Methods for improving the appearance of the periorbital area

Abstract

A method of providing one or more skin benefits to the periorbita area by applying a periorbital skin treatment composition comprised of extracts of Phellinus linteus and Angelica polymorpha var. sinensis. The provided cosmetic benefit(s) include: (a) reducing appearance of one or more of periorbital skin treatment 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. The inventive methods reduce the amount of hemoglobin and / or melanin and / or the volume of undereye bags.

Description

Methods for Improving the Appearance of the Periorbital Area BACKGROUND OF THE INVENTION

[0001] Skin around the human eyes is thinner and more delicate and contains less fat than other areas on the body. The periorbital area is also rich in capillaries. With age, the capillaries become increasingly fragile and dilated, blood flow slows, and the vasculature begins to leak hemoglobin (and related globins) into the surrounding tissues. Leaked hemoglobin degrades through reactive oxygen species (ROS) oxidative processes, producing by-products (including bilirubin and hemosiderin) that are darker than hemoglobin. With aging, blood flow and drainage of these by- products (through the skin’s lymph system) slow. As a consequence, 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. In a prone position, blood pressure in the rich vascular bed under the eyes increases, causing blood to pool, and producing a darker appearance in the periorbital area. Additionally, with aging, degradation of collagen, elastin and other extracellular matrix (ECM) proteins increases causing skin to become laxer and exhibit crepiness, fine lines, and wrinkles. The breakdown of ECM proteins can and do lead to formation of advanced glycation end- products (AGEs) which create a yellow coloring in aging skin. Other environmental factors that can contribute to puffiness, bags, and darks circles, include UV-induced damage, irritation of the eyes and the surrounding skin, lack of sleep, alcohol consumption. With aging, hemoglobin breakdown products and AGEs can likewise be seen on the back of the hands.

[0002] Over expression of Vascular Endothelial Growth Factor-A (VEGFA) can lead to capillary dilation and result in increased leakage from expanded blood vessels. Vrcek I, et al. (2016) J Cutan Aesthet Surg 9:65-72; Ryan T. (2004) Micron.35:161-171.

[0003] Expression of Ninjurin-1 (NINJ-1) can both strengthen blood vessels and support angiogenesis. Matsuki MM, et al. (2015). Circul J 79: 1363-1376.; Matsuo R, et al. (2022). JID Innovations 2:10014.

[0004] Skin barrier integrity is associated with expression of Late Cornified Envelop-3B (LCE3B); Low levels of LCE3B protein are associated with compromised barrier function and inflammation. Niehues H, et al. (2016) Brit J Dermatol 174:795-802.; Bashir S, et al. (2016) Adv Biomed Res 5:109.

[0005] Human cytoglobin (CYGB), one of four human globin proteins, is similar in structure to neuroglobin, and has been described in the literature as a potent antioxidant protein with superoxide dismutase-type activity that is expressed during times of cellular stress. Hankeln T, et al. (2004) IUBMB Life 56:671-679.; Mathai C, et al. (2020) Redox Biol 32:101468.; Tanaka Y, et al. (2024) Pigment Cell Melanoma Res 37:276-285.; Reeder BJ. (2023) Biochem Soc Trans 51:1907- 1919.; Zweler JL, et al. (2021) Proc Nat Acad Sci 118:e2105053118.

[0006] Like CYGB , Oxidative Stress Response Kinase-1 (OXSR1) is an antioxidant protein expressed in keratinocytes and immune cells that is upregulated during times of oxidative stress.

[0038] . Hortle E, et al. (2022) Life Sci Alliance 5:e202201476. OXSR1 has been associated with the sleep / wake cycles during which it interacts with melatonin. Yang HJ, et al. (2021) Kor J Physiol Pharmacol 25: 449-457.

