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Research ArticleThai Fruits Exhibit Antioxidant Activity and Induction ofAntioxidant Enzymes in HEK-293 Cells

Natthinee Anantachoke1 Pattamapan Lomarat2 Wasin Praserttirachai2

Ruksinee Khammanit3 and Supachoke Mangmool3

1Department of Pharmacognosy Faculty of Pharmacy Mahidol University Bangkok 10400 Thailand2Department of Food Chemistry Faculty of Pharmacy Mahidol University Bangkok 10400 Thailand3Department of Pharmacology Faculty of Pharmacy Mahidol University Bangkok 10400 Thailand

Correspondence should be addressed to Supachoke Mangmool supachokemanmahidolacth

Received 29 June 2016 Revised 25 October 2016 Accepted 31 October 2016

Academic Editor G K Jayaprakasha

Copyright copy 2016 Natthinee Anantachoke et alThis 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

The cellular antioxidant enzymes play the important role of protecting the cells and organisms from the oxidative damage Naturalantioxidants contained in fruits have attracted considerable interest because of their presumed safety and potential nutritionalvalue Even though antioxidant activities of many fruits have been reported the effects of phytochemicals contained in fruits on theinduction of antioxidant enzymes in the cells have not been fully defined In this study we showed that extracts from AntidesmaghaesembillaAverrhoa bilimbiMalpighia glabraMangifera indica Sandoricum koetjape Syzygiummalaccense and Ziziphus jujubainhibited H

2O2-induced intracellular reactive oxygen species production in HEK-293 cells Additionally these Thai fruit extracts

increased themRNAandprotein expressions of antioxidant enzymes catalase glutathione peroxidase-1 andmanganese superoxidedismutase The consumption of Thai fruits rich in phenolic compounds may reduce the risk of oxidative stress

1 Introduction

Oxidative stress is defined as an imbalance between endoge-nous antioxidant defense mechanisms and the production ofreactive oxygen species (ROS) which at high levels can causecell injury and damage through modifications of proteinslipids and DNA Increased oxidative stress is involved inthe pathophysiology of many diseases such as cardiovasculardiseases [1] neurodegenerative diseases [2] and diabetes [3]The cellular antioxidant enzymes play the important roleof protecting the cells and organisms from the oxidativedamage For instance superoxide dismutase (SOD) acts asa catalyst to convert superoxide radicals into oxygen andhydrogen peroxide [4] The hydrogen peroxide convertsinto water and oxygen by catalase to protect the cells fromaccumulation of H

2O2[5] Glutathione peroxidase (GPx) is

the important enzyme to remove H2O2by using glutathione

as a substrate [6] Heme oxygenase 1 (HO-1) is involved in

antistress of physiological conditions including antioxidantand anti-inflammatory effects [7]

Epidemiological studies have found that consumption offruits and vegetables has attracted growing interest becauseof their significant role in reducing the risk of cardiovasculardiseases and other chronic diseases [8] Consumption ofantioxidants through diet and supplements is expected toremove ROS from the living system and provide healthbenefits Several studies demonstrated that medicinal plantsand fruits are a rich source of antioxidant compounds suchas phenolics flavonoids quinones vitamins and alkaloidswhich can decrease the incidence of oxidative stress andassociated diseases [9ndash11] Previous studies showed that thephytochemicals especially phenolics in fruits are the majorbioactive compounds showing potent antioxidant effects [12ndash14]There was a direct relationship between the total phenoliccontents and the antioxidant activities in fruits [13ndash15]

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 6083136 14 pageshttpdxdoiorg10115520166083136

2 Evidence-Based Complementary and Alternative Medicine

Natural antioxidants that are presented in fruits haveattracted considerable interest because of their presumedsafety and potential nutritional and therapeutic value Theincreased interest in natural antioxidants has led to theantioxidant evaluation of many species of fruits Even thoughantioxidant activities of many fruits have been reported andthe results showed that some of them could be rich sources ofnatural antioxidants the effects of phytochemicals containedin fruits on the induction of antioxidant enzymes in the cellshave not been fully defined Therefore the objectives of thisstudy were to determine the profiles of total phenolics totalflavonoids the antioxidant activities ofThai fruit extracts andthe molecular mechanisms of Thai fruit extracts on H

2O2-

induced oxidative stress in HEK-293 cells

2 Materials and Methods

21 Chemicals 22-Diphenyl-1-picrylhydrazyl (DPPH) gallicacid ascorbic acid 6-hydroxy-2578-tetramethylchroman-2-carboxylic acid (Trolox) sodium nitrite aluminium chlo-ride hexahydrate sodium hydroxide hydrogen peroxide 3-[45-dimethylthiazol-2-yl]-25-diphenyl tetrazoliumbromide(MTT) and dichlorodihydrofluorescein (DCF) acetate werepurchased fromSigma-Aldrich (Saint LouisMO)DulbeccorsquosModified EagleMedium (DMEM) fetal bovine serum 025trypsin-EDTA solution and penicillin-streptomycin solutionwere purchased from Gibco (Grand Island NY) FolinndashCiocalteursquos phenol reagent was purchased fromMerck KGaA(Darmstadt Germany) Anhydrous sodium carbonate wasavailable from Ajax Finechem (Auckland New Zealand)

22 Plant Materials The fruits of Antidesma ghaesembillaGaertn (Phyllanthaceae) Artocarpus integer (Thumb) Merr(Moraceae) Averrhoa bilimbi L (Oxalidaceae) and Mangi-fera foetida Lour (Anacardiaceae) were collected from Ran-ong ProvinceThailand while the fruits ofDurio zibethinus Lcultivar Mon Thong (Malvaceae) Malpighia glabra L (Mal-pighiaceae) Mangifera indica L cultivar Aok Rong (Anac-ardiaceae) Musa paradisiaca cultivar Awak (Musaceae)Sandoricum koetjape (Burm f) Merr cultivar Tuptim (Meli-aceae) Syzygium malaccense (L) Merr amp Perry (Myrtaceae)and Ziziphus jujubaMill cultivar Milk Jujube (Rhamnaceae)were purchased from localmarkets inBangkokThailandTheplants were identified by Professor Wongsatit Chuakul thebotanist and lecturer in the Department of PharmaceuticalBotany Faculty of Pharmacy Mahidol University

23 Preparation of Extracts Edible portions of the freshfruits were run through a food chopper The finely choppedfruits were dried by freeze dryer (FreeZone Labconco)The coarsely ground dried fruits (50 g) were extracted withmethanol (300ndash500ml times 3 times) After filtration throughfilter paper (number 1 Whatman Inc) the solvent wasremoved under reduced pressure using a rotary evaporatorand the crude methanol extracts were stored at minus20∘C untilanalysis

24 Determination of Total Phenolic Content Total phenoliccontent of the fruit extracts was analyzed by the FolinndashCiocalteu colorimetricmethoddescribed previously [16]withminormodifications Stock solutions of the fruit extracts withconcentration of 15mgml were prepared in 50 methanolTwenty microliters of the stock solutions was mixed with50 120583l of 10 FolinndashCiocalteu reagent in 96-well plates andallowed to react for 3minThemixtureswere thenneutralizedby adding 80120583l of 75 of sodium carbonate solution Afterincubation at room temperature for 2 h the absorbance wasrecorded at 765 nm by an Infinite M200 Microplate Reader(Tecan) The assay was measured in triplicate and the resultswere calculated using a calibration curve for gallic acid (0625to 30 120583gml) and expressed as mg gallic acid equivalent(GAE) per 100 g fresh weight (mg GAE100 g FW)

25 Determination of Total Flavonoid Content Total flavon-oid content was determined by aluminium chloride colori-metric test modified from previous study [17] One hundredand sixty microliters of sterile water was added to the testtube followed by 40 120583l of the fruit extracts dissolved in sterilewater at the concentration of 1mgml and 15120583l of 5NaNO

2

After 5min 24120583l of 10AlCl3sdot6H2Owas addedThemixture

was allowed to react for 6min Lastly 80120583l of 1M NaOHand 80 120583l of sterile water were added and well mixed Then250 120583l of reaction mixture was transferred to 96-well plateThe plate was shaken for 30 sec and the absorbances wereread at 510 nm by microplate reader The total flavonoidcontent was expressed as quercetin equivalent per 100 g freshweight (mg QE100 g FW) which was calculated from theregression equation obtained from graph plotted betweenthe absorbance and the concentration of quercetin at variousconcentrations (4ndash1500120583gml) The assay was performed intriplicate

26 DPPH Radical-Scavenging Assay Free radical-scaveng-ing activity of the fruit extracts was determined based on thereduction of DPPH radicals by means of the modified spec-trophotometric method in 96-well plates The test sampleswere prepared at concentration of 2mgml in methanol andmixed with 002wv DPPH radical methanolic solution 1 1ratio in triplicateThe reactionmixtureswere allowed to standin the dark for 30min at room temperature The absorbancewas measured at 517 nm using Infinite M200 MicroplateReader (Tecan) The DPPH radical-scavenging ability wascalculated as percent inhibition by the following equation Inhibition = [(119860controlminus119860 sample)119860control]times100TheDPPHradical-scavenging activity of the fruit extracts was expressedas the IC

50values (120583gml) (inhibition of DPPH radical forma-

tion by 50 at concentration of 1mgml) 6-Hydroxy-2578-tetramethylchroman-2-carboxylic acid (Trolox) was used asa positive control

27 Cell Culture Human embryonic kidney-293 (HEK-293)cells were cultured usingDMEMsupplementedwith 10FBSand 1 (vv) penicillin-streptomycin solution in a humidifiedatmosphere of 5 CO

2at 37∘C as previously described [18]

Under serum starvation conditions cells were treated withcrude extract of Thai fruits

Evidence-Based Complementary and Alternative Medicine 3

28 MTT Assay Cell viability was performed by the MTTassay according to the method previously described [19]Cells were seeded in 96-well culture plates at a density of1 times 104 cellswell in a total volume of 200120583l of DMEMsupplemented with 1 FBS plus 1 penicillinstreptomycin

