lichenic extracts and metabolites as uv filters

Journal of Photochemistry and Photobiology B: Biology 120 (2013) 17–28

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Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier .com/locate / jphotobiol

Lichenic extracts and metabolites as UV filters

Françoise Lohézic-Le Dévéhat a,⇑,1, Béatrice Legouin a,⇑,1, Céline Couteau b, Joël Boustie a,Laurence Coiffard b

a PNSCM-UMR 6226; Faculté des Sciences Pharmaceutiques et Biologiques, Université Européenne de Bretagne, Université de Rennes 1, 2 Av. du Pr Léon Bernard,35043 Rennes Cedex, Franceb Université de Nantes, Nantes Atlantique Universités, LPiC, MMS, EA2160, Faculty of Pharmacy, 1 rue G. Veil – BP 53508, Nantes F-44000, France

a r t i c l e i n f o a b s t r a c t

Article history:Received 7 August 2012Received in revised form 13 December 2012Accepted 7 January 2013Available online 26 January 2013

Keywords:PhotoprotectionLichenSun Protection FactorProtection Factor-UVALasallia pustulataGyrophoric acid

1011-1344/$ - see front matter � 2013 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.jphotobiol.2013.01.009

Abbreviations: SPF, Sun Protection Factor; PF-UVA,oil-in-water; OMC, Octylmethoxycinnamate; PMMMOC, methyl b-orcinolcarboxylate; PIF, photo irritancyspecies.⇑ Corresponding authors. Address: Department of Ph

Pharmacy, 2 av. Pr Léon Bernard, 35043 Rennes Cedex,fax: +33 223234704.

E-mail addresses: [email protected]@univ-rennes1.fr (B. Legouin), laurenCoiffard).

1 These authors contributed equally to this paper.

Three lichen extracts and ten lichenic compounds have been screened for their photoprotective activities.The determination of their Sun Protection Factor (SPF) and Protection Factor-UVA (PF-UVA) values wasdone in vitro. Among them, a Lasallia pustulata extract and gyrophoric acid exhibited SPF values over 5,which is better than Homosalate (SPF � 4). Their photoprotective properties are only slightly modifiedafter a 2-hours period of irradiation. Salazinic acid and L. pustulata presented characteristics of a UVAbooster like the butyl-methoxydibenzoylmethane (Avobenzone) (PF-UVA � 2 vs. 2.8 for Avobenzone).Salazinic acid was a better anion superoxide scavenger than ascorbic acid and none of them exhibiteda photosensitizing cytotoxicity by exposing them on HaCaT cells to UVA radiations (photo-irritancy factorPIF < 5).

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

It is estimated that as many as 60,000 people worldwide die peryear from too much sun exposure and mostly from malignant skincancers. Malignant melanoma account for 48,000 of these deaths,and other types of skin cancers for 12,000. In France, alone, theincidence of melanoma doubles every 10 years. Chronic exposureas well as severe sunburns during childhood and teenage years in-crease the risk of skin cancer. Genetic risk factors also contribute tospecific sun sensitivity [1]. It is thus crucial to protect ourselvesfrom sun-DNA-damages. A valuable tool against the deleteriousUV radiation effects is photoprotection. Current methods of photo-protection include sun avoidance, seeking shade, using protectiveclothing and applying sunscreen. The use of sunscreens is nowa-days the most common protective strategy used by the population.

ll rights reserved.

UVA-Protection Factor; O/W,A, Polymethylmethacrylate;

factor; ROS, reactive oxygen

armacognosy-Mycology, Fac.France. Tel.: +33 223234817;

s1.fr (F. Lohézic-Le Dévéhat),[email protected] (L.

Future sunscreens should ideally offer an improved protectionwith broad-spectrum UVA–UVB coverage. It is also known thatthe long-term effects of UV-radiation cause DNA damages, immu-nosuppression and photoaging. UVB radiation is now considered tobe a complete carcinogen. It initiates a photo-oxidative reactionwhich impairs the antioxidant status and increases the level ofreactive oxygen species (ROS) accompanied by activation of signal-ling pathways [2]. So, an additional advantage for a pre-selectedsunscreen is to assist the natural antioxidant systems to preventthese deleterious effects in a topical or systemic treatment of skin.Biologically active natural products keep on inspiring the develop-ment of many new drugs and skin care products. Some of thesecompounds are isolated from plants [3–6], marine sources [7], fun-gi [8] and microorganisms [9]. Nature’s potential is still underex-plored and lichens are one example of a poorly explored source.Lichens are symbiotic organisms combining fungi and algae, whichare known to live in regions where the UV-radiation is particularlyintensive because of the altitude and the ozone rarefaction. In theArctic and Antarctica regions where the ozone depletion is thehighest, lichens represent a quantitatively important part of thephotosynthetically active biomass. Over time, they have developedsome protective tools that enable their survival under UV radia-tion. They synthesize pigments with antioxidant capacities andstrong absorption in the UV region. So, according to criteria re-ported by several authors, these lichens are good candidates tobe screened for UV-filters [10–14]. Some publications emphasize

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OH

OCH3

OH

CHOHO

CH3COOCH3

CH3

O

1

O

O OH

CHOHO

H3C

CH3

O

COOH

OCOCH=CHCOOH

5

CO O OHCH3

H3CO COOHCH3

OH

2

O

OO

H3C

HO

O

OOH3

O

OHOH2C

OH

CHOHO

H3C

O

O

HO

O

4

O

O

OH OH

H3CO CH3

6

OHCHO

HO

CH3COCH3

7

C13H27O O

HOOC

11bC13H27

O O

CH3HOOC

11aOH

CO O

CO O

CH3COOH

OH

OH

CH3

CH3

HO

10

OH OH

H3C

HO

OHH3C

O

O

CH3

OCH3

9

HO

O

O

H3COOC

8

Fig. 1. Structures of the lichen compounds.

