melinjo (gnetum gnemon l.) seed extract decreases serum uric

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 589169 9 pageshttpdxdoiorg1011552013589169

Research ArticleMelinjo (Gnetum gnemon L) Seed Extract DecreasesSerum Uric Acid Levels in Nonobese Japanese MalesA Randomized Controlled Study

Hiroyuki Konno1 Yoshiaki Kanai1 Mikiyuki Katagiri1

Tami Watanabe2 Akemi Mori2 Tomoki Ikuta2 Hiroko Tani2 Shinobu Fukushima2

Tomoki Tatefuji2 and Takuji Shirasawa1

1 Department of Aging Control Medicine Juntendo University Graduate School of Medicine Bunkyo-Ku Tokyo 113-0033 Japan2 Institute for Bee Products amp Health Science Yamada Bee Company Inc 194 Ichiba Kagamino-cho Okayama 708-0393 Japan

Correspondence should be addressed to Takuji Shirasawa shirasawashirasawa-aclnet

Received 27 September 2013 Accepted 24 November 2013

Academic Editor Syed Ibrahim Rizvi

Copyright copy 2013 Hiroyuki Konno 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

Melinjo (Gnetum gnemon L) seed extract (MSE) containing trans-resveratrol (3541015840-trihydroxy-trans-stilbene) and otherderivatives exerts various beneficial effects However its mechanism of action in humans remains unknown In this study weaimed to investigate beneficial effects of MSE in healthy adult males In this double-blind randomized controlled study 30 malesaged 35ndash70 years with le10 flow-mediated dilatation received placebo or 750mgMSE powder for 8 weeks and twenty-nine males(451 plusmn 88 years old) completed the trial There was a significant difference in the melinjo and placebo groups Compared withthe placebo control MSE significantly reduced serum uric acid at 4 weeks and 8 weeks (119899 = 14 and 15 resp) HDL cholesterolwas significantly increased in the melinjo group To clarify the mechanism of MSE for reducing uric acid we investigated xanthineoxidase inhibitory activity angiotensin II type 1 (AT1) receptor binding inhibition rate and agonistic activities for PPAR120572 andPPAR120574 MSE trans-resveratrol and a resveratrol dimer gnetin C (GC) significantly inhibit AT1 receptor binding and exhibit mildagonistic activities for PPAR120572 and PPAR120574 In conclusion MSE may decrease serum uric acid regardless of insulin resistance andmay improve lipid metabolism by increasing HDL cholesterol

1 Introduction

Melinjo (Gnetum gnemon L) belongs to the family Gne-taceae native to Indonesia The tree is small to mediumin size 15ndash20m tall with evergreen leaves The fruit-likestrobilus consists of little skin and a large nut-like seed thatis 2ndash4 cm long inside with both the fruits and leaves beingvery popular in Indonesian cuisines

Kato et al found that melinjo seed extract (MSE)contains various stilbenoids including trans-resveratrol(3541015840-trihydroxy-trans-stilbene) gnetin C (GC resveratroldimer) gnetin L (GC derivative) gnemonoside A (GC-diglucoside) gnemonoside C (GC-monoglucoside) andgnemonoside D (GC-monoglucoside) [1] These derivativesare collectively referred to as ldquoMelinjo resveratrolrdquo Recently

trans-resveratrol has attracted considerable attentionbecause it extended the lifespan of mice that were fed ahigh-calorie diet [2] Moreover human studies indicatedthat trans-resveratrol is beneficial in the management ofdiabetes [3] and cardiovascular diseases [4] Howeverseveral in vitro studies on the resveratrol derivatives in MSErevealed its nutraceutical effects such as the inhibition oflipase and amylase antibacterial properties [1] inhibitionof angiogenesis [5] and immunostimulatory effects [6] Inmice MSE was reported to suppress body weight gain andimprove insulin resistance [7] However the clinical efficacyof MSE remains unknown in humans Therefore to evaluatethe effects of MSE on humans we designed this clinicalstudy using healthy volunteers and evaluated the variousbiomarkers in association with metabolic syndrome

2 Evidence-Based Complementary and Alternative Medicine

2 Materials and Methods

21 Clinical Study Design The present study was a ran-domized double-blinded and placebo-controlled trial withparallel groups We conducted the study according to theguidelines in the Declaration of Helsinki All proceduresinvolving human participants were approved by the Shira-sawa Clinical Research Center Ethical Review Board Eachparticipant provided written and informed consent prior toparticipation

22 Participants Adult males aged 35ndash70 years with le10flow-mediated dilation (FMD) determined at the time ofscreening were recruited for this study from August toSeptember 2011 through newspaper advertisements at theShirasawa Clinical Research Center Tatebayashi city Gunmaprefecture Japan Exclusion criteria included the followingconditions consumption of functional foods related to lipidand glucose metabolism allergy to melinjo drinking redwine that contains trans-resveratrol regularly receivingmed-ication for hypertension diabetes or hyperlipidemia andpreexisting severe liver renal or heart disease In addition weexcluded the volunteers who participated in another clinicalstudy within two months and were not judged to meet theconditions by the doctor responsible for the study

The enrolled participants who met the inclusion crite-ria were randomly assigned into the melinjo and placebogroups by using computer-generated random numbers Eachparticipant in the melinjo group consumed five capsulescontaining 750mg MSE powder every morning (once daily)for eight weeks while each participant in the placebo groupconsumed five placebo capsules following the same protocolThe participants could not distinguish the difference betweenthe two types of capsules with respect to their shape sizeweight and color The participants were advised not to con-sume other health foods during the study They attended theShirasawa Clinical Research Center for clinical assessment atthe following three study time points baseline (0) 4 and 8weeks Finally 29 adult males (age 451 plusmn 88 years BMI244 plusmn 19 kgm2) participated in and completed the trial

23 Test Substances All MSE and placebo capsules weresupplied by the Yamada Bee Company Inc The seeds(endosperms) of melinjo were collected in Indonesia (DesaBangkok Kecamatan Gurah Kabupaten Kediri Kediri JawaTimur) in July 2009The dried endosperms ofmelinjo (250 g)were powdered and soaked in 55 EtOH (750mL) at roomtemperature for 3 days to obtain MSE (23 g) Dextrin (039 g)and water (5 g) were added to 625 g MSE and lyophilizedto prepare the MSE powder used for biological experimentsIn order to confirm the safety of the MSE powder theYamada Bee Company Inc conducted a repeated dose studyin humans Every morning for 28 days 44 healthy volunteersaged 32ndash49 years were administered a maximum of 5000mgMSE powderThroughout the study no clinically noteworthyabnormalities were observed (unpublished data)

In our study one melinjo capsule contained 150mg MSEpowder 100mg dextrin 29mg cellulose and 9mg sugar

ester The MSE powder contained gt20 the resveratrolderivatives Regarding trans-resveratrol the content ratio was01 On the other hand one placebo capsule contained250mg dextrin 29mg cellulose and 9mg sugar ester Theappearance of the capsules used for both groupswas identical

