Photoprotective activity of resveratrol analogues

Article abstract of DOI:10.1016/j.bmc.2012.11.052

Resveratrol is a promising agent for protecting human skin from UV radiation and to reduce the occurrence of cutaneous malignancies. We describe the photoprotective activity of six resveratrol analogues using the diffuse transmittance technique to determine the SPF and the protection against UVA radiation. The analogues presented a varied profile of photoprotection, the SPF ranging from 2 to 10 and the UVAPF from 0 to 9. Among the six compounds tested, the protection against UVB sunrays provided by compound B was more significant than the protection provided by resveratrol; compounds C, D, E and F show photoprotection similar to resveratrol.

Full text of DOI:10.1016/j.bmc.2012.11.052

Bioorganic & Medicinal Chemistry 21 (2013) 964–968
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Photoprotective activity of resveratrol analogues
Hudson Caetano Polonini a, Larissa Lavorato Lima b, Karla Mara Gonçalves a,
Antônio Márcio Resende do Carmo c, Adilson David da Silva b, Nádia Rezende Barbosa Raposo a,
⇑
a Núcleo de Pesquisa e Inovação em Ciências da saúde (NUPICS), Universidade Federal de Juiz de Fora, Campus Universitário, Juiz de Fora 36036-900, Brazil
b Departamento de Química, Universidade Federal de Juiz de Fora, Campus Universitário, Juiz de Fora 36036-900, Brazil
c Faculdade de Odontologia, Universidade Federal de Juiz de Fora, Campus Universitário, Juiz de Fora 36036-900, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Resveratrol is a promising agent for protecting human skin from UV radiation and to reduce the occur-
rence of cutaneous malignancies. We describe the photoprotective activity of six resveratrol analogues
using the diffuse transmittance technique to determine the SPF and the protection against UVA radiation.
The analogues presented a varied profile of photoprotection, the SPF ranging from 2 to 10 and the UVAPF
from 0 to 9. Among the six compounds tested, the protection against UVB sunrays provided by compound
B was more significant than the protection provided by resveratrol; compounds C, D, E and F show photo-
protection similar to resveratrol.
Received 12 October 2012
Revised 26 November 2012
Accepted 30 November 2012
Available online 20 December 2012
Keywords:
Photoprotective activity
Sunscreening agents/UV filters
Chemical synthesis
Ó 2012 Elsevier Ltd. All rights reserved.
Ultraviolet radiation
Resveratrol, Structure–activity relationship
1. Introduction
It is therefore essential to develop products that protect the hu-
man skin against UVR, both UVA and UVB. Resveratrol is an impor-
Ultraviolet radiation (UVR) from the sun is known to be the
main trigger for numerous pathological problems, notably skin
cancer.1 This fact becomes more relevant when one considers that
this is the most common cancer diagnosed worldwide, and it can
evolve into its most serious complication, the melanoma, which
has a high mortality rate.2
tant antioxidant which shows a variable anticancer activity and
suppresses, retards or reverses the deleterious effects of UV radia-
tion.7,8 Within this context, the present work aimed at developing
six analogues, which could act as sunscreening agents/UV filters,
and at determining their photoprotective activity.
UVR can be divided into three distinct regions: ultraviolet A
(UVA, 315–400 nm), ultraviolet B (UVB, 280–315 nm) and ultravi-
olet C (UVC, 100–280 nm). It is currently known that both UVA and
UVB are linked to cancer pathogenesis, because of the damage to
cellular DNA they can cause, either alone or synergistically.3,4 In
fact, it is estimated that at minimum 10% of all new cancer cases
would be prevented if people made proper and continuous use of
sunscreens.1 Yet, 78% of non-melanoma skin cancers are prevent-
able, when one uses these products correctly.5
2. Experimental
2.1. Chemicals
Analytical-grade glycerin (Vetec, Brazil) and ultrapure water
(18.2 MX cm) obtained from an aquaMAX–Ultra 370 Series water
purification system (YoungLin, Korea) were used throughout the
analysis. Square-shaped (50 ꢀ 50 mm) polymethylmethacrylate
(PMMA) Helioplate™ HD6 (HelioScreen, France) plates with rough-
The efficacy of such products is dependent on their capacity to
absorb radiant energy, which is proportional to the concentration
of active absorbing molecules, and to the absorption range of the
wavelength within which their maximum absorption occurs.
When using a combination of UVA and UVB filters, broad-spectrum
protection to the skin takes place.6
ened surface on one side (Sa ꢁ 6
lm) were used as the substrate
for the determination of photoprotection activity by diffuse trans-
mittance spectrophotometry.
2.2. Equipments
The in vitro photoprotection experiments were conducted in an
UV-2000S Ultraviolet Transmittance Analyzer (Labsphere, USA),
composed of two photodiode array spectrographs and equipped
with an integrating sphere and a xenon flashlamp, which emits a
⇑
Corresponding author.
E-mail address: hudson_polonini@hotmail.com (N.R.B. Raposo).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.052

H. C. Polonini et al. / Bioorg. Med. Chem. 21 (2013) 964–968
965
Table 1
continuous spectrum of radiation with no peaks and supplies en-
Spectral data of resveratrol analogues
ergy for a 290–450 nm spectral range, with a wavelength incre-
ment step of 1 nm, having low irradiance such that the
photostability of the product is not unduly challenged.
ꢀ
rnu C@N Melting point (°C) Yield (%)
Compounds d CH@N d C@N
A
B
C
D
E
F
8.90
8.62
8.96
8.50
8.69
8.63
157.18 1625.8
158.46 1622.0
160.77 1631.6
158.84 1614.3
159.73 1625.8
158.87 1623.9
160.0–161.0
90.1–92.0
149.0–150.3
118.0–119.0
90.8–91.5
78.0
66.0
90.0
50.0
83.0
72.0
A
long-arc xenon SuntestTM insolator, type CPS+ (Atlas,
Germany), filtered with its original UV short cut-off filter combined
with a Special UV Glass filter (limiting radiation at approximately
290 nm), providing a VIS + UVA + UVB spectrum, was used as the
artificial UV source for irradiation of the sunscreen samples. PMMA
plates were supported firmly throughout the irradiation by a Sun-
Tray holder which also provided a dark background behind each
plate to reduce the risk of any back exposure.
118.7–119.6
⁄
The NMR experiments were performed at 300 MHz for 1H and 75 MHz for 13C in
dimethyl sulfoxide (DMSO-d6) (ppm) and I.R. experiments were performed at KBr
support (cmꢂ1). The 1H NMR of compound B is available as Supplementary data, to
confirm the purity of the material.
An electronic analytical balance AY-220 (Shimadzu, Japan) and
a positive-displacement manual pipette (Mettler Toledo, USA)
were used for the preparation of the samples PMMA plates.
450 nm at 1 nm intervals on 9 different sites of each plate (total
measurement area = 2.0 cm2). The blank was prepared using the
HD6 plates covered with 15-lL of glycerin, because of its non-
2.3. Samples
fluorescence and UV transparency.
Six analogs of resveratrol were synthesized in the Laboratory of
Chemistry of the Federal University of Juiz de Fora (Juiz de Fora,
Minas Gerais, Brazil). As a reference standard, resveratrol (trans-
resveratrol 99.0%, Gamma, Brazil) was used. The resveratrol ana-
logues A–F were synthesized by the classical method of imine for-
mation involving condensation between aromatic amine (2-
aminophenol) with a variety of aromatic aldehydes in ethanol
(Scheme 1). All compounds were characterized by 1H and 13C nu-
clear magnetic resonance (NMR), infrared (IR) and melting point
(mp) (Table 1) and were in accordance with data in the literature.
The analogues were incorporated in a cosmetic neutral lotion at
15%, and then subjected to photoprotection assay.
Using the generated data, SPFin vitro was calculated using Eq. 1:
R
k¼400nm EðkÞ ꢀ IðkÞ ꢀ dk
k¼290nm
SPFin vitro
¼
ð1Þ
R
k¼400nm
k¼290nm
EðkÞ ꢀ IðkÞ ꢀ 10ꢂA ðkÞ ꢀ dk
0
where E(k) is the erythema action spectrum,10 I(k) is the spectral
irradiance of the UV source, A0(k) is the mean monochromatic
absorbance measurements per plate of the test product layer before
UV exposure, and dk is the wavelength step (1 nm).
In order to generate the UVAPF value, the coefficient of adjust-
ment ‘C’ was calculated as shown in Eq. 2 and using the SPF label as
the value generated by the UV-2000’s software.
SPFin vitro;adj ¼ SPF label
R
k¼400nm EðkÞ ꢀ IðkÞ ꢀ dk
2.4. Photoprotection assay
k¼290nm
¼
ð2Þ
R
k¼400nm
k¼290nm
EðkÞ ꢀ IðkÞ ꢀ 10ꢂA
ꢀ dk
0ðkÞꢀC
The samples were accurately and quickly weighed (to reduce
product evaporation and dryness) to satisfy the application rate
of 1.3 mg cmꢂ2 in each PMMA plate (actual quantity applied:
32.5 mg, determined by weighing the plates before and immedi-
ately after applying the products). They were directly weighed on
the plate surface, applied as a large number of small droplets of
approximately equal mass, and distributed in an even manner on
the roughened surface of the plate. Then, the products were spread
over the whole surface with a fingertip covered with a vinyl glove
and pre-saturated with the product, to prevent possible losses of
the amount weighed. The spreading was achieved in two steps:
(i) quick distribution of the product, without pressure (20–30 s);
and (ii) rubbing it into the rough surface using pressure (20–30 s
too). For each product, three plates were prepared, which were
kept protected from light exposure in a dark chamber at room tem-
perature (ꢁ20 °C) for 15 min, in order to facilitate the formation of
a standard stabilized sunscreen film.
Using the ‘C’ value, initial UVAPF was calculated using Eq. 3, and
the dose ‘D’ of UV irradiation was determined by Eq. 4.
R
k¼400nm PðkÞ ꢀ IðkÞ ꢀ dk
k¼320nm
UVAPF ¼
ð3Þ
ð4Þ
R
0
k¼400nm
k¼290nm
PðkÞ ꢀ IðkÞ ꢀ 10ꢂA
ꢀ dk
0ðkÞꢀC
D ¼ UVAPF0 ꢀ D0
where P(k) is the PPD action spectrum10 and D0 = 1.2 J cmꢂ2
.
The plates were inserted into the UV irradiation source (tem-
perature maintained below 40 °C) and then exposed to the calcu-
lated UV dose D. After that, new transmission measurements of
the sunscreen samples were conducted, for acquisition of the sec-
ond UV spectrum. The final UVAPF was calculated according to Eq.
5. If the coefficient of variation (CV) between the UVAPF’s of the
individual plates exceeded 20%, then further plates were measured
until the CV threshold was achieved.
After this period, the plates containing the product were placed
in the light-path of the transmittance analyzer. The transmission of
UV radiation through the sample was measured from 290 to
R
k¼400nm PðkÞ ꢀ IðkÞ ꢀ dk
k¼320nm
UVAPF ¼
ð5Þ
R
k¼400nm PðkÞ ꢀ IðkÞ ꢀ 10ꢂAðkÞꢀC ꢀ dk
k¼290nm
NH2
A. R3 = NO2
R
O
2
5
HO
B. R3 = OMe
3
1
OH
5
4
1
2
N
C. R2 = OH
4
R
3
D. R3 = NMe2
E. R = H
Aromatic
EtOH
Aldehydes
F. R2 = R3 = R4 = OMe
Scheme 1. Synthetic pathway for resveratrol analogues.

966
H. C. Polonini et al. / Bioorg. Med. Chem. 21 (2013) 964–968
where A(k) is the mean monochromatic absorbance of the test prod-
uct layer after UV exposure.
analyzed, in the hopes of identifying which ones could be used to
minimize its harmful effects on the human skin. The first substance
used with that purpose was the acidified quinine sulfate, by Wid-
mark in 1889. The same substance was incorporated in lotions and
ointments by Hammer in 1891, determining what today is consid-
ered the first chemical sunscreen in history.11 Since then, several
sunscreens were developed and commercially launched, evolving
into the products currently available.
For calculation of the critical wavelength value (kc), a series of
absorbance values were calculated for each of the three separate
plates to which the samples were applied. Absorbance at each
wavelength increment A(k) was calculated using Eq. 6, and the kc
using Eq. 7.
Ak ¼ logðCk=PkÞ
ð6Þ
The protection level against solar radiations provided by sunsc-
reens are currently represented by the Sun Protection Factor (SPF),
which is understood as a measure of the UV solar energy required
to produce sunburn on a skin area (generally on the back) pro-
tected with a sunscreen product, relative to the amount of solar en-
ergy required to produce the same sunburn on an unprotected skin
area. Alongside the in vivo methods, several studies were and have
been carried out in an attempt to elaborate and standardize an
in vitro method for the determination of the SPF. In vitro photopro-
tection studies are currently considered of utmost importance to
elucidate the effective performance of candidates for active sun-
screen ingredients or final products against UVR, before the more
expensive in vivo tests.12 They are also a safety issue, as only posi-
tive in vitro responses validate the submission of products to
in vivo experiments, virtually eliminating any risks of those tests
to human volunteers.13,14 In the present work, we used the diffuse
transmittance in vitro technique to determine the photoprotection
profile of six resveratrol analogues, because it can determine not
only the SPF, but also the protection against UVA radiation
(UVAPF), which is important since its role on the skin cancer path-
ogenesis is currently known.
where
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
n
n
Ck ¼ ðCk½1ꢃ ꢀ Ck½2ꢃ ꢀ ꢄꢄꢄ ꢀ Ck½nꢃÞ and Pk ¼ ðPk½1ꢃ ꢀ Pk½2ꢃ ꢀ ꢄꢄꢄ ꢀ Pk½nꢃÞ.
Z
Z
kc
400nm
Ak ꢀ dk ¼ 0:9
Ak ꢀ dk
ð7Þ
290nm
290nm
Finally, the UVA/UVB ratio was calculated as the ratio between
the final UVAPF and the SPF label.
The verification of the validity of the results was obtained using
the Cosmetics Europe Reference Sunscreen S2 (determined
SPF = 18 1.5, UVAPF = 12 1.1, kc = 381 nm, and UVA/UVB ra-
tio = 0.88). All results were expressed as a mean of 27 determina-
tions (3 plates, 9 readings each, at different sites) for lotions
containing 15% of each resveratrol analogue in isolation.
3. Results and discussion
Since the discovery of the UVR spectrum by Ritter, in 1801, and
of the sunlight role on skin burn by Sir Everard Home, in 1820,
many substances had their absorption potential for such radiation
Table 2
Photoprotection results of resveratrol analogues
Substance
SPF
UVAPF
—
kc (nm)
UVA/UVB ratio
—
OH
OH
OH
Analogue A
2
0.5
—
N
N
N
NO2
OMe
Analogue B
Analogue C
10 0.2
9
6
0.7
1.0
383
389
0.74
1.00
6
0.7
HO
OH
OH
OH
Analogue D
Analogue E
5
6
0.3
1.1
8
6
1.8
0.6
388
387
1.21
0.89
N
N
N
N
OMe
OMe
Analogue F
Resveratrol
6
7
1.1
1.7
6
2
0.3
0.2
387
362
0.90
0.46
OMe
OH
HO
OH
SPF: Sun Protection Factor. UVAPF: UVA Protection Factor.

H. C. Polonini et al. / Bioorg. Med. Chem. 21 (2013) 964–968
967
Table 3
Boots Star Rating criteria for classification of UVA protection in sunscreens
Initial mean UVA/UVB ratio
0.0–0.59
0.6–0.79
0.8–0.89
0.89 and over
Post exposure mean UVA/UVB ratio
0.0–0.56
No rating
No rating
No rating
No rating
0.57–0.75
0.76–0.85
0.86 and over
No rating
No rating
No rating
⁄ ⁄ ⁄
⁄ ⁄ ⁄
⁄ ⁄ ⁄
⁄ ⁄ ⁄
⁄ ⁄ ⁄
⁄ ⁄ ⁄ ⁄
⁄ ⁄ ⁄ ⁄
⁄ ⁄ ⁄ ⁄
⁄ ⁄ ⁄ ⁄ ⁄
Resveratrol was chosen as a model molecule as it is an impor-
tant antioxidant that shows a variable anticancer activity, includ-
ing on the skin cancer spectrum. Additionally, resveratrol
suppresses, retards or reverses the deleterious effects of UV radia-
tion.7,8 Unfortunately, this substance shows problems such as
instability as it converts to the cis-form (a less active form), partic-
ularly after exposure to UV light.15–17 According to Walle and col-
leagues, methylation of the polyphenols effectively blocks
metabolic conjugation reactions, thereby dramatically increasing
stability.17
of the spectrum, however, can be different according to the refer-
ence adopted. For instance, the US Food and Drug Administration
(FDA)21 classifies products on a scale consisting of five numerical
categories: 0 (kc < 325 nm), 1 (325 6 kc < 335), 2 (335 6 kc < 350),
3 (350 6 kc < 370), and 4 (370 6 kc). Under such classification, all
the analogues (except A) were rated ‘4’, the highest category. Res-
veratrol, in turn, is category ‘3’, and this ratifies the improvement
in the protection produced by the modifications in the chemical
structure. Another possible classification is the one created by
Springsteen et al.,22 who classifies broad-spectrum sunscreen
products as those which have a kc value greater than 370 nm. Thus,
the same, previously mentioned analogues may be considered
broad spectrum products by this classification, offering protection
against UVA and UVB.
