Efficacy, stability, and safety evaluation of new polyphenolic xanthones towards identification of bioactive compounds to fight skin photoaging

Article abstract of DOI:10.3390/molecules25122782

Antioxidants have long been used in the cosmetic industry to prevent skin photoaging, which is mediated by oxidative stress, making the search for new antioxidant compounds highly desirable in this field. Naturally occurring xanthones are polyphenolic compounds that can be found in microorganisms, fungi, lichens, and some higher plants. This class of polyphenols has a privileged scaffold that grants them several biological activities. We have previously identified simple oxygenated xanthones as promising antioxidants and disclosed as hit, 1,2-dihydroxyxanthone (1). Herein, we synthesized and studied the potential of xanthones with different polyoxygenated patterns as skin antiphotoaging ingredients. In the DPPH antioxidant assay, two newly synthesized derivatives showed IC₅₀ values in the same range as ascorbic acid. The synthesized xanthones were discovered to be excellent tyrosinase inhibitors and weak to moderate collagenase and elastase inhibitors but no activity was revealed against hyaluronidase. Their metal-chelating effect (FeCl₃ and CuCl₂) as well as their stability at different pH values were characterized to understand their potential to be used as future cosmetic active agents. Among the synthesized polyoxygenated xanthones, 1,2-dihydroxyxanthone (1) was reinforced as the most promising, exhibiting a dual ability to protect the skin against UV damage by combining antioxidant/metal-chelating properties with UV-filter capacity and revealed to be more stable in the pH range that is close to the pH of the skin. Lastly, the phototoxicity of 1,2-dihydroxyxanthone (1) was evaluated in a human keratinocyte cell line and no phototoxicity was observed in the concentration range tested.

Full text of DOI:10.3390/molecules25122782

molecules
Article
Efficacy, Stability, and Safety Evaluation of New
Polyphenolic Xanthones Towards Identification of
Bioactive Compounds to Fight Skin Photoaging
Diana I. S. P. Resende 1,2 , Mariana C. Almeida 1,2, Bruna Maciel 3, Helena Carmo 4
José Sousa Lobo 5, Carlotta Dal Pozzo 6, Sara M. Cravo 1,2 , Gonçalo P. Rosa 7,8
,
,
Aida Kane-Pagès 8, Maria do Carmo Barreto 7,8 , Isabel F Almeida 5,
* ,
Maria Emília de Sousa 1,2,
*
and Madalena M. M. Pinto 1,2
1
Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de
Farmácia, Universidade do Porto, 4050-313 Porto, Portugal; dresende@ff.up.pt (D.I.S.P.R.);
mariana_c_a@hotmail.com (M.C.A.); scravo@ff.up.pt (S.M.C.); madalena@ff.up.pt (M.M.M.P.)
CIIMAR-Centro Interdisciplinar de Investigação Marinha e Ambiental, Avenida General Norton de Matos,
S/N, 4450-208 Matosinhos, Portugal
Laboratório de Tecnologia Farmacêutica, Departamento do Medicamento, Faculdade de Farmácia,
Universidade do Porto, 4050-313 Porto, Portugal; brunademaciel@gmail.com
UCIBIO, REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de
Farmácia, Universidade do Porto, 4050-313 Porto, Portugal; helenacarmo@ff.up.pt
UCIBIO/REQUIMTE, MedTec-Laboratório de Tecnologia Farmacêutica, Departamento de Ciências do
Medicamento, Faculdade de Farmácia, Universidade do Porto, 4050-313 Porto, Portugal; slobo@ff.up.pt
University of Ferrara, Dept. of Life Sciences and Biotechnology, Via Fossato di Mortara,
17/1944121 Ferrara, Italy; carlotta.dalpozzo@student.unife.it
2
3
4
5
6
7
8
cE3c–Centre for Ecology, Evolution and Environmental Changes/Azorean Biodiversity Group,
9500-321 Ponta Delgada, Portugal; goncalo.p.rosa@uac.pt (G.P.R.); maria.cr.barreto@uac.pt (M.d.C.B.)
Faculdade de Ciências e Tecnologia, Universidade dos Açores, 9500-321 Ponta Delgada, Portugal;
aidakane.1@hotmail.com
*
Correspondence: ifalmeida@ff.up.pt (I.F.A.); esousa@ff.up.pt (M.E.d.S.);
Tel.: +351-220-428-689 (I.F.A. & M.E.d.S.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Diego Sampedro
Received: 22 May 2020; Accepted: 15 June 2020; Published: 16 June 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Antioxidants have long been used in the cosmetic industry to prevent skin photoaging,
which is mediated by oxidative stress, making the search for new antioxidant compounds highly
desirable in this field. Naturally occurring xanthones are polyphenolic compounds that can be
found in microorganisms, fungi, lichens, and some higher plants. This class of polyphenols has
a privileged scaffold that grants them several biological activities. We have previously identified
simple oxygenated xanthones as promising antioxidants and disclosed as hit, 1,2-dihydroxyxanthone
(1). Herein, we synthesized and studied the potential of xanthones with different polyoxygenated
patterns as skin antiphotoaging ingredients. In the DPPH antioxidant assay, two newly synthesized
derivatives showed IC50 values in the same range as ascorbic acid. The synthesized xanthones
were discovered to be excellent tyrosinase inhibitors and weak to moderate collagenase and elastase
inhibitors but no activity was revealed against hyaluronidase. Their metal-chelating effect (FeCl3 and
CuCl2) as well as their stability at different pH values were characterized to understand their potential
to be used as future cosmetic active agents. Among the synthesized polyoxygenated xanthones,
1,2-dihydroxyxanthone (1) was reinforced as the most promising, exhibiting a dual ability to protect
the skin against UV damage by combining antioxidant/metal-chelating properties with UV-filter
capacity and revealed to be more stable in the pH range that is close to the pH of the skin. Lastly,
the phototoxicity of 1,2-dihydroxyxanthone (1) was evaluated in a human keratinocyte cell line and
no phototoxicity was observed in the concentration range tested.
Molecules 2020, 25, 2782; doi:10.3390/molecules25122782
www.mdpi.com/journal/molecules