18 F. Lohézic-Le Dévéhat et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 17–28

the photoprotective properties of some lichens [15–20]. In theselatter, the evaluated compounds were some depsides (atranorin,gyrophoric acid, diffractaic acid, divaricatic acid), depsidones(lobaric acid, pannarin, 10-chloropannarin, vicanicin), a dibenzofu-ran like ((+)-usnic acid), diphenylethers (epiphorellic acids), a pul-vinic acid derivative (calycin) and a mycosporine (collemin)). Ourscreening aims at evaluating some lichen extracts and lichen com-pounds to find new natural photoprotective agents in these evolu-tionary conserved symbiotic organisms. In this screening, SunProtection Factor (SPF) and Protection Factor-UVA (PF UVA) weredetermined with an in vitro method, and we explored the activityof the lichenic compounds available in sufficient amount in ourlibrary: two depsides (atranorin (1), evernic acid (2)), threedepsidones (variolaric acid (3), salazinic acid (4), fumarprotocetra-ric acid (5)), an anthraquinone (parietin (6)), a monoaromatic com-pound (methyl b-orcinolcarboxylate MOC (7)) and a pulvinic acidderivative (vulpinic acid (8)) were explored (Fig. 1). And yet, thecosmetic industry uses more and more crude extracts as activeingredients [21–23] to formulate new skin products. This is firstfor economical reason so as to avoid purification processes to ob-tain pure compounds and secondly to adopt the REACH directivesconcerning the restrictive list of advisable solvents. So, we decided

to test crude extracts of three lichens, all exposed to UV radiationsbut living under different ecological habitats: the terricolous Ce-traria islandica (E1), the saxicolous Lasallia pustulata (E2) and thecorticolous Usnea hirta (E3). Nevertheless, it can appear that theactivity of an extract is simply due to one or two major com-pounds. So, it is of interest to compare the SPF values of the extractand of its major compounds to confirm if a purification processwhich is time consuming and expensive, is needed. We thus ex-plore the photoprotective properties of fumarprotocetraric acid(5), (+)-usnic acid (9) and gyrophoric acid (10) which are respec-tively the main secondary metabolites of C. islandica, U. hirta andL. pustulata. For the most active compounds, the SPF values afterirradiation, antioxidant and phototoxic capacities were performedto corroborate their interest as photoprotective and photochemo-preventive agents.

2. Experimental

2.1. General

All the chemicals (absolute ethanol, dimethylsulfoxyde) usedwere analytical reagent grade. Dimethicone (Abil� WE 09) was ob-

F. Lohézic-Le Dévéhat et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 17–28 19

tained from Goldschmidt (Montigny-le-Bretonneux, France). Ceti-ol� HE, stearic acid, glycerin, parabens, and triethanolamine(TEA) were purchased from Cooper (Melun, France). Xanthangum (Keltrol� BT) was obtained from Kelco (Lille Skensved, Den-mark). Homosalate (Eusolex� HMS), Avobenzone (Eusolex� 9020)and Octylmethoxycinnamate (Eusolex� 2292) were purchasedfrom Merck (Fontenay Sous Bois, France). Polymethylmethacrylate(PMMA) plates were purchased from Europlast (Aubervilliers,France). Powder-free latex finger cots were purchased from Cooper(Melun, France). Evernic acid (2) and Parietin (6) were purchasedfrom Extrasynthese (Genay, France). Methyl b-orcinolcarboxylate(7) as well as 1,1-diphenyl-2-picrylhydrazyl (DPPH, Fluka 43180),nitro blue tetrazolium (NBT, N-6876), NADH (N-8129), phenazinemethosulfate (PMS, Fluka 68600), neutral red (NR, 209-035-8)and phosphate buffer saline (PBS, P5493) were obtained from Sig-ma (St Quentin-Fallavier, France).

2.2. Lichens

2.2.1. Biological materialAll voucher specimens were deposited at the Faculty’s herbar-

ium of Rennes with a code (JB/year/number). Thalli of C. islandica(L.) Ach. (JB/03/08) and U. hirta (L.) Weber ex F.H. Wigg. (JB/02/03) were both collected in the Pyrenees (France) while thalli of L.pustulata (L.) Mérat (JB/06/77) had been harvested on rocks nearbyRennes (Brittany, France).

