citrus flavonoids with skin lightening effects – safety and efficacy

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12-2010 English Edition International Journal for Applied Science • Personal Care • Detergents • Specialties S. Kiefer, M. Weibel, J. Smits, M. Juch, J. Tiedtke, N. Herbst: Citrus Flavonoids with Skin Lightening Effects – Safety and Efficacy Studies

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Page 1: Citrus Flavonoids with Skin Lightening Effects – Safety and Efficacy

12-2010

English EditionInternational Journal for Applied Science

• Personal Care • Detergents • Specialties

S. Kiefer, M. Weibel, J. Smits, M. Juch,

J. Tiedtke, N. Herbst:

Citrus Flavonoids with Skin Lightening Effects –

Safety and Efficacy Studies

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COSMETICSSKIN L IGHTENING

tion of tyrosine to dopaquinone (4). Thesubsequent steps of melanin formationfrom dopaquinone can occur sponta-neously at physiological pH. Dopaquinoneis oxidized to dopachrome and throughfurther oxidation reacts to melanin.Many of the currently used depigment-ing agents 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 pro-duced other interesting targets for thetreatment of hyper-pigmentation, suchas the inhibition of translation and matu-ration of tyrosinase, stimulation of degra-dation and acceleration of skin turnover.The best known skin whitening agent ishydroquinone, which has been used forover 50 years now although some con-troversy about it has come up in recentyears. Long term use of high concentra-tions of hydroquinone can produce sideeffects like ochronosis, a thickening anddarkening of skin, especially of dark-skinned individuals (5) and general safe-ty concerns 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 es-tablished. The highest content of citrus flavonoidscan be found in unripe citrus fruits (7).

S. Kiefer, M. Weibel, J. Smits, M. Juch, J. Tiedtke, N. Herbst*

Citrus Flavonoids with Skin Lightening Effects –Safety and Efficacy Studies

� Introduction

With ageing of the skin pigmentary dis-orders such 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 seg-ment in the cosmetic industry and are apart of many anti-ageing products thatreach the market every year.

Melanin, the substance responsible forthe pigmentation of the skin, is producedto protect the DNA from harmful, muta-genic UV radiation. Age spots or solarlentiges are age-related and UV irradia-tion-induced pigmented spots that usu-ally occur on sunlight-exposed areassuch as the face, the back of the hands,and the upper chest. The rate-limiting enzyme in melanin for-mation is 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 in-gredients are used to lighten skin by the inhibition of tyrosinase, the

rate-limiting enzyme in melanin formation. Flavonoids, as major con-stituents of 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 irri-tation and mutagenic potential and exhibited a very good safety profile.Finally the product was studied in vivo on Caucasian skin for efficacy dur-ing 56 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.

Abstract

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These 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 ide-al composition of flavonoids from vari-ous citrus fruit extracts to use as an ef-fective skin lightening cosmetic ingredi-ent.

� Materials and Methods

All solvents from Merck, all other chemi-cals from Sigma Aldrich

Raw 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, bi-nary high pressure mixing pump, columnthermostat, and auto sampler. The HPLCwas coupled to a DAD detector and da-ta acquisition and processing was per-formed using ChromQuest Chromatog-raphy Data 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 (fi-nal concentration 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 in-cubated for 72 hours with test compoundand control. After incubation, culturemedium was removed and cells washedwith PBS. The tyrosinase enzyme was ex-tracted with Triton X in PBS and then in-cubated with 2 mM L-3,4-dihydroxyphe-nylalanine (L-DOPA) as substrate. Afterincubation enzymatic activity was mea-sured at 540 nm (ThermoMax micro platereader, Molecular Devices).

CytotoxicityNormal Human Epidermal Melanocytes(NHEM, Bioalternatives, France) wereincubated with concentrations from

0.0015 to 1.14 mg/ml of flavonoid mix-ture for 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 In-struments (Worcestershire, United King-dom).

