Journal of Photochemistry and Photobiology B: Biology 127 (2013) 153–160
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier .com/locate / jphotobiol
Efficacy of topical formulations containing Pimenta pseudocaryophyllusextract against UVB-induced oxidative stress and inflammationin hairless mice
1011-1344/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.jphotobiol.2013.08.007
⇑ Corresponding author. Address: Avenida Robert Koch, 60, Vila Operária, CEP86039-440 Londrina, Paraná, Brazil. Tel.: +55 43 33712475.
E-mail address: [email protected] (R. Casagrande).
Marcela Z. Campanini a, Felipe A. Pinho-Ribeiro b, Ana L.M. Ivan a, Vitor S. Ferreira a, Fernanda M.P. Vilela c,Fabiana T.M.C. Vicentini c, Renata M. Martinez a, Ana C. Zarpelon b, Maria J.V. Fonseca c, Terezinha J. Faria d,Marcela M. Baracat a, Waldiceu A. Verri Jr. b, Sandra R. Georgetti a, Rúbia Casagrande a,⇑a Departamento de Ciências Farmacêuticas, Universidade Estadual de Londrina-UEL, Avenida Robert Koch, 60, Hospital Universitário, 86039-440 Londrina, Paraná, Brazilb Departamento de Patologia, Universidade Estadual de Londrina-UEL, Rodovia Celso Garcia Cid, Km 380, PR445, Cx. Postal 10.011, 86051-980 Londrina, Paraná, Brazilc Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, Av. do Café s/n, 14049-903 Ribeirão Preto, São Paulo, Brazild Departamento de Química, Universidade Estadual de Londrina-UEL, Rodovia Celso Garcia Cid, Km 380, 86051-980 Londrina, Paraná, Brazil
a r t i c l e i n f o
Article history:Received 16 May 2013Received in revised form 27 July 2013Accepted 19 August 2013Available online 29 August 2013
Keywords:AntioxidantFree radicalOxidative stressPimenta pseudocaryophyllusTopical formulationUV-B irradiation
a b s t r a c t
Plants rich in antioxidant substances may be a promising strategy for preventing UV-induced oxidativeand inflammatory damage of the skin. Pimenta pseudocaryophyllus is native to Brazil and presents flavo-noids and other polyphenolic compounds in high concentration. Thus, the present study evaluated thepossible effects of topical formulations containing P. pseudocaryophyllus ethanolic extract (PPE) at inhib-iting UV-B irradiation-induced oxidative stress and inflammation. PPE was administered on the dorsalskin of hairless mice using two formulations: F1 (non-ionic emulsion with high lipid content) and F2(anionic emulsion with low lipid content) before and after UV-B irradiation. The following parameterswere evaluated in skin samples: edema, myeloperoxidase activity, cytokines levels, matrix metallopro-tease-9 (MMP-9) secretion/activity, reduced glutathione (GSH), superoxide anion and lipid peroxidationlevels, and mRNA expression for glutathione reductase and gp91phox. The UV-B irradiation increased allparameters, except for IL-10 levels and glutathione reductase mRNA expression, which were not altered,and GSH levels, which were reduced by exposure to UV-B light. Treatments with F1 and F2 containing PPEinhibited UV-B-induced edema formation (89% and 86%), myeloperoxidase activity (85% and 81%), IL-1bproduction (62% and 82%), MMP-9 activity (71% and 74%), GSH depletion (73% and 85%), superoxide anion(83% and 66%) and TBARS (100% and 100%) levels, increased glutathione reductase (2.54 and 2.55-fold)and reduced gp91phox (67% and 100%) mRNA expression, respectively. F2 containing PPE also increasedIL-10 levels. Therefore, this study demonstrates the effectiveness of topical formulations containing PPEin inhibiting UV-B irradiation-induced inflammation and oxidative stress of the skin.
� 2013 Elsevier B.V. All rights reserved.
1. Introduction
As a protective barrier of the body, skin is highly exposed tooxidative stress resulting from, among other sources, ultraviolet(UV) irradiation [1,2]. Reactive oxygen species (ROS) formed as aconsequence of UV irradiation may oxidize and damage cellularlipids, proteins and deoxyribonucleic acid (DNA), leading tochanges and often to destruction of skin structures, which can re-sult in inhibition of its regular function [3]. The exposure of theskin to UV induces an imbalance between ROS and endogenous
antioxidant (AO) systems, such as reduced glutathione (GSH)[4–6]. Furthermore, the release of a network of cytokines, whichparticipate in the onset of cutaneous inflammation, among themIL-1b, is certainly important [7,8]. These molecules cause vasodila-tation, widening of interendothelial junctions and separation ofendothelial cells, increasing microvascular protein and fluidleakage into interstitium resulting in edema [9,10]. Neutrophilsare also activated, stimulating the activity of myeloperoxidase(MPO), a ROS-generator enzyme [11]. UV irradiation also inducesthe activity of matrix metalloproteases (MMPs), which can be con-sidered the primary mediators of connective-tissue damage in skinexposed to UV irradiation and in the premature aging [12]. There isadditional evidence on the link between oxidative stress andinflammatory cytokines. For instance, IL-1b activates nicotinamideadenine dinucleotide phosphate (NADPH) oxidase generating
154 M.Z. Campanini et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 153–160
superoxide anion. In turn, superoxide anion activates nuclear fac-tor jB (NFjB) inducing the production of cytokines [13].
Therefore, various efforts have been made to prevent theseevents caused by sun exposure. In this context, antioxidants fromnatural sources may provide new possibilities for treatment andprevention of oxidative stress-mediated diseases. In recent years,many studies trying to establish and to characterize natural antiox-idants, including both isolated compounds and natural extracts, tobe topically applied have been performed [5,12,14,15]. Peppers andaromatic herbs have been subject of study due to their high AOproperties, generally attributed to the presence of polyphenoliccompounds, including flavonoids [16,17].