2.2.2. Extraction and isolationTo obtain the crude extracts, the ground air-dried thalli of li-

chen were extracted with solvents during three reflux phases of1 hour each. A mixture of acetone/dichloromethane (1:1) was usedfor C. islandica (10 g) and U. hirta (5 g) and acetone for L. pustulata(4.5 g). Crude extracts, called E1, E2, E3 and for C. islandica, L.pustulata and U. hirta respectively, were obtained after evaporatingthe solvents under reduced pressure.

ppm (t1)10.0

11.964

10.584

6.8536.647

6.2165.933

0.87

0.42

0.50

0.50

0.13

Fig. 2a. 1H NMR (500 MHz, DMSO-d6) of

In order to isolate the main secondary metabolites of these li-chens, a larger quantity of dried thalli were ground and extractedunder reflux thanks to successive extractions. C. islandica (375 g)was first extracted with n-heptane (3 � 1.5 L) and a mixture oflichesterinic (11a) and protolichesterinic (11b) acids precipitatedafter the evaporation of the solvent at room temperature. Theresidue of lichen powder was then extracted with acetone(3 � 1.5 L) to obtain a fumarprotocetraric acid (5) precipitate.L. pustulata (100 g) was extracted with solvents of increasingpolarity (n-heptane, dichloromethane, tetrahydrofuran (THF),3 � 500 mL for each) and in the last fraction obtained withTHF, gyrophoric acid (10) precipitated. (+)-Usnic acid (9) wasisolated by precipitation of the extraction of U. hirta (375 g) withn-heptane (3 � 1.5 L).

Atranorin (1), salazinic acid (4), variolaric acid (3) and vulpinicacid (8) were obtained from the laboratory’s chemical library andreferenced as JB/A/134, JB/A/106, JB/A/119 and JB/A/018 respec-tively. The identity and purity of the lichen compounds and the rel-ative amount of the main secondary metabolites in the extractswere assessed by NMR analysis (Bruker Avance 300 instrument(BioSpin, Wissembourg, France)) using DMSO-d6 or CDCl3 (withTMS as internal standard). Different shifts were observed for a gi-ven compound and were the result of the use of these different sol-vents. (Figs. 2–4). But UV spectra (Figs. 5–7) were also recorded inabsolute ethanol (Uvikon 931 UV–vis spectrophotometer with10 mm path length cells, Serlabo Technologies, Entraigues sur laSorgue, France).

2.3. Determination of photoprotective efficacy of lichen extracts

2.3.1. Preparation of the oil-in-water emulsionAn O/W emulsion was prepared by adding substance to

be tested to the formulation components. The composition of theemulsion is presented in Table 1. The hydrophilic-phase and theoil-phase were heated separately in a water bath until the ingredi-

0.05.0

0

500

1000

1500

5.3055.168

4.694

2.4552.4492.184

1.728

1.242

0.857

1.00

3.50

0.12

0.13

0.53

3.56

0.11

0.15

21.5

7

Cetraria islandica crude extract (E1).

ppm (t1)0.05.010.015.0

0

500

1000

1500

2000

11.965

10.582

6.8536.648

5.306

2.4562.441

1.00

0.98

0.56

0.58

0.59

3.49

-0.1

0

O

O OH

CHOHO

H3C

CH3

O

9'

COOH

OCOCH=CHCOOH

8

9 8'2''

5

3''

Fig. 2b. 1H NMR (500 MHz, DMSO-d6) of fumarprotocetraric acid (5).

ppm (f1)0.01.02.03.04.05.06.07.08.09.0

0

500

6.483

6.035

5.133

4.829

3.640

2.244

1.738

1.258

0.881

1.00

1.03

0.75

1.03

1.06

2.17

2.89

43.4

1

5.87

H2C O O

CH3HOOC

Lichesterinic acid

2

719

3

5

(H2C)11H3C

4 H2C O O

HOOC

(+)-Protolichesterinic acid

2

719

3

5

(H2C)11H3C

4

Fig. 2c. 1H NMR (500 MHz, CDCl3) of a mixture of lichesterinic (11a) and protolichesterinic acids (11b) obtained from the n-heptane fraction. The shifts in italic belong tolichesterinic acid, in plain to protolichesterinic acid while in bold, they belong to both molecules.


ents of each part were solubilised or melted. Next, the hydrophilicphase was added to the oil phase all at once stirring constantly. Fi-nally, in order to determine the in vitro SPF and PF-UVA values, the

extract was added to the preparation at a 10% (w/w) concentration.This percentage was corresponding to the maximum concentrationauthorized for many filters by the European regulation.

ppm (t1)0.05.010.0

0

500

1000

10.48610.31410.014

6.6856.6756.6296.6066.2456.233

2.3892.3732.363

1.00

0.89

0.38

0.56

4.65

0.46

0.48

0.46

Fig. 3a. 1H NMR (500 MHz, DMSO-d6) of Lasallia pustulata extract (E2).

ppm (t1)6.006.106.206.306.406.506.606.706.806.90

0

100

200

300

400

500

600

7006.6856.675

6.629

6.606

6.2456.233

1.00

0.89

0.38

0.56

Fig. 3b. Enlargement of 1H NMR (500 MHz, DMSO-d6) focused on the 6–7 ppm of Lasallia pustulata extract E2.


