research article phytochemical composition, antioxidant...

Research ArticlePhytochemical Composition Antioxidant Activity andthe Effect of the Aqueous Extract of Coffee (Coffea arabica L)Bean Residual Press Cake on the Skin Wound Healing

Regina Celis Lopes Affonso Ana Paula Lorenzen Voytena Simone FananHeloiacutesa Pitz Daniela Sousa Coelho Ana Luiza Horstmann Aline PereiraVirgiacutelio Gavicho Uarrota Maria Clara Hillmann Lucas Andre Calbusch VarelaRosa Maria Ribeiro-do-Valle and Marcelo Maraschin

Plant Morphogenesis and Biochemistry Laboratory Federal University of Santa Catarina 1346 Admar Gonzaga Road88048-000 Florianopolis SC Brazil

Correspondence should be addressed to Regina Celis Lopes Affonso reafonso147gmailcom

Received 12 July 2016 Revised 9 October 2016 Accepted 17 October 2016

Academic Editor Renata Szymanska

Copyright copy 2016 Regina Celis Lopes Affonso et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The world coffee consumption has been growing for its appreciated taste and its beneficial effects on health The residual biomassof coffee originated in the food industry after oil extraction from coffee beans called coffee beans residual press cake has attractedinterest as a source of compounds with antioxidant activityThis study investigated the chemical composition of aqueous extracts ofcoffee beans residual press cake (AE) their antioxidant activity and the effect of topical application on the skin wound healing inanimalmodel of hydrogels containing theAE chlorogenic acid (CGA) allantoin (positive control) and carbopol (negative control)The treatmentsrsquo performance was compared by measuring the reduction of the wound area with superior result (119901 lt 005) for thegreen coffee AE (7820) with respect to roasted coffee AE (5371) allantoin (7083) and carbopol (2356) CGA hydrogelsreduced significantly the wound area size on the inflammatory phase which may be associated with the well known antioxidantand anti-inflammatory actions of that compound The topic use of the coffee AE studied improved the skin wound healing andpoints to an interesting biotechnological application of the coffee bean residual press cake

1 Introduction

The imbalance between the production of reactive oxidizingagents and the ability of a biological system to counteract thereactive intermediates results in oxidative stress The reactiveoxidizing agents are formedduring normal physiological pro-cesses or under stress conditions that can damage biologicalsystem [1]

The coffee (Coffea arabicaL) beans press cake is a residualbiomass from the coffee beans oil extraction process claimedto be rich in bioactive compounds of interest for humanhealth and cosmetics There is considerable emphasis on therecovery of plant biomass originating from the food industryin order to target it to other industries [2] adding value andreducing eventual environmental damage In this sense the

large amount ofC arabica beans produced in Brazil gives riseto certain amounts of biomass not acceptable by the beveragemarket but with an important potential for the developmentof other products such as cosmetics

Coffee has been claimed as a functional beverage beingan important source of antioxidants in human diet especiallydue to the high amounts of phenolic compounds and caffeineThe chemical constituents of arabica coffee involve phenoliccompounds and their derivatives (such as chlorogenic acids)alkaloids (specially caffeine) diterpenoid alcohols (such ascafestol and kahweol) carbohydrates lipids and volatile andheterocyclic compounds [3] Chlorogenic acids (CGA) areesters of the trans-cinnamic acids [4] whose amounts ingreen coffees are significantly reduced during the roastingprocess [5] In the past decades polyphenolic compounds

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 1923754 10 pageshttpdxdoiorg10115520161923754

2 Oxidative Medicine and Cellular Longevity

have been proposed as one of the most effective functionalingredients in foods and beverages with antiaging propertiesand able to neutralize the effects of oxidative damage tothe skin for example [6] Studies with C arabica extractshave revealed a set of important biological activities forexample antibacterial [7] antiviral [8] anti-inflammatory[9 10] suppressive activity of metalloproteinase expression[11] and reduction of oxidative damage to macromolecules[12] Besides phenolic compounds coffee is well recognizedas a rich source of the alkaloids especially caffeine Suchsecondary metabolites have presented relevant biologicalactivities as for instance stimulation of the central nervoussystem diuretic and peripheral vasoconstriction [13] Caf-feine is the major alkaloid present in coffee beans and itscontent is correlated with the quality of the beverage alsocontributing to the brew bitterness [14]

Skin serves as a protective barrier against the environ-ment and also plays a fundamental role in the homeostasismaintenance [15] The loss of the skin integrity results ininjury that can lead to serious commitments to the body ifnot repairedThe tissue lesions trigger intracellular responsesthat coordinate the restoration of tissuersquos integrity and home-ostasis The ability to respond to the injury and tissue repairis a fundamental property for multicellular organisms Thecutaneous tissue repair occurs via tissue regeneration withthe recovery of tissue functionality or the healing and therestoration of tissue homeostasis [16]

The wound healing is a complex biological process afteran injury This process can be understood into three classicstages inflammation proliferation and remodeling Theinflammatory stage begins immediately after injury of thetissue and lasts for around 3 days with inflammation Subse-quently there are cellular proliferation migration of differentcell types angiogenesis and extracellular matrix componentsproduction especially collagen The proliferation stage maylast from 2 to 10 days and is characterized by the migrationand differentiation of various cell types being themost abun-dant fibroblasts The remodeling stage is the third and laststage of repair It begins 2 to 3 weeks after the injury and lastsfor about a year or more The majority of endothelial cellsmyofibroblasts and macrophages are destroyed by apoptosisand these changes result in wound contraction and scarformation [16] Inflammation and oxidative stress are closelyrelated at the wound site considering the influx of neutrophilsand macrophages producing reactive oxygen and nitrogenReactive oxygen and nitrogen species are involved in theredox regulation of cell functions and increasingly viewedas a major upstream component in the signalling cascadeinvolved in inflammatory responses and the stimulation ofadhesion molecule and chemoattractant production [15]Testing by oxygen radical absorbance capacity assay of thecoffee berry outperforms common antioxidants such as greentea extract pomegranate extract vitamin C and vitamin EPolymerase chain reaction microarray analysis on humancultured fibroblasts treated with multiple doses of 0001coffee berry revealed upregulation of gene expression forseveral collagens and connective tissue growth factor anddownregulation for metalloproteinases [17]

It is known that the effect of aging causes a temporaldelay in wound healing but not an actual impairment interms of the quality of healing [18 19] Delayed woundhealing in the aged is associated with an altered inflammatoryresponse such as delayed T-cell infiltration into the woundarea with alterations in chemokine production and reducedmacrophage phagocytic capacity [20] Retarded reepithelial-ization collagen synthesis and angiogenesis have also beenobserved in aged mice as compared with young ones [21]

The topical administration of antioxidant compoundson the skin is gaining prominence among dermatologistsbecause of their anti-inflammatory and anticarcinogenicactivity [22] In this context it has been claimed that woundrepair process could be helped by topically administratingantioxidant molecules that may control the oxidative stress[23] Phenolic compounds have been showed to be activeon skin tissues through hampering collagen destruction andcollagenase activation [24] Indeed CGA has been shown toaccelerate the skin wound healing and burn healing due toits antioxidant free radical scavenging anti-inflammatoryradioprotective antiulcerogenic and analgesic properties [1025ndash29] Importantly the effectiveness of polyphenols in therepair of cutaneous tissue is initially determined by theirphysicochemical properties and the ability to overcome theepidermal barrier to achieve appropriate receptors [6]

In this sense the working hypothesis of this study is basedon the assumption that a biologically compatible extract thatis the AE of coffee beans residual press cake is a rich sourceof polyphenolic compounds that topically administrated onthe damaged cutaneous tissue would ameliorate the woundhealing process Thus the phytochemical profile and theantioxidant activity of the AE of coffee beans (C arabica L)residual press cake were determined In a second approachthe in vivo effect of topically administrated hydrogels contain-ing AE and chlorogenic acid on the wound healing processwas investigated using a skin excision wound model in Swissalbino mice

