Research ArticleUVA-UVB Photoprotective Activity ofTopical Formulations Containing Morinda citrifolia Extract

Mairim Russo Serafini1 Cassia Britto Detoni2

Paula dos Passos Menezes1 Rose Nely Pereira Filho3 Vanessa Silveira Fortes4

Maria Joseacute Fonseca Vieira4 Siacutelvia Stanisccediluaski Guterres2

Ricardo Luiz Cavalcanti de Albuquerque Junior3 and Adriano Antunes de Souza Arauacutejo1

1 Departamento de Fisiologia Universidade Federal de Sergipe (UFS) Sao Cristovao SE Brazil2 Programa de Pos-Graduacao em Ciencias Farmaceuticas Universidade Federal do Rio Grande do Sul (UFRGS)Porto Alegre RS Brazil

3 Instituto de Tecnologia e Pesquisa (ITP) Aracaju SE Brazil4 Faculdade de Ciencias Farmaceuticas de Ribeirao Preto (FCFRP) Ribeirao Preto SP Brazil

Correspondence should be addressed to Mairim Russo Serafini maiserafinihotmailcom

Received 15 April 2014 Accepted 19 June 2014 Published 15 July 2014

Academic Editor Wan-Liang Lu

Copyright copy 2014 Mairim Russo Serafini et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Exposure to solar radiation particularly its ultraviolet (UV) component has a variety of harmful effects on human health Some ofthese effects include sunburn cell formations basal and squamous cell cancers melanoma cataracts photoaging of the skin andimmune suppression The beneficial photoprotective effects of topical formulations with the extract Morinda citrifolia have notbeen investigated This present study aims to investigate the potential benefits ofM citrifolia topical application on the dorsal skinofmice exposed to UVA-UVB light Using 7 days of treatment [before (baseline values) and 20 h after UV exposure] the thicknessskin barrier damage (TEWL) erythema and histological alterations were evaluated The results showed that the formulationscontaining the extract protected the skin against UV-induced damage

1 Introduction

Ultraviolet radiation (UVR) from the sun is divided intoUVC(200ndash290 nm) UVB (290ndash320 nm) and UVA (320ndash400 nm)[1] Skin is the body organ that is most exposed to UVRHuman skin is constantly exposed to potentially harmfulcompounds and radiation because it serves as a protectivebarrier between the environment and its internal organs thusmaking it liable to aging [2]

Photoaging is a long-term process that results fromchronic exposure of the skin to solar UV radiation UV radia-tion has been shown to cause a variety of lesions in DNAincluding pyrimidine dimmers of cyclobutane type pho-toadducts of the pyrimidine pyrimidine type and othernucleic acid base photoproducts such a single-strand breakand DNA-protein cross-links These photoproducts are

implicated in cellular lethality mutation and other biologicaleffects [3] Photoaging is characterized by thickening rough-ness coarse wrinkles mottled pigmentation and histologicchanges including damage to collagen fibers the excessivedeposition of abnormal elastic fibers and an increase inglycosaminoglycans [4ndash6] This phenotype is caused byalterations in cellular function as well as deterioration of theextracellular matrix of skin connective tissue (dermis) Thedermis provides structural support for the skin and chronicsolar UV exposure leads to fragmentation and disorganiza-tion of the primary structural protein elastin and collagen [7]

Several studies have evaluated the protective effect ofnatural products against damage induced by UVR in cellstissues animals and humans Photochemoprevention is theuse of synthetic or natural substances that prevent delayor reverse the damage caused by UVR [8 9] Within this

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 587819 10 pageshttpdxdoiorg1011552014587819

2 BioMed Research International

concept we can find a variety of polyphenolic compoundswith antioxidant anti-inflammatory immunomodulatoryand antimutagenic properties In general photochemopre-ventive agents act on two levels (a) prevention of the damagecaused by UVR and (b) modulation of different cellular res-ponses to UVR that can prevent stop or correct tumor pro-motion and progression [1 9 10]

Free radicals and reactive oxygen species can be generatednot only in metabolic reactions but also by UV radiationespecially by UVA (320ndash400 nm) radiation involving sensi-tized reactions There are many endogenous chromophoresin human skin which in the presence of UVA radiation cangenerate reactive oxygen species in the connective tissue ofskin [3] A decrease in the reactive oxygen species (ROS)load by efficient sunscreens andor other protective agentsmay represent a promising strategy in preventing or at leastminimizing ROS-induced cutaneous pathological states [11ndash13] It has been suggested that the beneficial effects of topicalantioxidants might be a successful strategy for diminishingUV radiation-mediated oxidative damage of the skin [14]Thus botanical antioxidants have shown good potential asphotoprotective agents [15]

The tropical plantMorinda citrifolia commonly known asldquoNonirdquo is widely distributed in areas of Micronesia HawaiiAustralia and Southeast AsiaThe genusMorinda belongingto the family Rubiaceae is indigenous to tropical countriesand is considered an important traditional folk medicine [1617]

The fruits roots barks and leaves of M citrifolia havebeen used as folk medicine for the treatment of various ill-nesses including burns cancer cold sores skin inflamma-tion and wounds among other things [18 19]

In previous study we demonstrated that the aque-ous extract from M citrifolia leaves contains alkaloidscoumarins flavonoids tannins saponins steroids and triter-penoids and showed relevant in vitro antioxidant activityagainst different radicals A carbomer gel base containing theethanol extract and juice pressed from the leaves was eva-luated in a UVB-induced erythema and the UVB doserequired to induce erythema was almost 35 times greaterthan with untreated skin [20] Based on this data we decidedto investigate the potential benefits of topical application ofaqueous extract from Morinda citrifolia on skin exposed toUVA-UVB light

2 Material and Methods

21 Preparation of Morinda citrifolia Extract M citrifo-lia leaves were collected in Sao Cristovao Sergipe Brazil[10∘18101584020710158401015840 (S) 36∘39101584010158407210158401015840 (W)] Herbarium voucher spec-imens (registry number 13503) were prepared and depositedat the Department of Biology Federal University of SergipeThe extract was prepared by boiling dry leaves in distilledwater (75wv) for 15minutes the solvent evaporated underreduced pressure and lyophilized

22 Preparation of Morinda citrifolia Formulations TheMorinda citrifolia extract (0 10 or 15 ww) was dis-solved in water (25mL) and in ethanol (05mL) Then the

formulation was suspended in hydroxyethylcellulose (2)Imidazolidinyl urea (20 120583L) was added at the end The con-trolled formulations did not contain the Morinda citrifoliaextract (0) The formulations were stored at 4∘C

23 Measurement of Spreadability The spreadability of theformulations was determined by compressing the sampleunder several glass plates of knownweight [21] Twenty plateswere subsequently placed over the sample at 1 min intervalsThe spreading areas reached by the sample were measured inmillimeters on vertical and horizontal axes The results wereexpressed in terms of the spreading area as a function of theapplied mass according to the following equation (1)

119878119894= 1198892

times120587

4(1)

in which 119878119894is the spreading area (mm2) resulting from the

applied mass 119894(119892) and 119889 is the mean diameter (mm) reachedby the sample The spreading area was plotted against theplate weight to obtain the spreading profiles The experimentwas repeated three times and the mean time was taken forcalculation

24 In Vitro Effectiveness The formulations were scannedunder UV light (200 to 400 nm) using a UV-1 800 PCspectrophotometer Samples were placed in a quartz cuvette(model 4-QHellmaGermany) presenting a capacity of 3mLand an optical path length of 1 cm To subtract the scatteringof the gel matrix a blank formulation (0 extract) preparedexclusively with hydroxyethylcellulose and ethanolwater wasused as the baseline [22]

25 In Vivo Effectiveness Male CF1 mice weighing 20ndash30 g(119899 = 10 per group) were housed in a temperature-controlledroomwith access to water and food ad libitum until useTheywere housed within cages with a 12 h light and 12 h dark cycleAll experiments were conducted in accordance with NationalInstitutes of Health guidelines for the welfare of experimentalanimals and with the approval of the Ethics Committee of theFaculty of Pharmaceutical Science Porto Alegre Rio Grandedo Sul (number 21804)

The animals were divided into five groups (119899 = 10)group 1 = nonirradiated control-NIC (the group was notirradiated and did not receive any topical treatment) group2 = irradiated control-IC (the group was irradiated but didnot receive any topical treatment) group 3 = irradiated andtreated with the formulation 10 group 4 = irradiated andtreated with the formulation 15 and group 5 = irradiatedand treated with the vehicle-IV (formulation 0)

Animals were anesthetized before formulation adminis-tration using intraperitoneal ketamine (80mgkg) and xila-sine (10mgkg)

26 Formulation Administration CF1 mice were randomlydesigned to different groups with 10 mice in each groupAfter the removal of dorsal hair (by trichotomy followed by2min exposure of the surface to Veet cream) the mice weretopically treated on the dorsal surface (2 times 2 cm) with 01 gof formulations daily The formulations were administrated

BioMed Research International 3

30min before the dorsal surface irradiation The untreatedcontrol groups irradiated and nonirradiated were included inthe experiments

27 Irradiation The UVA-UVB source of irradiation con-sisted of a Repti Glo 100 lamp (Exo Terra) emitting acontinuous spectrum between 270 and 400 nm 33 UVAand 10 UVB The lamp was mounted 20 cm above thesupport where the mice were placed The UVA-UVB outputwasmeasured using amodel UV-400 Research Radiometer (Icel) with a radiometer sensor forUVAandUVBThemajorityof the resulting wavelengths were in the UVB (290ndash320 nmabove 90) and UVA (less than 10) range and the peakemission was recorded at 314 nm The mice were irradiatedfor 7 days 2 hday with a daily dose of 19 Jcm2

28 Assessment of Biophysical Skin Properties The thicknessof irradiated skin wasmeasured by 7 days of treatment by dialthickness gage (Mitutoyo)

Transepidermal water loss (TEWL) was measured usinga Tewameter R TM 210 (Courage amp Khazaka ElectronicGmbH Germany) before (baseline values) and 20 h after UVexposure with 7 days of treatment Readings of TEWL wereregistered in gm2sdoth during 2min after 30 s probe equili-bration on the skin The UV-induced skin barrier damageassessed by TEWL measurements is due to abnormalitiesin the structures related to corneocytes adhesion and todisruption of the epidermal permeability barrier function[23]

Erythema was measured photometrically using a Mex-ameter R MX16 (Couragem amp Khazaka Eletronic GmbHCologne Germany) before (baseline values) and 20 h afterUV exposure over 7 days Six readings of the erythema indexwere recorded at each area at both points in time The degreeof erythema quantified as an erythema index is related to thehemoglobin content

29 Animal Sacrifice After treatment over 7 days the micewere sacrificed by cervical dislocation 2 h after the ultimateUVA-UVB exposure The full thickness of the dorsal skinswas removed and fixed in 10 formalin until histologicalmeasurements

210 Histological Measurements Skin biopsies were fixed in10 formalin embedded in paraffin according to routineprotocol of histological procedures Five micrometer thinsections of the paraffin-embedded skin were obtained andstained by means of hematoxylin eosin (HE) and Siriusred (SR) histochemical methods Morphological analysis ofthe histological sections was performed by light microscopyfollowed by a closed numerical protocol in such a manneras the pathologist was not aware of which group was beingevaluated until the end of the experiment

211 Measurement of Cytotoxicity Activity The B16F10(murine malignant melanoma ATCC CRL-6475) cellswere obtained from the Rio de Janeiro Cell Bank BrazilThey were routinely grown in 150 cm2 tissue culture flasksin DMEM supplemented with 1 (vv) of an antibiotic

solution containing 5mg of penicillin 5mg of streptomycinand 10mg of neomycin per mL and 75 or 100 (vv)heat-inactivated Fetal Bovine Serum at 37∘C under 5 CO

2

The sensitivity of the cells to the Morinda citrifoliaextract was determined by a standard spectrophotometric3-(45-dimethylthiazole-2-yl)-25-diphenyltetrazoliumbromide (MTT) assay [24] Cells were seeded at a density of105 cellswells into 96-well plates and incubated for 24 h at37∘C in an atmosphere of 95 air and 5 CO

2 Then 20120583L

of extract at different concentrations in a phosphate buffersaline (PBS) was added to the culture plates for 24 h Aftertreatment cells were rinsed once with PBS and a serum-freeculture medium without phenol red and were replaced in allwells Cells were then incubated for 4 h with MTT solution(5mgmL)

The yellow tetrazolium salt was metabolized by viablecells to form purple crystals of formazan The crystals weresolubilized overnight (12 h) in a mixture consisting of 20sodium dodecyl sulfate (SDS) in HCl (001M) The productwas quantified spectrophotometrically by measuring theabsorbency at 570 nmusing amicroplate reader (120583QuantTMBioTek Instruments Inc USA) The cellular viability wasexpressed as a percentage of viable cells compared to thecontrol group

The percentage of viable cells was calculated as follows

Viability

=Total number of viable cells

Total number of viable and nonviable cellstimes 100

(2)

212 Statistical Analysis Data were expressed as mean plusmnstandard errorof the mean (SEM) or plusmn standard deviation(SD) The data obtained was evaluated by one-way analysisof variance (ANOVA) followed by Tukeyrsquos test Data analyseswere performed using the GraphPad Prism 50 software Inall cases differences were considered significant if 119875 lt 005

3 Results

31 Measurement of Spreadability The spreadability is anessential characteristic of semisolid pharmaceutical dosageforms for dermal application Thus the spreadability fac-tor was measured (Table 1) The formulation contain-ing the extract at 10 presented the higher spreadabil-ity (1144mm2sdotgminus1) against the formulation containing theextract at 15 (0916mm2sdotgminus1)

32 In Vitro Effectiveness The absorbance spectrum of theformulations extracts (10 and 15) (Figure 1) showed bandsat 200 nm and at 240 nm and it showed absorbance in theUVA and UVB regions The blank formulation (0 extract)did not show absorbance in this spectra

33 Biophysical Skin Properties The results showed that thethickness TEWL values and erythema indexes were signifi-cantly decreased by treatment with formulations containingthe extract (Figures 2(a) 2(b) and 2(c) resp)

4 BioMed Research International

000020040

Abso

rban

ce (a

u)

060080100120140160180

2000 3000 4000 5000 6000Wavelength (nm)

7000 8000

15100

Figure 1 UV absorbance spectra of a blank formulation (0 extract) formulation extract 10 and formulation extract 15The formulationswere scanned under UV light (200 to 400 nm)

IV IC NIC 10 1500

05

10

15

20

25

Groups n = 10

Thic

knes

s (m

m)

lowastlowastlowastlowastlowastlowast lowastlowastlowast

(a)

IV IC NIC 10 150

10

20

30

40

Groups n = 10

lowastlowastlowastlowastlowastlowast lowastlowast

TEW

L (g

h m

2)

(b)

IV IC NIC 10 150

100

200

300

400

Groups n = 10

Eryt

hem

a ind

ex lowastlowast

lowastlowastlowast

(c)

Figure 2 (a) Thickness values (mm) (b) transepidermal water loss and (c) erythema obtained for the dorsal areas IV = irradiated andtreated with the vehicle (formulation 0) IC = irradiated control (the group was irradiated but did not receive any topical treatment) NIC= nonirradiated control (the group was not irradiated and did not receive any topical treatment) 10 = group irradiated and treated withthe formulation 10 15 = group irradiated and treated with the formulation 15 The values were obtained before (baseline values) and20 h after UV exposure during 7 days of treatment (119899 = 10 mice) 119875 lt 005 versus IC lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus IVANOVA followed by Tukeyrsquos test Data represents mean plusmn SEM

BioMed Research International 5

Table 1 Measurement of spreadability of formulations containingMorinda citrifolia extract (10 or 15 ww)

Formulations lowastSP1 lowastSP2 lowastSP3 lowastSP Mean lowastSP Mean plusmn SD CVExtract 10 1136 1044 1253 1144 1144 plusmn 107 94Extract 15 0955 0824 0969 0916 0916 plusmn 008 87lowastSpreadability factor (mm2

sdotgminus1)

IV IC NIC 10 15

Figure 3 Skin biophysical techniques Photoprotective effects of formulations onUVA-UVB-induced pathological changes in the skin surfaceon different groups after 7 days of treatment IV = irradiated and treated with the vehicle (formulation 0) IC = irradiated control (the groupwas irradiated but did not receive any topical treatment) NIC = nonirradiated control (the group was not irradiated and did not receive anytopical treatment) 10 = group irradiated and treated with the formulation 10 15 = group irradiated and treated with the formulation15

34 Pathological Changes in the Skin Surface Treated areaswith the formulation containing Morinda citrifolia extract(10 and 15) have enhanced protection against UV radiation(Figure 3)

35 Histological Measurements

Hematoxylin Eosin (HE) As demonstrated in Figure 4(a) thenonirradiated group showed that the epidermis was repre-sented by a thin continuous squamous epithelial liningwith an orthokeratotic surfaceThe connective tissue showedinterlaced collagen fibers associated with spindle and roundshaped stromal cells consistent with fibroblasts Some fewcapillary vessels and venules could also be seen as wellas numerous cutaneous appendages No inflammatory infil-trates were found in the dermal tissues Both irradiated andvehicle groups (Figures 4(b) and 4(c)) presented increasedepidermal thickness associated to hyperorthokeratosis Thepapillary dermis exhibited intense inflammatory infiltratemainly composed of lymphocytes mild interstitial edemaand prominent capillary and venular hyperemia Extensiveareas of elastotic alterations of the collagen bundles (baso-philic degeneration) were also observed throughout theconnective tissue (Figure 4(d))The treated groups presentedremarkable reduction in the inflammatory content as wellas less expressive hyperemia However epidermal thickeningwas more evident in 10 than in 15 formulation (Figures4(e) and 4(f)) Such similar histological features were quite

similar to those verified in the normal dermalepidermal tis-suesSirius Red (SR) The nonirradiated-NIC groups presentedinterlaced less compacted type I and III collagen fibers (Fig-ures 5(a) and 5(b)) In irradiated and vehicle groups thepapillary dermis showed the presence of gross thick parallel-arranged collagen fibers exhibiting intense yellow birefrin-gence (type I collagen) which were associated to shorter andthinner reddish fibrils perpendicularly disposed in relationto the surface Such collagen bundles were densely com-pacted showing sparse interfibrillar spaces (Figures 5(c) and5(d)) Both treated groups (10 and 15 formulations) pre-sented similar features regarding the collagenization patternshowing a mixture of yellowish type I (predominant) andgreenish type III collagen fibers These fibers were moredelicate slightly wavy with variable thicknesses lengths andexhibited interlaced arrangements (Figures 5(e) and 5(f))The interfibrillary spaces were more evident than in irradi-ated and vehicle groups Such morphological appearanceswere very similar to those observed in nonirradiated group(Figures 5(a) and 5(b))

36 Measurement of Cytotoxicity Activity The extract wasevaluated on murine malignant melanoma (cell line B16F10)in order to examine their cytotoxic effects on malignant cellsCytotoxicity of the extract on the growth of the B16F10 cellline is shown in Figure 6 Cell proliferation was analyzed

6 BioMed Research International

70120583m

(a)

70120583m

(b)

70120583m

(c)

70120583m

(d)

70120583m

(e)

70120583m

(f)

Figure 4 Hematoxylin-eosin stained histological sections (a) Nonirradiated group-NIC showing thin epidermal lining and absence ofdermal inflammation (b) Irradiated-IC and (c) vehicle groups-IV presenting epidermal thickening and intense lymphocytic infiltration in thedermal tissue (d) Elastotic change of the collagen (e) 10 and (f) 15 formulations showingmarked reduction of the inflammatory responseNote the thin epidermis in 15 formulation Short thick arrows epidermis Long thin arrows dermal connective tissues (Hematoxylin Eosin400x magnification)

24 h after B16F10 cells had been cultured with the extract atdifferent concentrations (005 01 02 04 08 16 32 64128 and 25mgmL) using the MTT assay The extract atconcentrations of 005 to 128mgmL did not show any cyto-toxic effects in the B16F10 cell line (showed no potential anti-cancer) A reduction in cell viability at high concentrationswas observed (25mgmL)

4 Discussion

The most distinguished harmful effect of solar exposure ispremature skin aging especially UV irradiation In recent

years thinning of the ozone layer has caused increased UVexposure Detrimental effects of UV on the skin involvethe generation of reactive oxygen species (ROS) [25] Com-ponents of the skin including DNA lipids and proteinscan be impaired by ROS which is the cause of erythemasunburn immune suppression and skin cancer [26] Most ofthe disorders were formerly regarded as having been causedbyUVBNevertheless recent studies showed that UVA is alsoan essential factor [27]

Accordingly use of antioxidants capable of scavengingand quenching ROS is another important approach to pre-vent UVB induced photoaging [27] Other studies have

BioMed Research International 7

70120583m

(a)

70120583m

(b)

70120583m

(c)

70120583m

(d)

70120583m

(e)

70120583m

(f)

Figure 5 Sirius Red stained histological sections (a b)Nonirradiated-NICgroups presenting interlaced less compacted type I and III collagenfibers (c) Irradiated-IC and (d) vehicle groups-IV showing dense disposition of parallel-arranged type I collagen fibers (e) 10 and (f) 15formulations presenting morphological and architectural collagen deposition pattern resembling that observed in the normal dermis (SiriusRedPolarization Light 400x magnification)

shown that botanical compounds with an antioxidative activ-ity are potential agents capable of reducing skin diseasesincluding photoaging [28 29]

The extract used in this work was the same studied bySerafini et al [19] and showed relevant in vitro antioxidantactivity against different radicals The extract was chemicallyanalyzed (qualitative method) The phytochemical screeningof extract showed the presence of alkaloids coumarins flavo-noids tannins saponins steroids and triterpenoids Totalphenolic content of the extract was 1968mg of phenolic equi-valents (gallic acid) per gram of extract Furthermore quali-tative analysis of extract was performed by HPLC revealingthe presence of 3 major compounds all showing ultraviolet

spectra typical of flavonols (quercetin-3-O-rutinoside orrutin and kaempferol glycosides) According to Deng et al[30] flavonol glycosides mainly rutin are the major com-pounds inM citrifolia leaves

The presence of rutin in the formulation could also besecondarily involved in this process of collagenization Ithas been demonstrated that the rutin is able to impair theglycation of collagen [31] a biochemical event involved in thepathogenesis of cell damaging in UV-irradiated tissue [32]

The use of botanical supplements to protect the humanskin from the adverse biological effects of solar radiationhas received great interest in recent years [33] Howevermost tests have been conducted by oral ingestion or topical

8 BioMed Research International

005 01 02 04 08 16 32 64 128 2500

50

100

150

Morinda citrifolia extract (mgmL)

Cel

l via

bilit

y (

)

Figure 6 Cytotoxic effect of theMorinda citrifolia extract in B16F10(murine malignant melanoma) cell line pretreated with the extractsat concentrations (005 to 25mgmL) for 24 h Each value representsthe mean plusmn SD

application of these ingredients in inappropriate vehicleswhich can alter the skin barrier To be of practical use todermatologists and to be safe and efficient extracts musthave physicochemical characteristics that permit their incor-poration in adequate concentrations in a skin-compatiblevehicle (eg gels moisturizing creams) [34] In this studythe capacity of the Morinda citrifolia extract added totopical formulations (gels) to prevent or to treat UVA-UVBirradiation-induced skin damage in mice was evaluated

First three formulations presenting different concen-trations (0 10 and 15) were evaluated in vitro byspectrophotometer UV absorbance spectra showed peaksat 200 nm and at 240 nm (UVC region) and absorbance inthe UVA (320ndash400 nm) and UVB (290ndash320 nm) regionsTheblank formulation (0 extract) did not show absorbance inthis spectra These data are compared to commercial organicfiltersThus this result suggests a photoprotective effect of theextractThe concentration of formulation (10 and 15ww)used in this assay was selected based on a pilot study

Second the gel formulation efficacy against UVA-UVBinduced skin damage was evaluated in vivo by measuring thefollowing parameters thickness values (mm) transepidermalwater loss (TEWL) and erythema

Moisturizing antifree radicals and sunscreen productshave been used largely to prevent skin disorders resultingfrom UV damage However other aspects of skin damageshould be considered such as erythema sunburn cell forma-tion epidermal hyperplasia and impairment of the skin bar-rier integrity which in turn is related to skin dryness skinaging and increased incidence of irritant contact dermatitis[35]

UV radiation damages the structures responsible for cor-neocyte adhesion and the functioning of the permeabilitybarrier which in turn alter thicknesses and TEWL measure-ments and accelerate the desquamation process [36]

The thickness TEWL and erythema were observed inour study and only the formulations containing M citri-folia extract protected mouse skin against the UV-induceddamages The pronounced protective effects provided by

both formulations (10 and 15) in the three parametersmeasured were not due to any extraneous raw material butwere shown by the unprotected groups treated by vehicle only(VI) Furthermore the results show that areas treated withthis extract (10 and 15) were statistically equivalent to theunirradiated control ones (NIC)which represented the intactskin areas

TEWL is an important parameter to be evaluated instudies of skin photoprotective effects considering that UVirradiation of mammalian skin disrupts the epidermal per-meability barrier function accompanied by an increase intransepidermal water loss (TEWL) and alterations in thestratum corneum lipid profile [33]

In the TEWL measurement the group 10 was differentfrom IC and the 15 was not As well as in the thickness andTEWL the protective effect of 10 was most pronouncedIt can also be seen in the photos of the dorsal of the ani-mal This observationcan be explained by differences in thespreadability Formulations containing extract at 10 pre-sented the higher spreadability

The spreadability is represented by the thickness of thefilm that the preparation leaves on the skin an importantfeature in cosmetics [37]Those producing thinner films thatis higher spreadability are naturally of greater interest [38]

Formulations containing extracts were shown to be effec-tive also in the presence of erythema another parameteroften used to evaluate photoprotection Extract formulationsreduced erythema formation when compared with the irra-diated control (IC) This statement corroborates [20] whichdemonstrated that the M citrifolia leaf extracts mitigate theeffects of UV light and provide some measure of defenseagainst localized inflammation (topical microswelling)

Third histological parameters were analyzed Epidermalthickening and inflammatory changes are common histo-logical changes observed in mice skin after short-term UVexposure [38 39] Such an increase in the epidermal thicknessis supposed to occur in response to cell proliferation inattempt to regenerate the tissue leading to a compensatorytype of hyperplasia However the thickened epidermis aswell as the thickened keratin layer (hyperkeratosis) could beobserved in this study

The presence of intense inflammatory infiltrate elastosisand the accumulation of parallel-arranged type I collagenfibers observed in IC and vehicle groups have also beendescribed as UV-induced dermal changes [31] Inflammationis one of the major histological signs of tissue damage andhas been reported in other studies as an early change of theconnective tissue after UV exposure [38 39] On the otherhand elastosis represents an actinic-induced accumulationof amorphous elastin material replacing the normal der-mis unrelated to preexisting or newly synthesized collagen[40] Moreover it has been proposed that UV irradiationimpairs ongoing collagen synthesis primarily through thedownregulation of types I and III procollagen expression[41] Therefore such impairment could likely promote delayin the collagen remodeling leading to the typical immatureparallel-arranged disposition of the fibers [31]

In this study only 15 formulation avoided compen-satory epidermal thickening However the reduction of the

BioMed Research International 9

major deleterious effects associated with the UV exposureon the animals treated with both 10 and 15 formulations isstrongly suggestive of photoprotective effect regardless of theextract concentration

Lastly the extract was evaluated on murine malignantmelanoma in order to examine its cytotoxic effects on malig-nant cells A reduction in cell viability was observed only atthe highest concentration (25mgmL) The results can sug-gest that the extract (in low concentrations) has only photo-protection effects and does not have antimelanoma activity

5 Conclusion

In conclusion present results strongly suggest the photo-protective effect of M citrifolia The results indicate thatphotoprotective effects are due to not only UV absorbancelike the UV filters but also biological effects which happenedmainly with the application of the formulation containing theMorinda citrifolia extract Further studies are necessary toclarify the precise nature of this photoprotective effect

