

[0007] Figure 1 This is an illustration of the presence of ceramides (visible via immunostaining) in human keratinocyte samples from skin explants treated with a formulation containing 0.5% barley extract and explant skin samples treated with the same formulation except for the presence of barley extract.

[0008] Figure 2 It is a diagram showing the expression of genes responsible for regulating the strength of the skin barrier.

[0009] Figure 3 It displays usage and about Figure 2 Another diagram analyzing the expression of genes responsible for controlling skin barrier strength using the same scheme discussed.

[0010] Figure 4 This is a diagram showing the expression of genes responsible for regulating skin hydration.

[0011] Figure 5 This is a graph showing the effect of barley extract on increasing ATP synthesis.

[0012] Figure 6 This is a graph showing the effect of barley extract on increasing collagen synthesis in fibroblasts, as assessed in vitro.

[0013] Figure 7 This is a graph showing gene expression in normal human fibroblasts cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 2% fetal bovine serum (FBS).

[0014] Figure 8 This is a graph showing the results of culturing normal human fibroblasts in DMEM (Dupuy's modified Eagle medium) containing 2% FBS (fetal bovine serum).

[0015] Figure 9 The graph shows the results of treating normal human skin explants with 0.5% barley for 24 hours at 37°C and 5% CO2. Detailed Implementation

[0016] The following detailed description includes reference to the accompanying drawings, which form part of the detailed description. The drawings schematically illustrate specific aspects in which the present disclosure may be practiced. These aspects are described in sufficient detail to enable those skilled in the art to practice the present disclosure, and are also referred to herein as “examples.” 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. Therefore, the following detailed description should not be considered limiting, and the scope of the present disclosure is defined by the appended claims and their equivalents.

[0017] In this document, the term "a" is used to include one or more, and the term "or" is used to mean a non-exclusive "or," unless otherwise specified. Furthermore, it should be understood that any wording or term used herein without further definition is for descriptive purposes only and not for limitation.

[0018] The "therapeutic effective amount" (or "effective amount") of a compound used in treatment refers to the amount of the compound in a formulation which, when administered as part of a desired dosing regimen (to a mammal, such as a human), relieves symptoms, improves condition, or slows the onset of disease symptoms for the condition or symptom to be treated or for cosmetic purposes, based on clinically acceptable criteria (e.g., a reasonable benefit / risk ratio applicable to any medical treatment).

[0019] Furthermore, all publications, patents, and patent documents mentioned in this document are incorporated herein by reference in their entirety as if they were individually cited. In the event of any inconsistency between the usage in this document and those cited documents, the usage in the incorporated references shall be considered supplementary to the usage in this document; in the case of irreconcilable inconsistencies, the usage in this document shall prevail.

[0020] Collagen synthesis and degradation play a role in common skin problems. A decrease in collagen I (the type of collagen in the skin) is associated with loss of skin firmness and elasticity, leading to age-related wrinkles. Collagen is a major structural protein in the body and gives skin strength, durability, and a smooth, plump appearance. It is produced by fibroblasts (specialized skin cells located in the dermis) in a process involving the conversion of early procollagen I into precollagen I and ultimately into procollagen (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 zinc-dependent proteases, including metalloproteinases (MMP-1, MMP-8, and MMP-13) and gelatinases (MMP-2 and MMP-9).

[0021] One aspect of the cosmetic formulation of the present invention includes the use of a barley extract formulation for increasing skin barrier strength and collagen synthesis, as identified by specific panel analysis, wherein the barley extract formulation has a molecular oxygen content of about 180 mbar partial pressure, as measured by a pulse oximeter, and a chlorophyll content of about 180 mg / kg. The barley extract aspect disclosed herein is obtained from barley grass as a leaf extract. Barley grass naturally contains a large amount of chlorophyll. The barley extract aspect also includes a large amount of chlorophyll obtained by increased photosynthesis due to specific light exposure. The barley grass used in the claimed barley extract aspect is harvested one week after sowing, or before the plant begins to produce flowers and seeds, when the young leaves have a peak chlorophyll content. The oily barley extract aspect includes chlorophyll and oxygen. This molecular oxygen generates energy in skin cells by increasing the production of the required adenosine triphosphate (ATP) to enhance the production of essential cellular proteins.

