12-2010 Citrus Flavonoids with Skin Lightening Effects ... - Hostpoint

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COSMETICSSKIN LIGHTENINGS. Kiefer, M. Weibel, J. Smits, M. Juch, J. Tiedtke, N. Herbst*Citrus Flavonoids with Skin Lightening Effects –Safety and Efficacy Studies IntroductionWith ageing of the skin pigmentary disorderssuch as age spots, melasma, anduneven skin tone become more frequent.An even, bright and unblemished skin isone of the main criteria for a younglooking appearance (1-3). Hence skinlightening products form a major segmentin the cosmetic industry and are apart of many anti-ageing products thatreach the market every year.AbstractMelanin, the substance responsible forthe pigmentation of the skin, is producedto protect the DNA from harmful, mutagenicUV radiation. Age spots or solarlentiges are age-related and UV irradiation-inducedpigmented spots that usuallyoccur on sunlight-exposed areassuch as the face, the back of the hands,and the upper chest.The rate-limiting enzyme in melanin formationis polyphenol oxidase tyrosinase(EC 1.14.18.1) which catalyses the oxida-Ayouthful appearance is not only related to the absence of wrinklesbut also to skin radiance and an even skin tone. Many cosmetic ingredientsare used to lighten skin by the inhibition of tyrosinase, therate-limiting enzyme in melanin formation. Flavonoids, as major constituentsof the human diet, have many well-known beneficial qualities.The aim of this study was to find the ideal composition of flavonoids fromvarious citrus extracts for an effective skin lightening cosmetic ingredient.A chosen blend of citrus extracts was tested for safety and in vitro efficacy.No cytotoxic effects up to 0.8mg/ml could be detected and inhibition ofhuman tyrosinase was 60% at 0.4mg/ml. The blend of citrus extracts wasliposomal-encapsulated, incorporated into a cosmetic formulation at 1%,and then tested for stability and skin irritation capacity, as well as eye irritationand mutagenic potential and exhibited a very good safety profile.Finally the product was studied in vivo on Caucasian skin for efficacy during56 days and showed marked skin brightening effects at a concentrationof 1% (0.4mg/ml).This liposomal-encapsulated citrus flavonoid blend is effective at 1% in acosmetic formulation to fade age spots and brighten skin tone.tion of tyrosine to dopaquinone (4). Thesubsequent steps of melanin formationfrom dopaquinone can occur spontaneouslyat physiological pH. Dopaquinoneis oxidized to dopachrome and throughfurther oxidation reacts to melanin.Many of the currently used depigmentingagents are tyrosinase inhibitors, asit seems the most straightforward wayto inhibit melanin formation. Althoughmore recent in-depth knowledge ofmelanocyte biology and the processesunderlying melanin formation has producedother interesting targets for thetreatment of hyper-pigmentation, suchas the inhibition of translation and maturationof tyrosinase, stimulation of degradationand acceleration of skin turnover.The best known skin whitening agent ishydroquinone, which has been used forover 50 years now although some controversyabout it has come up in recentyears. Long term use of high concentrationsof hydroquinone can produce sideeffects like ochronosis, a thickening anddarkening of skin, especially of darkskinnedindividuals (5) and general safetyconcerns have been brought up byregulatory agencies all over the world.Therefore there is still a need for safe andeffective skin lightening products andthe hunt for natural skin lighteners (6) isongoing.Flavonoids form major constituents ofthe human diet as they contribute to theflavour and colour of many fruits andvegetables. Their beneficial antioxidanteffects are thoroughly studied and established.The highest content of citrus flavonoidscan be found in unripe citrus fruits (7).46 SOFW-Journal | 136 | 12-2010