Formulation1% of the liposomal solution was incor-porated into a lotion (Table 1) for appli-cation studies (patch tests and in vivo ef-ficacy). 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 before

Component INCI Weight (g) Function

Water Aqua 78.6 Solvent

NaOH 10% Aqua, Sodium Hydroxide 0.5 pH-adjustment

Keltrol CG SFT Xanthan Gum 0.4 Emulsion stabilizer

Crodafos CES Cetearyl Alcohol, Dicethyl Phosphate, Ceteth-10 Phosphate 3.0 Emulsifier

Myritol 318 Caprylic/Capric Triglyceride 13.0 Skin-conditioning agent

Preservative Phenoxyethanol, Ethylhexylglycerin q.s. Preservative

Silicone Oil Dimethicone 0.5 Antifoaming agent

Shea Butter Butyrospermum Parkii (Shea Butter) 3.0 Skin-conditioning agent

Flavonoid mixture, Glycerin, Aqua, Lecithin, Citrus Paradisi (Grapefruit) Fruit Extract, 1.0 ActiveCitrolumine 8™ Citrus Aurantium Amara (Bitter Orange) Fruit Extract, Sodium

Ascorbate, Tocopherol, Heliantus Annus (Sunflower) Seed Oil

Total 100.0

Table 1 Frame formulation for the liposomal encapsulated citrus flavonoid blend, as used in the application studies

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product application and then 1% of li-posomal-encapsulated flavonoid mix-ture in a lotion was applied using a FinnChamber (7 mm). The product was left incontact with the skin for 48 hours (sin-gle patch test) or 24 hours and reapplied4 times (repeated patch test). The cuta-neous reactions were evaluated 15 min-utes, 1 hour, and 24 hours after removalof the product. One chamber with dis-tilled water was used as negative controland for the single patch test only a pos-itive control was applied with 0.5% sodi-um dodecyl sulphate (SDS).

Application studyCaucasian skin: Six healthy female vol-unteers of Caucasian skin type (all Fitz-patrick skin types II) were recruited forapplication of the lotion or a control(formulation without active ingredient).The cream was applied twice a day dur-ing 8 weeks on the back of the hand andthe face. Melanin Index was measured onthe back of the hand and the face witha Skin Pigment Analyzer, SPA99 (Courage& Khazaka, Köln, Germany) and pho-tographs were taken from the face andback of the hand with VisioFace Quick(Courage & Khazaka).Asian skin: Six healthy female volun-teers of Asian skin type were recruitedfor application of the lotion on the ex-ternal forearm. The lotion was appliedtwice daily during 8 weeks. Skin colorwas measured with a Chromameter CR300(Konica Minolta, Dietikon, Switzerland).All volunteers were measured and pho-tographed 3 times; at day 0 (baseline),day 28, and day 56.

� Results

Raw materialThe various citrus extracts were tested bychromatographic separation for theircontent of narirutin, naringin, hesperidin,and neohesperidin. Two extracts were chosen, one from un-ripe citrus paradisi (grapefruit) and onefrom unripe citrus aurantium amarum(bitter orange) fruit and mixed. The re-sulting mixture of raw materials result-ed in the following flavonoid contentof 4.9% narirutin, 22.0% naringin, 1.0%hesperidin, and 5.3% neohesperidin (Fig. 1).

Tyrosinase inhibition (mushroom tyrosinase)Inhibition of mushroom tyrosinase wastested in a concentration range from0.025 to 3 mg/ml. Up to 1 mg/ml inhibi-tion increased dose dependently up to40% of untreated control, in concentra-tion higher than 1 mg/ml there was nofurther increase possible (Fig. 2). Theflavonoid mixture showed an estimatedIC50 of 0.75mg/ml.

Cellular human Tyrosinase activity testTyrosinase activity was tested after incu-bation of NHEM with 0.004 to 0.4 mg/mlflavonoid mixture for 72 hours and sub-

sequent tyrosinase extraction of thecells. As a control 0.02 mg/ml of kojic acidwas used. A concentration of 0.004 mg/ml of flavonoid mixture reduced the ty-rosinase activity to 66%, 0.04 mg/ml to65%, and 0.4 mg/ml to 40%, whereas ko-jic acid reduced tyrosinase activity to61% at 0.02mg/ml (Fig. 3).