[0022] Barley used for barley extract was exposed to a 75-watt, 2400-lux light source. Individual samples were also tested using exposures of 60 watts, 600 lux, and 100 watts, 20000 lux. Data showed that barley exposed to 60 watts / 600 lux had an oxygen partial pressure of approximately 180 mbar; barley exposed to 75 watts / 2400 lux had an oxygen partial pressure of 183 mbar; and barley exposed to 100 watts / 20000 lux had an oxygen partial pressure of 178 mbar. The data indicates that the strongest light did not produce barley with the highest oxygen partial pressure. Barley exposed to 75 watts / 2400 lux produced the extract with the highest oxygen partial pressure.

[0023] The barley extract disclosed herein was obtained from barley cultivated under a specific type of light. The light used to prepare the barley extract was 2400 lux, as measured using a lux meter. One lux equals one lumen per square meter.

[0024] It has been found that oily barley extract exposed to high light intensity has been identified as having selective skin barrier strengthening and simultaneous energy generation properties. Compared to formulations without barley extract, this oily barley extract exhibits increased oxygenation.

[0025] Molecular oxygen from ambient air is adsorbed into the skin following the oxygen gradient.

[0026] Analysis of the ability of active ingredients to increase ATP synthesis and increase gene expression associated with upregulation of lipid transporters (ABCA12), ceramide synthesis (PNPLA1 and GBA), fatty acid synthesis (ELOVL1, ELOVL4, FASN, SCD), cholesterol synthesis (HMGCR, HMGSC1), and / or PPARα synthesis has recently been shown to be useful for selecting active ingredients that can improve the aesthetic appearance of the skin.

[0027] In addition, a combination of analyses assessing the ability of active substances to increase the expression of genes associated with keratinization capsule formation (IVL, SPRR1A, TGM1), stratum corneum adhesion (CDSN, CLDN4, OCLN), and peeling regulation (KLK5 and KLK7) has recently shown to be predictive in assessing the ability of active ingredients to improve aesthetic appearance.

[0028] Furthermore, a combination of analyses assessing the ability of active substances to upregulate genes associated with promoting skin hydration (AQP3, AQP9, HAS2) has recently shown to be predictive in assessing the ability of active ingredients to improve aesthetic appearance.

[0029] All oxygen-dependent living organisms obtain their energy from the respiratory system, where molecular oxygen is reduced to water and energy in the form of ATP. Every living organism requires a sufficient amount of energy to maintain metabolic activity, repair, and regenerate for cell division. In particular, human cells in the skin, directly exposed to sunlight, are highly dependent on energy to combat damage and cellular imbalances. Cellular energy levels decline with age, and these deficiencies are highly correlated with changes in human skin structure. This mechanism produces signs of aging.

[0030] Oxygen is an essential factor for collagen production. Adequate molecular oxygen concentrations are necessary to allow for the normal hydroxylation of collagen, and the accumulation of newly synthesized collagen increases in tissues with elevated PO(2). When oxygen enters fibroblasts, it increases their synthetic capacity.

[0031] Oxygen supply through the microcirculation and the amount of oxygen in the skin depend on factors such as age, sex, and risk of microvascular complications. Therefore, smokers have reduced amounts of oxygen in their skin. Male subjects typically have lower PO(2) levels than women. In women, PO(2) levels decline rapidly from age 30 to 45, then begin to increase again after age 50. This increase may be related to changes in skin thickness associated with hormonal changes and hormone replacement therapy. Aging is accompanied by a decrease in the amount of oxygen in the skin.

[0032] These aspects have been described in sufficient detail to enable those skilled in the art to practice this disclosure. Other aspects may be employed, and changes to the formulations and methods of use may be made without departing from the scope of this disclosure. Therefore, the detailed description should not be construed as limiting, and the scope of this disclosure is defined only by the full scope of the appended claims and their equivalents.

[0033] Those skilled in the art will understand that changes can be made to the above aspects without departing from its broad inventive concept. Therefore, it should be understood that this disclosure is not limited to the specific aspects disclosed, but is intended to cover modifications as defined in this specification within the spirit and scope of the disclosure.

