Thai Fruits Exhibit Antioxidant Activity and Induction of Antioxidant Enzymes in HEK-293 Cells

14 Pages·2016·1.55 MB·English

Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2016, Article ID 6083136, 14 pages
http://dx.doi.org/10.1155/2016/6083136
Research Article
Thai Fruits Exhibit Antioxidant Activity and Induction of
Antioxidant Enzymes in HEK-293 Cells
NatthineeAnantachoke,1PattamapanLomarat,2WasinPraserttirachai,2
RuksineeKhammanit,3andSupachokeMangmool3
1DepartmentofPharmacognosy,FacultyofPharmacy,MahidolUniversity,Bangkok10400,Thailand
2DepartmentofFoodChemistry,FacultyofPharmacy,MahidolUniversity,Bangkok10400,Thailand
3DepartmentofPharmacology,FacultyofPharmacy,MahidolUniversity,Bangkok10400,Thailand
CorrespondenceshouldbeaddressedtoSupachokeMangmool;[email protected]
Received29June2016;Revised25October2016;Accepted31October2016
AcademicEditor:G.K.Jayaprakasha
Copyright©2016NatthineeAnantachokeetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
Thecellularantioxidantenzymesplaytheimportantroleofprotectingthecellsandorganismsfromtheoxidativedamage.Natural
antioxidantscontainedinfruitshaveattractedconsiderableinterestbecauseoftheirpresumedsafetyandpotentialnutritional
value.Eventhoughantioxidantactivitiesofmanyfruitshavebeenreported,theeffectsofphytochemicalscontainedinfruitsonthe
inductionofantioxidantenzymesinthecellshavenotbeenfullydefined.Inthisstudy,weshowedthatextractsfromAntidesma
ghaesembilla,Averrhoabilimbi,Malpighiaglabra,Mangiferaindica,Sandoricumkoetjape,Syzygiummalaccense,andZiziphusjujuba
inhibitedH2O2-inducedintracellularreactiveoxygenspeciesproductioninHEK-293cells.Additionally,theseThaifruitextracts
increasedthemRNAandproteinexpressionsofantioxidantenzymes,catalase,glutathioneperoxidase-1,andmanganesesuperoxide
dismutase.TheconsumptionofThaifruitsrichinphenoliccompoundsmayreducetheriskofoxidativestress.
1.Introduction antistress of physiological conditions including antioxidant
andanti-inflammatoryeffects[7].
Oxidativestressisdefinedasanimbalancebetweenendoge-
Epidemiologicalstudieshavefoundthatconsumptionof
nousantioxidantdefensemechanismsandtheproductionof
fruits and vegetables has attracted growing interest because
reactiveoxygenspecies(ROS),whichathighlevelscancause
oftheirsignificantroleinreducingtheriskofcardiovascular
cell injury and damage through modifications of proteins,
diseases and other chronic diseases [8]. Consumption of
lipids, and DNA. Increased oxidative stress is involved in
antioxidants, through diet and supplements, is expected to
thepathophysiologyofmanydiseasessuchascardiovascular
remove ROS from the living system and provide health
diseases[1],neurodegenerativediseases[2],anddiabetes[3].
benefits.Severalstudiesdemonstratedthatmedicinalplants
The cellular antioxidant enzymes play the important role
andfruitsarearichsourceofantioxidantcompoundssuch
of protecting the cells and organisms from the oxidative
damage. For instance, superoxide dismutase (SOD) acts as as phenolics, flavonoids, quinones, vitamins, and alkaloids,
a catalyst to convert superoxide radicals into oxygen and which can decrease the incidence of oxidative stress and
hydrogen peroxide [4]. The hydrogen peroxide converts associated diseases [9–11]. Previous studies showed that the
into water and oxygen by catalase to protect the cells from phytochemicals,especiallyphenolics,infruitsarethemajor
accumulationofH2O2 [5].Glutathioneperoxidase(GPx)is bioactivecompoundsshowingpotentantioxidanteffects[12–
theimportantenzymetoremoveH2O2byusingglutathione 14].Therewasadirectrelationshipbetweenthetotalphenolic
as a substrate [6]. Heme oxygenase 1 (HO-1) is involved in contentsandtheantioxidantactivitiesinfruits[13–15].
2 Evidence-BasedComplementaryandAlternativeMedicine
Natural antioxidants that are presented in fruits have 2.4.DeterminationofTotalPhenolicContent. Totalphenolic
attracted considerable interest because of their presumed content of the fruit extracts was analyzed by the Folin–
safety and potential nutritional and therapeutic value. The Ciocalteucolorimetricmethoddescribedpreviously[16]with
increased interest in natural antioxidants has led to the minormodifications.Stocksolutionsofthefruitextractswith
antioxidantevaluationofmanyspeciesoffruits.Eventhough concentrationof1.5mg/mlwerepreparedin50%methanol.
antioxidantactivitiesofmanyfruitshavebeenreportedand Twenty microliters of the stock solutions was mixed with
theresultsshowedthatsomeofthemcouldberichsourcesof 50𝜇l of 10% Folin–Ciocalteu reagent in 96-well plates and
allowedtoreactfor3min.Themixtureswerethenneutralized
naturalantioxidants,theeffectsofphytochemicalscontained
byadding80𝜇lof7.5%ofsodiumcarbonatesolution.After
infruitsontheinductionofantioxidantenzymesinthecells
incubationatroomtemperaturefor2h,theabsorbancewas
havenotbeenfullydefined.Therefore,theobjectivesofthis
recorded at 765nm by an Infinite M200 Microplate Reader
studyweretodeterminetheprofilesoftotalphenolics,total
(Tecan).Theassaywasmeasuredintriplicateandtheresults
flavonoids,theantioxidantactivitiesofThaifruitextracts,and
werecalculatedusingacalibrationcurveforgallicacid(0.625
the molecular mechanisms of Thai fruit extracts on H2O2- to 30𝜇g/ml) and expressed as mg gallic acid equivalent
inducedoxidativestressinHEK-293cells.
