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The role of the aqueous extract Polypodium

Cite this: DOI: 10.1039/d0pp00124d
leucotomos in photoprotection
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Concepción Parrado,a Jimena Nicolas,b Angeles Juarranzb and

Salvador Gonzalez *c

Received 16th April 2020,
Accepted 13th May 2020
DOI: 10.1039/d0pp00124d
rsc.li/pps
Solar radiation in the ultraviolet (UV), visible (VIS), and infrared (IR) ranges produces different biological
effects in humans. Most of these, particularly those derived from ultraviolet radiation (UVR) are harmful to
the skin, and include cutaneous aging and increased risk of cutaneous diseases, particularly skin cancer.
Pharmacological photoprotection is mostly topical, but it can also be systemic. Oral photoprotectives
constitute a new generation of drugs to combat the deleterious effects of solar radiation. Among these,
an extract of Polypodium leucotomos (PL/Fernblock®, IFC Group, Spain) contains a high content of phe-
nolic compounds that endow it with antioxidant activity. PL can administered orally or topically and is
completely safe. PL complements and enhances endogenous antioxidant systems by neutralizing super-
oxide anions, hydroxyl radicals, and lipoperoxides. In addition to its antioxidant activity, PL also improves
DNA repair and modulates immune and inflammatory responses. These activities are likely due to its
ability to inhibit the generation and release of reactive oxygen species (ROS) by UVR, VIS, and IR radiation.
PL also prevents direct DNA damage by accelerating the removal of induced photoproducts and decreas-
ing UV-induced mutations. Oral PL increases the expression of active p53, decreases cell proliferation,
and inhibits UV-induced COX-2 enzyme levels. PL has been used to treat skin diseases such as photoder-
matoses and pigmentary disorders and recently as a complement of photodynamic phototherapy in
actinic keratoses. The photoprotective capability of PL has been proven in a multitude of in vitro and
in vivo studies, which include animal models and clinical trials with human subjects. Based on this evi-
dence, PL is a new generation photoprotector with antioxidant and anti-inflammatory properties that also
protects DNA integrity and enhances the immune response.

Beneficial and detrimental effects of
sunlight and photoprotection
The World Health Organization (WHO) has indicated that skin
exposure to ultraviolet radiation (UVR) is a major risk factor to
develop diverse types of skin cancers. In fact, solar UVR is a
potent carcinogen, because it causes not only initial damage,
but also mutations in the skin 1 and acts as an tumor promo-
ter, having numerous implications for public health.2 WHO
and local policies on UVR-induced skin damage are designed
to prevent the burden of diseases associated with overexposure
to solar radiation.1 In addition to UVR, solar radiation com-
prises visible light (VIS, 400–700 nm), and infrared (IR, above
800 nm) radiation.
a
Department of Histology and Pathology, Faculty of Medicine, University of Málaga,
Málaga, Spain
b tion, cell cycle, cell proliferation, and apoptosis.
Department of Biology, Faculty of Sciences, Autónoma University of Madrid, Spain
c
Medicine and Medical Specialties Department, Alcala University, Madrid, Spain.
E-mail: salvagonrod@gmail.com
Despite its deleterious effects, UVR also has healthy effects
on skin, for example it catalyses the synthesis of vitamin D.3
UVR is also used clinically to treat inflammatory skin con-
ditions, such as psoriasis and vitiligo, among others. The
benefits of UVR-based phototherapies in the treatment of skin
diseases are well established.4–6
They are three types of UVR, classified according to their
wavelength: UVA (315–400 nm), UVB (280–315 nm), and UVC
(100–280 nm). The ozone layer of the stratosphere completely
blocks UVC. Only a percentage of UVA and UVB rays reach the
surface of the earth. UVR – induced photoaging and photocar-
cinogenesis are due to the direct and indirect effects of UVR
on DNA.7 UVR directly promotes DNA damage, for example
thymidine dimers. Indirectly, UVR oxidizes lipids, producing
free radicals that generate oxidative stress, which is deleterious
at several levels, including inflammation, immunosuppres-
sion, and indirect DNA damage.8 At the same time, oxidative
stress activates signaling pathways that affect gene transcrip-
UVR is the main cause of photodamage; however, VIS and
IR radiation also contribute to skin aging.9,10 Recent reports