[0007] Angelica polymorpha var. sinensis root extract (commonly referred to as Dong Quai) has been used in the Ayurvedic traditional medicines and is rich in many bioactive compounds, including phthalides, coumarins, lignans, and terpenoids. Commun. Biol; 2023; 6:1198. Notably, A. polymorpha var. 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. Sarker SD et al., Curr Med Chem, 2004;11:1479

[0008] Mammalian studies (in rats and mice) have reported cytoprotective effects of A. sinensis: Hui et al. Int. J. Med. Sci.2006; 3:1 (administration of polysaccharides extracted from root of A. sinensis to mice reduces cyclophosphamide-induced toxicity in mice, including by increasing both VEGF expression and turnover of gastrointestinal mucosal and hemopoietic stem cells); Liu World J Gastroenterol 2003;9(12):2786-2790 (administration of polysaccharides extracted from root of A. sinensis to rats reduced immunologic injury induced by 2, 4, 6-trinitrobenzene sulfonic acid (TNBS), including by upregulating EGF expression); Liu et al. Front. Pharmacol.2022; 13; 900439 (ethanolic extract of A. sinensis improved wound healing in diabetic rats; in vitro, increased proliferation of human normalfibroblasts and upregulated expression of collagen I and collagen III). See also, Li et al. Front. Pharmacol.2022; 13; 928817.

[0009] Antioxidative, anti-inflammatory, and anti-aging benefits (decreased oxidative stress) of constituents in Phellinus linteus (also known as Black Hoof or Huang Sang mushroom) are reported in the literature. Chen et al. Molecules.2019; 24: 1888. For example, 3,4- dihydroxybenzalacetone from the fruiting body is reported to suppress protein expression of iNOS, COX-2, TNF-α, interleukin-1β, interleukin-6, MMP-2, and MMP-9, but also increased theexpression of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. Qin et al. Int. J. Biol. Macromol.2023; 123163. See also, Kou et al. Int. J. Biol. Macromol.2024; 258. Bioactive compounds in Phellinus linteus – notably, hispolon and hispidin – have been studied for anti-inflammatory, anti-oxidant, and anti-cancer properties (inducing apoptosis and inhibiting metastasis; Sarfraz A et al. Environ. Res.2020; 190: 110017.

[0010] Sunny Biodiscovery (Santa Ana, CA) reported that SBD.4A, a proprietary isolate of A. polymorpha var. sinensis, “rich in arginine and gallotanin”, improved dermal microcirculation. See, http: / / vampireskintherapy.com / wp-content / uploads / 2018 / 03 / SBD.4A-Cellulite-White- Paper.pdf (accessed on 2 / 27 / 2024).

[0011] 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.

[0012] Abandoned Chinese Patent Application CN108634317 teaches an orally-administered “health care product for strengthening body and invigorating Qi” comprised of 1-2 parts of an extract of a different Phellinus species, P. igniarius, and 5 parts of an extract of Angelica sinensis. Other ingredients in this “health care product” include Ganoderma lucidum (1-2 parts), Cordyceps sinensis (0.5-1 part), and 5 parts each of Codonopsis pilosula, Astragalus membranaceus and “liquorice” (genus Glycyrrhiza).

[0013] Lee et al have reported that topical application of Angelica sinensis improves pruritus and skin inflammation in mice with atopic dermatitis-like symptoms. J Med Food 2016; 19 (1): 98– 105.

[0014] Extracts of P. linteus have been reported to have vasodilatory effects in rat mesenteric arteries. Kwon Y et al. (2020) Molecules 25:3160.

[0015] Cambodian P. linteus extract has been reported to have anti-wrinkle effects. Cheon SJ et al. (2008). J Life Sci 12: 1718-1722.

[0016] There has been and remains a need for topically-applied products that improve the appearance of periorbital skin, including undereye bags, and fine lines and wrinkles in the corners of eye (crow’s feet). Desired improvements in the periorbital area include reducing the appearance of one or more of puffiness, dark circles, fine lines and / or wrinkles, improving skin firmness, increasing thickness, and / or reducing transepidermal water loss. There is also a need for topical compositions that reduce the amount of hemoglobin leakage and pooling under theeyes and / or the volume of bags under the eyes. Those needs are met by the compositions and methods of the present invention. BRIEF DESCRIPTION OF FIGURES

[0017] 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.