Cells were allowed to adhere to plate for 24 h beforebeing treatedwith solvent (vehicle control) and crude extractsat various concentrations (001ndash2000120583gml) for 24 h Theexperiments were performed in 2 replicate wells The relativenumber of viable cells was then determined at 24 h afterincubation by adding 2mgml of MTT solution and furtherincubating the cell for 4 h The formazan crystals formedwere then solubilized with DMSO The plate was read usingan Infinite M200 Microplate Reader (Tecan) at wavelengthof 570 nm The absorption value of the solution at 570 nmdirectly represents relative cell numbers The percentageof cell viability was calculated according to the followingequation

Theof cell viability

= [Absorbance of treated cellsAbsorbance of control cells

] times 100 (1)

29Measurement of Intracellular ROS Level The intracellularantioxidant activities of the crude extract of Thai fruitswere quantified using a fluorescent probe dichlorodihy-drofluorescein (DCF) diacetate (Sigma-Aldrich) to estimatethe intracellular ROS production in the cell as previouslydescribed [20] HEK-293 cells were seeded in a 12-well plate(5 times 104 cellswell) or 35-mm glass bottomed dishes (1 times105 cellsdish) and treated with Thai fruit extracts (10 and100 120583gml) for 12 h After 12 h the cells were then incubatedwith 100 120583MofH

2O2for 2 h andwashed oncewith phosphate

buffered saline (PBS) pH 74 Thereafter 10 120583M DCFH-DAwas added and incubated with the cells at 37∘C in the dark for30min The fluorescence intensity of DCF was determinedusing Multi-Detection Microplate Reader (BioTek Instru-ments) with an excitationwavelength of 485 nmand emissionwavelength of 530 nm To monitor the ROS production inliving cells HEK-293 cells were visualized using single-lineexcitation (488 nm) of an IX-81 fluorescence microscope(Olympus) with a 40x (NA 14) objective lens (Olympus)

210 mRNA Expression Analysis by RT-qPCR The extractionof RNA was performed by using the RNeasy Mini Kit(Qiagen) as previously described [21] RT-qPCRs for RNAexpression were performed by using the KAPA SYBR FASTOne-Step RT-qPCR Kit (KAPA biosystems) according to themanufacturerrsquos instructions RT-qPCRs were performed onMx 3005p Real-Time PCR System (Stratagene) We designedand synthesized the primers as follows human GPx-1 (sense51015840-ctcttcgagaagtgcgaggt-31015840 antisense 51015840-tcgatgtcaatggtctg-gaa-31015840) human catalase (sense 51015840-gcagatacctgtgaactgtc-31015840antisense 51015840-gtagaatgtccgcacctgag-31015840) human HO-1 (sense51015840-caggcagagaatgctgag-31015840 antisense 51015840-gcttcacatagcgctgca-31015840) humanMn-SOD (sense 51015840-gcacattaacgcgcagatca-31015840 anti-sense 51015840-agcctccagcaactctcctt-31015840) human CuZn-SOD (sense

51015840-aaggccgtgtgcgtgaa-31015840 antisense 51015840-caggtctccaacatgcctct-31015840) human GRe (sense 51015840-cagtgggactcacggaagat-31015840 anti-sense 51015840-ttcactgcaacagcaaaacc-31015840) and human GAPDH(sense 51015840-cgagatccctccaaaatcaa-31015840 antisense 51015840-gtcttctgggtg-gcagtgat-31015840)

211 Western Blotting Protein expression of antioxidantenzymes was performed by Western blotting as previouslydescribed [20] Following stimulation cells werewashed oncewith PBS and solubilized in lysis buffer containing 20mMTris-HCl pH 74 08 Triton X-100 150mM NaCl 2mMEDTA 10 glycerol 100 120583M PMSF 5 120583gml aprotinin and5 120583gml leupeptin Protein concentration of cell lysates wasdetermined using a Bio-Rad protein assay kit with bovineserumalbumin as standard Samplesweremixedwith loadingbuffer anddenatured by heating at 95∘C for 5minprior to sep-aration by SDS-PAGE Separated proteins were transferredto polyvinylidene fluoride (PVDF)membrane (Bio-Rad) andsubjected to immunoblotting with primary antibodies tocatalase (Cell Signaling) Mn-SOD (Cell Signaling) GPx-1(Abcam) and GAPDH (Cell Signaling) Immunoblots werevisualized with HRP-conjugated secondary antibodies and achemiluminescence detection system (GE Healthcare)

212 Statistical Analysis Data were presented as mean plusmnSEM The statistical analysis was determined using one-wayanalysis of variance (ANOVA) and Studentrsquos t-test and valuesof 119875 lt 005 were considered to be significant

3 Results

31 Yields of Thai Fruit Extracts Eleven fruit samples werecollected and bought from southern and central regions ofThailand and were authenticated by a botanist The scientificnames of the plants are shown in Table 1 Although thereare many kinds of fruits in Thailand in this study we choseonly 11 fruits that have a potential effect on inhibition ofoxidative stress Thai people consume significant amount ofthe investigated fruits at a ripe stage and the yields of thecrude methanol extracts of Thai fruits ranged from 268 to928 of fresh weight (edible part of fresh fruits weight)(Table 1)

32 Determination of Total Phenolic and Flavonoid Contentsof Thai Fruit Extracts The total phenolic contents weredetermined using the FolinndashCiocalteu method a simple andwidely used method which relied on the transfer of electronsfrom phenolic compounds to the FolinndashCiocalteu reagent inalkaline medium As shown in Table 1 the total phenoliccontents of 11 selectedThai fruits are expressed as mg of gallicacid equivalent (GAE) per 100 g of freshweight (FW) Amongall the fruits analyzed the highest phenolic content was foundinM glabra (72383 plusmn 3694mg GAE100 g FW) followed byS koetjape (24101 plusmn 1651) A ghaesembilla (9838 plusmn 648)M indica (4539 plusmn 133) Z jujuba (4529 plusmn 345) M foetida(3883 plusmn 315) A bilimbi (3377 plusmn 137) and S malaccense(2955 plusmn 091) (Table 1) Phenolic content was slightly lowerin A integer and D zibethinusM paradisiaca cultivar Awak


Table 1 Yield of methanol extracts total phenolic and flavonoid contents and DPPH scavenging activities of Thai fruit extracts

Fruits(ripe stage) Scientific name

Yield ofcrude extract

( FW)

Total phenoliccontent

(mg GAE100 g FW)

Total flavonoidcontent

(mg QE100 g FW)

DPPH scavenging activity( inhibition at 1mgmL)

Mao khai pla(Thai)

Antidesmaghaesembilla Gaertn 483 9838 plusmn 648 13406 plusmn 290 6765 plusmn 031

Champedak Artocarpus integer(Thumb) Merr 685 2145 plusmn 228 15831 plusmn 102 1542 plusmn 152

Bilimbi Averrhoa bilimbi L 268 3377 plusmn 137 5533 plusmn 070 3066 plusmn 196Durian Durio zibethinus L

cultivar MonThong 486 965 plusmn 071 9385 plusmn 036 680 plusmn 097Escobillo Malpighia glabra L 440 72383 plusmn 3694 19536 plusmn 014 9662 plusmn 044Horse mango Mangifera foetida

Lour 674 3883 plusmn 315 4613 plusmn 185 2563 plusmn 132Mango Mangifera indica L

cultivar Aok Rong 912 4539 plusmn 133 ND 4485 plusmn 071Pisang Awak Musa paradisiaca L

cultivar Awak 724 034 plusmn 015 370 plusmn 131 515 plusmn 115

SantolSandoricum koetjape(Burm f) Merrillcultivar Tuptim

928 24101 plusmn 1651 85149 plusmn 030 8473 plusmn 138Malay applePomerac

Syzygium malaccense(L) Merr amp Perry 428 2955 plusmn 091 399 plusmn 165 1675 plusmn 108

Jujube Ziziphus jujubaMillcultivar Milk Jujube 763 4529 plusmn 345 136 plusmn 167 445 plusmn 091

ND = not detected

had the lowest total phenolic content among the tested fruits(Table 1)

The total flavonoid contents of 11 selected Thai fruits areexpressed as mg of quercetin equivalent (QE) per 100 g offresh weight (mg QE100 g FW) As shown in Table 1 totalflavonoid contents of Thai fruit extracts ranged from 136 plusmn167 to 85149 plusmn 030mg QE100 g FWThe highest flavonoidcontent was found in S koetjape which had 85149 plusmn030mg QE100 g FW followed by M glabra A integer Aghaesembilla D zibethinus A bilimbi and M foetida thevalues of whichwere 19536plusmn 014 15831plusmn 102 13406plusmn 2909385 plusmn 036 5533 plusmn 070 and 4613 plusmn 185mg QE100 g FWrespectively Flavonoid content was lower in S malaccenseand M paradisiaca On the other hand Z jujuba had thelowest content of total flavonoid (136 plusmn 167) while that ofM indica was not detected

33 DPPH Scavenging Activities of Thai Fruit Extracts Wenext determined the antioxidant activity of fruit extractsusingDPPHmethodThis assay based on themeasurement ofthe antioxidant ability to scavenge the stable organic radicalDPPH and used Trolox as positive control with the IC

50of

412 plusmn 03 120583gml As shown in Table 1 the extract of Mglabra at the concentration of 1mgml exhibited the highestDPPH free radical-scavenging activity with 9662 plusmn 044 ofinhibition (IC

50of 4500 plusmn 190 120583gml) which related to the

high total phenolic content (72383 plusmn 3694mg GAE100 gFW) The extracts of S koetjape showed moderate DPPHscavenging activities 8473 plusmn 138 (IC

50of 4158 plusmn 15 120583gml)

followed by A ghaesembilla (6765 plusmn 031 IC50

of 6480 plusmn

62 120583gml) M indica (4485 plusmn 071) and A bilimbi (3066plusmn 196) The extracts of D zibethinus M paradisiaca andZ jujuba had DPPH scavenging activity less than 10 ofinhibition (Table 1)