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by Fundacao de Amparo a Pesquisado Estado de Sergipe (FAPITEC-SE) and Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) ofBrazil

References

[1] A M Garcıa-Bores and J G Avila ldquoProducts ldquoMolecularmechanisms in the photochemopreventionrdquordquo Revista LatinoAmericana de Quımica vol 36 no 3 pp 83ndash102 2008

[2] M D Adil P Kaiser N K Satti A M Zargar R A Vish-wakarma and S A Tasduq ldquoEffect of Emblica officinalis (fruit)against UVB-induced photo-aging in human skin fibroblastsrdquoJournal of Ethnopharmacology vol 132 no 1 pp 109ndash114 2010

[3] M Dalle Carbonare and M A Pathak ldquoSkin photosensitizingagents and the role of reactive oxygen species in photoagingrdquoJournal of Photochemistry and Photobiology B Biology vol 14no 1-2 pp 105ndash124 1992

[4] G J Fisher S Datta ZWang et al ldquoc-Jun-dependent inhibitionof cutaneous procollagen transcription following ultravioletirradiation is reversed by all-trans retinoic acidrdquoThe Journal ofClinical Investigation vol 106 no 5 pp 663ndash670 2000

[5] S Inomata Y Matsunaga S Amano et al ldquoPossible involve-ment of gelatinases in basementmembrane damage andwrinkleformation in chronically ultraviolet B-exposed hairless mouserdquoJournal of Investigative Dermatology vol 120 no 1 pp 128ndash1342003

[6] P S Peres V A Terra F A Guarnier R Cecchini and A LCecchini ldquoPhotoaging and chronological aging profile under-standing oxidation of the skinrdquo Journal of Photochemistry andPhotobiology B Biology vol 103 no 2 pp 93ndash97 2011

[7] Y Xu and G J Fisher ldquoUltraviolet (UV) light irradiationinduced signal transduction in skin photoagingrdquo Journal of Der-matological Science Supplement vol 1 no 2 pp S1ndashS8 2005

[8] S P Stratton R T Dorr and D S Alberts ldquoThe state-of-the-artin chemoprevention of skin cancerrdquoEuropean Journal of Cancervol 36 no 10 pp 1292ndash1297 2000

[9] F Afaq V M Adhami and H Mukhtar ldquoPhotochemopreven-tion of ultraviolet B signaling and photocarcinogenesisrdquoMuta-tion Research vol 571 no 1-2 pp 153ndash173 2005

[10] V M Adhami D N Syed N Khan and F Afaq ldquoPhytochem-icals for prevention of solar ultraviolet radiation-induced dam-agesrdquo Photochemistry and Photobiology vol 84 no 2 pp 489ndash500 2008

[11] C D Ropke T CH Sawada V V da Silva N SMichalany andS B DeMoraes Barros ldquoPhotoprotective effect of Pothomorpheumbellata root extract against ultraviolet radiation inducedchronic skin damage in the hairless mouserdquo Clinical andExperimental Dermatology vol 30 no 3 pp 272ndash276 2005

[12] M G D Melo J P A dos Santos M R Serafini et al ldquoRedoxproperties and cytoprotective actions of atranorin a lichensecondary metaboliterdquo Toxicology in Vitro vol 25 no 2 pp462ndash468 2011

[13] M R Serafini D M C Vergne T K Rabelo et al ldquoDetermi-nation of chemical and physical properties of Hyptis pectinataessential oil and their redox active profilerdquo Journal of Biotech-nology and Pharmaceutical Research vol 3 no 1 pp 1ndash9 2012

[14] R Casagrande S R Georgetti W A Verri Jr D J Dorta A Cdos Santos and M J V Fonseca ldquoProtective effect of topicalformulations containing quercetin against UVB-induced oxi-dative stress in hairless micerdquo Journal of Photochemistry andPhotobiology B Biology vol 84 no 1 pp 21ndash27 2006

[15] S Frsquoguyer F Afaq and H Mukhtar ldquoPhotochemopreventionof skin cancer by botanical agentsrdquo Photodermatology Photoim-munology and Photomedicine vol 19 no 2 pp 56ndash72 2003

[16] Y Chan-Blanco F Vaillant A Mercedes Perez M Reynes JBrillouet and P Brat ldquoThe noni fruit (Morinda citrifolia L) areview of agricultural research nutritional and therapeutic pro-pertiesrdquo Journal of Food Composition and Analysis vol 19 no 6-7 pp 645ndash654 2006

[17] H Kim M Kwon J Kim et al ldquoIdentification of novel fattyacid glucosides from the tropical fruit Morinda citrifolia LrdquoPhytochemistry Letters vol 3 no 4 pp 238ndash241 2010

[18] A Hirazumi E Furusawa S C Chou and Y Hokama ldquoImmu-nomodulation contributes to the anticancer activity ofMorindacitrifolia (noni) fruit juicerdquo Proceedings of the Western Pharma-cology Society vol 39 pp 7ndash9 1996

[19] M R Serafini R C Santos A G Guimaraes et al ldquoMorindacitrifolia linn leaf extract possesses antioxidant activities andreduces nociceptive behavior and leukocyte migrationrdquo Journalof Medicinal Food vol 14 no 10 pp 1159ndash1166 2011

[20] B J West S Deng A K Palu and C J Jensen ldquoMorinda citri-folia Linn (Rubiaceae) leaf extracts mitigate UVB-induced ery-themardquo Journal of Natural Medicines vol 63 no 3 pp 351ndash3542009

[21] A Garg D Aggarwal S Garg and A Singela ldquoSpreading ofsemisolid formulations pharmaceutical technologyrdquo Pharma-ceutical Technology vol 26 pp 84ndash105 2002

[22] K Paese A Jager F S Poletto et al ldquoSemisolid formulationcontaining a nanoencapsulated sunscreen effectiveness in vitrophotostability and immune responserdquo Journal of Biomedical andNanotechnology vol 5 no 3 pp 240ndash246 2009

10 BioMed Research International

[23] M Demerjian P Hachem E Tschachler et al ldquoAcute modula-tions in permeability barrier function regulate epidermal corni-ficationrdquo The American Journal of Pathology vol 172 no 1 pp86ndash97 2008

[24] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[25] H Sakurai H Yasui Y Yamada H Nishimura and M Shige-moto ldquoDetection of reactive oxygen species in the skin of livemice and rats exposed toUVA light a research reviewon chemi-luminescence and trials for UVA protectionrdquo Photochemical ampPhotobiological Sciences vol 4 no 9 pp 715ndash720 2005

[26] R Haywood P Wardman R Sanders and C Linge ldquoSun-screens inadequately protect against ultraviolet-A-induced freeradicals in skin implications for skin aging and melanomardquoJournal of Investigative Dermatology vol 121 no 4 pp 862ndash8682003

[27] F Afaq DN Syed AMalik et al ldquoDelphinidin an anthocyani-din in pigmented fruits and vegetables protects human HaCaTkeratinocytes and mouse skin against UVB-mediated oxidativestress and apoptosisrdquo Journal of Investigative Dermatology vol127 no 1 pp 222ndash232 2007

[28] J-Y Bae J-S Choi Y-J Choi et al ldquo(minus)Epigallocatechin gallatehampers collagen destruction and collagenase activation inultraviolet-B-irradiated humandermal fibroblasts Involvementof mitogen-activated protein kinaserdquo Food and Chemical Toxi-cology vol 46 no 4 pp 1298ndash1307 2008

[29] H Choi D Kim J W Kim S Ngadiran M R Sarmidi and CS Park ldquoLabisia pumila extract protects skin cells from photo-aging caused by UVB irradiationrdquo Journal of Bioscience andBioengineering vol 109 no 3 pp 291ndash296 2010

[30] S Deng B JWest andC J Jensen ldquoSimultaneous characterisa-tion and quantitation of flavonol glycosides and aglycones innoni leaves using a validated HPLC-UVMS methodrdquo FoodChemistry vol 111 no 2 pp 526ndash529 2008

[31] D Cervantes-Laurean D D Schramm E L Jacobson IHalaweish G G Bruckner and G A Boissonneault ldquoInhibi-tion of advanced glycation end product formation on collagenby rutin and its metabolitesrdquo Journal of Nutritional Biochem-istry vol 17 no 8 pp 531ndash540 2006

[32] Y M Fonseca C D Catini F T M C Vicentini J C CardosoR L Cavalcanti De Albuquerque Junior and M J Vieira Fon-seca ldquoEfficacy of marigold extract-loaded formulations againstUV-induced oxidative stressrdquo Journal of Pharmaceutical Sci-ences vol 100 no 6 pp 2182ndash2193 2011

[33] P K Vayalil C A Elments and S K Katiyar ldquoTreatment ofgreen tea polyphenols in hydrophilic cream prevents UVB-induced oxidation of lipids and proteins depletion of antioxi-dant enzymes and phosphorylation ofMAPKproteins in SKH-1hairless mouse skinrdquo Carcinogenesis vol 24 no 5 pp 927ndash9362003

[34] S E Dal Belo L R Gaspar and PM B GM Campos ldquoPhoto-protective effects of topical formulations containing a combi-nation of Ginkgo biloba and green tea extractsrdquo PhytotherapyResearch vol 25 no 12 pp 1854ndash1860 2011

[35] R Ghadially B E Brown S M Sequeira-Martin K R Fein-gold and P M Elias ldquoThe aged epidermal permeability barrierStructural functional and lipid biochemical abnormalities inhumans and a senescent murine modelrdquo Journal of ClinicalInvestigation vol 95 no 5 pp 2281ndash2290 1995

[36] M D Contreras and R Sanchez ldquoApplication of a factorialdesign to the study of the flow behavior spreadability and trans-parency of a Carbopol ETD 2020 gel Part IIrdquo International Jour-nal of Pharmaceutics vol 234 no 1-2 pp 149ndash157 2002

[37] BW BarryRheology ofDermatological VehiclesMarcelDekkerNew York NY USA 1983

[38] S U Kim S J Kim J Y Lee et al ldquoProtective effects of dietarysoy isoflavones against UV-induced skin-aging in hairlessmouse modelrdquo Journal of the American College of Nutrition vol23 no 2 pp 157ndash162 2004

[39] M Amouroux G Dıaz-Ayil W C P M Blondel G Bourg-Heckly A Leroux and F Guillemin ldquoClassification of ultra-violet irradiated mouse skin histological stages by bimodalspectroscopy multiple excitation autofluorescence and diffusereflectancerdquo Journal of Biomedical Optics vol 14 no 1 ArticleID 014011 2009

[40] K A Hwang B R Yi and K C Choi ldquoMolecular Mechanismsand In Vivo Mouse Models of Skin Aging Associated withDermal Matrix Alterationsrdquo Laboratory Animal Research vol27 no 1 pp 1ndash8 2011

[41] G J Fisher S Kang J Varani et al ldquoMechanisms of photoagingand chronological skin agingrdquoArchives of Dermatology vol 138no 11 pp 1462ndash1470 2002

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

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AntibioticsInternational Journal of

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StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

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Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

2 BioMed Research International

concept we can find a variety of polyphenolic compoundswith antioxidant anti-inflammatory immunomodulatoryand antimutagenic properties In general photochemopre-ventive agents act on two levels (a) prevention of the damagecaused by UVR and (b) modulation of different cellular res-ponses to UVR that can prevent stop or correct tumor pro-motion and progression [1 9 10]

Free radicals and reactive oxygen species can be generatednot only in metabolic reactions but also by UV radiationespecially by UVA (320ndash400 nm) radiation involving sensi-tized reactions There are many endogenous chromophoresin human skin which in the presence of UVA radiation cangenerate reactive oxygen species in the connective tissue ofskin [3] A decrease in the reactive oxygen species (ROS)load by efficient sunscreens andor other protective agentsmay represent a promising strategy in preventing or at leastminimizing ROS-induced cutaneous pathological states [11ndash13] It has been suggested that the beneficial effects of topicalantioxidants might be a successful strategy for diminishingUV radiation-mediated oxidative damage of the skin [14]Thus botanical antioxidants have shown good potential asphotoprotective agents [15]

The tropical plantMorinda citrifolia commonly known asldquoNonirdquo is widely distributed in areas of Micronesia HawaiiAustralia and Southeast AsiaThe genusMorinda belongingto the family Rubiaceae is indigenous to tropical countriesand is considered an important traditional folk medicine [1617]

The fruits roots barks and leaves of M citrifolia havebeen used as folk medicine for the treatment of various ill-nesses including burns cancer cold sores skin inflamma-tion and wounds among other things [18 19]

In previous study we demonstrated that the aque-ous extract from M citrifolia leaves contains alkaloidscoumarins flavonoids tannins saponins steroids and triter-penoids and showed relevant in vitro antioxidant activityagainst different radicals A carbomer gel base containing theethanol extract and juice pressed from the leaves was eva-luated in a UVB-induced erythema and the UVB doserequired to induce erythema was almost 35 times greaterthan with untreated skin [20] Based on this data we decidedto investigate the potential benefits of topical application ofaqueous extract from Morinda citrifolia on skin exposed toUVA-UVB light

2 Material and Methods

21 Preparation of Morinda citrifolia Extract M citrifo-lia leaves were collected in Sao Cristovao Sergipe Brazil[10∘18101584020710158401015840 (S) 36∘39101584010158407210158401015840 (W)] Herbarium voucher spec-imens (registry number 13503) were prepared and depositedat the Department of Biology Federal University of SergipeThe extract was prepared by boiling dry leaves in distilledwater (75wv) for 15minutes the solvent evaporated underreduced pressure and lyophilized

22 Preparation of Morinda citrifolia Formulations TheMorinda citrifolia extract (0 10 or 15 ww) was dis-solved in water (25mL) and in ethanol (05mL) Then the

formulation was suspended in hydroxyethylcellulose (2)Imidazolidinyl urea (20 120583L) was added at the end The con-trolled formulations did not contain the Morinda citrifoliaextract (0) The formulations were stored at 4∘C

23 Measurement of Spreadability The spreadability of theformulations was determined by compressing the sampleunder several glass plates of knownweight [21] Twenty plateswere subsequently placed over the sample at 1 min intervalsThe spreading areas reached by the sample were measured inmillimeters on vertical and horizontal axes The results wereexpressed in terms of the spreading area as a function of theapplied mass according to the following equation (1)

119878119894= 1198892

times120587

4(1)

in which 119878119894is the spreading area (mm2) resulting from the

applied mass 119894(119892) and 119889 is the mean diameter (mm) reachedby the sample The spreading area was plotted against theplate weight to obtain the spreading profiles The experimentwas repeated three times and the mean time was taken forcalculation

24 In Vitro Effectiveness The formulations were scannedunder UV light (200 to 400 nm) using a UV-1 800 PCspectrophotometer Samples were placed in a quartz cuvette(model 4-QHellmaGermany) presenting a capacity of 3mLand an optical path length of 1 cm To subtract the scatteringof the gel matrix a blank formulation (0 extract) preparedexclusively with hydroxyethylcellulose and ethanolwater wasused as the baseline [22]

25 In Vivo Effectiveness Male CF1 mice weighing 20ndash30 g(119899 = 10 per group) were housed in a temperature-controlledroomwith access to water and food ad libitum until useTheywere housed within cages with a 12 h light and 12 h dark cycleAll experiments were conducted in accordance with NationalInstitutes of Health guidelines for the welfare of experimentalanimals and with the approval of the Ethics Committee of theFaculty of Pharmaceutical Science Porto Alegre Rio Grandedo Sul (number 21804)

The animals were divided into five groups (119899 = 10)group 1 = nonirradiated control-NIC (the group was notirradiated and did not receive any topical treatment) group2 = irradiated control-IC (the group was irradiated but didnot receive any topical treatment) group 3 = irradiated andtreated with the formulation 10 group 4 = irradiated andtreated with the formulation 15 and group 5 = irradiatedand treated with the vehicle-IV (formulation 0)

Animals were anesthetized before formulation adminis-tration using intraperitoneal ketamine (80mgkg) and xila-sine (10mgkg)

26 Formulation Administration CF1 mice were randomlydesigned to different groups with 10 mice in each groupAfter the removal of dorsal hair (by trichotomy followed by2min exposure of the surface to Veet cream) the mice weretopically treated on the dorsal surface (2 times 2 cm) with 01 gof formulations daily The formulations were administrated

BioMed Research International 3

30min before the dorsal surface irradiation The untreatedcontrol groups irradiated and nonirradiated were included inthe experiments

27 Irradiation The UVA-UVB source of irradiation con-sisted of a Repti Glo 100 lamp (Exo Terra) emitting acontinuous spectrum between 270 and 400 nm 33 UVAand 10 UVB The lamp was mounted 20 cm above thesupport where the mice were placed The UVA-UVB outputwasmeasured using amodel UV-400 Research Radiometer (Icel) with a radiometer sensor forUVAandUVBThemajorityof the resulting wavelengths were in the UVB (290ndash320 nmabove 90) and UVA (less than 10) range and the peakemission was recorded at 314 nm The mice were irradiatedfor 7 days 2 hday with a daily dose of 19 Jcm2

28 Assessment of Biophysical Skin Properties The thicknessof irradiated skin wasmeasured by 7 days of treatment by dialthickness gage (Mitutoyo)

Transepidermal water loss (TEWL) was measured usinga Tewameter R TM 210 (Courage amp Khazaka ElectronicGmbH Germany) before (baseline values) and 20 h after UVexposure with 7 days of treatment Readings of TEWL wereregistered in gm2sdoth during 2min after 30 s probe equili-bration on the skin The UV-induced skin barrier damageassessed by TEWL measurements is due to abnormalitiesin the structures related to corneocytes adhesion and todisruption of the epidermal permeability barrier function[23]

Erythema was measured photometrically using a Mex-ameter R MX16 (Couragem amp Khazaka Eletronic GmbHCologne Germany) before (baseline values) and 20 h afterUV exposure over 7 days Six readings of the erythema indexwere recorded at each area at both points in time The degreeof erythema quantified as an erythema index is related to thehemoglobin content

29 Animal Sacrifice After treatment over 7 days the micewere sacrificed by cervical dislocation 2 h after the ultimateUVA-UVB exposure The full thickness of the dorsal skinswas removed and fixed in 10 formalin until histologicalmeasurements

210 Histological Measurements Skin biopsies were fixed in10 formalin embedded in paraffin according to routineprotocol of histological procedures Five micrometer thinsections of the paraffin-embedded skin were obtained andstained by means of hematoxylin eosin (HE) and Siriusred (SR) histochemical methods Morphological analysis ofthe histological sections was performed by light microscopyfollowed by a closed numerical protocol in such a manneras the pathologist was not aware of which group was beingevaluated until the end of the experiment

211 Measurement of Cytotoxicity Activity The B16F10(murine malignant melanoma ATCC CRL-6475) cellswere obtained from the Rio de Janeiro Cell Bank BrazilThey were routinely grown in 150 cm2 tissue culture flasksin DMEM supplemented with 1 (vv) of an antibiotic

solution containing 5mg of penicillin 5mg of streptomycinand 10mg of neomycin per mL and 75 or 100 (vv)heat-inactivated Fetal Bovine Serum at 37∘C under 5 CO

2

The sensitivity of the cells to the Morinda citrifoliaextract was determined by a standard spectrophotometric3-(45-dimethylthiazole-2-yl)-25-diphenyltetrazoliumbromide (MTT) assay [24] Cells were seeded at a density of105 cellswells into 96-well plates and incubated for 24 h at37∘C in an atmosphere of 95 air and 5 CO

2 Then 20120583L

of extract at different concentrations in a phosphate buffersaline (PBS) was added to the culture plates for 24 h Aftertreatment cells were rinsed once with PBS and a serum-freeculture medium without phenol red and were replaced in allwells Cells were then incubated for 4 h with MTT solution(5mgmL)

The yellow tetrazolium salt was metabolized by viablecells to form purple crystals of formazan The crystals weresolubilized overnight (12 h) in a mixture consisting of 20sodium dodecyl sulfate (SDS) in HCl (001M) The productwas quantified spectrophotometrically by measuring theabsorbency at 570 nmusing amicroplate reader (120583QuantTMBioTek Instruments Inc USA) The cellular viability wasexpressed as a percentage of viable cells compared to thecontrol group

The percentage of viable cells was calculated as follows

Viability

=Total number of viable cells

Total number of viable and nonviable cellstimes 100

(2)

212 Statistical Analysis Data were expressed as mean plusmnstandard errorof the mean (SEM) or plusmn standard deviation(SD) The data obtained was evaluated by one-way analysisof variance (ANOVA) followed by Tukeyrsquos test Data analyseswere performed using the GraphPad Prism 50 software Inall cases differences were considered significant if 119875 lt 005

3 Results

31 Measurement of Spreadability The spreadability is anessential characteristic of semisolid pharmaceutical dosageforms for dermal application Thus the spreadability fac-tor was measured (Table 1) The formulation contain-ing the extract at 10 presented the higher spreadabil-ity (1144mm2sdotgminus1) against the formulation containing theextract at 15 (0916mm2sdotgminus1)

32 In Vitro Effectiveness The absorbance spectrum of theformulations extracts (10 and 15) (Figure 1) showed bandsat 200 nm and at 240 nm and it showed absorbance in theUVA and UVB regions The blank formulation (0 extract)did not show absorbance in this spectra

33 Biophysical Skin Properties The results showed that thethickness TEWL values and erythema indexes were signifi-cantly decreased by treatment with formulations containingthe extract (Figures 2(a) 2(b) and 2(c) resp)

4 BioMed Research International

000020040

Abso

rban

ce (a

u)

060080100120140160180

2000 3000 4000 5000 6000Wavelength (nm)

7000 8000

15100

Figure 1 UV absorbance spectra of a blank formulation (0 extract) formulation extract 10 and formulation extract 15The formulationswere scanned under UV light (200 to 400 nm)

IV IC NIC 10 1500

05

10

15

20

25

Groups n = 10

Thic

knes

s (m

m)

lowastlowastlowastlowastlowastlowast lowastlowastlowast

(a)

IV IC NIC 10 150

10

20

30

40

Groups n = 10

lowastlowastlowastlowastlowastlowast lowastlowast

TEW

L (g

h m

2)

(b)

IV IC NIC 10 150

100

200

300

400

Groups n = 10

Eryt

hem

a ind

ex lowastlowast

lowastlowastlowast

(c)

Figure 2 (a) Thickness values (mm) (b) transepidermal water loss and (c) erythema obtained for the dorsal areas IV = irradiated andtreated with the vehicle (formulation 0) IC = irradiated control (the group was irradiated but did not receive any topical treatment) NIC= nonirradiated control (the group was not irradiated and did not receive any topical treatment) 10 = group irradiated and treated withthe formulation 10 15 = group irradiated and treated with the formulation 15 The values were obtained before (baseline values) and20 h after UV exposure during 7 days of treatment (119899 = 10 mice) 119875 lt 005 versus IC lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus IVANOVA followed by Tukeyrsquos test Data represents mean plusmn SEM

BioMed Research International 5

Table 1 Measurement of spreadability of formulations containingMorinda citrifolia extract (10 or 15 ww)

Formulations lowastSP1 lowastSP2 lowastSP3 lowastSP Mean lowastSP Mean plusmn SD CVExtract 10 1136 1044 1253 1144 1144 plusmn 107 94Extract 15 0955 0824 0969 0916 0916 plusmn 008 87lowastSpreadability factor (mm2

sdotgminus1)

IV IC NIC 10 15

Figure 3 Skin biophysical techniques Photoprotective effects of formulations onUVA-UVB-induced pathological changes in the skin surfaceon different groups after 7 days of treatment IV = irradiated and treated with the vehicle (formulation 0) IC = irradiated control (the groupwas irradiated but did not receive any topical treatment) NIC = nonirradiated control (the group was not irradiated and did not receive anytopical treatment) 10 = group irradiated and treated with the formulation 10 15 = group irradiated and treated with the formulation15

34 Pathological Changes in the Skin Surface Treated areaswith the formulation containing Morinda citrifolia extract(10 and 15) have enhanced protection against UV radiation(Figure 3)

35 Histological Measurements

Hematoxylin Eosin (HE) As demonstrated in Figure 4(a) thenonirradiated group showed that the epidermis was repre-sented by a thin continuous squamous epithelial liningwith an orthokeratotic surfaceThe connective tissue showedinterlaced collagen fibers associated with spindle and roundshaped stromal cells consistent with fibroblasts Some fewcapillary vessels and venules could also be seen as wellas numerous cutaneous appendages No inflammatory infil-trates were found in the dermal tissues Both irradiated andvehicle groups (Figures 4(b) and 4(c)) presented increasedepidermal thickness associated to hyperorthokeratosis Thepapillary dermis exhibited intense inflammatory infiltratemainly composed of lymphocytes mild interstitial edemaand prominent capillary and venular hyperemia Extensiveareas of elastotic alterations of the collagen bundles (baso-philic degeneration) were also observed throughout theconnective tissue (Figure 4(d))The treated groups presentedremarkable reduction in the inflammatory content as wellas less expressive hyperemia However epidermal thickeningwas more evident in 10 than in 15 formulation (Figures4(e) and 4(f)) Such similar histological features were quite

similar to those verified in the normal dermalepidermal tis-suesSirius Red (SR) The nonirradiated-NIC groups presentedinterlaced less compacted type I and III collagen fibers (Fig-ures 5(a) and 5(b)) In irradiated and vehicle groups thepapillary dermis showed the presence of gross thick parallel-arranged collagen fibers exhibiting intense yellow birefrin-gence (type I collagen) which were associated to shorter andthinner reddish fibrils perpendicularly disposed in relationto the surface Such collagen bundles were densely com-pacted showing sparse interfibrillar spaces (Figures 5(c) and5(d)) Both treated groups (10 and 15 formulations) pre-sented similar features regarding the collagenization patternshowing a mixture of yellowish type I (predominant) andgreenish type III collagen fibers These fibers were moredelicate slightly wavy with variable thicknesses lengths andexhibited interlaced arrangements (Figures 5(e) and 5(f))The interfibrillary spaces were more evident than in irradi-ated and vehicle groups Such morphological appearanceswere very similar to those observed in nonirradiated group(Figures 5(a) and 5(b))

36 Measurement of Cytotoxicity Activity The extract wasevaluated on murine malignant melanoma (cell line B16F10)in order to examine their cytotoxic effects on malignant cellsCytotoxicity of the extract on the growth of the B16F10 cellline is shown in Figure 6 Cell proliferation was analyzed

6 BioMed Research International

70120583m

(a)

70120583m

(b)

70120583m

(c)

70120583m

(d)

70120583m

(e)

70120583m

(f)

Figure 4 Hematoxylin-eosin stained histological sections (a) Nonirradiated group-NIC showing thin epidermal lining and absence ofdermal inflammation (b) Irradiated-IC and (c) vehicle groups-IV presenting epidermal thickening and intense lymphocytic infiltration in thedermal tissue (d) Elastotic change of the collagen (e) 10 and (f) 15 formulations showingmarked reduction of the inflammatory responseNote the thin epidermis in 15 formulation Short thick arrows epidermis Long thin arrows dermal connective tissues (Hematoxylin Eosin400x magnification)

24 h after B16F10 cells had been cultured with the extract atdifferent concentrations (005 01 02 04 08 16 32 64128 and 25mgmL) using the MTT assay The extract atconcentrations of 005 to 128mgmL did not show any cyto-toxic effects in the B16F10 cell line (showed no potential anti-cancer) A reduction in cell viability at high concentrationswas observed (25mgmL)

4 Discussion

The most distinguished harmful effect of solar exposure ispremature skin aging especially UV irradiation In recent

years thinning of the ozone layer has caused increased UVexposure Detrimental effects of UV on the skin involvethe generation of reactive oxygen species (ROS) [25] Com-ponents of the skin including DNA lipids and proteinscan be impaired by ROS which is the cause of erythemasunburn immune suppression and skin cancer [26] Most ofthe disorders were formerly regarded as having been causedbyUVBNevertheless recent studies showed that UVA is alsoan essential factor [27]