[0018] FIG.7 summarizes the results of gene expression testing (microarray) and protein expression testing (ELISA).

[0019] 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).

[0020] 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). Differences are statistically significant (p≤0.05).

[0021] 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. “A” images are taken before treatment (at baseline on day 0); “B” images are taken after four weeks of once-daily treatment with cream containing 1% PSB concentrate (on day 28).

[0024] FIG.13 presents electron ion chromatogram of three active constituents of PSB concentrate before and after heating to 80ºC for three hours.

[0025] 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. SUMMARY OF THE INVENTION

[0026] A method of providing one or more skin benefits to human skin, preferably, the periorbital area, by applying to periorbital skin (including undereye bags, crow’s feet and upper and lower lids) a topical composition comprised of extracts of Phellinus linteus and Angelica polymorpha var. sinensis. 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. The inventive compositions and methods reduce the amount of hemoglobin and / or melanin and / or the volume of bags under the eyes. DETAILED DESCRIPTION OF THE INVENTION

[0027] In one preferred embodiment, Angelica polymorpha var. sinensis extract is comprised of at least about 15% amino acids and at least about 7.5% phenolics.

[0028] In one preferred embodiments, 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. In certain preferred embodiments, 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.

[0029] In one preferred embodiment, 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. In certain preferred embodiments, the hispolon content of Phellinus linteus extract is from about 10 ppm to about 0.05ppm, preferably from about 2.0ppm to 0.1ppm.

[0030] 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.

[0031] The extracts of P. linteus extract to A. polymorpha var. sinensis are present in the PSB concentrate in a ratio of from about 10:1 to about 1:1; more preferably, from about 7:1 to about 3:1.

[0032] 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. In certain preferred embodiments, arginine content in the PSB concentrate is from about 10 ppm to about 0.05ppm, preferably from about 2.0ppm to 0.1ppm.

[0033] In certain embodiments, the PSB concentrate is added to an aqueous dispersion comprised of one or more thickening agents and a preservative system. In other embodiments, the PSB concentrate is added to an emulsion – namely, a cream, lotion, gel, or serum – comprised of one or more surfactants. While the finished formulations (topical compositions in the form of an aqueous dispersions or emulsion) are preferably applied to the periorbital area, 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).

[0034] 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. Surprisingly and unexpectedly, based on liquid chromatography coupled with mass spectrometry (LC-MS) after heating the blend to 80°C for three hours, 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.

[0035] 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.

[0036] To obtain at least one, preferably more than one, cosmetic benefit selected from (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, 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%.

[0037] 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. (Ashland, MA). 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. – are incorporated in pertinent part herein by reference.

[0038] NHEK Cell Culture Model: NHEKs are grown using EpiLife® Media, with 60 µM calcium (Thermo Fisher Scientific, Inc. Waltham, MA) supplemented with 0.2% v / v bovine pituitary extract, 1 µg / ml recombinant human insulin-like growth factor-I, 0.18 µg / ml hydrocortisone, 5 µg / ml bovine transferrin, 0.2 ng / ml human epidermal growth factor. The cells are cultured at 37±2°C and 5±1% CO2, seeded into 12-well plates and grown until confluent.

[0039] 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%.

[0040] In certain preferred embodiments, 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.

[0041] In still other preferred embodiments, 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..

[0042] In addition to the PSB concentrate, 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. Vol.53, pp.4290−4302 (2005) (oxygen radical absorbance capacity assay, Trolox equivalent antioxidant capacity assay); or protects endogenous antioxidant from degrading (e.g., ergothioneine; olive fruit extract).

[0044] Anti-inflammatory – reduces expression of one or more nuclear factor kappa β (NF-ƙβ)- dependent inflammatory mediators selected from the group consisting of IL-1β, IL-6, IL-8, cyclooxygenase (COX)-2, tumor necrosis factor alpha (TNF-α), or monocyte chemoattractant protein (MCP-1), preferably by at least 30%.