34 Correlation between Antioxidant Capacity and TotalPhenolic Content The correlation between total phenoliccontent and antioxidant activity using DPPH assay of fruitextracts was determined and shown in Figure 1 A positivecorrelation (1198772 = 06317) between the DPPH scavengingvalue and total phenolic content indicated that phenoliccompounds mainly contributed to the antioxidant activitiesof these fruits This result was in agreement with several pre-vious studies [15] However some fruits containing high totalflavonoid compounds such as A integer and D zibethinus(15831 plusmn 102 and 9385 plusmn 036mgQE100 g FW resp) did notexhibit strong antioxidant capacity In contrast S koetjapeand M glabra that contained higher flavonoid contentselicited strong DPPH scavenging activity with 8473 plusmn 138and 9662 plusmn 044 of inhibition respectively Thus thecorrelations between antioxidant activity and total flavonoidcontents of Thai fruit extracts were poor (1198772 = 03843)(Figure 1) According to their high antioxidants activities itcould be speculated that these fruits will be beneficial forinhibition of oxidative stress

35 Thai Fruit Extract Inhibits H2O2-Induced IntracellularROS Production in HEK-293 Cells We first assessed the cellcytotoxicity of crude extracts of 11 Thai fruits on HEK-293 cells by using MTT assay to determine the optimal


200 800600 10000 400Phenolic content (mg GAE100 g FW)

0

50

100

150

200

250

300D

PPH

scav

engi

ng (

inhi

bitio

n)

R2 = 06317

200 800600 10000 400Flavonoid content (mg QE100 g FW)

0

50

100

150

200

250

300

DPP

H sc

aven

ging

( in

hibi

tion)

R2 = 03843

Figure 1 Correlation between antioxidant capacities and total phenolic and flavonoid contents in 11 Thai fruits Antioxidant capacities ofThai fruit extracts were measured by the DPPH free radical-scavenging assay GAE gallic acid equivalent QE quercetin equivalent FWfresh weight

concentrations of the crude extract for measurement ofintracellular antioxidant activity We found that Thai fruitextracts at the concentration of 001ndash100120583gml were not toxicto HEK-293 cells (Figure 2) Upon treatment of the cells withthe crude extracts more than 100 120583gml the cell viabilitiesof HEK-293 were less than 80 The concentrations usedin further experiment were 10 and 100 120583gml for the fruitextracts in order to prevent the cytotoxic effect and allow atleast 80 cells survival

We next investigated whether Thai fruit extract inhibitsH2O2-induced intracellular ROS production in HEK-293

cells The intracellular ROS levels were measured by using afluorescent probe DCFH-DA After incubation with H

2O2

the levels of intracellular ROS significantly increased com-pared to that of control (vehicle) group (Figure 3) As shownin Figure 3(a) treatment with A ghaesembilla and A bilimbisignificantly inhibited H

2O2-induced ROS production in a

dose-dependent manner whereas treatment with A integerandD zibethinus did not show the inhibitory effect onH

2O2-

induced ROS production In addition the crude extracts ofM glabra and M indica showed a significant reduction inROS production which has effects similar to those of vitaminC (a potent antioxidant) whereas treatment with extracts ofM foetida and M paradisiaca had no effect (Figure 3(b))Moreover treatment with fruit extracts of S koetjape Smalaccense and Z jujuba also suppressed H

2O2-induced

ROS production in a dose-dependent manner (Figure 3(c))As shown in Figure 3 the fruit extracts ofA ghaesembilla

A bilimbi M glabra M indica S koetjape S malaccenseand Z jujuba have the antioxidant effects by preventing theproduction of intracellular ROS induced byH

2O2We further

quantified ROS production in intact HEK-293 cells usingthe fluorescent microscope Incubation of HEK-293 cellswith H

2O2induced a significant increase in the fluorescence

intensity of dichlorodihydrofluorescein (DCF) as shown ingreen color in intact cells of the control (vehicle) group(Figure 4) Treatment with fruit extracts of A ghaesembillaA bilimbi M glabra M indica S koetjape S malaccenseand Z jujuba decreased the fluorescence intensity of DCF

suggesting that these fruits suppressed the ROS productionin intact cells However treatment with fruits extracts ofA integer D zibethinus M foetida and M paradisiaca didnot reduce the fluorescence intensity of DCF inducing byH2O2compared to that of control Taken together these data

demonstrated that Thai fruits have the antioxidant effects bypreventing the production of ROS in HEK-293 cells

36 Induction of mRNA Expression of Antioxidant Enzymesby Thai Fruit Extracts Antioxidant enzymes (eg GPx-1catalase Mn-SOD CuZn-SOD and HO-1) are the majorcomponents of antioxidant signaling cascade in many celltypes Treatment withA bilimbi extract elicits the antioxidanteffect by increasing the activity of SOD and GPx both inblood and in tissues (eg liver kidney and heart) of rats [22]Moreover an aqueous extract ofM indica inhibited oxidativestress by enhancing the production of antioxidant enzymessuch as SOD and catalase [23] Consistent with these previousstudies we showed that treatment with fruit extracts of Mglabra S malaccense M indica and A bilimbi significantlyelevated themRNA level ofMn-SOD (Figure 5(a))Moreoverthe GPx-1 mRNA expression significantly increased whentreated with fruit extracts of S malaccense S koetjape Mindica A bilimbi Z jujuba andA ghaesembilla (Figure 5(b))In addition the mRNA level of catalase also increased aftertreatment with fruit extracts from M glabra M indicaZ jujuba and A ghaesembilla (Figure 5(c)) However allThai fruit extracts failed to induce the mRNA expressionsof CuZn-SOD GRe and HO-1 in HEK-293 cells (Figures5(d)ndash5(f) resp) Collectively our results showed that Thaifruit extracts play an important role in the prevention ofoxidative stress by enhancing the mRNA expression of theseantioxidant enzymes in HEK-293 cells

37 Thai Fruit Extracts Increased Protein Level of AntioxidantEnzymes As shown in Figure 5 the mRNA expressions ofMn-SOD GPx-1 and catalase were increased after treatmentwith the Thai fruit extracts We next investigated the effectsof these fruit extracts on the protein expressions of these


Artocarpus integer

1 200010 1000001 01 1000

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ce

ll vi

abili

ty

Averrhoa bilimbi

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1 200010 100 100001001

Malpighia glabra

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cell

viab

ility

01 1 10 100 1000 2000001

Mangifera foetida

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abili

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1 200010 100 100001001

Sandoricum koetjape

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abili

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10001 10 2000001 01 100

Vehicle

0

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1 200010 1000001 01 100

Antidesma ghaesembilla

1 200010 100 100001001Concentration (120583gml) Concentration (120583gml) Concentration (120583gml)

Concentration (120583gml) Concentration (120583gml) Concentration (120583gml)



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ll vi

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Durio zibethinus

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Mangifera indica

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1 200010 100 100001001

Musa paradisiaca

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Ziziphus jujuba

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Syzygium malaccense

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1 200010 1000001 01 100

Figure 2 Cytotoxicity profile of Thai fruit extracts in HEK-293 cells HEK-293 cells were treated with Thai fruit extracts (001ndash2000120583gml)for 36 h Cell viability was quantified expressed as a percentage of cell viability and shown as the mean plusmn SEM (119899 = 4)

antioxidant enzymes We found that treatment with fruitextracts of M glabra S malaccense M indica and Abilimbi at the concentration of 100 120583gml for 24 h significantlyelevated the protein level of Mn-SOD (Figure 6 upper-rightpanel) Similar to the mRNA expression experiments the

results of protein expression revealed that treatment withfruit extracts of S malaccense S koetjape M indica Abilimbi Z jujuba and A ghaesembilla caused an increase inthe GPx-1 protein level compared with the control (vehicle)(Figure 6 lower-left panel) Moreover the protein levels of


A integerA ghaesembilla Vit C10

A bilimbi D zibethinus100 10 100 10 100 10 100 10 100 Vehicle Vehicle

(120583gml) (120583gml) (120583gml) (120583gml) (120583M)+ H2O2

0

50

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ROS

prod

uctio

n (

)

lowast

(a)

M glabra M paradisiacaM foetida M indica Vit C10 100 10 100 10 100 10 100 10 100 Vehicle Vehicle


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prod

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(b)

Z jujubaS malaccenseS koetjapeVehicle Vehicle 100 10 100 10 100 10 100 10

Vit C(120583gml) (120583gml) (120583gml) (120583M)

+ H2O2

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prod

uctio

n (

)

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(c)

Figure 3 Effects ofThai fruit extracts on H2O2-induced ROS production in HEK-293 cells (andashc) Cells were treated with vehicle (control) 10

and 100120583M vitamin C or 10 and 100 120583gml fruit extracts for 6 h Cells were then incubated with 200120583MH2O2for 30min The intracellular

ROS production was quantified expressed as a percentage of the control and shown as the mean plusmn SEM (119899 = 4) lowast119875 lt 005 versus control119875 lt 005 versus H2O2


DCF fluorescence intensity

DCF

Phasecontrast

Vehicle VehicleA bilimbi M glabraA integer D zibethinusA ghaesembilla

(100120583gml) (100120583gml) (100120583gml)(100120583gml) (100120583gml)

+ H2O2

(a)


DCF

Phasecontrast

M foetida M indica Z jujubaS malaccenseM paradisiaca S koetjape Vit C(100120583gml) (100120583gml) (100120583gml) (100120583gml) 100120583M(100120583gml) (100120583gml)

+ H2O2

(b)

Figure 4Thai fruit extracts attenuated intracellular ROS production in HEK-293 cells (a-b) Cells were treated with vehicle (control) 10 and100120583M vitamin C or 10 and 100 120583gml fruit extracts for 6 h Cells were then incubated with 200120583MH

2O2for 30min Cells were washed and

incubated with 10120583MDCFH-DA Cells were visualized by green fluorescence for DCF and by bright field Scale bar 10120583m (119899 = 4)

catalase significantly increased after treatment with crudeextracts ofM glabra M indica and A ghaesembilla for 24 hwhereas the catalase protein level tended to increase aftertreatment with crude extract of Z jujuba (Figure 6 lower-right panel) These results indicated that Thai fruits preventthe oxidative stress by enhancing the protein synthesis ofantioxidant enzymes

4 Discussion

In this study we provide a new molecular mechanism ofThai fruits extracts on the inhibition of oxidative stress inHEK-293 cells We demonstrate that ethanol extracts fromA ghaesembilla A bilimbi M glabra M indica S koetjapeS malaccense and Z jujuba have antioxidant effects bysuppressing ROS production and inducing the synthesis ofantioxidant enzymes such as GPx-1 catalase and Mn-SODin HEK-293 cells