Viability/ CytotoxicityUp to 0.4 mg/ml flavonoid mixture via-bility was increased up to 130%, at1.12 mg/ml a reduction of viability to83% was measured (Fig. 4). A concen-tration of 3.3 mg/ml resulted in no re-maining viability (data not visible).

-Fig. 1 HPLC chromatogram of the flavonoid mixture

-Fig. 2 Mushroom tyrosinase inhibition of flavonoid mixture

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Safety testsAmes reverse mutation assay (OECD No.471) using Salmonella typhimurium andEscherichia coli showed no mutagenicpotential of the liposomal-encapsulatedflavonoid mixture.The bovine corneal opacity and perme-ability assay (BCOP, OECD No. 437) of theliposomal-encapsulated flavonoid mix-ture revealed no eye irritation potentialof the product.

Liposomal encapsulationLiposomes showed an average particlesize of 120 nm which was stable overseveral production batches. They formeda transparent, stable solution and nopreservation was necessary to keep thesolution from microbiological conta -mination. A Pharmacopoeia Europea 6preservation test for topical productswas performed and received a grade Bevaluation.

Stability of frame formulationThe formulation for the applicationstudy was stable for 6 month at roomtemperature, at 40°C, and at 2-8°C in therefrigerator.

Patch testsThe single and repeated human patchtests showed no irritation and sensitiza-tion potential of the liposomal-encapsu-lated flavonoid mixture in a lotion at 1%,which corresponds to 0.4 mg/ml offlavonoid mixture.

� Application Study

Caucasian SkinThe application study on Caucasian skin(n=6) showed skin lightening effects andage spots brightening after 28 and 56days of application compared to base-line. The effects on age spot brighteningwere slightly more apparent than thelightening effects on skin tone. Both onhand (Fig. 5) and face (Fig. 6) the re-sulting birightening effects were in thesame range. Fig. 7 shows the spot-fad-ing and skin brightening effects of aVisoFace Quick picture of the back of thehand on day 0 and day 56.

-Fig. 3 Tyrosinase inhibition of cellular tyrosinase extracted from NHEM comparedto untreated control

-Fig. 4 MTT viability assay with flavonoid mixture from 0.0015 mg/ml up to 1.116 mg/ml

-Fig. 5 Skin brightening effect of liposomal-encapsulated flavonoid mixture at 1%in a lotion (corresponding to 0.4 mg/ml flavonoid mixture) applied on the back ofthe hand twice daily for 28 and 56 days compared to baseline (day 0)

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Asian SkinAfter promising results on Caucasianskin an additional study on the externalforearm of Asian volunteers (n=3) wasperformed and showed a lightening ef-fect compared to baseline (day 0), whichwas significantly present after 56 days(Fig. 8).

� Discussion

The flavonoid mixture was effective invitro. Good inhibtion has been shown inthe mushroom tyrosinase enzyme inhi-bition test with an estimated IC50 ofabout 0.75 mg/ml. The cellular human tyrosinase inhibitionassay resulted in stronger tyrosinase in-hibition, there the estimated IC50 was0.24 mg/ml of flavonoid mixture. Origi-nally the potential lighthening effect ofcitrus flavonoids was discovered by mush-room tyrosinase inhibition assay (9). A range of tests showed that the citrusflavonoid blend is safe for application onskin. No cytotoxicity has been observedin concentrations that were applied. Inthe single and repeated patch tests no ir-ritation or sensitisation potential wasfound and also no mutagenic potential(as tested by Ames Test, data not shown)and no eye irritation was detectable(BCOP test, data not shown). These find-ings show the excellent safety profile ofthe flavonoid blend for use as a cosmet-ic ingredient.In vivo studies showed that it was veryeffective in brightening the skin tone andhad an even stronger effect on the fad-ing of age-spots, resulting in a more evenskin tone. To get more accurate data withlower standard deviations a bigger studywith at least 20 volunteers would be nec-essary. The high standard deviation wasdue to the small test groups (n=6 forCaucasian and n=3 for Asian Skin). After28 days effects were already visible andwere enhanced after 56 days, thus im-plying that stronger brightening effectscan be obtained after longer application.These data suggest that the liposomal-encapsulated citrus flavonoid blend(Citrolumine 8™ from Cosmetochem AG,Switzerland) can be used at 1% in a cos-metic lotion as a very safe and effectiveskin lightening ingredient.

