[go: up one dir, main page]

WO2025184553A1 - Procédés pour améliorer l'aspect de la zone périorbitaire - Google Patents

Procédés pour améliorer l'aspect de la zone périorbitaire

Info

Publication number
WO2025184553A1
WO2025184553A1 PCT/US2025/017918 US2025017918W WO2025184553A1 WO 2025184553 A1 WO2025184553 A1 WO 2025184553A1 US 2025017918 W US2025017918 W US 2025017918W WO 2025184553 A1 WO2025184553 A1 WO 2025184553A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
psb
skin
extract
comprised
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/017918
Other languages
English (en)
Inventor
James Vincent Gruber
Sebastien MASSARD
Nicole E. TERPAK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
VANTAGE SPECIALTY INGREDIENTS Inc
Original Assignee
VANTAGE SPECIALTY INGREDIENTS Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by VANTAGE SPECIALTY INGREDIENTS Inc filed Critical VANTAGE SPECIALTY INGREDIENTS Inc
Publication of WO2025184553A1 publication Critical patent/WO2025184553A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

Definitions

  • hemoglobin breakdown products accumulate in the fatty areas below the eyes the under-eye area manifesting as puffiness, pouch-like skin bags, and dark rings or circles. Dark circles are even more noticeable after sleeping.
  • blood pressure in the rich vascular bed under the eyes increases, causing blood to pool, and producing a darker appearance in the periorbital area.
  • ECM extracellular matrix
  • VAGFA Vascular Endothelial Growth Factor-A
  • NINJ-1 Ninjurin-1
  • Oxidative Stress Response Kinase-1 is an antioxidant protein expressed in keratinocytes and immune cells that is upregulated during times of oxidative stress. [38]. Hortle E, et al.
  • sinensis contains feruloylmethane (also known as dihydrozingerone), a derivative of curcumin, which has been reported in the literature to have potent antioxidant and healing benefits.
  • feruloylmethane also known as dihydrozingerone
  • curcumin a derivative of curcumin
  • 3,4- dihydroxybenzalacetone from the fruiting body is reported to suppress protein expression of iNOS, COX-2, TNF- ⁇ , interleukin-1 ⁇ , interleukin-6, MMP-2, and MMP-9, but also increased the expression of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase.
  • antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase.
  • Sunny Biodiscovery SBD.4A, a proprietary isolate of A. polymorpha var. sinensis, “rich in arginine and gallotanin”, improved dermal microcirculation.
  • CN106474012 discloses an eye cream for improving eye wrinkles comprised of extracts from several plants, each at a concentration of 0.005-0.015 parts by weight: Phellinus linteus; Burdock root extract; Purslane; Glycyrrhiza glabra; Paeonia root; Pueraria lobata; Cnidium officinale root; Kava leaf/root/stem.
  • FIGS.1-6 present the results of treatment with Phellinus linteus extract and Angelica polymorpha sinensis extract on expression of periorbital skin relevant genes and their corresponding proteins – namely, upregulation of CYGB, LCE3B, NINJ1 and OXSR1 by P. linteus; and upregulation of EGFR and downregulation of VEGFA by A. polymorpha var. sinensis.
  • FIG.7 summarizes the results of gene expression testing (microarray) and protein expression testing (ELISA).
  • FIG.8 presents Doppler flowmetry measurements on Day-14 and Day-28 after twice-daily application of a topical composition containing 1% periorbital skin benefit (PSB) concentrate of the present invention. Percentage changes versus baseline of a placebo cream (light green bars) are plotted versus a cream containing PSB concentrate at 1.0% concentration (dark green bars; labeled as “active”). Differences are statistically significant (p ⁇ 0.05).
  • FIG.9 plots changes in hydration of periorbital skin in Arbitrary Units (AU) at baseline (day 0) and after twice-daily application of a cream containing 1% PSB concentrate at the end of two and four weeks (days 14 and 28).
  • AU Arbitrary Units
  • FIG.10 maps changes in crow’s feet area (fine lines or wrinkles that form at the outer corners of the eyes, often as a result of aging and repeated facial expressions like smiling and squinting) after four weeks. "A" images are taken before treatment at baseline (on day 0); “B” images are taken after four weeks of twice-daily treatment with cream containing 1% PSB (on day 28). [0022] FIG.11 plots statistically significant changes in surface area and length of wrinkles in the crow’s feet area after twice-daily treatment of a cream containing 1% PSB concentrate. [0023] FIG.12 maps changes in upper lid sagging after four weeks.
  • FIG.13 presents electron ion chromatogram of three active constituents of PSB concentrate before and after heating to 80oC for three hours.
  • FIG.14 summarizes results of a questionnaire administered to subjects assigned to a test group and used an active formulation (with PSB concentrate) as directed for the duration of a 28- day study described in Paragraph [0099] below. Subjects in the test group provided one of four responses to nine questions – strongly agree; agree; disagree; or strongly disagree.
  • the provided cosmetic benefit(s) include: (a) reducing appearance of one or more of periorbital puffiness, dark circles, fine lines and/or wrinkles (crow’s feet), (b) improving skin firmness, smoothness and/or thickness, and/or (c) reducing transepidermal water loss.
  • Angelica polymorpha var. sinensis extract is comprised of at least about 15% amino acids and at least about 7.5% phenolics.
  • Angelica polymorpha var. sinensis extract is comprised of feruloylmethane at a concentration of at least about 1 ppm, preferably at least about 20 ppm, more preferably at least about 50 ppm, even more preferably at least about 100 ppm.