2 Material and Methods

21 Coffee Samples Chemicals and Cell Lines Samples ofgreen (ie nonroasted beans) and roasted coffee beans presscake residual were supplied by Cooxupe (Regional Coopera-tive of Coffee Growers Ltda at Guaxupe Minas Gerais Statesoutheastern Brazil) Green and roasted coffee bean presscake samples were obtained after mechanically pressing thebeans for the extraction of the oil fraction After extractionthe residual biomass was packed in polyethylene bags withsilica sachets and stored at minus20∘C for further analysis Ana-lytical grade methanol ethanol hydrochloric acid sodiumchloride and hydrogen peroxide were purchased from Vetec(Rio de Janeiro Brazil) The Folin-Ciocalteu reagent sodiumdodecyl sulphate (SDS) dimethyl sulfoxide (DMSO) 22-diphenyl-1-picrylhydrazyl (DPPH) (plusmn)-6-hydroxy-2578-tetramethylchroman-2-carboxylic acid (Trolox) potassiumphosphate buffer (TPK) Tween 20 Bradford reagent bovineserum albumin (BSA) methionine riboflavin nitrote-trazolium blue chloride (NBT) neutral red (NR) andthe analytical reference standards (chlorogenic acid gallic

Oxidative Medicine and Cellular Longevity 3

acid p-hydroxybenzoic acid ferulic acid caffeic acidsyringic acid p-coumaric acid caffeine theophylline theo-bromine trigonelline and allantoin) were purchased fromSigma-Aldrich (Missouri USA) Carbopol 940 NF poly-mer disodium ethylenediaminetetraacetic acid (EDTA) andaminomethyl propanol (AMP) were obtained from PharmaNostra (Campinas Brazil) Mouse fibroblasts L929 cell linewas purchased from Rio de Janeiro Cell Bank (BCRJ) asDulbeccorsquos modified eaglersquos medium (DMEM) fetal bovineserum (FBS) penicillin and streptomycin were obtainedfrom Sigma-Aldrich (Missouri USA)

22 Preparation of the Aqueous Extracts of Green andRoasted Coffee Bean Residual Press Cake AE extracts ofthe biomasses in study were obtained according to the coldextraction method from the Brazilian Pharmacopoeia [30]with modifications Samples (4 g dry weight) were addedof 100mL EtOH solution 70 (vv) at room temperaturefollowed by magnetic stirring during 1 h protected fromthe light and kept 18 h under refrigeration (2ndash8∘C) Theextracts were recovered by filtration on cellulose supportunder reduced pressure followed by concentration in arotatory evaporator (60∘C 82 rpm) Dried extracts wereresuspended in 10mL distilled-deionized water centrifuge(10min 4000 rpm 137 cm rotor radius) and the supernatantcollected for further analysis

23 Preparation of the Treatments for Topical Applicationon the Skin Wound Healing Carbopol hydrogel was pre-pared containing 1 (wv) carbopol 940 01 (wv) EDTA05 (wv) aminomethyl propanol and water (60mL) Thecarbopol hydrogels were used as a vehicle base and thetreatments were prepared as follows (1) carbopol base plus10 AE (vv) green coffee (2) carbopol base plus 10AE (vv) roasted coffee (3) carbopol base plus 3 (vw)chlorogenic acid (4) carbopol base plus 1 (vw) allantoin-positive control and (5) carbopol base-negative control Thechlorogenic acid concentration in the hydrogel (3 wv) waschosen according to the content of this compound in the AEof green coffee press cake previously determined by HPLCanalysis

24 Determination of Total Phenolic Contents The totalphenolic contents of the AE were determined using theFolin-Ciocalteu reagent according to Randhir et al [31] Forthat 1mL AE was diluted in 9mL distilled-deionized waterThe diluted sample (40 120583L) was added to 316mL distilled-deionized water and 200 120583L Folin-Ciocalteau reagent vor-texed stirring for 1min after which 600 120583L of sodiumcarbonate solutionwas added (20wv) vortexed and incu-bated for 2 h The blank solution was prepared as describedabove by replacing the test sample by distilled-deionizedwater The absorbance was measured at 750 120578m using a UVspectrophotometer (BEL LGS 53 BEL Engineering MonzaItaly) and the analysis was carried out in triplicateThe resultswere expressed asmean (mg equivalent gallic acidg biomass)plusmn standard error of the mean (sem) based on a calibrationcurve of gallic acid (100ndash700120583gsdotmLminus1 1199032 = 0983 119910 =0001119909)

Table 1

Time (min) Eluent A ( vv) Eluent B ( vv)0ndash5 85 155ndash45 0 10045ndash50 85 15

25 RP-HPLC Analysis of Phenolic Acids and AlkaloidsThe RP-HPLC analysis was adapted from Rodrigues andBragagnolo [32] that propose simultaneous determinationof caffeine and chlorogenic acids AE aliquots (60 120583L) wereinjected into a liquid chromatograph (HPLC Thermo Scien-tific UltiMate 3000 RSDual System) equipped with a reverse-phase column (Thermo Scientific C18 250mm times 46mm Oslash05 120583m particle 35∘C) and a diode array detector operatingat 240 nm 260 nm 280 nm and 320 nm The alkaloids andphenolic compounds were quantified at 280 nm and 320 nmrespectively according to previous recordsThemobile phaseconsisted of HCl acidified Milli-Q water (pH 23-Eluent A)and methanol (Eluent B) The chromatographic conditionsfor the gradient were as shown in Table 1

A flow rate at 1mLmin and a run time of 50min wereused Besides the AE was solubilized in 10mL 80methanoland diluted 9 1 (vv) previously to injection The quantifica-tion of phenolic compounds was based on the integration ofthe peak areas and a calibration curve of chlorogenic acid(detection range 10ndash400120583gsdotmLminus1 1199032 = 099 119910 = 0707119909)Similarly the amounts of alkaloids were determined basedon the integration of the peak areas and calibration curve ofcaffeine (detection range 100ndash1000120583gsdotmLminus1 1199032 = 099 119910 =0842119909) The analysis was carried out in triplicate The resultswere expressed as mean (mgsdotgminus1) plusmn standard deviation (sd)

26 DPPH Radical Scavenging Activity Assay The AE wasfirstly freeze-dried weighted and resuspended in 95methanol (vv) for a final concentration at 10mgmL AEdried mass Antioxidant activity of the green and roastedcoffee AE was determined by measuring their scaveng-ing capacity of the 221015840-diphenyl-1-picrylhydrazyl (DPPH)radical as previously described and adapted by Molyneux[33] The (plusmn)-6-hydroxy-2578-tetramethylchroman-2-car-boxylic acid (Trolox) was used as positive control andmethanol as the negative one The amount of 28120583L DPPHmethanolic solution (70120583M) was incubated with 972 120583Lsample test (from 1 to 30 120583gmL) for 20min at 25∘C (n= 3) DPPH scavenging activity which manifests itself as adecrease in absorbance was measured at 517 nm with a UV-Vis spectrophotometer (UV-2000 Instruterm) The DPPHradical scavenging activity () was calculated as follows

Radical scavenging activity

= 100 minus [Abs sample lowast 100Abs negative control

] (1)

The experiment was carried out in triplicate and data areexpressed as mean plusmn standard deviation (sd)