Regarding Pimenta pseudocaryophyllus, its anxiolytic, antimicro-bial, antinociceptive and anti-inflammatory activities have beendemonstrated [18,19]. Studies also show the presence of flavo-noids, tanins and other phenolic compounds with AO activity inhigh concentrations in its leaves [19,20]. Thus, the developmentof topical formulations with P. pseudocaryophyllus may representa promising strategy for skin protection against damages causedby UV irradiation.
The present study aimed to evaluate the in vivo efficacy of for-mulations containing P. pseudocaryophyllus ethanolic extract (PPE)in the prevention and/or treatment of oxidative damage caused byUV irradiation in the skin of hairless mice.
2. Materials and methods
2.1. Chemicals
Brilliant blue dye, hexadecyltrimethylammonium bromide(HTAB), o-dianisidine dihydrochloride, ethylene glycol bis (-ami-noethyl ether)-N,N,N0,N0-tetraacetic acid (EGTA), o-phthalalde-hyde (OPT), GSH, sodium dodecyl sulphate (SDS) and acrylamidewere obtained from Sigma Chemical Co. (St.Louis, MO, USA). Rawmaterials for formulations were obtained from Galena (Campinas,SP, Brazil) and are presented in the formulation section. All otherreagents used were of pharmaceutical grade.
2.2. Plant material and extract preparation
The leaves of P. pseudocaryophyllus were collected in December2007 at São Jerônimo da Serra (Paraná, Brazil). The plant specimenswere identified by A.O.S. Vieira, Departamento de Biologia Animale Vegetal (Centro de Ciências da Saúde) and a voucher specimenwas deposited at the Herbarium of Universidade Estadual de Lond-rina under code No. FUEL 43025. The plant material was dried at40 �C and coarsely powdered in industrial blender. The ethanolicextract (1:10) was obtained by exhaustive maceration at roomtemperature (RT) (25 �C) for 12 days. The extract was filtered andconcentrated under vacuum. The characterization of the extractby HPLC analysis can be obtained upon request.
2.3. Formulations
Formulations were developed varying the content of lipidic andemulsifying agent. Self-emulsifying wax Polawax� (cetostearylalcohol + polyoxyethylene derived of a fatty acid ester of sorbitan20E) was used in both formulations, although in major (10%) andminor (2%) proportions in formulation 1 (F1) and formulation 2(F2), respectively. Into F2, 0.18% of anionic hydrophilic colloid(carboxypolymethylene, Carbopol 940�) was also added as stabi-lizing agent. Caprylic/capric triglycerides (5%) were added as emol-lients, and propylene glycol (5%) as moisturizer. The preservativeused was a mixture of parabens (1%). Deionized water was usedfor the preparation of all formulations to complete 100%. PPE
was incorporated (5%) into the formulations at room temperature.All concentrations of the formulations raw materials wereexpressed as percentages weight/weight. Control formulationsdid not contain PPE.
2.4. Animals
Sex matched hairless mice (HRS/J), weighing 20–30 g, werehoused in a temperature-controlled room, with access to waterand food ad libitum until use. All experiments were conducted inaccordance with National Institutes of Health guidelines for thewelfare of experimental animals and with the approval of the Eth-ics Committee of State University of Londrina (34994/209).
2.5. Formulation administration
Hairless mice were randomly designed to different groups with5 mice in each of the following groups: (a) control (non-irradiatedgroup – NI), (b) irradiated, (c) irradiated treated with formulation 1without PPE, (d) irradiated treated with formulation 1 containingPPE 5%, (e) irradiated treated with formulation 2 without PPE,and (f) irradiated treated with formulation 2 containing PPE 5%.Mice received topical treatment on the dorsal surface with 0.5 gof formulation, which in the case of groups d and e correspondsto 6.92 lg of polyphenols/cm2 of skin. Formulations were adminis-trated 1 h before, 5 min before and right after the irradiation [21].Untreated control groups irradiated and non-irradiated were in-cluded in the experiments. Results are representative of 2 separateexperiments.
2.6. Irradiation
The UV-B source of irradiation consisted of a Philips TL40W/12RS lamp (Medical-Holand) emitting a continuous spectrum be-tween 270 and 400 nm with a peak emission at 313 nm. The lampwas mounted 20 cm above the table where the mice were placedon, resulting in an irradiation of 0.384 mW/cm2, as measured byan IL 1700 radiometer (Newburyport, MA, USA) with sensor forUV (SED005) and UV-B (SED240). The dose of UV-B used was4.14 J/cm2 [21,22]. The mice were killed by anesthesia overdose 2(Figs. 5B, 5C and 6) or 12 (Figs. 1–4 and 5A) h after the UV-B expo-sure, and the full thickness of the dorsal skins were removed andstored at �70 �C for further analysis.
2.7. Edema evaluation
The effect of F1 and F2 on UV-B-induced skin edema was mea-sured as an increase in dorsal skin weight. After dorsal skin re-moval, a constant area (6 mm diameter) was delimited with theaid of a punch, followed by weighing of this constant area[23,24]. The result was obtained comparing the weight of the skinbetween groups and the result was expressed in g of skin.
2.8. MPO activity
The UV-B-induced leukocyte migration to the skin was evalu-ated using the MPO kinetic-colorimetric assay as previously de-scribed [21,25]. Skins were collected in 400 lL of 50 mM K2HPO4
buffer (pH 6.0) containing 0.5% of HTAB, and homogenized usingTissue-Tearor (Biospec�). After that, homogenates were centri-fuged at 16.100g for 2 min at 4 �C. The supernatant was removedto assay. Briefly, 30 lL of sample was mixed with 200 lL of0.05 M K2HPO4 buffer (pH 6.0), containing 0.0167% o-dianisidinedihydrochloride and 0.05% hydrogen peroxide. The absorvancewas determined after 5 min at 450 nm (Asys Expert Plus, Bio-chrom). The MPO activity of samples was compared to a standard
Fig. 1. Formulations containing PPE inhibit UV-B irradiation-induced edema. Theskin edema was determined 12 h after the end of irradiation. Bars representmeans ± SEM of 2 separated experiments, 5 mice per group. �p < 0.001 compared tothe control (non-irradiated) group, ��p < 0.001 compared to irradiated group and F1control group, and ���p < 0.001 compared to irradiated group and F2 control group.Non-irradiated group (NI).