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have addressed the harmful role of VIS and IR radiation on the
skin.11 Prolonged exposure to IR-A radiation induces erythema,
telangiectasia, and alters the shape and density of dermal
fibers.12 At the molecular level, IR radiation increases the
expression of matrix metalloproteinases (MMPs).13 On the
other hand, several pathologies such as solar urticaria, poly-
morphic light eruption, chronic actinic dermatitis, actinic
prurigo and other phototoxic and photoallergic reactions have
all been related to the action of VIS radiation.14–18 A recent
report has suggested that VIS radiation synergizes with UVA to
cause skin damage.19
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For all these reasons, effective sun protection is essential
for humans, including physical protection, topical sunscreens,
and systemic photoprotectors.20,21
Topical photoprotection is an important step as the first
line of prevention to combat photoaging and skin cancer. But
despite the widespread use of topical sunscreens, sunburn
remains frequent. On the other hand, oral photoprotectors are
not very effective against erythema and other harmful effects
caused by the sun as they do not directly protect the skin from
the damage induced by high energy photons. Conversely, their
efficiency is not altered by external conditions; their half-lives
can be determined pharmacologically, and their effects
depend on their skin affinity.21
Most of these oral photoprotectors are phytochemicals that
contain one or more ingredients that activate different photo-
protection mechanisms, especially those that counter oxi-
dation.21 These substances act by increasing the antioxidant
threshold of the body after the loss of endogenous antioxi-
dants that occurs after exposure to UVR. Some of these sub-
stances also reduce photo-immunosuppression and decrease
DNA damage induced by solar radiation.
Among them, a standardized aqueous extract of Polypodium
leucotomos (PL/Fernblock®) improves the clinical and mole-
cular signs of photodamage. In this review, we present the ben-
eficial effects of PL.
This extract exerts beneficial effects through multiple
mechanisms, countering signalling pathways that are nega-
tively affected by UVR, such as collagen and elastin fibre degra-
dation through oxidative stress; and the activation of extra-
cellular metalloproteinases (MMP). Some evidence from our
group has demonstrated that PL is endowed with antioxidant,
anti-inflammatory, and immunomodulatory effects, preventing
DNA damage induced by UVR as well as VIS-IR radiation.22
Scientific evidence backing the
efficiency of the aqueous extract of
Polypodium leucotomos leaves
(Fernblock®)
In vitro and in vivo studies support the photoprotective effects
of PL extract on the skin damage induced by UVR and VIS_IR
radiation.23–41
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Fernblock® is a controlled aqueous extract of the leaves of
Polypodium leucotomos (also known as Phlebodium aureum).
This fern belongs to the Polypodiaceae family, which is native
to Central and South America. It has been used to treat skin
diseases like psoriasis and atopic dermatitis in traditional
medicine.35
PL is formulated to maintain the its phenolic content con-
stant. Phenolic compounds naturally occurring in PL include
vanillic acid, p-coumaric acid, protocatechuic acid, caffeic acid,
ferulic acid, and five chlorogenic acid isomers.42 Caffeic and
ferulic acids are the most potent antioxidants present in PL.
Studies in vitro show a permeability of PL that ranged between
70–100%, equivalent to human absorption after PL oral
administration.43
PL has been marketed in Europe and the United States
(Fernblock®, Heliocare).44 Studies on the pharmacology of PL
and its biological disposition have revealed that oral uptake of
240 mg of PL twice daily (for 60 days) is safe and significantly
reduces UVR-induced skin damage. PL is well tolerated and its
toxicity is negligible even at high doses.45
Strict quality examination has been used to control the
composition and effects of this specific formulation of the oral
PL capsule of this brand. In 2018, Gonzalez et al.24 reported a
comparative in vitro study of six different Polypodium leucoto-
mos extracts, including Fernblock® (sample 2 in that study).
The methodologies used in the study included antioxidant
assays, cell viability assays, and high-performance liquid
chromatography (HPLC). The results of the study support the
fact that the content of antioxidant polyphenols and the
photoprotective properties of oral PL formulation are superior
to other extracts.24 This fact is likely due to the use of leaves as
starting material, combined with aqueous extraction at basic
pH. Similar leave extraction at neutral or acidic pH had signifi-
cantly less efficiency. Also, rhizome extracts were much less
efficient regardless of the extraction method. The molecular
moiety of the aqueous, basic leave extract is richer in anti-
oxidant aromatic acids ( protocatechuic acid, vanillic acid,
caffeic acid, and ferulic acid) than those present in any
other formulation.42 PL also displayed the highest anti-
oxidant capacity, measured by two spectrophotometric