Oxidative stress has been implicated in the pathogenesisof various chronic diseases such as cancer heart diseases anddiabetes [1ndash3]The nonenzymatic antioxidant defense systemof the body is made up of some antioxidants such as vitaminC vitamin E vitamin K and glutathione The exogenousantioxidants are mainly comprised of synthetic and naturalantioxidantsMedicinal plants have been used to treat humandiseases for thousands of years People are becoming increas-ingly interested in medicinal plants including fruits becauseof their good therapeutic performance and low toxicity Fruitsare potential sources of natural antioxidants and have beenshown in epidemiological studies to be protective againstseveral chronic diseases associated with aging such as cancerimmune dysfunction and cardiovascular diseases [8] Thesenatural protective effects have been attributed to various com-ponents such as carotenoids vitamins C and E and phenoliccompounds [24 25] Thus the interest in phytochemicalsincluding phenolic compounds derived from fruits and theirroles in inhibition of oxidative stress are therefore increasing


Mn-SOD

Relat

ive M

n-SO

DG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilimglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast

lowast

M

(a)

Rela

tive G

Px-1

GA

PDH

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast lowast lowast

lowast

(b)

Rela

tive c

atal

ase

GA

PDH

Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast lowast

(c)

CuZn-SOD

Relat

ive C

uZn-

SOD

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

(d)

GRe

Relat

ive G

ReG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

mRN

A ex

pres

sion

100120583gml

(e)

HO-1

Rela

tive H

O-1

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

14

mRN

A ex

pres

sion

100120583gml

(f)

Figure 5 Effects of Thai fruit extracts on the mRNA expression of antioxidant enzymes (andashf) Cells were treated with vehicle (control) or100120583gml fruit extracts for 6 h After treatment the total RNAwas extracted from cells and the mRNA expression was analyzed using specificprimers The relative Mn-SOD (a) GPx-1 (b) catalase (c) CuZn-SOD (d) GRe (e) and HO-1 (f) mRNA levels were quantified and shownas the mean plusmn SEM (119899 = 4) lowast119875 lt 005 versus vehicle


lowast

lowast

lowastlowast lowast

lowast

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

GPx

-1G

APD

H (f

old

over

bas

al)

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

Mn-SOD22kD

GPx-122kD

Catalase60kD

GAPDH37kD

Mn-SOD

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

05

10

15

20

Mn-

SOD

GA

PDH

(fol

d ov

er b

asal

)

100120583gml

lowastlowast lowast lowast

Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

25

Cata

lase

GA

PDH

(fol

d ov

er b

asal

)

lowastlowast

lowast

Figure 6 Effects ofThai fruit extracts on the protein expression of antioxidant enzymes Cells were treated with vehicle (control) or 100 120583gmlfruit extracts for 24 h After treatment the cell lysates were immunoblotted with anti-Mn-SOD anti-GPx-1 or anti-catalase antibodies Theexpression of GAPDH served as a loading controlThe protein expression was quantified expressed as the fold increase over vehicle (control)level and shown as the mean plusmn SEM (119899 = 4) lowast119875 lt 005 versus control

The antioxidative effects of Thai fruit extracts could bedue to the presence of phenolic compounds Studies havealso shown that phenolic compounds from various sourcesare effective in improving antioxidant enzymes suggestingtheir role in the prevention and treatment of oxidative stress-related diseases Hence the consumption of phenolic-richfruits has been associated with reduced levels of ROS in ani-mal experiments [10 26] Likewise a high intake of phenolic-rich fruits has been reported to enhance the activities of theantioxidant enzymes [27] It was reported that the antioxidantactivity of raspberry and santol (S koetjape) was directlyrelated to the phenolic content [28] In addition the juiceof Ziziphus mauritiana that has a moderate total phenoliccontent and low content of vitamin C elicits the antioxidantactivity [29] Consistent with these previous studies ourstudy demonstrated that the extract from M glabra hasthe highest phenolic content followed by S koetjape AghaesembillaM indica Z jujubaM foetida A bilimbi andS malaccense (Table 1)TheseThai fruit extracts exhibited theantioxidant activity as determined by DPPH assay

The correlation between total antioxidant capacitiesobtained from DPPH assay is shown in Figure 1 There was adirect linear relationship between the phenolic contents and

total antioxidant activities in the tested 11 Thai fruits (R2 =06317) The higher total phenolic content in fruits resultedin higher total antioxidant activity Similarly previous studyreported a significant positive correlation between the antiox-idant capacity and the contents of total flavonoids and totalphenolics in celery [30] Moreover antioxidant activitiesassayed by DPPH and FRAP methods showed stronglypositive relationship with total phenolic content in Oxal-idaceae fruits [31] Taken together these results suggestedthat phenolic compounds may be the major contributor tothe total antioxidant activities of fruits However our resultsdemonstrated a weak correlation (R2 = 03843) betweenthe DPPH scavenging activity and total flavonoid contentsuggesting that flavonoid compounds could not be maincomponents responsible for DPPH scavenging activity ofsome fruits such asM indica S koetjape andM glabra

In our present study we found that among the tested11 Thai fruits tested 7 of them showed the ability to inhibitH2O2-induced oxidative stress in HEK-293 cells Previous

study demonstrated that the administration of phenolic acids(ie gentisic acid gallic acid ferulic acid and p-coumaricacid) in rat at a concentration of 100mgkg exhibited aninduction of hepatic antioxidant enzymes such as SOD and


GPx [10] This suggested that modulation of antioxidantenzymes and oxidative status in the liver by phenolic acidsmay play an important role in the protection against oxidativedamage Therefore further identification of phytochemicalscorresponded to antioxidant activities ofThai fruits are worthinvestigating

Interestingly the antioxidant effects of fruits might bedue to the induction of antioxidant genes such as SOD andGPx [32 33] Fruit extracts from Averrhoa carambola (starfruit) also induced the activities of enzymatic antioxidants(ie SOD and catalase) and nonenzymatic antioxidant andreduced glutathione in mice [34] Consistent with theseprevious studies we showed that treatment with the Thaifruit extracts from M glabra S malaccense M indica andA bilimbi induced the mRNA and protein expressions ofMn-SOD We also demonstrated that fruit extracts from Mglabra M indica and A ghaesembilla are able to induce themRNA and protein expressions of other antioxidant enzymessuch as catalase However our study is unlike the study byothers in which activities of these antioxidant enzymes werenot determined after treatment with fruit extracts in HEK-293 cells Further evaluation of theThai fruit extracts-inducedantioxidant enzyme activity in HEK-293 cells is required

There are two categories of antioxidative defenses thatprevent radical formation repair oxidative damage removeradicals before damage can occur eliminate damagedmolecules and prevent mutations These two categories areantioxidant enzymes and nonenzymatic antioxidant com-pounds [35] SOD catalase and GPx are enzymes thatdestroy the peroxides and play a significant role in providingantioxidant defenses to an organism GPx and catalase areinvolved in the elimination ofH

2O2 SODacts by dismutating

superoxide radical to H2O2 which is then acted upon by

GPx (Figure 7) The functions of all three enzymes areinterconnected and their insufficient activities result in theaccumulation of ROS production and increased oxidativestress in the cells

The previous study suggested that treatment with theaqueous extract of A bilimbi can partially reduce the imbal-ance between the ROS generation and the scavenging enzymeactivity in diabetic animals exposed to oxidative stress [33]Consistent with this previous study we demonstrated thatA bilimbi extract is able to inhibit H

2O2-induced ROS

production in HEK-293 cells by inducing the synthesis ofantioxidant enzymes such asMn-SOD and GPx-1 Accordingto this previous study and our present data A bilimbi couldbe a supplement as an antioxidant therapy and may bebeneficial for correcting the ROS production and preventingoxidative stress due to lipid peroxidation and free radicals

It has been reported thatmango peel extract contains sev-eral valuable compounds such as polyphenols carotenoidsterpenoids sterols fatty acids and dietary fiber [12] Themajor components such as mangiferin amentoflavonefriedelin daucosterol and beta-sitosterol have been alreadyidentified [36] The peel extract of M indica exhibitedhigh antioxidant capacity by effectively scavenging variousfree radical such as DPPH hydroxyl and peroxyl radicals[12] In addition treatment with aqueous extract of Mindica inhibited lipofundin-induced oxidative stress in rats

Mn-SOD

Oxidative stress

Lipid peroxidationDNA damageProtein oxidationApoptosis

CatalaseGPx

S koetjape

A ghaesembillaM indica

A bilimbi

S malaccenseZ jujuba

M glabraM indica

A ghaesembillaM glabraA bilimbi

M indicaS malaccense

Antioxidant effects of Thai fruits

synthesissynthesis synthesis

H2O2O2 O∙2minus H2O + O2

uarr Mn-SOD uarr Catalase uarr GPx

∙OH

Figure 7 Schematic diagram representing the antioxidant effectsof Thai fruits in HEK-293 cells Thai fruit extracts from Antidesmaghaesembilla Averrhoa bilimbi Malpighia glabra Mangifera indicaSandoricum koetjape Syzygium malaccense and Ziziphus jujubainhibit oxidative stress in HEK-293 cells by increasing the produc-tion of antioxidant enzymes such as catalase GPx and Mn-SOD

[23] This antioxidant effect is derived from the increasingactivities of SOD and catalase [23] We also showed in thisstudy that M indica extract has an antioxidant activity invitro and in HEK-239 cells This extract induced the mRNAand protein expressions of Mn-SOD GPx-1 and catalasein HEK-293 cells Thus the active compounds in mangoextract might exert the protective effects through stimulatingoxidative defense system in the cells

Malpighia glabra (Escobillo) is well known as an excellentdietary source of vitamin C and many phytochemicals suchas polyphenols anthocyanin carotenoids and tannins [37]Interestingly the phytochemical estimation in ten Indianfruits revealed thatM glabra has the highest phenolic contentin methanol extract (35574 plusmn 429mg GAE100 g FW) [37]Our data are consistent with this previous study showing thatamong 11 fruits the highest phenolic content was found inM glabra (72383 plusmn 3694mg GAE100 g FW)The differencein phenolic content may be due to the variation in the stateof maturity genotype and climatic factors Previous studyreported that aqueous extract of M glabra has a potentantioxidant activity determined by DPPH scavenging test[38] In addition our present study reported that treatmentwith extract from M glabra attenuated H