  • the feruloylmethane content of Angelica polymorpha var. sinensis extract is from about 150 ppm to about 1 ppm, preferably from about 50 ppm to about 3 ppm, and more preferably from about 20 ppm to about 5 ppm.
  • the Phellinus linteus extract contains hispolon at a concentration of at least about 0.05 ppm, preferably at least about 0.1 ppm, more preferably at least about 0.5 ppm, even more preferably at least about 1 ppm, still more preferably at least about 5 ppm, and most preferably at least about 10 ppm.
  • the hispolon content of Phellinus linteus extract is from about 10 ppm to about 0.05ppm, preferably from about 2.0ppm to 0.1ppm.
  • Phellinus linteus extract and Angelica polymorpha var. sinensis extract are preferably combined in a hydrophilic carrier – either water or a hydroalcoholic vehicle, a combination of water and a diol or triol, preferably glycerin (1,2,3-propanetriol) or butylene glycol (1,3- butanediol) – to form a periorbital skin benefit (“PSB”) concentrate.
  • PSB periorbital skin benefit
  • the PSB concentrate preferably contains arginine at a concentration of at least about 0.05 ppm, preferably at least about 0.1 ppm, more preferably at least about 0.5 ppm, even more preferably at least about 1 ppm, still more preferably at least about 5 ppm, and most preferably at least about 10 ppm.
  • arginine content in the PSB concentrate is from about 10 ppm to about 0.05ppm, preferably from about 2.0ppm to 0.1ppm.
  • the PSB concentrate is added to an aqueous dispersion comprised of one or more thickening agents and a preservative system.
  • the PSB concentrate is added to an emulsion – namely, a cream, lotion, gel, or serum – comprised of one or more surfactants.
  • the finished formulations topical compositions in the form of an aqueous dispersions or emulsion
  • the same formulations may be applied to other areas of the body, including for examples to areas prone to skin thinning and/or bruising (e.g., back of hands).
  • Emulsions are typically prepared by adding one or a combination of water-soluble ingredients to a continuous oil phase; or adding one or a combination of oil-soluble ingredients to a continuous water phase.
  • the addition and mixing of the oil- and water- phase ingredients is typically performed at elevated temperature (e.g., at least about 65°C), which can degrade “active” ingredients – here, extracts of P. linteus and A. polymorpha var. sinensis.
  • the PSB of the present invention is very thermal stable – de minimis degradation of three active constituents in the PSB concentrate – hispolon, feruloylmethane, and arginine – is observed. See FIG.13.
  • the inventive compositions and methods reduce the amount of hemoglobin and/or melanin and/or the volume of bags under the eyes and improve blood circulation under the eyes.
  • the PSB concentrate of the present invention is applied to the periorbital area of a human at a concentration ranging from about 0.1% to about 10%, preferably from about 0.5% to about 10%, and most preferably at least about 1%.
  • One aspect of the present invention is directed to a method of eliciting a change of at least about 1.3-fold in the level of gene expression (either increase or decrease) as quantified by DNA microarray analysis of each of four periorbital skin-relevant genes – CYGB, OXSR1, LCE3B, EGFR – by administering the PSB concentrate of the present invention for at least 24 hours in the constituent ratios set out in Paragraph [0031] above to a periorbital skin-relevant substrate – either a normal, human epidermal keratinocytes (“NHEK”) cell culture model described in Paragraph [0038] immediately below; or a human-relevant full thickness (“FT”) tissue comprised of NHEK and normal, human dermal fibroblasts available as EpiDerm FT from MatTek Corp.
  • NHEK normal, human epidermal keratinocytes
  • FT human-relevant full thickness
  • DNA microarray analysis is a technique known to persons having ordinary skill in the art and is described in Gruber et al. (2024). Int J Cosmet Sci 46:995-1003. DNA microarray of selected periorbital skin relevant genes are evaluated using microarrays known to the skilled artisan, including OneArray® from Phalanx Biotech Group (San Diego, CA) or INSERT from Agilent Technologie (Santa Clara, CA). EpiDerm FT from MatTek is described in Dumont et al. (Sept.2015). Applied In Vitro Toxicology 1(3):226-233. Both of the above publications – Gruber et al. and Dumont et al.
  • NHEK Cell Culture Model NHEKs are grown using EpiLife® Media, with 60 ⁇ M calcium (Thermo Fisher Scientific, Inc. Waltham, MA) supplemented with 0.2% v/v bovine pituitary extract, 1 ⁇ g/ml recombinant human insulin-like growth factor-I, 0.18 ⁇ g/ml hydrocortisone, 5 ⁇ g/ml bovine transferrin, 0.2 ng/ml human epidermal growth factor. The cells are cultured at 37 ⁇ 2°C and 5 ⁇ 1% CO 2 , seeded into 12-well plates and grown until confluent.
  • a second aspect of the present invention is directed to a method of eliciting a 20% (or greater) increase as quantified by Enzyme-Linked Immunosorbent Assay (“ELISA”) in the concentration of each of five periorbital skin-relevant proteins – namely, CYGB, OXSR1, LCE3B, NINJ1 and EGFR – by administering the PSB concentrate of the present invention in the constituent ratios set out in Paragraph [0031] to the NHEK cell culture model described immediately above in Paragraph [0038].
  • This aspect of the invention also elicits a decrease in the concentration of VEGFA by about 25%.
  • the second aspect of the present invention elicits a 25% (or greater) increase in the concentration of each of four periorbital skin relevant proteins – CYGB, OXSR1, LCE3B, NINJ1.