27 In Vitro Cell Viability The effect of the AE on the cellviability was determined in vitro according to the proto-col established by ICCVAM [34] with modifications Themethod is based on the assessment of cell viability in vitroafter exposure to the AE measuring the lysosomersquos uptakeof neutral red dye For that mouse L929 fibroblasts wereplated in 96-well plates (1 times 104 cellswell) and incubatedduring 24 h (37∘C 95 humidity and 5 CO2) After thisperiod cells were washed with phosphate buffered saline(PBS) and treatedwith increasing concentrations of coffeeAE(3 10 30 100 300 1000 3000 and 10000120583gmL) during 24 h(37∘C 95 humidity and 5 CO2) The protein denaturingagent SDS in the same concentrations of the coffee AE wasused as positive control With the treatment period elapsingcells were washed with PBS and incubated in the presenceof neutral red dye (25 120583gmL DMEM) for 3 h (37∘C 95humidity and 5 CO2) Subsequently cells were washedwith PBS and neutral red dye destaining solution was added(1 acetic acid 50 ethanol 49 distilled water vvv)Absorbance was measured at 540 nm using a microplatereader (SpectraMax ParadigmMulti-ModeMicroplateReaderMolecular Devices Sunnyvale USA) Cell viability wasexpressed as percentage of growth based on the controlcells and concentration of test samples which showed 50inhibition of cell grow (IC50) expressing the results in mgAEmLThe results were calculated according to the equationbelow

[(119862V) = [ (119862me minus 119861119898)(Ctr119898 minus 119861119898)] lowast 100] (2)

where 119862V is the cell viability () 119862me is the mean absorbanceof each concentration of the sample 119861119898 is the meanabsorbance of the blank and Ctr119898 is the mean absorbanceof control

28 In Vivo Skin Wound Healing Assays Swiss albino malemice (Mus musculus) 40ndash60 g 9 months old (assay 1) and2 months old (assay 2) from the Central Biotery of theFederal University of Santa Catarina (Florianopolis SantaCatarina State southern Brazil) were adapted to the labo-ratory conditions prior to the assay having free access tofood and water The animals were anesthetized using anintraperitoneal injection of 10 ketamine (75mgkg) and2 xylazine (10mgkg) The excisional wound model wasrealized according to Frank and Kampfer [35] Briefly thedorsal surface of the thoracic region was trichotomized andcleaned up with iodized alcohol A 10mm diameter skinwound was made with a scissor aid under aseptic conditionsHydrogels (01 ganimal) were applied topically and dailyCarbopol hydrogel (1 wv) was used as negative control ascarbopol hydrogel containing allantoin (1 wv) was chosenas positive control because of the recognized antisepticaction and ability to promote cell proliferation and woundhealing of that compound [36] The wound contractionwas evaluated and the wound area measured at the end ofthe experimental period that is day 14 The experimentalprotocol was approved by the Ethics Committee on the useof animals (Federal University of Santa Catarina PP0957)

281 Evaluation of Wound Healing 1 Treatment with Hydro-gels Containing Green and Roasted Coffee Bean AE The aimof this experiment was to determine the effect of the topicaltreatment with AE of green and roasted coffee beans presscake on the skin wound healing This investigation adoptedan in vivo assay with 9-month-old mice in order to verifythe wound healing potential of the AE in an animal groupwith delayedmetabolismdue to the advanced age Initially 24male 40ndash60 g Swiss albinomicewere used randomly dividedinto 4 groups (119899 = 6) according to the treatments withhydrogel enriched with (1) AE green coffee (2) AE roastedcoffee (3) allantoin (positive control) and (4) carbopol gel(negative control) Animalswere kept in individual cages overa 14-day-long experimental period Lesions located in theback of the animals were observed and treated daily afterthe initial surgical procedure to check the reepithelializationprocess The wound healing and the efficacy of a treatmentwere evaluated by assessing the percentage of wound areareduction along the experimental period The wound areawas measured using a digital caliper at the beginning andat the end of the experimental period calculating the injuryreduction percentage The wound area was recorded andexpressed as the percentage reduction in the original woundarea according to the equation

[(119882cp) = (1198821198890 minus119882119889119899)1198821198890 lowast 100] (3)

where119882cp is the wound contraction percentage ()1198821198890 thewound area on day ldquo0rdquo and119882119889119899 the wound area on day ldquo119899rdquo282 Evaluation of Wound Healing 2 Treatment with Hydro-gels Containing Chlorogenic Acid and Green Coffee Bean AEIn a second experimental approach the effectiveness of thetopical administration of hydrogels containing chlorogenicacid or AE of green coffee was determined on the skin woundhealing using Swiss albino mice 2 months old A total of 96male 40ndash60 g Swiss albino mice were randomly divided into3 groups (119899 = 32) considering different sampling times (3 7and 15 days) Subsequently each time group was separatedinto 4 groups (119899 = 8) according to the treatments withhydrogel as follows (1)AE green coffee (2)CGA (3) allantoin(positive control) and (4) carbopol gel (negative control)Animals were kept in individual cages over a 15-day-longexperimental period Lesions were observed and treated dailyafter the initial surgical procedure to check the reepithelial-ization process The skin wound was photographed using adigital camera placed in a standard distance from the lesionimmediately after surgery and at 3rd 7th and 15th days Thewound healing process was evaluated by assessing the size ofwound reduction in each sampling timeThewound area wasmeasured through scanned photographic images using thesoftware ImageJ The results were recorded and expressed asmeasurement of wound area Data are presented as means ofwound area (cm2) plusmnmean standard error (sem)

29 Determination of Oxidative Stress Markers Skin woundsamples of the second experimental approach were collectedfor biochemical analyses following the protocols of Bagdas


et al [37] and Pereira [38] Skin wound samples werecollected and the animals sacrificed immediately Sampleswere washed in 09 (wv) NaCl and immediately frozenat minus80∘C until use Tissue samples were cut into piecesand homogenized (ULTRA-TURRAX homogenizer) in a2mL potassium phosphate buffer solution (20mM pH 74)with TWEEN20 (1) and NaCl (150mM) The sampleswere vortexed for 10 s and centrifuged (4000 rpm 15min4∘C) and the supernatants recovered for further analysisProtein contents were determined according to the Bradfordmethod [39] The catalase (CAT) activity was measuredspectrophotometrically (120582 = 240 nm) following the methoddescribed by Pereira [38] For the test 25 120583L of supernatantwas added to 240120583L of potassium phosphate buffer (50mMpH 70) supplemented with hydrogen peroxide solution(10mM) The decomposition rate of hydrogen peroxide wasmeasured sequentially in intervals of 20 s until 10min Theresultswere expressed asmmolsdotminminus1mgproteinThe super-oxide dismutase (SOD) activity was assessed based on themethod of Giannopolitis and Ries [40] The determinationconsiders the enzymersquos ability to inhibit the photoreductionof nitrotetrazolium blue chloride (NBT) The activity wasdetermined adding 25 120583L sample to 240120583L work solution in96-well microplates The work solution was prepared with13mM methionine 75120583M NBT 100 nM EDTA and 2mMriboflavin in potassium phosphate buffer (50mM pH 78)The reaction was started by lighting the microplate in achamber composed of fluorescent tubes (15W) at roomtemperature After 5min incubation the end of catalysis wasforced by the interruption of light The content of the bluecompound (formazan) formed by the photoreduction ofNBTwas measured spectrophotometrically at 560 nm The samereaction was prepared and protected from the light One unitof SOD is defined as the enzyme activity required for 50inhibition of NBT photoreduction To calculate the specificactivity of the enzyme it was considered as the percentageof enzymatic inhibition obtained the sample volume andthe sample protein concentration (120583g protein) The resultswere expressed as U120583g protein The enzymatic experimentswere realized in triplicate and the data expressed as means plusmnstandard deviation (sd)

210 Statistical Analysis Data were collected and summa-rized following statistical analysis using one-way ANOVAand Tukeyrsquos test The results were considered statisticallysignificant when 119901 lt 005The values were expressed asmeanplusmn sd or mean plusmn sem as indicated in tables subtitles

3 Results and Discussions

In this study AE of residual biomasses from the green androasted coffee oil industry were chemically characterized andfurther investigated to determine the effectiveness of hydro-gels containing AE and chlorogenic acid on the cutaneoustissue repair process