Accordingly use of antioxidants capable of scavengingand quenching ROS is another important approach to pre-vent UVB induced photoaging [27] Other studies have

BioMed Research International 7

70120583m

(a)

70120583m

(b)

70120583m

(c)

70120583m

(d)

70120583m

(e)

70120583m

(f)

Figure 5 Sirius Red stained histological sections (a b)Nonirradiated-NICgroups presenting interlaced less compacted type I and III collagenfibers (c) Irradiated-IC and (d) vehicle groups-IV showing dense disposition of parallel-arranged type I collagen fibers (e) 10 and (f) 15formulations presenting morphological and architectural collagen deposition pattern resembling that observed in the normal dermis (SiriusRedPolarization Light 400x magnification)

shown that botanical compounds with an antioxidative activ-ity are potential agents capable of reducing skin diseasesincluding photoaging [28 29]

The extract used in this work was the same studied bySerafini et al [19] and showed relevant in vitro antioxidantactivity against different radicals The extract was chemicallyanalyzed (qualitative method) The phytochemical screeningof extract showed the presence of alkaloids coumarins flavo-noids tannins saponins steroids and triterpenoids Totalphenolic content of the extract was 1968mg of phenolic equi-valents (gallic acid) per gram of extract Furthermore quali-tative analysis of extract was performed by HPLC revealingthe presence of 3 major compounds all showing ultraviolet

spectra typical of flavonols (quercetin-3-O-rutinoside orrutin and kaempferol glycosides) According to Deng et al[30] flavonol glycosides mainly rutin are the major com-pounds inM citrifolia leaves

The presence of rutin in the formulation could also besecondarily involved in this process of collagenization Ithas been demonstrated that the rutin is able to impair theglycation of collagen [31] a biochemical event involved in thepathogenesis of cell damaging in UV-irradiated tissue [32]

The use of botanical supplements to protect the humanskin from the adverse biological effects of solar radiationhas received great interest in recent years [33] Howevermost tests have been conducted by oral ingestion or topical

8 BioMed Research International

005 01 02 04 08 16 32 64 128 2500

50

100

150

Morinda citrifolia extract (mgmL)

Cel

l via

bilit

y (

)

Figure 6 Cytotoxic effect of theMorinda citrifolia extract in B16F10(murine malignant melanoma) cell line pretreated with the extractsat concentrations (005 to 25mgmL) for 24 h Each value representsthe mean plusmn SD

application of these ingredients in inappropriate vehicleswhich can alter the skin barrier To be of practical use todermatologists and to be safe and efficient extracts musthave physicochemical characteristics that permit their incor-poration in adequate concentrations in a skin-compatiblevehicle (eg gels moisturizing creams) [34] In this studythe capacity of the Morinda citrifolia extract added totopical formulations (gels) to prevent or to treat UVA-UVBirradiation-induced skin damage in mice was evaluated

First three formulations presenting different concen-trations (0 10 and 15) were evaluated in vitro byspectrophotometer UV absorbance spectra showed peaksat 200 nm and at 240 nm (UVC region) and absorbance inthe UVA (320ndash400 nm) and UVB (290ndash320 nm) regionsTheblank formulation (0 extract) did not show absorbance inthis spectra These data are compared to commercial organicfiltersThus this result suggests a photoprotective effect of theextractThe concentration of formulation (10 and 15ww)used in this assay was selected based on a pilot study

Second the gel formulation efficacy against UVA-UVBinduced skin damage was evaluated in vivo by measuring thefollowing parameters thickness values (mm) transepidermalwater loss (TEWL) and erythema

Moisturizing antifree radicals and sunscreen productshave been used largely to prevent skin disorders resultingfrom UV damage However other aspects of skin damageshould be considered such as erythema sunburn cell forma-tion epidermal hyperplasia and impairment of the skin bar-rier integrity which in turn is related to skin dryness skinaging and increased incidence of irritant contact dermatitis[35]

UV radiation damages the structures responsible for cor-neocyte adhesion and the functioning of the permeabilitybarrier which in turn alter thicknesses and TEWL measure-ments and accelerate the desquamation process [36]

The thickness TEWL and erythema were observed inour study and only the formulations containing M citri-folia extract protected mouse skin against the UV-induceddamages The pronounced protective effects provided by

both formulations (10 and 15) in the three parametersmeasured were not due to any extraneous raw material butwere shown by the unprotected groups treated by vehicle only(VI) Furthermore the results show that areas treated withthis extract (10 and 15) were statistically equivalent to theunirradiated control ones (NIC)which represented the intactskin areas

TEWL is an important parameter to be evaluated instudies of skin photoprotective effects considering that UVirradiation of mammalian skin disrupts the epidermal per-meability barrier function accompanied by an increase intransepidermal water loss (TEWL) and alterations in thestratum corneum lipid profile [33]

In the TEWL measurement the group 10 was differentfrom IC and the 15 was not As well as in the thickness andTEWL the protective effect of 10 was most pronouncedIt can also be seen in the photos of the dorsal of the ani-mal This observationcan be explained by differences in thespreadability Formulations containing extract at 10 pre-sented the higher spreadability

The spreadability is represented by the thickness of thefilm that the preparation leaves on the skin an importantfeature in cosmetics [37]Those producing thinner films thatis higher spreadability are naturally of greater interest [38]

Formulations containing extracts were shown to be effec-tive also in the presence of erythema another parameteroften used to evaluate photoprotection Extract formulationsreduced erythema formation when compared with the irra-diated control (IC) This statement corroborates [20] whichdemonstrated that the M citrifolia leaf extracts mitigate theeffects of UV light and provide some measure of defenseagainst localized inflammation (topical microswelling)

Third histological parameters were analyzed Epidermalthickening and inflammatory changes are common histo-logical changes observed in mice skin after short-term UVexposure [38 39] Such an increase in the epidermal thicknessis supposed to occur in response to cell proliferation inattempt to regenerate the tissue leading to a compensatorytype of hyperplasia However the thickened epidermis aswell as the thickened keratin layer (hyperkeratosis) could beobserved in this study

The presence of intense inflammatory infiltrate elastosisand the accumulation of parallel-arranged type I collagenfibers observed in IC and vehicle groups have also beendescribed as UV-induced dermal changes [31] Inflammationis one of the major histological signs of tissue damage andhas been reported in other studies as an early change of theconnective tissue after UV exposure [38 39] On the otherhand elastosis represents an actinic-induced accumulationof amorphous elastin material replacing the normal der-mis unrelated to preexisting or newly synthesized collagen[40] Moreover it has been proposed that UV irradiationimpairs ongoing collagen synthesis primarily through thedownregulation of types I and III procollagen expression[41] Therefore such impairment could likely promote delayin the collagen remodeling leading to the typical immatureparallel-arranged disposition of the fibers [31]

In this study only 15 formulation avoided compen-satory epidermal thickening However the reduction of the

BioMed Research International 9

major deleterious effects associated with the UV exposureon the animals treated with both 10 and 15 formulations isstrongly suggestive of photoprotective effect regardless of theextract concentration

Lastly the extract was evaluated on murine malignantmelanoma in order to examine its cytotoxic effects on malig-nant cells A reduction in cell viability was observed only atthe highest concentration (25mgmL) The results can sug-gest that the extract (in low concentrations) has only photo-protection effects and does not have antimelanoma activity

5 Conclusion

In conclusion present results strongly suggest the photo-protective effect of M citrifolia The results indicate thatphotoprotective effects are due to not only UV absorbancelike the UV filters but also biological effects which happenedmainly with the application of the formulation containing theMorinda citrifolia extract Further studies are necessary toclarify the precise nature of this photoprotective effect

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by Fundacao de Amparo a Pesquisado Estado de Sergipe (FAPITEC-SE) and Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) ofBrazil

References

[1] A M Garcıa-Bores and J G Avila ldquoProducts ldquoMolecularmechanisms in the photochemopreventionrdquordquo Revista LatinoAmericana de Quımica vol 36 no 3 pp 83ndash102 2008

[2] M D Adil P Kaiser N K Satti A M Zargar R A Vish-wakarma and S A Tasduq ldquoEffect of Emblica officinalis (fruit)against UVB-induced photo-aging in human skin fibroblastsrdquoJournal of Ethnopharmacology vol 132 no 1 pp 109ndash114 2010

[3] M Dalle Carbonare and M A Pathak ldquoSkin photosensitizingagents and the role of reactive oxygen species in photoagingrdquoJournal of Photochemistry and Photobiology B Biology vol 14no 1-2 pp 105ndash124 1992

[4] G J Fisher S Datta ZWang et al ldquoc-Jun-dependent inhibitionof cutaneous procollagen transcription following ultravioletirradiation is reversed by all-trans retinoic acidrdquoThe Journal ofClinical Investigation vol 106 no 5 pp 663ndash670 2000

[5] S Inomata Y Matsunaga S Amano et al ldquoPossible involve-ment of gelatinases in basementmembrane damage andwrinkleformation in chronically ultraviolet B-exposed hairless mouserdquoJournal of Investigative Dermatology vol 120 no 1 pp 128ndash1342003

[6] P S Peres V A Terra F A Guarnier R Cecchini and A LCecchini ldquoPhotoaging and chronological aging profile under-standing oxidation of the skinrdquo Journal of Photochemistry andPhotobiology B Biology vol 103 no 2 pp 93ndash97 2011

[7] Y Xu and G J Fisher ldquoUltraviolet (UV) light irradiationinduced signal transduction in skin photoagingrdquo Journal of Der-matological Science Supplement vol 1 no 2 pp S1ndashS8 2005

[8] S P Stratton R T Dorr and D S Alberts ldquoThe state-of-the-artin chemoprevention of skin cancerrdquoEuropean Journal of Cancervol 36 no 10 pp 1292ndash1297 2000

[9] F Afaq V M Adhami and H Mukhtar ldquoPhotochemopreven-tion of ultraviolet B signaling and photocarcinogenesisrdquoMuta-tion Research vol 571 no 1-2 pp 153ndash173 2005

[10] V M Adhami D N Syed N Khan and F Afaq ldquoPhytochem-icals for prevention of solar ultraviolet radiation-induced dam-agesrdquo Photochemistry and Photobiology vol 84 no 2 pp 489ndash500 2008

[11] C D Ropke T CH Sawada V V da Silva N SMichalany andS B DeMoraes Barros ldquoPhotoprotective effect of Pothomorpheumbellata root extract against ultraviolet radiation inducedchronic skin damage in the hairless mouserdquo Clinical andExperimental Dermatology vol 30 no 3 pp 272ndash276 2005

[12] M G D Melo J P A dos Santos M R Serafini et al ldquoRedoxproperties and cytoprotective actions of atranorin a lichensecondary metaboliterdquo Toxicology in Vitro vol 25 no 2 pp462ndash468 2011

[13] M R Serafini D M C Vergne T K Rabelo et al ldquoDetermi-nation of chemical and physical properties of Hyptis pectinataessential oil and their redox active profilerdquo Journal of Biotech-nology and Pharmaceutical Research vol 3 no 1 pp 1ndash9 2012

[14] R Casagrande S R Georgetti W A Verri Jr D J Dorta A Cdos Santos and M J V Fonseca ldquoProtective effect of topicalformulations containing quercetin against UVB-induced oxi-dative stress in hairless micerdquo Journal of Photochemistry andPhotobiology B Biology vol 84 no 1 pp 21ndash27 2006

[15] S Frsquoguyer F Afaq and H Mukhtar ldquoPhotochemopreventionof skin cancer by botanical agentsrdquo Photodermatology Photoim-munology and Photomedicine vol 19 no 2 pp 56ndash72 2003

[16] Y Chan-Blanco F Vaillant A Mercedes Perez M Reynes JBrillouet and P Brat ldquoThe noni fruit (Morinda citrifolia L) areview of agricultural research nutritional and therapeutic pro-pertiesrdquo Journal of Food Composition and Analysis vol 19 no 6-7 pp 645ndash654 2006

[17] H Kim M Kwon J Kim et al ldquoIdentification of novel fattyacid glucosides from the tropical fruit Morinda citrifolia LrdquoPhytochemistry Letters vol 3 no 4 pp 238ndash241 2010

[18] A Hirazumi E Furusawa S C Chou and Y Hokama ldquoImmu-nomodulation contributes to the anticancer activity ofMorindacitrifolia (noni) fruit juicerdquo Proceedings of the Western Pharma-cology Society vol 39 pp 7ndash9 1996

[19] M R Serafini R C Santos A G Guimaraes et al ldquoMorindacitrifolia linn leaf extract possesses antioxidant activities andreduces nociceptive behavior and leukocyte migrationrdquo Journalof Medicinal Food vol 14 no 10 pp 1159ndash1166 2011

[20] B J West S Deng A K Palu and C J Jensen ldquoMorinda citri-folia Linn (Rubiaceae) leaf extracts mitigate UVB-induced ery-themardquo Journal of Natural Medicines vol 63 no 3 pp 351ndash3542009

[21] A Garg D Aggarwal S Garg and A Singela ldquoSpreading ofsemisolid formulations pharmaceutical technologyrdquo Pharma-ceutical Technology vol 26 pp 84ndash105 2002

[22] K Paese A Jager F S Poletto et al ldquoSemisolid formulationcontaining a nanoencapsulated sunscreen effectiveness in vitrophotostability and immune responserdquo Journal of Biomedical andNanotechnology vol 5 no 3 pp 240ndash246 2009

10 BioMed Research International

[23] M Demerjian P Hachem E Tschachler et al ldquoAcute modula-tions in permeability barrier function regulate epidermal corni-ficationrdquo The American Journal of Pathology vol 172 no 1 pp86ndash97 2008

[24] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[25] H Sakurai H Yasui Y Yamada H Nishimura and M Shige-moto ldquoDetection of reactive oxygen species in the skin of livemice and rats exposed toUVA light a research reviewon chemi-luminescence and trials for UVA protectionrdquo Photochemical ampPhotobiological Sciences vol 4 no 9 pp 715ndash720 2005

[26] R Haywood P Wardman R Sanders and C Linge ldquoSun-screens inadequately protect against ultraviolet-A-induced freeradicals in skin implications for skin aging and melanomardquoJournal of Investigative Dermatology vol 121 no 4 pp 862ndash8682003

[27] F Afaq DN Syed AMalik et al ldquoDelphinidin an anthocyani-din in pigmented fruits and vegetables protects human HaCaTkeratinocytes and mouse skin against UVB-mediated oxidativestress and apoptosisrdquo Journal of Investigative Dermatology vol127 no 1 pp 222ndash232 2007

[28] J-Y Bae J-S Choi Y-J Choi et al ldquo(minus)Epigallocatechin gallatehampers collagen destruction and collagenase activation inultraviolet-B-irradiated humandermal fibroblasts Involvementof mitogen-activated protein kinaserdquo Food and Chemical Toxi-cology vol 46 no 4 pp 1298ndash1307 2008

[29] H Choi D Kim J W Kim S Ngadiran M R Sarmidi and CS Park ldquoLabisia pumila extract protects skin cells from photo-aging caused by UVB irradiationrdquo Journal of Bioscience andBioengineering vol 109 no 3 pp 291ndash296 2010

[30] S Deng B JWest andC J Jensen ldquoSimultaneous characterisa-tion and quantitation of flavonol glycosides and aglycones innoni leaves using a validated HPLC-UVMS methodrdquo FoodChemistry vol 111 no 2 pp 526ndash529 2008

[31] D Cervantes-Laurean D D Schramm E L Jacobson IHalaweish G G Bruckner and G A Boissonneault ldquoInhibi-tion of advanced glycation end product formation on collagenby rutin and its metabolitesrdquo Journal of Nutritional Biochem-istry vol 17 no 8 pp 531ndash540 2006

[32] Y M Fonseca C D Catini F T M C Vicentini J C CardosoR L Cavalcanti De Albuquerque Junior and M J Vieira Fon-seca ldquoEfficacy of marigold extract-loaded formulations againstUV-induced oxidative stressrdquo Journal of Pharmaceutical Sci-ences vol 100 no 6 pp 2182ndash2193 2011

[33] P K Vayalil C A Elments and S K Katiyar ldquoTreatment ofgreen tea polyphenols in hydrophilic cream prevents UVB-induced oxidation of lipids and proteins depletion of antioxi-dant enzymes and phosphorylation ofMAPKproteins in SKH-1hairless mouse skinrdquo Carcinogenesis vol 24 no 5 pp 927ndash9362003

[34] S E Dal Belo L R Gaspar and PM B GM Campos ldquoPhoto-protective effects of topical formulations containing a combi-nation of Ginkgo biloba and green tea extractsrdquo PhytotherapyResearch vol 25 no 12 pp 1854ndash1860 2011

[35] R Ghadially B E Brown S M Sequeira-Martin K R Fein-gold and P M Elias ldquoThe aged epidermal permeability barrierStructural functional and lipid biochemical abnormalities inhumans and a senescent murine modelrdquo Journal of ClinicalInvestigation vol 95 no 5 pp 2281ndash2290 1995

[36] M D Contreras and R Sanchez ldquoApplication of a factorialdesign to the study of the flow behavior spreadability and trans-parency of a Carbopol ETD 2020 gel Part IIrdquo International Jour-nal of Pharmaceutics vol 234 no 1-2 pp 149ndash157 2002

[37] BW BarryRheology ofDermatological VehiclesMarcelDekkerNew York NY USA 1983

[38] S U Kim S J Kim J Y Lee et al ldquoProtective effects of dietarysoy isoflavones against UV-induced skin-aging in hairlessmouse modelrdquo Journal of the American College of Nutrition vol23 no 2 pp 157ndash162 2004

[39] M Amouroux G Dıaz-Ayil W C P M Blondel G Bourg-Heckly A Leroux and F Guillemin ldquoClassification of ultra-violet irradiated mouse skin histological stages by bimodalspectroscopy multiple excitation autofluorescence and diffusereflectancerdquo Journal of Biomedical Optics vol 14 no 1 ArticleID 014011 2009

[40] K A Hwang B R Yi and K C Choi ldquoMolecular Mechanismsand In Vivo Mouse Models of Skin Aging Associated withDermal Matrix Alterationsrdquo Laboratory Animal Research vol27 no 1 pp 1ndash8 2011

[41] G J Fisher S Kang J Varani et al ldquoMechanisms of photoagingand chronological skin agingrdquoArchives of Dermatology vol 138no 11 pp 1462ndash1470 2002

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

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StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

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Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 3

30min before the dorsal surface irradiation The untreatedcontrol groups irradiated and nonirradiated were included inthe experiments

27 Irradiation The UVA-UVB source of irradiation con-sisted of a Repti Glo 100 lamp (Exo Terra) emitting acontinuous spectrum between 270 and 400 nm 33 UVAand 10 UVB The lamp was mounted 20 cm above thesupport where the mice were placed The UVA-UVB outputwasmeasured using amodel UV-400 Research Radiometer (Icel) with a radiometer sensor forUVAandUVBThemajorityof the resulting wavelengths were in the UVB (290ndash320 nmabove 90) and UVA (less than 10) range and the peakemission was recorded at 314 nm The mice were irradiatedfor 7 days 2 hday with a daily dose of 19 Jcm2

28 Assessment of Biophysical Skin Properties The thicknessof irradiated skin wasmeasured by 7 days of treatment by dialthickness gage (Mitutoyo)

Transepidermal water loss (TEWL) was measured usinga Tewameter R TM 210 (Courage amp Khazaka ElectronicGmbH Germany) before (baseline values) and 20 h after UVexposure with 7 days of treatment Readings of TEWL wereregistered in gm2sdoth during 2min after 30 s probe equili-bration on the skin The UV-induced skin barrier damageassessed by TEWL measurements is due to abnormalitiesin the structures related to corneocytes adhesion and todisruption of the epidermal permeability barrier function[23]

Erythema was measured photometrically using a Mex-ameter R MX16 (Couragem amp Khazaka Eletronic GmbHCologne Germany) before (baseline values) and 20 h afterUV exposure over 7 days Six readings of the erythema indexwere recorded at each area at both points in time The degreeof erythema quantified as an erythema index is related to thehemoglobin content

29 Animal Sacrifice After treatment over 7 days the micewere sacrificed by cervical dislocation 2 h after the ultimateUVA-UVB exposure The full thickness of the dorsal skinswas removed and fixed in 10 formalin until histologicalmeasurements

210 Histological Measurements Skin biopsies were fixed in10 formalin embedded in paraffin according to routineprotocol of histological procedures Five micrometer thinsections of the paraffin-embedded skin were obtained andstained by means of hematoxylin eosin (HE) and Siriusred (SR) histochemical methods Morphological analysis ofthe histological sections was performed by light microscopyfollowed by a closed numerical protocol in such a manneras the pathologist was not aware of which group was beingevaluated until the end of the experiment

211 Measurement of Cytotoxicity Activity The B16F10(murine malignant melanoma ATCC CRL-6475) cellswere obtained from the Rio de Janeiro Cell Bank BrazilThey were routinely grown in 150 cm2 tissue culture flasksin DMEM supplemented with 1 (vv) of an antibiotic

solution containing 5mg of penicillin 5mg of streptomycinand 10mg of neomycin per mL and 75 or 100 (vv)heat-inactivated Fetal Bovine Serum at 37∘C under 5 CO

2

The sensitivity of the cells to the Morinda citrifoliaextract was determined by a standard spectrophotometric3-(45-dimethylthiazole-2-yl)-25-diphenyltetrazoliumbromide (MTT) assay [24] Cells were seeded at a density of105 cellswells into 96-well plates and incubated for 24 h at37∘C in an atmosphere of 95 air and 5 CO

2 Then 20120583L

of extract at different concentrations in a phosphate buffersaline (PBS) was added to the culture plates for 24 h Aftertreatment cells were rinsed once with PBS and a serum-freeculture medium without phenol red and were replaced in allwells Cells were then incubated for 4 h with MTT solution(5mgmL)

The yellow tetrazolium salt was metabolized by viablecells to form purple crystals of formazan The crystals weresolubilized overnight (12 h) in a mixture consisting of 20sodium dodecyl sulfate (SDS) in HCl (001M) The productwas quantified spectrophotometrically by measuring theabsorbency at 570 nmusing amicroplate reader (120583QuantTMBioTek Instruments Inc USA) The cellular viability wasexpressed as a percentage of viable cells compared to thecontrol group

The percentage of viable cells was calculated as follows

Viability

=Total number of viable cells

Total number of viable and nonviable cellstimes 100

(2)

212 Statistical Analysis Data were expressed as mean plusmnstandard errorof the mean (SEM) or plusmn standard deviation(SD) The data obtained was evaluated by one-way analysisof variance (ANOVA) followed by Tukeyrsquos test Data analyseswere performed using the GraphPad Prism 50 software Inall cases differences were considered significant if 119875 lt 005

3 Results

31 Measurement of Spreadability The spreadability is anessential characteristic of semisolid pharmaceutical dosageforms for dermal application Thus the spreadability fac-tor was measured (Table 1) The formulation contain-ing the extract at 10 presented the higher spreadabil-ity (1144mm2sdotgminus1) against the formulation containing theextract at 15 (0916mm2sdotgminus1)

32 In Vitro Effectiveness The absorbance spectrum of theformulations extracts (10 and 15) (Figure 1) showed bandsat 200 nm and at 240 nm and it showed absorbance in theUVA and UVB regions The blank formulation (0 extract)did not show absorbance in this spectra

33 Biophysical Skin Properties The results showed that thethickness TEWL values and erythema indexes were signifi-cantly decreased by treatment with formulations containingthe extract (Figures 2(a) 2(b) and 2(c) resp)

4 BioMed Research International

000020040

Abso

rban

ce (a

u)

060080100120140160180

2000 3000 4000 5000 6000Wavelength (nm)

7000 8000

15100

Figure 1 UV absorbance spectra of a blank formulation (0 extract) formulation extract 10 and formulation extract 15The formulationswere scanned under UV light (200 to 400 nm)

IV IC NIC 10 1500

05

10

15

20

25

Groups n = 10

Thic

knes

s (m

m)

lowastlowastlowastlowastlowastlowast lowastlowastlowast

(a)

IV IC NIC 10 150

10

20

30

40

Groups n = 10

lowastlowastlowastlowastlowastlowast lowastlowast

TEW

L (g

h m

2)

(b)

IV IC NIC 10 150

100

200

300

400

Groups n = 10

Eryt

hem

a ind

ex lowastlowast

lowastlowastlowast

(c)

Figure 2 (a) Thickness values (mm) (b) transepidermal water loss and (c) erythema obtained for the dorsal areas IV = irradiated andtreated with the vehicle (formulation 0) IC = irradiated control (the group was irradiated but did not receive any topical treatment) NIC= nonirradiated control (the group was not irradiated and did not receive any topical treatment) 10 = group irradiated and treated withthe formulation 10 15 = group irradiated and treated with the formulation 15 The values were obtained before (baseline values) and20 h after UV exposure during 7 days of treatment (119899 = 10 mice) 119875 lt 005 versus IC lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus IVANOVA followed by Tukeyrsquos test Data represents mean plusmn SEM

BioMed Research International 5

Table 1 Measurement of spreadability of formulations containingMorinda citrifolia extract (10 or 15 ww)

Formulations lowastSP1 lowastSP2 lowastSP3 lowastSP Mean lowastSP Mean plusmn SD CVExtract 10 1136 1044 1253 1144 1144 plusmn 107 94Extract 15 0955 0824 0969 0916 0916 plusmn 008 87lowastSpreadability factor (mm2

sdotgminus1)

IV IC NIC 10 15

Figure 3 Skin biophysical techniques Photoprotective effects of formulations onUVA-UVB-induced pathological changes in the skin surfaceon different groups after 7 days of treatment IV = irradiated and treated with the vehicle (formulation 0) IC = irradiated control (the groupwas irradiated but did not receive any topical treatment) NIC = nonirradiated control (the group was not irradiated and did not receive anytopical treatment) 10 = group irradiated and treated with the formulation 10 15 = group irradiated and treated with the formulation15

34 Pathological Changes in the Skin Surface Treated areaswith the formulation containing Morinda citrifolia extract(10 and 15) have enhanced protection against UV radiation(Figure 3)

35 Histological Measurements

Hematoxylin Eosin (HE) As demonstrated in Figure 4(a) thenonirradiated group showed that the epidermis was repre-sented by a thin continuous squamous epithelial liningwith an orthokeratotic surfaceThe connective tissue showedinterlaced collagen fibers associated with spindle and roundshaped stromal cells consistent with fibroblasts Some fewcapillary vessels and venules could also be seen as wellas numerous cutaneous appendages No inflammatory infil-trates were found in the dermal tissues Both irradiated andvehicle groups (Figures 4(b) and 4(c)) presented increasedepidermal thickness associated to hyperorthokeratosis Thepapillary dermis exhibited intense inflammatory infiltratemainly composed of lymphocytes mild interstitial edemaand prominent capillary and venular hyperemia Extensiveareas of elastotic alterations of the collagen bundles (baso-philic degeneration) were also observed throughout theconnective tissue (Figure 4(d))The treated groups presentedremarkable reduction in the inflammatory content as wellas less expressive hyperemia However epidermal thickeningwas more evident in 10 than in 15 formulation (Figures4(e) and 4(f)) Such similar histological features were quite

similar to those verified in the normal dermalepidermal tis-suesSirius Red (SR) The nonirradiated-NIC groups presentedinterlaced less compacted type I and III collagen fibers (Fig-ures 5(a) and 5(b)) In irradiated and vehicle groups thepapillary dermis showed the presence of gross thick parallel-arranged collagen fibers exhibiting intense yellow birefrin-gence (type I collagen) which were associated to shorter andthinner reddish fibrils perpendicularly disposed in relationto the surface Such collagen bundles were densely com-pacted showing sparse interfibrillar spaces (Figures 5(c) and5(d)) Both treated groups (10 and 15 formulations) pre-sented similar features regarding the collagenization patternshowing a mixture of yellowish type I (predominant) andgreenish type III collagen fibers These fibers were moredelicate slightly wavy with variable thicknesses lengths andexhibited interlaced arrangements (Figures 5(e) and 5(f))The interfibrillary spaces were more evident than in irradi-ated and vehicle groups Such morphological appearanceswere very similar to those observed in nonirradiated group(Figures 5(a) and 5(b))