(GAE)per100gfreshweight(mgGAE/100gFW).
2.MaterialsandMethods 2.5.DeterminationofTotalFlavonoidContent. Totalflavon-
oid content was determined by aluminium chloride colori-
2.1.Chemicals. 2,2-Diphenyl-1-picrylhydrazyl(DPPH),gallic metrictestmodifiedfrompreviousstudy[17].Onehundred
acid, ascorbic acid, 6-hydroxy-2,5,7,8-tetramethylchroman- and sixty microliters of sterile water was added to the test
2-carboxylicacid(Trolox(cid:2)),sodiumnitrite,aluminiumchlo- tubefollowedby40𝜇lofthefruitextractsdissolvedinsterile
ridehexahydrate,sodiumhydroxide,hydrogenperoxide,3- waterattheconcentrationof1mg/mland15𝜇lof5%NaNO2.
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide After5min,24𝜇lof10%AlCl3⋅6H2Owasadded.Themixture
(MTT),anddichlorodihydrofluorescein(DCF)acetatewere was allowed to react for 6min. Lastly, 80𝜇l of 1M NaOH
purchasedfromSigma-Aldrich(SaintLouis,MO).Dulbecco’s and 80𝜇l of sterile water were added and well mixed. Then
ModifiedEagleMedium(DMEM),fetalbovineserum,0.25% 250𝜇l of reaction mixture was transferred to 96-well plate.
trypsin-EDTAsolution,andpenicillin-streptomycinsolution The plate was shaken for 30sec and the absorbances were
were purchased from Gibco (Grand Island, NY). Folin– read at 510nm by microplate reader. The total flavonoid
Ciocalteu’sphenolreagentwaspurchasedfromMerckKGaA contentwasexpressedasquercetinequivalentper100gfresh
weight (mg QE/100g FW) which was calculated from the
(Darmstadt, Germany). Anhydrous sodium carbonate was
regression equation obtained from graph plotted between
availablefromAjaxFinechem(Auckland,NewZealand).
theabsorbanceandtheconcentrationofquercetinatvarious
concentrations(4–1,500𝜇g/ml).Theassaywasperformedin
2.2. Plant Materials. The fruits of Antidesma ghaesembilla triplicate.
Gaertn.(Phyllanthaceae),Artocarpusinteger(Thumb.)Merr.
(Moraceae), Averrhoa bilimbi L. (Oxalidaceae), and Mangi- 2.6. DPPH Radical-Scavenging Assay. Free radical-scaveng-
ferafoetidaLour.(Anacardiaceae)werecollectedfromRan- ingactivityofthefruitextractswasdeterminedbasedonthe
ongProvince,Thailand,whilethefruitsofDuriozibethinusL. reductionofDPPHradicalsbymeansofthemodifiedspec-
cultivarMonThong(Malvaceae),MalpighiaglabraL.(Mal- trophotometric method in 96-well plates. The test samples
pighiaceae), Mangifera indica L. cultivar Aok Rong (Anac- werepreparedatconcentrationof2mg/mlinmethanoland
ardiaceae), Musa paradisiaca cultivar Awak (Musaceae), mixedwith0.02%w/vDPPHradicalmethanolicsolution1:1
Sandoricumkoetjape(Burm.f.)Merr.cultivarTuptim(Meli- ratiointriplicate.Thereactionmixtureswereallowedtostand
aceae),Syzygiummalaccense(L.)Merr.&Perry(Myrtaceae), inthedarkfor30minatroomtemperature.Theabsorbance
andZiziphusjujubaMill.cultivarMilkJujube(Rhamnaceae) was measured at 517nm using Infinite M200 Microplate
werepurchasedfromlocalmarketsinBangkok,Thailand.The Reader (Tecan). The DPPH radical-scavenging ability was
calculated as percent inhibition by the following equation:
plants were identified by Professor Wongsatit Chuakul, the
% Inhibition=[(𝐴 −𝐴 )/𝐴 ]×100.TheDPPH
botanist and lecturer in the Department of Pharmaceutical control sample control
radical-scavengingactivityofthefruitextractswasexpressed
Botany,FacultyofPharmacy,MahidolUniversity.
astheIC50values(𝜇g/ml)(inhibitionofDPPHradicalforma-
tionby50%atconcentrationof1mg/ml).6-Hydroxy-2,5,7,8-
2.3. Preparation of Extracts. Edible portions of the fresh
tetramethylchroman-2-carboxylicacid(Trolox)wasusedas
fruitswererunthroughafoodchopper.Thefinelychopped
apositivecontrol.
fruits were dried by freeze dryer (FreeZone, Labconco).
The coarsely ground dried fruits (50g) were extracted with 2.7.CellCulture. Humanembryonickidney-293(HEK-293)
methanol (300–500ml × 3 times). After filtration through cellswereculturedusingDMEMsupplementedwith10%FBS
filter paper (number 1, Whatman Inc.), the solvent was and1%(v/v)penicillin-streptomycinsolutioninahumidified
removed under reduced pressure using a rotary evaporator atmosphereof5%CO2 at37∘Caspreviouslydescribed[18].
andthecrudemethanolextractswerestoredat−20∘Cuntil Under serum starvation conditions, cells were treated with
analysis. crudeextractofThaifruits.