  • the second aspect of the present invention elicits a 50% (or greater) increase in the concentration of each of three periorbital skin relevant proteins – OXSR1, LCE3B and NINJ1..
  • periorbital skin treatment compositions of the present invention can, and preferably do contain, one or more ingredients that provide skin benefit(s) by one or more of the following modes of action: [0043] Antioxidant – reduces oxidative damage (stress) and/or quenches free radicals as measured by FRAP assay or DPPH assay, or other methods known to the skilled artisan. Anal. Biochem. Vol.239 No.1, pp.70–76 (1996) (FRAP); Food Sci Technol, Vol.30, pp.609–615 (1997) (DPPH); J. Agric. Food Chem.
  • NF- ⁇ nuclear factor kappa ⁇
  • COX cyclooxygenase
  • TNF- ⁇ tumor necrosis factor alpha
  • MCP-1 monocyte chemoattractant protein
  • retinoids may help thicken the skin over the periorbital area, hiding the darker blood rich skin beneath, they are often irritating to the sensitive skin around the eyes. Accordingly, retinoids may or may not be included in under-eye compositions of the present invention; and, when present, may be included at low concentrations.
  • [0048] Supports production of hyaluronic acid and/or collagen; in certain embodiments, e.g., acetyl glucosamine. [0049] Reduces the appearance of fine lines or wrinkles. [0050] Reduces transepidermal water loss and/or improves skin barrier function. [0051] Strengthens elastin and/or collagen (reducing capillary leakage) and/or promotes angiogenesis.
  • a peptide comprised on three to seven amino acid residues; in certain embodiments Acetyl Hexapeptide-8 (sold under the tradename ARGIRELINE®) or Tripeptide-32.
  • Vitamins A, B, C, D, E and K and derivatives thereof selected from Vitamins A, B, C, D, E and K and derivatives thereof; preferably at least one of Vitamin C, Vitamin E, or their derivative(s), including, but not limited to, Tetrahexyldecyl Ascorbate; Aminopropyl Ascorbyl Phosphate; Tocopheryl Acetate; Niacinamide.
  • Caffeine or a source of caffeine e.g., green tea extract
  • Hyaluronic Acid or its derivative or other humectant; in certain embodiments Sodium Hyaluronate; in other embodiments multi-molecular weight hyaluronic acids (TRILURONIC® Acid from Vantage Specialty Ingredients, Inc.); in still other embodiments a pyrrolidone carboxylic acid (Sodium PCA or Lauryl PCA); a urea compound (e.g., Hydroxyethyl Urea).
  • a ferment in certain embodiments selected from Bifida Ferment Lysate; Lactobacillus Ferment; Thermus Thermophillus Ferment; Saccharomyces Ferment.
  • Licorice or its derivative – Dipotassium Glycyrrhizate A protein or hydrolyzed protein; in certain embodiments selected from Hydrolyzed Rice Protein; Hydrolyzed Wheat Protein; Hydrolyzed Yeast Protein e.g., whey protein, available under the tradename Whey Protein NXP from Glanbia).
  • Jojoba or its derivative in certain embodiments selected from Jojoba Esters (e.g., Isopropyl Jojobate) or Jojoba Alcohol.
  • a lipid or ceramide that supports and/or improves skin barrier function in certain embodiments Phytosphingosine [0063] A sugar or an ingredient rich in sugars: Saccharum Officinarum; Sorbitol; Sucrose; Trehalose [0064] A marine extract; in certain embodiments selected from Algae Extract; Artemia Extract; Hydrolyzed Algin.
  • plant derived ingredients that can be, and in certain embodiments are, included in under-eye compositions of the present invention include: Adansonia Digitata Seed Extract; Anthemis Nobilis (Chamomile) Flower Extract; Anthemis Nobilis Flower Oil; Ascophyllum Nodosum Extract; Asparagopsis Armata Extract; Betula Alba (Birch) Extract; Boswellia Serrata Extract; Camelina Sativa Seed Oil; Chamomilla Recutita (Matricaria) Flower Oil; Citrullus Lanatus (Watermelon) Fruit Extract; Citrus Aurantium Dulcis (Orange) Peel Extract; Cladosiphon Okamuranus Extract; Cucumis Sativus (Cucumber) Fruit Extract; Glycine Soja (Soybean) Seed Extract; Helianthus Annuus (Sunflower) Seed Extract; Hibiscus Sinensis Flower Extract; Hordeum Vulgare (Barley) Extract; Hypnea Musciformis Extract; Laminaria Digi
  • the periorbital skin treatment composition contains, Sodium Hyaluronate; in certain embodiments in combination with Tripeptide-32 and/or Tocopheryl Acetate; in other embodiments in combination with Moringa Oleifera Seed Extract and/or Opuntia Tuna Extract.
  • the periorbital skin treatment composition contains Tuber Melanosporum Extract, including in combination with Narcissus Tazetta Bulb Extract.
  • the periorbital skin treatment compositions include pigments of various colors to cover over or offset the darkness of the dark circles and reflect incident light, including TiO 2 and Iron Oxides.
  • the following examples are illustrative. Modifications will be apparent to, and can be readily made by, those skilled in the art without departing from the spirit and scope of the invention. The scope of the appended claims is not to be limited to the examples. (Unless indicated with an asterisk, supplier of ingredient is Vantage Specialty Ingredients, LLC).
  • the resulting cream has a viscosity of 85,000 cps (RVT, TC, 4 rpm, 1 min), a pH of about 5.8 and is stable when stored at 45C for twelve weeks and after three freeze/thaw cycles.
  • a Aqua (Water) 65.00 00 A Sodium Acrylate/Sodium Acryloyl Dimethyl Taurate Copolymer, Cetyl Alcohol, Glycol 4.00 Stearate, Glyceryl Stearate, Caprylic/Capric Triglyceride 0 0 0 0 0 0 ts one at a time and mix until uniform. Add Phase C and mix until uniform. Add Phase D and mix until uniform. Add Phase E and mix until uniform. Add Phase F and mix until uniform. Adjust pH to 5.5.