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Keywords: skin; photoaging; antioxidants; xanthones; 1,2-dihydroxyxanthone; DPPH; elastase;
collagenase; tyrosinase; phototoxicity
1. Introduction
Being constantly exposed, the skin is highly affected by external factors like air pollutants and
UV radiation. Besides that, food contaminants and toxic drugs can also have damaging effects on
the skin [1]. All these aspects, as well as the intrinsic production of reactive oxygen species (ROS), result
in oxidative processes that contribute to skin aging as well as to some pathological conditions like skin
cancer [2,3]. When the amount of ROS is too high and/or the skin’s intrinsic antioxidant mechanisms
are overwhelmed oxidative stress occurs [1]. In order to supplement the organism’s natural defenses,
it is important to include antioxidants in the diet. In the case of the skin, there is yet another way to
reinforce the cutaneous antioxidant system, which is via the application of topical antioxidant products.
It has been proved that the topical application of effective antioxidant formulations can improve several
skin parameters and is useful in the reduction of skin aging signs [4,5]. Furthermore, the introduction
of antioxidants in sunscreens has shown to promote an increase in photoprotection effectiveness and
UV-filter stability [6–8].
Repeated exposure to UV radiation leads to the formation of ROS, which triggers a series of
processes involved in photoaging [9]. Increased production of elastase, collagenase, and hyaluronidase
occurs when UV rays penetrate the dermal layers leading to degradation of collagen, elastin, and
hyaluronic acid, respectively. Common consequences are wrinkle formation and sagging of skin, since
these macromolecules are responsible for providing strength, elasticity, and moisture to the skin. [10,11].
Additionally, hyperpigmentation occurs due to excessive accumulation of melanin. Tyrosinase is
the key enzyme initiating skin pigmentation, and, therefore, the inhibition of this enzyme is one of
the most common strategies for reducing pigmentation in the skin [12]. Skin exposition to UV rays
also leads to an increase in cutaneous intracellular catalytic ferric levels establishing the potential of
ferric chelators for topical treatments [13]. Environmental contaminants, mainly generated by industry
and car exhaust, also have a negative impact on the skin, causing premature aging, pigmentation spots,
acne, or even more serious dermatological issues. These pollutants include, among others, polycyclic
aromatic hydrocarbons, volatile organic compounds, particulate matter, oxides, and heavy metals.
They enhance the production of ROS, reducing the content of antioxidants in the skin and causing
oxidative stress [11].
Polyphenols have long been recognized as one of the most important groups of antioxidants.
Particularly, simple oxygenated xanthones have been reported to have antioxidant activity that
varies with the degree of oxygenation as well as with the position of the substituents. This type of
naturally occurring xanthones has shown scavenging activity against ROS as well as metal-chelating
capacity and inhibition of lipid peroxidation [14–16]. By reacting directly with free radicals, they
neutralize them and prevent their deleterious effects in the organism. Structure–activity studies
suggested that simple xanthones with a catechol unit (vicinal diol groups) have promising antioxidant
activity [16–18]. The catechol unit is present in other known antioxidants and it had already been
linked to the antioxidant activity of other polyphenolic compounds [19]. One possible explanation is
that the cooperation between vicinal hydroxyl groups increases the strength of the hydrogen bond
that is formed between the xanthone derivative and its target [16]. Additionally, it has been suggested
that the radical derived from catechol could be stabilized by the vicinal hydroxyl group and by an
intramolecular hydrogen bond [16].
In a previous study developed by our group [16], a library of 20 mono and deoxygenated
xanthones was investigated for their antioxidant potential by the 2,2-diphenyl-1-picrylhydrazyl
(DPPH) and peroxyl radical scavenging effect as well as the inhibitory effect on the prooxidant
enzyme myeloperoxidase (MPO). From the antioxidant screening, 1,2-dihydroxyxanthone (1,2-DHX, 1,

Molecules 2020, 25, 2782
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Figure 1) emerged as a hit compound, that was further characterized for its chelating properties and
its cytotoxic effect on a human keratinocyte cell line [16]. The results suggested the possible efficacy
of xanthone derivatives as antioxidants with potential for skin care application. In order to further
explore the potential of 1,2-DHX (
melanoma epithelial cell line and its modulatory effects on the activity of the THP-1 macrophage cell
line, namely cytokine production. The results suggested a putative impact of 1,2-DHX ( ) in melanoma
1), we also investigated its effects on the growth of the A375-C5
1
treatment, not only due to a direct effect on cancer cells but also by modulation of macrophage
activity [20]. Toxicity was excluded by performing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) reduction, neutral red (NR) uptake, Alamar Blue reduction, and Trypan
Blue exclusion assays. 1,2-DHX (
1) did not promote a significant cytotoxic effect up to 50 µM. This
compound can be considered noncytotoxic since an 83% decrease in HaCaT cell viability in the neutral
red assay was observed. Additionally, 1,2-DHX (1) did not show any scavenging activity of NO
generated in a cell-free system [16,20].
Figure 1. Xanthones investigated in the scope of this work: 1,2-dihydroxy-9H-xanthen-9-one
), 3,4-dihydroxy-1-methyl-9H-xanthen-9-one ( ), 3,4,6-trihydroxy-1-methyl-9H-xanthen-9-one ( ),
1,4-dihydroxy-3-methoxy-9H-xanthen-9-one ( ), and 1,8-dihydroxy-3,6-dimethyl-9H-xanthen-9-one ( ).
(
1
2
3
4
5
In the light of these recent findings, the optimization of the xanthone structure aiming the discovery
of new antioxidants with potential as skin antiaging actives, led us to assemble a small library of
polyoxygenated xanthones with privileged patterns of antioxidant motifs such as catechol (
hydroquinone ( ), or peri-carbonyl hydroxyl groups ( , and ; Figure 1) and to investigate their
potential for skin care. Inhibition activities of enzymes related to skin photoaging (tyrosinase,
collagenase, elastase, and hyaluronidase), were for the first time investigated for 1,2-DHX ( ) and
1–3),
4
1,
4
5
1
related xanthones as well as DPPH reduction activity, metal-chelating effect, and stability at different
pH values. For the first time to the best of our knowledge, the phototoxic potential and prooxidant
effect of 1,2-DHX (1) was also evaluated in a human keratinocyte cell line.
2. Results and Discussion
2.1. Synthesis
Xanthones 1 [21]
modifications (Scheme 1). The synthesis of 1,2-DHX (
,
2 [22]
,
3 [22], and 4 [23] were prepared as described before with slight
; Scheme 1a) involved as starting materials methyl
1
2-bromobenzoate and 3,4-dimethoxyphenol; the resulting methyl 2-(3,4-dimethoxyphenoxy)benzoate,
after hydrolysis and subsequent cyclization of 2-(3,4-dimethoxyphenoxy)-benzoic acid with lithium
diisopropylamide (LDA) or with AcCl, and further demethylation afforded 1 [21]. Xanthones
2 and 3
were prepared via a Friedel–Crafts acylation of the appropriate methoxytoluene by 2-methoxybenzoyl
chloride, using aluminum chloride as an acid catalyst to give a benzophenone intermediate. Further
nucleophilic addition followed by the elimination of methanol under basic conditions and microwave
irradiation afforded 2 and 3 (Scheme 1b) [24]. Xanthone 4 was synthesized using a recently reported

