Major components of energy drinks (caffeine, taurine, and guarana) exert cytotoxic effects on human...

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2013 Article ID 791795 22 pageshttpdxdoiorg1011552013791795

Research ArticleMajor Components of Energy Drinks (Caffeine Taurine andGuarana) Exert Cytotoxic Effects on Human Neuronal SH-SY5YCells by Decreasing Reactive Oxygen Species Production

Fares Zeidaacuten-Chuliaacute1 Daniel Pens Gelain1 Eduardo Antocircnio Kolling1

Joseacute Luiz Rybarczyk-Filho12 Priscilla Ambrosi1 Silvia Resende Terra1

Andreacute Simotildees Pires1 Joatildeo Batista Teixeira da Rocha3

Guilherme Antocircnio Behr14 and Joseacute Claacuteudio Fonseca Moreira1

1 Department of Biochemistry Center of Oxidative Stress Research Institute of Basic Health SciencesFederal University of Rio Grande do Sul (UFRGS) 90035-003 Porto Alegre RS Brazil

2 Departamento de Fısica e Biofısica Instituto de Biociencias de Botucatu Universidade Estadual Paulista (UNESP)18618-970 Botucatu SP Brazil

3 Departamento de Quımica Centro de Ciencias Naturais e Exatas (CCNE) Universidade Federal de Santa Maria (UFSM)97105-900 Santa Maria RS Brazil

4Department of Psychiatry and Behavioral Neurosciences McMaster University Hamilton ON Canada L8P 3B6

Correspondence should be addressed to Fares Zeidan-Chulia fzchuliabiomedgmailcom

Received 26 January 2013 Accepted 16 March 2013

Academic Editor Felipe Dal-Pizzol

Copyright copy 2013 Fares Zeidan-Chulia 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

Scope To elucidate the morphological and biochemical in vitro effects exerted by caffeine taurine and guarana alone or incombination since they are major components in energy drinks (EDs)Methods and Results On human neuronal SH-SY5Y cellscaffeine (0125ndash2mgmL) taurine (1ndash16mgmL) and guarana (3125ndash50mgmL) showed concentration-dependent nonenzymaticantioxidant potential decreased the basal levels of free radical generation and reduced both superoxide dismutase (SOD)and catalase (CAT) activities especially when combined together However guarana-treated cells developed signs of neuritedegeneration in the form of swellings at various segments in a beaded or pearl chain-like appearance and fragmentation of suchneurites at concentrations ranging from 125 to 50mgmL Swellings but not neuritic fragmentation were detected when cells weretreated with 05mgmL (or higher doses) of caffeine concentrations that are present in EDs Cells treated with guarana also showedqualitative signs of apoptosis including membrane blebbing cell shrinkage and cleaved caspase-3 positivity Flow cytometricanalysis confirmed that cells treated with 125ndash50mgmL of guarana and its combinations with caffeine andor taurine underwentapoptosis Conclusion Excessive removal of intracellular reactive oxygen species to nonphysiological levels (or ldquoantioxidativestressrdquo) could be a cause of in vitro toxicity induced by these drugs

1 Introduction

Worldwide consumption of energy drinks (EDs) is exponen-tially increasing due to their stimulant effect on the centralnervous system and body and the purpose of enhancing bothcognitive and physical performances EDs are highly caf-feinated and may contain herbal supplements (eg guarana)

alkaloids found in different plants (eg yohimbine) vita-mins (eg B vitamins) and taurine (Figure 1) [1 2] Forinstance one single can of one of the most consumedbrand of EDs (Red Bull 250mL) contains 80mg of caffeine(or 032mgmL) and 1000mg of taurine (or 4mgmL) asmajor components Other brands commonly add guarana inthe composition (eg Monster Energy Rockstar and 180)

2 Oxidative Medicine and Cellular Longevity

Plant extract

Medicinal plants

YohimbinePausinystalia yohimbe

Snakeroot or sarpagandhaRauwolfia serpentina

Yohimbine

GuaranaPaullinia cupana

GinsengPanax ginseng

(1) Methylxanthines

TheobromineTheophylline

(2) Tannins(3) Saponins(4) Catechins(5) Epicatechins(6) Proanthocyanidins

Energy drinks

(1) SaponinsGinsenosides

(2) Other compounds

(7) Other compounds

N

N

N

N O

O

S

HO O

O

O

OHCaffeine

Vitamins

Taurine

L-carnitine

GinkgoGinkgo biloba

HH

OH

N

N

H

H

OO

Am

ino

acid

s

B vitamins

Oth

ers

(1) Flavonoids

(2) Terpenes

QuercetinKaempferolBiflavoneGinkgetinBilobetinSciadopitysin

Ginkgolide A Ginkgolide BGinkgolide CBilobalide

Caffeinelowast

N+

Ominus

NH2

CH3

CH3

H3C

Figure 1 Schematic representations illustrating the different components found in EDs EDs are highly caffeinated beverages which maycontain herbal supplements alkaloids from different plants vitamins or even amino acids In some cases the addition of herbal supplements(eg guarana) can increase the caffeine content of these beverages (lowast) For our in vitro study we selected caffeine taurine and guarana asmajor components of EDs

Furthermore it has been reported that the number of energydrinks consumed in one session reaches an average of 5 units(cans) in one single night [3]

The concentration of caffeine in EDs is significantlyhigher than those found in cola drinks and such amounts areknown to cause a variety of adverse health effects [4] Alsothe use of some herbal supplements in these beverages likeguarana increases the amount of caffeine and other activemethylxanthines that multiply any potential toxicity

The widespread consumption of EDs is becoming espe-cially popular among people below the age of 25 and adole-scents [1 2] Nonetheless only a limited number of studieshave tried to evaluate the short- and long-term effects of EDconsumption Most of them examined the cognitive effectsderived from the use of these drinks or some of their majorcomponents (eg caffeine or taurine) alone or in coadmin-istration with alcohol [5 6] Some other reports are high-lighting the possible link between the use of highly caffeinatedbeverages and the increased propensity for addiction to othersubstances like alcohol or even new-onset seizures in adults[7 8]

Different countries have already started to regulate thesale of highly caffeinated EDs to prevent potential healthproblems Denmark and France banned the sale of somebrands in Norway some EDs are only available in pharma-cies in general the European Union demands the labelingof highly caffeinated drinks [1 2] Major concerns are arisingwith the impact these beverages may have on children andadolescentsrsquo developing brains [9] Therefore in the presentstudy we used a well-known neurotoxicity cell culture model(human neuronal SH-SY5Y cells) [10] in order to elucidatethe potential toxicity (synergistic or not) of caffeine taurineand guarana three components that are commonly presentin EDs at high concentrations

2 Materials and Methods

21 Drugs Guarana (Paullinia cupana Mart) powder wasobtained fromLifar Ltd (PortoAlegre RS Brazil) Accordingto the manufacturer 50mg of guarana powder (obtainedfrom grounding of dried seeds) contains approximately 2mgof caffeine Both caffeine and taurine were purchased fromSigma Chemical Co (St Louis MO USA)

Oxidative Medicine and Cellular Longevity 3

22 Cell Culture and Treatments Human neuroblastoma-derived SH-SY5Y cells were purchased from Rio de JaneiroCell Bank ((BCRJ) Rio de Janeiro Brazil) The cells werecultured in a 1 1 mixture of Hamrsquos F12 and Dulbecco Mod-ified Eagle Medium (DMEM) supplemented with 10 heat-inactivated fetal bovine serum (FBS) 2mM of glutamine028mgmL of gentamicin and 250120583g of amphotericin Bin a 5 CO

2humidified incubator at 37∘C Cells were

subcultured until they reached 80ndash90 of confluence andthen trypsinized 24 hours after trypsinization approximatelyhalf of volume of old cell culture medium was replaced withfresh 10 FBS-DMEM F12 medium All treatments wereperformed when cells reached a confluence of approximately75 followed by 2-hour incubation in complete absenceof FBS except for one experiment (long-term effects oncell number after 24 hours of treatment with ED compo-nents or their combinations Figure 11) where no previousFBS deprivation was performed For each assay guarana(Gua) caffeine (Caf) and taurine (Tau) at concentrations of50mgmL 2mgmL and 16mgmL respectively were dis-solved in either DMEM F12 medium (without FBS) or 10FBS-DMEM F12 medium (Figure 11) and serial dilutionswere obtained from this stock solution

23 Phase ContrastMicroscopy (PCM)Analysis PCMmicro-graphs were taken by using an inverted microscope (NikonEclipse TE300) connected to a digital camera

24 Scanning Electron Microscopy (SEM) Analysis SH-SY5Ycells were exposed to each drug treatment for 4 hours fixedin 25 glutaraldehyde treated with osmium tetroxide indistilled water dehydrated with sequential washes in 3050 70 90 and 100 acetone and then dried in criticalpoint dryer by using a Balzers CPD-030 instrument beforecoating with gold in a Balzers SCD 050 sputter coater Sam-ples were visualized in a JEOL-JSM-6060 scanning electronmicroscope

25 Confocal Immunofluorescence Microscopy (CM) AnalysisCells were washed with phosphate-buffered saline (PBS) andfixed on chamber slides with 4 paraformaldehyde (PFA)in 4∘C for 15min Samples were sequentially treated with015 Triton X-100 (for permeabilization) for 10min and10 serum (to avoid nonspecific binding) for 30min Cellswere immunostained with specific antibodies after 4 hours oftreatment with guarana caffeine and taurine Primary anti-bodies were utilized as follows rabbit anticleaved caspase-3 (Cell Signaling Technology 9664 1 1000) mouse anti-120573-III tubulin (Novex 480011 1 1000) and fluorescent-labelingAlexa Fluor 488 goat anti-mouse (Molecular Probes A110011 1000) fluorescent-labeling Alexa Fluor 488 goat anti-rabbit (Molecular Probes A11008 1 1000) For nuclear stain-ing Prolong Gold Antifade Reagent with DAPI (MolecularProbes P36931) was used As a marker for cellular viabilitytogether with 120573-III tubulin (antineutron-specific antibody)cells were co-immunostained with propidium iodide (PI)(BD Pharmingen) Images were taken with an OlympusFluoView 1000 confocal microscope and subsequently ana-lyzed by using Olympus FluoView FV1000 Software ver 30

Thresholds discriminating between signal and backgroundwere selected by utilizing cells that were only stained withsecondary antibodies in order to discriminate any falsepositive result

26 Total Reactive Antioxidant Potential (TRAP) and TotalAntioxidant Reactivity (TAR) TRAP and TAR were mea-sured and calculated as previously described [11] BrieflyTRAP represents the nonenzymatic antioxidant capacity ofthe cells once they are treated (in this study with guaranacaffeine and taurine) This is determined by measuring theluminol chemiluminescence intensity of emission induced bythermolysis of 221015840-azobis (2-amidinopropane) hydrochlo-ride (AAPH) as free radical source The system was left tostabilize for 2 hours Then samples were added and thereadings monitored for 2 hours Results were transformedinto a percentile rank and the area under the curve (AUC)was calculated by utilizing theGraphPad software (SanDiegoCA USA) as previously described The smaller the AUC is(in comparison to the system) the higher the total reactiveantioxidant potential of the sample is TAR represents thetotal antioxidant properties of all antioxidants This param-eter is closely related to the quality of the antioxidants withinthe sample In our study TAR was calculated as the ratio oflight in the absence of samples (119868o)light intensity right aftersample addition (119868) The higher these values are the higherthe total antioxidant reactivity of the sample is

27 Intracellular Reactive Oxygen Species Production (DCFH-DA Assay) Intracellular reactive species production wasmeasured by the DCFH-DA assay as previously described[11] This technique is based on the capability of DCFH tobe oxidized to highly fluorescent dichlorofluorescein (DCF)in the presence of reactive oxygen species (ROS) This canbe used as an index to measure the level of free radicalproduction in cells Briefly SH-SY5Y cells were seeded in96-well plates 100 120583M DCFH-DA was then dissolved inmedium containing 1 FBS and added to each well Cellswere incubated for 2 hours in order to allow cellular incor-poration Thereafter this medium was discarded and cellswere treated with guarana (12mgmL) caffeine (05mgmL)taurine (4mgmL) and their different combinations H

2O2

1mM was used as positive control for DCF fluorescence Atthis endpoint DCF fluorescence was read at 37∘C in a fluo-rescence plate reader (Spectra Max M2 Molecular DevicesUSA) with an emission wavelength of 535 nm and an excita-tion wavelength of 485 nmThe results were expressed as per-centage of DCF fluorescence in relation to untreated control

28 Cellular Antioxidant EnzymeActivities In order to deter-mine the cellular antioxidant enzyme activities cells werecollected resuspended and homogenized in 50mM PBS atpH 74 The resulting cellular suspension was centrifugedat 3000timesg for 10min and the supernatant was collectedCellular superoxide dismutase (SOD) activity was asses-sed by quantifying the inhibition of superoxide-dependentadrenaline autooxidation in a spectrophotometer at 480 nmas previously described [12] Results were expressed as unitsof SODmg protein Cellular catalase (CAT) activity was


determined by measuring the rate of decrease in H2O2

absorbance in a spectrophotometer at 240 nm [12] CATactivity was expressed as units of CATmg protein Cellularglutathione peroxidase (GPx) activity was assessed by mea-suring the rate of NADPH oxidation in a spectrophotometerat 340 nm as previously described [12] GPx activity wasexpressed as units (nmol NADPH oxidizedmin)mg pro-tein Cellular glutathione S-transferase (GST) activity wasdetermined in a reaction mixture with 1mM 1-chloro-24-dinitrobenzene (CDNB) and 1mM glutathione as substratesas previously described [13] GST activity was expressed asunits of GSTmg protein

29 Measurement of Protein Thiol Content This assay isutilized for analyzing oxidative alterations in proteins bymeasuring the level of reduced thiol content (SH) in thesamples [14] Briefly each aliquot was diluted in 10mM 551015840-dithiobis-2-nitrobenzoic acid in ethanol After 60 minutes ofincubation at 25∘C the reaction gives rise to an intense yellowcolor which is then read in a spectrophotometer at 412 nmThe results were expressed as 120583mol SHmg protein

210 Measurement of Thiobarbituric Acid Reactive Species(TBARS) Formation of TBARS is widely accepted as anindex of lipid peroxidation as previously described [12]Briefly the samples were mixed with 1mL of trichloroaceticacid (TCA) 10 and 1mL of thiobarbituric acid (TBA) 067and then heated in a boiling water bath for 15min 1133-tetramethoxypropane (TMP) was used as a TBARS standardTBARS were determined by the absorbance at 535 nm andwere expressed as nmol TMPmg protein

211 Flow Cytometric (FC) Analyses Analysis of granularityversus size the side (SSC-H) and forward (FSC-H) scatterparameters distinguish the cells from one another based oninternal complexity and size respectively To examine thesemorphological characteristics SH-SY5Y cells were culturedin 6-well plates preincubated for 2 hours in FBS-free culturemedium and then treated with guarana caffeine and taurinefor 4 hours in DMEM F12 medium (without FBS) Thencells were analyzed by flow cytometry (BD FACSCaliburflow cytometer BD Biosciences) data were presented as dotplots (SSC-Hgranularity plotted against FSC-Hsize) andfinally analyzed by FlowJo (cytometric data analysis and pre-sentation software)

Analysis of cell death (necrosis versus apoptosis) SH-SY5Y cells were cultured in 6-well plates preincubated for2 hours in FBS-free culture medium and then treated withguarana caffeine taurine and their combinations for 4 hoursin DMEM F12 medium (without FBS)The FITC Annexin Vapoptosis detection kit (BD Pharmingen) was then utilized inorder to distinguish apoptotic from necrotic events Brieflycells were washed with PBS and resuspended in bindingbuffer Thereafter Annexin V and PI were added and leftfor incubation (15 minutes) at room temperature (25∘C) inthe complete absence of light Then cell death was measuredby using flow cytometry (BD FACSCalibur flow cytometerBD Biosciences) Data are presented as dot plots (PI plottedagainst Annexin V) and analyzed by FlowJo (cytometric

data analysis and presentation software) Ten thousand cellswere analyzed per sample and data were also reported asthe percentage of late necrotic cells (Q1 quadrant 1 upperleft) early necroticlate apoptotic cells (Q2 quadrant 2 upperright) early apoptotic (Q3 quadrant 3 lower right) andliving cells (Q4 quadrant 4 lower left)

Analysis of drug-induced long-term effects on cell num-ber for examining the in vitro long-term effects of guaranacaffeine taurine and their combinations SH-SY5Y cellswere cultured in 6-well plates and treated in the presenceof 10 FBS-DMEM F12 medium for 24 hours Then aftercollecting the cells the total cell count was determined byusing the flow cytometer (BD FACSCalibur flow cytometerBD Biosciences)

212 In Silico Network Development and Determination ofCentralities to Predict the Relevance of GenesProteins inthe Overall Architecture of the MEDRI Model The in siliconetwork model of interactions between ED componentswithin redoxnitric oxide (NO) [11] and apoptotic path-ways (KEGGpathway database httpwwwgenomejpkeggpathwayhtml) was developed by interconnecting 16 com-pounds (12 ED components hydrogen peroxide hydroxylradicals molecular oxygen and nitric oxide) and 144 proteins(87 apoptosis-related and 57 redoxNO-related proteins)based on their possible interactions through either ldquoactiva-tionrdquo ldquoinhibitionrdquo ldquocatalysisrdquo ldquobindingrdquo or ldquoreactionrdquo Thenetwork was generated by using the database resource searchtool STRING 90 (httpstring-dborg) for the retrieval ofinteracting genes [15] with ldquoDatabasesrdquo and ldquoExperimentsrdquoas input options and a confidence score of 0400 (mediumconfidence) STRING provides a public database with infor-mation about direct and indirect functional protein-proteinassociationsinteractions Proteins were identified by theHUGO Gene Symbol [16] and Ensembl protein ID [17]Then small molecule-small molecule and small molecule-protein interactions were found by using STITCH 30(httpstitchemblde) [18] with ldquoDatabasesrdquo and ldquoExper-imentsrdquo as input options and a confidence score of 0400(medium confidence)The links between two different nodes(protein-protein compound-compound and protein-com-pound) provided by STRING 90 and STITCH 30 aresaved in data files to be handled in the Medusa interface[19]

The complete list with gene symbols compoundnames and IDs (Ensembl protein IDs and Compound IDsresp) is additionally provided (see Supporting Informa-tion Tables S1 S2 and S3 available online at httpdxdoiorg1011552013791795)

For elucidating the topological network propertiesCytoscape (httpwwwcytoscapeorg) an open sourceplatform for complex network analysis and visualization wasused [20] Numeric values concerning the properties of eachnode are also provided (Supporting Information Table S4)Finally to visualize the topological network properties of eachnode within MEDRI network model numeric values wereprojected in a 2D color representation by utilizing ViaCom-plex software (httpliefifufrgsbrpubbiosoftwaresvia-complex) [21]


213 Protein Quantification The protein content of eachsample was measured by Lowry method [22]

214 Statistical Analysis Results were expressed as the meanplusmn SEM of three independent experiments (119899 = 3) Data wereanalyzed by a one-way analysis of variance (ANOVA) fol-lowed by Dunnettrsquos multiple comparison test Differenceswere considered to be significant when lowast119875 lt 005 lowastlowast119875 lt001 or lowastlowastlowast119875 lt 0001

3 Results

31 Distinct Morphological Alterations on Human NeuronalCells Triggered by Guarana Taurine and Caffeine Signs ofApoptosis andNeuriteDegeneration Firstly we characterizedthe morphological events induced by separate treatments ofhuman neuronal cells (SH-SY5Y) with increasing concentra-tions of either guarana (3125 125 and 50mgmL) caffeine(0125 05 and 2mgmL) or taurine (1 4 and 16mgmL)For each drug we selected a range of concentrations basedon (I) the content of these three compounds that one can findin different brands of EDs available for human consumptionin Brazil and according to the information provided in thelabel (ie caffeine and taurine concentrations in one popularbrand are 032 and 4mgmL resp) (II) that already invitro (Chinese hamster ovary cells (CHO cells)) doses ofaqueous extracts from guarana powder (Paullinia cupana)ranging from 10 to 40mgmL exhibited significant differencesin cytotoxicity [23] (III) that Bydlowski and colleagues(1988) [24] with the goal of finding new therapeutic uses ofguarana found that concentrations of 100mgmL of guaranadecreased thromboxane synthesis in blood platelets as wellas reversed and inhibited platelet aggregation and postulatedthat guarana could be useful in preventing cardiovasculardisease (IV) that any potential effects induced by the studiedconcentrations of guarana and caffeine in this and furtherexperiments of the current study could be compared since50mg of guarana (Lifar Ltd Porto Alegre RS Brazil) con-tains approximately 2mg of caffeine according to the manu-facturer (as indicated in Section 2)

Thus to perform such characterization we decided touse PCM analysis after short-term drug treatments (2 hours)and in order to look for further morphological features(changes in growth pattern cell morphology size granular-ity or any marker of interest) we consequently analyzed thesamples after longer treatments (medium-term treatment of 4hours) by using SEM FC (side scatter versus forward scatter)and CM