  • the test material used in the vitro testing examples below is comprised of PSB concentrate (1% or 0.1%) in water.
  • the in vivo testing example below is uses a thickened aqueous dispersion comprised of: 1% PSB concentrate, 5% thickener [Sodium Acrylate/Sodium Acryloyl Dimethyl Taurate Copolymer, Cetyl Alcohol, Glycol Stearate, Glyceryl Stearate, and Caprylic/Capric Triglyceride; commercially available as JEESPERSE® ICE-T LB-TNS from Vantage Specialty Ingredients, Inc.
  • EpiDerm FT Model Tissue samples are removed from the shipping tray, placed into a 6- well plate containing 2.5-5.0 ml of assay medium (37 ⁇ 2°C), and incubated for at least 24 hours at 37 ⁇ 2°C. and 5 ⁇ 1% CO 2 . After this initial incubation, the assay medium is replaced with 2.5-5.0 ml of fresh medium (37 ⁇ 2°C).25-50 ml of test material (extracts that are components of the PSB concentrate) and/or phosphate buffered saline (“PSB” as a negative control) is then applied directly onto the surface of the tissue. The 6-well plates are then incubated at 37 ⁇ 2°C. and 5 ⁇ 1% CO 2 for 24 hours.
  • assay medium 37 ⁇ 2°C
  • test material extracts that are components of the PSB concentrate
  • PBSB phosphate buffered saline
  • tissue samples are washed with 100 ml of PSB and placed into a 1.5 ml centrifuge tube containing 10-12 volumes of guanidinium thiocyanate lysis solution.
  • the tissues are minced with fine tipped scissors and homogenized until thoroughly disrupted. After homogenization, the tissues are centrifuged at 15,000 RPM for 10 minutes. The supernatant is transferred to a new tube. The pellet (tissue debris) is discarded, and the tissue homogenate is then stored at ⁇ 75°C until the RNA extraction process (described below) is completed.
  • NHEK Cell Culture Model Cell culture media for NHEKs prepared as described in Paragraph [0038] is removed and replaced with media containing test material (extracts that are components of the PSB concentrate). Cells are then incubated for 48 hours. At the end of the incubation period, the cells are washed once with PSB and then lysed with 100 ⁇ l of extraction buffer.
  • RNA Isolation RNA isolation was performed using the RNAqueous Kit from Ambion Inc. (Austin, Tex.). To the cell lysates or tissue homogenates prepared above, an equal volume of 64% ethanol is added, and the tubes are vortexed.
  • RNA bound to the glass fibers within the cartridge is then eluted by applying 30 ml of Tris-EDTA buffer (Sigma) (10 mM Tris-HCl, 1 mM EDTA (Sigma), preheated to 70-80°C, hereinbelow “TE buffer”) to the cartridge and centrifuging the cartridge in a new collection tube at 14,000 RPM for one minute.
  • Tris-EDTA buffer Sigma
  • TE buffer preheated to 70-80°C
  • RNA Concentration Assay Ribogreen reagent (Nanoprop Technologies, Wilmington, Del.) is provided as a stock solution in DMSO. Prior to use, the reagent is diluted 2000-fold in TE buffer. The RNA assay requires 200 ml of diluted Ribogreen reagent per sample to be tested and 1 ml of reagent as a standard. The diluted reagent is stored protected from light. A series of RNA standards are prepared by diluting purified ribosomal RNA derived from E.
  • RNA samples prepared above Prior to assaying, the RNA samples prepared above are diluted 1,000-fold in TE buffer. For the RNA assay, 100 ml of the diluted samples or standards are transferred to the wells of a black 96-well plate. The samples and standards are assayed in duplicate. After the samples/standards are added to the plate 100 ml of diluted Ribogreen assay reagent is added to the wells and the plate is gently mixed and allowed to incubate for 5-10 minutes protected from the light.
  • RNA Gel Electrophoresis A 1% RNA gel is prepared by adding 0.3 g agarose to 21.6 ml diethylpyrocarbonate (DEPC) treated water. The agarose is dissolved by boiling the water in a microwave oven.
  • DEPC diethylpyrocarbonate
  • the comb is removed, and the buffer chamber of the gel apparatus is filled with 150-175 ml 1 ⁇ MOPS (enough buffer is added to cover the gel with approximately 3 mm of buffer).
  • the cover is placed on the apparatus, the electrical leads are attached to the power source, and the empty gel is run at 40 V (4 V/cm) for 5-10 minutes.
  • the RNA samples are prepared by transferring approximately 1 mg of each sample RNA to a 600 ml PCR tube.
  • DEPC H 2 O is used to bring the total volume of all the samples to a common level and then 1-3 volumes of a gel-loading buffer (i.e.5% glycerol, 1 mM EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol FF, 20% formaldehyde, 50% formamide, 10 mg/ml ethidium bromide) are added.
  • a gel-loading buffer i.e.5% glycerol, 1 mM EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol FF, 20% formaldehyde, 50% formamide, 10 mg/ml ethidium bromide
  • the samples are denatured by placing them at 65-70°C for 5-15 minutes and then placed on ice to cool.
  • the samples are then carefully loaded into the lanes (each loading slot can hold 10-15 ml of sample, depending upon the thickness of the gel) and run on the gel at 40 V for 1-3
  • RNA is visualized by placing the gel on a UV transilluminator (Cleaver Scientific). An RNA sample is used for subsequent processing if both the 18S and 28S ribosomal bands are clearly visible and there is little or no staining below the 18S band.
  • Amplified mRNA Amplification (aRNA) mRNA is amplified using the MessageAmp, aRNA kit from Ambion Inc. (Austin, Tex.) as follows: [0081] First Strand cDNA Synthesis: To start the first strand synthesis, 5 mg of total RNA for each sample are added to 600 ml PCR tubes and the total volume of liquid in the tube is adjusted to 12 ml with DEPC H 2 O.