[0034] Example

[0035] refer to Figure 1 This diagram illustrates the presence of ceramides (visible via immunostaining) in human keratinocytes from skin explants treated with a formulation containing 0.5% barley extract and those treated with the same formulation except for the presence of barley extract. Normal human skin explants were treated with 0.5% barley for 24 hours and incubated at 37°C and 5% CO2. Subsequently, formaldehyde-fixed paraffin-embedded skin sections were immunostained for ceramides using an avidin / biotin amplifier system with a monoclonal anti-ceramide antibody and revealed via VIP (a substrate for peroxidase), producing a purple stain upon oxidation. The results are represented by the surface of the immunostain. The staining depth indicates that application of the formulation containing 0.5% barley extract resulted in a 28% increase in ceramide production in explant skin cells after 24 hours.

[0036] refer to Figure 2 This presents an analysis of the expression of genes responsible for regulating skin barrier strength. For example... Figure 2The results showed that the use of 0.5% barley extract led to increased expression of certain genes associated with skin barrier enhancement. The protocol used for gene expression involved culturing normal human keratinocytes in serum-free keratinocyte medium (KSFM). Barley was diluted 0.5% in the medium and applied to the cells for 48 hours, followed by incubation at 37°C and 5% CO2. Untreated cells were incubated under the same conditions. Total RNA was extracted using the NucleoMag RNA Kit (Macherey-Nagel) with a Kingfisher Flex (Thermo Fischer Scientific) and quantified spectrophotometer at 260 nm. First-strand cDNA synthesis was performed using a High cDNA reverse transcription kit. Real-time PCR was performed using a QuantStudio 7 Flex real-time PCR system (Thermo Fischer Scientific) with TaqMan primers and probes specific to the genes studied. Relative changes in gene expression were calculated using the 2-ΔΔCt method with 7300 Systemsoftware™.

[0037] Figure 2 The results shown are summarized in Table 1 below.

[0038] Table 1

[0039]

[0040] refer to Figure 3 Use and about Figure 2 The same approach described above is used to further analyze the expression of genes responsible for regulating skin barrier strength. For example... Figure 3 It can be seen that using 0.5% barley extract led to an increase in certain genes related to skin barrier enhancement.

[0041] Figure 3 The results shown are summarized in Table 2 below.

[0042] Table 2

[0043]

[0044] refer to Figure 4 This study presents an analysis of the expression of genes responsible for regulating skin hydration. (Usage and related information) Figure 2 The same scheme is discussed. For example... Figure 4 The results show that using 0.5% barley extract led to an increase in the expression of genes related to skin hydration.

[0045] Figure 4 The results shown are summarized in Table 3 below.

[0046] Table 3

[0047]

[0048] refer to Figure 5 The effect of barley extract on increasing ATP synthesis. Normal human keratinocytes were cultured in serum-free KGM-2 medium. Normal human fibroblasts were cultured in DMEM (Dubai modified Eagle medium) containing 2% FBS (fetal bovine serum). Barley was diluted 0.1% in the medium and applied to the cells for 72 hours, and the cells were incubated at 37°C and 5% CO2. Untreated cells were incubated under the same conditions. Cellular ATP content was assessed using a bioluminescent microplate assay. ATP levels were normalized by total cellular protein content. Figure 5 As shown, ATP synthesis in keratinocytes increased by 51% compared to the composition without barley extract, and ATP synthesis in fibroblasts increased by 14% compared to the composition without barley extract.

[0049] refer to Figure 6 This study demonstrated the effect of barley extract on increasing collagen synthesis in fibroblasts, as assessed in vitro. The results showed the potential of barley extract to evaluate the novel synthesis of collagen-I. To achieve this, in vitro tests were performed using cell cultures of normal human dermal fibroblasts. Barley extract was tested at different doses of 0.1%, 0.25%, and 0.5% and exposed to cells for 72 hours, with some untreated cells serving as a control. The dose could be increased beyond 0.5%. A positive control prepared from 50 μg / ml sodium ascorbate was tested in parallel.