31 Total Phenolic Contents Table 2 provides the total phe-nolic contents of AE of green and roasted coffee press cakeshowing appreciable and statistically different (119901 lt 005)

Table 2 Contents of total phenolic compounds (mg gallic acidequivalentssdotgminus1 biomass dry weight) of the AE of green and roastedcoffee beans press cake

Samples Total phenolic compounds (mg gallic acidequivalentsdotgminus1 biomass dry weight)

Green coffee 3539 plusmn 369 aRoasted coffee 2413 plusmn 145 bMean of extractions in triplicate plusmn sem Distinct letters denote significativedifference Tukeyrsquos test 119901 lt 005

amounts of that secondary metabolite in the samples Thehighest concentration of phenolic compounds was detectedin the green coffee AE Thus one could speculate that theextraction of the apolar fraction of the green coffee beanspress cake (ie oil fraction) allows obtaining a high yieldof phenolic compounds in the AE of the residual biomassIt is well known that the phenolic content in coffee beansmay vary largely according to the species variety degree ofmaturation postharvest processing and roasting After theharvest and along the coffee bean processing some phenoliccompounds can be isomerized hydrolyzed or degraded intolow molecular weight compounds The high temperaturesof coffee roasting process degrade part of the phenoliccompounds [41] Indeed lower amounts (119901 lt 005) ofphenolic compounds were found in the AE of roasted coffeebean sample comparatively to the nonroasted one provingthe negative effect of the roasting process on the contents ofthose bioactive compounds

32 Quantitation of Phenolic Acids and Alkaloids The useof the liquid chromatography with a gradient elution systemcontaining acidified water and methanol as mobile phaseis often in the analysis of phenolic compounds [42] How-ever the method adapted in this assay for simultaneousdetermination of phenolics and alkaloids allowed identifyinganalytes for both groups of compounds The results revealedchlorogenic acid (1111mgsdotgminus1) and caffeine (45mgsdotgminus1) arethe majors compounds in the AE of green coffee samples(Table 3) This finding is consistent with previous results forcoffee beans as mentioned by several authors [41 43ndash45]Again considering that phenolic acids and their derivativescan be degraded under high temperatures one could specu-late that the lowest concentration of these acids in the roastedcoffee press cake samples results from the thermal treatmentof that biomass during the roasting processThe alkaloid con-tents revealed important amounts of caffeine and trigonellinein both samples but low concentration of theophylline Theroasted coffee sample showed superior caffeine amountscomparatively to the other studied sampleTheobromine wasnot detected Besides the studied samples seemed to be quitediscrepant in their alkaloid contents suggesting differentpotentials regarding their biological effectsThe large amountof caffeine in the coffee extracts is responsible for severalbiological activities [46]

33 Antioxidant Potential DPPH Assay Reactive oxygenspecies (ROS) reactive nitrogen species (RNS) and reactive


Table 3 Concentrations (mgsdotgminus1 biomass dry weight) of phenolicacids and alkaloids of the green and roasted AE of coffee beansdetermined by RP-HPLC analysis

lowastPhenolic acid andalkaloids (mgsdotgminus1) Samples

Green coffee Roasted coffee280 nm

Chlorogenic acid 1111 plusmn 028 a 195 plusmn 031 bSyringic acid 096 plusmn 004 d 084 plusmn 008 cFerulic acid 140 plusmn 029 d 092 plusmn 016 cProtocatechuic acid 120 plusmn 0003 d 021 plusmn 002 dHydroxybenzoic acid 008 plusmn 41 e 005 plusmn 105 dCaffeine 45 plusmn 006 b 560 plusmn 008 a

320 nmCaffeic acid 001 plusmn 0002 e naTheophylline 001 plusmn 0003 e naTrigonelline 155 plusmn 051 c 102 plusmn 014 clowastMean of injections in triplicate plusmn sd Distinct letters in the column aresignificantly different Tukeyrsquos test 119901 lt 005 na = not available

Table 4 Maximum activity of DPPH radical scavenging of AEof green and roasted coffee beans press cake and Trolox (positivecontrol)

Sample (30 120583gsdot120583Lminus1) Maximum activity ()lowastAE green coffee bean press cake 9621 plusmn 126AE roasted coffee beans press cake 9535 plusmn 220Trolox 9636 plusmn 033lowastValues are shown as mean plusmn standard deviation

sulfur species (RSS) might react with lipid protein and DNAto cause inflammation cancer and ischemia for instanceDPPH is a stable free radical by virtue of the delocalizationof the spare electron over the molecule as a whole so thatthe molecules do not dimerise as would be the case of mostof the free radicals DPPH radical which shows absorptionat 517 nm has been used as a convenient tool for the radicalscavenge assay which is independent of any enzyme activity[33] The DPPH radical scavenging activity () of the AEin study calculated after 20min of incubation revealed amaximum activity achieved with 30 120583gAEmL similar to thepositive control (Trolox) activity (Table 4) The quantitationof phenolic compounds and alkaloids in samples revealed thepresence of secondary metabolites related to the antioxidantcapacity for example caffeine and chlorogenic acids It islong known that the roasting process degrades phenolic com-pounds however the alkaloids are more resistant and stableand caffeine has been shown to present expressive antioxi-dant activity [47] It is important to note that the methodherein used for measuring total phenolic compounds thatis Folin-Ciocalteau is based on a redox reaction as it canbe considered an evaluation of the antioxidant activity [48]The oxidative balance in physiological systems is regulatedby endogenous and exogenous mechanisms in which theexcess of free radicals is related to many diseases [49 50]The control of the excess of oxidative molecules includes the

intake or topical application of exogenous antioxidants oreven molecules that can stimulate the endogenous antioxi-dant [12]Thus the antioxidant activity of coffee bean extractsis related to the presence of several natural constituents andcompounds formed during processing for example caffeine[51] chlorogenic and hydroxycinnamic acids [52ndash55] andMaillard reaction products such as melanoidins [54 55]

34 Cell Viability Profile The cell viability decrease wasonly detected in cells treated with the roasted coffee AEat 3mgsdotmLminus1 with an IC50 value of 488mgsdotmLminus1 and aLD50 = 248200mgsdotkgminus1 as showed in Figure 1 Althoughcell viability decreased totally at 10mgsdotmLminus1 the LD50 valuecharacterized the AE as nontoxic because the cytotoxicityis considered for LD50 values lower than 2000mgsdotkgminus1Besides green coffee AE led to no reduction in cell via-bility in concentrations equal to or lower than 10mgmLTraditionally in vitro determination of cell viability profileand toxic effect of compounds is the first biological assay toperform aiming at a further application in human health forinstance If the toxicity of the molecule is not proved it canproceed to the next step in vivo assay Experimentally thishas been performed by counting viable cells after stainingthem with a vital dye The neutral red assay system is ameans of measuring living cells through the uptake of thevital neutral red dye [56] as adopted in this study As notedby Triglia et al (1991) the neutral red dye passes through theintact cellmembrane and becomes concentrated in lysosomesof viable cells Test agents that damage the cell surface andlysosomal membrane inhibit the incorporation of the reddye and the amount taken up by the cells is thereforeproportional to the number of viable ones The neutral redassay was developed by Borenfreund and Puerner [57] andhas been utilized extensively to study the toxic effect ofa number of compounds on different cell types grown inmonolayer cultures as L929 cells used in this study Thoughcell viability reduction showed the roasted coffee extract allthe extracts concentrations tested in this assay showed beingnoncytotoxic