Under the cell culture conditions utilized for the expan-sion of our in vitro model (see Section 2) aggregates of SH-SY5Y cells are rare and they usually grow inmonolayers withldquoflattened-likerdquo phenotype (Figures 2(a)(1) and 2(b)(1)) withsome cytoplasmic projections (Figure 2(b)(2)) However inthe presence of low concentrations of guarana (3125mgmL)contiguous tracts of cells were often observed within theculture (Figures 2(a)(2) and 2(b)(3)) Also the membraneof some cells looked disrupted in a higher or lower grade(Figure 2(b)(4)) In some cells single or very few membraneblebs were detected (Figure 2(b)(4b)) at such concentration

On the other hand at higher concentrations of guarana (125and 50mgmL) the majority of the cells started to aggregateinto clusters and shrunk (arrows Figures 2(a)(3) and 2(a)(4))Membrane integrity did not look compromised but bothnumber and shape of the blebs were considerably increased(Figures 2(b)(5)ndash2(b)(8)) Moreover the presence of neuriticabnormalities after treatment with 125 or 50mgmL ofguarana was remarkable Treated cells (125 and 50mgmL)displayed neuritic swellings and shortened neurites (by eitherretraction or lack of growth) (arrowheads in Figures 2(a)(3)and 2(a)(4)) Since the predominant signatures of apoptosisin addition to membrane blebbing include the loss of cellvolume and higher granularity we performed FC analysis(SSC-Hversus FSC-H) after treating the cells for 4 hourswithincreasing concentrations of guarana Qualitatively increas-ing concentrations of guarana correlated with a decreasein cell size (FSC-H) and increased granularity (SSC-H)(Figure 2(c)) Thus in order to search for further evidence ofapoptotic process we immunostained the cells with a specificanticleaved caspase-3 antibody together with DAPI (nuclearstaining) and then analyzed the cells by confocal microscopyCleaved caspase-3 expression was detected in cells treatedwith 125mgmL of guarana for 4 hours (Figure 2(d)(3))

Treatments with 0125mgmL of caffeine for either 2 or 4hours did not show any clearmorphological alteration in SH-SY5Y cells (black and white arrows in Figures 3(a)(1) 3(a)(2)and 3(b)(1)ndash3(b)(4)) On the other hand cells exposed to05 and 2mgmL of caffeine (2 or 4 hours) exhibited neu-ritic swellings (black and white arrowheads Figures 3(a)(3)3(a)(4) and 3(b)(5)ndash3(b)(8a)) When cells were treated with2mgmL of caffeine one could also find few cells with mildsigns of membrane blebbing (Figure 3(b)(8b)) However nosignificant changes in size granularity or cleaved caspase-3 expression were observed when compared to control cells(Figures 3(c) and 3(d))

In contrast to guarana and caffeine taurine-treated cellsexhibited healthy neuritic processes with long cytoplasmicprojections (Figures 4(a) and 4(b)) without changes in cellshape or complexity (Figure 4(c))Neither blebs (Figure 4(b))nor cleaved caspase-3 positive cells were found (Figure 4(d))after taurine treatment

Even though guarana-(125 and 50mgmL) and caffeine-(05 and 2mgmL) treated cells showed neuritic swellingsCM analysis with 120573-III tubulin antibody (neuron-specificmarker) and PI (marker for cellular viability) highlightedsome differences Formation of pearl chain-like structuresalong the neurite was present in both treatments (Fig-ures 5(b)(3)ndash5(b)(5) and 5(c)(3)ndash5(c)(5)) However guarana-treated neurites looked shrunken and fragmented (Figures5(b)(4) and 5(b)(5)) when compared to caffeine-treated cells(Figures 5(c)(4) and 5(c)(5)) In contrast to caffeine and tau-rine the viability of guarana-treated cells was compromisedas shown in the figure with PI positivity (Figure 6)

32 Antioxidant Potential and Intracellular ROS Scaveng-ing in SH-SY5Y Cells after Treatments with Guarana Caf-feine Taurine or Their Combinations Many of the com-ponents present in EDs (eg taurine guarana or carni-tine) are known to exert antioxidant activity in different


3125

125

50

PCM (2 hours)

Control

3125

125

3125

125

50

Control

SEM (4 hours)SEM (4 hours)

50

(a) (b) (d)(c)

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K800600400200Control

Gua 3125

Gua 125

Gua 50

0

1 K8006004002000

1 K8006004002000

1 K8006004002000

(3)

(4)

(3)

(4)

FC (granularity versus size)

SSC-

HSS

C-H

SSC-

HSS

C-H

FSC-H

(2) (2)

(1)

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Con

trol

Gua

12

5G

ua 1

25

Caspase 3

CM (4 hours)

Con

trol

(1)(1)

(2)

(3)

(4)

(1)

(3)

(5)

(7)

(2)

(4)

(6)

(8)

Control

Figure 2 Guarana-induced morphological alterations in human neuronal SH-SY5Y cells (a) Phase contrast microscopy (PCM) of SH-SY5Ycells after treatments with different concentrations of guarana (3125 125 and 50mgmL) for 2 hours in FBS-free culture medium Scalebar represents 30 120583m (b) Scanning electron microscopy (SEM) of SH-SY5Y cells after treatments with different concentrations of guarana(3125 125 and 50mgmL) for 4 hours in FBS-free culture medium Scale bars represent 20 or 10 120583m (for times700 or times2000 resp) (c) Analysisof cellular complexity or granularity (side scatter SSC-H) and size (forward scatter FSC-H) by flow cytometry (FC) after treatment withdifferent concentrations of guarana (3125 125 and 50mgmL) for 4 hours in FBS-free culture medium (d) Confocal immunofluorescencemicroscopy (CM) of cleaved caspase-3 expression (green fluorescence) and DAPI (nuclei) on SH-SY5Y cells after treatment with 125mgmLof guarana for 4 hours in FBS-free culture medium Scale bars represent 10 120583m

biological systems [25ndash27] Thus we evaluated the nonen-zymatic antioxidant potential in vitro exerted after 2hours of treatment with different concentrations of guarana(3125ndash50mgmL) caffeine (0125ndash2mgmL) and taurine (1ndash16mgmL) by using the TRAP and TAR assays (Figure 7) Byutilizing theTRAPassay (which ismore related to the amountof the antioxidant) an increase of nonenzymatic antioxidant

potential was observed in guarana-treated cells at all testedconcentrations when compared to control cells ( lowastlowastlowast119875 lt0001) as shown in the graph by the decrease in the AUC(Figures 7(a)(2) and 7(a)(4)) These effects were maintainedduring approximately 120minutes of analysisMoreover TARanalysis (more related to scavenger capacity or quality ofthe antioxidant) also showed significant differences between


Control

0125

05

(1) (1)

(2)

(5)

(7)

(6)

(8)

(3)

(3)

(4)

(1)

(2)

(3)

(4)

(1)

(2)

(3)

(4)

(4)

(2)

2

PCM (2 hours)

Control Control

0125

05

2 2

0125

05

SEM (4 hours) SEM (4 hours)1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K800600400200Control

Caf 0125

Caf 05

Caf 2

0

1 K8006004002000

1 K8006004002000

1 K8006004002000


SSC-

HSS

C-H

SSC-

HSS

C-H

FSC-H

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Caspase 3

CM (4 hours)

Con

trol

Con

trol

Caf 0

5Ca

f 05

(a) (b) (d)(c)

Figure 3 Caffeine-induced morphological alterations in human neuronal SH-SY5Y cells (a) Phase contrast microscopy (PCM) of SH-SY5Ycells after treatments with different concentrations of caffeine (0125 05 and 2mgmL) for 2 hours in FBS-free culture medium Scale barrepresents 30120583m (b) Scanning electron microscopy (SEM) of SH-SY5Y cells after treatments with different concentrations of caffeine (012505 and 2mgmL) for 4 hours in FBS-free culture medium Scale bars represent 20 or 10 120583m (for times700 or times2000 resp) (c) Analysis of cellularcomplexity or granularity (side scatter SSC-H) and size (forward scatter FSC-H) by flow cytometry (FC) after treatments with differentconcentrations of caffeine (0125 05 and 2mgmL) for 4 hours in FBS-free culture medium (d) Confocal immunofluorescence microscopy(CM) of cleaved caspase-3 expression (green fluorescence) and DAPI (nuclei) on SH-SY5Y cells after treatment with 05mgmL of caffeinefor 4 hours in FBS-free culture medium Scale bars represent 10 120583m

control and guarana-treated cells when cells were exposedto either 125 ( lowast119875 lt 005) 25 ( lowast119875 lt 005) or 50mgmL( lowastlowastlowast119875 lt 0001) (Figure 7(a)(5))

At concentrations of 025 ( lowastlowast119875 lt 001) 05 ( lowastlowastlowast119875 lt0001) 1 ( lowastlowastlowast119875 lt 0001) and 2mgmL ( lowastlowastlowast119875 lt 0001) of caf-feine an increase of nonenzymatic antioxidant potential wasalso detected in the cells (Figure 7(b)(2)) which lasted forapproximately 30minutes In this case a significant difference

in the total antioxidant reactivity of caffeine-treated cells wasobserved at the highest concentration of 2mgmL ( lowastlowast119875 lt001) (Figure 7(b)(5))

Interestingly 2 hours of treatment with the lowest con-centration of taurine (1mgmL) seems to exert prooxidanteffects ( lowastlowast119875 lt 001) on SH-SY5Y cells (Figure 7(c)(2))In contrast higher concentrations of the drug (8 and16mgmL) increased the nonenzymatic antioxidant potential


Control

16

(8)

(5)

(1)

(6)

(2)

(2) (3)

(3)

(4)

(4)(4)

(1)

(2)

(3)

(1)

(4)

(2)

(3)

(1)

(7)

1

4

PCM (2 hours)

Control Control

1 1

16 16

4 4

SEM (4 hours)SEM (4 hours)1 K

800

600

400

200

01 K800600400200

Control

Tau 1

Tau 4

Tau 16

0

1 K8006004002000

1 K8006004002000

1 K8006004002000


SSC-

H

1 K

800

600

400

200

0

SSC-

H

1 K

800

600

400

200

0

SSC-

H

1 K

800

600

400

200

0

SSC-

H

FSC-H

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Caspase 3

CM (4 hours)

Con

trol

Con

trol

Tau

4Ta

u 4

(a) (b) (d)(c)

Figure 4 Taurine-induced morphological alterations in human neuronal SH-SY5Y cells (a) Phase contrast microscopy (PCM) of SH-SY5Y cells after treatments with different concentrations of taurine (1 4 and 16mgmL) for 2 hours in FBS-free culture medium Scalebar represents 30 120583m (b) Scanning electron microscopy (SEM) of SH-SY5Y cells after treatments with different concentrations of taurine (14 and 16mgmL) for 4 hours in FBS-free culture medium Scale bars represent 20 or 10 120583m (for times700 or times2000 resp) (c) Analysis of cellularcomplexity or granularity (side scatter SSC-H) and size (forward scatter FSC-H) by flow cytometry (FC) after treatment with differentconcentrations of taurine (1 4 and 16mgmL) for 4 hours in FBS-free culture medium (d) Confocal immunofluorescence microscopy (CM)of cleaved caspase-3 expression (green fluorescence) andDAPI (nuclei) on SH-SY5Y cells after treatment with 4mgmL of taurine for 4 hoursin FBS-free culture medium Scale bars represent 10 120583m

( lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 resp) Such effect was main-tained for approximately 30 minutes of analysis Thereafterno significant changes were detected (Figure 7(c)(4)) TARassay showed that exclusively 8mgmL of taurine was able toincrease the total antioxidant reactivity ( lowast119875 lt 005) on thecells (Figure 7(c)(5))

In order to determine the potential effect of these drugson the production of intracellular ROS we used the DCFH-DA assay Cells were treated for 2 hours with 125mgmLof guarana 05mgmL of caffeine 4mgmL of taurineand their different combinations (125mgmL of guarana +05mgmL of caffeine 125mgmL of guarana + 4mgmL

Oxidative Medicine and Cellular Longevity 9C

ontro

lC

ontro

l

Light

(2)

(3)

(4)

(5)(1)

3D

3D

PI

PI120573-III-tubulinDAPI

(a)

Gua

12

5G

ua 1

25

Light

3D

PI


(2)

(3)

(4)

(5)(1)

3D

(b)

Caf 0

5Ca

f 05

Light

3D

3D

PI


(2)

(3)

(4)

(5)(1)

(c)

Figure 5 Qualitative analysis of drug-induced neuritic morphology Confocal immunofluorescence microscopy (CM) of 120573-III-tubulinexpression (green fluorescence) propidium iodide (PI) (red fluorescence) and DAPI (nuclei) on human neuronal SH-SY5Y cells (a) aftertreatment with 125mgmL of guarana (b) and 05mgmL of caffeine (c) for 4 hours in FBS-free culturemediumWhite and black arrow heads(in 2D and 3D representations) mark the presence of neuritic swelling after guarana (125mgmL) and caffeine (05mgmL) treatments Scalebars represent 10120583m

of taurine 05mgmL of caffeine + 4mgmL of taurine1mgmLof caffeine + 4mgmLof taurine 125mgmLof gua-rana + 05mgmL of caffeine + 4mgmL of taurine) All thetested treatments significantly decreased ( lowastlowastlowast119875 lt 0001) thebasal levels of free radical generation (endpoint 2 hours)(Figure 8(b)) PCM analysis confirmed the presence of neu-ritic swellings (arrowheads in Figure 8(a)) when cells wereexposed to 125mgmL of guarana 05mgmL of caffeineand also combinations of guarana (125mgmL) with caffeine(05mgmL) andor taurine (4mgmL)

33 Significant Decrease of SOD and CAT Activities in SH-SY5Y Cells Exposed to Guarana and Its Combinations withCaffeine andor Taurine For screening any potential vari-ability in the antioxidant enzyme activity of SH-SY5Y cells

after 2 hours of drug treatment with guarana caffeinetaurine and their combinations the cellular activities ofSOD CAT GPx and GST were analyzed Guarana-treatedcells showed an exponential decrease in SOD activity (Figure9(a)(1)) at concentrations ranging from 3125 to 50mgmL(3125 and 625mgmL lowastlowast119875 lt 001 125 25 and 50mgmLlowastlowastlowast

119875 lt 0001) Statistical analysis did not reveal significantdifferences for caffeine- or taurine-treated cells (Figures9(b)(1) and 9(c)(1)) Combinations of caffeine andor tau-rine with 125mgmL of guarana (125mgmL of guarana +05mgmL of caffeine 125mgmL of guarana + 4mgmLof taurine 125mgmL of guarana + 05mgmL of caffeine+ 4mgmL of taurine) also induced a significant decreasein SOD activity in vitro (Figure 10(a)) Similar results wereobtained for CAT with significant decrease in the enzymatic


ControlPI

PI120573

-III

-tubu

linD

API

(1)

(2)

(a)

PIPI

120573-I

II-tu

bulin

DA

PI

Gua 125(1)

(2)

(b)PI

PI120573

-III

-tubu

linD

API

Caf 05(1)

(2)

(c)

PIPI

120573-I

II-tu

bulin

DA

PI

Tau 4(1)

(2)

(d)

Figure 6 Qualitative analysis of drug-induced changes on cellular viability Confocal immunofluorescence microscopy (CM) of propidiumiodide (PI) (red fluorescence) 120573-III-tubulin expression (green fluorescence) and DAPI (nuclei) on human neuronal SH-SY5Y cells (a) aftertreatment with 125mgmL of guarana (b) 05mgmL of caffeine (c) and 4mgmL of taurine (d) for 4 hours in FBS-free culture mediumScale bars represent 10120583m

activity at concentrations of guarana ranging from 125 to50mgmL ( lowastlowast119875 lt 001) (Figure 9(a)(2)) but no significantdifferences were detected for both caffeine- and taurine-treated cells (Figures 9(b)(2) and 9(c)(2)) Again combina-tions of caffeine andor taurine with 125mgmL of guarana(125mgmL of guarana + 05mgmL of caffeine 125mgmLof guarana + 4mgmL of taurine 125mgmL of guarana +05mgmL of caffeine + 4mgmL of taurine) also decreasedCAT activity in the cells (Figure 10(b))

Analyses of GPx and GST activities under the sameconditions did not show significant variations except for adecrease in cellular GPx activity at 50mgmL of guaranatreatment (Figures 9(a)(3) 10(c) and 10(d)) Statistical anal-ysis of SODCAT + GPx ratio by ANOVA followed by Dun-nettrsquos multiple comparison test did not reveal any imbalance(data not shown)

34 Guarana Protects SH-SY5Y Cells against Oxidative Alter-ations of Proteins and Lipids It is well known that proteinthiols can maintain the cellular redox status by their cysteinecontent [28] Lipid peroxidation on the other hand is oneof the main causes of free radical-mediated damage tocellular membranes [28] Therefore we investigated the invitro effects of these drugs and their combinations againstoxidative alterations in both proteins and lipids Our resultsshowed that only guarana treatment protected against lipidperoxidation (125ndash50mgmL lowast119875 lt 005) (Figure 9(a)(6))Though ANOVA followed by Dunnettrsquos multiple comparisontest did not show significant differences in lipid peroxidationwhen cells were treated with the combination of guarana(125mgmL) caffeine (05mgmL) and taurine (4mgmL)(Figure 10(f)) the Studentrsquos 119905-test revealed on the other hand

a significant difference when compared to control cells (119875 =00162 statistical significance when 119875 lt 005) Additionallyconcentrations of guarana ranging from 625 to 50mgmLand all combinations where this drug was present decreased( lowast119875 lt 005 and lowastlowast119875 lt 001) the total reduced thiol content(Figures 9(a)(5) and 10(e)) demonstrating protective effectsagainst oxidative alterations of proteins

35 Increasing Concentrations of Guarana and Its Combi-nations with Caffeine andor Taurine Decrease the Numberof Viable SH-SY5Y Cells by Elevating the Percentage of LateApoptotic Cells For elucidating the drug-induced effects onthe cellular number cells were seeded in 6-well plates 48hours later treatments were performed in the presence of10 FBS-DMEM F12 medium for 24 hours Then cellswere collected and counted by using a flow cytometer Anadditional experimental control (control 1) was utilized (andcompared to control 2) to exclude any potential interferencetriggered by FBS replacement on the cell number Our resultsshowed a significant reduction ( lowastlowastlowast119875 lt 0001) in the totalcell count with increasing concentrations of the guarana aswell as combinations of guarana with caffeine andor taurine(Figure 11(a)) A similar pattern was observed in caffeine-treated cells especially with 2mgmL ( lowastlowastlowast119875 lt 0001)Taurine significantly reduced the total number of cells atconcentrations of 1 and 4mgmL ( lowastlowastlowast119875 lt 0001 andlowastlowast

119875 lt 001 resp) but not 16mgmL Representative PCMmicrographs of the cells were taken before proceeding withflow cytometric analysis (Figure 11(b)) Signs of generalizedcell death were observed in guarana-treated cells (Figures11(b)(3)ndash11(b)(5)) or its combinations with caffeine andortaurine (Figures 11(b)(12) 11(b)(13) and 11(b)(16)) In these


15

10

5

0

Time (min) Time (min)

TRAP TRAP

TAR

Con

trol

312

5

625

125 25 50

Con

trol

312

5

625

125 25 50

Control3125625

1252550

Control3125625

1252550

3125 625 125 25 500

120

100

80

60

40

20

0

120

100

80

60

40

20

0

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(mgmL)

(mgmL)

(mgmL)

lowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast lowastlowastlowast lowastlowastlowast

Gua

rana

120

90

60

30

00 5 10 15 20 25 30

Gua

(mg

mL)

200

150

100

50

00 30 60 90 120

Radi

cal p

rodu

ctio

n (

)

Gua

rana

(1)

(2)

(3)

(4)

(5)

TAR

(1198680119868

)

(a)

120

100

80

60

40

20

0

120

90

60

30

00 5 10 15 20 25 30


TRAPTRAP

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

Con

trol

012

5

025 0

5 1 2

(mgmL)

Con

trol

012

5

025 0

5 1 2

0125 025 05 1 20

(mgmL)

Control0125025

0512

Control0125025

0512

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

120

100

80

60

40

20

0

TAR

5

4

3

2

1

0

(mgmL)

Caffe

ine

Caffe

ine

TAR

(1198680119868

)

Radi

cal p

rodu

ctio

n (

)Pe

rcen

tage

of A

UC

(in re

latio

n to

cont

rol)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(1)

(2)

(3)

(4)

(5)

(b)

Figure 7 Continued


TAR

(1198680119868

)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Radi

cal p

rodu

ctio

n (

)

120

90

60

30

00 5 10 15 20 25 30

Time (min)

Con

trol 1 2 4 8 16

(mgmL)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Con

trol 1 2 4 8 16

120

100

80

60

40

20

0

(mgmL)

Time (min)

