Research ArticleAntioxidant-Enhancing Property of the PolarFraction of Mangosteen Pericarp Extract and Evaluation ofIts Safety in Humans

Wichit Suthammarak1 Pornpayom Numpraphrut1 Ratiya Charoensakdi1

Neelobol Neungton1 Vachara Tunrungruangtavee2 Nattapon Jaisupa2

Suwit Charoensak3 Primchanien Moongkarndi2 and Weerasak Muangpaisan4

1Department of Biochemistry Faculty of Medicine Siriraj Hospital Mahidol University Bangkok Thailand2Department of Microbiology Faculty of Pharmacy Mahidol University Bangkok Thailand3Department of Psychiatry Faculty of Medicine Siriraj Hospital Mahidol University Bangkok Thailand4Department of Preventive and Social Medicine Faculty of Medicine Siriraj Hospital Mahidol University Bangkok Thailand

Correspondence should be addressed to Primchanien Moongkarndi primchanienmoomahidolacthand Weerasak Muangpaisan drweerasakgmailcom

Received 28 April 2016 Revised 20 July 2016 Accepted 27 July 2016

Academic Editor Alessandra Ricelli

Copyright copy 2016 Wichit Suthammarak et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Crude extract from the pericarp of the mangosteen (mangosteen extract [ME]) has exhibited several medicinal properties in bothanimal models and human cell lines Interestingly the cytotoxic activities were always observed in nonpolar fraction of the extractwhereas the potent antioxidant was often found in polar fraction Although it has been demonstrated that the polar fraction of MEexhibited the antioxidant activity the safety of the polar fraction of ME has never been thoroughly investigated in humans In thisstudy we investigated the safety of oral administration of the polar fraction of ME in 11 healthyThai volunteers During a 24-weekperiod of the study only minor and tolerable side effects were reported no serious side effects were documented Blood chemistrystudies also showed no liver damage or kidney dysfunction in all subjects We also demonstrated antioxidant property of the polarfraction of ME both in vitro and in vivo Interestingly oral administration of the polar fraction of ME enhanced the antioxidantcapability of red blood cells and decreased oxidative damage to proteins within red blood cells and whole blood

1 Introduction

The fruit of the tropical evergreen tree Garcinia mangostanaor the mangosteen is regarded as the queen of fruits becauseof its particularly favorable taste All parts of the mangosteenincluding the seed pericarp leaves and bark have longbeen used for medicinal purposes such as treatment ofskin infections and diarrhea Several compounds in thepericarp of the mangosteen have recently been identifiedmainly polyphenolic compounds including anthocyaninsxanthones garcinone E 8-deoxygartanin gartanin preny-lated xanthones and condensed tannin [1 2] These com-pounds have been shown to have many biological activitiesIn particular extract from the pericarp of the mangosteen

(mangosteen extract [ME]) has been shown to exhibit anti-inflammatory [3ndash5] anticancer [6ndash10] antimicrobial [11ndash14]and antioxidant effects [15ndash17] both in vivo and in vitro (for acomprehensive review see [18])

Several studies have highlighted the protective effect ofME against oxidative stress A study by Moongkarndi et al[15] demonstrated that the water-soluble partition of MEprotects the SK-N-SH neuroblastoma cell line from beta-amyloid-induced oxidative stress and alters the proteomicprofile of the cells Sattayasai et al [19] performed anotherstudy that provided compelling evidence for the antioxidanteffect of ME They showed that ME protects SK-N-SH cellsfrom cytotoxicity caused by endogenous hydrogen peroxideand exogenous polychlorinated biphenyl both of which

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 1293036 10 pageshttpdxdoiorg10115520161293036

2 Oxidative Medicine and Cellular Longevity

induce the production of reactive oxygen species (ROS)Intriguingly supplementation of ME was also shown toprevent memory impairment in scopolamine-treated miceMoreover 200 to 800120583gmL of ME significantly suppressedacetylcholinesterase activity in neurons to approximately60 of the activity in control cells which is comparable tothe effect of donepezil at its therapeutic level for treatment ofpatients with Alzheimerrsquos disease

These findings suggest the potential use of ME as anovel cognitive enhancer in patients with neurodegenerativeconditions such as Alzheimerrsquos disease The use of ME-based food supplements has been gaining increasingly morepublic attention However little is known about whetherME supplementation is indeed safe for humans or benefitshuman health because most studies of ME supplementationhave been conducted using animal models or cell culturesThe cytotoxic activities were always observed in nonpolarfraction of the extract whereas the potent antioxidant wasfound in polar fraction In addition the safety of the polarfraction has never been thoroughly investigated in humansSeveral studies reported beneficial effects of the use of 120572-mangostin the most abundant nonpolar constituent in thepericarp of the mangosteen [20] while some studies showedcontradictory results For instance dietary 120572-mangostin wasshown to exacerbate experimental colitis in a mouse model[21] whereas various studies reported the anticancer andcytotoxic properties of 120572-mangostin [22] It has also beenreported that a single oral dose of 120572-mangostin at 100ndash1000mgkg did not exhibit any harmful effect on the kidneyand liver tissues of both male and female ICR mice [23]

Therefore we conducted the present study to determinethe safety of oral administration of thewater-soluble partitionor polar fraction of ME in human subjects Healthy Thaivolunteers ingested the polar fraction of ME in a capsulepreparation daily for 24 weeks During the study the volun-teers were interviewed regardingME adherence any possibleside effects any othermedication or supplements and clinicalsymptomsThey underwent physical examinations includingevaluation of vital signs body weight and clinical statusAll volunteers also underwent blood testing to evaluate theirliver function kidney function coagulation status completeblood count thyroid function and plasma electrolyte levelsElectrocardiographic examination and urinary analysis werealso performed The polar fraction of ME in this study wasprepared as described previously and possibly contained verylow level of 120572-mangostin [19] The polar fraction of ME wasalso analyzed by thin-layer chromatography (TLC) for thepreparation consistency whereas its antioxidant property wasdemonstrated by in vitro free radical scavenging assay Finallywe investigated the volunteersrsquo levels of ROS and proteinoxidative damage in whole blood and within red blood cells(RBCs)

2 Materials and Methods

21 ME Capsule Preparation The pericarp of mangosteenwas dried at 60∘C for 2 days and pulverized by a grinder It wasfurther extracted by ethanol for 4 daysThe extract was evap-orated at 45∘C by an evaporator The crude ethanol extract

was partitioned with ethyl acetate to 2 1 weight volume for2 days The sediment was dissolved with distilled water to1 1 or 1 2 volume volume at 55∘C to 60∘C for 4 to 6 hThe supernatant was collected by centrifugation at 320timesg for20min and then dried by a spray dryer The calculated MEpowder and cornstarch as an excipient were carefully mixedand placed into hard gelatin capsules (Capsule Products CoLtd BangkokThailand) using a semiautomatic capsule fillerat doses of 220 and 280mg of ME per capsule The capsuleswere automatically packed in aluminum foil by a capsuleblister-packing machine for moisture protection At 0 3 and6 months after storage the moisture content of the productswas analyzed using a moisture balance (Model MA45 Sar-torius Gottingen Germany) to ensure control of the capsuleproducts The physical appearance weight variation disinte-gration andmicrobiological contaminationwere determinedaccording to the standards of the British Pharmacopeia Thebiological activities (ie antioxidant activities as determinedby chemical and cell culture techniques and the fingerprintof ME by thin-layer chromatography and high-performanceliquid chromatography) were analyzed to confirm the efficacyof the mangosteen products

22 Subject Recruitment Healthy volunteers were recruitedfrom the Faculty of Medicine Siriraj Hospital MahidolUniversity Bangkok Thailand The interested volunteerswere selected according to inclusion and exclusion criteriaThe inclusion criteria were (1) age between 20 and 60 yearsand (2) no previous health problems The exclusion criteriawere (1) those with cardiac neurological psychiatric renaland liver diseases cancer pregnancy or lactation alcoholicdependence illicit drug use and diseases considered bythe physician not appropriate to participate in the studyand (2) the presence of the following laboratory profilesabsolute neutrophil count lt 2 times 109L platelet lt 100 times 109LASTALT gt 25x upper limit bilirubin gt 15x upper limitblood urea nitrogengt 15x upper limit or creatinine clearancelt 60mLmin (Cockcroft and Gault formula) All clinicalparameters were obtained at 0 1 4 12 16 and 24 weeks afterbeginning the oral ME administration according to standardprocedures performed by physicians participating in thisstudy Participants who developed intolerable side effect orwere unwilling to continue in the study or did not complywith the study protocol would be withdrawn from the studyThe study was conducted with the understanding and writteninformed consent of all volunteers This study was approvedby the Siriraj Institutional Review Board (ethical approvalnumber SI3332012)

23 ME Capsule Administration and Blood Sample CollectionA 1-month supply of ME capsules or an adequate numberof capsules to last until the next visit was given to eachvolunteer During the first 3months volunteers who weighedlt55 kg took a single 220mg ME capsule daily while thosewho weighed gt55 kg took a single 280mg ME capsule dailyDosing was calculated based on the previous study on micefrom Sattayasai et al [19] The dose in mice was convertedto the human equivalent dose according to ldquothe guidance for

Oxidative Medicine and Cellular Longevity 3

industry estimating themaximum safe starting dose in initialclinical trials for therapeutics in adult healthy volunteersrdquofrom US Department of Health and Human Services Foodand Drug Administration Center for Drug Evaluation andResearch CDER July 2005 After 3 months all of the volun-teers received a double dose daily All volunteers underwentcollection of 4mL blood samples at 0 1 4 12 16 and 24weeksafter beginning the oral ME administration

24 Separation of ME by TLC and Screening of AntioxidantActivity The slightly modified TLC was applied [24] Theextract was dissolved in methanol and then subjected ontoa 20 times 20 cm normal phase aluminum plate (silica gel 60F254 Merck St Louis MO) The plate was developed inthe mobile phase consisting of ethyl acetate 70 ethanolin a ratio of 10 1 volume volume The isolated bands werevisualized under UV light To screen the antioxidant activityof each isolated band of ME stable free radical 22-diphenyl-1-picrylhydrazyl radical (DPPH) was appliedThe developedplate was sprayed by 03mMDPPH in methanol The forma-tion of yellow color indicated the antioxidant activity on thebasis of the formation of reduced DPPH

25 In Vitro Assay for Antioxidant Property of ME Theassay was performed as described by Moongkarndi et al[25] In brief isolated band A from TLC separation wasdissolved in methanol to various concentrations (0 25 510 20 50 and 100 120583gmL) To 100 120583L of extract solution100 120583L of fresh 04mM DPPH solution in methanol wasadded The experiment was conducted in a 96-well plate(119899 = 3 independent samples) The reaction was incubatedin darkness at room temperature for 30min The absorbancewas measured at 517 nm by microplate reader (Anthos 2010Anthos Labtec Instruments GmbH) Vitamin C was usedas positive control The degree of discoloration indicatesthe scavenging potential of the antioxidant property Thescavenging activity was calculated by [1 minus (119860

1minus blank)119860

0minus

blank] times 100 where 1198601was the absorbance of DPPH in the

presence of the extracts 1198600represented the absorbance of

DPPH solution without extract addition and blank was theabsorbance without DPPH solution

26 Measurement of ROS in RBCs The heparinized bloodwas diluted with Ca2+- and Mg2+-free phosphate bufferedsaline (PBS) (Sigma St Louis MO USA) to a concentrationof 1 times 106 RBCsmL The RBCs were incubated with 04mMof 21015840-71015840-dichlorofluorescein diacetate (DCFH-DA) (Sigma)dissolved in methanol (Fluka Sigma-Aldrich St Louis MOUSA) After incubation at 37∘C for 15min in a humidifiedatmosphere of 5 carbon dioxide in air the cells were washedand resuspended in PBS to the original cell concentrationThe RBC suspension was analyzed by incubation with orwithout 2mM of freshly prepared hydrogen peroxide Afterincubation at room temperature for 20min the sampleswere analyzed by a fluorescence-activated cell sorter (FAC-SCalibur Becton-Dickinson Immunofluorometry SystemsMountain View CA USA) The RBCs were passed at a rateof about 1000 cellss A 488 nm argon laser beam was used

for excitation [26] The absolute fluorescence intensity of the2101584071015840-dichlorofluorescein (DCF) which represented the ROSlevel in the RBCs was interpreted on a two-parameter dotplot of the side light scatter and forward light scatter of thepopulation Cell fluorescence was measured using logarith-mic amplification Green fluorescence of 10000 cells was thenmeasured using linear amplification The arithmetic meanfluorescence channel was derived by CellQuest software(Becton-Dickinson)

27 Determination of Protein Oxidative Damage by Anti-4-Hydroxynonenal Antibody Staining To prepare RBCsforanti-4-hydroxynonenal (anti-HNE) antibody stainingwhole blood was mixed with an equal volume of PBS andthen centrifuged at 800timesg for 5min at 4∘C The pelletcontaining the RBCs was collected and washed three timeswith PBS Finally distilled water was added to suspend andlyse the cells and the solution was stored at minus80∘C until use

Either whole blood or RBCs were denatured in samplebuffer (100mM Tris-HCl pH 68 10 glycerol 4 SDS0002 bromophenol blue 100mM dithiothreitol and 52-mercaptoethanol) and then boiled at 95∘C for 5min Thesamples were dotted manually in duplicate (one for HNE andone for120573-actin detection) onto nitrocellulosemembranewithamount of total protein 05 120583g per dot based on Bradfordassay and left to dry in air The nitrocellulose membraneswere then blocked in 5 nonfat milk in PBS with 001Tween-20 (PBST) with 001 NaN

3at 4∘C for 1 h and

subsequently incubated with either primary antibody anti-HNE antibody (Millipore Billerica MA USA) or anti-actin-3 antibody (Santa Cruz Biotechnology Santa Cruz CA USA)for 2 hThen themembranes were washed 3 times with PBSTand subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz BiotechnologySanta Cruz CA USA) in the same solution without NaN

3at

room temperature for 2 h After incubation with secondaryantibody the membranes were washed with PBST for 3times A chemiluminescence substrate (SuperSignal WestPico Thermo Fisher Scientific Inc Rockford IL USA) wasused to develop the reactions The fluorescence generatedfrom the reaction was visualized by exposing to the filmThe band intensities of HNE and actin-3 from the sampleswere quantitated by ImageJ software (US National Institutesof Health Bethesda MD USA)

The negative control dots were also performed withoutprimary antibody for both whole blood and RBCs samplesno signal was detected

28 Data Analysis Analysis of variance was performed toanalyze groups of data for significant differences UnpairedStudentrsquos 119905-test was then used to calculate statistical signifi-cance of specific pairs if a difference was demonstrated withanalysis of variance

3 Results

31 Biosafety Assessment and Side Effects of Oral Administra-tion of ME The safety of oral administration of the polarfraction ofMEwas assessed in 11 healthy human subjectsThe

4 Oxidative Medicine and Cellular Longevity

Table 1 Demographic data of the subjects

Demographic data n Sex

Male 5 455Female 6 545

Age in years (119909 = 32)20ndash30 4 36431ndash40 6 545gt40 1 91

OccupationEmployee 2 182Government officer 1 91University worker 6 545Others 2 182

Alcohol consumptionStopped drinking for gt3mo 2 182Drinking less than oncemo 7 636Never 2 182

SmokingNever 11 1000

Herbvitamin supplementConsuming 1 91Never 10 909

demographic data of the subjects are shown in Table 1 Theplasma electrolyte levels lipid profiles liver function enzymelevels thyroid hormone levels coagulograms and completeblood counts were measured from whole blood samples ofall subjects at 0 1 4 12 16 and 24 weeks after begin-ning oral administration of the polar fraction of ME Theresults showed no significant alterations in these parameters(Table 2) Additionally the side effects of oral administrationof the polar fraction of ME were determined throughout thestudy All subjects reported minor and tolerable side effectsas shown in Table 3

32 Separation of ME by TLC and Screening of AntioxidantActivity TLC chromatogram of the polar fraction of MEvisualized under UV light at 254 nm revealed a remarkableband that had 119877

119891value of approximately 075 (band A)

After spraying with 03mM DPPH solution several yellowspots which indicated antioxidant properties were observed(Figure 1)

33 In Vitro Antioxidant Assay of ME Quantitative analysisof antioxidant activity of the polar fraction of ME wasperformedon the extract of bandAbyDPPHassay (Figure 2)Antioxidant activity was shown in dose-dependent mannerThe IC

50values of band A extract and vitamin C (positive

control) were 1603 plusmn 144 120583gmL and 516 plusmn 092 120583gmLrespectively

34 Antioxidant Capacity of RBCs The antioxidant capacityof RBCs was determined by measuring the fluorescent signalof DCF from the RBCs after incubation with hydrogen

Band A

(a) (b)

Figure 1 Separation ofME by TLC and antioxidant properties of itsconstituents (a)MEwas separated by TLC and visualized under UVat 254 nmMajor compounds appeared in a single band at119877

119891

= 075

(band A) (b) Screening of antioxidant activity was done by applyingDPPHonto theTLCplate Scavenging ofDPPHwas shown in yellowstaining indicating antioxidative activity of ME

ME-band AVitamin C

0

25

50

75

100

125

sc

aven

ging

75 10025 500Concentration (120583gmL)

Figure 2 Quantitative analysis of antioxidant activity ofME DPPHassay was performed on the extract of band A (ME-band A) fromTLC Vitamin C was used as a positive control for comparisonAntioxidant activities ( scavenging) of both ME and vitaminC calculated as described in Materials and Methods were dose-dependent The IC

50

values of ME and vitamin C were 1603 plusmn144 120583gmL and 516 plusmn 092 120583gmL respectively Error bars representSEM

peroxide which was used as an exogenous ROS The RBCsfrom the subjects taking the polar fraction of ME exhibited asignificant decrease in the mean fluorescent intensity of DCFstarting in week 12 of oral administration of ME (Figure 3)This suggests that ME increases the antioxidant capacity of

Oxidative Medicine and Cellular Longevity 5

Table 2 Subject data and biochemical parameters for assessment of safety of oral administration of ME Blood and urine samples werecollected from the subjects in 0 1 4 12 16 and 24 weeks after beginning oral administration of ME The biochemical parameters weremeasured as tabulated Body mass index and blood pressure were also determined All parameters were collected and calculated from 11healthy individuals

Test Subject data and laboratory safety assessment mean (SEM)Day 0 1 week 4 weeks 12 weeks 16 weeks 24 weeks

Glucose(mgdL) 8673 (164) 8500 (167) 8309 (145) 8145 (304) 8091 (146) 8500 (253)

Cholesterol(mgdL) 20654 (1310) 19600 (1295) 20109 (1432) 19345 (1034) 19063 (1361) 19245 (2474)

Triglycerides(mgdL) 7800 (1115) 7718 (1958) 8127 (2011) 8718 (1727) 10354 (3080) 9227 (2265)

HDL-CHOL(mgdL) 6290 (294) 6045 (252) 5945 (213) 5972 (208) 5781 (226) 5945 (195)

LDL-calculated(mgdL) 12803 (1200) 12010 (1062) 12538 (1234) 11629 (895) 11210 (913) 11454 (1080)

BUN(mgdL) 1284 (163) 1126 (058) 1243 (103) 1182 (082) 1223 (088) 1217 (118)

Creatinine(mgdL) 086 (006) 084 (004) 081 (005) 082 (004) 082 (004) 081 (004)

Sodium(mmolL) 14127 (057) 14154 (089) 14136 (051) 14009 (047) 14109 (041) 14018 (038)

Potassium(mmolL) 375 (006) 372 (009) 375 (007) 358 (008) 356 (006) 363 (008)

Chloride(mmolL) 10279 (045) 10308 (056) 10250 (050) 10179 (071) 10323 (055) 10293 (036)

Bicarbonate(mmolL) 2814 (085) 2492 (070) 2650 (104) 2543 (070) 2485 (101) 2414 (071)

Total bilirubin(mgdL) 063 (014) 060 (011) 049 (008) 059 (011) 060 (014) 057 (010)

Direct bilirubin(mgdL) 021 (004) 023 (003) 020 (002) 024 (004) 024 (004) 022 (003)

AST (SGOT)(UL) 2081 (189) 1945 (207) 3054 (804) 1800 (180) 1909 (146) 1763 (283)

ALT (SGPT)(UL) 1918 (412) 1700 (368) 3127 (1040) 1845 (454) 1563 (261) 2045 (771)

ALP(UL) 6072 (268) 5927 (314) 6363 (317) 6245 (205) 6409 (374) 6254 (314)

T3(ngdL) 9942 (327) 10462 (353) 9745 (316) 9735 (382) 9926 (633) 9262 (372)

TSH(120583IUmL) 215 (032) 201 (032) 209 (042) 203 (036) 207 (028) 216 (030)

FT4(ngdL) 132 (005) 128 (005) 126 (005) 122 (002) 118 (003) 122 (004)

Hemoglobin(gdL) 1360 (031) 1325 (039) 1345 (031) 1355 (037) 1350 (040) 1350 (043)

Hematocrit() 4219 (100) 4091 (104) 4132 (105) 4143 (110) 4051 (125) 4064 (121)

WBC count(times103120583L)

597 (036) 577 (033) 594 (026) 644 (032) 570 (037) 543 (029)

Platelet count(times103120583L) 24627 (1314) 24045 (1430) 25536 (1413) 24354 (1265) 23827 (1220) 23972 (1064)

Absoluteneutrophils(times103120583L)

309 (023) 319 (026) 327 (014) 388 (044) 312 (028) 284 (020)

6 Oxidative Medicine and Cellular Longevity

Table 2 Continued

Test Subject data and laboratory safety assessment mean (SEM)Day 0 1 week 4 weeks 12 weeks 16 weeks 24 weeks

PT(sec) 1214 (021) 1208 (021) 1199 (024) 1223 (019) 1248 (027) 1262 (022)

aPTT(sec) 2853 (060) 2819 (076) 2861 (069) 2897 (061) 2920 (081) 2970 (074)

Urine protein(Negndashtrace) 545 727 727 818 727 545

Body mass index 2174 (112) 2186 (112) 2164 (107) 2207 (114) 2191 (112) 2189 (113)Arterial bloodpressure(mmHg)

11772 11776 11777 11973 11872 11373

HDL-CHOL high-density lipoprotein cholesterol LDL low-density lipoprotein BUN blood urea nitrogen AST (SGOT) aspartate aminotransferase(serum glutamic oxaloacetic transaminase) ALT (SGPT) alanine aminotransferase (serum glutamic pyruvic transaminase) ALP alkaline phosphataseT3 triiodothyronine TSH thyroid-stimulating hormone FT4 free thyroxine WBC white blood cell PT prothrombin time aPTT activated partialthromboplastin time

Table 3 Side effects of oral administration ofMEThe subjects wereasked to report the side effects after oral administration of ME ateach visit The number of episodes of each side effect prior to andafter double doses was collected and calculated as the percentage ofthe total episodes of 110 (11 participants times 10 visits)

SymptomBefore (single dose) After (double doses)Number ofepisodes Number of

episodes

Tirednesssleepiness 4 36 0 00Constipation 4 36 1 09Dry throat 2 18 3 27Headache 1 09 4 36Flu-like symptoms 2 18 0 00Abnormal stool 4 36 0 00Abdominaldiscomfort 3 27 0 00

Cough 2 18 0 00Weight loss 2 18 2 18Nasal congestion 2 18 0 00Abdominal bloating 2 18 1 09Dizziness 1 09 0 00Salivation 1 09 0 00Nauseavomiting 1 09 1 09Diarrhea 1 09 1 09Skin rash 1 09 0 00Abnormal urination 1 09 0 00Malaise 1 09 0 00

RBCsThis effect was further accentuated through the end ofthe study

35 Oxidative Damage to Proteins in RBCs and Whole BloodOral administration of ME was associated with a significantdecrease in oxidative damage to proteins in both RBCs andwhole blood Quantification of oxidative damage to these

proteins was achieved by normalizing HNE staining to actinAs shown in Figure 4 a significant decrease in proteinoxidative damage appeared inweeks 4 and 24 for whole bloodand RBCs respectively

4 Discussion

Several compounds in the extract frommangosteen pericarphave recently been identified and shown to have beneficialeffects on human health However the cytotoxic activitieswere always observed in nonpolar fraction of the extract thatis 120572-mangostin whereas the potent antioxidant was foundin polar fraction In addition the safety of the polar fractionhas never been thoroughly investigated in humans In thisstudy we investigated the safety of oral administration of thepolar fraction of ME inThai subjects For the entire 24 weeksof the study only minor side effects were reported Thesecan be categorized into gastrointestinal skin respiratory andgeneral side effects The most frequently reported side effectswere related to the gastrointestinal systemWe suspected thatsuch side effects might be associated with an antibacterialproperty of the compounds from the mangosteen pericarpwhich may have led to the perturbation of gut microbesNonetheless these side effects accounted for only a fewepisodes and were reportedly tolerable by all subjects

The present study also showed that oral administra-tion of the polar fraction of ME poses no threat to vitalorgans including the liver kidney and thyroid gland Thesubjectsrsquo liver enzyme levels lipid profiles coagulogramparameters blood urea nitrogen level creatinine level urineprotein level plasma electrolyte levels and thyroid hormonelevels remained normal for the entire 24 weeks of thestudy Additionally all subjectsrsquo complete blood counts andblood pressures remained unchanged Although two subjectsshowed slightly increased liver enzymes in the fourth weekof the study the enzyme levels spontaneously returned tothe normal range thereafter Notably a study by Stern et al[27] revealed that obese subjects with a body mass index of30 to 40 kgm2 exhibited a significant net reduction in body

Oxidative Medicine and Cellular Longevity 7

0

1000

FSC

SSC

0

1000

SSC

0

1000

SSC

0

0

1000

1000

FSC0 1000

FSC0 1000

FSC0 1000

SSC

DCF

Cou

nts

100 101 102 103 104

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

DCF100 101 102 103 104

0

100

Cou

nts

0

100

0

1000

SSC

0

1000

SSC

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

0

1000

SSC

0

1000

SSC

0

1000

SSC

0

1000

SSC

FSC

0 1000

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

0

100

100

0

100

Stim

ulat

edSt

imul

ated

0

1000

FSC

SSC

0

0

1000

1000

FSC0 1000

SSC

DCF

DCF

Cou

nts

100 101 102 103 104

100 101 102 103 104

Cou

nts

W12 W16 W24

W0 W1 W44 + H(0)001

4 + H(20)001

4 + H(0)001

4 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001 M012 + H(0)001

M012 + H(20)001 M012 + H(20)001

M012 + H(0)001 M012 + H(0)001

M012 + H(20)001 M012 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001M012 + H(0)001 M012 + H(0)001

M012 + H(20)001M012 + H(20)001 M012 + H(20)001

0min

Stim

ulat

ed0min

20min

Stim

ulat

ed20min

(a)

W1

W4

W12

W16W

0

W24

Weeks of oral administration of ME

Mea

n flu

ores

cenc

e int

ensit

y

lowastlowast

lowast

40

30

20

10

50

(b)

Figure 3 Effect of oral administration of ME on antioxidant capacity of RBCs RBCs were isolated from the peripheral blood of the subjectsat 0 1 4 12 16 and 24 weeks after beginning oral administration of ME Hydrogen peroxide was an exogenous ROS used to stimulate theresponse of the RBC antioxidant system (a) A representative measurement of the level of ROS within the RBCs which was determined by themean fluorescent signal of DCF emitted from hydrogen peroxide-incubated RBCs (b) Quantitative analysis of the mean fluorescent signalof DCF from all samples during the entire period of the study Data are presented as means (119899 = 11) Error bars represent SEMThe asteriskindicates statistical significance 119901 lt 005 in comparison with week 0 (119905-test)

weight (374 kg 119901 lt 00001) after taking capsules containingSphaeranthus indicus and G mangostana for 8 weeks com-pared with subjects taking placebo The discrepancy in thesubjectsrsquo body weights between that study and the presentstudy might derive from the volunteers in our study havinga normal BMI or from the weight-loss property of S indicus

We performedTLC onME in order to test for preparationconsistency and to demonstrate its essential compounds Itwas shown that ME was composed of at least one groupof major compounds which consistently appeared as asingle band on the chromatogram at 119877