Molecules 2020, 25, 2782
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methodology via acyl radical intermediates starting from salicylaldehyde and 1,2,4-trimethoxybenzene
(Scheme 1c) [23]. Coupling of 2-(dimethoxymethyl)phenol (obtained from salicylaldehyde) with
2-bromo-5-methoxycyclohexa-2,5-diene-1,4-dione (obtained from 1,2,4-trimethoxybenzene), cyclization
with NBS and benzoyl peroxide and subsequent reduction with zinc and cyclization led to
the formation of 1,4-dihydroxy-3-methoxy-9H-xanthen-9-one (
4). The synthesis of a new oxygenated
xanthone (Scheme 1d) was performed through a modification to the classic Grover, Shah,
5
and Shah reaction involving the condensation between 2,6-dihydroxy-4-methylbenzoic acid and
5-methylbenzene-1,3-diol [25]. The use of Eaton’s reagent (phosphorus pentoxide and methanesulfonic
acid (P2O5/CH3SO3H)) as the coupling agent and catalyst instead of a mixture of a phosphorus
oxychloride and zinc chloride led to 1,8-dihydroxy-3,6-dimethyl-9H-xanthen-9-one (
5) in 7% yield. To
the best of our knowledge, derivatives 1–5 are not naturally occurring polyphenols.
Scheme 1. Synthesis of (a) 1,2-dihydroxy-9H-xanthen-9-one (1); (b) 3,4-dihydroxy-1-methyl-9H-
xanthen-9-one and 3,4,6-trihydroxy-1-methyl-9H-xanthen-9-one
(
2
)
(3
); 1,4-dihydroxy-3-
(c)
methoxy-9H-xanthen-9-one (4); and (d) 1,8-dihydroxy-3,6-dimethyl-9H-xanthen-9-one (5).
2.2. Antioxidant Activity
2.2.1. DPPH Reduction Activity
The screening of the reduction activity of xanthones
a decolorization assay using DPPH• as free radical (Table 1).
expressed as the maximum inhibition reached at 25
3,4-dihydroxy-1-methyl-9H-xanthen-9-one ( ) and 3,4,6-trihydroxy-1-methyl-9H-xanthen-9-one (
revealed DPPH reduction activity comparable to 1,2-dihydroxy-9H-xanthen-9-one ( ) and with an IC50
in the same range of the positive control ascorbic acid. Compound remained as the hit compound with
the highest radical scavenging activity [16]. For compounds and , it was not possible to determine
the IC50 since maximum reduction activity was lower than 50%, probably due to solubility issues.
1
–
5
was firstly estimated by
Antiradical activity was
µM. Among all tested xanthone derivatives,
2
3)
1
1
4
5

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Table 1. DPPH scavenging activity at 25
µM (mean value and standard deviation) and IC50 values of
the tested compounds and ascorbic acid as positive control.
DPPH Reduction Activity (%) at
Compound *
IC50 µM (at 60 min)
25 µM (at 60 min)
1
2
3
4
28.4 ± 0.2 a
31.2 ± 4.8 a
43.3 ± 1.5 a
36.8 ± 4.9 a,b
24.9 ± 1.3 c
34.6 ± 3.2 b,d
9.2 ± 2.4 e
47.3 ± 0.4 b
Not determined
Not determined
40.0 ± 0.8 c
5
28.9 ± 0.3 c,d
Ascorbic Acid
* Compounds 1, 2, and 3 were dissolved in ethanol 70%. Due to low solubility in ethanol 70%, compounds 4 and 5
were dissolved in a solution of ethanol 70% with 1% DMSO. In each column, different letters indicate significant
differences (p < 0.05).
2.2.2. Metal-Chelating Effect
Interactions between metal ions and organic molecules can easily be detected by UV/Vis.
The interaction of the tested xanthones with ferric and cupric ions was evaluated on the basis
of changes in the UV/Vis spectra of the free xanthones, following the addition of FeCl3 and CuCl2,
respectively. Based on the DPPH reduction activity, compounds presented the best results.
Although the antioxidant potential of was higher in comparison with , the low solubility of did
not allow the determination of the IC50. Hence, compounds , and 3 were selected to be further
1–
4
4
3
4
1,
2
investigated for their capacity to absorb UV radiation (Figure 2) and their spectra revealed high
absorbance in the UVB range (280–320 nm). UVB radiation is responsible for a series of damage effects
on the skin, either erythema and burns caused by short-term UV exposure or even photocarcinogenesis
after long-term exposure [26]. Sequential additions of FeCl3 and CuCl2 solutions to 1,2-DHX (1) resulted
in a significant change of the absorbance spectrum with bathochromic shifts (UV/Vis spectra) from
the original band, as previously reported by our group [16]. This shift substantiates the formation of
a complex in the presence of Fe (III) and Cu (II) ions due to the presence of a hydroxyl group peri-carbonyl
emphasizing the potential of 1,2-DHX (
study was repeated under the same conditions for compounds
3,4-dihydroxy-1-methyl-9H-xanthen-9-one ( ) and 3,4,6-trihydroxy-1-methyl-9H-xanthen-9-one (3)
1) to chelate metals. In order to serve as a comparison, this
2
and . On the other hand, for
3
2
bathochromic shifts were observed only with sequential additions of FeCl3 while no effect was observed
when CuCl2 was added. This fact validates the importance of a peri-carbonyl hydroxyl group in
xanthone
xanthones, we can infer that xanthone
by combining antioxidant/metal-chelating properties with UV-filter capacity.
1. Considering the preliminary results regarding the antioxidant activity of the selected
1
exhibits a dual ability to protect the skin against UV damage