24 Clinical Assessments The participants were instructed toarrive without having consumed anything for at least 8 h onthe examination day Before the initiation of the trial (week0) and again at four and eight weeks all participants wentthrough FMD pulse wave velocity (PWV) ankle-brachialindex (ABI) body weight fat percentage BMI and a generalexamination including blood pressure pulse rate and bloodchemistry analysis (levels of total protein albumin albuminglobulin ratio total bilirubin aspartate aminotransferase ala-nine transaminase 120574-GTP total cholesterol HDL cholesterollow-density lipoprotein (LDL) cholesterol arterioscleroticindex remnant-like particles (RLP) cholesterol triglyceridesuric acid urea nitrogen creatinine sodium potassiumchlorine HOMA-IR fasting immunoreactive insulin bloodsugar hemoglobin A1c total homocycteine and N-terminalprohormone of brain natriuretic peptide (NT-proBNP) thereactive oxygen metabolites-derived compounds (d-ROMs)test the Biological Antioxidant Potential (BAP) test whiteblood cell and red blood cell counts hemoglobin lev-els hematocrit value mean cell volume levels of meancell hemoglobin mean cell hemoglobin concentration andplatelets) and urinalysis (specific gravity pH protein glu-cose ketones blood bilirubin and urobilinogen) (Table 1)

For the evaluation of endothelial function in metabolicsyndrome FMD was measured in the right brachial arteryusing UNEXEF38G (UNEX Corporation Nagoya Japan)specialized in measuring FMD this device is a combinationof ultrasonography and a sphygmomanometer The probe ofthis device is composed of two probes to capture the minoraxis of the vessel and one probe to capture the long axis ofthe vessels during the two probes With the three probes theposition of a brachial artery and the long axis can be easilylocated and the vessel diameter can be accurately measuredIn addition the equipment can automatically measure FMDafter 5min of avascularizationThe participants were reclinedon the bed in a supine position during the FMD test Theywere fitted with a cuff which was positioned on the rightupper arm abutting the cubital fossa We maintained thelaboratory room temperature at 25plusmn1∘C taking into accountits effects on the examination [8]

The arteriosclerosis index was assessed using PWV andABI by BP-203RPE III (Omron Healthcare Co Ltd TokyoJapan) This device has four cuffs that can simultaneouslymeasure blood pressure levels in both arms and both legs andautomatically calculate ABI Moreover the device can recordpulsewaves via sensors in the cuffs calculate the transmissiondistance from the right arm to each ankle according to bodyheight and automatically compute and output the bilateralbrachial-ankle PWV (baPWV) values using the transmissiontime and distance

Evidence-Based Complementary and Alternative Medicine 3

Table 1 The effects of melinjo seed extract administration for 8 weeks in adult men

Week 0 Week 4 Week 8

119875Melinjo (119899 = 14) Placebo (119899 = 15) Melinjo (119899 = 14) Placebo (119899 = 15) Melinjo (119899 = 14) Placebo (119899 = 15)Means plusmn SD Means plusmn SD Means plusmn SD Means plusmn SD Means plusmn SD Means plusmn SD

Body weight (kg) 708 plusmn 95 704 plusmn 59 715 plusmn 99 710 plusmn 64 714 plusmn 100 708 plusmn 62 096Fat percentage () 221 plusmn 38 225 plusmn 34 229 plusmn 46 235 plusmn 32 229 plusmn 47 234 plusmn 34 0946BMI (kgm2) 245 plusmn 26 243 plusmn 11 248 plusmn 26 245 plusmn 13 247 plusmn 26 244 plusmn 12 0965Systolic bloodpressure (mmHg) 1157 plusmn 107 118 plusmn 147 1153 plusmn 92 1189 plusmn 130 1176 plusmn 105 1195 plusmn 133 0708

Diastolic bloodpressure (mmHg) 749 plusmn 93 77 plusmn 118 757 plusmn 70 764 plusmn 104 752 plusmn 68 765 plusmn 93 0756

Pulse (bpm) 606 plusmn 59 591 plusmn 87 613 plusmn 68 607 plusmn 86 629 plusmn 94 636 plusmn 118 0711FMD () 55 plusmn 26 58 plusmn 21 67 plusmn 16 61 plusmn 22 57 plusmn 24 68 plusmn 18 0233baPWV (right) (cms) 12104 plusmn 1459 1297 plusmn 1493 12365 plusmn 1127 12825 plusmn 1624 12835 plusmn 1563 13149 plusmn 1564 027baPWV (left) (cms) 12261 plusmn 1380 13241 plusmn 1737 12419 plusmn 1278 13051 plusmn 1569 12997 plusmn 1720 13375 plusmn 1755 0287Ankle-brachial index(right) 114 plusmn 006 117 plusmn 006 113 plusmn 007 116 plusmn 004 113 plusmn 007 118 plusmn 004 0549

Ankle-brachial index(left) 114 plusmn 005 116 plusmn 005 113 plusmn 007 114 plusmn 005 112 plusmn 008 116 plusmn 004 041

Total protein (gdL) 71 plusmn 04 70 plusmn 03 712 plusmn 03 71 plusmn 03 72 plusmn 03 71 plusmn 04 0156Albumin (gdL) 44 plusmn 02 44 plusmn 02 44 plusmn 02 44 plusmn 03 45 plusmn 02 44 plusmn 01 0771Total bilirubin(mgdL) 10 plusmn 04 10 plusmn 07 09 plusmn 03 10 plusmn 08 09 plusmn 04 09 plusmn 07 0956

Aspartateaminotransferase(UL)

281 plusmn 106 217 plusmn 74 228 plusmn 56 221 plusmn 52 227 plusmn 59 220 plusmn 56 0074

Alanineaminotransferase(UL)


Gamma-glutamyltranspeptidase (UL) 441 plusmn 338 363 plusmn 228 429 plusmn 350 429 plusmn 345 425 plusmn 407 421 plusmn 352 0289

Total cholesterol(mgdL) 1939 plusmn 448 2143 plusmn 267 1964 plusmn 459 2191 plusmn 281 1990 plusmn 378 2139 plusmn 297 0654

HDL cholesterol(mgdL) 524 plusmn 114 512 plusmn 129 541 plusmn 116 507 plusmn 114 574 plusmn 126 514 plusmn 137 0111

LDL cholesterol(mgdL) 1229 plusmn 389 1353 plusmn 278 1235 plusmn 422 1380 plusmn 243 1245 plusmn 347 1357 plusmn 300 0871

Triglycerides (mgdL) 1063 plusmn 650 1445 plusmn 1047 1147 plusmn 647 990 plusmn 477 1185 plusmn 745 1731 plusmn 1366 0381Arteriosclerotic index 29 plusmn 14 34 plusmn 10 28 plusmn 12 35 plusmn 10 26 plusmn 11 34 plusmn 11 0346Uric acid (mgdL) 67 plusmn 15 66 plusmn 11 63 plusmn 14 67 plusmn 09 61 plusmn 14 66 plusmn 11 0009lowast

Blood urea nitrogen(mgdL) 136 plusmn 37 138 plusmn 44 133 plusmn 40 133 plusmn 23 131 plusmn 37 129 plusmn 27 0992