119891= 075 Such a

high 119877119891suggested highly organic-volatile property We are

8 Oxidative Medicine and Cellular Longevity

Red blood cell Whole blood

W0

W1

W4

W12

W16

W24

Actin HNE Actin HNE

(a)

05

10

HNE-WBHNE-RBC

W1

W4

W12

W16W

0

W24

Weeks of oral administration of ME

Leve

l of H

NE

mod

ifica

tion

(rel

ativ

e to

W0) lowast

lowast

lowast

lowastlowast

(b)

Figure 4 Oral administration of ME resulted in decreased protein oxidative damage in RBCs and whole blood The RBCs and whole bloodof the subjects were collected at 0 1 4 12 16 and 24 weeks after beginning oral administration of ME (a) A representative blot showsHNE staining using a western blotting technique to determine the protein oxidative damage in RBCs and whole blood Actin was used asa loading control (b) Quantitative analysis of the extent of HNE modification to RBCs (HNE-RBC) and whole blood (HNE-WB) after oraladministration of ME was carried out as that relative to week 0 Data are presented as means (119899 = 11) Error bars represent SEMThe asteriskindicates statistical significance 119901 lt 005 in comparison with week 0 (119905-test)

currently identifying the chemical compounds in this band(data not shown) Further in vitro analysis of these majorcompounds revealed that they exhibited approximately one-third of antioxidant activity of vitamin C Therefore ME islikely to possess considerably high antioxidant activity

Antioxidant property of the polar fraction of MEobserved in our in vitro experiments was shown to exertbiological impactThe 24-week period of oral administrationof the polar fraction of ME in the present study resulted ina significant increase in the antioxidant capacity of RBCswhich was determined by the fluorescent signal from DCFemitted from hydrogen peroxide-incubated RBCs A signifi-cant increase in the antioxidant capacity (ie decreased DCFsignal) of RBCs could be detected from week 12 of oraladministration of the polar fraction of ME Therefore ourresults suggest for the first time that extract frommangosteenpericarp can augment the antioxidant system within humanRBCs probably due to its ability to neutralize ROS

We further investigated whether and how such anincreased antioxidant capacity affects protein oxidative dam-age in RBCs and whole blood Protein oxidative damagewas determined by the degree of HNE modification HNEis a byproduct of lipid peroxidation which is an indicatorof oxidative stress HNE can react with lysine histidine orcysteine residue in proteins to form adducts resulting instructural and functional changes to the proteins [28 29]HNE is also reportedly involved in the pathophysiology ofmany diseases [30ndash32] We speculated that if RBCs havean improved antioxidant capacity lipid peroxidation shouldbe reduced and HNE modification to proteins should thus

decrease Consistent with our speculation oral administra-tion of the polar fraction of ME led to a significant decreasein HNE modification in whole blood and RBCs starting inweeks 4 and 24 respectively Notably the significant decreasein HNE modification of whole blood proteins appearedearlier than that of RBC proteins This may be explained bythe faster turnover rate of albumin [33] the most abundantprotein in serum in comparison to the lifespan of RBCs[34] This result provides further supporting evidence inhuman subjects of the antioxidant property of extract frommangosteen pericarp

5 Conclusions

We have demonstrated for the first time in human subjectsthat the polar fraction of extract from mangosteen pericarpis adequately safe for oral administration for up to 24 weeksMajor components of the polar fraction of extract frommangosteen pericarp exhibited considerably high antioxidantactivity according to the in vitro antioxidant assay Addi-tionally the polar extract from mangosteen pericarp appearsto significantly increase the antioxidant capacity of RBCsand significantly decrease protein oxidative damage in RBCsand whole blood It might alleviate oxidative damage inother tissues as well Our results suggest that the antioxidantcapacity of the polar fraction of mangosteen pericarp extractcould come from its ability to neutralize ROS Howeverwhether some compounds in this extract can also upregulateantioxidant enzyme is still unknown It would be interestingto further quantitate the expression level of antioxidant

Oxidative Medicine and Cellular Longevity 9

enzymes In all the polar fraction of extract frommangosteenpericarp might benefit human patients with pathologicalconditions related to oxidative stress

Competing Interests

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

Acknowledgments

The authors are extremely grateful to Mrs Angkana Jong-sawaddipatana and all staff members of the Geriatric Clinicof Siriraj Hospital for their kind collaboration in recruit-ment of the participants This work was supported by theThai Traditional Medical Knowledge Fund Department forDevelopment of Thai Traditional and Alternative MedicineMinistry of Public Health Thailand

References

[1] S M Petiwala G Li A Ramaiya et al ldquoPharmacokineticcharacterization of mangosteen (Garcinia mangostana) fruitextract standardized to 120572-mangostin in C57BL6 micerdquo Nutri-tion Research vol 34 no 4 pp 336ndash345 2014

[2] Z Xu L Huang X-H Chen et al ldquoCytotoxic prenylated xan-thones from the pericarps of Garcinia mangostanardquo Moleculesvol 19 no 2 pp 1820ndash1827 2014

[3] F Gutierrez-Orozco C Chitchumroonchokchai G B Lesin-ski S Suksamrarn and M L Failla ldquo120572-Mangostin anti-inflammatory activity and metabolism by human cellsrdquo Journalof Agricultural and Food Chemistry vol 61 no 16 pp 3891ndash3900 2013

[4] H-Y Jang O-K Kwon S-R Oh H-K Lee K-S Ahn and Y-W Chin ldquoMangosteen xanthones mitigate ovalbumin-inducedairway inflammation in a mouse model of asthmardquo Food andChemical Toxicology vol 50 no 11 pp 4042ndash4050 2012

[5] S Syam A Bustamam R Abdullah et al ldquo120573 Mangostinsuppress LPS-induced inflammatory response in RAW 2647macrophages in vitro and carrageenan-induced peritonitis invivordquo Journal of Ethnopharmacology vol 153 no 2 pp 435ndash4452014

[6] J Yuan Y Wu and G Lu ldquoA-mangostin suppresseslipopolysaccharide-induced invasion by inhibiting matrixmetalloproteinase-29 and increasing E-cadherin expressionthrough extracellular signal-regulated kinase signaling inpancreatic cancer cellsrdquo Oncology Letters vol 5 no 6 pp1958ndash1964 2013

[7] M-A Shibata M Iinuma J Morimoto et al ldquo120572-Mangostinextracted from the pericarp of the mangosteen (Garcinia man-gostana Linn) reduces tumor growth and lymph node metas-tasis in an immunocompetent xenograft model of metastaticmammary cancer carrying a p53mutationrdquo BMCMedicine vol9 article 69 2011

[8] A F A Aisha K M Abu-Salah Z Ismail and A MajidldquoIn vitro and in vivo anti-colon cancer effects of Garciniamangostana xanthones extractrdquo BMC Complementary andAlternative Medicine vol 12 article 104 2012

[9] S Taokaew N Nunkaew P Siripong and M PhisalaphongldquoCharacteristics and anticancer properties of bacterial cellulose

films containing ethanolic extract of mangosteen peelrdquo Journalof Biomaterials Science Polymer Edition vol 25 no 9 pp 907ndash922 2014

[10] P Pan-In S Wanichwecharungruang J Hanes and A J KimldquoCellular trafficking and anticancer activity of Garcinia man-gostana extract-encapsulated polymeric nanoparticlesrdquo Inter-national Journal of Nanomedicine vol 9 no 1 pp 3677ndash36862014

[11] H R W Dharmaratne Y Sakagami K G P Piyasena and VThevanesam ldquoAntibacterial activity of xanthones fromGarciniamangostana (L) and their structure-activity relationship stud-iesrdquo Natural Product Research vol 27 no 10 pp 938ndash941 2013

[12] J-J Koh S Qiu H Zou et al ldquoRapid bactericidal action ofalpha-mangostin against MRSA as an outcome of membranetargetingrdquo Biochimica et Biophysica ActamdashBiomembranes vol1828 no 2 pp 834ndash844 2013

[13] M T Chomnawang S Surassmo K Wongsariya and N Bun-yapraphatsara ldquoAntibacterial activity of Thai medicinal plantsagainstmethicillin-resistant Staphylococcus aureusrdquo Fitoterapiavol 80 no 2 pp 102ndash104 2009

[14] S Suksamrarn N SuwannapochW Phakhodee et al ldquoAntimy-cobacterial activity of prenylated xanthones from the fruits ofGarcinia mangostanardquo Chemical and Pharmaceutical Bulletinvol 51 no 7 pp 857ndash859 2003

[15] P Moongkarndi C Srisawat P Saetun et al ldquoProtective effectof mangosteen extract against 120573-amyloid-induced cytotoxicityoxidative stress and altered proteome in SK-N-SH cellsrdquo Journalof Proteome Research vol 9 no 5 pp 2076ndash2086 2010

[16] Y-W Chin H-A Jung H Chai W J Keller and A DKinghorn ldquoXanthones with quinone reductase-inducing activ-ity from the fruits of Garcinia mangostana (Mangosteen)rdquoPhytochemistry vol 69 no 3 pp 754ndash758 2008

[17] H-A Jung B-N Su W J Keller R G Mehta and AD Kinghorn ldquoAntioxidant xanthones from the pericarp ofGarcinia mangostana (Mangosteen)rdquo Journal of Agriculturaland Food Chemistry vol 54 no 6 pp 2077ndash2082 2006

[18] F Gutierrez-Orozco and M L Failla ldquoBiological activities andbioavailability of mangosteen xanthones a critical review of thecurrent evidencerdquo Nutrients vol 5 no 8 pp 3163ndash3183 2013

[19] J Sattayasai P Chaonapan T Arkaravichie et al ldquoProtectiveeffects of mangosteen extract on H

2

O2

-induced cytotoxicity inSK-N-SH cells and scopolamine-induced memory impairmentin micerdquo PLoS ONE vol 8 no 12 Article ID e85053 2013

[20] E B Walker ldquoHPLC analysis of selected xanthones in mangos-teen fruitrdquo Journal of Separation Science vol 30 no 9 pp 1229ndash1234 2007

[21] F Gutierrez-Orozco J MThomas-Ahner L D Berman-Bootyet al ldquoDietary 120572-mangostin a xanthone from mangosteenfruit exacerbates experimental colitis and promotes dysbiosisin micerdquoMolecular Nutrition and Food Research vol 58 no 6pp 1226ndash1238 2014

[22] M Y IbrahimNMHashim A AMariod et al ldquo120572-Mangostinfrom Garcinia mangostana Linn an updated review of itspharmacological propertiesrdquo Arabian Journal of Chemistry vol9 no 3 pp 317ndash329 2016

[23] M Y Ibrahim N M Hashim S Mohan et al ldquo120572-Mangostinfrom Cratoxylum arborescens an in vitro and in vivo toxicolog-ical evaluationrdquo Arabian Journal of Chemistry vol 8 no 1 pp129ndash137 2015

[24] A Plazonic F Bucar Z Males A Mornar B Nigovic andN Kujundzic ldquoIdentification and quantification of flavonoids

10 Oxidative Medicine and Cellular Longevity

and phenolic acids in burr parsley (Caucalis platycarpos L)using high-performance liquid chromatography with diodearray detection and electrospray ionizationmass spectrometryrdquoMolecules vol 14 no 7 pp 2466ndash2490 2009

[25] P Moongkarndi N Jaisupa J Samer et al ldquoComparison of thebiological activity of two different isolates from mangosteenrdquoJournal of Pharmacy and Pharmacology vol 66 no 8 pp 1171ndash1179 2014

[26] E Fibach ldquoTechniques for studying stimulation of fetal hemo-globin production in human erythroid culturesrdquo Hemoglobinvol 22 no 5-6 pp 445ndash458 1998

[27] J S Stern J Peerson A T Mishra M V Sadasiva Rao andK P Rajeswari ldquoEfficacy and tolerability of a novel herbalformulation for weight managementrdquo Obesity vol 21 no 5 pp921ndash927 2013

[28] C M Spickett ldquoThe lipid peroxidation product 4-hydroxy-2-nonenal advances in chemistry and analysisrdquo Redox Biologyvol 1 no 1 pp 145ndash152 2013

[29] C Schneider N A Porter and A R Brash ldquoRoutes to 4-hydroxynonenal fundamental issues in themechanisms of lipidperoxidationrdquo Journal of Biological Chemistry vol 283 no 23pp 15539ndash15543 2008

[30] A Negre-Salvayre C Coatrieux C Ingueneau and R SalvayreldquoAdvanced lipid peroxidation end products in oxidative damageto proteins Potential role in diseases and therapeutic prospectsfor the inhibitorsrdquo British Journal of Pharmacology vol 153 no1 pp 6ndash20 2008

[31] M P Mattson ldquoRoles of the lipid peroxidation product 4-hydroxynonenal in obesity the metabolic syndrome and asso-ciated vascular and neurodegenerative disordersrdquo ExperimentalGerontology vol 44 no 10 pp 625ndash633 2009

[32] A Ayala M F Munoz and S Arguelles ldquoLipid peroxidationproduction metabolism and signaling mechanisms of malon-dialdehyde and 4-hydroxy-2-nonenalrdquo Oxidative Medicine andCellular Longevity vol 2014 Article ID 360438 31 pages 2014

[33] K Sterling ldquoThe turnover rate of serum albumin in manas measured by I131-tagged albuminrdquo The Journal of ClinicalInvestigation vol 30 no 11 pp 1228ndash1237 1951

[34] C J Lindsell R S Franco E P Smith C H Joiner and R MCohen ldquoA method for the continuous calculation of the age oflabeled red blood cellsrdquo American Journal of Hematology vol83 no 6 pp 454ndash457 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Oxidative Medicine and Cellular Longevity

induce the production of reactive oxygen species (ROS)Intriguingly supplementation of ME was also shown toprevent memory impairment in scopolamine-treated miceMoreover 200 to 800120583gmL of ME significantly suppressedacetylcholinesterase activity in neurons to approximately60 of the activity in control cells which is comparable tothe effect of donepezil at its therapeutic level for treatment ofpatients with Alzheimerrsquos disease

These findings suggest the potential use of ME as anovel cognitive enhancer in patients with neurodegenerativeconditions such as Alzheimerrsquos disease The use of ME-based food supplements has been gaining increasingly morepublic attention However little is known about whetherME supplementation is indeed safe for humans or benefitshuman health because most studies of ME supplementationhave been conducted using animal models or cell culturesThe cytotoxic activities were always observed in nonpolarfraction of the extract whereas the potent antioxidant wasfound in polar fraction In addition the safety of the polarfraction has never been thoroughly investigated in humansSeveral studies reported beneficial effects of the use of 120572-mangostin the most abundant nonpolar constituent in thepericarp of the mangosteen [20] while some studies showedcontradictory results For instance dietary 120572-mangostin wasshown to exacerbate experimental colitis in a mouse model[21] whereas various studies reported the anticancer andcytotoxic properties of 120572-mangostin [22] It has also beenreported that a single oral dose of 120572-mangostin at 100ndash1000mgkg did not exhibit any harmful effect on the kidneyand liver tissues of both male and female ICR mice [23]

Therefore we conducted the present study to determinethe safety of oral administration of thewater-soluble partitionor polar fraction of ME in human subjects Healthy Thaivolunteers ingested the polar fraction of ME in a capsulepreparation daily for 24 weeks During the study the volun-teers were interviewed regardingME adherence any possibleside effects any othermedication or supplements and clinicalsymptomsThey underwent physical examinations includingevaluation of vital signs body weight and clinical statusAll volunteers also underwent blood testing to evaluate theirliver function kidney function coagulation status completeblood count thyroid function and plasma electrolyte levelsElectrocardiographic examination and urinary analysis werealso performed The polar fraction of ME in this study wasprepared as described previously and possibly contained verylow level of 120572-mangostin [19] The polar fraction of ME wasalso analyzed by thin-layer chromatography (TLC) for thepreparation consistency whereas its antioxidant property wasdemonstrated by in vitro free radical scavenging assay Finallywe investigated the volunteersrsquo levels of ROS and proteinoxidative damage in whole blood and within red blood cells(RBCs)

2 Materials and Methods

21 ME Capsule Preparation The pericarp of mangosteenwas dried at 60∘C for 2 days and pulverized by a grinder It wasfurther extracted by ethanol for 4 daysThe extract was evap-orated at 45∘C by an evaporator The crude ethanol extract

was partitioned with ethyl acetate to 2 1 weight volume for2 days The sediment was dissolved with distilled water to1 1 or 1 2 volume volume at 55∘C to 60∘C for 4 to 6 hThe supernatant was collected by centrifugation at 320timesg for20min and then dried by a spray dryer The calculated MEpowder and cornstarch as an excipient were carefully mixedand placed into hard gelatin capsules (Capsule Products CoLtd BangkokThailand) using a semiautomatic capsule fillerat doses of 220 and 280mg of ME per capsule The capsuleswere automatically packed in aluminum foil by a capsuleblister-packing machine for moisture protection At 0 3 and6 months after storage the moisture content of the productswas analyzed using a moisture balance (Model MA45 Sar-torius Gottingen Germany) to ensure control of the capsuleproducts The physical appearance weight variation disinte-gration andmicrobiological contaminationwere determinedaccording to the standards of the British Pharmacopeia Thebiological activities (ie antioxidant activities as determinedby chemical and cell culture techniques and the fingerprintof ME by thin-layer chromatography and high-performanceliquid chromatography) were analyzed to confirm the efficacyof the mangosteen products

22 Subject Recruitment Healthy volunteers were recruitedfrom the Faculty of Medicine Siriraj Hospital MahidolUniversity Bangkok Thailand The interested volunteerswere selected according to inclusion and exclusion criteriaThe inclusion criteria were (1) age between 20 and 60 yearsand (2) no previous health problems The exclusion criteriawere (1) those with cardiac neurological psychiatric renaland liver diseases cancer pregnancy or lactation alcoholicdependence illicit drug use and diseases considered bythe physician not appropriate to participate in the studyand (2) the presence of the following laboratory profilesabsolute neutrophil count lt 2 times 109L platelet lt 100 times 109LASTALT gt 25x upper limit bilirubin gt 15x upper limitblood urea nitrogengt 15x upper limit or creatinine clearancelt 60mLmin (Cockcroft and Gault formula) All clinicalparameters were obtained at 0 1 4 12 16 and 24 weeks afterbeginning the oral ME administration according to standardprocedures performed by physicians participating in thisstudy Participants who developed intolerable side effect orwere unwilling to continue in the study or did not complywith the study protocol would be withdrawn from the studyThe study was conducted with the understanding and writteninformed consent of all volunteers This study was approvedby the Siriraj Institutional Review Board (ethical approvalnumber SI3332012)

23 ME Capsule Administration and Blood Sample CollectionA 1-month supply of ME capsules or an adequate numberof capsules to last until the next visit was given to eachvolunteer During the first 3months volunteers who weighedlt55 kg took a single 220mg ME capsule daily while thosewho weighed gt55 kg took a single 280mg ME capsule dailyDosing was calculated based on the previous study on micefrom Sattayasai et al [19] The dose in mice was convertedto the human equivalent dose according to ldquothe guidance for

Oxidative Medicine and Cellular Longevity 3

industry estimating themaximum safe starting dose in initialclinical trials for therapeutics in adult healthy volunteersrdquofrom US Department of Health and Human Services Foodand Drug Administration Center for Drug Evaluation andResearch CDER July 2005 After 3 months all of the volun-teers received a double dose daily All volunteers underwentcollection of 4mL blood samples at 0 1 4 12 16 and 24weeksafter beginning the oral ME administration

24 Separation of ME by TLC and Screening of AntioxidantActivity The slightly modified TLC was applied [24] Theextract was dissolved in methanol and then subjected ontoa 20 times 20 cm normal phase aluminum plate (silica gel 60F254 Merck St Louis MO) The plate was developed inthe mobile phase consisting of ethyl acetate 70 ethanolin a ratio of 10 1 volume volume The isolated bands werevisualized under UV light To screen the antioxidant activityof each isolated band of ME stable free radical 22-diphenyl-1-picrylhydrazyl radical (DPPH) was appliedThe developedplate was sprayed by 03mMDPPH in methanol The forma-tion of yellow color indicated the antioxidant activity on thebasis of the formation of reduced DPPH

25 In Vitro Assay for Antioxidant Property of ME Theassay was performed as described by Moongkarndi et al[25] In brief isolated band A from TLC separation wasdissolved in methanol to various concentrations (0 25 510 20 50 and 100 120583gmL) To 100 120583L of extract solution100 120583L of fresh 04mM DPPH solution in methanol wasadded The experiment was conducted in a 96-well plate(119899 = 3 independent samples) The reaction was incubatedin darkness at room temperature for 30min The absorbancewas measured at 517 nm by microplate reader (Anthos 2010Anthos Labtec Instruments GmbH) Vitamin C was usedas positive control The degree of discoloration indicatesthe scavenging potential of the antioxidant property Thescavenging activity was calculated by [1 minus (119860

1minus blank)119860

0minus

blank] times 100 where 1198601was the absorbance of DPPH in the

presence of the extracts 1198600represented the absorbance of

DPPH solution without extract addition and blank was theabsorbance without DPPH solution

26 Measurement of ROS in RBCs The heparinized bloodwas diluted with Ca2+- and Mg2+-free phosphate bufferedsaline (PBS) (Sigma St Louis MO USA) to a concentrationof 1 times 106 RBCsmL The RBCs were incubated with 04mMof 21015840-71015840-dichlorofluorescein diacetate (DCFH-DA) (Sigma)dissolved in methanol (Fluka Sigma-Aldrich St Louis MOUSA) After incubation at 37∘C for 15min in a humidifiedatmosphere of 5 carbon dioxide in air the cells were washedand resuspended in PBS to the original cell concentrationThe RBC suspension was analyzed by incubation with orwithout 2mM of freshly prepared hydrogen peroxide Afterincubation at room temperature for 20min the sampleswere analyzed by a fluorescence-activated cell sorter (FAC-SCalibur Becton-Dickinson Immunofluorometry SystemsMountain View CA USA) The RBCs were passed at a rateof about 1000 cellss A 488 nm argon laser beam was used

for excitation [26] The absolute fluorescence intensity of the2101584071015840-dichlorofluorescein (DCF) which represented the ROSlevel in the RBCs was interpreted on a two-parameter dotplot of the side light scatter and forward light scatter of thepopulation Cell fluorescence was measured using logarith-mic amplification Green fluorescence of 10000 cells was thenmeasured using linear amplification The arithmetic meanfluorescence channel was derived by CellQuest software(Becton-Dickinson)

27 Determination of Protein Oxidative Damage by Anti-4-Hydroxynonenal Antibody Staining To prepare RBCsforanti-4-hydroxynonenal (anti-HNE) antibody stainingwhole blood was mixed with an equal volume of PBS andthen centrifuged at 800timesg for 5min at 4∘C The pelletcontaining the RBCs was collected and washed three timeswith PBS Finally distilled water was added to suspend andlyse the cells and the solution was stored at minus80∘C until use

Either whole blood or RBCs were denatured in samplebuffer (100mM Tris-HCl pH 68 10 glycerol 4 SDS0002 bromophenol blue 100mM dithiothreitol and 52-mercaptoethanol) and then boiled at 95∘C for 5min Thesamples were dotted manually in duplicate (one for HNE andone for120573-actin detection) onto nitrocellulosemembranewithamount of total protein 05 120583g per dot based on Bradfordassay and left to dry in air The nitrocellulose membraneswere then blocked in 5 nonfat milk in PBS with 001Tween-20 (PBST) with 001 NaN

3at 4∘C for 1 h and

subsequently incubated with either primary antibody anti-HNE antibody (Millipore Billerica MA USA) or anti-actin-3 antibody (Santa Cruz Biotechnology Santa Cruz CA USA)for 2 hThen themembranes were washed 3 times with PBSTand subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz BiotechnologySanta Cruz CA USA) in the same solution without NaN

3at

room temperature for 2 h After incubation with secondaryantibody the membranes were washed with PBST for 3times A chemiluminescence substrate (SuperSignal WestPico Thermo Fisher Scientific Inc Rockford IL USA) wasused to develop the reactions The fluorescence generatedfrom the reaction was visualized by exposing to the filmThe band intensities of HNE and actin-3 from the sampleswere quantitated by ImageJ software (US National Institutesof Health Bethesda MD USA)

The negative control dots were also performed withoutprimary antibody for both whole blood and RBCs samplesno signal was detected

28 Data Analysis Analysis of variance was performed toanalyze groups of data for significant differences UnpairedStudentrsquos 119905-test was then used to calculate statistical signifi-cance of specific pairs if a difference was demonstrated withanalysis of variance

3 Results

31 Biosafety Assessment and Side Effects of Oral Administra-tion of ME The safety of oral administration of the polarfraction ofMEwas assessed in 11 healthy human subjectsThe

4 Oxidative Medicine and Cellular Longevity

Table 1 Demographic data of the subjects

Demographic data n Sex

Male 5 455Female 6 545

Age in years (119909 = 32)20ndash30 4 36431ndash40 6 545gt40 1 91

OccupationEmployee 2 182Government officer 1 91University worker 6 545Others 2 182

Alcohol consumptionStopped drinking for gt3mo 2 182Drinking less than oncemo 7 636Never 2 182

SmokingNever 11 1000

Herbvitamin supplementConsuming 1 91Never 10 909

demographic data of the subjects are shown in Table 1 Theplasma electrolyte levels lipid profiles liver function enzymelevels thyroid hormone levels coagulograms and completeblood counts were measured from whole blood samples ofall subjects at 0 1 4 12 16 and 24 weeks after begin-ning oral administration of the polar fraction of ME Theresults showed no significant alterations in these parameters(Table 2) Additionally the side effects of oral administrationof the polar fraction of ME were determined throughout thestudy All subjects reported minor and tolerable side effectsas shown in Table 3

32 Separation of ME by TLC and Screening of AntioxidantActivity TLC chromatogram of the polar fraction of MEvisualized under UV light at 254 nm revealed a remarkableband that had 119877

119891value of approximately 075 (band A)

After spraying with 03mM DPPH solution several yellowspots which indicated antioxidant properties were observed(Figure 1)

33 In Vitro Antioxidant Assay of ME Quantitative analysisof antioxidant activity of the polar fraction of ME wasperformedon the extract of bandAbyDPPHassay (Figure 2)Antioxidant activity was shown in dose-dependent mannerThe IC

50values of band A extract and vitamin C (positive

control) were 1603 plusmn 144 120583gmL and 516 plusmn 092 120583gmLrespectively

34 Antioxidant Capacity of RBCs The antioxidant capacityof RBCs was determined by measuring the fluorescent signalof DCF from the RBCs after incubation with hydrogen

Band A

(a) (b)

Figure 1 Separation ofME by TLC and antioxidant properties of itsconstituents (a)MEwas separated by TLC and visualized under UVat 254 nmMajor compounds appeared in a single band at119877

119891

= 075

(band A) (b) Screening of antioxidant activity was done by applyingDPPHonto theTLCplate Scavenging ofDPPHwas shown in yellowstaining indicating antioxidative activity of ME

ME-band AVitamin C

0

25

50

75

100

125

sc

aven

ging

75 10025 500Concentration (120583gmL)

Figure 2 Quantitative analysis of antioxidant activity ofME DPPHassay was performed on the extract of band A (ME-band A) fromTLC Vitamin C was used as a positive control for comparisonAntioxidant activities ( scavenging) of both ME and vitaminC calculated as described in Materials and Methods were dose-dependent The IC

50

values of ME and vitamin C were 1603 plusmn144 120583gmL and 516 plusmn 092 120583gmL respectively Error bars representSEM

peroxide which was used as an exogenous ROS The RBCsfrom the subjects taking the polar fraction of ME exhibited asignificant decrease in the mean fluorescent intensity of DCFstarting in week 12 of oral administration of ME (Figure 3)This suggests that ME increases the antioxidant capacity of

Oxidative Medicine and Cellular Longevity 5

Table 2 Subject data and biochemical parameters for assessment of safety of oral administration of ME Blood and urine samples werecollected from the subjects in 0 1 4 12 16 and 24 weeks after beginning oral administration of ME The biochemical parameters weremeasured as tabulated Body mass index and blood pressure were also determined All parameters were collected and calculated from 11healthy individuals