Fig. 2. Formulations containing PPE inhibit the UV-B irradiation-induced increaseof MPO activity. The MPO activity was determined 12 h after the end of irradiation.Bars represent means ± SEM of 2 separated experiments, 5 mice per group.�p < 0.001 compared to the control (non-irradiated) group, ��p < 0.001 comparedto irradiated group and F1 control group, and ���p < 0.001 compared to irradiatedgroup and F2 control group. Non-irradiated group (NI).
Fig. 3. Effect of formulations containing PPE on UV-B irradiation-induced cytokineproduction. Mice were treated with formulations F1 or F2 containing PPE orcontrols and were challenged with UV-B irradiation. The levels of IL-1b (A) and IL-10 (B) were determined at the 12 h after the end of irradiation. Bars representmeans ± SEM of 2 separated experiments, 5 mice per group. �p < 0.001 compared tothe control (non-irradiated) group, ��p < 0.001 compared to irradiated group and F1control group, and ���p < 0.001 compared to irradiated group and F2 control group.Non-irradiated group (NI).
M.Z. Campanini et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 153–160 155
curve of neutrophils. Protein levels in the skin homogenates wasmeasured using the Lowry method [25]. The results are presentedas MPO activity (number of total neutrophils/mg of protein).
2.9. IL-1b and IL-10 assays
Skin samples were removed and subsequently homogenized in500 lL of saline solution (NaCl 0.09%) using Tissue-Tearor (Bio-spec�). The homogenates were centrifuged at 2.000g for 15 minat 4 �C and stored at �70 �C until further use. Supernatants wereused to measure the cytokines. IL-1b and IL-10 contents weredetermined as described previously by Verri et al. [26] by an en-zyme-linked immunosorbent assay (ELISA) according to manufac-turer’s instructions (eBioscience). Absorbances were determined at450 nm (Victor X3, Perkin Elmer�) and the protein levels in theskin homogenates was measured using the Lowry method [27].The results are expressed as picograms (pg) of each cytocine/100 mg of protein.
2.10. Analysis of skin MMP-9 by substrate-embedded enzymography
SDS polyacrylamide gel electrophoresis substrate-embeddedenzymography was used to detect MMP-9, an enzyme with
gelatinase activity. Assays were carried out as previously described[12]. The total skins taken from each group (1:4, w/w dilution)were homogenized in 50 mM Tris–HCl buffer (pH 7.4) containing10 mM CaCl2 and 1% of protease inhibitor cocktail in Ultra Turrax(T 18 Basic, IKA�). The entire homogenates were centrifuged twiceat 12.000g for 10 min at 4 �C and the Lowry et al. [27] method wasused to measure protein levels in the supernatants. Aliquots mea-suring 50 lL were mixed with 10 lL of 100 mM Tris–HCl buffer(pH 7.4) containing 4% SDS, 20% glycerol, and 0.005% of xylenecyanol. 25 lL of the mixture (40 lg of protein) were taken forelectrophoresis in a gel containing acrylamide 10% and gelatin0.25%. After electrophoresis, the gels were incubated for 1 h with2.5% Triton X-100 under constant shaking, incubated overnight in0.05 M Tris–HCl (pH 7.4), 0.01 M CaCl2 and 0.02% sodium azideat 37 �C, and stained the following day with brilliant blue R. Afterdestaining in 20% acetic acid, zone of enzyme activity wereanalyzed by comparing the groups in the Image J� program.
2.11. GSH assay
Cutaneous GSH levels were determined using a fluorescenceassay as previously described [12]. Firstly, the skin (1:3, w/wdilution) was homogenized in 100 mM NaH2PO4 (pH 8.0) contain-ing 5 mM EDTA (buffer 1) using Ultra Turrax (IKA�). After that,homogenates were treated with 30% trichloroacetic acid andcentrifuged twice (at 1.940g for 6 min and at 485g for 10 min)
Fig. 4. Formulations containing PPE inhibit UV-B irradiation-induced increase of MMP-9 activity. The MMP-9 activity was determined 12 h after the end of irradiation. Barsrepresent means ± SEM of 2 separated experiments, 5 mice per group. �p < 0.001 compared to the control (non-irradiated) group, ��p < 0.001 compared to irradiated group andF1 control group, and ���p < 0.001 compared to irradiated group and F2 control group. Non-irradiated group (NI).
156 M.Z. Campanini et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 153–160
and the fluorescence of the resulting supernatant was measured ina RF-5301PC, Shimadzu� fluorescence spectrophotometer. Briefly,100 lL of the supernatant was mixed with 1 mL of buffer 1 and100 lL of OPT (1 mg/mL in methanol). The fluorescence was deter-mined after 15 min (kexc = 350 nm; kem = 420 nm). The standardcurve was prepared with different concentrations of GSH (0.0–75.0 lM). Protein levels in the skin homogenates was measuredusing the Lowry method [27]. Results are presented as lM ofGSH/mg of protein.
2.12. Lipid peroxidation
Firstly, the protein content of homogenate (10 mg/ml in 1.15%KCl) was measured using the Lowry method. Thiobarbituric acidreactive substances (TBARS) measurement was used to evaluate li-pid peroxidation as previously described [28]. For this assay, tri-chloroacetic acid (10%) was added to the homogenate toprecipitate proteins. This mixture was then centrifuged (3 min,1000g). The protein-free sample was extracted and thiobarbituricacid (0.67%) was added. The mixture was kept in water bath at100 �C for 15 min. Malondialdehyde (MDA), an intermediate prod-uct of lipoperoxidation, was determined by difference betweenabsorbances at 535 and 572 nm on a microplate spectrophotome-ter reader (Multiskan GO, Thermo Scientific) and the results arereported as nmol/mg of protein [29].
2.13. Superoxide anion production
The quantitation of superoxide anion production in tissuehomogenates (10 mg/ml in 1.15% KCl) was performed using thenitroblue tetrazolium assay (NBT) [30]. Briefly, 50 ll of homoge-nate was incubated with 100 ll of NBT (1 mg/ml) in 96-well platesat 37 �C for 1 h. The supernatant was then carefully removed andthe reduced formazan solubilized by adding 120 ll of 2 M KOHand 140 ll of DMSO. The NBT reduction was measured at600 nm using a microplate spectrophotometer reader (MultiskanGO, Thermo Scientific). The protein content was used for datanormalization.