2.3.2. Procedure used for in vitro determination of the SPF and PF-UVAThirty accurately weighed milligrams of emulsion were spread

across the entire surface (25 cm2) of a PMMA plate using a fingercot. After spreading, 15 mg remained on the finger cot. SPF andPF-UVA values of the emulsion were then measured in vitro. Three

plates were prepared for each product to be tested and ninemeasurements were performed on each plate. Transmissionmeasurements between 290 and 400 nm and between 320 and400 nm for SPF and PF-UVA respectively, were performed using aspectrophotometer equipped with an integrating sphere (UV

ppm (t1)0.05.010.015.0

0

500

1000

10.33310.124

6.6066.5876.2316.1826.126

2.398

1.00

0.42

0.38

0.88

1.02

0.00

04.

56

OH

CO O

CO O

MeCOOH

OH

OH

Me

Me

HO 3

53'

5''

3''

5'

2''

2'

2

Fig. 3c. 1H NMR (500 MHz, DMSO-d6) of gyrophoric acid (10).

ppm (t1)6.106.206.306.406.506.60

0

500

6.6066.587

6.231

6.182

6.126

1.00

0.42

0.38

1.02

ppm (t1)2.3502.400

0

1000

2000

3000

4000

5000

2.398

2.379

2.355

3.00

0.59

0.56

Fig. 3d. Enlargement of 1H NMR (500 MHz, DMSO-d6) in the range 2–3 ppm and 6–7 ppm of gyrophoric acid (10).


Transmittance analyser UV1000S (Labsphere, North Sutton, US).The calculations were carried out according to the followingequations:

SPF ¼

X400

290

EkSkDk

X400

290

EkSkTkDk

ð1Þ

PF-UVA ¼

X400

315

EkSkDk

X400

315

EkSkTkDk

ð2Þ

where Ek is the erythemal spectral effectiveness at k, Sk is the solarspectral irradiance at k and Tk is the spectral transmittance of thesample at k [24,25].

2.3.3. Influence of irradiation on effectiveness of photoprotectionThe plates were irradiated for various times with a solar simula-

tor apparatus equipped with a xenon arc lamp (1500 W) and specialglass filters to restrict the transmission of light below 290 nm(Suntest CPS+; Atlas, Moussy le Neuf, France). The temperature ofthe samples was kept low and constant using a tap water coolingcircuit on to the walls of the reactor. In order to eliminate the turbu-lence inside the Suntest chamber, we developed a system where theplates are blocked between two rails and covered with a quartzplate. The light source emission was maintained at 650 W/m2 in

ppm (t1)0.05.010.015.0

0

500

1000

13.368

11.330

6.3186.216

5.933

2.6772.6022.0101.746

1.00

0.84

1.04

3.00

2.90

3.30

3.08

0.14

0.11

Fig. 4a. 1H NMR (500 MHz, DMSO-d6) of Usnea hirta extract (E3).

ppm (t1)1.001.502.002.503.00

0

500

1000

1500

2000

2.677

2.602

2.010

1.746

3.00

2.90

3.30

3.08

Fig. 4b. Enlargement of 1H NMR (500 MHz, DMSO-d6) focused on the 1–3 ppm range of Usnea hirta extract.


accordance to global solar spectral irradiance at sea level measuredin accordance to CIE (Commission Internationale de l’Eclairage). The

SPF and the PF-UVA of the emulsions were measured in vitro beforeand after irradiation by the protocol previously described [26].

ppm (t1)5.010.0

0

500

1000

1500

13.368

11.331

6.318

2.6772.6012.0101.746

1.00

0.91

1.02

3.07

3.09

3.06

3.16

O OH

H3C

HO

OHH3C

O

O

CH3

OCH3 4

7

9

10

1215

14

Fig. 4c. 1H NMR (500 MHz, DMSO-d6) of (+)-usnic acid.

Table 1Formula of O/W emulsion.

Ingredients % (w/w)

Oil phaseParaffinum liquidum 17.00Cetiol HE� (PEG-7 glyceryl cocoate) 5.00Stearic acid 5.00Butylhydroxytoluene 0.01Eumulgin� B1 (Ceteareth-12) 1.50Eumulgin� B2 (Ceteareth-20) 1.50

Hydrophilic phaseGlycerin 4.00Rhodicare� T (Xanthan gum) 0.90Sodium propylparaben 0.05Sodium methylparaben 0.10TEA 0.30Distilled water qsp 100.00


2.4. Antioxidant and cytotoxicity studies

2.4.1. Antioxidant assaysTwo antioxidant assays were performed on the most photopro-

tective agents L. pustulata extract and gyrophoric and salazinicacids, one using the 1,10-Diphenyl-2-Picrylhydrazyl free radical as-say (DPPH) and the other based on the measurement of superoxideanion scavenging activity.

