Lichenic extracts and metabolites as UV lters

Franoise Lohzic-Le Dvhat

a,,1
, Batrice Legouin
a,,1
, Cline Couteau
b
, Jol Boustie
a
,
Laurence Coiffard
b
a
PNSCM-UMR 6226; Facult des Sciences Pharmaceutiques et Biologiques, Universit Europenne de Bretagne, Universit de Rennes 1, 2 Av. du Pr Lon Bernard,
35043 Rennes Cedex, France
b
Universit de Nantes, Nantes Atlantique Universits, 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
Article history:
Received 7 August 2012
Received in revised form 13 December 2012
Accepted 7 January 2013
Available online 26 January 2013
Keywords:
Photoprotection
Lichen
Sun Protection Factor
Protection Factor-UVA
Lasallia pustulata
Gyrophoric acid
a b s t r a c t
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 was
done 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 modied
after a 2-hours period of irradiation. Salazinic acid and L. pustulata presented characteristics of a UVA
booster 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 exhibited
a photosensitizing cytotoxicity by exposing them on HaCaT cells to UVA radiations (photo-irritancy factor
PIF < 5).
2013 Elsevier B.V. All rights reserved.
1. Introduction
It is estimated that as many as 60,000 people worldwide die per
year from too much sun exposure and mostly from malignant skin
cancers. Malignant melanoma account for 48,000 of these deaths,
and other types of skin cancers for 12,000. In France, alone, the
incidence of melanoma doubles every 10 years. Chronic exposure
as well as severe sunburns during childhood and teenage years in-
crease the risk of skin cancer. Genetic risk factors also contribute to
specic sun sensitivity [1]. It is thus crucial to protect ourselves
from sun-DNA-damages. A valuable tool against the deleterious
UV radiation effects is photoprotection. Current methods of photo-
protection include sun avoidance, seeking shade, using protective
clothing and applying sunscreen. The use of sunscreens is nowa-
days the most common protective strategy used by the population.
Future sunscreens should ideally offer an improved protection
with broad-spectrum UVAUVB coverage. It is also known that
the long-term effects of UV-radiation cause DNA damages, immu-
nosuppression and photoaging. UVB radiation is now considered to
be a complete carcinogen. It initiates a photo-oxidative reaction
which impairs the antioxidant status and increases the level of
reactive oxygen species (ROS) accompanied by activation of signal-
ling pathways [2]. So, an additional advantage for a pre-selected
sunscreen is to assist the natural antioxidant systems to prevent
these 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 these
compounds are isolated from plants [36], marine sources [7], fun-
gi [8] and microorganisms [9]. Natures potential is still underex-
plored and lichens are one example of a poorly explored source.
Lichens are symbiotic organisms combining fungi and algae, which
are known to live in regions where the UV-radiation is particularly
intensive because of the altitude and the ozone rarefaction. In the
Arctic and Antarctica regions where the ozone depletion is the
highest, lichens represent a quantitatively important part of the
photosynthetically active biomass. Over time, they have developed
some protective tools that enable their survival under UV radia-
tion. They synthesize pigments with antioxidant capacities and
strong absorption in the UV region. So, according to criteria re-
ported by several authors, these lichens are good candidates to
be screened for UV-lters [1014]. Some publications emphasize
1011-1344/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotobiol.2013.01.009
Abbreviations: SPF, Sun Protection Factor; PF-UVA, UVA-Protection Factor; O/W,
oil-in-water; OMC, Octylmethoxycinnamate; PMMA, Polymethylmethacrylate;
MOC, methyl b-orcinolcarboxylate; PIF, photo irritancy factor; ROS, reactive oxygen
species.

Corresponding authors. Address: Department of Pharmacognosy-Mycology, Fac.

Pharmacy, 2 av. Pr Lon Bernard, 35043 Rennes Cedex, France. Tel.: +33 223234817;
fax: +33 223234704.
E-mail addresses: francoise.le-devehat@univ-rennes1.fr (F. Lohzic-Le Dvhat),
beatrice.legouin@univ-rennes1.fr (B. Legouin), laurence.coiffard@univ-nantes.fr (L.
Coiffard).
1
These authors contributed equally to this paper.
Journal of Photochemistry and Photobiology B: Biology 120 (2013) 1728
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
j our nal homepage: www. el sevi er . com/ l ocat e/ j phot obi ol
the photoprotective properties of some lichens [1520]. In these
latter, the evaluated compounds were some depsides (atranorin,
gyrophoric acid, diffractaic acid, divaricatic acid), depsidones
(lobaric acid, pannarin, 1
0
-chloropannarin, vicanicin), a dibenzofu-
ran like ((+)-usnic acid), diphenylethers (epiphorellic acids), a pul-
vinic acid derivative (calycin) and a mycosporine (collemin)). Our
screening aims at evaluating some lichen extracts and lichen com-
pounds to nd new natural photoprotective agents in these evolu-
tionary conserved symbiotic organisms. In this screening, Sun
Protection Factor (SPF) and Protection Factor-UVA (PF UVA) were
determined with an in vitro method, and we explored the activity
of the lichenic compounds available in sufcient amount in our
library: two depsides (atranorin (1), evernic acid (2)), three
depsidones (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 acid
derivative (vulpinic acid (8)) were explored (Fig. 1). And yet, the
cosmetic industry uses more and more crude extracts as active
ingredients [2123] to formulate new skin products. This is rst
for economical reason so as to avoid purication processes to ob-
tain pure compounds and secondly to adopt the REACH directives
concerning the restrictive list of advisable solvents. So, we decided
to test crude extracts of three lichens, all exposed to UV radiations
but living under different ecological habitats: the terricolous Ce-
traria islandica (E1), the saxicolous Lasallia pustulata (E2) and the
corticolous Usnea hirta (E3). Nevertheless, it can appear that the
activity 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 extract
and of its major compounds to conrm if a purication process
which 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 and
L. pustulata. For the most active compounds, the SPF values after
irradiation, antioxidant and phototoxic capacities were performed
to corroborate their interest as photoprotective and photochemo-
preventive agents.
2. Experimental
2.1. General
All the chemicals (absolute ethanol, dimethylsulfoxyde) used
were analytical reagent grade. Dimethicone (Abil