36 Measurement of Cytotoxicity Activity The extract wasevaluated on murine malignant melanoma (cell line B16F10)in order to examine their cytotoxic effects on malignant cellsCytotoxicity of the extract on the growth of the B16F10 cellline is shown in Figure 6 Cell proliferation was analyzed

6 BioMed Research International

70120583m

(a)

70120583m

(b)

70120583m

(c)

70120583m

(d)

70120583m

(e)

70120583m

(f)

Figure 4 Hematoxylin-eosin stained histological sections (a) Nonirradiated group-NIC showing thin epidermal lining and absence ofdermal inflammation (b) Irradiated-IC and (c) vehicle groups-IV presenting epidermal thickening and intense lymphocytic infiltration in thedermal tissue (d) Elastotic change of the collagen (e) 10 and (f) 15 formulations showingmarked reduction of the inflammatory responseNote the thin epidermis in 15 formulation Short thick arrows epidermis Long thin arrows dermal connective tissues (Hematoxylin Eosin400x magnification)

24 h after B16F10 cells had been cultured with the extract atdifferent concentrations (005 01 02 04 08 16 32 64128 and 25mgmL) using the MTT assay The extract atconcentrations of 005 to 128mgmL did not show any cyto-toxic effects in the B16F10 cell line (showed no potential anti-cancer) A reduction in cell viability at high concentrationswas observed (25mgmL)

4 Discussion

The most distinguished harmful effect of solar exposure ispremature skin aging especially UV irradiation In recent

years thinning of the ozone layer has caused increased UVexposure Detrimental effects of UV on the skin involvethe generation of reactive oxygen species (ROS) [25] Com-ponents of the skin including DNA lipids and proteinscan be impaired by ROS which is the cause of erythemasunburn immune suppression and skin cancer [26] Most ofthe disorders were formerly regarded as having been causedbyUVBNevertheless recent studies showed that UVA is alsoan essential factor [27]

Accordingly use of antioxidants capable of scavengingand quenching ROS is another important approach to pre-vent UVB induced photoaging [27] Other studies have

BioMed Research International 7

70120583m

(a)

70120583m

(b)

70120583m

(c)

70120583m

(d)

70120583m

(e)

70120583m

(f)

Figure 5 Sirius Red stained histological sections (a b)Nonirradiated-NICgroups presenting interlaced less compacted type I and III collagenfibers (c) Irradiated-IC and (d) vehicle groups-IV showing dense disposition of parallel-arranged type I collagen fibers (e) 10 and (f) 15formulations presenting morphological and architectural collagen deposition pattern resembling that observed in the normal dermis (SiriusRedPolarization Light 400x magnification)

shown that botanical compounds with an antioxidative activ-ity are potential agents capable of reducing skin diseasesincluding photoaging [28 29]

The extract used in this work was the same studied bySerafini et al [19] and showed relevant in vitro antioxidantactivity against different radicals The extract was chemicallyanalyzed (qualitative method) The phytochemical screeningof extract showed the presence of alkaloids coumarins flavo-noids tannins saponins steroids and triterpenoids Totalphenolic content of the extract was 1968mg of phenolic equi-valents (gallic acid) per gram of extract Furthermore quali-tative analysis of extract was performed by HPLC revealingthe presence of 3 major compounds all showing ultraviolet

spectra typical of flavonols (quercetin-3-O-rutinoside orrutin and kaempferol glycosides) According to Deng et al[30] flavonol glycosides mainly rutin are the major com-pounds inM citrifolia leaves

The presence of rutin in the formulation could also besecondarily involved in this process of collagenization Ithas been demonstrated that the rutin is able to impair theglycation of collagen [31] a biochemical event involved in thepathogenesis of cell damaging in UV-irradiated tissue [32]

The use of botanical supplements to protect the humanskin from the adverse biological effects of solar radiationhas received great interest in recent years [33] Howevermost tests have been conducted by oral ingestion or topical

8 BioMed Research International

005 01 02 04 08 16 32 64 128 2500

50

100

150

Morinda citrifolia extract (mgmL)

Cel

l via

bilit

y (

)

Figure 6 Cytotoxic effect of theMorinda citrifolia extract in B16F10(murine malignant melanoma) cell line pretreated with the extractsat concentrations (005 to 25mgmL) for 24 h Each value representsthe mean plusmn SD

application of these ingredients in inappropriate vehicleswhich can alter the skin barrier To be of practical use todermatologists and to be safe and efficient extracts musthave physicochemical characteristics that permit their incor-poration in adequate concentrations in a skin-compatiblevehicle (eg gels moisturizing creams) [34] In this studythe capacity of the Morinda citrifolia extract added totopical formulations (gels) to prevent or to treat UVA-UVBirradiation-induced skin damage in mice was evaluated

First three formulations presenting different concen-trations (0 10 and 15) were evaluated in vitro byspectrophotometer UV absorbance spectra showed peaksat 200 nm and at 240 nm (UVC region) and absorbance inthe UVA (320ndash400 nm) and UVB (290ndash320 nm) regionsTheblank formulation (0 extract) did not show absorbance inthis spectra These data are compared to commercial organicfiltersThus this result suggests a photoprotective effect of theextractThe concentration of formulation (10 and 15ww)used in this assay was selected based on a pilot study

Second the gel formulation efficacy against UVA-UVBinduced skin damage was evaluated in vivo by measuring thefollowing parameters thickness values (mm) transepidermalwater loss (TEWL) and erythema

Moisturizing antifree radicals and sunscreen productshave been used largely to prevent skin disorders resultingfrom UV damage However other aspects of skin damageshould be considered such as erythema sunburn cell forma-tion epidermal hyperplasia and impairment of the skin bar-rier integrity which in turn is related to skin dryness skinaging and increased incidence of irritant contact dermatitis[35]

UV radiation damages the structures responsible for cor-neocyte adhesion and the functioning of the permeabilitybarrier which in turn alter thicknesses and TEWL measure-ments and accelerate the desquamation process [36]

The thickness TEWL and erythema were observed inour study and only the formulations containing M citri-folia extract protected mouse skin against the UV-induceddamages The pronounced protective effects provided by

both formulations (10 and 15) in the three parametersmeasured were not due to any extraneous raw material butwere shown by the unprotected groups treated by vehicle only(VI) Furthermore the results show that areas treated withthis extract (10 and 15) were statistically equivalent to theunirradiated control ones (NIC)which represented the intactskin areas

TEWL is an important parameter to be evaluated instudies of skin photoprotective effects considering that UVirradiation of mammalian skin disrupts the epidermal per-meability barrier function accompanied by an increase intransepidermal water loss (TEWL) and alterations in thestratum corneum lipid profile [33]

In the TEWL measurement the group 10 was differentfrom IC and the 15 was not As well as in the thickness andTEWL the protective effect of 10 was most pronouncedIt can also be seen in the photos of the dorsal of the ani-mal This observationcan be explained by differences in thespreadability Formulations containing extract at 10 pre-sented the higher spreadability

The spreadability is represented by the thickness of thefilm that the preparation leaves on the skin an importantfeature in cosmetics [37]Those producing thinner films thatis higher spreadability are naturally of greater interest [38]

Formulations containing extracts were shown to be effec-tive also in the presence of erythema another parameteroften used to evaluate photoprotection Extract formulationsreduced erythema formation when compared with the irra-diated control (IC) This statement corroborates [20] whichdemonstrated that the M citrifolia leaf extracts mitigate theeffects of UV light and provide some measure of defenseagainst localized inflammation (topical microswelling)

Third histological parameters were analyzed Epidermalthickening and inflammatory changes are common histo-logical changes observed in mice skin after short-term UVexposure [38 39] Such an increase in the epidermal thicknessis supposed to occur in response to cell proliferation inattempt to regenerate the tissue leading to a compensatorytype of hyperplasia However the thickened epidermis aswell as the thickened keratin layer (hyperkeratosis) could beobserved in this study

The presence of intense inflammatory infiltrate elastosisand the accumulation of parallel-arranged type I collagenfibers observed in IC and vehicle groups have also beendescribed as UV-induced dermal changes [31] Inflammationis one of the major histological signs of tissue damage andhas been reported in other studies as an early change of theconnective tissue after UV exposure [38 39] On the otherhand elastosis represents an actinic-induced accumulationof amorphous elastin material replacing the normal der-mis unrelated to preexisting or newly synthesized collagen[40] Moreover it has been proposed that UV irradiationimpairs ongoing collagen synthesis primarily through thedownregulation of types I and III procollagen expression[41] Therefore such impairment could likely promote delayin the collagen remodeling leading to the typical immatureparallel-arranged disposition of the fibers [31]

In this study only 15 formulation avoided compen-satory epidermal thickening However the reduction of the

BioMed Research International 9

major deleterious effects associated with the UV exposureon the animals treated with both 10 and 15 formulations isstrongly suggestive of photoprotective effect regardless of theextract concentration

Lastly the extract was evaluated on murine malignantmelanoma in order to examine its cytotoxic effects on malig-nant cells A reduction in cell viability was observed only atthe highest concentration (25mgmL) The results can sug-gest that the extract (in low concentrations) has only photo-protection effects and does not have antimelanoma activity

5 Conclusion

In conclusion present results strongly suggest the photo-protective effect of M citrifolia The results indicate thatphotoprotective effects are due to not only UV absorbancelike the UV filters but also biological effects which happenedmainly with the application of the formulation containing theMorinda citrifolia extract Further studies are necessary toclarify the precise nature of this photoprotective effect

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by Fundacao de Amparo a Pesquisado Estado de Sergipe (FAPITEC-SE) and Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) ofBrazil

References

[1] A M Garcıa-Bores and J G Avila ldquoProducts ldquoMolecularmechanisms in the photochemopreventionrdquordquo Revista LatinoAmericana de Quımica vol 36 no 3 pp 83ndash102 2008

[2] M D Adil P Kaiser N K Satti A M Zargar R A Vish-wakarma and S A Tasduq ldquoEffect of Emblica officinalis (fruit)against UVB-induced photo-aging in human skin fibroblastsrdquoJournal of Ethnopharmacology vol 132 no 1 pp 109ndash114 2010

[3] M Dalle Carbonare and M A Pathak ldquoSkin photosensitizingagents and the role of reactive oxygen species in photoagingrdquoJournal of Photochemistry and Photobiology B Biology vol 14no 1-2 pp 105ndash124 1992

[4] G J Fisher S Datta ZWang et al ldquoc-Jun-dependent inhibitionof cutaneous procollagen transcription following ultravioletirradiation is reversed by all-trans retinoic acidrdquoThe Journal ofClinical Investigation vol 106 no 5 pp 663ndash670 2000

[5] S Inomata Y Matsunaga S Amano et al ldquoPossible involve-ment of gelatinases in basementmembrane damage andwrinkleformation in chronically ultraviolet B-exposed hairless mouserdquoJournal of Investigative Dermatology vol 120 no 1 pp 128ndash1342003

[6] P S Peres V A Terra F A Guarnier R Cecchini and A LCecchini ldquoPhotoaging and chronological aging profile under-standing oxidation of the skinrdquo Journal of Photochemistry andPhotobiology B Biology vol 103 no 2 pp 93ndash97 2011

[7] Y Xu and G J Fisher ldquoUltraviolet (UV) light irradiationinduced signal transduction in skin photoagingrdquo Journal of Der-matological Science Supplement vol 1 no 2 pp S1ndashS8 2005

[8] S P Stratton R T Dorr and D S Alberts ldquoThe state-of-the-artin chemoprevention of skin cancerrdquoEuropean Journal of Cancervol 36 no 10 pp 1292ndash1297 2000

[9] F Afaq V M Adhami and H Mukhtar ldquoPhotochemopreven-tion of ultraviolet B signaling and photocarcinogenesisrdquoMuta-tion Research vol 571 no 1-2 pp 153ndash173 2005

[10] V M Adhami D N Syed N Khan and F Afaq ldquoPhytochem-icals for prevention of solar ultraviolet radiation-induced dam-agesrdquo Photochemistry and Photobiology vol 84 no 2 pp 489ndash500 2008

[11] C D Ropke T CH Sawada V V da Silva N SMichalany andS B DeMoraes Barros ldquoPhotoprotective effect of Pothomorpheumbellata root extract against ultraviolet radiation inducedchronic skin damage in the hairless mouserdquo Clinical andExperimental Dermatology vol 30 no 3 pp 272ndash276 2005

[12] M G D Melo J P A dos Santos M R Serafini et al ldquoRedoxproperties and cytoprotective actions of atranorin a lichensecondary metaboliterdquo Toxicology in Vitro vol 25 no 2 pp462ndash468 2011

[13] M R Serafini D M C Vergne T K Rabelo et al ldquoDetermi-nation of chemical and physical properties of Hyptis pectinataessential oil and their redox active profilerdquo Journal of Biotech-nology and Pharmaceutical Research vol 3 no 1 pp 1ndash9 2012

[14] R Casagrande S R Georgetti W A Verri Jr D J Dorta A Cdos Santos and M J V Fonseca ldquoProtective effect of topicalformulations containing quercetin against UVB-induced oxi-dative stress in hairless micerdquo Journal of Photochemistry andPhotobiology B Biology vol 84 no 1 pp 21ndash27 2006

[15] S Frsquoguyer F Afaq and H Mukhtar ldquoPhotochemopreventionof skin cancer by botanical agentsrdquo Photodermatology Photoim-munology and Photomedicine vol 19 no 2 pp 56ndash72 2003

[16] Y Chan-Blanco F Vaillant A Mercedes Perez M Reynes JBrillouet and P Brat ldquoThe noni fruit (Morinda citrifolia L) areview of agricultural research nutritional and therapeutic pro-pertiesrdquo Journal of Food Composition and Analysis vol 19 no 6-7 pp 645ndash654 2006

[17] H Kim M Kwon J Kim et al ldquoIdentification of novel fattyacid glucosides from the tropical fruit Morinda citrifolia LrdquoPhytochemistry Letters vol 3 no 4 pp 238ndash241 2010

[18] A Hirazumi E Furusawa S C Chou and Y Hokama ldquoImmu-nomodulation contributes to the anticancer activity ofMorindacitrifolia (noni) fruit juicerdquo Proceedings of the Western Pharma-cology Society vol 39 pp 7ndash9 1996

[19] M R Serafini R C Santos A G Guimaraes et al ldquoMorindacitrifolia linn leaf extract possesses antioxidant activities andreduces nociceptive behavior and leukocyte migrationrdquo Journalof Medicinal Food vol 14 no 10 pp 1159ndash1166 2011

[20] B J West S Deng A K Palu and C J Jensen ldquoMorinda citri-folia Linn (Rubiaceae) leaf extracts mitigate UVB-induced ery-themardquo Journal of Natural Medicines vol 63 no 3 pp 351ndash3542009

[21] A Garg D Aggarwal S Garg and A Singela ldquoSpreading ofsemisolid formulations pharmaceutical technologyrdquo Pharma-ceutical Technology vol 26 pp 84ndash105 2002

[22] K Paese A Jager F S Poletto et al ldquoSemisolid formulationcontaining a nanoencapsulated sunscreen effectiveness in vitrophotostability and immune responserdquo Journal of Biomedical andNanotechnology vol 5 no 3 pp 240ndash246 2009

10 BioMed Research International

[23] M Demerjian P Hachem E Tschachler et al ldquoAcute modula-tions in permeability barrier function regulate epidermal corni-ficationrdquo The American Journal of Pathology vol 172 no 1 pp86ndash97 2008

[24] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[25] H Sakurai H Yasui Y Yamada H Nishimura and M Shige-moto ldquoDetection of reactive oxygen species in the skin of livemice and rats exposed toUVA light a research reviewon chemi-luminescence and trials for UVA protectionrdquo Photochemical ampPhotobiological Sciences vol 4 no 9 pp 715ndash720 2005

[26] R Haywood P Wardman R Sanders and C Linge ldquoSun-screens inadequately protect against ultraviolet-A-induced freeradicals in skin implications for skin aging and melanomardquoJournal of Investigative Dermatology vol 121 no 4 pp 862ndash8682003

[27] F Afaq DN Syed AMalik et al ldquoDelphinidin an anthocyani-din in pigmented fruits and vegetables protects human HaCaTkeratinocytes and mouse skin against UVB-mediated oxidativestress and apoptosisrdquo Journal of Investigative Dermatology vol127 no 1 pp 222ndash232 2007

[28] J-Y Bae J-S Choi Y-J Choi et al ldquo(minus)Epigallocatechin gallatehampers collagen destruction and collagenase activation inultraviolet-B-irradiated humandermal fibroblasts Involvementof mitogen-activated protein kinaserdquo Food and Chemical Toxi-cology vol 46 no 4 pp 1298ndash1307 2008

[29] H Choi D Kim J W Kim S Ngadiran M R Sarmidi and CS Park ldquoLabisia pumila extract protects skin cells from photo-aging caused by UVB irradiationrdquo Journal of Bioscience andBioengineering vol 109 no 3 pp 291ndash296 2010

[30] S Deng B JWest andC J Jensen ldquoSimultaneous characterisa-tion and quantitation of flavonol glycosides and aglycones innoni leaves using a validated HPLC-UVMS methodrdquo FoodChemistry vol 111 no 2 pp 526ndash529 2008

[31] D Cervantes-Laurean D D Schramm E L Jacobson IHalaweish G G Bruckner and G A Boissonneault ldquoInhibi-tion of advanced glycation end product formation on collagenby rutin and its metabolitesrdquo Journal of Nutritional Biochem-istry vol 17 no 8 pp 531ndash540 2006

[32] Y M Fonseca C D Catini F T M C Vicentini J C CardosoR L Cavalcanti De Albuquerque Junior and M J Vieira Fon-seca ldquoEfficacy of marigold extract-loaded formulations againstUV-induced oxidative stressrdquo Journal of Pharmaceutical Sci-ences vol 100 no 6 pp 2182ndash2193 2011

[33] P K Vayalil C A Elments and S K Katiyar ldquoTreatment ofgreen tea polyphenols in hydrophilic cream prevents UVB-induced oxidation of lipids and proteins depletion of antioxi-dant enzymes and phosphorylation ofMAPKproteins in SKH-1hairless mouse skinrdquo Carcinogenesis vol 24 no 5 pp 927ndash9362003

[34] S E Dal Belo L R Gaspar and PM B GM Campos ldquoPhoto-protective effects of topical formulations containing a combi-nation of Ginkgo biloba and green tea extractsrdquo PhytotherapyResearch vol 25 no 12 pp 1854ndash1860 2011

[35] R Ghadially B E Brown S M Sequeira-Martin K R Fein-gold and P M Elias ldquoThe aged epidermal permeability barrierStructural functional and lipid biochemical abnormalities inhumans and a senescent murine modelrdquo Journal of ClinicalInvestigation vol 95 no 5 pp 2281ndash2290 1995

[36] M D Contreras and R Sanchez ldquoApplication of a factorialdesign to the study of the flow behavior spreadability and trans-parency of a Carbopol ETD 2020 gel Part IIrdquo International Jour-nal of Pharmaceutics vol 234 no 1-2 pp 149ndash157 2002

[37] BW BarryRheology ofDermatological VehiclesMarcelDekkerNew York NY USA 1983

[38] S U Kim S J Kim J Y Lee et al ldquoProtective effects of dietarysoy isoflavones against UV-induced skin-aging in hairlessmouse modelrdquo Journal of the American College of Nutrition vol23 no 2 pp 157ndash162 2004

[39] M Amouroux G Dıaz-Ayil W C P M Blondel G Bourg-Heckly A Leroux and F Guillemin ldquoClassification of ultra-violet irradiated mouse skin histological stages by bimodalspectroscopy multiple excitation autofluorescence and diffusereflectancerdquo Journal of Biomedical Optics vol 14 no 1 ArticleID 014011 2009

[40] K A Hwang B R Yi and K C Choi ldquoMolecular Mechanismsand In Vivo Mouse Models of Skin Aging Associated withDermal Matrix Alterationsrdquo Laboratory Animal Research vol27 no 1 pp 1ndash8 2011

[41] G J Fisher S Kang J Varani et al ldquoMechanisms of photoagingand chronological skin agingrdquoArchives of Dermatology vol 138no 11 pp 1462ndash1470 2002

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

4 BioMed Research International

000020040

Abso

rban

ce (a

u)

060080100120140160180

2000 3000 4000 5000 6000Wavelength (nm)

7000 8000

15100

Figure 1 UV absorbance spectra of a blank formulation (0 extract) formulation extract 10 and formulation extract 15The formulationswere scanned under UV light (200 to 400 nm)

IV IC NIC 10 1500

05

10

15

20

25

Groups n = 10

Thic

knes

s (m

m)

lowastlowastlowastlowastlowastlowast lowastlowastlowast

(a)

IV IC NIC 10 150

10

20

30

40

Groups n = 10

lowastlowastlowastlowastlowastlowast lowastlowast

TEW

L (g

h m

2)

(b)

IV IC NIC 10 150

100

200

300

400

Groups n = 10

Eryt

hem

a ind

ex lowastlowast

lowastlowastlowast

(c)

Figure 2 (a) Thickness values (mm) (b) transepidermal water loss and (c) erythema obtained for the dorsal areas IV = irradiated andtreated with the vehicle (formulation 0) IC = irradiated control (the group was irradiated but did not receive any topical treatment) NIC= nonirradiated control (the group was not irradiated and did not receive any topical treatment) 10 = group irradiated and treated withthe formulation 10 15 = group irradiated and treated with the formulation 15 The values were obtained before (baseline values) and20 h after UV exposure during 7 days of treatment (119899 = 10 mice) 119875 lt 005 versus IC lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus IVANOVA followed by Tukeyrsquos test Data represents mean plusmn SEM

BioMed Research International 5

Table 1 Measurement of spreadability of formulations containingMorinda citrifolia extract (10 or 15 ww)

Formulations lowastSP1 lowastSP2 lowastSP3 lowastSP Mean lowastSP Mean plusmn SD CVExtract 10 1136 1044 1253 1144 1144 plusmn 107 94Extract 15 0955 0824 0969 0916 0916 plusmn 008 87lowastSpreadability factor (mm2

sdotgminus1)

IV IC NIC 10 15

Figure 3 Skin biophysical techniques Photoprotective effects of formulations onUVA-UVB-induced pathological changes in the skin surfaceon different groups after 7 days of treatment IV = irradiated and treated with the vehicle (formulation 0) IC = irradiated control (the groupwas irradiated but did not receive any topical treatment) NIC = nonirradiated control (the group was not irradiated and did not receive anytopical treatment) 10 = group irradiated and treated with the formulation 10 15 = group irradiated and treated with the formulation15

34 Pathological Changes in the Skin Surface Treated areaswith the formulation containing Morinda citrifolia extract(10 and 15) have enhanced protection against UV radiation(Figure 3)

35 Histological Measurements

Hematoxylin Eosin (HE) As demonstrated in Figure 4(a) thenonirradiated group showed that the epidermis was repre-sented by a thin continuous squamous epithelial liningwith an orthokeratotic surfaceThe connective tissue showedinterlaced collagen fibers associated with spindle and roundshaped stromal cells consistent with fibroblasts Some fewcapillary vessels and venules could also be seen as wellas numerous cutaneous appendages No inflammatory infil-trates were found in the dermal tissues Both irradiated andvehicle groups (Figures 4(b) and 4(c)) presented increasedepidermal thickness associated to hyperorthokeratosis Thepapillary dermis exhibited intense inflammatory infiltratemainly composed of lymphocytes mild interstitial edemaand prominent capillary and venular hyperemia Extensiveareas of elastotic alterations of the collagen bundles (baso-philic degeneration) were also observed throughout theconnective tissue (Figure 4(d))The treated groups presentedremarkable reduction in the inflammatory content as wellas less expressive hyperemia However epidermal thickeningwas more evident in 10 than in 15 formulation (Figures4(e) and 4(f)) Such similar histological features were quite

similar to those verified in the normal dermalepidermal tis-suesSirius Red (SR) The nonirradiated-NIC groups presentedinterlaced less compacted type I and III collagen fibers (Fig-ures 5(a) and 5(b)) In irradiated and vehicle groups thepapillary dermis showed the presence of gross thick parallel-arranged collagen fibers exhibiting intense yellow birefrin-gence (type I collagen) which were associated to shorter andthinner reddish fibrils perpendicularly disposed in relationto the surface Such collagen bundles were densely com-pacted showing sparse interfibrillar spaces (Figures 5(c) and5(d)) Both treated groups (10 and 15 formulations) pre-sented similar features regarding the collagenization patternshowing a mixture of yellowish type I (predominant) andgreenish type III collagen fibers These fibers were moredelicate slightly wavy with variable thicknesses lengths andexhibited interlaced arrangements (Figures 5(e) and 5(f))The interfibrillary spaces were more evident than in irradi-ated and vehicle groups Such morphological appearanceswere very similar to those observed in nonirradiated group(Figures 5(a) and 5(b))

36 Measurement of Cytotoxicity Activity The extract wasevaluated on murine malignant melanoma (cell line B16F10)in order to examine their cytotoxic effects on malignant cellsCytotoxicity of the extract on the growth of the B16F10 cellline is shown in Figure 6 Cell proliferation was analyzed

6 BioMed Research International

70120583m

(a)

70120583m

(b)

70120583m

(c)

70120583m

(d)

70120583m

(e)

70120583m

(f)

Figure 4 Hematoxylin-eosin stained histological sections (a) Nonirradiated group-NIC showing thin epidermal lining and absence ofdermal inflammation (b) Irradiated-IC and (c) vehicle groups-IV presenting epidermal thickening and intense lymphocytic infiltration in thedermal tissue (d) Elastotic change of the collagen (e) 10 and (f) 15 formulations showingmarked reduction of the inflammatory responseNote the thin epidermis in 15 formulation Short thick arrows epidermis Long thin arrows dermal connective tissues (Hematoxylin Eosin400x magnification)

24 h after B16F10 cells had been cultured with the extract atdifferent concentrations (005 01 02 04 08 16 32 64128 and 25mgmL) using the MTT assay The extract atconcentrations of 005 to 128mgmL did not show any cyto-toxic effects in the B16F10 cell line (showed no potential anti-cancer) A reduction in cell viability at high concentrationswas observed (25mgmL)

4 Discussion

The most distinguished harmful effect of solar exposure ispremature skin aging especially UV irradiation In recent

years thinning of the ozone layer has caused increased UVexposure Detrimental effects of UV on the skin involvethe generation of reactive oxygen species (ROS) [25] Com-ponents of the skin including DNA lipids and proteinscan be impaired by ROS which is the cause of erythemasunburn immune suppression and skin cancer [26] Most ofthe disorders were formerly regarded as having been causedbyUVBNevertheless recent studies showed that UVA is alsoan essential factor [27]

Accordingly use of antioxidants capable of scavengingand quenching ROS is another important approach to pre-vent UVB induced photoaging [27] Other studies have

BioMed Research International 7

70120583m

(a)

70120583m

(b)

70120583m

(c)

70120583m

(d)

70120583m

(e)

70120583m

(f)

Figure 5 Sirius Red stained histological sections (a b)Nonirradiated-NICgroups presenting interlaced less compacted type I and III collagenfibers (c) Irradiated-IC and (d) vehicle groups-IV showing dense disposition of parallel-arranged type I collagen fibers (e) 10 and (f) 15formulations presenting morphological and architectural collagen deposition pattern resembling that observed in the normal dermis (SiriusRedPolarization Light 400x magnification)

shown that botanical compounds with an antioxidative activ-ity are potential agents capable of reducing skin diseasesincluding photoaging [28 29]