2.4.1.1. DPPH assay. The scavenging activity of the lichen com-pounds on DPPH was measured using the Matsukawa et al. [27]method with some modifications as previously described [28]. Areaction mixture containing 100 lL of DPPH (0.5 mM) in methanoland 10 lL of the lichen compound solutions in DMSO to give finalconcentrations of 500, 250, 125, 62.5, 31.25 lg/mL per well, wasdistributed in each microplate well. Ascorbic acid was used as a

positive control on each plate. Each concentration and all testswere done in triplicate and the results averaged. The percentageinhibition at steady state for each dilution was used to determinethe IC50 values graphically.

2.4.1.2. NBT assay. For the same compounds, measurements ofsuperoxide anion scavenging activity in 96-well microplates basedon the non-enzymatic method previously described with somemodifications were performed [28,29]. The reaction mixture inthe sample wells consisted of NADH (78 lM), Nitro-blue tetrazo-lium (NBT) (50 lM), Phenazine methosulfate (PMS) (10 lM), andlichen compounds (500, 250, 125, 62.5, 31.25 lg/mL). The reagentswere dissolved in 16 mM tris–hydrochloride buffer, at pH = 8 ex-cept for all the lichen compounds which were dissolved in DMSO.After 5 min of incubation at room temperature, the spectrophoto-metric measurement was performed at 560 nm against a blankwithout PMS and sample. Ascorbic acid was used as a positive con-trol. The percentage inhibition at steady state for each dilution wasused to calculate the IC50 values. This gave the amount of antioxi-dant required (measured as the concentration of the stock solutionadded to the reaction mixture) to scavenge 50% of O��2 : the lowest isthe values, the best is the efficiency in scavenging O��2 . All testswere done in triplicate and the results averaged.

2.4.2. Cytotoxicity assaysThe 3T3 Neutral Red Uptake (NRU) phototoxicity test was

developed and validated in Europe and has since been acceptedat the international level as a replacement for animal-based photo-toxicity studies. This test is based on the relative reduction in via-bility of cells exposed to the chemical in the presence vs. absenceof light. The cell viability is measured by degree to which theyare able to absorb the neutral red.

Cytotoxic and photocytotoxic activities of L. pustulata extract,gyrophoric and salazinic acids were evaluated according to the

290 310 330 350 370 390

λ (nm)

0

0.2

0.4

0.6

0.8

1

1.2

Abso

rban

ce

9E3

Fig. 5. Absorbance of Usnea hirta E3 and (+)-usnic acid 9 (20 lg/mL in ethanol).

0.4

0.5

0.6

0.7

0.8

orba

nce

10E2


OECD guideline [30] with some modifications. In brief, 100 lL perwell of a cell suspension of HaCaT cells (ATCC) at 8 � 105 cells/mLwere maintained in a RPMI culture medium with 5% of calf serumat 37 �C under 5% CO2 for 24 h for formation of monolayers. Att = 24 h, two 96-well plates per test chemical were preincubatedfor 1 h with nine different concentrations of the tested compound(in the range 0.01–200 lg/mL). Chlorpromazine was used as thepositive control. Thereafter, one of the two plates was irradiated(+UV) for 50 min with 5 J/m2 using a UV stratalinker 2400 (Strata-gene, USA) whereas the other plate (�UV) was kept in the dark. Inboth plates the treatment medium (PBS) was replaced by a culturemedium and after 24 h of incubation, cell viability is determined byNeutral Red Uptake. Cell viability was expressed as percentage ofuntreated cell controls and is calculated for each test concentra-tion. To predict the phototoxic potential, we determined thephoto-irritancy factor (PIF) thanks to the concentration responsecurves obtained in the presence and in the absence of irradiation.If PIF < 5, no phototoxic potential is predicted. If both IC50 (�UV)and (+UV) cannot be calculated, a formal PIF (PIF = ⁄1) is used pre-dicting no phototoxic potential [30].

0

0.1

0.2

0.3

290 310 330 350 370 390

Abs

λ (nm)

Fig. 6. Absorbance of Lasallia pustulata E2 and gyrophoric acid 10 (20 lg/mL inethanol).

0.15

0.2

0.25

ance

5

E1

11a + 11b

3. Results and discussion

3.1. Extraction yields

The solvents were chosen to extract a great variety of metabo-lites such as depsides or depsidones and to get the highest yields(Table 2). L. pustulata was the most extractable lichen with a yieldof about 10% (w/w) which is of interest for an industrial use.

3.2. Characterization of lichen extracts

1H NMR of the crude extract of C. islandica E1 (Fig. 2a) exhibitedthe characteristic signals of three major compounds. The twomethyl groups (d = 2.44, 2.45 ppm), the three aromatic hydrogens(d = 6.64, 6.85 ppm), the methylene group (d = 5.30 ppm), the alde-hyde function (d = 10.58 ppm) and the hydroxyl group(d = 11.96 ppm) were assigned to the depsidone fumarprotocetra-ric acid 5 (Fig. 2b) [31]. The proton signals (d = 4.69, 5.93,6.21 ppm) were assigned to protolichesterinic acid (11b) whiled = 5.16 ppm was attributed to lichesterinic acid (11a). Except forthe methyl group at d = 5.45 ppm attributed to 11a, all the othermethyl and methylene groups of the two butenolides overlapped(Fig. 2c) [32]. 11a, 11b, and 5 were found in the relative10:12:78 ratio, based on the relative integration of the respectivearomatic hydrogens.

The chemical shifts on the 1H NMR spectrum of the E2 L. pustu-lata extract (Figs. 3a and 3b) indicated the presence of gyrophoricacid 10 [33] with the aromatic hydrogens (d = 6.23, 6.59–6.68 ppm), the phenol groups (d = 10.01, 10.31, 10.48 ppm) andthe methyl groups (d = 2.36–2.39 ppm) (Figs. 3c and 3d). UsingNMR, some minor compounds were calculated to be less than 2%(w/w).

As seen on 1H NMR spectra (Fig. 4), four methyl groups (d = 1.74,2.01, 2.60, 2.67 ppm), aromatic hydrogen (d = 6.31 ppm) and

Table 2Extraction yield (%) of the lichens (E1–E3) and their main secondary metabolites.

Lichenextract

Extraction yield(w/w)

Main secondary metabolite

E1 2.7 Fumarprotocetraric acid, protolichesterinicacid, lichesterinic acid

E2 9.4 Gyrophoric acidE3 4.0 (+)-Usnic acid

hydroxyl groups (d = 11.33, 13.36 ppm) corresponding to (+)-usnicacid signals (Fig. 4c) were recognized in U. hirta extract E3 (Figs. 4aand 4b). It is indeed well-known that usnic acid is a major com-pound in Usnea genus [34]. Based on the relative integration ofthe aromatic protons in the 6–8 ppm zone of the Fig. 2a spectrum,additional minor compounds having aromatic protons were esti-mated less than 10% (w/w).

UV spectra of the extracts dissolved in ethanol were comparedwith those of their major compounds summarized in Table 2. Sim-ilar profiles were observed between U. hirta extract and usnic acid(Fig. 5) as well as for L. pustulata and gyrophoric acid (Fig. 6) and C.islandica and protolichesterinic, lichesterinic and fumarprotocetra-ric acids (Fig. 7). For the latter, a spectrum was calculated fromthose of pure metabolites according to the composition and ratiodeduced from 1H NMR data. A large overlap between the experi-mental and calculated spectra confirmed the composition of E3(Fig. 7).

3.3. Photoprotective activity

The absorbances of extracts and lichen compounds were re-corded between 290–315 nm and 315–400 nm corresponding to

0

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290 310 330 350 370 390

Abso

rb

λ (nm)

(11a + 11b + 5) calc

Fig. 7. Absorbance of Cetraria islandica extract E1 and its main metabolitesfumarprotocetraric acid 5 and the mixture of 11a and 11b (20 lg/mL in ethanol).The spectrum (11a + 11b + 5)calc has been obtained by calculation from puremetabolites according to the ratio in the extract.

Table 3UV spectral data of lichen compounds (1–10) and reference UV filters.

Compound kmax (nm) e (L mol�1 cm�1)

1 293 5700280 10,200

2 307 16,3003 267 91004 311 64005 320 52006 287 18,6007 303 35008 367 11,000

289 14,8009 284 18,60010 300 16,000Homosalatea 306 4600OMCa 309 24,700Avobenzonea 358 15,500

a UV filters.


the UVB and UVA regions respectively. Out of the eleven com-pounds, two (fumarprotocetraric acid (5), vulpinic acid (8)) exhib-ited a maximal absorbance (kmax) in the UVA range similar to thereference compound Avobenzone while the others exhibited max-imal absorbances between 280 and 311 nm (Table 3). Seven ofthem also possessed a molar extinction coefficient, e, superior to10,000 L mol�1 cm�1 which corresponds to one of criteria usedfor selecting UV filters [35]. The highest values of epsilon wasfound for parietin (6) and (+)-usnic acid (9) and were in the rangeof the OMC e value. Nevertheless, some authorized filters exhibitedlow e values such as Homosalate (e = 4600 L mol�1 cm�1). Some li-chen products such as fumarprotocetraric acid (5), MOC (7), salazi-nic acid (4) and variolaric acid (3) were in this range (5200–9100 L mol�1 cm�1).

The SPF value is the ratio between the minimal erythemal dose(MED) of protected and unprotected skin. One lichen compound,gyrophoric acid (10), exhibited a promising SPF value superior to5 which is better than Homosalate (Table 4). So, the applicationof this product should provide a sunburn protection for at least fivetimes longer than an unprotected skin [36].

If UVB protection is imperative, UVA protection is now recog-nized to be equally essential. One molecule (salazinic acid, (4))and one extract (L. pustulata, (E2)) can be good PF-UVA boosterscandidates (PF-UVA > 2).

Anyway, most of the approved active sunscreen ingredients arenever used alone in a galenic preparation but in combination to of-fer a maximal protective action in the whole UVA–UVB range. So,

Table 4Photoprotective results: SPF, PF-UVA values obtained with the lichen compounds,extracts and three commercial filters.

Product tested SPF ± SD PF-UVA ± SD

E1 1.55 ± 0.30 1.37 ± 0.28E2 5.52 ± 0.21 2.45 ± 0.15E3 1.67 ± 0.25 1.48 ± 0.201 1.52 ± 0.06 1.42 ± 0.052 2.42 ± 0.19 1.57 ± 0.093 2.09 ± 0.10 1.36 ± 0.034 2.56 ± 0.15 2.07 ± 0.105 1.91 ± 0.10 1.75 ± 0.086 1.94 ± 0.15 1.85 ± 0.137 2.03 ± 0.08 1.24 ± 0.028 2.55 ± 0.24 1.67 ± 0.109 1.62 ± 0.25 1.48 ± 0.2010 5.03 ± 0.76 1.77 ± 0.08Homosalatea 3.91 ± 0.44OMCa 11.16 ± 0.41Avobenzonea 2.76 ± 0.31

a Reported value for Homosalate and OMC 4 and Avobenzone 4.

most of the lichen compounds considered as good protectors in arestricted UV range but moderate protectors as far as broad-spec-trum is concerned, could be mixed with other filters to yield thebroad-spectrum coverage. Furthermore, the SPF and epsilon valuesdo not appear to be correlated: high values of epsilon do not nec-essarily lead to high SPF values (evernic acid, vulpinic acid), and alow value of epsilon do not preclude a significant value of SPF(Homosalate).

The (+)-usnic acid was found to be a weak photoprotector in ourexperiment. In contrast, many authors reported (+)-usnic acid as agood photoprotective agent [16,18,37]. Their methodologies weredifferent for in vitro assays either in solution or on cells with Mem-brane Protection Factor (MPF) measurements and also for in vivoassays on albino guinea pigs. Recently, Boehm et al. [20], in aphotophysical study, reported usnic acid as a potential sunscreenwithout determining any SPF values.

The same discussion could also stand for atranorin which ourresults proved to be a weak filter.

The gyrophoric acid was found to be the best UVB photoprotec-tor in our experiment which is in agreement with literature, even ifour value was lower. In a previous study, Fernandez et al. [37] re-ported an in vivo SPF value of 10 with a non-standardized methodon albino guinea pigs (the ISO 24444:2010 recommends in vivo as-says on human volunteers [38]).

Lichen extracts photoprotective profiles were found to be simi-lar to those of their major compounds i.e. gyrophoric acid for L.pustulata and (+)-usnic acid for U. hirta so no synergism on SPFactivity was highlighted for these extracts.

The structure of the most active compound, gyrophoric acid, isthe result of the esterification between three orsellinic acid units.Among the tested compounds, compound 2 also presented thismoiety unit but without any noticeable SPF. An attempt at correla-tion between chemical structure and SPF values failed. More activemolecules are needed to establish a structure relationship activity.

3.4. Influence of irradiation on the effectiveness of lichens compounds

L. pustulata and its main metabolite gyrophoric acid appeared tobe good UV-filters (SPF > 5 and PF-UVA � 2) and a further evalua-tion of their toughness against UV irradiation was required (Ta-ble 5). After 2 h of irradiation at 650 W/m2, more than 90%efficacy was conserved. These results are in agreement with Begoraexperiments who reported no degradation after UVA and UVBexposure of the gyrophoric acid contained in Umbilicaria deusta,U. mammulata and Punctelia borreri extracts [39]. This photostabil-ity is all the more remarkable than UV filters are not so stable [26].

3.5. Antioxidant activity of L. pustulata, gyrophoric acid and salazinicacid

Antioxidant properties of E2, 4 and 10 were evaluated so as tofind a new natural source of antioxidant. Because of its stabilityin the radical form and simplicity of the assay, DPPH radical is acommonly used substrate for a fast evaluation of antioxidantactivity. The principle behind this assay is the color change frompurple to yellow of the DPPH solution because the radical isquenched by the antioxidant. The colour changes can be measuredquantitatively by spectrophotometric absorbance at 540 nm. The

Table 5Influence of irradiation on effectiveness for Lasallia pustulata E2 and its mainmetabolite gyrophoric acid 10.

Product SPF0 FP-UVA0 SPF2h FP-UVA2h

E2 5.52 ± 0.21 2.45 ± 0.15 5.21 ± 0.48 2.38 ± 0.1610 5.03 ± 0.76 1.77 ± 0.08 5.34 ± 0.64 1.82 ± 0.18

Table 6Antioxidant, cytotoxic and phototoxic activities of 4, 10 and E2.

Compound Antioxidant activities Phototoxic activities on HaCaTcells

DPPHassay (%activityat500 lg/mL)

NBT assayIC50 ± SD(mg/mL)

IC50 ± SD (lg/mL) Photo-irritancyfactor(PIF)

Withoutirradiation

Withirradiation

4 0 3.9 ± 1.0 >200 >200 ⁄1d

10 25 ± 2.9 79.6 ± 16.4 168 ± 33 >200 –e

E2 0 72 ± 16 200 ± 40 118 ± 16 1.7f

Ascorbic acida 98.5 ± 0.5 6.2 ± 0.2 ncc ncc ncc

Chlorpromazineb ncc ncc 9.5 ± 1.5 0.48 ± 0.13 20f

a Antioxidant positive control.b Cytotoxic and phototoxic positive control.c nc: not concerned.d Formal PIF = Cmax(�UV)/Cmax(+UV).e No phototoxic potential.f PIF = IC50(�UV)/IC50(+UV).


compounds were screened for DPPH radical scavenging activityaccording to the method described and the results of the screeningare shown in Table 6. By comparison to ascorbic acid, no activitywas observed at the highest concentration for salazinic acid andgyrophoric acid and while the crude extract of L. pustulata can becategorized as a weak free radical scavenger because no IC50 canbe calculated. It backed up, the results obtained by Kumar aboutgyrophoric acid [40].

The superoxide radicals generated from dissolved oxygen byPMS-NADH coupling can be measured by their ability to reduceNBT. The decrease in absorbance at 560 nm with the tested com-pound or extract and the reference compound ascorbic acid indi-cated their abilities to quench superoxide radicals in the reactionmixture. As shown in Table 6, the IC50 values of salazinic acidand ascorbic acid on superoxide scavenging activity were3.9 ± 1 lg/mL and 6.2 ± 0.2 lg/mL, respectively. Such an activityreinforces the interest of this compound to be a candidate asUVA booster. L. pustulata extract and gyrophoric acid exhibited asimilar but moderate activity as superoxide anion scavengers.

3.6. Cytotoxicity and phototoxic activities of L. pustulata, gyrophoricacid and salazinic acid

Phototoxicity is defined as a toxic response after the exposureto light or to UV irradiation of a substance applied to the body or

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Fig. 8. Cytotoxicity and phototoxicity activities of 4, 10 and E2 on HaCaT cells. (a) (�UV)(+UV)-curve of mean values of the cell viability in % of the respective negative controlchlorpromazine or tested compounds. Points are means of three independent experime

after its systemic administration. It is well documented throughclinical and experimental studies that non-cytotoxic doses of manychemicals could cause phototoxic responses when exposed to non-phototoxic doses of UV radiation. So, this test is a comparison be-tween the cytotoxicity of a chemical compound when tested in thepresence and one tested in the absence of exposure to a non-cyto-toxic dose of UVA light. One day after treatment, cytotoxicity ismeasured as an inhibition of the capacity of the cell to take up avital dye, which is neutral red.

The cytotoxicity of the tested lichenic compounds on humankeratinocytes HaCaT is weak (Table 6) compared to the positivecontrol chlorpromazine (IC50 = 9.5 ± lg/mL). IC50 was over200 lg/mL for salazinic acid and indicated low cytotoxicity forgyrophoric acid (IC50 = 168 ± lg/mL) and L. pustulata extract(IC50 = 200 ± lg/mL), mean dose–response curves for cytotoxicityare shown in Fig. 8. Under 5 J/m2 UVA irradiation, the viability ofHaCaT cells was 100%. The cytotoxicities of salazinic acid (4) andgyrophoric acid (10) were not enhanced by UVA irradiation(IC50 > 200 lg/mL) counter to L. pustulata extract (IC50 = 118 ± lg/mL) (Fig. 8) but its PIF factor remained under 5 (Table 6) whichis the value of Ref. [30]. The NRU phototoxicity test identifies thesubstances likely to be phototoxic in vivo and reveals no phototox-icity for the tested compounds.

4. Conclusion

One extract L. pustulata and its major compound gyrophoricacid are valuable candidates as UV filters because of their efficacybefore and after UV irradiation. They are indeed promising agentsbecause they have very low phototoxicity even the maximal con-centration of the Lasallia extract to be used in cosmetic preparationshould be reduced. The gyrophoric acid could also be a usefulstructure for in silico modelisation in order to determine an effec-tive pattern for the activity as UV filter. Some of the other studiedmolecules have no high SPF values but can be used as filter boost-ers: variolaric acid, evernic acid and vulpinic acid and especiallysalazinic acid in term of UVA. In fact, it is of common use in cosme-tology to mix UV filters for the additivity of the SPF values. More-over, additivity or synergism of lichen compounds with knownsunscreens could be of interest [41].

UV radiations damage skin cells through indirect mechanismswith the formation of ROS. An overproduction of ROS results inan oxidative stress, a process that can serve as an important medi-ator of damage to cell structure including lipids and membranes,proteins and DNA. One approach to protect the skin against the

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-curve of mean values of the cell viability in % of the respective negative control. (b). In phototoxicity evaluation the cells were irradiated at 5 J/m2 in the presence ofnts (n = 9 for each concentration).


dangerous effects of UVs could be the use of phytochemicals withantioxidant properties. For example, vitamins and phenols havegained considerable attention as protective agents and are addedin preparations for topical applications in the recent years [4].Polyphenolic compounds such as salazinic acid could be good can-didates as antioxidant. Further studies as decreasing effects on nat-ural agents on UVB-induced damage and anti-inflammatory effectsshould be investigated to complement these valuable capacities[22].

Acknowledgements

The authors thank Aurélie Sauvager, Isabelle Rouaud and EvaPaparis for their technical assistance. We thank also the Researchteam Expression des gènes et oncogenèse of the UMR 6290 – IGDR(Rennes) for the access to the Stratalinker apparatus.

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lichenic extracts and metabolites as uv filters

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