Evidence-BasedComplementaryandAlternativeMedicine 3
󸀠 󸀠 󸀠
2.8. MTT Assay. Cell viability was performed by the MTT 5 -aaggccgtgtgcgtgaa-3; antisense, 5 -caggtctccaacatgcctct-
󸀠 󸀠 󸀠
assay according to the method previously described [19]. 3 ), human GRe (sense, 5 -cagtgggactcacggaagat-3 ; anti-
󸀠 󸀠
Cells were seeded in 96-well culture plates at a density of sense, 5 -ttcactgcaacagcaaaacc-3 ), and human GAPDH
1 × 104cells/well, in a total volume of 200𝜇l of DMEM (sense,5󸀠-cgagatccctccaaaatcaa-3󸀠;antisense,5󸀠-gtcttctgggtg-
󸀠
supplementedwith1%FBSplus1%penicillin/streptomycin. gcagtgat-3 ).
Cells were allowed to adhere to plate for 24h, before
beingtreatedwithsolvent(vehiclecontrol)andcrudeextracts 2.11. Western Blotting. Protein expression of antioxidant
at various concentrations (0.01–2000𝜇g/ml) for 24h. The enzymes was performed by Western blotting as previously
experimentswereperformedin2replicatewells.Therelative described[20].Followingstimulation,cellswerewashedonce
number of viable cells was then determined at 24h after with PBS and solubilized in lysis buffer containing 20mM
incubation,byadding2mg/mlofMTTsolutionandfurther Tris-HCl, pH 7.4, 0.8% Triton X-100, 150mM NaCl, 2mM
incubating the cell for 4h. The formazan crystals formed EDTA, 10% glycerol, 100𝜇M PMSF, 5𝜇g/ml aprotinin, and
werethensolubilizedwithDMSO.Theplatewasreadusing 5𝜇g/ml leupeptin. Protein concentration of cell lysates was
an Infinite M200 Microplate Reader (Tecan) at wavelength determined using a Bio-Rad protein assay kit with bovine
of 570nm. The absorption value of the solution at 570nm serumalbuminasstandard.Samplesweremixedwithloading
directly represents relative cell numbers. The percentage bufferanddenaturedbyheatingat95∘Cfor5minpriortosep-
of cell viability was calculated according to the following aration by SDS-PAGE. Separated proteins were transferred
equation: topolyvinylidenefluoride(PVDF)membrane(Bio-Rad)and
subjected to immunoblotting with primary antibodies to
The%ofcellviability catalase (Cell Signaling), Mn-SOD (Cell Signaling), GPx-1
(Abcam),andGAPDH(CellSignaling).Immunoblotswere
(1)
=[Absorbanceoftreatedcells]×100. visualizedwithHRP-conjugatedsecondaryantibodiesanda
Absorbanceofcontrolcells chemiluminescencedetectionsystem(GEHealthcare).
2.9.MeasurementofIntracellularROSLevel. Theintracellular 2.12. Statistical Analysis. Data were presented as mean ±
antioxidant activities of the crude extract of Thai fruits SEM.Thestatisticalanalysiswasdeterminedusingone-way
were quantified using a fluorescent probe dichlorodihy- analysisofvariance(ANOVA)andStudent’st-test,andvalues
drofluorescein(DCF)diacetate(Sigma-Aldrich)toestimate of𝑃<0.05wereconsideredtobesignificant.
the intracellular ROS production in the cell as previously
described[20].HEK-293cellswereseededina12-wellplate 3. Results
(5 × 104cells/well) or 35-mm glass bottomed dishes (1 ×
5
10 cells/dish) and treated with Thai fruit extracts (10 and 3.1. Yields of Thai Fruit Extracts. Eleven fruit samples were
100𝜇g/ml)for12h.After12h,thecellswerethenincubated collected and bought from southern and central regions of
with100𝜇MofH2O2for2handwashedoncewithphosphate Thailandandwereauthenticatedbyabotanist.Thescientific
buffered saline (PBS), pH 7.4. Thereafter, 10𝜇M DCFH-DA names of the plants are shown in Table 1. Although there
∘
wasaddedandincubatedwiththecellsat37 Cinthedarkfor aremanykindsoffruitsinThailand,inthisstudy,wechose
30min. The fluorescence intensity of DCF was determined only 11 fruits that have a potential effect on inhibition of
using Multi-Detection Microplate Reader (BioTek Instru- oxidativestress.Thaipeopleconsumesignificantamountof
ments)withanexcitationwavelengthof485nmandemission the investigated fruits at a ripe stage and the yields of the
wavelength of 530nm. To monitor the ROS production in crude methanol extracts of Thai fruits ranged from 2.68 to
living cells, HEK-293 cells were visualized using single-line 9.28% of fresh weight (edible part of fresh fruits weight)
excitation (488nm) of an IX-81 fluorescence microscope (Table1).
(Olympus)witha40x(NA1.4)objectivelens(Olympus).