Molecules 2020, 25, 2782
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(a)
(b)
(c)
(d)
(e)
(f)
(g)
) Typical metal (M) chelation sites in 1,2-dihydroxy- (
Figure 2.
b); (
(a
1
, a) and 3,4-dihydroxyxanthones (
2
–
3
)
,
b
) UV/Vis spectra of compound
UV/Vis spectra of compound in methanolic solution with sequential additions of CuCl2; (
spectra of compound in methanolic solution with sequential additions of FeCl3; ( ) UV/Vis spectra
of compound in methanolic solution with the sequential addition of CuCl2; ( ) UV/Vis spectra of
compound in methanolic solution with sequential additions of FeCl3; ( ) UV/Vis spectra of compound
3 in methanolic solution with the sequential addition of CuCl2.
1
in methanolic solution with sequential additions of FeCl3;
(
c
1
d) UV/Vis
2
e
2
f
3
g

Molecules 2020, 25, 2782
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2.3. Inhibition of Elastase, Collagenase, Hyaluronidase and Tyrosinase Activity
The inhibitory activities of xanthones
screened in vitro by colorimetric methods. Each enzyme was incubated with xanthones
1
–
3
against enzymes related to skin photoaging were
and after
1–3
the addition of the specific substrates, its activity was measured and compared to the activity of
a control without the xanthone derivative. The enzymes screened were tyrosinase, elastase, collagenase,
and hyaluronidase. and the results obtained are summarized in Table 2. The IC50 of compounds 1–3 in
each activity was calculated when the % of activity inhibition at 150 µM was higher than 50%.
Table 2.
Inhibitory activities of xanthones
1–3 against tyrosinase, elastase, collagenase,
and hyaluronidase.
Tyrosinase
Elastase
Collagenase
Hyaluronidase
% Inhibition
(150 µM)
% Inhibition
(150 µM)
% Inhibition
(150 µM)
% Inhibition
(150 µM)
Comp.
IC50 (µM)
IC50 (µM)
IC50 (µM)
1
2
3
96.17 ± 0.88
7.8 ± 0.07 a
8.93 ± 0.16 b
3.28 ± 0.01 c
12.81 ± 0.01 d
-
35.2 ± 1.84 a
n.d
n.d
n.d
-
35.77 ± 1.82 a
n.d
n.d
n.d
-
n.a.
n.a.
n.a.
-
-
-
10.85 ± 0.39 b
26.83 ± 1.45 b
84.05 ± 1.87
18.21 ± 0.71 c
24.91 ± 0.93 b
91.42 ± 0.23
Kojic acid
MAAPVCK
EDTA
-
-
-
-
-
-
-
-
-
0.26 ± 0.003
-
-
-
102.95 ± 5.30
n.a.—Not active (0% inhibition); n.d.—Not determined; Kojic acid (for tyrosinase), MAAPVCK (for elastase),
and EDTA (for collagenase) were used as positive controls. In each column, different letters indicate significant
differences (p < 0.05).
All the xanthone derivatives tested, 1,2-DHX (
and 3,4,6-trihydroxy-1-methyl-9H-xanthen-9-one (
inhibitors, with IC50 in the same order of magnitude and lower than the one obtained for the positive
control kojic acid. Besides reports of other xanthone derivatives, like 3-aryl substituted xanthones [27
1
), 3,4-dihydroxy-1-methyl-9H-xanthen-9-one (
2),
3
), were found to be extremely good tyrosinase
]
or pyranocycloartobiloxanthone A [28], being very active against tyrosinase, this is, as far as we were
able to confirm, the first time that the tyrosinase inhibition activity of simple oxygenated xanthones
such as compounds 1–3 is being reported.
Contrary to the observed for tyrosinase inhibition, compounds
elastase. Compound exhibited the strongest effect by inhibiting 35.2% of elastase activity at
the maximum concentration tested (150 M). For the collagenase inhibition assay, all three compounds
1–3 presented low activity against
1
µ
presented weak to moderate activity, inhibiting the enzymatic activity by 25–35% at 150
the compounds had inhibitory activity against hyaluronidase.
µM. None of
2.4. Stability
Considering the results obtained in the previous screening assays and their feasible synthesis
and potential to scale-up, xanthones (1,2-DHX) and were selected to proceed with additional
1
2
tests with the final purpose to be used in a skin care formulation. Since the quality of the active
ingredient can change with time under the influence of various environmental factors, it is a primary
concern to the formulator to assess physical and chemical stability as it affects the safety and efficacy of
the cosmetic product [29
versus time was observed in pre-established special conditions that accelerate the degradation of
compounds. Hence, (1,2-DHX) and were submitted under a range of pH buffers to test their
,30] To establish the stability of xanthones 1 (1,2-DHX) and 2, their behavior
1
2
pH stability for 21 days. As far as the efficacy of the skin care formulations is concerned, the pH is
regarded as a significant parameter. The pH of human skin normally ranges from 4.5 to 6.0. Therefore,
in order for a formulation present the highest skin compatibility, it should have a pH that is close to
this range [3,31,32]
The graphs depicting absorbance versus time (0, 1, 2, 24, 192, 360, and 504 h) at different pH
are overlapped for xanthones
the variation in the absorbance peak. Considering xanthone
1
(1,2-DHX) and
2
(Figure 3A,B, respectively) in order to understand
, a degradation of the compound for
2