[0050] Normal human fibroblasts were cultured in DMEM (Dupuy's modified Eagle medium) containing 2% FBS (fetal bovine serum). Barley was diluted 0.5% in the medium and applied to the cells for 24 hours, followed by incubation at 37°C and 5% CO2. Untreated cells were incubated under the same conditions. Cell proliferation was assessed by quantifying total DNA content using the CyQuant Cell Proliferation Assay Kit.

[0051] This assay included ELISA analysis of untreated cells, cells treated with a positive control, and cells treated with different doses of barley extract to measure collagen-I levels in cell cultures. Furthermore, the inclusion of barley showed a 61% increase in collagen synthesis compared to the regimen without barley.

[0052] refer to Figure 7The results show the culturing of normal human fibroblasts in DMEM (Dupuy's modified Eagle medium) containing 2% FBS (fetal bovine serum). Barley was diluted 0.5% in the medium and applied to the cells for 24 hours, and the cells were incubated at 37°C and 5% CO2. Untreated cells were incubated under the same conditions. Cell proliferation was assessed by quantifying total DNA content using the CyQuant Cell Proliferation Assay Kit.

[0053] See Figure 8 Normal human fibroblasts were cultured in DMEM (Dupuyeh modified Eagle medium) containing 2% FBS (fetal bovine serum). Barley was diluted 0.5% in the medium and applied to the cells for 48 hours, followed by incubation at 37°C and 5% CO2. Untreated cells were incubated under the same conditions. Total RNA was extracted using the NucleoMag RNA Kit (Macherey-Nagel) with a Kingfisher Flex (Thermo Fischer Scientific) and quantified spectrophotometer at 260 nm. First-strand cDNA was synthesized using the High cDNA reverse transcription kit. Real-time PCR was performed using the QuantStudio 7 Flex real-time PCR system (Thermo Fischer Scientific) with TaqMan primers and probes specific to the genes under study. Relative changes in gene expression were calculated using the 2-ΔΔCt method with 7300 Systemsoftware™.

[0054] refer to Figure 9 Normal human skin explants were treated with 0.5% barley for 24 hours and incubated at 37°C and 5% CO2. Subsequently, frozen skin sections were immunostained with polyclonal anti-AQP3 antibody and exposed using fluorescent secondary antibody. The results were represented by the immunostained surface.

Claims

1. A method for improving the aesthetic appearance of aging-affected human skin, said aging being caused by both reduced ATP synthesis and weakened skin barrier function, said method comprising topically applying a therapeutically effective amount of an active agent, said active agent being selected to meet the following criteria: when applied to fibroblasts at a sufficient concentration (w / w) in a cosmetically acceptable medium and sustained for a sufficient duration to achieve an improvement in the aesthetic appearance of said human skin, said active agent upregulates one or more genes listed in Figures 1 to 5 and upregulates ATP synthesis.

2. The method of claim 1, wherein a standard of at least 50% increase in collagen synthesis in fibroblasts is further achieved.

3. The method according to claim 1, wherein the activator comprises at least one barley extract.

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

5. A method for screening a putative active agent capable of effectively improving the aesthetic appearance of human skin, the method comprising culturing fibroblast cultures in the presence or absence of the putative active agent; measuring the relative levels of gene expression and ATP synthesis of one or more genes listed in Figures 1 to 5 in treated and untreated cells; and selecting the putative active agent for use if the putative active agent upregulates ATP synthesis of at least one of the genes relative to baseline.

6. A method for providing a personalized approach to improve the aesthetic appearance of skin, the method comprising obtaining a skin cell sample from an individual; assessing the gene expression level of at least one gene listed in Figures 1 to 5 and the level of ATP synthesis in a cultured sample of the obtained skin cells; and if the cultured sample shows an increase in ATP synthesis and elevated expression of at least one gene listed in Figures 1 to 5 relative to baseline, then recommending an aesthetic appearance improvement program to the individual, the aesthetic appearance improvement program comprising the use of a cosmetic comprising an active agent that, when applied to fibroblasts at a concentration of 0.50% (w / w), increases ATP synthesis and the expression of at least one gene listed in Figures 1 to 5.

7. The method according to any one of claims 1 to 6, wherein the analysis selected is any one of those described in Figures 1 to 5.

8. The method according to any one of claims 1 to 7, wherein the gene expression is increased by at least 10% relative to baseline.

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