3.2.DeterminationofTotalPhenolicandFlavonoidContents
2.10.mRNAExpressionAnalysisbyRT-qPCR. Theextraction of Thai Fruit Extracts. The total phenolic contents were
of RNA was performed by using the RNeasy Mini Kit determinedusingtheFolin–Ciocalteumethod,asimpleand
(Qiagen) as previously described [21]. RT-qPCRs for RNA widelyusedmethod,whichreliedonthetransferofelectrons
expressionwereperformedbyusingtheKAPASYBRFAST fromphenoliccompoundstotheFolin–Ciocalteureagentin
One-StepRT-qPCRKit(KAPAbiosystems)accordingtothe alkaline medium. As shown in Table 1, the total phenolic
manufacturer’s instructions. RT-qPCRs were performed on contentsof11selectedThaifruitsareexpressedasmgofgallic
Mx3005pReal-TimePCRSystem(Stratagene).Wedesigned acidequivalent(GAE)per100goffreshweight(FW).Among
andsynthesizedtheprimersasfollows:humanGPx-1(sense, allthefruitsanalyzed,thehighestphenoliccontentwasfound
5󸀠-ctcttcgagaagtgcgaggt-3󸀠; antisense, 5󸀠-tcgatgtcaatggtctg- inM.glabra(723.83±36.94mgGAE/100gFW),followedby
gaa-3󸀠), human catalase (sense, 5󸀠-gcagatacctgtgaactgtc-3󸀠; S.koetjape(241.01±16.51),A.ghaesembilla(98.38±6.48),
antisense, 5󸀠-gtagaatgtccgcacctgag-3󸀠), human HO-1 (sense, M.indica(45.39±1.33),Z.jujuba(45.29±3.45),M.foetida
5󸀠-caggcagagaatgctgag-3󸀠; antisense, 5󸀠-gcttcacatagcgctgca- (38.83 ± 3.15), A. bilimbi (33.77 ± 1.37), and S. malaccense
3󸀠),humanMn-SOD(sense,5󸀠-gcacattaacgcgcagatca-3󸀠;anti- (29.55±0.91)(Table1).Phenoliccontentwasslightlylower
󸀠 󸀠
sense,5 -agcctccagcaactctcctt-3 ),humanCuZn-SOD(sense, inA.integerandD.zibethinus.M.paradisiacacultivarAwak
4 Evidence-BasedComplementaryandAlternativeMedicine
Table1:Yieldofmethanolextracts,totalphenolicandflavonoidcontents,andDPPHscavengingactivitiesofThaifruitextracts.
Yieldof Totalphenolic Totalflavonoid
Fruits DPPHscavengingactivity
Scientificname crudeextract content content
(ripestage) (%inhibitionat1mg/mL)
(%FW) (mgGAE/100gFW) (mgQE/100gFW)
Maokhaipla Antidesma 4.83 98.38±6.48 134.06±2.90 67.65±0.31
(Thai) ghaesembillaGaertn.
Champedak Artocarpusinteger 6.85 21.45±2.28 158.31±1.02 15.42±1.52
(Thumb.)Merr.
Bilimbi AverrhoabilimbiL. 2.68 33.77±1.37 55.33±0.70 30.66±1.96
Durian DuriozibethinusL. 4.86 9.65±0.71 93.85±0.36 6.80±0.97
cultivarMonThong
Escobillo MalpighiaglabraL. 4.40 723.83±36.94 195.36±0.14 96.62±0.44
Horsemango Mangiferafoetida 6.74 38.83±3.15 46.13±1.85 25.63±1.32
Lour.
Mango MangiferaindicaL. 9.12 45.39 ±1.33 ND 44.85±0.71
cultivarAokRong
PisangAwak MusaparadisiacaL. 7.24 0.34±0.15 3.70±1.31 5.15±1.15
cultivarAwak
Sandoricumkoetjape
Santol (Burm.f.)Merrill 9.28 241.01±16.51 851.49±0.30 84.73±1.38
cultivarTuptim
Malayapple, Syzygiummalaccense 4.28 29.55±0.91 3.99±1.65 16.75±1.08
Pomerac (L.)Merr.&Perry
Jujube ZiziphusjujubaMill. 7.63 45.29±3.45 1.36±1.67 4.45±0.91
cultivarMilkJujube
ND=notdetected.
hadthelowesttotalphenoliccontentamongthetestedfruits 6.2𝜇g/ml), M. indica (44.85 ± 0.71), and A. bilimbi (30.66
(Table1). ± 1.96). The extracts of D. zibethinus, M. paradisiaca, and
Thetotalflavonoidcontentsof11selectedThaifruitsare Z. jujuba had DPPH scavenging activity less than 10% of
expressed as mg of quercetin equivalent (QE) per 100g of inhibition(Table1).
fresh weight (mg QE/100g FW). As shown in Table 1, total
flavonoidcontentsofThaifruitextractsrangedfrom1.36± 3.4. Correlation between Antioxidant Capacity and Total
1.67to851.49±0.30mgQE/100gFW.Thehighestflavonoid Phenolic Content. The correlation between total phenolic
content was found in S. koetjape, which had 851.49 ± content and antioxidant activity using DPPH assay of fruit
0.30mg QE/100g FW, followed by M. glabra, A. integer, A. extracts was determined and shown in Figure 1. A positive
ghaesembilla, D. zibethinus, A. bilimbi, and M. foetida, the correlation (𝑅2 = 0.6317) between the DPPH scavenging
valuesofwhichwere195.36±0.14,158.31±1.02,134.06±2.90, value and total phenolic content indicated that phenolic
93.85±0.36,55.33±0.70,and46.13±1.85mgQE/100gFW, compoundsmainlycontributedtotheantioxidantactivities
respectively. Flavonoid content was lower in S. malaccense ofthesefruits.Thisresultwasinagreementwithseveralpre-
and M. paradisiaca. On the other hand, Z. jujuba had the viousstudies[15].However,somefruitscontaininghightotal
lowestcontentoftotalflavonoid(1.36±1.67),whilethatof flavonoid compounds, such as A. integer and D. zibethinus
M.indicawasnotdetected. (158.31±1.02and93.85±0.36mgQE/100gFW,resp.)didnot
exhibit strong antioxidant capacity. In contrast, S. koetjape
3.3. DPPH Scavenging Activities of Thai Fruit Extracts. We and M. glabra that contained higher flavonoid contents
next determined the antioxidant activity of fruit extracts elicited strong DPPH scavenging activity with 84.73 ± 1.38
usingDPPHmethod.Thisassaybasedonthemeasurementof and 96.62 ± 0.44% of inhibition, respectively. Thus, the
theantioxidantabilitytoscavengethestableorganicradical correlationsbetweenantioxidantactivityandtotalflavonoid
DPPH and used Trolox as positive control with the IC50 of contents of Thai fruit extracts were poor (𝑅2 = 0.3843)
4.12 ± 0.3𝜇g/ml. As shown in Table 1, the extract of M. (Figure1).Accordingtotheirhighantioxidantsactivities,it
glabraattheconcentrationof1mg/mlexhibitedthehighest could be speculated that these fruits will be beneficial for
DPPHfreeradical-scavengingactivitywith96.62±0.44%of inhibitionofoxidativestress.