Creatinine (mgdL) 09 plusmn 01 09 plusmn 01 08 plusmn 01 09 plusmn 01 08 plusmn 01 09 plusmn 01 0283Sodium (mEqL) 1399 plusmn 21 1389 plusmn 18 1389 plusmn 22 1389 plusmn 26 1391 plusmn 23 1391 plusmn 24 0629Potassium (mEqL) 43 plusmn 05 42 plusmn 03 44 plusmn 04 45 plusmn 03 44 plusmn 03 45 plusmn 04 0284Chlorine (mEqL) 1025 plusmn 25 1022 plusmn 19 1011 plusmn 23 1019 plusmn 26 1013 plusmn 22 1019 plusmn 20 074Blood sugar (mgdL) 928 plusmn 137 926 plusmn 44 936 plusmn 135 935 plusmn 39 943 plusmn 187 959 plusmn 82 0837Hemoglobin A1c () 51 plusmn 05 50 plusmn 03 50 plusmn 07 50 plusmn 02 50 plusmn 07 51 plusmn 03 058Albumin globulinratio 17 plusmn 02 17 plusmn 02 16 plusmn 02 16 plusmn 03 16 plusmn 02 16 plusmn 02 0666

White blood cell(times104120583L) 48467 plusmn 11862 62667 plusmn 27281 47600 plusmn 10253 60667 plusmn 21380 48600 plusmn 12403 60400 plusmn 22334 0894


Table 1 Continued



Red blood cell(times104120583L) 4966 plusmn 316 4801 plusmn 247 5035 plusmn 346 4911 plusmn 224 5084 plusmn 283 4833 plusmn 219 0185

Hemoglobin (gdL) 152 plusmn 06 149 plusmn 08 155 plusmn 07 154 plusmn 07 157 plusmn 06 152 plusmn 07 0161

Hematocrit () 442 plusmn 21 432 plusmn 19 449 plusmn 20 446 plusmn 20 455 plusmn 17 440 plusmn 19 015

Mean cell volume (fL) 891 plusmn 27 903 plusmn 32 895 plusmn 31 909 plusmn 32 897 plusmn 35 913 plusmn 29 0862Mean cell hemoglobin(pg) 306 plusmn 14 310 plusmn 14 308 plusmn 15 315 plusmn 11 308 plusmn 15 315 plusmn 11 02

Mean cell hemoglobinconcentration (gdL) 343 plusmn 10 344 plusmn 08 344 plusmn 10 346 plusmn 07 344 plusmn 10 346 plusmn 07 0756

Platelets (times104120583L) 232 plusmn 48 218 plusmn 54 240 plusmn 41 224 plusmn 63 243 plusmn 42 235 plusmn 77 0685

Fasting IRI (120583UmL) 51 plusmn 29 49 plusmn 22 54 plusmn 34 70 plusmn 54 54 plusmn 35 71 plusmn 65 0407

HOMA-IR 12 plusmn 08 11 plusmn 05 13 plusmn 08 16 plusmn 13 13 plusmn 09 17 plusmn 17 0404

NT-proBNP (pgmL) 765 plusmn 1954 321 plusmn 200 260 plusmn 167 262 plusmn 222 203 plusmn 144 251 plusmn 231 0505RLP cholesterol(mgdL) 69 plusmn 48 89 plusmn 84 76 plusmn 53 130 plusmn 119 69 plusmn 49 110 plusmn 116 0242

Total homocysteine(mgdL) 135 plusmn 52 146 plusmn 84 117 plusmn 25 147 plusmn 89 112 plusmn 28 125 plusmn 46 0399

d-ROMs test(UCARR) 3308 plusmn 584 3316 plusmn 552 3380 plusmn 536 3456 plusmn 522 3565 plusmn 462 3650 plusmn 605 0781

BAP test (120583molL) 25853 plusmn 1742 24715 plusmn 1893 23411 plusmn 919 22770 plusmn 1157 24419 plusmn 1785 23294 plusmn 1799 0633

Urine PH 57 plusmn 09 57 plusmn 12 55 plusmn 05 62 plusmn 11 61 plusmn 09 56 plusmn 08 0008lowast

Urine specific gravity 1021 plusmn 0006 1019 plusmn 0008 1023 plusmn 0004 1019 plusmn 0008 1022 plusmn 0004 1018 plusmn 0007 0689BAP biological antioxidant potential d-ROMS reactive oxygen metabolites-derived compounds LDL low-density lipoprotein NT-proBNP amino-terminalprobrain natriuretic peptide RLP remnant-like particles Values are given as means plusmn SD 119875 for interaction lowast119875 lt 005

25 Statistical Analysis Statistical analyses were performedusing SPSS version 20 (IBM Corporation NY USA) Sta-tistical comparisons between groups were calculated usingtwo-way factorial ANOVA The factors were the assignmentand the survey period and the dependent variables werethe evaluation criteriaMultiple comparisonswere performedusing Tukeyrsquos HSD test Values of 119875 lt 005 were consideredsignificant Results are presented as means plusmn SD

26 In Vitro Experiments

261 Assay of Xanthine Oxidase Activity The assay mixtureconsisting of 50mL test solution and 50mL enzyme solution(005 unitsmL in 70mM phosphate buffer pH 75) wasprepared immediately before use After preincubation at 25∘Cfor 15min the reactionwas initiated by the addition of 100mLsubstrate solution (800mMxanthine in the same buffer)Theassay mixture was incubated at 25∘C for 30minThe reactionwas stopped by adding 20mL of 1MHCl and 20mL volumesof diluted 20mM potassium dihydrogenphosphate onto aSunniest RP-AQUQ column (46mm ID times 150mm) Themobile phasewas acetonitrile20mMpotassiumdihydrogen-phosphate (1 99 vv) at a flow rate of 08mLmin Furtheruric acid was detected by its UV absorbance at 290 nmThe retention time of uric acid on this system was 49min

A blank was prepared in the same way but the enzymesolution was added to the assay mixture after adding 1MHCl One unit of XO is defined as the amount of enzymerequired to produce 1mmol of uric acidmin at 25∘C TheXO inhibitory activity was expressed as the percentageinhibition of XO in the above assay system calculated as(1 minus 119861119860) times 100 where 119860 and 119861 are the activities of theenzyme without and with test material respectivelyThe IC

50

values were calculated from the mean values of data from thefour determinations The extracts were dissolved initially inEtOH followed by dilution with the buffer the final EtOHconcentration was lt5 Allopurinol a known XO inhibitorwas used as a positive control

262 Evaluation of the Binding Inhibition Rate of AngiotensinII Type 1 Receptor Evaluation of the affinity of compoundsfor the human angiotensin II type 1 (AT1) receptor in thetransfected HEK-293 cells was determined using a radioli-gand binding assay The cell membrane homogenates (8120583gprotein) were incubated for 120min at 37∘C with 005 nM[125I][Sar1-Ile8] angiotensin II both in the absence or pres-ence of the test compound in a buffer containing 50mMTris-HCl (pH 74) 5mM MgCl

2 1 mM EDTA and 01

BSA Nonspecific binding was determined in the presenceof 10 120583M angiotensin II Following incubation the samples


were rapidly filtered under vacuum through glass fiber filters(GFB Packard) presoaked with 03 PEI and rinsed severaltimes with ice-cold 50mM Tris-HCl using a 96-sample cellharvester (Unifilter Packard) Thereafter the filters weredried andmeasured for radioactivity in a scintillation counter(Topcount Packard) using a scintillation cocktail (Microscint0 Packard)The results were expressed as a percent inhibitionof the control radioligand specific binding The standardreference compound was saralasin which was tested in eachexperiment at several concentrations to obtain a competitioncurve from which its IC