  • T7 Oligo(dT) primer is added to each tube, and the tube is incubated at 70 ⁇ 2°C for 10 minutes to denature the RNA and is then placed on ice to allow the primer to anneal to the poly A ends of the mRNA.
  • 2 ml of 10 ⁇ first strand buffer, 1 ml of RNAse inhibitor and 4 ml of dNTP mix is added to each tube, and the tube is placed at 42°C.
  • 1 ml of reverse transcriptase is added, and the tubes are returned to 42 ⁇ 2°C for 2 hours.
  • the tubes are briefly centrifuged to collect all of the fluid at the bottom of the tube and then placed on ice.
  • Second Strand Synthesis and cDNA Purification For the synthesis of the second strand of cDNA the following ingredients are added sequentially to the tubes: 63 ml DEPC H 2 O, 10 ml 10 ⁇ second strand buffer, 4 ml dNTP mix, 2 ml DNA Polymerase and 1 ml of RNAse H. The tube is mixed and then incubated at 16 ⁇ 2°C for 2 hours. Towards the end of the 2-hour incubation, a sufficient quantity of DEPC H 2 O is warmed to 50 ⁇ 2°C, and a cDNA purification filter cartridge is equilibrated with 50 ml of cDNA binding buffer (one cartridge per sample) for at least 5 minutes.
  • cDNA binding buffer 250 ml of cDNA binding buffer are added to each tube and thoroughly mixed.
  • the contents of the PCR tube are then transferred to the cDNA purification filter cartridge.
  • the cartridge is then placed in a collection tube and centrifuged at 10,000 RPM for 1 minute.
  • the flow-through is discarded and 650 ml of cDNA wash solution is added to the cartridge.
  • the cartridge is centrifuged again, the flow-through is discarded, and is then centrifuged one additional time to ensure that the wash buffer has been completely emptied from the filter.
  • the cDNA is eluted by applying 10 ml of preheated DEPC H 2 O to the filter and centrifuging the filter in a new collection tube at 10,000 RPM for one minute.
  • In vitro transcription begins by adding the following to the cDNA solution: 4 ml each of T7 ATP solution, T7 CTP solution, T7 GTP solution, T7 UTP solution, 4 ml of 10 ⁇ Reaction buffer, and 4 ml of T7 enzyme mix. The tube is mixed and then incubated at 37 ⁇ 2°C for 6-14 hours.
  • a sufficient volume of Elution Solution is warmed to 50-60°C and an aRNA filter cartridge is equilibrated with 100 ml of aRNA binding buffer for at least 5 minutes.
  • 350 ml of aRNA binding buffer is added to the sample tubes and thoroughly mixed.
  • An additional 250 ml of absolute ethanol is also added to each tube.
  • the mixture is then transferred to an aRNA filter cartridge; the cartridge is then inserted into a collection tube and centrifuged at 10,000 RPM for 1 minute. The flow-through is discarded and 650 ml of aRNA wash buffer is added to the cartridge followed by centrifuging at 10,000 RPM for one minute.
  • the cartridge After discarding the flow-through, the cartridge is spun one final time to remove all traces of the wash buffer. The cartridge is then transferred to a new collection tube.25 ml of pre- warmed Elution Solution is added to the cartridge. The cartridge is incubated for 2 minutes at room temperature and then aRNA is eluted by centrifuging for 1 minute at 10,000 RPM. This elution is performed one additional time to give a total volume of 45-50 ml of aRNA solution. The final concentration of the aRNA is determined by the Ribogreen assay described above. In addition, the quality of the aRNA is checked via gel electrophoresis as described above. An aRNA sample is used for subsequent processing if a broad band of RNA is observed.
  • aRNA is labeled with fluorescent dyes using the PerkinElmer ASAP RNA Labeling Kit.
  • Two tubes are prepared for the labeling process—for the untreated sample Cy3 labeling (green), and for the treated sample Cy5 labeling (red).
  • To the Cy3 tube add 2 mg of aRNA prepared from the untreated/control sample and add enough DEPC H 2 O to bring the total volume up to 4 ml.
  • To the Cy5 tube add 2 mg of aRNA prepared from the sample treated with the test material and add enough DEPC H 2 O to bring the total volume up to 4 ml.
  • To both tubes add 5 ml of ASAP labeling buffer and 1 ml of the specific dye for the tube (Cy3 or Cy5).
  • a micron YM-30 filter column is inserted into a collection tube and filled with 400 ml of TE buffer. The Cy3 and Cy5 probes are combined (12.5 ml of each) and then added to the micron filter and thoroughly mixed with the TE buffer. The filter is centrifuged at 12,000 RPM for 8 minutes and the flow-through is discarded.
  • Microarray Hybridization and Washing For hybridization, 45 ml of 10 ⁇ control target RNA (supplied with Agilent Technologies In Situ Hybridization Kit) is mixed with 160 ml of DEPC H 2 O and 9 ml of 25 ⁇ Agilent Fragmentation Buffer. This mixture is incubated at 60°C for approximately 30 minutes in a hybridization oven.
  • Agilent Hybridization Buffer is added along with the fluorescent aRNA probes prepared above.
  • the mixture is then incubated at 70°C for 5-10 minutes in a water bath.
  • an Agilent SUREHYB hybridization chamber is prepared by inserting a glass gasket slide into the bottom half of the chamber.
  • the hybridization mixture (approximately 450 ml) is applied to the glass gasket slide and an Agilent Human 1A Oligo Microarray Chip is placed face down on top of the gasket such that the hybridization solution is sandwiched between the glass gasket slide and the microarray face of the chip.
  • the top half of the chamber is attached, and the connecting thumbscrew tightened. After verifying that there is good bubble formation in the chamber, it is placed into the hybridization oven for approximately 17 hours (60°C and rotating at 4 RPM).