[0045] Activates one or more of retinoic acid receptor (RAR), retinoid-x-receptor (RXR) ligand and / or peroxisomal proliferator activated receptor (PPAR) ligand. While 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 beincluded in under-eye compositions of the present invention; and, when present, may be included at low concentrations.

[0046] Stimulates epidermal differentiation.

[0047] Reduces melanocyte proliferation and / or tyrosinase levels.

[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.

[0052] Categories of skin benefit ingredients that provide one or more of the above skin benefits that may be, and preferably are, present in the periorbital skin treatment compositions of the present invention:

[0053] A peptide comprised on three to seven amino acid residues; in certain embodiments Acetyl Hexapeptide-8 (sold under the tradename ARGIRELINE®) or Tripeptide-32.

[0054] One or more vitamins, 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.

[0055] Caffeine or a source of caffeine (e.g., green tea extract)

[0056] 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).

[0057] A probiotic, prebiotic or postbiotic.

[0058] A ferment; in certain embodiments selected from Bifida Ferment Lysate; Lactobacillus Ferment; Thermus Thermophillus Ferment; Saccharomyces Ferment.

[0059] Licorice or its derivative – Dipotassium Glycyrrhizate

[0060] 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).

[0061] Jojoba or its derivative; in certain embodiments selected from Jojoba Esters (e.g., Isopropyl Jojobate) or Jojoba Alcohol.

[0062] 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.

[0065] Other 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 Digitata Extract; Laminaria Ochroleuca Extract; Lavandula Angustifolia (Lavender) Flower Extract; Lens Esculenta (Lentil) Fruit Extract; Limonium Vulgare Flower / Leaf / Stem Extract; Mimosa Tenuiflora Bark Extract; Moringa Oleifera Seed Extract; Morus Bombycis (Mulberry) Root Extract; Narcissus Tazetta Bulb Extract; Opuntia Tuna (Prickly Pear) Extract; Palmaria Palmata Extract; Polygonum Cuspidatum Root Extract; Poria Cocos Sclerotium Extract; Porphyridium Cruentum Extract; Prunus Mume Fruit Extract; Pyrus Malus (Apple) Fruit Extract; Salvia Sclarea (Clary) Extract; Scutellaria Baicalensis Root Extract; Sigesbeckia Orientalis (St. Paul's Wort) Extract; Silybum Marianum (Lady's Thistle) Extract; Triticum Vulgare (Wheat) Germ Extract or Germ Oil; Tuber Melanosporum Extract; Whey Protein Hu X et al., Clin Cosmet Invest Dermatol 2024;17:1943.

[0066] In certain preferred embodiments, 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.

[0067] In other preferred embodiments, the periorbital skin treatment composition contains Tuber Melanosporum Extract, including in combination with Narcissus Tazetta Bulb Extract.

[0068] In certain preferred embodiments, 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 TiO2and Iron Oxides.

[0069] 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).

[0070] Formulation Examples A Aqua (Water) 54.00 A Glycerin (and) Phospholipids 1.00

[0071] Add Phase A to main vessel and begin propeller mixing. Mix until uniform. Heat to 80°C. Premix Phase B ingredients and add to main vessel ; mix until uniform. Add Phase C ingredients and hydrate at 80°C for 25 minutes. Premix Phase D ingredients in a side vessel ; heat to 80-85°C until uniform. Mix water phase (combination of Phases A, B and C) with Silverson® Homogenizer at 6,000 RPM. Add Phase D under homogenization. Add Phase E and Phase F under homogenization until uniform. At 50°C, add Phase G. At 35°C, add Phase H. Cool batch to 30°C. 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 00A Sodium Acrylate / Sodium Acryloyl Dimethyl Taurate Copolymer, Cetyl Alcohol, Glycol 4.00 Stearate, Glyceryl Stearate, Caprylic / Capric Triglyceride 0 0 0 0 0 0 0ts 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.

[0073] 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. (Warren, NJ)], 1% preservative (Caprylyl Glycol, Glyceryl Laurate, and Glyceryl Undecylenate; commercially available as JEECIDE® CAP-7 LB-TNS from Vantage Specialty Ingredients, Inc.) and 93% water.