methods [2,2′-azinobis (3-ethylbenzothiazoline-6-sulphonic
acid)] free radical scavenging activity assay (ABTS) and Ferric
ion Reducing Antioxidant Power assay (FRAP)24 (Fig. 1). Also,
PL reduced the levels of γH2AX phosphorylation in response
to UVR in live cells more efficiently than any other formu-
lation tested (Fig. 1).24 γ-H2AX phosphorylation (Ser139) is a
canonical biomarker of DNA damage responses after UVB
exposure.46 Finally, we determined the effect of the polypo-
dium formulations in the formation of pyrimidine cyclo-
butane dimers (CPDs). CPDs constitute an additional marker
of UV-induced DNA damage.47 These experiments revealed
UVR-induced CPDs formation in 70% of the cells.24 PL was
the most efficient formulation in quenching this effect as
determined by flow cytometry24 (Fig. 1). Cellular survival cor-
related well with markers of DNA damage only with extreme
positivity. The aqueous extract of PL leaves was the most
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Fig. 1 PL antioxidant capacity (A) and PL DNA preservation properties
(B and C). A. The different antioxidant potential of the assayed
Polypodium leucotomos extracts by free radical scavenging activity
assay (ABTS) and Ferric ion Reducing Antioxidant Power assay (FRAP).
Antioxidant assays for each sample (#1–6). B. The assayed Polypodium
leucotomos extracts have a different effect on the phosphorylation of
the histone H2AX (γH2AX) upon UV irradiation. HaCaT transformed kera-
tinocytes (white bars). Data represents the percentage of γH2AX very
positive cells (γH2AXbright). C. The assayed Polypodium leucotomos
extracts have a different effect on the appearance of CPDs. Data rep-
resents the percentage of CPD-positive cells. In all graphs, data are the
mean percentage ± SD. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-
2-carboxylic acid) ( permission requested to Springer Japan, journalper-
missions@springernature.com from Gonzalez et al., J. Med. Plants Res.,
2018).
efficient photoprotector according to all the metrics evalu-
ated in this study. In contrast, the rest of the extracts offered
mixed effects.24 These differences likely underlie in the
method of extraction, the part of the plant used, and their
geographical origin.
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Scientific evidence of an aqueous
extract of Polypodium leucotomos
leaves (Fernblock®) as a systemic
photoprotector (Table 1)
Antioxidant activity
PL has intrinsic antioxidant capacity,24 but it also increases
the capacity of the endogenous antioxidant systems. PL
decreases superoxide anions, hydroxyl radicals, and lipoperox-
ides induced in the skin after exposure to solar
radiation.34,43,48,49 The most relevant difference between this
extract and conventional antioxidants is its ability to decrease
the levels of superoxide anion (O2•−) (Fig. 7).43,48
In vitro studies demonstrated that PL inhibited the
reduction of nitroblue tetrazolium (NTB) induced by O2•− rad-
icals generated by the hypoxanthine-xanthine oxidase system.
PL was an excellent quencher of superoxide anion, with
approximately 40–60% of the activity of superoxide dismutase
used as a positive control. It should be noted that PL did not
produce O2•− in the absence of the generation system. In the
presence of a buffer medium, PL showed a substantial 31%
reduction in the detection of O2•− radical.43,48
These results indicate that PL could act directly on O2•-,
which would lead to its inactivation, blocking the beginning of
the cascade that amplifies reactive oxygen species (ROS).
Additionally, PL also inhibits lipid peroxidation.
Administration of PL after UVA or UVB radiation significantly
inhibited lipid peroxidation in keratinocytes and fibroblasts in
non-irradiated and irradiated cells, consistent with a protective
effect.49
The antioxidant effect of PL has also been demonstrated
in vivo. PL decreased oxidized glutathione (GSSG) content in
dorsal skin in hairless mice irradiated with UVR (from 20 to
640 mJ cm−2; UVB/UVA lamps). In blood, PL decreased erythro-
cytic GSSG and increased plasma antioxidant capacity
(measured as acute induction of oxygen radical absorbance
capacity, ORAC).34 In parallel, when PL was orally administered
to hairless rats before irradiation (UVB/UVA ratio of 0.9 and a
peak at 312 nm 7 J cm−2) a decrease in GSSG content was
found in the rat dorsal skin and plasma, compared to irra-
diated rats in the absence of PL.50 These changes are likely to
reflect the activation of defensive, antioxidant systems to neu-
tralize increased ROS production triggered by the UVR50
(Fig. 2).
The proven antioxidant effect of PL in vivo and in vitro
suggests that PL could be an efficient therapeutic tool against
the generation of ROS induced by UVR since oxidative stress
causes cell damage related to photoaging and
photocarcinogenesis.
DNA damage prevention
Chronic exposure to solar radiation is the most important
factor involved in solar radiation-induced photoaging and
pathogenesis of skin cancers, especially actinic keratosis (AK)
and squamous cell carcinoma (SCC). DNA is the primary cellu-
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Table 1 Photoprotective effects of PL and its putative molecular targets (see graphic art – Fig. 7)