  • the microarray/glass gasket is removed from the SUREHYB chamber and placed in 50 ml of a first wash solution (room temperature, 6 ⁇ SSC, 0.005% Triton X-102). After the gasket has fallen away from the microarray, the array is transferred to 300 ml of fresh wash solution 1 on a magnetic stir plate.
  • microarray Scanning and Analysis The microarrays are scanned with an Axon GenePix 4100A Scanner with the scanning resolution set to 10 mm and analyzed with GenePix Pro software. During the initial scan the PMT gains for the scanner are adjusted such that the Cy5/Cy3 image count ratios are between 0.88 and 1.12.
  • the relative fluorescent units (RFU) versus the known RNA concentrations in mg/ml for the standards is plotted and subjected to regression analysis to establish the line that best fits these data points.
  • Mean RFU values for the test materials and untreated samples are then used to estimate the amount of RNA present in each sample.
  • the level of gene expression is related to the fluorescence intensity of the probed gene marker on the microarray. Fluorescence measurements between the Cy3 and Cy5 probes are normalized. The total fluorescent signal for both dyes is normalized with a correction factor such that the ratio of total intensities for both dyes equal to one.
  • Criteria for evaluating changes in gene expression include the following: (i) the ratio of Cy3/Cy5 (untreated/treated) fluorescence intensity is greater than 1.5 or less than 0.66, corresponding to a change in gene expression of at least +/ ⁇ 30%; (ii) the fluorescence intensity of the gene marker is greater than the background intensity; (iii) the gene feature is clearly marked specifically by the aRNA probes and is not due to non-specific fluorescence.
  • the first two criteria are filtered via computer analysis. The last criterion requires visual inspection of the array.
  • Cy3/Cy5 ratios of greater than about 1.3 are interpreted to indicate that a gene is upregulated by the treatment, whereas ratios of less than about 0.7 are interpreted to indicate a downregulated gene.
  • a ratio of 0.7 means that the treated value was 70% of the untreated value, indicating a 30% decrease in gene expression.
  • EpiDerm FT treated for 24 hours with 1% Phellinus linteus extract (hispolon content of at least about 0.05 ppm) exhibited Cy3/Cy5 ratio of >1.3 for the following genes: OSXR1, LCE3B and CYGB.
  • EpiDerm FT treated for 24 hours with 1% Angelica polymorpha sinensis extract (at least about 1 ppm of feruloylmethane) exhibited a Cy3/Cy5 ratio of >1.3 for EGFR gene.
  • EpiDerm FT treated for 24 hours with 1% Angelica polymorpha sinensis extract (at least about 1 ppm of feruloylmethane) exhibited a Cy3/Cy5 ratio of ⁇ 0.7 for VEGFA gene.
  • ELISA At the end of treatment, the periorbital skin-relevant substrate – normal, human epidermal keratinocytes (“NHEK”) cell culture model or EpiDerm® FT – is homogenized in radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (Thermo Fisher Scientific), centrifuged to pellet any insoluble material. Protein concentration of the supernatant is determined using BCA.
  • NHEK human epidermal keratinocytes
  • MTT Assay At the end of treatment period, the treated periorbital skin-relevant substrate is placed into a 6 well plate with 2.5 ml of DMEM supplemented with 1 mg/ml MTT ( (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The well plate is incubated for 3 hours at 37 ⁇ 2°C and 5 ⁇ 1% CO2. After the incubation, the tissues are rinsed with phosphate buffered saline (PSB) and placed into a 6-well plate containing 4 ml of isopropyl alcohol per well and are incubated for 2 hours at room temperature.
  • PSB phosphate buffered saline
  • BCA Protein Assay A series of protein standards is prepared using BSA ranging from 0 to 1,000 ⁇ g/ml. Ten microliter aliquots of the standards and 5 ⁇ l aliquots of the cell lysate samples are then transferred to the wells of a 96-well plate. Two hundred microliters of working BCA reagent are added to each well and the plate is incubated for 30 minutes at 37°C. At the end of the incubation period the well plate is read at 540 nm using a plate reader.
  • enzymatic signal generation requires catalysis of a substrate to produce a colored (or fluorescent). Colorimetric substrates form a colored product that accumulates over time relative to the amount of enzyme present in each well. A color generation reaction is allowed to proceed until differences in color intensity of are clearly discernable. When the desired color intensity is reached, 50 ⁇ l of a stop solution is added to each well, providing a fixed end point for the assay. The enzymatic signal is measured using a spectrophotometric plate reader at 460 nm.
  • ELISA assays 100 ⁇ l of standards or diluted lysates (10 ⁇ l lysate diluted with 90 ⁇ l of sample buffer) are added to the wells of an ELISA plate and the plate is incubated for 90 minutes at 37°C. At the end of the incubation period the ELISA plate is emptied and 100 ⁇ l of biotinylated detection antibody solution is added to each well. The plate is then incubated for 60 minutes at 37°C and then washed three times with a wash buffer. After the final wash was removed, 100 ⁇ l of an avidin-enzyme solution is added to each well and the plate is incubated for 30 minutes at 37°C.
  • Colorimetric change is expressed as Delta E using the L*a*b* three coordinate system CIELAB 1976 and can be measured using a chromameter (CR-400, Konica Minolta, Japan) or a handheld spectrophometer (CM2600-d, Konica Minolta, Japan).
  • CM2600-d Konica Minolta, Japan
  • Reduction in fine lines and wrinkles and pores is measured by computer image analysis, including with cross-polarized facial photographs, for example, using VISIA-CR (Canfield, USA).