35 Effect of AE of Green and Roasted Coffee Beans PressCake on the Wound Healing The hydrogel containing AE ofgreen coffee showed the best result on the wound reduction(7820) being similar to the positive control (7083) ascan be noted in Figure 2 In its turn the roasted coffeehydrogel appeared to be less effective in ameliorating thewound healing (5371) comparatively to the green coffeeAE A negligible reduction of the wound area was detectedfor the negative control (2356) statistically differing fromthe other treatments Thus it can be assumed that hydrogelsenriched with AE of coffee beans press cake improved thewound healing process revealing the positive effects onthe cutaneous tissue regeneration Indeed one could arguethat the superior performance of the hydrogel containinggreen coffee extract in the wound healing results from itshighest concentration of phenolic compounds according tothe phytochemical analysis Previous in vivo assays in miceon the effect of coffee extracts in the skin wound healingprocess were not found in literature so far In the present


Concentration (mgmL)

Cel

l via

bilit

y (

)

0

20

40

60

80

100

Postpressing roasted coffee

3e minus 03 3e + 00 1e + 01

Figure 1 In vitro cell viability after 24 h exposure to the AE ofroasted coffee press cake Important reductions of cell viability weredetected at AE concentration of 3mgmL or higher as determinedthrough the NRU assay Results are expressed as mean plusmn standarddeviation (sd)

Wou

nd h

ealin

g re

duct

ion

()

Treatments

c

ab

b

a

0

20

40

60

80

100

NCPC

PPRCPPGC

Figure 2 Reduction of thewound area () after 14 days of treatmentwith hydrogels applied daily in mice 9 months old NC (negativecontrol = 2356 c) PC (positive control = 7083 ab) PPGC(postpressing green coffee = 7820 a) and PPRC (postpressingroasted coffee = 5371 b) Data are presented as mean plusmn semDistinct letters denote significant differences at 119901 lt 005 (Tukeyrsquostest)

study it is worthmentioning that by using 9-month-old adultmice typically differing from other experimental approacheswhere younger animals have been used [58] the elapsingtime of 14 days was not enough to achieve full woundhealing although all treatments significantly decreased thewound area comparatively to negative control In fact thereare substantial differences in the wound healing responsebetween young and adult subjects generally lasting longer

in adults The adverse effects of aging on wound repair arewell known and were offset by treatments that acceleratedthe wound closure [59 60] Previous study by Bagdas etal [25] reported that systematical CGA application in skinflap surgeries can accelerate healing and flap survival inanimal of wound healing delay by diabetic condition Ithas been also suggested that topically CGA ointment hasa potent wound healing effect on nondiabetics rats [26]Thus our results are consistent with previous reports inliterature In this sense the findings are relevant since it canbe assumed that even for adult individuals the treatmentof skin lesions with coffee bean press cake extracts is trulyefficient to increase the physiological response amelioratingthe cutaneous regeneration process significantly

36 Effect of Chlorogenic Acid on the Wound Healing Thechlorogenic acid hydrogel daily applied on the skin woundappeared to reduce significantly the wound area size in theinflammatory phase (eg day 3) which can be associatedwith its antioxidant and anti-inflammatory activities Indeedhydrogel containing CGA resulted (057 cm2 plusmn 006 sem) ina similar effect regarding the positive control (055 cm2 plusmn006 sem) as can be noted in Table 5 Contrarily the post-pressing green coffee AE assayed under the same conditionsdid not show the same performance (075 cm2 plusmn 006 sem)at the third day especially taking into consideration thatthe wound area at the beginning of the experiment was072 cm2 plusmn 002 sem All the treatments achieved similarperformance after 7 days and 15 days was enough to achievefull wound healing It can be assumed that the physiologicalresponses in young mice are decisive for full wound healingconsidering the elapsing time of 15 days The significantdifferences at the third day can be attributed to the influenceof the treatments on the inflammatory response that beginsimmediately after injury of the tissue and lasts around 3days The inflammation helps wound heal with activation ofimmune system cellular components the blood coagulationcascade cytokines and the oxidative stress [16] Studies ofMoreira et al [9] showed that AE of green coffee has anti-inflammatory effect due to the presence of anti-inflammatoryand antioxidant compounds The use of chlorogenic acidimproving skin wound healing may be derived from itsinfluence on the inflammatory mediators involved in thisresponse and its antioxidant capacity

37 Oxidative StressMarkers The antioxidant defense systemhas been developed by the organism as a protective mech-anism against ROS formation Among the most reportedendogenous antioxidant systems are the activity of theenzymes SOD and CAT Wound healing needs a fine balancebetween the antioxidants activities because ROS are harmfulto cells and tissues in cutaneous injury [60] It is interestingto note the significant increasing of CAT activity in thehealed tissues when compared to the unhealed ones (Table 6)Besides the CAT activity was increased on the tissue treatedin all treatment groups along the wound healing processProbably the increase can be attributed to the higher ROSlevels on the wound bed resulting from the inflammatory


Table 5Wound area size (cm2) in the elapsing time of 15 days with daily application of green coffee press cake and chlorogenic acid hydrogels

Groups (119899 = 8) Day 0 Day 3 Day 7 Day 15GC 070 plusmn 002 a 075 plusmn 006 ab 042 plusmn 004 a 0002 plusmn 002 aCGA 070 plusmn 002 a 057 plusmn 002 a 041 plusmn 002 a 0002 plusmn 002 aPC 072 plusmn 002 a 055 plusmn 006 a 031 plusmn 004 a 000 plusmn 001 aNC 074 plusmn 002 a 085 plusmn 006 b 042 plusmn 004 a 0002 plusmn 002 aData are presented as means plusmn sem Distinct letters in the column denote significant differences (Tukey test 119901 lt 005) GC = green coffee CGA = chlorogenicacid PC = positive control and CN = negative control

Table 6 Activity of catalase (CAT) and superoxide dismutase (SOD) in the epithelial tissue of Swiss albino mice according to the treatmentwith AE of green coffee bean press cake and chlorogenic acid related to controls groups

Daytreatment GC CGA PC NCCAT (mmolsdotminminus1mg protein)

Day 0lowast 1641 plusmn 126 1641 plusmn 126 1641 plusmn 126 1641 plusmn 126Day 3 5975 plusmn 124 b 3606 plusmn 126 c 90935 plusmn 58 a 1747 plusmn 21 dDay 7 2607 plusmn 119 d 3995 plusmn 194 c 8466 plusmn 201 a 6837 plusmn 41 bDay 15 10050 plusmn 165 a 5066 plusmn 107 b 4087 plusmn 18 c 3000 plusmn 128 d

SOD (Umg protein)Day 0lowast 045 plusmn 002 045 plusmn 002 045 plusmn 002 045 plusmn 002Day 3 105 plusmn 008 b 107 plusmn 004 b 025 plusmn 001 c 233 plusmn 002 aDay 7 052 plusmn 002 a 029 plusmn 003 b 026 plusmn 002 b 020 plusmn 003 cDay 15 012 plusmn 001 b 017 plusmn 003 b 021 plusmn 002 a 025 plusmn 005 aData are presented as means plusmn sd Different letters in the line denote significant differences (Tukey test 119901 lt 005) lowastDay 0 = enzymatic activity on theundamaged tissue GC = green coffee CGA = chlorogenic acid PC = positive control and CN = negative control

response cellular signaling pathways to avoid the infectionrecruiting different kinds of cells and promoting cells divi-sions The increase in the CAT enzymatic activity uponthe green coffee AE and hydrogel containing CGA treat-ments appeared to be increased on day 3 (inflammationstage) following a reduction at the 7th day during the cellproliferation stage with a strong augment again until day15 Interestingly the positive control group also showed asimilar profile of CAT activity except at the end of theexperimental period on day 15 Importantly regardless of thetreatment the results revealed a superior CAT activity alongthe wound healing process in comparison to the basal level(day 0) suggesting that CAT activity is positively modulatedin damaged cutaneous tissues Finallymeaningful differences(119901 lt 005) were detected for each sampling time in alltreatments suggesting that a typical standard of CAT activityseems not to occur