50was calculated

263 Assay of Peroxisome Proliferator-Activated Receptor(PPAR) 120572 and 120574 Agonist Activity The COS-1 cells werecollected by processing of trypsin centrifuged at 1000 rpmfor 3min at 4∘C After removing the supernatant the cellswere seeded in 60mm culture dishes at a density of 5 times 105cellwell in a 2mL medium and cultured for 24 h at 37∘Cwith the presence of 5 CO

2 The Effectene Transfection

Reagent (QIAGEN Tokyo Japan) was used to transform thecells 150120583L Buffer EC 025 120583g pPPAR120572-Gal4 (or pPPAR120574-Gal4) 1 120583g pGal4-Luc 1 120583g pSEAP-control vector and 18 120583LEnhancer were added into a 15mL tube and the contents inthe tube were stirred with the vortex for 10 s Subsequentlyafter leaving for 3min at 25∘C 25 120583L Effectene was added tothe tubeThe contents were stirredwith the vortex for 10 s andwere left for 7min at 25∘CThemedium of the 60mm culturedish was removed and 4mL fresh medium was introducedthere during this time Subsequently 7min later 1mL culturemedium was added to the 15mL tube and was suspendedwith a pipette All the contents were dripped to the 60mmculture dish and the contents were incubated for 16 h at 37∘Cwith the presence of 5 CO

2

The transformed cells were collected by processingtrypsin The cells were centrifuged at 1000 rpm for 3minat 4∘C and the supernatant was removed The cells weresuspended in 10mL culturemedium andwere seeded in a 96-well plate with 125 120583L medium for each well The cells werethen cultured for 1-2 h at 37∘C with the presence of 5 CO

2

The test samples (125 120583L) were added to each well and werecultured with gentle stirring for 24 h at 37∘C in the presenceof 5 CO

2

The medium (25120583L) was removed from each well of the96-well plate and was transferred to each well of a 96-wellwhite plate Thereafter a solution for measurement of theluciferase activity was fused at 37∘C and the 100 120583L solutionwas added to the 100 120583Lmedium of the rest in each well Eachluminescence activity was measured after reacting for 35minin a dark place 25 120583L 1 times dilution buffer was added to each25 120583L medium which was collected from the 96-well plateIt was stirred gently and left for 30min at 65∘C Thereafterthey were cooled down to 4∘C and then back to 25∘C 90120583Lassay buffer was added to each well was gently stirred andwas left for 5min at 25∘C 10 120583LMUP solution was addedto each medium and it was gently stirred After reacting for60min at 25∘C in a dark place the fluorescence intensity (Ex=360 nm Em = 460 nm) based on 4-methylumbelliferone wasmeasured

3 Results

31 MSE Decreases the Serum Uric Acid Levels in HealthyVolunteers In order to study the beneficial effects of MSEwe designed a clinical trial of MSE with healthy volunteerswherein we evaluated the various biomarkers includingblood chemistry CBCs body weight blood pressure uri-nalysis pulse wave velocity (PWV) flow-mediated dilatation(FMD) and HOMA-IR (a biomarker of insulin sensitivity)(Table 1)

Healthy volunteers with administration of 750mg MSEpowder revealed a significant decrease in the uric acid levelsat four weeks (63 plusmn 14 versus 67 plusmn 09mgdL 119875 lt 005Figure 1(a)) and at eight weeks (61 plusmn 14 versus 66 plusmn 11mgdL119875 lt 005 Figure 1(a)) when compared to the placebo con-trol As presented in Figure 1(b) we confirmed a beneficialeffect of MSE at four weeks which was as well maintained ateight weeks suggesting the stable clinical benefit of MSE inthe long-term control of serum uric acid levels

Although a previous clinical study on trans-resveratroldemonstrated the improvements in insulin resistance andlipid profiles with human subjects [9] we failed to demon-strate these clinical benefits for MSE Interestingly in thisstudy we found a novel clinical benefit of MSE on uric acidBecause uric acid not only plays an important role as anantioxidant molecule but also as a biomarker for cardiovas-cular diseases and gout we explored the possibility that MSEconfers health benefits in the prevention of cardiovasculardiseases and gout

32 MSE May Inhibit AT1 Receptor Binding In order toclarify the mechanism of MSE in decreasing the serumuric acid levels we investigated the potential inhibition ofuric acid synthesis and uric acid reabsorption in the renaltubular epitheliaWith regard to the synthesis of uric acid wefirst investigated the inhibitory activity of MSE on xanthineoxidase Allopurinol a well-known chemical compound usedfor the treatment of gout effectively inhibited the xanthineoxidase activity with IC

50at a concentration of 023120583gmL

(Figure 2(a)) However MSE GC GC monoglucuronic acidconjugate and trans-resveratrol failed to demonstrate anyinhibitory activities on xanthine oxidase (Figures 2(b)ndash2(e)IC50

at the concentrations of 133 120583gmL 157120583gmL and350 120583gmL resp) suggesting that MSE decreases the serumuric acid levels by a mechanism other than the xanthine oxi-dase suppression Next we explored the possibility whetherMSE inhibits the reabsorption of uric acid in the renaltubules The inhibition of angiotensin decreases the serumuric acid levels by suppressing the reabsorption of uricacid from the renal tubular epithelia [10] In this paper weperformed in vitro investigation to evaluate the inhibitoryactivity of MSE on angiotensin as well as GC which revealedthat MSE and GC have a significant inhibitory activity onAT1 receptor binding whereas trans-resveratrol revealed noinhibitory activity These data suggest that MSE inhibits theangiotensin signal which then downregulates the transporterof uric acid however we cannot rule out the possibility thatother pathways are involved in the regulation of uric acid


90

80

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60

50

40

30

20

10

00

Time (weeks)

PlaceboMelinjo

Uric

acid

(mg

dL)

lowast

lowast

0 4 8

(a)

Time (weeks)

PlaceboMelinjo

lowastlowast

10

05

0

minus05

minus10

minus15

minus20

0 4 8

Uric

acid

(am

ount

of c

hang

e)(b)

Figure 1 The effects of MSE on the serum uric acid levels before and four or eight weeks after the administration of 750mgMSE or placebo(a) The serum uric acid levels significantly decreased in the melinjo group (119899 = 14) than in the placebo group (119899 = 15) (b) The changes inthe uric acid levels in the melinjo group were presented by an amount of changes in placebo group The effect of MSE at four weeks was aswell maintained at eight weeks Statistical significance was calculated using Tukeyrsquos HSD test Values are presented as means plusmn SD lowast119875 lt 005

because we have not evaluated all the relevant regulatorypathways

33 MSE May Increase the Serum HDL Cholesterol Levelsin Healthy Volunteers Although we failed to detect anybeneficial effects of MSE on LDL cholesterol we found asignificant increase in the HDL cholesterol levels in healthyvolunteers who consumed 750mg MSE powder for eightweeks as indicated in Figure 3(a) (524 plusmn 114 to 574 plusmn126mgdL 119875 lt 005) It is well known that HDL cholesteroltransports the deposited cholesterol from atheroscleroticlesions of blood vessels to the liver [11] and it also counteractsthe deleterious effect of LDL cholesterol in the pathogenesisof atherosclerosis In this context MSE may confer a benefitin the prevention of atherosclerosis without the alternation ofLDL metabolism