2O2-induced ROS

production in HEK-293 cells confirming that the fruits ofM glabra are rich in antioxidants especially polyphenolsThestudy from Singh and his colleagues showed the positive cor-relation for antioxidant capacity with phenolic compounds(R2 = 0845) [37] We also reported the strong correlationbetween antioxidant capacity and phenolic contents in Thaifruits including M glabra In addition we found that the


methanol extract of M glabra exhibited the antioxidanteffect in HEK-293 cells by increasing mRNA and proteinexpressions of Mn-SOD and catalase Hence the fruits ofM glabra could be a good resource of phytochemicals withhigh antioxidant activity However further studies on theidentification and isolation of individual active compoundsare required

A ghaesembilla is an indigenous species found in Thai-land A few observations demonstrated that the extract ofA ghaesembilla has antioxidant activity For instance Aghaesembillawas analyzed for their total antioxidant capacitythrough in vitro radical-scavenging assays such as DPPHand FRAP assays and antioxidant enzyme activity assay[32] This previous study reported that extract from Aghaesembilla has the highest DPPH (10206 AEAC mg100 gdry wt) and FRAP values (2114 120583M AEACg dry wt) Inaddition A ghaesembilla can activate the enzymatic activ-ities of SOD catalase and peroxidase [32] In the presentstudy we reported that the extract of A ghaesembilla atthe concentration of 1mgml exhibited moderate DPPH freeradical-scavenging activity with 6765 plusmn 031 of inhibitionwhich related to the total phenolic content (9838 plusmn 648mgGAE100 g FW) Moreover the extract of A ghaesembillashowed an antioxidant effect in HEK-293 cells by stimulatingthe synthesis of GPx-1 and catalase Interestingly adminis-tration of crude extract (dose 400ndash1000mgkg body weight)from A ghaesembilla for 21 days in diabetic rats resulted ina decrease of blood glucose level [39] As regards antioxidantand hypoglycemic actions A ghaesembilla can be served as agood source of natural supplement

The juice of Z mauritiana has moderate total phenoliccontent (39696 plusmn 777 120583g GAEg sample) while juice of Smalaccense has low total phenolic content (8151 plusmn 006 120583gGAEg sample) [29] Consistent with this previous studywe reported that fruit extract of Z jujuba exhibited higherphenolic content than S malaccense (4529 plusmn 345 versus2955 plusmn 091mg GAE100 g FW) The juice of Z mauritianaand S malaccense exhibited the great DPPH scavengingactivity (46058 plusmn 1793 and 13711 plusmn 207 120583g VCEACg sampleresp) [29] In addition our study showed that the methanolextracts of Z jujuba and S malaccense fruits also exhibitedthe DPPH scavenging activity Moreover these two fruitsattenuated H

2O2-induced ROS production and increased

the synthesis of GPx-1 and catalase in HEK-293 cells Theantioxidant activity of Z mauritiana might be contributedby p-hydroxybenzoic acid that was reported to be the mostabundant phenolic compound in the fruit extract [40]

The juice of S koetjape has high total phenolic content(61704 plusmn 223 120583g GAEg sample) [29] In addition wereported that higher phenolic content was also detected incrude methanol extract of S koetjape (24101 plusmn 1651mgGAE100 g FW) The antioxidant activities of S koetjapecould be associated with its high total phenolic content Thejuice of S koetjape exhibited the great DPPH scavengingactivity (41822 plusmn 517 120583g VCEACg sample) [29] Consistentwith this previous study we found that methanol extract of Skoetjape showed a high DPPH scavenging activity (Table 1)Moreover S koetjape extract elicited the antioxidant activity

in HEK-293 cells by attenuating H2O2-induced ROS produc-

tion Our present study demonstrated that the antioxidantactivity of S koetjape extract derived from the induction ofmRNA and protein expression of GPx-1 in HEK-293 cells

It has been reported that administration of phenolicacids (ie gentisic acid gallic acid ferulic acid and p-coumaric acid) at a dosage of 100mgkg for 14 consecutivedays in rats enhanced the mRNA expression of hepaticCuZn-SOD GPx and catalase [10] Moreover these phe-nolic acids also stimulated the enzyme activities In thepresent study we demonstrated that Thai fruits that havehigh phenolic contents showed the antioxidant effects byincreasing the synthesis of Mn-SOD GPx-1 and catalase inHEK-293 cells Thus polyphenolics found in fruits mightexert the antioxidant effect through stimulating synthe-sis of these antioxidant enzymes Although it is possiblethat phenolic acids contained in Thai fruits are involvedin the regulation of antioxidant enzyme synthesis furtherstudy is required to determine the active compounds nec-essary for antioxidant actions of Thai fruits in HEK-293cells

5 Conclusion

We have identified a new mechanism for fruit extractsmediating the inhibition of oxidative stress in HEK-293 cellsThai fruits provide an antioxidation which enhances themRNA and protein expressions of antioxidant enzymes suchas catalase GPx-1 and Mn-SOD (Figure 7) Our resultsprovide a better understanding of the antioxidant effects ofThai fruits on antioxidant enzyme production in the cells aswell as information regarding the intake of fruits for humanhealth

Competing Interests

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

Authorsrsquo Contributions

Natthinee Anantachoke performed the experiments and dataanalysis and wrote the manuscript Pattamapan Lomaratperformed the experiments and data analysis and wrotethe manuscript Wasin Praserttirachai performed the exper-iments Ruksinee Khammanit performed the experimentsand data analysis Supachoke Mangmool carried out theexperiments and data analysis participated in the studyplanning and wrote the manuscript

Acknowledgments

This work was supported by the Thailand Research FundGrant RDG5720041 (SupachokeMangmool)The authors aregrateful to Professor Wongsatit Chuakul for identification ofthe fruits


References

[1] I M Fearon and S P Faux ldquoOxidative stress and cardiovasculardisease novel tools give (free) radical insightrdquo Journal of Mo-lecular and Cellular Cardiology vol 47 no 3 pp 372ndash381 2009

[2] C Y Guo L Sun X P Chen andD S Zhang ldquoOxidative stressmitochondrial damage and neurodegenerative diseasesrdquoNeuralRegeneration Research vol 8 no 21 pp 2003ndash2014 2013

[3] D M Niedowicz and D L Daleke ldquoThe role of oxidative stressin diabetic complicationsrdquoCell Biochemistry and Biophysics vol43 no 2 pp 289ndash330 2005

[4] I Fridovich ldquoSuperoxide anion radical (119874∙2) superoxide dis-

mutases and related mattersrdquo Journal of Biological Chemistryvol 272 no 30 pp 18515ndash18517 1997

[5] B Halliwell ldquoAntioxidant defencemechanisms from the begin-ning to the end (of the beginning)rdquo Free Radical Research vol31 no 4 pp 261ndash272 1999

[6] F M Maiorino R Brigelius-Flohe K D Aumann A RoveriD Schomburg and L Flohe ldquoDiversity of glutathione peroxi-dasesrdquoMethods in Enzymology vol 252 pp 38ndash53 1995

[7] J A Araujo M Zhang and F Yin ldquoHeme oxygenase-1 oxida-tion inflammation and atherosclerosisrdquo Frontiers in Pharma-cology vol 3 article 119 2012

[8] H Boeing A Bechthold A Bub et al ldquoCritical reviewvegetables and fruit in the prevention of chronic diseasesrdquoEuropean Journal of Nutrition vol 51 no 6 pp 637ndash663 2012

[9] Y Cai Q Luo M Sun and H Corke ldquoAntioxidant activityand phenolic compounds of 112 traditional Chinese medicinalplants associated with anticancerrdquo Life Sciences vol 74 no 17pp 2157ndash2184 2004

[10] C-T Yeh and G-C Yen ldquoInduction of hepatic antioxidantenzymes by phenolic acids in rats is accompanied by increasedlevels of multidrug resistance-associated protein 3 mRNAexpressionrdquo Journal of Nutrition vol 136 no 1 pp 11ndash15 2006

[11] L Zhang A S Ravipati S R Koyyalamudi et al ldquoAntioxidantand anti-inflammatory activities of selected medicinal plantscontaining phenolic and flavonoid compoundsrdquo Journal ofAgricultural and FoodChemistry vol 59 no 23 pp 12361ndash123672011

[12] CM Ajila K A Naidu S G Bhat andU J S P Rao ldquoBioactivecompounds and antioxidant potential of mango peel extractrdquoFood Chemistry vol 105 no 3 pp 982ndash988 2007

[13] L Fu B-T Xu X-R Xu et al ldquoAntioxidant capacities and totalphenolic contents of 62 fruitsrdquo Food Chemistry vol 129 no 2pp 345ndash350 2011

[14] J Sun Y-F Chu X Wu and R H Liu ldquoAntioxidant andantiproliferative activities of common fruitsrdquo Journal of Agricul-tural and Food Chemistry vol 50 no 25 pp 7449ndash7454 2002

[15] L Fu B-T Xu X-R Xu X-S Qin R-Y Gan and H-B LildquoAntioxidant capacities and total phenolic contents of 56 wildfruits from South Chinardquo Molecules vol 15 no 12 pp 8602ndash8617 2010

[16] B Vongsak P Sithisarn S Mangmool S ThongpraditchoteY Wongkrajang and W Gritsanapan ldquoMaximizing total phe-nolics total flavonoids contents and antioxidant activity ofMoringa oleifera leaf extract by the appropriate extractionmethodrdquo Industrial Crops and Products vol 44 pp 566ndash5712013

[17] T J Herald P Gadgil and M Tilley ldquoHigh-throughputmicro plate assays for screening flavonoid content and DPPH-scavenging activity in sorghum bran and flourrdquo Journal of the