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biotechnology (AREA)
  • Botany (AREA)
  • Engineering & Computer Science (AREA)
  • Birds (AREA)
  • Epidemiology (AREA)
  • Dermatology (AREA)
  • Gerontology & Geriatric Medicine (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

L'invention concerne un procédé de fourniture d'un ou de plusieurs bénéfices pour la peau à la zone périorbitaire par application d'une composition de traitement de la peau périorbitaire constituée d'extraits de Phellinus linteus et d'Angelica polymorpha var. sinensis. L'avantage ou les avantages cosmétiques fournis comprennent : (a) la réduction de l'aspect d'un ou de plusieurs éléments parmi la facilité de traitement de la peau périorbitaire, les cernes, les ridules et/ou les rides (pattes d'oie), (b) l'amélioration de la fermeté, du lissé et/ou de l'épaisseur de la peau, et/ou (c) la réduction de la perte d'eau transépidermique. Les procédés de l'invention permettent de réduire la quantité d'hémoglobine et/ou de mélanine et/ou le volume de poches sous les yeux.
PCT/US2025/017918 2024-02-28 2025-02-28 Procédés pour améliorer l'aspect de la zone périorbitaire Pending WO2025184553A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463559003P 2024-02-28 2024-02-28
US63/559,003 2024-02-28