TRAPTRAP

Control12

4816

Control12

4816

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

TAR

1 2 4 8 160(mgmL)

5

4

3

2

1

0

Taur

ine

Taur

ine

160140120100

80604020

0

(1)

(2)

(3)

(4)

(5)

(c)

Figure 7 Total reactive antioxidant potential (TRAP) and total antioxidant reactivity (TAR) on human neuronal SH-SY5Y cells exposed tomajor components of EDs TRAP and TAR measurements on guarana-treated cells (2 hours) at concentrations of 3125 625 125 25 and50mgmL (a) caffeine-treated cells (2 hours) at concentrations of 0125 025 05 1 and 2mgmL (b) and taurine-treated cells (2 hours) atconcentrations of 1 2 4 8 and 16mgmL (c) TRAP is shown as a representative graphic of the area under the curve (AUC) for 2 hoursand TAR is calculated as the ratio of light in the absence of samples (119868

0)light intensity right after sample addition (119868) Values are expressed

as mean plusmn SEM of three independent experiments (119899 = 3) Statistical difference compared to control was determined by one-way ANOVAfollowed by Dunnettrsquos multiple comparison test ( lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

cultures cells seem to have lost the typical neuronal pheno-type and characteristic neuritic processes of SH-SY5Y cellsbecame rounded and thereafter lost their viability Mildersigns of the same morphological events were also observedin cells treated with 2mgmL of caffeine (Figure 11(b)(8)) Onthe contrary taurine (1 4 and 16mgmL) did not induce anymorphological sign of cell death after 24 hours of treatment(Figures 11(b)(9)ndash11(b)(11))

In order to assess the type of cell death induced by thesemajor components of EDs flow cytometric analyses withAnnexin V and PI were performed after 4 hours of treatmentBoth qualitative and quantitative data were obtained and thefraction (in ) of live (Q4) early apoptotic (Q3) late apo-ptotic (Q2) and necrotic (Q1) cells was given after counting10000 cells per case (Figure 12) Our results show thattreatments with increasing concentrations of guarana and itscombinations with caffeine andor taurine decrease the per-centage of viable cells (Figures 12(b)(1)ndash12(b)(3) 12(c)(1)12(c)(2) and 12(c)(5)) when compared to control cells (un-treated) (Figure 12(a)) Interestingly it seems that higherconcentrations of guarana (125 and 50mgmL) enrich the

fraction of cells undergoing late apoptosis (from 798 to952 resp) from the total percentage of unviable cells(Figure 12(d))

36 In Silico Development and Topological Analysis of aNetworkModel for the Interactions of ED Components throughRedoxNitric Oxide (NO) and Apoptotic Pathways (MEDRIModel) Highlight ldquoHydroxyl Radicalsrdquo as the Node with theHighest Stress Many of the components present in EDs (egtaurine guarana or carnitine) are known to exert antioxi-dant activity in different biological systems [25ndash27] Basedon this fact and the previous evidence of apoptosis andorneurite degeneration we decided to establish an in silicoevaluation of the general landscape of interactions betweenED components as well as redoxNO and apoptosis-relatedproteins by using systems biology tools For instance basedon the network centralities (eg connectivity and stress) itis virtually possible to identify ldquoessentialrdquo nodes in a newlydeveloped interaction network [29 30] Targeting essentialnodes with high centrality values can considerably disruptthe whole network integrity Moreover high stress value for


(1) (2)

(4) (5)

(9)(7) (8)

(6)

(3)

30 120583m

Gua 125 + Caf 05 Gua 125 + Tau 4

Gua 125 + Caf 05 + Tau 4

Control Gua 125 Caf 05

Tau 4

Caf 05 + Tau 4 Caf 1+ Tau 4

SH-S

Y5Y

cells

0

FBS

+co

mbi

ning

trea

tmen

t (m

gm

L 2

hour

s)

PCM

(400

x)

(a)

500

100

80

60

40

20

0

Intr

acel

lula

r RO

S pr

oduc

tion

( in

relat

ion

to co

ntro

l)

Con

trol

Gua

12

5

Caf 0

5

Tau

4

2 hours(mgmL)

H2O2

(1 m

Moplus

cont

rol)

lowastlowastlowast

lowastlowastlowastDCFH-DA

Gua

125+

Caf0

5

Gua

125+

Tau4

Caf0

5+

Tau4

Caf1

+Ta

u4

Gua

12

5+

Caf 0

5+

Tau

4

(b)

Figure 8 Morphological alterations of human neuronal SH-SY5Y cells treated with drug combinations (guarana caffeine and taurine) andeffects on the intracellular production of reactive oxygen species (ROS) (a) Phase contrast microscopy (PCM) of human neuronal SH-SY5Ycells after treatments with guarana (125mgmL) caffeine (05mgmL) taurine (4mgmL) and their combinations for 2 hours in FBS-freeculturemedium Scale bar represents 30 120583m (b) Intracellular ROS productionwas examined by incubating the cells for 2 hours with 100 120583MofDCFH-DA dissolved in 1 FBS-containing culture mediumThen medium was discarded and human neuronal SH-SY5Y cells were treatedwith H

2O2(1mM positive control for intracellular ROS production) guarana (125mgmL) caffeine (05mgmL) taurine (4mgmL) and

their combinations for 2 hours Finally DCF fluorescence was read (endpoint 2 hours) at 37∘C in a fluorescence plate reader (Spectra MaxM2 Molecular Devices USA) with an emission wavelength set at 535 nm and an excitation wavelength set at 485 nm Values are expressedas mean plusmn SEM of three independent experiments (119899 = 3) Statistical difference compared to control was determined by one-way ANOVAfollowed by Dunnettrsquos multiple comparison test ( lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

a given node can be an indicator of relevance of a pro-teincompound within a biological network and it is com-monly used tool in computational biology-based studies [3132]

Our in silico analysis gave rise to the MEDRI networkmodel (Figure 13(a)) where 87 apoptosis-related and 57redoxNO-related proteins interconnected with 12 ED com-ponents linked through 4 additional compounds hydroxylradicals molecular oxygen hydrogen peroxide and nitricoxide

Topological network properties (connectivity neighbor-hood connectivity stress and clustering coefficient) of thenodes (proteins and compounds) highlighted ldquohydroxyl rad-icalsrdquo as a highly interconnected node with the highest valueof stress centrality in the interaction model (MEDRI) (Figure13(b) and Supporting Information Table S4)

4 Discussion

Even though caffeine is considered themain active ingredientin EDs the presence of other substances such as taurineB vitamins carnitine and diverse herbal derivatives shouldbe considered since both acute and long-term effects of itscombined consumption with caffeine and related methylxan-thines are not well known

Here we used human neuronal SH-SY5Y cells to per-form morphological and cytotoxic evaluations of the mostcommon components of EDs (caffeine taurine and guarana)alone or in combination SH-SY5Y cell line is an immor-talized cell culture system with obvious limitations for datainterpretation mainly due to the differences in cell cycle stateand expression levels of neuronal markers that are observedin mature neurons However this and other catecholaminer-gic cell lines are widely used for toxicological studies at thecellular and molecular levels due to inherent advantages inrelation to primary neuronal culture systems for instancethe human origin of these cells high cellular homogeneity(rodent primary dopaminergic culture systems present only5of tyrosine hydroxylase-positive cells) and the presence ofmany biochemical and functional characteristics of neuronsthat are easily measurable and detectable such as neuriteextension 120573-III-tubulin and tyrosine hydroxylase expres-sion as well as catecholamine production and secretionMoreover these cells exhibit cellular responses to cytotoxiccompounds that are similar to those found in human primarycultures [10 33ndash35]

In general ROS are able to react with diverse cellularcomponents (eg DNA carbohydrates proteins and lipids)in a destructive way These entities include free radicals(superoxide nitric oxide and hydroxyl radicals) as well


50

40

30

20

10

0

03

125

625

125 25 50

03

125

625

125 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

Gua

rana

U S

OD

mg

prot

ein

SOD CAT

Antioxidant enzyme activity Antioxidant enzyme activity Antioxidant enzyme activity

GPx

GST

(1) (2) (3)

(4) (5) (6)

Oxidative damageOxidative damage

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowast

lowastlowast

lowastlowastlowastlowast

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

Gua

rana

U G

STm

g pr

otei

n

8

6

4

2

0

04

03

02

01

00

SH TBARS

lowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastTB

ARS

(nm

ol T

MP

mg

prot

ein)

75

50

25

0

020

015

010

005

000

Antioxidant enzyme activity120583

mol

SH

mg

prot

ein

(mgmL) (mgmL) (mgmL) 2 hours

(a)

50

40

30

20

10

0

00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2 00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2

Caffe

ine

U S

OD

mg

prot

ein

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

Caffe

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000


120583m

ol S

Hm

g pr

otei

n

Antioxidant enzyme activity Antioxidant enzyme activity Antioxidant enzyme activity(1) (2) (3)

(4) (5) (6)Oxidative damageOxidative damageAntioxidant enzyme activity

SOD CAT GPx

GST SH TBARS

(b)

Figure 9 Continued


50

40

30

20

10

0

Taur

ine

U S

OD

mg

prot

ein

0 1 2 4 8 16

0 1 2 4 8 16

0 1 2 4 8 16 0 1 2 4 8 16

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

(mgmL)

0 1 2 4 8 16

(mgmL)

0 1 2 4 8 16

(mgmL) 2 hours

Taur

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000

120583m

ol S

Hm

g pr

otei

n


(1) (2) (3)


SOD CAT GPx

GST SH TBARS

(c)

Figure 9 Antioxidant enzyme activity and oxidative damage parameters on human neuronal SH-SY5Y cells exposed to major componentsof EDs Enzymatic activity (SOD CAT GPx and GST) SH and TBARS measurements on guarana-treated cells (2 hours) at concentrationsof 3125 625 125 25 and 50mgmL (a) caffeine-treated cells (2 hours) at concentrations of 0125 025 05 1 and 2mgmL (b) and taurine-treated cells (2 hours) at concentrations of 1 2 4 8 and 16mgmL (c) Values are expressed as mean plusmn SEM of three independent experiments(119899 = 3) Statistical difference compared to control was determined by one-way ANOVA followed by Dunnettrsquos multiple comparison test( lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

as other molecular species (hydrogen peroxide and per-oxynitrite) In neurodegenerative processes it is believedthat neurological impairment is intrinsically linked to ROS-triggered neuronal apoptosis [28] Here in vitromorphologi-cal analyses showed clear signs of apoptosis such membraneblebbing cell shrinkage and cleaved caspase-3 positive cellsin guarana-treated cells (125ndash50mgmL) after 2ndash4 hours oftreatment (Figure 2) Then we characterized the completeredox profile of SH-SY5Y cells after treatments with ED com-ponents in order to see how changes in ROS nonenzymaticand enzymatic antioxidant activity and oxidative alterationsin proteins andor lipidsmay correlate with potential changesin the cellular viability Our analysis of different oxidativestress-related parameters showed strong antioxidant effectsinduced in SH-SY5Y cells when treatedwith ED componentsespecially with guarana and its combinations with caffeineandor taurine which are consistent with the strong invitro antioxidant potential exerted by guarana and observedin TRAP and TAR measurements (Figure 7) The decreaseof the basal levels of free radical generation after thesetreatments (Figure 8(b)) may result in down-regulation ofthe cellular enzymatic antioxidant defense (eg SOD and

CAT activities) (Figure 10) The concomitant loss of viabil-ity and induction of apoptotic parameters in cells underthe same treatments (Figures 11 and 12) indicate that thisstrong antioxidant effect is responsible at least in partfor the in vitro cytotoxic effect exerted by ED compo-nents

Although induction of cellular necrosis and apoptosis isgenerally associated with prooxidant conditions leading tooxidative stress it must be pointed out that physiologicalROS levels exert essential roles in the maintenance of cellularhomeostasis The regulation of protein activity by oxidationand reduction of lateral chains in certain residues has beenrecognized as one of the most important mechanisms of cellfunction regulation together with protein phosphorylation[28] Since ROS regulate themitogen-activated protein kinase(MAPK) and the phosphoinositide 3-kinase (PI3 K) [36] it isvery likely that excessive intracellular ROS scavenging afterexposure to drugs with high antioxidant potential (alone orin combinations) may disrupt the cellular redox homeostasisand thus proliferation and survival-related signaling path-ways Such a concept where a strong antioxidant-derivedredox imbalance can be as dangerous as oxidative stress


SOD50

40

30

20

10

0

U S

OD

mg

prot

ein

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowast

(a)

CAT4

3

2

1

0

U C

ATm

g pr

otei

n

lowastlowast lowastlowast lowastlowastlowast

(b)

GPx8

6

4

2

0

U G

Pxm

g pr

otei

n

(c)

GST04

03

02

01

00

U G

STm

g pr

otei

n

(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(d)

SH

lowastlowastlowastlowast lowastlowastlowast

(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

75

50

25

0

120583m

ol S

Hm

g pr

otei

n

(e)

TBARS

lowast

(mgmL)

020

015

010

005

000

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(f)

Figure 10 Antioxidant enzyme activity and oxidative damage parameters on human neuronal SH-SY5Y cells treated for 2 hours with drugcombinations (guarana caffeine and taurine) Measurements for SOD (a) CAT (b) GPx (c) GST (d) SH (e) and TBARS (f) at different drugconcentrations (mgmL) are shown Values are expressed as mean plusmn SEM of three independent experiments (119899 = 3) Statistical differencecompared to control was determined by one-way ANOVA followed by Dunnettrsquos multiple comparison test ( lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt0001)

is already known in the literature as ldquoantioxidative stressrdquo[37] (Figure 13) As a matter of fact in vitro induction ofcellular apoptosis associated with drug antioxidant activity isalready known for a number of substances (eg flavonoidspolyphenols and saponins) [38ndash40] Therefore the conceptof antioxidative stress would be particularly relevant inthe case of excessive consumption of nutritional supple-ments or beverages (like EDs) where diverse substanceswith antioxidant potential are mixed together at relativelyhigh concentrations For instance the Brazilian native plantguarana traditionally used as stimulant and aphrodisiac [41]has already been shown to exert antibacterial and antioxidantactivities in 3T3-L1 cells [25] One of its major componentscaffeine is an antagonist of adenosine receptors able to inhibitphosphodiesterase (PDE) GABA receptor-mediated effects[42 43] and has been suggested to have a role in preventingage-associated decline in cognitive function after its chronicingestion through the protection of the antioxidant systemin vivo [44] Finally taurine (2-aminoethanesulfonic acid) isa sulfur amino acid commonly found in electrically excitabletissues such as heart or brain [45] which is known to exertother diverse effects in organs and tissues like modulation ofcalcium levels stabilization of membranes and antioxidantand antiapoptotic effects able to improve wound healing [2745ndash47]

In this study we postulated that combined effect of exces-sive depletion of ROS-mediated cellular signaling togetherwith caffeine-mediated induction of apoptosis [48] wouldlead SH-SY5Y cells to lose their viability and undergo apop-tosis after guarana treatments alone or in combination withadditional caffeine andor taurine doses In fact inductionof apoptosis in SH-SY5Y cells by other classical antioxidantcompounds (eg sodium ascorbate or vitamin C) is alreadyknown [49]

It has been described that 24-hour treatment with lowconcentrations of caffeine induces p53-dependent apopto-sis in JB6 cells through the Bax and caspase-3 pathways[48] Additionally it is known that 1ndash10mM of caffeinetriggers calcium release from intracellular stores througha caffeine-sensitive ryanodine receptor and such increasecould lead to cell death in the central nervous system[50 51] Furthermore calcium itself could directly activatecaspases (eg caspase-3) or even proteases having a role inthe degeneration of neurofilaments [52ndash54] In our study24-hour treatment with 2mgmL of caffeine significantlydecreased the total cell number (Figure 11(a)) and inducedtypical morphological signs of decreased viability in vitro(Figure 11(b)(8)) Our data also showed signs of neuritedegeneration in the form of swellings at various segmentsin a beaded or pearl chain-like structure together with


Con

trol (

1)

Con

trol (

2)

(1) Without 10 FBS-medium replacement(2) With fresh 10 FBS medium

Gua

31

25

Gua

12

5

Gua

50

Caf 0

125

Caf 0

5

Caf 2

Tau

1

Tau

4

Tau

16

Gua

12

5+

Caf 0

5

Gua

12

5+

Tau

4

Caf 0

5+

Tau

4

Caf 1

+Ta

u 4

Gua

12

5+

Caf 0

5+

Tau

4

Drug(s) treatment with fresh 10 FBS medium (24 hours)

Tota

l cel

l cou

nt (times

106)

20

15

10

05

00

Control

Guarana Caffeine Taurine Combinations

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowastlowastlowast lowastlowast lowastlowast lowastlowastlowast

(mgmL)

(a)

PCM

(200

x)

Control (1) Control (2) Gua 3125 Gua 125

Gua 50 Caf 0125 Caf 05 Caf 2

Tau 1 Tau 4 Tau 16 Gua 125 + Caf 05

Gua 125 + Tau 4 Caf 05 + Tau 4 Caf 1 + Tau 4 Gua 125 + Caf 05 + Tau 4

(1) (3)(2) (4)

(5)

(10)(9) (11) (12)

(13)

(6) (8)(7)

SH-S

Y5Y

cells

10

FBS

+dr

ug(s

) tre

atm

ent (

mg

mL

24 h

ours

)

(14) (15) (16)

(b)

Figure 11 Analysis of drug-induced long-term effects on cell number (a) Flow cytometric analysis (FC) of the total number of humanneuronal SH-SY5Y cells after treatments with guarana (3125 125 and 50mgmL) caffeine (0125 05 and 2mgmL) taurine (1 4 and16mgmL) and their combinations for 24 hours in 10 FBS-containing culture medium Values are expressed as mean plusmn SEM of threeindependent experiments (119899 = 3) Statistical difference compared to control (1) was determined by one-way ANOVA followed by Dunnettrsquosmultiple comparison test ( lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001) (b) Phase contrast microscopy (PCM) of human neuronal SH-SY5Y cellsafter treatments with different concentrations of guarana (3125 125 and 50mgmL) caffeine (0125 05 and 2mgmL) taurine (1 4 and16mgmL) and their combinations for 24 hours in 10 FBS-containing culture medium Scale bar represents 30120583m


104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)

FC (PI versus Annexin V)

SH-SY5Y cells

Viable necrotic and apoptotic cells

Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)

Early apoptosisEarly necrosislate apoptosisLate necrosis

Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4

0 FBS + treatment (mgmL 4 hours)

Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4

Gua 125 + Caf 05 Gua 125 + Tau 4 Caf 05 + Tau 4 Caf 1 + Tau 4 Gua 125 + Caf 05 +Tau 4

Figure 12 Analysis of drug-induced effects on cellular viability Flow cytometric analysis (FC) of the fraction () of viable apoptotic andnecrotic human neuronal SH-SY5Y cells by propidium iodide (PI) versus AnnexinV after treatments with different concentrations of guarana(3125 125 and 50mgmL) caffeine (0125 05 and 2mgmL) taurine (1 4 and 16mgmL) and their combinations for 4 hours

neurite fragmentation in guarana-treated cells (125ndash50mgmL providing approximately 05 and 2mgmL of caffeineresp) as well as cells treated with combinations of guaranawith caffeine andor taurine (Figures 2 5 and 8(a)) Caffeine-treated cells did not exhibit fragmented neurites but thepresence of neuritic pearl chain-like structures was indeedabundant especially with 2mgmL (Figures 3 and 5) Thesestructures are similar to those found in a wide variety

of neurological diseases as axonal defects (and impairedaxonal transport) in the form of swellings that accumulateexcessive amounts of proteins organelles and vesicles [55] Incontrast healthy neuritic processes were observed in taurine-treated cells (1 to 16mgmL) as shown by PCM SEM and120573-III-tubulin immunofluorescence staining (Figures 4 and6) and confirmed after long-term taurine treatments (24hours) (Figure 11(b)) Interestingly in vitro taurine-derived


NodesCompoundProtein

EdgesReactionBindingPhenotypeNot specified

(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)

Figure 13 Model for the interactions of ED components through redoxNO and apoptotic pathways (a) In silico analysis of the interactionsof ED components through redox and NO pathways based on experimental data and database gave rise to a networkmodel (MEDRI) whereexactly 16 compounds (12 ED components hydrogen peroxide hydroxyl radicals molecular oxygen and nitric oxide) and 144 proteins(87 apoptosis-related and 57 redoxNO-related proteins) interconnected considering their potential interaction through either ldquoactivationrdquoldquoinhibitionrdquo ldquocatalysisrdquo ldquobindingrdquo or ldquoreactionrdquo (b) Analysis of the topological network properties by Cytoscape and its 2D projection byViaComplex Software show in a color-grading representation the areas of maximum values (the closer to red color the higher value) ofconnectivity neighborhood connectivity stress and clustering coefficient of each node

neuronal differentiation and its remarkable neuroprotectiverole in cerebellar granule cells have already been demon-strated and the mechanism would involve the regulation ofcytoplasmic free calcium and intramitochondrial calciumhomeostasis [56 57] This might explain the apparentdecrease in percentage of late apoptotic cells in taurine com-bination with guarana when compared to guarana-treatedcells and the absence of neuritic swellings in cells treatedwith 05mgmL of caffeine together with 4mgmL of taurine(Figure 8(a))