WE 09) was ob-

OH
O
CH
3
OH
CHO
HO
CH
3
COOCH
3
CH
3
O
1
O
O
OH
CHO
HO
H
3
C
CH
3
O
COOH
O
COCH=CHCOOH
5
CO O OH
CH
3
H
3
CO
COOH
CH
3
OH
2
O
O
O
H
3
C
HO
O
O
OH
3
O
O
HOH
2
C
OH
CHO HO
H
3
C
O
O
HO
O
4
O
O
OH OH
H
3
CO CH
3
6
OH
CHO
HO
CH
3
COCH
3
7
C
13
H
27
O
O
HOOC
11b
C
13
H
27
O
O
CH
3
HOOC
11a
OH
CO O
CO O
CH
3
COOH
OH
OH
CH
3
CH
3
HO
10
OH
OH
H
3
C
HO
OH
H
3
C
O
O
CH
3
O
CH
3
9
HO
O
O
H
3
COOC
8
Fig. 1. Structures of the lichen compounds.
18 F. Lohzic-Le Dvhat et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 1728
tained from Goldschmidt (Montigny-le-Bretonneux, France). Ceti-
ol

HE, stearic acid, glycerin, parabens, and triethanolamine

(TEA) were purchased from Cooper (Melun, France). Xanthan
gum (Keltrol

BT) was obtained from Kelco (Lille Skensved, Den-

mark). Homosalate (Eusolex

HMS), Avobenzone (Eusolex

9020)
and Octylmethoxycinnamate (Eusolex

2292) were purchased

from Merck (Fontenay Sous Bois, France). Polymethylmethacrylate
(PMMA) plates were purchased from Europlast (Aubervilliers,
France). Powder-free latex nger cots were purchased from Cooper
(Melun, France). Evernic acid (2) and Parietin (6) were purchased
from 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), phenazine
methosulfate (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 material
All voucher specimens were deposited at the Facultys 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.) Mrat (JB/06/77) had been harvested on rocks nearby
Rennes (Brittany, France).
2.2.2. Extraction and isolation
To obtain the crude extracts, the ground air-dried thalli of li-
chen were extracted with solvents during three reux phases of
1 hour each. A mixture of acetone/dichloromethane (1:1) was used
for 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 evaporating
the solvents under reduced pressure.
In order to isolate the main secondary metabolites of these li-
chens, a larger quantity of dried thalli were ground and extracted
under reux thanks to successive extractions. C. islandica (375 g)
was rst extracted with n-heptane (3 1.5 L) and a mixture of
lichesterinic (11a) and protolichesterinic (11b) acids precipitated
after the evaporation of the solvent at room temperature. The
residue 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 increasing
polarity (n-heptane, dichloromethane, tetrahydrofuran (THF),
3 500 mL for each) and in the last fraction obtained with
THF, gyrophoric acid (10) precipitated. (+)-Usnic acid (9) was
isolated by precipitation of the extraction of U. hirta (375 g) with
n-heptane (3 1.5 L).
Atranorin (1), salazinic acid (4), variolaric acid (3) and vulpinic
acid (8) were obtained from the laboratorys chemical library and
referenced 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 extracts
were assessed by NMR analysis (Bruker Avance 300 instrument
(BioSpin, Wissembourg, France)) using DMSO-d
6
or CDCl
3
(with
TMS 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. 24). But UV spectra (Figs. 57) were also recorded in
absolute ethanol (Uvikon 931 UVvis spectrophotometer with
10 mm path length cells, Serlabo Technologies, Entraigues sur la
Sorgue, France).
2.3. Determination of photoprotective efcacy of lichen extracts
2.3.1. Preparation of the oil-in-water emulsion
An O/W emulsion was prepared by adding substance to
be tested to the formulation components. The composition of the
emulsion is presented in Table 1. The hydrophilic-phase and the
oil-phase were heated separately in a water bath until the ingredi-
ppm (t1)
0.0 5.0 10.0
0
500
1000
1500
1
1
.
9
6
4
1
0
.
5
8
4
6
.
8
5
3
6
.
6
4
7
6
.
2
1
6
5
.
9
3
3
5
.
3
0
5
5
.
1
6
8
4
.
6
9
4
2
.
4
5
5
2
.
4
4
9
2
.
1
8
4
1
.
7
2
8
1
.
2
4
2
0
.
8
5
7
1
.
0
0
3
.
5
0
0
.
8
7
0
.
4
2
0
.
5
0
0
.
5
0
0
.
1
3
0
.
1
2
0
.
1
3
0
.
5
3
3
.
5
6
0
.
1
1
0
.
1
5
2
1
.
5
7
Fig. 2a.
1
H NMR (500 MHz, DMSO-d
6
) of Cetraria islandica crude extract (E1).
F. Lohzic-Le Dvhat et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 1728 19
ents of each part were solubilised or melted. Next, the hydrophilic
phase 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 concentration
authorized for many lters by the European regulation.
ppm (t1)
0.0 5.0 10.0 15.0
0
500
1000
1500
2000
1
1
.
9
6
5
1
0
.
5
8
2
6
.
8
5
3
6
.
6
4
8
5
.
3
0
6
2
.
4
5
6
2
.
4
4
1
1
.
0
0
0
.
9
8
0
.
5
6
0
.
5
8
0
.
5
9
3
.
4
9
-
0
.
1
0
O
O OH
CHO HO
H
3
C
CH
3
O
9'
COOH
O
COCH=CHCOOH
8
9
8'
2''
5
3''
Fig. 2b.
1
H NMR (500 MHz, DMSO-d
6
) of fumarprotocetraric acid (5).
ppm (f1)
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
0
500
6
.
4
8
3
6
.
0
3
5
5
.
1
3
3
4
.
8
2
9
3
.
6
4
0
2
.
2
4
4
1
.
7
3
8
1
.
2
5
8
0
.
8
8
1
1
.
0
0
1
.
0
3
0
.
7
5
1
.
0
3
1
.
0
6
2
.
1
7
2
.
8
9
4
3
.
4
1
5
.
8
7
H
2
C
O
O
CH
3 HOOC
Lichesterinic acid
2
7
19
3
5
(H
2
C)
11
H
3
C
4
H
2
C
O
O
HOOC
(+)-Protolichesterinic acid
2
7
19
3
5
(H
2
C)
11
H
3
C
4
Fig. 2c.
1
H NMR (500 MHz, CDCl
3
) of a mixture of lichesterinic (11a) and protolichesterinic acids (11b) obtained from the n-heptane fraction. The shifts in italic belong to
lichesterinic acid, in plain to protolichesterinic acid while in bold, they belong to both molecules.
20 F. Lohzic-Le Dvhat et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 1728
2.3.2. Procedure used for in vitro determination of the SPF and PF-UVA
Thirty accurately weighed milligrams of emulsion were spread
across the entire surface (25 cm
2
) of a PMMA plate using a nger
cot. After spreading, 15 mg remained on the nger cot. SPF and