inhibition (IC50of 45.00 ±1.90𝜇g/ml), which related to the
high total phenolic content (723.83 ± 36.94mg GAE/100g 3.5. Thai Fruit Extract Inhibits H O -Induced Intracellular
2 2
FW). The extracts of S. koetjape showed moderate DPPH ROSProductioninHEK-293Cells. Wefirstassessedthecell
scavengingactivities84.73±1.38(IC50 of415.8±1.5𝜇g/ml), cytotoxicity of crude extracts of 11 Thai fruits on HEK-
followed by A. ghaesembilla (67.65 ± 0.31; IC50 of 648.0 ± 293 cells by using MTT assay to determine the optimal
Evidence-BasedComplementaryandAlternativeMedicine 5
300 300
R2=0.6317 R2=0.3843
n) n)
o 250 o 250
biti biti
hi hi
n 200 n 200
% i % i
ng ( 150 ng ( 150
gi gi
n n
e e
v 100 v 100
a a
c c
H s H s
P 50 P 50
P P
D D
0 0
0 200 400 600 800 1000 0 200 400 600 800 1000
Phenolic content (mg GAE/100g FW) Flavonoid content (mg QE/100g FW)
Figure1:Correlationbetweenantioxidantcapacitiesandtotalphenolicandflavonoidcontentsin11Thaifruits.Antioxidantcapacitiesof
ThaifruitextractsweremeasuredbytheDPPHfreeradical-scavengingassay.GAE:gallicacidequivalent.QE:quercetinequivalent.FW:
freshweight.
concentrations of the crude extract for measurement of suggesting that these fruits suppressed the ROS production
intracellular antioxidant activity. We found that Thai fruit in intact cells. However, treatment with fruits extracts of
extractsattheconcentrationof0.01–100𝜇g/mlwerenottoxic A. integer, D. zibethinus, M. foetida, and M. paradisiaca did
toHEK-293cells(Figure2).Upontreatmentofthecellswith not reduce the fluorescence intensity of DCF inducing by
the crude extracts more than 100𝜇g/ml, the cell viabilities H2O2comparedtothatofcontrol.Takentogether,thesedata
of HEK-293 were less than 80%. The concentrations used demonstratedthatThaifruitshavetheantioxidanteffectsby
in further experiment were 10 and 100𝜇g/ml for the fruit preventingtheproductionofROSinHEK-293cells.
extractsinordertopreventthecytotoxiceffectandallowat
least80%cellssurvival. 3.6. Induction of mRNA Expression of Antioxidant Enzymes
We next investigated whether Thai fruit extract inhibits by Thai Fruit Extracts. Antioxidant enzymes (e.g., GPx-1,
H2O2-induced intracellular ROS production in HEK-293 catalase, Mn-SOD, CuZn-SOD, and HO-1) are the major
cells.TheintracellularROSlevelsweremeasuredbyusinga components of antioxidant signaling cascade in many cell
fluorescentprobe,DCFH-DA.AfterincubationwithH2O2, types.TreatmentwithA.bilimbiextractelicitstheantioxidant
the levels of intracellular ROS significantly increased com- effect by increasing the activity of SOD and GPx both in
paredtothatofcontrol(vehicle)group(Figure3).Asshown bloodandintissues(e.g.,liver,kidney,andheart)ofrats[22].
inFigure3(a),treatmentwithA.ghaesembillaandA.bilimbi Moreover,anaqueousextractofM.indicainhibitedoxidative
significantly inhibited H2O2-induced ROS production in a stress by enhancing the production of antioxidant enzymes
dose-dependent manner, whereas treatment with A. integer suchasSODandcatalase[23].Consistentwiththeseprevious
andD.zibethinusdidnotshowtheinhibitoryeffectonH2O2- studies, we showed that treatment with fruit extracts of M.
inducedROSproduction.Inaddition,thecrudeextractsof glabra,S.malaccense,M.indica,andA.bilimbisignificantly
M. glabra and M. indica showed a significant reduction in elevatedthemRNAlevelofMn-SOD(Figure5(a)).Moreover,
ROSproduction,whichhaseffectssimilartothoseofvitamin the GPx-1 mRNA expression significantly increased when
C(apotentantioxidant),whereastreatmentwithextractsof treated with fruit extracts of S. malaccense, S. koetjape, M.