COSMETICSSKIN LIGHTENINGThese unripe fruits can be collected bythinning out, which is necessary for agood harvest of ripe fruits later on (8).Whole ground fruits are extracted witha water methanol mixture to yield thehighest flavonoid content.The aim of this study was to find the idealcomposition of flavonoids from variouscitrus fruit extracts to use as an effectiveskin lightening cosmetic ingredient. Materials and MethodsAll solvents from Merck, all other chemicalsfrom Sigma AldrichRaw materialThe various citrus extracts were boughtfrom different extract manufacturersand analyzed by HPLC for their contentof naringin, narirutin, hesperidin, andneohesperidin.HPLC instrumentationHPLC separation was carried out on aSurveyor HPLC system with degasser, binaryhigh pressure mixing pump, columnthermostat, and auto sampler. The HPLCwas coupled to a DAD detector and dataacquisition and processing was performedusing ChromQuest ChromatographyData System (CDS) (all ThermoFisher Scientific).Separation of flavonoidsOn a C18 column (Utisphere OBD, 120Å,150 x 4.6 mm, 5µm) an isocratic elutionwith 75% water, 10% methanol, 10%acetonitrile, and 5% acetic acid 99%was performed with a flow of 1 ml/minand subsequent UV detection at 285 nm.Tyrosinase inhibitionMushroom tyrosinase (333 U/ml, EC1.14.18.1, Sigma Aldrich, Switzerland)was incubated in 18 mM phosphatebuffer, extracts and tyrosine solution (finalconcentration of 1 mM) were addedand absorbance was measured over 5hours at 490 nm.Cellular human tyrosinase activity testNormal human epidermal melanocytes(NHEM, Bioalternatives, France) were incubatedfor 72 hours with test compoundand control. After incubation, culturemedium was removed and cells washedwith PBS. The tyrosinase enzyme was extractedwith Triton X in PBS and then incubatedwith 2 mM L-3,4-dihydroxyphenylalanine(L-DOPA) as substrate. Afterincubation enzymatic activity was measuredat 540 nm (ThermoMax micro platereader, Molecular Devices).CytotoxicityNormal Human Epidermal Melanocytes(NHEM, Bioalternatives, France) wereincubated with concentrations from0.0015 to 1.14 mg/ml of flavonoid mixturefor 72 hours. Then a solution of0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)was added, after incubation at 37 ° C for4 hours, the media was discarded and100 ul DMSO was added to each well. Theoptical density of the dissolved residueswas measured at 540 nm (Microplatereader, Molecular devices).Liposomal encapsulationThe flavonoid mixture was mixed withglycerin, lecithin, tocopherol, sodiumascorbate, and homogenized with a highpressure homogenizer (Microfluidics,Lampertheim, Germany). Subsequentlythe liposome size was determined usinga Zetasizer Nano ZS90S from Malvern Instruments(Worcestershire, United Kingdom).Formulation1% of the liposomal solution was incorporatedinto a lotion (Table 1) for applicationstudies (patch tests and in vivo efficacy).The formulation was tested forstability towards temperature and time.Patch testsFor both, single and repeated patch tests25 healthy volunteers between 18 and60 years were selected. The dorsal skinwas cleaned with 70% alcohol beforeComponent INCI Weight (g) FunctionWater Aqua 78.6 SolventNaOH 10% Aqua, Sodium Hydroxide 0.5 pH-adjustmentKeltrol CG SFT Xanthan Gum 0.4 Emulsion stabilizerCrodafos CES Cetearyl Alcohol, Dicethyl Phosphate, Ceteth-10 Phosphate 3.0 EmulsifierMyritol 318 Caprylic/Capric Triglyceride 13.0 Skin-conditioning agentPreservative Phenoxyethanol, Ethylhexylglycerin q.s. PreservativeSilicone Oil Dimethicone 0.5 Antifoaming agentShea Butter Butyrospermum Parkii (Shea Butter) 3.0 Skin-conditioning agentFlavonoid mixture, Glycerin, Aqua, Lecithin, Citrus Paradisi (Grapefruit) Fruit Extract, 1.0 ActiveCitrolumine 8 Citrus Aurantium Amara (Bitter Orange) Fruit Extract, SodiumAscorbate, Tocopherol, Heliantus Annus (Sunflower) Seed OilTotal 100.0Table 1 Frame formulation for the liposomal encapsulated citrus flavonoid blend, as used in the application studies48 SOFW-Journal | 136 | 12-2010
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Page 7: COSMETICSSKIN LIGHTENINGAcknowledge

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