The extract used in this work was the same studied bySerafini et al [19] and showed relevant in vitro antioxidantactivity against different radicals The extract was chemicallyanalyzed (qualitative method) The phytochemical screeningof extract showed the presence of alkaloids coumarins flavo-noids tannins saponins steroids and triterpenoids Totalphenolic content of the extract was 1968mg of phenolic equi-valents (gallic acid) per gram of extract Furthermore quali-tative analysis of extract was performed by HPLC revealingthe presence of 3 major compounds all showing ultraviolet

spectra typical of flavonols (quercetin-3-O-rutinoside orrutin and kaempferol glycosides) According to Deng et al[30] flavonol glycosides mainly rutin are the major com-pounds inM citrifolia leaves

The presence of rutin in the formulation could also besecondarily involved in this process of collagenization Ithas been demonstrated that the rutin is able to impair theglycation of collagen [31] a biochemical event involved in thepathogenesis of cell damaging in UV-irradiated tissue [32]

The use of botanical supplements to protect the humanskin from the adverse biological effects of solar radiationhas received great interest in recent years [33] Howevermost tests have been conducted by oral ingestion or topical

8 BioMed Research International

005 01 02 04 08 16 32 64 128 2500

50

100

150

Morinda citrifolia extract (mgmL)

Cel

l via

bilit

y (

)

Figure 6 Cytotoxic effect of theMorinda citrifolia extract in B16F10(murine malignant melanoma) cell line pretreated with the extractsat concentrations (005 to 25mgmL) for 24 h Each value representsthe mean plusmn SD

application of these ingredients in inappropriate vehicleswhich can alter the skin barrier To be of practical use todermatologists and to be safe and efficient extracts musthave physicochemical characteristics that permit their incor-poration in adequate concentrations in a skin-compatiblevehicle (eg gels moisturizing creams) [34] In this studythe capacity of the Morinda citrifolia extract added totopical formulations (gels) to prevent or to treat UVA-UVBirradiation-induced skin damage in mice was evaluated

First three formulations presenting different concen-trations (0 10 and 15) were evaluated in vitro byspectrophotometer UV absorbance spectra showed peaksat 200 nm and at 240 nm (UVC region) and absorbance inthe UVA (320ndash400 nm) and UVB (290ndash320 nm) regionsTheblank formulation (0 extract) did not show absorbance inthis spectra These data are compared to commercial organicfiltersThus this result suggests a photoprotective effect of theextractThe concentration of formulation (10 and 15ww)used in this assay was selected based on a pilot study

Second the gel formulation efficacy against UVA-UVBinduced skin damage was evaluated in vivo by measuring thefollowing parameters thickness values (mm) transepidermalwater loss (TEWL) and erythema

Moisturizing antifree radicals and sunscreen productshave been used largely to prevent skin disorders resultingfrom UV damage However other aspects of skin damageshould be considered such as erythema sunburn cell forma-tion epidermal hyperplasia and impairment of the skin bar-rier integrity which in turn is related to skin dryness skinaging and increased incidence of irritant contact dermatitis[35]

UV radiation damages the structures responsible for cor-neocyte adhesion and the functioning of the permeabilitybarrier which in turn alter thicknesses and TEWL measure-ments and accelerate the desquamation process [36]

The thickness TEWL and erythema were observed inour study and only the formulations containing M citri-folia extract protected mouse skin against the UV-induceddamages The pronounced protective effects provided by

both formulations (10 and 15) in the three parametersmeasured were not due to any extraneous raw material butwere shown by the unprotected groups treated by vehicle only(VI) Furthermore the results show that areas treated withthis extract (10 and 15) were statistically equivalent to theunirradiated control ones (NIC)which represented the intactskin areas

TEWL is an important parameter to be evaluated instudies of skin photoprotective effects considering that UVirradiation of mammalian skin disrupts the epidermal per-meability barrier function accompanied by an increase intransepidermal water loss (TEWL) and alterations in thestratum corneum lipid profile [33]

In the TEWL measurement the group 10 was differentfrom IC and the 15 was not As well as in the thickness andTEWL the protective effect of 10 was most pronouncedIt can also be seen in the photos of the dorsal of the ani-mal This observationcan be explained by differences in thespreadability Formulations containing extract at 10 pre-sented the higher spreadability

The spreadability is represented by the thickness of thefilm that the preparation leaves on the skin an importantfeature in cosmetics [37]Those producing thinner films thatis higher spreadability are naturally of greater interest [38]

Formulations containing extracts were shown to be effec-tive also in the presence of erythema another parameteroften used to evaluate photoprotection Extract formulationsreduced erythema formation when compared with the irra-diated control (IC) This statement corroborates [20] whichdemonstrated that the M citrifolia leaf extracts mitigate theeffects of UV light and provide some measure of defenseagainst localized inflammation (topical microswelling)

Third histological parameters were analyzed Epidermalthickening and inflammatory changes are common histo-logical changes observed in mice skin after short-term UVexposure [38 39] Such an increase in the epidermal thicknessis supposed to occur in response to cell proliferation inattempt to regenerate the tissue leading to a compensatorytype of hyperplasia However the thickened epidermis aswell as the thickened keratin layer (hyperkeratosis) could beobserved in this study

The presence of intense inflammatory infiltrate elastosisand the accumulation of parallel-arranged type I collagenfibers observed in IC and vehicle groups have also beendescribed as UV-induced dermal changes [31] Inflammationis one of the major histological signs of tissue damage andhas been reported in other studies as an early change of theconnective tissue after UV exposure [38 39] On the otherhand elastosis represents an actinic-induced accumulationof amorphous elastin material replacing the normal der-mis unrelated to preexisting or newly synthesized collagen[40] Moreover it has been proposed that UV irradiationimpairs ongoing collagen synthesis primarily through thedownregulation of types I and III procollagen expression[41] Therefore such impairment could likely promote delayin the collagen remodeling leading to the typical immatureparallel-arranged disposition of the fibers [31]

In this study only 15 formulation avoided compen-satory epidermal thickening However the reduction of the

BioMed Research International 9

major deleterious effects associated with the UV exposureon the animals treated with both 10 and 15 formulations isstrongly suggestive of photoprotective effect regardless of theextract concentration

Lastly the extract was evaluated on murine malignantmelanoma in order to examine its cytotoxic effects on malig-nant cells A reduction in cell viability was observed only atthe highest concentration (25mgmL) The results can sug-gest that the extract (in low concentrations) has only photo-protection effects and does not have antimelanoma activity

5 Conclusion

In conclusion present results strongly suggest the photo-protective effect of M citrifolia The results indicate thatphotoprotective effects are due to not only UV absorbancelike the UV filters but also biological effects which happenedmainly with the application of the formulation containing theMorinda citrifolia extract Further studies are necessary toclarify the precise nature of this photoprotective effect

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by Fundacao de Amparo a Pesquisado Estado de Sergipe (FAPITEC-SE) and Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) ofBrazil

References

[1] A M Garcıa-Bores and J G Avila ldquoProducts ldquoMolecularmechanisms in the photochemopreventionrdquordquo Revista LatinoAmericana de Quımica vol 36 no 3 pp 83ndash102 2008

[2] M D Adil P Kaiser N K Satti A M Zargar R A Vish-wakarma and S A Tasduq ldquoEffect of Emblica officinalis (fruit)against UVB-induced photo-aging in human skin fibroblastsrdquoJournal of Ethnopharmacology vol 132 no 1 pp 109ndash114 2010

[3] M Dalle Carbonare and M A Pathak ldquoSkin photosensitizingagents and the role of reactive oxygen species in photoagingrdquoJournal of Photochemistry and Photobiology B Biology vol 14no 1-2 pp 105ndash124 1992

[4] G J Fisher S Datta ZWang et al ldquoc-Jun-dependent inhibitionof cutaneous procollagen transcription following ultravioletirradiation is reversed by all-trans retinoic acidrdquoThe Journal ofClinical Investigation vol 106 no 5 pp 663ndash670 2000

[5] S Inomata Y Matsunaga S Amano et al ldquoPossible involve-ment of gelatinases in basementmembrane damage andwrinkleformation in chronically ultraviolet B-exposed hairless mouserdquoJournal of Investigative Dermatology vol 120 no 1 pp 128ndash1342003

[6] P S Peres V A Terra F A Guarnier R Cecchini and A LCecchini ldquoPhotoaging and chronological aging profile under-standing oxidation of the skinrdquo Journal of Photochemistry andPhotobiology B Biology vol 103 no 2 pp 93ndash97 2011

[7] Y Xu and G J Fisher ldquoUltraviolet (UV) light irradiationinduced signal transduction in skin photoagingrdquo Journal of Der-matological Science Supplement vol 1 no 2 pp S1ndashS8 2005

[8] S P Stratton R T Dorr and D S Alberts ldquoThe state-of-the-artin chemoprevention of skin cancerrdquoEuropean Journal of Cancervol 36 no 10 pp 1292ndash1297 2000

[9] F Afaq V M Adhami and H Mukhtar ldquoPhotochemopreven-tion of ultraviolet B signaling and photocarcinogenesisrdquoMuta-tion Research vol 571 no 1-2 pp 153ndash173 2005

[10] V M Adhami D N Syed N Khan and F Afaq ldquoPhytochem-icals for prevention of solar ultraviolet radiation-induced dam-agesrdquo Photochemistry and Photobiology vol 84 no 2 pp 489ndash500 2008

[11] C D Ropke T CH Sawada V V da Silva N SMichalany andS B DeMoraes Barros ldquoPhotoprotective effect of Pothomorpheumbellata root extract against ultraviolet radiation inducedchronic skin damage in the hairless mouserdquo Clinical andExperimental Dermatology vol 30 no 3 pp 272ndash276 2005

[12] M G D Melo J P A dos Santos M R Serafini et al ldquoRedoxproperties and cytoprotective actions of atranorin a lichensecondary metaboliterdquo Toxicology in Vitro vol 25 no 2 pp462ndash468 2011

[13] M R Serafini D M C Vergne T K Rabelo et al ldquoDetermi-nation of chemical and physical properties of Hyptis pectinataessential oil and their redox active profilerdquo Journal of Biotech-nology and Pharmaceutical Research vol 3 no 1 pp 1ndash9 2012

[14] R Casagrande S R Georgetti W A Verri Jr D J Dorta A Cdos Santos and M J V Fonseca ldquoProtective effect of topicalformulations containing quercetin against UVB-induced oxi-dative stress in hairless micerdquo Journal of Photochemistry andPhotobiology B Biology vol 84 no 1 pp 21ndash27 2006

[15] S Frsquoguyer F Afaq and H Mukhtar ldquoPhotochemopreventionof skin cancer by botanical agentsrdquo Photodermatology Photoim-munology and Photomedicine vol 19 no 2 pp 56ndash72 2003

[16] Y Chan-Blanco F Vaillant A Mercedes Perez M Reynes JBrillouet and P Brat ldquoThe noni fruit (Morinda citrifolia L) areview of agricultural research nutritional and therapeutic pro-pertiesrdquo Journal of Food Composition and Analysis vol 19 no 6-7 pp 645ndash654 2006

[17] H Kim M Kwon J Kim et al ldquoIdentification of novel fattyacid glucosides from the tropical fruit Morinda citrifolia LrdquoPhytochemistry Letters vol 3 no 4 pp 238ndash241 2010

[18] A Hirazumi E Furusawa S C Chou and Y Hokama ldquoImmu-nomodulation contributes to the anticancer activity ofMorindacitrifolia (noni) fruit juicerdquo Proceedings of the Western Pharma-cology Society vol 39 pp 7ndash9 1996

[19] M R Serafini R C Santos A G Guimaraes et al ldquoMorindacitrifolia linn leaf extract possesses antioxidant activities andreduces nociceptive behavior and leukocyte migrationrdquo Journalof Medicinal Food vol 14 no 10 pp 1159ndash1166 2011

[20] B J West S Deng A K Palu and C J Jensen ldquoMorinda citri-folia Linn (Rubiaceae) leaf extracts mitigate UVB-induced ery-themardquo Journal of Natural Medicines vol 63 no 3 pp 351ndash3542009

[21] A Garg D Aggarwal S Garg and A Singela ldquoSpreading ofsemisolid formulations pharmaceutical technologyrdquo Pharma-ceutical Technology vol 26 pp 84ndash105 2002

[22] K Paese A Jager F S Poletto et al ldquoSemisolid formulationcontaining a nanoencapsulated sunscreen effectiveness in vitrophotostability and immune responserdquo Journal of Biomedical andNanotechnology vol 5 no 3 pp 240ndash246 2009

10 BioMed Research International

[23] M Demerjian P Hachem E Tschachler et al ldquoAcute modula-tions in permeability barrier function regulate epidermal corni-ficationrdquo The American Journal of Pathology vol 172 no 1 pp86ndash97 2008

[24] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[25] H Sakurai H Yasui Y Yamada H Nishimura and M Shige-moto ldquoDetection of reactive oxygen species in the skin of livemice and rats exposed toUVA light a research reviewon chemi-luminescence and trials for UVA protectionrdquo Photochemical ampPhotobiological Sciences vol 4 no 9 pp 715ndash720 2005

[26] R Haywood P Wardman R Sanders and C Linge ldquoSun-screens inadequately protect against ultraviolet-A-induced freeradicals in skin implications for skin aging and melanomardquoJournal of Investigative Dermatology vol 121 no 4 pp 862ndash8682003

[27] F Afaq DN Syed AMalik et al ldquoDelphinidin an anthocyani-din in pigmented fruits and vegetables protects human HaCaTkeratinocytes and mouse skin against UVB-mediated oxidativestress and apoptosisrdquo Journal of Investigative Dermatology vol127 no 1 pp 222ndash232 2007

[28] J-Y Bae J-S Choi Y-J Choi et al ldquo(minus)Epigallocatechin gallatehampers collagen destruction and collagenase activation inultraviolet-B-irradiated humandermal fibroblasts Involvementof mitogen-activated protein kinaserdquo Food and Chemical Toxi-cology vol 46 no 4 pp 1298ndash1307 2008

[29] H Choi D Kim J W Kim S Ngadiran M R Sarmidi and CS Park ldquoLabisia pumila extract protects skin cells from photo-aging caused by UVB irradiationrdquo Journal of Bioscience andBioengineering vol 109 no 3 pp 291ndash296 2010

[30] S Deng B JWest andC J Jensen ldquoSimultaneous characterisa-tion and quantitation of flavonol glycosides and aglycones innoni leaves using a validated HPLC-UVMS methodrdquo FoodChemistry vol 111 no 2 pp 526ndash529 2008

[31] D Cervantes-Laurean D D Schramm E L Jacobson IHalaweish G G Bruckner and G A Boissonneault ldquoInhibi-tion of advanced glycation end product formation on collagenby rutin and its metabolitesrdquo Journal of Nutritional Biochem-istry vol 17 no 8 pp 531ndash540 2006

[32] Y M Fonseca C D Catini F T M C Vicentini J C CardosoR L Cavalcanti De Albuquerque Junior and M J Vieira Fon-seca ldquoEfficacy of marigold extract-loaded formulations againstUV-induced oxidative stressrdquo Journal of Pharmaceutical Sci-ences vol 100 no 6 pp 2182ndash2193 2011

[33] P K Vayalil C A Elments and S K Katiyar ldquoTreatment ofgreen tea polyphenols in hydrophilic cream prevents UVB-induced oxidation of lipids and proteins depletion of antioxi-dant enzymes and phosphorylation ofMAPKproteins in SKH-1hairless mouse skinrdquo Carcinogenesis vol 24 no 5 pp 927ndash9362003

[34] S E Dal Belo L R Gaspar and PM B GM Campos ldquoPhoto-protective effects of topical formulations containing a combi-nation of Ginkgo biloba and green tea extractsrdquo PhytotherapyResearch vol 25 no 12 pp 1854ndash1860 2011

[35] R Ghadially B E Brown S M Sequeira-Martin K R Fein-gold and P M Elias ldquoThe aged epidermal permeability barrierStructural functional and lipid biochemical abnormalities inhumans and a senescent murine modelrdquo Journal of ClinicalInvestigation vol 95 no 5 pp 2281ndash2290 1995

[36] M D Contreras and R Sanchez ldquoApplication of a factorialdesign to the study of the flow behavior spreadability and trans-parency of a Carbopol ETD 2020 gel Part IIrdquo International Jour-nal of Pharmaceutics vol 234 no 1-2 pp 149ndash157 2002

[37] BW BarryRheology ofDermatological VehiclesMarcelDekkerNew York NY USA 1983

[38] S U Kim S J Kim J Y Lee et al ldquoProtective effects of dietarysoy isoflavones against UV-induced skin-aging in hairlessmouse modelrdquo Journal of the American College of Nutrition vol23 no 2 pp 157ndash162 2004

[39] M Amouroux G Dıaz-Ayil W C P M Blondel G Bourg-Heckly A Leroux and F Guillemin ldquoClassification of ultra-violet irradiated mouse skin histological stages by bimodalspectroscopy multiple excitation autofluorescence and diffusereflectancerdquo Journal of Biomedical Optics vol 14 no 1 ArticleID 014011 2009

[40] K A Hwang B R Yi and K C Choi ldquoMolecular Mechanismsand In Vivo Mouse Models of Skin Aging Associated withDermal Matrix Alterationsrdquo Laboratory Animal Research vol27 no 1 pp 1ndash8 2011

[41] G J Fisher S Kang J Varani et al ldquoMechanisms of photoagingand chronological skin agingrdquoArchives of Dermatology vol 138no 11 pp 1462ndash1470 2002

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 5

Table 1 Measurement of spreadability of formulations containingMorinda citrifolia extract (10 or 15 ww)

Formulations lowastSP1 lowastSP2 lowastSP3 lowastSP Mean lowastSP Mean plusmn SD CVExtract 10 1136 1044 1253 1144 1144 plusmn 107 94Extract 15 0955 0824 0969 0916 0916 plusmn 008 87lowastSpreadability factor (mm2

sdotgminus1)

IV IC NIC 10 15

Figure 3 Skin biophysical techniques Photoprotective effects of formulations onUVA-UVB-induced pathological changes in the skin surfaceon different groups after 7 days of treatment IV = irradiated and treated with the vehicle (formulation 0) IC = irradiated control (the groupwas irradiated but did not receive any topical treatment) NIC = nonirradiated control (the group was not irradiated and did not receive anytopical treatment) 10 = group irradiated and treated with the formulation 10 15 = group irradiated and treated with the formulation15

34 Pathological Changes in the Skin Surface Treated areaswith the formulation containing Morinda citrifolia extract(10 and 15) have enhanced protection against UV radiation(Figure 3)

35 Histological Measurements

Hematoxylin Eosin (HE) As demonstrated in Figure 4(a) thenonirradiated group showed that the epidermis was repre-sented by a thin continuous squamous epithelial liningwith an orthokeratotic surfaceThe connective tissue showedinterlaced collagen fibers associated with spindle and roundshaped stromal cells consistent with fibroblasts Some fewcapillary vessels and venules could also be seen as wellas numerous cutaneous appendages No inflammatory infil-trates were found in the dermal tissues Both irradiated andvehicle groups (Figures 4(b) and 4(c)) presented increasedepidermal thickness associated to hyperorthokeratosis Thepapillary dermis exhibited intense inflammatory infiltratemainly composed of lymphocytes mild interstitial edemaand prominent capillary and venular hyperemia Extensiveareas of elastotic alterations of the collagen bundles (baso-philic degeneration) were also observed throughout theconnective tissue (Figure 4(d))The treated groups presentedremarkable reduction in the inflammatory content as wellas less expressive hyperemia However epidermal thickeningwas more evident in 10 than in 15 formulation (Figures4(e) and 4(f)) Such similar histological features were quite

similar to those verified in the normal dermalepidermal tis-suesSirius Red (SR) The nonirradiated-NIC groups presentedinterlaced less compacted type I and III collagen fibers (Fig-ures 5(a) and 5(b)) In irradiated and vehicle groups thepapillary dermis showed the presence of gross thick parallel-arranged collagen fibers exhibiting intense yellow birefrin-gence (type I collagen) which were associated to shorter andthinner reddish fibrils perpendicularly disposed in relationto the surface Such collagen bundles were densely com-pacted showing sparse interfibrillar spaces (Figures 5(c) and5(d)) Both treated groups (10 and 15 formulations) pre-sented similar features regarding the collagenization patternshowing a mixture of yellowish type I (predominant) andgreenish type III collagen fibers These fibers were moredelicate slightly wavy with variable thicknesses lengths andexhibited interlaced arrangements (Figures 5(e) and 5(f))The interfibrillary spaces were more evident than in irradi-ated and vehicle groups Such morphological appearanceswere very similar to those observed in nonirradiated group(Figures 5(a) and 5(b))

36 Measurement of Cytotoxicity Activity The extract wasevaluated on murine malignant melanoma (cell line B16F10)in order to examine their cytotoxic effects on malignant cellsCytotoxicity of the extract on the growth of the B16F10 cellline is shown in Figure 6 Cell proliferation was analyzed

6 BioMed Research International

70120583m

(a)

70120583m

(b)

70120583m

(c)

70120583m

(d)

70120583m

(e)

70120583m

(f)

Figure 4 Hematoxylin-eosin stained histological sections (a) Nonirradiated group-NIC showing thin epidermal lining and absence ofdermal inflammation (b) Irradiated-IC and (c) vehicle groups-IV presenting epidermal thickening and intense lymphocytic infiltration in thedermal tissue (d) Elastotic change of the collagen (e) 10 and (f) 15 formulations showingmarked reduction of the inflammatory responseNote the thin epidermis in 15 formulation Short thick arrows epidermis Long thin arrows dermal connective tissues (Hematoxylin Eosin400x magnification)

24 h after B16F10 cells had been cultured with the extract atdifferent concentrations (005 01 02 04 08 16 32 64128 and 25mgmL) using the MTT assay The extract atconcentrations of 005 to 128mgmL did not show any cyto-toxic effects in the B16F10 cell line (showed no potential anti-cancer) A reduction in cell viability at high concentrationswas observed (25mgmL)

4 Discussion

The most distinguished harmful effect of solar exposure ispremature skin aging especially UV irradiation In recent

years thinning of the ozone layer has caused increased UVexposure Detrimental effects of UV on the skin involvethe generation of reactive oxygen species (ROS) [25] Com-ponents of the skin including DNA lipids and proteinscan be impaired by ROS which is the cause of erythemasunburn immune suppression and skin cancer [26] Most ofthe disorders were formerly regarded as having been causedbyUVBNevertheless recent studies showed that UVA is alsoan essential factor [27]

Accordingly use of antioxidants capable of scavengingand quenching ROS is another important approach to pre-vent UVB induced photoaging [27] Other studies have

BioMed Research International 7

70120583m

(a)

70120583m

(b)

70120583m

(c)

70120583m

(d)

70120583m

(e)

70120583m

(f)

Figure 5 Sirius Red stained histological sections (a b)Nonirradiated-NICgroups presenting interlaced less compacted type I and III collagenfibers (c) Irradiated-IC and (d) vehicle groups-IV showing dense disposition of parallel-arranged type I collagen fibers (e) 10 and (f) 15formulations presenting morphological and architectural collagen deposition pattern resembling that observed in the normal dermis (SiriusRedPolarization Light 400x magnification)

shown that botanical compounds with an antioxidative activ-ity are potential agents capable of reducing skin diseasesincluding photoaging [28 29]

The extract used in this work was the same studied bySerafini et al [19] and showed relevant in vitro antioxidantactivity against different radicals The extract was chemicallyanalyzed (qualitative method) The phytochemical screeningof extract showed the presence of alkaloids coumarins flavo-noids tannins saponins steroids and triterpenoids Totalphenolic content of the extract was 1968mg of phenolic equi-valents (gallic acid) per gram of extract Furthermore quali-tative analysis of extract was performed by HPLC revealingthe presence of 3 major compounds all showing ultraviolet

spectra typical of flavonols (quercetin-3-O-rutinoside orrutin and kaempferol glycosides) According to Deng et al[30] flavonol glycosides mainly rutin are the major com-pounds inM citrifolia leaves

The presence of rutin in the formulation could also besecondarily involved in this process of collagenization Ithas been demonstrated that the rutin is able to impair theglycation of collagen [31] a biochemical event involved in thepathogenesis of cell damaging in UV-irradiated tissue [32]

The use of botanical supplements to protect the humanskin from the adverse biological effects of solar radiationhas received great interest in recent years [33] Howevermost tests have been conducted by oral ingestion or topical

8 BioMed Research International

005 01 02 04 08 16 32 64 128 2500

50

100

150

Morinda citrifolia extract (mgmL)

Cel

l via

bilit

y (

)

Figure 6 Cytotoxic effect of theMorinda citrifolia extract in B16F10(murine malignant melanoma) cell line pretreated with the extractsat concentrations (005 to 25mgmL) for 24 h Each value representsthe mean plusmn SD

application of these ingredients in inappropriate vehicleswhich can alter the skin barrier To be of practical use todermatologists and to be safe and efficient extracts musthave physicochemical characteristics that permit their incor-poration in adequate concentrations in a skin-compatiblevehicle (eg gels moisturizing creams) [34] In this studythe capacity of the Morinda citrifolia extract added totopical formulations (gels) to prevent or to treat UVA-UVBirradiation-induced skin damage in mice was evaluated

First three formulations presenting different concen-trations (0 10 and 15) were evaluated in vitro byspectrophotometer UV absorbance spectra showed peaksat 200 nm and at 240 nm (UVC region) and absorbance inthe UVA (320ndash400 nm) and UVB (290ndash320 nm) regionsTheblank formulation (0 extract) did not show absorbance inthis spectra These data are compared to commercial organicfiltersThus this result suggests a photoprotective effect of theextractThe concentration of formulation (10 and 15ww)used in this assay was selected based on a pilot study

Second the gel formulation efficacy against UVA-UVBinduced skin damage was evaluated in vivo by measuring thefollowing parameters thickness values (mm) transepidermalwater loss (TEWL) and erythema

Moisturizing antifree radicals and sunscreen productshave been used largely to prevent skin disorders resultingfrom UV damage However other aspects of skin damageshould be considered such as erythema sunburn cell forma-tion epidermal hyperplasia and impairment of the skin bar-rier integrity which in turn is related to skin dryness skinaging and increased incidence of irritant contact dermatitis[35]

UV radiation damages the structures responsible for cor-neocyte adhesion and the functioning of the permeabilitybarrier which in turn alter thicknesses and TEWL measure-ments and accelerate the desquamation process [36]

The thickness TEWL and erythema were observed inour study and only the formulations containing M citri-folia extract protected mouse skin against the UV-induceddamages The pronounced protective effects provided by

both formulations (10 and 15) in the three parametersmeasured were not due to any extraneous raw material butwere shown by the unprotected groups treated by vehicle only(VI) Furthermore the results show that areas treated withthis extract (10 and 15) were statistically equivalent to theunirradiated control ones (NIC)which represented the intactskin areas

TEWL is an important parameter to be evaluated instudies of skin photoprotective effects considering that UVirradiation of mammalian skin disrupts the epidermal per-meability barrier function accompanied by an increase intransepidermal water loss (TEWL) and alterations in thestratum corneum lipid profile [33]

In the TEWL measurement the group 10 was differentfrom IC and the 15 was not As well as in the thickness andTEWL the protective effect of 10 was most pronouncedIt can also be seen in the photos of the dorsal of the ani-mal This observationcan be explained by differences in thespreadability Formulations containing extract at 10 pre-sented the higher spreadability

The spreadability is represented by the thickness of thefilm that the preparation leaves on the skin an importantfeature in cosmetics [37]Those producing thinner films thatis higher spreadability are naturally of greater interest [38]

Formulations containing extracts were shown to be effec-tive also in the presence of erythema another parameteroften used to evaluate photoprotection Extract formulationsreduced erythema formation when compared with the irra-diated control (IC) This statement corroborates [20] whichdemonstrated that the M citrifolia leaf extracts mitigate theeffects of UV light and provide some measure of defenseagainst localized inflammation (topical microswelling)

