Mycosporine-like amino acids:

Natural UV-screening compounds from red algae

to protect the skin against photoaging

D. Schmid, C. Schürch, F. Zülli, H.-P. Nissen, H. Prieur:Sonderdruckaus7 - 2003

Internationales Journalfür angewandte Wissenschaft■ Kosmetik ■ Haushalt ■ Spezialprodukte

synthetic filters

MAA’s

cream control

MAA’s

cream control

2 SÖFW-Journal, 129. Jahrgang 7-2003

D. Schmid, C. Schürch, F. Zülli*, H.-P. Nissen, H. Prieur**

Mycosporine-like amino acids: Natural UV-screening compounds from red algae to protect the skin against photoagingKeywords: UVA, photoaging, Mycosporine-like amino acids, Porphyra umbilicalis

Abstract

UV-screening compounds to reducedamage caused by ultraviolet (UV) radi-ation are almost ubiquitous in nature.The most active natural UV-absorbingsubstances are the mycosporine-likeamino acids (MAA's) that are pro-duced by certain algae, corals andcyanobacteria. The peak absorption ofMAA's is in the UVA range and theirabsorption coefficients are similar tothose of synthetic sunscreens. A hu-man study showed that a cream con-taining MAA's from the red alga Por-phyra umbilicalis efficiently protectsthe skin against UVA exposure on atypical working day.

Introduction

Solar UV radiation can induce acuteskin reactions like erythema but pro-duces also long term effects such aspremature skin aging (photoaging)and carcinogenesis. The solar UV spec-trum that reaches the earth's sur-face has been divided into UVB (290 –320 nm) and UVA (320 – 400 nm). Themore energetic UVB is absorbed in theepidermis where it can cause acute sun-burn, DNA mutation or even cancer. Al-though less energetic, the longer wave-lengths in the UVA region can pene-trate much deeper into the skin. UVAreaches the dermis where it is respon-sible for the premature skin aging ef-fects of sun light.UVA alters the expression of certaingenes by generation of reactive oxy-gen species (ROS) and / or stimulationof pro-inflammatory cytokines such as

IL-1β and TNF-α (1). It has been shownthat UVA activates the transcriptionfactors NF-κB and AP-1 (2). This resultsin the induction of a series of collagenand elastin degrading enzymes, theso called matrix metalloproteinases(MMP's). A decrease in collagen con-tent and fiber fragmentation ulti-mately leads to the typical signs ofphotoaging. ROS are formed after ab-sorption of UV through skin chro-mophors, like urocanic acid or DNA.When UV light is absorbed by trans-urocanic acid (absorption maximum at345 nm), singlet oxygen is generated.This highly reactive molecule can reactwith proteins or with lipids. The reac-tion products, such as lipid peroxides,are themselves ROS and have lost cel-lular functionality (3).While UVB is highly dependent on theseason, day-time, cloudiness and lati-tude, UVA is relatively constant duringthe year. In middle Europe erythemalUVB is of minor importance for dailyskin care products, but UVA is presentall year round in harmful doses thatare received indoors as well since UVAeasily penetrates window glass. To es-timate individual, daily UV exposure,test persons were equipped with smallUV dosimeters (4). Exposure was mea-sured on typical working days, takinginto account also indoor UV radiation.Their data show that indoors UVB is incontrast to UVA not of relevance. Adaily UVB dose of 2.5 MED's at openwindows on a sunny summer day is re-duced to 0.2 MED at closed windows.But about 40 % of the outdoor UVA ra-diation could be still received indoors.Of real relevance for daily skin care aretherefore not SPF factors but UVA pro-tection. Since synthetic filters might beproblematic for everyday applicationbecause of endocrine side effects (5),we analyzed the potential of naturalUVA-screening substances to protectthe skin against photoaging.The strongest UVA-absorbing com-pounds in nature are the mycosporine-like amino acids (MAA's). They werefound in different organisms such ascyanobacteria, red algae, dinoflagel-

lates, corals and many marine inverte-brates. The basic cyclohexanone or cy-clohexenimine chromophore is respon-sible for UV absorbance. Incorporationof various amino acids or iminoalcoholgroups results in a diversity of about20 MAA's (6). The red alga Porphyraumbilicalis is reported to produce theMAA's Porphyra-334 (Fig. 1) and Shi-norine (7). Their absorption coeffi-cients (εmolar) at 334 nm are 42 300 and44 700 respectively. Their filter capaci-ty is therefore similar to that of syn-thetic UVA sunscreens such as Parsol®1789 (εmolar 40 000) and Mexoryl® SX(εmolar 45 000).This article summarizes the study re-sults on the efficacy of a cream withisolated MAA's from Porphyra umbili-calis to prevent photoaging inducedby UVA irradiation.