Test Subject data and laboratory safety assessment mean (SEM)Day 0 1 week 4 weeks 12 weeks 16 weeks 24 weeks

Glucose(mgdL) 8673 (164) 8500 (167) 8309 (145) 8145 (304) 8091 (146) 8500 (253)

Cholesterol(mgdL) 20654 (1310) 19600 (1295) 20109 (1432) 19345 (1034) 19063 (1361) 19245 (2474)

Triglycerides(mgdL) 7800 (1115) 7718 (1958) 8127 (2011) 8718 (1727) 10354 (3080) 9227 (2265)

HDL-CHOL(mgdL) 6290 (294) 6045 (252) 5945 (213) 5972 (208) 5781 (226) 5945 (195)

LDL-calculated(mgdL) 12803 (1200) 12010 (1062) 12538 (1234) 11629 (895) 11210 (913) 11454 (1080)

BUN(mgdL) 1284 (163) 1126 (058) 1243 (103) 1182 (082) 1223 (088) 1217 (118)

Creatinine(mgdL) 086 (006) 084 (004) 081 (005) 082 (004) 082 (004) 081 (004)

Sodium(mmolL) 14127 (057) 14154 (089) 14136 (051) 14009 (047) 14109 (041) 14018 (038)

Potassium(mmolL) 375 (006) 372 (009) 375 (007) 358 (008) 356 (006) 363 (008)

Chloride(mmolL) 10279 (045) 10308 (056) 10250 (050) 10179 (071) 10323 (055) 10293 (036)

Bicarbonate(mmolL) 2814 (085) 2492 (070) 2650 (104) 2543 (070) 2485 (101) 2414 (071)

Total bilirubin(mgdL) 063 (014) 060 (011) 049 (008) 059 (011) 060 (014) 057 (010)

Direct bilirubin(mgdL) 021 (004) 023 (003) 020 (002) 024 (004) 024 (004) 022 (003)

AST (SGOT)(UL) 2081 (189) 1945 (207) 3054 (804) 1800 (180) 1909 (146) 1763 (283)

ALT (SGPT)(UL) 1918 (412) 1700 (368) 3127 (1040) 1845 (454) 1563 (261) 2045 (771)

ALP(UL) 6072 (268) 5927 (314) 6363 (317) 6245 (205) 6409 (374) 6254 (314)

T3(ngdL) 9942 (327) 10462 (353) 9745 (316) 9735 (382) 9926 (633) 9262 (372)

TSH(120583IUmL) 215 (032) 201 (032) 209 (042) 203 (036) 207 (028) 216 (030)

FT4(ngdL) 132 (005) 128 (005) 126 (005) 122 (002) 118 (003) 122 (004)

Hemoglobin(gdL) 1360 (031) 1325 (039) 1345 (031) 1355 (037) 1350 (040) 1350 (043)

Hematocrit() 4219 (100) 4091 (104) 4132 (105) 4143 (110) 4051 (125) 4064 (121)

WBC count(times103120583L)

597 (036) 577 (033) 594 (026) 644 (032) 570 (037) 543 (029)

Platelet count(times103120583L) 24627 (1314) 24045 (1430) 25536 (1413) 24354 (1265) 23827 (1220) 23972 (1064)

Absoluteneutrophils(times103120583L)

309 (023) 319 (026) 327 (014) 388 (044) 312 (028) 284 (020)

6 Oxidative Medicine and Cellular Longevity

Table 2 Continued

Test Subject data and laboratory safety assessment mean (SEM)Day 0 1 week 4 weeks 12 weeks 16 weeks 24 weeks

PT(sec) 1214 (021) 1208 (021) 1199 (024) 1223 (019) 1248 (027) 1262 (022)

aPTT(sec) 2853 (060) 2819 (076) 2861 (069) 2897 (061) 2920 (081) 2970 (074)

Urine protein(Negndashtrace) 545 727 727 818 727 545

Body mass index 2174 (112) 2186 (112) 2164 (107) 2207 (114) 2191 (112) 2189 (113)Arterial bloodpressure(mmHg)

11772 11776 11777 11973 11872 11373

HDL-CHOL high-density lipoprotein cholesterol LDL low-density lipoprotein BUN blood urea nitrogen AST (SGOT) aspartate aminotransferase(serum glutamic oxaloacetic transaminase) ALT (SGPT) alanine aminotransferase (serum glutamic pyruvic transaminase) ALP alkaline phosphataseT3 triiodothyronine TSH thyroid-stimulating hormone FT4 free thyroxine WBC white blood cell PT prothrombin time aPTT activated partialthromboplastin time

Table 3 Side effects of oral administration ofMEThe subjects wereasked to report the side effects after oral administration of ME ateach visit The number of episodes of each side effect prior to andafter double doses was collected and calculated as the percentage ofthe total episodes of 110 (11 participants times 10 visits)

SymptomBefore (single dose) After (double doses)Number ofepisodes Number of

episodes

Tirednesssleepiness 4 36 0 00Constipation 4 36 1 09Dry throat 2 18 3 27Headache 1 09 4 36Flu-like symptoms 2 18 0 00Abnormal stool 4 36 0 00Abdominaldiscomfort 3 27 0 00

Cough 2 18 0 00Weight loss 2 18 2 18Nasal congestion 2 18 0 00Abdominal bloating 2 18 1 09Dizziness 1 09 0 00Salivation 1 09 0 00Nauseavomiting 1 09 1 09Diarrhea 1 09 1 09Skin rash 1 09 0 00Abnormal urination 1 09 0 00Malaise 1 09 0 00

RBCsThis effect was further accentuated through the end ofthe study

35 Oxidative Damage to Proteins in RBCs and Whole BloodOral administration of ME was associated with a significantdecrease in oxidative damage to proteins in both RBCs andwhole blood Quantification of oxidative damage to these

proteins was achieved by normalizing HNE staining to actinAs shown in Figure 4 a significant decrease in proteinoxidative damage appeared inweeks 4 and 24 for whole bloodand RBCs respectively

4 Discussion

Several compounds in the extract frommangosteen pericarphave recently been identified and shown to have beneficialeffects on human health However the cytotoxic activitieswere always observed in nonpolar fraction of the extract thatis 120572-mangostin whereas the potent antioxidant was foundin polar fraction In addition the safety of the polar fractionhas never been thoroughly investigated in humans In thisstudy we investigated the safety of oral administration of thepolar fraction of ME inThai subjects For the entire 24 weeksof the study only minor side effects were reported Thesecan be categorized into gastrointestinal skin respiratory andgeneral side effects The most frequently reported side effectswere related to the gastrointestinal systemWe suspected thatsuch side effects might be associated with an antibacterialproperty of the compounds from the mangosteen pericarpwhich may have led to the perturbation of gut microbesNonetheless these side effects accounted for only a fewepisodes and were reportedly tolerable by all subjects

The present study also showed that oral administra-tion of the polar fraction of ME poses no threat to vitalorgans including the liver kidney and thyroid gland Thesubjectsrsquo liver enzyme levels lipid profiles coagulogramparameters blood urea nitrogen level creatinine level urineprotein level plasma electrolyte levels and thyroid hormonelevels remained normal for the entire 24 weeks of thestudy Additionally all subjectsrsquo complete blood counts andblood pressures remained unchanged Although two subjectsshowed slightly increased liver enzymes in the fourth weekof the study the enzyme levels spontaneously returned tothe normal range thereafter Notably a study by Stern et al[27] revealed that obese subjects with a body mass index of30 to 40 kgm2 exhibited a significant net reduction in body

Oxidative Medicine and Cellular Longevity 7

0

1000

FSC

SSC

0

1000

SSC

0

1000

SSC

0

0

1000

1000

FSC0 1000

FSC0 1000

FSC0 1000

SSC

DCF

Cou

nts

100 101 102 103 104

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

DCF100 101 102 103 104

0

100

Cou

nts

0

100

0

1000

SSC

0

1000

SSC

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

0

1000

SSC

0

1000

SSC

0

1000

SSC

0

1000

SSC

FSC

0 1000

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

0

100

100

0

100

Stim

ulat

edSt

imul

ated

0

1000

FSC

SSC

0

0

1000

1000

FSC0 1000

SSC

DCF

DCF

Cou

nts

100 101 102 103 104

100 101 102 103 104

Cou

nts

W12 W16 W24

W0 W1 W44 + H(0)001

4 + H(20)001

4 + H(0)001

4 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001 M012 + H(0)001

M012 + H(20)001 M012 + H(20)001

M012 + H(0)001 M012 + H(0)001

M012 + H(20)001 M012 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001M012 + H(0)001 M012 + H(0)001

M012 + H(20)001M012 + H(20)001 M012 + H(20)001

0min

Stim

ulat

ed0min

20min

Stim

ulat

ed20min

(a)

W1

W4

W12

W16W

0

W24

Weeks of oral administration of ME

Mea

n flu

ores

cenc

e int

ensit

y

lowastlowast

lowast

40

30

20

10

50

(b)

Figure 3 Effect of oral administration of ME on antioxidant capacity of RBCs RBCs were isolated from the peripheral blood of the subjectsat 0 1 4 12 16 and 24 weeks after beginning oral administration of ME Hydrogen peroxide was an exogenous ROS used to stimulate theresponse of the RBC antioxidant system (a) A representative measurement of the level of ROS within the RBCs which was determined by themean fluorescent signal of DCF emitted from hydrogen peroxide-incubated RBCs (b) Quantitative analysis of the mean fluorescent signalof DCF from all samples during the entire period of the study Data are presented as means (119899 = 11) Error bars represent SEMThe asteriskindicates statistical significance 119901 lt 005 in comparison with week 0 (119905-test)

weight (374 kg 119901 lt 00001) after taking capsules containingSphaeranthus indicus and G mangostana for 8 weeks com-pared with subjects taking placebo The discrepancy in thesubjectsrsquo body weights between that study and the presentstudy might derive from the volunteers in our study havinga normal BMI or from the weight-loss property of S indicus

We performedTLC onME in order to test for preparationconsistency and to demonstrate its essential compounds Itwas shown that ME was composed of at least one groupof major compounds which consistently appeared as asingle band on the chromatogram at 119877

119891= 075 Such a

high 119877119891suggested highly organic-volatile property We are

8 Oxidative Medicine and Cellular Longevity

Red blood cell Whole blood

W0

W1

W4

W12

W16

W24

Actin HNE Actin HNE

(a)

05

10

HNE-WBHNE-RBC

W1

W4

W12

W16W

0

W24

Weeks of oral administration of ME

Leve

l of H

NE

mod

ifica

tion

(rel

ativ

e to

W0) lowast

lowast

lowast

lowastlowast

(b)

Figure 4 Oral administration of ME resulted in decreased protein oxidative damage in RBCs and whole blood The RBCs and whole bloodof the subjects were collected at 0 1 4 12 16 and 24 weeks after beginning oral administration of ME (a) A representative blot showsHNE staining using a western blotting technique to determine the protein oxidative damage in RBCs and whole blood Actin was used asa loading control (b) Quantitative analysis of the extent of HNE modification to RBCs (HNE-RBC) and whole blood (HNE-WB) after oraladministration of ME was carried out as that relative to week 0 Data are presented as means (119899 = 11) Error bars represent SEMThe asteriskindicates statistical significance 119901 lt 005 in comparison with week 0 (119905-test)

currently identifying the chemical compounds in this band(data not shown) Further in vitro analysis of these majorcompounds revealed that they exhibited approximately one-third of antioxidant activity of vitamin C Therefore ME islikely to possess considerably high antioxidant activity

Antioxidant property of the polar fraction of MEobserved in our in vitro experiments was shown to exertbiological impactThe 24-week period of oral administrationof the polar fraction of ME in the present study resulted ina significant increase in the antioxidant capacity of RBCswhich was determined by the fluorescent signal from DCFemitted from hydrogen peroxide-incubated RBCs A signifi-cant increase in the antioxidant capacity (ie decreased DCFsignal) of RBCs could be detected from week 12 of oraladministration of the polar fraction of ME Therefore ourresults suggest for the first time that extract frommangosteenpericarp can augment the antioxidant system within humanRBCs probably due to its ability to neutralize ROS

We further investigated whether and how such anincreased antioxidant capacity affects protein oxidative dam-age in RBCs and whole blood Protein oxidative damagewas determined by the degree of HNE modification HNEis a byproduct of lipid peroxidation which is an indicatorof oxidative stress HNE can react with lysine histidine orcysteine residue in proteins to form adducts resulting instructural and functional changes to the proteins [28 29]HNE is also reportedly involved in the pathophysiology ofmany diseases [30ndash32] We speculated that if RBCs havean improved antioxidant capacity lipid peroxidation shouldbe reduced and HNE modification to proteins should thus

decrease Consistent with our speculation oral administra-tion of the polar fraction of ME led to a significant decreasein HNE modification in whole blood and RBCs starting inweeks 4 and 24 respectively Notably the significant decreasein HNE modification of whole blood proteins appearedearlier than that of RBC proteins This may be explained bythe faster turnover rate of albumin [33] the most abundantprotein in serum in comparison to the lifespan of RBCs[34] This result provides further supporting evidence inhuman subjects of the antioxidant property of extract frommangosteen pericarp

5 Conclusions

We have demonstrated for the first time in human subjectsthat the polar fraction of extract from mangosteen pericarpis adequately safe for oral administration for up to 24 weeksMajor components of the polar fraction of extract frommangosteen pericarp exhibited considerably high antioxidantactivity according to the in vitro antioxidant assay Addi-tionally the polar extract from mangosteen pericarp appearsto significantly increase the antioxidant capacity of RBCsand significantly decrease protein oxidative damage in RBCsand whole blood It might alleviate oxidative damage inother tissues as well Our results suggest that the antioxidantcapacity of the polar fraction of mangosteen pericarp extractcould come from its ability to neutralize ROS Howeverwhether some compounds in this extract can also upregulateantioxidant enzyme is still unknown It would be interestingto further quantitate the expression level of antioxidant

Oxidative Medicine and Cellular Longevity 9

enzymes In all the polar fraction of extract frommangosteenpericarp might benefit human patients with pathologicalconditions related to oxidative stress

Competing Interests

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

Acknowledgments

The authors are extremely grateful to Mrs Angkana Jong-sawaddipatana and all staff members of the Geriatric Clinicof Siriraj Hospital for their kind collaboration in recruit-ment of the participants This work was supported by theThai Traditional Medical Knowledge Fund Department forDevelopment of Thai Traditional and Alternative MedicineMinistry of Public Health Thailand

References

[1] S M Petiwala G Li A Ramaiya et al ldquoPharmacokineticcharacterization of mangosteen (Garcinia mangostana) fruitextract standardized to 120572-mangostin in C57BL6 micerdquo Nutri-tion Research vol 34 no 4 pp 336ndash345 2014

[2] Z Xu L Huang X-H Chen et al ldquoCytotoxic prenylated xan-thones from the pericarps of Garcinia mangostanardquo Moleculesvol 19 no 2 pp 1820ndash1827 2014

[3] F Gutierrez-Orozco C Chitchumroonchokchai G B Lesin-ski S Suksamrarn and M L Failla ldquo120572-Mangostin anti-inflammatory activity and metabolism by human cellsrdquo Journalof Agricultural and Food Chemistry vol 61 no 16 pp 3891ndash3900 2013

[4] H-Y Jang O-K Kwon S-R Oh H-K Lee K-S Ahn and Y-W Chin ldquoMangosteen xanthones mitigate ovalbumin-inducedairway inflammation in a mouse model of asthmardquo Food andChemical Toxicology vol 50 no 11 pp 4042ndash4050 2012

[5] S Syam A Bustamam R Abdullah et al ldquo120573 Mangostinsuppress LPS-induced inflammatory response in RAW 2647macrophages in vitro and carrageenan-induced peritonitis invivordquo Journal of Ethnopharmacology vol 153 no 2 pp 435ndash4452014

[6] J Yuan Y Wu and G Lu ldquoA-mangostin suppresseslipopolysaccharide-induced invasion by inhibiting matrixmetalloproteinase-29 and increasing E-cadherin expressionthrough extracellular signal-regulated kinase signaling inpancreatic cancer cellsrdquo Oncology Letters vol 5 no 6 pp1958ndash1964 2013

[7] M-A Shibata M Iinuma J Morimoto et al ldquo120572-Mangostinextracted from the pericarp of the mangosteen (Garcinia man-gostana Linn) reduces tumor growth and lymph node metas-tasis in an immunocompetent xenograft model of metastaticmammary cancer carrying a p53mutationrdquo BMCMedicine vol9 article 69 2011

[8] A F A Aisha K M Abu-Salah Z Ismail and A MajidldquoIn vitro and in vivo anti-colon cancer effects of Garciniamangostana xanthones extractrdquo BMC Complementary andAlternative Medicine vol 12 article 104 2012

[9] S Taokaew N Nunkaew P Siripong and M PhisalaphongldquoCharacteristics and anticancer properties of bacterial cellulose

films containing ethanolic extract of mangosteen peelrdquo Journalof Biomaterials Science Polymer Edition vol 25 no 9 pp 907ndash922 2014

[10] P Pan-In S Wanichwecharungruang J Hanes and A J KimldquoCellular trafficking and anticancer activity of Garcinia man-gostana extract-encapsulated polymeric nanoparticlesrdquo Inter-national Journal of Nanomedicine vol 9 no 1 pp 3677ndash36862014

[11] H R W Dharmaratne Y Sakagami K G P Piyasena and VThevanesam ldquoAntibacterial activity of xanthones fromGarciniamangostana (L) and their structure-activity relationship stud-iesrdquo Natural Product Research vol 27 no 10 pp 938ndash941 2013

[12] J-J Koh S Qiu H Zou et al ldquoRapid bactericidal action ofalpha-mangostin against MRSA as an outcome of membranetargetingrdquo Biochimica et Biophysica ActamdashBiomembranes vol1828 no 2 pp 834ndash844 2013

[13] M T Chomnawang S Surassmo K Wongsariya and N Bun-yapraphatsara ldquoAntibacterial activity of Thai medicinal plantsagainstmethicillin-resistant Staphylococcus aureusrdquo Fitoterapiavol 80 no 2 pp 102ndash104 2009

[14] S Suksamrarn N SuwannapochW Phakhodee et al ldquoAntimy-cobacterial activity of prenylated xanthones from the fruits ofGarcinia mangostanardquo Chemical and Pharmaceutical Bulletinvol 51 no 7 pp 857ndash859 2003

[15] P Moongkarndi C Srisawat P Saetun et al ldquoProtective effectof mangosteen extract against 120573-amyloid-induced cytotoxicityoxidative stress and altered proteome in SK-N-SH cellsrdquo Journalof Proteome Research vol 9 no 5 pp 2076ndash2086 2010

[16] Y-W Chin H-A Jung H Chai W J Keller and A DKinghorn ldquoXanthones with quinone reductase-inducing activ-ity from the fruits of Garcinia mangostana (Mangosteen)rdquoPhytochemistry vol 69 no 3 pp 754ndash758 2008

[17] H-A Jung B-N Su W J Keller R G Mehta and AD Kinghorn ldquoAntioxidant xanthones from the pericarp ofGarcinia mangostana (Mangosteen)rdquo Journal of Agriculturaland Food Chemistry vol 54 no 6 pp 2077ndash2082 2006

[18] F Gutierrez-Orozco and M L Failla ldquoBiological activities andbioavailability of mangosteen xanthones a critical review of thecurrent evidencerdquo Nutrients vol 5 no 8 pp 3163ndash3183 2013

[19] J Sattayasai P Chaonapan T Arkaravichie et al ldquoProtectiveeffects of mangosteen extract on H

2

O2

-induced cytotoxicity inSK-N-SH cells and scopolamine-induced memory impairmentin micerdquo PLoS ONE vol 8 no 12 Article ID e85053 2013

[20] E B Walker ldquoHPLC analysis of selected xanthones in mangos-teen fruitrdquo Journal of Separation Science vol 30 no 9 pp 1229ndash1234 2007

[21] F Gutierrez-Orozco J MThomas-Ahner L D Berman-Bootyet al ldquoDietary 120572-mangostin a xanthone from mangosteenfruit exacerbates experimental colitis and promotes dysbiosisin micerdquoMolecular Nutrition and Food Research vol 58 no 6pp 1226ndash1238 2014

[22] M Y IbrahimNMHashim A AMariod et al ldquo120572-Mangostinfrom Garcinia mangostana Linn an updated review of itspharmacological propertiesrdquo Arabian Journal of Chemistry vol9 no 3 pp 317ndash329 2016

[23] M Y Ibrahim N M Hashim S Mohan et al ldquo120572-Mangostinfrom Cratoxylum arborescens an in vitro and in vivo toxicolog-ical evaluationrdquo Arabian Journal of Chemistry vol 8 no 1 pp129ndash137 2015

[24] A Plazonic F Bucar Z Males A Mornar B Nigovic andN Kujundzic ldquoIdentification and quantification of flavonoids

10 Oxidative Medicine and Cellular Longevity

and phenolic acids in burr parsley (Caucalis platycarpos L)using high-performance liquid chromatography with diodearray detection and electrospray ionizationmass spectrometryrdquoMolecules vol 14 no 7 pp 2466ndash2490 2009

[25] P Moongkarndi N Jaisupa J Samer et al ldquoComparison of thebiological activity of two different isolates from mangosteenrdquoJournal of Pharmacy and Pharmacology vol 66 no 8 pp 1171ndash1179 2014

[26] E Fibach ldquoTechniques for studying stimulation of fetal hemo-globin production in human erythroid culturesrdquo Hemoglobinvol 22 no 5-6 pp 445ndash458 1998

[27] J S Stern J Peerson A T Mishra M V Sadasiva Rao andK P Rajeswari ldquoEfficacy and tolerability of a novel herbalformulation for weight managementrdquo Obesity vol 21 no 5 pp921ndash927 2013

[28] C M Spickett ldquoThe lipid peroxidation product 4-hydroxy-2-nonenal advances in chemistry and analysisrdquo Redox Biologyvol 1 no 1 pp 145ndash152 2013

[29] C Schneider N A Porter and A R Brash ldquoRoutes to 4-hydroxynonenal fundamental issues in themechanisms of lipidperoxidationrdquo Journal of Biological Chemistry vol 283 no 23pp 15539ndash15543 2008

[30] A Negre-Salvayre C Coatrieux C Ingueneau and R SalvayreldquoAdvanced lipid peroxidation end products in oxidative damageto proteins Potential role in diseases and therapeutic prospectsfor the inhibitorsrdquo British Journal of Pharmacology vol 153 no1 pp 6ndash20 2008

[31] M P Mattson ldquoRoles of the lipid peroxidation product 4-hydroxynonenal in obesity the metabolic syndrome and asso-ciated vascular and neurodegenerative disordersrdquo ExperimentalGerontology vol 44 no 10 pp 625ndash633 2009

[32] A Ayala M F Munoz and S Arguelles ldquoLipid peroxidationproduction metabolism and signaling mechanisms of malon-dialdehyde and 4-hydroxy-2-nonenalrdquo Oxidative Medicine andCellular Longevity vol 2014 Article ID 360438 31 pages 2014

[33] K Sterling ldquoThe turnover rate of serum albumin in manas measured by I131-tagged albuminrdquo The Journal of ClinicalInvestigation vol 30 no 11 pp 1228ndash1237 1951

[34] C J Lindsell R S Franco E P Smith C H Joiner and R MCohen ldquoA method for the continuous calculation of the age oflabeled red blood cellsrdquo American Journal of Hematology vol83 no 6 pp 454ndash457 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 3

industry estimating themaximum safe starting dose in initialclinical trials for therapeutics in adult healthy volunteersrdquofrom US Department of Health and Human Services Foodand Drug Administration Center for Drug Evaluation andResearch CDER July 2005 After 3 months all of the volun-teers received a double dose daily All volunteers underwentcollection of 4mL blood samples at 0 1 4 12 16 and 24weeksafter beginning the oral ME administration

24 Separation of ME by TLC and Screening of AntioxidantActivity The slightly modified TLC was applied [24] Theextract was dissolved in methanol and then subjected ontoa 20 times 20 cm normal phase aluminum plate (silica gel 60F254 Merck St Louis MO) The plate was developed inthe mobile phase consisting of ethyl acetate 70 ethanolin a ratio of 10 1 volume volume The isolated bands werevisualized under UV light To screen the antioxidant activityof each isolated band of ME stable free radical 22-diphenyl-1-picrylhydrazyl radical (DPPH) was appliedThe developedplate was sprayed by 03mMDPPH in methanol The forma-tion of yellow color indicated the antioxidant activity on thebasis of the formation of reduced DPPH

25 In Vitro Assay for Antioxidant Property of ME Theassay was performed as described by Moongkarndi et al[25] In brief isolated band A from TLC separation wasdissolved in methanol to various concentrations (0 25 510 20 50 and 100 120583gmL) To 100 120583L of extract solution100 120583L of fresh 04mM DPPH solution in methanol wasadded The experiment was conducted in a 96-well plate(119899 = 3 independent samples) The reaction was incubatedin darkness at room temperature for 30min The absorbancewas measured at 517 nm by microplate reader (Anthos 2010Anthos Labtec Instruments GmbH) Vitamin C was usedas positive control The degree of discoloration indicatesthe scavenging potential of the antioxidant property Thescavenging activity was calculated by [1 minus (119860

1minus blank)119860

0minus

blank] times 100 where 1198601was the absorbance of DPPH in the

presence of the extracts 1198600represented the absorbance of

DPPH solution without extract addition and blank was theabsorbance without DPPH solution

26 Measurement of ROS in RBCs The heparinized bloodwas diluted with Ca2+- and Mg2+-free phosphate bufferedsaline (PBS) (Sigma St Louis MO USA) to a concentrationof 1 times 106 RBCsmL The RBCs were incubated with 04mMof 21015840-71015840-dichlorofluorescein diacetate (DCFH-DA) (Sigma)dissolved in methanol (Fluka Sigma-Aldrich St Louis MOUSA) After incubation at 37∘C for 15min in a humidifiedatmosphere of 5 carbon dioxide in air the cells were washedand resuspended in PBS to the original cell concentrationThe RBC suspension was analyzed by incubation with orwithout 2mM of freshly prepared hydrogen peroxide Afterincubation at room temperature for 20min the sampleswere analyzed by a fluorescence-activated cell sorter (FAC-SCalibur Becton-Dickinson Immunofluorometry SystemsMountain View CA USA) The RBCs were passed at a rateof about 1000 cellss A 488 nm argon laser beam was used

for excitation [26] The absolute fluorescence intensity of the2101584071015840-dichlorofluorescein (DCF) which represented the ROSlevel in the RBCs was interpreted on a two-parameter dotplot of the side light scatter and forward light scatter of thepopulation Cell fluorescence was measured using logarith-mic amplification Green fluorescence of 10000 cells was thenmeasured using linear amplification The arithmetic meanfluorescence channel was derived by CellQuest software(Becton-Dickinson)

27 Determination of Protein Oxidative Damage by Anti-4-Hydroxynonenal Antibody Staining To prepare RBCsforanti-4-hydroxynonenal (anti-HNE) antibody stainingwhole blood was mixed with an equal volume of PBS andthen centrifuged at 800timesg for 5min at 4∘C The pelletcontaining the RBCs was collected and washed three timeswith PBS Finally distilled water was added to suspend andlyse the cells and the solution was stored at minus80∘C until use

Either whole blood or RBCs were denatured in samplebuffer (100mM Tris-HCl pH 68 10 glycerol 4 SDS0002 bromophenol blue 100mM dithiothreitol and 52-mercaptoethanol) and then boiled at 95∘C for 5min Thesamples were dotted manually in duplicate (one for HNE andone for120573-actin detection) onto nitrocellulosemembranewithamount of total protein 05 120583g per dot based on Bradfordassay and left to dry in air The nitrocellulose membraneswere then blocked in 5 nonfat milk in PBS with 001Tween-20 (PBST) with 001 NaN

3at 4∘C for 1 h and

subsequently incubated with either primary antibody anti-HNE antibody (Millipore Billerica MA USA) or anti-actin-3 antibody (Santa Cruz Biotechnology Santa Cruz CA USA)for 2 hThen themembranes were washed 3 times with PBSTand subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz BiotechnologySanta Cruz CA USA) in the same solution without NaN