Mechanism Results Model Ref.

Erythema Decreased erythema/increased MED Human 33 and 41

Oxidative stress Decreased lipid peroxidation In vitro 49
Human 48
DNA damage Decreased levels of 8-oxo-G, DNA mutations, CPDs Mouse 55
Human 19 and 41

Inflammation Decreased expression of TNF-α, iNOS, AP-1 NF-κB In vitro 39

Reduced infiltration Mouse 50
Reduced number of macrophages and neutrophils Mouse 55
Reduced number of mast cells Human 41 and 59
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Decreased COX-2, PGE2 expression Mouse 55

Human 19

Immunosuppression Reduced trans-UCA isomerization In vitro 61

Reduced gluthatione oxidation Mouse 34 and 50
Preservation of eLC population Mouse 50
Human 40 and 41
Reduced PMLE reaction Human 76 and 77

Photocarcinogenesis Increased the number of p53(+) cells Mouse 34 and 55

Delayed skin tumor development Mouse 34
Increased clearance/decreased recurrence of AKs Human 70
Increased MED in familial MM Human 33
Decreased epidermal cell proliferation Human 19 and 41
Decreased PCNA, cyclin D1 expression Human 19

ECM damage Increased expression of collagen I, III and V In vitro 37

Decreased MMP1 expression In vitro 37 and 49
Increased TIMP expression In vitro 37
Mouse 55

lar target in UVR-induced carcinogenesis.51 DNA absorbs UVB
photons, resulting in a structural rearrangement of nucleo-
tides that leads to defects in the DNA chain and replication.
CPDs are the main photoproduct of UVR-induced DNA
damage.
UVR also indirectly damages DNA. UVA photon absorption
mainly produces the free radical singlet anion oxygen (1O2).
Subsequently, 1O2 induces the oxidation of guanine residues
and production of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-
dG), which is a biochemical marker of DNA damage.52
A study reported the effect of PL in preventing DNA damage
induced by UVR in human subjects. Volunteers were exposed
to artificial UV radiation after the oral administration of PL
(7.5 mg kg−1). After 24 hours, skin biopsy samples were col-
lected from treated and untreated individuals with PL.41 PL-
treated skin clearly showed a significantly lower number of
sunburn cells when compared to untreated skin.
Immunohistochemistry studies demonstrated that the amount
of CPDs was significantly lower in PL-treated skin compared to
PL-untreated skin41 (Fig. 3). Since CPDs are mutagenic and
lead to carcinogenesis,53,54 erythema prevention and blockade
of UVR-induced DNA damage or boosting DNA repair are
highly desirable in photoprotection.
To better understand the possible photoprotective mecha-
nism of PL regarding DNA, we performed experiments in a
mouse model of xeroderma pigmentosum. Hairless Xpc+/−
mice display skin cancer that is highly comparable to mild
human xeroderma pigmentosum syndromes.55 Mice were irra-
diated with UVB (a single dose of 25 mJ cm−2) after receiving a
daily oral dose of 300 mg kg−1 of PL. Mice were sacrificed
before (non-irradiated), immediately after UV exposure (time
0), as well as 6, 24, 48, and 72 hours after exposure. Xpc+/−
mice irradiated and PL-fed showed significantly lower levels of
CPDs than those that did not receive treatment with PL as
early as 72 hours after irradiation.55 These experiments
demonstrate that PL enhanced DNA repair (Fig. 4). The effects
of PL in Xpc+/− mice were reproduced in wild type mice.55 The
same study reported that PL decreased UVR-induced oxidative
DNA damage. Hairless Xpc+/− mice irradiated and PL-sup-
plemented displayed a lower number of cells positive for 8-ox-
dG (a marker of oxidative damage) than those left untreated55
(Fig. 4).
Since PL accelerated the elimination of UV-induced
photoproducts (CPD) and decreased UVR-induced oxidative
damage to DNA (Fig. 4) we also examined the ability of PL
to control UVR-induced DNA mutations. Mice that were
not irradiated displayed only basal levels of mutations,
which increased fivefold five times after a single exposure
to UVR. However, the level of mutations decreased by 25%
in mice irradiated and fed with PL.55 Therefore, PL acceler-
ates the removal of UV-induced photoproducts (CPDs) and
decreases UVR-induced mutations. Related to the decrease
of CPDs and mutations, PL activated the tumor suppressor
p53 in irradiated Xpc+/− mice, which inversely correlated
with the levels of the pro-inflammatory enzyme cyclooxy-
genase 2 enzyme (COX-2).55