Publications (1)

Publication Number Publication Date
WO2025184553A1 true WO2025184553A1 (fr) 2025-09-04

Family

ID=96921724

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/017918 Pending WO2025184553A1 (fr) 2024-02-28 2025-02-28 Procédés pour améliorer l'aspect de la zone périorbitaire

Country Status (1)

Country Link
WO (1) WO2025184553A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104771350A (zh) * 2015-03-18 2015-07-15 王秀凤 一种中草药组合物的制备工艺及其应用
CN105943483A (zh) * 2016-06-14 2016-09-21 邬德明 Aob抗皱净面护肤(液)及制备工艺
CN116998723A (zh) * 2022-04-28 2023-11-07 江苏正元堂生物科技有限公司 一种利用桑黄提取物复方保健品及其制备工艺

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104771350A (zh) * 2015-03-18 2015-07-15 王秀凤 一种中草药组合物的制备工艺及其应用
CN105943483A (zh) * 2016-06-14 2016-09-21 邬德明 Aob抗皱净面护肤(液)及制备工艺
CN116998723A (zh) * 2022-04-28 2023-11-07 江苏正元堂生物科技有限公司 一种利用桑黄提取物复方保健品及其制备工艺

Similar Documents

Publication Publication Date Title
CN107875067B (zh) 皮肤细致组合物及其应用
US9044490B2 (en) Active ingredient obtained from Cichorium intybus for acting on the barrier function of the skin that is similar to that of vitamin D
CN103816095A (zh) 高级糖化终产物的去糖化
KR101716680B1 (ko) 자운고 발효 오일 및 이의 제조방법
CN107714531B (zh) 抗敏舒缓组合物及其应用
JP2010529092A (ja) ＭＣ−１Ｒ、ＭＣ−２Ｒ、および／またはμオピオイド受容体刺激
JPH1029929A (ja) 目の下の皮膚のくすみをとるための合成物と方法
TWI784229B (zh) 金銀花發酵物的製備方法及其改善皮膚外觀與抗老化的用途
CN101491490A (zh) 美白祛斑组合物
US8226956B2 (en) Extract of R. miehei
US9000033B2 (en) Composition for improving skin condition and appearance
CN111544372A (zh) 发酵产物复配植物提取物抗衰修复组合物及应用与含该组合物的精华乳
CN111265441A (zh) 可增强皮肤夜间修复、再生能力的组合物及其应用
CN116712370A (zh) 一种温和的具有美白保湿功效的化妆品组合物及其制备方法
WO2025184553A1 (fr) Procédés pour améliorer l'aspect de la zone périorbitaire
KR20050106710A (ko) 피부 외용제 조성물
CN107427453A (zh) 五味子果实的肽和糖苷提取物及其对皮肤神经系统反应的改善作用
CN111743798B (zh) 一种多效抗衰护肤品及其制备方法
CN119925201A (zh) 一种含羟基频哪酮9-顺式视黄酸酯的紧致抗皱组合物及其制备方法
CN113116798A (zh) 一种含抗初老组合物的面霜及其制备方法
Uwaya et al. Topical antiaging activity of a combination of Morinda citrifolia (noni) seed concentrate, fruit juice, leaf juice, and seed oil.
CN113456732B (zh) 一种淡化黄褐斑的组合物及其制备方法
EP3434256A1 (fr) Compositions d'herbes à usage topique
TWI754137B (zh) 白芷發酵物及其製備方法與用於改善皮膚外觀的用途
KR102468264B1 (ko) 국소 허브 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25762834

Country of ref document: EP

Kind code of ref document: A1