[0074] In Vitro Testing: Changes in gene and protein expression are tested on two periorbital skin-relevant substrate models – (i) cell culture of normal human epidermal keratinocytes (NHEK) from an adult donor or (ii) EpiDerm FT. Total protein content is assessed using bicinchoninic acid assay (BCA). ELISA is used to quantify expression of specific proteins (corresponding to the selected genes).

[0075] 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% CO2. 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 PSBconcentrate) 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% CO2for 24 hours. Thereafter, the 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.

[0076] 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 µl of extraction buffer.

[0077] 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. Up to 700 ml of the resulting mixture is transferred to a glass fiber filter cartridge, which is loaded into a 1.5 ml collection tube and the cartridge is centrifuged for 1 minute at 14,000 RPM. The flow-through is discarded. The remaining mixture is loaded into the filter cartridge and the centrifugation process is repeated until all of the mixture is processed. The filter is then washed to remove any residual cellular debris from the RNA bound to the glass fibers by applying 700 ml of a first wash solution (1 time) and 500 ml of a second wash solution (2 times) to the filter cartridge and centrifuging at 14,000 RPM for 1 minute to pass each wash through the cartridge. The flow-through is discarded after each wash. After the final wash, one final spin is performed without wash solution to remove any residual wash solution in the filter cartridge. The 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. For samples prepared from cell lysates and small tissues, the elution process is repeated with an additional 30 ml of preheated TE buffer. For samples prepared from larger (i.e., full thickness) tissues, the elution process is repeated two additional times. After the RNA is eluted, RNA concentration is quantified using a Ribogreen assay. RNA quality is assessed via gel electrophoresis.

[0078] 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. coli to the following concentrations: 2 mg / ml, 1 mg / ml, 200 ng / ml, 40 ng / ml, and 0 ng / ml (blank). 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. After this incubation, the plate is read with a fluorometer (Cole Parmer) using an excitation wavelength of 500 nm and an emission wavelength of 525 nm.

[0079] 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. After the solution is cooled to approximately 55°C, 5.4 ml of formaldehyde and 3.0 ml 10×MOPS (3-morpholinopropanesulfonic acid) (0.2 M MOPS [pH 7.0], 20 mM sodium acetate, 10 mM EDTA, made in DEPC H2O) is added and filter sterilized. After mixing, the agarose gel is cast in the horizontal gel apparatus with loading slots placed on the side of the gel closest to the negative terminal. The gel is allowed to set for at least 1 hour at room temperature. While the gel is setting, 175 ml of 1×MOPS is prepared by diluting the 10× stock. After the gel is set, 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. While the gel is running, the RNA samples are prepared by transferring approximately 1 mg of each sample RNA to a 600 ml PCR tube. DEPC H2O 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. 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 hours. At the end ofthe run, the 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.

[0080] 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 H2O. To each tube, 1 ml of T7 Oligo(dT) primer is added, 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. After cooling, 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. As soon as the tube is heated, 1 ml of reverse transcriptase is added, and the tubes are returned to 42±2°C for 2 hours. At the end of the two hours, the tubes are briefly centrifuged to collect all of the fluid at the bottom of the tube and then placed on ice.

[0082] 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 H2O, 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 H2O 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. After the samples are finished incubating, 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 H2O to the filter and centrifuging the filter in a new collection tube at 10,000 RPM for one minute. This elution is performed one additional time to give a total volume of 16-18 ml of cDNA solution.

[0083] In Vitro Transcription to Synthesize aRNA and aRNA Purification: 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 enzymemix. The tube is mixed and then incubated at 37±2°C for 6-14 hours. Towards the end of the incubation, 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. At the end of the incubation period, 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. 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.

[0084] Labeling and Purification of aRNA: 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 H2O 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 H2O 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). Incubate the tubes for 15 minutes at 85±2°C At the end of the 15 minutes, place the tubes on ice to cool and then add 2.5 ml of ASAP stop solution to each tube. The above proportions are sufficient for analyzing one microarray chip. If more chips are used, then the labeling is increased proportionately.