Molecules 2020, 25, 2782
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the range of pH tested overtime is observed. 1,2-DHX (
1) revealed to be more stable in the pH range that
is closest to the pH of the skin, thus presenting a potential to be used as a future cosmetic active agent.
0.35
1
0.8
0.6
0.4
0.2
0
0.25
0.15
0.05
-0.05
0
100
200
300
400
500
600
0
200
400
600
(a) Compound 1
(b) Compound 2
Figure 3. Stabilities of xanthones
1
((a
) 1,2-DHX; 390 nm) and
2
((b), 310 nm), given by variation in pH
buffers over the time of analysis (0, 1, 2, 24, 192, 360 and 504 h) at 200 µM.
2.5. Phototoxicity
Current regulatory guidelines demand preclinical photosafety evaluation within
the pharmaceutical and cosmetic industry framework. According to the animal testing banning
put in force by EU Regulation 1223/2009, only in vitro tests can be performed for cosmetic ingredients.
Phototoxicity was herein evaluated in a human keratinocyte cell line, using a method adapted from
the OECD 432 guideline and previously implemented in our lab (HaCaT, neutral red uptake assay
(NRU)) [33]. This provides a more realistic experimental model in comparison with the 3T3 NRU assay
preconized in the OECD guideline, performed with mice fibroblasts [34].
1,2-DHX (1) was not cytotoxic to HaCaT cells even after irradiation. The IC50 values and
consequently the photo-irritation factor (PIF) could not be obtained. However, as the cell viability
was not decreased after UV exposure (Figure 4), the compound is deemed nonphototoxic up
to 200
µM. 1,2-DHX (1) did not promote any significant increase in the fluorescence signal of
the dichloro-dihydro-fluorescein diacetate (DCFH-DA) probe, relative to solvent control, supporting
the absence of ROS generation upon incubation with HaCaT cells (Figure 5).
Figure 4. Cell viability of the HaCaT cell line exposed to 1,2-DHX (
the evaluation of phototoxicity by the HaCaT NRU assay. Data are presented as mean
relative to solvent control, with (+irr) = 100 20.5 (n = 3) and (-irr) = 100 16.9 (n = 3). Data were
analyzed using one-way ANOVA with a Dunnett post hoc test.
1; 12.5, 25, 50, 100, and 200
µM) for
±
SD (n = 3)
±
±

Molecules 2020, 25, 2782
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Figure 5. Fluorescence of HaCaT cell line exposed to 1,2-DHX (
determined by the DCFH-DA assay. Data are presented as mean
= 100 ± 1.7 (n = 3). Data were analyzed using one-way ANOVA with a Dunnett post hoc test.
1
; 12.5, 25, 50, 100, and 200
µM) as
±
SD (n = 3) relative to solvent control
In the same way as other catechol containing polyphenols [35], 1,2-DHX (1) did not generate
prooxidant effects, which are usually associated with the presence of one phenol group on the aromatic
ring, since the derived phenoxyl radicals tend to be very unstable.
3. Materials and Methods
3.1. Materials
All reagents and solvents were purchased from TCI (Tokyo Chemical Industry Co. Ltd., Chuo-ku,
Tokyo, Japan), Acros (Geel, Belgium), Sigma-Aldrich (Sigma-Aldrich Co. Ltd., Gillingham, UK), or
Alfa Aesar (Thermo Fisher GmbH, Kandel, Germany) and had no further purification process. Solvents
were evaporated using a rotary evaporator under reduced pressure, Buchi Waterchath B-480 (BÜCHI
Labortechnik AG, Switzerland). The reaction was monitored by TLC carried out on precoated plates
with 0.2 mm thickness using Merck (Darmstadt, Germany) silica gel 60 (GF254) with appropriate mobile
phases and detection at 254 and/or 365 nm. Purification of the synthesized compound was performed
by chromatography flash column using Merck silica gel 60 (0.040–0.063 mm). Melting point (mp) was
1
measured in a Köfler microscope (Wagner and Munz, Munich, Germany) and is uncorrected. H and
13C-NMR spectra were taken in CDCl3 at room temperature on a Bruker Avance 300 (Bruker Biosciences
Corporation, Billerica, MA, USA) instrument (300.13 or 500.13 MHz for 1H and 75.47 or 125.77 MHz for
13C). Chemical shifts are expressed in δ (ppm) values relative to tetramethylsilane (TMS) as an internal
reference. Coupling constants are reported in hertz (Hz). 13C-NMR assignments were made by 2D
HSQC and HMBC experiments (long-range C, H coupling constants were optimized to 7 and 1 Hz).
HRMS mass spectra were measured on a Bruker Daltonics micrOTOF Mass Spectrometer, recorded
as ESI (electrospray) mode in Centro de Apoio Científico e Tecnolóxico á Investigation (C.A.C.T.I.),
University of Vigo, Spain. Immortalized human keratinocyte (HaCaT) cell line was obtained from Cell
Lines Service (CLS, Eppelheim, Germany).
Tyrosinase, L-tyrosine, kojic acid, monosodium phosphate, sodium phosphate dibasic elastase,
N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide, N-methoxysuccinyl-Ala-Ala-Pro-chloro, 2-[4-(2-
hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala
(FALGPA), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES) sodium salt, ninhydrin,
citric acid, sodium citrate, EDTA, hyaluronidase, hyaluronic acid, calcium chloride, sodium
aurothiomalate, potassium metaborate, 4-dimethylaminobenzaldehyde (DMAB), sodium acetate,
acetic acid, and hydrochloric acid were purchased from Sigma-Aldrich. Collagenase was supplied by
Merck. Tin (II) chloride was obtained from Riedel-deHaën (Seelze, Germany). The water used in all
experiments was purified water obtained using a Direct-Q Water Purification System (Merck Millipore,
Burlington, Massachusetts, United States) with a reverse osmosis process.