In order to clarify the molecular mechanisms thatincrease the HDL cholesterol levels on MSE administrationwe evaluated the agonistic activities for PPAR120572 and PPAR120574because these receptors can increase theHDL cholesterol lev-els [12ndash15] As displayed in Figures 3(b)ndash3(d) MSE or grapesextract revealed mild agonistic activities for PPAR120572 andPPAR120574 (Figures 3(b) and 3(c)) which is similarly identifiedwith trans-resveratrol (Figure 3(d)) The agonistic activitiesdetected here revealed weaker signals compared with thepositive controls WY1643 for PPAR120572 and troglitazone forPPAR120574 (Figures 3(b) 3(c) and 3(d)) For a better understand-ing of the mechanisms further studies are warranted on themolecular mechanism involved in the metabolism of HDLcholesterol

4 Discussion

The results of this study suggest that the MSE decreases theserum uric acid levels by inhibiting the reabsorption of uricacid in the renal tubular epithelia as well as by increasingthe HDL cholesterol levels by PPAR agonistic activity In thispaper we demonstrated for the first time the novel actionsof MSE which is distinct from trans-resveratrol The actionsdemonstrated here have not been previously reported withtrans-resveratrol [16ndash19]

In metabolic syndrome insulin resistance causes hyper-insulinemia which then leads to upregulation of serumuric acid by enhancing the reabsorption of serum uric acidin the renal tubules [20] In addition hyperinsulinemiadownregulates GAPDH one of the key glycolysis enzymeswhich can then activate the pentose phosphorylation pathwaywith a concomitant increase of purine synthesis de novo [21]It is unlikely that MSE downregulates uric acid by improvinginsulin resistance because we failed to identify any signs ofimprovement on HOMA-IR in the present study This resultsuggests that MSE downregulates the serum uric acid levelsindependently of insulin resistance

GC gnemonoside C and gnemonoside D in MSE arereported to inhibit the 120572-amylase activity [1] This inhibitoryactivity could suppress the rapid postprandial insulin secre-tion which inhibits the reabsorption of uric acid in the renaltubules Notably dysfunction of the ATP-binding cassettetransporter subfamily G member 2 (ABCG2) suppresses theexcretion of uric acid into intestine [22] Another possibilityis that MSE enhances ABCG2 to secrete more uric acid intothe intestine thereby decreasing the serum uric acid levels


100

80

60

40

20

00001 001 01 1 10

Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()

Allopurinol (120583gmL)

IC50 = 023 120583gmL

(a)

100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()

MSE (120583gmL)

IC50 gt 1000 120583gmL

(b)

10 100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()

GC (120583gmL)

IC50 = 133 120583gmL

(c)

10 100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()

GC monoglucuronic acid conjugate (120583gmL)

IC50 = 157 120583gmL

(d)

10 100

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()

trans-Resveratrol (120583gmL)

IC50 = 350 120583gmL

(e)

80

70

60

50

40

30

20

10

0MSE

300 120583gmLtrans-Resveratrol

30 120583MGC

30 120583M

AT1

rece

ptor

bin

ding

inhi

bitio

n ra

te (

)

(f)

Figure 2 The effects of the 50 inhibitory concentration (IC50) of MSE GC GC monoglucuronic acid conjugate and trans-resveratrol on

the xanthine oxidase inhibitory activity Allopurinol was used as a positive control (a) Allopurinol (b)MSE (c) GC (d) GCmonoglucuronicacid conjugate (e) trans-Resveratrol (f) AT1 receptor binding inhibition rate of MSE GC and trans-resveratrol

Further clinical studies would be required to confirm theefficacy as well as the optimized MSE dose for an efficientcontrol of uric acid and HDL cholesterol

Regarding the influence of trans-resveratrol on uricacid a few studies reported that in animals [23 24] Inartificially induced hyperuricemia in mice trans-resveratrol

and its analogues decreased the serum uric acid levels andincreased the uric acid excretion by regulating the renalorganic ion transporters [23] In addition in diabetic ratstrans-resveratrol decreased the serum uric acid levels [24]These findings suggest that trans-resveratrol decreases theserum uric acid levels in the presence of insulin resistance


80

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60

50

40

30

20

10

00 4 8

Time (weeks)

PlaceboMelinjo

HD

L ch

oles

tero

l (m

gdL

)

lowast

(a)

lowast

lowast14

12

10

8

6

4

2

0

PPA

R120572ag

onist

activ

ity (R

LU)

Positivecontrol

(WY14643)

Negativecontrol

(DMSO)

MSE100 120583gmL

Grapesextract

10 120583gmL

GC20 120583M

trans-Resveratrol

1 120583M

times103

(b)

lowast

lowast16141210

86420

Positive

(troglitazone)10 120583M

Negative

(DMSO)

MSE100 120583gmL

Grapes extract10 120583gmL

PPA

R120574ag

onist

activ

ity (R

LU)

times103

control control

(c)

lowast

GC10 120583M

trans-Resveratrol

10 120583M

PPA

R120574ag

onist

activ

ity (R

LU)

25

20

15

10

5

0

times103

Positive


Negative

(DMSO)control control

(d)

Figure 3 (a)The effects of MSE on the HDL cholesterol levels before and four or eight weeks after the administration of 750mgMSE powderor placebo Statistical analysis is presented in Figure 1 lowast119875 lt 005 (b) The PPAR120572 agonist activity of MSE grapes extract GC and trans-resveratrol (c)The PPAR120574 agonist activity of MSE and grapes extract (d)The PPAR120574 agonist activity of GC and trans-resveratrol Values arepresented as means plusmn SD lowast119875 lt 005

Considering these results of studies trans-resveratrol wouldnot have influenced serum uric acid level in our study

About the effects of trans-resveratrol on HDL choles-terol some clinical trials have shown that trans-resveratrolincreased HDL cholesterol However Sahebkar recently con-cluded that trans-resveratrol does not have a significanteffect of resveratrol supplementation on plasma lipid con-centrations in the meta-analysis of randomized controlledtrials (RCT) [18] In addition the range of trans-resveratroldoses was between 10mgday and 1500mgday in the RCTs[18] while the dose in our study was about 075mgdayit was very less than the selected studies Therefore trans-resveratrol would not have contributed to the increase ofHDL cholesterol level in MSE group However it might bepossible that trans-resveratrol was one of the ingredients inMSE that affected the parameters in our study since the effectsof trans-resveratrol on serum uric acid and HDL-cholesterolstill remain unclear

5 Conclusions

Since the relation between hyperuricemia and metabolicsyndrome has been pointed out these days [25] our studyshed light on the possibility that MSE decreases the serumuric acid levels Furthermore MSE may improve the lipidmetabolism by increasing the HDL cholesterol levels Nev-ertheless further research would be required to understandthe molecular mechanism for regulating uric acid and HDLcholesterol which is more specific for MSE and distinct fromtrans-resveratrol

Acknowledgments

The authors wish to thank Matsuura Nobuyasu AssociateProfessor of Department of Life Science OkayamaUniversityof Science for the cooperation provided in the evaluationof the PPAR agonist activities This study was funded by


the Institute for Bee Products and Health Science YamadaBee Company Inc Okayama Japan TamiWatanabe AkemiMori Tomoki Ikuta Hiroko Tani Shinobu Fukushima andTomoki Tatefuji are employees of the Yamada Bee CompanyInc No other authors declare any potential conflict ofinterests

References

[1] E Kato Y Tokunaga and F Sakan ldquoStilbenoids isolated fromthe seeds of melinjo (Gnetum gnemon L) and their biologicalactivityrdquo Journal of Agricultural and Food Chemistry vol 57 no6 pp 2544ndash2549 2009