3at

room temperature for 2 h After incubation with secondaryantibody the membranes were washed with PBST for 3times A chemiluminescence substrate (SuperSignal WestPico Thermo Fisher Scientific Inc Rockford IL USA) wasused to develop the reactions The fluorescence generatedfrom the reaction was visualized by exposing to the filmThe band intensities of HNE and actin-3 from the sampleswere quantitated by ImageJ software (US National Institutesof Health Bethesda MD USA)

The negative control dots were also performed withoutprimary antibody for both whole blood and RBCs samplesno signal was detected

28 Data Analysis Analysis of variance was performed toanalyze groups of data for significant differences UnpairedStudentrsquos 119905-test was then used to calculate statistical signifi-cance of specific pairs if a difference was demonstrated withanalysis of variance

3 Results

31 Biosafety Assessment and Side Effects of Oral Administra-tion of ME The safety of oral administration of the polarfraction ofMEwas assessed in 11 healthy human subjectsThe

4 Oxidative Medicine and Cellular Longevity

Table 1 Demographic data of the subjects

Demographic data n Sex

Male 5 455Female 6 545

Age in years (119909 = 32)20ndash30 4 36431ndash40 6 545gt40 1 91

OccupationEmployee 2 182Government officer 1 91University worker 6 545Others 2 182

Alcohol consumptionStopped drinking for gt3mo 2 182Drinking less than oncemo 7 636Never 2 182

SmokingNever 11 1000

Herbvitamin supplementConsuming 1 91Never 10 909

demographic data of the subjects are shown in Table 1 Theplasma electrolyte levels lipid profiles liver function enzymelevels thyroid hormone levels coagulograms and completeblood counts were measured from whole blood samples ofall subjects at 0 1 4 12 16 and 24 weeks after begin-ning oral administration of the polar fraction of ME Theresults showed no significant alterations in these parameters(Table 2) Additionally the side effects of oral administrationof the polar fraction of ME were determined throughout thestudy All subjects reported minor and tolerable side effectsas shown in Table 3

32 Separation of ME by TLC and Screening of AntioxidantActivity TLC chromatogram of the polar fraction of MEvisualized under UV light at 254 nm revealed a remarkableband that had 119877

119891value of approximately 075 (band A)

After spraying with 03mM DPPH solution several yellowspots which indicated antioxidant properties were observed(Figure 1)

33 In Vitro Antioxidant Assay of ME Quantitative analysisof antioxidant activity of the polar fraction of ME wasperformedon the extract of bandAbyDPPHassay (Figure 2)Antioxidant activity was shown in dose-dependent mannerThe IC

50values of band A extract and vitamin C (positive

control) were 1603 plusmn 144 120583gmL and 516 plusmn 092 120583gmLrespectively

34 Antioxidant Capacity of RBCs The antioxidant capacityof RBCs was determined by measuring the fluorescent signalof DCF from the RBCs after incubation with hydrogen

Band A

(a) (b)

Figure 1 Separation ofME by TLC and antioxidant properties of itsconstituents (a)MEwas separated by TLC and visualized under UVat 254 nmMajor compounds appeared in a single band at119877

119891

= 075

(band A) (b) Screening of antioxidant activity was done by applyingDPPHonto theTLCplate Scavenging ofDPPHwas shown in yellowstaining indicating antioxidative activity of ME

ME-band AVitamin C

0

25

50

75

100

125

sc

aven

ging

75 10025 500Concentration (120583gmL)

Figure 2 Quantitative analysis of antioxidant activity ofME DPPHassay was performed on the extract of band A (ME-band A) fromTLC Vitamin C was used as a positive control for comparisonAntioxidant activities ( scavenging) of both ME and vitaminC calculated as described in Materials and Methods were dose-dependent The IC

50

values of ME and vitamin C were 1603 plusmn144 120583gmL and 516 plusmn 092 120583gmL respectively Error bars representSEM

peroxide which was used as an exogenous ROS The RBCsfrom the subjects taking the polar fraction of ME exhibited asignificant decrease in the mean fluorescent intensity of DCFstarting in week 12 of oral administration of ME (Figure 3)This suggests that ME increases the antioxidant capacity of

Oxidative Medicine and Cellular Longevity 5

Table 2 Subject data and biochemical parameters for assessment of safety of oral administration of ME Blood and urine samples werecollected from the subjects in 0 1 4 12 16 and 24 weeks after beginning oral administration of ME The biochemical parameters weremeasured as tabulated Body mass index and blood pressure were also determined All parameters were collected and calculated from 11healthy individuals

Test Subject data and laboratory safety assessment mean (SEM)Day 0 1 week 4 weeks 12 weeks 16 weeks 24 weeks

Glucose(mgdL) 8673 (164) 8500 (167) 8309 (145) 8145 (304) 8091 (146) 8500 (253)

Cholesterol(mgdL) 20654 (1310) 19600 (1295) 20109 (1432) 19345 (1034) 19063 (1361) 19245 (2474)

Triglycerides(mgdL) 7800 (1115) 7718 (1958) 8127 (2011) 8718 (1727) 10354 (3080) 9227 (2265)

HDL-CHOL(mgdL) 6290 (294) 6045 (252) 5945 (213) 5972 (208) 5781 (226) 5945 (195)

LDL-calculated(mgdL) 12803 (1200) 12010 (1062) 12538 (1234) 11629 (895) 11210 (913) 11454 (1080)

BUN(mgdL) 1284 (163) 1126 (058) 1243 (103) 1182 (082) 1223 (088) 1217 (118)

Creatinine(mgdL) 086 (006) 084 (004) 081 (005) 082 (004) 082 (004) 081 (004)

Sodium(mmolL) 14127 (057) 14154 (089) 14136 (051) 14009 (047) 14109 (041) 14018 (038)

Potassium(mmolL) 375 (006) 372 (009) 375 (007) 358 (008) 356 (006) 363 (008)

Chloride(mmolL) 10279 (045) 10308 (056) 10250 (050) 10179 (071) 10323 (055) 10293 (036)

Bicarbonate(mmolL) 2814 (085) 2492 (070) 2650 (104) 2543 (070) 2485 (101) 2414 (071)

Total bilirubin(mgdL) 063 (014) 060 (011) 049 (008) 059 (011) 060 (014) 057 (010)

Direct bilirubin(mgdL) 021 (004) 023 (003) 020 (002) 024 (004) 024 (004) 022 (003)

AST (SGOT)(UL) 2081 (189) 1945 (207) 3054 (804) 1800 (180) 1909 (146) 1763 (283)

ALT (SGPT)(UL) 1918 (412) 1700 (368) 3127 (1040) 1845 (454) 1563 (261) 2045 (771)

ALP(UL) 6072 (268) 5927 (314) 6363 (317) 6245 (205) 6409 (374) 6254 (314)

T3(ngdL) 9942 (327) 10462 (353) 9745 (316) 9735 (382) 9926 (633) 9262 (372)

TSH(120583IUmL) 215 (032) 201 (032) 209 (042) 203 (036) 207 (028) 216 (030)

FT4(ngdL) 132 (005) 128 (005) 126 (005) 122 (002) 118 (003) 122 (004)

Hemoglobin(gdL) 1360 (031) 1325 (039) 1345 (031) 1355 (037) 1350 (040) 1350 (043)

Hematocrit() 4219 (100) 4091 (104) 4132 (105) 4143 (110) 4051 (125) 4064 (121)

WBC count(times103120583L)

597 (036) 577 (033) 594 (026) 644 (032) 570 (037) 543 (029)

Platelet count(times103120583L) 24627 (1314) 24045 (1430) 25536 (1413) 24354 (1265) 23827 (1220) 23972 (1064)

Absoluteneutrophils(times103120583L)

309 (023) 319 (026) 327 (014) 388 (044) 312 (028) 284 (020)

6 Oxidative Medicine and Cellular Longevity

Table 2 Continued

Test Subject data and laboratory safety assessment mean (SEM)Day 0 1 week 4 weeks 12 weeks 16 weeks 24 weeks

PT(sec) 1214 (021) 1208 (021) 1199 (024) 1223 (019) 1248 (027) 1262 (022)

aPTT(sec) 2853 (060) 2819 (076) 2861 (069) 2897 (061) 2920 (081) 2970 (074)

Urine protein(Negndashtrace) 545 727 727 818 727 545

Body mass index 2174 (112) 2186 (112) 2164 (107) 2207 (114) 2191 (112) 2189 (113)Arterial bloodpressure(mmHg)

11772 11776 11777 11973 11872 11373

HDL-CHOL high-density lipoprotein cholesterol LDL low-density lipoprotein BUN blood urea nitrogen AST (SGOT) aspartate aminotransferase(serum glutamic oxaloacetic transaminase) ALT (SGPT) alanine aminotransferase (serum glutamic pyruvic transaminase) ALP alkaline phosphataseT3 triiodothyronine TSH thyroid-stimulating hormone FT4 free thyroxine WBC white blood cell PT prothrombin time aPTT activated partialthromboplastin time

Table 3 Side effects of oral administration ofMEThe subjects wereasked to report the side effects after oral administration of ME ateach visit The number of episodes of each side effect prior to andafter double doses was collected and calculated as the percentage ofthe total episodes of 110 (11 participants times 10 visits)

SymptomBefore (single dose) After (double doses)Number ofepisodes Number of

episodes

Tirednesssleepiness 4 36 0 00Constipation 4 36 1 09Dry throat 2 18 3 27Headache 1 09 4 36Flu-like symptoms 2 18 0 00Abnormal stool 4 36 0 00Abdominaldiscomfort 3 27 0 00

Cough 2 18 0 00Weight loss 2 18 2 18Nasal congestion 2 18 0 00Abdominal bloating 2 18 1 09Dizziness 1 09 0 00Salivation 1 09 0 00Nauseavomiting 1 09 1 09Diarrhea 1 09 1 09Skin rash 1 09 0 00Abnormal urination 1 09 0 00Malaise 1 09 0 00

RBCsThis effect was further accentuated through the end ofthe study

35 Oxidative Damage to Proteins in RBCs and Whole BloodOral administration of ME was associated with a significantdecrease in oxidative damage to proteins in both RBCs andwhole blood Quantification of oxidative damage to these

proteins was achieved by normalizing HNE staining to actinAs shown in Figure 4 a significant decrease in proteinoxidative damage appeared inweeks 4 and 24 for whole bloodand RBCs respectively

4 Discussion

Several compounds in the extract frommangosteen pericarphave recently been identified and shown to have beneficialeffects on human health However the cytotoxic activitieswere always observed in nonpolar fraction of the extract thatis 120572-mangostin whereas the potent antioxidant was foundin polar fraction In addition the safety of the polar fractionhas never been thoroughly investigated in humans In thisstudy we investigated the safety of oral administration of thepolar fraction of ME inThai subjects For the entire 24 weeksof the study only minor side effects were reported Thesecan be categorized into gastrointestinal skin respiratory andgeneral side effects The most frequently reported side effectswere related to the gastrointestinal systemWe suspected thatsuch side effects might be associated with an antibacterialproperty of the compounds from the mangosteen pericarpwhich may have led to the perturbation of gut microbesNonetheless these side effects accounted for only a fewepisodes and were reportedly tolerable by all subjects

The present study also showed that oral administra-tion of the polar fraction of ME poses no threat to vitalorgans including the liver kidney and thyroid gland Thesubjectsrsquo liver enzyme levels lipid profiles coagulogramparameters blood urea nitrogen level creatinine level urineprotein level plasma electrolyte levels and thyroid hormonelevels remained normal for the entire 24 weeks of thestudy Additionally all subjectsrsquo complete blood counts andblood pressures remained unchanged Although two subjectsshowed slightly increased liver enzymes in the fourth weekof the study the enzyme levels spontaneously returned tothe normal range thereafter Notably a study by Stern et al[27] revealed that obese subjects with a body mass index of30 to 40 kgm2 exhibited a significant net reduction in body

Oxidative Medicine and Cellular Longevity 7

0

1000

FSC

SSC

0

1000

SSC

0

1000

SSC

0

0

1000

1000

FSC0 1000

FSC0 1000

FSC0 1000

SSC

DCF

Cou

nts

100 101 102 103 104

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

DCF100 101 102 103 104

0

100

Cou

nts

0

100

0

1000

SSC

0

1000

SSC

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

0

1000

SSC

0

1000

SSC

0

1000

SSC

0

1000

SSC

FSC

0 1000

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

0

100

100

0

100

Stim

ulat

edSt

imul

ated

0

1000

FSC

SSC

0

0

1000

1000

FSC0 1000

SSC

DCF

DCF

Cou

nts

100 101 102 103 104

100 101 102 103 104

Cou

nts

W12 W16 W24

W0 W1 W44 + H(0)001

4 + H(20)001

4 + H(0)001

4 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001 M012 + H(0)001

M012 + H(20)001 M012 + H(20)001

M012 + H(0)001 M012 + H(0)001

M012 + H(20)001 M012 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001M012 + H(0)001 M012 + H(0)001

M012 + H(20)001M012 + H(20)001 M012 + H(20)001

0min

Stim

ulat

ed0min

20min

Stim

ulat

ed20min

(a)

W1

W4

W12

W16W

0

W24

Weeks of oral administration of ME

Mea

n flu

ores

cenc

e int

ensit

y

lowastlowast

lowast

40

30

20

10

50

(b)

Figure 3 Effect of oral administration of ME on antioxidant capacity of RBCs RBCs were isolated from the peripheral blood of the subjectsat 0 1 4 12 16 and 24 weeks after beginning oral administration of ME Hydrogen peroxide was an exogenous ROS used to stimulate theresponse of the RBC antioxidant system (a) A representative measurement of the level of ROS within the RBCs which was determined by themean fluorescent signal of DCF emitted from hydrogen peroxide-incubated RBCs (b) Quantitative analysis of the mean fluorescent signalof DCF from all samples during the entire period of the study Data are presented as means (119899 = 11) Error bars represent SEMThe asteriskindicates statistical significance 119901 lt 005 in comparison with week 0 (119905-test)

weight (374 kg 119901 lt 00001) after taking capsules containingSphaeranthus indicus and G mangostana for 8 weeks com-pared with subjects taking placebo The discrepancy in thesubjectsrsquo body weights between that study and the presentstudy might derive from the volunteers in our study havinga normal BMI or from the weight-loss property of S indicus

We performedTLC onME in order to test for preparationconsistency and to demonstrate its essential compounds Itwas shown that ME was composed of at least one groupof major compounds which consistently appeared as asingle band on the chromatogram at 119877

119891= 075 Such a

high 119877119891suggested highly organic-volatile property We are

8 Oxidative Medicine and Cellular Longevity

Red blood cell Whole blood

W0

W1

W4

W12

W16

W24

Actin HNE Actin HNE

(a)

05

10

HNE-WBHNE-RBC

W1

W4

W12

W16W

0

W24

Weeks of oral administration of ME

Leve

l of H

NE

mod

ifica

tion

(rel

ativ

e to

W0) lowast

lowast

lowast

lowastlowast

(b)

Figure 4 Oral administration of ME resulted in decreased protein oxidative damage in RBCs and whole blood The RBCs and whole bloodof the subjects were collected at 0 1 4 12 16 and 24 weeks after beginning oral administration of ME (a) A representative blot showsHNE staining using a western blotting technique to determine the protein oxidative damage in RBCs and whole blood Actin was used asa loading control (b) Quantitative analysis of the extent of HNE modification to RBCs (HNE-RBC) and whole blood (HNE-WB) after oraladministration of ME was carried out as that relative to week 0 Data are presented as means (119899 = 11) Error bars represent SEMThe asteriskindicates statistical significance 119901 lt 005 in comparison with week 0 (119905-test)

currently identifying the chemical compounds in this band(data not shown) Further in vitro analysis of these majorcompounds revealed that they exhibited approximately one-third of antioxidant activity of vitamin C Therefore ME islikely to possess considerably high antioxidant activity

Antioxidant property of the polar fraction of MEobserved in our in vitro experiments was shown to exertbiological impactThe 24-week period of oral administrationof the polar fraction of ME in the present study resulted ina significant increase in the antioxidant capacity of RBCswhich was determined by the fluorescent signal from DCFemitted from hydrogen peroxide-incubated RBCs A signifi-cant increase in the antioxidant capacity (ie decreased DCFsignal) of RBCs could be detected from week 12 of oraladministration of the polar fraction of ME Therefore ourresults suggest for the first time that extract frommangosteenpericarp can augment the antioxidant system within humanRBCs probably due to its ability to neutralize ROS

We further investigated whether and how such anincreased antioxidant capacity affects protein oxidative dam-age in RBCs and whole blood Protein oxidative damagewas determined by the degree of HNE modification HNEis a byproduct of lipid peroxidation which is an indicatorof oxidative stress HNE can react with lysine histidine orcysteine residue in proteins to form adducts resulting instructural and functional changes to the proteins [28 29]HNE is also reportedly involved in the pathophysiology ofmany diseases [30ndash32] We speculated that if RBCs havean improved antioxidant capacity lipid peroxidation shouldbe reduced and HNE modification to proteins should thus

decrease Consistent with our speculation oral administra-tion of the polar fraction of ME led to a significant decreasein HNE modification in whole blood and RBCs starting inweeks 4 and 24 respectively Notably the significant decreasein HNE modification of whole blood proteins appearedearlier than that of RBC proteins This may be explained bythe faster turnover rate of albumin [33] the most abundantprotein in serum in comparison to the lifespan of RBCs[34] This result provides further supporting evidence inhuman subjects of the antioxidant property of extract frommangosteen pericarp

5 Conclusions

We have demonstrated for the first time in human subjectsthat the polar fraction of extract from mangosteen pericarpis adequately safe for oral administration for up to 24 weeksMajor components of the polar fraction of extract frommangosteen pericarp exhibited considerably high antioxidantactivity according to the in vitro antioxidant assay Addi-tionally the polar extract from mangosteen pericarp appearsto significantly increase the antioxidant capacity of RBCsand significantly decrease protein oxidative damage in RBCsand whole blood It might alleviate oxidative damage inother tissues as well Our results suggest that the antioxidantcapacity of the polar fraction of mangosteen pericarp extractcould come from its ability to neutralize ROS Howeverwhether some compounds in this extract can also upregulateantioxidant enzyme is still unknown It would be interestingto further quantitate the expression level of antioxidant

Oxidative Medicine and Cellular Longevity 9

enzymes In all the polar fraction of extract frommangosteenpericarp might benefit human patients with pathologicalconditions related to oxidative stress

Competing Interests

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

Acknowledgments

The authors are extremely grateful to Mrs Angkana Jong-sawaddipatana and all staff members of the Geriatric Clinicof Siriraj Hospital for their kind collaboration in recruit-ment of the participants This work was supported by theThai Traditional Medical Knowledge Fund Department forDevelopment of Thai Traditional and Alternative MedicineMinistry of Public Health Thailand

References

[1] S M Petiwala G Li A Ramaiya et al ldquoPharmacokineticcharacterization of mangosteen (Garcinia mangostana) fruitextract standardized to 120572-mangostin in C57BL6 micerdquo Nutri-tion Research vol 34 no 4 pp 336ndash345 2014

[2] Z Xu L Huang X-H Chen et al ldquoCytotoxic prenylated xan-thones from the pericarps of Garcinia mangostanardquo Moleculesvol 19 no 2 pp 1820ndash1827 2014

[3] F Gutierrez-Orozco C Chitchumroonchokchai G B Lesin-ski S Suksamrarn and M L Failla ldquo120572-Mangostin anti-inflammatory activity and metabolism by human cellsrdquo Journalof Agricultural and Food Chemistry vol 61 no 16 pp 3891ndash3900 2013

[4] H-Y Jang O-K Kwon S-R Oh H-K Lee K-S Ahn and Y-W Chin ldquoMangosteen xanthones mitigate ovalbumin-inducedairway inflammation in a mouse model of asthmardquo Food andChemical Toxicology vol 50 no 11 pp 4042ndash4050 2012

[5] S Syam A Bustamam R Abdullah et al ldquo120573 Mangostinsuppress LPS-induced inflammatory response in RAW 2647macrophages in vitro and carrageenan-induced peritonitis invivordquo Journal of Ethnopharmacology vol 153 no 2 pp 435ndash4452014

[6] J Yuan Y Wu and G Lu ldquoA-mangostin suppresseslipopolysaccharide-induced invasion by inhibiting matrixmetalloproteinase-29 and increasing E-cadherin expressionthrough extracellular signal-regulated kinase signaling inpancreatic cancer cellsrdquo Oncology Letters vol 5 no 6 pp1958ndash1964 2013

[7] M-A Shibata M Iinuma J Morimoto et al ldquo120572-Mangostinextracted from the pericarp of the mangosteen (Garcinia man-gostana Linn) reduces tumor growth and lymph node metas-tasis in an immunocompetent xenograft model of metastaticmammary cancer carrying a p53mutationrdquo BMCMedicine vol9 article 69 2011

[8] A F A Aisha K M Abu-Salah Z Ismail and A MajidldquoIn vitro and in vivo anti-colon cancer effects of Garciniamangostana xanthones extractrdquo BMC Complementary andAlternative Medicine vol 12 article 104 2012

[9] S Taokaew N Nunkaew P Siripong and M PhisalaphongldquoCharacteristics and anticancer properties of bacterial cellulose

films containing ethanolic extract of mangosteen peelrdquo Journalof Biomaterials Science Polymer Edition vol 25 no 9 pp 907ndash922 2014

[10] P Pan-In S Wanichwecharungruang J Hanes and A J KimldquoCellular trafficking and anticancer activity of Garcinia man-gostana extract-encapsulated polymeric nanoparticlesrdquo Inter-national Journal of Nanomedicine vol 9 no 1 pp 3677ndash36862014

[11] H R W Dharmaratne Y Sakagami K G P Piyasena and VThevanesam ldquoAntibacterial activity of xanthones fromGarciniamangostana (L) and their structure-activity relationship stud-iesrdquo Natural Product Research vol 27 no 10 pp 938ndash941 2013

[12] J-J Koh S Qiu H Zou et al ldquoRapid bactericidal action ofalpha-mangostin against MRSA as an outcome of membranetargetingrdquo Biochimica et Biophysica ActamdashBiomembranes vol1828 no 2 pp 834ndash844 2013

[13] M T Chomnawang S Surassmo K Wongsariya and N Bun-yapraphatsara ldquoAntibacterial activity of Thai medicinal plantsagainstmethicillin-resistant Staphylococcus aureusrdquo Fitoterapiavol 80 no 2 pp 102ndash104 2009

[14] S Suksamrarn N SuwannapochW Phakhodee et al ldquoAntimy-cobacterial activity of prenylated xanthones from the fruits ofGarcinia mangostanardquo Chemical and Pharmaceutical Bulletinvol 51 no 7 pp 857ndash859 2003

[15] P Moongkarndi C Srisawat P Saetun et al ldquoProtective effectof mangosteen extract against 120573-amyloid-induced cytotoxicityoxidative stress and altered proteome in SK-N-SH cellsrdquo Journalof Proteome Research vol 9 no 5 pp 2076ndash2086 2010

[16] Y-W Chin H-A Jung H Chai W J Keller and A DKinghorn ldquoXanthones with quinone reductase-inducing activ-ity from the fruits of Garcinia mangostana (Mangosteen)rdquoPhytochemistry vol 69 no 3 pp 754ndash758 2008

[17] H-A Jung B-N Su W J Keller R G Mehta and AD Kinghorn ldquoAntioxidant xanthones from the pericarp ofGarcinia mangostana (Mangosteen)rdquo Journal of Agriculturaland Food Chemistry vol 54 no 6 pp 2077ndash2082 2006

[18] F Gutierrez-Orozco and M L Failla ldquoBiological activities andbioavailability of mangosteen xanthones a critical review of thecurrent evidencerdquo Nutrients vol 5 no 8 pp 3163ndash3183 2013

[19] J Sattayasai P Chaonapan T Arkaravichie et al ldquoProtectiveeffects of mangosteen extract on H

2

O2

-induced cytotoxicity inSK-N-SH cells and scopolamine-induced memory impairmentin micerdquo PLoS ONE vol 8 no 12 Article ID e85053 2013

[20] E B Walker ldquoHPLC analysis of selected xanthones in mangos-teen fruitrdquo Journal of Separation Science vol 30 no 9 pp 1229ndash1234 2007

[21] F Gutierrez-Orozco J MThomas-Ahner L D Berman-Bootyet al ldquoDietary 120572-mangostin a xanthone from mangosteenfruit exacerbates experimental colitis and promotes dysbiosisin micerdquoMolecular Nutrition and Food Research vol 58 no 6pp 1226ndash1238 2014

[22] M Y IbrahimNMHashim A AMariod et al ldquo120572-Mangostinfrom Garcinia mangostana Linn an updated review of itspharmacological propertiesrdquo Arabian Journal of Chemistry vol9 no 3 pp 317ndash329 2016

[23] M Y Ibrahim N M Hashim S Mohan et al ldquo120572-Mangostinfrom Cratoxylum arborescens an in vitro and in vivo toxicolog-ical evaluationrdquo Arabian Journal of Chemistry vol 8 no 1 pp129ndash137 2015

[24] A Plazonic F Bucar Z Males A Mornar B Nigovic andN Kujundzic ldquoIdentification and quantification of flavonoids

10 Oxidative Medicine and Cellular Longevity

and phenolic acids in burr parsley (Caucalis platycarpos L)using high-performance liquid chromatography with diodearray detection and electrospray ionizationmass spectrometryrdquoMolecules vol 14 no 7 pp 2466ndash2490 2009

[25] P Moongkarndi N Jaisupa J Samer et al ldquoComparison of thebiological activity of two different isolates from mangosteenrdquoJournal of Pharmacy and Pharmacology vol 66 no 8 pp 1171ndash1179 2014

[26] E Fibach ldquoTechniques for studying stimulation of fetal hemo-globin production in human erythroid culturesrdquo Hemoglobinvol 22 no 5-6 pp 445ndash458 1998

[27] J S Stern J Peerson A T Mishra M V Sadasiva Rao andK P Rajeswari ldquoEfficacy and tolerability of a novel herbalformulation for weight managementrdquo Obesity vol 21 no 5 pp921ndash927 2013

[28] C M Spickett ldquoThe lipid peroxidation product 4-hydroxy-2-nonenal advances in chemistry and analysisrdquo Redox Biologyvol 1 no 1 pp 145ndash152 2013

[29] C Schneider N A Porter and A R Brash ldquoRoutes to 4-hydroxynonenal fundamental issues in themechanisms of lipidperoxidationrdquo Journal of Biological Chemistry vol 283 no 23pp 15539ndash15543 2008

[30] A Negre-Salvayre C Coatrieux C Ingueneau and R SalvayreldquoAdvanced lipid peroxidation end products in oxidative damageto proteins Potential role in diseases and therapeutic prospectsfor the inhibitorsrdquo British Journal of Pharmacology vol 153 no1 pp 6ndash20 2008

[31] M P Mattson ldquoRoles of the lipid peroxidation product 4-hydroxynonenal in obesity the metabolic syndrome and asso-ciated vascular and neurodegenerative disordersrdquo ExperimentalGerontology vol 44 no 10 pp 625ndash633 2009

[32] A Ayala M F Munoz and S Arguelles ldquoLipid peroxidationproduction metabolism and signaling mechanisms of malon-dialdehyde and 4-hydroxy-2-nonenalrdquo Oxidative Medicine andCellular Longevity vol 2014 Article ID 360438 31 pages 2014

[33] K Sterling ldquoThe turnover rate of serum albumin in manas measured by I131-tagged albuminrdquo The Journal of ClinicalInvestigation vol 30 no 11 pp 1228ndash1237 1951

[34] C J Lindsell R S Franco E P Smith C H Joiner and R MCohen ldquoA method for the continuous calculation of the age oflabeled red blood cellsrdquo American Journal of Hematology vol83 no 6 pp 454ndash457 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Oxidative Medicine and Cellular Longevity