2.14. Quantitative polymerase chain reaction (qPCR)
qPCR was performed as previously described [31]. Sampleswere homogenized in TRIzol reagent, and total RNA was extractedby using the SV Total RNA Isolation System (Promega). All
reactions were performed in triplicate using the manufacturer cy-cling conditions: 50 �C for 2 min, 95 �C for 2 min, followed by 50cycles of 95 �C for 15 s and 60 �C for 30 s. qPCR was performed inan LightCycler� Nano Instrument (Roche, Mississauga, ON, USA)sequence detection system by using the SYBR-green fluorescence.The primers used were: glutathione reductase, sense: 50-TGCGTGAATGTTGGATGTGTACCC-30, antisense: 50-CCGGCATTCTCCAGTTCCTCG-30; gp91phox, sense: 50-AGCTATGAGGTGGTGATGTTAGTGG-30,antisense: 50-CACAATATTTGTACCAGACAGACTTGAG-30; b-actin,sense: 50-AGCTGCGTTTTACACCCTTT-30, antisense: 50-AAGCCATGC-CAATGTTGTCT-30. The expression of b-actin mRNA was used as acontrol for tissue integrity in all samples.
2.15. Statistical analysis
The bars in the figures indicate the mean values ± standard er-ror of the mean (SEM) of 2 separate experiments with n = 5 animalsper group. Data were statistically analyzed by one-way ANOVA fol-lowed by Bonferroni’s t test. Results were considered significantlydifferent when p < 0.05.
3. Results
3.1. Formulations containing PPE prevent UV-B-induced edema in theskin
Several studies show that exposure to UV-B light leads to skinedema, which can be considered a marker of skin inflammation[23,24,33,34]. Standard-sized punches of skin were weighted in or-der to evaluate the capacity of formulations to inhibit UV-B irradi-ation-induced edema. As shown in Fig. 1, UV-B exposure increasedapproximately 2.46-fold punch weight of untreated irradiated ani-mals and irradiated animals treated with control formulations. F1and F2 containing PPE were able to decrease edema formation tocontrol levels.
3.2. Formulations containing PPE prevent UV-B-induced MPO activityincrease in the skin
MPO plays an important role in the innate immune system andcan be used as a marker of the presence of neutrophils or inflam-mation [11]. Results show that UV-B irradiation induced anincrease of approximately 8.06-fold in the MPO activity of un-treated irradiated animals and irradiated animals treated with
Fig. 5. Formulations containing PPE inhibit UV-B irradiation-induced GSH deple-tion, superoxide anion production and lipid peroxidation. The GSH (A), superoxideanion (B, NBT assay) and lipid peroxidation (C, TBARS assay) were determined 5 (Band C) or 12 h (A) after the end of irradiation. Bars represent means ± SEM of 2separated experiments, 5 mice per group. �p < 0.001 compared to the control(non-irradiated) group, ��p < 0.01 compared to irradiated group and F1 controlgroup, and ���p < 0.05 compared to irradiated group and F2 control group.Non-irradiated group (NI).
Fig. 6. Formulations containing PPE increase glutathione reductase mRNA expres-sion and inhibit UV-B irradiation-induced gp91phox mRNA expression. The mRNAexpression for glutathione reductase (A) and gp91phox (B) was determined 5 hafter the end of irradiation. Bars represent means ± SEM of 2 separated experiments,5 mice per group. �p < 0.001 compared to the control (non-irradiated) group,��p < 0.01 compared to irradiated group and F1 control group, and ���p < 0.001compared to irradiated group and F2 control group. Non-irradiated group (NI).
M.Z. Campanini et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 153–160 157
control formulations. F1 and F2 containing PPE inhibited the MPOactivity to non-irradiated control levels (Fig. 2).
3.3. Influence of formulations containing PPE on cytokine after UV-Bexposure
UV-B light significantly increased IL-1b levels (approximately2.24-fold) of untreated irradiated animals and irradiated animalstreated with control formulations. F1 and F2 were able to decrease
IL-1b to control levels (Fig. 3A). F2 containing PPE also induced a2.06-fold increase in IL-10 levels when compared to control(Fig. 3B).
3.4. Formulations containing PPE prevent the UV-B-induced MMP-9activity increase
Results showed a significant increase in the expression/activityof gelatinase in hairless mice skin after UV-B irradiation. The semi-quantitative analysis of MMP-9 in the skin demonstrated that UV-Birradiation induced an increase of approximately 1.63-fold inMMP-9 activity of untreated irradiated animals and irradiated ani-mals treated with control formulations. F1 and F2 containing PPEwere again able to inhibit MMP-9 activity to control levels (Fig. 4).
3.5. Formulations containing PPE prevent UV-B-induced oxidativestress
In this study, UV-B irradiation induced a decrease of approxi-mately 1.95-fold of GSH levels in untreated irradiated animalsand irradiated animals treated with control formulations. Bothformulations containing PPE inhibited this depletion, maintaininglevels similar to control (non-irradiated) group (Fig. 5A). UV-B irra-diation also induced 1.96-fold increase of superoxide anion (NBTassay) levels compared to non-irradiated mice while F1 and F2containing PPE inhibited such production by 83% and 66%, respec-tively (Fig. 5B). Furthermore, UV-B irradiation induced 3.69-foldincrease of lipid peroxidation (TBARS assay) levels, which werereduced to control levels by treatment with both formulations
158 M.Z. Campanini et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 153–160
containing PPE (Fig. 5C). No effect was observed with both formu-lations without PPE regarding the levels of superoxide anion(Fig. 5B) and lipid peroxidation (Fig. 5C).