[2] J A Baur K J Pearson N L Price et al ldquoResveratrol improveshealth and survival of mice on a high-calorie dietrdquo Nature vol444 no 7117 pp 337ndash342 2006

[3] P Brasnyo GAMolnarMMohas et al ldquoResveratrol improvesinsulin sensitivity reduces oxidative stress and activates the Aktpathway in type 2 diabetic patientsrdquo British Journal of Nutritionvol 106 no 3 pp 383ndash389 2011

[4] R H X Wong P R C Howe J D Buckley A M CoatesI Kunz and N M Berry ldquoAcute resveratrol supplementa-tion improves flow-mediated dilatation in overweightobeseindividuals with mildly elevated blood pressurerdquo NutritionMetabolism and Cardiovascular Diseases vol 21 no 11 pp 851ndash856 2011

[5] K Kunimasa T Ohta H Tani et al ldquoResveratrol derivative-rich melinjo (Gnetum gnemon L) seed extract suppressesmultiple angiogenesis-related endothelial cell functions andtumor angiogenesisrdquo Molecular Nutrition and Food Researchvol 55 no 11 pp 1730ndash1734 2011

[6] H Kato M Samizo R Kawabata F Takano and T OhtaldquoStilbenoids from the melinjo (Gnetum gnemon L) fruit mod-ulate cytokine production inmurine peyerrsquos patch cells ex vivordquoPlanta Medica vol 77 no 10 pp 1027ndash1034 2011

[7] D Matsuura ldquoThe anti-metabolic syndrome activity of melinjo(Gnetum gnemon L) seed extract powderrdquo Food Style 21 vol 16no 4 pp 20ndash22 2012 (Japanese)

[8] M E Widlansky J A Vita M J Keyes et al ldquoRelation ofseason and temperature to endothelium-dependent flow-medi-ated vasodilation in subjects without clinical evidence of cardio-vascular disease (from the Framingham Heart Study)rdquo Ameri-can Journal of Cardiology vol 100 no 3 pp 518ndash523 2007

[9] S Timmers E Konings L Bilet et al ldquoCalorie restriction-likeeffects of 30 days of resveratrol supplementation on energymetabolism and metabolic profile in obese humansrdquo CellMetabolism vol 14 no 5 pp 612ndash622 2011

[10] A J Reyes ldquoCardiovascular drugs and serumuric acidrdquoCardio-vascular Drugs and Therapy vol 17 no 5-6 pp 397ndash414 2003

[11] P P Toth ldquoThe ldquogood cholesterolrdquo high-density lipoproteinrdquoCirculation vol 111 no 5 pp e89ndashe91 2005

[12] S Abourbih K B Filion L Joseph et al ldquoEffect of fibrates onlipid profiles and cardiovascular outcomes a systematic reviewrdquoThe American Journal of Medicine vol 122 no 10 pp 962e1ndash962e8 2009

[13] L Berthou N Duverger F Emmanuel et al ldquoOpposite regu-lation of human versus mouse apolipoprotein A-I by fibratesin human apolipoprotein A-I transgenic micerdquo The Journal ofClinical Investigation vol 97 no 11 pp 2408ndash2416 1996

[14] J A Dormandy B Charbonnel D J A Eckland et al ldquoSecond-ary prevention of macrovascular events in patients with type

2 diabetes in the PROactive Study (PROspective pioglitAzoneClinical Trial in macroVascular Events) a randomised con-trolled trialrdquoTheLancet vol 366 no 9493 pp 1279ndash1289 2005

[15] A Chawla W A Boisvert C-H Lee et al ldquoA PPAR120574-LXR-ABCA1 pathway inmacrophages is involved in cholesterol effluxand atherogenesisrdquoMolecular Cell vol 7 no 1 pp 161ndash171 2001

[16] O Vang N Ahmad C A Baile et al ldquoWhat is new for an oldmolecule systematic review and recommendations on the useof resveratrolrdquo PLoS ONE vol 6 no 6 Article ID e19881 2011

[17] S Timmers J Auwerx and P Schrauwen ldquoThe journey ofresveratrol from yeast to humanrdquo Aging vol 4 no 3 pp 146ndash158 2012

[18] A Sahebkar ldquoEffects of resveratrol supplementation on plasmalipids a systematic review and meta-analysis of randomizedcontrolled trialsrdquo Nutrition Reviews 2013

[19] O Vang ldquoWhat is new for resveratrol Is a new set of recom-mendations necessaryrdquo Annals of the New York Academy ofSciences vol 1290 no 1 pp 1ndash11 2013

[20] J C ter Maaten A Voorburg R J Heine P M terWee A J MDonker andRO B Gans ldquoRenal handling of urate and sodiumduring acute physiological hyperinsulinaemia in healthy sub-jectsrdquo Clinical Science vol 92 no 1 pp 51ndash58 1997

[21] F Leyva C S Wingrove I F Godsland and J C StevensonldquoThe glycolytic pathway to coronary heart disease a hypothe-sisrdquoMetabolism vol 47 no 6 pp 657ndash662 1998

[22] K Ichida H Matsuo T Takada et al ldquoDecreased extra-renalurate excretion is a common cause of hyperuricemiardquo NatureCommunications vol 3 pp 764ndash767 2012

[23] Y W Shi C P Wang L Liu et al ldquoAntihyperuricemic andnephroprotective effects of resveratrol and its analogues inhyperuricemic micerdquoMolecular Nutrition amp Food Research vol56 no 9 pp 1433ndash1444 2012

[24] P Palsamy and S Subramanian ldquoResveratrol a natural phytoa-lexin normalizes hyperglycemia in streptozotocin-nicotinam-ide induced experimental diabetic ratsrdquo Biomedicine and Phar-macotherapy vol 62 no 9 pp 598ndash605 2008

[25] X Sui T S Church R A Meriwether F Lobelo and S N BlairldquoUric acid and the development of metabolic syndrome inwomen and menrdquoMetabolism vol 57 no 6 pp 845ndash852 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


2 Materials and Methods

21 Clinical Study Design The present study was a ran-domized double-blinded and placebo-controlled trial withparallel groups We conducted the study according to theguidelines in the Declaration of Helsinki All proceduresinvolving human participants were approved by the Shira-sawa Clinical Research Center Ethical Review Board Eachparticipant provided written and informed consent prior toparticipation

22 Participants Adult males aged 35ndash70 years with le10flow-mediated dilation (FMD) determined at the time ofscreening were recruited for this study from August toSeptember 2011 through newspaper advertisements at theShirasawa Clinical Research Center Tatebayashi city Gunmaprefecture Japan Exclusion criteria included the followingconditions consumption of functional foods related to lipidand glucose metabolism allergy to melinjo drinking redwine that contains trans-resveratrol regularly receivingmed-ication for hypertension diabetes or hyperlipidemia andpreexisting severe liver renal or heart disease In addition weexcluded the volunteers who participated in another clinicalstudy within two months and were not judged to meet theconditions by the doctor responsible for the study