Molecules 2020, 25, 2782
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3.2. Synthesis of 1,4-dihydroxy-3-methoxy-9H-xanthen-9-one (5)
A mixture of phosphorus pentoxide (1.418 g, 5.000 mmol) and methanesulfonic acid (30 mL)
was heated at 90 ◦C until a clear solution was obtained (30 min). Then, a mixture of
5-methylresorcinol (0.517 g, 4.162 mmol) and 2,6-dihydroxy-4-methylbenzoic acid (0.700 g, 4.162 mmol)
was added to the reaction mixture. This mixture was stirred at reflux for 1 h and the progress
of the reaction was monitored by TLC. Then, the resulting mixture was slowly poured into
crushed ice and allowed to stand overnight in the fridge. The solid was collected by filtration,
washed with water, and dried. Purification by column chromatography using a mixture of
EtOAc/hexane (1:9) as mobile phase gave 1,4-dihydroxy-3-methoxy-9H-xanthen-9-one (
1,4-Dihydroxy-3-methoxy-9H-xanthen-9-one (
); White solid (72.7 mg, 7% yield); m.p. 171–173 ◦C.
1H-NMR (CDCl3, 300.13 MHz):
= 8.18 (d, 3J8,7 = 8.8 Hz, 1H, H-8), 6.94 (dd, 3J7,8 = 8.8 Hz, 4J7,5
2.4 Hz, 1H, H-7), 7.52 (d, 4J5,7 = 2.4 Hz, 1H, H-5), 4.06 (s, 3H, 3-OCH3), 4.03 (3H, s, 4-OCH3), 3.94 (3H, s,
6-OCH3), 3.06 (s, 3H, 1-CH3) ppm. 13C-NMR (CDCl3, 75.47 MHz):
= 176.9 (C-9), 164.8 (C-6), 156.6
5) in 7% yield.
5
δ
=
δ
(C-10a), 153.8 (C-3), 151.4 (C-4a), 139.7 (C-4), 136.8 (C-1), 134.6 (C-6), 128.5 (C-8), 118.0 (C-9a), 117.7 (8a),
116.5 (C-2), 113.6 (C-7), 99.7 (C-5), 61.9 (3-OCH3), 61.2 (4-OCH3), 55.9 (6-OCH3), 21.5 (1-CH3) ppm.
HRMS (ESI+): m/z [C17H15BrO5 + H]+ calcd. for [C17H16BrO5]: 379.0176; found 379.0161.
3.3. DPPH Reduction Assay
The reduction of the stable DPPH free radical was measured by monitoring the change in
absorbance at 517 nm. These experiments were made in a 96-well microplate. Compounds 1, 2 and
3
were dissolved in ethanol 70%, and compounds
70% with 1% DMSO. In each experiment, the compounds were tested in the concentration range
0.7812 M to 50 M, in triplicate. For each sample concentration, a blank with ethanol 70%, instead
of DPPH was made. Ascorbic acid was used as a positive control and tested in the same conditions.
4 and 5 were dissolved in a solution of ethanol
µ
µ
Blanks containing only ethanol 70% and control containing DPPH in ethanol 70% (190 µM) were
used. The absorbance at 517 nm was measured in a microplate reader BioTekTM Synergy HT (BioTeck
Instruments, Vermont, USA) after 60 min of incubation in the dark with a temperature-controlled
holder at 25 ◦C. The microplate was shaken before each reading.
The scavenging percentage of DPPH was calculated using the following equation:
!
Abssample+DPPH − Abssample blank
AbsDPPH − AbsEtOH
%scavenging of DPPH = 100 × 1 −
(1)
The IC50 value for each sample and for the ascorbic acid was obtained by linear regression.
3.4. Metal-Chelating Effect
The ultraviolet/visible (UV/Vis) spectra were recorded in methanol using a path length of
1 cm, at room temperature on a Varian CARY100 spectrophotometer (Agilent Technologies, Santa
Clara, California, United States) from a range of 200 nm to 500 nm (software CaryWin UV, v. 3.0).
The metal-chelating effect was evaluated by described procedures [36] with stock solutions (1.0
×
10−4 M) in methanol. Iron (III) and copper (II) solutions were prepared in a concentration of 1.0
×
10−3 M from FeCl3 and CuCl2 in deionized water. Briefly, sequential additions of 33
µL of metal ion
solution were performed to 3 mL of compounds 1, 2, and 3.
3.5. Inhibitory Activities of Enzyme Related with Skin Aging
3.5.1. Tyrosinase Inhibition Assay
This activity was assayed by an adaptation of the tyrosinase inhibition method described by
Shimizu et al. [37] and modified by Manosroi et al. [38] Briefly, 25 L of tyrosinase enzyme solution
(135 U/mL), 25 L of ten serial concentrations of the compounds (0.3–150 M dissolved in 0.1 M
µ
µ
µ