M. foetida and M. paradisiaca had no effect (Figure 3(b)). indica,A.bilimbi,Z.jujuba,andA.ghaesembilla(Figure5(b)).
Moreover, treatment with fruit extracts of S. koetjape, S. Inaddition,themRNAlevelofcatalasealsoincreasedafter
malaccense, and Z. jujuba also suppressed H2O2-induced treatment with fruit extracts from M glabra, M. indica,
ROSproductioninadose-dependentmanner(Figure3(c)). Z. jujuba, and A. ghaesembilla (Figure 5(c)). However, all
AsshowninFigure3,thefruitextractsofA.ghaesembilla, Thai fruit extracts failed to induce the mRNA expressions
A. bilimbi, M. glabra, M. indica, S. koetjape, S. malaccense, of CuZn-SOD, GRe, and HO-1 in HEK-293 cells (Figures
andZ.jujubahavetheantioxidanteffectsbypreventingthe 5(d)–5(f), resp.). Collectively our results showed that Thai
productionofintracellularROSinducedbyH2O2.Wefurther fruit extracts play an important role in the prevention of
quantified ROS production in intact HEK-293 cells using oxidativestressbyenhancingthemRNAexpressionofthese
the fluorescent microscope. Incubation of HEK-293 cells antioxidantenzymesinHEK-293cells.
withH2O2inducedasignificantincreaseinthefluorescence
intensity of dichlorodihydrofluorescein (DCF) as shown in 3.7.ThaiFruitExtractsIncreasedProteinLevelofAntioxidant
green color in intact cells of the control (vehicle) group Enzymes. As shown in Figure 5, the mRNA expressions of
(Figure4).TreatmentwithfruitextractsofA.ghaesembilla, Mn-SOD,GPx-1,andcatalasewereincreasedaftertreatment
A. bilimbi, M. glabra, M. indica, S. koetjape, S. malaccense, withtheThaifruitextracts.Wenextinvestigatedtheeffects
and Z. jujuba decreased the fluorescence intensity of DCF, of these fruit extracts on the protein expressions of these
6 Evidence-BasedComplementaryandAlternativeMedicine
Antidesma ghaesembilla Artocarpus integer Averrhoa bilimbi
120 120 120
100 100 100
bility 80 bility 80 bility 80
% cell via 4600 % cell via 4600 % cell via 4600
20 20 20
0 0 0
0.01 0.1 1 10 100 10002000 0.01 0.1 1 10 100 10002000 0.01 0.1 1 10 100 10002000
Concentration (𝜇g/ml) Concentration (𝜇g/ml) Concentration (𝜇g/ml)
Durio zibethinus Malpighia glabra Mangifera foetida
120 120 120
100 100 100
bility 80 bility 80 bility 80
% cell via 4600 % cell via 4600 % cell via 4600
20 20 20
0 0 0
0.01 0.1 1 10 100 10002000 0.01 0.1 1 10 100 10002000 0.01 0.1 1 10 100 10002000
Concentration (𝜇g/ml) Concentration (𝜇g/ml) Concentration (𝜇g/ml)
Mangifera indica Musa paradisiaca Sandoricum koetjape
120 120 120
100 100 100
bility 80 bility 80 bility 80
% cell via 4600 % cell via 4600 % cell via 4600
20 20 20
0 0 0
0.01 0.1 1 10 100 10002000 0.01 0.1 1 10 100 10002000 0.01 0.1 1 10 100 10002000
Concentration (𝜇g/ml) Concentration (𝜇g/ml) Concentration (𝜇g/ml)
Syzygium malaccense Ziziphus jujuba Vehicle
120 120 120
100 100 100
% cell viability 468000 % cell viability 846000 % cell viability 468000
20 20 20
0 0 0
0.01 0.1 1 10 100 10002000 0.01 0.1 1 10 100 10002000 0.01 0.1 1 10 100 10002000
Concentration (𝜇g/ml) Concentration (𝜇g/ml) Concentration (𝜇g/ml)
Figure2:CytotoxicityprofileofThaifruitextractsinHEK-293cells.HEK-293cellsweretreatedwithThaifruitextracts(0.01–2,000𝜇g/ml)
for36h.Cellviabilitywasquantified,expressedasapercentageofcellviability,andshownasthemean±SEM(𝑛=4).
antioxidant enzymes. We found that treatment with fruit results of protein expression revealed that treatment with
extracts of M. glabra, S. malaccense, M. indica, and A. fruit extracts of S. malaccense, S. koetjape, M. indica, A.
bilimbiattheconcentrationof100𝜇g/mlfor24hsignificantly bilimbi,Z.jujuba,andA.ghaesembillacausedanincreasein
elevatedtheproteinlevelofMn-SOD(Figure6,upper-right theGPx-1proteinlevelcomparedwiththecontrol(vehicle)
panel). Similar to the mRNA expression experiments, the (Figure 6, lower-left panel). Moreover, the protein levels of
Evidence-BasedComplementaryandAlternativeMedicine 7
250
∗
200
%)
on ( 150 # #
cti #
u
d
o
pr 100
S
O
R
50
0
VehicleVehicle 10 100 10 100 10 100 10 100 10 100
A. ghaesembilla A. integer A. bilimbi D. zibethinus Vit. C
(𝜇g/ml) (𝜇g/ml) (𝜇g/ml) (𝜇g/ml) (𝜇M)
+H2O2
(a)
250
∗
200
%)
on ( 150 # # #
cti # #
u
d
o
pr 100
S
O
R
50
0
VehicleVehicle 10 100 10 100 10 100 10 100 10 100
M. glabra M. foetida M. indica M. paradisiaca Vit. C
(𝜇g/ml) (𝜇g/ml) (𝜇g/ml) (𝜇g/ml) (𝜇M)
+H2O2
(b)
250
∗
200
n (%) 150 # # # #
uctio # #
d
o
pr 100
S
O
R
50
0
Vehicle Vehicle 10 100 10 100 10 100 10 100
S. koetjape S. malaccense Z. jujuba Vit. C
(𝜇g/ml) (𝜇g/ml) (𝜇g/ml) (𝜇M)
+H2O2
(c)
Figure3:EffectsofThaifruitextractsonH2O2-inducedROSproductioninHEK-293cells.(a–c)Cellsweretreatedwithvehicle(control),10
and100𝜇MvitaminC,or10and100𝜇g/mlfruitextractsfor6h.Cellswerethenincubatedwith200𝜇MH2O2for30min.Theintracellular
ROSproductionwasquantified,expressedasapercentageofthecontrol,andshownasthemean±SEM(𝑛=4).∗𝑃<0.05versuscontrol;
#𝑃<0.05versusH2O2.