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Fig. 2 Oxidized glutathione (GSSG) content in plasma (A) and dorsal
skin (B) of hairless rats irradiated with UVB/UVA and treated with oral
PL. A. Significant (P < 0.05) influence of PL treatment on each parameter
was calculated using the multivariate analysis. * PL Control vs. Control;
** PL irradiated vs. irradiated. B. Significant (P < 0.05) influence of PL
treatment on each parameter was calculated using the multivariate ana-
lysis. # PL Control vs. Control and irradiated; ## PL Controls vs. Control;
+irradiated vs. Control and PL irradiated; ++PL irradiated vs. irradiated.
Fig. 3 Histology from paired biopsy specimens of skin treated with UV
radiation (SS) alone (A) and with PL (B). PL-treated skin shows: less
cyclobutane pyrimidine dimers. (Permission requested to Elsevier
https://www.elsevier.com/about/policies/copyright/permissions from
Middelkamp-Hup et al., J. Am. Acad. Dermatol., 2004, pp. 910–918).
DNA damage correlates with phosphorylation of p53, which
inhibits its ability to prevent, or undo, DNA damage.56 On the
other hand, upregulation of active p53 plays an essential role in
the decrease in oxidative DNA damage and in reducing UV-
induced carcinogenesis. Also, activation of p53 accelerates the
removal of UVB-induced photoproducts, most importantly CPDs.57
In agreement with the overexpression of p53 mediated by
PL, we observed a decrease in epidermal cell proliferation
induced by UVR in human and experimental animals.34,41
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Recently, a clinical trial was carried out to investigate the
effect of oral PL (480 mg) on biomarkers associated to UV
damage in 22 volunteers with skin phototype I–III. (UVB MED
testing technique, 6 doses of 100, 150, 200, 250, 300, and
350 mJ cm−2).19 These biomarkers included parameters
related to DNA damage and apoptosis (CPDs), inflammation,
and cell proliferation. PL improved all these biomarkers. CPDs
displayed a 32% decrease in irradiated and treated individuals
compared to untreated subjects. Also, PL reduced the number
of cyclin D1 and PCNA-positive epidermal cells induced by
UVR.19
In summary, oral supplementation of PL shows the follow-
ing photoprotective effects by: (a) accelerates the removal of
UV-induced photoproducts (CPDs) and decreases UV-induced
mutations; (b) reduces UV-induced oxidative DNA damage; (c)
increases expression of active p53; (d) decreases cell prolifer-
ation, and (e) inhibits UV-induced COX-2 enzyme
levels.19,39,41,55
In vitro and in vivo studies suggest that the oral adminis-
tration of PL could have photoprotective effects, reducing the
DNA damage caused by UVR directly and indirectly, suggesting
its usefulness to prevent UV-induced skin cancer.
Langerhans cells preservation
UVR induces isomerization of urocanic acid (3-(1H-imidazole-
4-yl)-2-propenoic acid; UCA) and a decrease in the number of
epidermal Langerhans cells. UCA is an important epidermal
chromophore that undergoes trans to cis isomerization after
exposure to UVR. cis-UCA is inactive in terms of photoprotec-
tion and constitutes an immunosuppression marker. Also, the
immunosuppressive effects of UVR are due to the actions of
immunomodulatory molecules such as TNF-α, prostaglandin
PG E2, and IL-10 (Christensen et al., 2017).58
The Granstein group59 demonstrated the functional preser-
vation of LC population in C57BL/6 mice sensitized with oxa-
zolone. In this model, UVB radiation suppressed the induction
of contact hypersensitivity (CHS).60 Mice challenged with oxa-
zolone and subjected to UVB radiation (3500 J m−2 daily for 4
days) displayed significantly decreased levels of CHS compared
with non-irradiated mice. When PL was added to the drinking
water, the levels of CHS were similar to those observed in non-
irradiated animals. This finding suggests that PL adminis-
tration inhibits the ability of UVB radiation to suppress the
immune system.59 Hairless Xpc+/− mice also displayed a
marked decrease (about 21%) in the total number of LC upon
irradiation, but pre-treatment with PL partially blocked this
effect. A significantly higher number of LC was detected in the
group treated with PL and irradiated, compared to the vehicle-
treated group and irradiated (Fig. 5).59
We also studied whether PL protection of the LC population
was accompanied by an effect of PL on trans-UCA photoisome-
rization.61 In vitro experiments showed that UVB irradiation
(doses 4.8 J cm−2) induces the formation of cis-UCA in human
primary fibroblasts. When PL is added, significant inhibition
of cis-UCA formation was observed. The same results were also
obtained by irradiating human primary fibroblasts with UVA.
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Fig. 4 Effects of PL in DNA damage in vivo in Xpc+/− mouse skin A: Representative images of immunostained (green fluorescence) CPDs + cells in
Xpc+/− mice treated with PL or vehicle at 0, 6, 24, 48, and 72 hours after UV radiation. In vehicle-fed Xpc+/− mice, CPD positivity persisted up to
72 hours post-UVB, whereas by 72 hours there was noticeably less detectable CPDs remained in PL-fed Xpc+/− mouse skin (in epidermis as well as
dermis). B: Graphic representation of CPD + nuclei per 200 linear µm of epidermal length showed a statistically significant decrease of percent
remaining CPDs in PL-fed Xpc+/− mice at 72 hours. C: Representative images of immunostained (green fluorescence) 8-ox-dG (marker of oxidative
damage) positive cells in mice treated with PL or vehicle at 0, 6, and 24 hours after UV radiation. 8-ox-dG + cells were negligible at 48 and 72 hours
post-UVB. D: Graphic representation of the number of 8-ox-dG + cells per 200 linear µm of epidermal length showed a marked decrease of these
cells in PL-fed mice at 0, 6, and 24 hours after UV radiation. Dermal-epidermal junctions are outlined by dotted lines. Scale bar 200 µm. (Permission
requested to Elsevier https://www.elsevier.com/about/policies/copyright/permissions, from Zattra et al., Am. J. Pathol., 2009).

Our results also showed how PL inhibits the breakdown of UV-
mediated trans-UCA in the presence of strong oxidants such as
H2O2.61 This effect is partially based on the antioxidant effect
of PL, which had been demonstrated previously.34,43,48,49
In summary, PL efficiently inhibits the photodecomposition
of natural photoprotective molecules (such as trans-UCA) and
blocks the harmful effects of other reagents, which can induce
the formation of ROS in the skin when stimulated with UVR.
We have also confirmed the anti-immunosuppressive effect
of PL in humans. We performed a study in healthy volunteers
exposed to natural sunlight, first untreated and later treated
with PL, which was the first to show how an oral antioxidant
showed tended to preserve the number of Langerhans cells.48
Also, LC from untreated skin increased in size and showed
a loss of dendritic morphology, while LC from skin treated
with PL retained their size and dendritic appearance. Similar
observations had been made in PL-treated patients undergoing
PUVA therapy.41 Non-melanoma skin tumors such as SCC typi-
cally exhibit a severely reduced presence of LC in the tumor
and adjacent regions. Because dendritic cells can mediate anti-
tumor immune responses, LC preservation in the epidermis
could play a crucial role in immune surveillance of cancer.62