The SOD assay showed higher activity in the early stageof healing (day 3) for the green coffee AE- and CGA-treatedgroups followed by a gradual reduction Furthermore con-trary toCATactivity SODdecreased bellow the baseline in allexperimental groups at the end of the experiment suggestingthat such an enzymatic response to the oxidative stress seemsnot to be relevant to the cutaneous cells upon regeneration

4 Conclusions

The chemical analysis revealed the green coffee is a rawmaterial richer in chlorogenic acid than the roasted one

The present study shows that the daily topical application ofchlorogenic acid and coffee beans press cake AE enhancedthe skin wound healing in mice model especially the laterone that reduced more quickly the wound area Takinginto consideration that the experiments were performedwith plant extracts that is chemically complex matricesit is assumed that the effects herein described eventuallyresult from the synergistic action of their components Wesuggest that the highest amount of CGA and caffeine inthe extracts of coffee press cake presents beneficial effectson wound repair by its antioxidant potential enhancing thephysiological responses to achieve the wound closure Theformation of reactive oxygen radical species (ROS) can playan important role in delayed wound healing Antioxidantscan help controlling wound oxidative stress related to ROSgeneration and thus accelerate wound repair

Finally to the best of our knowledge this is the first studyinvestigating the effect of AE of coffee beans press cake onthe skin wound healing showing beneficial effects on woundrepair Antioxidant and radical scavenger activities of coffeebeans press cake AE and CGA can improve wound healingto control overexposure of oxidative stress in the wound bedThe coffee beans press cake has shown a lower commercialvalue however it might gain value as thought of as a valuablebiomass source of bioactive compounds interesting for thehuman health usage Indeed the residual coffee biomassesstudied are able to improve the regeneration of damage skintissue allowing new product development in the cosmeticaland pharmaceutical industries


Competing Interests

The authors declare there is no conflict of interest regardingthe publication of this paper

Acknowledgments

The authors are thankful to FAPESC (Fundacao de Amparo aPesquisa e Inovacao do Estado de Santa Catarina Brazil) andCooxupe (CoffeeGrowersCooperative) for financial supportThe research fellowship from CNPq on behalf of the laterauthor is acknowledged

References

[1] R Quek and C J Henry ldquoInfluence of polyphenols fromlingonberry cranberry and red grape on in vitro digestibility ofricerdquo International Journal of Food Sciences and Nutrition vol66 no 4 pp 378ndash382 2015

[2] G Laufenberg B Kunz and M Nystroem ldquoTransformation ofvegetable waste into value added products (A) the upgradingconcept (B) practical implementationsrdquo Bioresource Technol-ogy vol 87 no 2 pp 167ndash198 2003

[3] V Brezova A Slebodova and A Stasko ldquoCoffee as a source ofantioxidants an EPR studyrdquo Food Chemistry vol 114 no 3 pp859ndash868 2009

[4] M N Clifford ldquoChlorogenic acidsrdquo in Coffee Chemistry RJ Clarke and R Macrae Eds pp 153ndash202 Elsevier AppliedScience Publication London UK 1985

[5] L C Trugo and R Macrae ldquoA study of the effect of roasting onthe chlorogenic acid composition of coffee using HPLCrdquo FoodChemistry vol 15 no 3 pp 219ndash227 1984

[6] A Ratz-Yko J Arct S Majewski and K Pytkowska ldquoInfluenceof polyphenols on the physiological processes in the skinrdquoPhytotherapy Research vol 29 no 4 pp 509ndash517 2015

[7] A A P Almeida A Farah D A M Silva E A Nunan andM B A Gloria ldquoAntibacterial activity of coffee extracts andselected coffee chemical compounds against enterobacteriardquoJournal of Agricultural and Food Chemistry vol 54 no 23 pp8738ndash8743 2006

[8] H Utsunomiya M Ichinose M Uozaki K Tsujimoto HYamasaki and A H Koyama ldquoAntiviral activities of coffeeextracts in vitrordquo Food and Chemical Toxicology vol 46 no 6pp 1919ndash1924 2008

[9] D P Moreira M C Monteiro M Ribeiro-Alves C MDonangelo and L C Trugo ldquoContribution of chlorogenic acidsto the iron-reducing activity of coffee beveragesrdquo Journal ofAgricultural and Food Chemistry vol 53 no 5 pp 1399ndash14022005

[10] M D dos Santos M C Almeida N P Lopes and G EP de Souza ldquoEvaluation of the anti-inflammatory analgesicand antipyretic activities of the natural polyphenol chlorogenicacidrdquo Biological and Pharmaceutical Bulletin vol 29 no 11 pp2236ndash2240 2006

[11] H-M Chiang T-J Lin C-Y Chiu et al ldquoCoffea arabica extractand its constituents prevent photoaging by suppressing MMPsexpression and MAP kinase pathwayrdquo Food and ChemicalToxicology vol 49 no 1 pp 309ndash318 2011

[12] C Hoelzl S Knasmuller K-H Wagner et al ldquoInstant coffeewith high chlorogenic acid levels protects humans againstoxidative damage of macromoleculesrdquoMolecular Nutrition andFood Research vol 54 no 12 pp 1722ndash1733 2010

[13] C I Heck and E G De Mejia ldquoYerba mate tea (Ilexparaguariensis) a comprehensive review on chemistry healthimplications and technological considerationsrdquo Journal of FoodScience vol 72 no 9 pp R138ndashR151 2007

[14] A Farah M C Monteiro V Calado A S Franca and LC Trugo ldquoCorrelation between cup quality and chemicalattributes of Brazilian coffeerdquo Food Chemistry vol 98 no 2 pp373ndash380 2006

[15] C L Baum and C J Arpey ldquoNormal cutaneous woundhealing clinical correlation with cellular andmolecular eventsrdquoDermatologic Surgery vol 31 no 6 pp 674ndash686 2005

[16] G C Gurtner S Werner Y Barrandon and M T LongakerldquoWound repair and regenerationrdquoNature vol 453 no 7193 pp314ndash321 2008

[17] P Farris ldquoIdebenone green tea and Coffeeberry extract newand innovative antioxidantsrdquoDermatologicTherapy vol 20 no5 pp 322ndash329 2007

[18] AGosain and LADiPietro ldquoAging andwoundhealingrdquoWorldJournal of Surgery vol 28 no 3 pp 321ndash326 2004

[19] K T Keylock V J Vieira M A Wallig L A DiPietro MSchrementi and J A Woods ldquoExercise accelerates cutaneouswound healing and decreases wound inflammation in agedmicerdquo American Journal of PhysiologymdashRegulatory Integrativeand Comparative Physiology vol 294 no 1 pp R179ndashR1842008

[20] M E Swift A L Burns K L Gray and L A DiPietro ldquoAge-related alterations in the inflammatory response to dermalinjuryrdquo Journal of Investigative Dermatology vol 117 no 5 pp1027ndash1035 2001

[21] M E Swift H K Kleinman and L A DiPietro ldquoImpairedwound repair and delayed angiogenesis in aged micerdquo Labora-tory Investigation vol 79 no 12 pp 1479ndash1487 1999

[22] A Nkondjock ldquoCoffee consumption and the risk of cancer anoverviewrdquo Cancer Letters vol 277 no 2 pp 121ndash125 2009

[23] S D Fitzmaurice R K Sivamani and R R Isseroff ldquoAntioxi-dant therapies for wound healing a clinical guide to currentlycommercially available productsrdquo Skin Pharmacology and Phys-iology vol 24 no 3 pp 113ndash126 2011

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

[25] D Bagdas N Cinkilic H Y Ozboluk M O Ozyigit andM S Gurun ldquoAntihyperalgesic activity of chlorogenic acid inexperimental neuropathic painrdquo Journal of Natural Medicinesvol 67 no 4 pp 698ndash704 2013