[0085] To purify the labeled aRNA, 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. The column is washed twice with 400 ml of TE buffer, discarding the flow through each time. After the finalwash, the filter column is inverted, placed into a new collection tube, and centrifuged at 12,000 RPM for 2 minutes to collect the probe (the probe is concentrated in a volume of 2-30 ml of residual TE buffer).

[0086] 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 H2O and 9 ml of 25× Agilent Fragmentation Buffer. This mixture is incubated at 60°C for approximately 30 minutes in a hybridization oven. At the end of the incubation, 225 ml of 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. During this incubation period, an Agilent SUREHYB hybridization chamber is prepared by inserting a glass gasket slide into the bottom half of the chamber. At the end of the incubation, 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). At the end of the hybridization period, 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. The array is washed while the solution is mixed at medium speed for 10 minutes and is then transferred to 300 ml of wash solution 2 (0.1×SSX, 0.005% Triton X-102, 4°C) for 5 minutes. After the final wash, the array is centrifuged at 500 RPM for 5 minutes until dry.

[0087] 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.

[0088] To derive the standard curve for the Ribogreen assay, 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 probedgene 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.

[0089] Criteria for evaluating changes in gene expression are known to those of ordinary skill in the art and 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.

[0090] 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 1.3, where the treated value is 130% of the untreated value, indicates a 30% increase in gene expression. A ratio of 0.7 means that the treated value was 70% of the untreated value, indicating a 30% decrease in gene expression.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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 welland are incubated for 2 hours at room temperature. At the end of this incubation 200 µl of each sample of extracted MTT is transferred to a 96-well plate and read at 540 nm using isopropyl alcohol as the blank.

[0096] BCA Protein Assay: A series of protein standards is prepared using BSA ranging from 0 to 1,000 μg / ml. Ten microliter aliquots of the standards and 5 µl 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.

[0097] 100 µl of standards or diluted lysates (10 µl lysate diluted with 90 µl 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 µl of biotinylated detection antibody solution is added to each well. The plate is incubated for 60 minutes at 37˚C and is then washed three times with wash buffer. After the third wash is removed, 100 µl of an avidin-enzyme solution is added to each well; and the plate is incubated for 30 minutes at 37˚C. After this final incubation, the plate is washed five times with wash buffer. After the fifth wash is removed, 100 μl of substrate solution is added to each well. As is known in the art, 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 µl 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.

[0098] For the ELISA assays, 100 µl of standards or diluted lysates (10 µl lysate diluted with 90 µl 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 µl 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 µl of an avidin-enzyme solution is added to each well and the plate is incubated for 30 minutes at 37°C. After the final incubation, the plate is washed five times with a wash buffer. After the last wash is removed, 100 µl of substrate solution is added to each well.50 µl of stop solution is then added to each well and the plate is read at 460 nm.

[0099] Human Clinical Testing: Fifty individuals are recruited to participate in 28-day in vivo human study comparing a test formulation containing 1.0% PSB concentrate and placebo formulation identical to the test formulation without 1% PSB concentrate. Subjects in each of the placebo and test groups are instructed to apply the assigned formulations twice daily to the periorbital area (including the crow’s feet both the upper and lower eyelids).

[0100] Improvement in the periorbital area is observed in self-reported questionnaire is reported in several respects: improved appearance in terms of perceived reduction in the look of puffy eyes and / or fine lines and wrinkles; improved skin feel in term of eye area feeling firmer, softer and / or smoother.

[0101] Reduced intensity of dark circles under the eyes is scored by an experienced technician under standardized lighting using a six-point scoring scale (0=absence, 1=very light, 2=light, 3=moderate, 4=dark, 5=very dark).

[0102] 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).

[0103] 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).

[0104] Skin thickness is assessed using ultrasound imaging (Dermcup, Atys, France)

[0105] Hyperspectral image analysis of skin is conducted with SpectraCam® (Newtone Technologies, Lyon, France) to assess melanin as well as hemoglobin (oxy-hemoglobin and deoxy- hemoglobin) levels. Pigment Cell Res.2004; 17: 618-626. Skin Res Technol.2008;14(2):135-141; Skin Res Technol.2018;24 (1):99-107.

