Influence of hydroxypropyl-β-cyclodextrin on photo-induced free radical production by the sunscreen agent, butyl-methoxydibenzoylmethane

Article abstract of DOI:10.1211/002235702207

The aim of the study was to investigate the effect of hydroxypropyl-β-cyclodextrin (HP-β-CD) on the photo-induced production of free radicals by the sunscreen agent, butyl-methoxydibenzoylmethane (BMDBM). Spin-trapping/electron paramagnetic resonance spectroscopy was used to evaluate the formation of radicals and the extent of BMDBM photodegradation was measured by high-performance liquid chromatography. The stable 2,2,6,6-tetramethylpiperidine-1-oxyl, nitroxide radical (TEMPO) was used as spin-trap. Any free radicals generated during irradiation of the sunscreen agent will couple with the TEMPO radicals giving diamagnetic species and thus a decrease of the signal intensity in the electron paramagnetic resonance spectrum. Following 2-h illumination with simulated sunlight, the solution containing free BMDBM exhibited a 93.9% decrease of the intensity of the TEMPO signal. Under the same irradiation conditions, only a 12.2% reduction of the TEMPO concentration was measured in the sample containing BMDBM complexed with HP-β-CD. Moreover, the decrease of the spin-trap level observed for the HP-β-CD/BMDBM complex was not significantly different from that produced when solutions containing TEMPO only or TEMPO in the presence of HP-β-CD alone were subjected to irradiation. In addition, the photodegradation of the sunscreen agent was reduced by complexation with HP-β-CD (the extent of degradation was 27.6% for the complex compared with 63.1% for free BMDBM). The results obtained indicate that the free radicals generated by BMDBM when exposed to simulated sunlight are effectively scavenged by inclusion complexation of the sunscreen agent with HP-β-CD.

Full text of DOI:10.1211/002235702207

Journal of
JPP 2002, 54: 1553–1558
# 2002 The Authors
Received March 14, 2002
Accepted July 18, 2002
DOI 10.1211/002235702207
ISSN 0022-3573
In uence of hydroxypropyl-b-cyclodextrin on photo-
induced free radical production by the sunscreen agent,
butyl-methoxydibenzoylmethane
Santo Scalia, Silvia Simeoni, Andrea Barbieri and Silvana Sostero
Abstract
The aim of the study was to investigate the effect of hydroxypropyl- -cyclodextrin (HP- -CD) on the
photo-induced production of free radicals by the sunscreen agent, butyl-methoxydibenzoylmethane
(BMDBM). Spin-trapping/electron paramagnetic resonance spectroscopy was used to evaluate the
formation of radicals and the extent of BMDBM photodegradation was measured by high-
performance liquid chromatography. The stable 2,2,6,6-tetramethylpiperidine-1-oxyl, nitroxide
radical (TEMPO) was used as spin-trap. Any free radicals generated during irradiation of the sunscreen
agent will couple with the TEMPO radicals giving diamagnetic species and thus a decrease of the sig-
nal intensity in the electron paramagnetic resonance spectrum. Following 2-h illumination with simu-
lated sunlight, the solution containing free BMDBM exhibited a 93.9% decrease of the intensity of
the TEMPO signal. Under the same irradiation conditions, only a 12.2% reduction of the TEMPO
concentration was measured in the sample containing BMDBM complexed with HP- -CD. Moreover,
the decrease of the spin-trap level observed for the HP- -CD/BMDBM complex was not signiŽ cantly
different from that produced when solutions containing TEMPO only or TEMPO in the presence of HP-
-CD alone were subjected to irradiation. In addition, the photodegradation of the sunscreen agent
was reduced by complexation with HP- -CD (the extent of degradation was 27.6% for the complex
compared with 63.1% for free BMDBM). The results obtained indicate that the free radicals
generated by BMDBM when exposed to simulated sunlight are effectively scavenged by inclusion
complexation of the sunscreen agent with HP- -CD.
Introduction
It is well recognised that exposure of human skin to sunlight UV radiations
(290±400 nm) leads to various types of skin pathologies (National Institute of Health
1989) including acute in¯ ammatory responses (i.e., erythema, oedema) and chronic
eŒects such as actinic aging, immune suppression and cutaneous cancers (National
Institute of Health 1989 ; Ziegler et al 1994 ; Serre et al 1997 ; Urbach 1997 ; Tarras-
Wahlberg et al 1999). Awareness of the harmful eŒects of solar UV rays has promoted
the widespread use of topical photoprotective preparations (National Institute of
Health 1989 ; Green et al 1999). These products incorporate UV-absorbing organic
chemicals which lessen the dose of sunlight UV radiations reaching human skin.
The photoactivated sunscreen molecule dissipates the excitation energy by several
mechanisms, including thermal energy, ¯ uorescence, phosphorescence, interaction with
neighbouring molecules or photochemical reactions (Damiani et al 1999 ; Stokes &
DiŒey 1999). Although the shorter-wavelength portion (UV-B, 290±320 nm) of the
solar UV spectrum reaching the earth surface is regarded as the most deleterious
(National Institute of Health 1989), protection against the longer-wavelength UV
radiations (UV-A, 320±400 nm) by sunscreens has become very important due to
increasing evidence about their harmful eŒects on human skin, such as photoaging and
photocarcinogenesis (National Institute of Health 1989 ; Schwack & Rudolph 1995 ;
Tarras-Wahlberg et al 1999 ; Damiani et al 2000).
Department of Pharmaceutical
Sciences, University of Ferrara,
Via Fossato di Mortara 17,
44100 Ferrara, Italy
Santo Scalia, Silvia Simeoni
Institut for Organic Synthesis
and Photoreactivity, C.N.R.-
I.S.O.F. (FE), c/o Department of
Chemistry, University of Ferrara,
Via Borsari 46, 44100 Ferrara,
Italy
Andrea Barbieri, Silvana Sostero
Correspondence: S. Scalia,
Dipartimento di Scienze
Farmaceutiche, via Fossato di
Mortara 17, 44100 Ferrara, Italy.
E-mail: sls!unife.it
Butyl-methoxydibenzoylmethane (BMDBM; Figure 1A) is the most e cient and
frequently used UV-A ®lter (Roscher et al 1994 ; Schwack & Rudolph 1995 ;
1553

1554
Santo Scalia et al
Hayden et al 1998 ; Tarras-Wahlberg et al 1999 ; Damiani Japan) linked to an injection valve with a 20- L sample
et al 2000). It is approved by the regulatory authorities of loop (Model 7125 ; Rheodyne, Cotati, CA). The detector
Europe (EEC Directive 1976), USA (US Food and Drug was set at 350 nm. Data acquisition and processing were
Administration 1999), Japan and Australia (Hayden et al accomplished with a personal computer using Borwin
1998). However, one drawback of this sunscreen agent is software (JBMS Developpements, Le Fontanil, France).
its lack of photostability. Published studies have demon- Sample injections were eŒected with a Model 80365 syringe
strated that BMDBM undergoes decomposition under (10 L; Hamilton, Bonaduz, Switzerland). Separations
solar-simulated radiation, leading to a decrease of its were performed on a 5- m Nucleosil C18 column
UV-protective capacity (Stokes & DiŒey 1999 ; Tarras- (150 4 6 mm i.d. ; Macherey-Nagel, Duren, Germany)
¬ .
Wahlberg et al 1999). In addition, the photo-induced ®tted with a guard column and eluted isocratically, at a
1
-
modi®cation of the sunscreen agent yields an array of ¯ ow-rate of 1 mL min
with methanol±acetonitrile±
}
breakdown products (Roscher et al 1994 ; Schwack & tetrahydrofuran±water (60:15:10:15, v v). The mobile
Rudolph 1995 ; Tarras-Wahlberg et al 1999) whose toxi- phase was deaerated on-line by a Model ERC-3311 auto-
cological properties are unknown and which have been matic solvent degasser (Erma, Tokyo, Japan). The identity
associated to photocontact sensitization to BMDBM of the BMDBM peak was assigned by co-chromatography
(Parry et al 1995 ; Schmidt et al 1998). Reports from with the authentic standard. Quanti®cation was carried
Schwack & Rudolph (1995) and Damiani et al (1999, 2000) out by integration of the peak areas using the external
have demonstrated that BMDBM generates carbon- standardization method.
centred free radicals when illuminated with simulated sun-
light and, as a consequence, causes in-vitro strand breaks
in DNA (Damiani et al 1999) and oxidative modi®cations
in bovine serum albumin (Damiani et al 2000). Hence,
these results indicate that the most dangerous eŒect of the
photolability of this sunscreen agent is the radiation-
induced free radical formation rather than the reduction in
screening e ciency.
In an earlier investigation (Scalia et al 1998), we demon-
strated that the photodegradation of BMDBM was reduced
by inclusion complexation of the sunscreen agent with
hydroxypropyl- -cyclodextrin (HP- -CD). This study has
now been extended to evaluate the eŒect of HP- -CD as
complexing agent on the photo-induced production of free
Preparation of the inclusion complex
The inclusion complex was prepared at a 1:2 molar ratio of
BMDBM to HP- -CD. The complex was obtained by
.
adding 3 5 mL of puri®ed water containing 344 6 mg
(0 250 mmol) of HP- -CD to a solution of BMDBM
.
.
.
.
.
(38 8 mg, 0 125 mmol)in methanol (4 0 mL). The obtained
mixture was maintained under stirring for 24 h at room
temperature and shielded from light. The solvent was then
°
evaporated under vacuum at 40 C by rotary evaporation
and the residue was kept in a desiccator until used. The
content of BMDBM in the complex was determined by
HPLC after proper dilution.
}
radicals by BMDBM. Spin-trapping electron paramag-
netic resonance spectroscopy was used for the detection of
radical formation.
Thermal analysis
DiŒerential thermal analysis (DTA) and thermal gravi-
metric analysis (TGA) were carried out on a Netzsch STA
409 simultaneous thermal analyser (Netzsch Italiana,
Verona, Italy). The samples (6±7 mg) were accurately
weighed in platinum pans (Netzsch) and heated from 30 to
Materials and Methods
Materials
1
-
130 C, at a scanning rate of 10 C min .
°
°
Butyl-methoxydibenzoylmethane (BMDBM) was supplied
by HoŒmann-La Roche Ltd (Geneva, Switzerland).
Hydroxypropyl- -cyclodextrin (HP- -CD; average molar
X-ray diffractometry
.
substitution 0 6) was purchased from Aldrich Chimica
(Milan, Italy). Methanol, acetonitrile and water were
HPLC grade from Merck (Darmstadt, Germany). 2,2,6,6-
Tetramethylpiperidine-1-oxyl, nitroxide radical (TEMPO;
Figure 1B)was used without further puri®cation as received
from Fluka Chemie (Buchs, Switzerland). All other
chemicals were of analytical-reagent grade (Sigma, Milan,
Italy).
X-ray diŒraction patterns were recorded on a D 5000
powder diŒractometer (Siemens, Munich, Germany) using
a voltage of 45 kV and a current of 25 mA for the generator,
with Cu anode material. The wavelength of the graphite-
.
monocromated radiation was 1 5406 A. The diŒracto-
grams were recorded from 3 (2 ) to 50 (2 ) at an angular
°
°
°
speed of 1 (2 ) per minute using 1-1-1-0 15 slits.
.
°
High-performance liquid chromatography
UV spectrophotometry
The HPLC apparatus consisted of a modular chromato- UV spectra of BMDBM alone or its complex with HP- -
}
graphic system (Model 980-PU pump and Model 975-UV CD were recorded in ethanol on a UV VIS NIR Spec-
variable wavelength UV-Vis detector; Jasco, Tokyo, trometer (Lambda 19; Perkin Elmer, Norwalk, USA).
}

1555
Hydroxypropyl- -cyclodextrin effect on free radical formation
by comparing the peak areas of BMDBM from the
irradiated samples with those obtained by analysis of an
equivalent amount of the non-exposed preparation.
Statistical analysis
Statistical analyses were performed by analysis of variance
to assess the signi®cance of diŒerences between the sets
A
P
of data from the spin-trap EPR measurements. A
-
!
.
value 0 05 was considered signi®cant. All computations
were carried out using the statistical software GraphPad
Instat (GraphPad Software, San Diego, CA).
B
Results and Discussion
Figure 1 Chemical structures of butyl-methoxydibenzoylmethane
(A) and 2,2,6,6-tetramethylpiperidine-1-oxyl, free radical (B).
In an earlier study (Scalia et al 1998) we reported that the
photodegradation of BMDBM in solution was signi®cantly
.
.
reduced (from 70 4% to 49 2% ) by complexation with
HP- -CD. To investigate the in¯ uence of this system on
the production of free radicals by the UV-irradiated
EPR measurements
}
sunscreen agent, the HP- -CD BMDBM complex was
Electron paramagnetic resonance (EPR) measurements
were performed at room temperature on a Bruker EMX
spectrometer (Bruker, Karlsruhe, Germany) operating in
prepared as previously described (Scalia et al 1998) and
characterized using X-ray diŒraction and DTA. The ab-
sence of both the BMDBM crystalline peaks in the X-ray
diŒraction pattern and the UV-®lter melting peak in the
DTA thermogram (data not shown) provided evidence of
the inclusion of BMDBM into the HP- -CD cavity. In
addition, UV spectrophotometric analysis of BMDBM
and its complex with HP- -CD showed that the shape of
the spectrum and the degree of UV absorption of the
sunscreen agent (BMDBM speci®c absorbance at 356 nm,
.
the X band (9 71 GHz) equipped with a TE 201 resonator
(Bruker OR 4104, 100% optical transmittance), using
2 mW microwave power, 1 G modulation amplitude and
100 KHz ®eld modulation.
Photodegradation studies
}
Photodecomposition experiments were carried out in 5% 1132 ; BMDBM HP- -CD complex speci®c absorbance at
}
m
(v v) ethanol±water solutions containing 10
TEMPO 356 nm, 1138) werenot aŒected by complexation. Solutions
free or cyclodextrin-complexed BMDBM. containing BMDBM alone or complexed with HP- -CD
After thorough degassing with freeze-pump-thaw tech-
nique, 1 mL of the test sample was transferred via cannula of photolysis was followed, for the same sample, by spin-
m
and 100
were exposed for 2 h to simulated sunlight and the course
}
under Ar atmosphere into a quartz ¯ at cell and inserted trapping EPR measurements and HPLC determination of
into the microwave cavity of the EPR spectrometer. The the extent of sunscreen degradation. During the light-
samples were ¯ uxed with nitrogen stream and irradiated stability measurements, the applied UV energy corre-
directly in the cavity with a 350-W medium pressure Hg sponded to 20 Minimal Erythemal Doses (MED) which is
lamp ®tted with focusing lens. Irradiation wavelengths
were selected using an Oriel 59812 band pass ®lter Wahlberg et al 1999). The formation of radicals was
considered comparable with a daily solar emission (Tarras-
!
!
410 nm) coupled with an Oriel IR-block ®lter detected using TEMPO stable nitroxide free radical as
(290
to avoid thermal eŒects. The solar simulator emission was spin-trap. Any carbon-centred radicals generated during
measured by a Goldlux radiometer (Oriel Corporation, sunscreen irradiation will couple with the unpaired electron
2
USA) and was maintained at about 0 4 mW cm and ofthe nitroxide radical (Figure 1B)giving nonparamagnetic
-
.
2 5 mW cm for UV-B and UV-A, respectively. Samples
2
-
.
species and thus producing a decrease of its EPR signal.
weresubjected to 2-h illumination and whole EPR nitroxide Preliminary photodegradation studies performed in pure
radical spectra were recorded every 5 min. Concentration methanolic solutions indicated that this solvent interfered
values of remaining TEMPO were obtained from double with EPR measurements and consequently 5% ethanol in
integration of the spectra. Each batch of experiments was water was used for all subsequent experiments. In the
repeated at least three times. After the exposure interval, solution containing free BMDBM, the intensity of the
the ¯ at cell was removed and its content quantitatively
EPR signals showed a marked decrease (Figure 2A) with
.
transferred into a 10-mL calibrated ¯ ask, diluted to volume 93 9% of the nitroxide radical lost after 2 h (Figure 3). This
with methanol and assayed by HPLC for BMDBM. All indicated the formation of carbon-centred free radicals
samples were protected from light both before and after during illumination of the sunscreen agent with simulated
irradiation. The degree of photodegradation was measured sunlight, in accordance with the data reported by Schwack

1556
Santo Scalia et al
A
10
8
6
4
2
0
–2
–4
–6
–8
–10
3390 3400 3410 3420 3430 3440 3450
Magnetic field (G)
B
10
8
6
4
2
0
–2
–4
–6
–8
–10
3390 3400 3410 3420 3430 3440 3450
Magnetic field (G)
}
Figure 2 Evolution of TEMPO electron paramagnetic resonance (EPR) spectra in time in the presence of BMDBM (A) or BMDBM
HP- -CD complex (B). Spectra were recorded every 5 min during 2-h irradiation with simulated sunlight.

1557
Hydroxypropyl- -cyclodextrin effect on free radical formation
100
75
50
25
0
0
30
60
90
120
Time (min)
m
m
Figure 3 Percent consumption of TEMPO (initial concn, 10
) during 2-h irradiation in the presence of BMDBM (E; 100
);
BMDBM); BMDBM HP- -CD physical
m
HP- -CD); HP- -CD (U; 200 ); ¬, solvent only. Each point represents the mean³s.d. of at least
}
}
.
m
BMDBM HP- -CD inclusion complex (+; 3 4 mg in 10 mL, giving a ®nal concentrationof 100
m
m
mixture (_; 100
three experiments.
BMDBM, 200
& Rudolf (1995) and Damiani et al (1999). The pro®le of
the EPR signal versus time obtained for uncomplexed
BMDBM follows a two-state time course (Figure 3), the
decrease of the TEMPO concentration being satisfactorily
described, in the 40±120 min interval, by a single expo-
nential decay (r2 0 996).
¯ .
Under the same irradiation conditions, the sample con-
taining BMDBM complexed with HP- -CD exhibited only
a small decrease of the EPR peak heights (Figure 2B)
.
corresponding to a 12 2% loss of TEMPO radical after 2 h
(Figure 3). The reduction of the spin-trap concentration
}
observed for the HP- -CD BMDBM complex was not sig-
ni®cantly diŒerent (analysis of variance : F 2 3, df 7,
¯ .
¯
"
P
.
0 05) from that produced when solutions containing
only the nitroxide radical or the nitroxide in the presence of
HP- -CD alone were subjected to irradiation (Figure 3).
Futhermore, the EPR spectrum features (g-factor, hyper-
®ne coupling constants and line broadening) were not
aŒected by the presence of the cyclodextrin. Therefore the
possible inclusion of the nitroxide radicals into the cyclo-
dextrin cavity can be ruled out. Additional measurements
Figure 4 Primary photochemical process in the photodegradation
of BMDBM.
were performed on samples containing uncomplexed the variation was not as marked as in the case of free
BMDBM and HP- -CD (physical mixture) at the same BMDBM. The diŒerence between free BMDBM and its
concentrations used in the previous tests. A decline of the physical mixture with HP- -CD can be probably traced to
.
EPR signals (52 7% decrease after 2 h) was observed on the gradual formation of the complex through an equi-
UV illumination of the physical mixture (Figure 3), though librium process which is established in solution between

1558
Santo Scalia et al
Damiani, E., Carloni, P., Biondi, C., Greci, L. (2000) Increased
oxidative modi®cation of albumin when illuminated in vitro in the
presence of a commonsunscreeningredient: protectionby nitroxide
the sunscreen and the cyclodextrin during the EPR
measurements.
The results shown in Figure 3 demonstrate that the
photo-induced free radical production by BMDBM is
eŒectively reduced by inclusion complexation of the
sunscreen agent with HP- -CD.
Free Radic Biol Med
. . . 28: 193±201
radicals.
EEC Directive (1976) European Economic Community Council
}
Directive 76 768 Annex VII
Green, A., Williams, G., Neale, R., Hart, V., Leslie, D., Parsons, P.,
Marks, G. C., GaŒney, P., Battistutta, D., Frost, C., Lang, C.,
Russell, A. (1999) Daily sunscreen application and betacarotene
supplementation in prevention of basal-cell and squamous-cell
To gain insight into the action of the cyclodextrin as
}
complexing agent, the samples after spin-trapping EPR
evaluation were analysed by HPLC to measure the per-
centage loss of the sunscreen agent following irradiation.
In the solution containing BMDBM alone, the degree
Lancet
carcinomas of the skin: a randomised controlled trial.
723±729
354:
Hayden, C. G., Roberts, M. S., Benson H. A. E. (1998) Sunscreens:
Aust NZ J Med
. 28: 639±646
. ³ .