Materials and Methods

Extraction of MAA'sDried Porphyra umbilicalis material wassuspended in water at 3.3 % and ex-

Fig. 1 Chemical structure of the my-cosporine-like amino acid Porphyra-334

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tracted by incubation at 45 °C for 2hours under constant stirring. After re-moval of the algae material the extractwas clarified by ultrafiltration througha 10 kDa cut-off membrane. The testsubstance was prepared by mixing theclear extract with 3.3 % lecithin inform of liposomes and 0.4 % phenoxy-ethanol. The MAA concentration in thetest substance was adjusted to 0.1 %.

HPLC-Analysis of MAA'sSeparation of MAA's was done on aSuperspher 100 RP 18 column (4 µm,254 x 4 mm) with a LiChrospher 100 RP18 endcapt precolumn (5 µm). The mo-bile phase was 0.02 % acetic acid andthe flow rate 1.0 ml /min. The detectionwavelength was 334 nm. Identificationwas done by comparison with MAAstandards that were provided by Prof.Dr. D.-P. Häder (Friedrich-Alexander Uni-versity of Erlangen, Germany).

Cell culture experimentsMouse fibroblasts 3T3 were cultured in24 well plates in 100% DMEM for 24hours at 10 % confluence (1x104 cells /ml) in an incubator at 37 °C with 5 %carbon dioxide. After the medium wasreplaced by a mixture of 95 % basalmedium and 5 % DMEM, the test sub-stances were added. One series was ir-radiated with UVA for 1 min, the oth-er series stayed untreated as control.After 48 hours incubation, the cellswere fixed in 100 % methanol andstained in Coomassie blue staining so-lution. For determination of cell num-ber, the absorption at 580 nm wasmeasured.

Human studyIn a study over four weeks with 20women in the age of 36 to 54 the fol-lowing skin parameters were ana-lyzed: elasticity by means of Cutome-ter SEM 575 (Courage & KhazakaGmbH; Cologne, Germany), roughnessusing the digital micromirror devicePRIMOS (GFMesstechnik GmbH; Teltow,Germany), depth of wrinkles with theoptical 3D in vivo measurement sys-tem PRIMOS and lipid peroxidation byanalysis of squalenehydroperoxide for-mation (HPLC).The test products were a standardcream with 5 % MAA test substance(final MAA concentration of 0.005 %)and a standard cream with 4 % NeoHeliopan AV and 1 % Parsol 1789. Thestandard cream without actives servedas control. The test products were ap-plied twice daily on the inner side ofthe forearm and on the face (only thecream containing MAA's to measure

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Fig. 2 Protection of fibroblast cells by mycosporine-like amino acids against UVA

Fig. 3 Inhibition of lipid peroxidation compared to the beginning of the studyand the untreated area

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depth of wrinkles). The test areas onthe forearm were irradiated twiceweekly with UVA (10 J /cm2).

Results and Discussion

Analysis of Porphyra umbilicalisAnalysis by HPLC showed that Porphyraumbilicalis contained the MAA's Por-

phyra-334 and Shinorine, in a ratio of11.5 : 1. The total MAA concentrationwas 1.4 % of dry mass. This is consider-ably more than the reported typical con-centration range of 0.16 to 0.84 % (6).

Cell culture experimentsAfter UVA irradiation, growth of fi-broblast cells was reduced compared tonon-treated cells. The presence of 0.1to 5 µg/ml MAA's in the culture medi-um resulted in a concentration-depen-dent protection against the growth-limiting UVA irradiation (Fig. 2). The

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not-irradiated control assays showedno effect of MAA's in the concentra-tion range of 0.1 to 5 µg/ml. These fi-broblast cell culture assays demon-strate a specific UVA protection effectof MAA's. The effect was measurablealready at 0.1 µg/ml MAA's (0.00001%)in the culture medium.