8 Evidence-BasedComplementaryandAlternativeMedicine
DCF fluorescence intensity
+H2O2
A. ghaesembilla A. integer A. bilimbi D. zibethinus M. glabra
Vehicle Vehicle (100𝜇g/ml) (100𝜇g/ml) (100𝜇g/ml) (100𝜇g/ml) (100𝜇g/ml)
DCF
Phase
contrast
(a)
DCF fluorescence intensity
+H2O2
M. foetida M. indica M. paradisiaca S. koetjape S. malaccense Z. jujuba Vit. C
(100𝜇g/ml) (100𝜇g/ml) (100𝜇g/ml) (100𝜇g/ml) (100𝜇g/ml) (100𝜇g/ml) 100𝜇M
DCF
Phase
contrast
(b)
Figure4:ThaifruitextractsattenuatedintracellularROSproductioninHEK-293cells.(a-b)Cellsweretreatedwithvehicle(control),10and
100𝜇MvitaminC,or10and100𝜇g/mlfruitextractsfor6h.Cellswerethenincubatedwith200𝜇MH2O2for30min.Cellswerewashedand
incubatedwith10𝜇MDCFH-DA.CellswerevisualizedbygreenfluorescenceforDCFandbybrightfield.Scalebar,10𝜇m(𝑛=4).
catalase significantly increased after treatment with crude Oxidativestresshasbeenimplicatedinthepathogenesis
extractsofM.glabra,M.indica,andA.ghaesembillafor24h, ofvariouschronicdiseases,suchascancer,heartdiseases,and
whereas the catalase protein level tended to increase after diabetes[1–3].Thenonenzymaticantioxidantdefensesystem
treatment with crude extract of Z. jujuba (Figure 6, lower- ofthebodyismadeupofsomeantioxidants,suchasvitamin
rightpanel).TheseresultsindicatedthatThaifruitsprevent C, vitamin E, vitamin K, and glutathione. The exogenous
the oxidative stress by enhancing the protein synthesis of antioxidants are mainly comprised of synthetic and natural
antioxidantenzymes. antioxidants.Medicinalplantshavebeenusedtotreathuman
diseasesforthousandsofyears.Peoplearebecomingincreas-
inglyinterestedinmedicinalplants,includingfruitsbecause
4.Discussion oftheirgoodtherapeuticperformanceandlowtoxicity.Fruits
are potential sources of natural antioxidants and have been
In this study, we provide a new molecular mechanism of shown in epidemiological studies to be protective against
Thai fruits extracts on the inhibition of oxidative stress in severalchronicdiseasesassociatedwithagingsuchascancer,
HEK-293 cells. We demonstrate that ethanol extracts from immunedysfunction,andcardiovasculardiseases[8].These
A.ghaesembilla,A.bilimbi,M.glabra,M.indica,S.koetjape, naturalprotectiveeffectshavebeenattributedtovariouscom-
S. malaccense, and Z. jujuba have antioxidant effects by ponentssuchascarotenoids,vitaminsCandE,andphenolic
suppressing ROS production and inducing the synthesis of compounds [24, 25]. Thus, the interest in phytochemicals
antioxidant enzymes such as GPx-1, catalase, and Mn-SOD includingphenoliccompoundsderivedfromfruitsandtheir
inHEK-293cells. rolesininhibitionofoxidativestressarethereforeincreasing.
Evidence-BasedComplementaryandAlternativeMedicine 9
Mn-SOD GPx-1
0.8 0.8
H
D ∗ H
Mn-SOD/GAPNA expression 00..46 ∗ ∗ ∗ e GPx-1/GAPDNA expression 00..46 ∗ ∗ ∗ ∗ ∗ ∗
elative mR 0.2 RelativmR 0.2
R
0 0
Vehicle M. S. S. M. A. Z. A. Vehicle M. S. S. M. A. Z. A.
glabra malac koet indica bilim jujuba ghaes glabra malac koet indica bilim jujuba ghaes
100𝜇g/ml 100𝜇g/ml
(a) (b)
Catalase CuZn-SOD
1.2 1.2
1.0 H 1.0
H ∗ D
D ∗ P
Pn An
alase/GAexpressio 00..68 ∗ ∗ n-SOD/Gexpressio 00..68
atA ZA
Relative cmRN 00..24 elative CumRN 00..24
R
0 0
Vehicle M. S. S. M. A. Z. A. Vehicle M. S. S. M. A. Z. A.