Molecules 2020, 25, 2782
11 of 16
phosphate buffer pH 6.8 containing no more than 2.5% DMSO) and 100
µ
L phosphate buffer were
mixed in a 96-well plate and incubated at 37
±
2 ◦C for 20 min. Then, 50
µL of 1.66 mM of tyrosine
solution in 0.1 M phosphate buffer (pH 6.8) were added. The enzyme activity was measured at
490 nm every 10 min for 30 min in a Bio-Rad Model 680 Microplate Reader (Bio-Rad Laboratories,
Inc., Hercules, CA, USA). Kojic acid was used as positive control. The experiments were done in
triplicate. For each concentration, enzyme activity was calculated as a percentage of the velocities
compared to that of the assay using buffer without any inhibitor. The IC50 value was determined as
the concentration of a compound that inhibited 50% of enzyme activity.
3.5.2. Elastase Inhibition Assay
The compounds were assayed by the method described by Ndlovu et al. [39] with some
modifications. Briefly, 25
of the compounds (0.3–150
DMSO) and 125 L HEPES buffer were mixed in a 96-well plate and incubated at room temperature
for 20 min. Then, 50 L of N-Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (1 mM) were added.
µL of elastase enzyme solution (0.3 U/mL), 50
µL of ten serial concentrations
µM dissolved in 0.1 M HEPES buffer pH 7.5 containing no more than 2.5%
µ
µ
The enzyme activity was measured at 405 nm in the moment of substrate addition and after 40 min of
incubation at 25 ◦C in a Bio-Rad Model 680 Microplate Reader (Bio-Rad Laboratories, Inc., Hercules,
CA, USA). N-Methoxysuccinyl-Ala-Ala-Pro-Chloro was used as positive control. The experiments
were done in triplicate. For each concentration, enzyme activity was calculated as a percentage of
the velocities compared to that of the assay using buffer without any inhibitor. The IC50 value which
was the concentration of the sample that inhibited 50% of the enzyme activity was determined.
3.5.3. Collagenase Inhibition Assay
An adaptation of the method of Moore and Stein [40] with modifications by Mandl et al. [41
was used to determine anticollagenase activity. To 2 mL test tubes was added: 25 L of collagenase
solution (0.8 U/mL), 25 L TES buffer (50 mM) with 0.36 mM calcium chloride (pH 7.4) and 50 L of
M).
]
µ
µ
µ
test sample or the reference compound EDTA (with concentrations ranging between 9.4 and 150
µ
◦
The tubes were incubated in a water bath at 37 C for 20 min. Thereafter, 50
µ
L FALGPA (1 mM)
solution was added to the tubes and incubated further for 60 min at 37 ◦C. To all tubes, 200
µ
L of
a solution containing equal volumes of a 1.6 mg/mLTin chloride (II) solution in 200 mM citrate buffer
(pH 5) and 50 mg/mL ninhydrin solution in DMSO was added. All tubes were placed in a water bath
(100 ◦C) for 5 min and left to cool to room temperature before adding 200
µL of 50% isopropanol to each
tube. Contents in the tubes were then transferred to respective wells in 96-well plates. Absorbance was
detected at 550 nm in a Bio-Rad Model 680 Microplate Reader (Bio-Rad Laboratories, Inc., Hercules,
CA, USA). Percentage of collagenase inhibition was calculated as:
Abscontrol − Abssample
% collagenase inhibition =
× 100
(2)
Abscontrol
where Abscontrol is the absorbance of buffer + collagenase; Abssample is the absorbance of buffer +
collagenase + sample/standard. All assays were carried out in triplicate and results expressed as % of
collagenase inhibition and/or IC50, i.e., as the concentration yielding 50% of collagenase inhibition,
calculated by interpolation from the % collagenase inhibition vs concentration curve.
3.5.4. Hyaluronidase Inhibition Assay
The methods described by Ndlovu et al. [39] and Zhou et al. [42] were applied for the hyaluronidase
inhibition assay. Into 2 mL test tubes was placed: 50
samples or sodium aurothiomalate diluted in 0.1 M acetate buffer; pH 3.5 (with concentrations ranging
between 9.4 and 150 M), and 25 L hyaluronidase (0.5 mg/mL). The tubes were incubated in a water
bath (37 ◦C; 20 min) after which 50
L of the substrate hyaluronic acid (0.25 mg/mL in 0.1 M acetate
µL of calcium chloride (12.5 mM), 50 µL of test
µ
µ
µ

Molecules 2020, 25, 2782
12 of 16
buffer; pH 3.5) was added and the tubes incubated for further 40 min. Twenty-five microliters of KBO2
4 H2O (0.8 M) was added to all tubes which were placed in a water bath (100 ◦C) for 3 min, left to
cool to room temperature, and 800 L of DMAB (4 g DMAB in 40 mL acetic acid and 5 mL 10 N HCl)
•
µ
was added. The tubes were then incubated for 20 min and the contents transferred to respective wells
in a 96-well plate. Absorbance was detected at 585 nm in a Bio-Rad Model 680 Microplate Reader
(Bio-Rad Laboratories, Inc., Hercules, CA, USA). Percentage of hyaluronidase inhibition was calculated
as:
Abscontrol − Abssample
% hyaluronidase inhibition =
× 100
(3)
Abscontrol
where Abscontrol is the absorbance of buffer + hyaluronidase; Abssample is the absorbance of buffer +
hyaluronidase + sample/standard. All assays were carried out in triplicate and results expressed as
% of hyaluronidase inhibition and/or IC50, i.e., as the concentration yielding 50% of hyaluronidase
inhibition, calculated by interpolation from the % hyaluronidase inhibition vs concentration curve.
3.6. Stability
Buffer solutions at pH values of 2, 3, 4, 5, 6, 7, and 8 were prepared and the pH of each buffer was
measured with a pH meter. All the solutions were prepared following Portuguese Pharmacopeia 9.0
specifications (F.P. (VIII) 2008).
A solution at 10 mM in DMSO of each compound was prepared. One hundred microliters of
antioxidant solution were diluted to 5 mL of the different buffered pH solutions, prepared in triplicate.
Their absorbance measured by UV/Vis spectrophotometry in the wavelength range between 200 and
500 nm at time intervals of 0, 1, and 2 h and 1, 8, 15, and 21 d at room temperature. Each pH buffer
solution was used as a compensation solution.
3.7. Safety Evaluation
3.7.1. Cell Culture
The HaCaT cells were maintained at 37 ◦C in a humidified atmosphere of 95% air and 5% CO2 in
the incubator in dulbecco’s modified eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and
1% antibiotics. Using an inverted microscope, cell confluence was observed and if the cells reached
70–80% confluence, subculture was done to prevent cell death. For this purpose, the culture medium
was aspirated and the cells were washed with dulbecco’s phosphate buffered saline (DPBS), 2 mL of
trypsin-EDTA were added and incubated for 5–8 min at 37 ◦C in a 5% CO2 atmosphere. After cell
detaching, medium was added in order to block the trypsin action and cell suspension was centrifuged
at 416 g for 5 min. The supernatant was discarded, the pellet suspended in culture medium, and cells
were counted. For cell counting: 50 µL of cell suspension were added to 50 µL of Trypan Blue vital dye
(1:10), resuspended, placed in a Neubauer chamber (Hirschmann, Germany) and the viable cells were
counted. For cell freezing, DMSO (5% v/v) was used as a cryopreservative to prevent the formation of
crystals during the storage phase.
3.7.2. ROS Generation Assay
Cells (2
a 5% CO2 atmosphere for 24 h. Afterwards, the medium was removed, cells were washed with DPBS,
and complete DMEM containing 50 M DCFH-DA was added and incubated for 30 min. Afterwards,
the cells were washed once with DPBS and 1,2-DHX ( ; 12.5, 25, 50, 100, and 200 M) was added and
incubated for 24 h. H2O2 (700 M) was used as positive control, and untreated cells were taken as
×
104 cells/well) were seeded on 96-well tissue culture plates and incubated at 37 ◦C in
µ
1
µ
µ
the negative control. After incubation, the fluorescence was measured using a fluorescence plate reader
with a baseline of 485 nm excitation and 530 nm emission. The study of the putative interference of
compound and solvent fluorescence was previously performed. The results were calculated as the ratio
between the fluorescence of treated cells and untreated cells and expressed in percentage.