[0106] Changes in concentrations of globin(s) and / or melanin is measured using the BrightTex Bio-Photonics Clarity® 3D camera from BrightTex Bio-Photonics (San Jose, CA). The same system is used to measure changes in volume of periorbital skin treatment bags as well as the extent of fine lines and wrinkles (crow's feet).

[0107] Blood flow and skin hydration are measured using laser Doppler flowmetry and a standard corneometer, respectively. Changes in wrinkles (number, depth, area) are assessed based on before and after images captured with the BrightTex Bio-Photonics Clarity® 3D camera.

[0108] The 25 subjects in the test group assigned to the active formulation reported multiple skin benefits (improvements over baseline). See FIG.14.

Claims

Claims 1. A composition for topical application to human skin, preferably to the periorbital area of a human, comprised of a periorbital skin benefit (PSB) concentrate consisting essentially of two extracts, one each from the genera Phellinus, preferably P. linteus, and Angelica, preferably A. polymorpha var. sinensis, wherein after 24-hour application of a 1% PSB concentrate to a cell culture comprised of a confluent layer of normal human epidermal keratinocytes (NHEKs) expression of each of four periorbital skin-relevant genes – CYGB, OXSR1, LCE3B, and EGFR – as quantified by microarray analysis increases by at 1.3 fold.

2. The composition of claim 1 wherein after the 24-hour application of the 1% PSB concentrate to the cell culture comprised of a confluent layer of NHEKs the concentration of each of five periorbital skin-relevant proteins - CYGB, OXSR1, LCE3B, NINJ1 and EGFR - as quantified by Enzyme-Linked Immunosorbent Assay increases by at least 20%.

3. The composition of claim 2 wherein after the 24-hour application of the 1% PSB concentrate to the cell culture comprised of a confluent layer of NHEKs the concentration of each of CYGB, OXSR1, LCE3B, and NINJ1 increases by at least 25%.

4. The composition of claim 3 wherein after the 24-hour application of the 1% PSB concentrate to the cell culture comprised of a confluent layer of NHEKs the concentration of each of OXSR1, LCE3B and NINJ1 increases by at least 50%.

5. The composition of any of claims 2-4 after the 24-hour application of the 1% PSB concentrate to the cell culture comprised of a confluent layer of NHEKs the expression of VEGFA decreases by about 25%.

6. The composition of any of claims 2-5 wherein the ratio of the Phellinus extract to the Angelica extract is from about 10:1 to about 1:

1.

7. The composition of any of claims 6 wherein the Phellinus extract is Phellinus linteus species.

8. The composition of any of claim 7 wherein the Angelica extract is Angelica polymorpha var. sinensis.

9. The composition of any of claims 1-8 wherein the PSB is comprised of at least about 1 ppm of feruloylmethane.

10. The composition of claim 9 wherein the PSB is comprised of at least about 0.05 ppm hispolon.

11. The composition of claim 10 wherein the PSB is comprised of at least about 0.05 ppm of arginine.

12. The composition of any of claims 1-11 further comprising one or more of a peptide, caffeine, Vitamin C and / or E, hyaluronic acid, ergothioneine, glucosamine, olive fruit extract, or derivatives thereof.

13. A method of providing one or more skin benefits to the periorbital area comprised of the step of applying the composition of any of claims 1-12, where the cosmetic benefit(s) is / are selected from the group consisting of (a) reducing appearance of one or more of periorbital skin puffiness, dark circles, fine lines and / or wrinkles, (b) improving skin firmness, smoothness and / or thickness, and / or (c) reducing transepidermal water loss.

14. The method of claim 13 wherein the capillary microcirculation under the eyes is increased and the amount of hemoglobin and / or melanin under the eyes is reduced.

15. The method of claim 14 wherein the volume of periorbital skin treatment bag / puffiness is reduced.

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