Human studyNormal UVA exposure at non-protect-ed skin areas is between 3 and 20 J /cm2.Rudolph and Träger (4) measured anUVA exposure of 8 J /cm2 at the outsideof the forearm that is facing the win-dow, during a day that was cloudy and

Fig. 4 Improvement of skin firmness compared to the be-ginning of the study and the untreated area

Fig. 5 Improvement of skin smoothness compared to the be-ginning of the study and the untreated area

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sunny. The irradiation dose that was ap-plied in this study (two times 10 J/cm2

per week) corresponds therefore to anaverage UVA exposure at a non-pro-tected skin site. Thus, the study mimicsa daily photoaging of the skin, and itsprotection with creams.The study results demonstrate thatboth test products could reduce theprimary injure of UVA irradiation suchas lipid peroxidation by about 35 %(Fig. 3). Application of the standardcream without actives could minimal-ly inhibit (10 %) lipid peroxidation. Al-ready after two weeks application thetest products significantly reduced thesecondary effects of UVA exposureshown by improved skin conditionssuch as firmness and smoothness. Af-ter four weeks these parameters wereimproved by more than 10 % in areastreated with the MAA test cream andby about 6 % in zones treated withsynthetic filters (Fig. 4 and 5). The MAAtest product could reduce wrinkledepth by almost 20 % (Fig. 6).

Conclusion

The study clearly demonstrates that acream with 5 % MAA test substancecan neutralize the UVA effects as effi-cient as a cream with 1 % syntheticUVA filters and 4 % UVB filters. TheUVB sunscreens are not of relevance inthis study as only UVA irradiation wastested. But as outlined in the intro-duction this corresponds to normal

Fig. 6 Reduction of wrinkle depth compared to the beginning of the study andthe untreated area

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working day situations in middle Eu-rope where only UVA effects are im-portant. Considering the UVA-filteringcapacity, the cream with 1 % syntheticUVA filter is 200 times more activethan the MAA test product (0.005 %MAA's). The fact that this was not re-flected in the study results (or even inthe opposite way) might indicate thatthe application of 5 % UV-filters is byfar more than needed for an efficientprotection against photoaging duringnormal working days. Thus, MAA's area welcome, natural alternative to pro-tect our skin against daily UVA expo-sure (8.).

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Literature

(1) Tebbe B., Wu S., Geilen Ch. C., Eberle J., Kodel-ja V. and Orfanos C.E. (1997) L-Ascorbic AcidInhibits UVA-Induced Lipid Peroxidation andSecretion of IL-1α and IL-6 in Cultured HumanKeratinocytes In Vitro. J Invest Dermatol 108:302-306

(2) Djavaheri-Mergny M., Dubertret L. (2001) UV-A-induced AP-1 activation requires theRaf/ERK pathway in human NCTC 2544 ker-atinocytes. Exp Dermatol 10: 204-210

(3) Pinnell S.R. (2003) Cutaneous photodamage,oxidative stress, and topical antioxidant pro-tection. J Am Acad Dermetol 48: 1-19

(4) Rudolph T., Träger Ch., 22nd IFSCC CongressEdinburgh 2002, oral presentations, volume 2

(5) Schlumpf M., Cotton B., Conscience M., HallerV., Steinmann B. and Lichtensteiger W. (2001)In vitro and in vivo estrogenicity of UV screens.Environ Health Perspect 109: 239-244

(6) Cockell Ch. S., Knowland J. (1999) Ultravioletradiation screening compounds. Biol Rev 74:311-345

(7) Gröniger A., Sinha R. P., Klisch M. and HäderD.-P. (2000) Photoprotective compounds incyanobacteria, phytoplankton and macroal-gae – a database. J Photochem Photobiol. B:Biology 58: 115-122

(8) Schmid D., Schürch C. and Zülli F. (2003) Patentpending

Author’s addresses:*Daniel Schmid, Cornelia Schürch,Fred ZülliMibelle AG CosmeticsBolimattstrasse 15033 BuchsSwitzerlande-mail: daniel.schmid@mibelle.ch

** Hans-Peter Nissen, Hanspeter PrieurDerma Consult GmbHBrunnenstrasse 6153347 AlfterGermany

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