Third histological parameters were analyzed Epidermalthickening and inflammatory changes are common histo-logical changes observed in mice skin after short-term UVexposure [38 39] Such an increase in the epidermal thicknessis supposed to occur in response to cell proliferation inattempt to regenerate the tissue leading to a compensatorytype of hyperplasia However the thickened epidermis aswell as the thickened keratin layer (hyperkeratosis) could beobserved in this study

The presence of intense inflammatory infiltrate elastosisand the accumulation of parallel-arranged type I collagenfibers observed in IC and vehicle groups have also beendescribed as UV-induced dermal changes [31] Inflammationis one of the major histological signs of tissue damage andhas been reported in other studies as an early change of theconnective tissue after UV exposure [38 39] On the otherhand elastosis represents an actinic-induced accumulationof amorphous elastin material replacing the normal der-mis unrelated to preexisting or newly synthesized collagen[40] Moreover it has been proposed that UV irradiationimpairs ongoing collagen synthesis primarily through thedownregulation of types I and III procollagen expression[41] Therefore such impairment could likely promote delayin the collagen remodeling leading to the typical immatureparallel-arranged disposition of the fibers [31]

In this study only 15 formulation avoided compen-satory epidermal thickening However the reduction of the

BioMed Research International 9

major deleterious effects associated with the UV exposureon the animals treated with both 10 and 15 formulations isstrongly suggestive of photoprotective effect regardless of theextract concentration

Lastly the extract was evaluated on murine malignantmelanoma in order to examine its cytotoxic effects on malig-nant cells A reduction in cell viability was observed only atthe highest concentration (25mgmL) The results can sug-gest that the extract (in low concentrations) has only photo-protection effects and does not have antimelanoma activity

5 Conclusion

In conclusion present results strongly suggest the photo-protective effect of M citrifolia The results indicate thatphotoprotective effects are due to not only UV absorbancelike the UV filters but also biological effects which happenedmainly with the application of the formulation containing theMorinda citrifolia extract Further studies are necessary toclarify the precise nature of this photoprotective effect

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by Fundacao de Amparo a Pesquisado Estado de Sergipe (FAPITEC-SE) and Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) ofBrazil

References

[1] A M Garcıa-Bores and J G Avila ldquoProducts ldquoMolecularmechanisms in the photochemopreventionrdquordquo Revista LatinoAmericana de Quımica vol 36 no 3 pp 83ndash102 2008

[2] M D Adil P Kaiser N K Satti A M Zargar R A Vish-wakarma and S A Tasduq ldquoEffect of Emblica officinalis (fruit)against UVB-induced photo-aging in human skin fibroblastsrdquoJournal of Ethnopharmacology vol 132 no 1 pp 109ndash114 2010

[3] M Dalle Carbonare and M A Pathak ldquoSkin photosensitizingagents and the role of reactive oxygen species in photoagingrdquoJournal of Photochemistry and Photobiology B Biology vol 14no 1-2 pp 105ndash124 1992

[4] G J Fisher S Datta ZWang et al ldquoc-Jun-dependent inhibitionof cutaneous procollagen transcription following ultravioletirradiation is reversed by all-trans retinoic acidrdquoThe Journal ofClinical Investigation vol 106 no 5 pp 663ndash670 2000

[5] S Inomata Y Matsunaga S Amano et al ldquoPossible involve-ment of gelatinases in basementmembrane damage andwrinkleformation in chronically ultraviolet B-exposed hairless mouserdquoJournal of Investigative Dermatology vol 120 no 1 pp 128ndash1342003

[6] P S Peres V A Terra F A Guarnier R Cecchini and A LCecchini ldquoPhotoaging and chronological aging profile under-standing oxidation of the skinrdquo Journal of Photochemistry andPhotobiology B Biology vol 103 no 2 pp 93ndash97 2011

[7] Y Xu and G J Fisher ldquoUltraviolet (UV) light irradiationinduced signal transduction in skin photoagingrdquo Journal of Der-matological Science Supplement vol 1 no 2 pp S1ndashS8 2005

[8] S P Stratton R T Dorr and D S Alberts ldquoThe state-of-the-artin chemoprevention of skin cancerrdquoEuropean Journal of Cancervol 36 no 10 pp 1292ndash1297 2000

[9] F Afaq V M Adhami and H Mukhtar ldquoPhotochemopreven-tion of ultraviolet B signaling and photocarcinogenesisrdquoMuta-tion Research vol 571 no 1-2 pp 153ndash173 2005

[10] V M Adhami D N Syed N Khan and F Afaq ldquoPhytochem-icals for prevention of solar ultraviolet radiation-induced dam-agesrdquo Photochemistry and Photobiology vol 84 no 2 pp 489ndash500 2008

[11] C D Ropke T CH Sawada V V da Silva N SMichalany andS B DeMoraes Barros ldquoPhotoprotective effect of Pothomorpheumbellata root extract against ultraviolet radiation inducedchronic skin damage in the hairless mouserdquo Clinical andExperimental Dermatology vol 30 no 3 pp 272ndash276 2005

[12] M G D Melo J P A dos Santos M R Serafini et al ldquoRedoxproperties and cytoprotective actions of atranorin a lichensecondary metaboliterdquo Toxicology in Vitro vol 25 no 2 pp462ndash468 2011

[13] M R Serafini D M C Vergne T K Rabelo et al ldquoDetermi-nation of chemical and physical properties of Hyptis pectinataessential oil and their redox active profilerdquo Journal of Biotech-nology and Pharmaceutical Research vol 3 no 1 pp 1ndash9 2012

[14] R Casagrande S R Georgetti W A Verri Jr D J Dorta A Cdos Santos and M J V Fonseca ldquoProtective effect of topicalformulations containing quercetin against UVB-induced oxi-dative stress in hairless micerdquo Journal of Photochemistry andPhotobiology B Biology vol 84 no 1 pp 21ndash27 2006

[15] S Frsquoguyer F Afaq and H Mukhtar ldquoPhotochemopreventionof skin cancer by botanical agentsrdquo Photodermatology Photoim-munology and Photomedicine vol 19 no 2 pp 56ndash72 2003

[16] Y Chan-Blanco F Vaillant A Mercedes Perez M Reynes JBrillouet and P Brat ldquoThe noni fruit (Morinda citrifolia L) areview of agricultural research nutritional and therapeutic pro-pertiesrdquo Journal of Food Composition and Analysis vol 19 no 6-7 pp 645ndash654 2006

[17] H Kim M Kwon J Kim et al ldquoIdentification of novel fattyacid glucosides from the tropical fruit Morinda citrifolia LrdquoPhytochemistry Letters vol 3 no 4 pp 238ndash241 2010

[18] A Hirazumi E Furusawa S C Chou and Y Hokama ldquoImmu-nomodulation contributes to the anticancer activity ofMorindacitrifolia (noni) fruit juicerdquo Proceedings of the Western Pharma-cology Society vol 39 pp 7ndash9 1996

[19] M R Serafini R C Santos A G Guimaraes et al ldquoMorindacitrifolia linn leaf extract possesses antioxidant activities andreduces nociceptive behavior and leukocyte migrationrdquo Journalof Medicinal Food vol 14 no 10 pp 1159ndash1166 2011

[20] B J West S Deng A K Palu and C J Jensen ldquoMorinda citri-folia Linn (Rubiaceae) leaf extracts mitigate UVB-induced ery-themardquo Journal of Natural Medicines vol 63 no 3 pp 351ndash3542009

[21] A Garg D Aggarwal S Garg and A Singela ldquoSpreading ofsemisolid formulations pharmaceutical technologyrdquo Pharma-ceutical Technology vol 26 pp 84ndash105 2002

[22] K Paese A Jager F S Poletto et al ldquoSemisolid formulationcontaining a nanoencapsulated sunscreen effectiveness in vitrophotostability and immune responserdquo Journal of Biomedical andNanotechnology vol 5 no 3 pp 240ndash246 2009

10 BioMed Research International

[23] M Demerjian P Hachem E Tschachler et al ldquoAcute modula-tions in permeability barrier function regulate epidermal corni-ficationrdquo The American Journal of Pathology vol 172 no 1 pp86ndash97 2008

[24] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[25] H Sakurai H Yasui Y Yamada H Nishimura and M Shige-moto ldquoDetection of reactive oxygen species in the skin of livemice and rats exposed toUVA light a research reviewon chemi-luminescence and trials for UVA protectionrdquo Photochemical ampPhotobiological Sciences vol 4 no 9 pp 715ndash720 2005

[26] R Haywood P Wardman R Sanders and C Linge ldquoSun-screens inadequately protect against ultraviolet-A-induced freeradicals in skin implications for skin aging and melanomardquoJournal of Investigative Dermatology vol 121 no 4 pp 862ndash8682003

[27] F Afaq DN Syed AMalik et al ldquoDelphinidin an anthocyani-din in pigmented fruits and vegetables protects human HaCaTkeratinocytes and mouse skin against UVB-mediated oxidativestress and apoptosisrdquo Journal of Investigative Dermatology vol127 no 1 pp 222ndash232 2007

[28] J-Y Bae J-S Choi Y-J Choi et al ldquo(minus)Epigallocatechin gallatehampers collagen destruction and collagenase activation inultraviolet-B-irradiated humandermal fibroblasts Involvementof mitogen-activated protein kinaserdquo Food and Chemical Toxi-cology vol 46 no 4 pp 1298ndash1307 2008

[29] H Choi D Kim J W Kim S Ngadiran M R Sarmidi and CS Park ldquoLabisia pumila extract protects skin cells from photo-aging caused by UVB irradiationrdquo Journal of Bioscience andBioengineering vol 109 no 3 pp 291ndash296 2010

[30] S Deng B JWest andC J Jensen ldquoSimultaneous characterisa-tion and quantitation of flavonol glycosides and aglycones innoni leaves using a validated HPLC-UVMS methodrdquo FoodChemistry vol 111 no 2 pp 526ndash529 2008

[31] D Cervantes-Laurean D D Schramm E L Jacobson IHalaweish G G Bruckner and G A Boissonneault ldquoInhibi-tion of advanced glycation end product formation on collagenby rutin and its metabolitesrdquo Journal of Nutritional Biochem-istry vol 17 no 8 pp 531ndash540 2006

[32] Y M Fonseca C D Catini F T M C Vicentini J C CardosoR L Cavalcanti De Albuquerque Junior and M J Vieira Fon-seca ldquoEfficacy of marigold extract-loaded formulations againstUV-induced oxidative stressrdquo Journal of Pharmaceutical Sci-ences vol 100 no 6 pp 2182ndash2193 2011

[33] P K Vayalil C A Elments and S K Katiyar ldquoTreatment ofgreen tea polyphenols in hydrophilic cream prevents UVB-induced oxidation of lipids and proteins depletion of antioxi-dant enzymes and phosphorylation ofMAPKproteins in SKH-1hairless mouse skinrdquo Carcinogenesis vol 24 no 5 pp 927ndash9362003

[34] S E Dal Belo L R Gaspar and PM B GM Campos ldquoPhoto-protective effects of topical formulations containing a combi-nation of Ginkgo biloba and green tea extractsrdquo PhytotherapyResearch vol 25 no 12 pp 1854ndash1860 2011

[35] R Ghadially B E Brown S M Sequeira-Martin K R Fein-gold and P M Elias ldquoThe aged epidermal permeability barrierStructural functional and lipid biochemical abnormalities inhumans and a senescent murine modelrdquo Journal of ClinicalInvestigation vol 95 no 5 pp 2281ndash2290 1995

[36] M D Contreras and R Sanchez ldquoApplication of a factorialdesign to the study of the flow behavior spreadability and trans-parency of a Carbopol ETD 2020 gel Part IIrdquo International Jour-nal of Pharmaceutics vol 234 no 1-2 pp 149ndash157 2002

[37] BW BarryRheology ofDermatological VehiclesMarcelDekkerNew York NY USA 1983

[38] S U Kim S J Kim J Y Lee et al ldquoProtective effects of dietarysoy isoflavones against UV-induced skin-aging in hairlessmouse modelrdquo Journal of the American College of Nutrition vol23 no 2 pp 157ndash162 2004

[39] M Amouroux G Dıaz-Ayil W C P M Blondel G Bourg-Heckly A Leroux and F Guillemin ldquoClassification of ultra-violet irradiated mouse skin histological stages by bimodalspectroscopy multiple excitation autofluorescence and diffusereflectancerdquo Journal of Biomedical Optics vol 14 no 1 ArticleID 014011 2009

[40] K A Hwang B R Yi and K C Choi ldquoMolecular Mechanismsand In Vivo Mouse Models of Skin Aging Associated withDermal Matrix Alterationsrdquo Laboratory Animal Research vol27 no 1 pp 1ndash8 2011

[41] G J Fisher S Kang J Varani et al ldquoMechanisms of photoagingand chronological skin agingrdquoArchives of Dermatology vol 138no 11 pp 1462ndash1470 2002

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

6 BioMed Research International

70120583m

(a)

70120583m

(b)

70120583m

(c)

70120583m

(d)

70120583m

(e)

70120583m

(f)

Figure 4 Hematoxylin-eosin stained histological sections (a) Nonirradiated group-NIC showing thin epidermal lining and absence ofdermal inflammation (b) Irradiated-IC and (c) vehicle groups-IV presenting epidermal thickening and intense lymphocytic infiltration in thedermal tissue (d) Elastotic change of the collagen (e) 10 and (f) 15 formulations showingmarked reduction of the inflammatory responseNote the thin epidermis in 15 formulation Short thick arrows epidermis Long thin arrows dermal connective tissues (Hematoxylin Eosin400x magnification)

24 h after B16F10 cells had been cultured with the extract atdifferent concentrations (005 01 02 04 08 16 32 64128 and 25mgmL) using the MTT assay The extract atconcentrations of 005 to 128mgmL did not show any cyto-toxic effects in the B16F10 cell line (showed no potential anti-cancer) A reduction in cell viability at high concentrationswas observed (25mgmL)

4 Discussion

The most distinguished harmful effect of solar exposure ispremature skin aging especially UV irradiation In recent

years thinning of the ozone layer has caused increased UVexposure Detrimental effects of UV on the skin involvethe generation of reactive oxygen species (ROS) [25] Com-ponents of the skin including DNA lipids and proteinscan be impaired by ROS which is the cause of erythemasunburn immune suppression and skin cancer [26] Most ofthe disorders were formerly regarded as having been causedbyUVBNevertheless recent studies showed that UVA is alsoan essential factor [27]

Accordingly use of antioxidants capable of scavengingand quenching ROS is another important approach to pre-vent UVB induced photoaging [27] Other studies have

BioMed Research International 7

70120583m

(a)

70120583m

(b)

70120583m

(c)

70120583m

(d)

70120583m

(e)

70120583m

(f)

Figure 5 Sirius Red stained histological sections (a b)Nonirradiated-NICgroups presenting interlaced less compacted type I and III collagenfibers (c) Irradiated-IC and (d) vehicle groups-IV showing dense disposition of parallel-arranged type I collagen fibers (e) 10 and (f) 15formulations presenting morphological and architectural collagen deposition pattern resembling that observed in the normal dermis (SiriusRedPolarization Light 400x magnification)

shown that botanical compounds with an antioxidative activ-ity are potential agents capable of reducing skin diseasesincluding photoaging [28 29]

The extract used in this work was the same studied bySerafini et al [19] and showed relevant in vitro antioxidantactivity against different radicals The extract was chemicallyanalyzed (qualitative method) The phytochemical screeningof extract showed the presence of alkaloids coumarins flavo-noids tannins saponins steroids and triterpenoids Totalphenolic content of the extract was 1968mg of phenolic equi-valents (gallic acid) per gram of extract Furthermore quali-tative analysis of extract was performed by HPLC revealingthe presence of 3 major compounds all showing ultraviolet

spectra typical of flavonols (quercetin-3-O-rutinoside orrutin and kaempferol glycosides) According to Deng et al[30] flavonol glycosides mainly rutin are the major com-pounds inM citrifolia leaves

The presence of rutin in the formulation could also besecondarily involved in this process of collagenization Ithas been demonstrated that the rutin is able to impair theglycation of collagen [31] a biochemical event involved in thepathogenesis of cell damaging in UV-irradiated tissue [32]

The use of botanical supplements to protect the humanskin from the adverse biological effects of solar radiationhas received great interest in recent years [33] Howevermost tests have been conducted by oral ingestion or topical

8 BioMed Research International

005 01 02 04 08 16 32 64 128 2500

50

100

150

Morinda citrifolia extract (mgmL)

Cel

l via

bilit

y (

)

Figure 6 Cytotoxic effect of theMorinda citrifolia extract in B16F10(murine malignant melanoma) cell line pretreated with the extractsat concentrations (005 to 25mgmL) for 24 h Each value representsthe mean plusmn SD

application of these ingredients in inappropriate vehicleswhich can alter the skin barrier To be of practical use todermatologists and to be safe and efficient extracts musthave physicochemical characteristics that permit their incor-poration in adequate concentrations in a skin-compatiblevehicle (eg gels moisturizing creams) [34] In this studythe capacity of the Morinda citrifolia extract added totopical formulations (gels) to prevent or to treat UVA-UVBirradiation-induced skin damage in mice was evaluated

First three formulations presenting different concen-trations (0 10 and 15) were evaluated in vitro byspectrophotometer UV absorbance spectra showed peaksat 200 nm and at 240 nm (UVC region) and absorbance inthe UVA (320ndash400 nm) and UVB (290ndash320 nm) regionsTheblank formulation (0 extract) did not show absorbance inthis spectra These data are compared to commercial organicfiltersThus this result suggests a photoprotective effect of theextractThe concentration of formulation (10 and 15ww)used in this assay was selected based on a pilot study

Second the gel formulation efficacy against UVA-UVBinduced skin damage was evaluated in vivo by measuring thefollowing parameters thickness values (mm) transepidermalwater loss (TEWL) and erythema

Moisturizing antifree radicals and sunscreen productshave been used largely to prevent skin disorders resultingfrom UV damage However other aspects of skin damageshould be considered such as erythema sunburn cell forma-tion epidermal hyperplasia and impairment of the skin bar-rier integrity which in turn is related to skin dryness skinaging and increased incidence of irritant contact dermatitis[35]

UV radiation damages the structures responsible for cor-neocyte adhesion and the functioning of the permeabilitybarrier which in turn alter thicknesses and TEWL measure-ments and accelerate the desquamation process [36]

The thickness TEWL and erythema were observed inour study and only the formulations containing M citri-folia extract protected mouse skin against the UV-induceddamages The pronounced protective effects provided by

both formulations (10 and 15) in the three parametersmeasured were not due to any extraneous raw material butwere shown by the unprotected groups treated by vehicle only(VI) Furthermore the results show that areas treated withthis extract (10 and 15) were statistically equivalent to theunirradiated control ones (NIC)which represented the intactskin areas

TEWL is an important parameter to be evaluated instudies of skin photoprotective effects considering that UVirradiation of mammalian skin disrupts the epidermal per-meability barrier function accompanied by an increase intransepidermal water loss (TEWL) and alterations in thestratum corneum lipid profile [33]

In the TEWL measurement the group 10 was differentfrom IC and the 15 was not As well as in the thickness andTEWL the protective effect of 10 was most pronouncedIt can also be seen in the photos of the dorsal of the ani-mal This observationcan be explained by differences in thespreadability Formulations containing extract at 10 pre-sented the higher spreadability

The spreadability is represented by the thickness of thefilm that the preparation leaves on the skin an importantfeature in cosmetics [37]Those producing thinner films thatis higher spreadability are naturally of greater interest [38]

Formulations containing extracts were shown to be effec-tive also in the presence of erythema another parameteroften used to evaluate photoprotection Extract formulationsreduced erythema formation when compared with the irra-diated control (IC) This statement corroborates [20] whichdemonstrated that the M citrifolia leaf extracts mitigate theeffects of UV light and provide some measure of defenseagainst localized inflammation (topical microswelling)

Third histological parameters were analyzed Epidermalthickening and inflammatory changes are common histo-logical changes observed in mice skin after short-term UVexposure [38 39] Such an increase in the epidermal thicknessis supposed to occur in response to cell proliferation inattempt to regenerate the tissue leading to a compensatorytype of hyperplasia However the thickened epidermis aswell as the thickened keratin layer (hyperkeratosis) could beobserved in this study

The presence of intense inflammatory infiltrate elastosisand the accumulation of parallel-arranged type I collagenfibers observed in IC and vehicle groups have also beendescribed as UV-induced dermal changes [31] Inflammationis one of the major histological signs of tissue damage andhas been reported in other studies as an early change of theconnective tissue after UV exposure [38 39] On the otherhand elastosis represents an actinic-induced accumulationof amorphous elastin material replacing the normal der-mis unrelated to preexisting or newly synthesized collagen[40] Moreover it has been proposed that UV irradiationimpairs ongoing collagen synthesis primarily through thedownregulation of types I and III procollagen expression[41] Therefore such impairment could likely promote delayin the collagen remodeling leading to the typical immatureparallel-arranged disposition of the fibers [31]

In this study only 15 formulation avoided compen-satory epidermal thickening However the reduction of the

BioMed Research International 9

major deleterious effects associated with the UV exposureon the animals treated with both 10 and 15 formulations isstrongly suggestive of photoprotective effect regardless of theextract concentration

Lastly the extract was evaluated on murine malignantmelanoma in order to examine its cytotoxic effects on malig-nant cells A reduction in cell viability was observed only atthe highest concentration (25mgmL) The results can sug-gest that the extract (in low concentrations) has only photo-protection effects and does not have antimelanoma activity

5 Conclusion

In conclusion present results strongly suggest the photo-protective effect of M citrifolia The results indicate thatphotoprotective effects are due to not only UV absorbancelike the UV filters but also biological effects which happenedmainly with the application of the formulation containing theMorinda citrifolia extract Further studies are necessary toclarify the precise nature of this photoprotective effect

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by Fundacao de Amparo a Pesquisado Estado de Sergipe (FAPITEC-SE) and Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) ofBrazil

References

[1] A M Garcıa-Bores and J G Avila ldquoProducts ldquoMolecularmechanisms in the photochemopreventionrdquordquo Revista LatinoAmericana de Quımica vol 36 no 3 pp 83ndash102 2008

[2] M D Adil P Kaiser N K Satti A M Zargar R A Vish-wakarma and S A Tasduq ldquoEffect of Emblica officinalis (fruit)against UVB-induced photo-aging in human skin fibroblastsrdquoJournal of Ethnopharmacology vol 132 no 1 pp 109ndash114 2010

[3] M Dalle Carbonare and M A Pathak ldquoSkin photosensitizingagents and the role of reactive oxygen species in photoagingrdquoJournal of Photochemistry and Photobiology B Biology vol 14no 1-2 pp 105ndash124 1992

[4] G J Fisher S Datta ZWang et al ldquoc-Jun-dependent inhibitionof cutaneous procollagen transcription following ultravioletirradiation is reversed by all-trans retinoic acidrdquoThe Journal ofClinical Investigation vol 106 no 5 pp 663ndash670 2000

[5] S Inomata Y Matsunaga S Amano et al ldquoPossible involve-ment of gelatinases in basementmembrane damage andwrinkleformation in chronically ultraviolet B-exposed hairless mouserdquoJournal of Investigative Dermatology vol 120 no 1 pp 128ndash1342003

[6] P S Peres V A Terra F A Guarnier R Cecchini and A LCecchini ldquoPhotoaging and chronological aging profile under-standing oxidation of the skinrdquo Journal of Photochemistry andPhotobiology B Biology vol 103 no 2 pp 93ndash97 2011

[7] Y Xu and G J Fisher ldquoUltraviolet (UV) light irradiationinduced signal transduction in skin photoagingrdquo Journal of Der-matological Science Supplement vol 1 no 2 pp S1ndashS8 2005

[8] S P Stratton R T Dorr and D S Alberts ldquoThe state-of-the-artin chemoprevention of skin cancerrdquoEuropean Journal of Cancervol 36 no 10 pp 1292ndash1297 2000

[9] F Afaq V M Adhami and H Mukhtar ldquoPhotochemopreven-tion of ultraviolet B signaling and photocarcinogenesisrdquoMuta-tion Research vol 571 no 1-2 pp 153ndash173 2005

[10] V M Adhami D N Syed N Khan and F Afaq ldquoPhytochem-icals for prevention of solar ultraviolet radiation-induced dam-agesrdquo Photochemistry and Photobiology vol 84 no 2 pp 489ndash500 2008

[11] C D Ropke T CH Sawada V V da Silva N SMichalany andS B DeMoraes Barros ldquoPhotoprotective effect of Pothomorpheumbellata root extract against ultraviolet radiation inducedchronic skin damage in the hairless mouserdquo Clinical andExperimental Dermatology vol 30 no 3 pp 272ndash276 2005

[12] M G D Melo J P A dos Santos M R Serafini et al ldquoRedoxproperties and cytoprotective actions of atranorin a lichensecondary metaboliterdquo Toxicology in Vitro vol 25 no 2 pp462ndash468 2011

[13] M R Serafini D M C Vergne T K Rabelo et al ldquoDetermi-nation of chemical and physical properties of Hyptis pectinataessential oil and their redox active profilerdquo Journal of Biotech-nology and Pharmaceutical Research vol 3 no 1 pp 1ndash9 2012

[14] R Casagrande S R Georgetti W A Verri Jr D J Dorta A Cdos Santos and M J V Fonseca ldquoProtective effect of topicalformulations containing quercetin against UVB-induced oxi-dative stress in hairless micerdquo Journal of Photochemistry andPhotobiology B Biology vol 84 no 1 pp 21ndash27 2006

[15] S Frsquoguyer F Afaq and H Mukhtar ldquoPhotochemopreventionof skin cancer by botanical agentsrdquo Photodermatology Photoim-munology and Photomedicine vol 19 no 2 pp 56ndash72 2003

[16] Y Chan-Blanco F Vaillant A Mercedes Perez M Reynes JBrillouet and P Brat ldquoThe noni fruit (Morinda citrifolia L) areview of agricultural research nutritional and therapeutic pro-pertiesrdquo Journal of Food Composition and Analysis vol 19 no 6-7 pp 645ndash654 2006

[17] H Kim M Kwon J Kim et al ldquoIdentification of novel fattyacid glucosides from the tropical fruit Morinda citrifolia LrdquoPhytochemistry Letters vol 3 no 4 pp 238ndash241 2010

[18] A Hirazumi E Furusawa S C Chou and Y Hokama ldquoImmu-nomodulation contributes to the anticancer activity ofMorindacitrifolia (noni) fruit juicerdquo Proceedings of the Western Pharma-cology Society vol 39 pp 7ndash9 1996

[19] M R Serafini R C Santos A G Guimaraes et al ldquoMorindacitrifolia linn leaf extract possesses antioxidant activities andreduces nociceptive behavior and leukocyte migrationrdquo Journalof Medicinal Food vol 14 no 10 pp 1159ndash1166 2011

[20] B J West S Deng A K Palu and C J Jensen ldquoMorinda citri-folia Linn (Rubiaceae) leaf extracts mitigate UVB-induced ery-themardquo Journal of Natural Medicines vol 63 no 3 pp 351ndash3542009

[21] A Garg D Aggarwal S Garg and A Singela ldquoSpreading ofsemisolid formulations pharmaceutical technologyrdquo Pharma-ceutical Technology vol 26 pp 84ndash105 2002

[22] K Paese A Jager F S Poletto et al ldquoSemisolid formulationcontaining a nanoencapsulated sunscreen effectiveness in vitrophotostability and immune responserdquo Journal of Biomedical andNanotechnology vol 5 no 3 pp 240ndash246 2009

10 BioMed Research International

[23] M Demerjian P Hachem E Tschachler et al ldquoAcute modula-tions in permeability barrier function regulate epidermal corni-ficationrdquo The American Journal of Pathology vol 172 no 1 pp86ndash97 2008