The enrolled participants who met the inclusion crite-ria were randomly assigned into the melinjo and placebogroups by using computer-generated random numbers Eachparticipant in the melinjo group consumed five capsulescontaining 750mg MSE powder every morning (once daily)for eight weeks while each participant in the placebo groupconsumed five placebo capsules following the same protocolThe participants could not distinguish the difference betweenthe two types of capsules with respect to their shape sizeweight and color The participants were advised not to con-sume other health foods during the study They attended theShirasawa Clinical Research Center for clinical assessment atthe following three study time points baseline (0) 4 and 8weeks Finally 29 adult males (age 451 plusmn 88 years BMI244 plusmn 19 kgm2) participated in and completed the trial

23 Test Substances All MSE and placebo capsules weresupplied by the Yamada Bee Company Inc The seeds(endosperms) of melinjo were collected in Indonesia (DesaBangkok Kecamatan Gurah Kabupaten Kediri Kediri JawaTimur) in July 2009The dried endosperms ofmelinjo (250 g)were powdered and soaked in 55 EtOH (750mL) at roomtemperature for 3 days to obtain MSE (23 g) Dextrin (039 g)and water (5 g) were added to 625 g MSE and lyophilizedto prepare the MSE powder used for biological experimentsIn order to confirm the safety of the MSE powder theYamada Bee Company Inc conducted a repeated dose studyin humans Every morning for 28 days 44 healthy volunteersaged 32ndash49 years were administered a maximum of 5000mgMSE powderThroughout the study no clinically noteworthyabnormalities were observed (unpublished data)

In our study one melinjo capsule contained 150mg MSEpowder 100mg dextrin 29mg cellulose and 9mg sugar

ester The MSE powder contained gt20 the resveratrolderivatives Regarding trans-resveratrol the content ratio was01 On the other hand one placebo capsule contained250mg dextrin 29mg cellulose and 9mg sugar ester Theappearance of the capsules used for both groupswas identical

24 Clinical Assessments The participants were instructed toarrive without having consumed anything for at least 8 h onthe examination day Before the initiation of the trial (week0) and again at four and eight weeks all participants wentthrough FMD pulse wave velocity (PWV) ankle-brachialindex (ABI) body weight fat percentage BMI and a generalexamination including blood pressure pulse rate and bloodchemistry analysis (levels of total protein albumin albuminglobulin ratio total bilirubin aspartate aminotransferase ala-nine transaminase 120574-GTP total cholesterol HDL cholesterollow-density lipoprotein (LDL) cholesterol arterioscleroticindex remnant-like particles (RLP) cholesterol triglyceridesuric acid urea nitrogen creatinine sodium potassiumchlorine HOMA-IR fasting immunoreactive insulin bloodsugar hemoglobin A1c total homocycteine and N-terminalprohormone of brain natriuretic peptide (NT-proBNP) thereactive oxygen metabolites-derived compounds (d-ROMs)test the Biological Antioxidant Potential (BAP) test whiteblood cell and red blood cell counts hemoglobin lev-els hematocrit value mean cell volume levels of meancell hemoglobin mean cell hemoglobin concentration andplatelets) and urinalysis (specific gravity pH protein glu-cose ketones blood bilirubin and urobilinogen) (Table 1)

For the evaluation of endothelial function in metabolicsyndrome FMD was measured in the right brachial arteryusing UNEXEF38G (UNEX Corporation Nagoya Japan)specialized in measuring FMD this device is a combinationof ultrasonography and a sphygmomanometer The probe ofthis device is composed of two probes to capture the minoraxis of the vessel and one probe to capture the long axis ofthe vessels during the two probes With the three probes theposition of a brachial artery and the long axis can be easilylocated and the vessel diameter can be accurately measuredIn addition the equipment can automatically measure FMDafter 5min of avascularizationThe participants were reclinedon the bed in a supine position during the FMD test Theywere fitted with a cuff which was positioned on the rightupper arm abutting the cubital fossa We maintained thelaboratory room temperature at 25plusmn1∘C taking into accountits effects on the examination [8]

The arteriosclerosis index was assessed using PWV andABI by BP-203RPE III (Omron Healthcare Co Ltd TokyoJapan) This device has four cuffs that can simultaneouslymeasure blood pressure levels in both arms and both legs andautomatically calculate ABI Moreover the device can recordpulsewaves via sensors in the cuffs calculate the transmissiondistance from the right arm to each ankle according to bodyheight and automatically compute and output the bilateralbrachial-ankle PWV (baPWV) values using the transmissiontime and distance























Table 1 Continued





















50



2 1 mM EDTA and 01




50was calculated




2


2


2


3 Results









90

80

70

60

50

40

30

20

10

00

Time (weeks)

PlaceboMelinjo

Uric

acid

(mg

dL)

lowast

lowast

0 4 8

(a)

Time (weeks)

PlaceboMelinjo

lowastlowast

10

05

0

minus05

minus10

minus15

minus20

0 4 8

Uric

acid

(am

ount

of c

hang

e)(b)





4 Discussion





100

80

60

40

20

00001 001 01 1 10

Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()


IC50 = 023 120583gmL

(a)

100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()

MSE (120583gmL)

IC50 gt 1000 120583gmL

(b)

10 100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()

GC (120583gmL)

IC50 = 133 120583gmL

(c)

10 100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()


IC50 = 157 120583gmL

(d)

10 100

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()


IC50 = 350 120583gmL

(e)

80

70

60

50

40

30

20

10

0MSE


30 120583MGC

30 120583M

AT1

rece

ptor

bin

ding

inhi

bitio

n ra

te (

)

(f)







80

70

60

50

40

30

20

10

00 4 8

Time (weeks)

PlaceboMelinjo

HD

L ch

oles

tero

l (m

gdL

)

lowast

(a)

lowast

lowast14

12

10

8

6

4

2

0

PPA

R120572ag

onist

activ

ity (R

LU)

Positivecontrol

(WY14643)

Negativecontrol

(DMSO)

MSE100 120583gmL

Grapesextract

10 120583gmL

GC20 120583M

trans-Resveratrol

1 120583M

times103

(b)

lowast

lowast16141210

86420

Positive


Negative

(DMSO)

MSE100 120583gmL


PPA

R120574ag

onist

activ

ity (R

LU)

times103

control control

(c)

lowast

GC10 120583M

trans-Resveratrol

10 120583M

PPA

R120574ag

onist

activ

ity (R

LU)

25

20

15

10

5

0

times103

Positive


Negative


(d)




5 Conclusions


Acknowledgments




References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































Table 1 Continued





















50



2 1 mM EDTA and 01




50was calculated




2


2


2


3 Results









90

80

70

60

50

40

30

20

10

00

Time (weeks)

PlaceboMelinjo

Uric

acid

(mg

dL)

lowast

lowast

0 4 8

(a)

Time (weeks)

PlaceboMelinjo

lowastlowast

10

05

0

minus05

minus10

minus15

minus20

0 4 8

Uric

acid

(am

ount

of c

hang

e)(b)





4 Discussion





100

80

60

40

20

00001 001 01 1 10

Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()


IC50 = 023 120583gmL

(a)

100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()

MSE (120583gmL)

IC50 gt 1000 120583gmL

(b)

10 100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()

GC (120583gmL)

IC50 = 133 120583gmL

(c)

10 100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()


IC50 = 157 120583gmL

(d)

10 100

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()


IC50 = 350 120583gmL

(e)

80

70

60

50

40

30

20

10

0MSE


30 120583MGC

30 120583M

AT1

rece

ptor

bin

ding

inhi

bitio

n ra

te (

)

(f)







80

70

60

50

40

30

20

10

00 4 8

Time (weeks)