Table 1 Demographic data of the subjects

Demographic data n Sex

Male 5 455Female 6 545

Age in years (119909 = 32)20ndash30 4 36431ndash40 6 545gt40 1 91

OccupationEmployee 2 182Government officer 1 91University worker 6 545Others 2 182

Alcohol consumptionStopped drinking for gt3mo 2 182Drinking less than oncemo 7 636Never 2 182

SmokingNever 11 1000

Herbvitamin supplementConsuming 1 91Never 10 909

demographic data of the subjects are shown in Table 1 Theplasma electrolyte levels lipid profiles liver function enzymelevels thyroid hormone levels coagulograms and completeblood counts were measured from whole blood samples ofall subjects at 0 1 4 12 16 and 24 weeks after begin-ning oral administration of the polar fraction of ME Theresults showed no significant alterations in these parameters(Table 2) Additionally the side effects of oral administrationof the polar fraction of ME were determined throughout thestudy All subjects reported minor and tolerable side effectsas shown in Table 3

32 Separation of ME by TLC and Screening of AntioxidantActivity TLC chromatogram of the polar fraction of MEvisualized under UV light at 254 nm revealed a remarkableband that had 119877

119891value of approximately 075 (band A)

After spraying with 03mM DPPH solution several yellowspots which indicated antioxidant properties were observed(Figure 1)

33 In Vitro Antioxidant Assay of ME Quantitative analysisof antioxidant activity of the polar fraction of ME wasperformedon the extract of bandAbyDPPHassay (Figure 2)Antioxidant activity was shown in dose-dependent mannerThe IC

50values of band A extract and vitamin C (positive

control) were 1603 plusmn 144 120583gmL and 516 plusmn 092 120583gmLrespectively

34 Antioxidant Capacity of RBCs The antioxidant capacityof RBCs was determined by measuring the fluorescent signalof DCF from the RBCs after incubation with hydrogen

Band A

(a) (b)

Figure 1 Separation ofME by TLC and antioxidant properties of itsconstituents (a)MEwas separated by TLC and visualized under UVat 254 nmMajor compounds appeared in a single band at119877

119891

= 075

(band A) (b) Screening of antioxidant activity was done by applyingDPPHonto theTLCplate Scavenging ofDPPHwas shown in yellowstaining indicating antioxidative activity of ME

ME-band AVitamin C

0

25

50

75

100

125

sc

aven

ging

75 10025 500Concentration (120583gmL)

Figure 2 Quantitative analysis of antioxidant activity ofME DPPHassay was performed on the extract of band A (ME-band A) fromTLC Vitamin C was used as a positive control for comparisonAntioxidant activities ( scavenging) of both ME and vitaminC calculated as described in Materials and Methods were dose-dependent The IC

50

values of ME and vitamin C were 1603 plusmn144 120583gmL and 516 plusmn 092 120583gmL respectively Error bars representSEM

peroxide which was used as an exogenous ROS The RBCsfrom the subjects taking the polar fraction of ME exhibited asignificant decrease in the mean fluorescent intensity of DCFstarting in week 12 of oral administration of ME (Figure 3)This suggests that ME increases the antioxidant capacity of

Oxidative Medicine and Cellular Longevity 5

Table 2 Subject data and biochemical parameters for assessment of safety of oral administration of ME Blood and urine samples werecollected from the subjects in 0 1 4 12 16 and 24 weeks after beginning oral administration of ME The biochemical parameters weremeasured as tabulated Body mass index and blood pressure were also determined All parameters were collected and calculated from 11healthy individuals

Test Subject data and laboratory safety assessment mean (SEM)Day 0 1 week 4 weeks 12 weeks 16 weeks 24 weeks

Glucose(mgdL) 8673 (164) 8500 (167) 8309 (145) 8145 (304) 8091 (146) 8500 (253)

Cholesterol(mgdL) 20654 (1310) 19600 (1295) 20109 (1432) 19345 (1034) 19063 (1361) 19245 (2474)

Triglycerides(mgdL) 7800 (1115) 7718 (1958) 8127 (2011) 8718 (1727) 10354 (3080) 9227 (2265)

HDL-CHOL(mgdL) 6290 (294) 6045 (252) 5945 (213) 5972 (208) 5781 (226) 5945 (195)

LDL-calculated(mgdL) 12803 (1200) 12010 (1062) 12538 (1234) 11629 (895) 11210 (913) 11454 (1080)

BUN(mgdL) 1284 (163) 1126 (058) 1243 (103) 1182 (082) 1223 (088) 1217 (118)

Creatinine(mgdL) 086 (006) 084 (004) 081 (005) 082 (004) 082 (004) 081 (004)

Sodium(mmolL) 14127 (057) 14154 (089) 14136 (051) 14009 (047) 14109 (041) 14018 (038)

Potassium(mmolL) 375 (006) 372 (009) 375 (007) 358 (008) 356 (006) 363 (008)

Chloride(mmolL) 10279 (045) 10308 (056) 10250 (050) 10179 (071) 10323 (055) 10293 (036)

Bicarbonate(mmolL) 2814 (085) 2492 (070) 2650 (104) 2543 (070) 2485 (101) 2414 (071)

Total bilirubin(mgdL) 063 (014) 060 (011) 049 (008) 059 (011) 060 (014) 057 (010)

Direct bilirubin(mgdL) 021 (004) 023 (003) 020 (002) 024 (004) 024 (004) 022 (003)

AST (SGOT)(UL) 2081 (189) 1945 (207) 3054 (804) 1800 (180) 1909 (146) 1763 (283)

ALT (SGPT)(UL) 1918 (412) 1700 (368) 3127 (1040) 1845 (454) 1563 (261) 2045 (771)

ALP(UL) 6072 (268) 5927 (314) 6363 (317) 6245 (205) 6409 (374) 6254 (314)

T3(ngdL) 9942 (327) 10462 (353) 9745 (316) 9735 (382) 9926 (633) 9262 (372)

TSH(120583IUmL) 215 (032) 201 (032) 209 (042) 203 (036) 207 (028) 216 (030)

FT4(ngdL) 132 (005) 128 (005) 126 (005) 122 (002) 118 (003) 122 (004)

Hemoglobin(gdL) 1360 (031) 1325 (039) 1345 (031) 1355 (037) 1350 (040) 1350 (043)

Hematocrit() 4219 (100) 4091 (104) 4132 (105) 4143 (110) 4051 (125) 4064 (121)

WBC count(times103120583L)

597 (036) 577 (033) 594 (026) 644 (032) 570 (037) 543 (029)

Platelet count(times103120583L) 24627 (1314) 24045 (1430) 25536 (1413) 24354 (1265) 23827 (1220) 23972 (1064)

Absoluteneutrophils(times103120583L)

309 (023) 319 (026) 327 (014) 388 (044) 312 (028) 284 (020)

6 Oxidative Medicine and Cellular Longevity

Table 2 Continued

Test Subject data and laboratory safety assessment mean (SEM)Day 0 1 week 4 weeks 12 weeks 16 weeks 24 weeks

PT(sec) 1214 (021) 1208 (021) 1199 (024) 1223 (019) 1248 (027) 1262 (022)

aPTT(sec) 2853 (060) 2819 (076) 2861 (069) 2897 (061) 2920 (081) 2970 (074)

Urine protein(Negndashtrace) 545 727 727 818 727 545

Body mass index 2174 (112) 2186 (112) 2164 (107) 2207 (114) 2191 (112) 2189 (113)Arterial bloodpressure(mmHg)

11772 11776 11777 11973 11872 11373

HDL-CHOL high-density lipoprotein cholesterol LDL low-density lipoprotein BUN blood urea nitrogen AST (SGOT) aspartate aminotransferase(serum glutamic oxaloacetic transaminase) ALT (SGPT) alanine aminotransferase (serum glutamic pyruvic transaminase) ALP alkaline phosphataseT3 triiodothyronine TSH thyroid-stimulating hormone FT4 free thyroxine WBC white blood cell PT prothrombin time aPTT activated partialthromboplastin time

Table 3 Side effects of oral administration ofMEThe subjects wereasked to report the side effects after oral administration of ME ateach visit The number of episodes of each side effect prior to andafter double doses was collected and calculated as the percentage ofthe total episodes of 110 (11 participants times 10 visits)

SymptomBefore (single dose) After (double doses)Number ofepisodes Number of

episodes

Tirednesssleepiness 4 36 0 00Constipation 4 36 1 09Dry throat 2 18 3 27Headache 1 09 4 36Flu-like symptoms 2 18 0 00Abnormal stool 4 36 0 00Abdominaldiscomfort 3 27 0 00

Cough 2 18 0 00Weight loss 2 18 2 18Nasal congestion 2 18 0 00Abdominal bloating 2 18 1 09Dizziness 1 09 0 00Salivation 1 09 0 00Nauseavomiting 1 09 1 09Diarrhea 1 09 1 09Skin rash 1 09 0 00Abnormal urination 1 09 0 00Malaise 1 09 0 00

RBCsThis effect was further accentuated through the end ofthe study

35 Oxidative Damage to Proteins in RBCs and Whole BloodOral administration of ME was associated with a significantdecrease in oxidative damage to proteins in both RBCs andwhole blood Quantification of oxidative damage to these

proteins was achieved by normalizing HNE staining to actinAs shown in Figure 4 a significant decrease in proteinoxidative damage appeared inweeks 4 and 24 for whole bloodand RBCs respectively

4 Discussion

Several compounds in the extract frommangosteen pericarphave recently been identified and shown to have beneficialeffects on human health However the cytotoxic activitieswere always observed in nonpolar fraction of the extract thatis 120572-mangostin whereas the potent antioxidant was foundin polar fraction In addition the safety of the polar fractionhas never been thoroughly investigated in humans In thisstudy we investigated the safety of oral administration of thepolar fraction of ME inThai subjects For the entire 24 weeksof the study only minor side effects were reported Thesecan be categorized into gastrointestinal skin respiratory andgeneral side effects The most frequently reported side effectswere related to the gastrointestinal systemWe suspected thatsuch side effects might be associated with an antibacterialproperty of the compounds from the mangosteen pericarpwhich may have led to the perturbation of gut microbesNonetheless these side effects accounted for only a fewepisodes and were reportedly tolerable by all subjects

The present study also showed that oral administra-tion of the polar fraction of ME poses no threat to vitalorgans including the liver kidney and thyroid gland Thesubjectsrsquo liver enzyme levels lipid profiles coagulogramparameters blood urea nitrogen level creatinine level urineprotein level plasma electrolyte levels and thyroid hormonelevels remained normal for the entire 24 weeks of thestudy Additionally all subjectsrsquo complete blood counts andblood pressures remained unchanged Although two subjectsshowed slightly increased liver enzymes in the fourth weekof the study the enzyme levels spontaneously returned tothe normal range thereafter Notably a study by Stern et al[27] revealed that obese subjects with a body mass index of30 to 40 kgm2 exhibited a significant net reduction in body

Oxidative Medicine and Cellular Longevity 7

0

1000

FSC

SSC

0

1000

SSC

0

1000

SSC

0

0

1000

1000

FSC0 1000

FSC0 1000

FSC0 1000

SSC

DCF

Cou

nts

100 101 102 103 104

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

DCF100 101 102 103 104

0

100

Cou

nts

0

100

0

1000

SSC

0

1000

SSC

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

0

1000

SSC

0

1000

SSC

0

1000

SSC

0

1000

SSC

FSC

0 1000

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

0

100

100

0

100

Stim

ulat

edSt

imul

ated

0

1000

FSC

SSC

0

0

1000

1000

FSC0 1000

SSC

DCF

DCF

Cou

nts

100 101 102 103 104

100 101 102 103 104

Cou

nts

W12 W16 W24

W0 W1 W44 + H(0)001

4 + H(20)001

4 + H(0)001

4 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001 M012 + H(0)001

M012 + H(20)001 M012 + H(20)001

M012 + H(0)001 M012 + H(0)001

M012 + H(20)001 M012 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001M012 + H(0)001 M012 + H(0)001

M012 + H(20)001M012 + H(20)001 M012 + H(20)001

0min

Stim

ulat

ed0min

20min

Stim

ulat

ed20min

(a)

W1

W4

W12

W16W

0

W24

Weeks of oral administration of ME

Mea

n flu

ores

cenc

e int

ensit

y

lowastlowast

lowast

40

30

20

10

50

(b)

Figure 3 Effect of oral administration of ME on antioxidant capacity of RBCs RBCs were isolated from the peripheral blood of the subjectsat 0 1 4 12 16 and 24 weeks after beginning oral administration of ME Hydrogen peroxide was an exogenous ROS used to stimulate theresponse of the RBC antioxidant system (a) A representative measurement of the level of ROS within the RBCs which was determined by themean fluorescent signal of DCF emitted from hydrogen peroxide-incubated RBCs (b) Quantitative analysis of the mean fluorescent signalof DCF from all samples during the entire period of the study Data are presented as means (119899 = 11) Error bars represent SEMThe asteriskindicates statistical significance 119901 lt 005 in comparison with week 0 (119905-test)

weight (374 kg 119901 lt 00001) after taking capsules containingSphaeranthus indicus and G mangostana for 8 weeks com-pared with subjects taking placebo The discrepancy in thesubjectsrsquo body weights between that study and the presentstudy might derive from the volunteers in our study havinga normal BMI or from the weight-loss property of S indicus

We performedTLC onME in order to test for preparationconsistency and to demonstrate its essential compounds Itwas shown that ME was composed of at least one groupof major compounds which consistently appeared as asingle band on the chromatogram at 119877

119891= 075 Such a

high 119877119891suggested highly organic-volatile property We are

8 Oxidative Medicine and Cellular Longevity

Red blood cell Whole blood

W0

W1

W4

W12

W16

W24

Actin HNE Actin HNE

(a)

05

10

HNE-WBHNE-RBC

W1

W4

W12

W16W

0

W24

Weeks of oral administration of ME

Leve

l of H

NE

mod

ifica

tion

(rel

ativ

e to

W0) lowast

lowast

lowast

lowastlowast

(b)

Figure 4 Oral administration of ME resulted in decreased protein oxidative damage in RBCs and whole blood The RBCs and whole bloodof the subjects were collected at 0 1 4 12 16 and 24 weeks after beginning oral administration of ME (a) A representative blot showsHNE staining using a western blotting technique to determine the protein oxidative damage in RBCs and whole blood Actin was used asa loading control (b) Quantitative analysis of the extent of HNE modification to RBCs (HNE-RBC) and whole blood (HNE-WB) after oraladministration of ME was carried out as that relative to week 0 Data are presented as means (119899 = 11) Error bars represent SEMThe asteriskindicates statistical significance 119901 lt 005 in comparison with week 0 (119905-test)

currently identifying the chemical compounds in this band(data not shown) Further in vitro analysis of these majorcompounds revealed that they exhibited approximately one-third of antioxidant activity of vitamin C Therefore ME islikely to possess considerably high antioxidant activity

Antioxidant property of the polar fraction of MEobserved in our in vitro experiments was shown to exertbiological impactThe 24-week period of oral administrationof the polar fraction of ME in the present study resulted ina significant increase in the antioxidant capacity of RBCswhich was determined by the fluorescent signal from DCFemitted from hydrogen peroxide-incubated RBCs A signifi-cant increase in the antioxidant capacity (ie decreased DCFsignal) of RBCs could be detected from week 12 of oraladministration of the polar fraction of ME Therefore ourresults suggest for the first time that extract frommangosteenpericarp can augment the antioxidant system within humanRBCs probably due to its ability to neutralize ROS

We further investigated whether and how such anincreased antioxidant capacity affects protein oxidative dam-age in RBCs and whole blood Protein oxidative damagewas determined by the degree of HNE modification HNEis a byproduct of lipid peroxidation which is an indicatorof oxidative stress HNE can react with lysine histidine orcysteine residue in proteins to form adducts resulting instructural and functional changes to the proteins [28 29]HNE is also reportedly involved in the pathophysiology ofmany diseases [30ndash32] We speculated that if RBCs havean improved antioxidant capacity lipid peroxidation shouldbe reduced and HNE modification to proteins should thus

decrease Consistent with our speculation oral administra-tion of the polar fraction of ME led to a significant decreasein HNE modification in whole blood and RBCs starting inweeks 4 and 24 respectively Notably the significant decreasein HNE modification of whole blood proteins appearedearlier than that of RBC proteins This may be explained bythe faster turnover rate of albumin [33] the most abundantprotein in serum in comparison to the lifespan of RBCs[34] This result provides further supporting evidence inhuman subjects of the antioxidant property of extract frommangosteen pericarp

5 Conclusions

We have demonstrated for the first time in human subjectsthat the polar fraction of extract from mangosteen pericarpis adequately safe for oral administration for up to 24 weeksMajor components of the polar fraction of extract frommangosteen pericarp exhibited considerably high antioxidantactivity according to the in vitro antioxidant assay Addi-tionally the polar extract from mangosteen pericarp appearsto significantly increase the antioxidant capacity of RBCsand significantly decrease protein oxidative damage in RBCsand whole blood It might alleviate oxidative damage inother tissues as well Our results suggest that the antioxidantcapacity of the polar fraction of mangosteen pericarp extractcould come from its ability to neutralize ROS Howeverwhether some compounds in this extract can also upregulateantioxidant enzyme is still unknown It would be interestingto further quantitate the expression level of antioxidant

Oxidative Medicine and Cellular Longevity 9

enzymes In all the polar fraction of extract frommangosteenpericarp might benefit human patients with pathologicalconditions related to oxidative stress

Competing Interests

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

Acknowledgments

The authors are extremely grateful to Mrs Angkana Jong-sawaddipatana and all staff members of the Geriatric Clinicof Siriraj Hospital for their kind collaboration in recruit-ment of the participants This work was supported by theThai Traditional Medical Knowledge Fund Department forDevelopment of Thai Traditional and Alternative MedicineMinistry of Public Health Thailand

References

[1] S M Petiwala G Li A Ramaiya et al ldquoPharmacokineticcharacterization of mangosteen (Garcinia mangostana) fruitextract standardized to 120572-mangostin in C57BL6 micerdquo Nutri-tion Research vol 34 no 4 pp 336ndash345 2014

[2] Z Xu L Huang X-H Chen et al ldquoCytotoxic prenylated xan-thones from the pericarps of Garcinia mangostanardquo Moleculesvol 19 no 2 pp 1820ndash1827 2014

[3] F Gutierrez-Orozco C Chitchumroonchokchai G B Lesin-ski S Suksamrarn and M L Failla ldquo120572-Mangostin anti-inflammatory activity and metabolism by human cellsrdquo Journalof Agricultural and Food Chemistry vol 61 no 16 pp 3891ndash3900 2013

[4] H-Y Jang O-K Kwon S-R Oh H-K Lee K-S Ahn and Y-W Chin ldquoMangosteen xanthones mitigate ovalbumin-inducedairway inflammation in a mouse model of asthmardquo Food andChemical Toxicology vol 50 no 11 pp 4042ndash4050 2012

[5] S Syam A Bustamam R Abdullah et al ldquo120573 Mangostinsuppress LPS-induced inflammatory response in RAW 2647macrophages in vitro and carrageenan-induced peritonitis invivordquo Journal of Ethnopharmacology vol 153 no 2 pp 435ndash4452014

[6] J Yuan Y Wu and G Lu ldquoA-mangostin suppresseslipopolysaccharide-induced invasion by inhibiting matrixmetalloproteinase-29 and increasing E-cadherin expressionthrough extracellular signal-regulated kinase signaling inpancreatic cancer cellsrdquo Oncology Letters vol 5 no 6 pp1958ndash1964 2013

[7] M-A Shibata M Iinuma J Morimoto et al ldquo120572-Mangostinextracted from the pericarp of the mangosteen (Garcinia man-gostana Linn) reduces tumor growth and lymph node metas-tasis in an immunocompetent xenograft model of metastaticmammary cancer carrying a p53mutationrdquo BMCMedicine vol9 article 69 2011

[8] A F A Aisha K M Abu-Salah Z Ismail and A MajidldquoIn vitro and in vivo anti-colon cancer effects of Garciniamangostana xanthones extractrdquo BMC Complementary andAlternative Medicine vol 12 article 104 2012

[9] S Taokaew N Nunkaew P Siripong and M PhisalaphongldquoCharacteristics and anticancer properties of bacterial cellulose

films containing ethanolic extract of mangosteen peelrdquo Journalof Biomaterials Science Polymer Edition vol 25 no 9 pp 907ndash922 2014

[10] P Pan-In S Wanichwecharungruang J Hanes and A J KimldquoCellular trafficking and anticancer activity of Garcinia man-gostana extract-encapsulated polymeric nanoparticlesrdquo Inter-national Journal of Nanomedicine vol 9 no 1 pp 3677ndash36862014

[11] H R W Dharmaratne Y Sakagami K G P Piyasena and VThevanesam ldquoAntibacterial activity of xanthones fromGarciniamangostana (L) and their structure-activity relationship stud-iesrdquo Natural Product Research vol 27 no 10 pp 938ndash941 2013

[12] J-J Koh S Qiu H Zou et al ldquoRapid bactericidal action ofalpha-mangostin against MRSA as an outcome of membranetargetingrdquo Biochimica et Biophysica ActamdashBiomembranes vol1828 no 2 pp 834ndash844 2013

[13] M T Chomnawang S Surassmo K Wongsariya and N Bun-yapraphatsara ldquoAntibacterial activity of Thai medicinal plantsagainstmethicillin-resistant Staphylococcus aureusrdquo Fitoterapiavol 80 no 2 pp 102ndash104 2009

[14] S Suksamrarn N SuwannapochW Phakhodee et al ldquoAntimy-cobacterial activity of prenylated xanthones from the fruits ofGarcinia mangostanardquo Chemical and Pharmaceutical Bulletinvol 51 no 7 pp 857ndash859 2003

[15] P Moongkarndi C Srisawat P Saetun et al ldquoProtective effectof mangosteen extract against 120573-amyloid-induced cytotoxicityoxidative stress and altered proteome in SK-N-SH cellsrdquo Journalof Proteome Research vol 9 no 5 pp 2076ndash2086 2010

[16] Y-W Chin H-A Jung H Chai W J Keller and A DKinghorn ldquoXanthones with quinone reductase-inducing activ-ity from the fruits of Garcinia mangostana (Mangosteen)rdquoPhytochemistry vol 69 no 3 pp 754ndash758 2008

[17] H-A Jung B-N Su W J Keller R G Mehta and AD Kinghorn ldquoAntioxidant xanthones from the pericarp ofGarcinia mangostana (Mangosteen)rdquo Journal of Agriculturaland Food Chemistry vol 54 no 6 pp 2077ndash2082 2006

[18] F Gutierrez-Orozco and M L Failla ldquoBiological activities andbioavailability of mangosteen xanthones a critical review of thecurrent evidencerdquo Nutrients vol 5 no 8 pp 3163ndash3183 2013

[19] J Sattayasai P Chaonapan T Arkaravichie et al ldquoProtectiveeffects of mangosteen extract on H

2

O2

-induced cytotoxicity inSK-N-SH cells and scopolamine-induced memory impairmentin micerdquo PLoS ONE vol 8 no 12 Article ID e85053 2013

[20] E B Walker ldquoHPLC analysis of selected xanthones in mangos-teen fruitrdquo Journal of Separation Science vol 30 no 9 pp 1229ndash1234 2007

[21] F Gutierrez-Orozco J MThomas-Ahner L D Berman-Bootyet al ldquoDietary 120572-mangostin a xanthone from mangosteenfruit exacerbates experimental colitis and promotes dysbiosisin micerdquoMolecular Nutrition and Food Research vol 58 no 6pp 1226ndash1238 2014

[22] M Y IbrahimNMHashim A AMariod et al ldquo120572-Mangostinfrom Garcinia mangostana Linn an updated review of itspharmacological propertiesrdquo Arabian Journal of Chemistry vol9 no 3 pp 317ndash329 2016

[23] M Y Ibrahim N M Hashim S Mohan et al ldquo120572-Mangostinfrom Cratoxylum arborescens an in vitro and in vivo toxicolog-ical evaluationrdquo Arabian Journal of Chemistry vol 8 no 1 pp129ndash137 2015

[24] A Plazonic F Bucar Z Males A Mornar B Nigovic andN Kujundzic ldquoIdentification and quantification of flavonoids

10 Oxidative Medicine and Cellular Longevity

and phenolic acids in burr parsley (Caucalis platycarpos L)using high-performance liquid chromatography with diodearray detection and electrospray ionizationmass spectrometryrdquoMolecules vol 14 no 7 pp 2466ndash2490 2009

[25] P Moongkarndi N Jaisupa J Samer et al ldquoComparison of thebiological activity of two different isolates from mangosteenrdquoJournal of Pharmacy and Pharmacology vol 66 no 8 pp 1171ndash1179 2014

[26] E Fibach ldquoTechniques for studying stimulation of fetal hemo-globin production in human erythroid culturesrdquo Hemoglobinvol 22 no 5-6 pp 445ndash458 1998

[27] J S Stern J Peerson A T Mishra M V Sadasiva Rao andK P Rajeswari ldquoEfficacy and tolerability of a novel herbalformulation for weight managementrdquo Obesity vol 21 no 5 pp921ndash927 2013

[28] C M Spickett ldquoThe lipid peroxidation product 4-hydroxy-2-nonenal advances in chemistry and analysisrdquo Redox Biologyvol 1 no 1 pp 145ndash152 2013

[29] C Schneider N A Porter and A R Brash ldquoRoutes to 4-hydroxynonenal fundamental issues in themechanisms of lipidperoxidationrdquo Journal of Biological Chemistry vol 283 no 23pp 15539ndash15543 2008

[30] A Negre-Salvayre C Coatrieux C Ingueneau and R SalvayreldquoAdvanced lipid peroxidation end products in oxidative damageto proteins Potential role in diseases and therapeutic prospectsfor the inhibitorsrdquo British Journal of Pharmacology vol 153 no1 pp 6ndash20 2008

[31] M P Mattson ldquoRoles of the lipid peroxidation product 4-hydroxynonenal in obesity the metabolic syndrome and asso-ciated vascular and neurodegenerative disordersrdquo ExperimentalGerontology vol 44 no 10 pp 625ndash633 2009

[32] A Ayala M F Munoz and S Arguelles ldquoLipid peroxidationproduction metabolism and signaling mechanisms of malon-dialdehyde and 4-hydroxy-2-nonenalrdquo Oxidative Medicine andCellular Longevity vol 2014 Article ID 360438 31 pages 2014

[33] K Sterling ldquoThe turnover rate of serum albumin in manas measured by I131-tagged albuminrdquo The Journal of ClinicalInvestigation vol 30 no 11 pp 1228ndash1237 1951

[34] C J Lindsell R S Franco E P Smith C H Joiner and R MCohen ldquoA method for the continuous calculation of the age oflabeled red blood cellsrdquo American Journal of Hematology vol83 no 6 pp 454ndash457 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 5

Table 2 Subject data and biochemical parameters for assessment of safety of oral administration of ME Blood and urine samples werecollected from the subjects in 0 1 4 12 16 and 24 weeks after beginning oral administration of ME The biochemical parameters weremeasured as tabulated Body mass index and blood pressure were also determined All parameters were collected and calculated from 11healthy individuals

Test Subject data and laboratory safety assessment mean (SEM)Day 0 1 week 4 weeks 12 weeks 16 weeks 24 weeks

Glucose(mgdL) 8673 (164) 8500 (167) 8309 (145) 8145 (304) 8091 (146) 8500 (253)

Cholesterol(mgdL) 20654 (1310) 19600 (1295) 20109 (1432) 19345 (1034) 19063 (1361) 19245 (2474)

Triglycerides(mgdL) 7800 (1115) 7718 (1958) 8127 (2011) 8718 (1727) 10354 (3080) 9227 (2265)

HDL-CHOL(mgdL) 6290 (294) 6045 (252) 5945 (213) 5972 (208) 5781 (226) 5945 (195)

LDL-calculated(mgdL) 12803 (1200) 12010 (1062) 12538 (1234) 11629 (895) 11210 (913) 11454 (1080)

BUN(mgdL) 1284 (163) 1126 (058) 1243 (103) 1182 (082) 1223 (088) 1217 (118)

Creatinine(mgdL) 086 (006) 084 (004) 081 (005) 082 (004) 082 (004) 081 (004)