Finally, we have determined the UVA/UVB ratio, which provides
a good idea of which UV region is better blocked by the substances.
Again, the analogues had a greater value than the original com-
pound. The UVA/UVB ratio can also be used to provide the so-
called Boots Star Rating,23 which classifies the products into cate-
gories from 0 to 5 stars. Such classification should be done accord-
ing to Table 3.
Based on: (i) the concept of bioisosterism,19 the basic skeleton
of trans-stilbene was modified by replacing the central C@C link-
age with a C@N double bond; (ii) the knowledge that the products
presented significant antioxidant activity in a previous work;20 and
(iii) the fact that resveratrol has activity as a skin anticancer agent,
in this work we proposed six resveratrol analogues using hydroxyl
at position 2 of the aromatic ring and several group substituents.
The six analogues presented a varied profile of photoprotection,
the SPF ranging from 2 to 10 and the UVAPF from 0 to 9, as shown
in Table 2.
As one can see from Table 2, all the molecules, resveratrol and
its analogues, presented a significant photoprotection for a single
UV-filter substance, except for analogue A. Their SPF, that is, pro-
tection against UVB sunrays, was similar, but the activity in ana-
logue B was more significant than in resveratrol itself. This
demonstrates that the presence of a 4-methoxy group (electron
donating group) at the para position is relevant. On the other hand,
the presence of a nitro group (electron withdrawing) at the para
position of the aromatic ring of compound A considerately dimin-
ishes the value of photoprotection. One can also consider that the
compounds B (R = –OCH3) and D [R = –N(CH3)2] show similar
chemical structure, which would lead to similar SPF profiles, but
we must consider that there are differences between oxygen-based
compounds and nitrogen-based ones. For instance, amines have a
very ‘active’ lone pair, that is, they are much more basic, and this
basicity can influence the activity of hydrogen bonds [OꢂHꢄ ꢄ ꢄ:N
(29 kJ/mol or 6.9 kcal/mol) and OꢂHꢄ ꢄ ꢄ:O (21 kJ/mol or 5.0 kcal/
mol)], therefore possibly explaining the difference in the final
UVB absorption by these molecules.
Analogues from B to F also presented very similar protection
against UVA rays (although compound B is little more effective
than C–F), and it is important to highlight that the structural mod-
ifications led to a very significant improvement in activity in this
solar spectrum range, as the UVAPF increased from 2 in resveratrol
to 6–8 in the referred analogues (three-fold greater than the origi-
nal compound). This is of sheer relevance, as UVA rays are known
to play an important role on skin cancers, and the analogues were
more active in the protection from this type of radiation.
The Boots Star Rating is important to determine the stability of
the generated photoprotection values, since the components of the
sunscreens may degrade. Analogues C–F are, therefore, 5 stars; B is
3 stars; and resveratrol receives no stars, because its protection is
mainly focused on the UVB region.
As a final consideration, it is important to highlight that the
photoprotective activity achieved by those molecules is due to
the fact that they were capable of absorbing the short wavelength,
high-energized UV rays, and converting them into less energetic
radiations of the infrared region.24,25 This occurs because the UV
photon absorbs enough energy to cause the transfer of electrons
to a more energetic orbital in the molecule which contains this
chromophore group,25 that is, the absorption of UV leads to the
excitation of electrons found in orbital
p HOMO and their subse-
quent transference to the
p
ꢅ LUMO orbital.24
4. Conclusions
In conclusion, one can infer that the structural modifications
among the analogues B–F were of great value, as they played a role
in increasing the photoprotection of the original compound, resve-
ratrol, mainly in the UVA region, one of the primary causes for the
emergence of skin cancers.
Acknowledgments
This research was supported by CNPq, CAPES, FAPEMIG and
PROPESQ/UFJF.
Critical Wavelength is another parameter determined to mea-
sure the UVA protection, being defined as the wavelength at which
the integral of the area under the absorption spectrum of the sam-
ple reaches 90% of the total absorption, from 290 to 400 nm,10 and
thus the protection spectrum is measured (sunscreens with kc val-
ues near 400 nm are considered broad spectrum). The classification
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.11.052.

968
H. C. Polonini et al. / Bioorg. Med. Chem. 21 (2013) 964–968
14. Velasco, M. V. R.; Sarruf, F. D.; Salgado-Santos, I. M. N.; Haroutiounian-Filho, C.
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