[24] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[25] H Sakurai H Yasui Y Yamada H Nishimura and M Shige-moto ldquoDetection of reactive oxygen species in the skin of livemice and rats exposed toUVA light a research reviewon chemi-luminescence and trials for UVA protectionrdquo Photochemical ampPhotobiological Sciences vol 4 no 9 pp 715ndash720 2005

[26] R Haywood P Wardman R Sanders and C Linge ldquoSun-screens inadequately protect against ultraviolet-A-induced freeradicals in skin implications for skin aging and melanomardquoJournal of Investigative Dermatology vol 121 no 4 pp 862ndash8682003

[27] F Afaq DN Syed AMalik et al ldquoDelphinidin an anthocyani-din in pigmented fruits and vegetables protects human HaCaTkeratinocytes and mouse skin against UVB-mediated oxidativestress and apoptosisrdquo Journal of Investigative Dermatology vol127 no 1 pp 222ndash232 2007

[28] J-Y Bae J-S Choi Y-J Choi et al ldquo(minus)Epigallocatechin gallatehampers collagen destruction and collagenase activation inultraviolet-B-irradiated humandermal fibroblasts Involvementof mitogen-activated protein kinaserdquo Food and Chemical Toxi-cology vol 46 no 4 pp 1298ndash1307 2008

[29] H Choi D Kim J W Kim S Ngadiran M R Sarmidi and CS Park ldquoLabisia pumila extract protects skin cells from photo-aging caused by UVB irradiationrdquo Journal of Bioscience andBioengineering vol 109 no 3 pp 291ndash296 2010

[30] S Deng B JWest andC J Jensen ldquoSimultaneous characterisa-tion and quantitation of flavonol glycosides and aglycones innoni leaves using a validated HPLC-UVMS methodrdquo FoodChemistry vol 111 no 2 pp 526ndash529 2008

[31] D Cervantes-Laurean D D Schramm E L Jacobson IHalaweish G G Bruckner and G A Boissonneault ldquoInhibi-tion of advanced glycation end product formation on collagenby rutin and its metabolitesrdquo Journal of Nutritional Biochem-istry vol 17 no 8 pp 531ndash540 2006

[32] Y M Fonseca C D Catini F T M C Vicentini J C CardosoR L Cavalcanti De Albuquerque Junior and M J Vieira Fon-seca ldquoEfficacy of marigold extract-loaded formulations againstUV-induced oxidative stressrdquo Journal of Pharmaceutical Sci-ences vol 100 no 6 pp 2182ndash2193 2011

[33] P K Vayalil C A Elments and S K Katiyar ldquoTreatment ofgreen tea polyphenols in hydrophilic cream prevents UVB-induced oxidation of lipids and proteins depletion of antioxi-dant enzymes and phosphorylation ofMAPKproteins in SKH-1hairless mouse skinrdquo Carcinogenesis vol 24 no 5 pp 927ndash9362003

[34] S E Dal Belo L R Gaspar and PM B GM Campos ldquoPhoto-protective effects of topical formulations containing a combi-nation of Ginkgo biloba and green tea extractsrdquo PhytotherapyResearch vol 25 no 12 pp 1854ndash1860 2011

[35] R Ghadially B E Brown S M Sequeira-Martin K R Fein-gold and P M Elias ldquoThe aged epidermal permeability barrierStructural functional and lipid biochemical abnormalities inhumans and a senescent murine modelrdquo Journal of ClinicalInvestigation vol 95 no 5 pp 2281ndash2290 1995

[36] M D Contreras and R Sanchez ldquoApplication of a factorialdesign to the study of the flow behavior spreadability and trans-parency of a Carbopol ETD 2020 gel Part IIrdquo International Jour-nal of Pharmaceutics vol 234 no 1-2 pp 149ndash157 2002

[37] BW BarryRheology ofDermatological VehiclesMarcelDekkerNew York NY USA 1983

[38] S U Kim S J Kim J Y Lee et al ldquoProtective effects of dietarysoy isoflavones against UV-induced skin-aging in hairlessmouse modelrdquo Journal of the American College of Nutrition vol23 no 2 pp 157ndash162 2004

[39] M Amouroux G Dıaz-Ayil W C P M Blondel G Bourg-Heckly A Leroux and F Guillemin ldquoClassification of ultra-violet irradiated mouse skin histological stages by bimodalspectroscopy multiple excitation autofluorescence and diffusereflectancerdquo Journal of Biomedical Optics vol 14 no 1 ArticleID 014011 2009

[40] K A Hwang B R Yi and K C Choi ldquoMolecular Mechanismsand In Vivo Mouse Models of Skin Aging Associated withDermal Matrix Alterationsrdquo Laboratory Animal Research vol27 no 1 pp 1ndash8 2011

[41] G J Fisher S Kang J Varani et al ldquoMechanisms of photoagingand chronological skin agingrdquoArchives of Dermatology vol 138no 11 pp 1462ndash1470 2002

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 7

70120583m

(a)

70120583m

(b)

70120583m

(c)

70120583m

(d)

70120583m

(e)

70120583m

(f)

Figure 5 Sirius Red stained histological sections (a b)Nonirradiated-NICgroups presenting interlaced less compacted type I and III collagenfibers (c) Irradiated-IC and (d) vehicle groups-IV showing dense disposition of parallel-arranged type I collagen fibers (e) 10 and (f) 15formulations presenting morphological and architectural collagen deposition pattern resembling that observed in the normal dermis (SiriusRedPolarization Light 400x magnification)

shown that botanical compounds with an antioxidative activ-ity are potential agents capable of reducing skin diseasesincluding photoaging [28 29]

The extract used in this work was the same studied bySerafini et al [19] and showed relevant in vitro antioxidantactivity against different radicals The extract was chemicallyanalyzed (qualitative method) The phytochemical screeningof extract showed the presence of alkaloids coumarins flavo-noids tannins saponins steroids and triterpenoids Totalphenolic content of the extract was 1968mg of phenolic equi-valents (gallic acid) per gram of extract Furthermore quali-tative analysis of extract was performed by HPLC revealingthe presence of 3 major compounds all showing ultraviolet

spectra typical of flavonols (quercetin-3-O-rutinoside orrutin and kaempferol glycosides) According to Deng et al[30] flavonol glycosides mainly rutin are the major com-pounds inM citrifolia leaves

The presence of rutin in the formulation could also besecondarily involved in this process of collagenization Ithas been demonstrated that the rutin is able to impair theglycation of collagen [31] a biochemical event involved in thepathogenesis of cell damaging in UV-irradiated tissue [32]

The use of botanical supplements to protect the humanskin from the adverse biological effects of solar radiationhas received great interest in recent years [33] Howevermost tests have been conducted by oral ingestion or topical

8 BioMed Research International

005 01 02 04 08 16 32 64 128 2500

50

100

150

Morinda citrifolia extract (mgmL)

Cel

l via

bilit

y (

)

Figure 6 Cytotoxic effect of theMorinda citrifolia extract in B16F10(murine malignant melanoma) cell line pretreated with the extractsat concentrations (005 to 25mgmL) for 24 h Each value representsthe mean plusmn SD

application of these ingredients in inappropriate vehicleswhich can alter the skin barrier To be of practical use todermatologists and to be safe and efficient extracts musthave physicochemical characteristics that permit their incor-poration in adequate concentrations in a skin-compatiblevehicle (eg gels moisturizing creams) [34] In this studythe capacity of the Morinda citrifolia extract added totopical formulations (gels) to prevent or to treat UVA-UVBirradiation-induced skin damage in mice was evaluated

First three formulations presenting different concen-trations (0 10 and 15) were evaluated in vitro byspectrophotometer UV absorbance spectra showed peaksat 200 nm and at 240 nm (UVC region) and absorbance inthe UVA (320ndash400 nm) and UVB (290ndash320 nm) regionsTheblank formulation (0 extract) did not show absorbance inthis spectra These data are compared to commercial organicfiltersThus this result suggests a photoprotective effect of theextractThe concentration of formulation (10 and 15ww)used in this assay was selected based on a pilot study

Second the gel formulation efficacy against UVA-UVBinduced skin damage was evaluated in vivo by measuring thefollowing parameters thickness values (mm) transepidermalwater loss (TEWL) and erythema

Moisturizing antifree radicals and sunscreen productshave been used largely to prevent skin disorders resultingfrom UV damage However other aspects of skin damageshould be considered such as erythema sunburn cell forma-tion epidermal hyperplasia and impairment of the skin bar-rier integrity which in turn is related to skin dryness skinaging and increased incidence of irritant contact dermatitis[35]

UV radiation damages the structures responsible for cor-neocyte adhesion and the functioning of the permeabilitybarrier which in turn alter thicknesses and TEWL measure-ments and accelerate the desquamation process [36]

The thickness TEWL and erythema were observed inour study and only the formulations containing M citri-folia extract protected mouse skin against the UV-induceddamages The pronounced protective effects provided by

both formulations (10 and 15) in the three parametersmeasured were not due to any extraneous raw material butwere shown by the unprotected groups treated by vehicle only(VI) Furthermore the results show that areas treated withthis extract (10 and 15) were statistically equivalent to theunirradiated control ones (NIC)which represented the intactskin areas

TEWL is an important parameter to be evaluated instudies of skin photoprotective effects considering that UVirradiation of mammalian skin disrupts the epidermal per-meability barrier function accompanied by an increase intransepidermal water loss (TEWL) and alterations in thestratum corneum lipid profile [33]

In the TEWL measurement the group 10 was differentfrom IC and the 15 was not As well as in the thickness andTEWL the protective effect of 10 was most pronouncedIt can also be seen in the photos of the dorsal of the ani-mal This observationcan be explained by differences in thespreadability Formulations containing extract at 10 pre-sented the higher spreadability

The spreadability is represented by the thickness of thefilm that the preparation leaves on the skin an importantfeature in cosmetics [37]Those producing thinner films thatis higher spreadability are naturally of greater interest [38]

Formulations containing extracts were shown to be effec-tive also in the presence of erythema another parameteroften used to evaluate photoprotection Extract formulationsreduced erythema formation when compared with the irra-diated control (IC) This statement corroborates [20] whichdemonstrated that the M citrifolia leaf extracts mitigate theeffects of UV light and provide some measure of defenseagainst localized inflammation (topical microswelling)

Third histological parameters were analyzed Epidermalthickening and inflammatory changes are common histo-logical changes observed in mice skin after short-term UVexposure [38 39] Such an increase in the epidermal thicknessis supposed to occur in response to cell proliferation inattempt to regenerate the tissue leading to a compensatorytype of hyperplasia However the thickened epidermis aswell as the thickened keratin layer (hyperkeratosis) could beobserved in this study

The presence of intense inflammatory infiltrate elastosisand the accumulation of parallel-arranged type I collagenfibers observed in IC and vehicle groups have also beendescribed as UV-induced dermal changes [31] Inflammationis one of the major histological signs of tissue damage andhas been reported in other studies as an early change of theconnective tissue after UV exposure [38 39] On the otherhand elastosis represents an actinic-induced accumulationof amorphous elastin material replacing the normal der-mis unrelated to preexisting or newly synthesized collagen[40] Moreover it has been proposed that UV irradiationimpairs ongoing collagen synthesis primarily through thedownregulation of types I and III procollagen expression[41] Therefore such impairment could likely promote delayin the collagen remodeling leading to the typical immatureparallel-arranged disposition of the fibers [31]

In this study only 15 formulation avoided compen-satory epidermal thickening However the reduction of the

BioMed Research International 9

major deleterious effects associated with the UV exposureon the animals treated with both 10 and 15 formulations isstrongly suggestive of photoprotective effect regardless of theextract concentration

Lastly the extract was evaluated on murine malignantmelanoma in order to examine its cytotoxic effects on malig-nant cells A reduction in cell viability was observed only atthe highest concentration (25mgmL) The results can sug-gest that the extract (in low concentrations) has only photo-protection effects and does not have antimelanoma activity

5 Conclusion

In conclusion present results strongly suggest the photo-protective effect of M citrifolia The results indicate thatphotoprotective effects are due to not only UV absorbancelike the UV filters but also biological effects which happenedmainly with the application of the formulation containing theMorinda citrifolia extract Further studies are necessary toclarify the precise nature of this photoprotective effect

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by Fundacao de Amparo a Pesquisado Estado de Sergipe (FAPITEC-SE) and Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) ofBrazil

References

[1] A M Garcıa-Bores and J G Avila ldquoProducts ldquoMolecularmechanisms in the photochemopreventionrdquordquo Revista LatinoAmericana de Quımica vol 36 no 3 pp 83ndash102 2008

[2] M D Adil P Kaiser N K Satti A M Zargar R A Vish-wakarma and S A Tasduq ldquoEffect of Emblica officinalis (fruit)against UVB-induced photo-aging in human skin fibroblastsrdquoJournal of Ethnopharmacology vol 132 no 1 pp 109ndash114 2010

[3] M Dalle Carbonare and M A Pathak ldquoSkin photosensitizingagents and the role of reactive oxygen species in photoagingrdquoJournal of Photochemistry and Photobiology B Biology vol 14no 1-2 pp 105ndash124 1992

[4] G J Fisher S Datta ZWang et al ldquoc-Jun-dependent inhibitionof cutaneous procollagen transcription following ultravioletirradiation is reversed by all-trans retinoic acidrdquoThe Journal ofClinical Investigation vol 106 no 5 pp 663ndash670 2000

[5] S Inomata Y Matsunaga S Amano et al ldquoPossible involve-ment of gelatinases in basementmembrane damage andwrinkleformation in chronically ultraviolet B-exposed hairless mouserdquoJournal of Investigative Dermatology vol 120 no 1 pp 128ndash1342003

[6] P S Peres V A Terra F A Guarnier R Cecchini and A LCecchini ldquoPhotoaging and chronological aging profile under-standing oxidation of the skinrdquo Journal of Photochemistry andPhotobiology B Biology vol 103 no 2 pp 93ndash97 2011

[7] Y Xu and G J Fisher ldquoUltraviolet (UV) light irradiationinduced signal transduction in skin photoagingrdquo Journal of Der-matological Science Supplement vol 1 no 2 pp S1ndashS8 2005

[8] S P Stratton R T Dorr and D S Alberts ldquoThe state-of-the-artin chemoprevention of skin cancerrdquoEuropean Journal of Cancervol 36 no 10 pp 1292ndash1297 2000

[9] F Afaq V M Adhami and H Mukhtar ldquoPhotochemopreven-tion of ultraviolet B signaling and photocarcinogenesisrdquoMuta-tion Research vol 571 no 1-2 pp 153ndash173 2005

[10] V M Adhami D N Syed N Khan and F Afaq ldquoPhytochem-icals for prevention of solar ultraviolet radiation-induced dam-agesrdquo Photochemistry and Photobiology vol 84 no 2 pp 489ndash500 2008

[11] C D Ropke T CH Sawada V V da Silva N SMichalany andS B DeMoraes Barros ldquoPhotoprotective effect of Pothomorpheumbellata root extract against ultraviolet radiation inducedchronic skin damage in the hairless mouserdquo Clinical andExperimental Dermatology vol 30 no 3 pp 272ndash276 2005

[12] M G D Melo J P A dos Santos M R Serafini et al ldquoRedoxproperties and cytoprotective actions of atranorin a lichensecondary metaboliterdquo Toxicology in Vitro vol 25 no 2 pp462ndash468 2011

[13] M R Serafini D M C Vergne T K Rabelo et al ldquoDetermi-nation of chemical and physical properties of Hyptis pectinataessential oil and their redox active profilerdquo Journal of Biotech-nology and Pharmaceutical Research vol 3 no 1 pp 1ndash9 2012

[14] R Casagrande S R Georgetti W A Verri Jr D J Dorta A Cdos Santos and M J V Fonseca ldquoProtective effect of topicalformulations containing quercetin against UVB-induced oxi-dative stress in hairless micerdquo Journal of Photochemistry andPhotobiology B Biology vol 84 no 1 pp 21ndash27 2006

[15] S Frsquoguyer F Afaq and H Mukhtar ldquoPhotochemopreventionof skin cancer by botanical agentsrdquo Photodermatology Photoim-munology and Photomedicine vol 19 no 2 pp 56ndash72 2003

[16] Y Chan-Blanco F Vaillant A Mercedes Perez M Reynes JBrillouet and P Brat ldquoThe noni fruit (Morinda citrifolia L) areview of agricultural research nutritional and therapeutic pro-pertiesrdquo Journal of Food Composition and Analysis vol 19 no 6-7 pp 645ndash654 2006

[17] H Kim M Kwon J Kim et al ldquoIdentification of novel fattyacid glucosides from the tropical fruit Morinda citrifolia LrdquoPhytochemistry Letters vol 3 no 4 pp 238ndash241 2010

[18] A Hirazumi E Furusawa S C Chou and Y Hokama ldquoImmu-nomodulation contributes to the anticancer activity ofMorindacitrifolia (noni) fruit juicerdquo Proceedings of the Western Pharma-cology Society vol 39 pp 7ndash9 1996

[19] M R Serafini R C Santos A G Guimaraes et al ldquoMorindacitrifolia linn leaf extract possesses antioxidant activities andreduces nociceptive behavior and leukocyte migrationrdquo Journalof Medicinal Food vol 14 no 10 pp 1159ndash1166 2011

[20] B J West S Deng A K Palu and C J Jensen ldquoMorinda citri-folia Linn (Rubiaceae) leaf extracts mitigate UVB-induced ery-themardquo Journal of Natural Medicines vol 63 no 3 pp 351ndash3542009

[21] A Garg D Aggarwal S Garg and A Singela ldquoSpreading ofsemisolid formulations pharmaceutical technologyrdquo Pharma-ceutical Technology vol 26 pp 84ndash105 2002

[22] K Paese A Jager F S Poletto et al ldquoSemisolid formulationcontaining a nanoencapsulated sunscreen effectiveness in vitrophotostability and immune responserdquo Journal of Biomedical andNanotechnology vol 5 no 3 pp 240ndash246 2009

10 BioMed Research International

[23] M Demerjian P Hachem E Tschachler et al ldquoAcute modula-tions in permeability barrier function regulate epidermal corni-ficationrdquo The American Journal of Pathology vol 172 no 1 pp86ndash97 2008

[24] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[25] H Sakurai H Yasui Y Yamada H Nishimura and M Shige-moto ldquoDetection of reactive oxygen species in the skin of livemice and rats exposed toUVA light a research reviewon chemi-luminescence and trials for UVA protectionrdquo Photochemical ampPhotobiological Sciences vol 4 no 9 pp 715ndash720 2005

[26] R Haywood P Wardman R Sanders and C Linge ldquoSun-screens inadequately protect against ultraviolet-A-induced freeradicals in skin implications for skin aging and melanomardquoJournal of Investigative Dermatology vol 121 no 4 pp 862ndash8682003

[27] F Afaq DN Syed AMalik et al ldquoDelphinidin an anthocyani-din in pigmented fruits and vegetables protects human HaCaTkeratinocytes and mouse skin against UVB-mediated oxidativestress and apoptosisrdquo Journal of Investigative Dermatology vol127 no 1 pp 222ndash232 2007

[28] J-Y Bae J-S Choi Y-J Choi et al ldquo(minus)Epigallocatechin gallatehampers collagen destruction and collagenase activation inultraviolet-B-irradiated humandermal fibroblasts Involvementof mitogen-activated protein kinaserdquo Food and Chemical Toxi-cology vol 46 no 4 pp 1298ndash1307 2008

[29] H Choi D Kim J W Kim S Ngadiran M R Sarmidi and CS Park ldquoLabisia pumila extract protects skin cells from photo-aging caused by UVB irradiationrdquo Journal of Bioscience andBioengineering vol 109 no 3 pp 291ndash296 2010

[30] S Deng B JWest andC J Jensen ldquoSimultaneous characterisa-tion and quantitation of flavonol glycosides and aglycones innoni leaves using a validated HPLC-UVMS methodrdquo FoodChemistry vol 111 no 2 pp 526ndash529 2008

[31] D Cervantes-Laurean D D Schramm E L Jacobson IHalaweish G G Bruckner and G A Boissonneault ldquoInhibi-tion of advanced glycation end product formation on collagenby rutin and its metabolitesrdquo Journal of Nutritional Biochem-istry vol 17 no 8 pp 531ndash540 2006

[32] Y M Fonseca C D Catini F T M C Vicentini J C CardosoR L Cavalcanti De Albuquerque Junior and M J Vieira Fon-seca ldquoEfficacy of marigold extract-loaded formulations againstUV-induced oxidative stressrdquo Journal of Pharmaceutical Sci-ences vol 100 no 6 pp 2182ndash2193 2011

[33] P K Vayalil C A Elments and S K Katiyar ldquoTreatment ofgreen tea polyphenols in hydrophilic cream prevents UVB-induced oxidation of lipids and proteins depletion of antioxi-dant enzymes and phosphorylation ofMAPKproteins in SKH-1hairless mouse skinrdquo Carcinogenesis vol 24 no 5 pp 927ndash9362003

[34] S E Dal Belo L R Gaspar and PM B GM Campos ldquoPhoto-protective effects of topical formulations containing a combi-nation of Ginkgo biloba and green tea extractsrdquo PhytotherapyResearch vol 25 no 12 pp 1854ndash1860 2011

[35] R Ghadially B E Brown S M Sequeira-Martin K R Fein-gold and P M Elias ldquoThe aged epidermal permeability barrierStructural functional and lipid biochemical abnormalities inhumans and a senescent murine modelrdquo Journal of ClinicalInvestigation vol 95 no 5 pp 2281ndash2290 1995

[36] M D Contreras and R Sanchez ldquoApplication of a factorialdesign to the study of the flow behavior spreadability and trans-parency of a Carbopol ETD 2020 gel Part IIrdquo International Jour-nal of Pharmaceutics vol 234 no 1-2 pp 149ndash157 2002

[37] BW BarryRheology ofDermatological VehiclesMarcelDekkerNew York NY USA 1983

[38] S U Kim S J Kim J Y Lee et al ldquoProtective effects of dietarysoy isoflavones against UV-induced skin-aging in hairlessmouse modelrdquo Journal of the American College of Nutrition vol23 no 2 pp 157ndash162 2004

[39] M Amouroux G Dıaz-Ayil W C P M Blondel G Bourg-Heckly A Leroux and F Guillemin ldquoClassification of ultra-violet irradiated mouse skin histological stages by bimodalspectroscopy multiple excitation autofluorescence and diffusereflectancerdquo Journal of Biomedical Optics vol 14 no 1 ArticleID 014011 2009

[40] K A Hwang B R Yi and K C Choi ldquoMolecular Mechanismsand In Vivo Mouse Models of Skin Aging Associated withDermal Matrix Alterationsrdquo Laboratory Animal Research vol27 no 1 pp 1ndash8 2011

[41] G J Fisher S Kang J Varani et al ldquoMechanisms of photoagingand chronological skin agingrdquoArchives of Dermatology vol 138no 11 pp 1462ndash1470 2002

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

8 BioMed Research International

005 01 02 04 08 16 32 64 128 2500

50

100

150

Morinda citrifolia extract (mgmL)

Cel

l via

bilit

y (

)

Figure 6 Cytotoxic effect of theMorinda citrifolia extract in B16F10(murine malignant melanoma) cell line pretreated with the extractsat concentrations (005 to 25mgmL) for 24 h Each value representsthe mean plusmn SD

application of these ingredients in inappropriate vehicleswhich can alter the skin barrier To be of practical use todermatologists and to be safe and efficient extracts musthave physicochemical characteristics that permit their incor-poration in adequate concentrations in a skin-compatiblevehicle (eg gels moisturizing creams) [34] In this studythe capacity of the Morinda citrifolia extract added totopical formulations (gels) to prevent or to treat UVA-UVBirradiation-induced skin damage in mice was evaluated

First three formulations presenting different concen-trations (0 10 and 15) were evaluated in vitro byspectrophotometer UV absorbance spectra showed peaksat 200 nm and at 240 nm (UVC region) and absorbance inthe UVA (320ndash400 nm) and UVB (290ndash320 nm) regionsTheblank formulation (0 extract) did not show absorbance inthis spectra These data are compared to commercial organicfiltersThus this result suggests a photoprotective effect of theextractThe concentration of formulation (10 and 15ww)used in this assay was selected based on a pilot study

Second the gel formulation efficacy against UVA-UVBinduced skin damage was evaluated in vivo by measuring thefollowing parameters thickness values (mm) transepidermalwater loss (TEWL) and erythema

Moisturizing antifree radicals and sunscreen productshave been used largely to prevent skin disorders resultingfrom UV damage However other aspects of skin damageshould be considered such as erythema sunburn cell forma-tion epidermal hyperplasia and impairment of the skin bar-rier integrity which in turn is related to skin dryness skinaging and increased incidence of irritant contact dermatitis[35]

UV radiation damages the structures responsible for cor-neocyte adhesion and the functioning of the permeabilitybarrier which in turn alter thicknesses and TEWL measure-ments and accelerate the desquamation process [36]

The thickness TEWL and erythema were observed inour study and only the formulations containing M citri-folia extract protected mouse skin against the UV-induceddamages The pronounced protective effects provided by

both formulations (10 and 15) in the three parametersmeasured were not due to any extraneous raw material butwere shown by the unprotected groups treated by vehicle only(VI) Furthermore the results show that areas treated withthis extract (10 and 15) were statistically equivalent to theunirradiated control ones (NIC)which represented the intactskin areas

TEWL is an important parameter to be evaluated instudies of skin photoprotective effects considering that UVirradiation of mammalian skin disrupts the epidermal per-meability barrier function accompanied by an increase intransepidermal water loss (TEWL) and alterations in thestratum corneum lipid profile [33]

In the TEWL measurement the group 10 was differentfrom IC and the 15 was not As well as in the thickness andTEWL the protective effect of 10 was most pronouncedIt can also be seen in the photos of the dorsal of the ani-mal This observationcan be explained by differences in thespreadability Formulations containing extract at 10 pre-sented the higher spreadability

The spreadability is represented by the thickness of thefilm that the preparation leaves on the skin an importantfeature in cosmetics [37]Those producing thinner films thatis higher spreadability are naturally of greater interest [38]

Formulations containing extracts were shown to be effec-tive also in the presence of erythema another parameteroften used to evaluate photoprotection Extract formulationsreduced erythema formation when compared with the irra-diated control (IC) This statement corroborates [20] whichdemonstrated that the M citrifolia leaf extracts mitigate theeffects of UV light and provide some measure of defenseagainst localized inflammation (topical microswelling)

Third histological parameters were analyzed Epidermalthickening and inflammatory changes are common histo-logical changes observed in mice skin after short-term UVexposure [38 39] Such an increase in the epidermal thicknessis supposed to occur in response to cell proliferation inattempt to regenerate the tissue leading to a compensatorytype of hyperplasia However the thickened epidermis aswell as the thickened keratin layer (hyperkeratosis) could beobserved in this study

The presence of intense inflammatory infiltrate elastosisand the accumulation of parallel-arranged type I collagenfibers observed in IC and vehicle groups have also beendescribed as UV-induced dermal changes [31] Inflammationis one of the major histological signs of tissue damage andhas been reported in other studies as an early change of theconnective tissue after UV exposure [38 39] On the otherhand elastosis represents an actinic-induced accumulationof amorphous elastin material replacing the normal der-mis unrelated to preexisting or newly synthesized collagen[40] Moreover it has been proposed that UV irradiationimpairs ongoing collagen synthesis primarily through thedownregulation of types I and III procollagen expression[41] Therefore such impairment could likely promote delayin the collagen remodeling leading to the typical immatureparallel-arranged disposition of the fibers [31]

In this study only 15 formulation avoided compen-satory epidermal thickening However the reduction of the

BioMed Research International 9

major deleterious effects associated with the UV exposureon the animals treated with both 10 and 15 formulations isstrongly suggestive of photoprotective effect regardless of theextract concentration

Lastly the extract was evaluated on murine malignantmelanoma in order to examine its cytotoxic effects on malig-nant cells A reduction in cell viability was observed only atthe highest concentration (25mgmL) The results can sug-gest that the extract (in low concentrations) has only photo-protection effects and does not have antimelanoma activity

5 Conclusion

In conclusion present results strongly suggest the photo-protective effect of M citrifolia The results indicate thatphotoprotective effects are due to not only UV absorbancelike the UV filters but also biological effects which happenedmainly with the application of the formulation containing theMorinda citrifolia extract Further studies are necessary toclarify the precise nature of this photoprotective effect

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by Fundacao de Amparo a Pesquisado Estado de Sergipe (FAPITEC-SE) and Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) ofBrazil

References

[1] A M Garcıa-Bores and J G Avila ldquoProducts ldquoMolecularmechanisms in the photochemopreventionrdquordquo Revista LatinoAmericana de Quımica vol 36 no 3 pp 83ndash102 2008

[2] M D Adil P Kaiser N K Satti A M Zargar R A Vish-wakarma and S A Tasduq ldquoEffect of Emblica officinalis (fruit)against UVB-induced photo-aging in human skin fibroblastsrdquoJournal of Ethnopharmacology vol 132 no 1 pp 109ndash114 2010

[3] M Dalle Carbonare and M A Pathak ldquoSkin photosensitizingagents and the role of reactive oxygen species in photoagingrdquoJournal of Photochemistry and Photobiology B Biology vol 14no 1-2 pp 105ndash124 1992

[4] G J Fisher S Datta ZWang et al ldquoc-Jun-dependent inhibitionof cutaneous procollagen transcription following ultravioletirradiation is reversed by all-trans retinoic acidrdquoThe Journal ofClinical Investigation vol 106 no 5 pp 663ndash670 2000

[5] S Inomata Y Matsunaga S Amano et al ldquoPossible involve-ment of gelatinases in basementmembrane damage andwrinkleformation in chronically ultraviolet B-exposed hairless mouserdquoJournal of Investigative Dermatology vol 120 no 1 pp 128ndash1342003

[6] P S Peres V A Terra F A Guarnier R Cecchini and A LCecchini ldquoPhotoaging and chronological aging profile under-standing oxidation of the skinrdquo Journal of Photochemistry andPhotobiology B Biology vol 103 no 2 pp 93ndash97 2011

[7] Y Xu and G J Fisher ldquoUltraviolet (UV) light irradiationinduced signal transduction in skin photoagingrdquo Journal of Der-matological Science Supplement vol 1 no 2 pp S1ndashS8 2005

[8] S P Stratton R T Dorr and D S Alberts ldquoThe state-of-the-artin chemoprevention of skin cancerrdquoEuropean Journal of Cancervol 36 no 10 pp 1292ndash1297 2000

[9] F Afaq V M Adhami and H Mukhtar ldquoPhotochemopreven-tion of ultraviolet B signaling and photocarcinogenesisrdquoMuta-tion Research vol 571 no 1-2 pp 153ndash173 2005

[10] V M Adhami D N Syed N Khan and F Afaq ldquoPhytochem-icals for prevention of solar ultraviolet radiation-induced dam-agesrdquo Photochemistry and Photobiology vol 84 no 2 pp 489ndash500 2008

[11] C D Ropke T CH Sawada V V da Silva N SMichalany andS B DeMoraes Barros ldquoPhotoprotective effect of Pothomorpheumbellata root extract against ultraviolet radiation inducedchronic skin damage in the hairless mouserdquo Clinical andExperimental Dermatology vol 30 no 3 pp 272ndash276 2005

[12] M G D Melo J P A dos Santos M R Serafini et al ldquoRedoxproperties and cytoprotective actions of atranorin a lichensecondary metaboliterdquo Toxicology in Vitro vol 25 no 2 pp462ndash468 2011

[13] M R Serafini D M C Vergne T K Rabelo et al ldquoDetermi-nation of chemical and physical properties of Hyptis pectinataessential oil and their redox active profilerdquo Journal of Biotech-nology and Pharmaceutical Research vol 3 no 1 pp 1ndash9 2012

[14] R Casagrande S R Georgetti W A Verri Jr D J Dorta A Cdos Santos and M J V Fonseca ldquoProtective effect of topicalformulations containing quercetin against UVB-induced oxi-dative stress in hairless micerdquo Journal of Photochemistry andPhotobiology B Biology vol 84 no 1 pp 21ndash27 2006

[15] S Frsquoguyer F Afaq and H Mukhtar ldquoPhotochemopreventionof skin cancer by botanical agentsrdquo Photodermatology Photoim-munology and Photomedicine vol 19 no 2 pp 56ndash72 2003

[16] Y Chan-Blanco F Vaillant A Mercedes Perez M Reynes JBrillouet and P Brat ldquoThe noni fruit (Morinda citrifolia L) areview of agricultural research nutritional and therapeutic pro-pertiesrdquo Journal of Food Composition and Analysis vol 19 no 6-7 pp 645ndash654 2006

[17] H Kim M Kwon J Kim et al ldquoIdentification of novel fattyacid glucosides from the tropical fruit Morinda citrifolia LrdquoPhytochemistry Letters vol 3 no 4 pp 238ndash241 2010

[18] A Hirazumi E Furusawa S C Chou and Y Hokama ldquoImmu-nomodulation contributes to the anticancer activity ofMorindacitrifolia (noni) fruit juicerdquo Proceedings of the Western Pharma-cology Society vol 39 pp 7ndash9 1996

[19] M R Serafini R C Santos A G Guimaraes et al ldquoMorindacitrifolia linn leaf extract possesses antioxidant activities andreduces nociceptive behavior and leukocyte migrationrdquo Journalof Medicinal Food vol 14 no 10 pp 1159ndash1166 2011

[20] B J West S Deng A K Palu and C J Jensen ldquoMorinda citri-folia Linn (Rubiaceae) leaf extracts mitigate UVB-induced ery-themardquo Journal of Natural Medicines vol 63 no 3 pp 351ndash3542009

[21] A Garg D Aggarwal S Garg and A Singela ldquoSpreading ofsemisolid formulations pharmaceutical technologyrdquo Pharma-ceutical Technology vol 26 pp 84ndash105 2002

[22] K Paese A Jager F S Poletto et al ldquoSemisolid formulationcontaining a nanoencapsulated sunscreen effectiveness in vitrophotostability and immune responserdquo Journal of Biomedical andNanotechnology vol 5 no 3 pp 240ndash246 2009

10 BioMed Research International

[23] M Demerjian P Hachem E Tschachler et al ldquoAcute modula-tions in permeability barrier function regulate epidermal corni-ficationrdquo The American Journal of Pathology vol 172 no 1 pp86ndash97 2008

[24] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[25] H Sakurai H Yasui Y Yamada H Nishimura and M Shige-moto ldquoDetection of reactive oxygen species in the skin of livemice and rats exposed toUVA light a research reviewon chemi-luminescence and trials for UVA protectionrdquo Photochemical ampPhotobiological Sciences vol 4 no 9 pp 715ndash720 2005

[26] R Haywood P Wardman R Sanders and C Linge ldquoSun-screens inadequately protect against ultraviolet-A-induced freeradicals in skin implications for skin aging and melanomardquoJournal of Investigative Dermatology vol 121 no 4 pp 862ndash8682003

[27] F Afaq DN Syed AMalik et al ldquoDelphinidin an anthocyani-din in pigmented fruits and vegetables protects human HaCaTkeratinocytes and mouse skin against UVB-mediated oxidativestress and apoptosisrdquo Journal of Investigative Dermatology vol127 no 1 pp 222ndash232 2007

[28] J-Y Bae J-S Choi Y-J Choi et al ldquo(minus)Epigallocatechin gallatehampers collagen destruction and collagenase activation inultraviolet-B-irradiated humandermal fibroblasts Involvementof mitogen-activated protein kinaserdquo Food and Chemical Toxi-cology vol 46 no 4 pp 1298ndash1307 2008

[29] H Choi D Kim J W Kim S Ngadiran M R Sarmidi and CS Park ldquoLabisia pumila extract protects skin cells from photo-aging caused by UVB irradiationrdquo Journal of Bioscience andBioengineering vol 109 no 3 pp 291ndash296 2010

[30] S Deng B JWest andC J Jensen ldquoSimultaneous characterisa-tion and quantitation of flavonol glycosides and aglycones innoni leaves using a validated HPLC-UVMS methodrdquo FoodChemistry vol 111 no 2 pp 526ndash529 2008

[31] D Cervantes-Laurean D D Schramm E L Jacobson IHalaweish G G Bruckner and G A Boissonneault ldquoInhibi-tion of advanced glycation end product formation on collagenby rutin and its metabolitesrdquo Journal of Nutritional Biochem-istry vol 17 no 8 pp 531ndash540 2006

[32] Y M Fonseca C D Catini F T M C Vicentini J C CardosoR L Cavalcanti De Albuquerque Junior and M J Vieira Fon-seca ldquoEfficacy of marigold extract-loaded formulations againstUV-induced oxidative stressrdquo Journal of Pharmaceutical Sci-ences vol 100 no 6 pp 2182ndash2193 2011

[33] P K Vayalil C A Elments and S K Katiyar ldquoTreatment ofgreen tea polyphenols in hydrophilic cream prevents UVB-induced oxidation of lipids and proteins depletion of antioxi-dant enzymes and phosphorylation ofMAPKproteins in SKH-1hairless mouse skinrdquo Carcinogenesis vol 24 no 5 pp 927ndash9362003

[34] S E Dal Belo L R Gaspar and PM B GM Campos ldquoPhoto-protective effects of topical formulations containing a combi-nation of Ginkgo biloba and green tea extractsrdquo PhytotherapyResearch vol 25 no 12 pp 1854ndash1860 2011

[35] R Ghadially B E Brown S M Sequeira-Martin K R Fein-gold and P M Elias ldquoThe aged epidermal permeability barrierStructural functional and lipid biochemical abnormalities inhumans and a senescent murine modelrdquo Journal of ClinicalInvestigation vol 95 no 5 pp 2281ndash2290 1995

[36] M D Contreras and R Sanchez ldquoApplication of a factorialdesign to the study of the flow behavior spreadability and trans-parency of a Carbopol ETD 2020 gel Part IIrdquo International Jour-nal of Pharmaceutics vol 234 no 1-2 pp 149ndash157 2002

[37] BW BarryRheology ofDermatological VehiclesMarcelDekkerNew York NY USA 1983

[38] S U Kim S J Kim J Y Lee et al ldquoProtective effects of dietarysoy isoflavones against UV-induced skin-aging in hairlessmouse modelrdquo Journal of the American College of Nutrition vol23 no 2 pp 157ndash162 2004

[39] M Amouroux G Dıaz-Ayil W C P M Blondel G Bourg-Heckly A Leroux and F Guillemin ldquoClassification of ultra-violet irradiated mouse skin histological stages by bimodalspectroscopy multiple excitation autofluorescence and diffusereflectancerdquo Journal of Biomedical Optics vol 14 no 1 ArticleID 014011 2009

[40] K A Hwang B R Yi and K C Choi ldquoMolecular Mechanismsand In Vivo Mouse Models of Skin Aging Associated withDermal Matrix Alterationsrdquo Laboratory Animal Research vol27 no 1 pp 1ndash8 2011

[41] G J Fisher S Kang J Varani et al ldquoMechanisms of photoagingand chronological skin agingrdquoArchives of Dermatology vol 138no 11 pp 1462ndash1470 2002

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 9

major deleterious effects associated with the UV exposureon the animals treated with both 10 and 15 formulations isstrongly suggestive of photoprotective effect regardless of theextract concentration

Lastly the extract was evaluated on murine malignantmelanoma in order to examine its cytotoxic effects on malig-nant cells A reduction in cell viability was observed only atthe highest concentration (25mgmL) The results can sug-gest that the extract (in low concentrations) has only photo-protection effects and does not have antimelanoma activity

5 Conclusion

In conclusion present results strongly suggest the photo-protective effect of M citrifolia The results indicate thatphotoprotective effects are due to not only UV absorbancelike the UV filters but also biological effects which happenedmainly with the application of the formulation containing theMorinda citrifolia extract Further studies are necessary toclarify the precise nature of this photoprotective effect

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by Fundacao de Amparo a Pesquisado Estado de Sergipe (FAPITEC-SE) and Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) ofBrazil

References

[1] A M Garcıa-Bores and J G Avila ldquoProducts ldquoMolecularmechanisms in the photochemopreventionrdquordquo Revista LatinoAmericana de Quımica vol 36 no 3 pp 83ndash102 2008

[2] M D Adil P Kaiser N K Satti A M Zargar R A Vish-wakarma and S A Tasduq ldquoEffect of Emblica officinalis (fruit)against UVB-induced photo-aging in human skin fibroblastsrdquoJournal of Ethnopharmacology vol 132 no 1 pp 109ndash114 2010

[3] M Dalle Carbonare and M A Pathak ldquoSkin photosensitizingagents and the role of reactive oxygen species in photoagingrdquoJournal of Photochemistry and Photobiology B Biology vol 14no 1-2 pp 105ndash124 1992

[4] G J Fisher S Datta ZWang et al ldquoc-Jun-dependent inhibitionof cutaneous procollagen transcription following ultravioletirradiation is reversed by all-trans retinoic acidrdquoThe Journal ofClinical Investigation vol 106 no 5 pp 663ndash670 2000

[5] S Inomata Y Matsunaga S Amano et al ldquoPossible involve-ment of gelatinases in basementmembrane damage andwrinkleformation in chronically ultraviolet B-exposed hairless mouserdquoJournal of Investigative Dermatology vol 120 no 1 pp 128ndash1342003

[6] P S Peres V A Terra F A Guarnier R Cecchini and A LCecchini ldquoPhotoaging and chronological aging profile under-standing oxidation of the skinrdquo Journal of Photochemistry andPhotobiology B Biology vol 103 no 2 pp 93ndash97 2011

[7] Y Xu and G J Fisher ldquoUltraviolet (UV) light irradiationinduced signal transduction in skin photoagingrdquo Journal of Der-matological Science Supplement vol 1 no 2 pp S1ndashS8 2005

[8] S P Stratton R T Dorr and D S Alberts ldquoThe state-of-the-artin chemoprevention of skin cancerrdquoEuropean Journal of Cancervol 36 no 10 pp 1292ndash1297 2000

[9] F Afaq V M Adhami and H Mukhtar ldquoPhotochemopreven-tion of ultraviolet B signaling and photocarcinogenesisrdquoMuta-tion Research vol 571 no 1-2 pp 153ndash173 2005

[10] V M Adhami D N Syed N Khan and F Afaq ldquoPhytochem-icals for prevention of solar ultraviolet radiation-induced dam-agesrdquo Photochemistry and Photobiology vol 84 no 2 pp 489ndash500 2008

[11] C D Ropke T CH Sawada V V da Silva N SMichalany andS B DeMoraes Barros ldquoPhotoprotective effect of Pothomorpheumbellata root extract against ultraviolet radiation inducedchronic skin damage in the hairless mouserdquo Clinical andExperimental Dermatology vol 30 no 3 pp 272ndash276 2005

[12] M G D Melo J P A dos Santos M R Serafini et al ldquoRedoxproperties and cytoprotective actions of atranorin a lichensecondary metaboliterdquo Toxicology in Vitro vol 25 no 2 pp462ndash468 2011

[13] M R Serafini D M C Vergne T K Rabelo et al ldquoDetermi-nation of chemical and physical properties of Hyptis pectinataessential oil and their redox active profilerdquo Journal of Biotech-nology and Pharmaceutical Research vol 3 no 1 pp 1ndash9 2012

[14] R Casagrande S R Georgetti W A Verri Jr D J Dorta A Cdos Santos and M J V Fonseca ldquoProtective effect of topicalformulations containing quercetin against UVB-induced oxi-dative stress in hairless micerdquo Journal of Photochemistry andPhotobiology B Biology vol 84 no 1 pp 21ndash27 2006

[15] S Frsquoguyer F Afaq and H Mukhtar ldquoPhotochemopreventionof skin cancer by botanical agentsrdquo Photodermatology Photoim-munology and Photomedicine vol 19 no 2 pp 56ndash72 2003

[16] Y Chan-Blanco F Vaillant A Mercedes Perez M Reynes JBrillouet and P Brat ldquoThe noni fruit (Morinda citrifolia L) areview of agricultural research nutritional and therapeutic pro-pertiesrdquo Journal of Food Composition and Analysis vol 19 no 6-7 pp 645ndash654 2006

[17] H Kim M Kwon J Kim et al ldquoIdentification of novel fattyacid glucosides from the tropical fruit Morinda citrifolia LrdquoPhytochemistry Letters vol 3 no 4 pp 238ndash241 2010

[18] A Hirazumi E Furusawa S C Chou and Y Hokama ldquoImmu-nomodulation contributes to the anticancer activity ofMorindacitrifolia (noni) fruit juicerdquo Proceedings of the Western Pharma-cology Society vol 39 pp 7ndash9 1996

[19] M R Serafini R C Santos A G Guimaraes et al ldquoMorindacitrifolia linn leaf extract possesses antioxidant activities andreduces nociceptive behavior and leukocyte migrationrdquo Journalof Medicinal Food vol 14 no 10 pp 1159ndash1166 2011

[20] B J West S Deng A K Palu and C J Jensen ldquoMorinda citri-folia Linn (Rubiaceae) leaf extracts mitigate UVB-induced ery-themardquo Journal of Natural Medicines vol 63 no 3 pp 351ndash3542009

[21] A Garg D Aggarwal S Garg and A Singela ldquoSpreading ofsemisolid formulations pharmaceutical technologyrdquo Pharma-ceutical Technology vol 26 pp 84ndash105 2002

[22] K Paese A Jager F S Poletto et al ldquoSemisolid formulationcontaining a nanoencapsulated sunscreen effectiveness in vitrophotostability and immune responserdquo Journal of Biomedical andNanotechnology vol 5 no 3 pp 240ndash246 2009

10 BioMed Research International

[23] M Demerjian P Hachem E Tschachler et al ldquoAcute modula-tions in permeability barrier function regulate epidermal corni-ficationrdquo The American Journal of Pathology vol 172 no 1 pp86ndash97 2008

[24] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[25] H Sakurai H Yasui Y Yamada H Nishimura and M Shige-moto ldquoDetection of reactive oxygen species in the skin of livemice and rats exposed toUVA light a research reviewon chemi-luminescence and trials for UVA protectionrdquo Photochemical ampPhotobiological Sciences vol 4 no 9 pp 715ndash720 2005

[26] R Haywood P Wardman R Sanders and C Linge ldquoSun-screens inadequately protect against ultraviolet-A-induced freeradicals in skin implications for skin aging and melanomardquoJournal of Investigative Dermatology vol 121 no 4 pp 862ndash8682003

[27] F Afaq DN Syed AMalik et al ldquoDelphinidin an anthocyani-din in pigmented fruits and vegetables protects human HaCaTkeratinocytes and mouse skin against UVB-mediated oxidativestress and apoptosisrdquo Journal of Investigative Dermatology vol127 no 1 pp 222ndash232 2007

[28] J-Y Bae J-S Choi Y-J Choi et al ldquo(minus)Epigallocatechin gallatehampers collagen destruction and collagenase activation inultraviolet-B-irradiated humandermal fibroblasts Involvementof mitogen-activated protein kinaserdquo Food and Chemical Toxi-cology vol 46 no 4 pp 1298ndash1307 2008

[29] H Choi D Kim J W Kim S Ngadiran M R Sarmidi and CS Park ldquoLabisia pumila extract protects skin cells from photo-aging caused by UVB irradiationrdquo Journal of Bioscience andBioengineering vol 109 no 3 pp 291ndash296 2010

[30] S Deng B JWest andC J Jensen ldquoSimultaneous characterisa-tion and quantitation of flavonol glycosides and aglycones innoni leaves using a validated HPLC-UVMS methodrdquo FoodChemistry vol 111 no 2 pp 526ndash529 2008

[31] D Cervantes-Laurean D D Schramm E L Jacobson IHalaweish G G Bruckner and G A Boissonneault ldquoInhibi-tion of advanced glycation end product formation on collagenby rutin and its metabolitesrdquo Journal of Nutritional Biochem-istry vol 17 no 8 pp 531ndash540 2006

[32] Y M Fonseca C D Catini F T M C Vicentini J C CardosoR L Cavalcanti De Albuquerque Junior and M J Vieira Fon-seca ldquoEfficacy of marigold extract-loaded formulations againstUV-induced oxidative stressrdquo Journal of Pharmaceutical Sci-ences vol 100 no 6 pp 2182ndash2193 2011

[33] P K Vayalil C A Elments and S K Katiyar ldquoTreatment ofgreen tea polyphenols in hydrophilic cream prevents UVB-induced oxidation of lipids and proteins depletion of antioxi-dant enzymes and phosphorylation ofMAPKproteins in SKH-1hairless mouse skinrdquo Carcinogenesis vol 24 no 5 pp 927ndash9362003

[34] S E Dal Belo L R Gaspar and PM B GM Campos ldquoPhoto-protective effects of topical formulations containing a combi-nation of Ginkgo biloba and green tea extractsrdquo PhytotherapyResearch vol 25 no 12 pp 1854ndash1860 2011

[35] R Ghadially B E Brown S M Sequeira-Martin K R Fein-gold and P M Elias ldquoThe aged epidermal permeability barrierStructural functional and lipid biochemical abnormalities inhumans and a senescent murine modelrdquo Journal of ClinicalInvestigation vol 95 no 5 pp 2281ndash2290 1995

[36] M D Contreras and R Sanchez ldquoApplication of a factorialdesign to the study of the flow behavior spreadability and trans-parency of a Carbopol ETD 2020 gel Part IIrdquo International Jour-nal of Pharmaceutics vol 234 no 1-2 pp 149ndash157 2002

[37] BW BarryRheology ofDermatological VehiclesMarcelDekkerNew York NY USA 1983

[38] S U Kim S J Kim J Y Lee et al ldquoProtective effects of dietarysoy isoflavones against UV-induced skin-aging in hairlessmouse modelrdquo Journal of the American College of Nutrition vol23 no 2 pp 157ndash162 2004

[39] M Amouroux G Dıaz-Ayil W C P M Blondel G Bourg-Heckly A Leroux and F Guillemin ldquoClassification of ultra-violet irradiated mouse skin histological stages by bimodalspectroscopy multiple excitation autofluorescence and diffusereflectancerdquo Journal of Biomedical Optics vol 14 no 1 ArticleID 014011 2009

[40] K A Hwang B R Yi and K C Choi ldquoMolecular Mechanismsand In Vivo Mouse Models of Skin Aging Associated withDermal Matrix Alterationsrdquo Laboratory Animal Research vol27 no 1 pp 1ndash8 2011

[41] G J Fisher S Kang J Varani et al ldquoMechanisms of photoagingand chronological skin agingrdquoArchives of Dermatology vol 138no 11 pp 1462ndash1470 2002

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

10 BioMed Research International

[23] M Demerjian P Hachem E Tschachler et al ldquoAcute modula-tions in permeability barrier function regulate epidermal corni-ficationrdquo The American Journal of Pathology vol 172 no 1 pp86ndash97 2008

[24] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[25] H Sakurai H Yasui Y Yamada H Nishimura and M Shige-moto ldquoDetection of reactive oxygen species in the skin of livemice and rats exposed toUVA light a research reviewon chemi-luminescence and trials for UVA protectionrdquo Photochemical ampPhotobiological Sciences vol 4 no 9 pp 715ndash720 2005

[26] R Haywood P Wardman R Sanders and C Linge ldquoSun-screens inadequately protect against ultraviolet-A-induced freeradicals in skin implications for skin aging and melanomardquoJournal of Investigative Dermatology vol 121 no 4 pp 862ndash8682003

[27] F Afaq DN Syed AMalik et al ldquoDelphinidin an anthocyani-din in pigmented fruits and vegetables protects human HaCaTkeratinocytes and mouse skin against UVB-mediated oxidativestress and apoptosisrdquo Journal of Investigative Dermatology vol127 no 1 pp 222ndash232 2007

[28] J-Y Bae J-S Choi Y-J Choi et al ldquo(minus)Epigallocatechin gallatehampers collagen destruction and collagenase activation inultraviolet-B-irradiated humandermal fibroblasts Involvementof mitogen-activated protein kinaserdquo Food and Chemical Toxi-cology vol 46 no 4 pp 1298ndash1307 2008

[29] H Choi D Kim J W Kim S Ngadiran M R Sarmidi and CS Park ldquoLabisia pumila extract protects skin cells from photo-aging caused by UVB irradiationrdquo Journal of Bioscience andBioengineering vol 109 no 3 pp 291ndash296 2010

[30] S Deng B JWest andC J Jensen ldquoSimultaneous characterisa-tion and quantitation of flavonol glycosides and aglycones innoni leaves using a validated HPLC-UVMS methodrdquo FoodChemistry vol 111 no 2 pp 526ndash529 2008

[31] D Cervantes-Laurean D D Schramm E L Jacobson IHalaweish G G Bruckner and G A Boissonneault ldquoInhibi-tion of advanced glycation end product formation on collagenby rutin and its metabolitesrdquo Journal of Nutritional Biochem-istry vol 17 no 8 pp 531ndash540 2006

[32] Y M Fonseca C D Catini F T M C Vicentini J C CardosoR L Cavalcanti De Albuquerque Junior and M J Vieira Fon-seca ldquoEfficacy of marigold extract-loaded formulations againstUV-induced oxidative stressrdquo Journal of Pharmaceutical Sci-ences vol 100 no 6 pp 2182ndash2193 2011

[33] P K Vayalil C A Elments and S K Katiyar ldquoTreatment ofgreen tea polyphenols in hydrophilic cream prevents UVB-induced oxidation of lipids and proteins depletion of antioxi-dant enzymes and phosphorylation ofMAPKproteins in SKH-1hairless mouse skinrdquo Carcinogenesis vol 24 no 5 pp 927ndash9362003

[34] S E Dal Belo L R Gaspar and PM B GM Campos ldquoPhoto-protective effects of topical formulations containing a combi-nation of Ginkgo biloba and green tea extractsrdquo PhytotherapyResearch vol 25 no 12 pp 1854ndash1860 2011

[35] R Ghadially B E Brown S M Sequeira-Martin K R Fein-gold and P M Elias ldquoThe aged epidermal permeability barrierStructural functional and lipid biochemical abnormalities inhumans and a senescent murine modelrdquo Journal of ClinicalInvestigation vol 95 no 5 pp 2281ndash2290 1995

[36] M D Contreras and R Sanchez ldquoApplication of a factorialdesign to the study of the flow behavior spreadability and trans-parency of a Carbopol ETD 2020 gel Part IIrdquo International Jour-nal of Pharmaceutics vol 234 no 1-2 pp 149ndash157 2002

[37] BW BarryRheology ofDermatological VehiclesMarcelDekkerNew York NY USA 1983

[38] S U Kim S J Kim J Y Lee et al ldquoProtective effects of dietarysoy isoflavones against UV-induced skin-aging in hairlessmouse modelrdquo Journal of the American College of Nutrition vol23 no 2 pp 157ndash162 2004

[39] M Amouroux G Dıaz-Ayil W C P M Blondel G Bourg-Heckly A Leroux and F Guillemin ldquoClassification of ultra-violet irradiated mouse skin histological stages by bimodalspectroscopy multiple excitation autofluorescence and diffusereflectancerdquo Journal of Biomedical Optics vol 14 no 1 ArticleID 014011 2009

[40] K A Hwang B R Yi and K C Choi ldquoMolecular Mechanismsand In Vivo Mouse Models of Skin Aging Associated withDermal Matrix Alterationsrdquo Laboratory Animal Research vol27 no 1 pp 1ndash8 2011

[41] G J Fisher S Kang J Varani et al ldquoMechanisms of photoagingand chronological skin agingrdquoArchives of Dermatology vol 138no 11 pp 1462ndash1470 2002

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of