Science of Food and Agriculture vol 92 no 11 pp 2326ndash23312012

[18] W Parichatikanond D Pinthong and S Mangmool ldquoBlockadeof the renin-angiotensin system with delphinidin cyanin andquercetinrdquo Planta Medica vol 78 no 15 pp 1626ndash1632 2012

[19] C Chittasupho M Jaturanpinyo and S Mangmool ldquoPectinnanoparticle enhances cytotoxicity of methotrexate againsthepG2 cellsrdquo Drug Delivery vol 20 no 1 pp 1ndash9 2013

[20] S Mangmool P Hemplueksa W Parichatikanond and NChattipakorn ldquoEpac is required for GLP-1R-mediated inhi-bition of oxidative stress and apoptosis in cardiomyocytesrdquoMolecular Endocrinology vol 29 no 4 pp 583ndash596 2015

[21] B Vongsak S Mangmool and W Gritsanapan ldquoAntioxidantactivity and induction of mRNA expressions of antioxidantenzymes in HEK-293 cells of Moringa oleifera leaf extractrdquoPlanta Medica vol 81 no 12-13 pp 1084ndash1089 2015

[22] N Thamizhselvam I V Liji Y R Sanjayakumar C G SanalGopi K G Vasantha Kumar and G K Swamy ldquoEvaluationof antioxidant activity of Averrhoa bilimbi Linn fruit juice inparacetamol intoxicatedWistarAlbino ratsrdquoEnliven Toxicologyand Allied Clinical Pharmacology vol 1 no 1 pp 1ndash6 2015

[23] L D Roche and A F Perez ldquoProtective effects of Mangiferaindica L extract against lipofundin-induced oxidative stress inratsrdquo Pharmaceutical Crops vol 3 pp 94ndash98 2012

[24] S Li S-K Li R-Y Gan F-L Song L Kuang and H-B LildquoAntioxidant capacities and total phenolic contents of infusionsfrom 223 medicinal plantsrdquo Industrial Crops and Products vol51 pp 289ndash298 2013

[25] J Lako V C Trenerry M Wahlqvist N WattanapenpaiboonS Sotheeswaran and R Premier ldquoPhytochemical flavonolscarotenoids and the antioxidant properties of a wide selection ofFijian fruit vegetables and other readily available foodsrdquo FoodChemistry vol 101 no 4 pp 1727ndash1741 2007

[26] C Y Lim S Mat Junit M A Abdulla and A Abdul AzizldquoIn vivo biochemical and gene expression analyses of theantioxidant activities and hypocholesterolaemic properties ofTamarindus indica fruit pulp extractrdquo PLoS ONE vol 8 no 7Article ID e70058 2013

[27] A C D S Pereira A P Dionısio N J Wurlitzer et al ldquoEffectof antioxidant potential of tropical fruit juices on antioxidantenzyme profiles and lipid peroxidation in ratsrdquo Food Chemistryvol 157 pp 179ndash185 2014

[28] M Liu X Q Li C Weber C Y Lee J Brown and R H LiuldquoAntioxidant and antiproliferative activities of raspberriesrdquoJournal of Agricultural and Food Chemistry vol 50 no 10 pp2926ndash2930 2002

[29] S F Sulaiman and K L Ooi ldquoAntioxidant and 120572-glucosidaseinhibitory activities of 40 tropical juices from malaysia andidentification of phenolics from the bioactive fruit juices ofBarringtonia racemosa and Phyllanthus acidusrdquo Journal of Agri-cultural and FoodChemistry vol 62 no 39 pp 9576ndash9585 2014

[30] Y Yao W Sang M Zhou and G Ren ldquoPhenolic compositionand antioxidant activities of 11 celery cultivarsrdquo Journal of FoodScience vol 75 no 1 pp C9ndashC13 2010

[31] A Noor Asna and A Noriham ldquoAntioxidant activity andbioactive components of oxalidaceae fruit extractsrdquo MalaysianJournal of Analytical Sciences vol 18 no 1 pp 116ndash126 2014

[32] U C Basak A K Mahapatra and S Mishra ldquoAssessment ofprotective antioxidant mechanisms in some ethno-medicinallyimportant wild Edible fruits of Odisha Indiardquo Agrotechnololgyvol 2 article 116 2013


[33] S B Kurup and S Mini ldquoAttenuation of hyperglycemia andoxidative stress in streptozotocin-induced diabetic rats by aque-ous extract ofAverrhoa bilimbi LINN fruitsrdquo International Jour-nal of Pharmaceutical Sciences and Research vol 5 no 11 pp4981ndash4988 2014

[34] R Singh J Sharma and P K Goyal ldquoProphylactic role of Aver-rhoa carambola (Star Fruit) extract against chemically inducedhepatocellular carcinoma in Swiss albino micerdquo Advances inPharmacological Sciences vol 2014 Article ID 158936 8 pages2014

[35] V Lobo A Patil A Phatak and N Chandra ldquoFree radicalsantioxidants and functional foods impact on human healthrdquoPharmacognosy Reviews vol 4 no 8 pp 118ndash126 2010

[36] M H A Jahurul I S M Zaidul K Ghafoor et al ldquoMango(Mangifera indica L) by-products and their valuable compo-nents a reviewrdquo Food Chemistry vol 183 pp 173ndash180 2015

[37] D R Singh S Singh KM Salim andR C Srivastava ldquoEstima-tion of phytochemicals and antioxidant activity of underutilizedfruits ofAndaman Islands (India)rdquo International Journal of FoodSciences and Nutrition vol 63 no 4 pp 446ndash452 2012

[38] L Wang F Li C He Y Dong and Q Wang ldquoAntioxidantactivity and melanogenesis inhibitory effect of acerola fruit(Malpighia glabra L) aqueous extract and its safe use incosmeticsrdquo Asian Journal of Chemistry vol 27 no 3 pp 957ndash960 2015

[39] M F Gargantiel and M C Ysrael ldquoAntioxiant activity andhypoglycemic potential of Antidesma ghaesembilla Gaertn(Phyllantaceae)rdquo International Journal Science and TechnologyResearch vol 3 pp 422ndash431 2014

[40] M Muchuweti G Zenda A R Ndhlala and A KasiyamhuruldquoSugars organic acid and phenolic compounds of Ziziphusmauritiana fruitrdquo European Food Research and Technology vol221 no 3-4 pp 570ndash574 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



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OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Natural antioxidants that are presented in fruits haveattracted considerable interest because of their presumedsafety and potential nutritional and therapeutic value Theincreased interest in natural antioxidants has led to theantioxidant evaluation of many species of fruits Even thoughantioxidant activities of many fruits have been reported andthe results showed that some of them could be rich sources ofnatural antioxidants the effects of phytochemicals containedin fruits on the induction of antioxidant enzymes in the cellshave not been fully defined Therefore the objectives of thisstudy were to determine the profiles of total phenolics totalflavonoids the antioxidant activities ofThai fruit extracts andthe molecular mechanisms of Thai fruit extracts on H

2O2-

induced oxidative stress in HEK-293 cells

2 Materials and Methods

21 Chemicals 22-Diphenyl-1-picrylhydrazyl (DPPH) gallicacid ascorbic acid 6-hydroxy-2578-tetramethylchroman-2-carboxylic acid (Trolox) sodium nitrite aluminium chlo-ride hexahydrate sodium hydroxide hydrogen peroxide 3-[45-dimethylthiazol-2-yl]-25-diphenyl tetrazoliumbromide(MTT) and dichlorodihydrofluorescein (DCF) acetate werepurchased fromSigma-Aldrich (Saint LouisMO)DulbeccorsquosModified EagleMedium (DMEM) fetal bovine serum 025trypsin-EDTA solution and penicillin-streptomycin solutionwere purchased from Gibco (Grand Island NY) FolinndashCiocalteursquos phenol reagent was purchased fromMerck KGaA(Darmstadt Germany) Anhydrous sodium carbonate wasavailable from Ajax Finechem (Auckland New Zealand)

22 Plant Materials The fruits of Antidesma ghaesembillaGaertn (Phyllanthaceae) Artocarpus integer (Thumb) Merr(Moraceae) Averrhoa bilimbi L (Oxalidaceae) and Mangi-fera foetida Lour (Anacardiaceae) were collected from Ran-ong ProvinceThailand while the fruits ofDurio zibethinus Lcultivar Mon Thong (Malvaceae) Malpighia glabra L (Mal-pighiaceae) Mangifera indica L cultivar Aok Rong (Anac-ardiaceae) Musa paradisiaca cultivar Awak (Musaceae)Sandoricum koetjape (Burm f) Merr cultivar Tuptim (Meli-aceae) Syzygium malaccense (L) Merr amp Perry (Myrtaceae)and Ziziphus jujubaMill cultivar Milk Jujube (Rhamnaceae)were purchased from localmarkets inBangkokThailandTheplants were identified by Professor Wongsatit Chuakul thebotanist and lecturer in the Department of PharmaceuticalBotany Faculty of Pharmacy Mahidol University

23 Preparation of Extracts Edible portions of the freshfruits were run through a food chopper The finely choppedfruits were dried by freeze dryer (FreeZone Labconco)The coarsely ground dried fruits (50 g) were extracted withmethanol (300ndash500ml times 3 times) After filtration throughfilter paper (number 1 Whatman Inc) the solvent wasremoved under reduced pressure using a rotary evaporatorand the crude methanol extracts were stored at minus20∘C untilanalysis

24 Determination of Total Phenolic Content Total phenoliccontent of the fruit extracts was analyzed by the FolinndashCiocalteu colorimetricmethoddescribed previously [16]withminormodifications Stock solutions of the fruit extracts withconcentration of 15mgml were prepared in 50 methanolTwenty microliters of the stock solutions was mixed with50 120583l of 10 FolinndashCiocalteu reagent in 96-well plates andallowed to react for 3minThemixtureswere thenneutralizedby adding 80120583l of 75 of sodium carbonate solution Afterincubation at room temperature for 2 h the absorbance wasrecorded at 765 nm by an Infinite M200 Microplate Reader(Tecan) The assay was measured in triplicate and the resultswere calculated using a calibration curve for gallic acid (0625to 30 120583gml) and expressed as mg gallic acid equivalent(GAE) per 100 g fresh weight (mg GAE100 g FW)

25 Determination of Total Flavonoid Content Total flavon-oid content was determined by aluminium chloride colori-metric test modified from previous study [17] One hundredand sixty microliters of sterile water was added to the testtube followed by 40 120583l of the fruit extracts dissolved in sterilewater at the concentration of 1mgml and 15120583l of 5NaNO

2

After 5min 24120583l of 10AlCl3sdot6H2Owas addedThemixture

was allowed to react for 6min Lastly 80120583l of 1M NaOHand 80 120583l of sterile water were added and well mixed Then250 120583l of reaction mixture was transferred to 96-well plateThe plate was shaken for 30 sec and the absorbances wereread at 510 nm by microplate reader The total flavonoidcontent was expressed as quercetin equivalent per 100 g freshweight (mg QE100 g FW) which was calculated from theregression equation obtained from graph plotted betweenthe absorbance and the concentration of quercetin at variousconcentrations (4ndash1500120583gml) The assay was performed intriplicate

26 DPPH Radical-Scavenging Assay Free radical-scaveng-ing activity of the fruit extracts was determined based on thereduction of DPPH radicals by means of the modified spec-trophotometric method in 96-well plates The test sampleswere prepared at concentration of 2mgml in methanol andmixed with 002wv DPPH radical methanolic solution 1 1ratio in triplicateThe reactionmixtureswere allowed to standin the dark for 30min at room temperature The absorbancewas measured at 517 nm using Infinite M200 MicroplateReader (Tecan) The DPPH radical-scavenging ability wascalculated as percent inhibition by the following equation Inhibition = [(119860controlminus119860 sample)119860control]times100TheDPPHradical-scavenging activity of the fruit extracts was expressedas the IC

50values (120583gml) (inhibition of DPPH radical forma-

tion by 50 at concentration of 1mgml) 6-Hydroxy-2578-tetramethylchroman-2-carboxylic acid (Trolox) was used asa positive control

27 Cell Culture Human embryonic kidney-293 (HEK-293)cells were cultured usingDMEMsupplementedwith 10FBSand 1 (vv) penicillin-streptomycin solution in a humidifiedatmosphere of 5 CO

2at 37∘C as previously described [18]

Under serum starvation conditions cells were treated withcrude extract of Thai fruits






] times 100 (1)








3 Results







( FW)


(mg GAE100 g FW)


(mg QE100 g FW)


Mao khai pla(Thai)












ND = not detected




50of




50of 4158 plusmn 15 120583gml)


of 6480 plusmn






0

50

100

150

200

250

300D

PPH

scav

engi

ng (

inhi

bitio

n)

R2 = 06317


0

50

100

150

200

250

300

DPP

H sc

aven

ging

( in

hibi

tion)

R2 = 03843





2O2




2O2-


2O2-induced



2O2We further









Artocarpus integer

1 200010 1000001 01 1000

20

40

60

80

100

120

ce

ll vi

abili

ty

Averrhoa bilimbi

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Malpighia glabra

0

20

40

60

80

100

120

cell

viab

ility

01 1 10 100 1000 2000001

Mangifera foetida

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Sandoricum koetjape

0

20

40

60

80

100

120

ce

ll vi

abili

ty

10001 10 2000001 01 100

Vehicle

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100






0

20

40

60

80

100

120

ce

ll vi

abili

ty

Durio zibethinus

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100

Mangifera indica

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Musa paradisiaca

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Ziziphus jujuba

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100

Syzygium malaccense

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100








0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(a)



0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(b)


Vit C(120583gml) (120583gml) (120583gml) (120583M)

+ H2O2

0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(c)






DCF

Phasecontrast



+ H2O2

(a)


DCF

Phasecontrast


+ H2O2

(b)





4 Discussion




Mn-SOD

Relat

ive M

n-SO

DG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilimglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast

lowast

M

(a)

Rela

tive G

Px-1

GA

PDH

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast


lowast

(b)

Rela

tive c

atal

ase

GA

PDH

Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast lowast

(c)

CuZn-SOD

Relat

ive C

uZn-

SOD

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

(d)

GRe

Relat

ive G

ReG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

mRN

A ex

pres

sion

100120583gml

(e)

HO-1

Rela

tive H

O-1

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

14

mRN

A ex

pres

sion

100120583gml

(f)



lowast

lowast

lowastlowast lowast

lowast

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

GPx

-1G

APD

H (f

old

over

bas

al)

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

Mn-SOD22kD

GPx-122kD

Catalase60kD

GAPDH37kD

Mn-SOD

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

05

10

15

20

Mn-

SOD

GA

PDH

(fol

d ov

er b

asal

)

100120583gml


Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

25

Cata

lase

GA

PDH

(fol

d ov

er b

asal

)

lowastlowast

lowast















2O2-induced ROS



Mn-SOD

Oxidative stress


CatalaseGPx

S koetjape


A bilimbi


M glabraM indica







∙OH




2O2-induced ROS












5 Conclusion


Competing Interests




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















] times 100 (1)








3 Results







( FW)


(mg GAE100 g FW)


(mg QE100 g FW)


Mao khai pla(Thai)












ND = not detected




50of




50of 4158 plusmn 15 120583gml)


of 6480 plusmn






0

50

100

150

200

250

300D

PPH

scav

engi

ng (

inhi

bitio

n)

R2 = 06317


0

50

100

150

200

250

300

DPP

H sc

aven

ging

( in

hibi

tion)

R2 = 03843





2O2




2O2-


2O2-induced



2O2We further









Artocarpus integer

1 200010 1000001 01 1000

20

40

60

80

100

120

ce

ll vi

abili

ty

Averrhoa bilimbi

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Malpighia glabra

0

20

40

60

80

100

120

cell

viab

ility

01 1 10 100 1000 2000001

Mangifera foetida

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Sandoricum koetjape

0

20

40

60

80

100

120

ce

ll vi

abili

ty

10001 10 2000001 01 100

Vehicle

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100






0

20

40

60

80

100

120

ce

ll vi

abili

ty

Durio zibethinus

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100

Mangifera indica

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Musa paradisiaca

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Ziziphus jujuba

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100

Syzygium malaccense

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100








0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(a)



0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(b)


Vit C(120583gml) (120583gml) (120583gml) (120583M)

+ H2O2

0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(c)






DCF

Phasecontrast



+ H2O2

(a)


DCF

Phasecontrast


+ H2O2

(b)





4 Discussion




Mn-SOD

Relat

ive M

n-SO

DG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilimglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast

lowast

M

(a)

Rela

tive G

Px-1

GA

PDH

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast


lowast

(b)

Rela

tive c

atal

ase

GA

PDH

Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast lowast

(c)

CuZn-SOD

Relat

ive C

uZn-

SOD

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

(d)

GRe

Relat

ive G

ReG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

mRN

A ex

pres

sion

100120583gml

(e)

HO-1

Rela

tive H

O-1

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

14

mRN

A ex

pres

sion

100120583gml

(f)



lowast

lowast

lowastlowast lowast

lowast

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

GPx

-1G

APD

H (f

old

over

bas

al)

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

Mn-SOD22kD

GPx-122kD

Catalase60kD

GAPDH37kD

Mn-SOD

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

05

10

15

20

Mn-

SOD

GA

PDH

(fol

d ov

er b

asal

)

100120583gml


Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

25

Cata

lase

GA

PDH

(fol

d ov

er b

asal

)

lowastlowast

lowast















2O2-induced ROS



Mn-SOD

Oxidative stress


CatalaseGPx

S koetjape


A bilimbi


M glabraM indica







∙OH




2O2-induced ROS












5 Conclusion


Competing Interests




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















( FW)


(mg GAE100 g FW)


(mg QE100 g FW)


Mao khai pla(Thai)












ND = not detected




50of




50of 4158 plusmn 15 120583gml)


of 6480 plusmn






0

50

100

150

200

250

300D

PPH

scav

engi

ng (

inhi

bitio

n)

R2 = 06317


0

50

100

150

200

250

300

DPP

H sc

aven

ging

( in

hibi

tion)

R2 = 03843





2O2




2O2-


2O2-induced



2O2We further









Artocarpus integer

1 200010 1000001 01 1000

20

40

60

80

100

120

ce

ll vi

abili

ty

Averrhoa bilimbi

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Malpighia glabra

0

20

40

60

80

100

120

cell

viab

ility

01 1 10 100 1000 2000001

Mangifera foetida

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Sandoricum koetjape

0

20

40

60

80

100

120

ce

ll vi

abili

ty

10001 10 2000001 01 100

Vehicle

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100






0

20

40

60

80

100

120

ce

ll vi

abili

ty

Durio zibethinus

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100

Mangifera indica

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Musa paradisiaca

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Ziziphus jujuba

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100

Syzygium malaccense

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100








0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(a)



0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(b)


Vit C(120583gml) (120583gml) (120583gml) (120583M)

+ H2O2

0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(c)






DCF

Phasecontrast



+ H2O2

(a)


DCF

Phasecontrast


+ H2O2

(b)





4 Discussion




Mn-SOD

Relat

ive M

n-SO

DG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilimglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast

lowast

M

(a)

Rela

tive G

Px-1

GA

PDH

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast


lowast

(b)

Rela

tive c

atal

ase

GA

PDH

Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast lowast

(c)

CuZn-SOD

Relat

ive C

uZn-

SOD

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

(d)

GRe

Relat

ive G

ReG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

mRN

A ex

pres

sion

100120583gml

(e)

HO-1

Rela

tive H

O-1

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

14

mRN

A ex

pres

sion

100120583gml

(f)



lowast

lowast

lowastlowast lowast

lowast

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

GPx

-1G

APD

H (f

old

over

bas

al)

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

Mn-SOD22kD

GPx-122kD

Catalase60kD

GAPDH37kD

Mn-SOD

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

05

10

15

20

Mn-

SOD

GA

PDH

(fol

d ov

er b

asal

)

100120583gml


Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

25

Cata

lase

GA

PDH

(fol

d ov

er b

asal

)

lowastlowast

lowast















2O2-induced ROS



Mn-SOD

Oxidative stress


CatalaseGPx

S koetjape


A bilimbi


M glabraM indica







∙OH




2O2-induced ROS












5 Conclusion


Competing Interests




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















0

50

100

150

200

250

300D

PPH

scav

engi

ng (

inhi

bitio

n)