PF-UVA values of the emulsion were then measured in vitro. Three
plates were prepared for each product to be tested and nine
measurements were performed on each plate. Transmission
measurements between 290 and 400 nm and between 320 and
400 nm for SPF and PF-UVA respectively, were performed using a
spectrophotometer equipped with an integrating sphere (UV
ppm (t1)
0.0 5.0 10.0
0
500
1000
1
0
.
4
8
6
1
0
.
3
1
4
1
0
.
0
1
4
6
.
6
8
5
6
.
6
7
5
6
.
6
2
9
6
.
6
0
6
6
.
2
4
5
6
.
2
3
3
2
.
3
8
9
2
.
3
7
3
2
.
3
6
3
1
.
0
0
0
.
8
9
0
.
3
8
0
.
5
6
4
.
6
5
0
.
4
6
0
.
4
8
0
.
4
6
Fig. 3a.
1
H NMR (500 MHz, DMSO-d
6
) of Lasallia pustulata extract (E2).
ppm (t1)
6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90
0
100
200
300
400
500
600
700
6
.
6
8
5
6
.
6
7
5
6
.
6
2
9
6
.
6
0
6
6
.
2
4
5
6
.
2
3
3
1
.
0
0
0
.
8
9
0
.
3
8
0
.
5
6
Fig. 3b. Enlargement of
1
H NMR (500 MHz, DMSO-d
6
) focused on the 67 ppm of Lasallia pustulata extract E2.
F. Lohzic-Le Dvhat et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 1728 21
Transmittance analyser UV1000S (Labsphere, North Sutton, US).
The calculations were carried out according to the following
equations:
SPF
X
400
290
E
k
S
k
D
k
X
400
290
E
k
S
k
T
k
D
k
1
PF-UVA
X
400
315
E
k
S
k
D
k
X
400
315
E
k
S
k
T
k
D
k
2
where E
k
is the erythemal spectral effectiveness at k, S
k
is the solar
spectral irradiance at k and T
k
is the spectral transmittance of the
sample at k [24,25].
2.3.3. Inuence of irradiation on effectiveness of photoprotection
The plates were irradiated for various times with a solar simula-
tor apparatus equipped with a xenon arc lamp (1500 W) and special
glass lters to restrict the transmission of light below 290 nm
(Suntest CPS+; Atlas, Moussy le Neuf, France). The temperature of
the samples was kept low and constant using a tap water cooling
circuit on to the walls of the reactor. In order to eliminate the turbu-
lence inside the Suntest chamber, we developed a systemwhere the
plates are blocked between two rails and covered with a quartz
plate. The light source emission was maintained at 650 W/m
2
in
ppm (t1)
0.0 5.0 10.0 15.0
0
500
1000
1
0
.
3
3
3
1
0
.
1
2
4
6
.
6
0
6
6
.
5
8
7
6
.
2
3
1
6
.
1
8
2
6
.
1
2
6
2
.
3
9
8
1
.
0
0
0
.
4
2
0
.
3
8
0
.
8
8
1
.
0
2
0
.
0
0
0
4
.
5
6
OH
CO O
CO O
Me
COOH
OH
OH
Me
Me
HO
3
5
3'
5''
3''
5'
2''
2'
2
Fig. 3c.
1
H NMR (500 MHz, DMSO-d
6
) of gyrophoric acid (10).
ppm (t1)
6.10 6.20 6.30 6.40 6.50 6.60
0
500
6
.
6
0
6
6
.
5
8
7
6
.
2
3
1
6
.
1
8
2
6
.
1
2
6
1
.
0
0
0
.
4
2
0
.
3
8
1
.
0
2
ppm (t1)
2.350 2.400
0
1000
2000
3000
4000
5000
2
.
3
9
8
2
.
3
7
9
2
.
3
5
5
3
.
0
0
0
.
5
9
0
.
5
6
Fig. 3d. Enlargement of
1
H NMR (500 MHz, DMSO-d
6
) in the range 23 ppm and 67 ppm of gyrophoric acid (10).
22 F. Lohzic-Le Dvhat et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 1728
accordance to global solar spectral irradiance at sea level measured
in accordance to CIE (Commission Internationale de lEclairage). The
SPF and the PF-UVA of the emulsions were measured in vitro before
and after irradiation by the protocol previously described [26].
ppm (t1)
0.0 5.0 10.0 15.0
0
500
1000
1
3
.
3
6
8
1
1
.
3
3
0
6
.
3
1
8
6
.
2
1
6
5
.
9
3
3
2
.
6
7
7
2
.
6
0
2
2
.
0
1
0
1
.
7
4
6
1
.
0
0
0
.
8
4
1
.
0
4
3
.
0
0
2
.
9
0
3
.
3
0
3
.
0
8
0
.
1
4
0
.
1
1
Fig. 4a.
1
H NMR (500 MHz, DMSO-d
6
) of Usnea hirta extract (E3).
ppm (t1)
1.00 1.50 2.00 2.50 3.00
0
500
1000
1500
2000
2
.
6
7
7
2
.
6
0
2
2
.
0
1
0
1
.
7
4
6
3
.
0
0
2
.
9
0
3
.
3
0
3
.
0
8
Fig. 4b. Enlargement of
1
H NMR (500 MHz, DMSO-d
6
) focused on the 13 ppm range of Usnea hirta extract.
F. Lohzic-Le Dvhat et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 1728 23
2.4. Antioxidant and cytotoxicity studies
2.4.1. Antioxidant assays
Two antioxidant assays were performed on the most photopro-
tective agents L. pustulata extract and gyrophoric and salazinic
acids, one using the 1,1
0
-Diphenyl-2-Picrylhydrazyl free radical as-
say (DPPH) and the other based on the measurement of superoxide
anion 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 modications as previously described [28]. A
reaction mixture containing 100 lL of DPPH (0.5 mM) in methanol
and 10 lL of the lichen compound solutions in DMSO to give nal
concentrations of 500, 250, 125, 62.5, 31.25 lg/mL per well, was
distributed in each microplate well. Ascorbic acid was used as a
positive control on each plate. Each concentration and all tests
were done in triplicate and the results averaged. The percentage
inhibition at steady state for each dilution was used to determine
the IC
50
values graphically.
2.4.1.2. NBT assay. For the same compounds, measurements of
superoxide anion scavenging activity in 96-well microplates based
on the non-enzymatic method previously described with some
modications were performed [28,29]. The reaction mixture in
the sample wells consisted of NADH (78 lM), Nitro-blue tetrazo-
lium (NBT) (50 lM), Phenazine methosulfate (PMS) (10 lM), and
lichen compounds (500, 250, 125, 62.5, 31.25 lg/mL). The reagents
were dissolved in 16 mM trishydrochloride 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 blank
without PMS and sample. Ascorbic acid was used as a positive con-
trol. The percentage inhibition at steady state for each dilution was
used to calculate the IC
50
values. This gave the amount of antioxi-
dant required (measured as the concentration of the stock solution
added to the reaction mixture) to scavenge 50% of O