glabra malac koet indica bilim jujuba ghaes glabra malac koet indica bilim jujuba ghaes
100𝜇g/ml 100𝜇g/ml
(c) (d)
GRe HO-1
1.0 1.4
1.2
H 0.8 H
Relative GRe/GAPDmRNA expression 000...246 Relative HO-1/GAPDmRNA expression 0001....4680
0.2
0 0
Vehicle M. S. S. M. A. Z. A. Vehicle M. S. S. M. A. Z. A.
glabra malac koet indica bilim jujuba ghaes glabra malac koet indica bilim jujuba ghaes
100𝜇g/ml 100𝜇g/ml
(e) (f)
Figure5:EffectsofThaifruitextractsonthemRNAexpressionofantioxidantenzymes.(a–f)Cellsweretreatedwithvehicle(control)or
100𝜇g/mlfruitextractsfor6h.Aftertreatment,thetotalRNAwasextractedfromcellsandthemRNAexpressionwasanalyzedusingspecific
primers.TherelativeMn-SOD(a),GPx-1(b),catalase(c),CuZn-SOD(d),GRe(e),andHO-1(f)mRNAlevelswerequantifiedandshown
asthemean±SEM(𝑛=4).∗𝑃<0.05versusvehicle.
10 Evidence-BasedComplementaryandAlternativeMedicine
Mn-SOD
2.0
22kD Mn-SOD asal)
ver b 1.5 ∗ ∗ ∗ ∗
o
22kD GPx-1 old
H (f 1.0
D
P
60kD Catalase A
G
D/ 0.5
O
S
37kD GAPDH n-
M
0
Vehicle M. S. S. M. A. Z. A. Vehicle M. S. S. M. A. Z. A.
glabramalac koet indica bilim jujuba ghaes glabra malac koet indica bilim jujuba ghaes
100𝜇g/ml 100𝜇g/ml
GPx-1 Catalase
ver basal) 12..50 ∗ ∗ ∗ ∗ ∗ ∗ over basal) 22..05 ∗ ∗ ∗
DH (fold o 1.0 PDH (fold 11..05
P A
A G
x-1/G 0.5 alase/ 0.5
GP Cat
0 0
Vehicle M. S. S. M. A. Z. A. Vehicle M. S. S. M. A. Z. A.
glabra malac koet indica bilim jujuba ghaes glabra malac koet indica bilim jujuba ghaes
100𝜇g/ml 100𝜇g/ml
Figure6:EffectsofThaifruitextractsontheproteinexpressionofantioxidantenzymes.Cellsweretreatedwithvehicle(control)or100𝜇g/ml
fruitextractsfor24h.Aftertreatment,thecelllysateswereimmunoblottedwithanti-Mn-SOD,anti-GPx-1,oranti-catalaseantibodies.The
expressionofGAPDHservedasaloadingcontrol.Theproteinexpressionwasquantified,expressedasthefoldincreaseovervehicle(control)
level,andshownasthemean±SEM(𝑛=4).∗𝑃<0.05versuscontrol.
2
The antioxidative effects of Thai fruit extracts could be total antioxidant activities in the tested 11 Thai fruits (R =
due to the presence of phenolic compounds. Studies have 0.6317). The higher total phenolic content in fruits resulted
also shown that phenolic compounds from various sources inhighertotalantioxidantactivity.Similarly,previousstudy
are effective in improving antioxidant enzymes, suggesting reportedasignificantpositivecorrelationbetweentheantiox-
theirroleinthepreventionandtreatmentofoxidativestress- idantcapacityandthecontentsoftotalflavonoidsandtotal
related diseases. Hence, the consumption of phenolic-rich phenolics in celery [30]. Moreover, antioxidant activities
fruitshasbeenassociatedwithreducedlevelsofROSinani- assayed by DPPH and FRAP methods showed strongly
malexperiments[10,26].Likewise,ahighintakeofphenolic- positive relationship with total phenolic content in Oxal-
richfruitshasbeenreportedtoenhancetheactivitiesofthe idaceae fruits [31]. Taken together, these results suggested
antioxidantenzymes[27].Itwasreportedthattheantioxidant that phenolic compounds may be the major contributor to
activity of raspberry and santol (S. koetjape) was directly thetotalantioxidantactivitiesoffruits.However,ourresults
2
related to the phenolic content [28]. In addition, the juice demonstrated a weak correlation (R = 0.3843) between
of Ziziphus mauritiana that has a moderate total phenolic the DPPH scavenging activity and total flavonoid content
contentandlowcontentofvitaminCelicitstheantioxidant suggesting that flavonoid compounds could not be main
activity [29]. Consistent with these previous studies, our components responsible for DPPH scavenging activity of
study demonstrated that the extract from M. glabra has somefruits,suchasM.indica,S.koetjape,andM.glabra.
the highest phenolic content, followed by S. koetjape, A. In our present study, we found that, among the tested
ghaesembilla,M.indica,Z.jujuba,M.foetida,A.bilimbi,and 11 Thai fruits tested, 7 of them showed the ability to inhibit
S.malaccense(Table1).TheseThaifruitextractsexhibitedthe H2O2-induced oxidative stress in HEK-293 cells. Previous
antioxidantactivityasdeterminedbyDPPHassay. studydemonstratedthattheadministrationofphenolicacids
The correlation between total antioxidant capacities (i.e., gentisic acid, gallic acid, ferulic acid and p-coumaric
obtainedfromDPPHassayisshowninFigure1.Therewasa acid) in rat at a concentration of 100mg/kg exhibited an
directlinearrelationshipbetweenthephenoliccontentsand inductionofhepaticantioxidantenzymessuchasSODand