Perhaps the large number and mixture of active com-pounds in EDs at relatively high concentrations may rep-resent the biggest obstacle for designing efficient exper-imental plans to determine any potential toxicity at themolecular level of these beverages Nowadays systems biol-ogypharmacologytoxicology offers a number of computa-tional tools that allow researchers to work with large amountsof experimental knowledge and databases for elaboratingsimulations or supporting hypotheses that can be laterconfirmed in other models (eg in vivo or in vitro) [11 3158 59]

In this study our strategy consisted in developing anetwork model of interactions between ED components(Supporting Information Table S3) and proteins belonging to

the routes of interest in this case redoxNO and apoptoticpathways

Text mining or text analytics for constructing our in silicomodel were avoided whichmeans that instead of discussionsor hypotheses from the literature exclusively experimentaldata and databases were used as sources of information(from STRING 90 and STITCH 30) for constructing theMEDRI network Interestingly enough proteins of redoxNOand apoptotic pathways interconnected with ED compo-nents (Figure 13(a)) through reaction with highly stressedand connected (topologically speaking) ldquohydroxyl radicalsrdquoand ldquohydrogen peroxiderdquo nodes (Supporting InformationTable S4 and Figures 13(b)(1) and 13(b)(3)) in this model(Figure 13(a)) strongly supporting a redox component in themodulation of cell survivaldeath by ED components

5 Conclusion

Despite the limitations of SH-SY5Y cells as an in vitromodel our results suggest that guarana and caffeine aloneor in combination with taurine may exert neurotoxicologicaleffects in part by disruption of redox homeostasis (Figure 14)which is also consistent with the data obtained in the in silicoapproach (Figure 13)


Low

ldquoAntioxidativerdquo stress Oxidative stressOxidative stress

Endogenousantioxidant

defenses

Exogenoussources of stress

Consumption ofexogenous

antioxidants

Endogenoussources of stress

Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)

Homeostasis Disruptedphysiology

Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS

eg substanceintoxication lack ofoxygen and extreme

temperatures

eg SOD CATGPx and GST

eg syntheticantioxidants and

antioxidantbeverages (EDs)

eg lack of sleepdaily stress and

emotional shocks

Cellulardamage

Cell proliferationand survival

- Energy drink consumption and the concept of redox homeostasis

+

minus

Figure 14 EDs and the concept of redox homeostasis In an individual organism homeostasis is required for healthy development andnormal tissue functioning In our daily life one can be exposed to diverse sources of stress (endogenous andor exogenous) able to impair thephysiology of developing brains (eg during pregnancy children or adolescents) In these cases our antioxidant defenses shouldmaintain thehomeostatic levels of ROS in order to avoid an oxidative stress scenario Paradoxically the relevant role of ROS in regulating cell proliferationand survival signaling pathways is already knownTherefore an excessive antioxidant-derived redox imbalance (ldquoantioxidativerdquo stress) wouldalso impair homeostasis becoming as dangerous as oxidative stress itself In our study we show in vitro evidence that highlights the dangerof energy drink consumption and how their potential cytotoxicity could be partially attributed to the excessive depletion of intracellular ROSand disruption of redox homeostasis due to the combination of multiple components with antioxidant activity in these beverages

We believe that our results highlight the need for furtherin vivo and epidemiological studies to unravel the potentialfor serious adverse health effects of ED consumption

Abbreviations

EDs Energy drinksGua GuaranaCaf CaffeineTau TaurinePCM Phase contrast microscopySEM Scanning electron microscopy

CM Confocal microscopyFC Flow cytometry

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by the Brazilian researchfunding agencies FAPERGS (PqG 1008860 PqG 1008857ARD111893-7 and PRONEX 1000274) CAPES (PROCAD


0662007) CNPq PROPESQ-UFRGS and IBN-Net no01060842-00 Both SEM and CM were performed at theCenter of Electron Microscopy (CME) from Federal Univer-sity of Rio Grande do Sul (UFRGS) The authors thank MsMoema Queiroz and Mr Henrique Biehl for their technicalassistance at the CME

References

[1] C J Reissig E C Strain andR R Griffiths ldquoCaffeinated energydrinks a growing problemrdquo Drug and Alcohol Dependence vol99 no 1ndash3 pp 1ndash10 2009

[2] S M Seifert J L Schaechter E R Hershorin and S E Lip-shultz ldquoHealth effects of energy drinks on children adolescentsand young adultsrdquo Pediatrics vol 127 no 3 pp 511ndash528 2011

[3] N Gunja and J A Brown ldquoEnergy drinks health risks andtoxicityrdquoMedical Journal of Australia vol 196 no 1 pp 46ndash492012

[4] J P Higgins T D Tuttle and C L Higgins ldquoEnergy beveragescontent and safetyrdquo Mayo Clinic Proceedings vol 85 no 11 pp1033ndash1041 2010

[5] C Alford H Cox and R Wescott ldquoThe effects of Red BullEnergyDrink on human performance andmoodrdquoAminoAcidsvol 21 no 2 pp 139ndash150 2001

[6] C A Marczinski M T Fillmore M E Bardgett and M AHoward ldquoEffects of energy drinksmixedwith alcohol on behav-ioral control risks for college students consuming trendy cock-tailsrdquo Alcoholism Clinical and Experimental Research vol 35no 7 pp 1282ndash1292 2011

[7] S J P Iyadurai and S S Chung ldquoNew-onset seizures in adultspossible association with consumption of popular energydrinksrdquo Epilepsy and Behavior vol 10 no 3 pp 504ndash508 2007

[8] A M Arria and M OrsquoBrien ldquoThe ldquohighrdquo risk of energy drinksrdquoThe Journal of the American Medical Association vol 305 no 6pp 600ndash601 2011

[9] J L Temple ldquoCaffeine use in children what we know what wehave left to learn and why we should worryrdquo Neuroscience andBiobehavioral Reviews vol 33 no 6 pp 793ndash806 2009

[10] L Agholme T Lindstrom K Kgedal J Marcusson and MHallbeck ldquoAn in vitro model for neuroscience differentiationof SH-SY5Y cells into cells with morphological and biochem-ical characteristics of mature neuronsrdquo Journal of AlzheimerrsquosDisease vol 20 no 4 pp 1069ndash1082 2010

[11] T K Rabelo F Zeidan-Chulia L M Vasques et al ldquoRedoxcharacterization of usnic acid and its cytotoxic effect on humanneuron-like cells (SH-SY5Y)rdquo Toxicology in Vitro vol 26 no 2pp 304ndash314 2012

[12] M L da Frota Junior A S Pires F Zeidan-Chulia et al ldquoInvitro optimization of retinoic acid-induced neuritogenesis andTH endogenous expression in human SH-SY5Y neuroblastomacells by the antioxidant Troloxrdquo Molecular and Cellular Bio-chemistry vol 358 pp 325ndash334 2011

[13] W H Habig M J Pabst and W B Jakoby ldquoGlutathione S-transferasesThefirst enzymatic step inmercapturic acid forma-tionrdquo Journal of Biological Chemistry vol 249 no 22 pp 7130ndash7139 1974

[14] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[15] D Szklarczyk A Franceschini M Kuhn et al ldquoThe STRINGdatabase in 2011 functional interaction networks of proteins

globally integrated and scoredrdquo Nucleic Acids Res vol 39 sup-plement 1 pp D561ndashD568 2011

[16] H M Wain M J Lush F Ducluzeau V K Khodiyar and SPovey ldquoGenew the human gene nomenclature database 2004updatesrdquo Nucleic Acids Research vol 32 pp D255ndashD257 2004

[17] E Birney D Andrews M Caccamo et al ldquoEnsembl 2006rdquoNucleic Acids Research vol 34 pp D556ndashD561 2006

[18] MKuhn C vonMeringMCampillos L J Jensen and P BorkldquoSTITCH interaction networks of chemicals and proteinsrdquoNucleic Acids Research vol 36 no 1 pp D684ndashD688 2008

[19] S D Hooper and P Bork ldquoMedusa a simple tool for interactiongraph analysisrdquo Bioinformatics vol 21 no 24 pp 4432ndash44332005

[20] M E Smoot K Ono J Ruscheinski P L Wang and T IdekerldquoCytoscape 28 new features for data integration and networkvisualizationrdquo Bioinformatics vol 27 no 3 pp 431ndash432 2011

[21] M A A Castro J L Rybarczyk Filho R J S Dalmolin et alldquoViaComplex software for landscape analysis of gene expres-sion networks in genomic contextrdquo Bioinformatics vol 25 no11 pp 1468ndash1469 2009

[22] O H Lowry N J Rosebrough A L Farr and R J RandalldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[23] A SantaMaria A LopezMMDiaz DMunoz-Mingarro andJM Pozuelo ldquoEvaluation of the toxicity of guaranawith in vitrobioassaysrdquo Ecotoxicology and Environmental Safety vol 39 no3 pp 164ndash167 1998

[24] S P Bydlowski R L Yunker andMT R Subbiah ldquoAnovel pro-perty of an aqueous guarana extract (Paullinia cupana) inhi-bition of platelet aggregation in vitro and in vivordquo BrazilianJournal ofMedical and Biological Research vol 21 no 3 pp 535ndash538 1988

[25] A Basile L Ferrara M Del Pezzo et al ldquoAntibacterial andantioxidant activities of ethanol extract from Paullinia cupanaMartrdquo Journal of Ethnopharmacology vol 102 no 1 pp 32ndash362005

[26] I Gulcin ldquoAntioxidant and antiradical activities of L-carnitinerdquoLife Sciences vol 78 no 8 pp 803ndash811 2006

[27] S E Gultekin B Senguven A Sofuoglu L Taner andM KochldquoEffect of the topical use of the antioxidant taurine on the twobasement membrane proteins of regenerating oral gingival epi-theliumrdquo Journal of Periodontology vol 83 no 1 pp 127ndash1342012

[28] B Halliwel and J M C Gutteridge Free Radicals in BiologyandMedicine OxfordUniversity PressOxfordUK 4th edition2005

[29] S Wuchty and P F Stadler ldquoCenters of complex networksrdquoJournal of Theoretical Biology vol 223 no 1 pp 45ndash53 2003

[30] E Estrada ldquoVirtual identification of essential proteins withinthe protein interaction network of yeastrdquo Proteomics vol 6 no1 pp 35ndash40 2006

[31] F Zeidan-Chulia J L Rybarczyk-Filho M Gursoy et alldquoBioinformatical and in vitro approaches to essential oil-induced matrix metalloproteinase inhibitionrdquo PharmaceuticalBiology vol 50 no 6 pp 675ndash686 2012

[32] J O Rosado J P Henriques andD Bonatto ldquoA systems pharm-acology analysis of major chemotherapy combination regimensused in gastric cancer treatment predicting potential newprotein targets and drugsrdquo Current Cancer Drug Targets vol 11no 7 pp 849ndash869 2011


[33] J L Biedler L Helson and B A Spengler ldquoMorphology andgrowth tumorigenicity and cytogenetics of human neuroblas-toma cells in continuous culturerdquo Cancer Research vol 33 no11 pp 2643ndash2652 1973

[34] C Sanfeliu R Cristofol N Toran E Rodrıguez-Farre and SU Kim ldquoUse of human central nervous system cell cultures inneurotoxicity testingrdquo Toxicology in Vitro vol 13 no 4-5 pp753ndash759 1999

[35] A Morales-Hernandez F J Sanchez-Martın M P Hortigon-Vinagre F Henao and J M Merino ldquo2 3 7 8-tetrachloro-dibenzo-p-dioxin induces apoptosis by disruption of intracellu-lar calcium homeostasis in human neuronal cell line SHSY5YrdquoApoptosis vol 17 no 11 pp 1170ndash1181 2012

[36] P D Ray B W Huang and Y Tsuji ldquoReactive oxygen species(ROS) homeostasis and redox regulation in cellular signalingrdquoCellular Signalling vol 24 pp 981ndash990 2012

[37] B Poljsak and I Milisav ldquoThe neglected significance of lsquoantiox-idative stressrsquordquo Oxidative Medicine and Cellular Longevity vol2012 Article ID 480895 12 pages 2012

[38] N P Seeram L S Adams S M Henning et al ldquoIn vitro anti-proliferative apoptotic and antioxidant activities of punicala-gin ellagic acid and a total pomegranate tannin extract areenhanced in combination with other polyphenols as found inpomegranate juicerdquo Journal of Nutritional Biochemistry vol 16no 6 pp 360ndash367 2005

[39] C L Hsu and G C Yen ldquoInduction of cell apoptosis in 3T3-L1pre-adipocytes by flavonoids is associatedwith their antioxidantactivityrdquoMolecular Nutrition and Food Research vol 50 no 11pp 1072ndash1079 2006

[40] N W He Y Zhao L Guo J Shang and X B Yang ldquoAntioxi-dant antiproliferative and pro-apoptotic activities of a saponinextract derived from the roots of Panax notoginseng (Burk)FH Chenrdquo Journal of Medicinal Food vol 15 no 4 pp 350ndash359 2012

[41] H Fukumasu J L Avanzo M K Nagamine J A Barbuto KV Rao and M L Z Dagli ldquoPaullinia cupana Mart var sorbilisguarana reduces cell proliferation and increases apoptosis ofB16F10 melanoma lung metastases in micerdquo Brazilian Journalof Medical and Biological Research vol 41 no 4 pp 305ndash3102008

[42] L L Howell ldquoComparative effects of caffeine and selectivephosphodiesterase inhibitors on respiration and behavior inrhesus monkeysrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 266 no 2 pp 894ndash903 1993

[43] F Lopez L G Miller D J Greenblatt G B Kaplan and RI Shader ldquoInteraction of caffeine with the GABA(A) receptorcomplex alterations in receptor function but not ligand bind-ingrdquo European Journal of Pharmacology vol 172 no 6 pp 453ndash459 1989

[44] R V Abreu E M Silva-Oliveira M F Moraes G S Pereiraand T Moraes-Santos ldquoChronic coffee and caffeine ingestioneffects on the cognitive function and antioxidant system of ratbrainsrdquo Pharmacology Biochemistry and Behavior vol 99 no 4pp 659ndash664 2011

[45] R J Huxtable ldquoPhysiological actions of taurinerdquo PhysiologicalReviews vol 72 no 1 pp 101ndash163 1992

[46] Z Degim N Celebi H Sayan A Babul D Erdogan and GTake ldquoAn investigation on skin wound healing in mice with ataurine-chitosan gel formulationrdquoAmino Acids vol 22 pp 187ndash198 2002

[47] M Giris B Depboylu S Dogru-Abbasoglu et al ldquoEffectof taurine on oxidative stress and apoptosis-related protein

expression in trinitrobenzene sulphonic acid-induced colitisrdquoClinical and Experimental Immunology vol 152 no 1 pp 102ndash110 2008

[48] Z He W Y Ma T Hashimoto A M Bode C S Yang and ZDong ldquoInduction of apoptosis by caffeine is mediated by thep53 Bax and caspase 3 pathwaysrdquo Cancer Research vol 63 no15 pp 4396ndash4401 2003

[49] R Carosio G Zuccari I Orienti S Mangraviti and P G Mon-taldo ldquoSodium ascorbate induces apoptosis in neuroblastomacell lines by interfering with iron uptakerdquoMolecular Cancer vol6 article 55 2007

[50] S Orrenius and P Nicotera ldquoThe calcium ion and cell deathrdquoJournal of Neural Transmission Supplement vol 43 pp 1ndash111994

[51] M H Jang M C Shin I S Kang et al ldquoCaffeine inducesapoptosis in human neuroblastoma cell line SK-N-MCrdquo Journalof Korean Medical Science vol 17 no 5 pp 674ndash678 2002

[52] M Nordin-Andersson A Forsby N Heldring J DeJongh PKjellstrand and E Walum ldquoNeurite degeneration in differen-tiated human neuroblastoma cellsrdquo Toxicology in Vitro vol 12no 5 pp 557ndash560 1998

[53] M I Porn-Ares M P S Ares and S Orrenius ldquoCalcium sig-nalling and the regulation of apoptosisrdquo Toxicology in Vitro vol12 no 5 pp 539ndash543 1998

[54] M Pelletier L Oliver K Meflah and F M Vallette ldquoCaspase-3 can be pseudo-activated by a Ca2+-dependent proteolysis at anon-canonical siterdquoFEBSLetters vol 579 no 11 pp 2364ndash23682005

[55] G B Stokin C Lillo T L Falzone et al ldquoAxonopathy and trans-port deficits early in the pathogenesis of Alzheimerrsquos diseasesrdquoScience vol 307 no 5713 pp 1282ndash1288 2005

[56] P E Spoerri C G Caple and F J Roisen ldquoTaurine-inducedneuronal differentiation the influence of calcium and theganglioside GM1rdquo International Journal of Developmental Neu-roscience vol 8 no 4 pp 491ndash503 1990

[57] A El Idrissi ldquoTaurine increases mitochondrial buffering ofcalcium role in neuroprotectionrdquo Amino Acids vol 34 no 2pp 321ndash328 2008

[58] F Zeidan-Chulia J L Rybarczyk-Filho A B Salmina B Hde Oliveira M Noda and J C Moreira ldquoExploring the multi-factorial nature of autism through computational systems biol-ogy calcium and the Rho GTPase RAC1 under the spotlightrdquoNeuroMolecular Medicine vol 15 no 2 pp 364ndash383 2013

[59] F Zeidan-Chulia B H Neves de Oliveira M Gursoy et alldquoMMP-REDOXNO interplay in periodontitis and its inhibi-tion with Satureja hortensis L essential oilrdquo Chemistry amp Bio-diversity vol 10 no 4 pp 507ndash523 2013

Submit your manuscripts athttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

MediatorsinflaMMation

of

Diabetes ResearchJournal of


ISRN AIDS



Computational and Mathematical Methods in Medicine

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013Issue 1

GastroenterologyResearch and Practice

Clinical ampDevelopmentalImmunology


Volume 2013


ISRN Biomarkers

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal


Oxidative Medicine and Cellular Longevity

ISRN Addiction


International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

ISRN Anesthesiology


BioMed Research International



OncologyJournal of

Volume 2013

OphthalmologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch



Plant extract

Medicinal plants

YohimbinePausinystalia yohimbe

Snakeroot or sarpagandhaRauwolfia serpentina

Yohimbine

GuaranaPaullinia cupana

GinsengPanax ginseng

(1) Methylxanthines

TheobromineTheophylline

(2) Tannins(3) Saponins(4) Catechins(5) Epicatechins(6) Proanthocyanidins

Energy drinks

(1) SaponinsGinsenosides

(2) Other compounds

(7) Other compounds

N

N

N

N O

O

S

HO O

O

O

OHCaffeine

Vitamins

Taurine

L-carnitine

GinkgoGinkgo biloba

HH

OH

N

N

H

H

OO

Am

ino

acid

s

B vitamins

Oth

ers

(1) Flavonoids

(2) Terpenes

QuercetinKaempferolBiflavoneGinkgetinBilobetinSciadopitysin

Ginkgolide A Ginkgolide BGinkgolide CBilobalide

Caffeinelowast

N+

Ominus

NH2

CH3

CH3

H3C

Figure 1 Schematic representations illustrating the different components found in EDs EDs are highly caffeinated beverages which maycontain herbal supplements alkaloids from different plants vitamins or even amino acids In some cases the addition of herbal supplements(eg guarana) can increase the caffeine content of these beverages (lowast) For our in vitro study we selected caffeine taurine and guarana asmajor components of EDs

Furthermore it has been reported that the number of energydrinks consumed in one session reaches an average of 5 units(cans) in one single night [3]

The concentration of caffeine in EDs is significantlyhigher than those found in cola drinks and such amounts areknown to cause a variety of adverse health effects [4] Alsothe use of some herbal supplements in these beverages likeguarana increases the amount of caffeine and other activemethylxanthines that multiply any potential toxicity

The widespread consumption of EDs is becoming espe-cially popular among people below the age of 25 and adole-scents [1 2] Nonetheless only a limited number of studieshave tried to evaluate the short- and long-term effects of EDconsumption Most of them examined the cognitive effectsderived from the use of these drinks or some of their majorcomponents (eg caffeine or taurine) alone or in coadmin-istration with alcohol [5 6] Some other reports are high-lighting the possible link between the use of highly caffeinatedbeverages and the increased propensity for addiction to othersubstances like alcohol or even new-onset seizures in adults[7 8]

Different countries have already started to regulate thesale of highly caffeinated EDs to prevent potential healthproblems Denmark and France banned the sale of somebrands in Norway some EDs are only available in pharma-cies in general the European Union demands the labelingof highly caffeinated drinks [1 2] Major concerns are arisingwith the impact these beverages may have on children andadolescentsrsquo developing brains [9] Therefore in the presentstudy we used a well-known neurotoxicity cell culture model(human neuronal SH-SY5Y cells) [10] in order to elucidatethe potential toxicity (synergistic or not) of caffeine taurineand guarana three components that are commonly presentin EDs at high concentrations