2
: the lowest is
the values, the best is the efciency in scavenging O

2
. All tests
were done in triplicate and the results averaged.
2.4.2. Cytotoxicity assays
The 3T3 Neutral Red Uptake (NRU) phototoxicity test was
developed and validated in Europe and has since been accepted
at 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. absence
of light. The cell viability is measured by degree to which they
are able to absorb the neutral red.
Cytotoxic and photocytotoxic activities of L. pustulata extract,
gyrophoric and salazinic acids were evaluated according to the
ppm (t1)
5.0 10.0
0
500
1000
1500
1
3
.
3
6
8
1
1
.
3
3
1
6
.
3
1
8
2
.
6
7
7
2
.
6
0
1
2
.
0
1
0
1
.
7
4
6
1
.
0
0
0
.
9
1
1
.
0
2
3
.
0
7
3
.
0
9
3
.
0
6
3
.
1
6
O
OH
H
3
C
HO
OH
H
3
C
O
O
CH
3
O
CH
3 4
7
9
10
12
15
14
Fig. 4c.
1
H NMR (500 MHz, DMSO-d
6
) of (+)-usnic acid.
Table 1
Formula of O/W emulsion.
Ingredients % (w/w)
Oil phase
Parafnum liquidum 17.00
Cetiol HE

(PEG-7 glyceryl cocoate) 5.00

Stearic acid 5.00
Butylhydroxytoluene 0.01
Eumulgin

B1 (Ceteareth-12) 1.50
Eumulgin

B2 (Ceteareth-20) 1.50
Hydrophilic phase
Glycerin 4.00
Rhodicare

T (Xanthan gum) 0.90

Sodium propylparaben 0.05
Sodium methylparaben 0.10
TEA 0.30
Distilled water qsp 100.00
24 F. Lohzic-Le Dvhat et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 1728
OECD guideline [30] with some modications. In brief, 100 lL per
well of a cell suspension of HaCaT cells (ATCC) at 8 10
5
cells/mL
were maintained in a RPMI culture medium with 5% of calf serum
at 37 C under 5% CO
2
for 24 h for formation of monolayers. At
t = 24 h, two 96-well plates per test chemical were preincubated
for 1 h with nine different concentrations of the tested compound
(in the range 0.01200 lg/mL). Chlorpromazine was used as the
positive control. Thereafter, one of the two plates was irradiated
(+UV) for 50 min with 5 J/m
2
using a UV stratalinker 2400 (Strata-
gene, USA) whereas the other plate (UV) was kept in the dark. In
both plates the treatment medium (PBS) was replaced by a culture
mediumand after 24 h of incubation, cell viability is determined by
Neutral Red Uptake. Cell viability was expressed as percentage of
untreated cell controls and is calculated for each test concentra-
tion. To predict the phototoxic potential, we determined the
photo-irritancy factor (PIF) thanks to the concentration response
curves obtained in the presence and in the absence of irradiation.
If PIF < 5, no phototoxic potential is predicted. If both IC
50
(UV)
and (+UV) cannot be calculated, a formal PIF (PIF =

1) is used pre-
dicting no phototoxic potential [30].
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 yield
of about 10% (w/w) which is of interest for an industrial use.
3.2. Characterization of lichen extracts
1
H NMR of the crude extract of C. islandica E1 (Fig. 2a) exhibited
the characteristic signals of three major compounds. The two
methyl 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) while
d = 5.16 ppm was attributed to lichesterinic acid (11a). Except for
the methyl group at d = 5.45 ppm attributed to 11a, all the other
methyl and methylene groups of the two butenolides overlapped
(Fig. 2c) [32]. 11a, 11b, and 5 were found in the relative
10:12:78 ratio, based on the relative integration of the respective
aromatic hydrogens.
The chemical shifts on the
1
H NMR spectrum of the E2 L. pustu-
lata extract (Figs. 3a and 3b) indicated the presence of gyrophoric
acid 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) and
the methyl groups (d = 2.362.39 ppm) (Figs. 3c and 3d). Using
NMR, some minor compounds were calculated to be less than 2%
(w/w).
As seen on
1
H NMR spectra (Fig. 4), four methyl groups (d = 1.74,
2.01, 2.60, 2.67 ppm), aromatic hydrogen (d = 6.31 ppm) and
hydroxyl groups (d = 11.33, 13.36 ppm) corresponding to (+)-usnic
acid signals (Fig. 4c) were recognized in U. hirta extract E3 (Figs. 4a
and 4b). It is indeed well-known that usnic acid is a major com-
pound in Usnea genus [34]. Based on the relative integration of
the aromatic protons in the 68 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 compared
with those of their major compounds summarized in Table 2. Sim-
ilar proles 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 from
those of pure metabolites according to the composition and ratio
deduced from
1
H NMR data. A large overlap between the experi-
mental and calculated spectra conrmed the composition of E3
(Fig. 7).
3.3. Photoprotective activity
The absorbances of extracts and lichen compounds were re-
corded between 290315 nm and 315400 nm corresponding to
Table 2
Extraction yield (%) of the lichens (E1E3) and their main secondary metabolites.
Lichen
extract
Extraction yield
(w/w)
Main secondary metabolite
E1 2.7 Fumarprotocetraric acid, protolichesterinic
acid, lichesterinic acid
E2 9.4 Gyrophoric acid
E3 4.0 (+)-Usnic acid
290 310 330 350 370 390
(nm)
0
0.2
0.4
0.6
0.8
1
1.2
A
b
s
o
r
b
a
n
c
e
9
E3
Fig. 5. Absorbance of Usnea hirta E3 and (+)-usnic acid 9 (20 lg/mL in ethanol).
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
290 310 330 350 370 390
A
b
s
o
r
b
a
n
c
e
(nm)
10
E2
Fig. 6. Absorbance of Lasallia pustulata E2 and gyrophoric acid 10 (20 lg/mL in
ethanol).
0
0.05
0.1
0.15
0.2
0.25
290 310 330 350 370 390
A
b
s
o
r
b
a
n
c
e
(nm)
5
E1
11a + 11b
(11a + 11b + 5) calc
Fig. 7. Absorbance of Cetraria islandica extract E1 and its main metabolites
fumarprotocetraric 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 pure
metabolites according to the ratio in the extract.
F. Lohzic-Le Dvhat et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 1728 25
the UVB and UVA regions respectively. Out of the eleven com-
pounds, two (fumarprotocetraric acid (5), vulpinic acid (8)) exhib-
ited a maximal absorbance (k
max
) in the UVA range similar to the
reference compound Avobenzone while the others exhibited max-
imal absorbances between 280 and 311 nm (Table 3). Seven of
them also possessed a molar extinction coefcient, e, superior to
10,000 L mol
1
cm
1
which corresponds to one of criteria used
for selecting UV lters [35]. The highest values of epsilon was
found for parietin (6) and (+)-usnic acid (9) and were in the range
of the OMC e value. Nevertheless, some authorized lters exhibited
low 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 to
5 which is better than Homosalate (Table 4). So, the application
of this product should provide a sunburn protection for at least ve
times 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 boosters
candidates (PF-UVA > 2).
Anyway, most of the approved active sunscreen ingredients are
never used alone in a galenic preparation but in combination to of-
fer a maximal protective action in the whole UVAUVB range. So,
most of the lichen compounds considered as good protectors in a
restricted UV range but moderate protectors as far as broad-spec-
trum is concerned, could be mixed with other lters to yield the
broad-spectrum coverage. Furthermore, the SPF and epsilon values
do not appear to be correlated: high values of epsilon do not nec-
essarily lead to high SPF values (evernic acid, vulpinic acid), and a
low value of epsilon do not preclude a signicant value of SPF
(Homosalate).
The (+)-usnic acid was found to be a weak photoprotector in our
experiment. In contrast, many authors reported (+)-usnic acid as a
good photoprotective agent [16,18,37]. Their methodologies were
different for in vitro assays either in solution or on cells with Mem-
brane Protection Factor (MPF) measurements and also for in vivo
assays on albino guinea pigs. Recently, Boehm et al. [20], in a
photophysical study, reported usnic acid as a potential sunscreen
without determining any SPF values.
The same discussion could also stand for atranorin which our
results proved to be a weak lter.
The gyrophoric acid was found to be the best UVB photoprotec-
tor in our experiment which is in agreement with literature, even if
our value was lower. In a previous study, Fernandez et al. [37] re-
ported an in vivo SPF value of 10 with a non-standardized method
on albino guinea pigs (the ISO 24444:2010 recommends in vivo as-
says on human volunteers [38]).
Lichen extracts photoprotective proles 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 SPF
activity was highlighted for these extracts.
The structure of the most active compound, gyrophoric acid, is
the result of the esterication between three orsellinic acid units.
Among the tested compounds, compound 2 also presented this
moiety unit but without any noticeable SPF. An attempt at correla-
tion between chemical structure and SPF values failed. More active
molecules are needed to establish a structure relationship activity.
3.4. Inuence of irradiation on the effectiveness of lichens compounds
L. pustulata and its main metabolite gyrophoric acid appeared to
be good UV-lters (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/m
2
, more than 90%
efcacy was conserved. These results are in agreement with Begora
experiments who reported no degradation after UVA and UVB
exposure 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 lters are not so stable [26].
3.5. Antioxidant activity of L. pustulata, gyrophoric acid and salazinic
acid
Antioxidant properties of E2, 4 and 10 were evaluated so as to
nd a new natural source of antioxidant. Because of its stability
in the radical form and simplicity of the assay, DPPH radical is a
commonly used substrate for a fast evaluation of antioxidant
activity. The principle behind this assay is the color change from
purple to yellow of the DPPH solution because the radical is
quenched by the antioxidant. The colour changes can be measured
quantitatively by spectrophotometric absorbance at 540 nm. The
Table 3
UV spectral data of lichen compounds (110) and reference UV lters.
Compound k
max
(nm) e (L mol
1
cm
1
)
1 293 5700
280 10,200
2 307 16,300
3 267 9100
4 311 6400
5 320 5200
6 287 18,600
7 303 3500
8 367 11,000
289 14,800
9 284 18,600
10 300 16,000
Homosalate
a
306 4600
OMC
a
309 24,700
Avobenzone
a
358 15,500
a
UV lters.
Table 4
Photoprotective results: SPF, PF-UVA values obtained with the lichen compounds,
extracts and three commercial lters.
Product tested SPF SD PF-UVA SD
E1 1.55 0.30 1.37 0.28
E2 5.52 0.21 2.45 0.15
E3 1.67 0.25 1.48 0.20
1 1.52 0.06 1.42 0.05
2 2.42 0.19 1.57 0.09
3 2.09 0.10 1.36 0.03
4 2.56 0.15 2.07 0.10
5 1.91 0.10 1.75 0.08
6 1.94 0.15 1.85 0.13
7 2.03 0.08 1.24 0.02
8 2.55 0.24 1.67 0.10
9 1.62 0.25 1.48 0.20
10 5.03 0.76 1.77 0.08
Homosalate
a
3.91 0.44
OMC
a
11.16 0.41
Avobenzone
a
2.76 0.31
a
Reported value for Homosalate and OMC 4 and Avobenzone 4.
Table 5
Inuence of irradiation on effectiveness for Lasallia pustulata E2 and its main
metabolite gyrophoric acid 10.
Product SPF
0
FP-UVA
0
SPF
2h
FP-UVA
2h
E2 5.52 0.21 2.45 0.15 5.21 0.48 2.38 0.16
10 5.03 0.76 1.77 0.08 5.34 0.64 1.82 0.18
26 F. Lohzic-Le Dvhat et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 1728
compounds were screened for DPPH radical scavenging activity
according to the method described and the results of the screening
are shown in Table 6. By comparison to ascorbic acid, no activity
was observed at the highest concentration for salazinic acid and
gyrophoric acid and while the crude extract of L. pustulata can be
categorized as a weak free radical scavenger because no IC
50
can
be calculated. It backed up, the results obtained by Kumar about
gyrophoric acid [40].
The superoxide radicals generated from dissolved oxygen by
PMS-NADH coupling can be measured by their ability to reduce
NBT. 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 reaction
mixture. As shown in Table 6, the IC
50
values of salazinic acid
and ascorbic acid on superoxide scavenging activity were
3.9 1 lg/mL and 6.2 0.2 lg/mL, respectively. Such an activity
reinforces the interest of this compound to be a candidate as
UVA booster. L. pustulata extract and gyrophoric acid exhibited a
similar but moderate activity as superoxide anion scavengers.
3.6. Cytotoxicity and phototoxic activities of L. pustulata, gyrophoric
acid and salazinic acid
Phototoxicity is dened as a toxic response after the exposure
to light or to UV irradiation of a substance applied to the body or
after its systemic administration. It is well documented through
clinical and experimental studies that non-cytotoxic doses of many
chemicals 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 the
presence and one tested in the absence of exposure to a non-cyto-
toxic dose of UVA light. One day after treatment, cytotoxicity is
measured as an inhibition of the capacity of the cell to take up a
vital dye, which is neutral red.
The cytotoxicity of the tested lichenic compounds on human
keratinocytes HaCaT is weak (Table 6) compared to the positive
control chlorpromazine (IC
50
= 9.5 lg/mL). IC
50
was over
200 lg/mL for salazinic acid and indicated low cytotoxicity for
gyrophoric acid (IC
50
= 168 lg/mL) and L. pustulata extract
(IC
50
= 200 lg/mL), mean doseresponse curves for cytotoxicity
are shown in Fig. 8. Under 5 J/m
2
UVA irradiation, the viability of
HaCaT cells was 100%. The cytotoxicities of salazinic acid (4) and
gyrophoric acid (10) were not enhanced by UVA irradiation
(IC
50
> 200 lg/mL) counter to L. pustulata extract (IC
50
= 118 lg/
mL) (Fig. 8) but its PIF factor remained under 5 (Table 6) which
is the value of Ref. [30]. The NRU phototoxicity test identies the
substances 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 gyrophoric
acid are valuable candidates as UV lters because of their efcacy
before and after UV irradiation. They are indeed promising agents
because they have very low phototoxicity even the maximal con-
centration of the Lasallia extract to be used in cosmetic preparation
should be reduced. The gyrophoric acid could also be a useful
structure for in silico modelisation in order to determine an effec-
tive pattern for the activity as UV lter. Some of the other studied
molecules have no high SPF values but can be used as lter boost-
ers: variolaric acid, evernic acid and vulpinic acid and especially
salazinic acid in term of UVA. In fact, it is of common use in cosme-
tology to mix UV lters for the additivity of the SPF values. More-
over, additivity or synergism of lichen compounds with known
sunscreens could be of interest [41].
UV radiations damage skin cells through indirect mechanisms
with the formation of ROS. An overproduction of ROS results in
an 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
Table 6
Antioxidant, cytotoxic and phototoxic activities of 4, 10 and E2.
Compound Antioxidant activities Phototoxic activities on HaCaT
cells
DPPH
assay (%
activity
at
500 lg/
mL)
NBT assay
IC
50
SD
(mg/mL)
IC
50
SD (lg/mL) Photo-
irritancy
factor
(PIF)
Without
irradiation
With
irradiation
4 0 3.9 1.0 >200 >200

1
d
10 25 2.9 79.6 16.4 168 33 >200
e
E2 0 72 16 200 40 118 16 1.7
f
Ascorbic acid
a
98.5 0.5 6.2 0.2 nc
c
nc
c
nc
c
Chlorpromazine
b
nc
c
nc
c
9.5 1.5 0.48 0.13 20
f
a
Antioxidant positive control.
b
Cytotoxic and phototoxic positive control.
c
nc: not concerned.
d
Formal PIF = C
max
(UV)/C
max
(+UV).
e
No phototoxic potential.
f
PIF = IC
50
(UV)/IC
50
(+UV).
-1
10
20
30
40
50
60
70
80
90
100
110
120
0 50 100 150 200 250
%

c
e
l
l

s
u
r
v
i
v
a
l
Concentration (g/mL)
-UVA
E2
10
4
chlorpromazine
0
10
20
30
40
50
60
70
80
90
100
110
120
0 50 100 150 200 250
%

c
e
l
l

s
u
r
v
i
v
a
l
Concentration (g/mL)
+UVA
E2
10
4
chlorpromazine
(a) (b)
Fig. 8. Cytotoxicity and phototoxicity activities of 4, 10 and E2 on HaCaT cells. (a) (UV)-curve of mean values of the cell viability in % of the respective negative control. (b)
(+UV)-curve of mean values of the cell viability in % of the respective negative control. In phototoxicity evaluation the cells were irradiated at 5 J/m
2
in the presence of
chlorpromazine or tested compounds. Points are means of three independent experiments (n = 9 for each concentration).
F. Lohzic-Le Dvhat et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 1728 27
dangerous effects of UVs could be the use of phytochemicals with
antioxidant properties. For example, vitamins and phenols have
gained considerable attention as protective agents and are added
in 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-inammatory effects
should be investigated to complement these valuable capacities
[22].
Acknowledgements
The authors thank Aurlie Sauvager, Isabelle Rouaud and Eva
Paparis for their technical assistance. We thank also the Research
team Expression des gnes et oncogense of the UMR 6290 IGDR
(Rennes) for the access to the Stratalinker apparatus.
References
[1] S.E. Jones, M. Saraiya, J. Miyamoto, Z. Berkowitz, Trends in sunscreen use
among U.S. High School Students: 19992009, J. Adolescent Health 50 (2012)
304307.
[2] F. Afaq, M. Abu Zaid, N. Khan, M. Dreher, H. Mukhtar, Protective effect of
pomegranate-derived products on UVB-mediated damage in human
reconstituted skin, Exp. Dermatol. 18 (2009) 553561.
[3] M. Bobin, M. Raymond, M. Martini, UVA/UVB absorption properties of natural
products, Cosmet. Toilet. 109 (1994) 6370.
[4] S. Fguyer, F. Afaq, H. Mukhtar, Photochemoprevention of skin cancer by
botanical agents, Photodermatol. Photoimmunol. Photomed. 19 (2003) 5672.
[5] S. Gonzalez, Y. Gilaberte, N. Philips, Mechanistic insights in the use of a
Polypodium leucotomos extract as an oral and topical photoprotective agent,
Photochem. Photobiol. Sci. 9 (2010) 559563.
[6] F. Giampieri, J.M. Alvarez-Suarez, S. Tulipani, A.M. Gonzlez Params, C.
Santos-Buelga, S. Bompadre, J.L. Quiles, B. Mezzetti, M. Battino,
Photoprotective potential of strawberry (Fragaria x ananassa) extract against
UV-a irradiation damage on human broblasts, J. Agric. Food Chem. 60 (2012)
23222327.
[7] R. Rastogi, R. Richa, R. Sinha, S. Singh, D.-P. Hder, Photoprotective compounds
from marine organisms, J. Ind. Microbiol. Biotechnol. 37 (2010) 537558.
[8] W. Wong, J. Wu, I. Benzie, Photoprotective potential of Cordyceps
polysaccharides against ultraviolet B radiation-induced DNA damage to
human skin cells, Br. J. Dermatol. 164 (2011) 980986.
[9] L. Brandao, D. Linbkind, A. Vaz, L. Espirito Santo, M. Moline, V. De Garcia, M.
Van Broock, C. Rosa, Yeasts from an oligotrophic lake in Patagonia (Argentina):
diversity, distribution and synthesis of photoprotective compounds and
extracellular enzymes, FEMS Microbiol. Ecol. 76 (2011) 113.
[10] C. Cockwell, J. Knowland, Ultraviolet radiation screening compounds, Biol. Rev.
74 (1999) 311345.
[11] K.A. Solhaug, Y. Gauslaa, L. Nybakken, W. Bilger, UV-induction of sun-
screening pigments in lichens, New Phytol. 158 (2003) 91100.
[12] Y. Gauslaa, E. Ustvedt, Is parietin a UVB or a blue-light screening pigment in
the lichen Xanthoria parietina?, Photochem Photobiol. Sci. 2 (2003) 424432.
[13] L. Nybakken, K. Solhaug, W. Bilger, Y. Gauslaa, The lichens Xanthoria elegans
and Cetraria islandica maintain a high protection against UVB radiation in
arctic habitats, Oecologia 140 (2004) 211216.
[14] J. Rozema, L. Bjor, J. Bornman, A. Gaberscik, D. Hader, T. Trost, M. Germ, M.
Klish, A. Groniger, R. Siha, M. Lebert, Y. He, R. Buffoni-Hall, N. De Bakker, J. Van
de Staaij, B. Meijkamp, The role of UVB radiation in aquatic and terrestrial
ecosystems an experimental and functional analysis of the evolution of UV-
absorbing compounds, J. Photochem. Photobiol. B: Biol. 66 (2002) 212.
[15] M.E. Hidalgo, L. Bascunan, W. Quilhot, E. Fernandez, C. Rubio, Spectroscopic
and photochemical properties of the lichen compound lobaric acid,
Photochem. Photobiol. 81 (2005) 14471449.
[16] E. Fernandez, W. Quilhot, C. Rubio, M.E. Hidalgo, R. Diaz, J. Ojeda, Effects of UV
radiation on usnic acid in Xanthoparmelia microspora, Photochem. Photobiol.
82 (2006) 10651068.
[17] E. Fernandez, A. Reyes, M.E. Hidalgo, W. Quilhot, Photoprotector capacity of
lichen metabolites assessed through the inhibition of the 8-methoxypsoralen
photobinding to protein, J. Photochem. Photobiol. B: Biol. 42 (1998) 195201.
[18] F. Rancan, S. Rosan, K. Boehm, E. Fernandez, M.E. Hidalgo, W. Quilhot, W.
Rubio, F. Boehm, H. Piazena, U. Oltmanns, Protection against UVB irradiation
by natural lters extracted from lichens, J. Photochem. Photobiol. B: Biol. 68
(2002) 133139.
[19] A. Torres, M. Hochberg, I. Pergament, R. Smoun, V. Niddam, V. Dembitsky, M.
Temina, I. Dor, O. Lev, M. Srebnik, C. Enk, A new UVB absorbing mycosporine
with photoprotective activity from the lichenized ascomycete Collema
cristatum, Eur. J. Biochem. 271 (2004) 780784.
[20] F. Boehm, K. Clarke, R. Edge, E. Fernandez, S. Navaratnam, W. Quilhot, F.
Rancan, G. Truscott, Lichens photophysical studies of potential new
sunscreens, J. Photochem. Photobiol. B: Biol. 95 (2009) 4045.
[21] S. Saraf, Phytoconstituents as photoprotective novel cosmetic formulations,
Phcog. Rev. 4 (2010) 111.
[22] F. Afaq, Natural agents: cellular and molecular mechanisms of
photoprotection, Arch. Biochem. Biophys. 508 (2011) 144151.
[23] G. Soares dos Reis, A. Furtado Valadao, L. Ramos Paes de Lima, M. Lucy Moreira,
Preparacion de un protector solar y evaluacion de la accion fotoprotectora del
propoleo verde del Vale do aco, Minas Gerais, Brasil, BLACPMA 8 (2009) 282
288.
[24] B.L. Diffey, J. Robson, A new substrate to measure sunscreen protection factors
throughout the ultraviolet spectrum, J. Soc. Cosmet. Chem. 40 (1989) 127133.
[25] R.M. Sayre, P.P. Agin, G.J. Le Vee, E. Marlowe, A comparison of in vivo and
in vitro testing of sunscreening formulas, Photochem. Photobiol. 29 (1979)
559566.
[26] C. Couteau, A. Faure, J. Fortin, E. Paparis, L.J.M. Coiffard, Study of photostability
of 18 sunscreens in creams by measuring the SPF in vitro, J. Pharm. Biomed.
Anal. 44 (2007) 270273.
[27] R. Matsukawa, Z. Dubinsky, E. Kishimoto, K. Masaki, Y. Masuda, T. Takeuchi, M.
Chihara, Y. Yamamoto, E. Niki, I. Karube, A comparison of screening methods
for antioxidant activity in seaweeds, J. Appl. Phycol. 9 (1997) 2935.
[28] F. Lohzic-Le Dvhat, S. Tomasi, J. Elix, A. Bernard, I. Rouaud, P. Uriac, Stictic
acid derivatives from the lichen Usnea articulata and their antioxidant
activities, J. Nat. Prod. 70 (2007) 12181220.
[29] F. Ismed, F. Lohzic-Le Dvhat, O. Delalande, S. Sinbandhit, A. Bakhtiar, J.
Boustie, Lobarin from the Sumatran lichen, Stereocaulon halei, Fitoterapia 83
(2012) 16931698.
[30] OECD Guideline for testing chemicals, in: Guideline for Testing of Chemicals
Number 432: in vitro 3T3 NRU phototoxicity test, 2004.
[31] C. Bzivin, S. Tomasi, I. Rouaud, J.G. Delcros, J. Boustie, Cytotoxic activity of
compounds from the lichen: Cladonia convoluta, Planta Med. 70 (2004) 877
880.
[32] A.C. Le Lamer, a-Mthylne-c-lactones lichniques: extraction, isolement et
synthse danalogues, in: EA Substances lichniques et photoprotection, 2006,
PhD, Rennes 1: Rennes. pp. 162.
[33] B. Posner, G.B. Feige, S. Huneck, Phytochemical investigations on Western
European Lasallia species, Z. Naturforsch. C 45 (1990) 161165.
[34] Y. Kinoshita, Y. Yamamoto, I. Yoshimura, T. Kurosawa, Y. Yamada, Production
of usnic acid in cultured Usnea hirta, Biblioth. Lichenol. 53 (1993) 137146.
[35] C. Fournier, 70 ans de photoprotection, in: Pharmacie de Chatenay-Malabry,
2006, Universit de Paris-Sud 11: Practical PhD, Paris. pp. 82.
[36] D. Sambandan, D. Ratner, Sunscreens: an overview and update, J. Am. Acad.
Dermatol. 64 (2011) 748758.
[37] E. Fernandez, W. Quilhot, I. Gonzalez, M.E. Hidalgo, X. Molina, I. Meneses,
Lichen metabolites as UVB lters, Cosmet. Toilet. 111 (1996) 6974.
[38] Cosmetics-Sun protection test methods-in vivo determination of the sun
protection factor (SPF), in: ISO 24444. 2010.
[39] M. Begora, D. Fahselt, Photolability of secondary compounds in some lichen
species, Symbiosis 31 (2001) 322.
[40] S. Kumar, K. Muller, Lichen metabolites. I. Inhibitory action against leukotriene
B4 biosynthesis by a non-redox mechanism, J. Nat. Prod. 62 (1999) 817820.
[41] M. Ramos, E. Santos, C. Bizarri, H. Mattos, Preliminary studies towards
utilization of various plant extracts as antisolar agents, Int. J. Cosmet. Sci. 18
(1996) 87101.
28 F. Lohzic-Le Dvhat et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 1728