¯
of photodegradation was 63 1 4 7% (n 5), which de-
creased to 27 6 2 2% (n 5) in the sample containing
are Australian getting the good oil?
.
.
. ³ .
¯
Lucarini, M., Pedulli, G. F. (2000) EPR properties of two new cyclic
phosphinylhydrazyl radicals and of their inclusion complexes with
}
the BMDBM HP- -CD complex. These results are in
agreement with those obtained in a previous study (Scalia
et al 1998) and indicate that the eŒect of HP- -CD com-
plexation on BMDBM photodecomposition is smaller than
that measured on the light-induced free radical generation
(Figure 3). Since the photodegradation mechanism of
BMDBM (Schwack & Rudolf 1995 ; Damiani et al 1999)
proceeds through the initial formation of carbon-centred
free radicals (Figure 4), it is reasonable to assume that these
radicals either remain trapped or recombine in the cyclo-
dextrin cavity, or both (Lucarini & Pedulli 2000), and thus
their interaction with the nitroxide spin label is hindered, as
illustrated by the minor decrease of TEMPO EPR signals
(Figures 2B and 3). Moreover, e cient inclusion of the
photo-induced carbon-centred radicals by HP- -CD could
account for the enhanced photostability of the complex.
Additional research will be necessary to investigate whether
the free-radical scavenging achieved by complexation of
BMDBM with HP- -CD would apply to human skin
exposed to sunlight.
J Org Chem
. . . 65: 2723±2727
cyclodextrins.
National Institute of Health (1989) National Institute of Health
Consensus Statement Online. Sunlight, Ultraviolet Radiation, and
the Skin. 7: 1±29
Parry, E. J., Bilsland, D., Morley, W. N. (1995) Photocontact allergy
Contact
to 4-ter.butyl-4«-methoxy-dibenzoylmethane(Parsol 1789).
Dermatitis
32: 251±252
Roscher, N. M., Lindemann, M. K. O., Kong, S. B., Cho, C. G.,
Jiang, P. (1994) Photodecomposition of several compounds com-
J. Photochem.Photobiol. A: Chem.
monly used as sunscreen agents.
80: 417±421
Scalia, S., Villani, S., Scatturin, A., Vandelli, M. A., Forni, F. (1998)
Complexation of the sunscreen agent, butyl-methoxydibenzoyl-
Int J Pharmaceutics
methane, with hydroxypropyl- -cyclodextrin. . .
175: 205±213
Schmidt, T., Ring, J., Abeck, D. (1998) Photoallergic contact-
dermatitis due to combined UVB (4-methylbenzylidene camphor,
octylmethoxycinnamate) and UVA (benzophenone-3, butyl-
Dermatology
methoxydibenzoylmethane) absorber sensitization.
196: 354±357
Schwack, W., Rudolph, T. (1995) Photochemistry of dibenzoyl-
J. Photochem.Photobiol.B: Biol.
methane UVA ®lters.
28: 229±234
Serre, I., Cano, J. P., Picot, M. C., Meynadier, J., Meunier, L. (1997)
Immunosuppression induced by acute solar-simulated ultraviolet
exposure in humans: prevention by a sunscreen with a sun pro-
Conclusions
The results described in this study demonstrate that in
addition to enhancing the photostability of BMDBM, its
complexation with HP- -CD represents an eŒective
strategy to scavenge the free radicals produced by the sun-
screen agent upon illumination with simulated sunlight,
thereby minimizing the photo-induced damage in¯ icted
by BMDBM to important biological macromolecules.
J
Am Acad
. .
tection factor of 15 and high UVA protection.
Dermatol
.
. 37: 187±194
Stokes, R., DiŒey, B. (1999) In vitro assessment of sunscreen photo-
stability: the eŒect of radiation source, sunscreen applicationthick-
Int J Cosmet Sci
. 21: 341±351
ness and substrate.
.
.
.
Tarras-Wahlberg, N., Stenhagen, G., Larko, O., Rosen, A.,
Wennberg, A. M., Wennerstrom, O. (1999) Changes in ultraviolet
J
Invest
.
absorption of sunscreens after ultraviolet irradiation.
Dermatol
.
. 113: 547±553
Urbach, F. (1997) Ultraviolet radiation and skin cancer of human.
J Photochem Photobiol B Biol
. 40: 3±7
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