3.6. Formulations containing PPE induce glutathione reductase mRNAexpression and prevent UV-B-induced gp91phox mRNA expression
There was no difference of glutathione reductase mRNA expres-sion among non-irradiated, untreated UV-B irradiated and bothformulations without PPE groups. On the other hand, F1 and F2containing PPE induced approximately 2.54 and 2.55-fold increaseof glutathione reductase mRNA expression compared to all othergroups, respectively (Fig. 6A). Additionally, UV-B irradiation in-duced 1.56-fold increase of gp91phox mRNA expression, whichwas inhibited by both formulations containing PPE (67% F1 and100% F2), but not by formulations without PPE (Fig. 6B).
4. Discussion
In the present study, it was observed that topical formulationscontaining PPE inhibited the inflammatory and oxidative phenom-ena in the skin of hairless mice irradiated with UV-B irradiation.The formulations containing PPE with different lipid contentsinhibited UV-B irradiation-induced increase of skin edema, MPOactivity, MMP-9 activity, IL-1b production, GSH depletion, superox-ide anion production and lipid peroxidation as well as increasedthe mRNA expression for glutathione reductase and reduced themRNA expression of gp91phox. F2 also increased anti-inflamma-tory cytokine IL-10 levels. Therefore, these results show in a con-sistent manner that P. pseudocaryophyllus extract can bedelivered using topical formulations to reduce UV-B-inducedinflammation and oxidative stress of the skin, and a formulationwith less lipid content might be more suitable to deliver theextract.
Sunlight coupled with living in an oxygen-rich atmospherecauses unwanted and deleterious consequences to the skin, suchas cancer, wrinkling, scaling, dryness, and mottled pigment abnor-malities (hyper or hypopigmentation) [34]. Inflammatory chemicalmediators including cyclooxygenase derived metabolites of arachi-donic acid increase vascular permeability and blood flow [35–37].The spectrum of UV light that induces edema in hairless mice anderythema in humans is the same. Because of this similarity, achange in sensitivity to UV irradiation in mice as measured by skinedema should reliably predict a change in sensitivity in humans[32]. Our results clearly show that PPE incorporated to both formu-lations was able to inhibit skin edema in animals exposed to UVirradiation. In addition to the present data using an UV irradiationmodel of inflammation, a fraction of the P. pseudocaryophyllus eth-anolic extract inhibited the croton oil-induced ear edema [20].Therefore, P. pseudocaryophyllus extracts can be used in modelswith different triggering mechanisms, and, thus, have wider appli-cability. For comparison purposes, it is noteworthy to mention thatextracts from a different species of Pimenta, Pimenta racemosa, in-hibit paw and ear edema induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) and carrageenin in mice [38,39].
In general, neutrophils are the first cells recruited from periph-eral blood to inflammatory sites. ROS such as superoxide anion areessential for neutrophil recruitment to the inflammatory foci andROS production is triggered by UV-B irradiation [11,35,40]. Oneof the special neutrophils’ products is the heme enzyme MPO,which is stored in large amounts in azurophilic granules of thesecells [35]. MPO-derived hypochlorous acid (HOCl) reacts with pro-teins, DNA, and lipids to form long-lived oxidants, which have beenimplicated in processes like carcinogenesis, atherosclerosis, andchronic renal failure [35]. F1 and F2 containing PPE were able to
decrease UV-induced MPO activity to control levels, demonstratingits anti-inflammatory and protective activity. Both formulationscontaining PPE also inhibited the mRNA expression of NADPH oxi-dase sub-unity gp91phox and superoxide anion production, whichcould be a contributing mechanism for reducing neutrophilrecruitment and activity [35,41] and, therefore, MPO activity. Cor-roborating, Garcia et al. [38] and Fernandez et al. [39] also demon-strated the effectiveness of P. racemosa against TPA-induced MPOincrease in ear tissue.
In ROS-induced inflammation, nuclear factor kB (NF-kB) plays acrucial role. It binds to distinct promoter genes, which encode TNF-a, interleukins (IL-6 and IL-1), and several adhesion molecules,thus allowing their transcription [4,42]. In this context, UV-B stim-ulation of cultured human keratinocytes induces the expression ofcytokines, such as TNF-a, IL-1a, IL-1b and IL-6 [4]. IL-1b stimulatesneutrophils and other cells, increasing expression of adhesionmolecules, such as intercellular adhesion molecules (ICAMs) andL-selectins [43–45]. In this study, UV-B irradiation increasedIL-1b levels. Again, F1 and F2 containing PPE decreased IL-1b tocontrol levels, demonstrating the potential anti-inflammatory ef-fect of the extract. In this regard, it was demonstrated that UV irra-diation induces the expression of NOD1 and NOD2 receptors,which induce the production of pro-IL-1b [46]. UV irradiation alsoinduces ROS-dependent activation of inflammasomes resulting incaspase-1-dependent cleavage of pro-IL-1b to active IL-1b [47].Furthermore, the inhibition of the gp91phox mRNA expressionand superoxide anion production by both formulations containingPPE suggests that preventing ROS-mediated IL-1b productionmight be an important mechanism of PPE formulations. The inhibi-tion of IL-1b production by the formulations containing PPE linesup well with the inhibition of MPO activity, since IL-1b is chemo-tactic for neutrophils [48], which indicates that inhibition of IL-1b production could lead to reduced neutrophil recruitment and,therefore, reduced MPO activity. Regarding IL-10, UV-B irradiationhad no influence on its levels in the present experimental condi-tions. Nevertheless, F2 containing PPE increased the productionof IL-10. IL-10 is an anti-inflammatory cytokine, which inhibitsNF-kB and balances the activator and inhibitor signals of theinflammatory process by reducing the transcription and produc-tion of pro-inflammatory cytokines [42,43]. Thus, an additionalanti-inflammatory mechanism of F2 compared to F1 was theinduction of IL-10 production. It is possible that F2 presents betterlipid concentration than F1 for the release of active compoundsand/or a group of compounds from PPE at an adequate dose to in-duce IL-10 production. In fact, some flavonoids induce the produc-tion of IL-10 [49].