Sodium(mmolL) 14127 (057) 14154 (089) 14136 (051) 14009 (047) 14109 (041) 14018 (038)

Potassium(mmolL) 375 (006) 372 (009) 375 (007) 358 (008) 356 (006) 363 (008)

Chloride(mmolL) 10279 (045) 10308 (056) 10250 (050) 10179 (071) 10323 (055) 10293 (036)

Bicarbonate(mmolL) 2814 (085) 2492 (070) 2650 (104) 2543 (070) 2485 (101) 2414 (071)

Total bilirubin(mgdL) 063 (014) 060 (011) 049 (008) 059 (011) 060 (014) 057 (010)

Direct bilirubin(mgdL) 021 (004) 023 (003) 020 (002) 024 (004) 024 (004) 022 (003)

AST (SGOT)(UL) 2081 (189) 1945 (207) 3054 (804) 1800 (180) 1909 (146) 1763 (283)

ALT (SGPT)(UL) 1918 (412) 1700 (368) 3127 (1040) 1845 (454) 1563 (261) 2045 (771)

ALP(UL) 6072 (268) 5927 (314) 6363 (317) 6245 (205) 6409 (374) 6254 (314)

T3(ngdL) 9942 (327) 10462 (353) 9745 (316) 9735 (382) 9926 (633) 9262 (372)

TSH(120583IUmL) 215 (032) 201 (032) 209 (042) 203 (036) 207 (028) 216 (030)

FT4(ngdL) 132 (005) 128 (005) 126 (005) 122 (002) 118 (003) 122 (004)

Hemoglobin(gdL) 1360 (031) 1325 (039) 1345 (031) 1355 (037) 1350 (040) 1350 (043)

Hematocrit() 4219 (100) 4091 (104) 4132 (105) 4143 (110) 4051 (125) 4064 (121)

WBC count(times103120583L)

597 (036) 577 (033) 594 (026) 644 (032) 570 (037) 543 (029)

Platelet count(times103120583L) 24627 (1314) 24045 (1430) 25536 (1413) 24354 (1265) 23827 (1220) 23972 (1064)

Absoluteneutrophils(times103120583L)

309 (023) 319 (026) 327 (014) 388 (044) 312 (028) 284 (020)

6 Oxidative Medicine and Cellular Longevity

Table 2 Continued

Test Subject data and laboratory safety assessment mean (SEM)Day 0 1 week 4 weeks 12 weeks 16 weeks 24 weeks

PT(sec) 1214 (021) 1208 (021) 1199 (024) 1223 (019) 1248 (027) 1262 (022)

aPTT(sec) 2853 (060) 2819 (076) 2861 (069) 2897 (061) 2920 (081) 2970 (074)

Urine protein(Negndashtrace) 545 727 727 818 727 545

Body mass index 2174 (112) 2186 (112) 2164 (107) 2207 (114) 2191 (112) 2189 (113)Arterial bloodpressure(mmHg)

11772 11776 11777 11973 11872 11373

HDL-CHOL high-density lipoprotein cholesterol LDL low-density lipoprotein BUN blood urea nitrogen AST (SGOT) aspartate aminotransferase(serum glutamic oxaloacetic transaminase) ALT (SGPT) alanine aminotransferase (serum glutamic pyruvic transaminase) ALP alkaline phosphataseT3 triiodothyronine TSH thyroid-stimulating hormone FT4 free thyroxine WBC white blood cell PT prothrombin time aPTT activated partialthromboplastin time

Table 3 Side effects of oral administration ofMEThe subjects wereasked to report the side effects after oral administration of ME ateach visit The number of episodes of each side effect prior to andafter double doses was collected and calculated as the percentage ofthe total episodes of 110 (11 participants times 10 visits)

SymptomBefore (single dose) After (double doses)Number ofepisodes Number of

episodes

Tirednesssleepiness 4 36 0 00Constipation 4 36 1 09Dry throat 2 18 3 27Headache 1 09 4 36Flu-like symptoms 2 18 0 00Abnormal stool 4 36 0 00Abdominaldiscomfort 3 27 0 00

Cough 2 18 0 00Weight loss 2 18 2 18Nasal congestion 2 18 0 00Abdominal bloating 2 18 1 09Dizziness 1 09 0 00Salivation 1 09 0 00Nauseavomiting 1 09 1 09Diarrhea 1 09 1 09Skin rash 1 09 0 00Abnormal urination 1 09 0 00Malaise 1 09 0 00

RBCsThis effect was further accentuated through the end ofthe study

35 Oxidative Damage to Proteins in RBCs and Whole BloodOral administration of ME was associated with a significantdecrease in oxidative damage to proteins in both RBCs andwhole blood Quantification of oxidative damage to these

proteins was achieved by normalizing HNE staining to actinAs shown in Figure 4 a significant decrease in proteinoxidative damage appeared inweeks 4 and 24 for whole bloodand RBCs respectively

4 Discussion

Several compounds in the extract frommangosteen pericarphave recently been identified and shown to have beneficialeffects on human health However the cytotoxic activitieswere always observed in nonpolar fraction of the extract thatis 120572-mangostin whereas the potent antioxidant was foundin polar fraction In addition the safety of the polar fractionhas never been thoroughly investigated in humans In thisstudy we investigated the safety of oral administration of thepolar fraction of ME inThai subjects For the entire 24 weeksof the study only minor side effects were reported Thesecan be categorized into gastrointestinal skin respiratory andgeneral side effects The most frequently reported side effectswere related to the gastrointestinal systemWe suspected thatsuch side effects might be associated with an antibacterialproperty of the compounds from the mangosteen pericarpwhich may have led to the perturbation of gut microbesNonetheless these side effects accounted for only a fewepisodes and were reportedly tolerable by all subjects

The present study also showed that oral administra-tion of the polar fraction of ME poses no threat to vitalorgans including the liver kidney and thyroid gland Thesubjectsrsquo liver enzyme levels lipid profiles coagulogramparameters blood urea nitrogen level creatinine level urineprotein level plasma electrolyte levels and thyroid hormonelevels remained normal for the entire 24 weeks of thestudy Additionally all subjectsrsquo complete blood counts andblood pressures remained unchanged Although two subjectsshowed slightly increased liver enzymes in the fourth weekof the study the enzyme levels spontaneously returned tothe normal range thereafter Notably a study by Stern et al[27] revealed that obese subjects with a body mass index of30 to 40 kgm2 exhibited a significant net reduction in body

Oxidative Medicine and Cellular Longevity 7

0

1000

FSC

SSC

0

1000

SSC

0

1000

SSC

0

0

1000

1000

FSC0 1000

FSC0 1000

FSC0 1000

SSC

DCF

Cou

nts

100 101 102 103 104

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

DCF100 101 102 103 104

0

100

Cou

nts

0

100

0

1000

SSC

0

1000

SSC

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

0

1000

SSC

0

1000

SSC

0

1000

SSC

0

1000

SSC

FSC

0 1000

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

0

100

100

0

100

Stim

ulat

edSt

imul

ated

0

1000

FSC

SSC

0

0

1000

1000

FSC0 1000

SSC

DCF

DCF

Cou

nts

100 101 102 103 104

100 101 102 103 104

Cou

nts

W12 W16 W24

W0 W1 W44 + H(0)001

4 + H(20)001

4 + H(0)001

4 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001 M012 + H(0)001

M012 + H(20)001 M012 + H(20)001

M012 + H(0)001 M012 + H(0)001

M012 + H(20)001 M012 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001M012 + H(0)001 M012 + H(0)001

M012 + H(20)001M012 + H(20)001 M012 + H(20)001

0min

Stim

ulat

ed0min

20min

Stim

ulat

ed20min

(a)

W1

W4

W12

W16W

0

W24

Weeks of oral administration of ME

Mea

n flu

ores

cenc

e int

ensit

y

lowastlowast

lowast

40

30

20

10

50

(b)

Figure 3 Effect of oral administration of ME on antioxidant capacity of RBCs RBCs were isolated from the peripheral blood of the subjectsat 0 1 4 12 16 and 24 weeks after beginning oral administration of ME Hydrogen peroxide was an exogenous ROS used to stimulate theresponse of the RBC antioxidant system (a) A representative measurement of the level of ROS within the RBCs which was determined by themean fluorescent signal of DCF emitted from hydrogen peroxide-incubated RBCs (b) Quantitative analysis of the mean fluorescent signalof DCF from all samples during the entire period of the study Data are presented as means (119899 = 11) Error bars represent SEMThe asteriskindicates statistical significance 119901 lt 005 in comparison with week 0 (119905-test)

weight (374 kg 119901 lt 00001) after taking capsules containingSphaeranthus indicus and G mangostana for 8 weeks com-pared with subjects taking placebo The discrepancy in thesubjectsrsquo body weights between that study and the presentstudy might derive from the volunteers in our study havinga normal BMI or from the weight-loss property of S indicus

We performedTLC onME in order to test for preparationconsistency and to demonstrate its essential compounds Itwas shown that ME was composed of at least one groupof major compounds which consistently appeared as asingle band on the chromatogram at 119877

119891= 075 Such a

high 119877119891suggested highly organic-volatile property We are

8 Oxidative Medicine and Cellular Longevity

Red blood cell Whole blood

W0

W1

W4

W12

W16

W24

Actin HNE Actin HNE

(a)

05

10

HNE-WBHNE-RBC

W1

W4

W12

W16W

0

W24

Weeks of oral administration of ME

Leve

l of H

NE

mod

ifica

tion

(rel

ativ

e to

W0) lowast

lowast

lowast

lowastlowast

(b)

Figure 4 Oral administration of ME resulted in decreased protein oxidative damage in RBCs and whole blood The RBCs and whole bloodof the subjects were collected at 0 1 4 12 16 and 24 weeks after beginning oral administration of ME (a) A representative blot showsHNE staining using a western blotting technique to determine the protein oxidative damage in RBCs and whole blood Actin was used asa loading control (b) Quantitative analysis of the extent of HNE modification to RBCs (HNE-RBC) and whole blood (HNE-WB) after oraladministration of ME was carried out as that relative to week 0 Data are presented as means (119899 = 11) Error bars represent SEMThe asteriskindicates statistical significance 119901 lt 005 in comparison with week 0 (119905-test)

currently identifying the chemical compounds in this band(data not shown) Further in vitro analysis of these majorcompounds revealed that they exhibited approximately one-third of antioxidant activity of vitamin C Therefore ME islikely to possess considerably high antioxidant activity

Antioxidant property of the polar fraction of MEobserved in our in vitro experiments was shown to exertbiological impactThe 24-week period of oral administrationof the polar fraction of ME in the present study resulted ina significant increase in the antioxidant capacity of RBCswhich was determined by the fluorescent signal from DCFemitted from hydrogen peroxide-incubated RBCs A signifi-cant increase in the antioxidant capacity (ie decreased DCFsignal) of RBCs could be detected from week 12 of oraladministration of the polar fraction of ME Therefore ourresults suggest for the first time that extract frommangosteenpericarp can augment the antioxidant system within humanRBCs probably due to its ability to neutralize ROS

We further investigated whether and how such anincreased antioxidant capacity affects protein oxidative dam-age in RBCs and whole blood Protein oxidative damagewas determined by the degree of HNE modification HNEis a byproduct of lipid peroxidation which is an indicatorof oxidative stress HNE can react with lysine histidine orcysteine residue in proteins to form adducts resulting instructural and functional changes to the proteins [28 29]HNE is also reportedly involved in the pathophysiology ofmany diseases [30ndash32] We speculated that if RBCs havean improved antioxidant capacity lipid peroxidation shouldbe reduced and HNE modification to proteins should thus

decrease Consistent with our speculation oral administra-tion of the polar fraction of ME led to a significant decreasein HNE modification in whole blood and RBCs starting inweeks 4 and 24 respectively Notably the significant decreasein HNE modification of whole blood proteins appearedearlier than that of RBC proteins This may be explained bythe faster turnover rate of albumin [33] the most abundantprotein in serum in comparison to the lifespan of RBCs[34] This result provides further supporting evidence inhuman subjects of the antioxidant property of extract frommangosteen pericarp

5 Conclusions

We have demonstrated for the first time in human subjectsthat the polar fraction of extract from mangosteen pericarpis adequately safe for oral administration for up to 24 weeksMajor components of the polar fraction of extract frommangosteen pericarp exhibited considerably high antioxidantactivity according to the in vitro antioxidant assay Addi-tionally the polar extract from mangosteen pericarp appearsto significantly increase the antioxidant capacity of RBCsand significantly decrease protein oxidative damage in RBCsand whole blood It might alleviate oxidative damage inother tissues as well Our results suggest that the antioxidantcapacity of the polar fraction of mangosteen pericarp extractcould come from its ability to neutralize ROS Howeverwhether some compounds in this extract can also upregulateantioxidant enzyme is still unknown It would be interestingto further quantitate the expression level of antioxidant

Oxidative Medicine and Cellular Longevity 9

enzymes In all the polar fraction of extract frommangosteenpericarp might benefit human patients with pathologicalconditions related to oxidative stress

Competing Interests

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

Acknowledgments

The authors are extremely grateful to Mrs Angkana Jong-sawaddipatana and all staff members of the Geriatric Clinicof Siriraj Hospital for their kind collaboration in recruit-ment of the participants This work was supported by theThai Traditional Medical Knowledge Fund Department forDevelopment of Thai Traditional and Alternative MedicineMinistry of Public Health Thailand

References

[1] S M Petiwala G Li A Ramaiya et al ldquoPharmacokineticcharacterization of mangosteen (Garcinia mangostana) fruitextract standardized to 120572-mangostin in C57BL6 micerdquo Nutri-tion Research vol 34 no 4 pp 336ndash345 2014

[2] Z Xu L Huang X-H Chen et al ldquoCytotoxic prenylated xan-thones from the pericarps of Garcinia mangostanardquo Moleculesvol 19 no 2 pp 1820ndash1827 2014

[3] F Gutierrez-Orozco C Chitchumroonchokchai G B Lesin-ski S Suksamrarn and M L Failla ldquo120572-Mangostin anti-inflammatory activity and metabolism by human cellsrdquo Journalof Agricultural and Food Chemistry vol 61 no 16 pp 3891ndash3900 2013

[4] H-Y Jang O-K Kwon S-R Oh H-K Lee K-S Ahn and Y-W Chin ldquoMangosteen xanthones mitigate ovalbumin-inducedairway inflammation in a mouse model of asthmardquo Food andChemical Toxicology vol 50 no 11 pp 4042ndash4050 2012

[5] S Syam A Bustamam R Abdullah et al ldquo120573 Mangostinsuppress LPS-induced inflammatory response in RAW 2647macrophages in vitro and carrageenan-induced peritonitis invivordquo Journal of Ethnopharmacology vol 153 no 2 pp 435ndash4452014

[6] J Yuan Y Wu and G Lu ldquoA-mangostin suppresseslipopolysaccharide-induced invasion by inhibiting matrixmetalloproteinase-29 and increasing E-cadherin expressionthrough extracellular signal-regulated kinase signaling inpancreatic cancer cellsrdquo Oncology Letters vol 5 no 6 pp1958ndash1964 2013

[7] M-A Shibata M Iinuma J Morimoto et al ldquo120572-Mangostinextracted from the pericarp of the mangosteen (Garcinia man-gostana Linn) reduces tumor growth and lymph node metas-tasis in an immunocompetent xenograft model of metastaticmammary cancer carrying a p53mutationrdquo BMCMedicine vol9 article 69 2011

[8] A F A Aisha K M Abu-Salah Z Ismail and A MajidldquoIn vitro and in vivo anti-colon cancer effects of Garciniamangostana xanthones extractrdquo BMC Complementary andAlternative Medicine vol 12 article 104 2012

[9] S Taokaew N Nunkaew P Siripong and M PhisalaphongldquoCharacteristics and anticancer properties of bacterial cellulose

films containing ethanolic extract of mangosteen peelrdquo Journalof Biomaterials Science Polymer Edition vol 25 no 9 pp 907ndash922 2014

[10] P Pan-In S Wanichwecharungruang J Hanes and A J KimldquoCellular trafficking and anticancer activity of Garcinia man-gostana extract-encapsulated polymeric nanoparticlesrdquo Inter-national Journal of Nanomedicine vol 9 no 1 pp 3677ndash36862014

[11] H R W Dharmaratne Y Sakagami K G P Piyasena and VThevanesam ldquoAntibacterial activity of xanthones fromGarciniamangostana (L) and their structure-activity relationship stud-iesrdquo Natural Product Research vol 27 no 10 pp 938ndash941 2013

[12] J-J Koh S Qiu H Zou et al ldquoRapid bactericidal action ofalpha-mangostin against MRSA as an outcome of membranetargetingrdquo Biochimica et Biophysica ActamdashBiomembranes vol1828 no 2 pp 834ndash844 2013

[13] M T Chomnawang S Surassmo K Wongsariya and N Bun-yapraphatsara ldquoAntibacterial activity of Thai medicinal plantsagainstmethicillin-resistant Staphylococcus aureusrdquo Fitoterapiavol 80 no 2 pp 102ndash104 2009

[14] S Suksamrarn N SuwannapochW Phakhodee et al ldquoAntimy-cobacterial activity of prenylated xanthones from the fruits ofGarcinia mangostanardquo Chemical and Pharmaceutical Bulletinvol 51 no 7 pp 857ndash859 2003

[15] P Moongkarndi C Srisawat P Saetun et al ldquoProtective effectof mangosteen extract against 120573-amyloid-induced cytotoxicityoxidative stress and altered proteome in SK-N-SH cellsrdquo Journalof Proteome Research vol 9 no 5 pp 2076ndash2086 2010

[16] Y-W Chin H-A Jung H Chai W J Keller and A DKinghorn ldquoXanthones with quinone reductase-inducing activ-ity from the fruits of Garcinia mangostana (Mangosteen)rdquoPhytochemistry vol 69 no 3 pp 754ndash758 2008

[17] H-A Jung B-N Su W J Keller R G Mehta and AD Kinghorn ldquoAntioxidant xanthones from the pericarp ofGarcinia mangostana (Mangosteen)rdquo Journal of Agriculturaland Food Chemistry vol 54 no 6 pp 2077ndash2082 2006

[18] F Gutierrez-Orozco and M L Failla ldquoBiological activities andbioavailability of mangosteen xanthones a critical review of thecurrent evidencerdquo Nutrients vol 5 no 8 pp 3163ndash3183 2013

[19] J Sattayasai P Chaonapan T Arkaravichie et al ldquoProtectiveeffects of mangosteen extract on H

2

O2

-induced cytotoxicity inSK-N-SH cells and scopolamine-induced memory impairmentin micerdquo PLoS ONE vol 8 no 12 Article ID e85053 2013

[20] E B Walker ldquoHPLC analysis of selected xanthones in mangos-teen fruitrdquo Journal of Separation Science vol 30 no 9 pp 1229ndash1234 2007

[21] F Gutierrez-Orozco J MThomas-Ahner L D Berman-Bootyet al ldquoDietary 120572-mangostin a xanthone from mangosteenfruit exacerbates experimental colitis and promotes dysbiosisin micerdquoMolecular Nutrition and Food Research vol 58 no 6pp 1226ndash1238 2014

[22] M Y IbrahimNMHashim A AMariod et al ldquo120572-Mangostinfrom Garcinia mangostana Linn an updated review of itspharmacological propertiesrdquo Arabian Journal of Chemistry vol9 no 3 pp 317ndash329 2016

[23] M Y Ibrahim N M Hashim S Mohan et al ldquo120572-Mangostinfrom Cratoxylum arborescens an in vitro and in vivo toxicolog-ical evaluationrdquo Arabian Journal of Chemistry vol 8 no 1 pp129ndash137 2015

[24] A Plazonic F Bucar Z Males A Mornar B Nigovic andN Kujundzic ldquoIdentification and quantification of flavonoids

10 Oxidative Medicine and Cellular Longevity

and phenolic acids in burr parsley (Caucalis platycarpos L)using high-performance liquid chromatography with diodearray detection and electrospray ionizationmass spectrometryrdquoMolecules vol 14 no 7 pp 2466ndash2490 2009

[25] P Moongkarndi N Jaisupa J Samer et al ldquoComparison of thebiological activity of two different isolates from mangosteenrdquoJournal of Pharmacy and Pharmacology vol 66 no 8 pp 1171ndash1179 2014

[26] E Fibach ldquoTechniques for studying stimulation of fetal hemo-globin production in human erythroid culturesrdquo Hemoglobinvol 22 no 5-6 pp 445ndash458 1998

[27] J S Stern J Peerson A T Mishra M V Sadasiva Rao andK P Rajeswari ldquoEfficacy and tolerability of a novel herbalformulation for weight managementrdquo Obesity vol 21 no 5 pp921ndash927 2013

[28] C M Spickett ldquoThe lipid peroxidation product 4-hydroxy-2-nonenal advances in chemistry and analysisrdquo Redox Biologyvol 1 no 1 pp 145ndash152 2013

[29] C Schneider N A Porter and A R Brash ldquoRoutes to 4-hydroxynonenal fundamental issues in themechanisms of lipidperoxidationrdquo Journal of Biological Chemistry vol 283 no 23pp 15539ndash15543 2008

[30] A Negre-Salvayre C Coatrieux C Ingueneau and R SalvayreldquoAdvanced lipid peroxidation end products in oxidative damageto proteins Potential role in diseases and therapeutic prospectsfor the inhibitorsrdquo British Journal of Pharmacology vol 153 no1 pp 6ndash20 2008

[31] M P Mattson ldquoRoles of the lipid peroxidation product 4-hydroxynonenal in obesity the metabolic syndrome and asso-ciated vascular and neurodegenerative disordersrdquo ExperimentalGerontology vol 44 no 10 pp 625ndash633 2009

[32] A Ayala M F Munoz and S Arguelles ldquoLipid peroxidationproduction metabolism and signaling mechanisms of malon-dialdehyde and 4-hydroxy-2-nonenalrdquo Oxidative Medicine andCellular Longevity vol 2014 Article ID 360438 31 pages 2014

[33] K Sterling ldquoThe turnover rate of serum albumin in manas measured by I131-tagged albuminrdquo The Journal of ClinicalInvestigation vol 30 no 11 pp 1228ndash1237 1951

[34] C J Lindsell R S Franco E P Smith C H Joiner and R MCohen ldquoA method for the continuous calculation of the age oflabeled red blood cellsrdquo American Journal of Hematology vol83 no 6 pp 454ndash457 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Oxidative Medicine and Cellular Longevity

Table 2 Continued

Test Subject data and laboratory safety assessment mean (SEM)Day 0 1 week 4 weeks 12 weeks 16 weeks 24 weeks

PT(sec) 1214 (021) 1208 (021) 1199 (024) 1223 (019) 1248 (027) 1262 (022)

aPTT(sec) 2853 (060) 2819 (076) 2861 (069) 2897 (061) 2920 (081) 2970 (074)

Urine protein(Negndashtrace) 545 727 727 818 727 545

Body mass index 2174 (112) 2186 (112) 2164 (107) 2207 (114) 2191 (112) 2189 (113)Arterial bloodpressure(mmHg)

11772 11776 11777 11973 11872 11373

HDL-CHOL high-density lipoprotein cholesterol LDL low-density lipoprotein BUN blood urea nitrogen AST (SGOT) aspartate aminotransferase(serum glutamic oxaloacetic transaminase) ALT (SGPT) alanine aminotransferase (serum glutamic pyruvic transaminase) ALP alkaline phosphataseT3 triiodothyronine TSH thyroid-stimulating hormone FT4 free thyroxine WBC white blood cell PT prothrombin time aPTT activated partialthromboplastin time

Table 3 Side effects of oral administration ofMEThe subjects wereasked to report the side effects after oral administration of ME ateach visit The number of episodes of each side effect prior to andafter double doses was collected and calculated as the percentage ofthe total episodes of 110 (11 participants times 10 visits)

SymptomBefore (single dose) After (double doses)Number ofepisodes Number of

episodes

Tirednesssleepiness 4 36 0 00Constipation 4 36 1 09Dry throat 2 18 3 27Headache 1 09 4 36Flu-like symptoms 2 18 0 00Abnormal stool 4 36 0 00Abdominaldiscomfort 3 27 0 00

Cough 2 18 0 00Weight loss 2 18 2 18Nasal congestion 2 18 0 00Abdominal bloating 2 18 1 09Dizziness 1 09 0 00Salivation 1 09 0 00Nauseavomiting 1 09 1 09Diarrhea 1 09 1 09Skin rash 1 09 0 00Abnormal urination 1 09 0 00Malaise 1 09 0 00

RBCsThis effect was further accentuated through the end ofthe study

35 Oxidative Damage to Proteins in RBCs and Whole BloodOral administration of ME was associated with a significantdecrease in oxidative damage to proteins in both RBCs andwhole blood Quantification of oxidative damage to these

proteins was achieved by normalizing HNE staining to actinAs shown in Figure 4 a significant decrease in proteinoxidative damage appeared inweeks 4 and 24 for whole bloodand RBCs respectively

4 Discussion

Several compounds in the extract frommangosteen pericarphave recently been identified and shown to have beneficialeffects on human health However the cytotoxic activitieswere always observed in nonpolar fraction of the extract thatis 120572-mangostin whereas the potent antioxidant was foundin polar fraction In addition the safety of the polar fractionhas never been thoroughly investigated in humans In thisstudy we investigated the safety of oral administration of thepolar fraction of ME inThai subjects For the entire 24 weeksof the study only minor side effects were reported Thesecan be categorized into gastrointestinal skin respiratory andgeneral side effects The most frequently reported side effectswere related to the gastrointestinal systemWe suspected thatsuch side effects might be associated with an antibacterialproperty of the compounds from the mangosteen pericarpwhich may have led to the perturbation of gut microbesNonetheless these side effects accounted for only a fewepisodes and were reportedly tolerable by all subjects

The present study also showed that oral administra-tion of the polar fraction of ME poses no threat to vitalorgans including the liver kidney and thyroid gland Thesubjectsrsquo liver enzyme levels lipid profiles coagulogramparameters blood urea nitrogen level creatinine level urineprotein level plasma electrolyte levels and thyroid hormonelevels remained normal for the entire 24 weeks of thestudy Additionally all subjectsrsquo complete blood counts andblood pressures remained unchanged Although two subjectsshowed slightly increased liver enzymes in the fourth weekof the study the enzyme levels spontaneously returned tothe normal range thereafter Notably a study by Stern et al[27] revealed that obese subjects with a body mass index of30 to 40 kgm2 exhibited a significant net reduction in body

Oxidative Medicine and Cellular Longevity 7

0

1000

FSC

SSC

0

1000

SSC

0

1000

SSC

0

0

1000

1000

FSC0 1000

FSC0 1000

FSC0 1000

SSC

DCF

Cou

nts

100 101 102 103 104

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

DCF100 101 102 103 104

0

100

Cou

nts

0

100

0

1000

SSC

0

1000

SSC

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

0

1000

SSC

0

1000

SSC

0

1000

SSC

0

1000

SSC

FSC

0 1000

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

0

100

100

0

100

Stim

ulat

edSt

imul

ated

0

1000

FSC

SSC

0

0

1000

1000

FSC0 1000

SSC

DCF

DCF

Cou

nts

100 101 102 103 104

100 101 102 103 104

Cou

nts

W12 W16 W24

W0 W1 W44 + H(0)001

4 + H(20)001

4 + H(0)001

4 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001 M012 + H(0)001

M012 + H(20)001 M012 + H(20)001

M012 + H(0)001 M012 + H(0)001

M012 + H(20)001 M012 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001M012 + H(0)001 M012 + H(0)001

M012 + H(20)001M012 + H(20)001 M012 + H(20)001

0min

Stim

ulat

ed0min

20min

Stim

ulat

ed20min

(a)

W1

W4

W12

W16W

0

W24

Weeks of oral administration of ME

Mea

n flu

ores

cenc

e int

ensit

y

lowastlowast

lowast

40

30

20

10

50

(b)