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Fig. 5 Langerhans cells (LC) in epidermis of hairless rats irradiated with
UVB/UVA and treated with oral PL. Significant (P < 0.05) influence of PL
treatment on each parameter was calculated using the multivariate ana-
lysis. •Control vs. irradiated; irradiated vs. Control and PL irradiated; •••PL
irradiated vs. irradiated.
Photoaging prevention
Chronic exposure to solar radiation results in skin photoaging
and photocarcinogesis. Photo-exposed skin appears rough and
thick, with wrinkles and aberrant pigmentation.63
The histological features of the photo-damaged skin
include an accumulation of amorphous dermal elastic fibres
(solar elastosis) and fragmented and disorganized dermal col-
lagen. These fibrillar proteins support the structure and func-
tion of the skin, and their modifications alter wound healing
and cutaneous photoaging.64
UVR also increases the activity of matrix metalloproteases
(MMPs). MMP upregulation is also partially responsible for
skin photoaging, as MMP alters the homeostasis of the ECM,
promoting degradation of elastin and collagen. UVR-induced
ROS activate mitogen-activated protein kinase (MAPK) and
nuclear factor-κB (NF-κB), increasing MMPs transcription. NF-
κB acts as an inducible transcription factor that regulates ECM
degradation and stimulates inflammatory mediators.65
UVR radiation causes DNA damage, oxidation processes, and
alterations in the cell cycle in keratinocytes, and fibroblasts,
leading to photoaging and photocarcinogenesis.22,30,61,64 PL
acts by decreasing the signs of photoaging.26,36,37,49,55
A study in human subjects proved that PL prevented mito-
chondrial damage induced by UVA.36 which is a typical
markers of oxidative stress in photoaging.63 Specifically, that
study addressed the ability of PL to prevent the appearance of
common deletion (CD) in response to UVA irradiation. CD is
the most extensively studied deletion of mitochondrial DNA
and is a marker of chronic UVA.63 UVA increased the appear-
ance of CD. Pre-treatment with PL showed a non-statistically
significant tendency prevent this increase 24 hours after UVA
irradiation in a small cohort of subjects.36
Also, PL increased expression of collagen I, III, and V,37,49
repression of MMP-1,37,49 and increased expression of TIMP
(tissue inhibitor of MMPs).37 Similar effects were observed
after exposure to visible light (400–700 nm; VIS) and infrared
(IR) radiation.26
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A recent in vitro study addressed the effect of PL on the
photoaging effect of VIS and IR radiation in human skin26
using dermal fibroblast markers such as elastin (ELN) and
fibrillins 1 and 2 (FBN1 and FBN2), and enzymes involved in
ECM remodelling such as MMP and Cathepsin K.66,67 MMP-1
expression increased significantly 24 h after VIS exposure
(247.3 J cm−2). Pre-treatment with PL significantly decreased
MMP-1 expression in response to VIS radiation. IR-A radiation
(1.56 J cm−2) also caused a significant increase in the
expression of MMP-1.26 Incubation with PL before irradiation
with IR-A significantly prevented this increase (Fig. 6).
Cathepsin K (CTSK) levels also increased after exposure to
both VIS and IR-A radiations, but pretreatment with PL pre-
vented CTSK increase26 (Fig. 6).
According with our previous results,49 UV light repressed
expression FBN, and PL stimulated their expression in both
non-UV-exposed and UV-exposed keratinocytes. Interestingly,
the response to VIS and IR-A radiations was different. VIS
increased expression of FBN1 and 2, while IR-A significantly
decreased their expression.37 PL also stimulated the expression
of FBN1 and FBN2 per se and in the presence of either type of
radiation26 (Fig. 6). PL also increased the levels of ELN. VIS
irradiation increased the expression of ELN, without or with
pre-treatment with PL. Conversely, IR-A irradiation signifi-
cantly decreased the levels of ELN, and this effect was coun-
tered by PL (fivefold increase).26
Finally, a pilot human clinical trial showed the cutaneous
effects of oral PL after irradiation with VIS and IR. Volunteers
were irradiated with IR-VIS (600 J cm−2 and 200 J cm−2,
respectively). 960 mg day−1 of PL was administered for 21 days,
and then irradiation was performed.68
These data demonstrated that the levels of MMP-1
increased after irradiation with VIS or IR, while this increase
significantly decreased (51.7% compared to irradiated,
untreated controls) after treatment with PL.68
In summary, PL reversed the expression of ECM degradative
enzymes of the dermal ECM induced by sunlight. It also dis-
played a protective role of the ECM fibres akin to skin toler-
ance. The alteration of these proteins leads to premature skin
aging. Thus, PL improves photoaging by inhibiting the pro-
cesses that cause damage and improving repair processes.
Clinical implications
PL has been used to treat dermatologic disorders such as psor-
iasis, melasma, vitiligo, atopic dermatitis and immunologi-
cally-mediated photodermatoses. Here, we will discuss its
potential to treat photodermatoses, pigmentation disorders
and the latest evidence regarding the treatment of skin
cancer.33,38,69–81
Regarding its potential use, it is important to underscore
that oral PL is well tolerated and associated with a negligible
risk of side effects.27,32,44,45
In 2015 Winkelmann et al.,32 performed a retrospective
study based on a total of 19 human studies and 6 studies in
Photochem. Photobiol. Sci.

Paper
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Fig. 6 Effects of visible light (VIS) and infra-red radiation (wIRA) on the
expression of MMP-1 (A), cathepsin (CTSK) (B) in the presence of
Fernblock (FB). Expression of Fibrilins 1 and 2 (FBN1 and FBN2) (C) and
Elastin (ELN) (D) after VIS/wIRA(I) and FB + VIS/wIRA(II) in the presence
of FB. Cells were treated with FB and then irradiated as indicated. (A. * p
≤ 0.05, with respect to control; +p ≤ 0.05, with respect to VIS or 1.56 J
cm−2 wIRA. B * p ≤ 0.05, with respect to control; +p ≤ 0.05, with
respect to VIS or wIRA. C *p ≤ 0.05, with respect to Control; +p ≤ 0.05,
with respect to VIS or wIRA; ++ p ≤ 0.01, with respect to VIS or wIRA. D
* p ≤ 0.05, with respect to Control; +++ p ≤ 0.001, with respect to
wIRA). (Permission by the authors, from Zamarron et al., Int. J. Mol. Sci.
2018).
animal models. PL was administered at daily doses between
120 mg to 1080 mg. PL was well tolerated in all doses adminis-
tered and associated with an insignificant risk of side effects.
In humans, side effects were found only in a very small
Photochem. Photobiol. Sci.
View Article Online
Photochemical & Photobiological Sciences
Fig. 7 Schematic representation of the beneficial effects of PL. Red
lines indicate decreased or blocked by PL. Green lines indicate induced
by PL. Acronyms: t/c-UCA, trans/cis urocanic acid (this pathway, blocked
by PL, is responsible for the disappearance of eLC, hence PL favors eLC
survival likely through this pathway); Gltn, glutathione; eLC, epidermal
Langerhans cells, TNF-alpha, tumor necrosis factor-alpha; AP-1, activa-
tor protein-1; NF-κB, nuclear factor-κB; COX-2, cyclooxygenase-2;
PGE2, prostaglandin-2; CPDs, cyclobutane pyrimidine dimers, ECM,
extracellular matrix, TIMP, tissue inhibitor of metalloproteinases; MMP-1,
matrix metalloproteinase-1; COL, collagen.
number of patients [16/1016 (2%)], who reported mild to mod-
erate gastrointestinal discomfort and pruritus.
In a recent study in 2017 (Murbach et al., 2017) conducted
in Wistar Han rats, different of PL (0, 2000, 3500, and 5000 mg
per kg per bw per day) were administered orally for 28 consecu-
tive days.27 No mortality or toxic effects were observed, and no
alterations in target organs were identified. No adverse effects
were detected even at the dose of 5000 mg per kg per bw per
day, the highest dose tested.27
Human studies indicate that for humans, 240 mg of PL
taken twice daily provides photoprotection with an excellent
safety profile. Thus, oral PL could be considered as a photopro-
tector suitable for long term use.44
Polypodium leucotomos aqueous extract (Fernblock®) and
inflammatory conditions
Polymorphic light eruption (PMLE). PMLE is the most
common photosensitive disorder, characterized by an inter-
mittent eruption of vesicles, papules, or pruritic, erythematous
plaques that develop after a few hours of skin exposure to
UVR. This condition depends on genetic susceptibility,
environmental components, and type of exposure. Currently,
some new aspects in the pathogenesis of PMLE related to the
activation and promotion of the inflammatory process have
been highlighted, and new therapeutic and preventive strat-
egies are being developed.74,75 In a group of approximately 80
patients, PL exerted powerful antioxidant and immunomodu-
latory effects. PL (480 mg day−1) was administered the day
before sun exposure, which significantly reduced skin reac-
tions and subjective symptoms of PMLE.76,78 Another study
This journal is © The Royal Society of Chemistry and Owner Societies 2020

Photochemical & Photobiological Sciences
was performed in 35 patients with long-standing PMLE. The sub-
jects underwent irradiation with artificial UVB and UVA light,
receiving PL orally after irradiation. PL was administered for two
weeks (<55 kg: 620 mg d−1, 56–70 kg: 960 mg d−1 > 70 kg:
1200 mg d−1) and a second set of irradiation was performed after
one week while the patients were still receiving a constant dose of
PL.78 After PL treatment, 30% and 28% patients (first and second
irradiations, respectively), were unresponsive to repeated UVA and
UVB exposure. In the remaining 70% and 72% patients, the
average number of UVA and UVB irradiations required to induce
PMLE increased significantly. Interestingly, 74% of patients
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treated with PL during the summer displayed no PMLE-related
injuries.78 These results suggest that oral PL treatment might be
beneficial for the prevention to prevent PMLE.
Vitiligo. Vitiligo is an acquired chronic loss of skin pigmen-
tation, which corresponds to a substantial loss of
melanocytes.71,72 Treatment options for vitiligo include photo-
therapy: PUVA ( psoralen and exposure to UVA radiation) and
NUVB (Narrow-Band UVB radiation). The first achieves repig-
mentation of up to 100 percent in some patients, but it
requires several treatment sessions per week for many months
with an increased risk of skin cancer as the main side effect.73
The optimization of PUVA therapy is an important clinical aim
in the treatment of patients with vitiligo.
Our group has observed the beneficial effects of adding PL
to PUVA therapy to treat vitiligo.69 A randomized, double-
blind, placebo-controlled pilot study evaluated the combined
efficacy of PUVA plus PL versus PUVA plus placebo.69 The per-
centage of patients with a skin pigmentation greater than 50%
was significantly higher among patients treated with PUVA +
PL than patients treated only with PUVA and placebo. We also
have correlated the response of the regulatory T cells (Tregs).
The overall pigmentation response was inversely correlated
with the observed decrease in CD3 + CD25 + T cells.69
Regarding NUVB, a randomized, double-blind, placebo-con-
trolled study, showed that re-pigmentation was higher in the
PL vs. placebo group in the head and neck area (44% vs.
27%).38
Polypodium leucotomos aqueous extract (Fernblock®) and
skin cancers
PL inhibits oxidative stress induced by UVR. It also inhibits
DNA photodamage while hampering epidermal cell prolifer-
ation and increasing p53 tumor suppressor gene expression. In
vivo, preliminary studies in hairless mice exposed to chronic
UVB radiation PL treatment reduced the number of mice
showing skin tumors eight weeks after halting the UVB
irradiation protocol.79
Actinic keratosis (AK) is a common pathology that affects the
skin areas most exposed to the sun, and its appearance is attrib-
uted to cumulative damage from chronic exposure. AK is also
associated with the presence of subclinical lesions adjacent to
the tumor tissue, which has led to the use of the term “field can-
cerization.” Although treatments targeting injuries and fields
are currently available, many are associated with local side
effects and the recurrence of new lesions.81 Photodynamic
This journal is © The Royal Society of Chemistry and Owner Societies 2020
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Paper
therapy is one of the most effective treatments for AK. The
Auriemma group70 evaluated conventional MAL-PDT in humans
concurrent with oral uptake of PL. Patients with at least two AKs
on the scalp were included in this study, and divided into two
groups: one group (n = 17) received with PDT alone, while the
other (n = 17) underwent oral treatment with PL one week after
the last PDT session.70 Both groups were comparable in terms
of skin phototype and previous UV exposure. Both treatments
were successful in reducing the number of AK. However, sup-
plementation with PL reduced the recurrence rate compared to
PDT when evaluated six months after the protocol had ended,
suggesting that PL could be a suitable adjuvant agent to PDT in
the treatment of field cancerization.
Malignant melanoma (MM) high-risk patients
Melanoma is a devastating skin cancer related to early
exposure to ultraviolet radiation. Hereditary traits modify the
risk of melanoma of a given subject. Familial/hereditary mela-
nomas are widely used to describe families with a higher inci-
dence of melanoma. Only about 10% of MM occurs in a family
context, but most of these families bear germline mutations of
the cyclin-dependent kinase inhibitor 2A CDKN2A.80 In these
cases, individuals bearing this mutation generally have mul-
tiple family members with melanoma. Alterations to other
genes confer a moderate risk, such as the Melanocortin 1
receptor (MC1R) gene. MC1R gene variants control pigment
synthesis of a red/yellow pheomelanin and induce oxidative
cell damage.80 The natural expression of two gene variants sen-
sitizes melanocytes to the cytotoxic effect of UV and increases
the of DNA damage and oxidative stress.
The Puig group33 investigated the possible role of an oral
PL extract to improve systemic photoprotection in patients at
risk MM. The authors analysed the ability of PL to decrease
UV-induced erythema and the interaction among MC1R poly-
morphisms and CDKN2A status with the minimal erythema-
tous dose (MED) and how influenced their response to oral
PL.33
A total of 61 patients (20 with sporadic MM, 25 with famil-
ial and multiple MM, and 16 without a history of MM) were
exposed to UVB radiation. PL improved photoprotection by
decreasing UV-induced erythema and increasing by 30% at a
minimum erythematous dose (MED) in all patient patients.
Although not significant, oral PL had a greater effect on
MED in patients with familial MM than in sporadic MM (U =
273, p = 0.06). Among patients with familial MM, those that
exhibited a mutated CDKN2A and polymorphisms in MC1R
showed the most benefit from the effect of PL. These results
are promising, suggesting the need for further studies with
long-term administration of PL in patients at high risk of
developing MM and with long-term follow-up.33
Conclusions
Systemic photoprotection is now a reality against the detrimen-
tal effects of sunlight, including VIS and IR radiation. While it
Photochem. Photobiol. Sci.

Paper
will never substitute conventional photoprotection, it may be a
suitable complement. A standardized aqueous extract of
Polypodium leucotomos (PL/Fernblock®)82 is a photoprotector
with antioxidant and anti-inflammatory properties, DNA and
immune protective effects. Of particular relevance, PL needs to
be considered as an oral photoprotector in the following high-
risk populations: lighter skin phototypes; patients with photo-
dermatosis; patients with a clinical history of skin cancer and
patients undergoing phototherapy.
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Conflicts of interest
S. G. consults for Cantabria Labs, Madrid, Spain. The remain-
ing authors declare no conflict of interest. The funders had no
role in the design of the study; in the collection, analyses, or
interpretation of data; in the writing of the manuscript, or in
the decision to publish the results.
Acknowledgements
This research was funded by Instituto de Salud Carlos III,
MINECO grant number PI15/00974, Feder Funds.
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