Besides NF-kB, another transcription factor induced by UV irra-diation is the activation protein-1 (AP-1), which can be activatedby a series of mitogen-activated protein kinases. As a consequence,the induction of MMPs, which degrade the collagen framework ofskin, occurs [12,33]. MMP-9 (gelatinase B) is one of the primary en-zymes related to degradation of skin collagen and components ofthe elastic fibers network. It displays the greatest elastolytic andfibrillin-degrading activity [44]. MMPs are produced by fibroblasts,keratinocytes, mast cells, endothelial cells, and leukocytes, such asneutrophils, and are released from cytoplasmatic granules [45]. Inthe present study, UV-B irradiation clearly induced an increase ofMMP-9 activity. The capacity of formulations containing PPE toprevent the increase of MMP-9 activity was presented in qualita-tive and semi-quantitative manners. Similarly, Bellosta et al. [48]demonstrated the activity of the extract of Tristaniopsis calobuxus,which, as P. pseudocaryophyllus, also belongs to Myrtaceae family,to inhibit MMP-9 activity in mouse macrophages. The activationof MMPs results in elevated levels of degraded collagen, which ap-pears to downregulate type I procollagen synthesis. Thereby, Fons-eca et al. [12] suggests that a formulation that can decrease
M.Z. Campanini et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 153–160 159
procollagen synthesis might also be able to improve skincollagenization.
Importantly, UV-B irradiation also leads to an imbalance be-tween ROS and endogenous antioxidants, causing depletion ofendogenous antioxidants such as GSH, an epidermal marker thatis sensitive to oxidative stress caused by UV-B irradiation [4,50].Its sulfhydryl group (SH), highly polarizable, allows the removalof radicals directly by hydrogen transfer, which makes it an opti-mum nucleophile for reactions with electrophilic chemicals [6]. Inaddition, it acts as a cofactor for glutathione peroxidase and gluta-thione reductase, which reduce hydrogen peroxide and lipid hydro-peroxides [4,33]. In the present study, the levels of GSH andglutathione reductase mRNA expression in non irradiated controland in groups treated with F1 and F2 containing PPE were signifi-cantly higher than in the other three groups (p < 0.01), suggestingthat formulations containing PPE exerted strong AO activity bymaintaining the glutathione system despite UV-B exposure. Furthersupporting the AO effect of F1 and F2 containing PPE, these formu-lations inhibited lipid peroxidation as determined by TBARS assay.
Therefore, the present data demonstrate the beneficial effec-tiveness of P. pseudocaryophyllus on UV-B-induced skin oxidativestress and inflammation. Thus, these data suggest the possible use-fulness of PPE to prevent skin damages caused by UV-B irradiationand show the importance of performing further pre-clinical andclinical studies with this extract.
5. Abbreviations
AO
antioxidant AP-1 activation protein-1 DNA deoxyribonucleic acid EGTA ethylene glycol bis (-aminoethyl ether)-N,N,N0,N0-tetraacetic acid
Formulation 1 F1 Formulation 2 F2 GSH reduced glutathione HTAB hexadecyltrimethylammonium bromide HOCl hypochlorous acid ICAMs intercellular adhesion molecules IL interleukin MMP matrix metalloprotease MPO myeloperoxidase NADPH oxidase nicotinamide adenine dinucleotidephosphate-oxidase
NF-kB nuclear factor kB OPT o-phthalaldehyde PPE P. pseudocaryophyllus ethanolic extract ROS reactive oxygen species SDS sodium dodecyl sulphate SEM standard error mean SH sulfhydryl grouping TBARS thiobarbituric reactive substances TNF-a tumour necrosis factor-a TPA 12-O-tetradecanoylphorbol-13-acetate UV ultravioletAcknowledgments
This work was supported by Grants from Coordenação de Aper-feiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacion-al de Desenvolvimento Científico e Tecnológico (CNPq), Parana StateGovernment and SETI/Fundação Araucária. We thank the technicalassistance of Denise Duarte from Post-graduation Laboratory of UEL.
References
[1] L. Al Shaal, R. Shegokar, R.H. Müller, Production and characterization ofantioxidant apigenin nanocrystals as a novel UV skin protective formulation,Int. J. Pharm. 420 (2011) 133–140.
[2] F.T.M.C. Vicentini, R. Casagrande, S.R. Georgetti, M.V.L.B. Bentley, M.J.V.Fonseca, Influence of vehicle on antioxidant activity of quercetin: a liquidcrystalline formulation, Lat. Am. J. Pharm. 26 (2007) 805–810.
[3] M. Valko, D. Leibfritz, J. Moncol, M.T. Cronin, M. Mazur, J. Telser, Free radicalsand antioxidants in normal physiological functions and human disease, Int. J.Biochem. Cell Biol. 39 (2007) 44–84.
[4] M. Carini, G. Aldini, M. Piccone, R.M. Facino, Fluorescent probes as markers ofoxidative stress in keratinocyte cell lines following UV-B exposure, II Farmaco55 (2000) 526–534.
[5] R. Casagrande, S.R. Georgetti, W.A. Verri Jr., J.R. Jabor, A.C. Santos, M.J.V.Fonseca, Evaluation of functional stability of quercetin as a raw material and indifferent topical formulations by its antilipoperoxidative activity, AAPS Pharm.Sci. Technol. 7 (2006) E1–E8.
[6] P.C. Huber, W.P. Almeida, Glutationa e enzimas relacionadas: papel biológico eimportância em processos patológicos, Quim. Nova 31 (2008) 1170–1179.
[7] U.C. Obermüller-Jevic, B. Schlegel, A. Flaccus, H.K. Biesalski, The effect of L-carotene on the expression of interleukin-6 and heme oxygenase-1 in UV-irradiated human skin fibrobroblasts in vitro, FEBS Lett. 509 (2001) 186–190.
[8] N.P. Lopes, T. Guaratini, D.R. Callejon, D.C. Pires, J.N.C. Lopes, L.M. Lima, D.G.Neto, C. Sustovich, Fotoprotetores derivados de produtos naturais:perspectivas de mercado e interações entre o setor produtivo e centros depesquisa, Quim. Nova 32 (2009) 717–721.
[9] P.R. Kvietys, D.N. Granger, Role of reactive oxygen and nitrogen species in thevascular responses to inflammation, Free Radic. Biol. Med. 52 (2012) 556–592.