PlaceboMelinjo

HD

L ch

oles

tero

l (m

gdL

)

lowast

(a)

lowast

lowast14

12

10

8

6

4

2

0

PPA

R120572ag

onist

activ

ity (R

LU)

Positivecontrol

(WY14643)

Negativecontrol

(DMSO)

MSE100 120583gmL

Grapesextract

10 120583gmL

GC20 120583M

trans-Resveratrol

1 120583M

times103

(b)

lowast

lowast16141210

86420

Positive


Negative

(DMSO)

MSE100 120583gmL


PPA

R120574ag

onist

activ

ity (R

LU)

times103

control control

(c)

lowast

GC10 120583M

trans-Resveratrol

10 120583M

PPA

R120574ag

onist

activ

ity (R

LU)

25

20

15

10

5

0

times103

Positive


Negative


(d)




5 Conclusions


Acknowledgments




References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















50was calculated




2


2


2


3 Results









90

80

70

60

50

40

30

20

10

00

Time (weeks)

PlaceboMelinjo

Uric

acid

(mg

dL)

lowast

lowast

0 4 8

(a)

Time (weeks)

PlaceboMelinjo

lowastlowast

10

05

0

minus05

minus10

minus15

minus20

0 4 8

Uric

acid

(am

ount

of c

hang

e)(b)





4 Discussion





100

80

60

40

20

00001 001 01 1 10

Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()


IC50 = 023 120583gmL

(a)

100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()

MSE (120583gmL)

IC50 gt 1000 120583gmL

(b)

10 100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()

GC (120583gmL)

IC50 = 133 120583gmL

(c)

10 100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()


IC50 = 157 120583gmL

(d)

10 100

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()


IC50 = 350 120583gmL

(e)

80

70

60

50

40

30

20

10

0MSE


30 120583MGC

30 120583M

AT1

rece

ptor

bin

ding

inhi

bitio

n ra

te (

)

(f)







80

70

60

50

40

30

20

10

00 4 8

Time (weeks)

PlaceboMelinjo

HD

L ch

oles

tero

l (m

gdL

)

lowast

(a)

lowast

lowast14

12

10

8

6

4

2

0

PPA

R120572ag

onist

activ

ity (R

LU)

Positivecontrol

(WY14643)

Negativecontrol

(DMSO)

MSE100 120583gmL

Grapesextract

10 120583gmL

GC20 120583M

trans-Resveratrol

1 120583M

times103

(b)

lowast

lowast16141210

86420

Positive


Negative

(DMSO)

MSE100 120583gmL


PPA

R120574ag

onist

activ

ity (R

LU)

times103

control control

(c)

lowast

GC10 120583M

trans-Resveratrol

10 120583M

PPA

R120574ag

onist

activ

ity (R

LU)

25

20

15

10

5

0

times103

Positive


Negative


(d)




5 Conclusions


Acknowledgments




References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















90

80

70

60

50

40

30

20

10

00

Time (weeks)

PlaceboMelinjo

Uric

acid

(mg

dL)

lowast

lowast

0 4 8

(a)

Time (weeks)

PlaceboMelinjo

lowastlowast

10

05

0

minus05

minus10

minus15

minus20

0 4 8

Uric

acid

(am

ount

of c

hang

e)(b)





4 Discussion





100

80

60

40

20

00001 001 01 1 10

Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()


IC50 = 023 120583gmL

(a)

100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()

MSE (120583gmL)

IC50 gt 1000 120583gmL

(b)

10 100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()

GC (120583gmL)

IC50 = 133 120583gmL

(c)

10 100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()


IC50 = 157 120583gmL

(d)

10 100

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()


IC50 = 350 120583gmL

(e)

80

70

60

50

40

30

20

10

0MSE


30 120583MGC

30 120583M

AT1

rece

ptor

bin

ding

inhi

bitio

n ra

te (

)

(f)







80

70

60

50

40

30

20

10

00 4 8

Time (weeks)

PlaceboMelinjo

HD

L ch

oles

tero

l (m

gdL

)

lowast

(a)

lowast

lowast14

12

10

8

6

4

2

0

PPA

R120572ag

onist

activ

ity (R

LU)

Positivecontrol

(WY14643)

Negativecontrol

(DMSO)

MSE100 120583gmL

Grapesextract

10 120583gmL

GC20 120583M

trans-Resveratrol

1 120583M

times103

(b)

lowast

lowast16141210

86420

Positive


Negative

(DMSO)

MSE100 120583gmL


PPA

R120574ag

onist

activ

ity (R

LU)

times103

control control

(c)

lowast

GC10 120583M

trans-Resveratrol

10 120583M

PPA

R120574ag

onist

activ

ity (R

LU)

25

20

15

10

5

0

times103

Positive


Negative


(d)




5 Conclusions


Acknowledgments




References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

80

60

40

20

00001 001 01 1 10

Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()


IC50 = 023 120583gmL

(a)

100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()

MSE (120583gmL)

IC50 gt 1000 120583gmL

(b)

10 100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()

GC (120583gmL)

IC50 = 133 120583gmL

(c)

10 100 1000

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()


IC50 = 157 120583gmL

(d)

10 100

100

80

60

40

20

0Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

()


IC50 = 350 120583gmL

(e)

80

70

60

50

40

30

20

10

0MSE


30 120583MGC

30 120583M

AT1

rece

ptor

bin

ding

inhi

bitio

n ra

te (

)

(f)







80

70

60

50

40

30

20

10

00 4 8

Time (weeks)

PlaceboMelinjo

HD

L ch

oles

tero

l (m

gdL

)

lowast

(a)

lowast

lowast14

12

10

8

6

4

2

0

PPA

R120572ag

onist

activ

ity (R

LU)

Positivecontrol

(WY14643)

Negativecontrol

(DMSO)

MSE100 120583gmL

Grapesextract

10 120583gmL

GC20 120583M

trans-Resveratrol

1 120583M

times103

(b)

lowast

lowast16141210

86420

Positive


Negative

(DMSO)

MSE100 120583gmL


PPA

R120574ag

onist

activ

ity (R

LU)

times103

control control

(c)

lowast

GC10 120583M

trans-Resveratrol

10 120583M

PPA

R120574ag

onist

activ

ity (R

LU)

25

20

15

10

5

0

times103

Positive


Negative


(d)




5 Conclusions


Acknowledgments




References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















80

70

60

50

40

30

20

10

00 4 8

Time (weeks)

PlaceboMelinjo

HD

L ch

oles

tero

l (m

gdL

)

lowast

(a)

lowast

lowast14

12

10

8

6

4

2

0

PPA

R120572ag

onist

activ

ity (R

LU)

Positivecontrol

(WY14643)

Negativecontrol

(DMSO)

MSE100 120583gmL

Grapesextract

10 120583gmL

GC20 120583M

trans-Resveratrol

1 120583M

times103

(b)

lowast

lowast16141210

86420

Positive


Negative

(DMSO)

MSE100 120583gmL


PPA

R120574ag

onist

activ

ity (R

LU)

times103

control control

(c)

lowast

GC10 120583M

trans-Resveratrol

10 120583M

PPA

R120574ag

onist

activ

ity (R

LU)

25

20

15

10

5

0

times103

Positive


Negative


(d)




5 Conclusions


Acknowledgments




References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















melinjo (gnetum gnemon l.) seed extract decreases serum uric

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