[26] J Bravo L Arbillaga M P de Pena and C Cid ldquoAntioxidantand genoprotective effects of spent coffee extracts in humancellsrdquo Food and Chemical Toxicology vol 60 pp 397ndash403 2013

[27] N Cinkilic S K Cetintas T Zorlu et al ldquoRadioprotectionby two phenolic compounds chlorogenic and quinic acid onX-ray induced DNA damage in human blood lymphocytes invitrordquo Food and Chemical Toxicology vol 53 pp 359ndash363 2013

[28] A T Shimoyama J R Santin I D Machado et al ldquoAntiul-cerogenic activity of chlorogenic acid in different models ofgastric ulcerrdquoNaunyn-Schmiedebergrsquos Archives of Pharmacologyvol 386 no 1 pp 5ndash14 2013

[29] N Yun J-W Kang and S-M Lee ldquoProtective effects of chloro-genic acid against ischemiareperfusion injury in rat livermolecular evidence of its antioxidant and anti-inflammatory


propertiesrdquoThe Journal of Nutritional Biochemistry vol 23 no10 pp 1249ndash1255 2012

[30] ANVISA Agencia Nacional de Vigilancia Sanitaria Farma-copeia Brasileira 2010 httpwwwanvisagovbrhotsitecdfarmacopeiaindexhtm

[31] R Randhir P Shetty andK Shetty ldquoL-DOPAand total phenolicstimulation in dark germinated fava bean in response to peptideand phytochemical elicitorsrdquoProcess Biochemistry vol 37 no 11pp 1247ndash1256 2002

[32] N P Rodrigues andN Bragagnolo ldquoIdentification and quantifi-cation of bioactive compounds in coffee brews byHPLCndashDADndashMS119899rdquo Journal of Food Composition and Analysis vol 32 no 2pp 105ndash115 2013

[33] P Molyneux ldquoThe use of the stable free radical diphenylpicryl-hydrazyl (DPPH) for estimating antioxidant activityrdquo Songk-lanakarin Journal and Science Technology vol 26 no 2 pp 211ndash219 2004

[34] ICCVAM Inter-Agency Coordinating Committee on the Val-idation of Alternative Methods In Vitro Methods for AssessingAcute Systemic Toxicity 2001 httpiccvamniehsnihgovdocsFR6649686htm

[35] S Frank and H Kampfer ldquoExcisional wound healing an exper-imental approachrdquo in Wound Healing Methods and ProtocolsL A DiPietro and A L Burns Eds vol 78 of Methods inMolecular Medicine pp 3ndash15 Humana Press Totowa NJUSA 2003

[36] J R C Pereira ldquoTratamento de cicatrizes patologicas com aassociacao de cepalin heparina e alantoınardquo Revista Brasileirade Medicina vol 60 pp 603ndash608 2003

[37] D Bagdas B C Etoz Z Gul et al ldquoIn vivo systemic chlorogenicacid therapy under diabetic conditions wound healing effectsand cytotoxicitygenotoxicity profilerdquo Food and Chemical Toxi-cology vol 81 pp 54ndash61 2015

[38] A Pereira Avaliacao das atividades cicatrizante e antitumoralde extratos provenientes da casca de banana cultivar PrataAna (Musa spp) [Dissertacao] Universidade Federal de SantaCatarina Florianopolis Brazil 2010

[39] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[40] C N Giannopolitis and S K Ries ldquoSuperoxide dismutases Ioccurrence in higher plantsrdquo Plant Physiology vol 59 no 2 pp309ndash314 1977

[41] A Farah andCMDonangelo ldquoPhenolic compounds in coffeerdquoBrazilian Journal of Plant Physiology vol 18 no 1 pp 23ndash362006

[42] A Farah T de Paulis L C Trugo and P R Martin ldquoEffect ofroasting on the formation of chlorogenic acid lactones in coffeerdquoJournal of Agricultural and Food Chemistry vol 53 no 5 pp1505ndash1513 2005

[43] MW Cheong K H Tong J J M Ong S Q Liu P Curran andB Yu ldquoVolatile composition and antioxidant capacity ofArabicacoffeerdquo Food Research International vol 51 no 1 pp 388ndash3962013

[44] M C Monteiro and A Farah ldquoChlorogenic acids in BrazilianCoffea arabica cultivars from various consecutive cropsrdquo FoodChemistry vol 134 no 1 pp 611ndash614 2012

[45] M E D C Moreira R G F A Pereira D F Dias et al ldquoAnti-inflammatory effect of aqueous extracts of roasted and greenCoffea arabica Lrdquo Journal of Functional Foods vol 5 no 1 pp466ndash474 2013

[46] P Esquivel and V M Jimenez ldquoFunctional properties of coffeeand coffee by-productsrdquo Food Research International vol 46no 2 pp 488ndash495 2012

[47] J A Vignoli D G Bassoli and M T Benassi ldquoAntioxidantactivity polyphenols caffeine andmelanoidins in soluble coffeethe influence of processing conditions and raw materialrdquo FoodChemistry vol 124 no 3 pp 863ndash868 2011

[48] R L Prior X Wu and K Schaich ldquoStandardized methodsfor the determination of antioxidant capacity and phenolicsin foods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[49] J Bouayed and T Bohn ldquoExogenous antioxidantsmdashdouble-edged swords in cellular redox state health beneficial effectsat physiologic doses versus deleterious effects at high dosesrdquoOxidativeMedicine and Cellular Longevity vol 3 no 4 pp 228ndash237 2010

[50] L Su J-J Yin D Charles K Zhou J Moore and L Yu ldquoTotalphenolic contents chelating capacities and radical-scavengingproperties of black peppercorn nutmeg rosehip cinnamon andoregano leafrdquo Food Chemistry vol 100 no 3 pp 990ndash997 2007

[51] T P A Devasagayam J P Kamat HMohan and P C KesavanldquoCaffeine as an antioxidant inhibition of lipid peroxidationinduced by reactive oxygen speciesrdquo Biochimica et BiophysicaActa vol 1282 no 1 pp 63ndash70 1996

[52] C Gallardo L Jimenez and M-T Garcıa-Conesa ldquoHydrox-ycinnamic acid composition and in vitro antioxidant activity ofselected grain fractionsrdquo Food Chemistry vol 99 no 3 pp 455ndash463 2006

[53] J A Gomez-Ruiz D S Leake and J M Ames ldquoIn vitroantioxidant activity of coffee compounds and theirmetabolitesrdquoJournal of Agricultural and Food Chemistry vol 55 no 17 pp6962ndash6969 2007

[54] R C Borrelli A Visconti C Mennella M Anese and VFogliano ldquoChemical characterization and antioxidant proper-ties of coffee melanoidinsrdquo Journal of Agricultural and FoodChemistry vol 50 no 22 pp 6527ndash6533 2002

[55] C Delgado-Andrade J A Rufian-Henares and F J MoralesldquoAssessing the antioxidant activity of melanoidins from coffeebrews by different antioxidant methodsrdquo Journal of Agriculturaland Food Chemistry vol 53 no 20 pp 7832ndash7836 2005

[56] D Triglia S S Braa C Yonan and G K Naughton ldquoIn vitrotoxicity of various classes of test agents using the neutral redassay on a human three-dimensional physiologic skin modelrdquoIn Vitro Cellular amp Developmental BiologymdashAnimal vol 27 no3 pp 239ndash244 1991

[57] E Borenfreund and J A Puerner ldquoToxicity determined invitro by morphological alterations and neutral red absorptionrdquoToxicology Letters vol 24 no 2-3 pp 119ndash124 1985

[58] R D Galiano J Michaels V M Dobryansky J P Levine andG C Gurtner ldquoQuantitative and reproducible murine modelof excisional wound healingrdquoWound Repair and Regenerationvol 12 no 4 pp 485ndash492 2004