[10] S. Widyarini, N. Spinks, A.J. Husband, V.E. Reeve, Isoflavonoid compounds fromred clover (Trifolium pratense) protect from inflammation and immunesuppression induced by UV radiation, Photochem. Photobiol. 74 (2001) 465–470.
[11] W. Jantschko, P.G. Fortmuller, M. Zederbauer, K. Neugschwandter, I. Lehner, C.Jakopitsch, J. Arnhold, C. Obinger, Exploitation of the unusual thermodynamicproperties of human myeloperoxidase in inhibitor design, Biochem.Pharmacol. 69 (2005) 1149–1157.
[12] Y.M. Fonseca, C.D. Catini, F.T.M.C. Vicentini, J.C. Cardoso, R.L.C. Albuquerque-Junior, M.J.V. Fonseca, Efficacy of marigold extract-loaded formulations againstuv-induced oxidative stress, J. Pharm. Sci. 100 (2011) 2182–2193.
[13] W.A. Verri, Jr., F.T.M.C. Vicentini, M.M. Baracat, S.R. Georgetti, R.D.R. Cardoso,T.M. Cunha, S.H. Ferreira, F.Q. Cunha, M.J.V. Fonseca, R. Casagrande, in: Atta-ur-Rahman (Ed.), Studies in Natural Products Chemistry, Elsevier, Amsterdam,2012; Vol. 36, pp. 297�322.
[14] S.R. Georgetti, R. Casagrande, W.A. Verri Jr., R.F.V. Lopez, M.J.V. Fonseca,Evaluation of in vivo efficacy of topical formulations containing soybeanextract, Int. J. Pharm. 352 (2008) 189–196.
[15] F. Marquele-Oliveira, Y.M. Fonseca, O. Freitas, M.J.V. Fonseca, Developmentof topical functionalized formulations added with propolis extract:stability, cutaneous absorption and in vivo studies, Int. J. Pharm. 342 (2007)40–48.
[16] M. Suhaj, Spice antioxidants isolation and their antiradical activity: a review, J.Food. Compos. Anal. 19 (2006) 531–537.
[17] S.S. Chun, D.A. Vatem, Y.T. Lin, K. Shetty, Phenolic antioxidants from clonaloregano (Origanum vulgare) with antimicrobial activity against Helicobacterpylori, Process. Biochem. 40 (2005) 809–816.
[18] J.O. Fajemiroye, P.M. Galdino, S.F. Alves, J.A.M. de Paula, J.R. de Paula, P.C.Ghedini, E.A. Costa, Involvement of 5-HT1A in the anxiolytic-like effect ofdichloromethane fraction of Pimenta pseudocaryophyllus, J. Ethnopharmacol.141 (2012) 872–877.
[19] J.A.M. Paula, M.R.R. Silva, M.P. Costa, D.G.A. Diniz, F.A.S. Sá, S.F. Alves, E.A.Costa, R.C. Lino, J.R. Paula, Phytochemical Analysis and Antimicrobial,Antinociceptive, and Anti-Inflammatory Activities of Two Chemotypes ofPimenta pseudocaryophyllus (Myrtaceae), Evid-Based Compl. Alt., Article ID420715, 15 pages.
[20] J.A.M. Paula, J.R. Paula, M.T.F. Bara, M.H. Rezende, H.D. Ferreira, Estudofarmacognóstico das folhas de Pimenta pseudocaryophyllus (Gomes) L.R.Landrum – Myrtaceae, Rev. Bras. Farmacogn. 18 (2008) 265–278.
[21] R. Casagrande, S.R. Georgetti, W.A. Verri Jr., D.J. Dorta, A.C. Santos, M.J.V.J.Fonseca, Protective effect of topical formulations containing quercetin againstUV-B-induced oxidative stress in hairless mice, J. Photochem. Photobiol. B 84(2006) 21–27.
[22] Y. Shindo, E. Witt, D. Han, L. Packer, Dose-response effects of acute ultravioletirradiation on antioxidants and molecular markers of oxidation in murineepidermis and dermis, J. Invest. Dermatol. 102 (1994) 470–475.
[23] F. Afaq, M. Saleem, C.G. Krueger, J.D. Reed, H. Mukhtar, Anthocyanin- andhydrolyzable tannin-rich pomegranate fruit extract modulates MAPK and NF-kB pathways and inhibits skin tumorigenesis in CD-1 mice, Int. J. Cancer 113(2005) 423–433.
[24] N. Bhatia, T.A. Demmer, A.K. Sharma, I. Elcheva, V.S. Spiegelman, Role of b-TrCPubiquitin ligase receptor in UVB mediated responses in skin, Arch. Biochem.Biophys. 508 (2011) 178–184.
[25] P.P. Bradley, D.A. Priebat, R.D. Christensen, G. Rothstein, Measurement ofcutaneous inflammation: estimation of neutrophil content with an enzymemarker, J. Invest. Dermatol. 78 (1982) 206–209.
160 M.Z. Campanini et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 153–160
[26] W.A. Verri Jr., F.O. Souto, S.M. Vieira, S.C. Almeida, S.Y. Fukada, D. Xu, J.C. Alves-Filho, T.M. Cunha, A.T. Guerrero, R.B. Mattos-Guimaraes, F.R. Oliveira, M.M.Teixeira, J.S. Silva, I.B. Mcinnes, S.H. Ferreira, P. Louzada-Junior, F.Y. Liew, F.Q.Cunha, Ann. Rheum. Dis. 69 (2010) 1697–1703.
[27] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement withthe Folin-Phenol reagents, J. Biol. Chem. 193 (1951) 265–275.
[28] R.P. Guedes, L.D. Bosco, C.M. Teixeira, A.S. Araujo, S. Llesuy, A. Bello-Klein, M.F.Ribeiro, W.A. Partata, Neuropathic pain modifies antioxidant activity in ratspinal cord, Neurochem. Res. 31 (2006) 603–609.
[29] D.S. Barbosa, R. Cecchini, M.Z. El Kadri, M.A. Rodriguez, R.C. Burini, I. Dichi,Decreased oxidative stress in patients with ulcerative colitis supplementedwith fish oil omega-3 fatty acids, Nutrition 19 (2003) 837–842.
[30] T.J. Harrigan, I.F. Abdullaev, D. Jourd’heuil, A.A. Mongin, Activation of microgliawith zymosan promotes excitatory amino acid release via volume-regulatedanion channels: the role of NADPH oxidases, J. Neurochem. 106 (2008) 2449–2462.
[31] W.A. Verri Jr, A.T. Guerrero, S.Y. Fukada, D.A. Valerio, T.M. Cunha, D. Xu, S.H.Ferreira, F.Y. Liew, F.Q., Cunha, IL-33 mediates antigen-induced cutaneous andarticular hypernociception in mice, Proc. Natl. Acad. Sci. USA 105 (2008) 2723–2728.
[32] R.L. Sams II, L.H. Couch, B.J. Miller, C.V. Okerberg, A.R. Warbritton, W.G.Wamer, J.Z. Beer, P.C. Howard, Effects of a- and b-hydroxy acids on the edemalresponse induced in female SKH-1 mice by simulated solar light, Toxicol. Appl.Pharmacol. 184 (2002) 136–143.
[33] S.R. Pinnell, Cutaneous photodamage, oxidative stress, and topical antioxidantprotection, J. Am. Acad. Dermatol. 48 (2003) 1–22.
[34] C.M. Kim, S.M. Kang, H.J. Jeon, S.H. Shin, Production of Vibrio vulnificusmetalloprotease VvpE begins during the early growth phase: usefulness ofgelatin-zymography, J. Microbiol. Methods 70 (2007) 96–102.
[35] V. Witko-Sarsat, P. Rieu, B. Descamps-Latscha, P. Lesavre, L. Halbwachs-Mecarelli, Neutrophils: molecules, functions and pathophysiological aspects,Lab. Invest. 80 (2000) 617–653.
[36] W.A. Verri Jr, T.M. Cunha, S.H. Ferreira, X. Wei, B.P. Leung, A. Fraser, I.B.McInnes, F.Y. Liew, F.Q. Cunha, IL-15 mediates antigen-induced neutrophilmigration by triggering IL-18 production, Eur. J. Immunol. 37 (2007) 3373–3380.
[37] D.F. Woodward, D.A.A. Owen, Effect of H1- and H2-receptor antagonists oncutaneous inflammation evoked by histamine analogues and UV radiation,Eur. J. Pharmacol. 77 (1982) 103–112.
[38] M.D. García, M.A. Fernández, A. Alvarez, M.T. Saenz, Antinociceptive and anti-inflammatory effect of the aqueous extract from leaves of Pimenta racemosavar. ozua (Mirtaceae), J. Ethnopharmacol. 91 (2004) 69–73.
[39] A. Fernández, A. Álvarez, M.D. García, M.T. Sáenz, Anti-inflammatory effect ofPimenta racemosa var. ozua and isolation of the triterpene lupeol, Il Farmaco 56(2001) 335–338.
[40] M. Banerjee, M. Saxena, Interleukin-1 (IL-1) family of cytokines: role in type 2diabetes, Clin. Chim. Acta 413 (2012) 1163–1170.
[41] H. Hattori, K.K. Subramanian, J. Sakai, Y. Jia, Y. Li, T.F. Porter, F. Loison, B. Sarraj,A. Kasorn, H. Jo, C. Blanchard, D. Zirkle, D. McDonald, S.Y. Pai, C.N. Serhan, H.R.Luo, Small-molecule screen identifies reactive oxygen species as keyregulators of neutrophil chemotaxis, Proc. Natl. Acad. Sci. USA 107 (2010)3546–3551.
[42] K.A. Ribbons, J.H. Thompson, X. Liu, K. Pennline, D.A. Clark, M.J.S. Miller, Anti-inflammatory properties of interleukin-10 administration in hapten-inducedcolitis, Eur. J. Pharmacol. 323 (1997) 245–254.
[43] E. Weiss, A.J. Mamelak, S. La Morgia, B. Wang, C. Feliciani, A. Tulli, D.N. Sauder,The role of interleukin 10 in the pathogenesis and potential treatment of skindiseases, J. Am. Acad. Dermatol. 50 (2004) 657–675.
[44] G. Jenkins, Molecular mechanisms of skin ageing, Mech. Age. Dev. 123 (2002)801–810.
[45] J. Fuchs, T.M. Zollner, R. Kaufmann, M. Podda, Redox-modulated pathways ininflammatory skin diseases, Free Radic. Biol. Med. 30 (2001) 337–353.
[46] S. Benko, J. Tozser, G. Miklossy, A. Varga, J. Kadas, A. Csutak, A. Berta, E.Rajnavolgyi, Constitutive and UV-B modulated transcription of Nod-likereceptors and their functional partners in human corneal epithelial cells,Mol. Vis. 14 (2008) 1575–1583.
[47] A. Takeishi, E. Kuranaga, M. Miura, Sensing and reacting to dangers bycaspases: caspase activation via inflammasomes, Drug Discov. Ther. 2 (2008)14–23.
[48] S. Bellosta, M. Dell’Agli, M. Canavesi, N. Mitro, M. Monetti, M. Crestani, L.Verotta, N. Fuzzati, F. Bernini, E. Bosisio, Inhibition of metalloproteinase-9activity and gene expression by polyphenolic compounds isolated from thebark of Tristaniopsis calobuxus (Myrtaceae), Cell. Mol. Life Sci. 60 (2003) 1440–1448.
[49] S.M. Borghi, T.T. Carvalho, L. Staurengo-Ferrari, M.S. Hohmann, P. Pinge-Filho,R. Casagrande, W.A. Verri Jr., Vitexin inhibits inflammatory pain in mice bytargeting TRPV1, oxidative stress, and cytokines, J. Nat. Prod. 76 (2013) 1141–1149.
[50] F.R. Gruijl, Skin cancer and solar UV radiation, Eur. J. Cancer 35 (1999) 2003–2009.
Recommended