-Fig. 6 Skin brightening effect of liposomal-encapsulated flavonoid mixture at 1%in a lotion (corresponding to 0.4 mg/ml flavonoid mixture) applied to the face twicedaily after 28 and 56 days compared to baseline (day 0)

-Fig. 7 Skin brightening and spot-fading effect of liposomal-encapsulated flavonoidmixture at 1% in a lotion (corresponding to 0.4 mg/ml flavonoid mixture) appliedto the back of the hand twice daily for 56 days. Left picture baseline at day 0, rightpicture after twice daily application during 56 days

Day 0 Day 56

-Fig. 8 Skin brightening effect of liposomal-encapsulated flavonoid mixture at 1%in a lotion (corresponding to 0.4 mg/ml flavonoid mixture) applied to the externalforearm of asian volunteers twice daily after 28 and 56 days compared to baseline(day 0). After 56 days a sigificant brightening effect can be measured

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AcknowledgementsThese investigations were realized andfunded by Cosmetochem InternationalAG, Switzerland.The authors’ would like to thank Dr. Ru-di Wajda from Lipoid GmbH, Ludwigs-hafen, for liposomal encapsulation andmeasurements and Daniel Lisibach fromCosmetochems analytical lab for all theHPLC analyses.

References

(1) Fink B, Matts PJ. The effects of skin colour dis-tribution and topography cues on the percep-tion of female facial age and health. J Eur AcadDermatol Venereol 2008;22 (4):493-8

(2) Gunn DA, Rexbye H, Griffiths CE, Murray PG,Fereday A, Catt SD, Tomlin CC, StrongitharmBH, Perrett DI, Catt M, Mayes AE, MessengerAG, Green MR, van der Ouderaa F, Vaupel JW,Christensen K. Why some women look youngfor their age. PLoS One 2009;4 (12):e8021

(3) Matts PJ, Fink B, Grammer K, Burquest M. Col-or homogeneity and visual perception of age,health, and attractiveness of female facial skin.J Am Acad Dermatol 2007;57 (6):977-84

(4) Chang TS. An updated review of tyrosinase in-hibitors. Int J Mol Sci 2009;10 (6):2440-75

(5) Parvez S, Kang M, Chung HS, Cho C, Hong MC,Shin MK, Bae H. Survey and mechanism of skindepigmenting and lightening agents. Phy-tother Res 2006;20 (11):921-34

(6) Smit N, Vicanova J, Pavel S. The hunt for nat-ural skin whitening agents. Int J Mol Sci2009;10 (12):5326-49

(7) Kubo M, Fujita T, Nishimura S, Tokunaga M,Matsuda H, Tomohiro N, Sakai K, UtsunomiyaN. Seasonal variation in anti-allergic Activity ofCitrus fruit and Flavonone Glycoside Content.Nat Med 2004;58 (6):284-94

(8) Itoh K, Hirata N, Masuda M, Naruto S, MurataK, Wakabayashi K, Matsuda H. Inhibitory ef-fects of Citrus hassaku extract and its fla-vanone glycosides on melanogenesis. BiolPharm Bull 2009;32 (3):410-5

(9) Sasaki K, Yoshizaki F. Nobiletin as a tyrosinaseinhibitor from the peel of Citrus fruit. BiolPharm Bull 2002;25 (6):806-8

* Authors’ address:Sabine Kiefer, Michaela Weibel

Julian Smits, Mathias JuchJane Tiedtke, Norbert Herbst

Cosmetochem International AGSennweidstrasse 44/46

6312 SteinhausenSwitzerland

Email: [email protected]

SKIN L IGHTENING

COSMETICS