R2 = 06317


0

50

100

150

200

250

300

DPP

H sc

aven

ging

( in

hibi

tion)

R2 = 03843





2O2




2O2-


2O2-induced



2O2We further









Artocarpus integer

1 200010 1000001 01 1000

20

40

60

80

100

120

ce

ll vi

abili

ty

Averrhoa bilimbi

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Malpighia glabra

0

20

40

60

80

100

120

cell

viab

ility

01 1 10 100 1000 2000001

Mangifera foetida

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Sandoricum koetjape

0

20

40

60

80

100

120

ce

ll vi

abili

ty

10001 10 2000001 01 100

Vehicle

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100






0

20

40

60

80

100

120

ce

ll vi

abili

ty

Durio zibethinus

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100

Mangifera indica

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Musa paradisiaca

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Ziziphus jujuba

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100

Syzygium malaccense

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100








0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(a)



0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(b)


Vit C(120583gml) (120583gml) (120583gml) (120583M)

+ H2O2

0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(c)






DCF

Phasecontrast



+ H2O2

(a)


DCF

Phasecontrast


+ H2O2

(b)





4 Discussion




Mn-SOD

Relat

ive M

n-SO

DG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilimglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast

lowast

M

(a)

Rela

tive G

Px-1

GA

PDH

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast


lowast

(b)

Rela

tive c

atal

ase

GA

PDH

Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast lowast

(c)

CuZn-SOD

Relat

ive C

uZn-

SOD

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

(d)

GRe

Relat

ive G

ReG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

mRN

A ex

pres

sion

100120583gml

(e)

HO-1

Rela

tive H

O-1

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

14

mRN

A ex

pres

sion

100120583gml

(f)



lowast

lowast

lowastlowast lowast

lowast

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

GPx

-1G

APD

H (f

old

over

bas

al)

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

Mn-SOD22kD

GPx-122kD

Catalase60kD

GAPDH37kD

Mn-SOD

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

05

10

15

20

Mn-

SOD

GA

PDH

(fol

d ov

er b

asal

)

100120583gml


Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

25

Cata

lase

GA

PDH

(fol

d ov

er b

asal

)

lowastlowast

lowast















2O2-induced ROS



Mn-SOD

Oxidative stress


CatalaseGPx

S koetjape


A bilimbi


M glabraM indica







∙OH




2O2-induced ROS












5 Conclusion


Competing Interests




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Artocarpus integer

1 200010 1000001 01 1000

20

40

60

80

100

120

ce

ll vi

abili

ty

Averrhoa bilimbi

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Malpighia glabra

0

20

40

60

80

100

120

cell

viab

ility

01 1 10 100 1000 2000001

Mangifera foetida

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Sandoricum koetjape

0

20

40

60

80

100

120

ce

ll vi

abili

ty

10001 10 2000001 01 100

Vehicle

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100






0

20

40

60

80

100

120

ce

ll vi

abili

ty

Durio zibethinus

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100

Mangifera indica

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Musa paradisiaca

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 100 100001001

Ziziphus jujuba

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100

Syzygium malaccense

0

20

40

60

80

100

120

ce

ll vi

abili

ty

1 200010 1000001 01 100








0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(a)



0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(b)


Vit C(120583gml) (120583gml) (120583gml) (120583M)

+ H2O2

0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(c)






DCF

Phasecontrast



+ H2O2

(a)


DCF

Phasecontrast


+ H2O2

(b)





4 Discussion




Mn-SOD

Relat

ive M

n-SO

DG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilimglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast

lowast

M

(a)

Rela

tive G

Px-1

GA

PDH

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast


lowast

(b)

Rela

tive c

atal

ase

GA

PDH

Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast lowast

(c)

CuZn-SOD

Relat

ive C

uZn-

SOD

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

(d)

GRe

Relat

ive G

ReG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

mRN

A ex

pres

sion

100120583gml

(e)

HO-1

Rela

tive H

O-1

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

14

mRN

A ex

pres

sion

100120583gml

(f)



lowast

lowast

lowastlowast lowast

lowast

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

GPx

-1G

APD

H (f

old

over

bas

al)

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

Mn-SOD22kD

GPx-122kD

Catalase60kD

GAPDH37kD

Mn-SOD

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

05

10

15

20

Mn-

SOD

GA

PDH

(fol

d ov

er b

asal

)

100120583gml


Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

25

Cata

lase

GA

PDH

(fol

d ov

er b

asal

)

lowastlowast

lowast















2O2-induced ROS



Mn-SOD

Oxidative stress


CatalaseGPx

S koetjape


A bilimbi


M glabraM indica







∙OH




2O2-induced ROS












5 Conclusion


Competing Interests




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(a)



0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(b)


Vit C(120583gml) (120583gml) (120583gml) (120583M)

+ H2O2

0

50

100

150

200

250

ROS

prod

uctio

n (

)

lowast

(c)






DCF

Phasecontrast



+ H2O2

(a)


DCF

Phasecontrast


+ H2O2

(b)





4 Discussion




Mn-SOD

Relat

ive M

n-SO

DG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilimglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast

lowast

M

(a)

Rela

tive G

Px-1

GA

PDH

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast


lowast

(b)

Rela

tive c

atal

ase

GA

PDH

Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast lowast

(c)

CuZn-SOD

Relat

ive C

uZn-

SOD

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

(d)

GRe

Relat

ive G

ReG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

mRN

A ex

pres

sion

100120583gml

(e)

HO-1

Rela

tive H

O-1

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

14

mRN

A ex

pres

sion

100120583gml

(f)



lowast

lowast

lowastlowast lowast

lowast

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

GPx

-1G

APD

H (f

old

over

bas

al)

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

Mn-SOD22kD

GPx-122kD

Catalase60kD

GAPDH37kD

Mn-SOD

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

05

10

15

20

Mn-

SOD

GA

PDH

(fol

d ov

er b

asal

)

100120583gml


Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

25

Cata

lase

GA

PDH

(fol

d ov

er b

asal

)

lowastlowast

lowast















2O2-induced ROS



Mn-SOD

Oxidative stress


CatalaseGPx

S koetjape


A bilimbi


M glabraM indica







∙OH




2O2-induced ROS












5 Conclusion


Competing Interests




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















DCF

Phasecontrast



+ H2O2

(a)


DCF

Phasecontrast


+ H2O2

(b)





4 Discussion




Mn-SOD

Relat

ive M

n-SO

DG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilimglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast

lowast

M

(a)

Rela

tive G

Px-1

GA

PDH

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast


lowast

(b)

Rela

tive c

atal

ase

GA

PDH

Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast lowast

(c)

CuZn-SOD

Relat

ive C

uZn-

SOD

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

(d)

GRe

Relat

ive G

ReG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

mRN

A ex

pres

sion

100120583gml

(e)

HO-1

Rela

tive H

O-1

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

14

mRN

A ex

pres

sion

100120583gml

(f)



lowast

lowast

lowastlowast lowast

lowast

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

GPx

-1G

APD

H (f

old

over

bas

al)

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

Mn-SOD22kD

GPx-122kD

Catalase60kD

GAPDH37kD

Mn-SOD

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

05

10

15

20

Mn-

SOD

GA

PDH

(fol

d ov

er b

asal

)

100120583gml


Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

25

Cata

lase

GA

PDH

(fol

d ov

er b

asal

)

lowastlowast

lowast















2O2-induced ROS



Mn-SOD

Oxidative stress


CatalaseGPx

S koetjape


A bilimbi


M glabraM indica







∙OH




2O2-induced ROS












5 Conclusion


Competing Interests




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Mn-SOD

Relat

ive M

n-SO

DG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilimglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast

lowast

M

(a)

Rela

tive G

Px-1

GA

PDH

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

mRN

A ex

pres

sion

100120583gml

lowastlowast


lowast

(b)

Rela

tive c

atal

ase

GA

PDH

Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

lowastlowast

lowast lowast

(c)

CuZn-SOD

Relat

ive C

uZn-

SOD

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

mRN

A ex

pres

sion

100120583gml

(d)

GRe

Relat

ive G

ReG

APD

H

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

mRN

A ex

pres

sion

100120583gml

(e)

HO-1

Rela

tive H

O-1

GA

PDH

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

02

04

06

08

10

12

14

mRN

A ex

pres

sion

100120583gml

(f)



lowast

lowast

lowastlowast lowast

lowast

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

GPx

-1G

APD

H (f

old

over

bas

al)

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

Mn-SOD22kD

GPx-122kD

Catalase60kD

GAPDH37kD

Mn-SOD

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

05

10

15

20

Mn-

SOD

GA

PDH

(fol

d ov

er b

asal

)

100120583gml


Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

25

Cata

lase

GA

PDH

(fol

d ov

er b

asal

)

lowastlowast

lowast















2O2-induced ROS



Mn-SOD

Oxidative stress


CatalaseGPx

S koetjape


A bilimbi


M glabraM indica







∙OH




2O2-induced ROS












5 Conclusion


Competing Interests




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

lowast

lowastlowast lowast

lowast

GPx-1

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

GPx

-1G

APD

H (f

old

over

bas

al)

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

Mn-SOD22kD

GPx-122kD

Catalase60kD

GAPDH37kD

Mn-SOD

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

0

05

10

15

20

Mn-

SOD

GA

PDH

(fol

d ov

er b

asal

)

100120583gml


Catalase

Vehicle Mindica

Zjujuba

Smalac

Abilim

Mglabra

Skoet

Aghaes

100120583gml

0

05

10

15

20

25

Cata

lase

GA

PDH

(fol

d ov

er b

asal

)

lowastlowast

lowast















2O2-induced ROS



Mn-SOD

Oxidative stress


CatalaseGPx

S koetjape


A bilimbi


M glabraM indica







∙OH




2O2-induced ROS












5 Conclusion


Competing Interests




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























2O2-induced ROS



Mn-SOD

Oxidative stress


CatalaseGPx

S koetjape


A bilimbi


M glabraM indica







∙OH




2O2-induced ROS












5 Conclusion


Competing Interests




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























5 Conclusion


Competing Interests




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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