Molecules 2020, 25, 2782
13 of 16
3.7.3. Phototoxicity Evaluation
HaCaT cells were seeded onto 96-well tissue culture plates (Orange Scientific, Braine-l’Alleud,
◦
Belgium) at a 2
×
104 cells/well density and incubated at 37 C in a 5% CO2 atmosphere for 24 h.
Afterwards, the medium was removed, cells were washed with DPBS, and fresh DMEM without FBS
and phenol red containing the different concentrations of 1,2-DHX ( ; 12.5, 25, 50, 100, and 200 M)
1
µ
was added and incubated under the same conditions for 1 h. For each experiment, two identical
plates were seeded and incubated with the test compound. One plate was kept in the dark while
the other plate was irradiated for 10 min [OSRAM Ultra Vitalux 240V E27 lamp (Munich, Germany),
◦
UVA irradiation dose of 1.7 mW/cm2] with the temperature kept at 29–32 C. The cells were then
washed once again with DPBS and the medium was replaced with fresh DMEM without phenol red
and incubated for 18–22 h. After this incubation period, cells from both plates were washed with DPBS
and complete DMEM containing 50 µg/mL NR was added to each well and incubated for 3 h. Since
the NR could precipitate, NR solutions were prepared every second day and incubated overnight at
37 ◦C in a 5% CO2 atmosphere protected from light. Before the addition to the wells, the NR solution
was centrifuged at 1500 g for 10 min and filtered (5 µm). After this, the NR solution was removed, cells
were washed once with DPBS previously heated at 37 ◦C, and an NR desorb solution (50% ethanol/1%
acetic acid/49% distilled water) was added to extract the NR dye from the cells. For the reading
procedure, each plate was placed in a microplate shaker (BioTek, United States) for 10 min, at room
temperature and protected from light, to extract all the NR from the cells and obtain a homogeneous
solution. In this last step, the absorbance was measured at 540 nm. Within each plate, adequate solvent
controls were tested. Cell viability data obtained from each plate was expressed as the absorbance
ratio of treated to solvent control cells and was further used to estimate the IC50 values using linear
regression analysis. The PIF is the ratio between the IC50 value of the irradiated (Irr+) versus the IC50
value of the nonirradiated (Irr-) cells. According to the OECD 432 guideline, a PIF index < 2 predicts
the absence of a phototoxic effect, a PIF index between 2 and 5 predicts a probable phototoxic effect,
and a PIF > 5 predicts a phototoxic effect [34].
3.8. Statistical Analysis
The assays were carried out in triplicate. The results were expressed as mean values
deviations. The data was analyzed using a one-way ANOVA with Tukey’s test; p < 0.05 was considered
±
standard
statistically significant.
4. Conclusions
A series of five polyoxygenated xanthones were synthesized and their antioxidant activity was
evaluated by the DPPH assay. Further studies on their capacity to inhibit skin-degrading enzymes,
metal-chelating effect as well as the stability at different pH values led to one hit compound, 1,2-DHX
(1). Results of these studies indicated that 1,2-DHX (1) displays a dual ability to protect the skin against
UV damage by combining antioxidant/metal-chelating properties with UV-filter capacity, and revealed
to be more stable in the pH range that is closest to the pH of the skin, with no phototoxicity observed
in the concentration range tested. This study reinforces the safety and efficacy of a dihydroxyxanthone
as a potential antiaging compound paving the way to in vivo studies.
Author Contributions: M.E.d.S., I.F.A. and M.M.M.P. conceived and designed the experiments; M.E.d.S., D.I.S.P.R.,
and M.C.A. synthesized the compounds and elucidated their structure; M.C.A., C.D.P., and S.M.C. performed
the antioxidant activity assays, analyzed, and discussed the results with D.I.S.P.R. and I.F.A.; G.P.R. and A.K.-P.
performed the inhibitory activities of skin related enzyme assays, analyzed, and discussed the results with
M.d.C.B.; B.M. performed the phototoxicity assays, analyzed, and discussed the results with H.C. and I.A.;
D.I.S.P.R., M.C.A., and I.F.A. wrote the manuscript, while all authors gave significant contributions in discussion
and revision; M.E.d.S. and J.S.L. funding acquisition. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was supported by national funds through FCT, Foundation for Science and Technology,
within the scope of UIDB/04423/2020 and UIDP/04423/2020 under the project PTDC/SAU-PUB/28736/2017 (reference

Molecules 2020, 25, 2782
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POCI-01-0145-FEDER-028736), cofinanced by COMPETE 2020, Portugal 2020 and the European Union through
the ERDF and by FCT through national funds, as well as CHIRALBIOACTIVE-PI-3RL-IINFACTS-2019, and
supported by the Applied Molecular Biosciences Unit-UCIBIO which is financed by national funds from
FCT/MCTES (UID/Multi/04378/2019). Thanks are also due to FCT, the European Union, QREN, FEDER, COMPETE,
by funding cE3c center (Ref. UID/BIA/00329/2019) and Direção Regional da Ciência e Tecnologia (Azores
Government) by funding Azorean Biodiversity Group.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1-5 are available from the authors.
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