[59] S Guo and L A Di Pietro ldquoFactors affecting wound healingrdquoJournal of Dental Research vol 89 no 3 pp 219ndash229 2010

[60] S Suzuki Y Miyachi Y Niwa and N Isshiki ldquoSignificance ofreactive oxygen species in distal flap necrosis and its salvagewith liposomal SODrdquo British Journal of Plastic Surgery vol 42no 5 pp 559ndash564 1989

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


have been proposed as one of the most effective functionalingredients in foods and beverages with antiaging propertiesand able to neutralize the effects of oxidative damage tothe skin for example [6] Studies with C arabica extractshave revealed a set of important biological activities forexample antibacterial [7] antiviral [8] anti-inflammatory[9 10] suppressive activity of metalloproteinase expression[11] and reduction of oxidative damage to macromolecules[12] Besides phenolic compounds coffee is well recognizedas a rich source of the alkaloids especially caffeine Suchsecondary metabolites have presented relevant biologicalactivities as for instance stimulation of the central nervoussystem diuretic and peripheral vasoconstriction [13] Caf-feine is the major alkaloid present in coffee beans and itscontent is correlated with the quality of the beverage alsocontributing to the brew bitterness [14]

Skin serves as a protective barrier against the environ-ment and also plays a fundamental role in the homeostasismaintenance [15] The loss of the skin integrity results ininjury that can lead to serious commitments to the body ifnot repairedThe tissue lesions trigger intracellular responsesthat coordinate the restoration of tissuersquos integrity and home-ostasis The ability to respond to the injury and tissue repairis a fundamental property for multicellular organisms Thecutaneous tissue repair occurs via tissue regeneration withthe recovery of tissue functionality or the healing and therestoration of tissue homeostasis [16]

The wound healing is a complex biological process afteran injury This process can be understood into three classicstages inflammation proliferation and remodeling Theinflammatory stage begins immediately after injury of thetissue and lasts for around 3 days with inflammation Subse-quently there are cellular proliferation migration of differentcell types angiogenesis and extracellular matrix componentsproduction especially collagen The proliferation stage maylast from 2 to 10 days and is characterized by the migrationand differentiation of various cell types being themost abun-dant fibroblasts The remodeling stage is the third and laststage of repair It begins 2 to 3 weeks after the injury and lastsfor about a year or more The majority of endothelial cellsmyofibroblasts and macrophages are destroyed by apoptosisand these changes result in wound contraction and scarformation [16] Inflammation and oxidative stress are closelyrelated at the wound site considering the influx of neutrophilsand macrophages producing reactive oxygen and nitrogenReactive oxygen and nitrogen species are involved in theredox regulation of cell functions and increasingly viewedas a major upstream component in the signalling cascadeinvolved in inflammatory responses and the stimulation ofadhesion molecule and chemoattractant production [15]Testing by oxygen radical absorbance capacity assay of thecoffee berry outperforms common antioxidants such as greentea extract pomegranate extract vitamin C and vitamin EPolymerase chain reaction microarray analysis on humancultured fibroblasts treated with multiple doses of 0001coffee berry revealed upregulation of gene expression forseveral collagens and connective tissue growth factor anddownregulation for metalloproteinases [17]

It is known that the effect of aging causes a temporaldelay in wound healing but not an actual impairment interms of the quality of healing [18 19] Delayed woundhealing in the aged is associated with an altered inflammatoryresponse such as delayed T-cell infiltration into the woundarea with alterations in chemokine production and reducedmacrophage phagocytic capacity [20] Retarded reepithelial-ization collagen synthesis and angiogenesis have also beenobserved in aged mice as compared with young ones [21]

The topical administration of antioxidant compoundson the skin is gaining prominence among dermatologistsbecause of their anti-inflammatory and anticarcinogenicactivity [22] In this context it has been claimed that woundrepair process could be helped by topically administratingantioxidant molecules that may control the oxidative stress[23] Phenolic compounds have been showed to be activeon skin tissues through hampering collagen destruction andcollagenase activation [24] Indeed CGA has been shown toaccelerate the skin wound healing and burn healing due toits antioxidant free radical scavenging anti-inflammatoryradioprotective antiulcerogenic and analgesic properties [1025ndash29] Importantly the effectiveness of polyphenols in therepair of cutaneous tissue is initially determined by theirphysicochemical properties and the ability to overcome theepidermal barrier to achieve appropriate receptors [6]

In this sense the working hypothesis of this study is basedon the assumption that a biologically compatible extract thatis the AE of coffee beans residual press cake is a rich sourceof polyphenolic compounds that topically administrated onthe damaged cutaneous tissue would ameliorate the woundhealing process Thus the phytochemical profile and theantioxidant activity of the AE of coffee beans (C arabica L)residual press cake were determined In a second approachthe in vivo effect of topically administrated hydrogels contain-ing AE and chlorogenic acid on the wound healing processwas investigated using a skin excision wound model in Swissalbino mice

2 Material and Methods

21 Coffee Samples Chemicals and Cell Lines Samples ofgreen (ie nonroasted beans) and roasted coffee beans presscake residual were supplied by Cooxupe (Regional Coopera-tive of Coffee Growers Ltda at Guaxupe Minas Gerais Statesoutheastern Brazil) Green and roasted coffee bean presscake samples were obtained after mechanically pressing thebeans for the extraction of the oil fraction After extractionthe residual biomass was packed in polyethylene bags withsilica sachets and stored at minus20∘C for further analysis Ana-lytical grade methanol ethanol hydrochloric acid sodiumchloride and hydrogen peroxide were purchased from Vetec(Rio de Janeiro Brazil) The Folin-Ciocalteu reagent sodiumdodecyl sulphate (SDS) dimethyl sulfoxide (DMSO) 22-diphenyl-1-picrylhydrazyl (DPPH) (plusmn)-6-hydroxy-2578-tetramethylchroman-2-carboxylic acid (Trolox) potassiumphosphate buffer (TPK) Tween 20 Bradford reagent bovineserum albumin (BSA) methionine riboflavin nitrote-trazolium blue chloride (NBT) neutral red (NR) andthe analytical reference standards (chlorogenic acid gallic






Table 1







] (1)








[(119882cp) = (1198821198890 minus119882119889119899)1198821198890 lowast 100] (3)





























Cel

l via

bilit

y (

)

0

20

40

60

80

100


3e minus 03 3e + 00 1e + 01


Wou

nd h

ealin

g re

duct

ion

()

Treatments

c

ab

b

a

0

20

40

60

80

100

NCPC

PPRCPPGC















4 Conclusions





Competing Interests


Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Table 1







] (1)








[(119882cp) = (1198821198890 minus119882119889119899)1198821198890 lowast 100] (3)





























Cel

l via

bilit

y (

)

0

20

40

60

80

100


3e minus 03 3e + 00 1e + 01


Wou

nd h

ealin

g re

duct

ion

()

Treatments

c

ab

b

a

0

20

40

60

80

100

NCPC

PPRCPPGC















4 Conclusions





Competing Interests


Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















[(119882cp) = (1198821198890 minus119882119889119899)1198821198890 lowast 100] (3)





























Cel

l via

bilit

y (

)

0

20

40

60

80

100


3e minus 03 3e + 00 1e + 01


Wou

nd h

ealin

g re

duct

ion

()

Treatments

c

ab

b

a

0

20

40

60

80

100

NCPC

PPRCPPGC















4 Conclusions





Competing Interests


Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































Cel

l via

bilit

y (

)

0

20

40

60

80

100


3e minus 03 3e + 00 1e + 01


Wou

nd h

ealin

g re

duct

ion

()

Treatments

c

ab

b

a

0

20

40

60

80

100

NCPC

PPRCPPGC















4 Conclusions





Competing Interests


Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























4 Conclusions





Competing Interests


Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Competing Interests


Acknowledgments


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article phytochemical composition, antioxidant...

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