Figure 3 Effect of oral administration of ME on antioxidant capacity of RBCs RBCs were isolated from the peripheral blood of the subjectsat 0 1 4 12 16 and 24 weeks after beginning oral administration of ME Hydrogen peroxide was an exogenous ROS used to stimulate theresponse of the RBC antioxidant system (a) A representative measurement of the level of ROS within the RBCs which was determined by themean fluorescent signal of DCF emitted from hydrogen peroxide-incubated RBCs (b) Quantitative analysis of the mean fluorescent signalof DCF from all samples during the entire period of the study Data are presented as means (119899 = 11) Error bars represent SEMThe asteriskindicates statistical significance 119901 lt 005 in comparison with week 0 (119905-test)

weight (374 kg 119901 lt 00001) after taking capsules containingSphaeranthus indicus and G mangostana for 8 weeks com-pared with subjects taking placebo The discrepancy in thesubjectsrsquo body weights between that study and the presentstudy might derive from the volunteers in our study havinga normal BMI or from the weight-loss property of S indicus

We performedTLC onME in order to test for preparationconsistency and to demonstrate its essential compounds Itwas shown that ME was composed of at least one groupof major compounds which consistently appeared as asingle band on the chromatogram at 119877

119891= 075 Such a

high 119877119891suggested highly organic-volatile property We are

8 Oxidative Medicine and Cellular Longevity

Red blood cell Whole blood

W0

W1

W4

W12

W16

W24

Actin HNE Actin HNE

(a)

05

10

HNE-WBHNE-RBC

W1

W4

W12

W16W

0

W24

Weeks of oral administration of ME

Leve

l of H

NE

mod

ifica

tion

(rel

ativ

e to

W0) lowast

lowast

lowast

lowastlowast

(b)

Figure 4 Oral administration of ME resulted in decreased protein oxidative damage in RBCs and whole blood The RBCs and whole bloodof the subjects were collected at 0 1 4 12 16 and 24 weeks after beginning oral administration of ME (a) A representative blot showsHNE staining using a western blotting technique to determine the protein oxidative damage in RBCs and whole blood Actin was used asa loading control (b) Quantitative analysis of the extent of HNE modification to RBCs (HNE-RBC) and whole blood (HNE-WB) after oraladministration of ME was carried out as that relative to week 0 Data are presented as means (119899 = 11) Error bars represent SEMThe asteriskindicates statistical significance 119901 lt 005 in comparison with week 0 (119905-test)

currently identifying the chemical compounds in this band(data not shown) Further in vitro analysis of these majorcompounds revealed that they exhibited approximately one-third of antioxidant activity of vitamin C Therefore ME islikely to possess considerably high antioxidant activity

Antioxidant property of the polar fraction of MEobserved in our in vitro experiments was shown to exertbiological impactThe 24-week period of oral administrationof the polar fraction of ME in the present study resulted ina significant increase in the antioxidant capacity of RBCswhich was determined by the fluorescent signal from DCFemitted from hydrogen peroxide-incubated RBCs A signifi-cant increase in the antioxidant capacity (ie decreased DCFsignal) of RBCs could be detected from week 12 of oraladministration of the polar fraction of ME Therefore ourresults suggest for the first time that extract frommangosteenpericarp can augment the antioxidant system within humanRBCs probably due to its ability to neutralize ROS

We further investigated whether and how such anincreased antioxidant capacity affects protein oxidative dam-age in RBCs and whole blood Protein oxidative damagewas determined by the degree of HNE modification HNEis a byproduct of lipid peroxidation which is an indicatorof oxidative stress HNE can react with lysine histidine orcysteine residue in proteins to form adducts resulting instructural and functional changes to the proteins [28 29]HNE is also reportedly involved in the pathophysiology ofmany diseases [30ndash32] We speculated that if RBCs havean improved antioxidant capacity lipid peroxidation shouldbe reduced and HNE modification to proteins should thus

decrease Consistent with our speculation oral administra-tion of the polar fraction of ME led to a significant decreasein HNE modification in whole blood and RBCs starting inweeks 4 and 24 respectively Notably the significant decreasein HNE modification of whole blood proteins appearedearlier than that of RBC proteins This may be explained bythe faster turnover rate of albumin [33] the most abundantprotein in serum in comparison to the lifespan of RBCs[34] This result provides further supporting evidence inhuman subjects of the antioxidant property of extract frommangosteen pericarp

5 Conclusions

We have demonstrated for the first time in human subjectsthat the polar fraction of extract from mangosteen pericarpis adequately safe for oral administration for up to 24 weeksMajor components of the polar fraction of extract frommangosteen pericarp exhibited considerably high antioxidantactivity according to the in vitro antioxidant assay Addi-tionally the polar extract from mangosteen pericarp appearsto significantly increase the antioxidant capacity of RBCsand significantly decrease protein oxidative damage in RBCsand whole blood It might alleviate oxidative damage inother tissues as well Our results suggest that the antioxidantcapacity of the polar fraction of mangosteen pericarp extractcould come from its ability to neutralize ROS Howeverwhether some compounds in this extract can also upregulateantioxidant enzyme is still unknown It would be interestingto further quantitate the expression level of antioxidant

Oxidative Medicine and Cellular Longevity 9

enzymes In all the polar fraction of extract frommangosteenpericarp might benefit human patients with pathologicalconditions related to oxidative stress

Competing Interests

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

Acknowledgments

The authors are extremely grateful to Mrs Angkana Jong-sawaddipatana and all staff members of the Geriatric Clinicof Siriraj Hospital for their kind collaboration in recruit-ment of the participants This work was supported by theThai Traditional Medical Knowledge Fund Department forDevelopment of Thai Traditional and Alternative MedicineMinistry of Public Health Thailand

References

[1] S M Petiwala G Li A Ramaiya et al ldquoPharmacokineticcharacterization of mangosteen (Garcinia mangostana) fruitextract standardized to 120572-mangostin in C57BL6 micerdquo Nutri-tion Research vol 34 no 4 pp 336ndash345 2014

[2] Z Xu L Huang X-H Chen et al ldquoCytotoxic prenylated xan-thones from the pericarps of Garcinia mangostanardquo Moleculesvol 19 no 2 pp 1820ndash1827 2014

[3] F Gutierrez-Orozco C Chitchumroonchokchai G B Lesin-ski S Suksamrarn and M L Failla ldquo120572-Mangostin anti-inflammatory activity and metabolism by human cellsrdquo Journalof Agricultural and Food Chemistry vol 61 no 16 pp 3891ndash3900 2013

[4] H-Y Jang O-K Kwon S-R Oh H-K Lee K-S Ahn and Y-W Chin ldquoMangosteen xanthones mitigate ovalbumin-inducedairway inflammation in a mouse model of asthmardquo Food andChemical Toxicology vol 50 no 11 pp 4042ndash4050 2012

[5] S Syam A Bustamam R Abdullah et al ldquo120573 Mangostinsuppress LPS-induced inflammatory response in RAW 2647macrophages in vitro and carrageenan-induced peritonitis invivordquo Journal of Ethnopharmacology vol 153 no 2 pp 435ndash4452014

[6] J Yuan Y Wu and G Lu ldquoA-mangostin suppresseslipopolysaccharide-induced invasion by inhibiting matrixmetalloproteinase-29 and increasing E-cadherin expressionthrough extracellular signal-regulated kinase signaling inpancreatic cancer cellsrdquo Oncology Letters vol 5 no 6 pp1958ndash1964 2013

[7] M-A Shibata M Iinuma J Morimoto et al ldquo120572-Mangostinextracted from the pericarp of the mangosteen (Garcinia man-gostana Linn) reduces tumor growth and lymph node metas-tasis in an immunocompetent xenograft model of metastaticmammary cancer carrying a p53mutationrdquo BMCMedicine vol9 article 69 2011

[8] A F A Aisha K M Abu-Salah Z Ismail and A MajidldquoIn vitro and in vivo anti-colon cancer effects of Garciniamangostana xanthones extractrdquo BMC Complementary andAlternative Medicine vol 12 article 104 2012

[9] S Taokaew N Nunkaew P Siripong and M PhisalaphongldquoCharacteristics and anticancer properties of bacterial cellulose

films containing ethanolic extract of mangosteen peelrdquo Journalof Biomaterials Science Polymer Edition vol 25 no 9 pp 907ndash922 2014

[10] P Pan-In S Wanichwecharungruang J Hanes and A J KimldquoCellular trafficking and anticancer activity of Garcinia man-gostana extract-encapsulated polymeric nanoparticlesrdquo Inter-national Journal of Nanomedicine vol 9 no 1 pp 3677ndash36862014

[11] H R W Dharmaratne Y Sakagami K G P Piyasena and VThevanesam ldquoAntibacterial activity of xanthones fromGarciniamangostana (L) and their structure-activity relationship stud-iesrdquo Natural Product Research vol 27 no 10 pp 938ndash941 2013

[12] J-J Koh S Qiu H Zou et al ldquoRapid bactericidal action ofalpha-mangostin against MRSA as an outcome of membranetargetingrdquo Biochimica et Biophysica ActamdashBiomembranes vol1828 no 2 pp 834ndash844 2013

[13] M T Chomnawang S Surassmo K Wongsariya and N Bun-yapraphatsara ldquoAntibacterial activity of Thai medicinal plantsagainstmethicillin-resistant Staphylococcus aureusrdquo Fitoterapiavol 80 no 2 pp 102ndash104 2009

[14] S Suksamrarn N SuwannapochW Phakhodee et al ldquoAntimy-cobacterial activity of prenylated xanthones from the fruits ofGarcinia mangostanardquo Chemical and Pharmaceutical Bulletinvol 51 no 7 pp 857ndash859 2003

[15] P Moongkarndi C Srisawat P Saetun et al ldquoProtective effectof mangosteen extract against 120573-amyloid-induced cytotoxicityoxidative stress and altered proteome in SK-N-SH cellsrdquo Journalof Proteome Research vol 9 no 5 pp 2076ndash2086 2010

[16] Y-W Chin H-A Jung H Chai W J Keller and A DKinghorn ldquoXanthones with quinone reductase-inducing activ-ity from the fruits of Garcinia mangostana (Mangosteen)rdquoPhytochemistry vol 69 no 3 pp 754ndash758 2008

[17] H-A Jung B-N Su W J Keller R G Mehta and AD Kinghorn ldquoAntioxidant xanthones from the pericarp ofGarcinia mangostana (Mangosteen)rdquo Journal of Agriculturaland Food Chemistry vol 54 no 6 pp 2077ndash2082 2006

[18] F Gutierrez-Orozco and M L Failla ldquoBiological activities andbioavailability of mangosteen xanthones a critical review of thecurrent evidencerdquo Nutrients vol 5 no 8 pp 3163ndash3183 2013

[19] J Sattayasai P Chaonapan T Arkaravichie et al ldquoProtectiveeffects of mangosteen extract on H

2

O2

-induced cytotoxicity inSK-N-SH cells and scopolamine-induced memory impairmentin micerdquo PLoS ONE vol 8 no 12 Article ID e85053 2013

[20] E B Walker ldquoHPLC analysis of selected xanthones in mangos-teen fruitrdquo Journal of Separation Science vol 30 no 9 pp 1229ndash1234 2007

[21] F Gutierrez-Orozco J MThomas-Ahner L D Berman-Bootyet al ldquoDietary 120572-mangostin a xanthone from mangosteenfruit exacerbates experimental colitis and promotes dysbiosisin micerdquoMolecular Nutrition and Food Research vol 58 no 6pp 1226ndash1238 2014

[22] M Y IbrahimNMHashim A AMariod et al ldquo120572-Mangostinfrom Garcinia mangostana Linn an updated review of itspharmacological propertiesrdquo Arabian Journal of Chemistry vol9 no 3 pp 317ndash329 2016

[23] M Y Ibrahim N M Hashim S Mohan et al ldquo120572-Mangostinfrom Cratoxylum arborescens an in vitro and in vivo toxicolog-ical evaluationrdquo Arabian Journal of Chemistry vol 8 no 1 pp129ndash137 2015

[24] A Plazonic F Bucar Z Males A Mornar B Nigovic andN Kujundzic ldquoIdentification and quantification of flavonoids

10 Oxidative Medicine and Cellular Longevity

and phenolic acids in burr parsley (Caucalis platycarpos L)using high-performance liquid chromatography with diodearray detection and electrospray ionizationmass spectrometryrdquoMolecules vol 14 no 7 pp 2466ndash2490 2009

[25] P Moongkarndi N Jaisupa J Samer et al ldquoComparison of thebiological activity of two different isolates from mangosteenrdquoJournal of Pharmacy and Pharmacology vol 66 no 8 pp 1171ndash1179 2014

[26] E Fibach ldquoTechniques for studying stimulation of fetal hemo-globin production in human erythroid culturesrdquo Hemoglobinvol 22 no 5-6 pp 445ndash458 1998

[27] J S Stern J Peerson A T Mishra M V Sadasiva Rao andK P Rajeswari ldquoEfficacy and tolerability of a novel herbalformulation for weight managementrdquo Obesity vol 21 no 5 pp921ndash927 2013

[28] C M Spickett ldquoThe lipid peroxidation product 4-hydroxy-2-nonenal advances in chemistry and analysisrdquo Redox Biologyvol 1 no 1 pp 145ndash152 2013

[29] C Schneider N A Porter and A R Brash ldquoRoutes to 4-hydroxynonenal fundamental issues in themechanisms of lipidperoxidationrdquo Journal of Biological Chemistry vol 283 no 23pp 15539ndash15543 2008

[30] A Negre-Salvayre C Coatrieux C Ingueneau and R SalvayreldquoAdvanced lipid peroxidation end products in oxidative damageto proteins Potential role in diseases and therapeutic prospectsfor the inhibitorsrdquo British Journal of Pharmacology vol 153 no1 pp 6ndash20 2008

[31] M P Mattson ldquoRoles of the lipid peroxidation product 4-hydroxynonenal in obesity the metabolic syndrome and asso-ciated vascular and neurodegenerative disordersrdquo ExperimentalGerontology vol 44 no 10 pp 625ndash633 2009

[32] A Ayala M F Munoz and S Arguelles ldquoLipid peroxidationproduction metabolism and signaling mechanisms of malon-dialdehyde and 4-hydroxy-2-nonenalrdquo Oxidative Medicine andCellular Longevity vol 2014 Article ID 360438 31 pages 2014

[33] K Sterling ldquoThe turnover rate of serum albumin in manas measured by I131-tagged albuminrdquo The Journal of ClinicalInvestigation vol 30 no 11 pp 1228ndash1237 1951

[34] C J Lindsell R S Franco E P Smith C H Joiner and R MCohen ldquoA method for the continuous calculation of the age oflabeled red blood cellsrdquo American Journal of Hematology vol83 no 6 pp 454ndash457 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 7

0

1000

FSC

SSC

0

1000

SSC

0

1000

SSC

0

0

1000

1000

FSC0 1000

FSC0 1000

FSC0 1000

SSC

DCF

Cou

nts

100 101 102 103 104

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

DCF100 101 102 103 104

0

100

Cou

nts

0

100

0

1000

SSC

0

1000

SSC

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

0

1000

SSC

0

1000

SSC

0

1000

SSC

0

1000

SSC

FSC

0 1000

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

FSC0 1000

FSC0 1000

DCF100 101 102 103 104

DCF100 101 102 103 104

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

100

Cou

nts

0

0

100

100

0

100

Stim

ulat

edSt

imul

ated

0

1000

FSC

SSC

0

0

1000

1000

FSC0 1000

SSC

DCF

DCF

Cou

nts

100 101 102 103 104

100 101 102 103 104

Cou

nts

W12 W16 W24

W0 W1 W44 + H(0)001

4 + H(20)001

4 + H(0)001

4 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001 M012 + H(0)001

M012 + H(20)001 M012 + H(20)001

M012 + H(0)001 M012 + H(0)001

M012 + H(20)001 M012 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001

M012 + H(20)001

M012 + H(0)001M012 + H(0)001 M012 + H(0)001

M012 + H(20)001M012 + H(20)001 M012 + H(20)001

0min

Stim

ulat

ed0min

20min

Stim

ulat

ed20min

(a)

W1

W4

W12

W16W

0

W24

Weeks of oral administration of ME

Mea

n flu

ores

cenc

e int

ensit

y

lowastlowast

lowast

40

30

20

10

50

(b)

Figure 3 Effect of oral administration of ME on antioxidant capacity of RBCs RBCs were isolated from the peripheral blood of the subjectsat 0 1 4 12 16 and 24 weeks after beginning oral administration of ME Hydrogen peroxide was an exogenous ROS used to stimulate theresponse of the RBC antioxidant system (a) A representative measurement of the level of ROS within the RBCs which was determined by themean fluorescent signal of DCF emitted from hydrogen peroxide-incubated RBCs (b) Quantitative analysis of the mean fluorescent signalof DCF from all samples during the entire period of the study Data are presented as means (119899 = 11) Error bars represent SEMThe asteriskindicates statistical significance 119901 lt 005 in comparison with week 0 (119905-test)

weight (374 kg 119901 lt 00001) after taking capsules containingSphaeranthus indicus and G mangostana for 8 weeks com-pared with subjects taking placebo The discrepancy in thesubjectsrsquo body weights between that study and the presentstudy might derive from the volunteers in our study havinga normal BMI or from the weight-loss property of S indicus

We performedTLC onME in order to test for preparationconsistency and to demonstrate its essential compounds Itwas shown that ME was composed of at least one groupof major compounds which consistently appeared as asingle band on the chromatogram at 119877

119891= 075 Such a

high 119877119891suggested highly organic-volatile property We are

8 Oxidative Medicine and Cellular Longevity

Red blood cell Whole blood

W0

W1

W4

W12

W16

W24

Actin HNE Actin HNE

(a)

05

10

HNE-WBHNE-RBC

W1

W4

W12

W16W

0

W24

Weeks of oral administration of ME

Leve

l of H

NE

mod

ifica

tion

(rel

ativ

e to

W0) lowast

lowast

lowast

lowastlowast

(b)

Figure 4 Oral administration of ME resulted in decreased protein oxidative damage in RBCs and whole blood The RBCs and whole bloodof the subjects were collected at 0 1 4 12 16 and 24 weeks after beginning oral administration of ME (a) A representative blot showsHNE staining using a western blotting technique to determine the protein oxidative damage in RBCs and whole blood Actin was used asa loading control (b) Quantitative analysis of the extent of HNE modification to RBCs (HNE-RBC) and whole blood (HNE-WB) after oraladministration of ME was carried out as that relative to week 0 Data are presented as means (119899 = 11) Error bars represent SEMThe asteriskindicates statistical significance 119901 lt 005 in comparison with week 0 (119905-test)

currently identifying the chemical compounds in this band(data not shown) Further in vitro analysis of these majorcompounds revealed that they exhibited approximately one-third of antioxidant activity of vitamin C Therefore ME islikely to possess considerably high antioxidant activity

Antioxidant property of the polar fraction of MEobserved in our in vitro experiments was shown to exertbiological impactThe 24-week period of oral administrationof the polar fraction of ME in the present study resulted ina significant increase in the antioxidant capacity of RBCswhich was determined by the fluorescent signal from DCFemitted from hydrogen peroxide-incubated RBCs A signifi-cant increase in the antioxidant capacity (ie decreased DCFsignal) of RBCs could be detected from week 12 of oraladministration of the polar fraction of ME Therefore ourresults suggest for the first time that extract frommangosteenpericarp can augment the antioxidant system within humanRBCs probably due to its ability to neutralize ROS

We further investigated whether and how such anincreased antioxidant capacity affects protein oxidative dam-age in RBCs and whole blood Protein oxidative damagewas determined by the degree of HNE modification HNEis a byproduct of lipid peroxidation which is an indicatorof oxidative stress HNE can react with lysine histidine orcysteine residue in proteins to form adducts resulting instructural and functional changes to the proteins [28 29]HNE is also reportedly involved in the pathophysiology ofmany diseases [30ndash32] We speculated that if RBCs havean improved antioxidant capacity lipid peroxidation shouldbe reduced and HNE modification to proteins should thus

decrease Consistent with our speculation oral administra-tion of the polar fraction of ME led to a significant decreasein HNE modification in whole blood and RBCs starting inweeks 4 and 24 respectively Notably the significant decreasein HNE modification of whole blood proteins appearedearlier than that of RBC proteins This may be explained bythe faster turnover rate of albumin [33] the most abundantprotein in serum in comparison to the lifespan of RBCs[34] This result provides further supporting evidence inhuman subjects of the antioxidant property of extract frommangosteen pericarp

5 Conclusions

We have demonstrated for the first time in human subjectsthat the polar fraction of extract from mangosteen pericarpis adequately safe for oral administration for up to 24 weeksMajor components of the polar fraction of extract frommangosteen pericarp exhibited considerably high antioxidantactivity according to the in vitro antioxidant assay Addi-tionally the polar extract from mangosteen pericarp appearsto significantly increase the antioxidant capacity of RBCsand significantly decrease protein oxidative damage in RBCsand whole blood It might alleviate oxidative damage inother tissues as well Our results suggest that the antioxidantcapacity of the polar fraction of mangosteen pericarp extractcould come from its ability to neutralize ROS Howeverwhether some compounds in this extract can also upregulateantioxidant enzyme is still unknown It would be interestingto further quantitate the expression level of antioxidant

Oxidative Medicine and Cellular Longevity 9

enzymes In all the polar fraction of extract frommangosteenpericarp might benefit human patients with pathologicalconditions related to oxidative stress

Competing Interests

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

Acknowledgments

The authors are extremely grateful to Mrs Angkana Jong-sawaddipatana and all staff members of the Geriatric Clinicof Siriraj Hospital for their kind collaboration in recruit-ment of the participants This work was supported by theThai Traditional Medical Knowledge Fund Department forDevelopment of Thai Traditional and Alternative MedicineMinistry of Public Health Thailand

References

[1] S M Petiwala G Li A Ramaiya et al ldquoPharmacokineticcharacterization of mangosteen (Garcinia mangostana) fruitextract standardized to 120572-mangostin in C57BL6 micerdquo Nutri-tion Research vol 34 no 4 pp 336ndash345 2014

[2] Z Xu L Huang X-H Chen et al ldquoCytotoxic prenylated xan-thones from the pericarps of Garcinia mangostanardquo Moleculesvol 19 no 2 pp 1820ndash1827 2014

[3] F Gutierrez-Orozco C Chitchumroonchokchai G B Lesin-ski S Suksamrarn and M L Failla ldquo120572-Mangostin anti-inflammatory activity and metabolism by human cellsrdquo Journalof Agricultural and Food Chemistry vol 61 no 16 pp 3891ndash3900 2013

[4] H-Y Jang O-K Kwon S-R Oh H-K Lee K-S Ahn and Y-W Chin ldquoMangosteen xanthones mitigate ovalbumin-inducedairway inflammation in a mouse model of asthmardquo Food andChemical Toxicology vol 50 no 11 pp 4042ndash4050 2012

[5] S Syam A Bustamam R Abdullah et al ldquo120573 Mangostinsuppress LPS-induced inflammatory response in RAW 2647macrophages in vitro and carrageenan-induced peritonitis invivordquo Journal of Ethnopharmacology vol 153 no 2 pp 435ndash4452014

[6] J Yuan Y Wu and G Lu ldquoA-mangostin suppresseslipopolysaccharide-induced invasion by inhibiting matrixmetalloproteinase-29 and increasing E-cadherin expressionthrough extracellular signal-regulated kinase signaling inpancreatic cancer cellsrdquo Oncology Letters vol 5 no 6 pp1958ndash1964 2013

[7] M-A Shibata M Iinuma J Morimoto et al ldquo120572-Mangostinextracted from the pericarp of the mangosteen (Garcinia man-gostana Linn) reduces tumor growth and lymph node metas-tasis in an immunocompetent xenograft model of metastaticmammary cancer carrying a p53mutationrdquo BMCMedicine vol9 article 69 2011

[8] A F A Aisha K M Abu-Salah Z Ismail and A MajidldquoIn vitro and in vivo anti-colon cancer effects of Garciniamangostana xanthones extractrdquo BMC Complementary andAlternative Medicine vol 12 article 104 2012

[9] S Taokaew N Nunkaew P Siripong and M PhisalaphongldquoCharacteristics and anticancer properties of bacterial cellulose

films containing ethanolic extract of mangosteen peelrdquo Journalof Biomaterials Science Polymer Edition vol 25 no 9 pp 907ndash922 2014

[10] P Pan-In S Wanichwecharungruang J Hanes and A J KimldquoCellular trafficking and anticancer activity of Garcinia man-gostana extract-encapsulated polymeric nanoparticlesrdquo Inter-national Journal of Nanomedicine vol 9 no 1 pp 3677ndash36862014

[11] H R W Dharmaratne Y Sakagami K G P Piyasena and VThevanesam ldquoAntibacterial activity of xanthones fromGarciniamangostana (L) and their structure-activity relationship stud-iesrdquo Natural Product Research vol 27 no 10 pp 938ndash941 2013

[12] J-J Koh S Qiu H Zou et al ldquoRapid bactericidal action ofalpha-mangostin against MRSA as an outcome of membranetargetingrdquo Biochimica et Biophysica ActamdashBiomembranes vol1828 no 2 pp 834ndash844 2013

[13] M T Chomnawang S Surassmo K Wongsariya and N Bun-yapraphatsara ldquoAntibacterial activity of Thai medicinal plantsagainstmethicillin-resistant Staphylococcus aureusrdquo Fitoterapiavol 80 no 2 pp 102ndash104 2009

[14] S Suksamrarn N SuwannapochW Phakhodee et al ldquoAntimy-cobacterial activity of prenylated xanthones from the fruits ofGarcinia mangostanardquo Chemical and Pharmaceutical Bulletinvol 51 no 7 pp 857ndash859 2003

[15] P Moongkarndi C Srisawat P Saetun et al ldquoProtective effectof mangosteen extract against 120573-amyloid-induced cytotoxicityoxidative stress and altered proteome in SK-N-SH cellsrdquo Journalof Proteome Research vol 9 no 5 pp 2076ndash2086 2010

[16] Y-W Chin H-A Jung H Chai W J Keller and A DKinghorn ldquoXanthones with quinone reductase-inducing activ-ity from the fruits of Garcinia mangostana (Mangosteen)rdquoPhytochemistry vol 69 no 3 pp 754ndash758 2008

[17] H-A Jung B-N Su W J Keller R G Mehta and AD Kinghorn ldquoAntioxidant xanthones from the pericarp ofGarcinia mangostana (Mangosteen)rdquo Journal of Agriculturaland Food Chemistry vol 54 no 6 pp 2077ndash2082 2006

[18] F Gutierrez-Orozco and M L Failla ldquoBiological activities andbioavailability of mangosteen xanthones a critical review of thecurrent evidencerdquo Nutrients vol 5 no 8 pp 3163ndash3183 2013

[19] J Sattayasai P Chaonapan T Arkaravichie et al ldquoProtectiveeffects of mangosteen extract on H

2

O2

-induced cytotoxicity inSK-N-SH cells and scopolamine-induced memory impairmentin micerdquo PLoS ONE vol 8 no 12 Article ID e85053 2013

[20] E B Walker ldquoHPLC analysis of selected xanthones in mangos-teen fruitrdquo Journal of Separation Science vol 30 no 9 pp 1229ndash1234 2007

[21] F Gutierrez-Orozco J MThomas-Ahner L D Berman-Bootyet al ldquoDietary 120572-mangostin a xanthone from mangosteenfruit exacerbates experimental colitis and promotes dysbiosisin micerdquoMolecular Nutrition and Food Research vol 58 no 6pp 1226ndash1238 2014

[22] M Y IbrahimNMHashim A AMariod et al ldquo120572-Mangostinfrom Garcinia mangostana Linn an updated review of itspharmacological propertiesrdquo Arabian Journal of Chemistry vol9 no 3 pp 317ndash329 2016

[23] M Y Ibrahim N M Hashim S Mohan et al ldquo120572-Mangostinfrom Cratoxylum arborescens an in vitro and in vivo toxicolog-ical evaluationrdquo Arabian Journal of Chemistry vol 8 no 1 pp129ndash137 2015

[24] A Plazonic F Bucar Z Males A Mornar B Nigovic andN Kujundzic ldquoIdentification and quantification of flavonoids