2 Materials and Methods

21 Drugs Guarana (Paullinia cupana Mart) powder wasobtained fromLifar Ltd (PortoAlegre RS Brazil) Accordingto the manufacturer 50mg of guarana powder (obtainedfrom grounding of dried seeds) contains approximately 2mgof caffeine Both caffeine and taurine were purchased fromSigma Chemical Co (St Louis MO USA)











2O2


















3 Results










3125

125

50

PCM (2 hours)

Control

3125

125

3125

125

50

Control


50

(a) (b) (d)(c)

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K800600400200Control

Gua 3125

Gua 125

Gua 50

0

1 K8006004002000

1 K8006004002000

1 K8006004002000

(3)

(4)

(3)

(4)


SSC-

HSS

C-H

SSC-

HSS

C-H

FSC-H

(2) (2)

(1)

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Con

trol

Gua

12

5G

ua 1

25

Caspase 3

CM (4 hours)

Con

trol

(1)(1)

(2)

(3)

(4)

(1)

(3)

(5)

(7)

(2)

(4)

(6)

(8)

Control





Control

0125

05

(1) (1)

(2)

(5)

(7)

(6)

(8)

(3)

(3)

(4)

(1)

(2)

(3)

(4)

(1)

(2)

(3)

(4)

(4)

(2)

2

PCM (2 hours)

Control Control

0125

05

2 2

0125

05


800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K800600400200Control

Caf 0125

Caf 05

Caf 2

0

1 K8006004002000

1 K8006004002000

1 K8006004002000


SSC-

HSS

C-H

SSC-

HSS

C-H

FSC-H

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Caspase 3

CM (4 hours)

Con

trol

Con

trol

Caf 0

5Ca

f 05

(a) (b) (d)(c)







Control

16

(8)

(5)

(1)

(6)

(2)

(2) (3)

(3)

(4)

(4)(4)

(1)

(2)

(3)

(1)

(4)

(2)

(3)

(1)

(7)

1

4

PCM (2 hours)

Control Control

1 1

16 16

4 4


800

600

400

200

01 K800600400200

Control

Tau 1

Tau 4

Tau 16

0

1 K8006004002000

1 K8006004002000

1 K8006004002000


SSC-

H

1 K

800

600

400

200

0

SSC-

H

1 K

800

600

400

200

0

SSC-

H

1 K

800

600

400

200

0

SSC-

H

FSC-H

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Caspase 3

CM (4 hours)

Con

trol

Con

trol

Tau

4Ta

u 4

(a) (b) (d)(c)





ontro

lC

ontro

l

Light

(2)

(3)

(4)

(5)(1)

3D

3D

PI


(a)

Gua

12

5G

ua 1

25

Light

3D

PI


(2)

(3)

(4)

(5)(1)

3D

(b)

Caf 0

5Ca

f 05

Light

3D

3D

PI


(2)

(3)

(4)

(5)(1)

(c)







ControlPI

PI120573

-III

-tubu

linD

API

(1)

(2)

(a)

PIPI

120573-I

II-tu

bulin

DA

PI

Gua 125(1)

(2)

(b)PI

PI120573

-III

-tubu

linD

API

Caf 05(1)

(2)

(c)

PIPI

120573-I

II-tu

bulin

DA

PI

Tau 4(1)

(2)

(d)









15

10

5

0


TRAP TRAP

TAR

Con

trol

312

5

625

125 25 50

Con

trol

312

5

625

125 25 50

Control3125625

1252550

Control3125625

1252550

3125 625 125 25 500

120

100

80

60

40

20

0

120

100

80

60

40

20

0

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(mgmL)

(mgmL)

(mgmL)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


Gua

rana

120

90

60

30

00 5 10 15 20 25 30

Gua

(mg

mL)

200

150

100

50

00 30 60 90 120

Radi

cal p

rodu

ctio

n (

)

Gua

rana

(1)

(2)

(3)

(4)

(5)

TAR

(1198680119868

)

(a)

120

100

80

60

40

20

0

120

90

60

30

00 5 10 15 20 25 30


TRAPTRAP

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

Con

trol

012

5

025 0

5 1 2

(mgmL)

Con

trol

012

5

025 0

5 1 2

0125 025 05 1 20

(mgmL)

Control0125025

0512

Control0125025

0512

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

120

100

80

60

40

20

0

TAR

5

4

3

2

1

0

(mgmL)

Caffe

ine

Caffe

ine

TAR

(1198680119868

)

Radi

cal p

rodu

ctio

n (

)Pe

rcen

tage

of A

UC

(in re

latio

n to

cont

rol)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(1)

(2)

(3)

(4)

(5)

(b)

Figure 7 Continued


TAR

(1198680119868

)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Radi

cal p

rodu

ctio

n (

)

120

90

60

30

00 5 10 15 20 25 30

Time (min)

Con

trol 1 2 4 8 16

(mgmL)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Con

trol 1 2 4 8 16

120

100

80

60

40

20

0

(mgmL)

Time (min)

TRAPTRAP

Control12

4816

Control12

4816

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

TAR

1 2 4 8 160(mgmL)

5

4

3

2

1

0

Taur

ine

Taur

ine

160140120100

80604020

0

(1)

(2)

(3)

(4)

(5)

(c)









(1) (2)

(4) (5)

(9)(7) (8)

(6)

(3)

30 120583m

Gua 125 + Caf 05 Gua 125 + Tau 4

Gua 125 + Caf 05 + Tau 4


Tau 4


SH-S

Y5Y

cells

0

FBS

+co

mbi

ning

trea

tmen

t (m

gm

L 2

hour

s)

PCM

(400

x)

(a)

500

100

80

60

40

20

0

Intr

acel

lula

r RO

S pr

oduc

tion

( in

relat

ion

to co

ntro

l)

Con

trol

Gua

12

5

Caf 0

5

Tau

4

2 hours(mgmL)

H2O2

(1 m

Moplus

cont

rol)

lowastlowastlowast


Gua

125+

Caf0

5

Gua

125+

Tau4

Caf0

5+

Tau4

Caf1

+Ta

u4

Gua

12

5+

Caf 0

5+

Tau

4

(b)







4 Discussion





50

40

30

20

10

0

03

125

625

125 25 50

03

125

625

125 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

Gua

rana

U S

OD

mg

prot

ein

SOD CAT


GPx

GST

(1) (2) (3)

(4) (5) (6)



lowastlowast


4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

Gua

rana

U G

STm

g pr

otei

n

8

6

4

2

0

04

03

02

01

00

SH TBARS



ARS

(nm

ol T

MP

mg

prot

ein)

75

50

25

0

020

015

010

005

000


mol

SH

mg

prot

ein


(a)

50

40

30

20

10

0

00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2 00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2

Caffe

ine

U S

OD

mg

prot

ein

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

Caffe

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000


120583m

ol S

Hm

g pr

otei

n



SOD CAT GPx

GST SH TBARS

(b)

Figure 9 Continued


50

40

30

20

10

0

Taur

ine

U S

OD

mg

prot

ein

0 1 2 4 8 16

0 1 2 4 8 16

0 1 2 4 8 16 0 1 2 4 8 16

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

(mgmL)

0 1 2 4 8 16

(mgmL)

0 1 2 4 8 16

(mgmL) 2 hours

Taur

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000

120583m

ol S

Hm

g pr

otei

n


(1) (2) (3)


SOD CAT GPx

GST SH TBARS

(c)






SOD50

40

30

20

10

0

U S

OD

mg

prot

ein

lowastlowastlowast


lowastlowast

(a)

CAT4

3

2

1

0

U C

ATm

g pr

otei

n


(b)

GPx8

6

4

2

0

U G

Pxm

g pr

otei

n

(c)

GST04

03

02

01

00

U G

STm

g pr

otei

n

(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(d)

SH


(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

75

50

25

0

120583m

ol S

Hm

g pr

otei

n

(e)

TBARS

lowast

(mgmL)

020

015

010

005

000

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(f)






Con

trol (

1)

Con

trol (

2)


Gua

31

25

Gua

12

5

Gua

50

Caf 0

125

Caf 0

5

Caf 2

Tau

1

Tau

4

Tau

16

Gua

12

5+

Caf 0

5

Gua

12

5+

Tau

4

Caf 0

5+

Tau

4

Caf 1

+Ta

u 4

Gua

12

5+

Caf 0

5+

Tau

4


Tota

l cel

l cou

nt (times

106)

20

15

10

05

00

Control


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast



(mgmL)

(a)

PCM

(200

x)





(1) (3)(2) (4)

(5)

(10)(9) (11) (12)

(13)

(6) (8)(7)

SH-S

Y5Y

cells

10

FBS

+dr

ug(s

) tre

atm

ent (

mg

mL

24 h

ours

)

(14) (15) (16)

(b)



104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)


SH-SY5Y cells


Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)


Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4


Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4








(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch












2O2


















3 Results










3125

125

50

PCM (2 hours)

Control

3125

125

3125

125

50

Control


50

(a) (b) (d)(c)

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K800600400200Control

Gua 3125

Gua 125

Gua 50

0

1 K8006004002000

1 K8006004002000

1 K8006004002000

(3)

(4)

(3)

(4)


SSC-

HSS

C-H

SSC-

HSS

C-H

FSC-H

(2) (2)

(1)

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Con

trol

Gua

12

5G

ua 1

25

Caspase 3

CM (4 hours)

Con

trol

(1)(1)

(2)

(3)

(4)

(1)

(3)

(5)

(7)

(2)

(4)

(6)

(8)

Control





Control

0125

05

(1) (1)

(2)

(5)

(7)

(6)

(8)

(3)

(3)

(4)

(1)

(2)

(3)

(4)

(1)

(2)

(3)

(4)

(4)

(2)

2

PCM (2 hours)

Control Control

0125

05

2 2

0125

05


800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K800600400200Control

Caf 0125

Caf 05

Caf 2

0

1 K8006004002000

1 K8006004002000

1 K8006004002000


SSC-

HSS

C-H

SSC-

HSS

C-H

FSC-H

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Caspase 3

CM (4 hours)

Con

trol

Con

trol

Caf 0

5Ca

f 05

(a) (b) (d)(c)







Control

16

(8)

(5)

(1)

(6)

(2)

(2) (3)

(3)

(4)

(4)(4)

(1)

(2)

(3)

(1)

(4)

(2)

(3)

(1)

(7)

1

4

PCM (2 hours)

Control Control

1 1

16 16

4 4


800

600

400

200

01 K800600400200

Control

Tau 1

Tau 4

Tau 16

0

1 K8006004002000

1 K8006004002000

1 K8006004002000


SSC-

H

1 K

800

600

400

200

0

SSC-

H

1 K

800

600

400

200

0

SSC-

H

1 K

800

600

400

200

0

SSC-

H

FSC-H

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Caspase 3

CM (4 hours)

Con

trol

Con

trol

Tau

4Ta

u 4

(a) (b) (d)(c)





ontro

lC

ontro

l

Light

(2)

(3)

(4)

(5)(1)

3D

3D

PI


(a)

Gua

12

5G

ua 1

25

Light

3D

PI


(2)

(3)

(4)

(5)(1)

3D

(b)

Caf 0

5Ca

f 05

Light

3D

3D

PI


(2)

(3)

(4)

(5)(1)

(c)







ControlPI

PI120573

-III

-tubu

linD

API

(1)

(2)

(a)

PIPI

120573-I

II-tu

bulin

DA

PI

Gua 125(1)

(2)

(b)PI

PI120573

-III

-tubu

linD

API

Caf 05(1)

(2)

(c)

PIPI

120573-I

II-tu

bulin

DA

PI

Tau 4(1)

(2)

(d)









15

10

5

0


TRAP TRAP

TAR

Con

trol

312

5

625

125 25 50

Con

trol

312

5

625

125 25 50

Control3125625

1252550

Control3125625

1252550

3125 625 125 25 500

120

100

80

60

40

20

0

120

100

80

60

40

20

0

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(mgmL)

(mgmL)

(mgmL)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


Gua

rana

120

90

60

30

00 5 10 15 20 25 30

Gua

(mg

mL)

200

150

100

50

00 30 60 90 120

Radi

cal p

rodu

ctio

n (

)

Gua

rana

(1)

(2)

(3)

(4)

(5)

TAR

(1198680119868

)

(a)

120

100

80

60

40

20

0

120

90

60

30

00 5 10 15 20 25 30


TRAPTRAP

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

Con

trol

012

5

025 0

5 1 2

(mgmL)

Con

trol

012

5

025 0

5 1 2

0125 025 05 1 20

(mgmL)

Control0125025

0512

Control0125025

0512

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

120

100

80

60

40

20

0

TAR

5

4

3

2

1

0

(mgmL)

Caffe

ine

Caffe

ine

TAR

(1198680119868

)

Radi

cal p

rodu

ctio

n (

)Pe

rcen

tage

of A

UC

(in re

latio

n to

cont

rol)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(1)

(2)

(3)

(4)

(5)

(b)

Figure 7 Continued


TAR

(1198680119868

)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Radi

cal p

rodu

ctio

n (

)

120

90

60

30

00 5 10 15 20 25 30

Time (min)

Con

trol 1 2 4 8 16

(mgmL)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Con

trol 1 2 4 8 16

120

100

80

60

40

20

0

(mgmL)

Time (min)

TRAPTRAP

Control12

4816

Control12

4816

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

TAR

1 2 4 8 160(mgmL)

5

4

3

2

1

0

Taur

ine

Taur

ine

160140120100

80604020

0

(1)

(2)

(3)

(4)

(5)

(c)









(1) (2)

(4) (5)

(9)(7) (8)

(6)

(3)

30 120583m

Gua 125 + Caf 05 Gua 125 + Tau 4

Gua 125 + Caf 05 + Tau 4


Tau 4


SH-S

Y5Y

cells

0

FBS

+co

mbi

ning

trea

tmen

t (m

gm

L 2

hour

s)

PCM

(400

x)

(a)

500

100

80

60

40

20

0

Intr

acel

lula

r RO

S pr

oduc

tion

( in

relat

ion

to co

ntro

l)

Con

trol

Gua

12

5

Caf 0

5

Tau

4

2 hours(mgmL)

H2O2

(1 m

Moplus

cont

rol)

lowastlowastlowast


Gua

125+

Caf0

5

Gua

125+

Tau4

Caf0

5+

Tau4

Caf1

+Ta

u4

Gua

12

5+

Caf 0

5+

Tau

4

(b)







4 Discussion





50

40

30

20

10

0

03

125

625

125 25 50

03

125

625

125 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

Gua

rana

U S

OD

mg

prot

ein

SOD CAT


GPx

GST

(1) (2) (3)

(4) (5) (6)



lowastlowast


4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

Gua

rana

U G

STm

g pr

otei

n

8

6

4

2

0

04

03

02

01

00

SH TBARS



ARS

(nm

ol T

MP

mg

prot

ein)

75

50

25

0

020

015

010

005

000


mol

SH

mg

prot

ein


(a)

50

40

30

20

10

0

00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2 00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2

Caffe

ine

U S

OD

mg

prot

ein

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

Caffe

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000


120583m

ol S

Hm

g pr

otei

n



SOD CAT GPx

GST SH TBARS

(b)

Figure 9 Continued


50

40

30

20

10

0

Taur

ine

U S

OD

mg

prot

ein

0 1 2 4 8 16

0 1 2 4 8 16

0 1 2 4 8 16 0 1 2 4 8 16

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

(mgmL)

0 1 2 4 8 16

(mgmL)

0 1 2 4 8 16

(mgmL) 2 hours

Taur

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000

120583m

ol S

Hm

g pr

otei

n


(1) (2) (3)


SOD CAT GPx

GST SH TBARS

(c)






SOD50

40

30

20

10

0

U S

OD

mg

prot

ein

lowastlowastlowast


lowastlowast

(a)

CAT4

3

2

1

0

U C

ATm

g pr

otei

n


(b)

GPx8

6

4

2

0

U G

Pxm

g pr

otei

n

(c)

GST04

03

02

01

00

U G

STm

g pr

otei

n

(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(d)

SH


(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

75

50

25

0

120583m

ol S

Hm

g pr

otei

n

(e)

TBARS

lowast

(mgmL)

020

015

010

005

000

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(f)






Con

trol (

1)

Con

trol (

2)


Gua

31

25

Gua

12

5

Gua

50

Caf 0

125

Caf 0

5

Caf 2

Tau

1

Tau

4

Tau

16

Gua

12

5+

Caf 0

5

Gua

12

5+

Tau

4

Caf 0

5+

Tau

4

Caf 1

+Ta

u 4

Gua

12

5+

Caf 0

5+

Tau

4


Tota

l cel

l cou

nt (times

106)

20

15

10

05

00

Control


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast



(mgmL)

(a)

PCM

(200

x)





(1) (3)(2) (4)

(5)

(10)(9) (11) (12)

(13)

(6) (8)(7)

SH-S

Y5Y

cells

10

FBS

+dr

ug(s

) tre

atm

ent (

mg

mL

24 h

ours

)

(14) (15) (16)

(b)



104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)


SH-SY5Y cells


Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)


Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4


Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4








(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch

















3 Results










3125

125

50

PCM (2 hours)

Control

3125

125

3125

125

50

Control


50

(a) (b) (d)(c)

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K800600400200Control

Gua 3125

Gua 125

Gua 50

0

1 K8006004002000

1 K8006004002000

1 K8006004002000

(3)

(4)

(3)

(4)


SSC-

HSS

C-H

SSC-

HSS

C-H

FSC-H

(2) (2)

(1)

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Con

trol

Gua

12

5G

ua 1

25

Caspase 3

CM (4 hours)

Con

trol

(1)(1)

(2)

(3)

(4)

(1)

(3)

(5)

(7)

(2)

(4)

(6)

(8)

Control





Control

0125

05

(1) (1)

(2)

(5)

(7)

(6)

(8)

(3)

(3)

(4)

(1)

(2)

(3)

(4)

(1)

(2)

(3)

(4)

(4)

(2)

2

PCM (2 hours)

Control Control

0125

05

2 2

0125

05


800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K800600400200Control

Caf 0125

Caf 05

Caf 2

0

1 K8006004002000

1 K8006004002000

1 K8006004002000


SSC-

HSS

C-H

SSC-

HSS

C-H

FSC-H

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Caspase 3

CM (4 hours)

Con

trol

Con

trol

Caf 0

5Ca

f 05

(a) (b) (d)(c)







Control

16

(8)

(5)

(1)

(6)

(2)

(2) (3)

(3)

(4)

(4)(4)

(1)

(2)

(3)

(1)

(4)

(2)

(3)

(1)

(7)

1

4

PCM (2 hours)

Control Control

1 1

16 16

4 4


800

600

400

200

01 K800600400200

Control

Tau 1

Tau 4

Tau 16

0

1 K8006004002000

1 K8006004002000

1 K8006004002000


SSC-

H

1 K

800

600

400

200

0

SSC-

H

1 K

800

600

400

200

0

SSC-

H

1 K

800

600

400

200

0

SSC-

H

FSC-H

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Caspase 3

CM (4 hours)

Con

trol

Con

trol

Tau

4Ta

u 4

(a) (b) (d)(c)





ontro

lC

ontro

l

Light

(2)

(3)

(4)

(5)(1)

3D

3D

PI


(a)

Gua

12

5G

ua 1

25

Light

3D

PI


(2)

(3)

(4)

(5)(1)

3D

(b)

Caf 0

5Ca

f 05

Light

3D

3D

PI


(2)

(3)

(4)

(5)(1)

(c)







ControlPI

PI120573

-III

-tubu

linD

API

(1)

(2)

(a)

PIPI

120573-I

II-tu

bulin

DA

PI

Gua 125(1)

(2)

(b)PI

PI120573

-III

-tubu

linD

API

Caf 05(1)

(2)

(c)

PIPI

120573-I

II-tu

bulin

DA

PI

Tau 4(1)

(2)

(d)









15

10

5

0


TRAP TRAP

TAR

Con

trol

312

5

625

125 25 50

Con

trol

312

5

625

125 25 50

Control3125625

1252550

Control3125625

1252550

3125 625 125 25 500

120

100

80

60

40

20

0

120

100

80

60

40

20

0

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(mgmL)

(mgmL)

(mgmL)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


Gua

rana

120

90

60

30

00 5 10 15 20 25 30

Gua

(mg

mL)

200

150

100

50

00 30 60 90 120

Radi

cal p

rodu

ctio

n (

)

Gua

rana

(1)

(2)

(3)

(4)

(5)

TAR

(1198680119868

)

(a)

120

100

80

60

40

20

0

120

90

60

30

00 5 10 15 20 25 30


TRAPTRAP

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

Con

trol

012

5

025 0

5 1 2

(mgmL)

Con

trol

012

5

025 0

5 1 2

0125 025 05 1 20

(mgmL)

Control0125025

0512

Control0125025

0512

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

120

100

80

60

40

20

0

TAR

5

4

3

2

1

0

(mgmL)

Caffe

ine

Caffe

ine

TAR

(1198680119868

)

Radi

cal p

rodu

ctio

n (

)Pe

rcen

tage

of A

UC

(in re

latio

n to

cont

rol)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(1)

(2)

(3)

(4)

(5)

(b)

Figure 7 Continued


TAR

(1198680119868

)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Radi

cal p

rodu

ctio

n (

)

120

90

60

30

00 5 10 15 20 25 30

Time (min)

Con

trol 1 2 4 8 16

(mgmL)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Con

trol 1 2 4 8 16

120

100

80

60

40

20

0

(mgmL)

Time (min)

TRAPTRAP

Control12

4816

Control12

4816

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

TAR

1 2 4 8 160(mgmL)

5

4

3

2

1

0

Taur

ine

Taur

ine

160140120100

80604020

0

(1)

(2)

(3)

(4)

(5)

(c)









(1) (2)

(4) (5)

(9)(7) (8)

(6)

(3)

30 120583m

Gua 125 + Caf 05 Gua 125 + Tau 4

Gua 125 + Caf 05 + Tau 4


Tau 4


SH-S

Y5Y

cells

0

FBS

+co

mbi

ning

trea

tmen

t (m

gm

L 2

hour

s)

PCM

(400

x)

(a)

500

100

80

60

40

20

0

Intr

acel

lula

r RO

S pr

oduc

tion

( in

relat

ion

to co

ntro

l)

Con

trol

Gua

12

5

Caf 0

5

Tau

4

2 hours(mgmL)

H2O2

(1 m

Moplus

cont

rol)

lowastlowastlowast


Gua

125+

Caf0

5

Gua

125+

Tau4

Caf0

5+

Tau4

Caf1

+Ta

u4

Gua

12

5+

Caf 0

5+

Tau

4

(b)







4 Discussion





50

40

30

20

10

0

03

125

625

125 25 50

03

125

625

125 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

Gua

rana

U S

OD

mg

prot

ein

SOD CAT


GPx

GST

(1) (2) (3)

(4) (5) (6)



lowastlowast


4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

Gua

rana

U G

STm

g pr

otei

n

8

6

4

2

0

04

03

02

01

00

SH TBARS



ARS

(nm

ol T

MP

mg

prot

ein)

75

50

25

0

020

015

010

005

000


mol

SH

mg

prot

ein


(a)

50

40

30

20

10

0

00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2 00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2

Caffe

ine

U S

OD

mg

prot

ein

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

Caffe

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000


120583m

ol S

Hm

g pr

otei

n



SOD CAT GPx

GST SH TBARS

(b)

Figure 9 Continued


50

40

30

20

10

0

Taur

ine

U S

OD

mg

prot

ein

0 1 2 4 8 16

0 1 2 4 8 16

0 1 2 4 8 16 0 1 2 4 8 16

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

(mgmL)

0 1 2 4 8 16

(mgmL)

0 1 2 4 8 16

(mgmL) 2 hours

Taur

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000

120583m

ol S

Hm

g pr

otei

n


(1) (2) (3)


SOD CAT GPx

GST SH TBARS

(c)






SOD50

40

30

20

10

0

U S

OD

mg

prot

ein

lowastlowastlowast


lowastlowast

(a)

CAT4

3

2

1

0

U C

ATm

g pr

otei

n


(b)

GPx8

6

4

2

0

U G

Pxm

g pr

otei

n

(c)

GST04

03

02

01

00

U G

STm

g pr

otei

n

(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(d)

SH


(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

75

50

25

0

120583m

ol S

Hm

g pr

otei

n

(e)

TBARS

lowast

(mgmL)

020

015

010

005

000

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(f)






Con

trol (

1)

Con

trol (

2)


Gua

31

25

Gua

12

5

Gua

50

Caf 0

125

Caf 0

5

Caf 2

Tau

1

Tau

4

Tau

16

Gua

12

5+

Caf 0

5

Gua

12

5+

Tau

4

Caf 0

5+

Tau

4

Caf 1

+Ta

u 4

Gua

12

5+

Caf 0

5+

Tau

4


Tota

l cel

l cou

nt (times

106)

20

15

10

05

00

Control


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast



(mgmL)

(a)

PCM

(200

x)





(1) (3)(2) (4)

(5)

(10)(9) (11) (12)

(13)

(6) (8)(7)

SH-S

Y5Y

cells

10

FBS

+dr

ug(s

) tre

atm

ent (

mg

mL

24 h

ours

)

(14) (15) (16)

(b)



104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)


SH-SY5Y cells


Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)


Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4


Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4








(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch



3125

125

50

PCM (2 hours)

Control

3125

125

3125

125

50

Control


50

(a) (b) (d)(c)

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K800600400200Control

Gua 3125

Gua 125

Gua 50

0

1 K8006004002000

1 K8006004002000

1 K8006004002000

(3)

(4)

(3)

(4)


SSC-

HSS

C-H

SSC-

HSS

C-H

FSC-H

(2) (2)

(1)

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Con

trol

Gua

12

5G

ua 1

25

Caspase 3

CM (4 hours)

Con

trol

(1)(1)

(2)

(3)

(4)

(1)

(3)

(5)

(7)

(2)

(4)

(6)

(8)

Control





Control

0125

05

(1) (1)

(2)

(5)

(7)

(6)

(8)

(3)

(3)

(4)

(1)

(2)

(3)

(4)

(1)

(2)

(3)

(4)

(4)

(2)

2

PCM (2 hours)

Control Control

0125

05

2 2

0125

05


800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K800600400200Control

Caf 0125

Caf 05

Caf 2

0

1 K8006004002000

1 K8006004002000

1 K8006004002000


SSC-

HSS

C-H

SSC-

HSS

C-H

FSC-H

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Caspase 3

CM (4 hours)

Con

trol

Con

trol

Caf 0

5Ca

f 05

(a) (b) (d)(c)







Control

16

(8)

(5)

(1)

(6)

(2)

(2) (3)

(3)

(4)

(4)(4)

(1)

(2)

(3)

(1)

(4)

(2)

(3)

(1)

(7)

1

4

PCM (2 hours)

Control Control

1 1

16 16

4 4


800

600

400

200

01 K800600400200

Control

Tau 1

Tau 4

Tau 16

0

1 K8006004002000

1 K8006004002000

1 K8006004002000


SSC-

H

1 K

800

600

400

200

0

SSC-

H

1 K

800

600

400

200

0

SSC-

H

1 K

800

600

400

200

0

SSC-

H

FSC-H

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Caspase 3

CM (4 hours)

Con

trol

Con

trol

Tau

4Ta

u 4

(a) (b) (d)(c)





ontro

lC

ontro

l

Light

(2)

(3)

(4)

(5)(1)

3D

3D

PI


(a)

Gua

12

5G

ua 1

25

Light

3D

PI


(2)

(3)

(4)

(5)(1)

3D

(b)

Caf 0

5Ca

f 05

Light

3D

3D

PI


(2)

(3)

(4)

(5)(1)

(c)







ControlPI

PI120573

-III

-tubu

linD

API

(1)

(2)

(a)

PIPI

120573-I

II-tu

bulin

DA

PI

Gua 125(1)

(2)

(b)PI

PI120573

-III

-tubu

linD

API

Caf 05(1)

(2)

(c)

PIPI

120573-I

II-tu

bulin

DA

PI

Tau 4(1)

(2)

(d)









15

10

5

0


TRAP TRAP

TAR

Con

trol

312

5

625

125 25 50

Con

trol

312

5

625

125 25 50

Control3125625

1252550

Control3125625

1252550

3125 625 125 25 500

120

100

80

60

40

20

0

120

100

80

60

40

20

0

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(mgmL)

(mgmL)

(mgmL)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


Gua

rana

120

90

60

30

00 5 10 15 20 25 30

Gua

(mg

mL)

200

150

100

50

00 30 60 90 120

Radi

cal p

rodu

ctio

n (

)

Gua

rana

(1)

(2)

(3)

(4)

(5)

TAR

(1198680119868

)

(a)

120

100

80

60

40

20

0

120

90

60

30

00 5 10 15 20 25 30


TRAPTRAP

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

Con

trol

012

5

025 0

5 1 2

(mgmL)

Con

trol

012

5

025 0

5 1 2

0125 025 05 1 20

(mgmL)

Control0125025

0512

Control0125025

0512

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

120

100

80

60

40

20

0

TAR

5

4

3

2

1

0

(mgmL)

Caffe

ine

Caffe

ine

TAR

(1198680119868

)

Radi

cal p

rodu

ctio

n (

)Pe

rcen

tage

of A

UC

(in re

latio

n to

cont

rol)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(1)

(2)

(3)

(4)

(5)

(b)

Figure 7 Continued


TAR

(1198680119868

)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Radi

cal p

rodu

ctio

n (

)

120

90

60

30

00 5 10 15 20 25 30

Time (min)

Con

trol 1 2 4 8 16

(mgmL)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Con

trol 1 2 4 8 16

120

100

80

60

40

20

0

(mgmL)

Time (min)

TRAPTRAP

Control12

4816

Control12

4816

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

TAR

1 2 4 8 160(mgmL)

5

4

3

2

1

0

Taur

ine

Taur

ine

160140120100

80604020

0

(1)

(2)

(3)

(4)

(5)

(c)









(1) (2)

(4) (5)

(9)(7) (8)

(6)

(3)

30 120583m

Gua 125 + Caf 05 Gua 125 + Tau 4

Gua 125 + Caf 05 + Tau 4


Tau 4


SH-S

Y5Y

cells

0

FBS

+co

mbi

ning

trea

tmen

t (m

gm

L 2

hour

s)

PCM

(400

x)

(a)

500

100

80

60

40

20

0

Intr

acel

lula

r RO

S pr

oduc

tion

( in

relat

ion

to co

ntro

l)

Con

trol

Gua

12

5

Caf 0

5

Tau

4

2 hours(mgmL)

H2O2

(1 m

Moplus

cont

rol)

lowastlowastlowast


Gua

125+

Caf0

5

Gua

125+

Tau4

Caf0

5+

Tau4

Caf1

+Ta

u4

Gua

12

5+

Caf 0

5+

Tau

4

(b)







4 Discussion





50

40

30

20

10

0

03

125

625

125 25 50

03

125

625

125 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

Gua

rana

U S

OD

mg

prot

ein

SOD CAT


GPx

GST

(1) (2) (3)

(4) (5) (6)



lowastlowast


4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

Gua

rana

U G

STm

g pr

otei

n

8

6

4

2

0

04

03

02

01

00

SH TBARS



ARS

(nm

ol T

MP

mg

prot

ein)

75

50

25

0

020

015

010

005

000


mol

SH

mg

prot

ein


(a)

50

40

30

20

10

0

00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2 00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2

Caffe

ine

U S

OD

mg

prot

ein

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

Caffe

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000


120583m

ol S

Hm

g pr

otei

n



SOD CAT GPx

GST SH TBARS

(b)

Figure 9 Continued


50

40

30

20

10

0

Taur

ine

U S

OD

mg

prot

ein

0 1 2 4 8 16

0 1 2 4 8 16

0 1 2 4 8 16 0 1 2 4 8 16

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

(mgmL)

0 1 2 4 8 16

(mgmL)

0 1 2 4 8 16

(mgmL) 2 hours

Taur

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000

120583m

ol S

Hm

g pr

otei

n


(1) (2) (3)


SOD CAT GPx

GST SH TBARS

(c)






SOD50

40

30

20

10

0

U S

OD

mg

prot

ein

lowastlowastlowast


lowastlowast

(a)

CAT4

3

2

1

0

U C

ATm

g pr

otei

n


(b)

GPx8

6

4

2

0

U G

Pxm

g pr

otei

n

(c)

GST04

03

02

01

00

U G

STm

g pr

otei

n

(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(d)

SH


(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

75

50

25

0

120583m

ol S

Hm

g pr

otei

n

(e)

TBARS

lowast

(mgmL)

020

015

010

005

000

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(f)






Con

trol (

1)

Con

trol (

2)


Gua

31

25

Gua

12

5

Gua

50

Caf 0

125

Caf 0

5

Caf 2

Tau

1

Tau

4

Tau

16

Gua

12

5+

Caf 0

5

Gua

12

5+

Tau

4

Caf 0

5+

Tau

4

Caf 1

+Ta

u 4

Gua

12

5+

Caf 0

5+

Tau

4


Tota

l cel

l cou

nt (times

106)

20

15

10

05

00

Control


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast



(mgmL)

(a)

PCM

(200

x)





(1) (3)(2) (4)

(5)

(10)(9) (11) (12)

(13)

(6) (8)(7)

SH-S

Y5Y

cells

10

FBS

+dr

ug(s

) tre

atm

ent (

mg

mL

24 h

ours

)

(14) (15) (16)

(b)



104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)


SH-SY5Y cells


Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)


Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4


Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4








(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch



Control

0125

05

(1) (1)

(2)

(5)

(7)

(6)

(8)

(3)

(3)

(4)

(1)

(2)

(3)

(4)

(1)

(2)

(3)

(4)

(4)

(2)

2

PCM (2 hours)

Control Control

0125

05

2 2

0125

05


800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K

800

600

400

200

0

1 K800600400200Control

Caf 0125

Caf 05

Caf 2

0

1 K8006004002000

1 K8006004002000

1 K8006004002000


SSC-

HSS

C-H

SSC-

HSS

C-H

FSC-H

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Caspase 3

CM (4 hours)

Con

trol

Con

trol

Caf 0

5Ca

f 05

(a) (b) (d)(c)







Control

16

(8)

(5)

(1)

(6)

(2)

(2) (3)

(3)

(4)

(4)(4)

(1)

(2)

(3)

(1)

(4)

(2)

(3)

(1)

(7)

1

4

PCM (2 hours)

Control Control

1 1

16 16

4 4


800

600

400

200

01 K800600400200

Control

Tau 1

Tau 4

Tau 16

0

1 K8006004002000

1 K8006004002000

1 K8006004002000


SSC-

H

1 K

800

600

400

200

0

SSC-

H

1 K

800

600

400

200

0

SSC-

H

1 K

800

600

400

200

0

SSC-

H

FSC-H

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Caspase 3

CM (4 hours)

Con

trol

Con

trol

Tau

4Ta

u 4

(a) (b) (d)(c)





ontro

lC

ontro

l

Light

(2)

(3)

(4)

(5)(1)

3D

3D

PI


(a)

Gua

12

5G

ua 1

25

Light

3D

PI


(2)

(3)

(4)

(5)(1)

3D

(b)

Caf 0

5Ca

f 05

Light

3D

3D

PI


(2)

(3)

(4)

(5)(1)

(c)







ControlPI

PI120573

-III

-tubu

linD

API

(1)

(2)

(a)

PIPI

120573-I

II-tu

bulin

DA

PI

Gua 125(1)

(2)

(b)PI

PI120573

-III

-tubu

linD

API

Caf 05(1)

(2)

(c)

PIPI

120573-I

II-tu

bulin

DA

PI

Tau 4(1)

(2)

(d)









15

10

5

0


TRAP TRAP

TAR

Con

trol

312

5

625

125 25 50

Con

trol

312

5

625

125 25 50

Control3125625

1252550

Control3125625

1252550

3125 625 125 25 500

120

100

80

60

40

20

0

120

100

80

60

40

20

0

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(mgmL)

(mgmL)

(mgmL)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


Gua

rana

120

90

60

30

00 5 10 15 20 25 30

Gua

(mg

mL)

200

150

100

50

00 30 60 90 120

Radi

cal p

rodu

ctio

n (

)

Gua

rana

(1)

(2)

(3)

(4)

(5)

TAR

(1198680119868

)

(a)

120

100

80

60

40

20

0

120

90

60

30

00 5 10 15 20 25 30


TRAPTRAP

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

Con

trol

012

5

025 0

5 1 2

(mgmL)

Con

trol

012

5

025 0

5 1 2

0125 025 05 1 20

(mgmL)

Control0125025

0512

Control0125025

0512

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

120

100

80

60

40

20

0

TAR

5

4

3

2

1

0

(mgmL)

Caffe

ine

Caffe

ine

TAR

(1198680119868

)

Radi

cal p

rodu

ctio

n (

)Pe

rcen

tage

of A

UC

(in re

latio

n to

cont

rol)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(1)

(2)

(3)

(4)

(5)

(b)

Figure 7 Continued


TAR

(1198680119868

)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Radi

cal p

rodu

ctio

n (

)

120

90

60

30

00 5 10 15 20 25 30

Time (min)

Con

trol 1 2 4 8 16

(mgmL)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Con

trol 1 2 4 8 16

120

100

80

60

40

20

0

(mgmL)

Time (min)

TRAPTRAP

Control12

4816

Control12

4816

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

TAR

1 2 4 8 160(mgmL)

5

4

3

2

1

0

Taur

ine

Taur

ine

160140120100

80604020

0

(1)

(2)

(3)

(4)

(5)

(c)









(1) (2)

(4) (5)

(9)(7) (8)

(6)

(3)

30 120583m

Gua 125 + Caf 05 Gua 125 + Tau 4

Gua 125 + Caf 05 + Tau 4


Tau 4


SH-S

Y5Y

cells

0

FBS

+co

mbi

ning

trea

tmen

t (m

gm

L 2

hour

s)

PCM

(400

x)

(a)

500

100

80

60

40

20

0

Intr

acel

lula

r RO

S pr

oduc

tion

( in

relat

ion

to co

ntro

l)

Con

trol

Gua

12

5

Caf 0

5

Tau

4

2 hours(mgmL)

H2O2

(1 m

Moplus

cont

rol)

lowastlowastlowast


Gua

125+

Caf0

5

Gua

125+

Tau4

Caf0

5+

Tau4

Caf1

+Ta

u4

Gua

12

5+

Caf 0

5+

Tau

4

(b)







4 Discussion





50

40

30

20

10

0

03

125

625

125 25 50

03

125

625

125 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

Gua

rana

U S

OD

mg

prot

ein

SOD CAT


GPx

GST

(1) (2) (3)

(4) (5) (6)



lowastlowast


4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

Gua

rana

U G

STm

g pr

otei

n

8

6

4

2

0

04

03

02

01

00

SH TBARS



ARS

(nm

ol T

MP

mg

prot

ein)

75

50

25

0

020

015

010

005

000


mol

SH

mg

prot

ein


(a)

50

40

30

20

10

0

00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2 00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2

Caffe

ine

U S

OD

mg

prot

ein

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

Caffe

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000


120583m

ol S

Hm

g pr

otei

n



SOD CAT GPx

GST SH TBARS

(b)

Figure 9 Continued


50

40

30

20

10

0

Taur

ine

U S

OD

mg

prot

ein

0 1 2 4 8 16

0 1 2 4 8 16

0 1 2 4 8 16 0 1 2 4 8 16

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

(mgmL)

0 1 2 4 8 16

(mgmL)

0 1 2 4 8 16

(mgmL) 2 hours

Taur

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000

120583m

ol S

Hm

g pr

otei

n


(1) (2) (3)


SOD CAT GPx

GST SH TBARS

(c)






SOD50

40

30

20

10

0

U S

OD

mg

prot

ein

lowastlowastlowast


lowastlowast

(a)

CAT4

3

2

1

0

U C

ATm

g pr

otei

n


(b)

GPx8

6

4

2

0

U G

Pxm

g pr

otei

n

(c)

GST04

03

02

01

00

U G

STm

g pr

otei

n

(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(d)

SH


(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

75

50

25

0

120583m

ol S

Hm

g pr

otei

n

(e)

TBARS

lowast

(mgmL)

020

015

010

005

000

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(f)






Con

trol (

1)

Con

trol (

2)


Gua

31

25

Gua

12

5

Gua

50

Caf 0

125

Caf 0

5

Caf 2

Tau

1

Tau

4

Tau

16

Gua

12

5+

Caf 0

5

Gua

12

5+

Tau

4

Caf 0

5+

Tau

4

Caf 1

+Ta

u 4

Gua

12

5+

Caf 0

5+

Tau

4


Tota

l cel

l cou

nt (times

106)

20

15

10

05

00

Control


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast



(mgmL)

(a)

PCM

(200

x)





(1) (3)(2) (4)

(5)

(10)(9) (11) (12)

(13)

(6) (8)(7)

SH-S

Y5Y

cells

10

FBS

+dr

ug(s

) tre

atm

ent (

mg

mL

24 h

ours

)

(14) (15) (16)

(b)



104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)


SH-SY5Y cells


Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)


Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4


Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4








(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch



Control

16

(8)

(5)

(1)

(6)

(2)

(2) (3)

(3)

(4)

(4)(4)

(1)

(2)

(3)

(1)

(4)

(2)

(3)

(1)

(7)

1

4

PCM (2 hours)

Control Control

1 1

16 16

4 4


800

600

400

200

01 K800600400200

Control

Tau 1

Tau 4

Tau 16

0

1 K8006004002000

1 K8006004002000

1 K8006004002000


SSC-

H

1 K

800

600

400

200

0

SSC-

H

1 K

800

600

400

200

0

SSC-

H

1 K

800

600

400

200

0

SSC-

H

FSC-H

Caspase 3DAPI

Caspase 3DAPI

Caspase 3

Caspase 3

CM (4 hours)

Con

trol

Con

trol

Tau

4Ta

u 4

(a) (b) (d)(c)





ontro

lC

ontro

l

Light

(2)

(3)

(4)

(5)(1)

3D

3D

PI


(a)

Gua

12

5G

ua 1

25

Light

3D

PI


(2)

(3)

(4)

(5)(1)

3D

(b)

Caf 0

5Ca

f 05

Light

3D

3D

PI


(2)

(3)

(4)

(5)(1)

(c)







ControlPI

PI120573

-III

-tubu

linD

API

(1)

(2)

(a)

PIPI

120573-I

II-tu

bulin

DA

PI

Gua 125(1)

(2)

(b)PI

PI120573

-III

-tubu

linD

API

Caf 05(1)

(2)

(c)

PIPI

120573-I

II-tu

bulin

DA

PI

Tau 4(1)

(2)

(d)









15

10

5

0


TRAP TRAP

TAR

Con

trol

312

5

625

125 25 50

Con

trol

312

5

625

125 25 50

Control3125625

1252550

Control3125625

1252550

3125 625 125 25 500

120

100

80

60

40

20

0

120

100

80

60

40

20

0

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(mgmL)

(mgmL)

(mgmL)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


Gua

rana

120

90

60

30

00 5 10 15 20 25 30

Gua

(mg

mL)

200

150

100

50

00 30 60 90 120

Radi

cal p

rodu

ctio

n (

)

Gua

rana

(1)

(2)

(3)

(4)

(5)

TAR

(1198680119868

)

(a)

120

100

80

60

40

20

0

120

90

60

30

00 5 10 15 20 25 30


TRAPTRAP

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

Con

trol

012

5

025 0

5 1 2

(mgmL)

Con

trol

012

5

025 0

5 1 2

0125 025 05 1 20

(mgmL)

Control0125025

0512

Control0125025

0512

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

120

100

80

60

40

20

0

TAR

5

4

3

2

1

0

(mgmL)

Caffe

ine

Caffe

ine

TAR

(1198680119868

)

Radi

cal p

rodu

ctio

n (

)Pe

rcen

tage

of A

UC

(in re

latio

n to

cont

rol)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(1)

(2)

(3)

(4)

(5)

(b)

Figure 7 Continued


TAR

(1198680119868

)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Radi

cal p

rodu

ctio

n (

)

120

90

60

30

00 5 10 15 20 25 30

Time (min)

Con

trol 1 2 4 8 16

(mgmL)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Con

trol 1 2 4 8 16

120

100

80

60

40

20

0

(mgmL)

Time (min)

TRAPTRAP

Control12

4816

Control12

4816

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

TAR

1 2 4 8 160(mgmL)

5

4

3

2

1

0

Taur

ine

Taur

ine

160140120100

80604020

0

(1)

(2)

(3)

(4)

(5)

(c)









(1) (2)

(4) (5)

(9)(7) (8)

(6)

(3)

30 120583m

Gua 125 + Caf 05 Gua 125 + Tau 4

Gua 125 + Caf 05 + Tau 4


Tau 4


SH-S

Y5Y

cells

0

FBS

+co

mbi

ning

trea

tmen

t (m

gm

L 2

hour

s)

PCM

(400

x)

(a)

500

100

80

60

40

20

0

Intr

acel

lula

r RO

S pr

oduc

tion

( in

relat

ion

to co

ntro

l)

Con

trol

Gua

12

5

Caf 0

5

Tau

4

2 hours(mgmL)

H2O2

(1 m

Moplus

cont

rol)

lowastlowastlowast


Gua

125+

Caf0

5

Gua

125+

Tau4

Caf0

5+

Tau4

Caf1

+Ta

u4

Gua

12

5+

Caf 0

5+

Tau

4

(b)







4 Discussion





50

40

30

20

10

0

03

125

625

125 25 50

03

125

625

125 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

Gua

rana

U S

OD

mg

prot

ein

SOD CAT


GPx

GST

(1) (2) (3)

(4) (5) (6)



lowastlowast


4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

Gua

rana

U G

STm

g pr

otei

n

8

6

4

2

0

04

03

02

01

00

SH TBARS



ARS

(nm

ol T

MP

mg

prot

ein)

75

50

25

0

020

015

010

005

000


mol

SH

mg

prot

ein


(a)

50

40

30

20

10

0

00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2 00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2

Caffe

ine

U S

OD

mg

prot

ein

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

Caffe

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000


120583m

ol S

Hm

g pr

otei

n



SOD CAT GPx

GST SH TBARS

(b)

Figure 9 Continued


50

40

30

20

10

0

Taur

ine

U S

OD

mg

prot

ein

0 1 2 4 8 16

0 1 2 4 8 16

0 1 2 4 8 16 0 1 2 4 8 16

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

(mgmL)

0 1 2 4 8 16

(mgmL)

0 1 2 4 8 16

(mgmL) 2 hours

Taur

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000

120583m

ol S

Hm

g pr

otei

n


(1) (2) (3)


SOD CAT GPx

GST SH TBARS

(c)






SOD50

40

30

20

10

0

U S

OD

mg

prot

ein

lowastlowastlowast


lowastlowast

(a)

CAT4

3

2

1

0

U C

ATm

g pr

otei

n


(b)

GPx8

6

4

2

0

U G

Pxm

g pr

otei

n

(c)

GST04

03

02

01

00

U G

STm

g pr

otei

n

(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(d)

SH


(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

75

50

25

0

120583m

ol S

Hm

g pr

otei

n

(e)

TBARS

lowast

(mgmL)

020

015

010

005

000

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(f)






Con

trol (

1)

Con

trol (

2)


Gua

31

25

Gua

12

5

Gua

50

Caf 0

125

Caf 0

5

Caf 2

Tau

1

Tau

4

Tau

16

Gua

12

5+

Caf 0

5

Gua

12

5+

Tau

4

Caf 0

5+

Tau

4

Caf 1

+Ta

u 4

Gua

12

5+

Caf 0

5+

Tau

4


Tota

l cel

l cou

nt (times

106)

20

15

10

05

00

Control


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast



(mgmL)

(a)

PCM

(200

x)





(1) (3)(2) (4)

(5)

(10)(9) (11) (12)

(13)

(6) (8)(7)

SH-S

Y5Y

cells

10

FBS

+dr

ug(s

) tre

atm

ent (

mg

mL

24 h

ours

)

(14) (15) (16)

(b)



104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)


SH-SY5Y cells


Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)


Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4


Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4








(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch



ontro

lC

ontro

l

Light

(2)

(3)

(4)

(5)(1)

3D

3D

PI


(a)

Gua

12

5G

ua 1

25

Light

3D

PI


(2)

(3)

(4)

(5)(1)

3D

(b)

Caf 0

5Ca

f 05

Light

3D

3D

PI


(2)

(3)

(4)

(5)(1)

(c)







ControlPI

PI120573

-III

-tubu

linD

API

(1)

(2)

(a)

PIPI

120573-I

II-tu

bulin

DA

PI

Gua 125(1)

(2)

(b)PI

PI120573

-III

-tubu

linD

API

Caf 05(1)

(2)

(c)

PIPI

120573-I

II-tu

bulin

DA

PI

Tau 4(1)

(2)

(d)









15

10

5

0


TRAP TRAP

TAR

Con

trol

312

5

625

125 25 50

Con

trol

312

5

625

125 25 50

Control3125625

1252550

Control3125625

1252550

3125 625 125 25 500

120

100

80

60

40

20

0

120

100

80

60

40

20

0

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(mgmL)

(mgmL)

(mgmL)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


Gua

rana

120

90

60

30

00 5 10 15 20 25 30

Gua

(mg

mL)

200

150

100

50

00 30 60 90 120

Radi

cal p

rodu

ctio

n (

)

Gua

rana

(1)

(2)

(3)

(4)

(5)

TAR

(1198680119868

)

(a)

120

100

80

60

40

20

0

120

90

60

30

00 5 10 15 20 25 30


TRAPTRAP

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

Con

trol

012

5

025 0

5 1 2

(mgmL)

Con

trol

012

5

025 0

5 1 2

0125 025 05 1 20

(mgmL)

Control0125025

0512

Control0125025

0512

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

120

100

80

60

40

20

0

TAR

5

4

3

2

1

0

(mgmL)

Caffe

ine

Caffe

ine

TAR

(1198680119868

)

Radi

cal p

rodu

ctio

n (

)Pe

rcen

tage

of A

UC

(in re

latio

n to

cont

rol)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(1)

(2)

(3)

(4)

(5)

(b)

Figure 7 Continued


TAR

(1198680119868

)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Radi

cal p

rodu

ctio

n (

)

120

90

60

30

00 5 10 15 20 25 30

Time (min)

Con

trol 1 2 4 8 16

(mgmL)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Con

trol 1 2 4 8 16

120

100

80

60

40

20

0

(mgmL)

Time (min)

TRAPTRAP

Control12

4816

Control12

4816

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

TAR

1 2 4 8 160(mgmL)

5

4

3

2

1

0

Taur

ine

Taur

ine

160140120100

80604020

0

(1)

(2)

(3)

(4)

(5)

(c)









(1) (2)

(4) (5)

(9)(7) (8)

(6)

(3)

30 120583m

Gua 125 + Caf 05 Gua 125 + Tau 4

Gua 125 + Caf 05 + Tau 4


Tau 4


SH-S

Y5Y

cells

0

FBS

+co

mbi

ning

trea

tmen

t (m

gm

L 2

hour

s)

PCM

(400

x)

(a)

500

100

80

60

40

20

0

Intr

acel

lula

r RO

S pr

oduc

tion

( in

relat

ion

to co

ntro

l)

Con

trol

Gua

12

5

Caf 0

5

Tau

4

2 hours(mgmL)

H2O2

(1 m

Moplus

cont

rol)

lowastlowastlowast


Gua

125+

Caf0

5

Gua

125+

Tau4

Caf0

5+

Tau4

Caf1

+Ta

u4

Gua

12

5+

Caf 0

5+

Tau

4

(b)







4 Discussion





50

40

30

20

10

0

03

125

625

125 25 50

03

125

625

125 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

Gua

rana

U S

OD

mg

prot

ein

SOD CAT


GPx

GST

(1) (2) (3)

(4) (5) (6)



lowastlowast


4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

Gua

rana

U G

STm

g pr

otei

n

8

6

4

2

0

04

03

02

01

00

SH TBARS



ARS

(nm

ol T

MP

mg

prot

ein)

75

50

25

0

020

015

010

005

000


mol

SH

mg

prot

ein


(a)

50

40

30

20

10

0

00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2 00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2

Caffe

ine

U S

OD

mg

prot

ein

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

Caffe

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000


120583m

ol S

Hm

g pr

otei

n



SOD CAT GPx

GST SH TBARS

(b)

Figure 9 Continued


50

40

30

20

10

0

Taur

ine

U S

OD

mg

prot

ein

0 1 2 4 8 16

0 1 2 4 8 16

0 1 2 4 8 16 0 1 2 4 8 16

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

(mgmL)

0 1 2 4 8 16

(mgmL)

0 1 2 4 8 16

(mgmL) 2 hours

Taur

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000

120583m

ol S

Hm

g pr

otei

n


(1) (2) (3)


SOD CAT GPx

GST SH TBARS

(c)






SOD50

40

30

20

10

0

U S

OD

mg

prot

ein

lowastlowastlowast


lowastlowast

(a)

CAT4

3

2

1

0

U C

ATm

g pr

otei

n


(b)

GPx8

6

4

2

0

U G

Pxm

g pr

otei

n

(c)

GST04

03

02

01

00

U G

STm

g pr

otei

n

(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(d)

SH


(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

75

50

25

0

120583m

ol S

Hm

g pr

otei

n

(e)

TBARS

lowast

(mgmL)

020

015

010

005

000

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(f)






Con

trol (

1)

Con

trol (

2)


Gua

31

25

Gua

12

5

Gua

50

Caf 0

125

Caf 0

5

Caf 2

Tau

1

Tau

4

Tau

16

Gua

12

5+

Caf 0

5

Gua

12

5+

Tau

4

Caf 0

5+

Tau

4

Caf 1

+Ta

u 4

Gua

12

5+

Caf 0

5+

Tau

4


Tota

l cel

l cou

nt (times

106)

20

15

10

05

00

Control


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast



(mgmL)

(a)

PCM

(200

x)





(1) (3)(2) (4)

(5)

(10)(9) (11) (12)

(13)

(6) (8)(7)

SH-S

Y5Y

cells

10

FBS

+dr

ug(s

) tre

atm

ent (

mg

mL

24 h

ours

)

(14) (15) (16)

(b)



104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)


SH-SY5Y cells


Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)


Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4


Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4








(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch



ControlPI

PI120573

-III

-tubu

linD

API

(1)

(2)

(a)

PIPI

120573-I

II-tu

bulin

DA

PI

Gua 125(1)

(2)

(b)PI

PI120573

-III

-tubu

linD

API

Caf 05(1)

(2)

(c)

PIPI

120573-I

II-tu

bulin

DA

PI

Tau 4(1)

(2)

(d)









15

10

5

0


TRAP TRAP

TAR

Con

trol

312

5

625

125 25 50

Con

trol

312

5

625

125 25 50

Control3125625

1252550

Control3125625

1252550

3125 625 125 25 500

120

100

80

60

40

20

0

120

100

80

60

40

20

0

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(mgmL)

(mgmL)

(mgmL)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


Gua

rana

120

90

60

30

00 5 10 15 20 25 30

Gua

(mg

mL)

200

150

100

50

00 30 60 90 120

Radi

cal p

rodu

ctio

n (

)

Gua

rana

(1)

(2)

(3)

(4)

(5)

TAR

(1198680119868

)

(a)

120

100

80

60

40

20

0

120

90

60

30

00 5 10 15 20 25 30


TRAPTRAP

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

Con

trol

012

5

025 0

5 1 2

(mgmL)

Con

trol

012

5

025 0

5 1 2

0125 025 05 1 20

(mgmL)

Control0125025

0512

Control0125025

0512

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

120

100

80

60

40

20

0

TAR

5

4

3

2

1

0

(mgmL)

Caffe

ine

Caffe

ine

TAR

(1198680119868

)

Radi

cal p

rodu

ctio

n (

)Pe

rcen

tage

of A

UC

(in re

latio

n to

cont

rol)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(1)

(2)

(3)

(4)

(5)

(b)

Figure 7 Continued


TAR

(1198680119868

)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Radi

cal p

rodu

ctio

n (

)

120

90

60

30

00 5 10 15 20 25 30

Time (min)

Con

trol 1 2 4 8 16

(mgmL)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Con

trol 1 2 4 8 16

120

100

80

60

40

20

0

(mgmL)

Time (min)

TRAPTRAP

Control12

4816

Control12

4816

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

TAR

1 2 4 8 160(mgmL)

5

4

3

2

1

0

Taur

ine

Taur

ine

160140120100

80604020

0

(1)

(2)

(3)

(4)

(5)

(c)









(1) (2)

(4) (5)

(9)(7) (8)

(6)

(3)

30 120583m

Gua 125 + Caf 05 Gua 125 + Tau 4

Gua 125 + Caf 05 + Tau 4


Tau 4


SH-S

Y5Y

cells

0

FBS

+co

mbi

ning

trea

tmen

t (m

gm

L 2

hour

s)

PCM

(400

x)

(a)

500

100

80

60

40

20

0

Intr

acel

lula

r RO

S pr

oduc

tion

( in

relat

ion

to co

ntro

l)

Con

trol

Gua

12

5

Caf 0

5

Tau

4

2 hours(mgmL)

H2O2

(1 m

Moplus

cont

rol)

lowastlowastlowast


Gua

125+

Caf0

5

Gua

125+

Tau4

Caf0

5+

Tau4

Caf1

+Ta

u4

Gua

12

5+

Caf 0

5+

Tau

4

(b)







4 Discussion





50

40

30

20

10

0

03

125

625

125 25 50

03

125

625

125 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

Gua

rana

U S

OD

mg

prot

ein

SOD CAT


GPx

GST

(1) (2) (3)

(4) (5) (6)



lowastlowast


4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

Gua

rana

U G

STm

g pr

otei

n

8

6

4

2

0

04

03

02

01

00

SH TBARS



ARS

(nm

ol T

MP

mg

prot

ein)

75

50

25

0

020

015

010

005

000


mol

SH

mg

prot

ein


(a)

50

40

30

20

10

0

00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2 00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2

Caffe

ine

U S

OD

mg

prot

ein

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

Caffe

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000


120583m

ol S

Hm

g pr

otei

n



SOD CAT GPx

GST SH TBARS

(b)

Figure 9 Continued


50

40

30

20

10

0

Taur

ine

U S

OD

mg

prot

ein

0 1 2 4 8 16

0 1 2 4 8 16

0 1 2 4 8 16 0 1 2 4 8 16

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

(mgmL)

0 1 2 4 8 16

(mgmL)

0 1 2 4 8 16

(mgmL) 2 hours

Taur

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000

120583m

ol S

Hm

g pr

otei

n


(1) (2) (3)


SOD CAT GPx

GST SH TBARS

(c)






SOD50

40

30

20

10

0

U S

OD

mg

prot

ein

lowastlowastlowast


lowastlowast

(a)

CAT4

3

2

1

0

U C

ATm

g pr

otei

n


(b)

GPx8

6

4

2

0

U G

Pxm

g pr

otei

n

(c)

GST04

03

02

01

00

U G

STm

g pr

otei

n

(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(d)

SH


(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

75

50

25

0

120583m

ol S

Hm

g pr

otei

n

(e)

TBARS

lowast

(mgmL)

020

015

010

005

000

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(f)






Con

trol (

1)

Con

trol (

2)


Gua

31

25

Gua

12

5

Gua

50

Caf 0

125

Caf 0

5

Caf 2

Tau

1

Tau

4

Tau

16

Gua

12

5+

Caf 0

5

Gua

12

5+

Tau

4

Caf 0

5+

Tau

4

Caf 1

+Ta

u 4

Gua

12

5+

Caf 0

5+

Tau

4


Tota

l cel

l cou

nt (times

106)

20

15

10

05

00

Control


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast



(mgmL)

(a)

PCM

(200

x)





(1) (3)(2) (4)

(5)

(10)(9) (11) (12)

(13)

(6) (8)(7)

SH-S

Y5Y

cells

10

FBS

+dr

ug(s

) tre

atm

ent (

mg

mL

24 h

ours

)

(14) (15) (16)

(b)



104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)


SH-SY5Y cells


Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)


Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4


Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4








(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch



15

10

5

0


TRAP TRAP

TAR

Con

trol

312

5

625

125 25 50

Con

trol

312

5

625

125 25 50

Control3125625

1252550

Control3125625

1252550

3125 625 125 25 500

120

100

80

60

40

20

0

120

100

80

60

40

20

0

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(mgmL)

(mgmL)

(mgmL)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast


Gua

rana

120

90

60

30

00 5 10 15 20 25 30

Gua

(mg

mL)

200

150

100

50

00 30 60 90 120

Radi

cal p

rodu

ctio

n (

)

Gua

rana

(1)

(2)

(3)

(4)

(5)

TAR

(1198680119868

)

(a)

120

100

80

60

40

20

0

120

90

60

30

00 5 10 15 20 25 30


TRAPTRAP

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

Con

trol

012

5

025 0

5 1 2

(mgmL)

Con

trol

012

5

025 0

5 1 2

0125 025 05 1 20

(mgmL)

Control0125025

0512

Control0125025

0512

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

120

100

80

60

40

20

0

TAR

5

4

3

2

1

0

(mgmL)

Caffe

ine

Caffe

ine

TAR

(1198680119868

)

Radi

cal p

rodu

ctio

n (

)Pe

rcen

tage

of A

UC

(in re

latio

n to

cont

rol)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

(1)

(2)

(3)

(4)

(5)

(b)

Figure 7 Continued


TAR

(1198680119868

)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Radi

cal p

rodu

ctio

n (

)

120

90

60

30

00 5 10 15 20 25 30

Time (min)

Con

trol 1 2 4 8 16

(mgmL)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Con

trol 1 2 4 8 16

120

100

80

60

40

20

0

(mgmL)

Time (min)

TRAPTRAP

Control12

4816

Control12

4816

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

TAR

1 2 4 8 160(mgmL)

5

4

3

2

1

0

Taur

ine

Taur

ine

160140120100

80604020

0

(1)

(2)

(3)

(4)

(5)

(c)









(1) (2)

(4) (5)

(9)(7) (8)

(6)

(3)

30 120583m

Gua 125 + Caf 05 Gua 125 + Tau 4

Gua 125 + Caf 05 + Tau 4


Tau 4


SH-S

Y5Y

cells

0

FBS

+co

mbi

ning

trea

tmen

t (m

gm

L 2

hour

s)

PCM

(400

x)

(a)

500

100

80

60

40

20

0

Intr

acel

lula

r RO

S pr

oduc

tion

( in

relat

ion

to co

ntro

l)

Con

trol

Gua

12

5

Caf 0

5

Tau

4

2 hours(mgmL)

H2O2

(1 m

Moplus

cont

rol)

lowastlowastlowast


Gua

125+

Caf0

5

Gua

125+

Tau4

Caf0

5+

Tau4

Caf1

+Ta

u4

Gua

12

5+

Caf 0

5+

Tau

4

(b)







4 Discussion





50

40

30

20

10

0

03

125

625

125 25 50

03

125

625

125 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

Gua

rana

U S

OD

mg

prot

ein

SOD CAT


GPx

GST

(1) (2) (3)

(4) (5) (6)



lowastlowast


4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

Gua

rana

U G

STm

g pr

otei

n

8

6

4

2

0

04

03

02

01

00

SH TBARS



ARS

(nm

ol T

MP

mg

prot

ein)

75

50

25

0

020

015

010

005

000


mol

SH

mg

prot

ein


(a)

50

40

30

20

10

0

00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2 00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2

Caffe

ine

U S

OD

mg

prot

ein

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

Caffe

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000


120583m

ol S

Hm

g pr

otei

n



SOD CAT GPx

GST SH TBARS

(b)

Figure 9 Continued


50

40

30

20

10

0

Taur

ine

U S

OD

mg

prot

ein

0 1 2 4 8 16

0 1 2 4 8 16

0 1 2 4 8 16 0 1 2 4 8 16

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

(mgmL)

0 1 2 4 8 16

(mgmL)

0 1 2 4 8 16

(mgmL) 2 hours

Taur

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000

120583m

ol S

Hm

g pr

otei

n


(1) (2) (3)


SOD CAT GPx

GST SH TBARS

(c)






SOD50

40

30

20

10

0

U S

OD

mg

prot

ein

lowastlowastlowast


lowastlowast

(a)

CAT4

3

2

1

0

U C

ATm

g pr

otei

n


(b)

GPx8

6

4

2

0

U G

Pxm

g pr

otei

n

(c)

GST04

03

02

01

00

U G

STm

g pr

otei

n

(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(d)

SH


(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

75

50

25

0

120583m

ol S

Hm

g pr

otei

n

(e)

TBARS

lowast

(mgmL)

020

015

010

005

000

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(f)






Con

trol (

1)

Con

trol (

2)


Gua

31

25

Gua

12

5

Gua

50

Caf 0

125

Caf 0

5

Caf 2

Tau

1

Tau

4

Tau

16

Gua

12

5+

Caf 0

5

Gua

12

5+

Tau

4

Caf 0

5+

Tau

4

Caf 1

+Ta

u 4

Gua

12

5+

Caf 0

5+

Tau

4


Tota

l cel

l cou

nt (times

106)

20

15

10

05

00

Control


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast



(mgmL)

(a)

PCM

(200

x)





(1) (3)(2) (4)

(5)

(10)(9) (11) (12)

(13)

(6) (8)(7)

SH-S

Y5Y

cells

10

FBS

+dr

ug(s

) tre

atm

ent (

mg

mL

24 h

ours

)

(14) (15) (16)

(b)



104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)


SH-SY5Y cells


Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)


Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4


Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4








(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch



TAR

(1198680119868

)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Perc

enta

ge o

f AU

C(in

relat

ion

to co

ntro

l)

Radi

cal p

rodu

ctio

n (

)

120

90

60

30

00 5 10 15 20 25 30

Time (min)

Con

trol 1 2 4 8 16

(mgmL)

lowastlowast

lowastlowast

lowast

lowastlowastlowast

Con

trol 1 2 4 8 16

120

100

80

60

40

20

0

(mgmL)

Time (min)

TRAPTRAP

Control12

4816

Control12

4816

Caf (

mg

mL)

200

150

100

50

00 30 60 90 120

TAR

1 2 4 8 160(mgmL)

5

4

3

2

1

0

Taur

ine

Taur

ine

160140120100

80604020

0

(1)

(2)

(3)

(4)

(5)

(c)









(1) (2)

(4) (5)

(9)(7) (8)

(6)

(3)

30 120583m

Gua 125 + Caf 05 Gua 125 + Tau 4

Gua 125 + Caf 05 + Tau 4


Tau 4


SH-S

Y5Y

cells

0

FBS

+co

mbi

ning

trea

tmen

t (m

gm

L 2

hour

s)

PCM

(400

x)

(a)

500

100

80

60

40

20

0

Intr

acel

lula

r RO

S pr

oduc

tion

( in

relat

ion

to co

ntro

l)

Con

trol

Gua

12

5

Caf 0

5

Tau

4

2 hours(mgmL)

H2O2

(1 m

Moplus

cont

rol)

lowastlowastlowast


Gua

125+

Caf0

5

Gua

125+

Tau4

Caf0

5+

Tau4

Caf1

+Ta

u4

Gua

12

5+

Caf 0

5+

Tau

4

(b)







4 Discussion





50

40

30

20

10

0

03

125

625

125 25 50

03

125

625

125 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

Gua

rana

U S

OD

mg

prot

ein

SOD CAT


GPx

GST

(1) (2) (3)

(4) (5) (6)



lowastlowast


4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

Gua

rana

U G

STm

g pr

otei

n

8

6

4

2

0

04

03

02

01

00

SH TBARS



ARS

(nm

ol T

MP

mg

prot

ein)

75

50

25

0

020

015

010

005

000


mol

SH

mg

prot

ein


(a)

50

40

30

20

10

0

00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2 00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2

Caffe

ine

U S

OD

mg

prot

ein

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

Caffe

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000


120583m

ol S

Hm

g pr

otei

n



SOD CAT GPx

GST SH TBARS

(b)

Figure 9 Continued


50

40

30

20

10

0

Taur

ine

U S

OD

mg

prot

ein

0 1 2 4 8 16

0 1 2 4 8 16

0 1 2 4 8 16 0 1 2 4 8 16

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

(mgmL)

0 1 2 4 8 16

(mgmL)

0 1 2 4 8 16

(mgmL) 2 hours

Taur

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000

120583m

ol S

Hm

g pr

otei

n


(1) (2) (3)


SOD CAT GPx

GST SH TBARS

(c)






SOD50

40

30

20

10

0

U S

OD

mg

prot

ein

lowastlowastlowast


lowastlowast

(a)

CAT4

3

2

1

0

U C

ATm

g pr

otei

n


(b)

GPx8

6

4

2

0

U G

Pxm

g pr

otei

n

(c)

GST04

03

02

01

00

U G

STm

g pr

otei

n

(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(d)

SH


(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

75

50

25

0

120583m

ol S

Hm

g pr

otei

n

(e)

TBARS

lowast

(mgmL)

020

015

010

005

000

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(f)






Con

trol (

1)

Con

trol (

2)


Gua

31

25

Gua

12

5

Gua

50

Caf 0

125

Caf 0

5

Caf 2

Tau

1

Tau

4

Tau

16

Gua

12

5+

Caf 0

5

Gua

12

5+

Tau

4

Caf 0

5+

Tau

4

Caf 1

+Ta

u 4

Gua

12

5+

Caf 0

5+

Tau

4


Tota

l cel

l cou

nt (times

106)

20

15

10

05

00

Control


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast



(mgmL)

(a)

PCM

(200

x)





(1) (3)(2) (4)

(5)

(10)(9) (11) (12)

(13)

(6) (8)(7)

SH-S

Y5Y

cells

10

FBS

+dr

ug(s

) tre

atm

ent (

mg

mL

24 h

ours

)

(14) (15) (16)

(b)



104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)


SH-SY5Y cells


Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)


Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4


Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4








(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch



(1) (2)

(4) (5)

(9)(7) (8)

(6)

(3)

30 120583m

Gua 125 + Caf 05 Gua 125 + Tau 4

Gua 125 + Caf 05 + Tau 4


Tau 4


SH-S

Y5Y

cells

0

FBS

+co

mbi

ning

trea

tmen

t (m

gm

L 2

hour

s)

PCM

(400

x)

(a)

500

100

80

60

40

20

0

Intr

acel

lula

r RO

S pr

oduc

tion

( in

relat

ion

to co

ntro

l)

Con

trol

Gua

12

5

Caf 0

5

Tau

4

2 hours(mgmL)

H2O2

(1 m

Moplus

cont

rol)

lowastlowastlowast


Gua

125+

Caf0

5

Gua

125+

Tau4

Caf0

5+

Tau4

Caf1

+Ta

u4

Gua

12

5+

Caf 0

5+

Tau

4

(b)







4 Discussion





50

40

30

20

10

0

03

125

625

125 25 50

03

125

625

125 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

Gua

rana

U S

OD

mg

prot

ein

SOD CAT


GPx

GST

(1) (2) (3)

(4) (5) (6)



lowastlowast


4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

Gua

rana

U G

STm

g pr

otei

n

8

6

4

2

0

04

03

02

01

00

SH TBARS



ARS

(nm

ol T

MP

mg

prot

ein)

75

50

25

0

020

015

010

005

000


mol

SH

mg

prot

ein


(a)

50

40

30

20

10

0

00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2 00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2

Caffe

ine

U S

OD

mg

prot

ein

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

Caffe

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000


120583m

ol S

Hm

g pr

otei

n



SOD CAT GPx

GST SH TBARS

(b)

Figure 9 Continued


50

40

30

20

10

0

Taur

ine

U S

OD

mg

prot

ein

0 1 2 4 8 16

0 1 2 4 8 16

0 1 2 4 8 16 0 1 2 4 8 16

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

(mgmL)

0 1 2 4 8 16

(mgmL)

0 1 2 4 8 16

(mgmL) 2 hours

Taur

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000

120583m

ol S

Hm

g pr

otei

n


(1) (2) (3)


SOD CAT GPx

GST SH TBARS

(c)






SOD50

40

30

20

10

0

U S

OD

mg

prot

ein

lowastlowastlowast


lowastlowast

(a)

CAT4

3

2

1

0

U C

ATm

g pr

otei

n


(b)

GPx8

6

4

2

0

U G

Pxm

g pr

otei

n

(c)

GST04

03

02

01

00

U G

STm

g pr

otei

n

(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(d)

SH


(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

75

50

25

0

120583m

ol S

Hm

g pr

otei

n

(e)

TBARS

lowast

(mgmL)

020

015

010

005

000

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(f)






Con

trol (

1)

Con

trol (

2)


Gua

31

25

Gua

12

5

Gua

50

Caf 0

125

Caf 0

5

Caf 2

Tau

1

Tau

4

Tau

16

Gua

12

5+

Caf 0

5

Gua

12

5+

Tau

4

Caf 0

5+

Tau

4

Caf 1

+Ta

u 4

Gua

12

5+

Caf 0

5+

Tau

4


Tota

l cel

l cou

nt (times

106)

20

15

10

05

00

Control


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast



(mgmL)

(a)

PCM

(200

x)





(1) (3)(2) (4)

(5)

(10)(9) (11) (12)

(13)

(6) (8)(7)

SH-S

Y5Y

cells

10

FBS

+dr

ug(s

) tre

atm

ent (

mg

mL

24 h

ours

)

(14) (15) (16)

(b)



104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)


SH-SY5Y cells


Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)


Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4


Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4








(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch



50

40

30

20

10

0

03

125

625

125 25 50

03

125

625

125 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

03

125

625

125 25 50 0

312

56

2512

5 25 50

Gua

rana

U S

OD

mg

prot

ein

SOD CAT


GPx

GST

(1) (2) (3)

(4) (5) (6)



lowastlowast


4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

Gua

rana

U G

STm

g pr

otei

n

8

6

4

2

0

04

03

02

01

00

SH TBARS



ARS

(nm

ol T

MP

mg

prot

ein)

75

50

25

0

020

015

010

005

000


mol

SH

mg

prot

ein


(a)

50

40

30

20

10

0

00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2 00

125

025 0

5 1 2

00

125

025 0

5 1 2 00

125

025 0

5 1 2

Caffe

ine

U S

OD

mg

prot

ein

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

Caffe

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000


120583m

ol S

Hm

g pr

otei

n



SOD CAT GPx

GST SH TBARS

(b)

Figure 9 Continued


50

40

30

20

10

0

Taur

ine

U S

OD

mg

prot

ein

0 1 2 4 8 16

0 1 2 4 8 16

0 1 2 4 8 16 0 1 2 4 8 16

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

(mgmL)

0 1 2 4 8 16

(mgmL)

0 1 2 4 8 16

(mgmL) 2 hours

Taur

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000

120583m

ol S

Hm

g pr

otei

n


(1) (2) (3)


SOD CAT GPx

GST SH TBARS

(c)






SOD50

40

30

20

10

0

U S

OD

mg

prot

ein

lowastlowastlowast


lowastlowast

(a)

CAT4

3

2

1

0

U C

ATm

g pr

otei

n


(b)

GPx8

6

4

2

0

U G

Pxm

g pr

otei

n

(c)

GST04

03

02

01

00

U G

STm

g pr

otei

n

(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(d)

SH


(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

75

50

25

0

120583m

ol S

Hm

g pr

otei

n

(e)

TBARS

lowast

(mgmL)

020

015

010

005

000

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(f)






Con

trol (

1)

Con

trol (

2)


Gua

31

25

Gua

12

5

Gua

50

Caf 0

125

Caf 0

5

Caf 2

Tau

1

Tau

4

Tau

16

Gua

12

5+

Caf 0

5

Gua

12

5+

Tau

4

Caf 0

5+

Tau

4

Caf 1

+Ta

u 4

Gua

12

5+

Caf 0

5+

Tau

4


Tota

l cel

l cou

nt (times

106)

20

15

10

05

00

Control


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast



(mgmL)

(a)

PCM

(200

x)





(1) (3)(2) (4)

(5)

(10)(9) (11) (12)

(13)

(6) (8)(7)

SH-S

Y5Y

cells

10

FBS

+dr

ug(s

) tre

atm

ent (

mg

mL

24 h

ours

)

(14) (15) (16)

(b)



104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)


SH-SY5Y cells


Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)


Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4


Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4








(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch



50

40

30

20

10

0

Taur

ine

U S

OD

mg

prot

ein

0 1 2 4 8 16

0 1 2 4 8 16

0 1 2 4 8 16 0 1 2 4 8 16

4

3

2

1

0

U C

ATm

g pr

otei

n

U G

Pxm

g pr

otei

n

8

6

4

2

0

(mgmL)

0 1 2 4 8 16

(mgmL)

0 1 2 4 8 16

(mgmL) 2 hours

Taur

ine

U G

STm

g pr

otei

n

04

03

02

01

00

75

50

25

0

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

020

015

010

005

000

120583m

ol S

Hm

g pr

otei

n


(1) (2) (3)


SOD CAT GPx

GST SH TBARS

(c)






SOD50

40

30

20

10

0

U S

OD

mg

prot

ein

lowastlowastlowast


lowastlowast

(a)

CAT4

3

2

1

0

U C

ATm

g pr

otei

n


(b)

GPx8

6

4

2

0

U G

Pxm

g pr

otei

n

(c)

GST04

03

02

01

00

U G

STm

g pr

otei

n

(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(d)

SH


(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

75

50

25

0

120583m

ol S

Hm

g pr

otei

n

(e)

TBARS

lowast

(mgmL)

020

015

010

005

000

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(f)






Con

trol (

1)

Con

trol (

2)


Gua

31

25

Gua

12

5

Gua

50

Caf 0

125

Caf 0

5

Caf 2

Tau

1

Tau

4

Tau

16

Gua

12

5+

Caf 0

5

Gua

12

5+

Tau

4

Caf 0

5+

Tau

4

Caf 1

+Ta

u 4

Gua

12

5+

Caf 0

5+

Tau

4


Tota

l cel

l cou

nt (times

106)

20

15

10

05

00

Control


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast



(mgmL)

(a)

PCM

(200

x)





(1) (3)(2) (4)

(5)

(10)(9) (11) (12)

(13)

(6) (8)(7)

SH-S

Y5Y

cells

10

FBS

+dr

ug(s

) tre

atm

ent (

mg

mL

24 h

ours

)

(14) (15) (16)

(b)



104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)


SH-SY5Y cells


Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)


Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4


Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4








(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch



SOD50

40

30

20

10

0

U S

OD

mg

prot

ein

lowastlowastlowast


lowastlowast

(a)

CAT4

3

2

1

0

U C

ATm

g pr

otei

n


(b)

GPx8

6

4

2

0

U G

Pxm

g pr

otei

n

(c)

GST04

03

02

01

00

U G

STm

g pr

otei

n

(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(d)

SH


(mgmL)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

75

50

25

0

120583m

ol S

Hm

g pr

otei

n

(e)

TBARS

lowast

(mgmL)

020

015

010

005

000

TBA

RS(n

mol

TM

Pm

g pr

otei

n)

Con

trol

Gua

12

5Ca

f 05

Caf 1

Tau

4

Gua

12

5+

Caf 0

5G

ua 1

25+

Tau

4Ca

f 05+

Tau

4Ca

f 1+

Tau

4G

ua 1

25+

Caf 0

5+

Tau

4

(f)






Con

trol (

1)

Con

trol (

2)


Gua

31

25

Gua

12

5

Gua

50

Caf 0

125

Caf 0

5

Caf 2

Tau

1

Tau

4

Tau

16

Gua

12

5+

Caf 0

5

Gua

12

5+

Tau

4

Caf 0

5+

Tau

4

Caf 1

+Ta

u 4

Gua

12

5+

Caf 0

5+

Tau

4


Tota

l cel

l cou

nt (times

106)

20

15

10

05

00

Control


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast



(mgmL)

(a)

PCM

(200

x)





(1) (3)(2) (4)

(5)

(10)(9) (11) (12)

(13)

(6) (8)(7)

SH-S

Y5Y

cells

10

FBS

+dr

ug(s

) tre

atm

ent (

mg

mL

24 h

ours

)

(14) (15) (16)

(b)



104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)


SH-SY5Y cells


Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)


Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4


Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4








(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch



Con

trol (

1)

Con

trol (

2)


Gua

31

25

Gua

12

5

Gua

50

Caf 0

125

Caf 0

5

Caf 2

Tau

1

Tau

4

Tau

16

Gua

12

5+

Caf 0

5

Gua

12

5+

Tau

4

Caf 0

5+

Tau

4

Caf 1

+Ta

u 4

Gua

12

5+

Caf 0

5+

Tau

4


Tota

l cel

l cou

nt (times

106)

20

15

10

05

00

Control


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast



(mgmL)

(a)

PCM

(200

x)





(1) (3)(2) (4)

(5)

(10)(9) (11) (12)

(13)

(6) (8)(7)

SH-S

Y5Y

cells

10

FBS

+dr

ug(s

) tre

atm

ent (

mg

mL

24 h

ours

)

(14) (15) (16)

(b)



104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)


SH-SY5Y cells


Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)


Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4


Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4








(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch



104

103

102

101

100

104103102101100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100FL

3-H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H

104

103

102

101

100

FL3-

H104

103

102

101

100

FL3-

H

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-HGua 3125

104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H104103102101100

FL1-H

104103102101100

FL1-H104103102101100

FL1-H

Gua 125 Gua 50

Guarana (mgmL)

Caf 0125

Tau 1 Tau 16Tau 4

Caf 05 Caf 2

Control

Caffeine (mgmL)

Combinations (mgmL)

Taurine (mgmL)


SH-SY5Y cells


Cell

s (

)

10080

60

40

20

0

Con

trol

312

5

125 50

012

5

05 2 1 4 16

Viability

(a)

(1)

(4) (5)

(7) (8) (9)

(6)

(2) (3)

(1) (4) (5)(2) (3)

(b)

(c)(d)


Annexin V

PI

Gua125+

Caf05

Gua125+

Tau4

Caf05+

Tau4

Caf1+

Tau4


Taurine(mgmL)

Caffeine(mgmL)

Guarana(mgmL)

Combinations(mgmL)

Gua125+

Caf05+

Tau4








(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch





(a)

ProteinCompound

Level

1 2

3 4

00 02 04 06 08 10

(b)







5 Conclusion



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch



Low



defenses



antioxidants


Disruptedphysiology

Child

ren

adol

esce

nts

(imm

atur

e bra

in)


Dur

ing

preg

nanc

y(d

evel

opin

g br

ain)

High

Health

ROS


temperatures





emotional shocks

Cellulardamage



+

minus



Abbreviations





Acknowledgments




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch




References




































































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch




































of



ISRN AIDS





Volume 2013Issue 1




Volume 2013


ISRN Biomarkers





ISRN Addiction




Volume 2013

ISRN Anesthesiology





OncologyJournal of

Volume 2013


Journal of


ObesityJournal of

ISRN Allergy


PPARRe sea rch


Major components of energy drinks (caffeine, taurine, and guarana) exert cytotoxic effects on human...

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Transcript of Major components of energy drinks (caffeine, taurine, and guarana) exert cytotoxic effects on human...