10 Oxidative Medicine and Cellular Longevity

and phenolic acids in burr parsley (Caucalis platycarpos L)using high-performance liquid chromatography with diodearray detection and electrospray ionizationmass spectrometryrdquoMolecules vol 14 no 7 pp 2466ndash2490 2009

[25] P Moongkarndi N Jaisupa J Samer et al ldquoComparison of thebiological activity of two different isolates from mangosteenrdquoJournal of Pharmacy and Pharmacology vol 66 no 8 pp 1171ndash1179 2014

[26] E Fibach ldquoTechniques for studying stimulation of fetal hemo-globin production in human erythroid culturesrdquo Hemoglobinvol 22 no 5-6 pp 445ndash458 1998

[27] J S Stern J Peerson A T Mishra M V Sadasiva Rao andK P Rajeswari ldquoEfficacy and tolerability of a novel herbalformulation for weight managementrdquo Obesity vol 21 no 5 pp921ndash927 2013

[28] C M Spickett ldquoThe lipid peroxidation product 4-hydroxy-2-nonenal advances in chemistry and analysisrdquo Redox Biologyvol 1 no 1 pp 145ndash152 2013

[29] C Schneider N A Porter and A R Brash ldquoRoutes to 4-hydroxynonenal fundamental issues in themechanisms of lipidperoxidationrdquo Journal of Biological Chemistry vol 283 no 23pp 15539ndash15543 2008

[30] A Negre-Salvayre C Coatrieux C Ingueneau and R SalvayreldquoAdvanced lipid peroxidation end products in oxidative damageto proteins Potential role in diseases and therapeutic prospectsfor the inhibitorsrdquo British Journal of Pharmacology vol 153 no1 pp 6ndash20 2008

[31] M P Mattson ldquoRoles of the lipid peroxidation product 4-hydroxynonenal in obesity the metabolic syndrome and asso-ciated vascular and neurodegenerative disordersrdquo ExperimentalGerontology vol 44 no 10 pp 625ndash633 2009

[32] A Ayala M F Munoz and S Arguelles ldquoLipid peroxidationproduction metabolism and signaling mechanisms of malon-dialdehyde and 4-hydroxy-2-nonenalrdquo Oxidative Medicine andCellular Longevity vol 2014 Article ID 360438 31 pages 2014

[33] K Sterling ldquoThe turnover rate of serum albumin in manas measured by I131-tagged albuminrdquo The Journal of ClinicalInvestigation vol 30 no 11 pp 1228ndash1237 1951

[34] C J Lindsell R S Franco E P Smith C H Joiner and R MCohen ldquoA method for the continuous calculation of the age oflabeled red blood cellsrdquo American Journal of Hematology vol83 no 6 pp 454ndash457 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Oxidative Medicine and Cellular Longevity

Red blood cell Whole blood

W0

W1

W4

W12

W16

W24

Actin HNE Actin HNE

(a)

05

10

HNE-WBHNE-RBC

W1

W4

W12

W16W

0

W24

Weeks of oral administration of ME

Leve

l of H

NE

mod

ifica

tion

(rel

ativ

e to

W0) lowast

lowast

lowast

lowastlowast

(b)

Figure 4 Oral administration of ME resulted in decreased protein oxidative damage in RBCs and whole blood The RBCs and whole bloodof the subjects were collected at 0 1 4 12 16 and 24 weeks after beginning oral administration of ME (a) A representative blot showsHNE staining using a western blotting technique to determine the protein oxidative damage in RBCs and whole blood Actin was used asa loading control (b) Quantitative analysis of the extent of HNE modification to RBCs (HNE-RBC) and whole blood (HNE-WB) after oraladministration of ME was carried out as that relative to week 0 Data are presented as means (119899 = 11) Error bars represent SEMThe asteriskindicates statistical significance 119901 lt 005 in comparison with week 0 (119905-test)

currently identifying the chemical compounds in this band(data not shown) Further in vitro analysis of these majorcompounds revealed that they exhibited approximately one-third of antioxidant activity of vitamin C Therefore ME islikely to possess considerably high antioxidant activity

Antioxidant property of the polar fraction of MEobserved in our in vitro experiments was shown to exertbiological impactThe 24-week period of oral administrationof the polar fraction of ME in the present study resulted ina significant increase in the antioxidant capacity of RBCswhich was determined by the fluorescent signal from DCFemitted from hydrogen peroxide-incubated RBCs A signifi-cant increase in the antioxidant capacity (ie decreased DCFsignal) of RBCs could be detected from week 12 of oraladministration of the polar fraction of ME Therefore ourresults suggest for the first time that extract frommangosteenpericarp can augment the antioxidant system within humanRBCs probably due to its ability to neutralize ROS

We further investigated whether and how such anincreased antioxidant capacity affects protein oxidative dam-age in RBCs and whole blood Protein oxidative damagewas determined by the degree of HNE modification HNEis a byproduct of lipid peroxidation which is an indicatorof oxidative stress HNE can react with lysine histidine orcysteine residue in proteins to form adducts resulting instructural and functional changes to the proteins [28 29]HNE is also reportedly involved in the pathophysiology ofmany diseases [30ndash32] We speculated that if RBCs havean improved antioxidant capacity lipid peroxidation shouldbe reduced and HNE modification to proteins should thus

decrease Consistent with our speculation oral administra-tion of the polar fraction of ME led to a significant decreasein HNE modification in whole blood and RBCs starting inweeks 4 and 24 respectively Notably the significant decreasein HNE modification of whole blood proteins appearedearlier than that of RBC proteins This may be explained bythe faster turnover rate of albumin [33] the most abundantprotein in serum in comparison to the lifespan of RBCs[34] This result provides further supporting evidence inhuman subjects of the antioxidant property of extract frommangosteen pericarp

5 Conclusions

We have demonstrated for the first time in human subjectsthat the polar fraction of extract from mangosteen pericarpis adequately safe for oral administration for up to 24 weeksMajor components of the polar fraction of extract frommangosteen pericarp exhibited considerably high antioxidantactivity according to the in vitro antioxidant assay Addi-tionally the polar extract from mangosteen pericarp appearsto significantly increase the antioxidant capacity of RBCsand significantly decrease protein oxidative damage in RBCsand whole blood It might alleviate oxidative damage inother tissues as well Our results suggest that the antioxidantcapacity of the polar fraction of mangosteen pericarp extractcould come from its ability to neutralize ROS Howeverwhether some compounds in this extract can also upregulateantioxidant enzyme is still unknown It would be interestingto further quantitate the expression level of antioxidant

Oxidative Medicine and Cellular Longevity 9

enzymes In all the polar fraction of extract frommangosteenpericarp might benefit human patients with pathologicalconditions related to oxidative stress

Competing Interests

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

Acknowledgments

The authors are extremely grateful to Mrs Angkana Jong-sawaddipatana and all staff members of the Geriatric Clinicof Siriraj Hospital for their kind collaboration in recruit-ment of the participants This work was supported by theThai Traditional Medical Knowledge Fund Department forDevelopment of Thai Traditional and Alternative MedicineMinistry of Public Health Thailand

References

[1] S M Petiwala G Li A Ramaiya et al ldquoPharmacokineticcharacterization of mangosteen (Garcinia mangostana) fruitextract standardized to 120572-mangostin in C57BL6 micerdquo Nutri-tion Research vol 34 no 4 pp 336ndash345 2014

[2] Z Xu L Huang X-H Chen et al ldquoCytotoxic prenylated xan-thones from the pericarps of Garcinia mangostanardquo Moleculesvol 19 no 2 pp 1820ndash1827 2014

[3] F Gutierrez-Orozco C Chitchumroonchokchai G B Lesin-ski S Suksamrarn and M L Failla ldquo120572-Mangostin anti-inflammatory activity and metabolism by human cellsrdquo Journalof Agricultural and Food Chemistry vol 61 no 16 pp 3891ndash3900 2013

[4] H-Y Jang O-K Kwon S-R Oh H-K Lee K-S Ahn and Y-W Chin ldquoMangosteen xanthones mitigate ovalbumin-inducedairway inflammation in a mouse model of asthmardquo Food andChemical Toxicology vol 50 no 11 pp 4042ndash4050 2012

[5] S Syam A Bustamam R Abdullah et al ldquo120573 Mangostinsuppress LPS-induced inflammatory response in RAW 2647macrophages in vitro and carrageenan-induced peritonitis invivordquo Journal of Ethnopharmacology vol 153 no 2 pp 435ndash4452014

[6] J Yuan Y Wu and G Lu ldquoA-mangostin suppresseslipopolysaccharide-induced invasion by inhibiting matrixmetalloproteinase-29 and increasing E-cadherin expressionthrough extracellular signal-regulated kinase signaling inpancreatic cancer cellsrdquo Oncology Letters vol 5 no 6 pp1958ndash1964 2013

[7] M-A Shibata M Iinuma J Morimoto et al ldquo120572-Mangostinextracted from the pericarp of the mangosteen (Garcinia man-gostana Linn) reduces tumor growth and lymph node metas-tasis in an immunocompetent xenograft model of metastaticmammary cancer carrying a p53mutationrdquo BMCMedicine vol9 article 69 2011

[8] A F A Aisha K M Abu-Salah Z Ismail and A MajidldquoIn vitro and in vivo anti-colon cancer effects of Garciniamangostana xanthones extractrdquo BMC Complementary andAlternative Medicine vol 12 article 104 2012

[9] S Taokaew N Nunkaew P Siripong and M PhisalaphongldquoCharacteristics and anticancer properties of bacterial cellulose

films containing ethanolic extract of mangosteen peelrdquo Journalof Biomaterials Science Polymer Edition vol 25 no 9 pp 907ndash922 2014

[10] P Pan-In S Wanichwecharungruang J Hanes and A J KimldquoCellular trafficking and anticancer activity of Garcinia man-gostana extract-encapsulated polymeric nanoparticlesrdquo Inter-national Journal of Nanomedicine vol 9 no 1 pp 3677ndash36862014

[11] H R W Dharmaratne Y Sakagami K G P Piyasena and VThevanesam ldquoAntibacterial activity of xanthones fromGarciniamangostana (L) and their structure-activity relationship stud-iesrdquo Natural Product Research vol 27 no 10 pp 938ndash941 2013

[12] J-J Koh S Qiu H Zou et al ldquoRapid bactericidal action ofalpha-mangostin against MRSA as an outcome of membranetargetingrdquo Biochimica et Biophysica ActamdashBiomembranes vol1828 no 2 pp 834ndash844 2013

[13] M T Chomnawang S Surassmo K Wongsariya and N Bun-yapraphatsara ldquoAntibacterial activity of Thai medicinal plantsagainstmethicillin-resistant Staphylococcus aureusrdquo Fitoterapiavol 80 no 2 pp 102ndash104 2009

[14] S Suksamrarn N SuwannapochW Phakhodee et al ldquoAntimy-cobacterial activity of prenylated xanthones from the fruits ofGarcinia mangostanardquo Chemical and Pharmaceutical Bulletinvol 51 no 7 pp 857ndash859 2003

[15] P Moongkarndi C Srisawat P Saetun et al ldquoProtective effectof mangosteen extract against 120573-amyloid-induced cytotoxicityoxidative stress and altered proteome in SK-N-SH cellsrdquo Journalof Proteome Research vol 9 no 5 pp 2076ndash2086 2010

[16] Y-W Chin H-A Jung H Chai W J Keller and A DKinghorn ldquoXanthones with quinone reductase-inducing activ-ity from the fruits of Garcinia mangostana (Mangosteen)rdquoPhytochemistry vol 69 no 3 pp 754ndash758 2008

[17] H-A Jung B-N Su W J Keller R G Mehta and AD Kinghorn ldquoAntioxidant xanthones from the pericarp ofGarcinia mangostana (Mangosteen)rdquo Journal of Agriculturaland Food Chemistry vol 54 no 6 pp 2077ndash2082 2006

[18] F Gutierrez-Orozco and M L Failla ldquoBiological activities andbioavailability of mangosteen xanthones a critical review of thecurrent evidencerdquo Nutrients vol 5 no 8 pp 3163ndash3183 2013

[19] J Sattayasai P Chaonapan T Arkaravichie et al ldquoProtectiveeffects of mangosteen extract on H

2

O2

-induced cytotoxicity inSK-N-SH cells and scopolamine-induced memory impairmentin micerdquo PLoS ONE vol 8 no 12 Article ID e85053 2013

[20] E B Walker ldquoHPLC analysis of selected xanthones in mangos-teen fruitrdquo Journal of Separation Science vol 30 no 9 pp 1229ndash1234 2007

[21] F Gutierrez-Orozco J MThomas-Ahner L D Berman-Bootyet al ldquoDietary 120572-mangostin a xanthone from mangosteenfruit exacerbates experimental colitis and promotes dysbiosisin micerdquoMolecular Nutrition and Food Research vol 58 no 6pp 1226ndash1238 2014

[22] M Y IbrahimNMHashim A AMariod et al ldquo120572-Mangostinfrom Garcinia mangostana Linn an updated review of itspharmacological propertiesrdquo Arabian Journal of Chemistry vol9 no 3 pp 317ndash329 2016

[23] M Y Ibrahim N M Hashim S Mohan et al ldquo120572-Mangostinfrom Cratoxylum arborescens an in vitro and in vivo toxicolog-ical evaluationrdquo Arabian Journal of Chemistry vol 8 no 1 pp129ndash137 2015

[24] A Plazonic F Bucar Z Males A Mornar B Nigovic andN Kujundzic ldquoIdentification and quantification of flavonoids

10 Oxidative Medicine and Cellular Longevity

and phenolic acids in burr parsley (Caucalis platycarpos L)using high-performance liquid chromatography with diodearray detection and electrospray ionizationmass spectrometryrdquoMolecules vol 14 no 7 pp 2466ndash2490 2009

[25] P Moongkarndi N Jaisupa J Samer et al ldquoComparison of thebiological activity of two different isolates from mangosteenrdquoJournal of Pharmacy and Pharmacology vol 66 no 8 pp 1171ndash1179 2014

[26] E Fibach ldquoTechniques for studying stimulation of fetal hemo-globin production in human erythroid culturesrdquo Hemoglobinvol 22 no 5-6 pp 445ndash458 1998

[27] J S Stern J Peerson A T Mishra M V Sadasiva Rao andK P Rajeswari ldquoEfficacy and tolerability of a novel herbalformulation for weight managementrdquo Obesity vol 21 no 5 pp921ndash927 2013

[28] C M Spickett ldquoThe lipid peroxidation product 4-hydroxy-2-nonenal advances in chemistry and analysisrdquo Redox Biologyvol 1 no 1 pp 145ndash152 2013

[29] C Schneider N A Porter and A R Brash ldquoRoutes to 4-hydroxynonenal fundamental issues in themechanisms of lipidperoxidationrdquo Journal of Biological Chemistry vol 283 no 23pp 15539ndash15543 2008

[30] A Negre-Salvayre C Coatrieux C Ingueneau and R SalvayreldquoAdvanced lipid peroxidation end products in oxidative damageto proteins Potential role in diseases and therapeutic prospectsfor the inhibitorsrdquo British Journal of Pharmacology vol 153 no1 pp 6ndash20 2008

[31] M P Mattson ldquoRoles of the lipid peroxidation product 4-hydroxynonenal in obesity the metabolic syndrome and asso-ciated vascular and neurodegenerative disordersrdquo ExperimentalGerontology vol 44 no 10 pp 625ndash633 2009

[32] A Ayala M F Munoz and S Arguelles ldquoLipid peroxidationproduction metabolism and signaling mechanisms of malon-dialdehyde and 4-hydroxy-2-nonenalrdquo Oxidative Medicine andCellular Longevity vol 2014 Article ID 360438 31 pages 2014

[33] K Sterling ldquoThe turnover rate of serum albumin in manas measured by I131-tagged albuminrdquo The Journal of ClinicalInvestigation vol 30 no 11 pp 1228ndash1237 1951

[34] C J Lindsell R S Franco E P Smith C H Joiner and R MCohen ldquoA method for the continuous calculation of the age oflabeled red blood cellsrdquo American Journal of Hematology vol83 no 6 pp 454ndash457 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 9

enzymes In all the polar fraction of extract frommangosteenpericarp might benefit human patients with pathologicalconditions related to oxidative stress

Competing Interests

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

Acknowledgments

The authors are extremely grateful to Mrs Angkana Jong-sawaddipatana and all staff members of the Geriatric Clinicof Siriraj Hospital for their kind collaboration in recruit-ment of the participants This work was supported by theThai Traditional Medical Knowledge Fund Department forDevelopment of Thai Traditional and Alternative MedicineMinistry of Public Health Thailand

References

[1] S M Petiwala G Li A Ramaiya et al ldquoPharmacokineticcharacterization of mangosteen (Garcinia mangostana) fruitextract standardized to 120572-mangostin in C57BL6 micerdquo Nutri-tion Research vol 34 no 4 pp 336ndash345 2014

[2] Z Xu L Huang X-H Chen et al ldquoCytotoxic prenylated xan-thones from the pericarps of Garcinia mangostanardquo Moleculesvol 19 no 2 pp 1820ndash1827 2014

[3] F Gutierrez-Orozco C Chitchumroonchokchai G B Lesin-ski S Suksamrarn and M L Failla ldquo120572-Mangostin anti-inflammatory activity and metabolism by human cellsrdquo Journalof Agricultural and Food Chemistry vol 61 no 16 pp 3891ndash3900 2013

[4] H-Y Jang O-K Kwon S-R Oh H-K Lee K-S Ahn and Y-W Chin ldquoMangosteen xanthones mitigate ovalbumin-inducedairway inflammation in a mouse model of asthmardquo Food andChemical Toxicology vol 50 no 11 pp 4042ndash4050 2012

[5] S Syam A Bustamam R Abdullah et al ldquo120573 Mangostinsuppress LPS-induced inflammatory response in RAW 2647macrophages in vitro and carrageenan-induced peritonitis invivordquo Journal of Ethnopharmacology vol 153 no 2 pp 435ndash4452014

[6] J Yuan Y Wu and G Lu ldquoA-mangostin suppresseslipopolysaccharide-induced invasion by inhibiting matrixmetalloproteinase-29 and increasing E-cadherin expressionthrough extracellular signal-regulated kinase signaling inpancreatic cancer cellsrdquo Oncology Letters vol 5 no 6 pp1958ndash1964 2013

[7] M-A Shibata M Iinuma J Morimoto et al ldquo120572-Mangostinextracted from the pericarp of the mangosteen (Garcinia man-gostana Linn) reduces tumor growth and lymph node metas-tasis in an immunocompetent xenograft model of metastaticmammary cancer carrying a p53mutationrdquo BMCMedicine vol9 article 69 2011

[8] A F A Aisha K M Abu-Salah Z Ismail and A MajidldquoIn vitro and in vivo anti-colon cancer effects of Garciniamangostana xanthones extractrdquo BMC Complementary andAlternative Medicine vol 12 article 104 2012

[9] S Taokaew N Nunkaew P Siripong and M PhisalaphongldquoCharacteristics and anticancer properties of bacterial cellulose

films containing ethanolic extract of mangosteen peelrdquo Journalof Biomaterials Science Polymer Edition vol 25 no 9 pp 907ndash922 2014

[10] P Pan-In S Wanichwecharungruang J Hanes and A J KimldquoCellular trafficking and anticancer activity of Garcinia man-gostana extract-encapsulated polymeric nanoparticlesrdquo Inter-national Journal of Nanomedicine vol 9 no 1 pp 3677ndash36862014

[11] H R W Dharmaratne Y Sakagami K G P Piyasena and VThevanesam ldquoAntibacterial activity of xanthones fromGarciniamangostana (L) and their structure-activity relationship stud-iesrdquo Natural Product Research vol 27 no 10 pp 938ndash941 2013

[12] J-J Koh S Qiu H Zou et al ldquoRapid bactericidal action ofalpha-mangostin against MRSA as an outcome of membranetargetingrdquo Biochimica et Biophysica ActamdashBiomembranes vol1828 no 2 pp 834ndash844 2013

[13] M T Chomnawang S Surassmo K Wongsariya and N Bun-yapraphatsara ldquoAntibacterial activity of Thai medicinal plantsagainstmethicillin-resistant Staphylococcus aureusrdquo Fitoterapiavol 80 no 2 pp 102ndash104 2009

[14] S Suksamrarn N SuwannapochW Phakhodee et al ldquoAntimy-cobacterial activity of prenylated xanthones from the fruits ofGarcinia mangostanardquo Chemical and Pharmaceutical Bulletinvol 51 no 7 pp 857ndash859 2003

[15] P Moongkarndi C Srisawat P Saetun et al ldquoProtective effectof mangosteen extract against 120573-amyloid-induced cytotoxicityoxidative stress and altered proteome in SK-N-SH cellsrdquo Journalof Proteome Research vol 9 no 5 pp 2076ndash2086 2010

[16] Y-W Chin H-A Jung H Chai W J Keller and A DKinghorn ldquoXanthones with quinone reductase-inducing activ-ity from the fruits of Garcinia mangostana (Mangosteen)rdquoPhytochemistry vol 69 no 3 pp 754ndash758 2008

[17] H-A Jung B-N Su W J Keller R G Mehta and AD Kinghorn ldquoAntioxidant xanthones from the pericarp ofGarcinia mangostana (Mangosteen)rdquo Journal of Agriculturaland Food Chemistry vol 54 no 6 pp 2077ndash2082 2006

[18] F Gutierrez-Orozco and M L Failla ldquoBiological activities andbioavailability of mangosteen xanthones a critical review of thecurrent evidencerdquo Nutrients vol 5 no 8 pp 3163ndash3183 2013

[19] J Sattayasai P Chaonapan T Arkaravichie et al ldquoProtectiveeffects of mangosteen extract on H

2

O2

-induced cytotoxicity inSK-N-SH cells and scopolamine-induced memory impairmentin micerdquo PLoS ONE vol 8 no 12 Article ID e85053 2013

[20] E B Walker ldquoHPLC analysis of selected xanthones in mangos-teen fruitrdquo Journal of Separation Science vol 30 no 9 pp 1229ndash1234 2007

[21] F Gutierrez-Orozco J MThomas-Ahner L D Berman-Bootyet al ldquoDietary 120572-mangostin a xanthone from mangosteenfruit exacerbates experimental colitis and promotes dysbiosisin micerdquoMolecular Nutrition and Food Research vol 58 no 6pp 1226ndash1238 2014

[22] M Y IbrahimNMHashim A AMariod et al ldquo120572-Mangostinfrom Garcinia mangostana Linn an updated review of itspharmacological propertiesrdquo Arabian Journal of Chemistry vol9 no 3 pp 317ndash329 2016

[23] M Y Ibrahim N M Hashim S Mohan et al ldquo120572-Mangostinfrom Cratoxylum arborescens an in vitro and in vivo toxicolog-ical evaluationrdquo Arabian Journal of Chemistry vol 8 no 1 pp129ndash137 2015

[24] A Plazonic F Bucar Z Males A Mornar B Nigovic andN Kujundzic ldquoIdentification and quantification of flavonoids

10 Oxidative Medicine and Cellular Longevity

and phenolic acids in burr parsley (Caucalis platycarpos L)using high-performance liquid chromatography with diodearray detection and electrospray ionizationmass spectrometryrdquoMolecules vol 14 no 7 pp 2466ndash2490 2009

[25] P Moongkarndi N Jaisupa J Samer et al ldquoComparison of thebiological activity of two different isolates from mangosteenrdquoJournal of Pharmacy and Pharmacology vol 66 no 8 pp 1171ndash1179 2014

[26] E Fibach ldquoTechniques for studying stimulation of fetal hemo-globin production in human erythroid culturesrdquo Hemoglobinvol 22 no 5-6 pp 445ndash458 1998

[27] J S Stern J Peerson A T Mishra M V Sadasiva Rao andK P Rajeswari ldquoEfficacy and tolerability of a novel herbalformulation for weight managementrdquo Obesity vol 21 no 5 pp921ndash927 2013

[28] C M Spickett ldquoThe lipid peroxidation product 4-hydroxy-2-nonenal advances in chemistry and analysisrdquo Redox Biologyvol 1 no 1 pp 145ndash152 2013

[29] C Schneider N A Porter and A R Brash ldquoRoutes to 4-hydroxynonenal fundamental issues in themechanisms of lipidperoxidationrdquo Journal of Biological Chemistry vol 283 no 23pp 15539ndash15543 2008

[30] A Negre-Salvayre C Coatrieux C Ingueneau and R SalvayreldquoAdvanced lipid peroxidation end products in oxidative damageto proteins Potential role in diseases and therapeutic prospectsfor the inhibitorsrdquo British Journal of Pharmacology vol 153 no1 pp 6ndash20 2008

[31] M P Mattson ldquoRoles of the lipid peroxidation product 4-hydroxynonenal in obesity the metabolic syndrome and asso-ciated vascular and neurodegenerative disordersrdquo ExperimentalGerontology vol 44 no 10 pp 625ndash633 2009

[32] A Ayala M F Munoz and S Arguelles ldquoLipid peroxidationproduction metabolism and signaling mechanisms of malon-dialdehyde and 4-hydroxy-2-nonenalrdquo Oxidative Medicine andCellular Longevity vol 2014 Article ID 360438 31 pages 2014

[33] K Sterling ldquoThe turnover rate of serum albumin in manas measured by I131-tagged albuminrdquo The Journal of ClinicalInvestigation vol 30 no 11 pp 1228ndash1237 1951

[34] C J Lindsell R S Franco E P Smith C H Joiner and R MCohen ldquoA method for the continuous calculation of the age oflabeled red blood cellsrdquo American Journal of Hematology vol83 no 6 pp 454ndash457 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Oxidative Medicine and Cellular Longevity

and phenolic acids in burr parsley (Caucalis platycarpos L)using high-performance liquid chromatography with diodearray detection and electrospray ionizationmass spectrometryrdquoMolecules vol 14 no 7 pp 2466ndash2490 2009

[25] P Moongkarndi N Jaisupa J Samer et al ldquoComparison of thebiological activity of two different isolates from mangosteenrdquoJournal of Pharmacy and Pharmacology vol 66 no 8 pp 1171ndash1179 2014

[26] E Fibach ldquoTechniques for studying stimulation of fetal hemo-globin production in human erythroid culturesrdquo Hemoglobinvol 22 no 5-6 pp 445ndash458 1998

[27] J S Stern J Peerson A T Mishra M V Sadasiva Rao andK P Rajeswari ldquoEfficacy and tolerability of a novel herbalformulation for weight managementrdquo Obesity vol 21 no 5 pp921ndash927 2013

[28] C M Spickett ldquoThe lipid peroxidation product 4-hydroxy-2-nonenal advances in chemistry and analysisrdquo Redox Biologyvol 1 no 1 pp 145ndash152 2013

[29] C Schneider N A Porter and A R Brash ldquoRoutes to 4-hydroxynonenal fundamental issues in themechanisms of lipidperoxidationrdquo Journal of Biological Chemistry vol 283 no 23pp 15539ndash15543 2008

[30] A Negre-Salvayre C Coatrieux C Ingueneau and R SalvayreldquoAdvanced lipid peroxidation end products in oxidative damageto proteins Potential role in diseases and therapeutic prospectsfor the inhibitorsrdquo British Journal of Pharmacology vol 153 no1 pp 6ndash20 2008

[31] M P Mattson ldquoRoles of the lipid peroxidation product 4-hydroxynonenal in obesity the metabolic syndrome and asso-ciated vascular and neurodegenerative disordersrdquo ExperimentalGerontology vol 44 no 10 pp 625ndash633 2009

[32] A Ayala M F Munoz and S Arguelles ldquoLipid peroxidationproduction metabolism and signaling mechanisms of malon-dialdehyde and 4-hydroxy-2-nonenalrdquo Oxidative Medicine andCellular Longevity vol 2014 Article ID 360438 31 pages 2014

[33] K Sterling ldquoThe turnover rate of serum albumin in manas measured by I131-tagged albuminrdquo The Journal of ClinicalInvestigation vol 30 no 11 pp 1228ndash1237 1951

[34] C J Lindsell R S Franco E P Smith C H Joiner and R MCohen ldquoA method for the continuous calculation of the age oflabeled red blood cellsrdquo American Journal of Hematology vol83 no 6 pp 454ndash457 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom